HK1243417B - Pyridone and aza-pyridone compounds and methods of use - Google Patents

Description

Pyridone and azapyridone compounds and methods of use

The present application is a divisional application of Chinese patent application No. 201180033536.8 filed on May 6, 2011, entitled “Pyridone and Azapyridone Compounds and Methods of Use”.

Technical Field

The present invention generally relates to compounds useful for treating conditions mediated by Bruton's tyrosine kinase (Btk), including inflammation, immune disorders, and cancer, and more specifically to compounds that inhibit Btk activity. The present invention also relates to methods for diagnosing or treating mammalian cells or related pathological conditions in vitro, in situ, and in vivo using the compounds.

Background Art

Protein kinases are the largest family of human enzymes, comprising over 500 proteins. Bruton's tyrosine kinase (Btk) is a member of the Tec family of tyrosine kinases and is a regulator of early B cell development and mature B cell activation, signaling, and survival.

B cell signal transduction through the B cell receptor (BCR) can produce a wide range of biological output signals (biological output), and the signal in turn depends on the developmental stage of the B cell. The intensity and duration of the BCR signal must be accurately regulated. Abnormal BCR-mediated signal transduction can cause dysregulated B cell activation and/or form pathogenic autoantibodies that cause multiple autoimmune diseases and/or inflammatory diseases. Mutations in Btk in humans lead to X-linked agammaglobulinemia (XLA). This disease is related to impaired B cell maturation, reduced immunoglobulin production, impaired immune response independent of T cells, and a significant weakening of the calcium signal that persists when BCR stimulates.

Evidence that Btk plays a role in allergic and/or autoimmune and/or inflammatory diseases has been established in Btk-deficient mouse models. For example, in a standard murine preclinical model of systemic lupus erythematosus (SLE), Btk deficiency has been shown to result in a significant improvement in disease progression. Furthermore, Btk-deficient mice are also resistant to developing collagen-induced arthritis and are less susceptible to staphylococcal-induced arthritis.

A large body of evidence supports the role of B cells and the humoral immune system in the pathogenesis of autoimmune and/or inflammatory diseases. Protein-based therapeutics (such as Rituxan) that have been developed to deplete B cells represent an approach to treating many autoimmune and/or inflammatory diseases. Due to the role of Btk in B cell activation, Btk inhibitors can be used as inhibitors of B cell-mediated pathogenic activities (e.g., production of autoantibodies).

Btk is also expressed in osteoclasts, mast cells, and monocytes and has been shown to be important for the function of these cells. For example, Btk deficiency in mice is associated with impaired IgE-mediated mast cell activation (markedly reduced release of TNF-α and other inflammatory cytokines), and human Btk deficiency is associated with a significant reduction in TNF-α production by activated monocytes.

Therefore, inhibition of Btk activity can be used to treat allergic disorders and/or autoimmune diseases and/or inflammatory diseases, such as SLE, rheumatoid arthritis, polyangiitis, idiopathic thrombocytopenic purpura (ITP), myasthenia gravis, allergic rhinitis and asthma. In addition, it is reported that Btk plays a role in apoptosis; therefore, inhibition of Btk activity can be used for cancer and the treatment of B cell lymphoma and leukemia. Moreover, considering the role of Btk in osteoclast function, inhibition of Btk activity can be used to treat bone diseases such as osteoporosis.

Summary of the Invention

The present invention generally relates to compounds of Formula I having Bruton's tyrosine kinase (Btk) modulating activity.

The compound of formula I has the following structure:

The present invention includes stereoisomers, tautomers and pharmaceutically acceptable salts thereof. The definitions of various substituents are as follows.

One aspect of the present invention is a pharmaceutical composition comprising a compound of Formula I and a pharmaceutically acceptable carrier, glidant, diluent or excipient. The pharmaceutical composition may further comprise another therapeutic agent.

Another aspect of the present invention is a process for preparing a pharmaceutical composition, which comprises mixing a compound of formula I and a pharmaceutically acceptable carrier.

The present invention includes methods of treating a disease or condition comprising administering to a patient suffering from the disease or condition a therapeutically effective amount of a compound of Formula I, wherein the disease or condition is selected from immune disorders, cancer, cardiovascular disease, viral infection, inflammation, metabolic/endocrine dysfunction, and neurological disorders, and is mediated by Bruton's tyrosine kinase.

The present invention includes a kit for treating a condition mediated by Bruton's tyrosine kinase comprising: a) a first pharmaceutical composition comprising a compound of Formula I; and b) instructions for use.

The present invention includes compounds of formula I for use as medicaments and for the treatment of a disease or condition selected from immune disorders, cancer, cardiovascular disease, viral infection, inflammation, metabolic/endocrine dysfunction and neurological disease, and mediated by Bruton's tyrosine kinase.

The present invention includes the use of a compound of formula I in the preparation of a medicament for treating immune disorders, cancer, cardiovascular disease, viral infection, inflammation, metabolic/endocrine dysfunction and neurological disorders, and wherein the medicament modulates Bruton's tyrosine kinase.

The present invention includes processes for preparing compounds of formula I.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows an exemplary synthetic route for preparing 6-chloro, 4-aminopyridazinone compounds including 6-chloro-4-(5-methyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)pyridazin-3(2H)-one 101f from 3-nitropyrazole-5-carboxylic acid.

FIG2 shows an exemplary synthetic route for preparing tricyclic amide-phenylboronic acid ester compounds including 2-(2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 101m from 4,5,6,7-tetrahydro-1H-indole.

FIG3 shows an exemplary synthetic route for preparing tricyclic amide-phenyl bromide compounds including 2-bromo-6-(1-oxo-3,4,5,6,7,8-hexahydrobenzothieno[2,3-c]pyridin-2(1H)-yl)benzyl acetate 104h from 4,5,6,7-tetrahydrobenzo[b]thiophene-2-carboxylic acid.

FIG4 shows another exemplary synthetic route to prepare tricyclic amide-phenyl bromide compounds including 6,6-dimethyl-3,4,6,7-tetrahydro-5H-cyclopenta[4,5]thieno[2,3-c]pyridin-1(2H)-one 105i from 3-methylcyclopent-2-enone.

Figure 5 shows an exemplary synthetic route for preparing tricyclic (1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrido[3,4-b]indolizin-1(2H)-one compounds as boronic acid esters, including 2-(1-oxo-3,4,6,7,8,9-hexahydropyrido[3,4-b]indolizin-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 118f, from 5,6,7,8-tetrahydroindolizin-2-carboxylic acid.

6 shows an exemplary synthetic route for the preparation of intermediate 2-bromo-4-fluoro-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 198d from 1,3-dibromo-5-fluoro-2-iodobenzene.

Figure 7 shows an exemplary synthetic route for preparing intermediate 4-fluoro-2-(1-methyl-5-(5-(4-methylpiperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 198g.

8 shows an exemplary synthetic route for preparing intermediate 5-fluoro-2-(1-methyl-5-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 210e.

Figure 9 shows an exemplary synthetic route for preparing intermediate 5-[5-fluoro-2-(acetoxymethyl)-3-(1-methyl-5-{[5-(4-methylpiperazin-1-yl)pyridin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)phenyl]-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-6-one 212c.

DETAILED DESCRIPTION

Certain embodiments of the present invention are now described in detail, examples of which are illustrated in the accompanying structures and formulas. Although the present invention is described in conjunction with the embodiments listed, it should be understood that the present invention is not limited to those embodiments. On the contrary, the present invention is intended to encompass all alternatives, modifications and equivalences that may be included within the scope of the present invention defined by the claims. Those skilled in the art will recognize that many methods and materials similar or equivalent to those described herein can be used to implement the present invention. The present invention is in no way limited to the methods and materials described. In the event that one or more of the cited documents, patents and similar materials differ from or conflict with the present application (including but not limited to defined terms, term usage, described technology, etc.), the present application shall prevail.

definition

The term "alkyl" as used herein refers to a saturated, linear or branched, monovalent hydrocarbon radical having 1 to 12 carbon atoms (C ₁ -C ₁₂ ), wherein the alkyl radical may be optionally substituted independently with one or more substituents described below. In another embodiment, the alkyl radical has 1 to 8 carbon atoms (C ₁ -C ₈ ), or 1 to 6 carbon atoms (C ₁ -C ₆ ). Examples of alkyl groups include, but are not limited to, methyl (Me, -CH ₃ ), ethyl (Et, -CH ₂ CH ₃ ), 1-propyl (n-Pr, n-propyl, -CH ₂ CH ₂ CH ₃ ), 2-propyl (i-Pr, isopropyl, -CH(CH ₃ ) ₂ ), 1-butyl (n-Bu, n-butyl, -CH ₂ CH ₂ CH ₂ CH ₃ ), 2-methyl-1-propyl (i-Bu, isobutyl, -CH ₂ CH(CH ₃ ) ₂ ), 2-butyl (s-Bu, sec-butyl, -CH(CH ₃ )CH ₂ CH ₃ ), 2-methyl-2-propyl (t-Bu, tert-butyl, -C(CH ₃ ) ₃ ), 1-pentyl (n-pentyl, -CH ₂ CH ₂ CH ₂ CH ₂ CH ₃ ), 2-pentyl (-CH(CH ₃ )CH 2 CH ₂ CH _{3 ),} CH ₃ ), 3-pentyl (-CH(CH ₂ CH ₃ ) ₂ ), 2-methyl-2-butyl (-C(CH ₃ ) ₂ CH ₂ CH ₃ ), 3-methyl-2-butyl (-CH(CH ₃ )CH(CH ₃ ) ₂ ), 3-methyl-1-butyl (-CH ₂ CH ₂ CH(CH ₃ ) ₂ ), 2-methyl-1-butyl (-CH ₂ CH(CH ₃ )CH ₂ CH ₃ ), 1-hexyl (-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₃ ), 2-hexyl (-CH(CH ₃ )CH ₂ CH ₂ CH ₂ CH ₃ ), 3-hexyl (-CH(CH ₂ CH ₃ )(CH ₂ CH ₂ CH ₃ )), 2-methyl-2-pentyl (-C(CH ₃ ) ₂ CH ₂ CH ₂ CH ₃ ), 3-methyl-2-pentyl (-CH(CH ₃ )CH(CH ₃ )CH ₂ CH ₃ ), 4-methyl-2-pentyl (-CH(CH ₃ )CH ₂ CH(CH ₃ ) ₂ ), 3-methyl-3-pentyl (-C(CH ₃ )(CH ₂ CH ₃ ) ₂ ), 2-methyl-3-pentyl (-CH(CH ₂ CH ₃ )CH(CH ₃ ) ₂ ), 2,3-dimethyl-2-butyl (-C(CH ₃ ) ₂ CH(CH ₃ ) ₂ ), 3,3-dimethyl-2-butyl (-CH(CH ₃ )C(CH ₃ ) ₃ , 1-heptyl, 1-octyl, and the like.

The term "alkylene" as used herein refers to a saturated linear or branched divalent hydrocarbon group having 1 to 12 carbon atoms ( _C1 - _C12 ), wherein the alkylene group may be optionally substituted independently with one or more substituents described below. In another embodiment, the alkylene group has 1 to 8 carbon atoms ( _C1 - _C8 ), or 1 to 6 carbon atoms ( _C1 - _C6 ). Examples of alkylene groups include, but are _not limited to, methylene ( _-CH2- ), ethylene ( _-CH2CH2- ), propylene ( _{-CH2CH2CH2- )} , and the _like .

The terms "carbocycle,""carbocyclyl,""carbocyclicring," and "cycloalkyl" refer to a monovalent non-aromatic saturated or partially unsaturated ring having 3 to 12 carbon atoms ( _C3 - _C12 ) as a monocyclic ring or 7 to 12 carbon atoms as a bicyclic ring. Bicyclic carbocycles having 7-12 atoms may be arranged, for example, as bicyclo[4,5], [5,5], [5,6], or [6,6] systems, and bicyclic carbocycles having 9 or 10 ring atoms may be arranged as bicyclo[5,6] or [6,6] systems or as bridged ring systems, such as bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, and bicyclo[3.2.2]nonane. Examples of monocyclic carbocycles include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, cyclododecyl, and the like.

"Aryl" refers to a monovalent aromatic hydrocarbon radical having 6-20 carbon atoms ( _C6 - _C20 ) derived by removing a hydrogen atom from a single carbon atom of a parent aromatic ring system. Some aryl groups are represented as "Ar" in the example structures. Aryl groups include bicyclic radicals comprising an aromatic ring fused to a saturated, partially unsaturated ring, or aromatic carbocyclic ring. Typical aryl groups include, but are not limited to, radicals derived from benzene (phenyl), substituted benzenes, naphthalene, anthracene, biphenyl, indenyl, indanyl, 1,2-dihydronaphthalene, 1,2,3,4-tetrahydronaphthyl, and the like. Aryl groups are optionally substituted independently with one or more substituents described herein.

"Arylene" refers to a divalent aromatic hydrocarbon radical having 6-20 carbon atoms ( _C6 - _C20 ) derived by removing two hydrogen atoms from two carbon atoms of a parent aromatic ring system. Some arylene groups are represented as "Ar" in the example structures. Arylene groups include bicyclic groups comprising an aromatic ring fused to a saturated, partially unsaturated ring, or an aromatic carbocyclic ring. Typical arylene groups include, but are not limited to, groups derived from benzene (phenylene), substituted benzenes, naphthalene, anthracene, biphenylene, indenylene, indanylene, 1,2-dihydronaphthalene, 1,2,3,4-tetrahydronaphthyl, and the like. Arylene groups are optionally substituted.

The terms "heterocycle," "heterocyclyl," and "heterocyclic ring" are used interchangeably herein and refer to a saturated or partially unsaturated (i.e., having one or more double and/or triple bonds within the ring) carbocyclic group having 3 to about 20 ring atoms, at least one of which is a heteroatom selected from nitrogen, oxygen, phosphorus, sulfur, and silicon, and the remaining ring atoms being C, wherein one or more ring atoms are optionally substituted independently with one or more substituents described below. The heterocycle may be a monocyclic ring having 3 to 7 ring members (2 to 6 carbon atoms and 1 to 4 heteroatoms selected from N, O, P, and S), or a bicyclic ring having 7 to 10 ring members (4 to 9 carbon atoms and 1 to 6 heteroatoms selected from N, O, P, and S), for example: a bicyclic [4,5], [5,5], [5,6], or [6,6] system. Heterocycles are described in Paquette, Leo A.; "Principles of Modern Heterocyclic Chemistry" (W.A. Benjamin, New York, 1968), particularly Chapters 1, 3, 4, 6, 7, and 9; "The Chemistry of Heterocyclic Compounds, A series of Monographs" (John Wiley & Sons, New York, 1950), particularly Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82: 5566. "Heterocyclyl" also includes radicals in which a heterocyclyl radical is fused to a saturated, partially unsaturated ring, or an aromatic carbocyclic or heterocyclic ring. Examples of heterocycles include, but are not limited to, morpholin-4-yl, piperidin-1-yl, piperidonyl, oxopiperazinyl, piperazinyl, piperazin-4-yl-2-one, piperazin-4-yl-3-one, pyrrolidin-1-yl, thiomorpholin-4-yl, S-dioxothiomorpholin-4-yl, azocan-1-yl, azetidin-1-yl, octahydropyrido[1,2-a]pyrazin-2-yl, [1,4]diazepan-1-yl, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino, thiooxanyl, piperazinyl, homopiperazinyl 1.0]hexyl, 3-azabicyclo[4.1.0]heptyl, azabicyclo[2.2.2]hexyl, 3H-indolyl, quinolizinyl, and N-pyridylurea. Spirocyclic groups are also included within the scope of this definition. Examples of heterocyclic groups wherein 2 ring atoms are replaced by oxo (=0) groups are pyrimidinone and 1,1-dioxothiomorpholinyl. The heterocyclic groups herein are optionally substituted independently with one or more substituents described herein.

The term "heteroaryl" refers to a monovalent aromatic group of 5-membered, 6-membered or 7-membered rings, and also includes a fused ring system having 5-20 atoms (at least one of which is aromatic), wherein the monovalent aromatic group of the 5-membered, 6-membered or 7-membered rings and the fused ring system contain one or more heteroatoms independently selected from nitrogen, oxygen and sulfur. Examples of heteroaryl groups are pyridyl (including, for example, 2-hydroxypyridyl), imidazolyl, imidazopyridyl, pyrimidinyl (including, for example, 4-hydroxypyrimidinyl), pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furanyl, thienyl, isoxazolyl, thiazolyl, oxadiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, tetrahydroisoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, triazolyl, thiadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothienyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl. Heteroaryl groups are optionally substituted independently with one or more substituents described herein.

Where possible, the heterocycle or heteroaryl group may be carbon-bonded (carbon-linked) or nitrogen-bonded (nitrogen-linked). By way of example and not limitation, a carbon-bonded heterocycle or heteroaryl group is bonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5, or 6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position 2, 3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan, tetrahydrofuran, thiophene, pyrrole, or tetrahydropyrrole, position 2, 4, or 5 of an oxazole, imidazole, or thiazole, position 3, 4, or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of an aziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6, 7, or 8 of a quinoline, or position 1, 3, 4, 5, 6, 7, or 8 of an isoquinoline.

By way of example and not limitation, nitrogen-bonded heterocycles or heteroaryls are bonded at the 1-position of aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, ^1H -indazole, the 2-position of isoindole or isoindoline, the 4-position of morpholine, and the 9-position of carbazole or β-carboline.

The terms "treat" and "treatment" refer to therapeutic treatments, the purpose of which is to slow down (mitigate) undesirable physiological changes or illnesses, such as the formation or spread of arthritis or cancer. For purposes of the present invention, useful or desired clinical outcomes include, but are not limited to, relieving symptoms, reducing the scope of the disease, stabilizing the disease state (i.e., not worsening), delaying or slowing disease progression, improving or alleviating the disease state, and alleviating (whether partially or completely), whether detectable or undetectable. "Treatment" can also refer to extending survival time compared to the expected survival time if not receiving treatment. Those requiring treatment include those with illness or illness.

The phrase "therapeutically effective amount" refers to an amount of a compound of the present invention that (i) treats a specific disease, condition, or disorder described herein, (ii) alleviates, ameliorates, or eliminates one or more symptoms of the specific disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of the specific disease, condition, or disorder. In the case of cancer, a therapeutically effective amount of a drug can reduce the number of cancer cells; reduce tumor size; inhibit (i.e., slow down to some extent and preferably stop) cancer cell infiltration into surrounding organs; inhibit (i.e., slow down to some extent and preferably stop) tumor metastasis; inhibit tumor growth to some extent; and/or alleviate to some extent one or more symptoms associated with cancer. As long as a drug can prevent existing cancer cell growth and/or kill existing cancer cells, it can be cytostatic and/or cytotoxic. For cancer treatment, efficacy can be measured, for example, by assessing the time to disease progression (TTP) and/or determining the response rate (RR).

As used herein, "inflammatory disorder" may refer to any disease, disorder, or syndrome in which an excessive or unregulated inflammatory response results in excessive inflammatory symptoms, host tissue damage, or loss of tissue function. "Inflammatory disorder" also refers to a pathological state mediated by leukocyte influx and/or neutrophil chemotaxis.

"Inflammation" as used herein refers to a local protective response triggered by injury or destruction of tissue that serves to destroy, dilute, or shield (isolate) noxious substances and injured tissue. Inflammation is clearly associated with leukocyte influx and/or neutrophil chemotaxis. Inflammation can result from infection by pathogenic organisms and viruses, as well as non-infectious pathways such as trauma or reperfusion after myocardial infarction or stroke, immune responses to foreign antigens, and autoimmune responses. Thus, inflammatory conditions amenable to treatment with compounds of Formula I include conditions associated with responses of the specific defense system as well as responses of the nonspecific defense system.

"Specific defense system" refers to the component of the immune system that responds to the presence of specific antigens. Examples of inflammation caused by responses of the specific defense system include conventional responses to foreign antigens, autoimmune diseases, and delayed-type hypersensitivity reactions mediated by T cells. Other examples of inflammatory responses of the specific defense system include chronic inflammatory diseases, rejection of solid transplanted tissues and organs, such as kidney transplants and bone marrow transplants, and graft-versus-host disease (GVHD).

The term "non-specific defense system" as used herein refers to inflammatory conditions mediated by leukocytes (e.g., granulocytes and macrophages) that are not capable of immune memory. Examples of inflammation that is at least partially due to a response of the non-specific defense system include inflammation associated with conditions such as adult (acute) respiratory distress syndrome (ARDS) or multiple organ injury syndrome; reperfusion injury; acute glomerulonephritis; reactive arthritis; skin diseases with an acute inflammatory component; acute purulent meningitis or other central nervous system inflammatory conditions such as stroke; thermal injury; inflammatory bowel disease; granulocyte transfusion-associated syndrome; and cytokine-induced poisoning.

"Autoimmune disease" as used herein refers to any class of disorders in which tissue damage is associated with a humoral or cellular-mediated response to the body's own constituents.

"Allergic disease" as used herein refers to any symptom, tissue damage or loss of tissue function produced by an allergic reaction. "Arthritic disease" as used herein refers to any disease characterized by inflammatory damage to the joints attributable to various etiologies. "Dermatitis" as used herein refers to any of a large class of diseases characterized by skin inflammation attributable to various etiologies. "Transplant rejection" as used herein refers to any immune response to transplanted tissue, such as an organ or cell (e.g., bone marrow), characterized by loss of function of the transplanted tissue and surrounding tissue, pain, swelling, leukocytosis, and thrombocytopenia. The therapeutic methods of the present invention include methods for treating conditions associated with inflammatory cell activation.

"Inflammatory cell activation" refers to the induction of inflammatory responses by stimuli of proliferative cells (including but not limited to cytokines, antigens or autoantibodies), the production of soluble mediators (including but not limited to cytokines, oxygen free radicals, enzymes, prostaglandins or vasoactive amines), or the cell surface expression of new or increased mediators (including but not limited to major histocompatibility antigens or cell adhesion molecules) in inflammatory cells (including but not limited to monocytes, macrophages, T lymphocytes, B lymphocytes, granulocytes (i.e., polymorphonuclear leukocytes such as neutrophils, basophils and eosinophils), mast cells, dendritic cells, Langerhans cells and endothelial cells). It will be understood by those skilled in the art that activation of one or a combination of these phenotypes in these cells can contribute to the initiation, perpetuation or exacerbation of inflammatory conditions.

The term "NSAID," an acronym for "nonsteroidal anti-inflammatory drugs," is a therapeutic agent that has analgesic, antipyretic (lowers high body temperature and relieves pain without impairing sensation) and, in higher doses, anti-inflammatory properties (reduces inflammation). The term "nonsteroidal" is used to distinguish these drugs from steroids, which have similar eicosanoid-lowering, anti-inflammatory effects. As analgesics, NSAIDs are unusual in that they are non-narcotic. NSAIDs include aspirin, ibuprofen, and naproxen. NSAIDs are generally prescribed to treat acute or chronic conditions associated with pain and inflammation. NSAIDs are generally indicated for the symptomatic relief of the following conditions: rheumatoid arthritis, osteoarthritis, inflammatory arthritis (e.g., ankylosing spondylitis, psoriatic arthritis, Reiter's syndrome, acute gout, dysmenorrhea, metastatic bone pain, headaches and migraines, postoperative pain, mild to moderate pain due to inflammation and tissue damage, fever, intestinal obstruction, and renal colic. Most NSAIDs act as nonselective inhibitors of cyclooxygenases, inhibiting both the cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) isozymes. Cyclooxygenase catalyzes the formation of prostaglandins and thromboxanes from arachidonic acid (which itself is derived from the cellular phospholipid bilayer by phospholipase _A2 ). Prostaglandins act as messenger molecules, among other things, in inflammatory processes. COX-2 inhibitors include celecoxib, etoricoxib, lumiracoxib, parecoxib, rofecoxib, rofecoxib, and valdecoxib.

The term "cancer" refers to or describes a physiological condition in mammals that is typically characterized by dysregulated cell growth. A "tumor" includes one or more cancerous cells. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinomas (e.g., epithelial squamous cell carcinomas), lung cancers including small cell lung cancer, non-small cell lung cancer ("NSCLC"), lung adenomas, and squamous cell lung carcinomas, peritoneal cancer, hepatocellular carcinoma, gastric cancer including gastrointestinal cancer, pancreatic cancer, glioblastomas, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatomas, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, and head and neck cancer.

A "chemotherapeutic agent" is a compound used to treat cancer, regardless of mechanism of action. Classes of chemotherapeutic agents include, but are not limited to, alkylating agents, antimetabolites, spindle poison plant alkaloids, cytotoxic/antitumor antibiotics, topoisomerase inhibitors, antibodies, photosensitizers, and kinase inhibitors. Chemotherapeutic agents include compounds used in "targeted therapy" and conventional chemotherapy. Examples of chemotherapeutic agents include: erlotinib (Genentech/OSI Pharm.), docetaxel (Sanofi-Aventis), 5-FU (fluorouracil, 5-fluorouracil, CAS No. 51-21-8), gemcitabine (Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer), cisplatin (cis-diamine, platinum (II) dichloride, CAS No. 15663-27-1), carboplatin (CAS No. 41575-94-4), paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), trostuzumab (Genentech), temozolomide (4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]nona-2,7,9-triene-9-carboxamide, CAS No. 85622-93-1, Schering Plough), tamoxifen ((Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethylethanamine,) and doxorubicin (), Akti-1/2, HPPD, and rapamycin.

Further examples of chemotherapeutic agents include: oxaliplatin (Sanofi), bortezomib (Millennium Pharm.), sutent (SU11248, Pfizer), letrozole (Novartis), imatinib mesylate (Novartis), XL-518 (Mek inhibitor, Exelixis, WO 2007/044515), ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma, AstraZeneca), SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235 (PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), PTK787/ZK 222584 (Novartis), fulvestrant (AstraZeneca), leucovorin (folinic acid), rapamycin (sirolimus, Wyeth), lapatinib (GSK572016, GlaxoSmithKline Kline), lonafarnib (SARASAR ^™ , SCH 66336, Schering Plough), sorafenib (BAY43-9006, Bayer Labs), gefitinib (AstraZeneca), irinotecan (CPT-11, Pfizer), tipifarnib (ZARNESTRA ^™ , Johnson & Johnson), ABRAXANE ^™ (Cremophor-free), albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, 11), vandetanib (rINN, ZD6474, AstraZeneca), chloranmbucil, AG1478, AG1571 (SU 5271; Sugen), temsirolimus (Wyeth), pazopanib (GlaxoSmithKline), canfosfamide (Telik), thiotepa, and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquinone, meturedopa, and uredopa; ethyleneimines and methylamelamines including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (particularly bullatacin and bullatacinone); camptothecins (including the synthetic analogue topotecan); bryostatin; spongoside; CC-1065 (including its and synthetic analogs of adolesin, carzelesin, and biszelesin); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongin; nitrogen mustards such as chlorambucil, naphthyl mustard, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, chlorambucil hydrochloride, melphalan, chlorambucil, phenylephrine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, calicheamicin γ 1I, calicheamicin ω 1I (Angewandte) Chem.Intl.Ed.Engl.(1994)33:183-186); dynemicin, dynemicin A; bisphosphonates such as clodronate; esperamicins; and the neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclarubicin, actinomycin, authramycin, azoserine, bleomycin, actinomycin C, carabicin, daunorubicin, carmomycin, chromomycins, actinomycin D, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrroline-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, nelrubicin, marseromycin, mitomycins such as mitomycin C, mycophenolic acid, noramycin, olivomycin, Peplomycin, porfibrinocin, puromycin, triferric doxorubicin, rhodorubicin, streptozocin, streptozocin, tuberculin, ubenimex, zoloft, daunorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as leucovorin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamidine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as captestosterone, drostanolone propionate, cyclothiocarbamate, melastane, testolactone; antiadrenal agents such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as frolinic acid; aceglucuronolide; propionaldehydeoxyphosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatrexate; defofamine; colcemid; diacrazone; elfornithine; elliptonium acetate; epothilones; etoglucagon; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansines such as maytansine and ansamitocin; mitoxantrone; mitoxantrone; mopidarol; nitraerine; pentostatin; methambucil; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; lisoxine; sizolan; spirogermanamine; tenuisporic acid; triimidoquinone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, baculosporin A and serpentin); urethane; vindesine; dacarbazine; mannomustine; dibromomannitol; dibromodulcitol; pipobroman; gacytosine; arabinoside ("Ara-C");cyclophosphamide;thiotepa;6-thioguanine;mercaptopurine;methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; teniposide; edatrexate; daunorubicin; aminopterin; capecitabine (Roche); ibandronate; CPT-11; the topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing.

The definition of "chemotherapeutic agent" also includes: (i) antihormonal drugs used to modulate or inhibit the effects of hormones on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen, traloxifen, raloxifene, LY117018, onapristone, and toremifene citrate; (ii) aromatase inhibitors, such as 4-hydroxytamoxifen, which inhibit the aromatase enzyme that regulates estrogen production in the adrenal glands; (5)-imidazoles, aminoglutethimide, (megestrol acetate), (exemestane; Pfizer), formestane, pharoazole, (vorozole), (letrozole; Novartis), and (anarozole; AstraZeneca); (iii) antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and troxacitabine (a 1,3-dioxolane cytidine nucleoside analog); (iv) protein kinase inhibitors, such as MEK inhibitors (WO 2007/044515); (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, particularly those that inhibit the expression of genes in signal transduction pathways associated with abnormal cell proliferation, such as PKC-α, Raf and H-Ras, such as oblimersen (Genta Inc.); (vii) ribozymes, such as VEGF expression inhibitors (e.g., and HER2 expression inhibitors; (viii) vaccines, such as gene therapy vaccines, such as and rIL-2; topoisomerase 1 inhibitors, such as rmRH; (ix) anti-angiogenic agents, such as bevacizumab (Genentech); and pharmaceutically acceptable salts, acids and derivatives of any of the above.

The definition of "chemotherapeutic agent" also includes therapeutic antibodies, such as alemtuzumab (Campath), bevacizumab (Genentech); cetuximab (Imclone); panitumumab (Amgen), rituximab (Genentech/Biogen Idec), pertuzumab (OMNITARG ^™ , 2C4, Genentech), trastuzumab (Genentech), tositumomab (Bexxar, Corixia), and the antibody drug conjugate, gemtuzumab ozogamicin (Wyeth).

Humanized monoclonal antibodies with therapeutic efficacy used in combination with the Btk inhibitors of the present invention as chemotherapeutic agents include: alemtuzumab, apolizumab, aseluzumab, atlizumab, bapinezumab, bevacizumab, bivatuzumab mertansine, cantuzumab mertansine, and selezinumab. mertansine), cilizumab, beceretuzumab, cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, favuzumab, fontuzumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pacolizumab uzumab, pecfusituzumab, pectuzumab, pertuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, resyvizumab, rovizumab, lulizumab, sirolizumab, cilizumab, sontuzumab, taluzumab, tetuzumab, taduzumab, talizumab, tefizumab, tocilizumab, tocilizumab, trastuzumab, simolukurin, tucusituzumab, umavizumab, urolizumab, and visilizumab.

"Metabolites" are products produced by the metabolism of a particular compound or its salt in vivo. Metabolites of a compound can be identified using conventional techniques known in the art and their activity can be determined using assays such as those described herein. Such products can be produced, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, defatting, enzymatic hydrolysis, etc. of the administered compound. Thus, the present invention includes metabolites of compounds of the present invention, including compounds produced by a method comprising contacting a compound of Formula I of the present invention with a mammal for a period of time sufficient to produce a metabolic product thereof.

The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products.

The term "chiral" refers to molecules that have the property of non-superimposability of their mirror image pairs, whereas the term "achiral" refers to molecules that are superimposable on their mirror image pairs.

The term "stereoisomers" refers to compounds which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space.

"Diastereoisomers" refer to stereoisomers that have two or more chiral centers and whose molecules are not mirror images of one another. Diastereoisomers have different physical properties, such as melting points, boiling points, spectral properties, and reactivities. Mixtures of diastereoisomers can be separated by high-resolution analytical methods such as electrophoresis and chromatography.

"Enantiomers" refers to two stereoisomers of a compound that are non-superimposable mirror images of one another.

The stereochemical definitions and conventions used herein generally follow those of S. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., New York, 1994. The compounds of the present invention may contain asymmetric or chiral centers and therefore exist in different stereoisomeric forms. All stereoisomeric forms of the compounds of the present invention, including but not limited to diastereomers, enantiomers and atropisomers thereof, and mixtures thereof, such as racemic mixtures, are intended to form part of the present invention. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. In describing optically active compounds, the prefixes D and L, or R and S, are used to indicate the absolute configuration of the chiral center of the molecule. The prefixes d and l or (+) and (-) are used to indicate the sign by which a compound rotates plane polarized light, where (-) or l means that the compound is levorotatory. Compounds with the prefix (+) or d are dextrorotatory. For a particular chemical structure, these stereoisomers are identical except that they are mirror images of each other. Specific stereoisomers may also be referred to as enantiomers, and mixtures of such isomers are often referred to as enantiomeric mixtures. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which can occur when a chemical reaction or process lacks stereoselectivity or stereospecificity. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomers that lacks optical activity. In one aspect, the stereoisomers of the present invention can exist in a predominant form, for example, with greater than 50% ee (enantiomeric excess), greater than 80% ee, greater than 90% ee, greater than 95% ee, or greater than 99% ee.

The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that are interconvertible via a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions via migration of a proton, such as keto-enol and imine-enamine isomerizations. Valence tautomers include interconversions via reorganization of some of the bonding electrons.

The term "diastereomer" refers to stereoisomeric molecules that are not enantiomers. Diastereomers include cis-trans isomers and conformational isomers that have the same molecular formula but different geometric structures.

The term "pharmaceutically acceptable salt" is used herein to refer to a pharmaceutically acceptable organic or inorganic salt of a compound of the present invention. Exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate (i.e., 1,1'-methylene-bis(2-hydroxy-3-naphthoate)). Pharmaceutically acceptable salts may include the inclusion of another molecule such as acetate ion, succinate ion or other counter ion. The counter ion can be any organic or inorganic ion that stabilizes the charge on the parent compound. In addition, a pharmaceutically acceptable salt may have more than one charged atom in its structure. Where multiple charged atoms are part of a pharmaceutically acceptable salt, multiple counterions may be present. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and/or one or more counterions.

If the compound of the present invention is a base, the desired pharmaceutically acceptable salt can be prepared by any suitable method available in the art, for example, by treating the free base with an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, methanesulfonic acid, phosphoric acid, and the like, or with an organic acid such as acetic acid, trifluoroacetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, a pyranosidic acid such as glucuronic acid or galacturonic acid, an α-hydroxy acid such as citric acid or tartaric acid, an amino acid such as aspartic acid or glutamic acid, an aromatic acid such as benzoic acid or cinnamic acid, a sulfonic acid such as p-toluenesulfonic acid or ethanesulfonic acid, and the like.

If the compound of the present invention is an acid, the desired pharmaceutically acceptable salt can be prepared by any suitable method, for example, by treating the free acid with an inorganic or organic base such as an amine (primary, secondary or tertiary), an alkali metal hydroxide or an alkaline earth metal hydroxide, etc. Illustrative examples of suitable salts include, but are not limited to, organic salts derived from amino acids such as glycine and arginine, ammonia, primary, secondary and tertiary amines, and cyclic amines such as piperidine, morpholine and piperazine, and inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum and lithium.

The term "pharmaceutically acceptable" means that the substance or composition must be chemically and/or toxicologically compatible with the other ingredients comprising the formulation and/or the mammal to be treated therewith.

"Solvate" refers to an association or complex of one or more solvent molecules with a compound of the invention. Examples of solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.

The term "compounds of the present invention" includes compounds of Formula I and stereoisomers, tautomers, solvates, metabolites and pharmaceutically acceptable salts and prodrugs thereof.

Any molecular formula or structure shown herein, including compounds of Formula I, is also intended to represent hydrates, solvates, and polymorphs of these compounds, and mixtures thereof.

Any formula or structure shown herein, including compounds of Formula I, is also intended to represent unlabeled and isotopically labeled forms of the compounds. Isotopically labeled compounds have the structure shown in the molecular formula given herein, except that one or more atoms are replaced by atoms with a selected atomic mass or mass number. Examples of isotopes that may be included in the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine and chlorine, such as, but not limited to, 2H (deuterium, D), 3H (tritium), 11C, 13C, 14C, 15N, 18F, 31P, 32P, 35S, 36Cl and 125I. Various isotopically labeled compounds of the present invention, for example, those containing radioactive isotopes such as 3H, 13C and 14C. Such isotopically labeled compounds can be used in metabolic studies, reaction kinetic studies, detection or imaging techniques, such as positron emission tomography (PET) or single photon emission tomography (SPECT), including drug or substrate tissue distribution assays, or in radiotherapy of patients. Deuterium-labeled or substituted therapeutic compounds of the present invention may have improved DMPK (drug metabolism and pharmacokinetics) properties related to distribution, metabolism, and excretion (ADME). Substitution with heavier isotopes, such as deuterium, may provide certain therapeutic advantages due to greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements. 18F-labeled compounds can be used in PET or SPECT studies. Isotopically labeled compounds of the present invention and their prodrugs can generally be prepared by the methods disclosed in the schemes or examples and the preparation methods described below, substituting readily available isotopically labeled reagents for non-isotopically labeled reagents. In addition, substitution with heavier isotopes, particularly deuterium (i.e., 2H or D), may provide certain therapeutic advantages due to greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements or improved therapeutic index. It should be understood that deuterium is considered a substituent in the compounds of formula (I) in this case. The concentration of such heavier isotopes, particularly deuterium, can be defined by the isotopic enrichment factor. In the compounds of the present invention, any atom not explicitly designated as a particular isotope is intended to represent any stable isotope of that atom. Unless otherwise stated, when a position is explicitly designated with "H" or "hydrogen," it should be understood that the position has hydrogen in its natural abundance isotopic composition. Therefore, in the compounds of the present invention, any atom explicitly designated as deuterium (D) is intended to represent deuterium.

Pyridone and azapyridone compounds

The present invention provides pyridone and azapyridone compounds of Formula I, including Formulas Ia-bf, including stereoisomers, tautomers or pharmaceutically acceptable salts thereof, and pharmaceutical formulations thereof, which are effective for treating diseases, conditions and/or disorders mediated by Btk kinase.

in:

R ¹ is H, D, F, Cl, CN, —NH ₂ , —NHCH ₃ , —N(CH ₃ ) ₂ , —OH, —OCH ₃ , —OCH ₂ CH ₃ , —OCH ₂ CH ₂ OH, a heteroaryl group selected from imidazolyl and pyrazolyl, a heterocyclyl group selected from oxetanyl and azetidinyl, and C ₁ -C ₃ alkyl;

R ² , R ³ and R ⁴ are independently selected from H, D, F, Cl, -NH ₂ , -NHCH ₃ , -N(CH ₃ ) ₂ , -OH, -OCH ₃ , -OCH ₂ CH ₃ , -OCH ₂ CH ₂ OH and C ₁ -C ₃ alkyl;

^R5 is optionally substituted _C6 - _C20 aryl, _C3 - _C12 carbocyclyl, C2-C20 heterocyclyl, _C1 - _C20 heteroaryl, -( _C6 - _C20 aryl)-( _C2 - _C20 heterocyclyl), -(C1- _C20 heteroaryl)-( _C2 -C20 heterocyclyl), -( _C1 - _C20 heteroaryl)-( _C2 - _C20 heterocyclyl), -( _C1 - _C20 heteroaryl)-( _C1 - _C6 alkyl), or -( _C1 - _C20 heteroaryl)-C(＝O)-( _C2 - _C20 heterocyclyl);

R ⁶ is H, F, -NH ₂ , -OH, or optionally substituted C ₁ -C ₃ alkyl;

X is S, S(═O), S(═O) ₂ , N, NR ⁶ , O, or CR ⁷ ;

R ⁷ is independently selected from H, D, F, Cl, -CH ₃ , -CH ₂ CH ₃ , -CN, -CH ₂ F, -CHF ₂ , -CF ₃ , -NH ₂ , -OH, and -OCH ₃ ;

Y ¹ and Y ² are independently selected from CR ⁶ and N;

Z ¹ , Z ² , Z ³ and Z ⁴ are independently selected from C, CR ⁷ and N;

Z ⁵ is selected from -C(R ³ ) ₂ -, -C(=O)-, -N(R ⁶ )-, -C(R ³ ) ₂ C(R ³ ) ₂ -, -C(R ³ ) ₂ C(=O)-, -CR ³ =CR ³ -, -CR ³ =N-, -N(R ⁶ )C(R ³ ) ₂ -, -N(R ⁶ )C(R ³ ) ₂ C(R ³ ) ₂ - and -OC(R ³ ) ₂ C(R ³ ) ₂ -;

a pair of Z ¹ and Z ² or X and Z ¹ forms a five-membered, six-membered or seven-membered aryl ring, carbocyclyl ring, heterocyclyl ring or heteroaryl ring, wherein X is not S, S(═O) or S(═O) ₂ ;

wherein alkyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl are optionally independently selected from D, F, Cl, Br, I, _-CH3 , _-CH2CH3 , -CH2CH(CH3) ₂ , _{-CH2OH ,} -CH2CH2OH, -C( _CH3 ) _2OH , -CH _{(OH) CH (CH3)2 , -C ( CH3} ) _2CH2OH , -CH2CH2SO2CH3 _, -CH2OP ₍ O _{) (} OH _{)2 ,} -CN, _{-CH2F, -CHF2 , -CF3} , -CO2H, _{-COCH3 , -CO2CH3} , _-CO2C ( _CH3 ) ₃ , -COCH(OH) _{CH3 ,} -CONH2 _, -CONHCH3 _, -CON( _{CH3 )} ) ₂ , -C(CH ₃ ) ₂ CONH ₂ , -NO ₂ , -NH ₂ , -NHCH ₃ , -N(CH ₃ ) ₂ , -NHCOCH ₃ , -N(CH ₃ )COCH ₃ , -NHS(O) ₂ CH ₃ , -N(CH ₃ )C(CH ₃ ) ₂ CONH ₂ , -N(CH ₃ )CH ₂ CH ₂ S(O) ₂ CH ₃ , =O, -OH, -OCH ₃ , -OCH ₂ CH ₂ N(CH ₃ ) ₂ , -OP(O)(OH) ₂ , -S(O) ₂ N(CH ₃ ) ₂ , -SCH ₃ , -CH ₂ OCH ₃ , -S(O) ₂ CH ₃ , cyclopropyl, azetidinyl, 1-methylazetidin-3-yl)oxy, N-methyl-N-oxetan-3-ylamino, azetidin-1-ylmethyl, oxetanyl and morpholino are substituted by one or more groups.

In one aspect, the compound of Formula I is 3-amino-5-phenylpyridin-2(1H)-one Ia (wherein Y ¹ and Y ² are CR ⁶ ), 4-amino-6-phenylpyridazin-3(2H)-one Ib (wherein Y ¹ is N and Y ² is CR ⁶ ), and 3-amino-5-phenylpyrazin-2(1H)-one Ic (wherein Y ¹ is CR ⁶ and Y ² is N).

Exemplary embodiments of compounds of Formula I include compounds of Formulas Ia-Ibf:

Exemplary embodiments of compounds of Formula I include those wherein the group

The following structure is formed:

The wavy lines represent connection points.

Exemplary embodiments of compounds of Formula I include those in which the central phenyl ring group is substituted or unsubstituted, for example: (i) R ¹ , R ² , R ³ and R ⁴ are each H; (ii) or one or more of R ¹ , R ² , R ³ and R ⁴ are F; (iii) R ¹ is selected from F, -CH ₃ , -CH ₂ F, -CHF ₂ and -CF ₃ ; (iv) R ¹ is -CH ₂ OH; (v) R ³ is F; and (vi) R ¹ is -CH ₂ OH, R ² and R ⁴ are each H, and R ³ is F.

Exemplary embodiments of the compound of Formula I include those wherein R ⁵ is an optionally substituted C ₆ -C ₂₀ aryl group selected from phenyl and naphthyl.

Exemplary embodiments of compounds of Formula I include those wherein R ⁵ is an optionally substituted C ₃ -C ₁₂ carbocyclyl selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

Exemplary embodiments of compounds of Formula I include those wherein R ⁵ is an optionally substituted C ₂ -C ₂₀ heterocyclyl selected from oxetanyl, azetidinyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, morpholinyl, and tetrahydropyranyl.

Exemplary embodiments of compounds of Formula I include those wherein ^R5 is an optionally substituted C1-C20 heteroaryl selected from pyrazolyl, pyridinyl, pyrimidinyl, 5-methyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-yl, 5-acetyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-yl, 6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-2-yl, and 1-methyl-5-(5-(4-methylpiperazin-1-yl)pyridin- _{2 -} yl).

Exemplary embodiments of compounds of Formula I include those wherein R ⁵ is selected from the following structures:

The wavy lines represent connection points.

Exemplary embodiments of compounds of Formula I include those wherein R ⁶ is H.

Exemplary embodiments of compounds of Formula I include those wherein Y ¹ is CR ⁶ and Y ² is N.

Exemplary embodiments of compounds of Formula I include those wherein Y ¹ is N and Y ² is CR ⁶ .

Exemplary embodiments of compounds of Formula I include those wherein Y ¹ and Y ² are each CR ⁶ .

In the structures of compounds of formula I, in the five-membered ring formed by X, Z ¹ , Z ² , Z ³ and Z ⁴ , each solid/dashed line represents a single bond or a double bond, provided that any two double bonds in the ring are not adjacent.

The compounds of formula I of the present invention may contain asymmetric centers or chiral centers and thus exist in different stereoisomeric forms. All stereoisomeric forms of the compounds of the present invention, including but not limited to diastereomers, enantiomers and atropisomers thereof, and mixtures thereof, such as racemic mixtures, are intended to form part of the present invention.

In addition, the present invention encompasses all diastereomers, including cis-trans (geometric) isomers and conformational isomers. For example, if a compound of formula I contains a double bond or a fused ring, both the cis and trans forms, as well as mixtures thereof, are encompassed within the scope of the present invention.

In the structures shown herein, if the stereochemistry of any particular chiral atom is not specified, all stereoisomers are considered and included as compounds of the present invention. If stereochemistry is indicated by a solid or dashed wedge representing a specific configuration, then that stereoisomer is so indicated and defined.

The compounds of the present invention may exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like, and it is intended that the present invention encompass both solvated and unsolvated forms.

The compounds of the present invention may also exist in different tautomeric forms, and all such forms are encompassed within the scope of the present invention. The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that are interconvertible via a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include those that interconvert via the migration of a proton, such as keto-enol and imine-enamine isomerizations. Valence tautomers include those that interconvert via the reorganization of some of the bonding electrons.

Biological evaluation

The relative effectiveness of the compounds of Formula I as inhibitors of enzyme activity (or other biological activity) can be determined by measuring the concentration of each compound that inhibits the activity to a predetermined degree and then comparing the results. Generally, it is preferred to measure the concentration that inhibits 50% of the activity in a biochemical assay, i.e., the 50% inhibitory concentration or " _IC50 ". The determination of _IC50 values can be accomplished using conventional techniques known in the art. The _IC50 can generally be determined by measuring the activity of a specific enzyme in the presence of a range of concentrations of the inhibitor to be studied. The experimentally obtained enzyme activity values are then plotted against the inhibitor concentration used. The inhibitor concentration that shows 50% enzyme activity (compared to the activity in the absence of any inhibitor) is used as the _IC50 value. Similarly, other inhibitory concentrations can be determined by appropriately measuring the activity. For example, in some cases it may be desirable to determine a 90% inhibitory concentration, i.e., an _IC90 , etc.

Compounds of Formula I (Example 901) were tested by a standard biochemical Btk kinase assay.

A general standard cellular Btk kinase assay that can be used to test compounds of Formula I is the Ramos cell Btk assay (Example 902).

A standard cellular B cell proliferation assay can be used to test compounds of Formula I (Example 903) using B cells purified from the spleens of Balb/c mice.

A standard T cell proliferation assay can be used to test compounds of Formula I (Example 904) using T cells purified from the spleens of Balb/c mice.

To inhibit B cell activity, compounds of Formula I can be subjected to CD86 inhibition assays using whole mouse splenocytes purified from the spleens of 8-16 week old Balb/c mice (Example 905).

To determine the number of B-ALL cells that survive in culture, compounds of Formula I can be subjected to a B-ALL cell survival assay (Example 906).

To determine the ability of compounds to inhibit CD69 production by B lymphocytes activated by cross-linked surface IgM with sheep F(ab')2 anti-human IgM in human whole blood, compounds of Formula I can be subjected to a CD69 whole blood assay (Example 907).

The exemplary compounds of Formula I in Tables 1, 2 and 3, which have the following structures and corresponding names (ChemDraw Ultra, Version 9.0.1, and ChemBioDraw, Version 11.0, CambridgeSoft Corp., Cambridge MA), were prepared and characterized according to the methods of the present invention and tested for inhibition of Btk. In cases where more than one name is associated with a compound of Formula I or an intermediate, the compound should be defined by the chemical structure.

Table 1.

Table 2 .

Table 3.

Administration of compounds of formula I

The compound of the present invention can be administered by any route suitable for the disease to be treated. Suitable routes include oral, parenteral (including subcutaneous, intramuscular, intravenous, intraarterial, intradermal, intrathecal and epidural), transdermal, rectal, nasal, local (including buccal and sublingual), vaginal, intraperitoneal, intrapulmonary and intranasal. For local immunosuppressive treatment, the compound can be administered by intralesional administration (including perfusion or contacting the inhibitor with the transplant before transplantation). It should be understood that preferred routes can change with, for example, the condition of the recipient. In the case of oral administration of the compound, it can be prepared into pills, capsules, tablets, etc. together with a pharmaceutically acceptable carrier or excipient. In the case of parenteral administration of the compound, as described in detail below, it can be formulated in a unit dose injectable form together with a pharmaceutically acceptable parenteral carrier.

The dosage for treating human patients can be from about 10 mg to about 1000 mg of the compound of formula I. A typical dosage can be from about 100 mg to about 300 mg of the compound. Depending on the pharmacokinetic and pharmacodynamic properties of the specific compound, including absorption, distribution, metabolism and excretion, the dosage can be administered once a day (QID), twice a day (BID) or more frequently. In addition, toxicity factors can affect the dosage and dosing regimen. When administered orally, the pill, capsule or tablet can be swallowed daily or less frequently for a specified period of time. The regimen can be repeated for several treatment cycles.

Methods of treatment using compounds of formula I

The compounds of formula I of the present invention are used to treat human or animal patients suffering from diseases or conditions caused by abnormal cell growth, function or behavior associated with Btk kinase, such as immune disorders, cardiovascular diseases, viral infections, inflammation, metabolic/endocrine disorders or neurological diseases, and can therefore be treated by a method comprising administering to them a compound of the present invention as defined above. Human or animal patients suffering from cancer can also be treated by a method comprising administering to them a compound of the present invention as defined above. This can improve or ameliorate the patient's condition.

The compounds of formula I are useful for the diagnosis or treatment of mammalian cells, organisms or related pathological conditions in vitro, in situ or in vivo, such as systemic and local inflammation, immunoinflammatory diseases such as rheumatoid arthritis, immunosuppression, organ transplant rejection, allergies, ulcerative colitis, Crohn's disease, dermatitis, asthma, systemic lupus erythematosus, Sjogren's syndrome, multiple sclerosis, scleroderma/systemic sclerosis, idiopathic thrombocytopenic purpura (ITP), anti-neutrophil cytoplasmic antibodies (ANCA) vasculitis, chronic obstructive pulmonary disease (COPD), psoriasis, and overall joint protection.

The methods of the present invention also include treating diseases such as: joint diseases such as rheumatoid arthritis, monoarticular arthritis, osteoarthritis, gouty arthritis, spondylitis; Behcet's disease; sepsis, septic shock, endotoxic shock, gram-negative sepsis, gram-positive sepsis and toxic shock syndrome; multiple organ injury syndrome secondary to sepsis, trauma or hemorrhage; eye diseases such as allergic conjunctivitis, vernal conjunctivitis, uveitis and thyroid-related eye disease; eosinophilic granuloma; lung or respiratory tract disorders such as asthma, chronic bronchitis, allergic rhinitis, ARDS, chronic inflammatory lung diseases (such as chronic obstructive pulmonary disease), silicosis, pulmonary sarcoidosis, pleurisy, alveolitis, vasculitis, emphysema, pneumonia, bronchiectasis and pulmonary oxygen toxicity; reperfusion injury of the myocardium, brain or extremities; fibrosis such as cystic fibrosis; keloid formation or scar tissue formation; atherosclerosis; autoimmune diseases Examples include systemic lupus erythematosus (SLE), autoimmune thyroiditis, multiple sclerosis, some forms of diabetes, and Raynaud's syndrome; and transplant rejection disorders such as GVHD and allograft rejection; chronic glomerulonephritis; inflammatory bowel diseases such as chronic inflammatory bowel disease (CIBD), Crohn's disease, ulcerative colitis, and necrotizing enterocolitis; inflammatory skin diseases such as contact dermatitis, atopic dermatitis, psoriasis, or urticaria; fever and myalgia due to infection; inflammatory disorders of the central or peripheral nervous system such as meningitis, encephalitis, and brain or spinal cord injury due to minor trauma; Sjogren's syndrome; diseases involving leukocytic infiltration; alcoholic hepatitis; bacterial pneumonia; diseases mediated by antigen-antibody complexes; hypovolemic shock; type 1 diabetes; acute and delayed hypersensitivity reactions; conditions due to leukocyte dyscrasias and translocation; heat injury; granulocyte transfusion-related syndromes; and cytokine-induced poisoning.

The methods of the present invention can be used to treat subjects who have or may be susceptible to reperfusion injury (i.e., injury caused by a period of ischemia followed by reperfusion of a tissue or organ). The term "ischemia" refers to localized tissue anemia caused by a blockage of arterial blood flow. Brief ischemia followed by reperfusion characteristically results in neutrophil activation and migration across the endothelium of blood vessels in the affected area. The accumulation of activated neutrophils, in turn, leads to the production of reactive oxygen metabolites that damage components of the associated tissue or organ. This phenomenon of "reperfusion injury" is commonly associated with conditions such as vascular stroke (including global and focal ischemia), hemorrhagic shock, myocardial ischemia or infarction, organ transplantation, and cerebral vasospasm. For example, reperfusion injury occurs at the end of heart bypass surgery or during cardiac arrest (when the heart, which was previously blocked from receiving blood, begins to reperfuse). It is expected that inhibition of Btk activity may result in a reduction in the amount of reperfusion injury in such situations.

pharmaceutical preparations

In order to use the compounds of the present invention for treating mammals including humans, they are generally formulated into pharmaceutical compositions following standard pharmaceutical methods.According to this aspect of the present invention, there is provided a pharmaceutical composition comprising a compound of the present invention and a pharmaceutically acceptable diluent or carrier.

Typical formulations are prepared by mixing the compound of the present invention with a carrier, diluent or excipient. Suitable carriers, diluents and excipients are well known to those skilled in the art and include materials such as sugars, waxes, water-soluble and/or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, etc. The specific carrier, diluent or excipient used depends on the approach and purpose of applying the compound of the present invention. Solvents are generally selected based on solvents that are generally recognized by those skilled in the art as safe (GRAS) for use in mammals. Generally, safe solvents are non-toxic aqueous solvents such as water and other non-toxic solvents that are soluble in or miscible in water. Suitable aqueous solvents include water, ethanol, propylene glycol, polyethylene glycol (e.g., PEG 400, PEG 300), etc., and mixtures thereof. The preparation may also contain one or more buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, light-shielding agents, glidants, processing aids, colorants, sweeteners, aromas, flavorings and other known additives to give the drug (i.e., the compound of the present invention or its pharmaceutical composition) an elegant appearance or to facilitate the production of pharmaceutical products (i.e., medicines).

The formulations can be prepared using conventional dissolution and mixing methods. For example, a bulk drug substance (i.e., a compound of the invention or a stable form of the compound (e.g., a complex formed with a cyclodextrin derivative or other known complexing agent)) is dissolved in a suitable solvent in the presence of one or more of the above-mentioned excipients. The compounds of the invention are typically prepared in pharmaceutical dosage forms to facilitate control of drug dosage and enable patients to comply with the prescribed treatment regimen.

The pharmaceutical composition (or formulation) to be administered can be packaged in a variety of ways, depending on the method used to administer the drug. Articles typically used for distribution include containers in which the pharmaceutical formulation in an appropriate form is placed. Suitable containers are well known to those skilled in the art and include materials such as bottles (plastic and glass), pouches, ampoules, plastic bags, metal cylinders, etc. The container may also include an infection prevention device to prevent inadvertent contact with the contents of the package. In addition, the container is provided with a label indicating the contents of the container. The label may also include appropriate warnings.

The pharmaceutical preparation of the compounds of this invention can be prepared for various routes of administration and types. For example, the compound of formula I with desired purity can be optionally mixed into the form of lyophilized preparations, pulverized powders or aqueous solutions with pharmaceutically acceptable diluents, carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences (1980) the 16th edition, Osol, A.Ed.). Can be prepared by mixing together with a physiologically acceptable carrier (i.e., a carrier that is nontoxic to the recipient at the dosage and concentration used) with the desired purity at ambient temperature. The pH of the preparation depends primarily on specific purposes and the concentration of the compound, but can be about 3-about 8. Preparing in the acetate buffer of pH 5 is a suitable embodiment.

The compound can generally be stored in the form of a solid composition, a lyophilized formulation, or an aqueous solution.

The pharmaceutical compositions of the present invention can be prepared, administered, and administered in a manner consistent with good medical practice (i.e., dosage, concentration, timing, course of treatment, carrier, and route). Factors to be considered in this context include: the specific condition to be treated, the specific mammal to be treated, the clinical condition of the individual patient, the cause of the condition, the site of delivery of the agent, the method of administration, the timing of administration, and other factors known to medical practitioners. A "therapeutically effective amount" of the compound to be administered depends on these considerations and is the minimum amount required to ameliorate or treat a hyperproliferative condition.

As a general suggestion, the initial pharmaceutically effective amount of the inhibitor per parenteral dose may be about 0.01-100 mg/kg, ie, about 0.1-20 mg/kg patient body weight per day, with a typical initial range of 0.3-15 mg/kg/day of compound used.

Acceptable diluents, carriers, excipients, and stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (small The active pharmaceutical ingredient may be encapsulated in microcapsules (e.g., hydroxymethylcellulose ^{microcapsules} , gelatin microcapsules, and poly(methyl methacrylate ⁾ microcapsules) prepared by coacervation or interfacial polymerization. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The sustained-release preparation of the compound of formula I can be prepared. The applicable example of sustained-release preparation includes a semipermeable matrix of a solid hydrophobic polymer containing the compound of formula I, and the matrix is a shaped article such as a film or a microcapsule. The example of sustained-release matrix includes a polyester, a hydrogel (such as poly (2-hydroxyethyl methacrylate) or poly (vinyl alcohol)), polylactic acid (US3773919), a copolymer of L-glutamic acid and γ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymer such as LUPRON DEPOT ^™ (injectable microspheres comprising lactic acid-glycolic acid copolymer and leuprorelin acetate) and poly-D-(-)-3-hydroxybutyric acid.

The formulations include those suitable for the routes of administration described herein. The formulations can be conveniently in unit dosage form and can be prepared by any method known in the art of pharmaceutics. Techniques and formulations are generally found in Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, PA). Such methods include the step of combining the active ingredient with a carrier that constitutes one or more auxiliary ingredients. The formulations are generally prepared by uniformly and closely combining the active ingredient with a liquid carrier, a pulverized solid carrier, or both, and then optionally shaping the product.

Formulations of compounds of Formula I suitable for oral administration can be prepared as discrete units, such as pills, capsules, cachets, or tablets, each containing a predetermined amount of a compound of Formula I. Compressed tablets can be prepared by compacting a free-flowing active ingredient, such as a powder or granules, in a suitable machine, optionally mixed with a binder, lubricant, inert diluent, preservative, surfactant, or dispersant. Molded tablets can be prepared by molding a mixture of the crushed active ingredient moistened with an inert liquid diluent in a suitable machine. The tablets can optionally be coated and scored, and optionally formulated to provide a slow or controlled release of the active ingredient therefrom. Tablets, lozenges, pastilles, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, such as gelatin capsules, syrups, or elixirs can be prepared for oral administration. Formulations of the compound of formula I for oral administration can be prepared by any method known in the art for the preparation of pharmaceutical compositions, and such compositions may contain one or more substances including sweeteners, flavorings, coloring agents, and preservatives to provide a palatable formulation. Tablets containing the active ingredient mixed with non-toxic pharmaceutically acceptable excipients suitable for the preparation of tablets are acceptable. These excipients may be, for example: inert diluents such as calcium carbonate or sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrants such as corn starch or alginic acid; binders such as starch, gelatin, or gum arabic; and lubricants such as magnesium stearate, stearic acid, or talc. Tablets may be uncoated or may be coated by known techniques, including microencapsulation, to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained effect over a longer period of time. Time-delay materials such as glyceryl monostearate or glyceryl distearate may be used alone or in combination with a wax.

For treatment of the eye or other external tissues such as the mouth and skin, the formulation is preferably administered as a topical ointment or cream and contains the active ingredient in an amount of, for example, 0.075% to 20% w/w. When the active ingredient is formulated as an ointment, it may be used with a paraffinic or water-miscible ointment base. Alternatively, the active ingredient may be formulated as a cream with an oil-in-water cream base. If desired, the aqueous phase of the cream base may contain a polyol, i.e., an alcohol containing two or more hydroxyl groups, such as propylene glycol, 1,3-butylene glycol, mannitol, sorbitol, glycerol, and polyethylene glycol (including PEG 400), and mixtures thereof. Topical formulations may suitably contain compounds that enhance the absorption or penetration of the active ingredient through the skin or other affected area. Examples of such skin penetration enhancers include dimethyl sulfoxide and related analogs. The oil phase of the emulsions of the present invention may be formed in a known manner from known ingredients. While this phase may comprise solely an emulsifier, it suitably comprises a mixture of at least one emulsifier with a fat or oil, or with both a fat and an oil. Preferably, the emulsifier comprises a hydrophilic emulsifier and a lipophilic emulsifier as stabilizers. It is also preferably comprised of oils and fats. In summary, the emulsifier, together with the optional stabilizer, constitutes the so-called emulsifying wax, and the wax, together with the oil and fat, constitutes the so-called emulsifying ointment base, which forms the oily dispersed phase of the cream formulation. Suitable emulsifiers and emulsion stabilizers for use in the formulations of the present invention include 60, 80, cetearyl alcohol, benzyl alcohol, myristyl alcohol, glyceryl monostearate, and sodium lauryl sulfate.

The aqueous suspension of the compound of formula I comprises an active material mixed with an excipient suitable for preparing an aqueous suspension. Such excipients include: suspending agents such as sodium carboxymethylcellulose, cross-linked carboxymethylcellulose, povidone, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinyl pyrrolidone, gum tragacanth and gum arabic, and dispersants or wetting agents such as naturally occurring phospholipids (e.g., lecithin), condensation products of alkylene oxides and fatty acids (e.g., polyoxyethylene stearate), condensation products of ethylene oxide and long-chain fatty alcohols (e.g., heptadecaethyleneoxycetanol), condensation products of ethylene oxide and partial esters derived from fatty acids and anhydrohexitol (e.g., polyoxyethylene sorbitan monooleate). The aqueous suspension may also include one or more preservatives (e.g., ethyl parahydroxybenzoate or propyl parahydroxybenzoate), one or more coloring agents, one or more flavoring agents, and one or more sweeteners (e.g., sucrose or saccharin).

Pharmaceutical compositions of the compound of Formula I can be in the form of sterile injectable formulations, such as sterile injectable aqueous or oily suspensions. Such suspensions can be prepared using suitable dispersants or wetting agents and suspending agents as described above according to known techniques. Sterile injectable formulations can also be sterile injectable solutions or suspensions in a non-toxic, parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol, or prepared as a lyophilized powder. Acceptable carriers and solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are generally used as solvents or suspending media. For this purpose, any bland, fixed oil can be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid can also be used to prepare injectable formulations.

The amount of active ingredient that can be combined with a carrier material to prepare a single dosage form can vary depending on the host to be treated and the specific mode of administration. For example, a time-release formulation intended for oral administration to humans can contain about 1-1000 mg of active material and be mixed with a suitable and appropriate amount of carrier material that can account for about 5% by weight to about 95% by weight of the total composition. Pharmaceutical compositions can be prepared to easily determine the dosage. For example, an aqueous solution intended for intravenous infusion can contain about 3-500 μg of active ingredient/ml of solution, thereby enabling infusion of a suitable volume at a rate of about 30 mL/hr.

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may contain suspending agents and thickening agents.

Formulations suitable for topical administration to the eye also include eye drops in which the active ingredient is dissolved or suspended in a suitable carrier, particularly an aqueous solvent for the active ingredient. Preferably, the concentration of the active ingredient present in such a formulation is about 0.5%-20% w/w, for example about 0.5%-10% w/w, for example about 1.5% w/w.

Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored basis, usually sucrose and acacia or tragacanth; lozenges comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

Formulations for rectal administration may be presented as a suppository with a suitable base containing for example cocoa butter or a salicylate.

The particle size of the formulation suitable for intrapulmonary or intranasal administration is, for example, 0.1-500 microns (including particle sizes within the range of micron increments between 0.1-500 microns, such as 0.5, 1, 30 microns, 35 microns, etc.), administered by rapid inhalation through the nasal passages or by oral inhalation to reach the alveolar sacs. Suitable formulations include aqueous or oily solutions of the active ingredient. Formulations suitable for spray or dry powder administration can be prepared according to conventional methods and can be delivered together with other therapeutic agents, such as the compounds described above for treating or preventing the following conditions.

Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or sprays containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

The preparation can be packaged in unit dose or multidose containers, for example, sealed ampoules and vials, and can be stored under freeze-dried (lyophilized) conditions, in order to inject immediately before use, only need to add sterile liquid carriers such as water. The solutions and suspensions for temporary injection are made from sterile powders, granules and tablets of the above-mentioned kind. Preferred unit dose formulations are those comprising the active ingredient of daily dose or unit daily divided doses or their appropriate portion as described above.

The present invention also provides veterinary compositions comprising at least one of the above-mentioned active ingredients and a veterinary carrier. The veterinary carrier is a material used for the purpose of administering the composition and can be a solid, liquid, or gaseous substance that is inert or veterinarily acceptable and compatible with the active ingredient. These veterinary compositions can be administered parenterally, orally, or by any other desired route.

Combination therapy

The compound of formula I can be used alone or in combination with other therapeutic agents for treating diseases or conditions described herein, such as inflammation or hyperproliferative conditions (e.g., cancer). In some embodiments, in a pharmaceutical combination formulation or as a dosing regimen for combined therapy, the compound of formula I is combined with another therapeutic compound having anti-inflammatory or anti-hyperproliferative properties or for treating inflammation, immunoreactive conditions, or hyperproliferative conditions (e.g., cancer). The other therapeutic agent can be an NSAID anti-inflammatory drug. The other therapeutic agent can be a chemotherapeutic agent. The second compound of the pharmaceutical combination formulation or dosing method preferably has an activity complementary to that of the compound of formula I, so that they do not adversely affect each other. Such compounds are suitably present in an amount effective for the intended purpose. In one embodiment, the composition of the present invention comprises a compound of formula I or its stereoisomer, tautomer, solvate, metabolite, or pharmaceutically acceptable salt or prodrug, and a therapeutic agent such as an NSAID.

The combination therapy can be administered simultaneously or in a sequential manner. When administered sequentially, the combination can be administered in two or more administrations. Combination therapy includes administration of separate formulations or single pharmaceutical formulations simultaneously, and administration sequentially in any order, wherein preferably there is a time period during which two (or all) activating agents simultaneously exert their biological activity.

Suitable dosages for any of the above co-administered drugs are those currently used and may be lowered due to the combined (synergistic) action of the newly identified drug and other therapeutic agents or treatments.

Combination therapy may provide and prove to be "synergistic," i.e., the effect achieved by the active ingredients when taken together is greater than the sum of the effects produced when the compounds are taken separately. A synergistic effect may be achieved when the active ingredients are: (1) co-formulated in a combined unit dosage formulation and administered or delivered simultaneously; (2) delivered as separate formulations alternatingly or in parallel; or (3) by some other regimen. When delivered in alternation therapy, a synergistic effect may be achieved when the compounds are administered or delivered sequentially, for example, by separate injections in separate syringes, by separate pills or capsules, or by separate infusions. Typically, in alternation therapy, an effective dosage of each active ingredient is administered sequentially, i.e., serially, whereas in combination therapy, effective dosages of two or more active ingredients are administered together.

In a specific embodiment of treatment, the compound of Formula I, its stereoisomers, tautomers, solvates, metabolites, pharmaceutically acceptable salts or prodrugs can be combined with other therapeutic agents, hormones or antibodies such as those described herein, and can also be combined with surgical treatment and radiotherapy. Therefore, the combination therapy of the present invention comprises administering at least one compound of Formula I, its stereoisomers, tautomers, solvates, metabolites, pharmaceutically acceptable salts or prodrugs, and using at least one other cancer treatment method. In order to achieve the desired combination therapy effect, the amount of the compound of Formula I and the other pharmaceutically active therapeutic agent and the relative timing of administration are selected.

Metabolites of compounds of formula I

The in vivo metabolic products of the compounds of Formula I described herein are also within the scope of the present invention. Such products may be produced, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, defatting, enzymatic hydrolysis, and the like of the administered compound. Thus, the present invention includes metabolites of compounds of Formula I, including compounds produced by contacting a compound of the present invention with a mammal for a period of time sufficient to produce a metabolic product thereof.

Metabolites are typically identified by preparing a radioisotope (e.g., ¹⁴ C or ³ H) labeled compound of the invention, administering it parenterally to an animal, such as a rat, mouse, guinea pig, monkey, or human, at a detectable dose (e.g., greater than about 0.5 mg/kg), allowing sufficient time for metabolism (usually about 30 seconds to 30 hours), and then isolating the conversion products from urine, blood, or other biological samples. These products are easily isolated because they are labeled (others are isolated using antibodies that can bind to epitopes remaining in the metabolites). Metabolite structures are determined by conventional methods, such as by MS, LC/MS, or NMR analysis. Metabolite analysis is performed in the same manner as conventional drug metabolism studies known to those skilled in the art. Metabolites, as long as they are not found in vivo, can be used in diagnostic assays to therapeutically administer the compounds of the invention.

Products

In another embodiment of the present invention, there is provided an article or "kit" comprising materials for treating the above-mentioned diseases and conditions. In one embodiment, the kit comprises a container comprising a compound of formula I, its stereoisomers, tautomers, solvates, metabolites, pharmaceutically acceptable salts or prodrugs. The kit may also include a label or package insert on or accompanying the container. The term "package insert" refers to instructions typically included in the commercial packaging of a therapeutic product, which contains information on indications, usage, dosage, administration, contraindications and/or warnings for use of the therapeutic product. Suitable containers include, for example, bottles, vials, syringes, blister packs, etc. The container can be made of various materials such as glass and plastic. The container can hold a compound of formula I or its formulation that is effective for treating a condition and can have a sterile entrance (for example, the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). At least one active ingredient in the composition is a compound of formula I. The label or package insert indicates that the composition is used to treat a selected condition such as cancer. In addition, the label or package insert may indicate that the patient to be treated is a patient suffering from a condition such as a hyperproliferative disorder, neurodegeneration, cardiac hypertrophy, pain, migraine, or neurotraumatic disease or event. In one embodiment, the label or package insert indicates that the composition comprising the compound of Formula I can be used to treat a condition resulting from abnormal cell growth. The label or package insert may also indicate that the composition can be used to treat other conditions. Alternatively, or additionally, the product may also include a second container comprising a pharmaceutically acceptable buffer such as antibacterial water for injection (BWFI), phosphate buffered saline, Ringer's solution, and glucose solution. The product may also include other materials desirable for commerce and users, including other buffers, diluents, fillers, needles, and syringes.

The kit may further include instructions for administering the compound of Formula I and the second pharmaceutical formulation (if present). For example, if the kit includes a first composition containing a compound of Formula I and a second pharmaceutical formulation, the kit may further include instructions for administering the first pharmaceutical composition and the second pharmaceutical composition simultaneously, sequentially, or separately to a patient in need thereof.

In another embodiment, the medicine box is suitable for delivering a solid oral form of a compound of formula I, such as a tablet or capsule. Such a medicine box preferably includes a plurality of unit doses. Such a medicine box may include a card with a dosage positioned with their intended use. An example of such a medicine box is a "blister pack". Blister packs are well known in the packaging industry and are widely used for packaging pharmaceutical unit dosage forms. If desired, a memory aid can be provided in the form of numbers, letters or other markings or calendar inserts for specifying the days on which administration can be made in a treatment schedule.

According to one embodiment, the medicine box may include (a) a first container containing a compound of Formula I; and optionally (b) a second container containing a second pharmaceutical preparation, wherein the second pharmaceutical preparation comprises a second compound having anti-hyperproliferative activity. Alternatively, or additionally, the medicine box may further include a third container containing a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and glucose solution. The medicine box may also include other materials desirable for business and users, including other buffers, diluents, fillers, needles, and syringes.

In some other embodiments in which the kit includes a composition of a compound of formula I and another therapeutic agent, the kit may include a container for accommodating the separated compositions, such as a separate bottle or a separate foil package, but the separated compositions may also be contained in a single, unseparated container. Typically, the kit includes instructions for administering the separated components. The kit form is particularly advantageous in the following situations: when the separated components are preferably administered in different dosage forms (e.g., oral and parenteral) or are administered at different intervals, or when the prescribing physician desires to gradually increase the dosage of each component in the combination.

Preparation of compounds of formula I

Compounds of formula I can be synthesized by synthetic routes including methods analogous to those well known in the chemical art, specifically referring to the description herein, as well as those descriptions of other heterocycles in Comprehensive Heterocyclic Chemistry II, Katritzky and Rees, Elsevier, 1997, e.g., Volume 3; Liebigs Annalen der Chemie, (9): 1910-16, (1985); Helvetica Chimica Acta, 41: 1052-60, (1958); Arzneimittel-Forschung, 40(12): 1328-31, (1990) (each expressly incorporated herein by reference). Starting materials are generally available from commercial sources such as Aldrich Chemicals (Milwaukee, WI), or are readily prepared using methods well known to those skilled in the art (e.g., by methods outlined in Louis F. Fieser and Mary Fieser, Reagents for Organic Synthesis, vol. 1-23, Wiley, N.Y. (1967-2006 ed.), or in Beilsteins Handbuch der organischen Chemie, 4, Aufl. ed. Springer-Verlag, Berlin, including the appendix (also available through the Beilstein online database)).

Synthetic chemistry transformations and protecting group methods (protection and deprotection) and the required reagents and intermediates for synthesizing compounds of Formula I are known in the art and include, for example, those described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and PGM Wuts, Protective Groups in Organic Synthesis, ^3rd Ed., John Wiley and Sons (1999); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995) and subsequent editions.

Compounds of Formula I can be prepared individually or as a library of compounds comprising at least two, for example, 5-1,000 compounds or 10-100 compounds. Libraries of compounds of Formula I can be prepared by combinatorial 'split and mix' methods, or by multistage parallel synthesis, using liquid or solid phase chemistry, according to methods known to those skilled in the art. Thus, according to another aspect of the present invention, a library of compounds comprising at least two compounds or pharmaceutically acceptable salts thereof is provided.

The accompanying drawings and examples provide exemplary methods for preparing compounds of Formula I. Those skilled in the art will appreciate that other synthetic routes can be used to synthesize compounds of Formula I. Although specific raw materials and reagents are described and discussed in the accompanying drawings and examples, other raw materials and reagents can be easily substituted to provide various derivatives and/or reaction conditions. In addition, many of the example compounds prepared by the described methods can be further modified using conventional chemistry known to those skilled in the art with reference to this disclosure.

When preparing compound of formula I, it may be necessary to protect the distal functional group (such as primary amine or secondary amine) of the intermediate. The needs for this protection can change along with the property of the distal functional group and the conditions of the preparation method. Suitable amino protecting groups include acetyl, trifluoroacetyl, tert-butyloxycarbonyl (BOC), benzyloxycarbonyl (CBz) and 9-fluorenylmethyleneoxycarbonyl (Fmoc). Those skilled in the art easily determine the necessity of such protection. About the overview of protecting groups and their purposes, referring to T.W.Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 1991.

General preparation method

General Method A Suzuki Coupling

Suzuki-type coupling reactions are used to form carbon-carbon bonds to connect the rings of compounds of Formula I and intermediates such as A-3 (Suzuki (1991) Pure Appl. Chem. 63:419-422; Miyaura and Suzuki (1979) Chem. Reviews 95(7):2457-2483; Suzuki (1999) J. Organometal. Chem. 576:147-168). Suzuki coupling is a palladium-mediated cross-coupling reaction of an aryl halide such as B-2 or B-5 with a boronic acid such as A-1 or A-2. For example, B-2 can be mixed with about 1.5 equivalents of 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bis(1,3,2-dioxaborolane) and dissolved in about 3 equivalents of sodium carbonate (1 M aqueous solution) and an equal volume of acetonitrile. A catalytic amount or more of a low-valent palladium reagent such as bis(triphenylphosphine)palladium(II) dichloride is added. In some cases, potassium acetate is used instead of sodium carbonate to adjust the pH of the aqueous layer. The reaction is then heated to about 140-150°C under pressure in a microwave reactor such as a Biotage Optimizer (Biotage, Inc.) for 10-30 minutes. The contents are extracted with ethyl acetate or another organic solvent. After evaporation of the organic layer, the boronate ester A-1 can be purified on silica gel or by reverse phase HPLC. Substituents Y ¹ , Y ² , R ⁵ , and R ⁶ are as defined, or protected forms or precursors thereof. Similarly, the bromide intermediate B-5 can be boronic acid esterified to provide A-2. Substituents Y ¹ , Y ² , R ¹ , R ² , R ³ , R ⁴ , Z ¹ , Z ² , Z ³ , Z ⁴ , and X are as defined, or protected forms or precursors thereof.

Suzuki coupling of B-2 and A-2 or A-1 and B-5 produces a compound of Formula I or intermediate A-3. Boronic acid ester (or boronic acid) (1.5 eq) A-1 or A-2 and a palladium catalyst such as bis(triphenylphosphine)palladium(II) dichloride (0.05 eq) are added to a mixture of halogenated intermediate (1 eq) B-2 or B-5 in acetonitrile and 1 M aqueous sodium carbonate solution (equal volumes to acetonitrile). The reaction mixture is heated to about 150° C. for about 15 minutes in a microwave. LC/MS indicates when the reaction is complete. Water is added to the mixture, the precipitated product is filtered, and then purified by HPLC to give product A-3. Substituents R ^1′ , R ^2′ , and R ^4′ can be R ¹ , R ² , and R ⁴ as defined, or protected forms or precursors thereof.

A variety of palladium catalysts can be used in the Suzuki coupling step. Various low-valent Pd(II) and Pd(0) catalysts can be used for the Suzuki coupling reaction, including PdCl2(PPh ₃ ) ₂ , Pd(t-Bu) ₃ , PdCl ₂ dppf CH ₂ Cl ₂ , Pd(PPh ₃ ) ₄ , Pd(OAc)/PPh ₃ , Cl ₂ Pd[(Pet ₃ )] ₂ , Pd(DIPHOS) ₂ , Cl ₂ Pd(Bipy), [PdCl(Ph ₂ PCH ₂ PPh ₂ )] ₂ , Cl ₂ Pd[P(o-tol) ₃ ] ₂ , Pd ₂ (dba) ₃ /P(o-tol) ₃ , Pd ₂ (dba)/P(furyl) ₃ , and Cl ₂ Pd[P(Pet ₃ )] _{2 .} Pd( _PMePh2 ) ₂ , _Cl2Pd [P(4-F-Ph) ₃ ] ₂ , Cl2Pd[P( _C6F6 ) ₃ ] ₂ , _Cl2Pd [P(2-COOH- _Ph )(Ph) ₂ ] ₂ , _Cl2Pd [P(4-COOH-Ph)(Ph _{) 2} ] ₂ , and the encapsulated catalysts PdEnCat ^™ 30, PdEnCat ^™ TPP30, and Pd(II)EnCat ^™ BINAP30 (US 2004/0254066).

General Procedure B Buchwald reaction

The Buchwald reaction is used to aminate the 6-bromo intermediate B-1 (Wolf and Buchwald (2004) Org. Synth Coll. Vol. 10:423; Paul et al. (1994) Jour. Amer. Chem. Soc. 116:5969-5970). To a solution of the halogenated intermediate B-1 in DMF is added _the appropriate amine ^R5 - _NH2 (200 mol%), _Cs2CO3 (50 mol%), _Pd2 (dba) ₃ (5 mol%), and XANTPHOS (10 mol%). The reaction is heated to approximately 110°C under pressure in a Biotage Optimizer microwave reactor for approximately 30 min. The resulting solution is concentrated in vacuo to provide B-2. Other palladium catalysts and phosphine ligands may be used.

N-Arylamide intermediate B-5 can also be prepared from cyclic amide intermediate B-3 and aryl bromide B-4 via a Buchwald reaction.

FIG1 shows an exemplary synthetic route for preparing 6-chloro, 4-aminopyridazinone compounds including 6-chloro-4-(5-methyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)pyridazin-3(2H)-one 101f from 3-nitropyrazole-5-carboxylic acid.

FIG2 shows an exemplary synthetic route for preparing tricyclic amide-phenylboronic acid ester compounds including 2-(2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 101m from 4,5,6,7-tetrahydro-1H-indole.

FIG3 shows an exemplary synthetic route to tricyclic amide-phenyl bromides including 2-bromo-6-(1-oxo-3,4,5,6,7,8-hexahydrobenzothieno[2,3-c]pyridin-2(1H)-yl)benzyl acetate 104h from 4,5,6,7-tetrahydrobenzo[b]thiophene-2-carboxylic acid.

FIG4 shows another exemplary synthetic route to tricyclic amide-phenyl bromides including 6,6-dimethyl-3,4,6,7-tetrahydro-5H-cyclopenta[4,5]thieno[2,3-c]pyridin-1(2H)-one 105i from 3-methylcyclopent-2-enone.

Figure 5 shows an exemplary synthetic route for preparing tricyclic (1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrido[3,4-b]indolizin-1(2H)-one compounds as boronic acid esters, including 2-(1-oxo-3,4,6,7,8,9-hexahydropyrido[3,4-b]indolizin-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 118f, from 5,6,7,8-tetrahydroindolizin-2-carboxylic acid.

6 shows an exemplary synthetic route for the preparation of intermediate 2-bromo-4-fluoro-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 198d from 1,3-dibromo-5-fluoro-2-iodobenzene.

Figure 7 shows an exemplary synthetic route for preparing intermediate 4-fluoro-2-(1-methyl-5-(5-(4-methylpiperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 198g.

8 shows an exemplary synthetic route for preparing intermediate 5-fluoro-2-(1-methyl-5-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 210e.

Figure 9 shows an exemplary synthetic route for preparing intermediate 5-[5-fluoro-2-(acetoxymethyl)-3-(1-methyl-5-{[5-(4-methylpiperazin-1-yl)pyridin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)phenyl]-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-6-one 212c.

Separation method

In the method for preparing the compound of formula I, it is advantageous to separate the reaction products from each other and/or from the raw materials. The desired product of each step or successive step is separated and/or purified to the desired uniformity by conventional techniques in the art. Typically such separations include multiphase extraction, crystallization from a solvent or solvent mixture, distillation, sublimation, or chromatography. Chromatography can include many methods, including, for example, reverse phase and normal phase; size exclusion; ion exchange; high pressure, medium pressure, and low pressure liquid chromatography and apparatus; small scale analysis; simulated moving bed (SMB) and preparative thin layer chromatography or thick layer chromatography, as well as small scale thin layer techniques and flash chromatography.

Another type of separation method involves treating the mixture with a reagent selected to bind to or separable the desired product, unreacted starting materials, reaction by-products, etc. Such reagents include adsorbents or absorbents, such as activated carbon, molecular sieves, ion exchange media, etc. Alternatively, the reagent (which may be an acid in the case of alkaline materials or a base in the case of acidic materials) may be a binding agent, such as an antibody, a binding protein, a selective chelating agent such as a crown ether, a liquid/liquid ion exchanger (LIX), etc. The appropriate separation method is selected based on the properties of the materials involved, such as boiling point and molecular weight in distillation and sublimation, the presence or absence of polar functional groups in chromatography, the stability of the materials in acidic and alkaline media in multiphase extraction, etc.

Can be by methods well known to those skilled in the art, for example by chromatography and/or fractional crystallization, according to the physicochemical differences of diastereomers, diastereomeric mixtures are separated into their individual diastereomers. Enantiomers can be by reacting with suitable optically active compounds (for example chiral auxiliary such as chiral alcohol or Mosher acyl chlorides), enantiomeric mixtures are converted into diastereomeric mixtures, separated diastereomers, and individual diastereoisomers are converted (for example hydrolyzed) into corresponding pure enantiomers for separation. In addition, some compounds of the present invention can be atropisomers (for example substituted biaryls), and they are considered as part of the present invention. Enantiomers can also be separated using chiral HPLC columns.

Racemic mixtures can be resolved, for example, by diastereomeric formation with an optically active resolving agent to obtain a single stereoisomer, such as an enantiomer, substantially free of its stereoisomers (Eliel, E. and Wilen, S. "Stereochemistry of Organic Compounds," John Wiley & Sons, Inc., New York, 1994; Lochmuller, C.H., (1975) J. Chromatogr., 113(3):283-302). Racemic mixtures of the chiral compounds of the present invention can be separated and isolated by any suitable method, including: (1) formation of diastereomeric salts with chiral compounds and separation by fractional crystallization or other methods, (2) formation of diastereomeric compounds with a chiral derivatizing agent, separation of the diastereoisomers, and conversion to the pure stereoisomers, and (3) direct separation of the substantially pure or enriched stereoisomers under chiral conditions. See: "Drug Stereochemistry, Analytical Methods and Pharmacology," Irving W. Wainer, Ed., Marcel Dekker, Inc., New York (1993).

According to method (1), diastereomeric salts are formed by reacting an enantiomerically pure chiral base such as strychnine, quinine, ephedrine, strychnine, α-methyl-β-phenylethylamine (amphetamine), etc. with an asymmetric compound containing an acidic functional group such as a carboxylic acid or a sulfonic acid. The diastereomeric salts can be separated by fractional crystallization or ion chromatography. In order to separate the optical isomers of an amino compound, a chiral carboxylic acid or sulfonic acid such as camphorsulfonic acid, tartaric acid, mandelic acid, or lactic acid is added to form diastereomeric salts.

Alternatively, according to method (2), the substrate to be resolved is reacted with one enantiomer of a chiral compound to form a diastereomeric pair (E. and Wilen, S. "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., 1994, p. 322). The diastereomeric compound can be formed by reacting the asymmetric compound with an enantiomerically pure chiral derivatizing agent such as a menthyl derivative, followed by separation of the diastereomers and hydrolysis to produce a pure or enriched enantiomer. Methods for determining optical purity include preparing a chiral ester of the racemic mixture, for example, a menthyl ester such as (-) menthyl chloroformate, or preparing the Mosher ester, α-methoxy-α-(trifluoromethyl)phenyl acetate (Jacob III. J. Org. Chem. (1982) 47:4165), in the presence of a base, and then analyzing the ¹ H NMR spectrum to determine the presence of two atropisomeric enantiomers or diastereomers. Stable diastereomers of atropisomeric compounds can be separated and isolated by normal-phase and reverse-phase chromatography according to the method for separating atropisomeric naphthyl-isoquinolines (WO 96/15111). According to method (3), a racemic mixture of two enantiomers can be separated by chromatography using a chiral stationary phase ("Chiral Liquid Chromatograph" (1989) WJ Lough, Ed., Chapman and Hall, New York; Okamoto, J. Chromatogr., (1990) 513:375-378). Enriched or purified enantiomers can be distinguished by methods used to distinguish other chiral molecules with asymmetric carbon atoms, such as optical rotation and circular dichroism.

Example

Example 101 2-(2-methyl-3-(5-(5-methyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-6-oxo-1,6-dihydropyridazin-3-yl)phenyl)-3,4-dihydropyrazino[1,2-a]indol-1(2H)-one 101

Example 101a (3-Nitro-1H-pyrazol-5-yl)methanol 101a

A 3-L, three-necked round-bottom flask equipped with a mechanical stirrer, addition funnel, and nitrogen inlet was purged with nitrogen. 3-Nitropyrazole-5-carboxylic acid (28.0 g, 178 mmol) and THF (420 mL) were added and cooled to –5°C in an ice/acetone bath. Borane-THF complex solution (1.0 M, 535 mL, 535 mmol) was added at a rate that maintained the internal reaction temperature below 5°C. After the addition was complete, the cooling bath was removed, and the reaction was stirred at room temperature for 18 hours. The reaction was then cooled to –5°C in an ice/acetone bath, and water (70 mL) and 4N hydrochloric acid (70 mL) were added and stirred at reflux for 1 hour to disrupt the borane-pyrazole complex. The reaction was cooled to room temperature and concentrated under reduced pressure to a volume of approximately 30 mL. Ethyl acetate (175 mL) was added, and the mixture was stirred for 15 minutes. The aqueous layer was separated and extracted with ethyl acetate (4 x 200 mL). The combined organic layers were washed with saturated aqueous sodium bicarbonate solution (2×50 mL), brine (50 mL), and dried over sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure to afford 101a as a light yellow solid in 94% yield (24.0 g): ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 13.90 (br s, 1H), 6.87 (s, 1H), 5.58 (t, 1H, J=5.4 Hz), 4.53 (d, 2H, J=5.1 Hz); MS (ESI+) m/z 144.0 (M+H).

Example 101b (1-(2-bromoethyl)-3-nitro-1H-pyrazol-5-yl)methanol 101b

A 1-L, three-necked round-bottom flask equipped with a mechanical stirrer and thermostat was purged with nitrogen and charged with (3-nitro-1H-pyrazol-5-yl)methanol 101a (25.0 g, 175 mmol), DMF (250 mL), and cesium carbonate (70.0 g, 215 mmol). The mixture was heated at 104°C for 5 min _. The reaction mixture was then cooled to 0°C using an ice/acetone bath, and dibromoethane (329 g, 1.75 mol) was added portionwise (no exotherm). The reaction was stirred at 0°C for 1 h and then at room temperature for 4 h. A solution of _KH₂PO₄ (40 g) in water (400 mL) was then slowly added. The reaction mixture was stirred at room temperature for 30 min. Ethyl acetate (450 mL) was added, the aqueous layer was separated, and the mixture was extracted with ethyl acetate (2 x 100 mL). The combined organic layers were washed with water (200 mL), brine (200 mL), dried over sodium sulfate, and the drying agent was removed by filtration. The filtrate was concentrated under reduced pressure to give crude product 101b as an orange oil in 86% yield (37.5 g): ¹ H NMR (300 MHz, CDCl ₃ ) δ 6.85 (s, 1H), 4.82 (d, 2H, J=5.4 Hz), 4.66 (t, 2H, J=6.3 Hz), 3.83 (t, 2H, J=6.3 Hz); MS (ESI+) m/z 249.9 (M+H).

Example 101c 1-(2-bromoethyl)-5-(bromomethyl)-3-nitro-1H-pyrazole 101c

A 500 mL, three-necked round-bottom flask equipped with a magnetic stirrer, nitrogen inlet, and reflux condenser was purged with nitrogen and charged with (1-(2-bromoethyl)-3-nitro-1H-pyrazol-5-yl)methanol 101b (37.0 g, 148 mmol) and chloroform (160 mL). The reaction was cooled to –5°C using an ice/acetone bath and phosphorus tribromide (40.0 g, 148 mmol) was added portionwise. The cooling bath was removed and the reaction was stirred at reflux for 2 h. Thereafter, the reaction was cooled to –5°C and saturated aqueous sodium bicarbonate (250 mL) was added until the pH reached 8.5. The mixture was extracted with ethyl acetate (3 x 150 mL), and the combined organic layers were washed with saturated aqueous sodium carbonate (2 x 50 mL), brine (75 mL), dried over sodium sulfate, and the drying agent was removed by filtration. The filtrate was concentrated under reduced pressure to yield a yellow residue, which was dissolved in dichloromethane (60 mL) with gentle heating. To the 4-nitro-1H-pyrazoles (19.7g) of 1- (2- bromoethyl) -5- (bromomethyl) -3- nitro -1H- pyrazoles was added (2 × 20mL). The filtrate was evaporated and the combined solution was operated again to obtain an additional 9.70g of 1- (2- bromoethyl) -5- (bromomethyl) -3- nitro -1H- pyrazoles. The solids were combined and dried under high vacuum for 18 h to afford 101c in 57% yield (26.0 g) as white crystals: mp 95-97° C.; ¹ H NMR (300 MHz, CDCl ₃ ) δ 6.93 (s, 1H), 4.63 (t, 2H, J=6.0 Hz), 4.54 (s, 2H), 3.86 (t, 2H, J=6.0 Hz).

Example 101d 5-Methyl-2-nitro-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazine 101d

To a 1 L single-necked round-bottom flask equipped with a magnetic stirrer and a nitrogen inlet was added THF (350 mL), 101c (10.0 g, 32.2 mmol), and a 2M methylamine solution in THF (113 mL, 225 mmol) and stirred at room temperature for 72 h. The reaction was then concentrated to dryness under reduced pressure, and the resulting solid was stirred with a mixture of ethyl acetate (75 mL) and 10% aqueous potassium carbonate (75 mL). The aqueous layer was separated and extracted with ethyl acetate (2 x 75 mL). The combined organic extracts were washed with 10% aqueous potassium carbonate (75 mL) and then brine (50 mL) and dried over sodium sulfate. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure to afford 101d as a yellow solid in 97% yield (5.70 g): ¹ H NMR (300 MHz, CDCl ₃ ) δ 6.62 (s, 1H), 4.28 (t, 2H, J=5.4 Hz), 3.67 (s, 2H), 2.95 (t, 2H, J=5.4 Hz), 2.52 (s, 3H); MS (ESI+) m/z 183.0 (M+H).

Example 101e 5-Methyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-amine 101e

A 500 mL Parr reaction bottle was purged with nitrogen and 10% palladium on carbon (50% humidity, 800 mg dry weight) was added, along with a solution of 5-methyl-2-nitro-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazine 101d (4.00 g, 2.20 mmol) in ethanol (160 mL). The bottle was connected to a Parr hydrogenation reactor, vacuumized, filled with hydrogen to a pressure of 45 psi and shaken for 2 h. After this, hydrogen was extracted and nitrogen was charged into the bottle. Celite 521 (1.0 g) was added and the mixture was filtered through a Celite 521 pad. The filter cake was washed with ethanol (2×75 mL), and the combined filtrates were concentrated to dryness under reduced pressure to afford 101e (3.31 g) as an orange solid in 99% yield: ¹ H NMR (300 MHz, CDCl ₃ ) δ 5.34 (s, 1H), 3.98 (t, 2H, J=5.4 Hz), 3.52 (s, 3H), 2.84 (t, 2H, J=5.7 Hz), 2.45 (s, 3H); MS (ESI+) m/z 153.1 (M+H)

Example 101f 6-Chloro-4-(5-methyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)pyridazin-3(2H)-one 101f

To a 50-mL single-necked round-bottom flask equipped with a magnetic stirrer, a reflux condenser, and a nitrogen inlet, 1,4-dioxane (5.0 mL), 101e (152 mg, 1.00 mmol), 4-bromo-6-chloropyridazine-3(2H)-one (209 mg, 1.00 mmol), and 1M LiHMDS THF solution (5.0 mL, 5.00 mmol) were added. After nitrogen was bubbled through the resulting solution for 30 min, Xantphos (49 mg, 0.05 mmol) and tris(dibenzylideneacetone)dipalladium(0) (59 mg, 0.085 mmol) were added, and the reaction mixture was heated under reflux for 3 h. Afterwards, the reaction was cooled to room temperature and water (10 mL) was added. The pH was adjusted to 6.5 with 2N hydrochloric acid. The resulting precipitate was collected by vacuum filtration, washed with water (2×25 mL), absorbed onto silica gel and purified by flash chromatography to afford 74% yield (210 mg) of 101f as a light brown solid: ^1H NMR (300 MHz, DMSO-d ₆ ) δ 12.94 (s, 1H), 9.55 (s, 1H), 7.68 (s, 1H), 5.96 (s, 1H), 4.04 (t, 1H, J=5.7 Hz), 3.53 (s, 2H), 2.82 (t, 2H, J=5.7 Hz), 2.36 (s, 3H); MS (ESI+) m/z 281.1 (M+H)

Example 101g 2,2,2-trichloro-1-(4,5,6,7-tetrahydro-1H-indol-2-yl)ethanone 101g

A 100 mL single-necked round-bottom flask equipped with a magnetic stirrer, condenser, and nitrogen inlet was purged with nitrogen and charged with 4,5,6,7-tetrahydro-1H-indole (3.00 g, 24.8 mmol), trichloroacetyl chloride (13.5 g, 74.4 mmol), and 1,2-dichloroethane (50 mL). The solution was stirred at 85°C for 2 h. Thereafter, the reaction mixture was concentrated under reduced pressure to give 100% yield (6.50 g) of 101 g as a black semisolid: ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 11.94 (s, 1H), 7.05 (s, 1H), 2.62 (t, 2H, J=6.0 Hz), 2.47 (t, 2H, J=6.0 Hz), 1.80 (m, 2H), 1.65 (m, 2H); MS (ESI+) m/z 266.0 (M+H)

Example 101h Ethyl 4,5,6,7-tetrahydro-1H-indole-2-carboxylate 101h

A 100 mL single-necked round-bottom flask equipped with a magnetic stirrer and a nitrogen inlet was purged with nitrogen and charged with 101 g (6.50 g, 24.8 mmol), sodium ethoxide (17.0 mg, 0.25 mmol), and ethanol (40 mL). The solution was stirred at room temperature for 1 h. Thereafter, the reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography to afford 100% yield (4.80 g) of 101h as a brown solid: mp 70-72° C.; ¹ H NMR (300 MHz, CDCl ₃ ) δ 9.08 (s, 1H), 6.75 (s, 1H), 4.25 (q, 2H, J=7.2 Hz), 2.65 (t, 2H, J=6.0 Hz), 2.56 (t, 2H, J=6.0 Hz), 1.85 (m, 4H), 1.28 (t, 3H, J=7.2 Hz); MS (ESI+) m/z 194.1 (M+H)

Example 101i 1-(Cyanomethyl)-4,5,6,7-tetrahydro-1H-indole-2-carboxylic acid ethyl ester 101i

A 125 mL single-neck round-bottom flask equipped with a magnetic stirrer and a nitrogen inlet was purged with nitrogen, and 101h (5.76 g, 29.8 mmol) and DMF (50 mL) were added. The solution was cooled to 0 ° C using an ice bath. NaH (60% dispersion in mineral oil, 1.43 g, 35.8 mmol) was added. The resulting mixture was stirred at room temperature for 1 h. Thereafter, bromoacetonitrile (1.43 g, 35.8 mmol) was added. The mixture was stirred at room temperature for 14 h. Thereafter, the reaction mixture was concentrated under reduced pressure, and the residue was then partitioned between ethyl acetate (150 mL) and water (450 mL). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (3×150 mL). The combined organic layers were washed with brine, dried over sodium sulfate, and then concentrated under reduced pressure. The residue was purified by column chromatography to give 55% yield (3.80 g) of ethyl 1-(cyanomethyl)-4,5,6,7-tetrahydro-1H-indole-2-carboxylate 101i as a yellow semisolid: ¹ H NMR (300 MHz, CDCl ₃ ) δ 6.66 (s, 1H), 5.29 (s, 2H), 4.28 (q, 2H, J=7.2 Hz), 2.62 (t, 2H, J=6.3 Hz), 2.49 (t, 2H, J=6.3 Hz), 1.92 (m, 2H), 1.75 (m, 2H), 1.33 (t, 3H, J=7.2 Hz); MS (ESI+) m/z 233.1 (M+H)

Example 101j 1-(2-aminoethyl)-4,5,6,7-tetrahydro-1H-indole-2-carboxylic acid ethyl ester 101j

A 200 mL Parr reaction bottle was purged with nitrogen and charged with 10% palladium on carbon (50% humidity, 1.28 g dry weight), ethyl 1-(cyanomethyl)-4,5,6,7-tetrahydro-1H-indole-2-carboxylate 101i (3.00 g, 12.9 mmol), 12% hydrochloric acid (6.5 mL, 25 mmol), ethyl acetate (60 mL), and ethanol (40 mL). The bottle was connected to a Parr hydrogenation reactor, evacuated, filled with hydrogen to a pressure of 50 psi, and then shaken for 6 h. Thereafter, the hydrogen was removed and nitrogen was filled into the bottle. Celite 521 (4.0 g) was added and the mixture was filtered through a pad of Celite 521. The filter cake was washed with ethanol (2×20 mL), and the combined filtrate was then concentrated to dryness under reduced pressure. The residue was partitioned between ethyl acetate (150 mL) and 10% aqueous potassium carbonate solution (100 mL). The organic layer was separated and the aqueous layer was extracted with ethyl acetate (3 x 75 mL).The combined organic layers were dried over sodium sulfate and concentrated under reduced pressure. The residue was triturated with ethanol (5 mL) to give 71% yield (1.71 g) of ethyl 1-(2-aminoethyl)-4,5,6,7-tetrahydro-1H-indole-2-carboxylate 101j as a white solid: mp 102–104° C.; ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 6.61 (s, 1H), 6.22 (br, 2H), 4.15 (m, 4H), 2.77 (m, 2H), 2.59 (t, 2H, J=6.5 Hz), 2.42 (t, 2H, J=6.5 Hz), 1.70 (m, 2H), 1.62 (m, 2H), 1.23 (t, 3H, J=7.0 Hz); MS (APCI+) m/z 237.2 (M+H)

Example 101k 1-(2-aminoethyl)-4,5,6,7-tetrahydro-1H-indole-2-carboxylic acid ethyl ester 101k

A 100 mL single-necked round-bottom flask equipped with a magnetic stirrer and a nitrogen inlet was purged with nitrogen and 1-(2-aminoethyl)-4,5,6,7-tetrahydro-1H-indole-2-carboxylic acid ethyl ester 101j (1.80 g, 7.63 mmol), sodium ethoxide (1.55 g, 22.8 mmol) and ethanol (50 mL) were added. The mixture was stirred at 55 ° C for 5 h. Thereafter, the reaction mixture was concentrated under reduced pressure, and the residue was then partitioned between ethyl acetate (200 mL) and water (100 mL). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (2×100 mL). The combined organic layers were washed with brine, dried over sodium sulfate, and then concentrated under reduced pressure. The residue was purified by column chromatography to give 42% yield (605 mg) of ethyl 1-(2-aminoethyl)-4,5,6,7-tetrahydro-1H-indole-2-carboxylate 101k as a white solid: mp 207-209 °C; ^1H NMR (500 MHz, DMSO- _d6 ) δ 7.41 (s, 1H), 6.36 (s, 1H), 3.84 (t, 2H, J = 6.0 Hz), 3.42 (m, 2H), 2.51 (t, 2H, J = 6.0 Hz), 2.42 (t, 2H, J = 6.0 Hz), 1.76 (m, 2H), 1.65 (m, 2H); (APCI+) m/z 191.3 (M+H)

Example 1011 2-(3-bromo-2-methylphenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 1011

To a 50 mL three-necked round-bottom flask equipped with a reflux condenser, a magnetic stirrer, and a nitrogen inlet was added 1-(2-aminoethyl)-4,5,6,7-tetrahydro-1H-indole-2-carboxylic acid ethyl ester 101k (560 mg, 2.95 mmol), 2,6-dibromotoluene (1.47 g), cesium carbonate (1.92 g, 5.90 mmol), N,N'-dimethylethylenediamine (260 mg, 2.95 mmol), and 1,4-dioxane (25 mL). After nitrogen was bubbled through the resulting suspension for 30 min, copper N,N'-dimethylethylenediamine (260 mg, 2.95 mmol) was added, and the reaction mixture was heated at 105 ° C (oil bath temperature) for 14 h. Afterwards, the mixture was cooled to room temperature and filtered. The filtrate was diluted with ethyl acetate (100 mL) and water (20 mL). The organic layer was separated and the aqueous layer was extracted with ethyl acetate (3×30 mL). The combined organic layers were dried over sodium sulfate and then concentrated under reduced pressure. The residue was purified by column chromatography to afford 57% yield (600 mg) of 2-(3-bromo-2-methylphenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 1011 as a white solid: mp 163–165° C.; ¹ H NMR (500 MHz, DMSO-d ₆ )δ7.57(dd,1H,J＝8.0,0.5Hz),7.32(d,1H,J＝7.5Hz),7.21(t,1H,J＝8.0Hz),6.50(s,1H),4.11(m ,3H),3.75(m,1H),2.59(m,2H),2.45(m,2H),2.21(s,3H),1.78(m,2H),1.68(m,2H); (APCI+)m/z 358.6(M+H)

Example 101m 2-(2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 101m

To a 50-mL three-necked round-bottom flask equipped with a reflux condenser, a magnetic stirrer, and a nitrogen inlet was added 2-(3-bromo-2-methylphenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 1011 (600 mg, 1.67 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-di(1,3,2-dioxaborolane) (1.70 g, 6.70 mmol), potassium acetate (656 mg, 6.68 mmol), and 1,4-dioxane (25 mL). After nitrogen was bubbled through the resulting suspension for 30 min, XPhos (159 mg, 0.334 mmol) and tris(dibenzylideneacetone) were added. )dipalladium(0) (153 mg, 0.167 mmol) was added, and the reaction mixture was heated at 105°C (oil bath temperature) for 14 h. Thereafter, the mixture was cooled to room temperature and filtered. The filtrate was diluted with ethyl acetate (75 mL) and water (20 mL). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (3×25 mL). The combined organic layers were dried over sodium sulfate and then concentrated under reduced pressure. The residue was purified by column chromatography to give 109% crude yield (740 mg) of 2-(2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indole-1(2H)-one 101m as a yellow oil: ¹ H NMR (300MHz, CDCl ₃ )δ7.75(dd,1H,J＝6.6,2.1Hz),7.26(m,2H),6.80(s,1H),4.10(m,3H),3.75(m,1H) ,2.54(m,4H),2.45(s,3H),1.87(m,2H),1.75(m,2H),1.25(s,12H); MS(APCI+)m/z 407.7(M+H)

To a 100 mL three-necked round-bottom flask equipped with a reflux condenser, a magnetic stirrer and a nitrogen inlet was added 2-(2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 101m (740 mg, 1.67 mmol), 6-chloro-4-(5-methyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)pyridazin-3(2H)-one 101f (422 mg, 1.34 mmol), sodium carbonate (568 mg, 5.36 mmol), DMF (5 mL), water (5 mL) and 1,4-dioxane (20 mL). After bubbling nitrogen through the resulting suspension for 30 min, tetrakis(triphenylphosphine)palladium(0) (155 mg, 0.134 mmol) was added and the reaction mixture was heated at 100 °C for 15 h. Thereafter, the mixture was cooled to room temperature and diluted with ethyl acetate (100 mL) and water (30 mL). The organic layer was separated and the aqueous layer was extracted with ethyl acetate (3×25 mL). The combined organic layers were dried over sodium sulfate and then concentrated under reduced pressure. The residue was purified by column chromatography on silica gel to give 101 in 17% yield (120 mg) as an off-white solid: mp 195–197 °C; ¹ H NMR (500 MHz, DMSO-d ₆ )δ12.95(s,1H),9.19(s,1H),7.75(s,1H),7.34(m,2H),7.29(m,1H),6 .50(s,1H),5.96(s,1H),4.14(m,1H),4.08(m,2H),3.95(m,2H),3.77(m ,1H),3.51(s,2H),2.78(t,2H,J＝6.0Hz),2.60(m,2H),2.46(t,2H,J＝6. 0Hz),2.35(s,3H),2.07(s,3H),1.78(m,2H),1.68(m,2H); MS(ESI+)m/z 525.2(M+H)

Example 102 2-(2-methyl-3-(5-(5-methyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-6-oxo-1,6-dihydropyridazin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 102

Example 102a Ethyl 1-(cyanomethyl)-1H-indole-2-carboxylate 102a

A 250-mL single-necked round-bottom flask equipped with a magnetic stirrer and a nitrogen inlet was purged with nitrogen and charged with ethyl indole-2-carboxylate (10.0 g, 52.9 mmol) and DMF (100 mL). The solution was cooled to 0°C using an ice bath. NaH (60% suspension in mineral oil, 2.54 g, 63.5 mmol) was added. The resulting mixture was stirred at room temperature for 1 hour. Subsequently, bromoacetonitrile (7.62 g, 63.5 mmol) was added. The mixture was stirred at room temperature for 14 hours. The reaction mixture was then concentrated under reduced pressure, and the residue was partitioned between ethyl acetate (300 mL) and water (900 mL). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (3 x 300 mL). The combined organic layers were washed with brine, dried over sodium sulfate, and then concentrated under reduced pressure. The residue was purified by column chromatography to give 66% yield (8.00 g) of 102a as an off-white solid: mp 65-67 °C; ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.72 (d, 1H, J=8.1), 7.44 (m, 3H), 7.25 (m, 1H), 5.62 (s, 2H), 4.42 (q, 2H, J=7.2 Hz), 1.43 (t, 3H, J=7.2 Hz); MS (ESI+) m/z 229.1 (M+H).

Example 102b 3,4-dihydropyrazino[1,2-a]indol-1(2H)-one 102b

A 500-mL Parr reaction bottle was purged with nitrogen and charged with 10% palladium on carbon (50% humidity, 3.47 g dry weight), 102a (8.00 g, 35.0 mmol), 12% hydrochloric acid (17.5 mL, 70 mmol), ethyl acetate (150 mL), and ethanol (100 mL). The bottle was connected to a Parr hydrogenation reactor, evacuated, filled with hydrogen to a pressure of 50 psi, and then shaken for 6 h. Thereafter, the hydrogen was removed and nitrogen was filled into the bottle. Celite 521 (10.0 g) was added and the mixture was filtered through a pad of Celite 521. The filter cake was washed with ethanol (2×50 mL), and the combined filtrate was concentrated to dryness under reduced pressure. The residue was partitioned between ethyl acetate (400 mL) and 10% aqueous potassium carbonate solution (300 mL). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (3×200 mL). The combined organic layers were dried over sodium sulfate and then concentrated under reduced pressure. The residue was triturated with ethanol (5 mL) to give 70% yield (4.57 g) of 102b as an off-white solid: mp 228-230 °C; ¹ H NMR (300 MHz, CDCl ₃ ) 7.73 (d, 1H, J=8.1 Hz), 7.34 (m, 3H), 7.18 (m, 1H), 6.75 (br s, 1H), 4.29 (t, 2H, J=5.4 Hz), 3.84 (m, 2H); MS (ESI+) m/z 187.1 (M+H).

Example 102c 2-(3-bromo-2-methylphenyl)-3,4-dihydropyrazino[1,2-a]indol-1(2H)-one 102c

To a 100-mL three-necked round-bottom flask equipped with a reflux condenser, a magnetic stirrer, and a nitrogen inlet was added 102b (1.00 g, 5.37 mmol), (2.69 g, 10.7 mmol), cesium carbonate (3.49 g, 10.7 mmol), N,N'-dimethylethylenediamine (473 mg, 5.37 mmol), and 1,4-dioxane (45 mL). After bubbling nitrogen through the resulting suspension for 30 min., cuprous iodide (510 mg, 2.69 mmol) was added, and the reaction mixture was heated at 105 ° C (oil bath temperature) for 14 h. Thereafter, the mixture was cooled to room temperature and filtered. The filtrate was diluted with ethyl acetate (200 mL) and water (40 mL). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (3×50 mL). The combined organic layers were dried over sodium sulfate and then concentrated under reduced pressure. The residue was purified by column chromatography to afford 62% yield (1.18 g) of 102c as an off-white solid: mp 178-180 °C; ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.74 (dt, 1H, J=8.2, 1.0 Hz), 7.58 (dd, 1H, J=7.8, 1.2 Hz), 7.37 (m, 3H), 7.19 (m, 3H), 4.45 (m, 2H), 4.21 (m, 1H), 3.95 (m, 1H), 2.38 (s, 3H); MS (ESI+) m/z 355.0 (M+H).

Example 102d 2-(2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-3,4-dihydropyrazino[1,2-a]indol-1(2H)-one 102d

To a 100-mL three-necked round-bottom flask equipped with a reflux condenser, a magnetic stirrer, and a nitrogen inlet was added 102c (1.18 g, 3.32 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bis(1,3,2-dioxaborolane) (929 mg, 3.65 mmol), potassium acetate (491 mg, 4.98 mmol), and 1,4-dioxane (25 mL). After nitrogen was bubbled through the resulting suspension for 30 min, XPhos (317 mg, 0.664 mmol) and tris(dibenzylideneacetone)dipalladium(0) (561 mg, 0.332 mmol) were added, and the reaction mixture was heated at 105° C. (oil bath temperature) for 14 h. Thereafter, the mixture was cooled to room temperature and filtered. The filtrate was diluted with ethyl acetate (150 mL) and water (40 mL). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (3×50 mL). The combined organic layers were dried over sodium sulfate and then concentrated under reduced pressure. The residue was purified by column chromatography to afford 102d in 57% yield (760 mg) as an off-white solid: mp 193–195° C.; ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.80 (dd, 1 H, J=7.0, 1.7 Hz), 7.74 (d, 1 H, J=8.2 Hz), 7.37 (m, 3 H), 7.28 (m, 2 H), 7.19 (m, 1 H), 4.45 (m, 2 H), 4.22 (m, 1 H), 3.94 (m, 1 H), 2.50 (s, 3 H), 1.35 (s, 12 H); MS (ESI+) m/z 403.2 (M+H).

To a 25 mL three-necked round-bottom flask equipped with a reflux condenser, a magnetic stirrer, and a nitrogen inlet was added 102d (760 mg, 1.89 mmol), 101f (379 mg, 1.35 mmol), sodium carbonate (430 mg, 4.05 mmol), DMF (5 mL), water (2.5 mL), and 1,4-dioxane (8 mL). After nitrogen was bubbled through the resulting suspension for 30 min, tetrakis(triphenylphosphine)palladium(0) (437 mg, 0.378 mmol) was added, and the reaction mixture was heated under reflux for 14 h. Thereafter, the mixture was cooled to room temperature and diluted with ethyl acetate (100 mL) and water (30 mL). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (3×25 mL). The combined organic layers were dried over sodium sulfate and then concentrated under reduced pressure. The residue was purified by column chromatography on silica gel to give 26% yield (183 mg) of 102 as an off-white solid: mp 188-190 °C (dec); ¹ H NMR (300 MHz, DMSO-d ₆ )δ12.99(s,1H),9.23(s,1H),7.79(s,1H),7.71(d,1H,J＝8.1Hz),7.61(d,1H,J＝7.9Hz),7.38(m,4H),7.14(m,2H),5.97(s,1H), 4.63(m,1H),4.49(m,1H),4.27(m,1H),4.12(m,3H),3.51(s,2H),2.79(t,2H,J=5.1Hz),2.35(s,3H),2.18(s,3H); MS(ESI+)m/z 521.2(M+H).

Example 103 4-{2-methyl-3-[1-methyl-5-({5-methyl-4H,5H,6H,7H-pyrazolo[1,5-a]pyrazin-2-yl}amino)-6-oxo-1,6-dihydropyridazin-3-yl]phenyl}-7-thia-4-azatricyclo[6.4.0.0 ^2,6 ]dodecano-1(8),2(6),9,11-tetraen-5-one 103

Example 103a Methyl 3-(bromomethyl)benzo[b]thiophene-2-carboxylate 103a

A 250-mL single-necked round-bottom flask equipped with a magnetic stirrer, nitrogen inlet, and reflux condenser was purged with nitrogen and charged with methyl 3-methylbenzo[b]thiophene-2-carboxylate (2.00 g, 9.70 mmol) and benzene (20 mL). N-bromosuccinimide (1.72 g, 9.70 mmol) and 2,2'-azobisisobutyronitrile (160 mg, 1.00 mmol) were added, and the mixture was refluxed for 2 h. Afterwards, the mixture was cooled to room temperature and filtered. The filter cake was washed with carbon tetrachloride (20 mL), and the filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford 82% yield (2.27 g) of 103a as a white solid: mp 101-102 °C; ¹ H NMR (500 MHz, CDCl ₃ ) δ 7.97 (m, 1H), 7.86 (m, 1H), 7.51 (m, 2H), 5.22 (s, 2H), 3.97 (s, 3H).

Example 103b 3-((3-bromo-2-methylphenylamino)methyl)benzo[b]thiophene-2-carboxylic acid 103b

A 250-mL single-necked round-bottom flask equipped with a magnetic stirrer and reflux condenser was purged with nitrogen and charged with 103a (2.26 g, 7.92 mmol), 3-bromo-2-methylaniline (1.77 g, 9.51 mmol), and acetonitrile (50 mL). Cesium carbonate (7.75 g, 23.8 mmol) was added, and the mixture was stirred at 50°C for 14 h. The reaction mixture was then concentrated under reduced pressure, and the resulting residue was dissolved in THF (15 mL), methanol (15 mL), and water (15 mL) and treated with lithium hydroxide monohydrate (1.30 g, 31.0 mmol). After stirring at room temperature for 14 h, the solvent was removed under reduced pressure, and the resulting residue was acidified to pH 5 with 2M hydrochloric acid. The resulting mixture was extracted with ethyl acetate (3 x 30 mL), and the organic extracts were combined, dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by flash chromatography to afford 59% yield (1.70 g) of 103b as a white solid: mp 120-121 °C; ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 8.30 (d, 1H, J=12.5 Hz), 7.98 (d, 1H, J=12.5 Hz), 7.46 (m, 2H), 6.77 (m, 3H), 4.97 (s, 2H), 2.15 (s, 3H); MS (ESI+) m/z 378.9 (M+H).

Example 103c 2-((3-bromo-2-methylphenyl)-1H-benzothieno[2,3-c]pyrrol-3(2H)-one 103c

A 250 mL single-necked round-bottom flask equipped with a magnetic stirrer was purged with nitrogen and 103b (1.70 g, 4.51 mmol), triethylamine (914 mg, 9.00 mmol) and anhydrous DMF (25 mL) were added. Benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP, 2.60 g, 5.90 mmol) was added and the reaction was stirred at room temperature for 14 h. Thereafter, the reaction was diluted with water (20 mL) and the resulting suspension was filtered. The filter cake was dissolved in dichloromethane (40 mL) and the solution was washed with saturated aqueous sodium bicarbonate solution (10 mL), water (10 mL), and then dried over sodium sulfate. The desiccant was removed by filtration, and the solvent was evaporated under reduced pressure. The resulting residue was purified by flash chromatography to afford 78% yield of 103c (1.26 g) as a white semisolid: ¹ H NMR (500 MHz, CDCl ₃ ) δ 7.97 (m, 1H), 7.80 (m, 1H), 7.60 (d, 1H, J=8.0 Hz), 7.49 (m, 2H), 7.28 (d, 1H, J=8.0 Hz), 7.16 (t, 1H, J=8.0 Hz), 4.85 (s, 2H), 2.37 (s, 3H).

Example 103d 2-(2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-1H-benzothieno[2,3-c]pyrrol-3(2H)-one 103d

A 250 mL single-necked round-bottom flask equipped with a magnetic stirrer and a reflux condenser was purged with nitrogen and charged with 103c (1.26 g, 3.51 mmol), 4,4,4',4',-5,5,5',5'-octamethyl-2,2'-bis(1,3,2-dioxaborolane) (2.23 g, 8.80 mmol), potassium acetate (1.00 g, 10.20 mmol), and 1,4-dioxane (30 mL). A stream of nitrogen was passed through the resulting suspension for 30 min. [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (260 mg, 0.355 mmol) was added, and the reaction was stirred at reflux for 3 h. Afterwards, the mixture was cooled to ambient temperature, partitioned between water (25 mL) and ethyl acetate (50 mL), and then filtered through a pad of Celite 521. The organic phase was separated, dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by flash chromatography to give 103d in quantitative yield (1.84 g) as a yellow semisolid: ¹ H NMR (500 MHz, CDCl ₃ ) δ 7.97 (m, 1H), 7.80 (m, 2H), 7.47 (m, 2H), 7.35 (d, 1H, J=7.5 Hz), 7.28 (d, 1H, J=7.5 Hz), 4.86 (s, 2H), 2.48 (s, 3H), 1.35 (s, 12H).

Example 103e 4-Bromo-6-chloro-2-methylpyridazin-3(2H)-one 103e

A 250 mL single-necked round-bottom flask equipped with a magnetic stirrer was purged with nitrogen and 4-bromo-6-chloropyridazine-3 (2H) -one (1.00 g, 4.77 mmol) and DMF (15 mL) were added. Sodium hydride (60% by weight in oil, 229 mg, 5.73 mmol) was added at once. After stirring at room temperature for 10 minutes, iodomethane (1.02 g, 7.16 mmol) was added and the reaction was stirred at room temperature for 1.5 h. The reaction was then terminated with saturated aqueous sodium bicarbonate solution (10 mL) and the resulting solution was poured into water (150 mL). The mixture was then extracted with ethyl acetate (250 mL). The organic layer was dried over sodium sulfate. The drying agent was then removed by filtration, and the filtrate was concentrated under reduced pressure to a residue. Purification by column chromatography afforded 103e in 68% yield (722 mg) as a white solid: mp 107-108° C.; ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.62 (s, 1H), 3.81 (s, 3H).

Example 103f 6-Chloro-2-methyl-4-(5-methyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)pyridazin-3(2H)-one 103f

To a 250 mL three-necked round-bottom flask equipped with a reflux condenser, a magnetic stirrer, and a nitrogen inlet were added 4-bromo-6-chloro-2-methylpyridazin-3(2H)-one 103e (1.90 g, 8.53 mmol), 101e (1.18 g, 7.75 mmol), and 1,4-dioxane (40 mL). The flask was purged with nitrogen and cooled to 0°C. A 1 M solution of lithium hexamethyldisilazide in THF (39 mL, 39.0 mmol) was added. After bubbling nitrogen through the resulting suspension for 30 min, Xantphos (381 mg, 0.659 mmol) and tris(dibenzylideneacetone)dipalladium(0) (355 mg, 0.388 mmol) were added, and the reaction mixture was heated at reflux for 2 h. Thereafter, the mixture was cooled to room temperature and diluted with water (10 mL). The pH of the solution was adjusted to 7.6 with 2N hydrochloric acid. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (3×40 mL). The combined organic layers were dried over sodium sulfate and then concentrated under reduced pressure. The residue was purified by column chromatography on silica gel to afford 103f in 76% yield (1.74 g) as an off-white solid: mp 184–186°C; ^1H NMR (300 MHz, DMSO-d ₆ ) δ 9.62 (s, 1H), 7.72 (s, 1H), 6.00 (s, 1H), 4.04 (t, 2H, J=5.1 Hz), 3.65 (s, 3H), 3.53 (s, 2H), 2.82 (t, 2H, J=5.1 Hz), 2.37 (s, 3H); MS (ESI+) m/z 295.1 (M+H).

A 150-mL single-necked round-bottom flask equipped with a magnetic stirrer and reflux condenser was purged with nitrogen and charged with 103d (580 mg, 1.43 mmol), 103f (300 mg, 1.00 mmol), sodium carbonate (320 mg, 3.00 mmol), 1,4-dioxane (8 mL), DMF (5 mL), and water (2.5 mL). The mixture was degassed with nitrogen for 30 min. Tetrakis(triphenylphosphine)palladium (120 mg, 0.103 mmol) was added. After heating at reflux for 14 h, the reaction mixture was cooled to room temperature and partitioned between water (40 mL) and dichloromethane (100 mL). The layers were separated, and the aqueous phase was extracted with dichloromethane (2×50 mL). The organic extracts were combined, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by flash chromatography to afford 44% yield (240 mg) of 103 as an off-white solid: mp 175-176 °C; ^1H NMR (500 MHz, DMSO- _d6 ) 9.28 (s, 1H), 8.20 (m, 1H), 8.04 (s, 1H), 7.81 (s, 1H), 7.56 (m, 3H), 7.40 (m, 2H), 5.99 (s, 1H), 5.12 (s, 1H), 3.97 (t, 2H, J = 5.5 Hz), 3.76 (s, 3H), 3.51 (s, 2H), 2.79 (t, 2H, J = 8.5 Hz), 2.35 (s, 3H), 2.17 (s, 3H); MS (ESI+) m/z 538.2 (M+H).

Example 104 5-[2-(Hydroxymethyl)-3-[1-methyl-6-oxo-5-(pyrimidin-4-ylamino)-1,6-dihydropyridin-3-yl]phenyl]-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-6-one 104

Example 104a N-methoxy-N-methyl-4,5,6,7-tetrahydrobenzo[b]thiophene-2-carboxamide 104a

A 250-mL single-necked round-bottom flask equipped with a magnetic stirrer was purged with nitrogen and charged with 4,5,6,7-tetrahydrobenzo[b]thiophene-2-carboxylic acid (3.00 g, 16.5 mmol), dichloromethane (80 mL), and DMF (60 mg, 0.825 mmol) and cooled to 0°C. Oxalyl chloride (2.31 g, 18.2 mmol) was added dropwise to the resulting solution. After this addition was complete, the reaction was warmed to room temperature and stirred for 2 hours. The reaction was then concentrated to dryness under reduced pressure. The resulting white solid was dissolved in dichloromethane (80 mL), and the solution was cooled to 0°C. Triethylamine (5.00 g, 49.5 mmol) and N,O-dimethylhydroxylamine (1.61 g, 16.5 mmol) were then added. After the addition was complete, the cooling bath was removed, and the reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was then partitioned between water (100 mL) and ethyl acetate (200 mL). The layers were separated, and the aqueous phase was extracted with ethyl acetate (100 mL). The combined organic extracts were washed with water (100 mL) and then with brine (100 mL), then dried over sodium sulfate. The drying agent was removed by filtration, and the solvent was evaporated under reduced pressure. The resulting residue was purified by flash chromatography to afford 104a (3.29 g) in 88% yield as a white solid: mp 36–37 °C; ¹ H NMR (500 MHz, CDCl ₃ ) δ 7.79 (s, 1H), 3.76 (s, 3H), 3.34 (s, 3H), 2.78 (t, 2H, J = 6.0 Hz), 2.62 (t, 2H, J = 6.0 Hz), 1.82 (m, 4H); MS (APCI+) m/z 226.3 (M+H)

Example 104b 3-Chloro-1-(4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)propan-1-one 104b

A 100-mL single-necked round-bottom flask equipped with a magnetic stirrer was purged with nitrogen, and 104a (2.70 g, 12.0 mmol) and anhydrous THF (45 mL) were added, and the solution was cooled to -10 ° C with an acetone/ice bath. A 1.0 M solution of vinylmagnesium bromide in THF (13.2 mL, 13.2 mmol) was added dropwise, and the resulting reaction mixture was stirred at 0 ° C for 4 h. Thereafter, the reaction mixture was partitioned between ethyl acetate (100 mL) and 2M aqueous hydrochloric acid solution (40 mL). The layers were separated, and the aqueous phase was extracted with ethyl acetate (40 mL). The combined organic extracts were washed with water (100 mL) and then with brine (100 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure. The resulting residue was dissolved in dichloromethane (30 mL), and a 2M solution of hydrogen chloride in diethyl ether (15 mL) was added. After stirring at room temperature for 1 h, the solvent was removed under reduced pressure. The resulting residue was purified by column chromatography to afford 29% yield (804 mg) of 104b as an off-white solid: mp 57-58°C; ¹ H NMR (500 MHz, CDCl ₃ ) δ 7.41 (s, 1H), 3.89 (t, 2H, J = 7.0 Hz), 3.30 (t, 2H, J = 7.0 Hz), 2.81 (t, 2H, J = 6.0 Hz), 2.64 (t, 2H, J = 6.0 Hz), 1.83 (m, 4H); MS (ECI+) m/z 229.1 (M+H)

Example 104c 5,6,7,8-Tetrahydro-1H-benzo[b]cyclopenta[d]thiophen-3(2H)-one 104c

To a 50-mL single-necked round-bottom flask equipped with a magnetic stirrer was added 104b (800 mg, 3.51 mmol) and 98% sulfuric acid (8 mL). After stirring at 95°C for 16 h, the reaction mixture was poured into ice (50 g), and the resulting suspension was extracted with ethyl acetate (3 x 50 mL). The organic extracts were combined, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by flash chromatography to afford 104c in 47% yield (320 mg) as an off-white solid: mp 75–76°C; ¹ H NMR (500 MHz, CDCl ₃ ) δ 2.89 (m, 2H), 2.87–2.83 (m, 4H), 2.56 (t, 2H, J = 6.5 Hz), 1.84 (m, 4H).

Example 104d 5,6,7,8-Tetrahydro-1H-benzo[b]cyclopenta[d]thiophene-3(2H)-one oxime 104d

To a 100 mL single-necked round-bottom flask equipped with a mechanical stirrer and a nitrogen inlet was added hydroxylamine hydrochloride (573 mg, 8.25 mmol) and methanol (10 mL). The mixture was cooled to 0°C using an ice bath. Sodium acetate (677 mg, 8.25 mmol) was added. The mixture was stirred at 0°C for 30 min. Thereafter, 104c (319 mg, 1.65 mmol) was added, and the reaction was stirred at room temperature for 16 h. Thereafter, the mixture was concentrated, and the resulting residue was then powdered with water (10 mL). The resulting solid was collected and then dried in a vacuum oven at 45°C to afford 84% yield (287 mg) of 104d as an off-white solid: mp 173-174°C; ^1H NMR (500 MHz, DMSO- _d6 ) δ 10.38 (s, 1H), 2.97 (m, 2H), 2.77-2.73 (m, 4H), 2.47 (m, 2H), 1.75 (m, 4H); MS (APCI+) m/z 208.3 (M+H)

Example 104e 3,4,5,6,7,8-Hexahydrobenzothieno[2,3-c]pyridin-1(2H)-one 104e

To a 50 mL single-necked round-bottom flask equipped with a reflux condenser, a magnetic stirrer, and a nitrogen inlet was added 104d (285 mg, 1.38 mmol) and polyphosphoric acid (15 g). After stirring at 80°C for 16 h, the reaction mixture was cooled to room temperature and water (30 mL) was added. The resulting mixture was stirred for 30 min and then filtered. The filter cake was washed with water (20 mL) and then dried in a vacuum oven at 45°C to afford 104e in a 75% yield (215 mg) as an off-white solid: mp 203°C dec; ^1H NMR (500 MHz, CDCl ₃ ) δ 5.62 (s, 1H), 3.59 (t, 2H, J = 7.0 Hz), 2.81 (t, 2H, J = 6.0 Hz), 2.72 (t, 2H, J = 7.0 Hz), 2.48 (t, 2H, J = 6.0 Hz), 1.84 (m, 4H). MS (APCI+) m/z 208.3 (M+H)

Example 104f 2-Bromo-6-(1-oxo-3,4,5,6,7,8-hexahydrobenzothieno[2,3-c]pyridin-2(1H)-yl)benzyl acetate 104f

To a 100 mL three-necked round-bottom flask equipped with a reflux condenser, a magnetic stirrer, and a nitrogen inlet was added 104e (214 mg, 1.04 mmol), 2,6-dibromobenzyl acetate 104 g (519 mg, 2.08 mmol), cesium carbonate (678 mg, 2.08 mmol), N,N'-dimethylethylenediamine (92 mg, 1.04 mmol), and 1,4-dioxane (10 mL). After bubbling nitrogen through the resulting suspension for 30 min, cuprous iodide (99 mg, 0.520 mmol) was added, and the reaction mixture was heated at 100 ° C (oil bath temperature) for 16 h. Thereafter, the mixture was cooled to room temperature and filtered. The filtrate was diluted with ethyl acetate (100 mL) and water (50 mL). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (3×50 mL). The combined organic layers were dried over sodium sulfate and then concentrated under reduced pressure. The residue was purified by column chromatography to give 30% yield (138 mg) of 2-bromo-6-(1-oxo-3,4,5,6,7,8-hexahydrobenzothieno[2,3-c]pyridin-2(1H)-yl)benzyl acetate 104f as a yellow oil: ¹ H NMR (500 MHz, CDCl ₃ ) δ 7.59 (dd, 1H, J=7.0, 1.5 Hz), 7.30–7.24 (m, 2H), 5.23 (m, 2H), 4.06 (m, 1H), 3.77 (m, 1H), 2.98 (m, 1H), 2.83–2.77 (m, 3H), 2.50 (m, 2H), 2.04 (s, 3H), 1.85 (m, 4H)

Example 104 2,6-dibromobenzyl acetate 104g

A 250 mL single-necked round-bottom flask equipped with a magnetic stirrer, reflux condenser, and nitrogen inlet was purged with nitrogen and charged with 2,6-dibromotoluene (2.50 g, 10.0 mmol), N-bromosuccinimide (1.78 g, 10.0 mmol), and carbon tetrachloride (40 mL). The solution was heated to 80°C (oil bath temperature) and 2,2'-azobisisobutyronitrile (164 mg, 1.00 mmol) was added. The resulting mixture was refluxed for 14 h. Thereafter, the mixture was cooled to room temperature and filtered. The filter cake was washed with carbon tetrachloride (2 x 20 mL). The filtrate was diluted with ethyl acetate (200 mL) and washed with water (40 mL), saturated aqueous sodium bicarbonate solution (40 mL), and brine (40 mL). The organic layer was dried over sodium sulfate and then concentrated under reduced pressure to give a quantitative yield (3.28 g) of 1,3-dibromo-2-(bromomethyl)benzene as a yellow solid: mp 77–78° C.; ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.55 (d, 2H, J=8.1 Hz), 7.07 (t, 1H, J=8.1 Hz), 4.83 (s, 2H)

A 250 mL single-necked round-bottom flask equipped with a magnetic stirrer and a nitrogen inlet was purged with nitrogen and charged with 1,3-dibromo-2-(bromomethyl)benzene (3.28 g, 10.0 mmol), potassium acetate (3.93 g, 40.0 mmol), and DMF (100 mL). The solution was stirred at room temperature for 14 h. Thereafter, the reaction mixture was diluted with water (900 mL) and extracted with ethyl acetate (3 x 200 mL). The combined organic layers were washed with brine (100 mL), dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography to give 104 g of 2,6-dibromobenzyl acetate in 88% yield (2.70 g) as an off-white solid: mp 62-65° C.; ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.57 (d, 2H, J=8.0 Hz), 7.07 (t, 1H, J=7.9 Hz), 5.42 (s, 2H), 2.11 (s, 3H); MS (ESI+)

Example 104h 1-methyl-3-(pyrimidin-4-ylamino)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one 104h

A 100-mL single-necked round-bottom flask equipped with a magnetic stirrer and a thermostat was purged with nitrogen and charged with 5-bromo-1-methyl-3-(pyrimidin-4-ylamino)pyridin-2(1H)-one 109b (300 mg, 1.07 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bis(1,3,2-dioxaborolane) (534 mg, 2.14 mmol), potassium acetate (210 mg, 2.14 mmol), and 1,4-dioxane (10 mL). A stream of nitrogen was passed through the resulting suspension for 30 minutes. [1,1'-bis(diphenylphosphino)ferrocene]palladium(II) dichloride complex with dichloromethane ( _PddppfCl2 · _CH2Cl2 , 39 mg, 0.054 mmol) was then _added , and the reaction was stirred at 95°C for 30 minutes. After this time, the mixture was cooled to ambient temperature, partitioned between water (40 mL) and ethyl acetate (60 mL), and then filtered through a pad of Celite 521. The organic phase was separated, dried over sodium sulfate, filtered, and then concentrated under reduced pressure to afford crude 104h as a black oil, which was used directly in the next step without further purification. MS (ESI+) m/z 329.1 (M+H)

A 100-mL single-necked round-bottom flask equipped with a magnetic stirrer and reflux condenser was purged with nitrogen and charged with 104f (136 mg, 0.313 mmol), crude 104h (1.07 mmol, assumed quantitative yield), sodium carbonate (100 mg, 0.940 mmol), 1,4-dioxane (5 mL), and water (1 mL). The mixture was degassed with nitrogen for 30 min. A tetrakis(triphenylphosphine)palladium mixture (636 mg, 0.031 mmol) was added. After heating at 100°C for 3 h, the reaction was cooled to room temperature and partitioned between water (40 mL) and dichloromethane (100 mL). The layers were separated, and the aqueous phase was extracted with dichloromethane (2 x 50 mL). The organic extracts were combined, dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was dissolved in methanol (5 mL), and potassium carbonate (500 mg, 3.62 mmol) was added. After stirring at room temperature for 2 h, the reaction mixture was partitioned between water (20 mL) and dichloromethane (20 mL). The layers were separated, and the aqueous phase was extracted with dichloromethane (2×20 mL). The organic extracts were combined, dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by flash chromatography to afford 104 in 26% yield (42 mg) as a white solid: mp>250°C; ¹ H NMR (500 MHz, DMSO-d ₆ )δ9.17(s,1H),8.71(d,1H,J＝2.5Hz),8.64(s,1H),8.29(d,1H,J＝6.0Hz), 7.52(d,1H,J＝2.5Hz),7.45(t,1H,J＝7.5Hz),7.36–7.30(m,3H),4.84(t,1H ,J＝3.5Hz),4.36(m,2H),4.03(m,1H),3.87(m,1H),3.60(s,3H),2.96(m,1H ),2.85(m,1H),2.77(m,2H),2.58–2.46(m,2H),1.79(m,4H); MS(APCI+)m/z 514.2(M+H)

Example 105 10-[2-(Hydroxymethyl)-3-[1-methyl-6-oxo-5-(pyrimidin-4-ylamino)-1,6-dihydropyridin-3-yl]phenyl]-4,4-dimethyl-7-thia-10-azatricyclo[6.4.0.02,6]dodeca-1(8),2(6)-dien-9-one 105

Example 105a 3,3-Dimethylcyclopentanone 105a

A 1-L three-necked round-bottom flask equipped with a magnetic stirrer, addition funnel, and nitrogen inlet was purged with nitrogen and charged with diethyl ether (200 mL) and copper (I) iodide (54.46 g, 0.286 mol). The mixture was cooled to 0°C, and methyl lithium (1.6 M in diethyl ether, 357.5 mL, 0.572 mol) was added dropwise to the reaction mixture over 1.5 hours and stirred at 0°C for an additional 2 hours. Thereafter, a solution of 3-methylcyclopent-2-enone (25 g, 0.260 mol) in diethyl ether (150 mL) was added dropwise over 1.5 hours. The reaction mixture was then stirred at 0°C for 2 hours and then poured into sodium sulfate decahydrate (300 g). The resulting mixture was stirred for 30 minutes. Thereafter, the mixture was filtered and washed with diethyl ether (1000 mL). The filtrate was concentrated and distilled under reduced pressure to give 70% yield (20.5 g) of 3,3-dimethylcyclopentanone 105a as a colorless liquid: bp 50–55 °C (at 10 mmHg); ¹ H NMR (300 MHz, CDCl ₃ ) δ 2.31 (t, 2H, J = 7.8 Hz), 2.05 (s, 2H), 1.79 (t, 2H, J = 7.8 Hz); MS (ESI+) m/z 113.3 (M+H)

Example 105b Ethyl 5,5-dimethyl-5,6-dihydro-4H-cyclopenta[b]thiophene-2-carboxylate 105b

A 500-mL three-necked round-bottom flask equipped with a magnetic stirrer, reflux condenser, addition funnel, and nitrogen inlet was purged with nitrogen and charged with DMF (9.49 g, 0.100 mol) and dichloromethane (100 mL). The reaction mixture was cooled to 0°C and phosphorus oxychloride (14.1 g, 0.920 mol) was added dropwise to the reaction over 30 minutes. Once this addition was complete, the reaction was warmed to room temperature and stirred for 1 hour. Thereafter, a solution of 105a (11.2 g, 0.100 mol) in dichloromethane (100 mL) was added dropwise over 1 hour. The reaction was then stirred at reflux for 18 hours. The reaction mixture was cooled to room temperature and then poured into a mixture of crushed ice (400 mL) and sodium acetate (100 g, 1.22 mol). The resulting mixture was stirred for 45 minutes. Afterwards, the aqueous layer was separated and extracted with dichloromethane (2 x 500 mL). The combined organic layers were then washed with water (2 x 200 mL) followed by brine (200 mL) and dried over sodium sulfate. The desiccant was then removed by filtration, and the filtrate was concentrated to yield the crude product, 2-chloro-4,4-dimethylcyclopent-1-enecarbaldehyde, which was placed in a 500-mL three-necked round-bottom flask equipped with a mechanical stirrer, a reflux condenser, and a nitrogen inlet. Dichloromethane (200 mL), ethyl 2-mercaptoacetate (11.0 g, 0.092 mol), and triethylamine (30 g, 0.207 mol) were then added. The reaction mixture was then stirred at reflux for 6 h. Afterwards, the reaction was cooled to room temperature and concentrated to a thick orange residue. Ethanol (200 mL) and triethylamine (30.0 g, 0.207 mol) were added, and the reaction was heated at reflux for 12 h. The reaction was then cooled to room temperature and concentrated under reduced pressure, and the resulting residue was diluted with diethyl ether (600 mL). The resulting mixture was washed with 1 M hydrochloric acid (150 mL), brine (100 mL), dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The residue was purified by flash chromatography to afford 105b in 34% yield (7.70 g) as a colorless liquid: ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.48 (s, 1H), 4.33 (q, 2H, J=7.2 Hz), 2.72 (s, 2H), 2.56 (s, 2H), 1.38 (t, 3H, J=1.8 Hz), 1.17 (s, 6H); MS (ESI+) m/z 225.1

Example 105c 5,5-Dimethyl-5,6-dihydro-4H-cyclopenta[b]thiophene-2-carboxylic acid 105c

In a 250-mL single-necked round-bottom flask equipped with a magnetic stirrer and a reflux condenser, 105b (4.00 g, 17.8 mmol) was dissolved in ethanol (50 mL). THF (50 mL), water (50 mL), and lithium hydroxide (854 mg, 35.6 mmol) were added, and the mixture was stirred at 60°C for 4 h. The reaction was then cooled to room temperature and acidified to pH 1.5 with 2M hydrochloric acid, followed by extraction with ethyl acetate (2 x 200 mL). The organic layers were combined, washed with water (2 x 100 mL), then with brine (100 ml), and dried over sodium sulfate. The drying agent was then separated by filtration. After evaporation of the resulting filtrate, 105c was obtained in 91% yield (3.2 g) as a white solid: mp 170-172 °C; ¹ H NMR (300 MHz, CDCl ₃ ) δ 12.77 (s, 1H), 7.46 (s, 1H), 2.71 (s, 2H), 2.53 (s, 2H), 1.20 (s, 6H); MS (ESI–) m/z 195.0

Example 105d 5,5-Dimethyl-5,6-dihydro-4H-cyclopenta[b]thiophene-2-carboxylic acid 105d

To a 100-mL single-necked round-bottom flask equipped with a magnetic stirrer, a reflux condenser, and a bubbler placed on the condenser was added 105c (2.30 g, 11.6 mmol), toluene (25 mL), thionyl chloride (4.09 g, 34.9 mmol), and DMF (1 drop). The mixture was heated under reflux for 1 h and then evaporated under reduced pressure on a rotary evaporator at 45°C. The resulting acid chloride was diluted with dichloromethane (20 mL).

In another 250-mL three-necked round-bottom flask equipped with a magnetic stirrer, N,O-dimethylhydroxylamine hydrochloride (2.26 g, 23.2 mmol) and N,N-diisopropylethylamine (2.97 g, 23.0 mmol) were dissolved in anhydrous dichloromethane (20 mL) under nitrogen and cooled to 0°C in an ice/water bath. The acid chloride solution was added, and the reaction mixture was stirred at room temperature for 18 h. The reaction mixture was extracted with water (100 mL), 10% citric acid solution (50 mL), and a 1:1 mixture of saturated aqueous sodium bicarbonate and water (100 mL). The organic layer was dried over sodium sulfate and evaporated on a rotary evaporator under reduced pressure to give 93% yield (2.60 g) of 105d as a light yellow solid: ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.66 (s, 1H), 3.77 (s, 3H), 3.35 (s, 3H), 2.74 (s, 2H), 2.58 (s, 2H), 1.23 (s, 6H)

Example 105e 3-Chloro-1-(5,5-dimethyl-5,6-dihydro-4H-cyclopenta[b]thien-2-yl)propan-1-one 105e

A 100-mL single-necked round-bottom flask equipped with a magnetic stirrer was purged with nitrogen and charged with 105d (2.41 g, 10.0 mmol) and anhydrous THF (20 mL). The solution was cooled to -70°C and 1M vinylmagnesium bromide/THF (11 mL, 11.0 mmol) was added while maintaining the reaction temperature below -60°C. The reaction mixture was stirred at -13 to -7°C for 2 h and then warmed to room temperature over 30 min. The reaction was cooled again to -70°C and a 2M solution of hydrogen chloride in diethyl ether (22.5 ml, 45 mmol) was added. The reaction was then stored in a refrigerator at -10°C overnight. The mixture was then evaporated under reduced pressure on a rotary evaporator, and the resulting residue was partitioned between water (100 mL) and ether (100 mL). The ether extract was dried over sodium sulfate and then evaporated on a rotary evaporator under reduced pressure to give crude 105e (2.86 g, 118%) as a brown oil with a purity of about 75% (NMR): ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.45 (s, 1H), 3.89 (t, 2H, J = 6.9 Hz), 3.30 (t, 2H, J = 6.9 Hz), 2.75 (s, 2H), 2.59 (s, 2H), 1.24 (s, 6H)

Example 105f 6,6-Dimethyl-1,2,6,7-tetrahydrodicyclopenta[b,d]thiophene-3(5H)-one 105f

To a 100-mL single-necked round-bottom flask equipped with a magnetic stirrer was added crude 105e (2.86 g, 10.0 mmol, assuming quantitative yield) and 98% sulfuric acid. The reaction mixture was heated in a 90°C oil bath overnight. The reaction mixture was placed in an ice/acetone bath, and a cold solution (5°C) of potassium hydrogen phosphate (105 g, 0.603 mol) in water (300 mL) was added in one go. The resulting mixture was shaken with ethyl acetate (300 mL) and filtered. The filter cake was washed with ethyl acetate (100 mL). The ethyl acetate layer of the filtrate was separated, dried over sodium sulfate, and then evaporated under reduced pressure on a rotary evaporator. The resulting residue was purified by flash chromatography (silica gel, 80:20 hexanes/ethyl acetate) to afford 105f as a non-crystalline brown solid in 37% yield (683 mg) over two steps: mp 60-62°C; ¹ H NMR (500 MHz, CDCl ₃ ) δ 2.92-2.87 (m, 4H), 2.79 (s, 2H), 2.53 (s, 2H), 1.26 (s, 6H); MS (ESI+) m/z 207.0 (M+H)

Example 105g 6,6-dimethyl-1,2,6,7-tetrahydrodicyclopenta[b,d]thiophene-3(5H)-one 105g

To a 250-mL single-necked round-bottom flask equipped with a magnetic stirrer and a nitrogen inlet, hydroxylamine hydrochloride (688 mg, 9.90 mmol), sodium acetate (812 mg, 9.90 mmol) and methanol (10 mL) were added, and the mixture was stirred at room temperature for 30 min. Afterwards, a solution of 105f (680 mg, 3.30 mmol) was added dropwise at room temperature, and the reaction was stirred at room temperature for 14 h under a nitrogen atmosphere. Because the reaction was not complete, hydroxylamine hydrochloride (1.15 g, 16.5 mmol) and sodium acetate (1.35 g, 16.5 mmol) were added, and stirring was continued at room temperature for 58 h. Afterwards, the mixture was diluted with dichloromethane (150 mL) and water (100 mL), and the layers were separated. The organic layer was washed with brine (50 mL) and then dried over sodium sulfate. The drying agent was removed by filtration, and the filtrate was concentrated to give a quantitative yield (730 mg) of 105 g of crude product as a yellow semisolid, which was used in the next step without purification: mp 122-124 °C; ¹ H NMR (500 MHz, CDCl ₃ ) δ 3.13-3.11 (m, 2H), 2.85-2.83 (m, 2H), 2.77 (s, 2H), 2.49 (s, 2H), 1.24 (s, 6H) of the major isomer; MS (ESI+) m/z 222.0 (M+H)

Example 105h 6,6-Dimethyl-3,4,6,7-tetrahydro-5H-cyclopenta[4,5]thieno[2,3-c]pyridin-1(2H)-one 105h

To a 100-mL three-necked round-bottom flask equipped with a reflux condenser, a mechanical stirrer, and a nitrogen inlet was added 105g (700mg, 3.16mmol) and polyphosphoric acid (25g). The reaction mixture was stirred at 80°C for 13h under a nitrogen atmosphere. Thereafter, the mixture was cooled to 0°C and water (50mL) was carefully added dropwise while maintaining the internal temperature at 10–45°C. The mixture was diluted with 90:10 dichloromethane/methanol (100mL) and the layers were separated. The aqueous layer was extracted with 90:10 dichloromethane/methanol (50mL), and the combined organic layers were washed with saturated aqueous sodium bicarbonate solution (50mL), brine (150mL), and then dried over sodium sulfate. The desiccant was removed by filtration. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, 95:5 dichloromethane/methanol) to afford 90% yield (630 mg) of 6,6-dimethyl-3,4,6,7-tetrahydro-5H-cyclopenta[4,5]thieno[2,3-c]pyridin-1(2H)-one 105h as a non-crystalline off-white solid: mp 205-207 °C; ¹ H NMR (500 MHz, CDCl ₃ ) δ 5.51 (s, 1H), 3.60-3.56 (m, 2H), 2.76-2.73 (m, 4H), 2.49 (s, 2H), 1.26 (s, 6H); MS (ESI+) m/z 222.0 (M+H)

Example 105i (2-bromo-6-{4,4-dimethyl-9-oxo-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-10-yl}phenyl)methyl acetate 105i

To a 250-mL single-necked round-bottom flask equipped with a magnetic stirrer and a nitrogen inlet was added 105h (624 mg, 2.82 mmol), 2,6-dibromobenzyl acetate 104g (1.73 g, 5.65 mmol), cesium carbonate (1.84 g, 5.65 mmol), N,N'-dimethylethylenediamine (249 mg, 2.82 mmol) and 1,4-dioxane (15 mL). After bubbling nitrogen through the resulting suspension for 30 min, cuprous iodide (269 mg, 1.41 mmol) was added. A reflux condenser was connected to the flask, and the reaction mixture was heated at 90 ° C for 14 h. Thereafter, the mixture was cooled to room temperature and filtered. The filtrate was diluted with ethyl acetate (100 mL) and water (50 mL), and the layers were separated. The aqueous layer was extracted with ethyl acetate (2×30 mL), the combined organic layers were washed with brine (100 mL), and then dried over sodium sulfate. The drying agent was removed by filtration. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, 70:30 hexanes/ethyl acetate) to afford 105i in 52% yield (660 mg) as a white solid: mp 126-128°C; ^1H NMR (500 MHz, CDCl ₃ ) δ 7.60 (dd, J=7.5, 1.5 Hz, 1H), 7.29-7.24 (m, 2H), 5.24 (s, 2H), 4.05-3.99 (m, 1H), 3.78-3.74 (m, 1H), 3.06-2.99 (m, 1H), 2.84-2.80 (m, 1H), 2.77 (s, 2H), 2.52 (s, 2H), 2.05 (s, 3H), 1.27 (s, 6H); MS (ESI+) m/z 448.0(M+H)

To a 100-mL single-necked round-bottom flask equipped with a magnetic stirrer and a nitrogen inlet was added 105i (250 mg, 0.558 mmol), the crude 1-methyl-3-(pyrimidin-4-ylamino)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one 104h (400 mg, 0.951 mmol, assuming quantitative yield in the previous step), sodium carbonate (177 mg, 1.67 mmol), DMF (2 mL), water (2 mL), and 1,4-dioxane (10 mL). After bubbling nitrogen through the resulting suspension for 30 min, tetrakis(triphenylphosphine)palladium(0) (129 mg, 0.112 mmol) was added. A reflux condenser was attached to the flask, and the reaction mixture was heated at 100° C. for 12 h. Thereafter, the mixture was diluted with 90:10 dichloromethane/methanol (100 mL) and water (75 mL), and the layers were separated. The aqueous layer was extracted with 90:10 dichloromethane/methanol (2×50 mL), and the combined organic layers were washed with brine (100 mL) and dried over sodium sulfate. The desiccant was removed by filtration. The filtrate was concentrated under reduced pressure, and the resulting residue was dissolved in THF (5 mL), water (5 mL) and methanol (5 mL). Lithium hydroxide monohydrate (117 mg, 2.79 mmol) was added, and the mixture was stirred at room temperature for 2 h. Thereafter, the mixture was diluted with 90:10 dichloromethane/methanol (100 mL) and water (50 mL), and the layers were separated. The aqueous layer was extracted with 90:10 dichloromethane/methanol (2×75 mL), the combined organic layers were washed with brine (100 mL), and then dried over sodium sulfate. The desiccant was removed by filtration. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, 90:10 dichloromethane/methanol) and preparative HPLC (70:30 water/acetonitrile) to afford 105 in 14% yield (42 mg) as a non-crystalline off-white solid: mp 252-254 °C; ¹ H NMR (500 MHz, DMSO-d ₆ )δ9.18(s,1H),8.71(s,1H),8.64(s,1H),8.30(d,J＝6.0Hz,1H),7.53(s,1H) ,7.47(t,J＝7.5Hz,1H),7.37–7.31(m,3H),4.86–4.85(m,1H),4.40–4.32(m, 2H),4.05–4.00(m,1H),3.88–3.84(m,1H),3.60(s,3H),3.05–2.99(m,1H),2 .91–2.88(m,1H),2.75(s,2H),2.57–2.53(m,2H),1.23(s,6H); MS(ESI+)m/z 528.2(M+H)

Example 106 2-(3-(5-(5-cyclopropyl-1H-pyrazol-3-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 106

Example 106 was prepared using the same procedures as Example 136e and Example 136, except using 113a and 136d, to afford 20 mg of 106 as a white solid. MS (ESI+) m/z 525 (M+H).

Example 107 2-(2-(Hydroxymethyl)-3-(1-methyl-5-(5-morpholinopyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 107

Example 107 was prepared using the same method as Example 301 using 255c and 114a as starting materials to afford 122 mg of 107 as a white solid. MS (ESI+) m/z 622.4 (M+H).

Example 108 2-(3-(5-(5-acetyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 108

Example 108a 2-Nitro-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazine 108a

To a sealed tube equipped with a magnetic stirrer was added 1-(2-bromoethyl)-5-(bromomethyl)-3-nitro-1H-pyrazole 101c (4 g, 12.9 mmol) and a 0.5 M ammonia solution in dioxane (200 mL). The resulting mixture was carefully heated to 50°C overnight. The reaction mixture was then concentrated under reduced pressure, and _H₂O (50 mL) and EtOAc (50 mL) were added to the residue. The aqueous layer was separated and extracted with EtOAc (2 x 50 mL). The combined organic extracts were washed with brine (100 mL) and then dried over sodium sulfate. The resulting solution was concentrated under reduced pressure to provide crude 2-nitro-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazine 108a in 100% yield (2.1 g).

Example 108b 1-(2-nitro-6,7-dihydropyrazolo[1,5-a]pyrazin-5(4H)-yl)ethanone 108b

To a 200 mL round-bottom flask was added 2-nitro-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazine 108a (2.1 g, 12.9 mmol), triethylamine (5.5 mL, 38.7 mmol), acetyl chloride (1.1 mL, 15.5 mmol), and _CH₂Cl₂ (100 _mL ). The mixture was stirred at room temperature overnight. Afterwards, the reaction mixture was concentrated under reduced pressure, and _H₂O (50 mL) and EtOAc (50 mL) were added to the residue. The aqueous layer was separated and extracted with EtOAc (2 x 50 mL). The combined organic extracts were washed with brine (100 mL). The combined aqueous extracts were back-extracted with 9:1 _CH₂Cl₂ :MeOH (2 x 50 mL). The combined organic extracts were dried _over sodium sulfate. The resulting residue was purified by column chromatography eluting with _a gradient _{of CH2Cl2-9:1 CH2Cl2 : MeOH} to afford 84% yield (2.3 g) of 1-(2-nitro-6,7-dihydropyrazolo[1,5-a]pyrazin-5(4H)-yl)ethanone 108b.

Example 108c 1-(2-amino-6,7-dihydropyrazolo[1,5-a]pyrazin-5(4H)-yl)ethanone 108c

To a 500-mL Parr hydrogenation bottle was added 1-(2-nitro-6,7-dihydropyrazolo[1,5-a]pyrazin-5(4H)-yl)ethanone 108b (2.3 g, 10.9 mmol), 10% palladium on carbon (50% wet, 570 mg dry weight), and ethanol (100 mL). The bottle was evacuated, filled with hydrogen to 50 psi, and shaken on the Parr hydrogenation apparatus for 2 h. The catalyst was removed by filtration through a pad _of Celite 521 and washed with 1:1 _CH₂Cl₂ :MeOH (500 mL). The resulting solution was concentrated under reduced pressure to provide crude 1-(2-amino-6,7-dihydropyrazolo[1,5-a]pyrazin-5(4H)-yl)ethanone 108c in a 95% yield (1.9 g).

Example 108d 3-(5-acetyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-5-bromo-1-methylpyridin-2(1H)-one 108d

To a sealed tube equipped with a magnetic stirrer was added 1-(2-amino-6,7-dihydropyrazolo[1,5-a]pyrazin-5(4H)-yl)ethanone 108c (860 mg, 4.8 mmol), 3,5-dibromo-1-methylpyridin-2(1H)-one (1.8 g, 6.7 mmol), cesium carbonate (3.4 g, 10.5 mmol), and 1,4-dioxane (67 mL). After nitrogen was bubbled through the solution for 30 min, Xantphos (330 mg, 0.6 mmol) and tris(dibenzylideneacetone)dipalladium(0) (300 mg, 0.3 mmol) were added, and the reaction mixture was heated to 100° C. for 16 h. Afterwards, H ₂ O (50 mL) and EtOAc (50 mL) were added. The aqueous layer was separated and extracted with EtOAc (2×50 mL). The combined organic extracts were washed with brine (100 mL) and dried over sodium sulfate. The resulting residue was purified by column chromatography using a gradient of _CH2Cl2-60 : ₃₅ :5 _CH2Cl2 : _Et2O :MeOH _to afford 41% yield (720 mg) of 3-(5-acetyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-5-bromo-1-methylpyridin-2(1H)-one 108d.

Example 108e 2-(5-(5-acetyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 108e

To a microwave tube equipped with a magnetic stirrer was added 3-(5-acetyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-5-bromo-1-methylpyridin-2(1H)-one 108d (120 mg, 0.3 mmol), 2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 114a (200 mg, 0.4 mmol), DME (4 mL), and 1 M aqueous sodium carbonate solution (1 mL). After bubbling nitrogen for 15 min, Pd(PPh ₃ ) ₄ (19 mg, 0.02 mmol) was added. The mixture was heated to 130° C. in a microwave for 15 min. To the 4-thiazolyl-2-ylamino)-6-( _1- oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol- ₂ ( _{1H )} -yl)benzyl acetate 108e was obtained in a 33% yield ( ₆₉ mg).

To a 25 mL round-bottom flask equipped with a magnetic stirrer was added 2-(5-(5-acetyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 108e (69 mg, 0.11 mmol), lithium hydroxide (8 mg, 0.3 mmol), THF (1 mL), i-PrOH (1 mL) and water (2 mL). The mixture was stirred at rt for 2 h. Thereafter, EtOAc (3 mL) and water (3 mL) were added. The separated aqueous layer was extracted with EtOAc (2 x 5 mL). The combined organic phase was washed with brine (10 mL), dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by column chromatography eluting with a gradient _{of CH2Cl2-75} :18: _7CH2Cl2 : _Et2O :MeOH to afford 63% yield (40 mg) of 2-(3-(5-(5-acetyl-4,5,6,7-tetrahydropyrazolo[1,5-a _] pyrazin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 108. MS (ESI+) m/z 582.3 (M+H).

Example 109 2-(2-(Hydroxymethyl)-3-(1-methyl-6-oxo-5-(pyrimidin-4-ylamino)-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 109

Example 109a 2-Bromo-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 109a

To a 100-mL three-necked round-bottom flask equipped with a reflux condenser, a magnetic stirrer, and a nitrogen inlet was added 3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 101k (720 mg, 3.78 mmol), 2,6-dibromobenzyl acetate 104 g (2.33 g, 7.57 mmol), cesium carbonate (2.47 g, 7.57 mmol), N,N'-dimethylethylenediamine (333 mg, 3.78 mmol), and 1,4-dioxane (31 mL). After bubbling nitrogen through the resulting suspension for 30 min, cuprous iodide (360 mg, 1.89 mmol) was added, and the reaction mixture was heated at 105°C (oil bath temperature) for 3 days. Thereafter, the mixture was cooled to room temperature and filtered. The filtrate was diluted with ethyl acetate (200 mL) and water (40 mL). The organic layer was separated and the aqueous layer was extracted with ethyl acetate (3×50 mL). The combined organic layers were dried over sodium sulfate and then concentrated under reduced pressure. The residue was purified by column chromatography to afford 2-bromo-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 109a in a 31% yield (490 mg).

Example 109b 5-Bromo-1-methyl-3-(pyrimidin-4-ylamino)pyridin-2(1H)-one 109b

A solution of pyrimidin-4-amine (2.0 g, 21 mmol), 3,5-dibromo-1-methylpyridin-2(1H)-one (6.2 g, 23.1 mmol), tris(dibenzylideneacetone)dipalladium(0) (0.96 g, 1.1 mmol), Xantphos (1.03 g, 1.79 mmol), and cesium carbonate (7.5 g, 23 mmol) in dioxane (25 mL) was heated at 130° C. for 18 hours in a sealed tube. The mixture was diluted with CH ₂ Cl ₂ (100 mL) and filtered through a pad of Celite. The solution was concentrated under vacuum on a rotary evaporator. The material was then dissolved in a minimum of CH ₂ Cl ₂ (5 mL) and triturated with Et ₂ O (80 mL). The product was then filtered and washed with Et ₂ O (100 mL) to give 2.9 g (49%) of 109b.

Example 109c 1-Methyl-3-(pyrimidin-4-ylamino)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one 109c

A solution of 5-bromo-1-methyl-3-(pyrimidin-4-ylamino)pyridin-2(1H)-one 109b (600 mg, 2.13 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bis(1,3,2-dioxaborolane) (596 mg, 2.35 mmol), [1,1'-bis(diphenylphosphino)ferrocene]palladium(II) dichloride complex with dichloromethane (174 mg, 0.213 mmol), and potassium acetate (628 mg, 6.40 mmol) in dioxane (4.5 mL) was sealed in a microwave tube and heated to 130° C. for 10 minutes. The solution was filtered through a pad of celite and concentrated. The crude 1-methyl-3-(pyrimidin-4-ylamino)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one 109c was used in the next step without purification.

A solution of 109c (crude, 2.13 mmol), 109a (300 mg, 0.719 mmol), tetrakis(triphenylphosphine)palladium(0) (83 mg, 0.0719 mmol), and sodium carbonate (229 mg, 2.16 mmol) in dioxane (4.5 mL) and water (2.3 mL) was sealed in a microwave tube and heated to 130° C. for 10 minutes. The organic layer was separated from the aqueous layer and filtered through a pad of celite. The material was then adsorbed onto celite and purified by silica gel chromatography using 0-100% ethyl acetate in hexanes followed by a switch to 0-15% methanol in dichloromethane to afford 105 mg (27%) of 109.

Example 110 2-(3-(5-(6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 110

Example 110a 2-Nitro-6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazine 110a

To a 250-mL single-necked round-bottom flask equipped with a magnetic stirrer and a reflux condenser was added 101c (3.00 g, 9.59 mmol) and 4M aqueous hydrobromic acid solution (120 mL), and the resulting mixture was heated under reflux for 24 h. Thereafter, the reaction mixture was concentrated under reduced pressure to a volume of approximately 6 mL, and the residue was stirred in a 2M aqueous sodium hydroxide solution (40 mL) for 2 h. Thereafter, dichloromethane (40 mL) was added and the mixture was stirred for 15 min. The aqueous layer was separated and extracted with dichloromethane (2×50 mL). The combined organic extracts were washed with brine (100 mL) and then dried over sodium sulfate. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure to give 62% yield (1.01 g) of 110a as a white solid: mp 110-112 °C; ¹ H NMR (300 MHz, CDCl ₃ ) 6.68 (s, 1H), 4.87 (s, 2H), 4.28 (t, 2H, J=5.4 Hz), 4.20 (t, 2H, J=5.1 Hz); MS (ESI+) m/z 170.0 (M+H).

Example 110b 6,7-Dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-2-amine 110b

A 500-mL Parr hydrogenation bottle was purged with nitrogen and 110a (1.01 g, 5.92 mmol), 10% palladium on carbon (50% humidity, 125 mg dry weight), and ethanol (50 mL) were added. The bottle was evacuated, filled with hydrogen to a pressure of 25 psi, and shaken on a Parr hydrogenation apparatus for 2 h. The hydrogen was then removed and nitrogen was introduced into the bottle. The catalyst was removed by filtration through a Celite 521 pad, and the filtrate was then concentrated under reduced pressure. The resulting residue was purified by column chromatography using 400 cc of silica gel and eluted with 3% methanol/dichloromethane. Fractions containing 110b were collected and concentrated under reduced pressure to give 73% yield (601 mg) of 110b as a yellow solid: mp 74-76°C ¹ H NMR (300 MHz, 5.37 (s, 1H), 4.72 (s, 2H), 4.07 (t, 2H, J = 5.1 Hz), 3.98 (t, 2H, J = 5.1 Hz), 3.57 (br s, 2H); MS (ESI+) m/z 140.4 (M+H).

Example 110c 5-Bromo-3-(6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-2-ylamino)-1-methylpyridin-2(1H)-one 110c

To a 50-mL three-necked round-bottom flask equipped with a magnetic stirrer, a reflux condenser, and a nitrogen inlet was added 1,4-dioxane (20 mL), 110b (600 mg, 4.31 mmol), 3,5-dibromo-1-methylpyridin-2(1H)-one (1.44 g, 5.40 mmol), and cesium carbonate (3.08 g, 9.48 mmol). After bubbling nitrogen through the resulting solution for 30 minutes, Xantphos (300 mg, 0.52 mmol) and tris(dibenzylideneacetone)dipalladium(0) (320 mg, 0.35 mmol) were added, and the reaction mixture was heated under reflux for 2 hours. Afterwards, the reaction was cooled to room temperature, partitioned between ethyl acetate (75 mL) and water (75 mL), and then filtered. The aqueous layer was separated and extracted with ethyl acetate (2×25 mL). The organic layers were combined, washed with brine (50 mL), and then dried over sodium sulfate. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography using 500 cc of silica gel and eluting with 1% methanol/dichloromethane. Fractions containing 110c were collected and concentrated under reduced pressure to afford 110c in a 31% yield (433 mg) as a green solid: mp 195–197° C.; ¹ H NMR (300 MHz, CDCl ₃ ) 7.92 (d, 1 H, J=2.4 Hz), 7.44 (s, 1 H), 6.90 (d, 1 H, J=2.4 Hz), 5.65 (s, 1 H), 4.80 (s, 2 H), 4.13 (s, 2 H), 3.61 (s, 5 H); MS (ESI+) m/z 324.9 (M+H).

Example 110d 2-(5-(6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 110d

To a microwave tube equipped with a magnetic stirrer was added 5-bromo-3-(6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-2-ylamino)-1-methylpyridin-2(1H)-one 110c (130 mg, 0.4 mmol), 2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 114a (220 mg, 0.5 mmol), DME (4 mL) and 1 M aqueous sodium carbonate solution (1.2 mL). After _N2 was bubbled for 15 min, Pd( _PPh3 ) ₄ (23 mg, 0.02 mmol) was added. The mixture was heated to 130°C in a microwave for 20 min. After this time, EtOAc (5 mL) and water (5 mL) were added. The separated aqueous layer was extracted with EtOAc (2 x 5 mL). The combined organic phase was washed with brine (10 mL), dried over sodium _sulfate , filtered, and then concentrated under reduced pressure. The resulting residue was purified by column chromatography using a gradient of _CH2Cl2-60 :35:5 _CH2Cl2 : _Et2O :MeOH _to afford 110d in 89% yield (210 mg).

To a 25 mL round-bottom flask equipped with a magnetic stirrer was added 110d (210 mg, 0.4 mmol), lithium hydroxide (45 mg, 1.1 mmol), THF (3.6 mL), i-PrOH (3.6 mL), and water (7.2 mL). The mixture was stirred at room temperature for 3 h. Thereafter, EtOAc (10 mL) and water (10 mL) were added. The separated aqueous layer was extracted with EtOAc (2 x 10 mL). The combined organic phases were washed with brine (20 mL), dried over _sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by column chromatography using a gradient of _CH₂Cl₂ to ₆₀ :35:5 _CH₂Cl₂ : _Et₂O :MeOH to afford 110 in 57% yield (110 mg). MS (ESI+) m/z 541.3 (M+H).

Example 111 5-[2-(Hydroxymethyl)-3-[4-methyl-5-oxo-6-(pyridin-3-ylamino)-4,5-dihydropyrazin-2-yl]phenyl]-8-thia-5-azatricyclo[7.4.0.02,7]trideca-1(9),2(7)-diene-6-one 111

Example 111a 2-(1-oxo-3,4,5,6,7,8-hexahydrobenzothieno[2,3-c]pyridin-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 111a

A 100-mL single-necked round-bottom flask equipped with a magnetic stirrer and a reflux condenser was purged with nitrogen and charged with 104f (1.18 g, 2.72 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-di(1,3,2-dioxaborolane) (2.04 g, 8.16 mmol), potassium acetate (800 mg, 8.16 mmol) and 1,4-dioxane (20 mL). A stream of nitrogen was passed through the resulting suspension for 30 min. [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (398 mg, 0.544 mmol) was then added and the reaction was stirred at 90°C for 8 h. Thereafter, the mixture was cooled to ambient temperature, partitioned between water (25 mL) and ethyl acetate (50 mL), and then filtered through Celite The organic phase was separated, dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by flash chromatography to give 111a in 77% yield (1.01 g) as a brown solid: ¹ H NMR (500 MHz, CDCl ₃ )δ7.81(d,1H,J＝7.0Hz),7.41(t,1H,J＝7.0Hz),7.37(d,1H,J＝7.0Hz),5.49(d,1H,J＝11.5Hz),5.23(d,1H,J＝11.5Hz),4.0 1(m,1H),3.76(m,1H),2.96(m,1H),2.81–2.75(m,3H),2.50(m,2H),1.99(s,3H),1.86(m,4H),1.33(s,12H); MS(ESI+)m/z 482.2(M+H).

Example 111b 5-Bromo-1-methyl-3-(pyridin-3-ylamino)pyrazin-2(1H)-one 111b

To a 100-mL single-necked round-bottom flask equipped with a magnetic stirrer, reflux condenser, and nitrogen inlet was added THF (15 mL), 3,5-dibromo-1-methylpyrazin-2(1H)-one (1.00 g, 3.73 mmol), 3-aminopyridine (351 mg, 3.73 mmol), and sodium tert-butoxide (789 mg, 8.21 mmol). After bubbling nitrogen through the resulting solution for 30 minutes, di-μ-bromobis(tri-tert-butylphosphine)dipalladium(I) (29 mg, 0.037 mmol) was added, and the reaction mixture was stirred at room temperature for 2.5 hours. The reaction was then partitioned between ethyl acetate (50 mL) and water (50 mL) and filtered. The aqueous layer was separated and extracted with ethyl acetate (2 x 25 mL). The combined organic layers were washed with brine (50 mL) and then dried over sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to give 35% yield (370 mg) of 111b as a brown solid: mp>250°C; ¹ H NMR (500 MHz, DMSO-d ₆ ) 9.75 (s, 1H), 9.08 (d, 1H, J=2.5 Hz), 8.32 (m, 1H), 8.24 (dd, 1H, J=5.0, 1.5 Hz), 7.40 (s, 1H), 7.36 (dd, 1H, J=8.5, 4.5 Hz), 3.45 (s, 3H); MS (APCI+) m/z 281.0 (M+H).

A 100-mL single-necked round-bottom flask equipped with a magnetic stirrer and reflux condenser was purged with nitrogen and charged with 111a (134 mg, 0.279 mmol), 111b (71 mg, 0.253 mmol), sodium carbonate (80 mg, 0.759 mmol), 1,4-dioxane (5 mL), and water (1 mL). The mixture was degassed with nitrogen for 30 min. Tetrakis(triphenylphosphine)palladium (29 mg, 0.025 mmol) was added. After heating at 100°C for 3 h, the reaction mixture was cooled to room temperature and partitioned between water (40 mL) and dichloromethane (100 mL). The layers were separated, and the aqueous phase was extracted with dichloromethane (2 x 50 mL). The organic extracts were combined, dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was dissolved in methanol (5 mL), and potassium carbonate (500 mg, 3.62 mmol) was added. After stirring at room temperature for 2 h, the reaction mixture was partitioned between water (20 mL) and dichloromethane (20 mL). The layers were separated, and the aqueous phase was extracted with dichloromethane (2×20 mL). The organic extracts were combined, dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by flash chromatography to afford 111 in 38% yield (49 mg) as a white solid: mp 164–165° C.; ¹ H NMR (500 MHz, DMSO-d ₆ )δ9.54(s,1H),9.14(d,1H,J＝2.5Hz),8.42(m,1H),8.18(dd,1H,J＝4.5,1.5Hz),7.54(d,1H, J＝8.0Hz),7.49–7.46(m,2H),7.34(d,1H,J＝8.0Hz),7.30(dd,1H,J＝8.0,4.5Hz),4.81(m,1H) ,4.49(dd,1H,J＝11.0,3.5Hz),4.43(dd,1H,J＝11.0,6.5Hz),4.02(m,1H),3.86(m,1H),3.56( s,3H),2.95(m,1H),2.86(m,1H),2.77(m,2H),2.58–2.46(m,2H),1.79(m,4H); MS(APCI+)m/z 514.2(M+H).

Example 112 2-(2-(Hydroxymethyl)-3-(1-methyl-5-(5-methyl-1H-pyrazol-3-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 112

Example 112a 5-Bromo-1-methyl-3-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2(1H)-one 112a

To a 250ml sealed tube with a magnetic stirrer was added 5-methyl-1H-pyrazole-3-amine (0.91g, 9.36mmol), 3,5-dibromo-1-methyl-1H-pyridin-2-one (2.1g, 7.87mmol), cesium carbonate (7.6g, 23.56mmol) and 1,4-dioxane (78mL). After degassing for 10 minutes, tris(dibenzylideneacetone)dipalladium(0) (0.72g, 0.8mmol) and Xantphos (0.91g, 1.57mmol) were added. The reaction mixture was heated at 115°C for 48 hours. The mixture was then cooled to room temperature and distributed between dichloromethane (50mL) and water (30mL). The organic phase was separated and the aqueous layer was extracted with dichloromethane (3X 30mL). The combined organic phases were dried over sodium sulfate and then concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, 9:1 dichloromethane/methanol) to afford 85% yield (1.88 g) of 112a as a solid: MS (ESI+) m/z 285.0 (M+H).

To a 10 mL microwave vial equipped with a magnetic stirrer was added 2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl)benzyl acetate 114a (181 mg, 0.39 mmol), 112a (85 mg, 0.3 mmol), 1 M sodium carbonate solution (1.2 mL, 1.2 mmol) and 1,2-dimethoxyethane (3 mL). After nitrogen was bubbled through the resulting suspension for 10 min, tetrakis(triphenylphosphine)palladium(0) (18 mg, 0.015 mmol) was added. The reaction mixture was heated at 130 ° C for 10 minutes in a microwave reactor. Ethyl acetate (10 mL) and water (5 mL) were then added and the layers were separated. The aqueous layer was extracted with ethyl acetate (2×10 mL), and the combined organic layers were washed with brine and then dried over sodium sulfate. The desiccant was removed by filtration. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, 60:35:5 dichloromethane/ether/methanol) to give 100 mg of a mixture of 112b and 112 as a yellow residue. The residue was dissolved in a mixture of THF (1 mL), water (0.5 mL) and isopropanol (1 mL). Lithium hydroxide monohydrate (31 mg, 0.74 mmol) was added, and the mixture was stirred at room temperature for 2 h. Thereafter, the mixture was diluted with 90:10 dichloromethane/methanol (10 mL) and water (5 mL), and the layers were separated. The aqueous layer was extracted with 90:10 dichloromethane/methanol (2×10 mL), and the combined organic layers were washed with brine and then dried over sodium sulfate. The desiccant was removed by filtration. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, 60:35:5 dichloromethane/ether/methanol) to afford 18% yield (2 steps, 28 mg) of 2-(2-(hydroxymethyl)-3-(1-methyl-5-(5-methyl-1H-pyrazol-3-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 112 as a white solid: MS (ESI+) m/z 499.3 (M+H).

Example 113 2-(2-(Hydroxymethyl)-3-(1-methyl-5-(5-(4-methylpiperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 113

Example 113a 2-Bromo-6-(1-oxo-3,4,6,7,8,9-hexahydro-1H-pyrazino[1,2-a]indol-2-yl)benzyl acetate 113a

To a 350-mL sealed tube equipped with a magnetic stir bar was added 3,4,6,7,8,9-hexahydro-2H-pyrazino[1,2-a]indol-1-one 101k (5.0 g, 0.026 mol), 2,6-dibromobenzyl acetate 104 g (16.2 g, 0.052 mol), N,N'-dimethylethylenediamine (2.6 mL, 0.026 mol), Cs ₂ CO ₃ (17.0 g, 0.052 mol), and 1,4-dioxane (80 mL). After a nitrogen stream was passed through the resulting suspension for 30 min, CuI (2.5 g, 0.013 mol) was added, and the resulting reaction mixture was stirred at 95° C. for 16 h. The mixture was then cooled to room temperature, partitioned between EtOAc (50 mL) and water (50 mL), and the organic phase was extracted with EtOAc (30 mL x 3). The combined organic phases were washed with water (20 mL x 2) and brine (20 mL x 1), dried ( _Na2SO4 ), and concentrated.The crude product was purified by flash chromatography (dichloromethane:MeOH, 97:3) _to afford 35% yield (4.5 g) of 113a as a white solid.

Example 113b 2-{1-methyl-5-[5-(4-methylpiperazin-1-yl)pyridin-2-ylamino]-6-oxo-1,6-dihydropyridin-3-yl}-6-(1-oxo-3,4,6,7,8,9-hexahydro-1H-pyrazino[1,2-a]indol-2-yl)benzyl acetate 113b

In a 10-mL glass vessel equipped with a magnetic stir bar, 1-methyl-3-[5-(4-methylpiperazin-1-yl)pyridin-2-ylamino]-5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-pyridin-2-one 197f (210 mg, 0.49 mmol), 113a (250 mg, 0.60 mmol), Pd(PPh ₃ ) ₄ (50 mg, 0.043 mmol) in 2N Na ₂ CO ₃ (2 mL), and 1,2-dimethoxyethane (5 mL) were added. The vessel was sealed with a stopper and placed in a microwave cavity. The reaction mixture was stirred at 125° C. for 7 min and then purified by flash chromatography (dichloromethane:methanol, 85:15) to afford 34% (105 mg) of solid 2-{1-methyl-5-[5-(4-methylpiperazin-1-yl)pyridin-2-ylamino]-6-oxo-1,6-dihydropyridin-3-yl}-6-(1-oxo-3,4,6,7,8,9-hexahydro-1H-pyrazino[1,2-a]indol-2-yl)benzyl acetate 113b.

To a 100-mL single-necked round-bottom flask equipped with a magnetic stir bar was added 113b (105 mg, 0.17 mmol), LiOH·H ₂ O (35 mg, 0.83 mmol), THF (2 mL), isopropanol (2 mL), and water (1 mL). The reaction mixture was stirred at room temperature for 3 h, then partitioned between dichloromethane (5 mL) and water (5 mL), and the organic phase was extracted with dichloromethane (5 mL x 3). The combined organic phases were washed with water (5 mL x 2) and brine (5 mL x 1), dried (Na ₂ SO ₄ ), and then concentrated. The crude product was redissolved in dichloromethane (3 mL). Hexane (10 mL) was added to the solution, and the resulting precipitate was filtered to give 80% yield (79 mg) of 2-(2-hydroxymethyl-3-{1-methyl-5-[5-(4-methylpiperazin-1-yl)pyridin-2-ylamino]-6-oxo-1,6-dihydropyridin-3-yl}phenyl)-3,4,6,7,8,9-hexahydro-2H-pyrazino[1,2-a]indol-1-one 113; MS (ESI ⁺ ) m/z 594.3 (M+H).

Example 114 2-(3-(6-(1-cyclopropyl-1H-pyrazol-4-ylamino)-4-methyl-5-oxo-4,5-dihydropyrazin-2-yl)-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 114

Example 114a 2-(2-(acetoxymethyl)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 114a

To a round-bottom flask equipped with a stir bar was added 2-(3-bromo-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 113a (1.96 g, 4.70 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bis(1,3,2-dioxaborolane) (1.79 g, 7.05 mmol), _Cl2Pd (dppf) ₂ · _CH2Cl2 (306.9 mg, 0.376 mmol), KOAc (4.77 g, 18.79 mmol) and dioxane (33.6 mL). The mixture was heated at 95 _° C overnight. The resulting mixture was filtered through Celite and washed with ethyl acetate (200 mL). The organic phase was washed with water (50 mL), dried over MgSO ₄ , and the solvent was removed in vacuo to afford the crude product 114a , which was used directly as the starting material for further synthesis.

Example 114b 5-Bromo-3-(1-cyclopropyl-1H-pyrazol-4-ylamino)-1-methylpyrazin-2(1H)-one 114b

To a sealed tube equipped with a stir bar was added 1-cyclopropyl-1H-pyrazol-4-amine (600 mg, 4.87 mmol), 3,5-dibromo-1-methylpyrazin-2(1H)-one (1.96 g, 7.31 mmol), _Pd ( _dba ) (223.1 mg, 0.244 mmol), XantPhos (225.5 mg, 0.390 mmol), _{CsCO (} 5.25 g, 16.08 mmol), and dioxane (12 mL). The tube was sealed and heated at 100°C overnight. _CHCl (200 _mL ) was added to the resulting mixture, and the _CHCl solution was washed with water (30 mL x 3 ₎ . The precipitate in the aqueous phase was filtered to obtain the pure product 114b. The organic phase was dried over _MgSO , filtered, and the solvent was removed in vacuo. _CH2Cl2 /ether (1: ₂ , 3 mL) was added to the residue and the mixture was sonicated. The precipitate was filtered, combined with the solid filtered from the aqueous phase, and dried to afford 5-bromo-3-(1-cyclopropyl-1H-pyrazol-4-ylamino)-1-methylpyrazin-2(1H)-one 114b as a yellow solid.

Example 114c 2-(6-(1-cyclopropyl-1H-pyrazol-4-ylamino)-4-methyl-5-oxo-4,5-dihydropyrazin-2-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 114c

To a microwave tube equipped with a stir bar was added 114b (200 mg, 0.645 mmol), 114a (0.903 mmol), Pd(PPh ₃ ) ₄ , aqueous Na ₂ CO ₃ solution (1.0 N, 2.13 mL, 2.13 mmol), and DME (2.0 mL). The mixture was reacted in a microwave at 135° C. for 15 min. DCM (200 mL) was added, and the resulting mixture was washed with water (30 mL x 3), brine (30 mL x 1), dried over MgSO ₄ , filtered, and the solvent removed in vacuo. Silica gel column chromatography (MeOH:DCM=5:95) gave 2-(6-(1-cyclopropyl-1H-pyrazol-4-ylamino)-4-methyl-5-oxo-4,5-dihydropyrazin-2-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 114c, which was used directly in the next step.

To a round-bottom flask equipped with a stir bar was added 114c in THF (1.25 mL), i-PrOH (1.25 mL), _H₂O (1.25 mL), and LiOH/ _H₂O (135 mg). The resulting mixture was stirred at room temperature for 1 hr. All solvents were removed in vacuo, and silica gel column chromatography (MeOH:DCM = 10:90) afforded 38.6 mg of 2-(3-(6-(1-cyclopropyl-1H-pyrazol-4-ylamino)-4-methyl-5-oxo-4,5-dihydropyrazin-2-yl)-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 114 as a yellow solid. MS (ESI+) m/z 526.3 (M+H).

Example 115 5-[5-fluoro-2-(hydroxymethyl)-3-[1-methyl-6-oxo-5-(pyrimidin-4-ylamino)-1,6-dihydropyridin-3-yl]phenyl]-8-thia-5-azatricyclo[7.4.0.02,7]trideca-1(9),2(7)-dien-6-one 115

Example 115b tert-Butyl(2-bromo-4-fluoro-6-(1-oxo-3,4,5,6,7,8-hexahydrobenzothieno[2,3-c]pyridin-2(1H)-yl)benzyloxy)dimethylsilane 115b

To a sealed tube equipped with a magnetic stirrer was added 104e (1.11 g, 5.37 mmol), 115a (4.24 g, 10.7 mmol), cesium carbonate (3.49 g, 10.7 mmol), N,N'-dimethylethylenediamine (0.47 g, 5.37 mmol) and 1,4-dioxane (45 mL). After degassing for 10 minutes, cuprous iodide (0.51 g, 2.68 mmol) was added, and the reaction mixture was heated at 105 ° C for 2 days. Another portion of N,N'-dimethylethylenediamine (0.47 g, 5.37 mmol) and cuprous iodide (0.51 g, 2.68 mmol) were added, and the reaction mixture was heated at 105 ° C for another 5 hours. Thereafter, the mixture was cooled to room temperature and filtered. The filtrate was diluted with ethyl acetate (50 mL) and water (40 mL). The combined organic layers were dried over sodium sulfate and then concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, ethyl acetate/hexanes) to give 40% yield (1.14 g) of compound 115b as a yellow solid: MS (ESI+) m/z 524.1 (M+H).

To a 10 mL microwave vial equipped with a magnetic stirrer was added 115b (157 mg, 0.3 mmol), 1-methyl-3-(pyrimidin-4-ylamino)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl)pyridin-2(1H)-one 109c (148 mg, 0.45 mmol), 1 M sodium carbonate solution (1.2 mL, 1.2 mmol) and 1,2-dimethoxyethane (3 mL). After nitrogen was bubbled through the resulting suspension for 10 min, tetrakis(triphenylphosphine)palladium(0) (18 mg, 0.015 mmol) was added. The reaction mixture was heated at 130 ° C for 15 minutes in a microwave reactor. The reaction was repeated on the same scale and the reaction mixtures were combined. Ethyl acetate (20 mL) and water (10 mL) were added and the layers were separated. The aqueous layer was extracted with ethyl acetate (2×10 mL), and the combined organic layers were washed with brine and then dried over sodium sulfate. The desiccant was removed by filtration. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, 60:35:5 dichloromethane/ether/methanol) to obtain 400 mg of a mixture of compounds 115c and 115 as a yellow residue. The above residue was dissolved in methanol (5 mL). 10-Camphorsulfonic acid (350 mg, 1.5 mmol) was added, and the mixture was stirred at room temperature for 1 h. Thereafter, the mixture was alkalized with saturated sodium bicarbonate. The aqueous layer was extracted with dichloromethane (2×10 mL), and the combined organic layers were washed with brine and then dried over sodium sulfate. The desiccant was removed by filtration. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (NH-silica gel, ethyl acetate/hexane) to give 33% yield (88 mg) of compound 115 as a light yellow solid: MS (ESI+) m/z 532.2 (M+H)

Example 116 5-[3-(6-{[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]amino}-4-methyl-5-oxo-4,5-dihydropyrazin-2-yl)-2-(hydroxymethyl)phenyl]-8-thia-5-azatricyclo[7.4.0.02,7]trideca-1(9),2(7)-diene-6-one 116

Example 116a 1-(2-(tert-Butyldimethylsilyloxy)ethyl)-4-nitro-1H-pyrazole 116a

A 100-mL single-necked round-bottom flask equipped with a reflux condenser and a magnetic stirrer was purged with nitrogen and charged with 4-nitro-1H-pyrazole (500 mg, 4.42 mmol), (2-bromoethoxy)(tert-butyl)dimethylsilane (2.12 g, 8.85 mmol), cesium carbonate (5.76 g, 17.7 mmol), and anhydrous DMF (5 mL). After heating at 70°C for 1 hour, the mixture was cooled to room temperature and then diluted with dichloromethane (50 mL) and water (30 mL). The organic layer was separated, and the aqueous layer was extracted with dichloromethane (2 × 30 mL). The combined organic layers were dried over sodium sulfate and then concentrated under reduced pressure. The residue was purified by column chromatography to give 92% yield (1.11 g) of 116a as a white solid: mp 63-64 °C; ¹ H NMR (500 MHz, CDCl ₃ ) δ 8.20 (s, 1H), 8.08 (s, 1H), 4.24 (t, 2H, J=4.5 Hz), 3.95 (t, 2H, J=4.5 Hz), 0.84 (s, 9H), -0.04 (s, 6H).

Example 116b 1-(2-(tert-Butyldimethylsilyloxy)ethyl)-1H-pyrazol-4-amine 116b

A 250-mL Parr reaction bottle was purged with nitrogen and 10% palladium on carbon (50% humidity, 150 mg dry weight) and a solution of 116a (1.11 g, 4.10 mmol) in ethanol (20 mL) were added. The bottle was connected to a Parr hydrogenation reactor, evacuated, filled with hydrogen to a pressure of 50 psi and shaken for 3 h. After this, the hydrogen was removed and nitrogen was charged into the bottle. Celite 521 (1.00 g) was added and the mixture was filtered through a Celite 521 pad. The filter cake was washed with ethanol (2×25 mL), and the combined filtrates were concentrated to dryness under reduced pressure to afford 116b (985 mg) in 100% yield as an orange oil: ¹ H NMR (500 MHz, CDCl ₃ ) 7.18 (s, 1H), 7.11 (s, 1H), 4.09 (t, 2H, J=5.5 Hz), 3.89 (t, 2H, J=5.5 Hz), 3.25 (br s, 2H), 0.86 (s, 9H), -0.32 (s, 6H). MS (ESI+) m/z 242.2 (M+H).

Example 116c 5-Bromo-3-(1-(2-(tert-butyldimethylsilyloxy)ethyl)-1H-pyrazol-4-ylamino)-1-methylpyrazin-2(1H)-one 116c

To a 100-mL three-necked round-bottom flask equipped with a reflux condenser, a magnetic stirrer, and a nitrogen inlet was added 116b (400 mg, 1.66 mmol), 3,5-dibromo-1-methylpyrazin-2(1H)-one (443 mg, 1.66 mmol), cesium carbonate (1.19 g, 3.65 mmol), and 1,4-dioxane (20 mL). After nitrogen was bubbled through the resulting suspension for 30 min, Xantphos (144 mg, 0.249 mmol) and tris(dibenzylideneacetone)dipalladium(0) (152 mg, 0.166 mmol) were added, and the reaction mixture was heated under reflux for 4 h. Afterwards, the mixture was cooled to room temperature, filtered, and the filter cake was washed with dichloromethane (2×20 mL). The combined filtrates were concentrated under reduced pressure, and the resulting residue was purified by column chromatography to afford 116c in 51% yield (353 mg) as a yellow solid: mp 172-173 °C; ¹ H NMR (500 MHz, CDCl ₃ ) δ 8.17 (s, 1H), 8.06 (s, 1H), 7.65 (s, 1H), 6.70 (s, 1H), 4.28 (t, 2H, J=5.0 Hz), 3.97 (t, 2H, J=5.5 Hz), 3.51 (s, 3H), 0.85 (s, 9H), -0.79 (s, 6H); MS (APCI+) m/z 428.3 (M+H).

A 100-mL single-necked round-bottom flask equipped with a magnetic stirrer and reflux condenser was purged with nitrogen and charged with 116c (150 mg, 0.312 mmol), 111a (133 mg, 0.312 mmol), sodium carbonate (99 mg, 0.936 mmol), 1,4-dioxane (5 mL), and water (1 mL). The mixture was degassed with nitrogen for 30 min. Tetrakis(triphenylphosphine)palladium (36 mg, 0.031 mmol) was added. After heating at 100°C for 3 h, the reaction mixture was cooled to room temperature and then partitioned between water (40 mL) and dichloromethane (100 mL). The layers were separated, and the aqueous phase was extracted with dichloromethane (2 x 50 mL). The organic extracts were combined, dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was dissolved in methanol (5 mL), and potassium carbonate (500 mg, 3.62 mmol) was added. After stirring at room temperature for 2 h, the reaction mixture was partitioned between water (20 mL) and dichloromethane (20 mL). The layers were separated and the aqueous phase was extracted with dichloromethane (2×20 mL). The organic extracts were combined, dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was redissolved in THF (5 mL) and tetrabutylammonium fluoride trihydrate (500 mg, 1.58 mmol) was added. After stirring at room temperature for 3 h, the solvent was removed under reduced pressure and the resulting residue was washed with water (10 mL). The resulting solid was purified by flash chromatography to give 27% yield (47 mg) of 116 as an off-white solid: mp 171–172°C; ¹ H NMR (500 MHz, DMSO-d ₆ )δ9.56(s,1H),8.16(s,1H),7.75(s,1H),7.58(d,1H,J＝8.0Hz),7.44(t,1H,J＝8.0Hz),7.33–7.32 (m,2H),4.86(m,1H),4.81(t,1H,J＝5.5Hz),4.51(dd,1H,J＝11.0,6.5Hz),4.44(dd,1H,J＝11.0,6.5 Hz), 4.06 (t, 2H, J = 5.5Hz), 4.02 (m, 1H), 3.68 (t, 2H, J = 5.5Hz), 3.88 (m, 1H), 3.68 (q, 2H, J = 5.5Hz), 3.52(s,3H),2.98(m,1H),2.86(m,1H),2.77(m,2H),2.58–2.46(m,2H),1.79(m,4H); MS(APCI+)m/z 547.2(M+H)

Example 117 2-(2-methyl-3-(5-(5-methyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-6-oxo-1,6-dihydropyridazin-3-yl)phenyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-one 117

Example 117a 9-((2-(Trimethylsilyl)ethoxy)methyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-one 117a

A 25-mL single-necked round-bottom flask equipped with a magnetic stirrer was purged with nitrogen, and anhydrous DMF (4 mL) and a 60% sodium hydride/mineral oil suspension (181 mg, 4.52 mmol) were added and the reaction mixture was cooled to 0°C. 1,2,3,4-Tetrahydro-1-oxo-β-carboline (841 mg, 4.52 mmol) was added, and the reaction was stirred at 0°C for 15 minutes. Thereafter, 2-(Trimethylsilyl)ethoxymethyl chloride (829 mg, 4.97 mmol) was added, and the reaction was stirred at 0°C for 1 hour. The reaction mixture was then partitioned between water (10 mL) and ethyl acetate (20 mL). The layers were separated, and the aqueous phase was extracted with ethyl acetate (2 x 10 mL). The organic extracts were combined, dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by flash chromatography to afford 57% yield (818 mg) of 117a as a colorless oil: ^1H NMR (500 MHz, CDCl ₃ ) 7.67 (d, 1H, J=8.0 Hz), 7.65 (d, 1H, J=8.0 Hz), 7.46 (t, 1H, J=8.0 Hz), 7.27 (t, 1H, J=8.0 Hz), 6.14 (s, 2H), 5.72 (br s, 1H), 3.75 (t, 2H, J=7.0 Hz), 3.66 (t, 2H, J=8.0 Hz), 3.14 (t, 2H, J=7.0 Hz), 0.96 (t, 2H, J=8.0 Hz), -0.25 (s, 9H).

Example 117b 2-(3-bromo-2-methylphenyl)-9-((2-(trimethylsilyl)ethoxy)methyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-one 117b

To a 100-mL single-necked round-bottom flask equipped with a magnetic stirrer and a nitrogen inlet was added 117a (400 mg, 1.27 mmol), 2,6-dibromotoluene (633 mg, 2.53 mmol), cesium carbonate (828 mg, 2.54 mmol), N-methylethylenediamine (112 mg, 1.27 mmol) and 1,4-dioxane (20 mL). After nitrogen was bubbled through the resulting suspension for 30 min, cuprous iodide (121 mg, 2.54 mmol) was added. A reflux condenser was connected to the flask, and the reaction mixture was heated at 100 ° C for 16 h. Thereafter, the mixture was cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure and the resulting residue was purified by flash chromatography to give 41% yield (251 mg) of 117b as a yellow oil: ¹ H NMR (500 MHz, CDCl ₃ ) δ 7.71 (d, 1H, J=8.0 Hz), 7.68 (d, 1H, J=8.0 Hz), 7.63 (d, 1H, J=8.0 Hz), 7.47 (t, 1H, J=8.0 Hz), 7.32-7.28 (m, 2H), 7.21 (t, 1H, J=8.0 Hz), 6.13 (s, 2H), 4.15 (m, 1H), 3.93 (m, 1H), 3.69 (m, 2H), 3.29 (m, 2H), 2.45 (s, 3H), 0.95 (t, 2H, J=8.0 Hz), -0.07 (s, 9H).

Example 117c 2-(2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-9-((2-(trimethylsilyl)ethoxy)methyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-one 117c

To a 100-mL single-necked round-bottom flask equipped with a magnetic stirrer, a reflux condenser, and a nitrogen inlet was added 117b (250 mg, 0.515 mmol), 4,4,4',4',-5,5,5',5'-octamethyl-2,2'-di(1,3,2-dioxaborolane) (157 mg, 0.619 mmol), potassium acetate (152 mg, 1.55 mmol), and 1,4-dioxane (5 mL). After nitrogen was bubbled through the resulting suspension for 30 min, [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium(II)/ _CH2Cl2 was added _. (38 mg, 0.052 mmol), and the reaction mixture was heated at 95°C for 5 h. Thereafter, the mixture was diluted with ethyl acetate (20 mL) and water (20 mL), and the layers were separated. The aqueous layer was extracted with ethyl acetate (50 mL), and the combined organic layers were washed with brine (50 mL) and then dried over sodium sulfate. The desiccant was removed by filtration. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography to afford 117c in 97% yield (242 mg) as a colorless oil: ¹ H NMR (500 MHz, CDCl ₃ )δ7.85(d,1H,J＝8.0Hz),7.72(d,1H,J＝8.0Hz),7.68(d,1H,J＝8.0Hz),7.4 6(t,1H,J＝8.0Hz),7.39–7.28(m,2H),7.21(t,1H,J＝8.0Hz),6.19(d,1H,J＝ 10.5Hz),6.15(d,1H,J=10.5Hz),4.16(m,1H),3.97(m,1H),3.68(m,2H),3. 29(m,2H),2.45(s,3H),0.95(t,2H,J＝8.0Hz),1.34(s,12H),-0.07(s,9H).

Example 117d 4-Bromo-6-chloro-2-((2-(trimethylsilyl)ethoxy)methyl)pyridazin-3(2H)-one 117d

A 500-mL single-necked round-bottom flask equipped with a magnetic stirrer was purged with nitrogen and charged with anhydrous DMF (150 mL) and 4-bromo-6-chloro-pyridazin-3(2H)-one (10.0 g, 47.8 mmol). The reaction mixture was cooled to 0°C and sodium hydride was added. The reaction was stirred at 0°C for 20 minutes. Afterwards, 2-(trimethylsilyl)ethoxymethyl chloride (11.9 g, 71.6 mmol) was added, the cooling bath was removed, and the reaction was stirred at room temperature for 3 hours. The reaction was then quenched with saturated aqueous sodium bicarbonate solution (30 mL). The mixture was extracted with ethyl acetate (2 x 300 mL). The extract was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by flash chromatography to afford 56% yield (9.00 g) of 117d as a yellow oil: ¹ H NMR (300 MHz, CDCl ₃ ) δ 8.02 (s, 1H), 5.42 (s, 2H), 3.79 (t, 2H, J=5.4 Hz), 0.96 (t, 2H, J=5.4 Hz), 0.01 (s, 9H).

Example 117e 6-chloro-4-(5-methyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-2-((2-(trimethylsilyl)ethoxy)methyl)pyridazin-3(2H)-one 117e

117d (1.12 g, 3.29 mmol), 103e (500 mg, 3.29 mmol), cesium carbonate (3.22 g, 9.87 mmol) and 1,4-dioxane (25 mL) were added to a 100-mL single-necked round-bottom flask equipped with a magnetic stirrer and a reflux condenser. After nitrogen was bubbled through the resulting solution for 30 min, Xantphos (286 mg, 0.494 mmol) and tris(dibenzylideneacetone)dipalladium (0) (301 mg, 0.329 mmol) were added and the reaction mixture was heated under reflux for 3 h. After this, the reaction mixture was cooled to room temperature and distributed between water (30 mL) and dichloromethane (60 mL). Each layer was separated and aqueous phase was extracted with dichloromethane (60 mL). The organic extracts were combined, dried over sodium sulfate, filtered, and then desolvated from the filtrate under reduced pressure. The resulting residue was purified by column chromatography to give 72% yield (977 mg) of 117e as a yellow solid: mp 68-69°C; ¹ H NMR (500 MHz, CDCl ₃ ) δ 7.85 (s, 1H), 7.56 (s, 1H), 5.70 (s, 1H), 5.47 (s, 2H), 4.16 (t, 2H, J=5.5 Hz), 3.73 (m, 2H), 3.63 (s, 2H), 2.94 (t, 2H, J=5.5 Hz), 2.51 (s, 3H), 0.98 (m, 2H), 0.12 (s, 9H); MS (ESI+) m/z 411.2 (M+H).

Example 117f 2-(2-methyl-3-(5-(5-methyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-6-oxo-1-((2-(trimethylsilyl)ethoxy)methyl)-1,6-dihydropyridazin-3-yl)phenyl)-9-((2-(trimethylsilyl)ethoxy)methyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-one 117f

1,4-dioxane (5 mL), water (1 mL) and sodium carbonate (143 mg, 1.35 mmol) were added to a 50-mL single-necked round-bottom flask equipped with a magnetic stirrer, a reflux condenser and a nitrogen inlet. After nitrogen was bubbled through the resulting mixture for 30 min, 117c (240 mg, 0.451 mmol), 117e (185 mg, 0.451 mmol) and tetrakis(triphenylphosphine)palladium(0) (52 mg, 0.045 mmol) were added and the reaction mixture was heated at 100 ° C for 3 h. Afterwards, the reaction was cooled to room temperature and distributed between water (25 mL) and dichloromethane (50 mL). The organic layer was separated, washed with brine (75 mL), and then dried over sodium sulfate. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure to a black oil, which was purified by flash chromatography to give 89% yield (315 mg) of 117f as a yellow oil: ¹ H NMR (500 MHz, CDCl ₃ ) δ 7.88 (s, 1H), 7.66 (d, 1H, J=8.0 Hz), 7.45 (m, 3H), 7.39 (t, 1H, J=8.0 Hz), 7.34 (d, 1H, J=8.0 Hz), 7.24 (t, 2H, J=8.0 Hz), 6.12 (d, 1H, J=10.5 Hz), 6.05 (d, 1H, J=10.5 Hz), 5.69 (s, 1H), 5.63 (d, 1H, J=10.5 Hz), 5.52 (d, 1H,J＝10.5Hz),4.09(m,2H),3.88(m,2H),3.75(m,2H),3.68(s,3H),3.59(m,2H),3.26–3.17(m,4H),2.61( m,2H),2.29(s,3H),0.98(t,2H,J＝8.0Hz),0.85(t,2H,J＝8.0Hz),-0.01(s,9H),-0.07(s,9H); MS(ESI+)m/z 781.4(M+H).

A 50-mL single-necked round-bottom flask equipped with a magnetic stirrer was purged with nitrogen and 117f (315 mg, 0.404 mmol), anisole (438 mg, 4.04 mmol), dichloromethane (3 mL) and trifluoroacetic acid (3 mL) were added. After stirring at room temperature for 2 h, the reaction mixture was concentrated and the resulting residue was partitioned between 1 M aqueous potassium dihydrogen phosphate solution (10 mL) and dichloromethane (20 mL). The layers were separated and the aqueous phase was extracted with dichloromethane (2×10 mL). The organic extracts were combined, dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by flash chromatography to give 117 in 23% yield (49 mg) as a white solid: mp 254°C dec; ¹ H NMR (500 MHz, DMSO-d ₆ )δ12.97(s,1H),11.74(s,1H),9.21(s,1H),7.79(s,1H),7.65(d,1H,J＝8.0Hz) ,7.44–7.37(m,3H),7.34(d,1H,J＝8.0Hz),7.26(t,1H,J＝8.0Hz),7.08(t,1H,J＝ 8.0Hz),5.75(s,1H),4.17(m,1H),3.97(t,2H,J＝5.0Hz),3.84(m,1H),3.51(s, 2H),3.17(m,2H),2.79(t,2H,J=5.0Hz),2.35(s,3H),2.18(s,3H);MS(ESI+)m/z 521.2(M+H)

Example 118 2-(2-(Hydroxymethyl)-3-(1-methyl-6-oxo-5-(pyrimidin-4-ylamino)-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrido[3,4-b]indolizine-1(2H)-one 118

Example 118a Methyl 5,6,7,8-tetrahydroindolizine-2-carboxylate 118a

A 500-mL round-bottom flask equipped with a magnetic stirrer and nitrogen inlet was purged with nitrogen and charged with 5,6,7,8-tetrahydroindolizine-2-carboxylic acid (30.4 g, 184 mmol), DMF (1.00 g, 13.6 mmol), and dichloromethane (300 mL). The solution was cooled to 0°C in an ice bath. Oxalyl chloride (28.0 g, 221 mmol) was added dropwise, and the reaction mixture was then warmed to room temperature over 30 minutes and stirred for 5 hours. The resulting solution was then concentrated to yield a brown solid. This solid was dissolved in anhydrous methanol (400 mL), and the solution was cooled to 0°C. Triethylamine (57 g, 552 mmol) was added to the reaction mixture, and the mixture was stirred at room temperature for an additional 2 hours. The reaction mixture was then concentrated to dryness under reduced pressure. The residue was diluted with dichloromethane (300 mL) and washed with water (200 mL) and saturated aqueous sodium bicarbonate solution (200 mL). The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was triturated with hexanes (200 mL) to afford 118a in 58% yield (19.1 g) as a white solid: mp 72–74° C.; ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.13 (s, 1H), 6.23 (s, 1H), 3.93 (t, 2H, J=6.0 Hz), 3.77 (s, 3H), 2.75 (t, 2H, J=6.0 Hz), 1.93 (m, 2H), 1.80 (m, 2H); (APCI+) m/z 180.1 (M+H)

Example 118b Methyl 3-(cyanomethyl)-5,6,7,8-tetrahydroindolizine-2-carboxylate 118b

To a 500-mL three-necked round-bottom flask equipped with an addition funnel and a thermometer was added 118a (6.70 g, 37.4 mmol), iodoacetonitrile (12.5 g, 74.9 mmol), iron (II) sulfate heptahydrate (5.20 g, 18.7 mmol), and dimethyl sulfoxide (250 mL). Hydrogen peroxide (35%, 18.2 g, 187 mmol) was added dropwise to the mixture via a syringe pump over 1 hour at room temperature using a water bath. Iron (II) sulfate heptahydrate (2–3 equiv) was added portionwise to the reaction mixture, maintaining the temperature at 25–35°C, until the reaction mixture turned deep red. If TLC indicated the reaction was incomplete, additional hydrogen peroxide (2–3 equiv) and iron (II) sulfate heptahydrate (1–2 equiv) were added in the same manner until the reaction was complete. The reaction mixture was then partitioned between saturated sodium bicarbonate solution (200 mL) and ethyl acetate (400 mL). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (2×100 mL). The combined organic layers were washed with saturated sodium thiosulfate solution (50 mL), dried over sodium sulfate, and then concentrated under reduced pressure. The residue was purified by column chromatography to afford 118b in 78% yield (6.40 g) as a yellow oil: ¹ H NMR (500 MHz, CDCl ₃ ) δ 6.23 (s, 1H), 4.23 (s, 2H), 3.94 (t, 2H, J=6.5 Hz), 3.81 (s, 3H), 2.74 (t, 2H, J=6.5 Hz), 2.00 (m, 2H), 1.83 (m, 2H); (APCI+) m/z 219.3 (M+H)

Example 118c 3-(2-aminoethyl)-5,6,7,8-tetrahydroindolizine-2-carboxylic acid methyl ester hydrochloride 118c

Intermediate 118b was hydrogenated with platinum oxide catalyst under 50 psi of hydrogen in ethanol and ethyl acetate in the presence of hydrogen chloride at room temperature overnight to afford 118c (380 mg, 1.74 mmol) which was used directly in the next step.

Example 118d 3,4,6,7,8,9-hexahydropyrido[3,4-b]indolizine-1(2H)-one 118d

A 100-mL single-necked round-bottom flask equipped with a magnetic stirrer and a nitrogen inlet was purged with nitrogen and 3-(2-aminoethyl)-5,6,7,8-tetrahydroindolizine-2-carboxylic acid methyl ester hydrochloride 118c (estimated 1.74 mmol, assuming quantitative yield), sodium ethoxide (354 mg, 5.22 mmol) and ethanol (20 mL) were added. The mixture was stirred at 55 ° C for 5 h. Thereafter, the reaction mixture was concentrated under reduced pressure and the residue was partitioned between ethyl acetate (200 mL) and water (100 mL). The organic layer was separated and the aqueous layer was extracted with ethyl acetate (2×100 mL). The combined organic layers were washed with brine, dried over sodium sulfate, and then concentrated under reduced pressure. The residue was purified by column chromatography to give 118d in 67% yield (220 mg) as a white solid: mp 195-197 °C; ^1H NMR (500 MHz, DMSO-d ₆ ) δ 6.76 (s, 1H), 5.89 (s, 1H), 3.78 (t, 2H, J=6.5 Hz), 3.35 (m, 2H), 2.66 (m, 4H), 1.87 (m, 2H), 1.72 (m, 2H); (APCI+) m/z 191.3 (M+H)

Example 118e 2-Bromo-6-(1-oxo-3,4,6,7,8,9-hexahydropyrido[3,4-b]indolizine-2(1H)-yl)benzyl acetate 118e

A 250-mL three-necked round-bottom flask equipped with a reflux condenser and a magnetic stirrer was purged with nitrogen and charged with 118d (1.50 g, 7.89 mmol), 104 g (4.80 g, 15.7 mmol) of 2,6-dibromobenzyl acetate, cesium carbonate (5.11 g, 15.7 mmol), N,N'-dimethylethylenediamine (695 mg, 7.89 mmol), and 1,4-dioxane (100 mL). After bubbling nitrogen through the resulting suspension for 20 min, cuprous iodide (752 mg, 3.95 mmol) was added, and the reaction mixture was heated at 95°C (oil bath temperature) for 12 h. N,N'-dimethylethylenediamine (695 mg, 7.89 mmol) and cuprous iodide (752 mg, 3.95 mmol) were then added and heated at 95 °C for another 12 h. This operation was repeated until most of 118d was converted to 118e, about 48 h. Afterwards, the mixture was cooled to room temperature and then filtered. The filtrate was diluted with ethyl acetate (300 mL) and water (100 mL). The organic layer was separated and the aqueous layer was extracted with ethyl acetate (3×100 mL). The combined organic layers were dried over sodium sulfate and then concentrated under reduced pressure. The residue was purified by flash chromatography to afford 27% yield (905 mg) of 118e as an off-white solid: mp 176-178 °C; ^1H NMR (300 MHz, DMSO- _d6 ) δ 7.64 (dd, 1H, J = 7.5, 1.8 Hz), 7.37 (m, 2H), 5.99 (s, 1H), 5.00 (d, 2H, J = 6.0 Hz), 4.00 (m, 1H), 3.85 (m, 2H), 3.62 (m, 1H), 2.93 (t, 2H, J = ^6.1 Hz), 2.67 (t, 2H, J = ^6.1 Hz), 2.00 (d, 3H, J = 6.0 Hz), 1.90 (m, 2H), 1.75 (m, 2H); MS (ESI+) m/z 417.0 (M+H)

Example 118f 2-(1-oxo-3,4,6,7,8,9-hexahydropyrido[3,4-b]indolizine-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 118f

To a 100-mL three-necked round-bottom flask equipped with a reflux condenser, a magnetic stirrer, and a nitrogen inlet was added 118e (1.20 g, 2.88 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bis(1,3,2-dioxaborolane) (2.20 g, 8.65 mmol), potassium acetate (1.13 g, 11.5 mmol), and 1,4-dioxane (50 mL). After bubbling nitrogen through the resulting suspension for 20 min, [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (210 mg, 0.288 mmol) was added, and the reaction mixture was heated at 95 ° C for 8 h. Thereafter, the mixture was cooled to room temperature and then filtered. The filter cake was washed with ethyl acetate (40 mL). The filtrate was diluted with ethyl acetate (150 mL) and water (40 mL). The organic layer was separated and the aqueous layer was extracted with ethyl acetate (3 x 50 mL).The combined organic layers were dried over sodium sulfate and concentrated under reduced pressure to give 100% yield (1.35 g) of crude 118f as a yellow oil.

To a 100-mL three-necked round-bottom flask equipped with a magnetic stirrer and a reflux condenser were added 118f (400 mg, 0.862 mmol), 5-bromo-1-methyl-3-(pyrimidin-4-ylamino)pyridin-2(1H)-one 109b (241 mg, 0.862 mmol), sodium carbonate (365 mg, 3.45 mmol), water (4 mL), and 1,4-dioxane (20 mL). After nitrogen was bubbled through the resulting suspension for 20 min, tetrakis(triphenylphosphine)palladium(0) (100 mg, 0.086 mmol) was added, and the reaction mixture was heated at 100°C for 2 h. Thereafter, the reaction mixture was cooled to room temperature, filtered, and the filter cake was washed with a 1:10 mixture of methanol and dichloromethane (30 mL). The filtrate was concentrated under reduced pressure to obtain a brown residue. To another 50-mL single-necked round-bottom flask equipped with a magnetic stirrer and a reflux condenser was added the above residue, THF (5 mL), ethanol (5 mL), water (5 mL) and lithium hydroxide (83.0 mg, 3.45 mmol). The mixture was stirred at 50 ° C for 2 h. Thereafter, the reaction mixture was concentrated under reduced pressure. The resulting residue was purified by flash chromatography to give 23% (106 mg) of 118 as an off-white solid: mp 173–175 ° C; ¹ H NMR (500 MHz, DMSO-d ₆ )δ7.16(s,1H),8.72(s,1H),8.64(s,1H),8.29(d,1H,J＝6.0Hz),7.55(s,1H), 7.45(t,1H,J＝7.5Hz),7.29(m,3H),6.00(s,1H),4.75(t,1H,J＝5.0Hz),4.31(d ,2H,J＝5.0Hz),4.00(m,1H),3.96(m,1H),3.81(m,2H),3.60(s,3H),3.00(m,1 H),2.91(m,1H),2.71(t,2H,J=5.5Hz),1.92(m,2H),1.75(m,2H); MS(ESI+)m/z 497.2 (M+H)

Example 119 5-[2-(Hydroxymethyl)-3-(5-{[5-(2-hydroxypropane-2-yl)-1-methyl-1H-pyrazol-3-yl]amino}-1-methyl-6-oxo-1,6-dihydropyridazin-3-yl)phenyl]-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-6-one 119

Example 119a Methyl 3-amino-1-methyl-1H-pyrazole-5-carboxylate 119a

To a 250mL Parr hydrogenation bottle was added 1-methyl-3-nitro-1H-pyrazole-5-carboxylic acid methyl ester (530mg, 2.9mmol), dissolved in ethyl acetate (156mL) and ethanol (15mL), to which was added 10% Pd/C (Degussa type) (100mg). The mixture was placed on a Parr apparatus and pressurized to 50psi with hydrogen and shaken for 2.5hr. The reaction solution was filtered through a celite pad and washed with ethyl acetate. The solvent was removed under vacuum to obtain 119a as a white solid (450mg, 2.9mmol, quantitative yield).

Example 119b 3-(6-chloro-2-methyl-3-oxo-2,3-dihydropyridazin-4-ylamino)-1-methyl-1H-pyrazole-5-carboxylic acid methyl ester 119b

To a three-necked RBF was added 119a (500 mg, 3.2 mmol), 4-bromo-6-chloro-2-methylpyridazin-3(2H)-one (725 mg, 3.2 mmol), cesium carbonate (2.3 g, 7.0 mmol), and Xantphos (160 mg, 8.5 mol%). The flask was evacuated and filled with nitrogen 3X. Dioxane (20 ml) was added and the mixture was degassed by bubbling nitrogen for 25 min. Tris(dibenzylideneacetone)dipalladium(0) (150 mg, 5 mol%) was then added, and the reaction was heated to 100°C for 6 hr. The reaction was cooled and diluted with EtOAc (125 mL) and saturated aqueous _NaHCO3 (50 mL). The layers were separated and extracted with EtOAc ( ₂ x 100 mL). The organic extract was washed 3X with brine, dried over _Na2SO4 , filtered, and then concentrated under reduced pressure. The residue was purified by chromatography (ISCO 40 g silica gel, EtOAc/hexanes) to afford 119b.

Example 119c 6-Chloro-4-(5-(2-hydroxypropan-2-yl)-1-methyl-1H-pyrazol-3-ylamino)-2-methylpyridazin-3(2H)-one 119c

Under nitrogen, anhydrous THF (20 mL) and anhydrous toluene (5 mL) were added to a 100 mL round-bottom flask containing 119b (500 mg, 1.7 mmol) and the mixture was stirred and cooled to -20 to -30°C. 3.0 M methylmagnesium bromide/diethyl ether (1.6 mL, 4.75 mmol) was then slowly added. After the addition, the reaction was slowly warmed to room temperature and stirred for approximately 3 hours, after which it was quenched with 1 N HCl. The mixture was concentrated to remove THF, diluted with ethyl acetate and water, and then the pH was adjusted to ~6-7 with 1 M NaOH. The mixture was separated and extracted 2X with ethyl acetate, washed with brine, dried over Na ₂ SO ₄ , filtered, and then concentrated under reduced pressure. The residue was purified by chromatography (ISCO 24 g silica gel, ethyl acetate/hexane) to provide 119c.

Example 119d 2-(5-(5-(2-hydroxypropan-2-yl)-1-methyl-1H-pyrazol-3-ylamino)-1-methyl-6-oxo-1,6-dihydropyridazin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 119d

To a microwave-safe vial were added 119c (150 mg, 0.50 mmol) and 111a (240 mg, 0.50 mmol), along with DME (4 mL) and then _{1N Na₂CO₃} (1.1 mL). After degassing with argon for 5 min, tetrakis(triphenylphosphine)palladium(0) (29 mg, 5 mol%) was added, and the mixture was heated at 130°C for 15 min in a microwave reactor. An additional 40 mg of 111a was added, and the mixture was heated for an additional ₁₀ min. The reaction was diluted with ethyl acetate and water, and the ethyl acetate layer was separated, washed with brine, dried over _Na₂SO₄ , filtered, and then concentrated under reduced pressure. The residue was purified by chromatography (ISCO 12 g silica gel, eluting with methanol and _CH₂Cl₂ ) _to afford 119d (170 mg, 56% yield).

To a vial containing 119d (170 mg, 0.28 mmol) dissolved in THF (1.5 mL) and isopropanol (1.5 ml) was added 1N LiOH/water (1.4 mL, 1.4 mmol) and the mixture was stirred overnight. The reaction was then judged to be complete by LC-MS. The mixture was concentrated, then diluted with ethyl acetate and water, and the pH was adjusted to 7 by adding 1N HCl and 1N NaOH. The ethyl acetate layer was separated, washed with brine, dried _{over Na₂SO₄} , filtered, and then concentrated under reduced pressure. The residue was triturated with ethyl acetate, and the solid was collected by filtration to afford 119 (98 mg, 61% yield). MS (ESI+) m/z 575.2 (M+H).

Example 120 5-[5-fluoro-2-(hydroxymethyl)-3-[4-methyl-5-oxo-6-(1,2,3,4-tetrahydroisoquinolin-6-ylamino)pyrazin-2-yl]phenyl]-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-diene-6-one 120

Example 120a tert-Butyl 6-(6-bromo-4-methyl-3-oxo-3,4-dihydropyrazin-2-ylamino)-3,4-dihydroisoquinoline-2(1H)-carboxylate 120a

A mixture of tert-butyl 6-amino-3,4-dihydroisoquinoline-2(1H)-carboxylate (3 g, 12 mmol), 3,5-dibromo-1-methylpyrazin-2(1H)-one (2.68 g, 10 mmol), and triethylamine (1.5 g, 15 mmol) in IPA (50 mL) was heated at 70°C for 15 h. The mixture was cooled to room temperature. The resulting yellow solid was collected by filtration and then dried in vacuo to afford 120a as a yellow solid (2.83 g, 65%). MS: [M+H] ⁺ 435.

Example 120b 4-Fluoro-2-[4-methyl-5-oxo-6-(1,2,3,4-tetrahydroisoquinolin-6-ylamino)pyrazin-2-yl]-6-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-5-yl}phenyl}methyl acetate 120b

According to Example 150b, 482 mg of 218b and 435 mg of 120a were reacted to give 362 mg (51%) of 120b as a yellow solid. MS: [M+H] ⁺ 711.

According to Example 149, 200 mg of 120b was converted to 120 as a white solid (78 mg, 42%). ¹ H NMR (500 MHz, MeOD) δ 7.72 (s, 1H), 7.58 (d, J = 8.0, 1H), 7.39 (m, 2H), 7.21 (ss, J = 9.5, 1H), 7.10 (d, J = 8.5, 1H), 4.62 (d, J = 12, 1H), 4.50 (d, J = 12, 1H), 4.11 (m, 3H), 3.98 (m, 1H), 3.64 (s, 3H), 3.28 (s, 2H), 2.95 (m, 4H), 2.85 (s, 2H), 2.60 (m, 2H), 1.9 (m, 4H).

Example 121 5-[2-(Hydroxymethyl)-3-(4-methyl-5-oxo-6-{[4-(piperidin-4-yl)phenyl]amino}-4,5-dihydropyrazin-2-yl)phenyl]-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-diene-6-one 121

Example 121a tert-Butyl 4-(4-(6-bromo-4-methyl-3-oxo-3,4-dihydropyrazin-2-ylamino)phenyl)piperidine-1-carboxylate 121a

Compound 121a was synthesized using the same method as Example 112a, except that tert-butyl 4-(4-aminophenyl)piperidine-1-carboxylate (0.83 g, 3.0 mmol), 3,5-dibromo-1-methylpyrazin-2(1H)-one (0.88 g, 3.3 mmol), cesium carbonate (1.27 g, 3.9 mmol), tris(dibenzylideneacetone)dipalladium(0) (0.275 g, 0.3 mmol), Xantphos (0.26 g, 0.45 mmol), and 1,4-dioxane (30 mL) were used. The reaction mixture was heated at 100° C. overnight. Workup and purification by flash column chromatography (silica gel, ethyl acetate/hexane) gave 121a as a solid in 80% yield (1.1 g): MS (ESI+) m/z 465.0 (M+H).

Example 121b 5-[2-(Acetoxymethyl)-3-(4-methyl-5-oxo-6-{[4-(piperidin-4-yl)phenyl]amino}-4,5-dihydropyrazin-2-yl)phenyl]-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-6-one 121b

To a 10 mL microwave vial equipped with a magnetic stirrer was added 111a (173 mg, 0.36 mmol), 121a (139 mg, 0.3 mmol), 1 M sodium carbonate solution (1.2 mL, 1.2 mmol) and 1,2-dimethoxyethane (3 mL). After nitrogen was bubbled through the resulting suspension for 10 min, tetrakis(triphenylphosphine)palladium (0) (18 mg, 0.015 mmol) was added. The reaction mixture was heated at 130 ° C for 15 minutes in a microwave reactor. Thereafter, ethyl acetate (15 mL) and water (10 mL) were added, and the layers were separated. The aqueous layer was extracted with ethyl acetate (2 × 20 mL), and the combined organic layers were washed with brine and then dried over sodium sulfate. The desiccant was removed by filtration. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, ethyl acetate / hexane) to give compound 121b as a yellow oil (240 mg).

Compound 121b was dissolved in dichloromethane (10 mL). Trifluoroacetic acid (0.3 mL, 3.9 mmol) was added, and the mixture was stirred at room temperature for 4 h. Thereafter, the mixture was alkalized with saturated sodium bicarbonate, and the aqueous layer was extracted with dichloromethane (2×15 mL). The combined organic layers were washed with brine and then dried over sodium sulfate. The desiccant was removed by filtration. The filtrate was concentrated, and the resulting residue 121c was dissolved in a mixture of THF (1.5 mL), water (0.8 mL) and isopropanol (1.5 mL). Lithium hydroxide monohydrate (56 mg, 1.32 mmol) was added, and the mixture was stirred at room temperature for 3 h. Thereafter, the mixture was diluted with 90:10 dichloromethane/methanol (10 mL) and water (5 mL), and the layers were separated. The aqueous layer was extracted with 90:10 dichloromethane/methanol (2×10 mL), and the combined organic layers were washed with brine and then dried over sodium sulfate. The desiccant was removed by filtration. The filtrate was purified by flash chromatography (silica gel, dichloromethane/methanol) to give 35% yield (3 steps, 63 mg) of compound 121 as a light pink solid: MS (ESI+) m/z 596.3 (M+H).

Example 122 5-[5-fluoro-2-(hydroxymethyl)-3-(4-methyl-6-{[4-(morpholin-4-yl)phenyl]amino}-5-oxo-4,5-dihydropyrazin-2-yl)phenyl]-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-6-one 122

Example 122a 5-Bromo-1-methyl-3-(4-morpholinophenylamino)pyrazin-2(1H)-one (3-3) 122a

To a microwave vial equipped with a magnetic stirrer was added 3,5-dibromo-1-methylpyrazin-2(1H)-one (1.97 g, 7.4 mmol), 4-morpholinoaniline (1.97 g, 11.1 mmol), and isopropanol (25 mL). The system was evacuated and then refilled with N ₂ . The mixture was heated at 90 ° C for 16 h. The mixture was then cooled to room temperature and concentrated under reduced pressure. The residue was purified by flash chromatography eluting with 5:1 petroleum ether/ethyl acetate to afford 122a (2.3 g, 85%). LCMS: [M+H] ⁺ 365.

Example 122b 4-Methyl-6-(4-morpholinophenylamino)-5-oxo-4,5-dihydropyrazin-2-ylboronic acid 122b

To a microwave-safe vial equipped with a magnetic stirrer was added 122a (764 mg, 2.1 mmol), (PinB) _₂ (2.75 g, 10 mmol), Pd(dppf) _Cl₂ (0.1 g, 0.13 mmol), KOAc (0.6 g, 6 mmol), and DMF (5 mL). The system was evacuated and then refilled with _N₂ . The reaction mixture was then heated at 105°C for 1 h, cooled to room temperature, and filtered. The filtrate was concentrated under reduced pressure to afford crude product 122b, which was used without further purification. LCMS: [M+H] ^⁺ 331.

Example 122c [4-Fluoro-2-(4-methyl-6-{[4-(morpholin-4-yl)phenyl]amino}-5-oxopyrazin-2-yl)-6-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-5-yl}phenyl]methyl acetate 122c

To a 25 mL vial was added (2-bromo-4-fluoro-6-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-5-yl}phenyl)methyl acetate 212a (300 mg, 0.67 mmol) and 122b (440 mg, 1.33 mmol) suspended in 1,2-dimethoxyethane (15 mL) and water (1 mL). The resulting orange solution was heated in a Biotage microwave reactor at 130°C for 30 minutes. After the reaction, the residue was purified by reverse phase Combi-flash chromatography eluting with 0.3% NH ₄ HCO ₃ /1:6 water/CH ₃ CN to afford 122c as a brown solid (200 mg, 46%). MS: (M+H) ⁺ 658.

To a solution of 122c (220 mg, 0.33 mmol) in propan-2-ol (7 mL), tetrahydrofuran (7 mL), and water (2 mL) was added LiOH (804 mg, 33 mmol). The mixture was stirred at 30°C for 2 h. The residue was then evaporated under reduced pressure and purified by preparative HPLC to afford 122 as a yellow solid (82 mg, 40%). MS: (M+H) ⁶¹⁶ . ¹ H NMR(500MHz,MeOD)δ1.89(s,5H),2.55-2.63(m,2H),2.94(s,2H),3.11-3.13(t,5H),3.64(s,3H),3.82-3.84(t,4H),3.93-3.98(m, 1H),4.07-4.14(m,1H),4.43-4.53(m,2H),6.97-6.99(d,2H),7.18-7.20(d,1H),7.29(s,1H),7.36-7.38(d,1H),7.62-7.64(d,2H).

Example 123 5-(3-{5-[(5-cyclopropyl-1H-pyrazol-3-yl)amino]-1-methyl-6-oxo-1,6-dihydropyridin-3-yl}-2-(hydroxymethyl)phenyl)-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-6-one 123

Example 123a 1-Cyclopropyl-3-nitro-1H-pyrazole 123a

A 250-mL three-necked round-bottom flask equipped with a reflux condenser and a magnetic stirrer was purged with nitrogen and charged with 3-nitro-(1H)pyrazole (1.30 g, 11.5 mmol), cyclopropylboronic acid (1.98 g, 23.0 mmol), sodium carbonate (3.66 g, 34.5 mmol), 2,2'-bipyridine (3.58 g, 23.0 mmol), dichloroethane (60 mL), and copper(II) acetate (2.08 g, 11.5 mmol). The reaction mixture was heated at 70°C (oil bath temperature) for 3 h. An additional portion of cyclopropylboronic acid (1.98 g, 23.0 mmol) was then added, and the mixture was heated for 3 h. The mixture was then cooled to room temperature and filtered. The filtrate was diluted with ethyl acetate (350 mL) and water (40 mL). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (3 x 150 mL). The combined organic layers were dried over sodium sulfate and then concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, 0%-100% ethyl acetate/hexanes) to afford 85% yield (1.49 g) of 123a as a colorless oil: ¹ H NMR (500 MHz, CDCl ₃ ) δ 7.53 (d, 1H, J=2.4 Hz), 6.87 (d, 1H, J=2.5 Hz), 3.71 (m, 1H), 1.25 (m, 2H), 1.12 (m, 2H); MS (APCI+) m/z 154.1 (M+H).

Example 123b 1-Cyclopropyl-1H-pyrazol-3-amine 123b

A 250-mL Parr reaction bottle was purged with nitrogen and added with 10% palladium on carbon (50% humidity, 137 mg dry weight) and a solution of 123a (600 mg, 3.92 mmol) in ethanol (70 mL). The bottle was connected to a Parr hydrogenation reactor, evacuated, filled with hydrogen to a pressure of 50 psi and shaken for 3 h. Thereafter, the hydrogen was removed and nitrogen was introduced into the bottle. Celite 521 (1.00 g) was added and the mixture was filtered through a pad of Celite 521. The filter cake was washed with ethanol (2 x 25 mL), and the combined filtrates were concentrated to dryness under reduced pressure to afford 93% yield of 123b (450 mg) as a purple oil: ¹ H NMR (300 MHz, CDCl ₃ ) 7.17 (d, 1H, J=2.4 Hz), 5.55 (d, 1H, J=2.4 Hz), 3.43 (m, 1H), 2.92 (br s, 2H), 1.01 (m, 2H), 0.93 (m, 2H); MS (ESI+) m/z 124.1 (M+H).

Example 123c 5-Bromo-3-(1-cyclopropyl-1H-pyrazol-3-ylamino)-1-methylpyridin-2(1H)-one 123c

To a 250-mL three-necked round-bottom flask equipped with a reflux condenser, a magnetic stirrer, and a nitrogen inlet was added 123b (444 mg, 3.61 mmol), 2,5-dibromo-1-methylpyrazin-6-one (1.06 g, 3.97 mmol), cesium carbonate (3.52 g, 10.8 mmol), and 1,4-dioxane (45 mL). After nitrogen was bubbled through the resulting suspension for 30 min, Xantphos (177 mg, 0.306 mmol) and tris(dibenzylideneacetone)dipalladium(0) (165 mg, 0.180 mmol) were added, and the reaction mixture was heated under reflux for 3 h. Thereafter, the mixture was cooled to room temperature and diluted with ethyl acetate (150 mL) and water (30 mL). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (3×150 mL). The combined organic layers were dried over sodium sulfate and then concentrated under reduced pressure. The residue was triturated with methanol (20 mL) to give 63% yield (700 mg) of 123c as an off-white solid: mp 161-163 °C; ^1H NMR (300 MHz, DMSO- _d6 ) δ 8.42 (s, 1H), 8.00 (d, 1H, J = 2.5 Hz), 7.57 (d, 1H, J = 2.4 Hz), 7.38 (d, 1H, J = 2.5 Hz), 6.05 (d, 1H, J = 2.4 Hz), 3.61 (m, 1H), 3.49 (s, 1H), 0.95 (m, 4H); MS (ESI+) m/z 309.0 (M+H).

To a 150-mL single-necked round-bottom flask equipped with a reflux condenser, a magnetic stirrer, and a nitrogen inlet was added 123c (247 mg, 0.800 mmol), 111a (770 mg, 1.60 mmol), sodium carbonate (254 mg, 2.40 mmol), DMF (5 mL), water (2.5 mL), and 1,4-dioxane (8 mL). After bubbling nitrogen through the resulting suspension for 30 min, tetrakis(triphenylphosphine)palladium(0) (93 mg, 0.080 mmol) was added, and the reaction mixture was heated under reflux for 14 h. Thereafter, the mixture was cooled to room temperature and then diluted with ethyl acetate (150 mL) and water (30 mL). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (3×150 mL). The combined organic layers were dried over sodium sulfate and then concentrated under reduced pressure. The residue was dissolved in a mixture of THF (8 mL), methanol (4 mL), and water (4 mL). To the resulting solution was added lithium hydroxide monohydrate (167 mg, 3.40 mmol). The mixture was stirred at room temperature for 2 h and then concentrated in vacuo. The residue was partitioned between ethyl acetate (150 mL) and water (30 mL). The organic layer was separated and the aqueous layer was extracted with a 20% (v/v) solution of methanol/dichloromethane (3×150 mL). The combined organic layers were dried over sodium sulfate and then concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, 0%-10% methanol/dichloromethane) to give 19% yield (84 mg) of 123 as an off-white solid: mp 200–201°C; ¹ H NMR (500 MHz, DMSO-d ₆ )δ11.75(s,1H),7.98(s,1H),7.92(s,1H),7.418(t,1H,J=8.0Hz),7.31(d ,1H,J＝7.5Hz),7.27(d,1H,J＝7.5Hz),7.21(s,1H),5.78(s,1H),4.79(m,1 H),4.36(m,2H),4.12(m,1H),3.80(m,1H),3.56(s,3H),2.95(m,1H),2.80 (m,1H),2.78(s,2H),1.80(m,5H),0.87(m,2H),0.62(m,2H);MS(ESI+)m/z 542.1(M+H).

Example 124 5-[5-Fluoro-2-(hydroxymethyl)-3-{1-oxo-1H,2H,3H,4H,6H,7H,8H,9H-pyrazino[1,2-a]indol-2-yl}phenyl]-1-methyl-3-[(5-methyl-1H-pyrazol-3-yl)amino]-1,2-dihydropyridin-2-one 124

Example 124a 1-Methyl-3-(5-methyl-1H-pyrazol-3-ylamino)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one 124a

To a microwave-safe vial equipped with a magnetic stirrer was added 5-bromo-1-methyl-3-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2(1H)-one 112a (2.3 g, 8.3 mmol), (PinB) ( ₁₁ g, 41 mmol), Pd(dppf) _Cl (0.4 g, 0.5 mmol), KOAc (2.4 g, 25 mmol), and 1,4-dioxane (150 mL). The system was evacuated and then refilled with _N. The reaction mixture was heated at 100°C under microwave irradiation for 0.5 h. The mixture was then cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by flash chromatography eluting with 5:1 petroleum ether/ethyl acetate to afford 124a (0.57 g, 21%). LCMS: [M+H] ³³¹ .

Example 124b 4-Fluoro-2-(1-methyl-5-(5-methyl-1H-pyrazol-3-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 124b

A mixture of 124a (330 mg, 1 mmol), 2-bromo-4-fluoro-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 197d (434 mg, 1 mmol), _PdCl2 (dppf) (82 mg, 0.1 mmol), and 2.0 _{M Na2CO3} (2.0 equiv) in DME (10 mL) was heated at 120°C under microwave irradiation for 0.5 h. The solvent was evaporated in vacuo. The residue was purified by reverse phase Combi-flash chromatography to give the title compound (160 mg, 29%). LCMS: [M+H] ⁺ 559

A mixture of 124b (150 mg, 0.27 mmol) and LiOH (324 mg, 14 mmol) in isopropanol/THF (1:1, 10 mL) and water (3 mL) was stirred at 30°C for 2 h. The mixture was evaporated in vacuo and the residue was extracted with ethyl acetate (10 mL x 2). The combined ethyl acetate extracts were concentrated under reduced pressure and the residue was purified by preparative HPLC to afford 124 (60 mg, 43%). LCMS: [M+H] ⁺ 517. ^1H NMR (500MHz, DMSO) δ11.74(s,1H),8.00(m,2H),7.30(m,2H),7.16(dd,J=9.5,1H),6.52(s,1H),5.87(s,1H),4.87 (m,1H),4.33(m,2H),4.12(m,3H),3.87(m,1H),3.56(s,3H),2.59(m,2H),2.47(m,2H),2.45(s,3H),1.70(m,4H).

Example 125 3-[(5-ethyl-1H-pyrazol-3-yl)amino]-5-[2-(hydroxymethyl)-3-{1-oxo-1H,2H,3H,4H,6H,7H,8H,9H-pyrazino[1,2-a]indol-2-yl}phenyl]-1-methyl-1,2-dihydropyridin-2-one 125

Example 125a 5-Bromo-3-(5-ethyl-1H-pyrazol-3-ylamino)-1-methylpyridin-2(1H)-one 125a

To a 350-mL sealed tube equipped with a magnetic stir bar was added 3,5-dibromo-1-methyl-1H-pyridin-2-one (3.2 g, 0.012 mol), 5-ethyl-1H-pyrazol-3-amine (2.0 g, 0.018 mol), Pd ₂ (dba) ₃ (0.55 g, 0.60 mmol), 9,9-dimethyl-4,5-bis(diphenylphosphino)xanthene (0.49 g, 0.00084 mol), Cs ₂ CO ₃ (7.8 g, 0.024 mol), and 1,4-dioxane (80 mL). The reaction mixture was stirred at 105° C. for 16 h, then cooled to room temperature, partitioned between dichloromethane (50 mL) and water (30 mL), and the organic phase was extracted with dichloromethane (30 mL x 3). The combined organic phases were washed with water (30 mL x 2) and brine (20 mL x 1), dried (Na ₂ SO ₄ ), filtered through a Celite pad, and the filtrate was concentrated. Dichloromethane (20 mL) and ether (100 mL) were added to the crude product. The mixture was sonicated for 10 min, and the resulting precipitate was filtered to afford 5-bromo-3-(5-ethyl-1H-pyrazol-3-ylamino)-1-methylpyridin-2(1H)-one (125a) as a solid in a 42% yield (1.5 g).

Example 125b 2-(5-(5-ethyl-1H-pyrazol-3-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 125b

In a 10-mL glass vessel equipped with a magnetic stir bar, 125a (116 mg, 0.39 mmol), 2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 114a (200 mg, 0.43 mmol), and Pd( _PPh3 ) ₄ (30 mg, 0.0.26 mmol) were added to _2NNa2CO3 (2 _mL ) and 1,2-dimethoxyethane (5 mL). The vessel was sealed with a stopper and placed in a microwave cavity. The reaction mixture was stirred at 125°C for 7 minutes and then purified by flash chromatography (dichloromethane:methanol, 85:15) to afford 125b as a solid in 23% yield (50 mg).

To a 25-mL single-necked round-bottom flask equipped with a magnetic stir bar was added 125b (50 mg, 0.090 mmol), LiOH· _H₂O (20 mg, 0.83 mmol), THF (2 mL), isopropanol (2 mL), and water (2 mL). The reaction mixture was stirred at room temperature for 3 h, then partitioned between dichloromethane (5 mL) and water (5 mL), and the organic phase was extracted with dichloromethane (5 mL x 3). The combined organic phases were washed with water (5 mL x 2) and brine (5 mL x ₁ ), dried ( _Na₂SO₄ ), and then concentrated. The crude product was redissolved in dichloromethane (3 mL). To this solution was added hexanes (10 mL) and the resulting precipitate was filtered to give 50% yield (23 mg) of 2-(3-(5-(5-ethyl-1H-pyrazol-3-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one. MS (ESI ⁺ ) m/z 513.3 (M+H).

Example 126 2-(2-(hydroxymethyl)-3-(1-methyl-6-oxo-5-(4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 126

Example 126a 5-Bromo-1-methyl-3-(4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)pyridin-2(1H)-one 126a

To a 50 mL round-bottom flask equipped with a magnetic stirrer and a reflux condenser was added 3-(5-acetyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-5-bromo-1-methylpyridin-2(1H)-one 108d (250 mg, 0.7 mmol), aqueous NaOH solution (5N, 6 mL), and ethanol (6 mL). The mixture was stirred at reflux for 30 min. Thereafter, ethyl acetate (5 mL) and water (5 mL) were added. The separated aqueous layer was extracted with ethyl acetate (2 x 5 mL). The combined organic phases were washed with brine (10 mL), dried over sodium sulfate, filtered, and then concentrated under reduced pressure to afford crude 5-bromo-1-methyl-3-(4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)pyridin-2(1H)-one (126a) in a 91% yield (200 mg).

Example 126b 2-(1-methyl-6-oxo-5-(4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 126b

To a microwave tube equipped with a magnetic stirrer was added 126a (210 mg, 0.65 mmol), 2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 114a (330 mg, 0.7 mmol), DME (6 mL), and 1 M aqueous sodium carbonate solution (1.9 mL). After bubbling _N for 15 min, Pd(PPh ₃ ) ₄ (38 mg, 0.03 mmol) was added. The mixture was heated to 135° C. in a microwave for 15 min. Thereafter, ethyl acetate (10 mL) and water (10 mL) were added. The separated aqueous layer was extracted with ethyl acetate (2 x 10 mL). The combined organic phases were washed with brine (20 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure.The resulting residue was purified by column chromatography eluting with a gradient of _CH2Cl2 to ₉ :1 _CH2Cl2 :MeOH _to afford 126b in 36% yield (140 mg).

To a 25 mL round-bottom flask equipped with a magnetic stirrer was added 126b (140 mg, 0.24 mmol), lithium hydroxide (50 mg, 1.2 mmol), THF (1.2 mL), isopropanol (1.2 mL), and water (2.4 mL). The mixture was stirred at rt for 1 h. Ethyl acetate (5 mL) and water (5 mL) were then added. The separated aqueous layer was extracted with ethyl acetate (2 x 5 mL). The combined organic phases were washed with brine (10 mL), dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by column chromatography using a gradient of _CH₂Cl₂ to ₆₀ :35:5 _CH₂Cl₂ : _Et₂O :MeOH _to afford 126 in 33% yield (42 mg). MS (ESI+) m/z 540.3 (M+H).

Example 127 2-(5-fluoro-2-(hydroxymethyl)-3-(4-methyl-6-(4-morpholinophenylamino)-5-oxo-4,5-dihydropyrazin-2-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 127

Example 127a 4-Fluoro-2-(4-methyl-6-(4-morpholinophenylamino)-5-oxo-4,5-dihydropyrazin-2-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 127a

A mixture of 4-methyl-6-(4-morpholinophenylamino)-5-oxo-4,5-dihydropyrazin-2-ylboronic acid 122b (330 mg, 1 mmol), 2-bromo-4-fluoro-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 197d (434 mg, 1 mmol), _PdCl₂ (dppf) (82 mg, 0.1 mmol), and 2.0 _{M Na₂CO₃} (1 mL, 2.0 equiv) in DME (10 mL) was heated at 130° C. under microwave irradiation for 0.5 h. The solvent was evaporated in vacuo, and the residue was purified by reverse phase Combi-flash chromatography to afford 127a (200 mg, 45%). LCMS: [M+H] ^⁺ 641.

A mixture of 127a (200 mg, 0.31 mmol) and LiOH (372 mg, 16 mmol) in ^iPrOH /THF (1:1, 10 mL) and H ₂ O (3 mL) was stirred at 30° C. for 2 h. The mixture was then evaporated in vacuo, and the residue was extracted with ethyl acetate (10 mL x 2). The combined extracts were concentrated under reduced pressure, and the residue was purified by preparative HPLC to afford 127 (58 mg, 33%). LCMS:[M+H] ⁺ 599 ¹ H NMR(500MHz,DMSO)δ9.13(s,1H),7.81(m,2H),7.43(s,1H),7.34(m,2H),6.89(m,2H),6.52(s,1H),4. 85(s,1H),4.14(m,1H),3.72(m,3H),3.56(m,3H),3.07(m,4H),2.63(m,3H),2.47(m,2H),1.75(m,4H).

Example 128 2-(3-(5-(5-(3-hydroxyazetidin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 128

Example 128a 1-(6-nitropyridin-3-yl)azetidin-3-ol 128a

To a 50 mL round-bottom flask equipped with a magnetic stirrer and a reflux condenser were added 3-hydroxyazetidine·HCl (2 g, 18.4 mmol), 5-bromo-2-nitropyridine (2.1 g, 10.2 mmol), diisopropylethylamine (5.4 mL, 30.7 mmol), tetrabutylammonium iodide (5.7 g, 15.4 mmol), and N,N-dimethylacetamide (10 mL). The mixture was stirred at 120 ° C for 16 h. Thereafter, the mixture was cooled and ethyl acetate (25 mL) and water (25 mL) were added. The separated aqueous layer was extracted with ethyl acetate (2 x 10 mL). The combined organic phases were washed with brine (20 mL), dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by column chromatography using a gradient elution of 4:1 hexane:ethyl acetate-100 ethyl acetate to give 128a in 52% yield (1.9 g).

Example 128b 1-(6-aminopyridin-3-yl)azetidin-3-ol 128b

To a 500-mL Parr hydrogenation bottle was added 128a (1.9 g, 9.6 mmol), 10% palladium on carbon (50% wet, 570 mg dry weight), and ethanol (100 mL). The bottle was evacuated, filled with hydrogen to 50 psi, and shaken on a Parr hydrogenation apparatus for 24 h. The catalyst was removed by filtration through a pad _of Celite 521 and washed with 1:1 _CH₂Cl₂ :MeOH (500 mL). The resulting residue was purified by column chromatography using a gradient of 100% DCM to 100% 3:1 DCM:MeOH to afford 128b in 70% yield (1.1 g).

Example 128c 5-Bromo-3-(5-(3-hydroxyazetidin-1-yl)pyridin-2-ylamino)-1-methylpyridin-2(1H)-one 128c

To a sealed tube equipped with a magnetic stirrer was added 128b (375 mg, 2.3 mmol), 3,5-dibromo-1-methylpyridin-2(1H)-one (848 g, 3.2 mmol), cesium carbonate (1.7 g, 5 mmol), and 1,4-dioxane (24 mL). After nitrogen was bubbled through the solution for 30 min, Xantphos (160 mg, 0.3 mmol) and tris(dibenzylideneacetone)dipalladium(0) (150 mg, 0.2 mmol) were added, and the reaction mixture was heated at 100°C for 5 days. Thereafter, _H₂O (20 mL) and EtOAc (20 mL) were added. The aqueous layer was separated and extracted with ethyl acetate (2 x 20 mL). The combined organic extracts were washed with brine (50 mL) and then dried over sodium sulfate. The resulting residue was purified by column chromatography eluting with a gradient of _CH2Cl2 to ₉ :1 _CH2Cl2 :MeOH _to afford 128c in 39% yield (430 mg).

Example 128d 2-(5-(5-(3-hydroxyazetidin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 128d

To a microwave tube equipped with a magnetic stirrer was added 128c (220 mg, 0.6 mmol), 2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 114a (350 mg, 0.8 mmol), DME (7 mL), and 1 M aqueous sodium carbonate (1.9 mL). After bubbling _N for 15 min, Pd(PPh ₃ ) ₄ (36 mg, 0.03 mmol) was added. The mixture was heated to 135° C. in a microwave for 15 min. Thereafter, ethyl acetate (10 mL) and water (10 mL) were added. The separated aqueous layer was extracted with ethyl acetate (2 x 10 mL). The combined organic phases were washed with brine (20 mL), dried _over sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by column chromatography eluting with a gradient _{of CH2Cl2-60} :35:5 _CH2Cl2 :ether:MeOH to afford 128d in 30% yield (110 mg).

To a 25 mL round-bottom flask equipped with a magnetic stirrer was added 128d (110 mg, 0.2 mmol), lithium hydroxide (38 mg, 0.9 mmol), THF (0.9 mL), isopropanol (0.9 mL), and water (1.8 mL). The mixture was stirred at room temperature (rt) for 1 hr. Thereafter, ethyl acetate (5 mL) and water (5 mL) were added. The separated aqueous layer was extracted with ethyl acetate (2 x 5 mL). The combined organic phases were washed with brine (10 mL), dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by column chromatography using a gradient of _CH₂Cl₂ to ₆₀ :35:5 _CH₂Cl₂ : _diethyl ether:MeOH to afford 128d in 12% yield (12 mg). MS (ESI+) m/z 567.2 (M+H).

Example 129 2-(5-fluoro-2-(hydroxymethyl)-3-(4-methyl-5-oxo-6-(1,2,3,4-tetrahydroisoquinolin-6-ylamino)-4,5-dihydropyrazin-2-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 129

Example 129a tert- Butyl 6-(6-(2-(acetoxymethyl)-5-fluoro-3-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)phenyl)-4-methyl-3-oxo-3,4-dihydropyrazin-2-ylamino)-3,4-dihydroisoquinoline-2(1H)-carboxylate 129a

A mixture of 482 mg of 4-fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 210d and 435 mg of tert-butyl 6-(6-bromo-4-methyl-3-oxo-3,4-dihydropyrazin-2-ylamino)-3,4-dihydroisoquinoline _- 2(1H)-carboxylate 120a, _PdCl2 (dppf) (1110 mg, 0.015 mmol), 2M _Na2CO3 solution (3 mL) in DME (16 mL) was heated under microwave irradiation at 120°C for 0.5 h. The solvent was evaporated in vacuo and the residue was purified by reverse phase Combi flash chromatography to afford 129a as a yellow solid (362 mg, 51%). MS: [M+H] ⁺ 711.

Example 129b 4-Fluoro-2-(4-methyl-5-oxo-6-(1,2,3,4-tetrahydroisoquinolin-6-ylamino)-4,5-dihydropyrazin-2-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 129b

To a solution of 129a (360 mg 0.51 mmol) in DCM (30 mL) was added 3M HCl/dioxane (8 mL) at room temperature. The mixture was stirred at room temperature for 5 h. After the reaction was complete, the solvent was removed under reduced pressure to give 129b as a yellow solid (310 mg, 99%).

According to Example 149, 250 mg of 129b was converted to 129 as a white solid (98 mg, 42%). ^1H NMR(500MHz,MeOD)δ7.62(s,1H),7.55(dd,J＝7.0,1H),7.41(dd,J＝9.5,1H) ,7.38(s,1H),7.20(dd,J＝9.5,1H),7.04(d,J＝8.5,1H),6.71(s,1H),4.59( d,J＝12,1H),4.8(d,J＝11.5,1H),4.20(m,3H),4.00(m,3H),3.64(s,3H),3. 13(m,2H),2.86(m,2H),2.64(m,2H),2.54(m,2H),1.88(m,2H),1.78(m,2H).

Example 130 2-(2-(hydroxymethyl)-3-(1-methyl-5-(5-(1-methylpiperidin-4-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 130

Example 130a 5-(1-methyl-1,2,3,6-tetrahydropyridin-4-yl)-2-nitropyridine 130a

To a round-bottom flask equipped with a stir bar was added 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,2,3,6-tetrahydropyridine (1.50 g, 6.72 mmol), 5-bromo-2-nitropyridine (1.64 g, 8.07 mmol), Pd(PPh ₃ ) ₄ (388 mg, 0.336 mmol), aqueous Na ₂ CO ₃ solution (1.0 N, 20.2 mL, 20.2 mmol), and dioxane (60.6 mL). The reaction mixture was heated at 100° C. for 10 hr. CH ₂ Cl ₂ (200 mL) was added to the resulting mixture, which was then washed with water (30 mL x 3). _CH2Cl2 (200 _mL ) was added, and the resulting mixture was washed with water (30 mL x 3), brine (30 mL x 1), dried over _MgSO4 , filtered, and the solvent removed in vacuo. Silica gel column chromatography (MeOH:DCM = 5:95) gave 5-(1-methyl-1,2,3,6-tetrahydropyridin-4-yl)-2-nitropyridine (130a) as a yellow solid.

Example 130b 5-(1-methylpiperidin-4-yl)pyridin-2-amine 130b

In a hydrogenation bottle, 130a (1.25 g, 5.73 mmol), EtOH (100 mL), and 10% Pd/C (304 mmol, 0.286 mmol) were added. The mixture was hydrogenated at 55 psi for 2 hr, filtered through celite, and washed with MeOH (20 mL). The solvent was removed in vacuo to obtain 5-(1-methylpiperidin-4-yl)pyridin-2-amine 130b (1.13 g, 100%) as an off-white solid.

Example 130c 5-Bromo-1-methyl-3-(5-(1-methylpiperidin-4-yl)pyridin-2-ylamino)pyridin-2(1H)-one 130c

To a round-bottom flask equipped with a stir bar was added 130b (1.08 g, 5.65 mmol), 3,5-dibromo-1-methylpyridin-2(1H)-one (2.26 g, 8.47 mmol) _{, Pd(dba) (517 mg, 0.565 mmol), XantPhos (523 mg, 0.903 mmol), CsCO ( 6.07 g} , 18.6 mmol), and dioxane (28.3 mL). The reaction mixture was heated at 100°C overnight. _{CHCl (} 200 mL) was added to the resulting mixture, which was then washed with water (30 mL x 3). _{CHCl (} 200 mL) was added, and the resulting mixture was washed with water (30 mL x 3), brine (30 mL x 1), _dried over MgSO, filtered, and the solvent removed in vacuo. CH ₂ Cl ₂ /ether (1:2, 5 mL) was added, followed by sonication, and the precipitate was filtered and then dried to obtain compound 130c as a green solid, 784 mg (37%).

Example 130d 2-(1-methyl-5-(5-(1-methylpiperidin-4-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 130d

To a microwave tube equipped with a stir bar were added 130c (250 mg, 0.663 mmol), 2-(2-(hydroxymethyl)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 114a (338 mg, 0.729 mmol), Pd( _PPh3 ) ₄ (38.3 mg, 0.033 mmol), _{aqueous Na2CO3} solution (1.0 N, 2.19 mL, 2.19 mmol), and DME (2 mL). The mixture was reacted in a microwave at 135°C for 15 min. CH ₂ Cl ₂ (200 mL) was added, and the resulting mixture was washed with water (30 mL×3), brine (30 mL×1), dried over MgSO ₄ , filtered, and then the solvent was removed in vacuo . Silica gel column chromatography (MeOH:DCM=5:95) gave 130d.

To a round-bottom flask equipped with a stir bar was added 130d THF (1.25 mL), i-PrOH (1.25 mL), _H₂O (1.25 mL), and LiOH/ _H₂O (135 mg). The resulting mixture was stirred at room temperature for 1 hr. The solvent was removed in vacuo, and the resulting residue was added to _CH₂Cl₂ (200 _mL ). The solution was washed with water (30 mL x 3), brine (30 mL x 1), dried over _MgSO₄ , filtered, and the solvent was removed in vacuo. Column chromatography on silica gel (MeOH: _CH₂Cl₂ = _10:90 ) afforded 130 as an off-white solid, 39 mg. MS (ESI+) m/z 593.4 (M+H).

Example 131 5-(3-(6,6-dimethyl-3,4,6,7-tetrahydro-5H-cyclopenta[4,5]thieno[2,3-c]pyridin-1(2H)-yl)-2-(hydroxymethyl)phenyl)-1-methyl-3-(1-ethyl-1H-pyrazol-4-ylamino)pyrazin-2(1H)-one 131

Example 131a 2-(6,6-dimethyl-3,4,6,7-tetrahydro-5H-cyclopenta[4,5]thieno[2,3-c]pyridin-1(2H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 131a

To a 250-mL single-necked round-bottom flask equipped with a magnetic stirrer and a nitrogen inlet were added 105i (411 mg, 0.917 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-di(1,3,2-dioxaborolane) (698 mg, 2.75 mmol), potassium acetate (360 mg, 3.66 mmol) and 1,4-dioxane (15 mL). After nitrogen was bubbled through the resulting suspension for 30 min, Pd(dppf) _Cl2 · _CH2Cl2 (35 _mg , 0.047 mmol) was added. A reflux condenser was attached to the flask, and the reaction mixture was heated at 90°C for 14 h. Thereafter, more Pd(dppf) _Cl2 · _CH2Cl2 was added _. (70 mg, 0.094 mmol) was added and the reaction was stirred at 90°C for 4 h. Thereafter, the mixture was diluted with ethyl acetate (100 mL) and water (75 mL), and the layers were separated. The aqueous layer was extracted with ethyl acetate (50 mL), and the combined organic layers were washed with brine (50 mL) and then dried over sodium sulfate. The desiccant was removed by filtration. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, 70:30 hexane/ethyl acetate) to afford 131a in 82% yield (373 mg) as a non-crystalline off-white solid: ¹ H NMR (500 MHz, CDCl ₃ )δ7.81(dd,J＝7.5,1.5Hz,1H),7.41(t,J＝7.5Hz,1H),7.35(dd,J＝7.0,1.5 Hz,1H),5.51(d,J＝11.5Hz,1H),5.26(d,J＝11.5Hz,1H),4.03–3.98(m,1H), 3.77–3.72(m,1H),3.05–2.98(m,1H),2.82–2.76(m,1H),2.76(s,2H),2.52 (s,2H),1.99(s,3H),1.33(s,12H),1.27(s,3H),1.26(s,3H);MS(ESI+)m/z 496.2(M+H).

Example 131b 5-Bromo-3-(1-ethyl-1H-pyrazol-4-ylamino)-1-methylpyrazin-2(1H)-one 131b

According to Example 111b, 1-ethyl-1H-pyrazol-4-amine (500 mg, 4.50 mmol) and 3,5-dibromo-1-methylpyrazin-2(1H)-one (1.33 g, 4.95 mmol) were reacted to give 131b as an off-white solid in 75% yield (1.01 g): mp 237-239°C; ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 9.90 (s, 1H), 8.02 (s, 1H), 7.73 (s, 1H), 7.20 (s, 1H), 4.11 (q, 2H, J=7.5 Hz), 3.41 (s, 3H), 1.34 (t, 3H, J=7.3 Hz); MS (ESI+) m/z 298.0 (M+H).

To a 100-mL single-necked round-bottom flask equipped with a magnetic stirrer and a nitrogen inlet were added 131b (200 mg, 0.670 mmol), boronate ester 131a (365 mg, 0.737 mmol), sodium carbonate (184 mg, 1.73 mmol), DMF (2 mL), water (2 mL) and 1,4-dioxane (10 mL). After bubbling nitrogen through the resulting suspension for 30 min, tetrakis(triphenylphosphine)palladium(0) (134 mg, 0.116 mmol) was added. A reflux condenser was connected to the flask, and the reaction mixture was heated at 100 ° C for 14 h. Thereafter, it was diluted with a mixture of 90:10 dichloromethane/methanol (100 mL) and water (75 mL), and the layers were separated. The aqueous layer was extracted with 90:10 dichloromethane/methanol (2×50 mL), and the combined organic layers were washed with brine (100 mL) and then dried over sodium sulfate. The desiccant was removed by filtration. The filtrate was concentrated under reduced pressure, and the resulting residue was dissolved in a mixture of THF (5 mL), water (5 mL) and methanol (5 mL). Lithium hydroxide monohydrate (121 mg, 2.89 mmol) was added, and the mixture was stirred at room temperature for 2 h. Thereafter, the mixture was diluted with 90:10 dichloromethane/methanol (100 mL) and water (50 mL), and the layers were separated. The aqueous layer was extracted with 90:10 dichloromethane/methanol (2×75 mL), and the combined organic layers were washed with brine (100 mL) and then dried over sodium sulfate. The desiccant was removed by filtration. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, 90:10 dichloromethane/methanol) to obtain 131 in 26% yield (96 mg) as a non-crystalline yellow solid: mp 138–140°C; ¹ HNMR (500 MHz, DMSO-d ₆ )δ9.56(s,1H),8.16(s,1H),7.73(s,1H),7.56(dd,J＝7.5,1.0Hz,1H),7.46(t,J＝8.0Hz,1H ),7.34(dd,J＝7.5,1.0Hz,1H),7.31(s,1H),4.85–4.82(m,1H),4.56–4.53(m,1H),4.47–4.4 4(m,1H),4.08–4.02(m,3H),3.90–3.86(m,1H),3.52(s,3H),3.03–3.00(m,1H),2.92–2.87( m,1H),2.75(s,2H),2.54(d,J＝5.0Hz,2H),1.32(t,J＝7.5Hz,3H),1.23(s,6H); MS(ESI+)m/z 545.1(M+H).

Example 132 2-(2-(Hydroxymethyl)-3-(5-(5-(2-hydroxypropan-2-yl)-1-methyl-1H-pyrazol-3-ylamino)-1-methyl-6-oxo-1,6-dihydropyridazin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 132

Example 132a 2-(2-(acetoxymethyl)-3-(5-(5-(2-hydroxypropan-2-yl)-1-methyl-1H-pyrazol-3-ylamino)-1-methyl-6-oxo-1,6-dihydropyridazin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 132a

According to Example 119d, 119c (150 mg, 0.50 mmol), 113a (257 mg, 0.55 mmol), 1N Na ₂ CO ₃ (1.1 mL) and tetrakis(triphenylphosphine)palladium(0) (29 mg, 5 mol%) were reacted to give 132a (160 mg, 53% yield).

According to Example 132, 132a (160 mg, 0.27 mmol), 1N LiOH (1.3 mL), THF (2 mL) and isopropanol (2 mL) were reacted to give 132 as a white solid (118 mg, 78% yield). MS (ESI+) m/z 558.3 (M+H).

Example 133 2-(2-(Hydroxymethyl)-3-(6-oxo-5-(pyrimidin-4-ylamino)-1,6-dihydropyridazin-3-yl)phenyl)-3,4,5,6,7,8-hexahydrobenzothieno[2,3-c]pyridin-1(2H)-one 133

Example 133a 6-Chloro-4-(pyrimidin-4-ylamino)pyridazin-3(2H)-one 133a

To a 1-L three-necked round-bottom flask equipped with a mechanical stirrer, a nitrogen inlet, and a reflux condenser was added 4-bromo-6-chloropyridazin-3(2H)-one (7.30 g, 35.0 mmol), 2-aminopyrimidine (3.33 g, 35.0 mmol), cesium carbonate (25.0 g, 76.8 mmol), and 1,4-dioxane (345 mL). After nitrogen was bubbled through the resulting solution for 30 minutes, Xantphos (1.71 g, 2.96 mmol) and tris(dibenzylideneacetone)dipalladium(0) (1.60 g, 1.74 mmol) were added, and the reaction mixture was heated under reflux for 3 h. Thereafter, the reaction was cooled to room temperature and filtered. The filter cake was washed with dichloromethane (3×50 mL) and water (3×20 mL), then dried in a vacuum oven at 45° C. overnight to afford 133a as a tan solid (5.54 g, 71%): mp>300° C.; ¹ H NMR (500 MHz, DMSO-d ₆ ) 13.28 (br s, 1H), 9.90 (br s, 1H), 8.91 (s, 1H), 8.51 (d, 1H, J=6.0 Hz), 8.39 (s, 1H), 7.53 (dd, 1H, J=1.5, 6.0 Hz); MS (ESI+) m/z 224.1 (M+H).

According to Example 119d, 133a (145 mg, 0.650 mmol) and 111a (313 mg, 0.650 mmol) were used to obtain 133 as a pink solid in 18% yield (60 mg): mp 150-151 °C; ¹ H NMR (500 MHz, DMSO-d ₆ )δ13.30(s,1H),9.81(s,1H),8.79(s,1H),8.66(s,1H),8.47(s,1H),7.48(m,2H),7.43(m,2H),4.66(m,1H),4.44(m,1H ),4.38(m,1H),4.04(m,1H),3.85(m,1H),2.94(m,1H),2.87(m,1H),2.77(m,2H),2.53(m,1H),1.79(m,4H); MS(ESI+)m/z 501.1(M+H).

Example 134 2-(2-(Hydroxymethyl)-3-(5-(5-(2-hydroxypropan-2-yl)-1-methyl-1H-pyrazol-3-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 134

Example 134a 3-(5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)-1-methyl-1H-pyrazole-5-carboxylic acid methyl ester 134a

Following Example 119a, 3,5-dibromo-1-methylpyridin-2(1H)-one (2.0 g, 7.5 mmol) was converted to 134a.

Example 134b 5-Bromo-3-(5-(2-hydroxypropan-2-yl)-1-methyl-1H-pyrazol-3-ylamino)-1-methylpyridin-2(1H)-one 134b

According to Example 119b, 132a (330 mg, 0.97 mmol) and 3.0 M MeMgBr/ether (5.8 mmol, 1.9 mL) were reacted in THF (10 mL) to give 134b (270 mg, 82% yield).

Example 134c 2-(5-(5-(2-hydroxypropan-2-yl)-1-methyl-1H-pyrazol-3-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 134c

According to Example 119, 113b (120 mg, 0.35 mmol), 113a (180 mg, 0.39 mmol), 1N Na ₂ CO ₃ (0.8 mL) and palladium tetrakis (20 mg, 5 mol%) were reacted to give 134c (85 mg, 40% yield).

Following Example 119, 134c (80 mg, 0.13 mmol), 1N LiOH (0.7 mL), THF (1.5 mL), and isopropanol (1.5 mL) were reacted. The product was purified by column chromatography (silica gel, MeOH/CH ₂ Cl ₂ ) and then pulverized with EtOAc to afford 134 (18 mg, 25% yield). MS (ESI+) m/z 557.3 (M+H).

Example 135 2-(3-(5-(5-cyclopropyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 135

Example 135a 5-Cyclopropyl-2-nitro-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazine 135a

A mixture of 1-(2-bromoethyl)-5-(bromomethyl)-3-nitro-1H-pyrazole 101c (4 g, 12.9 mmol) and cyclopropylamine (7.35 g, 129 mmol) in THF (40 mL) was stirred at 30°C overnight. After the reaction was complete, the mixture was filtered and the solid was washed with THF (100 mL). The filtrate was concentrated under reduced pressure to afford 135a (2.68 g, 99%). MS: [M+H] ⁺ 209.

Example 135b 5-Cyclopropyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-amine 135b

A mixture of 5-cyclopropyl-2-nitro-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazine 135a (2.68 g, 12.9 mmol), Fe (3.6 g, 64.4 mmol), and _NH₄Cl (4.1 g, 77.4 mmol) in ethanol (30 mL) and water (5 mL) was heated under reflux for 2 h. After the reaction was complete, the mixture was filtered. The solid was washed with ethanol (150 mL). The filtrate was evaporated in vacuo, and the residue was extracted with methanol/dichloromethane (1/7). The combined extracts were dried _{over Na₂SO₄} and then evaporated. The residue was purified by reverse phase Combi-flash chromatography to afford 135b (1.8 g, 75%). MS: [M+H] ^⁺ 179.

Example 135c 5-Bromo-3-(5-cyclopropyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-1-methylpyridin-2(1H)-one 135c

A mixture of 5-cyclopropyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-amine 135b (1.39 g, 7.8 mmol), XantPhos (450 mg, 0.78 mmol), Pd ₂ dba ₃ (476 mg, 0.52 mmol), 3,5-dibromo-1-methylpyridin-2(1H)-one (1.72 g, 6.5 mmol), and Cs ₂ CO ₃ (6.3 mg, 19.5 mmol) in 1,4-dioxane (30 mL) was heated under reflux for 1 h. After completion of the reaction, the mixture was filtered and the solid washed with methanol (60 mL). The filtrate was evaporated in vacuo, and the residue was purified by reverse phase Combi flash chromatography to afford 135c (0.84 g, 30%). MS: [M+H] ⁺ 364.

Example 135d 2-(5-(5-cyclopropyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-4-fluoro-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 135d

A mixture of 4-fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 210d (105 mg, 0.26 mmol), 135c (100 mg, 0.28 mmol), PdCl₂(dppf) ₍ 29 mg, 0.039 mmol), _{K₃PO₄ (} 100 mg), and NaOAc (50 mg) in MeCN (10 mL) and water (3 mL) was heated at 110° C. for 2 h. The solvent was evaporated in vacuo. The residue was purified by reverse phase Combi-flash chromatography to afford 135d (100 mg, 60%). MS: [M+H] ^⁺ 640.

A mixture of 135d (100 mg, 0.16 mmol) and LiOH hydrate (100 mg, 2.3 mmol) in isopropanol (10 mL) and water (3 mL) was stirred at 30°C for 2 h. The mixture was evaporated in vacuo, and the residue was extracted with ethyl acetate (10 mL x 2). The combined extracts were concentrated under reduced pressure, and the residue was purified by preparative HPLC to afford 135 (40 mg, 42%). MS: [M+H] ⁺ 598. ¹ H NMR (500MHz, MeOD) δ7.89 (s, 1H), 7.26 (s, 1H), 7.20 (d, J = 9.0, 2H), 6.72 (s, 1H) ),5.88(s,1H),4.52-4.44(m,2H),4.22-4.18(m,3H),4.03-3.97(m,3H),3.81 (s,2H),3.69(s,3H),3.15-3.13(m,2H),2.67-2.61(m,2H),2.57-2.51(m,2H) ,1.96-1.87(m,3H),1.81-1.75(m,2H),0.62-0.58(m,2H),0.53-0.49(m,2H).

Example 136 5-(3-{5-[(5-cyclopropyl-1H-pyrazol-3-yl)amino]-1-methyl-6-oxo-1,6-dihydropyridin-3-yl}-5-fluoro-2-(hydroxymethyl)phenyl)-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-6-one 136

Example 136a 3-Cyclopropyl-3-oxopropionitrile 136a

To a solution of CH ₃ CN (0.34 mL, 6.58 mmol) in THF (3 mL) was added dropwise LDA (3.3 mL, 6.58 mmol) at -78°C under N ₂ protection. The reaction mixture was stirred at -78°C for 3 h. Ethyl cyclopropanecarboxylate (0.5 g, 4.38 mmol) in THF (2 mL) was then added, and the mixture was allowed to warm to room temperature over 1 h. Water (2 mL) was added, and the solvent was removed under reduced pressure. CH ₂ Cl ₂ (2 mL) was added, and the pH of the mixture was adjusted to 5 with 2N HCl. The product was then extracted with CH ₂ Cl ₂ (5 mL x 2), dried over Na ₂ SO ₄ , and concentrated to afford 136a as a yellow oil, which was used in the next step without further purification.

Example 136b 3-Cyclopropyl-1H-pyrazol-5-amine 136b

_To a solution of 136a (477 mg, 4.38 mmol) in MeOH (5 mL) was added _{N₂H₄.H₂O} (80%) (5 mL). The reaction mixture was heated at 75 _° C for 15 h. The _MeOH was removed under reduced pressure. The residue was extracted with _CH₂Cl₂ (2 x 8 _mL ), dried over _Na₂SO₄ , and concentrated. The residue was purified by flash column chromatography using 100:1 _CH₂Cl₂ /MeOH to afford 136b as _a yellow oil (37% over two steps). LCMS: (M+H) ¹²⁴ .

Example 136c tert-Butyl 5-amino-3-cyclopropyl-1H-pyrazole-1-carboxylate 136c

To a mixture _of 136b (0.25 g, 2 mmol) and _K₂CO₃ (0.828 g, 6 mmol) in THF (5 mL) was added (Boc) _₂O (0.436 g, 2 mmol) in THF (5 mL). The reaction mixture was stirred at room temperature for 15 h, then filtered and concentrated. The residue was purified by flash column chromatography using 6:1 petroleum ether/ethyl acetate as the eluent to afford 136c (240 mg, 54%) as a white solid. LCMS: (M-Boc) ¹²⁴ .

Example 136d 5-Bromo-3-(3-cyclopropyl-1H-pyrazol-5-ylamino)-1-methylpyridin-2(1H)-one 136d

To a 100-mL single-necked round-bottom flask equipped with a magnetic stirrer and a reflux condenser were added 1,4-dioxane (15 mL), 136c (455 mg, 1.95 mmol), 3,5-dibromo-1-methylpyridin-2(1H)-one (0.40 g, 1.5 mmol) and cesium carbonate (1.22 g, 3.75 mmol). After nitrogen was bubbled through the resulting mixture for 30 minutes, XantPhos (87 mg, 0.15 mmol) and tris(dibenzylideneacetone)dipalladium(0) (70 mg, 0.075 mmol) were added, and the reaction mixture was heated under reflux for 15 h. Thereafter, the reaction was cooled to room temperature, partitioned between ethyl acetate (30 mL) and water (30 mL), and then filtered. The aqueous layer was separated and extracted with ethyl acetate (2 × 50 mL). The organic layers were combined, washed with brine (50 mL), and then dried over Na ₂ SO ₄ . The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by flash column chromatography using 50:1 CH ₂ Cl ₂ /MeOH as the eluent to afford 136d (320 mg, 50%) as a yellow solid. LCMS: (M+H) ⁺ 309. ¹ H NMR (500 MHz, DMSO) δ 11.85 (s, 1H), 8.23 (s, 1H), 8.02 (d, J=2.5, 1H), 7.35 (d, J=2.5, 1H), 5.77 (d, J=2, 1H), 3.46 (s, 3H), 1.83 (m, 1H), 0.90 (m, 2H), 0.64 (m, 2H)

Example 136e (2-{5-[(5-cyclopropyl-1H-pyrazol-3-yl)amino]-1-methyl-6-oxopyridin-3-yl}-4-fluoro-6-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]tridec-1(9),2(7)-dien-5-yl}phenyl)methyl acetate 136e

To a 25 mL sealed vial was added (4-fluoro-6-{6-oxo-8-thia-5-azatricyclo[ ^7.4.0.02,7 ]trideca-1(9),2(7)-dien-5-yl}-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-methyl acetate 212b (580 mg, 1.16 mmol), 136d (300 mg, 0.97 mmol), _CH3COONa (160 mg, 1.94 mmol), _K3PO4 (410 mg, 1.94 mmol), _PdCl2 (dppf) (100 mg, 0.12 mmol), _CH3CN (12 mL) and _H2O (1 mL). _The mixture was heated at 110°C for 2 h. The reaction mixture was evaporated and the residue was purified by flash column chromatography eluting with 50:1 dichloromethane/methanol containing 0.5% triethylamine to afford 136e as a black solid (300 mg, 52%).

To a solution of 136e (300 mg, 0.50 mmol) in propan-2-ol (3 mL), tetrahydrofuran (3 mL), and water (3 mL) was added LiOH (1.0 g, 25 mmol). The mixture was stirred at 30°C for 2 h. 20 mL of _H₂O was then added and extracted with EA (30 _mL x 3). The combined organic layers were dried over _Na₂SO₄ and concentrated to afford a yellow solid, which was further purified by preparative HPLC to afford 136e (200 mg, 70%) as a white solid. LCMS: (M+H) ⁺ 560 ¹ H NMR (500MHz, DMSO) δ7.78 (s, 1H), 7.25 (d, J = 2.5, 1H), 7.21 (m, 2H), 5.79 (s, 1H), 4.48 (m, 2H), 4.15 (m, 1H), 4.00 ( m,1H),3.69(s,3H),3.07(m,1H),2.95(m,1H),2.85(m,2H),2.61(m,2H),1.89(m,5H),0.96(m,2H),0.73(m,2H).

Example 137 5-[5-fluoro-2-(hydroxymethyl)-3-[1-methyl-5-(5-(oxetane-3-yl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-6-oxopyridin-3-yl]phenyl]-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-diene-6-one 137

Example 137a 5-[5-fluoro-3-[1-methyl-5-(5-(oxetan-3-yl)-4,5,6,7-tetrahydropyrazolo-[1,5-a]pyrazin-2-ylamino)-6-oxopyridin-3-yl]benzyl acetate]-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-6-one 137a

To a 25 mL sealed tube was added (4-fluoro-2-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-5-yl}-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methyl acetate 212b (990 mg, 2 mmol), 5-bromo-1-methyl-3-(5-(oxetan-3-yl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)pyridin-2(1H)-one 252a (500 mg, 1.3 mmol), CH ₃ COONa (220 mg, 2.6 mmol), K ₃ PO ₄ (700 mg, 2.6 mmol) and PdCl ₂ (dppf) (110 mg, 0.13 mmol) was suspended in CH ₃ CN (25 mL) and H ₂ O (1 mL). The mixture was stirred at 110° C. for 2 h. The solvent was then evaporated and the residue was purified on a silica gel column using 20:1 CH ₂ Cl ₂ /methanol as eluent to afford 137a (300 mg, 35%) as a brown solid. MS: (M+H) ⁺ 673.

To a solution of 137a (270 mg, 0.4 mmol) in propan-2-ol (8 mL), tetrahydrofuran (8 mL), and water (1.5 mL) was added LiOH (964 mg, 40 mmol). The mixture was stirred at 30°C for 2 h. The mixture was then evaporated under reduced pressure, and the residue was purified by preparative HPLC to afford 137 as a yellow solid (84 mg, 33%). MS: (M+H) ⁺ 631. ^1H NMR(500MHz,MeOD)δ1.87(s,4H),2.52-2.56(d,2H),2.83-2.90(d,5H),3.01(s,1H),3.56-3.74(t,6H),3.96-4.0 1(t,4H),4.44-4.48(t,2H),4.63(s,2H),4.74(s,2H),5.87(s,1H),7.16-7.18(d,2H),7.25(s,1H),7.89(s,1H).

Example 138 2-(3-(5-(5-(4-ethylpiperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 138

Example 138a 1-Ethyl-4-(6-nitropyridin-3-yl)piperazine 138a

To a sealed tube equipped with a stir bar was added 5-bromo-2-nitropyridine (3.00 g, 14.78 mmol), 1-ethylpiperazine (5.06 g, 44.34 mmol), tetrabutylammonium iodide (273 mmol, 0.739 mmol), K ₂ CO ₃ (6.128 g, 44.34 mmol), and DMSO (30 mL). The tube was sealed and heated at 90° C. overnight. Water (200 mL) was added, and the precipitate was filtered to afford 1.24 g of 138a as a yellow solid.

Example 138b 5-(4-ethylpiperazin-1-yl)pyridin-2-amine 138b

In a hydrogenation bottle, 138a (2.59 g, 10.96 mmol), EtOH (100 mL), and 10% Pd/C (580 mg, 0.55 mmol) were added. The mixture was hydrogenated at 55 psi for 2 hr, then filtered through Celite and washed with MeOH (20 mL). The solvent was removed in vacuo to afford 138b (2.51 g, 82%) as a pink solid.

Example 138c 5-Bromo-3-(5-(4-ethylpiperazin-1-yl)pyridin-2-ylamino)-1-methylpyridin-2(1H)-one 138c

To a round-bottom flask equipped with a stir bar was added 138b (2.52 g, 12.22 mmol), 3,5-dibromo-1-methylpyridin-2(1H)-one (4.89 g, 18.32 mmol), _Pd (dba) ₍ 1.12 g, 1.22 mmol), XantPhos (1.13 mg, 1.96 mmol), _CsCO (13.14 g, 40.33 mmol), and _dioxane (50 mL). The reaction mixture was heated at 100°C overnight. _{CHCl (} 200 mL) was added to the resulting mixture, which was washed with water (30 mL x 3). _{CHCl (} 200 mL) was added, and the resulting mixture was washed with water (30 mL x 3), brine (30 mL x 1), dried over MgSO _, filtered, and the solvent removed in vacuo. CH ₂ Cl ₂ /diethyl ether (1:2, 5 mL) was added, followed by sonication, and the precipitate was filtered to give 138c as a yellow solid, 2.718 g (57%).

Example 138d 2-(5-(5-(4-ethylpiperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 138d

To a microwave tube equipped with a stir bar were added 138c (250 mg, 0.637 mmol), 2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 114a (325.5 mg, 0.701 mmol), Pd(PPh ₃ ) ₄ (36.8 mg, 0.0319 mmol), aqueous Na ₂ CO ₃ solution (1.0 N, 2.10 mL, 2.10 mmol), and DME (2.0 mL). The mixture was reacted in a microwave at 135° C. for 15 min. _CH₂Cl₂ (200 _mL ) was added, and the resulting mixture was washed with water (30 mL x 3), brine (30 mL x 1), dried over _MgSO₄ , filtered, and the solvent removed in vacuo. Silica gel column chromatography (MeOH: _CH₂Cl₂ = 5:95) afforded 2-(5-(5-(4- _{ethylpiperazin} -1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 138d.

To a round-bottom flask equipped with a stir bar was added 138d, THF (5.0 mL), i-PrOH (5.0 mL), _H₂O (5.0 mL), and LiOH/ _H₂O (200 mg). The resulting mixture was stirred at room temperature for 2 hr. All solvents were removed in vacuo, and the resulting residue was added to _CH₂Cl₂ (200 _mL ). The solution was washed with water (30 mL x 3), brine (30 mL x 1), dried over _MgSO₄ , filtered, and the solvent was removed in vacuo. Silica gel column chromatography (MeOH: _CH₂Cl₂ = _10:90 ) afforded 60 mg of 138 as a gray solid. MS (ESI+) m/z 608.3 (M+H).

Example 139 3-{[4-(3-hydroxy-3-methylazetidin-1-yl)phenyl]amino}-5-[2-(hydroxymethyl)-3-{1-oxo-1H,2H,3H,4H,6H,7H,8H,9H-pyrazino[1,2-a]indol-2-yl}phenyl]-1-methyl-1,2-dihydropyrazin-2-one 139

According to Example 301, 1-(4-aminophenyl)-3-methylazetidin-3-ol was converted to 99 mg of white solid 139. MS (ESI+) m/z 581 (M+H).

Example 140 2-(3-(6-(1-(2-hydroxyethyl)-1H-pyrazol-4-ylamino)-4-methyl-5-oxo-4,5-dihydropyrazin-2-yl)-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 140

Example 140a 5-Bromo-3-(1-(2-hydroxyethyl)-1H-pyrazol-4-ylamino)-1-methylpyrazin-2(1H)-one 140a

To a flask equipped with a magnetic stirrer was added 1-(2-(tert-butyldimethylsilyloxy)ethyl)-1H-pyrazol-4-amine 116b (1.7 g, 7.1 mmol), 3,5-dibromo-1-methylpyrazin-2(1H)-one (1.25 g, 4.7 mmol), and IPA (25 mL). The system was evacuated and then refilled with N ₂ . The reaction mixture was heated at 90° C. for 6 h. The mixture was then cooled to room temperature and concentrated under reduced pressure. The residue was purified by flash chromatography eluting with petroleum ether/ethyl acetate to afford 140a (1.7 g, 78%). LCMS: [M+H] ³¹⁴ .

Example 140b 2-(6-(1-(2-hydroxyethyl)-1H-pyrazol-4-ylamino)-4-methyl-5-oxo-4,5-dihydropyrazin-2-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 140b

A mixture of 140a (595 mg, 1.9 mmol), 2-( _{1 -} oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 114a (882 mg, 1.9 mmol), CH3COONa (309 mg, 3.8 mmol), _PdCl2 (dppf) (153 mg, 0.19 mmol) and _K3PO4 (1 g, 3.8 mmol) suspended in _CH3CN (30 mL) and _H2O (2 mL) was heated at 110°C for 15 h under argon atmosphere. After the reaction, CH ₃ CN was evaporated and the residue was purified by reverse phase Combi-flash chromatography eluting with 1:4 water/CH ₃ CN containing 0.3% NH ₄ HCO ₃ to give 140b as a brown solid (477 mg, 44%). LCMS: [M+H] ⁺ 572.

A mixture of 140b (410 mg, 0.72 mmol) and LiOH (372 mg, 16 mmol) in ^iPrOH /THF (1:1, 10 mL) and H ₂ O (3 mL) was stirred at 30° C. for 2 h. The mixture was evaporated in vacuo, and the residue was extracted with EtOAc (10 mL x 2). The combined extracts were concentrated under reduced pressure, and the residue was purified by preparative HPLC to give 140 (200 mg, 54%). LCMS:[M+H] ⁺ 530 ¹ H NMR(500MHz, CDCl3)δ7.71(s,1H),7.68(s,1H),7.49(s,1H),7.36(m,2H),7.27(m,1H),6.92(s,1H),6.78(s,1H),5.2 9(s,1H),4.43(d,J=12,1H),4.21(m,4H),4.04(m,2H),4.01(m,3H),3.63(s,3H),2.55(dt,J=14.5,4H),1.78(m,4H).

Example 141 2-(2-(Hydroxymethyl)-3-(1-methyl-5-(5-methyl-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 141

Example 141a 5-Methyl-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridin-2-amine 141a

A solution of 1-methyl-4-piperidone (11.3 g, 100 mmol) in 2-propanol (80 mL) was heated to 50°C. To this solution was added a solution of cyanamide (4.2 g, 100 mmol) in 2-propanol (25 mL) and sulfur powder (3.2 g). After adding a catalytic amount of pyrrolidine (1.3 mL), the resulting mixture was stirred at 50°C or above for 2 hours. The reaction mixture was allowed to cool to room temperature and then stirred overnight. The resulting mixture was cooled to 10°C or below in an ice-water bath and stirred at the same temperature for 1 hour. The precipitated crystals were collected by filtration and washed with 2-propanol (20 mL). The wet crystals were dried under reduced pressure to afford 141a (10 g, 60%). LCMS: [M+H] ⁺ 170 ¹ H NMR (500MHz, DMSO) δ6.70 (s, 2H), 3.31 (s, 2H), 2.61 (t, J = 5.5, 2H), 2.45 (m,, 2H), 2.33 (s, 3H).

Example 141b 5-Bromo-1-methyl-3-(5-methyl-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridin-2-ylamino)pyridin-2(1H)-one 141b

According to Example 110c, 3 g of 141a and 4 g of 3,5-dibromo-1-methylpyridin-2(1H)-one were reacted to give 141b as a yellow solid (2.8 g, 52%). LCMS: [M+H] ⁺ 357

Example 141c 2-(1-methyl-5-(5-methyl-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 141c

According to Example 147b, 232 mg of 114a and 178 mg of 141b were reacted to give 141c as a yellow solid (240 mg, 80%). LCMS: [M+H] ⁺ 613

According to Example 148, 240 mg of 141c was converted to 141 as a white solid (112 mg, 50%). LCMS: [M+H] ⁺ 514.

Example 142 2-(3-(5-(1,5-dimethyl-1H-pyrazol-3-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 142

Example 142a 5-Bromo-3-(1,5-dimethyl-1H-pyrazol-3-ylamino)-1-methylpyridin-2(1H)-one 142a

A solution of 5-bromo-1-methyl-3-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2(1H)-one 112a (2.8 g, 9.9 mmol) in anhydrous DMF (10 mL) was treated with a 60% suspension of NaH in mineral oil (0.5 g, 13 mmol) while stirring under nitrogen. After bubbling, the reaction was stirred for an additional 30 minutes. The reaction was then treated with MeI (0.98 g, 7 mmol) and stirred under nitrogen for an additional 2 hours. Water (50 mL) was slowly added, the mixture was filtered, and then concentrated. The residue was purified by flash chromatography eluting with petroleum ether/ethyl acetate to afford 142a (0.7 g, 24%), which was used without further purification. LCMS: (M+H)+297.

Example 142b 2-(5-(1,5-dimethyl-1H-pyrazol-3-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 142b

A mixture of 142a (510 mg, 1.7 mmol), 2-( _{1 -} oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 114a (882 mg, 1.9 mmol), CH3COONa (309 mg, 3.8 mmol), _PdCl2 (dppf) (153 mg, 0.19 mmol) and _K3PO4 (1 g, 3.8 mmol) suspended in _CH3CN (30 mL) and _H2O (2 mL) was heated at 110°C for 15 h under argon atmosphere. It was then evaporated and the residue was purified by reverse phase Combi-flash chromatography using 1:4 water/CH ₃ CN containing 0.3% NH ₄ HCO ₃ as eluent to afford 142b as a brown solid (200 mg, 21%). LCMS: [M+H] ⁺ 555.

A mixture of 142b (210 mg, 0.38 mmol) and LiOH (372 mg, 16 mmol) in ^iPrOH /THF (1:1, 10 mL) and water (3 mL) was stirred at 30°C for 2 h. The mixture was evaporated in vacuo, and the residue was extracted with EtOAc (10 mL x 2). The combined extracts were concentrated under reduced pressure, and the residue was purified on preparative HPLC to afford 142 (95 mg, 50%). LCMS: [M+H] ⁺ 530. ¹ H NMR (500MHz, CDCl3) δ7.88(s,1H),7.43(m,2H),7.38(s,1H),7.30(m,1H),7.21(m,1H),6.84(s,1H),5.71(s,1H),4.58(d,J＝ 11.5,1H),4.38(d,J＝11.5,,1H),4.13(m,3H),3.94(m,1H),3.67(s,3H),3.64(s,3H),2.57(m,4H),2.21(s,3H),1.78(m,4H).

Example 143 2-(5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-(5-(oxetan-3-yl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 143

Example 143a 1-methyl-5-(5-(oxetan-3-yl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-ylboronic acid 143a

To a solution of 5-bromo-1-methyl-3-(5-(oxetan-3-yl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)pyridin-2(1H)-one 252a (1 g, 2.64 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bis(1,3,2-dioxaborolane) (2 g, 7.92 mmol) in dioxane (40 mL) was added _PdCl2 (dppf) (215 mg, 0.26 mmol) and _CH3COOK (776 mg, 7.92 mmol). The mixture was stirred at 100°C under argon atmosphere for 6 h. After the reaction, the mixture was filtered and then evaporated in vacuo. The residue was purified by reverse phase Combi flash chromatography eluting with 1:3 water/CH ₃ CN containing 0.3% NH ₄ HCO ₃ to afford 143a as a white solid (300 mg, 33%). MS: (M+H) ⁺ 346.

Example 143b 4-Fluoro-2-(1-methyl-5-(5-(oxetan-3-yl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 143b

To a 25 mL vial was added 143a (238 mg, 0.7 mmol), 2-bromo-4-fluoro-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 197d ( ₃₀₀ mg, 0.7 mmol), _Na₂CO₃ (147 mg, 1.4 mmol), and _PdCl₂ (dppf) (56 mg, 0.07 mmol) suspended in DME (15 mL) and _H₂O (1 mL). The resulting orange mixture was heated in a Biotage microwave reactor at 130° C. for 30 minutes. After the reaction, the residue was purified by reverse phase Combi-flash chromatography eluting with ₁ :7 water/ _CH₃CN containing 0.3% _NH₄HCO₃ to afford 143b (150 mg, 33%) as a brown solid. MS:(M+H) ⁺ 656.

To a solution of 4-fluoro-2-(1-methyl-5-(5-(oxetan-3-yl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 143b (120 mg, 0.18 mmol) in propan-2-ol (5 mL), tetrahydrofuran (5 mL) and water (1.5 mL) was added LiOH (440 mg, 18 mmol). The mixture was stirred at 30° C. for 2 h. The reaction mixture was then evaporated and the residue was purified by preparative HPLC to give 143 as a yellow solid (50 mg, 45%). MS: (M+H) ⁺ 614. ¹ H NMR (500MHz, MeOD) δ1.79 (s, 2H), 1.90 (s, 2H), 2.54-2.56 (t, J = 6.5Hz, 2H), 2.63-2. 67(m,2H),2.86-2.88(t,J＝6Hz,2H),3.59(s,2H),3.70(s,3H),3.76-3.79(m,1H),4. 00-4.07(m,3H),4.21(s,3H),4.48-4.53(m,2H),4.64-4.67(t,J＝6.5Hz,2H),4.75- 4.78(t,J=7Hz,2H),5.90(s,1H),6.72(s,1H),7.20-7.27(m,3H),7.91-7.92(d,1H).

Example 144 5-[5-Fluoro-2-(hydroxymethyl)-3-[1-methyl-5-(5-cyclopropyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-6-oxopyridin-3-yl]phenyl]-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-diene-6-one 144

Example 144a 3-(5-cyclopropyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one 144a

A mixture of 5-bromo-3-(5-cyclopropyl-4,5,6,7-tetrahydropyrazolo[1,5-a]-pyrazin-2-ylamino)-1-methylpyridin-2(1H)-one 135c (0.9 g, 2.48 mmol), bis(pinacolato)diborate (1.26 g, 4.96 mmol), _PdCl₂ (dppf) (272 mg, 0.37 mmol), and KOAc (486 mg, 4.96 mmol) in 1,4-dioxane (40 mL) was heated under reflux for 15 h. After completion of the reaction, the mixture was filtered and washed with ethyl acetate (100 mL). The filtrate was evaporated in vacuo, and the residue was purified on a silica gel column to afford 144a (407 mg, 40%). MS: [M+H] ^⁺ 412.

Example 144b 5-[5-fluoro-3-[1-methyl-5-(5-cyclopropyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-6-oxopyridin-3-yl]benzyl acetate]-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-diene-6-one 144b

A mixture of 144a (300 mg, 0.73 mmol), (2-bromo-4-fluoro-6-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-5-yl}phenyl)methyl acetate (212a) (297 mg, 0.66 mmol), PdCl ₂ (dppf) (73 mg, 0.1 mmol), and 2M Na ₂ CO ₃ solution (2 mL) in DME (8 mL) was heated at 120° C. for 0.5 h under microwave irradiation. The solvent was evaporated in vacuo, and the residue was purified by reverse phase Combi flash chromatography to give 144b (173 mg, 40%). MS: [M+H] ⁺ 657.

A mixture of 144b (170 mg, 0.26 mmol) and LiOH hydrate (104 mg, 2.6 mmol) in ⁱ PrOH (15 mL) and H ₂ O (3 mL) was stirred at 30° C. for 2 h. The mixture was evaporated in vacuo, and the residue was extracted with EtOAc (20 mL×2). The combined extracts were concentrated under reduced pressure. The residue was purified by preparative HPLC to give 144 (50 mg, 31%). MS: [M+H] ⁺ 615. ¹ H NMR(500MHz,MEOD)δ7.89(d,J＝2.0,1H),7.26(d,J＝2.0,1H),7.22-7.18(m,2H),5 .89(s,1H),4.52-4.46(m,2H),4.17-4.11(m,1H),4.02-3.97(m,3H),3.82(s,2H), 3.69(s,3H),3.16-3.14(m,2H),3.10-3.03(m,1H),2.96-2.90(m,1H),2.87-2.85( m,2H),2.65-2.53(m,2H),1.97-1.85(m,5H),0.62-0.58(m,2H),0.53-0.50(m,2H)

Example 145 2-(2-(Hydroxymethyl)-3-(4-methyl-5-oxo-6-(4-(piperidin-4-yl)phenylamino)-4,5-dihydropyrazin-2-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 145

Example 145a tert-Butyl 4-(4-(6-bromo-4-methyl-3-oxo-3,4-dihydropyrazin-2-ylamino)phenyl)piperidine-1-carboxylate 145a

Compound 145a was synthesized using the same method as Example 112a, except that tert-butyl 4-(4-aminophenyl)piperidine-1-carboxylate (0.83 g, 3.0 mmol), 3,5-dibromo-1-methylpyrazin-2(1H)-one (0.88 g, 3.3 mmol), cesium carbonate (1.27 g, 3.9 mmol), tris(dibenzylideneacetone)dipalladium(0) (0.275 g, 0.3 mmol), Xantphos (0.26 g, 0.45 mmol), and 1,4-dioxane (30 mL) were used. The reaction mixture was heated at 100° C. overnight. Workup and purification by flash column chromatography (silica gel, ethyl acetate/hexanes) afforded tert-butyl 4-(4-(6-bromo-4-methyl-3-oxo-3,4-dihydropyrazin-2-ylamino)phenyl)-piperidine-1-carboxylate as a solid 145a in 80% yield (1.1 g): MS (ESI+) m/z 465.0 (M+H).

Example 145b

Compound mixture 145b was synthesized by the same method as Example 121b, except that 2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 114a (162 mg, 0.35 mmol), tert-butyl 4-(4-(6-bromo-4-methyl-3-oxo-3,4-dihydropyrazin-2-ylamino)phenyl)piperidine-1-carboxylate 145a (135 mg, 0.3 mmol), 1 M sodium carbonate solution (1.2 mL, 1.2 mmol), tetrakis(triphenylphosphine)palladium(0) (18 mg, 0.015 mmol) and 1,2-dimethoxyethane (3 mL) were used. Workup and flash column chromatography (silica gel, ethyl acetate/hexanes) afforded 145b as a yellow oil (120 mg).

Example 145c

A mixture of compounds was synthesized using the same method as Example 121c except that 145b (120 mg), trifluoroacetic acid (0.5 mL, 6.5 mmol) and dichloromethane (7 mL) were used. Workup and concentration to dryness afforded compound 145c as a yellow oil, which was used in the next step without purification.

Compound 145 was synthesized using the same procedure as Example 121, except that a mixture of THF (1 mL), water (0.5 mL), and isopropanol (1 mL), compound 145c, and lithium hydroxide monohydrate (50 mg, 1.30 mmol) was used. Workup and flash column chromatography (silica gel, dichloromethane/methanol containing 20% TEA) gave a yellow solid (68 mg), which was eluted from a basic aluminum column (ethyl acetate) to give 145 as a white solid in 15% yield (3 steps, 25 mg): MS (ESI+) m/z 579.4 (M+H).

Example 146 2-(2-(Hydroxymethyl)-3-(1-methyl-5-(5-methyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 146

Example 146a 5-Bromo-1-methyl-3-(5-methyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)pyridin-2(1H)-one 146a

A suspension of 5-methyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-amine 101e (1 g, 6.6 mmol), 3,5-dibromo-1-methylpyridin-2(1H)-one (1.7 g, 6.6 mmol), XantPhos (380 mg, 0.66 mmol), Pd ₂ (dba) ₃ (602 mg, 0.66 mmol), and Cs ₂ CO ₃ (4 g, 13.2 mmol) in dioxane (30 mL) was heated in a sealed tube under nitrogen at 120° C. for 2 h. After the reaction, the solvent was filtered, and the filtrate was evaporated in vacuo to afford a yellow solid. The yellow solid was washed with EtOAc (10 mL x 3) to afford 146a (1 g, 45%) as a yellow solid, which was used without further purification. MS: (M+H) ⁺ 338.

Example 146b 2-(1-methyl-5-(5-methyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 146b

To a 25 mL vial were added 5-bromo-1-methyl-3-(5-methyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)pyridin-2(1H)-one 145a (500 mg, 1.48 mmol), 2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 114a (664 mg, 1.48 mmol), CH ₃ COONa (243 mg, 2.96 mmol), PdCl ₂ (dppf) (121 mg, 0.148 mmol) and K ₃ PO ₄ (790 mg, 2.96 mmol) and suspended in CH _{3 .} CN (50 mL) and H ₂ O (3 mL). The mixture was then heated at 110° C. for 12 h under an argon atmosphere. After the reaction, the mixture was evaporated and the residue was purified by reverse-phase Combi-Flash chromatography, eluting with 1:5 water/CH ₃ CN containing 0.3% NH ₄ HCO ₃ to afford 146b (200 mg, 23%) as a brown solid. MS: (M+H) ⁺ 596.

To a solution of 2-(1-methyl-5-(5-methyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 146b (180 mg, 0.3 mmol) in propan-2-ol (9 mL), tetrahydrofuran (9 mL), and water (3 mL) was added LiOH (726 mg, 30 mmol). The mixture was stirred at 30° C. for 2 h. After the reaction, the mixture was evaporated, and the residue was purified by preparative HPLC to give 146 as a white solid (67 mg, 41%). MS: (M+H) ⁺ 554. ^1H NMR(500MHz,MeOD)δ1.80(s,2H),1.90(s,2H),2.49(s,3H),2.54-2.57(t,2H), 2.63-2.67(m,2H),2.93-2.96(t,J＝5.5Hz,2H),3.64(s,2H),3.70(s,3H),4.02- 4.06(m,3H),4.18-4.23(m,3H),4.49-4.57(m,2H),5.89(s,1H),6.72(s,1H),7. 23(s,1H),7.36-7.38(d,1H),7.41-7.42(d,1H),7.49-7.52(t,1H),7.1(s,1H).

Example 147 2-(3-(5-(6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridazin-3-yl)-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 147

Example 147a 6-Chloro-4-(6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-2-ylamino)-2-methylpyridazin-3(2H)-one 147a

A mixture of 6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-2-amine 110b (0.8 g, 5.76 mmol), xantophos (360 mg, 0.623 mmol), Pd ₂ dba ₃ (384 mg, 0.42 mmol), 4-bromo-6-chloro-2-methylpyridazin-3(2H)-one 103e (1.28 g, 5.76 mmol), and Cs ₂ CO ₃ (5.05 g, 17.3 mmol) in 1,4-dioxane (40 mL) was heated at reflux for 2 h. After completion of the reaction, the mixture was filtered and washed with MeOH (60 mL). The filtrate was evaporated in vacuo. The residue was purified by reverse phase Combi-flash chromatography to afford 147a (1.3 g, 81%). MS: [M+H] ⁺ 282.

Example 147b 2-(5-(6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridazin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 147b

A mixture of 6-chloro-4-(6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-2-ylamino)-2-methylpyridazin-3(2H)-one 147a (400 mg, 1.42 mmol), 2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 114a (660 mg, 1.42 mmol), _PdCl2 (dppf) (155 mg, 0.21 mmol), _{K3PO4 (} 150 mg) and NaOAc (50 mg) in MeCN (20 mL) and _H2O (4 mL) was heated in a sealed tube at 110°C for 2 h. The solvent was evaporated in vacuo and the residue was purified by reverse phase Combi-flash chromatography to afford 147b (273 mg, 33%). MS: [M+H] ⁺ 584.

A mixture of 2-(5-(6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridazin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 147b (410 mg, 0.7 mmol) and LiOH hydrate (590 mg, 14 mmol) in ⁱ PrOH (20 mL) and H ₂ O (4 mL) was stirred at 30° C. for 2 h. The mixture was evaporated in vacuo, and the residue was extracted with ethyl acetate (20 mL×2). The combined extracts were concentrated under reduced pressure. The residue was purified by preparative HPLC to give 147 (120 mg, 32%). MS: [M+H] ⁺ 542. ¹ H NMR(500MHz,DMSO)δ9.30(s,1H),7.90(s,1H),7.49-7.46(m,1H),7.41-7.37(m,2 H),6.51(s,1H),6.04(s,1H),4.74(s,2H),4.62-4.60(m,1H),4.48-4.45(m,1H),4 .39-4.35(m,1H),4.18-4.06(m,3H),4.04-3.95(m,4H),3.90-3.85(m,1H),3.75(s ,3H),2.60-2.56(m,2H),2.51-2.45(m,2H),1.82-1.74(m,2H),1.73-1.64(m,2H).

Example 148 2-(3-(5-(5-fluoropyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 148

Example 148a 5-Bromo-3-(5-fluoropyridin-2-ylamino)-1-methylpyridin-2(1H)-one 148a

To a 100-mL single-necked round-bottom flask equipped with a magnetic stirrer and a reflux condenser was added 1,4-dioxane (50 mL), 5-fluoropyridin-2-amine (0.67 g, 6 mmol), 3,5-dibromo-1-methylpyridin-2(1H)-one (1.34 g, 5 mmol) and cesium carbonate (4.89 g, 15 mmol). After nitrogen was bubbled through the resulting solution for 30 minutes, XantPhos (576 mg, 1 mmol) and tris(dibenzylideneacetone)dipalladium(0) (460 mg, 0.5 mmol) were added, and the reaction mixture was heated under reflux for 15 h. The reaction was then cooled to room temperature, partitioned between ethyl acetate (100 mL) and water (100 mL), and then filtered. The aqueous layer was separated and extracted with ethyl acetate (50 mL × 2). The organic layers were combined, washed with brine (50 mL), and then dried over sodium sulfate. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was washed with acetonitrile (30 mL) and then filtered to give 148a (900 mg, 61%). MS: [M+H] ⁺ 298.

Example 148b 2-(5-(5-fluoropyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 148b

To a sealed tube was added a mixture of 2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 114a (232 mg, 0.5 mmol), 148a (148 mg, 0.50 mmol), Pd(dppf) _Cl₂ (25 mg, 0.03 _mmol ), _{K₃PO₄.3H₂O (} 266 mg, 1.0 mmol), and NaOAc (82 mg, 1.0 mmol) in _CH₃CN (18 mL). The system was evacuated and refilled with _N₂ . The reaction mixture was heated at 110°C for 2 h, then cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure and the resulting residue was purified by flash chromatography using 30:1 DCM/MeOH as eluent to afford 148b as a yellow solid (200 mg, 72%). MS: [M+H] ⁺ 556.

To a solution of 148b (200 mg, 0.36 mmol) in THF/iPA/ _H₂O (6 mL/6 mL/2 mL) was added LiOH (87 mg, 3.6 mmol) at room temperature with stirring. The mixture was stirred for 0.5 h. 20 mL of _H₂O was then added, and the mixture was extracted with ethyl acetate (30 mL x 3). The combined organic layers were dried over _{Na₂SO₄ and} concentrated to afford a yellow solid, which was further purified by preparative HPLC to afford 148 (110 mg, 59%) as a white solid. LCMS: [M+H] ^⁺ 514. ¹ H NMR (500MHz, DMSO) δ8.77(s,1H),8.62(d,J＝2.0,1H),8.16(d,J＝2.5,1H),7.59(m,,1H),7.47(t,J＝7.5, ¹ H),7.39-7.14(m,2H),7.34(m,2H),6.52(s,1H),4.85(s,1H),4.35(d,J＝4.0,2H),4.17(m,2H),4 .10(m,1H),3.90(m,1H),3.61(s,3H),2.60(m,2H),2.48(m,2H),1.80(t,J=5.5,2H),1.70(m,2H).

Example 149 2-(3-(5-(6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 149

Example 149a 2-(5-(6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-4-fluoro-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 149a

A mixture of 2-(acetoxymethyl)-5-fluoro-3-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)phenylboronic acid 210d (150 mg, 0.38 mmol), 5-bromo-3-(6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-2-ylamino)-1-methylpyridin-2(1H)-one 110c (122 mg, 0.38 mmol), _PdCl2 (dppf) (41 mg, 0.056 mmol), _{K3PO4 (} 100 mg) and NaOAc (50 mg) in MeCN (10 mL) and _H2O (3 mL) was heated in a sealed tube at 110°C for 2 h. The solvent was evaporated in vacuo and the residue was purified by reverse phase Combi flash chromatography to give 149a (120 mg, 53%). MS: [M+H] ⁺ 601.

A mixture of 149a (120 mg, 0.2 mmol) and LiOH hydrate (84 mg, 2 mmol) in ⁱ PrOH (10 mL) and H ₂ O (3 mL) was stirred at 30° C. for 2 h. The mixture was evaporated in vacuo, and the residue was extracted with ethyl acetate (10 mL x 2). The combined extracts were concentrated under reduced pressure, and the residue was purified by preparative HPLC to afford 149 (60 mg, 54%). MS: [M+H] ⁺ 559. ¹ H NMR(500MHz,MeOD)δ7.82(s,1H),7.16(s,1H),7.10(d,J＝9.0,2H),6.62(s,1H),5.77(s,1H),4.67(s,2H),4.43-4.35(m,2H),4.11-4.07 (m,3H),4.00-3.99(m,2H),3.93-3.90(m,3H),3.60(s,3H),2.55-2.52(m,2H),2.46-2.43(m,2H),1.82-1.78(m,2H),1.71-1.67(m,2H).

Example 150 5-[5-Fluoro-2-(hydroxymethyl)-3-(1-methyl-6-oxo-5-{4H,6H,7H-pyrazolo[3,2-c][1,4]oxazin-2-ylamino}pyridin-3-yl)phenyl]-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-diene-6-one 150

Example 150a 3-(6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-2-ylamino)-1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one 150a

A mixture of 5-bromo-3-(6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-2-ylamino)-1-methylpyridin-2(1H)-one 110c (1.3 g, 4.0 mmol), bis(pinacolato)diborate (2.03 g, 8 mmol), _PdCl₂ (dppf) (439 mg, 0.6 mmol), and KOAc (784 mg, 8.0 mmol) in 1,4-dioxane (60 mL) was heated under reflux for 15 h. After completion of the reaction, the mixture was filtered and washed with EtOAc (100 mL). The filtrate was evaporated in vacuo, and the residue was purified on a silica gel column to afford 150a (446 mg, 30%). MS: [M+H] ^⁺ 373.

Example 150b [4-Fluoro-2-(1-methyl-6-oxo-5-{4H,6H,7H-pyrazolo[3,2-c][1,4]oxazin-2-ylamino}pyridin-3-yl)-6-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-5-yl}phenyl]methyl acetate 150b

A mixture of 150a (260 mg, 0.70 mmol), (2-bromo-4-fluoro-6-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-5-yl}phenyl)methyl acetate 218a (225 mg, 0.50 mmol), PdCl ₂ (dppf) (55 mg, 0.075 mmol), and 2M Na ₂ CO ₃ solution (1.5 mL) in DME (8 mL) was heated at 120° C. for 0.5 h under microwave irradiation. The solvent was evaporated in vacuo, and the residue was purified by reverse phase Combi flash chromatography to afford 150b (154 mg, 50%). MS: [M+H] ⁺ 618.

A mixture of 150b (150 mg, 0.24 mmol) and LiOH hydrate (96 mg, 2.4 mmol) in ^iPrOH (15 mL) and H ₂ O (3 mL) was stirred at 30°C for 2 h. The mixture was evaporated in vacuo, and the residue was extracted with ethyl acetate (20 mL x 2). The combined extracts were concentrated under reduced pressure. The residue was purified by preparative HPLC to afford 150 (70 mg, 51%). MS: [M+H] ⁺ 576. ¹ H NMR(500MHz,DMSO)δ8.22(s,1H),8.00(s,1H),7.32-7.29(m,2H),7.17-7.15(m,1H),5.93(s,1H),4.86-4.85(m,1H),4. 71(s,2H),4.36-4.28(m,2H),4.10-3.84(m,7H),3.57(s,3H),3.02-2.92(m,1H),2.90-2.76(m,3H),1.86-1.74(m,4H).

Example 151 2-(3-(5-(5-(azetidin-3-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 151

Example 151a 2-(5-(5-(azetidin-3-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 151a

In a 44-mL sealed tube equipped with a magnetic stir bar, 3-(5-(azetidin-3-yl)pyridin-2-ylamino)-5-bromo-1-methylpyridin-2(1H)-one 155n (60 mg, 0.18 mmol), 2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 114a (110 mg, 0.23 mmol), Pd( _PPh3 ) ₄ (21 mg, 0.0.18 mmol) were added to 2N _Na2CO3 (3 _mL ), DME (2 mL) and dioxane (3 mL). After the reaction mixture was stirred at 100 ° C for 14 h, it was distributed between dichloromethane (5 mL) and water (5 mL), and the organic phase was extracted with dichloromethane (5 mL x 3). The combined organic phase was washed with water (5 mL x 2) and brine (5 mL x 1), dried (Na ₂ SO ₄ ), and then concentrated. The crude product was purified by flash chromatography (dichloromethane: MeOH, 85: 15) to obtain 40% (40 mg) of solid 2- (5- (5- (azetidine -3- bases) pyridin-2-ylamino) -1-methyl -6- oxo -1,6- dihydropyridine -3- bases) -6- (1- oxo -3,4,6,7,8,9- hexahydropyrazino [1,2-a] indole -2 (1H) - base) benzyl acetate 151a.

To a 25-mL single-necked round-bottom flask equipped with a magnetic stir bar was added 151a (40 mg, 0.068 mmol), LiOH· _H₂O (20 mg, 0.48 mmol), THF (2 mL), i-PrOH (2 mL), and water (2 mL). The reaction mixture was stirred at room temperature for 3 h, then partitioned between dichloromethane (5 mL) and water (5 mL), and the organic phase was extracted with dichloromethane (5 mL x 3). The combined organic phases were washed with water (5 mL x 2) and brine (5 mL x 1), dried ( _Na₂SO₄ ), and then concentrated. The crude product was redissolved in dichloromethane (3 mL). Hexane (10 mL) was _added to this solution, and the resulting precipitate was filtered to provide 151 in an 88% yield (33 mg); MS ( ^ESI⁺ ) m/z 551.3 (M+H).

Example 152 2-(2-(Hydroxymethyl)-3-(1-methyl-6-oxo-5-(pyrimidin-4-ylamino)-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]benzimidazol-1-one 152

Example 152a N-(2-Hydroxyethyl)-1H-benzo[d]imidazole-2-carboxamide 152a

A 100-mL single-neck round-bottom flask equipped with a magnetic stirrer and a reflux condenser was purged with nitrogen and ^1H -benzo[d]imidazole-2-carboxylic acid (1.50 g, 9.26 mmol) and thionyl chloride (10 mL) were added. After heating under reflux for 16 h, the suspension was cooled to room temperature and filtered. The filter cake was washed with toluene (10 mL) and then dried under vacuum at room temperature for 5 h. The resulting solid was added to a 100-mL single-neck round-bottom flask equipped with a magnetic stirrer and a reflux condenser, followed by the addition of chloroform (10 mL) and 2-hydroxyethylamine (559 mg, 9.17 mmol). After stirring at reflux for 16 h, the reaction mixture was concentrated under reduced pressure and the resulting residue was powdered with water (20 mL) and then dried in a vacuum oven at 45° C. to give 152a as a white solid in 73% yield (1.41 g): mp 194-195° C.; ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 13.13 (s, 1H), 8.74 (s, 1H), 7.71-7.29 (m, 4H), 4.80 (s, 1H), 3.55 (dd, 2H, J=10.5, 5.5 Hz), 3.38 (dd, 2H, J=10.5, 5.5 Hz); MS (APCI+) m/z 206.6 (M+H).

Example 152b 3,4-dihydropyrazino[1,2-a]benzimidazol-1-one 152b

A 100-mL single-necked round-bottom flask equipped with a magnetic stirrer and a reflux condenser was purged with nitrogen, and 152a (1.41 g, 6.84 mmol) and DMF (10 mL) were added, and the reaction mixture was cooled to 0°C. A solution of thionyl chloride (896 mg, 7.53 mmol) in DMF (5 mL) was added dropwise. The reaction was heated at 150°C for 2 h. Thereafter, the solvent was removed under reduced pressure. The resulting residue was partitioned between water (20 mL) and dichloromethane (20 mL). The layers were separated, and the aqueous phase was extracted with dichloromethane (2×20 mL). The organic extracts were combined, dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by flash chromatography to afford 152b as a brown solid in 52% yield (672 mg): mp >250°C; ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 8.53 (s, 1H), 7.76 (d, 1H, J=9.0 Hz), 7.66 (d, 1H, J=9.0 Hz), 7.40 (t, 1H, J=9.0 Hz), 7.31 (t, 1H, J=9.0 Hz), 4.41 (t, 2H, J=6.0 Hz), 3.71 (m, 2H); MS (APCI+) m/z 188.4 (M+H).

Example 152c 3,4,6,7,8,9-hexahydropyrazino[1,2-a]benzimidazol-1-one 152c

To a 250-mL stainless steel pressure reactor was added a solution of 10% palladium on carbon (50% moisture, 150 mg dry weight) and 152b (670 mg, 3.58 mmol) in acetic acid (25 mL). The reactor was evacuated, filled with hydrogen to a pressure of 350 psi and stirred at 95°C for 16 h. Thereafter, the hydrogen was evacuated and nitrogen was introduced into the reactor. Celite 521 (1.00 g) was added and the mixture was filtered through a pad of Celite 521. The filter cake was washed with ethanol (2×25 mL) and the combined filtrates were concentrated to dryness under reduced pressure. Water (10 mL) was added to the resulting residue, and the pH was adjusted to 9 by the addition of potassium carbonate. The mixture was extracted with dichloromethane (4×20 mL), and the combined organic extracts were dried over sodium sulfate, filtered, and concentrated under reduced pressure to afford 152c (487 mg) as a white solid in 71% yield: mp>250°C; ¹ H NMR (500 MHz, DMSO-d ₆ ) 7.86 (s, 1H), 3.98 (t, 2H, J=6.5 Hz), 3.51 (m, 2H), 2.50 (m, 4H), 1.75 (m, 4H); MS (APCI+) m/z 192.6 (M+H).

Example 152d 2-Bromo-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]benzimidazol-2-yl)benzyl acetate 152d

To a 100-mL three-necked round-bottom flask equipped with a reflux condenser, a magnetic stirrer, and a nitrogen inlet was added 152c (485 mg, 2.54 mmol), 104 g (1.56 g, 5.08 mmol), cesium carbonate (1.66 g, 5.08 mmol), N,N'-dimethylethylenediamine (447 mg, 5.08 mmol), and 1,4-dioxane (20 mL). After nitrogen was bubbled through the resulting suspension for 30 min, cuprous iodide (484 mg, 2.54 mmol) was added, and the reaction mixture was heated at 100 ° C (oil bath temperature) for 16 h. Thereafter, the mixture was cooled to room temperature and filtered. The filtrate was diluted with ethyl acetate (100 mL) and water (50 mL). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (3×50 mL). The combined organic layers were dried over sodium sulfate and then concentrated under reduced pressure. The residue was purified by column chromatography to obtain a yellow oil, which was dissolved in dichloromethane (5 mL). Acetyl chloride (506 mg, 2.54 mmol) and triethylamine (1.28 g, 12.7 mmol) were added, and the reaction mixture was stirred at room temperature for 2 h. Thereafter, the reaction mixture was diluted with dichloromethane (50 mL) and saturated aqueous sodium bicarbonate solution (20 mL). The organic layer was separated, and the aqueous layer was extracted with dichloromethane (3×50 mL). The combined organic layers were dried over sodium sulfate and concentrated under reduced pressure to give 13% yield (140 mg) of yellow oil 152d: ^1H NMR (500 MHz, CDCl ₃ ) 7.60 (d, 1H, J=8.0 Hz), 7.27 (t, 1H, J=8.0 Hz), 7.18 (d, 1H, J=8.0 Hz), 5.26 (d, 1H, J=12.0 Hz), 5.14 (d, 1H, J=12.0 Hz), 4.32 (m, 1H), 4.24 (m, 1H), 4.14 (m, 1H), 3.87 (m, 1H), 2.75 (m, 2H), 2.56 (m, 2H), 2.07 (s, 3H), 1.83 (m, 4H); MS (APCI+) m/z 418.8 (M+H).

A 100-mL single-necked round-bottom flask equipped with a magnetic stirrer and reflux condenser was purged with nitrogen and charged with 152d (140 mg, 0.335 mmol), 104h (140 mg, 0.426 mmol), sodium carbonate (106 mg, 1.00 mmol), 1,4-dioxane (5 mL), and water (1 mL). The mixture was degassed with nitrogen for 30 min. Tetrakis(triphenylphosphine)palladium (39 mg, 0.033 mmol) was added. After heating at 100°C for 5 h, the reaction mixture was cooled to room temperature and partitioned between water (40 mL) and dichloromethane (100 mL). The layers were separated, and the aqueous phase was extracted with dichloromethane (2 x 50 mL). The combined organic extracts were dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was dissolved in methanol (5 mL), and potassium carbonate (500 mg, 3.62 mmol) was added. After stirring at room temperature for 2 h, the reaction mixture was partitioned between water (20 mL) and dichloromethane (20 mL). The layers were separated, and the aqueous phase was extracted with dichloromethane (2×20 mL). The organic extracts were combined, dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by flash chromatography to afford 152 as a yellow solid in 9% yield (15 mg): mp 205–206° C.; ¹ H NMR (500 MHz, DMSO-d ₆ )δ9.18(s,1H),8.70(d,1H,J＝2.5Hz),8.64(m,1H),8.30(d,1H,J＝6.0Hz),7 .51–7.47(m,2H),7.39(d,1H,J＝8.0Hz),7.35(d,1H,J＝8.0Hz),7.31(d,1H, J＝6.0Hz),4.92(t,1H,J＝4.5Hz),4.36(m,2H),4.27(m,2H),4.19(m,1H),3. 97(m,1H),3.61(s,3H),2.63(m,2H),2.52(m,2H),1.87(m,4H); MS(ESI+)m/z 498.2(M+H).

Example 153 2-(2-(Hydroxymethyl)-3-(4-methyl-5-oxo-6-(1,2,3,4-tetrahydroisoquinolin-6-ylamino)-4,5-dihydropyrazin-2-yl)phenyl)-3,4,5,6,7,8-hexahydrobenzothieno[2,3-c]pyridin-1(2H)-one 153

Example 153a 5-Bromo-1-methyl-3-(1,2,3,4-tetrahydroisoquinolin-6-ylamino)pyrazin-2(1H)-one 153a

A 100-mL single-necked round-bottom flask equipped with a magnetic stirrer and a nitrogen inlet was purged with nitrogen and charged with 120a (1.14 g, 2.62 mmol), dichloromethane (10 mL), and trifluoroacetic acid (10 mL). The solution was stirred at room temperature for 2 h. Thereafter, the solution was concentrated under reduced pressure. The residue was partitioned between dichloromethane (100 mL) and 1 M aqueous potassium hydrogen phosphate (30 mL). The aqueous layer was extracted with a 20% (v/v) solution of methanol/dichloromethane (3 x 100 mL). The organic extracts were combined, washed with brine (20 mL), and then dried over sodium sulfate. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure to afford 153a as an off-white solid in 68% yield (600 mg): mp 170-171 °C; ^1H NMR (300 MHz, DMSO- _d6 ) δ 9.30 (s, 1H), 7.62 (m, 2H), 7.31 (s, 1H), 6.97 (d, 1H, J = 7.8 Hz), 4.10 (br s, 1H), 3.83 (s, 2H), 3.43 (s, 3H), 2.96 (t, 2H, J = 5.7 Hz), 2.68 (t, 2H, J = 5.7 Hz); MS (ESI+) m/z 334.0 (M+H).

153a (168 mg, 0.500 mmol), 111a (289 mg, 0.600 mmol), sodium carbonate (159 mg, 1.50 mmol), DMF (5 mL), water (2.5 mL) and 1,4-dioxane (8 mL) were added to a 50-mL three-necked round-bottom flask equipped with a reflux condenser, a magnetic stirrer and a nitrogen inlet. After nitrogen was bubbled through the resulting suspension for 30 min, tetrakis (triphenylphosphine) palladium (0) (58 mg, 0.050 mmol) was added and the reaction mixture was heated under reflux for 14 h. Afterwards, the mixture was cooled to room temperature and methanol (2 mL), water (2 mL) and lithium hydroxide monohydrate (42 mg, 1.00 mmol) were added. The mixture was stirred at room temperature for 4 h and then concentrated in vacuo. The residue was distributed between ethyl acetate (150 mL) and water (30 mL). The organic layer was separated, and the aqueous layer was extracted with a 20% (v/v) methanol/dichloromethane solution (3×150 mL). The combined organic layers were dried over sodium sulfate and then concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, 0%-10% methanol/dichloromethane) to afford 153 as an off-white solid in 4% yield (12 mg): 209–210°C dec; ¹ H NMR (500 MHz, DMSO-d ₆ )δ9.04(s,1H),7.74(s,1H),7.61(d,1H,J＝7.4Hz),7.53(d,1H,J＝8.0Hz),7.4 6(t,1H,J＝8.0Hz),7.40(s,1H),7.32(d,1H,J＝7.4Hz),6.90(d,1H,J＝8.3Hz),4 .77(m,1H),4.53(m,1H),4.43(m,1H),4.02(m,1H),3.87(m,1H),3.77(s,2H), 3.58(s,3H),2.91(m,4H),2.79(s,2H),2.63(m,2H),1.78(m,4H); MS(ESI+)m/z 568.1(M+H).

Example 154 2-(2-(Hydroxymethyl)-3-(6-oxo-5-(pyrimidin-4-ylamino)-1,6-dihydropyridin-3-yl)phenyl)-3,4,5,6,7,8-hexahydrobenzothieno[2,3-c]pyridin-1(2H)-one 154

Example 154a 5-Bromo-3-(pyrimidin-4-ylamino)pyridin-2(1H)-one 154a

To a 250-mL three-necked round-bottom flask equipped with a reflux condenser, a magnetic stirrer, and a nitrogen inlet was added 2-aminopyrimidine (376 mg, 3.95 mmol), 3,5-dibromopyridin-2(1H)-one (1.00 g, 3.95 mmol), 1M lithium hexamethyldisilazide THF solution (20 mL, 20.0 mmol), and 1,4-dioxane (25 mL). After nitrogen was bubbled through the resulting suspension for 30 minutes, Xantphos (194 mg, 0.211 mmol) and tris(dibenzylideneacetone)dipalladium(0) (165 mg, 0.197 mmol) were added, and the reaction mixture was heated under reflux for 3 hours. Thereafter, the mixture was cooled to room temperature and diluted with ethyl acetate (150 mL) and water (30 mL). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (3×150 mL). The combined organic extracts were dried over sodium sulfate and then concentrated under reduced pressure. The residue was purified by flash chromatography and then triturated with ethyl acetate (20 mL) to give 154a as an off-white solid in 24% yield (250 mg): mp 150-151 °C; ^1H NMR (500 MHz, DMSO- _d6 ) δ 12.03 (s, 1H), 9.27 (s, 1H), 8.54 (s, 1H), 8.20 (d, 1H, J = 6.0 Hz), 7.85 (s, 1H), 6.64 (d, 1H, J = 6.0 Hz); MS (ESI+) m/z 268.2 (M+H).

154a (175 mg, 0.655 mmol), 111a (315 mg, 0.655 mmol), sodium carbonate (208 mg, 2.00 mmol), DMF (2.5 mL), water (1.2 mL) and 1,4-dioxane (4 mL) were added to a 50-mL three-necked round-bottom flask equipped with a reflux condenser, a magnetic stirrer and a nitrogen inlet. After nitrogen was bubbled through the resulting suspension for 30 min, tetrakis (triphenylphosphine) palladium (0) (76 mg, 0.065 mmol) was added and the reaction mixture was heated under reflux for 14 h. Afterwards, the mixture was cooled to room temperature, methanol (2 mL), water (2 mL) and lithium hydroxide monohydrate (82 mg, 1.95 mmol) were added. The mixture was stirred at room temperature for 2 h and then concentrated in vacuo. The residue was distributed between ethyl acetate (150 mL) and water (30 mL). The organic layer was separated and the aqueous layer was extracted with a 20% (v/v) methanol/dichloromethane solution (3×150 mL). The combined organic extracts were dried over sodium sulfate and then concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, 0%-10% methanol/dichloromethane) to afford 154 as an off-white solid in 5% yield (15 mg): mp 200–201°C; ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 11.99 (s, 1H), 9.34 (s, 1H), 8.57 (s, 1H), 8.21 (d, 1H, J=6.0 Hz), 8.00 (s, 1H), 7.62 (s, 1H), 7.44 (t, 1H, J=7.5 Hz), 7.35 (d, 1H, J=7.0 Hz), 6.70 (d, 1H, J=5.5 Hz), 5.05 (br s,1H),4.28(t,1H,J＝11.0Hz),4.18(d,1H,J＝10.0Hz),3.96(m,1H),3.90 (m,1H),2.94(m,1H),2.84(m,1H),2.77(m,2H),1.76(m,4H); MS(ESI+)m/z 500.1(M+H).

Example 155 5,5,6,6,7,7-hexadeuterated-2-(2-(hydroxymethyl)-3-(1-methyl-5-(5-(1-methylazetidin-3-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,5,6,7,8-hexahydrobenzothieno[2,3-c]pyridin-1(2H)-one 155

Example 155c 4,4,5,5,6,6-hexadeuterated-4,5,6,7-tetrahydrobenzo[b]thiophene-2-carboxylic acid ethyl ester 155c

Intermediate 155a (1.53 g) was reacted with ethyl 2-mercaptoacetate (1.57 g) using the same general procedure as described in Example 105b to afford Intermediate 155c as a clear oil in 51% yield (1.46 g): ¹ H NMR (500 MHz, CDCl ₃ ) 7.46 (s, 1H), 4.31 (q, 2H, J=7.5 Hz), 2.77 (s, 2H), 1.35 (t, 3H, J=7.5 Hz); MS (ESI+) m/z 217 (M+H).

Example 155d 4,4,5,5,6,6-hexadeuterated-4,5,6,7-tetrahydrobenzo[b]thiophene-2-carboxylic acid 155d

Intermediate 155d (1.34 g) was saponified using the same general procedure as described in Example 105c to afford 94% yield (1.10 g) of white solid Intermediate 155d: mp 192-193° C.; ¹ H NMR (500 MHz, DMSO-d ₆ ) 12.75 (brs, 1H), 7.40 (s, 1H), 2.73 (s, 2H).

Example 155e 4,4,5,5,6,6-hexadeuterated-N-methoxy-N-methyl-4,5,6,7-tetrahydrobenzo[b]thiophene-2-carboxamide 155e

A 250-mL single-necked round-bottom flask equipped with a magnetic stirrer was purged with nitrogen, and 155d (5.67 g, 30.1 mmol), dichloromethane (100 mL), and DMF (110 mg, 1.50 mmol) were added and cooled to 0°C. Oxalyl chloride (4.21 g, 33.1 mmol) was added dropwise to the resulting solution. After this addition was complete, the reaction was allowed to warm to room temperature and stirred for 3 h. Thereafter, the reaction was concentrated to dryness under reduced pressure. The residue was dissolved in dichloromethane (100 mL), and the resulting solution was cooled to 0°C. Triethylamine (9.15 g, 90.3 mmol) and N,O-dimethylhydroxylamine hydrochloride (3.23 g, 33.1 mmol) were added. After the addition was complete, the cooling bath was removed, and the reaction mixture was stirred at room temperature for 14 h. Thereafter, the reaction mixture was partitioned between water (100 mL) and ethyl acetate (200 mL). The layers were separated, and the aqueous phase was extracted with ethyl acetate (100 mL). The combined organic extracts were washed with water (100 mL) followed by brine (100 mL) and then dried over sodium sulfate. The drying agent was removed by filtration, and the solvent was evaporated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, 0%-100% ethyl acetate/hexanes) to afford 155e (6.29 g) as a white solid in 90% yield: mp 47–48°C; ^1H NMR (500 MHz, CDCl ₃ ) δ 7.62 (s, 1H), 3.76 (s, 3H), 3.31 (s, 3H), 2.77 (s, 2H); MS (ESI+) m/z 232.1 (M+H).

Example 155f 1-(4,4,5,5,6,6-Hexadeuterated-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)prop-2-en-1-one 155f

A 250-mL single-necked round-bottom flask equipped with a magnetic stirrer was purged with nitrogen and charged with 155e (6.29 g, 27.2 mmol) and anhydrous THF (60 mL). The resulting solution was cooled to -25°C using an acetone/ice bath. A 1.0 M solution of vinylmagnesium bromide in THF (32.3 mL, 32.6 mmol) was added dropwise, and the resulting reaction mixture was stirred at 0°C for 1 h. Afterwards, the reaction mixture was partitioned between ethyl acetate (250 mL) and 2 M hydrochloric acid (50 mL). The layers were separated, and the aqueous phase was extracted with ethyl acetate (40 mL). The combined organic extracts were washed with water (100 mL) followed by brine (100 mL), dried over sodium sulfate, filtered, and then concentrated under reduced pressure to afford a quantitative yield of crude semisolid 155f (5.39 g). This material was used in the next step without further purification.

Example 155g 3-chloro-1-(4,4,5,5,6,6-hexadeuterated-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)propan-1-one 155g

A 250-mL single-necked round-bottom flask equipped with a magnetic stirrer was purged with nitrogen and charged with 155f (5.39 g, 27.2 mmol) and dichloromethane (60 mL). The resulting solution was cooled to 0°C. A 2M solution of hydrogen chloride in diethyl ether (34 mL) was added. After stirring at room temperature for 3 h, the solvent was removed under reduced pressure. The resulting residue was purified by column chromatography (silica gel, 0%-50% ethyl acetate/hexanes) to afford 155f in quantitative yield (6.38 g) as an off-white solid: mp 51–53°C; ^1H NMR (300 MHz, CDCl ₃ ) δ 7.41 (s, 1H), 3.89 (t, 2H, J = 7.0 Hz), 3.30 (t, 2H, J = 7.0 Hz), 2.79 (s, 2H); MS (ESI+) m/z 235.1 (M+H).

Example 155h 6,6,7,7,8,8-hexadeuterated-5,6,7,8-tetrahydro-1H-benzo[b]cyclopenta[d]thiophene-3(2H)-one 155h

To a 250-mL single-necked round-bottom flask equipped with a magnetic stirrer was added 155 g (6.38 g, 27.2 mmol) and 98% sulfuric acid (50 mL). After stirring at 95°C for 14 h, the reaction mixture was poured into ice (50 g), and the resulting suspension was extracted with ethyl acetate (3 x 50 mL). The organic extracts were combined, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, 0%-50% ethyl acetate/hexane) to afford 155 h in a 56% yield (3.03 g) of an off-white solid: mp 43–44°C; ¹ H NMR (300 MHz, CDCl ₃ ) δ 2.91 (m, 6H).

Example 155i 6,6,7,7,8,8-Hexadeuterated-5,6,7,8-tetrahydro-1H-benzo[b]cyclopenta[d]thiophene-3(2H)-one oxime 155i

To a 250-mL single-necked round-bottom flask equipped with a mechanical stirrer and a nitrogen inlet was added hydroxylamine hydrochloride (1.59 g, 22.9 mmol) and methanol (40 mL). The mixture was cooled to 0°C using an ice bath. Sodium acetate trihydrate (3.19 g, 22.9 mmol) was added. The mixture was stirred at 0°C for 30 min. Thereafter, 155h (3.03 g, 15.3 mmol) was added, and the reaction was stirred at room temperature for 14 h. Thereafter, the mixture was concentrated and redissolved in dichloromethane (200 mL) and water (30 mL). The organic layer was separated, and the aqueous layer was extracted with dichloromethane (3 x 150 mL). The combined organic layers were dried over sodium sulfate and concentrated under reduced pressure to afford 84% yield (2.72 g) of off-white solid 155i: mp 174-176°C; ¹ H NMR (300 MHz, CDCl ₃ ) δ 3.12 (m, 2H), 2.82 (m, 4H); MS (ESI+) m/z 214.1 (M+H).

Example 155j 5,5,6,6,7,7-Hexadeuterated-3,4,5,6,7,8-hexahydrobenzothieno[2,3-c]pyridin-1(2H)-one 155j

To a 50-mL, single-necked, round-bottom flask equipped with a reflux condenser, a magnetic stirrer, and a nitrogen inlet was added 155i (2.72 g, 12.8 mmol) and polyphosphoric acid (150 g). After stirring at 80°C for 14 h, the reaction mixture was cooled to room temperature and water (300 mL) was added. The resulting mixture was stirred for 30 min and then filtered. The filter cake was washed with water (20 mL) and then dried in a vacuum oven at 45°C to afford 155j in a 74% yield (2.00 g) as an off-white solid: mp 204–205°C; ^1H NMR (300 MHz, CDCl ₃ ) δ 5.58 (s, 1H), 3.58 (m, 2H), 2.79 (s, 2H), 2.71 (t, 2H, J = 7.0 Hz); MS (ESI+) m/z 214.1 (M+H).

Example 155k 2-Bromo-6-(5,5,6,6,7,7-hexadeuterated-1-oxo-3,4,5,6,7,8-hexahydrobenzothieno[2,3-c]pyridin-2(1H)-yl)benzyl acetate 155k

To a 250-mL three-necked round-bottom flask equipped with a reflux condenser, a magnetic stirrer, and a nitrogen inlet was added 155j (1.00 g, 4.69 mmol), 104 g (2.89 g, 9.38 mmol), cesium carbonate (4.59 g, 14.1 mmol), N,N'-dimethylethylenediamine (412 mg, 4.69 mmol), and 1,4-dioxane (35 mL). After bubbling nitrogen through the resulting suspension for 30 min, cuprous iodide (447 mg, 2.35 mmol) was added, and the reaction mixture was heated at 80 ° C (oil bath temperature) for 20 h. Thereafter, the mixture was cooled to room temperature and then filtered. The filtrate was diluted with ethyl acetate (150 mL) and water (30 mL). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (3×100 mL). The combined organic layers were dried over sodium sulfate and then concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, 0%-40% ethyl acetate/hexanes) to provide 155k in 35% yield (715 mg) of an off-white solid: mp 74-75° C.; ¹ H NMR (500 MHz, CDCl ₃ ) δ 7.59 (m, 1H), 7.31 (m, 2H), 5.15 (d, 1H, J=12.0 Hz), 5.04 (d, 1H, J=12.0 Hz), 3.99 (m, 1H), 3.65 (m, 1H), 2.81 (m, 1H), 2.74 (s, 2H), 2.06 (s, 3H); MS (ESI+) m/z 440.1 (M+H).

Example 1551 2-(5,5,6,6,7,7-hexadeuterated-1-oxo-3,4,5,6,7,8-hexahydrobenzothieno[2,3-c]pyridin-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 1551

A 100-mL single-necked round-bottom flask equipped with a magnetic stirrer and a reflux condenser was purged with nitrogen and charged with 155k (710 mg, 1.61 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bis(1,3,2-dioxaborolane) (819 mg, 3.22 mmol), potassium acetate (474 mg, 4.83 mmol), and 1,4-dioxane (12 mL). After bubbling nitrogen through the resulting suspension for 30 minutes, [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (118 mg, 0.161 mmol) was added, and the reaction mixture was heated at reflux for 2 hours. Thereafter, the mixture was cooled to room temperature and diluted with ethyl acetate (150 mL) and water (30 mL). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (3 x 150 mL). The combined organic layers were dried over sodium sulfate and then concentrated under reduced pressure to give a quantitative yield of crude 1551 (785 mg).This material was used in the next step without further purification.

Example 155m tert-Butyl 3-(6-(6-bromo-2-methyl-3-oxo-2,3-dihydropyridin-4-ylamino)pyridin-3-yl)azetidine-1-carboxylate 155m

To a 250-mL single-necked round-bottom flask equipped with a magnetic stirrer and a reflux condenser was added tert-butyl 3-(6-aminopyridin-3-yl)azetidine-1-carboxylate (333 mg, 1.33 mmol), 3,5-dibromo-1-methylpyridine-2 (1H) -one (350 mg, 1.33 mmol), cesium carbonate (870 mg, 2.70 mmol) and 1,4-dioxane (10 mL). After nitrogen was bubbled through the resulting solution for 30 min, Xantphos (66 mg, 0.114 mmol) and tris(dibenzylideneacetone)dipalladium (0) (61 mg, 0.066 mmol) were added and the reaction mixture was heated at 105 ° C for 3 h. Thereafter, the mixture was cooled to room temperature and then filtered. The filter cake was washed with dichloromethane (2 × 10 mL) and the combined filtrate was then concentrated under reduced pressure. The resulting residue was purified by column chromatography on silica gel to give 79% yield (460 mg) of a green solid 155m: mp 134-136°C; ^1H NMR (500 MHz, DMSO- _d6 ) δ 8.75 (s, 1H), 8.65 (s, 1H), 8.19 (s, 1H), 7.66 (dd, 1H, J = 8.5, 2.0 Hz), 7.51 (s, 1H), 7.35 (d, 1H, J = 8.5 Hz), 4.21 (t, 2H, J = 8.0 Hz), 3.81 (m, 2H), 3.51 (s, 3H), 1.40 (s, 9H); MS (ESI+) m/z 436.1 (M+H).

Example 155n 3-(5-(azetidin-3-yl)pyridin-2-ylamino)-5-bromo-1-methylpyridin-2(1H)-one 155n

A 100-mL single-necked round-bottom flask equipped with a magnetic stirrer and a nitrogen inlet was purged with nitrogen and 155 mL (1.20 g, 2.76 mmol), dichloromethane (10 mL), and trifluoroacetic acid (10 mL) were added. The solution was stirred at room temperature for 2 h. Thereafter, the solution was concentrated under reduced pressure. The residue was partitioned between dichloromethane (100 mL) and 1 M aqueous potassium hydrogen phosphate (30 mL). The aqueous layer was extracted with a 20% (v/v) solution of methanol/dichloromethane (3 x 100 mL). The organic extracts were combined, washed with brine (20 mL), and then dried over sodium sulfate. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure to give 155 as an off-white solid in quantitative yield (920 mg): mp 123-124 °C; ^1H NMR (300 MHz, DMSO- _d6 ) 8.72 (s, 1H), 8.65 (d, 1H, J = 2.3 Hz), 8.19 (d, 1H, J = 2.1 Hz), 7.71 (dd, 1H, J = 8.6, 2.2 Hz), 7.51 (d, 1H, J = 2.3 Hz), 7.33 (d, 1H, J = 8.2 Hz), 3.74 (m, 3H), 3.58 (m, 2H), 3.52 (s, 3H); MS (ESI+) m/z 335.1 (M+H).

Example 155o 2-(5-(5-(azetidin-3-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-6-(5,5,6,6,7,7-hexadeuterated-1-oxo-3,4,5,6,7,8-hexahydrobenzothieno[2,3-c]pyridin-2(1H)-yl)benzyl acetate 155o

To a 100-mL three-necked round-bottom flask equipped with a reflux condenser, a magnetic stirrer, and a nitrogen inlet was added crude 155l (785 mg, assumed 1.61 mmol), 155n (450 mg, 1.34 mmol), sodium carbonate (426 mg, 4.02 mmol), DMF (10 mL), water (5 mL), and 1,4-dioxane (16 mL). After bubbling nitrogen through the resulting suspension for 30 min, tetrakis(triphenylphosphine)palladium(0) (155 mg, 0.134 mmol) was added, and the reaction mixture was heated under reflux for 14 h. The residue was partitioned between ethyl acetate (150 mL) and water (30 mL). The organic layer was separated, and the aqueous layer was extracted with a 20% (v/v) solution of methanol/dichloromethane (3×150 mL). The combined organic layers were dried over sodium sulfate and then concentrated under reduced pressure to afford crude 155o. This material was used in the next step without further purification.

Example 155p 2-(3-(5-(5-(azetidin-3-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-2-(hydroxymethyl)phenyl)-5,5,6,6,7,7-hexadeuterated-3,4,5,6,7,8-hexahydrobenzothieno[2,3-c]pyridin-1(2H)-one 155p

To a 100-mL single-necked round-bottom flask (equipped with) was added the crude product 155o from the previous step, THF (8 mL), methanol (4 mL), water (4 mL) and lithium hydroxide monohydrate (420 mg, 10.0 mmol). The mixture was stirred at room temperature for 4 h and then concentrated in vacuo. The residue was partitioned between ethyl acetate (150 mL) and water (30 mL). The organic layer was separated and the aqueous layer was extracted with a 20% (v/v) solution of methanol/dichloromethane (3×150 mL). The combined organic layers were dried over sodium sulfate and then concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, 0%-10% methanol/dichloromethane) to give 15% yield (140 mg) of an off-white solid 155p: mp 140–142°C; ¹ H NMR (500 MHz, DMSO-d ₆ )δ8.65(d,1H,J＝2.0Hz),8.53(s,1H),8.10(d,1H,J＝2.4Hz),7.67(m,1H),7.45(t,1H,J＝8.0Hz),7.31(m,4H),4.81(t,1H,J＝5.0Hz) ,4.35(m,2H),4.04(m,1H),3.88(m,1H),3.71(m,3H),3.59(s,3H),3.55(m,2H),2.96(m,1H),2.86(m,1H),2.77(s,2H); MS(ESI+)m/z 574.3(M+H).

A 150-mL single-neck round-bottom flask equipped with a magnetic stirrer was purged with nitrogen and 155p (140 mg, 0.244 mmol), a 37% solution of formaldehyde/water (26 mg, 0.317 mmol) and methanol (10 mL) were added. A suspension of sodium cyanoborohydride (48 mg, 0.732 mmol) and zinc chloride (50 mg, 0.366 mmol) in methanol (4 mL) was added and the reaction was stirred at room temperature for 16 h. Thereafter, the reaction mixture was concentrated in vacuo. The residue was partitioned between ethyl acetate (150 mL) and water (30 mL). The organic layer was separated and the aqueous layer was extracted with a 20% (v/v) solution of methanol/dichloromethane (3×150 mL). The combined organic layers were dried over sodium sulfate and then concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, 0%-10% methanol/dichloromethane) to give 45% yield (65 mg) of 155 as a white solid: mp 190-192 °C; ¹ H NMR (500 MHz, DMSO-d ₆ )δ8.65(d,1H,J＝2.5Hz),8.58(s,1H),8.15(d,1H,J＝2.5Hz),7.67(dd,1H,J＝8.5,2.5Hz),7.46(t,1H,J＝8.0Hz),7.33(m,4H),4.81(t,1H,J＝4.0Hz ),4.35(m,2H),4.02(m,1H),3.85(m,3H),3.64(m,1H),3.59(s,3H),3.42 (m,2H),2.98(m,1H),2.87(m,1H),2.76(s,2H),2.47(s,3H); MS(ESI+)m/z 588.2(M+H).

Example 156 2-(3-(5-(5-((3,3-difluoroazetidin-1-yl)methyl)-1-methyl-1H-pyrazol-3-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 156

Example 156a Methyl 5-nitro-1H-pyrazole-3-carboxylate 156a

A 50-mL single-necked round-bottom flask equipped with a magnetic stirrer and reflux condenser was purged with nitrogen and charged with 5-nitro-1H-pyrazole-3-carboxylic acid (0.86 g, 5.5 mmol) and anhydrous methanol (10 mL). The reaction mixture was cooled to 0°C in an ice/water bath. To the resulting solution was added thionyl chloride (1.7 g, 14.4 mmol) dropwise. After the addition was complete, the ice bath was removed, and the reaction was heated at reflux for 3 h. Afterwards, the reaction was concentrated to dryness under reduced pressure to afford 156a (0.94 g) as a white solid in quantitative yield: MS (ESI-) m/z 170 (M-H).

Example 156b Methyl 1-methyl-3-nitro-1H-pyrazole-5-carboxylate 156b

A 50-mL single-necked round-bottom flask equipped with a magnetic stirrer was purged with nitrogen and charged with methyl 5-nitro-1H-pyrazole-3-carboxylate (156a) (0.94 g, 5.5 mmol), anhydrous N,N-dimethylformamide (11 mL), iodomethane (0.85 g, 6 mmol), and potassium carbonate (0.83 g, 6.1 mmol). The reaction was stirred at room temperature for 16 h. The reaction was then diluted with water (40 mL) and extracted with dichloromethane (3 x 25 mL). The combined organic extracts were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, ethyl acetate/hexane) to afford methyl 1-methyl-3-nitro-1H-pyrazole-5-carboxylate 156b as a white solid in a 66% yield (0.67 g): MS (ESI+) m/z 186.0 (M+H). The regioisomer, methyl 1-methyl-5-nitro-1H-pyrazole-3-carboxylate, was also isolated in 15% yield (0.15 g) as a white solid: MS (ESI-) m/z 186.0 (M+H).

Example 156c (1-Methyl-3-nitro-1H-pyrazol-5-yl)methanol 156c

A 100-mL three-necked round-bottom flask equipped with a magnetic stirrer, addition funnel, and nitrogen inlet was purged with nitrogen, and 156b (0.67 g, 3.6 mmol) and THF (20 mL) were added and cooled to 0°C with an ice bath. 2M lithium borohydride solution (3.6 mL, 7.2 mmol) was added dropwise at a rate to maintain the internal reaction temperature below 5°C. After the addition was complete, the cooling bath was removed, and the reaction was stirred at room temperature for 3 h. The reaction was cooled to 0°C with an ice bath, and saturated aqueous sodium bicarbonate solution (30 mL) was added dropwise. The layers were separated, and the aqueous layer was extracted with ethyl acetate (3×30 mL). The combined organic layers were washed with brine (30 mL), dried over sodium sulfate, and then concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, ethyl acetate/hexanes) to give a quantitative yield (0.56 g) of (1-methyl-3-nitro-1H-pyrazol-5-yl)methanol (156c) as an off-white solid: MS (ESI+) m/z 158.1 (M+H).

Example 156d 5-(Bromomethyl)-1-methyl-3-nitro-1H-pyrazole 156d

A 25-mL round-bottom flask equipped with a magnetic stirrer and reflux condenser was purged with nitrogen and charged with 156c (0.56 g, 3.6 mmol) and chloroform (10 mL). The reaction was cooled to 0°C with an ice bath, and phosphorus tribromide (0.98 g, 3.6 mmol) was then added dropwise. The cooling bath was removed, and the reaction was stirred at reflux for 3 h. Afterwards, the reaction was cooled to 0°C and diluted with dichloromethane (25 mL). Saturated aqueous sodium bicarbonate was added until the pH reached 8.5. The layers were separated, and the aqueous layer was extracted with dichloromethane (3 x 25 mL). The combined organic layers were washed with brine (30 mL), dried over sodium sulfate, and then concentrated under reduced pressure to afford 156d as an off-white solid in quantitative yield (0.79 g): MS (ESI+) m/z 222.1 (M+H).

Example 156e 5-((3,3-difluoroazetidin-1-yl)methyl)-1-methyl-3-nitro-1H-pyrazole 156e

To a sealed tube equipped with a magnetic stirrer was added DMF (5 mL), 5-(bromomethyl)-1-methyl-3-nitro-1H-pyrazole 156d (0.39 g, 1.78 mmol), 3,3-difluoroazetidine hydrochloride (276 mg, 2.13 mmol), and DIPEA (0.8 mL, 4.45 mmol). The reaction mixture was heated at 65°C for 3-5 h. The reaction was then concentrated to dryness under reduced pressure, and the resulting residue was diluted with a mixture of ethyl acetate (15 mL) and water (15 mL). The aqueous layer was separated and extracted with ethyl acetate (2 x 15 mL). The combined organic extracts were dried over sodium sulfate and then concentrated under reduced pressure to afford 156e as a yellow oil in quantitative yield, which was used in the next step without further purification. MS (ESI+) m/z 233.0 (M+H).

Example 156f 5-((3,3-difluoroazetidin-1-yl)methyl)-1-methyl-1H-pyrazol-3-amine 156f

A Parr reaction flask was purged with nitrogen and a solution of 10% palladium on carbon (30% humidity, 150 mg dry weight) and 156e (1.78 mmol) in ethanol (25 mL) was added. The flask was connected to a Parr hydrogenation reactor, evacuated, filled with hydrogen to a pressure of 40 psi and shaken for 2 h. Thereafter, the hydrogen was removed and the reaction mixture was filtered through a pad of Celite 521. The filter cake was washed with ethanol (2×25 mL) and the combined filtrate was concentrated to dryness under reduced pressure to give 156f (360 mg) as a yellow oil in quantitative yield: MS (ESI+) m/z 203.1 (M+H).

Example 156g 5-bromo-3-(5-((3,3-difluoroazetidin-1-yl)methyl)-1-methyl-1H-pyrazol-3-ylamino)-1-methylpyridin-2(1H)-one 156g

Intermediate 156g was synthesized using the same procedure as Example 112a, except using 156f (360 mg, 1.78 mmol), 3,5-dibromo-1-methyl-1H-pyridin-2-one (0.43 g, 1.6 mmol), cesium carbonate (1.56 g, 4.8 mmol), tris(dibenzylideneacetone)dipalladium(0) (0.15 g, 0.16 mmol), Xantphos (0.18 g, 0.32 mmol), and 1,4-dioxane (18 mL). The reaction mixture was heated at 115°C for 24 hours. Workup and flash column chromatography (silica gel, 60:35:5 dichloromethane/diethyl ether/methanol) afforded 156g as a green solid in a 38% yield (0.23 g): MS (ESI+) m/z 390.1 (M+H).

According to Example 121c, except using 2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 114a (165 mg, 0.35 mmol), 156g (115 mg, 0.3 mmol), 1M sodium carbonate solution (1.2 mL, 1.2 mmol), tetrakis(triphenylphosphine)palladium(0) (18 mg, 0.015 mmol), and 1,2-dimethoxyethane (3 mL), workup and flash column chromatography (silica gel, 60:35:5 dichloromethane/diethyl ether/methanol) gave 156h (a mixture of compound 156 and its acetate) as a yellow oil (190 mg). The mixture (0.3 mmol) was deprotected with a mixture of THF (2 mL), water (1 mL), and isopropanol (2 mL), along with lithium hydroxide monohydrate (80 mg, 1.90 mmol). Workup and flash column chromatography (silica gel, 60:35:5 dichloromethane/diethyl ether/methanol) afforded 156 as a white solid in 12% yield (2 steps, 22 mg): MS (ESI+) m/z 604.4 (M+H).

Example 157 2-(2-(Hydroxymethyl)-3-(5-(5-methoxy-1H-pyrazol-3-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 157

Example 157a 5-Bromo-3-(5-methoxy-1H-pyrazol-3-ylamino)-1-methylpyridin-2(1H)-one 157a

To a sealed tube equipped with a magnetic stirrer were added 5-methoxy-1H-pyrazol-3-amine (1.9 g, 17 mmol), 3,5-dibromo-1-methylpyridin-2(1H)-one (4.9 g, 18 mmol), cesium carbonate (12 g, 37 mmol), and 1,4-dioxane (160 mL). After nitrogen was bubbled through the solution for 30 min, Xantphos (1.1 g, 2 mmol) and tris(dibenzylideneacetone)dipalladium(0) (1.1 g, 1.2 mmol) were added, and the reaction mixture was heated to 100° C. for 16 hours. Thereafter, H ₂ O (50 mL) and EtOAc (50 mL) were added. The aqueous layer was separated and extracted with EtOAc (2×50 mL). The combined organic extracts were washed with brine (100 mL) and then dried over sodium sulfate. The resulting solution was concentrated to almost dryness under reduced pressure, and the desired product precipitated from the solution. Filtration and washing with _Et2O (10 mL) gave 12% yield (590 mg) of crude 157a.

Example 157b 2-(5-(5-methoxy-1H-pyrazol-3-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 157b

To a microwave tube equipped with a magnetic stirrer was added 157a (94 mg, 0.3 mmol), 2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 114a (180 mg, 0.4 mmol), DME (4 mL), and 1 M aqueous sodium carbonate solution (0.9 mL). After bubbling _N for 15 min, Pd(PPh ₃ ) ₄ (18 mg, 0.02 mmol) was added. The mixture was heated to 130° C. in a microwave for 25 min. Thereafter, EtOAc (5 mL) and water (5 mL) were added. The separated aqueous layer was extracted with EtOAc (2 x 5 mL). The combined organic phases were washed with brine (10 mL), dried over sodium _sulfate , filtered, and concentrated under reduced pressure. The resulting residue was purified by column chromatography eluting with a gradient of _CH2Cl2-60 :35:5 _CH2Cl2 : _Et2O :MeOH _to afford 157b in 19% yield (33 mg).

To a 25 mL round-bottom flask equipped with a magnetic stirrer was added 157b (33 mg, 0.06 mmol), lithium hydroxide (10 mg, 1.2 mmol), THF (0.3 mL), i-PrOH (0.3 mL), and water (0.6 mL). The mixture was stirred at rt for 1 h. Thereafter, EtOAc (5 mL) and water (5 mL) were added. The separated aqueous layer was extracted with EtOAc (2 x 5 mL). The combined organic phases were washed with brine (10 mL), dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by column chromatography using a gradient of _CH₂Cl₂ to ₆₀ :35:5 _CH₂Cl₂ : _Et₂O :MeOH _to afford 157 in 76% yield (23 mg). MS (ESI+) m/z 515.4 (M+H).

Example 158 5-[2-(Hydroxymethyl)-3-[1-methyl-6-oxo-5-(5,6,7,8-tetrahydro-1,6-naphthyridin-2-ylamino)-1,6-dihydropyridin-3-yl]phenyl]-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-6-one 158

Example 158a tert-Butyl 2-(diphenylmethyleneamino)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate 158a

To a round-bottom flask equipped with a stir bar was added tert-butyl 2-chloro-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate (1.09 g, 4.05 mmol), diphenylmethylamine 26 (2.20 g, 12.14 mmol), Pd(OAc) (181.6 mg, 0.809 mmol), BINAP (503.8 mg, 0.809 mmol), _CsCO (6.59 _g , 20.23 mmol), and toluene (16 mL). The reaction mixture was heated at 110° C. for 2 days. The reaction mixture was filtered, and the solvent was removed in vacuo. The residue 158a was used directly in the next step.

Example 158b tert -Butyl 2-amino-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate 158b

Intermediate 158a was added to a round-bottom flask. MeOH (50 mL) and NH ₂ OH HCl (1.76 g, 25.3 mmol) were added. The resulting mixture was stirred at room temperature for 5 hr. The solvent was removed in vacuo, and then purified on a silica gel column (MeOH:DCM = 10:90) to afford 158b as a black oil, 851 mg (84%, 2 steps).

Example 158c tert-Butyl 2-(5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate 158c

To a round-bottom flask equipped with a stir bar was added 158b (851 mg, 3.41 mmol), 3,5-dibromo-1-methylpyridin-2(1H)-one (1.37 g, 5.12 mmol), _Pd2 (dba) ₃ (312.5 mg, 0.341 mmol), XantPhos (316 mg, 0.546 mmol), _Cs2CO3 (3.67 g, 11.3 mmol), and _dioxane (17 mL). The reaction mixture was heated at 100°C overnight. DCM (200 mL) was added to the resulting mixture, which was then washed with water (30 mL x 3). DCM (200 mL) was added, and the resulting mixture was washed with water (30 mL x 3), brine (30 mL x 1), dried over _MgSO4 , filtered, and the solvent removed in vacuo. DCM/diethyl ether (1:2, 5 mL) was added, followed by sonication, and the precipitate was filtered to give 158c as a green solid, 865 mg (58%).

Example 158d 5-[2-(acetoxymethyl)-3-[1-methyl-6-oxo-5-(5,6,7,8-tetrahydro-1,6-naphthyridin-2-ylamino)-1,6-dihydropyridin-3-yl]phenyl]-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-6-one 158d

To a microwave tube equipped with a stir bar was added 158c (300 mg, 0.689 mmol), boronate ester 111a (365 mg, 0.758 mmol), Pd(PPh ₃ ) ₄ (39.8 mg, 0.034 mmol), aqueous Na ₂ CO ₃ solution (1.0 N, 2.27 mL, 2.27 mmol), and 1,2-dimethoxyethane (3.0 mL). The mixture was reacted in a microwave at 130° C. for 10 min. Dichloromethane (200 mL) was added, and the resulting mixture was washed with water (30 mL x 3) and brine (30 mL x 1), dried over MgSO ₄ , filtered, and the solvent removed in vacuo. Silica gel column chromatography (MeOH:dichloromethane = 5:95) afforded 158d.

To a round-bottom flask equipped with a stir bar was added 158d, THF (5 mL), isopropanol (5 mL), water (5 mL), and LiOH monohydrate (278 mg). The resulting mixture was stirred at room temperature for ₁ hr. The solvent was removed in vacuo, and the resulting residue was added to dichloromethane (200 mL). The solution was washed with water (30 mL x 3), brine (30 mL x 1), dried over MgSO₄, filtered, and the solvent was removed in vacuo. Silica gel column chromatography (methanol:dichloromethane = 10:90) afforded 158 (145 mg) as a yellow solid. MS (ESI+) m/z 568.3 (M+H).

Example 159 2-(2-(Hydroxymethyl)-3-(1-methyl-6-oxo-5-(pyrimidin-4-ylamino)-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrido[3,4-b]imidazo[4,5-c]pyridin-1-one 159

Example 159a 3-Nitro-4-(piperidin-1-yl)pyridine 159a

A 500-mL single-necked round-bottom flask equipped with a reflux condenser and a magnetic stirrer was purged with nitrogen and charged with 3-nitro-4-chloro-pyridine (10.0 g, 63.3 mmol), piperidine (16.2 g, 190 mmol), and ethanol (200 mL). After heating at 70°C for 2 h, the solvent was removed under reduced pressure, and the residue was then partitioned between ethyl acetate (200 mL) and 10% aqueous potassium carbonate solution (100 mL). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (2 x 100 mL). The combined organic layers were dried over sodium sulfate and then concentrated under reduced pressure. The residue was purified by column chromatography to give 100% yield (15.2 g) of yellow oil 159a: ¹ H NMR (500 MHz, CDCl ₃ ) δ 8.81 (s, 1H), 8.31 (d, 1H, J=6.0 Hz), 6.86 (d, 1H, J=6.0 Hz), 3.22 (t, 4H, J=4.5 Hz), 1.72 (m, 6H); MS (APCI+) m/z 208.4 (M+H).

Example 159b 6,7,8,9-tetrahydropyrido[3,4-b]imidazo[4,5-c]pyridine 159b

A 500-mL single-necked round-bottom flask equipped with a reflux condenser and a magnetic stirrer was purged with nitrogen and charged with 159a (6.00 g, 29.0 mmol) and triethyl phosphite (200 mL). After heating at 110°C for 24 h, the triethyl phosphite was removed under reduced pressure, and the residue was partitioned between dichloromethane (200 mL) and water (100 mL). The organic layer was separated, and the aqueous layer was extracted with dichloromethane (2 x 100 mL). The combined organic layers were dried over sodium sulfate and then concentrated under reduced pressure. The residue was purified by column chromatography to give 159b as a yellow solid in 53% yield (2.66 g): mp 94-95° C.; ¹ H NMR (500 MHz, CDCl ₃ ) 8.98 (s, 1H), 8.37 (d, 1H, J=6.0 Hz), 7.22 (d, 1H, J=6.0 Hz), 4.09 (t, 2H, J=6.0 Hz), 3.11 (t, 2H, J=6.0 Hz), 2.15 (m, 2H), 2.05 (m, 2H); MS (APCI+) m/z 174.1 (M+H).

Example 159c 6,7,8,9-tetrahydropyrido[3,4-b]imidazo[4,5-c]pyridine 2-oxide 159c

To a 100-mL single-necked round-bottom flask equipped with a reflux condenser and a magnetic stirrer were added 159b (2.50 g, 14.4 mmol) and dichloromethane (50 mL), and the reaction mixture was cooled to 0° C. 3-Chloroperbenzoic acid (4.99 g, 28.9 mmol) was added, and the mixture was stirred at room temperature for 1.5 h. After that, the solvent was removed under reduced pressure, and the residue was purified by column chromatography to give 159c as a yellow solid in 83% yield (2.26 g): mp 178-179° C.; ¹ H NMR (500 MHz, CDCl ₃ ) 8.71 (s, 1H), 8.15 (d, 1H, J=6.0 Hz), 7.19 (d, 1H, J=6.0 Hz), 4.12 (t, 2H, J=6.0 Hz), 3.12 (t, 2H, J=6.0 Hz), 2.18 (m, 2H), 2.05 (m, 2H); MS (APCI+) m/z 190.4 (M+H).

Example 159d 6,7,8,9-tetrahydropyrido[3,4-b]imidazo[4,5-c]pyridin-1-one 159d

A 250-mL single-necked round-bottom flask equipped with a reflux condenser and a magnetic stirrer was purged with nitrogen and charged with 159c (2.26 g, 12.0 mmol) and acetic anhydride (90 mL). After heating at 140°C for 1.5 h, the solvent was removed under reduced pressure, and the residue was purified by column chromatography to afford 159d as a yellow solid in 62% yield (1.40 g): mp>250°C; ^1H NMR (500 MHz, CDCl ₃ ) 11.3 (s, 1H), 7.24 (d, 1H, J=7.0 Hz), 6.39 (d, 1H, J=6.0 Hz), 4.07 (t, 2H, J=6.0 Hz), 3.11 (t, 2H, J=6.0 Hz), 2.13 (m, 2H), 2.02 (m, 2H); MS (APCI+) m/z 190.7 (M+H).

Example 159e 3,4,6,7,8,9-hexahydropyrido[3,4-b]imidazo[4,5-c]pyridin-1-one 159e

To a 250-mL stainless steel pressure reactor was added 10% palladium on carbon (50% humidity, 250 mg dry weight) and a solution of 159d (1.07 g, 5.66 mmol) in acetic acid (50 mL). The reactor was evacuated, filled with hydrogen to a pressure of 350 psi and stirred at 85°C for 48 h. Thereafter, the hydrogen was removed and nitrogen was filled into the reactor. Celite 521 (1.00 g) was added and the mixture was filtered through a pad of Celite 521. The filter cake was washed with ethanol (2×25 mL) and the combined filtrate was concentrated to dryness under reduced pressure. Water (10 mL) was added to the resulting residue, and potassium carbonate was then added to adjust the pH to 9. The mixture was extracted with dichloromethane (4×20 mL), and the combined organic extracts were dried over sodium sulfate, filtered, and then concentrated under reduced pressure to afford 159e (749 mg) as a white solid in 69% yield: mp>250° C.; ¹ H NMR (500 MHz, CDCl ₃ ) 5.28 (s, 1H), 3.85 (t, 2H, J=6.0 Hz), 3.62 (m, 2H), 2.92 (t, 2H, J=6.0 Hz), 2.81 (t, 2H, J=6.0 Hz), 2.03 (m, 2H), 1.94 (m, 2H); MS (APCI+) m/z 192.7 (M+H).

Example 159f 2-Bromo-6-(1-oxo-3,4,6,7,8,9-hexahydropyrido[3,4-b]imidazo[4,5-c]pyridin-2-yl)benzyl acetate 159f

To a 100-mL three-necked round-bottom flask equipped with a reflux condenser, a magnetic stirrer, and a nitrogen inlet was added 159e (745 mg, 3.90 mmol), 104 g (2.40 g, 7.80 mmol), cesium carbonate (2.54 g, 7.80 mmol), cuprous iodide (743 mg, 3.90 mmol), and 1-methyl-2-pyrrolidone (7 mL). After heating at 120°C (oil bath temperature) for 16 h, the mixture was cooled to room temperature and then partitioned between dichloromethane (100 mL) and water (50 mL). The organic layer was separated, and the aqueous layer was extracted with dichloromethane (3 × 50 mL). The combined organic layers were dried over sodium sulfate and then concentrated under reduced pressure. The residue was purified by column chromatography to give a yellow oil, which was used in dichloromethane (5 mL). Acetyl chloride (306 mg, 3.90 mmol) and triethylamine (1.97 g, 19.5 mmol) were added, and the reaction mixture was stirred at room temperature for 2 h. The reaction mixture was then diluted with dichloromethane (50 mL) and saturated aqueous sodium bicarbonate solution (20 mL). The organic layer was separated, and the aqueous layer was extracted with dichloromethane (3 x 50 mL). The combined organic layers were dried over sodium sulfate and concentrated under reduced pressure to afford 6% yield (100 mg) of 159f as a pink oil: ^1H NMR (500 MHz, CDCl ₃ ) 7.60 (d, 1H, J=8.0 Hz), 7.34 (t, 1H, J=8.0 Hz), 7.16 (d, 1H, J=8.0 Hz), 5.18 (m, 2H), 4.18 (m, 1H), 4.04 (m, 1H), 3.87 (m, 1H), 3.79 (m, 1H), 3.16 (m, 1H), 3.05 (m, 1H), 2.95 (m, 2H), 2.09-1.99 (m, 7H); MS (APCI+) m/z 418.9 (M+H).

A 100-mL single-necked round-bottom flask equipped with a magnetic stirrer and reflux condenser was purged with nitrogen and charged with 159f (100 mg, 0.239 mmol), 104h (350 mg, 1.07 mmol), sodium carbonate (183 mg, 1.72 mmol), 1,4-dioxane (8 mL), and water (2 mL). The mixture was degassed with nitrogen for 30 min. Tetrakis(triphenylphosphine)palladium (66 mg, 0.057 mmol) was added. After heating at 100°C for 5 h, the reaction mixture was cooled to room temperature and then partitioned between water (40 mL) and dichloromethane (100 mL). The layers were separated, and the aqueous phase was extracted with dichloromethane (2 x 50 mL). The organic extracts were combined, dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was dissolved in methanol (5 mL), and potassium carbonate (500 mg, 3.62 mmol) was added. After stirring at room temperature for 2 h, the reaction mixture was partitioned between water (20 mL) and dichloromethane (20 mL). The layers were separated, and the aqueous phase was extracted with dichloromethane (2×20 mL). The organic extracts were combined, dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by flash chromatography to afford 159 as a light brown solid in 10% yield (12 mg): mp>250°C; ¹ H NMR (500 MHz, DMSO-d ₆ )δ9.16(s,1H),8.71(d,1H,J＝2.0Hz),8.64(s,1H),8.29(dd,1H,J＝6.0, 1.5Hz),7.53(s,1H),7.45(td,1H,J＝6.0,1.5Hz),7.31(m,3H),4.75(m,1 H),4.32(m,2H),4.02(m,2H),3.88(m,2H),3.60(s,3H),3.05(m,1H),2. 96(m,1H),2.76(t,2H,J=5.5Hz),1.95(m,2H),1.86(m,2H);MS(ESI+)m/z 498.2(M+H).

Example 160 3-({4-[(2R)-1,4-dimethyl-3-oxopiperazin-2-yl]phenyl}amino)-5-[2-(hydroxymethyl)-3-{1-oxo-1H,2H,3H,4H,6H,7H,8H,9H-pyrazino[1,2-a]indol-2-yl}phenyl]-1-methyl-1,2-dihydropyrazin-2-one 160

According to Example 121b, 2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 114a (167 mg, 0.36 mmol), 5-bromo-3-(4-(1,4-dimethyl-3-oxopiperazin-2-yl)phenylamino)-1-methylpyrazin-2(1H)-one 160a (WO 2009/039397; 122 mg, 0.3 mmol), 1 M sodium carbonate solution (0.9 mL, 0.9 mmol), tetrakis(triphenylphosphine)palladium(0) (18 mg, 0.015 mmol) and 1,2-dimethoxyethane (3 mL) were used. Workup and flash column chromatography (silica gel, 60:35:5 dichloromethane/ether/methanol) afforded a yellow oily mixture of 2-(5-(4-(1,4-dimethyl-3-oxopiperazin-2-yl)phenylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 160b and 160. The mixture (0.3 mmol) was deprotected with a mixture of THF (1 mL), water (0.5 mL), and isopropanol (1 mL) and lithium hydroxide monohydrate (80 mg, 1.90 mmol). Workup and flash column chromatography (silica gel, 60:35:5 dichloromethane/ether/methanol) afforded 37% yield of compound 160 as a white solid (2 steps, 70 mg): MS (ESI+) m/z 622.4 (M+H).

Example 161 2-(3-(5-(1-(2-hydroxyethyl)-5-methyl-1H-pyrazol-3-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 161

Example 161a 5-Bromo-3-(1-(2-(tert-butyldimethylsilyloxy)ethyl)-5-methyl-1H-pyrazol-3-ylamino)-1-methylpyridin-2(1H)-one 161a

A solution of 5-bromo-1-methyl-3-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2(1H)-one 112a (1.08 g, 3.8 mmol) in anhydrous DMF (10 mL) was treated with a 60% suspension of NaH in mineral oil (0.17 g, 4.3 mmol) while stirring under nitrogen. After bubbling, the reaction was stirred for an additional 30 minutes. The reaction was then treated with (2-bromoethoxy)(tert-butyl)dimethylsilane (15-1) (0.908 g, 3.8 mmol) and stirred under nitrogen for another 10 hours. Following the reaction, water (50 mL) was slowly added, and the mixture was filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by flash chromatography eluting with petroleum ether/ethyl acetate to afford 161a (1 g, 35%), which was used without further purification. LCMS: (M+H) 443.

Example 161b 2-(5-(1-(2-(tert-butyldimethylsilyloxy)ethyl)-5-methyl-1H-pyrazol-3-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydro-pyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 161b

A mixture of 161a (750 mg, 1.7 mmol), 2-( _{1 -} oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 114a (882 mg, 1.9 mmol), CH3COONa (309 mg, 3.8 mmol), _PdCl2 (dppf) (153 mg, 0.19 mmol) and _K3PO4 (1 g, 3.8 mmol) suspended in _CH3CN (30 mL) and _H2O (2 mL) was heated at 110°C for 12 h under argon atmosphere. After the reaction, the mixture was evaporated and the residue was purified by reverse phase Combi-flash chromatography eluting with 1:4 water/CH ₃ CN containing 0.3% NH ₄ HCO ₃ to give 161b as a brown solid (210 mg, 18%). LCMS: [M+H] ⁺ 699.

Example 161c 2-(3-(5-(1-(2-(tert-butyldimethylsilyloxy)ethyl)-5-methyl-1H-pyrazol-3-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 161c

A mixture of 161b (210 mg, 0.4 mmol) and LiOH (372 mg, 16 mmol) in isopropanol/THF (1:1, 10 mL) and water (3 mL) was stirred at 30°C for 2 h. The mixture was evaporated in vacuo, and the residue was extracted with ethyl acetate (2 x 10 mL). The combined extracts were concentrated under reduced pressure, and the residue was purified on preparative HPLC to afford 161c. LCMS: [M+H] ⁺ 657

A solution of 161c and camphorsulfonic acid (330 mg, 1.5 mmol) in methanol (30 mL) was stirred at room temperature for 3 h. After the reaction, the methanol was evaporated and the residue was purified by preparative HPLC to afford 161 as a brown solid (63 mg, 29%, 2 steps). LCMS: [M+H] ⁺ 543. ¹ H NMR(500MHz,DMSO)δ7.96(m,1H),7.45(m,1H),7.33(m,2H),7.24(m,1H),6.51(s,1H),5.87(s,1H),4.86(m,1H),4.77(m,1 H),4.36(m,2H),4.15(m,3H),3.90(m,3H),3.64(m,2H),3.57(s,3H),2.51(m,2H),2.46(m,2H),2.19(s,3H),1.79(m,4H).

Example 162 2-(2-(Hydroxymethyl)-3-(1-methyl-5-(2-methylpyrimidin-4-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 162

Example 162a 2-Methylpyrimidin-4-amine 162a

To a pressure-resistant container made of stainless steel with an inner volume of 500 mL was added 3-methoxypropionitrile (10 g, 120 mmol), 1.1.1-trimethoxyethane (39 g, 324 mmol) and 40.0 g (560 mmol, 24 wt% ammonia-methanol solution). The mixture was stirred at 130 ° C for 8 hours. After the reaction was completed, it was filtered and then concentrated to obtain a yellow solid. The solid was washed with ethyl acetate (50 mL) and dried in vacuo to give 162a (7.8 g, 60%). LCMS: [M+H] ⁺ 110

Example 162b 5-Bromo-1-methyl-3-(2-methylpyrimidin-4-ylamino)pyridin-2(1H)-one 162b

According to Example 148a, 2.0 g of 162a and 4.0 g of 3,5-dibromo-1-methyl-pyridin-2(1H)-one were reacted to give 162b (2.3 g, 50%) as a yellow solid. LCMS: [M+H] ⁺ 357 ¹ H NMR (500 MHz, DMSO) δ 9.20 (s, 1H), 8.78 (s, 1H), 8.26 (d, J=4.5, 1H), 7.68 (s, 1H), 7.18 (d, J=4.5, 1H), 3.59 (s, 3H), 2.52 (s, 3H).

Example 162c 2-(1-methyl-5-(2-methylpyrimidin-4-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 162c

According to Example 148b, 464 mg of 2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 114a was reacted with 443 mg of 162b to give 162c (386 mg, 70%) as a yellow solid. LCMS: [M+H] ⁺ 553

According to Example 148, 160 mg of 162c was hydrolyzed to give 162 as a white solid (90 mg, 60%). LCMS: [M+H] ⁺ 514. ¹ H NMR (500MHz, DMSO) δ9.04(s,1H),8.87(s,1H),8.21(s,1H),7.55(s,1H),7.48(s,1H),7.36(t,J＝6.5,1H),7.12(s,1H),6.52(s,H),4. 85(s,1H),4.40(s,1H),4.17-4.10(m,3H),3.92(m,1H),3.61(s,3H),2.60(m,2H),2.47(m,2H),2.42(S,3H),1.80(m,2H),1.69(m,2H).

Example 163 2-(3-(5-(1-(2-hydroxyethyl)-1H-pyrazol-3-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 163

Example 163a 5-Bromo-3-(1-(2-(tert-butyldimethylsilyloxy)ethyl)-1H-pyrazol-3-ylamino)-1-methylpyridin-2(1H)-one 163a

A mixture of 1-(2-(tert-butyldimethylsilyloxy)ethyl)-1H-pyrazol-3-amine 116b (1.2 g, 5 mmol), 3,5-dibromo-1-methylpyridin-2(1H)-one (1.3 g, 5 mmol), XantPhos (300 mg, 0.5 mmol), Pd ₂ (dba) ₃ (460 mg, 0.5 mmol), and Cs ₂ CO ₃ (4 g, 2.5 mmol) in dioxane (30 mL) was heated in a sealed tube under nitrogen at 120° C. for 2 h. After the reaction, the mixture was filtered, and the filtrate was evaporated in vacuo to give a yellow solid. The solid was then washed with ethyl acetate (6 mL×3) to give 163a (0.80 g, 38%) as a yellow solid, which was used without further purification. MS: (M+H) ⁺ 427.

Example 163b 2-(5-(1-(2-(tert-butyldimethylsilyloxy)ethyl)-1H-pyrazol-3-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 163b

A mixture of 163a (800 mg, 1.88 mmol), 2-( ₁ -oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 114a (1.3 g, 2.82 mmol), sodium acetate (308 mg, 3.76 mmol), PdCl2(dppf) (153 mg, 0.188 mmol) and _K3PO4 (1 g, 3.76 mmol) suspended in _CH3CN (50 mL) and water ( ₃ mL) was heated at 110°C for 12 h under argon atmosphere. The mixture was then evaporated and the residue was purified by reverse phase Combi-flash chromatography eluting with 1:5 water/CH ₃ CN containing 0.3% NH ₄ HCO ₃ to afford 163b as a brown solid (350 mg, 29%). MS: (M+H) ⁺ 685

Example 163c 2-(3-(5-(1-(2-(tert-butyldimethylsilyloxy)ethyl)-1H-pyrazol-3-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 163c

To a solution of 163b (460 mg, 0.67 mmol) in propan-2-ol (15 mL), tetrahydrofuran (15 mL), and water (5 mL) was added LiOH monohydrate (1.6 g, 67 mmol). The mixture was stirred at 30°C for 2 h. It was then evaporated and the residue was purified by reverse phase Combi-flash chromatography using ₁ :4 water/ _CH3CN containing 0.3% _NH4HCO3 to afford 163c as a brown solid (300 mg, 70%). MS: (M+H) ⁺ 643

A solution of 163c (300 mg, 0.50 mmol) and camphorsulfonic acid (330 mg, 1.5 mmol) in methanol (30 mL) was stirred at room temperature for 3 h. It was then evaporated and the residue was purified by preparative HPLC to give 163 as a brown solid (140 mg, 57%). MS: (M+H) ⁺ 529. ¹ H NMR (500 MHz, MeOD) δ 1.79 (s, 2H), 1.89 (s, 2H), 2.54-2.56 (t, J = 5.5 Hz, 2H), 2.62-2.66 (m, 2H), 3.70 (s, 3H), 3.84-3.86 (t, J = 5.5 Hz, 2H), 4.01-4.02 (m, 1H), 4.09-4.11 (t, J = 5. 5Hz,2H),4.17-4.22(m,3H),4.49-4.57(m,2H),6.05-6.06(d,1H),6.71(s,1H),7.22-7 .23(d,1H),7.35-7.37(d,1H),7.40-7.42(d,1H),7.48-7.51(m,2H),7.94-7.95(d,1H).

Example 164 5-[3-(5-{[5-(azetidin-3-yl)-1H-pyrazol-3-yl]amino}-1-methyl-6-oxopyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl]-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-diene-6-one 164

Example 164b tert-Butyl 3-(2-cyanoacetyl)azetidine-1-carboxylate 164b

Following Example 136a, 1-tert-butyl 3-ethylazetidine-1,3-dicarboxylate 164a was converted to a yellow oil 164b (crude). LCMS: (M+H) ^{424. 1H} NMR (500 MHz, DMSO) δ 12.1 (dd, J = 2, 1H), 8.38 (s, 1H), 8.04 (d, J = 2.5, 1H), 7.36 (s, J = 2.5, 1H), 6.06 (d, J = 2.5, 1H), 4.18 (s, 2H), 3.80 (m, 1H), 3.49 (s, 3H), 1.39 (s, 9H).

Example 164c tert-Butyl 3-(5-amino-1H-pyrazol-3-yl)azetidine-1-carboxylate 164c

164b was converted to 164c according to Example 136b and used in the next step without further purification.

Example 164d tert -Butyl 5-amino-3-(1-(tert-butoxycarbonyl)azetidin-3-yl)-1H-pyrazole-1-carboxylate 164d

Following Example 136c, 164c was converted to 164d in 26% yield.

Example 164e tert-Butyl 3-(5-(5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)-1H-pyrazol-3-yl)azetidine-1-carboxylate 164e

Following Example 136d, 164d was converted to 164e in 50% yield.

Example 164e [2-(5-{[5-(azetidin-3-yl)-1H-pyrazol-3-yl]tert-butoxycarbonylamino}-1-methyl-6-oxopyridin-3-yl)-4-fluoro-6-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]tridec-1(9),2(7)-dien-5-yl}phenyl]methyl acetate 164e

According to Example 136e, 164d was reacted with (4-fluoro-2-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-5-yl}-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methyl acetate 112b to give 164e in 39% yield.

Example 164f [2-(5-{[5-(azetidin-3-yl)-1H-pyrazol-3-yl]amino}-1-methyl-6-oxopyridin-3-yl)-4-fluoro-6-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]tridec-1(9),2(7)-dien-5-yl}phenyl]methyl acetate 164f

To a solution of 164e (300 mg, 0.42 mmol) in dioxane (2 mL) was added HCl/dioxane (4 M, 6 mL) dropwise at room temperature. The reaction mixture was stirred for 1 h. After completion of the reaction, the mixture was concentrated to afford 164f as a black solid (crude product), which was used in the next step without purification. LCMS: (M+H) ⁺ 617

According to Example 136, 164f was converted to 164 in 20% yield. LCMS: (M+H) ^{575. 1H} NMR (500 MHz, DMSO) δ 7.78 (s, 1H), 7.28 (d, J=1.5, 1H), 7.223 (s, 1H), 7.21 (s, 1H), 6.21 (s, 1H), 4.50 (m, 2H), 4.14 (m, 1H), 4.00 (m, 3H), 3.90 (m, 2H), 3.71 (s, 3H), 3.09 (m, 1H), 2.96 (m, 1H), 2.86 (m, 2H), 2.60 (m, 2H), 1.88 (m, 4H).

Example 165 2-(3-(5-(5-(azetidin-3-yl)-1H-pyrazol-3-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 165

Example 165a tert- Butyl 3-(3-(5-(2-(acetoxymethyl)-5-fluoro-3-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)phenyl)-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)-1H-pyrazol-5-yl)azetidine-1-carboxylate 165a

According to Example 164f, 164e was reacted with 4-fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 210d to afford 165a in 20% yield. LCMS: (M+H) ⁺ 700

Example 165b 2-(5-(5-(azetidin-3-yl)-1H-pyrazol-3-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-4-fluoro-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 165b

According to Example 164g, 165a was converted to crude 165b, which was used in the next step without further purification. LCMS: (M+H) ⁺ 600

According to Example 164, 165b was converted to 165 in 40% (2 steps). LCMS: (M+H) ⁺ 575. ^1H NMR (500 MHz, DMSO) δ 7.78 (s, 1H), 7.28 (d, J=1.5, 1H), 7.22 (s, 1H), 7.21 (s, 1H), 6.21 (s, 1H), 4.50 (m, 2H), 4.14 (m, 1H), 4.00 (m, 3H), 3.90 (m, 2H), 3.71 (s, 3H), 3.09 (m, 1H), 2.96 (m, 1H), 2.86 (m, 2H), 2.60 (m, 2H), 1.88 (m, 4H).

Example 166 2-(2-(Hydroxymethyl)-3-(1-methyl-6-oxo-5-(5,6,7,8-tetrahydro-1,6-naphthyridin-2-ylamino)-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 166

Example 166a 2-(5-(2-(acetoxymethyl)-3-(1-oxo-3,4,6,7,8,9-hexahydro-pyrazino[1,2-a]indol-2(1H)-yl)phenyl)-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylic acid tert-butyl ester 166a

To a microwave tube equipped with a stir bar was added tert-butyl 2-(5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate 158c (300 mg, mmol), 2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 114a (352 mg, 0.758 mmol), Pd( _PPh3 ) ₄ (39.8 mg, 0.035 mmol), _{aqueous Na2CO3} solution (1.0 N, 2.27 mL, 2.27 mmol) and 1,2-dimethoxyethane (3.5 mL). The mixture was reacted in a microwave at 130°C for 10 min. Dichloromethane (200 mL) was added, and the resulting mixture was washed with water (30 mL x 3), brine (30 mL x 1), dried over _MgSO4 , filtered, and the solvent was removed in vacuo. Silica gel column chromatography (methanol:dichloromethane = 5:95) afforded 166a.

To a round-bottom flask equipped with a stir bar was added 166a and dichloromethane (10 mL). The solution was cooled to 0°C in an ice-water bath. TFA (1 mL) was added and the resulting solution was stirred overnight. All volatiles were removed in vacuo, and THF (10 mL), i-PrOH (10 mL), _H₂O (10 mL), and LiOH _H₂O (1.00 g) were added to the flask. The resulting mixture was stirred at room temperature for 1 hr. All solvents were removed in vacuo, and the resulting residue was added to dichloromethane (200 mL). The solution was washed with water (×30 mL), brine (30 mL), dried over _MgSO₄ , filtered, and the solvent was removed in vacuo. Silica gel column chromatography (methanol:dichloromethane = 10:90) afforded 166 as a light brown solid (53 mg). MS (ESI+) m/z 551.3 (M+H).

Example 167 10-[2-(Hydroxymethyl)-3-[1-methyl-6-oxo-5-(pyrimidin-4-ylamino)-1,6-dihydropyridin-3-yl]phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 167

Example 167a (E)-Ethyl 3-(2-chloro-4,4-dimethylcyclopent-1-enyl)acrylate 167a

The following two methods were modified from Organic Preparations and Procedures Int., 29(4):471-498. 2-Chloro-4,4-dimethylcyclopent-1-enecarbaldehyde (38 g, 240 mmol) was added to benzene (240 mL) in a 500-mL single-necked round-bottom flask equipped with a magnetic stirrer and a nitrogen inlet. To this solution was added ethoxycarbonylmethylenetriphenylphosphine (84 g, 240 mmol). The mixture was stirred for 14 h. Thereafter, the solvent was evaporated and the residue was pulverized with hexane (2 L) to extract and separate the PPh ₃ byproduct from the product. The organic layer was dried over sodium sulfate and then concentrated in vacuo. The residue was purified by column chromatography using a 100% hexane-1:1 hexane/ethyl acetate gradient to give 167a in 37% yield (20 g).

Example 167b Ethyl 5,5-dimethyl-1,4,5,6-tetrahydrocyclopenta[b]pyrrole-2-carboxylate 167b

To a 250-mL single-necked round-bottom flask equipped with a magnetic stirrer and a nitrogen inlet was added 167a (17 g, 74 mmol) and DMSO (100 mL). To this solution was added sodium azide (9.6 g, 150 mmol). The mixture was then heated to 75°C and stirred for 8 h. After cooling to rt, _H₂O (100 mL) and _CH₂Cl₂ (200 _mL ) were added, and the organic layer was separated. The aqueous layer was extracted with _CH₂Cl₂ (50 _mL ). The combined organic layers were washed with brine, dried over sodium sulfate, and then concentrated in vacuo. The residue was purified by column chromatography using a 100% hexane to 1:1 hexane/ethyl acetate gradient to afford 167b in 37% yield (5.7 g).

Example 167c Ethyl 1-(cyanomethyl)-5,5-dimethyl-1,4,5,6-tetrahydrocyclopenta[b]pyrrole-2-carboxylate 167c

To a 250-mL single-necked round-bottom flask equipped with a magnetic stirrer and a nitrogen inlet was added 167b (6.2 g, 30 mmol) and DMF (57 mL). To this solution was added NaH (80% suspension in mineral oil, 1.26 g, 42.1 mmol). The resulting mixture was stirred at rt for 90 min. Thereafter, bromoacetonitrile (2.94 mL, 42 mmol) was added. The mixture was stirred for 14 h. Thereafter, water (100 mL) and ethyl acetate (200 mL) were added, and the organic layer was separated. The aqueous layer was extracted with ethyl acetate (2 × 50 mL). The combined organic layers were washed with brine, dried over sodium sulfate, and then concentrated in vacuo. The residue was purified by column chromatography to obtain 167c in a 95% yield (7 g).

Example 167d 1-(2-aminoethyl)-5,5-dimethyl-1,4,5,6-tetrahydrocyclopenta[b]pyrrole-2-carboxylic acid ethyl ester hydrochloride 167d

A 500-mL Parr reaction bottle was purged with nitrogen and charged with 10% palladium on carbon (50% humidity, 2.0 g dry weight), 167c (4.5 g, 18 mmol), 12% hydrochloric acid (9.2 mL, 37 mmol), ethyl acetate (80 mL), and ethanol (52 mL). The bottle was connected to a Parr hydrogenation reactor, evacuated, filled with hydrogen to 50 psi, and shaken for 6 h. Thereafter, the hydrogen was removed and nitrogen was filled into the bottle. Celite 521 (10.0 g) was added, and the mixture was filtered through a pad of Celite 521. The filter cake was washed with ethanol (2×50 mL), and the combined filtrates were concentrated to dryness under reduced pressure. The crude residue 167d was used in the next step without further purification.

Example 167e 4,4-Dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 167e

A 100-mL single-necked round-bottom flask equipped with a magnetic stirrer and a nitrogen inlet was purged with nitrogen and crude 167d (~18 mmol), sodium ethoxide (6.2 g, 92 mmol) and ethanol (120 mL) were added. The mixture was stirred at 55 ° C overnight. Thereafter, the reaction mixture was concentrated under reduced pressure, and the residue was partitioned between ethyl acetate (200 mL) and water (100 mL). The solution was filtered. The solid was washed with ethyl acetate (15 mL) to obtain 850 mg of 167e. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (2×100 mL). The combined organic layers were dried over sodium sulfate and then concentrated to near dryness under reduced pressure. The solution was filtered, and the solid (1.44 g) was washed with ethyl acetate (15 mL). The combined solid was dried under vacuum to obtain 167e in a 61% yield (2.3 g).

Example 167f 2-Bromo-6-(9-oxo-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-10-yl)benzyl acetate 167f

To a microwave tube equipped with a magnetic stirrer was added 167e (301 mg, 1.47 mmol), 2,6-dibromobenzyl acetate 104 g (1.1 g, 3.0 mmol), CuI (140 mg, 0.7 mmol), _{CsCO (} 961 mg, 3.0 mmol), N',N',N',N'-tetramethylethylenediamine (0.22 mL, 1.5 mmol), and 1,2-dimethoxyethane (4.1 mL). The mixture was heated in a microwave at 150°C for 3 h. Afterwards, the mixture was filtered, and the resulting solid was washed with 9:1 _CH2Cl2 /MeOH (50 mL). The combined organic phases were washed with brine (20 mL), dried over _sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by column chromatography eluting with a hexane-ethyl acetate gradient to afford 167f in a 32% yield (200 mg).

Example 167g 10-[2-(acetoxymethyl)-3-[1-methyl-6-oxo-5-(pyrimidin-4-ylamino)-1,6-dihydropyridin-3-yl]phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 167g

To a microwave tube equipped with a magnetic stirrer was added 1-methyl-3-(pyrimidin-4-ylamino)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one 109c (210 mg, 0.64 mmol), 167f (140 mg, 0.3 mmol), 1,2-dimethoxyethane (4 mL), and 1 M aqueous sodium carbonate solution (1 mL). After bubbling _N for 15 min, Pd( _PPh ) ₍ 18 mg, 0.02 mmol) was added. The mixture was heated in a microwave at 130° C. for 25 min. Thereafter, ethyl acetate (5 mL) and water (5 mL) were added. The separated aqueous layer was extracted with ethyl acetate (2 x 5 mL). The combined organic phases were washed with brine (10 mL), dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by column chromatography eluting with a gradient of _CH2Cl2 - ₆₀ :35: _{5 CH2Cl2} : ether: methanol to afford 167 g in 53% yield (93 mg).

To a 25 mL round-bottom flask equipped with a magnetic stirrer was added 167 g (93 mg, 0.17 mmol), lithium hydroxide (35 mg, 0.8 mmol), THF (0.8 mL), isopropyl alcohol (0.8 mL), and water (1.7 mL). The mixture was stirred at room temperature for 1 h. Thereafter, ethyl acetate (5 mL) and water (5 mL) were added. The separated aqueous layer was extracted with ethyl acetate (2 x 5 mL). The combined organic phases were washed with brine (10 mL), dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by column chromatography using a gradient of _CH₂Cl₂ to ₆₀ :35:5 _CH₂Cl₂ : _diethyl ether:methanol to afford 167 in 76% yield (23 mg). MS (ESI+) m/z 511.8 (M+H).

Example 168 2-(2-(Hydroxymethyl)-3-(1-methyl-5-(5-(1-methylazetidin-3-yl)-1H-pyrazol-3-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 168

Example 168a 3-(5-(azetidin-3-yl)-1H-pyrazol-3-ylamino)-5-bromo-1-methylpyridin-2(1H)-one 168a

To a solution of tert-butyl 3-(3-(5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)-1H-pyrazol-5-yl)azetidine-1-carboxylate 164e (1 g, 2.36 mmol) in dioxane (10 ml) was added dropwise HCl/dioxane (4 M, 20 mL) at room temperature. The reaction mixture was then stirred at room temperature for 1 h. After the reaction was complete, the mixture was concentrated to give 168a as a yellow solid, which was used in the next step without purification. LCMS: (M+H) ⁺ 325

Example 168b 5-Bromo-1-methyl-3-(5-(1-methylazetidin-3-yl)-1H-pyrazol-3-ylamino)pyridin-2(1H)-one 168b

To a solution of 3-(5-(azetidin-3-yl)-1H-pyrazol-3-ylamino)-5-bromo-1-methylpyridin-2(1H)-one 168a (crude, 2.36 mmol) in methanol (30 mL) and acetic acid (5 mL) was added CH ₂ O (30 wt % H ₂ O solution) (12 g, 120 mmol) at 0° C., followed by the addition of NaBH ₄ (1.8 g, 47.2 mmol) in small portions over 1 h at 0° C. After the reaction was complete, the mixture was adjusted to pH>7 with 2N aqueous NaOH. The product was then extracted with dichloromethane (60 mL×3), dried over Na ₂ SO ₄ , and concentrated to afford a yellow solid, which was further purified by flash column chromatography eluting with 50:1 dichloromethane/methanol containing 0.5% triethylamine to afford 168b as a yellow solid (50% over 2 steps).

Example 168c 2-(1-methyl-5-(5-(1-methylazetidin-3-yl)-1H-pyrazol-3-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 168c

Following Example 136e, 168b was reacted with 2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 114a to afford 168c in 20% yield.

According to Example 136, 168c was converted to 168 in 54% yield. LCMS: (M+H) ⁺ 554. ¹ H NMR (500MHz, DMSO) δ8.05(s,1H),8.01(s,1H),7.45(t,J＝8,1H),7.31(m,2H),7.24(d,J＝2.5,1H),6.50(s,1H),6.02(s,1H),4.37(m,2H),4.1 4(m,3H),3.88(m,1H),3.57(s,3H),3.53(m,2H),3.50(m,2H),3.03(m, 2H),2.61(m,2H),2.47(m,3H),2.23(s,3H),1.78(m,2H),1.69(m,2H).

Example 169 5-[2-(Hydroxymethyl)-3-[1-methyl-6-oxo-5-({4H,6H,7H-pyrazolo[3,2-c][1,4]oxazin-2-yl}amino)-1,6-dihydropyridin-3-yl]phenyl]-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-6-one 169

Example 169a {2-[1-ethyl-6-oxo-5-({4H,6H,7H-pyrazolo[3,2-c][1,4]oxazin-2-yl}amino)1,6-dihydropyridin-3-yl]-6-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-5-yl}phenyl}methyl acetate 169a

A mixture of 5-bromo-3-(6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-2-ylamino)-1-methylpyridin-2(1H)-one 110c (500 mg, 1.54 mmol), (2-{6-oxo-8-thia-5-azatricyclo[ ^7.4.0.02,7 ]trideca-1(9),2(7)-dien-5-yl}-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methyl acetate 111a (750 mg, 1.56 mmol), _PdCl2 (dppf) (170 mg, 0.23 mmol), _{K3PO4 (} 150 mg) and sodium acetate (60 mg) in MeCN (25 mL) and water (5 mL) was heated at reflux for 2 h. The solvent was evaporated in vacuo and the residue was purified by reverse phase Combi flash chromatography to afford 169a (369 mg, 40%). MS: [M+H] ⁺ 600.

A mixture of 169a (440 mg, 0.73 mmol) and LiOH hydrate (308 mg, 7.3 mmol) in isopropanol (20 mL) and _H₂O (4 mL) was stirred at 30°C for 2 h. The mixture was evaporated in vacuo, and the residue was extracted with ethyl acetate (20 mL x 2). The combined extracts were concentrated under reduced pressure. The residue was purified by preparative HPLC to afford 169 (104 mg, 26%). MS: [M+H] ^⁺ 558. ¹ H NMR(500MHz, CDCl3)δ7.93(s,1H),7.46-7.39(m,3H),7.31-7.24(m,3H),5.73(s,1H),4.78(s,2H),4.61(d,J＝11.5,1H),4.4 2-4.20(m,2H),4.14-3.98(m,4H),3.90-3.82(m,1H),3.69(s,3H),3.04-2.80(m,4H),2.60-2.46(m,2H),1.94-1.82(m,4H).

Example 170 5-[2-(Hydroxymethyl)-3-[1-methyl-5-({5-[(morpholin-4-yl)carbonyl]pyridin-2-yl}amino)-6-oxo-1,6-dihydropyridin-3-yl]phenyl]-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-6-one 170

Example 170a (6-aminopyridin-3-yl)(morpholino)methanone 170a

To a solution of morpholine (9.00 g, 103 mmol) in EtOH (400 mL) were added EDCI (10.0 g, 52.2 mmol), HOBt (7.00 g, 51.8 mmol), and 6-aminonicotinic acid (6.00 g, 43.4 mmol). After stirring for 18 h, the resulting suspension was filtered. The solid was triturated with a mixture of MeOH (100 mL) and dichloromethane (100 mL) to afford 170a as a white solid (2.7 g, 30%). LCMS: (M+H) ²⁰⁸

Example 170b 5-Bromo-1-methyl-3-(5-(morpholine-4-carbonyl)pyridin-2-ylamino)pyridin-2(1H)-one 170b

Following Example 136e, 170a was reacted with 3,5-dibromo-1-methylpyridin-2(1H)-one to afford 170b in 21% yield. LCMS: (M+H) ^{394. 1H} NMR (500 MHz, MeOD) δ 8.84 (d, J = 2.5, 1H), 8.42 (d, J = 2, 1H), 7.72 (m, 1H), 7.42 (d, J = 2, 1H), 7.11 (d, J = 8.5, 1H), 3.72 (m, 8H), 3.63 (s, 3H).

Example 170c {2-[1-methyl-5-({5-[(morpholin-4-yl)carbonyl]pyridin-2-yl}amino)-6-oxo-1,6-dihydropyridin-3-yl]-6-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-5-yl}phenyl}methyl acetate 170c

According to Example 136e, 170b was reacted with (2-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-5-yl}-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methyl acetate 111a to give 170c in 65% yield. LCMS: (M+H) ⁺ 668

According to Example 148, 170c was converted to 170 in 71% yield. LCMS: (M+H) ^{626. 1H} NMR (500 MHz, DMSO) δ 8.95 (s, 1H), 8.72 (d, J=2, 1H), 8.25 (d, J=2, 1H), 7.64 (dd, J=8.5, 1H), 7.45 (m, 2H), 7.35 (m, 3H), 4.85 (t, J=4, 1H), 4.35 (m, 2H), 4.03 (m, 1H), 3.88 (m, 1H), 3.59 (m, 7H), 3.49 (m, 4H), 2.98 (m, 1H), 2.85 (m, 1H), 2.77 (m, 2H), 2.54 (m, 2H), 1.79 (m, 4H).

Example 171 2-(2-(hydroxymethyl)-3-(1-methyl-5-(5-(morpholine-4-carbonyl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 171

Example 171a 2-(1-methyl-5-(5-(morpholine-4-carbonyl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 171a

According to Example 136d, 5-bromo-1-methyl-3-(5-(morpholine-4-carbonyl)pyridin-2-ylamino)pyridin-2(1H)-one 170a and 2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 114a were reacted to give 171a in 27% yield. LCMS: (M+H) ⁶⁵¹

According to Example 170, 171a was converted to 171 in 60% yield. LCMS: (M+H) ^{609. 1H} NMR (500 MHz, DMSO) δ 8.85 (d, J=2, 1H), 8.32 (d, J=1.5, 1H), 7.71 (dd, J=8.5, 1H), 7.53 (m, 1H), 7.43 (m, 3H), 7.35 (m, 3H), 7.11 (d, J=8, 1H), 6.72 (s, 1H), 4.56 (m, 2H), 4.21 (s, 3H), 4.03 (m, 1H), 3.71 (m, ^1H ), 2.65 (m, 2H), 2.55 (m, 2H), 1.89 (m, 2H), 1.80 (m, 1H).

Example 172 2-(2-(hydroxymethyl)-3-(1-methyl-5-(5-(1-methylazetidin-3-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 172

Example 172a 5-Bromo-1-methyl-3-(5-(1-methylazetidin-3-yl)pyridin-2-ylamino)pyridin-2(1H)-one 172a

A mixture of 3-(5-(azetidin-3-yl)pyridin-2-ylamino)-5-bromo-1-methylpyridin-2(1H)-one 155n (469 mg, 1.4 mmol), 37% aqueous formaldehyde (4 g, 50 mmol), _NaBH₃CN (261 mg, 4.2 mmol), and 1M zinc chloride in 1,2-diethoxyethane (4 mL, 4.2 mmol) and methanol (40 mL) was stirred at room temperature for 2 hours. The mixture was added to water (20 mL) and extracted with dichloromethane (50 mL x 3). The organic layer was concentrated under reduced pressure. The residue was purified by column chromatography eluting with 10:1 dichloromethane/methanol to afford 172a (0.3 g, 83%) as a yellow solid. MS: [M+H] ^⁺ 348.

Example 172b 2-(1-methyl-5-(5-(1-methylazetidin-3-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 172b

To a sealed tube was added a mixture of 172a (167 mg, 0.48 mmol), 2-(2-(hydroxymethyl)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 114a (231 mg, 0.48 mmol), Pd(dppf) _Cl₂ (39 mg, 0.048 mmol), _{and Na₂CO₃} (101 mg, 0.96 mmol) in N,N-dimethylformamide (18 mL). The system was evacuated and then refilled with _N₂ . The reaction mixture was heated at 150° C. under microwave irradiation for 1 h. The mixture was then cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by column chromatography, eluting with 15:1 dichloromethane/methanol to afford 172b as a brown solid (140 mg, 50%). MS: [M+H] ⁺ 606.

To a solution of 172b (150 mg, 0.25 mol) in THF/isopropyl acetate/ _H₂O (6 mL/6 mL/2 mL) was added LiOH (346 mg, 14 mmol) while stirring at room temperature. The mixture was stirred for 2 h. Then, 20 mL of water was added, and the mixture was extracted with ethyl acetate (60 mL x 3 ₎ . The combined organic layers were dried over _Na₂SO₄ and concentrated to give a yellow solid. Further purification by preparative HPLC afforded 172 (50 mg, 35%) as a white solid. MS: [M+H] ^⁺ 565. ¹ H NMR (500MHz, DMSO) δ8.66(s,1H),8.54(s,1H),8.10(s,1H),7.65(d,J＝8,1H),7.46(m,1H),7.31(m,4H),6.51(s,1H),4.84(s,1H),4.33(m ,2H),4.13(m,3H),3.85(m,1H),3.59(s,3H),3.53(m,2H),3.35(m,1H),3.02(m,2H),2.60(m,2H),2.46(m,2H),2.23(s,3H),1.77(m,4H).

Example 173 10-[2-(Hydroxymethyl)-3-(1-methyl-5-{[5-(4-methylpiperazin-1-yl)pyridin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 173

Example 173a 10-[2-(acetoxymethyl)-3-(1-methyl-5-{[5-(4-methylpiperazin-1-yl)pyridin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 173a

In a 10-mL glass container equipped with a magnetic stir bar, 1-methyl-3-[5-(4-methylpiperazin-1-yl)pyridin-2-ylamino]-5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-pyridin-2-one 197e (210 mg, 0.49 mmol), 10-[2-(acetoxymethyl)-3-bromophenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 167f (143 mg, 0.33 mmol), and Pd(PPh ₃ ) ₄ (30 mg, 0.0.26 mmol) were added to 2N Na ₂ CO ₃ (2 mL) and 1,2-dimethoxyethane (5 mL). The vessel was sealed with a stopper and placed in a microwave cavity. After the reaction mixture was stirred at 125° C. for 7 min, it was purified by flash chromatography (dichloromethane:methanol, 85:15) to afford 26% (50 mg) of 173a as a solid.

To a 100-mL single-necked round-bottom flask equipped with a magnetic stir bar was added 173a (50 mg, 0.077 mmol), LiOH· _H₂O (20 mg, 0.83 mmol), THF (2 mL), isopropanol (2 mL), and water (2 mL). The reaction mixture was stirred at room temperature for 3 h, then partitioned between dichloromethane (5 mL) and water (5 mL), and the organic phase was extracted with dichloromethane (3 x 5 mL). The combined organic phases were washed with water (5 mL x 2) and brine (5 mL x ₁ ), dried ( _Na₂SO₄ ), and then concentrated. The crude product was redissolved in dichloromethane (3 mL). To this solution was added hexane (10 mL), and the resulting precipitate was filtered to provide 173 in a 75% yield (35 mg). MS ( ^ESI⁺ ) m/z 608.4 (M+H).

Example 174 2-(2-(Hydroxymethyl)-3-(4-methyl-5-oxo-6-(1,2,3,4-tetrahydroisoquinolin-6-ylamino)-4,5-dihydropyrazin-2-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 174

Example 174a tert- Butyl 6-(6-(2-(acetoxymethyl)-3-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)phenyl)-4-methyl-3-oxo-3,4-dihydropyrazin-2-ylamino)-3,4-dihydroisoquinoline-2(1H)-carboxylate 174a

To a microwave tube equipped with a stir bar was added tert-butyl 6-(6-bromo-4-methyl-3-oxo-3,4-dihydropyrazin-2-ylamino)-3,4-dihydroisoquinoline-2(1H)-carboxylate 120a (300 mg, 0.689 mmol), 114a (352 mg, 0.758 mmol), Pd(PPh ₃ ) ₄ (39.8 mg, 0.0345 mmol), aqueous Na ₂ CO ₃ solution (1.0 N, 2.27 mL, 2.27 mmol), and 1,2-dimethoxyethane (3.5 mL). The mixture was reacted in a microwave at 130° C. for 10 min. Dichloromethane (200 mL) was added, and the resulting mixture was washed with water (30 mL × 3), brine (30 mL × 1), dried over MgSO ₄ , filtered, and the solvent removed in vacuo. Silica gel column chromatography (methanol:dichloromethane=5:95) gave 174a.

To a round-bottom flask equipped with a stir bar was added 174a and dichloromethane (50 mL). The solution was cooled to 0°C in an ice-water bath. TFA (1 mL) was added and the resulting solution was stirred overnight. All volatiles were removed in vacuo, and THF (10 mL), isopropanol (10 mL), water (10 mL), and LiOH monohydrate (1.00 g) were added to the flask. The resulting mixture was stirred at room temperature for 1 hr. The solvent was removed in vacuo, and the resulting residue was added to dichloromethane (200 mL). The solution was washed with water (330 mL) and brine (30 mL), dried over _MgSO₄ , filtered, and then the solvent was removed in vacuo. Silica gel column chromatography (methanol:dichloromethane = 10:90) gave 28 mg of 174 as a yellow solid. MS (ESI+) m/z 551.4 (M+H).

Example 175 10-[2-(Hydroxymethyl)-3-(1-methyl-6-oxo-5-{4H,6H,7H-pyrazolo[3,2-c][1,4]oxazin-2-ylamino}-1,6-dihydropyridin-3-yl)phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 175

Example 175a 3-(6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-2-ylamino)-1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one 175a

To a sealed tube equipped with a magnetic stirrer was added 5-bromo-3-(6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-2-ylamino)-1-methylpyridin-2(1H)-one (110c) (250 mg, 0.77 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bis(1,3,2-dioxaborolane) (290 mg, 1.2 mmol), potassium acetate (230 mg, 2.3 mmol) and 1,4-dioxane (5.5 mL). After nitrogen was bubbled through the resulting suspension for 30 minutes, [1,1'-bis(diphenylphosphino)ferrocene]palladium(II) dichloride and _{CH2Cl2 complex} (PdCl2dppf: _CH2Cl2 (1: ₁ ), 63 mg, 0.08 mmol ₎ was added and the reaction was stirred at 105°C for 90 minutes. Afterwards, the mixture was cooled to ambient temperature and partitioned between water (20 mL) and ethyl acetate (20 mL). The separated aqueous layer was extracted with ethyl acetate (2 x 10 mL). The combined organic phases were washed with brine (30 mL), dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting crude 175a was used in the next step without further purification.

Example 175b 10-[2-(acetoxymethyl)-3-(1-methyl-6-oxo-5-{4H,6H,7H-pyrazolo[3,2-c][1,4]oxazin-2-ylamino}-1,6-dihydropyridin-3-yl)phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 175b

To a microwave tube equipped with a magnetic stirrer was added 175a (120 mg, 0.31 mmol), 167f (130 mg, 0.3 mmol), 1,2-dimethoxyethane (4 mL), and 1 M aqueous sodium carbonate solution (1 mL). After blowing _N2 for 15 min, Pd( _PPh3 ) ₄ (18 mg, 0.02 mmol) was added. The mixture was heated in a microwave at 130°C for 10 min. Thereafter, ethyl acetate (5 mL) and water (5 mL) were added. The separated aqueous layer was extracted with ethyl acetate (2 x 5 mL). The combined organic phases were washed with brine (10 mL), dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The residue was purified by column chromatography eluting with a gradient of _CH2Cl2-60 : ₃₅ : _{5 CH2Cl2} :ether:methanol to afford 175b in 74% yield (140 mg).

To a 25 mL round-bottom flask equipped with a magnetic stirrer was added 175b (140 mg, 0.23 mmol), lithium hydroxide (49 mg, 1.2 mmol), THF (1.2 mL), i-PrOH (1.2 mL), and water (2.4 mL). The mixture was stirred at rt for 90 min. Thereafter, ethyl acetate (5 mL) and water (5 mL) were added. The separated aqueous layer was extracted with ethyl acetate (2 x 5 mL). The combined organic phases were washed with brine (10 mL), dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by column chromatography using a gradient of _CH₂Cl₂ to ₆₀ :35:5 _CH₂Cl₂ :diethyl ether: _methanol to afford 175 in 79% yield (100 mg). MS (ESI+) m/z 555.3 (M+H).

Example 176 2-(2-(Hydroxymethyl)-3-(5-(5-(methoxymethyl)-1-methyl-1H-pyrazol-3-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 176

Example 176a 5-(Methoxymethyl)-1-methyl-3-nitro-1H-pyrazole 176a

To a 250-mL single-necked round-bottom flask equipped with a magnetic stirrer and a reflux condenser were added _CH₃OH (50 mL), 5-(bromomethyl)-1-methyl-3-nitro-1H-pyrazole 156d (8.8 g, 40 mmol), and _CH₃ONa (4.3 g, 80 mmol). The reaction mixture was heated under reflux for 2 h. The reaction was then cooled to room temperature and concentrated. The residue was partitioned between ethyl acetate (60 mL) and water (60 mL). The aqueous layer was separated and extracted with ethyl acetate (50 mL x 2). The organic layers were combined, washed with brine (50 mL), and then dried over sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure to afford 176a (6.1 g, 90%) as a yellow oil. LCMS: [M+H] ^⁺ 172.

Example 176b 5-(Methoxymethyl)-1-methyl-1H-pyrazol-3-amine 176b

To a 250-mL single-necked round-bottom flask equipped with a magnetic stirrer was added 176a (4 g, 23 mmol), Pd/C (1 g), and C ₂ H ₅ OH (100 mL). The mixture was hydrogenated at room temperature for 15 h. The mixture was then filtered, and the filtrate was concentrated under reduced pressure to afford 176b as a yellow oil (3.3 g, 99%), which was used in the next step without further purification. MS: [M+H] ⁺ 142.

Example 176c 5-Bromo-3-(5-(methoxymethyl)-1-methyl-1H-pyrazol-3-ylamino)-1-methylpyridin-2(1H)-one 176c

Following Example 148a, 1.7 g of 176b and 3.2 g of 3,5-dibromo-1-methylpyridin-2(1H)-one were reacted to afford 176c (2.8 g, 70%) as a yellow solid. MS: [M+H] ⁺ 327. ¹ H NMR (500 MHz, CDCl 3 ) δ 7.86 (d, J = 2.5, 1H), 7.38 (s, 1H), 6.88 (d, J = 2.5, 1H), 5.86 (s, 1H), 4.41 (s, 2H), 3.82 (s, 3H), 3.58 (s, 3H), 3.36 (s, 3H).

Example 176d 2-(5-(5-(methoxymethyl)-1-methyl-1H-pyrazol-3-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 176d

According to Example 148b, 557 mg of 2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 114a and 327 mg of 5-bromo-3-(5-(methoxymethyl)-1-methyl-1H-pyrazol-3-ylamino)-1-methylpyridin-2(1H)-one 176c were reacted to give 176d (420 mg, 72%) as a yellow solid. LCMS: [M+H] ⁺ 585

According to Example 148, 240 mg of 176d was converted to 176 (133 mg, 60%) as a white solid. LCMS: [M+H] ⁺ 542. ¹ H NMR (500 MHz, CDCl 3 ) δ 7.91 (d, J=2, 1H), 7.43 (m, 3H), 7.30 (d, J=2, 1H), 7.23 (s, 1H), 6.85 (s, ^1H ), 5.96 (s, 1H), 4.60 (d, J=6, 1H), 4.40 (m, 3H), 4.16 (m, 3H), 3.94 (m, 1H), 3.76 (s, 3H), 3.69 (s, 3H), 3.34 (s, 3H), 2.60 (m, 4H), 1.89 (m, 2H), 1.78 (m, 2H).

Example 177 5-[2-(Hydroxymethyl)-3-(5-{[5-(methoxymethyl)-1-methyl-1H-pyrazol-3-yl]amino}-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)phenyl]-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-6-one 177

Example 177a [2-(5-{[5-(methoxymethyl)-1-methyl-1H-pyrazol-3-yl]amino}-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-6-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]tridec-1(9),2(7)-dien-5-yl}phenyl]methyl acetate 177a

According to Example 176d, 327 mg of 5-bromo-3-(5-(methoxymethyl)-1-methyl-1H-pyrazol-3-ylamino)-1-methylpyridin-2(1H)-one 176b was reacted with 481 mg of 111a to give 177a (420 mg, 70%) as a yellow solid. LCMS: [M+H] ⁺ 602

According to Example 176, 240 mg of 177a was converted to 177 (112 mg, 50%) as a white solid. LCMS: [M+H] ⁺ 560. ¹ H NMR (500 MHz, CDCl 3 ) δ 7.90 (s, 1H), 7.44-7.38 (m, 3H), 7.30-7.24 (m, 2H), 5.94 (s, 1H), 4.62 (d, J=11.5, 1H), 4.37 (m, 3H), 4.26 (d, J=10.5, 1H), 4.05 (m, 1H), 3.87 (m, 1H), 3.75 (s, 3H), 3.68 (s, 3H), 3.34 (s, 3H), 2.85 (m, 4H), 2.54 (s, 2H), 1.89 (s, 5H).

Example 178 2-(5-fluoro-2-(hydroxymethyl)-3-(5-(5-(methoxymethyl)-1-methyl-1H-pyrazol-3-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 178

Example 178a 4-Fluoro-2-(5-(5-(methoxymethyl)-1-methyl-1H-pyrazol-3-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 178a

To a sealed tube was added a mixture of 4-fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 210d (230 mg, 0.48 mmol), 5-bromo-3-(5-(methoxymethyl)-1-methyl-1H-pyrazol-3-ylamino)-1-methylpyridin-2(1H)-one 176c (156 mg, 0.48 mmol), Pd(dppf) _Cl₂ (39 mg, 0.048 mmol), _{and Na₂CO₃} (101 mg, 0.96 mmol) in DMF (18 mL). The system was evacuated and then refilled with _N₂ . The reaction mixture was heated at 110°C for 2 h. The mixture was then cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure and the resulting residue was purified by column chromatography eluting with 15:1 dichloromethane/methanol to afford 178a (160 mg, 53%) as a brown solid. MS: [M+H] ⁺ 603.

To a solution of 178a (160 mg, 0.27 mol) in THF/iPA/ _H₂O (6 mL/6 mL/ ₂ mL) was added LiOH (346 mg, 14 mmol) while stirring at room temperature. The mixture was stirred for 2 h. Then, 20 mL of water was added, and the mixture was extracted with ethyl acetate (60 mL x 3). The combined organic layers were dried over _Na₂SO₄ and concentrated to give a yellow solid. Further purification by preparative HPLC afforded 178 (60 mg, 40%) as a white solid. LCMS: [M+H] ^⁺ 561. ¹ H NMR(500MHz,DMSO)δ8.16(s,1H),7.99(m,1H),7.32(m,2H),7.18(m,1H),6.52(s,1H),6.11(s,1H),4.89(m,1H),4.38(m,2 H),4.31(m,2H),4.17(m,3H),3.90(m,1H),3.64(s,3H),3.57(s,3H),3.26(s,3H),2.63(m,2H),2.51(m,2H),1.70(m,4H).

Example 179 5-[2-(Hydroxymethyl)-3-(1-methyl-5-{[5-(1-methylazetidin-3-yl)pyridin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)phenyl]-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-6-one 179

Example 179a [2-(1-methyl-5-{[5-(1-methylazetidin-3-yl)pyridin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)-6-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]tridec-1(9),2(7)-dien-5-yl}phenyl]methyl acetate 179a

According to Example 176d, 5-bromo-1-methyl-3-(5-(1-methylazetidin-3-yl)pyridin-2-ylamino)pyridin-2(1H)-one 172a and (2-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-5-yl}-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methyl acetate 111a were reacted to give 179a in 55% yield. LCMS: (M+H) ⁺ 624

According to Example 176, 179a was converted to 179 in 42% yield. LCMS: (M+H) ^{582. 1H} NMR (500 MHz, MEOD) δ 8.73 (s, 1H), 8.17 (s, 1H), 7.74 (m, 1H), 7.51 (m, 1H), 7.40 (m, 3H), 7.10 (d, J=3.5, 1H), 4.55 (m, 2H), 4.31 (m, 2H), 4.13 (m, 1H), 4.06 (s, 3H), 3.99 (m, 1H), 3.71 (s, 3H), 3.05 (m, 1H), 2.94 (m, 6H), 2.59 (m, 2H), 1.89 (m, 4H).

Example 180 6-[2-(Hydroxymethyl)-3-{1-oxo-1H,2H,3H,4H,6H,7H,8H,9H-pyrazino[1,2-a]indol-2-yl}phenyl]-2-methyl-4-[(5-{[methyl(propan-2-yl)amino]methyl}pyridin-2-yl)amino]-2,3-dihydropyridazin-3-one 180

Example 180a Methyl 6-(6-chloro-2-methyl-3-oxo-2,3-dihydropyridazin-4-ylamino)nicotinate 180a

According to Example 186b, 4-bromo-6-chloro-2-methylpyridazin-3(2H)-one 103e (4.0 g, 17.9 mmol), 186a (2.7 g, 17.9 mmol), cesium carbonate (12.8 g, 39.4 mmol) and Xantphos (880 mg, 8.5 mol%), dioxane (120 ml) and tris(dibenzylideneacetone)dipalladium(0) (820 mg, 5 mol%) were reacted to give 180a (3.0 g, 57% yield).

Example 180b 6-Chloro-4-(5-(hydroxymethyl)pyridin-2-ylamino)-2-methylpyridazin-3(2H)-one 180b

According to Example 186c, 180a (3.0 g, 10.2 mmol), dichloromethane (100 mL) and 1.0 M DIBAL-H were reacted in dichloromethane (30.5 mL, 30.5 mmol) to give 180b (2.3 g, 86% yield).

Example 180c (6-(6-chloro-2-methyl-3-oxo-2,3-dihydropyridazin-4-ylamino)pyridin-3-yl)methyl methanesulfonate 180c

Intermediate 180b was treated with diisopropylethylamine and methanesulfonyl chloride in dichloromethane at 0°C to afford 180c.

Example 180d 6-Chloro-4-(5-((isopropyl(methyl)amino)methyl)pyridin-2-ylamino)-2-methylpyridazin-3(2H)-one 180d

Following Example 186c, 180c was reacted with methylisopropylamine to give 180d.

Example 180e 2-(5-(5-((isopropyl(methyl)amino)methyl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridazin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 180e

According to Example 179a, 180d (125 mg, 0.39 mmol), 114a (215 mg, 0.47 mmol), 1N _Na2CO3 (0.86 mL) and _tetrakis (triphenylphosphine)palladium(0) (23 mg, 5 mol%) were reacted to give 180e (150 mg, 61% yield).

According to Example 179, 180e (150 mg, 0.24 mmol), 1N LiOH (1.2 mL), THF (2 mL), and isopropanol (2 mL) were reacted, and the mixture was purified by column chromatography (silica gel, methanol/dichloromethane) to give 180 (105 mg, 75% yield). MS (ESI+) m/z 582.5 (M+H).

Example 181 5-[3-(5-{[5-(4-ethylpiperazin-1-yl)pyridin-2-yl]amino}-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-2-(hydroxymethyl)phenyl]-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-6-one 181

Example 181a 5-[3-(5-{[5-(4-ethylpiperazin-1-yl)pyridin-2-yl]amino}-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-2-(acetoxymethyl)phenyl]-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-6-one 181a

To a microwave tube equipped with a stir bar was added 5-bromo-3-(5-(4-ethyl-piperazin-1-yl)pyridin-2-ylamino)-1-methylpyridin-2(1H)-one 138c (250 mg, 0.637 mmol), boronate ester 111a (308 mg, 0.701 mmol), Pd(PPh ₃ ) ₄ (36.8 mg, 0.0319 mmol), aqueous Na ₂ CO ₃ solution (1.0 N, 2.10 mL, 2.10 mmol), and 1,2-dimethoxyethane (3.0 mL). The mixture was reacted in a microwave at 130° C. for 10 min. Dichloromethane (200 mL) was added, and the resulting mixture was washed with water (3×30 mL), brine (30 mL×1), dried over MgSO ₄ , filtered, and the solvent removed in vacuo. Silica gel column chromatography (methanol:dichloromethane=5:95) gave 181a.

To a round-bottom flask equipped with a stir bar was added 181a, THF (5 mL), isopropanol (5 mL), water (5 mL), and LiOH monohydrate (300 mg). The resulting mixture was stirred at room temperature for 1 hr. All solvents were removed in vacuo, and the resulting residue was added to dichloromethane (200 mL). The solution was washed with water (30 mL x 3), brine (30 mL x 1), dried over _MgSO₄ , filtered, and the solvent was removed in vacuo. Silica gel column chromatography (methanol:dichloromethane = 10:90) afforded 181 as a brick-red solid, 101 mg, MS (ESI+) m/z 625.4 (M+H).

Example 182 5-(3-(6,6-dimethyl-3,4,6,7-tetrahydro-5H-cyclopenta[4,5]thieno[2,3-c]pyridin-1(2H)-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-1-methyl-3-(pyrimidin-4-ylamino)pyridin-2(1H)-one 182

Example 182a tert-Butyl (2,6-dibromo-4-fluorobenzyloxy) dimethylsilane 182a

A 250-mL single-necked round-bottom flask equipped with a magnetic stirrer was purged with nitrogen and 197b (5.00 g, 17.6 mmol), imidazole (6.00 g, 88.0 mmol) and dichloromethane (125 mL) were added. Tert-butyldimethylsilyl chloride (7.96 g, 52.8 mmol) was added and the reaction mixture was stirred at room temperature for 16 h. Thereafter, the reaction was diluted with water (100 mL) and dichloromethane (100 mL). The layers were separated and the aqueous phase was extracted with dichloromethane (100 mL). The organic extracts were combined and dried over sodium sulfate. The desiccant was removed by filtration and the solvent was evaporated under reduced pressure. The resulting residue was purified by flash chromatography to afford 96% yield (6.75 g) of 182a as a colorless oil: ¹ H NMR (500 MHz, CDCl ₃ ) 7.30 (d, 2H, J=8.0 Hz), 4.93 (s, 2H), 0.93 (s, 9H), 0.15 (s, 6H).

Example 182b 2-(3-bromo-2-((tert-butyldimethylsilyloxy)methyl)-5-fluorophenyl)-6-(6,6-dimethyl-3,4,6,7-tetrahydro-5H-cyclopenta[4,5]thieno[2,3-c]pyridin-1(2H)-one 182b

To a 250-mL single-necked round-bottom flask equipped with a magnetic stirrer and a nitrogen inlet were added 105h (400 mg, 1.81 mmol), 182a (1.44 g, 3.62 mmol), cesium carbonate (1.18 g, 3.62 mmol), N,N'-dimethylethylenediamine (159 mg, 1.81 mmol) and 1,4-dioxane (15 mL). After bubbling nitrogen through the resulting suspension for 30 min, cuprous iodide (174 mg, 0.905 mmol) was added. A reflux condenser was connected to the flask, and the reaction mixture was heated at 100 ° C for 16 h. Thereafter, the mixture was cooled to room temperature and filtered. The filtrate was diluted with ethyl acetate (100 mL) and water (50 mL), and the layers were separated. The aqueous layer was extracted with ethyl acetate (2×30 mL), and the combined organic layers were washed with brine (100 mL) and then dried over sodium sulfate. The desiccant was removed by filtration. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography to afford 182b as a white solid in 57% yield (554 mg): mp 38-39°C; ¹ H NMR (500 MHz, CDCl ₃ ) δ 7.32 (dd, 1H, J=8.0, 2.5 Hz), 7.00 (dd, 1H, J=8.0, 2.5 Hz), 4.74 (s, 2H), 3.93 (m, 2H), 2.98 (m, 1H), 2.80 (m, 1H), 2.77 (s, 2H), 2.52 (s, 2H), 1.28 (s, 3H), 1.27 (s, 3H), 0.89 (s, 9H), 0.11 (s, 6H).

Example 182c 2-((tert-Butyldimethylsilyloxy)methyl)-5-fluorophenyl-6-(6,6-dimethyl-3,4,6,7-tetrahydro-5H-cyclopenta[4,5]thieno[2,3-c]pyridin-1(2H)-yl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-one 182c

To a 100-mL single-necked round-bottom flask equipped with a magnetic stirrer and a nitrogen inlet was added 182b (554 mg, 1.03 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bis(1,3,2-dioxaborolane) (785 mg, 3.09 mmol), potassium acetate (404 mg, 4.12 mmol) and 1,4-dioxane (8 mL). After nitrogen was bubbled through the resulting suspension for 30 min, Pd(dppf)Cl ₂ ·CH ₂ Cl ₂ (75 mg, 0.103 mmol) was added. A reflux condenser was attached to the flask, and the reaction mixture was heated under reflux for 8 h. Thereafter, the mixture was diluted with ethyl acetate (100 mL) and water (75 mL), and the layers were separated. The aqueous layer was extracted with ethyl acetate (2×50 mL), and the combined organic layers were washed with brine (100 mL) and then dried over sodium sulfate. The drying agent was removed by filtration. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography to give 182c as a white solid in 91% yield (600 mg): mp 41-42°C; ¹ H NMR (500 MHz, CDCl ₃ ) δ 7.52 (dd, 1H, J=8.0, 2.5 Hz), 7.02 (dd, 1H, J=8.0, 2.5 Hz), 5.14 (d, 1H, J=10.5 Hz), 5.70 (d, 1H, J=10.5 Hz), 3.93 (m, 2H), 2.98 (m, 1H), 2.80 (m, 1H), 2.77 (s, 2H), 2.52 (s, 2H), 1.34 (s, 3H), 1.33 (s, 3H), 1.26 (s, 12H), 0.84 (s, 9H), 0.04 (s, 6H).

A 100-mL single-necked round-bottom flask equipped with a magnetic stirrer and a reflux condenser was purged with nitrogen and charged with 182c (300 mg, 0.513 mmol), 109b (111 mg, 0.394 mmol), sodium carbonate (125 mg, 1.18 mmol), 1,4-dioxane (8 mL), and water (2 mL). The mixture was degassed with nitrogen for 30 min. Tetrakis(triphenylphosphine)palladium(0) (46 mg, 0.039 mmol) was added. After heating at reflux for 2 h, the reaction mixture was cooled to room temperature and then partitioned between water (40 mL) and dichloromethane (100 mL). The layers were separated, and the aqueous phase was extracted with dichloromethane (2 x 50 mL). The organic extracts were combined, dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was dissolved in THF (3 mL), and tetrabutylammonium fluoride trihydrate (372 mg, 1.18 mmol) was added. After stirring at room temperature for 2 h, the reaction mixture was partitioned between water (20 mL) and dichloromethane (20 mL). The layers were separated, and the aqueous phase was extracted with dichloromethane (2×20 mL). The organic extracts were combined, dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by flash chromatography to afford 182 as an off-white solid in 41% yield (88 mg): mp>250°C; ¹ H NMR (500 MHz, DMSO-d ₆ )δ9.22(s,1H),8.72(d,1H,J＝2.0Hz),8.65(s,1H),8.29(d,1H,J＝6.0Hz),7.57( d,1H,J＝2.0Hz),7.34(dd,1H,J＝8.5,3.0Hz),7.31(d,1H,J＝6.0Hz),7.29(dd,1H ,J＝8.5,3.0Hz),4.87(m,1H),4.31(m,2H),4.04(m,1H),3.84(m,1H),3.60(s,3H ),3.03(m,1H),2.89(m,1H),2.75(s,2H),2.53(m,2H),1.23(s,6H); MS(ESI+)m/z 546.2(M+H).

Example 183 5-(3-(6,6-dimethyl-3,4,6,7-tetrahydro-5H-cyclopenta[4,5]thieno[2,3-c]pyridin-1(2H)-yl)-2-(hydroxymethyl)phenyl)-1-methyl-3-(6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-3-ylamino)pyridin-2(1H)-one 183

According to Example 184, 131a (300 mg, 0.606 mmol) and 110c (151 mg, 0.466 mmol) were reacted to give 183 as an off-white solid in 51% yield (129 mg): mp 167-168 °C; ¹ H NMR (500 MHz, DMSO-d ₆ )δ8.18(s,1H),7.95(d,1H,J＝2.0Hz),7.44(t,1H,J＝8.0Hz),7.33(dd,1H,J＝8.0, 1.0Hz),7.29(dd,1H,J＝8.0,1.0Hz),7.23(d,1H,J＝2.0Hz),5.92(s,1H),4.81(m, 1H),4.71(s,2H),4.36(m,2H),4.01(m,3H),3.94(m,2H),3.85(m,1H),3.57(s,3H ),3.02(m,1H),2.86(m,1H),2.75(m,2H),2.53(m,2H),1.23(s,6H); MS(ESI+)m/z 572.3(M+H).

Example 184 5-(3-(6,6-dimethyl-3,4,6,7-tetrahydro-5H-cyclopenta[4,5]thieno[2,3-c]pyridin-1(2H)-yl)-2-(hydroxymethyl)phenyl)-1-methyl-3-(pyridin-2-ylamino)pyridin-2(1H)-one 184

Example 184a 5-Bromo-1-methyl-3-(pyridin-2-ylamino)pyridin-2(1H)-one 184a

To a 100-mL single-necked round-bottom flask equipped with a magnetic stirrer, a nitrogen inlet, and a reflux condenser was added 3,5-dibromo-1-methylpyridin-2(1H)-one (936 mg, 3.51 mmol), 2-aminopyridine (300 mg, 3.19 mmol), cesium carbonate (3.11 g, 9.57 mmol), and 1,4-dioxane (20 mL). After nitrogen was bubbled through the resulting solution for 20 minutes, Xantphos (184 mg, 0.319 mmol) and tris(dibenzylideneacetone)dipalladium(0) (146 mg, 0.160 mmol) were added, and the reaction mixture was heated at 100° C. for 3 h. Afterwards, the reaction was cooled to room temperature, filtered, and then concentrated under reduced pressure. The resulting residue was purified by flash chromatography to afford 184a as an off-white solid in 42% yield (376 mg): mp 153-154 °C; ¹ H NMR (300 MHz, DMSO-d ₆ ) 8.75 (s, 1H), 8.69 (d, 1H, J=2.4 Hz), 8.26 (dd, 1H, J=5.4, 1.5 Hz), 7.61 (m, 1H), 7.54 (d, 1H, J=2.4 Hz), 7.33 (d, 1H, J=5.4 Hz), 6.86 (m, 1H), 3.45 (s, 3H).

A 100-mL single-necked round-bottom flask equipped with a magnetic stirrer and reflux condenser was purged with nitrogen and charged with 131a (300 mg, 0.606 mmol), 184a (131 mg, 0.466 mmol), sodium carbonate (148 mg, 1.40 mmol), 1,4-dioxane (8 mL), and water (2 mL). The mixture was degassed with nitrogen for 30 min. Tetrakis(triphenylphosphine)palladium (54 mg, 0.047 mmol) was added. After heating at 100°C for 3 h, the reaction mixture was cooled to room temperature and then partitioned between water (40 mL) and dichloromethane (100 mL). The layers were separated, and the aqueous phase was extracted with dichloromethane (2 x 50 mL). The organic extracts were combined, dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was dissolved in methanol (5 mL), and potassium carbonate (500 mg, 3.62 mmol) was added. After stirring at room temperature for 2 h, the reaction mixture was partitioned between water (20 mL) and dichloromethane (20 mL). The layers were separated, and the aqueous phase was extracted with dichloromethane (2×20 mL). The organic extracts were combined, dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by flash chromatography to give 184 as a white solid in 49% yield (118 mg): mp 150–151° C.; ¹ H NMR (500 MHz, DMSO-d ₆ )δ8.66(d,1H,J＝2.0Hz),8.57(m,1H),8.16(dd,1H,J＝5.0,1.0Hz),7.57(m,1H),7.45 (t,1H,J＝8.0Hz),7.38(d,1H,J＝2.0Hz),7.36–7.31(m,2H),7.28(d,1H,J＝8.0Hz),6.7 8(dd,1H,J＝11.0,5.0Hz),4.82(m,1H),4.35(m,2H),4.02(m,1H),3.86(m,1H),3.59( s,3H),3.01(m,1H),2.87(m,1H),2.75(s,2H),2.53(m,2H),1.23(s,6H); MS(ESI+)m/z 527.2(M+H).

Example 185 5-(3-(6,6-dimethyl-3,4,6,7-tetrahydro-5H-cyclopenta[4,5]thieno[2,3-c]pyridin-1(2H)-yl)-2-(hydroxymethyl)phenyl)-1-methyl-3-(1,5-dimethyl-1H-pyrazol-4-ylamino)pyridin-2(1H)-one 185

Using the same general procedure as described for the preparation of 184, 131a (300 mg, 0.606 mmol) was reacted with 142a (138 mg, 0.466 mmol) to afford 185 in 41% yield (104 mg) as a yellow solid: mp 164-165 °C; ¹ H NMR (500 MHz, DMSO-d ₆ )δ7.93–7.92(m,2H),7.44(t,1H,J＝7.5Hz),7.33(dd,1H,J＝7.5,1.0Hz),7.2 9(dd,1H,J＝7.5,1.0Hz),7.21(d,1H,J＝2.0Hz),5.88(s,1H),4.83(m,1H),4.3 6(m,2H),4.02(m,1H),3.87(m,1H),3.57(s,3H),3.56(s,3H),3.03(m,1H),2 .86(m,1H),2.75(m,2H),2.53(m,2H),2.17(s,3H),1.23(s,6H); MS(ESI+)m/z 544.2(M+H).

Example 186 2-(2-(Hydroxymethyl)-3-(5-(5-((isopropyl(methyl)amino)methyl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 186

Example 186a Methyl 6-(5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)nicotinate 186a

To a 300 mL sealed tube was added 3,5-dibromo-1-methylpyridin-2(1H)-one (4.0 g, 15.0 mmol), methyl 6-aminonicotinate (2.3 g, 15.0 mmol), cesium carbonate (10.7 g, 33 mmol), and Xantphos (740 mg, 8.5 mol%). The flask was evacuated and filled with nitrogen 3X. Dioxane (100 ml) was added, and the mixture was degassed by bubbling nitrogen for 25 minutes. Tris(dibenzylideneacetone)dipalladium(0) (690 mg, 5 mol%) was then added, the vessel sealed, and the reaction heated at 120°C overnight. The reaction was allowed to cool and diluted with ethyl acetate (500 mL) and saturated _NaHCO₃ solution (150 mL). The layers were separated and extracted 2X with ethyl acetate. The organic phase was washed 3X with brine, dried _{over Na₂SO₄} , filtered, and then concentrated under reduced pressure. The residue was purified by chromatography (ISCO 80 g silica gel, ethyl acetate/hexanes) to afford 186a.

Example 186b 5-Bromo-3-(5-(hydroxymethyl)pyridin-2-ylamino)-1-methylpyridin-2(1H)-one 186b.

In a flask, 186a (500 mg, 1.5 mmol) was suspended in dichloromethane under _N₂ and cooled to -78°C in a dry ice/acetone bath. 1.0 M DIBAL-H in dichloromethane (4.4 mL, 4.4 mmol) was slowly added via syringe. After the addition was complete, the reaction was allowed to warm to -20°C, at which point the dry ice/acetone bath was replaced with an ice/water bath. The reaction was stirred for an additional 0.5 hr, at which point it was slowly quenched with 1N HCl (~5 mL). The reaction was then diluted with ethyl acetate, stirred, and allowed to warm to room temperature over ~1 hr. The pH was adjusted to ~7 with 1N NaOH, and 50 mL of a saturated aqueous solution of Rochelle's salt was added. The mixture was heated and stirred at 40°C for 1 hr _, during which time the mixture clarified. The layers were separated, and the organic phase was washed with brine, dried over _Na₂SO₄ , filtered, and concentrated to afford 186b (420 mg, 90% yield) as a white solid.

Example 186d 5-Bromo-3-(5-((isopropyl(methyl)amino)methyl)pyridin-2-ylamino)-1-methylpyridin-2(1H)-one 186d

In a flask, 186b (350 mg, 1.13 mmol) was suspended in dichloromethane (20 mL) under _N₂ , followed by the addition of diisopropylethylamine (0.89 mL, 5.09 mmol) and the reaction cooled to 0°C in an ice/water bath. Methanesulfonyl chloride (518 mg, 0.35 mL, 4.52 mmol) was added, and the reaction was allowed to warm to room temperature and stirred for ~1 hr. The reaction was diluted with dichloromethane and saturated aqueous sodium bicarbonate was added. The layers were separated, and the organic phase was washed with sodium bicarbonate, dried over anhydrous magnesium sulfate, filtered, and concentrated to afford the mesylate 186c, which was used directly to prepare 186d. 186c was dissolved in DMF (10 mL) and the solution was transferred to a pressure flask. N-methylpropane-2-amine (1.2 mL, 830 mg, 11.3 mmol) was then added. The flask was sealed and heated at 80°C overnight. The reaction was cooled and then diluted with ethyl acetate and water and the layers were separated. The organic phase was washed 4X with brine, dried _{over Na2SO4} , filtered, and concentrated to afford 186d.

Example 186f 2-(5-(5-((isopropyl(methyl)amino)methyl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 186f

Step 1: To a pressure flask was added 186d (205 mg, 0.56 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bis(1,3,2-dioxaborolane) (213 mg, 0.84 mmol), potassium acetate (220 mg, 2.24 mmol), and 1,1'-bis(diphenylphosphino)ferrocenedichloropalladium(II) dichloromethane complex (817 mg, 0.1 mol%). The flask was evacuated and filled with _N2 3X, and dioxane (5 mL) was added. The vessel was sealed and heated at 90°C for ~3 hr. The reaction was allowed to cool, then diluted with ethyl acetate, filtered through a pad of Celite, and concentrated under reduced pressure to afford 186e, which was used directly in the next step.

Step 2: 186e was dissolved in DME (5 mL) and transferred to a pressure flask containing 109a (187 mg, 0.45 mmol ₎ , 1N _Na₂CO₃ (1.2 ml), and tetrakis(triphenylphosphine)palladium(0) (32 mg, 5 mol%). The flask was sealed and heated at 100°C overnight. The reaction was then diluted with ethyl acetate and water, separated, washed with brine, dried over _Na₂SO₄ , filtered, and then concentrated under reduced pressure. The residue was purified by chromatography (ISCO 12 g silica gel, eluting with methanol and _CH₂Cl₂ ) to afford 186f ₍ 110 mg, 32% over ₂ steps).

According to Example 119, 186f (110 mg, 0.18 mmol), 1N LiOH (0.88 mL), THF (2 mL), and isopropanol (2 mL) were reacted and purified by column chromatography (ISCO 12 g silica gel, methanol/dichloromethane) to give 186 (80 mg, 77% yield). MS (ESI+) m/z 581.4 (M+H).

Example 187 2-(2-(Hydroxymethyl)-3-(5-(5-(methoxymethyl)-1-methyl-1H-pyrazol-3-ylamino)-1-methyl-6-oxo-1,6-dihydropyridazin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 187

Example 187a 6-Chloro-4-(5-(methoxymethyl)-1-methyl-1H-pyrazol-3-ylamino)-2-methylpyridazin-3(2H)-one 187a

A mixture of 5-(methoxymethyl)-1-methyl-1H-pyrazol-3-amine 176b (600 mg, 4.26 mmol), XantPhos (300 mg, 0.51 mmol), Pd ₂ dba ₃ (310 mg, 0.34 mmol), 4-bromo-6-chloro-2-methylpyridazin-3(2H)-one (103e) (1.22 g, 5.53 mmol), and Cs ₂ CO ₃ (4.2 g, 12.8 mmol) in 1,4-dioxane (40 mL) was heated under reflux for 2 h. After completion of the reaction, the mixture was filtered and then washed with methanol (100 mL). The filtrate was evaporated in vacuo, and the residue was purified by reverse phase Combi flash chromatography to afford 187a (1.13 g, 94%). MS: [M+H] ⁺ 284.

Example 187b 2-(5-(5-(methoxymethyl)-1-methyl-1H-pyrazol-3-ylamino)-1-methyl-6-oxo-1,6-dihydropyridazin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 187b

A mixture of 6-chloro-4-(5-(methoxymethyl)-1-methyl-1H-pyrazol-3-ylamino)-2-methylpyridazin-3(2H)-one 187a (300 mg, 1.06 mmol), 2-(acetoxymethyl)-3-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)phenylboronic acid 114a (500 mg, 1.3 mmol), _PdCl2 (dppf) (116 mg, 0.16 mmol), _K3PO4 ( ₁₀₀ mg), and NaOAc (50 mg) in MeCN (15 mL) and water (3 mL) was heated at reflux for 2 h. The solvent was evaporated in vacuo, and the residue was purified by reverse phase Combi-flash chromatography to afford 187b (310 mg, 50%). MS: [M+H] ⁺ 586.

A mixture of 187b (310 mg, 0.53 mmol) and LiOH hydrate (222 mg, 5.3 mmol) in isopropanol (20 mL) and water (4 mL) was stirred at 30°C for 2 h. The mixture was evaporated in vacuo, and the residue was extracted with ethyl acetate (20 mL x 2). The combined extracts were concentrated under reduced pressure. The residue was purified by preparative HPLC to give 187 (103 mg, 36%). MS: [M+H] ⁺ 544. ¹ H NMR(500MHz,DMSO)δ9.25(s,1H),7.89(s,1H),7.50-7.47(m,1H),7.42-7.38(m, 2H),6.51(s,1H),6.22(s,1H),4.63-4.60(m,1H),4.48-4.45(m,1H),4.40-4.35( m,3H),4.20-4.05(m,3H),3.90-3.86(m,1H),3.75(s,3H),3.67(s,3H),3.26(s, 3H),2.65-2.54(m,2H),2.48-2.42(m,2H),1.84-1.74(m,2H),1.74-1.64(m,2H).

Example 188 2-(2-(hydroxymethyl)-3-(1-methyl-5-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 188

Example 188a tert-Butyl 4-(6-nitropyridin-3-yl)piperazine-1-carboxylate 188a

To a solution of 5-bromo-2-nitropyridine (30 g, 148 mmol) in DMSO (1 L) were added K ₂ CO ₃ (40 g, 296 mmol) and tert-butyl piperazine-1-carboxylate (28 g, 148 mmol). The mixture was stirred at 65°C overnight. After cooling, it was poured into water (2 L). The precipitated solid was collected and dried under vacuum. It was further purified by flash column chromatography using 20:1 petroleum ether/ethyl acetate followed by dichloromethane to afford 188a (17 g, 37%) as a yellow solid. MS: [M+H] ⁺ 309.

Example 188b tert-Butyl 4-(6-aminopyridin-3-yl)piperazine-1-carboxylate 188b

A 500-mL bottle was purged with nitrogen and charged with 188a (3.1 g, 10 mmol), 10% palladium on carbon (50% wet, 1.0 g), and ethanol (100 mL). The mixture was evacuated, filled with hydrogen, and stirred at room temperature for 16 h. The hydrogen was then removed and nitrogen was introduced into the bottle. The catalyst was removed by filtration through a pad of Celite, and the filtrate was concentrated under reduced pressure to afford 188b (2.7 g, 97%). MS: [M+H] ⁺ 279

Example 188c tert-Butyl 4-(6-(5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)pyridin-3-yl)piperazine-1-carboxylate 188c

To a 100-mL single-necked round-bottom flask equipped with a magnetic stirrer and a reflux condenser were added 1,4-dioxane (50 mL), 188b (1.3 g, 4.7 mmol), 3,5-dibromo-1-methylpyridin-2(1H)-one (1.24 g, 4.7 mmol), and cesium carbonate (3.8 g, 12 mmol). After nitrogen was bubbled through the resulting mixture for 30 minutes, XantPhos (272 mg, 0.47 mmol) and tris(dibenzylideneacetone)dipalladium(0) (430 mg, 0.47 mmol) were added, and the reaction mixture was heated under reflux for 3 hours. The reaction was then cooled to room temperature, partitioned between ethyl acetate (100 mL) and water (100 mL), and then filtered. The aqueous layer was separated and extracted with ethyl acetate (50 mL×2). The organic layers were combined, washed with brine (50 mL), and then dried over sodium sulfate. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by flash column chromatography using 50:1 dichloromethane/methanol as eluent to afford 188c (1.3 g, 59%). MS: [M+H] ⁺ 464.

Example 188d 5-Bromo-1-methyl-3-(5-(piperazin-1-yl)pyridin-2-ylamino)pyridin-2(1H)-one 188d

A mixture of 188c (3.6 g, 7.8 mmol) and 4.0 M HCl/dioxane (10 mL) was stirred at room temperature for 5 h. The mixture was then concentrated under reduced pressure. The residue was basified with 1.0 M aqueous NaOH and extracted with dichloromethane. The combined organic layers were washed with water and concentrated under reduced pressure to afford 188d (2.46 g, 87%). MS: [M+H] ⁺ 364.

Example 188e 5-Bromo-1-methyl-3-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)pyridin-2(1H)-one 188e

A mixture of 188d (2.75 g, 7.5 mmol), oxetane-3-one (1.6 g, 22.7 mmol), NaBH ₃ CN (4.75 g, 22.5 mmol), and zinc chloride (3 g, 22.7 mmol) in methanol (125 mL) was stirred at 50°C for 5 hours. The mixture was added to water and extracted three times with dichloromethane. The organic layer was concentrated under reduced pressure. The residue was purified by column chromatography eluting with 25:1 dichloromethane/methanol to afford 188e (1.92 g, 61%). MS: [M+H] ⁺ 420. ¹ H NMR (500MHz, DMSO) δ8.58(d,J＝2.5,1H),8.55(s,1H),7.94(d,J＝3,1H),7.54(d,J＝2.5,1H),7.39(dd,J＝3,1H), 7.25 (d, J = 4, 1H), 4.56 (t, J = 6.5, 2H), 4.46 (t, J = 6.5, 2H), 3.50 (s, 3H), 3.43 (m, 1H), 3.01 (m, 4H), 2.40 (m, 4H).

Example 188f 2-(1-methyl-5-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 188f

According to Example 148b, 464 mg of 2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 114a was reacted with 420 mg of 188e to give 188f as a yellow solid (360 mg, 54%). LCMS: [M+H] ⁺ 678

According to Example 148, 270 mg of 188f was converted to a white solid 188 (144 mg, 54%). LCMS: [M+H] ⁺ 635. ¹ H NMR (500 MHz, CDCl 3 ) δ: 8.55 (d, J=2.5, 1H), 7.89 (d, J=3, 1H), 7.80 (s, ¹ H),7.47-7.41(m,3H),7.26-7.21(m,2H),6.85(s,1H),6.83(d,J＝8.5,1H),4.72-4.65(m,3H),4.59(d,J＝10.5,1H),4.43-4.36(m,2 H),4.20-4.13(m,3H),3.93-3.89(m,1H),3.59-3.57(m,1H),3.15(s,3H),3.05(m,1H),2.6-2.51(m,7H),1.89(s,2H),1.78(S,2H).

Example 189 10-[5-fluoro-2-(hydroxymethyl)-3-[1-methyl-6-oxo-5-(pyrimidin-4-ylamino)-1,6-dihydropyridin-3-yl]phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 189

Example 189a 2-Bromo-4-fluoro-6-(9-oxo-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-10-yl)benzyl acetate 189a

In a sealed tube equipped with a magnetic stirrer, 167e (740 mg, 3.6 mmol), 2,6-dibromo-4-fluorobenzyl acetate 197c (2.4 g, 7.2 mmol), and cesium carbonate (2.6 g, 7.9 mmol) were added to 1,4-dioxane (36 mL). After nitrogen was bubbled through the solution for 30 min, Xantphos (250 mg, 0.43 mmol) and tris(dibenzylideneacetone)dipalladium(0) (260 mg, 0.29 mmol) were added, and the reaction mixture was heated at 100°C for 16 h. Thereafter, _H₂O (50 mL) and ethyl acetate (50 mL) were added. The aqueous layer was separated and extracted with ethyl acetate (2×50 mL). The combined organic extracts were washed with brine (100 mL) and then dried over sodium sulfate. The resulting residue was purified by column chromatography eluting with a gradient of 100% hexanes to 100% ethyl acetate to afford 189a in 56% yield (910 mg).

Example 189b 10-[5-fluoro-2-(acetoxymethyl)-3-[1-methyl-6-oxo-5-(pyrimidin-4-ylamino)-1,6-dihydropyridin-3-yl]phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 189b

To a microwave tube equipped with a magnetic stirrer was added 1-methyl-3-(pyrimidin-4-ylamino)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one 109c (170 mg, 0.5 mmol), 189a (150 mg, 0.33 mmol), 1,2-dimethoxyethane (4 mL) and 1 M aqueous sodium carbonate solution (1 mL). After bubbling _N for 15 min, Pd( _PPh ) ₍ 19 mg, 0.02 mmol) was added. The mixture was heated in a microwave at 130° C. for 10 min. Thereafter, ethyl acetate (5 mL) and water (5 mL) were added. The separated aqueous layer was extracted with ethyl acetate (2 x 5 mL). The combined organic phases were washed with brine (10 mL), dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by column chromatography eluting with a gradient of _{CH2Cl2 -} 60:35:5 dichloromethane: ether: methanol to afford 189b in 37% yield (71 mg).

To a 25 mL round-bottom flask equipped with a magnetic stirrer was added 189b (71 mg, 0.12 mmol), lithium hydroxide (26 mg, 0.6 mmol), THF (0.6 mL), isopropanol (0.6 mL), and water (1.2 mL). The mixture was stirred at room temperature for 1 h. Afterwards, ethyl acetate (5 mL) and water (5 mL) were added. The separated aqueous layer was extracted with ethyl acetate (2 x 5 mL). The combined organic phases were washed with brine (10 mL), dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by column chromatography using a gradient of dichloromethane:60:35:5 _CH₂Cl₂ : _diethyl ether:methanol to afford 189 in 58% yield (38 mg). MS (ESI+) m/z 529.7 (M+H).

Example 190 10-[3-(5-{[5-(4-ethylpiperazin-1-yl)pyridin-2-yl]amino}-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-2-(hydroxymethyl)phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 190

Example 190a 3-(5-(4-ethylpiperazin-1-yl)pyridin-2-ylamino)-1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one 190a

To a round-bottom flask equipped with a stir bar was added 5-bromo-3-(5-(4-ethylpiperazin-1-yl)pyridin-2-ylamino)-1-methylpyridin-2(1H)-one 138c (305.6 mg, 0.696 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bis(1,3,2-dioxaborolane) (185.4 g, 0.730 mmol), _Pd2 (dba) ₃ (63.7 mg, 0.070 mmol), X-Phos (66.3 mg, 0.139 mmol), KOAc (102.4 mg, 1.043 mmol), and dioxane (5 mL). The mixture was heated at 90°C for 4 hr. The resulting mixture was filtered through celite and washed with ethyl acetate (200 mL). The organic phase was washed with water (50 mL), dried over MgSO ₄ , and the solvent was removed in vacuo to give the crude product 190a which was used directly in the next step.

Example 190b 10-[3-(5-{[5-(4-ethylpiperazin-1-yl)pyridin-2-yl]amino}-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-2-(acetoxymethyl)phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 190b

To a microwave tube equipped with a stir bar was added bromide 167f (150 mg, 0.348 mmol), 190a (306 mg, 0.696 mmol), Pd(PPh ₃ ) ₄ (40.2 mg, 0.0348 mmol), aqueous Na ₂ CO ₃ solution (1.0 N, 1.15 mL, 1.15 mmol), and DME (4 mL). The mixture was reacted in a microwave at 130° C. for 10 min. Dichloromethane (200 mL) was added, and the resulting mixture was washed with water (3 x 30 mL), brine (30 mL x 1), dried over MgSO ₄ , filtered, and the solvent removed in vacuo. Silica gel column chromatography (methanol:dichloromethane = 5:95) afforded 190b.

To a round-bottom flask equipped with a stir bar was added 190b, THF (3 mL), isopropanol (3 mL), water (3 mL), and LiOH monohydrate (200 mg). The resulting mixture was stirred at room temperature for 1 hr. All solvents were removed in vacuo, and the resulting residue was added to dichloromethane (200 mL). The solution was washed with water (3 x 30 mL), brine (30 mL), dried over _MgSO₄ , filtered, and the solvent was removed in vacuo. Silica gel column chromatography (MeOH:dichloromethane = 10:90) afforded 190 as a yellow solid (16 mg). MS (ESI+) m/z 622.5 (M+H).

Example 191 10-[3-(5-{[5-(4-ethylpiperazin-1-yl)pyridin-2-yl]amino}-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-2-(hydroxymethyl)phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 191

Example 191a 10-[3-(5-{[5-(4-ethylpiperazin-1-yl)pyridin-2-yl]amino}-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-2-(hydroxymethyl)phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 191a

To a microwave tube equipped with a magnetic stirrer was added 3-(6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-2-ylamino)-1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one 150a (200 mg, 0.5 mmol), 189a (150 mg, 0.34 mmol), 1,2-dimethoxyethane (4 mL), and 1 M aqueous sodium carbonate solution (1 mL). After bubbling _N for 15 min, Pd(PPh ₃ ) ₄ (19 mg, 0.02 mmol) was added. The mixture was heated in a microwave at 130° C. for 10 min. Thereafter, ethyl acetate (5 mL) and water (5 mL) were added. The separated aqueous layer was extracted with ethyl acetate (2 x 5 mL). The combined organic phases were washed with brine (10 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by column chromatography eluting with a gradient of dichloromethane-60:35: _{5 CH2Cl2} :ether:methanol to afford 191a in 59% yield (125 mg).

To a 25 mL round-bottom flask equipped with a magnetic stirrer was added 191a (130 mg, 0.2 mmol), lithium hydroxide (43 mg, 1.0 mmol), THF (1 mL), isopropanol (1 mL), and water (2 mL). The mixture was stirred at rt for 30 min. Thereafter, ethyl acetate (5 mL) and water (5 mL) were added. The separated aqueous layer was extracted with ethyl acetate (2 x 5 mL). The combined organic phases were washed with brine (10 mL), dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by column chromatography eluting with a gradient of dichloromethane:60:35: _{5 CH₂Cl₂} :diethyl ether:methanol to afford 191 in 59% yield (68 mg). MS (ESI+) m/z 573.4 (M+H).

Example 192 6-(3-(5-(6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-2,3-(5,5-dimethyl-5,6-dihydro-4H-cyclopentadienyl)-5,6-dihydrothieno[2,3-c]pyridin-7(4H)-one 192

To a 100-mL three-necked round-bottom flask equipped with a magnetic stirrer and a reflux condenser were added 182c (300 mg, 0.513 mmol), 110c (167 mg, 0.513 mmol), sodium carbonate (218 mg, 2.05 mmol), water (6 mL), and 1,4-dioxane (30 mL). After nitrogen was bubbled through the resulting suspension for 20 min, tetrakis(triphenylphosphine)palladium(0) (60.0 mg, 0.051 mmol) was added, and the reaction mixture was heated at 100°C for 4 h. Thereafter, the reaction mixture was cooled to room temperature, filtered, and the filter cake was washed with a 1:10 mixture of methanol and dichloromethane (30 mL). The filtrate was concentrated under reduced pressure to obtain a brown residue. The residue obtained, tetrabutylammonium fluoride (1.0 M in THF, 2 mL, 2.00 mmol), and THF (6 mL) were added to another 50-mL single-necked round-bottom flask equipped with a magnetic stirrer and a reflux condenser. The mixture was stirred at room temperature for 1.5 h. Thereafter, the reaction mixture was concentrated under reduced pressure. The resulting residue was purified by flash chromatography to give 192 as an off-white solid in 36% (110 mg): mp 170-172° C.; ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 8.21 (s, 1H), 7.99 (d, 1H, J=2.0 Hz), 7.32 (dd, 1H, J=9.0, 3.0 Hz), 7.28 (d, 1H, J=2.0 Hz), 7.15 (dd, 1H, J=9.0, 3.0 Hz), 5.93 (s, 1H), 4.86 (t, 1H, J=5.0 Hz)

Example 193 5-(2-(Hydroxymethyl)-3-(6,6-dimethyl-3,4,6,7-tetrahydro-5H-cyclopentadienyl[4,5]thieno[2,3-c]pyridin-1(2H)-yl))-1-methyl-3-(pyridin-3-ylamino)pyrazin-2(1H)-one 193

According to Example 204, 111b (132 mg, 0.466 mmol) and 131a (300 mg, 0.606 mmol) were reacted to give 193 as an off-white solid in 31% yield (75 mg): mp 135-136° C.; ¹ H NMR (500 MHz, DMSO-d ₆ )δ9.53(s,1H),9.15(d,1H,J＝1.9Hz),8.42(m,1H),8.17(dd,1H,J＝4.5,1.4Hz) ,7.54(dd,1H,J＝7.4,1.0Hz),7.47(m,2H),7.34(dd,1H,J＝8.0,1.5Hz),7.30(m, 1H),4.82(m,1H),4.46(m,2H),4.01(m,1H),3.86(m,1H),3.56(s,3H),3.02(m, 1H), 2.88 (m, 1H), 2.75 (s, 2H), 2.54 (d, 2H, J = 6.0Hz), 1.23 (s, 6H); MS (ESI+) m/z 528.2(M+H).

Example 194 5-(2-(Hydroxymethyl)-3-(6,6-dimethyl-3,4,6,7-tetrahydro-5H-cyclopenta[4,5]thieno[2,3-c]pyridin-1(2H)-yl)-1-methyl-3-(5-(4-methylpiperazin-1-yl)pyridin-2-ylamino)pyridin-2(1H)-one 194

According to Example 204, 197e (176 mg, 0.466 mmol) and 131a (300 mg, 0.606 mmol) were reacted to give a brown residue. To a 100-mL single-necked round-bottom flask equipped with a magnetic stirrer were added the above crude residue, THF (8 mL), methanol (4 mL), water (4 mL) and lithium hydroxide monohydrate (196 mg, 4.66 mmol). The mixture was stirred at room temperature for 2 h and then concentrated in vacuo. The residue was distributed between a 20% (v/v) solution of methanol/dichloromethane (150 mL) and water (30 mL). The organic layer was separated and the aqueous layer was extracted with a 20% (v/v) solution of methanol/dichloromethane (3×30 mL). The combined organic layers were dried over sodium sulfate and then concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, 0%-10% methanol/dichloromethane) to give 33% yield (95 mg) of 194 as an off-white solid: mp 142-143 °C; ¹ H NMR (500 MHz, DMSO-d ₆ )δ8.54(d,1H,J＝2.5Hz),8.33(s,1H),7.85(d,1H,J＝3.0Hz),7.45(t,1H,J＝ 7.4Hz),7.35(m,2H),7.32(m,2H),7.20(d,1H,J＝9.3Hz),4.84(m,1H),4.32( m,2H),4.03(m,1H),3.85(m,1H),3.58(s,3H),3.03(m,5H),2.87(m,1H),2. 75(s,2H),2.53(m,2H),2.43(m,4H),2.20(s,3H),1.23(s,6H); MS(ESI+)m/z 625.3(M+H).

Example 195 5-[2-(Hydroxymethyl)-3-{1-oxo-1H,2H,3H,4H,6H,7H,8H,9H-pyrazino-[1,2-a]indol-2-yl}phenyl]-1-methyl-3-({5-[4-(propan-2-yl)piperazin-1-yl]pyridin-2-yl}amino)-1,2-dihydropyridin-2-one 195

According to Example 138, 5-bromo-1-methyl-3-(5-(4-(1-methylethyl)piperazin-1-yl)pyridin-2-ylamino)pyridin-2(1H)-one 220a was converted to 75 mg of a white solid 195. MS (ESI+) m/z 622 (M+H).

Example 196 10-(3-{5-[(1,5-dimethyl-1H-pyrazol-3-yl)amino]-1-methyl-6-oxo-1,6-dihydropyridin-3-yl}-5-fluoro-2-(hydroxymethyl)phenyl)-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 196

Example 196a 3-(1,5-dimethyl-1H-pyrazol-3-ylamino)-1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one 196a

To a sealed tube equipped with a magnetic stirrer was added 5-bromo-3-(1,5-dimethyl-1H-pyrazol-3-ylamino)-1-methylpyridin-2(1H)-one 142a (380 mg, 1.4 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bis(1,3,2-dioxaborolane) (690 mg, 2.7 mmol), potassium acetate (401 mg, 4.1 mmol) and 1,4-dioxane (34 mL). After nitrogen was bubbled through the resulting suspension for 30 min, [1,1'-bis(diphenylphosphino)ferrocene]palladium(II) dichloride and dichloromethane complex ( _PdCl2dppf : _CH2Cl2 (1:1), 110 mg, 0.14 mmol) _was added and the reaction was stirred at 105°C for 16 h. After this, the mixture is cooled to environment temperature, distributed between water (20mL) and ethyl acetate (20mL).The aqueous layer separated by ethyl acetate (2x 10mL) extraction.The organic phase merged is washed with salt water (30mL), dried over sodium sulfate, filtered, then concentrated under reduced pressure.The crude product 196a obtained is used for the next step without further purification.

Example 196b 10-(3-{5-[(1,5-dimethyl-1H-pyrazol-3-yl)amino]-1-methyl-6-oxo-1,6-dihydropyridin-3-yl}-5-fluoro-2-(acetoxymethyl)phenyl)-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 196b

To a microwave tube equipped with a magnetic stirrer was added 196a (450 mg, 1.3 mmol), 189a (240 mg, 0.5 mmol), DME (4 mL) and 1 M aqueous sodium carbonate solution (1.5 mL). After blowing _N for 15 min, Pd(PPh ₃ ) ₄ (31 mg, 0.03 mmol) was added. The mixture was heated in a microwave at 130 ° C for 10 min. Thereafter, EtOAc (5 mL) and water (5 mL) were added. The separated aqueous layer was extracted with EtOAc (2 x 5 mL). The combined organic phases were washed with brine (10 mL), dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by column chromatography using a gradient elution of dichloromethane-60:35:5 dichloromethane: ether: methanol to give 196b in 83% yield (260 mg).

To a 25 mL round-bottom flask equipped with a magnetic stirrer was added 196b (260 mg, 0.5 mmol), lithium hydroxide (94 mg, 2.2 mmol), THF (2 mL), isopropanol (2 mL) and water (5 mL). The mixture was stirred at rt for 30 min. Thereafter, ethyl acetate (5 mL) and water (5 mL) were added. The separated aqueous layer was extracted with ethyl acetate (2 x 5 mL). The combined organic phases were washed with brine (10 mL), dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by column chromatography using a Biotage KPNH 12+M column eluting with a hexane-ethyl acetate gradient to give 196 in 46% yield (110 mg). MS (ESI+) m/z 545.4 (M+H).

Example 197 2-(5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-(5-(4-methylpiperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 197

Example 197a 2,6-Dibromo-4-fluorobenzaldehyde 197a

To a solution of 1,3-dibromo-5-fluoro-2-iodobenzene (50 g, 132 mmol) in anhydrous toluene (300 mL) cooled to –35°C was added isopropylmagnesium chloride solution (84 mL, 171 mmol, 2.0 M in diethyl ether) over 30 minutes, while maintaining the internal temperature below –25°C. A clear brown solution was obtained. Stirring was continued for 1.5 hours. Anhydrous DMF (34 mL, 436 mmol) was then added over 30 minutes. The temperature of the reaction mixture was increased to –19°C. The reaction mixture was warmed to 10°C (room temperature) over 1 hour and stirred at this temperature for 1.5 hours. The reaction mixture was quenched with saturated aqueous _NH₄Cl (100 mL), filtered, and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (eluting with petroleum ether/ethyl acetate from 50:1 to 20:1) to afford 197a (20 g, 54% yield) as a yellow solid.

Example 197b (2,6-dibromo-4-fluorophenyl)methanol 197b

To a solution of 197a (20 g, 71 mmol) in ethanol (500 mL) was added _NaBH₄ (10 g, 284 mmol). The mixture was stirred at room temperature (10°C) for 4 h. TLC indicated the disappearance of the starting material. The reaction was quenched with HCl solution (150 mL, 1 M). Most of the ethanol was evaporated under reduced pressure. The residue was extracted with ethyl acetate (3 x 500 mL). The organic layers were combined, dried _over anhydrous _Na₂SO₄ , and then evaporated in vacuo. The residue was purified by silica gel column chromatography (eluting with petroleum ether/ethyl acetate from 50:1 to 20:1) to afford 197b (15 g, 75% yield) as a white solid.

Example 197c 2,6-dibromo-4-fluorobenzyl acetate 197c

To a 500-mL single-necked round-bottom flask equipped with a magnetic stirrer and a nitrogen inlet was added 197b (23.0 g, 81.0 mmol), triethylamine (25.0 g, 247 mmol), and anhydrous dichloromethane (100 mL). Acetic anhydride (10.0 g, 98.0 mmol) was added, and the mixture was stirred at room temperature for 16 h. Thereafter, the mixture was diluted with dichloromethane (100 mL) and washed with saturated aqueous sodium bicarbonate (100 mL). The layers were separated, and the aqueous layer was extracted with dichloromethane (2 x 20 mL). The organic extracts were combined and dried over sodium sulfate. The desiccant was removed by filtration. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, 0%-50% ethyl acetate/hexane) to afford 197c as a white solid in 87% yield (23.0 g).

Example 197d 2-Bromo-4-fluoro-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 197d

To a 250-mL single-necked round-bottom flask equipped with a magnetic stirrer were added ethyl 1-(2-aminoethyl)-4,5,6,7-tetrahydro-1H-indole-2-carboxylate 101k (3.8 g, 20 mmol), 197c (20 g, 60 mmol), Xantphos (1.2 g, 2 mmol), tris(dibenzylideneacetone)dipalladium(0) (1.8 g, 2 mmol), Cs ₂ CO ₃ (16 g, 50 mmol), and 1,4-dioxane (120 mL). The system was evacuated and then refilled with N ₂ . A reflux condenser was attached to the flask, and the reaction mixture was heated at 100° C. for 16 h. It was then cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography eluting with 5:1 petroleum ether/ethyl acetate to afford 197d as a white solid in 60% yield (5.2 g). MS: [M+H] ⁺ 435. ¹ H NMR (500MHz, DMSO) δ7.70(dd,J＝3,1H),7.48(dd,J＝3,1H),6.52(s,1H),5.01(m,2H),4.18(m,2 H),4.02(m,1H),3.73(m,1H),2.60(m,2H),2.45(m,2H),1.98(s,3H),1.77(m,2H),1.68(m,2H)

Example 197f 1-Methyl-3-[5-(4-methylpiperazin-1-yl)-pyridin-2-ylamino]-5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-pyridin-2-one 197f

A 1-L single-necked round-bottom flask equipped with a magnetic stirrer and a temperature regulator was purged with nitrogen and charged with 197e (10.0 g, 0.027 mol), bis(pinacolato)borate (8.06 g, 0.032 mol), potassium acetate (10.4 g, 0.11 mol), and 1,4-dioxane (200 mL), prepared according to US2009/0318448. After a 30-minute stream of nitrogen was passed through the resulting suspension, Pd(dppf) _{Cl₂CH₂Cl₂} (582 _mg , 0.795 mmol) was added. The resulting reaction mixture _was stirred at reflux for 3 h. The mixture was then cooled to room temperature, partitioned between water (400 mL) and ethyl acetate (600 mL), and filtered through a pad of Celite. The organic phase was extracted, dried over sodium sulfate, filtered, and concentrated. The residue was triturated with a mixture of ether (50 mL) and hexanes (250 mL), and the suspension was filtered. The filter cake was dried under vacuum at room temperature to afford 197f as a brown solid in 27% yield (3.04 g).

Example 197g 4-Fluoro-2-(1-methyl-5-(5-(4-methylpiperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 197g

To a 10-mL glass vessel equipped with a magnetic stir bar was added 197f (244 mg, 0.58 mmol), 197d (200 mg, 0.46 mmol), Pd(PPh ₃ ) ₄ (33 mg, 0.030 mmol), 2N Na ₂ CO ₃ (1 mL), and DME (2 mL). The vessel was sealed with a stopper and placed in a microwave cavity. The reaction mixture was stirred at 125° C. for 7 min and then purified by flash chromatography (dichloromethane:methanol 85:15) to afford 197g as a solid (30% yield, 90 mg).

To a 100-mL single-necked round-bottom flask equipped with a magnetic stir bar was added 197 g (90 mg, 0.14 mmol), LiOH hydrate (60 mg, 1.4 mmol), THF (2 mL), i-PrOH (2 mL), and water (2 mL). After stirring the reaction mixture at room temperature for 3 h, it was partitioned between dichloromethane (5 mL) and water (5 mL), and the organic phase was extracted with dichloromethane (5 mL x 3). The combined organic phases were washed with water (5 mL x 2) and brine (5 mL x 1), dried (Na ₂ SO ₄ ), and then concentrated. The crude product was redissolved in dichloromethane (3 mL). To this solution was added hexane (10 mL), and the resulting precipitate was filtered to obtain 197 in 81% yield (69 mg). MS (ESI+) m/z 612.5 (M+H)

Example 198 10-[5-Fluoro-2-(hydroxymethyl)-3-(1-methyl-5-{[5-(4-methylpiperazin-1-yl)pyridin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^{2 ,6} ]dodeca-2(6),7-dien-9-one 198

Example 198a 2,6-Dibromo-4-fluorobenzaldehyde 198a

A solution of 1,3-dibromo-5-fluoro-2-iodobenzene (50 g, 132 mmol) in anhydrous toluene (300 mL) was cooled to -35°C, and isopropylmagnesium chloride solution (84 mL, 171 mmol, 2.0 M _Et₂O solution) was added over 30 minutes while maintaining the internal temperature below -25°C (Figure 6). A clear brown solution was obtained. Stirring was continued for 1.5 hours. Anhydrous DMF (34 mL, 436 mmol) was then added over 30 minutes. The temperature of the reaction mixture was increased to -19°C. The reaction mixture was warmed to 10°C (room temperature) over 1 hour and stirred at this temperature for 1.5 hours. The reaction was quenched with saturated aqueous _NH₄Cl (100 mL), filtered, and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (eluting with petroleum ether/ethyl acetate from 50:1 to 20:1) to afford 198a (20 g, 54% yield) as a yellow solid.

Example 198b (2,6-dibromo-4-fluorophenyl)methanol 198b

To a solution of 198a (20 g, 71 mmol) in EtOH (500 mL) was added _NaBH₄ (10 g, 284 mmol). The mixture was stirred at room temperature (10°C) for 4 h. TLC showed the disappearance of the starting material. The reaction was quenched with HCl solution (150 mL, 1 M). Most of the EtOH was evaporated under reduced pressure. The residue was extracted with ethyl acetate (3 x 500 mL). The organic layers were combined, dried _over anhydrous _Na₂SO₄ , and evaporated in vacuo. The residue was purified by silica gel column chromatography (eluting with petroleum ether/ethyl acetate from 50:1 to 20:1) to afford 198b (15 g, 75% yield) as a white solid.

Example 198c 2,6-dibromo-4-fluorobenzyl acetate 198c

To a 500-mL single-necked round-bottom flask equipped with a magnetic stirrer and a nitrogen inlet was added 198b (23.0 g, 81.0 mmol), triethylamine (25.0 g, 247 mmol), and anhydrous dichloromethane (100 mL). Acetic anhydride (10.0 g, 98.0 mmol) was added, and the mixture was stirred at room temperature for 16 h. The mixture was then diluted with dichloromethane (100 mL) and washed with saturated aqueous sodium bicarbonate (100 mL). The layers were separated, and the aqueous layer was extracted with dichloromethane (2 x 20 mL). The organic extracts were combined and dried over sodium sulfate. The desiccant was removed by filtration. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, 0%-50% ethyl acetate/hexane) to afford 198c as a white solid in 87% yield (23.0 g).

Example 198d 2-Bromo-4-fluoro-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 198d

To a 250-mL single-necked round-bottom flask equipped with a magnetic stirrer was added ethyl 1-(2-aminoethyl)-4,5,6,7-tetrahydro-1H-indole-2-carboxylate 101k (3.8 g, 20 mmol), 198c (20 g, 60 mmol), Xantphos (1.2 g, 2 mmol), tris(dibenzylideneacetone)dipalladium(0) (1.8 g, 2 mmol), Cs ₂ CO ₃ (16 g, 50 mmol), and 1,4-dioxane (120 mL). The system was evacuated and then refilled with N ₂ . A reflux condenser was attached to the flask, and the reaction mixture was heated at 100° C. for 16 h. It was then cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography eluting with 5:1 PE/EA to afford 198d as a white solid in 60% yield (5.2 g). MS: [M+H] ⁺ 435. ^1H NMR (500MHz, DMSO) δ7.70(dd,J＝3,1H),7.48(dd,J＝3,1H),6.52(s,1H),5.01(m,2H),4.18(m,2 H),4.02(m,1H),3.73(m,1H),2.60(m,2H),2.45(m,2H),1.98(s,3H),1.77(m,2H),1.68(m,2H)

Example 198e 5-Bromo-1-methyl-3-[5-(4-methylpiperazin-1-yl)-pyridin-2-ylamino]-1H-pyridin-2-one 198e

Step 1: 1-Methyl-4-(6-nitropyridin-3-yl)piperazine

According to US 2009/0318448, to a solution of 5-bromo-2-nitropyridine (2.0 g, 9.85 mmol) in DMSO (10 mL) was added K ₂ CO ₃ (2.72 g, 19.7 mmol) and 1-methylpiperazine (1.64 mL, 14.8 mmol) and tetrabutylammonium iodide (36 mg). The mixture was stirred at 120° C. overnight. It was allowed to cool and acidified with 1N HCl. The mixture was extracted with DCM. The aqueous layer was basified with saturated Na ₂ CO ₃ and then extracted with DCM. The combined organic layers were dried over Na ₂ SO ₄ and then concentrated to give a brown solid, which was washed with a small amount of water. The solid was dried under vacuum to give 2.16 g of 1-methyl-4-(6-nitropyridin-3-yl)piperazine as a yellow powder (99% yield). LCMS: (M+H) ⁺ 223

Step 2: 5-(4-methylpiperazin-1-yl)pyridin-2-amine

A suspension of 1-methyl-4-(6-nitropyridin-3-yl)piperazine (5 g, 22.5 mmol), NH ₄ Cl (12 g, 225 mmol) and Fe (5 g, 5 mmol) in EtOH/H ₂ O (1:1) (100 mL) was stirred at 80° C. for 3 h. TLC showed that the starting material 1-methyl-4-(6-nitropyridin-3-yl)piperazine disappeared. After filtration on a Celite pad, the solvent was removed under vacuum. EA and brine were added to the mixture, the organic layer was separated, dried over Na ₂ SO ₄ , and then concentrated under reduced pressure to give 2.5 g of 5-(4-methylpiperazin-1-yl)pyridin-2-amine (yield 60%). LCMS: (M+H) ⁺ 193

Step 3: 5-(4-Methylpiperazin-1-yl)pyridin-2-amine (1 g, 10.5 mmol), 3,5-dibromo-1-methylpyridin-2(1H)-one (1.87 g, 7 mmol) and Cs ₂ CO ₃ (5 g, 21 mmol) were added to dioxane (30 mL) in a sealed tube. After nitrogen was bubbled through the resulting solution for 15 minutes, Xantphos (405 mg, 0.7 mmol) and tris(dibenzylideneacetone)dipalladium(0) (320 mg, 0.35 mmol) were added, and the reaction mixture was stirred at 100° C. for 0.5 h. After filtration, the solid was washed with warm EA, and the filtrate was concentrated to give the crude product as a black solid. The crude product was recrystallized from MeOH to give 1.26 g (64%) of 198e. LCMS: (M+H) ⁺ 380. ^1H NMR (500MHz, DMSO) δ8.57(d,J＝2.5,1H),8.54(s,1H),7.93(d,J＝3,1H),7.44(d,J＝2.5,1H),7 .38(dd,J＝3.5,2H),7.24(d,J＝4.5,1H),3.50(s,3H),3.07(m,4H),2.44(m,4H),2.21(s,3H).

Example 198f 1-Methyl-3-[5-(4-methylpiperazin-1-yl)pyridin-2-ylamino]-5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-pyridin-2-one 198f

A 1-L single-necked round-bottom flask equipped with a magnetic stirrer and a temperature regulator was purged with nitrogen and charged with 198e (10.0 g, 0.027 mol), bis(pinacolato)borate (8.06 g, 0.032 mol), potassium acetate (10.4 g, 0.11 mol), and 1,4-dioxane (200 mL). After a nitrogen stream was passed through the resulting suspension for 30 min, Pd(dppf) _{Cl₂CH₂Cl₂} (582 mg, 0.795 mmol) was _added . The resulting reaction mixture _was stirred at reflux for 3 h. It was then cooled to room temperature, partitioned between water (400 mL) and ethyl acetate (600 mL), and filtered through a pad of Celite. The organic phase was extracted, dried over sodium sulfate, filtered, and then concentrated. The residue was pulverized with a mixture of ether (50 mL) and hexane (250 mL), and the suspension was filtered. The filter cake was dried under vacuum at room temperature to afford 198f as a brown solid in 27% yield (3.04 g).

Example 198g 4-Fluoro-2-(1-methyl-5-(5-(4-methylpiperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 198g

To a 10-mL glass vessel equipped with a magnetic stir bar was added 198f (244 mg, 0.58 mmol), 198d (200 mg, 0.46 mmol), Pd(PPh ₃ ) ₄ (33 mg, 0.030 mmol), 2N Na ₂ CO ₃ (1 mL), and DME (2 mL). The vessel was sealed with a stopper and placed in a microwave cavity. The reaction mixture was stirred at 125° C. for 7 min and then purified by flash chromatography (dichloromethane:MeOH 85:15) to afford 198g as a solid (30% yield, 90 mg).

According to Example 148, 198 g was converted to 75 mg of a white solid 198. MS (ESI+) m/z 626.6 (M+H).

Example 199 5-[5-fluoro-2-(hydroxymethyl)-3-[1-methyl-5-({5-[(1-methylazetidin-3-yl)oxy]pyridin-2-yl}amino)-6-oxo-1,6-dihydropyridin-3-yl]phenyl]-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-6-one 199

Example 199a tert-Butyl 3-iodoazetidine-1-carboxylate 199a

A solution of tert-butyl 3-hydroxyazetidine-1-carboxylate (25-3) (3.5 g, 0.02 mol) in toluene (200 mL) was treated with imidazole (4.08 g, 0.06 mol), PPh _{3 (} 0.6 g, 0.04 mol) and I 2 (7.62 g, 0.03 mol). The mixture was heated at 100 ° C for 1 h and then cooled to room temperature. It was then poured into a saturated NaHCO ₃ solution (30 mL). Excess PPh ₃ was destroyed by adding iodine until I ₂ coloration remained in the organic layer. The mixture was washed with 5% Na ₂ SO ₃ solution, dried over Na ₂ SO ₄ , and then evaporated in vacuo. The residue was purified by silica gel column to give 199a (5.31 g, 93%). MS: [M+H] ⁺ 284.

Example 199b tert-Butyl 3-(6-nitropyridin-3-yloxy)azetidine-1-carboxylate 199b

A mixture of 199a (2.24 g, 7.9 mmol), 6-nitropyridin-3-ol (1 g, 7.2 mmol), and Cs ₂ CO ₃ (2.6 g, 7.9 mmol) in DMF (8 mL) was heated overnight at 125° C. in a sealed tube. The mixture was filtered and washed with ethyl acetate (20 mL x 2). The filtrate was evaporated in vacuo, and the residue was purified by reverse-phase Combi-flash chromatography to afford 199b (1.25 g, 59%). MS: [M+H] ⁺ 296.

Example 199c tert-Butyl 3-(6-aminopyridin-3-yloxy)azetidine-1-carboxylate 199c

A 100-mL Parr hydrogenation bottle was purged with nitrogen and charged with 199b (1.07 g, 3.6 mmol), 10% palladium on carbon (50% humidity, 0.3 g), and methanol (60 mL). The bottle was evacuated, filled with hydrogen to 25 psi, and shaken on a Parr hydrogenation apparatus for 2 h. The hydrogen was then removed and nitrogen was introduced into the bottle. The catalyst was removed by filtration through a pad of Celite, and the filtrate was concentrated under reduced pressure to afford 199c (0.95 g, 99%). MS: [M+H] ⁺ 266.

Example 199d tert-Butyl 3-(6-(5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)pyridin-3-yloxy)azetidine-1-carboxylate 199d

A mixture of 199c (950 mg, 3.6 mmol), XantPhos (125 mg, 0.29 mmol), Pd ₂ dba ₃ (260 mg, 0.29 mmol), 3,5-dibromo-1-methylpyridin-2(1H)-one (1.03 g, 3.9 mmol), and Cs ₂ CO ₃ (1.8 g, 7.2 mmol) in 1,4-dioxane (20 mL) was heated under reflux for 2 h. After completion of the reaction, the mixture was filtered and then washed with methanol (100 mL). The filtrate was evaporated in vacuo. The residue was purified by reverse phase Combi-flash chromatography to afford 199d (1.46 g, 91%). MS: [M+H] ⁺ 451.

Example 199e 3-(5-(azetidin-3-yloxy)pyridin-2-ylamino)-5-bromo-1-methylpyridin-2(1H)-one hydrochloride 199e

A mixture of 199d (1.46 g, 3.2 mmol) and HCl/1,4-dioxane (3.2 mL, 12.8 mmol, 4 M) in methanol (20 mL) was heated at 80° C. for 1 h. The mixture was concentrated under reduced pressure to afford 199e (1.24 g, 99%). MS: [M+H] ⁺ 351.

Example 199f 5-Bromo-1-methyl-3-(5-(1-methylazetidin-3-yloxy)pyridin-2-ylamino)pyridin-2(1H)-one 199f

A mixture of 199e (1.24 g, 3.2 mmol), HCHO solution (15 mL, 37% aqueous solution), acetic acid (1 mL), and NaBH(OAc) _₃ (1.36 g, 6.4 mmol) in methanol (10 mL) was stirred at room temperature for 4 h. The solvent was evaporated in vacuo, and the residue was extracted with ethyl acetate (20 mL x 3). The combined extracts were dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by reverse-phase Combi-flash chromatography to afford 199f (940 mg, 80%). MS: [M+H] ^⁺ 365.

Example 199g {4-fluoro-2-[1-methyl-5-({5-[(1-methylazetidin-3-yl)oxy]pyridin-2-yl}amino)-6-oxo-1,6-dihydropyridin-3-yl]-6-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]tridec-1(9),2(7)-dien-5-yl}phenyl}methyl acetate 199g

A mixture of 199f (434 mg, 1.2 mmol), (4-fluoro-2-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-5-yl}-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methyl acetate 212b (600 mg, 1.2 mmol), PdCl ₂ (dppf) (134 mg, 0.18 mmol), K ₃ PO ₄ (150 mg), and NaOAc (100 mg) in MeCN (8 mL) and water (2 mL) was heated in a sealed tube at 110° C. for 2 h. The solvent was evaporated in vacuo. The residue was purified by reverse phase Combi flash chromatography to afford 199g (320 mg, 41%). MS: [M+H] ⁺ 658.

A mixture of 199g (320mg, 0.49mmol) and LiOH hydrate (409mg, 9.8mmol) in isopropanol (20mL) and water (3mL) was stirred at 30°C for 2h. The mixture was evaporated in vacuo, and the residue was extracted with ethyl acetate (20mL x 2). The combined extracts were concentrated under reduced pressure. The residue was purified on preparative HPLC to give 199 (30mg, 10%). MS: [M+H] ⁺ 616. ^1H NMR (500MHz, CDCl3) δ8.52(d,J＝2.0,1H),7.82(s,1H),7.75(d,J＝2.5,1H),7.48(d,J＝2.0,1H ),7.16-7.14(m,1H),7.11-7.09(m,1H),6.98-6.96(m,1H),6.79(d,J＝9.0,1H),4.76-7.72(m ,1H),4.56(d,J＝11.5,1H),4.32-4.25(m,1H),4.19-4.07(m,2H),3.97-3.80(m,3H),3.69(s, 3H),3.23-3.15(m,2H),3.00-2.81(m,4H),2.59-2.50(m,2H),2.47(s,2H),1.93-1.83(m,4H).

Example 200 2-(3-(5-(5-(azetidin-1-ylmethyl)-1-methyl-1H-pyrazol-3-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 200

Example 200a 5-(azetidin-1-ylmethyl)-1-methyl-3-nitro-1H-pyrazole 200a

Following Example 156e, 5-(bromomethyl)-1-methyl-3-nitro-1H-pyrazole 156d (1.76 g, 8 mmol), azetidine (550 mg, 9.6 mmol), diisopropylethylamine (1.75 mL, 10 mmol), and dichloromethane (40 mL) were stirred at room temperature for 3-5 hours. Workup and concentration under reduced pressure afforded 5-(azetidin-1-ylmethyl)-1-methyl-3-nitro-1H-pyrazole 200a as a yellow oil (quantitative), which was used in the next step without further purification. MS (ESI+) m/z 197.1 (M+H).

Example 200b 5-(azetidin-1-ylmethyl)-1-methyl-1H-pyrazol-3-amine 200b

Following Example 156f, 10% palladium on carbon (30% wet, 480 mg dry weight) and a solution of 200a (1.6 g, 8 mmol) in ethanol (50 mL) were reacted. The reaction mixture was filtered through a pad of Celite 521. The filter cake was washed with ethanol (2 x 30 mL), and the combined filtrates were concentrated to dryness under reduced pressure to afford 5-(azetidin-1-ylmethyl)-1-methyl-1H-pyrazol-3-amine 200b as a yellow oil (quantitative). MS (ESI+) m/z 167.1 (M+H).

Example 200c 3-(5-(azetidin-1-ylmethyl)-1-methyl-1H-pyrazol-3-ylamino)-5-bromo-1-methylpyridin-2(1H)-one 200c

According to Example 156g, 200b (350 mg, 2 mmol), 3,5-dibromo-1-methyl-1H-pyridin-2-one (0.54 g, 2 mmol), cesium carbonate (1.3 g, 4 mmol), tris(dibenzylideneacetone)dipalladium(0) (0.18 g, 0.2 mmol), Xantphos (0.23 g, 0.4 mmol), and 1,4-dioxane (20 mL) were heated at 100°C for 8 hours. Workup and flash column chromatography (silica gel, 3:1 dichloromethane/methanol) gave 3-(5-(azetidin-1-ylmethyl)-1-methyl-1H-pyrazol-3-ylamino)-5-bromo-1-methylpyridin-2(1H)-one 200c in 98% yield (0.69 g) as a yellow solid. MS (ESI+) m/z 354.1 (M+H).

Example 200d 2-(5-(5-(azetidin-1-ylmethyl)-1-methyl-1H-pyrazol-3-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-4-fluoro-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 200d

[0269] Following Example 121b, 4-fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 210d (202 mg, 0.42 mmol), 3-(5-(azetidin-1-ylmethyl)-1-methyl-1H-pyrazol-3-ylamino)-5-bromo-1-methylpyridin-2(1H)-one 200c (105 mg, 0.3 mmol), 1 M sodium carbonate solution (1.2 mL, 1.2 mmol), tetrakis(triphenylphosphine)palladium(0) (18 mg, 0.015 mmol) and 1,2-dimethoxyethane (3 mL) were heated in a microwave reactor at 130 °C for 10 minutes. After workup, the resulting residue was purified by flash chromatography (silica gel, 3:1 dichloromethane/methanol) to afford 180 mg of compound 200d (quantitative, containing 3% 200) as a yellow residue: MS (ESI+) m/z 628.1 (M+H).

Intermediate 200d (0.3 mmol) was deprotected using Example 121 with THF (1 mL), water (0.5 mL), isopropanol (1 mL), and lithium hydroxide monohydrate (80 mg, 1.93 mmol). Workup and purification of the resulting residue by flash chromatography (NH-silica gel, ethyl acetate/hexanes) afforded 200d as a light pink solid in 25% yield (2 steps, 45 mg): MS (ESI+) m/z 586.6 (M+H).

Example 201 2-(5-(5-(4-ethylpiperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl alcohol 201

Example 201b 2-(5-(5-(4-ethylpiperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 201b

To a 44-mL sealed tube equipped with a magnetic stirrer was added 138e (190 mg, 0.49 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bis(1,3,2-dioxaborolane) (200 mg, 0.79 mmol), potassium acetate (190 mg, 2.0 mmol), and 1,4-dioxane (5 mL). After the mixture was degassed for 30 minutes, Pd ₍ dppf) _Cl₂ · _CH₂Cl₂ (40 mg, 0.049 mmol) was added. The resulting reaction mixture was stirred at 105°C for 6 hours. It was then cooled to room temperature and filtered through a Celite pad. The filtrate was concentrated, and the crude boronolate product was redissolved in DME (2 mL) and transferred to a 10-mL microwave reaction vessel. To this solution was added 2-bromo-4-fluoro-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 197d (171 mg, 0.39 mmol), Pd(PPh ₃ ) ₄ (30 mg, 0.025 mmol), and 2N Na ₂ CO ₃ (2 mL). The reaction mixture was then degassed for 5 minutes and then placed in a microwave cavity. After stirring the reaction mixture at 125° C. for 7 minutes, it was purified by flash chromatography (dichloromethane:MeOH 85:15) to afford 46% (120 mg) of 201b as a solid.

To a 100-mL single-necked round-bottom flask equipped with a magnetic stir bar was added 2-(5-(5-(4-ethylpiperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 201b (120 mg, 0.18 mmol), LiOH·H ₂ O (80 mg, 1.8 mmol), THF (2 mL), isopropanol (2 mL), and water (2 mL). After stirring the reaction mixture at room temperature for 3 h, it was partitioned between dichloromethane (5 mL) and water (5 mL), and the organic phase was extracted with dichloromethane (3×5 mL). The combined organic phases were washed with water (2 x 5 mL) and brine (5 mL), dried ( _Na2SO4 ), and concentrated. The crude product was redissolved in dichloromethane (3 _mL ). Hexanes (10 mL) were added to this solution, and the resulting precipitate was filtered to afford 201 in 40% yield (45 mg); MS (ESI ⁺ ) m/z 626.4 (M+H).

Example 202 5-[5-Fluoro-2-(hydroxymethyl)-3-{1-oxo-1H,2H,3H,4H,6H,7H,8H,9H-pyrazino[1,2-a]indol-2-yl}phenyl]-1-methyl-3-{[5-(1-methylpyrrolidin-2-yl)pyridin-2-yl]amino}-1,2-dihydropyridin-2-one 202

Example 202a 5-Bromo-1-methyl-3-(5-(1-methylpyrrolidin-2-yl)pyridin-2-ylamino)pyridin-2(1H)-one 202a

Following Example 142a, 5-(1-methylpyrrolidin-2-yl)pyridin-2-amine (500 mg, 2 mmol), 3,5-dibromo-1-methylpyridin-2(1H)-one (0.54 g, 2 mmol), cesium carbonate (1.9 g, 6 mmol), tris(dibenzylideneacetone)dipalladium(0) (0.18 g, 0.2 mmol), Xantphos (0.23 g, 0.4 mmol) and 1,4-dioxane (20 mL) were heated at 100° C. for 8 hours. Workup and flash column chromatography (silica gel, 9:1 dichloromethane/methanol) afforded 82% yield (0.6 g) of a green solid 5-bromo-1-methyl-3-(5-(1-methylpyrrolidin-2-yl)pyridin-2-ylamino)pyridin-2(1H)-one 202a: MS (ESI+) m/z 365.0 (M+H).

According to Example 121b, 4-fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 210d (338 mg, 0.7 mmol), 202a (181 mg, 0.5 mmol), 1 M sodium carbonate solution (2 mL, 2 mmol), tetrakis(triphenylphosphine)palladium(0) (29 mg, 0.025 mmol) and 1,2-dimethoxyethane (5 mL) were reacted. Workup and flash column chromatography (silica gel, 3:1 dichloromethane/methanol) afforded a mixture of 4-fluoro-2-(1-methyl-5-(5-(1-methylpyrrolidin-2-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate and 202 (200 mg) as a yellow residue.

The above residue was deprotected according to Example 121, except using a mixture of THF (1 mL), water (0.5 mL), isopropanol (1 mL), and lithium hydroxide monohydrate (55 mg, 1.2 mmol). Workup and flash column chromatography (NH-silica gel, ethyl acetate/hexane) gave 202 as a yellow solid in 32% yield (60 mg): MS (ESI+) m/z 597.4 (M+H).

Example 203 5-(3-(6,6-dimethyl-5,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-c]pyridin-1(2H)-yl)-2-(hydroxymethyl)phenyl)-1-methyl-3-(pyrimidin-4-ylamino)pyridin-2(1H)-one 203

To a 100-mL single-necked round-bottom flask equipped with a magnetic stirrer and a nitrogen inlet was added 105i (226 mg, 0.506 mmol), 109c (200 mg, 0.610 mmol), sodium carbonate (160 mg, 1.51 mmol), water (2 mL) and 1,4-dioxane (8 mL). After bubbling nitrogen through the resulting suspension for 30 min, tetrakis(triphenylphosphine)palladium(0) (60 mg, 0.052 mmol) was added. A reflux condenser was connected to the flask, and the reaction mixture was heated at 100 ° C for 2 h. Thereafter, the mixture was diluted with 90:10 dichloromethane/methanol (100 mL) and water (75 mL), and the layers were separated. The aqueous layer was extracted with 90:10 dichloromethane/methanol (2×50 mL), and the combined organic layers were washed with brine (100 mL) and then dried over sodium sulfate. The desiccant was removed by filtration. The filtrate was concentrated under reduced pressure, and the resulting residue was dissolved in a mixture of THF (3 mL), water (3 mL), and methanol (3 mL). Lithium hydroxide monohydrate (61 mg, 1.45 mmol) was added, and the reaction was stirred at room temperature for 2 h. Thereafter, the mixture was diluted with 90:10 dichloromethane/methanol (100 mL) and water (50 mL), and the layers were separated. The aqueous layer was extracted with 90:10 dichloromethane/methanol (2×75 mL), and the combined organic layers were washed with brine (100 mL) and then dried over sodium sulfate. The drying agent was removed by filtration. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, 0%-10% methanol/dichloromethane) followed by powdering with methanol (10 mL), yielding 203 in 37% yield (57 mg) as a non-crystalline white solid: mp 170–171°C; ¹ H NMR (500 MHz, DMSO-d ₆ )δ9.18(s,1H),8.74(d,1H,J＝3.5Hz),8.65(s,1H),8.29(d,1H,J＝10.0Hz),7 .93(d,1H,J＝10.0Hz),7.57(d,1H,J＝3.5Hz),7.43(t,1H,J＝13.0Hz),7.32(m, 2H),7.24(d,1H,J＝13.0Hz),7.19(d,1H,J＝13.0Hz),4.83(m,1H),4.38(d,2H ,J＝6.5Hz),3.60(s,3H),2.90(s,2H),2.74(s,2H),1.28(s,6H); MS(ESI+)m/z 526.2(M+H).

Example 204 5-(5-fluoro-2-(hydroxymethyl)-3-(6,6-dimethyl-3,4,6,7-tetrahydro-5H-cyclopenta[4,5]thieno[2,3-c]pyridin-1(2H)-yl)-1-methyl-3-(5-(4-methylpiperazin-1-yl)pyridin-2-ylamino)pyridine-2(1H)204

To a 100-mL three-necked round-bottom flask equipped with a reflux condenser, a magnetic stirrer, and a nitrogen inlet was added 197e (170 mg, 0.450 mmol), 182c (300 mg, 0.513 mmol), sodium carbonate (143 mg, 1.35 mmol), water (2 mL), and 1,4-dioxane (8 mL). After bubbling nitrogen through the resulting suspension for 30 min, tetrakis(triphenylphosphine)palladium(0) (52 mg, 0.045 mmol) was added, and the reaction mixture was heated under reflux for 2 h. Thereafter, the mixture was cooled to room temperature and diluted with dichloromethane (100 mL) and water (30 mL). The organic layer was separated, and the aqueous layer was extracted with dichloromethane (3×30 mL). The combined organic layers were dried over sodium sulfate and then concentrated under reduced pressure to give a brown residue.

A 100-mL single-necked round-bottom flask equipped with a magnetic stirrer was purged with nitrogen and the crude residue, THF (10 mL) and 1 M tetrabutylammonium fluoride/THF solution (4.50 mmol, 4.5 mL) were added. The resulting mixture was stirred at room temperature for 1 h. Thereafter, the mixture was diluted with dichloromethane (100 mL) and washed with water (30 mL). The organic layer was separated, and the aqueous layer was extracted with dichloromethane (3×30 mL). The combined organic layers were dried over sodium sulfate and then concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, 0%-10% methanol/dichloromethane) to give 53% yield (154 mg) of an off-white solid 204: mp 143–145°C; ¹ H NMR (500 MHz, DMSO-d ₆ )δ8.54(d,1H,J＝2.5Hz),8.35(s,1H),7.86(d,1H,J＝3.0Hz),7.34(m,3H), 7.21(d,1H,J＝8.9Hz),7.17(dd,1H,J＝9.3,3.0Hz),4.85(m,1H),4.32(m,2 H),4.04(m,1H),3.85(m,1H),3.58(s,3H),3.03(m,5H),2.87(m,1H),2.75 (s,2H),2.53(m,2H),2.43(m,4H),2.20(s,3H),1.23(s,6H);MS(ESI+)m/z 643.3(M+H).

Example 205 2-(5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-(6-methyl-5,6,7,8-tetrahydro-1,6-naphthyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 205

Example 205a 5-Bromo-1-methyl-3-(5,6,7,8-tetrahydro-1,6-naphthyridin-2-ylamino)pyridin-2(1H)-one 205a

Tert-butyl 2-(5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate 158c (3.2 g, 7.4 mmol) was suspended in dichloromethane (20 mL) and saturated hydrogen chloride/dioxane (20 mL) was added dropwise. The reaction mixture was stirred for 20 minutes and then concentrated under reduced pressure to afford 205a, which was used in the next step without further purification. LC/MS: m/z 336 (M+H) ⁺

Example 205b 5-Bromo-1-methyl-3-(6-methyl-5,6,7,8-tetrahydro-1,6-naphthyridin-2-ylamino)pyridin-2(1H)-one 205b

A mixture of 205a (2.75 g, 7.5 mmol), formaldehyde (37% aqueous solution, 30 ml, 375 mmol), NaBH(OAc) ₃ (4.75 g, 22.5 mmol), and acetic acid (25 ml, 150 mmol) in methanol (125 ml) was stirred at room temperature for 4 hours. The mixture was then made basic with saturated NaOH solution and extracted with ethyl acetate. The organic layer was dried over Na ₂ SO ₄ and then evaporated under reduced pressure. The residue was purified by column chromatography eluting with 4:1 ethyl acetate/methanol to afford 205b (2.0 g, 77%). LC/MS: m/z 349 (M+H) ⁺

Example 205c 4-Fluoro-2-(1-methyl-5-(6-methyl-5,6,7,8-tetrahydro-1,6-naphthyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 205c

To a 15 mL microwave reaction vial equipped with a magnetic stir bar was added 205b (0.35 g, 1 mmol), 4-fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 210d (0.48 g, 1 mmol), potassium phosphate (0.54 g, 2 mmol), sodium acetate (0.17 g, 2 mmol), acetonitrile (10 mL), water (1 mL), and 1,1'-bis(diphenylphosphino)ferrocenepalladium(II) chloride in dichloromethane (0.08 g, 0.1 mmol). The mixture was heated at 110° C. under an argon atmosphere for 2 hours. Afterwards, the mixture was cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography using 40:1 DMC/methanol as eluent to afford 205c (0.22 g, 36%). LC/MS: m/z 625 (M+H) ⁺

To a 25 mL round-bottom flask equipped with a magnetic stirrer was added compound 4-fluoro-2-(1-methyl-5-(6-methyl-5,6,7,8-tetrahydro-1,6-naphthyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 205c (0.2 g, 0.32 mmol), LiOH (2 g, 48 mmol), THF (5 mL), isopropanol (5 mL), and water (2 mL). The reaction mixture was stirred at room temperature for 30 minutes and then filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by preparative HPLC to afford 205 (100 mg, 53%). LC/MS: m/z 583 (M+H) ⁺ . ¹ H NMR (500MHz, DMSO) δ8.79(d,J＝2,1H),8.45(s,1H),7.44(d,J＝2,1H),7.31(m,2H),7.20(m,1H),7.08(d,J＝9,1H),6.52(s,1H),4.89(m, 1H),4.34(m,2H),4.17(m,3H),3.89(m,1H),3.59(s,3H),3.38(m,2H),2.75(m,2H),2.62(m,4H),2.51(m,2H),2.33(s,3H),1.70(m,4H).

Example 206 2-(2-(Hydroxymethyl)-3-(1-methyl-5-(1-(2-(methylamino)ethyl)-1H-pyrazol-3-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 206

Example 206a 2-(2-(3-nitro-1H-pyrazol-1-yl)ethyl)isoindoline-1,3-dione 206a

A solution of 4-nitro-1H-pyrazole (10 g, 88.5 mmol) in anhydrous DMF (250 mL) was treated with a 60% suspension of NaH in mineral oil (4.6 g, 115 mmol) while stirring under nitrogen. After bubbling, the reaction was stirred for an additional 30 minutes. The reaction was then treated with 2-(2-bromoethyl)isoindoline-1,3-dione (18-2) (25 g, 97.3 mmol) and stirred under nitrogen for an additional 3 hours. Water (50 mL) was then slowly added, and the mixture was filtered to afford 206a (20 g, 80%) as a white solid, which was used without further purification. LCMS: (M+H) 287.

Example 206b 2-(3-nitro-1H-pyrazol-1-yl)ethanamine 206b

To a solution of 206a (2.0 g, 7.0 mmol) in ethanol (30 mL) was added hydrazine hydrate (1 mL, 21 mmol) and the mixture was stirred at room temperature for 3 hours. The mixture was evaporated and the residue was purified on a silica gel column using 10:1 dichloromethane/methanol as the eluent to afford 206b (0.8 g, 80%) as a white solid. MS: (M+H) ⁺ 157.

Example 206c tert-Butyl 2-(3-nitro-1H-pyrazol-1-yl)ethylaminocarboxylate 206c

To a solution of 206b (18 g, 115 mmol) in dichloromethane (400 mL) was added (Boc) _2O (50 g, 230 mmol) and triethylamine (35 g, 346 mmol). The mixture was stirred at room temperature for 5 hours. It was then evaporated and purified on a silica gel column eluting with 1:1 petroleum ether/ethyl acetate to afford 206c (24 g, 80%) as a white solid. MS: (M+H) ²⁵⁷ .

Example 206d tert-Butyl methyl (2-(3-nitro-1H-pyrazol-1-yl)ethyl)aminocarboxylate 206d

To a solution of 206c (5 g, 19.5 mmol) in THF (40 mL) was added NaH (938 mg, 23.4 mmol). The reaction mixture was stirred at room temperature for 30 minutes. Iodomethane (1.7 mL, 25.4 mmol) was added, and the mixture was stirred for an additional 15 hours. The mixture was then evaporated and purified on a silica gel column using 2:1 petroleum ether/ethyl acetate as the eluent to afford 206d (4.5 g, 85%) as a white solid. MS: (M+H) ²⁷¹ .

Example 206e tert- Butyl 2-(3-amino-1H-pyrazol-1-yl)ethyl(methyl)aminocarboxylate 206e

To a solution of 206d (4.5 g, 1.7 mmol) in ethanol (40 mL) were added Fe (4.7 g, 8.5 mmol) and NH ₄ Cl (900 mg, 17 mmol). The reaction mixture was stirred at room temperature for 4 hours. It was then evaporated and purified on a silica gel column using 2:1 petroleum ether/ethyl acetate as the eluent to afford 206e (3.2 g, 80%) as a brown solid. MS: (M+H) ⁺ 241.

Example 206f tert-Butyl 2-(3-(5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)-1H-pyrazol-1-yl)ethyl(methyl)aminocarboxylate 206f

A mixture of 206e (2 g, 8.3 mmol), 3,5-dibromo-1-methylpyridin-2(1H)-one (2.2 g, 8.3 mmol), XantPhos (482 mg, 0.83 mmol), Pd ₂ (dba) ₃ (762 mg, 0.83 mmol) and Cs ₂ CO ₃ (6.8 g, 21 mmol) in dioxane (80 mL) was heated at 100° C. for 15 h under nitrogen. It was then filtered, evaporated in vacuo, and purified by silica gel column eluting with 1:2 petroleum ether/ethyl acetate to give 206f (2.7 g, 77%) as a yellow solid. MS: (M+H) ⁺ 426.

Example 206g 2-(5-(1-(2-(tert-butoxycarbonyl(methyl)amino)ethyl)-1H-pyrazol-3-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 206g

A mixture of 206f (800 mg, 1.9 mmol), 2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate (114a) (873 mg, 1.9 mmol), _CH3COONa (309 mg, 3.8 mmol), _PdCl2 ( _dppf ) (153 mg, 0.19 mmol) and _K3PO4 (1 g, 3.8 mmol) was suspended in _CH3CN (30 _mL ) and H2O (2 mL), which was heated at 110°C under argon atmosphere for 15 h. It was then evaporated and the residue was purified by reverse phase Combi flash chromatography using 1:4 water/CH ₃ CN containing 0.3% NH ₄ HCO ₃ as eluent to give a brown solid 206 g (800 mg, 63%). MS: (M+H) ⁺ 684

Example 206h tert- Butyl 2-(3-(5-(2-(hydroxymethyl)-3-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)phenyl)-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)-1H-pyrazol-1-yl)ethyl(methyl)aminocarboxylate 206h

To a solution of 206g (750mg, 1.1mmol) in propan-2-ol (15mL), tetrahydrofuran (15mL) and water (5mL) was added LiOH (2.6g, 110mmol). The mixture was stirred at 30°C for 2h. It was then evaporated and the residue was purified by reverse phase Combi-flash chromatography using ₁ :3 water/ _CH3CN containing 0.3% _NH4HCO3 to give 206h (500mg, 71%) as a brown solid. MS: (M+H) ⁺ 642

A solution of 206h (500 mg, 0.78 mmol) in HCl/ethyl acetate (5 mL) was stirred at room temperature for 2 h. It was then evaporated and the residue was purified by preparative HPLC to give 206 as a brown solid (134 mg, 35%). MS: (M+H) ⁺ 542. ¹ HNMR(500MHz,MeOD)δ1.79(s,2H),1.89(s,2H),2.35(s,3H),2.54-2.56(t,J＝ 6Hz,2H),2.63-2.67(m,2H),2.97-2.99(t,J＝6Hz,2H),3.71(s,3H),4.00-4.0 3(m,1H),4.14-4.23(m,5H),4.50-4.60(m,2H),6.06-6.07(d,1H),6.71(s,1H ),7.23-7.24(d,1H),7.37-7.43(m,2H),7.49-7.52(m,2H),8.01-8.02(d,1H).

Example 207 2-(3-(5-(5-(3-hydroxy-3-methylazetidin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 207

Example 207a 3-Methyl-1-(6-nitropyridin-3-yl)azetidin-3-ol 207a

A mixture of 5-bromo-2-nitropyridine (3.28 g, 16.3 mmol), XantPhos (1.13 g, 1.96 mmol), Pd ₂ dba ₃ (1.19 g, 1.30 mmol), 3-methylazetidin-3-ol hydrochloride (2 g, 16.3 mmol), and Cs ₂ CO ₃ (15.9 g, 48.9 mmol) in 1,4-dioxane (70 mL) was heated under reflux for 2 h. After completion of the reaction, the mixture was filtered and washed with MeOH (100 mL). The filtrate was evaporated in vacuo, and the residue was purified by reverse phase Combi-flash chromatography to afford 207a (2.5 g, 71%). MS: [M+H] ⁺ 210.

Example 207b 1-(6-aminopyridin-3-yl)-3-methylazetidin-3-ol 207b

A 100-mL Parr hydrogenation bottle was purged with nitrogen and charged with 207a (2.3 g, 11 mmol), 10% palladium on carbon (50% humidity, 1.0 g), and methanol (100 mL). The bottle was evacuated, filled with hydrogen to 25 psi, and shaken on a Parr hydrogenation apparatus for 2 h. The hydrogen was then removed and nitrogen was introduced into the bottle. The catalyst was removed by filtration through a pad of Celite, and the filtrate was concentrated under reduced pressure to afford 207b (1.85 g, 92%). MS: [M+H] ⁺ 180.

Example 207c 5-Bromo-3-(5-(3-hydroxy-3-methylazetidin-1-yl)pyridin-2-ylamino)-1-methylpyridin-2(1H)-one 207c

A mixture of 207b (1.84 g, 10.3 mmol), XantPhos (714 mg, 1.24 mmol), Pd ₂ dba ₃ (755 mg, 0.82 mmol), 3,5-dibromo-1-methylpyridin-2(1H)-one (3.27 g, 12.3 mmol), and Cs ₂ CO ₃ (10 g, 30.9 mmol) in 1,4-dioxane (50 mL) was heated under reflux for 2 h. After completion of the reaction, the mixture was filtered and washed with MeOH (100 mL). The filtrate was evaporated in vacuo, and the residue was purified by reverse phase Combi flash chromatography to afford 207c (2.11 g, 54%). MS: [M+H] ⁺ 365.

Example 207d 2-(5-(5-(3-hydroxy-3-methylazetidin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 207d

A mixture of 207c (364 mg, 1.0 mmol), 2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 114a (510 mg, 1.1 mmol), PdCl ₂ (dppf) (110 mg, 0.15 mmol), K ₃ PO ₄ (100 mg), and NaOAc (50 mg) in MeCN (20 mL) and water (4 mL) was heated at reflux for 2 h. The solvent was evaporated in vacuo, and the residue was purified by reverse phase Combi flash chromatography to afford 207d (267 mg, 43%). MS: [M+H] ⁺ 623.

A mixture of 207d (350 mg, 0.56 mmol) and LiOH hydrate (236 mg, 5.6 mmol) in ⁱ PrOH (20 mL) and water (4 mL) was stirred at 30°C for 2 h. The mixture was evaporated in vacuo, and the residue was extracted with ethyl acetate (20 mL x 2). The combined extracts were concentrated under reduced pressure. The residue was purified by preparative HPLC to give 207 (130 mg, 40%). MS: [M+H] ⁺ 581. ¹ H NMR(500MHz,DMSO)δ8.48(s,1H),8.25(s,1H),7.48-7.44(m,2H),7.34-7.28(m,3H),7.17(d ,J＝8.5,1H),6.89-6.87(m,1H),6.51(s,1H),5.47(s,1H),4.84-4.83(m,1H),4.33(d,J＝4.5, 2H),4.18-4.06(m,3H),3.90-3.86(m,1H),3.70(d,J＝7.5,2H),3.58(s,3H),3.53-3.52(m,2 H),2.64-2.54(m,2H),2.47-2.45(m,2H),1.82-1.75(m,2H),1.73-1.65(m,2H),1.43(s,3H).

Example 208 2-(3-(5-(6-ethyl-5,6,7,8-tetrahydro-1,6-naphthyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 208

Example 208a 2-(5-(6-ethyl-5,6,7,8-tetrahydro-1,6-naphthyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-4-fluoro-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 208a

According to Example 136e, 5-bromo-3-(6-ethyl-5,6,7,8-tetrahydro-1,6-naphthyridin-2-ylamino)-1-methylpyridin-2(1H)-one and 4-fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 210d were converted to 208a in 42% yield. LCMS: (M+H) ⁺ 639

208a was converted to 208 in 40% yield according to Example 136. LCMS: (M+H) ⁺ 597. ¹ H NMR (500MHz, DMSO) δ8.80(d,J＝2,1H),8.45(s,1H),7.45(d,J＝2,1H),7.31(m,2H),7.20(dd,J＝10,1H),7.07(d,J＝9,1H),6.53(s,1H),4.36(s, 2H),4.14(m,3H),3.90(m,1H),3.60(s,3H),2.74(m,2H),2.68(m,2H),2 .59(m,2H),2.47(m,3H),1.79(m,2H),1.69(m,2H),1.08(t,J=7.5,3H).

Example 209 5-[2-(Hydroxymethyl)-3-[1-methyl-5-({5-[4-(oxetan-3-yl)piperazin-1-yl]pyridin-2-yl}amino)-6-oxo-1,6-dihydropyridin-3-yl]phenyl]-8-thia-5-azatricyclo[7.4.0.0 ^{2 ,7} ]trideca-1(9),2(7)-dien-6-one 209

Example 209a {2-[1-methyl-5-({5-[4-(oxetan-3-yl)piperazin-1-yl]pyridin-2-yl}amino)-6-oxopyridin-3-yl]-6-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]tridec-1(9),2(7)-dien-5-yl}phenyl}methyl acetate 209a

According to Example 148c, 481 mg of 111a and 420 mg of 5-bromo-1-methyl-3-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)pyridin-2(1H)-one 188e were reacted to give 209a (347 mg, 50%) as a yellow solid. MS: [M+H] ⁺ 695

According to Example 148, 230 mg of 209a was converted to a white solid 209 (108 mg, 50%). MS: [M+H] ⁺ 653. ¹ H NMR (500 MHz, DMSO) δ 8.56 (d, J = 2.5, 1H), 8.37 (s, 1H), 7.86 (d, J = 3.0, 1H), 7.45 (d, J = 7.5, 1H), 7.36-7.30 (m, 4H), 7.23 (d, J = 9.0, 1H), 4.82 (s, 1H), 4.55 (t, J = 6.5, 2H), 4.45 (d, J = 7.5, 1H). (t,J＝6.0,2H),4.35(s,2H),4.02(m,1H),3.82(m,1H),3.58(s,3H),3.42(m,2H),3.06 (t,J=4.5,4H),2.92-2.80(m,2H),2.78(s,2H),2.53(m,1H),2.38(s,4H),1.80(s,4H).

Example 210 2-(5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 210

Example 210a 5-Bromo-1-methyl-3-[5-(4-tert-butoxycarbonylpiperazin-1-yl)-pyridin-2-ylamine 210a

Step 1: tert-Butyl 4-(6-nitropyridin-3-yl)piperazine-1-carboxylate

To a solution of 5-bromo-2-nitropyridine (30 g, 148 mmol) in DMSO (1 L) was added K ₂ CO ₃ (40 g, 296 mmol) and tert-butyl piperazine-1-carboxylate (28 g, 148 mmol). The mixture was stirred at 65 ° C overnight. After cooling, it was poured into water (2 L). The precipitated solid was collected and then dried under vacuum. It was then purified by flash column chromatography using PE:EA (20:1) followed by DCM to give 17 mg of tert-butyl 4-(6-nitropyridin-3-yl)piperazine-1-carboxylate as a yellow solid (37% yield). MS: [M+H] ⁺ 309

Step 2: tert-Butyl 4-(6-aminopyridin-3-yl)piperazine-1-carboxylate

A 500-mL bottle was purged with nitrogen and added with tert-butyl 4-(6-nitropyridin-3-yl)piperazine-1-carboxylate (3.1 g, 10 mmol), 10% palladium/carbon (50% humidity, 1 g), and ethanol (100 mL). The bottle was evacuated, filled with hydrogen, and stirred at room temperature for 16 h. The hydrogen was then removed and nitrogen was introduced into the bottle. The catalyst was removed by filtration through a Celite pad, and the filtrate was then concentrated under reduced pressure to give tert-butyl 4-(6-aminopyridin-3-yl)piperazine-1-carboxylate (2.7 g, 97%). MS: [M+H] ⁺ 279

Step 3: A 100-mL single-necked round-bottom flask equipped with a magnetic stirrer and a reflux condenser was charged with 1,4-dioxane (50 mL), tert-butyl 4-(6-aminopyridin-3-yl)piperazine-1-carboxylate (1.3 g, 4.7 mmol) prepared according to US 7456168, 3,5-dibromo-1-methylpyridin-2(1H)-one (1.24 g, 4.7 mmol), and cesium carbonate (3.8 g, 12 mmol). After nitrogen was bubbled through the resulting solution for 30 min, Xantphos (272 mg, 0.47 mmol) and tris(dibenzylideneacetone)dipalladium(0) (430 mg, 0.47 mmol) were added, and the reaction mixture was heated under reflux for 3 h. Afterwards, the reaction was cooled to room temperature, partitioned between ethyl acetate (100 mL) and water (100 mL), and then filtered. The aqueous layer was separated and extracted with ethyl acetate (2 x 50 mL). The combined organic layers were washed with brine (50 mL) and then dried over sodium sulfate. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by flash column chromatography eluting with DCM:MeOH (50:1) to afford 210a (1.3 g, 59%). MS: [M+H] ⁺ 464.

Example 210b 5-Bromo-1-methyl-3-[5-(piperazin-1-yl)-pyridin-2-ylamino]-1H-pyridin-2-one 210b

Compound 210a (3.6 g, 7.8 mmol) was suspended in 4.0 M HCl/dioxane (10 mL). The reaction mixture was stirred at room temperature for 5 h and then concentrated under reduced pressure. The desired product was basified with 1.0 M aqueous NaOH and extracted with dichloromethane. The combined organic layers were washed with _H₂O and then concentrated under reduced pressure to give 2.46 g of 210b, 87% yield. MS: [M+H] ^⁺ 364.

Example 210c 5-Bromo-1-methyl-3-[5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino]-1H-pyridin-2-one 210c

A mixture of 210b (2.75 g, 7.5 mmol), oxetane-3-one (1.6 g, 22.7 mmol), NaBH ₃ CN (4.75 g, 22.5 mmol), and zinc chloride (3 g, 22.7 mmol) in methanol (125 mL) was stirred at 50°C for 5 hours. The mixture was added to water and extracted three times with dichloromethane. The organic layer was concentrated under reduced pressure. The residue was purified by column chromatography using dichloromethane:methanol = 25:1 to afford 210c (1.92 g, 61%). MS: [M+H] ⁺ 420. ¹ H NMR (500MHz, DMSO) δ8.58(d,J＝2.5,1H),8.55(s,1H),7.94(d,J＝3,1H),7.54(d,J＝2.5,1H),7.39(dd,J＝3,1H), 7.25 (d, J = 4, 1H), 4.56 (t, J = 6.5, 2H), 4.46 (t, J = 6.5, 2H), 3.50 (s, 3H), 3.43 (m, 1H), 3.01 (m, 4H), 2.40 (m, 4H).

Example 210d 2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-4-fluoro-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 210d

To a 250-mL single-necked round-bottom flask equipped with a magnetic stirrer was added 197d (3.8 g, 8.65 mmol), bis(pinacol)borate (11 g, 43.25 mmol), Pd(dppf) _Cl₂ (0.4 g, 0.5 mmol), KOAc (2.5 g, 26 mmol), and 1,4-dioxane (150 mL). The system was evacuated and then refilled with _N₂ . A reflux condenser was attached to the flask, and the reaction mixture was heated at 100°C for 15 h. It was then cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography using 5:1 petroleum ether/ethyl acetate as the eluent to afford 210d as a yellow solid in 77% yield (3.2 g). MS: [M+H] ^⁺ 483.

Example 210e 5-Fluoro-2-(1-methyl-5-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 210e

To a sealed tube was added a _mixture of 210d (337 mg, 0.7 mmol), 210c (294 mg, 0.7 mmol), Pd(dppf) _Cl₂ ( ₃₃ mg, 0.04 mmol), _{K₃PO₄.3H₂O} (372 mg, 1.4 mmol), and NaOAc (115 mg, 1.4 mmol) in 20 mL of _CH₃CN . The system was evacuated and refilled with _N₂ . The reaction mixture was heated at 110°C for 2 h, then cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography using 30:1 DCM/MeOH to afford 210e as a yellow solid in 54% yield (263 mg). MS: [M+H] ^⁺ 696.

To a solution of 210e (250 mg, 0.36 mol) in THF/isopropanol/water (6 mL/6 mL/2 mL) was added LiOH (87 mg, 3.6 mmol) at room temperature while stirring. The mixture was stirred for 0.5 h. Then, 20 mL of water was added and extracted with ethyl acetate (30 mL x 3). The combined organic layers were dried over Na ₂ SO ₄ and concentrated to give a yellow solid, which was further purified by preparative HPLC to give 210 as a white solid (134 mg, 57% yield). LCMS: [M+H] ⁺ 654. ¹ H NMR (50 0MHz, MEOD) δ8.53(d,J＝2.5,1H),7.93(d,J＝3,1H),7.42(dd,J＝3,1H),7.34(d,J＝ 2.5,1H),7.23(s,1H),7.21(s,1H),7.03(d,J＝4.5,1H),6.72(s,1H),4.73(t,J＝2 ,2H),4.64(t,J＝1.5,2H),4.51(m,2H),4.21(s,3H),4.03(m,1H),3.71(s,3H),3. 57(m,1H),3.17(t,J＝4.5,4H),2.65(m,2H),2.55(m,6H),1.90(m,2H),1.79(m,2H)

Example 211 5-[5-fluoro-2-(hydroxymethyl)-3-[1-methyl-5-({5-[4-(oxetan-3-yl)piperazin-1-yl]pyridin-2-yl}amino)-6-oxo-1,6-dihydropyridin-3-yl]phenyl]-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-6-one 211

Example 211a {4-fluoro-2-[1-methyl-5-({5-[4-(oxetan-3-yl)piperazin-1-yl]pyridin-2-yl}amino)-6-oxo-1,6-dihydropyridin-3-yl]-6-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-5-yl}phenyl}methyl acetate 211a

According to Example 148h, 400 mg of (4-fluoro-2-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-5-yl}-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methyl acetate 212b and 336 mg of 5-bromo-1-methyl-3-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)pyridin-2(1H)-one 210c were reacted to give a yellow solid 211a (319 mg, 56%). LCMS: (M+H) ⁷¹³

According to Example 148, 270 mg of 211a was converted into a white solid 211 (120 mg, 48%). LCMS: [M+H] ⁺ 671. ¹ H NMR (500MHz, DMSO) δ8.56 (s, 1H), 8.39 (s, 1H), 7.87 (s, ¹ H), 7.38-7.16 (m, 5H), 4.8 (s, ¹ H), 4.56 (t, J = 6.5 2H),4.46(t,J＝6.5,2H),4.33(s,1H),4.05(m, ¹ H), 3.87 (m, 1H), 3.58 (s, 3H), 3.43 (t, J = 6, 2H), 3.06 (m, 4H), 2.88-2.78 (m, 4H), 2.38 (t, J = 5, 1H), 1.79 (m, 4H).

Example 212 5-[5-Fluoro-2-(hydroxymethyl)-3-(1-methyl-5-{[5-(4-methylpiperazin-1-yl)pyridin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)phenyl]-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-6-one 212

Example 212a 2-Bromo-4-fluoro-6-(1-oxo-3,4,5,6,7,8-hexahydrobenzothieno[2,3-c]pyridin-2(1H)-yl)benzyl acetate 212a

A solution of 104e (3 g, 14.5 mmol), 2,6-dibromo-4-fluorobenzyl acetate 197c (14 g, 43.5 mmol), Xantphos (839 mg, 1.45 mmol), Pd ₂ (dba) ₃ (1.33 g, 1.45 mmol), and Cs ₂ CO ₃ (9.4 g, 29 mmol) in dioxane (200 mL) was heated at 100° C. for 15 h under nitrogen. After filtration, the filtrate was evaporated in vacuo and purified by flash column chromatography using ethyl acetate/petroleum ether (1:1) as the eluent to afford 212a (5 g, 77% yield) as a yellow solid. LCMS: (M+H) ⁺ 452. ¹ H NMR(500MHz,DMSO)δ7.71(dd,J＝2.5,1H),7.51(dd,J＝3,1H),5.04(m,1H),4.10(m, 1H),3.68(m,1H),2.86(m,2H),2.77(m,2H),2.55(m,3H),1.98(s,3H),1.78(m,4H)

Example 212b 2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-4-fluoro-6-(1-oxo-3,4,5,6,7,8-hexahydrobenzothieno[2,3-c]pyridin-2(1H)-yl)benzyl acetate 212b

To a solution of 212a (3 g, 6.65 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bis(1,3,2-dioxaborolane) (10 g, 40 mmol) in dioxane (160 mL) was added _PdCl2 (dppf) (543 mg, 0.66 mmol) and _CH3COOK (3.9 g, 40 mmol). The mixture was stirred at 100° for 15 h under argon atmosphere. The mixture was filtered, evaporated in vacuo, and purified by flash column chromatography using ethyl acetate/petroleum ether (1:2) as the eluent to afford 212b (2.5 g, 76% yield) as a yellow solid. LCMS: (M+H) ⁺ 500

Example 212c [4-Fluoro-2-(1-methyl-5-{[5-(4-methylpiperazin-1-yl)pyridin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)-6-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]tridec-1(9),2(7)-dien-5-yl}phenyl]methyl acetate 212c

To a 25 mL sealed tube was added 212b (990 mg, 2 mmol), 5-bromo-1-methyl-3-(5-(4-methylpiperazin-1-yl)pyridin-2-ylamino)pyridin-2(1H)-one 198e (500 mg, 1.3 mmol), _CH3COONa (220 mg, 2.6 mmol), _K3PO4 (700 mg, 2.6 mmol) _, and _PdCl2 (dppf) (110 mg, 0.13 mmol) and suspended in _CH3CN (25 mL) and water (1 mL). The mixture was heated at 110°C for 2 h. The residue was evaporated and purified by silica gel column eluting with 20:1 dichloromethane/methanol to give 212c (500 mg, 56%) as a brown solid. LCMS: [M+H] ⁺ 670

To a solution of 212c (500 mg, 0.75 mmol) in propan-2-ol (8 mL), tetrahydrofuran (8 mL), and water (1.5 mL) was added LiOH (964 mg, 40 mmol). The mixture was stirred at 30°C for 2 h. Then, 20 mL of _H₂O was added, and the mixture was extracted with ethyl acetate (3 x ₃₀ mL). The combined organic layers were dried over _Na₂SO₄ and concentrated to afford a yellow solid, which was further purified by preparative HPLC to afford 212 as a white solid (200 mg, 48%). LCMS: [M+H] ^⁺ 629. ¹ H NMR (500MHz, DMSO-d ₆ )δ8.56(d,J＝2,1H),8.37(s,1H),7.85(d,J＝2.5,1H),7.34(m,3H),7.19(m,2H),4.86(s,1H),4.32(s,2H),4.05(m,1H),3.87( m,1H),3.57(s,3H),3.02(m,4H),2.96(m,1H),2.87(m,1H),2.77(m,2H),2.54(m,1H),2.43(m,4H),2.19(s,3H),1.79(m,4H).

Example 213 2-(5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-(5-(1-methylazetidin-3-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 213

Example 213a 4-Fluoro-2-(1-methyl-5-(5-(1-methylazetidin-3-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 213a

According to Example 148b, 482 mg of 4-fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 210d and 350 mg of 5-bromo-1-methyl-3-(5-(1-methylazetidin-3-yl)pyridin-2-ylamino)pyridin-2(1H)-one 172a were reacted to give 213a (324 mg, 52%) as a yellow solid. MS: [M+H] ⁺ 625

Following the method described for 148, starting from 220 mg of 213a, compound 213 (82 mg, 40%) was obtained as a white solid. MS: [M+H] ⁺ 583. ¹ H NMR (500 MHz, CDCl3) δ 8.64 (d, J = 2.0, 1H), 8.13 (d, J = 2.5, 1H), 7.89 (s, 1H), 7.58 (m, 1H), 7.50 (d, J = 2.5, 1H), 7.16 (dd, J = 9.0, 1H), 6.96 (dd, J = 8.5, 1H), 6.86 (s, 1H), 6.83 (d, J = 8.5,1H),4.55(d,J＝11.5,1H),4.40(s,1H),4.30(s,1H),4.16(m,2H),3.91(m,1H),3.7 2(m,5H),3.60(m,1H),3.14(t,J＝7.0,2H),2.59(m,4H),2.38(s,3H),1.90-1.79(m,5H).

Example 214 2-(5-fluoro-2-(hydroxymethyl)-3-(4-methyl-6-(4-(1-(oxetan-3-yl)piperidin-4-yl)phenylamino)-5-oxo-4,5-dihydropyrazin-2-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 214

Example 214a 5-Bromo-1-methyl-3-(4-(piperidin-4-yl)phenylamino)pyrazin-2(1H)-one 214a

Compound 214a was synthesized using the same procedure as 121c, except that tert-butyl 4-(4-(6-bromo-4-methyl-3-oxo-3,4-dihydropyrazin-2-ylamino)phenyl)piperidine-1-carboxylate (121a) (0.53 g, 1.08 mmol), trifluoroacetic acid (0.9 mL, 10.8 mmol), and dichloromethane (20 mL) were used. Workup and concentration gave 214a as a yellow oil in quantitative yield (390 mg), which was used in the next step without purification.

Example 214b 5-Bromo-1-methyl-3-(4-(1-(oxetan-3-yl)piperidin-4-yl)phenylamino)-pyrazin-2(1H)-one 214b

A 100-mL sealed tube equipped with a magnetic stirrer was purged with nitrogen and 214a (390 mg, 1.08 mmol), oxetane-3-one (800 mg, 11 mmol) and methanol (10 mL) were added. A suspension of sodium cyanoborohydride (208 mg, 3.3 mmol) and zinc chloride (225 mg, 1.65 mmol) in methanol (10 mL) was added and heated at 48 ° C for 12 hours. Afterwards, the reaction mixture was concentrated and the residue was partitioned between ethyl acetate (50 mL) and 10% aqueous potassium carbonate solution (10 mL). The aqueous layer was extracted with ethyl acetate (3×30 mL). The combined organic layers were dried over sodium sulfate and then concentrated under reduced pressure. The resulting residue was purified by column chromatography (silica gel, 60:35:5 dichloromethane/diethyl ether/methanol) to give 60% yield (270 mg) of 5-bromo-1-methyl-3-(4-(1-(oxetan-3-yl)piperidin-4-yl)phenylamino)pyrazin-2(1H)-one (214b) as a yellow solid: MS (ESI+) m/z 421.2 (M+H).

Example 214c 4-Fluoro-2-(4-methyl-6-(4-(1-(oxetan-3-yl)piperidin-4-yl)phenylamino)-5-oxo-4,5-dihydropyrazin-2-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 214c

[0266] Following Example 121b, 4-fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 210d (217 mg, 0.45 mmol), 214b (158 mg, 0.375 mmol), 1 M sodium carbonate solution (1.5 mL, 1.5 mmol), tetrakis(triphenylphosphine)palladium(0) (22 mg, 0.019 mmol) and 1,2-dimethoxyethane (3.5 mL) were heated in a microwave reactor at 130 °C for 15 minutes. Workup and flash column chromatography (silica gel, 60:35:5 dichloromethane/ether/methanol) afforded 214c as a brown oil in 62% yield (160 mg): MS (ESI+) m/z 696.1 (M+H).

Compound 214 was synthesized using the same procedure as 121, except that a mixture of THF (1 mL), water (0.5 mL), and isopropanol (1 mL), 214c (160 mg, 0.23 mmol), and lithium hydroxide monohydrate (80 mg, 2 mmol) was used. Workup and flash column chromatography (NH-silica gel, ethyl acetate/hexane) gave 214 as a yellow solid in 34% yield (52 mg): MS (ESI+) m/z 653.6 (M+H).

Example 215 10-[5-fluoro-2-(hydroxymethyl)-3-(4-methyl-6-{[4-(1-methylazetidin-3-yl)phenyl]amino}-5-oxo-4,5-dihydropyrazin-2-yl)phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^{2 ,6} ]dodeca-2(6),7-dien-9-one 215

Example 215a 10-[5-fluoro-2-(acetoxymethyl)-3-(4-methyl-6-{[4-(1-methylazetidin-3-yl)phenyl]amino}-5-oxo-4,5-dihydropyrazin-2-yl)phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 215a

To a 44-mL sealed tube equipped with a magnetic stirrer was added 298c (160 mg, 0.48 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi-1,3,2-dioxaborolane (180 mg, 0.71 mmol), potassium acetate (188 mg, 1.9 mmol), and 1,4-dioxane (5 mL). After the mixture was degassed for 30 minutes, Pd ₍ dppf) _Cl₂ · _CH₂Cl₂ (39 mg, 0.048 mmol) was added. The resulting reaction mixture was stirred at 105°C for 4 hours. It was then cooled to room temperature and filtered through a Celite pad. The filtrate was concentrated, and the crude mixture was redissolved in 1,2-dimethoxyethane (2 mL) and transferred to a 10-mL microwave reaction vessel. To this solution was added 10-[2-(acetoxymethyl)-3-bromo-5-fluorophenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 189a (172 mg, 0.38 mmol), Pd(PPh ₃ ) ₄ (30 mg, 0.024 mmol), and 2N Na ₂ CO ₃ (2 mL). The reaction mixture was then degassed for 5 minutes and placed in a microwave cavity. After stirring the reaction mixture at 125° C. for 10 minutes, it was purified by flash chromatography (dichloromethane:methanol, 3:1) to afford 20% (49 mg) of 215a.

To a 25-mL single-necked round-bottom flask equipped with a magnetic stir bar was added 215a (49 mg, 0.077 mmol), LiOH· _H₂O (16 mg, 0.38 mmol), THF (2 mL), isopropanol (2 mL), and water (2 mL). The reaction mixture was stirred at room temperature for 3 h, then partitioned between dichloromethane (5 mL) and water (5 mL), and the organic phase was extracted with dichloromethane (3 x 5 mL). The combined organic phases were washed with water (2 x 5 mL) and brine (5 _mL ), dried ( _Na₂SO₄ ), and then concentrated. The crude product was redissolved in dichloromethane (3 mL). Hexane (10 mL) was added to this solution, and the resulting precipitate was filtered to provide 215 in a 74% yield (34 mg). MS ( ^ESI⁺ ) m/z 597.5 (M+H).

Example 216 Disodium 2-(1-methyl-6-oxo-5-(pyrimidin-4-ylamino)-1,6-dihydropyridin-3-yl)-6-(6,6-dimethyl-3,4,6,7-tetrahydro-5H-cyclopenta[4,5]thieno[2,3-c]pyridin-1(2H)-yl)benzyl phosphate

Example 216a Bis(2-cyanoethyl)2-(1-methyl-6-oxo-5-(pyrimidin-4-ylamino)-1,6-dihydropyridin-3-yl)-6-(6,6-dimethyl-3,4,6,7-tetrahydro-5H-cyclopenta[4,5]thieno[2,3-c]pyridin-1(2H)-yl)benzyl phosphate 216a

To a 250-mL single-necked round-bottom flask equipped with a magnetic stirrer and a nitrogen inlet was added 105 (850 mg, 1.61 mmol), 1H-tetrazole (451 mg, 6.44 mmol), and dichloromethane (20 mL). A solution of bis(2-cyanoethyl)-NN-diisopropylphosphoramidite (1.31 g, 4.83 mmol) in dichloromethane (5 mL) was added at room temperature, and the reaction mixture was stirred under a nitrogen atmosphere for 3 h. Afterwards, the reaction mixture was cooled to 0°C, and a 5.5 M solution of tert-butyl hydroperoxide in decane (2.00 mL, 11.0 mmol) was added dropwise, and the reaction mixture was stirred at 0°C under a nitrogen atmosphere for 3 h. After that, the mixture was concentrated under reduced pressure to a volume of 5 mL, and the resulting suspension was loaded onto a silica gel column and eluted with 90:10 dichloromethane/methanol to give 216a as a white semisolid in 39% yield (450 mg): ¹ H NMR (500 MHz, CDCl ₃ ) δ 8.77 (d, J = 0.5 Hz, 1H), 8.73 (d, J = 2.0 Hz, 1H), 8.36 (d, J = 5.5 Hz, 1H), 8.17 (s, 1H), 7.55 (t, J = 7.5 Hz, 1H), 7.38-7.35 (m, 2H), 7.09 (d, J = 2.0 Hz, 1H), 6.76 (dd, J = 6.0, 1.5 Hz, 1H), 5.21- 5.14(m,2H),4.15–4.04(m,5H),3.86–3.83(m,1H),3.72(s,3H),3.21–3.18(m,1H),2.90–2. 82(m,1H),2.79(d,J＝3.5Hz,2H),2.63–2.48(m,6H),1.28(s,3H),1.27(s,3H); MS(ESI+)m/z 714.2(M+H).

Example 216b 2-(1-methyl-6-oxo-5-(pyrimidin-4-ylamino)-1,6-dihydropyridin-3-yl)-6-(6,6-dimethyl-3,4,6,7-tetrahydro-5H-cyclopenta[4,5]thieno[2,3-c]pyridin-1(2H)-yl)benzyl diammonium phosphate 216b

To a 250-mL single-necked round-bottom flask equipped with a magnetic stirrer and a nitrogen inlet was added 216a (450 mg, 0.631 mmol) and acetonitrile (8 mL), and the mixture was cooled to 0° C. Triethylamine (4 mL) was added, followed by bistrimethylsilyltrifluoroacetamide (4 mL), and the mixture was stirred at room temperature for 40 h. After this time, the mixture was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, 40:20:40 chloroform/methanol/ammonia) to afford 216b as a non-crystalline yellow solid in 52% yield (210 mg): mp 260-262 °C d; ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 9.12 (s, 1H), 8.66 (s, 1H), 8.62 (d, J=2.0 Hz, 1H), 8.27 (d, J=4.0 Hz, 1H), 7.54 (s, 1H), 7.43 (t, J=7.5 Hz, 1H), 7.32-7.27 (m, 3H), 7.15 (br s,2H),4.69(d,J＝9.0Hz,1H),4.46(d,J＝7.0Hz,1H),3.87–3.83(m,3H),3.57(s,3H),3.36–3.17(br s,8H),2.77–2.63(m,3H),1.23(s,3H),1.21(s,3H); MS(ESI+)m/z 608.2(M+H for C ₂₉ H ₃₀ N ₅ O ₆ PS).

To a 200-mL single-necked round-bottom flask equipped with a magnetic stirrer were added 216b (210 mg, 0.327 mmol), methanol (10 mL), and water (5 mL). 0.1 M aqueous sodium hydroxide solution (6.50 mL, 0.65 mmol) was added and the mixture was stirred for 30 min at room temperature. Afterwards, the mixture was concentrated under reduced pressure and then dried under vacuum at 45°C for 14 h to afford 216 in 92% yield (197 mg) as a non-crystalline yellow solid: mp 256–258°C dec; ¹ H NMR (500 MHz, DMSO-d ₆ /D ₂ O(15:1))δ8.65(s,1H),8.63(d,J＝1.5Hz,1H),8.28(d,J＝6.0Hz,1H),8.09(d,J＝1.5Hz,1H),7 .47(t,J＝7.5Hz,1H),7.38(d,J＝7.5Hz,1H),7.24(d,J＝7.5Hz,1H),7.16(d,J＝6.0Hz,1H),4.72 (d,J＝9.5Hz,1H),4.45(d,J＝8.0Hz,1H),4.23–4.21(m,1H),3.83–3.78(m,1H),3.71(s,3H),3. 48–3.41(m,1H),2.78–2.73(m,3H),2.59–2.49(m,2H),1.23(s,3H),1.22(s,3H); MS(ESI+)m/z 608.2(M+H for C ₂₉ H ₃₀ N ₅ O ₆ PS).

Example 217 5-(5-fluoro-2-(hydroxymethyl)-3-(6,6-dimethyl-3,4,6,7-tetrahydro-5H-cyclopenta[4,5]thieno[2,3-c]pyridin-1(2H)-yl)-3-(5-(4-(2-fluoroethyl)piperazin-1-yl)pyridin-2-ylamino)-1-methylpyridin-2(1H)-one 217

Example 217a 1-(6-nitropyridin-3-yl)piperazine 217a

2-nitro-5-bromopyridine (5.00 g, 24.6 mmol), piperazine (5.66 g, 65.7 mmol) and acetonitrile (70 mL) were added to a 250-mL three-necked round-bottom flask equipped with a magnetic stirrer and a reflux condenser. The reaction mixture was heated under reflux for 20 h. Thereafter, the reaction mixture was cooled to room temperature and then concentrated under reduced pressure. The residue was diluted with ethyl acetate (200 mL) and then washed with saturated sodium bicarbonate aqueous solution (100 mL). The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was triturated with hexanes/ethyl acetate (1:1, 30 mL) to afford 217a as a yellow solid in 41% yield (2.10 g): mp 113-115°C; ^1H NMR (500 MHz, DMSO- _d6 ) δ 8.23 (d, 1H, J = 3.5 Hz), 8.13 (d, 1H, J = 9.0 Hz), 7.44 (dd, 1H, J = 9.0, 3.5 Hz), 3.41 (t, 4H, J = 5.0 Hz), 2.82 (t, 4H, J = 5.0 Hz), 1.90 (s, 1H); MS (ESI+) m/z 209.1 (M+H).

Example 217b 1-(2-fluoroethyl)-4-(6-nitropyridin-3-yl)piperazine 217b

To a 250-mL single-necked round-bottom flask equipped with a magnetic stirrer, a reflux condenser, and a nitrogen inlet was added 217a (598 mg, 2.87 mmol), 1-bromo-2-fluoroethane (1.09 g, 8.61 mmol), potassium carbonate (1.19 g, 8.61 mmol), THF (10 mL), and DMF (10 mL). The reaction mixture was heated at 85 ° C for 14 h under a nitrogen atmosphere. Thereafter, the mixture was diluted with 90:10 dichloromethane/methanol (100 mL) and water (75 mL), and the layers were separated. The aqueous layer was extracted with 90:10 dichloromethane/methanol (50 mL), and the combined organic layers were washed with brine (100 mL) and then dried over sodium sulfate. The desiccant was removed by filtration. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, 95:5 dichloromethane/methanol) to give 217b as a yellow semisolid in 63% yield (460 mg): ¹ H NMR (500 MHz, CDCl ₃ ) δ 8.16 (d, J = 9.0 Hz, 1H), 8.14 (d, J = 3.0 Hz, 1H), 7.21 (dd, J = 9.0, 2.0 Hz, 1H), 4.67 (t, J = 5.0 Hz, 1H), 4.57 (t, J = 5.0 Hz, 1H), 3.49 (t, J = 5.0 Hz, 4H), 2.81 (t, J = 5.0 Hz, 1H), 2.75 (t, J = 5.0 Hz, 1H), 2.73 (t, J = 5.5 Hz, 4H); MS (ESI+) m/z 255.1(M+H).

Example 217c 5-(4-(2-fluoroethyl)piperazin-1-yl)pyridin-2-amine 217c

A 250-mL Parr reaction bottle was purged with nitrogen and a solution of 10% palladium on carbon (50% humidity, 75 mg dry weight) and 217b (455 mg, 1.79 mmol) in ethanol (20 mL) was added. The bottle was connected to a Parr hydrogenation reactor, evacuated, filled with hydrogen to a pressure of 50 psi and shaken for 3 h. Thereafter, the hydrogen was removed and nitrogen was introduced into the bottle. Celite 521 (3.50 g) was added and the mixture was filtered through a Celite 521 pad. The filter cake was washed with ethanol (2×50 mL), and the combined filtrates were concentrated to dryness under reduced pressure to afford 217c (425 mg) as a yellow semisolid in quantitative yield: ¹ H NMR (500 MHz, CDCl ₃ ) δ 7.75 (d, J=3.0 Hz, 1H), 7.20 (dd, J=9.0, 3.0 Hz, 1H), 6.51 (d, J=8.5 Hz, 1H), 4.66 (t, J=5.0 Hz, 1H), 4.56 (t, J=5.0 Hz, 1H), 4.35–4.15 (br s, 2H), 3.08 (t, J=5.0 Hz, 4H), 2.79 (t, J=5.0 Hz, 1H), 2.74 (t, J=4.5 Hz, 1H), 2.71 (t, J=5.0 Hz, 4H); MS (ESI+) m/z 225.1(M+H).

Example 217d 5-Bromo-3-(5-(4-(2-fluoroethyl)piperazin-1-yl)pyridin-2-ylamino)-1-methylpyridin-2(1H)-one 217d

217c (402 mg, 1.79 mmol), 3,5-dibromo-1-methyl-pyridin-2(1H)-one (478 mg, 1.79 mmol), cesium carbonate (1.75 g, 5.37 mmol) and 1,4-dioxane (15 mL) were added to a 100-mL single-necked round-bottom flask equipped with a magnetic stirrer and a nitrogen inlet. After nitrogen was bubbled through the resulting suspension for 30 min, Xantphos (93 mg, 0.161 mmol) and tris(dibenzylideneacetone)dipalladium(0) (82 mg, 0.090 mmol) were added. A reflux condenser was connected to the flask, and the reaction mixture was heated at 100 ° C for 3 h. Thereafter, the mixture was cooled to room temperature and diluted with 90:10 dichloromethane/methanol (100 mL) and water (75 mL), and the layers were separated. The aqueous layer was extracted with 90:10 dichloromethane/methanol (2 x 50 mL), and the combined organic layers were washed with brine and then dried over sodium sulfate. The drying agent was removed by filtration. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, 95:5 dichloromethane/methanol) to afford 217d in 62% yield (459 mg) as a non-crystalline yellow solid: mp 141–143°C; ¹ H NMR (500 MHz, CDCl ₃ )δ8.57(d,J＝2.0Hz,1H),7.99(d,J＝3.0Hz,1H),7.74(s,1H),7.24(d,J＝3.0Hz,1H),6.93(d,J＝2.0Hz,1H),6.76(d,J＝9.0Hz,1H),4.67(t,J＝5.0Hz ,1H),4.57(t,J＝5.0Hz,1H),3.59(s,3H),3.16(t,J＝5.0Hz,4H),2.80(t,J＝5.0Hz,1H),2.75(t,J＝5.0Hz,1H),2.73(t,J＝5.5Hz,4H); MS(ESI+)m/z 410.1(M+H)

To a 100-mL single-necked round-bottom flask equipped with a magnetic stirrer and a nitrogen inlet was added 217d (177 mg, 0.431 mmol), 182c (315 mg, 0.539 mmol), sodium carbonate (100 mg, 0.862 mmol), water (2 mL) and 1,4-dioxane (10 mL). After bubbling nitrogen through the resulting suspension for 30 min, tetrakis(triphenylphosphine)palladium(0) (100 mg, 0.086 mmol) was added. A reflux condenser was connected to the flask, and the reaction mixture was heated at 90 ° C for 1 h. Thereafter, the mixture was diluted with 90:10 dichloromethane/methanol (100 mL) and water (75 mL), and the layers were separated. The aqueous layer was extracted with 90:10 dichloromethane/methanol (2×50 mL), and the combined organic layers were washed with brine (100 mL) and then dried over sodium sulfate. The desiccant was removed by filtration. The filtrate was concentrated under reduced pressure, and the resulting residue was dissolved in a mixture of THF. A 1.0 M tetrabutylammonium fluoride/THF solution (1.10 mL, 1.10 mmol) was added, and the mixture was stirred at room temperature for 3 h. Thereafter, the mixture was diluted with 90:10 dichloromethane/methanol (75 mL) and water (50 mL), and the layers were separated. The aqueous layer was extracted with 90:10 dichloromethane/methanol (2×50 mL), and the combined organic layers were washed with brine (100 mL) and then dried over sodium sulfate. The drying agent was removed by filtration. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, 95:5 dichloromethane/methanol) to give a 25% yield (73 mg) of a non-crystalline yellow solid 217: mp 158–160°C; ¹ H NMR (500 MHz, DMSO-d ₆ )δ8.55(d,J＝2.0Hz,1H),8.35(s,1H),7.86(d,J＝3.0Hz,1H),7.37–7.31(m,3H),7.22(d,J＝9.0Hz,1H),7.17( dd,J＝9.5,3.0Hz,1H),4.85(t,J＝4.0Hz,1H),4.61(t,J＝5.0Hz,1H),4.51(t,J＝5.0Hz,1H),4.35–4.30(m,2H) ,4.08–4.02(m,1H),3.87–3.82(m,1H),3.58(s,3H),3.05–2.99(m,5H),2.91–2.87(m,1H),2.75(s,2H),2.68 (t,J＝5.0Hz,1H),2.62(t,J＝4.5Hz,1H),2.57–2.53(m,4H),2.52(d,J＝4.5Hz,2H),1.23(s,6H); MS(ESI+)m/z 675.3(M+H).

Example 218 5-(3-{5-[(6-ethyl-5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl)amino]-1-methyl-6-oxo-1,6-dihydropyridin-3-yl}-5-fluoro-2-(hydroxymethyl)phenyl)-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-6-one 218

Example 218a (2-{5-[(6-ethyl-5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl)amino]-1-methyl-6-oxo-1,6-dihydropyridin-3-yl}-4-fluoro-6-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]tridec-1(9),2(7)-dien-5-yl}phenyl)methyl acetate 218a

To a 25 mL sealed tube were added (2-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-5-yl}-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methyl acetate (212b) (893 mg, 1.8 mmol), 5-bromo-3-(6-ethyl-5,6,7,8-tetrahydro-1,6-naphthyridin-2-ylamino)-1-methylpyridin-2(1H)-one (500 mg, 1.4 mmol), CH ₃ COONa (226 mg, 2.75 mmol), K ₃ PO ₄ (733 mg, 2.75 mmol), and PdCl ₂ (dppf) (112 mg, 0.14 mmol), and the mixture was suspended in CH ₃ CN (25 mL) and H ₂ O (1 mL). The mixture was stirred at 110° C. for 2 h. After the reaction, the solvent was evaporated and the residue was purified on a silica gel column eluting with 20:1 dichloromethane/methanol to give 218a as a brown solid (400 mg, 44%). MS: (M+H) ⁺ 656.

To a solution of 218a (350 mg, 0.53 mmol) in propan-2-ol (10 mL), tetrahydrofuran (10 mL), and water (2 mL) was added LiOH (1.28 g, 53 mmol). The mixture was stirred at 30°C for 2 h. After the reaction, the mixture was evaporated and the residue was purified by preparative HPLC to give 218 as a yellow solid (124 mg, 38%). MS: (M+H) ⁺ 614. ¹ H NMR (500MHz, DMSO) δ1.06-1.09 (t, J＝7.5Hz, 3H), 1.80 (s, 4H), 2.55-2.58 (m, 2H), 2.67-2. 68(d,2H),2.74-2.79(d,4H),2.85-2.98(m,2H),3.43(s,2H),3.60(s,3H),3.87-3.89(m, 1H),4.05-4.07(m,1H),4.37-4.38(d,2H),4.87-4.89(t,J＝4.5Hz,1H),7.07-7.09(d,1H) ,7.19-7.21(m,1H),7.29-7.34(m,2H),7.45-7.46(d,1H),8.46(s,1H),8.79-8.80(d,1H).

Example 219 5-[5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-{[6-(oxetane-3-yl)-5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)phenyl]-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-diene-6-one 219

Example 219a 5-Bromo-1-methyl-3-(6-(oxetan-3-yl)-5,6,7,8-tetrahydro-1,6-naphthyridin-2-ylamino)pyridin-2(1H)-one 219a

A mixture of 5-bromo-1-methyl-3-(5,6,7,8-tetrahydro-1,6-naphthyridin-2-ylamino)pyridin-2(1H)-one hydrochloride 205a (800 mg, 2.16 mmol), oxetane-3-one (778 mg, 10.8 mmol), _NaBH₃CN (681 mg, 10.8 mmol), and zinc chloride (1.47 g, 10.8 mmol) in methanol (30 mL) was stirred at 50°C for 4 hours. The mixture was added to _H₂O (30 mL) and extracted with DCM (50 mL x 3). The organic layer was concentrated under reduced pressure. The residue was purified by column chromatography eluting with 10:1 ethyl acetate/methanol to afford 219a (750 mg, 89%) as a yellow solid. MS: [M+H] ^⁺ 391.

Example 219b [4-Fluoro-2-(1-methyl-5-{[6-(oxetan-3-yl)-5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)-6-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^{2 ,7} ]tridec-1(9),2(7)-dien-5-yl}phenyl]methyl acetate 219b

To a 25 mL sealed tube was added (2-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-5-yl}-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methyl acetate 212b (512 mg, 1.0 mmol), 219a (400 mg, 1.0 mmol), CH ₃ COONa (168 mg, 2.0 mmol), K ₃ PO ₄ (546 mg, 2.0 mmol) and PdCl ₂ (dppf) (84 mg, 0.1 mmol) and suspended in CH ₃ CN (25 mL) and H ₂ O (1 mL). The mixture was stirred at 110° C. for 2 hours. After the reaction, the mixture was evaporated and the residue was purified by column chromatography using 10:1 dichloromethane/methanol as eluent to give 219b as a brown solid (450 mg, 64%). MS: (M+H) ⁺ 684.

To a solution of 219b (400 mg, 0.58 mmol) in propan-2-ol (12 mL), tetrahydrofuran (12 mL), and water (2 mL) was added LiOH (1.4 g, 58 mmol). The mixture was stirred at 30°C for 2 h. After the reaction, the mixture was evaporated and the residue was purified by preparative HPLC to give 219 as a yellow solid (213 mg, 57%). MS: (M+H) ⁺ 642. ¹ H NMR(500MHz,DMSO)δ1.80(s,4H),2.55-2.58(m,3H),2.77-2.79(d,4H),2.85-2.89(m,1H),2.96- 2.98(m,1H),3.36(s,2H),3.60-3.61(d,4H),3.87-3.89(m,1H),4.05-4.07(m,1H),4.37-4.38(d, 2H),4.50-4.52(t,J＝5Hz,2H),4.59-4.62(t,J＝6.5Hz,2H),4.87-4.90(t,J＝4Hz,2H),7.08-7.10 (d,1H),7.19-7.21(m,1H),7.29-7.34(m,2H),7.45-7.46(d,1H),8.46(s,1H),8.79-8.80(d,1H).

Example 220 5-[5-fluoro-2-(hydroxymethyl)-3-[1-methyl-6-oxo-5-({5-[4-(propan-2-yl)piperazin-1-yl]pyridin-2-yl}amino)-1,6-dihydropyridin-3-yl]phenyl]-8-thia-5-azatricyclo[7.4.0.02,7]trideca-1(9),2(7)-dien-6-one 220

Example 220a 5-Bromo-3-(5-(4-isopropylpiperazin-1-yl)pyridin-2-ylamino)-1-methylpyridin-2(1H)-one 220a

A mixture of 5-bromo-1-methyl-3-(5-(piperazin-1-yl)pyridin-2-ylamino)pyridin- ₂ (1H)-one 188d (1.2 g, 3.3 mmol) and acetone (1.0 g, 16.5 mmol) in methanol/acetic acid (30 mL/3 mL) was stirred at room temperature for 5 minutes, followed by the addition of NaBH(OAc) (3.5 g, 16.5 mmol). The mixture was stirred at 50°C for 2 hours. The mixture was cooled to room temperature, and _H₂O (50 mL) was added. The mixture was extracted three times with DCM (50 mL). The organic layer was concentrated under reduced pressure, and the residue was purified by column chromatography eluting with 25:1 DCM/methanol to afford 220a (2.97 g, 81%). MS: [M+H] ^⁺ 406. ^1H NMR (500MHz, DMSO) δ8.58(d,J＝3.0,1H),8.53(s,1H),7.92(d,J＝3.0,1H),7.44(d,J＝2.5,1H),7.37(d,J＝2. 5,1H),7.25(d,J＝8,1H),3.51(s,3H),3.06(t,J＝4.5,4H),2.69(s,1H),2.57-2.50(m,4H),1.00(d,J＝7,6H).

Example 220b {4-fluoro-2-[1-methyl-6-oxo-5-({5-[4-(propan-2-yl)piperazin-1-yl]pyridin-2-yl}amino)-1,6-dihydropyridin-3-yl]-6-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]tridec-1(9),2(7)-dien-5-yl}phenyl}methyl acetate 220b

According to the method described for 148b, starting from 400 mg of (4-fluoro-2-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-5-yl}-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methyl acetate 212b and 340 mg of 5-bromo-3-(5-(4-isopropylpiperazin-1-yl)pyridin-2-ylamino)-1-methylpyridin-2(1H)-one 220a, compound 220b was obtained as a yellow solid (446 mg, 82%). MS: [M+H] ⁺ 699

Following the method described for 148, starting from 200 mg of 220b, compound 220 (70 mg, 37%) was obtained as a white solid. MS: [M+H] ⁺ 657. ¹ H NMR (500 MHz, DMSO) δ 8.56 (d, J = 2.5, 1H), 8.37 (s, 1H), 7.85 (d, J = 2.5, ^1H ), 7.34 (m, 3H), 7.23 (m, 2H), 4.68 (s, 1H), 4.32 (s, 2H), 4.06 (s, 1H), 3.85 (s, ^1H ), 3.58 (s, 3H), 3.02 (s, 4H), 2.78 (m, 4H), 2.55 (m, 7H), 1.80 (d, J = 3.5, 4H), 1.00 (d, J = 7, 6H).

Example 221 5-[5-Fluoro-2-(hydroxymethyl)-3-{4-methyl-6-[(2-methyl-1,2,3,4-tetrahydroisoquinolin-6-yl)amino]-5-oxo-4,5-dihydropyrazin-2-yl}phenyl]-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-6-one 221

Example 221a 5-Bromo-1-methyl-3-(1,2,3,4-tetrahydroisoquinolin-6-ylamino)pyrazin-2(1H)-one 221a

To a solution of tert-butyl 6-(6-bromo-4-methyl-3-oxo-3,4-dihydropyrazin-2-ylamino)-3,4-dihydroisoquinoline-2(1H)-carboxylate 120a (3.6 g, 8.3 mmol) in dioxane (10 mL) was added dropwise a saturated solution of hydrogen chloride/dioxane (20 mL). The reaction mixture was stirred for 20 minutes and then concentrated under reduced pressure to afford 221a, which was used in the next step without further purification. LC/MS: m/z 336 (M+H) ⁺

Example 221b 5-Bromo-1-methyl-3-(2-methyl-1,2,3,4-tetrahydroisoquinolin-6-ylamino)pyrazin-2(1H)-one 221b

A mixture of 221a (2.75 g, 7.5 mmol), formaldehyde (37% _H₂O solution, 30 mL, 375 mmol), NaBH(OAc) _₃ (4.75 g, 22.5 mmol), and AcOH (25 mL, 150 mmol) in methanol (125 mL) was stirred at room temperature for 4 hours. The mixture was then made basic with saturated NaOH solution and extracted with ethyl acetate. The organic layer was dried _{over Na₂SO₄} and evaporated under reduced pressure. The residue was purified by column chromatography eluting with 4:1 ethyl acetate/methanol to give 221b (0.9 g, 34.4%). LC/MS: m/z 350 (M+H) ⁺

Example 221c (4-Fluoro-2-{4-methyl-6-[(2-methyl-1,2,3,4-tetrahydroisoquinolin-6-yl)amino]-5-oxo-4,5-dihydropyrazin-2-yl}-6-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]tridec-1(9),2(7)-dien-5-yl}phenyl)methyl acetate 221c

To a 15 mL microwave-safe vial equipped with a magnetic stirrer was added 5-bromo-1-methyl-3-(2-methyl-1,2,3,4-tetrahydroisoquinolin-6-ylamino)pyrazin-2(1H)-one (0.42 g, 1.2 mmol), 212b (0.6 g, 1.2 mmol), potassium phosphate (0.64 g, 2.4 mmol), sodium acetate (0.2 g, 2.4 mmol), acetonitrile (10 mL), water (1 mL), and 1,1'-bis(diphenylphosphino)ferrocenepalladium(II) dichloride in dichloromethane (0.1 g, 0.12 mmol). The reaction mixture was heated at 110°C under an argon atmosphere for 2 hours. Afterwards, the mixture was cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography eluting with 40:1 DMC/methanol to afford 221c (0.667 g, 86.6%). LC/MS:m/z 642(M+H) ⁺

To a 25 mL round-bottom flask equipped with a magnetic stirrer was added 221c (0.647 g, 1.0 mmol), LiOH (2.1 g, 50 mmol), THF (10 mL), isopropanol (10 mL), and water (5 mL). The reaction mixture was stirred at room temperature for 30 minutes and then filtered. The filtrate was concentrated under reduced pressure. The residue was purified by preparative HPLC to afford 221. LC/MS: m/z 600 (M+H) ⁺ . ^1H NMR (500MHz, DMSO) δ9.13(s,1H),7.80(s,1H),7.62(d,J＝8.5,1H),7.51(s,1H),7.39(d,J＝10,1H),7.30(d,J＝6.5,,1H),6.97(d,J＝8.5, 1H),4.86(m,1H),44.51(m,3H),4.41(m,1H),4.05(m,1H),3.87(m,1H),3.55(m,6H),2.77(m,3H),2.55(m,3H),2.31(m,3H),1.80(m,4H).

Example 222 5-[5-fluoro-2-(hydroxymethyl)-3-(4-methyl-6-{[2-(oxetan-3-yl)-1,2,3,4-tetrahydroisoquinolin-6-yl]amino}-5-oxo-4,5-dihydropyrazin-2-yl)phenyl]-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-6-one 222

Example 222a 5-Bromo-1-methyl-3-(2-(oxetan-3-yl)-1,2,3,4-tetrahydroisoquinolin-6-ylamino)pyrazin-2(1H)-one 222a

A mixture of 5-bromo-1-methyl-3-(1,2,3,4-tetrahydroisoquinolin-6-ylamino)pyrazin-2(1H)-one 221a (2.75 g, 7.5 mmol), oxetane-3-one (2.5 g, 37.5 mmol), NaBH ₃ CN (4.75 g, 22.5 mmol), and zinc chloride (5 g, 37.5 mmol) in methanol (125 ml) was stirred at room temperature for 4 hours. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography eluting with 20:1 DCM/methanol to afford 222a (0.76 g, 25.9%). LC/MS: m/z 392 (M+H) ⁺

Example 222b [4-fluoro-2-(4-methyl-6-{[2-(oxetan-3-yl)-1,2,3,4-tetrahydroisoquinolin-6-yl]amino}-5-oxo-4,5-dihydropyrazin-2-yl)-6-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]tridec-1(9),2(7)-dien-5-yl}phenyl]methyl acetate 222b

To a 250 mL round-bottom flask equipped with a magnetic stirrer was added 222a (0.31 g, 0.8 mmol), (4-fluoro-2-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-5-yl}-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methyl acetate 212b (0.4 g, 0.8 mmol), potassium phosphate (0.43 g, 1.6 mmol), sodium acetate (0.13 g, 1.6 mmol), acetonitrile (30 mL), water (2 mL) and 1,1′-bis(diphenylphosphino)ferrocenepalladium(II) chloride dichloromethane (0.07 g, 0.08 mmol). The reaction mixture was refluxed under an argon atmosphere overnight. Afterwards, the mixture was cooled to room temperature and then filtered. The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography using 40:1 dichloromethane/methanol as eluent to afford 222b (0.491 g, 89.3%). LC/MS: m/z 684 (M+H) ⁺

To a 25 mL round-bottom flask equipped with a magnetic stirrer was added 222b (0.471 g, 0.69 mmol), LiOH (1.45 g, 34.5 mmol), THF (10 mL), isopropanol (10 mL), and water (5 mL). The reaction mixture was stirred at room temperature for 30 minutes and then filtered. The filtrate was concentrated under reduced pressure. The residue was purified by preparative HPLC to afford 222 as a white solid (20 mg, 11%). LC/MS: m/z 642 (M+H) ⁺ . ¹ H NMR (500MHz, DMSO) δ9.14 (s, 1H), 7.82 (s, 1H), 7.63 (d, J = 8.5, 1H), 7.51 (s ,1H),7.39(d,J＝10,1H),7.30(d,J＝6.5,,1H),6.97(d,J＝8.5,1H),4.86(m, 1H),4.84(m,2H),4.51(m,3H),4.41(m,1H),4.05(m,1H),3.87(m,1H),3.5 5(m,5H),2.88(m,1H),2.77(m,1H),2.53(m,4H),1.80(m,4H),1.23(m,2H).

Example 223 5-[5-Fluoro-2-(hydroxymethyl)-3-{1-methyl-5-[(6-methyl-5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl)amino]-6-oxo-1,6-dihydropyridin-3-yl}phenyl]-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-6-one 223

Example 223a (4-Fluoro-2-{1-methyl-5-[(6-methyl-5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl)amino]-6-oxo-1,6-dihydropyridin-3-yl}-6-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]tridec-1(9),2(7)-dien-5-yl}phenyl)methyl acetate 223a

According to the method described for 136e, starting from 5-bromo-1-methyl-3-(6-methyl-5,6,7,8-tetrahydro-1,6-naphthyridin-2-ylamino)pyridin-2(1H)-one 205b and (4-fluoro-2-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-5-yl}-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methyl acetate 212b, compound 223a was obtained in 50% yield.

Compound 223 was obtained in 33% yield from 223a following the procedure described for compound 136f. LCMS: (M+H) ⁺ 600. ¹ H NMR (500MHz, MEOD) δ8.87(d,J＝2,1H),7.40(d,J＝2.5,1H),7.35(d,J＝8,1H),7.23(s,1H),7.21(s,1H),6.88(d,J＝8,1H),4.56(m,2H) ,4.16(m,1H),4.01(m,1H),3.72(s,3H),3.57(s,2H),3.05(m,1H),2.95(m,3H),2.84(m,4H),2.59(m,2H),2.49(s,3H),1.90(m,4H).

Example 224 10-[5-fluoro-2-(hydroxymethyl)-3-{1-methyl-5-[(5-{[methyl(propan-2-yl)amino]methyl}pyridin-2-yl)amino]-6-oxo-1,6-dihydropyridin-3-yl}phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 224

Example 224a 10-[5-fluoro-2-(acetoxymethyl)-3-{1-methyl-5-[(5-{[methyl(propan-2-yl)amino]methyl}pyridin-2-yl)amino]-6-oxo-1,6-dihydropyridin-3-yl}phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 224a

Following Example 186f, 186e (365 mg, 0.64 mmol) and 189 (230 mg, 0.51 mmol) were reacted to give 224a (80 mg, 20% yield).

According to Example 186, 224a (80 mg, 0.12 mmol), 1N LiOH (0.61 mL), THF (2 mL), and isopropanol (2 mL) were reacted and then pulverized with EtOAc to give 224 (57 mg, 76% yield). MS (ESI+) m/z 613.6 (M+H).

Example 225 2-(2-(Hydroxymethyl)-3-(1-methyl-5-(5-methyl-1H-pyrazol-3-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrido[3,4-b]indolizine-1(2H)-one 225

Using the same general procedure as described for 118, 112a (200 mg, 0.706 mmol) and 118f (330 mg, 0.710 mmol) were reacted to afford 225 as a yellow-green solid in 17% yield (60 mg): mp 199-200 °C; ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 11.69 (s, 1H), 7.99 (s, 1H), 7.93 (s, 1H), 7.41 (t, 1H, J=8.0 Hz), 7.25 (d, 3H, J=8.5 Hz), 6.00 (s, 1H), 5.86 (s, 1H), 4.69 (br s,1H),4.31(m,2H),3.97(m,1H),3.91(m,1H),3.79(m,2H),3.56(s,3H),3.01(m,1 H),2.90(m,1H),2.70(m,2H),2.15(s,3H),1.90(m,2H),1.74(m,2H); MS(ESI+)m/z 499.2(M+H).

Example 226 2-(2-(Hydroxymethyl)-3-(1-methyl-6-oxo-5-(5,6,7,8-tetrahydro-2,6-naphthyridin-3-ylamino)-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrido[3,4-b]indolizine-1(2H)-one 226

Example 226a tert-Butyl 7-(5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)-3,4-dihydro-2,6-naphthyridine-2(1H)-carboxylate

To a 100-mL three-necked round-bottom flask equipped with a magnetic stirrer, a nitrogen inlet, and a reflux condenser was added 3,5-dibromo-1-methylpyridin-2(1H)-one (536 mg, 2.01 mmol), tert-butyl 7-amino-3,4-dihydro-2,6-naphthyridine-2(1H)-carboxylate (500 mg, 2.01 mmol), cesium carbonate (1.44 g, 4.40 mmol), and 1,4-dioxane (30 mL). After nitrogen was bubbled through the resulting mixture for 20 minutes, Xantphos (98.4 mg, 0.170 mmol) and tris(dibenzylideneacetone)dipalladium(0) (91.6 mg, 0.100 mmol) were added, and the reaction mixture was heated at 100° C. for 6 h. Afterwards, the reaction was cooled to room temperature, filtered, and then concentrated under reduced pressure. The resulting residue was purified by flash chromatography to afford 226a as a yellow solid in 96% yield (840 mg): mp 126-128° C.; ¹ H NMR (500 MHz, CDCl ₃ ) 8.60 (d, 1H, J=2.5 Hz), 8.10 (s, 1H), 7.79 (s, 1H), 6.95 (d, 1H, J=2.5 Hz), 6.55 (s, 1H), 4.53 (s, 2H), 3.64 (t, 2H, J=5.0 Hz), 3.59 (s, 3H), 2.77 (t, 2H, J=5.0 Hz), 1.49 (s, 9H); MS (ESI+) m/z 435.0 (M+H).

Example 226b 5-Bromo-1-methyl-3-(5,6,7,8-tetrahydro-2,6-naphthyridin-3-ylamino)pyridin-2(1H)-one 226b

A 50-mL single-necked round-bottom flask equipped with a magnetic stirrer was purged with nitrogen and 226a (840 mg, 1.94 mmol) and dichloromethane (12 mL) were added. Trifluoroacetic acid (6 mL) was added. The reaction was stirred at room temperature for 2 h. Thereafter, the reaction mixture was evaporated under reduced pressure. The residue 226b was used directly in the next step.

To a 100-mL three-necked round-bottom flask equipped with a magnetic stirrer and a reflux condenser was added 1/2 the amount of crude product 226b (assumed 0.898 mmol), 118f (416 mg, 0.898 mmol), sodium carbonate (381 mg, 3.59 mmol), water (4 mL), and 1,4-dioxane (20 mL). After nitrogen was bubbled through the resulting suspension for 20 min, tetrakis(triphenylphosphine)palladium(0) (104 mg, 0.090 mmol) was added, and the reaction mixture was heated at 100°C for 4 h. Thereafter, the reaction mixture was cooled to room temperature, filtered, and the filter cake was washed with a 1:10 mixture of methanol and dichloromethane (30 mL). The filtrate was concentrated under reduced pressure to give a brown residue. To another 50-mL single-necked round-bottom flask equipped with a magnetic stirrer and a reflux condenser was added the residue obtained above, THF (10 mL), ethanol (10 mL), water (10 mL) and lithium hydroxide (86.0 mg, 3.59 mmol). The mixture was stirred at 50°C for 2 h. Thereafter, the reaction mixture was concentrated under reduced pressure. The resulting residue was purified by flash chromatography to give 226 as an off-white solid in a yield of 7.7% (38 mg): mp 197–199°C; ¹ H NMR (500 MHz, DMSO-d ₆ )δ8.53(d,1H,J＝2.0Hz),8.23(s,1H),7.90(s,1H),7.43(t,1H,J＝7.5Hz),7.35(d,1H,J＝2.0H z),7.28(s,1H),7.27(s,1H),6.93(s,1H),6.00(s,1H),4.72(t,1H,J＝5.0Hz),4.32(d,2H,J＝5 .0Hz),3.96(m,1H),3.90(m,1H),3.82(m,2H),3.75(s,2H),3.58(s,3H),3.00(m,1H),2.94(m ,1H),2.90(t,2H,J=5.0Hz),2.70(m,2H),2.56(m,2H),1.92(m,2H),1.76(m,2H); MS(ESI+)m/z 551.2(M+H).

Example 227 2-(3-(5-(5-(azetidin-3-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrido[3,4-b]indolizin-1(2H)-one 227

To a 100-mL single-necked round-bottom flask equipped with a magnetic stirrer and a nitrogen inlet was added 155n (312 mg, 0.931 mmol), 118f (472 mg, 1.12 mmol), sodium carbonate (296 mg, 2.79 mmol), DMF (2 mL), water (2 mL) and 1,4-dioxane (10 mL). After bubbling nitrogen through the resulting suspension for 30 min, tetrakis(triphenylphosphine)palladium(0) (215 mg, 0.186 mmol) was added. A reflux condenser was connected to the flask, and the reaction mixture was heated at 100 ° C for 14 h. Thereafter, the mixture was diluted with 90:10 dichloromethane/methanol (100 mL) and water (75 mL), and the layers were separated. The aqueous layer was extracted with 90:10 dichloromethane/methanol (2×50 mL), and the combined organic layers were washed with brine (100 mL) and then dried over sodium sulfate. The desiccant was removed by filtration. The filtrate was concentrated under reduced pressure, and the resulting residue was dissolved in a mixture of THF (5 mL), water (5 mL), and methanol (5 mL). Lithium hydroxide monohydrate (195 mg, 4.66 mmol) was added, and the mixture was stirred at room temperature for 3 h. Thereafter, the mixture was diluted with 90:10 dichloromethane/methanol (100 mL) and water (50 mL), and the layers were separated. The aqueous layer was extracted with 90:10 dichloromethane/methanol (2X75 mL), and the combined organic layers were washed with brine (100 mL) and then dried over sodium sulfate. The drying agent was removed by filtration. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, 90:10 dichloromethane/methanol) to give a 7% yield (36 mg) of a non-crystalline off-white solid 227: mp 227–229 °C; ¹ H NMR (500 MHz, DMSO-d ₆ )8.66(d,J＝1.5Hz,1H),8.53(s,1H),8.11(s,1H),7.68(d,J＝7.0Hz,1H),7.44(t,J ＝8.0Hz,1H),7.39(s,1H),7.29(m,3H),6.01(s,1H),4.72(t,J＝4.5Hz,1H),4.33(s, 2H),3.99–3.88(m,3H),3.84–3.75(m,2H),3.59(m,3H),3.51(m,5H),3.02–2.91(m ,2H),2.72(t,J＝5.5Hz,2H),1.92(t,J＝5.0Hz,2H),1.77–1.73(m,2H); MS(ESI+)m/z 551.2(M+H).

Example 228 2-(2-(Hydroxymethyl)-3-(4-methyl-5-oxo-6-(1,2,3,4-tetrahydroisoquinolin-7-ylamino)-4,5-dihydropyrazin-2-yl)phenyl)-3,4,6,7,8,9-hexahydropyrido[3,4-b]indolizin-1(2H)-one 228

Using the same general procedure as described for the preparation of 226, 153a (400 mg, 1.19 mmol) and 118f (718 mg, 1.55 mmol) were reacted and then hydrolyzed to give 228 as a white solid in 12% yield (88 mg): mp 218-220 °C; ^1H NMR (500 MHz, DMSO- _d6 ) δ 9.03 (s, 1H), 7.73 (s, 1H), 7.63 (dd, 1H, J = 7.5, 2.0 Hz), 7.52 (dd, 1H, J = 7.5, 2.0 Hz), 7.43 (t, 2H, J = 8.0 Hz), 7.25 (dd, 1H, J = 7.5, 1.0 Hz), 6.90 (d, 1H, J = 8.0 Hz), 6.00 (s, 1H), 4.71 (dd, 1H ,J＝7.0,3.5Hz),4.46(m,2H),3.94(m,2H),3.82(m,2H),3.77(s,2H),3.54(s,3H),3.00(m,1 H),2.93(m,1H),2.90(t,2H,J＝5.0Hz),2.70(t,2H,J＝6.0Hz),2.62(t,2H,J＝6.0Hz),2.45(m, ¹ H), 1.92 (m, 2H), 1.76 (m, 2H); MS (ESI+) m/z 551.2 (M+H).

Example 229 2-(2-(Hydroxymethyl)-3-(1-methyl-5-(5-(1-methylpyrrolidin-2-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrido[3,4-b]indolizine-1(2H)-one 229

Example 229a 5-Bromo-1-methyl-3-(5-(1-methylpyrrolidin-2-yl)pyridin-2-ylamino)pyridin-2(1H)-one 229a

Using the same general procedure as described for the preparation of 226b, 3,5-dibromo-1-methylpyridin-2(1H)-one (1.51 g, 5.65 mmol) and 5-(1-methylpyrrolidin-2-yl)-2-aminopyridine (1.00 g, 5.65 mmol) were reacted to afford 229a as a yellow solid in 81% yield (1.65 g): mp 143–145 °C; ¹ H NMR (300 MHz, DMSO-d ₆ )δ8.73(s,1H),8.68(d,1H,J＝2.4Hz),8.15(d,1H,J＝2.1Hz),7.55(dd,1H,J＝8.9,2.1Hz),7.52(s,1H),7.30(d,1H,J＝8.9Hz),3.52( s,3H),3.13(t,1H,J＝7.5Hz),2.99(t,1H,J＝7.5Hz),2.19(m,1H),2.10(m,1H),2.05(s,3H),1.79(m,2H),1.71(m,1H); MS(ESI+)m/z 363.3(M+H).

Using the same general procedure as described for the preparation of 226, 229a (500 mg, 1.38 mmol) and 118f (705 mg, 1.52 mmol) were reacted and then hydrolyzed to give 229 as a white solid in 27% yield (215 mg): mp 146-148 °C; ¹ H NMR (500 MHz, DMSO-d ₆ )δ8.67(dd,1H,J＝2.0Hz),8.52(s,1H),8.05(s,1H),7.53(dd,1H,J＝8.5,2.5Hz),7.44(t,1H,J＝8.0Hz),7.39(d ,1H,J＝2.5Hz),7.29-7.24(m,3H),6.00(s,1H),4.72(t,1H,J＝5.0Hz),4.32(d,2H,J＝5.0Hz),3.99(m,1H),3.90 (m,1H),3.81(m,2H),3.58(s,3H),3.11(t,1H,J＝8.0Hz),3.00(m,1H),2.93(m,2H),2.71(t,2H,J＝6.0Hz),2.17 (q,1H,J＝9.0Hz),2.06(m,1H),2.03(s,3H),1.92(t,2H,J＝6.0Hz),1.83-1.72(m,4H),1.59(m,1H);MS(ESI+)m/z 579.2(M+H).

Example 230 10-[3-(5-{[5-(azetidin-1-ylmethyl)-1-methyl-1H-pyrazol-3-yl]amino}-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 230

Example 230a 2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-4-fluoro-6-(9-oxo-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-10-yl)benzyl acetate 230a

According to Example 211d, 2-bromo-4-fluoro-6-(9-oxo-4,4-dimethyl-1,10-diazatricyclo[ ^6.4.0.02,6 ]dodeca-2(6),7-dien-10-yl)benzyl acetate 189a (450 mg, 1.0 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bis(1,3,2-dioxaborolane) (635 mg, 2.5 mmol), potassium acetate (393 mg, 4.0 mmol), bis(diphenylphosphino)ferrocene] _{dichloropalladium} (II) complex with _CH2Cl2 ( _PdCl2dppf : _CH2Cl2 (1:1), 66 mg, 0.08 mmol) and 1,4-dioxane (20 mL) were heated at 100 _° C for 5 h. The reaction mixture was cooled to room temperature and then filtered through a pad of Celite 521. The filter cake was washed with ethyl acetate (2×25 mL), and the combined filtrates were concentrated to dryness under reduced pressure to afford compound 230a as a black oil (quantitative yield), which was used directly in the next step. MS (ESI+) m/z 497.3 (M+H).

Example 230b 10-[3-(5-{[5-(azetidin-1-ylmethyl)-1-methyl-1H-pyrazol-3-yl]amino}-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(acetoxymethyl)phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 230b

Example 230b was synthesized using the same procedure as 121b, except that compound 230a (223 mg, 0.45 mmol), 3-(5-(azetidin-1-ylmethyl)-1-methyl-1H-pyrazol-3-ylamino)-5-bromo-1-methylpyridin-2(1H)-one (200c) (160 mg, 0.45 mmol), 1 M sodium carbonate solution (1.5 mL, 1.5 mmol), tetrakis(triphenylphosphine)palladium(0) (26 mg, 0.022 mmol), and 1,2-dimethoxyethane (4.5 mL) were used. The reaction mixture was heated at 130° C. in a microwave reactor for 15 minutes. Workup and flash column chromatography (silica gel, 9:1 dichloromethane/methanol) afforded compound 230b as a brown oil in 43% yield (120 mg): MS (ESI+) m/z 643.1 (M+H).

Compound 230b (120 mg, 0.2 mmol) and lithium hydroxide monohydrate (50 mg, 1.2 mmol) were reacted in the same manner as 121, except using a mixture of THF (1 mL), water (0.5 mL), and isopropanol (1 mL). Workup and flash column chromatography (NH-silica gel, ethyl acetate/hexane) afforded a 30% yield (36 mg) of light green compound 230: MS (ESI+) m/z 600.6 (M+H).

Example 231 2-(5-fluoro-2-(hydroxymethyl)-3-(4-methyl-6-(2-methylisoindolin-5-ylamino)-5-oxo-4,5-dihydropyrazin-2-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 231

Example 231a 5-Nitroisoindoline 231a

To a suspension of isoindoline (2 g, 16.8 mmol) in 98% _H₂SO₄ (10 _mL ) at 0°C was added a mixture of concentrated nitric acid (2 mL) and 98% sulfuric acid (2 mL) dropwise. The mixture was stirred at 0°C for 45 minutes. The resulting yellow solution was then poured into ice water. The white solid precipitate was collected by filtration. The solid was washed with water (20 mL x 2) and then dried in vacuo to afford 231a (700 mg, 51%) as a white solid. MS: [M+H]+165.

Example 231b tert-Butyl 5-nitroisoindoline-2-carboxylate 231b

A mixture of 5-nitroisoindoline (600 mg, 3.66 mmol) and (Boc) ₂ O (1.6 g, 7.31 mmol) in THF (30 mL) was stirred at room temperature for 2 h. The resulting red solution was then concentrated under reduced pressure, and the residue was purified by flash chromatography using 5:1 petroleum ether/ethyl acetate as the eluent to afford 231b (750 mg, 80%) as a yellow solid. MS: [M+H] ⁺ 209.

Example 231c tert-Butyl 5-aminoisoindoline-2-carboxylate 231c

To a 100-mL single-necked round-bottom flask equipped with a magnetic stirrer was added tert-butyl 5-nitroisoindoline-2-carboxylate (650 mg, 2.5 mmol) and _C₂H₅OH (50 mL). The solution was hydrogenated over Pd/C (160 _mg ) at room temperature for 15 h. The mixture was then filtered, and the filtrate was concentrated under reduced pressure to afford 231c as a yellow oil (585 mg, 99%), which was used in the next step without further purification. MS: [M+H] ^⁺ 179.

Example 231d tert-Butyl 5-(6-bromo-4-methyl-3-oxo-3,4-dihydropyrazin-2-ylamino)isoindoline-2-carboxylate 231d

Following the procedure described for 129a, starting from 600 mg of tert-butyl 5-aminoisoindoline-2-carboxylate (231c) and 685 mg of 3,5-dibromo-1-methylpyrazin-2(1H)-one, 231d (732 mg, 68%) was obtained as a yellow solid. MS: [M+H] ⁺ 421.

Example 231e 5-Bromo-3-(isoindolin-5-ylamino)-1-methylpyrazin-2(1H)-one 231e

According to the method described for 129b, starting from 460 mg of 5-(6-bromo-4-methyl-3-oxo-3,4-dihydropyrazin-2-ylamino)isoindoline-2-carboxylic acid tert-butyl ester, 231e was obtained as a yellow solid (352 mg, 99%). MS: [M+H] ⁺ 321

Example 231f 5-Bromo-1-methyl-3-(2-methylisoindolin-5-ylamino)pyrazin-2(1H)-one 231f

Following the method described for 220a, starting from 336 mg of 5-bromo-3-(isoindolin-5-ylamino)-1-methylpyrazin-2(1H)-one, compound 231f (237 mg, 75%) was obtained as a yellow solid. MS: [M+H] ⁺ 337. ¹ H NMR (500 MHz, CDCl 3 ) δ 8.31 (s, 1H), 7.67 (s, 1H), 7.52 (t, J = 7.0, 1H), 7.18 (d, J = 8.0, 1H), 6.74 (s, 1H), 3.94 (s, 2H), 3.89 (s, 2H), 3.52 (s, 3H), 2.60 (s, 3H).

Example 231g 4-Fluoro-2-(4-methyl-6-(2-methylisoindolin-5-ylamino)-5-oxo-4,5-dihydropyrazin-2-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 231g

Following the method described for compound 148h, starting from 400 mg of 4-fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 210d and 278 mg of 5-bromo-1-methyl-3-(2-methylisoindolin-5-ylamino)pyrazin-2(1H)-one 231f, compound 231g (258 mg, 51%) was obtained as a yellow solid. MS: [M+H] ⁺ 611.

According to Example 148, 231 g was converted to a white solid 231 (116 mg, 50%). ¹ H NMR (500 MHz, DMSO) δ 9.23 (s, 1H), 7.90 (s, 1H), 7.73 (d, J = 8, 1H), 7.51 (s, 1H), 7.37 (dd, J = 10.0, 1H), 7.31 (dd, J = 10.0, 1H), 7.13 (d, J = 8.0, 1H), 6.53 (s, 1H), 4.88 (s, 1H),4.50(d,J＝8.0,1H),4.50(d,J＝11.5,1H),4.40(m,1H),4.13(m,3H),3.89(m,1 H),3.78(m,4H),3.52(s,3H),2.59(m,2H),2.48(m,5H),1.79(s,2H),1.70(s,2H).

Example 232 5-[5-Fluoro-2-(hydroxymethyl)-3-{4-methyl-6-[(2-methyl-2,3-dihydro-1H-isoindol-5-yl)amino]-5-oxo-4,5-dihydropyrazin-2-yl}phenyl]-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-6-one 232

Example 232a (4-Fluoro-2-{4-methyl-6-[(2-methyl-2,3-dihydro-1H-isoindol-5-yl)amino]-5-oxo-4,5-dihydropyrazin-2-yl}-6-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-5-yl}phenyl)methyl acetate 232a

According to the method described for 148h, starting from 499 mg of (4-fluoro-2-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-5-yl}-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methyl acetate 212b and 335 mg of 5-bromo-1-methyl-3-(2-methylisoindolin-5-ylamino)pyrazin-2(1H)-one, compound 232a (320 mg, 51%) was obtained as a yellow solid. MS: [M+H] ⁺ 628.

Starting from 300 mg of 232a, the method described for 148 was followed to obtain compound 232 (117 mg, 42%) as a white solid. ¹ H NMR (500MHz, DMSO) δ9.23(s,1H),7.90(s,1H),7.72(s,1H),7.51(d,J＝8.0,1H),7.36(s,1H),7.31(s,1H),7.13(s,1H),4.85(s ,1H),4.50(s,1H),4.40(m,1H),4.05(s,1H),3.87(s,1H),3.78(d,J＝6.5,4H),3.56(s,3H),3.33(s,3H),2.97(s,1H),2.87(s, ^1H ),2.79(s,2H),2.50(m,3H),1.80(s,4H).

Example 233 5-[3-(5-{[5-(1-ethylazetidin-3-yl)pyridin-2-yl]amino}-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl]-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-diene-6-one 233

Example 233a [2-(5-{[5-(1-ethylazetidin-3-yl)pyridin-2-yl]amino}-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-4-fluoro-6-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]tridec-1(9),2(7)-dien-5-yl}phenyl]methyl acetate 233a

According to the method described for 136e, starting from 5-bromo-3-(5-(1-ethylazetidin-3-yl)pyridin-2-ylamino)-1-methylpyridin-2(1H)-one (250a) and (4-fluoro-2-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-5-yl}-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methyl acetate 212b, compound 233a was obtained in 67% yield. LCMS: (M+H) ⁺ 597

Compound 233 was obtained in 58% yield using 233a as starting material according to the method described for 136. LCMS: (M+H) ⁺ 614. ¹ H NMR (500 MHz, DMSO) δ 8.66 (d, J = 2, 1H), 8.56 (s, 1H), 8.11 (d, J = 2, 1H), 7.65 (dd, J = 9, 1H), 7.41 (d, J = 2, 1H), 7.33 (dd, J = 9.5, 1H), 7.28 (d, J = 8, 1H), 7.18 (dd, J = 9, 1H), 4.86 (t, J = 4,1H),4.32(d,J＝4.5,2H),4.05(m,1H),3.86(m,1H),3.59(s,3H),3.49(m,3H),2.97(m, 3H), 2.86 (m, 1H), 2.78 (m, 2H), 2.56 (m, 1H), 2.40 (m, 2H), 1.79 (m, 4H), 0.87 (t, J = 7, 3H).

Example 234 2-(5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-(5-methyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 234

Example 234a {4-fluoro-2-[1-methyl-5-({5-methyl-4H,5H,6H,7H-pyrazolo[1,5-a]pyrazin-2-yl}amino)-6-oxo-1,6-dihydropyridin-3-yl]-6-{1-oxo-1H,2H,3H,4H,-6H,7H,8H,9H-pyrazino[1,2-a]indol-2-yl}phenyl}methyl acetate 234a

To a 25 mL sealed tube were added 4-fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate (210d) (500 mg, 1.0 mmol), 5-bromo-1-methyl-3-(5-methyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)pyridin-2(1H)-one (146a) (350 mg, 1.0 mmol), CH ₃ COONa (170 mg, 2.0 mmol), K ₃ PO ₄ (552 mg, 2.0 mmol), and PdCl ₂ (dppf) (85 mg, 0.1 mmol), and the mixture was suspended in CH ₃ CN (25 mL) and H ₂ O (1 mL). The mixture was heated at 110° C. for 2 h. It was then evaporated and the residue was purified on a silica gel column using 20:1 dichloromethane/methanol as eluent to afford 234a (400 mg, 63%) as a brown solid. MS: (M+H) ⁺ 614.

To a solution of 234a (400 mg, 0.65 mmol) in propan-2-ol (10 mL), tetrahydrofuran (10 mL), and water (2 mL) was added LiOH (1.57 g, 65 mmol). The mixture was stirred at 30°C for 2 h. It was then evaporated and the residue was purified by preparative HPLC to afford 234 as a yellow solid (119 mg, 32%). MS: (M+H) ⁺ 572. ¹ H NMR (500MHz, MEOD) δ1.78-1.80(t,2H),1.89-1.90(d,2H),2.49(s,3H),2.54-2.56(t,J＝6Hz,2H),2.64-2.66(m,2H),2.94-2.96(t,2H),3.64(s,2 H),3.67(s,3H),4.04-4.06(m,3H),4.20(s,3H),4.46-4.51(m,2H),5.89 (s,1H),6.72(s,1H),7.20-7.22(d,2H),7.26-7.27(d,1H),7.91(s,1H).

Example 235 5-(5-fluoro-2-(hydroxymethyl)-3-(6,6-dimethyl-3,4,6,7-tetrahydro-5H-cyclopenta[4,5]thieno[2,3-c]pyridin-1(2H)-yl)phenyl)-1-methyl-3-(2-methylisoindolin-5-ylamino)pyrazin-2(1H)-one 235

To a 100-mL three-necked round-bottom flask equipped with a reflux condenser, a magnetic stirrer, and a nitrogen inlet was added 231e (150 mg, 0.447 mmol), 131a (340 mg, 0.581 mmol), sodium carbonate (142 mg, 1.34 mmol), water (2 mL), and 1,4-dioxane (8 mL). After bubbling nitrogen through the resulting suspension for 30 min, tetrakis(triphenylphosphine)palladium(0) (52 mg, 0.045 mmol) was added, and the reaction mixture was heated under reflux for 2 h. Thereafter, the mixture was cooled to room temperature and diluted with dichloromethane (100 mL) and water (30 mL). The organic layer was separated, and the aqueous layer was extracted with dichloromethane (3×30 mL). The combined organic layers were dried over sodium sulfate and then concentrated under reduced pressure to give a brown residue. A 100-mL single-necked round-bottom flask equipped with a magnetic stirrer was purged with nitrogen and the crude residue, THF (10 mL) and 1 M tetrabutylammonium fluoride/THF solution (4.50 mmol, 4.5 mL) were added. The resulting mixture was stirred at room temperature for 2 h. Thereafter, the mixture was diluted with dichloromethane (100 mL) and washed with water (30 mL). The organic layer was separated, and the aqueous layer was extracted with dichloromethane (3×30 mL). The combined organic layers were dried over sodium sulfate and then concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, 0%-10% methanol/dichloromethane) to give 44% yield (120 mg) of an off-white solid 235: mp 218–220°C (dec); ¹ H NMR (500 MHz, DMSO-d ₆ )δ9.21(s,1H),7.89(d,1H,J＝1.0Hz),7.70(dd,1H,J＝8.1,2.0Hz),7.50(s,1H),7.34(dd ,1H,J＝9.9,3.0Hz),7.30(dd,1H,J＝9.1,2.5Hz),7.13(d,1H,J＝8.0Hz),4.82(m,1H),4.5 0(m,1H),4.04(m,1H),4.04(m,1H),3.84(m,1H),3.75(m,4H),3.54(s,3H),3.02(m,1H), 2.89(m,1H),2.75(s,2H),2.53(d,2H,J=6.5Hz),2.46(s,3H),1.23(s,6H); MS(ESI+)m/z 600.2(M+H).

Example 236 5-(5-fluoro-2-(hydroxymethyl)-3-(6,6-dimethyl-3,4,6,7-tetrahydro-5H-cyclopenta[4,5]thieno[2,3-c]pyridin-1(2H)-yl)-3-(5-(3-methylazetidin-1-yl)pyridin-2-ylamino)-1-methylpyridin-2(1H)-one 236

Example 236a 5-(3-methylazetidin-1-yl)-2-nitropyridine 236a

3-methylazetidine benzenesulfonate (4.02g, 17.5mmol), 2-nitro-5-bromopyridine (3.56g, 17.5mmol), cesium carbonate (28.5g, 87.7mmol) and 1,4-dioxane (50mL) are added into a 500-mL single-necked round-bottom flask equipped with a magnetic stirrer and a nitrogen inlet. After nitrogen is bubbled through the resulting suspension for 30min, Xantphos (862mg, 1.50mmol) and tris (dibenzylideneacetone) dipalladium (0) (800mg, 0.900mmol) are added. Reflux condensing tube is connected to the flask, and the reaction mixture is heated under reflux for 3h. After this, the mixture is cooled to room temperature and diluted with 90:10 dichloromethane/methanol (100mL) and water (75mL), and each layer is then separated. The aqueous layer was extracted with 90:10 dichloromethane/methanol (2 x 50 mL) and the combined organic extracts were washed with brine and then dried over sodium sulfate. The drying agent was removed by filtration. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, 0%-80% ethyl acetate/hexanes) to afford 236a as a non-crystalline yellow solid in 69% yield (2.35 g): mp 141-143° C.; ¹ H NMR (500 MHz, CDCl ₃ ) δ 8.12 (d, 1 H, J=9.0 Hz), 7.60 (d, 1 H, J=2.5 Hz), 6.66 (dd, 1 H, J=9.0, 2.5 Hz), 4.23 (t, 2 H, J=8.5 Hz), 3.68 (m, 2 H), 2.99 (m, 1 H), 1.36 (d, 3 H, J=7.0 Hz; MS (ESI+) m/z 194.0 (M+H)

Example 236b 5-(3-methylazetidin-1-yl)pyridin-2-amine 236b

A 250-mL Parr reaction bottle was purged with nitrogen and a solution of 10% palladium on carbon (50% humidity, 800 mg dry weight) and 236a (2.58 g, 13.4 mmol) in a mixture of ethanol (25 mL) and ethyl acetate (25 mL) was added. The bottle was connected to a Parr hydrogenation reactor, evacuated, filled with hydrogen to a pressure of 50 psi and shaken for 3 h. After this, the hydrogen was removed and nitrogen was charged into the bottle. Celite 521 (3.50 g) was added and the mixture was then filtered through a Celite 521 pad. The filter cake was washed with ethanol (2×50 mL), and the combined filtrates were concentrated to dryness under reduced pressure to give 236b as a purple oil in quantitative yield (2.21 g): ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 7.19 (d, 1H, J=3.0 Hz), 6.66 (dd, 1H, J=9.0, 3.0 Hz), 6.36 (d, 1H, J=8.5 Hz), 5.13 (br s, 2H), 3.81 (t, 2H, J=7.5 Hz), 3.21 (t, 2H, J=6.5 Hz), 2.70 (m, 1H), 1.18 (d, 3H, J=7.0 Hz); MS (ESI+) m/z 164.1 (M+H).

Example 236c 5-Bromo-1-methyl-3-(5-(3-methylazetidin-1-yl)pyridin-2-ylamino)pyridin-2(1H)-one 236c

Using the same method as described for the preparation of 121a, 236b (1.04 g, 6.40 mmol) and 3,5-dibromo-1-methylpyridin-2(1H)-one (1.90 g, 7.12 mmol) were reacted to give 236c as a brown solid in 44% yield (980 mg): mp 135-136 °C; ¹ H NMR (500 MHz, DMSO-d ₆ )δ8.49(d,1H,J＝5.5Hz),8.42(s,1H),7.50(d,1H,J＝4.5Hz),7.41(d,1H,J＝7.0Hz),7.20(d,1H,J＝14.5Hz),6.85(dd,1H ,J＝14.5,5.0Hz),3.91(m,2H),3.49(s,3H),3.34(m,2H),2.75(m,1H),1.21(d,3H,J＝11.5Hz; MS(ESI+)m/z350.2(M+H).

236c (140 mg, 0.400 mmol), 182c (304 mg, 0.520 mmol), sodium carbonate (130 mg, 1.23 mmol), water (2 mL) and 1,4-dioxane (10 mL) were added to a 100-mL single-necked round-bottom flask equipped with a magnetic stirrer and a nitrogen inlet. After nitrogen was bubbled through the resulting suspension for 30 min, tetrakis(triphenylphosphine)palladium(0) (50 mg, 0.043 mmol) was added. A reflux condensing tube was connected to the flask, and the reaction mixture was heated under reflux for 2 h. After this, the mixture was diluted with 90:10 dichloromethane/methanol (100 mL) and water (75 mL), and the layers were then separated. The aqueous layer was extracted with 90:10 dichloromethane/methanol (2 × 50 mL), the combined organic layer was washed with brine (100 mL), and then dried over sodium sulfate. The desiccant was removed by filtration. The filtrate was concentrated under reduced pressure, and the resulting residue was dissolved in THF. 1M tetrabutylammonium fluoride/THF solution (1.20 mL, 1.20 mmol) was added, and the mixture was stirred at room temperature for 14 h. Thereafter, the mixture was diluted with 90:10 dichloromethane/methanol (75 mL) and water (50 mL), and the layers were separated. The aqueous layer was extracted with 90:10 dichloromethane/methanol (2X 50 mL), and the combined organic extracts were washed with brine (100 mL) and then dried over sodium sulfate. The desiccant was removed by filtration. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, 0%-10% dichloromethane/methanol) to obtain a 14% yield (35 mg) of a non-crystalline brown solid 236: mp 170–171 ° C; ¹ H NMR (500 MHz, DMSO-d ₆ )δ8.45(d,1H,J＝2.5Hz),8.22(s,1H),7.31(m,2H),7.15(m,2H),6.83(dd,1H,J＝9.0,3.0Hz),4.83(t,1H,J＝4. 5Hz),4.32(m,2H),4.05(m,1H),3.88(m,3H),3.02(m,1H),2.89(m,1H),2.75(s,3H),2.54(m,4H); MS(ESI+)m/z 614.3(M+H).

Example 237 6-(3-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-2,3-(5,5-dimethyl-5,6-dihydro-4H-cyclopentyl)-5,6-dihydrothieno[2,3-c]pyridin-7(4H)-one 237

To a 100-mL three-necked round-bottom flask equipped with a magnetic stirrer and a reflux condenser were added 210c (160 mg, 0.382 mmol), 182c (300 mg, 0.512 mmol), sodium carbonate (162 mg, 1.53 mmol), water (6 mL), and 1,4-dioxane (30 mL). After nitrogen was bubbled through the resulting suspension for 20 min, tetrakis(triphenylphosphine)palladium(0) (44.0 mg, 0.038 mmol) was added, and the reaction mixture was heated at 100°C for 4 h. Thereafter, the reaction mixture was cooled to room temperature, filtered, and the filter cake was washed with a 1:10 mixture of methanol and dichloromethane (30 mL). The filtrate was concentrated under reduced pressure to obtain a brown residue. To another 50-mL single-necked round-bottom flask equipped with a magnetic stirrer and a reflux condenser was added the residue obtained above, tetrabutylammonium fluoride (1.0 M THF solution, 1.54 mL, 1.54 mmol) and THF (10 mL). The mixture was stirred at room temperature for 1.5 h. Thereafter, the reaction mixture was concentrated under reduced pressure. The resulting residue was purified by flash chromatography to give 237 as a yellow solid in 24% (86 mg) yield: mp 175–177 ° C; ¹ H NMR (500 MHz, DMSO-d ₆ )δ8.55(d,1H,J＝2.0Hz),8.36(s,1H),7.86(d,1H,J＝3.0Hz),7.35(m,3H),7.22(d,1H,J＝9.0 Hz), 7.16 (dd, 1H, J = 9.0, 3.0Hz), 4.85 (t, 1H, J = 5.0Hz), 4.55 (t, 2H, J = 6.5Hz), 4.45 (t, 2H, J = 6.0Hz),4.32(m,2H),4.04(m,1H),3.86(m,1H),3.58(s,3H),3.44(m,1H),3.06(t,4H,J＝4.5 Hz), 3.03 (m, 1H), 2.89 (m, 1H), 2.75 (s, 2H), 2.52 (m, 2H), 2.38 (t, 4H, J = 4.5Hz); MS (ESI+) m/z 685.4(M+H).

Example 238 10-[3-(5-{[5-(4-ethylpiperazin-1-yl)pyridin-2-yl]amino}-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 238

Example 238a 10-[3-(5-{[5-(4-ethylpiperazin-1-yl)pyridin-2-yl]amino}-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(acetoxymethyl)phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 238a

To a microwave tube equipped with a stir bar were added 189a (381.9 mg, 0.850 mmol), 3-(5-(4-ethylpiperazin-1-yl)pyridin-2-ylamino)-1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one (201a) (560 mg, 1.275 mmol), Pd( _PPh3 ) ₄ (49.1 mg, 0.043 mmol), _{aqueous Na2CO3} solution (1.0 N, 2.81 mL, 2.81 mmol), and 1,2-dimethoxyethane (4 mL). The mixture was reacted in a microwave at 130°C for 10 min. Dichloromethane (200 mL) was added, and the resulting mixture was washed with water (30 mL x 3), brine (30 mL x 1), dried over MgSO ₄ , filtered, and the solvent was removed in vacuo. Silica gel column chromatography (methanol:dichloromethane=5:95) afforded 238a.

To a round-bottom flask equipped with a stir bar was added 238a, THF (5 mL), i-PrOH (5 mL), _H₂O (5 mL), and LiOH/ _H₂O (200 mg). The resulting mixture was stirred at room temperature for 1 hr. The solvent was removed in vacuo, and the resulting residue was added to dichloromethane (200 mL). The solution was washed with water (30 mL x 3), brine (30 mL x 1), dried over _MgSO₄ , filtered, and the solvent was removed in vacuo. Silica gel column chromatography (MeOH:dichloromethane/10:90) afforded 238a as a pink solid (53,143 mg). MS (ESI+) m/z 640.6 (M+H).

Example 239 5-(5-fluoro-2-(hydroxymethyl)-3-(6,6-dimethyl-3,4,6,7-tetrahydro-5H-cyclopenta[4,5]thieno[2,3-c]pyridin-1(2H)-yl)-3-(5-(4-isopropylpiperazin-1-yl)pyridin-2-ylamino)-1-methylpyridin-2(1H)-one 239

Using the same procedure as described for 236, 220a (165 mg, 0.410 mmol) and 182c (312 mg, 0.533 mmol) were reacted to afford 239 as an off-white solid in 21% yield (57 mg): mp 191-192 °C; ¹ H NMR (500 MHz, DMSO-d ₆ )δ8.54(d,1H,J＝2.0Hz),8.34(s,1H),7.85(d,1H,J＝3.0Hz),7.33(m,3H),7.20(d,1H,J＝9.0Hz),7.15(dd,1H,J＝9.0, 3.0Hz),4.84(t,1H,J＝4.0Hz),4.32(m,2H),4.04(m,1H),3.84(m,1H),3.57(s,3H),3.02(m,5H),2.87(m,1H),2.75(br s,2H),2.63(m,1H),2.55(m,6H),1.22(s,6H),0.98(d,6H,J=3.0Hz); MS(ESI+)m/z 671.3(M+H).

Example 240 5-(3-(6,6-dimethyl-3,4,6,7-tetrahydro-5H-cyclopenta[4,5]thieno[2,3-c]pyridin-1(2H)-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-1-methyl-3-(6-methyl-5,6,7,8-tetrahydro-1,6-naphthyridin-2-ylamino)pyridin-2(1H)-one 240

A 100-mL single-necked round-bottom flask equipped with a magnetic stirrer and reflux condenser was purged with nitrogen and charged with 182c (300 mg, 0.513 mmol), 205b (138 mg, 0.394 mmol), sodium carbonate (125 mg, 1.18 mmol), 1,4-dioxane (8 mL), and water (2 mL). The mixture was degassed with nitrogen for 30 min. Tetrakis(triphenylphosphine)palladium (46 mg, 0.039 mmol) was added. After heating at reflux for 2 h, the reaction mixture was cooled to room temperature and then partitioned between water (40 mL) and dichloromethane (100 mL). The layers were separated, and the aqueous phase was extracted with dichloromethane (2 x 50 mL). The organic extracts were combined, dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was dissolved in THF (3 mL), and tetrabutylammonium fluoride trihydrate (372 mg, 1.18 mmol) was added. After stirring at room temperature for 2 h, the reaction mixture was partitioned between water (20 mL) and dichloromethane (20 mL). The layers were separated and the aqueous phase was extracted with dichloromethane (2 x 20 mL). The organic extracts were combined, dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by flash chromatography to give 22% yield (53 mg) of a yellow solid 240: mp 142–143 °C; ¹ H NMR (500 MHz, DMSO-d ₆ )δ8.77(d,1H,J＝2.0Hz),8.43(s,1H),7.44(d,1H,J＝2.0Hz),7.31(dd,1H,J＝9.5,2.5H z),7.27(d,1H,J＝8.0Hz),7.19(dd,1H,J＝9.5,2.5Hz),7.06(d,1H,J＝8.0Hz),4.86(m,1 H),4.38(m,2H),4.04(m,1H),3.84(m,1H),3.59(s,3H),3.38(s,2H),3.03(m,1H),2.89 (m,1H),2.75(s,4H),2.62(m,2H),2.53(m,2H),2.34(s,3H),1.23(s,6H); MS(ESI+)m/z 614.3(M+H).

Example 241 5-(3-(6,6-dimethyl-3,4,6,7-tetrahydro-5H-cyclopenta[4,5]thieno[2,3-c]pyridin-1(2H)-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-1-methyl-3-(6-ethyl-5,6,7,8-tetrahydro-1,6-naphthyridin-2-ylamino)pyridin-2(1H)-one 241

Using the same general procedure as described for the preparation of 240, 5-bromo-3-(6-ethyl-5,6,7,8-tetrahydro-1,6-naphthyridin-2-ylamino)-1-methylpyridin-2(1H)-one (143 mg, 0.394 mmol) and 182c (300 mg, 0.513 mmol) gave 28% yield (70 mg) of 241 as a yellow solid: mp 134-135 °C; ¹ H NMR (500 MHz, DMSO-d ₆ )δ8.78(d,1H,J＝2.0Hz),8.43(s,1H),7.44(d,1H,J＝2.0Hz),7.31(dd,1H,J＝9.5,2.5Hz),7. 28(d,1H,J＝8.0Hz),7.18(dd,1H,J＝9.5,2.5Hz),7.06(d,1H,J＝8.0Hz),4.86(m,1H),4.38(m, 2H),4.04(m,1H),3.84(m,1H),3.59(s,3H),3.43(s,2H),3.03(m,1H),2.89(m,1H),2.75(s, 4H), 2.62 (m, 2H), 2.53 (m, 2H), 2.50 (m, 2H), 1.23 (s, 6H), 1.08 (t, 3H, J = 7.0Hz); MS (ESI+) m/z 614.3(M+H).

Example 242 5-[5-Fluoro-2-(hydroxymethyl)-3-{1-oxo-1H,2H,3H,4H,6H,7H,8H,9H-pyrazino[1,2-a]indol-2-yl}phenyl]-1-methyl-3-{[5-(1-methylpiperidin-4-yl)pyridin-2-yl]amino}-1,2-dihydropyridin-2-one 242

Example 242a 4-Fluoro-2-(1-methyl-5-(5-(1-methylpiperidin-4-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 242a

To a microwave tube equipped with a stir bar were added 5-bromo-1-methyl-3-(5-(1-methylpiperidin-4-yl)pyridin-2-ylamino)pyridin-2(1H)-one (130c) (240 mg, 0.636 mmol), 4-fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 210d (336 mg, 0.763 mmol), Pd( _PPh3 ) ₄ (37 mg, 0.0318 mmol), _{aqueous Na2CO3} solution (1.0 N, 2.1 mL, 2.1 mmol), and 1,2-dimethoxyethane (4.2 mL). The mixture was reacted in a microwave at 130°C for 10 min. Dichloromethane (200 mL) was added and the resulting mixture was washed with water (3 x 30 mL), brine (30 mL), dried over _MgSO4 , filtered, and the solvent removed in vacuo. Silica gel column chromatography (methanol:dichloromethane = 5:95) afforded 242a.

To a round-bottom flask equipped with a stir bar was added 242a, THF (5 mL), i-PrOH (5 mL), _H₂O (5 mL), and LiOH/ _H₂O (300 mg). The resulting mixture was stirred at room temperature for 1 hr. All solvents were removed in vacuo, and the resulting residue was added to dichloromethane (200 mL). The solution was washed with water (30 mL x 3), brine (30 mL x 1), dried over _MgSO₄ , filtered, and the solvent was removed in vacuo. Silica gel column chromatography (MeOH:dichloromethane = 10:90) afforded 242 as a brown solid (56 mg). MS (ESI+) m/z 611.5 (M+H).

Example 243 10-[5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-{[5-(1-methylpyrrolidin-2-yl)pyridin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 243

Example 243a 10-[5-fluoro-2-(acetoxymethyl)-3-(1-methyl-5-{[5-(1-methylpyrrolidin-2-yl)pyridin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 243a

Example 243a was synthesized using the same procedure as 121b, except using compound 230a (446 mg, 0.9 mmol), 5-bromo-1-methyl-3-(5-(1-methylpyrrolidin-2-yl)pyridin-2-ylamino)pyridin-2(1H)-one (229a) (281 mg, 0.8 mmol), 1 M sodium carbonate solution (2.7 mL, 2.7 mmol), tetrakis(triphenylphosphine)palladium(0) (47 mg, 0.040 mmol), and 1,2-dimethoxyethane (6.5 mL). The reaction mixture was heated at 130°C in a microwave reactor for 15 minutes. Workup and flash column chromatography (silica gel, 9:1 dichloromethane/methanol) afforded 243a in 67% yield (350 mg) as a brown oil: MS (ESI+) m/z 653.1 (M+H).

According to Example 121, except using a mixture of THF (2 mL), water (1 mL), and isopropanol (2 mL), compound 243a (350 mg, 0.54 mmol), and lithium hydroxide monohydrate (150 mg, 3.5 mmol), post-processing and flash column chromatography (NH-silica gel, ethyl acetate/hexane) gave compound 243 as a light yellow solid in a 29% yield (95 mg): MS (ESI+) m/z 611.5 (M+H).

Example 244 10-[5-fluoro-2-(hydroxymethyl)-3-[4-methyl-6-({4-[1-(oxetan-3-yl)piperidin-4-yl]phenyl}amino)-5-oxo-4,5-dihydropyrazin-2-yl]phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 244

Example 244a 10-[5-fluoro-2-(acetoxymethyl)-3-[4-methyl-6-({4-[1-(oxetan-3-yl)piperidin-4-yl]phenyl}amino)-5-oxo-4,5-dihydropyrazin-2-yl]phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 244a

Following Example 121b, compound 230a (188 mg, 0.38 mmol), 5-bromo-1-methyl-3-(4-(1-(oxetan-3-yl)piperidin-4-yl)phenylamino)pyrazin-2(1H)-one 214b (160 mg, 0.38 mmol), 1 M sodium carbonate solution (1.5 mL, 1.5 mmol), tetrakis(triphenylphosphine)palladium(0) (22 mg, 0.019 mmol), and 1,2-dimethoxyethane (5 mL) were used. The reaction mixture was heated at 130° C. in a microwave reactor for 15 minutes. Workup and flash column chromatography (silica gel, 60:35:5 dichloromethane/diethyl ether/methanol) afforded a yellow oily mixture of compounds 244a and 244 (270 mg).

The mixture from 244a (270 mg) was deprotected using the same method as 121, except using a mixture of THF (2 mL), water (1 mL), and isopropanol (2 mL) and lithium hydroxide monohydrate (85 mg, 2 mmol). Workup and flash column chromatography (NH-silica gel, ethyl acetate/hexanes) gave compound 244 in 63% yield (167 mg) as a light yellow solid: MS (ESI+) m/z 667.6 (M+H).

Example 245 10-[5-fluoro-2-(hydroxymethyl)-3-[1-methyl-6-oxo-5-({5-[4-(propan-2-yl)piperazin-1-yl]-pyridin-2-yl}amino)-1,6-dihydropyridin-3-yl]phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 245

Example 245a 10-[5-fluoro-2-(acetoxymethyl)-3-[1-methyl-6-oxo-5-({5-[4-(propan-2-yl)piperazin-1-yl]-pyridin-2-yl}amino)-1,6-dihydropyridin-3-yl]phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 245a

To a 44-mL sealed tube equipped with a magnetic stirrer were added 220a (400 mg, 1.0 mmol), 7 (508 mg, 2.0 mmol), potassium acetate (392 mg, 4.0 mmol), and 1,4-dioxane (10 mL). After degassing the mixture for 30 minutes, Pd(dppf) _Cl₂ · _CH₂Cl₂ (82 mg, 0.10 mmol) was _added . The resulting reaction mixture was stirred at 105°C for 6 hours. It was then cooled to room temperature and filtered through a Celite pad. The filtrate was concentrated, and the crude mixture was redissolved in DME (2 mL) and transferred to a 10-mL microwave reaction vessel. To this solution was added 10-[2-(acetoxymethyl)-3-bromo-5-fluorophenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one (189a) (270 mg, 0.60 mmol), Pd(PPh ₃ ) ₄ (50 mg, 0.050 mmol), and 2N Na ₂ CO ₃ (2 mL). The reaction mixture was then degassed for 5 minutes and placed in a microwave cavity. After stirring the reaction mixture at 125° C. for 7 minutes, it was purified by flash chromatography (dichloromethane:methanol, 85:15) to afford 49% (150 mg) of 245a.

To a 100-mL single-necked round-bottom flask equipped with a magnetic stir bar was added 245a (150 mg, 0.22 mmol), LiOH· _H₂O (100 mg, 2.2 mmol), THF (2 mL), isopropanol (2 mL), and water (2 mL). The reaction mixture was stirred at room temperature for 3 h, then partitioned between dichloromethane (5 mL) and water (5 mL), and the organic phase was extracted with dichloromethane (5 mL x 3). The combined organic phases were washed with water (5 mL x 2) and brine (5 mL x 1), dried ( _Na₂SO₄ ), and concentrated. The crude product was redissolved in dichloromethane (3 mL). Hexane (10 mL) was added to this solution, and the resulting precipitate was filtered to provide 245 in a 49% yield (70 mg). _MS ( ^ESI⁺ ) m/z 654.6 (M+H).

Example 246 10-[5-fluoro-2-(hydroxymethyl)-3-[1-methyl-5-({5-methyl-4H,5H,6H,7H-pyrazolo[1,5-a]pyrazin-2-yl}amino)-6-oxo-1,6-dihydropyridin-3-yl]phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 246

Example 246a 10-[5-fluoro-2-(acetoxymethyl)-3-[1-methyl-5-({5-methyl-4H,5H,6H,7H-pyrazolo[1,5-a]pyrazin-2-yl}amino)-6-oxo-1,6-dihydropyridin-3-yl]phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 246a

To a microwave tube equipped with a magnetic stirrer was added 5-bromo-1-methyl-3-(5-methyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)pyridin-2(1H)-one 146a (210 mg, 0.6 mmol), 230a (460 mg, 0.9 mmol), 1,2-dimethoxyethane (8 mL) and 1 M aqueous sodium carbonate solution (2 mL). After bubbling _N for 15 min, Pd(PPh ₃ ) ₄ (36 mg, 0.03 mmol) was added. The mixture was heated to 135° C. in a microwave for 15 min. Thereafter, ethyl acetate (5 mL) and water (5 mL) were added. The separated aqueous layer was extracted with ethyl acetate (2×5 mL). The combined organic phases were washed with brine (10 mL), dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by column chromatography eluting with a gradient of dichloromethane to 60:35:5 dichloromethane/ether/methanol to afford 246a in 37% yield (140 mg).

To a 25 mL round-bottom flask equipped with a magnetic stirrer was added 246a (140 mg, 0.2 mmol), lithium hydroxide (200 mg, 4.8 mmol), THF (3 mL), isopropanol (3 mL) and water (2 mL). The mixture was stirred at rt for 1 h. Thereafter, ethyl acetate (5 mL) and water (5 mL) were added. The separated aqueous layer was extracted with ethyl acetate (2 x 5 mL). The combined organic phases were washed with brine (10 mL), dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by column chromatography using a Biotage KPNH 12+M column eluted with a gradient of hexane-ethyl acetate to afford 246 in 86% yield (120 mg). MS (ESI+) m/z 586.6 (M+H).

Example 247 10-[5-Fluoro-2-(hydroxymethyl)-3-{4-methyl-6-[(2-methyl-1,2,3,4-tetrahydroisoquinolin-6-yl)amino]-5-oxo-4,5-dihydropyrazin-2-yl}phenyl]-4,4-dimethyl-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-9-one 247

Example 247a (2-bromo-6-{4,4-dimethyl-9-oxo-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-10-yl}-4-fluorophenyl)methyl acetate 247a

A mixture of 4,4-dimethyl-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-9-one (105h) (2 g, 9.05 mmol), 2,6-dibromo-4-fluorobenzyl acetate (197c) (8.8 g, 27.15 mmol), XantPhos (524 mg, 0.9 mmol), Pd ₂ (dba) ₃ (828 mg, 0.9 mmol), and Cs ₂ CO ₃ (5.9 g, 18 mmol) in dioxane (200 mL) was heated at 100° C. under nitrogen for 15 h. The reaction mixture was filtered, and the filtrate was evaporated in vacuo. The residue was purified on a silica gel column eluted with 1:1 ethyl acetate/petroleum ether to afford 247a (3 g, 71%) as a yellow solid. MS: (M+H) ⁺ 466.

Example 247b (2-{4,4-dimethyl-9-oxo-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-10-yl}-4-fluoro-6-(tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methyl acetate 247b

To a solution of 247a (3 g, 6.45 mmol) and 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bis(1,3,2-dioxaborolane) (9.8 g, 38.7 mmol) in dioxane (160 mL) were added _PdCl2 (dppf) (525 mg, 0.65 mmol) and _CH3COOK (3.8 g, 38.7 mmol). The mixture was stirred at 100°C under an argon atmosphere for 15 h. After the reaction, the mixture was filtered and evaporated in vacuo. The residue was purified on a silica gel column eluted with 1:2 ethyl acetate/petroleum ether to afford 247b (2.5 g, 76%) as a yellow solid. MS: (M+H) ⁺ 514.

Example 247c (2-{4,4-dimethyl-9-oxo-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-10-yl}-4-fluoro-6-{4-methyl-6-[(2-methyl-1,2,3,4-tetrahydroisoquinolin-6-yl)amino]-5-oxo-4,5-dihydropyrazin-2-yl}phenyl)methyl acetate 247c

To a 25 mL sealed tube was added 247b (590 mg, 1.15 mmol), 5-bromo-1-methyl-3-(2-methyl- _1,2,3,4 -tetrahydroisoquinolin-6-ylamino)pyrazin-2(1H)-one 221b (400 mg, 1.15 mmol), _CH3COONa (189 mg, 2.3 mmol), _K3PO4 (611 mg, 2.3 mmol), and _PdCl2 (dppf) (94 mg, 0.11 mmol) and suspended in _CH3CN (25 mL) and water (1 mL). The mixture was heated at 110°C for 2 h. The mixture was evaporated and the residue was purified on a silica gel column eluting with 20:1 dichloromethane/methanol to afford 247c (400 mg, 53%) as a brown solid. MS: (M+H) ⁺ 656.

To a solution of 247c (400 mg, 0.61 mmol) in propan-2-ol (8 mL), tetrahydrofuran (8 mL) and water (1.5 mL) was added LiOH (1.46 g, 61 mmol). The mixture was stirred at 30 °C for 2 h. It was then evaporated and the residue was purified by preparative HPLC to give 247 (124 mg, 33%) as a yellow solid. MS: (M+H) ⁺ 614. ¹ H NMR (500 MHz, MeOD) δ 1.29 (s, 6H), 2.46 (s, 3H), 2.59-2.60 (d, 2H), 2.74-2.76 (t, 2H), 2.81 (s, 2H), 2.93-2.98 (m, 3H), 3.07-3.09 (m, 1H), 3.59 (s, 2H), 3.64 (s, 3H), 3 .95-3.97(m,1H),4.01-4.12(m,1H),4.48-4.51(d,1H),4.58-4.61(d,1H),7.03-7 .05(d,1H),7.18-7.21(m,1H),7.38-7.41(m,2H),7.52-7.54(d,1H),7.64(s,1H).

Example 248 10-(3-{6-[(2-ethyl-1,2,3,4-tetrahydroisoquinolin-6-yl)amino]-4-methyl-5-oxo-4,5-dihydropyrazin-2-yl}-5-fluoro-2-(hydroxymethyl)phenyl)-4,4-dimethyl-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-9-one 248

Example 248a 5-Bromo-3-(2-ethyl-1,2,3,4-tetrahydroisoquinolin-6-ylamino)-1-methylpyrazin-2(1H)-one 248a

According to the method described for 220a, starting from 385 mg of 5-bromo-1-methyl-3-(1,2,3,4-tetrahydroisoquinolin-6-ylamino)pyrazin-2(1H)-one (221a), 248a (292 mg, 70%) was obtained as a yellow solid. MS: [M+H] ⁺ 364.

Example 248b 2-{4,4-dimethyl-9-oxo-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-10-yl}-6-{6-[(2-ethyl-1,2,3,4-tetrahydroisoquinolin-6-yl)amino]-4-methyl-5-oxo-4,5-dihydropyrazin-2-yl}-4-fluorophenyl)methyl acetate 248b

According to the method described for 129a, starting from 400 mg of (2-{4,4-dimethyl-9-oxo-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-10-yl}-4-fluoro-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methyl acetate (247b) and 283 mg of 5-bromo-3-(2-ethyl-1,2,3,4-tetrahydroisoquinolin-6-ylamino)-1-methyl-pyrazin-2(1H)-one, 248b was obtained as a yellow solid (260 mg, 50%).

Following the procedure described for 148, starting from 230 mg of 248b, 127 mg of 248 (59%) was obtained as ^a white solid. HNMR(500MHz,DMSO)δ9.15(s,1H),7.81(s,1H),7.63(dd,J=8,1H),7.52(s,1H),7 .39(dd,J＝9.5,1H),7.31(d,J＝9.5,1H),6.97(d,J＝8.0,1H),4.88(s,1H),4.50(s ,1H),4.45(s,1H),4.04(m,1H),3.84(m,1H),3.54(s,3H),3.47(s,2H),3.03(m,1 H), 2.91 (m, 1H), 2.76 (s, 3H), 2.62-2.45 (m, 6H), 1.23 (s, 6H), 1.08 (t, J = 7.5, 3H).

Example 249 10-[5-fluoro-2-(hydroxymethyl)-3-(4-methyl-6-{[2-(oxetan-3-yl)-1,2,3,4-tetrahydroisoquinolin-6-yl]amino}-5-oxo-4,5-dihydropyrazin-2-yl)phenyl]-4,4-dimethyl-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-9-one 249

Example 249a (2-{4,4-dimethyl-9-oxo-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-10-yl}-4-fluoro-6-(4-methyl-6-{[2-(oxetane-3-yl)-1,2,3,4-tetrahydroisoquinolin-6-yl]amino}-5-oxo-4,5-dihydropyrazin-2-yl)phenyl)methyl acetate 249a

Starting from 247b and 305 mg of 222a according to the procedure described for 148b, 277 mg of 249a was obtained as a yellow solid (51%). MS: [M+H] ⁺ 698.

Starting from 250 mg of 249a, the procedure described for compound 148 gave 117 mg of 249 as a white solid (50%). ¹ H NMR(500MHz,DMSO)δ9.17(s,1H),7.85(d,J＝1.5,1H),7.65(dd,J＝8,1H),7 .52(s,1H),7.39(dd,J＝9.5,1H),7.31(d,J＝9.5,1H),6.97(d,J＝8.0,1H),4 .80(s,1H),4.61(m,2H),4.52(m,1H),4.43(m,1H),4.04(m,1H),3.85(m,1 H),3.56(m,4H),3.34(s,2H),3.01-2.57(m,6H),2.53(m,5H),1.23(s,6H).

Example 250 2-(3-(5-(5-(1-ethylazetidin-3-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 250

Example 250a 5-Bromo-3-(5-(1-ethylazetidin-3-yl)pyridin-2-ylamino)-1-methylpyridin-2(1H)-one 250a

To a solution of 3-(5-(azetidin-3-yl)pyridin-2-ylamino)-5-bromo-1-methylpyridin-2(1H)-one 155n (crude, 4.6 mmol) in methanol (50 mL) and acetic acid (5 mL) was added _CH₃CHO (40 wt% solution in _H₂O ) (10 g, 92 mmol) at 0°C, followed by the addition of NaBH( _CH₃O ) _₃ (20 g, 92 mmol) in small portions over 1 h. After the reaction was complete, the mixture was adjusted to pH >7 with 2N NaOH. The mixture was then extracted with DCM (80 mL x ₃ ), dried over _Na₂SO₄ , and concentrated to afford a yellow solid, which was purified by flash column chromatography eluting with 50:1 DCM/MeOH containing 0.5% _Et₃N to afford 250a as a yellow solid (43% over 2 steps). LCMS: (M+H) ^⁺ 364. ¹ H NMR (500MHz, DMSO) δ8.72(s,1H),8.66(d,J＝3,1H),8.21(d,J＝3,1H),7.68(dd,J＝9,1H),7.5 2(d,J=2.5,1H),7.32(d,J=9,1H),3.54(m,6H),2.99(m,2H),2.42(m,2H),0.89(t,J=7,3H).

Example 250b 2-(5-(5-(1-ethylazetidin-3-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-4-fluoro-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 250b

According to Example 136e, starting from 250a and 4-fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 210d, 250b was obtained in 65% yield. LCMS: (M+H) ⁺ 639

According to Example 135, 250b was used as the starting material to obtain 250 in a 63% yield. LCMS: (M+H) ⁺ 597. ¹ H NMR (500 MHz, DMSO) δ 8.66 (d, J = 2, 1H), 8.56 (s, 1H), 8.11 (d, J = 1.5, 1H), 7.66 (dd, J = 8.5, 1H), 7.42 (d, J = 2.5, 1H), 7.33 (dd, J = 9.5, 1H), 7.28 (d, J = 8.5, 1H), 7.19 (dd, J = 9, 1H), 6.53 ( s,1H),4.87(t,J＝4,1H),4.31(d,J＝4.5,2H),4.15(m,3H),3.89(m,1H),3.59(s,3H),3.47( m,3H),2.97(m,2H),2.62(m,3H),2.40(m,3H),1.79(m,2H),1.69(m,2H),0.88(t,J=7,3H).

Example 251 5-(5-fluoro-2-(hydroxymethyl)-3-(6,6-dimethyl-3,4,6,7-tetrahydro-5H-cyclopenta[4,5]thieno[2,3-c]pyridin-1(2H)-yl)-3-(5-(1-methylazetidin-3-yloxy))-1-methylpyridin-2(1H)-one 251

Example 251a tert-Butyl 3-(6-nitropyridin-3-yloxy)azetidine-1-carboxylate 251a

2-nitro-5-hydroxypyridine (2.00 g, 14.3 mmol), 3-iodine azetidine-1-carboxylic acid tert-butyl ester (6.06 g, 21.5 mmol), sodium hydride (800 mg, 20.2 mmol) and DMF (30 mL) were added to a 100-mL single-necked round-bottom flask equipped with a magnetic stirrer and a reflux condenser. The reaction mixture was heated at 110 ° C for 24 h. Afterwards, the reaction mixture was cooled to room temperature and then concentrated under reduced pressure. The residue was diluted with ethyl acetate (200 mL) and then washed with saturated sodium bicarbonate aqueous solution (100 mL). The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by flash chromatography to afford 95% yield (4.00 g) of yellow oil 251a: ¹ H NMR (500 MHz, CDCl ₃ ) δ 8.27 (d, 1H, J=9.0 Hz), 8.14 (d, 1H, J=3.0 Hz), 7.26 (dd, 1H, J=9.0, 3.0 Hz), 5.04 (m, 1H), 4.39 (m, 2H), 4.06 (m, 2H), 1.44 (s, 9H); MS (ESI+) m/z 196.1 (M-Boc+H).

Example 251b tert-Butyl 3-(6-aminopyridin-3-yloxy)azetidine-1-carboxylate 251b

A 250-mL Parr reaction bottle was purged with nitrogen and platinum oxide (200 mg), 251a (2.00 g, 6.78 mmol), ethyl acetate (30 mL) and ethanol (30 mL) were added. The bottle was connected to a Parr hydrogenation reactor, vacuumized, and filled with hydrogen to a pressure of 50 psi and shaken for 20 h. After this, the hydrogen was removed and nitrogen was charged into the bottle. Celite 521 (10.0 g) was added and the mixture was then filtered through a Celite 521 pad. The filter cake was washed with methanol (2×50 mL), and the combined filtrates were concentrated to dryness under reduced pressure to afford 251b as a white solid in 100% yield (2.01 g): mp 83–84° C.; ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 7.53 (d, 1 H, J=3.0 Hz), 7.04 (dd, 1 H, J=9.0, 3.0 Hz), 6.41 (d, 1 H, J=9.0 Hz), 5.32 (br s, 2 H), 4.21 (t, 2 H, J=6.5 Hz), 3.75 (t, 2 H, J=6.5 Hz), 3.25 (m, 1 H), 1.37 (s, 9 H); MS (ESI+) m/z 266.2 (M+H).

Example 251c tert-Butyl 3-(6-(5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)pyridin-3-yloxy)azetidine-1-carboxylate 251c

Using the same method as described for the preparation of 250a, 251b (1.01 g, 3.80 mmol) and 3,5-dibromo-1-methylpyridin-2(1H)-one (1.01 g, 3.80 mmol) were reacted to give 251c as a brown solid in 81% yield (1.40 mg): mp 120-121 °C; ¹ H NMR (500 MHz, DMSO-d ₆ )δ8.70(s,1H),8.57(d,1H,J＝2.5Hz),7.86(d,1H,J＝3.0Hz),7.48(d,1H,J＝2.5Hz),7.35(d,1H,J＝ 9.0Hz),7.28(m,1H),4.95(m,1H),4.3(m,2H),3.80(m,2H),3.50(s,3H),1.38(s,9H); MS(ESI+)m/z 452.3(M+H).

Example 251d 3-(5-(azetidin-3-yloxy)pyridin-2-ylamino)-5-bromo-1-methylpyridin-2(1H)-one 251d

To a 250-mL single-necked round-bottom flask equipped with a magnetic stirrer was added 251c (1.14 g, 3.10 mmol), dichloromethane (10 mL), and trifluoroacetic acid (5 mL). The reaction mixture was stirred at room temperature for 2 h. Thereafter, the mixture was concentrated under reduced pressure. The resulting residue was diluted with dichloromethane (100 mL) and then washed with saturated aqueous sodium bicarbonate solution (50 mL). The aqueous layer was extracted with dichloromethane (2×50 mL), and the combined organic extracts were dried over sodium sulfate, filtered, and then concentrated to afford 251d as a colorless oil in a 92% yield (1.10 g). This compound was used in the next step without any purification: ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 8.65 (s, 1H), 8.55 (d, 1H, J=2.5 Hz), 7.82 (d, 1H, J=3.0 Hz), 7.48 (d, 1H, J=2.5 Hz), 7.30 (d, 1H, J=9.0 Hz), 7.21 (m, 1H), 4.95 (t, 1H, J=6.0 Hz), 3.76 (m, 1H), 3.48 (m, 5H); MS (ESI+) m/z 353.0 (M+H).

Example 251e 5-Bromo-1-methyl-3-(5-(1-methylazetidin-3-yloxy)pyridin-2-ylamino)pyridin-2(1H)-one 251e

A 250-mL single-necked round-bottom flask equipped with a magnetic stirrer was purged with nitrogen and charged with 251d (1.10 g, 2.95 mmol), 37% aqueous formaldehyde (111 mg, 3.70 mmol), and methanol (15 mL). A suspension of sodium cyanoborohydride (540 mg, 8.60 mmol) and zinc chloride (600 mg, 4.40 mmol) in methanol (10 mL) was added and the reaction was stirred at room temperature for 4 h. Afterwards, the reaction mixture was concentrated and the residue was partitioned between 90:10 dichloromethane/methanol (250 mL) and 10% aqueous potassium carbonate (100 mL). The aqueous layer was extracted with 90:10 dichloromethane/methanol (3×150 mL). The combined organic extracts were dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by column chromatography (silica gel, 0%-50% methanol/dichloromethane) to afford 251e as a brown solid in 80% yield (830 mg): mp 100-101° C.; ¹ H NMR (500 MHz, DMSO-d ₆ )δ8.66(s,1H),8.55(d,1H,J＝2.5Hz),7.86(d,1H,J＝3.0Hz),7.47(d,1H,J＝2.5Hz),7.31(d,1H,J＝9.0Hz),7.24(dd, 1H, J＝9.5, 3.5Hz), 4.76 (m, 1H), 3.77 (t, 2H, J＝7.5Hz), 3.50 (s, 3H), 3.03 (t, 2H, J＝7.5Hz), 2.26 (s, 3H); MS (ESI+) m/z 366.0(M+H).

Using the same procedure as described for the preparation of 239, 251e (145 mg, 0.400 mmol) and 182c (304 mg, 0.520 mmol) were reacted to give 251 in 15% yield (37 mg) as an off-white solid: mp 160-161 °C; ^1H NMR (500 MHz, DMSO- _d6 ) δ 8.53 (d, 1H, J = 2.5 Hz), 8.49 (s, 1H), 7.77 (d, 1H, J = 2.5 Hz), 7.36 (d, 1H, J = 2.0 Hz), 7.32 (dd, 1H, J = 9.0, 2.5 Hz), 7.27 (d, 1H, J = 9.0, 2.5 Hz), 7.22 (dd, 1H, J = 9.0, 2.5 Hz), 7.16 (dd, 1H, J = 9.0,2.5Hz),4.84(t,1H,J＝4.0Hz),4.69(m,1H),4.30(m,2H),3.84(m,1H),3.69(m,2H),3 .58(s,3H),3.02(m,1H),2.92(m,3H),2.75(m,2H),2.54(m,2H),2.27(s,3H); MS(ESI+)m/z 630.3(M+H).

Example 252 10-[5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-{[5-(oxetan-3-yl)-4H,5H,6H,7H-pyrazolo[1,5-a]pyrazin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 252

Example 252a 5-Bromo-1-methyl-3-(5-(oxetan-3-yl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)pyridin-2(1H)-one 252a

According to Example 214b, 5-bromo-1-methyl-3-(4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)pyridin-2(1H)-one 126a (250 mg, 0.78 mmol) and oxetan-3-one (600 mg, 8.3 mmol) were dissolved in methanol (8 mL). Sodium cyanoborohydride (148 mg, 3 mmol) and zinc chloride (165 mg, 1.5 mmol) in methanol (8 mL) were added, and the reaction was heated at 48°C for 12 hours. Workup and flash column chromatography (silica gel, 60:35:5 dichloromethane/diethyl ether/methanol) afforded 252a as a light green solid in a 34% yield (100 mg): MS (ESI+) m/z 382.1 (M+H).

Following Example 121b, 230a (135 mg, 0.26 mmol), 252a (100 mg, 0.26 mmol), 1 M sodium carbonate solution (1 mL, 1 mmol), tetrakis(triphenylphosphine)palladium(0) (15 mg, 0.013 mmol), and 1,2-dimethoxyethane (2.5 mL) were used. The reaction mixture was heated at 130°C for 15 minutes in a microwave reactor. Workup and flash column chromatography (silica gel, 60:35:5 dichloromethane/ether/methanol) afforded a mixture of 252b and 252 (80 mg) as a yellow oil. This mixture (80 mg) was deprotected using the same procedure as for 121, except a mixture of THF (1 mL), water (0.5 mL), and isopropanol (1 mL) and lithium hydroxide monohydrate (25 mg, 0.6 mmol) were used. Workup and flash column chromatography (NH-silica gel, ethyl acetate/hexane) gave 252 as a white solid in 40% yield (30 mg): MS (ESI+) m/z 628.5 (M+H).

Example 253 5-[5-Fluoro-2-(hydroxymethyl)-3-{1-oxo-1H,2H,3H,4H,6H,7H,8H,9H-pyrazino[1,2-a]indol-2-yl}phenyl]-1-methyl-3-({5-[(2R)-1-methylpyrrolidin-2-yl]pyridin-2-yl}amino)-1,2-dihydropyridin-2-one 253

Example 254 5-[5-Fluoro-2-(hydroxymethyl)-3-{1-oxo-1H,2H,3H,4H,6H,7H,8H,9H-pyrazino[1,2-a]indol-2-yl}phenyl]-1-methyl-3-({5-[(2S)-1-methylpyrrolidin-2-yl]pyridin-2-yl}amino)-1,2-dihydropyridin-2-one 254

Examples 253 and 254 are enantiomers of racemate 202. Chiral separation of racemic mixture 202 was performed on a Chiralpak AD, 4.6 x 50 mm, 3 μm column (mobile phase 55% isopropanol (w/0.1% triethylamine)/45% _CO2 , flow rate 5 mL/min) at 40°C to afford the individual enantiomers, with 254 eluting first, followed by 253. Example 253 MS (ESI+) m/z 597.4 (M+H). Example 254 MS (ESI+) m/z 597.4 (M+H).

Example 255 5-[5-fluoro-2-(hydroxymethyl)-3-{1-oxo-1H,2H,3H,4H,6H,7H,8H,9H-pyrazino[1,2-a]indol-2-yl}phenyl]-1-methyl-3-{[5-(morpholin-4-yl)pyridin-2-yl]amino}-1,2-dihydropyridin-2-one 255

Example 255a 4-(6-nitropyridin-3-yl)morpholine 255a

Following the procedure described for 188a, starting from 2 g of 5-bromo-2-nitropyridine and 968 mg of morpholine, 255a was obtained as a yellow solid (1 g, 50%). MS: [M+H] ⁺ 210. ¹ H NMR (500 MHz, CDCl3) δ 8.18 (d, J = 9.0, 1H), 8.15 (d, J = 3.0, 1H), 7.22 (dd, J = 3.0, 1H), 3.90 (t, J = 5.0, 4H), 3.42 (t, J = 5.0, 4H).

Example 255b 5-Morpholinopyridin-2-amine 255b

According to the method described for 188b, starting from 1 g of 4-(6-nitropyridin-3-yl)morpholine, 255b was obtained as a yellow solid (840 mg, 98%). MS: [M+H] ⁺ 210

Example 255c 5-Bromo-1-methyl-3-(5-morpholinopyridin-2-ylamino)pyridin-2(1H)-one 255c

According to the method described for 188c, starting from 3,5-dibromo-1-methylpyridin-2(1H)-one (1.15 g, 4.3 mmol) and 5-morpholinopyridin-2-amine (770 mg, 4.3 mmol), 255c (986 mg, 63%) was obtained as a yellow solid. MS: [M+H] ⁺ 365

Example 255d 4-Fluoro-2-(1-methyl-5-(5-morpholinopyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 255d

According to the method described for 148c, starting from 400 mg of 4-fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 210d and 302 mg of 5-bromo-1-methyl-3-(5-morpholinopyridin-2-ylamino)pyridin-2(1H)-one, 255d was obtained as a yellow solid (281 mg, 53%). MS: [M+H] ⁺ 641

Following the procedure described for 148, starting from 270 mg of 4-fluoro-2-(1-methyl-5-(5-morpholinopyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate, 255 was obtained as a white solid (103 mg, 41%). MS: [M+H] ⁺ 599. ¹ H NMR (500MHz, DMSO) δ8.56 (d, J = 2.0, 1H), 8.38 (s, 1H), 7.87 (d, J = 3.0, ¹ H),7.36-7.32(m,3H),7.25(d,J＝9.5,,2H),7.19(d,J＝9.0,1H),6.52(s,1H),4.87(s,1H),4.32(d,J＝4,2H),4.15(m,3H) ,3.82(m,1H),3.72(t,J＝4.0,1H),3.50(s,3H),3.02(t,J＝4.5,3H),2.61(m,2H),2.47(m,2H),1.80(s,2H),1.68(S,2H).

Example 256 10-[5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-{[5-(morpholin-4-yl)pyridin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)phenyl]-4,4-dimethyl-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-9-one 256

Example 256a (2-{4,4-dimethyl-9-oxo-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-10-yl}-4-fluoro-6-(1-methyl-5-{[5-(morpholin-4-yl)pyridin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)phenyl)methyl acetate 256a

According to Example 148b, starting from 400 mg of (2-{4,4-dimethyl-9-oxo-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-10-yl}-4-fluoro-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methyl acetate 247b and 284 mg of 5-bromo-1-methyl-3-(5-morpholinopyridin-2-ylamino)pyridin-2(1H)-one 255c, 277 mg of a yellow solid 256a was obtained (53%). MS: [M+H] ⁺ 672

According to Example 148, starting from 270 mg of 256a, 103 mg of 256 as a white solid was obtained (41%). MS: [M+H] ⁺ 630. ¹ H NMR (500MHz, DMSO) δ8.56 (d, J = 2.0, 1H), 8.38 (s, 1H), 7.88 (d, J = 3.0, ¹ H),7.36-7.32(m,3H),7.25(d,J＝9.5,,2H),7.19(d,J＝9.0,1H),4.85(s,1H),4.32(d,J＝4,2H),4.07(m,3H),3.86 (m,1H),3.72(t,J＝4.0,1H),3.50(s,3H),3.02(t,J＝4.5,3H),2.89(m,1H),2.76(s,2H),2.53(m,2H),1.23(s,6H).

Example 257 10-[3-(5-{[5-(1-ethylazetidin-3-yl)pyridin-2-yl]amino}-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl]-4,4-dimethyl-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-9-one 257

Example 257a (2-{4,4-dimethyl-9-oxo-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-10-yl}-6-(5-{[5-(1-ethylazetidin-3-yl)pyridin-2-yl]amino}-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-4-fluorophenyl)methyl acetate 257a

According to Example 136a, starting from 5-bromo-3-(5-(1-ethylazetidin-3-yl)pyridin-2-ylamino)-1-methylpyridin-2(1H)-one 250a and (2-{4,4-dimethyl-9-oxo-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-10-yl}-4-fluoro-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methyl acetate 247b, compound 257a was obtained in 37% yield.

According to Example 136, compound 257a was used as the starting material to obtain compound 257 in a 46% yield. LCMS: (M+H) ⁺ 628. ¹ H NMR (500 MHz, DMSO) δ 8.66 (d, J = 2, 1H), 8.55 (s, 1H), 8.12 (d, J = 2.5, 1H), 7.65 (dd, J = 8.5, 1H), 7.42 (d, J = 2, 1H), 7.33 (dd, J = 9.5, 1H), 7.28 (d, J = 9, 1H), 7.19 (dd, J = 9, 1H), 4.86 (t, J = 4, 1H), 4.33(m,2H),4.05(m,1H),3.86(m,1H),3.59(s,3H),3.49(m,3H),3.04(m,1H),2.97(m,2H),2. 89(m,1H),2.76(s,2H),2.55(m,2H),2.40(m,2H),1.79(m,4H),1.23(s,6H),0.88(t,J=7,3H).

Example 258 2-(5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-(2-(oxetan-3-yl)-1,2,3,4-tetrahydroisoquinolin-6-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 258

Example 258a tert-Butyl 6-(5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)-3,4-dihydroisoquinoline-2(1H)-carboxylate

To a 100 mL round-bottom flask was added tert-butyl 6-amino-3,4-dihydroisoquinoline-2(1H)-carboxylate (2 g, 8 mmol), 3,5-dibromo-1-methylpyridin-2(1H)-one (1.8 g, 6.7 mmol), cesium carbonate (4.4 g, 13.4 mmol), XantPhos (0.41 g, 0.7 mmol), tris(dibenzylideneacetone)dipalladium(0) (0.64 g, 0.7 mmol) and 1,4-dioxane (50 mL). The reaction mixture was refluxed under an argon atmosphere for 2 h. Thereafter, the mixture was cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography eluting with 25:1 dichloromethane/methanol to give 258a (2.18 g, 63%). LC/MS: m/z 435 (M+H) ⁺

Example 258b 5-Bromo-1-methyl-3-(1,2,3,4-tetrahydroisoquinolin-6-ylamino)pyridin-2(1H)-one 258b

Compound 258a (2.18 g, 5 mmol) was suspended in dioxane (10 mL). A saturated solution of hydrogen chloride in dioxane (20 mL) was added dropwise. The reaction mixture was stirred for 20 minutes and then concentrated under reduced pressure to afford compound 258b, which was used in the next step without further purification. LC/MS: m/z 335 (M+H) ⁺

Example 258c 5-Bromo-1-methyl-3-(2-(oxetan-3-yl)-1,2,3,4-tetrahydroisoquinolin-6-ylamino)pyridin-2(1H)-one 258c

A mixture of 258b (1.7 g, 5 mmol), oxetane-3-one (1.8 g, 25 mmol), NaBH ₃ CN (1 g, 15 mmol), and zinc chloride (3.4 g, 25 mmol) in methanol (50 mL) was stirred at room temperature for 4 hours. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography eluting with 25:1 dichloromethane/methanol to afford 258c (1.8 g, 92%). LC/MS: m/z 391 (M+H) ⁺

Example 258d 4-Fluoro-2-(1-methyl-5-(2-(oxetan-3-yl)-1,2,3,4-tetrahydroisoquinolin-6-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 258d

To a 15 mL microwave reaction vial equipped with a magnetic stirrer was added 258c (0.39 g, 1 mmol), 4-fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 210d (0.48 g, 1 mmol), potassium phosphate (0.54 g, 2 mmol), sodium acetate (0.17 g, 2 mmol), acetonitrile (10 mL), water (1 mL), and 1,1'-bis(diphenylphosphino)ferrocenepalladium(II) dichloride in dichloromethane (0.08 g, 0.1 mmol). The reaction mixture was heated at 110°C under an argon atmosphere for 2 hours. Thereafter, the mixture was cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography using 40:1 dichloromethane/methanol as eluent to afford 258d (0.3 g, 50%). LC/MS: m/z 666 (M+H) ⁺

To a 25 mL round-bottom flask equipped with a magnetic stirrer was added 258d (0.6 g, 0.9 mmol), LiOH (2 g, 48 mmol), THF (5 mL), isopropanol (5 mL), and water (2 mL). The reaction mixture was stirred at room temperature for 30 minutes and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by preparative HPLC to afford 258. LC/MS: m/z 624 (M+H) ⁺ . ¹ H NMR(500MHz,DMSO)δ7.62(s,1H),7.82(s,1H),7.34(m,3H),7.20(m,1H),7.09(m,2H),6.94(m,1H),4.86(m,1H),4.96(m,1H),4.60(m,2 H),4.51(m,2H),4.31(m,2H),4.14(m,3H),3.87(m,1H),3.58(m,4H),3.33(m,3H),2.75(m,2H),2.60(m,3H),2.50(m,3H),1.78(m,4H).

Example 259 5-[2-(Hydroxymethyl)-3-[1-methyl-5-({5-methyl-4H,5H,6H,7H-pyrazolo[1,5-a]pyrazin-2-yl}amino)-6-oxo-1,6-dihydropyridin-3-yl]phenyl]-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-6-one 259

Example 259a {2-[1-methyl-5-({5-methyl-4H,5H,6H,7H-pyrazolo[1,5-a]pyrazin-2-yl}amino)-6-oxo-1,6-dihydropyridin-3-yl]-6-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]tridec-1(9),2(7)-dien-5-yl}phenyl}methyl acetate 259a

To a 25 mL sealed tube were added (2-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-5-yl}-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methyl acetate 111a (571 mg, 1.2 mmol), 5-bromo-1-methyl-3-(5-methyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)pyridin-2(1H)-one 146a (400 mg, 1.2 mmol), CH ₃ COONa (195 mg, 2.4 mmol), K ₃ PO ₄ (631 mg, 2.4 mmol), and PdCl ₂ (dppf) (97 mg, 0.12 mmol), and the mixture was suspended in CH ₃ CN (25 mL) and H ₂ O (1 mL). The mixture was heated at 110° C. for 2 h. It was then evaporated and the residue was purified by column chromatography using 20:1 dichloromethane/methanol as eluent to afford 259a (500 mg, 69%) as a brown solid. MS: (M+H) ⁺ 613.

To a solution of 259a (500 mg, 0.82 mmol) in propan-2-ol (10 mL), tetrahydrofuran (10 mL), and water (2 mL) was added LiOH (1.96 g, 82 mmol). The mixture was stirred at 30°C for 2 h. It was then evaporated and the residue was purified by preparative HPLC to afford 259 as a yellow solid (159 mg, 34%). MS: (M+H) ⁺ 571. ¹ H NMR(500MHz,DMSO)δ1.79(s,4H),2.35(s,2H),2.53-2.55(m,1H),2.76-2.79(m ,4H),2.84-2.88(m,1H),2.96-2.98(m,1H),3.48(s,2H),3.57(s,3H),3.87-3. 93(m,3H),3.99-4.03(m,1H),4.35(s,2H),4.81(s,1H),5.88(s,1H),7.23-7.2 4(d,2H),7.29-7.34(m,2H),7.43-7.46(d,1H),7.97-7.98(d,1H),8.10(s,1H).

Example 260 10-[5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-{[5-(1-methylpiperidin-4-yl)pyridin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^{2 ,6} ]dodeca-2(6),7-dien-9-one 260

Example 260a 10-[5-fluoro-2-(acetoxymethyl)-3-(1-methyl-5-{[5-(1-methylpiperidin-4-yl)pyridin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 260a

To a microwave tube equipped with a stir bar was added 5-bromo-1-methyl-3-(5-(1-methylpiperidin-4-yl)pyridin-2-ylamino)pyridin-2(1H)-one 130c (162 mg, 0.429 mmol), boronate ester 212b (347 mg, 0.700 mmol), Pd(PPh ₃ ) ₄ (24.8 mg, 0.022 mmol), aqueous Na ₂ CO ₃ solution (1.0 N, 1.42 mL, 1.42 mmol), and 1,2-dimethoxyethane (4.0 mL). The mixture was reacted in a microwave at 130° C. for 15 min. Dichloromethane (200 mL) was added, and the resulting mixture was washed with water (3×30 mL), brine (30 mL×1), dried over MgSO ₄ , filtered, and the solvent removed in vacuo. Silica gel column chromatography (methanol:dichloromethane=5:95) gave 260a.

To a round-bottom flask equipped with a stir bar was added 260a, THF (5 mL), i-PrOH (5 mL), _H₂O (5 mL), and LiOH/ _H₂O (300 mg). The resulting mixture was stirred at room temperature for 1 hr. All solvents were removed in vacuo, and the resulting residue was added to dichloromethane (200 mL). The solution was washed with water (3 x 30 mL), brine (30 mL), dried over _MgSO₄ , filtered, and the solvent was removed in vacuo. Silica gel column chromatography (MeOH:dichloromethane = 10:90) afforded 48 mg of the alcohol 260 as a yellow solid. MS (ESI+) m/z 625.5 (M+H).

Example 261 10-[5-fluoro-2-(hydroxymethyl)-3-{1-methyl-5-[(6-methyl-5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl)amino]-6-oxo-1,6-dihydropyridin-3-yl}phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 261

Example 261a 10-[5-fluoro-2-(acetoxymethyl)-3-{1-methyl-5-[(6-methyl-5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl)amino]-6-oxo-1,6-dihydropyridin-3-yl}phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 261a

In a 10-mL microwave reaction vessel equipped with a magnetic stir bar were added 5-bromo-1-methyl-3-(6-methyl-5,6,7,8-tetrahydro-1,6-naphthyridin-2-ylamino)pyridin-2(1H)-one 205b (150 mg, 0.43 mmol), 10-[2-(acetoxymethyl)-3-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-5-fluorophenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 230a (248 mg, 0.50 mmol), Pd(PPh ₃ ) ₄ (30 mg, 0.026 mmol), 2N Na ₂ CO ₃ (2 mL) and 1,2-dimethoxyethane (2 mL). After the reaction mixture was stirred at 125° C. for 10 min, it was purified by flash chromatography (dichloromethane:methanol, 3:1) to afford 36% (100 mg) of 261a as a solid.

To a 25-mL single-necked round-bottom flask equipped with a magnetic stir bar was added 261a (100 mg, 0.16 mmol), LiOH· _H₂O (200 mg, 4.8 mmol), THF (2 mL), isopropanol (2 mL), and water (2 mL). The reaction mixture was stirred at room temperature for 2 h, then partitioned between dichloromethane (5 mL) and water (5 mL), and the organic phase was extracted with dichloromethane (5 mL x 3 ₎ . The combined organic phases were washed with water (2 x 5 mL) and brine (5 mL), dried ( _Na₂SO₄ ), and then concentrated. The crude product was redissolved in dichloromethane (3 mL). Hexane (10 mL) was added to this solution, and the resulting precipitate was filtered to provide 261 in a 36% yield (34 mg). MS ( ^ESI⁺ ) m/z 597.5 (M+H).

Example 262 10-[3-(5-{[5-(1-ethylazetidin-3-yl)pyridin-2-yl]amino}-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 262

Examples 262a and 262b 262b was prepared according to Example 271a using 250a (305 mg, 0.84 mmol) in step 1 and 189a (302 mg, 0.67 mmol) in step 2 of Example 271b. The product was purified by column chromatography (ISCO 12 g silica gel, 0-10% MeOH) to give the desired product 262b (105 mg, 19% yield).

According to Example 119, 262a (105 mg, 0.36 mmol), 1N LiOH (0.8 mL), THF (2 mL) and isopropanol (2 mL) were reacted to give 262 (85 mg, 87% yield). MS (ESI+) m/z 611.5 (M+H).

Example 263 2-(5-fluoro-2-(hydroxymethyl)-3-(5-(2-methoxypyrimidin-4-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 263

Example 263a 5-Bromo-3-(2-methoxypyrimidin-4-ylamino)-1-methylpyridin-2(1H)-one 263a

Following Example 112a, 2-methoxypyrimidin-4-amine (0.625 g, 5 mmol), 3,5-dibromo-1-methyl-1H-pyridin-2-one (1.34 g, 5 mmol), cesium carbonate (4.88 g, 15 mmol), tris(dibenzylideneacetone)dipalladium(0) (0.465 g, 0.5 mmol), Xantphos (0.58 g, 1 mmol), and 1,4-dioxane (50 mL) were heated at 100° C. for 24 hours. The mixture was cooled to room temperature and filtered through a pad of Celite 521. The filter cake was washed with 9:1 dichloromethane/methanol (2×25 mL), and the combined filtrates were concentrated to dryness. The residue was dissolved in dichloromethane, diethyl ether was added, and the resulting precipitate was filtered to provide 263a as a green solid in quantitative yield (1.57 g): MS (ESI+) m/z 313.1 (M+H).

Example 263b 4-Fluoro-2-(5-(2-methoxypyrimidin-4-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 263b

[0263] Following Example 121b, 263a (158 mg, 0.5 mmol), 4-fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 210d (242 mg, 0.5 mmol), 1 M sodium carbonate solution (2 mL, 2 mmol), tetrakis(triphenylphosphine)palladium(0) (29 mg, 0.025 mmol) and 1,2-dimethoxyethane (5 mL) were heated in a microwave reactor at 130 °C for 15 minutes. Workup and flash column chromatography (silica gel, 60:35:5 dichloromethane/ether/methanol) afforded 263b as a yellow oil in 98% yield (290 mg): MS (ESI+) m/z 587.6 (M+H).

Following Example 121, a mixture of THF (2 mL), water (1 mL), and isopropanol (2 mL), 263b (290 mg, 0.5 mmol), and lithium hydroxide monohydrate (105 mg, 2.5 mmol) were reacted. Workup and flash column chromatography (NH-silica gel, ethyl acetate/hexanes) afforded 263 as a solid in 26% yield (70 mg): MS (ESI+) m/z 545.1 (M+H).

Example 264 10-[5-fluoro-2-(hydroxymethyl)-3-[1-methyl-5-({5-[1-(oxetan-3-yl)piperidin-4-yl]pyridin-2-yl}amino)-6-oxo-1,6-dihydropyridin-3-yl]phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 264

Example 264a tert-Butyl 4-(6-(5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)pyridin-3-yl)piperidine-1-carboxylate 264a

Following Example 112a, tert-butyl 4-(6-aminopyridin-3-yl)piperidine-1-carboxylate (1.9 g, 5.5 mmol), 3,5-dibromo-1-methylpyridin-2(1H)-one (1.47 g, 5.5 mmol), cesium carbonate (5.36 g, 16.5 mmol), tris(dibenzylideneacetone)dipalladium(0) (0.53 g, 0.55 mmol), Xantphos (0.63 g, 1.1 mmol), and 1,4-dioxane (50 mL) were heated at 100°C for 8 hours. Workup followed by concentration to dryness. The resulting solid was washed with diethyl ether and filtered to afford 264a as a gray solid in a 56% yield (1.4 g): MS (ESI+) m/z 465.2 (M+H).

Example 264b 5-Bromo-1-methyl-3-(5-(piperidin-4-yl)pyridin-2-ylamino)pyridin-2(1H)-one 264b

Following Example 121c, 264a (0.58 g, 1.25 mmol), trifluoroacetic acid (0.96 mL, 12.5 mmol), and dichloromethane (20 mL) were reacted. Workup and concentration afforded 264b as a yellow oil in quantitative yield (454 mg), which was used in the next step without purification. MS (ESI+) m/z 365.2 (M+H).

Example 264c 5-Bromo-1-methyl-3-(5-(1-(oxetan-3-yl)piperidin-4-yl)pyridin-2-ylamino)pyridin-2(1H)-one 264c

Following Example 214b, 264b (450 mg, 1.25 mmol) and oxetan-3-one (800 mg, 11.1 mmol) were mixed in methanol (10 mL). Sodium cyanoborohydride (236 mg, 3.75 mmol) and zinc chloride (256 mg, 1.88 mmol) in methanol (10 mL) were added, and the reaction was heated at 48°C for 12 hours. Workup and flash column chromatography (silica gel, 60:35:5 dichloromethane/ether/methanol) afforded 264c as a light green solid in a 30% yield (160 mg): MS (ESI+) m/z 421.1 (M+H).

Following Example 121b, 264c (160 mg, 0.38 mmol), 230a (188 mg, 0.38 mmol), 1 M sodium carbonate solution (1.5 mL, 1.5 mmol), tetrakis(triphenylphosphine)palladium(0) (22 mg, 0.019 mmol), and 1,2-dimethoxyethane (3.5 mL) were heated in a microwave reactor at 130°C for 15 minutes. Workup and flash column chromatography (silica gel, 9:1 dichloromethane/methanol) afforded a mixture of compounds 264c and 264 (110 mg) as a yellow oil. The mixture (110 mg) was deprotected using the same procedure as for 121, except that a mixture of THF (1 mL), water (0.5 mL), and isopropanol (1 mL) and lithium hydroxide monohydrate (50 mg, 1.2 mmol) were used. After workup and flash column chromatography (NH-silica gel, ethyl acetate/hexane), Compound 264 was obtained as a yellow solid in 56% yield (60 mg): MS (ESI+) m/z 667.6 (M+H).

Example 265 2-(5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-(5-(1-(oxetan-3-yl)piperidin-4-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 265

Example 265a 4-Fluoro-2-(1-methyl-5-(5-(1-(oxetan-3-yl)piperidin-4-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 265a

Following Example 121b, 264c (210 mg, 0.5 mmol), 4-fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 210d (242 mg, 0.5 mmol), 1 M sodium carbonate solution (2 mL, 2 mmol), tetrakis(triphenylphosphine)palladium(0) (29 mg, 0.025 mmol) and 1,2-dimethoxyethane (5 mL) were heated in a microwave reactor at 130 °C for 15 minutes. Workup and flash column chromatography (silica gel, 9:1 dichloromethane/methanol) afforded 265a in 69% yield (240 mg) as a brown oil: MS (ESI+) m/z 695.5 (M+H).

Following Example 121, a mixture of THF (2 mL), water (1 mL), and isopropanol (2 mL), 265a (240 mg, 0.35 mmol), and lithium hydroxide monohydrate (100 mg, 2.5 mmol) were reacted. Workup and flash column chromatography (NH-silica gel, ethyl acetate/hexanes) afforded 265 as a yellow solid in a 30% yield (70 mg): MS (ESI+) m/z 653.6 (M+H).

Example 266 10-[5-fluoro-2-(hydroxymethyl)-3-{4-methyl-6-[(2-methyl-1,2,3,4-tetrahydroisoquinolin-6-yl)amino]-5-oxo-4,5-dihydropyrazin-2-yl}phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 266

Example 266a 5-Bromo-1-methyl-3-(2-methyl-1,2,3,4-tetrahydroisoquinolin-6-ylamino)pyridin-2(1H)-one 266a.

To a 100 mL round-bottom flask equipped with a magnetic stirrer was added 258b (350 mg, 0.8 mmol), formaldehyde (37% aqueous solution, 4.1 mmol), _NaBH₃CN (150 mg, 2.4 mmol), _ZnCl₂ (170 mg, 1.2 mmol), and methanol (46 mL). The mixture was stirred at room temperature for 16 h. Thereafter, the mixture was concentrated under reduced pressure and added with 10% _{aqueous K₂CO₃} (25 mL). The desired product precipitated from the mixture and was then filtered. The product was washed with diethyl ether (20 mL) to afford 266a in an 85% yield (240 mg).

Example 266b 10-[5-fluoro-2-(acetoxymethyl)-3-{4-methyl-6-[(2-methyl-1,2,3,4-tetrahydroisoquinolin-6-yl)amino]-5-oxo-4,5-dihydropyrazin-2-yl}phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 266b

To a microwave tube equipped with a magnetic stirrer was added 266a (240 mg, 0.7 mmol), 210d (460 mg, 0.9 mmol), 1,2-dimethoxyethane (8 mL), and 1 M aqueous sodium carbonate solution (2 mL). After blowing _N2 for 15 min, Pd( _PPh3 ) ₄ (40 mg, 0.03 mmol) was added. The mixture was heated to 120°C in a microwave for 10 min. Thereafter, ethyl acetate (5 mL) and water (5 mL) were added. The separated aqueous layer was extracted with ethyl acetate (2 x 5 mL). The combined organic phases were washed with brine (10 mL), dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by column chromatography eluting with a gradient of dichloromethane to 9:1 dichloromethane:methanol to afford 266b in 27% yield (120 mg).

To a 25 mL round-bottom flask equipped with a magnetic stirrer was added 266b (120 mg, 0.2 mmol), lithium hydroxide (40 mg, 0.9 mmol), THF (1 mL), isopropanol (1 mL) and water (2 mL). The mixture was stirred at rt for 30 min. Thereafter, ethyl acetate (5 mL) and water (5 mL) were added. The separated aqueous layer was extracted with ethyl acetate (2 x 5 mL). The combined organic phases were washed with brine (10 mL), dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by column chromatography using a Biotage KPNH 12+M column eluted with a gradient of hexane-ethyl acetate to give 266 in 45% yield (50 mg). MS (ESI+) m/z 597.4 (M+H).

Example 267 : 5-[5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-{[5-(1-methylpiperidin-4-yl)pyridin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)phenyl]-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-diene-6-one 267

Example 267a [4-Fluoro-2-(1-methyl-5-{[5-(1-methylpiperidin-4-yl)pyridin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)-6-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]tridec-1(9),2(7)-dien-5-yl}phenyl]methyl acetate 267a

To a sealed tube was added a mixture of 5-bromo-1-methyl-3-(5-(1-methylpiperidin-4-yl)pyridin-2-ylamino)pyridin-2(1H)-one 130c (400 mg, 1.0 mmol), (4-fluoro-2-{6-oxo-8-thia-5-azatricyclo[ ^7.4.0.02,7 ]trideca-1(9),2(7)-dien-5-yl}-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methyl acetate 247b (534 mg, 1.0 mmol), Pd(dppf) _Cl2 ( _{87 mg, 0.1 mmol), K3PO4.3H2O ( 569} mg, 2.0 mmol) and NaOAc (175 mg, 2.0 mmol) in _CH3CN (20 mL). The system was evacuated and then refilled with N ₂ . The reaction mixture was heated at 110° C. for 2 h. The mixture was then cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by column chromatography using 10:1 dichloromethane/methanol to afford 267a (350 mg, 49%) as a brown solid. MS: [M+H] ⁺ 670.

To a solution of 267a (100 mg, 0.15 mmol) in propan-2-ol (5 mL), tetrahydrofuran (5 mL), and water (1 mL) was added LiOH (358 mg, 15 mmol). The mixture was stirred at 30°C for 2 h. It was then evaporated and the residue was purified by preparative HPLC to afford 267 as a yellow solid (18 mg, 19%). MS: (M+H) ⁺ 628. ¹ H NMR(500MHz,MEOD)δ1.83-1.96(m,8H),2.53-2.66(m,8H),2.85(t,2H),2.92-2.97(m,1H),3.04-3.07(m,1H),3.23-3.26(d,2H),3.98-4.0 1(m,1H),4.12-4.15(m,1H),4.48-4.55(m,2H),7.05(d,1H),7.20-7. 23(m,2H),7.39(d,1H),7.55-7.57(m,1H),8.12(s,1H),8.65(s,1H).

Example 268 5-{3-[5-({5-[2-(dimethylamino)ethoxy]pyridin-2-yl}amino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl]-5-fluoro-2-(hydroxymethyl)phenyl}-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-6-one 268

Example 268a N,N-dimethyl-2-(6-nitropyridin-3-yloxy)ethanamine 268a

A mixture of 6-nitropyridin-3-ol (0.5 g, 3.57 mmol), 2-chloro-N,N-dimethyl-ethanamine hydrochloride (0.61 g, 4.29 mmol), and Cs ₂ CO ₃ (2.56 g, 7.85 mmol) in DMF (7 mL) was stirred at 120°C for 15 hours in a sealed tube. The mixture was filtered, and the filtrate was worked up with ethyl acetate and water. The aqueous phase was extracted with ethyl acetate. The combined extracts were dried over sodium sulfate and then concentrated under reduced pressure. The residue was purified on a flash column to afford 268a (0.46 g, 61%). MS: [M+H] ⁺ 212.

Example 268b 5-(2-(Dimethylamino)ethoxy)pyridin-2-amine 268b

A 100-mL Parr hydrogenation bottle was purged with nitrogen and charged with 268a (0.92 g, 4.36 mmol), 10% palladium on carbon (50% humidity, 0.2 g), and methanol (30 mL). The bottle was evacuated, filled with hydrogen to 25 psi, and shaken on a Parr hydrogenation apparatus for 2 h. The hydrogen was then removed and nitrogen was introduced into the bottle. The catalyst was removed by filtration through a pad of Celite, and the filtrate was concentrated under reduced pressure to afford 268b (0.66 g, 83%). MS: [M+H] ⁺ 182.

Example 268c 5-Bromo-3-(5-(2-(dimethylamino)ethoxy)pyridin-2-ylamino)-1-methyl-pyridin-2(1H)-one 268c

A mixture of 268b (570 mg, 3.15 mmol), XantPhos (109 mg, 0.19 mmol), Pd ₂ dba ₃ (230 mg, 0.25 mmol), 3,5-dibromo-1-methylpyridin-2(1H)-one (918 mg, 3.46 mmol), and Cs ₂ CO ₃ (2.05 g, 6.3 mmol) in 1,4-dioxane (20 mL) was heated under reflux for 2 h. After completion of the reaction, the mixture was filtered and then washed with methanol (100 mL). The filtrate was evaporated in vacuo. The residue was purified by reverse phase Combi-flash chromatography to afford 268c (922 mg, 80%). MS: [M+H] ⁺ 367.

Example 268d {2-[5-({5-[2-(dimethylamino)ethoxy]pyridin-2-yl}amino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl]-4-fluoro-6-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-5-yl}phenyl}methyl acetate 268d

A mixture of 268c (360 mg, 0.99 mmol), (4-fluoro-2-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-5-yl}-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methyl acetate 212b (494 mg, 0.99 mmol), PdCl ₂ (dppf) (86 mg, 0.12 mmol), K ₃ PO ₄ (150 mg) and NaOAc (50 mg) in MeCN (6 mL) and water (2 mL) was heated in a sealed tube at 110° C. for 2 h. The solvent was evaporated in vacuo. The residue was purified by reverse phase Combi flash chromatography to give 268d (367 mg, 60%). MS: [M+H] ⁺ 660.

A mixture of 268d (700 mg, 1.06 mmol) and LiOH hydrate (446 mg, 10.6 mmol) in isopropanol (15 mL) and water (5 mL) was stirred at 30°C for 2 h. The mixture was evaporated in vacuo, and the residue was extracted with ethyl acetate (2 x 20 mL). The combined extracts were concentrated under reduced pressure. The residue was purified by preparative HPLC to give 268 (94 mg, 14%). MS: [M+H] ⁺ 618. ¹ H NMR (500MHz, CDCl3) δ8.54 (d, J = 2.0, 1H), 7.95 (d, J = 3.0, 1H), 7.80 (s, 1H), 7.48 (d, J = 2. 5,1H),7.23-7.21(m,1H),7.16-7.14(m,1H),6.98-6.96(m,1H),6.81(d,J＝9.0,1H),4.57 (d,J＝11.5,1H),4.30(t,J＝10.5,1H),4.20-4.02(m,4H),3.87-3.80(m,1H),3.69(s,3H), 3.00-2.84(m,4H),2.75-2.73(m,2H),2.59-2.48(m,2H),2.37(s,6H),1.94-1.83(m,4H).

Example 269 5-[3-(5-{[5-(4-ethylpiperazin-1-yl)pyridin-2-yl]amino}-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl]-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-6-one 269

Example 269a 5-[3-(5-{[5-(4-ethylpiperazin-1-yl)pyridin-2-yl]amino}-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl]-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-6-one 269a

To a microwave tube equipped with a stir bar was added 5-bromo-3-(5-(4-ethyl-piperazin-1-yl)pyridin-2-ylamino)-1-methylpyridin-2(1H)-one 138c (300 mg, 0.765 mmol), boronate ester 212b (420.1 mg, 0.841 mmol), Pd(PPh ₃ ) ₄ (44.2 mg, 0.038 mmol), aqueous Na ₂ CO ₃ solution (1.0 N, 2.52 mL, 2.52 mmol), and 1,2-dimethoxyethane (4.0 mL). The mixture was reacted in a microwave at 130° C. for 15 min. Dichloromethane (200 mL) was added, and the resulting mixture was washed with water (3×30 mL), brine (30 mL), dried over MgSO ₄ , filtered, and the solvent removed in vacuo. Silica gel column chromatography (methanol:dichloromethane=5:95) gave acetate 269a.

To a round-bottom flask equipped with a stir bar was added 269a, THF (5 mL), isopropanol (5 mL), water (5 mL), and _LiOHH₂O (300 mg). The resulting mixture was stirred at room temperature for 1 hr. The solvent was removed in vacuo, and the resulting residue was added to dichloromethane (200 mL). The solution was washed with water (3 x 30 mL), brine (30 mL), dried over _MgSO₄ , filtered, and the solvent was removed in vacuo. Column chromatography on silica gel (methanol:dichloromethane = 10:90) afforded 158 mg of 269 as a light brown solid. MS (ESI+) m/z 643.4 (M+H).

Example 270 3-(5-((2-(dimethylamino)ethyl)(methyl)amino)pyridin-2-ylamino)-5-(5-fluoro-2-(hydroxymethyl)-3-(6,6-dimethyl-3,4,6,7-tetrahydro-5H-cyclopenta[4,5]thieno[2,3-c]pyridin-1(2H)-yl)phenyl)-1-methylpyridin-2(1H)-one 270

Example 270a N ¹ ,N ¹ ,N ² -trimethyl-N ² -(6-nitropyridin-3-yl)ethane-1,2-diamine 270a

To a 100-mL round-bottom flask equipped with a magnetic stirrer was added 1e (3.00 g, 14.8 mmol), N ¹ , N ¹ , N ² -trimethylethane-1,2-diamine (2.26 g, 22.2 mmol), and N,N-dimethylacetamide (10 mL). N,N-diisopropylethylamine (3.69 g, 28.6 mmol) and tetrabutylammonium iodide (8.18 g, 22.8 mmol) were added, and the reaction mixture was stirred at 90°C for 14 h. Afterwards, the reaction was allowed to cool to room temperature and then poured into water (20 mL). The resulting mixture was extracted with ethyl acetate (100 mL). The organic layer was separated and then dried over sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to give 75% yield (2.50 g) of yellow oil 270a: ¹ H NMR (300 MHz, CDCl ₃ ) δ 8.15 (d, 1H, J=9.1 Hz), 7.98 (d, 1H, J=3.2 Hz), 7.00 (dd, 1H, J=9.1, 3.2 Hz), 3.58 (t, 1H, J=6.9 Hz), 3.14 (s, 3H), 2.52 (t, 2H, J=7.1 Hz), 2.29 (s, 6H); MS (ESI+) m/z 225.1 (M+H).

Example 270b N ⁵ -(2-(Dimethylamino)ethyl)-N ⁵ -methylpyridine-2,5-diamine 270b

A 250-mL Parr reaction bottle was purged with nitrogen and a solution of 10% palladium on carbon (50% humidity, 1.00 g dry weight) and 270a (2.20 g, 9.80 mmol) in ethanol (150 mL) was added. The bottle was connected to a Parr hydrogenation reactor, evacuated, filled with hydrogen to a pressure of 50 psi and shaken for 3 h. Thereafter, the hydrogen was removed and nitrogen was introduced into the bottle. Celite 521 (1.00 g) was added and the mixture was then filtered through a Celite 521 pad. The filter cake was washed with ethanol (2×25 mL), and the combined filtrates were concentrated to dryness under reduced pressure to afford 270b (1.91 g) as a purple oil in quantitative yield: ¹ H NMR (500 MHz, CDCl ₃ ) 7.67 (d, 1 H, J=2.9 Hz), 7.05 (dd, 1 H, J=8.5, 2.9 Hz), 6.48 (dd, 1 H, J=8.9, 0.5 Hz), 4.04 (br s, 2 H), 3.28 (t, 2 H, J=7.1 Hz), 2.84 (s, 3 H), 2.43 (t, 2 H, J=7.4 Hz), 2.26 (s, 6 H); MS (ESI+) m/z 195.2 (M+H).

Example 270c 5-Bromo-3-(5-((2-(dimethylamino)ethyl)(methyl)amino)pyridin-2-ylamino)-1-methylpyridin-2(1H)-one 270c

270b (1.00 g, 5.20 mmol), 3,5-dibromo-1-methylpyridine-2 (1H) -one (1.53 g, 5.72 mmol), cesium carbonate (5.09 g, 15.6 mmol) and 1,4-dioxane (52 mL) were added to a 100-mL double-necked round-bottom flask equipped with a reflux condenser, a magnetic stirrer and a nitrogen inlet. After nitrogen was bubbled through the resulting suspension for 30 min, Xantphos (257 mg, 0.442 mmol) and tris (dibenzylideneacetone) dipalladium (0) (239 mg, 0.260 mmol) were added and the reaction mixture was heated at reflux for 3 h. After this, the mixture was cooled to room temperature and diluted with ethyl acetate (150 mL) and water (30 mL). The organic layer was separated and the aqueous layer was extracted with ethyl acetate (3 × 150 mL). The combined organic layer was dried over sodium sulfate and then concentrated under reduced pressure. The residue was triturated with methanol (20 mL) to give 270c as an off-white solid in 63% yield (700 mg): mp 161-163 °C; ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 8.50 (d, 1H, J=2.3 Hz), 8.39 (s, 1H), 7.75 (d, 1H, J=2.4 Hz), 7.40 (d, 1H, J=2.4 Hz), 7.17 (m, 2H), 3.50 (s, 3H), 3.56 (t, 2H, J=6.9 Hz), 2.86 (s, 3H), 2.35 (t, 2H, J=7.1 Hz), 2.16 (s, 6H); MS (ESI+) m/z 380.1 (M+H).

Example 270d 3-(5-((2-(dimethylamino)ethyl)(methyl)amino)pyridin-2-ylamino)-1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one 270d

270c (500 mg, 1.32 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-di-1,3,2-dioxaborolane (668 mg, 2.63 mmol), potassium acetate (389 mg, 3.96 mmol) and 1,4-dioxane (10 mL) were added to a 100-mL single-necked round-bottom flask equipped with a reflux condenser, a magnetic stirrer and a nitrogen inlet. After nitrogen was bubbled through the resulting suspension for 30 min, [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (97 mg, 0.132 mmol) was added and the reaction mixture was heated under reflux for 1 h. Afterwards, the mixture was cooled to room temperature and diluted with ethyl acetate (150 mL) and water (30 mL). The organic layer was separated and the aqueous layer was extracted with ethyl acetate (3 x 150 mL). The combined organic layers were dried over sodium sulfate and then concentrated under reduced pressure to afford the crude product 270d in quantitative yield. The residue was used without further purification.

To a 50-mL three-necked round-bottom flask equipped with a reflux condenser, a magnetic stirrer, and a nitrogen inlet was added 247a (420 mg, 0.901 mmol), 270d (290 mg, 0.690 mmol), sodium carbonate (220 mg, 2.07 mmol), water (2 mL), and 1,4-dioxane (8 mL). After bubbling nitrogen through the resulting suspension for 30 min, tetrakis(triphenylphosphine)palladium(0) (80 mg, 0.069 mmol) was added, and the reaction mixture was heated under reflux for 2 h. Thereafter, the mixture was cooled to room temperature and diluted with ethyl acetate (150 mL) and water (30 mL). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (3×150 mL). The combined organic layers were dried over sodium sulfate and then concentrated under reduced pressure. The residue was dissolved in a mixture of THF (10 mL), methanol (10 mL), and water (10 mL). Lithium hydroxide monohydrate (420 mg, 10.0 mmol) was added to the resulting solution. The mixture was stirred at room temperature for 2 h and then concentrated in vacuo. The residue was partitioned between ethyl acetate (150 mL) and water (30 mL). The organic layer was separated and the aqueous layer was extracted with a 20% (v/v) solution of methanol/dichloromethane (3×150 mL). The combined organic layers were dried over sodium sulfate and then concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, 0%-20% methanol/dichloromethane) to give a 14% yield (53 mg) of an off-white solid 270: mp 106–107°C; ¹ H NMR (500 MHz, DMSO-d ₆ )δ8.45(d,1H,J＝2.5Hz),8.20(s,1H),7.69(d,1H,J＝2.5Hz),7.32(m,2H ),7.17(m,3H),4.84(t,1H,J＝4.0Hz),4.33(m,2H),4.04(m,1H),3.84(m ,1H),3.58(s,3H),3.03(m,1H),2.89(m,1H),2.75(s,2H),2.53(d,2H,J ＝8.9Hz),2.32(t,2H,J＝7.2Hz),2.14(s,6H),1.23(s,6H); MS(ESI+)m/z 645.3(M+H).

Example 271 10-[5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-{[5-(1-methylazetidin-3-yl)pyridin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 271

Example 271a 10-[5-fluoro-2-(acetoxymethyl)-3-(1-methyl-5-{[5-(1-methylazetidin-3-yl)pyridin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 271a

To a microwave tube equipped with a magnetic stirrer was added 5-bromo-1-methyl-3-(5-(1-methylazetidin-3-yl)pyridin-2-ylamino)pyridin-2(1H)-one (172a) (216 mg, 0.6 mmol), 230a (390 mg, 0.8 mmol), 1,2-dimethoxyethane (8 mL) and 1 M aqueous sodium carbonate solution (2 mL). After bubbling _N for 15 min, Pd(PPh ₃ ) ₄ (36 mg, 0.03 mmol) was added. The mixture was heated to 130° C. in a microwave for 15 min. Thereafter, ethyl acetate (5 mL) and water (5 mL) were added. The separated aqueous layer was extracted with ethyl acetate (2 x 5 mL). The combined organic phase was washed with brine (10 mL), dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by column chromatography eluting with a gradient of dichloromethane to 9:1 dichloromethane:methanol to afford 271a in 30% yield (120 mg).

271a (110 mg, 0.2 mmol), lithium hydroxide (40 mg, 0.9 mmol), THF (1 mL), isopropyl alcohol (1 mL) and water (2 mL) were added to a 25 mL round-bottom flask equipped with a magnetic stirrer. The mixture was stirred for 30 min at rt. Afterwards, ethyl acetate (5 mL) and water (5 mL) were added. The separated aqueous layer was extracted with ethyl acetate (2 x 5 mL). The combined organic phase was washed with brine (10 mL), dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by column chromatography using a Biotage KPNH 12+M column with a gradient elution of hexane-ethyl acetate to obtain 271 in 88% yield (94 mg). MS (ESI+) m/z 597.4 (M+H).

Example 272 10-[3-(5-{[5-(4-ethylpiperazin-1-yl)pyridin-2-yl]amino}-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl]-4,4-dimethyl-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-9-one 272

Example 272a 10-[3-(5-{[5-(4-ethylpiperazin-1-yl)pyridin-2-yl]amino}-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(acetoxymethyl)phenyl]-4,4-dimethyl-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-9-one 272a

To a microwave tube equipped with a stir bar were added 182b (270 mg, 0.579 mmol), 3-(5-(4-ethylpiperazin-1-yl)pyridin-2-ylamino)-1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one 201a (382 mg, 0.868 mmol), Pd(PPh ₃ ) ₄ (34 mg, 0.029 mmol), aqueous Na ₂ CO ₃ solution (1.0 N, 1.91 mL, 1.91 mmol), and 1,2-dimethoxyethane (4.0 mL). The mixture was reacted in a microwave at 130° C. for 15 min. Dichloromethane (200 mL) was added, and the resulting mixture was washed with water (30 mL x 3), brine (30 mL x 1), dried over MgSO ₄ , filtered, and the solvent was removed in vacuo. Silica gel column chromatography (methanol:dichloromethane=5:95) afforded 272a.

To a round-bottom flask equipped with a stir bar was added 272a, THF (5 mL), isopropanol (5 mL), water (5 mL), and _LiOHH₂O (300 mg). The resulting mixture was stirred at room temperature for 1 hr. The solvent was removed in vacuo, and the resulting residue was added to dichloromethane (200 mL). The solution was washed with water (30 mL x 3), brine (30 mL x 1), dried over _MgSO₄ , filtered, and the solvent was removed in vacuo. Silica gel column chromatography (methanol:dichloromethane = 10:90) afforded 143 mg of 272 as a yellow solid. MS (ESI+) m/z 657.6 (M+H).

Example 273 10-{5-fluoro-3-[5-({5-[4-(2-fluoroethyl)piperazin-1-yl]pyridin-2-yl}amino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl]-2-(hydroxymethyl)phenyl}-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 273

Example 273b 10-{5-fluoro-3-[5-({5-[4-(2-fluoroethyl)piperazin-1-yl]pyridin-2-yl}amino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl]-2-(acetoxymethyl)phenyl}-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 273b

Following Example 186f, 273a (prepared in situ from 217d using conditions similar to those used to prepare 270d) (200 mg, 0.49 mmol) and 189a (176 mg, 0.39 mmol) were reacted to give 273b (115 mg, 33% yield).

Following Example 186, 273b (115 mg, 0.16 mmol), 1N LiOH (0.82 mL), THF (3 mL), and isopropanol (3 mL) were reacted and purified by HPLC (acetonitrile/water/TFA). To the residue were added ethyl acetate and saturated aqueous sodium bicarbonate solution, and the mixture was stirred for ~15 min. The layers were separated, and the organic phase was washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to afford 273 (23 mg, 22% yield). MS (ESI+) m/z 658.5 (M+H).

Example 274 10-[5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-{[5-(morpholin-4-ylmethyl)pyridin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 274

Example 274a 5-Bromo-1-methyl-3-(5-(morpholinomethyl)pyridin-2-ylamino)pyridin-2(1H)-one 274a

According to Example 186d, Compound 274a was prepared.

Example 274c 10-[5-fluoro-2-(acetoxymethyl)-3-(1-methyl-5-{[5-(morpholin-4-ylmethyl)pyridin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 274c

Following Examples 186e and 186f, 274b was obtained in situ from 274a (300 mg, 0.79 mmol) in step 1 and then reacted with 189a (284 mg, 0.63 mmol) in step 2 to give 274c (250 mg, 46% yield).

According to Example 119, 274c (250 mg, 0.36 mmol), 1N LiOH (1.5 mL), THF (3 mL), and isopropanol (3 mL) were reacted and purified by column chromatography (ISCO 12 g silica gel, 0-10% methanol), followed by powdering with dichloromethane/diethyl ether to afford 274 (89 mg, 39% yield). MS (ESI+) m/z 627.5 (M+H).

Example 275 2-(2-(Hydroxymethyl)-3-(1-methyl-5-(5-(4-methylpiperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrido[3,4-b]indolizine-1(2H)-one 275

To a 100-mL single-necked round-bottom flask equipped with a magnetic stirrer and a nitrogen inlet was added 197e (210 mg, 0.560 mmol), 118f (330 mg, 0.710 mmol), sodium carbonate (175 mg, 1.70 mmol), water (2 mL) and 1,4-dioxane (8 mL). After bubbling nitrogen through the resulting suspension for 30 min, tetrakis(triphenylphosphine)palladium(0) (64 mg, 0.055 mmol) was added. A reflux condenser was connected to the flask, and the reaction mixture was heated at 100°C for 2 h. Thereafter, the mixture was diluted with 90:10 dichloromethane/methanol (100 mL) and water (75 mL), and the layers were separated. The aqueous layer was extracted with 90:10 dichloromethane/methanol (2×50 mL), and the combined organic extracts were washed with brine (100 mL) and then dried over sodium sulfate. The desiccant was removed by filtration. The filtrate was concentrated under reduced pressure, and the resulting residue was dissolved in a mixture of THF (3 mL), water (3 mL), and methanol (3 mL). Lithium hydroxide monohydrate (100 mg, 2.40 mmol) was added, and the reaction was stirred at room temperature for 2 h. Thereafter, the mixture was diluted with 90:10 dichloromethane/methanol (100 mL) and water (50 mL), and the layers were separated. The aqueous layer was extracted with 90:10 dichloromethane/methanol (2×75 mL), and the combined organic extracts were dried over sodium sulfate. The drying agent was removed by filtration. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, 0%-10% methanol/dichloromethane) to give an 8% yield (35 mg) of a non-crystalline off-white solid 275: mp 180–181°C; ¹ H NMR (500 MHz, DMSO-d ₆ )δ8.54(d,1H,J＝2.5Hz),8.31(s,1H),7.84(d,1H,J＝2.5Hz),7.42(t,1H,J＝8.0Hz),7.35(m,1H ),7.26(d,2H,J＝8.0Hz),7.19(d,1H,J＝9.0Hz),6.00(s,1H),4.69(t,1H,J＝5.0Hz),4.31(d,2H, J＝4.5Hz),3.99(m,1H),3.95(m,1H),3.88(m,2H),3.57(s,3H),3.05(m,5H),2.90(m,1H),2.70( t,2H,J＝6.0Hz),2.42(m,5H),2.19(s,3H),1.91(m,2H),2.73(m,2H); MS(ESI+)m/z594.3(M+H).

Example 276 10-[5-fluoro-2-(hydroxymethyl)-3-{1-methyl-5-[(5-methyl-1H-pyrazol-3-yl)amino]-6-oxo-1,6-dihydropyridin-3-yl}phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 276

Example 276a 10-[5-fluoro-2-(hydroxymethyl)-3-{1-methyl-5-[(5-methyl-1H-pyrazol-3-yl)amino]-6-oxo-1,6-dihydropyridin-3-yl}phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 276a

To a microwave tube equipped with a magnetic stirrer was added 5-bromo-1-methyl-3-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2(1H)-one 112a (150 mg, 0.5 mmol), 230a (350 mg, 0.7 mmol), 1,2-dimethoxyethane (6.4 mL) and 1 M aqueous sodium carbonate solution (1.6 mL). After blowing _N for 15 min, Pd(PPh ₃ ) ₄ (31 mg, 0.03 mmol) was added. The mixture was heated to 130° C. in a microwave for 15 min. Thereafter, ethyl acetate (5 mL) and water (5 mL) were added. The separated aqueous layer was extracted with ethyl acetate (2 x 5 mL). The combined organic phase was washed with brine (10 mL), dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by column chromatography eluting with a gradient of dichloromethane to 60:35:5 dichloromethane:diethyl ether:methanol to afford 276a in 16% yield (50 mg).

276a (50 mg, 0.1 mmol), lithium hydroxide (18 mg, 0.4 mmol), THF (0.4 mL), isopropyl alcohol (0.4 mL) and water (1 mL) were added into a 25 mL round-bottom flask equipped with a magnetic stirrer. The mixture was stirred for 30 min at rt. After this, ethyl acetate (5 mL) and water (5 mL) were added. The aqueous layer separated by extraction with ethyl acetate (2 x 5 mL) was used. The combined organic phase was washed with salt water (10 mL), dried over sodium sulfate, and then filtered. The mixture was concentrated to near dryness under reduced pressure, and the desired product was precipitated and then filtered. 276 was obtained in 87% yield (40 mg) through washing with ether (10 mL). MS (ESI+) m/z 531.4 (M+H).

Example 277 5-[5-fluoro-2-(hydroxymethyl)-3-{1-methyl-5-[(5-{[methyl(propan-2-yl)amino]methyl}pyridin-2-yl)amino]-6-oxo-1,6-dihydropyridin-3-yl}phenyl]-8-thia-5-azatricyclo[7.4.0.0 ^{2 ,7} ]trideca-1(9),2(7)-dien-6-one 277

Example 277a 5-[5-fluoro-2-(acetoxymethyl)-3-{1-methyl-5-[(5-{[methyl(propan-2-yl)amino]methyl}pyridin-2-yl)amino]-6-oxo-1,6-dihydropyridin-3-yl}phenyl]-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-6-one 277a

Following Example 186, 186e (270 mg, 0.74 mmol) prepared in situ was reacted with 212a (267 mg, 0.59 mmol) in step 2. Purification by column chromatography (ISCO 12 g silica gel, 50-100% ethyl acetate/ _hexanes then 0-15% MeOH/ _CH2Cl2 ) gave 277a (270 mg, 55% yield).

According to Example 119, 277a (270 mg, 0.41 mmol), 1N LiOH (2.0 mL), THF (4 mL), and isopropanol (4 mL) were reacted and then powdered with diethyl ether to give 277 (185 mg, 73% yield). MS (ESI+) m/z 616.4 (M+H).

Example 278 10-[5-fluoro-2-(hydroxymethyl)-3-(4-methyl-6-{[4-(1-methylpiperidin-4-yl)phenyl]amino}-5-oxo-4,5-dihydropyrazin-2-yl)phenyl]-4,4-dimethyl-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-9-one 278

Example 278a 10-[5-fluoro)-3-(4-methyl-6-{[4-(1-methylpiperidin-4-yl)phenyl]amino}-5-oxo-4,5-dihydropyrazin-2-yl)benzyl acetate]-4,4-dimethyl-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-9-one 278a

Following Example 136d, 282c was reacted with (2-{4,4-dimethyl-9-oxo-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-10-yl}-4-fluoro-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methyl acetate 247b to afford 278a in 62% yield. LCMS: (M+H) ⁺ 684

278a was converted to 278 in 39% yield according to Example 136. LCMS: (M+H) ⁺ 642. ¹ H NMR(500MHz,DMSO)δ9.22(s,1H),7.90(s,1H),7.87(s,1H),7.48(s,1H),7.36 (dd,J＝9.5,1H),7.31(dd,J＝9,1H),7.16(s,1H),7.14(s,1H),4.46(m,2H),4. 05(m,2H),3.85(m,2H),3.54(s,3H),3.01(m,1H),2.92(m,3H),2.75(s,2H),2 .53(m,2H),2.44(m,1H),2.27(s,3H),2.13(m,2H),1.70(m,4H),1.22(s,6H).

Example 279 10-[5-fluoro-2-(hydroxymethyl)-3-[1-methyl-5-({5-methyl-4H,5H,6H,7H-pyrazolo[1,5-a]pyrazin-2-yl}amino)-6-oxo-1,6-dihydropyridin-3-yl]phenyl]-4,4-dimethyl-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-9-one 279

Example 279a (2-{4,4-dimethyl-9-oxo-7-thia-10-azatricyclo[6.4.0.0 ^2,6- dodeca-1(8),2(6)-dien-10-yl}-4-fluoro-6-[1-methyl-5-({5-methyl-4H,5H,6H,7H-pyrazolo[1,5-a]pyrazin-2-yl}amino)-6-oxo-1,6-dihydropyridin-3-yl]phenyl)methyl acetate 279a

To a 25 mL sealed tube were added 5-bromo-1-methyl-3-(5-methyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)pyridin-2(1H)-one 146a (340 mg, 1.0 mmol), (2-{4,4-dimethyl-9-oxo-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-10-yl}-4-fluoro-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methyl acetate 247b (480 mg, 1.0 mmol), CH ₃ COONa (168 mg, 2.0 mmol), K ₃ PO ₄ (546 mg, 2.0 mmol), PdCl ₂ (dppf) (84 mg, 0.1 mmol) was suspended in CH ₃ CN (25 mL) and water (1 mL). The mixture was heated at 110° C. for 2 h, evaporated, and the residue was purified by column chromatography using 15:1 dichloromethane/methanol as eluent to afford 279a (300 mg, 46%) as a brown solid. MS: (M+H) ⁺ 645.

To a solution of 279a (300 mg, 0.46 mmol) in propan-2-ol (10 mL), tetrahydrofuran (10 mL), and water (2 mL) was added LiOH (1.1 g, 57 mmol). The mixture was stirred at 30°C for 2 h. The residue was then evaporated and purified by preparative HPLC to afford 279 (99 mg, 35%) as a white solid. MS: (M+H) ⁺ 603. ¹ H NMR(500MHz,MEOD)δ1.29(d,6H),2.49(s,3H),2.59(d,2H),2.81(s,2H),2.94-2.97(m,3H),3.07-3.14(m,1H),3.64(s,2H),3.70(s,3H) ,3.97-3.99(m,1H),4.05-4.07(t,2H),4.12-4.15(m,1H),4.47-4.54(m,2H),5.89(s,1H),7.19-7.22(m,2H),7.26(d,1H),7.91(s,1H).

Example 280 10-[5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-{[6-(oxetane-3-yl)-5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)phenyl]-4,4-dimethyl-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-9-one 280

Example 280a (2-{4,4-dimethyl-9-oxo-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-10-yl}-4-fluoro-6-(1-methyl-5-{[6-(oxetane-3-yl)-5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)phenyl)methyl acetate 280a

To a 25 mL sealed tube were added 5-bromo-1-methyl-3-(6-(oxetan-3-yl)-5,6,7,8-tetrahydro-1,6-naphthyridin-2-ylamino)pyridin-2(1H)-one 219a (400 mg, 1.0 mmol), (2-{4,4-dimethyl-9-oxo-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-10-yl}-4-fluoro-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methyl acetate 247b (493 mg, 1.0 mmol), CH ₃ COONa (168 mg, 2.0 mmol), K ₃ PO ₄ (546 mg, 2.0 mmol), PdCl ₂ (dppf) (84 mg, 0.1 mmol) was suspended in CH ₃ CN (25 mL) and H ₂ O (1 mL). The mixture was heated at 110° C. for 2 h. The mixture was then evaporated and the residue was purified by column chromatography using 20:1 dichloromethane/methanol (20:1) to afford 280a (400 mg, 56%) as a brown solid. MS: (M+H) ⁺ 698.

To a solution of 280a (400 mg, 0.57 mmol) in propan-2-ol (10 mL), tetrahydrofuran (10 mL) and water (2 mL) was added LiOH (1.4 g, 57 mmol). The mixture was stirred at 30°C for 2 h. It was then evaporated and the residue was purified by preparative HPLC to give 280 as a white solid (52 mg, 14%). MS: (M+H) ⁺ 656. ¹ H NMR (500 MHz, MEOD) δ 1.29 (d, 6H), 2.56-2.63 (q, 2H), 2.70-2.72 (t, 2H), 2.81 (s, 2H), 2.93-2.99 (m, 3H), 3.08-3.12 (m, 1H), 3.47 (s, 2H), 3.72-3.74 (m, 4H), 3.96- 4.01(m,1H),4.13-4.18(m,1H),4.51-4.60(q,2H),4.68-4.71(t,2H),4.76-4.7 9(t,2H),6.89(d,1H),7.21-7.23(d,2H),7.34(d,1H),7.40(s,1H),8.78(s,1H).

Example 281 5-[5-Fluoro-2-(hydroxymethyl)-3-(4-methyl-6-{[4-(1-methylpiperidin-4-yl)phenyl]amino}-5-oxo-4,5-dihydropyrazin-2-yl)phenyl]-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-diene-6-one 281

Example 281a [4-Fluoro-2-(4-methyl-6-{[4-(1-methylpiperidin-4-yl)phenyl]amino}-5-oxo-4,5-dihydropyrazin-2-yl)-6-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-5-yl}phenyl]methyl acetate 281a

According to Example 136d, 282c) was reacted with (4-fluoro-2-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-5-yl}-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methyl acetate 212b to give 281a in 73% yield. LCMS: (M+H) ⁺ 670

281a was converted to 281 in 70% yield according to Example 136. LCMS: (M+H) ⁺ 628. ¹ H NMR(500MHz,DMSO)δ9.21(s,1H),7.87(s,1H),7.85(s,1H),7.47(s,1H),7.3 5(dd,J＝10,1H),7.30(dd,J＝9,1H),7.16(s,1H),7.14(s,1H),4.84(m,1H),4. 48(m,1H),4.40(m,1H),3.86(m,1H),3.53(s,3H),2.96(m,1H),2.87(m,4H),2 .78(m,2H),2.42(m,2H),2.19(s,3H),1.96(m,2H),1.80(m,4H),1.62(m,5H).

Example 282 2-(5-fluoro-2-(hydroxymethyl)-3-(4-methyl-6-(4-(1-methylpiperidin-4-yl)phenylamino)-5-oxo-4,5-dihydropyrazin-2-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 282

Example 282a tert-Butyl 4-(4-(6-bromo-4-methyl-3-oxo-3,4-dihydropyrazin-2-ylamino)phenyl)piperidine-1-carboxylate 282a

A mixture of tert-butyl 4-(4-aminophenyl)piperidine-1-carboxylate (2.5 g, 9.06 mmol) and 3,5-dibromo-1-methylpyrazin-2(1H)-one (2.2 g, 8.23 mmol) in isopropanol (30 mL) was heated at 85°C for 15 h. After the reaction was complete, the mixture was filtered and the solid was washed with isopropanol to give 282a as a white solid (2.9 g, 80%). LCMS: (M+H) ⁺ 463

Example 282b 5-Bromo-1-methyl-3-(4-(piperidin-4-yl)phenylamino)pyrazin-2(1H)-one 282b

Following Example 247a, 282a was converted to 282b in 99% yield.

Example 282c 5-Bromo-1-methyl-3-(4-(1-methylpiperidin-4-yl)phenylamino)pyrazin-2(1H)-one 282c

Following Example 247b, 282b was converted to 282c in 65% yield.

Example 282d 4-Fluoro-2-(4-methyl-6-(4-(1-methylpiperidin-4-yl)phenylamino)-5-oxo-4,5-dihydropyrazin-2-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 282d

Following Example 247c, 282c was reacted with 4-fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 210d to give 282d in 41% yield.

According to Example 136, 282d was converted to 282 in 36% yield. LCMS: (M+H) ⁺ 611. ¹ H NMR(500MHz,DMSO)δ9.22(s,1H),7.90(s,1H),7.88(s,1H),7.49(s,1H),7 .36(dd,J＝9.5,1H),7.30(dd,J＝9.5,1H),7.17(s,1H),7.15(s,1H),6.54(s ,1H),4.47(m,2H),4.15(m,3H),3.88(m,2H),2.91(m,2H),2.59(m,2H),3.0 5(m,1H),2.46(m,3H),2.25(s,3H),2.07(m,2H),1.80(m,2H),1.70(m,6H).

Example 283 2-(5-fluoro-2-(hydroxymethyl)-3-(4-methyl-6-(2-methyl-1,2,3,4-tetrahydroisoquinolin-6-ylamino)-5-oxo-4,5-dihydropyrazin-2-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 283

Example 283a 4-Fluoro-2-(4-methyl-6-(2-methyl-1,2,3,4-tetrahydroisoquinolin-6-ylamino)-5-oxo-4,5-dihydropyrazin-2-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 283a

According to Example 148b, 290 mg of 4-fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 210d and 209 mg of 5-bromo-1-methyl-3-(2-methyl-1,2,3,4-tetrahydroisoquinolin-6-ylamino)pyrazin-2(1H)-one 221b were reacted to give 283a (217 mg, 58%) as a yellow solid. MS: [M+H] ⁺ 699

According to Example 148, 283a was converted to a white solid 283 (84 mg, 43%). ¹ H NMR (500 MHz, DMSO) δ 9.15 (s, 1H), 7.82 (s, 1H), 7.65 (d, J = 8, 1H), 7.52 (s, 1H), 7.39 (dd, J = 10, 1H), 7.31 (dd, J = 10, 1H), 6.96 (d, J = 8.0, 1H), 6.53 (s, 1H), 4.87 (s, 1H), 4.49(m,1H),4.42(m,1H),4.13(m,3H),3.88(m,1H),3.54(s,3H),3.41(s,2H),3.3 0(s,1H),2.76(d,J＝5.5,6H),2.58(m,5H),23.2(s,3H),1.79(s,2H),1.70(s,2H).

Example 284 10-(3-{5-[(6-ethyl-5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl)amino]-1-methyl-6-oxo-1,6-dihydropyridin-3-yl}-5-fluoro-2-(hydroxymethyl)phenyl)-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 284

In a 48-mL sealed tube equipped with a magnetic stir bar were added 5-bromo-1-methyl-3-(6-ethyl-5,6,7,8-tetrahydro-1,6-naphthyridin-2-ylamino)pyridin-2(1H)-one (143 mg, 0.4 mmol), 10-[2-(acetoxymethyl)-3-(4,4,5,5-tetramethyl[1,3,2]-dioxaborolan-2-yl)-5-fluorophenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 230a (496 mg, 1.0 mmol), Pd(PPh ₃ ) ₄ (23 mg, 0.020 mmol), 2N Na ₂ CO ₃ (4 mL) and DME (4 mL). After the reaction mixture was stirred at 100° C. for 1 h, it was purified by flash chromatography (dichloromethane:methanol, 3:1) to give 7% (18 mg) of 284 MS (ESI ⁺ ) m/z 611.5 (M+H).

Example 285 10-{3-[5-({5-ethyl-4H,5H,6H,7H-pyrazolo[1,5-a]pyrazin-2-yl}amino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl]-5-fluoro-2-(hydroxymethyl)phenyl}-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 285

Example 285a 5-Ethyl-2-nitro-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazine 285a

To a sealed tube equipped with a magnetic stirrer was added 1-(2-bromoethyl)-5-(bromomethyl)-3-nitro-1H-pyrazole 101c (2.8 g, 9 mmol) and a 2M ethylamine/THF solution (27 mL). The resulting mixture was heated at 35°C overnight. Thereafter, the reaction mixture was concentrated under reduced pressure, and water (50 mL) and ethyl acetate (50 mL) were added to the residue. The aqueous layer was separated and then extracted with ethyl acetate (2 x 50 mL). The combined organic extracts were washed with brine (100 mL) and then dried over sodium sulfate. The resulting solution was concentrated under reduced pressure to give a 100% yield (1.8 g) of crude product 285a.

Example 285b 5-Ethyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-amine 285b

To a 500-mL Parr hydrogenation bottle was added 285a (1.8 g, 9 mmol), 10% palladium on carbon (50% humidity, 500 mg dry weight), and ethanol (100 mL). The bottle was evacuated, filled with hydrogen to 50 psi, and shaken on a Parr hydrogenation apparatus for 1 h. The catalyst was removed by filtration through a pad of Celite 521 and washed with 1:1 dichloromethane:methanol (500 mL). The resulting solution was concentrated under reduced pressure to afford a 71% yield (1.7 g) of crude 285b.

Example 285c 5-Bromo-3-(5-ethyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-1-methylpyridin-2(1H)-one 285c

To a sealed tube equipped with a magnetic stirrer were added 285b (1.1 g, 6.4 mmol), 3,5-dibromo-1-methylpyridin-2(1H)-one 5 (1.7 g, 6.4 mmol), cesium carbonate (4.6 g, 14 mmol), and 1,4-dioxane (64 mL). After nitrogen was bubbled through the solution for 30 min, Xantphos (440 mg, 0.8 mmol) and tris(dibenzylideneacetone)dipalladium(0) (400 mg, 0.5 mmol) were added, and the reaction mixture was heated at 100°C for 16 h. Thereafter, water (50 mL) and ethyl acetate (50 mL) were added. The aqueous layer was separated and then extracted with ethyl acetate (2×50 mL). The combined organic extracts were washed with brine (100 mL) and then dried over sodium sulfate. The resulting residue was purified by column chromatography eluting with a gradient of dichloromethane to 60:35:5 dichloromethane:diethyl ether:methanol to afford 285c in 28% yield (620 mg).

Example 285d 10-{3-[5-({5-ethyl-4H,5H,6H,7H-pyrazolo[1,5-a]pyrazin-2-yl}amino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl]-5-fluoro-2-(acetoxymethyl)phenyl}-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 285d

To a microwave tube equipped with a magnetic stirrer was added 285c (190 mg, 0.5 mmol), 230a (350 mg, 0.7 mmol), 1,2-dimethoxyethane (6.4 mL), and 1 M aqueous sodium carbonate solution (1.6 mL). After blowing _N2 for 15 min, Pd( _PPh3 ) ₄ (31 mg, 0.03 mmol) was added. The mixture was heated to 130°C in a microwave for 15 min. Thereafter, ethyl acetate (5 mL) and water (5 mL) were added. The separated aqueous layer was extracted with ethyl acetate (2 x 5 mL). The combined organic phases were washed with brine (10 mL), dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by column chromatography eluting with a gradient of dichloromethane to 60:35:5 dichloromethane:ether:methanol to afford 285d in 35% yield (120 mg).

285d (120 mg, 0.2 mmol), lithium hydroxide (40 mg, 1 mmol), THF (1 mL), isopropyl alcohol (1 mL) and water (2 mL) were added into a 25 mL round-bottom flask equipped with a magnetic stirrer. The mixture was stirred for 30 min at rt. After this, ethyl acetate (5 mL) and water (5 mL) were added. The aqueous layer separated by extraction with ethyl acetate (2 × 5 mL) was used. The organic phase merged was washed with salt water (10 mL), dried over sodium sulfate, and then filtered. The mixture was concentrated to near dryness under reduced pressure, and the desired product was precipitated and then filtered. 285 was obtained in a 78% yield (90 mg) by washing with ether (10 mL). MS (ESI+) m/z 600.6 (M+H).

Example 286 2-(3-(5-(5-((2-(dimethylamino)ethyl)(methyl)amino)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-3,4,5,6,7,8-hexahydrobenzothieno[2,3-c]pyridin-1(2H)-one 286

According to Example 270, 212a (550 mg, 1.20 mmol) and 270d (428 mg, 1.00 mmol) were reacted to give 286 as an off-white solid in 8% yield (49 mg): mp 122-123 °C; ¹ H NMR (500 MHz, DMSO-d ₆ )δ8.45(d,1H,J＝2.0Hz),8.20(s,1H),7.69(d,1H,J＝2.4Hz),7.31(m,2H), 7.17(m,3H),4.83(t,1H,J＝4.6Hz),4.32(d,2H,J＝4.3Hz),4.06(m,1H),3.8 7(m,1H),3.58(s,3H),2.96(m,1H),2.86(m,1H),2.83(s,3H),2.78(m,2H), 2.54(m,1H),2.32(t,2H,J=7.0Hz),2.14(s,6H),1.80(m,4H);MS(ESI+)m/z 631.3(M+H).

Example 287 5-(5-fluoro-2-(hydroxymethyl)-3-(6,6-dimethyl-3,4,6,7-tetrahydro-5H-cyclopenta[4,5]thieno[2,3-c]pyridin-1(2H)-yl)phenyl)-1-methyl-3-(5-(1-methylpyrrolidin-3-yl)pyridin-2-ylamino)pyridin-2(1H)-one 287

Example 287a 1-Methyl-3-(5-(1-methylpyrrolidin-3-yl)pyridin-2-ylamino)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one 287a

To a 100-mL single-necked round-bottom flask equipped with a magnetic stirrer and a nitrogen inlet was added 202a (481 mg, 1.33 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-di-1,3,2-dioxaborolane (675 mg, 2.66 mmol), potassium acetate (392 mg, 3.99 mmol), and 1,4-dioxane (12 mL). After nitrogen was bubbled through the resulting suspension for 30 min, Pd(dppf) _Cl₂ (97.0 mg, 0.133 mmol) was added. A reflux condenser was attached to the flask, and the reaction mixture was heated at 100°C for 3 h. Thereafter, the mixture was diluted with ethyl acetate (100 mL) and water (75 mL), and the layers were separated. The aqueous layer was extracted with ethyl acetate (2 x 50 mL), and the combined organic layers were washed with brine (100 mL) and then dried over sodium sulfate. The drying agent was removed by filtration. The filtrate was concentrated under reduced pressure to give a quantitative yield (548 mg) of crude 287a as a brown semisolid. The crude mixture was used in the next reaction without further purification: MS (ESI+) m/z 411.2 (M+H).

To a 250-mL single-necked round-bottom flask equipped with a magnetic stirrer and a nitrogen inlet was added crude 287a (274 mg, 0.668 mmol, assumed quantitative yield), 247a (420 mg, 0.865 mmol), sodium carbonate (207 mg, 1.99 mmol), water (2 mL), and 1,4-dioxane (10 mL). After bubbling nitrogen through the resulting suspension for 30 min, tetrakis(triphenylphosphine)palladium(0) (151 mg, 0.133 mmol) was added. A reflux condenser was attached to the flask, and the reaction mixture was heated at 90°C for 1 h. Thereafter, the mixture was diluted with 90:10 dichloromethane/methanol (100 mL) and water (75 mL), and the layers were separated. The aqueous layer was extracted with 90:10 dichloromethane/methanol (2×50 mL), and the combined organic layers were washed with brine (100 mL) and then dried over sodium sulfate. The drying agent was removed by filtration. The filtrate was concentrated under reduced pressure, and the resulting residue was dissolved in THF (5 mL), water (5 mL) and methanol (5 mL). Lithium hydroxide monohydrate (47 mg, 1.12 mmol) was added, and the mixture was stirred at room temperature for 2 h. Thereafter, the mixture was diluted with 90:10 dichloromethane/methanol (100 mL) and water (50 mL), and the layers were separated. The aqueous layer was extracted with 90:10 dichloromethane/methanol (2X75 mL), and the combined organic layers were washed with brine (100 mL) and then dried over sodium sulfate. The desiccant was removed by filtration. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography (silica gel, 80:20 dichloromethane/methanol) to give a 27% yield (110 mg) of a non-crystalline off-white solid 287: mp 181–183 ° C; ¹ H NMR (500 MHz, DMSO-d ₆ )δ8.64(d,J＝2.0Hz,1H),8.49(s,1H),8.07(d,J＝2.5Hz,1H),7.54(dd,J＝8.5,2.5Hz,1H),7.40(d,J＝2.0Hz,1H),7.34(dd,J＝9. 0,2.5Hz,1H),7.25(d,J＝8.5Hz,1H),7.19(dd,J＝9.5,2.5Hz,1H),4.86(t,J＝4.0Hz,1H),4.34–4.32(m,2H),4.10–4.03(m,1H),3 .87–3.83(m,1H),3.59(s,3H),3.25–3.20(m,1H),3.05–2.99(m,1H),2.91–2.87(m,1H),2.81–2.77(m,1H),2.75(s,2H),2.60–2 .58(m,2H),2.55–2.50(m,2H),2.49–2.32(m,1H),2.27(s,3H),2.22–2.17(m,1H),1.71–1.67(m,1H),1.23(s,6H); MS(ESI+)m/z 628.3(M+H).

Example 288 2-(5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-(5-(1-methylpyrrolidin-2-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,5,6,7,8-hexahydrobenzothieno[2,3-c]pyridin-1(2H)-one 288

According to Example 286, 212a (450 mg, 1.04 mmol) and 287b (328 mg, 0.810 mmol) were reacted to give 26% yield (130 mg) of an off-white solid 288: mp 164-166°C; ^1H NMR (500 MHz, DMSO- _d6 ) δ 8.67 (d, 1H, J = 2.0 Hz), 8.55 (s, 1H), 8.06 (d, 1H, J = 2.4 Hz), 7.53 (dd, 1H, J = 8.5, 2.4 Hz), 7.41 (d, 1H, J = 2.4 Hz), 7.32 (dd, 1H, J = 9.1, 2.1 Hz), 7.26 (d, 1H, J = 9.0 Hz), 7.19 (dd, 1H, J = 9.0, 2.0 Hz), 4. 85(t,1H,J＝4.2Hz),4.32(m,2H),4.04(m,1H),3.87(m,1H),3.59(s,3H),3.13(m,1H),2.95(m,2H) ,2.84(m,1H),2.78(m,2H),2.54(m,1H),2.18(q,1H,J＝4.0Hz),2.04(m,4H),1.80(m,6H),1.58(m, ¹ H); MS (ESI+) m/z 614.3 (M+H).

Example 289 2-(5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-(5-morpholinylpyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,5,6,7,8-hexahydrobenzothieno[2,3-c]pyridin-1(2H)-one 289

Example 289a 1-Methyl-3-(5-morpholinylpyridin-2-ylamino)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one 289a

To a 250-mL single-necked round-bottom flask equipped with a magnetic stirrer and a nitrogen inlet was added 255c (610 mg, 1.70 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-di-1,3,2-dioxaborolane (850 mg, 3.40 mmol), potassium acetate ( ₄₉₂ mg, 5.00 mmol), and 1,4-dioxane (20 mL). After nitrogen was bubbled through the resulting suspension for 30 min, Pd(dppf) _Cl2 / _CH2Cl2 (122 mg, 0.200 mmol) was added. A reflux condenser was attached to the flask, and the reaction mixture was heated at 90°C for 2 h. Thereafter, the mixture was diluted with ethyl acetate (100 mL) and water (75 mL), and the layers were separated. The aqueous layer was extracted with ethyl acetate (50 mL), and the combined organic extracts were washed with brine (50 mL) and then dried over sodium sulfate. The drying agent was removed by filtration. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by trituration with hexane/ethyl acetate (80:20, 25 mL) to afford 289a as a brown solid in quantitative yield (688 mg): mp 100-101° C.; ¹ H NMR (500 MHz, DMSO-d ₆ ) 8.45 (d, 1 H, J=1.5 Hz), 8.21 (s, 1 H), 7.91 (s, 1 H), 7.89 (d, 1 H, J=3.0 Hz), 7.66 (d, 1 H, J=6.5 Hz), 7.14 (d, 1 H, J=9.0 Hz), 3.73 (t, 4 H, J=4.5 Hz), 3.55 (s, 3 H), 3.05 (t, 4 H, J=4.5 Hz), 1.29 (s, 12 H); MS (ESI+) m/z 413.0 (M+H)

A 100-mL single-necked round-bottom flask equipped with a magnetic stirrer and reflux condenser was purged with nitrogen and charged with 289a (485 mg, 0.415 mmol), 247a (488 mg, 1.10 mmol), sodium carbonate (264 mg, 2.50 mmol), 1,4-dioxane (8 mL), and water (2 mL). The mixture was degassed with nitrogen for 30 min. Tetrakis(triphenylphosphine)palladium (96 mg, 0.083 mmol) was added. After heating at 100°C for 2 h, the reaction mixture was cooled to room temperature and then partitioned between water (40 mL) and dichloromethane (100 mL). The layers were separated, and the aqueous phase was extracted with dichloromethane (2 x 50 mL). The organic extracts were combined, dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was dissolved in methanol (5 mL), and potassium carbonate (745 mg, 5.40 mmol) was added. After stirring at room temperature for 1 h, the reaction mixture was partitioned between water (20 mL) and dichloromethane (20 mL). The layers were separated, and the aqueous phase was extracted with dichloromethane (2×20 mL). The organic extracts were combined, dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, 0%-15% methanol/dichloromethane) to give 289 as a yellow solid in 51% yield (130 mg): mp 220–221°C; ¹ H NMR (500 MHz, DMSO-d ₆ )δ8.55(d,1H,J＝2.0Hz),8.37(s,1H),7.87(d,1H,J＝3.0Hz),7.34(m,3H),7.23(d ,1H,J＝9.0Hz),7.16(dd,1H,J＝9.5,3.0Hz),4.83(t,1H,J＝4.0Hz),4.32(m,2H),4 .05(m,1H),3.86(m,1H),3.71(t,4H,J＝4.5Hz),3.58(s,3H),3.01(t,4H,J＝4.5Hz ),2.98(m,1H),2.87(m,1H),2.77(m,2H),2.54(m,1H),1.79(m,4H); MS(ESI+)m/z 616.2(M+H).

Example 290 10-[5-fluoro-2-(hydroxymethyl)-3-[1-methyl-5-({5-[(2S)-1-methylpyrrolidin-2-yl]pyridin-2-yl}amino)-6-oxo-1,6-dihydropyridin-3-yl]phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 290

Example 291 10-[5-fluoro-2-(hydroxymethyl)-3-[1-methyl-5-({5-[(2R)-1-methylpyrrolidin-2-yl]pyridin-2-yl}amino)-6-oxo-1,6-dihydropyridin-3-yl]phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 291

Compounds 290 and 291 are enantiomers of racemate 243. Chiral separation of the racemic mixture 243 was performed on a Chiralpak AD, 4.6 x 50 mm, 3 μm column (mobile phase 45% isopropanol (w/0.1% triethylamine)/55% CO ₂ , flow rate 5 mL/min) at 40° C. to afford the individual enantiomers, with 290 eluting first: MS (ESI+) m/z 611.5 (M+H) and example 291 eluting last: MS (ESI+) m/z 611.5 (M+H).

Example 292 10-[5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-{[6-(oxetane-3-yl)-5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 292

Example 292a 10-[5-fluoro-2-(acetoxymethyl)-3-(1-methyl-5-{[6-(oxetane-3-yl)-5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 292a

A 10-mL microwave reaction vessel equipped with a magnetic stir bar was charged with 5-bromo-1-methyl-3-(6-(oxetan-3-yl)-5,6,7,8-tetrahydro-1,6-naphthyridin-2-ylamino)pyridin-2(1H)-one 219a (250 mg, 0.64 mmol), 10-[2-(acetoxymethyl)-3-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-5-fluorophenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 230a (635 mg, 1.3 mmol), Pd(PPh ₃ ) ₄ (37 mg, 0.03 mmol) and 2N Na ₂ CO ₃ (2 mL) and 1,2-dimethoxyethane (2 mL). The reaction mixture was stirred at 125°C for 10 min and then purified by flash chromatography (dichloromethane:methanol, 3:1) to afford 12% (50 mg) of 292a as a solid.

To a 25-mL single-necked round-bottom flask equipped with a magnetic stir bar was added 292a (50 mg, 0.078 mmol), LiOH· _H₂O (50 mg, 1.2 mmol), THF (2 mL), i-PrOH (2 mL), and water (2 mL). The reaction mixture was stirred at room temperature for 2 h and then partitioned between dichloromethane (5 mL) and water (5 mL), and the organic phase was extracted with dichloromethane (5 mL x 3). The combined organic phases were washed with water (5 mL x 2) and brine (5 mL _x 1), dried ( _Na₂SO₄ ), and concentrated. The crude product was redissolved in dichloromethane (3 mL). Hexanes (10 mL) were added to this solution, and the resulting precipitate was filtered to afford 292 in a 12% yield (6 mg). MS ( ^ESI⁺ ) m/z 639.5 (M+H).

Example 293 2-(5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-(5-(1-methylazetidin-3-yloxy)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 293

Example 293a 4-Fluoro-2-(1-methyl-5-(5-(1-methylazetidin-3-yloxy)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 293a

A mixture of 5-bromo-1-methyl-3-(5-(1-methylazetidin-3-yloxy)pyridin-2-ylamino)pyridin-2(1H)-one 199f (200 mg, 0.55 mmol), 4-fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate _210d (270 mg, 0.56 mmol), _PdCl2 (dppf) (40 mg, 0.055 mmol), _K3PO4 (150 mg), NaOAc (50 mg) in MeCN (8 mL) and water (2 mL) was heated in a sealed tube at 110°C for 2 h. The solvent was evaporated in vacuo. The residue was purified by reverse phase Combi flash chromatography to give 293a (307 mg, 70%). MS: [M+H] ⁺ 641.

A mixture of 293a (287 mg, 0.45 mmol) and LiOH hydrate (188 mg, 4.5 mmol) in isopropanol (25 mL) and water (5 mL) was stirred at 30°C for 2 h. The mixture was evaporated in vacuo, and the residue was extracted with ethyl acetate (20 mL x 2). The combined extracts were concentrated under reduced pressure. The residue was purified by preparative HPLC to give 293 (60 mg, 25%). MS: [M+H] ⁺ 599. ¹ H NMR(500MHz, CDCl3)δ8.51(s,1H),7.81(s,1H),7.74(s,1H),7.48(s,1H),7.16-7.14(m,1H ),7.11-7.09(m,1H),6.95-6.93(m,1H),6.86(s,1H),6.79(d,J＝9.0,1H),4.76-4.72(m,1H ),4.53(d,J＝8.5,1H),4.34-4.25(m,2H),4.20-4.13(m,3H),3.95-3.84(m,3H),3.69(s,3H ),3.24-3.13(m,2H),2.64-2.52(m,4H),2.46(s,3H),1.93-1.84(m,2H),1.81-1.75(m,2H).

Example 294 10-[5-fluoro-2-(hydroxymethyl)-3-(4-methyl-6-{[4-(1-methylpiperidin-4-yl)phenyl]amino}-5-oxo-4,5-dihydropyrazin-2-yl)phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 294

Example 294a (2-{4,4-dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-10-yl}-4-fluoro-6-(4-methyl-6-{[4-(1-methylpiperidin-4-yl)phenyl]amino}-5-oxo-4,5-dihydropyrazin-2-yl)phenyl)methyl acetate 294a

Following Example 150b, 282c was reacted with (2-{4,4-dimethyl-9-oxo-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-10-yl}-4-fluoro-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methyl acetate 230a to afford 294a in 81% yield. LCMS: (M+H) ⁺ 667

According to Example 150, 294a was converted to 294 in 47% yield. LCMS: (M+H) ⁺ 625. ¹ H NMR (500 MHz, DMSO) δ 9.20 (s, 1H), 7.89 (s, 1H), 7.87 (s, 1H), 7.48 (s, 1H), 7.36 (dd, J = 10, 1H), 7.31 (dd, J = 9.5, 1H), 7.17 (s, 1H), 7.15 (s, 1H), 6.52 (s, 1H), 4.85 (m, 1H), 4.49 (m, 1H), 4 .40(m,1H),4.20(m,2H),4.12(m,1H),3.87(m,1H),3.54(s,3H),2.84(d,J＝10.5,1H),2.57 (s,2H),2.42(s,2H),2.38(m,1H),2.18(s,3H),1.93(t,J=9,2H),1.66(m,4H),1.22(s,6H).

Example 295 10-[5-Fluoro-2-(hydroxymethyl)-3-(1-methyl-5-{[5-(1-methylpiperidin-4-yl)pyridin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)phenyl]-4,4-dimethyl-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-9-one 295

Example 295a (2-{4,4-dimethyl-9-oxo-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-10-yl}-4-fluoro-6-(1-methyl-5-{[5-(1-methylpiperidin-4-yl)pyridin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)phenyl)methyl acetate 295a

To a sealed tube were added 5-bromo-1-methyl-3-(5-(1-methylpiperidin-4-yl)pyridin-2-ylamino)pyridin-2(1H)-one 282c (350 mg, 0.93 mmol), (2-{4,4-dimethyl-9-oxo-7-thia-10-azatricyclo[6.4.0.0 ^{2 ,6} ]dodeca-1(8),2(6)-dien-10-yl}-4-fluoro-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methyl acetate 247b (448 mg, 0.93 mmol), Pd(dppf)Cl ₂ (76 mg, 0.09 mmol), K ₃ PO ₄ .3H ₂ A mixture of 2-[4-(2-[2-( ₂ - ^oxo -1-yl)-1 _- oxo ...

To a solution of 295a (250 mg, 0.37 mmol) in propan-2-ol (8 mL), tetrahydrofuran (8 mL), and water (1 mL) was added LiOH (878 mg, 37 mmol) and stirred at 30°C for 2 h. The mixture was then evaporated and the residue was purified by preparative HPLC to give 295 (76 mg, 32%) as a yellow solid. MS: (M+H) ⁺ 642. ¹ H NMR(500MHz,MEOD)δ1.29(d,6H),1.94-2.02(m,2H),2.18(d,2H),2.56-2.64(m,2H),2.81(s,2H),2.94-3.03(m,5H),3.08-3.19(m,3H), 3.66(d,1H),3.74(s,3H),3.92-3.97(m,1H),4.14-4.19(m,1H),4.50(s,1H),7.21-7.28(m,2H),7.84(s,1H),7.95(d,1H),8.13(s,1H).

Example 296 10-{3-[5-({5-[2-(dimethylamino)ethoxy]pyridin-2-yl}amino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl]-5-fluoro-2-(hydroxymethyl)phenyl}-4,4-dimethyl-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-9-one 296

Example 296a (2-{4,4-dimethyl-9-oxo-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-10-yl}-6-[5-({5-[2-(dimethylamino)ethoxy]pyridin-2-yl}amino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl]-4-fluorophenyl)methyl acetate 296a

5-Bromo-3-(5-(2-(dimethylamino)ethoxy)pyridin-2-ylamino)-1-methylpyridin-2(1H)-one 268c (244 mg, 0.67 mmol), (2-{4,4-dimethyl-9-oxo-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-10-yl}-4-fluoro-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methyl acetate 247b (342 mg, 0.67 mmol), PdCl ₂ (dppf) (59 mg, 0.08 mmol), K ₃ PO ₄ A mixture of 2-[ ^4- [ ...

A mixture of 296a (186 mg, 0.28 mmol) and LiOH hydrate (116 mg, 2.8 mmol) in isopropanol (25 mL) and water (5 mL) was stirred at 30°C for 2 h. The mixture was evaporated in vacuo, and the residue was extracted with ethyl acetate (20 mL x 2). The combined extracts were concentrated under reduced pressure, and the residue was purified by preparative HPLC to afford 296 (126 mg, 71%). MS: [M+H] ⁺ 632.

Example 297 2-(3-(5-(5-(2-(dimethylamino)ethoxy)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 297

Example 297a 2-(5-(5-(2-(dimethylamino)ethoxy)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-4-fluoro-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 297a

A mixture of 5-bromo-3-(5-(2-(dimethylamino)ethoxy)pyridin-2-ylamino)-1-methylpyridin-2(1H)-one 268c (205 mg, 0.56 mmol), 4-fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 210d (270 mg, 0.56 mmol), _PdCl2 (dppf) (51 mg, 0.07 mmol), _{K3PO4 (} 100 mg) and NaOAc (40 mg) in MeCN (6 mL) and water (2 mL) was heated in a sealed tube at 110°C for 2 h. The solvent was evaporated in vacuo. The residue was purified by reverse phase Combi flash chromatography to give 297a (206 mg, 50%). MS: [M+H] ⁺ 643.

A mixture of 297a (186 mg, 0.29 mmol) and LiOH hydrate (122 mg, 2.9 mmol) in isopropanol (15 mL) and water (3 mL) was stirred at 30°C for 2 h. The mixture was evaporated in vacuo, and the residue was extracted with ethyl acetate (20 mL x 2). The combined extracts were concentrated under reduced pressure, and the residue was purified on preparative HPLC to give 297 (84 mg, 48%). MS: [M+H] ⁺ 601. ¹ H NMR (500MHz, CDCl3) δ8.53(d,J＝2.5,1H),7.94(d,J＝3.0,1H),7.80(s,1H),7.47(d,J＝2.0,1H),7 .23-7.21(m,1H),7.16-7.14(m,1H),6.95-6.93(m,1H),6.86(s,1H),6.81(d,J＝9.0,1H),4.55(d ,J＝10.5,1H),4.40-4.26(m,2H),4.22-4.15(m,3H),4.15-4.04(m,2H),3.94-3.86(m,1H),3.69( s,3H),2.75-2.73(m,2H),2.62-2.54(m,4H),2.37(s,6H),1.92-1.86(m,2H),1.82-1.76(m,2H).

Example 298 10-[5-fluoro-2-(hydroxymethyl)-3-(4-methyl-6-{[4-(1-methylazetidin-3-yl)phenyl]amino}-5-oxopyrazin-2-yl)phenyl]-4,4-dimethyl-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-9-one 298

Example 298a tert-Butyl 3-(4-aminophenyl)azetidine-1-carboxylate 298a

A mixture of tert-butyl 3-iodoazetidine-1-carboxylate (1 g, 3.53 mmol), 4-aminophenylboronic acid (630 mg, 4.59 mmol), NiI ₂ (66 mg, 0.212 mmol), NaHMDS (1.94 g, 10.6 mmol), and (1R,2S)-2-aminocyclohexanol (24 mg, 0.212 mmol) in isopropanol (8 mL) was stirred in a microwave at 150°C for 2 h. The mixture was then evaporated, and the residue was dissolved in water and ethyl acetate. The aqueous phase was separated and extracted with ethyl acetate. The combined extracts were dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography to afford 298a (360 mg, 40%). MS: [M+H] ⁺ 249.

Example 298b tert-Butyl 3-(4-(6-bromo-4-methyl-3-oxo-3,4-dihydropyrazin-2-ylamino)phenyl)azetidine-1-carboxylate 298b

A mixture of 298a (500 mg, 2.02 mmol), 3,5-dibromo-1-methylpyrazin-2(1H)-one (536 mg, 2.02 mmol), and triethylamine (0.6 mL, 4.04 mmol) in isopropanol (10 mL) was heated at reflux for 4 days. The solvent was evaporated in vacuo, and the residue was purified by reverse phase Combi-flash chromatography to afford 298b (690 mg, 66%). MS: [M+H] ⁺ 435.

Example 298c 3-(4-(azetidin-3-yl)phenylamino)-5-bromo-1-methylpyrazin-2(1H)-one hydrochloride 298c

A mixture of 298b (690 mg, 1.6 mmol) and HCl/1,4-dioxane (8 M, 8 mL) in methanol (30 mL) was stirred at room temperature overnight. The mixture was evaporated in vacuo, and the residue was purified by reverse phase Combi-flash chromatography to afford 298c (100 mg, 17%). MS: [M+H] ⁺ 335.

Example 298d 5-Bromo-1-methyl-3-(4-(1-methylazetidin-3-yl)phenylamino)pyrazin-2(1H)-one 298d

A mixture of 298c (100 mg, 0.24 mmol), NaBH(OAc) ₍ 100 mg, 0.48 mmol), HCHO (10 mL), and acetic acid (1 mL) in methanol (15 mL) was stirred at room temperature for 4 h. The solvent was evaporated in vacuo, and the residue was neutralized with _NaHCO solution until pH 8 was reached. The mixture was extracted with ethyl acetate. The combined extracts were dried over sodium sulfate and then concentrated under reduced pressure to give 298d (100 mg), which was used in the next step without further purification. MS: [M+H] ³⁴⁹ .

Example 298e (2-{4,4-dimethyl-9-oxo-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-10-yl}-4-fluoro-6-(4-methyl-6-{[4-(1-methylazetidin-3-yl)phenyl]amino}-5-oxo-4,5-dihydropyrazin-2-yl)phenyl)methyl acetate 298e

To a sealed tube was added a mixture of (2-{4,4-dimethyl-9-oxo-7-thia-10-azatricyclo[ ^6.4.0.02,6 ]dodeca-1(8),2(6)-dien-10-yl}-4-fluoro-6-(tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methyl acetate 247b (100 mg, 0.19 mmol), 5-bromo-1-methyl-3-(4-(1-methylazetidin-3-yl)phenylamino)pyrazin-2(1H)-one 298d (68 mg, 0.19 mmol), _PdCl2 (dppf) (14 mg, 0.019 mmol), _K3PO4 ( ₆₀ mg) and NaOAc (30 mg) in MeCN (5 mL) and water (1 mL). The mixture was heated at 110°C for 2 h. The solvent was evaporated in vacuo and the residue was purified by reverse phase Combi-flash chromatography to give 298e (100 mg, 82%). MS: [M+H] ⁺ 656.

A mixture of 298e (100 mg, 0.16 mmol) and LiOH hydrate (66 mg, 1.6 mmol) in isopropanol (10 mL) and water (2 mL) was stirred at 30°C for 2 h. The mixture was evaporated in vacuo, and the residue was extracted with ethyl acetate (2 x 20 mL). The combined extracts were concentrated under reduced pressure. The residue was purified on preparative HPLC to give 298 (60 mg, 63%). MS: [M+H] ⁺ 614. ¹ H NMR (500MHz, CDCl3) δ8.29 (s, 1H), 7.72 (d, J = 7.5, 2H), 7.55 (s, 1H), 7.45-7.43 (m, 1H) ,7.30(s,1H),7.01-6.99(m,1H),4.56(d,J＝11.5,1H),4.41-4.30(m,2H),4.10-4.05(m ,1H),3.90-3.85(m,1H),3.82-3.72(m,2H),3.64(s,3H),3.22-3.10(m,2H),3.04-2.9 7(m,1H),2.93-2.88(m,1H),2.79(s,2H),2.57-2.50(m,2H),2.39(s,3H),1.27(s,6H).

Example 299 10-[5-fluoro-2-(hydroxymethyl)-3-[1-methyl-5-({5-[1-(oxetan-3-yl)piperidin-4-yl]pyridin-2-yl}amino)-6-oxo-1,6-dihydropyridin-3-yl]phenyl]-4,4-dimethyl-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-9-one 299

According to Example 121b, compound 247b (256 mg, 0.5 mmol), 5-bromo-1-methyl-3-(5-(1-(oxetan-3-yl)piperidin-4-yl)pyridin-2-ylamino)pyridin-2(1H)-one 264e (210 mg, 0.5 mmol), 1M sodium carbonate solution (2 mL, 2 mmol), tetrakis(triphenylphosphine)palladium(0) (29 mg, 0.025 mmol), and 1,2-dimethoxyethane (5 mL) were reacted. The reaction mixture was heated at 130°C in a microwave reactor for 15 minutes. Workup and flash column chromatography (silica gel, 9:1 dichloromethane/methanol) afforded a yellow oily mixture of compounds 299a and 299.

The above residue (0.5 mmol) was deprotected using the same method as 121, except that a mixture of THF (2 mL), water (1 mL), and isopropanol (2 mL) and lithium hydroxide monohydrate (105 mg, 2.5 mmol) were used. Workup and flash column chromatography (NH-silica gel, ethyl acetate/hexane) gave 299 as a light yellow solid in 12% yield (40 mg): MS (ESI+) m/z 684.5 (M+H).

Example 300 10-[5-fluoro-2-(hydroxymethyl)-3-[1-methyl-5-({5-[1-(oxetan-3-yl)piperidin-4-yl]pyridin-2-yl}amino)-6-oxo-1,6-dihydropyridin-3-yl]phenyl]-4,4-dimethyl-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-9-one 300

Example 300a 5-Bromo-3-(2-methoxypyrimidin-4-ylamino)-1-methylpyridin-2(1H)-one 300a

Following Example 121a, 2-methoxypyrimidin-4-amine (0.625 g, 5 mmol), 3,5-dibromo-1-methyl-1H-pyridin-2-one (1.34 g, 5 mmol), cesium carbonate (4.88 g, 15 mmol), tris(dibenzylideneacetone)dipalladium(0) (0.465 g, 0.5 mmol), Xantphos (0.58 g, 1 mmol), and 1,4-dioxane (50 mL) were reacted. The reaction mixture was heated at 100° C. for 24 hours, then cooled to room temperature and filtered through a pad of Celite 521. The filter cake was washed with 9:1 dichloromethane/methanol (2×25 mL), and the combined filtrates were concentrated to dryness. The residue was dissolved in dichloromethane, ether was added, and the resulting precipitate was filtered to give a quantitative yield (1.57 g) of 300a as a green solid: MS (ESI+) m/z 313.1 (M+H).

Example 300b 10-[5-fluoro-2-(acetoxymethyl)-3-[1-methyl-5-({5-[1-(oxetan-3-yl)piperidin-4-yl]pyridin-2-yl}amino)-6-oxo-1,6-dihydropyridin-3-yl]phenyl]-4,4-dimethyl-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-9-one 300b

According to Example 121b, 300a (242 mg, 0.5 mmol), 230a (210 mg, 0.5 mmol), 1 M sodium carbonate solution (2 mL, 2 mmol), tetrakis(triphenylphosphine)palladium(0) (29 mg, 0.025 mmol), and 1,2-dimethoxyethane (5 mL) were used. The reaction mixture was heated in a microwave reactor at 130°C for 15 minutes. Workup and flash column chromatography (silica gel, 60:35:5 dichloromethane/diethyl ether/methanol) gave 300b in 67% yield (200 mg) as a yellow solid: MS (ESI+) m/z 602.4 (M+H).

According to Example 121, a mixture of THF (2 mL), water (1 mL), and isopropanol (2 mL), 300b (200 mg, 0.33 mmol), and lithium hydroxide monohydrate (105 mg, 2.5 mmol) were used. Workup and flash column chromatography (NH-silica gel, ethyl acetate/hexane) gave Compound 300 as a white solid in 29% yield (55 mg): MS (ESI+) m/z 559.4 (M+H).

Example 301 2-(2-(hydroxymethyl)-3-(1-methyl-6-oxo-5-(5-(piperazin-1-yl)pyridin-2-ylamino)-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 301

Example 301a tert-Butyl 4-(6-(5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)pyridin-3-yl)piperazine-1-carboxylate 301a

To a round-bottom flask equipped with a stir bar was added tert-butyl 4-(6-aminopyridin-3-yl)piperazine-1-carboxylate (2.00 g, 7.18 mmol), 3,5-dibromo-1-methylpyridin-2(1H)-one (2.87 g, 10.77 mmol), _Pd2 (dba) ₃ (657 mg, 0.718 mmol), XantPhos _{(665 mg, 1.15 mmol), Cs2CO3 (} 7.72 g, 23.7 mmol), and dioxane (40 mL). The reaction mixture was heated at 100°C for 40 hr. Ethyl acetate (200 mL) was added, and the resulting mixture was washed with water (30 mL x 3), brine (30 mL x 1), dried over _MgSO4 , filtered, and the solvent removed in vacuo. Dichloromethane/diethyl ether (1:2, 5 mL) was added, followed by sonication, and the precipitate was filtered to obtain a yellow solid 301a, 1.946 g (58%).

Example 301b tert- Butyl 4-(6-(5-(2-(acetoxymethyl)-3-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)phenyl)-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)pyridin-3-yl)piperazine-1-carboxylate 301b

To a microwave tube equipped with a stir bar were added 301a (500 mg, 1.077 mmol), 2-(2-(hydroxymethyl)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 114a (546 mg, 1.292 mmol), Pd(PPh ₃ ) ₄ (62 mg, 0.054 mmol), aqueous Na ₂ CO ₃ solution (1.0 N, 3.55 mL, 3.55 mmol), and 1,2-dimethoxyethane (4.3 mL). The mixture was reacted in a microwave at 130° C. for 10 min. Dichloromethane (200 mL) was added, and the resulting mixture was washed with water (3×30 mL), brine (30 mL), dried over MgSO ₄ , filtered, and the solvent was removed in vacuo. Silica gel column chromatography (methanol:dichloromethane=5:95) afforded 301b.

To a round-bottom flask equipped with a stir bar was added 301b and dichloromethane (10 mL). The solution was cooled to 0°C in an ice-water bath. TFA (1 mL) was added, and the resulting solution was stirred overnight. All volatiles were removed in vacuo, and THF (5 mL), isopropanol (5 mL), _H₂O (5 mL), and LiOH monohydrate (300 mg) were then added to the flask. The resulting mixture was stirred at room temperature for 1 hr. All solvents were removed in vacuo, and dichloromethane (200 mL) was added to the resulting residue. The solution was washed with water (3 x 30 mL) and brine (30 mL), dried over _MgSO₄ , filtered, and the solvent was removed in vacuo. Silica gel column chromatography (methanol:dichloromethane = 10:90) followed by preparative HPLC afforded 301 (9 mg) as a yellow solid. MS (ESI+) m/z 580.4 (M+H).

Example 302 10-(3-{5-[(1-ethyl-5-methyl-1H-pyrazol-3-yl)amino]-1-methyl-6-oxo-1,6-dihydropyridin-3-yl}-5-fluoro-2-(hydroxymethyl)phenyl)-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 302

Example 302a 5-Bromo-3-(1-ethyl-5-methyl-1H-pyrazol-3-ylamino)-1-methylpyridin-2(1H)-one 302a

1-ethyl-5-methyl-1H-pyrazole-3-amine (870mg, 7mmol), 3,5-dibromo-1-methylpyridine-2 (1H) -one (1.9g, 7mmol) and cesium carbonate (5g, 15mmol) and 1,4-dioxane (69mL) are added into the sealed tube equipped with a magnetic stirrer. After nitrogen is bubbled through the solution for 30min, Xantphos (480mg, 0.8mmol) and tris (dibenzylideneacetone) dipalladium (0) (450mg, 0.5mmol) are added, and the reaction mixture is heated to 100 DEG C and continues for 16h. After this, water (50mL) and ethyl acetate (50mL) are added. The aqueous layer is separated and then extracted with ethyl acetate (2X 50mL). The organic extracts combined are washed with salt water (100mL), then dried over sodium sulfate. The mixture is concentrated under reduced pressure to near dryness, and now the desired product is precipitated and then filtered. Washing with ether (10 mL) afforded 302a in 40% yield (870 mg).

Example 302b 10-(3-{5-[(1-ethyl-5-methyl-1H-pyrazol-3-yl)amino]-1-methyl-6-oxo-1,6-dihydropyridin-3-yl}-5-fluoro-2-(acetoxymethyl)phenyl)-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 302b

To a microwave tube equipped with a magnetic stirrer was added 302a (170 mg, 0.5 mmol), 230a (350 mg, 0.7 mmol), 1,2-dimethoxyethane (4 mL), and 1 M aqueous sodium carbonate solution (1.6 mL). After blowing _N for 15 min, Pd(PPh ₃ ) ₄ (31 mg, 0.03 mmol) was added. The mixture was heated to 130° C. in a microwave for 15 min. Thereafter, ethyl acetate (5 mL) and water (5 mL) were added. The separated aqueous layer was extracted with ethyl acetate (2 x 5 mL). The combined organic phases were washed with brine (10 mL), dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by column chromatography eluting with a gradient of dichloromethane to 60:35:5 dichloromethane:ether:methanol to afford 302b in 60% yield (190 mg).

302b (190 mg, 0.3 mmol), lithium hydroxide (70 mg, 1.6 mmol), THF (1.6 mL), isopropyl alcohol (1.6 mL) and water (3.2 mL) were added to a 25 mL round-bottom flask equipped with a magnetic stirrer. The mixture was stirred at room temperature for 2 h. Afterwards, ethyl acetate (5 mL) and water (5 mL) were added. The aqueous layer separated was extracted with ethyl acetate (2 × 5 mL). The combined organic phase was washed with salt water (10 mL), dried over sodium sulfate, and then filtered. The mixture was concentrated under reduced pressure to near dryness, at which point the desired product was precipitated and then filtered. 302 was obtained in a 51% yield (90 mg) by washing with ether (10 mL). MS (ESI+) m/z 559.4 (M+H).

Example 303 5-[5-fluoro-2-(hydroxymethyl)-3-[1-methyl-5-({5-[1-(oxetan-3-yl)piperidin-4-yl]pyridin-2-yl}amino)-6-oxo-1,6-dihydropyridin-3-yl]phenyl]-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-6-one 303

Compound 212b was converted to 303 according to Example 299. MS (ESI+) m/z 670.3 (M+H).

Example 304 10-[5-fluoro-2-(hydroxymethyl)-3-[1-methyl-5-({5-[4-(oxetan-3-yl)piperazin-1-yl]pyridin-2-yl}amino)-6-oxo-1,6-dihydropyridin-3-yl]phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 304

Example 304a (2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl-6-{4,4-dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.02,6]dodeca-2(6),7-dien-10-yl}phenyl)methyl acetate 304a

According to Example 211d, (2-bromo-6-{4,4-dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.0 ^{2 ,6} ]dodeca-2(6),7-dien-10-yl}phenyl)methyl acetate 167f was converted to 304a in 85% yield. LCMS: (M+H) ⁺ 479.

Example 304b (2-{4,4-dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-10-yl}-6-[1-methyl-5-({5-[4-(oxetane-3-yl)piperazin-1-yl]pyridin-2-yl}amino)-6-oxo-1,6-dihydropyridin-3-yl]phenyl)methyl acetate 304b

To a sealed tube was added a mixture of 304a (335 mg, 0.7 mmol), 5-bromo-1-methyl-3-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)pyridin-2(1H)-one 188e (294 mg, 0.7 mmol), Pd(dppf) _Cl2 (33 mg, _0.04 mmol), _K3PO4.3H2O (372 _mg , 1.4 mmol), and NaOAc (115 mg, 1.4 mmol) in _CH3CN (20 mL). The system was evacuated and refilled with _N2 . The reaction mixture was heated at 110°C for 2 h. It was then cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography eluting with 30:1 dichloromethane/methanol to afford 304b (208 mg, 43%) as a yellow solid. MS:[M+H] ⁺ 696.

To a solution of 304b (200 mg, 0.29 mol) in THF/isopropanol/water (6 mL/6 mL/2 mL) was added LiOH (70 mg, 2.9 mmol) at room temperature with stirring. The mixture was stirred for 0.5 h. Then, 20 mL of water was added, and the mixture was extracted with ethyl acetate ( ₃ x 30 mL). The combined organic layers were dried over _Na₂SO₄ and concentrated to give a yellow solid, which was further purified by preparative HPLC to give 304 as a white solid (100 mg, 53%). LCMS: [M+H] ⁺ 635. ¹ H NMR (500MHz, DMSO) δ1.22 (s, 6H), 2.37-2.42 (m, 6H), 2.56 (d, J = 11.0Hz, 1H), 3.06 (d, J = 4.5 Hz,,4H),3.41-3.44(m,2H),3.59(s,3H),3.85-3.90(m,1H),4.08-4.21(m,3H),4.35(d,J＝ 4.5Hz,2H),4.45-4.47(m,2H),4.54-4.57(m,2H),4.84-4.86(m,1H),6.45(s,1H),7.22(d, J＝9.0Hz,1H),7.31-7.47(m,4H),7.86(d,J＝3.0Hz,1H),8.37(s,1H),8.56(d,J＝2.0Hz,1H).

Example 305 5-[2-(Hydroxymethyl)-3-[4-methyl-6-({4-[1-(oxetan-3-yl)piperidin-4-yl]phenyl}amino)-5-oxo-4,5-dihydropyrazin-2-yl]phenyl]-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-diene-6-one 305

Example 305a {2-[4-methyl-6-({4-[1-(oxetan-3-yl)piperidin-4-yl]phenyl}amino)-5-oxo-4,5-dihydropyrazin-2-yl]-6-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-5-yl}phenyl}methyl acetate 305a

According to Example 136e, 5-bromo-1-methyl-3-(4-(1-(oxetan-3-yl)piperidin-4-yl)phenylamino)pyrazin-2(1H)-one 214b and (2-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-5-yl}-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methyl acetate 111a were used to give 305a in 68% yield. LCMS: (M+H) ⁺ 694

According to Example 136, 305a was converted to 305 in 51% yield. LCMS: (M+H) ⁺ 652. ¹ H NMR (500 MHz, DMSO) δ 9.18 (s, 1H), 7.88 (s, 1H), 7.86 (s, 1H), 7.52 (d, J = 7.5, 1H), 7.46 (t, J = 7.5, 1H), 7.38 (s, 1H), 7.17 (s, 1H), 7.32 (d, J = 7.5, 1H), 7.15 (d, J = 8.5, 2H), 4.78 (m, 1H),4.52(m,3H),4.43(m,3H),4.01(m,1H),3.87(m,1H),3.54(s,3H),3.37(m,1H),2.9 6(m,1H),2.86(m,1H),2.78(m,4H),2.43(m,2H),1.82(s,6H),1.72(m,2H),1.60(m,2H).

Example 306 10-[2-(Hydroxymethyl)-3-[1-methyl-6-oxo-5-(pyrimidin-4-ylamino)-1,6-dihydropyridazin-3-yl]phenyl]-4,4-dimethyl-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-9-one 306

Example 306b 10-[2-(acetoxymethyl)-3-[1-methyl-6-oxo-5-(pyrimidin-4-ylamino)-1,6-dihydropyridazin-3-yl]phenyl]-4,4-dimethyl-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-9-one 306b

Step 1: In a pressure flask, 5-chloro-1-methyl-3-(pyrimidin-4-ylamino)pyridazin-2(1H)-one (300 mg, 1.26 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bis(1,3,2-dioxaborolane) (577 mg, 2.27 mmol), potassium acetate (247 mg, 2.32 mmol), X-Phos(2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl) (120 mg, 20 mol%), and tris(dibenzylideneacetone)dipalladium(0) (115 mg, 10 mol%) were added. The flask was evacuated and filled with _N2 3X, dioxane (12 mL) was added, the vessel was sealed, and then heated to 90°C for 2 hr. The reaction was allowed to cool, then diluted with ethyl acetate, filtered through a pad of Celite, and concentrated under reduced pressure to afford 306a, which was used directly in the next step.

Step 2: 306a was dissolved in dioxane (7 mL) and transferred to a _pressure flask containing 189a (448 mg, 1.0 mmol), 10% _K₂CO₃ /water (2.5 ml), and bis(di-tert-butyl(4-dimethylaminophenyl)phosphine)palladium(II) dichloride (45 mg, 5 mol%). The flask was sealed and heated to 100°C overnight. The reaction was then diluted with ethyl acetate and water, separated, washed 3X with brine, dried over _Na₂SO₄ , filtered, and concentrated under reduced pressure. The residue was purified by chromatography (ISCO 24 g silica gel, eluting with 50-100% ethyl acetate/hexanes) _to afford 306b (250 mg, 35% over 2 steps).

Following Example 119, 306b (250 mg, 0.44 mmol), 1N LiOH (2.2 mL), THF (4.5 mL), and isopropanol (4.5 mL) were reacted. The product was powdered with ether and then dried under vacuum to afford 306 as an off-white solid (185 mg, 80% yield). MS (ESI+) m/z 529.3 (M+H).

Example 307 2-(2-(Hydroxymethyl)-3-(1-methyl-5-(5-(4-methylpiperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridazin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 307.

Example 307a 6-Chloro-2-methyl-4-(5-(4-methylpiperazin-1-yl)pyridin-2-ylamino)pyridazin-3(2H)-one 307a

According to Example 119b, 4-bromo-6-chloro-2-methylpyridazin-3(2H)-one (1.5 g, 6.7 mmol), 5-(4-methyl-piperazin-1-yl)-pyridin-2-ylamine (1.3 g, 6.7 mmol), cesium carbonate (4.8 g, 39.4 mmol), Xantphos (330 mg, 8.5 mol%), dioxane (50 ml), and tris(dibenzylideneacetone)dipalladium(0) (307 mg, 5 mol%) were reacted. Purification by column chromatography (ISCO 40 g silica gel, 50-100% ethyl acetate/hexanes followed by 0-10% methanol) gave 307a (1.2 g, 53%) as a light tan solid.

Example 307c 2-(2-(acetoxymethyl)-3-(1-methyl-5-(5-(4-methylpiperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridazin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 307c

Following Example 306b, 307a (422 mg, 1.26 mmol) was converted to the boron intermediate 307b, which was coupled with 109a (420 mg, 1.0 mmol) under Suzuki conditions. The crude product was purified by column chromatography (ISCO 24 g silica gel, 50-100% ethyl acetate/hexanes followed by 0-10% MeOH) to afford 307c (285 mg, 36% yield).

According to Example 119, 307c (285 mg, 0.45 mmol), 1N LiOH (2.2 mL), THF (5 mL), and isopropanol (5 mL) were reacted, the mixture was powdered with ethyl acetate, and then dried under vacuum to give 307 as a pale yellow solid (115 mg, 43% yield). MS (ESI+) m/z 595.6 (M+H).

Example 308 5-[5-Fluoro-2-(hydroxymethyl)-3-(1-methyl-5-{[5-(1-methylpyrrolidin-3-yl)pyridin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)phenyl]-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-6-one 308

Following Example 270, boronate ester 212b was reacted with bromide 202a to afford 308. MS (ESI+) m/z 614.3 (M+H).

Example 309 5-[2-(Hydroxymethyl)-3-{1-oxo-1H,2H,3H,4H,6H,7H,8H,9H-pyrido[3,4-b]indolizin-2-yl}phenyl]-1-methyl-3-{4H,6H,7H-pyrazolo[3,2-c][1,4]oxazin-2-ylamino}-1,2-dihydropyridin-2-one 309

Following Example 270, boronate ester 113a was reacted with bromide 110c to afford 309. MS (ESI+) m/z 541.2 (M+H).

Example 310 3-{[5-(4-ethylpiperazin-1-yl)pyridin-2-yl]amino}-5-[2-(hydroxymethyl)-3-{1-oxo-1H,2H,3H,4H,6H,7H,8H,9H-pyrido[3,4-b]indolizin-2-yl}phenyl]-1-methyl-1,2-dihydropyridin-2-one 310

Following Example 270, boronate ester 113a was reacted with bromide 138c to afford 310. MS (ESI+) m/z 608.3 (M+H).

Example 311 10-[2-(Hydroxymethyl)-3-[1-methyl-5-({5-[4-(oxetan-3-yl)piperazin-1-yl]pyridin-2-yl}amino)-6-oxo-1,6-dihydropyridin-3-yl]phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 311

Example 311a (2-{4,4-dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-10-yl}-6-[1-methyl-5-({5-[4-(oxetan-3-yl)piperazin-1-yl]pyridin-2-yl}amino)-6-oxo-1,6-dihydropyridin-3-yl]phenyl)methyl acetate 311a

To a sealed tube was added (2-{4,4-dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-10-yl}-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methyl acetate 304a (335 mg, 0.7 mmol), 5-bromo-1-methyl-3-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)pyridin-2(1H)-one 188e (294 mg, 0.7 mmol), Pd(dppf)Cl ₂ (33 mg, 0.04 mmol), K ₃ PO ₄ .3H ₂ O (372 mg, 1.4 mmol) and NaOAc (115 mg, 1.4 mmol) in CH 4 . The mixture was added to ₃ % ethanol (20 mL) of 4-nitropropane-2-nitropropane (311a). The system was evacuated and refilled with _nitrogen . The reaction mixture was heated at 110°C for 2 h. It was then cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography using 30:1 dichloromethane/methanol as the eluent to afford 311a (208 mg, 43%) as a yellow solid. MS: [M+H] ⁺ 696.

To a solution of 311a (200 mg, 0.29 mol) in THF/isopropanol/water (6 mL/6 mL/2 mL) was added LiOH (70 mg, 2.9 mmol) at room temperature with stirring. The mixture was stirred for 0.5 h. Then, 20 mL of water was added, and the mixture was extracted with ethyl acetate ( ₃ x 30 mL). The combined organic layers were dried over _Na₂SO₄ and concentrated to give a yellow solid, which was further purified by preparative HPLC to give 311 (100 mg, 53%) as a white solid. LCMS: [M+H] ⁺ 635. ¹ H NMR (500MHz, DMSO) δ1.22 (s, 6H), 2.37-2.42 (m, 6H), 2.56 (d, J = 11.0Hz, 1H), 3.06 (d, J = 4.5 Hz,,4H),3.41-3.44(m,2H),3.59(s,3H),3.85-3.90(m,1H),4.08-4.21(m,3H),4.35(d,J＝ 4.5Hz,2H),4.45-4.47(m,2H),4.54-4.57(m,2H),4.84-4.86(m,1H),6.45(s,1H),7.22(d, J＝9.0Hz,1H),7.31-7.47(m,4H),7.86(d,J＝3.0Hz,1H),8.37(s,1H),8.56(d,J＝2.0Hz,1H).

Example 312 10-[2-(Hydroxymethyl)-3-[1-methyl-5-({5-[4-(oxetan-3-yl)piperazin-1-yl]pyridin-2-yl}amino)-6-oxo-1,6-dihydropyridin-3-yl]phenyl]-4,4-dimethyl-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-9-one 312

Example 312a (2-{4,4-dimethyl-9-oxo-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-10-yl}-6-[1-methyl-5-({5-[4-(oxetane-3-yl)piperazin-1-yl]pyridin-2-yl}amino)-6-oxo-1,6-dihydropyridin-3-yl]phenyl)methyl acetate 312a

Following Example 136e, 5-bromo-1-methyl-3-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)pyridin-2(1H)-one (24-7) and (2-{4,4-dimethyl-9-oxo-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-10-yl}-6-(tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methyl acetate 131a were converted to 312a in 63% yield. LCMS: (M+H) ⁷⁰⁹

312a was converted to 312 in 60% yield according to Example 136. LCMS: (M+H) ⁺ 667. ^1H NMR(500MHz,MEOD)δ8.52(s,1H),7.93(m,1H),7.50(m,1H),7.42(m,3H),7 .30(s,1H),7.05(m,1H),4.73(t,J＝6.5,2H),4.64(t,J＝6,2H),4.56(m,2H) ,4.14(m,1H),3.98(m,1H),3.71(s,3H),3.67(m,1H),3.16(m,4H),3.11(m, 1H), 2.96 (m, 1H), 2.81 (s, 2H), 2.60 (m, 2H), 2.53 (s, 4H), 1.29 (d, J = 3, 6H).

Example 313 5-[5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-{[5-(2S)-(1-methylpyrrolidin-2-yl)pyridin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)phenyl]-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-6-one 313

Example 314 5-[5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-{[5-(2R)-(1-methylpyrrolidin-2-yl)pyridin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)phenyl]-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-6-one 314

Compounds 313 and 314 are enantiomers of racemate 288. Chiral separation of the racemic mixture 288 was performed on a Chiralpak AD, 4.6x50 mm, 3 mm column (mobile phase 55% isopropanol (w/0.1% triethylamine)/45% _CO2 , flow rate 5 mL/min) at 40°C to afford the individual enantiomers, with 313 eluting first and 314 eluting last.

Example 315 5-[2-(Hydroxymethyl)-3-(1-methyl-5-{[5-(1-methylpiperidin-4-yl)pyridin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)phenyl]-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-6-one 315

Example 315a [2-(1-methyl-5-{[5-(1-methylpiperidin-4-yl)pyridin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)-6-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]tridec-1(9),2(7)-dien-5-yl}phenyl]methyl acetate 315a

To a sealed tube was added a mixture of 5-bromo-1-methyl-3-(5-(1-methylpiperidin-4-yl)pyridin-2-ylamino)pyridin-2(1H)-one 130c (400 mg, 1.06 mmol), (2-{6-oxo-8-thia-5-azatricyclo[ ^7.4.0.02,7 ]trideca-1(9),2(7)-dien-5-yl}-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methyl acetate 111a (512 mg, 1.06 mmol), Pd(dppf) _{Cl2 (87 mg, 0.1 mmol), K3PO4.3H2O ( 566} mg, 2.12 mmol) and NaOAc (174 mg, 2.12 mmol) in _CH3CN (25 mL). The system was evacuated and then refilled with N ₂ . The reaction mixture was heated at 110° C. for 2 h. The mixture was then cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography using 10:1 dichloromethane/methanol as eluent to afford 315a as a brown solid (300 mg, 43%). MS: [M+H] ⁺ 652

To a solution of 315a (250 mg, 0.38 mmol) in propan-2-ol (10 mL), tetrahydrofuran (10 mL) and water (1.5 mL) was added LiOH (922 mg, 38 mmol). The mixture was stirred at 30°C for 2 h. It was then evaporated and the residue was purified by reverse phase Combi-flash chromatography using ₁ :4 water/ _CH3CN containing 0.3% _NH4HCO3 to give 315 (37 mg, 16%) as a white solid. MS: (M+H) ⁺ 610. ¹ H NMR(500MHz,MeOD)δ1.74-1.84(m,4H),1.87-1.95(m,4H),2.13-2.18(t,J＝11.5,2H) ,2.32(s,3H),2.52-2.61(m,3H),2.85-2.87(t,J＝4.5,2H),2.92-3.06(m,4H),3.71( s,3H),3.99-4.03(m,1H),4.12-4.17(m,1H),4.52-4.59(m,2H),7.03(d,1H),7.34(d ,1H),7.39(d,1H),7.42(d,1H),7.51(d,1H),7.56(d,1H),8.08(s,1H),8.62(s,1H).

Example 316 5-[5-Fluoro-2-(hydroxymethyl)-3-{1-oxo-1H,2H,3H,4H,6H,7H,8H,9H-pyrazino[1,2-a]indol-2-yl}phenyl]-1-methyl-3-[(4-(1-methylazetidin-3-yl)phenyl)amino)-1,2-dihydropyrazin-2-one 316

Example 316a 4-Fluoro-2-(4-methyl-6-(4-(1-methylazetidin-3-yl)phenylamino)-5-oxo-4,5-dihydropyrazin-2-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 316a

To a sealed tube was added a mixture of 5-bromo-1-methyl-3-(4-(1-methylazetidin-3-yl)phenylamino)pyrazin-2(1H)-one 298c (200 mg, 0.58 mmol), 4-fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 210d (280 mg, 0.58 mmol), Pd( _dppf ) _Cl2 (40 mg, 0.06 mmol), _K3PO4.3H2O ( ₃₀₀ mg, 1.16 mmol) and NaOAc (100 mg, 1.16 mmol) in _CH3CN (20 mL). The system was evacuated and then refilled with N ₂ . The reaction mixture was heated at 110° C. for 3 h. The mixture was then cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography using 15:1 dichloromethane/methanol as the eluent to afford 216a (150 mg, 42%) as a brown solid. MS: [M+H] ⁺ 625

To a solution of 316a (130 mg, 0.21 mmol) in propan-2-ol (7 mL), tetrahydrofuran (7 mL), and water (1 mL) was added LiOH (500 mg, 21 mmol). The mixture was stirred at 30°C for 2 h. After evaporation, the residue was purified by preparative HPLC to afford 316 as a white solid (14 mg, 12%). MS: (M+H) ⁵⁸³ . ¹ H NMR (500MHz, MEOD) δ1.77-1.81(m,2H),1.89-1.95(m,2H),2.46(s,3H),2.55(t,J＝6,2H),2.60-2.68(m,2H),3.64(s,3H),3.73-3.77(m,1 H),3.85(s,2H),4.16-4.27(m,1H),4.46-4.59(m,2H),6.72(s,1H),7.19-7.21(d,1H),7.8-7.30(d,2H),7.31-7.41(dd,2H),7.78(d,2H).

Example 317 10-[2-(Hydroxymethyl)-3-(1-methyl-5-{[5-(oxetan-3-yl)-4H,5H,6H,7H-pyrazolo[1,5-a]pyrazin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)phenyl]-4,4-dimethyl-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-9-one 317

Example 317a (2-{4,4-dimethyl-9-oxo-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-10-yl}-6-(1-methyl-5-{[5-(oxetane-3-yl)-4H,5H,6H,7H-pyrazolo[1,5-a]pyrazin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)phenyl)methyl acetate 317a

To a sealed tube were added 5-bromo-1-methyl-3-(5-(oxetane-3-yl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)pyridin-2(1H)-one 252a (275 mg, 0.7 mmol), (2-{4,4-dimethyl-9-oxo-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-10-yl}-4-fluoro-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methyl acetate 131a (356 mg, 0.7 mmol), Pd(dppf)Cl ₂ (59 mg, 0.07 mmol), K ₃ PO ₄ .3H _{2 A} mixture of 1,4-dihydro- ^1,4 -dihydro- ₂ -oxo- ...

To a solution of 317a (300 mg, 0.5 mol) in THF/isopropanol/water (6 mL/6 mL/2 mL) was added LiOH (70 mg, 2.9 mmol) at room temperature with stirring. The mixture was stirred for 0.5 h. 20 mL of _H₂O was then added, and the mixture was extracted with ethyl acetate (3 _x 30 mL). The combined organic layers were dried over _Na₂SO₄ and concentrated to afford a yellow solid, which was further purified by preparative HPLC to afford 317 (93 mg, 28%) as a white solid. MS: [M+H] ⁺ 627. ¹ H NMR (500MHz, DMSO) δ1.24 (s, 6H), 2.65 (s, 1H), 2.75 (d, J = 8.5Hz, 4H), 2.88-2.91 (m, 1H), 3.00-3.03 (m, 1 H),3.50(s,2H),3.57(s,3H),3.67(t,J＝6.0Hz,1H),3.84-3.89(m,1H),3.93(t,J＝6.0Hz,2H),4.00-4.05 (m,1H),4.34-4.36(m,2H),4.49(t,J＝6.0Hz,2H),4.59(t,J＝7.0Hz,2H),4.83(d,J＝5.0Hz,1H),5.91(s, 1H), 7.24 (d, J = 2.0Hz, 1H), 7.24-7.35 (m, 2H), 7.45 (t, J = 7.0Hz, 1H), 7.98 (d, J = 2.0Hz, 1H), 8.13 (s, 1H).

Example 318 10-[5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-{[5-(oxetane-3-yl)-4H,5H,6H,7H-pyrazolo[1,5-a]pyrazin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)phenyl]-4,4-dimethyl-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-9-one 318

Example 318a (2-{4,4-dimethyl-9-oxo-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-10-yl}-4-fluoro-6-(1-methyl-5-{[5-(oxetane-3-yl)-4H,5H,6H,7H-pyrazolo[1,5-a]pyrazin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)phenyl)methyl acetate 318a

To a sealed tube were added (2-{4,4-dimethyl-9-oxo-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-10-yl}-4-fluoro-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methyl acetate 247b (300 mg, 0.584 mmol), 5-bromo-1-methyl-3-(5-(oxetane-3-yl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)pyridin-2(1H)-one 252a (222 mg, 0.584 mmol), Pd(dppf)Cl ₂ (48 mg, 0.0584 mmol), K ₃ PO ₄ .3H ₂ A mixture of 1,4-dihydro-1,4-dihydro-2 _- oxo-1,4-dihydro-1,4-dihydro- ₂ -oxo-1,4-dihydro-1,4-dihydro-2-oxo-1,4-dihydro-2-oxo-1,4-dihydro-1,4-dihydro ^- 2 ...

To a solution of 318a (300 mg, 0.44 mol) in THF/isopropanol/water (6 mL/6 mL/2 mL) was added LiOH (100 mg, 4.16 mmol) at room temperature. The mixture was stirred for 0.5 h. Then, 20 mL of _H₂O was added, and the mixture was extracted with ethyl acetate (30 mL x 3). _The combined organic layers were dried over _Na₂SO₄ and concentrated to give a yellow solid, which was further purified by preparative HPLC to give 318 (68.7 mg, 52.3%) as a white solid. ¹ H NMR (500MHz, DMSO) δ8.17(s,1H),7.98(d,J=6Hz,1H),7.30(t,2H),7.17(t,1H),5.91(s,1H),4.88(t,1H),4.58(t,2H),4.48(t,2H),4.32 (d,1H),4.05(t,1H),4.02(m,3H),3.94(m,2H),3.92(s,1H),3.83(s,1H),3.30(s,1H),3.02(m,1H),2.88(s,4H),2.73(s,2H),1.20(s,6H)

Example 319 2-(2-(Hydroxymethyl)-3-(1-methyl-5-(5-(oxetan-3-yl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 319

Example 319a [2-(1-methyl-5-{[5-(oxetan-3-yl)-4H,5H,6H,7H-pyrazolo[1,5-a]pyrazin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)-6-{1-oxo-1H,2H,3H,4H,6H,7H,8H,9H-pyrazino[1,2-a]indol-2-yl}phenyl]methyl acetate 319a

To a sealed tube were added 5-bromo-1-methyl-3-(5-(oxetan-3-yl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)pyridin-2(1H)-one 252a (265 mg, 0.7 mmol), 2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 114a (320 mg, 0.7 mmol), Pd(dppf) _Cl2 (56 mg, 0.07 mmol) _{, K3PO4.3H2O ( 367} mg, 1.4 mmol) and NaOAc (113 mg, 1.4 mmol) in _CH3 The mixture was added to 4% paraformaldehyde (20 mL) in 4% paraformaldehyde (CHCl) (20 mL). The system was evacuated and refilled with _N₂ . The reaction mixture was heated at 110°C for 2 h. It was then cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography using 30:1 dichloromethane/methanol to afford 319a (200 mg, 38%) as a yellow solid. MS: [M+H] ^⁺ 638.

To a solution of 319a (150 mg, 0.24 mol) in THF/isopropanol/water (6 mL/6 mL/2 mL) was added LiOH (70 mg, 2.9 mmol) at room temperature with stirring. The mixture was stirred for 0.5 h. Then, 20 mL of water was added, and the mixture was extracted with ethyl acetate ( ₃ x 30 mL). The combined organic layers were dried over _Na₂SO₄ and concentrated to give a yellow solid, which was further purified by preparative HPLC to give 319 (50 mg, 28%) as a white solid. MS: [M+H] ⁺ 596. ¹ H NMR (500MHz, DMSO) δ1.23 (t, J = 7.0Hz, 2H), 1.70-2.08 (m, 5H), 2.74-2.76 (m, 2H), 3. 50(s,2H),3.57(s,3H),3.67(s,1H),3.91-3.94(m,3H),4.03-4.16(m,4H),4.35(s,2 H),4.48-4.50(m,2H),4.58-4.60(m,2H),4.61(s,1H),5.91(s,1H),6.51(s,1H),7. 23(s,1H),7.30-7.34(m,2H),7.44-7.47(m,1H),7.98(d,J＝2.0Hz,1H),8.12(s,1H).

Example 320 2-(5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 320

Example 320a N-methyl(1-methyl-3-nitro-1H-pyrazol-5-yl)methanamine 320a

To a solution of MeNH ₂ (30 wt% H ₂ O) (2.5 g, 20 mmol) in acetone (10 mL) was added K ₂ CO ₃ (415 mg, 3 mmol) with stirring at 0° C. (ice bath), followed by the dropwise addition of a solution of 5-(bromomethyl)-1-methyl-3-nitro-1H-pyrazole (220 mg, 1 mmol) in acetone (5 mL). The reaction mixture was then warmed to room temperature and stirred for 3 h. The solvent was removed, and the residue was extracted with dichloromethane (15 mL×3), dried over Na ₂ SO ₄ , and then concentrated to afford 320a (170 mg, 99%) as a yellow oil, which was used in the next step without further purification. LCMS: (M+H) ⁺ 171

Example 320b N-methyl-N-((1-methyl-3-nitro-1H-pyrazol-5-yl)methyl)oxetane-3-amine 320b

To a mixture of 320a (170 mg, 1 mmol) in methanol (4 mL) was added ZnCl₂ ( ₁ mmol/L in diethyl ether) (2 mL, 2 mmol) and oxetane-3-one (150 mg, 2 mmol) at room temperature under nitrogen, followed by _NaBH₃CN (130 mg, 2 mmol). The reaction mixture was warmed to 50°C and stirred for 3 h. The mixture was then cooled to room temperature and the solvent removed. The residue was purified by flash column chromatography using 50:1 dichloromethane/methanol as the eluent to afford 320b (180 mg, 80% over 2 steps) as a yellow solid. LCMS: (M+H) ²²⁷ . ¹ H NMR (500MHz, DMSO) δ6.99 (s, 1H), 4.52 (t, J = 6.5, 2H), 4.42 (t, J = 6, 2H), 3.98 (s, 3H), 3.63 (m, 1H), 3.50 (s, 2H), 2.03 (s, 3H).

Example 320c 1-Methyl-5-((methyl(oxetan-3-yl)amino)methyl)-1H-pyrazol-3-amine 320c

To a solution of 320b (1.8 g, 7.96 mmol) in ethanol (20 mL) and water (20 mL) were added NH ₄ Cl (3.3 g, 63.6 mmol) and iron powder (1.80 g, 31.8 mmol). The reaction mixture was heated at 70°C for 2 h. Thereafter, the mixture was cooled to room temperature and filtered. The filtrate was evaporated, and the residue was extracted with dichloromethane (30 mL×3), dried over Na ₂ SO ₄ , and then concentrated to give the crude product, which was purified on a flash column eluted with 50:1 dichloromethane/methanol containing 0.5% triethylamine to give 320c (1.3 g, 83%) as a yellow oil. LCMS: (M+H) ⁺ 197

Example 320d 5-Bromo-1-methyl-3-(1-methyl-5-((methyl(oxetan-3-yl)amino)methyl)-1H-pyrazol-3-ylamino)pyridin-2(1H)-one 320d

Following Example 136d, 320c was reacted with 3,5-dibromo-1-methylpyridin-2(1H)-one to afford 320d in 63% yield. LCMS: (M+H) ^{383. 1H} NMR (500 MHz, DMSO) δ 8.35 (s, 1H), 7.99 (d, J = 2.5, 1H), 7.36 (d, J = 2.5, 1H), 5.99 (s, 1H), 4.50 (t, J = 7, 2H), 4.40 (t, J = 6.5, 2H), 3.77 (s, 3H), 3.57 (m, 1H), 3.49 (s, 3H), 3.35 (s, 2H), 2.01 (s, 3H).

Example 320e 4-Fluoro-2-(1-methyl-5-(1-methyl-5-((methyl(oxetan-3-yl)amino)methyl)-1H-pyrazol-3-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 320e

Following Example 136e, 210d was converted to 320e in 71% yield.

320e was converted to 320 in 66% yield according to Example 136. LCMS: (M+H) ⁺ 616. ¹ H NMR (500MHz, DMSO) δ8.07 (s, 1H), 7.97 (s, 1H), 7.30 (m, 2H), 7.17 (d, J = 9. 5,1H),6.53(s,1H),6.02(s,1H),4.88(m,1H),4.50(t,J＝6.5,2H),4.40( t,J＝5.5,2H),4.32(s,2H),4.14(m,3H),3.89(m,1H),3.70(s,3H),3.57( m,4H),2.59(m,2H),2.47(m,2H),2.00(s,3H),1.80(m,2H),1.70(m,2H).

Example 321 10-[5-fluoro-2-(hydroxymethyl)-3-{1-methyl-5-[(1-methyl-5-{[methyl(oxetan-3-yl)amino]methyl}-1H-pyrazol-3-yl)amino]-6-oxo-1,6-dihydropyridin-3-yl}phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 321

Example 321a (2-{4,4-dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-10-yl}-4-fluoro-6-{1-methyl-5-[(1-methyl-5-{[methyl(oxetan-3-yl)amino]methyl}-1H-pyrazol-3-yl)amino]-6-oxo-1,6-dihydropyridin-3-yl}phenyl)methyl acetate 321a

Following Example 136e, 5-bromo-1-methyl-3-(1-methyl-5-((methyl(oxetan-3-yl)amino)methyl)-1H-pyrazol-3-ylamino)pyridin-2(1H)-one 320d and 230a were reacted to afford 321a in 65% yield. LCMS: (M+H) ⁺ 672

According to Example 136, 321a was converted to 321 in 59% yield. LCMS: (M+H) ⁺ 630. ^1H NMR (500 MHz, DMSO) δ 8.07 (s, 1H), 7.97 (d, J = 2.5, 1H), 7.33 (d, J = 2.5, 1H), 7.31 (d, J = 3, 1H), 7.29 (d, J = 2, 1H), 7.17 (dd, J = 9.5, 1H), 6.51 (s, 1H), 6.02 (s, 1H), 4.89 (t, J = 4.5, 1H) ,4.50(t,J＝6.5,2H),4.40(t,J＝6,2H),4.33(d,J＝4.5,2H),4.21(m,2H),4.13(m,1H),3. 88(m,1H),3.70(s,3H),3.57(m,4H),2.57(s,2H),2.42(s,2H),2.00(s,3H),1.22(s,6H).

Example 322 10-[5-fluoro-2-(hydroxymethyl)-3-{1-methyl-5-[(1-methyl-5-{[methyl(oxetan-3-yl)amino]methyl}-1H-pyrazol-3-yl)amino]-6-oxo-1,6-dihydropyridin-3-yl}phenyl]-4,4-dimethyl-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-9-one 322

Example 322a (2-{4,4-dimethyl-9-oxo-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-10-yl}-4-fluoro-6-{1-methyl-5-[(1-methyl-5-{[methyl(oxetan-3-yl)amino]methyl}-1H-pyrazol-3-yl)amino]-6-oxo-1,6-dihydropyridin-3-yl}phenyl)methyl acetate 322a

To a sealed tube were added 5-bromo-1-methyl-3-(1-methyl-5-((methyl(oxetan-3-yl)amino)methyl)-1H-pyrazol-3-ylamino)pyridin-2(1H)-one 320d (250 mg, 0.65 mmol), (2-{4,4-dimethyl-9-oxo-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-10-yl}-4-fluoro-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methyl acetate 247b (334 mg, 0.65 mmol), Pd(dppf)Cl ₂ (55 mg, 0.07 mmol), K ₃ PO ₄ .3H ₂ A mixture of 1,4-dihydro-1,4-dihydro-2 _- oxo-1,4-dihydro-1,4-dihydro- ₂ -oxo-1,4-dihydro-1,4-dihydro-2-oxo-1,4-dihydro-2-oxo-1,4-dihydro-1,4-dihydro-2 ...2,4-dihydro-1,4-dihydro-2,4-dihydro-1,4-dihydro-2,4-dihydro-1,4-dihydro-2,4-dihydro-2,4-dihydro-1,4-dihydro-2,4-dihydro-2,4-dihydro-1,4-dihydro-2,4-dihydro-2,4-dihydro-1,4-dihydro-2,4-dihydro-2,4-dihydro ^-1,4-

To a solution of 322a (250 mg, 0.35 mol) in THF/isopropanol/water (6 mL/6 mL/2 mL) was added LiOH (70 mg, 2.9 mmol) at room temperature with stirring. The mixture was stirred for 0.5 h. Then, 20 mL of water was added, and the mixture was extracted with ethyl acetate (30 _mL x 3). The combined organic layers were dried over _Na₂SO₄ and concentrated to give a yellow solid, which was further purified by preparative HPLC to give 322 (94 mg, 36%) as a white solid. MS: [M+H] ⁺ 647. ¹ H NMR(500MHz,DMSO)δ1.23(s,6H),2.01(s,3H),2.50-2.52(m,2H),2.76(s,2H),2.88- 2.91(m,1H),3.04(s,1H),3.55-3.57(m,4H),3.70(s,3H),3.86(t,J＝6.0Hz,1H),4.0 6(d,J＝5.5Hz,1H),4.33-4.41(m,4H),4.50(t,J＝6.5Hz,2H),4.88(t,J＝5.0Hz,1H),6 .02(s,1H),7.16-7.18(m,1H),7.29-7.34(m,2H),7.97(d,J＝2.5Hz,1H),8.07(s,1H).

Example 323 10-[2-(Hydroxymethyl)-3-(1-methyl-5-{[5-(1-methylpiperidin-4-yl)pyridin-2-yl]amino}-6-oxopyridin-3-yl)phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-9-one 323

Example 323a (2-{4,4-dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.0 ^2,6 ]dodeca-2(6),7-dien-10-yl}-6-(1-methyl-5-{[5-(1-methylpiperidin-4-yl)pyridin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)phenyl)methyl acetate 323a

To a 25 mL sealed tube were added 5-bromo-1-methyl-3-(5-(1-methylpiperidin-4-yl)pyridin-2-ylamino)pyridin-2(1H)-one 282c (376 mg, 1.0 mmol), (2-{4,4-dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.0 ^{2 ,6} ]dodeca-2(6),7-dien-10-yl}-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methyl acetate (478 mg, 1.0 mmol), CH ₃ COONa (168 mg, 2.0 mmol), K ₃ PO ₄ (546 mg, 2.0 mmol), and PdCl ₂ (dppf) (84 mg, 0.1 mmol), which were suspended in CH ₃ CN (25 mL) and H ₂ The mixture was added to 1 mL of 4-[ ^...

To a solution of 323a (200 mg, 0.3 mmol) in propan-2-ol (10 mL), tetrahydrofuran (10 mL), and water (2 mL) was added LiOH (1.1 g, 57 mmol). The mixture was stirred at 30°C for 2 h. The residue was then evaporated and purified by preparative HPLC to afford 323 (41 mg, 22%) as a white solid. MS: (M+H) ⁺ 607. ¹ H NMR(500MHz,DMSO)δ8.65(s,1H),8.50(s,1H),8.05(s,1H),7.51(m,2H),7. 46(m,3H),7.31(d,J＝8.5,1H),6.50(s,1H),4.83(m,1H),4.39(m,2H),4.13( m,1H),3.85(m,1H),3.59(s,3H),3.03(m,1H),2.91(m,3H),2.84(m,2H),2. 64(m,1H),2.46(m,1H),2.17(s,3H),1.80(m,2H),1.60(m,4H),1.22(s,6H).

Example 324 5-[5-fluoro-2-(hydroxymethyl)-3-{1-methyl-5-[(1-methyl-5-{[methyl(oxetan-3-yl)amino]methyl}-1H-pyrazol-3-yl)amino]-6-oxo-1,6-dihydropyridin-3-yl}phenyl]-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-6-one 324

Example 324a (4-fluoro-2-{1-methyl-5-[(1-methyl-5-{[methyl(oxetan-3-yl)amino]methyl}-1H-pyrazol-3-yl)amino]-6-oxo-1,6-dihydropyridin-3-yl}-6-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]tridec-1(9),2(7)-dien-5-yl}phenyl)methyl acetate 324a

To a sealed tube were added 5-bromo-1-methyl-3-(1-methyl-5-((methyl(oxetan-3-yl)amino)methyl)-1H-pyrazol-3-ylamino)pyridin-2(1H)-one 320d (150 mg, 0.39 mmol), (4-fluoro-2-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7)-dien-5-yl}-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methyl acetate 247b (195 mg, 0.39 mmol), Pd(dppf)Cl ₂ (33 mg, 0.04 mmol), K ₃ PO ₄ .3H ₂ To a mixture of 1,4-dihydro-1,4-dihydro- _{2 -} oxo- ...2,4-dihydro-1,4-dihydro-2,4-dihydro-1,4-dihydro-2,4 ^- dihydro-

To a solution of 324a (150 mg, 0.22 mol) in THF/isopropanol/water (6 mL/6 mL/2 mL) was added LiOH (70 mg, 2.9 mmol) at room temperature with stirring. The mixture was stirred for 0.5 h. Then, 20 mL of H ₂ O was added and the mixture was extracted with ethyl acetate (3 x 30 mL). The combined organic layers were dried over Na ₂ SO ₄ and concentrated to give a yellow solid, which was further purified by preparative HPLC to give 324 (43 mg, 31%) as a white solid. MS: [M+H] ⁺ 633. ¹ H NMR(500MHz,DMSO)δ1.75-1.80(m,4H),2.00(s,4H),2.85(s,4H),2.36(s,1H),2.64(s,1H),2.7 8(s,2H),2.83-2.89(m,1H),2.94-3.01(m,1H),3.58(s,3H),3.69(s,3H),3.86-3.89(m,1H),4.0 2-4.07(m,1H),4.32(d,J＝3.0Hz,2H),4.39(t,J＝6.5Hz,2H),4.49(t,J＝6.5Hz,2H),4.85(d,J＝4. 5Hz,1H),6.01(s,1H),7.15-7.18(m,1H),7.28-7.32(m,2H),7.96(d,J＝2.0Hz,1H),8.06(s,1H).

Example 325 10-[2-(Hydroxymethyl)-3-(1-methyl-5-{[5-(1-methylpiperidin-4-yl)pyridin-2-yl]amino}-6-oxopyridin-3-yl)phenyl]-4,4-dimethyl-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-9-one 325

Example 325a (2-{4,4-dimethyl-9-oxo-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodeca-1(8),2(6)-dien-10-yl}-6-(1-methyl-5-{[5-(1-methylpiperidin-4-yl)pyridin-2-yl]amino}-6-oxopyridin-3-yl)phenyl)methyl acetate 325a

To a 25 mL sealed tube were added 5-bromo-1-methyl-3-(5-(1-methylpiperidin-4-yl)pyridin-2-ylamino)pyridin-2(1H)-one 282c (376 mg, 1.0 mmol), (2-{4,4-dimethyl-9-oxo-7-thia-10-azatricyclo[6.4.0.0 ^2,6 }]dodeca-1(8),2(6)-dien-10-yl}-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methyl acetate 131a (495 mg, 1.0 mmol), CH ₃ COONa (168 mg, 2.0 mmol), K ₃ PO ₄ (546 mg, 2.0 mmol), and PdCl ₂ (dppf) (84 mg, 0.1 mmol), and the mixture was suspended in CH ₃ The product was dissolved in CN (25 mL) and water (1 mL). The mixture was heated at 110°C for 2 h. The product was then evaporated and the residue was purified by column chromatography using 50:1 dichloromethane/methanol as eluent to afford 325a (278 mg, 42%) as a brown solid. MS: (M+H) ⁺ 666.

To a solution of 325a (200 mg, 0.3 mmol) in isopropanol (10 mL), tetrahydrofuran (10 mL), and water (2 mL) was added LiOH (1.1 g, 57 mmol). The mixture was stirred at 30°C for 2 h. It was then evaporated and the residue was purified by preparative HPLC to afford 325 as a white solid (54 mg, 29%). MS: (M+H) ⁺ 624. ¹ H NMR (500MHz, DMSO) δ8.66(s,1H),8.50(s,1H),8.10(s,1H),7.51(m,2H),7.46(m,3H),7.31(d,J＝8.5,1H),4.83(m,1H),4.39(m,2H),4.13(m,1H) ,3.85(m,1H),3.59(s,3H),3.03(m,1H),2.91(m,3H),2.84(m,2H),2.64( m,1H),2.46(m,1H),2.23(s,3H),1.80(m,2H),1.66(m,4H),1.20(s,6H).

Example 326 2-(5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-(5-(4-methylpiperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4-dihydropyrazino[1,2-a]indol-1(2H)-one 326

Example 326a 4-Fluoro-2-(1-oxo-3,4-dihydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 326a

326a was prepared according to Examples 102c and 102d.

Example 326b 4-Fluoro-2-(1-methyl-5-(5-(4-methylpiperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4-dihydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 326b

According to Example 148h, 478 mg of 326a and 378 mg of 5-bromo-1-methyl-3-(5-(4-methylpiperazin-1-yl)pyridin-2-ylamino)pyridin-2(1H)-one 197d were reacted to give 326b (324 mg, 50%) as a yellow solid. MS: [M+H] ⁺ 650

According to Example 148, 260 mg of 326b was converted to a white solid 326 (100 mg, 41%). MS: [M+H] ⁺ 607. ¹ H NMR (500 MHz, DMSO) δ 8.58 (d, J = 2.0, 1H), 8.39 (s, 1H), 7.87 (d, J = 3.0, 1H), 7.73 (d, J = 8.0, 1H), 7.63 (d, J = 8.0, 1H), 7.46 (dd, J = 9.0, 1H), 7.36 (m, 3H), 7.23 (m, 2H), 7.17 (m, 2H), 4.99 (d, J = 4.5, 1H), 4.65 (m, 1H), 4.50 (m, 1H), 4.38 (s, 2H), 4.29 (m, 1H), 4.10 (m, ^1H). H), 3.59 (s, 3H), 3.04 (t, J = 4.0, 4H), 4.36 (t, J = 4.0, 4H), 2.20 (s, 3H).

Example 327 2-(5-fluoro-2-(hydroxymethyl)-3-(4-methyl-6-(4-(4-(oxetan-3-yl)piperazin-1-yl)phenylamino)-5-oxo-4,5-dihydropyrazin-2-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 327

Example 327a tert-Butyl 4-(4-nitrophenyl)piperazine-1-carboxylate 327a

According to Example 188a, 1-fluoro-4-nitrobenzene (2.8 g, 20 mmol) and N-Boc-piperazine (11 g, 60 mmol) were reacted to give 327a as a yellow solid (3 g, 51%). MS: [M+H] ⁺ 308.

Example 327b tert-Butyl 4-(4-aminophenyl)piperazine-1-carboxylate 327b

According to Example 188b, 327a was reduced to a yellow solid 327b (1.35 g, 99%). MS: [M+H] ⁺ 278.

Example 327c tert- Butyl 4-(4-(6-bromo-4-methyl-3-oxo-3,4-dihydropyrazin-2-ylamino)phenyl)piperazine-1-carboxylate 327c

According to Example 188c, 327b (2.6 g, 9.4 mmol) and 3,5-dibromo-1-methylpyrazin-2(1H)-one (2.5 g, 9.4 mmol) were reacted to give 327c (2.96 g, 68%) as a yellow solid. MS: [M+H] ⁺ 464.

Example 327d 5-Bromo-1-methyl-3-(4-(piperazin-1-yl)phenylamino)pyrazin-2(1H)-one 327d

According to Example 188d, 327c was deprotected to give 327d (1.57 g, 99%) as a yellow solid. MS: [M+H] ⁺ 364.

Example 327e 5-Bromo-1-methyl-3-(4-(4-(oxetan-3-yl)piperazin-1-yl)phenyl-amino)pyrazin-2(1H)-one 327e

According to Example 188e, 1.2 g (3 mmol) of 327d was converted into a yellow solid 327e (943 mg, 75%). MS: [M+H] ⁺ 420.

Example 327f 4-Fluoro-2-(4-methyl-6-(4-(4-(oxetan-3-yl)piperazin-1-yl)phenylamino)-5-oxo-4,5-dihydropyrazin-2-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 327f

According to Example 148h, 300 mg of 4-fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 210d was reacted with 287 mg of 327e to give 327f (220 mg, 51%) as a yellow solid. LCMS: [M+H] ⁺ 696

According to Example 148, 220 mg of 327f was converted to a white solid 327 (87 mg, 42%). ¹ H NMR (500 MHz, DMSO) δ 9.11 (s, 1H), 7.81 (d, J = 8.5, 1H), 7.43 (s, 1H), 7.35 (t, J = 7.0, 1H), 7.29 (d, J = 9.0, 1H), 6.88 (d, J = 8.5, 2H), 6.53 (s, 1H), 4.86 (s, 1H), 4.56 (t, J = 6.5, 2H), 4. 67(d,t＝5.5,3H),4.42(m,1H),4.11(m,3H),3.89(m,1H),3.53(s,3H),3.43(s,1H),3.10 (t,J＝4.5,4H),2.62(m,2H),2.46(m,2H),2.39(t,J＝4.5,4H),1.79(s,2H),1.70(s,2H).

Example 328 2-(5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridazin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 328

Example 328a tert-Butyl 4-(6-(6-chloro-2-methyl-3-oxo-2,3-dihydropyridazin-4-ylamino)pyridin-3-yl)piperazine-1-carboxylate 328a

To a 100-mL single-necked round-bottom flask equipped with a magnetic stirrer and a reflux condenser was added 1,4-dioxane (50 mL), tert-butyl 4-(6-aminopyridin-3-yl)piperazine-1-carboxylate 188b (2.0 g, 7.2 mmol), 4-bromo-6-chloro-2-methyl-pyridin-3(2H)-one (1.6 g, 7.2 mmol), and cesium carbonate (4.7 g, 14.4 mmol). After nitrogen was bubbled through the resulting mixture for 30 minutes, XantPhos (500 mg, 0.9 mmol) and tris(dibenzylideneacetone)dipalladium(0) (450 mg, 0.45 mmol) were added, and the reaction mixture was heated under reflux for 3 hours. Afterwards, the reaction was cooled to room temperature, partitioned between ethyl acetate (100 mL) and water (100 mL), and then filtered. The aqueous layer was separated and then extracted with ethyl acetate (50 mL x 2). The organic layers were combined, washed with brine (50 mL), and then dried over sodium sulfate. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by flash column chromatography using petroleum ether/ethyl acetate as eluent to afford 328a (1.4 g, 43%). MS: [M+H] ⁺ 421

Example 328b 6-Chloro-2-methyl-4-(5-(piperazin-1-yl)pyridin-2-ylamino)pyridazin-3(2H)-one hydrochloride 328b

Intermediate 328a (1.4 g, 3.3 mmol) was suspended in 4.0 M HCl/dioxane (10 mL). The reaction mixture was stirred at room temperature for 2 h and then concentrated under reduced pressure to afford 328b (1.1 g, 96%). MS: [M+H] ⁺ 321.

Example 328c 6-Chloro-2-methyl-4-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)pyridazin-3(2H)-one 328c

A mixture of 328b (1.2 g, 3.5 mmol), oxetane-3-one (0.5 g, 7.0 mmol), NaBH ₃ CN (0.44 g, 7.0 mmol), and zinc chloride (0.93 g, 7.0 mmol) in methanol (125 ml) was stirred at 50° C. for 3.5 hours. The mixture was added to water and extracted with dichloromethane. The organic layers were combined and concentrated under reduced pressure. The residue was purified by column chromatography eluting with 25:1 dichloromethane/methanol to afford 328c (0.63 g, 50%). MS: [M+H] ⁺ 377.

Example 328d 4-Fluoro-2-(1-methyl-5-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridazin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 328d

To a sealed tube was added a mixture of 4-fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 210d (420 mg, 0.9 mmol), 328c (300 mg, 0.78 mmol), Pd(dppf) _Cl₂ (66 mg, 0.078 mmol), _{K₃PO₄.3H₂O ( 360} mg, 1.56 mmol), and NaOAc (130 mg, 1.56 mmol) in _CH₃CN (20 ml). The system was evacuated and then refilled with _N₂ . The reaction mixture was heated at 110°C for 2 h. The mixture was then cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure and the resulting residue was purified by flash chromatography using 30:1 dichloromethane/methanol as eluent to afford 328d as a yellow solid (400 mg, 72%). MS: [M+H] ⁺ 696.

To a solution of 328d (350 mg, 0.5 mol) in THF/iPA/H ₂ O (6 ml/6 ml/2 ml) was added LiOH (600 mg, 25 mmol) at room temperature with stirring. The mixture was stirred for 0.5 h. Then, 20 mL of water was added, and the resulting mixture was extracted with ethyl acetate (30 mL ^x 3). The combined organic layers were dried over Na ₂ SO ₄ and concentrated to give a yellow solid, which was further purified by preparative HPLC to give 328 (100 mg, 27%) as a white solid. NMR (500MHz, DMSO) δ9.25 (s, 1H), 8.41 (s, 1H), 7.97 (d, J = 3.5Hz, 1H), 7.39-7.45 (m, 3H), 7.25-7.28 ( m,1H),6.52(s,2H),4.66(t,J＝5.0Hz,2H),4.55(t,J＝6.5Hz,2H),4.41-4.46(m,3H),4.31-4.34(m,1 H),4.11-4.18(m,3H),3.85-3.87(m,1H),3.77(s,3H),3.43(t,J＝6.5Hz,1H),3.12(t,J＝5.0Hz,4H), 2.54-2.63(m,2H),2.46(t,J＝6.0Hz,2H),2.39(t,J＝5.0Hz,4H),1.79(t,J＝5.5Hz,2H),1.69(s,2H).

Example 329 5-[5-Fluoro-2-(hydroxymethyl)-3-(1-methyl-5-{[5-(4-methylpiperazin-1-yl)pyridin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)phenyl]-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7),10,12-tetraen-6-one 329

Example 329a (2Z)-3-(1-Benzothiophene-3-yl)prop-2-enoic acid 329a

A suspension of benzo[b]thiophene-3-carboxaldehyde (4.9 g, 30 mol), malonic acid (6.6 g, 60 mmol), and piperidine (1 mL) in 100 mL of anhydrous pyridine was heated at 110°C overnight. The reaction mixture was cooled to room temperature, and the solvent was removed in vacuo. The residue was dissolved in 100 mL of water, and the pH of the solution was adjusted to approximately 3 by adding 1N hydrochloric acid. The resulting suspension was filtered, and the yellow solid was collected, washed with water (3 x 30 mL), and dried in vacuo to afford 329a (5.5 g, 89%) as a yellow solid. MS: [M+H] ⁺ 205.

Example 329b 3-(1-Benzothiophene-3-yl)propanoic acid 329b

A suspension of 329a (5.5 g, 27 mmol) and 10% Pd/C (600 mg) in 1:1 methanol/ethyl acetate (100 mL) was hydrogenated in a Parr apparatus at 50 psi overnight. The mixture was filtered through a pad of Celite, and the filtrate was concentrated under reduced pressure to afford crude 329b (5.0 g, 90%) as a brown solid, which was used in the next step without further purification. MS: [M+H] ⁺ 207. ¹ H NMR (500 MHz, CDCl 3 ) δ 2.81-2.84 (t, J=8.5, 2H), 3.18-3.21 (m, 2H), 7.16 (s, 1H), 7.34-7.41 (m, 2H), 7.74-7.76 (d, 1H), 7.85-7.87 (d, 1H).

Example 329c 3-(1-Benzothiophene-3-yl)propionyl chloride 329c

Intermediate 329b (5.0 g, 24 mmol) was heated in thionyl chloride (15 mL) at 100°C for 4 hours. The mixture was cooled to room temperature, and the solvent was evaporated in vacuo. 30 mL of dichloromethane was added to the residue, and the resulting mixture was evaporated under reduced pressure to afford 329c as a brown solid (5.3 g, 97%), which was used in the next step without further purification. MS: [M+H] ⁺ 225.

Example 329d 7-thiatricyclo[6.4.0.0 ^2,6 ]dodecano-1(8),2(6),9,11-tetraen-5-one 329d

To 329c (5.0 g, 22.3 mmol) in dichloromethane (50 mL) was added anhydrous aluminum chloride (5.9 g, 44.6 mmol). The mixture was stirred at room temperature overnight. The reaction solution was evaporated in vacuo, and the residue was purified by column chromatography using 20:1 dichloromethane/methanol as the eluent to afford 329d (2.3 g, 55%) as a yellow solid. MS: [M+H] ⁺ 189. ¹ H NMR (500 MHz, CDCl3) δ 3.05-3.07 (m, 2H), 3.21-3.23 (m, 2H), 7.45-7.52 (m, 2H), 7.88-7.91 (m, 2H).

Example 329e N-[(5Z)-7-thiatricyclo[6.4.0.0 ^2,6 ]dodecano-1(8),2(6),9,11-tetraen-5-ylidene]hydroxylamine 329e

To a solution of _NH₂OH -HCl (18.4 g, 266 mmol) in MeOH (600 mL) at 0°C was slowly added _CH₃COONa (21.8 g, 266 mmol). After stirring at 0°C for 30 min, 329d (10 g, 53 mmol) was added. The reaction solution was allowed to warm to room temperature and stirred overnight. It was then evaporated in vacuo. 100 mL of water was added to the residue, and the resulting mixture was extracted with ethyl acetate (500 mL x 3). The organic layers were combined, washed with brine (50 mL), and dried over sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure to afford 329e (10 g, 92%) as a yellow solid. MS: [M+H] ^⁺ 203.

Example 329f 8-Thia-5-azatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7),10,12-tetraen-6-one 329f

A solution of 329e (5 g, 25 mmol) in PPA (80 mL) was stirred in an oil bath at 150°C overnight. The reaction was allowed to cool to room temperature and diluted with 150 mL of water/ice. The solid was collected by filtration, washed with 3 x 50 mL of water, and then dried in a vacuum oven under reduced pressure to give the crude product, which was further purified by column chromatography eluting with 1:1 ethyl acetate/petroleum ether to give 329f (3 g, 60%) as a yellow solid. MS: [M+H] ⁺ 204

Example 329g (2-bromo-4-fluoro-6-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]tridecyl-1(9),2(7),10,12-tetraen-5-yl}phenyl)methyl acetate 329g

A mixture of 329f (700 mg, 3.4 mmol), 197c (3.35 g, 10.2 mmol), XantPhos (200 mg, 0.34 mmol), Pd ₂ (dba) ₃ (316 mg, 0.34 mmol), and Cs ₂ CO ₃ (2.25 g, 6.8 mmol) in dioxane (50 mL) was heated at 100° C. for 15 h under an argon atmosphere. The mixture was then filtered and evaporated in vacuo. The residue was purified by column chromatography eluting with 1:3 ethyl acetate/petroleum ether to afford 329 g (500 mg, 33%) as a white solid. MS: [M+H] ⁺ 448.

Example 329h [4-Fluoro-2-(1-methyl-5-{[5-(4-methylpiperazin-1-yl)pyridin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)-6-{6-oxo-8-thia-5-azatricyclo[7.4.0.0 ^2,7 ]tridecyl-1(9),2(7),10,12-tetraen-5-yl}phenyl]methyl acetate 329h

To a sealed tube was added a mixture of 329 g (300 mg, 0.66 mmol), 1-methyl-5-(5-(4-methylpiperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-ylboronic acid 197f (690 mg, 1.32 mmol), Pd(dppf) _Cl₂ (54 mg, 0.066 mmol), and _{Na₂CO₃ (} 141 mg, 1.32 mmol) in DMF (22 mL). The system was evacuated and then refilled with _N₂ . The reaction mixture was heated in a microwave at 130°C for 1 h. The mixture was then cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by column chromatography eluting with 15:1 dichloromethane/methanol to afford 329h (300 mg, 67%) as a brown solid. MS: [M+H] ^⁺ 667.

To a solution of 329h (250 mg, 0.38 mol) in THF/isopropanol/water (10 mL/10 mL/2 mL) was added LiOH (90 mg, 38 mmol) with stirring at room temperature. The mixture was stirred for 2 h. Then, 20 mL of water was added, and the resulting mixture was extracted with ethyl acetate (30 mL x 3 ₎ . The combined organic layers were dried over _Na₂SO₄ and concentrated to afford a yellow solid, which was further purified by preparative HPLC to afford 329 (37 mg, 16%) as a brown solid. MS: [M+H] ^⁺ 625. ¹ H NMR(500MHz,MEOD)δ1.31(s,3H),2.39(s,4H),2.65-2.69(m,4H),3.30-3 .33(m,2H),3.68(s,3H),4.11-4.16(m,1H),4.24-4.30(m,1H),4.50-4.5 6(m,2H),6.99(d,1H),7.20(d,1H),7.22(d,1H),7.30(s,1H),7.36-7.38 (m,1H),7.47-7.54(m,2H),7.89-7.95(m,2H),7.97(d,1H),8.53(s,1H).

Example 330 2-(5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-(6-(4-(oxetan-3-yl)piperazin-1-yl)pyridazin-3-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 330

Example 330a tert-Butyl 4-(6-aminopyridazin-3-yl)piperazine-1-carboxylate 330a

To a 100-mL single-necked round-bottom flask equipped with a magnetic stirrer was added 6-chloropyridazin-3-amine (2.58 g, 20 mmol) and N-Boc-piperazine (22.3 g, 120 mmol). The reaction mixture was heated at 140°C and stirred for 2 h. It was then cooled to room temperature, and the resulting solid was washed with ethyl acetate (100 mL). The residue was purified by flash chromatography, eluting with 30:1 dichloromethane/methanol to afford 330a (3.5 g, 60%) as a yellow solid. MS: [M+H] ⁺ 280.

Example 330b tert-Butyl 4-(6-(5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)pyridazin-3-yl)piperazine-1-carboxylate 330b

According to Example 188c, 3,5-dibromo-1-methylpyridin-2(1H)-one (536 mg, 2.0 mmol) and 330a (558 mg, 2.0 mmol) were reacted to give 330b (560 mg, 60%) as a yellow solid. MS: [M+H] ⁺ 467

Example 330c 5-Bromo-1-methyl-3-(6-(piperazin-1-yl)pyridazin-3-ylamino)pyridin-2(1H)-one 330c

According to Example 188d, 560 mg (1.2 mmol) of 330b was deprotected to give a yellow solid 330c (440 mg, 99%). MS: [M+H] ⁺ 367

Example 330d 5-Bromo-1-methyl-3-(6-(4-(oxetan-3-yl)piperazin-1-yl)pyridazin-3-ylamino)pyridin-2(1H)-one 330d

According to Example 210a, 400 mg (1.1 mmol) of 330c was converted to a yellow solid 330d (312 mg, 68%). MS: [M+H] ⁺ 421

Example 330e 4-Fluoro-2-(1-methyl-5-(6-(4-(oxetan-3-yl)piperazin-1-yl)pyridazin-3-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 330e

According to Example 210a, 207 mg of 4-fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 210d (0.43 mmol) and 180 mg of 330d (0.43 mmol) were reacted to give 330e (156 mg, 51%) as a yellow solid. MS: [M+H] ⁺ 697.

According to Example 210, 330e (150 mg 0.22 mmol) was hydrolyzed to give 330 as a yellow solid (60 mg, 42%). LCMS: [M+H] ⁺ 655. ¹ H NMR (500MHz, CDCl3) δ8.67(d,J＝2.5,1H),7.77(s,1H),7.48(d,J＝2.0,1H),7.12(dd,J＝8.5,1H),6.98(m,3H),6.86(s,1H),4.69(m ,4H),4.51(d,J＝11,1H),4.20(m,5H),3.86(m,1H),3.71(s,3H),3.56(m,5H),2.60(m,4H),2.47(m,4H),1.90(s,2H),1.80(s,2H).

Example 331 2-(5-fluoro-2-(hydroxymethyl)-3-(4-methyl-6-(6-(4-methylpiperazin-1-yl)pyridin-3-ylamino)-5-oxo-4,5-dihydropyrazin-2-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 331

Example 331a 5-Bromo-1-methyl-3-(6-(4-methylpiperazin-1-yl)pyridin-3-ylamino)pyrazin-2(1H)-one 331a

A mixture of 6-(4-methylpiperazin-1-yl)pyridin-3-amine (1 g, 5.2 mmol), 3,5-dibromo-1-methylpyrazin-2(1H)-one (1.7 g, 6.2 mmol), and isopropanol (20 mL) was stirred at reflux for 16 h. After completion of the reaction, the solvent was removed to afford 331a as a brown solid (500 mg, 30%). MS: [M+H] ³⁷⁹ .

Example 331b 4-Fluoro-2-(4-methyl-6-(6-(4-methylpiperazin-1-yl)pyridin-3-ylamino)-5-oxo-4,5-dihydropyrazin-2-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 331b

To a sealed tube was added a mixture of 331a (400 mg, 1 mmol), 4-fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 210d (578 mg, 1.2 mmol), Pd(dppf) _Cl2 (82 mg, 0.1 mmol), _K3PO4.3H2O ( ₇₆₀ mg, 2 mmol) and NaOAc (164 mg, 2 mmol) in _{CH3CN (} 10 mL). The system was evacuated and then refilled with _N2 and the reaction mixture was heated at 110°C for 2 h. The mixture was then cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography using 50:1 dichloromethane/methanol as eluent to afford 331b as a yellow solid (100 mg, 14%). MS: [M+H] ⁺ 655.

To a solution of 331b (100 mg, 0.15 mmol) in THF/isopropanol/water (2 mL/2 mL/1 mL) was added LiOH (15 mg, 0.75 mmol). The reaction mixture was stirred at 30°C for 1 h. Then, 20 mL of water was added, and the mixture was extracted with ethyl acetate (90 _mL x 3). The combined organic layers were dried over _Na₂SO₄ and concentrated to give a yellow solid. Purification by preparative HPLC gave 331 (42 mg, 45%) as a yellow solid. MS: [M+H] ^⁺ 613. ¹ H NMR(500MHz,DMSO)δ9.26(s,1H),8.67(d,J＝2.5,1H),8.35(q,J＝2.5,1H),7.44 (s,1H),7.32(m,2H),6.82-6.80(d,J＝9,1H),6.52(s,1H),4.46-4.36(m,2H),4 .16-4.09(m,3H),3.88-3.87(m,1H),3.52(s,3H),3.40-3.34(m,5H),2.60-2.5 7(m,2H),2.51-2.45(m,2H),2.39-2.37(m,4H),2.20(s,3H),1.79-1.69(m,4H).

Example 332 2-(5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-(5-(4-methyl-2-oxopiperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 332

Example 332a tert-Butyl 3-oxopiperazine-1-carboxylate 332a

To a 100 mL dry single-necked round-bottom flask equipped with a stirring bar was added piperazin-2-one (5.0 g, 50 mmol), anhydrous dichloromethane (60 mL) and Et ₃ N (3.2 mL, 22.5 mmol) and the reaction mixture was stirred at room temperature for 1 h. The organic layer was concentrated under reduced pressure to give 332a (10 g, 99%).

Example 332b tert- Butyl 4-(6-nitropyridin-3-yl)-3-oxopiperazine-1-carboxylate 332b

To a microwave-safe vial were added 332a (1.5 g, 7.5 mmol), 5-bromo-2-nitropyridine (1.27 g, 6.25 mmol), palladium diacetate (71.8 mg, 0.32 mmol), Xantphos (278 mg, 0.48 mmol), and Cs ₂ CO ₃ (2.04 g, 6.25 mmol) in 30 mL of anhydrous 1,4-dioxane. After three cycles of vacuum/argon purge, the mixture was heated at 120° C. under microwave irradiation for 1.5 h. The reaction mixture was filtered, and the filtrate was evaporated in vacuo. The residue was purified by silica gel column chromatography eluting with 3:1 petroleum ether/ethyl acetate to afford 332b (1.1 g, 54%) as a yellow solid. MS: [M+H] ⁺ 323. ¹ H NMR (500MHz, CDCl ₃ ) δ 8.68 (d, J = 2.5 Hz, 1H), 8.31 (d, J = 9 Hz, 1H), 8.15 (m, 1H), 4.33 (s, 2H), 3.91 (m, 4H).

Example 332c tert-Butyl 4-(6-aminopyridin-3-yl)-3-oxopiperazine-1-carboxylate 332c

To a solution of 332b (1.1 g, 3.4 mmol) in methanol (20 mL) was added Pd/C (10%) (250 mg). After three cycles of vacuum/H ₂ purge, the mixture was stirred at room temperature under H ₂ for 10 h. The mixture was filtered, and the filtrate was evaporated in vacuo to afford 332c (946 mg, 95%), which was used in the next step without further purification. MS: [M+H] ⁺ 293.

Example 332d tert-Butyl 4-(6-(5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)pyridin-3-yl)-3-oxopiperazine-1-carboxylate 332d

To a reaction vessel was added 332c (946 mg, 3.2 mmol), 3,5-dibromo-1-methylpyridin-2(1H)-one (1.11 g, 4.2 mmol), tris(dibenzylideneacetone)dipalladium(0) (146 mg, 0.16 mmol), Xantphos (185 mg, 0.32 mmol), and Cs ₂ CO ₃ (2.6 g, 8.0 mmol) in 40 mL of anhydrous 1,4-dioxane. After three cycles of vacuum/argon purge, the mixture was stirred at 110° C. for 4 h. The reaction mixture was filtered, and the filtrate was evaporated in vacuo. The crude product was purified by silica gel column chromatography eluting with 50:1 dichloromethane/methanol to afford 332d (706 mg, 46%) as a yellow solid. MS: [M+H] ⁺ 479.

Example 332e 1-(6-(5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)pyridin-3-yl)piperazin-2-one 332e

To a 50-mL round-bottom flask equipped with a magnetic stirrer and reflux condenser was added HCl/1,4-dioxane (20 mL) and 332d (706 mg, 1.47 mmol). The mixture was stirred at 65°C for 6 h. It was then concentrated under reduced pressure to afford 332e as a yellow solid (500 mg, 90%). MS: [M+H] ⁺ 379.

Example 332f 1-(6-(5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)pyridin-3-yl)-4-methylpiperazin-2-one 332f

To a solution of 1-(6-(5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)pyridin-3-yl)piperazin-2-one (332e) (500 mg, 1.32 mmol) in methanol (20 mL) was added Na(OAc) ₃ BH (2.0 g, 13.2 mmol), formaldehyde (30% aqueous solution, 8 mL), and acetic acid (2.7 mL, 45 mmol). The mixture was stirred for 4 h. The pH of the reaction mixture was adjusted to 11-13 by the addition of NaOH (1 M). The mixture was then extracted three times with dichloromethane. The combined organic extracts were dried over anhydrous Na ₂ SO ₄ , filtered, and evaporated in vacuo. The residue was purified on a silica gel column eluting with 50:1 dichloromethane/methanol to afford 332f (312 mg, 60%) as a gray solid. MS: [M+H] ⁺ 393. ¹ H NMR (500MHz, CDCl ₃ )δ8.61(d,J＝2.5Hz,1H),8.17(d,J＝2.5Hz,1H),7.88(s,1H),7.45(dd,J＝9Hz,1H),6.92(d,J＝2. 5Hz, 1H), 6.74 (d, J = 9Hz, 1H), 3.64 (m, 2H), 3.53 (s, 3H), 3.22 (s, 2H), 2.74 (m, 2H), 2.35 (s, 3H).

Example 332g 4-Fluoro-2-(1-methyl-5-(5-(4-methyl-2-oxopiperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 332g

To the reaction vessel were added 1-(6-(5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)pyridin-3-yl)-4-methylpiperazin-2-one (332f) (0.3 g, 0.76 mmol), 4-fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 210d (0.37 g, 0.76 mmol), PdCl ₂ (dppf) (65 mg, 0.076 mmol), K ₃ PO ₄ A mixture of 4-[ ^...

A mixture of 332g (350 mg, 0.52 mmol) and LiOH hydrate (1.1 g, 26 mmol) in isopropanol (3 mL), THF (3 mL), and water (3 mL) was stirred at 30°C for 1 h. The mixture was evaporated in vacuo, and the residue was extracted with dichloromethane (30 mL x 3). The combined extracts were concentrated under reduced pressure, and the residue was purified on a silica gel column eluting with 50:1 dichloromethane/methanol to afford 332 (120 mg, 32%) as a yellow solid. MS: [M+H] ⁺ 626. ¹ H NMR (500MHz, CDCl ₃ )δ8.78(d,J＝2Hz,1H),8.17(d,J＝2.5Hz,1H),7.58(dd,J＝8.5Hz,1H),7.42 (d,J＝2.5Hz,1H),7.23(s,1H),7.21(s,1H),7.12(d,J＝9Hz,1H),6.73(s,1 H),4.53(m,2H),4.22(s,2H),4.03(m,1H),3.73(s,4H),3.27(s,2H),2.87 (m,2H),2.65(m,1H),2.55(m,2H),2.43(s,3H),1.89(m,2H),1.79(m,2H).

Example 333 2-(5-fluoro-2-(methoxymethyl)-3-(1-methyl-5-(5-(4-methylpiperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 333

To a solution of 2-(5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-(5-(4-methylpiperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 197 (200 mg, 0.33 mmol) and _K₂CO₃ (135 mg, 0.99 mmol) in DMF (30 mL) was added a solution _of iodomethane (70 mg, 0.50 mmol) in DMF (5 mL) at room temperature. The reaction mixture was stirred for 1 h. It was then filtered, and the residue was purified by preparative HPLC to afford 333 (165 mg, 80%) as a yellow solid. MS: [M+H] ^⁺ 626. ¹ H NMR (500MHz, DMSO) δ8.59(d,J＝2.0,1H),8.49(s,1H),7.97(d,J＝2.0,1H),7.44(s,1H),7.39(s,1H),7.32(m,2H),7.17(m,1H),6.52(s,1H),4. 31(s,2H),4.14(m,3H),3.88(s,1H),3.56(s,3H),3.54(s,4H),3.44(s ,4H),3.12(s,6H),2.58(m,2H),2.46(s,2H),1.79(s,2H),1.68(s,2H).

Example 334 {4-fluoro-2-[1-methyl-5-({5-[4-(oxetan-3-yl)piperazin-1-yl]pyridin-2-yl}amino)-6-oxo-1,6-dihydropyridin-3-yl]-6-{7-oxo-3,6-diazatetracyclo[9.2.1.0 ^2,10 .0 ^3,8 ]tetradec-2(10),8-dien-6-yl}phenyl}methyl acetate 334

Example 334a 3-Chlorobicyclo[2.2.1]hept-2-ene-2-carbaldehyde 334a

A 250-mL three-necked round-bottom flask equipped with a magnetic stirrer and reflux condenser was purged with nitrogen and charged with anhydrous 1,2-dichloroethane (24 mL) and anhydrous DMF (9.12 g, 125 mmol). The reaction mixture was cooled to 0°C, and phosphorus oxychloride (15.3 g, 100 mmol) was added over 5 minutes while maintaining the reaction temperature at 0-10°C. The cooling bath was removed, and the reaction was stirred at room temperature for 30 minutes. A solution of bicyclo[2.2.1]heptan-2-one (5.50 g, 50.0 mmol) in 1,2-dichloroethane (10 mL) was added, and the resulting mixture was heated at 80°C overnight. Thereafter, the reaction solution was poured into a solution of potassium hydrogen phosphate (43.5 g, 250 mmol) in water (200 mL) and stirred for 15 minutes. The organic layer was separated and then concentrated under reduced pressure. The residue was dissolved in dichloromethane (300 mL) and washed with water (2 x 50 mL). The dichloromethane layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The residue was purified by column chromatography eluting with 1:100 ethyl acetate/petroleum ether to afford 334a as a yellow oil (2.2 g, 28%). MS: [M+H] ⁺ 157. ¹ H NMR (500 MHz, CDCl3) δ 9.80 (s, 1H), 3.42 (s, 1H), 3.07 (d, J = 1.7 Hz, 1H,), 1.95-1.77 (m, 2H), 1.67 (t, J = 8.9 Hz, 1H), 1.41-1.17 (m, 3H).

Example 334b (E)-ethyl 3-(3-chlorobicyclo[2.2.1]hept-2-en-2-yl)acrylate 334b

To a solution of 334a (9.0 g, 57.7 mmol) in dichloromethane (250 mL) was added ethyl 2-(triphenyl-λ ⁵ -phosphanylidene) acetate (20 g, 57.7 mmol). The mixture was stirred at room temperature overnight. After the reaction, the reaction ^solution was evaporated in vacuo. The residue was purified by column chromatography using 1:100 ethyl acetate/petroleum ether to afford 334b (6.0 g, 46%) as a yellow oil. MS: [M+H] ⁺ 227.

Example 334c Ethyl 3-azatricyclo[5.2.1.0 ^2,6 ]dec-2(6),4-diene-4-carboxylate 334c

To a solution of 334b (5.0 g, 22 mmol) in DMSO (30 mL) was added _NaN₃ (2.2 g, 33 mmol), and the mixture was heated at 105°C for 6 h. Water (13 mL) was added to the reaction mixture, and after cooling to room temperature, the resulting mixture was extracted with dichloromethane (50 mL x ₃ ). The organic phase was dried ( _Na₂SO₄ ) and then evaporated to dryness. The residue was purified by column chromatography eluting with 20:1 dichloromethane/methanol to afford 334c (2.7 g, 60%) as a brown solid. MS: [M+H] ^⁺ 206. ¹ H NMR(500MHz, CDCl3)δ11.51(s,1H),6.45(dd,1H),4.14-4.19(m,2H,),3.25(d,2H) ,),1.79-1.82(m,2H),1.73(d,1H),1.22-1.25(t,J=6.5,3H),0.89-0.91(m,2H).

Example 334d Ethyl 3-(cyanomethyl)-3-azatricyclo[5.2.1.0 ^2,6 ]dec-2(6),4-diene-4-carboxylate 334d

To a solution of 334c (3.0 g, 14.6 mmol) in anhydrous DMF (30 mL) was added NaH (880 mg, 22 mmol). The mixture was stirred at room temperature for 30 minutes. 2-Bromoacetonitrile (3.5 g, 29.3 mmol) was added and the resulting mixture was heated at 65 ° C for 1 hour. It was then cooled to room temperature and stirred overnight. After the reaction, water (30 mL) was added and the reaction mixture was extracted with ethyl acetate (3×200 mL). The organic phase was evaporated to dryness. The residue was purified by column chromatography eluting with 20:1 dichloromethane/methanol to give 334d (2.6 g, 72%) as a brown solid. MS: [M+H] ⁺ 245.

Example 334e 3-(2-aminoethyl)-3-azatricyclo[5.2.1.0 ^2,6 ]dec-2(6),4-diene-4-carboxylic acid ethyl ester 334e

A suspension of 334d (4.0 g, 16 mmol) and Raney nickel (400 mg) in methanol (60 mL) was hydrogenated overnight at 50 psi in a Parr apparatus. The mixture was filtered through a pad of Celite, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography eluting with 20:1 dichloromethane/methanol to afford 334e (2 g, 50%) as a yellow solid. MS: [M+H] ⁺ 249.

Example 334f 3,6-diazatetracyclo[9.2.1.0 ^2,10 .0 ^3,8 ]tetradeca-2(10),8-dien-7-one 334f

To a solution of ethyl 3-(2-aminoethyl)-3-azatricyclo[5.2.1.0 ^2,6 ]dec-2(6),4-diene-4-carboxylate (334e) (1.8 g, 7.2 mmol) in ethanol (40 mL) was added sodium methoxide (2.5 g, 36 mmol). The mixture was heated at 65°C for 12 hours. It was then cooled to room temperature. The solvent was evaporated to dryness. The residue was purified by column chromatography eluting with 20:1 dichloromethane/methanol to give 334f (800 mg, 53%) as a brown solid. MS: [M+H] ⁺ 203.

Example 334g (2-bromo-4-fluoro-6-{7-oxo-3,6-diazatetracyclo[9.2.1.0 ^2,10 .0 ^3,8 ]tetradeca-2(10),8-dien-6-yl}phenyl)methyl acetate 334g

To a sealed tube were added 334f (800 mg, 4 mmol) and 197c (3.8 g, 12 mmol) in dioxane (25 mL) and cesium carbonate (2.6 g, 8 mmol). After bubbling nitrogen through the resulting solution for 30 min, XantPhos (230 mg, 0.4 mmol) and tris(dibenzylideneacetone)dipalladium(0) (362 mg, 0.4 mmol) were added, and the reaction mixture was heated at 100 ° C for 4 h. Thereafter, the reaction was cooled to room temperature and then partitioned between ethyl acetate (100 mL) and water (100 mL). The aqueous layer was separated and then extracted with ethyl acetate (50 mL×3). The organic layers were combined, washed with brine (20 mL×3), and then dried over sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography eluting with 1:5 ethyl acetate/petroleum ether to give 334g (1.3 g, 72%) as a brown solid. MS:[M+H] ⁺ 447.

Example 334h (4-fluoro-2-{7-oxo-3,6-diazatetracyclo[9.2.1.0 ^2,10 .0 ^3,8 ]tetradeca-2(10),8-dien-6-yl}-6-(tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methyl acetate 334h

To a solution of 334g (450 mg, 1.0 mmol) and 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bis(1,3,2-dioxaborolane) (770 mg, 3.0 mmol) in dioxane (40 mL) was added _PdCl2 (dppf) (82 mg, 0.1 mmol) and _CH3COOK (593 mg, 6 mmol). The mixture was stirred at 100°C under an argon atmosphere for 12 h. After the reaction, the mixture was filtered and then evaporated in vacuo. The residue was purified by column chromatography eluting with 1:4 ethyl acetate/petroleum ether to give 334h (400 mg, 80%) as a brown solid. MS: [M+H] ⁺ 495.

Example 334i {4-fluoro-2-[1-methyl-5-({5-[4-(oxetan-3-yl)piperazin-1-yl]pyridin-2-yl}amino)-6-oxo-1,6-dihydropyridin-3-yl]-6-{7-oxo-3,6-diazatetracyclo[9.2.1.0 ^2,10 .0 ^3,8 ]tetradec-2(10),8-dien-6-yl}phenyl}methyl acetate 334i

To a sealed tube were added 334h (300 mg, 0.6 mmol), 188e (254 mg, 0.6 mmol), _Na₂CO₃ (129 mg, 1.2 mmol), and _PdCl₂ (dppf) (50 mg, 0.06 mmol) _and suspended in DMF (20 mL) and _H₂O (1 mL). The mixture was stirred at 80°C for 5 h. After the reaction, the mixture was partitioned between ethyl acetate and water. The organic phase was washed with water (2×), dried over sodium sulfate, and concentrated. The residue was purified by reverse-phase Combi-flash chromatography eluting with ₁ :5 water/ _CH₃CN containing 0.3% _NH₄HCO₃ to afford 334i (300 mg, 70%) as a brown solid. MS: (M+H) ^⁺ 708.

To a solution of 334i (280 mg, 0.4 mol) in THF/isopropanol/water (10 mL/10 mL/2 mL) was added LiOH (950 mg, 40 mmol) with stirring at room temperature. The mixture was stirred for 2 h. Then, 20 mL of water was added, and the resulting mixture was extracted with ethyl acetate (3 x 30 mL). _The combined organic layers were dried over _Na₂SO₄ and concentrated to afford a yellow solid, which was further purified by reverse- _phase Combi-flash column chromatography using 1:4 water/ _CH₃CN containing 0.3% _NH₄HCO₃ as the eluent to afford 334 (160 mg, 67%) as a white solid. MS: [M+H] ^⁺ 666. ¹ H NMR (500MHz, MEOD) δ8.53(s,1H),7.93(d,1H),7.43(d,1H),7.34(s,1H),7.20-7.22(m,2H),7.04(d,1H),6.67(s ¹ H),4.72-4.75(t,J＝6.5,1H),4.63-4.66(t,J＝6,2H),4.47-4.56(M,2H),4.21-4.39(m,3H),3.98-4.06(m,1H),3.71(s,3H),3.56-3.60(m, 1H),3.48(s,1H),3.17-3.19(t,J＝4.5,4H),2.71(s,1H),2.53-2.55( t,J＝5,4H),1.88-1.95(m,3H),1.67-1.69(m,1H),1.04-1.17(m,2H).

Example 335 2-(5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-(5-(5-methylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 335

Example 335a 5-(6-nitropyridin-3-yl)-hexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylic acid tert-butyl ester 335a

According to Example 188a, 404 mg of tert-butyl hexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (2 mmol) and 566 mg of 5-fluoro-2-nitropyridine (1.1 mmol) were reacted to give 335a (234 mg, 35%) as a yellow solid. MS: [M+H] ⁺ 335. ¹ H NMR (500 MHz, DMSO) δ 8.18 (d, J = 9.0, 1H), 7.87 (d, J = 3.5, 1H), 7.06 (dd, J = 9.0, 1H), 3.66 (s, 2H), 3.55 (s, 2H), 3.36 (s, 2H), 3.18 (s, 2H), 3.05 (s, 2H), 1.39 (s, 9H).

Example 335b tert-Butyl 5-(6-aminopyridin-3-yl)hexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate 335b

According to Example 188b, 234 mg of 335a (0.7 mmol) was reduced to give 335b (213 mg, 99%) as a yellow solid. MS: [M+H] ⁺ 305

Example 335c tert-Butyl 5-(6-(5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)pyridin-3-yl)hexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate 335c

According to Example 188c, 3,5-dibromo-1-methylpyridin-2(1H)-one (212 mg, 0.79 mmol) and 335b (200 mg, 0.66 mmol) were reacted to give 335c as a yellow solid (194 mg, 60%). MS: [M+H] ⁺ 492

Example 335d 5-Bromo-3-(5-(hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)pyridin-2-ylamino-1-methylpyridin-2(1H)-one 335d

According to Example 188d, 194 mg (0.4 mmol) of 335c was deprotected to give a yellow solid 335d (154 mg, 99%). MS: [M+H] ⁺ 392

Example 335e 5-Bromo-1-methyl-3-(5-(5-methylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)pyridin-2-ylamino)pyridin-2(1H)-one 335e

According to Example 188e, 154 mg (0.4 mmol) of 335d was methylated to give 335e as a yellow solid (119 mg, 75%). MS: [M+H] ⁺ 404.

Example 335f 4-Fluoro-2-(1-methyl-5-(5-(5-methylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 335f

According to Example 148h, 142 mg of 4-fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 210d (0.29 mmol) and 119 mg of 335e (0.29 mmol) were reacted to give 335f as a yellow solid (100 mg, 51%). LCMS: [M+H] ⁺ 680

According to Example 148, 100 mg of 335f (0.15 mmol) was used to obtain 335 as a white solid (39 mg, 42%). ¹ H NMR (500 MHz, CDCl ₃ ) δ 8.45 (d, J = 2.0, 1H), 7.70 (d, J = 2.5, 1H), 7.67 (s, 1H), 7.42 (d, J = 2.5, 1H), 7.17 (m, 1H), 7.04 (dd, J = 9.0, 1H), 6.95 (dd, J = 6.0, 1H), 6.86 (s, 1H), 6.79 (d, J = 9.0, 1H), 4.54 (d, J = 11 .0,1H),4.31(m,1H),4.16(m,3H),3.91(m,1H),3.70(s,3H),3.27(s,2H),3.18(s,2H),2. 99(s,2H),2.83(s,2H),2.59(m,4H),2.46(m,2H),2.38(s,3H),1.90(s,2H),1.80(s,2H).

Example 336 2-(5-fluoro-3-(5-(5-(3-(fluoromethyl)-4-methylpiperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 336

Example 336a 1-tert-Butyl 2-methyl 4-benzylpiperazine-1,2-dicarboxylate 336a

To a dry 100 mL round-bottom flask equipped with a stir bar under _N₂ was added 1-tert-butyl 2-methylpiperazine-1,2-dicarboxylate (5 g, 20.5 mmol), anhydrous acetonitrile (60 mL), benzyl bromide (2.7 mL, 22.5 mmol), and triethylamine (8.5 mL, 61.5 mmol). The reaction mixture was heated at 71°C for 45 minutes and then concentrated under reduced pressure. It was then diluted with dichloromethane _and washed with water and brine. The organic layer was dried over _Na₂SO₄ , filtered, and then concentrated under reduced pressure. The crude compound was purified by flash column chromatography eluting with 8:1 petroleum ether/ethyl acetate to afford 336a (4.5 g, 66%). MS: [M+H] ^⁺ 335. ¹ H NMR (500MHz, CDCl ₃ )δ7.28(m,5H),4.60(m,1H),3.78(m,4H),3.57(m,1H),3.43(m,1H),3.28 (m,2H),2.77(m,1H),2.19(m,1H),2.11(m,1H),1.47(s,5H),1.42(s,4H).

Example 336b (4-Benzyl-1-methylpiperazin-2-yl)methanol 336b

To a solution of 336a (1 g, 2.99 mmol) in 100 mL of anhydrous tetrahydrofuran was slowly added lithium aluminum hydride (342 mg, 8.98 mmol) at 0°C and the mixture was stirred for 30 min. The mixture was then refluxed for 3 h, and the reaction mixture was poured portionwise onto ice. The resulting mixture was filtered, and the filtrate was evaporated in vacuo. 100 mL of brine was added to the residue, and the mixture was extracted with dichloromethane (100 mL x 3). The combined organic layers were dried over Na ₂ SO ₄ , filtered, and then concentrated to give 336b (0.60 g, 91%) as a yellow oil. MS: [M+H] ⁺ 221.

Example 336c 4-Benzyl-2-(fluoromethyl)-1-methylpiperazine 336c

To an ice-cold solution of diethylaminosulfur trifluoride (10.8 mL, 81.8 mmol) in dichloromethane was added dropwise a solution of 336b (9.0 g, 40.9 mmol) in dichloromethane under _N₂ . The yellow solution was stirred at 0°C for 1 hour and then at room temperature for 15 hours. The reaction mixture was then diluted with _NaHCO₃ , and _the organic layer was separated and dried over _Na₂SO₄ . The crude product was purified by silica gel column chromatography eluting with 50:1 dichloromethane/methanol to afford 336c (3.0 g, 33%) as a yellow oil. MS: [M+H] ^⁺ 223. ¹ HNMR(500MHz,DMSO)δ7.28(m,5H),4.50(m,0.5H),4.43(m,1H),4.35(m,0.5H),3.45(s,2H), 2.67(m,2H),2.60(m,1H),2.29(m,1H),2.24(s,3H),2.20(m,1H),2.11(m,1H),1.94(m,1H).

Example 336d 2-(Fluoromethyl)-1-methylpiperazine 336d

To a 250-mL single-necked round-bottom flask equipped with a magnetic stirrer was added 336c (3.0 g, 13.5 mmol), methanol (80 mL), and Pd/C (10%) (300 mg). The reaction mixture was stirred at 25°C under _H₂ for 15 h. After completion of the reaction, the mixture was filtered and concentrated to afford 336d (1.6 g, 90%) as a yellow oil.

Example 336e 2-(Fluoromethyl)-1-methyl-4-(6-nitropyridin-3-yl)piperazine 336e

To a 100-mL single-necked round-bottom flask equipped with a magnetic stirrer and a reflux condenser was added 1,4-dioxane (50 mL), 336d (1.6 g, 12.1 mmol), 5-bromo-2-nitropyridine (3.7 g, 18.2 mmol), and cesium carbonate (9.9 g, 30.2 mmol). After bubbling nitrogen through the resulting solution for 30 min, Xantphos (700 mg, 0.12 mmol) and tris(dibenzylideneacetone)dipalladium(0) (550 mg, 0.06 mmol) were added. After three cycles of vacuum/argon purge, the reaction mixture was heated at reflux for 15 h. Thereafter, the reaction was cooled to room temperature, filtered, and concentrated to afford the crude product as a black solid, which was further purified by column chromatography eluting with 100:1 dichloromethane/methanol to afford 336e (2.6 g, 76%) as a yellow solid. MS: [M+H] ⁺ 255. ¹ H NMR(500MHz,MeOD)δ8.20(m,2H),7.52(dd,J=9,1H),4.72(m,0.5H),4.64(m,1H),4.56(m,0.5H ),4.01(m,1H),3.94(m,1H),3.19(m,1H),3.06(m,1H),3.00(m,1H),2.50(m,2H),2.46(s,3H).

Example 336f 5-(3-(Fluoromethyl)-4-methylpiperazin-1-yl)pyridin-2-amine 336f

To a 250-mL single-necked round-bottom flask equipped with a magnetic stirrer was added 336e (2.6 g, 10.2 mmol), methanol (50 mL), and Pd/C (10%) (260 mg). The reaction mixture was stirred under _H₂ for 15 h. After completion of the reaction, the mixture was filtered and concentrated to afford 336f as a black oil, which was used in the next step without further purification. MS: [M+H] ^⁺ 225.

Example 336g 5-Bromo-3-(5-(3-(fluoromethyl)-4-methylpiperazin-1-yl)pyridin-2-ylamino)-1-methylpyridin-2(1H)-one 336g

To a 100-mL single-necked round-bottom flask equipped with a magnetic stirrer and a reflux condenser was added 1,4-dioxane (60 mL), 336f (crude, 14.1 mmol), 3,5-dibromo-1-methylpyridin-2(1H)-one (4.5 g, 16.9 mmol), and cesium carbonate (11.5 g, 35.2 mmol). After bubbling nitrogen through the resulting solution for 30 min, Xantphos (820 mg, 1.41 mmol) and tris(dibenzylideneacetone)dipalladium(0) (645 mg, 0.7 mmol) were added. After three cycles of vacuum/argon purge, the reaction mixture was heated at reflux for 15 h. The reaction was allowed to cool to room temperature, filtered, and then concentrated to give a black solid, which was purified by column chromatography eluting with dichloromethane/methanol (100:1 to 50:1) to give 336g (3.1 g, 50%) as a yellow solid. MS:[M+H] ⁺ 410.

Example 336h 4-Fluoro-2-(5-(5-(3-(fluoromethyl)-4-methylpiperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 336h

To a reaction vessel was added 336 g (1 g, 2.4 mmol), 4-fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl acetate 210d (1.3 g, 2.68 mmol), PdCl ₂ (dppf) (190 mg, 0.24 mmol), K ₃ PO ₄ (1 g, 4.8 mmol) and NaOAc (390 mg, 4.8 mmol) in MeCN (15 mL) and water (1.5 mL). The mixture was purged with nitrogen for 30 min and then heated at 110° C. for 3 h. The solvent was evaporated in vacuo and the residue was purified by column chromatography on silica gel eluting with 5:1 dichloromethane/methanol to give 336h (0.80 g, 45%). MS: [M+H] ⁺ 686.

A mixture of 336h (750 mg, 1.09 mmol) and LiOH hydrate (2.3 g, 55 mmol) in isopropanol (10 mL), THF (10 mL) and water (10 mL) was stirred at 30 ° C for 1 h. The mixture was evaporated in vacuo, and the residue was extracted with dichloromethane (30 mL x 3). The combined extracts were concentrated under reduced pressure, and the residue was purified on a silica gel column eluted with 50:1 dichloromethane/methanol to give 336 (700 mg, 93%) as a yellow solid. MS: [M+H] ⁺ 644. ¹ H NMR(500MHz,MeOD)δ8.54(d,J＝2Hz,1H),7.92(d,J＝1.5Hz,1H),7.42(m,1H),7.33(m,1H) ,7.22(s,1H),7.20(s,1H),7.02(m,1H),6.72(s,1H),4.46-4.69(m,4H),4.20(s,3H),4. 02(m,1H),3.70(s,3H),3.49(d,J＝11.5Hz,1H),3.42(d,J＝11.5Hz,1H),2.94(m,1H),2.8 5(m,1H),2.71(m,1H),2.64(m,2H),2.56(m,4H),2.44(s,3H),1.88(m,2H),1.79,(m,2H).

Example 337 6-[5-Fluoro-2-(hydroxymethyl)-3-{1-methyl-6-oxo-5-[(pyrimidin-4-yl)amino]-1,6-dihydropyridin-3-yl}phenyl]-3,6-diazatetracyclo[9.2.1.0 ^2,10 .0 ^3,8 ]tetradeca-2(10),8-dien-7-one 337

Example 337a (4-Fluoro-2-{1-methyl-6-oxo-5-[(pyrimidin-4-yl)amino]-1,6-dihydro-pyridin-3-yl}-6-{7-oxo-3,6-diazatetracyclo[9.2.1.0 ^2,10 .0 ^3,8 ]tetradec-2(10),8-dien-6-yl}phenyl)methyl acetate 337a

To a sealed tube were added (2-bromo-4-fluoro-6-{7-oxo-3,6-diazatetracyclo[9.2.1.0 ^2,10 .0 ^3,8 ]tetradeca-2(10),8-dien-6-yl}phenyl)methyl acetate 334 g (400 mg, 0.9 mmol), 1-methyl-3-(pyrimidin-4-ylamino)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one 109c (294 mg, 0.9 mmol), Na ₂ CO ₃ (190 mg, 1.8 mmol), and PdCl ₂ (dppf) (73 mg, 0.09 mmol) and suspended in DMF (20 mL) and water (1 mL). The mixture was stirred at 60° C. for 6 hours and then partitioned between water and ethyl acetate. The organic phase was washed with water and then evaporated to dryness. The residue was purified by column chromatography using 15:1 dichloromethane/methanol as eluent to afford 337a as a brown solid (300 mg, 58%). MS: [M+H] ⁺ 569.

To a solution of 337a (270 mg, 0.47 mol) in THF/isopropanol/water (10 mL/10 mL/2 mL) was added LiOH (1.1 g, 47 mmol) at room temperature with stirring. The mixture was stirred for 2 h. Then, 20 mL of water was added, and the resulting mixture was extracted with ethyl acetate (30 mL x 3 ₎ . The combined organic layers were dried over _Na₂SO₄ and concentrated to afford a yellow solid, which was further purified by reverse- _phase Combi-flash chromatography using 1:5 water/ _CH₃CN containing 0.3% _NH₄HCO₃ as the eluent to afford 337 (138 mg, 48%) as a white solid. MS: [M+H] ^⁺ 527. ¹ H NMR(500MHz,MEOD)δ8.87(s,1H),8.67(s,1H),8.29(d,1H),7.55(s,1H),7 .22-7.24(m,2H),7.10(d,1H),6.68(d,1H),4.48-4.55(m,2H),4.18-4.40 (m,3H),3.98-4.06(m,1H),3.72(s,3H),3.45-3.48(m,1H),1.88-1.98(m, 3H),1.67-1.69(m,1H),1.30(m,1H),1.12-1.17(m,1H),1.02-1.07(m,1H).

Example 338 2-(5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-(5-(4-methyl-1,4-diazepan-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 338

Example 338a 1-Methyl-4-(6-nitropyridin-3-yl)-1,4-diazepane 338a

To a solution of 1-methyl-1,4-diazepane (1 g, 8.8 mmol) in DMSO (20 mL) was added _{K₂CO₃ (} 2.4 g, 18 mmol) and 5-bromo-2-nitropyridine (51-7) (1.78 g, 8.8 mmol). The mixture was stirred at 65°C overnight. The mixture was allowed to cool to room temperature and then poured into water. The resulting solid was collected and dried under vacuum. The solid was further purified by flash column chromatography using 3:1 petroleum ether/ethyl acetate followed by dichloromethane to afford 338a (800 mg, 69%) as a yellow solid. MS: [M+H] ⁺ 236.

Example 338b 1-Methyl-4-(6-nitropyridin-3-yl)-1,4-diazepane 338b

A 500-mL bottle was purged with nitrogen and charged with 338a (0.8 g, 3.4 mmol), 10% palladium on carbon (50% humidity, 100 mg), and methanol (100 mL). The bottle was evacuated, filled with hydrogen, and stirred at room temperature for 16 h. The hydrogen was then removed and nitrogen was introduced into the bottle. The catalyst was removed by filtration through a pad of Celite, and the filtrate was concentrated under reduced pressure to afford 338b (0.6 g, crude product). MS: [M+H] ⁺ 207.

Example 338c 5-Bromo-1-methyl-3-(5-(4-methyl-1,4-diazepan-1-yl)pyridin-2-ylamino)pyridin-2(1H)-one 338c

338b (968 mg, 4.7 mmol), 3,5-dibromo-1-methylpyridine-2 (1H) -one (1.24 g, 4.7 mmol) and cesium carbonate (3.8 g, 12 mmol) were added to a 100-mL single-necked round-bottom flask equipped with a magnetic stirrer and a reflux condenser. After nitrogen was bubbled through the resulting solution for 30 minutes, XantPhos (272 mg, 0.47 mmol) and tris (dibenzylideneacetone) dipalladium (0) (430 mg, 0.47 mmol) were added. The reaction mixture was heated at reflux for 3 h. After this, the reaction was cooled to room temperature, distributed between ethyl acetate (100 mL) and water (100 mL), and then filtered. The aqueous layer was separated and extracted with ethyl acetate (2 x 50 mL). The organic layers were combined, washed with brine (50 mL), and then dried over sodium sulfate. The drying agent was removed by filtration, and the filtrate was then concentrated under reduced pressure. The residue was purified by flash column chromatography using petroleum ether/ethyl acetate as eluent to afford 338c (918 mg, 50%). MS: [M+H] ⁺ 392.

Example 338d 4-Fluoro-2-(1-methyl-5-(5-(4-methyl-1,4-diazepan-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 338d

To a sealed tube was added a mixture _of 338c (274 mg, 0.7 mmol), 210d (337 mg, 0.7 mmol), _Pd (dppf) _Cl₂ (33 mg, 0.04 mmol), _{K₃PO₄.3H₂O} (372 mg, 1.4 mmol), and NaOAc (115 mg, 1.4 mmol) in _CH₃CN (20 mL). The system was evacuated and then refilled with _N₂ . The reaction mixture was heated at 110°C under microwave irradiation for 3 h. The mixture was cooled to room temperature and then filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography eluting with 30:1 dichloromethane/methanol to afford 338d (120 mg, 43%) as a yellow solid. MS: [M+H] ^⁺ 668.

To a solution of 338d (120 mg, 0.18 mmol) in propan-2-ol (10 mL), tetrahydrofuran (10 mL), and water (2 mL) was added LiOH (1.1 g, 57 mmol). The mixture was stirred at 30°C for 2 h. The product was then evaporated and the residue was purified by preparative HPLC to afford 338d as a white solid (60 mg, 54%). MS: (M+H) ⁺ 626. ¹ H NMR(500MHz,MeOD)δ8.18(s,1H),7.62(s,1H),7.16(s,1H),7.06(m,3H),6.88(d,J＝11.5,2H),6.59(s,1H),4.38(m,2H),4.07 (m3H),3.87(m,1H),3.57(s,3H),3.42(m,2H),3.31(m,2H),3.20(s,3H),2.60(m,2H),2.50(m,4H),2.42(m,2H),1.75(m,6H).

Example 339 5-[5-fluoro-2-(hydroxymethyl)-3-[1-methyl-5-({5-[4-(oxetan-3-yl)piperazin-1-yl]pyridin-2-yl}amino)-6-oxo-1,6-dihydropyridin-3-yl]phenyl]-8-thia-4,5-diazatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7),3(4)-triene-6-one 339

Example 339a [4-Fluoro-2-(1-methyl-5-{[5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)-6-{6-oxo-8-thia-4,5-diazatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7),3-trien-5-yl}phenyl]methyl acetate 339a

According to Example 210e, 150 mg (0.30 mmol) of (4-fluoro-2-{6-oxo-8-thia-4,5-diazatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7),3-trien-5-yl}-6-(tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methyl acetate 341b was converted to a yellow solid 339a (129 mg, 60%). MS: [M+H] ⁺ 712

According to Example 148, 120 mg of 339a (0.17 mmol) was converted to a white solid 339 (56 mg, 50%). MS: [M+H] ⁺ 700. ¹ H NMR (500MHz, DMSO) δ8.56 (d, J＝3.0Hz, 1H), 8.48 (s, 1H), 8.41 (s, 1H), 7.88 (d, J＝ 3.0Hz,1H),7.37(m,4H),7.23(d,J＝9.5Hz,1H),4.60(t,J＝10.0Hz,1H),4.56(t,J ＝13.0Hz,2H),4.46(t,J＝12.0Hz,2H),4.28(s,2H),3.58(s,3H),3.43(m,1H),3. 07(t,J＝9.0Hz,4H)2.93(s,2H),2.85(s,2H),2.38(t,J＝9.0Hz,4H),1.87(m,4H).

Example 340 2-(5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-(5-(4-methylpiperazin-1-ylpyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydro-6,9-methanopyrazino[1,2-a]indol-1(2H)-one 340

Example 340a {4-fluoro-2-[1-methyl-5-({5-[4-methylpiperazin-1-yl]pyridin-2-yl}amino)-6-oxo-1,6-dihydropyridin-3-yl]-6-{7-oxo-3,6-diazatetracyclo[9.2.1.0 ^2,10 .0 ^3,8 ]tetradec-2(10),8-dien-6-yl}phenyl}methyl acetate 340a

To a microwave-safe vial equipped with a magnetic stirrer was added (2-bromo-4-fluoro-6-{7-oxo-3,6-diazatetracyclo[9.2.1.0 ^2,10 .0 ^3,8 ]tetradec-2(10),8-dien-6-yl}phenyl)methyl acetate 334g (293 mg, 0.65 mmol), 1-methyl-5-(5-(4-methylpiperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-ylboronic acid 197f (450 mg, 1.3 mmol), PdCl ₂ (dppf) (54 mg, 0.065 mmol), 2.0 M Na ₂ CO ₃ (2.0 equivalents) and 1,2-dimethoxyethane (18 mL). After three cycles of vacuum/argon purge, the mixture was heated at 130° C. under microwave irradiation for 0.5 h. The mixture was then filtered and the filtrate was evaporated in vacuo. The residue was purified by flash column chromatography using 10:1 dichloromethane/methanol as eluent to afford 340a as a brown solid (130 mg, 33%). LCMS: [M+H] ⁺ 666

A mixture of 340a (130 mg, 0.20 mmol) and LiOH (470 mg, 20.0 mmol) in isopropanol/THF (1:1, 10 mL) and water (3 mL) was stirred at 30°C for 2 h. The mixture was evaporated in vacuo, and the residue was extracted with ethyl acetate (30 mL x 2). The combined ethyl acetate extracts were concentrated under reduced pressure, and the residue was purified by preparative HPLC to give 340 (50 mg, 41%). LCMS: [M+H] ⁺ 624. ¹ H NMR(500MHz,MEOD)δ8.54(s,1H),7.93(s,1H),7.41-7.43(m,1H),7.34(s,1H),7.20-7.22( m,2H),7.02-7.04(d,J＝9.0Hz,1H),6.67-6.68(d,J＝3.0Hz,1H),4.46-4.55(m,2H),4.18-4 .38(m,3H),3.98-4.06(m,1H),3.71(s,3H),3.46-3.48(m,1H),3.14-3.18(m,4H),2.62-2. 64(t,J＝4.5Hz,4H),2.36(s,3H),1.88-1.95(m,3H),1.67-1.69(m,1H),1.01-1.30(m,2H).

Example 341 5-[5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-{[5-(4-methylpiperazin-1-yl)pyridin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)phenyl]-8-thia-4,5-diazatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7),3-triene-6-one 341

Example 341a (2-Bromo-4-fluoro-6-{6-oxo-8-thia-4,5-diazatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7),3-trien-5-yl}phenyl)methyl acetate 341a

To a 100-mL single-necked round-bottom flask equipped with a magnetic stirrer were added 8-thia-4,5-diazatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7),3-triene-6-one (1 g, 4.85 mmol), 2,6-dibromo-4-fluorobenzyl acetate 197c (4.8 g, 14.6 mmol), copper(I) iodide (553 mg, 2.9 mmol), N ¹ ,N ² -dimethylethane-1,2-diamine (512 mg, 5.82 mmol), Cs ₂ CO ₃ (3.2 g, 9.7 mmol), and 1,4-dioxane (50 mL). The system was evacuated and then refilled with N ₂ . A reflux condenser was attached to the flask, and the reaction mixture was heated at 100° C. for 16 h. It was then cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by flash chromatography using 5:1 petroleum ether/ethyl acetate as eluent to afford 341a as a yellow solid (437 mg, 20%). MS: [M+H] ⁺ 451.

Example 341b (4-Fluoro-2-{6-oxo-8-thia-4,5-diazatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7),3-trien-5-yl}-6-(tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methyl acetate 341b

According to Example 210d, 341a (400 mg 0.88 mmol) was converted to a yellow solid 341b (353 mg, 80%). MS: [M+H] ⁺ 499

Example 341c [4-Fluoro-2-(1-methyl-5-{[5-(4-methylpiperazin-1-yl)pyridin-2-yl]amino}-6-oxo-1,6-dihydropyridin-3-yl)-6-{6-oxo-8-thia-4,5-diazatricyclo[7.4.0.0 ^2,7 ]trideca-1(9),2(7),3-trien-5-yl}phenyl]methyl acetate 341c

According to Example 210a, 180 mg (0.36 mmol) of 341b was converted to a yellow solid 341c (120 mg, 50%). MS: [M+H] ⁺ 670

According to Example 148, 120 mg of 341c (0.18 mmol) was converted to a white solid 341 (46 mg, 41%). MS: [M+H] ⁺ 628. ¹ H NMR (500 MHz, CDCl3) δ 8.58 (d, J = 2.5, 1H), 8.26 (s, 1H), 7.95 (d, J = 3.0, 1H), 7.77 (s, 1H), 7.45 (d, J = 2.0, 1H), 7.31 (m, 1H), 7.25 (m, 1H), 7.11 (dd, J = 8.0, 1H), 6.82 ...6.82 (d, J = 3.0, 1H), 6.82 (s, 1H), 6.82 (d, J = 3.0, 1H), 6.82 (m, 1H), 6.82 (d, J = 3.0, 1H), 6.82 (m, 1H), 6. (d,J＝9.0,1H),4.31(s,1H),4.01(s,1H),3.71(s,3H),3.15(t,J＝4.5,4H),2.99( t, J = 5.0, 2H), 2.87 (t, J = 5.5, 2H), 2.60 (t, J = 5.0, 4H), 2.37 (s, 3H), 1.99 (s, 4H).

Example 342 2-(5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-(6-(4-methylpiperazin-1-yl)pyridazin-3-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 342

Example 342a 6-(4-methylpiperazin-1-yl)pyridazin-3-amine 342a

To a sealed tube equipped with a magnetic stirrer was added 6-chloro-pyridazin-3-amine (1.3 g, 10 mmol) and 1-methylpiperazine (15 mL). The reaction mixture was heated at 170°C for 3 h. It was then cooled to room temperature and concentrated under reduced pressure. The resulting residue was washed with _CH₃CN (20 mL) to afford 342a (1.5 g, 78%) as a yellow solid. MS: [M+H] ^⁺ 194. ^1H NMR (500 MHz, DMSO) δ 7.12 (d, J = 9.0 Hz, 1H), 6.74 (d, J = 9.0 Hz, 1H), 5.72 (s, 2H), 3.27 (t, J = 4.5 Hz, 4H), 2.40 (t, J = 4.5 Hz, 4H), 2.20 (s, 3H).

Example 342b 5-Bromo-1-methyl-3-(6-(4-methylpiperazin-1-yl)pyridazin-3-ylamino)pyridin-2(1H)-one 342b

According to Example 188c, 342a (580 mg, 3.0 mmol) was converted to a yellow solid 342b (920 mg, 80%). MS: [M+H] ⁺ 381

Example 342c 4-Fluoro-2-(1-methyl-5-(6-(4-methylpiperazin-1-yl)pyridazin-3-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzyl acetate 342c

According to Example 188f, 482 mg of 342b (1.0 mmol) and 379 mg of 5-bromo-1-methyl-3-(6-(4-methylpiperazin-1-yl)pyridazin-3-ylamino)pyridin-2(1H)-one (1.0 mmol) were reacted to give 342c (261 mg, 40%) as a yellow solid. MS: [M+H] ⁺ 655.

According to Example 188, 342c (150 mg 0.23 mmol) was converted to white solid 342 (73 mg, 52%). LCMS: [M+H] ⁺ 613. ¹ H NMR (500MHz, CDCl ₃ )δ8.68(d,J＝2.5Hz,1H),7.76(s,1H),7.46(d,J＝2.0Hz,1H),7.12(dd,J＝9.0Hz,1H),7.0(m,2H),6.90(dd,J＝9.0Hz,1H),6.86(s,1H),4.50 (dd,J＝11.0Hz,1H),4.27(m,2H),4.16(m,3H),3.88(m,1H),3.71(s,3H),3.54(m,4H),2.58(m,7H),2.35(s,3H),1.90(m,2H),1.71(m,2H).

Example 901 Btk biochemical assay

The general method for a standard biochemical assay for Btk kinase that can be used to test compounds of Formula I is as follows. Prepare a master mix containing 1X Cell Signaling Kinase Buffer (25 mM Tris-HCl, pH 7.5, 5 mM β-glycerophosphate, ₂ mM dithiothreitol, 0.1 mM _Na₃VO₄ , 10 mM _MgCl₂ ), 0.5 μM Promega PTK Biotinylated Peptide Substrate 2, and 0.01% BSA without Btk enzyme. Prepare a master mix containing 1X Cell Signaling Kinase Buffer, 0.5 μM PTK Biotinylated Peptide Substrate 2, 0.01% BSA, and 100 ng/well (0.06 mU/well) of Btk enzyme with Btk enzyme. The Btk enzyme was prepared as follows: full-length human wild-type Btk (accession number NM-000061) with a C-terminal V5 and 6x His tag was subcloned into the pFastBac vector to prepare baculovirus carrying this epitope-tagged Btk. Baculovirus was prepared according to the instructions detailed in Invitrogen's published experimental protocol "Bac-toBac Baculovirus Expression Systems" (Cat. Nos. 10359-016 and 10608-016). Passage 3 virus was used to infect Sf9 cells to overexpress recombinant Btk protein. The Btk protein was then purified to homogeneity using a Ni-NTA column. The final protein preparation had a purity greater than 95% based on the sensitive Sypro-Ruby staining method. A 200 μM ATP solution was prepared in water and adjusted to pH 7.4 with 1 N NaOH. 1.25 μL of a 5% DMSO solution of the compound was transferred to a 96-well half-area Costar polystyrene plate. Compounds were tested individually and using an 11-point dose-response curve (starting concentration was 10 μM; 1:2 dilution). An amount of 18.75 μL of the enzyme-free premix (as a negative control) and the enzyme-added premix was transferred to the appropriate wells of a 96-well half-area Costar polystyrene plate. 5 μL of 200 μM ATP was added to the mixture in the 96-well half-area Costar polystyrene plate to a final ATP concentration of 40 μM. The reaction was incubated at room temperature for 1 hour. The reaction was stopped with Perkin Elmer 1X detection buffer containing 30 mM EDTA, _{20 nM SA-APC, and 1 nM PT66Ab. The plate was read using time-resolved fluorescence using a Perkin Elmer Envision with an excitation filter of 330 nm, an emission filter of 665 nm, and a secondary emission filter of 615 nm. IC 50} values were then calculated. Alternatively, Btk activity can be assessed by quantifying its phosphorylated peptide product using the Lanthascreen assay. The FRET (fluorescence resonance energy transition) between the fluorescein on the peptide product and the terbium on the detection antibody decreases with the addition of a Btk inhibitor that reduces the phosphorylation of the peptide. In a final reaction volume of 25 uL, Btk (h) (0.1 ng/25 uL reaction) was incubated with 50 mM Hepes pH 7.5, 10 mM MgCl ₂ , 2 mM MnCl ₂ , 2 mM DTT, 0.2 mM NaVO ₄ , 0.01% BSA, and 0.4 uM fluorescein poly-GAT. The reaction was initiated by adding ATP to 25 uM (Km of ATP). After incubation at room temperature for 60 minutes, the reaction was terminated by adding the detection antibody at a final concentration of 2 nM Tb-PY20 in 60 mM EDTA for 30 minutes at room temperature. Detection was performed on a Perkin Elmer Envision using excitation at 340 nm and emission at 495 nm and 520 nm. Example Btk inhibition IC50 values are shown in Tables 1, 2, and 3.

Example 902 Ramos cell Btk assay

Another general method for a standard cell Btk kinase assay that can be used to test compounds of Formula I is as follows. Ramos cells are cultured at a density of 0.5×10 ⁷ cells/ml in the presence of the test compound at 37° C. for 1 hr. The cells are then stimulated by incubating with 10 μg/ml anti-human IgM F(ab) ₂ at 37° C. for 5 minutes. The cells are pelleted, lysed, and the clear lysate is subjected to protein assay. Each sample of equal protein amount is subjected to SDS-PAGE and Western blotting is performed with anti-phospho-Btk (Tyr223) antibody (Cell Signaling Technology #3531; Epitomics, cat. #2207-1) or phospho-Btk (Tyr551) antibody (BD Transduction Labs #558034) to assess Btk autophosphorylation or with anti-Btk antibody (BD Transduction Labs #611116) to control the total amount of Btk in each lysate.

Example 903 B cell proliferation assay

The general method for a standard cell B cell proliferation assay that can be used to test compounds of Formula I is as follows. B cells were purified from the spleens of 8-16 week old Balb/c mice using a B cell isolation kit (Miltenyi Biotech, Cat#130-090-862). Test compounds were diluted in 0.25% DMSO and incubated with 2.5×10 ⁵ purified mouse splenic B cells for 30 minutes, followed by the addition of 10 μg/ml anti-mouse IgM antibody (Southern Biotechnology Associates Cat#1022-01) in a final volume of 100 μl. After 24 hours of incubation, 1 μCi ³ H-thymidine was added, and the plates were incubated for an additional 36 hours before harvesting using the manufacturer's protocol for the SPA [ ³ H] Thymidine Uptake Assay System (Amersham Biosciences #RPNQ 0130). SPA bead-based fluorescence was counted in a microbeta counter (Wallace Triplex 1450, Perkin Elmer).

Example 904 T cell proliferation assay

The general method for a standard T cell proliferation assay that can be used to test compounds of Formula I is as follows. T cells were purified from the spleens of 8-16 week old Balb/c mice using a complete T cell isolation kit (Miltenyi Biotech, Cat#130-090-861). Test compounds were diluted in 0.25% DMSO and cultured with 2.5×10 ⁵ purified mouse spleen T cells in a final volume of 100 μl in a clear flat-bottom plate pre-coated with 10 μg/ml each of anti-CD3 (BD#553057) and anti-CD28 (BD#553294) antibodies at 37°C for 90 min. After 24 hours of incubation, 1 μCi ³ H-thymidine was added, the plate was incubated for another 36 hours, and then harvested using the manufacturer's protocol for the SPA [ ³ H] thymidine uptake assay system (Amersham Biosciences#RPNQ 0130). SPA bead-based fluorescence was counted in a microbeta counter (Wallace Triplex 1450, Perkin Elmer).

Example 905 CD86 inhibition assay

The general method of the standard assay for the inhibition of B cell activity that can be used to test the compound of formula I is as follows.Total mouse splenocytes were purified from the spleen of 8-16 week-old Balb/c mice by erythrocyte lysis (BD Pharmingen #555899).In a transparent flat-bottom plate (Falcon 353072), the test compound was diluted in 0.5% DMSO and cultured with 1.25 × ¹⁰⁶ splenocytes in a final volume of 200 μl at 37°C for 60 minutes. 15 μg/ml IgM (Jackson ImmunoResearch 115-006-020) was then added to stimulate the cells, and the cells were cultured at 37°C, 5% _CO2 for 24 hours. After 24 hours of culture, the cells were transferred to a conical bottom transparent 96-well plate and pelleted by centrifugation at 1200x gx 5min. Cells were preblocked with CD16/CD32 (BD Pharmingen #553142) and then triple stained with CD19-FITC (BD Pharmingen #553785), CD86-PE (BD Pharmingen #553692), and 7AAD (BD Pharmingen #51-68981E). Cells were sorted on a BD FACSCalibur and gated for the CD19 ⁺ /7AAD ⁻ population. The level of CD86 surface expression on the gated population was measured corresponding to the concentration of the test compound. Exemplary results are shown in Table 4.

Example 906 B-ALL cell survival assay

The following is a standard B-ALL (acute lymphoblastic leukemia) cell survival study method, which uses an XTT reader to measure the number of viable cells. This assay can be used to test the ability of Formula I compounds to inhibit the survival of B-ALL cells in culture. A human acute B-cell lymphoblastic leukemia line that can be used is SUP-B15, a human pre-B cell ALL line available from ATCC.

SUP-B15 pre-B-ALL cells were seeded at a concentration of 5× ¹⁰ cells/ml in 100 μl of Iscove's medium + 20% FBS in multiple 96-well microtiter plates. Test compounds were then added to a final concentration of 0.4% DMSO. Cells were cultured at 37°C and 5% _CO₂ for up to 3 days. After three days, the cells were split 1:3 into fresh 96-well plates containing the test compounds and allowed to grow for an additional 3 days. After each 24-hour period, 50 μl of XTT solution was added to a replicate 96-well plate, and absorbance readings were taken at 2, 4, and 20 hours according to the manufacturer's instructions. OD readings were then taken for cells treated with DMSO alone within the linear range of the assay (0.5-1.5), and the percentage of viable cells in the compound-treated wells relative to the DMSO-only cells was measured.

Example 907 CD69 whole blood assay

Human blood was obtained from healthy volunteers with the following restrictions: no drugs for 1 week, no smoking. Blood (approximately 20 ml for 8 compounds tested) was collected by venipuncture into tubes containing sodium heparin (Becton, Dickinson and Co.).

A 10 mM solution of the compound of Formula I in DMSO was diluted 1:10 in 100% DMSO and then serially diluted threefold in 100% DMSO for a 10-point dose-response curve. The compound was further diluted 1:10 in PBS, and 5.5 μl aliquots of each compound were added in duplicate to a 2 ml 96-well plate; 5.5 μl of 10% DMSO/PBS was added to control and unstimulated wells. Human whole blood (HWB) (100 μl) was added to each well. After mixing, the plate was incubated at 37°C, 5% _CO₂ , and 100% humidity for 30 minutes. Goat F(ab')2 anti-human IgM (10 μl of a 500 μg/ml solution, 50 μg/ml final) was added to each well (except the unstimulated well) and mixed. The plate was incubated for an additional 20 hours. At the end of the 20-hour incubation period, samples were incubated with fluorescently labeled antibodies for 30 minutes at 37°C, 5% _CO2 , and 100% humidity. Induction controls, unstained and single-stained samples for compensation adjustments, and initial voltage settings were included. Samples were then lysed with PharM Lyse ^™ (BD Biosciences Pharmingen) according to the manufacturer's instructions. Samples were then transferred to 96-well plates adapted for use on a BD Biosciences HTS 96-well system on an LSRII instrument. Data and mean fluorescence intensity were acquired using BD Biosciences DIVA software. Results were initially analyzed using FACS analysis software (Flow Jo). The IC50 of the test compound was defined as the concentration that reduced the number of CD69-positive cells that were also CD20-positive after anti-IgM stimulation by 50% (average of 8 control wells after subtracting the average of 8 wells with no stimulation). IC50 values were calculated using nonlinear regression curve fitting using Prism version 5.

Exemplary IC50 values for compounds selected from Tables 1, 2, and 3 in the CD69 whole blood assay include:

Compound number IC50 (micromolar) 105 0.088 197 0.023 210 0.035 313 0.044 324 0.053 325 0.024 343 0.053 345 0.024

Claims (27)

1. A compound selected from Formula I or a pharmaceutically acceptable salt thereof: in: ^R1 is a _C1 - _C3 alkyl group; ^R2 , ^R3 , and ^R4 are independently selected from H, D, F, Cl, _-NH2 , _-NHCH3 , -N( _CH3 ) ₂ , -OH, _-OCH3 , _{-OCH2CH3 , -OCH2CH2OH} , and _{C1 -C3 alkyl} groups; R ⁵ is optionally substituted _C6 - _C20 aryl, _C3 - _C12 carbocyclic, _C2 -C20 heterocyclic, _C1 - _C20 heteroaryl, -(C6- _C20 aryl)-( _C2 - _C20 heterocyclic), -( _C1 - _C20 heteroaryl)-( _C2 - _C20 heterocyclic), -( _C1 - _C20 heteroaryl)-( _C1 - _C6 alkyl), or -( _C1 - _C20 heteroaryl)-C(=O)-( _{C2 - C20 heterocyclic} ). ^R6 is H or a _C1 - _C3 alkyl group; ^Y1 and ^Y2 are independently selected from ^CR6 and N; The alkyl, carbocyclic, heterocyclic, aryl, and heteroaryl groups are optionally substituted by one or more groups independently selected from the following: D, F, Cl, Br, _I , _-CH3 , _-CH2CH3 , -CH2CH(CH3) _{2 ,} -CH2OH, _-CH2CH2OH , _{-C(CH3)2OH, -CH(OH)CH(CH3)2 , -C ( CH3 ) 2CH2OH , -CH2CH2SO2CH3 , -CH2OP(O)(OH)2 , -CN , -CH2F , -CHF2} , _-CF3 , _-CO2H , _-COCH3 , -CO2CH3, _-CO2C ( _{CH3 ) 3} , _-COCH ( _{OH ) CH3} , _-CONH2 , _-CONHCH3 , -CON(CH ₃ ) ₂ , -C(CH ₃ ) ₂ CONH ₂ , -NO ₂ , -NH ₂ , -NHCH ₃ , -N(CH ₃ ) ₂ , -NHCOCH ₃ , -N(CH ₃ )COCH ₃ , -NHS(O) ₂ CH ₃ , -N(CH ₃ )C(CH ₃ ) ₂ CONH ₂ , -N(CH ₃ )CH ₂ CH ₂ S(O) ₂ CH ₃ , =O, -OH, -OCH ₃ , -OCH ₂ CH ₂ N(CH ₃ ) ₂ , -OP(O)(OH) ₂ , -S(O) ₂ N(CH ₃ ) ₂ , -SCH ₃ , -CH ₂ OCH ₃ , -S(O) ₂ CH ₃ Cyclopropyl, aziridine, (1-methylaziridine-3-yl)oxy, N-methyl-N-oxetane-3-ylamino, aziridine-1-ylmethyl, oxetane and morpholino; Among them, groups: The following structure is formed: The wavy line represents the connection point. 2. The compound of claim 1, wherein the compound is selected from formulas Ia-c: 3. The compound of claim 1, wherein ^R2 , ^R3 and ^R4 are each H. 4. The compound of claim 1, wherein ^R1 is selected from _-CH3 , _-CH2F , _-CHF2 and _-CF3 . 5. The compound of claim 1, wherein ^R1 is _-CH2OH . 6. The compound of claim 1, wherein ^R3 is F. 7. The compound of claim 1, wherein ^R1 is _-CH2OH , ^R2 and ^R4 are each H, and ^R3 is F. 8. The compound of claim 1, wherein ^R5 is selected from optionally substituted phenyl and naphthyl groups. 9. The compound of claim 1, wherein ^R5 is selected from optionally substituted cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl. 10. The compound of claim 1, wherein ^R5 is selected from optionally substituted oxobutyranyl, azabutyranyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, morpholinyl, and tetrahydropyranyl. 11. The compound of claim 1, wherein R ⁵ is selected from optionally substituted pyrazolyl, pyridyl, pyrimidinyl, 5-methyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-yl, 5-acetyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-yl, 6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-2-yl and 1-methyl-5-(5-(4-methylpiperazin-1-yl)pyridin-2-yl). 12. The compound of claim 11, wherein ^R5 is substituted by one or more groups selected from F, Cl, _-CH3 , cyclopropyl, aziridine, oxadiazine, and morpholino. 13. The compound of claim 1, wherein ^R5 is selected from the following structures: The wavy line represents the connection point. 14. The compound of claim 1, wherein ^R6 is H. 15. The compound of claim 1, wherein ^Y1 is ^CR6 and ^Y2 is N. 16. The compound of claim 1, wherein ^Y1 is N and ^Y2 is ^CR6 . 17. The compound of claim 1, wherein ^Y1 and ^Y2 are each ^CR6 . 18. A compound, which is: 4-{2-methyl-3-[1-methyl-5-({5-methyl-4H,5H,6H,7H-pyrazolo[1,5-a]pyrazin-2-yl}amino)-6-oxo-1,6-dihydropyridazin-3-yl]phenyl}-7-thia-4-azatricyclo[6.4.0.0 ^2,6 ]dodecyl-1(8),2(6),9,11-tetraen-5-one. 19. The compound of claim 1, wherein the compound is selected from: [(2-{4,4-dimethyl-9-oxo-7-thia-10-azatricyclo[6.4.0.0 ^2,6 ]dodecyl-1(8),2(6)-dien-10-yl}-6-[1-methyl-6-oxo-5-(pyrimidin-4-ylamino)-1,6-dihydropyridin-3-yl]phenyl)methoxy]phosphonic acid; 5-[2-(hydroxymethyl)-3-{1-oxo-1H,2H,3H,4H,6H,7H,8H,9H-pyrido[3,4-b] indin-2-yl}phenyl]-1-methyl-3-[(5-methyl-1H-pyrazol-3-yl)amino]-1,2-dihydropyridin-2-one; 5-[2-(hydroxymethyl)-3-{1-oxo-1H,2H,3H,4H,6H,7H,8H,9H-pyrido[3,4-b] indin-2-yl}phenyl]-1-methyl-3-(5,6,7,8-tetrahydro-2,6-naphthidin-3-ylamino)-1,2-dihydropyridin-2-one; 3-{[5-(azacyclobutane-3-yl)pyridin-2-yl]amino}-5-[2-(hydroxymethyl)-3-{1-oxo-1H,2H,3H,4H,6H,7H,8H,9H-pyrido[3,4-b]-indene-2-yl}phenyl]-1-methyl-1,2-dihydropyridin-2-one; 5-[2-(hydroxymethyl)-3-{1-oxo-1H,2H,3H,4H,6H,7H,8H,9H-pyrido[3,4-b] indin-2-yl}phenyl]-1-methyl-3-(1,2,3,4-tetrahydroisoquinoline-6-ylamino)-1,2-dihydropyrazin-2-one; 5-[2-(hydroxymethyl)-3-{1-oxo-1H,2H,3H,4H,6H,7H,8H,9H-pyrido[3,4-b] indin-2-yl}phenyl]-1-methyl-3-{[5-(1-methylpyrrolidone-2-yl)pyridin-2-yl]amino}-1,2-dihydropyridin-2-one; 5-[2-(hydroxymethyl)-3-{1-oxo-1H,2H,3H,4H,6H,7H,8H,9H-pyrido[3,4-b] indin-2-yl}phenyl]-1-methyl-3-{[5-(4-methylpiperazin-1-yl)pyridin-2-yl]amino}-1,2-dihydropyridin-2-one; 5-[2-(hydroxymethyl)-3-{1-oxo-1H,2H,3H,4H,6H,7H,8H,9H-pyrido[3,4-b]-indene-2-yl}phenyl]-1-methyl-3-{4H,6H,7H-pyrazolo[3,2-c][1,4]oxazin-2-ylamino}-1,2-dihydropyridin-2-one; 3-{[5-(4-ethylpiperazin-1-yl)pyridin-2-yl]amino}-5-[2-(hydroxymethyl)-3-{1-oxo-1H,2H,3H,4H,6H,7H,8H,9H-pyrido[3,4-b]indene-2-yl}phenyl]-1-methyl-1,2-dihydropyridin-2-one. 20. A pharmaceutical composition comprising a compound of any one of claims 1 to 19 and a pharmaceutically acceptable carrier, gliding agent, diluent, or excipient. 21. The pharmaceutical composition of claim 20, further comprising another therapeutic agent. 22. A method for preparing a pharmaceutical composition, comprising mixing a compound of any one of claims 1 to 19 with a pharmaceutically acceptable carrier. 23. A kit for treating conditions mediated by Bruton's tyrosine kinase, comprising: a) a first pharmaceutical composition comprising the compound of any one of claims 1 to 19; and b) Instruction manual. 24. Use of the compound of any one of claims 1 to 19 in the preparation of a medicament for treating a disease or condition selected from immune disorders, cancer, cardiovascular disease, viral infection, inflammation, metabolic/endocrine dysfunction, and neurological disorders, wherein said medicament modulates Bruton's tyrosine kinase. 25. The use of claim 24, wherein the disease or condition is systemic and localized inflammation, arthritis, immunosuppression-related inflammation, organ transplant rejection, allergic reactions, ulcerative colitis, Crohn's disease, dermatitis, asthma, systemic lupus erythematosus, Sjögren's syndrome, multiple sclerosis, scleroderma/systemic sclerosis, idiopathic thrombocytopenic purpura (ITP), antineutrophil cytoplasmic antibody (ANCA) vasculitis, chronic obstructive pulmonary disease (COPD), or psoriasis. 26. The use of claim 24, wherein said disease or condition is selected from the following cancers: Breast cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, genitourinary tract cancer, esophageal cancer, glioblastoma, neuroblastoma, gastric cancer, skin cancer, keratoacanthoma, epidermoid carcinoma, large cell carcinoma, non-small cell lung cancer (NSCLC), small cell lung cancer, lung adenocarcinoma, bone cancer, colon cancer, adenoma, pancreatic cancer, thyroid cancer, follicular carcinoma, undifferentiated carcinoma, papillary carcinoma, seminoma, melanoma, sarcoma, bladder cancer, biliary tract cancer, kidney cancer, pilonidal carcinoma, oral cancer, nasopharyngeal carcinoma, pharyngeal cancer, lip cancer, tongue cancer, small intestine cancer, large intestine cancer, rectal cancer, Hodgkin's lymphoma, bronchial cancer, intrahepatic bile duct cancer, hepatocellular carcinoma, glioma, endometrial cancer, renal pelvis cancer, uterine corpus cancer, multiple myeloma, lymphocytic leukemia, myeloid leukemia, non-Hodgkin's lymphoma, and colonic villonoma. 27. The use of claim 24, wherein the medicament is used in combination with other therapeutic agents selected from anti-inflammatory drugs, compounds for treating immune-reactive diseases, chemotherapeutic agents, neurotrophic factors, agents for treating cardiovascular diseases, agents for treating liver diseases, antiviral agents, agents for treating blood diseases, agents for treating diabetes, and agents for treating immunodeficiency diseases.