HK1233260B - Oxepan-2-yl-pyrazol-4-yl-heterocyclyl-carboxamide compounds and methods of use - Google Patents

Description

Oxepan-2-yl-pyrazol-4-yl-heterocyclyl-carboxamide compounds and methods of use

Technical Field

The present application generally relates to oxepan-2-yl-pyrazol-4-yl-heterocyclyl-carboxamide compound inhibitors that are useful for treating conditions mediated by Pim kinases (Pim-1, Pim-2, and/or Pim-3) and are therefore useful as cancer therapies. The present application also relates to compositions, and more specifically pharmaceutical compositions, comprising these compounds and methods of using the compounds, alone or in combination, to treat various forms of cancer and hyperproliferative disorders, and methods of using the compounds to diagnose or treat mammalian cells or related pathological conditions in vitro, in situ, and in vivo.

Background Art

The Pim kinases are a family of three highly related serine and threonine protein kinases encoded by the genes Pim-1, Pim-2, and Pim-3. The gene name is derived from the phrase Proviral Insertion ( M oloney), which is a common integration site of murine Moloney virus, where the insertion results in overexpression of Pim kinases and de novo formation of T-cell lymphoma or a significant acceleration of tumorigenesis in a transgenic Myc-driven lymphoma model (Cuypers et al. (1984) Cell, vol. 37(1) pp. 141-50; Selten et al. (1985) EMBO J. vol. 4(7) pp. 1793-8; van der Lugt et al. (1995) EMBO J. vol. 14(11) pp. 2536-44; Mikkers et al. (2002) Nature Genetics, vol. 32(1) pp. 153-9; van Lohuizen et al. (1991) Cell, vol. 65(5) pp. 737-52). These experiments showed synergy with the oncogene c-Myc and suggested that inhibition of Pim kinases could have therapeutic benefit.

Mouse genetics suggest that antagonizing Pim kinases may have an acceptable safety profile; Pim-1-/-, Pim-2-/-, Pim-3-/- knockout mice are viable, although slightly smaller than wild-type littermates (Mikkers et al. (2004) Mol Cell Biol vol. 24(13) pp. 6104-154). The three genes produce six protein isoforms that contain a protein kinase domain and apparently have no identifiable regulatory domain. All six isoforms are constitutively active protein kinases that do not require post-translational modification for activity, so Pim kinases are primarily regulated at the transcriptional level (Qian et al. (2005) J Biol Chem, vol. 280(7) pp. 6130-7). Pim kinase expression is highly inducible by cytokines and growth factor receptors and Pim is a direct transcriptional target of Stat proteins, including Stat3 and Stat5. For example, Pim-1 requires the Stat3 proliferation signal mediated by gp130 (Aksoy et al. (2007) Stem Cells, vol. 25(12) pp. 2996-3004; Hirano et al. (2000) Oncogene vol. 19(21) pp. 2548-56; Shirogane et al. (1999) Immunity vol. 11(6) pp. 709-19).

The function of Pim kinases in cell proliferation and survival pathways is parallel to the PI3k/Akt/mTOR signaling axis (Hammerman et al. (2005) Blood vol. 105 (11) pp. 4477-83). In fact, several phosphorylation targets of the PI3k axis (including Bad and eIF4E-BP1) are cell growth and apoptosis regulators and are also phosphorylation targets of Pim kinases (Fox et al. (2003) Genes Dev vol. 17 (15) pp. 1841-54; Macdonald et al. (2006) Cell Biol vol. 7 pp. 1; Aho et al. (2004) FEBS Letters vol. 571 (1-3) pp. 43-9; Tamburini et al. (2009) Blood vol. 114 (8) pp. 1618-27). Pim kinases can affect cell survival because phosphorylation of Bad increases the activity of Bcl-2 and thus promotes cell survival. Similarly, phosphorylation of eIF4E-BP1 by mTOR or Pim kinases leads to inhibition of eIF4E, which promotes mRNA translation and cell growth. In addition, it has been recognized that Pim-1 promotes cell cycle progression by phosphorylating CDC25A, p21, and Cdc25C (Mochizuki et al. (1999) J Biol Chem vol. 274 (26) pp. 18659-66; Bachmann et al. (2006) Int J Biochem Cell Biol vol. 38 (3) pp. 430-43; Wang et al. (2002) Biochim Biophys Acta vol. 1593 (1) pp. 45-55).

Pim kinases have been shown to act synergistically in transgenic mouse models with c-Myc-driven and Akt-driven tumors (Verbeek et al. (1991) Mol Cell Biol vol. 11(2) pp. 1176-9; Allen et al. Oncogene (1997) vol. 15(10) pp. 1133-41; Hammerman et al. (2005) Blood vol. 105(11) pp. 4477-83). Pim kinases are involved in the transforming activity of oncogenes identified in acute myeloid leukemia (AML), including Flt3-ITD, BCR-abl, and Tel-Jak2. Expression of these oncogenes in BaF3 cells results in upregulation of Pim-1 and Pim-2 expression, which leads to IL-3-independent growth and subsequent Pim inhibition leads to apoptosis and cell growth arrest (Adam et al. (2006) Cancer Research 66(7) 3828-35). Pim overexpression and deregulation have also been noted as common events in a variety of hematopoietic cancers, including leukemias and lymphomas (Amson et al. (1989) Proc Natl Acad Sci USA 86(22)8857-61); Cohen et al. (2004) Leuk Lymphoma 45(5)951-5; Hüttmann et al. (2006) Leukemia 20(10)1774-82) and multiple myeloma (Claudio et al. (2002) Blood 100(6)2175-86). Multiple myeloma (MM) is a clonal B lymphocyte malignancy characterized by the accumulation of terminally differentiated antibody-producing cells in the bone marrow.

Pim-1 has been shown to be overexpressed and associated with the progression of prostate cancer (Cibull et al. (2006) J Clin Pathol 59 (3) 285-8; Dhanasekaran et al. (2001) Nature 412 (6849) 822-6). Pim-1 expression increases with disease progression in mouse models (Kim et al. (2002) Proc Natl Acad Sci USA 99 (5) 2884-9). Pim-1 has been reported to be the most highly overexpressed mRNA in a subset of human prostate tumor samples with a gene signature driven by c-Myc (Ellwood-Yen et al. (2003) Cancer Cell 4 (3) 223-38). Pim-3 has also been shown to be overexpressed in pancreatic cancer and hepatocellular carcinoma and to have a functional role (Li et al. (2006) Cancer Research 66(13): 6741-7; Fujii et al. (2005) Int J Cancer, 114(2): 209-18).

In addition to oncology therapeutic and diagnostic applications, Pim kinases may play a role in normal immune system function and inhibition of Pims may treat a variety of different immunological conditions, including tumorigenesis (Nawijn et al. (2011) Nature Rev. 11:23-34), inflammation, autoimmune diseases, allergies, and immunosuppression for organ transplantation (Aho et al. (2005) Immunology 116(1):82-8).

Summary of the Invention

The present application relates to oxepan-2-yl-pyrazol-4-yl-heterocyclyl-carboxamide compounds, which are compounds of formula I, for use in treating disorders mediated by Pim kinases (Pim-1, Pim-2 and/or Pim-3).

The compound of formula I has the following structure:

wherein X is thiazolyl, pyrazinyl, pyridinyl or pyrimidinyl;

and their stereoisomers, geometric isomers, tautomers and pharmaceutically acceptable salts. Each substituent including R ¹ , R ² and X is as defined in the present application.

One aspect of the present application 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 chemotherapeutic agent.

The present application includes a method for treating a disease or condition comprising administering to a patient suffering from the disease or condition a therapeutically effective amount of a compound of Formula I, wherein the disease or condition is selected from cancer, immune disorders, cardiovascular disease, viral infection, inflammation, metabolic/endocrine dysfunction, and neurological disorders, and diseases or conditions mediated by Pim kinases. The method further comprises administering an additional therapeutic agent selected from a chemotherapeutic agent, an anti-inflammatory agent, an immunomodulator, a neurotropic factor, a drug for treating cardiovascular disease, a drug for treating liver disease, an antiviral agent, a drug for treating a blood disorder, a drug for treating diabetes, and a drug for treating an immunodeficiency disorder.

The present application includes the use of a compound of Formula I in the preparation of a medicament for treating cancer, immune disorders, cardiovascular disease, viral infection, inflammation, metabolic/endocrine dysfunction, and neurological disorders, wherein the medicament mediates Pim kinases.

The present application includes kits for treating conditions mediated by Pim kinases, comprising: a) a first pharmaceutical composition comprising a compound of Formula I; and b) instructions for use.

The present application includes compounds of Formula I for use as medicaments and for treating diseases or conditions selected from cancer, immune disorders, cardiovascular diseases, viral infections, inflammation, metabolic/endocrine dysfunction and neurological disorders and diseases or conditions mediated by Pim kinases.

The present application includes methods for preparing compounds of Formula I.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows an exemplary synthesis of 4-aminopyrazole compound 5 from nitro-1H-pyrazole 1.

FIG2 shows an exemplary synthesis of 2-substituted thiazole-4-carboxylic acid compound 6 from thiazole-4-carboxylate compound 7.

FIG3 shows an exemplary synthesis of N-(1,5-disubstituted-1H-pyrazol-4-yl)-2-substituted thiazole-4-carboxamide compound 10 by coupling 1,5-disubstituted-1H-pyrazol-4-amine compound 5 with 2-substituted thiazole-4-carboxylic acid compound 6.

FIG4 shows an exemplary synthesis of 6-(4-nitro-1H-pyrazol-5-yl)oxepan-3-amine compound 19 from 5-chloro-4-nitro-1H-pyrazole compound 3.

FIG5 shows an exemplary synthesis of 5-(5-azido-6-fluorooxepan-2-yl)-1-substituted-4-nitro-1H-pyrazole compound 26 from 1-substituted-4-nitro-1H-pyrazole compound 2.

Figure 6 shows the exemplary synthesis of 5-(5-azido-4-fluorooxepan-2-yl)-1-substituted-4-nitro-1H-pyrazole compound 31 and 5-(4-azido-5-fluorooxepan-2-yl)-1-substituted-4-nitro-1H-pyrazole compound 32 from 5-(1-(allyloxy)pent-4-enyl)-1-substituted-4-nitro-1H-pyrazole compound 22.

FIG7 shows an exemplary synthesis of 5-(5-azido-6-fluorooxepan-2-yl)-1-substituted-4-nitro-1H-pyrazole compound 35 from 1-substituted-4-nitro-5-(2,3,4,7-tetrahydrooxepin-2-yl)-1H-pyrazole compound 23.

FIG8 shows an exemplary synthesis of 2-methyl-N-(2-substituted-7-(1-substituted-4-nitro-1H-pyrazol-5-yl)oxepan-4-yl)propane-2-sulfenamide compound 40 from 5-chloro-4-nitro-1H-pyrazole compound 3.

9 shows an exemplary synthesis of tert-butyl (2R,3R,4S,5R)-5-hydroxy-3,5-dimethyl-2-(1-substituted-4-nitro-1H-pyrazol-5-yl)tetrahydro-2H-pyran-4-ylcarbamate compound 47 from 1-substituted-4-nitro-1H-pyrazole-5-carbaldehyde compound 36.

FIG10 shows an exemplary synthesis of tert-butyl 3-methoxy-2-methyl-7-(1-substituted-4-nitro-1H-pyrazol-5-yl)oxepan-4-ylcarbamate compound 52 from 1-(1-substituted-4-nitro-1H-pyrazol-5-yl)pent-4-en-1-ol compound 21.

DETAILED DESCRIPTION

Some embodiments of the present application are now described in detail, examples of which are illustrated in the accompanying structures and molecular formulas. Although the present application is described in conjunction with the enumerated embodiments, it should be understood that the present application is not limited to those embodiments. On the contrary, the present application is intended to cover all alternative forms, modifications and equivalent forms that may be included within the scope of the present application defined by the claims. Those skilled in the art will recognize that a variety of methods and materials similar to or equivalent to those described in the present application can be used to implement the present application. The present application is in no way limited to the described methods and materials. In the event that one or more of the incorporated documents, patents and similar materials differ or conflict with the present application (including but not limited to defined terms, term usage, described technology, etc.), the present application shall prevail. Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as those generally understood by those skilled in the art to which the present application belongs. Although methods and materials similar to or equivalent to those described in the present application can be used to implement or test the present application, appropriate methods and materials are described below. All publications, patent applications, patents and other references mentioned in the present application are incorporated herein by reference. Unless otherwise stated, the nomenclature used in the present application is based on the IUPAC systematic nomenclature.

definition

When specifying the number of substituents, the term "one or more" refers to one substituent to the maximum possible number of substitutions, i.e., replacing one hydrogen to replacing all hydrogens with substituents. The term "substituent" refers to an atom or group of atoms that replaces a hydrogen atom on the parent molecule. The term "substituted" means that the specified group carries 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 specified group does not carry a substituent. The term "optionally substituted" means that the specified group is unsubstituted or substituted by one or more substituents independently selected from the possible substituents. When specifying the number of substituents, the term "one or more" refers to one substituent to the maximum possible number of substitutions, i.e., replacing one hydrogen to replacing all hydrogens with substituents.

The term "alkyl" as used herein refers to a saturated, linear or branched, monovalent hydrocarbon group having 1 to 12 carbon atoms (C ₁ -C ₁₂ ), wherein the alkyl group may be optionally substituted independently with one or more substituents as described below. In another embodiment, the alkyl group has 1 to 8 carbon atoms (C ₁ -C ₈ ) or 1 to 6 carbon atoms (C ₁ -C ₆ ). Examples of alkyl groups include, but are _not limited to, methyl (Me, _-CH3 ), ethyl (Et, _-CH2CH3 ), 1-propyl (n-Pr, n _- propyl _{, -CH2CH2CH3} ), 2-propyl (i-Pr, isopropyl, -CH( _CH3 ) ₂ ), 1-butyl (n-Bu, n _{- butyl} , _{-CH2CH2CH2CH3 )} , 2-methyl-1-propyl (i-Bu, isobutyl, _-CH2CH ( _{CH3)2 )} , 2-butyl (s-Bu, sec _- butyl, -CH( _CH3 ) _CH2CH3 ), 2-methyl-2-propyl (t-Bu, tert-butyl, -C( _CH3 ) ₃ ) _{, 1 -} pentyl ( _n -pentyl, _{-CH2CH2CH2CH2CH3} ), 2-pentyl (-CH( _{CH3 ) CH2CH2CH 3} ), 3-pentyl (-CH(CH ₂ CH ₃ ) ₂ ), 2-methyl-2-butyl (-C(CH ₃ ) ₂ CH ₂ CH ₃ ), 3-methyl-2-butyl (-CH(CH ₃ )CH(CH ₃ ) ₂ ), 3-methyl-1-butyl (-CH ₂ CH ₂ CH(CH ₃ ) ₂ ), 2-methyl-1-butyl (-CH ₂ CH(CH ₃ )CH ₂ CH ₃ ), 1-hexyl (-CH ₂ CH ₂ CH ₂ CH _{2 CH 3} ), 2-hexyl (-CH(CH ₃ )CH ₂ CH ₂ CH ₂ CH ₃ ), 3-hexyl (-CH(CH ₂ CH ₃ )(CH ₂ CH ₂ CH ₃ )), 2-methyl-2-pentyl (-C(CH ₃ ) ₂ CH ₂ CH ₂ CH ₃ ), 3-methyl-2-pentyl (-CH(CH ₃ )CH(CH ₃ )CH ₂ CH ₃ ), 4-methyl-2-pentyl (-CH(CH ₃ )CH ₂ CH(CH ₃ ) ₂ ), 3-methyl-3-pentyl (-C(CH ₃ )(CH ₂ CH ₃ ) ₂ ), 2-methyl-3-pentyl (-CH(CH ₂ CH ₃ )CH(CH ₃ ) ₂ ), 2,3-dimethyl-2-butyl (-C(CH ₃ ) ₂ CH(CH ₃ ) ₂ ), 3,3-dimethyl-2-butyl (-CH(CH ₃ )C(CH ₃ ) ₃ , 1-heptyl, 1-octyl, and the like.

The term "alkylene" as used herein refers to a saturated, linear or branched, divalent hydrocarbon group having 1 to 12 carbon atoms ( _C1 - _C12 ), wherein the alkylene group may be optionally substituted independently with one or more substituents described below. In another embodiment, the alkylene group has 1 to 8 carbon atoms ( _C1 - _C8 ) or 1 to 6 carbon atoms ( _C1 - _C6 ). Examples of alkylene groups include, but are not limited to, methylene ( _-CH2- ), _ethylene ( _{-CH2CH2- )} , propylene ( _-CH2CH2CH2- ), and the _like .

The term "alkenyl" refers to a linear or branched monovalent hydrocarbon group having 2 to 8 carbon atoms ( _C2 - _C8 ) with at least one site of unsaturation, i.e., a carbon-carbon ^sp2 double bond, wherein the alkenyl group may be optionally substituted independently with one or more substituents as described herein and includes groups with "cis" and "trans" orientations or "E" and "Z" orientations. Examples include, but are not limited to, vinyl (-CH= _CH2 ), allyl ( _-CH2CH = _CH2 ), and the like.

The term "alkenylene" refers to a straight or branched divalent hydrocarbon group having 2 to 8 carbon atoms ( _C2 - _C8 ) with at least one site of unsaturation, i.e., a carbon-carbon ^sp2 double bond, wherein the alkenylene group may be optionally substituted independently with one or more substituents as described herein and includes groups with "cis" and "trans" orientations or "E" and "Z" orientations. Examples include, but are not limited to, vinylene (-CH=CH-), allylene ( _-CH2CH =CH-), and the like.

The term "alkynyl" refers to a linear or branched monovalent hydrocarbon group having 2-8 carbon atoms ( _C2 - _C8 ) with at least one site of unsaturation, i.e., a carbon-carbon sp triple bond, wherein the alkynyl group may be optionally substituted independently with one or more substituents as described herein. Examples include, but are not limited to, ethynyl (-C≡CH), propynyl (propargyl, _-CH2C≡CH ), and the like.

The term "alkynylene" refers to a straight or branched divalent hydrocarbon group having 2 to 8 carbon atoms ( _C2 - _C8 ) with at least one site of unsaturation, i.e., a carbon-carbon sp triple bond, wherein the alkynylene group may be optionally substituted independently with one or more substituents described herein. Examples include, but are not limited to, ethynylene (-C≡C-), propynylene (propargylene, _-CH2C≡C- ), and the like.

The terms "carbocycle,""carbocyclyl,""carbocycle," and "cycloalkyl" refer to a monovalent, non-aromatic, saturated or partially unsaturated ring having 3 to 12 carbon atoms ( _C3 - _C12 ) as a monocyclic ring or 7 to 12 carbon atoms as a bicyclic ring. Bicyclic carbocycles having 7 to 12 atoms may be arranged, for example, as bicyclic [4,5], [5,5], [5,6], or [6,6] systems, while bicyclic carbocycles having 9 or 10 ring atoms may be arranged as bicyclic [5,6] or [6,6] systems or may be arranged as bridged ring systems such as 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, etc. Carbocyclyl groups are optionally substituted independently with one or more substituents described herein.

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

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

The terms "heterocycle", "heterocyclyl" and "heterocycle" are used interchangeably herein and refer to a saturated or partially unsaturated (i.e., having one or more double and/or triple bonds in the ring) carbocyclic group having 3 to about 20 ring atoms, at least one of which is a heteroatom selected from nitrogen, oxygen, phosphorus and sulfur and the remaining ring atoms being C, wherein one or more ring atoms are optionally substituted independently with one or more substituents described below. The heterocycle may be a monocycle having 3-7 ring members (2-6 carbon atoms and 1-4 heteroatoms selected from N, O, P and S) or a bicycle having 7-10 ring members (4-9 carbon atoms and 1-6 heteroatoms selected from N, O, P and S), such as a bicyclic [4,5], [5,5], [5,6] or [6,6] system. Heterocycles are described in Paquette, Leo A.; "Principles of Modern Heterocyclic Chemistry" (W.A. Benjamin, New York, 1968), particularly Chapters 1, 3, 4, 6, 7, and 9; "The Chemistry of Heterocyclic Compounds, A series of Monographs" (John Wiley & Sons, New York, 1950 to present), particularly Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82: 5566. "Heterocyclyl" also includes groups in which a heterocyclyl is fused to a saturated ring, a partially unsaturated ring, an aromatic carbocycle, or an aromatic heterocycle. 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, azocan-1-yl, azetidin-1-yl, octahydropyrido[1,2-a]pyrazin-2-yl, [1,4]diazepan-1-yl, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino, thiooxanyl, piperazinyl, homopiperazinyl, azetidinyl, oxo Heterocyclic groups include alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, alkyl, Examples of heterocyclic groups wherein a ring atom is substituted with an oxo (=O) moiety are pyrimidinone and 1,1-dioxothiomorpholinyl. The heterocyclic groups herein are optionally substituted independently with one or more substituents described herein.

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

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

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

The term "treatment" refers to therapeutic treatment and preventive measures, the purpose of which is to prevent or slow down (mitigate) undesirable physiological changes or the formation or spread of disease such as cancer. For the purposes of this application, useful or desired clinical results include but are not limited to relieving symptoms, narrowing the scope of the disease, stabilizing (i.e., not worsening) disease state, delaying or slowing down disease progression, improving or alleviating disease state and diminishing (whether partially or completely alleviated), whether detectable or undetectable. "Treatment" can also refer to a survival time extended compared to the survival time expected in the absence of treatment. Those objects in need of treatment include those objects with disease or disorder and those objects tending to have disease or disorder or those objects in which disease or disorder are to be prevented.

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

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

"Chemotherapeutic agents" are compounds that can be 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 therapies" and conventional chemotherapy. Examples of chemotherapeutic agents include: erlotinib (Genentech/OSIPharm.), docetaxel (Sanofi-Aventis), 5-FU (fluorouracil, 5-fluorouracil, CAS No. 51-21-8), gemcitabine (Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer), cisplatin (cis-diamine, dichloroplatinum (II), CAS No. 15663-27-1), carboplatin (CAS No. 41575-94-4), paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), trastuzumab (Genentech), temozolomide (4-methyl-5-oxo-2,3,4,6,8-pentaazabicyclo[4.3.0]nona-2,7,9-triene-9-carboxamide, CAS No. 85622-93-1, Schering Plough), tamoxifen ((Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethylethanamine), doxorubicin Akti-1/2, HPPD, and rapamycin.

Other examples of chemotherapeutic agents include: oxaliplatin (Sanofi), bortezomib (Millennium Pharm.), sunitinib (SU11248, Pfizer), letrozole (Novartis), imatinib mesylate (Novartis), XL-518 (Mek inhibitor, Exelixis, WO2007/044515), ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma, AstraZeneca), SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235 (PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), PTK787/ZK 222584 (Novartis), fulvestrant (AstraZeneca), leucovorin (folinic acid), rapamycin (sirolimus, Wyeth), rapamycin analogs, mTOR inhibitors such as everolimus, MEK inhibitors (GDC-0973), Bcl-2 inhibitors such as navitoclax (ABT-263 or ABT-199), lapatinib (GSK572016, Glaxo Smith Kline), lonafarnib (SARASAR ^™ , SCH 66336, Schering Plough), sorafenib (BAY43-9006, Bayer Labs), gefitinib (AstraZeneca), irinotecan (CPT-11, Pfizer), tipifarnib (ZARNESTRA ^™ , Johnson & Johnson), ABRAXANE ^™ (Cremophor-free), albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, 11), vandetanib (rINN, ZD6474, AstraZeneca), chlorambucil, AG1478, AG1571 (SU 5271; Sugen), temsirolimus (Wyeth), pazopanib (GlaxoSmithKline), canfosfamide (Telik), thiotepa and alkyl cyclophosphamide sulfonates, such as busulfan, improsulfan and piposulfan; aziridines, such as benzodepa, carboquinone, metodepa and uredepa; ethyleneimines and methylaminoacridines, including hexamethylmelamine, triimidazole, triethylenephosphoramide, triethylenephosphoramide sulfide and trihydroxymethylmelamine; annonaceous lactones (particularly bratacin and bratacinone); camptothecins (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its synthetic analogues adolesine, carzelesin and biszelesin); nostoc cyclic peptides (particularly nostoc cyclic peptide 1 and Nostoc cyclic peptide 8); dolastatin; duocarmycin (including synthetic analogs KW-2189 and CB1-TM1); arbutin; hyoscyamine; sarcodictyin; spongestatin; nitrogen mustards such as chlorambucil, naphthiazolin, clofosamide, estramustine, ifosfamide, mechlorethamine, oxazolidinone hydrochloride, melphalan, new nitrogen mustard, phenylephrine, prednimustine, trofosfamide, uracil mustard; nitroureas such as carmustine, chlorozolin, fotemustine, lomustine, nimustine and ranimustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, calicheamicin gamma 1I, calicheamicin omega 1I (Angew Chem. Intl. Ed. Engl. (1994) 33:183-186); anthracycline antibiotics, dynemicin A; bisphosphonates, such as clodronate; esperamicin; new tumor suppressor protein chromophores and related chromoprotein enediyne antibiotic chromophores, aclacinomysin, actinomycin, authramycin, azaserine, bleomycin, actinomycin C, carabicin, daunorubicin, carcinoglossin, chromomycin, actinomycin D, daunorubicin, detoxibacin, 6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin, epirubicin, esorubicin, idarubicin, nemorubicin, mexilomycin, mitomycins such as mitomycin C, mycophenolic acid, noramycin, olivomycin, peplomycin, porfibrinocin, puromycin, triferon-adriamycin, rhodorubicin, streptozocin, streptozocin, tuberculin, ubenimex, zoloft, doxycycline; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as leucovorin, methotrexate, Pteroxetine, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thioimidazole, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azaguanosine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as captestosterone, methylandrostanolone propionate, cyclothiosteroid, melastane, testolactone; antiadreners such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as folinic acid; aceglucuronolide; aldophosphamide glycosides; amino Levulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; idarucizumab; defosfamide; colcemid; diazocine; eflornithine; elliptonium acetate; epothilones; etoglucagon; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansines, such as maytansine and ansamitocin; mitoguanidine; mitoxantrone; mopidanmol; rhizobactam; pentostatin; methambucil; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazine; procarbazine; polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizolan; spirogermanium; tenuisporic acid; triamin; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin, and chloramphenicol) A, bacitracin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; dibromomannitol; dibromodulcitol; pipobroman; gacytosine; cytarabine ("Ara-C");cyclophosphamide;thiotepa;6-thioguanine;mercaptopurine;methotrexate; platinum analogs, such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; teniposide; idarucizumab; daunorubicin; aminopterin; capecitabine (Roche); ibandronate; CPT-11; the topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; and pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing.

Also included within the definition of "chemotherapeutic agent" are: (i) antihormonal drugs used to modulate or inhibit the effects of hormones on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen, troloxifene, raloxifene, LY117018, onapristone, and tomiphene citrate; (ii) aromatase inhibitors that inhibit the aromatase enzyme, which regulates estrogen production in the adrenal glands, such as 4(5)-imidazole, aminoglutethimide, megestrol acetate, exemestane, formestan, fadroxil, vodoxil, and valproate; (chlorazole; Novartis), letrozole (letrozole; Novartis), and anastrozole (anastrozole; AstraZeneca); (iii) antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) protein kinase inhibitors, such as MEK inhibitors (WO2007/044515); (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, particularly those that inhibit the expression of genes in signaling pathways involved in abnormal cell proliferation, such as PKC-α, Raf, and H-Ras, such as oblimersen (Genta Inc.); (vii) ribozymes, such as VEGF expression inhibitors (e.g., HER2 expression inhibitors); (viii) vaccines, such as gene therapy vaccines such as IL-2 and rIL-2; topoisomerase 1 inhibitors, such as rmRH; (ix) anti-angiogenic drugs, such as bevacizumab (Genentech); and pharmaceutically acceptable salts, acids and derivatives of any of the above substances.

Also included within the definition of "chemotherapeutic agent" are therapeutic antibodies such as alemtuzumab, bevacizumab (Genentech); cetuximab (Imclone); panitumumab (Amgen), rituximab (Genentech/Biogen Idec), pertuzumab (2C4, Genentech), trastuzumab (Genentech), and tositumomab (Corixa, GlaxoSmithKline).

Humanized monoclonal antibodies with therapeutic potential as chemotherapeutic agents for use in combination with the compounds of Formula I of the present application include: alemtuzumab, apolizumab, aseluzumab, atlizumab, bapineuzumab, bevacizumab, mobivacizumab, mocantuzumab, cilizumab, certolizumab pegol, cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, panvituzumab, fontuzumab, ogitozumab, inotuzumab, ipilimumab, labetuzumab, lebrikizumab, and levofloxacin. mab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab, pectuzumab, pertuzumab, pexelizumab, ralivizumab, ranibizumab, reslizumab, reslizumab, resyvizumab, rovizumab, lulizumab, sirolizumab, cilizumab, sotuzumab, tacilizumab,

tadocizumab, talizumab, tefeizumab, tocilizumab, tocilizumab, trastuzumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab, urolizumab, and visilizumab.

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

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

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

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

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

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

The stereochemical definitions and conventional terminology used herein generally follow those of S.P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., New York, 1994. The compounds of the present invention may contain asymmetric or chiral centers and therefore exist in different stereoisomeric forms. All stereoisomeric forms of the compounds of the present invention, including but not limited to diastereomers, enantiomers and atropisomers, and mixtures thereof, such as racemic mixtures, form part of this application. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. When describing an optically active compound, the prefixes D and L or R and S are used to indicate the absolute configuration of the molecule about its one or more chiral centers. The prefixes d and l or (+) and (-) are used as symbols to indicate the rotation of plane polarized light caused by a compound, wherein (-) or l indicates that the compound is left-handed. Compounds prefixed with (+) or d are right-handed. 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 the isomers are generally referred to as enantiomeric mixtures. A 50:50 mixture of enantiomers is referred to as a racemic mixture or racemate, which may occur when a chemical reaction or method does not have stereoselectivity or stereospecificity. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric substances that is optically inactive. Enantiomers can be separated from a racemic mixture by chiral separation methods such as supercritical fluid chromatography (SFC). Assignments of configuration at chiral centers in separated enantiomers may be tentative and are depicted in the structures in Table 1 for illustrative purposes, while the stereochemistry is to be determined, for example, by x-ray crystallographic data.

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

The term "pharmaceutically acceptable salts" 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 must be chemically and/or toxicologically compatible with the other ingredients of the formulation and/or the mammal to be treated therewith.

The term "pharmaceutically acceptable acid addition salts" refers to those pharmaceutically acceptable salts formed with 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 of organic acids 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, ethanesulfonic acid, p-toluenesulfonic acid and salicylic acid.

The term "pharmaceutically acceptable base addition salt" refers to 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 primary, secondary, and tertiary amines, substituted amines including natural 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, hydroxylamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, and polyamine resins.

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

The term " _EC50 " is half maximal effective concentration and represents the plasma concentration of a particular compound required to obtain 50% of the maximum value of a specific effect in vivo.

The term "Ki" is the inhibition constant and represents the absolute binding affinity of a particular inhibitor for a receptor. It is measured using a competition binding assay and is equal to the concentration at which the particular inhibitor occupies 50% of the receptors in the absence of a competing ligand (e.g., a radioligand). Ki values can be converted logarithmically to pKi values (-log Ki), where higher values represent exponentially greater potency.

The term " _IC50 " is the half-maximal inhibitory concentration and represents the concentration of a particular compound required to achieve 50% inhibition of a biological process in vitro. _IC50 values can be converted logarithmically to _pIC50 values ( _-logIC50 ), where higher values represent exponentially greater potency. _IC50 values are not absolute values, but rather depend on experimental conditions, such as the concentration used, and can be converted to an absolute inhibition constant (Ki) using the Cheng-Prusoff equation (Biochem. Pharmacol. (1973) 22:3099).

The terms "compounds of the present application" and "compounds of Formula I" include compounds of Formula I and stereoisomers, geometric isomers, tautomers, solvates, metabolites, 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 said compound, or mixtures thereof.

Any formula or structure (including compounds of Formula I) provided herein is also intended to represent the unlabeled form and isotopically labeled form of the compound. Isotopically labeled compounds have the structure described by the structural formula provided herein, but one or more atoms are replaced by atoms with a selected atomic mass or mass number. Examples of isotopes that can be introduced into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, such as, but not limited to, ² H (deuterium, D), ³ H (tritium), ¹¹ C, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁸ F, ³¹ P, ³² P, ³⁵ S, ³⁶ Cl, and ¹²⁵ I. Various isotopically labeled compounds of the present invention are, for example, those compounds of the present invention into which radioactive isotopes such as ³ H, ¹³ C, and ¹⁴ C are introduced. The isotopically labeled compounds can be used for metabolic studies, reaction kinetic 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 radiotherapy performed on patients. Deuterium-labeled or substituted therapeutic compounds of the present application may have improved DMPK (drug metabolism and pharmacokinetics) properties involving distribution, metabolism and excretion (ADME). Substitution with heavier isotopes such as deuterium can provide some therapeutic benefits such as extended in vivo half-life or reduced dosage requirements due to greater metabolic stability. ¹⁸ F-labeled compounds can be used for PET or SPECT studies. Isotopically labeled compounds of the present application and their prodrugs can generally be prepared as follows: the processes disclosed in the following schemes or examples and preparations are carried out using readily available isotopically labeled reagents instead of non-isotopically labeled reagents. In addition, substitution with heavier isotopes, particularly deuterium (i.e. ² H or D) can obtain some therapeutic benefits such as extended in vivo half-life or reduced dosage requirements or improved therapeutic index due to greater metabolic stability. It should be understood that deuterium in this context is considered to be a substituent in the compound of formula (I). The concentration of the heavier isotope (particularly deuterium) can be defined by the isotopic enrichment factor. In the present application's compounds, atoms that are not specifically designated as specific isotopes are intended to represent any stable isotope of the atom. Unless otherwise stated, when a position is specifically designated as "H" or "hydrogen", the position should be understood to have hydrogen in its natural abundance isotopic composition. Therefore, in the present application's compounds, any atom that is specifically designated as deuterium (D) is intended to represent deuterium.

Oxepan-2-yl-pyrazol-4-yl-heterocyclyl-carboxamide compounds

The present application provides oxepan-2-yl-pyrazol-4-yl-heterocyclyl-carboxamide compounds of Formula I (including Formula Ia-i) and pharmaceutical preparations thereof, which can be used to treat diseases, conditions and/or disorders regulated by Pim kinases.

The compound of formula I has the following structure:

and stereoisomers, geometric isomers, tautomers or pharmaceutically acceptable salts thereof, wherein:

^R1 is selected from H, _C1 - _C12 alkyl, _C2 - _C12 alkenyl, _C2 - _C12 alkynyl, _C6 - _C20 aryl, _C3 - _C12 carbocyclyl, _C2 - _C20 heterocyclyl, _C1 - _C20 heteroaryl and -( _C1 - _C12 alkylene)-( _C2 - _C20 heterocyclyl);

R ² is independently selected from F, Cl, Br, I, -CH ₃ , -CH ₂ CH ₃ , -CH(CH ₃ ) ₂ , -C(CH ₃ ) ₃ , -CH ₂ CH(CH ₃ ) ₂ , -CH=CH ₂ , -CH=C(CH ₃ ) ₂ , =CH ₂ , -CH ₂ F, -CHF ₂ , -CF ₃ , -CH ₂ OH, -CH ₂ OCH ₃ , -CH ₂ NH ₂ , -CH ₂ NHCH ₃ , -CH ₂ CH ₂ NH ₂ , -CH ₂ CHCH ₂ NH ₂ , -CH ₂ CH(CH ₃ )NH ₂ , -CH ₂ OH, -CH ₂ CH ₂ OH, -C(CH ₃ ) ₂ OH, -CH(OH)CH(CH ₃ ) ₂ ,-C(CH ₃ ) ₂ CH ₂ OH, -CH ₂ CH ₂ SO ₂ CH ₃ , -CN, -CO ₂ H, -COCH ₃ , -COCH ₂ NH ₂ , -CO ₂ CH ₃ , -CO ₂ C(CH ₃ ) ₃ , -COCH(OH)CH ₃ , -CONH ₂ , -CONHCH ₃ , -CON(CH ₃ ) ₂ , -C(CH ₃ ) ₂ CONH ₂ , -NO ₂ , -NH ₂ , -NHCH ₃ , -N(CH ₃ ) ₂ , -NHCH ₂ CHF ₂ , -NHCH ₂ CF ₃ , -NHCH ₂ CH ₂ OH, -NHCOCH ₃ , -N(CH ₃ )COCH ₃ , -NHC(O)OCH ₂ CH ₃ , -NHC(O)OCH ₂ Cl ₃ , -NHC(O)OC ₆ H ₅ , -NHS(O) ₂ CH ₃ , -N(CH ₃ )C(CH ₃ ) ₂ CONH ₂ , -N(CH ₃ )CH ₂ CH ₂ S(O) ₂ CH ₃ , =O, -OH, -OCH ₃ , -OCHF ₂ , -OCH ₂ F, -OCH ₂ CH ₃ , -OCH(CH ₃ ) ₂ , -OCH ₂ CH(CH ₃ ) ₂ , -OC(CH ₃ ) ₃ , -S(O) ₂ N(CH ₃ ) ₂ , -SCH ₃ , -CH ₂ OCH ₃ , -S(O) ₂ CH ₃ , cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, azetidinyl, azepanyl, oxetan-3-ylmethylamino, (3-methyloxetan-3-yl)methylamino, pyrrolidinyl, piperazinyl, piperidinyl, (piperidin-4-yl)ethyl, pyranyl, (piperidin-4-yl)methyl, morpholinomethyl, and morpholino;

n is 1, 2, 3, 4, 5 or 6;

X is selected from the following structures:

where wavy lines represent connection points; and

^R3 is selected from H, Cl, Br, _C1 - _C12 alkyl, -O-( _C1 - _C12 alkyl), -( _C1 - _C12 alkylene)-( _C3 - _C12 carbocyclyl), -( _C1 - _{C12 alkylene)-(C2-C20 heterocyclyl )} , -( _C2 - _C8 alkenylene)-( _C3 - _C12 carbocyclyl), -( _C2 -C8 alkenylene)-( _C2 - _{C20 heterocyclyl), C6-C20 aryl ,} -( _C6 - _C20 arylene)-( _C2 - _C20 heterocyclyl), -( _C6 - _C20 arylene)-( _C6 - _C20 arylene), -( _C1 - _C12 alkylene)-( _{C2 - C20 C 20} heterocyclyl), -(C ₆ -C ₂₀ arylene)-O-(C ₂ -C ₂₀ heterocyclyl), -(C ₆ -C ₂₀ arylene)-O-(C ₁ -C ₁₂ alkyl), C ₃ -C ₁₂ carbocyclyl, C ₂ -C ₂₀ heterocyclyl, C ₁ -C ₂₀ heteroaryl, -(C ₁ -C ₂₀ heteroaryl)-(C ₂ -C ₂₀ heterocyclyl) and -(C ₁ -C ₂₀ heteroaryl)-(C ₁ -C ₁₂ alkyl); wherein alkyl, alkenyl, alkynyl, alkylene, carbocyclyl, heterocyclyl, aryl and heteroaryl are optionally substituted with one or more groups independently selected from the group consisting of F, Cl, Br, I, -CH ₃ , -CH ₂ CH ₃ , -CH(CH ₃ ) ₂ , -CH ₂ CH(CH ₃ ) ₂ , -CH ₂ NH ₂ , -CH ₂ CH ₂ NH ₂ , -CH ₂ CHCH ₂ NH ₂ , -CH ₂ CH(CH ₃ )NH ₂ , -CH ₂ OH, -CH ₂ CH ₂ OH, -CH(CH ₂ OH) ₂ , -C(CH ₂ OH) ₃ , -CH(CH ₃ )OH, -C(CH ₃ ) ₂ OH, -CH(OH)CH(CH ₃ ) ₂ , -C(CH ₃ ) ₂ CH ₂ OH, -CH ₂ CH ₂ SO ₂ CH ₃ , -CN, -CF ₃ , -CHF ₂ , -CH ₂ F, -CO ₂ H, -COCH ₃ , -COCH(CH ₃ ) ₂ , -CO ₂ CH ₃ , -CO ₂ C(CH ₃ ) ₃ ,-COCH(OH)CH ₃ , -CONH ₂ , -CONHCH ₃ , -CON(CH ₃ ) ₂ , -C(CH ₃ ) ₂ CONH ₂ , -NO ₂ , -NH ₂ , -NHCH ₃ , -N(CH ₃ ) ₂ , -NHCOCH ₃ , -N(CH ₃ )COCH ₃ , -NHS(O) ₂ CH ₃ , -N(CH ₃ )C(CH ₃ ) ₂ CONH ₂ , -N(CH ₃ )CH ₂ CH ₂ S(O) ₂ CH ₃ , =O, -OH, -OCH ₃ , -OCF ₃ , -OCH(CH ₃ ) ₂ , -S(O) ₂ N(CH ₃ ) ₂ , -SCH ₃ , -CH ₂ OCH ₃ , -S(O) ₂ CH ₃ , cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, azetidinyl, azepanyl, oxetanyl, phenyl, pyrrolidinyl, piperazinyl, piperidinyl, (piperidin-4-yl)ethyl, pyranyl, (piperidin-4-yl)methyl, morpholinomethyl, and morpholino.

Exemplary embodiments of compounds of Formula I include those wherein R ¹ is H.

Exemplary embodiments of the compound of Formula I include those wherein R ¹ is C ₁ -C ₁₂ alkyl or C ₃ -C ₁₂ carbocyclyl.

Exemplary embodiments of compounds of Formula I include those wherein R ¹ is selected from the group consisting of —CH ₃ , —CH ₂ CH ₃ , —CH ₂ CHF ₂ , and —CH ₂ CF ₃ .

Exemplary embodiments of compounds of Formula I include those wherein ^R2 is independently selected from F, Cl, -OH, _-CH3 , _-CH2CH3 , _-CF3 , _-NH2 , _-NHCH3 , -N( _CH3 ) ₂ , _-NHCH2CHF2 , _{-NHCH2CF3 , -CH2NHCH3 ,} and _-OCH3 ; and n _is 1, 2, or ₃ .

Exemplary embodiments of compounds of Formula I include those wherein R ³ is C ₆ -C ₂₀ aryl, including phenyl substituted with one or more F groups.

Exemplary embodiments of compounds of Formula I include structures of Formulas Ia-d:

Exemplary embodiments of compounds of Formula I include the structure of Formula Ie:

Exemplary embodiments of compounds of Formula Ie include those wherein R ² is F or OCH ₃ .

Exemplary embodiments of compounds of Formula Ie include those wherein X is thiazolyl.

Exemplary embodiments of compounds of Formula I include structures of Formula If:

Exemplary embodiments of compounds of Formula If include those wherein R ³ is C ₆ -C ₂₀ aryl, including those wherein R ³ is phenyl or pyridyl, wherein the phenyl or pyridyl is optionally substituted with one or more groups selected from the group consisting of F, Cl, Br, I, -CH ₃ , -CH ₂ CH ₃ , -CH(CH ₃ ) ₂ , -CH ₂ CH(CH ₃ ) ₂ , -CH ₂ NH ₂ , -CH ₂ CH ₂ NH ₂ , -CH ₂ CHCH ₂ NH ₂ , -CH ₂ CH(CH ₃ )NH ₂ , -CH ₂ OH , -CH ₂ CH ₂ OH , -CH(CH ₂ OH) ₂ , -C(CH ₂ OH) ₃ , -CH(CH ₃ )OH , -C(CH ₃ ) ₂ OH , -CH(OH)CH(CH ₃ ) ₂ , -C(CH ₃ ) ₂ CH ₂ OH, -CH ₂ CH ₂ SO ₂ CH ₃ , -CN, -CF ₃ , -CHF ₂ , -CH ₂ F, -CO ₂ H, -COCH ₃ , -COCH(CH ₃ ) ₂ , -CO ₂ CH ₃ , -CO ₂ C(CH ₃ ) ₃ , -COCH(OH)CH ₃ , -CONH ₂ , -CONHCH ₃ , -CON(CH ₃ ) ₂ , -C(CH ₃ ) ₂ CONH ₂ , -NO ₂ , -NH ₂ , -NHCH ₃ , -N(CH ₃ ) ₂ , -NHCOCH ₃ , -N(CH ₃ )COCH ₃ , -NHS(O) ₂ CH ₃ , -N(CH ₃ )C(CH ₃ ) ₂ CONH ₂ , -N(CH ₃ )CH ₂ CH ₂ S(O) ₂ CH ₃ , ＝O, -OH, -OCH ₃ , -OCF ₃ , -OCH(CH ₃ ) ₂ , -S(O) ₂ N(CH ₃ ) ₂ , -SCH ₃ , -CH ₂ OCH ₃ , -S(O) ₂ CH ₃ , cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, azetidinyl, azepanyl, oxetanyl, phenyl, pyrrolidinyl, piperazinyl, piperidinyl, (piperidin-4-yl)ethyl, pyranyl, (piperidin-4-yl)methyl, morpholinomethyl, and morpholino.

Exemplary embodiments of compounds of Formula If include those wherein R ³ is selected from phenyl, 2-fluorophenyl, 2,6-difluorophenyl, 2,6-difluoro-4-methylphenyl, 2,4,6-trifluorophenyl, 2,4-difluorophenyl, 2-fluoro-4-hydroxyphenyl, and 3-methylpyridin-2-yl.

Biological evaluation

The Pim kinase activity of the compounds of Formula I can be determined by a variety of direct and indirect detection methods. The Pim kinase binding activity of some exemplary compounds described herein, including isoforms Pim-1, Pim-2, and Pim-3 (Example 901) and in vitro activity against tumor cells (Example 902) were determined. The Pim binding activity IC ₅₀ values of some exemplary compounds of the present application were less than about 1 micromolar concentration (μM). Some compounds of the present invention have tumor cell-based activity _EC50 values of less than about 1 micromolar concentration (μM), for example, against the cell line BaF3 [a murine interleukin-3-dependent pre-B cell line that can be used as a model system to evaluate the efficacy and downstream signaling of kinase oncogenes ("Ba/F3 cells and their use in kinase drug discovery", Warmuth, M et al. (January 2007) Current Opinion in Oncology, Vol 19(1):55-60] and against MM1.S [a multiple myeloma cell line that can be used as a model system to evaluate the efficacy of Pim inhibitors in the treatment of multiple myeloma patients (Greenstein et al. (2003) Exper. Hematol. 31(4):271-282)]. In the assays described in Examples 901 and 902, the compounds have Ki/ _IC50 /EC50 values of less than 1 μM. Compounds of formula I of ₅₀ are therapeutically useful as inhibitors of Pim kinases (Pim-1, Pim-2 and/or Pim-3).

hERG (human Ether-à-go-go related gene) is a gene (KCNH2) that encodes a protein called K _v 11.1 (the alpha subunit of the potassium ion channel). This ion channel (sometimes referred to as 'hERG') is known for its contribution to the electrical activity of the heart that coordinates the beating of the heart (i.e., the hERG channel mediates the repolarization I _Kr current in the cardiac action potential). When the ability of this channel to conduct current through the cell membrane is inhibited or impaired due to the administration of drugs or due to rare mutations in some families (Hedley PL et al. (2009) Human Mutation 30 (11): 1486-511), it can lead to a potentially fatal condition called long QT syndrome; a variety of clinically successful marketed drugs have a tendency to inhibit hERG and are accompanied by the risk of sudden death as a side effect, making hERG inhibition an important counter-target that must be avoided in drug development (Sanguinetti MC, Tristani-Firouzi M (March 2006) Nature 440 (7083): 463-9). hERG has been implicated in regulating the function of some nervous system cells (Chiesa N et al. (June 1997) J. Physiol. (Lond.). 501(Pt 2)(2):313-8; Overholt JL et al. (2000) Adv. Exp. Med. Biol. 475:241-8) and in establishing and maintaining cancer-like features in leukemia cells. hERG assays were performed according to Example 903.

The exemplary compounds of Formula I in Table 1 were prepared, characterized, and tested for inhibition of Pim kinases according to the methods of the present application and have the following structures and corresponding names (ChemBioDraw Ultra, Version 11.0, CambridgeSoft Corp., Cambridge MA). Some compounds in Table 1 with chiral atoms are not fully characterized with respect to stereochemistry. Tentative assignments of stereochemistry or stereochemical relationships with other groups may be described in the structure. Means for separation of stereoisomers and characterization data are given in the Examples.

Table 1

Administration of compounds of formula I

The administration of the compounds of the present invention (hereinafter referred to as "active compounds") can be carried out by any method that can deliver the compound to the site of action. These methods include oral routes, intraduodenal routes, parenteral injection (including intravenous, subcutaneous, intramuscular, intravascular or infusion), topical administration, inhalation administration and rectal administration.

The amount of the active compound administered will depend on the severity of the subject being treated, the disease or the condition, the rate of administration, the disposal of the compound and the judgment of the prescribing physician. However, the effective dose is about 0.001 to about 100 mg/kg body weight/day, preferably about 1 to about 35 mg/kg/day, with a single dose or a divided dose. For a person weighing 70 kg, the effective dose can amount to about 0.05 to 7 g/day, preferably about 0.05 to about 2.5 g/day. In some cases, the dosage level below the lower limit of the above range can be enough, and in other cases, a larger dosage can be used without causing any harmful side effects, provided that the above-mentioned larger dosage is first divided into several small dosages for administration throughout the day.

The active compounds can be administered as a monotherapy or in combination with one or more chemotherapeutic agents such as those described herein. Such combination therapy may be achieved by way of simultaneous, sequential or separate administration of the individual components of the treatment.

The application's formula I compound can be administered by any approach suitable for condition to be treated.Suitable approach includes oral, parenteral (including subcutaneous, intramuscular, intravenous, intra-arterial, intradermal, intrathecal and epidural), percutaneous, rectal, nasal, local (including buccal and sublingual), vagina, intraperitoneal, intrapulmonary and intranasal.For local immunosuppressive therapy, the compound can be administered by intralesional administration (including perfusion or contacting the graft with an inhibitor before transplantation).It should be understood that preferred approach can change with the situation of, for example, the recipient.When the compound is orally administered, it can be formulated into pills, capsules, tablets, etc. together with pharmaceutical carriers or excipients.When the compound is parenteral, it can be formulated into unit dose injection form together with medicinal parenteral vehicles as described below.

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

Methods of treatment using compounds of formula I

The compounds of the present application can be used to treat hyperproliferative diseases, conditions and/or disorders, including but not limited to those characterized by overexpression of Pim kinases such as Pim-1, Pim-2 and Pim-3 kinases. Therefore, another aspect of the present application includes a method for treating or preventing a disease or condition that can be treated or prevented by inhibiting Pim kinases. In one embodiment, the method comprises administering a therapeutically effective amount of a compound of formula I or its stereoisomers, geometric isomers, tautomers or pharmaceutically acceptable salts to a mammal in need thereof. In one embodiment, a human patient is treated with a compound of formula I and a pharmaceutical carrier, excipient or vehicle, wherein the compound of formula I is present in an amount that detectably inhibits Pim kinase activity.

The present application includes compositions (e.g., pharmaceutical compositions) comprising a compound of formula I and/or a solvate, hydrate, and/or salt thereof and a carrier (pharmaceutical carrier). The present application also includes compositions (e.g., pharmaceutical compositions) comprising a compound of formula I and/or a solvate, hydrate, and/or salt thereof and a carrier (pharmaceutical carrier), further comprising a second chemotherapeutic agent such as those described herein. The present application compositions can be used to inhibit abnormal cell growth or treat hyperproliferative conditions such as cancer in mammals (e.g., humans). For example, the present application compounds and compositions can be used to treat multiple myeloma, lymphoma, acute myeloid leukemia, prostate cancer, breast cancer, hepatocellular carcinoma, pancreatic cancer, and/or colorectal cancer in mammals (e.g., humans).

The present application includes methods for inhibiting abnormal cell growth or treating hyperproliferative disorders such as cancer in mammals (e.g., humans), comprising administering to the mammal a therapeutically effective amount of a compound of formula I and/or its solvate, hydrate and/or salt or a composition thereof. For example, the present application includes methods for treating multiple myeloma, lymphoma, acute myeloid leukemia, prostate cancer, breast cancer, hepatocellular carcinoma, pancreatic cancer and/or colorectal cancer in mammals (e.g., humans), comprising administering to the mammal a therapeutically effective amount of a compound of formula I and/or its solvate, hydrate and/or salt or a composition thereof.

Cancers that can be treated according to the methods of the present application include, but are not limited to, breast cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, genitourinary tract cancer, esophageal cancer, laryngeal cancer, stomach cancer, skin cancer, lung cancer, colon cancer, pancreatic cancer, liver cancer, bile duct cancer, kidney cancer, pharyngeal (oral) cancer, lip cancer, tongue cancer, oral cancer, pharyngeal cancer, small intestine cancer, colorectal cancer, large intestine cancer, rectal cancer, brain cancer, and central nervous system cancer. Cancer types that can be treated according to the methods of the present application include, but are not limited to, multiple myeloma, glioblastoma, neuroblastoma, keratoacanthoma, epidermoid carcinoma, large cell carcinoma, non-small cell lung cancer (NSCLC), small cell carcinoma, lung adenocarcinoma, adenoma, adenocarcinoma, thyroid cancer, follicular carcinoma, undifferentiated carcinoma, papillary carcinoma, seminoma, melanoma, sarcoma, bladder cancer, liver cancer, and kidney cancer, myeloid disorders, lymphoid disorders, hairy cell cancer, oral cancer, and Hodgkin's cancer and leukemia.

The present application includes methods for inhibiting abnormal cell growth or treating hyperproliferative disorders such as cancer in mammals (e.g., humans), comprising administering to the mammal a therapeutically effective amount of a compound of Formula I and/or its solvates, hydrates and/or salts thereof in combination with a second chemotherapeutic agent, such as those described herein. For example, the present application includes methods for treating multiple myeloma, lymphoma, acute myeloid leukemia, prostate cancer, breast cancer, hepatocellular carcinoma, pancreatic cancer and/or colorectal cancer in mammals (e.g., humans), comprising administering to the mammal a therapeutically effective amount of a compound of Formula I and/or its solvates, hydrates and/or salts thereof in combination with a second chemotherapeutic agent, such as those described herein.

The present application includes a method of treating lymphoma in a mammal (e.g., a human), comprising administering to the mammal a therapeutically effective amount of a compound of Formula I and/or a solvate, hydrate, and/or salt thereof, or a combination thereof, alone or in combination with a second chemotherapeutic agent, such as an anti-B cell antibody therapeutic (e.g., and/or dacetuzumab), gemcitabine, a corticosteroid (e.g., prednisolone and/or dexamethasone), a chemotherapy cocktail (e.g., CHOP (cyclophosphamide, doxorubicin, vincristine, prednisolone), and/or ICE (ifosfamide, cyclophosphamide, etoposide), combinations of biologics and chemotherapy (e.g., -ICE, dacetuzumab--ICE, R-Gem, and/or D-R-Gem), Akt inhibitors, PI3K inhibitors (e.g., GDC-0941 (Genentech) and/or GDC-0980 (Genentech)), rapamycin, rapamycin analogs, mTOR inhibitors such as everolimus or sirolimus, MEK inhibitors (GDC-0973), and Bcl-2 inhibitors (ABT-263 or ABT-199).

The present application includes a method of treating multiple myeloma in a mammal (e.g., a human) comprising administering to the mammal a therapeutically effective amount of a compound of Formula I and/or a solvate, hydrate, and/or salt thereof, or a combination thereof, alone or in combination with a second chemotherapeutic agent, such as melphalan, "Imids" (immunomodulators such as thalidomide, lenalidomide, and/or pomalidomide), corticosteroids (e.g., dexamethasone and/or prednisolone), and bortezomib or other proteasome inhibitors.

The present application includes a method of treating multiple myeloma, chronic lymphocytic leukemia (CLL) or acute myeloid leukemia (AML) in a mammal (e.g., a human), comprising administering to the mammal a therapeutically effective amount of a compound of Formula I and/or a solvate, hydrate and/or salt thereof, or a combination thereof, alone or in combination with a second chemotherapeutic agent, such as cytarabine (araC), an anthracycline antibiotic (e.g., daunorubicin and/or idarubicin), an anti-myeloid antibody therapeutic drug (e.g., SGN-33), an anti-myeloid antibody-drug conjugate (e.g., ).

The present application includes a method of treating chronic lymphocytic leukemia (CLL) in a mammal (e.g., a human) comprising administering to the mammal a therapeutically effective amount of a compound of Formula I and/or a solvate, hydrate, and/or salt thereof, or a combination thereof, alone or in combination with a second chemotherapeutic agent, such as fludarabine, cyclophosphamide, an anti-B cell antibody therapeutic (e.g., and/or dacetuzumab).

The present application includes a method of treating chronic myeloid leukemia (CML) in a mammal (e.g., a human), comprising administering to the mammal a therapeutically effective amount of a compound of Formula I and/or a solvate, hydrate and/or salt thereof, or a combination thereof, alone or in combination with a second chemotherapeutic agent, such as a BCR-abl inhibitor (e.g., imatinib, nilotinib and/or dasatinib).

The present application includes a method for treating myeloproliferative disorders (MDS) and myeloproliferative disorders (including polycythemia vera (PV), essential thrombocythemia (ET) or myelofibrosis (MF)) in mammals (e.g., humans), comprising administering to the mammal a therapeutically effective amount of a compound of Formula I and/or its solvate, hydrate and/or salt, or a combination thereof, alone or in combination.

The present application includes methods of using the compounds of the present application in vitro, in situ and in vivo to diagnose or treat mammalian cells, organisms or related pathological conditions.

Another aspect of the present application provides compounds of the present application for use in treating the diseases or conditions described herein in mammals, such as humans, suffering from the diseases or conditions described herein. The present application also provides the use of compounds of the present application in the preparation of medicaments for treating the diseases and conditions described herein in warm-blooded animals, such as mammals, such as humans, suffering from the diseases and conditions described herein.

pharmaceutical preparations

In order to use the compound of formula (I) for therapeutic treatment (including prophylactic treatment) of mammals (including humans), it is generally formulated into a pharmaceutical composition according to standard pharmaceutical practice. In this aspect of the application, a pharmaceutical composition is provided, which comprises the compound of the application and a pharmaceutically acceptable diluent or carrier.

The pharmaceutical composition can be in a form suitable for oral administration, for example, as a tablet, capsule, pill, powder, sustained-release formulation, solution, suspension, or a form suitable for parenteral administration, as a sterile solution, suspension, or emulsion, or a form suitable for topical administration, or as a suppository, or a form suitable for rectal administration. The pharmaceutical composition can be in a unit dosage form suitable for single administration of a precise dose. The pharmaceutical composition will comprise conventional pharmaceutical carriers or excipients and a compound of the present invention as an active ingredient. In addition, it may comprise other medical or pharmaceutical substances, carriers, excipients, and the like.

Exemplary parenteral administration forms include solutions or suspensions of a compound of Formula I in sterile aqueous solution, such as aqueous propylene glycol or dextrose solution. Such dosage forms can be suitably buffered as needed.

Suitable pharmaceutical carriers include inert diluents or fillers, water, and various organic solvents. Pharmaceutical compositions may contain additional ingredients such as flavoring agents, binders, excipients, etc. as needed. Thus, for oral administration, tablets containing various excipients such as citric acid can be used together with various disintegrants such as starch, alginic acid, and some complex silicates, and binders such as sucrose, gelatin, and gum arabic. In addition, lubricants such as magnesium stearate, sodium lauryl sulfate, and talc are often used for tableting purposes. Similar types of solid compositions can also be used in soft and hard-filled gelatin capsules. Therefore, preferred materials include lactose and high molecular weight polyethylene glycols. When aqueous suspensions or elixirs are used for oral administration, the active compound therein may be combined with various sweeteners or flavoring agents, coloring substances or dyes, and (as needed) emulsifiers or suspending agents and diluents such as water, ethanol, propylene glycol, glycerol, or a combination thereof.

Methods for preparing various pharmaceutical compositions having specific amounts of active compounds are known or will be apparent to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Ester, Pa., 15.sup.th Edition (1975).

Typical preparations are prepared by mixing the compound of formula I 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 water-swellable polymers, hydrophilic or hydrophobic substances, gelatin, oils, solvents, water. The specific carrier, diluent or excipient used will depend on the mode and purpose of administering the application's compound. Solvents are generally selected based on those skilled in the art as being safe solvents (GRAS) for administering mammals. Typically, safe solvents are nontoxic aqueous solvents such as water and other nontoxic solvents that can be dissolved or miscible in water. Suitable aqueous solvents include water, ethanol, propylene glycol, polyethylene glycol (such as PEG 400, PEG 300) and mixtures thereof. The formulation may also include one or more buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifiers, glidants, processing aids, colorants, sweeteners, aromas, flavorings and other known additives to give the drug (i.e., the compound of the present application or its pharmaceutical composition) a high-quality appearance or to facilitate the manufacture of pharmaceutical products (i.e., medicines).

The formulations can be prepared using conventional dissolution and mixing procedures. For example, a bulk drug substance (i.e., a compound of the present application or a stabilized form of a compound of Formula I (e.g., a complex with a cyclodextrin derivative or other known complexing agent)) is dissolved in a suitable solvent in the presence of one or more of the above-mentioned excipients. The compounds of the present application are typically formulated into pharmaceutical dosage forms to provide easily controllable drug dosages and enable patients to comply with prescribed regimens.

The pharmaceutical composition (or preparation) for administration can be packaged in a variety of ways depending on the method of administering the drug. Typically, the product for distribution includes a container in which a pharmaceutical preparation in a suitable form is stored. Suitable containers are well known to those skilled in the art and include, for example, bottles (plastic and glass), pouches, ampoules, plastic bags, metal cylinders and other items. The container may also include a pry-proof device to prevent inadvertent access to the package contents. In addition, the container may have a label describing the container contents. The label may also include suitable warning information.

Pharmaceutical preparations of the present application compounds can be prepared for various routes of administration and types. For example, the compound of formula I with the desired purity can be optionally mixed with a pharmaceutical diluent, carrier, excipient or stabilizer into a lyophilized preparation, ground powder or aqueous solution form (Remington's Pharmaceutical Sciences (1980) 16th edition, Osol, A.Ed.). Preparation can be carried out as follows: at ambient temperature at a suitable pH with a suitable purity and a physiologically acceptable carrier, i.e., a carrier that is nontoxic to the recipient at the dosage and concentration used. The pH of the preparation depends primarily on the specific use and compound concentration, but can be from about 3 to about 8. Preparations in acetate buffer at a pH of 5 are suitable embodiments.

The compounds of the present invention used herein are preferably sterile. Specifically, formulations for in vivo administration must be sterile. Such sterilization is readily achieved by filtration through a sterile filtration membrane.

The compound can typically be stored as a solid composition, a lyophilized formulation, or an aqueous solution.

The present application pharmaceutical composition comprising compound of formula I will be prepared, determined dosage and administration in a manner consistent with good medical practice (i.e. dosage, concentration, time arrangement, course of treatment, vehicle and approach). Factors to be considered in this context include the specific illness for treatment, the specific mammal for treatment, the clinical condition of individual patients, the cause of illness, the position of drug delivery, the method for administration, the time arrangement of administration and other factors known to medical practitioners. " Therapeutically effective amount " of compound to be administered will depend on the above-mentioned factors for consideration and is the minimum amount needed for preventing, improving or treating the illness mediated by coagulation factors. The above amount is preferably less than the amount that is toxic to the host or makes the host obviously more prone to bleeding.

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

Acceptable diluents, carriers, excipients, and stabilizers are nontoxic to recipients at the dosages and concentrations employed and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (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 such as TWEEN ^™ , PLURONICS ^™ or polyethylene glycol (PEG). The active pharmaceutical ingredient can also be entrapped in microcapsules prepared by, for example, coacervation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions. Such techniques are described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release formulations of the compound of formula I can be prepared. Suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing the compound of formula I, in the form of shaped articles such as films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactides (US3773919), copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT ^™ (microspheres for injection consisting of lactic acid-glycolic acid copolymer and leuprorelin acetate), and poly-D-(-)-3-hydroxybutyric acid.

The formulations include those suitable for the routes of administration described herein. The formulations can be suitably provided in unit dosage form and can be prepared by any method known in the pharmaceutical field. Techniques and formulations are generally described in Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, PA). The above method includes the step of combining the active ingredient with a carrier that serves as one or more auxiliary ingredients. Typically, the formulation is prepared by uniformly and tightly combining the active ingredient with a liquid carrier or a finely dispersed solid carrier or both, and then molding the product as needed.

Formulations of compounds 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 by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with one or more excipients selected from binders, lubricants, inert diluents, fillers, disintegrants, preservatives, surfactants and dispersants. Molded tablets may be prepared by molding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent. The tablets may optionally be coated or scored and are optionally formulated so as to provide slow or controlled release of the active ingredient therefrom.

Tablets, lozenges, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules such as gelatin capsules, syrups or elixirs can be prepared for oral administration. Formulations of the compound of formula I intended for oral administration can be prepared according to any method known in the art for preparing pharmaceutical compositions and such compositions may contain one or more substances including sweeteners, flavorings, colorants and preservatives to provide a palatable preparation. Tablets containing the active ingredient mixed with non-toxic physiologically acceptable excipients suitable for tablet preparation are acceptable. These excipients may be, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrants such as corn starch or alginic acid; binders such as starch, gelatin or gum arabic; and lubricants such as magnesium stearate, stearic acid or talc. Tablets may be uncoated or may be coated by known techniques including microencapsulation to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained effect over a longer period of time. For example, a time delay material such as glyceryl monostearate or glyceryl distearate alone or with a wax may be employed.

For treatment of the eye or other external tissues, such as the mouth and skin, the formulation may preferably be applied as a topical ointment or 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 a paraffinic or water-miscible ointment base. Alternatively, the active ingredient may be formulated as a cream with an oil-in-water cream base.

If desired, the aqueous phase of the cream base may contain polyols, i.e., alcohols having two or more hydroxyl groups, such as propylene glycol, butane-1,3-diol, mannitol, sorbitol, glycerol, and polyethylene glycol (including PEG 400), and mixtures thereof. Topical formulations may contain compounds that enhance the absorption or penetration of the active ingredient through the skin or other area of action, as desired. Examples of such skin penetration enhancers include dimethyl sulfoxide and related analogs.

The oil phase of the emulsion of the present application can be composed of known ingredients in a known manner. When the phase may contain only an emulsifier, it may contain at least one emulsifier and a fat or oil or a mixture of both a fat and an oil as needed. Preferably, a hydrophilic emulsifier is included together with the lipophilic emulsifier as a stabilizer. It is also preferred to include both oil and fat. At the same time, the emulsifier with or without a stabilizer constitutes a so-called emulsifying wax, and the wax, together with the oil and fat, constitutes a so-called emulsifying cream base, which forms the oily dispersed phase of the cream. Emulsifiers and emulsion stabilizers suitable for the formulation of the present application include cetearyl alcohol, benzyl alcohol, myristyl alcohol, glyceryl monostearate, and sodium lauryl sulfate.

Aqueous suspensions of the compound of formula I contain a mixture of the active substance and excipients suitable for preparing aqueous suspensions. Such excipients include suspending agents such as sodium carboxymethylcellulose, cross-linked carboxymethylcellulose, povidone, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinyl pyrrolidone, gum tragacanth, and gum arabic; and dispersants or wetting agents such as naturally occurring phospholipids (e.g., lecithin), condensation products of alkylene oxides with fatty acids (e.g., polyoxyethylene stearate), condensation products of ethylene oxide with long-chain fatty alcohols (e.g., heptadecaethyleneoxycetanol), and condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyoxyethylene sorbitan monooleate). The aqueous suspension may also contain one or more preservatives such as ethyl or n-propyl p-hydroxybenzoate, one or more colorants, one or more flavoring agents, and one or more sweeteners such as sucrose or saccharin.

The pharmaceutical composition of the compound of formula I can be in the form of a sterile injection, such as a sterile injection aqueous or oily suspension. The suspension can be prepared using the above-mentioned suitable dispersants or wetting agents and suspending agents according to methods known in the art. The sterile injection can also be a sterile injection solution or suspension in a non-toxic parenteral acceptable diluent or solvent, such as a solution in 1,3-butanediol, or prepared as a lyophilized powder. Acceptable vehicles and solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils can generally be used as solvents or suspending media. For this purpose, any mild fixed oil can be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid can also be used to prepare injections.

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

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

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

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

Formulations 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, for example, a particle size of 0.1 to 500 microns (inclusive between 0.1 and 500 microns and in increments of, for example, 0.5, 1, 30, 35 microns, etc.) and are administered by rapid inhalation through the nasal passages or by inhalation through the mouth to reach the alveolar sacs. Suitable formulations include aqueous or oily solutions of the active ingredient. Formulations suitable for aerosol or dry powder administration can be prepared according to conventional methods and can be delivered with other therapeutic agents, such as compounds heretofore used to treat or prevent the following conditions.

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

Preparation can be packaged in unit dose or multidose container such as sealed ampoule or bottle and can be stored under freeze drying (lyophilization) state, and it only needs to add sterile liquid carrier such as water for injection before use. Instant injection solution and suspension are prepared by sterile powder, granule and tablet of the above-mentioned kind. Preferred unit dose preparation is those preparations containing the active ingredient of the above-mentioned daily dose of the application or unit daily subdose or its suitable fraction.

The present application also provides veterinary compositions comprising at least one active ingredient as described above and a veterinary carrier. A veterinary carrier is a substance useful for the purpose of administering the composition and can be a solid, liquid, or gaseous substance that is inert or acceptable in veterinary medicine and compatible with the active ingredient. These veterinary compositions can be administered parenterally, orally, or by any other desired route.

Combination therapy

Formula I compound can be used alone or in combination with other therapeutic agents for treating diseases or conditions such as hyperproliferative disorders (such as cancer) described herein. In some embodiments, the formula I compound is combined with a second compound with anti-hyperproliferative properties or a second compound that can be used to treat hyperproliferative disorders (such as cancer) in a pharmaceutical combination formulation or as a dosing regimen for combined therapy. The second compound of the pharmaceutical combination formulation or dosing regimen preferably has an activity complementary to the formula I compound, so that they do not adversely affect each other. The above-mentioned compounds are suitably present in an effective amount combination for the intended purpose. In one embodiment, the present application composition comprises a combination of a formula I compound and a chemotherapeutic agent described herein.

Combination therapy can be administered in a simultaneous or sequential manner. When administered sequentially, the combination can be administered in two or more administrations. Combination administration includes co-administration using separate formulations or a single pharmaceutical formulation and sequential administration in any order, preferably with a period of time during which both (or all) active agents simultaneously exert their biological activities.

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

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

In a specific embodiment of the anticancer therapy, the compound of formula I or its stereoisomers, geometric isomers, tautomers, solvates, metabolites or pharmaceutically acceptable salts or prodrugs can be combined with other chemotherapeutic agents, hormone drugs or antibody drugs such as those described herein and in combination with surgical therapy and radiotherapy. The present application combination therapy thus includes administering at least one compound of formula I or its stereoisomers, geometric isomers, tautomers, solvates, metabolites or pharmaceutically acceptable salts or prodrugs and using at least one other cancer treatment method. The amount of the compound of formula I and other pharmaceutically active chemotherapeutic agents and the associated administration schedule will be selected to achieve the desired combined therapeutic effect.

Metabolites of compounds of formula I

The in vivo metabolic products of Formula I described herein also fall within the scope of this application. Such products may result from, for example, oxidation, reduction, hydrolysis, amidation, deamidation, esterification, deesterification, enzymatic cleavage, etc. of the administered compound. Thus, this application includes metabolites of compounds of Formula I, including compounds produced by a method comprising contacting a compound of this application with a mammal for a period of time sufficient to produce a metabolic product thereof.

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

Products

Another embodiment of the present application provides an article or "kit" containing substances that can be used to treat the above-mentioned diseases and conditions. The kit includes a container containing a compound of Formula I. The kit may also include a label or package insert on or associated with the container. The term "package insert" is used to refer to instructions typically included in the commercial packaging of a therapeutic product, which contains information about the indications, usage, dosage, administration, contraindications and/or precautions involved in using the above-mentioned therapeutic product. Suitable containers include, for example, bottles, vials, syringes, blister packs, etc. The container can be formed from a variety of materials such as glass or plastic. The container can hold a compound of Formula I or II or a formulation thereof that can effectively treat the condition and can have a sterile interface (for example, the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). At least one active agent in the composition is a compound of Formula I. The label or package insert indicates that the composition is used to treat a selected condition, such as cancer. In addition, the label or package insert may indicate that the patient to be treated is a patient suffering from a condition such as a hyperproliferative condition, neurodegeneration, cardiac hypertrophy, pain, migraine, or a neurotraumatic disease or event. In one embodiment, the label or package insert indicates that the composition comprising the compound of Formula I can be used to treat a condition resulting from abnormal cell growth. The label or package insert may also indicate that the composition can be used to treat other conditions. Alternatively or additionally, the article 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. It may also comprise other substances desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

The kit may further comprise instructions for administering the compound of Formula I and the second pharmaceutical formulation (if present). For example, if the 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 thereof.

In another embodiment, the kit is suitable for delivering a solid oral form of a compound of Formula I, such as a tablet or capsule. The kit preferably comprises a plurality of unit doses. The kit may comprise a card having doses arranged in the order in which they are intended to be used. An example of a kit is a "blister pack". Blister packs are known in the packaging industry and are widely used for packaging pharmaceutical unit dosage forms. A memory aid may be provided as needed, for example in the form of numbers, letters or other markings or with a calendar indicating the days on which administration may be performed in a treatment arrangement.

According to one embodiment, the kit may comprise (a) a first container containing a compound of Formula I therein; and optionally (b) a second container containing 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 glucose solution. It may also comprise other substances desired from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

In some other embodiments where the kit comprises a composition of Formula I and a second therapeutic agent, the kit may comprise a container, such as a separate bottle or a separate foil package, for accommodating the separated compositions, but the separated compositions may also be contained in a single, unseparated container. Typically, the kit comprises instructions for administering the separated components. When the separated components are preferably administered in different dosage forms (e.g., oral and parenteral) or at different dosage intervals, or when the attending physician needs to titrate the combined components, the kit form is particularly advantageous.

Preparation of compounds of formula I

The compounds of formula I can be synthesized by the following synthetic routes, which include methods analogous to those known in the chemical art and particularly those referenced in the present specification, as well as those for other heterocycles, such as those described in 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. Starting materials are generally available from commercial sources such as Aldrich Chemicals (Milwaukee, WI) or readily prepared using methods known to those skilled in the art (e.g., by methods outlined in Louis F. Fieser and Mary Fieser, Reagents for Organic Synthesis, v. 1-23, Wiley, N.Y. (1967-2006 ed.) or Beilsteins Handbuch der organischen Chemie, 4, Aufl. ed. Springer-Verlag, Berlin (including supplements) (also available from the Beilstein online database)).

The synthetic chemistry transformations and protecting group methodology (protection and deprotection) and the necessary reagents and intermediates that can be used to synthesize compounds of Formula I are known in the art and are described, for example, in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and PGM Wuts, Protective Groups in Organic Synthesis, ^3rd Ed., John Wiley and Sons (1999); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995) and subsequent editions.

Compounds of formula I can be prepared individually or as a library comprising at least two, 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 by a combined "split and mix" approach or by multiple parallel syntheses using solution phase or solid phase chemistry. Thus, another aspect of the present application provides a library of compounds comprising at least two compounds or pharmaceutically acceptable salts thereof.

The general procedures and examples provide exemplary methods for preparing compounds of Formula I. Those skilled in the art will recognize that other synthetic routes can be used to synthesize compounds of Formula I. Although specific raw materials and reagents are described and discussed in the figures, intermediates, and examples, other raw materials and reagents can be easily substituted to provide a variety of derivatives and/or reaction conditions. In addition, the various exemplary compounds prepared by the methods described can be further modified based on the present disclosure using conventional chemical methods known to those skilled in the art.

When preparing compound of formula I, it may be necessary to protect the remote functional group (such as primary amine or secondary amine) of the intermediate. The needs for the above protection will vary with the properties of the remote functional group and the conditions of the preparation method. Suitable amino protecting groups include acetyl, trifluoroacetyl, tert-butyloxycarbonyl (BOC), benzyloxycarbonyl (CBz) and 9-fluorenylmethyleneoxycarbonyl (Fmoc). The needs for the above protection are easily determined by those 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.

In the process for preparing the compound of formula I, it may be advantageous to separate the reaction products from each other and/or from the starting materials. The desired product of each step or steps is separated and/or purified to the desired uniformity by conventional techniques in the art. Typically, the separation involves multiphase extraction, crystallization from a solvent or solvent mixture, distillation, sublimation, or chromatography. Chromatography can involve a variety of methods, including, for example: reverse phase and normal phase chromatography; size exclusion chromatography; ion exchange chromatography; high, medium, and low pressure liquid chromatography methods and apparatus; small-scale analytical chromatography; simulated moving bed (SMB) and preparative thin or thick layer chromatography and small-scale thin layer and flash chromatography.

Another type of separation method involves treating the mixture with a reagent selected to bind to or otherwise render the desired product, unreacted starting materials, reaction by-products, etc. separable. Such reagents include adsorbents or absorbents such as activated carbon, molecular sieves, ion exchange media, etc. 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 extraction reagent (LIX), etc. The choice of an appropriate separation method depends on the properties of the substances involved, such as boiling point and molecular weight (in distillation and sublimation), the presence or absence of polar functional groups (in chromatography), the stability of the substance in acidic and basic media (in multiphase extraction), etc.

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

Individual stereoisomers, such as enantiomers, substantially free of their stereoisomers can be obtained by resolution of the racemic mixture using, for example, an optically active resolving agent to form diastereomers (Eliel, E. and Wilen, S. "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., New York, 1994; Lochmuller, C. H., (1975) J. Chromatogr., 113(3):283-302). Racemic mixtures of the chiral compounds of the present invention can be separated by any suitable method, including: (1) formation of ionic diastereomeric salts with chiral compounds and separation by fractional crystallization or other methods; (2) formation of diastereomeric compounds with chiral derivatizing reagents, separation of the diastereomers and conversion to pure stereoisomers; and (3) direct separation of the substantially pure or enriched stereoisomers under chiral conditions, such as by HPLC or SFC (supercritical fluid chromatography) on a chiral adsorbent, see White and Burnett (2005) Jour. of Chrom. A 1074: 175-185; and "Drug Stereochemistry, Analytical Methods and Pharmacology", (1993) Irving W. Wainer, Ed., Marcel Dekker, Inc., New York.

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

Alternatively, the substrate to be resolved is reacted with one enantiomer of a chiral compound by method (2) to form a diastereomeric pair (E. and Wilen, S. "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., 1994, p. 322). Diastereomeric compounds can be formed by reacting an asymmetric compound with an enantiomerically pure chiral derivatizing reagent, such as a menthyl derivative, followed by separation of the diastereomers and hydrolysis to yield the pure or enriched enantiomer. Methods for determining optical purity involve preparing a chiral ester of the racemic mixture [e.g., preparing a menthyl ester such as (-) menthyl chloroformate or preparing Mosher's ester, α-methoxy-α-(trifluoromethyl)phenyl acetate (Jacob III. J. Org. Chem. (1982) 47:4165) in the presence of a base and analyzing the ¹ H NMR spectrum for the presence of two atropisomeric enantiomers or diastereomers. Stable diastereomers of atropisomeric compounds can be separated by normal-phase and reverse-phase chromatography following methods for separating atropisomeric naphthyl-isoquinolines (WO 1996/15111). By method (3), a racemic mixture of two enantiomers can be separated by chromatography using a chiral stationary phase ("Chiral Liquid Chromatography" (1989) WJ Lough, Ed., Chapman and Hall, New York; Okamoto, J. Chromatogr., (1990) 513:375-378). The enriched or purified enantiomers can be distinguished by methods used to distinguish other chiral molecules with asymmetric carbon atoms, such as optical rotation or circular dichroism.

General preparation procedures

FIG1 shows an exemplary synthesis of 4-aminopyrazole compound 5. 4-Nitro-1H-pyrazole 1 is converted to 1-substituted-4-nitro-1H-pyrazole compound 2 by treatment with a base in a suitable solvent or neat followed by the addition of an alkylating agent such as dimethyl sulfate. Compound 2 can be converted to 5-chloro-4-nitro-1H-pyrazole 3 by treatment with a base such as lithium bis(trimethylsilyl)amide (LHMDS) or nBuLi (butyllithium) in a suitable solvent such as THF (tetrahydrofuran) at an appropriate temperature, such as −78° C. Compound 3 can be converted to compound 4 by direct SnAr or by a transition metal-catalyzed cross-coupling reaction such as Suzuki, Sonogashira, Heck, Buchwald, or Goldberg conditions under known methods. 4-Aminopyrazole 5 can be synthesized from 4 by a suitable reduction method, such as treatment with zinc powder and ammonium formate in tetrahydrofuran or hydrogenation with _H and a transition metal catalyst such as palladium on carbon. The ^R2 group of the reagents ^R2Br and ^R2H is a precursor to the formation of intermediates for the preparation of compounds of formula I.

Figure 2 shows an exemplary synthesis of 2-substituted thiazole-4-carboxylic acid compound 6 from thiazole-4-carboxylate compound 7. 7 is brominated to provide 2-bromothiazole-4-carboxylate compound 8, which reacts via a Suzuki reaction with palladium catalysis and a procatalyst and an ^R3 -X reagent wherein ^R3 is an aryl or heteroaryl group and X is a boronic acid or boronic acid ester group to provide 2-substituted thiazole-4-carboxylate compound 9. Aqueous base hydrolysis of the ester provides 6.

Buchwald coupling reactions can be carried out under Buchwald palladium catalytic conditions using the Buchwald procatalyst palladacycle complexes and ligand reagents listed in the table below and described in the following literature: Biscoe et al. (2008) J. Am. Chem. Soc. 130:6686-6687; Kinzel et al. (2010) J. Am. Chem. Soc. 132:14073-14075; Molander et al. (2012) J. Am. Chem. Soc. 134:11 667-11673; Walker et al. (2004) Angew. Chem. Int. Ed. 43:1871; Billingsley et al. (2007) Angew. Chem. Int. Ed. 46:5359-5363; US Pat. No. 6,946,560; US Pat. No. 7,026,498; US Pat. No. 7,247,731; US Pat. No. 7,560,582; US Pat. No. 6,307,087; US Pat. No. 6,395,916; US Pat. No. 7,223,879; US Pat. No. 7,858,784, which are incorporated by reference. The above reagents are commercially available (Johnson Matthey Inc., Wayne, PA; Sigma Aldrich Fine Chemical, St. Louis, MO; Strem Chemicals, Inc., Newburyport, MA).

Figure 2 shows an exemplary synthesis of 2-substituted-4-carboxy-5-aminothiazoles 6 by C-2 bromination of 5-aminothiazole-4-carboxylates, such as 7, followed by a Suzuki reaction. Bromination of 5-aminothiazole-4-carboxylates, such as 7, with a brominating agent in a suitable solvent, such as NBS (N-bromosuccinimide) in dichloromethane, affords 8. Suzuki-type coupling reactions can be used to attach heterocyclic or heteroaryl groups in the synthesis of compounds of Formula I by replacing the halogen at the 2-position of the thiazole, pyridyl, pyrazinyl, or pyrimidinyl ring. For example, 2-bromo(or chloro)thiazole 8 can be reacted with approximately 1.5 equivalents of an aryl, heterocyclic, or heteroaryl boronic acid or boronic ester reagent, R ³ -X, and an excess of aqueous sodium carbonate in acetonitrile. A catalytic amount or greater of a low-valent palladium reagent, such as bis(triphenylphosphine)palladium(II) dichloride, is added. Various boronic acids or boronic esters can be used as the X group of the reagent R ³ -X. Boronic acid esters include pinacol esters (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl). In addition, the nitrogen atom of the heterocyclic or heteroaryl group can be protected as, for example, N-THP. The R ³ group of the reagent R ³ -X is as defined in the compound of formula I or a useful precursor for preparing the compound of formula I. In some cases, potassium acetate is used instead of sodium carbonate to adjust the pH of the aqueous layer. The reaction mixture is heated to about 140-150° C. under pressure in a microwave reactor such as a Biotage Optimizer (Biotage, Inc.) and maintained for 10 to 30 minutes. The contents are extracted with ethyl acetate or another organic solvent. After evaporation of the organic layer, the Suzuki coupling product 9 or 6 can be purified on silica gel or by reverse phase HPLC.

A variety of palladium catalysts can be used in the Suzuki coupling step to form exemplary Formula I compounds. Low-valent Pd(II) and Pd(0) catalysts can be used for Suzuki coupling reactions, including PdCl ₂ (PPh ₃ ) ₂ , Pd(t-Bu) ₃ , PdCl ₂ dppfCH ₂ Cl ₂ , Pd(PPh ₃ ) ₄ , Pd(OAc)/PPh ₃ , Cl ₂ Pd[(Pet ₃ )] ₂ , Pd(DIPHOS) ₂ , Cl ₂ Pd(Bipy), [PdCl(Ph ₂ PCH ₂ PPh ₂ )] ₂ , Cl ₂ Pd[P(p-toluene) ₃ ] ₂ , Pd ₂ (dba) ₃ /P(p-toluene) ₃ , Pd ₂ (dba)/P(furyl) ₃ , Cl ₂ Pd[P(furyl) ₃ ] ₂ , Cl ₂ Pd(PmePh ₂ ) ₂ , Cl ₂ Pd[P(4-F-Ph) ₃ ] ₂ , Cl ₂ Pd[P(C ₆ F ₆ ) ₃ ] ₂ , Cl ₂ Pd[P(2-COOH-Ph)(Ph) ₂ ] ₂ , Cl ₂ Pd[P(4-COOH-Ph)(Ph) ₂ ] ₂ , and the encapsulated catalysts PdEnCat ^™ 30, Pd EnCat ^™ TPP30, and Pd(II)EnCat ^™ BINAP30 ( US2004/0254066 ).

Various solid adsorptive palladium scavengers can be used to remove palladium after Suzuki, Suzuki-Miyaura, or Buchwald reactions. Exemplary embodiments of palladium scavengers include thiols and thioureas. Other palladium scavengers include silica gel, controlled pore glass (TosoHaas), and derivatized low-crosslinked polystyrene QuadraPure ^™ AEA, QuadraPure ^™ IMDAZ, QuadraPure ^™ MPA, QuadraPure ^™ TU (Reaxa Ltd., Sigma-Aldrich Chemical Co.).

Figure 3 shows an exemplary synthesis of coupled pyrazole-thiazole compounds 10. 4-Aminopyrazole compound 5 is coupled with 2-substituted-4-carboxy-5-aminothiazole 6 using an amide-forming (peptide) coupling reagent such as dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), HATU (O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate), HBTU (O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate or PyBOP ((benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate) in a suitable solvent such as dichloromethane or DMF to form the amide bond in 10 (Hermanson, G., Bioconjugate Techniques, 2nd ed. (2008) Academic Press, 1996). Press, San Diego). Boc and other protecting groups of 5 can be removed under conventional conditions, such as HCl/dioxane and water or trifluoroacetic acid/dichloromethane to remove Boc, Fmoc or other acid-labile protecting groups from the 4-amino group of 5.

Figure 4 shows an exemplary synthesis of 6-(4-nitro-1H-pyrazol-5-yl)oxepan-3-amine compound 19, such as ^6- (1-methyl-4-nitro-1H-pyrazol-5-yl)oxepan-3-amine, wherein R is methyl, from 5-chloro-4-nitro-1H-pyrazol compound 3. Displacement of the chlorine from 3 with dimethyl malonate in the presence of a base such as potassium carbonate in a suitable solvent such as DMSO or by similar methods described in the literature provides 2-(1-substituted-4-nitro-1H-pyrazol-5-yl) malonate compound 11. Decarboxylation of 11 under basic, acidic, or a combination of these conditions as described in the literature provides alkyl 2-(4-nitro-1H-pyrazol-5-yl)acetate compound 12. Allylation of 14 using a suitable base such as sodium hydride in a suitable solvent such as DMF or by methods described in the literature provides alkyl 2-(4-nitro-1H-pyrazol-5-yl)pent-4-enoate compound 13. Reduction of 13 using a suitable reducing agent such as DIBAL in a suitable solvent such as THF or by methods described in the literature provides 2-(4-nitro-1H-pyrazol-5-yl)pent-4-en-1-ol compound 14. Allylation of compound of formula 14 using a suitable base such as sodium hydride in a suitable solvent such as DMF or by methods described in the literature provides 5-(1-(allyloxy)pent-4-en-2-yl)-4-nitro-1H-pyrazole compound 15. An isotopic cyclization of 15 using Grubb's or related ruthenium catalysts (RCM = ruthenium-catalyzed isotopic reaction) under appropriate conditions can provide 4-nitro-5-(2,3,4,7-tetrahydrooxan-3-yl)-1H-pyrazole compound 16. Isomerization of 18 using Grubb's or Wilkinson's catalysts can provide 4-nitro-5-(2,3,4,5-tetrahydrooxan-3-yl)-1H-pyrazole compound 17. Compound 15 can be directly converted to 17 in a one-pot procedure using literature-described isotopic cyclization conditions. Hydroboration of 17 using conditions described in the literature provides 6-(4-nitro-1H-pyrazol-5-yl)oxepan-3-ol 18, which can be oxidized to the ketone followed by reductive amination to give 6-(4-nitro-1H-pyrazol-5-yl)oxepan-3-amine 19, or sulfonylated and then displaced with an amine reagent.

Figure 5 shows an exemplary synthesis of 5-(5-azido-6-fluorooxepan-2-yl)-1-substituted-4-nitro-1H-pyrazole compound 26 from 1-substituted-4-nitro-1H-pyrazole compound 2. 2 is reacted with pent-4-enal 20 with a suitable base such as lithium bis(trimethylsilyl)amide in a suitable solvent such as THF at the desired temperature or by procedures described in the literature to provide 1-(1-substituted-4-nitro-1H-pyrazol-5-yl)pent-4-en-1-ol compound 21. 21 is heated with diallyl carbonate in the presence of a suitable catalyst such as tris(dibenzylideneacetone)dipalladium(0) and triphenylphosphine in a solvent such as dioxane or using procedures described in the literature to provide 5-(1-(allyloxy)pent-4-enyl)-1-substituted-4-nitro-1H-pyrazole compound 22. Cyclization of 22 by heating with a suitable catalyst, such as the first generation Grubbs catalyst, in a suitable solvent, such as toluene (RCM), or by methods described in the literature, affords 1-substituted-4-nitro-5-(2,3,4,7-tetrahydrooxin-2-yl)-1H-pyrazole compounds 23. Treatment of 23 with an epoxidizing reagent, such as m-CPBA (m-chloroperbenzoic acid), in a solvent such as dichloromethane, or by similar methods described in the literature, affords 5-(3,8-dioxabicyclo[5.1.0]octan-4-yl)-1-substituted-4-nitro-1H-pyrazole compounds 24. Opening of the epoxy ring of 24 with sodium azide according to literature procedures affords 4-azido-7-(1-substituted-4-nitro-1H-pyrazol-5-yl)oxepan-3-ol compounds 25. Fluorination of 25 with a reagent, such as in a suitable solvent, such as DCM, or by methods described in the literature affords 26.

Figure 6 shows an exemplary synthesis of 5-(5-azido-4-fluorooxepan-2-yl)-1-substituted-4-nitro-1H-pyrazole compounds 31 and 5-(4-azido-5-fluorooxepan-2-yl)-1-substituted-4-nitro-1H-pyrazole compounds 32 from 5-(1-(allyloxy)pent-4-enyl)-1-substituted-4-nitro-1H-pyrazole compound 22. Cyclization of 22 by heating with a suitable catalyst, such as 2nd generation Grubbs catalyst, in a suitable solvent, such as dichloromethane (RCM) or by methods described in the literature, affords 1-substituted-4-nitro-5-(2,3,6,7-tetrahydrooxa-2-yl)-1H-pyrazole compounds 27. Epoxidation of 27 with an epoxidizing agent such as m-CPBA in a solvent such as dichloromethane or by similar methods described in the literature provides 5-(4,8-dioxabicyclo[5.1.0]octan-3-yl)-1-substituted-4-nitro-1H-pyrazole compound 28. Treatment of 28 with an azide reagent (azidation) can provide a mixture of the ring-opened compounds 5-azido-2-(1-substituted-4-nitro-1H-pyrazol-5-yl)oxepan-4-ol 29 and 5-azido-7-(1-substituted-4-nitro-1H-pyrazol-5-yl)oxepan-4-ol 30. Fluorination of 29 and 30 with a fluorinating agent such as (Sigma-Aldrich) in a suitable solvent such as DCM or by methods described in the literature provides 33 and 34, respectively.

Figure 7 shows an exemplary synthesis of 5-(5-azido-6-fluorooxepan-2-yl)-1-substituted-4-nitro-1H-pyrazole compound 35 from 1-substituted-4-nitro-5-(2,3,4,7-tetrahydrooxepan-2-yl)-1H-pyrazole compound 23. Treatment of 23 with N-bromosuccinimide and acetic acid in a suitable solvent such as dichloromethane in the presence of molecular sieves, followed by treatment with potassium carbonate in a suitable solvent such as methanol, or by methods described in the literature, affords 5-(3,8-dioxabicyclo[5.1.0]octan-4-yl)-1-substituted-4-nitro-1H-pyrazole compound 33. Opening the epoxy ring of 33 with sodium azide according to literature procedures affords 4-azido-7-(1-substituted-4-nitro-1H-pyrazol-5-yl)oxepan-3-ol compound 34. Fluorination of 34 with a fluorinating agent, for example in a solvent such as DCM or by methods described in the literature, affords 35.

Figure 8 shows an exemplary synthesis of 2-methyl-N-(2-substituted-7-(1-substituted-4-nitro-1H-pyrazol-5-yl)oxepan-4-yl)propane-2-sulfenamide compound 40 from 5-chloro-4-nitro-1H-pyrazole compound 3. 3 is subjected to a Suzuki reaction by heating with potassium vinyl trifluoroborate and cesium carbonate in solvents such as DMF and water in the presence of a suitable catalyst such as 1,1′-bis(diphenylphosphino)ferrocene-palladium(II) dichloromethane complex, followed by treatment of the resulting olefin with ozone in a suitable solvent such as dichloromethane or using methods described in the literature to provide 1-substituted-4-nitro-1H-pyrazole-5-carboxaldehyde compound 36. Compound 36 is treated with (R)-trimethyl(1-(1-(trimethylsilyloxy)cyclopropyl)propan-2-yloxy)silane compound 37 and trimethylsilyl trifluoromethanesulfonate in a suitable solvent such as dichloromethane or using methods described in the literature (Minbiole et al. (2005) Org. Lett. 7:515) to provide 5-((5R,7R)-7-substituted-4,6-dioxaspiro[2.5]octan-5-yl)-1-substituted-4-nitro-1H-pyrazole compound 38. Treatment of compound 38 with a suitable Lewis acid such as titanium tetrachloride in a solvent such as dichloromethane or using methods described in the literature provides the rearranged product, (2R,7R)-2-substituted-7-(1-substituted-4-nitro-1H-pyrazol-5-yl)oxepan-4-one compound 39. Reductive amination of 39 affords 40 by heating with (R)-2-methylpropane-2-sulfenamide in the presence of a suitable Lewis acid such as titanium(IV) ethoxide in a solvent such as THF and subsequent treatment with sodium borohydride in a suitable solvent or using literature procedures.

9 shows an exemplary synthesis of tert-butyl (2R,3R,4S,5R)-5-hydroxy-3,5-dimethyl-2-(1-substituted-4-nitro-1H-pyrazol-5-yl)tetrahydro-2H-pyran-4-ylcarbamate compound 47 from 1-substituted-4-nitro-1H-pyrazole-5-carbaldehyde compound 36. Heating 36 with diene ((1E,3Z)-1-methoxy-2-methylpenta-1,3-dien-3-yloxy)trimethylsilane 48 in the presence of Resolve-Al ^™ EuFOD (europium(III) tris(1,1,1,2,2,3,3-heptafluoro-7,7-dimethyl-4,6-octanedionato), Sievers' reagent, europium tris(6,6,7,7,8,8,8-heptafluoro-2,2-dimethyl-3,5-octanedionato), Sigma-Aldrich Product No. 160938, CAS No. 17631-68-4) in a suitable solvent such as chloroform or using literature methods gives 3,5-dimethyl-2-(1-substituted-4-nitro-1H-pyrazol-5-yl)-2H-pyran-4(3H)-one compound 41. Treatment of 41 with a suitable reducing agent such as sodium borohydride in the presence of cerium(III) chloride heptahydrate in a suitable solvent such as methanol or using similar methods described in the literature provides 3,5-dimethyl-2-(1-substituted-4-nitro-1H-pyrazol-5-yl)-3,4-dihydro-2H-pyran-4-ol compound 42. Heating 42 with p-toluenesulfonic acid in methanol or using methods described in the literature provides the rearrangement product, 5-(6-methoxy-3,5-dimethyl-3,6-dihydro-2H-pyran-2-yl)-1-substituted-4-nitro-1H-pyrazole compound 43. Treatment of 43 with a Lewis acid such as boron trifluoride etherate and a reducing agent such as triethylsilane in a suitable solvent such as dichloromethane or using methods described in the literature provides 5-(3,5-dimethyl-3,6-dihydro-2H-pyran-2-yl)-1-substituted-4-nitro-1H-pyrazole compound 44. Epoxidation of 44 with an epoxidizing agent such as m-CPBA or by similar methods described in the literature provides 5-(1,5-dimethyl-3,7-dioxabicyclo[4.1.0]hept-4-yl)-1-substituted-4-nitro-1H-pyrazole compound 45. Opening of the epoxy ring of 45 with sodium azide according to literature procedures provides 4-azido-3,5-dimethyl-6-(1-substituted-4-nitro-1H-pyrazol-5-yl)tetrahydro-2H-pyran-3-ol compound 46. Staudinger azide reduction of 46 by heating with trimethylphosphine in THF and water followed by protection of the resulting amine with a suitable protecting group such as a Boc protecting group using those methods outlined or described in the literature provides 47.

Figure 10 shows an exemplary synthesis of tert-butyl 3-methoxy-2-methyl-7-(1-substituted-4-nitro-1H-pyrazol-5-yl)oxepan-4-ylcarbamate compound 52 from 1-(1-substituted-4-nitro-1H-pyrazol-5-yl)pent-4-en-1-ol compound 21. Palladium-catalyzed O-alkylation of 21 with 3-chlorobut-1-ene or but-3-en-2-yl acetate afforded 5-(1-(but-3-en-2-yloxy)pent-4-enyl)-1-substituted-4-nitro-1H-pyrazole compound 49. Grubbs catalyst cyclization of 49 afforded 1-substituted-5-(7-methyl-2,3,4,7-tetrahydrooxan-2-yl)-4-nitro-1H-pyrazole compound 50. Olefin epoxidation of 50 with m-chloroperbenzoic acid followed by azide epoxy ring opening affords 4-azido-2-methyl-7-(1-substituted-4-nitro-1H-pyrazol-5-yl)oxepan-3-ol compound 51. Hydroxymethylation of 51 with iodomethane, azide reduction with triphenylphosphine, and Boc protection affords 52, which is useful as an intermediate in the preparation of compounds of Formula I.

Example

Intermediate 1 5-chloro-1-methyl-4-nitro-1H-pyrazole

To a 500 mL round-bottom flask containing 4-nitro-1H-pyrazole (5 g, 44.2 mmol) was added sodium hydroxide (1 M, 200 mL) and dimethyl sulfate (31 mL, 330 mmol). The mixture was stirred at room temperature for 72 h and extracted with CH ₂ Cl ₂ (2×150 mL). The organic layer was separated and the solvent was distilled off to provide 1-methyl-4-nitro-1H-pyrazole as a white solid (4.30 g, 76%).

Following WO2007/99326, 1-methyl-4-nitro-1H-pyrazole (4.30 g, 33.8 mmol) and THF (12 mL) were added to a 500 mL 3-necked round-bottom flask. The mixture was cooled to -78 ° C and lithium bis(trimethylsilyl)amide/THF (1 M, 88.4 mL, 90 mmol) was added dropwise via an addition funnel over 20 min. The brown mixture was stirred for 30 min and warmed to -45 ° C over 30 min. The mixture was cooled back to -78 ° C and hexachloroethane (10.5 g, 44.2 mmol) dissolved in THF (20 mL) was added via an addition funnel over 15 min. The mixture was stirred for 2.5 h, warmed from -78 ° C to -40 ° C, and the reaction was monitored by LCMS. After completion of the reaction, the reaction mixture was quenched with saturated NH ₄ Cl solution (150 mL) and ethyl acetate (100 mL) was added. The organic layer was separated and the aqueous layer was extracted with ethyl acetate (100 mL). The combined organic layers were washed with water (150 mL), dried over Na ₂ SO ₄ , and the organic solvent was distilled off. The crude product was purified by flash chromatography (CH ₂ Cl ₂ /7% MeOH) to give 5-chloro-1-methyl-4-nitro-1H-pyrazole as a white solid (1.40 g, 20%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.13 (s, 1H), 3.92 (s, 3H); ESIMS m/z = 162.0 (M+1)

Intermediate 2 ethyl 2-amino-2-cyanoacetate

To a stirred solution of (E)-ethyl 2-cyano-2-(hydroxyimino)acetate (20 g, 0.14 mol) in water (250 mL) was added saturated aqueous NaHCO ₃ solution (160 mL) followed by Na ₂ S ₂ O ₄ (60 g, 0.423 mol). The reaction mixture was warmed to 35 ° C and stirred for an additional 2 h. It was then saturated with NaCl (150 g) and extracted with DCM (3×350 mL). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ , filtered, and concentrated in vacuo to afford ethyl 2-amino-2-cyanoacetate as a red oil (7.8 g, 43%), which was used in the next step without further purification. ¹ H-NMR (CDCl ₃ , 500MHz) δ (ppm): 4.45 (s, 1H), 4.34 (q, J = 7.0Hz, 2H), 1.36 (t, J = 7.0Hz, 3H); MS (ESI) m/z: 129 [M+H] ⁺ .

Intermediate 3 ethyl 2-benzamido-2-cyanoacetate

To a stirred solution of ethyl 2-amino-2-cyanoacetate (0.64 g, 5 mmol) in DCM (15 mL) was added saturated aqueous _NaHCO₃ (15 mL). Benzoyl chloride (0.84 g, 6 mmol) was added with vigorous stirring. The reaction mixture was stirred at ambient temperature for an additional 30 min, at which time it was extracted with DCM (3 × 15 mL). The combined organic layers were washed with brine (20 mL) and dried _{over Na₂SO₄} , filtered, and concentrated in vacuo. The resulting residue was purified by silica gel column chromatography (5:1 PE/EtOAc) to give ethyl 2-benzamido-2-cyanoacetate (0.25 g, 22%) as a white solid: ¹ H-NMR (CDCl ₃ , 500 MHz) δ (ppm): 7.83-7.85 (m, 2H), 7.59 (t, J=7.5 Hz, 1H), 7.49 (t, J=7.5 Hz, 2H), 7.02 (d, J=7.0 Hz, 1H), 5.72 (d, J=7.5 Hz, 1H), 4.40 (q, J=7.5 Hz, 2H), 1.39 (t, J=7.0 Hz, 3H); MS (ESI) m/z: 233 [M+H] ⁺ .

Intermediate 4 2-(4-cyclopropyl-2-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane

Step A: 3-Fluoro-4-nitrophenyl trifluoromethanesulfonate

To a stirred solution of 3-fluoro-4-nitrophenol (10.00 g, 63.65 mmol) and trifluoromethanesulfonic anhydride (20.0 mL, 119 mmol, 1.87 equiv) in anhydrous DCM (100.0 mL) was added triethylamine (33.27 mL, 238.7 mmol, 3.75 equiv) at 0 ° C. The resulting brown reaction mixture was stirred at 0 ° C for 2 h and then at ambient temperature for 16 h. The reaction mixture was slowly quenched with water and extracted with DCM (3 × 100 mL). The combined organic layer was washed with brine (1 ×), dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The crude oil was purified by flash column chromatography eluting with 0 to 65% DCM / hexane to give 15.67 g (85.1%) of 3-fluoro-4-nitrophenyl trifluoromethanesulfonate as an oil. ¹ H NMR (500MHz, CDCl ₃ ) δ8.23 (t, J = 8.52Hz, 1H), 7.34-7.27 (m, 2H).

Step B: 4-Cyclopropyl-2-fluoro-1-nitrobenzene

A mixture of 3-fluoro-4-nitrophenyl trifluoromethanesulfonate (7.15 g, 24.73 mmol), cyclopropylboronic acid (2.55 g, 29.67 mmol), [1,1'-bis(diphenylphosphino)-ferrocene]dichloropalladium(II) complexed with dichloromethane (1:1) (1.62 g, 1.98 mmol), and 2M aqueous cesium carbonate (19.8 mL, 39.56 mmol) in toluene (39.5 mL) was degassed for 20 min. The reaction mixture was stirred at 90°C under _N₂ for 2.5 h. The reaction mixture was cooled to room temperature, diluted with ethyl acetate (200 mL), and filtered through a pad of Celite. The filtrate was washed with brine, dried over _Na₂SO₄ , _filtered , and concentrated under reduced pressure. The crude residue was purified by flash column chromatography eluting with 0-75% DCM/hexanes to afford 4.11 g (91.7%) of 4-cyclopropyl-2-fluoro-1-nitrobenzene as an oil. ¹ H NMR (400 MHz, MeOD) δ 7.98 (dd, J=10.2, 6.6 Hz, 1 H), 7.12-7.02 (m, 2H), 2.11-1.97 (m, 1 H), 1.20-1.11 (m, 2H), 0.89-0.82 (m, 2H).

Step C: 4-Cyclopropyl-2-fluoroaniline

A mixture of 4-cyclopropyl-2-fluoro-1-nitrobenzene (3.36 g, 18.55 mmol), iron powder (4.35 g, 77.9 mmol), 2M ammonium chloride/water (19.8 mL), and 3:2:1 v/v EtOH:THF:H ₂ O (86 mL) was stirred at reflux under N ₂ for 17 h. The reaction mixture was cooled to room temperature and filtered through a pad of Celite. The Celite pad was rinsed thoroughly with ethyl acetate (approximately 50 mL). Saturated aqueous NaHCO ₃ solution was slowly added to the filtrate to neutralize the reaction mixture. The reaction mixture was extracted with ethyl acetate (3×200 mL). The combined organic layers were washed with water and brine, dried over Na ₂ SO ₄ , filtered, and concentrated under reduced pressure. The crude residue was purified by flash column chromatography eluting with 0 to 75% ethyl acetate/hexane to give 2.80 g (99%) of an orange oil that solidified at 20°C. ¹ H NMR (400MHz, CDCl ₃ ) δ6.75-6.63(m,3H),3.57(s,2H),1.87-1.72(m,1H),0.93-0.83(m,2H),0.64-0.51(m,2H); MS(ESI)m/z:152.3[M+H] ⁺ .

Step D: 4-Cyclopropyl-2-fluoro-1-iodobenzene

To a stirred mixture of 4-cyclopropyl-2-fluoroaniline (1.63 g, 10.78 mmol) in water (20 mL) was added concentrated sulfuric acid (8.6 mL, 15.0 eq) at 0°C while keeping the temperature constant at 0°C. A solution of sodium nitrite (781.0 mg, 11.32 mmol, 1.05 eq) in water (2.7 mL) was added and stirred for 5 minutes. The resulting reaction mixture was then added to a solution of potassium iodide (3.76 g, 22.64 mmol, 2.1 eq) in water (9.7 mL) and the reaction mixture was stirred at 60°C for 3 h. DCM (400 mL) was added to the cooled reaction mixture. The two phases were separated and the aqueous layer was extracted with DCM (2×150 mL). The combined organic layers were washed with saturated aqueous Na ₂ S ₂ O ₄ solution, water, and brine, dried over Na ₂ SO ₄ , filtered, and concentrated under reduced pressure. The crude residue was purified by flash column chromatography eluting with 100% heptane to afford 2.01 g (71.28%) of 4-cyclopropyl-2-fluoro-1-iodobenzene as a clear oil. ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.57 (dd, J=8.0, 6.9 Hz, 1H), 6.76 (dd, J=9.4, 1.9 Hz, 1H), 6.64 (dd, J=8.2, 1.9 Hz, 1H), 1.94-1.77 (m, 1H), 1.09-0.95 (m, 2H), 0.79-0.56 (m, 2H).

Step E: Place 4-cyclopropyl-2-fluoro-1-iodobenzene (1.32 g, 5.04 mmol), dipinacolatoboron (1.53 g, 6.04 mmol), potassium acetate (1.98 g, 20.15 mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (368.5 mg, 0.50 mmol), and N,N-dimethylformamide (35 mL) in a high-pressure tube. Degas the reaction mixture with _N₂ for 15 minutes. The vessel was sealed and the reaction mixture was stirred at 90°C for 16 hours. The cooled reaction mixture was diluted with ethyl acetate (75 mL) and water (25 mL), then filtered through a pad of Celite. The two layers were separated and the organic layer was washed with water and brine, dried over _Na₂SO₄ , _filtered , and concentrated under reduced pressure. The crude residue was purified by flash column chromatography eluting with 0-75% EA/heptane to afford 859.0 mg (65.1%) of 2-(4-cyclopropyl-2-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane as a clear oil. ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.58 (s, 1H), 6.83 (d, J=7.7 Hz, 1H), 6.68 (d, J=10.8 Hz, 1H), 1.91-1.81 (m, 1H), 1.33 (s, 12H), 0.98 (dd, J=8.3, 2.0 Hz, 2H), 0.74-0.66 (m, 2H).

Intermediate 5 5-chloro-1-ethyl-4-nitro-1H-pyrazole

Following the procedure of Example 1, starting from 1-ethyl-4-nitropyrazole, 5-chloro-1-ethyl-4-nitro-1H-pyrazole was obtained as a colorless solid (1.3 g, 74%). ¹ H-NMR (400 MHz, CDCl ₃ ) δ 8.16 (s, 1H), 4.26 (q, J=7 Hz, 2H), 1.50 (t, J=7 Hz, 3H).

Intermediate 6 5-chloro-1-cyclopropylmethyl-4-nitro-1H-pyrazole

Following the procedure of Example 1, starting from 1-cyclopropylmethyl-4-nitropyrazole, 5-chloro-1-cyclopropylmethyl-4-nitro-1H-pyrazole was obtained as a colorless oil (1.16 g, 56%). ¹ H-NMR (400 MHz, CDCl ₃ ) δ 8.17 (s, 1H), 4.07 (d, J=7 Hz, 2H), 1.39-1.28 (m, 1H), 0.66-0.59 (m, 2H), 0.50-0.40 (m, 2H).

Intermediate 7 5-chloro-1-cyclopropyl-4-nitro-1H-pyrazole

Following the procedure of Example 1, starting from 1-cyclopropyl-4-nitropyrazole, 5-chloro-1-cyclopropyl-4-nitro-1H-pyrazole was obtained as a colorless solid (0.23 g, 63%). ¹ H-NMR (400 MHz, CDCl ₃ ) δ 8.09 (s, 1H), 3.62-3.54 (m, 1H), 1.38-1.28 (m, 2H), 1.25-1.13 (m, 2H).

Intermediate 8 5-chloro-1-(2,2-difluoroethyl)-4-nitro-1H-pyrazole

To a stirred solution of 1-(2,2-difluoroethyl)-4-nitro-1H-pyrazole (1.0 g, 5.13 mmol) in anhydrous THF (20 mL) cooled to -70°C was added dropwise a solution of lithium bis(trimethylsilyl)amide (1 M in THF, 8.47 mL, 8.47 mmol). After stirring at -70°C for 40 min, the reaction mixture was warmed to -55°C over 20 min. After recooling to -70°C, a solution of perchloroethane (1.74 g, 7.34 mmol) in THF (10 mL) was slowly added, and the reaction mixture was stirred at -70°C for 1.5 h. Saturated aqueous ammonium chloride (30 mL) and then water (15 mL) were added, and the mixture was extracted with EtOAc (3 x 100 mL). The combined organic layers were dried over _MgSO₄ , and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (0-100% EtOAc/isohexane) to give 5-chloro-1-(2,2-difluoroethyl)-4-nitro-1H-pyrazole as an off-white solid (438 mg, 37%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.24 (s, 1H), 6.18 (tt, J=54.8, 4.2 Hz, 1H), 4.58 (td, J=12.8, 4.2 Hz, 2H).

Intermediate 9 5-chloro-1-cyclopropyl-4-nitro-1H-pyrazole

Following Example 37, 1-cyclopropyl-4-nitropyrazole was chlorinated to give 5-chloro-1-cyclopropyl-4-nitro-1H-pyrazole as a colorless solid (0.23 g, 63%). ¹ H-NMR (400 MHz, CDCl ₃ ) δ 8.09 (s, 1H), 3.62-3.54 (m, 1H), 1.38-1.28 (m, 2H), 1.25-1.13 (m, 2H).

Intermediate 10 5-chloro-1-(4-methoxybenzyl)-4-nitro-1H-pyrazole

Following Example 37, 1-(4-methoxybenzyl)-4-nitro-1H-pyrazole was chlorinated to give 5-chloro-1-(4-methoxybenzyl)-4-nitro-1H-pyrazole as a yellow solid (536 mg, 46%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.17 (s, 1H), 7.25 (d, J=8.3 Hz, 2H), 6.89 (d, J=8.3 Hz, 2H), 5.30 (s, 2H), 3.80 (s, 3H).

Intermediate 11 5-Bromo-4-nitro-1-(2,2,2-trifluoroethyl)-1H-pyrazole

To a stirred solution of 1-(2,2,2-trifluoroethyl)-1H-pyrazole-5-amine (990mg, 6.0mmol) in acetic acid (5mL) was added acetic anhydride (0.57mL, 6.0mmol) and the mixture was stirred at room temperature for 16h. More acetic anhydride (0.57mL, 6.0mmol) was added to the reaction mixture, which was cooled in an ice bath to dropwise add fuming nitric acid (0.28mL, 6mmol). The reaction mixture was stirred at room temperature for 7h and the solvent was removed under reduced pressure. The residue was dissolved in EtOH (15mL) and concentrated hydrochloric acid (10mL) was added. The mixture was heated to reflux for 16h. After concentration under reduced pressure, the residue was distributed between DCM (50mL) and 5% NaHCO ₃ aqueous solution (100mL). The mixture was filtered and the aqueous layer was extracted with DCM (100mL). The organic layers were combined, dried over MgSO ₄ and the solvent was removed under reduced pressure to give a light orange solid (540mg). The solid (540 mg, 2.57 mmol) was dissolved in bromoform (2.9 mL, 33 mmol) and tert-butyl nitrile (0.92 mL, 7.71 mmol) was added dropwise to the solution. The reaction mixture was stirred at room temperature for 15 min and then heated at 145°C for 1.5 h. The solvent was removed under reduced pressure and the residue was purified by silica gel column chromatography (0-100% EtOAc/isohexane) to afford 5-bromo-4-nitro-1-(2,2,2-trifluoroethyl)-1H-pyrazole as a pale yellow solid (536 mg, 33% over four steps). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.30 (s, 1H), 4.86 (q, J = 7.8 Hz, 2H).

Intermediate 12 5-chloro-1-ethyl-4-nitro-1H-pyrazole

Following the procedure of Intermediate 5, starting from 1-ethyl-4-nitropyrazole, 5-chloro-1-ethyl-4-nitro-1H-pyrazole was obtained as a colorless solid (1.3 g, 74%). ¹ H-NMR (400 MHz, CDCl ₃ ) δ 8.16 (s, 1H), 4.26 (q, J=7 Hz, 2H), 1.50 (t, J=7 Hz, 3H).

Intermediate 13 1-((3-methyloxetan-3-yl)methyl)-1H-pyrazol-4-amine

A mixture of 4-nitropyrazole (1.13 g, 10 mmol) and K ₂ CO ₃ (3.4 g, 25 mmol) in MeCN (50 mL) was stirred at room temperature for 15 min, and then 3-(bromomethyl)-3-methyloxetane (1.8 g, 11 mmol) was added. The reaction mixture was stirred at room temperature for 18 h, filtered, and the filter cake was washed with MeCN. The filtrate was concentrated under reduced pressure and the residue was purified by silica gel column chromatography (0-100% EtOAc/isohexane) to give 1-((3-methyloxetane-3-yl)methyl)-4-nitro-1H-pyrazole as a colorless solid (1.43 g, 73%). A portion of the solid (206 mg, 1.04 mmol) dissolved in MeOH (20 mL) was treated with ammonium formate (260 mg, 4.13 mmol) and 10% palladium on carbon (50 mg). The mixture was heated at 80°C for 1.5 h, cooled, filtered through celite, and the filtrate was concentrated under reduced pressure to give 1-((3-methyloxetan-3-yl)methyl)-1H-pyrazol-4-amine as a pale pink gum (160 mg, 92%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.15 (s, 1H), 6.97 (s, 1H), 4.66 (d, J=6.1 Hz, 2H), 4.37 (d, J=6.1 Hz, 2H), 4.19 (s, 2H), 2.91 (s, 2H), 1.23 (s, 3H).

Intermediate 14 5-chloro-1-cyclopropylmethyl-4-nitro-1H-pyrazole

Following the procedure of Intermediate 5, starting from 1-cyclopropylmethyl-4-nitropyrazole, 5-chloro-1-cyclopropylmethyl-4-nitro-1H-pyrazole was obtained as a colorless oil (1.16 g, 56%). ¹ H-NMR (400 MHz, CDCl ₃ ) δ 8.17 (s, 1H), 4.07 (d, J=7 Hz, 2H), 1.39-1.28 (m, 1H), 0.66-0.59 (m, 2H), 0.50-0.40 (m, 2H).

Intermediate 15 5-(3,4-dihydro-2H-pyran-6-yl)-1-methyl-4-nitro-1H-pyrazole

A mixture of 5-chloro-1-methyl-4-nitro-1H-pyrazole (200 mg, 1.25 mmol), potassium fluoride dihydrate (235 mg, 2.5 mmol) and 3,4-dihydro-2H-pyran-6-boronic acid pinacol ester (394 mg, 1.88 mmol) in THF (3 mL) was degassed by bubbling nitrogen for 15 min. A mixture of tris(dibenzylideneacetone)dipalladium/tri-tert-butylphosphonium tetrafluoroborate (molar ratio: 1/1.2, 151 mg, 0.13 mmol) was added and the mixture was degassed for another 10 min and then heated in a microwave at 85 ° C for 2 h. Water (10 mL) was added and the mixture was extracted with EtOAc (3×5 mL). The combined organic layer was passed through a phase separation cartridge and concentrated under reduced pressure. Purification via silica gel chromatography (0-5% EtOAc/isohexane) gave 5-(3,4-dihydro-2H-pyran-6-yl)-1-methyl-4-nitro-1H-pyrazole as a yellow solid (215 mg, 82%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.04 (s, 1H), 5.22 (t, J=3.9 Hz, 1H), 4.20 (t, J=5.1 Hz, 2H), 3.88 (s, 3H), 2.31-2.24 (m, 2H), 2.05-1.96 (m, 2H).

Intermediate 16 2-Methyl-4-nitro-pyrazole-3-carboxaldehyde

Nitrogen was bubbled through a solution of 3-chloro-2-methyl-4-nitro-pyrazole (16 g, 100 mmol), potassium vinyl trifluoroborate (18 g, 134 mmol), and cesium carbonate (3.7 M in water, 50 mL, 190 mmol) in DMF (100 mL). 1,1'-Bis(diphenylphosphino)ferrocene-palladium(II) dichloromethane complex (900 mg, 1.10 mmol) was added and degassing continued for 30 min. The reaction mixture was heated at 110°C for 18 h. More 1,1'-Bis(diphenylphosphino)ferrocene-palladium(II) dichloromethane complex (900 mg, 1.10 mmol) was added and heating continued for 24 h. More 1,1'-Bis(diphenylphosphino)ferrocene-palladium(II) dichloromethane complex (400 mg, 0.49 mmol) was added and heating continued for 4 h. The reaction mixture was cooled to room temperature and brine (200 mL) and EtOAc (500 mL) were added. The organic layer was washed with water (4 × 300 mL), separated, dried over Na ₂ SO ₄ and concentrated under reduced pressure. Purified via silica gel column chromatography (0-40% EtOAc/isohexane) to obtain 1- methyl -4- nitro -5 vinyl -1H- pyrazole as a colorless solid (9.1 g). Ozone was bubbled through a solution of the solid (9.1 g, 59 mmol) in DCM (400 mL) cooled to -78 ° C. When the solution turned blue, ozone was stopped. Nitrogen was passed through the solution until the blue disappeared. The mixture was warmed to room temperature and flushed with nitrogen for 15 min. Anhydrous dimethyl sulfide (5 mL) was added and the mixture was warmed to room temperature. After stirring for 12 h, the solvent was removed under reduced pressure. DCM (150 mL) was added and the mixture was washed with water (50 mL). The aqueous layer was extracted with DCM (3×100 mL) and the combined organic layers were washed with brine (100 mL), separated, dried over Na ₂ SO ₄ and concentrated to give 2-methyl-4-nitro-pyrazole-3-carbaldehyde as a yellow-orange solid (6.6 g, 43% over two steps). ¹ H NMR (400 MHz, CDCl ₃ ) δ 10.51 (s, 1H), 8.11 (s, 1H), 4.23 (s, 3H).

Intermediate 17 1-Methyl-5-(5-methyl-6,8-dioxaspiro[2.5]octan-7-yl)-4-nitro-pyrazole

To a solution of 1-(2-hydroxypropyl)cyclopropanol (2.0 g, 17.2 mmol) in DCM (35 mL) was added 2,6-lutidine (5 mL, 42.9 mmol) followed by trimethylsilyl trifluoromethanesulfonate (6 mL, 32.9 mmol) at 0°C. The reaction mixture was warmed to room temperature and stirred for 18 h. Additional amounts of 2,6-lutidine (5 mL, 42.9 mmol) and trimethylsilyl trifluoromethanesulfonate (6 mL, 32.9 mmol) were added at 0°C. The mixture was stirred for 1 h and quenched with saturated aqueous _NaHCO₃ (30 mL). The mixture was extracted with DCM (50 mL) and the organic layer was washed with 0.1 M aqueous HCl (2 x 15 mL) and passed through a phase separation cartridge. To this solution was added 2-methyl-4-nitro-pyrazole-3-carbaldehyde (1.40 g, 9.03 mmol) and the resulting solution was cooled to -78 ° C and trimethylsilyl trifluoromethanesulfonate (4.11 mL, 22.6 mmol) was added. The mixture was warmed to 0 ° C, stirred for 3 h, cooled to -78 ° C and additional trimethylsilyl trifluoromethanesulfonate (4.11 mL, 22.6 mmol) was added. The mixture was warmed to 0 ° C and stirred for 1 h and solid sodium carbonate (2.5 g) was added. The reaction mixture was stirred for 10 min, then saturated aqueous NaHCO ₃ solution (100 mL) was added. The organic layer was washed with water (100 mL) and brine (100 mL), separated, dried over Na ₂ SO ₄ and concentrated under reduced pressure. Purification via silica gel column chromatography (0-100% EtOAc/isohexane) gave 1-methyl-5-(5-methyl-6,8-dioxaspiro[2.5]octan-7-yl)-4-nitro-pyrazole as a colorless solid (1.0 g, 44% over two steps). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.00 (s, 1H), 6.59 (s, 1H), 4.29-4.02 (m, 4H), 2.33-2.23 (m, 1H), 1.33 (d, J=6.2 Hz, 3H), 1.17-1.12 (m, 1H), 1.02-0.89 (m, 2H), 0.70-0.52 (m, 2H).

Intermediate 18 2-methyl-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-one

To a solution of 1-methyl-5-(5-methyl-6,8-dioxaspiro[2.5]octan-7-yl)-4-nitro-pyrazole (1.0 g, 3.95 mmol) in DCM (20 mL) was added titanium tetrachloride (6.6 mL, 59.3 mmol) dropwise at -78 ° C. When halfway was added, the reaction mixture became difficult to stir, so more DCM (10 mL) was added. The brown slurry was warmed to 0 ° C and stirred for 1 h. Solid sodium carbonate (5 g) was added carefully, followed by saturated NaHCO ₃ aqueous solution (100 mL) and DCM (100 mL). The organic layer was washed with saturated NaHCO ₃ aqueous solution (100 mL) and brine (100 mL), separated, dried over Na ₂ SO ₄ and concentrated under reduced pressure. Purification via silica gel column chromatography (0-100% EtOAc/isohexane) gave 2-methyl-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-one as a colorless solid (749 mg, 75%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.02 (s, 1H), 5.69 (dd, J=11.0, 2.4 Hz, 1H), 4.21-4.14 (m, 1H), 4.01 (s, 3H), 3.07-2.97 (m, 1H), 2.79-2.63 (m, 3H), 2.19-2.07 (m, 1H), 2.07-1.91 (m, 1H), 1.30 (d, J=6.2 Hz, 3H).

Intermediate 19 2-methyl-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-ol

To a solution of 2-methyl-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-one (65 mg, 0.26 mmol) in THF (1 mL) cooled to -78°C was added a solution of L-selectride (1 M in THF, 0.28 mL, 0.28 mmol) dropwise under nitrogen. After 1 h, the mixture was quenched with MeOH (1 mL) and allowed to warm to room temperature. EtOAc (10 mL) and brine (10 mL) were added, and the layers were separated. The aqueous layer was extracted with EtOAc (3 x 10 mL), and the combined organic layers were washed with brine (10 mL), separated, _dried over _Na₂SO₄ , and concentrated under reduced pressure. Purification via silica gel column chromatography (0-100% EtOAc/isohexane) gave 2-methyl-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-ol as a colorless solid (54 mg, 81%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.01 (2 s, 1 H), 5.63-5.59 and 5.56-5.50 (2 m, 1 H), 4.26-4.01 (m, 5 H), 3.88-3.73 (m, 1 H), 2.21-1.72 (m, 4 H), 1.28-1.23 (m, 3 H), 0.99-0.81 (m, 2 H).

Intermediate 20 5-(1-allyloxypent-4-enyl)-1-methyl-4-nitro-pyrazole

To a solution of 1-methyl-4-nitro-pyrazole (9.7 g, 76.7 mmol) and pent-4-enal (10.0 g, 84.4 mmol) in THF (250 mL) was added a solution of LiHMDS in THF (1 M, 192 mL, 191.7 mmol) at -78 ° C. The reaction mixture was warmed to -40 ° C and stirred for 4 h. The reaction mixture was quenched with saturated ammonium chloride solution (100 mL), warmed to room temperature and the solvent was removed under reduced pressure. The residue was dissolved in EtOAc (100 mL) and washed with water (30 mL). The organic layer was separated, dried over MgSO ₄ and concentrated under reduced pressure. Purified via silica gel column chromatography (0-30% EtOAc/isohexane) to give a clear oil. Under nitrogen, the oil (7.1 g, 33.6 mmol), diallyl carbonate (14.33 g, 100.9 mmol) and triphenylphosphine (880 mg, 3.35 mmol) were dissolved in dioxane (236 mL), followed by the addition of tris(dibenzylideneacetone)-dipalladium(0) (780 mg, 0.84 mmol). The reaction mixture was heated at 50 ° C for 1 h and the solvent was removed under reduced pressure. Purification via silica gel column chromatography (0-40% EtOAc/isohexane) gave 5-(1-allyloxypent-4-enyl)-1-methyl-4-nitro-pyrazole as a yellow oil (8.35 g, 43% over two steps). ¹ H NMR (400MHz, CDCl ₃ )δ8.06(s,1H),5.90-5.73(m,2H),5.46(dd,J＝8.8,5.1Hz,1H),5.29-5.16(m,2H),5.10-5.00(m,2H),4.04(s,3H),3.9 2(d,J＝5.8Hz,2H),2.37-2.25(m,1H),2.22-2.09(m,1H),2.09-1.96(m,1H),1.84(dddd,J＝13.7,9.2,6.9,5.1Hz,1H).

Intermediate 21 1-Methyl-4-nitro-5-(2,3,4,7-tetrahydrooxa-2-yl)pyrazole

5-(1-allyloxypent-4-enyl)-1-methyl-4-nitro-pyrazole (5 g, 19.92 mmol) was dissolved in toluene (1 L) and the mixture was degassed for 30 min. Then, benzalkonium-bis(tricyclohexylphosphine)ruthenium dichloride (bis(tricyclohexylphosphine)benzylideneruthenium(IV) dichloride, "1st Generation Grubbs Catalyst", CAS No. 172222-30-9, Sigma-Aldrich Product No. 579726, US Pat. No. 6111121) (878 mg, 0.99 mmol) was added. The reaction mixture was degassed for an additional 20 min, then heated at reflux for 2 h, cooled to room temperature, and filtered through celite. The filtrate was concentrated under reduced pressure. The residue was dissolved in EtOAc (200 mL) and washed with 1M aqueous HCl (150 mL), water (150 mL), saturated aqueous NaHCO ₃ (2×150 mL) and brine (150 mL). The organic layer was separated, dried over MgSO ₄ and concentrated under reduced pressure. Purification via silica gel column chromatography (0-20% EtOAc/isohexane) gave 1-methyl-4-nitro-5-(2,3,4,7-tetrahydrooxa-2-yl)pyrazole as a clear oil (3.3 g, 75%). ¹ H NMR (400MHz, CDCl ₃ )δ8.02(s,1H),5.99-5.91(m,1H),5.83-5.76(m,1H),5.59(dd,J＝9.4,3.0Hz,1H),4.42(dd,J＝15.8,5.5Hz,1H),4.24- 4.17(m,1H),4.06(s,3H),2.58-2.48(m,1H),2.46-2.36(m,1H),2.14(ddt,J=14.1,6.8,3.5Hz,1H),1.99-1.88(m,1H).

Intermediate 22 7-(2-methyl-4-nitro-pyrazol-3-yl)oxepane-3,4-diol

To a solution of AD-mix α (1.51 g) in tert-butanol (5.4 mL) and water (5.5 mL) was added a solution of 1-methyl-4-nitro-5-(2,3,4,7-tetrahydrooxa-2-yl)pyrazole (240 mg, 1.08 mmol) in tert-butanol (0.8 mL) at 0 ° C. The reaction mixture was stirred at 0 ° C for 1 h, and then solid sodium thiosulfate (1.4 g) was slowly added. The mixture was stirred for another 1 h and diluted with EtOAc (20 mL). The aqueous layer was extracted with EtOAc (4 × 15 mL) and the organic layers were combined, dried over MgSO ₄ and concentrated under reduced pressure. Purified via silica gel column chromatography (0-2.5% MeOH / EtOAc) to obtain 7- (2-methyl-4-nitro-pyrazole-3-yl) oxepane-3,4-diol as a colorless solid (30 mg, 10%). ¹ H NMR (400MHz, CDCl ₃ )δ8.06-7.98(m,1H),5.49(dd,J＝8.9,5.7Hz,1H),4.20(dd,J＝13.7,3.2Hz,1H),4.16-4.10(m,1H),4.09(s,3H),4.04-3.97(m,1H),3.73(d d,J＝13.7,2.5Hz,1H),2.53-2.46(m,1H),2.32(dtd,J＝14.3,8.8,4.9Hz,1H),2.23(d,J＝5.8Hz,1H),2.19-2.01(m,2H),1.84-1.75(m,1H).

Intermediate 23 1-Methyl-5-(5-ethyl-6,8-dioxaspiro[2.5]octan-7-yl)-4-nitro-pyrazole

To a solution of (3R)-3-hydroxybutyric acid ethyl ester (2.5g, 18.9mmol) in THF (100mL) was added a solution of titanium (IV) isopropoxide (6.02mL, 19.9mmol) in THF (15mL) and a solution of ethyl magnesium bromide in diethyl ether (3M, 30.2mL, 90.7mmol) over 2h under nitrogen. The reaction mixture was stirred for another 2h, then cooled to 0°C and quenched by slowly adding a saturated aqueous ammonium chloride solution (75mL). The solution was filtered and the filtrate was extracted with DCM (3×20mL). The combined organic layer was washed with brine (75mL), separated, dried over _MgSO4 and concentrated under reduced pressure. Purified via silica gel column chromatography (0-100% EtOAc/isohexane) to obtain 1-[(2R)-2-hydroxybutyl]-cyclopropanol as a yellow oil (1.50g). To a solution of the oil (900 mg, 7.76 mmol) in DCM (15 mL) cooled to 0°C were added 2,6-lutidine (2.26 mL, 19.40 mmol) followed by trimethylsilyl trifluoromethanesulfonate (3.1 mL, 17.10 mmol). The reaction mixture was stirred at 0°C for 2 h, then additional 2,6-lutidine (2.26 mL, 19.40 mmol) and trimethylsilyl trifluoromethanesulfonate (3.1 mL, 17.10 mmol) were added. The reaction mixture was warmed to room temperature and stirred for 18 h. The mixture was cooled to 0°C, quenched with 0.1 M aqueous HCl (15 mL), and extracted with DCM (50 mL). The organic layer was washed with 0.1 M aqueous HCl (2 x 15 mL) and passed through a phase separation cartridge. To this solution was added 2-methyl-4-nitro-pyrazole-3-carboxaldehyde (1.90 g, 7.13 mmol) and the resulting solution was cooled to -78 ° C, followed by the dropwise addition of trimethylsilyl trifluoromethanesulfonate (0.64 mL, 3.56 mmol). The mixture was warmed to 0 ° C and stirred for 3 h, then cooled to -78 ° C and additional trimethylsilyl trifluoromethanesulfonate (1 mL, 5.49 mmol) was added. After stirring at 0 ° C for 3 h, the operation was repeated. The reaction mixture was stirred at 0 ° C for another 2 h, followed by the addition of solid sodium carbonate (2.5 g). The reaction mixture was stirred for 10 min and saturated NaHCO ₃ solution (10 mL) was added. The organic layer was washed with water (10 mL) and brine (10 mL), separated, dried over Na ₂ SO ₄ and concentrated under reduced pressure. Purification via silica gel column chromatography (0-100% EtOAc/isohexane) gave 1-methyl-5-(5-ethyl-6,8-dioxaspiro[2.5]octan-7-yl)-4-nitro-pyrazole as a colorless solid (655 mg, 4% over three steps). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.00 (s, 1H), 6.58 (s, 1H), 4.13 (s, 3H), 4.00-3.92 (m, 1H), 2.29 (t, J=12.4 Hz, 1H), 1.75-1.47 (m, 3H), 1.00-0.87 (m, 5H), 0.67-0.53 (m, 2H).

Intermediate 24 2-ethyl-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-one

Following the procedure of Intermediate 5, starting from 1-methyl-5-(5-ethyl-6,8-dioxaspiro[2.5]octan-7-yl)-4-nitro-pyrazole, 2-ethyl-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-one was obtained as a colorless solid (240 mg, 13% over two steps). ¹ H NMR (400MHz, CDCl ₃ )δ8.04(s,1H),5.67(dd,J＝11.0,2.4Hz,1H),4.02(s,3H),3.94(dd,J＝10.2,5.2Hz,1H),3.04(td,J＝13.3,3. 3Hz,1H),2.79-2.63(m,3H),2.20-2.12(m,1H),2.05-1.92(m,1H),1.67-1.57(m,2H),0.94(t,J＝7.4Hz,3H).

Intermediate 25 N-(2-ethyl-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-yl)-2-methyl-propane-2-sulfenamide

To a solution of 2-ethyl-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-one (120 mg, 0.45 mmol) in THF (3 mL) was added (R)-2-methylpropane-2-sulfenamide (70 mg, 0.58 mmol) and titanium (IV) ethoxide (0.30 mL, 1.12 mmol). The reaction mixture was heated to reflux for 4 h and then cooled to room temperature. The crude solution was added dropwise to a solution of sodium borohydride (69 mg, 1.80 mmol) in THF (3 mL) at -60 ° C. The reaction mixture was warmed to 0 ° C, quenched with MeOH (3 mL) and brine (50 mL) and stirred at room temperature for 18 h. The mixture was filtered through celite washed with EtOAc (200 mL). The aqueous layer was extracted with EtOAc (3×50 mL) and the combined organic layer was washed with brine (150 mL), separated, dried over MgSO ₄ and concentrated under reduced pressure. Purification via silica gel column chromatography (0-10% MeOH/DCM) gave N-(2-ethyl-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-yl)-2-methyl-propane-2-sulfenamide as a mixture of diastereomers (5:2 ratio) as a colorless solid (118 mg, 71% over two steps). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.03 and 8.02 (2s, 1H), 5.60-5.51 (m, 1H), 4.08 and 4.06 (2s, 3H), 3.83-3.66 (m, 2H), 3.62-3.50 (m, 1H), 3.22 and 3.15 (d, J=6.2 and 4.0 Hz, 1H), 2.11-1.96 (m, 4H), 1.76 (s, 1H), 1.63-1.54 (m, 2H), 1.28-1.15 (m, 9H), 0.91 (td, J=7.4, 2.4 Hz, 3H).

Intermediate 26 N-(2-methyl-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-yl)-2-methyl-propane-2-sulfenamide

Following the procedure of Intermediate 13, starting from 2-methyl-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-one, N-(2-methyl-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-yl)-2-methyl-propane-2-sulfenamide was obtained as an off-white solid (208 mg, 40% over two steps). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.01 (s, 1H), 5.63-5.51 (m, 1H), 4.05 (s, 3H), 3.86-3.72 (m, 2H), 3.19-3.11 (m, 1H), 2.22-1.69 (m, 6H), 1.29-1.20 (m, 12H).

Intermediate 27 3-Allyloxy-3-(2-methyl-4-nitro-pyrazol-3-yl)propionic acid

To a suspension of zinc powder (<10 μm, 10.3 g, 159 mmol) in anhydrous Et ₂ O (120 mL) was added a few drops of trimethylsilyl chloride to initiate the reaction. The reaction mixture was then heated to reflux for 5 minutes, and a few drops of 1,2-dibromoethane were carefully added. A solution of tert-butyl 2-bromoacetate (18.8 mL, 127 mmol) was added dropwise, and the reaction mixture was heated to reflux for 1 hour. A solution of 2-methyl-4-nitro-pyrazole-3-carbaldehyde (77 wt% in DMSO, 6.4 g, 31.8 mmol) in THF (120 mL) was added at room temperature, and stirring was continued for 150 minutes. The reaction mixture was diluted with EtOAc (200 mL) and saturated ammonium chloride/1M HCl (100 mL/100 mL) and stirred for 18 hours. The layers were separated, and the aqueous layer was extracted with EtOAc (3 x 200 mL). The combined organic layer was washed with brine (100 mL), separated, dried over Na ₂ SO ₄ and concentrated under reduced pressure. Purified via silica gel column chromatography (0-100% EtOAc/isohexane) to obtain tert-butyl 3-hydroxy-3-(1-methyl-4-nitro-1H-pyrazol-5-yl)propanoate as a colorless solid (6.52 g, 77%). Diallyl carbonate (10.2 g, 72 mmol) was added to a solution of the solid (6.52 g, 24 mmol) in dioxane (168 mL). The reaction mixture was degassed with nitrogen for 30 min. Tris(dibenzylideneacetone)-dipalladium (0) (557 mg, 0.60 mmol) and triphenylphosphine (630 mg, 2.40 mmol) were added once and degassed for 15 min. The reaction mixture was heated at 65 ° C for 1 h and cooled to room temperature. Brine (100mL) and EtOAc (150mL) were added and the layers were separated. The aqueous layer was extracted with EtOAc (3×150mL) and the combined organic layer was washed with brine (100mL), separated, dried over Na ₂ SO ₄ and concentrated under reduced pressure. Purified via silica gel column chromatography (0-100% EtOAc/isohexane) to obtain tert-butyl 3-(allyloxy)-3-(1-methyl-4-nitro-1H-pyrazol-5-yl)propanoate as a colorless solid (7.7g, 99%). TFA (40mL) was added to a solution of the solid (7.7g, 24mmol) in DCM (80mL) and the mixture was stirred at room temperature for 18h. After cooling to 0°C, sodium carbonate (5g), saturated NaHCO ₃ aqueous solution (100mL) and DCM (200mL) were carefully added until bubbling stopped. Concentrated HCl was slowly added until the solution was pH 4. The aqueous layer was extracted with DCM (3×200 mL) and the combined organic layers were separated, dried over Na ₂ SO ₄ and concentrated under reduced pressure to give 3-allyloxy-3-(2-methyl-4-nitro-pyrazol-3-yl)propanoic acid as a yellow oil (4.33 g, 55% over three steps). ¹ H NMR (400 MHz, CDCl ₃ ) δ 11.5-10.3 (br s, 1H), 8.08 (s, 1H), 5.90-5.78 (m, 3H), 5.28-5.18 (m, 1H), 4.07 (s, 3H), 4.06-3.96 (m, 2H), 2.99 (dd, J=16.2, 9.3 Hz, 1H), 2.87 (dd, J=16.2, 4.3 Hz, 1H).

Intermediate 28 2-(2-methyl-4-nitro-pyrazol-3-yl)-3,7-dihydro-2H-oxa-4-one

To a solution of 3-allyloxy-3-(2-methyl-4-nitro-pyrazol-3-yl)propionic acid (4.33 g, 17 mmol) in DCM (48 mL) was added oxalyl chloride (4.37 mL, 51 mmol) at 0 ° C under nitrogen, followed by the careful addition of DMF (0.05 mL) to initiate the acylation reaction. The reaction mixture was stirred at room temperature for 3 h and concentrated under reduced pressure. The residue was dissolved in DME (28 mL), vinyltributyltin (2.48 mL, 8.50 mmol) was added and the mixture was degassed with nitrogen for 30 min. Trans-benzyl(chloro)-bis(triphenylphosphine)palladium(II) (129 mg, 0.17 mmol) was added and degassed for 10 min. The reaction mixture was heated to 65 ° C and maintained for 1 h and cooled to room temperature. The mixture was concentrated under reduced pressure and purified by silica gel column chromatography (0-100% EtOAc/isohexane) to give 5-(allyloxy)-5-(1-methyl-4-nitro-1H-pyrazol-5-yl)pent-1-ene-3-one as a yellow syrup (2.76 g, 61%). A solution of the syrup (250 mg, 0.94 mmol) in toluene (90 mL) was degassed with nitrogen for 30 min at 35 ° C. Zhan 1B catalyst (26 mg, 0.04 mmol) dissolved in toluene (2 mL) was added to the reaction mixture and degassed for 15 min. After stirring at 35 ° C for 1 h, the reaction mixture was cooled to room temperature and concentrated under reduced pressure. Purification via silica gel column chromatography (0-100% EtOAc/isohexane) gave 2-(2-methyl-4-nitro-pyrazol-3-yl)-3,7-dihydro-2H-oxa-4-one as a colorless solid (107 mg, 30% over three steps). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.05 (s, 1H), 6.44 (ddd, J=12.8, 3.4, 2.3 Hz, 1H), 6.15 (m, 1H), 6.01 (dd, J=11.1, 3.4 Hz, 1H), 4.72 (ddd, J=19.8, 3.4, 1.7 Hz, 1H), 4.61 (ddd, J=19.6, 2.4 Hz, 1H), 4.08 (s, 3H), 3.20-3.12 (m, 2H).

Intermediate 29 5-(6-Azido-4,4-difluoro-oxepan-2-yl)-1-methyl-4-nitro-pyrazole (Diastereomer 1)

To a solution of 2-(2-methyl-4-nitro-pyrazol-3-yl)-3,7-dihydro-2H-oxa-4-one (440 mg, 0.42 mmol) in MeCN (3 mL) was added Amberlite IRA 900F resin (79 mg, 0.19 mmol) and trimethylsilyl azide (1.2 mL, 9.35 mmol). The reaction mixture was heated at 65 ° C behind a blast shield for 24 h, cooled to room temperature and concentrated under reduced pressure. Purification via silica gel column chromatography (0-100% EtOAc / isohexane) gave pure 6-azido-2-(1-methyl-4-nitro-1H-pyrazol-5-yl)oxepan-4-one and mixed fractions containing product and starting material. It was concentrated under reduced pressure and subjected to the same reaction conditions again. Finally, it was purified by silica gel column chromatography (0-100% EtOAc/isohexane) to give 6-azido-2-(1-methyl-4-nitro-1H-pyrazol-5-yl)oxepan-4-one as a colorless solid (449 mg). To this solid (449 mg, 1.60 mmol) was added (50% in THF, 5 mL) and the mixture was stirred at room temperature for 18 h. DCM (50 mL) was added and the reaction mixture was cooled to 0 ° C. Saturated NaHCO ₃ aqueous solution (30 mL) was then carefully added. The aqueous layer was extracted with DCM (3 × 30 mL) and the combined organic layers were dried over Na ₂ SO ₄ and concentrated under reduced pressure. Purification via silica gel column chromatography (0-100% EtOAc/isohexane) gave 5-(6-azido-4,4-difluoro-oxepan-2-yl)-1-methyl-4-nitro-pyrazole (diastereomer 1-major) as a colorless solid (264 mg, 47% over two steps). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.03 (s, 1H), 5.67-5.58 (m, 1H), 4.18-3.91 (m, 3H), 4.08 (s, 3H), 2.79-2.63 (m, 1H), 2.63-2.40 (m, 3H).

Intermediate 30 5-(6-Azido-4,4-difluoro-oxepan-2-yl)-1-methyl-4-nitro-pyrazole (Diastereomer 2)

Following the procedure of Intermediate 17, 5-(6-azido-4,4-difluoro-oxepan-2-yl)-1-methyl-4-nitro-pyrazole (diastereomer 2-minor) was also obtained as a colorless solid (69 mg, 12% over two steps). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.02 (s, 1H), 5.73 (dd, J=10.9, 4.5 Hz, 1H), 4.34-4.29 (m, 1H), 4.01 (s, 3H), 4.01-3.93 (m, 1H), 3.53 (dd, J=11.4, 11.4 Hz, 1H), 2.71-2.49 (m, 4H).

Intermediate 32 5-(5,8-Dioxabicyclo[5.1.0]octan-4-yl)-1-methyl-4-nitro-pyrazole

To a solution of 1-methyl-4-nitro-5-(2,3,4,7-tetrahydrooxa-2-yl)pyrazole (1.00 g, 4.74 mmol) in DCM (25 mL) was added m-CPBA (70-75%, 1.75 g, 7.11 mmol) and the reaction mixture was stirred at room temperature for 18 h. The reaction mixture was diluted with DCM (50 mL), washed with saturated NaHCO ₃ aqueous solution (50 mL), water (50 mL) and brine (50 mL). The organic layer was separated, dried over MgSO ₄ and concentrated under reduced pressure. Purified via silica gel column chromatography (0-30% EtOAc/isohexane) to obtain racemic 5- (5,8-dioxabicyclo [5.1.0] octane -4- bases) -1-methyl-4-nitro-pyrazole as a colorless solid (490 mg, 43%). ¹ H NMR (400MHz, CDCl ₃ )δ8.22-7.87(m,1H),5.07(d,J＝9.9Hz,1H),4.50(dd,J＝14.5,3.1Hz,1H),4.05(s,3H),3.93(d,J＝14.4Hz,1H),3.35(t,J＝ 4.5Hz,1H),3.13(t,J＝3.6Hz,1H),2.55-2.47(m,1H),2.31-2.21(m,1H),2.16-2.04(m,1H),1.79(dd,J＝14.4,1.8Hz,1H).

Intermediate 33 tert- Butyl N-(4-methoxy-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-3-yl)carbamate

A solution of 5-(5,8-dioxabicyclo[5.1.0]octan-4-yl)-1-methyl-4-nitro-pyrazole (220 mg, 0.92 mmol) in MeOH/water (6 mL/1.2 mL) was treated with ammonium chloride (122 mg, 2.30 mmol) and sodium azide (299 mg, 4.60 mmol), and the mixture was heated at 70°C behind a blast shield for 16 h. The reaction mixture was extracted with EtOAc (100 mL), and the organic layer was washed with water (2 x 50 mL), dried over _MgSO₄ , and concentrated under reduced pressure. The residue (500 mg, 1.77 mmol) was dissolved in anhydrous DMF (15 mL), cooled to 0°C, and sodium hydride (60% in mineral oil, 106 mg, 2.66 mmol) was added, and the mixture was stirred for 15 min. Iodomethane (0.17 mL, 2.66 mmol) was added, and the reaction mixture was allowed to warm to room temperature and stirred for 16 h. Water (20 mL) was added and the mixture was extracted with EtOAc (2 × 150 mL). The combined organic layers were dried over MgSO ₄ and concentrated under reduced pressure. Purified via silica gel column chromatography (0-30% EtOAc/isohexane) to give 4-azido-5-methoxy-1-(1-methyl-4-nitro-1H-pyrazole-5-yl)oxepane as a yellow oil (280 mg). A solution of the oil (270 mg, 0.91 mmol) in THF/water (13 mL/2.5 mL) was treated with triphenylphosphine (263 mg, 1.00 mmol) and the reaction mixture was heated at 70 ° C behind a blast shield for 18 h. The reaction mixture was concentrated under reduced pressure. The residue was dissolved in anhydrous DCM (15 mL) at 0 ° C and di-tert-butyl dicarbonate (238 mg, 1.09 mmol) and DIPEA (0.66 mL, 4.55 mmol) were added successively. The reaction mixture is warmed to room temperature and stirred for 3h, then quenched with water (20mL) and extracted with DCM (100mL). The organic layer is separated, dried over _MgSO4 and concentrated under reduced pressure. Purified via silica gel column chromatography (0-40% EtOAc/isohexane), four diastereomers are separated. The minor fraction obtains N-(4-methoxy-7-(2-methyl-4-nitro-pyrazole-3-yl)oxepane-3-yl) tert-butyl carbamate (racemate), which is a colorless solid (60mg, 17% over four steps). ¹ H NMR (400MHz, CDCl ₃ ) δ8.02 (s, 1H), 5.58-5.51 (m, 1H), 4.82 (br s, 1H), 4.31 (dd, J = 12.7, 3.0Hz, 1H), 4.02 (s, 3H), 3.87 (br s,1H),3.62-3.48(m,2H),3.41(s,3H),2.28-2.09(m,2H),2.03-1.83(m,2H),1.47(s,9H).

Intermediate 34 tert-Butyl ((3S,4R,7S)-3-methoxy-7-(1-methyl-4-nitro-1H-pyrazol-5-yl)oxepan-4-yl)carbamate

Following the procedure of Intermediate 20, the isolated major fraction (290 mg) was further purified via chiral SFC to afford tert-butyl ((3S,4R,7S)-3-methoxy-7-(1-methyl-4-nitro-1H-pyrazol-5-yl)oxepan-4-yl)carbamate as a colorless solid (101 mg, 29% over four steps). ¹ H NMR (400MHz, CDCl ₃ ) δ8.02 (s, 1H), 5.39 (dd, J = 10.6, 3.6Hz, 1H), 4.75 (br s,1H),4.33(dd,J＝14.2,1.9Hz,1H),4.06(s,3H),3.91-3.83(m,1H),3.75(dd,J＝14.2,3.2Hz,1H),3 .43(s,3H),3.39-3.34(m,1H),2.22-2.12(m,1H),2.12-2.03(m,1H),2.03-1.82(m,2H),1.47(s,9H).

Intermediate 35 tert-Butyl ((3R,4S,7R)-3-methoxy-7-(1-methyl-4-nitro-1H-pyrazol-5-yl)oxepan-4-yl)carbamate

Following the procedure of Intermediate 21, tert-butyl ((3R,4S,7R)-3-methoxy-7-(1-methyl-4-nitro-1H-pyrazol-5-yl)oxepan-4-yl)carbamate was also obtained as a colorless solid (101 mg, 29% over four steps). ¹ HNMR (400MHz, CDCl ₃ ) δ8.05-7.99 (m, 1H), 5.39 (dd, J = 10.6, 3.6Hz, 1H), 4.75 (br s,1H),4.33(dd,J＝14.2,1.9Hz,1H),4.06(s,3H),3.90-3.82(m,1H),3.75(dd,J＝14.2,3.2Hz,1H),3.43 (s,3H),3.42-3.31(m,1H),2.22-2.12(m,1H),2.12-2.03(m,1H),2.03-1.83(m,2H),1.62-1.29(m,9H).

Intermediate 36 tert- Butyl N-(3-fluoro-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-yl)carbamate

A solution of 5-(5,8-dioxabicyclo[5.1.0]octan-4-yl)-1-methyl-4-nitro-pyrazole (130 mg, 0.54 mmol) in MeOH/water (3 mL/0.6 mL) was treated with ammonium chloride (72 mg, 1.35 mmol) and sodium azide (177 mg, 2.72 mmol), and the mixture was heated at 70°C behind a blast shield for 18 h. The reaction mixture was extracted with EtOAc (100 mL), and the organic layer was washed with water (3 x 20 mL), brine (20 mL), separated, dried over _MgSO₄ , and concentrated under reduced pressure. To a solution of the resulting residue (100 mg, 0.35 mmol) in DCM (3 mL) was added 50% in THF (0.32 mL, 0.89 mmol), and the mixture was stirred at room temperature for 16 h. The mixture was diluted with DCM (30 mL), cooled in an ice/water bath and quenched by the dropwise addition of saturated NaHCO ₃ aqueous solution (30 mL). The resulting mixture was stirred for 10 min. The organic layer was separated, dried over Na ₂ SO ₄ and the solvent was removed under reduced pressure. Purification via silica gel column chromatography (0-40% EtOAc/isohexane) gave an oil (90 mg). A solution of the oil (90 mg, 0.35 mmol) in THF/water (4 mL/0.8 mL) was treated with triphenylphosphine (92 mg, 0.35 mmol) and the reaction mixture was heated at 70 ° C behind a blast shield for 18 h. The mixture was concentrated under reduced pressure. The residue was dissolved in anhydrous DCM (7 mL) at 0 ° C and di-tert-butyl dicarbonate (84 mg, 0.38 mmol) and DIPEA (0.22 mL, 1.6 mmol) were added. The reaction mixture was warmed to room temperature and stirred for 3 h. Water (10 mL) was added and the mixture was extracted with DCM (20 mL). The organic layer was separated, dried over _MgSO4 and concentrated under reduced pressure. Purification via silica gel column chromatography (0-40% EtOAc/isohexane) gave tert-butyl N-(3-fluoro-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-yl)carbamate as a mixture of enantiomers as an off-white solid (70 mg, 36% over four steps). ¹ H NMR (400MHz, CDCl ₃ )δ8.01(s,1H),5.55-5.49(m,1H),5.10-4.92(m,2H),4.36-4.09(m,2H),4.02(s,3H),3.9 7-3.83(m,1H),2.32-2.18(m,1H),2.02-1.89(m,2H),1.83(d,J=14.0Hz,1H),1.47(s,9H).

Intermediate 37 tert-Butyl ((3R,4R,7S)-3-fluoro-7-(1-methyl-4-nitro-1H-pyrazol-5-yl)oxepan-4-yl)carbamate

Tert-butyl N-(3-fluoro-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-yl)carbamate was further purified via chiral SFC to afford tert-butyl ((3R,4R,7S)-3-fluoro-7-(1-methyl-4-nitro-1H-pyrazol-5-yl)oxepan-4-yl)carbamate as an off-white solid (52 mg). ¹ H NMR (400MHz, CDCl ₃ )δ8.01(s,1H),5.55-5.49(m,1H),5.09-4.91(m,2H),4.36-4.10(m,2H),4.01(s,3H),3.91(ddd,J＝ 26.6,14.4,2.2Hz,1H),2.31-2.19(m,1H),2.02-1.95(m,2H),1.83(d,J＝13.9Hz,1H),1.47(s,9H).

Intermediate 38 tert- Butyl ((3S,4S,7R)-3-fluoro-7-(1-methyl-4-nitro-1H-pyrazol-5-yl)oxepan-4-yl)carbamate

Following the procedure of Intermediate 24, tert-butyl ((3S,4S,7R)-3-fluoro-7-(1-methyl-4-nitro-1H-pyrazol-5-yl)oxepan-4-yl)carbamate was also obtained as an off-white solid (61 mg). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.01 (s, 1H), 5.55-5.49 (m, 1H), 5.10-4.92 (m, 2H), 4.36-4.09 (m, 2H), 4.02 (s, 3H), 3.97-3.83 (m, 1H), 2.32-2.18 (m, 1H), 2.02-1.89 (m, 2H), 1.83 (d, J=14.0 Hz, 1H), 1.47 (s, 9H).

Intermediate 39 5-(4,8-dioxabicyclo[5.1.0]octan-5-yl)-1-methyl-4-nitro-pyrazole

5-(1-allyloxypent-4-enyl)-1-methyl-4-nitro-pyrazole (7.08 g, 28.2 mmol) was dissolved in DCM (910 mL) and the mixture was degassed for 30 min, and then the second generation Grubbs catalyst (1.19 g, 1.41 mmol) was added. The reaction mixture was heated at 40 ° C for 18 h and concentrated under reduced pressure. It was purified by silica gel column chromatography (0-10% EtOAc/isohexane) and reverse phase preparative HPLC to obtain a mixture of isomers of 1-methyl-4-nitro-5-(tetrahydrooxa-2-yl)pyrazole (66/34) as a clear oil (2.3 g). To a solution of the oil (2.3 g, 10.31 mmol) in DCM (50 mL) was added m-CPBA (70-75%, 3.56 g, 14.40 mmol) and the reaction mixture was stirred at room temperature for 4 h. The reaction mixture was diluted with DCM (50 mL) and the organic layer was washed with saturated aqueous NaHCO ₃ (2×50 mL), water (50 mL) and brine (50 mL), dried over MgSO ₄ and concentrated under reduced pressure. Purification via silica gel column chromatography (0-30% EtOAc/isohexane) gave 5-(4,8-dioxabicyclo[5.1.0]octan-5-yl)-1-methyl-4-nitro-pyrazole as a colorless solid (1.0 g, 14% over two steps). ¹ H NMR (400MHz, CDCl ₃ )δ8.02(s,1H),5.51-5.44(m,1H),4.02(s,3H),3.93(dt,J＝12.7,3.4Hz,1H) ,3.62-3.53(m,1H),3.35-3.27(m,2H),2.58-2.51(m,1H),2.41-2.25(m,3H).

Intermediate 40 5-azido-2-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-ol

To a solution of 5-(4,8-dioxabicyclo[5.1.0]octan-5-yl)-1-methyl-4-nitro-pyrazole (1.04 g, 4.35 mmol) in 4:1 MeOH:water (30 mL) was added ammonium chloride (0.58 g, 10.88 mmol) and sodium azide (1.41 g, 21.75 mmol). The mixture was heated at 70 ° C behind a blast shield for 16 h. MeOH was removed under reduced pressure and EtOAc (20 mL) was added. The organic layer was washed with saturated NaHCO ₃ aqueous solution (20 mL), passed through a phase separation column and concentrated under reduced pressure. Purified via silica gel chromatography (0-60% EtOAc/isohexane) to obtain 5-azido-2-(2-methyl-4-nitro-pyrazole-3-yl)oxepane-4-ol as a light yellow gum (718 mg, 58% yield). ¹ H NMR (400MHz, CDCl ₃ )δ8.03(s,1H),5.76(dd,J＝9.3,3.2Hz,1H),4.18-4.10(m,1H),4.08-4.04(m,4H),3.91(ddd,J＝9.4,6.6,6.2Hz,1H),3.79(ddd,J＝12.6,8.6,3 .5Hz,1H),2.44(ddd,J＝15.3,9.4,3.8Hz,1H),2.37-2.29(m,1H),2.24(d,J＝3.2Hz,1H),2.12(ddd,J＝15.3,5.7,3.2Hz,1H),2.06-1.96(m,1H).

Intermediate 41 5-azido-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-ol

Following the procedure of Intermediate 26, 5-azido-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-ol was also obtained as a light yellow gum (285 mg, 23% yield). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.04 (s, 1H), 5.64 (dd, J = 10.8, 1.4 Hz, 1H), 4.06-3.96 (m, 4H), 3.95-3.83 (m, 2H), 3.72 (ddd, J = 10.8, 9.0, 4.9 Hz, 1H), 2.43 (d, J = 2.5 Hz, 1H), 2.28 (ddd, J = 14.1, 4.9, 1.4 Hz, 1H), 2.21-2.12 (m, 2H), 2.09-2.00 (m, 1H).

Intermediate 42 tert-Butyl N-(5-fluoro-2-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-yl)carbamate

To a solution of 5-azido-2-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-ol (282 mg, 1.00 mmol) in DCM (6 mL) cooled to 0 ° C was added a solution of 50% in THF, 0.46 mL, 1.25 mmol). The mixture was warmed to room temperature and stirred for 16 h. Additional (50% in THF, 0.23 mL, 0.63 mmol) was added and the mixture was stirred at room temperature for 5 h. After cooling in an ice bath, saturated NaHCO ₃ aqueous solution (10 mL) was slowly added. The organic layer was passed through a phase separation column and concentrated under reduced pressure. The residue was purified by silica gel chromatography (0-50% EtOAc/isohexane) to give the fluoro compound as a clear gum (205 mg). To a solution of the gum (200 mg, 0.70 mmol) in THF (5 mL) and water (1 mL) was added triphenylphosphine (202 mg, 0.77 mmol) and the mixture was heated at 60 ° C for 2 h. The mixture was diluted with EtOAc (10 mL) and washed with brine (2 × 5 mL). The organic layer was passed through a phase separation column and concentrated under reduced pressure. The residue was dissolved in DCM (2 mL) and DIPEA (0.24 mL, 1.40 mmol) and di-tert-butyl dicarbonate (183 mg, 0.84 mmol) were added. The mixture was stirred at room temperature for 2 h. Water (2 mL) was added and the mixture was extracted with DCM (3 × 2 mL). The combined organic layer was passed through a phase separation column and concentrated under reduced pressure. Purification via silica gel chromatography (0-50% EtOAc/isohexane) gave tert-butyl N-(5-fluoro-2-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-yl)carbamate as a clear gum (240 mg, 66% over three steps). ¹ H NMR (400MHz, CDCl ₃ )δ8.02(s,1H),5.62(dd,J＝11.3,2.3Hz,1H),5.28-4.79(m,2H),4.29-4.19(m,1H),4.15-4.07(m,1H),4 .04(s,3H),3.77(ddd,J＝12.9,8.1,4.5Hz,1H),2.41-2.07(m,3H),2.04(d,J＝10.8Hz,1H),1.44(s,9H).

Intermediate 43 tert- Butyl N-(5-methoxy-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-yl)carbamate

To a solution of 5-azido-2-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-ol (352 mg, 1.25 mmol) in anhydrous THF (6 mL) cooled to 0°C under nitrogen was added sodium hydride (60% in mineral oil, 55 mg, 1.38 mmol). After stirring for 20 min, iodomethane (0.09 mL, 1.38 mmol) was added, and the reaction mixture was warmed to room temperature and stirred for 90 min. The mixture was cooled to 0°C again, and more sodium hydride (60% in mineral oil, 55 mg, 1.38 mmol) was added. After stirring for 20 min, more iodomethane (0.09 mL, 1.38 mmol) was added, and the reaction mixture was warmed to room temperature and stirred for 5 h. Water (5 mL) was added, and the mixture was extracted with EtOAc (3 x 5 mL). The combined organic layers were passed through a phase separation cartridge and concentrated under reduced pressure. The intermediate methyl ester is purified by silica gel column chromatography (0-50% EtOAc/isohexane) to obtain a clear jelly (155 mg). A solution of the jelly (154 mg, 0.52 mmol) in THF/water (5 mL/1 mL) is treated with triphenylphosphine (150 mg, 0.57 mmol) and the reaction mixture is heated at 60 ° C behind a blast screen for 2 h. The mixture is diluted with EtOAc (10 mL) and washed with brine (2 × 5 mL). The organic layer is passed through a phase separation column and concentrated under reduced pressure. The residue is dissolved in anhydrous DCM (2 mL) and DIPEA (0.18 mL, 1.04 mmol) and di-tert-butyl dicarbonate (136 mg, 0.62 mmol) are added. The reaction mixture is stirred at room temperature for 3 h. Water (2 mL) is added and the mixture is extracted with DCM (3 × 2 mL). The organic layer is passed through a phase separation column and concentrated under reduced pressure. Purification via silica gel column chromatography (0-50% EtOAc/isohexane) gave tert-butyl N-(5-methoxy-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-yl)carbamate as a clear gum (190 mg, 41% over three steps). ¹ H NMR (400MHz, CDCl ₃ )δ8.03(s,1H),5.67(dd,J＝10.5,2.0Hz,1H),4.96(s,1H),4.33(s,1H),4.06(s,3H),4.02-3.84(m,2H),3.62(d,J＝5 .2Hz,1H),3.44(s,3H),2.52(dddd,J=15.1,9.9,7.5,2.1Hz,1H),2.20-2.01(m,2H),1.90-1.78(m,1H),1.48(s,9H).

Intermediate 44 2-(2-methyl-4-nitro-pyrazol-3-yl)tetrahydropyran-4-one

To a solution of 2-methyl-4-nitro-pyrazole-3-carbaldehyde (600 mg, 3.87 mmol) in CDCl ₃ (20 mL) was added Danishefsky's diene (836 mg, 5.81 mmol) and Resolve-Al ^™ EuFOD (157 mg, 0.39 mmol). The reaction mixture was heated at 80° C. in a sealed tube for 24 h. Additional Resolve-Al ^™ EuFOD (250 mg, 0.62 mmol) was added and heating continued for another 24 h. The reaction mixture was concentrated under reduced pressure. Purification via silica gel column chromatography (0-100% EtOAc/isohexane) gave 2-(1-methyl-4-nitro-1H-pyrazol-5-yl)-2H-pyran-4(3H)-one as a yellow solid (710 mg, 82%). A portion of this solid (300 mg, 1.35 mmol) was dissolved in THF (10 mL) under nitrogen and cooled to -78°C. A solution of L-selectride (1 M in THF, 1.48 mL, 1.48 mmol) was added dropwise, and the mixture was stirred at -78°C for 30 min. The mixture was quenched with MeOH (2 mL) and warmed to room temperature. EtOAc (30 mL) and brine (30 mL) were added, and the layers were separated. The aqueous layer was extracted with EtOAc (3 x 20 mL), and the combined organic layers were washed with brine ( ₃₀ mL), separated, dried over _Na₂SO₄ , and concentrated under reduced pressure. Purification via silica gel column chromatography (0-100% EtOAc/isohexane) gave 2-(2-methyl-4-nitro-pyrazol-3-yl)tetrahydropyran-4-one as a colorless solid (224 mg, 61% over two steps). ¹ H NMR (400MHz, CDCl ₃ )δ8.06(s,1H),5.70(dd,J＝11.8,3.3Hz,1H),4.49(ddd,J＝11.8,7.5,1.3Hz ,1H),4.15(s,3H),3.94-3.86(m,1H),2.83-2.63(m,3H),2.58-2.50(m,1H).

Intermediate 45 7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-ol

To a solution of 2-(2-methyl-4-nitro-pyrazol-3-yl)tetrahydropyran-4-one (300 mg, 1.33 mmol) in DCM (12 mL) was added dropwise boron trifluoride etherate (0.75 mL, 1.73 mmol) followed by a solution of (trimethylsilyl)diazomethane (2 M in hexane, 0.87 mL, 1.73 mmol) at -70 °C. The reaction mixture was stirred at -70 °C for 90 min, quenched with water (10 mL), diluted with DCM (12 mL), and allowed to warm to room temperature. The organic layer was passed through a phase separation cartridge and concentrated under reduced pressure. Purification via silica gel column chromatography (0-100% EtOAc / isohexane) afforded 7-(1-methyl-4-nitro-1H-pyrazol-5-yl)oxepan-4-one as a colorless solid (121 mg) and its regioisomer 2-(1-methyl-4-nitro-1H-pyrazol-5-yl)oxepan-4-one (151 mg). To a solution of 7-(1-methyl-4-nitro-1H-pyrazol-5-yl)oxepan-4-one (121 mg, 0.51 mmol) in MeOH (5 mL) was added portionwise NaBH ₄ (23 mg, 0.61 mmol) at 0° C. Stirring was continued for 1 h and the reaction mixture was quenched with 1 M HCl (5 mL) and EtOAc (10 mL). The aqueous layer was extracted with EtOAc (3×20 mL) and the combined organic layers were washed with brine (30 mL), separated, dried over Na ₂ SO ₄ and concentrated under reduced pressure to afford 7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-ol as a 1:1 mixture of diastereomers as a colorless oil (85 mg, 27% over two steps). The product was used as a 1/1 mixture of diastereomers without further purification. ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.02 and 8.01 (s, 1H), 5.61-5.56 and 5.54-5.50 (m, 1H), 4.26-4.14 (m, 1H), 4.07 and 4.04 (s, 3H), 3.90-3.80 and 3.81-3.63 (m, 1H), 2.20-1.80 (m, 8H).

Intermediate 46 5-(5,8-Dioxabicyclo[5.1.0]octan-4-yl)-1-methyl-4-nitro-pyrazole

To a solution of 1- methyl -4- nitro -5- (2,3,4,7- tetrahydrooxa -2- bases) pyrazole (1.0 g, 4.5 mmol) in DCM (18 mL) was added molecular sieves, followed by NBS (0.80 g, 4.48 mmol) and acetic acid (0.26 mL, 4.48 mol). The reaction mixture was stirred at room temperature for 60 h. The mixture was diluted with DCM (30 mL) and washed with water (15 mL), saturated NaHCO ₃ aqueous solution (15 mL) and brine (15 mL). The organic layer was separated, dried over MgSO ₄ and the solvent was removed under reduced pressure. Purified via silica gel column chromatography (0-100% EtOAc/ isohexane) to obtain intermediate bromoacetate, which is a mixture of regioisomers, which is a clear oil (1.17 g). Repeat the operation to obtain more material. To a solution of the oil (1.55 g, 4.3 mmol) in MeOH (60 mL) was added K ₂ CO ₃ (2.66 g, 19.2 mmol) in one portion. The mixture was stirred for 1 h, then water (50 mL) was added. EtOAc (150 mL) was added and the layers were separated. The organic layer was dried over MgSO ₄ and the solvent was removed under reduced pressure to afford 5-(5,8-dioxabicyclo[5.1.0]octan-4-yl)-1-methyl-4-nitro-pyrazole as a clear oil (0.86 g, 61% over two steps). ¹ H NMR (400MHz, CDCl ₃ )δ8.02(s,1H),5.53-5.45(m,1H),4.53(dd,J＝13.5,5.2Hz,1H),4.07(s,3H) ,3.58-3.48(m,1H),3.36-3.25(m,2H),2.55-2.42(m,1H),2.07-1.87(m,3H).

Intermediate 47 tert- Butyl N-(3-fluoro-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-yl)carbamate

Following the procedure of Intermediate 23, starting from 5-(5,8-dioxabicyclo[5.1.0]octan-4-yl)-1-methyl-4-nitro-pyrazole (Intermediate 33), tert-butyl N-(3-fluoro-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-yl)carbamate (290 mg, 53% over four steps) was obtained as an off-white solid. ¹ H NMR (400MHz, CDCl ₃ )δ8.02(s,1H),5.50(dd,J＝9.9,3.8Hz,1H),4.96-4.73(m,2H),4.14-3.9 5(m,3H),4.03(s,3H),2.30-2.16(m,3H),1.95-1.84(m,1H),1.47(s,9H).

Intermediate 48 5-(6-methoxy-3,5-dimethyl-3,6-dihydro-2H-pyran-2-yl)-1-methyl-4-nitro-pyrazole

To a solution of 2-methyl-4-nitro-pyrazole-3-carbaldehyde (487 mg, 3.14 mmol) in CDCl ₃ (12 mL) was added [(Z)-1-[(E)-2-methoxy-1-methyl-vinyl]prop-1-enyloxy]-trimethyl-silane (944 mg, 4.71 mmol) and Resolve-Al ^™ EuFOD (127 mg, 0.31 mmol). The reaction mixture was heated at 80° C. in a sealed tube for 18 h. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. Purification via silica gel column chromatography (0-100% EtOAc/isohexane) gave 3,5-dimethyl-2-(2-methyl-4-nitro-pyrazol-3-yl)-2,3-dihydropyran-4-one as a mixture of diastereomers as a yellow oil (829 mg). A solution of the oil (829 mg, 3.14 mmol) and cerium (III) chloride heptahydrate (4.8 g, 12.56 mmol) in MeOH (10 mL) was stirred at room temperature for 15 min. After cooling to 0 ° C, sodium borohydride (143 mg, 3.8 mmol) was added portionwise and the mixture was stirred at 0 ° C for 1 h. The reaction mixture was quenched with 1M HCl aqueous solution (10 mL) and extracted with EtOAc (50 mL). The organic layer was separated, dried over MgSO ₄ and concentrated under reduced pressure. The residue was dissolved in MeOH (40 mL) and treated with p-toluenesulfonic acid monohydrate (87 mg). The mixture was heated to reflux for 18 h and concentrated under reduced pressure. The residue was dissolved in DCM (30 mL) and the organic layer was washed with _aqueous NaHCO3 (2×20 mL), washed with brine (20 mL), passed through a phase separation cartridge and concentrated under reduced pressure to give 5-(6-methoxy-3,5-dimethyl-3,6-dihydro-2H-pyran-2-yl)-1-methyl-4-nitro-pyrazole as a yellow oil (558 mg, 51% over three steps). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.15-7.98 (m, 1H), 5.90 (d, J=3.6 Hz) and 5.78 (d, J=3.2 Hz) (1H), 5.72 (d, J=5.6 Hz) and 5.64 (d, J=10.8 Hz) (1H), 4.80 and 4.76 (2s, 1H), 4.16 and 4.06 (2s, 3H), 3.42 and 3.40 (2s, 3H), 2.65-2.58 (m, 1H), 1.77 and 1.65 (2s, 3H), 0.90 (d, J=7.2 Hz) and 0.83 (d, J=7.2 Hz) (3H).

Intermediate 49 5-(2,6-Dimethyl-4,7-dioxabicyclo[4.1.0]hept-3-yl)-1-methyl-4-nitro-pyrazole

To a solution of 5-(6-methoxy-3,5-dimethyl-3,6-dihydro-2H-pyran-2-yl)-1-methyl-4-nitro-pyrazole (100 mg, 0.38 mmol) in DCM (1 mL) cooled to -78 ° C was added boron trifluoride etherate (0.14 mL, 1.13 mmol) and triethylsilane (0.36 mL), 2.68 mmol). After stirring at -78 ° C for 1 h, the reaction mixture was warmed to room temperature and stirred for 18 h. Saturated NaHCO ₃ aqueous solution (5 mL) and DCM (5 mL) were added and the organic layer was passed through a phase separation column and concentrated under reduced pressure. Purified via silica gel column chromatography (0-60% EtOAc/isohexane) to obtain 5-(3,5-dimethyl-3,6-dihydro-2H-pyran-2-yl)-1-methyl-4-nitro-pyrazole as a yellow oil. The reaction was repeated to obtain more material. To a solution of 5-(3,5-dimethyl-3,6-dihydro-2H-pyran-2-yl)-1-methyl-4-nitro-pyrazole (305 mg, 1.29 mmol) in DCM (6.5 mL) cooled to 0 ° C was added m-CPBA (70-75%, 382 mg, 1.54 mmol) and the mixture was stirred at 0 ° C for 90 min. More m-CPBA (70-75%, 191 mg, 0.774 mmol) was added and the mixture was slowly warmed to room temperature over 6 h. The mixture was filtered through celite washed with DCM (15 mL) and the filtrate was washed with saturated NaHCO ₃ aqueous solution (2 × 10 mL). The organic layer was passed through a phase separation column and concentrated under reduced pressure. Purification via silica gel column chromatography (0-60% EtOAc/isohexane) afforded 5-(2,6-dimethyl-4,7-dioxabicyclo[4.1.0]hept-3-yl)-1-methyl-4-nitro-pyrazole as a single diastereomer as an off-white solid (189 mg, 53% over two steps). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.08 (s, 1H), 5.32-5.28 (m, 1H), 4.15-4.08 (m, 1H), 4.06 (s, 3H), 3.78 (d, J=12.9 Hz, 1H), 3.30 (d, J=5.6 Hz, 1H), 2.71-2.61 (m, 1H), 1.38 (s, 3H), 0.92 (d, J=7.0 Hz, 3H).

Intermediate 50 4-azido-3,5-dimethyl-6-(2-methyl-4-nitro-pyrazol-3-yl)tetrahydropyran-3-ol

Following the procedure of Intermediate 27, starting from 5-(2,6-dimethyl-4,7-dioxabicyclo[4.1.0]hept-3-yl)-1-methyl-4-nitro-pyrazole, 4-azido-3,5-dimethyl-6-(2-methyl-4-nitro-pyrazol-3-yl)tetrahydropyran-3-ol was obtained as an off-white solid (140 mg, 63%). ¹ H NMR (400MHz, CDCl ₃ )δ8.09(s,1H),5.74(d,J＝2.9Hz,1H),4.14(s,2H),3.79-3.64(m,3H),3.58(s,1H), 2.58(qdd,J＝7.6,2.9,2.2Hz,1H),1.81(s,1H),1.25(s,3H),1.18(d,J＝7.6Hz,3H).

Intermediate 51 tert- Butyl N-[5-hydroxy-3,5-dimethyl-2-(2-methyl-4-nitro-pyrazol-3-yl)tetrahydropyran-4-yl]carbamate

A solution of 4-azido-3,5-dimethyl-6-(2-methyl-4-nitro-pyrazol-3-yl)tetrahydropyran-3-ol (140 mg, 0.47 mmol) in THF/water (1 mL/0.2 mL) was treated with triphenylphosphine (373 mg, 1.42 mmol), and the reaction mixture was heated at 65°C behind a blast shield for 18 h. More THF (1 mL) and a solution of trimethylphosphine (1 M in toluene, 1 mL, 1.0 mmol) were added. The mixture was heated at 65°C behind a blast shield for 3 h. The solvent was removed under reduced pressure, and the residue was dissolved in anhydrous DCM (4 mL). Di-tert-butyl dicarbonate (115 mg, 0.53 mmol) and DIPEA (0.18 mL, 1.05 mmol) were added, and the reaction mixture was stirred at room temperature for 72 h. The mixture was concentrated under reduced pressure. Purification via silica gel column chromatography (0-60% EtOAc/isohexane) gave tert-butyl N-[5-hydroxy-3,5-dimethyl-2-(2-methyl-4-nitro-pyrazol-3-yl)tetrahydropyran-4-yl]carbamate as an off-white solid (112 mg, 64% over two steps). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.05 (s, 1H), 5.52 (d, J=2.7 Hz, 1H), 4.11-4.01 (m, 6H), 2.67-2.58 (m, 1H), 2.54 (s, 1H), 1.61 (s, 1H), 1.48 (s, 9H), 1.36 (s, 3H), 0.98 (d, J=7.2 Hz, 3H).

Intermediate 52 2-(2-methyl-4-nitro-pyrazol-3-yl)tetrahydropyran-4-ol

Following the procedure of Intermediate 30, 2-(2-methyl-4-nitro-pyrazol-3-yl)tetrahydropyran-4-ol was also obtained as a mixture of diastereomers as a yellow gum (91 mg, 12% over two steps). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.06 and 8.03 (2s, 1H), 5.88 (dd, J=8.3, 6.1 Hz) and 5.70 (dd, J=11.8, 3.2 Hz) (1H), 4.49 (dd, J=11.8, 7.4 Hz) and 4.40 (s) (1H), 4.15-3.72 (m, 2H), 4.15 and 4.09 (s, 3H), 2.85-2.55 (m, 1H), 2.03-1.89 (m, 3H), 1.79-1.66 (m, 1H).

Intermediate 53 1-tert-Butyl 3-methyl 2-(1-methyl-4-nitro-1H-pyrazol-5-yl)malonate

Potassium carbonate (15.40 g, 111.42 mmol) was added in one portion to a stirred solution of 5-chloro-1-methyl-4-nitro-pyrazole (6.0 g, 37.140 mmol) and tert-butyl methyl malonate (8.74 g, 50.139 mmol) in anhydrous DMSO (100 mL) under nitrogen at room temperature. The mixture was heated at 75°C for 3 hours, then cooled and allowed to stand at room temperature overnight. The mixture was poured into water (500 mL), acidified with 2N HCl (80 mL, pH 5), and extracted with EtOAc (2 x 250 mL, 2 x 200 mL). The combined organics were dried (MgSO ₄ ) and the solvent removed under reduced pressure. The residue was purified via silica gel chromatography (0-30% EtOAc/heptane) to provide 1-tert-butyl 3-methyl 2-(1-methyl-4-nitro-1H-pyrazol-5-yl)malonate as a colorless solid (10.3 g, 92.7%).

Intermediate 54 2-(2-methyl-4-nitro-pyrazol-3-yl)acetic acid methyl ester

A mixture of 1-tert-butyl 3-methyl 2-(1-methyl-4-nitro-1H-pyrazol-5-yl)malonate (6.92 g, 23.1 mmol) and formic acid (100 mL) was heated at 50°C for 5 hours and then cooled to room temperature. The formic acid was removed under reduced pressure, and the residue was diluted with brine and extracted three times with DCM. The combined organics were dried ( _MgSO4 ) and the solvent removed under reduced pressure. The residue was purified via silica gel chromatography (0-60% EtOAc/heptane) to give methyl 2-(2-methyl-4-nitro-pyrazol-3-yl)acetate (4.15 g, 90%).

Intermediate 55 2-(2-methyl-4-nitro-pyrazol-3-yl)pent-4-enoic acid methyl ester

To a solution of methyl 2-(2-methyl-4-nitro-pyrazol-3-yl)acetate (869 mg, 4.36 mmol) in anhydrous DMF (10 mL) at 0°C was added sodium hydride (218 mg, 5.45 mmol, 60% by mass), causing the mixture to immediately turn dark red. After stirring at 0°C for 15 min, allyl bromide (0.57 mL, 6.54 mmol) was slowly added, followed by stirring at 0°C for 10 min and then at room temperature for 1 h. The reaction mixture was quenched with water (20 mL) and extracted with EA (200 mL, 50 mL). The combined organic layers were washed with water (15 x 3 mL), brine ( ₁₀ mL), dried (MgSO₄), and the solvent removed under reduced pressure. The residue was purified via silica gel chromatography (0-100% EtOAc/heptane) to afford methyl 2-(2-methyl-4-nitro-pyrazol-3-yl)pent-4-enoate (713 mg, 68%). ¹ H NMR (400MHz, CDCl3) δ8.10 (s, 1H), 5.71-5.54 (m, 1H), 5.01 (d, J = 13.1Hz, 2H), 4.43 (d d,J＝9.8,5.5Hz,1H),3.86(s,3H),3.72(s,3H),3.14-3.02(m,1H),2.79-2.62(m,1H).

Intermediate 56 2-(2-methyl-4-nitro-pyrazol-3-yl)pent-4-en-1-ol

A solution of DIBAL-H (1.0 mol/L) in toluene (16.03 mmol, 16 mL) was added to a solution of methyl 2-(2-methyl-4-nitro-pyrazol-3-yl)pent-4-enoate (959 mg, 4.01 mmol) in THF (16 mL) at 0°C under a nitrogen atmosphere. The mixture was stirred at 0°C for 30 min. 1N HCl (25 mL) was slowly added to the reaction mixture at 0°C, followed by ethyl acetate (30 mL). After separation, the organic layer was washed with saturated _NaHCO solution (30 mL) and brine (30 mL). The combined aqueous layers were extracted with ethyl acetate until the desired product was no longer present in the aqueous layer. The organic layers were combined, subsequently dried ( _Na2SO4 ), filtered, and evaporated _to give a light brown oil (610 mg). The crude material was purified on silica gel with 0-100% ethyl acetate/heptane to afford 2-(2-methyl-4-nitro-pyrazol-3-yl)pent-4-en-1-ol as a light yellow solid (676 mg, 80%).

Intermediate 57 5-[1-(allyloxymethyl)but-3-enyl]-1-methyl-4-nitro-pyrazole

To a solution of 2-(2-methyl-4-nitro-pyrazol-3-yl)pent-4-en-1-ol (91 mg, 0.43) in anhydrous DMF (5 mL) was added sodium hydride (20 mg, 0.49 mmol, 60% by mass) at 0 ° C. After stirring at 0 ° C for 15 min, allyl bromide (79 mg, 0.64 mmol) was slowly added, stirred at 0 ° C for 10 min, then warmed to room temperature and maintained for 2 h. The reaction mixture was quenched with water (10 ml) and extracted with EA (3 × 50 ml). The combined organic layers were washed with brine and concentrated to dryness. The residue was purified by silica gel chromatography (0-100% EtOAc/heptane) to give 5-[1-(allyloxymethyl)but-3-enyl]-1-methyl-4-nitro-pyrazole (84 mg, 78%).

Intermediate 58 1-Methyl-4-nitro-5-(2,3,4,5-tetrahydrooxa-3-yl)pyrazole

A solution of 1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene)dichloro(phenylmethylene)(tricyclohexylphosphine)ruthenium ("2nd Generation Grubb Catalyst", CAS Reg. No. 246047-72-3, Sigma-Aldrich Product No. 569747, US Pat. No. 6111121, US Pat. No. 7329758) (375 mg, 0.42 mmol) in toluene (15 ml) was added to a solution of 5-[1-(allyloxymethyl)but-3-enyl]-1-methyl-4-nitro-pyrazole (527 mg, 2.10 mmol) in toluene (115 mL). The resulting solution was heated to reflux (120° C.) for 2.5 h. After cooling to room temperature, the solvent was removed under reduced pressure and the residue was purified via silica gel chromatography (0-100% EtOAc/heptane) to give 1-methyl-4-nitro-5-(2,3,4,5-tetrahydrooxin-3-yl)pyrazole (133 mg, 30%).

Intermediate 59 6-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-3-ol

A solution of borane dimethyl sulfide complex (2.0 mol / L) in THF (0.91 mL, 1.82 mmol) was added to a solution of 1-methyl-4-nitro-5-(2,3,4,5-tetrahydrooxa-3-yl) pyrazole (204 mg, 0.91 mmol) in anhydrous THF (8 mL) at 0 ° C. The mixture was stirred at 0 ° C for 15 min, then warmed to room temperature and kept for 2 h. 1M NaOH (1.5 mL) and hydrogen peroxide (30% by mass in water, 0.8 mL) were added and the mixture was stirred at room temperature for 2 h. The reaction mixture was quenched with water and extracted with DCM (2 ×) and EA (1 ×). The combined organic layer was washed with brine (10 ml) and concentrated to dryness. The residue was purified via silica gel chromatography (0-100% EtOAc/heptane) to give 6-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-3-ol (53 mg, 24%).

Intermediate 60 6-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-3-one

To a solution of 6-(2-methyl-4-nitro-pyrazole-3-yl)oxepane-3-ol (53 mg, 0.22 mmol) in DCM (6 mL) was added Dess-Martin periodinane (192 mg, 0.44 mmol) and sodium bicarbonate (93 mg, 1.10 mmol). The mixture was stirred at room temperature overnight, quenched with water and extracted with DCM (3 ×). The combined organic layers were concentrated to dryness and purified via silica gel chromatography (0-100% EtOAc/heptane) to give 6-(2-methyl-4-nitro-pyrazole-3-yl)oxepane-3-one (53 mg, quantitative).

Intermediate 61 N-[6-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-3-yl]carbamic acid tert-butyl ester

6-(2-Methyl-4-nitro-pyrazol-3-yl)oxepan-3-one (53 mg, 0.23 mmol), ammonium acetate (219 mg, 2.76 mmol), sodium cyanoborohydride (38 mg, 0.57 mmol), and a few molecular sieves were dissolved in methanol (2 mL). Acetic acid (35 mg, 0.57 mmol) was added, and the mixture was stirred at room temperature under a nitrogen atmosphere for 3 days. The reaction mixture was quenched with saturated sodium bicarbonate and extracted with DCM (3×). The combined organic layers were dried (MgSO ₄ ) and the solvent was removed under reduced pressure. The residue was dissolved in DCM (5 mL), and di-tert-butyl dicarbonate (63 mg, 0.69 mmol) and DIPEA (0.067 mL, 0.38 mmol) were added. The mixture was stirred at room temperature overnight and then purified via silica gel chromatography (0-100% EtOAc/heptane) to give tert-butyl N-[6-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-3-yl]carbamate (53 mg, 81%).

Intermediate 62 N-[4-Fluoro-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-3-yl]carbamic acid tert-butyl ester and N-[3-Fluoro-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-yl]carbamic acid tert-butyl ester

Following the procedure of Intermediate 23, starting from 5-(5,8-dioxabicyclo[5.1.0]octan-4-yl)-1-methyl-4-nitro-pyrazole (Intermediate 33), an inseparable mixture of tert-butyl N-[4-fluoro-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-3-yl]carbamate and tert-butyl N-[3-fluoro-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-yl]carbamate was obtained as an oil (290 mg, 53% over four steps). ¹ H NMR (400MHz, CDCl ₃ )δ8.02(s,1H),5.58-5.47(m,1H),4.96-4.73(m,2H),4.14-3.93(m,5H),2.30-2.16(m,3H),2.04-1.83(m,2H),1.47(s,9H).

Intermediate 63 N-[5-fluoro-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-yl]carbamic acid tert-butyl ester

A solution of deoxo-Fluor (50% in THF, 0.576 mL, 1.56 mmol) was added dropwise to an ice-cold solution of 5-azido-2-(2-methyl-4-nitro-pyrazole-3-yl)oxepane-4-ol (353 mg, 1.25 mmol, intermediate 27) in DCM (6 mL). The mixture was warmed to room temperature while stirring for 16 h, then cooled in an ice bath and saturated _NaHCO3 aqueous solution (10 mL) was slowly added. The organic layer was passed through a phase separation column and concentrated under reduced pressure. Purified via silica gel chromatography (0-50% EtOAc/isohexane) to give 5-(5-azido-4-fluorooxepane-2-yl)-1-methyl-4-nitro-1H-pyrazole as a clear gum. To a solution of the gum (145 mg, 0.51 mmol) in THF (5 mL) and water (1 mL) was added triphenylphosphine (147 mg, 0.56 mmol) and the mixture was heated at 60 ° C for 2 h. The mixture was diluted with EtOAc (10 mL) and washed with brine (2×5 mL). The organic layer was passed through a phase separation column and concentrated under reduced pressure. The residue was dissolved in DCM (2 mL) and DIPEA (0.178 mL, 1.02 mmol) and di-tert-butyl dicarbonate (134 mg, 0.61 mmol) were added. The mixture was stirred at room temperature for 2 h. Water (2 mL) was added and the mixture was extracted with DCM (3×2 mL). The combined organic layers were passed through a phase separation cartridge, concentrated under reduced pressure and the residue purified via silica gel chromatography (0-50% EtOAc/isohexane) to give tert-butyl N-[5-fluoro-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-yl]carbamate as a clear gum (180 mg, 39% over three steps). ¹ H NMR (400MHz, CDCl ₃ )δ8.01(s,1H),5.54(dd,J＝10.5,4.2Hz,1H),5.10-4.92(m,2H),4.21-4.09(m,2H),4.05(s,3 H),3.74-3.62(m,1H),2.57-2.38(m,1H),2.35-2.15(m,2H),1.91-1.81(m,1H),1.46(s,9H).

Intermediate 64 4-azido-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-3-one

To a solution of 5-(5,8-dioxabicyclo[5.1.0]octan-4-yl)-1-methyl-4-nitro-pyrazole (2.85 g, 11.9 mmol, Intermediate 19) in MeOH (60 mL) and water (11.5 mL) was added _NH₄Cl (1.58 g, 29.8 mmol) followed by sodium azide (3.87 g, 59.5 mmol). The reaction mixture was heated at 70°C for 18 h and then cooled to room temperature. The solvent was removed under reduced pressure and the residue was dissolved in EtOAc (150 mL). The organic layer was washed with brine (50 mL), separated, dried over _MgSO₄ , and concentrated under reduced pressure to afford the azido alcohol as an orange oil, which was an 80/20 mixture of regioisomers. To the solution of the oil (1.9g, 6.7mmol) in DCM (40mL) was added Dess-Martin periodinane (1.8g, 4.26mmol) and the mixture was stirred at room temperature for 3h. Saturated _NaHCO3 aqueous solution (50mL) and 20% sodium thiosulfate solution (50mL) were added and the reaction mixture was stirred for 30min until the salt was observed to be completely dissolved. The mixture was diluted with DCM (50mL) and the organic layer was separated, dried over _MgSO4 and concentrated under reduced pressure. Purified via silica gel column chromatography (0-50% EtOAc/isohexane) to obtain 4-azido-7-(2-methyl-4-nitro-pyrazole-3-yl)oxepane-3-one, which was an oil (1.05g, 86% over two steps). ¹ H NMR (400MHz, CDCl ₃ ) δ 8.05 (s, 1H), 5.38 (dd, J = 10.1, 2.7Hz, 1H), 4.63-4.51 (m, 2H), 4.30-4.20 (m, 1H), 4.08 (s, 3H), 2.29-2.16 (m, 4H).

Intermediate 65 N-[3,3-difluoro-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-yl]carbamic acid tert-butyl ester

To a solution of 4-azido-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-3-one (440 mg, 1.57 mmol, intermediate 55) in DCM (10 mL) was added (50% in THF, 1.42 mL, 3.92 mmol) and the mixture was stirred at room temperature for 18 h. DCM (20 mL) was added, the mixture was cooled to 0 ° C and saturated aqueous NaHCO ₃ solution (20 mL) was carefully added. The aqueous layer was extracted with DCM (3×20 mL) and the combined organic layers were dried over Na ₂ SO ₄ and concentrated under reduced pressure. Purification via silica gel column chromatography (0-30% EtOAc/isohexane) gave 5-(5-azido-6,6-difluorooxepan-2-yl)-1-methyl-4-nitro-1H-pyrazole as an oil (280 mg). A solution of the oil (280 mg, 0.93 mmol) in THF/water (10 mL/1.8 mL) was treated with triphenylphosphine (267 mg, 1.02 mmol) and the reaction mixture was heated at 70 ° C behind a blast shield for 18 h. The reaction mixture was concentrated under reduced pressure. The residue was dissolved in anhydrous DCM (15 mL), cooled to 0 ° C and di-tert-butyl dicarbonate (243 mg, 1.12 mmol) and DIPEA (0.15 mL, 1.12 mmol) were added successively. The reaction mixture was warmed to room temperature and stirred for 72 h. Water (20 mL) was added and the mixture was extracted with DCM (100 mL). The organic layer was separated, dried over MgSO ₄ and concentrated under reduced pressure. Purification via silica gel column chromatography (0-35% EtOAc/isohexane) gave tert-butyl N-[3,3-difluoro-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-yl]carbamate as a clear oil (310 mg, 59% over three steps). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.03 (s, 1H), 5.48-5.42 (m, 1H), 5.10-5.01 (m, 1H), 4.49-4.35 (m, 2H), 4.04 (s, 3H), 3.99-3.80 (m, 1H), 2.17-1.98 (m, 4H), 1.48 (s, 9H).

Intermediate 66 4-azido-7-(1-methyl-4-nitro-1H-pyrazol-5-yl)oxepan-3-ol

To a solution of 4-azido-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-3-one (Intermediate 55) (1 g, 3.57 mmol) in anhydrous THF (25 mL) cooled to -78°C under nitrogen was added L-selectride (1 M in THF, 4.3 mL, 4.3 mmol) and the mixture was stirred at -78°C for 45 min. The mixture was warmed to room temperature and water (10 mL) was added. The solvent was removed under reduced pressure and the residue was dissolved in EtOAc (100 mL). The organic layer was washed with water (40 mL) and brine (40 mL), _dried over _Na2SO4 , and concentrated under reduced pressure. Purification via silica gel column chromatography (0-60% EtOAc/isohexane) gave racemic 4-azido-7-(1-methyl-4-nitro-1H-pyrazol-5-yl)oxepan-3-ol (relative stereochemistry as shown above) as a yellow oil (580 mg, 58%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.02 (s, 1H), 5.63 (dd, J=10.6, 3.5 Hz, 1H), 4.21-4.14 (m, 3H), 4.01 (s, 3H), 3.69-3.58 (m, 1H), 2.45-2.33 (m, 1H), 2.27-2.08 (m, 2H), 2.01-1.84 (m, 2H).

Intermediate 67 tert- Butyl N-[3-methoxy-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-yl]carbamate

To a solution of 4-azido-7-(1-methyl-4-nitro-1H-pyrazol-5-yl)oxepan-3-ol (Intermediate 57) (182 mg, 0.65 mmol) in anhydrous DMF (5 mL) was added sodium hydride (60% dispersion in mineral oil, 39 mg, 0.97 mmol) portionwise over 10 min under nitrogen. After an additional 45 min, methyl iodide (0.06 mL, 0.97 mmol) was added dropwise, and the mixture was stirred at room temperature for 18 h. Sodium hydride (60% dispersion in mineral oil, 39 mg, 0.97 mmol) was then added, followed by methyl iodide (0.06 mL, 0.97 mmol), and the mixture was stirred at room temperature for 48 h. The mixture was quenched with water (20 mL) and extracted with EtOAc (3 x 20 mL). The combined organic layer was washed with water (20 mL) and brine (20 mL), separated, dried over MgSO ₄ and the solvent was removed under reduced pressure. Purified via silica gel column chromatography (0-50% EtOAc/isohexane) to obtain 5- (5- azido -6- methoxy oxacycloheptan -2- bases) -1- methyl -4- nitro -1H- pyrazole, which is an oil (100 mg). A solution of the oil (100 mg, 0.37 mmol) in THF/ water (5 mL/1 mL) was treated with triphenylphosphine (97 mg, 0.37 mmol) and the reaction mixture was heated at 70 ° C behind a blast screen for 18 h. The mixture was concentrated under reduced pressure. The residue was dissolved in anhydrous DCM (3 mL) at 0 ° C and di-tert-butyl dicarbonate (89 mg, 0.4 mmol) and DIPEA (0.18 mL, 1.02 mmol) were added. The reaction mixture was warmed to room temperature and stirred for 3 h. To the _4- thiazolinyl group-2-nitro-1H-pyrazoles-5-yl) oxepane-4-yl) t-butyl carbamate (relative stereochemistry as shown above) was obtained as a clear oil (119 mg, 47% over three steps). ¹ H NMR (400MHz, CDCl ₃ ) δ8.02 (s, 1H), 5.39 (dd, J = 10.6, 3.6Hz, 1H), 4.75 (br s,1H),4.33(dd,J＝14.2,1.9Hz,1H),4.06(s,3H),3.91-3.83(m,1H),3.75(dd,J＝14.2,3.2Hz,1H),3 .43(s,3H),3.39-3.34(m,1H),2.22-2.12(m,1H),2.12-2.03(m,1H),2.03-1.82(m,2H),1.47(s,9H).

Intermediate 68 1-(1-methyl-4-nitro-1H-pyrazol-5-yl)pent-4-en-1-ol

A solution of 1-methyl-4-nitro-1H-pyrazole (9.7 g, 76.7 mmol) and 4-pentenal (10 g, 84.4 mmol) in anhydrous THF (250 mL) was cooled to -78 ° C and stirred under nitrogen. A solution of LiHMDS (1 M in THF, 192 mL, 191.7 mmol) was added dropwise over 3 h. The reaction mixture was warmed to -40 ° C and stirred for 2 h, quenched by the dropwise addition of saturated ammonium chloride solution (100 mL), warmed to room temperature and diluted with EtOAc (200 mL). The organic layer was washed with saturated ammonium chloride solution (50 mL), separated, dried over MgSO ₄ and the solvent was removed under reduced pressure. Purification by silica gel chromatography (0-100% EtOAc/DCM) followed by silica gel chromatography (0-100% EtOAc/isohexane) gave 1-(1-methyl-4-nitro-1H-pyrazol-5-yl)pent-4-en-1-ol as a pale yellow oil (5.75 g, 36%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.06 (s, 1H), 5.85-5.78 (m, 1H), 5.32-5.26 (m, 1H), 5.12-5.04 (m, 2H), 3.98 (s, 3H), 3.45 (d, J=8.7 Hz, 1H), 2.92-2.09 (m, 3H), 1.90-1.86 (m, 1H).

Intermediate 69 5-(5-(iodomethyl)tetrahydrofuran-2-yl)-1-methyl-4-nitro-1H-pyrazole

To a stirred solution of 1-(1-methyl-4-nitro-1H-pyrazol-5-yl)pent-4-en-1-ol (0.84 g, 3.98 mmol, Intermediate 59) in anhydrous THF (25 mL) was added iodine (1.52 g, 5.97 mmol) under nitrogen. After stirring for 5 min, Na ₂ CO ₃ (0.63 g, 5.97 mmol) and silver trifluoromethanesulfonate (3.07 g, 11.94 mmol) were added, and the dark red solution turned yellow. The mixture was stirred at room temperature for 1 h, diluted with THF (25 mL), and filtered through celite. The yellow solid was washed with THF/DCM, and the filtrate was concentrated under reduced pressure. Purification via silica gel chromatography (0-40% EtOAc/DCM) gave 5-(5-(iodomethyl)tetrahydrofuran-2-yl)-1-methyl-4-nitro-1H-pyrazole as a light yellow gum (640 mg, 48%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.03 (s, 1H), 5.91-5.87 (m, 1H), 4.39-4.35 (m, 1H), 4.02 (s, 3H), 3.37-3.30 (m, 2H), 2.69-2.67 (m, 1H), 2.45-2.41 (m, 1H), 2.05-1.89 (m, 2H).

Intermediate 70 5-(5-(Azidomethyl)tetrahydrofuran-2-yl)-1-methyl-4-nitro-1H-pyrazole

To a solution of 5-(5-(iodomethyl)tetrahydrofuran-2-yl)-1-methyl-4-nitro-1H-pyrazole (640 mg, 1.90 mmol, intermediate 60) in anhydrous DMF (10 mL) was added sodium azide (250 mg, 3.80 mmol) and the mixture was stirred at room temperature for 36 h. The mixture was diluted with EtOAc (25 mL) and washed with water (2×10 mL) and brine (20 mL). The organic layer was passed through a phase separation cartridge and concentrated under reduced pressure to give 5-(5-(azidomethyl)tetrahydrofuran-2-yl)-1-methyl-4-nitro-1H-pyrazole as a yellow oil (480 mg, 100%). ¹ H NMR (400MHz, CDCl ₃ )δ8.05(s,1H),5.84-5.70(m,1H),4.49-4.45(m,1H),4.03(s,3H),3.56 -3.39(m,2H),2.66-2.65(m,1H),2.29-2.22(m,1H),2.02-1.92(m,2H).

Intermediate 71 tert- Butyl ((5-(1-methyl-4-nitro-1H-pyrazol-5-yl)tetrahydrofuran-2-yl)methyl)carbamate

A solution of 5-(5-(azidomethyl)tetrahydrofuran-2-yl)-1-methyl-4-nitro-1H-pyrazole (520 mg, 2.07 mmol, Intermediate 61) in THF/water (20 mL/4 mL) was treated with triphenylphosphine (600 mg, 2.28 mmol) and the reaction mixture was heated at 70°C behind a blast shield for 1.5 h. The mixture was cooled to room temperature and the organic solvent was removed under reduced pressure. The aqueous layer was extracted with DCM (40 mL) and the organic layer was passed through a phase separation cartridge and concentrated under reduced pressure to give a pale yellow oil. This oil was dissolved in DCM (20 mL) and DIPEA (0.72 mL, 4.14 mmol) was added, followed by a solution of di-tert-butyl dicarbonate (540 mg, 2.48 mmol) in DCM (1 mL) in two portions. The reaction mixture was stirred at room temperature for 1 h. Water (10 mL) was added and the organic layer was passed through a phase separation cartridge and concentrated under reduced pressure. Purification via silica column chromatography (0-60% EtOAc/isohexane) gave tert-butyl ((5-(1-methyl-4-nitro-1H-pyrazol-5-yl)tetrahydrofuran-2-yl)methyl)carbamate as a colourless gum (145 mg, 21% over two steps). ¹ H NMR (400MHz, CDCl ₃ ) δ8.04(s,1H),5.80-5.76(m,1H),4.85(br s,1H),4.35(br s,1H),4.01(s,3H),3.50-3.40(m,1H),3.25-3.19(m,1H),2.65-2.55(m,1H),2.25-2.20(m,1H),2.00-1.80(m,2H),1.46(s,9H).

Intermediate 72 2-Azido-5-fluoro-5-(1-methyl-4-nitro-1H-pyrazol-5-yl)cycloheptanol

A solution of 5-(4-fluoro-8-oxabicyclo[5.1.0]octan-4-yl)-1-methyl-4-nitro-1H-pyrazole (2.75 g, 10.8 mmol, Intermediate 155) in DMF/water (35 mL/10 mL) was treated with ammonium chloride (1.43 g, 27.0 mmol) and sodium azide (3.5 g, 53.9 mmol) and the mixture was heated at 100° C. behind a blast shield for 18 h. The reaction mixture was extracted with EtOAc (200 mL) and the organic layer was washed with water (8×30 mL), brine (30 mL), separated, dried over MgSO ₄ and concentrated under reduced pressure. Purification via silica gel column chromatography (30-40% EtOAc/isohexane) gave 2-azido-5-fluoro-5-(1-methyl-4-nitro-1H-pyrazol-5-yl)cycloheptanol as the second eluting isomer as a white solid (2.16 g, 67%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.06 and 8.05 (2s, 1H), 4.08 and 4.06 (2s, 3H), 3.88-3.78 (m, 1H), 3.65-3.58 (m, 1H), 2.87-2.55 (m, 2H), 2.31-2.21 (m, 2H), 2.18-2.00 (m, 3H), 1.98-1.85 (m, 2H).

Intermediate 73 tert- Butyl N-[5-fluoro-2-hydroxy-5-(2-methyl-4-nitro-pyrazol-3-yl)cycloheptyl]carbamate

A solution of 2-azido-5-fluoro-5-(1-methyl-4-nitro-1H-pyrazol-5-yl)cycloheptanol (300 mg, 1.05 mmol, intermediate 63) in THF/water (15 mL/3 mL) was treated with triphenylphosphine (290 mg, 1.11 mmol) and the mixture was heated at 60 ° C behind a blast shield for 18 h. Brine (5 mL) was added and the mixture was extracted with EtOAc (2×50 mL). The organic layers were combined, dried over MgSO ₄ and concentrated under reduced pressure. To a solution of the resulting oil in anhydrous DCM (20 mL) was slowly added DIPEA (0.88 mL, 5.03 mmol) under nitrogen, followed by a solution of di-tert-butyl dicarbonate (263 mg, 1.21 mmol) in anhydrous DCM (10 mL). The reaction mixture was stirred at room temperature for 4 days. Water (30 mL) was added and the mixture was extracted with DCM (80 mL). The organic layer was separated, dried over _MgSO4 and concentrated under reduced pressure. Purification via silica gel column chromatography (40-50% EtOAc/isohexane) gave tert-butyl N-[5-fluoro-2-hydroxy-5-(2-methyl-4-nitro-pyrazol-3-yl)cycloheptyl]carbamate as a colorless oil (218 mg, 58% over two steps). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.06 and 8.05 (2s, 1H), 4.86 (br s, 1H), 4.08 and 4.06 (2s, 3H), 3.88-3.79 (m, 1H), 3.75-3.67 (m, 2H), 2.77-2.48 (m, 2H), 2.40-2.30 (m, 1H), 2.21-1.95 (m, 3H), 1.95-1.67 (m, 2H), 1.47 (s, 9H).

Intermediate 74 (5-ethyl-2-(1-methyl-4-nitro-1H-pyrazol-5-yl)-1,3-dioxan-5-yl)methanol (trans isomer)

To a solution of 2-methyl-4-nitro-pyrazole-3-carboxaldehyde (370 mg, 2.39 mmol, intermediate 3) in toluene (50 mL) was added 2-ethyl-2-(hydroxymethyl)propane-1,3-diol (315 mg, 2.35 mmol) and p-toluenesulfonic acid (20 mg, 0.10 mmol). The reaction mixture was heated at reflux for 36 h while azeotropically removing water. The mixture was cooled to room temperature and concentrated under reduced pressure. Purification by silica gel column chromatography (0-100% EtOAc/isohexane) gave (5-ethyl-2-(1-methyl-4-nitro-1H-pyrazol-5-yl)-1,3-dioxane-5-yl)methanol (trans isomer), which was the first eluting isomer as a colorless solid (244 mg, 38%). ¹ H NMR (400MHz, CDCl ₃ )δ8.02(s,1H),6.38(s,1H),4.16(s,3H),4.02(d,J＝11.5Hz,2H),3.97(d ,J＝5.2Hz,2H),3.42(d,J＝3.8Hz,2H),1.90(m,3H),0.99(t,J＝7.6Hz,3H).

Intermediate 75 (5-ethyl-2-(1-methyl-4-nitro-1H-pyrazol-5-yl)-1,3-dioxan-5-yl)methanol (cis isomer)

Following the procedure of Intermediate 66, (5-ethyl-2-(1-methyl-4-nitro-1H-pyrazol-5-yl)-1,3-dioxan-5-yl)methanol (cis isomer) was also obtained as the second eluting isomer as a colorless solid (118 mg, 18%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.02 (s, 1H), 6.37 (s, 1H), 4.13 (s, 3H), 4.12 (d, J=12.8 Hz, 2H), 3.98 (d, J=3.9 Hz, 2H), 3.73 (d, J=11.8 Hz, 2H), 1.74 (br s, 1H), 1.31 (q, J=7.7 Hz, 2H), 0.89 (t, J=7.7 Hz, 3H).

Intermediate 76 (2-(1-methyl-4-nitro-1H-pyrazol-5-yl)-1,3-dioxan-5-yl)methanol (trans isomer)

2-(Hydroxymethyl)propane-1,3-diol (700 mg, 6.73 mmol) and p-toluenesulfonic acid (88 mg, 0.463 mmol) were added to a solution of 2-methyl-4-nitro-pyrazole-3-carbaldehyde (718 mg, 4.63 mmol, intermediate ₃ ) in toluene (100 mL). The reaction mixture was heated to reflux for 18 h while azeotropically removing water. The mixture was cooled to room temperature and concentrated under reduced pressure. The residue was diluted with DCM (50 mL) and washed with saturated aqueous NaHCO3 (50 mL). The organic layer was washed with water (20 mL) and brine (20 mL), separated, _dried over _Na2SO4 , and concentrated under reduced pressure. Purification via silica gel column chromatography (0-100% EtOAc/isohexane) gave (2-(1-methyl-4-nitro-1H-pyrazol-5-yl)-1,3-dioxan-5-yl)methanol (trans isomer) as the first eluting isomer as a colorless solid (220 mg, 20%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.02 (s, 1H), 6.42 (s, 1H), 4.34 (dd, J=11.6, 4.7 Hz, 2H), 4.12 (s, 3H), 3.81 (t, J=11.5 Hz, 2H), 3.56 (t, J=5.1 Hz, 2H), 2.53-2.38 (m, 1H), 1.67 (t, J=4.6 Hz, 1H).

Intermediate 77 (2-(1-methyl-4-nitro-1H-pyrazol-5-yl)-1,3-dioxan-5-yl)methanol (cis isomer)

Following the procedure of Intermediate 68, (2-(1-methyl-4-nitro-1H-pyrazol-5-yl)-1,3-dioxan-5-yl)methanol (cis isomer) was also obtained as a colorless solid (268 mg, 24%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.02 (s, 1H), 6.49 (s, 1H), 4.28 (d, J=11.9 Hz, 2H), 4.20 (d, J=3.3 Hz, 2H), 4.12 (s, 3H), 4.06 (dd, J=7.8, 3.7 Hz, 2H), 1.82 (t, J=4.9 Hz, 1H), 1.78-1.71 (m, 1H).

Intermediate 78 (5-methyl-2-(1-methyl-4-nitro-1H-pyrazol-5-yl)-1,3-dioxan-5-yl)methanol (trans isomer)

Following the procedure of Intermediate 68, starting from 2-methyl-2-(hydroxymethyl)propane-1,3-diol, (5-methyl-2-(1-methyl-4-nitro-1H-pyrazol-5-yl)-1,3-dioxan-5-yl)methanol was obtained as the first eluting isomer as a colorless solid (167 mg, 13%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.03 (s, 1H), 6.38 (s, 1H), 4.19 (s, 3H), 4.02 (d, J=11.3 Hz, 2H), 3.89 (d, J=11.3 Hz, 2H), 3.43 (d, J=4.5 Hz, 2H), 1.65-1.40 (m, 1H), 1.36 (s, 3H).

Intermediate 79 (5-methyl-2-(1-methyl-4-nitro-1H-pyrazol-5-yl)-1,3-dioxan-5-yl)methanol (cis isomer)

Following the procedure of Intermediate 70, (5-methyl-2-(1-methyl-4-nitro-1H-pyrazol-5-yl)-1,3-dioxan-5-yl)methanol (cis isomer) was also obtained as the second eluting isomer (480 mg, 38%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.02 (s, 1H), 6.40 (s, 1H), 4.16-4.06 (m, 5H), 3.91 (s, 2H), 3.72 (d, J=11.9 Hz, 2H), 0.85 (s, 3H). OH was not observed.

Intermediate 80 tert-Butyl N-[(4R,7S)-3,3-difluoro-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-yl]carbamate

tert-Butyl N-[3,3-difluoro-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-yl]carbamate (Intermediate 56) was further purified via chiral SFC to afford tert-butyl N-[(4R,7S)-3,3-difluoro-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-yl]carbamate as the second eluting isomer as an off-white solid (57 mg, 47%). ¹ H NMR (400MHz, CDCl ₃ )δ8.04(s,1H),5.48-5.42(m,1H),5.06(d,J=9.5Hz,1H),4.49-4.38(m,2H),4.05(s,3H),3.98-3.82(m,1H),2.18-2.00(m,4H),1.48(s,9H).

Intermediate 81 tert-Butyl N-[(4S,7R)-3,3-difluoro-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-yl]carbamate

Following the procedure of Intermediate 72, tert-butyl N-[(4S,7R)-3,3-difluoro-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-yl]carbamate was also obtained as the first eluting isomer as an off-white solid (65 mg, 53%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.04 (s, 1H), 5.48-5.42 (m, 1H), 5.05 (d, J=9.2 Hz, 1H), 4.50-4.36 (m, 2H), 4.05 (s, 3H), 3.98-3.84 (m, 1H), 2.18-2.00 (m, 4H), 1.48 (s, 9H).

Intermediate 82 Methanesulfonic acid (5-ethyl-2-(1-methyl-4-nitro-1H-pyrazol-5-yl)-1,3-dioxan-5-yl)methyl ester

To a solution of (5-ethyl-2-(1-methyl-4-nitro-1H-pyrazol-5-yl)-1,3-dioxan-5-yl)methanol (trans isomer) (610 mg, 2.25 mmol, Intermediate 66) in anhydrous DCM (15 mL) was added _Et3N (0.45 mL, 3.38 mmol) followed by methanesulfonyl chloride (0.21 mL, 2.70 mmol) at 0°C. The reaction mixture was slowly warmed to room temperature over 1.5 h. The mixture was recooled to 0°C and diluted with 1 M aqueous HCl (10 mL) and DCM (20 mL). The organic layer was washed with saturated aqueous _NaHCO₃ (15 mL) and water (15 mL), separated, dried over _Na₂SO₄ , and concentrated under reduced pressure to give ( ₅ -ethyl-2-(1-methyl-4-nitro-1H-pyrazol-5-yl)-1,3-dioxan-5-yl)methyl methanesulfonate as a white solid (816 mg, quantitative). ¹ H NMR (400 MHz, _CDCl₃ ) δ 8.01 (s, 1H), 6.38 (s, 1H), 4.14 (s, 3H), 4.05-3.88 (m, 6H), 3.22-2.92 (m, 3H), 1.96 (q, J = 7.6 Hz, 2H), 1.03 (t, J = 7.6 Hz, 3H).

Intermediate 83 2-((5-ethyl-2-(1-methyl-4-nitro-1H-pyrazol-5-yl)-1,3-dioxan-5-yl)methyl)isoindoline-1,3-dione

To a solution of (5-ethyl-2-(1-methyl-4-nitro-1H-pyrazol-5-yl)-1,3-dioxan-5-yl)methyl methanesulfonate (816 mg, 2.25 mmol, Intermediate 74) in anhydrous DMSO (10 mL) was added potassium phthalimide (2.1 g, 11.3 mmol) in one portion. The reaction mixture was heated at 180° C. for 5 h, cooled to room temperature, and diluted with EtOAc (50 mL) and water (30 mL). The organic layer was washed with water (3×30 mL), 2N NaOH (2×20 mL), and water (20 mL), separated, dried over Na ₂ SO ₄ , and concentrated under reduced pressure. Purification via silica gel column chromatography (0-100% EtOAc/isohexane) gave 2-((5-ethyl-2-(1-methyl-4-nitro-1H-pyrazol-5-yl)-1,3-dioxan-5-yl)methyl)isoindoline-1,3-dione as a colourless solid (317 mg, 35%). ¹ H NMR (400MHz, CDCl ₃ )δ8.00(s,1H),7.93-7.88(m,2H),7.82-7.76(m,2H),6.31(s,1H),4.14(s,3H),4.06(d,J＝11 .8Hz, 2H), 3.85 (d, J = 11.8Hz, 2H), 3.51 (s, 2H), 1.92 (q, J = 7.6Hz, 2H), 1.14 (t, J = 7.6Hz, 3H).

Intermediate 84 tert- Butyl N-[7-(2-methyl-4-nitro-pyrazol-3-yl)-3-(trideuterated methoxy)oxepan-4-yl]carbamate

A solution of 5-(5,8-dioxabicyclo[5.1.0]octan-4-yl)-1-methyl-4-nitro-pyrazole (400 mg, 1.67 mmol, Intermediate 19) in MeOH/water (9 mL/1.7 mL) was treated with ammonium chloride (221 mg, 4.2 mmol) and sodium azide (544 mg, 8.37 mmol), and the mixture was heated at 70°C behind a blast shield for 18 h. The reaction mixture was extracted with EtOAc (100 mL), and the organic layer was washed with water (3 x 20 mL) and brine (20 mL), separated, dried over _MgSO₄ , and concentrated under reduced pressure. To a solution of the residue (310 mg, 1.1 mmol) in anhydrous DMF (5 mL) was added sodium hydride (60% dispersion in mineral oil, 53 mg, 1.32 mmol) portionwise at room temperature under nitrogen over 10 min. After another 45 min, trideuteromethyl iodide (0.21 mL, 3.3 mmol) was added dropwise and the mixture was stirred at room temperature for 18 h. More sodium hydride (60% dispersion in mineral oil, 310 mg, 1.1 mmol) was added, followed by more trideuteromethyl iodide (0.21 mL, 3.3 mmol), and the mixture was stirred at room temperature for 48 h. Water (20 mL) was added and the mixture was extracted with EtOAc (3 × 20 mL). The combined organic layers were washed with water (20 mL) and brine (20 mL), dried over _MgSO₄ , and concentrated under reduced pressure. Purification via silica gel column chromatography (0-40% EtOAc/isohexane) gave 5-[5-azido-6-(trideuteromethoxy)oxepan-2-yl]-1-methyl-4-nitro-pyrazole as an oil (140 mg). A solution of the oil (140 mg, 0.47 mmol) in THF/water (5 mL/0.9 mL) was treated with triphenylphosphine (135 mg, 0.52 mmol) and the reaction mixture was heated at 70 ° C behind a blast shield for 18 h. The mixture was concentrated under reduced pressure. The resulting residue was dissolved in anhydrous DCM (9 mL) at 0 ° C and di-tert-butyl dicarbonate (123 mg, 0.56 mmol) and DIPEA (0.25 mL, 1.41 mmol) were added. The reaction mixture was warmed to room temperature and stirred for 3 h. Water (10 mL) was added and the mixture was extracted with DCM (20 mL). The organic layer was separated, dried over MgSO ₄ and concentrated under reduced pressure. Purification via silica gel column chromatography (0-60% EtOAc/isohexane) gave racemic tert-butyl N-[7-(2-methyl-4-nitro-pyrazol-3-yl)-3-(trideuteriomethoxy)oxepan-4-yl]carbamate (relative stereochemistry shown above) as an off-white solid (125 mg, 28% over four steps). ¹ H NMR (400MHz, CDCl ₃ )δ8.01(s,1H),5.39(dd,J＝10.6,3.6Hz,1H),4.85-4.67(m,1H),4.32(dd,J＝14.2,1.9Hz,1H),4.06(s,3 H),3.90-3.82(m,1H),3.75(dd,J＝14.2,3.2Hz,1H),3.40-3.33(m,1H),2.20-1.82(m,4H),1.46(m,9H).

Intermediate 85 tert- Butyl N-[(3R,4S,7R)-7-(2-methyl-4-nitro-pyrazol-3-yl)-3-(trideuterated methoxy)oxepan-4-yl]carbamate

tert-Butyl N-[7-(2-methyl-4-nitro-pyrazol-3-yl)-3-(trideuteriomethoxy)oxepan-4-yl]carbamate was further purified via chiral SFC to afford tert-butyl N-[(3R,4S,7R)-7-(2-methyl-4-nitro-pyrazol-3-yl)-3-(trideuteriomethoxy)oxepan-4-yl]carbamate as the first eluting isomer as an off-white solid (54 mg, 43%). ¹ H NMR (400MHz, CDCl ₃ )δ8.01(s,1H),5.39(dd,J＝10.6,3.6Hz,1H),4.85-4.68(m,1H),4.32(dd,J＝14.2,1.9Hz,1H),4.06(s,3 H),3.90-3.82(m,1H),3.75(dd,J＝14.0,3.2Hz,1H),3.40-3.33(m,1H),2.20-1.83(m,4H),1.46(s,9H).

Intermediate 86 tert-Butyl N-[(3S,4R,7S)-7-(2-methyl-4-nitro-pyrazol-3-yl)-3-(trideuterated methoxy)oxepan-4-yl]carbamate

Following the procedure of Intermediate 77, tert-butyl N-[(3S,4R,7S)-7-(2-methyl-4-nitro-pyrazol-3-yl)-3-(trideuteriomethoxy)oxepan-4-yl]carbamate was also obtained as the second eluting isomer as an off-white solid (52 mg, 41%). ¹ H NMR (400MHz, CDCl ₃ )δ8.02(s,1H),5.39(dd,J＝10.6,3.6Hz,1H),4.85-4.66(m,1H),4.33(dd,J＝14.2,1.9Hz,1H),4.07(s,3 H),3.90-3.83(m,1H),3.75(dd,J＝14.2,3.2Hz,1H),3.40-3.33(m,1H),2.21-1.83(m,4H),1.47(m,9H).

Intermediate 87 5-(5-(Azidomethyl)-5-methyl-1,3-dioxan-2-yl)-1-methyl-4-nitro-1H-pyrazole (trans isomer)

To a solution of (5-methyl-2-(1-methyl-4-nitro-1H-pyrazol-5-yl)-1,3-dioxan-5-yl)methanol (trans isomer) (248 mg, 1.02 mmol, Intermediate 66) in anhydrous DCM (10 mL) at 0°C was added _Et3N (0.20 mL, 1.53 mmol) followed by methanesulfonyl chloride (0.10 mL, 1.22 mmol). The reaction mixture was slowly warmed to room temperature over 1.5 h. The mixture was recooled to 0°C and 1 M aqueous HCl (5 mL) and DCM (20 mL) were added. The organic layer was washed with saturated aqueous _NaHCO3 (10 mL) and water (10 mL), separated, dried _{over Na2SO4} , and concentrated under reduced pressure to give a colorless oil. The oil was dissolved in DMF (20 mL) and sodium azide (400 mg, 6.12 mmol) was added. The reaction mixture was heated at 140°C behind a blast shield for 18 h. The reaction mixture was cooled to room temperature and diluted with water (20 mL) and EtOAc (50 mL). The organic layer was washed with water (3 x 20 mL ₎ , separated, dried over _Na₂SO₄ , and concentrated under reduced pressure to afford 5-(5-(azidomethyl)-5-methyl-1,3-dioxan-2-yl)-1-methyl-4-nitro-1H-pyrazole as a colorless solid (300 mg, quantitative over two steps). ¹ H NMR (400 MHz, _CDCl₃ ) δ 8.03 (s, 1H), 6.36 (s, 1H), 4.17 (s, 3H), 3.88 (s, 4H), 3.20 (s, 2H), 1.40 (s, 3H).

Intermediate 88 tert-Butyl N-[(3S,4R,7S)-3-fluoro-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-yl]carbamate

To a solution of 4-azido-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-3-ol (660 mg, 2.34 mmol, intermediate 57) in DCM (12 mL) was added (50% in THF, 2.12 mL) and the mixture was stirred at room temperature for 18 h. The mixture was diluted with DCM (22 mL), cooled to 0 ° C and carefully added saturated aqueous NaHCO ₃ solution (20 mL). The aqueous layer was extracted with DCM (3×20 mL) and the combined organic layers were dried over Na ₂ SO ₄ and concentrated under reduced pressure. Purification via silica gel column chromatography (0-30% EtOAc / isohexane) gave 5-(5-azido-6-fluorooxepan-2-yl)-1-methyl-4-nitro-1H-pyrazole as an oil (440 mg). A solution of the oil (440 mg, 1.54 mmol) in THF/water (15 mL/2.8 mL) was treated with triphenylphosphine (487 mg, 1.86 mmol) and the reaction mixture was heated at 70 ° C behind a blast shield for 18 h. The reaction mixture was concentrated under reduced pressure. The residue was dissolved in anhydrous DCM (15 mL), cooled to 0 ° C and di-tert-butyl dicarbonate (402 mg, 1.84 mmol) and DIPEA (0.8 mL, 4.62 mmol) were added. The reaction mixture was warmed to room temperature and stirred for 18 h. Water (20 mL) was added and the mixture was extracted with DCM (100 mL). The organic layer was separated, dried over MgSO ₄ and concentrated under reduced pressure. Purification by silica gel column chromatography (0-35% EtOAc/isohexane) followed by chiral preparative SFC gave tert-butyl N-[(3S,4R,7S)-3-fluoro-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-yl]carbamate as a white solid (223 mg, 27% over three steps). ¹ H NMR (400MHz, CDCl ₃ ) δ8.02 (s, 1H), 5.37 (dd, J = 10.5, 3.0Hz, 1H), 4.89 (br s,1H),4.61(ddd,J＝49.1,7.7,3.2Hz,1H),4.44(dd,J＝22.2,15.0Hz,1H),4.07( s,3H),3.98-3.80(m,1H),3.49(d,J＝5.3Hz,1H),2.15-1.90(m,4H),1.47(s,9H).

Intermediate 89 tert-Butyl N-[(3R,4S,7R)-3-fluoro-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-yl]carbamate

Following the procedure of Intermediate 80, tert-butyl N-[(3R,4S,7R)-3-fluoro-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-yl]carbamate was also obtained as a white solid (247 mg, 91%). ¹ H NMR (400MHz, CDCl ₃ ) δ8.05 (s, 1H), 5.39 (dd, J = 10.7, 2.9Hz, 1H), 4.85 (br s,1H),4.61(ddd,J＝49.3,7.7,3.17Hz,1H),4.52-4.40(m,1H),4.07(s,3H) ,3.97-3.84(m,1H),3.49(d,J＝5.3Hz,1H),2.15-1.88(m,4H),1.49(s,9H).

Intermediate 90 5-(5-(Azidomethyl)-5-methyl-1,3-dioxan-2-yl)-1-methyl-4-nitro-1H-pyrazole (cis isomer)

Following the procedure of Intermediate 79, starting from (5-methyl-2-(1-methyl-4-nitro-1H-pyrazol-5-yl)-1,3-dioxan-5-yl)methanol (cis isomer, Intermediate 67), 5-(5-(azidomethyl)-5-methyl-1,3-dioxan-2-yl)-1-methyl-4-nitro-1H-pyrazole was obtained as a colorless solid (519 mg, 87% over two steps). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.02 (s, 1H), 6.39 (s, 1H), 4.14 (s, 3H), 4.04 (d, J=12.0 Hz, 2H), 3.73 (d, J=12.0 Hz, 2H), 3.70 (s, 2H), 0.87 (s, 3H).

Intermediate 91 2,2,2-Trifluoro-N-((5-methyl-2-(1-methyl-4-nitro-1H-pyrazol-5-yl)-1,3-dioxan-5-yl)methyl)acetamide (cis isomer)

To a solution of 5-(5-(azidomethyl)-5-methyl-1,3-dioxan-2-yl)-1-methyl-4-nitro-1H-pyrazole (cis isomer) (519 mg, 1.84 mmol, Intermediate 82) in anhydrous MeOH (25 mL) and THF (10 mL) was added ammonium formate (300 mg, 4.76 mmol) and 10% Pd/C (300 mg, 0.28 mmol). The mixture was heated at reflux for 30 min and then cooled to room temperature. The suspension was filtered through Celite and the filter cake was washed with EtOAc (200 mL). The filtrate was concentrated under reduced pressure and the crude residue was dissolved in anhydrous THF (11 mL) and DCM (2 mL) and cooled to 0°C. _Et3N (0.38 mL, 2.86 mmol) was added followed by trifluoroacetic anhydride (0.30 mL, 2.10 mmol). The reaction mixture was slowly warmed to room temperature and stirred for 18 h. The mixture was recooled to 0 ° C and quenched with water (10 mL) and extracted with EtOAc (20 mL). The organic layer was washed with brine (10 mL), separated, dried over Na ₂ SO ₄ and concentrated under reduced pressure. Purified via silica gel column chromatography (0-100% EtOAc/isohexane) to obtain 2,2,2-trifluoro-N-((5-methyl-2-(1-methyl-4-nitro-1H-pyrazole-5-yl)-1,3-dioxane-5-yl)methyl)acetamide as a colorless oil (410 mg, 63% over two steps). ¹ H NMR (400MHz, CDCl ₃ )δ7.95(s,1H),7.24(s,1H),6.33(s,1H),4.17(s,3H),3.92(d,J＝12.0Hz,2H),3.75(d,J＝6.8Hz,2H),3.71(d,J＝12.0Hz,2H),0.80(s,3H).

Intermediate 92 2,2,2-Trifluoro-N-((5-methyl-2-(1-methyl-4-nitro-1H-pyrazol-5-yl)-1,3-dioxan-5-yl)methyl)acetamide (trans isomer)

To a solution of 5-(5-(azidomethyl)-5-methyl-1,3-dioxan-2-yl)-1-methyl-4-nitro-1H-pyrazole (trans isomer; 300 mg, 1.02 mmol, Intermediate 79) in THF (3 mL) and water (0.3 mL) was added triphenylphosphine (322 mg, 1.22 mmol). The reaction mixture was heated at 70°C for 1 h. The mixture was cooled to room temperature and concentrated under reduced pressure. To a solution of the crude residue in anhydrous THF (10 mL) at 0°C were added _Et₃N (0.20 mL, 1.53 mmol) followed by trifluoromethanesulfonic anhydride (0.16 mL, 1.12 mmol). The reaction mixture was slowly warmed to room temperature and stirred for 18 h. The mixture was recooled to 0°C and more _Et₃N (0.20 mL, 1.53 mmol) and trifluoromethanesulfonic anhydride (0.16 mL, 1.12 mmol) were added. The reaction mixture was slowly warmed to room temperature and stirred for 6h. The mixture was recooled to 0 ° C, quenched with water (10mL) and extracted with DCM (20mL). The organic layer was passed through a phase separation column and concentrated under reduced pressure. Purified via silica gel column chromatography (0-100% EtOAc/isohexane) to obtain 2,2,2-trifluoro-N-((5-methyl-2-(1-methyl-4-nitro-1H-pyrazole-5-yl)-1,3-dioxane-5-yl)methyl)acetamide as a colorless solid (171mg, 0.49mmol).

Intermediate 93 5-(5,6-dimethyl-4-((triethylsilyl)oxy)-3,6-dihydro-2H-pyran-2-yl)-1-methyl-4-nitro-1H-pyrazole

To a solution of (E)-3-methylpent-3-en-2-one (2.69 mL, 24.1 mmol) in DCM (200 mL) cooled to 0°C was added _Et3N (10.5 mL, 79.5 mmol) followed by TESOTf (6.0 mL, 26.5 mmol). The mixture was warmed to room temperature and stirred for 18 h. Saturated aqueous _NaHCO3 (100 mL) and DCM (200 mL) were added. The aqueous layer was extracted with DCM (3 x 200 mL) and the combined organic layers were washed with brine (100 mL), separated, dried over _MgSO4 , and concentrated under reduced pressure to afford (E)-triethyl((3-methylpenta-1,3-dien-2-yl)oxy)silane. To a solution of 2-methyl-4-nitro-pyrazole-3-carbaldehyde (1.0 g, 8 mmol, Intermediate ₃ ) in CDCl₃ (28 mL) was added (E)-triethyl((3-methylpenta-1,3-dien-2-yl)oxy)silane (1.6 g, 7.55 mmol) followed by EuFOD (220 mg, 0.50 mmol). The reaction mixture was heated in a pressure tube at 65° C. behind a blast shield for 18 h. The mixture was cooled to room temperature and concentrated under reduced pressure. Purification via silica gel column chromatography (0-100% EtOAc/isohexane) gave 5-(5,6-dimethyl-4-((triethylsilyl)oxy)-3,6-dihydro-2H-pyran-2-yl)-1-methyl-4-nitro-1H-pyrazole as a colorless oil (2.92 g, quantitative). ¹ H NMR (400MHz, CDCl ₃ )δ8.07(s,1H),5.64(dd,J＝10.9,3.6Hz,1H),4.33-4.28(m,1H),4.25-3.94(m,3H),2.50-2.41 (m,1H),2.31(m,1H),1.61(s,3H),1.31(d,J＝6.4Hz,3H),1.05-0.97(m,6H),0.73-0.61(m,9H).

Intermediate 94 3-azido-2,3-dimethyl-6-(1-methyl-4-nitro-1H-pyrazol-5-yl)dihydro-2H-pyran-4(3H)-one

To a solution of 5-(5,6-dimethyl-4-((triethylsilyl)oxy)-3,6-dihydro-2H-pyran-2-yl)-1-methyl-4-nitro-1H-pyrazole (507 mg, 1.38 mmol, Intermediate 85) in anhydrous MeCN (3.5 mL) cooled to -20°C was added sodium azide (404 mg, 6.22 mmol), followed by the dropwise addition of a solution of ceric ammonium nitrate (2.27 g, 4.15 mmol) in _CH3CN (10.4 mL). The reaction mixture was stirred at -20°C for 1 h, slowly warmed to 0°C over 1 h, then quenched with water (20 mL) and extracted with EtOAc (20 mL). The organic layer was washed with water (10 mL) and brine (10 mL), separated, _dried over _Na2SO4 , and concentrated under reduced pressure. Purification via silica gel column chromatography (0-100% EtOAc/isohexane) gave 3-azido-2,3-dimethyl-6-(1-methyl-4-nitro-1H-pyrazol-5-yl)dihydro-2H-pyran-4(3H)-one as a white solid (187 mg, 46%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.05 (s, 1H), 5.78 (dd, J=12.3, 3.2 Hz, 1H), 4.21 (s, 3H), 3.73 (dd, J=12.3, 6.2 Hz, 1H), 3.13 (dd, J=14.6, 12.3 Hz, 1H), 2.73 (dd, J=14.6, 3.2 Hz, 1H), 1.44 (s, 3H), 1.41 (d, J=6.1 Hz, 3H).

Intermediate 95 5-(5-azido-4,4-difluoro-5,6-dimethyltetrahydro-2H-pyran-2-yl)-1-methyl-4-nitro-1H-pyrazole

To a solution of 3-azido-2,3-dimethyl-6-(1-methyl-4-nitro-1H-pyrazol-5-yl)dihydro-2H-pyran-4(3H)-one (335 mg, 1.14 mmol, Intermediate 86) in anhydrous DCM (10 ml) was added a solution of 50% in THF (830 mg, 1.88 mmol) and the mixture was stirred at room temperature for 18 h. Saturated aqueous _NaHCO₃ solution (20 mL) and DCM (20 mL) were added. The aqueous layer was extracted with DCM (3 x 20 mL) and the combined organic layers were washed with brine (20 mL), separated, dried over _MgSO₄ , and concentrated under reduced pressure. Purification via silica gel column chromatography (20% EtOAc/isohexane) gave 5-(5-azido-4,4-difluoro-5,6-dimethyltetrahydro-2H-pyran-2-yl)-1-methyl-4-nitro-1H-pyrazole (contaminated with some vinyl fluoride) as a pale yellow oil (157 mg, 44%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.04 (s, 1H), 5.69 (dd, J=12.2, 2.9 Hz, 1H), 4.13 (s, 3H), 3.76 (qd, J=6.3, 1.6 Hz, 1H), 2.59-2.40 (m, 1H), 2.38-2.28 (m, 1H), 1.48 (s, 3H), 1.32 (d, J=6.2 Hz, 3H).

Intermediate 96 tert-Butyl (2-(1-methyl-4-nitro-1H-pyrazol-5-yl)tetrahydro-2H-pyran-4-yl)carbamate

To a solution of 2-(2-methyl-4-nitro-pyrazol-3-yl)tetrahydropyran-4-ol (450 mg, 1.98 mmol, Intermediate 39) in anhydrous DCM (24 mL) at 0°C was added _Et₃N (0.33 mL, 2.97 mmol) followed by MsCl (0.44 mL, 4.0 mmol). The reaction mixture was stirred at 0°C for 30 min and then at room temperature for 18 h. The mixture was recooled to 0°C and quenched with saturated aqueous _NaHCO₃ (10 mL). The organic layer was washed with 0.1 M HCl (5 mL), passed through a phase separation cartridge, and concentrated under reduced pressure to yield a colorless oil. This oil was dissolved in DMF (10 mL) and sodium azide (660 mg, 10 mmol) was added. The reaction mixture was heated at 110°C behind a blast shield for 2 h. The reaction mixture was cooled to room temperature, diluted with water (20 mL), and extracted with EtOAc (50 mL). The organic layer was washed with water (3 x 20 mL), separated, _dried over _Na₂SO₄ , and concentrated under reduced pressure to yield a colorless solid (220 mg). To a solution of this solid (220 mg, 0.87 mmol) in THF (2.5 mL) and water (0.5 mL) was added triphenylphosphine (344 mg, 1.31 mmol). The reaction mixture was heated at 65°C behind a blast shield for 4 h. The mixture was recooled to room temperature and concentrated under reduced pressure. The residue was dissolved in DCM (5 mL), treated with di-tert-butyl dicarbonate (287 mg, 1.31 mmol) and DIPEA (0.44 mL, 2.62 mmol), and the reaction mixture was stirred at room temperature for 16 h. The mixture was concentrated under reduced pressure. Purification via silica gel column chromatography (30% EtOAc/isohexane) gave tert-butyl (2-(1-methyl-4-nitro-1H-pyrazol-5-yl)tetrahydro-2H-pyran-4-yl)carbamate as a yellow oil (155 mg, 24% over four steps). ¹ H NMR (400MHz, CDCl ₃ )δ8.03(s,1H),5.44(d,J＝11.6Hz,1H),4.52(s,1H),4.19(dd,J＝11.9,4.6Hz,1H),4.06(s,3H),3.68-3 .60(m,1H),2.29(d,J＝12.6Hz,1H),2.03(d,J＝8.4Hz,1H),1.75(s,1H),1.61-1.47(m,2H),1.45(s,9H).

Intermediate 97 (3S,4R,7S)-4-azido-7-(1-methyl-4-nitro-1H-pyrazol-5-yl)oxepan-3-ol

To a solution of 5-(5,8-dioxabicyclo[5.1.0]octan-4-yl)-1-methyl-4-nitro-pyrazole (2.7 g, 11.3 mmol, intermediate 19) in MeOH/water (60 mL/15 mL) was added ammonium chloride (1.51 g, 28.3 mmol) and sodium azide (3.67 g, 56.5 mmol). The mixture was heated at 70 ° C behind a blast shield for 4 h. MeOH was removed under reduced pressure and the aqueous residue was extracted with EtOAc (100 mL). The organic layer was washed with aqueous NaHCO ₃ (3×20 mL), passed through a phase separation cartridge and concentrated under reduced pressure. Purification by silica gel column chromatography (0-100% EtOAc/isohexane) followed by chiral SFC chromatography gave (3S,4R,7S)-4-azido-7-(1-methyl-4-nitro-1H-pyrazol-5-yl)oxepan-3-ol as the second eluting isomer as a clear gum (1.4 g, 41%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.03 (s, 1H), 5.43-5.37 (m, 1H), 4.18 (dd, J=13.9, 2.1 Hz, 1H), 4.06 (s, 3H), 3.97-3.77 (m, 3H), 2.45 (d, J=3.9 Hz, 1H), 2.32-2.09 (m, 2H), 2.10-1.85 (m, 2H).

Intermediate 98 (4R,7S)-4-azido-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-3-one

To a solution of (3S, 4R, 7S) -4- azido -7- (1- methyl -4- nitro -1H- pyrazole -5- bases) oxepan -3- alcohol (1.4 g, 4.96 mmol, intermediate 90) in DCM (35 mL) was added Dess-Martin periodinane (2.52 g, 5.96 mmol) and the mixture was stirred at room temperature for 2 h. Saturated NaHCO ₃ aqueous solution (60 mL) and 20% sodium thiosulfate solution (50 mL) were added and the reaction mixture was stirred for 30 min until the salt was observed to be completely dissolved. The organic layer was separated, dried over MgSO ₄ and the solvent was removed under reduced pressure. Purified via silica gel column chromatography (0-40% EtOAc / isohexane) to obtain (4R, 7S) -4- azido -7- (2- methyl -4- nitro - pyrazole -3- bases) oxepan -3- one as an off-white solid (1.1 g, 82%). ¹ H NMR (400MHz, CDCl ₃ )δ8.05(s,1H),5.38(dd,J＝10.2,2.7Hz,1H),4.62-4.49(m,2H),4.31-4.22(m,1H),4.08(s,3H),2.31-2.17(m,3H),2.15-2.04(m,1H).

Intermediate 99 (3R,4R,7S)-4-azido-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-3-ol

Following the procedure of Intermediate 57, starting from (4R,7S)-4-azido-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-3-one, (3R,4R,7S)-4-azido-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-3-ol was obtained as a dark orange oil (850 mg, 74%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.02 (s, 1H), 5.68-5.60 (m, 1H), 4.24-4.14 (m, 3H), 4.01 (s, 3H), 3.72-3.58 (m, 1H), 2.45-2.31 (m, 1H), 2.30-2.09 (m, 2H), 2.01-1.81 (m, 2H).

Intermediate 100 tert- Butyl N-[(3R,4R,7S)-3-methoxy-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-yl]carbamate

Following the procedure of Intermediate 58, starting from (3R,4R,7S)-4-azido-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-3-ol, tert-butyl N-[(3R,4R,7S)-3-methoxy-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-yl]carbamate was obtained as a colorless oil (357 mg, 32% over three steps). ¹ H NMR (400MHz, CDCl ₃ )δ8.00(s,1H),5.60-5.53(m,1H),5.12-5.02(m,1H),4.21-4.08(m,2H),4.01(s,3H),3.79(dd,J＝13.2,4.4H z,1H),3.75-3.70(m,1H),3.41(s,3H),2.28-2.07(m,1H),1.97-1.89(m,2H),1.80-1.72(m,1H),1.47(s,9H).

Intermediate 101 tert-Butyl ((3S,4R,7S)-3-hydroxy-7-(1-methyl-4-nitro-1H-pyrazol-5-yl)oxepan-4-yl)carbamate

To a solution of (3S,4R,7R)-4-azido-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-3-ol (Intermediate 90) (1.19 g, 4.22 mmol) in THF (50 mL) and water (10 mL) was added triphenylphosphine (1.22 g, 4.64 mmol) and the mixture was heated at 70 ° C for 24 h. The mixture was diluted with EtOAc (100 mL) and washed with brine (2×25 mL). The organic layer was separated, dried over MgSO ₄ and concentrated under reduced pressure. The residue was passed through an SCX column washed with MeOH and eluted with 3% 7N NH ₃ in MeOH/DCM to give an oil. The oil was dissolved in DCM (13.5 mL) and DIPEA (1.08 mL, 6.21 mmol) and di-tert-butyl dicarbonate (1.36 g, 6.21 mmol) were added. The mixture was stirred at room temperature for 3 h, then concentrated under reduced pressure. Purification via silica gel chromatography (0-60% EtOAc/isohexane) gave tert-butyl ((3S,4R,7S)-3-hydroxy-7-(1-methyl-4-nitro-1H-pyrazol-5-yl)oxepan-4-yl)carbamate (contaminated with some triphenylphosphine oxide) as a clear gum (895 mg, 60% over two steps). ¹ H NMR (400MHz, CDCl ₃ )δ8.02(s,1H),5.42-5.36(m,1H),4.83(d,J＝6.7Hz,1H),4.22(d,J＝13.4Hz,2H),4 .08(s,3H),3.86-3.76(m,3H),2.18-2.07(m,1H),2.02-1.89(m,3H),1.47(s,9H).

Intermediate 102 N-[(3R,4R,7S)-7-[4-[(6-bromo-5-fluoro-pyridine-2-carbonyl)amino]-2-methyl-pyrazol-3-yl]-3-methoxy-oxepan-4-yl]carbamic acid tert-butyl ester

Following the procedure of Example 65, starting from tert-butyl N-[(3R,4R,7S)-3-methoxy-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-yl]carbamate (Intermediate 93) and replacing 2-bromo-5-(tert-butoxycarbonylamino)thiazole-4-carboxylic acid with 6-bromo-5-fluoro-pyridine-2-carboxylic acid (see US2010/56576A1), tert-butyl N-[(3R,4R,7S)-7-[4-[(6-bromo-5-fluoro-pyridine-2-carbonyl)amino]-2-methyl-pyrazol-3-yl]-3-methoxy-oxepan-4-yl]carbamate was obtained (contaminated with tetramethylurea) as a clear oil (169 mg, 30% over two steps). ¹ H NMR (400MHz, CDCl ₃ ) δ10.37(s,1H),8.26-8.17(m,2H),7.63-7.55(m,1H),5.02(br s,1H),4.96(dd,J＝9.0,3.6Hz,1H),4.32(dd,J＝13.2,4.4Hz,1H),4.05-3.94(m,2H),3.85- 3.80(m,1H),3.78(s,3H),3.47(s,3H),2.10-1.91(m,3H),1.86-1.78(m,1H),1.45(s,9H).

Intermediate 103 tert-Butyl ((3S,4R,7S)-7-(4-(2-bromothiazole-4-carboxamido)-1-methyl-1H-pyrazol-5-yl)-3-fluorooxepan-4-yl)carbamate

Following the procedure of Intermediate 65, starting from tert-butyl ((3S,4R,7S)-3-fluoro-7-(1-methyl-4-nitro-1H-pyrazol-5-yl)oxepan-4-yl)carbamate (Intermediate 80) and replacing 2-bromo-5-(tert-butoxycarbonylamino)thiazole-4-carboxylic acid with 2-bromothiazole-4-carboxylic acid (commercially available), tert-butyl ((3S,4R,7S)-7-(4-(2-bromothiazole-4-carboxamido)-1-methyl-1H-pyrazol-5-yl)-3-fluorooxepan-4-yl)carbamate was obtained.

Intermediate 104 tert- Butyl ((3R,4R,7S)-7-(4-(2-bromothiazole-4-carboxamido)-1-methyl-1H-pyrazol-5-yl)-3-fluorooxepan-4-yl)carbamate

Following the procedure of Intermediate 65, starting from tert-butyl ((3R,4R,7S)-3-fluoro-7-(1-methyl-4-nitro-1H-pyrazol-5-yl)oxepan-4-yl)carbamate (Intermediate 24) and replacing 2-bromo-5-(tert-butoxycarbonylamino)thiazole-4-carboxylic acid with 2-bromothiazole-4-carboxylic acid (commercially available), tert-butyl ((3R,4R,7S)-7-(4-(2-bromothiazole-4-carboxamido)-1-methyl-1H-pyrazol-5-yl)-3-fluorooxepan-4-yl)carbamate was obtained.

Intermediate 105 tert-Butyl ((3R,4R,7S)-7-(4-(2-bromothiazole-4-carboxamido)-1-methyl-1H-pyrazol-5-yl)-3-methoxyoxepan-4-yl)carbamate

Following the procedure of Intermediate 65, starting from tert-butyl ((3R,4R,7S)-3-methoxy-7-(1-methyl-4-nitro-1H-pyrazol-5-yl)oxepan-4-yl)carbamate (Intermediate 93) and replacing 2-bromo-5-(tert-butoxycarbonylamino)thiazole-4-carboxylic acid with 2-bromothiazole-4-carboxylic acid (commercially available), tert-butyl ((3R,4R,7S)-7-(4-(2-bromothiazole-4-carboxamido)-1-methyl-1H-pyrazol-5-yl)-3-methoxyoxepan-4-yl)carbamate was obtained.

Intermediate 106 tert- Butyl ((3S,4R,7S)-7-(4-(2-bromothiazole-4-carboxamido)-1-methyl-1H-pyrazol-5-yl)-3-methoxyoxepan-4-yl)carbamate

Following the procedure of Intermediate 65, starting from tert-butyl ((3S,4R,7S)-3-methoxy-7-(1-methyl-4-nitro-1H-pyrazol-5-yl)oxepan-4-yl)carbamate (Intermediate 21) and replacing 2-bromo-5-(tert-butoxycarbonylamino)thiazole-4-carboxylic acid with 2-bromothiazole-4-carboxylic acid (commercially available), tert-butyl ((3S,4R,7S)-7-(4-(2-bromothiazole-4-carboxamido)-1-methyl-1H-pyrazol-5-yl)-3-methoxyoxepan-4-yl)carbamate was obtained.

Intermediate 107 tert-Butyl ((3S,4R,7S)-7-(4-(6-bromo-5-fluoropyridine-2-carboxamido)-1-methyl-1H-pyrazol-5-yl)-3-fluorooxepan-4-yl)carbamate

Following the procedure of Example 65, starting from tert-butyl N-[(3S,4R,7S)-3-fluoro-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-yl]carbamate (Intermediate 80) and replacing 2-bromo-5-(tert-butoxycarbonylamino)thiazole-4-carboxylic acid with 6-bromo-5-fluoro-pyridine-2-carboxylic acid (see US2010/56576A1) gave tert-butyl ((3S,4R,7S)-7-(4-(6-bromo-5-fluoropyridine-2-carboxamido)-1-methyl-1H-pyrazol-5-yl)-3-fluorooxepan-4-yl)carbamate.

Intermediate 108 2-(2,6-difluoro-4-methoxyphenyl)thiazole-4-carboxylic acid

To a solution of methyl 2-bromothiazole-4-carboxylate (3.27 mmol, 741 mg) in tetrahydrofuran (15 mL) and water (1.5 mL) was added 2,6-difluoro-4-methoxyphenylboronic acid (1.8 eq, 5.88 mmol, 1160 mg) and potassium fluoride (3.3 eq, 10.8 mmol, 627 mg). The mixture was degassed with nitrogen, and tris(dibenzylideneacetone)dipalladium(0) (0.2 eq, 0.654 mmol, 617 mg) and tri-tert-butylphosphine (1.0 M in toluene; 0.4 eq, 1.31 mmol, 1.3 mL) were added and the reaction mixture was heated in a microwave at 100° C. for 30 minutes. The reaction mixture was concentrated and the residue was purified on silica gel eluting with 0 to 50% EtOAc/heptane to give methyl 2-(2,6-difluoro-4-methoxy-phenyl)thiazole-4-carboxylate (2.40 mmol, 685 mg, 74% yield).

To a solution of 2- (2,6-difluoro-4-methoxy-phenyl) thiazole-4-formic acid methyl ester (2.403mmol, 685.5mg) in methanol (15mL) and water (5mL) is added lithium hydroxide (1.9 equivalents, 4.54mmol, 111mg). The reaction mixture is stirred at room temperature overnight. The reaction mixture is quenched with 1N HCl (aqueous solution) and then distributed between EtOAc and saline. The organic layer is concentrated. The residue is dried under high vacuum to obtain 2- (2,6-difluoro-4-methoxy-phenyl) thiazole-4-formic acid (650mg, quantitative) as a brown solid.

Intermediate 109 2-(2-fluoro-4-methoxyphenyl)thiazole-4-carboxylic acid

Methyl 2-bromothiazole-4-carboxylate is reacted with 2-fluoro-4-methoxyphenylboronic acid to provide the title compound.

Intermediate 110 1-(3,5-difluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)ethanol

To a solution of 1-(3,5-difluorophenyl)ethanol (10.2 mmol, 1660 mg, commercially available) in tetrahydrofuran (100 mL) was added dropwise a solution of n-butyllithium (2.5 mol/L) in hexanes (2.4 equiv, 24.4 mmol, 9.8 mL) at -78 °C. The mixture was stirred at -78 °C for 2 hours. 2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (2.50 equiv, 25.4 mmol, 5.29 mL) was added and the reaction mixture was stirred overnight and allowed to warm to room temperature. The reaction mixture was quenched with saturated _NaHCO₃ (aq) and extracted with ethyl acetate. The organic layer was washed with brine, dried _{over Na₂SO₄} , filtered, and the filtrate was concentrated to give the desired product, which was used without further purification.

Intermediate 111 2-(3,5-difluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)propan-2-ol

2-Isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane was reacted with 2-(3,5-difluorophenyl)propan-2-ol (see US 2012/225062) to give the title compound.

Intermediate 112 1-(3,5-difluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)cyclobutanol

2-Isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane was reacted with 1-(3,5-difluorophenyl)cyclobutanol (see US 2012/225062) to give the title compound.

Intermediate 113 2-(2,6-difluoro-4-(1-hydroxyethyl)phenyl)thiazole-4-carboxylic acid

Methyl 2-bromothiazole-4-carboxylate was reacted with 1-(3,5-difluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)ethanol to give the title compound.

Intermediate 114 2-(2,6-difluoro-4-(1-hydroxycyclobutyl)phenyl)thiazole-4-carboxylic acid

Methyl 2-bromothiazole-4-carboxylate was reacted with 1-(3,5-difluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)cyclobutanol to provide the title compound.

Intermediate 115 2-(2,6-difluoro-4-(2-hydroxypropan-2-yl)phenyl)thiazole-4-carboxylic acid

Methyl 2-bromothiazole-4-carboxylate was reacted with 2-(3,5-difluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)propan-2-ol to give the title compound.

Intermediate 116 2-(2-(difluoromethyl)phenyl)thiazole-4-carboxylic acid

Reaction of methyl 2-bromothiazole-4-carboxylate with (2-(difluoromethyl)phenyl)boronic acid afforded the title compound.

Intermediate 117 2-(3-fluoropyridin-4-yl)thiazole-4-carboxylic acid

Reaction of methyl 2-bromothiazole-4-carboxylate with (3-fluoropyridin-4-yl)boronic acid affords the title compound.

Intermediate 118 2-(2,5-difluorophenyl)thiazole-4-carboxylic acid

Methyl 2-bromothiazole-4-carboxylate was reacted with (2,5-difluorophenyl)boronic acid to provide the title compound.

Intermediate 119 2-(5-chloro-2-fluorophenyl)thiazole-4-carboxylic acid

Methyl 2-bromothiazole-4-carboxylate is reacted with (5-chloro-2-fluorophenyl)boronic acid to provide the title compound.

Intermediate 120 2-(2,6-difluoro-3-methylphenyl)thiazole-4-carboxylic acid

Methyl 2-bromothiazole-4-carboxylate was reacted with (2,6-difluoro-3-methylphenyl)boronic acid to give the title compound.

Intermediate 121 (R)-2-(2,6-difluoro-4-(1-hydroxyethyl)phenyl)thiazole-4-carboxylic acid

Methyl 2-bromothiazole-4-carboxylate was reacted with (R)-1-(3,5-difluorophenyl)ethanol (commercial source) to provide the title compound.

Intermediate 122 (S)-2-(2,6-difluoro-4-(1-hydroxyethyl)phenyl)thiazole-4-carboxylic acid

Methyl 2-bromothiazole-4-carboxylate was reacted with (S)-1-(3,5-difluorophenyl)ethanol (commercial source) to provide the title compound.

Intermediate 123 2-(2,3-difluorophenyl)thiazole-4-carboxylic acid

Methyl 2-bromothiazole-4-carboxylate was reacted with (2,3-difluorophenyl)boronic acid to give the title compound.

Intermediate 124 2-(5-ethyl-2-fluorophenyl)thiazole-4-carboxylic acid

Methyl 2-bromothiazole-4-carboxylate is reacted with (5-ethyl-2-fluorophenyl)boronic acid to give the title compound.

Intermediate 125 2-(3-chloro-2-fluorophenyl)thiazole-4-carboxylic acid

Methyl 2-bromothiazole-4-carboxylate is reacted with (3-chloro-2-fluorophenyl)boronic acid to provide the title compound.

Intermediate 126 2-(2-chloro-3-fluorophenyl)thiazole-4-carboxylic acid

Methyl 2-bromothiazole-4-carboxylate is reacted with (2-chloro-3-fluorophenyl)boronic acid to provide the title compound.

Intermediate 127 2-(5-cyclopropyl-2-fluorophenyl)thiazole-4-carboxylic acid

Methyl 2-bromothiazole-4-carboxylate is reacted with (5-cyclopropyl-2-fluorophenyl)boronic acid to provide the title compound.

Intermediate 128 2-(2-(trifluoromethyl)phenyl)thiazole-4-carboxylic acid

Reaction of methyl 2-bromothiazole-4-carboxylate with (2-(trifluoromethyl)phenyl)boronic acid afforded the title compound.

Intermediate 129 2-(2,6-difluoro-4-methylphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane

Reaction of 1,3-difluoro-5-methylbenzene with butyl lithium and 2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane afforded the title compound.

Intermediate 130 2-(2,6-difluoro-4-methylphenyl)thiazole-4-carboxylic acid

Reaction of methyl 2-bromothiazole-4-carboxylate with 2-(2,6-difluoro-4-methylphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane afforded the title compound.

Intermediate 131 2-(4-chloro-2-fluorophenyl)thiazole-4-carboxylic acid

Methyl 2-bromothiazole-4-carboxylate is reacted with (4-chloro-2-fluorophenyl)boronic acid to provide the title compound.

Intermediate 132 2-(2-Fluoro-6-methylphenyl)thiazole-4-carboxylic acid

Methyl 2-bromothiazole-4-carboxylate is reacted with (2-fluoro-6-methylphenyl)boronic acid to provide the title compound.

Intermediate 133 2-(5-bromo-2-fluorophenyl)thiazole-4-carboxylic acid

A solution of 5-bromo-2-fluoro-benzonitrile (12.4 mmol, 2470 mg) in pyridine (6.5 mL) was treated with ammonium sulfide (40% by mass in water, 1.1 equivalents, 13.6 mmol, 2.32 mL) and triethylamine (1.1 equivalents, 13.6 mmol, 1.90 mL). The reaction mixture was heated at 50 ° C for 3 hours and then cooled to room temperature. The reaction mixture was partitioned between EtOAc and water. The organic layer was washed with water (3×) and brine (3×), dried over MgSO ₄ , and then concentrated. The residue was purified on silica gel eluted with 0 to 50% EtOAc/heptane to give 5-bromo-2-fluoro-thiobenzamide (2.84 g, 94% yield).

A mixture of 5-bromo-2-fluoro-benzamide (11.8 mmol, 2840 mg) and ethyl bromopyruvate (1.05 equiv, 12.4 mmol, 1.56 mL) in ethanol (30 mL) was heated overnight at 80° C. The mixture was concentrated and the residue was purified on silica gel eluted with 0 to 20% EtOAc/heptane to give ethyl 2-(5-bromo-2-fluoro-phenyl)thiazole-4-carboxylate (2960 mg, 76.14% yield) as a clear oil.

To a solution of ethyl 2-(5-bromo-2-fluoro-phenyl)thiazole-4-carboxylate (8.97mmol, 2960mg) in methanol (40mL) and water (10mL) was added lithium hydroxide (1.6 equivalents, 14.2mmol, 347mg). The reaction mixture was stirred at 50 ° C for 2 hours. The reaction mixture was cooled to room temperature, concentrated, suspended in water, and then quenched with 2N HCl (aqueous solution). The solid was collected, washed with water and dried under high vacuum to give 2-(5-bromo-2-fluoro-phenyl)thiazole-4-carboxylic acid (2410mg, 89% yield) as a white solid.

Intermediate 134 2-(6-(Trifluoromethyl)pyridin-2-yl)thiazole-4-carboxylic acid

Following the procedure of Intermediate 133, substituting 6-(trifluoromethyl)pyridine-2-carbonitrile for 5-bromo-2-fluoro-benzonitrile, the title compound was obtained.

Intermediate 135 2-(2-Fluoro-4-methylphenyl)thiazole-4-carboxylic acid

Following the procedure of Intermediate 133, substituting 2-fluoro-4-methylbenzonitrile for 5-bromo-2-fluoro-benzonitrile, the title compound was obtained.

Intermediate 136 6-(2,6-difluoro-4-(2-hydroxypropan-2-yl)phenyl)-5-fluoropicolinic acid

2-(3,5-difluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)propan-2-ol was reacted with methyl 6-bromo-5-fluoropicolinate (see US 2012/225062) to give the title compound.

Intermediate 137 6-(2,6-difluoro-4-(1-hydroxycyclobutyl)phenyl)-5-fluoropicolinic acid

1-(3,5-Difluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)cyclobutanol was reacted with methyl 6-bromo-5-fluoropicolinate (see US 2012/225062) to give the title compound.

Intermediate 138 6-(2,6-difluoro-4-(1-hydroxyethyl)phenyl)-5-fluoropyridine-2-carboxylic acid

1-(3,5-Difluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)ethanol was reacted with methyl 6-bromo-5-fluoropicolinate (see US 2012/225062) to give the title compound.

Intermediate 139 6-(2,6-difluoro-4-hydroxyphenyl)-5-fluoropyridine-2-carboxylic acid

(2,6-Difluoro-4-hydroxyphenyl)boronic acid was reacted with methyl 6-bromo-5-fluoropicolinate (see US 2012/225062) to give the title compound.

Intermediate 140 6-(2,6-difluoro-4-(1-methoxyethyl)phenyl)-5-fluoropicolinic acid

To a solution of methyl 6-[2,6-difluoro-4-(1-hydroxyethyl)phenyl]-5-fluoro-pyridine-2-carboxylate (1.21 mmol, 376 mg; the penultimate intermediate in the synthetic route of Intermediate 136) in N,N-dimethylformamide (50 mL) was added sodium hydride (60% by mass in mineral oil, 1.5 eq, 1.81 mmol, 72.5 mg) at 0°C. The mixture was stirred for 2 minutes, then iodomethane (3.0 eq, 3.62 mmol, 0.226 mL) was added. The reaction mixture was stirred at room temperature for 2 days. The reaction mixture was quenched with water and extracted with EtOAc. _The organic layer was washed with brine, dried ( _Na2SO4 ), and concentrated. The residue was purified on silica gel eluted with 0 to 50% EtOAc/heptane to give methyl 6-(2,6-difluoro-4-(1-methoxyethyl)phenyl)-5-fluoropyridine-2-carboxylate (392 mg, 63%). The ester was diluted with MeOH (15 mL) and water (5 mL) and lithium hydroxide (60 mg) was added. The mixture was stirred at room temperature overnight. The reaction mixture was quenched by adding 1N HCl (aqueous solution), then the mixture was diluted with EtOAc and washed with brine. The organic extract was dried (Na ₂ SO ₄ ) and concentrated in vacuo to give the title compound (quantitative), which was used without purification.

Intermediate 141 Cyclopropyl(3,5-difluorophenyl)methanol

A solution of 3,5-difluorobenzaldehyde (1.0 g, 7.0 mmol) in tetrahydrofuran (10 mL) was cooled in an ice bath. Cyclopropylmagnesium bromide (0.5 M in THF, 1.2 eq, 8.4 mmol) was slowly added and the mixture was stirred at 0°C for 60 min. The reaction mixture was quenched with saturated ammonium chloride and extracted twice with EtOAc. The combined organic extracts were dried _{over Na₂SO₄} , filtered, and concentrated to afford the title compound, which was sufficiently pure to be used directly.

Intermediate 141 Cyclopropyl(3,5-difluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methanol

The title compound was obtained by reacting 2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane, butyl lithium and cyclopropyl(3,5-difluorophenyl)methanol.

Intermediate 142 6-(4-(cyclopropyl(hydroxy)methyl)-2,6-difluorophenyl)-5-fluoropicolinic acid

Cyclopropyl(3,5-difluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methanol was reacted with methyl 6-bromo-5-fluoropicolinate (see US 2012/225062) to give the title compound.

Intermediate 143 3-(3,5-difluorophenyl)tetrahydrofuran-3-ol

To a solution of 1-bromo-3,5-difluoro-benzene (4.00g, 20.7mmol) in tetrahydrofuran (70mL) was added magnesium (6.0 equivalents, 124mmol) under nitrogen and the solution was heated at 85°C for 3 hours. The solution was cooled to room temperature and a solution of 3-oxotetrahydrofuran (1 equivalent, 20.726mmol) in THF (20mL) was added via a syringe. The mixture was stirred at room temperature for 3 days. The reaction mixture was quenched with saturated NaHCO ₃ , extracted with EtOAc and washed with brine. Purified by CombiFlash (0 to 100% EtOAc/heptane) to give the title compound (405mg, 9.7%).

Intermediate 144 3-(3,5-difluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)tetrahydrofuran-3-ol

Reaction of 3-(3,5-difluorophenyl)tetrahydrofuran-3-ol, butyl lithium and 2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane afforded the title compound.

Intermediate 145 2-(2,6-difluoro-4-(3-hydroxytetrahydrofuran-3-yl)phenyl)thiazole-4-carboxylic acid

Methyl 2-bromothiazole-4-carboxylate was reacted with 3-(3,5-difluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)tetrahydrofuran-3-ol to give the title compound.

Intermediate 146 2-(2,6-difluoro-4-(tetrahydrofuran-3-yl)phenyl)thiazole-4-carboxylic acid

To a solution of methyl 2-[2,6-difluoro-4-(3-hydroxytetrahydrofuran-3-yl)phenyl]thiazole-4-carboxylate (250 mg, 0.732 mmol) in dichloromethane (1 mL) was added trifluoroacetic acid (1 mL). The mixture was heated in a microwave at 120 ° C for 2 h. After vacuum concentration, purification by (0 to 100% EtOAC/heptane) gave methyl 2-(4-(2,5-dihydrofuran-3-yl)-2,6-difluorophenyl)thiazole-4-carboxylate (57 mg, 24% yield) as a mixture of olefin isomers.

The mixture was diluted with 30 mL of MeOH and passed through an H-cube hydrogenator (1 mL/min, 60 bar, 70° C.). After concentration, methyl 2-(2,6-difluoro-4-(tetrahydrofuran-3-yl)phenyl)thiazole-4-carboxylate (44 mg) was obtained. The ester was diluted with THF (3 mL) and water (1.5 mL), and LiOH (6.5 mg, 2.0 equiv) was added. After stirring at room temperature for 2.5 hours, the mixture was neutralized with 1N HCl (aq.), diluted with EtOAc, and washed with brine. The organic extract was dried (Na ₂ SO ₄ ) and concentrated in vacuo to yield the title compound (42 mg, quantitative).

Intermediate 147 Methyl 2-(2,6-difluoro-4-hydroxyphenyl)thiazole-4-carboxylate

To a suspension of methyl 2-bromothiazole-4-carboxylate (500 mg, 2.16 mmol), 2,6-difluoro-4-hydroxyphenylboronic acid (2 eq., 767 mg) and potassium fluoride (3.3 eq., 414 mg) in tetrahydrofuran (10 mL) and water (1 mL) was added bis(tri-tert-butylphosphine)palladium(0) (0.1 eq., 110 mg) and the mixture was heated to 120° C. in a microwave reactor for 15 min. After concentration in vacuo, the reaction mixture was purified by CombiFlash (0 to 100% EtOAc/heptane) to give 241 mg of the title compound as a 1:1 mixture with methyl 2-bromothiazole-4-carboxylate, which was used directly without further purification.

Intermediate 148 (R)-2-(2,6-difluoro-4-((tetrahydrofuran-3-yl)oxy)phenyl)thiazole-4-carboxylic acid

To a solution of methyl 2-(2,6-difluoro-4-hydroxy-phenyl)thiazole-4-carboxylate (207 mg, 0.763 mmol) and (R)-3-hydroxytetrahydrofuran (3 eq., 206 mg) in tetrahydrofuran (5 mL) were added triphenylphosphine (3 eq., 600 mg) and diisopropyl azodicarboxylate (3 eq., 0.45 mL). The mixture was stirred at room temperature for 2 days. The mixture was concentrated and partitioned between EtOAc and water. The organic layer was washed with saturated _NaHCO₃ , brine, dried _{over Na₂SO₄} , and concentrated. The residue was diluted with THF (3 mL) and water (1 mL), and LiOH (36 mg) was added. After stirring at room temperature for 2.5 hours, the reaction mixture was neutralized with 1N HCl (aq.), diluted with EtOAc, and washed with brine. The organic extracts were dried ( _Na2SO4 ) and concentrated in vacuo _to afford the title compound, contaminated with triphenylphosphine oxide and other by-products, which was used without further purification.

Intermediate 149 (S)-2-(2,6-difluoro-4-((tetrahydrofuran-3-yl)oxy)phenyl)thiazole-4-carboxylic acid

Following the procedure of Intermediate 147, substituting (S)-3-hydroxytetrahydrofuran for (R)-3-hydroxytetrahydrofuran, the title compound was obtained.

Intermediate 150 1-Methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-(trifluoromethyl)-1H-pyrazole

4-Bromo-1-methyl-5-(trifluoromethyl)pyrazole (520 mg, 2.27 mmol, commercially available), bis(pinacolato)diboron (1.3 equiv., 749 mg), bis(triphenylphosphine)palladium(II) dichloride (0.05 equiv., 79 mg), and potassium acetate (2 equiv., 4445 mg) were dissolved in toluene (15 mL) in a microwave reaction vial. The mixture was heated to 150° C. in a microwave reactor for 10 min. After cooling to room temperature, the mixture was filtered through celite (EtOAc rinse). The filtrate was concentrated to give the title compound, which was sufficiently pure to be used directly.

Intermediate 151 5-Fluoro-1,3-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole

4-Bromo-5-fluoro-1,3-dimethyl-1H-pyrazole (commercially available), bis(pinacolato)diboron, bis(triphenylphosphine)palladium(II) dichloride and potassium acetate were reacted in toluene to give the title compound.

Intermediate 152 2-(2,6-difluoro-4-(3-fluorooxetan-3-yl)phenyl)thiazole-4-carboxylic acid

Methyl 2-bromothiazole-4-carboxylate was reacted with 3-(3,5-difluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)oxetane-3-ol (see US2012/225062) to provide the title compound after the addition of the following fluorination step and subsequent ester hydrolysis: A solution of methyl 2-(2,6-difluoro-4-(3-hydroxyoxetane-3-yl)phenyl)thiazole-4-carboxylate (50 mg) in dichloromethane (5 mL) was cooled to -78°C, and deoxo-fluor (1.5 equivalents, 50 wt% in toluene) was added. The mixture was slowly warmed to room temperature over 30 minutes. The reaction mixture was then quenched by the addition of saturated NaHCO ₃ (aq.), which was then diluted with EtOAc and washed with brine. The organic extracts were dried ( _Na2SO4 ) and concentrated in vacuo. Purification by CombiFlash (0 to 100% EtOAc/heptane) afforded methyl 2-(2,6-difluoro-4-(3-fluorooxetan-3-yl)phenyl)thiazole-4 _- carboxylate.

Intermediate 153 tert-Butyl ((2R*,3S*,4R*,6R*)-6-(4-amino-1-methyl-1H-pyrazol-5-yl)-3-hydroxy-2,3-dimethyltetrahydro-2H-pyran-4-yl)carbamate (racemic)

Prepared in a similar manner to tert-butyl ((2R*,3S*,4R*,6R*)-6-(3-aminopyridin-4-yl)-3-hydroxy-2,3-dimethyltetrahydro-2H-pyran-4-yl)carbamate (WO2012/004217), substituting 1-methyl-4-nitro-1H-pyrazole-5-carbaldehyde for 3-nitropyridine-4-carbaldehyde.

Table 1 Compounds of Formula I

Example 101 N-(5-((2S,5R,6S)-5-amino-6-fluorooxepan-2-yl)-1-methyl-1H-pyrazol-4-yl)-2-(2,6-difluoro-4-((S)-1-fluoroethyl)phenyl)thiazole-4-carboxamide 101

The compound was prepared according to the procedures and intermediates of this application. MS (M+H/1) = 498.18

Example 102 N-[5-[(2S,5R,6S)-5-amino-6-fluoro-oxepan-2-yl]-1-methyl-pyrazol-4-yl]-2-(2-fluoro-6-hydroxy-phenyl)thiazole-4-carboxamide 102

The compound was prepared according to the procedures and intermediates of this application. MS (M+H/1) = 450.1643

Example 103 N-[5-[(2R,5S,6R)-5-amino-6-fluoro-oxepan-2-yl]-1-cyclopropyl-pyrazol-4-yl]-2-(2,6-difluorophenyl)thiazole-4-carboxamide 103

The compound was prepared according to the procedures and intermediates of this application. MS (M+H/1) = 478.1722

Example 104 N-[5-[(2S,5R,6S)-5-amino-6-fluoro-oxepan-2-yl]-1-methyl-pyrazol-4-yl]-2-(2,3-dihydrobenzofuran-5-yl)thiazole-4-carboxamide 104

The compound was prepared according to the procedures and intermediates of this application. MS (M+H/1) = 458.1878

Example 105 N-[5-[(2S,5R,6S)-5-amino-6-fluoro-oxepan-2-yl]-1-methyl-pyrazol-4-yl]-2-(pyrazin-2-yl)-thiazole-4-carboxamide 105

The compound was prepared according to the procedures and intermediates of this application. MS (M+H/1) = 418.1565

Example 106 N-(5-((2S,5R,6S)-5-amino-6-fluorooxepan-2-yl)-1-methyl-1H-pyrazol-4-yl)-2-(2,6-difluoro-4-((R)-1-fluoroethyl)phenyl)thiazole-4-carboxamide 106

The compound was prepared according to the procedures and intermediates of this application. MS (M+H/1) = 498.18

Example 107 N-(5-((2R,5S,6R,7S)-5-amino-6-methoxy-7-methyloxepan-2-yl)-1-methyl-1H-pyrazol-4-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide 107

The compound was prepared according to the procedures and intermediates of this application. MS (M+H/1) = 478.1956

Example 108 N-[5-[(2S,5R,6S)-5-amino-6-fluoro-oxepan-2-yl]-1-methyl-pyrazol-4-yl]-2-(5-chloro-2-fluoro-4-methoxy-phenyl)thiazole-4-carboxamide 108

The compound was prepared according to the procedures and intermediates of this application. MS (M+H/1) = 498.1722

Example 109 N-[5-[(2S,5R,6S)-5-amino-6-fluoro-oxepan-2-yl]-1-methyl-pyrazol-4-yl]-2-(5-fluoro-1H-indazol-6-yl)thiazole-4-carboxamide 109

The compound was prepared according to the procedures and intermediates of this application. MS (M+H/1) = 474.1643

Example 110 N-[5-[(2S,5R,6S)-5-amino-6-fluoro-oxepan-2-yl]-1-methyl-pyrazol-4-yl]-2-(5-methylpyrazin-2-yl)thiazole-4-carboxamide 110

The compound was prepared according to the procedures and intermediates of this application. MS (M+H/1) = 432.1722

Example 111 N-(5-((2S,5R,6S)-5-amino-6-fluorooxepan-2-yl)-1-methyl-1H-pyrazol-4-yl)-2-((S)-6,8-difluoro-4-hydroxychroman-7-yl)thiazole-4-carboxamide 111

The compound was prepared according to the procedures and intermediates of this application. MS (M+H/1) = 524.1878

Example 112 N-(5-((2S,5R,6S)-5-amino-6-fluorooxepan-2-yl)-1-methyl-1H-pyrazol-4-yl)-2-((R)-6,8-difluoro-4-hydroxychroman-7-yl)thiazole-4-carboxamide 112

The compound was prepared according to the procedures and intermediates of this application. MS (M+H/1) = 524.1878

Example 113 N-[5-[(2S,5R,6S)-5-amino-6-fluoro-oxepan-2-yl]-1-methyl-pyrazol-4-yl]-2-(1,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)thiazole-4-carboxamide 113

The compound was prepared according to the procedures and intermediates of this application. MS (M+H/1) = 446.1878

Example 114 N-[5-[(2R,5S,6R)-5-amino-6-fluoro-oxepan-2-yl]-1-cyclopropyl-pyrazol-4-yl]-6-(2,6-difluorophenyl)-5-fluoro-pyridine-2-carboxamide 114

The compound was prepared according to the procedures and intermediates of this application. MS (M+H/1) = 490.18

Example 115 N-(5-((2S,5R,6S)-5-amino-6-fluorooxepan-2-yl)-1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide 115

The compound was prepared according to the procedures and intermediates of this application. MS (M+H/1) = 501.47

Example 116 N-(5-((2R,5S,6R)-5-amino-6-fluorooxepan-2-yl)-1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide 116

The compound was prepared according to the procedures and intermediates of this application. MS (M+H/1) = 501.47

Example 117 N-[5-[(2S,5R,6S,7S)-5-amino-6-methoxy-7-methyl-oxepan-2-yl]-1-methyl-pyrazol-4-yl]-2-(2,6-difluorophenyl)thiazole-4-carboxamide 117

Step 1: Preparation of 1-(2-methyl-4-nitro-pyrazol-3-yl)pent-4-en-1-ol and separation of the two enantiomers by supercritical fluid chromatography (SFC) on a chiral stationary phase using the following conditions:

Column: Chiralpak AD-H (5×25 cm; 5 μm)

Mobile phase: CO ₂ :MeOH (+0.2% Et ₂ NH)=55:45

Flow rate: 180g/min

Back pressure: 100 bar

Column temperature: 35°C

The two enantiomers eluted at 2.82 and 3.98 min, and the latter peak was later identified as the desired enantiomer, (1S)-1-(2-methyl-4-nitro-pyrazol-3-yl)pent-4-en-1-ol 117a, and used for all subsequent chemistries. To a solution of (1S)-1-(2-methyl-4-nitro-pyrazol-3-yl)pent-4-en-1-ol 117a (101.8 g, 481.9 mmol) in anhydrous tetrahydrofuran (109 mL) was added diethylzinc in hexanes (1 mol/L, 240.9 mL, 240.9 mmol, 1.10 equiv) dropwise at 0°C. The reaction mixture was allowed to warm to room temperature and stirred for 2 hours, during which time most of the evolving gas had subsided, but was still being produced. To the mixture was added 1-methylallyl acetate (25.0 g, 219.0 mmol), palladium (II) acetate (2.46 g, 10.95 mmol, 0.05 eq), 2-(di-tert-butylphosphino)biphenyl (4.95 g, 16.43 mmol, 0.075 eq), ammonium acetate (16.88 g, 219.0 mmol, 1.00 eq) and anhydrous tetrahydrofuran (219 mL) and stirred at room temperature for 3 days. The mixture was diluted with water and ethyl acetate, filtered through celite, extracted 5 times with EtOAc, dried over MgSO ₄ , filtered and evaporated. The residue was divided into 4 batches and purified by silica gel chromatography (330 g, 0-20% EtOAc/heptane, 26.6 min gradient, Rf of product in 4:1 heptane:EtOAc under UV was about 0.3, Rf of starting material in 4:1 heptane:EtOAc under UV was about 0.1) to give the desired product 1-methyl-5-[(1S)-1-(1-methylallyloxy)pent-4-enyl]-4-nitro-pyrazole as a mixture of two diastereomers 117b (19.17 g, 33% yield). ¹ H NMR (400 MHz, CDCl 3 ) δ 8.07-8.00 (m, 1H), 5.88-5.41 (m, 3H), 5.24-4.89 (m, 4H), 4.09-4.01 (m, 3H), 3.94-3.58 (m, 1H), 2.38-1.64 (m, 4H), 1.31-1.18 (m, 3H). LCMS: m/z = 266.2 (M+H). 89.46 g of starting material was also recovered.

Step 2: To a solution of 1-methyl-5-[(1S)-1-(1-methylallyloxy)pent-4-enyl]-4-nitro-pyrazole 117b (2.50 g, 9.42 mmol) in anhydrous dichloromethane (942 mL) was added (1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene)dichloro(o-isopropoxyphenylmethylene)ruthenium (2nd generation Grubbs catalyst, CAS Reg. No. 301224-40-8, 825 mg, 0.94 mmol, 0.10 equiv). The mixture was then heated at 42° C. and stirred for 3 days. The reaction mixture was concentrated and purified by chromatography on silica gel (80 g, 0-20% EtOAc/heptane, 25 min gradient, Rf of the product in 4:1 heptane:EtOAc was approximately 0.4 and 0.3 under UV or _KMnO staining). The sample was subjected to SFC purification (conditions shown below) to separate the diastereomers to afford 1-methyl-5-[(2S,7S)-7-methyl-2,3,4,7-tetrahydrooxan-2-yl]-4-nitro-pyrazole 117c (0.54 g, 24%) and 1-methyl-5-[(2S,7R)-7-methyl-2,3,4,7-tetrahydrooxan-2-yl]-4-nitro-pyrazole 117d (0.49 g, 22%). 1-Methyl-5-[(2S,7S)-7-methyl-2,3,4,7-tetrahydrooxin-2-yl]-4-nitro-pyrazole ^1H NMR (400 MHz, DMSO) δ 8.21 (s, 1H), 5.89-5.81 (m, 1H), 5.66-5.61 (m, 1H), 5.57 (dd, J = 9.6, 3.7 Hz, 1H), 4.45-4.36 (m, 1H), 4.01 (s, 3H), 2.56-2.44 (m, 1H), 2.37-2.24 (m, 1H), 2.07-1.97 (m, 1H), 1.96-1.86 (m, 1H), 1.26 (d, J = 6.7 Hz, 3H). LCMS: m/z = 238.12 (M+H). 1-Methyl-5-[(2S,7R)-7-methyl-2,3,4,7-tetrahydrooxin-2-yl]-4-nitro-pyrazole ^1H NMR (400 MHz, DMSO) δ 8.21 (s, 1H), 5.79-5.70 (m, 2H), 5.53-5.47 (m, 1H), 4.90-4.80 (m, 1H), 4.01 (s, 3H), 2.63-2.52 (m, 1H), 2.34-2.18 (m, 2H), 2.01-1.90 (m, 1H), 1.20 (d, J = 6.8 Hz, 3H). LCMS: m/z = 238.12 (M+H).

SFC conditions:

Column: Phenomenex Cellulose-1 (4.6×50mm; 3μm)

Mobile phase: CO ₂ :MeOH (+0.1% formic acid) = 95:5

Flow rate: 4 mL/min

Back pressure: 120 bar

Column temperature: 40°C

Retention time: 0.588 min for 1-methyl-5-[(2S,7S)-7-methyl-2,3,4,7-tetrahydrooxa-2-yl]-4-nitro-pyrazole; 0.678 min for 1-methyl-5-[(2S,7R)-7-methyl-2,3,4,7-tetrahydrooxa-2-yl]-4-nitro-pyrazole

Step 3: To a solution of 1-methyl-5-[(2S,7S)-7-methyl-2,3,4,7-tetrahydrooxin-2-yl]-4-nitro-pyrazole 117c (0.20 g, 0.84 mmol) in anhydrous dichloromethane (16 mL) was added m-chloroperbenzoic acid (727 mg, 4.215 mmol, 5.0 equiv). The sample was stirred at room temperature for 3 days. The solvent was evaporated and the residue was chromatographed on silica gel (40 g, 0-30% EtOAc/heptane, 28 min gradient, Rf of the product in 2:1 heptane:EtOAc under UV was approximately 0.3) to afford 1-methyl-5-[(1S,4S,6S,7R)-6-methyl-5,8-dioxabicyclo[5.1.0]octan-4-yl]-4-nitro-pyrazole 117e (213.5 mg, 66%). ^1H NMR(400MHz, CDCl3)δ8.00(s,1H),5.16(dd,J＝9.8,1.3Hz,1H),4.14-4.08(m,1H),4.05(s,3H),3.30-3.25(m,1H),2.9 8(d,J＝4.4Hz,1H),2.52-2.43(m,1H),2.32-2.22(m,1H),2.15-2.04(m,1H),1.82-1.75(m,1H),1.48(d,J＝6.6Hz,3H). LCMS: m/z=254.4(M+H).

Step 4: Combine 1-methyl-5-[(1S,4S,6S,7R)-6-methyl-5,8-dioxabicyclo[5.1.0]octan-4-yl]-4-nitro-pyrazole 117e (0.14 g, 0.56 mmol), methanol (5 mL), and water (1 mL). Add ammonium chloride (149 mg, 2.78 mmol, 5.0 equiv) followed by sodium azide (182.5 mg, 2.780 mmol, 5.0 equiv). Heat the mixture at 70°C overnight. LC-MS appears to indicate mostly product, but a small amount of starting material still appears to be present. Add ammonium chloride (149 mg, 2.78 mmol, 5.0 equiv) and sodium azide (182.5 mg, 2.780 mmol, 5.0 equiv) and heat the mixture at 70°C overnight. After cooling to room temperature, the reaction mixture was diluted with water and extracted three times with CH ₂ Cl _2. The combined organic phases were dried over MgSO ₄ , filtered, evaporated, and chromatographed on silica gel (4 g, 0-100% EtOAc/heptane, 11 min gradient, Rf -0.3 under UV in 1:1 heptane:EtOAc) to afford (2S,3S,4R,7S)-4-azido-2-methyl-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-3-ol 117 g (148 mg, 90%). ¹ H NMR(400MHz, CDCl3)δ8.02(s,1H),5.53(dd,J＝10.3,4.3Hz,1H),4.06(s,3H),3.99-3.92(m,1H),3.84-3.78(m,1H),3 .75-3.70(m,1H),2.31-2.23(m,1H),2.19-2.16(m,1H),2.16-2.10(m,1H),2.07-1.82(m,2H),1.29(d,J＝6.6Hz,3H). LCMS: m/z=297.3(M+H).

Following the same procedures as steps 3 and 4, 1-methyl-5-[(2S,7R)-7-methyl-2,3,4,7-tetrahydrooxin-2-yl]-4-nitro-pyrazole was converted to (2R,3S,4R,7S)-4-azido-2-methyl-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-3-ol 117h.

Step 5: To a solution of (2S,3S,4R,7S)-4-azido-2-methyl-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-3-ol 117 g (0.15 g, 0.50 mmol) in anhydrous tetrahydrofuran (4 mL) was added sodium hydride (60% in mineral oil, 60.0 mg, 1.50 mmol, 3.0 equiv) at 0°C. The mixture was stirred at 0°C for 1 hour, then iodomethane (0.15 mL, 2.50 mmol, 5.0 equiv) was added dropwise. The reaction mixture was slowly warmed to room temperature and stirred overnight. Water was added dropwise and the mixture was extracted three times with EtOAc, dried over _MgSO4 , filtered, evaporated and chromatographed on silica gel (4 g, 0-30% EtOAc/heptane, 22 min gradient, Rf of the product was about 0.3 under UV in 2:1 heptane:EtOAc) to afford 5-[(2S,5R,6S,7S)-5-azido-6-methoxy-7-methyl-oxepan-2-yl]-1-methyl-4-nitro-pyrazole 117i (121 mg, 78%). ¹ HNMR(400MHz, CDCl3)δ8.01(s,1H),5.54(dd,J＝10.0,4.5Hz,1H),4.06(s,3H),3.95-3.84(m,2H),3.55(s,3H) ,3.22(dd,J＝5.8,2.6Hz,1H),2.29-2.20(m,1H),2.15-2.07(m,1H),2.01-1.79(m,2H),1.29(d,J＝6.5Hz,3H). LCMS: m/z=311.1(M+H).

Step 6: Combine 5-[(2S,5R,6S,7S)-5-azido-6-methoxy-7-methyl-oxepan-2-yl]-1-methyl-4-nitro-pyrazole 117i (0.12 g, 0.39 mmol), triphenylphosphine (123 mg, 0.47 mmol, 1.20 equiv), tetrahydrofuran (5 mL), and water (1 mL), heat at 60°C, and stir for 4 days. The reaction mixture was diluted with _H₂O and EtOAc, extracted three times with EtOAc, dried over _MgSO₄ , filtered, and evaporated. The residue was redissolved in anhydrous dichloromethane (5 mL). N,N-diisopropylethylamine (0.14 mL, 0.78 mmol, 2.0 equiv) and di-tert-butyl dicarbonate (112 mg, 0.51 mmol, 1.30 equiv) were added and stirred for 1 hour. The reaction mixture was evaporated and purified by chromatography on silica gel (12 g, 0-40% EtOAc/heptane, 22 min gradient, Rf -0.2 under UV in 2:1 heptane:EtOAc) to afford tert-butyl N-[(2S,3S,4R,7S)-3-methoxy-2-methyl-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-yl]carbamate 117j (116 mg, 77%). ¹ HNMR(400MHz, CDCl3)δ8.01(s,1H),5.48(dd,J＝9.7,4.1Hz,1H),4.72(s,1H),4.08(s,3H),3.99-3.85(m,2H ), 3.53 (s, 3H), 3.29-3.24 (m, 1H), 2.20-2.01 (m, 2H), 2.00-1.82 (m, 2H), 1.47 (s, 9H), 1.29 (d, J = 6.5Hz, 3H). LCMS: m/z=385.3(M+H).

Step 7: tert-Butyl N-[(2S,3S,4R,7S)-3-methoxy-2-methyl-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-4-yl]carbamate 117j was reduced with 10% Pd/charcoal in MeOH and the intermediate amine, tert-butyl (2S,3S,4R,7S)-7-(4-amino-1-methyl-1H-pyrazol-5-yl)-3-methoxy-2-methyloxepan-4-ylcarbamate, was coupled with 2-(2,6-difluorophenyl)thiazole-4-carboxylic acid and _PyBOP in DIPEA and _CH2Cl2 . The resulting coupled intermediate was treated with 4M HCl/dioxane, MeOH to remove the Boc group to afford N-[5-[(2S,5R,6S,7S)-5-amino-6-methoxy-7-methyl-oxepan-2-yl]-1-methyl-pyrazol-4-yl]-2-(2,6-difluorophenyl)thiazole-4-carboxamide 117. ^1H NMR (400MHz, DMSO) δ10.08(s,1H),8.60(s,1H),7.93(s,1H),7.71-7.62(m,1H),7.38-7.30(m,2H),5.04-4.99(m,1H),4.05-3.98(m,1H),3.7 2(s,3H),3.07-3.02(m,1H),2.94(s,3H),2.84-2.80(m,1H),2.26-2.1 7(m,1H),1.76-1.67(m,1H),1.66-1.43(m,4H),1.15(d,J＝6.5Hz,3H). LCMS: m/z=478.2(M+H).

Example 118 N-(5-((2S,5R,6S,7R)-5-amino-6-methoxy-7-methyloxepan-2-yl)-1-methyl-1H-pyrazol-4-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide 118

Following the procedure of Example 11117, (2R,3S,4R,7S)-4-azido-2-methyl-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-3-ol was O-methylated to afford 5-((2S,5R,6S,7R)-5-azido-6-methoxy-7-methyloxepan-2-yl)-1-methyl-4-nitro-1H-pyrazole 118b, which was reduced and Boc-protected to afford tert-butyl (2R,3S,4R,7S)-3-methoxy-2-methyl-7-(1-methyl-4-nitro-1H-pyrazol-5-yl)oxepan-4-ylcarbamate 118c.

Following step 7 of example 117, intermediate 118c was converted to N-[5-[(2S,5R,6S,7R)-5-amino-6-methoxy-7-methyl-oxepan-2-yl]-1-methyl-pyrazol-4-yl]-2-(2,6-difluorophenyl)thiazole-4-carboxamide 118. ¹ H NMR (400MHz, DMSO) δ10.02(s,1H),8.63(s,1H),7.77(s,1H),7.71-7.63(m,1H),7.39-7.32(m,2H),5.00-4.94(m,1H),3.9 6(s,2H),3.85-3.75(m,4H),3.42(s,3H),3.02-2.88(m,2H),1.95-1.82(m,3H),1.64-1.51(m,1H),1.21(d,J＝6.5Hz,3H). LCMS: m/z=478.17(M+H).

Example 119 N-[5-[(2S,5R,6S)-5-amino-6-fluoro-oxepan-2-yl]-1-methyl-pyrazol-4-yl]-2-(5-methoxy-3-methylpyridin-2-yl)thiazole-4-carboxamide 119

The compound was prepared according to the procedures and intermediates of this application. MS (M+H/1) = 461.1956

Example 120 N-[5-[(2S,5R,6S)-5-amino-6-fluoro-oxepan-2-yl]-1-methyl-pyrazol-4-yl]-2-(quinolin-8-yl)thiazole-4-carboxamide 120

The compound was prepared according to the procedures and intermediates of this application. MS (M+H/1) = 467.18

Example 121 N-(5-((2S,5R,6R,7S)-5-amino-6-fluoro-7-methyloxepan-2-yl)-1-methyl-1H-pyrazol-4-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide 121

To a solution of (2S,3S,4R,7S)-4-azido-2-methyl-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-3-ol 117 g (0.085 g, 0.286 mmol) in anhydrous dichloromethane (6 mL) was added bis(2-methoxyethyl)aminosulfur trifluoride (Deoxo-Fluor, CAS Reg. No. 202289-38-1, 50% by mass in toluene) (0.37 mL, 0.858 mmol, 3.0 equiv). The reaction mixture was stirred at room temperature for 1 hour and then slowly quenched _with saturated _Na2CO3 . The mixture was extracted three times with _CH2Cl2 , _dried over _MgSO4 , filtered, evaporated and purified by chromatography on silica gel (4 g, 0-100% EtOAc/heptane, 22 min gradient, Rf of the product in 2:1 heptane:EtOAc under UV was approximately 0.3) to afford 5-((2S,5R,6R,7S)-5-azido-6-fluoro-7-methyloxepan-2-yl)-1-methyl-4-nitro-1H-pyrazole 121a (44.6 mg, 52%). ¹ HNMR (400MHz, CDCl3) δ8.01 (s, 1H), 5.73-5.68 (m, 1H), 4.59 (ddd, J = 48.6, 4.2, 1.6Hz, 1H), 4.14-3 .88(m,5H),2.48-2.33(m,1H),2.21-2.12(m,1H),2.02-1.83(m,2H),1.33(dd,J=6.6,1.2Hz,3H). LCMS: m/z=299.2(M+H).

Following the procedures in steps 6 and 7 of Example 117, 121a was converted to N-(5-((2S,5R,6R,7S)-5-amino-6-fluoro-7-methyloxepan-2-yl)-1-methyl-1H-pyrazol-4-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide 121. ¹ H NMR(400MHz,DMSO)δ10.03(s,1H),8.64(s,1H),7.92(s,1H),7.72-7.63(m, 1H),7.40-7.32(m,2H),5.06(dd,J＝8.7,3.8Hz,1H),4.44(ddd,J＝49.0,5.2 ,2.4Hz,1H),4.11-3.98(m,1H),3.72(s,3H),3.37-3.22(m,1H),2.21-2.11 (m,1H),1.92-1.80(m,1H),1.72-1.55(m,2H),1.15(dd,J=6.5,1.3Hz,3H). LCMS: m/z=466.2 (M+H).

Example 122 N-(5-((2S,5R,6S)-5-amino-6-fluorooxepan-2-yl)-1-methyl-1H-pyrazol-4-yl)-2-(imidazo[1,2-a]pyrazin-6-yl)thiazole-4-carboxamide 122

The compound was prepared according to the procedures and intermediates of this application. MS (M+H/1) = 457.1643

Example 123 N-(5-((2R,5S,6R)-5-amino-6-fluorooxepan-2-yl)-1-methyl-1H-pyrazol-4-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide 123

The compound was prepared according to the procedures and intermediates of this application. MS (M+H/1) = 452.1565

Example 124 N-[5-[(2S,5R,6S,7S)-5-amino-6-fluoro-7-methyl-oxepan-2-yl]-1-methyl-pyrazol-4-yl]-2-(2,6-difluorophenyl)thiazole-4-carboxamide 124

To a solution of (2S,3S,4R,7S)-4-azido-2-methyl-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-3-ol 117 g (0.59 g, 1.98 mmol) in anhydrous dichloromethane (12 mL) were added sodium bicarbonate (831 mg, 9.89 mmol, 5.0 equiv) followed by Dess-Martin periodinane (1.27 g, 2.966 mmol, 1.5 equiv). The mixture was stirred at room temperature for 1 hour, then diluted with water, extracted three times with dichloromethane, dried over _MgSO4 , filtered, evaporated and chromatographed on silica gel (40 g, 0-40% EtOAc/heptane, 28 min gradient, Rf of the product was about 0.5 under UV in 1:1 heptane:EtOAc) to give (2S,4R,7S)-4-azido-2-methyl-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-3-one 124a (525 mg, 90%). ^1H NMR (400MHz, CDCl3) δ8.04(s,1H),5.40-5.36(m,1H),4.52(dd,J＝11.9,2.7Hz,1H),4.22(q,J＝7.0H z,1H),4.08(s,3H),2.31-2.24(m,1H),2.23-2.12(m,2H),2.11-2.00(m,1H),1.44(d,J=7.0Hz,3H). LCMS: m/z=295.5(M+H).

To a solution of (2S,4R,7S)-4-azido-2-methyl-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-3-one 124a (525 mg, 1.78 mmol) in anhydrous tetrahydrofuran (16 mL) was added lithium tri-sec-butylborohydride (1.0 M in THF, 2.1 mL, 2.1 mmol, 1.2 equiv) dropwise at -78°C. The resulting solution was stirred at -78°C for 1 hour and quenched with _H2O . The mixture was extracted three times with EtOAc, dried over _MgSO4 , filtered, evaporated, and purified by chromatography on silica gel (4 g, 0-50% EtOAc/heptane, 22 min gradient). (2S,3R,4R,7S)-4-azido-2-methyl-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-3-ol 124b (Rf -0.5 under UV in 1:1 heptane:EtOAc) was obtained (294 mg, 55%). ¹ H NMR (400MHz, CDCl3) δ8.01 (s, 1H), 5.68 (dd, J = 11.0, 3.5Hz, 1H), 4.04-3.97 (m, 4H), 3.78-3.73 (m, 1H), 3.73-3.6 8(m,1H),2.46-2.34(m,1H),2.23-2.15(m,1H),2.13(d,J＝4.9Hz,1H),1.97-1.80(m,2H),1.32(d,J＝6.3Hz,3H). LCMS: m/z=297.3(M+H). Another isomer, (2S,3S,4R,7S)-4-azido-2-methyl-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-3-ol 117 g (Rf about 0.3 under UV in 1:1 heptane:EtOAc) was also obtained (178 mg, 34%). ^1H NMR (400MHz, CDCl3) δ8.01 (s, 1H), 5.68 (dd, J = 11.0, 3.6Hz, 1H), 4.04-3.97 (m, 4H), 3.78-3.73 (m, 1H), 3.72-3.6 8(m,1H),2.46-2.35(m,1H),2.23-2.15(m,1H),2.10(d,J＝4.9Hz,1H),1.97-1.80(m,2H),1.32(d,J＝6.3Hz,3H).

To a solution of (2S,3R,4R,7S)-4-azido-2-methyl-7-(2-methyl-4-nitro-pyrazol-3-yl)oxepan-3-ol 124b (294 mg, 0.991 mmol) in anhydrous dichloromethane (20 mL) was added bis(2-methoxyethyl)aminosulfur trifluoride (50% by mass in toluene, 1.29 mL, 2.97 mmol, _3.0 equiv). The mixture was stirred at room temperature for 1 hour and then carefully quenched with saturated _Na2CO3 . The resulting solution was extracted three times with _CH2Cl2 , _dried over _MgSO4 , filtered, evaporated and purified by chromatography on silica gel (4 g, 0-20% EtOAc/heptane, 33 min gradient, Rf of the product in 2:1 heptane:EtOAc under UV was about 0.3) to afford 5-[(2S,5R,6S,7S)-5-azido-6-fluoro-7-methyl-oxepan-2-yl]-1-methyl-4-nitro-pyrazole 124c (26.5 mg, 9%). ^1H NMR (400 MHz, CDCl3) δ 8.02 (s, 1H), 5.53 (dd, J = 10.7, 3.9 Hz, 1H), 4.51 (ddd, J = 47.7, 6.3, 2.6 Hz, 1H), 4.06 (s, 3H), 4.05-3.87 (m, 2H), 2.31-2.22 (m, 1H), 2.14-1.83 (m, 3H), 1.34 (dd, J = 6.7, 2.4 Hz, 3H). LCMS: m/z = 299.4 (M+H). Other isomers and rearranged products were also observed.

Following the procedures of steps 6 and 7 of Example 117, 5-((2S,5R,6S,7S)-5-azido-6-fluoro-7-methyloxepan-2-yl)-1-methyl-4-nitro-1H-pyrazole 124c was reduced and Boc-protected to afford (2S,3S,4R,7S)-3-fluoro-2-methyl-7-(1-methyl-4-nitro-1H-pyrazol-5-yl)oxepan-2-yl)-1-methyl-4-nitro-1H-pyrazole. tert-Butyl heterocycloheptan-4-ylcarbamate 124d was reduced with 10% Pd/charcoal in MeOH and the intermediate amine, tert-butyl (2S,3S,4R,7S)-7-(4-amino-1-methyl-1H-pyrazol-5-yl)-3-fluoro-2-methyloxepan-4-ylcarbamate, was coupled with 2-(2,6-difluorophenyl)thiazole-4-carboxylic acid and PyBOP in DIPEA _{and CH2Cl2} . The resulting coupling intermediate, tert-butyl (2S,3S,4R,7S)-7-(4-(2-(2,6-difluorophenyl)thiazole-4-carboxamido)-1-methyl-1H-pyrazol-5-yl)-3-fluoro-2-methyloxepan-4-ylcarbamate, was treated with 4M HCl/dioxane in MeOH to remove the Boc group to afford N-[5-[(2S,5R,6S,7S)-5-amino-6-fluoro-7-methyl-oxepan-2-yl]-1-methyl-pyrazol-4-yl]-2-(2,6-difluorophenyl)thiazole-4-carboxamide 124. ¹ H NMR (400MHz, DMSO) δ9.96(s,1H),8.63(s,1H),7.86(s,1H),7.69-7.61(m,1H),7.36-7.29(m,2H),4.95(dd,J＝9.1,4.2Hz, 1H), 4.24-3.97 (m, 2H), 3.75 (s, 3H), 3.20-3.09 (m, 1H), 2.16-2.05 (m, 1H), 1.77-1.58 (m, 5H), 1.15 (dd, J = 6.6, 2.3Hz, 3H). LCMS: m/z=466.2(M+H).

Example 125 N-(5-((2S,5R,6S)-5-amino-6-fluorooxepan-2-yl)-1-methyl-1H-pyrazol-4-yl)-2-(imidazo[1,2-a]pyrazin-2-yl)thiazole-4-carboxamide 125

The compound was prepared according to the procedures and intermediates of this application. MS (M+H/1) = 457.1643

Example 126 N-(5-((2R,5S,6R)-5-amino-6-fluorooxepan-2-yl)-1H-pyrazol-4-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide 126

The compound was prepared according to the procedures and intermediates of this application. MS (M+H/1) = 438.1409

Example 127 N-(5-((2R,5S,6R)-5-amino-6-fluorooxepan-2-yl)-1H-pyrazol-4-yl)-2-(3-methylpyridin-2-yl)thiazole-4-carboxamide 127

The compound was prepared according to the procedures and intermediates of this application. MS (M+H/1) = 417.1643

Example 128 N-(5-((2S,5R,6S)-5-amino-6-fluorooxepan-2-yl)-1H-pyrazol-4-yl)-2-(3-methylpyridin-2-yl)thiazole-4-carboxamide 128

The compound was prepared according to the procedures and intermediates of this application. MS (M+H/1) = 417.1643

Example 129 N-(5-((2S,5R,6S)-5-amino-6-fluorooxepan-2-yl)-1H-pyrazol-4-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide 129

The compound was prepared according to the procedures and intermediates of this application. MS (M+H/1) = 438.1409

Example 901 Pim kinase binding activity

PIM-1, -2, and -3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J.F., and He, Q.Y. (2005) Expert Rev. Proteomics, 2:649-657). Fluorescently labeled Pim-specific peptide substrates were custom synthesized by American Peptide Company (Sunnyvale, CA). The reaction buffer contained 10 mM HEPES (pH 7.2), 10 mM MgCl ₂ , 0.01% Tween 20, and 2 mM DTT. The stop buffer contained 190 mM HEPES (pH 7.2), 0.015% Brij-35, 0.2% coating reagent 3 (Caliper Life Sciences, Hopkinton, MA), and 20 mM EDTA. The separation buffer contained 100 mM HEPES (pH 7.2), 0.015% Brij-35, 0.1% coating reagent 3, 1:200 coating reagent 8 (Caliper Life Sciences, Hopkinton, MA), 10 mM EDTA, and 5% DMSO.

PIM reactions were performed in 384-well plates with a final volume of 10 μL per well. Standard enzyme reactions were initiated by adding 5 μL of 2× ATP and test compound to 5 μL of 2× enzyme and FAM-peptide in reaction buffer containing 20 pM PIM1, 50 pM PIM2, or 55 pM PIM3, 1 μM FAM-peptide, and 10 μM ATP. After incubation at room temperature for 90 minutes, the phosphorylation reaction was stopped by adding 10 μL of stop buffer. The product and substrate in each individual reaction were separated using a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, MA) running on a Caliper (Caliper Life Sciences, Hopkinton, MA). The separation of product and substrate was optimized by selecting voltage and pressure using Caliper's Optimizer software (Hopkinton, MA). Separation conditions used a downstream voltage of -500 V, an upstream voltage of -2150 V, and a screening pressure of -1.2 psi. Product and substrate fluorophores were excited at 488 nm and detected at 530 nm. Substrate conversion was calculated from the electropherogram using HTS Well Analyzer software (Caliper Life Sciences, Hopkinton, MA). Ki values for the test compounds were calculated. Representative PIM1LC3K Ki values (in micromolar concentrations) for exemplary compounds are shown in Table 1.

Example 902 In vitro cell proliferation potency assay

The BaF3 parental cell line was obtained from the DSMZ depository. BaF3 cell lines transfected with PIM1 or PIM2 were generated. Mouse IL-3 was purchased from R&D Systems. G418 was purchased from Clontech. The culture medium for the BaF3 parental cell line contained RPMI, 10% FBS, 2 mM L-glutamine, and 2 ng/mL mIL-3. The culture medium for the BaF3PIM1 and BaF3PIM2 cell lines contained RPMI, 10% FBS, 2 mM L-glutamine, and 250 μg/mL mIL-3. The culture medium for the MM1.S (multiple myeloma) cell line contained RPMI, 10% FBS, and 2 mM L-glutamine.

BaF3 (murine interleukin-3-dependent pre-B cell line) parental cells, BaF3PIM1 cells, BaF3PIM2 cells, and MM1.S (multiple myeloma) cells were seeded in 384-well plates at 2k/well, 5k/well, 5k/well, and 10k/well, respectively, using 45 μL/well. Test compounds were added at 5 μL/well. BaF3 cells (parental and transfected) were incubated overnight, while MM1.S cells were incubated at 37°C, 5% _CO₂ for 72 hours. CELL TITER reagent (Promega) was added at 50 μL/well, the plates were incubated for 30 minutes, and luminescence values were read on an HT Analyst. _IC₅₀ / _EC₅₀ values of the test compounds were calculated.

Representative compounds of the present application were tested as described above and found to exhibit Ki/ _IC50 / _EC50 [in μM (micromolar)] as shown in Table 2 below.

Table 2

Example 903 hERG assay

hERG assay (2 points) was performed as follows:

The in vitro potency of selected compounds of the present invention for inhibiting hERG (human ether-à-go-go related gene) potassium channel currents was evaluated according to standard laboratory procedures (ChanTest, Cleveland, OH). Briefly, after addition of the test compound, hERG-expressing HEK-293 cells (n = 2/concentration) were evaluated at 1 and 10 mM for 5 minutes in an automated PatchXpress 7000A system (Molecular Devices, Sunnyvale, CA). Data from the hERG assay (2 points) are expressed as a percentage of the maximum current.

The hERG assay ( _IC50 ) was performed as follows:

The in vitro efficacy of inhibiting hERG potassium channel current was evaluated according to the standard operation of the research unit (ChanTest, Cleveland, OH). In brief, after adding the test substance, hERG inhibition (% max) was determined in the HEK-293 cells (n=2/concentration) expressing hERG using an automated PatchXpress 7000A system (Molecular Devices, Sunnyvale, CA). Based on hERG inhibition calculation _IC50 values when the test substance concentration was 0.01, 0.1, 1, 10, 30 and 100 μM. The hERG IC ₅₀ and IC ₂₀ values of some compounds of the present application were measured and compared with the compound 5-amino-N-(5-((4R,5R)-4-amino-5-fluoroazepan-1-yl)-1-methyl-1H-pyrazol-4-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide [No. 139 in a series of PIM inhibitors wherein the corresponding R ² is a heterocyclic group linked via nitrogen or a carbocyclic group linked via carbon (US2013/0079321)]. The hERG data suggest that the compounds of the present application may reduce the likelihood of susceptibility to QTc prolongation. Excessively prolonged QTc intervals can lead to severe ventricular arrhythmias and sudden death (De Bruin, ML et al. (2005) European Heart Journal, 26: 590-597; Redfern, WS et al. (2003) Cardiovascular Research, 58: 32-45).

The words “comprises” and “comprising” when used in this specification and the appended claims are intended to specify the presence of stated features, integers, components or steps, but they do not exclude or preclude the presence or addition of one or more other features, integers, components, steps or combinations thereof.

Although the above invention has been described in detail to a certain extent by way of illustration and embodiment for the purpose of understanding clarity, said description and embodiment should not be construed as limiting the scope of this application. The entire disclosures of all patents and scientific literature cited in this application are expressly incorporated herein by reference.

Claims (11)

1. A compound selected from: N-(5-((2S,5R,6S)-5-amino-6-fluorooxetane-2-yl)-1-methyl-1H-pyrazol-4-yl)-2-(2,6-difluoro-4-((S)-1-fluoroethyl)phenyl)thiazole-4-carboxamide; N-[5-[(2S,5R,6S)-5-amino-6-fluoro-oxetane-2-yl]-1-methyl-pyrazol-4-yl]-2-(2-fluoro-6-hydroxy-phenyl)thiazol-4-carboxamide; N-[5-[(2R,5S,6R)-5-amino-6-fluoro-oxetane-2-yl]-1-cyclopropyl-pyrazol-4-yl]-2-(2,6-difluorophenyl)thiazol-4-carboxamide; N-[5-[(2S,5R,6S)-5-amino-6-fluoro-oxetane-2-yl]-1-methyl-pyrazol-4-yl]-2-(2,3-dihydrobenzofuran-5-yl)thiazolyl-4-carboxamide; N-[5-[(2S,5R,6S)-5-amino-6-fluoro-oxetane-2-yl]-1-methyl-pyrazol-4-yl]-2-(pyrazin-2-yl)-thiazolyl-4-carboxamide; N-(5-((2S,5R,6S)-5-amino-6-fluorooxetane-2-yl)-1-methyl-1H-pyrazol-4-yl)-2-(2,6-difluoro-4-((R)-1-fluoroethyl)phenyl)thiazole-4-carboxamide; N-(5-((2R,5S,6R,7S)-5-amino-6-methoxy-7-methyloxetane-2-yl)-1-methyl-1H-pyrazol-4-yl)-2-(2,6-difluorophenyl)thiazol-4-carboxamide; N-[5-[(2S,5R,6S)-5-amino-6-fluoro-oxetane-2-yl]-1-methyl-pyrazol-4-yl]-2-(5-chloro-2-fluoro-4-methoxy-phenyl)thiazol-4-carboxamide; N-[5-[(2S,5R,6S)-5-amino-6-fluoro-oxetane-2-yl]-1-methyl-pyrazol-4-yl]-2-(5-fluoro-1H-indazol-6-yl)thiazolyl-4-carboxamide; N-[5-[(2S,5R,6S)-5-amino-6-fluoro-oxetane-2-yl]-1-methyl-pyrazol-4-yl]-2-(5-methylpyrazin-2-yl)thiazolyl-4-carboxamide; N-(5-((2S,5R,6S)-5-amino-6-fluorooxetane-2-yl)-1-methyl-1H-pyrazol-4-yl)-2-((S)-6,8-difluoro-4-hydroxy-7-yl)thiazolyl-4-carboxamide; N-(5-((2S,5R,6S)-5-amino-6-fluorooxetane-2-yl)-1-methyl-1H-pyrazol-4-yl)-2-((R)-6,8-difluoro-4-hydroxychroman-7-yl)thiazolyl-4-carboxamide; N-[5-[(2S,5R,6S)-5-amino-6-fluoro-oxetane-2-yl]-1-methyl-pyrazol-4-yl]-2-(1,4,5,6-tetrahydrocyclopentano[c]pyrazol-3-yl)thiazole-4-carboxamide; N-[5-[(2R,5S,6R)-5-amino-6-fluoro-oxetane-2-yl]-1-cyclopropyl-pyrazol-4-yl]-6-(2,6-difluorophenyl)-5-fluoro-pyridine-2-carboxamide; N-(5-((2S,5R,6S)-5-amino-6-fluorooxetane-2-yl)-1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide; N-(5-((2R,5S,6R)-5-amino-6-fluorooxetane-2-yl)-1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide; N-[5-[(2S,5R,6S,7S)-5-amino-6-methoxy-7-methyl-oxetane-2-yl]-1-methyl-pyrazol-4-yl]-2-(2,6-difluorophenyl)thiazol-4-carboxamide; N-(5-((2S,5R,6S,7R)-5-amino-6-methoxy-7-methyloxetane-2-yl)-1-methyl-1H-pyrazol-4-yl)-2-(2,6-difluorophenyl)thiazol-4-carboxamide; N-[5-[(2S,5R,6S)-5-amino-6-fluoro-oxetane-2-yl]-1-methyl-pyrazol-4-yl]-2-(5-methoxy-3-methylpyridin-2-yl)thiazolyl-4-carboxamide; N-[5-[(2S,5R,6S)-5-amino-6-fluoro-oxetane-2-yl]-1-methyl-pyrazol-4-yl]-2-(quinolin-8-yl)thiazolyl-4-carboxamide; N-(5-((2S,5R,6R,7S)-5-amino-6-fluoro-7-methyloxetane-2-yl)-1-methyl-1H-pyrazol-4-yl)-2-(2,6-difluorophenyl)thiazol-4-carboxamide; N-(5-((2S,5R,6S)-5-amino-6-fluorooxetane-2-yl)-1-methyl-1H-pyrazol-4-yl)-2-(imidazo[1,2-a]pyrazin-6-yl)thiazolyl-4-carboxamide; N-(5-((2R,5S,6R)-5-amino-6-fluorooxetane-2-yl)-1-methyl-1H-pyrazol-4-yl)-2-(2,6-difluorophenyl)thiazol-4-carboxamide; N-[5-[(2S,5R,6S,7S)-5-amino-6-fluoro-7-methyl-oxetane-2-yl]-1-methyl-pyrazol-4-yl]-2-(2,6-difluorophenyl)thiazol-4-carboxamide; N-(5-((2S,5R,6S)-5-amino-6-fluorooxetane-2-yl)-1-methyl-1H-pyrazol-4-yl)-2-(imidazo[1,2-a]pyrazin-2-yl)thiazolyl-4-carboxamide; N-(5-((2R,5S,6R)-5-amino-6-fluorooxetane-2-yl)-1H-pyrazol-4-yl)-2-(2,6-difluorophenyl)thiazol-4-carboxamide; N-(5-((2R,5S,6R)-5-amino-6-fluorooxetane-2-yl)-1H-pyrazol-4-yl)-2-(3-methylpyridin-2-yl)thiazolyl-4-carboxamide; N-(5-((2S,5R,6S)-5-amino-6-fluorooxetane-2-yl)-1H-pyrazol-4-yl)-2-(3-methylpyridin-2-yl)thiazolyl-4-carboxamide; or N-(5-((2S,5R,6S)-5-amino-6-fluorooxetane-2-yl)-1H-pyrazol-4-yl)-2-(2,6-difluorophenyl)thiazole-4-carboxamide. 2. A pharmaceutical composition comprising the compound of claim 1 and a pharmaceutical carrier, flow aid, diluent, or excipient. 3. The pharmaceutical composition of claim 2, further comprising a chemotherapeutic agent. 4. The pharmaceutical composition of claim 2, for treating diseases or conditions selected from those mediated by Pim kinase, including: cancer, immune disorders, cardiovascular diseases, viral infections, inflammation, metabolic/endocrine disorders, and neurological disorders. 5. A kit for treating symptoms mediated by Pim kinase, comprising: a) the pharmaceutical composition of claim 2; and b) Instructions for use. 6. Use of the compound of claim 1 in the preparation of a medicament for treating diseases or conditions mediated by Pim kinase selected from: cancer, immune disorders, cardiovascular diseases, viral infections, inflammation, metabolic/endocrine disorders, and neurological disorders. 7. The use of claim 6, wherein the disease or condition is selected from the following cancers: multiple myeloma, breast cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, genitourinary tract cancer, esophageal cancer, laryngeal cancer, glioblastoma, neuroblastoma, gastric cancer, skin cancer, keratoacanthoma, lung cancer, epidermoid carcinoma, large cell carcinoma, small cell carcinoma, bone cancer, adenoma, adenocarcinoma, thyroid cancer, follicular carcinoma, undifferentiated carcinoma, papillary carcinoma, seminoma, melanoma, sarcoma, bladder cancer, liver cancer and biliary tract cancer, kidney cancer, myeloid diseases, lymphoma, pilocarcinoma, oral cancer, nasopharyngeal carcinoma, pharyngeal cancer, lip cancer, tongue cancer, small intestine cancer, large intestine cancer, central nervous system cancer, Hodgkin's cancer, leukemia, bronchial cancer, glioma, endometrial cancer, renal pelvis cancer, uterine corpus cancer, non-Hodgkin's lymphoma, and villous colonic adenoma. 8. The use of claim 6, wherein the disease or condition is selected from cancers including: gastric cancer, pancreatic cancer, bladder cancer, and oral and pharyngeal cancer. 9. The use of claim 6, wherein the disease or condition is selected from cancers including cervical cancer, liver and intrahepatic bile duct cancer, hepatocellular carcinoma and oral cancer. 10. The use of claim 6, wherein the disease or condition is selected from the following cancers: non-small cell lung cancer (NSCLC), pancreatic cancer, lung adenocarcinoma, colorectal cancer, colon cancer, rectal cancer, brain cancer, acute myeloid leukemia, chronic myeloid leukemia, lymphocytic leukemia, and myeloid leukemia. 11. The compound of claim 1, for treating diseases or conditions selected from those mediated by Pim kinase, including: cancer, immune disorders, cardiovascular diseases, viral infections, inflammation, metabolic/endocrine disorders, and neurological disorders.