HK1197401B - Alkylated piperazine compounds as inhibitors of btk activity - Google Patents

Description

Alkylated piperazine compounds as inhibitors of BTK activity

Cross Reference to Related Applications

This non-provisional application was filed according to 37CFR § 1.53(b), the benefit of us provisional application 61/555,395 filed on 3/11/2011, according to 35USC § 119(e), the entire contents of which are incorporated herein by reference.

Technical Field

The present invention relates generally to compounds useful for treating conditions mediated by Bruton's tyrosine kinase (Btk) including inflammation, immunological conditions, and cancer, and more particularly to compounds that inhibit Btk activity. The invention also relates to methods of using the compounds to reference in vitro, in situ, and in vivo diagnosis or treatment of mammalian cells or associated pathological conditions.

Background

Protein kinases are the largest family of human enzymes, including 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, signal transduction, and survival.

B cell signaling through the B Cell Receptor (BCR) can produce a wide range of biological output signals that in turn depend on the developmental stage of the B cell. The strength and duration of the BCR signal must be accurately adjusted. Aberrant BCR-mediated signal transduction can lead to dysregulated B-cell activation and/or the formation of pathogenic autoantibodies that lead to a variety of autoimmune and/or inflammatory diseases. Mutations in Btk in humans result in X-linked agammaglobulinemia (XLA). This disease is associated with impaired B cell maturation, decreased immunoglobulin production, an impaired T cell-independent immune response, and a marked reduction in sustained calcium signaling upon BCR stimulation. Evidence for a role for Btk in allergic and/or autoimmune and/or inflammatory diseases has been established in Btk-deficient mouse models. For example, Btk deficiency has been shown to cause significant improvement in disease progression in a standard murine preclinical model of Systemic Lupus Erythematosus (SLE). Moreover, Btk deficient mice are also resistant to developing collagen-induced arthritis and can be less susceptible to staphylococcal-induced arthritis. A large body of evidence supports the role of the B cell and 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 a method of treating many autoimmune and/or inflammatory diseases. Because of the role of Btk in B cell activation, Btk inhibitors can be used as inhibitors of B cell-mediated pathogenic activity (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, mouse Btk deficiency is associated with impaired IgE-mediated mast cell activation (significantly reduced release of TNF- α and other inflammatory cytokines), and human Btk deficiency is associated with greatly reduced TNF- α production by activated monocytes.

Thus, inhibition of Btk activity can be useful for treating allergic conditions and/or autoimmune and/or inflammatory diseases, such as: SLE, rheumatoid arthritis, polyangiitis, Idiopathic Thrombocytopenic Purpura (ITP), myasthenia gravis, allergic rhinitis, and asthma (Di Paolo et al (2011) Nature chem. biol.7(1): 41-50; Liu et al (2011) journal.of pharm. and expert. Ther.338(1): 154-. Furthermore, Btk has been reported to play a role in apoptosis; thus, inhibition of Btk activity can be useful in cancer as well as in the treatment of B cell lymphomas, leukemias, and other hematologic malignancies. Moreover, given the role of Btk in osteoclast function, inhibition of Btk activity can be useful in the treatment of bone diseases such as osteoporosis. Specific Btk inhibitors have been reported (Liu (2011) Drug Metab. AND dispensing 39(10): 1840. 1849; US7884108, WO 2010/056875; US 7405295; US 7393848; WO 2006/053121; US 7947835; US 2008/0139557; US 7838523; US 2008/0125417; US 2011/0118233; PCT/US2011/050034, PYRIDINONES/PYRAZINONES, METHOD OF MAKING, METHOD USEHEREOF "; PCT/US2011 05031, 7/2011 8/31, PCT/US 2011/013," PYRIDAZINONES, 2011 OF MAKING, AND METHOD OF THEEOF "; US13/102720," PYRIRINOOF AND OF AND-PYRINOOF "; US13/102720, 5/6, 3, 2011).

Disclosure of Invention

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

The compounds of formula I have the following structure:

including stereoisomers, tautomers or pharmaceutically acceptable salts thereof. The various substituents are as defined below.

One aspect of the 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 a second therapeutic agent.

Another aspect of the invention is a process for preparing a pharmaceutical composition comprising admixing a compound of formula I and a pharmaceutically acceptable carrier.

The invention includes a method of treating a disease or disorder selected from immune disorders, cancer, cardiovascular disease, viral infection, inflammation, metabolism/endocrine function disorders, and neurological disorders, mediated by Bruton's tyrosine kinase, comprising administering to a patient having a disease or disorder a therapeutically effective amount of a compound of formula I.

The present invention includes a kit for treating a disorder 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 diseases or disorders selected from immune disorders, cancer, cardiovascular diseases, viral infections, inflammation, metabolism/endocrine function disorders and neurological disorders, and mediated by Bruton's tyrosine kinase.

The invention includes the use of a compound of formula I in the manufacture of a medicament for the treatment of immune disorders, cancer, cardiovascular disease, viral infection, inflammation, metabolism/endocrine function disorders, and neurological disorders, and wherein the medicament modulates Bruton's tyrosine kinase.

The invention includes a process for preparing a compound of formula I.

Drawings

Figure 1 shows the preparation of (S) -2- (5-fluoro-2- (hydroxymethyl) -3- (1-methyl-5- (5- (2-methyl-4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-ylamino) -6-oxo-1, 6-dihydropyridin-3-yl) -phenyl) -3,4,6,7,8, 9-hexahydropyrido [3,4-b ] indolizin-1 (2H) -one 101, starting with intermediate 2, 6-dibromo-4-fluorobenzaldehyde 101 a.

FIG. 2 shows the preparation of (S) -5- [ 5-fluoro-2- (hydroxymethyl) -3 (1-methyl-5- (5- (2-methyl-4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-ylamino) -6-oxo-1, 6-dihydropyridin-3-yl) phenyl ] -8-thia-4, 5-diaza-tricyclo [7.4.0.02,7] tridec-1 (9),2(7), 3-trien-6-one 102 from the intermediate (S) -acetic acid [ 4-fluoro-2- (1-methyl-5- (5- (2-methyl-4- (oxetan-3-yl) -piperazine-1- Yl) pyridin-2-ylamino) -6-oxo-1, 6-dihydropyridin-3-yl) -6- { 6-oxo-8-thia-4, 5-diazacyclo [7.4.0.02,7] tridec-1 (9),2(7), 3-trien-5-yl } phenyl ] methyl ester 102 a.

FIG. 3 shows (2S) -10- [ 5-fluoro-2- (hydroxymethyl) -3- [ 1-methyl-5- ({5- [ 2-methyl-4- (oxetan-3-yl) piperazin-1-yl]Pyridin-2-yl } amino) -6-oxo-1, 6-dihydropyridin-3-yl]-phenyl radical]-4, 4-dimethyl-1, 10-diazacyclo [6.4.0.0^2,6]Preparation of dodeca-2 (6), 7-dien-9-one 103, starting from the intermediate ethyl (E) -3- (2-chloro-4, 4-dimethylcyclopent-1-enyl) acrylate 103 a.

FIG. 4a shows the preparation of 2- (3- (5- (5- ((2S,5R) -2, 5-dimethyl-4- (oxetan-3-yl) piperazin-1-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 104, starting from intermediate (2R,5S) -2, 5-dimethyl-4- (6-nitropyridin-3-yl) piperazine-1-carboxylic acid tert-butyl ester 104 a.

Figure 4b shows the preparation of acetic acid 4-fluoro-2- (1-oxo-3, 4,6,7,8, 9-hexahydro-pyrazino [1,2-a ] indol-1 (2H) -one 104j from 3,4,6,7,8, 9-hexahydro-pyrazino [1,2-a ] indol-2 (1H) -yl) -6- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzyl ester 104 o.

FIG. 5 shows the preparation of (S) -2- (3- (5- (5- (2-ethyl-4- (oxetan-3-yl) piperazin-1-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 105 from intermediate (S) -acetic acid 2- (5- (5- (2-ethyl-4- (oxetan-3-yl) piperazin-1-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 ester 105 a.

FIG. 6 shows the preparation of (S) -2- (5-fluoro-2- (hydroxymethyl) -3- (1-methyl-5- (5- (2-methyl-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 106, as an intermediate (S) -acetic acid 4-fluoro-2- (1-methyl-5- (5- (2-methyl-4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-yl Amino) -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 ester 106 a.

Figure 7 shows the preparation of (S) -2- (3- (5- (5- (3, 4-dimethylpiperazin-1-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 107, starting with intermediate (S) -tert-butyl 2-methyl-4- (6-nitropyridin-3-yl) piperazine-1-carboxylate 107 a.

Figure 8 shows the preparation of (R) -2- (3- (5- (5- (3, 4-dimethylpiperazin-1-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 108 starting from intermediate (R) -tert-butyl 2-methyl-4- (6-nitropyridin-3-yl) piperazine-1-carboxylate 108 a.

Figure 9 shows the preparation of (R) -2- (3- (5- (5- (2, 4-dimethylpiperazin-1-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 109, starting with intermediate (R) -3-methyl-4- (6-nitropyridin-3-yl) piperazine-1-carboxylic acid tert-butyl ester 109 a.

FIG. 10 shows the preparation of (S) -2- (3- (5- (5- (2, 4-dimethylpiperazin-1-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 110, starting from intermediate (S) -5-bromo-3- (5- (2, 4-dimethylpiperazin-1-yl) pyridin-2-ylamino) -1-methylpyridin-2 (1H) -one 110 a.

Figure 11 shows the preparation of 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 111 starting from intermediate 4-benzylpiperazine-1, 2-dicarboxylic acid 1-tert-butyl ester.2methyl ester 111 a.

FIG. 12 shows the preparation of 2- (5-fluoro-2- (hydroxymethyl) -3- (1-methyl-5- (5- (9-methyl-7-oxa-3, 9-diaza-bicyclo [3.3.1] non-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 112, starting with intermediate N, N-dibromobenzenesulfonamide 112 a.

FIG. 13 shows the preparation of (S) -acetic acid 2- (7, 7-difluoro-1-oxo-3, 4,6,7,8, 9-hexahydropyrazino [1,2-a ] indol-2 (1H) -yl) -4-fluoro-6- (1-methyl-5- (5- (2-methyl-4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-ylamino) -6-oxo-1, 6-dihydropyridin-3-yl) benzyl ester 113q, starting from intermediate (3S) -3-methyl-4- (6-nitropyridin-3-yl) piperazine-1-carboxylic acid tert-butyl ester 113 a.

Figure 14 shows the preparation of 113o from intermediate 1H-pyrrole-2-carboxylic acid ethyl ester.

FIG. 15 shows the preparation of 2-bromo-6- {4, 4-dimethyl-9-oxo-7-thia-10, 11-diazacyclo [6.4.0.0 ] from intermediate 3-methylcyclopent-2-enone^2,6]Dodeca-1 (8),2(6), 11-trien-10-yl } -4-fluorobenzaldehyde 131 i.

Detailed Description

Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the accompanying structures and molecular formulae. While the invention will be described in conjunction with the enumerated embodiments, it will be understood that the invention is not limited to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications and equivalents, which may be included within the scope of the invention as defined by the appended claims. Those skilled in the art will recognize that many methods and materials similar or equivalent to those described herein can be used in the practice of 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 or contradict this application (including but not limited to defined terms, usage of terms, described techniques, etc.), this application controls. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. Unless otherwise indicated, the nomenclature used in this application is based on the IUPAC systematic nomenclature.

Definition of

When stating the number of substituents, the term "one or more" refers to the range from one substituent to the maximum possible number of substituents, i.e., replacement of one hydrogen by a substituent to replacement of all hydrogens. The term "substituent" means an atom or group of atoms that replaces a hydrogen atom on a parent molecule. The term "substituted" means that the specified group bears one or more substituents. When any group can carry multiple substituents and a variety of possible substituents are provided, the substituents are independently selected and need not be the same. The term "unsubstituted" means that the indicated group bears no substituents. The term "optionally substituted" means that the specified group is unsubstituted or substituted with one or more substituents independently selected from possible substituents. When stating the number of substituents, the term "one or more" means from one substituent up to the maximum possible number of substituents, i.e., replacement of one hydrogen by a substituent to replacement of all hydrogens.

The term "alkyl" as used herein means having 1 to 12 carbon atoms (C)₁-C₁₂) Wherein the alkyl group may be optionally independently substituted with one or more substituents described below. In another embodiment, the alkyl group has 1 to 8 carbon atoms (C)₁-C₈) Or 1 to 6 carbon atoms (C)₁-C₆). Examples of alkyl groups include, but are not limited to: methyl (Me, -CH)₃) Ethyl (Et-CH)₂CH₃) 1-propyl (n-Pr, n-propyl, -CH)₂CH₂CH₃) 2-propyl (i-Pr, isopropyl, -CH (CH)₃)₂) 1-butyl (n-Bu, n-butyl, -CH)₂CH₂CH₂CH₃) 2-methyl-1-propyl (i-Bu, isobutyl, -CH)₂CH(CH₃)₂) 2-butyl (s-Bu, sec-butyl, -CH (CH)₃)CH₂CH₃) 2-methyl-2-propyl (t-Bu, tert-butyl, -C (CH)₃)₃) 1-pentyl (n-pentyl, -CH)₂CH₂CH₂CH₂CH₃) 2-pentyl (-CH (CH)₃)CH₂CH₂CH₃) 3-pentyl (-CH (CH)₂CH₃)₂) 2-methyl-2-butyl (-C (CH)₃)₂CH₂CH₃) 3-methyl-2-butyl (-CH (CH)₃)CH(CH₃)₂) 3-methyl-1-butyl (-CH)₂CH₂CH(CH₃)₂) 2-methyl-1-butyl (-CH)₂CH(CH₃)CH₂CH₃) 1-hexyl (-CH)₂CH₂CH₂CH₂CH₂CH₃) 2-hexyl (-CH (CH)₃)CH₂CH₂CH₂CH₃) 3-hexyl (-CH (CH)₂CH₃)(CH₂CH₂CH₃) 2-methyl-2-pentyl (-C (CH))₃)₂CH₂CH₂CH₃) 3-methyl-2-pentyl group(s) (ii)-CH(CH₃)CH(CH₃)CH₂CH₃) 4-methyl-2-pentyl (-CH (CH)₃)CH₂CH(CH₃)₂) 3-methyl-3-pentyl (-C (CH)₃)(CH₂CH₃)₂) 2-methyl-3-pentyl (-CH (CH)₂CH₃)CH(CH₃)₂)2, 3-dimethyl-2-butyl (-C (CH)₃)₂CH(CH₃)₂)3, 3-dimethyl-2-butyl (-CH (CH)₃)C(CH₃)₃1-heptyl, 1-octyl, and the like.

The term "alkylene" as used herein means having from 1 to 12 carbon atoms (C)₁-C₁₂) Wherein said alkylene group may be optionally independently substituted with one or more substituents described below. In another embodiment, the alkylene group has 1 to 8 carbon atoms (C)₁-C₈) Or 1 to 6 carbon atoms (C)₁-C₆). Examples of alkylene groups include, but are not limited to, methylene (-CH)₂-) ethylene (-CH₂CH₂-) propylene (-CH)₂CH₂CH₂-) and the like.

The term "alkenyl" refers to a group having 2 to 8 carbon atoms (C)₂-C₈) Having at least one site of unsaturation, i.e. carbon-carbon sp²A double bond, wherein said alkenyl group may be optionally independently substituted with one or more substituents as described herein, and includes groups having "cis" and "trans" orientations, or "E" and "Z" orientations. Examples include, but are not limited to, ethenyl (-CH ═ CH)₂) Allyl (-CH)₂CH＝CH₂) And the like.

The term "alkenylene" refers to a compound having 2-8 carbon atoms (C)₂-C₈) Having at least one site of unsaturation, i.e. carbon-carbon sp²A double bond, wherein said alkenylene group may be optionally independently substituted with one or more substituents as described herein, and includes groups having "cis" and "trans" orientations, or "E" and "Z" orientations. Examples include, but are not limited to, ethenylene (-CH-), allyl (-CH)₂CH-) and the like.

The term "alkynyl" refers to a compound having 2-8 carbon atoms (C)₂-C₈) Straight or branched chain monovalent hydrocarbon radical having at least one site of unsaturation, i.e., a carbon-carbon sp triple bond, wherein said alkynyl radical may be optionally independently substituted with one or more substituents described herein. Examples include, but are not limited to, ethynyl (-C ≡ CH), propynyl (propargyl, -CH)₂C.ident.CH) and the like.

The term "alkynylene" refers to a compound having 2-8 carbon atoms (C)₂-C₈) A straight or branched chain divalent hydrocarbon radical having at least one site of unsaturation, i.e., a carbon-carbon sp triple bond, wherein said alkynylene radical may be optionally independently substituted with one or more substituents described herein. Examples include, but are not limited to, ethynylene (-C ≡ C-), propynyl (propargylene, -CH)₂C.ident.C-), and the like.

The terms "carbocyclic", "carbocyclyl", "carbocyclic ring" and "cycloalkyl" refer to a group having 3 to 12 carbon atoms (C)₃-C₁₂) A monovalent non-aromatic saturated or partially unsaturated ring in the form of a monocyclic ring or in the form of a bicyclic ring having 7 to 12 carbon atoms. Bicyclic carbocyclic rings having 7 to 12 atoms may be arranged, for example, as bicyclo [4,5]]、[5,5]、[5,6]Or [6, 6]]The bicyclic carbocyclic rings having 9 or 10 ring atoms of the system may be arranged as bicyclo [5,6]Or [6, 6]]The systems may alternatively be arranged as bridged ring systems, e.g. bicyclo [2.2.1]Heptane, bicyclo [2.2.2]Octane and bicyclo [3.2.2]Nonane. Spirocyclic moieties are also included within the scope of this definition. 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. Carbocyclyl is optionally independently substituted with one or more substituents described herein.

' FangBy "radical" is meant a radical having from 6 to 20 carbon atoms (C) obtained by removing one hydrogen atom from a single carbon atom of a parent aromatic ring system₆-C₂₀) A monovalent aromatic hydrocarbon group of (2). Some aryl groups are represented in the exemplary structures as "Ar". Aryl includes bicyclic groups comprising an aromatic ring fused to a saturated, partially unsaturated ring or an aromatic carbocyclic ring. Typical aryl groups include, but are not limited to, groups derived from benzene (phenyl), substituted benzene, naphthalene, anthracene, biphenyl, indenyl, indanyl, 1, 2-dihydronaphthalene, 1,2,3, 4-tetrahydronaphthyl, and the like. Aryl is optionally independently substituted with one or more substituents described herein.

"arylene" means having 6 to 20 carbon atoms (C) by removal of two hydrogen atoms from two carbon atoms of a parent aromatic ring system₆-C₂₀) A divalent aromatic hydrocarbon group of (1). Some arylene groups are represented in the exemplary structures as "Ar". Arylene includes bicyclic groups comprising an aromatic ring fused to a saturated, partially unsaturated ring or aromatic carbocyclic ring. Typical arylene groups include, but are not limited to, groups derived from benzene (phenylene), substituted benzenes, naphthalene, anthracene, biphenylene, indenylene, indanene, 1, 2-dihydronaphthalene, 1,2,3, 4-tetrahydronaphthyl, and the like. The arylene group is optionally substituted with one or more substituents described herein.

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 from 3 to about 20 ring atoms in which at least one ring atom is a heteroatom selected from nitrogen, oxygen, phosphorus and sulfur and the remaining ring atoms are C, wherein one or more ring atoms are optionally independently substituted with one or more substituents described below. The heterocyclic ring may be a single 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: bicyclo [4,5]]、[5,5]、[5,6]Or [6, 6]]Provided is a system. Heterocycles are described in Paquette, Leo a.; "Principles of Modern heterocyclic chemistry" (W.A.B.enjamin, New York, 1968), in particular chapters 1,3, 4,6,7 and 9; "The chemistry of Heterocyclic compounds, A series of monograms" (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 groups in which a heterocyclyl group is fused to a saturated, partially unsaturated ring or to an aromatic carbocyclic or heterocyclic ring. Examples of heterocycles include, but are not limited to: morpholin-4-yl, piperidin-1-yl, piperazinyl, piperazin-4-yl-2-one, piperazin-4-yl-3-one, pyrrolidin-1-yl, thiomorpholin-4-yl, S-dioxothiomorpholin-4-yl, azetidin-1-yl, octahydropyrido [1,2-a ] base]Pyrazin-2-yl, [1,4 ]]Diazepan-1-yl, pyrrolidinyl, tetrahydrofuryl, dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino, thioxanyl, piperazinyl, homopiperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thietanyl, oxazepinyl, and oxazepinylRadical diazaRadical, sulfur nitrogen heteroYl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1, 3-dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuryl, pyrazolidinyl, imidazolinyl, imidazolidinyl, 3-azabicyclo [3.1.0]Hexyl, 3-azabicyclo [4.1.0]Heptyl, azabicyclo [2.2.2]Hexyl, 3H-indolyl, quinolizinyl and N-pyridylurea. Spirocyclic moieties are also included within the scope of this definition. Examples of heterocyclyl groups in which 2 ring atoms are partially replaced by oxo (═ O) are pyrimidinone groups and 1, 1-dioxothiomorpholinyl groups. Heterocyclic radical in this applicationOptionally independently substituted with one or more substituents described herein.

The term "heteroaryl" refers to a 5-, 6-or 7-membered monovalent aromatic group, and also includes fused ring systems (at least one of which is aromatic) having 5 to 20 atoms, the 5-, 6-or 7-membered monovalent aromatic group and the fused ring system containing 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, quinolyl, isoquinolyl, tetrahydroisoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, triazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothienyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridyl. Heteroaryl groups are optionally independently substituted with one or more substituents described herein.

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

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

The terms "treatment" and "treatment" refer to a therapeutic treatment with the purpose of slowing (alleviating) the formation or spread of an undesired physiological change or disorder, such as arthritis or cancer. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. "treatment" may also refer to an increase in survival time compared to the expected survival time if treatment was not received. Those in need of treatment include those with a condition or disorder.

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

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

"inflammation" as used herein refers to a local protective response triggered by the damage or destruction of tissue, which acts to destroy, dilute, or shield (sequester) harmful 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 following myocardial infarction or stroke, immune and autoimmune responses to foreign antigens. Thus, inflammatory disorders amenable to treatment with the compounds of formula I include those associated with a response to a specific defense system as well as a response to a non-specific defense system.

By "specific defense system" is meant a component of the immune system that reacts to the presence of a specific antigen. Examples of inflammation caused by the response of a specific defense system include a general response to a foreign antigen, autoimmune diseases, and delayed-type hypersensitivity mediated by T cells. Other examples of inflammatory responses of the specific defense system are chronic inflammatory diseases, rejection of solid transplanted tissues and organs such as kidney 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 incapable of immunological memory. Examples of inflammation arising at least in part from 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 disorders with an acute inflammatory component; acute purulent meningitis or other central nervous system inflammatory disorders such as stroke; heat damage; inflammatory bowel disease; a granulocyte transfusion-related syndrome; and cytokine-induced toxicity.

"autoimmune disease" as used herein refers to any type of condition in which tissue damage is associated with a humoral or cell-mediated response to the body's own components.

"allergic disease" as used herein refers to any symptom resulting from an allergy, tissue damage or loss of tissue function. "arthritic disease" as used herein refers to any disease characterized by arthritic damage attributable to various etiologies. "dermatitis" as used herein refers to any of a broad class of diseases characterized by skin inflammation attributable to various etiologies. "transplant rejection" as used herein refers to any immune response against a transplanted tissue, such as an organ or cell (e.g., bone marrow), characterized by loss of function of the transplanted and surrounding tissues, pain, swelling, leukocytosis, and thrombocytopenia. The therapeutic methods of the invention include methods for treating conditions associated with inflammatory cell activation.

By "inflammatory cell activation" is meant induction by the production of stimuli of a proliferative cell response (including but not limited to cytokines, antigens, or autoantibodies), soluble mediators (including but not limited to cytokines, oxygen radicals, enzymes, prostaglandins, or vasoactive amines), or 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 is understood by those skilled in the art that activation of one or a combination of these phenotypes in these cells can promote initiation, perpetuation, or exacerbation of an inflammatory disorder.

The term "NSAID" is an acronym for "nonsteroidal anti-inflammatory drug," a therapeutic agent that has analgesic, antipyretic (lowers body hyperthermia and relieves pain without compromising perception) and anti-inflammatory efficacy (reduces inflammation) at higher doses. The term "non-steroidal" is used to distinguish these drugs from steroidal phases that have similar eicosanoid-lowering, anti-inflammatory effects (a wide range of other effects). NSAIDs are unusual as analgesics in that they are non-narcotic. NSAIDs include aspirin, ibuprofen, and naproxen. NSAIDs are generally indicated for the treatment of acute or chronic conditions with pain and inflammation. NSAIDs are generally indicated for symptomatic relief of the following conditions: rheumatoid arthritis, osteoarthritis, inflammatory joint diseases (e.g., ankylosing spondylitis, psoriatic arthritis, reiter's syndrome, acute gout, dysmenorrhea, metastatic bone pain, headache and migraine, post-operative pain, mild to moderate pain due to inflammation and tissue injury, fever, ileus, and renal colic.) most NSAIDs are used as non-selective inhibitors of cyclooxygenase enzymes, cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) isoenzymes cyclooxygenase catalyzes the formation of prostaglandins and thromboxanes from arachidonic acid, itself derived from a cellular phospholipid bilayer by phospholipase A2.

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 cancer 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 cancer (e.g., epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer ("NSCLC"), adenoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or pancreatic cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, and head and neck cancer.

"hematological malignancies" (the uk spelling "Haematological" malignancies) are the type of cancer that affects the blood, bone marrow and lymph nodes. Since three of them are closely linked by the immune system, diseases affecting one of the three often also affect the other two: although lymphoma is a disease of lymph nodes, it often spreads to the bone marrow, affecting the blood. Hematological malignancies are malignancies ("cancers") and are essentially treated by hematological and/or oncological experts. In some centers "hematology/oncology" is a separate sub-discipline of medical medicine, while in other centers it is considered a different part (surgical and radiation oncologists also exist). Hematological disorders are not all malignant ("cancerous"); these other hematological disorders can also be managed by hematologists. Hematological malignancies can arise from two major blood cell lineages: myeloid and lymphoid cell lines. Myeloid cell lines normally produce granulocytes, erythrocytes, platelets, macrophages and mast cells; lymphoid cell lines give rise to B, T, NK and plasma cells. Lymphomas, lymphocytic leukemias and myelomas are from lymphoid cell lines, while acute and chronic myelogenous leukemias, myelodysplastic syndromes and myeloproliferative diseases are of myeloid cell origin. Leukemias include Acute Lymphocytic Leukemia (ALL), Acute Myelogenous Leukemia (AML), Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), acute monocytic leukemia (AMOL), and Small Lymphocytic Lymphoma (SLL). Lymphomas include hodgkin's lymphoma (all four subtypes) and non-hodgkin's lymphoma (all subtypes).

"chemotherapeutic agents" are compounds used to treat cancer, regardless of the 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: ere (Chinese character) deviceLotinib (A), (B), (C), (Genentech/OSI Pharm), docetaxelSanofi-Aventis), 5-FU (fluorouracil, 5-fluorouracil, CAS number 51-21-8), gemcitabine (Gemcitabine)Lilly), PD-0325901(CAS number 391210-10-9, Pfizer), cisplatin (cis-diamine, platinum (II) dichloride, CAS number 15663-27-1), carboplatin (CAS number 41575-94-4), paclitaxel (r) (paclitaxel: (r)Bristol-Myers Squibb Oncology, Princeton, N.J.), trastuzumab (Genentech), temozolomide (4-methyl-5-oxo-2, 3,4,6, 8-pentaazabicyclo [4.3.0]Nonane-2, 7, 9-triene-9-carboxamide, CAS number 85622-93-1,schering Plough), tamoxifen ((Z) -2- [4- (1, 2-diphenylbut-1-enyl) phenoxy]-N, N-dimethylethylamine,) And doxorubicin (c) ((c))) Akti-1/2, HPPD and rapamycin.

Other examples of chemotherapeutic agents include: oxaliplatin (oxaliplatin) ((oxaliplatin))Sanofi), bortezomib (bortezomib), (b), (c), (d), (Millennium Pharm.), sunitinib (sutent), (a mixture of them)SU11248, Pfizer), letrozole (letrozole), (L-Toxole)Novartis), imatinib mesylate (imatinib mesylate), (I) and (II) a pharmaceutically acceptable salt thereofNovartis), XL-518(MEK inhibitor, Exelixis, WO2007/044515), ARRY-886(MEK inhibitor, AZD6244, Array BioPharma, Astra Zeneca), SF-1126(PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235(PI3K inhibitor, Novartis), XL-147(PI3K inhibitor, Exelixis), PTK787/ZK222584(Novartis), fulvestrant (fulvestrant) ((fulvestrant)AstraZeneca), leucovorin (leucovorin), rapamycin (sirolimus, and folic acid,Wyeth), lapatinib (lapatinib)GSK572016,GlaxoSmith Kline)、lonafarnib(SARASAR^TMSCH66336, Schering Plough), Sorafenib (sorafenib) ((Schering Plough)BAY43-9006, Bayer Labs), gefitinib (gefitinib) ((B)AstraZeneca), irinotecan (irinotecan), (CPT-11,Pfizer)、tipifarnib(ZARNESTRA^TM,Johnson&Johnson)、ABRAXANE^TM(Cremophor-free), albumin engineered nanoparticle formulations of paclitaxel (albumin-engineered nanoparticles formulations of paclitaxel) (American Pharmaceutical Patners, Schaumberg, Il.), vandetanib (rINN, ZD6474,AstraZeneca)、chloranmbucil、AG1478、AG1571(SU5271；Sugen)、temsirolimus(Wyeth)、pazopanib(GlaxoSmithKline)、canfosfamide(telik), thiotepa and cyclophosphamide (cyclophosphamide) ((Telik)) (ii) a Alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines (aziridines) such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimine (ethylenimine) and methylaminoacridine (methyamelamine), including hexamethylmelamine, triimizine (triethyleneemimine), triethylenephosphoramide (triethylenephosphoramide), triethylenephosphoramide (triethylenethiophosphamide), and trimethymelamine; annonaceous acetogenin (especially bullatacin and bullatacin); camptothecin (including the synthetic analog topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin (adozelesin), carvelesin (carzelesin), and bizelesin (bizelesin) synthetic analogs); cryptophycins (especially cryptophycin1 and cryptophycin 8); dolastatin (dolastatin); duocarmycins (including the synthetic analogs KW-2189 and CB1-TM 1); eiscosahol (eleutherobin); pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as phenylbutyric acid nitrogenMustard, naphazoline, chlorophosphamide (chlorophosphamide), estramustine, ifosfamide, mechlorethamine (mechlorethamine), mechlorethamine hydrochloride (mechlorethamine oxide hydrochloride), melphalan, neomustard (novembichin), benzene mustard cholesterol (phenesterine), prednimustine, trofosfamide, uracil mustard (uracil musard); nitroureas such as carmustine, chlorozotocin (chlorozotocin), fotemustine, lomustine, nimustine and ranimustine; antibiotics such as enediyne (enediyne) antibiotics (e.g., calicheamicin gamma 1I, calicheamicin omega I1(Angew chem. Intl. Ed. Engl. (1994)33:183-186), anthracyclines (dynemicin), dynemicin A, bisphosphonates (bisphosphates) such as clodronate, esperamicin (esperamicin), and neooncostatin chromophores (neocarzinostatin chromophoropterin) and related chromogenes enediyne (enediyne chromophores), acrinomycin, actinomycin (ubomycin), aureomycin, serazaserine (azacine), desmomycin, actinomycin C, actinomycin (5-carboxymycin), actinomycin (5-6-erythromycin), normycin (monocrotamycin-6), monocrotamycin (monocrotamycin-5-6), monocrotamycin (monocrotamycin-6-5-D), monocrotamycin (monocrotamycin-6-D), monocrotamycin (monocrotamycin-D), monocrotamycin (monocrotamycin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolidono-doxorubicin and deoxydoxorubicin), epirubicin (epirubicin), esorubicin, idarubicin, nemorubicin (nemorubicin), mariomycin (marcelomycin), mitomycins such as mitomycin C, mycophenolic acid (mycophenolic acid), norramycin (nogalamycin), olivomycin (olivomycin), pelomycin (pelomycin), peplomycin (peplomycin), pofiromycin (porfiromycin), puromycin (puromycin), triformycin (quelamycin), rodobicin (rodorubicin), streptomycin (streptanigrin), streptozocin (streptozotocin), tubercidin (tubicin), ubenimex (ubenimex), stastatin (zostatin), zotocin (zotocin); antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate,Pteropterin (pteropterin), trimetrexate (trimetrexate); purine analogs such as fludarabine (fludarabine), 6-mercaptopurine, thiamiprine (thiamiprine), thioguanine (thioguanine); pyrimidine analogs such as ancitabine (ancitabine), azacitidine (azacitidine), 6-azaguanosine (6-azauridine), carmofur (carmofur), cytarabine (cytarabine), dideoxyuridine (dideoxyuridine), doxifluridine (doxifluridine), enocitabine (enocitabine), floxuridine (floxuridine); androgens such as carposterone (calusterone), methyl androsterone propionate (dromostanolone propionate), epitioandrostanol (epitiostanol), mepiquane (mepiquitane), testolactone (testolactone); anti-adrenaline (anti-adrenaline) such as aminoglutethimide, mitotane, trilostane; folic acid replenisher (folic acid replenisher) such as folinic acid (frilic acid); acetoglucurolactone (acegultone); (ii) an aldophosphamide glycoside; aminolevulinic acid (aminolevulinic acid); eniluracil (eniluracil); amsacrine (amsacrine); bestrabuucil; bisantrene; idazot (edatraxate); desphosphamide (defofamine); colchicine (demecolcine); diazaquinone (diaziqutone); elfornitine; ammonium etitanium acetate; epothilone (epothilone); etoglut (etoglucid); gallium nitrate (gallium nitrate); hydroxyurea (hydroxyurea); lentinan (lentinan); lonidamine (lonidainine); maytansinol (maytansinoids) such as maytansinoids (maytansine) and ansamitocins (ansamitocins); mitoguazone (mitoguzone); mitoxantrone (mitoxantrone); mopidanmol; rhizobia (nitrarine); pentostatin (pentostatin); methionine mustard (phenamett); pirarubicin (pirarubicin); losoxantrone (losoxantrone); podophyllinic acid (podophyllinic acid); 2-ethyl hydrazine; procarbazine (procarbazine);polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane (rizoxane); rhizomycin (rhizoxin); sizofuran (sizofiran); germanium spiroamines (spirogyranium); tenuazonic acid (tenuazonic acid); triimine quinone (triaziquone); 2,2' -trichlorotriethylamine; sheetTelesporin (trichothecene) (especially T-2 toxin, veracurin a, bacillocin a and anguidine); uratan; vindesine; dacarbazine (dacarbazine); mannomustine (mannomustine); dibromomannitol (mitobronitol); dibromodulcitol (mitolactol); pipobromane (pipobroman); a polycytidysine; cytarabine (arabine) ("Ara-C"); cyclophosphamide; thiotepa; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine (A)) (ii) a Norfloxacin (novantrone); teniposide (teniposide); idazocide (edatrexate); daunorubicin; aminopterin (aminopterin); capecitabine (capecitabine) (capecitabine)Roche); ibandronate (ibandronate); CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids (retinoids) such as retinoic acid (retinoic acid); and pharmaceutically acceptable salts, acids and derivatives of any of the foregoing.

The following are also included in the definition of "chemotherapeutic agents": (i) anti-hormonal agents, such as anti-estrogen agents (anti-estrogens) and Selective Estrogen Receptor Modulators (SERMs), including for example tamoxifen (including tamoxifen) for modulating or inhibiting the effects of hormones on tumorsTamoxifen citrate), raloxifene, droloxifene, 4-hydroxy tamoxifen, trioxifene (trioxifene), raloxifene (keoxifene), LY117018, onapristone (onapristone), and(toremifene citrate); (ii) inhibition of aromatase (modulation of estrogen production in the adrenal gland)) Aromatase inhibitors of (2), for example 4(5) -imidazole, aminoglutethimide,(megestrol acetate)) (a salt of megestrol acetate),(exemestane; Pfizer), formestanie, fadrozole,(vorozole) and (C) a salt thereof,(letrozole; Novartis) and(anastrozole; AstraZeneca), (iii) antiandrogen drugs (anti-androgen) such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin (goserelin) and troxacitabine (1, 3-dioxolane nucleoside cytosine analogues), (iv) protein kinase inhibitors such as MEK inhibitors (WO2007/044515), (v) lipid kinase inhibitors, (vi) antisense oligonucleotides, particularly those that inhibit gene expression in signal transduction pathways involved in abnormal cell proliferation, such as PKC- α, Raf and H-Ras, such as OBLIMERSEN (anti-androgen), such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin (goserelin), and troxacitabine (1, 3-dioxolane nucleoside cytosine analogues), (iv) protein kinase inhibitors such as MEK inhibitors (WO2007/044515), (v) lipid kinase inhibitors, (vi) antisense oligonucleotides, particularly those that inhibit gene expression in signal transduction pathways involved in abnormal cell proliferation, such as PKC- α, Raf and H-Genta Inc.); (vii) ribozymes such as VEGF expression inhibitors (e.g.) And inhibitors of HER2 expression; (viii) vaccines, e.g. gene therapy vaccines, e.g.AndrIL-2; topoisomerase 1 inhibitors such asrmRH; (ix) anti-angiogenic drugs such as bevacizumab (Genentech); and pharmaceutically acceptable salts, acids and derivatives of any of the foregoing.

Also included in the definition of "chemotherapeutic agent" are therapeutic antibodies such as alemtuzumab (Campath), bevacizumab (b)Genentech); cetuximab (Imclone); palizumab (panitumumab,amgen), rituximab (Genentech/Biogen Idec), pertuzumab (pertuzumab,2C4, Genentech), trastuzumab (Genentech), tositumomab (Bexxar, Corixia), and the antibody drug conjugate orituzumab (gemtuzumab ozogamicin,Wyeth)。

humanized monoclonal antibodies having therapeutic potential as chemotherapeutic agents for use in combination with the Btk inhibitors of the invention include: alemtuzumab (alemtuzumab), aprezumab (apilizumab), aselizumab (aselizumab), atlizumab, bapineuzumab, bevacizumab (bevacizumab), mabuzumab (bivatuzumab), mocatuzumab (cantuzumab mertansine), celizumab (cedenzumab), certuzumab (cetuzumab), seduzumab (cetuzumab), ciduzumab (ciduzumab), ciduzumab, daclizumab (daclizumab), eculizumab (efalizumab), epratuzumab (epatuzumab), epratuzumab (epuzumab), epruzumab (erbuzumab), panzeuzumab (fertuzumab), aryltuzumab (gemumab), aprezumab (epuzumab), epratuzumab (epuzumab), epruzumab (epuzumab), rituzumab (erbuzumab), tumumab (periuzumab), rituzumab (zerumab), rituzumab (tumumab), rituximab (zerumab (perizumab (perizozumab), rituximab (perizomab), rituximab (zemab), ritukumab), ritumab), rituximab (perizumab (perizomab), ritukumab (zemab), ritukumab), yab (zemab), ritukumab), yamab (zemab), zemab (zemab), zemazomab (zemab), zemazomab (zemazomab), zemab), zemazomab (zemazomab), zema, Paclobutrazumab (paclobulizumab), pecfuzumab, petuuzumab, pertuzumab (pertuzumab), pelizumab (pelizumab), relilizumab (rolizumab), ralvizumab, ranibizumab (ranibizumab), rayleigh-zumab (relilizumab), rayleigh-mab (relilizumab), resyvizumab (rovelizumab), lullizumab (rulizumab), sibutrumab (sibutrumab), sibutrumab (siplizumab), solozuzumab (solvumab), tacurizumab (tracathuzumab), taduzumab (taluzumab), talocuzumab (tallizumab), taluzumab (talilizumab), teubazumab (tebazumab), toluzumab (tuzumab), tuzumab (tulizumab), urotuzumab (tuzumab), tulizumab (tuzumab), tuzumab (tuzumab), tussitussimab (tuzumab (tussimab), tussimab (tussizumab).

"metabolites" are products produced by the metabolism of a particular compound or salt thereof in the body. Metabolites of compounds can be identified using conventional techniques known in the art and their activity determined using assays as described herein. The products may result, for example, from oxidation, reduction, hydrolysis, amidation, deamidation, esterification, deesterification, enzymatic cleavage, etc. of the administered compound. Accordingly, the present invention includes metabolites of the 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 metabolite thereof.

The term "package insert" refers to instructions typically included in commercial packages of therapeutic products that contain information regarding the indications, usage, dosage, administration, contraindications, and/or precautions relating to the use of the therapeutic products described above.

The term "chiral" refers to a molecule that has the property that its mirror partners (mirror image partner) are non-overlapping, while the term "achiral" refers to a molecule that can overlap its mirror partners.

The term "stereoisomers" refers to compounds having the same chemical composition but differing in the spatial arrangement of the orientation of the atoms or groups.

"diastereomer" refers to a stereoisomer having two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers have different physical properties, such as melting points, boiling points, spectral properties, and reactivities. Mixtures of diastereomers can be separated by high resolution analytical procedures such as electrophoresis and chromatography.

"enantiomer" refers to two stereoisomers of a compound that are non-superimposable mirror images of each other.

The stereochemical definitions and conventional terminology (convention) used herein generally follow the general definitions 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 invention may contain asymmetric or chiral centers and thus exist in different stereoisomeric forms. It is contemplated that all stereoisomeric forms of the compounds of the present invention, including but not limited to diastereomers, enantiomers, and atropisomers (atropisomers) and mixtures thereof, such as racemic mixtures, form part of the present invention. Many organic compounds exist in an optically active form, 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 denote the absolute configuration of a molecule with respect to one or more of its chiral centers. The prefixes d and l or (+) and (-) are used to designate the sign of the rotation of plane polarized light by the compound, where (-) or l denotes that the compound is left-handed. Compounds prefixed with (+) or d are dextrorotatory. For a given 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 commonly referred to as enantiomeric mixtures. A50: 50 mixture of enantiomers is referred to as a racemic mixture or racemate, which may occur when there is no stereoselectivity or stereospecificity in the chemical reaction or process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric species, which are not optically active. Enantiomers can be separated from racemic mixtures by chiral separation methods, such as Supercritical Fluid Chromatography (SFC). The arrangement of the configuration at the chiral center in the separated enantiomers can be tentative and is depicted in the structure of table 1 for illustrative purposes, while stereochemical determination is pending, such as x-ray crystallographic data.

The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that can be interconverted by a low energy barrier (low energy barrier). For example, proton tautomers (also known as prototropic tautomers) include interconversions by proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers (valenctautomers) include interconversion by recombination of some of the bonding electrons.

The term "pharmaceutically acceptable salt" refers to salts that are not biologically or otherwise undesirable. Pharmaceutically acceptable salts include acid and base addition salts. The phrase "pharmaceutically acceptable" indicates that the substance or composition is to be compatible chemically and/or toxicologically, with the other ingredients comprising the formulation and/or the mammal being treated therewith.

The term "pharmaceutically acceptable acid addition salts" denotes those pharmaceutically acceptable salts formed with the following acids: inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, carbonic acid, phosphoric acid, and organic acids selected from the aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes, such as formic acid, acetic acid, propionic acid, glycolic acid, gluconic acid, lactic acid, pyruvic acid, oxalic acid, malic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, aspartic acid, ascorbic acid, glutamic acid, anthranilic acid, benzoic acid, cinnamic acid, mandelic acid, pamoic acid, phenylacetic acid, methanesulfonic acid, "methanesulfonic acid", ethanesulfonic acid, p-toluenesulfonic acid, and salicylic acid.

The term "pharmaceutically acceptable base addition salts" denotes those pharmaceutically acceptable salts formed with organic or inorganic bases. Examples of acceptable inorganic bases include sodium, potassium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of the following compounds: primary, secondary and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethylamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine and polyamine resins.

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

The term "EC₅₀"is the half maximal effective concentration and means 50 of the maximum at which a particular effect is obtained in vivo% plasma concentration of the particular compound desired.

The term "Ki" is an inhibition constant and denotes the absolute affinity of a particular inhibitor for a receptor. The measurement is using a competitive binding assay and is equal to the concentration at which a particular inhibitor occupies 50% of the receptors when no competing ligand (e.g., radioligand) is present. Ki values can be converted logarithmically to pKi values (-log Ki), with higher values indicating greater potency of the index.

The term "IC₅₀"is the half maximal inhibitory concentration and means the concentration of a particular compound required to obtain 50% inhibition of a biological process in vitro. IC (integrated circuit)₅₀The values can be logarithmically converted into pIC₅₀Value (-log IC)₅₀) Where higher values indicate greater efficacy of the index. IC (integrated circuit)₅₀The values are not absolute values, but depend on experimental conditions, such as the concentrations used, and can be converted to absolute inhibition constants (Ki) using the Cheng-Prusoff equation (biochem. Pharmacol. (1973)22: 3099). Other percentage rejection parameters may be calculated, e.g. IC₇₀、IC₉₀And the like.

The terms "the compounds of the invention" and "the compounds of formula I" include the compounds of formula I and stereoisomers, geometric isomers, tautomers, solvates, metabolites and pharmaceutically acceptable salts and prodrugs thereof.

Any formula or structure given herein, including compounds of formula I, is also intended to represent hydrates, solvates and polymorphs of the compounds, or mixtures thereof.

Any formula or structure given herein, including compounds of formula I, is also intended to represent unlabeled forms and isotopically labeled forms of the compounds. Isotopically-labeled compounds have the structure depicted in the formulae given herein, except that one or more atoms are replaced by an atom having a selected atomic mass or mass number. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine and chlorine, such as, but not limited to²H (deuterium D),³H (tritium),¹¹C、¹³C、¹⁴C、¹⁵N、¹⁸F、³¹P、³²P、³⁵S、³⁶Cl and¹²⁵I. various isotopically-labelled compounds of the invention, for example those into which a radioactive isotope is incorporated, for example³H、¹³C and¹⁴those of C. The isotopically labeled compounds can be used in metabolic studies, reaction kinetics studies, detection or imaging techniques such as Positron Emission Tomography (PET) or Single Photon Emission Computed Tomography (SPECT), including drug or substrate tissue distribution assays, or in the radioactive treatment of patients. Deuterium labeled or deuterium substituted compounds of the invention may have improved DMPK (drug metabolism and pharmacokinetics) properties, relating to distribution, metabolism and excretion (ADME). Substitution with heavier isotopes such as deuterium may afford some therapeutic benefit resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements.¹⁸The F-labeled compounds are useful for PET or SPECT studies. Isotopically labeled compounds of the present invention and prodrugs thereof can be prepared by carrying out the procedures disclosed in the examples and the preparative methods described below in a manner that readily available isotopically labeled reagents are substituted for non-isotopically labeled reagents. In addition, the heavy isotopes, particularly deuterium (i.e.,²h or D) substitution may result in some therapeutic benefit due to greater metabolic stability, such as increased in vivo half-life or reduced dose requirements or improved therapeutic index. It is to be understood that deuterium in this context is considered as a substituent in the compound of formula (I). The concentration of the heavier isotopes, in particular deuterium, can be defined by the isotopic enrichment factor (isotopic enrichment factor). In the compounds of the present invention, an atom not specifically designated as a specific isotope is any stable isotope representing the atom. Unless otherwise indicated, when a position is specifically designated as "H" or "hydrogen," the position is to be understood as hydrogen having a natural abundance isotopic composition. Thus, any atom designated specifically as deuterium (D) in the compounds of the present invention is intended to represent deuterium.

Alkylated piperazine compounds

The present invention provides alkylated piperazine compounds of formula I and pharmaceutical formulations thereof, which are useful for treating diseases, conditions, and/or disorders modulated by Btk kinase.

Exemplary embodiments of the compounds of formula I include

Or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, wherein:

R¹、R²and R³Independently selected from H, F, Cl, -NH₂、-NHCH₃、-N(CH₃)₂、-OH、-OCH₃、-OCH₂CH₃、-OCH₂CH₂OH and C₁-C₃An alkyl group;

R⁴selected from H, F, Cl, CN, -CH₂OH、-CH(CH₃)OH、-C(CH₃)₂OH、-CH(CF₃)OH、-CH₂F、-CHF₂、-CH₂CHF₂、-CF₃、-C(O)NH₂、-C(O)NHCH₃、-C(O)N(CH₃)₂、-NH₂、-NHCH₃、-N(CH₃)₂、-NHC(O)CH₃、-OH、-OCH₃、-OCH₂CH₃、-OCH₂CH₂OH, cyclopropyl, cyclopropylmethyl, 1-hydroxycyclopropyl, imidazolyl, pyrazolyl, 3-hydroxy-oxetan-3-yl, oxetan-3-yl and azetidin-1-yl;

R⁵is selected from-CH₃、-CH₂CH₃、-CH₂OH、-CH₂F、-CHF₂、-CF₃-CN and-CH₂CH₂OH；

Or two R⁵The groups form a 3-, 4-, 5-, or 6-membered carbocyclic or heterocyclic ring;

or R⁵Group and R⁸The groups form a 3-, 4-, 5-, or 6-membered carbocyclic or heterocyclic ring;

n is 1,2,3 or 4;

R⁶selected from H, -CH₃、-CH₂CH₃、-CH₂CH₂OH、-NH₂and-OH;

R⁷selected from the following structures:

wherein the wavy line indicates the attachment site;

R⁸is selected from-CH₃、-S(O)₂CH₃Cyclopropyl, azetidin-3-yl, oxetan-3-yl and morpholin-4-yl;

X¹is CR⁹Or N, wherein R⁹Selected from H, F, Cl, -CH₃、-CH₂CH₃、-CH₂CH₂OH、-NH₂、-NHCH₃、-N(CH₃)₂、-OH、-OCH₃、-OCH₂CH₃and-OCH₂CH₂OH；

X²Is CR¹⁰Or N, wherein R¹⁰Selected from H, -CH₃、-CH₂CH₃and-CH₂CH₂OH; and is

Y¹And Y²Independently selected from CH and N, wherein Y¹And Y²Not N at the same time.

Exemplary embodiments of compounds of formula I include wherein X is CR⁹And R is⁹Is H.

Exemplary embodiments of compounds of formula I include wherein X is N.

Exemplary embodiments of the compounds of formula I includeR⁴is-CH₂OH。

Exemplary embodiments of compounds of formula I include those wherein R²Is F.

Exemplary embodiments of compounds of formula I include those wherein R¹And R³Is H.

Exemplary embodiments of compounds of formula I include those wherein R⁶Is CH₃。

The compounds of formula I according to the invention may contain asymmetric 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, as well as mixtures thereof, e.g., racemic mixtures, are intended to form part of the present invention.

Furthermore, the present invention encompasses all diastereomers, including cis-trans (geometric) isomers and conformational isomers. For example, if the compounds of formula I contain double bonds or fused rings, the cis and trans forms, as well as mixtures thereof, are encompassed within the scope of the present invention.

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

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

The compounds of the invention may also exist in different tautomeric forms and all such forms are contemplated to be within the scope of the invention. The term "tautomer" or "tautomeric form" refers to energetically different structural isomers that can be transformed into each other through a low energy barrier. For example, proton tautomers (also referred to as proton transfer tautomers) include interconversions by migration of protons, such as keto-enol and imine-enamine isomerizations. Valence tautomers include interconversions through recombination of some of the bonding electrons.

Biological evaluation

The relative potency of a compound of formula I as an inhibitor of enzyme activity (or other biological activity) can be determined by determining the concentration of each compound at which it inhibits activity to a predetermined extent and then comparing the results. Generally, it is preferred to determine the concentration at which 50% of the activity is inhibited in a biochemical assay, i.e., the 50% inhibitory concentration or "IC₅₀". The IC may be completed using conventional techniques known in the art₅₀And (4) measuring the value. IC can generally be determined by measuring the activity of a particular enzyme in the presence of a range of concentrations of the inhibitor to be investigated₅₀. The experimentally obtained enzyme activity values are then plotted against the inhibitor concentration used. The concentration of inhibitor at which 50% enzyme activity is exhibited (compared to the activity in the absence of any inhibitor) is taken as the IC₅₀The value is obtained. Similarly, other inhibitory concentrations can be determined by appropriate measurement of activity. For example, in some cases it may be desirable to determine the 90% inhibitory concentration, i.e., IC₉₀And the like.

Compounds of formula I (example 901) were tested by standard biochemical Btk kinase assays.

A general method for a standard cellular Btk kinase assay that can be used to test compounds of formula I is the Ramos cellular Btk assay (example 902).

A standard cell B cell proliferation assay can be used to test compounds of formula I with B cells purified from the spleen of Balb/c mice (example 903).

A standard T cell proliferation assay can be used to test compounds of formula I with T cells purified from the spleen of Balb/c mice (example 904).

For inhibition of B cell activity, compounds of formula I can be assayed for CD86 inhibition by whole mouse spleen cells purified from 8-16 week old Balb/c mouse spleens (example 905).

To measure the number of B-ALL cells that survived in culture, a B-ALL cell survival assay can be performed on the compound pair of formula I (example 906).

A CD69 whole blood assay can be performed on compounds of formula I to measure the ability of the compounds to inhibit the production of CD69 by B lymphocytes in human whole blood activated by cross-linking surface IgM with goat F (ab') 2 anti-human IgM (example 907). CD69 is a type II C lectin involved in lymphocyte migration and cytokine secretion. CD69 expresses one of the earliest available indicators of leukocyte activation and its rapid induction occurs through transcriptional activation (Vazquez et al (2009) journal.of Immunology, published 10.19.2009, doi: 10.4049/jimmmunol.0900839). Concentration-dependent inhibition of antigen receptor stimulation by selective Btk inhibitors induces cell surface expression of the lymphocyte activation marker CD69 (Honigberg et al (2010) Proc. Natl. Acad. Sci.107(29): 13075-13080). Thus, CD69 inhibition by selective Btk inhibitors may be correlated with the therapeutic efficacy of certain B cell disorders. CD69Hu Blood FACS IC70 values for exemplary compounds of formula I are shown in tables 1 and 2.

The cytotoxic or cytostatic activity of exemplary compounds of formula I can be measured as follows: establishing a proliferating mammalian tumor cell line in a cell culture medium, adding a compound of formula I, and culturing the cells for a period of about 6 hours to about 5 days; and cell viability was measured (example 908). Cell-based in vitro assays for measuring viability, i.e. proliferation (IC)₅₀) Cytotoxicity (EC)₅₀) And induction of apoptosis (caspase activation) and can be used to predict clinical efficacy against hematologic malignancies and solid tumors.

The in vitro efficacy of a combination of a compound of formula I and a chemotherapeutic agent can be measured as follows: the cell proliferation assay of example 908; commercially available from Promega Corp, Madison, WisAnd (4) luminous cell viability assay. The homogenization assay is based on recombinant expression of a Coleoptera luciferase (US 5583024; US 5674713; US5700670) andthe number of viable cells in culture was measured by quantification of the presence of ATP (an indicator of metabolically active cells) (Crouch et al (1993) J.Immunol.meth.160: 81-88; US 6602677).Assays were performed in 96 or 384 well format, allowing automated High Throughput Screening (HTS) (Cree et al (1995) AntiCancer Drugs6: 398-404). The homogeneous assay procedure comprises a single reagent(s) (ii)Reagent) was added directly to the cells cultured in serum-supplemented medium. No cell washing, media removal and multiple pipetting steps are required. Within 10 minutes after adding the reagents and mixing, the system detects as few as 15 cells/well in 384-well format.

The homogeneous "add-mix-measure" mode results in cell lysis and the production of a luminescent signal proportional to the amount of ATP present. The amount of ATP is directly proportional to the number of cells present in the culture.The assay produces a "glow-type" luminescent signal by luciferase, which has a half-life typically greater than five hours, depending on the cell type and culture medium used. Viable cells are reflected in Relative Luminescence Units (RLU). The substrate, Beetle Luciferin (Beetle luciferase), is oxidatively decarboxylated by recombinant firefly luciferase, with conversion of ATP to AMP and production of photons. The extended half-life eliminates the need for using reagent syringes and provides flexibility for continuous or batch mode processing of multiple plates. The cell proliferation assay can be used in various multiwell formats, such as 96 or 384 well formats. Data may be recorded by a photometer or a CCD camera imaging device. The luminescence output (luminescence output) is expressed as Relative Light Units (RLU) measured over time.

By passingThe assay (example 908) measures the antiproliferative efficacy of exemplary compounds of formula I and combinations with chemotherapeutic agents on certain hematological tumor cell lines. Establishing EC for tested compounds and combinations₅₀The value is obtained.

Exemplary compounds of formula I in tables 1 and 2 were prepared and characterized according to the methods of the present invention and tested for their inhibition of Btk, having the following structure and corresponding names (ChemDraw Ultra, version 9.0.1, and chemdbiodraw, version 11.0, Cambridge soft corp., Cambridge MA). Where more than one name is associated with a compound of formula I or an intermediate, the compound should be defined in terms of chemical structure.

Table 1.

Table 2.

Administration of Compounds of formula I

The compounds of the invention may be administered by any route suitable for the condition to be treated. Suitable routes include oral, parenteral (including subcutaneous, intramuscular, intravenous, intraarterial, intradermal, intrathecal and epidural), transdermal, rectal, nasal, topical (including buccal and sublingual), vaginal, intraperitoneal, intrapulmonary and intranasal. For local immunosuppressive therapy, the compound may be administered by administration to the injury area (including perfusion or contacting the graft with the inhibitor prior to transplantation). It will be appreciated that the preferred route may vary with, for example, the conditions of the receptor. When the compound is administered orally, it can be formulated with pharmaceutically acceptable carriers or excipients into pills, capsules, tablets, and the like. When the compound is administered parenterally, it may be formulated with a pharmaceutically acceptable parenteral vehicle and in unit dose injectable form as described in detail below.

The dose for treating a human patient may be from about 10mg to about 1000 mg of a compound of formula I. A typical dose may be from about 100mg to about 300mg of the compound. The dose may be administered once daily (QID), twice daily (BID), or more frequently, depending on the pharmacokinetic and pharmacodynamic properties, including absorption, distribution, metabolism, and excretion of the particular compound. In addition, toxicity factors may affect the dosage and dosing regimen. When administered orally, the pills, capsules or tablets may be taken daily, or less frequently, over a specified period of time. The regimen may be repeated for a plurality of treatment cycles.

Methods of treatment with compounds of formula I

The compounds of formula I of the invention are useful for treating a human or animal patient suffering from a disease or condition arising from abnormal cell growth, function or behaviour associated with Btk kinase such as an immune condition, cardiovascular disease, viral infection, inflammation, metabolic/endocrine disorder or neurological disorder and may therefore be treated by a method comprising administering thereto a compound of the invention as defined above. A human or animal patient suffering from cancer may also be treated by a method comprising administering thereto a compound of the invention as defined above. Whereby the condition of the patient may be improved or ameliorated.

The compounds of formula I are useful for in vitro, in situ, and in vivo diagnosis or treatment of mammalian cells, organisms, or associated pathological conditions, such as systemic and localized inflammation, immunoinflammatory diseases such as rheumatoid arthritis, immunosuppression, organ transplant rejection, allergy, 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 antibody (ANCA) vasculitis, Chronic Obstructive Pulmonary Disease (COPD), psoriasis, and overall joint protection efficacy.

The methods of the invention also include treating diseases such as: joint diseases such as rheumatoid arthritis, monoarthritis, 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-associated eye diseases; eosinophilic granuloma; pulmonary or respiratory tract disorders such as asthma, chronic bronchitis, allergic rhinitis, ARDS, chronic pulmonary inflammatory diseases (e.g. chronic obstructive pulmonary disease), silicosis, pulmonary sarcoidosis, pleuritis, alveolitis, vasculitis, emphysema, pneumonia, bronchiectasis, and pulmonary acidosis; reperfusion injury of the myocardium, brain or extremities; fibrosis such as cystic fibrosis; keloid formation or scar tissue formation; atherosclerosis; autoimmune diseases such as Systemic Lupus Erythematosus (SLE), autoimmune thyroiditis, multiple sclerosis, some forms of diabetes, and raynaud's syndrome; and graft 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 dermatoses such as contact dermatitis, atopic dermatitis, psoriasis or rubella; fever and myalgia due to infection; inflammatory disorders of the central or peripheral nervous system such as meningitis, encephalitis, and brain or spinal cord injuries resulting from minor trauma; sjogren's syndrome; diseases involving leukocyte extravasation; alcoholic hepatitis; bacterial pneumonia; antigen-antibody complex mediated diseases; hypovolemic shock; type I diabetes; acute and delayed hypersensitivity reactions; disease states resulting from leukocyte dyscrasia and metastasis; heat damage; syndrome associated with transfusion of granulocytes; and cytokine-induced toxicity.

The methods of the invention also include treating a cancer selected from the group consisting of: breast, ovarian, cervical, prostate, testicular, genitourinary tract, esophageal, laryngeal, glioblastoma, neuroblastoma, gastric, skin, keratoacanthoma, lung, epidermoid, large cell, non-small cell lung cancer (NSCLC), small cell, lung adenocarcinoma, bone, colon, adenoma, pancreatic, adenocarcinoma, thyroid, follicular, undifferentiated, papillary, seminoma, melanoma, sarcoma, bladder, liver and biliary tract, kidney, pancreatic, myeloid disorders, lymphoma, hairy cell, oral, nasopharyngeal, pharyngeal, lip, tongue, mouth, small intestine, colorectal, large intestine, rectal, brain and central nervous system, hodgkin's lymphoma, leukemia, bronchial, thyroid, liver and intrahepatic bile duct, hepatocellular, colon, or rectum, colon cancer, gastric cancer, glioma/glioblastoma, endometrial cancer, melanoma, renal and renal pelvis cancer, bladder cancer, uterine corpus cancer, cervical cancer, multiple myeloma, acute myelogenous leukemia, chronic myelogenous leukemia, lymphocytic leukemia, Chronic Lymphocytic Leukemia (CLL), myeloid leukemia, oral and pharyngeal cancer, non-hodgkin's lymphoma, melanoma, and villous colon adenoma.

The methods of the invention are useful for treating subjects who are or may be suffering from reperfusion injury (i.e., injury caused by a tissue or organ undergoing a period of ischemia followed by reperfusion). The term "ischemia" refers to ischemia of local tissue caused by the occlusion of arterial blood inflow. Transient ischemia followed by reperfusion characteristically results in neutrophil activation and transmigration across the endothelium of the vessels in the affected area. 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 a bypass surgery or during cardiac arrest (when reperfusion is initiated in a heart that has been prevented from receiving blood). It is expected that inhibition of Btk activity may result in a reduction in the amount of reperfusion injury in such a situation.

Pharmaceutical preparation

For use of the compounds of the present invention in the therapeutic treatment of mammals, including humans, they are generally formulated in accordance with standard pharmaceutical practice as pharmaceutical compositions. According to this aspect of the invention there is provided a pharmaceutical composition comprising a compound of the invention in association with a pharmaceutically acceptable diluent or carrier.

Typical formulations are prepared by mixing a compound of the 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 carbohydrates, waxes, water soluble and/or swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water and the like. The particular carrier, diluent or excipient employed will depend upon the mode and purpose for which the compounds of the present invention are to be employed. Solvents are generally selected based on the solvents considered safe for administration to mammals by those skilled in the art (GRAS). Generally, safe solvents are non-toxic aqueous solvents such as water and other non-toxic solvents that are soluble or miscible in water. Suitable aqueous solvents include water, ethanol, propylene glycol, polyethylene glycol (e.g., PEG400, PEG300), and the like, and mixtures thereof. The formulation may also include one or more of the following: buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifiers (opacifying agents), glidants, processing aids, colorants, sweeteners, fragrances, flavors, and other known additives that provide a superior appearance to a drug (i.e., a compound of the present invention or a pharmaceutical composition thereof) or aid in the manufacture of a pharmaceutical product (i.e., a drug).

The formulations may be prepared using conventional dissolution and mixing operations. For example, bulk pharmaceutical products (i.e., a stabilized form of a compound of the invention (e.g., a complex with a cyclodextrin derivative or other known complexation agent)) are dissolved in a suitable solvent in the presence of one or more of the above-mentioned excipients. The compounds of the present invention are typically formulated into pharmaceutical dosage forms that provide readily controllable dosages of the drug and enable patient compliance with a given regimen.

Depending on the method used to administer the drug, the pharmaceutical composition (or formulation) for administration may be packaged in a variety of ways. Generally, articles for dispensing include a container having a pharmaceutical formulation in a suitable form stored therein. Suitable containers are well known to those skilled in the art and include materials such as bottles (plastic and glass), sachets (sachets), ampoules, plastic bags, metal cylinders and the like. The container may also include a tamper-proof device (tag-proof) to prevent inadvertent access to the contents of the package. Further, there is a label on the container that describes the contents of the container. The label may also include appropriate precautions.

Pharmaceutical formulations of the compounds of the present invention may be prepared for a variety of routes and types of administration. For example, a compound of formula I having a desired purity may be optionally mixed with a pharmaceutically acceptable diluent, carrier, excipient or stabilizer (Remington's pharmaceutical Sciences (1980)16th edition, Osol, a.ed.) in the form of a lyophilized formulation, a finely divided powder or an aqueous solution. The formulation can be carried out as follows: mixed at ambient temperature, at an appropriate pH, and in an appropriate purity, with a physiologically acceptable carrier, i.e., a carrier that is non-toxic to the recipient at the dosages and concentrations employed. The pH of the formulation depends primarily on the particular use and concentration of the compound, but can range from about 3 to about 8. Formulations with a pH of 5 in acetate buffer are suitable embodiments.

The compounds may generally be stored as solid compositions, lyophilized formulations or aqueous solutions.

The pharmaceutical compositions of the present invention will be formulated, dosed and administered in a manner consistent with good medical practice (i.e., amount, concentration, schedule, procedure, vehicle and route of administration). Factors considered in this context include the particular condition being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the condition, the site of delivery of the drug, the method of administration, the schedule of administration, and other factors known to medical practitioners. A "therapeutically effective amount" of a compound administered will be governed by such considerations and is the minimum amount required to ameliorate or treat the hyperproliferative disorder.

As is generally recommended, an initial pharmaceutically effective amount of inhibitor per dose administered parenterally is about 0.01-100 mg/kg daily, i.e., about 0.1 to 20 mg/kg body weight of the patient, with a typical initial range of compounds used being 0.3 to 15 mg/kg/day.

Acceptable diluents, carriers, excipients and stability are nontoxic to recipients at the dosages and concentrations employed, and include buffering agents such as phosphates, citrates and other organic acids; antioxidants, including ascorbic acid and methionine (methionine); preservatives (e.g. octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens, e.g. methylparaben or paraben)Propyl benzoate; catechol; resorcinol (resorcinol); cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants, e.g. TWEEN^TM、PLURONICS^TMOr polyethylene glycol (PEG). The active pharmaceutical ingredient may also be embedded in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly- (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems such as liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules (nanocapsules) or in macroemulsions (macroemulsions). The technique is disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A.Ed. (1980).

Sustained release formulations of the compounds of formula I may be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing a compound of formula I, which matrices are present in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl methacrylate) or poly (vinyl alcohol)), polylactides (US3,773,919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT^TM(injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D- (-) -3-hydroxybutyric acid.

The formulations include those suitable for the routes of administration detailed herein. The formulations may suitably be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Techniques and formulations are generally described in Remington's pharmaceutical Sciences (Mack Publishing Co., Easton, Pa.). The process comprises the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients (access ingredients). Typically the formulation is prepared as follows: the active ingredient is homogeneously and intimately associated with liquid carriers or finely divided solid carriers or both, and then, if necessary, the product is shaped.

Formulations of a compound of formula I suitable for oral administration may 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 may be prepared as follows: the active ingredient in free-flowing form (e.g., powder or granules) and, optionally, admixed with a binder, lubricant, inert diluent, preservative, surfactant or dispersant, is compacted in a suitable machine. Molded tablets may be prepared as follows: the mixture of powdered active ingredient moistened with an inert liquid diluent is moulded in a suitable machine. The tablets may optionally be coated or scored and optionally formulated to provide slow or controlled release of the active ingredient therefrom. Tablets, troches (troche), lozenges, aqueous or oily suspensions, dispersible powders or dispersible granules, emulsions, hard or soft capsules such as gelatin capsules, syrups or elixirs may be prepared for oral administration. Formulations of compounds of formula I intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions which may contain one or more agents, including sweeteners, flavoring agents, colorants and preservatives, in order to provide a palatable preparation. Tablets containing the active ingredient in admixture with non-toxic physiologically acceptable excipients which are suitable for the manufacture of tablets are acceptable. These excipients may be, for example, inert diluents, such as calcium or sodium carbonate, lactose, calcium or sodium phosphate; granulating and disintegrating agents (granulating and disintegrating agents), such as corn starch or alginic acid; binding agents, such as starch, gelatin or acacia; and lubricating agents, such as magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques, including microencapsulation, to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a timing delay material such as glyceryl monostearate or glyceryl distearate alone or with a wax may be employed.

For the treatment of the eye or other external tissues such as mouth and skin, the formulations are preferably applied as a topical ointment (ointment) or cream (cream) containing the active ingredient in an amount of, for example, 0.075 to 20% w/w. When formulated as an ointment, the active ingredient may be employed with either a paraffinic (parafinic) or a 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 include polyhydric alcohols, i.e., alcohols having two or more hydroxyl groups, such as propylene glycol, butane-1, 3-diol, mannitol, sorbitol, glycerol, and polyethylene glycols (including PEG400) and mixtures of such alcohols. Topical formulations may include compounds that enhance the absorption or penetration of the active ingredient through the skin or other affected area. Examples of such skin permeation enhancers include dimethyl sulfoxide and related analogs. The oil phase of the emulsions of the invention may be constituted by known ingredients in a known manner. While the phase may comprise emulsifiers alone, it is contemplated that it comprises a mixture of at least one emulsifier with a fat or oil or with both a fat and an oil. Preferably, a hydrophilic emulsifier may be included as well as a lipophilic emulsifier as a stabilizer. It also preferably includes both oil and fat. At the same time, the emulsifiers, with or without stabilizers, constitute the so-called emulsifying waxes (emulsifying wax), which, together with oils and fats, constitute the so-called emulsifying ointment base which forms the oily dispersed phase of the ointment formulation. Emulsifiers and emulsion stabilizers suitable for use in the formulations of the present invention include60、80. Stearyl/cetyl alcohol (cetostearyl alcohol), benzyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulphate.

Aqueous suspensions of the compounds of formula I contain the active material in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include suspending agents, such as sodium carboxymethylcellulose, croscarmellose, povidone, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, as well as dispersing or wetting agents (dispersing or wetting agents) such as naturally occurring phosphatides (e.g., lecithin), condensation products of alkylene oxides with fatty acids (e.g., polyoxyethylene stearate), condensation products of ethylene oxide with long chain aliphatic alcohols (e.g., heptadecaethyleneoxycetanol), condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyoxyethylene sorbitan monooleate). The aqueous suspensions may also contain one or more preservatives, for example ethyl or n-propyl p-hydroxybenzoate, one or more colouring agents, one or more flavouring agents and one or more sweetening agents, such as sucrose or saccharin.

Pharmaceutical compositions of the compounds of formula I may be presented as sterile injectable preparations, such as sterile injectable aqueous or oleaginous suspension formulations. This suspension may be formulated according to the methods known in the art using suitable dispersing or wetting agents and suspending agents that have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in1, 3-butanediol, or as a lyophilized powder. Acceptable vehicles and solvents that may be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils (sterile fixed oils) are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, a time release formulation intended for oral administration to humans may contain from about 1 to 1000 milligrams of active substance in admixture with a suitable and convenient amount of a carrier material which may comprise from about 5 to about 95% of the total composition (weight: weight). The pharmaceutical composition can be prepared to provide an amount that is readily measured upon administration. For example, an aqueous solution intended for intravenous infusion may contain about 3 to 500 μ g of active ingredient per ml of solution, so that an appropriate volume of infusion occurs 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 include suspending agents and thickening agents.

Formulations suitable for topical administration to the eye also include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the active ingredient. The concentration of active ingredient present in the formulation is preferably from about 0.5 to 20% w/w, for example from about 0.5 to 10% w/w, for example about 1.5% w/w.

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

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

Formulations suitable for intrapulmonary or nasal administration have a particle size, for example, of from 0.1 to 500 microns (including between 0.1 and 500 microns, in increments of particle size, for example, of 0.5, 1, 30 microns, 35 microns, etc.), which are administered by rapid inhalation through the nasal passage or by oral inhalation, in order to reach the alveolar sacs (alveolars sacs). Suitable formulations include aqueous or oily solutions of the active ingredient. Formulations suitable for aerosol or dry powder administration may be prepared according to conventional methods and may be delivered with other therapeutic agents, such as compounds heretofore used in the treatment or prevention of the conditions described below.

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

The formulations may be packaged in unit-dose or multi-dose containers, for example sealed ampoules or vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water, for injections, immediately prior to use. Extemporaneous injection solutions (injections solutions and suspensions) and suspensions are prepared from sterile powders, granules and tablets of the kind previously described. Preferred unit dosage formulations are those containing a daily dose or unit sub-dose as herein above described, or an appropriate fraction thereof, of the active ingredient.

The invention also provides a veterinary composition (veterinary composition) whereby it contains at least one active ingredient as defined above together with a veterinary carrier. Veterinary carriers are substances used for the purpose of administering the composition and may be solid, liquid or gaseous substances which are either inert or acceptable in the veterinary art and which are compatible with the active ingredient. These veterinary combinations may be administered parenterally, orally or by any other desired route.

Combination therapy

The compounds of formula I may be used alone or in combination with other therapeutic agents for the treatment of diseases or disorders described herein, such as inflammatory or hyperproliferative disorders (e.g., cancer). In some embodiments, the compound of formula I is combined with a second therapeutic compound that is anti-inflammatory or anti-hyperproliferative or is useful for the treatment of inflammation, an immunoreactive disorder or a hyperproliferative disorder (e.g., cancer), in a pharmaceutical combination formulation or dosage regimen as a combination therapy. The second therapeutic agent may be an NSAID anti-inflammatory. The second therapeutic agent may be a chemotherapeutic agent. The second compound of the pharmaceutical combination formulation or method of administration preferably has complementary activity to the compound of formula I such that they do not adversely affect each other. Such compounds are suitably present in combination in an amount effective for the intended purpose. In one embodiment, the compositions of the present invention comprise a compound of formula I, or a stereoisomer, tautomer, solvate, metabolite, or pharmaceutically acceptable salt or prodrug thereof, in combination with a therapeutic agent, e.g., an NSAID.

The combination therapy may be administered as a simultaneous or sequential regimen. When administered sequentially, the compositions may be administered in two or more administrations. Combination administration includes simultaneous administration using separate formulations or a single pharmaceutical formulation, and sequential administration in either order, wherein it is preferred that there be a period of time during which both (or all) active agents exert their biological activities simultaneously.

Suitable dosages for any of the above-mentioned co-administered drugs are those presently used and may be reduced due to the combined effect (synergy) of the newly identified drug and the other therapeutic agent or treatment.

Combination therapy can provide "synergy" and "synergy", i.e., the effect achieved when the active ingredients are used together is greater than the sum of the effects that would result from the separate use of these compounds. When the active ingredients are: (1) when formulated simultaneously and administered or delivered simultaneously in a combined unit dose formulation; (2) when delivered alternately or in parallel as separate formulations; or (3) when administered by some other regimen, a synergistic effect may be achieved. When delivered in alternation therapy, a synergistic effect may be achieved when the compounds are administered or delivered sequentially, e.g. by separate injections in separate syringes, by separate pills or capsules or by separate infusions. Generally, an effective dose of each active ingredient is administered sequentially (i.e., sequentially) during alternation therapy, while in combination therapy, an effective dose of two or more active ingredients are administered together.

In a particular embodiment of treatment, a compound of formula I, or a stereoisomer, tautomer, solvate, metabolite, or pharmaceutically acceptable salt or prodrug thereof, may be used in combination with other therapeutic agents, hormonal agents, or antibody agents (e.g., agents described herein), or in combination with surgical treatment and radiation therapy. The combination therapy according to the present invention thus comprises the administration of at least one compound of formula I, or a stereoisomer, tautomer, solvate, metabolite or pharmaceutically acceptable salt or prodrug thereof, as well as the use of at least one other cancer treatment method. The amounts of the compound of formula I and the other pharmaceutically active therapeutic agent and the associated timing of administration will be selected so as to achieve the desired therapeutic effect in combination.

Metabolites of compounds of formula I

The in vivo metabolites of formula I described herein also fall within the scope of the present invention. The product may be caused, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, defatting, enzymatic cleavage, etc. of the administered compound. Accordingly, the present invention includes metabolites of the compounds of formula I, including compounds produced by a method comprising contacting a compound of the present invention with a mammal for a period of time sufficient to produce a metabolite thereof.

Metabolites are typically identified as follows: preparation of a radiolabelled compound of the invention (for example,¹⁴c or³H) Isotopes, which are administered parenterally to an animal, such as a rat, mouse, guinea pig, monkey, or to a human in detectable doses (e.g., greater than about 0.5 mg/kg), allowed sufficient time for metabolism to occur (typically about 30 seconds to 30 hours), and then their conversion products are separated from urine, blood samples, or other biological samples. These products are easy to isolate because they are labelled (others are isolated by using antibodies that are capable of binding to epitopes that survive in the metabolite). Metabolite structure is determined in a conventional manner, e.g. by MS, LC/MS or NMR analysis. In general, analysis of metabolites is accomplished in the same manner as in conventional drug metabolism studies well known to those skilled in the art. The metabolites, as long as they are not in vivoAnd, where present, for use in diagnostic assays for therapeutic dosages of the compounds of the present invention.

Article of manufacture

In another embodiment of the invention, there are provided articles of manufacture and "kits" containing materials useful for the treatment of the diseases and conditions described above. In one embodiment, the kit comprises a container comprising a compound of formula I or a stereoisomer, tautomer, solvate, metabolite, or pharmaceutically acceptable salt or prodrug thereof. The kit may further comprise a label or package insert attached to or in the container. The term "package insert" is used to refer to instructions typically included in commercial packages of therapeutic products containing information regarding the indications, usage, dosage, administration, contraindications and/or precautions relating to the use of the therapeutic product. Suitable containers include, for example, bottles, vials, syringes, blister packs (etc.). The container may be formed from a variety of materials, such as glass or plastic. The container may contain a compound of formula I or a formulation thereof effective to treat the condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is a compound of formula I. The label or package insert indicates that the composition is for use in treating a selected condition, such as cancer. Further, the label or package insert may indicate that the patient to be treated is a patient suffering from a condition such as a hyperproliferative condition, neurodegeneration, cardiac hypertrophy, pain, migraine or a neurotrauma disease or event. In one embodiment, the label or package insert indicates that compositions comprising compounds of formula I are useful for treating conditions resulting from abnormal cell growth. The label or package insert may also indicate that the composition may be used to treat other conditions. Alternatively or additionally, the article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, ringer's solution, and dextrose solution. The kit may also include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

The kit may further comprise instructions for administering the compound of formula I and, if present, the second pharmaceutical formulation. For example, if a kit comprises a first composition comprising a compound of formula I and a second pharmaceutical formulation, the kit may further comprise instructions for administering the first and second pharmaceutical compositions simultaneously, sequentially or separately to a patient in need of such formulation.

In another embodiment, the kit is suitable for delivering a compound of formula I in a solid oral form, such as a tablet or capsule. Such kits preferably comprise a plurality of unit doses. The kit may include a dosage card for the purpose of the intended use. One example of such a kit is a "blister pack". Blister packs are well known in the packaging industry and are widely used for packaging pharmaceutical unit dose forms. If desired, a memory aid (memory aid) may be provided, which may be in the form of, for example, numbers, letters or other indicia, or with a calendar insert, which specifies the number of days in a treatment schedule during which the dose may be administered.

According to one embodiment, a kit may comprise (a) a first container having a compound of formula I therein; and optionally (b) a second container having a second pharmaceutical formulation therein, wherein the second pharmaceutical formulation comprises a second compound having anti-hyperproliferative activity. Alternatively or additionally, the kit may further comprise a third container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, ringer's solution, and dextrose solution. It may also include other substances desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

In certain other embodiments of kits comprising a composition of formula I and a second therapeutic agent, the kit may comprise containers for holding separate compositions, such as separate bottles or separate foil packets, however, separate compositions may also be held in a single, undivided container. Typically, the kit contains instructions for administering the separate components. The kit form is particularly advantageous when the separate components are preferably administered in different dosage forms (e.g., oral and parenteral), when administered at different dosage intervals, or when titration of the individual components of the combination is desired by the attending physician.

Preparation of Compounds of formula I

The compounds of formula I can be synthesized by synthetic routes that include methods analogous to those well known in the chemical arts, particularly in light of the description contained herein, as well as methods directed to such other heterocycles: comprehensive Heterocyclic Chemistry II, Editors 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 of which is expressly incorporated by reference. The starting materials are generally available from commercial sources such as Aldrich Chemicals (Milwaukee, Wis.) or are readily prepared using methods well known to those skilled in the art (e.g., by Louis F. Fieser and Mary Fieser, Reagents for Organic Synthesis, v.1-23, Wiley, N.Y. (1967. d. 2006.), or Bellsteins Handbuch der organischen Chemie,4, of autofl. ed. Springer-Verlag, Berlin, including the procedures generally described in the appendix (also available via the Beilstein Web database)).

Synthetic chemical transformations and protecting group methodologies (protection and deprotection) and necessary reagents and intermediates useful in the synthesis of 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 P.G.M.Wuts, protective groups in Organic Synthesis,3rd Ed., John Wiley and Sons (1999); and L.Patquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995) and subsequent versions thereof.

The compounds of formula I may be prepared alone or as a library of compounds comprising at least 2, for example 5 to 1,000 or 10 to 100 compounds. Libraries of compounds of formula I can be prepared by procedures known to those skilled in the art, using solution-phase or solid-phase chemistry, by combinatorial "split and mix" routes, or by multiple parallel syntheses. Thus according to another method of the present invention there is provided a library of compounds comprising at least 2 compounds or pharmaceutically acceptable salts thereof.

The figures and examples provide exemplary methods for preparing compounds of formula I. It will be appreciated by those skilled in the art that other synthetic routes may be used to synthesize the compounds of formula I. Although specific starting materials and reagents are described and discussed in the figures and examples, other starting materials and reagents can be readily substituted to provide a variety of derivatives and/or reaction conditions. In addition, a variety of exemplary compounds prepared by the methods described can be further modified in light of this disclosure using conventional chemistry well known to those skilled in the art.

Protection of remote functional groups (e.g., primary or secondary amines) of intermediates may be necessary in the preparation of compounds of formula I. The need for such protection will vary with the nature of the remote functionality and the conditions of the preparation process. Suitable amino protecting groups include acetyl, trifluoroacetyl, tert-Butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ) and 9-fluorenylmethoxycarbonyl (Fmoc). The need for such protection is readily determined by one skilled in the art. For a general description of protecting Groups and their use, see t.w. greene, Protective Groups in organic synthesis, John Wiley & Sons, New York, 1991.

Experimental procedures, intermediates AND reagents for the preparation OF COMPOUNDS OF formula I can be found in US13/102720, "PYRIDONE AND AZA-PYRIDONE Compounds AND METHODS OF USE", filed 5/6.2011, the entire contents OF which are incorporated herein by reference.

FIGS. 1-12 depict the synthesis of an exemplary embodiment of the compounds of formula I101-125, described more fully in example 101-112, and can be used to prepare other compounds of formula I.

General preparation method

General procedure: suzuki coupling

Suzuki-type coupling reactions are used to form carbon-carbon bonds to link the compounds of formula I and intermediates such as the ring of A-3 (Suzuki (1991) Pure appl. chem.63: 419-422; Miyaura and Suzuki (1979) chem.Reviewss 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-4, 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 (1M in water) and an equal volume of acetonitrile. A catalytic amount or more of a palladium suboxide reagent such as bis (triphenylphosphine) palladium (II) dichloride is added. In some cases, potassium acetate was used in place of sodium carbonate to adjust the pH of the aqueous layer. The reaction mixture was then heated to about 140-150 ℃ under pressure in a microwave reactor (Biotage AB, Uppsala, Sweden) for 10-30 minutes. The contents are extracted with ethyl acetate or another organic solvent. After evaporation of the organic layer, boronic ester a-1 can be purified on silica gel or by reverse phase HPLC. The substituents are as defined, or a protected form or precursor thereof. Likewise, bromide intermediate B-4 may be borated to provide A-2.

Suzuki coupling of B-2 and A-2 or A-1 and B-4 gives the compounds of the formula I or intermediates A-3. The boronic acid ester (or boronic acid) (1.5 equivalents) a-1 or a-2 and a palladium catalyst such as bis (triphenylphosphine) palladium (II) dichloride (0.05 equivalents) are added to a mixture of the halogenated intermediate (1 equivalent) B-2 or B-4 in acetonitrile and 1M aqueous sodium carbonate (equal volume to acetonitrile). The reaction mixture was heated to about 150 ℃ in a microwave and held for about 15 minutes. LC/MS indicated when the reaction was complete. Water was added to the mixture and the precipitated product was filtered and then purified by HPLC to give product a-3. The substituents are as defined, or a protected form or precursor thereof.

Various palladium catalysts can be used in the Suzuki coupling step. Various low-valence Pd (II) and Pd (0) catalysts can be used in Suzuki coupling reactions, including PdCl2 (PPh)₃)₂、Pd(t-Bu)₃、PdCl₂dppfCH₂Cl₂、Pd(PPh₃)₄、Pd(OAc)/PPh₃、Cl₂Pd[(Pet₃)]₂、Pd(DIPHOS)₂、Cl₂Pd(Bipy)、[PdCl(Ph₂PCH₂PPh₂)]₂、Cl₂Pd[P(o-tol)₃]₂、Pd₂(dba)₃/P(o-tol)₃、Pd₂(dba)/P(furyl)₃、Cl₂Pd[P(furyl)₃]₂、Cl₂Pd(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]₂And an encapsulated catalyst Pd EnCat^TM30、Pd EnCat^TMTPP30 and Pd (II) EnCat^TMBINAP30(US2004/0254066)。

General procedure: buchwald reaction

The Buchwald reaction was used to ammoniate the 6-bromo intermediate B-1(Wolf and Buchwald (2004) org. SynthColl. Vol.10: 423; Paul et al (1994) journal. Amer. chem. Soc.116: 5969-. To a solution of the 3, 5-dihalo-pyridone intermediate B-1 in DMF is added a suitable 5- (piperazin-1-yl) pyridin-2-amine, e.g. 104B, or 5- (piperazin-1-yl) pyrazin-2-amine (200 mol%), Cs₂CO₃(50mol％)、Pd₂(dba)₃(5 mol%) and4, 5-bis (diphenylphosphino) -9, 9-dimethylxanthene (Xantphos, CAS registry number 161265-03-8, 10 mol%). The reaction mixture was heated to about 110 ℃ under pressure and held for about 30 minutes in a microwave reactor (Biotage AB, Uppsala, Sweden). The resulting solution was concentrated in vacuo to afford B-2. Other palladium catalysts and phosphine ligands may be used.

N-aryl amide intermediate B-4 Cyclic amide intermediate (R)⁷) For example, 3,4,6,7,8, 9-hexahydropyrazino [1,2-a ] is]Indol-1 (2H) -one 101e and aryl bromide B-3 were prepared under Buchwald conditions.

Separation method

In the process for preparing the compounds of the formula I, it may be advantageous to separate the reaction products from one another and/or from the starting materials. The desired product in each step or steps is isolated and/or purified to the desired degree of homogeneity by techniques common in the art. Typically the separation involves heterogeneous extraction, crystallization from a solvent or solvent mixture, distillation, sublimation or chromatography. Chromatography may involve any number of methods, including, for example: reverse phase and normal phase; size exclusion (size exclusion); ion exchange; high, medium and low pressure liquid chromatography methods and apparatus; small-scale analysis; simulated Moving Bed (SMB) and preparative thin or thick layer chromatography, as well as small scale thin layer and flash chromatography techniques.

Another class of separation methods involves treating the mixture with selected reagents to combine with or separate the desired product, unreacted starting materials, reaction byproducts, etc. The reagent comprises an adsorbent (adsorbent) or absorbent (adsorbent), such as activated carbon, molecular sieves, ion exchange media, and the like. Alternatively, the reagent may be an acid (in the case of a basic substance), a base (in the case of an acidic substance), a binding agent such as an antibody, a binding protein, a selective chelating agent such as a crown ether, a liquid/liquid ion exchange reagent (LIX), or the like. The choice of an appropriate separation method depends on the nature of the substances involved. For example, boiling point and molecular weight (in distillation and sublimation), presence or absence of polar functional groups (in chromatography), stability of the material in acidic and basic media (in heterogeneous extraction), and the like.

Diastereomeric mixtures can be separated into their individual diastereomers on the basis of their physicochemical differences, by methods well known to those skilled in the art (e.g., chromatography and/or fractional crystallization). Enantiomers can be separated by: the enantiomeric mixtures are converted into diastereomeric mixtures by reaction with an appropriate optically active compound (e.g., a chiral auxiliary such as a chiral alcohol or Mosher's acid chloride), the diastereomers are separated, and the individual diastereomers are then converted (e.g., hydrolyzed) into the corresponding pure enantiomers. In addition, some of the compounds of the present invention may be atropisomers (e.g., substituted biaryl) and are considered part of the present invention. Enantiomers can also be separated by using a chiral HPLC column.

A single stereoisomer, e.g., an enantiomer substantially free of its stereoisomer, can be obtained by: the racemic mixture is resolved with an optically active resolving agent using methods such as diastereomer formation (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-. The racemic mixture of chiral compounds of the present invention can be separated and isolated by any suitable method, including: (1) ionic diastereomeric salts with chiral compounds and subsequent separation by fractional crystallization or other means, (2) formation of diastereomeric compounds with chiral derivatizing reagents, separation of the diastereomers and subsequent conversion to pure stereoisomers, and (3) direct separation of 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).

In the case of process (1), diastereomeric salts may be formed by: enantiomerically pure chiral bases such as brucine, quinine, ephedrine, brucine, strychnine, alpha-methyl-beta-phenylethylamine (amphetamine), etc., are reacted with asymmetric compounds having acidic functional groups such as carboxylic and sulfonic acids. Diastereomeric salt separation can be induced by fractional crystallization or ion chromatography. For the separation of the optical isomers of amino compounds, the addition of chiral carboxylic or sulfonic acids, such as camphorsulfonic, tartaric, mandelic or lactic acids, can cause the formation of salts of diastereomers.

Alternatively, by process (2), the substrate to be resolved is reacted with one enantiomer of a chiral compound to form diastereomeric pairs (Eliel, E.and Wilen, S. "Stereochemistry of organic Compounds", John Wiley&Sons, Inc.,1994, p.322.) diastereoisomeric compounds may be formed by reacting an asymmetric compound with an enantiomerically pure chiral derivatizing agent such as a menthyl derivative, followed by separation of the diastereomers, followed by hydrolysis to give the pure or enriched enantiomer the method of determining optical purity involves preparing a chiral ester of the racemic mixture, such as menthyl esters such as (-) menthyl chloroformate, or Mosher ester, acetic acid α -methoxy- α - (trifluoromethyl) phenyl ester (Jacob III.J.org.chem. (1982)47:4165) in the presence of a base, and then analyzing for the presence of the two atropisomers or diastereomers¹H NMR spectrum. The stable diastereoisomers of atropisomeric compounds can be separated and isolated by normal and reverse phase chromatography following the procedure for separating the atropisomeric naphthyl-isoquinoline (WO 1996/15111). By method (3), racemic mixtures of the two enantiomers can be separated by Chromatography using a Chiral stationary phase ("Chiral Liquid Chromatography" (1989) W.J.Lough, Ed., Chapman and Hall, New York; Okamoto, J.Chromatogr., 1990)513: 375-. Enriched or purified enantiomers can be distinguished by methods for distinguishing other chiral molecules with asymmetric carbon atoms, such as optical rotation or circular dichroism.

Examples

Example 101a2, 6-dibromo-4-fluorobenzaldehyde 101a

A solution of 1, 3-dibromo-5-fluoro-2-iodobenzene (50g, 132mmol) in dry toluene (300mL) was cooled to-35 ℃. A solution of isopropyl magnesium chloride (84mL, 171mmol, 2.0M in ether) was added over a period of 30 minutes while maintaining the internal temperature below-25 deg.C. See fig. 1. A clear brown solution was obtained. Stirring was continued for 1.5 h. Then anhydrous DMF (34mL, 436mmol) was added over a period of 30 minutes. The temperature of the reaction mixture rose to-19 ℃. The reaction mixture was warmed to 10 ℃ over 1h (room temperature) and stirred at this temperature for 1.5 h. With saturated NH₄The reaction mixture was quenched with aqueous Cl (100mL), filtered and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (eluting with petroleum ether/ethyl acetate: 50:1 to 20:1) to give 101a (20g, yield 54%) as a yellow solid.

Example 101b2, 6-dibromo-4-fluorophenyl) methanol 101b

To a solution of 2, 6-dibromo-4-fluorobenzaldehyde 101a (20g, 71mmol) in ethanol (500mL) was added NaBH₄(10g, 284 mmol.) the mixture was stirred at room temperature (10 ℃ C.) for 4 h.TLC showed disappearance of starting material, the reaction mixture was quenched with HCl solution (150mL, 1M), most of the ethanol was evaporated under reduced pressure, the residue was extracted by ethyl acetate (3 × 500mL), the organic layers were combined, dried over anhydrous Na₂SO₄Dried and evaporated under vacuum. By passingThe residue was purified by silica gel column chromatography (eluting with petroleum ether/ethyl acetate: 50:1 to 20:1) to give 101b (15g, yield 75%) as a white solid.

Example 101cAcetic acid 2, 6-dibromo-4-fluorobenzyl ester 101c

A500-mL single-neck round bottom flask equipped with a magnetic stirrer and a nitrogen inlet was charged with a solution of 101b (23.0g, 81.0mmol), triethylamine (25.0g, 247mmol) in dry dichloromethane (100 mL). Acetic anhydride (10.0g, 98.0mmol) was added and the mixture stirred at room temperature for 16 h. After this time, the mixture was diluted with dichloromethane (100mL) and washed with saturated aqueous sodium bicarbonate (100 mL). The layers were separated and the aqueous layer was extracted with dichloromethane (2X 20 mL). The organic extracts were combined and 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 column chromatography (silica, 0% to 50% ethyl acetate/hexanes) to give 101c (23.0g) as a white solid in 87% yield.

Example 101d5,6,7, 8-Tetrahydroindazine-2-carboxylic acid methyl ester 112d

A500-mL round-bottom flask equipped with a magnetic stirrer and nitrogen inlet was purged with nitrogen and charged with 5,6,7, 8-tetrahydroindazine-2-carboxylic acid (30.4g, 184mmol), DMF (1.00g, 13.6mmol), and dichloromethane (300 mL). The solution was cooled to 0 ℃ with an ice bath. Oxalyl chloride (28.0g, 221mmol) was added dropwise and the reaction mixture was warmed to room temperature over 30 min and stirred for 5 h. After this time, the resulting solution was concentrated to give a brown solid. The solid was dissolved in anhydrous methanol (400mL) and the solution was cooled to 0 ℃. Mixing three ethylAmine (57g, 552mmol) was added to the reaction mixture and stirred at room temperature for an additional 2 h. After this time, the reaction mixture was concentrated to dryness under reduced pressure. The residue was washed with dichloromethane (300mL) and water (200mL) and saturated aqueous sodium bicarbonate (200 mL). The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. Titration of the resulting residue with hexanes (200mL) gave 101d (19.1g) as a white solid in 58% yield: mp 72-74 ℃;¹H NMR(300MHz，DMSO-d₆)7.13(s，1H)，6.23(s，1H)，3.93(t，2H，J＝6.0Hz)，3.77(s，3H)，2.75(t，2H，J＝6.0Hz)，1.93(m，2H)，1.80(m，2H)；(APCI+)m/z180.1(M+H)

example 101e3- (cyanomethyl) -5,6,7, 8-tetrahydroindazine-2-carboxylic acid methyl ester 101e

A 500-mL three-necked round bottom flask was equipped with an addition funnel, thermometer and charged with methyl 5,6,7, 8-tetrahydroindazine-2-carboxylate 101d (6.70g, 37.4mmol), iodoacetonitrile (12.5g, 74.9mmol), ferrous sulfate heptahydrate (5.20g, 18.7mmol) and dimethyl sulfoxide (250 mL). within 1h at room temperature, hydrogen peroxide (35%, 18.2g, 187mmol) was added dropwise to the mixture via a syringe pump using a water bath, ferrous sulfate heptahydrate (2 to 3 equivalents) was added portionwise to the reaction mixture maintaining the temperature between 25 ℃ and 35 ℃ until the color of the reaction mixture was dark red if TLC showed that the reaction was not complete, hydrogen peroxide (2-3 equivalents) and ferrous sulfate heptahydrate (1-2 equivalents) were added in the same manner until the reaction was complete.the reaction, then the reaction mixture was partitioned between saturated aqueous sodium bicarbonate solution (200mL) and ethyl acetate (400mL) and the saturated aqueous layer was extracted with ethyl acetate (2 × 100mL) to obtain a combined saturated aqueous solution of sodium thiosulfate and the yield was purified by column chromatography (40.78:):¹H NMR(500MHz，CDCl₃)6.23(s，1H)，4.23(s，2H)，3.94(t，2H，J＝6.5Hz)，3.81(s，3H)，2.74(t，2H，J＝6.5Hz)，2.00(m，2H)，1.83(m，2H)；(APCI+)m/z219.3(M+H)

example 101f3- (2-aminoethyl) -5,6,7, 8-tetrahydroindazine-2-carboxylic acid methyl ester hydrochloride 101f

Hydrogenation of methyl 3- (cyanomethyl) -5,6,7, 8-tetrahydroindazine-2-carboxylate 101e over night at room temperature under 50psi of hydrogen using a platinum oxide catalyst in ethanol and ethyl acetate in the presence of hydrogen chloride gave 101f (380mg, 1.74mmol) which was used directly in the next step.

Example 101g3,4,6,7,8, 9-hexahydropyrido [3,4-b ]]Indolizin-1 (2H) -one 101g

Purging a 100-mL single-neck round-bottom flask equipped with a magnetic stirrer and nitrogen inlet with nitrogen and charging 3- (2-aminoethyl) -5,6,7, 8-tetrahydroindazine-2-carboxylic acid methyl ester hydrochloride 101f (prepared above, estimated 1.74mmol, assuming quantitative yield), sodium ethoxide (354mg, 5.22mmol) and ethanol (20mL) 55 ℃ stir the mixture for 5h then concentrate the reaction mixture under reduced pressure and partition the residue between ethyl acetate (200mL) and water (100 mL.) the organic layer is separated and the aqueous layer is extracted with ethyl acetate (2 × 100 mL.) the combined organic layer is washed with brine, dried over sodium sulfate and concentrated under reduced pressure the residue is purified by column chromatography to give a white solid with 67% yield (220mg) of 101 g: 195-197 ℃;¹HNMR(500MHz，DMSO-d₆)6.76(s，1H)，5.89(s，1H)，3.78(t，2H，J＝6.5Hz)，3.35(m，2H)，2.66(m，4H)，1.87(m，2H)，1.72(m，2H)；(APCI+)m/z191.3(M+H)

example 101hAcetic acid 2-bromo-4-fluoro-6- (1-oxo-3, 4,6,7,8, 9-hexahydropyrido [3, 4-b)]-indolizin-2 (1H) -yl) benzyl ester 101H

To a 100-mL single-neck round-bottom flask equipped with a magnetic stirrer and reflux condenser was charged 1, 4-dioxane (60mL), 101c (1.9g, 6.0mmol), 101g (400mg, 2.0mmol) and cesium carbonate (1.3g, 4.0mmol), after bubbling nitrogen through the resulting mixture for 30 minutes, Xantphos (120mg, 0.2mmol) and tris (dibenzylideneacetone) dipalladium (0) (180mg, 0.2mmol) were added and the reaction mixture was heated at 100 ℃ for 12H, after which the reaction mixture was cooled to room temperature, partitioned between ethyl acetate (40mL) and water (40mL) and filtered the aqueous layer was separated and extracted with ethyl acetate (70mL × 3). the combined organic layers were washed with brine (30mL) and dried over sodium sulfate, the desiccant was removed by filtration and the filtrate was concentrated under reduced pressure.the residue was purified by flash column (eluting with 2:1 PE/EA) to give 101H (421mg, 46% MS: [ H + [ 421M ] + H ] (as a yellow solid)]⁺435。¹H NMR(500MHz，MeOD)7.52-7.50(m，1H)，7.20-7.23(m，1H)，6.14(s，1H)，5.10-5.20(m，2H)，4.06-4.12(m，1H)，3.92-3.97(m，1H)，3.83-3.88(m，1H)，3.75-3.79(m，1H)，3.03-3.10(m，1H)，2.94-2.99(m，1H)，2.75-2.83(m，2H)，2.00-2.05(m，5H)，1.83-1.88(m，2H)

Example 101i(S) -acetic acid 4-fluoro-2- (1-methyl-5- (5- (2-methyl-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-hexahydropyrido [3,4-b ] amino]Indolizin-2 (1H) -yl) benzyl ester 101i

Heating at 100 ℃ for 101H (300mg, 0.70mmol), (S) -1-methyl-3- (5- (2-methyl-4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-ylamino) -5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyridin-2 (1H) -one (332mg, 0.70mmol), CH₃COONa(114mg，1.4mmol)、K₃PO₄(368mg，1.4mmol)、PdCl₂(dppf)(56mg，0.07mmol)、CH₃CN (25mL) and H₂Mixture of O (1mL) for 3 hours. The residue was then evaporated and purified by column on silica gel (eluting with dichloromethane/methanol (30: 1)) to give 101i as a brown solid (293mg, 41% yield). MS (M + H)⁺710。

Example 101(S) -2- (5-fluoro-2- (hydroxymethyl) -3- (1-methyl-5- (5- (2-methyl-4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-ylamino) -6-oxo-1, 6-dihydropyridin-3-yl) -phenyl) -3,4,6,7,8, 9-hexahydropyrido [3,4-b]Indolizin-1 (2H) -ones 101

Add LiOH (914mg, 38mmol) to a solution of 101i (270mg, 0.38mmol) in propan-2-ol (8mL), tetrahydrofuran (8mL) and water (1.5 mL). The mixture was stirred at 30 ℃ for 2 h. Evaporation and purification of the residue by reverse phase preparative HPLC gave 101 as a white solid (127mg, yield 50%). MS: (M + H)⁺669。¹H NMR(500MHz，DMSO)8.58(t，J＝2.5，1H)，8.40(s，1H)，7.85(d，J＝3.0，1H)，7.35-7.38(m，2H)，7.22-7.25(m，2H)，7.13-7.16(m，1H)，6.01(s，1H)，4.76(s，1H)，4.57-4.54(m，2H)，4.47(t，J＝6.0，1H)，4.42(t，J＝6.5，1H)，4.29(s，2H)，3.98-4.04(m，1H)，3.89-3.94(m，1H)，3.78-3.83(m，2H)，3.67(s，1H)，3.58(s，3H)，3.37-3.42(m，1H)，3.00-3.10(m，2H)，2.90-2.95(m，2H)，2.71(t，J＝6.0，2H)，2.52-2.55(m，1H)，2.28-2.35(m，2H)，2.18(t，J＝8.5，1H)，1.89-1.94(m，2H)，1.72-1.78(m，2H)，0.93(t，J＝6.5，3H)

Example 102a(S) -acetic acid [ 4-fluoro-2- (1-methyl-5- (5- (2-methyl-4- (oxetan-3-yl) -piperazin-1-yl) pyridin-2-ylamino) -6-oxo-1, 6-dihydropyridin-3-yl) -6- { 6-oxo-8-thia-4, 5-diazacyclo [7.4.0.0^2,7]Thirteen-1 (9),2(7), 3-trien-5-yl) phenyl]Methyl ester 102a

Following the procedure described for 101i and using acetic acid (4-fluoro-2- { 6-oxo-8-thia-4, 5-diazacyclo [7.4.0.0 ]^2,7]Tridec-1 (9),2(7), 3-trien-5-yl } -6- (tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) methyl ester (250mg) and 5-bromo-1-methyl-3- (3-methyl-5- (4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-ylamino) pyridin-2 (1H) -one (218mg) gave 102a (225mg, 62%) as a yellow solid. LCMS: [ M + H ]]⁺726. See fig. 2.

Example 102(S) -5- [ 5-fluoro-2- (hydroxymethyl) -3 (1-methyl-5- (5- (2-methyl-4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-ylamino) -6-oxo-1, 6-dihydropyridin-3-yl) phenyl]-8-thia-4, 5-diazacyclo [7.4.0.0^2,7]Thirteen-1 (9),2(7), 3-trien-6-one 102

Hydrolysis of 102a (210mg, 0.29mmol) with lithium hydroxide as described in 101 gave 102(117mg, 85%) as a white solid. LCMS: [ M + H ]]⁺684。¹H NMR(500MHz，CDCl3)8.61(d，J＝2.5，1H)，8.26(s1H)，7.99(d，J＝2.5，1H)，7.83(s，1H)，7.46(d，J＝2.0，1H)，7.29-7.32(m，2H)，7.11(dd，J＝2.0，8.0，1H)，6.82(d，J＝9.0，1H)，4.62-4.73(m，4H)，4.31(d，J＝6.5，2H)，4.02(t，J＝6.5，1H)，3.71(s，3H)，3.52-3.55(m，1H)，3.45-3.49(m，1H)，3.09(t，J＝4.5，2H)，2.99(t，J＝4.5，2H)，2.87(t，J＝4.5，2H)，2.56(dd，J＝3.0，11.0，1H)，2.46-2.49(m，2H)，2.19-2.25(m，1H)，1.96-2.01(m，4H)，1.00(d，J＝6.0，3H)

Example 103a(E) Ethyl-3- (2-chloro-4, 4-dimethylcyclopent-1-enyl) acrylate 103a

The following two methods are adjusted from Organic Preparations and products int, 29(4), 471-498. A500-mL single-neck round bottom flask equipped with a magnetic stirrer and a nitrogen inlet was charged with a solution of 2-chloro-4, 4-dimethylcyclopent-1-enecarbaldehyde (38g, 240mmol) in benzene (240 mL). See fig. 3. To this solution was added ethoxycarbonylmethylene triphenylphosphine (84g, 240 mmol). The mixture was stirred for 14 h. Then, the solvent was evaporated and the product extracted with hexane (2L) to remove PPh₃By-products. The organic layer was dried over sodium sulfate and concentrated in vacuo. The residue was purified by column chromatography using a 100% hexane-1: 1 hexane/ethyl acetate gradient to give 103a in 37% yield (20 g).

Example 103b5, 5-dimethyl-1, 4,5, 6-tetrahydrocyclopenta [ b ]]Pyrrole-2-carboxylic acid ethyl ester 103b

A250-mL single-neck round bottom flask equipped with a magnetic stirrer and a nitrogen inlet was charged with a solution of 103a (17g, 74mmol) in DMSO (100 mL). To the solution was added sodium azide (9.6g, 150 mmol). The mixture was then heated to 75 ℃ and stirred for 8 h. After cooling to room temperature, H was added₂O (100mL) and CH₂Cl₂(200mL) and the organic layer was separated. By CH₂Cl₂The aqueous layer was extracted (50 mL). The combined organic layers were washed with brine, dried over sodium sulfate and concentrated in vacuo. By passingColumn chromatography the residue was purified using a 100% hexane-1: 1 hexane/ethyl acetate gradient to give 103b in 37% yield (5.7 g).

Example 103c1- (cyanomethyl) -5, 5-dimethyl-1, 4,5, 6-tetrahydrocyclopenta [ b)]Pyrrole-2-carboxylic acid ethyl ester 103c

A250-mL single-neck round bottom flask equipped with a magnetic stirrer and a nitrogen inlet was charged with a solution of 103b (6.2g, 30mmol) in DMF (57 mL). To this solution was added NaH (80% dispersion in mineral oil, 1.26g, 42.1 mmol). The resulting mixture was stirred at room temperature for 90 minutes. Bromoacetonitrile (2.94mL, 42mmol) was then added. The mixture was stirred for 14 h. Then, water (100mL) and ethyl acetate (200mL) were added and the organic layer was separated. The aqueous layer was extracted with ethyl acetate (2X50 mL). The combined organic layers were washed with brine, dried over sodium sulfate and concentrated in vacuo. The residue was purified by column chromatography to give 103c in 95% yield (7 g).

Example 103d1- (2-aminoethyl) -5, 5-dimethyl-1, 4,5, 6-tetrahydrocyclopenta [ b)]Pyrrole-2-carboxylic acid ethyl ester hydrochloride 103d

A500-mL Parr reactor vial was purged with nitrogen and charged with 10% palladium on charcoal (50% wet, 2.0g dry weight), 103c (4.5g, 18mmol), 12% hydrochloric acid (9.2mL, 37mmol), ethyl acetate (80mL), and ethanol (52 mL). The bottle was connected to a Parr hydrogenator, evacuated, charged with hydrogen to 50psi and shaken for 6 h. Thereafter, the hydrogen was evacuated and the bottle was filled with nitrogen. Adding diatomite filter agent (521,10.0g) and the mixture was filtered through a pad of celite 521 the filter cake was washed with ethanol (2 × 50mL) and the combined filtrates were concentrated to dryness under reduced pressure the next step was carried out with the crude residue 103d without further purification.

Example 103e4, 4-dimethyl-1, 10-diazacyclo [6.4.0.0 ]^2,6]Dodeca-2 (6), 7-dien-9-one 103e

A100-mL single-neck round bottom flask equipped with a magnetic stirrer and nitrogen inlet was purged with nitrogen and charged with crude 103d (. about.18 mmol), sodium ethoxide (6.2g, 92mmol) and ethanol (120 mL). The mixture was stirred at 55 ℃ overnight. The reaction mixture was then concentrated under reduced pressure and the residue was partitioned between ethyl acetate (200mL) and water (100 mL). The solution was filtered. The solid was washed with ethyl acetate (15mL) to give 850mg of the desired product, 103 e. The organic layer was separated and the aqueous layer was extracted with ethyl acetate (2X 100 mL). The combined organic layers were dried over sodium sulfate and concentrated to near dryness under reduced pressure. The solution was filtered and the solid (1.44g) was washed with ethyl acetate (15 mL). The combined solids were dried in vacuo to give 103e in 61% yield (2.3 g).

Example 103fAcetic acid 2-bromo-4-fluoro-6- (9-oxo-4, 4-dimethyl-1, 10 diazacyclo [6.4.0.0 ]^{2 ,6}]Dodecyl-2 (6), 7-dien-10-yl) benzyl ester 103f

The sealed tube was equipped with a magnetic stirrer and charged with a solution of 103e (740mg, 3.6mmol), 2, 6-dibromo-4-fluorobenzyl acetate 101c (2.4g, 7.2mmol) and cesium carbonate (2.6g, 7.9mmol) in1, 4-dioxane (36mL)And (4) liquid. After bubbling nitrogen through the solution for 30 min, Xantphos (250mg, 0.43mmol) and tris (dibenzylideneacetone) dipalladium (0) (260mg, 0.29mmol) were added and the reaction mixture was heated to 100 ℃ and held for 16 h. Thereafter adding H₂O (50mL) and ethyl acetate (50mL), the aqueous layer was separated and extracted with ethyl acetate (2 × 50mL), the combined organic extracts were washed with brine (100mL) and dried over sodium sulfate, and the resulting residue was purified by column chromatography (eluting with a 100% hexane-100% ethyl acetate gradient) to give 103f in 56% yield (910 mg).

Example 103gAcetic acid 2- (4,4,5, 5-tetramethyl- [1,3,2 ]]Dioxaborolan-2-yl) -4-fluoro-6- (9-oxo-4, 4-dimethyl-1, 10 diazacyclo [6.4.0.0^2,6]103g of (E) -dodeca-2 (6), 7-dien-10-yl) benzyl ester

The reaction mixture was purified by mixing compound 103f (450mg, 1.0mmol), 4,4,4',4',5,5,5',5' -octamethyl-2, 2' -bis (1,3, 2-dioxaborolane) (635mg, 2.5mmol), potassium acetate (393mg, 4.0mmol), bis (diphenylphosphino) -ferrocene]Palladium (II) dichloride and CH₂Cl₂Complex of (Pd Cl)₂dppf:CH₂Cl₂(1:1), 66mg, 0.08mmol) and 1, 4-dioxane (20mL) were mixed and heated at 100 ℃ for 5H cooling the reaction mixture to room temperature and filtered through a pad of celite 521 the filter cake was washed with ethyl acetate (2 × 25mL) and the combined filtrates were concentrated under reduced pressure to dryness to give 103g of a black oil (quantitative yield) which was used directly in the next step MS (ESI +) M/z497.3(M + H).

Example 103h(2S) -acetic acid (2- {4, 4-dimethyl-9-oxo-1, 10-diazacyclo- [ 6.4.0.0)^2,6]Dodeca-2 (6), 7-dien-10-yl } -4-fluoro-6- [ 1-methyl-5- ({5- [ 2-methyl-4- (oxetan-3-yl) piperazin-1-yl]Pyridin-2-yl } amino) -6-oxo-1, 6-dihydropyridin-3-yl]Phenyl) -methyl ester 103h

Acetic acid (2- {4, 4-dimethyl-9-oxo-1, 10-diazacyclo [ 6.4.0.0) was purified according to the procedure described for Compound 101i^2,6]Dodeca-2 (6), 7-dien-10-yl } -6- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) methyl ester (229mg, 0.46mmol) was reacted with (S) -5-bromo-1-methyl-3- (5- (2-methyl-4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-ylamino) pyridin-2 (1H) -one (200mg, 0.46mmol) to give 103H (80mg, 24%) as a yellow solid. MS: [ M + H ]]⁺724。

Example 103(2S) -10- [ 5-fluoro-2- (hydroxymethyl) -3- [ 1-methyl-5- ({5- [ 2-methyl-4- (oxetan-3-yl) piperazin-1-yl]Pyridin-2-yl } amino) -6-oxo-1, 6-dihydropyridin-3-yl]-phenyl radical]-4, 4-dimethyl-1, 10-diazacyclo [6.4.0.0^2,6]Dodeca-2 (6), 7-dien-9-one 103

Hydrolysis of intermediate 103h (80mg, 0.11mmol) with lithium hydroxide as described for 101 gave 103(40mg, 53%) as a yellow solid. LCMS: [ M + H ]]⁺682。¹H NMR(500MHz，CDCl₃)8.56(dd，J＝2.0，7.0，1H)，7.95(t，J＝3.0，1H)，7.82(d，J＝3.0，1H)，7.47(t，J＝3.0，1H)，7.31(dd，J＝3.0，9.0，1H)，7.17-7.14(m，1H)，6.95(dd，J＝2.5，9.0，1H)，6.82-6.80(m，2H)，4.71-4.61(m，4H)，4.56-4.53(m，1H)，4.41-4.37(m，1H)，4.33-4.28(m，1H)，4.23-4.15(m，3H)，3.91-3.86(m，1H)，3.70(s，3H)，3.55-3.44(m，2H)，3.08-3.06(m，2H)，2.56-2.46(m，7H)，2.21-2.16(m，1H)，1.27(s，6H)，0.98-0.97(m，3H)

Example 104a(2R,5S) -2, 5-dimethyl-4- (6-nitropyridin-3-yl) piperazine-1-carboxylic acid tert-butyl ester 104a

Tert-butyl (2R,5S) -4-chloro-2, 5-dimethylpiperazine-1-carboxylate (1.5g, 6.0mmol) was reacted with 5-bromo-2-nitropyridine (1212mg, 6.0mol) as described for compound 108a to give 104a (1500mg, 75%) as a yellow solid. LCMS: [ M + H ]]⁺337. See fig. 4.

Example 104b(2R,5S) -4- (6-Aminopyridin-3-yl) -2, 5-dimethylpiperazine-1-carboxylic acid tert-butyl ester 104b

104a (1.5g4.46mmol) was reacted as described for compound 108b to give 104b as a yellow solid (1130mg, 83%). LCMS: [ M + H ]]⁺307

Example 104c(2R,5S) -4- (6- (5-bromo-1-methyl-2-oxo-1, 2-dihydropyridin-3-ylamino) pyridin-3-yl) -2, 5-dimethylpiperazine-1-carboxylic acid tert-butyl ester 104c

Tert-butyl (2R,5S) -2, 5-dimethylpiperazine-1-carboxylate (332mg, 1.08mmol) was reacted with 3, 5-dibromo-1-methylpyridin-2 (1H) -one (294mg, 1.08mmol) as described for compound 108c to give 104c (402mg, 75%) as a yellow solid. LCMS: [ M + H ]]⁺492

Example 104d(2R,5S) -4- (6- (5-bromo-1-methyl-2-oxo-1, 2-dihydropyridin-3-ylamino) pyridin-3-yl) -2, 5-dimethylpiperazine-1-carboxylic acid tert-butyl ester 104d

Acid hydrolysis of 104c (402mg, 0.82mmol) to remove the boc group as described for compound 108d gave 104d as a yellow solid (300mg, 94%). LCMS: [ M + H ]]⁺392

Example 104e5-bromo-3- (5- ((2S,5R) -2, 5-dimethyl-4- (oxetan-3-yl) piperazin-1-yl) -pyridin-2-ylamino) -1-methylpyridin-2 (1H) -one 104e

Following the procedure as described for compound 108e and starting with 5-bromo-3- (5- ((2S,5R) -2, 5-dimethylpiperazin-1-yl) pyridin-2-ylamino) -1-methyl-pyridin-2 (H) -one (300mg, 0.77mmol), 104e was obtained as a yellow solid (320mg, 93%). LCMS: [ M + H ]]⁺448

Example 104f2,2, 2-trichloro-1- (4,5,6, 7-tetrahydro-1H-indol-2-yl) ethanone 104f

A100-mL, single-necked, round-bottomed flask equipped with a magnetic stirrer, condenser, and nitrogen inlet was purged with nitrogen and charged with 4,5,6, 7-tetrahydro-1H-indole (3.00g, 24.8mmol), trichloroacetyl chloride (13.5g, 74.4mmol), and 1, 2-dichloroethane (50 mL). The solution was stirred at 85 ℃ for 2 h. The reaction mixture was then concentrated under reduced pressure to give 104f as a black semi-solid in 100% yield (6.50 g):¹H NMR(500MHz，DMSO-d₆)11.94(s，1H)，7.05(s，1H)，2.62(t，2H，J＝6.0Hz)，2.47(t，2H，J＝6.0Hz)，1.80(m，2H)，1.65(m，2H)；MS(ESI+)m/z266.0(M+H)

example 104g104g of 4,5,6, 7-tetrahydro-1H-indole-2-carboxylic acid ethyl ester

A100-mL, single-necked, round-bottomed flask equipped with a magnetic stirrer and nitrogen inlet was purged with nitrogen and charged with 2,2, 2-trichloro-1- (4,5,6, 7-tetrahydro-1H-indol-2-yl) ethanone 104f (6.50g, 24.8mmol), sodium ethoxide (17.0mg, 0.25mmol) and ethanol (40 mL). The solution was stirred at room temperature for 1 h. Then, the reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography to give 100% yield (4.80g) of ethyl 4,5,6, 7-tetrahydro-1H-indole-2-carboxylate 104g as a brown solid: mp 70-72 ℃;¹H NMR(300MHz，CDCl₃)9.08(s，1H)，6.75(s，1H)，4.25(q，2H，J＝7.2Hz)，2.65(t，2H，J＝6.0Hz)，2.56(t，2H，J＝6.0Hz)，1.85(m，4H)，1.28(t，3H，J＝7.2Hz)；MS(ESI+)m/z194.1(M+H)

example 104h1- (cyanomethyl) -4,5,6, 7-tetrahydro-1H-indole-2-carboxylic acid ethyl ester 104H

A125-mL single neck round bottom flask equipped with a magnetic stirrer and nitrogen inlet was purged with nitrogen and charged with 104g (5.76g, 29.8mmol) of ethyl 4,5,6, 7-tetrahydro-1H-indole-2-carboxylate and DMF (50 mL). The solution was cooled to 0 ℃ with an ice bath. Sodium hydride NaH (60% dispersion in mineral oil, 1.43g, 35.8mmol) was added. The resulting mixture was stirred at room temperature for 1 h. Bromoacetonitrile (1.43g, 35.8mmol) was then added. The mixture was stirred at room temperature for 14 h. The reaction mixture was then concentrated under reduced pressure and the residue was partitioned between ethyl acetate (150mL) and water (450 mL). Separating the organic layer with a second solventThe aqueous layer was extracted with ethyl acetate (3 × 150mL), the combined organic layers were washed with brine, dried over sodium sulfate and concentrated under reduced pressure, the residue was purified by column chromatography to give 55% yield (3.80g) as a yellow semi-solid over 104 h:¹H NMR(300MHz，CDCl₃)6.66(s，1H)，5.29(s，2H)，4.28(q，2H，J＝7.2Hz)，2.62(t，2H，J＝6.3Hz)，2.49(t，2H，J＝6.3Hz)，1.92(m，2H)，1.75(m，2H)，1.33(t，3H，J＝7.2Hz)；MS(ESI+)m/z233.1(M+H)

example 104i1- (2-aminoethyl) -4,5,6, 7-tetrahydro-1H-indole-2-carboxylic acid ethyl ester 104i

Purging a 200-mL Parr reactor bottle with nitrogen and charging 10% palladium on charcoal (50% wet, 1.28g dry), 104h (3.00g, 12.9mmol), 12% hydrochloric acid (6.5mL, 25mmol), ethyl acetate (60mL), and ethanol (40mL), connecting the bottle to a Parr hydrogenator, evacuating, charging hydrogen to a pressure of 50psi and shaking for 6h, after which, evacuating hydrogen, and charging nitrogen to the bottle, adding diatomaceous earth 521(4.0g), and filtering the mixture through a pad of diatomaceous earth 521, washing the filter cake with ethanol (2 × 20mL), and concentrating the combined filtrate under reduced pressure to dryness, partitioning the residue between ethyl acetate (150mL) and 10% aqueous potassium carbonate (100mL), separating the organic layer, and extracting the aqueous layer with ethyl acetate (3 × 75mL), drying the combined organic layer over sodium sulfate, and concentrating under reduced pressure, milling the residue with ethanol (5mL) to give a yield of 71% white solid (104.71% wet, 104: 102 g);¹H NMR(500MHz，DMSO-d₆)6.61(s，1H)，6.22(br，2H)，4.15(m，4H)，2.77(m，2H)，2.59(t，2H，J＝6.5Hz)，2.42(t，2H，J＝6.5Hz)，1.70(m，2H)，1.62(m，2H)，1.23(t，3H，J＝7.0Hz)；MS(APCI+)m/z237.2(M+H)

example 104j3,4,6,7,8, 9-sixHydropyrazino [1,2-a ]]Indol-1 (2H) -ones 104j

Purging a 100-mL single-neck round-bottom flask equipped with a magnetic stirrer and nitrogen inlet with nitrogen and charging 104i (1.80g, 7.63mmol), sodium ethoxide (1.55g, 22.8mmol) and ethanol (50 mL). stirring the mixture at 55 ℃ for 5 h. then, concentrating the reaction mixture under reduced pressure and partitioning the residue between ethyl acetate (200mL) and water (100 mL.) the organic layer is separated and the aqueous layer is extracted with ethyl acetate (2 × 100 mL). the combined organic layers are washed with brine, dried over sodium sulfate and concentrated under reduced pressure, the residue is purified by column chromatography to give a 42% yield (605mg) of 104 j: mp 207-209 ℃ as a white solid;¹H NMR(500MHz，DMSO-d₆)7.41(s，1H)，6.36(s，1H)，3.84(t，2H，J＝6.0Hz)，3.42(m，2H)，2.51(t，2H，J＝6.0Hz)，2.42(t，2H，J＝6.0Hz)，1.76(m，2H)，1.65(m，2H)；(APCI+)m/z191.3(M+H)

example 104k2, 6-dibromo-4-fluorobenzaldehyde 104k

To a solution of-35 ℃ cooled 1, 3-dibromo-5-fluoro-2-iodobenzene (50g, 132mmol) in anhydrous toluene (300mL) was added an isopropyl magnesium chloride solution (84mL, 171mmol, Et) over 30 minutes while maintaining the internal temperature below-25 ℃₂2.0M in O). A clear brown solution was obtained and stirring was continued at-25 ℃ for 1.5 h. Then anhydrous DMF (34mL, 436mmol) was added over a period of 30 minutes. The reaction mixture was warmed to 10 ℃ over 1h (room temperature) and stirred at this temperature for 1.5 h. Then quenched with 3.0M HCl and then ethyl acetate was added. The organic layer was separated and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (eluting with petroleum ether/ethyl acetate (50:1 to 20: 1)) to give 104k (20g, 54%) as a white solid.¹H NMR(500MHz，CDCl₃)10.23(s，1H)，7.48(d，J＝7.5，2H)。

Example 104l(2, 6-dibromo-4-fluorophenyl) methanol 104l

Add NaBH to a solution of 104k (20g, 71mmol) in EtOH (500mL)₄(10g, 284 mmol.) the mixture was stirred at room temperature (10 ℃ C.) for 4h and TLC showed disappearance of starting material quenching the reaction mixture by aqueous HCl (150mL, 1M) and evaporating in vacuo until most of the EtOH was distilled the residue was extracted by ethyl acetate (500mL × 3.) the organic layers were combined, Na was used₂SO₄Dried and evaporated in vacuo. The residue was purified by column chromatography on silica gel (eluting with petroleum ether/ethyl acetate (50:1 to 20: 1)) to give 104l (15g, 75%) as a white solid. MS: [ M-OH]⁺267。¹H NMR(500MHz，DMSO-d₆)7.68(d，J＝8.5，2H)，5.23(s，1H)，4.71(s，2H)。

Example 104mAcetic acid 2, 6-dibromo-4-fluorobenzyl ester 104m

To (2, 6-dibromo-4-fluorophenyl) methanol (104l) (20g, 71mmol) in CH at 0 deg.C₂Cl₂To a solution in (500mL) was added pyridine (8.4g, 107mmol) and acetyl chloride (8.3g, 107 mmol). The mixture was stirred at room temperature for 5 h. TLC showed the disappearance of starting material. The reaction mixture was evaporated in vacuo and the residue was purified by silica gel column chromatography (eluting with petroleum ether/ethyl acetate (50:1 to 20: 1)) to give 104m (20g, 87%) as a white solid. MS: [ M-Oac]⁺267。¹H NMR(500MHz，CDCl₃)7.36(d，J＝7.5，2H)，5.38(s，2H)，2.10(s，3H)

Example 104nAcetic acid 2-bromo-4-fluoro-6- (1-oxo-3, 4,6,7,8, 9-hexahydropyrazino [1, 2-a)]Indol-2 (1H) -yl) benzyl ester 104n

A250-mL single-neck round bottom flask equipped with a magnetic stirrer was charged with 104j (3.8g, 20mmol), 104m (20g, 60mmol), XantPhos (1.2g, 2mmol), tris (dibenzylideneacetone) dipalladium (0) (1.8g, 2mmol), Cs₂CO₃(16g，50mmol) and 1, 4-dioxane (120 mL). Evacuating the system and then using N₂And (6) refilling. A reflux condenser was attached to the flask and the reaction mixture was heated at 100 ℃ for 16 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 column chromatography (eluting with 5:1 petroleum ether/ethyl acetate) to give 104n (5.2g, 60%) as a white solid. MS: [ M + H ]]⁺435。¹H NMR(500MHz，DMSO-d₆)7.70(dd，J＝3，1H)，7.48(dd，J＝3，1H)，6.52(s，1H)，5.01(m，2H)，4.18(m，2H)，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 104oAcetic acid 4-fluoro-2- (1-oxo-3, 4,6,7,8, 9-hexahydro-pyrazino [1, 2-a)]Indol-2 (1H) -yl) -6- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzyl ester 104o

A250-mL single-neck round-bottom flask equipped with a magnetic stirrer was charged with 104n (3.8g, 8.65mmol), (PinB)₂(11g，43.25mmol)、Pd(dppf)Cl₂(0.4g, 0.5mmol), KOAc (2.5g, 26mmol) and 1, 4-dioxane (150 mL). Evacuating the system and then using N₂And (6) refilling. A reflux condenser was attached to the flask and the reaction mixture was heated at 100 ℃ for 15 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 column chromatography (eluting with 5:1 petroleum ether/ethyl acetate) to give 104o (3.2g, 77%) as a yellow solid. MS: [ M + H ]]⁺483。

Example 104pAcetic acid 2- (5- (5- ((2S,5R) -2, 5-dimethyl-4- (oxetan-3-yl) piperazin-1-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 ] amino]Indol-2 (1H) -yl) benzyl ester 104p

104e (268mg, 0.60mmol) and 104o (289mg, 0.60mmol) were reacted as described for compound 108f to give 104p (300mg, 85%) as a yellow solid. LCMS: [ M + H ]]⁺724

Example 1042- (3- (5- (5- ((2S,5R) -2, 5-dimethyl-4- (oxetan-3-yl) piperazin-1-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 ] S]Indol-1 (2H) -ones 104

Hydrolysis of 104p acetate (288mg, 0.40mmol) with lithium hydroxide as in example 101i gave 104 as a white solid (80mg, 25%). LCMS: [ M + H ]]⁺682。¹HNMR(500MHz，CDCl₃)8.60(d，J＝2.0，1H)，8.02(d，J＝2.5，1H)，7.87(d，J＝1.5，1H)，7.49-7.48(m，1H)，7.37(d，J＝9.0，1H)，7.16(d，J＝9.0，1H)，6.96(d，J＝8.5，1H)，6.87(s，1H)，6.81(d，J＝9.0，1H)，4.77-4.61(m，4H)，4.54(d，J＝11.5，1H)，4.39-4.31(m，2H)，4.19-4.15(m，3H)，3.92-3.91(m，1H)，3.77-3.74(m，1H)，3.71(s，3H)，3.18(s，1H)，2.92-2.89(m，1H)，2.73-2.70(m，2H)，2.61-2.56(m，4H)，2.48(s，1H)，1.98-1.79(m，5H)，0.91-0.89(m，6H)

Example 105a(S) -acetic acid 2- (5- (5- (2-ethyl-4- (oxetan-3-yl) piperazin-1-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 ] amino]Indol-2 (1H) -yl) benzyl ester 105a

Charging (S) -5-bromo-3- (5- (2-ethyl-4- (oxy)) into a sealed tube equipped with a magnetic stirrerAzetidin-3-yl) piperazin-1-yl) pyridin-2-ylamino) -1-methylpyridin-2 (1H) -one (203mg, 0.45mmol), acetic acid 4-fluoro-2- (1-oxo-3, 4,6,7,8, 9-hexahydropyrazino [1,2-a [ -1]Indol-2 (1H) -yl) -6- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzyl ester (216mg, 0.45mmol), Pd (dppf) Cl₂(18mg，0.0225mmol)、NaOAc(74mg，0.90mmol)、K₃PO₄(191mg, 0.90mmol) and acetonitrile (3 mL). See fig. 5. After three cycles of vacuum/argon flushing, the mixture was heated at 100 ℃ for 1 h. Then filtered and the filtrate evaporated in vacuo. The residue was purified by silica gel column chromatography (eluting with dichloromethane/methanol (25:1, V/V)) to give 105a (220mg, 87%) as a brown solid. LCMS: [ M + H ]]⁺724

Example 105(S) -2- (3- (5- (5- (2-ethyl-4- (oxetan-3-yl) piperazin-1-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 ] amino]Indol-1 (2H) -ones 105

At 30 ℃ 105a (220mg, 0.30mmol) and LiOH (72mg, 3.0mmol) were stirred inⁱPrOH/THF (1:1, 4mL) and H₂Mixture in O (1mL) for 1 h. The mixture was evaporated in vacuo and the residue was extracted with EtOAc (10mL X2). The combined EtOAc extracts were concentrated under reduced pressure and the residue was purified by reverse phase preparative HPLC to give 105 as a white solid (54mg, 25%). LCMS: [ M + H ]]⁺682。¹H NMR(500MHz，CDCl₃)8.54(t，J＝2.5，1H)，7.91(s，1H)，7.80(s，1H)，7.47(d，J＝1.5，1H)，7.26-7.24(m，1H)，7.18-7.15(m，1H)，6.95(dd，J＝2.5，9.0，1H)，6.87(s，1H)，6.81(d，J＝7.5，1H)，4.72-4.61(m，4H)，4.54(d，J＝11.5，1H)，4.33-4.30(m，2H)，4.20-4.14(m，3H)，3.92-3.90(m，1H)，3.71(s，3H)，3.53(t，J＝5.5，1H)，3.31(s，1H)，3.12(s，2H)，2.61-2.56(m，5H)，2.44(s，2H)，2.35(s,1H)，1.91-1.89(m，2H)，1.80-1.79(m，2H)，1.67(s，1H)，1.43-1.37(m，1H)，0.82(t，J＝5.5，3H)

Example 106a(S) -acetic acid 4-fluoro-2- (1-methyl-5- (5- (2-methyl-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 ] amino]Indol-2 (1H) -yl) benzyl ester 106a

Charging acetic acid 4-fluoro-2- (1-oxo-3, 4,6,7,8, 9-hexahydropyrazino [1,2-a ] into a sealed tube]Indol-2 (1H) -yl) -6- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzyl ester ((337mg, 0.7mmol), (S) -5-bromo-1-methyl-3- (3-methyl-5- (4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-ylamino) pyridin-2 (1H) -one (303mg, 0.7mmol), Pd (dppf) Cl₂(33mg，0.04mmol)、K₃PO₄.3H₂O (372mg, 1.4mmol) and NaOAc (115mg, 1.4mmol) in CH₃Mixture in CN (20 mL). See fig. 6. Evacuating the system and using N₂And (6) refilling. The reaction mixture was heated at 110 ℃ 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 column chromatography (eluting with 30:1 DCM/MeOH) to afford 106a as a yellow solid (258mg, 52%). LCMS: [ M + H ]]⁺710

Example 106(S) -2- (5-fluoro-2- (hydroxymethyl) -3- (1-methyl-5- (5- (2-methyl-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 ] amino]Indol-1 (2H) -ones 106

To 106a (153mg0.22mmol) in THF/iPA/H with stirring at room temperature₂To a solution in O (6mL/6mL/2mL) was added LiOH (70mg, 2.9 mmol). The mixture was stirred for 0.5 h. Then H is added₂O (20mL) and the mixture extracted with EA (30mL X3). With Na₂SO₄The combined organic layers were dried and concentrated to give a yellow solid which was further purified by reverse phase preparative HPLCThe reaction mixture was purified to obtain 106(60mg, 42% yield) as a white solid. LCMS: [ M + H ]]⁺668。¹H NMR(500MHz，CDCl3)8.57(dd，J＝2.0，7.0，1H)，7.97(s，1H)，7.85(s，1H)，7.48(t，J＝2.5，1H)，7.33(d，J＝7.0，1H)，7.16(d，J＝8.0，1H)，6.96(dd，J＝2.5，9.0，1H)，6.87(s，1H)，6.83(d，J＝9.0，1H)，4.69-4.72(m，4H)，4.55(d，J＝11.5，1H)，4.29-4.38(m，2H)，4.14-4.20(m，3H)，3.90-3.93(m，1H)，3.71(s，3H)，3.57(s，1H)，3.48(s，1H)，3.09-3.14(m，2H)，2.52-2.61(m，7H)，2.23(s,1H)，1.88-1.91(m，2H)，1.79-1.81(m，2H)，0.99(d，J＝5.0，3H)

Example 107a(S) -2-methyl-4- (6-nitropyridin-3-yl) piperazine-1-carboxylic acid tert-butyl ester 107a

5-bromo-2-nitropyridine (1.5g) was reacted with tert-butyl (S) -2-methylpiperazine-1-carboxylate (2.3g) as described for compound 108a to give 107a (1.5g, 40%) as a yellow solid. MS: [ M + H ]]⁺323. See fig. 7.

Example 107b(S) -4- (6-Aminopyridin-3-yl) -2-methyl-piperazine-1-carboxylic acid tert-butyl ester 107b

Reduction of 107a (4.0g) as described for compound 108b gave 107b (1.23g, 97%) as a yellow solid. MS: [ M + H ]]⁺293

Example 107c(S) -4- (6- (5-bromo-1-methyl-2-oxo-)1, 2-dihydro-pyridin-3-ylamino) pyridin-3-yl) -2-methylpiperazine-1-carboxylic acid tert-butyl ester 107c

The reaction of 3, 5-dibromo-1-methylpyridin-2 (1H) -one (83mg) and 107b (90mg) was carried out as described for compound 108c to give 107c (120mg, 81%) as a yellow solid. MS: [ M + H ]]⁺480

Example 107d(S) -5-bromo-1-methyl-3- (5- (3-methylpiperazin-1-yl) -pyridin-2-yl-amino) pyridin-2 (1H) -one 107d

Acid hydrolysis of the boc group of 107c (120mg) as described for compound 108d gave 107d (100mg, 90%) as a yellow solid. MS: [ M + H ]]⁺380

Example 107e(S) -5-bromo-3- (5- (3, 4-dimethylpiperazin-1-yl) pyridin-2-ylamino) -1-methylpyridin-2 (1H) -one 107e

Methylation of 107d (100mg) as described for compound 108e gave 107e as a yellow solid (60mg, 59%). MS: [ M + H ]]⁺394

Example 107f(S) -acetic acid 2- (5- (5- (3, 4-dimethylpiperazin-1-yl) pyridin-2-ylamino) -1-methyl-6-oxo-1, 6-dihydropyridin-3-yl) -4-fluoro-6- (1-oxo-34,6,7,8, 9-hexahydropyrazino- [1,2-a]Indol-2 (1H) -yl) benzyl ester 107f

Acetic acid 4-fluoro-2- (1-oxo-3, 4,6,7,8, 9-hexahydropyrazino [1,2-a ] according to the process described for compound 108f]Indol-2 (1H) -yl) -6- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzyl ester (74mg) was reacted with 107e (60mg) to give 107f (60mg, 59%) as a yellow solid. LCMS: [ M + H ]]⁺668

Example 107(S) -2- (3- (5- (5- (3, 4-dimethylpiperazin-1-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 ] amino]Indol-1 (2H) -ones 107

Hydrolysis of 107f (60mg) with lithium hydroxide as in example 108 gave 107(20mg, 36%) as a white solid. LCMS: [ M + H ]]⁺626。¹H NMR(500MHz，CDCl3):8.55(s，1H)，7.90(d，J＝2.5，1H)，7.77(s，1H)，7.46(d，J＝2.5，1H)，7.26(dd，J＝3.0，9.5，1H)，7.17(dd，J＝3.0，9.5，1H)，6.95(dd，J＝3.0，9.0，1H)，6.87(s，1H)，6.81(d，J＝9.0，1H)，4.55(d，J＝10.5，1H)，4.31-4.36(m，2H)，4.14-4.20(m，3H)，3.90-3.92(m，1H)，3.70(s，3H)，3.38(d，J＝11，1H)，3.32(d，J＝11.5，1H)，2.94(s，2H)，2.56-2.61(m，5H)，2.49(s，1H)，2.38(s，4H)，1.89-1.91(m，2H)，1.79-1.80(m，2H)，1.17(d，J＝5.0，3H)

Example 108a(R) -2-methyl-4- (6-nitropyridin-3-yl) piperazine-1-carboxylic acid tert-butyl ester 108a

A100-mL single-neck round bottom flask equipped with a magnetic stirrer and a reflux condenser was charged with methylsulfinylmethane (50mL), 5-bromo-2-Nitropyridine (2.2g, 11mmol), (R) -tert-butyl 2-methylpiperazine-1-carboxylate (2.2g, 11mmol) and potassium carbonate (3.08g, 22mmol) see FIG. 8. the system was flushed with vacuum/nitrogen for three cycles and heated at 65 ℃ for 15H, then cooled to room temperature and partitioned between ethyl acetate (100mL) and water (20 mL.) the aqueous layer was separated and extracted with ethyl acetate (50mL × 2). the combined organic layers were washed with brine (50mL) and dried over anhydrous 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 2:1(V/V) petroleum ether/ethyl acetate to give 108a (1.75g, 50%) as a yellow solid]⁺323

Example 108b(R) -4- (6-Aminopyridin-3-yl) -2-methylpiperazine-1-carboxylic acid tert-butyl ester 108b

Add 10% Palladium on charcoal (100mg) to a mixture of 108a (1.0g, 3.1mmol) in methanol (15 mL). The mixture was stirred at room temperature under a hydrogen atmosphere overnight. At the end of the reaction, the filtrate was filtered and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (eluting with DCM/MeOH (10:1, V/V)) to give 108b as a brown solid (800mg, 88%). LCMS: [ M + H ]]⁺293

Example 108c(R) -4- (6- (5-bromo-1-methyl-2-oxo-1, 2-dihydropyridin-3-ylamino) pyridin-3-yl) -2-methylpiperazine-1-carboxylic acid tert-butyl ester 108c

To a 50-mL single neck round bottom flask equipped with a magnetic stirrer and reflux condenser was charged 1, 4-dioxane (15mL), 108b (800mg, 2.7mmol), 3, 5-dibromo-1-methylpyridin-2 (1H) -one (720mg, 2.7mmol), and cesium carbonate (1.76g, 5.4 mmol). after bubbling nitrogen through the suspension for 3 minutes, Xantphos (78mg, 0.14mmol) and tris (dibenzylideneacetone) dipalladium (0) (128mg, 0.14mmol) were added and the system was flushed with vacuum/argon for three cycles and heated at reflux for 3H, then cooled to room temperature and filtered.the filtrate was partitioned between ethyl acetate (30mL) and water (30 mL). the aqueous layer was separated and extracted with ethyl acetate (50mL × 2). brine (50mL ×) was usedmL) the combined organic layers were washed and dried over anhydrous 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 10:1 dichloromethane/methanol) to afford 108c as a yellow solid (624mg, 47%). LCMS: [ M + H ]]⁺480

Example 108d(R) -5-bromo-1-methyl-3- (5- (3-methylpiperazin-1-yl) pyridin-2-ylamino) pyridin-2 (1H) -one 108d

To a mixture of 108c (624mg, 1.3mmol) in dichloromethane (8mL) was added trifluoroacetic acid (300mg, 2.6mmol) dropwise at room temperature then the mixture stirred overnight, 2.0N NaOH was added to adjust the pH to greater than 10 then the mixture was extracted with ethyl acetate (50mL × 2), the combined organic layers were washed with brine (50mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give 108d (300mg, 61%) as a brown solid LCMS: [ M + H ] LCMS]⁺380

Example 108e(R) -5-bromo-3- (5- (3, 4-dimethylpiperazin-1-yl) pyridin-2-ylamino) -1-methylpyridin-2 (1H) -one 108e

Add two drops of acetic acid, 30% formaldehyde solution (0.5mL) and sodium triacetoxyborohydride (354mg, 1.6mmol) to a solution of 108d (300mg, 0.8mmol) in methanol (8mL) at RT then stir the mixture at RT for 1H at the end of the reaction, add water (10mL) and extract the mixture with ethyl acetate (20mL × 2). the combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure to give 108e as a brown solid (280mg, 90%). LCMS: [ M + H ], [ M]⁺392

Example 108f(R) -acetic acid 2- (5- (5- (3, 4-dimethylpiperazin-1-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 ] amino]Indol-2 (1H) -yl) benzyl ester 108f

To sealed tubes equipped with magnetic stirrers108e (280mg, 0.71mmol) and acetic acid 4-fluoro-2- (1-oxo-3, 4,6,7,8, 9-hexahydropyrazino [1,2-a ] were charged]Indol-2 (1H) -yl) -6- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzyl ester (344mg, 0.71mmol), PdCl₂(dppf) (58mg, 0.071mmol), 1.0M NaOAc (2.0 equiv.), 1.0M K₃PO₄(2.0 equiv.) and dioxane (5 mL). After three cycles of vacuum/argon flushing, the mixture was heated at 110 ℃ for 2 h. Then filtered and the filtrate evaporated in vacuo. The residue was purified by silica gel column chromatography (eluting with dichloromethane/methanol (10:1, V/V)) to give 108f as a white solid (250mg, 58%). LCMS: [ M + H ]]⁺668

Example 108(R) -2- (3- (5- (5- (3, 4-dimethylpiperazin-1-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 ] amino]Indol-1 (2H) -ones 108

Stirring 108f (250mg, 0.375mmol) and LiOH. H at 30 deg.C₂O (98mg, 2.0mmol) inⁱPrOH/THF (1:1, 3mL) and H₂Mixture in O (1mL) for 2 h. The mixture was evaporated in vacuo and the residue was extracted with EtOAc (10mL X2). The combined EtOAc extracts were concentrated under reduced pressure and the residue was purified by reverse phase preparative HPLC to give 108 as a white solid (62mg, 26%). LCMS: [ M + H ]]⁺626。¹H NMR(500MHz，CDCl₃)8.53(s，1H)，7.89(d，J＝2.5，1H)，7.76(s，1H)，7.45(d，J＝2，1H)，7.26(s，1H)，7.14-7.16(m，1H)，6.92-6.94(m，1H)，6.86(s,1H)，6.79(d，J＝9，1H)，4.52-4.54(m，1H)，4.30-4.32(m，2H)，4.14-1.16(m，3H)，3.90-3.91(m，1H)，3.69(s，3H)，3.34-3.35(m，2H)，2.91-2.92(m，2H)，2.60-2.38(m，10H)，1.83-1.85(m，2H)，1.78-1.79(m，2H)，1.17(s，3H)

Example 109a(R) -3-methyl-4- (6-nitropyridin-3-yl) piperazine-1-carboxylic acid tert-butyl ester 109a

To a 100-mL single neck round bottom flask equipped with a magnetic stirrer and reflux condenser was charged 1, 4-dioxane (60mL), 5-bromo-2-nitropyridine (2.0g, 10.0mmol), (R) -3-methylpiperazine-1-carboxylic acid tert-butyl ester (2.0g, 10.0mmol), and cesium carbonate (6.5g, 20 mmol.) see fig. 9. after bubbling nitrogen through the resulting mixture for 30 minutes, XantPhos (579mg, 1.0mmol) and tris (dibenzylideneacetone) dipalladium (0) (915mg, 1.0mmol) were added and the reaction mixture was heated at 100 ℃ for 15H before cooling to room temperature and filtering the filtrate was partitioned between ethyl acetate (100mL) and water (100 mL.) the aqueous layer was separated and extracted with ethyl acetate (150mL × 3.) the combined organic layers were washed with brine (50mL) and dried over sodium sulfate, the filtrate was removed by filtration and the residue was concentrated under reduced pressure by flash column eluting with DCM (30: 1.44g) to obtain yellow solid (6.44H)]⁺323。¹H NMR(500MHz，DMSO)8.21(d，J＝3.5，1H)，8.18(d，J＝9.0，1H)，7.43-7.45(m，1H)，4.33(s，1H)，3.92-3.99(m，1H)，3.80(d，J＝12.5，2H)，3.06-3.23(m，3H)，1.43(s，9H)，1.09(d，J＝6.5，3H)。

Example 109b(R) -4- (6-Aminopyridin-3-yl) -3-methylpiperazine-1-carboxylic acid tert-butyl ester 109b

The 500-mL flask was purged with nitrogen and charged with 109a (1.5g, 4.6mmol), 10% palladium on charcoal (50% wet, 200mg) and methanol (70 mL). Vacuum was applied, hydrogen was charged and stirred at room temperature for 10 h. The hydrogen was then evacuated and the flask was charged with nitrogen. The catalyst was removed by filtration through a pad of celite and the filtrate was concentrated under reduced pressure to give 109b (1.1g, 81%) as a brown solid. MS: [ M + H ]]⁺293

Example 109c(R) -4- (6- (5-bromo-1-methyl-2-oxo-1, 2-dihydro-pyridin-3-ylamino) pyridin-3-yl) -3-methylpiperazine-1-carboxylic acid tert-butyl ester 109c

To a 100-mL single neck round bottom flask equipped with a magnetic stirrer and reflux condenser was charged 1, 4-dioxane (50mL), 109b (1.0g, 3.4mmol), 3, 5-dibromo-1-methylpyridin-2 (1H) -one (2.7g, 10.2mmol) and cesium carbonate (2.2g, 6.8mmol), after bubbling nitrogen through the resulting mixture for 30 minutes, XantPhos (198mg, 0.34mmol) and tris (dibenzylideneacetone) -dipalladium (0) (313mg, 0.34mmol) were added, and the reaction mixture was heated at 100 ℃ for 5H, after which the reaction mixture was cooled to room temperature and filtered, the filtrate was partitioned between ethyl acetate (100mL) and water (100mL), the aqueous layer was separated and extracted with ethyl acetate (100mL × 3), the combined organic layers were washed with brine (50mL) and dried over sodium sulfate, the drying agent was removed by filtration and the filtrate was concentrated under reduced pressure by a flash column to purify the residue (30: 1H/1M) to obtain DCM (109 g, 1.63%) as a solid]⁺478。

Example 109d(R) -5-bromo-1-methyl-3- (5- (2-methylpiperazin-1-yl) pyridin-2-ylamino) pyridin-2 (1H) -one 109d

To a mixture of 109c (600mg, 1.26mmol) in methanol (20mL) was added HCl/dioxane (4.0M, 4 mL). The reaction mixture was stirred at room temperature for 4 h. Then concentrated under reduced pressure. The residue was basified with 1.0M aqueous NaOH and extracted with DCM. By H₂The combined organic layers were washed and concentrated under reduced pressure to afford 109d (450mg, 95%) as a yellow solid. MS:[M+H]⁺378。

example 109e(R) -5-bromo-3- (5- (2, 4-dimethylpiperazin-1-yl) pyridin-2-ylamino) -1-methylpyridin-2 (1H) -one 109e

A mixture of 109d (500mg, 1.3mmol) and 30% formaldehyde (6.5mmol) in methanol/HOAc (30mL/3mL) was stirred at room temperature for 5 min, followed by the addition of NaBH₃CN (120mg, 1.9 mmol). The mixture was stirred at room temperature for 4 h. Cooled to room temperature and H was added₂O (20 mL). The mixture was extracted three times with DCM (50 mL). The combined organic layers were concentrated under reduced pressure and the residue was purified by column chromatography (eluting with 30:1 DCM/methanol) to give 109e (473mg, 83%) as a yellow solid. MS: [ M + H ]]⁺392。

Example 109f(R) -acetic acid 2- (5- (5- (2, 4-dimethylpiperazin-1-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 ] amino]Indol-2 (1H) -yl) benzyl ester 109f

Heating 109e (400mg, 1.0mmol) and acetic acid 4-fluoro-2- (1-oxo-3, 4,6,7,8, 9-hexahydropyrazino [1,2-a ] at 100 deg.C]Indol-2 (1H) -yl) -6- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzyl ester (490mg, 1.0mmol), PdCl₂(dppf)(80mg，0.1mmol)、2.0M Na₂CO₃(2.0 equiv.) mixture in DMF (4mL) for 2 h. Brine was added and the mixture was extracted three times with EA (50 mL). The combined organic layers were concentrated under reduced pressure. The residue was purified by flash column chromatography (eluting with 30:1 DCM/MeOH) to give 109f as a brown solid (354mg, 52%). LCMS: [ M +H]⁺668

Example 109(R) -2- (3- (5- (5- (2, 4-dimethylpiperazin-1-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 ] amino]Indol-1 (2H) -ones 109

Hydrolysis of 109f (337mg,0.5mmol) with lithium hydroxide as described for compound 108 gave 109(152mg, 48%) as a yellow solid. LCMS (M + H)⁺626。¹H NMR(500MHz，DMSO)8.57(t，J＝2.5，1H)，8.39(s，1H)，7.83(d，J＝3.0，1H)，7.31-7.36(m，3H)，7.23(d，J＝9.0，1H)，7.17-7.20(m，1H)，6.53(s，1H)，4.86-4.88(m，1H)，4.32(d，J＝4.5，2H)，4.09-4.20(m，3H)，3.87-3.91(m，1H)，3.66(s，1H)，3.59(s，3H)，3.04-3.08(m，1H)，2.90-2.94(m，1H)，2.57-2.65(m，3H)，2.47(t，J＝5.5，2H)，2.35-2.42(m，2H)，2.19-2.21(m，1H)，2.18(s，3H)，1.79(t，J＝6.0，2H)，1.66-1.69(m，2H)，0.91(d，J＝6.0，3H)

Example 110a(S) -5-bromo-3- (5- (2, 4-dimethylpiperazin-1-yl) pyridin-2-ylamino) -1-methylpyridin-2 (1H) -one 110a

(S) -5-bromo-1-methyl-3- (5- (2-methylpiperazin-1-yl) pyridin-2-ylamino) pyridin-2 (1H) -one (377mg, 1mmol) was methylated by reductive formylation as described for compound 109e to give 110a (294mg, 75%) as a white solid. LCMS: [ M + H ]]⁺393. See fig. 10.

Example 110b(S) -acetic acid 2- (5- (5- (2, 4-dimethylpiperazin-1-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 ] amino]Indol-2 (1H) -yl) benzyl ester 110b

According to the method as described for compound 109f110a (391mg, 1mmol) was reacted with acetic acid 4-fluoro-2- (1-oxo-3, 4,6,7,8, 9-hexahydropyrazino [1,2-a ] via a Suzuki reaction]Indol-2 (1H) -yl) -6- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzyl ester (482mg, 1mmol) was coupled to give 110b as a white solid (334mg, 50%). LCMS: [ M + H ]]⁺668

Example 110(S) -2- (3- (5- (5- (2, 4-dimethylpiperazin-1-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 ] amino]Indol-1 (2H) -ones 110

The acetate ester of 110b (667mg, 1.0mmol) was hydrolyzed according to the method described in example 109 to give 110 as a white solid (75mg, 12%). LCMS: [ M + H ]]⁺626。¹H NMR(500MHz，CDCl₃)8.57(d，J＝8，1H)，7.96(s，1H)，7.82(s，1H)，7.47(d，J＝1.5，1H)，7.33(s，1H)，7.15-7.16(m，1H)，6.94-6.96(m，1H)，6.86(s,1H)，6.80(d，J＝8.5，1H)，4.52-4.54(m，1H)，4.31-4.33(m，2H)，4.18-4.19(m，3H)，3.90-3.91(m，2H)，3.70(s，3H)，3.49-3.50(m，1H)，3.06-3.07(m，2H)，2.58-2.60(m，7H)，2.34(s，3H)，1.88-1.89(m，2H)，1.78-1.79(m，2H)，0.96(s，3H)

Example 111a4-Benzylpiperazine-1, 2-dicarboxylic acid 1-tert-butyl 2-methyl ester 111a

N₂Next, to a 100ml dry single neck round bottom flask equipped with a stir bar was added piperazine-1, 2-dicarboxylic acid 1-tert-butyl 2-methyl ester (5g, 20.5mmol) (FIG. 1). Anhydrous acetonitrile (60mL) was added followed by BnBr (2.7mL, 22.5mmol) and triethylamine (8.5mL, 61.5 mmol). A condenser was then fitted to the flask and the reaction mixture was heated at 71 ℃ for 45 minutes. The reaction mixture was brought to room temperatureAnd concentrated under reduced pressure. Then diluted with dichloromethane and washed with water and brine. Through Na₂SO₄The organic layer was dried, filtered and concentrated under reduced pressure. Purification of the crude compound using flash column (PE: EA ═ 8:1) gave 4.5g (66%) of 111 a. MS: [ M + H ]]+：335

Example 111b(4-benzyl-1-methylpiperazin-2-yl) methanol 111b

Compound 111a (1g, 2.99mmol) was dissolved in 100mL of anhydrous tetrahydrofuran, lithium aluminum hydride (342mg, 8.98mmol) was added carefully at 0 deg.C and the mixture was stirred for 30 min. It was then refluxed for 3h and the reaction mixture was poured in portions onto ice. Then filtered and the filtrate evaporated in vacuo. After addition of 100mL of brine, it was extracted with dichloromethane (100mL x 3). Through Na₂SO₄The combined organic layers were dried, filtered and concentrated to give 111b (0.6g, 91% yield) as a yellow oil

Example 111c4-benzyl-2- (fluoromethyl) -1-methylpiperazine 111c

N₂Next, a solution of 111b (9g, 40.9mmol) in methylene chloride was added dropwise to an ice-cooled solution of N, N' -dimethylaminosulfur trifluoride (10.8ml, 81.8mmol) in methylene chloride. The yellow solution was stirred at 0 ℃ for 1 hour, warmed to room temperature and stirred for 15 hours. With NaHCO₃The reaction mixture was diluted, the organic layer was separated and washed with Na₂SO₄And (5) drying. The crude product was purified over silica gel (DCM: MeOH ═ 50:1) to give 111c as a yellow oil (3g, yield 33%). MS: [ M + H ]]+：223

Example 111d2- (fluoromethyl) -1-methylpiperazine 111d

A250-mL single-neck round-bottom flask equipped with a magnetic stirrer was charged with 111C (3g, 13.5mmol) and MeOH (80mL), and to the resulting mixture was added Pd/C (10%) (300 mg). Hydrogen (H)₂) The reaction mixture was stirred for 15 h. After completion of the reaction, filtration and concentration gave 111d as a yellow oil (1.6g, yield 90%).

Example 111e2- (fluoromethyl) -1-methyl-4- (6-nitropyridin-3-yl) piperazine 111e

A100-mL single-neck round bottom flask equipped with a magnetic stirrer and a reflux condenser was charged with 111d (1.6g, 12.1mmol), 5-bromo-2-nitropyridine (3.7g, 18.2mmol), and cesium carbonate (9.9g, 30.2 mmol). After bubbling nitrogen through the resulting solution for 30 min, Xantphos (700mg, 0.12mmol) and tris (dibenzylideneacetone) dipalladium (0) (550mg, 0.06mmol) were added and the reaction mixture was heated at reflux for 15 h. After this time the reaction mixture was cooled to room temperature, filtered and concentrated to give a black solid as crude product. Then purified over silica gel (DCM: MeOH ═ 100:1) to give 111e (2.6g, yield 76%) as a yellow solid.¹H NMR(500MHz，MeOD)8.20(dd，J＝12.5Hz，2H)，7.51(dd，J＝9.0Hz，1H)，4.74-4.54(m，2H)，4.03-3.92(m，2H)，3.20(m，1H)，3.09-2.99(m，2H)，2.50(m，2H)，2.46(s，3H)，

Example 111f5- (3- (fluoromethyl) -4-methylpiperazin-1-yl) pyridin-2-amine 111f

A250-mL single-neck round-bottom flask equipped with a magnetic stirrer was charged with 111e (2.6g, 10.2mmol) and MeOH (50mL), and to the resulting mixture was added Pd/C (10%) (260 mg). H₂The reaction mixture was stirred for 15 h. After completion of the reaction, filtration and concentration gave 111f, which was used in the next step without purification.

Example 111g111g of 5-bromo-3- (5- (3- (fluoromethyl) -4-methylpiperazin-1-yl) pyridin-2-ylamino) -1-methylpyridin-2 (1H) -one

A100-mL single-neck round bottom flask equipped with a magnetic stirrer and a reflux condenser was charged with 1, 4-dioxane (60mL), 111e (crude, 14.1mmol), 3, 5-dibromo-1-methylpyridin-2 (1H) -one (4.5g, 16.9mmol), and cesium carbonate (11.5g, 35.2 mmol). After bubbling nitrogen through the resulting solution for 30 min, Xantphos (820mg, 1.41mmol) and tris (dibenzylideneacetone) dipalladium (0) (645mg, 0.7mmol) were added, and the reaction mixture was heated at reflux for 15 h. After this time the reaction mixture was cooled to room temperature, filtered and concentrated to give a black solid as crude product. Purification over silica gel (DCM: MeOH: 100:1 to 50:1) then afforded 111g (3.1g, yield 50%) as a yellow solid.

Example 111hAcetic acid 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 ] amino]Indol-2 (1H) -yl) benzyl ester 111H

111g (1g, 2.4mmol) of acetic acid 4-fluoro-2- (1-oxo-3, 4,6,7,8, 9-hexahydropyrazino [1,2-a ] are heated at 110 DEG C]Indol-2 (1H) -yl) -6- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzyl ester (1.3g, 2.68mmol), PdCl₂(dppf)(190mg，0.24mmol)、K₃PO₄(1g, 4.8mmol) and NaOAc (390mg, 4.8mmol) in MeCN (15mL) and H₂Mixture in O (1.5mL) for 3 h. The solvent was evaporated in vacuo. The residue was purified by silica gel column (DCM: MeOH ═ 50:1) to give 111h (0.8g, yield 45%).

Example 1112- (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 ] amino]Indol-1 (2H) -ones 111

Stirring at 30 ℃ for 111H (750mg, 1.09mmol) and LiOH hydrate (2.3g, 55mmol) in iPrOH (10mL), THF (10mL) and H₂Mixture in O (10mL) for 1 h. The mixture was evaporated in vacuo and the residue was extracted with DCM (3 × 30 mL). The combined extracts were concentrated under reduced pressure. And the residue was purified by silica gel column (DCM: MeOH ═ 50:1) to give 111(700mg, 93%) as a yellow solid. MS: [ M + H ]]⁺644。¹H NMR(500MHz，MeOD)8.54(d，J＝2.0Hz，1H)，7.93(d，J＝1.5Hz，1H)，7.41(m，1H)，7.33(d，J＝2.0Hz，1H)，7.21(d，J＝9.0Hz，2H)，7.02(d，J＝9.0Hz，1H)，6.72(s，1H)，4.67-4.46(m，4H)，4.19(s，3H)，4.02(m，1H)，3.70(s，3H)，3.51(d，J＝11.5Hz，1H)，3.42(d，J＝11.Hz，1H)，3.36(s，1H)，2.96(d，J＝11.5Hz，1H)，2.85(m，1H)，2.73-2.50(m，7H)，2.48(s，3H)，1.88(m，2H)，1.78(m，2H)

Example 112aN, N-dibromobenzenesulfonamide 112a

Benzenesulfonamide (115g, 731.4mmol), KOH (82.8g, 1.48mol), and water (500mL) were placed in a 1000-mL three-necked flask (FIG. 12). Bromine (230g,1.48mol) was then added dropwise with vigorous stirring. The resulting precipitate was filtered, washed with water and filtered to give 112a (207g, 90%) as a yellow powder

Example 112b(S) -ethyl 2-bromo-3- (N- ((R) -2-bromo-3-ethoxy-3-oxopropyl) phenyl-sulfonylamino) propionate 112b

Add Ethyl acrylate (331.2g, 3.29mol) to a solution of 112a (207g, 658.26mmol) in DCM (500 mL). The mixture was stirred to reflux and the mercury lamp was turned on to ensure that the reaction proceeded under a mercury arc for 4 h. The reaction mixture was then purified by silica gel column (elution with PE: EA ═ 15:1 to 10: 1) to give 112b (20g, 6%) as a white solid. MS: [ M + H ] +: 538

Example 112c(2R,6S) -1-benzyl-4- (phenylsulfonyl) piperazine-2, 6-dicarboxylic acid diethyl ester 112c

Add BnNH to a solution of 112b (20g, 38.8mmol) in toluene (100mL)₂(12.48g, 116.5 mmol). The reaction mixture was stirred at 90 ℃ for 3 h. The mixture was then purified by silica gel column (elution with PE: EA ═ 50:1 to 10: 1) to give 112c (10.7g, 60%) MS as white crystals: [ M + H ]]+：461

Example 112d(1-benzyl-4- (phenylsulfonyl) piperazine-2, 6-diyl) dimethanol 112d

Cooling and stirring LiAIH₄1M solution in THF (70 ml; 70mmol) was added dropwise to a solution of 112c (10.7 g; 23.2mmol) in THF (275 ml). The reaction mixture was refluxed for 20 minutes and saturated Na was poured in₂CO₃To the solution and extracted three times with TBME. Through Na₂S0₄The combined organic phases were dried and evaporated to dryness to give a colourless solid, which was washed with TBME to give 112d as white crystals (7g, 80%). MS: [ M + H ]]+：377

Example 112e9-benzyl-3- (phenylsulfonyl) -7-oxa-3, 9-diaza-bicyclo [3.3.1]Nonane 112e

SOCl was added at room temperature with stirring₂A solution (1.34 ml; 18.5mmol) in toluene (14ml) was added quickly dropwise to a solution of 112e (7.0g, 18.57mmol) in DMF (276 ml). The reaction mixture was heated under reflux in an oil bath (170 ℃ C.) for 5 h. The reaction mixture was evaporated and taken up in saturated Na₂CO₃The solution was extracted three times with EtOAc. Through Na₂SO₄The combined organic phases were dried, filtered and evaporated to dryness and purified by silica gel column (elution with PE: EA ═ 10:1 to 5: 1) to give 112e (3.0g, 45%) MS as colorless crystals: [ M + H ]]+：413

Example 112f3- (phenylsulfonyl) -7-oxa-3, 9-diaza-bicyclo [3.3.1]Nonane 112f

Add a solution of Pd/C (0.5g) in MeOH (30mL) to a solution of 112e (3.0g, 8.37mmol) in EtOH (100 mL). The reaction mixture was hydrogenated under atmospheric hydrogen at 50 ℃ overnight. The catalyst was then filtered off, washed with ethanol, and the solvent was evaporated to give 112f (2.0g, 90%) MS as a colorless solid: [ M + H ] +: 268

Example 112g9-methyl-3- (phenylsulfonyl) -7-oxa-3, 9-diaza-bicyclo [3.3.1]Nonane 112g

To a solution of 112f (2.0g, 7.5mmol) in MeCN (60ml) was added HCHO (1.4ml, 16mmol) and 5 drops of AcOH at RT. Then adding NaCNBH₃(1g, 16mmol) and the mixture was stirred for 2 h. It was poured into water and extracted with EA (100 × 3). Through Na₂SO₄The organic layer was dried, filtered off, evaporated and purified by silica gel column (elution with PE: EA ═ 5:1 to 1:1) to give 112g (1.5g, 72%) MS as a colourless oil: [ M + H ]]+：283

Example 112h9-methyl-7-oxa-3, 9-diaza-bicyclo [3.3.1]Nonane hydrochloride 112h

To a solution of 112g (1.5g, 5.3mmol) in a mixture of toluene (15ml) and THF (15ml) was added LiAlH₄(0.42g, 10.8 mmol). The reaction mixture was heated at 110 ℃ overnight. It was then poured into HCl (2 mol/l). The organic layer was partitioned, the aqueous layer evaporated to dryness and dissolved in methanol. The resulting suspension was filtered and the filtrate was evaporated to give 1 as a colorless oil12h(800mg，43％)。MS：[M+H]+：143

Example 112i9-methyl-3- (6-nitropyridin-3-yl) -7-oxa-3, 9-diaza-bicyclo [3.3.1]Nonane 112i

Add Cs to a solution of 112h (800mg, 5.6mmol) and 5-bromo-2-nitropyridine (1.37g, 6.86mmol) in DMSO (50mL)₂CO₃(5g) And t-BuNH₄I (catalyst). The reaction mixture was stirred at 120 ℃ overnight. Then cooled to room temperature and extracted with EtOAc (300 ml). The organic layer was washed with water (3X100ml) and brine (150ml) and washed with Na₂SO4 was dried, filtered and concentrated to give the crude product, which was purified by silica gel column (eluting with DCM: MeOH ═ 100:1 to 50:1) to give 112i (850mg, 72%) MS as a yellow solid: [ M + H ]]+：265

Example 112j5- (9-methyl-7-oxa-3, 9-diaza-bicyclo [3.3.1]Non-3-yl) pyridin-2-amine 112j

To a solution of 112i (800mg, 3.0mmol) in THF (80ml) was added a solution of Pd/C (50mg) in MeOH (20ml) at room temperature. The reaction mixture was hydrogenated at atmospheric hydrogen pressure and stirred at room temperature overnight. The mixture was filtered and the filtrate was evaporated to give 112j (680mg, 90%) as a colorless oil. MS: [ M + H ] +: 235

Example 112k5-bromo-1-methyl-3- (5- (9-methyl-7-oxa-3, 9-diaza-bicyclo [ 3.3.1)]-nonan-3-yl) pyridin-2-ylamino) pyridin-2 (1H) -one 112k

Add Xantphos (58mg, 0.1mmol), Cs to a solution of 112j (500mg, 2.1mmol) and 3, 5-dibromo-1-methylpyridin-2 (1H) -one (852mg, 3.2mmol) in dioxane (100ml)₂CO₃(2.1g, 6.4mmol) and Pd₂(dba)₃(110mg, 0.1 mmol). The reaction mixture was stirred at 105 ℃ overnight. The mixture was cooled to RT, filtered, concentrated, and the crude product was purified by silica gel column (eluting with MeOH: DCM ═ 0 to 1: 5) to give 112k (500mg, 46.4%). MS: [ M + H ]]+：421

Example 112lAcetic acid 4-fluoro-2- (1-methyl-5- (5- (9-methyl-7-oxa-3, 9-diaza-bicyclo- [ 3.3.1)]Nonan-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 ] amino]Indol-2 (1H) -yl) benzyl ester 112l

Heating 112l (500mg, 1.2mmol) and acetic acid 4-fluoro-2- (1-oxo-3, 4,6,7,8, 9-hexahydropyrazino [1,2-a ] at 110 deg.C]Indol-2 (1H) -yl) -6- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzyl ester (636mg, 1.32mmol), PdCl₂(dppf)(95mg，0.12mmol)、K₃PO₄(500mg, 2.4mmol) and NaOAc (195mg, 2.4mmol) in MeCN (8mL) and H₂Mixture in O (0.8mL) for 3 h. The solvent was evaporated in vacuo. The residue was purified by column on silica gel (elution with DCM: MeOH ═ 50:1) to give 112l (340mg, yield 41%). MS: [ M + H ]]+：696

Example 1122- (5-fluoro-2- (hydroxymethyl) -3- (1-methyl-5- (5- (9-methyl-7-oxa-3, 9-diaza-bicyclo [ 3.3.1)]Non-3-yl) pyridin-2-ylamino) -6-oxo-1, 6-dihydropyridin-3-yl) benzeneYl) -3,4,6,7,8, 9-hexahydropyrazino [1,2-a ] ester]Indol-1 (2H) -ones 112

A mixture of 112l (340mg, 0.489mmol) and LiOH hydrate (445mg, 24.45mmol) in i-PrOH (4mL), THF (4mL) and H2O (4mL) was stirred at 30 ℃ for 1H. The mixture was evaporated in vacuo and the residue was purified by preparative HPLC to give 112 as a pale yellow solid (105mg, 32.85%). MS: [ M + H ]]⁺654。¹H NMR(500MHz，CDCl³)8.46(d，J＝2.5Hz，1H)，7.87(d，J＝3.0Hz，1H)，7.69(s，1H)，7.42(d，J＝2.0Hz，1H)，7.23-7.14(m，2H)，6.94(d，J＝2.5Hz，1H)，6.84(m，2H)，4.52(d，J＝11.5Hz，1H)，4.31(m，2H)，4.12(m，5H)，3.89(m，3H)，3.69(s，3H)，3.43(m，4H)，2.84(s，2H)，5.62-2.54(m，7H)，1.88(m，2H)，1.78(m，2H)

Example 113a(3S) -3-methyl-4- (6-nitropyridin-3-yl) piperazine-1-carboxylic acid tert-butyl ester 113a

The procedure described in example 104 and starting from tert-butyl (3S) -3-methylpiperazine-1-carboxylate (10.0g, 50mmol) and 5-bromo-2-nitropyridine (10.5g, 50mmol) gave 113a (8.05g, 50%) as a yellow solid. See fig. 13. MS-ESI: [ M + H ]]⁺323

Example 113b(3S) -4- (6-Aminopyridin-3-yl) -3-methylpiperazine-1-carboxylic acid tert-butyl ester 113b

The procedure described in example 104 and starting from 113a (5.8g, 18mmol) gave 113b as a brown solid (4.9g, 93%). See fig. 13. MS-ESI: [ M + H ]]⁺293

Example 113c(3S) -4- (6- (5-bromo-1-methyl-2-oxo-1, 2-dihydropyridin-3-ylamino) pyridin-3-yl) -3-methylPiperazine-1-carboxylic acid tert-butyl ester 113c

The procedure described in example 104 and starting from 113b (4.0g, 13.7mmol) and 3, 5-dibromo-1-methylpyridin-2 (1H) -one (5.5g, 20.6mmol) gave 113c as a yellow solid (5.4g, 83%). See fig. 13. MS-ESI: [ M + H ]]⁺478

Example 113d(3S) -5-bromo-1-methyl-3- (5- (2-methylpiperazin-1-yl) pyridin-2-ylamino) pyridin-2 (1H) -one 113d

The procedure described in example 104 and starting with 113c (3.1g, 6.5mmol) gave 113d (2.3g, 94%) as a yellow solid. See fig. 13. MS-ESI: [ M + H ]]⁺378。

Example 113e(S) -5-bromo-1-methyl-3- (5- (2-methyl-4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-ylamino) pyridin-2 (1H) -one 113e

113d (40.0g, 106mmol), oxetan-3-one (11.4g, 159mmol), NaBH were stirred at 50 deg.C₃A mixture of CN (10.0g, 159mmol) and zinc chloride (21.3g, 159mmol) in methanol (700mL) was used for 5 hours. The mixture was added to water (100mL) and concentrated under reduced pressure. The residue was extracted with dichloromethane (3X200 mL). The combined organic layers were concentrated under reduced pressure and the residue was purified by silica gel column chromatography (eluting with 40:1 dichloromethane/methanol) to give 113e (35g, 73%). See fig. 13. MS: [ M + H ]]⁺434。

Example 113f(3S) -1-methyl-3- (5- (2-methyl-4- (oxetan-3-yl) piperazin-1-yl) -pyridin-2-ylamino) -5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyridin-2 (1H) -one 113f

Equipped with magnetic stirringA100-mL, single-necked, round-bottomed flask with vessel and reflux condenser was charged with 113e (1.0g, 1.0 eq., 2.3mmol), Pin₂B₂(1.46g, 2.50 equiv., 5.75mmol), Pd₂(dba)₃(105mg, 0.05 eq, 0.125mmol), X-Phos (93mg, 0.1 eq, 0.23mmol), potassium acetate (676mg, 3.0 eq, 6.9mmol) and dioxane (50 mL). After three cycles of vacuum/argon flushing, the mixture was heated at 90 ℃ for 4 h. Then cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure and the resulting residue was washed with 3:1 petroleum ether/ethyl acetate (80mL) to give 113f (1.0g, 90%) as a yellow solid. See fig. 13. MS: [ M + H ]]⁺482。

Example 113g 4- [5- (ethoxycarbonyl) -1H-pyrrol-3-yl]-113 g of 4-oxobutanoic acid

To a 3000-mL 4-necked round bottom flask was added a solution of oxacyclopentane-2, 5-dione (100g, 999.27mmol, 2.00 equiv.) in1, 2-dichloroethane (690mL) and AlCl₃(400.5g, 3.00mol, 6.00 eq.) A solution of ethyl 1H-pyrrole-2-carboxylate (69g, 495.86mmol, 1.00 eq.) in1, 2-dichloroethane (660mL) was then added dropwise at room temperature with stirring over 20 minutes (FIG. 14). The resulting solution was stirred at room temperature for 3h and quenched by the addition of 3kg of water/ice. The solid was collected by filtration, washed with 1x1000mL water and dried in a vacuum oven to give 113g of 105g (89%) as a white solid.

Examples113h4- [5- (ethoxycarbonyl) -1H-pyrrol-3-yl]Butyric acid 113h

To a 2000-mL 4-necked round bottom flask was added 113g (105g, 438.92mmol, 1.00 eq.) of a solution in CF₃A solution in COOH (1000mL) was then added triethylsilane (204g, 1.75mol, 4.00 eq.) dropwise over 30 min at room temperature with stirring (FIG. 14). The resulting solution was stirred at room temperature for 8h, concentrated in vacuo and diluted with 500mL of water and 500mL of ethyl acetate. The pH of the solution was adjusted to 7 with saturated aqueous sodium bicarbonate. The resulting solution was extracted with 3x500mL ethyl acetate. Drying the combined extract with anhydrous sodium sulfateThe organic layer was concentrated in vacuo to give 30g (30%) as a light brown solid for 113 h.

Example 113i7-oxo-4, 5,6, 7-tetrahydro-1H-indole-2-carboxylic acid ethyl ester 113i

Into a 1000-mL round bottom flask was added 113h (30g, 133.19mmol, 1.00 eq.) in CF₃COOH (500mL) and trifluoroacetyl 2,2, 2-trifluoroacetate (42g, 199.97mmol, 1.50 equivalents). See fig. 14. The resulting solution was stirred at room temperature for 60 minutes, concentrated in vacuo, diluted with 500mL of water and 500mL of EA, and extracted with 3 × 500mL ethyl acetate. The combined organic layers were washed with 1x500mL saturated aqueous potassium carbonate and 1x500mL brine, dried over anhydrous sodium sulfate and concentrated in vacuo to give 24g (87%) of 113i as a light brown solid.

Example 113j6, 6-difluoro-7-oxo-4, 5,6, 7-tetrahydro-1H-indole-2-carboxylic acid ethyl ester 113j

To a 2000-mL4 necked round bottom flask purged and maintained with a nitrogen inert atmosphere was added a solution of 113i (24g, 115.82mmol, 1.00 equiv.) in tetrahydrofuran (200mL), followed by dropwise addition of LiHMDS (406mL, 3.50 equiv.) over 30 minutes at-78 deg.C with stirring (FIG. 14). A solution of N- (phenylsulfonyl) -S-phenyl-fluoroalkanesulfonamide (109.5g, 347.24mmol, 3.00 equiv.) in tetrahydrofuran (500mL) was added dropwise thereto over 30 minutes with stirring and at-78 ℃. The resulting solution was stirred at room temperature overnight by the addition of 200mL of saturated NH₄Aqueous Cl was quenched and extracted with 3x200mL ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by column on silica gel (elution with petroleum ether/ethyl acetate (30: 1)) to give 9.5g (34%) of 113j as a yellow solid.

Example 113k6, 6-difluoro-7-hydroxy-4, 5,6, 7-tetrahydro-1H-indole-2-carboxylic acid ethyl ester 113k

To a 250-mL 3-necked round bottom flask was added113j (18g, 74.01mmol, 1.00 equiv.) in ethanol (100mL), followed by the addition of NaBH in several portions at 0 deg.C₄(2.8g, 74.02mmol, 1.00 equiv) (FIG. 14). The resulting solution was stirred at 5 ℃ for 10 minutes by adding 50mL of saturated NH₄Aqueous Cl was quenched, concentrated in vacuo and extracted with 3x50mL ethyl acetate. The combined organic layers were washed with 1x100mL water and 1x100mL brine, dried over anhydrous sodium sulfate and concentrated in vacuo to give 18g (99%) of 113k as a white solid.

Example 113l6, 6-difluoro-4, 5,6, 7-tetrahydro-1H-indole-2-carboxylic acid ethyl ester 113l

To a 500-mL 3-necked round bottom flask was added 113k (18g, 73.40mmol, 1.00 equiv.) in dichloromethane (200mL) and CF₃A solution in COOH (41.9g, 367.48mmol, 5.00 equiv.) was added dropwise with triethylsilane (25.6g, 220.16mmol, 3.00 equiv.) over 20 min at 0 ℃ with stirring (FIG. 14). The resulting solution was stirred at room temperature for 4h, adjusted to pH 7 with saturated aqueous sodium bicarbonate and extracted with 1x200mL dichloromethane. The combined organic layers were washed with 1x100mL water and 1x100mL brine, dried over anhydrous sodium sulfate and concentrated in vacuo. Purification of the crude product by flash preparative HPLC afforded 113l of 10g (59%) as a white solid.

Example 113m1- (cyanomethyl) -6, 6-difluoro-4, 5,6, 7-tetrahydro-1H-indole-2-carboxylic acid ethyl ester 113m

To a 250-mL 3-necked round bottom flask was added 113l (5.0g, 21.81mmol, 1.00 equiv.) of a solution in N, N-dimethylformamide (50mL), followed by the addition of sodium hydride (1.3g, 54.17mmol, 1.40 equiv., 60%) in several portions over 10 minutes at 0 deg.C (FIG. 14). 2-bromoacetonitrile (3.7g, 30.85mmol, 1.40 equiv.) was added dropwise thereto over 10 min while stirring at 20 ℃. The resulting solution was stirred at room temperature for 7h, diluted with 100mL of water and extracted with 3 × 50mL ethyl acetate. The combined organic layers were washed with 1x100mL water and 1x100mL brine, dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by column on silica gel (elution with ethyl acetate/petroleum ether (1: 20)) to give 3.3g (56%) of 113m as a pale yellow oil.

Example 113n1- (2-aminoethyl) -6, 6-difluoro-4, 5,6, 7-tetrahydro-1H-indole-2-carboxylic acid ethyl ester 113n

A100-mL round bottom flask was charged with a solution of 113m (3.3g, 12.30mmol, 1.00 eq.) in ethanol (30mL) and NiCl₂.6H₂O (3.2g, 13.45mmol, 1.10 equiv.), followed by the addition of NaBH in several portions at 0 deg.C₄(1.4g, 37.01mmol, 3.00 equiv) (FIG. 14). The resulting solution was stirred at room temperature for 24 h. The solid was filtered off and the filtrate was concentrated in vacuo. The resulting solution was diluted with 30mL of ethyl acetate and washed with 1x30mL hydrochloric acid (2N). The solution was adjusted to pH 9 with saturated aqueous sodium bicarbonate and extracted with 2x30mL ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate and concentrated in vacuo to give 0.7g (21%) of 113n as a pale yellow oil.

Example 113o7, 7-difluoro-1H, 2H,3H,4H,6H,7H,8H, 9H-pyrazino [1,2-a ]]Indol-1-one 113o

To a 250-mL round bottom flask was added a solution of 113n (10g, 36.73mmol, 1.00 equiv.) in toluene (100mL) and acetic acid (1.1g, 18.32mmol, 0.50 equiv.). See fig. 14. The resulting solution was heated to reflux for 2h, cooled and concentrated in vacuo. The residue was triturated in 100mL dry ether. The crude product was purified by recrystallization from ethanol to yield 4.43g (53%) of 113o as a white solid. MS-ESI: [ M + H ]]⁺227。¹H NMR(300MHz，DMSO)2.10-2.24(2H，m)，2.59-2.64(2H，m)，3.17-3.27(2H，m)，3.43-3.48(2H，m)，3.88-3.92(2H，m)，6.44(1H，s)，7.56(1H，s)。

Example 113pAcetic acid 2-bromo-6- (7, 7-difluoro-1-oxo-3, 4,6,7,8, 9-hexahydropyrazino [1,2-a ]]Indol-2 (1H) -yl) -4-fluorobenzyl ester 113p

To 100 equipped with a reflux condenserA-mL round-bottom flask was charged with 1, 4-dioxane (40mL), 113o (890mg, 3.94mmol), 2, 6-dibromo-4-fluorobenzyl acetate 101c (3847mg, 11.8mmol), Pd₂(dba)₃(180mg, 0.197mmol), XantPhos (227mg, 0.394mmol) and cesium carbonate (2.57g, 7.88 mmol). After three cycles of vacuum/argon flushing, the mixture was heated at 100 ℃ for 16 h. Then filtered and the filtrate evaporated under reduced pressure. The residue was purified by silica gel column chromatography (eluting with 3:1 petroleum ether/ethyl acetate) to give 113p (1100mg, 62%) as a white solid. MS-ESI: [ M + H ]]⁺471.1。

Example 113q(S) -acetic acid 2- (7, 7-difluoro-1-oxo-3, 4,6,7,8, 9-hexahydropyrazino [1,2-a ] ester]Indol-2 (1H) -yl) -4-fluoro-6- (1-methyl-5- (5- (2-methyl-4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-ylamino) -6-oxo-1, 6-dihydropyridin-3-yl) benzyl ester 113q

A sealed tube was charged with 113p (47mg, 0.10mmol), 113f (47mg, 0.10mmol), Pd (dppf) Cl₂(4mg, 0.005mmol), sodium acetate (16mg, 0.2mmol), K₃PO₄(43mg, 0.2mmol), acetonitrile (2mL), and water (0.2 mL). After three cycles of vacuum/argon flushing, the mixture was heated at 100 ℃ for 1 h. Then filtered and the filtrate evaporated under reduced pressure. The residue was purified by silica gel column chromatography (eluting with 25:1 dichloromethane/methanol) to give 113q (37mg, 50%) as a brown solid. MS: [ M + H ]]⁺746.3

Example 113(S) -7, 7-difluoro-2- (5-fluoro-2- (hydroxymethyl) -3- (1-methyl-5- (5- (2-methyl-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 ] o]Indol-1 (2H) -ones 113

A mixture of 113q (37mg, 0.05mmol) and lithium hydroxide (12mg, 0.5mmol) in isopropanol/THF (1:1, 4mL) and water (1mL) was stirred at 30 ℃ for 1 h. The mixture was evaporated under reduced pressure. Water (10mL) was added to the residue and the resulting mixture was extracted with ethyl acetate (2X10 mL). Vacuum concentrating warpThe combined organic layers and the residue was purified by reverse phase preparative HPLC to give 113 as a white solid (20mg, 57%). MS: [ M + H ]]⁺704.3。¹H NMR(500MHz，CDCl₃)8.57-8.54(m，1H)，7.95(d，J＝3.0Hz，1H)，7.82(d，J＝3.5Hz，1H)，7.45-7.41(m，1H)，7.32(d，J＝8.5Hz，1H)，7.19-7.16(m，1H)，6.99-6.95(m,1H)，6.88(d，J＝6.5Hz，1H)，6.82(d，J＝8.5Hz，1H)，5.97(d，J＝11.5Hz，1H)，4.71-4.64(m，4H)，4.54(d，J＝12.0Hz，1H)，4.35-4.18(m，5H)，3.99-3.95(m，1H)，3.71(s，3H)，3.53(t，J＝6.0Hz，1H)，3.47-3.45(m，1H)，3.17(t，J＝12.0Hz，1H)，3.08(t，J＝5.0Hz，2H)，2.90(t，J＝8.5Hz，1H)，2.79(t，J＝6.0Hz,1H)，2.66-2.63(m,1H)，2.56(d，J＝11.0Hz，1H)，2.49-2.47(m，2H)，2.30-2.20(m，2H)，0.99(d，J＝6.0Hz，3H)。

Example 114a(S) -3-Ethyl-4- (6-nitropyridin-3-yl) piperazine-1-carboxylic acid tert-butyl ester

A250-mL single-neck round bottom flask equipped with a magnetic stirrer and a reflux condenser was charged with 1, 4-dioxane (50mL), 5-bromo-2-nitropyridine (2.02g, 10mmol), (S) -3-ethylpiperazine-1-carboxylic acid tert-butyl ester (2.14g, 10.0mmol), Pd₂(dba)₃(458mg, 0.50mmol), XantPhos (576mg, 1.0mmol) and cesium carbonate (6.52g, 20 mmol). After three cycles of vacuum/argon flushing, the mixture was heated at 100 ℃ overnight. The reaction mixture was then cooled to room temperature. Then filtered and the filtrate evaporated under reduced pressure. The residue was purified by silica gel column chromatography (eluting with 3:1 petroleum ether/ethyl acetate) to give 114a (700mg, 22%) as a yellow solid. MS: [ M + H ]]⁺336

Example 114b(S) -4- (6-Aminopyridin-3-yl) -3-ethylpiperazine-1-carboxylic acid tert-butyl esterEster 114b

A100-mL single-neck round bottom flask was purged with nitrogen and charged with 114a (0.7g, 2.08mmol), 10% palladium on charcoal (50% wet, 208mg) and methanol (40 mL). The mixture was evacuated, charged with hydrogen and stirred at room temperature for 6 h. The hydrogen was then evacuated and the flask was charged with nitrogen. The catalyst was removed by filtration through a pad of celite and the filtrate was concentrated under reduced pressure to give 114b (568mg, 89%). MS: [ M + H ]]⁺306

Example 114c(S) -4- (6- (5-bromo-1-methyl-2-oxo-1, 2-dihydropyridin-3-ylamino) pyridin-3-yl) -3-ethylpiperazine-1-carboxylic acid tert-butyl ester 114c

A100-mL, single-necked, round-bottomed flask equipped with a magnetic stirrer and a reflux condenser was charged with 1, 4-dioxane (50mL), 114b (568mg, 1.86mmol), 3, 5-dibromo-1-methylpyridin-2 (1H) -one (498mg, 1.86mmol), Pd₂(dba)₃(85mg, 0.093mmol), XantPhos (107mg, 0.186mmol) and cesium carbonate (1.198g, 3.72 mmol). After three cycles of vacuum/argon flushing, the mixture was heated at 100 ℃ for 6 h. Then filtered and the filtrate evaporated under reduced pressure. The residue was purified by silica gel column chromatography (eluting with 100:1 dichloromethane/methanol) to give 114c as a yellow solid (502mg, 55%). MS: [ M + H ]]⁺492。

Example 114d(S) -5-bromo-3- (5- (2-ethylpiperazin-1-yl) pyridin-2-ylamino) -1-methylpyridin-2 (1H) -one 114d

A mixture of 114c (502mg, 1.02mmol), dichloromethane (2mL) and 4.0M HCl/dioxane (4mL) was stirred at room temperature for 5 h. Concentration under reduced pressure then afforded crude 114d (263mg, 66%) as a yellow solid, which was used in the next step without purification. MS: [ M + H ]]⁺392。

Example 114e(S) -5-bromo-3- (5- (2-ethyl-4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-ylamino) -1-methylpyridin-2 (1H) -one 114e

114d (263mg, 0.67mmol), oxetan-3-one (96mg, 1.34mmol), NaBH were stirred at 50 deg.C₃A mixture of CN (104mg, 1.68mmol) and zinc chloride (227mg, 1.68mmol) in methanol (10mL) was used for 5 h. Water (10mL) was then added to the reaction mixture. The resulting mixture was concentrated under reduced pressure. The residue was extracted three times with dichloromethane. The combined organic layers were concentrated under reduced pressure and the residue was purified by silica gel column chromatography (eluting with 50:1 dichloromethane/methanol) to give 114e (203mg, 68%). MS: [ M + H ]]⁺448。

Example 114f(S) -3- (5- (2-ethyl-4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-ylamino) -1-methyl-5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyridin-2 (1H) -one 114f

A100-mL, single-necked, round-bottomed flask equipped with a magnetic stirrer and a reflux condenser was charged with 114e (3219mg, 7.20mmol), Pin₂B₂(9072mg，36.0mmol)、Pd₂(dba)₃(329mg, 0.36mmol), X-phos (302mg, 0.72mmol), potassium acetate (2117mg, 21.6mmol) and dioxane (50 mL). After three cycles of vacuum/argon flushing, the mixture was heated at 60 ℃ for 16 h. Then cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure and the resulting residue was washed with 8:1 petroleum ether/ethyl acetate (80mL) to give 114f (3.0g, 84%) as a yellow solid. MS: [ M + H ]]⁺496.4。

Example 114gAcetic acid (2- {4, 4-dimethyl-9-oxo-1, 10-diazacyclo [6.4.0.0 ]^2,6]Dodeca-2 (6), 7-dien-10-yl } -6- [5- ({5- [ (2S) -2-ethyl-4- (oxetan-3-yl) piperazin-1-yl]Pyridin-2-yl } amino) -1-methyl-6-oxo-1, 6-dihydropyridin-3-yl]114g of (e) -4-fluorophenyl) methyl ester

A25-mL, single-neck, round-bottom flask equipped with a magnetic stirrer and reflux condenser was charged with 114f (180mg, 0.40mmol), acetic acid (2- {4, 4-dimethyl-9-oxo-1, 10-diazacyclo [6.4.0.0 ]^2,6]Dodeca-2 (6), 7-dien-10-yl } -4-fluoro-6- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) methyl ester 103g (198mg, 0.40mmol), Pd (dppf) Cl₂(29mg，0.04mmol)、K₃PO₄(170mg, 0.8mmol), sodium acetate (66mg, 0.8mmol), acetonitrile (5mL), and water (1.0 mL). After three cycles of vacuum/argon purge, the mixture was heated at reflux for 2 h. Then cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure and the resulting residue was purified by silica gel column chromatography (eluting with 30:1 dichloromethane/methanol) to give 114g (115mg, 39%) as a yellow solid. MS: [ M + H ]]⁺738.4

Example 1142- (3- {5- [5- ((S) -2-ethyl-4-oxetan-3-yl-piperazin-1-yl) -pyridin-2-ylamino]-1-methyl-6-oxo-1, 6-dihydro-pyridin-3-yl } -5-fluoro-2-hydroxymethyl-phenyl) -7, 7-dimethyl-3, 4,7, 8-tetrahydro-2H, 6H-cyclopenta [4,5]Pyrrolo [1,2-a]Pyrazin-1-ones 114

A mixture of 114g (115mg, 0.16mmol) and lithium hydroxide (38mg, 1.6mmol) in isopropanol/THF (1:1, 4mL) and water (1mL) was stirred at 30 ℃ for 1 h. The mixture was evaporated under reduced pressure and the residue was extracted with ethyl acetate (2X10 mL). The combined ethyl acetate extracts were concentrated under reduced pressure and the residue was purified by reverse phase preparative HPLC to give 114 as a white solid (45mg, 40%). MS: [ M + H ]]⁺696.4。¹H NMR(500MHz，CDCl₃)8.55-8.54(m，1H)，7.91(s，1H)，7.80(d，J＝1.5Hz，1H)，7.47(s，1H)，7.26(d，J＝3.0Hz，1H)，7.17(d，J＝9.0Hz，1H)，6.95(dd，J＝2.5，8.5Hz，1H)，6.83-6.81(m，2H)，4.71(t，J＝6.5Hz，2H)，4.67(t，J＝6.0Hz，1H)，4.62(t，J＝6.0Hz，1H)，4.57-4.55(m，1H)，4.41-4.38(m，1H)，4.32-4.30(m，1H)，4.24-4.14(m，3H)，3.92-3.87(m，1H)，3.71(s，3H)，3.56-3.50(m，1H)，3.33-3.29(m，1H)，3.13-3.11(m，2H)，2.58-2.56(m，3H)，2.52(s，2H)，2.44-2.43(m，2H)，2.38-2.32(m，1H)，1.66-1.64(m，1H)，1.42-1.37(m，1H)，1.28(s，6H)，0.82(t，J＝7.0Hz，3H)。

Example 115a(R) -3-methyl-4- (6-nitropyridin-3-yl) piperazine-1-carboxylic acid tert-butyl ester 115a

To a 250-mL single neck round bottom flask equipped with a magnetic stirrer and reflux condenser was charged 1, 4-dioxane (60mL), 5-bromo-2-nitropyridine (2.0g, 10.0mmol), (R) -3-methylpiperazine-1-carboxylic acid tert-butyl ester (2.0g, 10.0mmol), and cesium carbonate (6.5g, 20 mmol). After bubbling nitrogen through the resulting mixture for 10 minutes, tris (dibenzylideneacetone) dipalladium (0) (915mg, 1.0mmol) and XantPhos (579mg, 1.0mmol) were added. The system was flushed through three cycles with vacuum/argon and heated at 100 ℃ for 15 h. The reaction mixture was then cooled to room temperature and filtered. The filtrate was partitioned between ethyl acetate (100mL) and water (100 mL). The aqueous layer was separated and extracted with ethyl acetate (3X50 mL). The combined organic layers were washed with brine (100mL) and dried over sodium sulfate. The drying agent was removed by filtration and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluting with 30:1 dichloromethane/methanol) to give 115a as a yellow solid (1.6g, 44%). MS-ESI: [ M + H ]]⁺323。¹H NMR(500MHz，DMSO-d₆)8.21(d，J＝3.5Hz，1H)，8.18(d，J＝9.0Hz，1H)，7.45-7.43(m，1H)，4.34-4.33(m，1H)，3.92-3.99(m，1H)，3.80(d，J＝12.5Hz，2H)，3.06-3.23(m，3H)，1.43(s，9H)，1.09(d，J＝6.5Hz，3H)。

Example 115b(R) -4- (6-Aminopyridin-3-yl) -3-methylpiperazine-1-carboxylic acid tert-butyl ester 115b

A250-mL flask was purged with nitrogen and charged with 115a (1.5g, 4.6mmol), 10% palladium on charcoal (50% wet)200mg) and methanol (70 mL). Vacuum was applied, hydrogen was charged and stirred at room temperature for 10 h. The hydrogen was then evacuated and the flask was charged with nitrogen. The catalyst was removed by filtration through a pad of celite and the filtrate was concentrated under reduced pressure to give 115b as a brown solid (1.1g, 81%). MS-ESI: [ M + H ]]⁺293

Example 115c(R) -4- (6- (5-bromo-1-methyl-2-oxo-1, 2-dihydropyridin-3-ylamino) pyridin-3-yl) -3-methylpiperazine-1-carboxylic acid tert-butyl ester 115c

A100-mL single-neck round bottom flask equipped with a magnetic stirrer and a reflux condenser was charged with 1, 4-dioxane (40mL), 115b (1.0g, 3.4mmol), 3, 5-dibromo-1-methylpyridin-2 (1H) -one (2.7g, 10.2mmol), and cesium carbonate (2.2g, 6.8 mmol). After bubbling nitrogen through the resulting mixture for 10 min, XantPhos (198mg, 0.34mmol) and tris (dibenzylideneacetone) dipalladium (0) (313mg, 0.34mmol) were added. The reaction mixture was flushed with vacuum/argon for three cycles and heated at 100 ℃ for 5 h. The reaction mixture was then cooled to room temperature and filtered. The filtrate was partitioned between ethyl acetate (50mL) and water (50 mL). The aqueous layer was separated and extracted with ethyl acetate (3X30 mL). The combined organic layers were washed with brine (50mL) and dried over sodium sulfate. The drying agent was removed by filtration and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluting with 30:1 dichloromethane/methanol) to give 115c as a yellow solid (1.1g, 63%). MS-ESI: [ M + H ]]⁺478。

Example 115d(R) -5-bromo-1-methyl-3- (5- (2-methylpiperazin-1-yl) pyridin-2-ylamino) pyridin-2 (1H) -one 115d

To a mixture of 115c (600mg, 1.26mmol) in methanol (20mL) was added HCl/dioxane (4M, 4 mL). The reaction mixture was stirred at room temperature for 4 h. Then concentrated under reduced pressure. The residue was basified with 1M aqueous NaOH and extracted with dichloromethane (3X30 mL). The combined organic layers were washed with brine and concentrated under reduced pressure to give 115d (450mg, 95%) as a yellow solid. MS-ESI: [ M + H ]]⁺378。

Example 115e(R) -5-bromo-3- (5- (2, 4-dimethylpiperazin-1-yl) pyridin-2-ylamino) -1-methylpyridin-2 (1H) -one 115e

A mixture of 115d (500mg, 1.3mmol) and 30% formaldehyde (650mg, 6.5mmol) in methanol/acetic acid (30mL/3mL) was stirred at room temperature for 5 min, followed by the addition of NaBH₃CN (120mg, 1.9 mmol). The mixture was stirred at room temperature for 4 h. Water (20mL) was added and the resulting mixture was concentrated under reduced pressure. The residue was extracted with dichloromethane (3X30 mL). The combined organic layers were concentrated under reduced pressure and the residue was purified by silica gel column chromatography (eluting with 30:1 dichloromethane/methanol) to give 115e (473mg, 92%) as a yellow solid. MS-ESI: [ M + H ]]⁺392。

Example 115f(R) -5-bromo-1-methyl-3- (5- (2-methyl-4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-ylamino) pyridin-2 (1H) -one 115f

115e (40.0g, 106mmol), oxetan-3-one (11.4g, 159mmol), NaBH was stirred at 50 deg.C₃A mixture of CN (10.0g, 159mmol) and zinc chloride (21.3g, 159mmol) in methanol (700mL) was used for 5 hours. Water (50mL) was added to the mixture and concentrated under reduced pressure. The residue was extracted with dichloromethane (3X200mL) and the combined organic layers were concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (eluting with 40:1 dichloromethane/methanol) to give 115f (35g, 73%). MS: [ M + H ]]⁺434。

Example 115g115g of 2,2, 2-trichloro-1- (4,5,6, 7-tetrahydro-1H-indol-2-yl) ethanone

A100-mL, single-necked, round-bottomed flask equipped with a magnetic stirrer, condenser, and nitrogen inlet was purged with nitrogen and charged with 4,5,6, 7-tetrahydro-1H-indole (3.00g, 24.8mmol), trichloroacetyl chloride (13.5g, 74.4mmol), and 1, 2-dichloroethane (50 mL). The solution was stirred at 85 ℃ for 2 h. Then, the reaction mixture was concentrated under reduced pressure to obtain a black color115g of semisolid 100% yield (6.50 g):¹H NMR(500MHz，DMSO-d₆)11.94(s，1H)，7.05(s，1H)，2.62(t，2H，J＝6.0Hz)，2.47(t，2H，J＝6.0Hz)，1.80(m，2H)，1.65(m，2H)；MS(ESI+)m/z266.0(M+H)

example 115h4,5,6, 7-tetrahydro-1H-indole-2-carboxylic acid ethyl ester 115H

A100-mL, single-necked, round-bottomed flask equipped with a magnetic stirrer and a nitrogen inlet was purged with nitrogen and charged with 115g (6.50g, 24.8mmol), sodium ethoxide (17.0mg, 0.25mmol) and ethanol (40 mL). The solution was stirred at room temperature for 1 h. Then, the reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography to give 100% yield (4.80g) as a brown solid over 115 h: mp 70-72 ℃;¹H NMR(300MHz，CDCl₃)9.08(s，1H)，6.75(s，1H)，4.25(q，2H，J＝7.2Hz)，2.65(t，2H，J＝6.0Hz)，2.56(t，2H，J＝6.0Hz)，1.85(m，4H)，1.28(t，3H，J＝7.2Hz)；MS(ESI+)m/z194.1(M+H)

example 115i1- (cyanomethyl) -4,5,6, 7-tetrahydro-1H-indole-2-carboxylic acid ethyl ester 115i

A 125-mL single neck round bottom flask equipped with a magnetic stirrer and nitrogen inlet was purged with nitrogen and charged with 115h (5.76g, 29.8mmol) and DMF (50mL), the solution was cooled to 0 ℃ with an ice bath, NaH (60% dispersion in mineral oil, 1.43g, 35.8mmol) was added, the resulting mixture was stirred at room temperature for 1h then bromoacetonitrile (1.43g, 35.8mmol) was added, the mixture was stirred at room temperature for 14h then the reaction mixture was concentrated under reduced pressure and the residue partitioned between ethyl acetate (150mL) and water (450mL) the organic layer was separated and the aqueous layer was extracted with ethyl acetate (3 × 150mL) the combined organic layer was washed with brine, dried over sodium sulfate and concentrated under reduced pressure.¹H NMR(300MHz，CDCl₃)6.66(s，1H)，5.29(s，2H)，4.28(q，2H，J＝7.2Hz)，2.62(t，2H，J＝6.3Hz)，2.49(t，2H，J＝6.3Hz)，1.92(m，2H)，1.75(m，2H)，1.33(t，3H，J＝7.2Hz)；MS(ESI+)m/z233.1(M+H)

Example 115j1- (2-aminoethyl) -4,5,6, 7-tetrahydro-1H-indole-2-carboxylic acid ethyl ester 115j

Purging a 200-mL Parr reactor bottle with nitrogen and charging 10% palladium on charcoal (50% wet, 1.28g dry), 115i (3.00g, 12.9mmol), 12% hydrochloric acid (6.5mL, 25mmol), ethyl acetate (60mL), and ethanol (40mL), connecting the bottle to a Parr hydrogenator, evacuating, charging hydrogen to a pressure of 50psi and shaking for 6h, after which, evacuating hydrogen, and charging nitrogen to the bottle, adding diatomaceous earth 521(4.0g), and filtering the mixture through a pad of diatomaceous earth 521, washing the filter cake with ethanol (2 × 20mL), and concentrating the combined filtrate under reduced pressure to dryness, partitioning the residue between ethyl acetate (150mL) and 10% aqueous potassium carbonate (100mL), separating the organic layer, and extracting the aqueous layer with ethyl acetate (3 × 75mL), drying the combined organic layer over sodium sulfate, and concentrating under reduced pressure, triturating the residue with ethanol (5mL) to give a yield of 71% white solid (115.71% mp: 102 g);¹H NMR(500MHz，DMSO-d₆)6.61(s，1H)，6.22(br，2H)，4.15(m，4H)，2.77(m，2H)，2.59(t，2H，J＝6.5Hz)，2.42(t，2H，J＝6.5Hz)，1.70(m，2H)，1.62(m，2H)，1.23(t，3H，J＝7.0Hz)；MS(APCI+)m/z237.2(M+H)

example 115k3,4,6,7,8, 9-hexahydropyrazino [1,2-a ] or a pharmaceutically acceptable salt thereof]Indol-1 (2H) -one 115k

A100-mL, single-necked, round-bottomed flask equipped with a magnetic stirrer and a nitrogen inlet was purged with nitrogen and charged with ethyl 1- (2-aminoethyl) -4,5,6, 7-tetrahydro-1H-indole-2-carboxylate 115j (1.80g, 7.63mmol), sodium ethoxide (1.55g, 22.8mmol), and ethanol (50 mL). the mixture was stirred at 55 deg.C for 5HThe combined organic layers were dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography to give 115k in 42% yield (605mg) as a white solid: mp 207-209 ℃;¹H NMR(500MHz，DMSO-d₆)7.41(s，1H)，6.36(s，1H)，3.84(t，2H，J＝6.0Hz)，3.42(m，2H)，2.51(t，2H，J＝6.0Hz)，2.42(t，2H，J＝6.0Hz)，1.76(m，2H)，1.65(m，2H)；(APCI+)m/z191.3(M+H)

example 115lAcetic acid 2-bromo-4-fluoro-6- (1-oxo-3, 4,6,7,8, 9-hexahydropyrazino [1, 2-a)]Indol-2 (1H) -yl) benzyl ester 115l

A250-mL single-neck round-bottom flask equipped with a magnetic stirrer was charged with 115k (3.8g, 20mmol), 2, 6-dibromo-4-fluorobenzyl acetate 101c (20.0g, 61mmol), XantPhos (1.16g, 2.0mmol), tris (dibenzylideneacetone) dipalladium (0) (1.83g, 2.0mmol), Cs₂CO₃(16.3g, 50mmol) and 1, 4-dioxane (120 mL). Evacuating the system and then using N₂And (6) refilling. A reflux condenser was attached to the flask and the reaction mixture was heated at 100 ℃ for 16 h. The mixture was cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure and the resulting residue was purified by silica gel column chromatography (eluting with 5:1 petroleum ether/ethyl acetate) to give 115l (5.2g, 60%) as a white solid. MS: [ M + H ]]⁺435。¹H NMR(500MHz，DMSO-d₆)7.71-7.69(m，1H)，7.49-7.47(m，1H)，6.52(s，1H)，5.01(m，2H)，4.18(m，2H)，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 115mAcetic acid 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 ester 115m

A250-mL single-neck round-bottom flask equipped with a magnetic stirrer was charged with 115l (3.8g, 8.8mmol), (PinB)₂(10.9g，43mmol)、Pd(dppf)Cl₂(0.37g, 0.50mmol), acetic acidPotassium (2.55g, 26mmol) and 1, 4-dioxane (150 mL). Evacuating the system and then using N₂And (6) refilling. A reflux condenser was attached to the flask and the reaction mixture was heated at 100 ℃ for 15 h. The mixture was cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure and the resulting residue was purified by silica gel column chromatography (eluting with 5:1 petroleum ether/ethyl acetate) to give 115m (3.2g, 75%) as a yellow solid. MS: [ M + H ]]⁺483。

Example 115n(R) -acetic acid 4-fluoro-2- (1-methyl-5- (5- (2-methyl-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 ] amino]Indol-2 (1H) -yl) benzyl ester 115n

The procedure described in example 102 was followed, starting from 115e (220mg, 0.50mmol, 1.0 equiv.), 115m (482mg, 1.0mmol, 2.0 equiv.) to give 115n (195mg, 55%) as a yellow solid. MS: [ M + H ] +710.4

Example 115(R) -2- (5-fluoro-2- (hydroxymethyl) -3- (1-methyl-5- (5- (2-methyl-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 ] amino]Indol-1 (2H) -ones 115

The procedure as in example 102 was followed, starting with 115n (190mg, 0.27mmol), to give 115 as a white solid (47mg, 26%). MS: [ M + H ]]+668.4。¹H NMR(500MHz，DMSO-d₆)8.58(s，1H)，8.42(s，1H)，7.84(d，J＝3.0Hz，1H)，7.36-7.32(m，3H)，7.25-7.18(m，2H)，6.52(s，1H)，4.88(s，1H)，4.56-4.42(m，4H)，4.31-4.30(m，2H)，4.18-4.13(m，3H)，3.89-3.88(m，1H)，3.68-3.67(m，1H)，3.58(s，3H)，3.39-3.38(m，1H)，3.08-3.07(m，1H)，2.94-2.93(m，1H)，2.51-2.45(m，5H)，2.33-2.32(m，2H)，2.19-2.18(m，1H)，1.79-1.69(m，4H)，0.93-0.92(m，3H)。

Example 116aAcetic acid {2- [5- ({5- [ (2S,5R) -2, 5-dimethyl-4- (oxetan-3-yl) piperazin-1-yl]Pyridin-2-yl } amino) -1-methyl-6-oxo-1, 6-dihydropyridin-3-yl]-6- {4, 4-dimethyl-9-oxo-1, 10-diazacyclo [6.4.0.0^2,6]Dodeca-2 (6), 7-dien-10-yl } -4-fluorophenyl } methyl ester 116a

Into a 25-mL single-necked round bottom flask equipped with a magnetic stirrer and a reflux condenser was charged 103g (298 mg) of 5-bromo-3- (5- ((2S,5R) -2, 5-dimethyl-4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-ylamino) -1-methylpyridin-2 (1H) -one 104e (134mg, 0.30mmol), 1-methyl-3- (5- (4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-ylamino) -5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyridin-2 (1H) -one, 0.6mmol), Pd (dppf) Cl₂(22mg，0.03mmol)、K₃PO₄(127mg, 0.6mmol), sodium acetate (49mg, 0.6mmol), acetonitrile (5mL), and water (1.0 mL). After three cycles of vacuum/argon purge, the mixture was heated at reflux for 2 h. Then cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure and the resulting residue was purified by silica gel column chromatography (eluting with 30:1 dichloromethane/methanol) to give 116a (150mg, 68%) as a yellow solid. MS: [ M + H ] ]⁺738.3

Example 1162- (3- {5- [5- ((2S,5R) -2, 5-dimethyl-4-oxetan-3-yl-piperazin-1-yl) -pyridin-2-ylamino]-1-methyl-6-oxo-1, 6-dihydro-pyridin-3-yl } -5-fluoro-2-hydroxymethyl-phenyl) -7, 7-dimethyl-3, 4,7, 8-tetrahydro-2H, 6H-cyclopenta [4,5]Pyrrolo [1,2-a]Pyrazin-1-ones 116

Stirring at 30 ℃ 116a (150 m)g, 0.20mmol) and lithium hydroxide (48mg, 2.0mmol) in isopropanol/THF (1:1, 4mL) and water (1mL) for 1 h. The mixture was evaporated under reduced pressure. The resulting residue was extracted with ethyl acetate (2X10 mL). The combined organic layers were concentrated under reduced pressure and the residue was purified by reverse phase preparative HPLC to give 116 as a white solid (57mg, 41%). MS: [ M + H ]]⁺696.3。¹H NMR(500MHz，CDCl₃)8.60(dd，J＝2.0，6.5Hz，1H)，8.02(d，J＝2.5Hz，1H)，7.88(s，1H)，7.50-7.49(m，1H)，7.37(d，J＝8.5Hz，1H)，7.16(d，J＝9.0Hz，1H)，6.96(dd，J＝2.5，9.0Hz，1H)，6.83-6.81(m，2H)，4.77-4.74(m，2H)，4.66-4.62(m，2H)，4.57-4.55(m，1H)，4.33-4.31(m，1H)，4.23-4.14(m，3H)，3.92-3.89(m，1H)，3.78-3.76(m，1H)，3.71(s，3H)，3.22-3.20(m，1H)，2.93-2.91(m，1H)，2.75-2.73(m，2H)，2.58(s，2H)，2.52(s，3H)，1.97-1.90(m，2H)，1.28(s，6H)，0.91(d，J＝5.5Hz，6H)。

Example 117aN-tert-butyl-4, 5,6, 7-tetrahydrobenzo [ b ]]Thiophene-2-carboxamide 117a

4,5,6, 7-tetrahydrobenzo [ b ]]A mixture of thiophene-2-carboxylic acid (500g, 2.75mol, 1.0 equiv.) and thionyl chloride (655g, 5.5mol, 2.0 equiv.) was boiled under reflux for 3 h. Excess thionyl chloride was removed by distillation under reduced pressure. The residue was taken up in dichloromethane (1.0L) and a solution of tert-butylamine (402g, 5.5mol, 2.0 eq) in dichloromethane (500mL) was added with stirring, keeping the temperature of the mixture below 10 ℃. The resulting solution was stirred at 25 ℃ for 16 h. Most of the solvent was removed under reduced pressure. The residue was cooled in an ice bath and 2M KOH solution was added slowly with stirring to adjust the pH to 11. The suspension was filtered and the solid was collected, washed three times with water and dried in vacuo to give 117a as a white solid (580g, 80% over two steps). MS: [ M + H ]]⁺238。¹H NMR(500MHz，CDCl₃)7.02(s，1H)，5.77(s，1H)，2.65(t，J＝6.0Hz，1H)，2.47(t，J＝6.0Hz，1H)，1.74-1.70(m，4H)，1.35(s，9H)。

Example 117bN-tert-butyl-3- (diazenylmethyl) -4,5,6, 7-tetrahydrobenzo [ b]Thiophene-2-carboxamide 117b

To a solution of 117a (100g, 0.42mol, 1.0 eq.) in THF (500mL) was slowly added n-BuLi (672mL, 2.5M in THF, 1.68mol, 4.0 eq.) at-78 deg.C under argon. The mixture was stirred for 2 h. DMF (306g, 4.2mol, 10.0 equivalents) was added to the mixture while maintaining the temperature at-78 ℃. After an additional 2.0h, the reaction mixture was quenched with methanol (500mL) at-78 ℃. Stir at rt for 0.50 h. 80% aqueous hydrazine hydrate (131g, 2.1mol) was added and the mixture was refluxed at 65 ℃ overnight. The organic solvent was removed under reduced pressure. The residue was filtered and the collected yellow solid was washed with water. The solid was dried in vacuo to give crude 117b, which was used in the next step without purification. MS: [ M + H ]]⁺280。

Example 117c8-thia-4, 5-diazacyclo [7.4.0.0 ]^2,7]Trideca- (9),2(7), 3-trien-6-one 117c

117b (40g, 144mmol) in H at 105 deg.C₂SO₄The mixture in (30% aqueous, 3L) was refluxed for 24 h. It was then filtered and the filtrate extracted with dichloromethane (3x 1L). Through Na₂SO₄The combined extracts were dried and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (eluting with 100:1 dichloromethane/methanol) to give 117c as a white solid (9.0g, 31%). MS: [ M + H ]]⁺207。¹H NMR(500MHz，CDCl₃)8.15(s，1H)，2.96-2.94(m，2H)，2.86-2.84(m，2H)，1.96-1.94(m，4H)。

Example 117dAcetic acid (2-bromo-4-fluoro-6- { 6-oxo-8-thia-4, 5-diazacyclo [7.4.0.0 ]^2,7]Thirteen-layer rubber-1(9),2(7), 3-trien-5-yl } phenyl) methyl ester 117d

A100-mL single-neck round-bottom flask equipped with a magnetic stirrer was charged with 117c (1.0g, 4.85mmol), 2, 6-dibromo-4-fluorobenzyl acetate 101c (4.8g, 14.6mmol), copper (I) iodide (553mg, 2.9mmol), N¹,N²Dimethylethane-1, 2-diamine (512mg, 5.82mmol), Cs₂CO₃(3.2g, 9.7mmol) and 1, 4-dioxane (50 mL). Evacuating the system and then using N₂And (6) refilling. A reflux condenser was attached to the flask and the reaction mixture was heated at 100 ℃ for 16 h. Then cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure and the resulting residue was purified by silica gel column chromatography (eluting with 5:1 petroleum ether/ethyl acetate) to give 117d (437mg, 20%) as a yellow solid. MS: [ M + H ]]⁺451。

Example 117eAcetic acid (4-fluoro-2- { 6-oxo-8-thia-4, 5-diazacyclo [7.4.0.0 ]^2,7]Tridec-1 (9),2(7), 3-trien-5-yl } -6- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) methyl ester 117e

Following the procedure in example 104 and starting with 117d (400mg0.88mmol), 117e was obtained as a yellow solid (353mg, 80%). MS: [ M + H ]]⁺499

Example 117fAcetic acid { 4-fluoro-2- [ 1-methyl-5- ({5- [ (2R) -2-methyl-4- (oxetan-3-yl) piperazin-1-yl]Pyridin-2-yl } amino) -6-oxopyridin-3-yl]-6- { 6-oxo-8-thia-4, 5-diazacyclo [7.4.0.0^2,7]Tridec-1 (9),2(7), 3-trien-5-yl } phenyl } methyl ester 117f

The procedure was as in example 102 and (R) -5-bromo-1-methyl-3- (5- (2-methyl-4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-ylamino) pyridine-Starting from 2(1H) -one 115f (217mg, 0.50mmol) and 117e (249mg, 0.50mmol), 117f (174mg, 48%) was obtained as a yellow solid. MS: [ M + H ]]⁺726。

Example 1173- (5-fluoro-2-hydroxymethyl-3- { 1-methyl-5- [5- ((R) -2-methyl-4-oxetan-3-yl-piperazin-1-yl) -pyridin-2-ylamino]-6-oxo-1, 6-dihydro-pyridin-3-yl } -phenyl) -6,7,8, 9-tetrahydro-3H-benzo [4,5]Thieno [2,3-d ]]Pyridazin-4-ones 117

Following the method in example 102 and starting with 117f (72mg, 0.10mmol), 117 was obtained as a yellow solid (35mg, 51%). LCMS: [ M + H ]]⁺684。¹H NMR(500MHz，DMSO-d₆)8.56(d，J＝2.0Hz，1H)，8.48(s，1H)，8.43(s，1H)，7.85(d，J＝2.5Hz，1H)，7.37-7.32(m，4H)，7.24(d，J＝9.0Hz，1H)，4.60(t，J＝5.5Hz，1H)，4.57-4.53(m，2H)，4.47-4.41(m，2H)，4.28-4.27(d，J＝4.0Hz，2H)，3.68-3.66(m，1H)，3.58(s，3H)，3.40-3.38(m，1H)，3.10-3.07(m，1H)，2.93-2.91(m，3H)，2.84-2.82(m，2H)，2.54-2.52(m，1H)，2.32-2.30(m，2H)，2.19-2.18(m，1H)，1.89-1.84(m，4H)，0.93(t，J＝6.5Hz，2H)。

Example 118aAcetic acid {2- [5- ({5- [ (2S,5R) -2, 5-dimethyl-4- (oxetan-3-yl) piperazin-1-yl]Pyridin-2-yl } amino) -1-methyl-6-oxo-1, 6-dihydropyridin-3-yl]-4-fluoro-6- { 6-oxo-8-thia-4, 5-diazacyclo [7.4.0.0^2,7]Tridec-1 (9),2(7), 3-trien-5-yl } phenyl } methyl ester 118a

A25-mL single-neck round bottom flask equipped with a magnetic stirrer and a reflux condenser was charged with 5-bromo-3- (5- ((2S,5R) -2, 5-dimethyl-4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-ylamino) -1-methylpyridin-2 (1H) -one104e (179mg, 0.40mmol), acetic acid (4-fluoro-2- { 6-oxo-8-thia-4, 5-diazacyclo [7.4.0.0 ]^2,7]Tridec-1 (9),2(7), 3-trien-5-yl } -6- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) methyl ester 117d (200mg, 0.40mmol), Pd (dppf) Cl₂(29mg，0.04mmol)、K₃PO₄(170mg, 0.8mmol), sodium acetate (66mg, 0.8mmol), acetonitrile (5mL), water (1.0 mL). After three cycles of vacuum/argon purge, the mixture was heated at reflux for 2 h. Then cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure and the resulting residue was purified by silica gel column chromatography (eluting with 30:1 dichloromethane/methanol) to give 118a (100mg, 34%) as a yellow solid. MS: [ M + H ]]⁺740.3

Example 1183- (3- {5- [5- ((2S,5R) -2, 5-dimethyl-4-oxetan-3-yl-piperazin-1-yl) -pyridin-2-ylamino]-1-methyl-6-oxo-1, 6-dihydro-pyridin-3-yl } -5-fluoro-2-hydroxymethyl-phenyl) -6,7,8, 9-tetrahydro-3H-benzo [4,5]Thieno [2,3-d ]]Pyridazin-4-ones 118

A mixture of 118a (100mg, 0.135mmol) and lithium hydroxide (33mg, 1.35mmol) in isopropanol/THF (1:1, 4mL) and water (1mL) was stirred at 30 ℃ for 1 h. The mixture was evaporated under reduced pressure. The resulting residue was extracted with ethyl acetate (2X10 mL). The combined organic layers were concentrated under reduced pressure and the residue was purified by reverse phase preparative HPLC to give 118 as a white solid (36mg, 38%). MS: [ M + H ]]⁺698.3。¹H NMR(500MHz，CDCl₃)8.64(d，J＝2.0Hz，1H)，8.27(s，1H)，8.05(d，J＝2.0Hz，1H)，7.88(s，1H)，7.48(d，J＝2,5Hz，1H)，7.37(d，J＝5.5Hz，1H)，7.30(dd，J＝2.5，9.0Hz，1H)，7.11(dd，J＝2.5，8.5Hz，1H)，6.82(d，J＝9.0Hz，1H)，4.79-4.73(m，2H)，4.67-4.61(m，2H)，4.31(s，2H)，3.77(t，J＝7.0Hz，1H)，3.72(s，3H)，3.21-3.19(m，1H)，2.99-2.98(m，2H)，2.94-2.91(m，1H)，2.88-2.86(m，2H)，2.78-2.71(m，2H)，2.51-2.49(m，1H)，1.99-1.96(m，6H)，0.92-0.90(m，6H)。

Example 119aAcetic acid 2-bromo-4-fluoro-6- (10-fluoro-1-oxo-3, 4,6,7,8, 9-hexahydropyrazino [1,2-a ]]Indol-2 (1H) -yl) benzyl ester 119a

Example 119a10-bromo-1H, 2H,3H,4H,6H,7H,8H, 9H-pyrazino [1,2-a ]]Indol-1-one 119a

To a 250-mL 3-necked round bottom flask was added a solution of 1H,2H,3H,4H,6H,7H,8H, 9H-pyrazino [1,2-a ] indol-1-one 104j (9.5g, 49.94mmol, 1.00 equiv.) in N, N-dimethylformamide (100mL), followed by N-bromosuccinimide (9.8g, 55.06mmol, 1.10 equiv.) in several portions at 0 ℃. The resulting solution was stirred at room temperature for 2h and diluted with 500mL of water. The precipitate was filtered and dried in a vacuum oven to give 9.5g (71%) of 119a as a light brown solid.

Example 119b10-fluoro-1H, 2H,3H,4H,6H,7H,8H, 9H-pyrazino [1,2-a ]]Indol-1-one 119b

To a 2-L4 necked round bottom flask purged and maintained with a nitrogen inert atmosphere was added a solution of 119a (40g, 148.62mmol, 1.00 equiv.) in tetrahydrofuran (200mL), followed by n-BuLi (2.4M) (218mL, 3.50 equiv.) dropwise with stirring at-78 ℃. The resulting solution was stirred at-40 ℃ for 3 h. To this was added dropwise, while stirring, a solution of N-fluorobenzenesulfonylimide (98.7g, 313.33mmol, 2.10 equiv.) in tetrahydrofuran (200mL) at-78 ℃. The resulting solution was stirred at room temperature for 3h, quenched by addition of 200mL water and extracted with 3 × 500mL ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate and concentrated in vacuo. The crude product (30g) was purified by preparative HPLC using the following conditions (mobile phase, A: 0.05% trifluoroacetic acid/water; B: CH)₃CN; gradient: 10% B-25% B) to give 5.05g (16%) of 119B as a white solid. MS: [ M + H ]]⁺209。1H NMR(300MHz，CDCl₃)6.16(br，1H)，3.90-3.86(m，2H)，3.65-3.62(m，2H)，2.53-2.47(m，4H)，1.88-1.80(m，2H)，1.77-1.72(m，2H)。

Example 119cAcetic acid 2-bromo-4-fluoro-6- (10-fluoro-1-oxo-3, 4,6,7,8, 9-hexahydropyrazino [1,2-a ]]Indol-2 (1H) -yl) benzyl ester 119c

A100-mL single-neck round bottom flask equipped with a magnetic stirrer and a reflux condenser was charged with 1, 4-dioxane (60mL), 2, 6-dibromo-4-fluorobenzyl acetate 101c (2.34g, 7.2mmol), 119b (500mg, 2.4mmol), and cesium carbonate (1.6g, 4.8 mmol). After bubbling nitrogen through the resulting mixture for 30 min, Xantphos (140mg, 0.24mmol) and tris (dibenzylideneacetone) dipalladium (0) (220mg, 0.24mmol) were added and the reaction mixture was heated at 100 ℃ for 12 h. The reaction mixture was then cooled to room temperature and filtered. The filtrate was partitioned between ethyl acetate (40mL) and water (40 mL). The aqueous layer was separated and extracted with ethyl acetate (3X70 mL). The combined organic layers were washed with brine (30mL) and dried over sodium sulfate. The drying agent was removed by filtration and the filtrate was concentrated under reduced pressure. The residue was purified by column on silica gel (eluting with 3:1 petroleum ether/ethyl acetate) to give 119c as a yellow solid (632mg, 58%). MS: [ M + H ]]⁺453.2

Example 119d(S) -acetic acid 4-fluoro-2- (10-fluoro-1-oxo-3, 4,6,7,8, 9-hexahydropyrazino [1, 2-a)]Indol-2 (1H) -yl) -6- (1-methyl-5- (5- (2-methyl-4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-ylamino) -6-oxo-1, 6-dihydropyridin-3-yl) benzyl ester 119d

To a 50-mL single-neck round bottom flask equipped with a magnetic stirrer and a reflux condenser was charged 119c (150mg, 1.0 eq, 0.33mmol), (S) -1-methyl-3- (5- (2-methyl-4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-ylamino) -5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyridin-2 (1H) -one 113f (160mg, 1.0 eq, 0.33mmol), K₃PO₄(210mg, 3.0 equiv., 0.99mmol), PdCl₂(dppf) (27.0mg, 0.10 eq.)0.033mmol), THF (20mL), and water (0.1 mL). After three cycles of vacuum/argon purge, the mixture was heated at reflux for 2 h. Then cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure and the resulting residue was purified by silica gel column chromatography (eluting with 40:1 dichloromethane/methanol) to give 119d (90mg, 37%) as a yellow solid. MS: [ M + H ]]⁺728.3。

Example 119(S) -10-fluoro-2- (5-fluoro-2- (hydroxymethyl) -3- (1-methyl-5- (5- (2-methyl-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 ] o]Indol-1 (2H) -ones 119

A50-mL single-neck round-bottom flask equipped with a magnetic stirrer was charged with 119d (90mg, 1.0 equiv., 0.12mmol), lithium hydroxide (9.0mg, 3.0 equiv., 0.37mmol), isopropanol (3mL), THF (3mL), and water (2 mL). The mixture was stirred at room temperature for 1 h. Then filtered and concentrated. The residue was purified by reverse phase preparative HPLC to give 119(42mg, 49%). MS: [ M + H ]]⁺686.3。¹H NMR(500MHz，CDCl₃)8.54(dd，J＝2.0Hz，9.0，1H)，7.94-7.93(m，1H)，7.81(d，J＝4.0Hz，1H)，7.45-7.44(m，1H)，7.31(dd，J＝3.0，9.0Hz，1H)，7.15-7.14(m，1H)，6.94(dd，J＝2.0，9.0Hz，1H)，6.81(d，J＝8.5Hz，1H)，4.71-4.61(m，4H)，4.53(d，J＝9.5Hz，1H)，4.32-4.31(m，2H)，4.15-4.08(m，3H)，3.89-3.86(m，1H)，3.69(s，3H)，3.55-3.43(m，2H)，3.07(m，2H)，2.57-2.46(m，7H)，2.20-2.16(m，1H)，1.88-1.76(m，4H)，0.98-096(m，3H)。

Example 120aAcetic acid {2- [5- ({5- [ (2S) -2-ethyl-4- (oxetan-3-yl) piperazin-1-yl]Pyridin-2-yl } amino) -1-methyl-6-oxo-1, 6-dihydropyridin-3-yl]-4-fluoro-6- { 6-oxo-8-thia-4, 5-diazacyclo [7.4.0.0^2,7]Tridec-1 (9),2(7), 3-trien-5-yl } phenyl } methyl ester 120a

Into a 25-mL single-necked round bottom flask equipped with a magnetic stirrer and a reflux condenser was charged (S) -5-bromo-3- (5- (2-ethyl-4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-ylamino) -1-methylpyridin-2 (1H) -one 114e (179mg, 0.40mmol), acetic acid (4-fluoro-2- { 6-oxo-8-thia-4, 5-diazacyclo [7.4.0.0 [ ] -^2,7]Tridec-1 (9),2(7), 3-trien-5-yl } -6- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) methyl ester 117e (200mg, 0.40mmol), Pd (dppf) Cl₂(29mg，0.04mmol)、K₃PO₄(170mg, 0.8mmol), sodium acetate (66mg, 0.8mmol), acetonitrile (5mL), and water (1.0 mL). After three cycles of vacuum/argon purge, the mixture was heated at reflux for 2 h. Then cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure and the resulting residue was purified by silica gel column chromatography (eluting with 30:1 dichloromethane/methanol) to give 120a (120mg, 41%) as a yellow solid. MS: [ M + H ]]⁺740.3

Example 1203- (3- {5- [5- ((S) -2-ethyl-4-oxetan-3-yl-piperazin-1-yl) -pyridin-2-ylamino]-1-methyl-6-oxo-1, 6-dihydro-pyridin-3-yl } -5-fluoro-2-hydroxymethyl-phenyl) -6,7,8, 9-tetrahydro-3H-benzo [4,5]Thieno [2,3-d ]]Pyridazin-4-ones 120

A mixture of 120a (120mg, 0.16mmol) and lithium hydroxide (38mg, 1.6mmol) in isopropanol/THF (1:1, 4mL) and water (1mL) was stirred at 30 ℃ for 1 h. The mixture was evaporated under reduced pressure. The resulting residue was extracted with ethyl acetate (2X10 mL). The combined organic layers were concentrated under reduced pressure and the residue was purified by reverse phase preparative HPLC to give 120 as a white solid (73mg, 65%). MS: [ M + H ]]⁺698.3。¹H NMR(500MHz，CDCl₃)8.58(d，J＝1.5Hz，1H)，8.26(s，1H)，7.94(d，J＝2.5Hz，1H)，7.80(s，1H)，7.45(d，J＝2.5Hz，1H)，7.30(d，J＝9.0Hz，1H)，7.27(d，J＝9.0Hz，1H)，7.11(dd，J＝2.5，8.0Hz，1H)，6.82(d，J＝9.0Hz，1H)，4.72-4.62(m，4H)，4.31(s，2H)，4.01(bs，1H)，3.71(s，3H)，3.53(t，J＝6.0Hz，1H)，3.34-3.32(m，1H)，3.13(t，J＝6.0Hz，2H)，2.99(t，J＝5.0Hz，2H)，2.87(t，J＝5.0Hz，2H)，2.60-2.56(m，1H)，2.46-2.44(m，2H)，2.36-2.34(m，1H)，2.01-1.96(m，4H)，1.71-1.68(m，1H)，1.44-1.36(m，1H)，0.83(t，J＝7.5Hz，3H)。

Example 121a4,5,6, 7-tetrahydro-1H-indole-2-carboxylic acid ethyl ester 121a

To a mixture of ethyl 3- (2-chlorocyclohex-1-enyl) acrylate (21.4g, 100mmol) in DMSO (100mL) was added sodium azide (9.75g, 150 mmol). The reaction mixture was heated at 105 ℃ for 4 h. After cooling to room temperature, the mixture was poured into ice water. The resulting precipitate was collected by filtration to yield 121a (18.0g, 93.3%). MS-ESI: [ M + H ]]⁺194。

Example 121b1- (2, 2-diethoxyethyl) -4,5,6, 7-tetrahydro-1H-indole-2-carboxylic acid ethyl ester 121b

To a suspension of NaH (1.44g, 60.2mmol) in N, N-Dimethylformamide (DMF) (30mL) was slowly added 121a (5.80g, 30.1mmol) at 0 deg.C. The resulting mixture was stirred at room temperature for 0.5h, followed by addition of 2-bromo-1, 1-diethoxyethane (11.9g, 60.2 mmol). The reaction mixture was heated at 70 ℃ for 30h and quenched with water (100 mL). The mixture was then extracted with ethyl acetate (3 × 100 mL). The combined organic phases were concentrated under reduced pressure and the residue was purified by silica gel column chromatography eluting with 40:1 petroleum ether/ethyl acetate to give 121b (4.7g, 51%). MS-ESI: [ M-ethanol + H ]]⁺264。¹H NMR(500MHz，DMSO-d₆)6.65(s，1H)，4.59(t，J＝5.0Hz，1H)，4.17-4.16(m，4H)，3.59-3.57(m，2H)，3.27-3.26(m，2H)，2.61(t，J＝6.0Hz，2H)，2.51(t，J＝6.0Hz，2H)，1.73-1.71(m，2H)，1.63-1.61(m，2H)，1.25(t，J＝7.0Hz，3H)，1.02(t，J＝7.0Hz，6H)。

Example 121c1- (2, 2-diethoxyethyl) -4,5,6, 7-tetrahydro-1H-indole-2-carboxylic acid 121c

To a mixture of 121b (4.7g, 15.2mmol) in a mixed solvent of ethanol (20mL), tetrahydrofuran (20mL) and water (30mL) was added sodium hydroxide (3.0g, 75.0 mmol). The reaction mixture was heated at 75 ℃ for 2 days and concentrated under reduced pressure. The residue was suspended in water and neutralized with dilute aqueous citric acid. The mixture was extracted with ethyl acetate (3X100mL) and the combined organic phases were concentrated under reduced pressure to give 121c (3.32g, 78%). MS-ESI: [ M-ethanol + H ]]⁺236。

Example 121d1- (2, 2-diethoxyethyl) -4,5,6, 7-tetrahydro-1H-indole-2-carboxamide 121d

Add O- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HATU) (5.7g, 15.0mmol), triethylamine (1.5g, 15.0mmol) and DMAP (128mg, 1.0mmol) to a mixture of 121c (2.8g, 10.0mmol) in N, N-dimethylformamide (30 mL). The reaction mixture was stirred at room temperature overnight. Saturated ammonium hydroxide (30mL) was added and the resulting mixture was stirred for an additional 2 h. Then diluted with water (100mL) and extracted with ethyl acetate (3X100 mL). The combined organic phases were concentrated under reduced pressure and the residue was purified by column chromatography on silica gel (eluting with petroleum ether/ethyl acetate (6:1 to 3: 1)) to give 121d (2.7g, 96%). MS-ESI: [ M-ethanol + H ]]⁺235。¹H NMR(500MHz，DMSO)7.35(bs，1H)，6.70(bs，1H)，6.60(s，1H)，4.60(t，J＝5.5Hz，1H)，4.18(d，J＝4.0Hz，2H)，3.57-3.56(m，2H)，3.25(m，2H)，2.57(t，J＝6.0Hz，2H)，2.40(t，J＝6.0Hz，2H)，1.71(t，J＝5.0Hz，2H)，1.64(t，J＝5.0Hz，2H)，1.01(t，J＝7.0Hz，6H)。

Example 121e6,7,8, 9-tetrahydropyrazino [1,2-a ] s]Indol-1 (2H) -one 121e

A mixture of 121d (2.7g, 9.6mmol) and acetic acid (10mL) was heated at 110 ℃ for 2 h. The mixture was cooled to room temperature, neutralized with aqueous sodium carbonate solution and extracted with ethyl acetate (3X30 mL). The combined organic phases were concentrated under reduced pressure to give 121e as a yellow solid (1.6g, 88%). MS-ESI: [ M + H ]]⁺189.3。¹H NMR(500MHz，DMSO-d₆)10.28(s，1H)，7.02(d，J＝5.5Hz，1H)，6.63(s，1H)，，6.52(pt，J＝5.5Hz,1H)，2.66(t，J＝6.0Hz，2H)，2.57(t，J＝6.0Hz，2H)，1.83-1.82(m，2H)，1.73-1.72(m，2H)。

Example 121f2-bromo-4-fluoro-6- (1-oxo-6, 7,8, 9-tetrahydropyrazino [1, 2-a)]Indol-2 (1H) -yl) benzaldehyde 121f

A100-mL single-neck round-bottom flask equipped with a magnetic stirrer and a reflux condenser was charged with 121e (500mg, 2.66mmol), 2, 6-dibromo-4-fluorobenzaldehyde (1.50g, 5.32mmol), and potassium acetate (521mg, 5.32 mmol). Argon was bubbled through the suspension for 30 min, 4, 7-dimethoxy-1, 10-phenanthroline (638.4mg, 2.66mmol) and cuprous iodide (506mg, 2.66mmol) were added. The system was flushed through three cycles of vacuum/argon and then heated at 100 ℃ for 16 h. Then cooled to room temperature and filtered. The solid was washed with dichloromethane (2X100 ml). The combined filtrates were concentrated under reduced pressure and the residue was purified by silica gel column chromatography (eluting with petroleum ether/ethyl acetate (10:1 to 3: 1)) to give 121f as a yellow solid (510mg, 49%). MS: [ M + H ]]⁺389。

Example 121g3- (5- ((2S,5R) -2, 5-dimethyl-4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-ylamino) -1-methyl-5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyridin-2 (1H) -one 121g

A100-mL single-necked round bottom flask equipped with a magnetic stirrer and a reflux condenser was charged with 5-bromo-3- (5- ((2S,5R) -2, 5-dimethyl-4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-ylamino) -1-methylpyridin-2 (1H) -one 104e (3.0g, 6.70mmol), Pin₂B₂(8442mg，33.5mmol)、Pd₂(dba)₃(311mg, 0.34mmol), X-phos (319mg, 0.67mmol), potassium acetate (1970mg, 20.1mmol) and dioxane (50 mL). After three cycles of vacuum/argon flushing, the mixture was heated at 60 ℃ for 16 h. Then cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was washed with 8:1 petroleum ether/ethyl acetate (80mL) to give 121g (3g, 90%) as a yellow solid. MS: [ M + H ]]⁺496.4。

Example 121h2- (5- (5- ((2S,5R) -2, 5-dimethyl-4- (oxetan-3-yl) piperazin-1-yl) -pyridin-2-ylamino) -1-methyl-6-oxo-1, 6-dihydropyridin-3-yl) -4-fluoro-6- (1-oxo-6, 7,8, 9-tetrahydropyrazino [1, 2-a)]Indol-2 (1H) -yl) benzaldehyde 121H

Add 121f (194mg, 0.5mmol), 121g (347.0mg, 0.7mmol), potassium acetate (98.0mg, 1.0mmol), 1' -bis (diphenylphosphino) ferrocene-palladium (II) dichloride (20.4mg, 0.025mmol), water (0.5mL), and acetonitrile (20mL) to a 50-mL round bottom flask, rinse the system with vacuum/argon for 3 cycles and heat at 100 deg.C under argon for 3h LCMS analysis of the reaction mixture showing a small amount of starting material remaining, cool the reaction mixture to room temperature and filter, dilute the filtrate with dichloromethane (50mL) and water (50mL), separate the aqueous layer and extract with dichloromethane (3 × 20mL), Na₂SO₄The combined organic layers were dried, filtered, and concentrated under reduced pressure. The dark residue was purified by silica gel column chromatography (eluting with dichloromethane/methanol (80/1 to 30/1)) to give 121g (182mg, 54%) as a yellow solid. MS: [ M + H ]]⁺678。

Example 1212- (3- (5- (5- ((2S,5R) -2, 5-dimethyl)4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-ylamino) -1-methyl-6-oxo-1, 6-dihydropyridin-3-yl) -5-fluoro-2- (hydroxymethyl) phenyl) -6,7,8, 9-tetrahydropyrazino [1,2-a]Indol-1 (2H) -ones 121

Add NaBH to a solution of 121h (150mg, 0.22mmol) in methanol (10mL) at RT₄(41.8mg, 1.1 mmol). After stirring the reaction mixture for 1h, LCMS indicated complete reaction. The mixture was poured into water (30mL) and concentrated under reduced pressure. The residue was extracted with dichloromethane (3X30 mL). The combined organic layers were washed with brine (30mL) and Na₂SO₄Dried, filtered, and concentrated under reduced pressure. Purification of the residual solid by preparative HPLC gave 121 as a white solid (60.3mg, 40%). MS: [ M + H ]]⁺680。¹H NMR(500MHz，DMSO-d₆)8.63(d，J＝2.0Hz，1H)，8.51(s，1H)，7.90(d，J＝3.0Hz，1H)，7.41(m，2H)，7.31-7.23(m，4H)，6.78(d，J＝1.0Hz，2H)，4.80(m，1H)，4.55-4.54(m，2H)，4.49-4.4.48(m，2H)，4.28-4.20(m，2H)，3.69-3.64(m，1H)，3.60(s，3H)，3.29-3.27(m，2H)，2.89-2.88(m，1H)，2.75-2.67(m，4H)，2.61(m，2H)，1.93-1.86(m，3H)，1.59(m，2H)，0.85-0.81(m，6H)。

Example 122a(S) -3- (5- (2-ethyl-4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-ylamino) -1-methyl-5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyridin-2 (1H) -one 122a

Into a 100-mL single-necked round bottom flask equipped with a magnetic stirrer and a reflux condenser was charged (S) -5-bromo-3- (5- (2-ethyl-4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-ylamino) -1-methylpyridin-2 (1H) -one 114e (3219mg, 7.20mmol), Pin₂B₂(9072mg，36.0mmol)、Pd₂(dba)₃(329mg, 0.36mmol), X-phos (302mg, 0.72mmol), Potassium acetate (211 mg, 0.72mmol)7mg, 21.6mmol) and dioxane (50 mL). After three cycles of vacuum/argon flushing, the mixture was heated at 60 ℃ for 16 h. Then cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure and the resulting residue was washed with 8:1 petroleum ether/ethyl acetate (80mL) to give 122a (3.0g, 84%) as a yellow solid.

Example 122b(S) -2- (5- (5- (2-ethyl-4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-ylamino) -1-methyl-6-oxo-1, 6-dihydropyridin-3-yl) -4-fluoro-6- (1-oxo-6, 7,8, 9-tetrahydropyrazino [1, 2-a)]Indol-2 (1H) -yl) benzaldehyde 122b

Charging round-bottom flask with 2-bromo-4-fluoro-6- (1-oxo-6, 7,8, 9-tetrahydropyrazino [1,2-a ]]Indol-2 (1H) -yl) benzaldehyde 121f (159mg, 0.41mmol), 122a (213mg, 0.43mmol), PdCl₂(dppf)(29mg，0.04mmol)、K₃PO₄(182mg, 0.86mmol), sodium acetate (71mg, 0.86mmol), acetonitrile (15mL), and water (1.5 mL). After three cycles of vacuum/argon flushing, the mixture was heated at 80 ℃ for 3 h. Then filtered and the filtrate evaporated under reduced pressure. The residue was purified by silica gel column chromatography (eluting with 1:20 methanol/dichloromethane) to give 122b as a red solid (120mg, 43%). MS: [ M + H ]]⁺678.3

Example 122(S) -2- (3- (5- (5- (2-ethyl-4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-ylamino) -1-methyl-6-oxo-1, 6-dihydropyridin-3-yl) -5-fluoro-2- (hydroxymethyl) phenyl) -6,7,8, 9-tetrahydropyrazino [1,2-a]Indol-1 (2H) -ones 122

122b (100mg, 0.15mmol), NaBH was stirred at 25 deg.C₄(22mg, 0.60) and methanol (10mL) for 1 h. Then quenched with water (5mL) and concentrated under reduced pressure. The residue was extracted with dichloromethane (2X10mL) and the combined organic layers were concentrated under reduced pressure. The residue was purified by reverse phase preparative HPLC to give 122(32mg, 31%). MS: [ M + H ]]⁺680.3。¹H NMR(500MHz，CDCl₃)8.56(s，1H)，7.91(s，1H)，7.80(s，1H)，7.53(s，1H)，7.28-7.26(m，2H)，7.05(s，1H)，6.97-6.93(m，2H)，6.81(d，J＝8.5Hz，1H)，6.45(d，J＝5.5Hz，1H)，4.71-4.61(m，4H)，4.38(d，J＝12.0Hz，1H)，4.36(d，J＝11.5Hz，1H)，3.70(s，3H)，3.52(bs，1H)，3.31(d，J＝5.5Hz，1H)，3.13-3.10(m，2H)，2.75-2.70(m，4H)，2.56-2.43(m，4H)，1.98-1.96(m，2H)，1.85-1.84(m，2H)，1.39-1.36(m，2H)，0.82(t，J＝7.0Hz，3H)。

Example 123aAcetic acid 2- (5- (5- (2-ethyl-4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-ylamino) -1-methyl-6-oxo-1, 6-dihydropyridin-3-yl) -4-fluoro-6- (1-oxo-3, 4,6,7,8, 9-hexahydropyrido [3,4-b [)]Indolizin-2 (1H) -yl) benzyl ester 123a

A100-mL single-neck round bottom flask was charged with acetic acid 2-bromo-4-fluoro-6- (1-oxo-3, 4,6,7,8, 9-hexahydropyrido [3,4-b ]]Indolizin-2 (1H) -yl) benzyl ester 101H (120mg, 0.27mmol), (S) -3- (5- (2-ethyl-4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-ylamino) -1-methyl-5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyridin-2 (1H) -one 122a (158.4mg, 0.32mmol), pd (dppf) Cl₂(24.5mg，0.03mmol)、K₃PO₄(114.5mg, 0.54mmol), sodium acetate trihydrate (73.4mg, 0.54mmol), water (0.5mL), and acetonitrile (20 mL). Evacuating the system and using N₂And (6) refilling. The reaction mixture was heated at 100 ℃ for 2 h. Then cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure and the resulting residue was purified by silica gel column chromatography (eluting with 25:1 dichloromethane/methanol) to give 123a (105mg, 53%) as a yellow-brown solid. MS: [ M + H ]]⁺724.3。

Example 123(S) -2- (3- (5- (5- (2-ethyl-4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-ylamino) -1-methyl-6-oxo-1, 6-dihydropyridin-3-yl) -5-fluoro-2- (hydroxy)Ylmethyl) phenyl) -3,4,6,7,8, 9-hexahydropyrido [3,4-b ]]Indolizin-1 (2H) -ones 123

A mixture of 123a (105mg, 0.15mmol) and lithium hydroxide (36mg, 1.5mmol) in isopropanol/THF (1:1, 4mL) and water (1mL) was stirred at 30 ℃ for 1 h. The mixture was evaporated under reduced pressure and the residue was extracted with ethyl acetate (2X10 mL). The combined ethyl acetate extracts were concentrated under reduced pressure and the residue was purified by reverse phase preparative HPLC to give 123 as a pale pink solid (40mg, 40%). MS: [ M + H ]]⁺682.3。¹H NMR(500M，CHCl₃)8.55(s，1H)，7.93(s，1H)，7.82(s，1H)，7.51(s，1H)，7.29(d，J＝3.5Hz，1H)，7.15-7.12(m，1H)，6.94(dd，J＝3.5Hz，10.5，1H)，6.81(d，J＝10.0Hz，1H)，6.30(s，1H)，4.77-4.74(m，4H)，4.72-4.70(m，1H)，4.57-4.55(m，1H)，4.29-4.24(m，1H)，4.12-4.05(m，1H)，3.90-3.78(m，4H)，3.70(s，3H)，3.52-3.50(m，1H)，3.32-3.30(m，1H)，3.12-3.10(m，2H)，3.04-2.83(m，2H)，2.81(m，2H)，2.57-2.50(m，1H)，2.43-2.33(m，2H)，2.05-2.00(m，2H)，1.87-1.85(m，2H)，1.49-1.35(m，2H)，0.81(t，J＝8.5Hz，3H)。

Example 124aAcetic acid (2- {4, 4-dimethyl-9-oxo-1, 10-diazacyclo [6.4.0.0 ]^2,6]Dodeca-2 (6), 7-dien-10-yl } -4-fluoro-6- [ 1-methyl-5- ({5- [ (2R) -2-methyl-4- (oxetan-3-yl) piperazin-1-yl]Pyridin-2-yl } amino) -6-oxo-1, 6-dihydropyridin-3-yl]Phenyl) methyl ester 124a

Into a round bottom flask equipped with a magnetic stirrer and reflux condenser was charged (R) -5-bromo-1-methyl-3- (5- (2-methyl-4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-ylamino) pyridin-2 (1H) -one 115f (152mg, 0.35mmol), acetic acid (2- {4, 4-dimethyl-9-oxo-1, 10-diazacyclo [6.4.0.0 ]^2,6]Dodeca-2 (6), 7-dien-10-yl } -4-fluoro-6- (4,4,5, 5-tetramethy l)Yl-1, 3, 2-dioxaborolan-2-yl) phenyl) methyl ester 103g (206mg, 0.415mmol), PdCl₂(dppf)(28mg，0.04mmol)、K₃PO₄(147mg, 0.69mmol), sodium acetate (57mg, 0.69mmol), acetonitrile (20mL) and water (2 mL). After three cycles of vacuum/argon flushing, the mixture was heated at 100 ℃ for 3 h. Then filtered and the filtrate evaporated under reduced pressure. The residue was purified by silica gel column chromatography (eluting with 1:20 methanol/dichloromethane) to give 124a as a red solid (70mg, 28%). MS: [ M + H ]]⁺724.2

Example 1242- (5-fluoro-2-hydroxymethyl-3- { 1-methyl-5- [5- ((R) -2-methyl-4-oxetan-3-yl-piperazin-1-yl) -pyridin-2-ylamino]-6-oxo-1, 6-dihydro-pyridin-3-yl } -phenyl) -7, 7-dimethyl-3, 4,7, 8-tetrahydro-2H, 6H-cyclopenta [4,5]Pyrrolo [1,2-a]Pyrazin-1-ones 124

A mixture of 124a (59mg, 0.080mmol), lithium hydroxide (19mg, 0.80mmol), THF (10mL), isopropanol (8mL) and water (10mL) was stirred at room temperature for 1.5 h. Then concentrated under reduced pressure and the residue extracted with dichloromethane (2X10 mL). The combined dichloromethane extracts were concentrated under reduced pressure. The residue was purified by reverse phase preparative HPLC to give 124(43mg, 79%). MS: [ M + H ]]⁺682.3。¹HNMR(500MHz，DMSO-d₆)8.58-8.57(m，1H)，8.41(s，1H)，7.84(d，J＝3.0Hz，1H)，7.37-7.31(m，3H)，7.22(d，J＝9.5Hz，1H)，7.19-7.16(m，1H)，6.50(s，1H)，4.87(d，J＝2.0Hz，1H)，4.57-4.53(m，2H)，4.46(t，J＝6.0Hz，1H)，4.41(t，J＝6.0Hz，1H)，4.31(d，J＝3.0Hz，2H)，4.21-4.18(m，2H)，4.15-4.10(m，1H)，3.88-3.85(m，1H)，3.67(d，J＝2.0Hz，1H)，3.58(s，3H)，3.41-3.37(m，2H)，3.10-3.07(m，1H)，2.95-2.91(m，1H)，2.56(d，J＝1.5Hz，2H)，2.41(s，2H)，2.32-2.28(m，2H)，2.17-2.15(m,1H)，1.21(s，6H)，0.91(d，J＝6.5Hz，3H)

Example 125a3, 3-dimethyl-4- (6-nitropyridin-3-yl) piperazine-1-carboxylic acid tert-butyl esterEster 125a

To a 100-mL single neck round bottom flask equipped with a magnetic stirrer and reflux condenser was charged 5-bromo-2-nitropyridine (5.6g, 28.0mmol), 3-dimethyl-piperazine-1-carboxylic acid tert-butyl ester (3.0g, 14.0mmol), cesium carbonate (9.1g, 28mmol) and 1, 4-dioxane (50mL), nitrogen was bubbled through the resulting solution for 30 minutes, Binap (870mg, 1.4mmol) and tris (dibenzylideneacetone) dipalladium (0) (1.2g, 1.4mmol) were added, the reaction mixture was flushed with vacuum/argon for three cycles and stirred at 120 ℃ for 24H, after which the reaction mixture was cooled to room temperature, filtered and the filtrate was partitioned between ethyl acetate (200mL) and water (50mL), the aqueous layer was separated and extracted with ethyl acetate (3 × 50mL), the combined organic layers were washed with brine (50mL) and dried over sodium sulfate, the desiccant was removed by filtration and the filtrate was concentrated by chromatography [ lcm ] to obtain a residue [ lcm ] silica gel column (27.27.27%: petroleum ether: [ 1.0g, 27.1.2% ]]⁺337.2。

Example 125b4- (6-Aminopyridin-3-yl) -3, 3-dimethylpiperazine-1-carboxylic acid tert-butyl ester 125b

A50-mL round bottom flask was purged with nitrogen and charged with 125a (1100mg, 3.2mmol), 10% palladium on charcoal (10% wet, 110mg), and methanol (20 mL). Then, vacuum was applied, hydrogen gas was introduced, and the mixture was stirred at room temperature for 5 hours. The hydrogen was evacuated and the flask was purged with nitrogen. The catalyst was removed by filtration through a pad of celite and the filtrate was concentrated under reduced pressure to give 125b (950mg, 94%). LCMS: [ M + H ]]⁺307.3

Example 125c4- (6- (5-bromo-1-methyl-2-oxo-1, 2-dihydropyridin-3-ylamino) pyridin-3-yl) -3, 3-dimethylpiperazine-1-carboxylic acid tert-butyl ester 125c

A100-mL single-neck round bottom flask equipped with a magnetic stirrer and a reflux condenser was charged with 125b (950mg, 3.1mmol), 3, 5-dibromo-1-methylpyridin-2 (1H) -one (1240mg, 4.6mmol), 1, 4-dioxane (30mL), and cesium carbonate (2015mg, 6.2 mmol). Bubbling nitrogen gas through the obtainedAfter 5 minutes of solution, Xantphos (179mg, 0.31mmol) and tris (dibenzylideneacetone) dipalladium (0) (283mg, 0.31mmol) were added the reaction mixture was washed with vacuum/argon for three cycles and heated at reflux for 10H, after which the reaction mixture was cooled to room temperature and filtered, the filtrate was partitioned between ethyl acetate (50mL) and water (10mL), the aqueous layer was separated and extracted with ethyl acetate (3 × 20mL), the combined organic layers were washed with brine (30mL) and dried over sodium sulfate, the drying agent was removed by filtration and the filtrate was concentrated under reduced pressure, the residue was purified by silica gel column chromatography (eluting with 4:1 petroleum ether/ethyl acetate) to give 125c (1.21g, 79%). LCMS: [ M + H ] acetone]⁺492.1。

Example 125d5-bromo-3- (5- (2, 2-dimethylpiperazin-1-yl) pyridin-2-ylamino) -1-methylpyridin-2 (1H) -one 125d

Add 3M HCl in diethyl ether (15mL) to a solution of 125c (1.19g, 1.9mmol) in dichloromethane (20 mL). The reaction mixture was stirred at room temperature for 4 h. Then concentrated under reduced pressure to give 125d (900mg, 95%). LCMS: [ M + H ]]⁺392.1。

Example 125e5-bromo-3- (5- (2, 2-dimethyl-4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-ylamino) -1-methylpyridin-2 (1H) -one 125e

125d (900mg, 2.3mmol), oxetan-3-one (497mg, 6.9mmol), NaBH were stirred at 50 deg.C₃A mixture of CN (435mg, 6.9mmol) and zinc chloride (311mg, 2.3mmol) in methanol (30mL) was used for 4 h. Then concentrated under reduced pressure. Water (10mL) was added to the residue and CHCl was used₃(3X50mL) the mixture was extracted. The combined organic layers were concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (eluting with 50:1 dichloromethane/methanol) to give 125e (800mg, 78%). LCMS: [ M + H ]]⁺448.1。¹H NMR(500MHz，CDCl₃)8.65(d，J＝2.0Hz，1H)，8.11(d，J＝2.5Hz，1H)，7.85(s，1H)，7.37-7.34(m，1H)，6.96(d，J＝2.5Hz，1H)，6.72(d，J＝8.5Hz，1H)，4.69-4.61(m，4H)，3.60(s，3H)，3.50-3.14(m，3H)，2.43-2.17(m，4H)，1.06(s，6H)。

Example 125fAcetic acid 2- (5- (5- (2, 2-dimethyl-4- (oxetan-3-yl) piperazin-1-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 ] amino]Indol-2 (1H) -yl) benzyl ester 125f

A50-mL, single-necked, round-bottomed flask equipped with a magnetic stirrer and a reflux condenser was charged with 125e (190mg, 1.0 eq., 0.42mmol), acetic acid 4-fluoro-2- (1-oxo-3, 4,6,7,8, 9-hexahydropyrazino [1,2-a ] acetic acid]Indol-2 (1H) -yl) -6- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzyl ester 115m (405mg, 2.0 eq, 0.84mmol), PdCl₂(dppf) (33mg, 0.10 eq, 0.040mmol), K₃PO₄(178mg, 2.0 equiv., 0.84mmol), sodium acetate (69mg, 2.0 equiv., 0.84mmol), acetonitrile (20mL), and water (0.1 mL). After three cycles of vacuum/argon flushing, the mixture was heated at 90 ℃ for 2 h. Then cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure and the resulting residue was purified by silica gel column chromatography (eluting with 50:1 dichloromethane/ethanol) to give 125f (90mg, 29%) as a yellow solid. MS: [ M + H ]]⁺724.3。

Example 1252- (3- (5- (5- (2, 2-dimethyl-4- (oxetan-3-yl) piperazin-1-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 ] amino]Indol-1 (2H) -one 125

A50-mL single-neck round bottom flask was equipped with a magnetic stirrer and charged with 125f (85mg, 1 equivalent, 0.11mmol), lithium hydroxide (14mg, 5 equivalents, 0.55mmol), isopropanol (3mL), THF (3mL) and water (2 mL). The mixture was stirred at 30 ℃ for 1 h. Then filteringAnd the residue was concentrated under reduced pressure. The residue was purified by reverse phase preparative HPLC to give 125(43mg, 57%). MS: [ M + H ]]⁺682.4。¹HNMR(500MHz，DMSO)8.62(d，J＝2.0Hz，1H)，8.55(s，1H)，7.94(d，J＝2.5Hz，1H)，7.41-7.39(m，2H)，7.33-7.31(m，1H)，7.23-7.17(m，2H)，6.52(s，1H)，4.86(brs，1H)，4.54(t，J＝6.5Hz，2H)，4.42(t，J＝6.0Hz，2H)，4.30(s，2H)，4.17-4.11(m，3H)，3.89-3.86(m，1H)，3.58(m，3H)，3.39-3.36(m，1H)，3.08-2.98(m，2H)，2.63-2.56(m，2H)，2.46(t，J＝6.0Hz，2H)，2.33-2.12(m，4H)，1.80-1.67(m，4H)，0.96(s，6H)。

Example 126aAcetic acid 2- (5- (5- ((2S,5R) -2, 5-dimethyl-4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-ylamino) -1-methyl-6-oxo-1, 6-dihydropyridin-3-yl) -4-fluoro-6- (1-oxo-3, 4,6,7,8, 9-hexahydropyrido [3,4-b ] amino]Indolizin-2 (1H) -yl) benzyl ester 126a

A100-mL single-neck round bottom flask was charged with acetic acid 2-bromo-4-fluoro-6- (1-oxo-3, 4,6,7,8, 9-hexahydropyrido [3,4-b ]]Indolizin-2 (1H) -yl) benzyl ester 101H (347mg, 0.80mmol), 3- (5- ((2S,5R) -2, 5-dimethyl-4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-ylamino) -1-methyl-5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyridin-2 (1H) -one 121g (792mg, 1.6mmol), pd (dppf) Cl₂(32.7mg，0.040mmol)、K₃PO₄(340.0mg, 1.6mmol), sodium acetate trihydrate (217.6mg, 1.6mmol), water (0.5mL), and acetonitrile (50 mL). Evacuating the system and using N₂And (6) refilling. The reaction mixture was heated at 100 ℃ for 2 h. Then cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure and the resulting residue was purified by silica gel column chromatography (eluting with 25:1 dichloromethane/methanol) to give 126a (200mg, 34.6%) as a yellow-brown solid. MS: [ M + H ]]⁺724.5。

Example 1262- [3- [5- [ [5- [ (2S,5R) -2, 5-dimethyl-4- (oxetan-3-yl) piperazin-1-yl]-2-pyridyl]Amino group]-1-methyl-6-oxo-3-pyridinyl]-5-fluoro-2- (hydroxymethyl) phenyl]-3,4,6,7,8, 9-hexahydropyrido [3,4-b ]]Indolizin-1-ones 126

A mixture of 126a (150mg, 0.20mmol) and lithium hydroxide (72mg, 3.0mmol) in isopropanol/THF (5:3, 8.0mL) and water (2.0mL) was stirred at 30 ℃ for 1 h. The mixture was evaporated under reduced pressure and the residue was extracted with ethyl acetate (2X20 mL). The combined ethyl acetate extracts were concentrated under reduced pressure and the residue was purified by preparative HPLC to give 126(45mg, 33%) as a white solid. MS: [ M + H ]]⁺682.9。¹H NMR(500MHz，CHCl₃)8.60(dd，J＝2，5Hz，1H)，8.03(s，1H)，7.87(s，1H)，7.54(s，1H)，7.37-7.35(m，1H)，7.14-7.12(m，1H)，6.96-6.94(m，1H)，6.82-6.80(m，1H)，6.31(s，1H)，4.77-4.71(m，2H)，4.67-4.62(m，2H)，4.58-4.55(m，1H)，4.41-4.40(m，1H)，4.29-4.25(m，1H)，4.13-4.09(m，1H)，3.91-3.80(m，3H)，3.77-3.74(m，1H)，3.70(s，3H)，3.18(d，J＝5.0Hz，1H)，3.06-3.01(m，1H)，2.98-2.90(m，2H)，2.83(m，2H)，2.77-2.70(m，2H)，2.47(m，1H)，2.06-2.01(m，2H)，1.97-1.93(m，1H)，1.88-1.87(m，2H)，0.91-0.89(m，6H)。

Example 127a2- (hydroxy (pyridin-2-yl) meth) acrylate 127a

A250-mL single-neck round bottom flask was charged with chloroform (100mL), pyridine carboxaldehyde (10.7g, 0.10mol), methyl acrylate (8.60g, 0.10mol), and 1, 4-diazabicyclo [2.2.2 ]]Octane (0.560g, 5.00 mmol). The reaction mixture was stirred at room temperature for 48 h. After this time the reaction mixture is concentrated under reduced pressure and the residue is purified by column chromatography on silica gel (replacement with 3:1 petroleum ether)Ethyl acetate elution) afforded 127a (11.6g, 60%) as a dark yellow oil. MS-ESI: (M + H)⁺194.2。¹H NMR(500MHz，CDCl₃)8.54(d，J＝5.0Hz，1H)，7.69-7.66(m，1H)，7.42(d，J＝8.0Hz，1H)，7.22-7.20(m，1H)，6.36(s，1H)，5.97(s，1H)，5.62(s，1H)，4.85(s，1H)，3.74(s，3H)。

Example 127bIndolizine-2-carboxylic acid methyl ester 127b

To a 250-mL single neck round bottom flask equipped with a magnetic stirrer and reflux condenser was charged acetic anhydride (80mL) and 127a (6.68g, 34.6mmol), the reaction mixture was heated under reflux and nitrogen for 4H, after which the reaction mixture was cooled to room temperature, poured into a mixture of ice (100g) and saturated aqueous sodium bicarbonate (200mL), and stirred for 1H, the resulting solution was neutralized with saturated aqueous sodium bicarbonate and extracted with dichloromethane (3 × 200mL), the combined organic extracts were dried over sodium sulfate and concentrated under reduced pressure, the residue was purified by silica gel column chromatography (eluting with 10:1 petroleum ether/ethyl acetate (10: 1)) to give 127b (2.1g, 35%) as a white solid, MS-ESI (M + H)⁺176.2。¹H NMR(500MHz，CDCl₃)7.86-7.84(m，1H)，7.79(d，J＝1.0Hz，1H)，7.36-7.34(m，1H)，6.82(s，1H)，6.70-6.66(m，1H)，6.55-6.51(m，1H)，3.88(s，3H)。

Example 127c5,6,7, 8-Tetrahydroindazine-2-carboxylic acid methyl ester 127c

A250-mL round bottom flask was purged with nitrogen and charged with 127b (2.0g, 11.4mmol), 10% palladium on charcoal (50% wet, 200)mg) and methanol (50 mL). Vacuum-pumping, charging hydrogen and stirring at 5atm hydrogen and room temperature for 8 h. The hydrogen was then evacuated and the flask was charged with nitrogen. By passingThe catalyst was removed by pad filtration and the filtrate was concentrated under reduced pressure to give 127c (1.1g, 81%) as a white solid. MS-ESI: [ M + H ]]⁺180.3。¹H NMR(500MHz，DMSO-d₆)7.25(d，J＝2.0Hz，1H)，6.09(s，1H)，3.93(t，J＝6.0Hz，2H)，3.66(s，3H)，2.67(t，J＝6.0Hz，2H)，1.87-1.83(m，2H)，1.75-1.70(m，2H)。

Example 127d3-formyl-5, 6,7, 8-tetrahydroindazine-2-carboxylic acid methyl ester 127d

A100-mL round-bottom flask equipped with a magnetic stirrer was purged with nitrogen and charged with anhydrous dichloroethane (20mL) and anhydrous DMF (0.70mL, 9.0 mmol). To the cooled mixture at 0 ℃ was added phosphorus oxychloride (0.70mL, 7.3mmol) over a period of 2 minutes, and the reaction temperature was maintained between 0 and 10 ℃. The cooling bath was removed and the reaction mixture was stirred at room temperature for 1 hour. A solution of methyl 5,6,7, 8-tetrahydroindazine-2-carboxylate (127c) (1.0g, 5.6mmol) in acetonitrile (10mL) was added and the reaction mixture was stirred at room temperature for 3 hours. Thereafter, the mixture was concentrated under reduced pressure. With saturated NaHCO₃The aqueous solution (20mL) absorbed the oily residue and was extracted with ethyl acetate (3 × 50 mL). The combined organic layers were washed with water (50mL) and Na₂SO₄Dried and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (eluting with 1:5 ethyl acetate/petroleum ether) to give 127d (703mg, 58%) as a white solid. MS-ESI: (M + H)⁺208.3。¹H NMR(500MHz，DMSO-d₆)10.14(s，1H)，6.40(s，1H)，4.27(t，J＝6.0Hz，2H)，3.78(s，3H)，2.78(t，J＝6.0Hz，2H)，1.94-1.85(m，2H)，1.78-1.69(m，2H)。

Example 127e6,7,8, 9-Tetrahydropyridazo [4,5-b ]]Indolizin-1 (2H) -one 127e

To a 100-mL single-neck round-bottom flask equipped with a reflux condenser was charged methyl 3-formyl-5, 6,7, 8-tetrahydroindazine-2-carboxylate (127d) (600mg, 2.9mmol) and hydrazine hydroxide (20 mL). The reaction mixture was heated at 100 ℃ for 4 hours. The reaction mixture was then cooled to room temperature and filtered to give 127e (413mg, 75%) as a yellow solid. MS-ESI: (M + H)⁺190.1。¹H NMR(500MHz，DMSO-d₆)12.17(s，1H)，8.24(s，1H)，6.33(s，1H)，4.16(t，J＝6.0Hz，2H)，2.88(t，J＝6.5Hz，2H)，2.00-1.96(m，2H)，1.84-1.79(m，2H)。

Example 127f2-bromo-4-fluoro-6- (1-oxo-6, 7,8, 9-tetrahydropyridazino [4, 5-b)]Indolizin-2 (1H) -yl) benzaldehyde 127f

A100-mL single-neck round bottom flask equipped with a magnetic stirrer and a reflux condenser was charged with 127e (450mg, 2.4mmol), 2, 6-dibromo-4-fluorobenzaldehyde (2.0g, 7.2mmol), cesium carbonate (1.6g, 4.8mmol), and 1, 4-dioxane (50 mL). After bubbling nitrogen through the resulting mixture for 10 minutes, copper (I) iodide (450mg, 2.4mmol) and 4, 7-dimethoxy-1, 10-phenanthroline (571mg, 2.4mmol) were added, and the reaction mixture was heated at 90 ℃ for 12 h. The reaction mixture was then cooled to room temperature and filtered. The filtrate was partitioned between dichloromethane (40mL) and water (40 mL). The aqueous layer was separated and extracted with dichloromethane (3X30 mL). The combined organic layers were washed with brine (50mL) and dried over sodium sulfate. Removing the dried matter by filtrationDrying agent and concentrating the filtrate under reduced pressure. The residue was purified by silica gel column chromatography (eluting with 1:2 ethyl acetate/petroleum ether) to give 127f (251mg, 31%) as a brown solid. MS-ESI: (M + H)⁺390.0。

Example 127g4-fluoro-2- (1-methyl-5- (5- (2-methyl-4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-ylamino) -6-oxo-1, 6-dihydropyridin-3-yl) -6- (1-oxo-6, 7,8, 9-tetrahydropyridazino [4, 5-b)]Indolizin-2 (1H) -yl) benzaldehyde 127g

Into a 100-mL round bottom flask equipped with a magnetic stirrer and a reflux condenser was charged 127f (125.0mg, 0.32mmol), 1-methyl-3- (5- (2-methyl-4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-ylamino) -5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyridin-2 (1H) -one 113f (155.0mg, 0.32mmol), sodium acetate (53.0mg, 0.64mmol), K₃PO₄(135.7.0mg，0.64mmol)、PdCl₂(dppf) (50.0mg, 0.06mmol), acetonitrile (25mL) and water (1 mL). The system was flushed through 3 cycles with vacuum/argon and heated at 100 ℃ for 3 hours. Then evaporated under reduced pressure and the residue purified by silica gel column chromatography (eluting with 30:1 dichloromethane/methanol) to give 127g of compound (108mg, 51%) as a brown solid. MS: [ M + H ]]⁺665.4。

Example 1272- [ 5-fluoro-2- (hydroxymethyl) -3- [ 1-methyl-5- [ [5- [ (2S) -2-methyl-4- (oxetan-3-yl) piperazin-1-yl]-2-pyridyl]Amino group]-6-oxo-3-pyridinyl]Phenyl radical]-6,7,8, 9-tetrahydropyridazino [4,5-b]Indolizin-1-ones 127

Add NaBH to a solution of 127g (100.0mg, 0.15mmol) in methanol (20mL)₄(17.0mg, 0.45 mmol). The mixture was stirred at room temperature for 2 h. Then quenched with water (1mL) and the mixture was evaporated under reduced pressure. The residue was purified by reverse phase preparative HPLC to give 127 as a white solid (56mg, yield 56%). MS: [ M + H ]]⁺667.4。¹H NMR(500MHz，DMSO-d₆)8.58(d，J＝2.0，1H)，8.47(s，1H)，8.40(s，1H)，7.85(d，J＝2.5，1H)，7.39(d，J＝2.0，1H)，7.37-7.34(m，1H)，7.30-7.28(m，1H)，7.25-7.22(m，2H)，6.48(s，1H)，4.57-4.53(m，2H)，4.46(m，2H)，4.41(m，1H)，4.25(m，2H)，4.20(s，2H)，3.69-3.64(m，1H)，3.58(s，3H)，3.41-3.36(m，1H)，3.10-3.07(m，1H)，2.96-2.92(m，3H)，2.54-2.50(m，1H)，2.35-2.28(m，2H)，2.18(m，1H)，2.04-2.00(m，2H)，1.87-1.82(m，2H)，0.92(d，J＝6.0，3H)。

Example 128a2-bromo-6- {4, 4-dimethyl-9-oxo-1, 10-diazacyclo [6.4.0.0 ]^2,6]Dodeca-2 (6), 7-dien-10-yl } -4-fluorobenzaldehyde 128a

A100-mL round-bottom flask equipped with a reflux condenser was charged with 4, 4-dimethyl-1, 10-diazacyclo [6.4.0.0^2,6]Dodeca-2 (6), 7-dien-9-one 103e (1.0g, 4.90mmol, 1.0 eq.), 2-bromo-6-chloro-4-fluorobenzaldehyde (2.76g, 9.8mmol, 2.0 eq.), Pd₂(dba)₃(224mg, 0.24mmol, 0.050 equiv.), xanthphos (283mg, 0.49mmol, 0.10 equiv.), potassium acetate (1.44g, 14.7mmol, 3.0 equiv.), and 1, 4-dioxane (50 mL). Evacuating the system and using N₂And (6) refilling. The reaction mixture was heated at 80 ℃ for 5 h. The reaction mixture was then cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by column on silica gel (eluting with 80:1 dichloromethane/methanol) to give 128a (992mg, 50%) as a yellow solid. MS: [ M + H ]]⁺405.1

Example 128b2- {4, 4-dimethyl-9-oxo-1, 10-diazacyclo [6.4.0.0 ]^2,6]Dodeca-2 (6), 7-dien-10-yl } -4-fluoro-6- [ 1-methyl-5- ({5- [ (2S) -2-methyl-4- (oxetan-3-yl) piperazin-1-yl]Pyridin-2-yl } amino) -6-oxo-1, 6-dihydropyridin-3-yl]Benzaldehyde 128b

A50-mL round-bottom flask equipped with a magnetic stirrer and a reflux condenser was charged with 128a (303mg, 0.75mmol), 1-methyl-3- ({5- [ (2S) -2-methyl-4- (oxetan-3-yl) piperazin-1-yl]Pyridin-2-yl } amino) -5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxa-borolan-2-yl) -1, 2-dihydropyridin-2-one 113f (385mg, 0.80mmol), Pd (dppf) Cl₂(68.6mg, 0.075mmol), potassium acetate (147mg, 1.50mmol), K₃PO₄(327mg, 1.50mmol), acetonitrile (15mL), and water (6 drops). After three cycles of vacuum/argon flushing, the mixture was heated at 100 ℃ for 2 h. The reaction mixture was then cooled to room temperature. Then filtered and the filtrate evaporated under reduced pressure. The residue was purified by silica gel column chromatography (eluting with 15:1 ethyl acetate/methanol) to give 128b as a black solid (382mg, 77%). MS-ESI: [ M + H ]]⁺680.3

Example 1282- (7, 7-dimethyl-4-oxo-1, 2,6, 8-tetrahydrocyclopenta [3,4 ]]Pyrrolo [3,5-b]Pyrazin-3-yl) -4-fluoro-6- [ 1-methyl-5- [ [5- [ (2S) -2-methyl-4- (oxetan-3-yl) piperazin-1-yl]-2-pyridyl]Amino group]-6-oxo-3-pyridinyl]Benzoic acid 128

Add 2-methyl-2-butene (9.0mL, 107mmol) to a mixture of 128b (190mg, 0.28mmol), t-butanol (7mL) and dichloromethane (0.5 mL). NaClO is added dropwise at-10 DEG C₂(53mg, 0.59mmol) and NaH₂PO₄2 aqueous solution (2mL) of water (135.7mg, 0.87 mmol). The mixture was stirred for 1h at-10 ℃. Then treated with water (20mL) and extracted with ethyl acetate (4X50 mL). Over MgSO₄The combined organic extracts were dried, filtered and evaporated under reduced pressure. The residue was purified by reverse phase preparative HPLC to give 128(33mg, 17%) as a light yellow solid. MS-ESI: [ M + H ]]⁺696.2。¹H NMR(500MHz，DMSO-d₆)13.19-13.17(m，1H)，8.60(s，1H)，8.36(s，1H)，7.86(d，J＝2.5Hz，1H)，7.37-7.35(m，2H)，7.24-.7.22(m，3H)，6.46(s，1H)，4.57-4.54(m，2H)，4.48-4.47(m，1H)，4.42-4.41(m，1H)，4.10-4.09(m，2H)，3.95-3.92(m，1H)，3.68-3.67(m，1H)，3.56(s，3H)，3.42-3.39(m，2H)，3.09-3.07(m，1H)，2.96-2.94(m，1H)，2.92(s，3H)，2.41-2.40(m，2H)，2.35-2.31(m，2H)，2.20-2.17(m，1H)，1.21-1.20(m，6H)，0.93(d，J＝6.5Hz，3H)。

Example 129a2-bromo-6- {4, 4-dimethyl-9-oxo-1, 10-diazacyclo [6.4.0.0 ]^2,6]Dodeca-2 (6), 7-dien-10-yl } -4-fluorobenzoic acid 129a

To 2-bromo-6- {4, 4-dimethyl-9-oxo-1, 10-diazacyclo [6.4.0.0 ]^2,6]To a mixture of dodeca-2 (6), 7-dien-10-yl } -4-fluorobenzaldehyde 128a (810mg, 2.0mmol), tert-butanol (50mL) and dichloromethane (3mL) was added 2-methyl-2-butene (22mL, 262 mmol). NaClO is dripped at 0 DEG C₂(1.8g, 20.0mmol) and NaH₂PO₄Aqueous solution (20mL) of dihydrate (2.2g, 14.0 mmol). The mixture was stirred at 0 deg.C for 1h, then treated with water (30mL) and extracted with ethyl acetate (4 × 90 mL.) over MgSO₄The combined organic extracts were dried and concentrated under reduced pressure to give 129a (930mg, 84%) as a yellow solid. MS-ESI: [ M + H ]]⁺421.1

Example 129b2-bromo-6- {4, 4-dimethyl-9-oxo-1, 10-diazacyclo [6.4.0.0 ]^2,6]Dodeca-2 (6), 7-dien-10-yl } -4-fluoro-N-methylbenzamide 129b

A25-mL single neck round bottom flask equipped with a magnetic stirrer was charged with DMF (8mL), 129a (160mg, 0.38mmol), HATU (505mg, 1.33mmol), DMAP (46mg, 0.38mmol) and triethylamine (1.0 mL). The mixture was heated at 25 ℃ for 0.5 h. Then MeNH is added₂HCl (266mg, 3.8mmol) and the resulting mixture stirred at 25 ℃ for 2.5 h. The reaction mixture was then cooled to room temperature. Then filtered and the filtrate evaporated under reduced pressure. The residue was purified by preparative TLC (1: 20 methanol/dichloro)Methane developed) yielded 129b as a black solid (116mg, 70%). MS-ESI: [ M + H ]]⁺434.0

Example 1292- (7, 7-dimethyl-4-oxo-1, 2,6, 8-tetrahydrocyclopenta [3,4 ]]Pyrrolo [3,5-b]Pyrazin-3-yl) -4-fluoro-N-methyl-6- [ 1-methyl-5- [ [5- [ (2S) -2-methyl-4- (oxetan-3-yl) piperazin-1-yl]-2-pyridyl]Amino group]-6-oxo-3-pyridinyl]Benzamide 129

A25-mL single-neck round bottom flask equipped with a magnetic stirrer and a reflux condenser was charged with 129b (116mg, 0.27mmol), 1-methyl-3- ({5- [ (2S) -2-methyl-4- (oxetan-3-yl) piperazin-1-yl]Pyridin-2-yl } amino) -5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1, 2-dihydropyridin-2-one 113f (260mg, 0.54mmol), Pd (dppf) Cl₂(26mg, 0.030mmol), potassium acetate (53mg, 0.54mmol), K₃PO₄(117mg, 0.54mmol), acetonitrile (5mL), and water (0.50 mL). After three cycles of vacuum/argon purge, the mixture was heated at reflux for 2 h. The reaction mixture was then cooled to room temperature. Then filtered and the filtrate evaporated under reduced pressure. The residue was purified by reverse phase preparative HPLC to give 129 as a light yellow solid (80mg, 42%). MS-ESI: [ M + H ]]⁺709.5。¹H NMR(500MHz，DMSO-d₆)8.69(d，J＝2.0Hz，1H)，8.39(s，1H)，8.11(d，J＝4.5Hz，1H)，7.89(d，J＝2.5Hz，1H)，7.38-7.36(m，1H)，7.32-7.30(m，2H)，7.28-7.26(m，1H)，7.23-7.21(m，1H)，6.47(s，1H)，4.58-4.54(m，2H)，4.48-4.46(m，1H)，4.43-4.41(m，1H)，4.05-3.91(m，4H)，3.67-3.66(m，1H)，3.57(s，3H)，3.41-3.38(m，1H)，3.10-3.08(m，1H)，2.97-2.94(m，1H)，2.55-2.54(m，3H)，2.48-2.47(m，3H)，2.40-2.39(m，2H)，2.36-2.28(m，2H)，2.22-2.19(m，1H)，1.21-1.20(m，6H)，0.93(d，J＝6.5Hz，3H)。

Example 130a2-bromo-6- { 6-oxo-8-thia-4, 5-diazacyclo [7.4.0.0^2,7]Tridec-1 (9),2(7), 3-trien-5-yl) benzylAldehyde 130a

A25-mL, single-necked, round-bottomed flask equipped with a magnetic stirrer and reflux condenser was charged with 1, 4-dioxane (20mL), 8-thia-4, 5-diazacyclo [7.4.0.0 [^2,7]Tridec-1 (9),2(7), 3-trien-6-one 117c (618mg, 3.0mmol), 2, 6-dibromobenzaldehyde (1980mg, 7.5mmol), CuBr (215mg, 1.5mmol), sarcosine (267mg, 3.0mmol) and K₂CO₃(828mg, 6.0 mmol). After three cycles of vacuum/argon flushing, the mixture was heated at 100 ℃ for 16 h. The reaction mixture was then cooled to room temperature. Then filtered and the filtrate evaporated under reduced pressure. The residue was purified by silica gel column chromatography (eluting with 30:1 dichloromethane/methanol) to give 130a as a yellow solid (702mg, 60%). MS: [ M + H ]]⁺389.0

Example 130b2- [ 1-methyl-5- ({5- [ (2S) -2-methyl-4- (oxetan-3-yl) piperazin-1-yl]Pyridin-2-yl } amino) -6-oxopyridin-3-yl]-6- { 6-oxo-8-thia-4, 5-diazacyclo [7.4.0.0^2,7]Tridec-1 (9),2(7), 3-trien-5-yl } benzaldehyde 130b

Into a sealed tube equipped with a magnetic stirrer were charged 130a (160mg, 0.40mmol), (S) -1-methyl-5- (5- (2-methyl-4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-ylamino) -6-oxo-1, 6-dihydropyridin-3-ylboronic acid 113f (160mg, 0.40mmol), Pd (dppf) Cl₂(32mg, 0.040mmol), sodium acetate (66mg, 0.80mmol), K₃PO₄(170mg, 0.80mmol) and acetonitrile (6 mL). After three cycles of vacuum/argon flushing, the mixture was heated at 100 ℃ for 2 h. Then filtered and the filtrate evaporated under reduced pressure. The residue was purified by silica gel column chromatography (eluting with 30:1 dichloromethane/methanol) to give 130b as a yellow solid (123mg, 46%). LCMS: [ M + H ]]⁺664.3

Example 1303- [2- (hydroxymethyl) -3- [ 1-methyl-5- [ [5- [ (2S) -2-methyl-4- (oxetan-3-yl) piperazin-1-yl]-2-pyridyl]Amino group]-6-oxo-3-pyridinyl]Phenyl radical]-6,7,8, 9-tetrahydrobenzothiazolo 2,3-d]Pyridazin-4-ones 130

To a solution of 130b (120mg, 0.18mmol) in methanol (5mL) was added sodium borohydride (20mg, 0.54mmol) at 0 deg.C. The reaction mixture was stirred for 30 minutes. Then quenched with water (1mL) and concentrated under reduced pressure. The residue was purified by reverse phase preparative HPLC to give 130(70mg, 59%). LCMS: [ M + H ]]⁺666.3。¹H NMR(500MHz，CDCl₃)8.62(d，J＝2.0Hz，1H)，8.26(s，1H)，7.98(s，1H)，7.84(s，1H)，7.56-7.54(m，2H)，7.42(d，J＝2.5Hz，1H)，7.37(dd，J＝2.0，7.0Hz，1H)，7.31(d，J＝8.0Hz，1H)，6.83(d，J＝8.5Hz，1H)，4.72-4.66(m，4H)，4.36(d，J＝5.0Hz，2H)，4.05(t，J＝5.5Hz，1H)，3.72(s，3H)，3.55-3.54(m，1H)，3.46-3.45(m，1H)，3.08-3.06(m，2H)，2.99(t，J＝5.0Hz，2H)，2.87(t，J＝5.0Hz，2H)，2.60-2.59(m，1H)，2.49-2.48(m，2H)，2.20-2.19(m，1H)，2.02-1.96(m，4H)，0.99(d，J＝6.0Hz，3H)。

Example 131a3, 3-dimethylcyclopentanone 131a

To a suspension of CuI (81.0g, 420mmol) in dry ether (500mL) cooled to 0 deg.C was added a solution of methyllithium in ether (430mL, 860mmol, 2.0M) over 30 minutes. See fig. 15. The mixture was stirred at 0 ℃ for 2 h. To the above mixture was added dropwise 3-methylcyclopent-2-enone (33.6g, 350mmol) at 0 ℃ over 1 h. The resulting mixture was stirred for an additional 2h at 0 ℃. Then with saturated NH₄Cl (300mL) was quenched and filtered. The filtrate was extracted with diethyl ether (2X200 mL). By anhydrous Mg₂SO₄The combined organic layers were dried and filtered. The filtrate was evaporated under reduced pressure to give 131a (28g, 71%) as a colorless oil.¹H NMR(500MHz，DMSO-d₆)2.31(t，J＝8.0Hz，2H)，2.05(s，2H)，1.79(t，J＝8.0Hz，2H)，1.12(s，6H)。

Example 131b2-chloro-4, 4-dimethylcyclopent-1-enecarbaldehyde 131b

To a 0 deg.C cooled solution of DMF (18.3g, 250mmol) in dichloromethane (300mL) was added POCl over a period of 10 minutes₃(40.5g, 250 mmol). See fig. 15. The mixture was stirred at 20 ℃ for 1 h. To the above mixture was added 131a (28.0g, 250mmol) dropwise over a period of 20 minutes. The resulting mixture was heated at reflux for 20 h. The reaction mixture was cooled to room temperature and poured into a solution of sodium acetate (60g) in ice-water (400 g). The mixture was extracted with dichloromethane (2X300 mL). The combined organic layers were washed with water (2X200mL), anhydrous Mg₂SO₄Dried and filtered. The filtrate was evaporated under reduced pressure to give 131b (33.0g, crude) as a colorless oil.¹H NMR(500MHz，DMSO-d₆)9.99(s，1H)，2.62(d，J＝2.0Hz，2H)，2.38(d，J＝2.0Hz，2H)，1.15(s，6H)。

Example 131c5, 5-dimethyl-5, 6-dihydro-4H-cyclopenta [ b)]Thiophene-2-carboxylic acid ethyl ester 131c

Add 2-mercaptoacetic acid ethyl ester (19.2g, 160mmol) to a solution of 131b (33.0g, crude) in dichloromethane (400mL) and triethylamine (60g, 600 mmol). See fig. 15. The reaction mixture was heated at reflux for 6 h. Then concentrated under reduced pressure. The residue was dissolved in ethanol (400mL) and triethylamine (60g, 600 mmol). The mixture was heated at reflux for 12 h. Again concentrated under reduced pressure and the residue purified by silica gel column chromatography (eluting with 40:1 petroleum ether/ethyl acetate) to give 131c as a yellow solid (18.0g, 32% over two steps). MS-ESI: [ M + H ]]⁺225.3。¹H NMR(500MHz，DMSO-d₆)7.49(s，1H)，4.32(q，J＝7.0Hz，2H)，2.72(s，2H)，2.56(s，2H)，1.35(t，J＝7.0Hz，3H)，1.22(s，6H)。

Example 131d5, 5-dimethyl-5, 6-dihydro-4H-cyclopenta [ b)]Thiophene-2-carboxylic acid 131d

To a solution of 131c (16.0g, 71.0mmol) in 2-propanol (200mL), tetrahydrofuran (200mL) and water (200mL) was added lithium hydroxide (6.82g, 284 mmol). See fig. 15. The reaction mixture was heated at 65 ℃ for 5 h. The organic solvent was removed under reduced pressure. The pH of the residue was adjusted to 1.0 with hydrochloric acid (12M). The precipitate was collected by filtration and dried in vacuo to give 131d (12.0g, 86%) as a white solid. MS-ESI: [ M + H ]]⁺196.9

Example 131eN-tert-butyl-5, 5-dimethyl-5, 6-dihydro-4H-cyclopenta [ b)]Thiophene-2-carboxamide 131e

131d (12.0g, 61.0mmol) in SOCl were heated at 65 deg.C₂Suspension in (80mL) for 2 h. The reaction mixture was concentrated under reduced pressure. See fig. 15. The residue was washed with dichloromethane (20mL) and added to a solution of 2-methylpropan-2-amine (4.45g, 61.0mmol) and triethylamine (18.0g, 180mmol) in dichloromethane (180 mL). The resulting mixture was stirred for 16h and diluted with dichloromethane (200 mL). Washed with water (3X50mL) over anhydrous Mg₂SO₄Drying, filtration and evaporation under reduced pressure gave 131e as a yellow solid (15.0g, 97%). MS-ESI: [ M + H ]]⁺252.0

Example 131fN-tert-butyl-3-formyl-5, 5-dimethyl-5, 6-dihydro-4H-cyclopenta [ b]Thiophene-2-carboxamide 131f

To a solution of 131e (1.5g, 6.0mmol) in anhydrous THF (60mL) cooled to-70 deg.C was added over a period of 5 minutes an n-butyllithium solution (10.0mL, 25mmol, 2.5M in hexanes). See fig. 15. Stirring at-70 ℃ for 6 h. DMF (1.3g, 18.0mmol) was added over 5 minutes and the resulting mixture was stirred at room temperature overnight. Then with saturated NH₄Cl (40mL) was quenched and concentrated under reduced pressure. The residue was extracted with ethyl acetate (2X30 mL). By anhydrous Mg₂SO₄The combined organic layers were dried and filtered. The filtrate was evaporated under reduced pressure to give 131f (1.34g, 80%) as a yellow solid. MS-ESI: [ M + H ]]⁺280.3

Example 131gN-tert-butyl-3- (hydrazonomethyl) -5, 5-dimethyl-5, 6-dihydro-4H-cyclopenta [ b]Thiophene-2-carboxamide 131g

To a solution of 85% aqueous hydrazine (10mL) in THF (180mL) was added a solution of 131f (5.6g, 20.0mmol) in dry THF (20mL) over a period of 5 minutes. See fig. 15. Stirred for 3h at 20 ℃. The reaction mixture was concentrated under reduced pressure to obtain 131g (6.0g, yield: 95%, purity: 95%) as a black solid. MS-ESI: [ M + H ]]⁺294.0

Example 131h4, 4-dimethyl-7-thia-10, 11-diazacyclo [6.4.0.0 ]^2,6]131h of dodeca-1 (8),2(6), 11-trien-9-one

131g (3.8g, 13.0mmol) were heated at reflux in 30% H₂SO₄(100mL) for 16 h. See fig. 15. The reaction mixture was cooled to room temperature and extracted with dichloromethane (3 × 200 mL). The combined organic layers were concentrated under reduced pressure and the residue was purified by silica gel column chromatography (eluting with 100:1 dichloromethane/methanol) to give 131h (1.72g, 60%) as a yellow solid. MS-ESI: [ M + H ]]⁺221.0

Example 131i2-bromo-6- {4, 4-dimethyl-9-oxo-7-thia-10, 11-diazacyclo [6.4.0.0 ]^{2 ,6}]Dodeca-1 (8),2(6), 11-trien-10-yl } -4-fluorobenzaldehyde 131i

Into a 100-mL round-bottom flask equipped with a magnetic stirrer and a reflux condenser were charged 131h (330mg, 1.5mmol), 2, 6-dibromo-4-fluorobenzaldehyde (1.26g, 4.5mmol), CuBr (113mg, 0.8mmol), sarcosine (142mg, 1.6mmol), K₂CO₃(420mg, 3.0mmol) and dioxane (20 mL). See fig. 15. After three cycles of vacuum/argon flushing, the mixture was heated at 95 ℃ for 15 h. Then cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure and the resulting residue was purified by silica gel column chromatography (using 5:1 petroleum ether/ethyl acetate) gave 131i as a white solid (380mg, 60%). MS-ESI: [ M + H ]]⁺420.6

Example 131j2- {4, 4-dimethyl-9-oxo-7-thia-10, 11-diazacyclo [6.4.0.0^2,6]Dodeca-1 (8),2(6), 11-trien-10-yl } -4-fluoro-6- [ 1-methyl-5- ({5- [ (2S) -2-methyl-4- (oxetan-3-yl) piperazin-1-yl]Pyridin-2-yl } amino) -6-oxo-1, 6-dihydropyridin-3-yl]Benzaldehyde 131j

Into a 50-mL round bottom flask equipped with a magnetic stirrer and a reflux condenser were charged 131i (421mg, 1.0mmol), (S) -1-methyl-3- (5- (2-methyl-4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-ylamino) -5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyridin-2 (1H) -one 113f (580mg, 1.2mmol), Pd (dppf) Cl₂(59mg，0.080mmol)、K₃PO₄Trihydrate (360mg, 1.6mmol), water (6 drops) and tetrahydrofuran (20 mL). After three cycles of vacuum/argon flushing, the mixture was heated at reflux for 6 h. Then cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure and the residue was washed with 1:1 petroleum ether/ethyl acetate (20mL) to give 131j (556mg, 80%) as a white solid. MS-ESI: [ M + H ]]⁺696.3

Example 1313- [ 5-fluoro-2- (hydroxymethyl) -3- [ 1-methyl-5- [ [5- [ (2S) -2-methyl-4- (oxetan-3-yl) piperazin-1-yl]-2-pyridyl]Amino group]-6-oxo-3-pyridinyl]Phenyl radical]-7, 7-dimethyl-6, 8-dihydrocyclopenta [3,4 ]]Thieno [1,3-d ]]Pyridazin-4-one 131

To a solution of 131j (520mg, 0.75mmol) in methanol (10mL) was added sodium borohydride (110mg, 3.0mmol) at 20 ℃. The reaction mixture was stirred for 30 minutes and quenched with water (3 mL). Then concentrating under reduced pressure and preparing with reverse phaseHPLC purification of the residue gave 131(340mg, 65%). MS-ESI: [ M + H ]]⁺698.3。¹H NMR(500MHz，DMSO-d₆)8.55(d，J＝2.5Hz，1H)，8.46(s，1H)，8.41(s，1H)，7.85(d，J＝2.5Hz，1H)，7.40-7.30(m，4H)，7.23(d，J＝9.0Hz，1H)，4.60(t，J＝5.0Hz，1H)，4.57-4.53(m，2H)，4.46(t，J＝6.0Hz，1H)，4.41(t，J＝6.0Hz，1H)，4.30-4.29(m，2H)，3.68-3.65(m，1H)，3.58(s，3H)，3.40-3.38(m，1H)，3.10-3.07(m，1H)，2.95-2.94(m，1H)，2.91-2.89(m，2H)，2.80-2.78(m，2H)，2.54-2.52(m，1H)，2.34-2.30(m，2H)，2.20-2.16(m，1H)，1.27(s，6H)，0.93(d，J＝6.5Hz，3H)。

Example 901Biochemical assay for Btk

A general method for a standard biochemical Btk kinase assay that can be used to test compounds of formula I is as follows.1 Xcell signal transduction kinase-containing buffer (25mM Tris-HCl, pH7.5, 5mM β -phosphoglycerol, 2mM dithiothreitol, 0.1mM Na₃VO₄、10mM MgCl₂) 0.5 μ M Promega PTK biotinylated peptide substrate 2 and 0.01% BSA premixed reagent without Btk enzyme (master mix). Premixed reagents containing Btk enzyme were prepared containing 1X cell signal transduction kinase buffer, 0.5 μ M PTK biotinylated peptide substrate 2, 0.01% BSA, and 100 ng/well (0.06 mU/well) of Btk enzyme. The Btk enzyme was prepared as follows: full-length human wild-type Btk (accession No. NM-000061) with C-terminal V5 and a 6 × His tag was subcloned into the pFastBac vector for the preparation of baculoviruses carrying this epitope-tagged Btk. Baculovirus was prepared according to the instructions detailed by Invitrogen in the published experimental protocol "Bac-to-Baculoviral 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. According to the sensitive Sypro-Ruby staining method, the purity of the final protein product is more than 95%. A200. mu.M ATP solution was prepared in water and adjusted to pH7.4 with 1N NaOH. Transfer a 1.25. mu.L amount of compound in 5% DMSO to 96-well half-zone Costar polystyreneAnd (3) a plate. Compounds were tested individually and with an 11-point dose response curve (starting concentration 10. mu.M; 1:2 dilution). An amount of 18.75 μ L of enzyme-free premixed reagent (as a negative control) and enzyme-containing premixed reagent were transferred to appropriate wells in a 96-well half-zone Costar polystyrene plate. mu.L of 200. mu.M ATP was added to the mixture in 96-well half-zone Costar polystyrene plates to give a final ATP concentration of 40. mu.M. The reaction mixture was incubated at room temperature for 1 hour. The reaction was stopped with Perkin Elmer1X assay buffer containing 30mM EDTA, 20nM SA-APC and 1nM PT66 Ab. Time resolved fluorescence read plates were used with a Perkin elmer envision using an excitation filter 330nm, an emission filter 665nm, and a second emission filter 615 nm. Then calculate the IC₅₀The value is obtained. Alternatively, the Lanthascreen assay can be used to assess Btk activity by quantifying its phosphorylated peptide product. The FRET (fluorescence resonance energy transition) that occurs between fluorescein on the peptide product and terbium on the detection antibody decreases with the addition of Btk inhibitors that decrease phosphorylation of the peptide. Btk (h) (0.1ng/25uL reaction) was reacted with 50mM Hepes pH7.5, 10mM MgCl in a final reaction volume of 25uL₂、2mMMnCl₂、2mM DTT、0.2mM NaVO₄0.01% BSA and 0.4uM fluorescein poly-GAT. The reaction was initiated by adding ATP to 25uM (Km of ATP). After incubation at room temperature for 60 minutes, the reaction was stopped by adding a detection antibody at a final concentration of 2nMTb-PY20 in 60mM EDTA for 30 minutes at room temperature. Detection was performed on a Perkin Elmer Envision using excitation at 340nm and emission at 495nm and 520 nm. Exemplary Btk inhibition IC50 values are shown in tables 1,2, and 3.

Example 902Ramos cell Btk assay

Another general method for a standard cellular Btk kinase assay that can be used to test compounds of formula I is as follows. At 0.5x10⁷The Ramos cells were cultured at a density of individual cells/ml in the presence of the test compound at 37 ℃ for 1 hour. Then by using 10. mu.g/ml of anti-human IgM F (ab)₂Cells were stimulated by incubation at 37 ℃ for 5 minutes. The cells were pelleted, lysed, and the clear lysate was subjected to protein assay. Peer-to-peerEach sample of protein amounts was subjected to SDS-PAGE and Western blotted with anti-Btk phosphate (Tyr223) antibody (CellSignaling Technology # 3531; Epitomics, cat. #2207-1) or Btk phosphate (Tyr551) antibody (BD Transduction Labs #558034) to assess Btk autophosphorylation or to control the total amount of Btk in each solution with anti-Btk antibody (BD Transduction Labs # 611116).

Example 903B cell proliferation assay

A general method for a standard cellular 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. su. 090. su. 862). Test compounds were diluted in 0.25% DMSO with 2.5x10⁵Purified mouse splenic B cells were cultured for 30 minutes, then 10. mu.g/ml anti-mouse IgM antibody (Southern Biotechnology Associates Cat #1022-01) was added, and the final volume was 100. mu.l. After 24 hours of incubation, 1. mu. Ci was added³H-thymidine, the plate was cultured for another 36 hours, and then SPA [2 ] was used as the manufacturer's reference³H]Protocol collection for thymidine uptake assay system (Amersham Biosciences # RPNQ 0130). SPA bead-based fluorescence was counted in a Microbeta counter (Wallace Triplex1450, Perkinelmer).

Example 904T cell proliferation assay

A 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 whole T cell isolation kit (Miltenyi Biotech, Cat # 130-. Test compounds were diluted in 0.25% DMSO and incubated with 2.5x10⁵Individual purified mouse splenic T cells were cultured together in a final volume of 100 μ l in clear flat bottom plates pre-coated with anti-CD 3(BD #553057) and anti-CD 28(BD #553294) antibodies each at 10 μ g/ml for 90 minutes at 37 ℃. After 24 hours of incubation, 1. mu. Ci was added³H-thymidine, the plate was cultured for another 36 hours, and then SPA [2 ] was used as the manufacturer's reference³H]Protocol collection for thymidine uptake assay system (Amersham Biosciences # RPNQ 0130). SPA bead-based fluorescence was counted in a Microbeta counter (Wallace Triplex1450, Perkin Elmer).

Example 905CD86 inhibition assay

A general method for a standard assay that can be used to test compounds of formula I for their B cell inhibitory activity is as follows. Total mouse splenocytes were purified from the spleens of 8-16 week old Balb/c mice by red blood cell lysis (BD Pharmingen # 555899). Test compounds were diluted in 0.5% DMSO and incubated with 1.25x10 in a clear flat bottom plate (Falcon353072)⁶Individual splenocytes were cultured in a final volume of 200 μ l for 60 minutes at 37 ℃. Cells were then stimulated with 15. mu.g/ml IgM (Jackson Immunoresearch115-006-₂And culturing for 24 hours. After 24 hours of culture, cells were transferred to conical-bottom clear 96-well plates and pelleted by centrifugation at 1200x g x5 min. Cells were pre-blocked (preblock) with CD16/CD32(BDPharmingen #553142) followed by triple staining with CD19-FITC (BD Pharmingen #553785), CD86-PE (BD Pharmingen #553692) and 7AAD (BD Pharmingen # 51-68981E). Cell sorting on BDFACSCalibur and targeting CD19⁺The/7 AAD-group is provided with a gate (gated). The level of surface expression of CD86 on the population corresponding to the concentration of the test compound was measured.

Example 906B-ALL cell survival assay

The following is a method of a standard B-ALL (acute lymphocytic leukemia) cell survival study using an XTT reader to measure the number of viable cells. This assay can be used to test the ability of compounds of formula I to inhibit the survival of B-ALL cells in culture. One human B-cell acute lymphocytic leukemia line that can be used is SUP-B15, a human pre-B-cell ALL line available from ATCC.

At 5x10⁵Concentration of individual cells/mlSUP-B15 pre-B-ALL cells were seeded in 100. mu.l Iscove medium + 20% FBS in multiple 96-well microtiter plates. Test compounds were then added to give a final concentration of 0.4% DMSO. Cells were incubated at 37 ℃ and 5% CO₂Incubate for up to 3 days. Three days later, cells were divided 1:3 into fresh 96-well plates containing test compounds and allowed to grow for an additional 3 days. After every 24h period, 50ul of XTT solution was added to one replicate 96-well plate and absorbance readings were taken at 2, 4 and 20 hours as instructed by the manufacturer. OD readings were then taken for cells treated with DMSO only within the linear range of the assay (0.5-1.5), and the percentage of viable cells in compound-treated wells relative to DMSO-only treated cells was measured.

Example 907CD69 Whole blood assay

Human blood was obtained from healthy volunteers with the following limitations: no medicine is taken and no smoke is drawn after 1 week. By intravenous puncture containing heparin sodium(Becton, Dickinson and Co.) tubes collect blood (approximately 20ml to test 8 compounds).

A 10mM solution of the compound of formula I in DMSO was diluted 1:10 in 100% DMSO, then diluted in three-fold serial dilutions in 100% DMSO for a 10-point dose-response curve. Compounds were further diluted 1:10 in PBS and then 5.5 μ l aliquots of each compound were added in duplicate to 2ml96 well plates; 5.5 μ l of 10% DMSO/PBS was added as a control and no wells were stimulated. Human whole blood-HWB (100. mu.l) was added to each well. After mixing, the plates were incubated at 37 ℃ with 5% CO₂And incubated at 100% humidity for 30 minutes. Goat F (ab') 2 anti-human IgM (10. mu.l of 500. mu.g/ml solution, 50. mu.g/ml final) was added to each well (except for non-stimulated wells) and mixed, and the plates were incubated for another 20 hours. At the end of the 20 hour incubation, the sample was incubated with fluorescently labeled antibody at 37 ℃ with 5% CO₂Incubate at 100% humidity for 30 minutes. Controls for Compensation including InductionDyed and single dyed, and initial voltage settings. Then PharM Lyse was used according to the manufacturer's instructions^TM(BD Biosciences Pharmingen) the sample was dissolved. The samples were then transferred to 96-well plates suitable for working on the BDBiosciences HTS96 well system on the LSRII instrument. The collected data and the mean fluorescence intensity values were obtained using BD Biosciences DIVA software. The results were analyzed primarily by FACS analysis software (Flow Jo). The inhibitory concentration of the test compound (IC50, IC70, IC90, etc.) is defined as the concentration at which CD69 positive cells that are also CD20 positive by anti-IgM stimulation are reduced by, for example, 50% (average of 8 control wells after subtraction of average of 8 wells without stimulating background). IC70 values were calculated by Prism version 5 using a non-linear regression curve fit, and the results are shown in tables 1 and 2.

Example 908In vitro cell proliferation assay

The efficacy of the compounds of formula I was measured by a cell proliferation assay using the following protocol (Mendoza et al (2002) Cancer Res.62: 5485-.The luminescent cell viability assay (including reagents and protocols) is commercially available (Promega corp., Madison, WI, Technical Bulletin TB 288). This assay assesses the ability of a compound to enter cells and inhibit cell proliferation. The assay principle is based on determining the number of viable cells present by quantifying the ATP present in a homogeneous assay, wherein the addition of Cell-Titer Glo reagent results in lysis of the cells and the generation of a luminescent signal by the luciferase reaction. The luminescent signal is proportional to the amount of ATP present.

A panel of B-cell lymphoma cell lines (BJAB, SUDHL-4, TMD8, OCI-Ly10, OCI-Ly3, WSU-DLCL2) were placed in 384-well plates of normal growth medium and serial dilutions of BTK inhibitor or DMSO were added individually to each well. After incubation for 96 hours by(Promega) to assess cell viability. Data can be expressed as relative cell viability of BTK inhibitor treated cells relative to DMSO treated control cells. Data points are the average of 4 replicates for each dose level. Error bars represent SD of the mean.

The procedure is as follows: day 1-seeded cell plates (384-well black, clear-bottomed (slightly clear) TC plates with lids from Falcon # 353962), cells were harvested and seeded at 1000 cells/54 μ Ι/well into 384-well cell plates for 3-day assays. Cell culture medium: RPMI or DMEM high glucose, 10% fetal bovine serum, 2mM L-glutamine, P/S. Incubate O/N at 37 ℃ under 5% CO 2.

Day 2-drug was added to cells, compounds were diluted, DMSO plates (1: 2, 9 spots in succession), and 10mM of 20 μ l of compound was added in column 2 of 96-well plates. For a total of 9 spots, 1:2 serial dilutions (10. mu.l + 20. mu.l 100% DMSO) were performed throughout the plate using Precision. The culture substrate was a 96-well conical-bottom polypropylene plate (1:50 dilution) from Nunc (catalog No. 249946). 147. mu.l of medium was added to all wells. 3 μ l of DMSO + compound from each well of the DMSO plate was transferred to each corresponding well on the culture plate using Rapidplate.

Drugs were added to the cells, the cell plates (1:10 dilution), and 6 μ l of medium + compounds were added directly to the cells (54 μ l of medium already on the cells). Incubate for 3 days at 37C, 5% CO2 in an incubator that is not often open.

Day 5-incubate plates, Thaw Cell Titer Glo buffer at room temperature. The cell plates were removed from 37 ℃ and equilibrated to room temperature for about 30 minutes. Cell Titer Glo buffer was added to Cell Titer Glo substrate (bottle to bottle). Mu.l of Cell Titer Glo reagent (Promega catalog number G7572) was added to each well of cells. Leave on the plate shaker for about 30 minutes. Luminescence (half second/well) was read on an Analyst HT plate reader.

Cell viability assay and combination assay: cells were seeded into 384-well plates at 1000-. The following day, 9 times 1:2 in DMSO in 96-well platesThe compounds were serially diluted. Compounds were further diluted in growth medium using a Rapidplate automated instrument (Zymark corp., Hopkinton, MA). The diluted compounds were then added in quadruplicate to wells of 384-well cell plates and incubated at 37 ℃ and 5% CO 2. After 4 days, the relative number of viable cells was measured using Cell-Titer Glo (Promega) luminescence according to the manufacturer's instructions and read on a wallammultilabel reader (PerkinElmer, Foster City). Use of4.0 software (GraphPad, San Diego) calculate EC50 values. The compound of formula I and chemotherapeutic agent are added simultaneously or 4 hours apart (one before the other) in all assays.

Additional exemplary in vitro cell proliferation assays include the following steps:

1. will contain about 10 in the culture medium⁴Aliquots of 100. mu.l of cell culture of individual cells were placed into each well of a 384-well opaque wall plate.

2. Control wells containing media without cells were prepared.

3. Compounds were added to the experimental wells and incubated for 3-5 days.

4. The plate was equilibrated to room temperature for about 30 minutes.

5. The CellTiter-Glo reagent was added in the same volume as the volume of cell culture medium present in each well.

6. The contents were mixed on an orbital shaker for 2 minutes to induce cell lysis.

7. The plate was incubated at room temperature for 10 minutes to stabilize the luminescence signal.

8. Luminescence was recorded and reported in the figure as RLU ═ relative luminescence units.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the description and example should not be construed as limiting the scope of the invention. Accordingly, all suitable modifications and equivalents may be resorted to, falling within the scope of the invention as defined by the claims that follow. The disclosures of all patent and scientific documents cited in this application are expressly incorporated herein by reference in their entirety.

Claims (18)

1. A compound selected from formula I:

or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, wherein:

R¹and R³Independently is H;

R²h and F;

R⁴is selected from-CH₂OH and-C (O) NHCH₃；

R⁵Is selected from-CH₃、-CH₂CH₃and-CH₂F；

Or two R⁵The groups form a 6-membered heterocyclic ring;

n is 1 or 2;

R⁶is selected from-CH₃；

R⁷Selected from the following structures:

wherein the wavy line indicates the attachment site;

R⁸is selected from-CH₃And oxetan-3-yl;

X¹is CR⁹Wherein R is⁹Is selected from H;

X²is CR¹⁰Wherein R is¹⁰Is selected from H; and is

Y¹And Y²Independently selected from CH;

with the proviso that the compound is not:

2. the compound of claim 1, wherein R⁴is-CH₂OH。

3. The compound of claim 2, wherein R²Is F.

4. The compound of claim 1 selected from the group consisting of:

(S) -2- (5-fluoro-2- (hydroxymethyl) -3- (1-methyl-5- (5- (2-methyl-4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-ylamino) -6-oxo-1, 6-dihydropyridin-3-yl) phenyl) -3,4,6,7,8, 9-hexahydropyrido [3,4-b ] indolizin-1 (2H) -one;

(S) -5- [ 5-fluoro-2- (hydroxymethyl) -3 (1-methyl-5- (5- (2-methyl-4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-ylamino) -6-oxo-1, 6-dihydropyridin-3-yl) phenyl ] -8-thia-4, 5-diazacyclo [7.4.0.02,7] tridec-1 (9),2(7), 3-trien-6-one;

(2S) -10- [ 5-fluoro-2- (hydroxymethyl) -3- [ 1-methyl-5- ({5- [ 2-methyl-4- (oxetan-3-yl) piperazin-1-yl ] pyridin-2-yl } amino) -6-oxo-1, 6-dihydropyridin-3-yl ] phenyl ] -4, 4-dimethyl-1, 10-diazacyclo [6.4.0.02,6] dodeca-2 (6), 7-dien-9-one;

2- (3- (5- (5- ((2S,5R) -2, 5-dimethyl-4- (oxetan-3-yl) piperazin-1-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;

(S) -2- (3- (5- (5- (2-ethyl-4- (oxetan-3-yl) piperazin-1-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;

(S) -2- (5-fluoro-2- (hydroxymethyl) -3- (1-methyl-5- (5- (2-methyl-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;

(S) -2- (3- (5- (5- (3, 4-dimethylpiperazin-1-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;

(R) -2- (3- (5- (5- (3, 4-dimethylpiperazin-1-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- α ] indol-1 (2H) -one;

(R) -2- (3- (5- (5- (2, 4-dimethylpiperazin-1-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;

(S) -2- (3- (5- (5- (2, 4-dimethylpiperazin-1-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;

(S) -7, 7-difluoro-2- (5-fluoro-2- (hydroxymethyl) -3- (1-methyl-5- (5- (2-methyl-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;

2- (3- {5- [5- ((S) -2-ethyl-4-oxetan-3-yl-piperazin-1-yl) -pyridin-2-ylamino ] -1-methyl-6-oxo-1, 6-dihydro-pyridin-3-yl } -5-fluoro-2-hydroxymethyl-phenyl) -7, 7-dimethyl-3, 4,7, 8-tetrahydro-2H, 6H-cyclopenta [4,5] pyrrolo [1,2-a ] pyrazin-1-one;

(R) -2- (5-fluoro-2- (hydroxymethyl) -3- (1-methyl-5- (5- (2-methyl-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;

2- (3- {5- [5- ((2S,5R) -2, 5-dimethyl-4-oxetan-3-yl-piperazin-1-yl) -pyridin-2-ylamino ] -1-methyl-6-oxo-1, 6-dihydro-pyridin-3-yl } -5-fluoro-2-hydroxymethyl-phenyl) -7, 7-dimethyl-3, 4,7, 8-tetrahydro-2H, 6H-cyclopenta [4,5] pyrrolo [1,2-a ] pyrazin-1-one;

3- (5-fluoro-2-hydroxymethyl-3- { 1-methyl-5- [5- ((R) -2-methyl-4-oxetan-3-yl-piperazin-1-yl) -pyridin-2-ylamino ] -6-oxo-1, 6-dihydro-pyridin-3-yl } -phenyl) -6,7,8, 9-tetrahydro-3H-benzo [4,5] thieno [2,3-d ] pyridazin-4-one;

3- (3- {5- [5- ((2S,5R) -2, 5-dimethyl-4-oxetan-3-yl-piperazin-1-yl) -pyridin-2-ylamino ] -1-methyl-6-oxo-1, 6-dihydro-pyridin-3-yl } -5-fluoro-2-hydroxymethyl-phenyl) -6,7,8, 9-tetrahydro-3H-benzo [4,5] thieno [2,3-d ] pyridazin-4-one;

(S) -10-fluoro-2- (5-fluoro-2- (hydroxymethyl) -3- (1-methyl-5- (5- (2-methyl-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;

3- (3- {5- [5- ((S) -2-ethyl-4-oxetan-3-yl-piperazin-1-yl) -pyridin-2-ylamino ] -1-methyl-6-oxo-1, 6-dihydro-pyridin-3-yl } -5-fluoro-2-hydroxymethyl-phenyl) -6,7,8, 9-tetrahydro-3H-benzo [4,5] thieno [2,3-d ] pyridazin-4-one;

2- (3- (5- (5- ((2S,5R) -2, 5-dimethyl-4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-ylamino) -1-methyl-6-oxo-1, 6-dihydropyridin-3-yl) -5-fluoro-2- (hydroxymethyl) phenyl) -6,7,8, 9-tetrahydropyrazino [1,2-a ] indol-1 (2H) -one;

(S) -2- (3- (5- (5- (2-ethyl-4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-ylamino) -1-methyl-6-oxo-1, 6-dihydropyridin-3-yl) -5-fluoro-2- (hydroxymethyl) phenyl) -6,7,8, 9-tetrahydropyrazino [1,2-a ] indol-1 (2H) -one;

(S) -2- (3- (5- (5- (2-ethyl-4- (oxetan-3-yl) piperazin-1-yl) pyridin-2-ylamino) -1-methyl-6-oxo-1, 6-dihydropyridin-3-yl) -5-fluoro-2- (hydroxymethyl) phenyl) -3,4,6,7,8, 9-hexahydropyrido [3,4-b ] indolizin-1 (2H) -one;

2- (5-fluoro-2-hydroxymethyl-3- { 1-methyl-5- [5- ((R) -2-methyl-4-oxetan-3-yl-piperazin-1-yl) -pyridin-2-ylamino ] -6-oxo-1, 6-dihydro-pyridin-3-yl } -phenyl) -7, 7-dimethyl-3, 4,7, 8-tetrahydro-2H, 6H-cyclopenta [4,5] pyrrolo [1,2-a ] pyrazin-1-one;

2- (3- (5- (5- (2, 2-dimethyl-4- (oxetan-3-yl) piperazin-1-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.

5. A pharmaceutical composition comprising a compound according to any one of claims 1 to 4, and a pharmaceutically acceptable carrier, glidant, diluent, or excipient.

6. The pharmaceutical composition of claim 5, further comprising a therapeutic agent.

7. A process for preparing a pharmaceutical composition comprising admixing a compound of any one of claims 1-4 and a pharmaceutically acceptable carrier.

8. Use of the pharmaceutical composition of claim 5 in the manufacture of a medicament for treating a disease or disorder selected from immune disorders, cancer, cardiovascular disease, viral infection, inflammation, metabolism/endocrine function disorders, and neurological disorders, and mediated by Bruton's tyrosine kinase.

9. The use of claim 8, wherein the disease or disorder is an immune disorder.

10. The use of claim 9, wherein the immune disorder is rheumatoid arthritis.

11. Use of a pharmaceutical composition according to claim 5 in the manufacture of a medicament for the treatment of a disease or disorder that is systemic and local inflammation, arthritis, inflammation associated with immune suppression, organ transplant rejection, allergy, 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.

12. The use of claim 11, wherein the disease or condition is systemic and local inflammation, arthritis, inflammation associated with immunosuppression, organ transplant rejection and systemic lupus erythematosus.

13. The use of claim 8, wherein the disease or condition is a cancer selected from the group consisting of: ovarian cancer, cervical cancer, testicular cancer, cancer of the genitourinary tract, cancer of the esophagus, cancer of the larynx, neuroblastoma, stomach cancer, skin cancer, keratoacanthoma, lung cancer, epidermoid carcinoma, large cell carcinoma, small cell carcinoma, bone cancer, colon cancer, adenoma, adenocarcinoma, follicular carcinoma, undifferentiated carcinoma, papillary carcinoma, seminoma, melanoma, sarcoma, bladder cancer, liver cancer and cancer of the biliary tract, kidney cancer, myeloid disorders, lymphoma, hairy cell carcinoma, oral cavity cancer, lip cancer, tongue cancer, mouth cancer, small intestine cancer, colorectal cancer, large intestine cancer, rectal cancer, brain and central nervous system cancer, leukemia, bronchial cancer, cancer of the liver and intrahepatic bile duct cancer, glioma/glioblastoma, cancer of the kidney and renal pelvis, cancer of the corpus uteri, oral cavity and pharyngeal cancer.

14. The use of claim 8, wherein the disease or condition is a cancer selected from the group consisting of: breast cancer, prostate cancer, lung adenocarcinoma, villous adenoma of the colon, non-small cell lung cancer (NSCLC), hepatocellular carcinoma, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, nasopharyngeal carcinoma, lymphocytic leukemia, myeloid leukemia, and endometrial carcinoma.

15. The use of claim 8, wherein the disease or condition is a cancer selected from the group consisting of: chronic Lymphocytic Leukemia (CLL), acute myelogenous leukemia, and chronic myelogenous leukemia.

16. The use of claim 13, wherein the disease or disorder is pancreatic cancer and thyroid cancer.

17. The use of claim 8, further comprising administering an additional therapeutic agent selected from the group consisting of: anti-inflammatory agents, immunomodulators, 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 disorders.

18. A kit for treating a disorder mediated by Bruton's tyrosine kinase, comprising:

a) the pharmaceutical composition of claim 5; and

b) instructions for use.