Tetrazole-substituted pyrazolopyrimidine inhibitors of JAK kinases and uses thereof

Abstract

Compounds of formula (I) useful as JAK kinase inhibitors TIFF2022536805000144.tif65170 (in the formula, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 (wherein R is as defined herein), and salts thereof. Also provided are pharmaceutical compositions comprising such JAK inhibitors and a pharmaceutically acceptable carrier, adjuvant, or vehicle, as well as methods for treating or lessening the severity of a disease or condition in a patient that responds to the inhibition of Janus kinase activity.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to International Application No. PCT / CN2019 / 091709, filed June 18, 2019, and U.S. Provisional Application No. 63 / 036,046, filed June 8, 2020, the disclosures of which are incorporated herein by reference.

[0002] The present invention relates to compounds that are inhibitors of Janus kinases, such as JAK1 and JAK2, as well as compositions containing these compounds and methods of use, including but not limited to, the diagnosis or treatment of patients suffering from conditions that respond to the inhibition of JAK kinases. [Background technology]

[0003] Cytokine pathways mediate a wide range of biological functions, including many aspects of inflammation and immunity. Janus kinases (JAKs), including JAK1, JAK2, JAK3, and TYK2, are cytoplasmic protein kinases that associate with type I and type II cytokine receptors and regulate cytokine signaling. Cytokine engagement with its cognate receptor triggers activation of receptor-associated JAKs, which leads to JAK-mediated tyrosine phosphorylation of signal transducer and activator of transcription (STAT) proteins and ultimately transcriptional activation of a specific set of genes (Schindler et al., 2007, J. Biol. Chem. 282:20059-63). JAK1, JAK2, and TYK2 exhibit widespread gene expression patterns, whereas JAK3 expression is restricted to leukocytes. Cytokine receptors typically function as heterodimers, such that two or more JAK kinases are usually associated with a cytokine receptor complex. The specific JAKs that associate with different cytokine receptor complexes are often determined by genetic studies and supported by other experimental evidence. Exemplary therapeutic benefits of inhibiting JAK enzymes are discussed, for example, in WO 2013 / 014567.

[0004] JAK1 was first identified in a screen for novel kinases (Wilks AF, 1989, Proc. Natl. Acad. Sci. USA 86:1603-1607). Genetic and biochemical studies have shown that JAK1 is functionally and physically associated with type I interferons (e.g., IFN-alpha), type II interferons (e.g., IFN-gamma), and IL-2 and IL-6 cytokine receptor complexes (Kisseleva et al., 2002, gene 285:1-24; Levy et al., 2005, Nat. Rev. Mol. Cell Biol. 3:651-662; O'Shea et al., 2002, Cell, 109(suppl.):S121-S131). JAK1 knockout mice die perinatally due to defects in LIF receptor signaling (Kisseleva et al., 2002, gene 285:1-24; O'Shea et al., 2002, Cell, 109(suppl.):S121-S131). Characterization of tissues derived from JAK1 knockout mice demonstrated a critical role for this kinase in the IFN, IL-10, IL-2 / IL-4 and IL-6 pathways. A humanized monoclonal antibody (tocilizumab) targeting the IL-6 pathway has been approved by the European Commission for the treatment of moderate to severe rheumatoid arthritis (Scheinecker et al., 2009, Nat. Rev. Drug Discov. 8:273-274).

[0005] CD4 T cells play a key role in the pathogenesis of asthma through the production of TH2 cytokines in the lungs, including IL-4, IL-9, and IL-13 (Cohn et al., 2004, Annu. Rev. Immunol. 22:789-815). IL-4 and IL-13 induce increased mucus production, recruitment of eosinophils to the lungs, and increased production of IgE (Kasaian et al., 2008, Biochem. Pharmacol. 76(2):147-155). IL-9 leads to mast cell activation, which exacerbates asthma symptoms (Kearley et al., 2011, Am. J. Resp. Crit. Care Med., 183(7):865-875). The IL-4Rα chain activates JAK1, which binds either IL-4 or IL-13 when bound to the common gamma chain or the IL-13Rα1 chain, respectively (Pernis et al., 2002, J. Clin. Invest. 109(10):1279-1283). The common gamma chain can also bind IL-9 in combination with IL-9Rα, which also activates JAK1 (Demoulin et al., 1996, Mol. Cell Biol. 16(9):4710-4716). The common gamma chain activates JAK3, but JAK1 predominates over JAK3, and inhibition of JAK1 has been shown to be sufficient to inactivate signaling through the common gamma chain despite JAK3 activity (Haan et al., 2011, Chem. Biol. 18(3):314-323). Inhibition of IL-4, IL-13 and IL-9 signaling by blocking the JAK / STAT signaling pathway can alleviate asthma symptoms in preclinical models of pulmonary inflammation (Mathew et al., 2001, J. Exp. Med. 193(9):1087-1096; Kudlacz et.al., 2008, Eur. J. Pharmacol. 582(1-3):154-161).

[0006] Biochemical and genetic studies have shown the association of JAK2 with the single chain (e.g., EPO), IL-3 and interferon gamma cytokine receptor families (Kisseleva et al., 2002, gene 285:1-24; Levy et al., 2005, Nat. Rev. Mol. Cell Biol. 3:651-662; O'Shea et al., 2002, Cell, 109(suppl.):S121-S131). Consistent with this, JAK2 knockout mice die of anemia (O'Shea et al., 2002, Cell, 109(suppl.):S121-S131). Kinase-activating mutations in JAK2 (e.g., JAK2 V617F) have been linked to myeloproliferative disorders in humans. In addition, JAK2 associates with receptors for cytokines such as IL-5 and thymic stromal lymphopoietin (TSLP). IL-5 is a key cytokine responsible for eosinophil differentiation, growth, activation, survival, and recruitment to the airways (Pelaia et al., 2019, Front. Physiol., 10:1514; Stirling et al., 2001, Am. J. Respir. Crit. Care Med., 164:1403-9; Fulkerson and Rothenberg, 2013, Nat. Rev. Drug Discov., 12:117-9.; Varricchi and Canonica, 2016, Expert. Rev. Clin. Immunol., 12:903-5). Three monoclonal antibody drugs targeting either IL-5 (mepolizumab, reslizumab) or the alpha chain of its receptor (benralizumab) have been approved for the treatment of asthma with an eosinophilic phenotype. TSLP is an epithelial cell-derived cytokine that plays a key role in regulating type II immunity and acts as an alarmin upstream of TH2 cytokine production (Kitajima et al., 2011, Eur J Immunol., 41:1862-71). Tezepelumab is an antagonist antibody against TSLP. Phase 2 trial results show that it successfully reduced asthma exacerbations in both patients with and without type 2 high signature (Corren et al., 2017, 377:936-46).

[0007] JAK3 associates exclusively with the gamma common cytokine receptor chain present in the IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21 cytokine receptor complexes. JAK3 is important for lymphoid cell development and proliferation, and mutations in JAK3 lead to severe combined immunodeficiency (SCID) (O'Shea et al., 2002, Cell, 109(suppl.):S121-S131). Based on its role in regulating lymphocytes, JAK3 and JAK3-mediated pathways have been targeted for immunosuppressive indications (e.g., transplant rejection and rheumatoid arthritis) (Baslund et al., 2005, Arthritis & Rheumatism 52:2686-2692; Changelian et al., 2003, Science 302:875-878).

[0008] TYK2 associates with type I interferon (e.g., IFN alpha), IL-6, IL-10, IL-12 and IL-23 cytokine receptor complexes (Kisseleva et al., 2002, gene 285:1-24; Watford, WT & O'Shea, JJ, 2006, Immunity 25:695-697). Consistent with this, primary cells derived from TYK2-deficient humans are defective in type I interferon, IL-6, IL-10, IL-12 and IL-23 signaling. A fully human monoclonal antibody (ustekinumab) targeting the shared p40 subunit of the IL-12 and IL-23 cytokines was recently approved by the European Commission for the treatment of moderate to severe plaque psoriasis (Krueger et al., 2007, N. Engl. J. Med. 356:580-92; Reich et al., 2009, Nat. Rev. Drug Discov. 8:355-356). In addition, antibodies targeting the IL-12 and IL-23 pathways have undergone clinical trials for treating Crohn's disease (Mannon et al., 2004, N. Engl. J. Med. 351:2069-79).

[0009] WO 2010 / 051549, WO 2011 / 003065, WO 2015 / 177326 and WO 2017 / 089390 describe certain pyrazolopyrimidine compounds that are reportedly useful as inhibitors of one or more Janus kinases. Data is presented therein for certain compounds that show inhibition of JAK1 as well as JAK2, JAK3 and / or TYK2 kinases.

[0010] Currently, there remains a need for additional compounds that are inhibitors of Janus kinases. For example, there is a need for compounds that have useful potency as inhibitors of one or more Janus kinases (e.g., JAK1 and JAK2) in combination with other pharmacological properties necessary to achieve a useful therapeutic benefit. For example, there is a need for potent compounds that demonstrate selectivity for one Janus kinase over other kinases in general (e.g., selectivity for JAK1 and / or JAK2 over other kinases such as leucine-rich repeat kinase 2 (LRRK2)). There is also a need for potent compounds that demonstrate selectivity for one Janus kinase over other Janus kinases (e.g., selectivity for JAK1 and / or JAK2 over JAK3 and / or TYK2). Compounds that demonstrate selectivity for both JAK1 and JAK2 over JAK3 and TYK2 may provide therapeutic benefit in conditions that respond to inhibition of JAK1. Additionally, there is currently a need for potent JAK1 inhibitors that have other properties (e.g., melting point, pK, solubility, etc.) necessary for formulation and administration by inhalation. Such compounds would be particularly useful in treating conditions such as asthma.

[0011] Thus, there is a need in the art for additional or alternative treatments for conditions mediated by JAK kinases, such as those conditions described above, and in particular for JAK1 and JAK2 kinase inhibitors that can be used for inhaled delivery in the treatment of airway inflammatory indications such as asthma. Summary of the Invention

[0012] Provided herein are pyrazolopyrimidines that inhibit JAK kinases, such as those selected from compounds of formula (I), or stereoisomers or salts thereof, such as pharma- ceutically acceptable salts thereof. The JAK kinase may be JAK1, JAK2, or both.

[0013] One embodiment is a compound of formula (I): TIFF0007682110000001.tif64170(I) or a stereoisomer or a pharma- ceutically acceptable salt thereof, During the ceremony, R 1 is hydroxyl-C 1 ~C 6 Alkyl;-(CR a1 R a2 ) m -het 1 ;-(CR a1 R a2 ) n -NR b R c ; or -(CR a1 R a2 ) m -C 3~6 Cycloalkyl, C 3~6 The cycloalkyl moiety is R d is substituted once with; R 2 Ha, Halo; Halo C 1 ~C 6 Alkoxy;C 1 ~C 6 Alkylthio;-SF 2 ; or C 3 ~C 6 is cycloalkyl; R 3 is hydrogen; or C 1 ~C 6 is alkyl; R 4 is hydrogen; or C 1 ~C 6 is alkyl; R 5 is hydrogen; or C 1 ~C 6 is alkyl; R 6 is hydrogen; or C 1 ~C 6 is alkyl; Or R 2 and R 6 can form, together with the atom to which they are attached, a six-membered ring containing two heteroatoms each independently selected from O, N, and S; m is 0 to 2; n is 0 to 3; Each R a1 are independently hydrogen; or C 1 ~C 6 is alkyl; Each R a2 are independently hydrogen; halo; or C 1 ~C 6 is alkyl; R b is hydrogen; or C 1 ~C 6 is alkyl; R c is hydrogen; C 1 ~C 6 alkyl; an amino protecting group; or unsubstituted or C 1 ~C 6 azetidinyl optionally substituted once with alkyl; het 1 is a heterocyclyl selected from azetidinyl; pyrrolidinyl; piperazinyl; piperidinyl; morpholinyl; and oxetanyl; each of which may be unsubstituted or R d Once at R g may be substituted once or twice with; R d is -(CR a1 R a2 ) p -het 2 ;-(CR a1 R a2 ) q -NR e R f ; or -(CR a1 R a2 ) p -C 3~6Cycloalkyl, C 3~6 The cycloalkyl moiety is -NR e R f is substituted once with; p is 0 to 2; q is 0 to 4; R e is hydrogen; or C 1 ~C 6 is alkyl; R f is hydrogen; C 1 ~C 6 Alkyl; or -CH 2 C 2 N(CH 3 ) 2 and; Each R g is C 1 ~C 6 alkyl; or halo; and Het 2 is a heterocycle selected from tetrahydropyranyl; azetidinyl; and pyrrolidinyl; each of which may be unsubstituted or selected from C 1 ~C 6 Alkyl or -NR e R f It may be substituted once by

[0014] In certain embodiments, R 1 is C 1 ~C 6 Alkyl; Hydroxyl-C 1 ~C 6 Alkyl;-(CR a1 R a2 ) m -het 1 ; or (CR a1 R a2 ) n -NR b R c And het 1 may be unsubstituted, or R d It may be substituted once by

[0015] Also provided is a pharmaceutical composition comprising a JAK inhibitor described herein, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier, diluent, or excipient.

[0016] Also provided is the use of a JAK inhibitor as described herein, or a pharma- ceutically acceptable salt thereof, in therapy, such as the treatment of an inflammatory disease (e.g., asthma).Also provided is the use of a JAK inhibitor as described herein, or a pharma- ceutically acceptable salt thereof, to prepare a medicament for the treatment of an inflammatory disease.Also provided is a method of preventing, treating, or reducing the severity of a disease or condition responsive to inhibition of Janus kinase activity in a patient, comprising administering to the patient a therapeutically effective amount of a JAK inhibitor as described herein, or a pharma- ceutically acceptable salt thereof.

[0017] The most validated cytokines in asthma (IL-4, IL-5, IL-9, IL-13, and TSLP) all signal through JAK1 and / or JAK2. The compounds of the present invention are active against both JAK1 and JAK2. Some of these compounds optimally have balanced co-activity against both JAK1 and JAK2, or have slightly higher affinity for JAK1 than JAK2, rather than having much higher activity against one of these kinases than the other. The subject compounds also have good selectivity against off-target kinases such as LRRK2, which are associated with pulmonary toxicity.

[0018] Although many compounds may exhibit high affinity for both JAK1 and JAK2 in simple biochemical assays, not all such compounds are effective in mediating the relevant cytokines associated with JAK1 and JAK2. Certain compounds of the present invention, in addition to being active against both JAK1 and JAK2, have also been shown to be effective in mediating asthma-related cytokines associated with JAK1 and JAK2 in cell-based assays.

[0019] The compounds of the present invention also exhibit favorable pharmacokinetic (PK) properties in lung tissue, making them useful for inhalation therapy. When administered via inhalation route using techniques such as dry powder inhalation (DPI) or intranasal (IN) delivery, certain compounds unexpectedly exhibit sustained retention in lung tissue, with much lower concentrations in the systemic circulation. Such improved PK properties may advantageously result in lower dosages and less frequent dosing requirements for effective treatment. Certain compounds exhibit unexpected improved solubility, which also provides demonstrated efficacy in the lung. Certain compounds of the present invention also exhibit unexpected reduced cytotoxicity compared to other JAK inhibitors. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] definition "Halogen" or "halo" refers to F, Cl, Br, or I. Additionally, terms such as "haloalkyl" are meant to include monohaloalkyl and polyhaloalkyl, where one or more halogens replace a hydrogen(s) on the alkyl group.

[0021] The term "alkyl" refers to a saturated linear or branched monovalent hydrocarbon group, where the alkyl group may be optionally substituted. In one example, an alkyl radical is an alkyl group having 1 to 18 carbon atoms (C 1 ~C 18 In another embodiment, the alkyl radical is 0 ~C 6 , C 0 ~C 5 , C 0 ~C 3 , C 1 ~C 12 , C 1 ~C 10、 C 1 ~C 8 , C 1 ~C 6 , C 1 ~C 5 , C 1 ~C 4 , or C 1 ~C 3 C 0Alkyl refers to a bond. Examples of alkyl groups are methyl (Me, -CH 3 ), ethyl (Et, -CH 2 CH 3 ), 1-propyl (n-Pr, n-propyl, -CH 2 CH 2 CH 3 ), 2-propyl (i-Pr, i-propyl, -CH(CH 3 ) 2 ), 1-Butyl (n-Bu, n-Butyl, -CH 2 CH 2 CH 2 CH 3 ), 2-methyl-1-propyl (i-Bu, i-butyl, -CH 2 CH(CH 3 ) 2 ), 2-Butyl (s-Bu, s-Butyl, -CH(CH 3 )CH 2 CH 3 ), 2-methyl-2-propyl (t-Bu, t-butyl, -C(CH 3 ) 3 ), 1-pentyl (n-pentyl, -CH 2 CH 2 CH 2 CH 2 CH 3 ), 2-pentyl (-CH(CH 3 )CH 2 CH 2 CH 3 ), 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 CH3 ), 1-hexyl (-CH 2 CH 2 CH 2 CH 2 CH 2 CH 3 ), 2-hexyl (-CH(CH 3 )CH 2 CH 2 CH 2 CH 3 ), 3-hexyl (-CH(CH 2 CH 3 )(CH 2 CH 2 CH 3 )), 2-methyl-2-pentyl (-C(CH 3 ) 2 CH 2 CH 2 CH 3 ) 3-Methyl-2-pentyl (-CH(CH 3 )CH(CH 3 )CH 2 CH 3 ), 4-methyl-2-pentyl (-CH(CH 3 )CH 2 CH(CH 3 ) 2 ), 3-methyl-3-pentyl (-C(CH 3 )(CH 2 CH 3 ) 2 ), 2-methyl-3-pentyl (-CH(CH 2 CH 3 )CH(CH 3 ) 2 ), 2,3-dimethyl-2-butyl (-C(CH 3 ) 2 CH(CH 3 ) 2 ), 3,3-dimethyl-2-butyl (-CH(CH 3 )C(CH 3 ) 3 , 1-heptyl, and 1-octyl. In some embodiments, the substituents of "optionally substituted alkyl" include F, Cl, Br, I, OH, SH, CN, NH 2 , N.H.C.H. 3 , N(CH 3 ) 2, NO 2 , N 3 , 、 C(O)CH 3 , COOH, CO 2 CH 3 , methyl, ethyl, propyl, iso-propyl, butyl, isobutyl, cyclopropyl, methoxy, ethoxy, propoxy, oxo, trifluoromethyl, difluoromethyl, sulfonylamino, methanesulfonylamino, SO, SO 2 , phenyl, piperidinyl, piperidinyl and pyrimidinyl, and the alkyl, phenyl and heterocyclic portions thereof may be optionally substituted with 1 to 4 examples of substituents selected from this same list, etc.

[0022] The term "alkenyl" refers to a linear or branched monovalent hydrocarbon radical having at least one site of unsaturation, i.e., a carbon-carbon double bond; the alkenyl radical may be optionally substituted and includes radicals having "cis" and "trans" orientations, or alternatively, "E" and "Z" orientations. In one example, an alkenyl group is an alkyl group having 2 to 18 carbon atoms (C 2 ~C 18 In another embodiment, the alkenyl radical is 2 ~C 12 , C 2 ~C 10、 C 2 ~C 8 , C 2 ~C 6 , or C 2 ~C 3 Examples are ethenyl or vinyl (-CH=CH 2 ), prop-1-enyl (-CH=CHCH 3 ), prop-2-enyl (-CH 2 CH=CH 2), 2-methylprop-1-enyl, but-1-enyl, but-2-enyl, but-3-enyl, buta-1,3-dienyl, 2-methylbuta-1,3-diene, hex-1-enyl, hex-2-enyl, hex-3-enyl, hex-4-enyl, and hex-1,3-dienyl. In some embodiments, the substituents of "optionally substituted alkenyl" include, but are not limited to, F, Cl, Br, I, OH, SH, CN, NH 2 , N.H.C.H. 3 , N(CH 3 ) 2 , NO 2 , N 3 , C(O)CH 3 , COOH, CO 2 CH 3 , methyl, ethyl, propyl, iso-propyl, butyl, isobutyl, cyclopropyl, methoxy, ethoxy, propoxy, oxo, trifluoromethyl, difluoromethyl, sulfonylamino, methanesulfonylamino, SO, SO 2 , phenyl, piperidinyl, piperidinyl and pyrimidinyl, and the alkyl, phenyl and heterocyclic portions thereof may be optionally substituted with 1 to 4 examples of substituents selected from this same list, etc.

[0023] The term "alkynyl" refers to a linear or branched monovalent hydrocarbon radical having at least one site of unsaturation, i.e., a carbon-carbon triple bond, and the alkynyl radical may be optionally substituted. In one example, an alkynyl radical is one having 2 to 18 carbon atoms (C 2 ~C 18 In other examples, the alkynyl radical is C 2 ~C 12 , C 2 ~C 10、 C 2 ~C 8 , C 2 ~C 6 , or C 2 ~C 3 Examples include ethynyl (-C≡CH), prop-1-ynyl (-C≡CCH 3), prop-2-ynyl (propargyl, -CH 2 C≡CH), but-1-ynyl, but-2-ynyl, and but-3-ynyl. In some embodiments, the substituents of "optionally substituted alkynyl" include F, Cl, Br, I, OH, SH, CN, NH 2 , N.H.C.H. 3 , N(CH 3 ) 2 , NO 2 , N 3 , C(O)CH 3 , COOH, CO 2 CH 3 , methyl, ethyl, propyl, iso-propyl, butyl, isobutyl, cyclopropyl, methoxy, ethoxy, propoxy, oxo, trifluoromethyl, difluoromethyl, sulfonylamino, methanesulfonylamino, SO, SO 2 , phenyl, piperidinyl, piperidinyl and pyrimidinyl, and the alkyl, phenyl and heterocyclic portions thereof may be optionally substituted with 1 to 4 examples of substituents selected from this same list, etc.

[0024] "Alkylene" refers to a saturated, branched, or straight-chain hydrocarbon group having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkane. In one example, a divalent alkylene group is an alkylene group having 1 to 18 carbon atoms (C 1 ~C 18 In another example, the divalent alkylene group is 0 ~C 6 , C 0 ~C 5 , C 0 ~C 3 , C 1 ~C 12 , C 1 ~C 10、 C 1 ~C 8 , C 1 ~C 6 , C 1 ~C 5 , C 1 ~C 4 , or C1 ~C 3 Group C 0 Alkylene refers to a bond. Exemplary alkylene groups include methylene (-CH 2 -), 1,1-ethyl (-CH(CH 3 )-), (1,2-ethyl(-CH 2 CH 2 -), 1,1-propyl (-CH(CH 2 CH 3 )-), 2,2-propyl (-C(CH 3 ) 2 -), 1,2-propyl (-CH(CH 3 )CH 2 -), 1,3-propyl (-CH 2 CH 2 CH 2 -), 1,1-dimethyleth-1,2-yl (-C(CH 3 ) 2 CH 2 -), 1,4-butyl (-CH 2 CH 2 CH 2 CH 2 -) etc.

[0025] The term "heteroalkyl" refers to a linear or branched monovalent hydrocarbon radical consisting of the specified number of carbon atoms, or up to 18 carbon atoms if not specified, and one to five heteroatoms selected from the group consisting of O, N, Si, and S, where the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized. In some embodiments, the heteroatoms are selected from O, N, and S, where the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized. The heteroatom(s) may be attached to the position at which the alkyl group is attached to the remainder of the molecule (e.g., -O-CH 2 -CH 3 ) can be placed at any interior position of the heteroalkyl group. Examples include CH 2 -CH 2 -O-CH 3 , -CH 2 -CH 2 -NH-CH3 , -CH 2 -CH 2 -N(CH 3 )-CH 3 , -CH 2 -S-CH 2 -CH 3 , -S(O)-CH 3 , -CH 2 -CH 2 -S(O) 2 -CH 3 , -Si(CH 3 ) 3 , and -CH 2 -CH=N-OCH 3 For example, -CH 2 -NH-OCH 3 , and -CH 2 -O-Si(CH 3 ) 3 Up to two heteroatoms may be consecutive. Heteroalkyl groups may be optionally substituted. In some embodiments, the substituents of "optionally substituted heteroalkyl" include F, Cl, Br, I, OH, SH, CN, NH 2 , N.H.C.H. 3 , N(CH 3 ) 2 , NO 2 , N 3 , C(O)CH 3 , COOH, CO 2 CH 3 , methyl, ethyl, propyl, iso-propyl, butyl, isobutyl, cyclopropyl, methoxy, ethoxy, propoxy, oxo, trifluoromethyl, difluoromethyl, sulfonylamino, methanesulfonylamino, SO, SO 2 , phenyl, piperidinyl, piperidinyl and pyrimidinyl, and the alkyl, phenyl and heterocyclic portions thereof may be optionally substituted with 1 to 4 examples of substituents selected from this same list, etc.

[0026] "Amino" refers to an optionally substituted primary (i.e., -NH 2), secondary (i.e., -NRH), tertiary (i.e., -NRR), and quaternary (i.e., -N(+)RRR) amines, where each R is the same or different and is selected from alkyl, cycloalkyl, aryl, and heterocyclyl, where the alkyl, cycloalkyl, aryl, and heterocyclyl groups are as defined herein. Particular secondary and tertiary amines are alkylamines, dialkylamines, arylamines, diarylamines, aralkylamines, and diaralkylamines, where the alkyl and aryl moieties may be optionally substituted. Particular secondary and tertiary amines are methylamine, ethylamine, propylamine, isopropylamine, phenylamine, benzylamine, dimethylamine, diethylamine, dipropylamine, and diisopropylamine. In some embodiments, the R groups of the quaternary amines are each independently an optionally substituted alkyl group.

[0027] "Aryl" refers to a carbocyclic aromatic group having the specified number of carbon atoms, or up to 14 carbon atoms if no number is specified, whether or not fused to one or more groups. An example is an aryl group having 6 to 14 carbon atoms. Another example is an aryl group having 6 to 10 carbon atoms. Examples of aryl groups include phenyl, naphthyl, biphenyl, phenanthrenyl, naphthacenyl, 1,2,3,4-tetrahydronaphthalenyl, 1H-indenyl, 2,3-dihydro-1H-indenyl, and the like (see, e.g., Lang's Handbook of Chemistry (Dean, JA, ed.) vol. 13). th ed.Table 7-2,

[1985] A particular aryl is phenyl. Substituted phenyl or aryl can be any of F, Cl, Br, I, OH, SH, CN, NH 2 , N.H.C.H. 3 , N(CH 3 ) 2 , NO 2 , N 3 , C(O)CH 3 , COOH, CO 2 CH3 , methyl, ethyl, propyl, isopropyl, butyl, isobutyl, cyclopropyl, methoxy, ethoxy, propoxy, oxo, trifluoromethyl, difluoromethyl, sulfonylamino, methanesulfonylamino, SO, SO 2By this definition is meant a phenyl or aryl group substituted by 1, 2, 3, 4 or 5 substituents, for example 1-2, 1-3 or 1-4 substituents, as selected from the groups explicitly set forth herein, such as phenyl, piperidinyl, piperidinyl and pyrimidinyl, wherein the alkyl, phenyl and heterocyclic portions thereof may be optionally substituted, such as by 1-4 instances of substituents selected from this same list.Examples of the term "substituted phenyl" include mono- or di(halo)phenyl groups, such as 2-chlorophenyl, 2-bromophenyl, 4-chlorophenyl, 2,6-dichlorophenyl, 2,5-dichlorophenyl, 3,4-dichlorophenyl, 3-chlorophenyl, 3-bromophenyl, 4-bromophenyl, 3,4-dibromophenyl, 3-chloro-4-fluorophenyl, 2-fluorophenyl, 2,4-difluorophenyl; mono- or di(hydroxy)phenyl groups, such as 4-hydroxyphenyl, 3-hydroxyphenyl, 2,4-dihydroxyphenyl, and protected hydroxy derivatives thereof; nitrophenyl groups, such as 3- or 4-nitrophenyl; cyanophenyl groups, e.g., 4-cyanophenyl; mono- or di(alkyl)phenyl groups, such as 4-methylphenyl, 2,4-dimethylphenyl, 2-methylphenyl, 4-(isopropyl)phenyl, 4-ethylphenyl, 3-(n-propyl)phenyl; includes di(alkoxy)phenyl groups such as 3,4-dimethoxyphenyl, 3-methoxy-4-benzyloxyphenyl, 3-ethoxyphenyl, 4-(isopropoxy)phenyl, 4-(t-butoxy)phenyl, 3-ethoxy-4-methoxyphenyl, and the like; 3- or 4-trifluoromethylphenyl; mono- or dicarboxyphenyl or (protected carboxy)phenyl groups (such as 4-carboxyphenyl), mono- or di(hydroxymethyl)phenyl or (protected hydroxymethyl)phenyl (such as 3-(protected hydroxymethyl)phenyl or 3,4-di(hydroxymethyl)phenyl); mono- or di(aminomethyl)phenyl or (protected aminomethyl)phenyl (such as 2-(aminomethyl)phenyl or 2,4-(protected aminomethyl)phenyl); or mono- or di(N-(methylsulfonylamino))phenyl, such as 3-(N-methylsulfonylamino))phenyl.The term "substituted phenyl" also refers to disubstituted phenyl groups with different substituents, such as 3-methyl-4-hydroxyphenyl, 3-chloro-4-hydroxyphenyl, 2-methoxy-4-bromophenyl, 4-ethyl-2-hydroxyphenyl, 3-hydroxy-4-nitrophenyl, 2-hydroxy-4-chlorophenyl, 2-chloro-5-difluoromethoxy, etc., as well as trisubstituted phenyl groups with different substituents, such as 3-methoxy-4-benzyloxy-6-methylsulfonylamino, 3-methoxy-4-benzyloxy-6-phenylsulfonylamino, and tetrasubstituted phenyl groups with different substituents, such as 3-methoxy-4-benzyloxy-5-methyl-6-phenylsulfonylamino. In some embodiments, the substituents of aryls, such as phenyl, include amides. For example, aryl (e.g., phenyl) substituents include -(CH. 2 ) 0~4 CONR′R″, where R′ and R″ are each independently, e.g., hydrogen; unsubstituted C 1~ C 6 Alkyl; halogen, OH, CN, unsubstituted C 1 ~C 6 Alkyl, unsubstituted C 1 ~C 6 C substituted with alkoxy, oxo or NR′R″ 1~ C 6 Alkyl; Unsubstituted C 1~ C 6 Heteroalkyl; halogen, OH, CN, unsubstituted C 1 ~C 6 Alkyl, unsubstituted C 1 ~C 6 C substituted with alkoxy, oxo or NR′R″ 1~ C 6 Heteroalkyl; Unsubstituted C 6~ C 10 Aryl; halogen, OH, CN, unsubstituted C 1 ~C 6 Alkyl, unsubstituted C 1 ~C 6 C substituted with alkoxy or NR'R'' 6~ C 10aryl; unsubstituted 3- to 11-membered heterocyclyl (e.g., 5- to 6-membered heteroaryl containing 1-4 heteroatoms selected from O, N, and S, or 4- to 11-membered heterocycloalkyl containing 1-4 heteroatoms selected from O, N, and S); and 3- to 11-membered heterocyclyl (e.g., 5- to 6-membered heteroaryl containing 1-4 heteroatoms selected from O, N, and S) or halogen, OH, CN, unsubstituted C 1 ~C 6 Alkyl, unsubstituted C 1 ~C 6 or R′ and R″ may be combined with the nitrogen atom to form a 3-, 4-, 5-, 6-, or 7-membered ring, the ring atoms of which are optionally substituted with N, O, or S, and the ring is optionally substituted with halogen, OH, CN, unsubstituted C, 1 ~C 6 Alkyl, unsubstituted C 1 ~C 6 Optionally substituted with alkoxy, oxo or NR'R''.

[0028] "Cycloalkyl" refers to a non-aromatic saturated or partially unsaturated hydrocarbon ring group, where the cycloalkyl group may be optionally substituted independently with one or more substituents described herein. In one example, the cycloalkyl group has 3 to 12 carbon atoms ( C3~12 In another embodiment, the cycloalkyl is 3 ~C 8 , C 3 ~C 10 , or C 5 ~C 10 In another embodiment, the cycloalkyl group, as a single ring, is 3 ~C 8 , C 3 ~C 6 Or C 5 ~C 6 In another example, the cycloalkyl group, as a bicycle, is 7 ~C 12 In another example, the cycloalkyl group can be, as a spiro system, C 5 ~C12 Examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, perdeuteriocyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, and cyclododecyl. Exemplary configurations of bicyclic cycloalkyls having 7 to 12 ring atoms include, but are not limited to, [4,4], [4,5], [5,5], [5,6], or [6,6] ring systems. Exemplary bridged bicyclic cycloalkyls include, but are not limited to, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, and bicyclo[3.2.2]nonane. Examples of spirocycloalkyls include spiro[2.2]pentane, spiro[2.3]hexane, spiro[2.4]heptane, spiro[2.5]octane, and spiro[4.5]decane. In some embodiments, the substituents of "optionally substituted cycloalkyl" include F, Cl, Br, I, OH, SH, CN, NH 2 , N.H.C.H. 3 , N(CH 3 ) 2 , NO 2 , N 3 , C(O)CH 3 , COOH, CO 2 CH 3 , methyl, ethyl, propyl, iso-propyl, butyl, isobutyl, cyclopropyl, methoxy, ethoxy, propoxy, oxo, trifluoromethyl, difluoromethyl, sulfonylamino, methanesulfonylamino, SO, SO 2Examples of cycloalkyl substituents include 1 to 4 of the following: phenyl, piperidinyl, piperidinyl, and pyrimidinyl, and the alkyl, aryl, and heterocyclic portions thereof may be optionally substituted with 1 to 4 examples of substituents selected from this same list, etc. In some embodiments, cycloalkyl substituents include amido. For example, cycloalkyl substituents include -(CH 2 ) 0~4 CONR′R″, where R′ and R″ are each independently, for example, hydrogen; unsubstituted C 1~ C 6 Alkyl; halogen, OH, CN, unsubstituted C 1 ~C 6 Alkyl, unsubstituted C 1 ~C 6 C substituted with alkoxy, oxo or NR′R″ 1~ C 6 Alkyl; Unsubstituted C 1~ C 6 Heteroalkyl; halogen, OH, CN, unsubstituted C 1 ~C 6 Alkyl, unsubstituted C 1 ~C 6 C substituted with alkoxy, oxo or NR′R″ 1~ C 6 Heteroalkyl; Unsubstituted C 6~ C 10 Aryl; halogen, OH, CN, unsubstituted C 1 ~C 6 Alkyl, unsubstituted C 1 ~C 6 C substituted with alkoxy or NR'R'' 6~ C 10 aryl; unsubstituted 3-11 membered heterocyclyl (e.g., 5-6 membered heteroaryl containing 1-4 heteroatoms selected from O, N, and S, or 4-11 membered heterocycloalkyl containing 1-4 heteroatoms selected from O, N, and S); and halogen, OH, CN, unsubstituted C 1 ~C 6 Alkyl, unsubstituted C 1 ~C 6or R′ and R″ may be combined with the nitrogen atom to form a 3-, 4-, 5-, 6-, or 7-membered ring, the ring atoms of which are optionally substituted with N, O, or S, and the ring is optionally substituted with halogen, OH, CN, unsubstituted C, or OH. 1 ~C 6 Alkyl, unsubstituted C 1 ~C 6 Optionally substituted with alkoxy, oxo or NR'R''.

[0029] The terms "heterocyclic group", "heterocyclic", "heterocycle", "heterocyclyl", or "heterocyclo" are used interchangeably and refer to any monocyclic, bicyclic, tricyclic, or spiro ring system, saturated or unsaturated, aromatic (heteroaryl) or non-aromatic (e.g., heterocycloalkyl) ring system having 3 to 20 ring atoms (e.g., 3 to 10 ring atoms), where the ring atoms are carbon and at least one atom of the ring or ring system is a heteroatom selected from nitrogen, sulfur, or oxygen. If any ring atom of the ring system is a heteroatom, the system is a heterocyclic ring, regardless of the point of attachment of the ring system to the rest of the molecule. In one example, a heterocyclyl contains 3 to 11 ring atoms ("members"), including monocyclic, bicyclic, tricyclic, and spiro ring systems, where the ring atoms are carbon and at least one atom in the ring or ring system is a heteroatom selected from nitrogen, sulfur, or oxygen. In one example, a heterocyclyl contains 1 to 4 heteroatoms. In one example, a heterocyclyl includes 1-3 heteroatoms. In another example, a heterocyclyl includes a 3-7 membered monocyclic ring having 1-2, 1-3, or 1-4 heteroatoms selected from nitrogen, sulfur, or oxygen. In another example, a heterocyclyl includes a 4-6 membered monocyclic ring having 1-2, 1-3, or 1-4 heteroatoms selected from nitrogen, sulfur, or oxygen. In another example, a heterocyclyl includes a 3 membered monocyclic ring. In another example, a heterocyclyl includes a 4 membered monocyclic ring. In another example, a heterocyclyl includes a 5-6 membered monocyclic ring, such as a 5-6 membered heteroaryl. In another example, a heterocyclyl includes a 3-11 membered heterocycloalkyl, such as a 4-11 membered heterocycloalkyl. In some embodiments, a heterocycloalkyl includes at least one nitrogen. In one example, a heterocyclyl group includes 0-3 double bonds. Any nitrogen or sulfur heteroatom may be optionally oxidized (e.g., NO, SO, SO 2 ), any nitrogen heteroatom may be optionally quaternized (e.g., [NR 4 ] + Cl - , [NR 4 ] + OH -Exemplary heterocycles are oxiranyl, aziridinyl, thiiranyl, azetidinyl, oxetanyl, thietanyl, 1,2-dithietanyl, 1,3-dithietanyl, pyrrolidinyl, dihydro-1H-pyrrolyl, dihydrofuranyl, tetrahydrofuranyl, dihydrothienyl, tetrahydrothienyl, imidazolidinyl, piperidinyl, piperazinyl, isoquinolinyl, tetrahydroisoquinolinyl, morpholinyl, thiomorpholinyl, 1,1-dioxo-thiomorpholinyl, dihydropyranyl, tetrahydropyranyl, hexahydrothio ... Dropyrimidinyl, oxazinanyl, thiazinanyl, thioxanyl, homopiperazinyl, homopiperidinyl, azepanyl, oxepanyl, thiepanyl, oxazepinyl, oxazepanyl, diazepanyl, 1,4-diazepanyl, diazepinyl, thiazepinyl, thiazepanyl, tetrahydrothiopyranyl, oxazolidinyl, thiazolidinyl, isothiazolidinyl, 1,1-dioxoisothiazolidinonyl, oxazolidinonyl, imidazolidinonyl, 4,5,6,7-tetrahydro[2H]indazolyl, tetrahydrobenzimidazolyl, 4,5,6, 7-Tetrahydrobenzo[d]imidazolyl, 1,6-dihydroimidazole[4,5-d]pyrrolo[2,3-b]pyridinyl, thiazinyl, oxazinyl, thiadiazinyl, oxadiazinyl, dithiazinyl, dioxazinyl, oxathiazinyl, thiatriazinyl, oxatriazinyl, dithiadiazinyl, imidazolinyl, dihydropyrimidyl, tetrahydropyrimidyl, 1-pyrrolinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, thiapyranyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl , pyrazolidinyl, dithianyl, dithiolanyl, pyrimidinonyl, pyrimidindionyl, pyrimidine-2,4-dionyl, piperazinonyl, piperazinedionyl, pyrazolidinylimidazolinyl, 3-azabicyclo[3.1.0]hexanyl, 3,6-diazabicyclo[3.1.1]heptanyl, 6-azabicyclo[3.1.1]heptanyl, 3-azabicyclo[3.1.1]heptanyl, 3-azabicyclo[4.1.0]heptanyl, azabicyclo[2.2.2]hexanyl, 2-azabicyclo[3.2.1]octanyl, 8-azabicyclo[3.2.1] Octanyl, 2-azabicyclo[2.2.2]octanyl, 8-azabicyclo[2.2.2]octanyl, 7-oxabicyclo[2.2.1]heptane, azaspiro[3.5]nonanyl, azaspiro[2.5]octanyl, azaspiro[4.5]decanyl, 1-azaspiro[4.5]decan-2-onyl (only), azaspiro[5.5]undecanyl, tetrahydroindolyl, octahydroindolyl, tetrahydroisoindolyl, tetrahydroindazolyl, 1,1-dioxohexahydrothiopyranyl. Examples of 5-membered heterocycles containing a sulfur or oxygen atom and 1 to 3 nitrogen atoms include thiazolyl containing thiazol-2-yl and thiazol-2-yl N-oxide, thiadiazolyl containing 1,3,4-thiadiazol-5-yl and 1,2,4-thiadiazol-5-yl, oxazolyl (e.g., oxazol-2-yl), and oxadiazolyl such as 1,3,4-oxadiazol-5-yl and 1,2,4-oxadiazol-5-yl. Examples of 5-membered ring heterocycles containing 2 to 4 nitrogen atoms include imidazolyl such as imidazol-2-yl, triazolyl such as 1,3,4-triazol-5-yl, 1,2,3-triazol-5-yl, 1,2,4-triazol-5-yl, and tetrazolyl such as 1H-tetrazol-5-yl. Examples of benzo-fused 5-membered heterocycles are benzoxazol-2-yl, benzthiazol-2-yl, and benzimidazol-2-yl. Examples of 6-membered heterocycles contain 1 to 3 nitrogen atoms and optionally a sulfur or oxygen atom, such as pyridyl (pyrid-2-yl, pyrid-3-yl, pyrid-4-yl, etc.); pyrimidyl (pyrimid-2-yl, pyrimid-4-yl, etc.); triazinyl (1,3,4-triazin-2-yl, 1,3,5-triazin-4-yl, etc.); pyridazinyl, especially pyridazin-3-yl and pyrazinyl. Pyridine N-oxide, pyridazine N-oxide, and pyridyl, pyrimid-2-yl, pyrimid-4-yl, pyridazinyl, and 1,3,4-triazin-2-yl groups are other exemplary heterocyclic groups. The heterocycle may be optionally substituted. For example, substituents of the "optionally substituted heterocycle" include F, Cl, Br, I, OH, SH, CN, NH. 2 , NHCH 3, N(CH 3 ) 2 , NO 2 , N 3 , C(O)CH 3 , COOH, CO 2 CH 3 , methyl, ethyl, propyl, iso-propyl, butyl, isobutyl, cyclopropyl, methoxy, ethoxy, propoxy, oxo, trifluoromethyl, difluoromethyl, sulfonylamino, methanesulfonylamino, SO, SO 2 Examples of the heterocyclic group include 1 to 4 of the following: phenyl, piperidinyl, piperidinyl, and pyrimidinyl, and the alkyl, aryl, and heterocyclic portions thereof may be optionally substituted with 1 to 4 examples of substituents selected from this same list, etc. In some embodiments, the substituents of a heterocyclic group, such as heteroaryl or heterocycloalkyl, include amide. For example, a heterocyclic (e.g., heteroaryl or heterocycloalkyl) substituent may be -(CH 2 ) 0~4 CONR′R″, where R′ and R″ are each independently, e.g., hydrogen; unsubstituted C 1~ C 6 Alkyl; halogen, OH, CN, unsubstituted C 1 ~C 6 Alkyl, unsubstituted C 1 ~C 6 C substituted with alkoxy, oxo or NR′R″ 1~ C 6 Alkyl; Unsubstituted C 1- C 6 Heteroalkyl; halogen, OH, CN, unsubstituted C 1 ~C 6 Alkyl, unsubstituted C 1 ~C 6 C substituted with alkoxy, oxo or NR′R″ 1~ C 6 Heteroalkyl; Unsubstituted C 6~ C 10 Aryl; halogen, OH, CN, unsubstituted C 1 ~C 6 Alkyl, unsubstituted C 1 ~C 6 C substituted with alkoxy or NR'R'' 6~ C10 aryl; unsubstituted 3- to 11-membered heterocyclyl (e.g., 5- to 6-membered heteroaryl containing 1-4 heteroatoms selected from O, N, and S, or 4- to 11-membered heterocycloalkyl containing 1-4 heteroatoms selected from O, N, and S); and 3- to 11-membered heterocyclyl (e.g., 5- to 6-membered heteroaryl containing 1-4 heteroatoms selected from O, N, and S) or halogen, OH, CN, unsubstituted C 1 ~C 6 Alkyl, unsubstituted C 1 ~C 6 or R′ and R″ may be combined with the nitrogen atom to form a 3-, 4-, 5-, 6-, or 7-membered ring, the ring atoms of which are optionally substituted with N, O, or S, and the ring is optionally substituted with halogen, OH, CN, unsubstituted C, 1 ~C 6 Alkyl, unsubstituted C 1 ~C 6 Optionally substituted with alkoxy, oxo or NR'R''.

[0030] "Heteroaryl" refers to any monocyclic, bicyclic, or tricyclic ring system in which at least one ring is a 5- or 6-membered aromatic ring containing 1 to 4 heteroatoms selected from nitrogen, oxygen, and sulfur, and in an exemplary embodiment, at least one heteroatom is nitrogen. See, for example, Lang's Handbook of Chemistry (Dean, JA, ed.) 13 thed. Table 7-2

[1985] . This definition includes any bicyclic group in which any of the above heteroaryl rings are fused to an aryl ring, and either the aryl ring or the heteroaryl ring is attached to the remainder of the molecule. In one embodiment, heteroaryl includes 5-6 membered monocyclic aromatic groups in which one or more ring atoms is nitrogen, sulfur, or oxygen. Exemplary heteroaryl groups include thienyl, furyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, thiadiazolyl, oxadiazolyl, tetrazolyl, thiatriazolyl, oxatriazolyl, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazinyl, tetrazinyl, tetrazolo[1,5-b]pyridazinyl, imidazol[1,2-a]pyrimidinyl and purinyl, as well as benzo-fused derivatives such as benzoxazolyl, benzofuryl, benzothiazolyl, benzothiadiazolyl, benzotriazolyl, benzimidazolyl, and indolyl. Heteroaryl groups may be optionally substituted. In some embodiments, the substituents of "optionally substituted heteroaryl" include F, Cl, Br, I, OH, SH, CN, NH 2 , N.H.C.H. 3 , N(CH 3 ) 2 , NO 2 , N 3 , C(O)CH 3 , COOH, CO 2 CH 3 , methyl, ethyl, propyl, iso-propyl, butyl, isobutyl, cyclopropyl, methoxy, ethoxy, propoxy, trifluoromethyl, difluoromethyl, sulfonylamino, methanesulfonylamino, SO, SO 2 Examples of heteroaryl substituents include 1-4 of -(CH, -CH- ... 2 ) 0~4CONR′R″, where R′ and R″ are each independently, for example, hydrogen; unsubstituted C 1~ C 6 Alkyl; halogen, OH, CN, unsubstituted C 1 ~C 6 Alkyl, unsubstituted C 1 ~C 6 Unsubstituted C substituted with alkoxy, oxo or NR′R″ 1- C 6 Alkyl; Unsubstituted C 1~ C 6 Heteroalkyl; halogen, OH, CN, unsubstituted C 1 ~C 6 Alkyl, unsubstituted C 1 ~C 6 C substituted with alkoxy, oxo or NR′R″ 1~ C 6 Heteroalkyl; Unsubstituted C 6~ C 10 Aryl; halogen, OH, CN, unsubstituted C 1 -C 6 Alkyl, unsubstituted C 1 -C 6 C substituted with alkoxy or NR'R'' 6~ C 10 aryl; unsubstituted 3-11 membered heterocyclyl (e.g., 5-6 membered heteroaryl containing 1-4 heteroatoms selected from O, N, and S, or 4-11 membered heterocycloalkyl containing 1-4 heteroatoms selected from O, N, and S); and halogen, OH, CN, unsubstituted C 1 ~C 6 Alkyl, unsubstituted C 1 ~C 6 or R′ and R″ may be combined with the nitrogen atom to form a 3-, 4-, 5-, 6-, or 7-membered ring, the ring atoms of which are optionally substituted with N, O, or S, and the ring is optionally substituted with halogen, OH, CN, unsubstituted C, or OH. 1 ~C6 Alkyl, unsubstituted C 1 ~C 6 Optionally substituted with alkoxy, oxo or NR'R''.

[0031] In certain embodiments, the heterocyclyl group is bonded to a carbon atom of the heterocyclyl group. For example, carbon-bonded heterocyclyl groups include bonding positions at the 2-, 3-, 4-, 5-, or 6-position of the pyridine ring, the 3-, 4-, 5-, or 6-position of the pyridazine ring, the 2-, 4-, 5-, or 6-position of the pyrimidine ring, the 2-, 3-, 5-, or 6-position of the pyrazine ring, the 2-, 3-, 4-, or 5-position of the furan, tetrahydrofuran, thiofuran, thiophene, pyrrole, or tetrahydropyrrole ring, the 2-, 4-, or 5-position of the oxazole, imidazole, or thiazole ring, the 3-, 4-, or 5-position of the isoxazole, pyrazole, or isothiazole ring, the 2-, or 3-position of the aziridine ring, the 2-, 3-, or 4-position of the azetidine ring, the 2-, 3-, 4-, 5-, 6-, 7-, or 8-position of the quinoline ring, or the 1-, 3-, 4-, 5-, 6-, 7-, or 8-position of the isoquinoline ring.

[0032] In certain embodiments, the heterocyclyl group is N-linked. For example, nitrogen-linked heterocyclyl or heteroaryl groups include attachment 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, 2-position of isoindole or isoindoline, 4-position of morpholine, and 9-position of carbazole or β-carboline.

[0033] The term "alkoxy" refers to a straight or branched monovalent radical represented by the formula -OR, where R is alkyl as defined herein. Alkoxy groups include methoxy, ethoxy, propoxy, isopropoxy, mono-, di-, and tri-fluoromethoxy, and cyclopropoxy.

[0034] "Acyl" means a carbonyl containing a substituent represented by the formula -C(O)-R, where R is hydrogen, alkyl, cycloalkyl, aryl, or heterocyclyl, where alkyl, cycloalkyl, aryl, and heterocyclyl are as defined herein. Acyl groups include alkanoyl (e.g., acetyl), aroyl (e.g., benzoyl), and heteroaroyl (e.g., pyridinoyl).

[0035] Unless otherwise specified, "optionally substituted" means that a group can be unsubstituted or substituted with one or more (e.g., 0, 1, 2, 3, 4, or 5 or more, or any of these range variables) of the substituents listed for that group, which can be the same or different. In one embodiment, an optionally substituted group has one substituent. In another embodiment, an optionally substituted group has two substituents. In another embodiment, an optionally substituted group has three substituents. In another embodiment, an optionally substituted group has four substituents. In another embodiment, an optionally substituted group has five substituents.

[0036] The alkyl radicals, either alone or as part of another substituent (e.g., alkoxy), and the optional substituents of alkylenyl, alkenyl, alkynyl, heteroalkyl, heterocycloalkyl, and cycloalkyl (again, each alone or as part of another substituent) can be various groups such as those described herein, including halogen; oxo; CN; NO; N 3 ;-OR'; Perfluoro-C 1~ C 4 Alkoxy; Unsubstituted C 3 ~C 7 Cycloalkyl; halogen, OH, CN, unsubstituted C 1 ~C 6 Alkyl, unsubstituted C 1 ~C 6 C substituted with alkoxy, oxo or NR′R″ 3 ~C 7 Cycloalkyl; Unsubstituted C 6 ~C10 Aryl (e.g., phenyl); halogen, OH, CN, unsubstituted C 1 ~C 6 Alkyl, unsubstituted C 1 ~C 6 C substituted with alkoxy or NR'R'' 6 ~C 10 Aryl; unsubstituted 3-11 membered heterocyclyl (e.g., 5-6 membered heteroaryl containing 1-4 heteroatoms selected from O, N and S, or 4-11 membered heterocycloalkyl containing 1-4 heteroatoms selected from O, N and S); halogen, OH, CN, unsubstituted C 1 ~C 6 Alkyl, unsubstituted C 1 ~C 6 3-11 membered heterocyclyl substituted with alkoxy, oxo or NR'R'' (e.g. 5-6 membered heteroaryl containing 1-4 heteroatoms selected from O, N and S or 4-11 membered heterocycloalkyl containing 1-4 heteroatoms selected from O, N and S); -NR'R''; -SR'; -SiR'R''R'''; -OC(O)R'; -C(O)R'; -CO 2 R';-CONR'R'';-OC(O)NR'R'';-NR''C(O)R';-NR'''C(O)NR'R'';-NR''C(O) 2 R';-S(O) 2 R';-S(O) 2 NR'R'';-NR'S(O) 2 R'';-NR'''S(O) 2 NR'R'';Amidinyl;Guanidinyl;-(CH 2 ) 1~4 -OR';-(CH 2 ) 1~4 -NR'R'';-(CH 2 ) 1~4 -SR';-(CH 2 ) 1~4 -SiR'R''R''';-(CH 2 ) 1~4 -OC(O)R';-(CH 2 ) 1~4 -C(O)R';-(CH 2 ) 1-4 -CO 2R'; and -(CH 2 ) 1-4 CONR'R'', or a combination thereof ranging from 0 to (2m'+1), where m' is the total number of carbon atoms in such group. R' and R'' can each be independently selected from the group consisting of hydrogen; unsubstituted C 1~ C 6 Alkyl; halogen, OH, CN, unsubstituted C 1 ~C 6 Alkyl, unsubstituted C 1 -C 6 C substituted with alkoxy, oxo or NR′R″ 1~ C 6 Alkyl; Unsubstituted C 1~ C 6 Heteroalkyl; halogen, OH, CN, unsubstituted C 1 ~C 6 Alkyl, unsubstituted C 1 ~C 6 C substituted with alkoxy, oxo or NR′R″ 1~ C 6 Heteroalkyl; Unsubstituted C 6~ C 10 Aryl; halogen, OH, CN, unsubstituted C 1 ~C 6 Alkyl, unsubstituted C 1 ~C 6 C substituted with alkoxy or NR'R'' 6~ C 10 aryl; unsubstituted 3-11 membered heterocyclyl (e.g., 5-6 membered heteroaryl containing 1-4 heteroatoms selected from O, N, and S, or 4-11 membered heterocycloalkyl containing 1-4 heteroatoms selected from O, N, and S); and halogen, OH, CN, unsubstituted C 1 ~C 6 Alkyl, unsubstituted C 1 ~C 6It refers to a group containing a 3-11 membered heterocyclyl (e.g., a 5-6 membered heteroaryl containing 1-4 heteroatoms selected from O, N, and S, or a 4-11 membered heterocycloalkyl containing 1-4 heteroatoms selected from O, N, and S) optionally substituted with alkoxy, oxo, or NR'R''. When R' and R'' are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 3-, 4-, 5-, 6-, or 7-membered ring, the ring atoms of which are optionally substituted with N, O, or S, and the ring can be optionally substituted with halogen, OH, CN, unsubstituted C 1 ~C 6 Alkyl, unsubstituted C 1 ~C 6 It is optionally substituted with alkoxy, oxo or NR'R''. For example, -NR'R'' is meant to include 1-pyrrolidinyl and 4-morpholinyl.

[0037] Similarly, optional substituents for aryl and heteroaryl groups are variable. In some embodiments, substituents for aryl and heteroaryl groups are halogen; CN; NO; N 3 ;-OR'; Perfluoro-C 1~ C 4 Alkoxy; Unsubstituted C 3 ~C 7 Cycloalkyl; halogen, OH, CN, unsubstituted C 1 ~C 6 Alkyl, unsubstituted C 1 ~C 6 C substituted with alkoxy, oxo or NR′R″ 3 ~C 7 Cycloalkyl; Unsubstituted C 6 ~C 10 Aryl (e.g., phenyl); halogen, OH, CN, unsubstituted C 1 ~C 6 Alkyl, unsubstituted C 1 ~C 6 C substituted with alkoxy or NR'R'' 6 ~C 10Aryl; unsubstituted 3-11 membered heterocyclyl (e.g., 5-6 membered heteroaryl containing 1-4 heteroatoms selected from O, N and S, or 4-11 membered heterocycloalkyl containing 1-4 heteroatoms selected from O, N and S); halogen, OH, CN, unsubstituted C 1 ~C 6 Alkyl, unsubstituted C 1 ~C 6 3-11 membered heterocyclyl substituted with alkoxy, oxo or NR'R'' (e.g. 5-6 membered heteroaryl containing 1-4 heteroatoms selected from O, N and S or 4-11 membered heterocycloalkyl containing 1-4 heteroatoms selected from O, N and S); -NR'R''; -SR'; -SiR'R''R'''; -OC(O)R'; -C(O)R'; -CO 2 R';-CONR'R'';-OC(O)NR'R'';-NR''C(O)R';-NR'''C(O)NR'R'';-NR''C(O) 2 R';-S(O) 2 R';-S(O) 2 NR'R'';-NR'S(O) 2 R'';-NR'''S(O) 2 NR'R'';Amidinyl;Guanidinyl;-(CH 2 ) 1~4 -OR';-(CH 2 ) 1~4 -NR'R'';-(CH 2 ) 1~4 -SR';-(CH 2 ) 1~4 -SiR'R''R''';-(CH 2 ) 1~4 -OC(O)R';-(CH 2 ) 1~4 -C(O)R';-(CH 2 ) 1-4 -CO 2 R'; and -(CH 2 ) 1-4 R′ and R″ are each independently selected from the group consisting of hydrogen; unsubstituted C 1~ C 6Alkyl; halogen, OH, CN, unsubstituted C 1 ~C 6 Alkyl, unsubstituted C 1 -C 6 C substituted with alkoxy, oxo or NR′R″ 1~ C 6 Alkyl; Unsubstituted C 1~ C 6 Heteroalkyl; halogen, OH, CN, unsubstituted C 1 ~C 6 Alkyl, unsubstituted C 1 ~C 6 C substituted with alkoxy, oxo or NR′R″ 1~ C 6 Heteroalkyl; Unsubstituted C 6~ C 10 Aryl; halogen, OH, CN, unsubstituted C 1 ~C 6 Alkyl, unsubstituted C 1 ~C 6 C substituted with alkoxy or NR'R'' 6~ C 10 aryl; unsubstituted 3-11 membered heterocyclyl (e.g., 5-6 membered heteroaryl containing 1-4 heteroatoms selected from O, N, and S, or 4-11 membered heterocycloalkyl containing 1-4 heteroatoms selected from O, N, and S); and halogen, OH, CN, unsubstituted C 1 ~C 6 Alkyl, unsubstituted C 1 ~C 6 It refers to a group containing a 3-11 membered heterocyclyl (e.g., a 5-6 membered heteroaryl containing 1-4 heteroatoms selected from O, N, and S, or a 4-11 membered heterocycloalkyl containing 1-4 heteroatoms selected from O, N, and S) optionally substituted with alkoxy, oxo, or NR'R''. When R' and R'' are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 3-, 4-, 5-, 6-, or 7-membered ring, the ring atoms of which are optionally substituted with N, O, or S, and the ring can be optionally substituted with halogen, OH, CN, unsubstituted C 1 ~C 6 Alkyl, unsubstituted C 1 ~C 6It is optionally substituted with alkoxy, oxo or NR'R''. For example, -NR'R'' is meant to include 1-pyrrolidinyl and 4-morpholinyl.

[0038] The term "oxo" means =O or (=O) 2 Refers to...

[0039] As used herein, a wavy line crossing a bond in a chemical structure TIFF0007682110000002.tif6170 shows the attachment points of atoms in chemical structures where wavy bonds attach them to the rest of the molecule, or to the rest of a fragment of a molecule. In some embodiments, arrows with asterisks are used as wavy lines to show the attachment points.

[0040] In certain embodiments, a divalent group is described generally without a specific bond structure. Unless otherwise specified, it is understood that the general description is meant to include both bond structures. For example, the group R 1 -R 2 -R 3 In the formula, the group R 2 Ga-CH 2 When depicted as C(O)-, unless otherwise specified, the group is R 1 -CH 2 C(O)-R 3 and R 1 -C(O)CH 2 -R 3 It is understood that both are combinable.

[0041] Terms such as "compound(s) of the invention" and "compound(s) of the present invention" include compounds of formula (I) herein, such as compounds 1-18, which are sometimes referred to as JAK inhibitors, including stereoisomers (including atropisomers), geometric isomers, tautomers, solvates, metabolites, isotopes, salts (e.g., pharma- ceutically acceptable salts), and prodrugs thereof, unless otherwise specified. In some embodiments, solvates, metabolites, isotopes, or prodrugs, or any combination thereof, are excluded.

[0042] The phrase "pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other side effects when properly administered to an animal, such as a human.

[0043] The compounds of the present invention can be in the form of salts, such as pharma- ceutically acceptable salts. "Pharmaceutically acceptable salts" includes both acid and base addition salts. "Pharmaceutically acceptable acid addition salts" refers to salts which retain the biological effectiveness and properties of the free base and which are biologically or otherwise desirable, formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, carbonic acid, phosphoric acid, and the like, and the organic acids can be selected from the aliphatic, alicyclic, aromatic, araliphatic, heterocyclic, carboxylic, and sulfonic acid 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, maloneic acid, succinic acid, fumaric acid, tartaric acid, citric acid, aspartic acid, ascorbic acid, glutamic acid, anthranilic acid, benzoic acid, cinnamic acid, mandelic acid, embonic acid, phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like.

[0044] "Pharmaceutically acceptable base addition salts" include salts derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, etc. Specific base addition salts are ammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, naturally occurring substituted amines, cyclic amines, and substituted amines including basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, tromethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purine, piperidine, piperidine, N-ethylpiperidine, polyamine resins, and the like. Specific organic non-toxic bases include isopropylamine, diethylamine, ethanolamine, tromethamine, dicyclohexylamine, choline, and caffeine.

[0045] In some embodiments, the salt is a hydrochloride, hydrobromide, trifluoroacetate, sulfate, phosphate, acetate, fumarate, maleate, tartrate, lactate, citrate, pyruvate, succinate, oxalate, methanesulfonate, p-toluenesulfonate, bisulfate, benzenesulfonate, ethanesulfonate, malonate, xinafoate, ascorbate, oleate, nicotinate, saccharate, adipate, formate, glycolate, palmitate, L-lactate, D-lactate, aspartate, malate, L-tartrate, D-tartrate, stearate, furoate (e.g., 2-furoate or 3-furoate), napadisylate (naphthalene-1,5-disulfonate, or naphthalene-1(sulfonic acid)-5-sulfonate), edisylate (ethane-1,2-disulfonic acid), salt, or ethane-1-(sulfonic acid)-2-sulfonate), isethionate (2-hydroxyethylsulfonate), 2-mesitylenesulfonate, 2-naphthalenesulfonate, 2,5-dichlorobenzenesulfonate, D-mandelate, L-mandelate, cinnamate, benzoate, adipate, esylate, malonate, mesitylate (2-mesitylenesulfonate), napsylate (2-naphthalenesulfonate), phthalenesulfonate), camsylate (camphor 10-sulfonate, e.g. (1S)-(+)-10-camphor-sulfonate), glutamate, glutarate, hippurate (2-(benzoylamino)acetate), orotate, xylate (p-xylene-2-sulfonate), and pamoate (2,2'-dihydroxy-1,1'-dinaphthylmethane-3,3'-dicarboxylate).

[0046] A "sterile" preparation is aseptic or free of all viable microorganisms and their spores.

[0047] "Stereoisomers" refer to compounds which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space. Stereoisomers include diastereomers, enantiomers, conformers, and the like.

[0048] "Chiral" refers to molecules that have the property of being non-superimposable on their mirror image partners, while the term "achiral" refers to molecules that are superimposable on their mirror image partners.

[0049] "Diastereomer" refers to a stereoisomer that has more than one chiral center and whose molecules are not mirror images of one another. Diastereomers have different physical properties, such as melting points, boiling points, spectroscopic properties, or biological activity. Mixtures of diastereomers can separate under high resolution analytical procedures such as electrophoresis and chromatography such as HPLC.

[0050] "Enantiomers" refers to two stereoisomers of a compound which are non-superimposable mirror images of one another.

[0051] Stereochemical definitions and conventions used herein generally follow 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. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. In describing optically active compounds, the prefixes D and L, or R and S, are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and l, or (+) and (-), are used to denote the sign of rotation of plane-polarized light by the compound, with (-) or 1 meaning that the compound is levorotatory. Compounds with the prefix (+) or D are dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of each other. A specific stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which may occur where there has been no stereoselection or stereospecificity in a chemical reaction or process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric species, devoid of optical activity.

[0052] The term "tautomer" or "tautomeric form" refers to structural isomers with different energies that are interconvertible via a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions via migration of a proton, such as keto-enol and imine-enamine isomerizations. Valence tautomers include interconversions via reorganization of some of the bonding electrons.

[0053] Certain compounds of the present invention can exist in unsolvated as well as solvated forms, including hydrated forms. "Solvate" refers to an association or complex of one or more solvent molecules and a compound of the present invention. Examples of solvents that form solvates include water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine. Certain compounds of the present invention can exist in multiple crystalline or amorphous forms. In general, all physical forms are intended to be within the scope of the present invention. The term "hydrate" refers to a complex in which the solvent molecule is water.

[0054] "Metabolite" refers to a product produced through metabolism in the body of a specified compound or a salt thereof. Such products may result, for example, from the oxidation, reduction, hydrolysis, amidation, deamidation, esterification, deesterification, enzymatic cleavage, and the like, of the administered compound.

[0055] The metabolites are typically radiolabeled (e.g., 14 C or 3 H) isotopes are prepared, administered to animals such as rats, mice, guinea pigs, monkeys, or humans at detectable doses (e.g., greater than about 0.5 mg / kg), allowed sufficient time (typically about 30 seconds to 30 hours) for metabolism to occur, and identified by isolating the conversion products from urine, blood, or other biological samples. Such products are easily isolated because they are labeled (others are isolated by the use of antibodies that can bind to epitopes remaining in the metabolites). The structures of the metabolites are determined by conventional methods, such as MS, LC / MS, or NMR analysis. In general, the analysis of the metabolites is performed in the same manner as conventional drug metabolism studies well known to those skilled in the art. The metabolites are useful in diagnostic assays for therapeutic administration of the compounds of the invention, unless otherwise found in vivo.

[0056] A "subject," "individual," or "patient" is a vertebrate. In certain embodiments, the vertebrate is a mammal. Mammals include, but are not limited to, farm animals (such as cows), sport animals, pets (such as guinea pigs, cats, dogs, rabbits, and horses), primates, mice, and rats. In certain embodiments, the mammal is a human. In embodiments involving administering to a patient a JAK inhibitor or a pharma- ceutically acceptable salt thereof as described herein, the patient may be in need thereof.

[0057] The term "Janus kinase" refers to JAK1, JAK2, JAK3, and TYK2 protein kinases. In some embodiments, a Janus kinase may be further defined as one of JAK1, JAK2, JAK3, or TYK2. In any embodiment, any one of JAK1, JAK2, JAK3, and TYK2 may be specifically excluded as a Janus kinase. In some embodiments, a Janus kinase is JAK1. In some embodiments, a Janus kinase is a combination of JAK1 and JAK2.

[0058] The terms "inhibit" and "reduce," or any variation of these terms, include any measurable reduction or complete inhibition to achieve a desired result. For example, there may be a decrease in activity (e.g., JAK1 activity) compared to normal, of about, up to about, or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more, or any variable of these ranges.

[0059] "Therapeutically effective amount" refers to an amount of a compound or salt (e.g., a pharma- ceutically acceptable salt thereof) of the present invention that (i) treats or prevents a particular disease, condition, or disorder, or (ii) attenuates, ameliorates, or eliminates one or more symptoms of a particular disease, condition, or disorder, and optionally (iii) prevents or delays the onset of one or more symptoms of a particular disease, condition, or disorder described herein. In some embodiments, a therapeutically effective amount is an amount sufficient to reduce or alleviate symptoms of an autoimmune or inflammatory disease (e.g., asthma). In some embodiments, a therapeutically effective amount is an amount of a chemical entity described herein sufficient to significantly reduce the activity or number of B cells. In the case of cancer, a therapeutically effective amount of an agent can reduce the number of cancer cells, reduce tumor size, inhibit (i.e., slow to a certain degree, and preferably stop) the invasion of cancer cells into peripheral organs, inhibit (i.e., slow to a certain degree, and preferably stop) tumor metastasis, inhibit (i.e., slow to a certain degree, and preferably stop) tumor growth to a certain degree, or reduce to a certain degree one or more symptoms associated with cancer. To the extent the drug may prevent growth of or kill existing cancer cells, it may be cytostatic or cytotoxic. With respect to cancer therapy, efficacy can be measured, for example, by assessing the time to disease progression (TTP) or determining the response rate (RR).

[0060] "Treatment" (and variations such as "treat" or "treating") refers to a clinical intervention to alter the natural course of the individual or cell being treated, and can be performed to prevent or during the course of a clinical disease state. Desired effects of treatment include prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, stable (i.e., not worsening) state of disease, slowing of the rate of disease progression, amelioration or alleviation of disease symptoms, increased survival compared to expected survival in the absence of treatment, and remission or improved prognosis. In some embodiments, the compounds of the invention, or salts thereof (e.g., pharma- ceutically acceptable salts thereof), are used to delay the development of a disease or disorder or to slow the progression of a disease or disorder. Subjects in need of treatment include those already having the condition or disorder, and those who are predisposed to having the condition or disorder (e.g., due to genetic mutations) or those in whom the condition or disorder is to be prevented.

[0061] "Inflammatory disorder" refers to any disease, disorder, or syndrome in which an excessive or unregulated inflammatory response leads to excessive inflammatory symptoms, host tissue damage, or loss of tissue function. "Inflammatory disorder" also refers to a pathological condition mediated by the influx of leukocytes or neutrophil chemotaxis.

[0062] "Inflammation" refers to a local defense response induced by tissue injury or destruction, which serves to destroy, dilute, or block (sequester) both the injurious agent and the injured tissue. Inflammation is particularly associated with the influx of leukocytes or chemotaxis of neutrophils. Inflammation can result from infection with pathogenic organisms and viruses, as well as from trauma or reperfusion after myocardial infarction or stroke, immune responses to foreign antigens, and autoimmune responses. Thus, inflammatory disorders suitable for treatment with the compounds of the present invention or salts thereof (e.g., pharma- ceutically acceptable salts thereof) include disorders associated with specific defense system reactions as well as non-specific defense system reactions.

[0063] "Specific defense system" refers to the components of the immune system that respond to the presence of specific antigens. Examples of inflammation resulting from a response of the specific defense system include classical responses to foreign antigens, autoimmune diseases, and delayed hypersensitivity reactions mediated by T cells. Chronic inflammatory diseases, rejection of solid transplanted tissues and organs, such as kidney and bone marrow transplants, and graft-versus-host disease (GVHD) are further examples of inflammatory reactions of the specific defense system.

[0064] The term "non-specific defense system" refers to inflammatory disorders mediated by leukocytes (e.g., granulocytes and macrophages) that are incapable of immunological memory. Examples of inflammation resulting at least in part from a reaction of the non-specific defense system include inflammation associated with conditions such as adult (acute) respiratory distress syndrome (ARDS) or multiple organ injury syndrome; reperfusion injury; acute glomerulonephritis; reactive arthritis; skin diseases with an acute inflammatory component; other central nervous system inflammatory disorders such as acute purulent meningitis or stroke; burns; inflammatory bowel disease; granulocyte transfusion-associated syndrome; and cytokine-induced toxicity.

[0065] "Autoimmune disease" refers to any of a group of disorders in which tissue damage is associated with humoral or cell-mediated responses to the body's own components. Non-limiting examples of autoimmune diseases include rheumatoid arthritis, lupus, and multiple sclerosis.

[0066] As used herein, "allergic disease" refers to any symptom, tissue damage, or loss of tissue function resulting from allergies. As used herein, "arthritic disease" refers to any disease characterized by inflammatory lesions of the joints resulting from a variety of etiologies. As used herein, "dermatitis" refers to any of a large family of skin diseases characterized by inflammation of the skin resulting from a variety of etiologies. As used herein, "transplant rejection" refers to any immune response against transplanted tissue, such as an organ or cells (e.g., bone marrow), characterized by loss of function of the transplanted tissue and surrounding tissue, pain, swelling, leukocytosis, and thrombocytopenia. The therapeutic methods of the present invention include methods for treating disorders associated with inflammatory cell activation.

[0067] "Inflammatory cell activation" refers to the induction by stimuli of a proliferative cell response (including, but not limited to, cytokines, antigens or autoantibodies), the production of soluble mediators (including, but not limited to, cytokines, oxygen radicals, enzymes, prostaglandins, or vasoactive amines), or the cell surface expression of new or increased numbers of mediators (including, but not limited to, major histocompatibility antigens or cell adhesion molecules) in inflammatory cells (including, but not limited to, monocytes, macrophages, T lymphocytes, B lymphocytes, granulocytes (i.e., polymorphonuclear leukocytes such as neutrophils, basophils, eosinophils, etc.), mast cells, dendritic cells, Langerhans cells, and endothelial cells). One of ordinary skill in the art will understand that activation of one or a combination of these phenotypes in these cells can contribute to the initiation, persistence, or exacerbation of inflammatory disorders.

[0068] In some embodiments, inflammatory disorders that can be treated according to the methods of the invention include, but are not limited to, asthma, rhinitis (e.g., allergic rhinitis), allergic airway syndrome, atopic dermatitis, bronchitis, rheumatoid arthritis, psoriasis, contact dermatitis, chronic obstructive pulmonary disease (COPD), and delayed-type hypersensitivity reactions.

[0069] The terms "cancer" and "cancerous," "neoplasm," and "tumor," and related terms, refer to or describe a physiological condition in mammals that is typically characterized by unregulated growth of cells. A "tumor" comprises one or more cancer cells. Examples of cancer include carcinoma, blastoma, sarcoma, seminoma, glioblastoma, melanoma, leukemia, and myeloid or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), and lung cancer (including small cell lung cancer, non-small cell lung cancer ("NSCLC"), adenocarcinoma of the lung, and squamous cell carcinoma of the lung). Other cancers include skin cancer, keratoacanthoma, follicular carcinoma, hairy cell leukemia, oral cavity cancer, pharyngeal (mouth) cancer, lip cancer, tongue cancer, mouth cancer, salivary gland cancer, esophageal cancer, laryngeal cancer, hepatocellular carcinoma, gastric cancer, stomach cancer, gastrointestinal cancer, small intestine cancer, large intestine cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, genitourinary cancer, biliary tract cancer, thyroid cancer, papillary cancer, hepatic cancer, uterine cancer, uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, testicular cancer, vulvar cancer, peritoneal cancer, anal cancer, penile cancer, bone cancer, multiple myeloma, B cell lymphoma, central nervous system, brain cancer, head and neck cancer, Hodgkin's disease, and associated metastases. Examples of neoplastic disorders include myeloproliferative disorders such as polycythemia vera, essential thrombocytosis, myelofibrosis such as primary myelofibrosis, and chronic myelogenous leukemia (CML).

[0070] A "chemotherapeutic agent" is an agent useful in the treatment of a given disorder, such as cancer or an inflammatory disorder. Examples of chemotherapeutic agents are known in the art and include examples such as those described in U.S. Patent Application Publication No. 2010 / 0048557, which is incorporated herein by reference. Additionally, chemotherapeutic agents include any pharma- ceutically acceptable salt, acid, or derivative of the chemotherapeutic agent, and combinations of two or more thereof.

[0071] "Package insert" is used to refer to instructions customarily included in commercial packaging of a therapeutic agent that contain information about the indications, usage, dosage, administration, contraindications, or warnings concerning such therapeutic agent's use.

[0072] Unless otherwise stated, structures depicted herein include compounds which differ only in the presence of one or more isotopically enriched atoms. Exemplary isotopes that can be incorporated into the compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, and iodine, respectively, e.g. 2 H, 3 H, 11 C. 13 C. 14 C. 13 N, 15 N, 15 O. 17 O. 18 O. 32 P, 33 P, 35 S, 18 F, 36 Cl, 123 I, and 125 Isotopically labeled compounds (e.g., 3 H and 14 C-labeled compounds) can be useful in compound or substrate tissue distribution assays. 3 H) and carbon-14 (i.e. 14 C) isotopes can be useful because of their ease of preparation and detectability. Additionally, heavier isotopes, such as deuterium (i.e., 2 Substitution with, for example, H, may result in greater metabolic stability and, therefore, certain therapeutic advantages (e.g., longer in vivo half-life or reduced dosage requirements). In some embodiments, one or more hydrogen atoms may be replaced by 2 H or 3 H or one or more carbon atoms are replaced 13 C- or 14 Replaced by C-enriched carbon. 15 O. 13 N, 11 C, and 18Positron emitting isotopes such as F are useful for positron emission tomography (PET) studies to investigate substrate receptor occupancy. Isotopically labeled compounds can generally be prepared by procedures similar to those described in the schemes or examples herein, substituting an isotopically labeled reagent for a non-isotopically labeled reagent.

[0073] It is specifically contemplated that any limitation discussed with respect to one embodiment of the invention may be applied to any other embodiment of the invention. Furthermore, any compound or salt thereof (e.g., a pharma- ceutically acceptable salt thereof) or composition of the invention may be used in any method of the invention, and any method of the invention may be used to make or utilize any compound or salt thereof (e.g., a pharma- ceutically acceptable salt thereof) or composition of the invention.

[0074] Use of the term "or" means "and / or" notwithstanding that the present disclosure supports a definition that refers to alternatives only and "and / or," unless expressly indicated to refer to alternatives only or that the alternatives are mutually exclusive.

[0075] Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device or method being employed to measure the value.

[0076] As used herein, unless otherwise specified, "a" or "an" means one or more. As used herein, "another" means at least a second, or more.

[0077] The headings used herein are intended for organizational purposes only. JANUS kinase inhibitors One embodiment is a compound of formula (I): TIFF0007682110000003.tif62170(I) or a stereoisomer or a pharma- ceutically acceptable salt thereof, During the ceremony, R 1 is hydroxyl-C 1 ~C 6 Alkyl;-(CR a1 R a2 ) m -het 1 ;-(CR a1 R a2 ) n -NR b R c ; or -(CR a1 R a2 ) m -C 3~6 Cycloalkyl, C 3~6 The cycloalkyl moiety is R d is substituted once with; R 2 Ha, Halo; Halo C 1 ~C 6 Alkoxy;C 1 ~C 6 Alkylthio;-SF 2 ; or C 3 ~C 6 is cycloalkyl; R 3 is hydrogen; or C 1 ~C 6 is alkyl; R 4 is hydrogen; or C 1 ~C 6 is alkyl; R 5 is hydrogen; or C 1 ~C 6 is alkyl; R 6 is hydrogen; or C 1 ~C 6 is alkyl; Or R 2 and R 6 can form, together with the atom to which they are attached, a six-membered ring containing two heteroatoms each independently selected from O, N, and S; m is 0 to 2; n is 0 to 3; Each R a1 are independently hydrogen; or C 1 ~C6 is alkyl; Each R a2 are independently hydrogen; halo; or C 1 ~C 6 is alkyl; R b is hydrogen; or C 1 ~C 6 is alkyl; R c is hydrogen; C 1 ~C 6 alkyl; an amino protecting group; or may be unsubstituted or C 1 ~C 6 azetidinyl optionally substituted once with alkyl; het 1 is a heterocyclyl selected from azetidinyl; pyrrolidinyl; piperazinyl; piperidinyl; morpholinyl; and oxetanyl; each of which may be unsubstituted or R d Once at R g may be substituted once or twice with; R d is -(CR a1 R a2 ) p -het 2 ;-(CR a1 R a2 ) q -NR e R f ; or -(CR a1 R a2 ) p -C 3~6 Cycloalkyl, C 3~6 The cycloalkyl moiety is -NR e R f is substituted once with; p is 0 to 2; q is 0 to 4; R e is hydrogen; or C 1 ~C 6 is alkyl; R f is hydrogen; C 1 ~C 6 Alkyl; or CH 2 C2 N(CH 3 ) 2 and; Each R g is C 1 ~C 6 alkyl; or halo; and Het 2 is a heterocycle selected from tetrahydropyranyl; azetidinyl; and pyrrolidinyl; each of which may be unsubstituted or selected from C 1 ~C 6 Alkyl or -NR e R f It may be substituted once by

[0078] In certain embodiments, R 1 is C 1 ~C 6 Alkyl; Hydroxyl-C 1 ~C 6 Alkyl;-(CR a1 R a2 ) m -het 1 ; or (CR a1 R a2 ) n -NR b R c And het 1 may be unsubstituted, or R d It may be substituted once by

[0079] In certain embodiments, R 1 is C 1 ~C 6 Alkyl;-(CR a1 R a2 ) m -het 1 ; or -(CHR a ) n -NR b R c And het 1 .

[0080] In certain embodiments, R 1 is -(CR a1 R a2 ) m-het 1 ; or -(CR a1 R a2 ) n -NR b R c And het 1 may be unsubstituted, or R d It may be substituted once by

[0081] In certain embodiments, R 1 is -(CHR a ) m -het 1 And het 1 may be unsubstituted or R d It may be substituted once by

[0082] In certain embodiments, R 1 CR a1 R a2 ) n -NR b R c It is.

[0083] In certain embodiments, R 2 Ha, Halo; Halo C 1 ~C 6 Alkoxy; or C 1 ~C 6 It is alkylthio.

[0084] In certain embodiments, R 2 is a halo.

[0085] In certain embodiments, R 2 is HaroC 1 ~C 6 It is an alkoxy.

[0086] In certain embodiments, R 2 is C 1 ~C 6 It is alkylthio.

[0087] In certain embodiments, R 2is chloro; difluoromethoxy; methylethio; or cyclopropyl.

[0088] In certain embodiments, R 2 is chloro.

[0089] In certain embodiments, R 2 is difluoromethoxy.

[0090] In certain embodiments, R 2 is methylthio.

[0091] In certain embodiments, R 3 is hydrogen.

[0092] In certain embodiments, R 4 is hydrogen.

[0093] In certain embodiments, R 5 is hydrogen.

[0094] In certain embodiments, R 6 is hydrogen.

[0095] In certain embodiments, R 2 and R 6 together with the atoms to which they are attached form a six-membered ring containing two heteroatoms each independently selected from O, N and S.

[0096] In certain embodiments, m is 0. When m is 0, R 1 Het 1 In an embodiment, 1 The bond connecting the to the tetrazole ring is a het rather than a heteroatom. 1 It should be understood that the aryl group is made up of carbon atoms.

[0097] In certain embodiments, m is 0.

[0098] In certain embodiments, m is 1.

[0099] In certain embodiments, m is 2.

[0100] In certain embodiments, n is 0.

[0101] In certain embodiments, n is 1.

[0102] In certain embodiments, n is 2.

[0103] In certain embodiments, R a1 is hydrogen.

[0104] In certain embodiments, R a2 is hydrogen.

[0105] In certain embodiments, R b is hydrogen.

[0106] In certain embodiments, R b is C 1 ~C 6 It is an alkyl.

[0107] In certain embodiments, R c is hydrogen.

[0108] In certain embodiments, R c is C 1 ~C 6 It is an alkyl.

[0109] In certain embodiments, R c is 1-methyl-azetidin-3-yl.

[0110] In certain embodiments, het 1 may be unsubstituted or R d is azetidinyl optionally substituted once by

[0111] In certain embodiments, het 1 may be unsubstituted or R dand pyrrolidinyl which is optionally substituted once by.

[0112] In certain embodiments, het 1 may be unsubstituted or R d and piperazinyl which is optionally substituted once by.

[0113] In certain embodiments, het 1 may be unsubstituted or R d and piperidinyl optionally substituted once by.

[0114] In certain embodiments, het 1 is morpholinyl.

[0115] In certain embodiments, het 1 is oxetanyl.

[0116] In certain embodiments, p is 0.

[0117] In certain embodiments, p is 1.

[0118] In certain embodiments, p is 2.

[0119] In certain embodiments, q is 0.

[0120] In certain embodiments, q is 2.

[0121] In certain embodiments, q is 3.

[0122] In certain embodiments, q is 4.

[0123] In certain embodiments, R e is hydrogen.

[0124] In certain embodiments, R e is C 1 ~C 6 It is an alkyl.

[0125] In certain embodiments, R f is hydrogen.

[0126] In certain embodiments, R f is C 1 ~C 6 It is an alkyl.

[0127] In certain embodiments, Het 2 is tetrahydropyranyl.

[0128] In certain embodiments, Het 2 may be unsubstituted or C 1 ~C 6 It is azetidinyl optionally substituted once with alkyl.

[0129] In certain embodiments, Het 2 may be unsubstituted or C 1 ~C 6 Pyrrolidinyl optionally substituted once with alkyl.

[0130] In certain embodiments, R 1 teeth, Selected from TIFF0007682110000004.tif246170TIFF0007682110000005.tif77170. In certain embodiments, R 1 teeth, Selected from TIFF0007682110000006.tif179170.

[0131] In certain embodiments, the subject compound has formula (II): TIFF0007682110000007.tif61170(II) or a stereoisomer or a pharma- ceutically acceptable salt thereof, wherein R 1 , R 2 , and R 6 is as defined herein.

[0132] In certain embodiments, the subject compound has formula (III): TIFF0007682110000008.tif61170(III) or a stereoisomer or a pharma- ceutically acceptable salt thereof, wherein R 1 , R 2 , and R 6 is as defined herein.

[0133] In certain embodiments, the subject compound has formula (IV): TIFF0007682110000009.tif60170(IV) or a stereoisomer or a pharma- ceutically acceptable salt thereof, In the formula, X is -O- or -S-; R g is hydrogen or C 1 ~C 6 is alkyl, R 1 is as defined herein.

[0134] In certain embodiments, the subject compound has formula (V): TIFF0007682110000010.tif60170(V) or a stereoisomer or a pharma- ceutically acceptable salt thereof, In the formula, R 2 and R d is as defined herein.

[0135] In certain embodiments, the subject compound has formula (VI): TIFF0007682110000011.tif60170(VI) or a stereoisomer or a pharma- ceutically acceptable salt thereof, In the formula, R 2 and R d is as defined herein.

[0136] Also provided is a pharmaceutical composition comprising a JAK inhibitor described herein, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier, diluent, or excipient.

[0137] Also provided is the use of a JAK inhibitor as described herein, or a pharma- ceutically acceptable salt thereof, in therapy, such as the treatment of an inflammatory disease (e.g., asthma).Also provided is the use of a JAK inhibitor as described herein, or a pharma- ceutically acceptable salt thereof, to prepare a medicament for the treatment of an inflammatory disease.Also provided is a method of preventing, treating, or reducing the severity of a disease or condition responsive to inhibition of Janus kinase activity in a patient, comprising administering to the patient a therapeutically effective amount of a JAK inhibitor as described herein, or a pharma- ceutically acceptable salt thereof.

[0138] In one embodiment, the disease or condition for treatment is cancer, polycythemia vera, essential thrombocytosis, myelofibrosis, chronic myelogenous leukemia (CML), rheumatoid arthritis, inflammatory bowel syndrome, Crohn's disease, psoriasis, contact dermatitis, or delayed hypersensitivity reactions.

[0139] In one embodiment, there is provided a use of a JAK inhibitor as described herein, or a pharma- ceutically acceptable salt thereof, for the treatment of cancer, polycythemia vera, essential thrombocytosis, myelofibrosis, chronic myelogenous leukemia (CML), rheumatoid arthritis, inflammatory bowel syndrome, Crohn's disease, psoriasis, contact dermatitis, or delayed hypersensitivity reactions.

[0140] In one embodiment, a composition formulated for administration by inhalation is provided.

[0141] In one embodiment, a metered dose inhaler is provided comprising a compound of the invention or a pharma- ceutically acceptable salt thereof.

[0142] In one embodiment, the JAK inhibitors described herein, or pharma- ceutically acceptable salts thereof, are at least 10 times more potent as inhibitors of JAK1 than as inhibitors of LRRK2.

[0143] In one embodiment, a method is provided for treating hair loss in a mammal, comprising administering to the mammal a JAK inhibitor described herein, or a pharma- ceutically acceptable salt thereof.

[0144] In one embodiment, there is provided a use of a JAK inhibitor, or a pharma- ceutically acceptable salt thereof, as described herein for the treatment of hair loss.

[0145] In one embodiment, there is provided a use of a JAK inhibitor, or a pharma- ceutically acceptable salt thereof, as described herein for the preparation of a medicament for treating hair loss in a mammal.

[0146] The compounds of the present invention may contain one or more asymmetric carbon atoms. Thus, the compounds may exist as diastereomers, enantiomers, or mixtures thereof. The synthesis of the compounds may use racemates, diastereomers, or enantiomers as starting materials or intermediates. A mixture of specific diastereomeric compounds may be separated or enriched into one or more specific diastereomers by chromatographic or crystallization techniques. Similarly, the same techniques, or other techniques known in the art, may be used to separate or enantiomerically enrich an enantiomeric mixture. Each of the asymmetric carbon or nitrogen atoms may be in the R or S configuration, and both of these configurations are within the scope of the present invention.

[0147] In the structures depicted herein, where the stereochemistry of any particular chiral atom is not specified, all stereoisomers are contemplated and included as the compounds of the present invention. Where stereochemistry is specified by a solid wedge or dashed line representing a particular configuration, that stereoisomer is so specified and defined. Unless otherwise indicated, relative stereochemistry is intended when a solid wedge or dashed line is used.

[0148] Another embodiment includes prodrugs of the compounds described herein that contain known amino- and carboxy-protecting groups that are released, e.g., hydrolyzed, under physiological conditions to yield the compounds of the invention.

[0149] The term "prodrug" refers to a precursor or derivative form of a pharmaceutically active substance that is less active in patients compared to the parent drug and can be activated by enzymes or hydrolysis or converted to a more active parent form. See, for example, Wilman, ‘‘Prodrugs in Cancer Chemotherapy’’ Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) and Stella et al., ‘‘Prodrugs: A Chemical Approach to Targeted Drug Delivery,’’ Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267, Humana Press (1985). Prodrugs include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, β-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, and 5-fluorocytosine and 5-fluorouridine prodrugs.

[0150] A particular class of prodrugs are compounds in which the nitrogen atom in the amino, amidino, aminoalkyleneamino, iminoalkyleneamino or guanidino group is replaced with a hydroxy group, an alkylcarbonyl (-CO-R) group, an alkoxycarbonyl (-CO-OR) or an acyloxyalkyl-alkoxycarbonyl (-CO-ORO-CO-R) group, where R is a monovalent or divalent group, such as alkyl, alkylene or aryl, or a group of the formula -C(O)-O-CP1P2-haloalkyl, where P1 and P2 are the same or different and are hydrogen, alkyl, alkoxy, cyano, halogen, alkyl or aryl. In a particular embodiment, the nitrogen atom is one of the nitrogen atoms of the amidino group. Prodrugs can be prepared by reacting a compound with an activated group, such as an acyl group, for example, by binding the nitrogen atom in the compound to the exemplary carbonyl of the activated acyl group. Examples of activated carbonyl compounds are compounds containing a leaving group attached to the carbonyl group, such as acyl halides, acyl amines, acyl pyridinium salts, acyl alkoxides, acyl phenoxides (e.g., p-nitrophenoxyacyl, dinitrophenoxyacyl, fluorophenoxyacyl, and difluorophenoxyacyl). The reaction is generally carried out in an inert solvent at low temperatures, such as from -78°C to about 50°C. The reaction can also be carried out in the presence of an inorganic base, such as potassium carbonate or sodium bicarbonate, or an organic base, such as an amine, including pyridine, trimethylamine, triethylamine, triethanolamine, and the like.

[0151] Additional types of prodrugs are also included. For example, the free carboxyl group of the JAK inhibitors described herein can be derivatized as an amide or alkyl ester. As another example, compounds of the present invention that contain a free hydroxy group can be derivatized as a prodrug by converting the hydroxy group to a group such as, but not limited to, a phosphate ester, a hemisuccinate, a dimethylaminoacetate, or a phosphoryloxymethyloxycarbonyl group, as outlined in Fleisher, D. et al., (1996) Improved oral drug delivery: solubility limitations overcome by the use of prodrugs (Advanced Drug Delivery Reviews, 19:115). Carbamate prodrugs of hydroxy and amino groups are also included, as are carbonate prodrugs, sulfonate esters, and sulfate esters of hydroxy groups. Derivatization of hydroxy groups as (acyloxy)methyl and (acyloxy)ethyl ethers is also included, where the acyl group can be an alkyl ester optionally substituted with groups including, but not limited to, ether, amine, and carboxylic acid functionalities, or where the acyl group is an amino acid ester as described above. This type of prodrug is described in J. Med. Chem., (1996), 39:10. More specific examples include prodrugs in which the hydrogen atom of the alcohol group is replaced with (C 1~ C 6 ) alkanoyloxymethyl, 1-((C 1~ C 6 )alkanoyloxy)ethyl, 1-methyl-1-((C 1~ C 6 )alkanoyloxy)ethyl, (C 1~ C 6 ) alkoxycarbonyloxymethyl, N-(C 1~ C 6 ) alkoxycarbonylaminomethyl, succinoyl, (C 1~ C 6 ) Alkanoyl, alpha-amino (C 1~ C 4) alkanoyl, arylacyl, and alpha-aminoacyl or alpha-aminoacyl-alpha-aminoacyl (each alpha-aminoacyl group is independently selected from the naturally occurring L-amino acids, P(O)(OH) 2 , -P(O)(O(C 1~ C 6 )Alkyl) 2 or glycosyl (a group resulting from removal of a hydroxyl group from the hemiacetal form of a carbohydrate).

[0152] "Leaving group" refers to a portion of a first reactant in a chemical reaction that is displaced from that first reactant. Examples of leaving groups include, but are not limited to, halogen atoms, alkoxy, and sulfonyloxy groups. Exemplary sulfonyloxy groups include, but are not limited to, alkylsulfonyloxy groups (e.g., methylsulfonyloxy (mesylate group) and trifluoromethylsulfonyloxy (triflate group)), and arylsulfonyloxy groups (e.g., p-toluenesulfonyloxy (tosylate group) and p-nitrosulfonyloxy (nosylate group)). Synthesis of JANUS kinase inhibitor compounds

[0153] The compounds may be synthesized by the synthetic routes described herein. In certain embodiments, processes well known in the chemical arts may be used in addition to or in light of the descriptions contained herein. Starting materials are generally available from commercial sources such as Aldrich Chemicals (Milwaukee, Wisconsin) or are readily prepared using methods well known to those skilled in the art (e.g., prepared by methods generally described in Louis F. Fieser and Mary Fieser, Reagents for Organic Synthesis, v.1-19, Wiley, NY (1967-1999 ed.), Beilsteins Handbuch der organischen Chemie, 4, Aufl. ed. Springer-Verlag, Berlin (also available via the Beilstein online database), including supplements), or Comprehensive Heterocyclic Chemistry, Editors Katrizky and Rees, Pergamon Press, 1984.

[0154] The compounds may be prepared singly or as a compound library comprising at least two, for example 5-1,000 compounds, or 10-100 compounds. Libraries of compounds may be prepared by procedures known to those skilled in the art, by combinatorial "split and mix" approaches, or by multiple parallel synthesis using either solution phase or solid phase chemistry. Thus, according to a further aspect of the invention, there is provided a compound library comprising at least two compounds of the invention.

[0155] For illustrative purposes, the reaction schemes shown below provide routes for the synthesis of the compounds of the present invention as well as key intermediates. For more detailed descriptions of the individual reaction steps, see the Examples section below. Those skilled in the art will appreciate that other synthetic routes can be used. Although some specific starting materials and reagents are described in the schemes and discussed below, other starting materials and reagents can be substituted to provide a variety of derivatives or reaction conditions. In addition, many of the compounds prepared by the methods described below can be further modified in light of this disclosure using conventional chemistry well known to those skilled in the art.

[0156] In the preparation of the compounds of the present invention, protection of remote functionality (e.g., primary or secondary amines) of intermediates may be necessary. The need for such protection will vary with the nature of the remote functionality and the conditions of the preparation method. Suitable amino-protecting groups include acetyl, trifluoroacetyl, benzyl, phenylsulfonyl, t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz), and 9-fluorenylmethylenoxycarbonyl (Fmoc). The need for such protection is readily determined by one skilled in the art. For a general description of protecting groups and their uses, see TW Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, New York (1991).

[0157] Other transformations commonly used in the synthesis of compounds of the invention, which can be carried out using a variety of reagents and conditions, include the following:

[0158] (1) Reaction of carboxylic acids with amines to form amides. Such a transformation can be achieved using a variety of reagents known to those skilled in the art, but a comprehensive review can be found in Tetrahedron, 2005, 61, 10827-10852.

[0159] (2) The reaction of primary or secondary amines with aryl halides or pseudohalides, such as triflates, commonly known as the "Buchwald-Hartwig cross-coupling", can be accomplished using a variety of catalysts, ligands and bases. A review of these methods is provided in Comprehensive Organic Name Reactions and Reagents, 2010, 575-581.

[0160] (3) Palladium cross-coupling reaction between aryl halides and vinylboronic acids or boronic esters. This transformation is a member of the “Suzuki-Miyaura cross-coupling” reaction class, which has been thoroughly reviewed in Chemical Reviews, 1995, 95(7), 2457-2483.

[0161] (4) Hydrolysis of esters to give the corresponding carboxylic acids is well known to one skilled in the art and conditions include: for methyl and ethyl esters, use of a strong aqueous base such as lithium hydroxide, sodium hydroxide or potassium hydroxide, or a strong aqueous mineral acid such as HCl; for tert-butyl esters, hydrolysis will be carried out using an acid such as HCl in dioxane or trifluoroacetic acid (TFA) in dichloromethane (DCM). TIFF0007682110000012.tif200170

[0162] Reaction scheme 1 illustrates the synthesis of compounds of the invention. Compound 1 can be arylated under palladium catalyzed conditions to produce compound 2. The nitro group of compound 2 can be reduced under conditions such as iron, ammonium chloride, to produce aminoaniline 3. Amide bond coupling of commercially available pyrazolo[1,5-a]pyrimidine-3-carboxylic acid with an organic base such as, but not limited to, DIPEA and DMAP in an organic solvent such as, but not limited to, DMF in the presence of a coupling reagent such as, but not limited to, PyAOP gives compound 4. Removal of the SEM protecting group of compound 4 using an acid such as, but not limited to, HCl in a solvent such as, but not limited to, 1,4-dioxane gives compound 5. Compound 5 can then be N-alkylated with a protected tetrazole compound. In certain embodiments, the protecting group PG can be tetrahydropyranyl, so that the tetrazole reagent is 5-(chloromethyl)-2-(tetrahydro-2H-pyran-2-yl)-2H-tetrazole, to give compound 6. Deprotection using HCl or other acid gives tetrazole compound 7. Compound 7 is then reacted with R 1 Upon N-alkylation by reaction with -X (wherein X is halo, such as iodo), compounds 8 and 9 are compounds of formula (I) according to the invention. TIFF0007682110000013.tif132170

[0163] Reaction Scheme 2 illustrates the synthesis of a compound of formula VII therein. Commercially available 4-(difluoromethoxy)phenol can be treated with a brominating agent, such as, but not limited to, NBS, in a solvent, such as, but not limited to, acetic acid, to give 12. Difluoromethylation of 12 to form compound 13 can be achieved by treatment of compound 12 with diethyl(bromodifluoromethyl)phosphonate in a solvent, such as, but not limited to, acetonitrile, using a base, such as, but not limited to, aqueous potassium hydroxide. Compound 13 can be treated with 4-nitro-1-[[2-(trimethylsilyl)ethoxy]methyl]-1 H-pyrazole 4-bromo-1-(difluoromethoxy)-2-iodobenzene in a solvent, such as, but not limited to, DMA, under palladium catalyzed conditions using a base, such as, but not limited to, potassium carbonate, to give compound 14. The nitro group of compound 14 can be reduced with conditions such as iron, ammonium chloride, to give aminopyrazole 15. Amide bond coupling of compound 15 with commercially available pyrazolo[1,5-a]pyrimidine-3-carboxylic acid with an organic base, such as, but not limited to, DIPEA, and DMAP in a solvent, such as, but not limited to, DMF, in the presence of a coupling reagent, such as, but not limited to, PyAOP, provides compound 16. Removal of the SEM protecting group of compound 16 can be accomplished with an acid, such as, but not limited to, HCl, in an organic solvent, such as, but not limited to, 1,4-dioxane, to generate intermediate compound 5, which can be used to prepare compounds of the present invention, as shown in Reaction Scheme 1.

[0164] It will be appreciated that, where appropriate functional groups are present, the compounds of the various formulae, or any intermediates used in their preparation, may be further derivatized by one or more standard synthetic methods using condensation, substitution, oxidation, reduction or cleavage reactions. Particular substitution approaches include conventional alkylation, arylation, heteroarylation, acylation, sulfonylation, halogenation, nitration, formylation and coupling procedures.

[0165] In a further example, primary or secondary amine groups can be converted to amide groups (-NHCOR' or -NRCOR') by acylation. Acylation can be achieved by reaction with a suitable acid chloride in the presence of a base, such as triethylamine, in a suitable solvent, such as dichloromethane, or by reaction with a suitable carboxylic acid in the presence of a suitable coupling agent, such as HATU (O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate), in a suitable solvent, such as dichloromethane. Similarly, amine groups can be converted to sulfonamide groups (-NHSO) by reaction with a suitable sulfonyl chloride in the presence of a suitable base, such as triethylamine, in a suitable solvent, such as dichloromethane. 2 R' or -NR''SO 2 The primary or secondary amine groups can be converted to urea groups (-NHCONR'R'' or -NRCONR'R'') by reaction with a suitable isocyanate in the presence of a suitable base, such as triethylamine, in a suitable solvent, such as dichloromethane.

[0166] Amine (-NH 2 ) can be converted to nitro(-NO) by catalytic hydrogenation, for example using hydrogen, in the presence of a metal catalyst, for example palladium on a support such as carbon in a solvent such as ethyl acetate or an alcohol, for example methanol. 2 Alternatively, the conversion may be carried out by chemical reduction using a metal such as tin or iron in the presence of an acid such as hydrochloric acid.

[0167] Further examples include amines (-CH 2 NH 2 ) groups can be reacted at a suitable temperature, e.g., about -78 o It can be obtained by reduction of a nitrile (-CN) by, for example, catalytic hydrogenation using hydrogen in the presence of a metal catalyst, for example palladium on a support such as carbon, or Raney nickel, in a solvent such as an ether, for example a cyclic ether such as tetrahydrofuran, at the reflux temperature of the solvent.

[0168] Further examples include amines (-NH 2 ) group is a carboxylic acid group (-CO 2 H) to the corresponding acyl azide (-CON 3 ), followed by Curtius rearrangement and hydrolysis of the resulting isocyanate (-N=C=O).

[0169] Aldehyde groups (-CHO) can be converted to amine groups (-CH) by reductive amination using an amine and a borohydride, such as sodium triacetoxyborohydride or sodium cyanoborohydride, in a solvent such as a halogenated hydrocarbon, for example dichloromethane, or an alcohol, such as ethanol, optionally in the presence of an acid, such as acetic acid, at about ambient temperature. 2 NR'R'').

[0170] In a further example, aldehyde groups can be converted to alkenyl groups (-CH=CHR') by use of the Wittig or Wadsworth-Emmons reaction using the appropriate phosphorane or phosphonate under standard conditions known to those skilled in the art.

[0171] The aldehyde group can be converted to an ester group (-CO) using diisobutylaluminum hydride in a suitable solvent such as toluene. 2 Aldehyde groups can be obtained by reduction of an aldehyde group (such as -Et) or a nitrile (-CN). Alternatively, aldehyde groups can be obtained by oxidation of an alcohol group using any suitable oxidizing agent known to those skilled in the art.

[0172] Ester group (-CO 2 Depending on the nature of R, R') can be converted to the corresponding acid group (-CO 2 H). When R is t-butyl, acid catalyzed hydrolysis can be achieved, for example, by treatment with an organic acid such as trifluoroacetic acid in an aqueous solvent, or by treatment with an inorganic acid such as hydrochloric acid in an aqueous solvent.

[0173] Carboxylic acid group (-CO 2H) can be converted to an amide (CONHR' or -CONR'R'') by reaction with a suitable amine in the presence of a suitable coupling agent such as HATU in a suitable solvent such as dichloromethane.

[0174] In a further example, a carboxylic acid can be converted to the corresponding acid chloride (-COCl) by subsequent Arndt-Eistert synthesis, to give a carboxylic acid with one carbon (i.e., -CO 2 H to-CH 2 CO 2 H).

[0175] In a further example, the -OH group can be converted to the corresponding ester (e.g., -CO) by reduction, for example using a complex metal hydride such as lithium aluminum hydride in diethyl ether or tetrahydrofuran, or sodium borohydride in a solvent such as methanol. 2 Alternatively, the alcohol can be prepared from the corresponding acid (-CO R) using, for example, lithium aluminum hydride in a solvent such as tetrahydrofuran, or using borane in a solvent such as tetrahydrofuran. 2 H).

[0176] The alcohol group can be converted to a leaving group such as a halogen atom or a sulfonyloxy group, for example an alkylsulfonyloxy, for example a trifluoromethylsulfonyloxy or an arylsulfonyloxy, for example a p-toluenesulfonyloxy group, using conditions known to those skilled in the art. For example, the alcohol can be reacted with thioyl chloride in a halogenated hydrocarbon (for example dichloromethane) to give the corresponding chloride. A base (for example triethylamine) can also be used in the reaction.

[0177] In another example, alcohol, phenol or amide groups may be alkylated by coupling the phenol or amide with the alcohol in the presence of a phosphine, such as triphenylphosphine, and an activating agent, such as diethyl-, diisopropyl- or dimethylazodicarboxylate, in a solvent such as tetrahydrofuran. Alternatively, alkylation can be achieved by deprotonation using a suitable base, such as sodium hydride, followed by addition of an alkylating agent, such as an alkyl halide.

[0178] The aromatic halogen substituents in the compounds may be reacted in a solvent such as tetrahydrofuran, optionally at low temperature, for example at about -78 o At C, it can be subjected to halogen-metal exchange by treatment with a base, for example a lithium base such as n-butyl or t-butyl lithium, followed by quenching with an electrophile to introduce the desired substituent. Thus, for example, a formyl group can be introduced by using N,N-dimethylformamide as the electrophile. Alternatively, the aromatic halogen substituent can be subjected to a metal (e.g., palladium or copper) catalyzed reaction to introduce, for example, an acid, ester, cyano, amide, aryl, heteroaryl, alkenyl, alkynyl, thio or amino substituent. Suitable procedures that can be used include those described by Heck, Suzuki, Stille, Buchwald or Hartwig.

[0179] Aromatic halogen substituents may also undergo nucleophilic displacement following reaction with appropriate nucleophiles such as amines or alcohols. Advantageously, such reactions may be carried out at elevated temperatures in the presence of microwave irradiation.

[0180] Separation method In each of the exemplary schemes, it may be advantageous to separate the reaction products from one another or from the starting materials. The desired products of each step or sequence of steps are separated or purified (hereinafter, separated) to the desired degree of homogeneity by techniques common in the art. Typically, such separations include multiphase extraction from a solvent or solvent mixture, crystallization or trituration, distillation, sublimation or chromatography. Chromatography may involve any number of methods, including, for example, reversed-phase and normal-phase, size exclusion, ion exchange, supercritical fluid, high, medium, and low pressure liquid chromatography methods and apparatus, small-scale analytical, simulated moving bed (SMB) and preparative thin or thick layer chromatography, and small-scale thin layer and flash chromatography techniques.

[0181] Another class of separation methods involves treating the mixture with a selected reagent to bind or otherwise render separable the desired product, unreacted starting materials, reaction by-products, etc. Such reagents include adsorbents or absorbents such as activated carbon, molecular sieves, ion exchange media, etc. Alternatively, the reagent can be an acid in the case of basic substances, a base in the case of acidic substances, a binding reagent such as an antibody, a binding protein, a selective chelating agent such as crown ethers, liquid-liquid ion extraction reagents (LIX), etc.

[0182] The selection of an appropriate separation method will depend on the properties of the materials involved. Examples of separation methods include boiling point and molecular weight in distillation and sublimation, the presence or absence of polar functional groups in chromatography, the stability of materials in acidic and basic media in multiphase extraction, etc. The skilled artisan will apply the technique most likely to achieve the desired separation.

[0183] Diastereomeric mixtures can be separated into their individual diastereoisomers based on their physical chemical differences by methods well known to those skilled in the art, such as chromatography or fractional recrystallization. Enantiomers can be separated by converting the enantiomeric mixture 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 acid chloride), separating the diastereoisomers, and converting the individual diastereoisomers into the corresponding pure enantiomers (e.g., hydrolysis). Some of the compounds of the present invention may also be atropisomers (e.g., substituted biaryls) and are considered as part of the present invention. Enantiomers can also be separated by using chiral HPLC columns or supercritical fluid chromatography.

[0184] A single stereoisomer, e.g., an enantiomer, can be obtained substantially free of its stereoisomer by resolving the racemic mixture using methods such as the formation of diastereomers with optically active resolving agents (Eliel, E. and Wilen, S., Stereochemistry of Organic Compounds, John Wiley & Sons, Inc., New York, 1994; Lochmuller, C.H., J. Chromatogr., 113(3):283-302(1975)). The racemic mixture of the chiral compounds of the present invention can be separated and isolated by any suitable method, including: (1) formation of ionic diastereomeric salts with chiral compounds and separation by fractional recrystallization or other methods, (2) formation of diastereomeric compounds with chiral derivatizing agents, separation of the diastereomers, and conversion to pure stereoisomers, and (3) separation of substantially pure or enriched stereoisomers under chiral conditions. See Drug Stereochemistry, Analytical Methods and Pharmacology, Irving W. Wainer, Ed., Marcel Dekker, Inc., New York (1993).

[0185] Diastereomeric salts can be formed by reaction of enantiomerically pure chiral bases, such as brucine, quinine, ephedrine, strychnine, α-methyl-β-phenylethylamine (amphetamine), with asymmetric compounds containing acidic functional groups, such as carboxylic and sulfonic acids. The diastereomeric salts can be induced to separate by fractional recrystallization or ionic chromatography. To separate the optical isomers of amino compounds, addition of chiral carboxylic or sulfonic acids, such as camphorsulfonic acid, tartaric acid, mandelic acid, or lactic acid, can form diastereomeric salts.

[0186] Alternatively, the substrate to be resolved is reacted with one enantiomer of a chiral compound to form a diastereomeric pair (Eliel, E. and Wilen, S., Stereochemistry of Organic Compounds, John Wiley & Sons, Inc., New York, 1994, p. 322). Diastereomeric compounds can be formed by reacting the asymmetric compound with an enantiomerically pure chiral derivatizing agent such as a menthyl derivative, followed by separation and hydrolysis of the diastereomers to obtain the pure or enriched enantiomer. Methods for determining optical purity include making a chiral ester such as the menthyl ester of the racemic mixture (e.g., (-) menthyl chloroformate) in the presence of a base or Mosher's ester, α-methoxy-α-(trifluoromethyl)phenylacetate (Jacob, J. Org. Chem. 47:4165 (1982)), and analyzing the NMR spectrum for the presence of the two atropisomeric enantiomers or diastereomers. Stable diastereomers of atropisomeric compounds can be separated and isolated by normal and reverse phase chromatography following the method for separating atropisomeric naphthyl-isoquinolines (WO 96 / 15111, incorporated herein by reference). Racemic mixtures of the two enantiomers can be separated by chromatography using chiral stationary phases according to method (3) (Chiral Liquid Chromatography WJ Lough, Ed., Chapman and Hall, New York, (1989); Okamoto, J. of Chromatogr. 513:375-378 (1990)). Enriched or purified enantiomers can be separated by methods used to distinguish other chiral molecules with asymmetric carbon atoms, such as optical rotation and circular dichroism. The absolute stereochemistry of the chiral centers and enantiomers can be determined by X-ray crystallography.

[0187] Regioisomers and intermediates for their synthesis can be observed by characterization methods such as NMR and analytical HPLC. For certain compounds where the energy barrier for interconversion is high enough, E and Z isomers can be separated, for example, by preparative HPLC. Pharmaceutical Compositions and Administration

[0188] The compounds with which the present invention relates are JAK kinase inhibitors, such as JAK1 inhibitors, and are useful in the treatment of a number of diseases, for example inflammatory diseases such as asthma.

[0189] Accordingly, another embodiment provides pharmaceutical compositions or medicaments containing a compound of the invention, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier, diluent or excipient, as well as methods of preparing such compositions and medicaments using the compounds of the invention.

[0190] In one example, the compounds of the present disclosure or pharma- ceutically acceptable salts thereof can be formulated into galenical dosage forms by mixing at ambient temperature, at an appropriate pH, and at the desired degree of purity with a physiologically acceptable carrier, i.e., a carrier that is non-toxic to the recipient at the dosage and concentration employed. The pH of the formulation will depend primarily on the particular use and concentration of the compound, but typically falls anywhere from about 3 to about 8. In one example, the compounds of the present invention or pharma- ceutically acceptable salts thereof are formulated in acetate buffer at pH 5. In another embodiment, the compounds of the present invention are sterile. The compounds may be stored, for example, as solid or amorphous compositions, as lyophilized formulations, or as aqueous solutions.

[0191] The compositions are formulated, dosed, and administered in a manner consistent with good medical practice. Factors to consider in this regard include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the administration schedule, and other factors known to medical practitioners.

[0192] It will be understood that the specific dose level of any particular patient will depend on a variety of factors, including the activity of the particular compound used, age, body weight, general health, sex, diet, time of administration, route of administration, excretion rate, drug combination, and the severity of the particular disease being treated. Optimal dose levels and frequency of administration will be determined by clinical trials, as required in the pharmaceutical field. In general, the daily dose range for oral administration is in the range of about 0.001 mg to about 100 mg per kg of human body weight, often 0.01 mg to about 50 mg per kg, for example 0.1 to 10 mg per kg, in single or divided doses. In general, the daily dose range for inhalation administration is in the range of about 0.1 μg to about 1 mg per kg of human body weight, preferably 0.1 μg to 50 μg per kg, in single or divided doses. However, in some cases, it may be necessary to use doses outside these limits.

[0193] The compounds of the present invention or their pharma- ceutically acceptable salts can be administered by any suitable means, including oral, topical (including buccal and sublingual), rectal, vaginal, transdermal, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intradermal, intrathecal, inhalation, and epidural and intranasal, and, if desired for localized treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some embodiments, inhalation administration is used.

[0194] The compounds of the present invention or pharma- ceutically acceptable salts thereof may be administered in any convenient dosage form, such as tablets, powders, capsules, lozenges, granules, liquids, dispersions, suspensions, syrups, sprays, vapors, suppositories, gels, emulsions, patches, etc. Such compositions may contain ingredients conventional in pharmaceutical preparations, such as diluents (e.g., glucose, lactose or mannitol), carriers, pH adjusters, buffers, sweeteners, bulking agents, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifying agents, glidants, processing aids, colorants, fragrances, flavorings, other known additives, as well as additional active agents.

[0195] Suitable carriers and excipients are well known to those skilled in the art and are described in detail, for example, in Ansel, Howard C., et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems. Philadelphia: Lippincott, Williams & Wilkins, 2004; Gennaro, Alfonso R., et al. Remington: The Science and Practice of Pharmacy. Philadelphia: Lippincott, Williams & Wilkins, 2000; and Rowe, Raymond C. Handbook of Pharmaceutical Excipients. Chicago, Pharmaceutical Press, 2005. For example, carriers include solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal), isotonicity agents, absorption delaying agents, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, such similar materials, and combinations thereof, as known to those skilled in the art (see, e.g., Remington's Pharmaceutical Sciences, pp 1289-1329, 1990). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated. Exemplary excipients include dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, or combinations thereof. The pharmaceutical composition may contain different types of carriers or excipients depending on whether it is to be administered in solid, liquid, or aerosol form, and whether it needs to be sterile for such route of administration.

[0196] For example, tablets and capsules for oral administration may be in unit dose presentation form and may contain conventional excipients such as binders, e.g., syrup, acacia, gelatin, sorbitol, tragacanth or polyvinylpyrrolidone; fillers, e.g., lactose, sugar, corn starch, calcium phosphate, sorbitol or glycine; tableting lubricants, e.g., magnesium stearate, talc, polyethylene glycol or silica; disintegrants, e.g., potato starch or acceptable wetting agents such as sodium lauryl sulfate. Tablets may be coated according to methods well known in normal pharmaceutical practice. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives, such as suspending agents, for example, sorbitol, syrup, methyl cellulose, glucose syrup, gelatin hydrogenated edible fats; emulsifying agents, for example, lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example, almond oil, fractionated coconut oil, oily esters such as glycerin, propylene glycol, or ethyl alcohol; preservatives, for example, methyl or propyl p-hydroxybenzoate, or sorbic acid, and, optionally, conventional flavorings or colorants.

[0197] For topical application to the skin, the compound can be made into a cream, lotion or ointment.The cream or ointment formulations that can be used for the drug are conventional formulations well known in the art, for example as described in standard textbooks of pharmacology such as the British Pharmacopoeia.

[0198] The compound of the present invention or its pharma- ceutically acceptable salt can also be formulated for inhalation, for example, as a nasal spray or dry powder or aerosol inhaler.For delivery by inhalation, the compound is typically in the form of microparticles, which can be prepared by a variety of techniques, including spray drying, freeze drying and atomization.Aerosol generation can be achieved, for example, by using a pressure-driven jet nebulizer or ultrasonic nebulizer, for example, by using propellant-driven metered aerosol or propellant-free administration of micronized compound from an inhalation capsule or other "dry powder" delivery system.

[0199] By way of example, the compositions of the invention may be formulated as suspensions for delivery from a nebulizer, or as aerosols in liquid propellants, for example for use in pressurized metered dose inhalers (PMDIs). Propellants suitable for use in PMDIs are known to those skilled in the art and include CFC-12, HFA-134a, HFA-227, HCFC-22 (CCl 2 F 2 ) and HFA-152(CH 4 F 2 and isobutane).

[0200] In some embodiments, the compositions of the invention are in dry powder form for delivery using a dry powder inhaler (DPI). Many types of DPIs are known.

[0201] For delivery by administration, microparticles can be formulated with excipients that aid delivery and release.For example, in dry powder formulations, microparticles can be formulated with large carrier particles that aid flow from DPI to lungs.Suitable carrier particles are known and include lactose particles, which can have a mass aerodynamic diameter of, for example, greater than 90 μm.

[0202] For aerosol-based formulations, an example is as follows: Compound of the invention* 24mg / canister Lecithin, NF Liquid Concentration 1.2mg / canister Trichlorofluoromethane, NF 4.025g / canister Dichlorodifluoromethane, NF 12.15g / canister * or a pharma- ceutically acceptable salt thereof

[0203] The compound of the present invention or its pharmaceutically acceptable salt can be administered as described according to the inhalation system used.In addition to the compound, the dosage form can further contain excipients as mentioned above, or for example, propellants (e.g., Frigen in the case of metered aerosol), surfactants, emulsifiers, stabilizers, preservatives, flavorings, fillers (e.g., lactose in the case of powder inhalers), or if necessary, additional active compounds.

[0204] For inhalation, a large number of systems are available that can generate and administer aerosols of optimal particle size using an inhalation technique suitable for the patient. In addition to the use of adapters (spacers, inflators) and pear-shaped containers (e.g., Nebulator®, Volumatic®), as well as automatic devices that release puffer sprays (Autohaler®), several technical solutions are available for metered aerosols, especially powder inhalers (e.g., Diskhaler®, Rotadisk®, Turbohaler® or inhalers, as described in U.S. Pat. No. 5,263,475, which is incorporated herein by reference). Furthermore, the compounds of the present invention or pharma- ceutically acceptable salts thereof can be delivered in multi-chamber devices, thus allowing the delivery of combination drugs.

[0205] Compound or its pharmaceutically acceptable salt can also be administered parenterally in a sterile medium.Depending on the vehicle and concentration used, compound can be suspended or dissolved in the vehicle.Advantageously, adjuvants such as local anesthetics, preservatives or buffers can be dissolved in the vehicle.

[0206] Targeted Inhaled Drug Delivery The compounds of the present invention can be used for targeted inhalation delivery. Optimization of drugs for delivery to the lungs by local (inhalation) administration has been recently reviewed (Cooper, AE et al. Curr. Drug Metab. 2012, 13, 457-473).

[0207] Due to limitations of delivery devices, the dose of inhaled drugs may be limited in humans, requiring highly potent molecules with good pulmonary pharmacokinetic properties. High potency against the intended target is particularly important for inhaled drugs due to factors such as the limited amount of drug that can be delivered in one puff from an inhaler and safety concerns associated with high aerosol load in the lungs (e.g., coughing or irritation). For example, in some embodiments, a Ki of about 0.5 nM or less in the JAK1 biochemical assay described herein and an IC50 of about 20 nM or less in the JAK1-dependent cell-based assay described herein may be desirable for an inhaled JAK1 inhibitor. In other embodiments, the predicted human dose of the compound of the present invention or a pharmaceutically acceptable salt thereof is at least half the predicted human dose of a compound known in the art. Thus, in some embodiments, the compounds described herein (or a pharmaceutically acceptable salt thereof) exhibit such potency values. The following procedure was used to evaluate the subject compounds for potential use as inhaled drugs.

[0208] IL13 signaling. IL13 signaling is strongly implicated in the pathogenesis of asthma. IL13 is a cytokine that requires active JAK1 for signaling. Thus, inhibition of JAK1 also inhibits IL13 signaling, which may provide benefit to asthma patients. Inhibition of IL13 signaling in animal models (e.g., mouse models) may predict future benefit for human asthma patients. Thus, it may be beneficial for inhaled JAK1 inhibitors to show suppression of IL13 signaling in animal models. Methods for measuring such suppression are known in the art. For example, as discussed herein and known in the art, JAK1-dependent STAT6 phosphorylation is known to be a downstream consequence of IL13 stimulation. Thus, in some embodiments, the compounds described herein (or pharma- ceutically acceptable salts thereof) demonstrate inhibition of pSTAT6 induction in the lung. To examine the pharmacodynamic effects on pSTAT6 levels, the compounds of the present invention were co-dosed with 1 μg of IL13 intranasally in female Balb / c mice. Compounds were formulated in 0.2% (v:v) Tween 80 in saline and mixed 1:1 (v:v) with IL13 immediately prior to dosing. Intranasal doses were administered to lightly anesthetized (isoflurane) mice by dispensing a metered volume (50 μL) by pipette directly into the nostrils to achieve the target dose levels (3 mg / kg, 1 mg / kg, 0.3 mg / kg, 0.1 mg / kg). 0.25 h after dosing, blood samples (approximately 0.5 mL) were collected by cardiac puncture and plasma was generated by centrifugation (1500 g, 10 min, +4° C.). Lungs were perfused with chilled phosphate-buffered saline (PBS), weighed, and flash frozen in liquid nitrogen. All samples were stored at −80° C. until analysis. Thawed lung samples were weighed and homogenized after adding 2 mL of HPLC grade water for each gram of tissue using an Omni-Prep Bead Ruptor at 4° C. Plasma and lung samples were extracted by protein precipitation with 3 volumes of acetonitrile containing tolbutamide (50 ng / mL) and labetalol (25 ng / mL) as analytical internal standards.After vortex mixing and centrifugation at 3200g and 4°C for 30 min, the supernatant was diluted appropriately (e.g., 1:1 v:v) with HPLC grade water in a 96-well plate. Representative aliquots of plasma and lung samples were assayed for parent compound by LC-MS / MS against a series of matrix-matched calibration and quality control standards. Standards were prepared by spiking aliquots of control Balb / c mouse plasma or lung homogenate (2:1 in HPLC grade water) with test compound and extracting as described for the experimental samples. Lung:plasma ratios were determined as the ratio of mean lung concentration (μM) to mean plasma concentration (μM) at the sampling time (0.25 h).

[0209] To measure pSTAT6 levels, mouse lungs were cryopreserved at -80 °C until assay and homogenized in 0.6 ml of ice-cold cell lysis buffer (Cell Signalling Technologies, catalog number 9803S) supplemented with 1 mM PMSF and a cocktail of protease (Sigma Aldrich, catalog number P8340) and phosphatase (Sigma Aldrich, catalog numbers P5726 and P0044) inhibitors. Samples were centrifuged at 16060 × g for 4 minutes at 4 °C to remove tissue debris and protein concentration of the homogenate determined using the Pierce BCA Protein Assay Kit (catalog number 23225). Samples were diluted to a protein concentration of 5 mg / ml in ice-cold distilled water and assayed for pSTAT6 levels by Meso Scale Discovery electrochemiluminescence immunoassay. Briefly, 5 μl / well of 150 μg / ml STAT6 capture antibody (R&D Systems, catalog number MAB 2169) was coated onto 96-well Meso Scale Discovery High Binding Plates (catalog number L15XB-3) and air-dried at room temperature for 5 hours. Plates were blocked by the addition of 150 μl / well of 30 mg / ml Meso Scale Discovery Blocker A (catalog number R93BA-4) and incubation at room temperature for 2 hours on a microplate shaker. The blocked plates were washed 4 times with Meso Scale Discovery TRIS wash buffer (catalog number R61TX-1) and then 50 μl / well of lung homogenate was transferred to achieve a protein loading of 250 μg / well. The assay plates were incubated overnight at 4 °C, washed 4 times with TRIS wash buffer, and then 25 μl / well of 2.5 μg / ml sulfo-tag labeled pSTAT6 detection antibody (BD Pharmingen, catalog number 558241) was added for 2 hours at room temperature on a microplate shaker. The plates were washed 4 times with TRIS wash buffer and 150 μl / well of 1X Meso Scale Discovery Read Buffer T (catalog number R92TC-1) was added.Lung homogenate pSTAT6 levels were quantified by electrochemiluminescence detection on a Meso Scale Discovery SECTOR S 600 instrument.

[0210] JAK and JAK2 inhibitor compounds that inhibit both JAK1 and JAK2 may be useful in treating different types of asthma. Selectivity between JAK1 and JAK2 may also be important for inhaled JAK1 inhibitors. For example, GMCSF (granulocyte macrophage colony stimulating factor) is a cytokine that signals exclusively through JAK2. Neutralization of GMCSF activity is associated with pulmonary alveolar proteinosis (PAP) in the lung. However, submaximal JAK2 inhibition does not appear to be associated with PAP. Thus, even moderate JAK1 vs. JAK2 selectivity, or near-equal inhibition of JAK1 and JAK2, may be beneficial to avoid complete suppression of the GMCSF pathway and avoid PAP. For example, in certain embodiments, compounds that are equipotential to JAK1 and JAK2 are desirable. In other embodiments, compounds with approximately 2- to 5-fold selectivity for JAK1 over JAK2 may be beneficial for inhaled JAK1 inhibitors. Thus, in some embodiments, the compounds described herein (or pharma- ceutically acceptable salts thereof) exhibit such selectivity. Methods for measuring JAK1 and JAK2 selectivity are known in the art and information can be found in the Examples herein.

[0211] Kinase profiling. In addition, it may be desirable for the inhaled JAK1 or JAK1 / JAK2 inhibitor to be selective against one or more other kinases, reducing the possibility of potential toxicity due to off-target kinase pathway inhibition. Thus, it may be beneficial for the inhaled JAK1 inhibitor to be selective against a broad range of non-JAK kinases, such as in protocols available from ThermoFisher Scientific's SelectScreen™ Biochemical Kinase Profiling Service using the Adapta™ Screening Protocol Assay Conditions (revised July 29, 2016), LanthaScreen™ Eu Kinase Binding Assay Screening Protocol and Assay Conditions (revised June 7, 2016), and / or Z'LYTE™ Screening Protocol and Assay Conditions (revised September 16, 2016). For example, the compound of the present invention or a pharma- ceutically acceptable salt thereof exhibits at least 50-fold selectivity for JAK1 against a panel of non-JAK kinases. Thus, in some embodiments, the compounds described herein (or pharma- ceutically acceptable salts thereof) exhibit such selectivity.

[0212] Cytotoxicity Assay Hepatotoxicity, general cytotoxicity or cytotoxicity of unknown mechanism are undesirable characteristics for potential drugs, including inhaled drugs. It may be beneficial for inhaled JAK1 or JAK1 / JAK2 inhibitors to have low intrinsic cytotoxicity against various cell types. Typical cell types used to assess cytotoxicity include both primary cells, such as human hepatocytes, and proliferative established cell lines, such as Jurkat and HEK-293. Thus, in some embodiments, the compounds described herein (or pharma- ceutically acceptable salts thereof) exhibit such values. Methods for measuring cytotoxicity are known in the art. In some embodiments, the compounds described herein were tested as follows: (a) Jurkat and HEK293T cells were maintained at subconfluent density in T175 flasks. Cells were seeded at 450 cells / 45 μl of medium in Greiner 384-well black / clear tissue culture treated plates. (Greiner catalogue no. 781091). After dispensing the cells, the plates were allowed to equilibrate at room temperature for 30 minutes. After 30 minutes at room temperature, the cells were incubated at 4°C for 30 minutes in a CO 2 The cells were incubated overnight at 37° C. in a humidity-controlled incubator. The next day, cells were treated with compounds diluted in 100% DMSO (final DMSO concentration on cells=0.5%) in a 10-point dose-response curve with a top concentration of 50 μM. Cells and compounds were then ... 2 and overnight at 37° C. in a humidity controlled incubator for 72 hours. After 72 hours of incubation, viability was measured for all wells using CellTiterGlo® (Promega Cat# G7572). After 20 minutes of incubation at room temperature, plates were read on an EnVision™ (Perkin Elmer Life Sciences) using luminescence mode. (b) Use of human primary hepatocytes: Test compounds were prepared as 10 mM solutions in DMSO. Additionally, a positive control such as chlorpromazine was prepared as a 10 mM solution in DMSO. Test compounds were typically evaluated using a 7-point dose-response curve with 2-fold dilutions. Typically, the highest concentration tested was 50-100 μM. The highest concentration was typically determined by the solubility of the test compound. Cryopreserved primary human hepatocytes (BioreclamationIVT) (Lot IZT) were thawed at 37°C, pelleted, and resuspended in InVitroGro™ HT Thawing Medium (BioreclamationIVT). Hepatocyte viability was assessed by trypan blue exclusion, and cells were seeded in black-walled BioCoat™ collagen 384-well plates (Corning BD) at a density of 13,000 cells / well in InVitroGro™ CP plating medium supplemented with 1% Torpedo™ Antibiotic Mix (Bioreclamation IVT) and 5% fetal bovine serum. Cells were incubated at 37° C., 5% CO for 18 h before treatment. 2) overnight. After 18 hours of incubation, plating medium was removed and hepatocytes were treated with compounds diluted in InVitroGro™ HI incubation medium containing 1% Torpedo™ Antibiotic Mix and 1% DMSO (serum-free conditions). Hepatocytes were treated with test compounds at concentrations such as 0.78, 1.56, 3.12, 6.25, 12.5, 25 and 50 μM in a final volume of 50 μL. Positive controls (e.g., chlorpromazine) were included in the assay, typically at the same concentration as the test compounds. Additional cells were treated with 1% DMSO as a vehicle control. All treatments were incubated for 48 hours (37° C., 5% CO 2 Cell viability assays were performed at 37 °C (at 4 °C) with each treatment condition performed in triplicate. After 48 h of compound treatment, ATP content was measured as a determination of cell viability using the CellTiter-Glo® Cell Viability Assay (Promega) as an end-point assay. Assays were performed according to the manufacturer's instructions. Luminescence was measured on an EnVision™ Muliplate Reader (PerkinElmer, Waltham, MA, USA). Luminescence data was normalized to vehicle (1% DMSO) control wells. Inhibition curves and IC 50 Estimates were generated by nonlinear regression of log-transformed inhibitor concentration (7-point serial dilutions including vehicle) versus normalized response, with a variable Hill slope and constrained to constant values ​​of 100 and 0 above and below, respectively (GraphPad Prism™, GraphPad Software, Lahore, CA, USA).

[0213] hERG Inhibition. Inhibition of the hERG (human ether-a-go-go-related gene) potassium channel can result in long QT syndrome and cardiac arrhythmias. Although plasma levels of inhaled JAK1 or JAK1 / JAK2 inhibitors are expected to be low, lung-deposited compounds that leave the lungs in the bloodstream via pulmonary absorption circulate directly to the heart. Thus, local cardiac concentrations of inhaled JAK1 inhibitors may be transiently higher than total plasma levels, especially immediately after administration. Thus, it may be beneficial to minimize hERG inhibition of inhaled JAK1 inhibitors. For example, in some embodiments, a hERG IC50 of more than 30-fold the free drug plasma Cmax is preferred. Thus, in some embodiments, the compounds of the present invention (or pharma- ceutically acceptable salts thereof) exhibit minimized hERG inhibition under the following conditions: (a) hERG 2pt automated patch clamp conditions were used to investigate the in vitro effects of compounds on hERG expressed in mammalian cells, assessed at room temperature using an automated parallel patch clamp system, QPatch HT® (Sophion Bioscience A / S, Denmark). In some cases, compounds were tested at only one or two concentrations, such as 1 or 10 uM. In other cases, broader concentration-response relationships were established to allow estimation of IC50. For example, test compound concentrations were selected to span a range of approximately 10-90% inhibition in half-log increments. Each test article concentration was tested in two or more cells (n≧2). The duration of exposure to each test article concentration was a minimum of 3 minutes; and / or (b) As described in the example of WO 2014 / 074775, "Effects on cloned hERG potassium channels expressed in mammalian cells", ChanTest™, Charles River Company, protocol, with the following modifications: Cells stably expressing hERG were held at -80 mV. Onset and steady-state inhibition of hERG potassium current by compounds was measured using a pulse pattern with constant amplitude (conditioning prepulse: +20 mV for 1 s; repolarization test ramping to 90 mV (-0.5 V / s) repeated at 5 s intervals). Each recording was terminated by a final application of the highest concentration of the reference substance E-4021 (500 nM) (Charles River Company). Residual uninhibited currents were digitally subtracted from the data offline to determine the potency of test substances on hERG inhibition.

[0214] CYP (Cytochrome P450) Inhibition Assay. CYP inhibition may not be a desirable feature for an inhaled JAK1 or JAK1 / JAK2 inhibitor. For example, a reversible or time-dependent CYP inhibitor may cause an undesirable increase in its own plasma level or that of other co-administered drugs (drug-drug interactions). Furthermore, time-dependent CYP inhibition may be caused by biotransformation of the parent drug into reactive metabolites. Such reactive metabolites may covalently modify proteins and result in toxicity. Therefore, minimizing reversible and time-dependent CYP inhibition may be beneficial for an inhaled JAK1 inhibitor. Thus, in some embodiments, the compounds of the present invention (or pharma- ceutically acceptable salts thereof) show minimal or no reversible and / or time-dependent CYP inhibition. Methods for measuring CYP inhibition are known in the art. CYP inhibition of the compounds described herein was evaluated using pooled (n=150) human liver microsomes (Corning, Tewksbury, MA) over a concentration range of 0.16-10 uM using previously reported methods (Halladay et al., Drug Metab. Lett. 2011, 5, 220-230). Incubation periods and protein concentrations were dependent on the CYP isoform and probe substrate / metabolite evaluated. The following substrates / metabolites and incubation times and protein concentrations for each CYP were used: CYP1A2, phenacetin / acetaminophen, 30 min, 0.03 mg / ml protein; CYP2C9, warfarin / 7-hydroxywarfarin, 30 min, 0.2 mg / ml protein; CYP2C19, mephenytoin / 4-hydroxymephenytoin, 40 min, 0.2 mg / ml protein; CYP2D6, dextromethorphan / dextrorphan, 10 min, 0.03 mg / ml protein; CYP3A4, midazolam / 1-hydroxymidazolam, 10 min, 0.03 mg / ml protein, and CYP3A4 testosterone / 6-hydroxytestosterone, 10 min, 0.06 mg / ml protein. These conditions were previously determined to be linear production rates for the CYP-specific metabolites.All reactions were initiated with 1 mM NADPH and terminated by the addition of 0.1% formic acid in acetonitrile containing the appropriate stably labeled internal standard. Samples were analyzed by LC-MS / MS.

[0215] Mouse Lung Tissue Binding. A high binding fraction or percentage of JAK1 / JAK2 inhibitors to lung tissue may be undesirable as it may reduce the amount of free drug available to inhibit JAK1 or JAK2.

[0216] (a) Tissue binding experiments were performed in triplicate (n=3) using disposable RED plates according to standard protocols. First, individual drugs were spiked into tissue homogenate (pH 7.4) to achieve a final concentration of 1 μM, and then 300 μL of the drug-tissue homogenate mixture was transferred to the donor wells of the RED plate, which was pre-filled with 500 μL of phosphate-buffered saline (133 mM) on the receiver wells. The RED plate was sealed with a gas-permeable membrane and incubated at 37 °C for 24 h at 5% CO. 2 The plates were placed in a 37°C shaking incubator (450 rpm, VWR Symphony®) containing 0.5% ethanol for 6 hours. At the end of the incubation, a 30 μL sample aliquot was removed from the RED device to obtain an equal volume of tissue homogenate or matrix equalized with buffer, and the resulting sample was then immediately quenched with ice-cold acetonitrile (sample:acetonitrile 1:4) containing either propranolol or labetalol as an internal standard. After shaking at 500 rpm for 15 minutes on a Thermo Scientific Compact Digital MicroPlate Shaker, all samples were then subjected to centrifugation (Beckman Coulter Allegra X 12 R) at 3700 rpm for 15 minutes to remove plasma proteins. The supernatant was then collected and then diluted with an equal volume of water prior to LC-MS / MS analysis.

[0217] (b) In another procedure, the extent of lung tissue binding of the test compound to mouse lung homogenate can also be determined by equilibrium dialysis using a Pierce RED (rapid equilibrium dialysis) apparatus (Fisher Scientific 89811&89809). A 10 mM solution of the compound in DMSO was prepared and diluted to 1 mM with DMSO. This 1 mM aliquot (4 μL) was added to the lung homogenate (dilution factor 1:9, lung tissue: potassium phosphate buffer (0.05 M, pH 7.4)) to obtain a final compound incubation concentration of 5 μM containing a solvent that occupies 0.5% (v / v) of the final incubation volume.

[0218] For each assay, the percentage of bound lung tissue was determined in triplicate. Lung homogenate (200 μL) was filled in triplicate on one side of the RED apparatus insert, and 350 μL of potassium phosphate buffer was filled on the other side. The RED apparatus was sealed and incubated at 37 °C (about 150 rpm) on an orbital shaker for 4 hours.

[0219] After incubation, aliquots of the lung homogenate (8 μL) and the dialysate (72 μL) were matrix-matched prior to analysis (lung homogenate containing 72 μL of phosphate buffer, dialysate containing 8 μL of lung homogenate). 160 μL of acetonitrile containing an internal standard was added to precipitate the protein from the sample. For the evaluation of mass balance, the same matrix matching and protein precipitation procedures were performed on the lung homogenate aliquot sampled at the start of the experiment (t = 0 minute sample). The quenched samples were centrifuged (4000 rpm, 30 minutes, 4 °C), and the resulting supernatant was diluted with water (3:1 (v / v), supernatant: water), and the sample was analyzed for the parent compound by liquid chromatography-mass spectrometry assay.

[0220] The unbound fraction (fu) in the lung homogenate was determined from the ratio of the dialysate to the homogenate peak area, corrected to account for the lung homogenate dilution (D) to enable the estimation of total lung tissue binding using the following equation: Undiluted fu = (1 / D) / [((1 / apparent fu) - 1) + (1 / D)] Corrected binding fraction (%) = (1-undiluted fu)*100

[0221] Kinetic solubility. Good water solubility of JAK1 / JAK2 inhibitors for inhalation delivery may be desirable. In one procedure for measuring kinetic solubility, 4 μL of a 10 mM DMSO stock solution of the test compound is added to 196 μL of pH 7.4 phosphate buffered saline in a Millipore Multiscreen® 96-well filter plate to obtain a test concentration of 200 μM with 2% residual DMSO. The filter plate is sealed with aluminum sealing film and shaken at room temperature for 24 hours, and then the mixture is vacuum filtered into a clean 96-well plate. The filtrate sample is diluted 2-fold using pH 7.4 phosphate buffered saline, and then 5 μL of the resulting solution is analyzed by ultra-high performance liquid chromatography (UHPLC) with chemiluminescence nitrogen detection (CLND) and ultraviolet (UV) detection at a wavelength of 254 nm. Sample concentration is typically quantified by CLND intensity, which is related to the number of nitrogens in the compound. UV detection is used primarily to confirm sample purity, except in rare cases where the test compound does not contain nitrogen. In those cases, a compound-specific calibration curve is collected based on UV absorbance, and this curve is then used to determine sample concentrations.

[0222] Lipophilicity: Lipophilicity is generally related to the solubility, absorption, tissue penetration, protein binding, distribution, and ADME and PK properties of a potential drug. Thus, the calculated logP (cLogP), which is the logarithm of the partition coefficient of a compound between n-octanol and water (i.e., log(concentration of compound in n-octanol / concentration of compound in water), may be an important consideration for JAK1 / JAK inhibitors for inhalation delivery.

[0223] Liver microsomal stability. To minimize systemic exposure of inhaled JAK1 / JAK2 inhibitors, it may be beneficial to optimize rapid metabolism in the liver. The liver microsomal stability assay was performed on a BioCel 1200 liquid handling workstation (Agilent Technologies, Santa Clara, CA). The compound (1.0 μM) was incubated in 100 μL of reaction mixture containing 100 mM phosphate buffer (pH 7.4), 0.5 mg / mL liver microsomes and 1 mM NADPH at 37 o °C for 5 minutes. At different time intervals (0, 20, 40, and 60 minutes), an aliquot of 20 μL of the reaction mixture was removed and mixed with 4 volumes of acetonitrile (ACN) containing 0.1 μM propranolol as an internal standard to stop the metabolic reaction. The samples were then centrifuged at 3250 × g for 40 minutes to remove the precipitated protein. The supernatant was then transferred to a new 96-well plate, diluted 2-fold using deionized water, and then subjected to LC-MS / MS analysis using an ABI Sciex 5500 QTRAP® mass spectrometer (Applied Biosystems, Foster City, CA) coupled with an Agilent 1260 HPLC (Agilent Technologies, Santa Clara, CA). The residual rate was calculated using the peak area ratio of the test compound to the internal standard at different time points compared to the control (T = 0 minutes). See B. Williamson, C. Wilson, g. Dagnell, RJ Riley. Harmonised high throughput microsomal stability assay. J. Pharmacol. Toxicol. Methods. 2017;84:31-36.

[0224] Solid state properties. For compounds to be delivered by dry powder inhalation, it is also necessary to be able to produce a crystalline form of the compound that can be micronized to a size between 1 and 5 μm. Particle size is an important determinant of pulmonary deposition of inhaled compounds. Particles with a diameter less than 5 microns (μm) are typically defined as respirable. Particles with a diameter greater than 5 μm are more likely to be deposited in the oropharynx and correspondingly less likely to be deposited in the lungs. Also, fine particles with a diameter less than 1 μm are more likely to remain suspended in the air and correspondingly more likely to be exhaled from the lungs than larger particles. Thus, a particle size between 1 and 5 μm may be beneficial for inhaled drugs whose site of action is in the lungs. Typical methods used to measure particle size include laser diffraction and cascade impaction. Typical values ​​used to define particle size include: Included are D10, D50, and D90, which are particle size measurements below which 10%, 50%, or 90% of the sample falls, respectively. For example, a D50 of 3 μm indicates that 50% of the sample is less than 3 μm in size.

[0225] Mass Mean Aerodynamic Diameter (MMAD). MMAD is the diameter at which 50% of the particles are larger and 50% are smaller, by mass. MMAD is a measure of central tendency.

[0226] Geometric Standard Deviation (GSD) GSD is a measure of the degree of dispersion from the MMAD, or the spread of the aerodynamic particle size distribution.

[0227] A typical formulation for inhaled pharmaceuticals is a dry powder formulation that includes the active pharmaceutical ingredient (API) blended with a carrier such as lactose with or without additional additives such as magnesium stearate. In such formulations, it may be beneficial for the API itself to have properties that allow it to be milled to a respirable particle size of 1-5 μm. Particle agglomeration should be avoided, which can be measured by methods known in the art, such as by examining the D90 values ​​under various pressure conditions. Thus, in some embodiments, the compounds of the invention (or pharma- ceutically acceptable salts thereof) can be prepared in a respirable particle size such that there is little or no agglomeration.

[0228] With regard to crystallinity, for some formulations of inhaled drugs, including lactose blends, it is important that a particular crystalline form of the API is used. Crystallinity and crystalline form can affect many parameters associated with inhaled drugs, including but not limited to chemical and aerodynamic stability over time, compatibility with inhaled formulation components such as lactose, hygroscopicity, lung retention and lung irritation. Thus, a stable and reproducible crystalline form can be beneficial for inhaled drugs. Furthermore, the techniques used to mill compounds to the desired particle size are often energetic and can convert low melting crystalline forms to other crystalline forms or become fully or partially amorphous. Crystalline forms with melting points below 150°C may be incompatible with milling, while crystallographic forms with melting points below 100°C may not be compatible with milling. Thus, it can be beneficial for inhaled drugs to have a melting point at least above 100°C, ideally above 150°C. Thus, in some embodiments, the compounds described herein (or pharma- ceutically acceptable salts thereof) exhibit such properties.

[0229] Furthermore, minimizing molecular weight may help to reduce the effective dose of inhaled JAK1 inhibitor.Lower molecular weight leads to a corresponding higher number of molecules per unit mass of active pharmaceutical ingredient (API).Therefore, it may be beneficial to find the inhaled JAK1 inhibitor with the minimum molecular weight that retains all other desirable properties of inhaled drug.

[0230] Finally, a compound must maintain sufficient concentration in the lung for a given period of time so that it can exert a desired duration of pharmacological effect and have low systemic exposure for those targets for which systemic inhibition of the pharmacological target is undesirable.Since the lung is inherently highly permeable to both large molecules (proteins, peptides) and small molecules with associated short lung half-lives, it may be necessary to attenuate the pulmonary absorption rate by modifying one or more characteristics of the compound, such as minimizing membrane permeability, optimizing pKa, cLogP, solubility, dissolution rate, or introducing a degree of basicity into the compound (e.g., introducing amines) or enhancing binding to phospholipid-rich lung tissue via trapping in acidic intracellular compartments (pH 5), such as lysosomes.Methods for measuring such characteristics are known in the art.

[0231] Thus, in some embodiments, the compounds of the present invention (or their pharma- ceutically acceptable salts) preferably exhibit one or more of the above characteristics.Furthermore, in some embodiments, the compounds of the present invention advantageously exhibit one or more of these characteristics compared to compounds known in the art, which may be particularly true for compounds in the art intended as oral drugs for inhalation.For example, compounds with rapid oral absorption typically have little retention in the lungs when inhaled.

[0232] Methods of treatment with JANUS kinase inhibitors and uses thereof The compounds of the present invention or pharma- ceutically acceptable salts thereof inhibit the activity of Janus kinases, such as JAK1 kinase. For example, the compounds or pharma- ceutically acceptable salts thereof inhibit the phosphorylation of signal transducers and activators of transcription (STAT) by JAK1 kinase as well as STAT-mediated cytokine production. The compounds of the present invention are useful for inhibiting JAK1 kinase activity in cells via cytokine pathways, such as IL-6, IL-15, IL-7, IL-2, IL-4, IL-9, IL-10, IL-13, IL-21, G-CSF, IFN alpha, IFN beta, or IFN gamma pathways. Thus, in one embodiment, a method is provided for inhibiting Janus kinase activity (e.g., JAK1 activity) in a cell by contacting the cell with a compound of the present invention or a pharma- ceutically acceptable salt thereof.

[0233] The compounds can be used to treat immunological disorders caused by aberrant IL-6, IL-15, IL-7, IL-2, IL-4, IL 9, IL-10, IL-13, IL-21, G-CSF, IFN alpha, IFN beta, or IFN gamma cytokine signaling.

[0234] Thus, one embodiment includes a compound of the invention, or a pharma- ceutically acceptable salt thereof, for use in therapy.

[0235] In some embodiments, the use of the compound of the present invention or a pharma- ceutically acceptable salt thereof in the treatment of an inflammatory disease is provided. The use of the compound of the present invention or a pharma- ceutically acceptable salt thereof for the preparation of a medicament for the treatment of an inflammatory disease, such as asthma, is further provided. The compound of the present invention or a pharma- ceutically acceptable salt thereof for use in the treatment of an inflammatory disease, such as asthma, is also provided.

[0236] Another embodiment includes a method of preventing, treating, or reducing the severity of a disease or condition, such as asthma, that responds to inhibition of Janus kinase activity, such as JAK1 kinase activity, in a patient. The method may include administering to the patient a therapeutically effective amount of a compound of the invention, or a pharma- ceutically acceptable salt thereof. In one embodiment, the disease or condition that responds to inhibition of Janus kinase, such as JAK1 kinase, is asthma.

[0237] In one embodiment, the disease or condition is cancer, stroke, diabetes, hepatomegaly, cardiovascular disease, multiple sclerosis, Alzheimer's disease, cystic fibrosis, a viral disease, an autoimmune disease, atherosclerosis, restenosis, psoriasis, rheumatoid arthritis, inflammatory bowel disease, asthma, an allergic disorder, inflammation, a neurological disorder, a hormone-related disease, a condition associated with organ transplantation (e.g., transplant rejection), an immunodeficiency disorder, a destructive bone disorder, a proliferative disorder, an infectious disease, a condition associated with cell death, thrombin-induced platelet aggregation, a liver disease, a pathological immune condition involving T-cell activation, a CNS disorder, or a myeloproliferative disorder.

[0238] In one embodiment, the inflammatory disease is rheumatoid arthritis, psoriasis, asthma, inflammatory bowel disease, contact dermatitis or delayed hypersensitivity reaction, hi one embodiment, the autoimmune disease is rheumatoid arthritis, lupus or multiple sclerosis.

[0239] In another embodiment, the compounds of the present invention or pharma- ceutically acceptable salts thereof may be used to treat pulmonary diseases such as fibrotic lung diseases or interstitial lung diseases (e.g., interstitial pneumonia). In some embodiments, the compounds of the present invention or pharma- ceutically acceptable salts thereof may be used to treat idiopathic pulmonary fibrosis (IPF), systemic sclerosis interstitial lung disease (SSc-ILD), non-specific interstitial pneumonia (NSIP), rheumatoid arthritis-associated interstitial lung disease (RA-ILD), sarcoidosis, hypersensitivity pneumonitis, or ILDs secondary to connective tissue diseases beyond scleroderma (e.g., polymyositis, dermatomyositis, rheumatoid arthritis, systemic lupus erythematosus (SLE), or mixed connective tissue disease).

[0240] In one embodiment, the cancer is cancer of the breast, ovary, cervix, prostate, testis, penis, genitourinary tract, seminoma, esophagus, larynx, gastric, stomach, gastrointestinal, skin, keratoacanthoma, follicular carcinoma, melanoma, lung, small cell lung carcinoma, non-small cell lung carcinoma (NSCLC), lung adenocarcinoma, squamous cell carcinoma of the lung, colon, pancreas, thyroid, nipple, bladder, liver, bile duct, kidney, bone, myeloid disorder, lymphatic disorder, hair cell, buccal cavity and pharynx (oral cavity), lips, tongue, mouth, salivary gland, pharynx, small intestine, colon, rectum, anus, kidney, prostate, vulva, thyroid, large intestine, endometrium, uterus, brain, central nervous system, peritoneum, hepatocellular carcinoma, head cancer, neck cancer, Hodgkin's or leukemia.

[0241] In one embodiment, the disease is a myeloproliferative disorder. In one embodiment, the myeloproliferative disorder is polycythemia vera, essential thrombocytosis, myelofibrosis, or chronic myelogenous leukemia (CML).

[0242] Another embodiment includes the use of a compound of the invention or a pharma- ceutically acceptable salt thereof for the manufacture of a medicament for treating a disease as described herein (e.g., an inflammatory disorder, an immunological disorder, or cancer).In one embodiment, the invention provides a method of treating a disease or condition as described herein, e.g., an inflammatory disorder, an immunological disorder, or cancer, by targeting inhibition of a JAK kinase, e.g., JAK1. Combination therapy

[0243] The above compounds, or their salts, can be used alone or in combination with other agents for treatment. The second or additional (e.g., third) compounds of the pharmaceutical composition or dosing regimen typically have complementary activities to the compounds of the present invention, so that they do not adversely affect each other. Such agents are suitably present in combination in amounts effective for the intended purpose. The compounds may be administered together in a combined pharmaceutical composition, or may be administered separately, and when administered separately, this may occur simultaneously or sequentially. Such sequential administration may be close in time or distant in time.

[0244] For example, other compounds may be combined with the compounds of the present invention, or pharma- ceutically acceptable salts thereof, for the prevention or treatment of inflammatory diseases such as asthma. Therapeutic agents suitable for combination therapy include, but are not limited to, adenosine A2A receptor antagonists; anti-infectives; nonsteroidal glucocorticoid receptor (GR receptor) agonists; antioxidants; beta2 adrenergic receptor agonists; CCR1 antagonists; chemokine antagonists (not CCR1); corticosteroids; CRTh2 antagonists; DP1 antagonists; formyl peptide receptor antagonists; histone deacetylase activators; chloride channel hCLCA1 blockers; epithelial sodium channel blockers (ENAC blockers; intercellular adhesion molecule 1 blockers (ICAM blockers); IKK2 inhibitors; JNK inhibitors; transient receptor potential ankyrin 1 (TRPA1) inhibitors; Bruton's tyrosine kinase (BTK) inhibitors (e.g., fenebrutinib); spleen tyrosine kinase (SYK) inhibitors; tryptase-beta antibodies; ST2 receptor antibodies (e.g., AMG 282); cyclooxygenase inhibitors (COX inhibitors); lipoxygenase inhibitors; leukotriene receptor antagonists; dual beta2 adrenergic receptor agonists / M3 receptor antagonists (MABA compounds); MEK-1 inhibitors; myeloperoxidase inhibitors (MPO inhibitors); muscarinic antagonists; p38 MAPK inhibitors; phosphodiesterase PDE4 inhibitors; phosphatidylinositol 3-kinase delta inhibitors (PI3-kinase delta inhibitors); phosphatidylinositol 3-kinase gamma inhibitors (PI3-kinase gamma inhibitors); peroxisome proliferator-activated receptor agonists (PPAR gamma agonists); protease inhibitors; retinoic acid receptor modulators (RAR gamma modulators); statins; thromboxane antagonists; TLR7 receptor agonists; or vasodilators.

[0245] Furthermore, the compounds of the present invention or pharma- ceutically acceptable salts thereof may be combined with: (1) corticosteroids, such as alclometasone dipropionate, amelomethasone, beclomethasone dipropionate, budesonide, butycocort propionate, bicresonide, clobetasol propionate, deisobutylsilcyclosonide, dexamethasone, etiprednol dicloacetate, fluocinolone acetonide, fluticasone furoate, fluticasone propionate, loteprednol etabonate (topical), or mometasone furoate; (2) β2-adrenergic receptors, such as salbutamol, albuterol, terbutaline, fenoterol, bitolterol, carbuterol, clenbuterol, pirbuterol, rimoterol, terbutaline, tretoquinol, tulobuterol, etc. (3) salmeterol / fluticasone propionate (also sold as Advair®, Seretide®), formoterol / budesonide (Symbicort®), formoterol / fluticasone propionate (Flutiform®), formoterol / ciclesonide, formoterol / mometasone furoate, indacaterol / mometasone furoate, bromodate furoate (BREO ... (4) anticholinergics, e.g., muscarinic-3 (M3) receptor antagonists such as ipratropium bromide, tiotropium bromide, aclidinium bromide (LAS-34273), glycopyrronium bromide, or umeclidinium bromide;(5) M3-anticholinergic / β2-adrenergic receptor agonist combinations such as vilanterol / umeclidinium (Anoro® Ellipta®), olodaterol / tiotropium bromide, glycopyrronium bromide / indacaterol (Ultibro®, also sold as Xoterna®), fenoterol hydrobromide / ipratropium bromide (Berodual®), albuterol sulfate / ipratropium bromide (Combivent®), formoterol fumarate / glycopyrrolate, or aclidinium bromide / formoterol. (6) Dual pharmacological M3-anticholinergic / β2-adrenergic receptor agonists such as batefenterol succinate, AZD-2115 or LAS-190792; (7) Leukotriene modulators, e.g. leukotriene antagonists such as montelukast, zafirlast or pranlukast, or leukotriene biosynthesis inhibitors such as zileuton, or LTB4 antagonists such as amervant, or FLAP inhibitors such as fiboflavon, GSK-2190915; (8) Roflumilast, cilomilast, oglemilast, rolipram, tetomilast, AVE-8112, revamilast, CHF 6001, orally or by inhalation; (9) antihistamines, e.g., selective histamine-1 (H1) receptor antagonists, such as fexofenadine, citirizine, loratadine or astemizole, or GSK 835726 or GSK (10) antitussives such as codeine or dextramorphan; (11) mucolytics, such as N-acetylcysteine ​​or fudosteine; (12) expectorants / mucodynamic regulators, such as ambroxol, hypertonic solutions (e.g., saline or mannitol) or detergents; (13) peptide mucolytics, such as recombinant human deoxyribonuclease I (dornase alfa and rhDNase) or helicidin; (14) antibiotics, such as azithromycin, tobramycin or aztreonam; (15) nonselective COX-1 / COX-2 inhibitors, such as ibuprofen or ketoprofen; (16) COX-2 inhibitors, such as celecoxib or rofecoxib;(17) VLA-4 antagonists, such as those described in WO 97 / 03094 and WO 97 / 02289, each of which is incorporated herein by reference; (18) TACE inhibitors and TNF-α inhibitors, e.g., anti-TNF monoclonal antibodies such as Remicade® and CDP-870, and TNF receptor immunoglobulin molecules such as Enbrel®; (19) inhibitors of matrix metalloproteinases, e.g., MMP-12; (20) BAY-85-8501 or human neutrophil elastase inhibitors such as those described in WO 2005 / 026124, WO 2003 / 053930 and WO 2006 / 082412; (21) A2b antagonists such as those described in WO 2002 / 42298, which is incorporated herein by reference; (22) modulators of chemokine receptor function, such as antagonists of CCR3 and CCR8; (23) compounds that modulate the action of other prostanoid receptors, such as thromboxane A; 2antagonists; DP1 antagonists such as laropiprant or asaprotan CRTH2 antagonists such as OC000459, fevipiprant, ADC 3680 or ARRY 502; (24) PPAR agonists, including PPAR alpha agonists (such as fenofibrate), PPAR delta agonists, PPAR gamma agonists such as pioglitazone, rosiglitazone and balaglitazone; (25) methylxanthines such as theophylline or aminophylline, and theophylline / budesonide, theophylline / fluticasone propionate, theophylline / Methylxanthine / corticosteroid combinations such as ciclesonide, theophylline / mometasone furoate and theophylline / beclomethasone dipropionate; (26) A2a agonists such as those described in European Patent No. 1052264 and European Patent No. 1241176; (27) CXCR2 or IL-8 antagonists such as AZD-5069, AZD-4721, or danilixin; (28) Kineret and ACZ (29) IL-R signaling modulators such as ABN-912; (30) p38 MAPK inhibitors such as BCT197, JNJ49095397, losmapimod or PH-797804; (31) TLR7 receptor agonists such as AZD 8848; (32) PI3-kinase inhibitors such as RV1729 or GSK2269557 (nemiralisib); (33) triple combination products such as TRELEGY ELLIPTA (fluticasone furoate, umeclidinium bromide, and vilanterol); or (34) small molecule inhibitors of TRPA1, BTK or SYK.

[0246] In some embodiments, the compounds of the invention or pharma- ceutically acceptable salts thereof can be used in combination with one or more additional drugs, such as anti-hyperproliferative, anti-cancer, cytostatic, cytotoxic, anti-inflammatory or chemotherapeutic agents, such as those agents disclosed in U.S. Patent Application Publication No. 2010 / 0048557, which is incorporated herein by reference. The compounds of the invention or pharma- ceutically acceptable salts thereof can also be used in combination with radiation therapy or surgery, as known in the art.

[0247] Combinations of any of the foregoing with a compound of the invention, or a pharma- ceutically acceptable salt thereof, are specifically contemplated.

[0248] manufactured goods Another embodiment includes an article of manufacture (e.g., a kit) for treating a disease or disorder responsive to the inhibition of a Janus kinase, such as JAK1 kinase. The kit includes: (a) a first pharmaceutical composition comprising a compound of the invention or a pharma- ceutical acceptable salt thereof; (b) instructions for use. In another embodiment, the kit comprises: (c) a second pharmaceutical composition, eg, a pharmaceutical composition comprising a treatment as described above, eg, a treatment for an inflammatory disorder or a chemotherapeutic agent.

[0249] In one embodiment, the instructions describe the simultaneous, sequential or separate administration of said first and second pharmaceutical compositions to a patient in need thereof.

[0250] In one embodiment, the first and second compositions are contained in separate containers. In another embodiment, the first and second compositions are contained in the same container.

[0251] Examples of containers for use include bottles, vials, syringes, blister packs, and the like. The container can be formed from various materials such as glass or plastic. The container contains a compound of the present invention or a pharmaceutically acceptable salt thereof that is effective in treating the condition and can have a sterile access port (e.g., the container can be an intravenous solution bag or vial having a stopper pierceable by a hypodermic needle). The label or package insert indicates that the compound is used to treat a selected condition such as asthma or cancer. In one embodiment, the label or package insert indicates that the compound can be used to treat a disorder. Further, the label or package insert can indicate that the patient to be treated has a disorder characterized by hyperactive or irregular Janus kinase activity, e.g., hyperactive or irregular JAK1 activity. The label or package insert may also indicate that the compound can be used to treat other disorders.

[0252] Alternatively or additionally, the kit may further comprise a second (or third) container containing a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, or dextrose solution. Other materials desirable from a commercial and user perspective may be further included, including other buffers, diluents, filters, needles, and syringes.

[0253] The following examples are included to illustrate the invention. However, it should be understood that these examples are not meant to limit the invention, but are meant only to suggest a method of carrying out the invention. Those skilled in the art will recognize that the described chemical reactions can be easily adapted to prepare other compounds of the invention, and alternative methods for preparing compounds are within the scope of the invention. For example, the synthesis of non-exemplary compounds according to the invention can be successfully carried out by making modifications obvious to those skilled in the art, such as by properly protecting intervening groups, by using other suitable reagents known in the art other than those described, or by making routine changes to the reaction conditions. Alternatively, other reactions disclosed herein or known in the art will be recognized as having applicability for preparing other compounds of the invention. EXAMPLES

[0254] Representative compounds in Table 1 below were prepared using procedures similar to those described in the schemes and examples herein. The absolute stereochemistry of each compound below may not be shown. Thus, structures may appear more than once, each representing a single stereoisomer. LC-MS methods, retention times and m / z are also provided in Table 1. [Table 1] TIFF0007682110000015.tif235170TIFF0007682110000016.tif248170TIFF0007682110000017.tif207170TIFF0007682110000018.tif232170TIFF000 7682110000019.tif252170TIFF0007682110000020.tif254170TIFF0007682 110000021.tif240170TIFF0007682110000022.tif210170TIFF00076821100 00023.tif221170TIFF0007682110000024.tif246170TIFF0007682110000025.tif247170TIFF0007682110000026.tif245170TIFF0007682110000027.t if234170TIFF0007682110000028.tif249170TIFF0007682110000029.tif245170TIFF0007682110000030.tif207170TIFF0007682110000031.tif203170

[0255] General Experimental Details All solvents and commercial reagents were used as received unless otherwise stated. Where products were purified by chromatography on silica, this was done using either glass columns manually packed with silica gel (Kieselgel 60, 220-440 mesh, 35-75 μm) or Isolute® SPE Si II cartridges. "Isolute SPE Si cartridge" refers to a packed polypropylene column containing unbonded activated silica with irregular particles having an average diameter of 50 μm and a nominal porosity of 60 Å. Where Isolute® SCX-2 cartridges were used, "Isolute® SCX-2 cartridge" refers to a packed polypropylene column containing a non-endcapped propylsulfonic acid functionalized silica strong cation exchange sorbent.

[0256] LCMS conditions Method A Experiments were performed on a SHIMADZU LCMS-2020 equipped with a C18 reversed-phase column (50 × 3 mm Shim-Pack XR-ODS, particle size 2.2 μm) and eluted with Solvent A: water + 0.05% trifluoroacetic acid; Solvent B: acetonitrile + 0.05% trifluoroacetic acid. gradient: TIFF0007682110000032.tif26170 Detection - UV (220 and 254 nm) and ELSD

[0257] Method B Experiments were performed on a SHIMADZU LCMS-2020 equipped with a C18 reversed-phase column (50 × 3 mm Shim-Pack XR-ODS, particle size 2.2 μm) and eluted with Solvent A: water + 0.05% trifluoroacetic acid; Solvent B: acetonitrile + 0.05% trifluoroacetic acid. gradient: TIFF0007682110000033.tif31170 Detection - UV (220 and 254 nm) and ELSD

[0258] Method C Experiments were performed on a SHIMADZU LCMS-2020 equipped with a C18 reversed-phase column (50 × 3 mm Shim-Pack XR-ODS, particle size 2.2 μm) and eluted with Solvent A: water + 0.05% trifluoroacetic acid; Solvent B: acetonitrile + 0.05% trifluoroacetic acid. gradient: TIFF0007682110000034.tif26170 Detection - UV (220 and 254 nm) and ELSD

[0259] Method D Experiments were performed on a SHIMADZU LCMS-2020 equipped with a C18 reversed-phase column (50 x 3 mm Shim-Pack XR-ODS, particle size 2.2 μm) and eluted with Solvent A: water + 0.05% trifluoroacetic acid; Solvent B: acetonitrile + 0.05% trifluoroacetic acid. Gradient: TIFF0007682110000035.tif31170 Detection - UV (220 and 254 nm) and ELSD

[0260] Method E Experiments were performed on a SHIMADZU LCMS-2020 equipped with a C18 reversed-phase column (50 x 3 mm Shim-Pack XR-ODS, particle size 2.2 μm) and eluted with Solvent A: water + 0.05% trifluoroacetic acid; Solvent B: acetonitrile + 0.05% trifluoroacetic acid. Gradient: TIFF0007682110000036.tif31170 Detection - UV (220 and 254 nm) and ELSD

[0261] Method F Experiments were performed on a SHIMADZU LCMS-2020 equipped with a C18 reversed-phase column (50 x 3 mm Shim-Pack XR-ODS, particle size 2.2 μm) and eluted with Solvent A: water + 0.05% trifluoroacetic acid; Solvent B: acetonitrile + 0.05% trifluoroacetic acid. Gradient: TIFF0007682110000037.tif31170 Detection - UV (220 and 254 nm) and ELSD

[0262] Method G The experiment was performed on a SHIMADZU 20A HPLC equipped with a C18 reverse phase column (50×2.1 mm Ascentis Express C18, 2.7 μm particle size) and eluted with Solvent A: water + 0.05% trifluoroacetic acid; Solvent B: acetonitrile + 0.05% trifluoroacetic acid. Gradient: TIFF0007682110000038.tif26170 Detection - UV (220 and 254 nm) and ELSD

[0263] Method H: Experiments were performed on a SHIMADZU LCMS-2020 equipped with a C18 reversed-phase column (50 x 3 mm Shim-Pack XR-ODS, particle size 2.2 μm) and eluted with Solvent A: water + 0.05% trifluoroacetic acid; Solvent B: acetonitrile + 0.05% trifluoroacetic acid. Gradient: TIFF0007682110000039.tif26170 Detection - UV (220 and 254 nm) and ELSD

[0264] Method I: Poroshell HPH-C 18 The experiments were carried out on a SHIMADZU 20 A HPLC equipped with a column (50 × 3 mm, particle size 2.7 μm), and solvent A: water / 5 mM NH 4 HCO 3 Solvent B: Eluted with acetonitrile. Gradient: TIFF0007682110000040.tif27170 Detection - UV (220 and 254 nm) and ELSD

[0265] Method J: C18 reversed phase column (50×3mm Kinetex XB-C 18 Experiments were performed on a SHIMADZU LCMS-2020 equipped with a column chromatography column (particle size 2.6 μm) and eluted with Solvent A: water + 0.05% trifluoroacetic acid; Solvent B: acetonitrile + 0.05% trifluoroacetic acid. Gradient: TIFF0007682110000041.tif26170 Detection - UV (220 and 254 nm) and ELSD

[0266] Method K Experiments were performed on a SHIMADZU LCMS-2020 equipped with a C18 reversed-phase column (50 × 3 mm Shim-Pack XR-ODS, particle size 2.2 μm) and eluted with Solvent A: water + 0.05% trifluoroacetic acid; Solvent B: acetonitrile + 0.05% trifluoroacetic acid. gradient: TIFF0007682110000042.tif27170 Detection - UV (220 and 254 nm) and ELSD

[0267] Method L C18 reversed phase column (50×2.1mm Kinetex XB-C 18 The experiment was performed on a SHIMADZU LCMS-2020 equipped with a column separator (100A, particle size 2.6 μm) and eluted with Solvent A: water + 0.05% trifluoroacetic acid; Solvent B: acetonitrile + 0.05% trifluoroacetic acid. Gradient: TIFF0007682110000043.tif26170 Detection - UV (220 and 254 nm) and ELSD

[0268] Method M Experiments were performed on a SHIMADZU LCMS-2020 equipped with a C18 reversed-phase column (30 x 2.1 mm Kinetex C18-100A, 1.7 μm particle size) and eluted with Solvent A: water + 0.05% trifluoroacetic acid; Solvent B: acetonitrile + 0.05% trifluoroacetic acid. Gradient: TIFF0007682110000044.tif26170 Detection - UV (220 and 254 nm) and ELSD

[0269] Method N Experiments were performed on a SHIMADZU LCMS-2020 equipped with a C18-reversed phase column (50×3.0 mm Poroshell HPH-C18, particle size 2.7 μm) and eluted with Solvent A: water + 5 mM ammonium bicarbonate; Solvent B: acetonitrile. Gradient: TIFF0007682110000045.tif26170 Detection - UV (220 and 254 nm) and ELSD

[0270] Method O Experiments were performed on a SHIMADZU LCMS-2020 equipped with a C18-reversed phase column (50×3.0 mm Titank C18, 3.0 μm particle size) and eluted with Solvent A: water + 5 mM ammonium bicarbonate; Solvent B: acetonitrile. Gradient: TIFF0007682110000046.tif26170 Detection - UV (220 and 254 nm) and ELSD

[0271] Method P Experiments were performed on a SHIMADZU LCMS-2020 equipped with a C18 reversed-phase column (30×2.1 mm Halo C18, 2.0 μm particle size) and eluted with Solvent A: water + 0.05% trifluoroacetic acid; Solvent B: acetonitrile + 0.05% trifluoroacetic acid. Gradient: TIFF0007682110000047.tif26170 Detection - UV (220 and 254 nm) and ELSD

[0272] Method Q Experiments were performed on a SHIMADZU LCMS-2020 equipped with a C18 reversed-phase column (50 x 3.0 mm YMC-Triart C18, particle size 2.5 μm) and eluted with Solvent A: water + 0.1% formic acid; Solvent B: acetonitrile + 0.1% formic acid. Gradient: TIFF0007682110000048.tif26170 Detection - UV (220 and 254 nm) and ELSD

[0273] Method R Experiments were performed on a SHIMADZU LCMS-2020 equipped with a C18 reversed-phase column (50 x 3 mm Shim-Pack XR-ODS, particle size 2.2 μm) and eluted with Solvent A: water + 0.05% trifluoroacetic acid; Solvent B: acetonitrile + 0.05% trifluoroacetic acid. Gradient: TIFF0007682110000049.tif26170 Detection - UV (220 and 254 nm) and ELSD

[0274] Method S Experiments were performed on a SHIMADZU LCMS-2020 equipped with a C18-reversed phase column (50×3.0 mm Poroshell HPH-C18, particle size 2.7 μm) and eluted with Solvent A: water + 5 mM ammonium bicarbonate; Solvent B: acetonitrile. Gradient: TIFF0007682110000050.tif31170 Detection - UV (220 and 254 nm) and ELSD

[0275] Method T The experiment was performed on a SHIMADZU 20A HPLC equipped with a C18 reverse phase column (50×2.1 mm Ascentis Express C18, 2.7 μm particle size) and eluted with Solvent A: water + 0.05% trifluoroacetic acid; Solvent B: acetonitrile + 0.05% trifluoroacetic acid. Gradient: TIFF0007682110000051.tif26170 Detection - UV (220 and 254 nm) and ELSD

[0276] Method U Experiments were performed on a SHIMADZU LCMS-2020 equipped with a C18 reversed-phase column (50 x 3 mm Shim-Pack XR-ODS, particle size 2.2 μm) and eluted with Solvent A: water + 0.05% trifluoroacetic acid; Solvent B: acetonitrile + 0.05% trifluoroacetic acid. Gradient: TIFF0007682110000052.tif31170 Detection - UV (220 and 254 nm) and ELSD

[0277] Method V Experiments were performed on a SHIMADZU LCMS-2020 equipped with a C18-reversed phase column (50×3.0 mm Poroshell HPH-C18, particle size 2.7 μm) and eluted with Solvent A: water + 5 mM ammonium bicarbonate; Solvent B: acetonitrile. Gradient: TIFF0007682110000053.tif31170 Detection - UV (220 and 254 nm) and ELSD

[0278] Method W Experiments were performed on a SHIMADZU LCMS-2020 equipped with a C18 reversed-phase column (50×2.1 mm Waters Acquity BEH, 1.7 μm particle size) and eluted with Solvent A: water + 0.1% formic acid; Solvent B: acetonitrile + 0.1% formic acid. Gradient: TIFF0007682110000054.tif26170 Detection - UV (220 and 254 nm) and ELSD

[0279] Method X Experiments were performed on an Agilent 1290 UHPLC coupled to an Agilent MSD (6140) mass spectrometer using ESI as the ionization source. LC separation used a Phenomenex XB-C18, 1.7um, 50x2.1mm column at a flow rate of 0.4ml / min. Mobile phase A was water with 0.1% formic acid and mobile phase B was acetonitrile with 0.1% formic acid. The gradient started at 2% B over 7 minutes, ended at 98% B and held at 98% B for 1.5 minutes after equilibration for 1.5 minutes. The LC column temperature was 40°C. UV absorbance was collected at 220nm and 254nm and mass spectrometry full scan was applied for all experiments.

[0280] List of common abbreviations ACN Acetonitrile Brine Saturated sodium chloride solution CH 3 OD Deuterated Methanol CDCl 3 Deuterated chloroform DCM Dichloromethane DIEA or DIPEA Diisopropylethylamine DMA Dimethylacetamide DMAP 4-Dimethylaminopyridine DMF Dimethylformamide DMSO Dimethyl sulfoxide DMSO-d6 Deuterated dimethyl sulfoxide DTAD Di-tert-butyl azodicarboxylate EDC or EDCI 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide ESI Electrospray Ionization EtOAc Ethyl acetate EtOH Ethanol FA Formic Acid HOAc Acetic acid g grams h time HATU (O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate) HCl Hydrochloric acid HOBt Hydroxybenzotriazole HPLC High Performance Liquid Chromatography IMS Industrial Methanol Denatured Alcohol L Liter LCMS: Liquid chromatography-mass spectrometry LiHMDS or LHMDS Lithium hexamethyldisilazide MDAP Mass directed automated purification MeCN Acetonitrile MeOH Methanol μm micrometer min mg milligram mL Milliliters mm millimeters M Molar concentration nm nanometer NMR nuclear magnetic resonance Pd 2 (dba) 3 .CHCl 3 Tris(dibenzylideneacetone)dipalladium(0)-chloroform adduct PE Petroleum Ether Preparative HPLC Preparative High Performance Liquid Chromatography PyAOP (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate SCX-2 Strong cation exchange TBAF Tetra-n-butylammonium fluoride THF Tetrahydrofuran TFA Trifluoroacetic acid Xantphos 4,5-bis(diphenylphosphino)-9,9-dimethylxanthine ZnCl 2 Zinc chloride

[0281] Intermediate 1 TIFF0007682110000055.tif51170N-[3-[2,5-bis(difluoromethoxy)phenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide Step 1: Synthesis of 1-(benzyloxy)-4-(difluoromethoxy)benzene TIFF0007682110000056.tif29170 A 3000 mL round-bottom flask purged with nitrogen and maintained under an inert atmosphere of nitrogen was charged with N,N-dimethylformamide (1500 mL), 4-(benzyloxy)phenol (200 g, 999 mmol), and cesium carbonate (651 g, 1.99 mol). The reaction vessel was filled with CO 2 A discharge outlet was fitted. Following this, sodium 2-chloro-2,2-difluoroacetate (228 g, 1.50 mol, 1.50 equiv.) was added in several batches at 120° C. The reaction was stirred in an oil bath at 120° C. until gas evolution ceased (approximately 1 h) and then cooled to room temperature. The reaction mixture was slowly added to 3000 mL of water / ice with stirring. The resulting mixture was extracted with ethyl acetate (3×4000 mL). The organic layers were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel eluting with ethyl acetate / petroleum ether (1 / 19). The appropriate fractions were combined and concentrated under reduced pressure. This reaction was repeated four times. This gave a total of 450 g (36%) of 1-(benzyloxy)-4-(difluoromethoxy)benzene as a white solid.

[0282] Step 2: Synthesis of 4-(difluoromethoxy)phenol TIFF0007682110000057.tif28170 A 3000 mL round bottom flask was charged with methanol (1500 mL), 1-(benzyloxy)-4-(difluoromethoxy)benzene (140 g, 559 mmol) and 10% palladium on carbon (15 g, 141 mmol). The resulting mixture was stirred overnight at room temperature under hydrogen (about 45 psi). The catalyst was filtered off. The filtrate was concentrated under reduced pressure. This reaction was repeated three times. This gave 300 g (78%) of 4-(difluoromethoxy)phenol as a yellow oil.

[0283] Step 3: Synthesis of 2-bromo-4-(difluoromethoxy)phenol TIFF0007682110000058.tif31170A 1000 mL round bottom flask was charged with acetic acid (500 mL), 4-(difluoromethoxy)phenol (50 g, 312 mmol) and NBS (55.6 g, 312 mmol). The reaction mixture was stirred at 15° C. for 1 h. The resulting mixture was then slowly added to 1000 mL of water / ice with stirring. The resulting solution was extracted with ethyl acetate (3×1000 mL). The organic layers were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel eluting with dichloromethane / petroleum ether (30 / 70). Appropriate fractions were collected and concentrated under reduced pressure. This gave 50 g (67%) of 2-bromo-4-(difluoromethoxy)phenol as a pale yellow oil.

[0284] Step 4: Synthesis of 2-bromo-1,4-bis(difluoromethoxy)benzene TIFF0007682110000059.tif381702000mL round bottom flask, CH 3CN (500 mL), water (500 mL), 2-bromo-4-(difluoromethoxy)phenol (54 g, 226 mmol) and potassium hydroxide (94 g, 1.68 mol) were charged. The flask was placed in an ice batch and the reaction mixture was stirred in the ice batch for 30 min. Diethyl(bromodifluoromethyl)phosphonate (120 g, 449 mmol) was then added dropwise to the reaction mixture at 0° C. Upon completion of the addition of diethyl(bromodifluoromethyl)phosphonate, the reaction mixture was stirred in a water / ice bath for 1 h. The resulting solution was extracted with ethyl acetate (3×300 mL). The organic layers were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel flash chromatography eluting with ethyl acetate / petroleum ether (1 / 19) and the appropriate fractions were collected and concentrated under reduced pressure. This gave 54 g (83%) of 2-bromo-1,4-bis(difluoromethoxy)benzene as a pale yellow oil.

[0285] Step 5: Synthesis of 5-[2,5-bis(difluoromethoxy)phenyl]-4-nitro-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazole TIFF0007682110000060.tif37170In a 1000 mL round bottom flask purged with nitrogen and maintained under an inert atmosphere of nitrogen, add DMA (500 mL), potassium carbonate (112 g, 810 mmol), 4-nitro-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazole (66 g, 271 mmol), 2-bromo-1,4-bis(difluoromethoxy)benzene (79 g, 273 mmol), 2,2-dimethylpropanoic acid (8.3 g, 81.3 mmol), and Pd(OAc). 2(6.0 g, 26.7 mmol) and bis(adamantan-1-yl)(butyl)phosphane (19 g, 52.9 mmol, 0.195 equiv.) were charged. The reaction mixture was stirred overnight at 120° C. in an oil bath and cooled to room temperature. The reaction mixture was then added to 1000 mL of water / ice with stirring. The resulting solution was extracted with ethyl acetate (3×1000 mL). The organic layers were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel eluting with ethyl acetate / petroleum ether (1 / 1). Appropriate fractions were collected and concentrated under reduced pressure. This gave 100 g (82%) of 5-[2,5-bis(difluoromethoxy)phenyl]-4-nitro-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazole as a solid. LC / MS (Method H, ESI): [M+H] + =452.1,R T =1.49 minutes

[0286] Step 6: Synthesis of 5-[2,5-bis(difluoromethoxy)phenyl]-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazol-4-amine TIFF0007682110000061.tif38170 In a 3,000 mL three-neck round-bottom flask was added ethanol (1500 mL), water (150 mL), 5-[2,5-bis(difluoromethoxy)phenyl]-4-nitro-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazole (100 g, 221 mmol), iron powder (124 g, 2.22 mol), and NH 4Cl (59.2 g, 1.11 mol) was charged. The resulting mixture was stirred at reflux in an oil bath for 2 h and then cooled to room temperature. The solid was filtered off and washed with ethanol. The filtrate was concentrated under reduced pressure. The residue was dissolved in 3000 mL of ethyl acetate. The ethyl acetate solution was washed with 1×1000 mL of brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. This gave 100 g of crude 5-[2,5-bis(difluoromethoxy)phenyl]-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazol-4-amine as a pale yellow oil, which was used directly without purification. LC / MS (Method H, ESI): [M+H] + =422.1,R T =1.25 minutes

[0287] Step 7: Synthesis of N-[5-[2,5-bis(difluoromethoxy)phenyl]-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide TIFF0007682110000062.tif51170 A 2000 mL round bottom flask was charged with DMA (1000 mL), 5-[2,5-bis(difluoromethoxy)phenyl]-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazol-4-amine, pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (58.1 g, 356 mmol), 7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP) (186 g, 356 mmol), 4-dimethylaminopyridine (2.90 g, 23.7 mmol) and DIPEA (92.0 g, 712 mmol). The resulting solution was stirred overnight at 65° C. in a water bath. The reaction mixture was then slowly added to 2000 mL of water with stirring. The resulting solution was extracted with ethyl acetate (3 x 2000 mL). The combined organic phases were washed with 1000 mL of brine, dried over anhydrous sodium sulfate and concentrated under pressure. The residue was purified by flash chromatography on silica gel eluting with ethyl acetate / petroleum ether (40 / 60). The appropriate fractions were combined and concentrated under reduced pressure to give 120 g of N-[5-[2,5-bis(difluoromethoxy)phenyl]-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide as a white solid. LC / MS (Method G, ESI): [M+H] + =567.2,R T =1.05 minutes

[0288] Step 8: Synthesis of N-[3-[2,5-bis(difluoromethoxy)phenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide TIFF0007682110000063.tif50170 A 2000 mL round bottom flask was charged with methanol (800 mL), concentrated hydrochloric acid (400 mL, 12N) and N-[5-[2,5-bis(difluoromethoxy)phenyl]-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (80 g, 141 mmol). The resulting solution was stirred at 25° C. for 4 hours. The solid was collected by filtration. The solid was added to a 1 L flask and diluted with H 2 200 mL of saturated NaHCO was added until the solution reached a pH of about 8. 3 The aqueous solution was added dropwise with stirring. The solid was collected by filtration, washed with water, and dried to give 55 g (89%) of N-[3-[2,5-bis(difluoromethoxy)phenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide as a pale yellow solid. 1 H NMR (300 MHz, CD 3 OD)δ 9.08(dd,J=7.2,1.5Hz,1H),8.65-8.61(m,2H),8.28(s,1H),7.46(d,J=9.0Hz,1H),7.40(d,J=3.0Hz,1H),7.34(dd,J LC / MS (Method H, ESI): [M+H] + =437.1,R T =1.12 minutes

[0289] Intermediate 2 TIFF0007682110000064.tif64170N-(5-(5-bromo-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide Step 1: Synthesis of 4-bromo-1-(difluoromethoxy)-2-iodobenzene To a solution of 4-bromo-2-iodophenol (282 g, 943 mmol) in N,N-dimethylformamide (2000 mL) and water (500 mL) was added sodium 2-chloro-2,2-difluoroacetate (216 g, 1.42 mol) and cesium carbonate (617 g, 1.89 mol). 2 An outlet was fitted for gas release. The resulting mixture was stirred at 120° C. overnight, cooled to room temperature and poured into ice water (3000 mL). The resulting solution was extracted with ethyl acetate (3×1500 mL) and the organic layers were combined. The ethyl acetate extract was washed with brine (1000 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel eluting with ethyl acetate / petroleum ether (1 / 10) to give 300 g (91%) of 4-bromo-1-(difluoromethoxy)-2-iodobenzene as a yellow oil. 1 H NMR (300 MHz, CDCl 3 )δ 7.96(dd,J=5.7Hz,2.4Hz,1H),7.45(dd,J=8.7Hz,2.4Hz,1H),7.03(d,J=8.7Hz,1H),6.39(t,J=72.9Hz,1H).

[0290] Step 2: Synthesis of 5-[5-bromo-2-(difluoromethoxy)phenyl]-4-nitro-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazole TIFF0007682110000066.tif34170A solution of 4-nitro-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazole (100 g, 411 mmol) in anhydrous THF (1000 mL) was added dropwise to a solution of LiHMDS (490 mL, 1.0 mol / L in THF) under nitrogen with stirring at -70°C. The resulting solution was stirred at -50°C for 1 h and then cooled to -70°C. ZnCl 2(500 mL, 0.7 mol / L in THF) was added dropwise at -70 °C. The resulting solution was allowed to warm to room temperature and stirred at room temperature for 1 h. The mixture was treated with 4-bromo-1-(difluoromethoxy)-2-iodobenzene (150 g, 860 mmol), Pd(PPh 3 ) 4( 24.0 g, 20.8 mmol) was added. The resulting solution was heated at reflux overnight, cooled to room temperature, and concentrated under reduced pressure. This reaction was repeated one more time at this scale, and the crude products from the two runs were combined for purification. The residue was purified by flash chromatography on silica gel eluting with ethyl acetate / petroleum ether (1 / 20). The appropriate fractions were combined and concentrated under reduced pressure. This gave 300 g (79%) of 5-[5-bromo-2-(difluoromethoxy)phenyl]-4-nitro-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazole as a pale yellow solid overall. 1 H NMR (300 MHz, CDCl 3 )δ 8.27(s,1H),7.68(dd,J=8.7,2.4Hz,1H),7.62(d,J=2.4Hz,1H),7.19(d,J=8.4Hz,1H),6.3 9(t,J=72.5Hz,1H),5.44-5.19(m,2H),3.72-3.54(m,2H),0.94-0.89(m,2H),0.02(s,9H).

[0291] Step 3: Synthesis of 5-(5-bromo-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-amine TIFF0007682110000067.tif341705-(5-Bromo-2-(difluoromethoxy)phenyl)-4-nitro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole (50.1 g, 108 mmol) in ethanol (2000 mL) and water (200 mL) was added to a solution of iron powder (60.1 g, 1.07 mol) and NH 4Cl (28.0 g, 0.523 mol) was added. The reaction mixture was stirred under nitrogen at reflux temperature for 3 h. The solid was filtered off and washed with ethanol (100 mL). The filtrate was concentrated under reduced pressure. The residue was dissolved in 3000 mL of ethyl acetate. The ethyl acetate solution was washed with 1×500 mL of brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure to give 50.1 g of crude 5-(5-bromo-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-amine as a yellow oil. The crude product was used in the next step without further purification. LC / MS (Method G, ESI): [M+H] + =434.2,R T =0.93 minutes

[0292] Step 4: Synthesis of N-(5-(5-bromo-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide TIFF0007682110000068.tif55170To a solution of 5-(5-bromo-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-amine (50.1 g, 115 mmol) in DMA (1500 mL) was added pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (32.1 g, 196.0 mmol), PyAOP (102 g, 196 mmol), DMAP (1.41 g, 11.0 mmol) and DIPEA (44.1 g, 0.341 mol). The resulting solution was stirred at 60° C. in an oil bath for 3 h and then cooled to room temperature. The reaction mixture was then partitioned between water / ice (2000 mL) and ethyl acetate (2000 mL). The aqueous phase was extracted with ethyl acetate (2x). The organic layers were combined, washed with brine (1000 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel eluting with ethyl acetate / petroleum ether (4:1). The appropriate fractions were combined and concentrated under reduced pressure. Water (150 mL) was added to the residue and the mixture was stirred in water at room temperature for 1 h. The solid was collected by filtration and air-dried to give 60.1 g (91%) of N-(5-(5-bromo-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide as a pale yellow solid. LC / MS (Method G, ESI): [M+H] + =579.1&581.1,R T =1.10 minutes 1 H NMR (300 MHz, CDCl 3 )δ 9.62(s,1H),8.80(dd,J=6.9,1.7Hz,1H),8.73(s,1H),8.53(dd,J=4.2,1.7H z,1H),8.38(s,1H),7.79(d,J=2.4Hz,1H),7.67(dd,J=8.8,2.5Hz,1H),7.29( d,J=1.4Hz,1H),7.00(dd,J=6.9,4.2Hz,1H),6.43(t,J=72.6Hz,1H),5.53-5. 27(m,2H),3.73-3.50(m,2H),0.88(ddd,J=9.5,6.4,4.4Hz,2H),0.00(s,9H).

[0293] Intermediate 3: TIFF0007682110000069.tif51170N-[3-[5-bromo-2-(difluoromethoxy)phenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide N-[5-[5-bromo-2-(difluoromethoxy)phenyl]-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (Intermediate 2, 5.00 g, 8.63 mmol) was treated with HCl / dioxane (150 mL, 4 M) at room temperature overnight. The mixture was concentrated under reduced pressure. This gave 3.80 g of N-[3-[5-bromo-2-(difluoromethoxy)phenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide as a yellow solid. The purity of the intermediate was sufficient to be used in the next step without further purification. LC / MS (Method I, ESI): [M+H] + =449.0,R T =1.02 minutes 1 H NMR (400 MHz, CD 3 OD)δ 9.11(dd,J=6.8,1.6Hz,1H),8.67-8.64(m,2H),8.32(s,1H),7.80(d,J=2.4Hz,1H),7.72(dd, J=8.8,2.4Hz,1H),7.37(d,J=8.8Hz,1H),7.23(dd,J=7.0,4.2Hz,1H),6.81(t,J=73.2Hz,1H).

[0294] Intermediate 4 TIFF0007682110000070.tif51170N-[3-[2-(difluoromethoxy)-5-iodophenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide Step 1: Synthesis of N-[5-[2-(difluoromethoxy)-5-iodophenyl]-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide To a solution of N-[5-[5-bromo-2-(difluoromethoxy)phenyl]-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (100 mg, 0.173 mmol) in t-BuOH (2 mL) was added N,N-dimethylethane-1,2-diamine (2.28 mg, 0.0259 mmol), NaI (155 mg, 1.04 mmol), and CuI (4.93 mg, 0.026 mmol) under nitrogen. The resulting solution was stirred at 120 °C in an oil bath under nitrogen for 14 h and then cooled to room temperature. The mixture was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel eluting with ethyl acetate / petroleum ether (1 / 1) to give 80 mg (74%) of N-[5-[2-(difluoromethoxy)-5-iodophenyl]-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide as a yellow solid. LC / MS (Method J, ESI): [M+H] + =627.1,R T =1.31 minutes

[0295] Step 2: Synthesis of N-[3-[2-(difluoromethoxy)-5-iodophenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide TIFF0007682110000072.tif51170N-[5-[2-(difluoromethoxy)-5-iodophenyl]-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (80.0 mg, 0.128 mmol) was dissolved in CF 3 CO 2H (3.0 mL) for 30 min at room temperature. The mixture was concentrated under reduced pressure. The residue was dissolved in water. Saturated sodium bicarbonate was added slowly until the pH of the solution was adjusted to about 8. The solid was collected by filtration. The solid was purified by flash chromatography on silica gel eluting with ethyl acetate / petroleum ether (2 / 1) to give 23.0 mg (36%) of N-[3-[2-(difluoromethoxy)-5-iodophenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide as a pale yellow solid. LC / MS (Method K, ESI): [M+H] + =497.1,R T =1.74 minutes 1 H NMR (300 MHz, CD 3 OD)δ 9.08(dd,J=6.9,1.5Hz,1H),8.65-8.61(m,2H),8.27(s,1H),7.94(s,1H),7.87(d,J=8.7Hz,1H),7.21-7.18(m,2H),6.78(t,J=73.2Hz,1H).

[0296] Intermediate 5 TIFF0007682110000073.tif51170N-[3-[5-cyclopropyl-2-(difluoromethoxy)phenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide Step 1: Synthesis of N-[3-[5-cyclopropyl-2-(difluoromethoxy)phenyl]-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide TIFF0007682110000074.tif52170To a solution of N-(5-(5-bromo-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (Intermediate 2, 1.40 g, 2.41 mmol) in dioxane (15 mL) and water (3.0 mL), cyclopropylboronic acid (314 mg, 3.66 mmol), Pd(dppf)Cl 2 -CH2 Cl 2( To the reaction mixture were added 200 mg, 0.245 mmol) and cesium carbonate (1.56 g, 4.79 mmol) under nitrogen. The reaction mixture was stirred at 80° C. under nitrogen overnight. The mixture was concentrated under reduced pressure. The residue was passed through a short pad of silica gel eluting with dichloromethane / methanol (94 / 6). The appropriate fractions were combined and concentrated under reduced pressure to give 1.40 g (purity=approximately 85% at 254 nm) of N-[3-[5-cyclopropyl-2-(difluoromethoxy)phenyl]-1-[[2-(trimethylsilyl)ethoxy]methoxy]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide as a dark red solid. LC / MS (Method G, ESI): [M+H] + =541.2,R T = 1.12 min. The intermediate was used without further purification.

[0297] Step 2: Synthesis of N-[3-[5-cyclopropyl-2-(difluoromethoxy)phenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide TIFF0007682110000075.tif52170N-[3-[5-cyclopropyl-2-(difluoromethoxy)phenyl]-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (145 mg) from the previous step was treated with HCl / dioxane (5.0 mL, 4 M) for 2 hours at 25°C. The solution was concentrated under reduced pressure. The residue was purified using the following: Column: Xbridge C18, 19*150 mm, 5 um; Mobile phase A: water / 0.05% NH 4 HCO 3 Purification by preparative HPLC under the following conditions: Mobile phase B: ACN; Flow rate: 30 mL / min; Gradient: 20% B to 85% B in 10 min; 254 nm afforded 44.9 mg (41%) of N-[3-[5-cyclopropyl-2-(difluoromethoxy)phenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrazolopyrimidine-3-carboxamide as a yellow solid. LC / MS (Method H, ESI): [M+H] + =411.2,RT =1.14 minutes 1 H NMR (300 MHz, CD 3 OD)δ 9.09(dd,J=6.9,1.5Hz,1H),8.63-8.61(m,2H),8.27(s,1H),7.28-7.25(m,3H),7.20(dd,J=7. 2,4.2Hz,1H),6.68(t,J=73.8Hz,1H),2.04-1.95(m,1H),1.03-0.97(m,2H),0.79-0.71(m,2H).

[0298] Intermediate 6 TIFF0007682110000076.tif51170 Pyrazolo[l,5-a]pyrimidine-3-carboxylic acid [3-(5-chloro-2-difluoromethoxyphenyl)-1H-pyrazol-4-yl]amide Step 1: Synthesis of 2-bromo-4-chloro-1-(difluoromethoxy)benzene TIFF0007682110000077.tif26170To a solution of 2-bromo-4-chlorophenol (4.98 g, 24.0 mmol) in DMF (25 mL) was added sodium chlorodifluoroacetate (8.42 g, 55.2 mmol), cesium carbonate (10.97 g, 33.67 mmol) and water (2.5 mL). The reaction was stirred at 100 °C for 16 h. The reaction mixture was partitioned between ethyl acetate and water and the organic portion was washed with brine and dried (MgSO 4 ) and evaporated. The crude product was purified by flash chromatography on silica eluting with 0-20% EtOAc in heptane to give 2-bromo-4-chloro-1-(difluoromethoxy)benzene as a clear, colorless oil (2.98 g, 48%). 1H NMR (400 MHz, DMSO-d 6 )δ:(ppm)7.90(d,1H),7.54(dd,1H),7.38(d,1H),7.28(t,1H).

[0299] Step 2: Synthesis of 5-(5-chloro-2-difluoromethoxyphenyl)-4-nitro-1-(2-trimethylsilanylethoxymethyl)-1H-pyrazole TIFF0007682110000078.tif40170To a solution of 4-nitro-1-(2-trimethylsilanylethoxymethyl)-1H-pyrazole (preparation described in WO2011003065) (46.5 g, 191 mmol) in DMA (350 mL) was added 2-bromo-4-chloro-1-difluoromethoxybenzene (64.0 g, 248 mmol), palladium(II) acetate (2.15 g, 9.6 mmol), di-(adamantyl)-n-butylphosphine (5.0 g, 13.4 mmol), potassium carbonate (79.2 g, 573 mmol) and trimethylacetic acid (5.27 g, 51.6 mmol). The mixture was degassed with nitrogen for 10 min and then heated at 130° C. for 8 h. The reaction mixture was allowed to cool to room temperature, diluted with ethyl acetate, washed with water and brine, and dried (MgSO 4 ), filtered and evaporated. The crude material was purified by flash chromatography on silica eluting with 0-10% EtOAc in cyclohexane to give 5-(5-chloro-2-difluoromethoxyphenyl)-4-nitro-1-(2-trimethylsilanylethoxymethyl)-1H-pyrazole (62.4 g, 78%). 1 H NMR (400 MHz, CDCl 3 )δ:(ppm)8.24(s,1H),7.52-7.53(m,2H),6.39(t,1H),5.29-5.30(m,2H),3.63-3.64(m,2H),0.90(s,9H).

[0300] Step 3: Synthesis of 5-(5-chloro-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-amine TIFF0007682110000079.tif34170 To a solution of 5-(5-chloro-2-difluoromethoxyphenyl)-4-nitro-1-(2-trimethylsilyllanylethoxymethyl)-1H-pyrazole (62 g, 148 mmol) in ethanol (600 mL) was added water (200 mL), ammonium chloride (32 g, 590 mmol) and iron powder (41 g, 740 mmol). The mixture was heated at 80° C. for 2 h and then cooled to room temperature. Residual solids were removed by filtration through Celite®. The filtrate was evaporated under reduced pressure, diluted with water and extracted twice with DCM. The combined organic extracts were washed with water and brine, dried (MgSO 4 ) and evaporated to give a dark oil. The oil was purified by flash chromatography on silica eluting with 0-25% EtOAc in DCM. Appropriate fractions were collected and the solvent removed in vacuo to give 5-(5-chloro-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-amine as a brown oil (30.8 g, 54%). 1 H NMR(400MHz,CDC13)δ:(ppm)7.56(d,1H),7.44(dd,1H),7.34(s,1H),7.30-7. 25(m,1H),6.37(t,1H),5.29(s,2H),3.56(t,2H),0.88(dd,2H),0.00(s,9H).

[0301] Step 4: Synthesis of N-(5-(5-chloro-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide TIFF0007682110000080.tif48170A solution of 5-(5-chloro-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-amine (60.0 g, 154 mmol) in THF (100 mL) was added dropwise over 30 min to an ice / water cooled mixture of pyrazolo[l,5-a]pyrimidine-3-carbonyl chloride (27.8 g, 153 mmol) and DIPEA (49.5 g, 383 mmol) in THF (300 mL). Once addition was complete the mixture was left stirring at room temperature for 1 h. The solvent was evaporated and the residue was diluted with 0.5 N aqueous HCl and extracted with ethyl acetate. The combined organic extracts were passed through Celite® to remove residual solids, and the filtrate was washed with 1M aqueous potassium carbonate, water and brine, dried (sodium sulfate) and evaporated to give a red solid. The solid was triturated with 10% diethyl ether in cyclohexane. The solid was collected by filtration, washed with 1:1 diethyl ether in cyclohexane and air-dried to give N-(5-(5-chloro-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide as an off-white solid (59.2 g, 73%). 1 H NMR (300 MHz, CDCl 3 ):δ(ppm)9.61(s,1H),8.77-8.78(m,1H),8.51(dd,1H),8.36(s,1H),7.65(d,1H),7.52(dd,1H),7.36(d,1H), 7.29(s,1H),7.01(dd,1H),6.42(t,1H),5.39-5.41(m,2H),3.60-3.64(m,2H),0.87-0.89(m,2H),0.09(s,9H).

[0302] Step 5: Synthesis of N-(3-(5-chloro-2-(difluoromethoxy)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide TIFF0007682110000081.tif54170 A suspension of N-(5-(5-chloro-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (59.0 g, 110 mmol) in methanol (420 mL) was treated with 6N HCl (80 mL) and the mixture was heated at 60° C. for 4 h. The solvent was evaporated and the residue triturated with water. The solid was collected by filtration, washed with water and air-dried. The solid was triturated with a minimum amount of acetonitrile, collected by filtration, washed with diethyl ether and dried under high vacuum at 60° C. to give the title compound as a yellow solid (42.9 g, 96%) 1 H NMR (400 MHz, DMSO-d 6 )δ:(ppm)9.71(s,1H),9.34(dd,1H),8.68-8.69(m,1H),8.66(s,1H),8.25(s,1H),7.62(dd,2H),7.43-7.46(m,1H),7.29(dd,1H),7.23(d,1H).

[0303] Intermediate 7 TIFF0007682110000082.tif54170N-(3-(2-(difluoromethoxy)-5-(methylthio)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide Step 1: Synthesis of 5-(2-(difluoromethoxy)-5-(methylthio)phenyl)-4-nitro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole TIFF0007682110000083.tif40170In a 1000 mL round bottom flask purged with nitrogen and maintained under an inert atmosphere of nitrogen, add toluene (500 mL), 5-[5-bromo-2-(difluoromethoxy)phenyl]-4-nitro-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazole (60 g, 129 mmol), NaSMe (26 g, 371 mmol), Pd 2 (dba) 3 .CHCl 3(6.7 g, 6.47 mmol) and XantPhos (7.5 g, 12.96 mmol) were added. The resulting mixture was stirred at 85 °C overnight. The resulting mixture was concentrated under vacuum. This reaction was repeated three times. The residue was subjected to a silica gel column eluting with ethyl acetate / petroleum ether (1:20). Appropriate fractions were combined and concentrated under vacuum. Thereby, 171 g of 5-(2-(difluoromethoxy)-5-(methylthio)phenyl)-4-nitro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole was obtained as a yellow solid in total. LC / MS (Method F, ESI): [M+H]+ = 432.1, RT = 1.23 min; 1 H NMR (300 MHz, CDC13) δ: (ppm) 8.25 (s, 1H), 7.42 (dd, J = 8.7, 2.4 Hz, 1H), 7.34 (d, J = 2.1 Hz, 1H), 7.23 (d, J = 8.7 Hz, 1H), 6.39 (t, J = 72.9 Hz, 1H), 5.36 - 5.22 (m, 2H), 3.74 - 3.55 (m, 2H), 2.51 (s, 3H), 0.94 - 0.90 (m, 2H), 0.02 (s, 9H).

[0304] Step 2: Synthesis of 5-(2-(difluoromethoxy)-5-(methylthio)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-amine TIFF0007682110000084.tif351705-[2-(difluoromethoxy)-5-(methoxysulfanyl)phenyl]-4-nitro-1-[[2- (trimethylsilyl)ethoxy]methoxy]-1H-pyrazole (171 g, 408 mmol), ethanol (2000 mL), water (200 mL) of the mixture was added with iron powder (228 g, 4.08 mol), NH 4 Cl (120 g, 2.24 mol). The reaction The mixture was stirred at reflux under nitrogen for 3 h and cooled to room temperature. The solid was filtered. The filtrate was concentrated under vacuum. The residue was dissolved in 3000 mL ethyl acetate and washed with 1×500 mL brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under vacuum. This gave 148 g of 5-(2-(difluoromethoxy)-5-(methylthio)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-amine as a yellow oil. LC / MS (Method F, ESI): [M+H]+=402.1, R T =0.93 minutes

[0305] Step 3: Synthesis of N-(5-(2-(difluoromethoxy)-5-(methylthio)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide TIFF0007682110000085.tif47170 A 3000 mL three-neck round bottom flask was charged with DMA (1500 mL), 5-(2-(difluoromethoxy)-5-(methoxythio)phenyl)-1-((2-(trimethylsilyl)ethoxy)methoxy)-1H-pyrazol-4-amine (148 g), pyrazolo[l,5-a]pyrimidine-3-carboxylic acid (102 g), HATU (325 g), 4-dimethylaminopyridine (4.5 g), and DIPEA (142 g). The resulting solution was stirred at 60° C. for 3 h, poured into ice water (2000 mL), extracted with 3×2000 mL of ethyl acetate, and the organic layers were combined. The resulting mixture was washed with 1×1000 mL of brine. The mixture was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was applied to a silica gel column eluted with ethyl acetate / petroleum ether (4:1) to give 200 g of N-(5-(2-(difluoromethoxy)-5-(methoxythio)phenyl)-1-((2-(trimethylsilyl)ethoxy)methoxy)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide as a pale yellow solid. LC / MS (Method A, ESI): [M+H]+=547.2, RT=1.10 min; 1 H NMR (300 MHz, CDC1 3)δ:(ppm)9.63(s,1H),8.77(dd,J=7.0,1.7Hz,1H),8.73(s,1H),8.51(dd,J=4. 2,1.8Hz,1H),8.38(s,1H),7.50(d,J=2.4Hz,1H),7.39(dd,J=8.7,2.4Hz,1H), 7.30(d,J=8.7Hz,1H),6.98(dd,J=6.9,4.2Hz,1H),6.39(t,J=73.2Hz,1H),5.4 6-5.38(m,2H),3.70-3.59(m,2H),2.52(s,3H),0.92-0.85(m,2H),0.03(s,9H).

[0306] Step 4: Synthesis of N-(3-(2-(difluoromethoxy)-5-(methylthio)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide TIFF0007682110000086.tif53170 To a solution of N-(5-(2-(difluoromethoxy)-5-(methoxythio)phenyl)-1-((2-(trimethylsilyl)ethoxy)methoxy)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (60 g) in methanol (600 mL) was added concentrated HCl solution (300 mL). The resulting solution was stirred at 35° C. overnight. The resulting mixture was concentrated under vacuum. The solid was collected by filtration. The solid was suspended in 200 mL of water. The pH value of the solution was adjusted to 8 with saturated sodium bicarbonate solution. The product was collected by filtration and dried to give 30 g (66%) of N-(3-(2-(difluoromethoxy)-5-(methoxythio)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide as a pale yellow solid. LC / MS (Method G, ESI): [M+H]+=417.0, R T =0.80 minutes; 1 H NMR (300 MHz, DMSO-d 6 ) δ: (ppm) 13.02 (s, 1H),9.71(s,1H),9.33(dd,J=6.9,1.5Hz,1H),8.68(dd,J=4.1,1.4Hz,1H),8.66(s,1H), 8.24(s,1H),7.47-7.36(m,3H),7.27(dd,J=6.9,4.2Hz,1H),7.17(t,J=73.8Hz,1H),2.51(s,3H).

[0307] Intermediate 8 TIFF0007682110000087.tif45170N-(5-(5-bromo-4-chloro-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide Step 1: Synthesis of 4-bromo-5-chloro-2-iodophenol TIFF0007682110000088.tif16170A solution of 5-chloro-2-iodophenol (100 g, 393 mmol) in acetonitrile (1000 mL) was diluted with CuBr 2 (264 g, 1.18 mol) was added in several batches with stirring at 70° C. The resulting mixture was stirred at 70° C. for 4 h, cooled to room temperature and concentrated under vacuum. The residue was then quenched by addition of 3000 mL water / ice, extracted with 3×2000 mL ethyl acetate and the organic layers combined. The extracts were washed with 1×1000 mL brine, dried over sodium sulfate and concentrated under vacuum. The residue was purified by flash chromatography on silica gel eluting with ethyl acetate / petroleum ether (1:30). Appropriate fractions were collected and concentrated under vacuum. The reaction was repeated one more time. This gave 140 g (53%) of 4-bromo-5-chloro-2-iodophenol as a white solid. 1 H NMR (400 MHz, CDCl 3 ):δ(ppm)7.89(s,1H),7.14(s,1H),5.32(s,1H).

[0308] Step 2: Synthesis of l-bromo-2-chloro-4-(difluoromethoxy)-5-iodobenzene TIFF0007682110000089.tif17170To a solution of 4-bromo-5-chloro-2-iodophenol (140 g, 420 mmol) in DMF (1200 mL) was added sodium 2-chloro-2,2-difluoroacetate (95.8 g, 628 mmol), cesium carbonate (274 g, 840 mmol). The reaction mixture was stirred in an oil bath at 120° C. for 2 h, cooled to room temperature, and then quenched by the addition of 2500 mL of water / ice. The resulting solution was extracted with 3×2000 mL of ethyl acetate and the organic layers were combined. The extracts were washed with 1×1000 mL of brine, dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was purified by flash chromatography on silica gel eluting with ethyl acetate / petroleum ether (1 / 30). The appropriate fractions were combined and concentrated in vacuo to give 130 g (81%) of l-bromo-2-chloro-4-(difluoromethoxy)-5-iodobenzene as a pale yellow solid.

[0309] Step 3: Synthesis of 5-(5-bromo-4-chloro-2-(difluoromethoxy)phenyl)-4-nitro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole TIFF0007682110000090.tif33170To a solution of 4-nitro-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazole (67.0 g, 275 mmol) in tetrahydrofuran (1000 mL) was added LiHMDS (340 mL, 1 M in THF) dropwise with stirring at −70° C. under nitrogen. The resulting solution was stirred at −70° C. for 1 h. To this solution was added ZnCl 2 (400 mL, 0.7 M in THF) was added dropwise. The resulting solution was stirred at -70 °C under nitrogen for 1 h. The mixture was treated with 1-bromo-2-chloro-4-(difluoromethoxy)-5-iodobenzene (105 g, 274 mmol), Pd(PPh 3 ) 4(16.0 g, 13.9 mmol) was added under nitrogen. The resulting solution was stirred at 90° C. overnight, allowed to cool to room temperature and concentrated under vacuum. The residue was purified by flash chromatography on silica gel eluting with ethyl acetate / petroleum ether (1:20). Appropriate fractions were collected and concentrated under vacuum. This gave 115 g (84%) of 5-[5-bromo-4-chloro-2-(difluoromethoxy)phenyl]-4-nitro-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazole as a pale yellow solid. LC / MS (Method B, ESI): [M+H]+=498.0&500.0, Rt=1.27 min.

[0310] Step 4: Synthesis of 5-[5-bromo-4-chloro-2-(difluoromethoxy)phenyl]-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazol-4-amine TIFF0007682110000091.tif32170To a solution of 5-(5-bromo-4-chloro-2-(difluoromethoxy)phenyl)-4-nitro-1-((2-(trimethylsilyl)ethoxy)methoxy)-1H-pyrazole (102 g, 205 mmol) in ethanol (1000 mL) and water (100 mL) was added iron powder (102 g, 1.82 mol) and ammonium chloride (53 g, 1.00 mol). The reaction mixture was stirred in an oil bath at 100° C. for 3 h. The solids were filtered. The filtrate was concentrated under vacuum. The residue was dissolved in 2000 mL of ethyl acetate. The organic solution was washed with 1×500 mL of brine, dried over anhydrous sodium sulfate, and concentrated under vacuum. This gave 102 g of (crude) 5-[5-bromo-4-chloro-2-(difluoromethoxy)phenyl]-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazol-4-amine as a pale yellow oil. LC / MS (Method E, ESI): [M+H]+=467.9&469.9, R T =1.29 minutes

[0311] Step 5: Synthesis of N-(5-(5-bromo-4-chloro-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide TIFF0007682110000092.tif45170To a solution of 5-[5-bromo-4-chloro-2-(difluoromethoxy)phenyl]-1-[[2-(trimethylsilyl)ethoxy]methoxy]-1H-pyrazol-4-amine (100 g, 213 mmol) in DMA (800 mL) was added pyrazolo[l,5-a]pyrimidine-3-carboxylic acid (52.0 g, 319 mmol), PyAOP (166 g, 319 mmol), DIPEA (82.3 g, 638 mmol) and 4-dimethylaminopyridine (2.59 g, 21.2 mmol). The resulting solution was stirred overnight at 60° C. in a water bath. The reaction was then quenched by adding 2000 mL of water / ice. The resulting solution was extracted with 3×1500 mL of ethyl acetate and the organic layers were combined. The combined organic layers were washed with 500 mL of brine, dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified by flash chromatography on silica gel eluting with ethyl acetate / petroleum ether (2:1). The appropriate fractions were combined and concentrated under vacuum. The residue was suspended in water (800 mL) and stirred for 1 h. The solid was collected by filtration. This gave 113 g (86%) of N-(5-(5-bromo-4-chloro-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide as an off-white solid. LC / MS (Method A, ESI): [M+H]+=613.2&615.2, RT=2.29 min. 1 H NMR (400 MHz, CDCl 3): δ (ppm) 9.56 (s, 1H), 8.81 (dd, J = 6.8, 1.5 Hz, 1H), 8.73 (s, 1H), 8.53 (d, J = 4.0 Hz, 1H), 8.33 (s, 1H), 7.92 (s, 1H), 7.54 (s, 1H), 7.03 (dd, J = 6.8 Hz, 4.0 Hz, 1H), 6.45 (t, J = 72.2 Hz, 1H), 5.43 (d, J = 11.2 Hz, 1H), 5.35 (d, J = 11.2 Hz, 1H), 3.68 - 3.56 (m, 2H), 0.94 - 0.84 (m, 2H), 0.00 (s, 9H).

[0312] Intermediate 9 TIFF0007682110000093.tif 48170 N-(3-(6-(Difluoromethoxy)-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide Step 1: Synthesis of tert-butyl (2-(2-chloro-4-(difluoromethoxy)-5-(4-(pyrazolo[1,5-a]pyrimidine-3-carboxamide)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-5-yl)phenoxy)ethyl)carbamate TIFF0007682110000094.tif 55170 To a solution of Intermediate 8 (200 mg, 0.326 mmol) in toluene (10 mL) under nitrogen, tert-butyl N-(2-hydroxyethyl)carbamate (105 mg, 0.651 mmol), [PdCl(allyl)] 2(6.01 mg, 0.0161 mmol), t-BuBrettPhos (16.0 mg, 0.0329 mmol) and cesium carbonate (213 mg, 0.654 mmol) were added. The resulting solution was stirred at 60° C. for 4 h and concentrated in vacuo. The residue was purified by silica gel flash chromatography eluting with dichloromethane / methanol (19 / 1). The appropriate fractions were combined and concentrated in vacuo to give 182 mg (80%) of tert-butyl (2-(2-chloro-4-(difluoromethoxy)-5-(4-(pyrazolo[1,5-a]pyrimidine-3-carboxamido)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-5-yl)phenoxy)ethyl)carbamate as a yellow oil. LC / MS (Method C, ESI): [M+H]+=694.1, Rt=1.54 min

[0313] Step 2: Synthesis of tert-butyl 6-(difluoromethoxy)-7-(4-(pyrazolo[1,5-a]pyrimidine-3-carboxamido)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-5-yl)-2,3-dihydro-4H-benzo[b][1,4]oxazine-4-carboxylate To a solution of tert-butyl (2-(2-chloro-4-(difluoromethoxy)-5-(4-(pyrazolo[1,5-a]pyrimidine-3-carboxamido)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-5-yl)phenoxy)ethyl)carbamate (182 mg, 0.270 mmol) in t-BuOH (15 mL) was added BrettPhos Palladacycle Gen.3 (CAS 1470372-59-8, vendor J&K Scientific Ltd) (48.0 mg, 0.0530 mmol), BrettPhos (56.0 mg, 0.104 mmol) and potassium carbonate (73.0 mg, 0.528 mmol) under nitrogen. The resulting solution was stirred at 110° C. for 20 h, cooled to room temperature and concentrated in vacuo. The residue was purified by flash chromatography on silica gel eluting with ethyl acetate / petroleum ether (1 / 1). Appropriate fractions were combined and concentrated in vacuo to give 95.0 mg (53%) of tert-butyl 6-(difluoromethoxy)-7-(4-(pyrazolo[1,5-a]pyrimidine-3-carboxamido)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-5-yl)-2,3-dihydro-4H-benzo[b][1,4]oxazine-4-carboxylate as a yellow solid. LC / MS (Method B, ESI): [M+H]+=658.1, Rt=1.17 min.

[0314] Step 3: Synthesis of N-(3-(6-(difluoromethoxy)-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide TIFF0007682110000096.tif49170 To a solution of tert-butyl 6-(difluoromethoxy)-7-(4-(pyrazolo[1,5-a]pyrimidine-3-carboxamido)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-5-yl)-2,3-dihydro-4H-benzo[b][1,4]oxazine-4-carboxylate (80.0 mg, 0.122 mmol) in methanol (8.0 mL) was added aqueous HCl (6 mol / L in water) (4.0 mL). The resulting solution was stirred at 25° C. for 4 hours and concentrated under vacuum. The crude product (50.0 mg) was purified using the following: Column, XBridge Shield RP18 OBD Column, 19*150 mm, 5 um; Mobile phase, 10 mM NH in water and acetonitrile; 4 HCO 3 (10.0% acetonitrile to 38.0% in 10 min); detection at UV 254 nm, purification by preparative HPLC gave 16.1 mg (24%) of N-(3-(6-(difluoromethoxy)-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide as a yellow solid. LC / MS (Method D, ESI): [M+H]+=428.0, Rt=2.05 min; 1 H NMR(400 MHz,CD3OD):δ(ppm)8.98(d,J=6.8Hz,1H),8.57-8.51(m,2H),8.12(s,1H),7.10(dd,J=7.0,4.2 Hz,1H),6.79(s,1H),6.51(s,1H),6.40(t,J=75.2Hz,1H),4.13-4.11(m,2H),3.39-3.32(m,2H).

[0315] Intermediate 10 TIFF0007682110000097.tif56170N-(1-((2H-tetrazol-5-yl)methyl)-3-(2,5-bis(difluoromethoxy)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide Step 1: Synthesis of N-(3-(2,5-bis(difluoromethoxy)phenyl)-1-((2-(tetrahydro-2H-pyran-2-yl)-2H-tetrazol-5-yl)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide and N-(3-(2,5-bis(difluoromethoxy)phenyl)-1-((1-(tetrahydro-2H-pyran-2-yl)-1H-tetrazol-5-yl)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide as red oils (mixture of regioisomers) TIFF0007682110000098.tif58170To a suspension of N-[3-[2,5-bis(difluoromethoxy)phenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (Intermediate 1, 10.0 g, 22.9 mmol), cesium carbonate (22.3 g, 68.6 mmol) and TBAI (423 mg, 1.15 mmol) in N,N-dimethylformamide (100 mL) was added 5-(chloromethyl)-2-tetrahydropyran-2-yl-tetrazole (11.6 g, 57.3 mmol) at room temperature. The reaction mixture was stirred for 1.5 h, then diluted with brine (300 mL) and extracted with ethyl acetate (3×300 mL). The organic layers were combined, washed with brine (300 mL), dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was purified by silica gel flash chromatography eluting with DCM (1% TEA) / EA (1 / 2) to give a mixture of N-(3-(2,5-bis(difluoromethoxy)phenyl)-1-((2-(tetrahydro-2H-pyran-2-yl)-2H-tetrazol-5-yl)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide and N-(3-(2,5-bis(difluoromethoxy)phenyl)-1-((1-(tetrahydro-2H-pyran-2-yl)-1H-tetrazol-5-yl)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide as a red oil. LC / MS (Method I, ESI): [M+H]+=603.25, Rt=1.02 min

[0316] Step 2: Synthesis of N-(1-((2H-tetrazol-5-yl)methyl)-3-(2,5-bis(difluoromethoxy)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide A solution of N-[3-[2,5-bis(difluoromethoxy)phenyl]-1-[(2-tetrahydropyran-2-yltetrazol-5-yl)methyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (mixture of regioisomers, 4.21 g, 6.98 mmol) in HCl / MeOH (60.0 mL, 240 mmol) was stirred at room temperature overnight. The residue was purified by flash chromatography on a C18 column eluting with 38.7% ACN / water to give N-(1-((2H-tetrazol-5-yl)methyl)-3-(2,5-bis(difluoromethoxy)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (2.50 g, 4.83 mmol, 69.2% yield) as a white solid. LC / MS (Method I, ESI): [M+H]+=519.3, Rt=0.71 min.

[0317] Intermediate 11 TIFF0007682110000100.tif52170N-(1-((2H-tetrazol-5-yl)methyl)-3-(2-(difluoromethoxy)-5-(methylthio)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide Step 1: Synthesis of N-(3-(2-(difluoromethoxy)-5-(methylthio)phenyl)-1-((2-(tetrahydro-2H-pyran-2-yl)-2H-tetrazol-5-yl)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (along with tetrazole alkylated isomers) TIFF0007682110000101.tif55170A solution of N-[3-[2-(difluoromethoxy)-5-methylsulfanyl-phenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (intermediate 7, 2.02 g, 4.85 mmol) in N,N-dimethylformamide (20 mL) was stirred at room temperature. Then, 5-(chloromethyl)-2-tetrahydropyran-2-yl-tetrazole (2.81 g, 13.9 mmol), cesium carbonate (4.71 g, 14.5 mmol), TBAI (88.7 mg, 0.24 mmol) were added and stirred at room temperature for 1.5 h. After filtration, the filtrate was diluted with water (50 mL). The resulting solution was extracted with EA (50×3 mL) and the organic layers were combined. The organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified by flash chromatography on silica gel eluting with EA / DCM (1% TEA) (46%) to give a mixture of N-[3-[2-(difluoromethoxy)-5-methylsulfanyl-phenyl]-1-[(2-tetrahydropyran-2-yltetrazol-5-yl)methyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide with tetrazole alkylation regioisomer (3.51 g, 6.02 mmol) as a white solid. LC / MS (Method M, ESI): [M+H]+=583.1, Rt=0.66 min.

[0318] Step 2: Synthesis of N-(1-((2H-tetrazol-5-yl)methyl)-3-(2-(difluoromethoxy)-5-(methylthio)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide TIFF0007682110000102.tif52170 A solution of N-[3-[2-(difluoromethoxy)-5-methylsulfanyl-phenyl]-1-[(2-tetrahydropyran-2-yltetrazol-5-yl)methyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (mixture of regioisomers, 8.34 g, 14.3 mmol) in methanol (30 mL) was stirred at room temperature. Then 1,4-dioxane (80 mL) (4 M HCl) was added and the mixture was stirred at room temperature for 2 h. The solvent was concentrated under vacuum and the crude product was used without further purification. LC / MS (Method M, ESI): [M+H]+=499.1, RT=0.61 min.

[0319] Intermediate 12 TIFF0007682110000103.tif52170N-(1-((2H-tetrazol-5-yl)methyl)-3-(5-chloro-2-(difluoromethoxy)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide Step 1: Synthesis of N-[3-[5-chloro-2-(difluoromethoxy)phenyl]-1-[(2-tetrahydropyran-2-yltetrazol-5-yl)methyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (along with tetrazole alkylated isomers) TIFF0007682110000104.tif51170To a solution of N-[3-[5-chloro-2-(difluoromethoxy)phenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (3.2 g, 7.92 mmol) in N,N-dimethylformamide (30 mL) was added cesium carbonate (5.2 g, 15.99 mmol) and TBAI (178 mg, 0.480 mmol) and 5-(chloromethyl)-2-tetrahydropyran-2-yl-tetrazole (4.05 g, 20.0 mmol) at room temperature. The resulting solution was stirred at room temperature for 2 h. The reaction mixture was diluted with water (150 mL). The resulting solution was extracted with EA (300*3 mL) and the organic layers were combined. The organic layers were concentrated under vacuum. The residue was purified by silica gel flash chromatography eluting with EA / DCM (24%) to give a mixture of N-[3-[5-chloro-2-(difluoromethoxy)phenyl]-1-[(2-tetrahydropyran-2-yltetrazol-5-yl)methyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (3.20 g, 5.33 mmol, 67.3% yield) with the tetrazole alkylation regioisomer as a yellow oil. LC / MS (Method M, ESI): [M+H]+=471.1, RT=0.66 min.

[0320] Step 2: Synthesis of N-(1-((2H-tetrazol-5-yl)methyl)-3-(5-chloro-2-(difluoromethoxy)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide A solution of N-[3-[5-chloro-2-(difluoromethoxy)phenyl]-1-[(2-tetrahydropyran-2-yltetrazol-5-yl)methyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (mixture of regioisomers, 3.21 g, 4.5 mmol) in TIFF0007682110000105.tif511704M HCl / MeOH (40 mL, 4.5 mmol) was stirred at room temperature for 2 h. The organic layer was concentrated under vacuum. The residue was purified by elution with ACN / H 2Purification by flash chromatography on C18 gel eluted with O(TFA) (35%) gave N-[3-[5-chloro-2-(difluoromethoxy)phenyl]-1-(2H-tetrazol-5-ylmethyl)pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (1.81 g, 3.35 mmol, 74.4% yield) as a yellow solid. LC / MS (Method H, ESI): [M+H]+=487.1, RT=1.12 min.

[0321] Intermediate 13 TIFF0007682110000106.tif51170N-(1-((2H-tetrazol-5-yl)methyl)-3-(5-cyclopropyl-2-(difluoromethoxy)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide Step 1: Synthesis of N-(3-(5-cyclopropyl-2-(difluoromethoxy)phenyl)-1-((2-(tetrahydro-2H-pyran-2-yl)-2H-tetrazol-5-yl)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide TIFF0007682110000107.tif52170 Step 1: Synthesis of N-[3-[5-cyclopropyl-2-(difluoromethoxy)phenyl]-1-[(2-tetrahydropyran-2-yltetrazol-5-yl)methyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide To a solution of N-[3-[5-cyclopropyl-2-(difluoromethoxy)phenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (intermediate 5, 3.02 g, 7.35 mmol), TBAI (271 mg, 0.740 mmol) and cesium carbonate (7.18 g, 22.1 mmol) in N,N-dimethylformamide (40 mL) was added 5-(chloromethyl)-2-tetrahydropyran-2-yl-tetrazole (3.75 g, 18.5 mmol) at room temperature. The resulting solution was stirred at room temperature for 1.5 h. The residue was filtered through Celite® and the filtrate was diluted with water (80 mL). The resulting mixture was extracted with EA (80*3 mL). The organic layer was washed with brine (100*3 mL), dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified by flash chromatography on silica gel eluting with EA / PE (0.1% TEA)=4 / 1 to give a mixture of N-[3-[5-cyclopropyl-2-(difluoromethoxy)phenyl]-1-[(2-tetrahydropyran-2-yltetrazol-5-yl)methyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide and the tetrazole alkylated regioisomer (3.26 g, 5.65 mmol, 76.8% yield) as a yellow oil. LC / MS (Method G, ESI): [M+H]+=577.2, RT=0.98 min.

[0322] Step 2: Synthesis of N-[3-[5-cyclopropyl-2-(difluoromethoxy)phenyl]-1-[(2-tetrahydropyran-2-yltetrazol-5-yl)methyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide A solution of N-[3-[5-cyclopropyl-2-(difluoromethoxy)phenyl]-1-[(2-tetrahydropyran-2-yltetrazol-5-yl)methyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (mixture of regioisomers, 3.26 g, 5.65 mmol) in TIFF0007682110000108.tif511704M HCl / methanol (20 mL) was stirred at room temperature for 2 h. The resulting solution was concentrated in vacuo. The resulting residue was purified by reverse phase chromatography (acetonitrile 0-45 / 0.1% TFA in water) to give N-[3-[5-cyclopropyl-2-(difluoromethoxy)phenyl]-1-(2H-tetrazol-5-ylmethyl)pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (1.85 g, 3.8 mmol, 66.5% yield) as a pale yellow solid. LC / MS (Method H, ESI): [M+H]+=493.2, RT=1.16 min.

[0323] Working Example Example 1 TIFF0007682110000109.tif52170N-(3-(5-chloro-2-(difluoromethoxy)phenyl)-1-((2-(2-hydroxyethyl)-2H-tetrazol-5-yl)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide A 100 mL round bottom flask was charged with N-[3-[5-chloro-2-(difluoromethoxy)phenyl]-1-(2H-1,2,3,4-tetrazol-5-ylmethyl)-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (Intermediate 12,200 mg, 0.412 mmol), 1,3-dioxolan-2-one (122 mg, 1.38 mmol, 3.35 equiv), sodium hydroxide (44 mg, 1.10 mmol, 2.67 equiv), and N,N-dimethylformamide (30 mL). The resulting solution was stirred in an oil bath at 120° C. for 4 hours. The resulting mixture was concentrated under vacuum. The residue was applied to a silica gel column with dichloromethane / petroleum ether (11.5:1). The crude product was purified by chiral-preparative HPLC (Prep HPLC-009) using the following conditions: column, Phenomenex Lux 5u Cellulose-4 XIA Packed, 2.12*25cm, 5um; mobile phase, hexane and ethanol (retained 60.0% ethanol at 32 min); detector, UV 220 / 254 nm. This gave 19.6 mg (9%) of N-[3-[5-chloro-2-(difluoromethoxy)phenyl]-1-[[2-(2-hydroxyethyl)-2H-1,2,3,4-tetrazol-5-yl]methyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide as a white solid. LC / MS (Method N, ESI): [M+H]+=531.2, RT=1.33 min. 1 H NMR (300 MHz, DMSO-d 6 ):δ(ppm)9.76(s,1H),9.35(dd,J=6.9,1.5Hz,1H),8.68-8.66(m,2H),8.52(s,1H),7.64(dd,J=8.9,2.9Hz,1H),7.57-6.99(m,4 H),5.78(s,2H),5.07(t,J=5.6Hz,1H),4.72(t,J=5.1Hz,2H),3.94-3.88(m,2H).

[0324] Example 21 N-(3-(2,5-bis(difluoromethoxy)phenyl)-1-((2-(1-(2-(dimethylamino)ethyl)azetidin-3-yl)-2H-tetrazol-5-yl)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide TIFF0007682110000110.tif52170 Step 1: Synthesis of tert-butyl 3-(5-((3-(2,5-bis(difluoromethoxy)phenyl)-4-(pyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-1-yl)methyl)-2H-tetrazol-2-yl)azetidine-1-carboxylate TIFF0007682110000111.tif611701-Boc-3-iodoazetidine (2.42 g, 8.55 mmol) was added to a mixture of N-[3-[2,5-bis(difluoromethoxy)phenyl]-1-(2H-tetrazol-5-ylmethyl)pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (Intermediate 1, 1.87 g, 3.61 mmol) and potassium carbonate (2.43 g, 17.6 mmol) in N,N-dimethylformamide (20 mL). The resulting solution was stirred at 60° C. for 3 h and at 75° C. overnight. The mixture was brought to room temperature and filtered through Celite®. The filtrate was diluted with brine (120 mL). The resulting mixture was extracted with EA (3×50 mL) and the organic layers were combined. The organic layer was washed with brine (2*50 mL). The residue was purified by silica gel flash chromatography eluting with DCM (1% TEA) / EA (1% TEA) (1 / 2) to give tert-butyl 3-[5-[[3-[2,5-bis(difluoromethoxy)phenyl]-4-(pyrazolo[1,5-a]pyrimidine-3-carbonylamino)pyrazol-1-yl]methyl]tetrazol-2-yl]azetidine-1-carboxylate (1.4 g, 92%) as a yellow oil and 330 mg of tert-butyl 3-[5-[[3-[2,5-bis(difluoromethoxy)phenyl]-4-(pyrazolo[1,5-a]pyrimidine-3-carbonylamino)pyrazol-1-yl]methyl]tetrazol-2-yl]azetidine-1-carboxylate (mixture of diastereomers) as a yellow oil.

[0325] 1.4 g of tert-butyl 3-[5-[[3-[2,5-bis(difluoromethoxy)phenyl]-4-(pyrazolo[1,5-a]pyrimidine-3-carbonylamino)pyrazol-1-yl]methyl]tetrazol-2-yl]azetidine-1-carboxylate was purified by reverse phase chromatography (acetonitrile 0-60 / 0.1% NH 4 HCO 3aqueous solution) to give tert-butyl 3-[5-[[3-[2,5-bis(difluoromethoxy)phenyl]-4-(pyrazolo[1,5-a]pyrimidine-3-carbonylamino)pyrazol-1-yl]methyl]tetrazol-2-yl]azetidine-1-carboxylate (1.00 g, 1.5 mmol, 41.2% yield) as a white solid. LC / MS (Method C, ESI): [M+H]+=674.2 RT=2.63 min.

[0326] 330 mg of the mixture of diastereomers was purified by silica gel flash chromatography eluting with DCM (1% TEA) / EA (1% TEA) (1 / 2) to give tert-butyl 3-[5-[[3-[2,5-bis(difluoromethoxy)phenyl]-4-(pyrazolo[1,5-a]pyrimidine-3-carbonylamino)pyrazol-1-yl]methyl]tetrazol-1-yl]azetidine-1-carboxylate (140 mg, 0.208 mmol, 5.8% yield) as a yellow solid. LC / MS (Method G, ESI): [M+H]+=674.2 RT=0.97 min.

[0327] Step 2: Synthesis of N-(1-((2-(azetidin-3-yl)-2H-tetrazol-5-yl)methyl)-3-(2,5-bis(difluoromethoxy)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide TIFF0007682110000112.tif541702,2,2-Trifluoroacetic acid (3 mL) / dichloromethane (12 mL) of tert-butyl 3-(5-((3-(2,5-bis(difluoromethoxy)phenyl)-4-(pyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-1-yl)methyl)-2H-tetrazol-2-yl)azetidine-1-carboxylate (1.05 g, 1.56 mmol) was stirred at room temperature for 2 h. The reaction mixture was concentrated under vacuum and the product obtained was used without further purification. LC / MS (Method R, ESI): [M+H]+ = 574.2 RT = 1.58 min.

[0328] Step 3: Synthesis of tert-butyl (2-(3-(5-((3-(2,5-bis(difluoromethoxy)phenyl)-4-(pyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-1-yl)methyl)-2H-tetrazol-2-yl)azetidin-1-yl)ethyl)carbamate TIFF0007682110000113.tif55170To a solution of N-(1-((2-(azetidin-3-yl)-2H-tetrazol-5-yl)methyl)-3-(2,5-bis(difluoromethoxy)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (821 mg, 1.43 mmol) in 1,4-dioxane (30 mL), potassium carbonate (593 mg, 4.29 mmol) was added at room temperature. The resulting solution was stirred for 10 min, after which tert-butyl 2-bromoethylcarbamate (1.29 g, 5.73 mmol) was added. The resulting reaction mixture was stirred at 70° C. overnight. The mixture was concentrated under vacuum and the residue was dissolved in 42% ACN / NH 4 HCO 3 Purification by flash chromatography on a C18 column eluting with (0.05%) afforded tert-butyl (2-(3-(5-((3-(2,5-bis(difluoromethoxy)phenyl)-4-(pyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-1-yl)methyl)-2H-tetrazol-2-yl)azetidin-1-yl)ethyl)carbamate (715 mg, 0.998 mmol, 69.8% yield) as a yellow oil. LC / MS (Method M, ESI): [M+H]+=717.4, RT=0.67 min.

[0329] Step 4: Synthesis of N-(1-((2-(1-(2-aminoethyl)azetidin-3-yl)-2H-tetrazol-5-yl)methyl)-3-(2,5-bis(difluoromethoxy)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide TIFF0007682110000114.tif54170 A solution of tert-butyl (2-(3-(5-((3-(2,5-bis(difluoromethoxy)phenyl)-4-(pyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-1-yl)methyl)-2H-tetrazol-2-yl)azetidin-1-yl)ethyl)carbamate (720 mg, 1 mmol) in dichloromethane (4 mL) and trifluoroacetic acid (1 mL) was stirred at room temperature for 2 h. The reaction mixture was concentrated in vacuo. The residue was purified by flash chromatography on a C18 column eluting with 45% ACN / water (0.05% HCl) to give N-(1-((2-(1-(2-aminoethyl)azetidin-3-yl)-2H-tetrazol-5-yl)methyl)-3-(2,5-bis(difluoromethoxy)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (615 mg, 0.10 mmol, 99.3% yield) as a yellow oil. LC / MS (Method M, ESI): [M+H]+=617.4, RT=0.54 min.

[0330] Step 5: Synthesis of N-(3-(2,5-bis(difluoromethoxy)phenyl)-1-((2-(1-(2-(dimethylamino)ethyl)azetidin-3-yl)-2H-tetrazol-5-yl)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide TIFF0007682110000115.tif54170A solution of N-[1-[[2-[1-(2-aminoethyl)azetidin-3-yl]tetrazol-5-yl]methyl]-3-[2,5-bis(difluoromethoxy)phenyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (615 mg, 1 mmol) in methanol (10 mL) at room temperature was diluted with HCHO / H 2 NaBH(AcO) (256 mg, 3.15 mmol) was added. The resulting solution was stirred at 25° C. for 2 h. 3 (846 mg, 3.99 mmol) was added and the reaction mixture was stirred at 25° C. for 3 h, after which it was concentrated in vacuo. The residue was purified by distillation with ACN / water (0.05% NH4 HCO 3 The product was purified by flash chromatography on silica gel eluting with 500 cc (1000 cc) of 1,2,5-tetrafluoroethanediamine (1,2,5-bis(difluoromethoxy)phenyl)-1-[[2-[1-[2-(dimethylamino)ethyl]azetidin-3-yl]tetrazol-5-yl]methyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (433 mg, 0.663 mmol, 66.4% yield) as a white solid. LC / MS (Method X, ESI): [M+H]+=645.3, RT=3.38 min. 1 H NMR (400 MHz, DMSO-d 6 ):δ(ppm)9.76(s,1H),9.34(dd,J=7.2,1.6Hz,1H),8.67-8.65(m,2H),8.52(s,1H),7.49-7.46(m,1H),7.40-6.99(m,5 H),5.80(s,2H),5.58-5.54(m,1H),3.83-3.79(m,2H),3.57-3.53(m,2H),2.60-2.56(m,2H),2.22-2.20(m,2H),2.12(s,6H).

[0331] Example 10 TIFF0007682110000116.tif52170N-(3-(2,5-bis(difluoromethoxy)phenyl)-1-((2-(1-methylazetidin-3-yl)-2H-tetrazol-5-yl)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide N-[1-[[2-(azetidin-3-yl)tetrazol-5-yl]methyl]-3-[2,5-bis(difluoromethoxy)phenyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (670.5 mg, 1.17 mmol) in methanol (10 mL) and HCHO / H 2 A solution of 2H2O (113 mg, 1.4 mmol) was stirred at 25 °C for 2 h. Then, NaBH(CH 3 COO) 3(301 mg, 1.42 mmol) was added and stirred at 25° C. overnight. The solvent was concentrated under vacuum and the resulting residue was diluted with water (20 mL). The resulting solution was extracted with EA (50×3 mL) and the organic layers were combined. The organic layers were dried over anhydrous sodium sulfate and concentrated under vacuum. The product was purified using the following: Column: XBridge Prep OBD C18 Column 30×150 mm 5 um; Mobile phase A: Water (10 mmol / L NH 4 HCO 3 ), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 27% B to 37% B in 10 min; 254 / 220 nm; Rt: 13 min to give N-[3-[2,5-bis(difluoromethoxy)phenyl]-1-[[2-(1-methylazetidin-3-yl)tetrazol-5-yl]methyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (507 mg, 0.858 mmol, 73.4% yield) as a white solid. LC / MS (Method E, ESI): [M+H]+=588.2, RT=2.28 min. 1 H NMR (400 MHz, DMSO-d 6 ):δ(ppm)9.77(s,1H),9.35(dd,J=7.2,1.2Hz,1H),8.68-8.64(m,2H),8.52(s,1H),7.50-7.0 0(m,6H),5.80(s,2H),5.58-5.52(m,1H),3.84-3.80(m,2H),3.59-3.52(m,2H),2.33(s,3H).

[0332] Example 12 TIFF0007682110000117.tif49170N-(1-((1-(azetidin-3-yl)-1H-tetrazol-5-yl)methyl)-3-(2,5-bis(difluoromethoxy)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide A solution of tert-butyl 3-[5-[[3-[2,5-bis(difluoromethoxy)phenyl]-4-(pyrazolo[1,5-a]pyrimidine-3-carbonylamino)pyrazol-1-yl]methyl]tetrazol-1-yl]azetidine-1-carboxylate (200 mg, 0.30 mmol) in dichloromethane (5 mL) was stirred at room temperature. TFA (1 mL, 0.30 mmol) was then added and stirred at room temperature for 2 h. The solvent was concentrated under vacuum. The residue was purified by HPLC with H 2 Purification by silica gel flash chromatography eluting with O (0.1% TFA) / ACN (69%) gave N-[1-[[1-(azetidin-3-yl)tetrazol-5-yl]methyl]-3-[2,5-bis(difluoromethoxy)phenyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (184 mg) as a yellow oil. LC / MS (Method H, ESI): [M+H]+=574.2, RT=0.97 min. 1 H NMR (300 MHz, DMSO-d 6 ):δ(ppm)9.78(s,1H),9.36(dd,J=6.9,1.5Hz,1H),8.70-8.64(m,2H),8.59( s,1H),7.61-6.97(m,6H),6.10(s,2H),5.94-5.88(m,1H),4.47-4.16(m,4H).

[0333] Example 78 TIFF0007682110000118.tif55170N-(3-(2,5-bis(difluoromethoxy)phenyl)-1-((2-(1'-methyl-[1,3'-biazetidin]-3-yl)-2H-tetrazol-5-yl)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide Step 1: Synthesis of tert-butyl 3-(5-((3-(2,5-bis(difluoromethoxy)phenyl)-4-(pyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-1-yl)methyl)-2H-tetrazol-2-yl)-[1,3'-biazetidine]-1'-carboxylate TIFF0007682110000119.tifA solution of N-[1-[[2-(azetidin-3-yl)tetrazol-5-yl]methyl]-3-[2,5-bis(difluoromethoxy)phenyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (1.0 g, 1.74 mmol) and 1-boc-3-azetidinone (896 mg, 5.23 mmol) in a mixture of methanol (20 mL) and acetic acid (1 mL) was stirred at 25 °C for 2 h. Then, NaBH(OAc) 3 (1.1 g, 5.19 mmol) was added and the mixture was stirred at room temperature for 3 h. When the reaction was complete, the solvent was removed under vacuum. The residue was purified by reverse phase separation column [mobile phase A: water (0.1% NH 4 HCO 3 ), mobile phase B: acetonitrile; gradient: 10% B to 70% B in 35 min] to give tert-butyl 3-(5-((3-(2,5-bis(difluoromethoxy)phenyl)-4-(pyrazolo[1,5-a]pyrimidine-3-carboxamide)-1H-pyrazol-1-yl)methyl)-2H-tetrazol-2-yl)-[1,3’-bi-azetidine]-1’-carboxylate (650 mg, 0.890 mmol, 50.1% yield) as a pale yellow solid. LC / MS (method P, ESI): [M+H]+ = 729.15, RT = 0.85 min Step 2: Synthesis of N-(1-((2-([1,3’-bi-azetidine]-3-yl)-2H-tetrazol-5-yl)methyl)-3-(2,5-bis(difluoromethoxy)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide TIFF0007682110000120.tif54170 A solution of tert-butyl 3-[3-[5-[[3-[2,5-bis(difluoromethoxy)phenyl]-4-(pyrazolo[1,5-a]pyrimidine-3-carbonylamino)pyrazol-1-yl]methyl]tetrazol-2-yl]azetidin-1-yl]azetidine-1-carboxylate (650 mg, 0.890 mmol) in dichloromethane (6 mL) and 2,2,2-trifluoroacetic acid (2 mL) was stirred at room temperature for 3 h. Upon completion of the reaction, the solvent was removed under vacuum to give 600 mg of crude product as a yellow solid. LC / MS (Method G, ESI): [M+H]+=629.2, RT=0.92 min. Step 3: Synthesis of N-(3-(2,5-bis(difluoromethoxy)phenyl)-1-((2-(1'-methyl-[1,3'-biazetidin]-3-yl)-2H-tetrazol-5-yl)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide TIFF0007682110000121.tif55170 A solution of N-(1-((2-([1,3'-biazetidin]-3-yl)-2H-tetrazol-5-yl)methyl)-3-(2,5-bis(difluoromethoxy)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (600 mg, 0.950 mmol) in a mixture of acetic acid (1 mL) and methanol (10 mL) and formaldehyde / water (2 mL) was stirred at room temperature for 1 h. Then, NaBH(OAc) 3 (1.0 g, 4.72 mmol) was added and the mixture was stirred for 3 h. Upon completion of the reaction, the solvent was removed under vacuum. The residue was purified by reverse phase separation column [mobile phase A: water (0.1% NH 4 HCO 3), mobile phase B: acetonitrile; gradient: 10% B to 70% B in 30 min] to give N-(3-(2,5-bis(difluoromethoxy)phenyl)-1-((2-(1'-methyl-[1,3'-biazetidin]-3-yl)-2H-tetrazol-5-yl)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (400 mg, 0.603 mmol, 63.3% yield) as a pale yellow solid. LC / MS (Method O, ESI): [M+H]+=643.3, RT=1.42 min. 1 H NMR (300MHz, DMSO-d 6 ):δ(ppm)9.78(s,1H),9.35(dd,J=7.2,1.7Hz,1H),8.67-8.64(m,2H),8.53(s,1H),7.5 3-6.96(m,6H),5.82(s,2H),5.64-5.59(m,1H),3.87-3.82(m,2H),3.71-3.35(m,10H).

[0334] Example 51 TIFF0007682110000122.tif55170N-[3-[2,5-bis(difluoromethoxy)phenyl]-1-[[2-[1-[3-(dimethylamino)propyl]azetidin-3-yl]tetrazol-5-yl]methyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide Step 1: Synthesis of tert-butyl (3-(3-(5-((3-(2,5-bis(difluoromethoxy)phenyl)-4-(pyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-1-yl)methyl)-2H-tetrazol-2-yl)azetidin-1-yl)propyl)carbamate TIFF0007682110000123.tif56170 A solution of N-[1-[[2-(azetidin-3-yl)tetrazol-5-yl]methyl]-3-[2,5-bis(difluoromethoxy)phenyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (500 mg, 0.870 mmol), acetic acid (104 mg, 1.74 mmol) and tert-butyl N-(3-oxopropyl)carbamate (302 mg, 1.74 mmol) in methanol (8 mL) at room temperature. The resulting solution was stirred at room temperature for 2 h. Then, NaBH(AcO) 3 (555 mg, 2.62 mmol) was added and stirred at room temperature for 3 h. The reaction mixture was concentrated in vacuo. The residue was dissolved in ACN / water (0.05% NH 4 HCO 3 ) (35%) to give tert-butyl N-[3-[3-[5-[[3-[2,5-bis(difluoromethoxy)phenyl]-4-(pyrazolo[1,5-a]pyrimidine-3-carbonylamino)pyrazol-1-yl]methyl]tetrazol-2-yl]azetidin-1-yl]propyl]carbamate (470 mg, 0.643 mmol, 73.8% yield) as a yellow oil. LC / MS (Method Q, ESI): [M+H]+=731.3, RT=1.35 min.

[0335] Step 2: Synthesis of N-(1-((2-(1-(3-aminopropyl)azetidin-3-yl)-2H-tetrazol-5-yl)methyl)-3-(2,5-bis(difluoromethoxy)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide TIFF0007682110000124.tif55170 A solution of tert-butyl N-[3-[3-[5-[[3-[2,5-bis(difluoromethoxy)phenyl]-4-(pyrazolo[1,5-a]pyrimidine-3-carbonylamino)pyrazol-1-yl]methyl]tetrazol-2-yl]azetidin-1-yl]propyl]carbamate (152 mg, 0.210 mmol) in dichloromethane (4 mL) and 2,2,2-trifluoroacetic acid (1 mL) at room temperature. The resulting solution was stirred at room temperature for 2 hours. The reaction mixture was concentrated in vacuo. The residue was purified by flash chromatography on a C18 column eluting with ACN / water (0.05% HCl) (45%) to give N-[1-[[2-[1-(3-aminopropyl)azetidin-3-yl]tetrazol-5-yl]methyl]-3-[2,5-bis(difluoromethoxy)phenyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (80.2 mg, 0.127 mmol, 61.1% yield) as a yellow oil. LC / MS (Method M, ESI): [M+H]+=631.4, RT=0.55 min.

[0336] Step 3: Synthesis of N-[3-[2,5-bis(difluoromethoxy)phenyl]-1-[[2-[1-[3-(dimethylamino)propyl]azetidin-3-yl]tetrazol-5-yl]methyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide TIFF0007682110000125.tif55170HCHO / H in methanol (5 mL) at room temperature 2 A solution of N-[1-[[2-[1-(3-aminopropyl)azetidin-3-yl]tetrazol-5-yl]methyl]-3-[2,5-bis(difluoromethoxy)phenyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (85.1 mg, 0.130 mmol) was added to the reaction mixture. The resulting solution was stirred at 25° C. for 2 h. Then, NaBH(AcO) 3 (114 mg, 0.540 mmol) was added and stirred at 25° C. for 3 h. Then, NaBH 3CN (14 mg, 0.23 mmol) was added and the mixture was stirred for 1 hour. The reaction mixture was concentrated under vacuum. The residue was purified by HPLC under the following conditions: column: XBridge Prep OBD C18 Column, 19 * 250 mm, 5 um; mobile phase A: water (10 mmol / L NH 4 HCO 3 ), mobile phase B: ACN; flow rate: 25 mL / min; gradient: 20B to 50B in 7 minutes; 254 nm; RT1: 5.88; to give N-[3-[2,5-bis(difluoromethoxy)phenyl]-1-[[2-[1-[3-(dimethylamino)propyl]azetidin-3-yl]tetrazol-5-yl]methyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (25.4 mg, 0.038 mmol, 27.9% yield) as a yellow solid. LC / MS (method A, ESI): [M + H]+ = 659.3, RT = 1.17 min 1 1H NMR (300 MHz, DMSO-d 6 6): δ (ppm) 9.78 (s, 1H), 9.36 (dd, J = 9.4, 2.2 Hz, 1H), 8.68 - 8.64 (m, 2H), 8.53 (s, 1H), 7.54 - 6.95 (m, 6H), 5.81 (s, 2H), 5.57 (m, 1H), 3.82 - 3.77 (m, 2H), 3.52 - 3.48 (m, 2H), 2.21 - 2.16 (m, 2H), 2.08 (s, 6H), 1.49 - 1.32 (m, 2H).

[0337] Example 80 TIFF0007682110000126.tif52170N-(3-(2,5-bis(difluoromethoxy)phenyl)-1-((2-(1-(2-(1-(dimethoxyamino)cyclopropyl)ethyl)azetidin-3-yl)-2H-tetrazol-5-yl)methoxy)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide Step 1: Synthesis of tert-butyl (1-(2-(3-(5-((3-(2,5-bis(difluoromethoxy)phenyl)-4-(pyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-1-yl)methyl)-2H-tetrazol-2-yl)azetidin-1-yl)ethyl)cyclopropyl)carbamate TIFF0007682110000127.tif54170 A solution of tert-butyl N-[1-(2-oxoethyl)cyclopropyl]carbamate (69.4 mg, 0.350 mmol), N-[1-[[2-(azetidin-3-yl)tetrazol-5-yl]methyl]-3-[2,5-bis(difluoromethoxy)phenyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (Intermediate 1, 200 mg, 0.350 mmol) and acetic acid (62.6 mg, 1.04 mmol) in methanol (10 mL) was stirred at room temperature for 1 h. Then, NaBH(OAc) 3 (148 mg, 0.700 mmol) was added and the mixture was stirred at room temperature for an additional hour. Then, NaBH 3 CN (21.9 mg, 0.350 mmol) was added and the mixture was stirred at room temperature for another hour. Upon completion of the reaction, the solvent was removed in vacuo. The residue was purified by reverse phase separation column [mobile phase A: water (0.1% TFA), mobile phase B: acetonitrile; gradient: 30% B to 70% B in 30 min] to give tert-butyl (1-(2-(3-(5-((3-(2,5-bis(difluoromethoxy)phenyl)-4-(pyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-1-yl)methyl)-2H-tetrazol-2-yl)azetidin-1-yl)ethyl)cyclopropyl)carbamate (136 mg, 0.18 mmol, 51.5% yield) as a pale yellow solid. LC / MS (Method H, ESI): [M+H]+=757.3, RT=1.10 min

[0338] Synthesis of N-(1-((2-(1-(2-(1-aminocyclopropyl)ethyl)azetidin-3-yl)-2H-tetrazol-5-yl)methyl)-3-(2,5-bis(difluoromethoxy)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide A solution of tert-butyl N-[1-[2-[3-[5-[[3-[2,5-bis(difluoromethoxy)phenyl]-4-(pyrazolo[1,5-a]pyrimidin-3-carbonylamino)pyrazol-1-yl]methyl]tetrazol-2-yl]azetidin-1-yl]ethyl]cyclopropyl]carbamate (126 mg, 0.170 mmol) in dichloromethane (4 mL) and 2,2,2-trifluoroacetic acid (1 mL) was stirred at room temperature for 2 hours. The solvent was concentrated under vacuum. The product was used without further purification. LC / MS (method H, ESI): [M+H]+ = 657.3, RT = 0.94 min

[0339] Synthesis of N-(1-((2-(1-(2-(1-aminocyclopropyl)ethyl)azetidin-3-yl)-2H-tetrazol-5-yl)methyl)-3-(2,5-bis(difluoromethoxy)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide A solution of N-(1-((2-(1-(2-(1-aminocyclopropyl)ethyl)azetidin-3-yl)-2H-tetrazol-5-yl)methyl)-3-(2,5-bis(difluoromethoxy)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (120 mg, 0.180 mmol) and formaldehyde (43.1 mg, 0.570 mmol) in methanol (4 mL) was stirred at room temperature for 2 hours. Then, NaBH(OAc) 3 (154 mg, 0.730 mmol) was added and the mixture was stirred at 35 °C for 3 hours. Finally, NaBH 3CN (9.3 mg, 0.150 mmol) was added and stirred at 35° C. for 1 h. The extract was washed with water and brine, dried over anhydrous sodium sulfate, and then concentrated in vacuo. The reaction was repeated on the same scale and the combined crude product was dissolved in: YMC-Actus Triart C18, 30*250, 5 um; Mobile phase A: water (10 mmol / L NH 4 HCO 3 ), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 38B to 44B in 7 min; purified by HPLC at 220 nm to give N-(3-(2,5-bis(difluoromethoxy)phenyl)-1-((2-(1-(2-(1-(dimethylamino)cyclopropyl)ethyl)azetidin-3-yl)-2H-tetrazol-5-yl)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (15 mg, 0.022 mmol, 6% yield). LC / MS (Method H, ESI): [M+H]+=685.3, RT=0.94 min. 1 H NMR (400 MHz, DMSO-d 6 ):δ(ppm)9.78(s,1H),9.38-9.38(m,1H),8.67-8.64(m,2H),8.52(s,1H),7.47-7.01(m,6H),5.80(s,2H),5 .60-5.55(m,1H),3.78-3.74(m,2H),3.49-3.46(m,2H),2.50-2.44(m,2H),2.21(s,6H),1.50-1.23(m,3H).

[0340] Example 83 TIFF0007682110000130.tif54170N-(3-(2,5-bis(difluoromethoxy)phenyl)-1-((2-(1-(3-(dimethylamino)-2-fluoropropyl)azetidin-3-yl)-2H-tetrazol-5-yl)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide Step 1: Synthesis of tert-butyl (3-(3-(5-((3-(2,5-bis(difluoromethoxy)phenyl)-4-(pyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-1-yl)methyl)-2H-tetrazol-2-yl)azetidin-1-yl)-2-fluoropropyl)carbamate TIFF0007682110000131.tif54170 A solution of N-[1-[[2-(azetidin-3-yl)tetrazol-5-yl]methyl]-3-[2,5-bis(difluoromethoxy)phenyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (Intermediate 1, 300 mg, 0.520 mmol) and tert-butyl N-(2-fluoro-3-oxo-propyl)carbamate (220 mg, 1.15 mmol) in a mixture of acetic acid (0.50 mL) and methanol (5 mL) was stirred at room temperature for 1 h. Then, NaBH(OAc) 3 (333 mg, 1.57 mmol) was added and the mixture was stirred for an additional 2 h. Upon completion of the reaction, the organic solvent was removed under vacuum. The residue was purified by reverse phase separation column [mobile phase A: water (0.1% NH 4 HCO 3 ), mobile phase B: acetonitrile; gradient: 20% B to 70% B in 30 min] to give tert-butyl (3-(3-(5-((3-(2,5-bis(difluoromethoxy)phenyl)-4-(pyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-1-yl)methyl)-2H-tetrazol-2-yl)azetidin-1-yl)-2-fluoropropyl)carbamate (290 mg, 0.387 mmol, 74% yield) as a pale yellow solid. LC / MS (Method Q, ESI): [M+H]+=749.25, RT=1.98 min.

[0341] Step 2: Synthesis of N-(1-((2-(1-(3-amino-2-fluoropropyl)azetidin-3-yl)-2H-tetrazol-5-yl)methyl)-3-(2,5-bis(difluoromethoxy)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide A solution of tert-butyl (3-(3-(5-((3-(2,5-bis(difluoromethoxy)phenyl)-4-(pyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-1-yl)methyl)-2H-tetrazol-2-yl)azetidin-1-yl)-2-fluoropropyl)carbamate (290 mg, 0.390 mmol) in a mixture of 2,2,2-trifluoroacetic acid (1 mL) and dichloromethane (3 mL) was stirred at room temperature for 3 h. Upon completion of the reaction, the solvent was concentrated under vacuum. The residue was purified by preparative chiral HPLC [Column: Chiralpak ID-2, 2×25 cm, 5 um; Mobile phase A: MTBE (0.3% IPA), Mobile phase B: MeOH; Flow rate: 20 mL / min; Gradient: 10B to 10B in 26 min; 220 / 254 nm; RT1: 18.047; RT2: 22.229; Injection volume: 0.6 mL; Runs: 12] to give two stereoisomers (65 mg (peak 1) and 52 mg (peak 2)). LC / MS (Method I, ESI): [M+H]+=649.3, RT=0.96 min.

[0342] Step 3: Synthesis of N-(3-(2,5-bis(difluoromethoxy)phenyl)-1-((2-(1-(3-(dimethylamino)-2-fluoropropyl)azetidin-3-yl)-2H-tetrazol-5-yl)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide TIFF0007682110000133.tif54170A solution of the second eluting enantiomer (peak 2), N-(1-((2-(1-(3-amino-2-fluoropropyl)azetidin-3-yl)-2H-tetrazol-5-yl)methyl)-3-(2,5-bis(difluoromethoxy)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (52 mg, 0.080 mmol) and HCHO (60 mg, 0.80 mmol) in a mixture of methanol (2 mL) and acetic acid (0.20 mL) was stirred at room temperature for 1 h. NaBH(OAc) 3 (51 mg, 0.24 mmol) was added and the mixture was stirred at room temperature for 1 h.3 CN (5.0 mg, 0.08 mmol) was added and the mixture was further stirred at room temperature for 1 h. The solvent was removed under vacuum. The residue was purified by preparative HPLC [Column: Xselect CSH OBD Column 30*150 mm 5 um; Mobile phase A: water (10 mmol / L NH 4 HCO 3 ), mobile phase B: ACN:MeOH=4:1; flow rate: 60 mL / min; gradient: 27B to 49B in 11 min; 220 nm; RT 1:10.08 to give N-(3-(2,5-bis(difluoromethoxy)phenyl)-1-((2-(1-(3-(dimethylamino)-2-fluoropropyl)azetidin-3-yl)-2H-tetrazol-5-yl)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (10.6 mg, 0.016 mmol, 19.3% yield) as a pale yellow solid. LC / MS (Method A, ESI): [M+H]+=677.2, RT=1.24 min. 1 H NMR (400 MHz, CDCl 3 ):δ(ppm)9.87(s,1H),8.80(dd,J=7.2,1.6Hz,1H),-9.38(m,1H),8.72(s,1H),8.54-8.5 2(m,2H),7.47(d,J=2.8Hz,1H),7.36-7.33(m,1H),7.25-7.22(m,1H),7.03-7.00(m,1H), 6.73-6.29(m,2H),5.67(s,2H),5.56-5.25(m,1H),4.80-4.60(m,1H),4.04-4.02(m,2H), 3.82-3.78(m,2H),2.92-2.82(m,2H),2.62-2.58(m,1H),2.52-2.44(m,1H),2.30(s,6H).

[0343] Assay Test substances Samples of the test substances were prepared as solutions at a concentration of 10 mM in dimethylsulfoxide (DMSO) and stored in the dark at room temperature before use.

[0344] JAK1 and JAK2 biochemical assays The in vitro biochemical assay quantifies JAK-catalyzed phosphorylation of a synthetic peptide detected using a Lab Chip® EZ Reader II microfluidic mobility shift device (PerkinElmer; Waltham, MA). The substrate peptide Y-1B has the sequence 5-FAM-VALVDGYFRLTT-NH 2 Y-1B is fluorescently labeled with 5-FAM (5-carboxyfluorescein) at the N-terminus and contains a single tyrosine residue (Y) that can be phosphorylated by JAK activity. Substrate peptide stocks are prepared in DMSO at 5 mM. Purified recombinant human JAK1 kinase domain protein (residues 854-1154) was expressed in insect cells and purchased from Proteros Biostructures GmbH (Martinsried, Germany). Recombinant human JAK2 kinase domain protein (residues 812-1132) was expressed in insect cells and purified at Genentech, Inc. (South San Francisco, CA).

[0345] Kinase reaction mixtures contained 100 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 7.2), 10 mM magnesium chloride, 0.015% Brij® 35, 4 mM dithiothreitol, 1.5 μM Y-1B peptide substrate, 25 μM adenosine triphosphate (ATP), 1 nM total JAK1 or 0.2 nM total JAK2, and up to 1000 nM test compound at a final concentration of 2% (volume to volume [v / v]) DMSO. In each titration experiment, test compounds were tested in duplicate at each of 12 concentrations. Blank reactions contained ATP, peptide, and DMSO but no JAK or test compound, while uninhibited control reactions contained ATP, peptide, JAK, and DMSO but no test compound.

[0346] The peptide+ATP mixture (24 μL) was added to 1 μL of test compound in DMSO (or DMSO alone). The reaction was started by adding 25 μL of JAK enzyme to the inhibitor / peptide / ATP mixture before thoroughly mixing the resulting solution. The reactions were incubated at room temperature (22° C.-23° C.) in a final volume of 50 μL per well in a 384-well plate. After a 30-minute incubation, the reaction was stopped by adding 25 μL of 150 mM ethylenediaminetetraacetic acid in 100 mM HEPES buffer (pH 7.2) containing 0.015% Brij 35 to each well.

[0347] In each reaction, the residual Y-1B substrate and the resulting phospho-peptide product were separated using an EZ Reader II instrument. Electrophoretic separation of product molecules from substrate molecules was achieved using downstream and upstream voltages of -500V and -2600V, respectively, at an operating pressure of -1.3 psi. The 5-FAM group present in both the substrate and product peptides was excited at 488 nm, fluorescence was detected at 530 nm, and peak heights were reported. Data Analysis: The extent (or percentage) of conversion of substrate to product was calculated from the corresponding peak heights in the electropherogram using HTS Well Analyzer software, version 5.2 (PerkinElmer) and the following equation (Equation 1): Equation 1: % conversion = [P÷(S+P)]×100

[0348] where S and P represent the peak heights of the substrate and product, respectively. After any baseline signal from blank wells containing no JAK was subtracted from the signals of all test wells, the % converted data was converted to fractional activity as shown in Equation 2, v i and v o are the % conversion in the presence and absence of test compound, respectively. The % conversion observed in the uninhibited control reaction containing JAK and DMSO vehicle but no test compound is calculated as fractional activity = 1 (in the absence of inhibitor, v i =v o), whereas blank wells containing no JAK were defined as having fractional activity 0 = 0. Percent activity was plotted versus test compound concentration and data was analyzed to calculate the tight-binding apparent inhibition constant (K i app ) was fitted to a quadratic equation (see Equation 2) (Williams JW, Morrison JF. The kinetics of reversible tight-binding inhibition. Methods Enzymol 1979;63:437-67.) and used to calculate the fractional activity and K i app was calculated. TIFF0007682110000134.tif40170

[0349] In the formula, [E] T and [I] T are the total concentrations of active enzyme (initial estimates of 0.15 nM for JAK1 and 0.048 nM for JAK2) and inhibitor (variable parameter), respectively. Finally, K i app From K i was calculated (Equation 3). equation 3 K i =K i app / (1+[ATP] / K m app )

[0350] where [ATP] is the concentration of ATP = 25 μM and K m app The apparent ATP Michaelis constant for JAK1 is 32.1 μM, and for JAK2, K m app = 11.7 μM. By applying equation 2 for tight-binding and equation 3 for competitive-inhibition relationships to account for any depletion of inhibitors, the sensitivity of the assay is calculated to be 0.008 nM for JAK1 and 0.0015 nM for JAK2. ican be expanded at least to

[0351] Kinase selectivity In vitro kinase selectivity of test substances was assessed in a panel of recombinant human kinase activity and binding assays including cytoplasmic and receptor tyrosine kinases, serine / threonine kinases, and lipid kinases (SelectScreen® Kinase Profiling Services, ThermoFisher Scientific, Madison, WI) at a concentration of 1 μM. Kinase activity assays measure peptide phosphorylation (Z'-LYTE®) or ADP production (Adapta®), while binding assays monitor displacement of an ATP site-binding probe (LanthaScreen®). ATP concentrations used in activity assays are typically determined based on the experimentally determined apparent Michaelis constant (K m app ) values, the competitive binding tracer concentrations used in the binding assays were generally within 2-fold of the experimentally determined dissociation constant (K d The inhibition rates were within 3-fold of the IC value. Inhibitors were tested in duplicate for each kinase, and the average % inhibition values ​​are reported. For kinases that were inhibited near or above 50% at the initial 1-μM test concentration, the inhibitor concentration causing 50% inhibition (IC 50 A 10-point inhibitor titration was performed using the same assay to determine IC1 activity. The total JAK1 concentration used in this assay panel was 75 nM. If 100% of the 75 nM JAK1 protein was catalytically active, the limit of JAK1 inhibitor sensitivity from the vendor's JAK1 assay would theoretically be an IC1 of 37.5 nM. 50 However, the SelectScreen® JAK1 assay showed JAK1 IC values ​​for several inhibitors that were much lower than 37.5 nM, consistent with our internal determinations. 50Thus, the active JAK1 enzyme concentration in the SelectScreen® assay must be much lower than the 75 nM total nominal JAK1 protein concentration used in the assay, and the observed sensitivity of this assay is greater than the theoretical sensitivity IC of 37.5 nM. 50 Much better than the limit.

[0352] Data analysis To fit the data to concentration-kinase inhibition plots, SelectScreen® Kinase Profiling Services used XLfit software (IDBS), model number 205 (sigmoidal concentration-response model), a 4-parameter logistic fit model described by Equation 4. Equation 4:

[0001] y=A+{(BA)÷[1+(C÷x) D ]}

[0353] where x is the inhibitor concentration, y is the % inhibition observed, A is the minimum y-value, B is the maximum y-value, and C is the IC 50 where is the value of σ and D is the Hill slope. In certain cases, a 3-parameter logistic fit was used. For example, if the plateau of the curve at infinitely low inhibitor concentration did not fit between -20% and 20% inhibition, the lower plateau was set to 0% inhibition, whereas if the plateau of the curve at infinite inhibitor concentration did not fit between 70% and 130% inhibition, the higher plateau was set to 100% inhibition.

[0354] TF-1 cell line phospho-STAT JAK1 and JAK2 pathway selectivity assay TF-1 human erythroleukemia cells (ATCC®; Manassas, VA; catalog number CRL-2003®) were grown in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 ng / mL granulocyte-macrophage colony-stimulating factor, 1× non-essential amino acids (NEAA), and 1 mM sodium pyruvate. The day before the assay, cultures were transferred to Opti-MEM®, 1× NEAA, 1 mM sodium pyruvate, and 0.5% charcoal-stripped FBS (starvation medium). Inhibitor stock solutions (5 mM in DMSO) were serially diluted 1:2 in DMSO to generate 10-point concentration titrations (at 500× test concentration), which were further diluted by 50-fold dilution in assay medium (RPMI containing 1× NEAA and 1 mM sodium pyruvate) to generate 10× concentration titrations (in 2% DMSO). Cells (300,000 cells / well in 35 μL of assay medium) were seeded into 384-well Greiner plates. 10× concentration of diluted inhibitor (5 μL) was added to the cells and the plates were incubated at 37° C. for 30 minutes in a humidified incubator. Cells were incubated at their respective EC 90Cells were stimulated with human recombinant cytokines at concentrations of 0.01 mM. For the phosphorylated signal transduction and activator of transcription 6 (P-STAT6) TF-1 + interleukin-13 (IL-13) assay, 10 μL of 250 ng / mL IL-13 (R&D Systems; Minneapolis, MN) was added to the cells, which were then incubated at 37° C. for 10 minutes. For the P-STAT5 TF-1 + erythropoietin (EPO) assay, 10 μL of 110 IU / mL EPO (Gibco Life Technologies, Catalog No. PHC2054) was added to the cells, which were then incubated at 37° C. for 30 minutes. For both assays, after incubation, 5 μL of ice-cold 10× cell lysis buffer (Cell Signaling Technologies; Danvers, MA; Catalog No. 9803S) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) was added to the cells. Assay plates were frozen at -80°C for a minimum of 1 hour. For IL-13 assays, P-STAT6 was measured by coating goat anti-rabbit (GAR) plates (Meso Scale Discovery [MSD]; Rockville, MD; catalog number MSD L21RA-1) with rabbit anti-human total STAT6 antibody (Cell Signaling Technologies; catalog number 9362S), incubating cell lysates in the coated plates overnight at 4°C, and then detecting with mouse anti-P-STAT6 (Tyr641) clone 16E12 antibody (MilliporeSigma; Burlington, MA; catalog number 05-590, custom labeled by MSD with a SULFO-tag) using standard MSD plate processing, washing, and detection protocols. For EPO assays, P-STAT5 was detected using the phospho-STAT5a,b Whole Cell Lysate Kit (MSD; catalog number K150IGD-1). The electrochemiluminescence (ECL) signals of the wells were read on a MESO SECTOR S600 (MSD) reader.

[0355] Data analysis The average ECL value of the negative controls (cytokine-stimulated cells and cells treated with 20 μM control inhibitor) was subtracted from the ECL values ​​of all wells, and the ratio of the control to the average ECL value of the positive controls (cytokine-stimulated cells and DMSO-treated cells) was determined, and the IC of the test compound was calculated using a 4-parameter logistic fit model shown in Equation 4. 50 Data analysis was performed by determining

[0356] P-STAT6 BEAS-2B + IL-13 cell assay To study the effects of JAK1 inhibitors in cell lines relevant to the cell biology of human asthma, we developed an IL-13-stimulated STAT6 phosphorylation assay in the human lung bronchial epithelial BEAS-2B cell line.

[0357] BEAS-2B cells (®CRL-9609™) were grown in Bronchial Epithelial Growth Medium (BEGM) (Lonza Catalog No. CC-3170; Walkersville, MD; or PromoCell Catalog No. C-21060; Heidelberg, Germany). Test compound stock solutions (0.5 mM in DMSO) were serially diluted 1:2 in DMSO to generate a 10-point concentration curve (at 500× test concentration), which was further diluted by 50-fold dilution steps in BEGM to generate a 10× concentration curve (in 2% DMSO). Cells were seeded at 100,000 cells / well in 200 μL of BEGM in 96-well plates and incubated at 37° C. for 48 hours in a humidified incubator. The medium was aspirated from the cells and replaced with 70 μL of fresh BEGM. Diluted test compounds (10 μL; or 2% DMSO in assay medium) were added to the cells and the plates were incubated for 1 hour at 37° C. in a humidified incubator. 20 μL of 250 ng / mL human recombinant IL-13 (Bio Techne Cat. No. 213-ILB) was then added to the cells and incubated for 15 minutes at 37° C. Media was aspirated from the cells and 60 μL of ice-cold 1× cell lysis buffer (Cell Signaling Technologies; Cat. No. 9803S) containing 1 mM PMSF was added to the cells. Assay plates were incubated at −80° C. for at least 1 hour. P-STAT6 was measured by coating GAR plates (MSD; Cat. No. L45RA-1) with rabbit anti-human total STAT6 antibody (Cell Signaling Technologies; Cat. No. 9362 S), incubating cell lysates in the coated plates overnight at 4° C., and then detecting with mouse anti-phospho-STAT6 (Tyr641) clone 16E12 antibody (Millipore; Cat. No. 05-590, custom labeled by MSD with a SULFO-tag) using standard MSD plate treatment, washing, and detection protocols. Plates were read on a MESO SECTOR S600.

[0358] Data analysis Data analysis was performed by subtracting the negative control values ​​from all wells and determining the percentage of control using the positive control values; IC 50 was determined using a 4-parameter logistic fitting model shown in Equation 4.

[0359] P-STAT6 BEAS-2B + IL-13 cell assay with inhibitor washout (WO) To assess the ability of JAK1 inhibitors to retain their ability to inhibit IL-13-stimulated STAT6 phosphorylation after cell washing to remove free unbound inhibitor, an inhibitor washout (WO) assay in the human lung bronchial epithelial BEAS-2B cell line was developed. Retention of inhibitory activity after inhibitor washout is consistent with persistent binding of the inhibitor to the JAK1 protein and / or retention of the inhibitor molecule within the cells after washout.

[0360] BEAS-2B cells were grown in bronchial epithelial growth medium (BEGM) as in the standard BEAS-2B cell assay (see above). Test compound stock solutions (0.5 mM in DMSO) were serially diluted 1:2 in DMSO to generate a 10-point concentration curve (at 500× test concentration), which was further diluted by 50-fold dilution steps in BEGM to generate a 10× concentration curve (in 2% DMSO). Cells were seeded at 100,000 cells / well in 200 μL of BEGM in 96-well plates and incubated at 37° C. for 48 hours in a humidified incubator. Media was aspirated from the cells and replaced with 70 μL of fresh BEGM. Diluted test compounds (10 μL; or 2% DMSO in assay medium) were added to the cells and the plates were incubated at 37° C. for 1 hour in a humidified incubator. The medium was aspirated from the cells and replaced with 80 μL of fresh BEGM to wash the inhibitors from the cells, and the cell plate was then incubated at 37° C. in a humidified incubator for 10 min. This washout procedure was repeated two more times. After the third wash step, the cell plate was returned to the humidified incubator at 37° C. and incubated for 1 h. Then, 20 μL of 250 ng / mL IL-13 was added to the cells and incubated at 37° C. for 15 min. The medium was aspirated from the cells and 60 μL of ice-cold 1× cell lysis buffer (Cell Signaling Technologies; Cat. No. 9803S) containing 1 mM PMSF was added to the cells. The assay plate was incubated at −80° C. for at least 1 h. P-STAT6 was measured by coating GAR plates (MSD; Cat. No. L45RA-1) with rabbit anti-human total STAT6 antibody (Cell Signaling Technologies; Cat. No. 9362S), incubating cell lysates in the coated plates overnight at 4° C., and then detecting with mouse anti-phospho-STAT6 (Tyr641) clone 16E12 antibody (Millipore; Cat. No. 05-590, custom labeled by MSD with a SULFO-tag) using standard MSD plate treatment, washing, and detection protocols. Plates were read on a MESO SECTOR S600.

[0361] Data analysis Data analysis was performed by subtracting the negative control values ​​from all wells and determining the percentage of control using the positive control values; IC 50 was determined using a 4-parameter logistic fitting model shown in Equation 4.

[0362] Cell cytotoxicity assay A549 (ATCC® CCL-185™), Jurkat clone E6-1 (ATCC® TIB-152™) and HEK-293T (ATCC® CRL-1573™) cells maintained at subconfluent density in T175 flasks were used. Exponentially growing cells were seeded (450 cells in 45 μL of medium) into Greiner 384-well black / clear tissue culture treated plates (Greiner catalog no. 781091). After dispensing the cells, the plates were allowed to equilibrate at room temperature for 30 minutes, after which the cell plates were incubated at 37° C. in CO 2 and placed in a humidity-controlled incubator overnight. The following day, cells were treated with test substances diluted in 100% DMSO (0.5% final DMSO concentration on cells) in a 10-point titration and with a top concentration of 50 μM. Cells and compounds were then incubated at 37° C., CO 2 Plates were incubated for 72 hours in a humidity-controlled incubator, after which cell viability was measured by adding CellTiter-Glo® (Promega G7572) reagent to all wells. Plates were incubated for 20 minutes at room temperature, and then the wells were read for luminescence on an EnVision plate reader (Perkin Elmer Life Sciences).

[0363] Data from the above JAK1 and JAK2 assays for the compounds of Table 1 are shown below in Table 2. [Table 2] TIFF0007682110000136.tif252170TIFF0007682110000137.tif58170

[0364] As can be seen from Table 2, the compounds of the present invention have good and balanced affinity for both JAK1 and JAK2, and many of the compounds are active in cell-based assays.

[0365] Animal models Mouse House Dust Mite (HDM) Model Female C57BL / 6J mice, 7-8 weeks of age, were purchased from Jackson West. Mice were immunized on days 0 and 14 by intraperitoneal administration of house dust mites (HDM, D. Pteronyssinus, purchased from Greer Laboratories, normalized to 0.918ug DerP1 content per mouse) mixed with 2mg alum (Thermo Scientific) diluted in sterile PBS. On days 21 and 24, mice were challenged with HDM (again normalized to 0.918ug DerP1 content) in PBS administered by intratracheal inhalation. Prior to each inhalation HDM challenge (and in a subset of groups on days 22 and 23), animals receive test compound by nose-only inhalation (using a dry powder inhaler from Electro-Medical Measurement Systems (EMMS) with a Wright dust feeder and 4-tier / 24-port or 2-tier / 12-port, directed flow, nose-only inhalation tower) ending 1 hour prior to challenge. Control animals receive nose-only inhalation of air only. 24 hours after the final treatment, mice are retro-orbitally bled for plasma PK and then euthanized by CO2 inhalation. After euthanasia, BAL fluid is collected for total cell counts (by FACS using known amounts of spike-in reference beads) and differential cell counts (by Wright Giemsa stained cytospins). Lungs and spleens are collected, weighed, and frozen for PK. There were 5 or 6 animals per group.

[0366] Additionally, to verify the pulmonary delivery dose, PK satellite groups of three naive animals are dosed with the test compound by nose-only inhalation for one or four consecutive days, respectively. Immediately after the final inhalation dose, the PK satellite animals are bled retro-orbitally for plasma PK and then CO 2Mice are euthanized by inhalation. Lungs and spleens are collected and weighed for PK analysis.

[0367] Rat OVA model Six-week-old male Brown Norway rats from Charles River-Kingston. Rats are immunized on day 0 by intraperitoneal administration of 150ug OVA (Sigma) mixed with 40mg alum (Thermo Scientific) diluted in sterile PBS. 28 days after sensitization, rats are challenged with 2% OVA in PBS aerosolized via a nebulizer for 30 minutes on three consecutive days. Prior to each OVA challenge, animals receive JAK1 / JAK2 test compound by nose-only inhalation (using a dry powder inhaler from Electro-Medical Measurement Systems (EMMS) with a Wright dust feeder and a 4-tier, 24-port, directed flow, nose-only inhalation tower) ending 1 hour before challenge. Control animals receive MCT buffer orally or nose-only inhalation of air only. 24 hours after the final treatment, rats are challenged with CO 2 Animals are euthanized by inhalation. Blood is collected from the abdominal aorta for plasma PK and whole blood FACS analysis. After euthanasia, BAL fluid is collected for total cell counts (by FACS using known amounts of spike-in reference beads) and differential cell counts (by Wright Giemsa stained cytospins). Lungs are collected, weighed, and frozen for PK. Spleens are weighed and cut in half for PK and FACS analysis. Blood and spleen samples are analyzed by FACS for total cell counts and % NK cells (CD161a positive). There are 6 animals per group, except for the naive control group, which contains 5 animals.

[0368] Additionally, to validate the pulmonary delivery dose, PK satellite groups of three naive animals were administered JAK1 / JAK2 test compounds by nose-only inhalation for one or three days, respectively. Immediately after the final inhalation dose, the PK satellite animals were administered CO 2 Mice were euthanized by inhalation. Blood was collected from the abdominal aorta for plasma PK. Lungs and spleens were collected and weighed for PK analysis.

[0369] The plasma and lung levels of test compounds and their ratios are determined in the following manner. BALB / c mice from Charles River Laboratories are used in the assay. Test compounds are formulated individually in 0.2% Tween 80 in saline, and the administration solution is introduced into the trachea of ​​the mouse by oral aspiration. At various times after administration (typically 0.167, 2, 6, 24 hours), blood samples are taken by cardiac puncture, and the intact lungs are excised from the mouse. Blood samples are centrifuged at approximately 12,000 rpm at 4°C for 4 minutes (Eppendorf centrifuge, 5804R) to collect plasma. Lungs are padded dry, weighed, and homogenized in 1:3 dilution in sterile water. Plasma and lung levels of test compounds are determined by LC-MS analysis against analytical standards constructed in a standard curve in test matrix. The lung-to-plasma ratio was determined as the ratio of lung AUC in microg hr / g to plasma AUC in microg hr / mL, where AUC is conventionally defined as the area under the curve of test compound concentration versus time.

[0370] Pharmacokinetics in plasma and lungs in mice The plasma and lung levels of test compounds and their ratios are determined in the following manner. BALB / c mice from Charles River Laboratories are used in the assay. Test compounds are formulated individually in 20% propylene glycol in pH 4 citrate buffer at a concentration of 0.2 mg / mL, and 50 uL of the administration solution is introduced into the trachea of ​​the mice by oral aspiration. At various times after administration (typically 0.167, 2, 6, 24 hours), blood samples are taken by cardiac puncture and the intact lungs are excised from the mice. Blood samples are centrifuged at approximately 12,000 rpm at 4°C for 4 minutes (Eppendorf centrifuge, 5804R) to collect plasma. Lungs are padded dry, weighed, and homogenized in sterile water at a dilution of 1:3. Plasma and lung levels of test compounds are determined by LC-MS analysis against analytical standards constructed in a standard curve in test matrix. The lung-to-plasma ratio was determined as the ratio of lung AUC in microg hr / g to plasma AUC in microg hr / mL, where AUC is conventionally defined as the area under the curve of test compound concentration versus time. Pharmacokinetics in plasma and lungs in mice

[0371] The pharmacokinetics of the compounds are determined in female Balb / c mice following administration of a target dose of 0.3 mg / kg formulated in 0.2% Tween 80 in saline by a single intranasal (IN) bolus solution / suspension administration. Female Balb / c mice aged 7-8 weeks can be purchased from Charles River. Mice are housed under specific pathogen-free conditions until use in the study.

[0372] Do not fast the animals before administration. Blood samples are collected from three animals into EDTA-coated microtainers via cardiac puncture under anesthesia (intraperitoneal injection of pentobarbital) at 0.083, 2, 7, and 24 hours after administration. The blood samples are centrifuged (1500 g, 10 minutes at 4°C) to separate the plasma. The plasma samples are frozen at approximately -80°C. After intranasal administration and before lung perfusion, the spleen is removed, weighed, and snap-frozen. After confirmation of death, the lungs of the administered animals are perfused with chilled PBS to remove residual blood from the pulmonary vasculature. The lungs are then excised and weighed (all weights are recorded). All tissue samples are frozen by immersion in liquid nitrogen. The tissue samples are stored frozen until analysis (approximately -80°C).

[0373] Before PK analysis, thawed tissue samples (spleen and lung) are weighed and homogenized using an Omni-Prep Bead Ruptor (Omni Inc., Kennesaw, GA) at 4°C with 4 mL of HPLC-grade water added per gram of tissue. Plasma and tissue homogenate samples are extracted using protein precipitation with 4 volumes of acetonitrile containing tolbutamide (200 ng / mL) or labetalol (100 ng / mL) as the internal standard. The samples are mixed and centrifuged at 3200 g and 4°C for 30 minutes to remove the precipitated protein, and the supernatant is appropriately diluted with HPLC-grade water in a 96-well plate (e.g., 1:1, v / v). Representative aliquots of plasma, spleen, and lung samples are assayed for compound concentration by LC-MS / MS in positive ion mode using a Waters Xevo TQ-S (Waters, Elstree, UK) against matrix-matched calibration curves and quality control standards. The standards are prepared by adding the compound to aliquots of control plasma, spleen, and lung tissue homogenates and are extracted as described for the experimental samples. The assay limit of detection was 0.168 mg / mL to 4000 ng / mL in all matrices.

[0374] Concentrations below the lower limit of quantification (LLOQ) are treated as 0 in the calculation of the mean and SD. The mean concentrations measured in the samples are used to construct semi-logarithmic concentration-time curve profiles. Pharmacokinetic (PK) analysis is performed using non-compartmental methods in Biobook (E-Workbook IDBS).

[0375] A Mouse Model of Alternaria alternata-Induced Eosinophilic Inflammation in the Lungs Airway eosinophilia is a hallmark of human asthma. Alternaria alternata is a fungal aeroallergen that can exacerbate asthma in humans and induces eosinophilic inflammation in the lungs of mice (Havaux et al. Clin Exp Immunol. 2005,139(2):179-88). In mice, it has been demonstrated that Alternaria indirectly activates tissue-resident type 2 innate lymphoid cells in the lungs, which release JAK-dependent cytokines (e.g., IL-5 and IL-13) in response to IL-2 and IL-7, and modulate eosinophilic inflammation (Bartemes et al. J Immunol. 2012,188(3):1503-13).

[0376] Seven to nine week old male C57 mice from Taconic are used for the study. On the day of the study, animals are lightly anesthetized with isoflurane and either vehicle or test compound is administered via oropharyngeal aspiration. Animals are placed in lateral recumbency after administration and monitored for complete recovery from anesthesia before being returned to their home cage. One hour later, animals are briefly anesthetized again and challenged with either vehicle or Alternaria extract via oropharyngeal aspiration, then monitored for recovery from anesthesia and returned to their home cage. Forty-eight hours after alternaria administration, bronchoalveolar lavage fluid (BALF) is collected and eosinophils in the BALF are counted using an Advia 120 Hematology System (Siemens).

[0377] Compound activity in the model is demonstrated by a reduction in the level of eosinophils present in the BALF of treated animals at 48 hours compared to vehicle-treated, alternaria-challenged control animals. Data is expressed as percent inhibition of vehicle-treated, alternaria-challenged BALF eosinophil response. To calculate percent inhibition, the number of BALF eosinophils for each condition is converted to a percentage of the average vehicle-treated, alternaria-challenged BALF eosinophils and subtracted from 100%.

Claims

[Claim 1] and 1. A compound selected from the group consisting of:

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