Tetrazolyl-substituted pyrazolopyrimidine JAK kinase inhibitors and uses thereof

Abstract

Described herein are compounds of formula (I) wherein R 1 、R\R 3 , R4, R 5 and R 6 As defined herein; and salts thereof, the compounds of formula (I) and their salts are used as JAK kinase inhibitors. Also provided herein: pharmaceutical compositions comprising such JAK inhibitors and pharmaceutically acceptable carriers, adjuvants or vehicles, and methods for treating a patient's disease or condition that responds to inhibition of Janus kinase activity or reducing the severity of the disease or condition.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This patent application claims priority to International Patent Application Serial No. PCT / CN2019 / 091709 filed on June 18, 2019 and U.S. Provisional Patent Application No. 63 / 036,046 filed on June 8, 2020, the disclosures of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to compounds that are inhibitors of Janus kinases (such as JAK1 and JAK2), compositions comprising these compounds, and methods of use including, but not limited to, diagnosing or treating patients suffering from disorders responsive to inhibition of JAK kinases. Background Art

[0004] 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 signal transduction. The binding of cytokines to cognate receptors triggers the activation of JAKs associated with receptors, and this leads to JAK-mediated tyrosine phosphorylation of signal transducers and transcription activators (STAT) proteins and the final transcriptional activation of specific gene sets (Schindler et al., 2007, J. Biol. Chem. 282: 20059-63). JAK1, JAK2, and TYK2 exhibit a wide range of gene expression patterns, while JAK3 expression is limited to leukocytes. Cytokine receptors usually function as heterodimers, and therefore usually more than one type of JAK kinase is associated with the cytokine receptor complex. Specific JAKs that associate with different cytokine receptor complexes have been identified in many cases through genetic studies and confirmed by other experimental evidence. Exemplary therapeutic benefits of inhibiting JAK enzymes are discussed, for example, in International Patent Application No. WO 2013 / 014567.

[0005] JAK1 was originally identified during a screen for new kinases (Wilks AF, 1989, Proc. Natl. Acad. Sci. USA 86: 1603-1607). Genetic and biochemical studies have shown that JAK1 functionally and physically associates with type I interferons (e.g., IFNα), type II interferons (e.g., IFNγ), 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 in the perinatal period 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 from JAK1 knockout mice demonstrated the key role of this kinase in the IFN, IL-10, IL-2 / IL-4 and IL-6 pathways. A humanized monoclonal antibody targeting the IL-6 pathway (tocilizumab) was 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).

[0006] CD4 T cells play an important role in the pathogenesis of asthma by producing TH2 cytokines including IL-4, IL-9 and IL-13 in the lungs (Cohn et al., 2004, Annu. Rev. Immunol. 22: 789-815). The induction of IL-4 and IL-13 increases mucus production, eosinophil recruitment to the lungs, and IgE production (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). When combined with the common γ chain or IL-13Rα1 chain, respectively, the IL-4Rα chain activates JAK1 and binds to IL-4 or IL-13 (Pernis et al., 2002, J. Clin. Invest. 109 (10): 1279-1283). The common γ chain can also be combined with IL-9Rα to bind IL-9, and IL-9Rα also activates JAK1 (Demoulin et al., 1996, Mol. Cell Biol. 16 (9): 4710-4716). Although the common γ chain activates JAK3, it has been shown that JAK1 is superior to JAK3, and although JAK3 is active, inhibition of JAK1 is sufficient to inactivate signal transduction via the common γ chain (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 lung inflammation models (Mathew et al., 2001, J. Exp. Med. 193(9):1087-1096; Kudlaczet. al., 2008, Eur. J. Pharmacol. 582(1-3):154-161).

[0007] Biochemical and genetic studies have shown the association between JAK2 and single chain (e.g., EPO), IL-3 and interferon gamma cytokine receptor family (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, mice knocked out of JAK2 died of anemia (O'Shea et al., 2002, Cell, 109 (suppl.): S121-S131). JAK2 kinase activating mutations (e.g., JAK2V617F) are associated with human myeloproliferative disorders. 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 the differentiation, growth, activation, survival, and recruitment of eosinophils 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 IL-5 (Mepolizumab, Reslizumab) or the α 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 an important role in regulating type II immunity and acts as an upstream alarm protein for TH2 cytokine production (Kitajima et al., 2011, Eur J Immunol., 41: 1862-71). Tezepelumab is an antagonist antibody to TSLP. The results of the Phase 2 trial indicate that it successfully reduced asthma exacerbations in patients with and without type 2 high markers (Corren et al., 2017, 377: 936-46).

[0008] JAK3 only associates with the γ common cytokine receptor chain, which is present in the IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21 cytokine receptor complexes. JAK3 is essential for the development and proliferation of lymphoid cells, and JAK3 mutations 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).

[0009] TYK2 associates with type I interferon (e.g., IFNα), 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 signal transduction. A fully human monoclonal antibody (ustekinumab) targeting the common 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 been clinically tested for the treatment of Crohn's disease (Mannon et al., 2004, N. Engl. J. Med. 351:2069-79).

[0010] International Patent Application Publication Nos. WO 2010 / 051549, WO 2011 / 003065, WO 2015 / 177326, and WO 2017 / 089390 discuss certain pyrazolopyrimidine compounds that are reportedly useful as inhibitors of one or more Janus kinases. Data are provided for certain specific compounds that show inhibitory effects on JAK1, JAK2, JAK3, and / or TYK2 kinases.

[0011] At present, there is still a need for other compounds that are inhibitors of Janus kinases. For example, there is a need for compounds that have useful efficacy as inhibitors of one or more Janus kinases (e.g., JAK1 and JAK2) and a combination of other pharmacological properties necessary to achieve useful therapeutic benefits. For example, in general, it is necessary to show a higher selectivity for one Janus kinase relative to other kinases (e.g., a higher selectivity for JAK1 and / or JAK2 relative to other kinases such as leucine-rich repeat kinase 2 (LRRK2)). Effective compounds that show a higher selectivity for one Janus kinase relative to other Janus kinases (e.g., a higher selectivity for JAK1 and / or JAK2 relative to JAK3 and / or TYK2) are also needed. Compounds that show a higher selectivity for JAK1 and JAK2 relative to JAK3 and TYK2 can provide therapeutic benefits for conditions that respond to JAK1 inhibition. In addition, there is currently a need for effective JAK1 inhibitors with other properties necessary for formulation and inhalation administration (e.g., melting point, pK, solubility, etc.). Such compounds are particularly useful for treating conditions such as asthma.

[0012] Therefore, there is a need in the art for additional or alternative treatments for JAK kinase-mediated conditions such as those described above. There is a particular need for JAK1 and JAK2 kinase inhibitors that can be used for inhaled delivery to treat airway inflammatory indications such as asthma. Summary of the invention

[0013] Provided herein are pyrazolopyrimidines that inhibit JAK kinases, such as selected from compounds of formula (I), stereoisomers or salts thereof, such as pharmaceutically acceptable salts thereof. The JAK kinase may be JAK1, JAK2, or both.

[0014] One embodiment provides a compound of formula (I):

[0015]

[0016] or a stereoisomer or a pharmaceutically acceptable salt thereof,

[0017] in:

[0018] R 1 Yes: Hydroxy-C1-C6 alkyl; -(CR a1 R a2 ) m -het 1 ;-(CR a1 R a2 ) n -NR b R c ; or -(CR a1 R a2 ) m -C3-6 Cycloalkyl, wherein C 3-6 The cycloalkyl part is R d Replace once;

[0019] R 2 is: halo; halo C1-C6 alkoxy; C1-C6 alkylthio; -SF2; or C3-C6 cycloalkyl;

[0020] R 3 is: hydrogen; or C1-C6 alkyl;

[0021] R 4 is: hydrogen; or C1-C6 alkyl;

[0022] R 5 is: hydrogen; or C1-C6 alkyl;

[0023] R 6 is: hydrogen; or C1-C6 alkyl;

[0024] or R 2 and R 6 Together with the atoms to which it is attached, it can form a six-membered ring containing two heteroatoms each independently selected from O, N and S;

[0025] m is 0 to 2;

[0026] n is 0 to 3;

[0027] Each R a1 are independently: hydrogen; or C1-C6 alkyl;

[0028] Each R a2 are independently: hydrogen; halo; or C1-C6 alkyl;

[0029] R b is: hydrogen; or C1-C6 alkyl;

[0030] R c is: hydrogen; C1-C6 alkyl; an amino protecting group; or azetidinyl, which may be unsubstituted or substituted once with C1-C6 alkyl;

[0031] het 1 is a heterocyclic group selected from the following: azetidinyl; pyrrolidinyl; piperazinyl; piperidinyl; morpholinyl; and oxetanyl; each of which may be unsubstituted or substituted with R d Replace once and pass R g Replace once or twice;

[0032] R d Yes:-(CR a1 R a2 ) p -het2 ;-(CR a1 R a2 ) q -NR e R f ; or -(CR a1 R a2 ) p -C 3-6 Cycloalkyl, wherein C 3-6 The cycloalkyl part is -NR e R f Replace once;

[0033] p is 0 to 2;

[0034] q is 0 to 4;

[0035] R e is: hydrogen; or C1-C6 alkyl;

[0036] R f is: hydrogen; C1-C6 alkyl; or -CH2C2N(CH3)2;

[0037] Each R g is: C1-C6 alkyl; or halogenated; and

[0038] Het 2 is a heterocycle selected from the group consisting of tetrahydropyran; azetidinyl; and pyrrolidinyl; each of which may be unsubstituted or substituted with C1-C6 alkyl or -NR e R f Alkyl substituted once.

[0039] In certain embodiments, R 1 Yes: C1-C6 alkyl; hydroxy-C1-C6 alkyl; -(CR a1 R a2 ) m -het 1 ; or (CR a1 R a2 ) n -NR b R c , where 1 May be unsubstituted or substituted with R d Replace once.

[0040] Also provided is a pharmaceutical composition comprising the JAK inhibitor described herein or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent or excipient.

[0041] Also provided is the use of the JAK inhibitors described herein or pharmaceutically acceptable salts thereof in treatment, such as in the treatment of inflammatory diseases (e.g., asthma). Also provided is the use of the JAK inhibitors described herein or pharmaceutically acceptable salts thereof in the preparation of a medicament for treating inflammatory diseases. Also provided is a method for preventing, treating or reducing the severity of a disease or condition responsive to inhibition of Janus kinase activity in a patient, the method comprising administering to the patient a therapeutically effective amount of a JAK inhibitor as described herein or pharmaceutically acceptable salts thereof.

[0042] The most potent cytokines in asthma (IL-4, IL-5, IL-9, IL-13, and TSLP) all signal through JAK1 and / or JAK2. The compounds of the invention are active against JAK1 and JAK2. Certain of the compounds optimally have well-balanced co-activity for both JAK1 and JAK2 or have a slightly higher affinity for JAK1 than JAK2, rather than having much greater activity for one of the kinases than the other. The subject compounds also have good selectivity for off-target kinases such as LRRK2 that have been associated with pulmonary toxicity.

[0043] Although many compounds can show 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 effectively mediate asthma-related cytokines associated with JAK1 and JAK2 in cell-based assays.

[0044] The compounds of the present invention also show favorable pharmacokinetic (PK) properties in lung tissue and can be used for inhalation therapy. When administered via an inhalation route using a technique such as dry powder inhalation (DPI) or intranasal (IN) delivery, some compounds unexpectedly show sustained retention in lung tissue, with much lower concentrations in the systemic circulation. Such improved PK properties can advantageously reduce the dose of effective therapy and reduce the frequency of administration requirements. The solubility of some compounds shows an unexpected increase, which again improves the efficacy in the lungs. Some compounds of the present invention show an unexpected reduction in cytotoxicity compared to other JAK inhibitors. DETAILED DESCRIPTION

[0045] definition

[0046] "Halogen" or "halo" refers to fluorine, chlorine, bromine or iodine. Additionally, terms such as "haloalkyl" are meant to include monohaloalkyl and polyhaloalkyl, wherein one or more halogens replace one or more hydrogens of the alkyl group.

[0047] The term "alkyl" refers to a saturated straight or branched monovalent hydrocarbon group, wherein the alkyl group may be optionally substituted. In one example, the alkyl group is 1 to 18 carbon atoms (C1-C 18 In other examples, the alkyl group is C0-C6, C0-C5, C0-C3, C1-C 12 、C1-C 10, C1-C8, C1-C6, C1-C5, C1-C4 or C1-C3. C0 alkyl refers to a bond. Examples of alkyl groups include methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1-propyl (n-Pr, n-propyl, -CH2CH2CH3), 2-propyl (i-Pr, isopropyl, -CH(CH3)2), 1-butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl-1-propyl (i-Bu, isobutyl, -CH2CH(CH3)2), 2-butyl (s-Bu, sec-butyl, -CH(CH3)CH2CH3), 2-methyl-2-propyl (t-Bu, tert-butyl, -C(CH3)3), 1-pentyl (n-pentyl, -CH2CH2CH2CH 2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3-methyl-1-butyl (-CH2CH2CH(CH3)2), 2-methyl-1-butyl (-CH2CH(CH3)CH2CH3), 1-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH 2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3)CH(CH3)CH2CH3), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl ((-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3, 1-heptyl and 1 -octyl. In some embodiments, substituents for "optionally substituted alkyl" include one to four examples of F, Cl, Br, I, OH, SH, CN, NH2, NHCH3, N(CH3)2, NO2, N3, C(O)CH3, COOH, CO2CH3, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, cyclopropyl, methoxy, ethoxy, propoxy, oxo, trifluoromethyl, difluoromethyl, sulfonylamino, methylsulfonylamino, SO, SO2, phenyl, piperidinyl, piperazinyl, and pyrimidinyl, wherein their alkyl, phenyl, and heterocyclic portions may be optionally substituted, such as by one to four substituents selected from the same list.

[0048] The term "alkenyl" refers to a straight or branched monovalent hydrocarbon radical having at least one site of unsaturation (i.e., a carbon-carbon double bond), wherein the alkenyl group may be optionally substituted, and includes groups having "cis" and "trans" orientations, or alternatively "E" and "Z" orientations. In one example, an alkenyl group is 2 to 18 carbon atoms (C2-C 18 In other examples, alkenyl is C2-C 12 、C2-C 10 , C2-C8, C2-C6 or C2-C3. Examples include, but are not limited to, ethenyl (vinyl) (-CH=CH2), prop-1-enyl (-CH=CHCH3), prop-2-enyl (-CH2CH=CH2), 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, substituents for “optionally substituted alkenyl” include one to four examples of F, Cl, Br, I, OH, SH, CN, NH2, NHCH3, N(CH3)2, NO2, N3, C(O)CH3, COOH, CO2CH3, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, cyclopropyl, methoxy, ethoxy, propoxy, oxo, trifluoromethyl, difluoromethyl, sulfonylamino, methylsulfonylamino, SO, SO2, phenyl, piperidinyl, piperazinyl, and pyrimidinyl, wherein their alkyl, phenyl, and heterocyclic portions may be optionally substituted, such as with one to four substituents selected from the same list.

[0049] The term "alkynyl" refers to a straight or branched monovalent hydrocarbon radical having at least one site of unsaturation (i.e., a carbon-carbon triple bond), wherein the alkynyl radical may be optionally substituted. In one example, the alkynyl radical is two to eighteen carbon atoms (C2-C 18 In other examples, the alkynyl group is C2-C 12 、C2-C 10, C2-C8, C2-C6 or C2-C3. Examples include, but are not limited to, ethynyl (-CCH), prop-1-ynyl (-C≡CCH3), prop-2-ynyl (propargyl, -CH2C≡CH), but-1-ynyl, but-2-ynyl and but-3-ynyl. In some embodiments, substituents for “optionally substituted alkynyl” include one to four examples of F, Cl, Br, I, OH, SH, CN, NH2, NHCH3, N(CH3)2, NO2, N3, C(O)CH3, COOH, CO2CH3, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, cyclopropyl, methoxy, ethoxy, propoxy, oxo, trifluoromethyl, difluoromethyl, sulfonylamino, methylsulfonylamino, SO, SO2, phenyl, piperidinyl, piperazinyl, and pyrimidinyl, wherein their alkyl, phenyl, and heterocyclic portions may be optionally substituted, such as with one to four substituents selected from the same list.

[0050] "Alkylene" refers to a saturated branched or straight chain hydrocarbon group having two monovalent free radical centers derived from the same or two different carbon atoms of a parent alkane. In one example, a divalent alkylene group is 1 to 18 carbon atoms (C1-C 18 In other examples, the divalent alkylene group is C0-C6, C0-C5, C0-C3, C1-C 12 、C1-C 10 , C1-C8, C1-C6, C1-C5, C1-C4 or C1-C3. The group C0 alkylene refers to a bond. Exemplary alkylene groups include methylene (-CH2-), 1,1-ethyl (-CH(CH3)-), (1,2-ethyl (-CH2CH2-), 1,1-propyl (-CH(CH2CH3)-), 2,2-propyl (-C(CH3)2-), 1,2-propyl (-CH(CH3)CH2-), 1,3-propyl (-CH2CH2CH2-), 1,1-dimethyl-2-1,2-yl (-C(CH3)2CH2-), 1,4-butyl (-CH2CH2CH2CH2-) and the like.

[0051] The term "heteroalkyl" refers to a straight or branched monovalent hydrocarbon radical consisting of the indicated number of carbon atoms (or, if not indicated, up to 18 carbon atoms) and 1 to 5 heteroatoms selected from the group consisting of O, N, Si and S, and wherein 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, wherein the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized. One or more heteroatoms may be located at any interior position of the heteroalkyl group, including where the alkyl group is attached to the rest of the molecule (e.g., -O-CH2-CH3). Examples include -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -S(O)-CH3, -CH2-CH2-S(O)2-CH3, -Si(CH3)3 and -CH2-CH=N-OCH3. Up to two heteroatoms may be consecutive, for example -CH2-NH-OCH3 and -CH2-O-Si(CH3)3. The heteroalkyl group may be optionally substituted. In some embodiments, substituents for “optionally substituted heteroalkyl” include one to four examples of F, Cl, Br, I, OH, SH, CN, NH2, NHCH3, N(CH3)2, NO2, N3, C(O)CH3, COOH, CO2CH3, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, cyclopropyl, methoxy, ethoxy, propoxy, oxo, trifluoromethyl, difluoromethyl, sulfonylamino, methylsulfonylamino, SO, SO2, phenyl, piperidinyl, piperazinyl, and pyrimidinyl, wherein their alkyl, phenyl, and heterocyclic portions may be optionally substituted, such as with one to four substituents selected from the same list.

[0052] "Amino" refers to primary amines (i.e., -NH2), secondary amines (i.e., -NRH), tertiary amines (i.e., -NRR), and quaternary amines (i.e., -N(+)RRR), which are optionally substituted, wherein each R is the same or different and is selected from alkyl, cycloalkyl, aryl, and heterocyclyl, wherein the alkyl, cycloalkyl, aryl, and heterocyclyl groups are as defined herein. Specific secondary and tertiary amines are alkylamines, dialkylamines, arylamines, diarylamines, aralkylamines, and diaralkylamines, wherein the alkyl and aryl portions may be optionally substituted. Specific secondary and tertiary amines are methylamine, ethylamine, propylamine, isopropylamine, aniline, benzylamine, dimethylamine, diethylamine, dipropylamine, and diisopropylamine. In some embodiments, the R groups of the quaternary amines are each independently an optionally substituted alkyl group.

[0053] "Aryl" refers to a carbocyclic aromatic group of up to 14 carbon atoms, whether or not fused to one or more groups. An example includes an aryl group with 6-14 carbon atoms. Another example includes an aryl group with 6-10 carbon atoms. Examples of aryl groups include phenyl, naphthyl, biphenyl, phenanthrenyl, tetraphenyl, 1,2,3,4-tetrahydronaphthyl, 1H-indenyl, 2,3-dihydro-1H-indenyl, etc. (see, for example, "Lan's Handbook of Chemistry" (Dean, JA ed.) 13th edition Table 7-2

[1985] ). A specific aryl is phenyl. Substituted phenyl or substituted aryl refers to a phenyl group or aryl group substituted with one, two, three, four or five substituents (e.g., 1-2, 1-3 or 1-4 substituents), such as selected from the groups specified herein (see the definition of "optionally substituted"), such as one to four examples of F, Cl, Br, I, OH, SH, CN, NH2, NHCH3, N(CH3)2, NO2, N3, C(O)CH3, COOH, CO2CH3, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, cyclopropyl, methoxy, ethoxy, propoxy, oxo, trifluoromethyl, difluoromethyl, sulfonylamino, methylsulfonylamino, SO, SO2, phenyl, piperidinyl, piperazinyl and pyrimidinyl, wherein their alkyl, phenyl and heterocyclic portions may be optionally substituted, such as with one to four 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, etc.; mono- or di-(hydroxy)phenyl groups such as 4-hydroxyphenyl, 3-hydroxyphenyl, 2,4-dihydroxyphenyl, their hydroxyl-protected derivatives, etc.; nitrophenyl groups such as 3-nitrophenyl or 4-nitrophenyl; cyanophenyl groups, for example 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; phenyl, for example 3-(hydroxymethyl)phenyl or (protected hydroxymethyl)phenyl or 3,4-di(hydroxymethyl)phenyl; mono- or di-(aminomethyl)phenyl or (protected hydroxymethyl)phenyl, such as 2-(aminomethyl)phenyl or 2,4-(aminomethyl)phenyl; or mono- or di-(N-(methylsulfonylamino)phenyl, for example 3-(N-(methylsulfonylamino)phenyl. In addition, the term "substituted phenyl" means a disubstituted phenyl group 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.; and a trisubstituted phenyl group with different substituents, such as 3-methoxy-4-benzyloxy-6-methylsulfonylamino, 3-methoxy-4-benzyloxy-6-phenylsulfonylamino; and a tetrasubstituted phenyl group with different substituents, such as 3-methoxy-4-benzyloxy-5-methyl-6-phenylsulfonylamino. In certain embodiments, the substituent of aryl, such as phenyl, comprises amide. For example, aryl (e.g., phenyl) substituent can be -(CH2). 0- 4CONR'R", wherein R' and R" each independently refer to a group including, for example, hydrogen; unsubstituted C 1- C6 alkyl; halogen, OH, CN, unsubstituted C 1- C6 alkyl, unsubstituted C 1- C6 alkoxy, oxo or NR'R'' substituted C 1- C6 alkyl; unsubstituted C1- C6 heteroalkyl; halogen, OH, CN, unsubstituted C 1- C6 alkyl, unsubstituted C 1- C6 alkoxy, oxo or NR'R" substituted C 1- C6 heteroalkyl; unsubstituted C 6- C 10 Aryl; halogen, OH, CN, unsubstituted C 1- C6 alkyl, unsubstituted C 1- C6 alkoxy or NR'R" substituted C 6- C 10 aryl; unsubstituted 3-11 membered heterocyclic group (e.g., 5-6 membered heteroaryl containing 1 to 4 heteroatoms selected from O, N and S or 4-11 membered heterocycloalkyl containing 1 to 4 heteroatoms selected from O, N and S); and halogen, OH, CN, unsubstituted C 1- C6 alkyl, unsubstituted C 1- C6 alkoxy, oxo or NR'R "substituted 3-11 membered heterocyclyl (for example, containing 1 to 4 heteroatoms selected from O, N and S 5-6 membered heteroaryl or containing 1 to 4 heteroatoms selected from O, N and S 4-11 membered heterocycloalkyl); or R' and R "can be combined with the nitrogen atom to form a 3-membered ring, a 4-membered ring, a 5-membered ring, a 6-membered ring or a 7-membered ring, wherein the ring atoms are optionally substituted with N, O or S, and wherein the ring is optionally substituted with halogen, OH, CN, unsubstituted C 1- C6 alkyl, unsubstituted C 1- C6 alkoxy, oxo or NR'R" substituted.

[0054] "Cycloalkyl" refers to a non-aromatic saturated or partially unsaturated hydrocarbon ring group, wherein the cycloalkyl group may be optionally substituted independently by one or more substituents described herein. In one example, the cycloalkyl group is 3 to 12 carbon atoms (C3-C 12 In other embodiments, the cycloalkyl group is C3-C8, C3-C 10 or C5-C 10 In other examples, the cycloalkyl group as a monocyclic ring is C3-C8, C3-C6 or C5-C6. In another example, the cycloalkyl group as a bicyclic ring is C7-C 12 In another embodiment, the cycloalkyl group as a spiro ring system is C5-C 12. Examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, perdeuterated cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohexyl-2-enyl, 1-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, and cyclododecyl. Exemplary arrangements 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 spirocycloalkyl 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, substituents for "optionally substituted cycloalkyl" include one to four examples of F, Cl, Br, I, OH, SH, CN, NH2, NHCH3, N(CH3)2, NO2, N3, C(O)CH3, COOH, CO2CH3, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, cyclopropyl, methoxy, ethoxy, propoxy, oxo, trifluoromethyl, difluoromethyl, sulfonylamino, methylsulfonylamino, SO, SO2, aryl, piperidinyl, piperazinyl and pyrimidinyl, wherein their alkyl, aryl and heterocyclic moieties may be optionally substituted, such as substituted by one to four substituents selected from the same list. In some embodiments, the substituents of cycloalkyl include amides. For example, the cycloalkyl substituent may be -(CH2) 0-4 CONR'R", wherein R' and R" each independently refer to a group including, for example, hydrogen; unsubstituted C 1- C6 alkyl; halogen, OH, CN, unsubstituted C 1- C6 alkyl, unsubstituted C 1- C6 alkoxy, oxo or NR'R'' substituted C 1- C6 alkyl; unsubstituted C 1- C6 heteroalkyl; halogen, OH, CN, unsubstituted C 1- C6 alkyl, unsubstituted C 1- C6 alkoxy, oxo or NR'R" substituted C 1- C6 heteroalkyl; unsubstituted C 6- C 10 Aryl; halogen, OH, CN, unsubstituted C 1- C6 alkyl, unsubstituted C 1- C6 alkoxy or NR'R" substituted C 6- C 10aryl; unsubstituted 3-11 membered heterocyclic group (e.g., 5-6 membered heteroaryl containing 1 to 4 heteroatoms selected from O, N and S or 4-11 membered heterocycloalkyl containing 1 to 4 heteroatoms selected from O, N and S); and halogen, OH, CN, unsubstituted C 1- C6 alkyl, unsubstituted C 1- C6 alkoxy, oxo or NR'R "substituted 3-11 membered heterocyclyl (for example, containing 1 to 4 heteroatoms selected from O, N and S 5-6 membered heteroaryl or containing 1 to 4 heteroatoms selected from O, N and S 4-11 membered heterocycloalkyl); or R' and R "can be combined with the nitrogen atom to form a 3-membered ring, a 4-membered ring, a 5-membered ring, a 6-membered ring or a 7-membered ring, wherein the ring atoms are optionally substituted with N, O or S, and wherein the ring is optionally substituted with halogen, OH, CN, unsubstituted C 1- C6 alkyl, unsubstituted C 1- C6 alkoxy, oxo or NR'R'' substituted.

[0055] "Heterocyclic group", "heterocyclic", "heterocycle", "heterocyclyl" or "heterocyclo" are used interchangeably and refer to any monocyclic, bicyclic, tricyclic or spirocyclic ring system having from 3 to 20 ring atoms (e.g., 3-10 ring atoms), saturated or unsaturated, aromatic (heteroaryl) or non-aromatic (e.g., heterocycloalkyl) ring systems, wherein 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. If any of the ring atoms in the ring system is a heteroatom, then the system is a heterocycle regardless of the point of attachment of the ring system to the rest of the molecule. In one example, a heterocyclic group includes 3-11 ring atoms ("members") and includes monocyclic, bicyclic, tricyclic and spirocyclic ring systems, wherein the ring atoms are carbon, wherein at least one atom in the ring or ring system is a heteroatom selected from nitrogen, sulfur or oxygen. In one example, a heterocyclic group contains from 1 to 4 heteroatoms. In one example, the heterocyclic group includes 1 to 3 heteroatoms. In another example, the heterocyclic group includes 3 to 7-membered monocycles with 1-2, 1-3 or 1-4 heteroatoms selected from nitrogen, sulfur or oxygen. In another example, the heterocyclic group includes 4 to 6-membered monocycles with 1-2, 1-3 or 1-4 heteroatoms selected from nitrogen, sulfur or oxygen. In another example, the heterocyclic group includes 3-membered monocycles. In another example, the heterocyclic group includes 4-membered monocycles. In another example, the heterocyclic group includes 5-6-membered monocycles, such as 5-6-membered heteroaryls. In another example, the heterocyclic group includes 3-11-membered heterocycloalkyls, such as 4-11-membered heterocycloalkyls. In some embodiments, the heterocycloalkyl includes at least one nitrogen. In one example, the heterocyclic group includes 0 to 3 double bonds. Any nitrogen or sulfur heteroatom can be optionally oxidized (e.g., NO, SO, SO2), and any nitrogen heteroatom can be optionally quaternized (e.g., [NR4] + Cl - 、[NR4] + OH -). Exemplary heterocycles are oxirane, aziridinyl, thiirane, azetidinyl, oxetanyl, thietanyl, 1,2-dithietanyl, 1,3-dithietanyl, pyrrolidinyl, dihydro-1H-pyrrolyl, dihydrofuranyl, tetrahydrofuranyl, dihydrothiophenyl, tetrahydrothiophenyl, imidazolidinyl, piperidinyl, piperazinyl, isoquinolinyl, tetrahydroisoquinolinyl, morpholinyl, thiomorpholinyl, 1,1-dioxo-thiomorpholinyl, dihydropyranyl, tetrahydropyranyl, hexahydrothiopyranyl, hexahydropyrimidinyl, oxazinyl, thiazinyl, thiazinyl, homopiperazinyl, homopiperidinyl, azepanyl, oxepanyl , thiepanyl, oxazepinyl, oxazepinyl, diazepanyl, 1,4-diazepanyl, diazepinyl, thiazepinyl, thiazepanyl, tetrahydrothiopyranyl, oxazolidinyl, thiazolidinyl, isothiazolidinyl, 1,1-dioxoisothiazolidinone, oxazolidinone, imidazolinone, 4,5,6,7-tetrahydro[2H]indazolyl, tetrahydrobenzimidazolyl, 4,5,6,7-tetrahydrobenzo[d]imidazolyl, 1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridinyl, thiazinyl, oxazinyl, thiadiazinyl, oxadiazinyl, dithiazinyl, dioxazinyl, oxathiazinyl, thiatriazinyl azinyl, oxatriazinyl, dithiadiazinyl, imidazolinyl, dihydropyrimidinyl, tetrahydropyrimidinyl, 1-pyrrolinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolyl, thiopyranyl, 2H-pyranyl, 4H-pyranyl, dialkyl, 1,3-dioxolanyl, pyrazolinyl, pyrazolidinyl, dithianyl, dithiolanyl, pyrimidinyl, pyrimidinedione, pyrimidine-2,4-dione, piperazinyl, piperazindione, pyrazolidinimidazolinyl, 3-azabicyclo[3.1.0]-hexyl, 3,6-diazabicyclo[3.1.1]heptyl, 6-azabicyclo[3.1.1]heptyl, 3-azabicyclo-[3.1.1]heptyl , 3-azabicyclo[4.1.0]heptyl, azabicyclo[2.2.2]hexyl, 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]nonyl, azaspiro[2.5]-octanyl, azaspiro[4.5]decyl, 1-azaspiro[4.5]decan-2-one, azaspiro[5.5]undecyl, tetrahydroindolyl, octahydroindolyl, tetrahydroisoindolyl, tetrahydroindazolyl, 1,1-dioxohexahydrothiopyranyl.Examples of 5-membered heterocycles containing sulfur or oxygen atoms and one to three nitrogen atoms are thiazolyl, including thiazol-2-yl and thiazol-2-yl N-oxide; thiadiazolyl, including 1,3,4-thiadiazol-5-yl and 1,2,4-thiadiazol-5-yl; oxazolyl, such as oxazol-2-yl; and oxadiazolyl, such as 1,3,4-oxadiazol-5-yl and 1,2,4-oxadiazol-5-yl. Exemplary 5-membered 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-tetrazolyl. Exemplary benzo-fused 5-membered heterocycles are benzooxazole-2-yl, benzothiazol-2-yl and benzimidazol-2-yl. Exemplary 6-membered heterocycles contain one to three nitrogen atoms and optional sulfur or oxygen atoms, for example pyridyl, such as pyridin-2-yl, pyridin-3-yl and pyridin-4-yl; pyrimidinyl, such as pyrimidin-2-yl and pyrimidin-4-yl; triazinyl, such as 1,3,4-triazin-2-yl and 1,3,5-triazin-4-yl; pyridazinyl, especially pyridazin-3-yl and pyrazinyl. Pyridine N-oxide and pyridazine N-oxide and pyridinyl, pyrimidin-2-yl, pyrimidin-4-yl, pyridazinyl and 1,3,4-triazin-2-yl groups are other exemplary heterocyclic groups. The heterocycle may be optionally substituted. For example, the substituents for "optionally substituted heterocycle" include one to four examples of F, Cl, Br, I, OH, SH, CN, NH2, NHCH3, N(CH3)2, NO2, N3, C(O)CH3, COOH, CO2CH3, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, cyclopropyl, methoxy, ethoxy, propoxy, oxo, trifluoromethyl, difluoromethyl, sulfonylamino, methylsulfonylamino, SO, SO2, aryl, piperidinyl, piperazinyl and pyrimidinyl, wherein their alkyl, aryl and heterocyclic moieties can be optionally substituted, such as substituted by one to four substituents selected from the same list. In some embodiments, the substituents of the heterocyclic group (such as heteroaryl or heterocycloalkyl) include amides. For example, the heterocyclic (e.g., heteroaryl or heterocycloalkyl) substituent substituent can be (CH2). 0-4 CONR'R", wherein R' and R" each independently refer to a group including, for example, hydrogen; unsubstituted C 1- C6 alkyl; halogen, OH, CN, unsubstituted C 1- C6 alkyl, unsubstituted C 1- C6 alkoxy, oxo or NR'R'' substituted C 1- C6 alkyl; unsubstituted C 1- C6 heteroalkyl; halogen, OH, CN, unsubstituted C 1- C6 alkyl, unsubstituted C 1-C6 alkoxy, oxo or NR'R" substituted C 1- C6 heteroalkyl; unsubstituted C 6- C 10 Aryl; halogen, OH, CN, unsubstituted C 1- C6 alkyl, unsubstituted C 1- C6 alkoxy or NR'R" substituted C 6- C 10 aryl; unsubstituted 3-11 membered heterocyclic group (e.g., 5-6 membered heteroaryl containing 1 to 4 heteroatoms selected from O, N and S or 4-11 membered heterocycloalkyl containing 1 to 4 heteroatoms selected from O, N and S); and halogen, OH, CN, unsubstituted C 1- C6 alkyl, unsubstituted C 1- C6 alkoxy, oxo or NR'R "substituted 3-11 membered heterocyclyl (for example, containing 1 to 4 heteroatoms selected from O, N and S 5-6 membered heteroaryl or containing 1 to 4 heteroatoms selected from O, N and S 4-11 membered heterocycloalkyl); or R' and R "can be combined with the nitrogen atom to form a 3-membered ring, a 4-membered ring, a 5-membered ring, a 6-membered ring or a 7-membered ring, wherein the ring atoms are optionally substituted with N, O or S, and wherein the ring is optionally substituted with halogen, OH, CN, unsubstituted C 1- C6 alkyl, unsubstituted C 1- C6 alkoxy, oxo or NR'R'' substituted.

[0056] "Heteroaryl" refers to any monocyclic, bicyclic or tricyclic ring system, wherein 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, Table 7-2 of the 13th edition of "Lan's Handbook of Chemistry" (Dean, JA ed.)

[1985] . This definition includes any bicyclic group, wherein any of the above heteroaromatic rings is fused to an aromatic ring, wherein the aromatic ring or heteroaromatic ring is connected to the rest of the molecule. In one embodiment, heteroaryl includes a 5-6-membered monocyclic aromatic group, wherein one or more ring atoms are nitrogen, sulfur or oxygen. Exemplary heteroaryl groups include thienyl, furanyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, thiadiazolyl, oxadiazolyl, tetrazolyl, thiatriazolyl, oxatriazolyl, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazinyl, tetrazinyl, tetrazo[1,5-b]pyridazinyl, imidazo[1,2-a]pyrimidyl, and purinyl, as well as benzo-fused derivatives such as benzoxazolyl, benzofuranyl, benzothiazolyl, benzothiadiazolyl, benzotriazolyl, benzimidazolyl, and indolyl. The heteroaryl groups may be optionally substituted. In some embodiments, substituents for "optionally substituted heteroaryl" include one to four examples of F, Cl, Br, I, OH, SH, CN, NH2, NHCH3, N(CH3)2, NO2, N3, C(O)CH3, COOH, CO2CH3, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, cyclopropyl, methoxy, ethoxy, propoxy, trifluoromethyl, difluoromethyl, sulfonylamino, methylsulfonylamino, SO, SO2, phenyl, piperidinyl, piperazinyl, and pyrimidinyl, wherein their alkyl, phenyl, and heterocyclic moieties may be optionally substituted, such as by one to four substituents selected from the same list. In some embodiments, substituents for heteroaryl include amides. For example, heteroaryl substituents may be -(CH2) 0-4 CONR'R", wherein R' and R" each independently refer to a group including, for example, hydrogen; unsubstituted C 1- C6 alkyl; halogen, OH, CN, unsubstituted C 1- C6 alkyl, unsubstituted C 1- C6 alkoxy, oxo or NR'R'' substituted C 1- C6 alkyl; unsubstituted C 1- C6 heteroalkyl; halogen, OH, CN, unsubstituted C 1- C6 alkyl, unsubstituted C 1- C6 alkoxy, oxo or NR'R" substituted C 1- C6 heteroalkyl; unsubstituted C 6- C 10 Aryl; halogen, OH, CN, unsubstituted C 1-C6 alkyl, unsubstituted C 1- C6 alkoxy or NR'R" substituted C 6- C 10 aryl; unsubstituted 3-11 membered heterocyclic group (e.g., 5-6 membered heteroaryl containing 1 to 4 heteroatoms selected from O, N and S or 4-11 membered heterocycloalkyl containing 1 to 4 heteroatoms selected from O, N and S); and halogen, OH, CN, unsubstituted C 1- C6 alkyl, unsubstituted C 1- C6 alkoxy, oxo or NR'R "substituted 3-11 membered heterocyclyl (for example, containing 1 to 4 heteroatoms selected from O, N and S 5-6 membered heteroaryl or containing 1 to 4 heteroatoms selected from O, N and S 4-11 membered heterocycloalkyl); or R' and R "can be combined with the nitrogen atom to form a 3-membered ring, a 4-membered ring, a 5-membered ring, a 6-membered ring or a 7-membered ring, wherein the ring atoms are optionally substituted with N, O or S, and wherein the ring is optionally substituted with halogen, OH, CN, unsubstituted C 1- C6 alkyl, unsubstituted C 1- C6 alkoxy, oxo or NR'R'' substituted.

[0057] In certain embodiments, the heterocyclyl group is attached at a carbon atom of the heterocyclyl group. For example, carbon-bonded heterocyclyl groups include bond arrangements at the following positions: 2, 3, 4, 5, or 6 of a pyridine ring, 3, 4, 5, or 6 of a pyridazine ring, 2, 4, 5, or 6 of a pyrimidine ring, 2, 3, 5, or 6 of a pyrazine ring, 2, 3, 4, or 5 of a furan, tetrahydrofuran, thiofuran, thiophene, pyrrole, or tetrahydropyrrole ring, 2, 4, or 5 of an oxazole, imidazole, or thiazole ring, 3, 4, or 5 of an isoxazole, pyrazole, or isothiazole ring, 2 or 3 of an aziridine ring, 2, 3, or 4 of an azetidine ring, 2, 3, 4, 5, 6, 7, or 8 of a quinoline ring, or 1, 3, 4, 5, 6, 7, or 8 of an isoquinoline ring.

[0058] In certain embodiments, the heterocyclyl group is N-linked. For example, nitrogen-bonded heterocyclyl or heteroaryl groups include the following bonding arrangements: position 1 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; position 2 of isoindole or isoindolinone; position 4 of morpholine; and position 9 of carbazole or β-carboline.

[0059] The term "alkoxy" refers to a straight or branched monovalent group represented by the formula -OR, wherein R is an alkyl group as defined herein. Alkoxy groups include methoxy, ethoxy, propoxy, isopropoxy, monofluoromethoxy, difluoromethoxy and trifluoromethoxy, and cyclopropyloxy.

[0060] "Acyl" refers to a carbonyl-containing substituent represented by the formula -C(O)-R, wherein R is hydrogen, alkyl, cycloalkyl, aryl, or heterocyclyl, wherein alkyl, cycloalkyl, aryl, and heterocyclyl are as defined herein. Acyl groups include alkanoyl (e.g., acetyl), aroyl (e.g., benzoyl), and heteroaroyl (e.g., picolinyl).

[0061] Unless otherwise indicated, "optionally substituted" means that the group can be unsubstituted or substituted by one or more (e.g., 0, 1, 2, 3, 4, or 5 or more, or any range derivable therein) substituents listed for the group, wherein the substituents may be the same or different. In one embodiment, the optionally substituted group has 1 substituent. In another embodiment, the optionally substituted group has 2 substituents. In another embodiment, the optionally substituted group has 3 substituents. In another embodiment, the optionally substituted group has 4 substituents. In another embodiment, the optionally substituted group has 5 substituents.

[0062] The optional substituents for alkyl, alone or as part of another substituent (e.g., alkoxy), and alkylene, alkenyl, alkynyl, heteroalkyl, heterocycloalkyl, and cycloalkyl, also alone or as part of another substituent, can be various groups as described herein, as well as groups selected from the group consisting of: halogen; oxo; CN; NO; N3; -OR'; perfluoro-C 1- C4 alkoxy; unsubstituted C3-C7 cycloalkyl; C3-C7 cycloalkyl substituted by halogen, OH, CN, unsubstituted C1-C6 alkyl, unsubstituted C1-C6 alkoxy, oxo or NR'R"; unsubstituted C6-C 10 Aryl (e.g., phenyl); C6-C6 substituted by halogen, OH, CN, unsubstituted C1-C6 alkyl, unsubstituted C1-C6 alkoxy, or NR'R'' 10aryl; unsubstituted 3-11 membered heterocyclyl (e.g., 5-6 membered heteroaryl containing 1 to 4 heteroatoms selected from O, N and S or 4-11 membered heterocycloalkyl containing 1 to 4 heteroatoms selected from O, N and S); 3-11 membered heterocyclyl (e.g., 5-6 membered heteroaryl containing 1 to 4 heteroatoms selected from O, N and S or 4-11 membered heterocycloalkyl containing 1 to 4 heteroatoms selected from O, N and S) substituted by halogen, OH, CN, unsubstituted C1-C6 alkyl, unsubstituted C1-C6 alkoxy, oxo or NR'R " 4-11 membered heterocycloalkyl); -NR'R"; -SR'; -SiR'R"R"'; -OC(O)R'; -C(O)R'; -CO2R'; -CONR'R"; -OC(O)NR'R"; -NR"C(O)R'; -NR"'C(O)NR'R"; -NR"C(O)2R'; -S(O)2R'; -S(O)2NR'R"; -NR'S(O)2R"; -NR"'S(O)2NR'R"; amidino; guanidino; -(CH2) 1-4 -OR';-(CH2) 1-4 -NR'R"; -(CH2) 1-4 -SR'; -(CH2) 1-4 -SiR'R"R"'; -(CH2) 1-4 -OC(O)R'; -(CH2) 1-4 -C(O)R'; -(CH2) 1-4 -CO2R'; and -(CH2) 1-4 CONR'R", or a combination thereof, the amount of the optional substituent is in the range of 0 to (2m'+1), wherein m' is the total number of carbon atoms in such a group. R', R'' and R''' each independently refer to a group including, for example, hydrogen; unsubstituted C 1- C6 alkyl; halogen, OH, CN, unsubstituted C 1- C6 alkyl, unsubstituted C 1- C6 alkoxy, oxo or NR'R'' substituted C 1- C6 alkyl; unsubstituted C 1- C6 heteroalkyl; halogen, OH, CN, unsubstituted C 1- C6 alkyl, unsubstituted C 1- C6 alkoxy, oxo or NR'R" substituted C 1- C6 heteroalkyl; unsubstituted C 6- C 10 Aryl; halogen, OH, CN, unsubstituted C 1- C6 alkyl, unsubstituted C 1- C6 alkoxy or NR'R" substituted C 6- C 10aryl; unsubstituted 3-11 membered heterocyclic group (e.g., 5-6 membered heteroaryl containing 1 to 4 heteroatoms selected from O, N and S or 4-11 membered heterocycloalkyl containing 1 to 4 heteroatoms selected from O, N and S); and halogen, OH, CN, unsubstituted C 1- C6 alkyl, unsubstituted C 1- C6 alkoxy, oxo or NR'R" substituted 3-11 membered heterocyclyl (for example, a 5-6 membered heteroaryl containing 1 to 4 heteroatoms selected from O, N and S or a 4-11 membered heterocycloalkyl containing 1 to 4 heteroatoms selected from O, N and S). When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 3-membered ring, a 4-membered ring, a 5-membered ring, a 6-membered ring or a 7-membered ring, wherein the ring atoms are optionally substituted with N, O or S, and wherein the ring can be optionally substituted with halogen, OH, CN, unsubstituted C1-C6 alkyl, unsubstituted C1-C6 alkoxy, oxo or NR'R". For example, -NR'R" is meant to include 1-pyrrolidinyl and 4-morpholinyl.

[0063] Similarly, the optional substituents for aryl and heteroaryl groups are numerous. In some embodiments, the substituents for aryl and heteroaryl groups are selected from the group consisting of: halogen; oxo; CN; NO; N3; -OR'; perfluoro-C 1- C4 alkoxy; unsubstituted C3-C7 cycloalkyl; C3-C7 cycloalkyl substituted by halogen, OH, CN, unsubstituted C1-C6 alkyl, unsubstituted C1-C6 alkoxy, oxo or NR'R''; unsubstituted C6-C 10 Aryl (e.g., phenyl); C6-C6 substituted by halogen, OH, CN, unsubstituted C1-C6 alkyl, unsubstituted C1-C6 alkoxy, or NR'R'' 10aryl; unsubstituted 3-11 membered heterocyclyl (e.g., 5-6 membered heteroaryl containing 1 to 4 heteroatoms selected from O, N and S or 4-11 membered heterocycloalkyl containing 1 to 4 heteroatoms selected from O, N and S); 3-11 membered heterocyclyl (e.g., 5-6 membered heteroaryl containing 1 to 4 heteroatoms selected from O, N and S or 4-11 membered heterocycloalkyl containing 1 to 4 heteroatoms selected from O, N and S) substituted by halogen, OH, CN, unsubstituted C1-C6 alkyl, unsubstituted C1-C6 alkoxy, oxo or NR'R " 4-11 membered heterocycloalkyl); -NR'R"; -SR'; -SiR'R"R"'; -OC(O)R'; -C(O)R'; -CO2R'; -CONR'R"; -OC(O)NR'R"; -NR"C(O)R'; -NR"'C(O)NR'R"; -NR"C(O)2R'; -S(O)2R'; -S(O)2NR'R"; -NR'S(O)2R"; -NR"'S(O)2NR'R"; amidino; guanidino; -(CH2) 1-4 -OR';-(CH2) 1-4 -NR'R"; -(CH2) 1-4 -SR'; -(CH2) 1-4 -SiR'R"R"'; -(CH2) 1-4 -OC(O)R'; -(CH2) 1-4 -C(O)R'; -(CH2) 1-4 -CO2R'; and (CH2) 1-4 CONR'R", or a combination thereof, the amount of the optional substituent is in the range of 0 to (2m'+1), wherein m' is the total number of carbon atoms in such a group. R', R'' and R''' each independently refer to a group including, for example, hydrogen; unsubstituted C 1- C6 alkyl; halogen, OH, CN, unsubstituted C 1- C6 alkyl, unsubstituted C 1- C6 alkoxy, oxo or NR'R'' substituted C 1- C6 alkyl; unsubstituted C 1- C6 heteroalkyl; halogenated, OH, CN, unsubstituted C 1- C6 alkyl, unsubstituted C 1- C6 alkoxy, oxo or NR'R" substituted C 1- C6 heteroalkyl; unsubstituted C 6- C 10 Aryl; halogen, OH, CN, unsubstituted C 1- C6 alkyl, unsubstituted C 1- C6 alkoxy or NR'R" substituted C 6- C 10aryl; unsubstituted 3-11 membered heterocyclic group (e.g., 5-6 membered heteroaryl containing 1 to 4 heteroatoms selected from O, N and S or 4-11 membered heterocycloalkyl containing 1 to 4 heteroatoms selected from O, N and S); and halogen, OH, CN, unsubstituted C 1- C6 alkyl, unsubstituted C 1- C6 alkoxy, oxo or NR'R" substituted 3-11 membered heterocyclyl (for example, a 5-6 membered heteroaryl containing 1 to 4 heteroatoms selected from O, N and S or a 4-11 membered heterocycloalkyl containing 1 to 4 heteroatoms selected from O, N and S). When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 3-membered ring, a 4-membered ring, a 5-membered ring, a 6-membered ring or a 7-membered ring, wherein the ring atoms are optionally substituted with N, O or S, and wherein the ring can be optionally substituted with halogen, OH, CN, unsubstituted C1-C6 alkyl, unsubstituted C1-C6 alkoxy, oxo or NR'R". For example, -NR'R" is meant to include 1-pyrrolidinyl and 4-morpholinyl.

[0064] The term "oxo" refers to =0 or (=0)2.

[0065] As used herein, a wavy line intersecting a bond in a chemical structure Indicates the point of connection of the atom connected to the wavy bond in the chemical structure with the rest of the molecule or with the rest of the molecular fragment. In some embodiments, an arrow and an asterisk are used together in a wavy line to indicate a point of connection.

[0066] In certain embodiments, a divalent group is generally described as not containing a specific bonding configuration. It should be understood that unless otherwise specified, the general description is intended to include both bonding configurations. For example, unless otherwise specified, in the group R 1 –R 2 –R 3 If the group R 2 is described as -CH2C(O)-, it is understood that this group can be used as R 1 –CH2C(O)–R 3 and R 1 –C(O)CH2–R 3 Bonding.

[0067] Unless otherwise indicated, the term "compound(s) of the invention / compound(s) of the present invention" and the like include compounds of formula (I) herein, such as compounds 1-18, sometimes referred to as JAK inhibitors, including stereoisomers (including atropisomers), geometric isomers, tautomers, solvates, metabolites, isotopes, salts (e.g., pharmaceutically acceptable salts), and prodrugs thereof. In some embodiments, solvates, metabolites, isotopes or prodrugs and any combination thereof are excluded.

[0068] The term "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when administered to animals (eg, humans), as appropriate.

[0069] The compounds of the present invention can be in the form of salts, such as pharmaceutically acceptable salts. "Pharmaceutically acceptable salts" include acid addition salts or base addition salts. "Pharmaceutically acceptable acid addition salts" refer to salts formed with inorganic acids (such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, carbonic acid, phosphoric acid, etc.) and organic acids that retain the biological effectiveness and properties of free alkalis and are not undesirable biologically or otherwise, and the organic acid can be selected from aliphatic, cycloaliphatic, aromatic, aromatic aliphatic, heterocyclic, carboxylic and sulfonic acid 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, pamoic acid, phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc.

[0070] "Pharmaceutically acceptable base addition salts" include those derived from inorganic bases, such as sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, and the like. Specific base addition salts are ammonium salts, potassium salts, sodium salts, calcium salts, and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include primary, secondary, and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines, and basic ion exchange resins (such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, tromethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like). Specific organic non-toxic bases include isopropylamine, diethylamine, ethanolamine, tromethamine, dicyclohexylamine, choline and caffeine.

[0071] In some embodiments, the salt is selected from hydrochloride, hydrobromide, trifluoroacetate, sulfate, phosphate, acetate, fumarate, maleate, tartrate, lactate, citrate, pyruvate, succinate, oxalate, methanesulfonate, p-toluenesulfonate, disulfate, benzenesulfonate, ethanesulfonate, malonate, xinafoate, ascorbate, oleate, nicotinate, saccharinate, adipate, formate, glycolate, palmitate, L-lactate, D-lactate, aspartate, malate, L-tartrate, D-tartrate, stearate, furoate (e.g., 2-furoate or 3-furoate), naphthalene disulfonate (naphthalene-1,5-disulfonate or naphthalene-1-(sulfonic acid)-5-sulfonate), ethane disulfonate ( The salts of the compounds include benzoate, adipate, ethanesulfonate, malonate, trimethylbenzenesulfonate (2-mesitylenesulfonate), naphthoate (2-naphthalenesulfonate), camphorate (camphor-10-sulfonate, such as (1S)-(+)-10-camphorsulfonate), glutamate, glutarate, hippurate (2-(benzamido)acetate), orotate, ditoluenesulfonate (p-xylene-2-sulfonate), and pamoate (2,2'-dihydroxy-1,1'-dinaphthomethane-3,3'-dicarboxylate).

[0072] A "sterile" preparation is sterile or free from all living microorganisms and their spores.

[0073] "Stereoisomers" refer to compounds that have the same chemical composition but differ in the arrangement of atoms or groups in space. Stereoisomers include diastereomers, enantiomers, conformers, etc.

[0074] "Chiral" refers to the property of a molecule that its mirror image counterpart is non-superimposable, whereas the term "achiral" refers to the molecule being superimposable onto its mirror image counterpart.

[0075] "Diastereomers" refers to stereoisomers with two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers have different physical properties, such as melting points, boiling points, spectral properties or biological activities. Mixtures of diastereomers can be separated by high resolution analytical procedures such as electrophoresis and chromatography (e.g., HPLC).

[0076] "Enantiomers" refers to two stereoisomers of a compound that are non-superimposable mirror images of one another.

[0077] Stereochemical definitions and conventions used herein generally follow SP 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. When describing optically active compounds, the prefixes D and L or R and S are used to indicate the absolute configuration of the molecule around its chiral center. The prefixes d and l or (+) and (-) are used to indicate the sign of the rotation of the compound to plane polarized light, where (-) or 1 indicates that the compound is left-handed. Compounds with the (+) or d prefix are right-handed. For a given chemical structure, these stereoisomers are identical except that they are mirror images of each other. Specific stereoisomers may also be referred to as enantiomers, and mixtures of such isomers are often referred to as enantiomeric mixtures. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which may occur without stereoselection or stereospecificity in a chemical reaction or process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two optically inactive enantiomeric species.

[0078] The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that interconvert via a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions via proton migration, such as keto-enol and imine-enamine isomerizations. Valence tautomers include interconversions by reorganization of some of the bonding electrons.

[0079] Certain compounds of the invention may exist in unsolvated forms as well as solvated forms, including hydrated forms. "Solvate" refers to an association or complex of one or more solvent molecules with a compound of the invention. Examples of solvents that form solvates include water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine. Certain compounds of the invention may exist in a variety of crystalline or amorphous forms. In general, all physical forms are intended to be included within the scope of the invention. The term "hydrate" refers to a complex in which the solvent molecule is water.

[0080] "Metabolite" refers to a product obtained by metabolism of a specified compound or a salt thereof in vivo. Such products may result from, for example, oxidation, reduction, hydrolysis, amidation, deamidation, esterification, deesterification, enzymatic cleavage, etc. of the administered compound.

[0081] Metabolites are usually identified by preparing a radioisotope (e.g., 14C or 3H) labeled compound of the invention; administering the radioisotope labeled compound of the invention to an animal, such as a rat, mouse, guinea pig, monkey or human, at a detectable dose (e.g., greater than about 0.5 mg / kg), thereby allowing sufficient time for metabolism to occur (usually about 30 seconds to 30 hours); and isolating its conversion products from urine, blood or other biological samples. Since these products are labeled, they are easily separated (by using antibodies that can bind to epitopes that survive in metabolites to separate other products). The structure of the metabolite is determined in a conventional manner, for example, by MS, LC / MS or NMR analysis. Typically, the analysis of metabolites is performed in the same manner as conventional drug metabolism studies well known to those skilled in the art. Metabolites can be used for diagnostic determination of therapeutic doses of the compounds of the invention as long as they are not found in vivo.

[0082] 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 cattle), sports 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 comprising administering a JAK inhibitor as described herein, or a pharmaceutically acceptable salt thereof, to a patient, the patient may be in need of the JAK inhibitor, or a pharmaceutically acceptable salt thereof.

[0083] The term "Janus kinase" refers to JAK1, JAK2, JAK3 and TYK2 protein kinases. In some embodiments, Janus kinases may be further defined as one of JAK1, JAK2, JAK3 or TYK2. In any embodiment, any of JAK1, JAK2, JAK3 and TYK2 may be specifically excluded as a Janus kinase. In some embodiments, Janus kinase is JAK1. In some embodiments, Janus kinase is a combination of JAK1 and JAK2.

[0084] 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 about, at most 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 of a reduction in activity (e.g., JAK1 activity) compared to normal, or a reduction in any range derivable therein.

[0085] "Therapeutically effective amount" refers to the amount of a compound of the present invention or a salt thereof (e.g., a pharmaceutically acceptable salt thereof) used for the following: (i) treating or preventing a specific disease, disorder or condition, or (ii) reducing, improving or eliminating one or more symptoms of a specific disease, disorder or condition, and optionally (iii) preventing or delaying the onset of one or more symptoms of a specific disease, disorder or condition described herein. In some embodiments, the therapeutically effective amount is an amount sufficient to reduce or alleviate the symptoms of an autoimmune disease or inflammatory disease (e.g., asthma). In some embodiments, the 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 a drug can reduce the number of cancer cells; reduce tumor size; inhibit (i.e., slow down and preferably stop to some extent) cancer cells from infiltrating surrounding organs; inhibit (i.e., slow down and preferably stop to some extent) tumor metastasis; inhibit tumor growth to some extent; or alleviate one or more symptoms associated with cancer to some extent. To the extent that a drug can prevent the growth of existing cancer cells or kill existing cancer cells, it may inhibit cell growth or be cytotoxic. For cancer therapy, for example, efficacy can be measured by assessing the time to disease progression (TTP) or determining the efficacy rate (RR).

[0086] "Treatment" (and variations thereof such as "treat" or "treating") refers to clinical intervention that attempts to alter the natural course of the individual or cell being treated, and can be performed for prevention or in the course of clinical pathology. Expected therapeutic effects include preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, stabilizing (i.e., not worsening) the condition, reducing the rate of disease progression, improving or alleviating the condition, prolonging survival (compared to the expected survival if not receiving treatment), and alleviating or improving prognosis. In some embodiments, the compounds of the invention or salts thereof (e.g., pharmaceutically acceptable salts thereof) are used to delay the development of a disease or disorder or slow the progression of a disease or disorder. Individuals in need of treatment include individuals already suffering from the disorder or disorder as well as those susceptible to the disorder or disorder (e.g., through genetic mutation) or those individuals in whom the disorder or disorder is to be prevented.

[0087] "Inflammatory disorder" refers to any disease, disorder or syndrome in which an excessive or uncontrolled inflammatory response results in excessive inflammatory symptoms, host tissue damage or loss of tissue function."Inflammatory disorder" also refers to a pathological state mediated by leukocyte influx or neutrophil chemotaxis.

[0088] "Inflammation" refers to a local protective response caused by tissue damage or injury, which has the effect of destroying, diluting or isolating (isolating) damaging factors and damaged tissues. Inflammation is significantly associated with leukocyte influx or neutrophil chemotaxis. Inflammation may be caused by pathogenic organisms and viral infections as well as non-infectious means, such as trauma or reperfusion after myocardial infarction or stroke, immune responses to exogenous antigens, and autoimmune responses. Therefore, inflammatory diseases suitable for treatment with the compounds of the present invention or salts thereof (e.g., pharmaceutically acceptable salts thereof) include diseases associated with specific defense system responses and diseases associated with non-specific defense system responses.

[0089] "Specific defense system" refers to the components of the immune system that respond to the presence of specific antigens. Examples of inflammation caused by specific defense system responses include classical responses to exogenous antigens, autoimmune diseases, and delayed hypersensitivity reactions mediated by T cells. Chronic inflammatory diseases, rejection of solid transplanted tissues and organs (e.g., kidney and bone marrow transplants), and graft-versus-host disease (GVHD) are further examples of inflammatory responses of the specific defense system.

[0090] The term "nonspecific defense system" refers to inflammatory conditions mediated by leukocytes (e.g., granulocytes and macrophages) that do not have immune memory. Examples of inflammation caused at least in part by the response of the nonspecific defense system include inflammation associated with the following conditions: such as adult (acute) respiratory distress syndrome (ARDS) or multiple organ injury syndrome; reperfusion injury; acute glomerulonephritis; reactive arthritis; skin diseases with acute inflammatory components; acute purulent meningitis or other central nervous system inflammatory conditions, such as stroke; thermal injury; inflammatory bowel disease; granulocyte transfusion-related syndrome; and cytokine-induced toxicity.

[0091] "Autoimmune disease" refers to any of a group of disorders in which tissue damage is associated with a humoral or cell-mediated response to the body's own components. Non-limiting examples of autoimmune diseases include rheumatoid arthritis, lupus, and multiple sclerosis.

[0092] As used herein, "allergic disease" refers to any symptom, tissue damage or loss of tissue function caused by allergy. As used herein, "arthritis" refers to a disease characterized by inflammatory damage to the joints attributable to a variety of causes. As used herein, "dermatitis" refers to any of a large family of skin diseases, characterized by skin inflammation attributable to a variety of causes. As used herein, "transplant rejection" refers to any immune response to transplanted tissue, such as an organ or cell (e.g., bone marrow), characterized by loss of function of the transplanted tissue and surrounding tissue, pain, swelling, leukocytosis and thrombocytopenia. The therapeutic methods of the present invention include methods for treating disorders associated with inflammatory cell activation.

[0093] "Inflammatory cell activation" refers to the induction of a proliferative cell response by stimulation (including but not limited to cytokines, antigens or autoantibodies), the production of soluble mediators (including but not limited to cytokines, oxygen free radicals, enzymes, prostaglandins or vasoactive amines), or cell surface expression of new or increased amounts 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 and eosinophils), mast cells, dendritic cells, Langerhans cells and endothelial cells). It will be appreciated by those skilled in the art that activation of one or more of these phenotypes in these cells can contribute to the initiation, maintenance or exacerbation of inflammatory disorders.

[0094] In some embodiments, inflammatory disorders that may 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 hypersensitivity.

[0095] The terms "cancer" and "cancerous," "tumor" and "neoplasm" and related terms refer to or describe the physiological condition in mammals that is typically characterized by uncontrolled cell growth. A "tumor" comprises one or more cancer cells. Examples of cancer include malignancies, blastomas, sarcomas, seminomas, glioblastomas, melanomas, leukemias, and myeloid or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinomas (e.g., epithelial squamous cell carcinomas) and lung cancers, including small cell lung cancer, non-small cell lung cancer ("NSCLC"), lung adenocarcinomas, and lung squamous cell carcinomas. Other cancers include skin cancer, keratoacanthoma, follicular carcinoma, hairy cell leukemia, buccal cancer, pharyngeal (oral) cancer, lip cancer, tongue cancer, oral 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, hepatocellular carcinoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, genitourinary tract cancer, biliary tract cancer, thyroid cancer, papillary cancer, liver cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, testicular cancer, vulvar cancer, peritoneal cancer, anal cancer, penis cancer, bone cancer, multiple myeloma, B-cell lymphoma, central nervous system cancer, brain cancer, head and neck cancer, Hodgkin's disease and related metastases. Examples of neoplastic disorders include myeloproliferative diseases such as polycythemia vera, essential thrombocythemia, myelofibrosis (such as primary myelofibrosis), and chronic myeloid leukemia (CML).

[0096] A "chemotherapeutic agent" is an agent used to treat a given condition, such as cancer or an inflammatory disease. Examples of chemotherapeutic agents are well known in the art and include, for example, those disclosed in U.S. Patent Application No. 2010 / 0048557, which is incorporated herein by reference. In addition, chemotherapeutic agents include pharmaceutically acceptable salts, acids or derivatives of any chemotherapeutic agent and combinations of two or more thereof.

[0097] "Package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications, and warnings concerning the use of such therapeutic products.

[0098] Unless otherwise stated, structures depicted herein include compounds that differ only in the presence of one or more isotopically enriched atoms. Exemplary isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, and iodine, such as 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 I. Isotope-labeled compounds (e.g., 3 H and 14 C-labeled compounds) can be used in compound and / or substrate tissue distribution assays. Tritiated (i.e. 3 H) and carbon-14 (i.e. 14 C) because they are easy to prepare and detect. In addition, heavier isotopes such as deuterium (i.e. 2 H) substitution may provide certain therapeutic advantages by virtue of its greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements). In some embodiments, one or more hydrogen atoms are replaced by 2 H or 3 H, or one or more carbon atoms are replaced by 13 C or 14 C substitution. Positron-emitting isotopes such as 15 O. 13 N. 11 C and 18F can be used in positron emission tomography (PET) studies to examine substrate receptor occupancy. Isotopically labeled compounds can generally be prepared by following procedures analogous to those disclosed in the Schemes or Examples herein, by substituting an isotopically labeled reagent for a non-isotopically labeled reagent.

[0099] It is specifically contemplated that any limitation discussed with respect to one embodiment of the present invention may be applicable to any other embodiment of the present invention. In addition, any compound of the present invention or its salt (e.g., its pharmaceutically acceptable salt) may be used in any method of the present invention, and any method of the present invention may be used to produce or utilize any compound of the present invention or its salt (e.g., its pharmaceutically acceptable salt).

[0100] Unless explicitly stated to refer to only alternatives or the alternatives are mutually exclusive, use of the term "or" means "and / or," although the present invention supports definitions referring to only alternatives and "and / or."

[0101]

[0043] The term "about" is used throughout this application to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0102] Unless expressly stated otherwise, as used herein, "a" or "an" means one or more. As used herein, "another" means at least a second or more.

[0103] The headings used herein are for organizational purposes only.

[0104] Inhibitors of Janus kinase

[0105] One embodiment provides a compound of formula (I):

[0106]

[0107] or a stereoisomer or a pharmaceutically acceptable salt thereof,

[0108] in:

[0109] R 1 Yes: Hydroxy-C1-C6 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, wherein the C 3-6 The cycloalkyl part is R d Replace once;

[0110] R 2 is: halo; halo C1-C6 alkoxy; C1-C6 alkylthio; -SF2; or C3-C6 cycloalkyl;

[0111] R 3 is: hydrogen; or C1-C6 alkyl;

[0112] R 4 is: hydrogen; or C1-C6 alkyl;

[0113] R 5 is: hydrogen; or C1-C6 alkyl;

[0114] R 6 is: hydrogen; or C1-C6 alkyl;

[0115] or R 2 and R 6 Together with the atoms to which it is attached, it can form a six-membered ring containing two heteroatoms each independently selected from O, N and S;

[0116] m is 0 to 2;

[0117] n is 0 to 3;

[0118] Each R a1 are independently: hydrogen; or C1-C6 alkyl;

[0119] Each R a2 are independently: hydrogen; halo; or C1-C6 alkyl;

[0120] R b is: hydrogen; or C1-C6 alkyl;

[0121] R c is: hydrogen; C1-C6 alkyl; an amino protecting group; or azetidinyl, which may be unsubstituted or substituted once with C1-C6 alkyl;

[0122] het 1 is a heterocyclic group selected from the following: azetidinyl; pyrrolidinyl; piperazinyl; piperidinyl; morpholinyl; and oxetanyl; each of which may be unsubstituted or substituted with R d Replace once and pass R g Replace once or twice;

[0123] R d Yes:-(CR a1 R a2 ) p -het 2 ;-(CR a1 R a2 ) q -NRe R f ; or -(CR a1 R a2 ) p -C 3-6 Cycloalkyl, wherein the C 3-6 The cycloalkyl part is -NR e R f Replace once;

[0124] p is 0 to 2;

[0125] q is 0 to 4;

[0126] R e is: hydrogen; or C1-C6 alkyl;

[0127] R f is: hydrogen; C1-C6 alkyl; or -CH2C2N(CH3)2;

[0128] Each R g is: C1-C6 alkyl; or halogenated; and

[0129] Het 2 is a heterocycle selected from the group consisting of tetrahydropyran; azetidinyl; and pyrrolidinyl; each of which may be unsubstituted or substituted with C1-C6 alkyl or -NR e R f Alkyl substituted once.

[0130] In certain embodiments, R 1 Yes: C1-C6 alkyl; hydroxy-C1-C6 alkyl; -(CR a1 R a2 ) m -het 1 ; or (CR a1 R a2 ) n -NR b R c , where 1 May be unsubstituted or substituted with R d Replace once.

[0131] In certain embodiments, R 1 Yes: C1-C6 alkyl; -(CR a1 R a2 ) m -het 1 ; or -(CHR a ) n -NR b R c , where 1

[0132] In certain embodiments, R 1 Yes:-(CR a1 R a2 ) m -het 1 ; or -(CR a1 R a2 ) n -NR b R c , where 1 May be unsubstituted or substituted with R d Replace once.

[0133] In certain embodiments, R 1 Yes-(CHR a ) m -het 1 , where 1 May be unsubstituted or d Replace once.

[0134] In certain embodiments, R 1 Yes-(CR a1 R a2 ) n -NR b R c .

[0135] In certain embodiments, R 2 It is: halo; halo C1-C6 alkoxy; or C1-C6 alkylthio.

[0136] In certain embodiments, R 2 It is halogenated.

[0137] In certain embodiments, R 2 It is a halogenated C1-C6 alkoxy group.

[0138] In certain embodiments, R 2 It is C1-C6 alkylthio.

[0139] In certain embodiments, R 2 is: chloro; difluoromethoxy; methylethio; or cyclopropyl.

[0140] In certain embodiments, R 2 It's chlorine.

[0141] In certain embodiments, R 2 It is difluoromethoxy.

[0142] In certain embodiments, R 2 It's methyl ethyl sulfide.

[0143] In certain embodiments, R3 It's hydrogen.

[0144] In certain embodiments, R 4 It's hydrogen.

[0145] In certain embodiments, R 5 It's hydrogen.

[0146] In certain embodiments, R 6 It's hydrogen.

[0147] In certain embodiments, R 2 and R 6 Together with the atoms to which they are attached, they form a six-membered ring containing two heteroatoms each independently selected from O, N and S.

[0148] In certain embodiments, m is 0. In the case where m is 0 and R 1 Yes 1 In the embodiment of 1 The bond to the tetrazole ring is het 1 It is composed of carbon atoms rather than heteroatoms.

[0149] In certain embodiments, m is 0.

[0150] In certain embodiments, m is 1.

[0151] In some embodiments, m is 2.

[0152] In certain embodiments, n is 0.

[0153] In certain embodiments, n is 1.

[0154] In some embodiments, n is 2.

[0155] In certain embodiments, R a1 It's hydrogen.

[0156] In certain embodiments, R a2 It's hydrogen.

[0157] In certain embodiments, R b It's hydrogen.

[0158] In certain embodiments, R b It is a C1-C6 alkyl group.

[0159] In certain embodiments, R c It's hydrogen.

[0160] In certain embodiments, R c It is a C1-C6 alkyl group.

[0161] In certain embodiments, Rc It is 1-methyl-azetidin-3-yl.

[0162] In certain embodiments, 1 is an azetidinyl group which may be unsubstituted or substituted with R d Replace once.

[0163] In certain embodiments, 1 is a pyrrolidinyl group, which may be unsubstituted or substituted with R d Replace once.

[0164] In certain embodiments, 1 is piperazinyl, which may be unsubstituted or substituted by R d Replace once.

[0165] In certain embodiments, 1 is piperidinyl, which may be unsubstituted or substituted with R d Replace once.

[0166] In certain embodiments, 1 It is a morpholinyl group.

[0167] In certain embodiments, 1 It is an oxetane group.

[0168] In certain embodiments, p is 0.

[0169] In certain embodiments, p is 1.

[0170] In some embodiments, p is 2.

[0171] In certain embodiments, q is 0.

[0172] In some embodiments, q is 2.

[0173] In some embodiments, q is 3.

[0174] In some embodiments, q is 4.

[0175] In certain embodiments, R e It's hydrogen.

[0176] In certain embodiments, R e It is a C1-C6 alkyl group.

[0177] In certain embodiments, R f It's hydrogen.

[0178] In certain embodiments, R f It is a C1-C6 alkyl group.

[0179] In certain embodiments, Het2 It is tetrahydropyranyl.

[0180] In certain embodiments, Het 2 is azetidinyl, which may be unsubstituted or substituted once with C1-C6 alkyl.

[0181] In certain embodiments, Het 2 is a pyrrolidinyl group which may be unsubstituted or substituted once with a C1-C6 alkyl group.

[0182] In certain embodiments, R 1 Selected from:

[0183] as well as

[0184] In certain embodiments, R 1 Selected from:

[0185] as well as

[0186] In certain embodiments, the target compound is a compound of formula (II)

[0187]

[0188] or a stereoisomer or a pharmaceutically acceptable salt thereof, wherein R 1 , R 2 and R 6 As defined herein.

[0189] In certain embodiments, the target compound is a compound of formula (III)

[0190]

[0191] or a stereoisomer or a pharmaceutically acceptable salt thereof, wherein R 1 , R 2 and R 6 As defined herein.

[0192] In certain embodiments, the target compound is a compound of formula (IV)

[0193]

[0194] or a stereoisomer or a pharmaceutically acceptable salt thereof,

[0195] Where X is -O- or -S-, R g is hydrogen or C1-C6 alkyl, and R 1 As defined herein.

[0196] In certain embodiments, the target compound is a compound of formula (V)

[0197]

[0198] or a stereoisomer or a pharmaceutically acceptable salt thereof,

[0199] Where R 2 and R d As defined herein.

[0200] In certain embodiments, the target compound is a compound of formula (VI)

[0201]

[0202] or a stereoisomer or a pharmaceutically acceptable salt thereof,

[0203] Where R 2 and R d As defined herein.

[0204] Also provided is a pharmaceutical composition comprising the JAK inhibitor described herein or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent or excipient.

[0205] Also provided is the use of the JAK inhibitors described herein or pharmaceutically acceptable salts thereof in treatment, such as in the treatment of inflammatory diseases (e.g., asthma). Also provided is the use of the JAK inhibitors described herein or pharmaceutically acceptable salts thereof in the preparation of a medicament for treating inflammatory diseases. Also provided is a method for preventing, treating or reducing the severity of a disease or condition responsive to inhibition of Janus kinase activity in a patient, the method comprising administering to the patient a therapeutically effective amount of a JAK inhibitor as described herein or pharmaceutically acceptable salts thereof.

[0206] In one embodiment, the disease or condition treated is cancer, polycythemia vera, essential thrombocythemia, myelofibrosis, chronic myeloid leukemia (CML), rheumatoid arthritis, inflammatory bowel syndrome, Crohn's disease, psoriasis, contact dermatitis, or delayed hypersensitivity.

[0207] In one embodiment, provided is a use of the JAK inhibitor described herein or a pharmaceutically acceptable salt thereof in the treatment of cancer, polycythemia vera, essential thrombocythemia, myelofibrosis, chronic myeloid leukemia (CML), rheumatoid arthritis, inflammatory bowel syndrome, Crohn's disease, psoriasis, contact dermatitis or delayed hypersensitivity.

[0208] In one embodiment, a composition is provided that is formulated for administration by inhalation.

[0209] In one embodiment, a metered dose inhaler is provided, the metered dose inhaler comprising a compound of the present invention or a pharmaceutically acceptable salt thereof.

[0210] In one embodiment, a JAK inhibitor described herein, or a pharmaceutically acceptable salt thereof, is at least ten times more potent as a JAK1 inhibitor than as a LRRK2 inhibitor.

[0211] In one embodiment, a method for treating hair loss in a mammal is provided, the method comprising administering the JAK inhibitor described herein or a pharmaceutically acceptable salt thereof to the mammal.

[0212] In one embodiment, provided is a use of the JAK inhibitor described herein or a pharmaceutically acceptable salt thereof for treating hair loss.

[0213] In one embodiment, provided is a use of the JAK inhibitor described herein or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating hair loss in a mammal.

[0214] The compounds of the present invention may contain one or more asymmetric carbon atoms. Therefore, the compounds may exist in the form of diastereomers, enantiomers or mixtures thereof. The synthesis of 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 chromatography or crystallization. Similarly, the same technology or other technologies known in the art may be used to separate or enrich enantiomeric mixtures. Each asymmetric carbon or nitrogen atom may be in R or S configuration, and both configurations are within the scope of the present invention.

[0215] In the structures shown herein, where the stereochemistry of any particular chiral atom is not specified, all stereoisomers are contemplated and included as compounds of the present invention. Where stereochemistry is specified by a solid wedge or dashed line representing a specific configuration, that stereoisomer should be specified and defined. Unless otherwise indicated, relative stereochemistry is indicated when a solid wedge or dashed line is used.

[0216] Another aspect includes prodrugs of the compounds described herein, including known amino protecting groups and carboxyl protecting groups, which are released (eg, hydrolyzed) under physiological conditions to produce the compounds of the invention.

[0217] The term "prodrug" refers to a precursor or derivative form of a pharmaceutically active substance that is less effective in patients than the parent drug and can be activated or converted to a more active parent form by enzymatic or hydrolytic lysis. 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., 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.

[0218] A particular class of prodrugs is one in which the nitrogen atom in an amino, amidino, aminoalkyleneamino, iminoalkyleneamino or guanidino group is substituted with a hydroxyl group, an alkylcarbonyl (-CO-R) group, an alkoxycarbonyl (-CO-OR) or an acyloxyalkyl-alkoxycarbonyl (-CO-ORO-CO-R) group, wherein R is a monovalent or divalent group, for example, an alkyl, alkylene or aryl group or a group having the formula -C(O)-O-CP1P2-haloalkyl, wherein 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 the compound with an activated group such as an acyl group, for example, to bond the nitrogen atom in the compound to an exemplary carbonyl group of an activated acyl group. Examples of activated carbonyl compounds are those containing a leaving group bonded to the carbonyl group, and include, for example, acyl halides, amides, acyl pyridinium salts, acyl alcoholates, acyl phenolates such as p-nitrophenoxy acyl, dinitrophenoxy acyl, fluorophenoxy acyl, and difluorophenoxy acyl. The reaction is typically carried out in an inert solvent at a reduced temperature such as -78°C to about 50°C. The reaction can also be carried out in the presence of an inorganic base (e.g., potassium carbonate or sodium bicarbonate) or an organic base (such as an amine, including pyridine, trimethylamine, triethylamine, triethanolamine, etc.).

[0219] Other types of prodrugs are also contemplated. For example, the free carboxyl groups of the JAK inhibitors described herein can be derived as amides or alkyl esters. As another example, the compounds of the present invention containing free hydroxyl groups can be derived as prodrugs by converting the hydroxyl groups into groups such as, but not limited to, phosphates, hemisuccinates, dimethylaminoacetates or phosphoryloxymethyloxycarbonyl groups, as described in Fleisher, D. et al., (1996) Improved oral drug delivery: solubility limitations overcome by the use of prodrugs Advanced Drug Delivery Reviews, 19: 115. Also included are carbamate prodrugs of hydroxyl and amino groups, such as carbonate prodrugs, sulfonates and sulfates of hydroxyl groups. Also contemplated are hydroxyl groups derived as (acyloxy) methyl and (acyloxy) ethyl ethers, wherein the acyl groups can be alkyl esters optionally substituted by some groups, including but not limited to ethers, amines and carboxylic acid functional groups, or wherein the acyl groups are amino acid esters as described above. Such prodrugs are described in J. Med. Chem., (1996), 39:10. More specific examples include substitution of the hydrogen atom of the alcohol group with a group such as: (C 1- C6) alkanoyloxymethyl, 1-((C 1- C6) alkanoyloxy)ethyl, 1-methyl-1-((C 1- C6) alkanoyloxy)ethyl, (C 1- C6) alkoxycarbonyloxymethyl, N-(C 1- C6) alkoxycarbonylaminomethyl, succinyl, (C 1- C6) alkanoyl, α-amino (C 1- C4 alkanoyl, aryl and α-aminoacyl or α-aminoacyl-α-aminoacyl, wherein each α-aminoacyl is independently selected from natural L-amino acids, P(O)(OH)2, -P(O)(O(C 1- C6) alkyl)2 or glycosyl (free radical generated by removal of hydroxyl group in the hemiacetal form of carbohydrate).

[0220] "Leaving group" refers to a part of the first reactant in a chemical reaction, which is displaced from the first reactant in the chemical reaction. Examples of leaving groups include, but are not limited to, halogen atoms, alkoxy groups, and sulfonyloxy groups. Examples of sulfonyloxy groups include, but are not limited to, alkylsulfonyloxy groups (e.g., methylsulfonyloxy (mesylate) and trifluoromethylsulfonyloxy (trifluoroformate)) and arylsulfonyloxy groups (e.g., p-toluenesulfonyloxy (toluenesulfonate) and p-nitrosulfonyloxy (nitrobenzenesulfonic acid)).

[0221] Synthesis of Janus kinase inhibitor compounds

[0222] Compounds can be synthesized by synthetic pathways as described herein. In certain embodiments, in addition to or according to the descriptions contained herein, methods known in the chemical field can be used. Starting materials are generally available from commercial sources, such as Aldrich Chemicals (Milwaukee, Wis.), or are easily prepared using methods known to those skilled in the art (e.g., prepared by the following generally described methods: Louis F. Fieser and Mary Fieser, Reagents for Organic Synthesis, v. 1-19, Wiley, NY (1967-1999 edition), Beilsteins Handbuch der organischen Chemie, 4, Aufl. Edited Springer-Verlag, Berlin, including supplements (also available through the Beilstein online database)), or Comprehensive Heterocyclic Chemistry, edited by Katrizky and Rees, Pergamon Press, 1984.

[0223] Compounds can be prepared individually or as a library of compounds comprising at least 2, e.g., 5 to 1,000 compounds or 10 to 100 compounds. Libraries of compounds can be prepared by methods known to those skilled in the art, by a combinatorial "split and mix" approach or by a variety of parallel synthesis methods using solution phase or solid phase chemistry. Therefore, according to another aspect of the present invention, a library of compounds comprising at least two compounds of the present invention is provided.

[0224] For exemplary purposes, the reaction scheme described below provides a route for synthesizing compounds of the present invention and key intermediates. For detailed description of each reaction step, see the example section below. It will be appreciated by those skilled in the art that other synthetic routes can be used. Although some specific raw materials and reagents are shown and described below in the scheme, other raw materials and reagents can be replaced to provide various derivatives or reaction conditions. In addition, according to the present invention, conventional chemical methods known to those skilled in the art can be used to further modify many compounds prepared by the method described below.

[0225] When preparing the compounds of the present invention, it may be necessary to protect the distal functionality (e.g., primary amine or secondary amine) of the intermediate. The demand for such protection will vary according to the properties of the distal functionality and the conditions of the preparation method. Suitable amino protecting groups include acetyl, trifluoroacetyl, benzyl, benzylsulfonyl, tert-butyloxycarbonyl (BOC), benzyloxycarbonyl (Cbz) and 9-fluorenylmethyleneoxycarbonyl (Fmoc). Whether such protection is needed is easily determined by those skilled in the art. For conventional descriptions of protecting groups and their uses, see TW Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 1991.

[0226] Other transformations commonly used in the synthesis of compounds of the invention may be performed using a variety of reagents and conditions, including the following:

[0227] (1) Reaction of carboxylic acids with amines to form amides. Various reagents known to those skilled in the art can be used to achieve this transformation, but a review can be found in Tetrahedron, 2005, 61, 10827-10852.

[0228] (2) The reaction of primary or secondary amines with aryl halides or quasi-halides (e.g., trifluoromethanesulfonates) can be achieved using a variety of catalysts, ligands, and bases, often referred to as "Buchwald-Hartwig cross-coupling." A review of these methods is provided in Comprehensive Organic Name Reactions and Reagents, 2010, 575-581.

[0229] (3) Palladium cross-coupling reaction between aryl halides and vinyl boronic acids or boronic esters. This transformation is a type of "Suzuki-Miyaura cross-coupling", and this type of reaction has been described in detail in Chemical Reviews, 1995, 95(7), 2457-2483.

[0230] (4) The hydrolysis of esters to form the corresponding carboxylic acids is well known to those skilled in the art, and the conditions include: for methyl and ethyl esters, using a strong base aqueous solution, such as lithium hydroxide, sodium hydroxide or potassium hydroxide, or a strong inorganic acid aqueous solution, such as HCl; for tert-butyl esters, using an acid (e.g., HCl in dioxane or trifluoroacetic acid (TFA) in dichloromethane (DCM)) for hydrolysis.

[0231] Reaction Scheme 1

[0232]

[0233] Reaction Scheme 1 shows the synthesis of compounds of the present invention. Compound 1 can be arylated under palladium catalysis to produce compound 2. The nitro group of compound 2 can be reduced under conditions such as iron and ammonium chloride to produce aminoaniline 3. Coupling of the amide bond with commercially available pyrazolo[1,5-a]pyrimidine-3-carboxylic acid in the presence of a coupling reagent such as but not limited to PyAOP with an organic base such as but not limited to DIPEA and DMAP in an organic solvent such as but not limited to DMF provides compound 4. The SEM protecting group of compound 4 is removed using an acid such as but not limited to HCl in a solvent such as but not limited to 1,4-dioxane to give compound 5. Compound 5 can then be N-alkylated with a protected tetrazole compound. In certain embodiments, the protecting group PG can be tetrahydropyranyl, such that the tetrazole reagent is 5-(chloromethyl)-2-(tetrahydro-2H-pyran-2-yl)-2H-tetrazole to give compound 6. Deprotection using HCl or other acids gives tetrazole compound 7. Compound 7 was then reacted with R 1 -X (wherein X is halogenated, such as iodinated) to carry out N-alkylation to provide compounds 8 and 9 of formula (I) according to the present invention.

[0234] Reaction Scheme 2

[0235]

[0236] Reaction Scheme 2 shows the synthesis of compounds 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 accomplished by treating compound 12 with diethyl (bromodifluoromethyl)phosphonate with a base such as but not limited to aqueous potassium hydroxide in a solvent such as but not limited to acetonitrile. Compound 13 can be treated with 4-nitro-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazole 4-bromo-1-(difluoromethoxy)-2-iodobenzene under palladium catalysis with a base such as but not limited to potassium carbonate in a solvent such as but not limited to DMA to produce compound 14. The nitro group of compound 14 can be reduced under conditions such as iron and ammonium chloride to produce aminopyrazole 15. Coupling of the amide bond of compound 15 with commercially available pyrazolo[1,5-a]pyrimidine-3-carboxylic acid in the presence of a coupling reagent such as but not limited to PyAOP with an organic base such as but not limited to DIPEA and DMAP in a solvent such as but not limited to DMF 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.

[0237] It should be understood that compounds of various formulae or any intermediates used in their preparation may be further derivatized by one or more standard synthetic methods employing condensation, substitution, oxidation, reduction or cleavage reactions, where appropriate functional groups are present. Specific substitution methods include conventional alkylation, arylation, heteroarylation, acylation, sulfonylation, halogenation, nitration, formylation and coupling procedures.

[0238] In another example, a primary or secondary amine group can be converted to an amide group (-NHCOR' or -NRCOR') by acylation. Acylation can be achieved by reaction with an appropriate acyl chloride in the presence of a base such as triethylamine in an appropriate solvent such as dichloromethane, or with an appropriate carboxylic acid in the presence of an appropriate coupling agent such as HATU (O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate). Similarly, an amine group can be converted to a sulfonamide group (-NHS02R' or -NR"S02R') by reaction with an appropriate sulfonyl chloride in the presence of a suitable base such as triethylamine in a suitable solvent such as dichloromethane. A primary or secondary amine group can be converted to a urea group (-NHCONR'R" or -NRCONR'R") by reaction with an appropriate isocyanate in the presence of a suitable base such as triethylamine in a suitable solvent such as dichloromethane.

[0239] Amines (NH2) can be obtained by reducing nitro (-NO2) groups, for example, by catalytic hydrogenation, for example, using hydrogen, in the presence of a metal catalyst (e.g., palladium), in a solvent (such as ethyl acetate or an alcohol, for example, methanol) on a support (such as carbon). Alternatively, the conversion can be carried out by chemical reduction using, for example, a metal (e.g., tin or iron) in the presence of an acid (such as hydrochloric acid).

[0240] In another example, the amine (—CH2NH2) group can be obtained by reducing the nitrile (—CN), for example, by catalytic hydrogenation, for example, using hydrogen, in the presence of a metal catalyst, such as 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 a suitable temperature, for example, from about -78°C to the reflux temperature of the solvent.

[0241] In another example, an amine (-NH2) group can be obtained by conversion of a carboxylic acid group (-CO2H) to the corresponding acyl azide (-CON3), Curtius rearrangement, and hydrolysis of the resulting isocyanate (-N=C=O).

[0242] An aldehyde group (-CHO) can be converted to an amine group (-CH2NR'R"') by a reductive amination reaction of an amine and a borohydride (e.g., sodium triacetoxyborohydride or sodium cyanoborohydride) in a solvent such as a halogenated hydrocarbon (e.g., dichloromethane), or an alcohol such as ethanol, where necessary in the presence of an acid (such as acetic acid) at near ambient temperature.

[0243] In another example, an aldehyde group can be converted to an alkenyl group (-CH=CHR') by using a Wittig or Wadsworth-Emmons reaction using an appropriate phosphane or phosphonate under standard conditions known to those skilled in the art.

[0244] The aldehyde group can be obtained by reducing an ester group (such as -CO2Et) or a nitrile (-CN) using diisobutylaluminum hydride in a suitable solvent (such as toluene). Alternatively, the aldehyde group can be obtained by oxidation of an alcohol group using any suitable oxidizing agent known to those skilled in the art.

[0245] The ester group (-CO2R') can be converted into the corresponding acid group (-CO2H) by acid-catalyzed hydrolysis or base-catalyzed hydrolysis, depending on the nature of R. If R is tert-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.

[0246] The carboxylic acid group (-CO2H) can be converted to an amide (CONHR' or -CONR'R") by reaction with an appropriate amine in the presence of a suitable coupling agent such as HATU in a suitable solvent such as dichloromethane.

[0247] In another example, a carboxylic acid can be homologated through one carbon (ie, -CO2H to -CH2CO2H) by conversion to the corresponding acid chloride (-COCl) followed by Arndt-Eistert synthesis.

[0248] In another example, the -OH group can be generated by reduction of the corresponding ester (e.g., -CO2R') or aldehyde (-CHO), 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. Alternatively, the alcohol can be prepared by reduction of the corresponding acid (-CO2H) using, for example, lithium aluminum hydride in a solvent such as tetrahydrofuran, or borane in a solvent such as tetrahydrofuran.

[0249] Conditions well known to persons skilled in the art can be used to convert alcohol groups into leaving groups (such as halogen atoms) or sulfonyloxy groups (such as alkylsulfonyloxy), for example, trifluoromethylsulfonyloxy or arylsulfonyloxy, for example, p-toluenesulfonyloxy groups. For example, alcohol and thionyl chloride are reacted in halogenated hydrocarbons (for example, methylene chloride) to produce corresponding chlorides. Bases (for example, triethylamine) can also be used in the reaction.

[0250] In another example, the alcohol, phenol or amide group can be alkylated by coupling the phenol or amide with the alcohol in the presence of a phosphine (e.g., triphenylphosphine) and an activator (such as diethyl azodicarboxylate, diisopropyl azodicarboxylate or dimethyl azodicarboxylate) in a solvent (such as tetrahydrofuran). Alternatively, alkylation can be achieved by deprotonation using an appropriate base (e.g., sodium hydride) followed by the addition of an alkylating agent (such as an alkyl halide).

[0251] Aromatic halogen substituents in compounds can be treated with a base, for example, in a lithium base such as n-butyllithium or tert-butyllithium, optionally at low temperatures (e.g., about -78°C), halogen metal exchange in a solvent such as tetrahydrofuran, and then quenched with an electrophilic reagent to introduce the desired substituent. Thus, for example, a formyl group can be introduced by using N,N-dimethylformamide as an electrophilic reagent. Aromatic halogen substituents can also be subjected to metal (e.g., palladium or copper) catalyzed reactions to introduce, for example, acids, esters, cyano groups, amides, aryls, heteroaryls, alkenyls, alkynyls, thio substituents or amino substituents. Suitable procedures that can be used include those described by Heck, Suzuki, Stille, Buchwald or Hartwig.

[0252] Aromatic halogen substituents may also undergo nucleophilic substitution following reaction with an appropriate nucleophile such as, for example, an amine or an alcohol. Advantageously, this reaction may be carried out at elevated temperature in the presence of microwave irradiation.

[0253] Separation methods

[0254] In each exemplary scheme, it may be advantageous to separate the reaction products from each other or from the raw materials. The desired product of each step or series of steps is separated or purified (hereinafter after separation) to the desired uniformity by techniques common in the art. Typically such separations involve multiphase extraction, crystallization or trituration from a solvent or solvent mixture, distillation, sublimation or chromatography. Chromatography may involve any method, including, for example: reverse phase and normal phase chromatography; size exclusion chromatography; ion exchange chromatography; supercritical fluid chromatography; high pressure, medium pressure and low pressure liquid chromatography methods and apparatus; small scale analytical chromatography; simulated moving bed (SMB) and preparative thin layer or thick layer chromatography, as well as small scale thin layer and flash chromatography techniques.

[0255] Another class of separation methods involves treating the mixture with a reagent selected to bind to or otherwise render the desired product, unreacted starting material, reaction byproduct, etc. separable. Such reagents include adsorbents or absorbents such as activated carbon, molecular sieves, ion exchange media, etc. Alternatively, the reagent may be an acid (in the case of a basic material); a base (in the case of an acidic material); a binding agent such as an antibody, a binding protein, a selective chelating agent such as a crown ether; a liquid / liquid ion extraction reagent (LIX), etc.

[0256] The choice of an appropriate separation method depends on the nature of the materials involved. Exemplary 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 the material in acidic and basic media (in multiphase extraction), etc. One skilled in the art will apply the technique most likely to achieve the desired separation.

[0257] A mixture of diastereomers can be separated into its individual diastereomers based on the physicochemical differences of the diastereomers by methods well known to those skilled in the art, such as by chromatography and / or fractional crystallization. Enantiomers can be separated by converting the mixture of enantiomers into a mixture of diastereomers by reacting the mixture of enantiomers with an appropriate optically active compound (e.g., a chiral auxiliary such as a chiral alcohol or Mosher's acid chloride), separating the diastereomers, and converting (e.g., hydrolyzing) the individual diastereomers to the corresponding pure enantiomers. In addition, some of the compounds of the invention may be atropisomers (e.g., substituted biaryl compounds) and are considered part of the invention. Enantiomers can also be separated using chiral HPLC columns or supercritical fluid chromatography.

[0258] Single stereoisomers, such as enantiomers substantially free of their stereoisomers, can be obtained by resolving racemic mixtures using methods such as diastereoisomer formation using optically active resolving agents (Eliel, E. and Wilen, S., Stereochemistry of Organic Compounds, John Wiley & Sons, Inc., New York, 1994; Lochmuller, CH, J. Chromatogr., 113(3):283-302 (1975)). Racemic mixtures of chiral compounds of the invention can be separated and isolated by any suitable method, including: (1) forming ionic diastereomeric salts with chiral compounds and separating by fractional crystallization or other methods; (2) forming diastereomeric compounds with chiral derivatizing agents, separating the diastereoisomers, and converting to pure stereoisomers; and (3) direct separation of substantially pure or enriched stereoisomers under chiral conditions. See: Drug Stereochemistry, Analytical Methods and Pharmacology, Irving W. Wainer, editor, Marcel Dekker, Inc., New York (1993).

[0259] Diastereomeric salts can be formed by reacting enantiomerically pure chiral bases such as brucine, quinine, ephedrine, strychnine, α-methyl-β-phenylethylamine (amphetamine), etc. with asymmetric compounds bearing acidic functional groups such as carboxylic acids and sulfonic acids. The separation of diastereomeric salts can be induced by fractional crystallization or ion chromatography. For the separation of optical isomers of amino compounds, the addition of chiral carboxylic or sulfonic acids such as camphorsulfonic acid, tartaric acid, mandelic acid, or lactic acid can cause the formation of diastereomeric salts.

[0260] 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 an asymmetric compound with an enantiomerically pure chiral derivatizing reagent such as a menthyl derivative, followed by separation of the diastereomers and hydrolysis to obtain a pure or enriched enantiomer. Methods for determining optical purity involve preparing a chiral ester of the racemic mixture, such as a menthyl ester, for example, (-) menthyl chloroformate, or Mosher's ester, α-methoxy-α-(trifluoromethyl)phenyl acetate (Jacob, J. Org. Chem. 47:4165 (1982)), in the presence of a base, and analyzing the NMR spectrum for the presence of two atropisomeric enantiomers or diastereomers. Stable diastereomers of atropisomeric compounds can be separated and isolated by normal and reverse phase chromatography following methods for the separation of atropisomeric naphthyl-isoquinolines (WO 96 / 15111, incorporated herein by reference). By method (3), a racemic mixture of two enantiomers can be separated by chromatography using a chiral stationary phase (Chiral Liquid Chromatography WJ Lough, Ed., Chapman and Hall, New York, (1989); Okamoto, J. of Chromatogr. 513: 375-378 (1990)). The enriched or purified enantiomer can be distinguished by methods used to distinguish other chiral molecules with asymmetric carbon atoms (such as optical rotation or circular dichroism). The absolute stereochemistry of chiral centers and enantiomers can be determined by X-ray crystallography.

[0261] Positional isomers and intermediates used in 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, the E and Z isomers can be separated, for example, by preparative HPLC.

[0262] Pharmaceutical compositions and administration

[0263] The compounds to which the present invention relates are JAK kinase inhibitors, such as JAK1 inhibitors, and are useful in the treatment of several diseases, for example inflammatory diseases such as asthma.

[0264] Therefore, another embodiment provides a pharmaceutical composition or drug containing a compound of the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent or excipient, and a method of preparing such a composition and drug using the compound of the present invention.

[0265] In one example, the compound of the present invention or its pharmaceutically acceptable salt can be formulated as a galenical administration form by mixing with a physiologically acceptable carrier (i.e., a carrier that is non-toxic to the recipient at the dosage and concentration used) at an appropriate pH value and desired purity at ambient temperature. The pH value of the preparation depends primarily on the specific use and concentration of the compound, but is generally in the range of about 3 to about 8. In one example, the compound of the present invention or its pharmaceutically acceptable salt is formulated in an acetate buffer at pH 5. In another embodiment, the compound of the present invention is sterile. The compound can be stored, for example, as a solid or amorphous composition, as a lyophilized preparation, or as an aqueous solution.

[0266] The compositions are formulated, dosed, and administered in a manner consistent with good medical practice. Factors to be considered in this context 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 timing of administration, and other factors known to practitioners.

[0267] It should be understood that the specific dosage level of any particular patient will depend on a variety of factors, including the activity of the specific compound used, age, body weight, general health, sex, diet, administration time, administration route, excretion rate, drug combination and the severity of the specific disease being treated. The optimized dosage level and administration frequency will be determined by clinical trials, as required in the pharmaceutical field. Usually, the daily dosage range for oral administration will be in the following range: about 0.001 mg to about 100 mg per kg of human body weight, usually 0.01 mg to about 50 mg per kg of human body weight, such as 0.1 to 10 mg per kg of human body weight, in a single dose or divided dose. In general, the daily dosage range for inhalation administration will be in the following range: about 0.1 μg to about 1 mg per kg of human body weight, preferably 0.1 μg to 50 μg per kg of human body weight, in a single dose or divided dose. On the other hand, it may be necessary to use dosages exceeding these limits in some cases.

[0268] The compounds of the present invention or pharmaceutically acceptable salts thereof 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 necessary for local treatment) intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. In some embodiments, inhalation is used.

[0269] The compounds of the present invention or pharmaceutically acceptable salts thereof can be administered in any convenient administration form, such as tablets, powders, capsules, lozenges, granules, solutions, dispersions, suspensions, syrups, sprays, vapors, suppositories, gels, emulsions, patches, etc. Such compositions may contain conventional components in pharmaceutical preparations, such as diluents (e.g., glucose, lactose or mannitol), carriers, pH modifiers, buffers, sweeteners, fillers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifiers, glidants, processing aids, colorants, aromatics, flavoring agents, other known additives and other active agents.

[0270] Suitable carriers and excipients are well known to those skilled in the art and are described, 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 agents, antifungal agents), isotonic agents, absorption delay agents, salts, preservatives, drugs, drug stabilizers, gels, adhesives, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, similar materials and combinations thereof, as known to those of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Sciences, pp. 1289-1329, 1990). Except for any conventional carriers that are incompatible with the active ingredient, the use of the carrier in the treatment or pharmaceutical composition is predictable. Exemplary excipients include dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, or a combination thereof. Pharmaceutical compositions can contain different types of carriers or excipients, depending on whether they are administered in solid, liquid, or aerosol form, and whether they need to be sterile for these administration routes.

[0271] For example, tablets and capsules for oral administration can be in unit dose presentation form and can contain conventional excipients such as binders, such as syrup, gum arabic, gelatin, sorbitol, tragacanth or polyvinyl pyrrolidone; fillers, such as lactose, sugar, corn starch, calcium phosphate, sorbitol or glycine; tableting lubricants, such as magnesium stearate, talc, polyethylene glycol or silicon dioxide; disintegrants, such as potato starch, or acceptable wetting agents such as sodium lauryl sulfate. Tablets can be coated according to methods well known in normal pharmaceutical practice. Oral liquid preparations can be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or can be presented as dry products for reconstitution with water or other suitable vehicles before use. Such liquid preparations may contain conventional additives such as suspending agents, for example, sorbitol, syrup, methylcellulose, glucose syrup, gelatin, hydrogenated edible fats; emulsifiers, for example, lecithin, sorbitan monooleate or acacia; non-aqueous vehicles (which may include edible oils), for example, almond oil, fractionated coconut oils, oily esters such as glycerol, propylene glycol or ethanol; preservatives, for example, methyl or propyl paraben or sorbic acid, and, if desired, conventional flavoring or coloring agents.

[0272] For topical administration to the skin, the compounds may be formulated into creams, lotions or ointments. Pharmaceutically acceptable cream or ointment formulations are conventional formulations well known in the art, for example as described in standard pharmaceutical textbooks such as the British Pharmacopoeia.

[0273] The compounds of the present invention or their pharmaceutically acceptable salts can also be formulated for inhalation, for example as nasal sprays, or for dry powder or aerosol inhalers. For delivery by inhalation, the compound is generally in the form of microparticles, which can be prepared by a variety of techniques including spray drying, freeze drying and micronization. Aerosol generation can be performed using, for example, a pressure-driven jet nebulizer or an ultrasonic nebulizer, for example, by using a propellant-driven metered aerosol or a propellant-free administration of a micronized compound from, for example, an inhalation capsule or other "dry powder" delivery system.

[0274] As an example, compositions of the present invention can be prepared as the suspension for sending from a nebulizer or as the aerosol in the liquid propellant, for example, in a pressurized metered dose inhaler (PMDI), use. The propellant that is suitable for PMDI is well known to those skilled in the art, and comprises CFC-12, HFA-134a, HFA-227, HCFC-22 (CCl F ) and HFA-152 (CH F and isobutane).

[0275] 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.

[0276] Microparticles delivered by administration can be formulated with excipients that aid delivery and release. For example, in a dry powder formulation, the microparticles can be formulated with large carrier particles that aid flow from the DPI into the lungs. Suitable carrier particles are known and include lactose particles; they can have, for example, a mass median aerodynamic diameter greater than 90 μm.

[0277] In the case of aerosol-based formulations, examples are:

[0278] Compound of the present invention*24 mg / can

[0279] Lecithin, NF liquid concentration 1.2mg / can

[0280] Trichlorofluoromethane, NF 4.025g / can

[0281] Dichlorodifluoromethane, NF 12.15g / can.

[0282] * or its pharmaceutically acceptable salt

[0283] Depending on the inhaler system used, the compounds of the invention or their pharmaceutically acceptable salts can be administered as described. In addition to the compounds, the administration forms may also contain excipients as described above, or for example propellants (e.g. Frigen in the case of metered aerosols), surfactants, emulsifiers, stabilizers, preservatives, flavoring agents, fillers (e.g. lactose in the case of powder inhalers) or, if appropriate, further active compounds.

[0284] For inhalation purposes, a number of systems are available that allow the generation and administration of an aerosol of optimized particle size using an inhalation technique that is appropriate for the patient. In addition to the use of adapters (spacers, expanders) and pear-shaped containers (e.g. ) and automatic devices for emitting air-blowing sprays In addition, for metered sprays, especially in the case of powder inhalers, a variety of technical solutions can be used (e.g. or, for example, an inhaler as described in U.S. Patent No. 5,263,475, which is incorporated herein by reference.) Additionally, the compounds of the invention, or pharmaceutically acceptable salts thereof, may be delivered in a multi-chamber device, thereby allowing for the delivery of a combination dose.

[0285] The compound or its pharmaceutically acceptable salt can also be administered parenterally in a sterile medium. Depending on the vehicle and concentration used, the compound can be suspended or dissolved in the vehicle. Advantageously, adjuvants such as local anesthetics, preservatives or buffers can be dissolved in the vehicle.

[0286] Targeted inhaled drug delivery

[0287] The compounds of the present invention are contemplated for targeted inhalation delivery. The optimization of drugs delivered to the lungs by topical (inhalation) administration has been recently reviewed (Cooper, AE et al. Curr. Drug Metab. 2012, 13, 457-473).

[0288] Due to the limitations of the delivery device, the dosage of the inhaled drug may be a limited human dose, which requires an efficient molecule with good lung pharmacokinetic properties. Due to factors such as the limited amount of drug that can be delivered in one puff from the inhaler, and safety issues related to high lung aerosol load (e.g., cough or irritation), high efficacy against the target target is particularly important for inhaled drugs. For example, in some embodiments, for inhaled JAK1 inhibitors, it can be expected that the Ki in the JAK1 biochemical assay as described herein is about 0.5nM or less, and the IC50 in the JAK1-dependent cell-based assay as described herein is about 20nM or less. In other embodiments, the expected human dose of the compound of the present invention or its pharmaceutically acceptable salt is at least twice less than the expected human dose of the compound known in the art. Therefore, in some embodiments, the compound described herein (or its pharmaceutically acceptable salt) exhibits such efficacy values. The following procedure is used to evaluate the potential of the target compound as an inhaled drug.

[0289] IL13 signaling.IL13 signal transduction is closely related to the onset of asthma. IL13 is a cytokine that requires active JAK1 for signal transduction. Therefore, inhibiting JAK1 will also inhibit IL13 signal transduction, which will benefit asthma patients. Inhibition of IL13 signal transduction in animal models (e.g., mouse models) can predict future benefits to human asthma patients. Therefore, inhaled JAK1 inhibitors may be beneficial in showing IL13 signal transduction inhibition in animal models. Methods for measuring this inhibition 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 result of IL13 stimulation. Therefore, in some embodiments, the compounds described herein (or pharmaceutically acceptable salts thereof) exhibit inhibition of lung pSTAT6 induction. In order to examine the pharmacodynamic effect on pSTAT6 levels, the compounds of the present invention were co-administered intranasally to female Balb / c mice with 1 μg IL13. The compound was formulated in a saline solution containing 0.2% (v: v) Tween 80 and mixed with IL13 at 1: 1 (v: v) before administration. The intranasal dose was administered to mice under mild anesthesia (isoflurane) by dispensing a fixed volume (50 μL) directly into the nostrils with a pipette to reach the target dose level (3 mg / kg, 1 mg / kg, 0.3 mg / kg, 0.1 mg / kg). 0.25 h after administration, blood samples (about 0.5 mL) were collected by cardiac puncture, and plasma was produced by centrifugation (1500 g, 10 min, +4 ° C). The lungs were perfused with cold phosphate buffered saline (PBS), weighed and flash frozen in liquid nitrogen. All samples were stored at about -80 ° C until used for analysis. After adding 2 mL of HPLC grade water per gram of tissue, the defrosted lung samples were weighed and homogenized using an Omni-Prep Bead Ruptor at 4 ° C. Plasma and lung samples were extracted by protein precipitation, using three volumes of acetonitrile containing tolbutamide (50 ng / mL) and labetalol (25 ng / mL) as analytical internal standards. After vortex mixing and centrifugation at 3200 g and 4 ° C for 30 minutes, the supernatant was appropriately diluted (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 compounds by LC-MS / MS, with reference to a series of matrix-matched calibration and quality control standards. Standards were prepared by spiking test compounds into aliquots of control Balb / c mouse plasma or lung homogenate (2:1, in HPLC grade water) and extracting as described for experimental samples. Lung: plasma ratio was determined as the ratio of the average lung concentration (μM) to the average plasma concentration (μM) at the sampling time (0.25 h).

[0290] To measure pSTAT6 levels, mouse lungs were stored frozen at -80°C until assayed and homogenized in 0.6 ml ice-cold cell lysis buffer (Cell Signaling Technologies, catalog number 9803S), supplemented with 1 mM PMSF and a mixture of protease (Sigma Aldrich, catalog number P8340) and phosphatase (Sigma Aldrich, catalog number P5726 and P0044) inhibitors. The samples were centrifuged at 4°C for 4 minutes at 16060 x g to remove tissue debris, and the protein concentration of the homogenate was determined using a Pierce BCA protein assay kit (catalog number 23225). The samples were diluted to a protein concentration of 5 mg / ml in ice-cold distilled water, and pSTAT6 levels were determined by Meso Scale Discovery electrochemiluminescent immunoassay. In brief, 5 μ l / hole 150 μ g / ml STAT6 capture antibody (R & D Systems, catalog number MAB2169) was coated on a 96-well Meso Scale Discovery high binding plate (catalog number L15XB-3) and air-dried for 5 hours at room temperature. The plate was blocked by adding 150 μ l / hole 30 mg / ml Meso Scale Discovery blocking agent A (catalog number R93BA-4) and incubated on a microplate oscillator at room temperature for 2 hours. The closed plate was washed 4 times with Meso Scale Discovery TRIS washing buffer (catalog number R61TX-1), and then 50 μ l / hole lung homogenate was transferred to reach a protein load of 250 μ g / hole. The assay plate was incubated overnight at 4°C and washed 4 times with TRIS wash buffer, then 25 μl / well 2.5 μg / ml thio-tagged pSTAT6 detection antibody (BD Pharmingen, catalog number 558241) was added for 2 hours on a microplate shaker at room temperature. The plate was washed 4 times with TRIS wash buffer and 150 μl / well 1X Meso Scale Discovery Reading Buffer T (catalog number R92TC-1) was added. The lung homogenate pSTAT6 level was quantified by detecting electrochemiluminescence on a Meso Scale Discovery SECTOR S 600 instrument.

[0291] JAK and JAK2 InhibitionCompounds that inhibit both JAK1 and JAK2 are potentially useful for treating different types of asthma. The 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 specifically transduces signals through JAK2. Neutralization of GMCSF activity is associated with pulmonary alveolar proteinosis (PAP) in the lungs. However, submaximal JAK2 inhibition appears to be unrelated to PAP. Therefore, even moderate JAK1 and JAK2 selectivity or inhibition of JAK1 and JAK2 that is approximately equivalent may be beneficial to avoid complete inhibition of the GMCSF pathway and avoid PAP. For example, in certain embodiments, compounds with equal potency for JAK1 and JAK2 are desirable. In other embodiments, compounds with selectivity for JAK1 that are about 2-5 times that of JAK2 may be beneficial for inhaled JAK1 inhibitors. Therefore, in some embodiments, the compounds described herein (or pharmaceutically acceptable salts thereof) exhibit such selectivity. Methods for measuring JAK1 and JAK2 selectivity are known in the art, and information can also be found in the Examples herein.

[0292] Kinase profiling. Additionally, it may be desirable for an inhaled JAK1 or JAK1 / JAK2 inhibitor to be selective for one or more other kinases to reduce the likelihood of potential toxicity due to inhibition of off-target kinase pathways. Thus, it may also be beneficial for an inhaled JAK1 inhibitor to be selective for multiple non-JAK kinases, such as in the SelectScreen® available from ThermoFisherScientific TM Biochemical Kinase Profiling Service Using Adapta TM Screening protocol assay conditions (revised on July 29, 2016), LanthaScreen TM Eu Kinase Binding Assay Screening Protocol and Assay Conditions (revised June 7, 2016) and / or Z'LYTE TM The screening protocol and assay conditions (revised on September 16, 2016) are described in the present invention. For example, the compounds of the present invention or pharmaceutically acceptable salts thereof exhibit at least 50-fold selectivity for JAK1 over a group of non-JAK kinases. Therefore, in some embodiments, the compounds described herein (or pharmaceutically acceptable salts thereof) exhibit such selectivity.

[0293] Cytotoxicity assay.Hepatotoxicity (general cytotoxicity or cytotoxicity of unknown mechanism) is an undesirable feature of potential drugs (including inhaled drugs). It may be beneficial that inhaled JAK1 or JAK1 / JAK2 inhibitors have low intrinsic cytotoxicity to various cell types. Typical cell types for assessing cytotoxicity include primary cells (such as human hepatocytes) and established proliferating cell lines (such as Jurkat and HEK-293). Therefore, in some embodiments, the compounds described herein (or pharmaceutically acceptable salts thereof) exhibit such values. Methods for measuring cytotoxicity are known in the art. In some embodiments, the compounds described herein are tested as follows:

[0294] (a) Jurkat and HEK293T cells were maintained at subconfluent density in T175 flasks. Cells were plated at 450 cells / 45 μl of medium in Greiner 384-well black / clear tissue culture treated plates. (Greiner catalog number 781091). After dispensing the cells, the plates were equilibrated at room temperature for 30 minutes. After 30 minutes at room temperature, the cells were incubated overnight at 37°C in a CO2 and humidity controlled incubator. The next day, the cells were treated with compounds diluted in 100% DMSO (final DMSO concentration for cells = 0.5%) at a 10-point dose response curve with a maximum concentration of 50 μM. The cells and compounds were then incubated overnight at 37°C in a CO2 and humidity controlled incubator for 72 hours. After 72 hours of incubation, the cells were treated with (Promega catalog number G7572). After incubation at room temperature for 20 minutes, the viability of all wells was measured using the EnVision TM (Perkin Elmer Life Sciences);

[0295] (b) Using primary human hepatocytes: Prepare the test compound as a 10 mM solution in DMSO. In addition, prepare a positive control (such as chlorpromazine) as a 10 mM solution in DMSO. Typically, a 7-point dose-response curve is used to evaluate the test compound at 2-fold dilution. Typically, the maximum concentration tested is 50-100 μM. The highest concentration is usually determined by the solubility of the test compound. Cryopreserved primary human hepatocytes (Bioreclamation IVT) (lot number IZT) are placed in an InVitroGro TM The cells were thawed at 37°C in Bioreclamation IVT HT thawing medium, pelleted and resuspended. Hepatocyte viability was assessed by trypan blue exclusion and cells were plated at a density of 13,000 cells / well in black-walled BioCoat TM InVitroGro in Collagen 384-well Plates (Corning BD) TMPlated in CP medium supplemented with 1% Torpedo TM Antibiotic cocktail (Bioreclamation IVT) and 5% fetal bovine serum. Before treatment, cells were incubated overnight for 18 hours (37°C, 5% CO2). After 18 hours of incubation, the plating medium was removed and the cells were plated in 1% Torpedo TM InVitroGro with antibiotic cocktail and 1% DMSO (serum-free conditions) TM Hepatocytes were treated with compounds diluted in HI incubation medium. 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. A positive control (e.g., chlorpromazine) was included in the assay, usually at the same concentration as the test compound. Additional cells were treated with 1% DMSO as a vehicle control. All treatments were performed for 48 hours (at 37°C, 5% CO2), and each treatment condition was performed in triplicate. After 48 hours of compound treatment, CellTiter- The cell viability assay (Promega) was used as an endpoint assay to measure ATP levels as a measure of cell viability. The assay was performed according to the manufacturer's instructions. TM Luminescence assays were performed on a multiplate reader (PerkinElmer, Waltham, MA, USA). Luminescence data were normalized to vehicle (1% DMSO) control wells. Inhibition curves and IC were generated by nonlinear regression of log-transformed inhibitor concentrations (7-point serial dilutions including vehicle) to normalized responses with variable Hill slopes. 50 The estimated values ​​were constrained to constant values ​​of 100 and 0, respectively (GraphPad Prism TM , GraphPad Software, La Jolla, CA, USA).

[0296] hERG inhibition.Inhibition of hERG (human ether-à-go-go-related gene) potassium channels may lead to long QT syndrome and arrhythmias. Although plasma levels of inhaled JAK1 or JAK1 / JAK2 inhibitors are expected to be low, lung-deposited compounds that leave the lungs and enter the bloodstream through pulmonary absorption will circulate directly to the heart. Therefore, local cardiac concentrations of inhaled JAK1 inhibitors can be transiently higher than total plasma levels, especially immediately after administration. Therefore, it can be beneficial to minimize hERG inhibition of inhaled JAK1 inhibitors. For example, in some embodiments, it is preferred that the hERG IC50 exceeds 30 times the free drug plasma Cmax. Therefore, in some embodiments, the compounds of the present invention (or pharmaceutically acceptable salts thereof) exhibit minimal hERG inhibition under conditions such as the following:

[0297] (a) hERG 2pt automated patch clamp conditions were used to examine the in vitro effects of compounds on hERG expressed in mammalian cells at room temperature using the automated parallel patch clamp system QPatch (Sophion Bioscience A / S, Denmark). In some cases, the compound is tested at only one or two concentrations, such as 1 or 10 uM. In other cases, a more extensive concentration-response relationship is established to allow estimation of IC50. For example, the concentrations of the test compound are selected to span the range of inhibition from approximately 10% to 90% in half-log increments. Each concentration of the test article is tested in two or more cells (n≥2). The exposure duration for each test article concentration is a minimum of 3 minutes; and / or

[0298] (b) The conditions described in WO 2014 / 074775 in the example of “Effect on Cloned hERG Potassium Channels Expressed in Mammalian Cells” were used for the ChanTest of Charles River Company. TM The scheme has been changed as follows: the cells stably expressing hERG are maintained at -80mV. The pulse mode of fixed amplitude (adjusting pre-pulse: +20mV lasts 1s; Repeating repolarization test ramepto-90mV (-0.5V / s) at intervals of 5s) is used to measure the initial and steady state inhibition of the hERG potassium current caused by the compound. Each recording ends with the final application of the super-large concentration of reference material E-4021 (500nM) (Charles River Company). From the data, digitally deduct the remaining uninhibited current offline, to determine the hERG inhibition efficacy of the test substance.

[0299] CYP (cytochrome P450) inhibition assay. For inhaled JAK1 or JAK1 / JAK2 inhibitors, CYP inhibition may not be a desired feature. For example, a reversible or time-dependent CYP inhibitor may lead to an undesirable increase in its own plasma level or the plasma level of other co-administered drugs (drug-drug interaction). In addition, time-dependent CYP inhibition is sometimes caused by the biotransformation of the parent drug to reactive metabolites. Such reactive metabolites may covalently modify proteins, thereby potentially causing toxicity. Therefore, minimizing reversible and time-dependent CYP inhibition may be beneficial to inhaled JAK1 inhibitors. Therefore, in some embodiments, the compounds of the present invention (or pharmaceutically acceptable salts thereof) exhibit minimal or no reversible and / or time-dependent CYP inhibition. Methods for measuring CYP inhibition are known in the art. Compounds described herein were assessed for CYP inhibition using previously reported methods (Halladay et al., Drug Metab. Lett. 2011, 5, 220-230) using pooled (n=150) human liver microsomes (Corning, Tewksbury, MA) over a compound concentration range of 0.16-10 uM. Incubation duration and protein concentration depended on the CYP isoform and probe substrate / metabolite being assessed. For each CYP, the following substrates / metabolites, as well as incubation times and protein concentrations 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 have been previously determined to be linear formation rates for CYP-specific metabolites. All reactions were initiated with 1 mM NADPH and terminated by the addition of 0.1% formic acid in acetonitrile (containing appropriate stable labeled internal standards). Samples were analyzed by LC-MS / MS.

[0300] Mouse lung tissue binding. A high bound fraction or percentage of a JAK1 / JAK2 inhibitor to lung tissue may be desirable because it may reduce the amount of free drug available to inhibit JAK1 or JAK2.

[0301] (a) Tissue binding experiments were performed in triplicate (n=3) using disposable RED plates by following a standard protocol. Initially, individual drugs were added to tissue homogenate (pH 7.4) to achieve a final concentration of 1 μM, and then 300 μL of drug-tissue homogenate was transferred to the donor well of the RED plate, which was pre-loaded with 500 μL of phosphate buffered saline (133 mM) in the receptor well. The RED plate was sealed with a gas permeable membrane and placed in a shaking incubator (450 rpm, VWR Symphony TM ) at 37°C, 5% CO2 for 6h. After the incubation, 30 μL sample aliquots were taken from the RED device and subjected to matrix equilibration with an equal volume of tissue homogenate or buffer, and then the resulting samples were immediately quenched with ice-cold acetonitrile containing propranolol or labetalol (sample: acetonitrile is 1:4) as internal standards. After shaking at 500 rpm for 15 min on a Thermo Scientific Compact Digital MicroPlate oscillator, all samples were then centrifuged at 3700 rpm for 15 min (Beckman Coulter Allegra X 12R) to remove plasma proteins. Subsequently, the supernatant was collected and then diluted with an equal volume of water before LC-MS / MS analysis.

[0302] (b) In an alternating procedure, the extent of lung tissue binding of test compounds to mouse lung homogenates can also be determined by equilibrium dialysis using a Pierce RED (Rapid Equilibrium Dialysis) device (Fisher Scientific 89811 & 89809). A 10 mM solution of the compound was prepared in DMSO 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, wherein the solvent accounted for 0.5% (v / v) of the final incubation volume.

[0303] For each assay, the percentage of bound lung tissue was determined in triplicate. Lung homogenate (200 μL) was loaded onto one side of the RED device insert in triplicate, and 350 μL of potassium phosphate buffer was loaded onto the other side. The RED device was sealed and incubated at approximately 37° C. on an orbital shaker (approximately 150 rpm) for 4 hours.

[0304] After incubation, lung homogenate aliquots (8 μL) and dialysate aliquots (72 μL) were matrix matched (lung homogenate with 72 μL phosphate buffer, dialysate with 8 μL lung homogenate) before analysis. Proteins were precipitated from the samples by adding 160 μL acetonitrile containing internal standard. The same matrix matching and protein precipitation procedures were performed on the lung homogenate aliquots collected at the beginning of the experiment (t=0 min samples) for assessing mass balance. The quenched samples were centrifuged (4000 rpm, 30 min, 4 ° C) and the resulting supernatant was diluted with water (3: 1 (v / v), supernatant: water), and the parent compound of the sample was analyzed by liquid chromatography mass spectrometry.

[0305] The unbound fraction (fu) in the lung homogenate was determined from the dialysate to homogenate peak area ratio, corrected to account for lung homogenate dilution (D), allowing estimation of whole lung tissue binding using the following equation:

[0306] Undiluted fu = (1 / D) / [((1 / surface fu)-1)+(1 / D)]

[0307] Corrected bound fraction (%) = (1-undiluted FU) * 100

[0308] Kinetic solubility. Good aqueous solubility of JAK1 / JAK2 inhibitors for inhalation delivery is desirable. In one procedure to measure kinetic solubility, 4 μL of a 10 mM DMSO stock solution of the test compound was added to a Millipore 196 μL pH 7.4 phosphate buffered saline solution in a 96-well filter plate to obtain a 200 μM test concentration and 2% residual DMSO. The filter plate is sealed with an aluminum seal 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 with a pH 7.4 phosphate buffered saline solution by a factor of 2, and then 5 μL of the resulting solution is analyzed by ultra-high performance liquid chromatography (UHPLC) using chemiluminescent nitrogen detection (CLND) and ultraviolet (UV) detection at a wavelength of 254 nm. The sample concentration is usually quantified by the CLND intensity, which is related to the amount of nitrogen in the compound. UV detection is mainly used to confirm sample purity, except for a few cases where the test sample does not contain nitrogen. In those cases, a compound-specific calibration curve is collected based on UV absorbance. The curve is then used to determine the sample concentration.

[0309] Lipophilic.Lipophilicity is often associated with the solubility, absorption, tissue permeability, protein binding, distribution, and ADME and PK properties of a potential drug. The calculated logP (cLogP) 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)), and therefore can be an important consideration for JAK1 / JAK inhibitors for inhaled delivery.

[0310] Hepatic microsomal stability. In order to minimize the systemic exposure of inhaled JAK1 / JAK2 inhibitors, it can be beneficial to optimize the rapid metabolism in the liver. Liver microsomal stability assay was performed on the BioCel 1200 liquid handling workbench (Agilent Technologies, Santa Clara, CA). The compound (1.0 μM) was incubated for 5 min at 37 ° C in 100 μL of a reaction mixture containing 100 mM phosphate buffer (pH 7.4) and 0.5 mg / mL liver microsomes and 1 mM NADPH. At different time intervals (0, 20, 40 and 60 min), aliquots of 20 μL of the reaction mixture were taken out and mixed with 4 volumes of acetonitrile (ACN) containing 0.1 μM propranolol as an internal standard to terminate the metabolic reaction. The sample was then centrifuged at 3250 x g for 40 min to remove precipitated protein. The supernatant was then transferred to a new 96-well plate and diluted 2-fold with deionized water before analysis using an ABI Sciex 5500 coupled to an Agilent 1260 HPLC (Agilent Technologies, Santa Clara, CA). Mass spectrometer (Applied Biosystems, Foster City, CA) was used for LC-MS / MS analysis. The percentage of the remaining part was calculated using the peak area ratio of the test compound to the internal standard relative to the control (T = 0 min) at different time points. See B. Williamson, C. Wilson, G. Dagnell, RJ Riley. Harmonised high throughput microsomal stability assay. J. Pharmacol. Toxicol. Methods. 2017; 84: 31-36.

[0311] Solid state properties.For compounds intended 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 of 1-5 μm. Particle size is an important determinant of lung deposition of inhaled compounds. Particles with a diameter of less than 5 microns (μm) are generally defined as inhalable. Particles with a diameter greater than 5 μm are more likely to be deposited in the oropharynx and are therefore less likely to be deposited in the lungs. In addition, fine particles with a diameter of less than 1 μm are more likely to remain suspended in the air than larger particles and are therefore more likely to be exhaled from the lungs. Therefore, for inhaled drugs whose site of action is in the lungs, a particle size of 1-5 μm can be beneficial. Typical methods for measuring particle size include laser diffraction and cascade impact. Typical values ​​used to define particle size include:

[0312] • D10, D50 and D90. These values ​​are measurements of particle size, indicating that 10%, 50% or 90% of the sample is below that value, respectively. For example, a D50 of 3 μm indicates that 50% of the sample is below 3 μm in size.

[0313] • Mass mean aerodynamic diameter (MMAD). MMAD is the diameter above which 50% of the particles by mass are larger and 50% are smaller. MMAD is a measure of central tendency.

[0314] • Geometric Standard Deviation (GSD). GSD is a measure of the dispersion value or aerodynamic particle size distribution spread according to MMAD.

[0315] A common formulation for inhaled drugs is a dry powder formulation comprising an active pharmaceutical ingredient (API) blended with a carrier (such as lactose) with (or without) additional additives (such as magnesium stearate). For this and other formulations, it may be beneficial for the API itself to have properties that allow it to be ground into a respirable particle size of 1-5 μm. Agglomeration of particles should be avoided, which can be measured by methods known in the art, such as checking the D90 value under different pressure conditions. Therefore, in some embodiments, the compounds of the present invention (or their pharmaceutically acceptable salts) can be prepared to have such a respirable particle size with little or no agglomeration.

[0316] As for crystallinity, for some inhaled drug formulations (including lactose blends), it is important to use APIs in a specific crystalline form. Crystallinity and crystalline form may affect many parameters related to inhaled drugs, including but not limited to: chemical and aerodynamic stability over time, compatibility with inhaled formulation components such as lactose, hygroscopicity, lung accumulation and lung irritation. Therefore, a stable, reproducible crystalline form can be beneficial for inhaled drugs. In addition, the technology used to grind the compound to the desired particle size is usually high-energy, and can convert low-melting crystalline forms into other crystalline forms, or into completely or partially amorphous. Crystalline forms with a melting point below 150°C may not be suitable for grinding, and crystalline forms with a melting point below 100°C may be incompatible with grinding. Therefore, the melting point of the inhaled drug is at least greater than 100°C, and ideally greater than 150°C can be beneficial. Therefore, in some embodiments, the compound described herein (or its pharmaceutically acceptable salt) exhibits this property.

[0317] Additionally, minimizing the molecular weight may help reduce the effective dose of an inhaled JAK1 inhibitor. The lower the molecular weight, the higher the number of molecules per unit mass of the active pharmaceutical ingredient (API). Therefore, it may be beneficial to find an inhaled JAK1 inhibitor of the smallest molecular weight that retains all other desirable properties of an inhaled drug.

[0318] Finally, the compound needs to maintain sufficient concentration in the lungs over a given period of time so that the pharmacological effect can be exerted for the desired duration and with low systemic exposure to the pharmacological target (in the case where systemic inhibition of the target is not desired). The lungs have an inherently high permeability to large molecules (proteins, peptides) and small molecules with short lung half-lives, so it may be necessary to attenuate the lung absorption rate by changing one or more characteristics of the compound: minimizing membrane permeability, optimal pKa, cLogP, solubility, dissolution rate, or introducing a degree of alkalinity (e.g., introducing amines) into the compound to enhance binding to phospholipid-rich lung tissue, or by trapping in acidic subcellular compartments such as lysosomes (pH 5). Methods for measuring such properties are known in the art.

[0319] Therefore, in some embodiments, the compounds of the present invention (or pharmaceutically acceptable salts thereof) advantageously exhibit one or more of the above-mentioned characteristics. In addition, in some embodiments, the compounds of the present invention advantageously exhibit one or more of these characteristics relative to compounds known in the art, especially for compounds of the art intended for use as oral drugs rather than inhaled drugs. For example, compounds with rapid oral absorption are generally difficult to retain in the lungs when inhaled.

[0320] Methods of treatment using Janus kinase inhibitors and uses thereof

[0321] The compounds of the present invention or pharmaceutically acceptable salts thereof inhibit the activity of Janus kinases such as JAK1 kinases. For example, the compound or pharmaceutically acceptable salt thereof inhibits the phosphorylation of signal transduction and transcription activator (STAT) by JAK1 kinase and STAT-mediated cytokine production. The compounds of the present invention can be used to inhibit the JAK1 kinase activity in cells through cytokine pathways such as IL-6, IL-15, IL-7, IL-2, IL-4, IL-9, IL-10, IL-13, IL-21, G-CSF, IFNα, IFNβ or IFNγ pathways. Therefore, in one embodiment, a method is provided for contacting a cell with a compound of the present invention or a pharmaceutically acceptable salt thereof to inhibit the activity of Janus kinases (e.g., JAK1 activity) in the cell.

[0322] The compounds can be used to treat immune diseases driven by aberrant IL-6, IL-15, IL-7, IL-2, IL-4, IL-9, IL-10, IL-13, IL-21, G-CSF, IFNα, IFNβ or IFNγ cytokine signaling.

[0323] Thus, one embodiment includes a compound of the invention or a pharmaceutically acceptable salt thereof for use in therapy.

[0324] In some embodiments, the use of the compounds of the present invention or pharmaceutically acceptable salts thereof in the treatment of inflammatory diseases is provided. Further provided is the use of the compounds of the present invention or pharmaceutically acceptable salts thereof in the preparation of a medicament for treating inflammatory diseases such as asthma. Also provided is a compound of the present invention or pharmaceutically acceptable salts thereof for treating inflammatory diseases such as asthma.

[0325] Another embodiment includes a method of preventing, treating, or reducing the severity of a disease or condition (such as asthma) in a patient that is responsive to inhibition of Janus kinase activity, such as JAK1 kinase activity. The method may include the step of administering to the patient a therapeutically effective amount of a compound of the invention or a pharmaceutically acceptable salt thereof. In one embodiment, the disease or condition that is responsive to inhibition of Janus kinase, such as JAK1 kinase, is asthma.

[0326] In one embodiment, the disease or disorder is cancer, stroke, diabetes, hepatomegaly, cardiovascular disease, multiple sclerosis, Alzheimer's disease, cystic fibrosis, viral disease, autoimmune disease, atherosclerosis, restenosis, psoriasis, rheumatoid arthritis, inflammatory bowel disease, asthma, allergic disease, inflammation, nervous system disease, hormone-related disease, disorder associated with organ transplantation (e.g., transplant rejection), immunodeficiency disease, destructive bone disease, proliferative disease, infectious disease, disorder associated with cell death, thrombin-induced platelet aggregation, liver disease, pathological immune disorder involving T cell activation, CNS disease or myeloproliferative disease.

[0327] In one embodiment, the inflammatory disease is rheumatoid arthritis, psoriasis, asthma, inflammatory bowel disease, contact dermatitis, or delayed hypersensitivity. In one embodiment, the autoimmune disease is rheumatoid arthritis, lupus, or multiple sclerosis.

[0328] In another embodiment, the compound of the present invention or its pharmaceutically acceptable salt can be used for treating lung diseases such as fibrotic lung disease or interstitial lung disease (for example, interstitial pneumonia). In certain embodiments, the compound of the present invention or its pharmaceutically acceptable salt can be used for treating idiopathic pulmonary fibrosis (IPF), systemic sclerosis interstitial lung disease (SSc-ILD), nonspecific interstitial pneumonia (NSIP), rheumatoid arthritis related interstitial lung disease (RA-ILD), sarcoidosis, hypersensitivity pneumonitis or scleroderma and are secondary to the ILD (for example, polymyositis, dermatomyositis, rheumatoid arthritis, systemic lupus erythematosus (SLE) or mixed connective tissue disease) of connective tissue disease.

[0329] In one embodiment, the cancer is breast cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, penile cancer, genitourinary tract cancer, seminoma, esophageal cancer, laryngeal cancer, gastric cancer, stomach cancer, gastrointestinal cancer, skin cancer, keratoacanthoma, follicular carcinoma, melanoma, lung cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), lung adenocarcinoma, lung squamous cell carcinoma, colon cancer, pancreatic cancer, thyroid cancer, papillary cancer, bladder cancer, liver cancer, biliary tract cancer, kidney cancer, bone cancer, myeloid disorders, lymphoid disorders, hairy cell cancer, buccal and pharyngeal (oral) cancer, lip cancer, tongue cancer, oral cancer, salivary gland cancer, pharyngeal cancer, small intestine cancer, colon cancer, rectum cancer, anal cancer, kidney cancer, vulva cancer, thyroid cancer, large intestine cancer, endometrial cancer, uterine cancer, brain cancer, central nervous system cancer, peritoneal cancer, hepatocellular carcinoma, head cancer, neck cancer, Hodgkin's disease, or leukemia.

[0330] In one embodiment, the disease is a myeloproliferative disease. In one embodiment, the myeloproliferative disease is polycythemia vera, essential thrombocythemia, myelofibrosis or chronic myeloid leukemia (CML).

[0331] Another embodiment includes the use of a compound of the invention or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating a disease described herein (e.g., an inflammatory disorder, an immune disease, or a cancer). In one embodiment, the invention provides a method of treating a disease or disorder as described herein (e.g., an inflammatory disorder, an immune disease, or a cancer) by targeted inhibition of a JAK kinase such as JAK1.

[0332] Combination therapy

[0333] The compound can be used alone or in combination with other agents for treatment. The second compound of the pharmaceutical composition or dosage regimen or in addition (e.g., the third) generally has an activity complementary to the compound of the present invention so that they do not adversely affect each other. Such agents are appropriately present in an amount combination effective for the intended purpose. The compound can be administered together or separately in a single pharmaceutical composition, and when administered separately, it can be administered simultaneously or sequentially. This sequential administration can be close in time or spaced farther apart.

[0334] For example, other compounds can be used in combination with the compounds of the present invention or pharmaceutically acceptable salts thereof for the prevention or treatment of inflammatory diseases, such as asthma. Suitable therapeutic agents for combined therapy include, but are not limited to, adenosine A2A receptor antagonists; anti-infectives; nonsteroidal glucocorticoid receptor (GR receptor) agonists; antioxidants; β2 adrenergic receptor agonists; CCR1 antagonists; chemokine antagonists (non-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., fenbrutinib); spleen tyrosine kinase (SYK) inhibitors; tryptase-β 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 (PPARγ agonists); protease inhibitors; retinoic acid receptor modulators (RARγ modulators); statins; thromboxane antagonists; TLR7 receptor agonists; or vasodilators.

[0335] In addition, the compounds of the present invention or their pharmaceutically acceptable salts may be combined with the following substances: (1) corticosteroids such as alclometasone dipropionate, amelometasone, beclomethasone dipropionate, budesonide, butixocortpropionate, biclesonide, clobetasol propionate, desisobutyrylciclesonide, dexamethasone, etiprednol dicloacetate, fluocinolone acetonide, fluticasone furoate, fluticasone propionate, loteprednol etabonate (topical) or mometasone furoate; (2) β2-adrenergic receptor agonists, such as salbutamol, albuterol, terbutaline, fenoterol, bitolterol, carbuterol, clenbuterol, pirbuterol, rimoterol, terbutaline, tretoquinol, tulobuterol, ) and long-acting 2-adrenoceptor agonists such as metaproterenol, isoproterenol, isoprenaline, salmeterol, indacaterol, formoterol (including formoterol fumarate), arformoterol, carmoterol, abediterol, vilanterol trifenate or olodaterol; (3) corticosteroid / long-acting 2-agonist combination products such as salmeterol / fluticasone propionate ( Also Sales), Formoterol / Budesonide Formoterol / Fluticasone Propionate formoterol / ciclesonide, formoterol / mometasone furoate, indacaterol / mometasone furoate, vilanterol trifenate / fluticasone furoate (BREO ELLIPTA), or arformoterol / ciclesonide; (4) anticholinergic agents, for example, 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 combination products, such as vilanterol / umeclidinium Ototerol / tiotropium, glycopyrrolate / indacaterol ( Also Sales), fenoterol hydrobromide / ipratropium bromide Albuterol sulfate / ipratropium bromide Formoterol fumarate / glycopyrrolate or aclidinium / formoterol; (6) dual pharmacology M3-anticholinergic / β2-adrenergic receptor agonists (such as batefenterol succinate) succinate), AZD-2115 or LAS-190792; (7) leukotriene modifiers, for example, leukotriene antagonists, such as montelukast, zafirulast or pranlukast, or leukotriene biosynthesis inhibitors, such as zileuton, or LTB4 antagonists, such as amelubant, or FLAP inhibitors, such as fiboflapon, GSK-2190915; (8) phosphodiesterase-IV (PDE-IV) inhibitors (oral or inhaled), such as roflumilast, cilomilast, oglemilast, rolipram, tetomilast, AVE-8112, revamilast, CHF 6001; (9) antihistamines, for example, selective histamine-1 (H1) receptor antagonists, such as fexofenadine, citirizine, loratidine or astemizole, or dual H1 / H3 receptor antagonists, such as GSK 835726 or GSK 1004723; (10) antitussives, such as codeine or dextramorphan; (11) mucolytics, for example, N-acetylcysteine ​​or fudostein; (12) expectorants / viscoelastic modifiers, for example, ambroxol, hypertonic solutions (e.g., saline or mannitol), or surfactants; (13) mucolytic peptides, for example, recombinant human deoxyribonuclease I (dornase-α and rhDNase) or spirocidin; (14 ) antibiotics, such as azithromycin, tobramycin or aztreonam; (15) non-selective COX-1 / COX-2 inhibitors, such as ibuprofen or ketoprofen; (16) COX-2 inhibitors, such as celecoxib and 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, for example, anti-TNF monoclonal antibodies, such as; and CDP-870, as well as TNF receptor immunoglobulin molecules, e.g. (19) Matrix metalloproteinase inhibitors, such as MMP-12; (20) Human neutrophil elastase inhibitors, such as BAY-85-8501 or those described in WO2005 / 026124, WO2003 / 053930 and WO2006 / 082412, each of which is incorporated herein by reference; (21) A2b antagonists, such as those described in WO2002 / 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 A2 antagonists; DP1 antagonists, such as laropiprant or asapiprant; CRTH2 antagonists, such as OC000459, fevipiprant, ADC 3680 or ARRY 502; (24) PPARα agonists, including PPARα agonists (such as fenofibrate), PPARδ agonists, PPARγ agonists (such as pioglitazone, rosiglitazone and balaglitazone); (25) methylxanthines, such as theophylline or aminophylline, and methylxanthine / corticosteroid combinations, such as theophylline / budesonide, theophylline / fluticasone propionate, theophylline / ciclesonide, theophylline / mometasone furoate and theophylline / beclometasone dipropionate dipropionate); (26) A2a agonists, such as those described in EP1052264 and EP1241176; (27) CXCR2 or IL-8 antagonists, such as AZD-5069, AZD-4721, danirixin; (28) IL-R signaling modulators, such as kineret and ACZ 885; (29) MCP-1 antagonists, 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.

[0336] In some embodiments, the compounds of the present invention or pharmaceutically acceptable salts thereof can be used in combination with one or more other drugs, such as anti-hyperproliferative drugs, anticancer drugs, cytostatic agents, cytotoxic agents, anti-inflammatory agents or chemotherapeutic agents, such as those disclosed in U.S. Published Application No. 2010 / 0048557, which is incorporated herein by reference. As known in the art, the compounds of the present invention or pharmaceutically acceptable salts thereof can also be used in combination with radiotherapy or surgery.

[0337] Combinations of any of the foregoing with a compound of the invention or a pharmaceutically acceptable salt thereof are specifically envisioned.

[0338] Products

[0339] Another embodiment includes an article of manufacture (e.g., a kit) for treating a disease or condition that responds to inhibition of a Janus kinase, such as JAK1 kinase. The kit may include:

[0340] (a) a first pharmaceutical composition comprising a compound of the present invention or a pharmaceutically acceptable salt thereof; and

[0341] (b) Instructions for use.

[0342] In another embodiment, the kit further comprises:

[0343] (c) a second pharmaceutical composition, such as a pharmaceutical composition comprising a therapeutic agent as described above, such as an agent for treating an inflammatory disorder, or a chemotherapeutic agent.

[0344] In one embodiment, the instructions describe administering the first pharmaceutical composition and the second pharmaceutical composition simultaneously, sequentially, or separately to a patient in need thereof.

[0345] In one embodiment, the first composition and the second composition are contained in separate containers. In another embodiment, the first composition and the second composition are contained in the same container.

[0346] The containers used include, for example, bottles, vials, syringes, blister packs, and the like. The container may be formed of a variety of materials such as glass or plastic. The container contains a compound of the present invention or a pharmaceutically acceptable salt thereof for effective treatment of a condition, and the container may have a sterile access port (for example, the container may be an intravenous infusion bag or a vial with 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 certain disease. In addition, the label or package insert may indicate that the patient to be treated is a patient with a disease characterized by overactive or irregular Janus kinase activity (such as overactive or irregular JAK1 activity). The label or package insert may also indicate that the compound can be used to treat other diseases.

[0347] Alternatively or additionally, the kit may further include a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution or dextrose solution. The article may also include other substances required from a commercial and user perspective, including other buffers, diluents, filters, needles and syringes.

[0348] The following examples are included to illustrate the present invention. However, it should be understood that these examples do not limit the present invention, but are intended only to suggest methods for implementing the present invention. Those skilled in the art will recognize that the chemical reactions described can be easily adjusted to prepare other compounds of the present invention, and alternative methods for preparing compounds are also within the scope of the present invention. For example, the synthesis of non-exemplary compounds according to the present invention can be successfully achieved by modifications that are obvious to those skilled in the art, such as, by appropriately protecting interfering groups, utilizing other suitable reagents known in the art, or by routine modifications to reaction conditions. Alternatively, other reactions disclosed herein or known in the art will be considered to be suitable for preparing other compounds of the present invention.

[0349] Examples

[0350] The following representative compounds of Table 1 were prepared using procedures similar to those described in the Schemes and Examples herein. The absolute stereochemistry of each of the following compounds may not have been depicted: therefore, structures may appear more than once, each representing a single stereoisomer. Table 1 also shows the LC-MS method, retention time, and m / z.

[0351] Table 1: Exemplary JAK inhibitors

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369] Table 1

[0370] General Experiment Details

[0371] Unless otherwise stated, all solvents and commercial reagents were used as received. In the case of purification of the product by silica gel chromatography, a glass column manually filled with silica gel (Kieselgel 60, 220-440 mesh, 35-75 μm) or This operation was performed using an SPE Si II column. "Isolute SPE Si column" refers to a column containing a 50 μm average particle size and a nominal Prepacked polypropylene columns with irregular porosity and unbonded activated silica. In the case of the SCX-2 column, "SCX-2 column" refers to a prepacked polypropylene column containing a non-terminated propylsulfonic acid functionalized silica strong cation exchange adsorbent.

[0372] LCMS conditions

[0373] Method A

[0374] The experiment was performed on a SHIMADZU LCMS-2020 using a C18 reverse phase column (50x3mm Shim-Pack XR-ODS, 2.2μm particle size) and the eluent was: solvent A: water + 0.05% trifluoroacetic acid; solvent B: acetonitrile + 0.05% trifluoroacetic acid. Gradient:

[0375]

[0376] Detection - UV (220 and 254 nm) and ELSD

[0377] Method B

[0378] The experiment was performed on a SHIMADZU LCMS-2020 using a C18 reverse phase column (50x3mm Shim-Pack XR-ODS, 2.2μm particle size) and the eluent was: solvent A: water + 0.05% trifluoroacetic acid; solvent B: acetonitrile + 0.05% trifluoroacetic acid. Gradient:

[0379]

[0380] Detection - UV (220 and 254 nm) and ELSD

[0381] Method C

[0382] The experiment was performed on a SHIMADZU LCMS-2020 using a C18 reverse phase column (50x3mm Shim-Pack XR-ODS, 2.2μm particle size) and the eluent was: solvent A: water + 0.05% trifluoroacetic acid; solvent B: acetonitrile + 0.05% trifluoroacetic acid. Gradient:

[0383]

[0384] Detection - UV (220 and 254 nm) and ELSD

[0385] Method D

[0386] The experiment was performed on a SHIMADZU LCMS-2020 using a C18 reverse phase column (50x3mm Shim-Pack XR-ODS, 2.2μm particle size) and the eluent was: solvent A: water + 0.05% trifluoroacetic acid; solvent B: acetonitrile + 0.05% trifluoroacetic acid. Gradient:

[0387]

[0388]

[0389] Detection - UV (220 and 254 nm) and ELSD

[0390] Method E

[0391] The experiment was performed on a SHIMADZU LCMS-2020 using a C18 reverse phase column (50x3mm Shim-Pack XR-ODS, 2.2μm particle size) and the eluent was: solvent A: water + 0.05% trifluoroacetic acid; solvent B: acetonitrile + 0.05% trifluoroacetic acid. Gradient:

[0392]

[0393] Detection - UV (220 and 254 nm) and ELSD

[0394] Method F

[0395] The experiment was performed on a SHIMADZU LCMS-2020 using a C18 reverse phase column (50x3mm Shim-Pack XR-ODS, 2.2μm particle size) and the eluent was: solvent A: water + 0.05% trifluoroacetic acid; solvent B: acetonitrile + 0.05% trifluoroacetic acid. Gradient:

[0396]

[0397] Detection - UV (220 and 254 nm) and ELSD

[0398] Method G

[0399] The experiment was conducted on a SHIMADZU 20A HPLC using a C18 reverse phase column (50x2.1 mm Ascentis ExpressC18, 2.7 μm particle size) and the eluents were: solvent A: water + 0.05% trifluoroacetic acid; solvent B: acetonitrile + 0.05% trifluoroacetic acid. Gradient:

[0400]

[0401] Detection - UV (220 and 254 nm) and ELSD

[0402] Method H

[0403] The experiment was performed on a SHIMADZU LCMS-2020 using a C18 reverse phase column (50x3mm Shim-Pack XR-ODS, 2.2μm particle size) and the eluent was: solvent A: water + 0.05% trifluoroacetic acid; solvent B: acetonitrile + 0.05% trifluoroacetic acid. Gradient:

[0404]

[0405] Detection - UV (220 and 254 nm) and ELSD

[0406] Method I

[0407] The experiments were performed on a SHIMADZU 20A HPLC using a Poroshell HPH-C 18 Column (50x3mm, 2.7μm particle size), eluent: solvent A: water / 5mM NH4HCO3; solvent B: acetonitrile. Gradient:

[0408]

[0409] Detection - UV (220 and 254 nm) and ELSD

[0410] Method J

[0411] The experiment was performed on a SHIMADZU LCMS-2020 using a C18 reverse phase column (50x3mm Kinetex XB-C 18 , 2.6 μm particle size), eluent: solvent A: water + 0.05% trifluoroacetic acid; solvent B: acetonitrile + 0.05% trifluoroacetic acid. Gradient:

[0412]

[0413]

[0414] Detection - UV (220 and 254 nm) and ELSD

[0415] Method K

[0416] The experiment was performed on a SHIMADZU LCMS-2020 using a C18 reverse phase column (50x3mm Shim-Pack XR-ODS, 2.2μm particle size) and the eluent was: solvent A: water + 0.05% trifluoroacetic acid; solvent B: acetonitrile + 0.05% trifluoroacetic acid. Gradient:

[0417]

[0418] Detection - UV (220 and 254 nm) and ELSD

[0419] Method L

[0420] The experiment was performed on a SHIMADZU LCMS-2020 using a C18 reverse phase column (50x2.1mm Kinetex XB-C 18 100A, 2.6 μm particle size), eluent: solvent A: water + 0.05% trifluoroacetic acid; solvent B: acetonitrile + 0.05% trifluoroacetic acid. Gradient:

[0421]

[0422] Detection - UV (220 and 254 nm) and ELSD

[0423] Method M

[0424] The experiment was performed on a SHIMADZU LCMS-2020 using a C18 reverse phase column (30x2.1mm Kinetex C18-100A, 1.7μm particle size) and the eluents were: solvent A: water + 0.05% trifluoroacetic acid; solvent B: acetonitrile + 0.05% trifluoroacetic acid. Gradient:

[0425]

[0426]

[0427] Detection - UV (220 and 254 nm) and ELSD

[0428] Method N

[0429] The experiment was performed on a SHIMADZU LCMS-2020 using a C18 reverse phase column (50x3.0mm Poroshell HPH-C18, 2.7μm particle size) and the eluent was: solvent A: water + 5mM ammonium bicarbonate; solvent B: acetonitrile. Gradient:

[0430]

[0431] Detection - UV (220 and 254 nm) and ELSD

[0432] Method O

[0433] The experiment was performed on a SHIMADZU LCMS-2020 using a C18 reverse phase column (50x3.0mm Titank C18, 3.0μm particle size) and the eluent was: solvent A: water + 5mM ammonium bicarbonate; solvent B: acetonitrile. Gradient:

[0434]

[0435] Detection - UV (220 and 254 nm) and ELSD

[0436] Method P

[0437] The experiment was performed on a SHIMADZU LCMS-2020 using a C18 reverse phase column (30x2.1mm Halo C18, 2.0μm particle size) and the eluent was: solvent A: water + 0.05% trifluoroacetic acid; solvent B: acetonitrile + 0.05% trifluoroacetic acid. Gradient:

[0438]

[0439] Detection - UV (220 and 254 nm) and ELSD

[0440] Method Q

[0441] The experiment was performed on a SHIMADZU LCMS-2020 using a C18 reverse phase column (50x3.0mm YMC-Triart C18, 2.5μm particle size) and the eluents were: solvent A: water + 0.1% formic acid; solvent B: acetonitrile + 0.1% formic acid. Gradient:

[0442]

[0443] Detection - UV (220 and 254 nm) and ELSD

[0444] Method R

[0445] The experiment was performed on a SHIMADZU LCMS-2020 using a C18 reverse phase column (50x3mm Shim-Pack XR-ODS, 2.2μm particle size) and the eluent was: solvent A: water + 0.05% trifluoroacetic acid; solvent B: acetonitrile + 0.05% trifluoroacetic acid. Gradient:

[0446]

[0447] Detection - UV (220 and 254 nm) and ELSD

[0448] Method S

[0449] The experiment was performed on a SHIMADZU LCMS-2020 using a C18 reverse phase column (50x3.0mm Poroshell HPH-C18, 2.7μm particle size) and the eluent was: solvent A: water + 5mM ammonium bicarbonate; solvent B: acetonitrile. Gradient:

[0450]

[0451] Detection - UV (220 and 254 nm) and ELSD

[0452] Method T

[0453] The experiment was conducted on a SHIMADZU 20A HPLC using a C18 reverse phase column (50x2.1 mm Ascentis ExpressC18, 2.7 μm particle size) and the eluents were: solvent A: water + 0.05% trifluoroacetic acid; solvent B: acetonitrile + 0.05% trifluoroacetic acid. Gradient:

[0454]

[0455] Detection - UV (220 and 254 nm) and ELSD

[0456] Method U

[0457] The experiment was performed on a SHIMADZU LCMS-2020 using a C18 reverse phase column (50x3mm Shim-Pack XR-ODS, 2.2μm particle size) and the eluent was: solvent A: water + 0.05% trifluoroacetic acid; solvent B: acetonitrile + 0.05% trifluoroacetic acid. Gradient:

[0458]

[0459] Detection - UV (220 and 254 nm) and ELSD

[0460] Method V

[0461] The experiment was performed on a SHIMADZU LCMS-2020 using a C18 reverse phase column (50x3.0mm Poroshell HPH-C18, 2.7μm particle size) and the eluent was: solvent A: water + 5mM ammonium bicarbonate; solvent B: acetonitrile. Gradient:

[0462]

[0463] Detection - UV (220 and 254 nm) and ELSD

[0464] Method W

[0465] The experiment was performed on a SHIMADZU LCMS-2020 using a C18 reverse phase column (50x2.1mm Waters AcquityBEH, 1.7μm particle size) and the eluents were: solvent A: water + 0.1% formic acid; solvent B: acetonitrile + 0.1% formic acid. Gradient:

[0466]

[0467] Detection - UV (220 and 254 nm) and ELSD

[0468] Method X

[0469] The experiment used an Agilent 1290UHPLC coupled to an Agilent MSD (6140) mass spectrometer system, which used ESI as the ion source. A Phenomenex XB-C18, 1.7m, 50×2.1mm column was used for LC separation 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 with 2% B and ended with 98% B for 7min, and maintained at 98% B for 1.5min after equilibration for 1.5min. The LC column temperature was 40°C. UV absorbance was collected at 220nm and 254nm, and all experiments used full mass spectrometry scans.

[0470] List of common abbreviations

[0471] ACN Acetonitrile

[0472] Brine saturated sodium chloride aqueous solution

[0473] CH-3OD Deuterated methanol

[0474] CDCl3 deuterated chloroform

[0475] DCM Dichloromethane

[0476] DIEA or DIPEA Diisopropylethylamine

[0477] DMA Dimethylacetamide

[0478] DMAP 4-dimethylaminopyridine

[0479] DMF Dimethylformamide

[0480] DMSO Dimethyl sulfoxide

[0481] DMSO-d6 Dimethyl sulfoxide

[0482] DTAD Di-tert-butyl azodicarboxylate

[0483] EDC or EDCI 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

[0484] ESI Electrospray ionization

[0485] EtOAc Ethyl acetate

[0486] EtOH

[0487] FA Formic acid

[0488] HOAc Acetic acid

[0489] g

[0490] h hour

[0491] HATU O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate

[0492] HCl

[0493] HOBt Hydroxybenzotriazole

[0494] HPLC High Performance Liquid Chromatography

[0495] IMS Industrial Methylated Spirits

[0496] L Liter

[0497] LCMS liquid chromatography-mass spectrometry

[0498] LiHMDS or LHMDS Lithium Hexamethyldisilazide

[0499] MDAP mass spectrometry-guided automated purification

[0500] MeCN Acetonitrile

[0501] MeOH Methanol

[0502] μm micrometer

[0503] min

[0504] mg milligram

[0505] mL milliliters

[0506] mm Millimeters

[0507] M Moore

[0508] Nm nanometer

[0509] NMR Nuclear Magnetic Resonance Spectroscopy

[0510] Pd2(dba)3.CHCl3 Chloroform adduct Tris(dibenzylideneacetone)dipalladium(0)-chloroform adduct

[0511] PE Petroleum Ether

[0512] Preparative-HPLC Preparative High Performance Liquid Chromatography

[0513] PyAOP (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate

[0514] SCX-2 Strong Cation Exchange

[0515] TBAF Tetra-n-butylammonium fluoride

[0516] THF Tetrahydrofuran

[0517] TFA Trifluoroacetic acid

[0518] Xantphos 4,5-bis(diphenylphosphino)-9,9-dimethylxanthine

[0519] ZnCl2 Zinc Chloride

[0520] Intermediate 1

[0521]

[0522] N-[3-[2,5-bis(difluoromethoxy)phenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0523] Step 1: Synthesis of 1-(benzyloxy)-4-(difluoromethoxy)benzene

[0524]

[0525] N, N-dimethylformamide (1500mL), 4-(benzyloxy)phenol (200g, 999mmol), cesium carbonate (651g, 1.99mol) are put into a 3000mL round-bottom flask, which is purged and maintained with a nitrogen inert atmosphere. The reaction vessel is equipped with an outlet for CO2 release. Subsequently, 2-chloro-2,2-difluoroacetic acid sodium (228g, 1.50mol, 1.50 equivalents) is added several times at 120°C. The reactant is stirred in an oil bath at 120°C until gas stops escaping (about 1h), and then cooled to room temperature. The reaction mixture is slowly added to 3000mL water / ice under stirring. The resulting mixture is extracted with ethyl acetate (3x4000mL). The organic layers are merged, and dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue is purified by flash chromatography on silica gel, eluted with ethyl acetate / petroleum ether (1 / 19). The appropriate fractions were combined and concentrated under reduced pressure. This reaction was repeated four times. A total of 450 g (36%) of 1-(benzyloxy)-4-(difluoromethoxy)benzene as a white solid was produced.

[0526] Step 2: Synthesis of 4-(difluoromethoxy)phenol

[0527]

[0528] Methanol (1500 mL), 1-(benzyloxy)-4-(difluoromethoxy)benzene (140 g, 559 mmol), 10% palladium on carbon (15 g, 141 mmol) were placed in a 3000 mL round-bottom flask. The resulting mixture was stirred overnight under hydrogen (about 45 psi) at room temperature. The catalyst was filtered out. The filtrate was concentrated under reduced pressure. The reaction was repeated three times. 300 g (78%) of 4-(difluoromethoxy)phenol was obtained as a yellow oil.

[0529] Step 3: Synthesis of 2-bromo-4-(difluoromethoxy)phenol

[0530]

[0531] Acetic acid (500mL), 4-(difluoromethoxy)phenol (50g, 312mmol) and NBS (55.6g, 312mmol) are put into a 1000mL round-bottom flask. The reaction mixture is stirred for 1h at 15°C. Then the resulting mixture is slowly added to 1000mL water / ice under stirring. The resulting solution is extracted with ethyl acetate (3x1000mL). The organic layers are merged, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue is purified by flash chromatography on silica gel, eluted with dichloromethane / petroleum ether (30 / 70). Appropriate fractions are collected and concentrated under reduced pressure. 50g (67%) of light yellow oily 2-bromo-4-(difluoromethoxy)phenol is obtained.

[0532] Step 4: Synthesis of 2-bromo-1,4-bis(difluoromethoxy)benzene

[0533]

[0534] CH3CN (500mL), water (500mL), 2-bromo-4-(difluoromethoxy)phenol (54g, 226mmol) and potassium hydroxide (94g, 1.68mol) were placed in a 2000mL round-bottom flask. The flask was placed in an ice bath and the reaction mixture was stirred in an ice bath for 30min. Then diethyl (bromodifluoromethyl)phosphonate (120g, 449mmol) was added dropwise to the reaction mixture at 0°C. After the addition of diethyl (bromodifluoromethyl)phosphonate was completed, the reaction mixture was stirred in a water / ice bath for 1h. The resulting solution was extracted with ethyl acetate (3x300mL). The organic layers were merged, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel, eluted with ethyl acetate / petroleum ether (1 / 19), and appropriate fractions were collected and concentrated under reduced pressure. 54g (83%) of light yellow oily 2-bromo-1,4-bis (difluoromethoxy)benzene were obtained.

[0535] Step 5: Synthesis of 5-[2,5-bis(difluoromethoxy)phenyl]-4-nitro-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazole

[0536]

[0537] DMA (500mL), potassium carbonate (112g, 810mmol), 4-nitro-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazole (66g, 271mmol), 2-bromo-1,4-bis(difluoromethoxy)benzene (79g, 273mmol), 2,2-dimethylpropionic acid (8.3g, 81.3mmol), Pd(OAc)2 (6.0g, 26.7mmol) and bis(adamantan-1-yl)(butyl)phosphine (19g, 52.9mmol, 0.195 equivalents) were placed in a 1000mL round-bottom flask, which was purged and maintained with a nitrogen inert atmosphere. The reaction mixture was stirred overnight in an oil bath at 120°C and then cooled to room temperature. The reaction mixture was then added to 1000mL water / ice under stirring. The resulting solution was extracted with ethyl acetate (3x1000mL). 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). The appropriate fractions were collected and concentrated under reduced pressure. 100 g (82%) of 5-[2,5-bis(difluoromethoxy)phenyl]-4-nitro-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazole was obtained as a solid. LC / MS (Method H, ESI): [M+H] + =452.1,R T =1.49min

[0538] Step 6: Synthesis of 5-[2,5-bis(difluoromethoxy)phenyl]-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazol-4-amine

[0539]

[0540] Ethanol (1500mL), water (150mL), 5-[2,5-bis(difluoromethoxy)phenyl]-4-nitro-1-[[2]-(trimethylsilyl)ethoxy]methyl]-1H-pyrazole (100g, 221mmol), iron powder (124g, 2.22mol) and NH4Cl (59.2g, 1.11mol) were placed in a 3000mL 3-necked round-bottom flask. The resulting mixture was stirred in an oil bath at reflux temperature for 2h and then cooled to room temperature. The solid was filtered out and washed with ethanol. The filtrate was concentrated under reduced pressure. The residue was dissolved in 3000mL ethyl acetate. The ethyl acetate solution was washed with 1x1000mL brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure. 100 g of crude 5-[2,5-bis(difluoromethoxy)phenyl]-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazol-4-amine was obtained as a light yellow oil, which was used directly without purification. LC / MS (Method H, ESI): [M+H] + =422.1,R T =1.25min.

[0541] 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

[0542]

[0543] DMA (1000 mL), 5-[2,5-bis(difluoromethoxy)phenyl]-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazole-4-amine, pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (58.1 g, 356 mmol), 7-azabenzotriazol-1-yloxy)tripyrrolidinephosphonium hexafluorophosphate (PyAOP) (186 g, 356 mmol), 4-dimethylaminopyridine (2.90 g, 23.7 mmol) and DIPEA (92.0 g, 712 mmol) were placed in a 2000 mL round-bottom flask. The resulting solution was stirred overnight in an oil bath at 65 ° C. The reaction mixture was then slowly added to 2000 mL of water under stirring. The resulting solution was extracted with ethyl acetate (3x2000 mL). The combined organic phase was washed with 1000 mL of brine, 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 (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 =1.05min.

[0544] Step 8: Synthesis of N-(3-(2,5-bis(difluoromethoxy)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0545]

[0546] 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) were placed in a 2000 mL round-bottom flask. The resulting solution was stirred at 25 °C for 4 h. The solid was collected by filtration. The solid was added to a 1 L flask and H2O (200 mL) was added. Saturated NaHCO3 aqueous solution was added dropwise with stirring until the pH of the solution was about 8. 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 light yellow solid. 1H NMR (300MHz, CD3OD) δ9.08(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 = 8.9, 2.9Hz, 1H), 7.19 (dd, J = 6.7, 4.4Hz, 1H), 6.87 (t, J = 73.7Hz, 1H), 6.73 (t, J = 73.7Hz, 1H). LC / MS (Method H, ESI): [M+H] + =437.1,R T =1.12min.

[0547] Intermediate 2

[0548]

[0549] N-(5-(5-bromo-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0550] Step 1: Synthesis of 4-bromo-1-(difluoromethoxy)-2-iodobenzene

[0551]

[0552] 2-chloro-2,2-difluoroacetic acid sodium (216g, 1.42mol) and cesium carbonate (617g, 1.89mol) are added to a solution of 4-bromo-2-iodophenol (282g, 943mmol) in N,N-dimethylformamide (2000mL) and water (500mL). The reaction vessel is equipped with a gas outlet for CO2 release. The resulting mixture is stirred overnight at 120°C, cooled to room temperature and poured into ice water (3000mL). The resulting solution is extracted with ethyl acetate (3x1500mL) and the organic layer is combined. The ethyl acetate extract is washed with brine (1000mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue is purified by flash chromatography on silica gel, eluted with ethyl acetate / petroleum ether (1 / 10), to obtain 300g (91%) of yellow oily 4-bromo-1-(difluoromethoxy)-2-iodobenzene. 1 H NMR (300MHz, CDCl3) δ7.96 (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).

[0553] Step 2: Synthesis of 5-[5-bromo-2-(difluoromethoxy)phenyl]-4-nitro-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazole

[0554]

[0555] LiHMDS (490 mL, 1.0 mol / L THF) solution was added dropwise to a solution of 4-nitro-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazole (100 g, 411 mmol) in anhydrous THF (1000 mL) under stirring at -70 °C under nitrogen. The resulting solution was stirred at -50 °C for 1 h and then cooled to -70 °C. ZnCl2 (500 mL, 0.7 mol / L THF solution) was added dropwise at -70 °C. The resulting solution was warmed to room temperature and stirred at room temperature for 1 h. 4-bromo-1-(difluoromethoxy)-2-iodobenzene (150 g, 860 mmol), Pd(PPh3)4 (24.0 g, 20.8 mmol) were added to the mixture. The resulting solution was heated at reflux temperature overnight, cooled to room temperature, and concentrated under reduced pressure. The reaction of this scale was repeated once more, and the crude product from both runs was combined and purified. 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. A total of 300 g (79%) of 5-[5-bromo-2-(difluoromethoxy)phenyl]-4-nitro-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazole was obtained as a light yellow solid. 1 H NMR (300MHz, CDCl3) δ8.27(s,1H),7.68(dd,J=8.7,2.4Hz,1H),7.62(d,J=2.4Hz,1H),7.19(d,J=8.4H z,1H),6.39(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).

[0556] Step 3: Synthesis of 5-(5-bromo-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-amine

[0557]

[0558] To a solution of 5-(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) were added iron powder (60.1 g, 1.07 mol) and NH4Cl (28.0 g, 0.523 mol). The reaction mixture was stirred at reflux temperature under nitrogen 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 1x500 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-pyrazole-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 =0.93min.

[0559] 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

[0560]

[0561] To 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 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 drying 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 light yellow solid. LC / MS (Method G, ESI): [M+H] + =579.1&581.1,R T =1.10min. 1 H NMR (300MHz, CDCl3) δ9.62 (s, 1H), 8.80 (dd, J = 6.9, 1.7Hz, 1H), 8.73 (s, 1H), 8.53 ( dd,J=4.2,1.7Hz,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).

[0562] Intermediate 3

[0563]

[0564] N-[3-[5-Bromo-2-(difluoromethoxy)phenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0565] 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) overnight at room temperature. The resulting mixture was concentrated under reduced pressure. 3.80 g of N-[3-[5-bromo-2-(difluoromethoxy)phenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide was obtained as a yellow solid. The intermediate was pure enough for the next step without further purification. LC / MS (Method I, ESI): [M+H] + =449.0,R T =1.02min. 1 HNMR(400MHz,CD3OD)δ9.11(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).

[0566] Intermediate 4

[0567]

[0568] N-[3-[2-(Difluoromethoxy)-5-iodophenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0569] 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

[0570]

[0571] 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) under nitrogen was added N,N-dimethylethane-1,2-diamine (2.28 mg, 0.0259 mmol), NaI (155 mg, 1.04 mmol), CuI (4.93 mg, 0.026 mmol). The resulting solution was stirred at 120° C. under nitrogen in an oil bath for 14 h and then cooled to room temperature. The resulting 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 =1.31min.

[0572] Step 2: Synthesis of N-[3-[2-(difluoromethoxy)-5-iodophenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0573]

[0574] N-[5-[2-(difluoromethoxy)-5-iodophenyl]-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazol-4-yl]pyrazolo[1,5a]pyrimidine-3-carboxamide (80.0 mg, 0.128 mmol) was treated with CF3CO2H (3.0 mL) at room temperature for 30 min. The resulting mixture was concentrated under reduced pressure. The residue was dissolved in water. Saturated sodium bicarbonate was slowly added until the pH of the solution was adjusted to about 8. The solid was collected by filtration. The residue 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 light yellow solid. LC / MS (Method K, ESI): [M+H] + =497.1,R T =1.74min. 1H NMR(300MHz,CD3OD)δ9.08(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).

[0575] Intermediate 5

[0576]

[0577] N-[3-[5-Cyclopropyl-2-(difluoromethoxy)phenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0578] 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

[0579]

[0580] 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 (Intermediate 2, 1.40 g, 2.41 mmol) in dioxane (15 mL) and water (3.0 mL) was added cyclopropylboronic acid (314 mg, 3.66 mmol), Pd(dppf)Cl2-CH2Cl2 (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 resulting mixture was concentrated under reduced pressure. The residue was passed through a short silica gel pad and eluted with dichloromethane / methanol (94 / 6). The appropriate fractions were combined and concentrated under reduced pressure to give 1.40 g (purity = about 85% at 254 nm) of N-[3-[5-cyclopropyl-2-(difluoromethoxy)phenyl]-1-[[2-(trimethylsilyl)ethoxy]methyl]-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.

[0581] Step 2: Synthesis of N-[3-[5-cyclopropyl-2-(difluoromethoxy)phenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0582]

[0583] N-[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 h at 25° C. The solution was concentrated under reduced pressure. The residue was purified by Prep-HPLC under the following conditions: column: Xbridge C18, 19*150mm, 5um; mobile phase A: water / 0.05% NH4HCO3, mobile phase B: ACN; flow rate: 30mL / min; gradient: 20% B to 85% B in 10min; 254nm to obtain 44.9mg (41%) of N-[3-[5-cyclopropyl-2-(difluoromethoxy)phenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide as a yellow solid. LC / MS (Method H, ESI): [M+H] + =411.2,R T =1.14min. 1 H NMR(300MHz,CD3OD)δ9.09(dd,J=6.9,1.5Hz,1H),8.63–8.61(m,2H),8.27(s,1H),7.28–7.25(m,3H),7.2 0(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).

[0584] Intermediate 6

[0585]

[0586] Pyrazolo[l,5-a]pyrimidine-3-carboxylic acid [3-(5-chloro-2-difluoromethoxyphenyl)-1H-pyrazol-4-yl]amide

[0587] Step 1: Synthesis of 2-bromo-4-chloro-1-(difluoromethoxy)benzene

[0588]

[0589] To 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 reactant was stirred at 100 ° C for 16 hours. The reaction mixture was partitioned between ethyl acetate and water, the organic portion was diluted with brine, dried (MgSO4), and evaporated. The residue was purified by flash chromatography on silica gel, eluted with 0-20% EtOAc in heptane to give 2-bromo-4-chloro-1-(difluoromethoxy)benzene (2.98 g, 48%) as a clear, colorless oil. 1H NMR (400 MHz, DMSO-d6) δ: (ppm) 7.90 (d, 1H), 7.54 (dd, 1H), 7.38 (d, 1H), 7.28 (t, 1H).

[0590] Step 2: Synthesis of 5-(5-chloro-2-difluoromethoxyphenyl)-4-nitro-1-(2-trimethylsilylethoxymethyl)-1H-pyrazole

[0591]

[0592] To a solution of 4-nitro-1-(2-trimethylsilylethoxymethyl)-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 cooled to room temperature, diluted with ethyl acetate, washed with water and brine, dried (MgSO4), filtered and evaporated. The resulting crude material was purified by flash chromatography on silica gel eluting with 0-10% EtOAc in cyclohexane to afford 5-(5-chloro-2-difluoromethoxyphenyl)-4-nitro-1-(2-trimethylsilanylethoxymethyl)-1H-pyrazole (62.4 g, 78%). 1 H NMR (400MHz, CDCl3) 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).

[0593] Step 3: Synthesis of 5-(5-chloro-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-amine

[0594]

[0595] To a solution of 5-(5-chloro-2-difluoromethoxyphenyl)-4-nitro-1-(2-trimethylsilylethoxymethyl)-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 hours and then allowed to cool to room temperature. The residual solid was removed by using The 5-(5-chloro-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-4-amine (30.8g, 54%) was added to the 4-(4-chloro-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-4-amine (30.8g, 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).

[0596] 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

[0597]

[0598] A 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 minutes to an ice / water cooled mixture of pyrazolo[1,5-a]pyrimidine-3-carbonyl chloride (27.8 g, 153 mmol) and DIPEA (49.5 g, 383 mmol) in THF (300 mL). After the addition was complete, the mixture was stirred 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 The residual solid was removed and the filtrate was washed with 1M aqueous potassium carbonate solution, water and brine, dried (sodium sulfate) and evaporated to give a red solid. The solid was triturated with 10% ether in cyclohexane. The solid was collected by filtration, washed with 1:1 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 (59.2 g, 73%) as an off-white solid. 1 H NMR (300MHz, CDCl3): 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), 7.3 6(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).

[0599] Step 5: Synthesis of N-(3-(5-chloro-2-(difluoromethoxy)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0600]

[0601] 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 was triturated with water. The solid was collected by filtration, washed with water and air-dried. The solid was triturated with a minimum volume of acetonitrile, the solid was collected by filtration, washed with ether and dried at 60 °C under high vacuum to give the title compound (42.9 g, 96%) as a yellow solid. 1 H NMR(400MHz,DMSO-d6)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).

[0602] Intermediate 7

[0603]

[0604] N-(3-(2-(difluoromethoxy)-5-(methylthio)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0605] Step 1: Synthesis of 5-(2-(difluoromethoxy)-5-(methylthio)phenyl)-4-nitro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole

[0606]

[0607] 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), Pd2(dba)3.CHCl3 (6.7 g, 6.47 mmol), XantPhos (7.5 g, 12.96 mmol) were placed in a 1000 mL round-bottom flask, which was purged and maintained with an inert atmosphere of nitrogen. 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 applied to a silica gel column and eluted with ethyl acetate / petroleum ether (1:20). The appropriate fractions were combined and concentrated under vacuum. A total of 171 g of 5-(2-(difluoromethoxy)-5-(methylthio)phenyl)-4-nitro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole was obtained as a yellow solid. LC / MS (Method F, ESI): [M+H]+=432.1, RT=1.23 min; 1H NMR (300MHz, CDC13) δ: (ppm) 8.25 (s, 1H), 7.42 (dd, J = 8.7, 2.4Hz, 1H), 7.34 (d, J = 2.1Hz, 1H), 7.23 (d, J = 8.7Hz, 1H ), 6.39(t,J=72.9Hz,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).

[0608] Step 2: Synthesis of 5-(2-(difluoromethoxy)-5-(methylthio)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-amine

[0609]

[0610] Iron powder (228 g, 4.08 mol) and NH4Cl (120 g, 2.24 mol) were added to a mixture of 5-[2-(difluoromethoxy)-5-(methylsulfanyl)phenyl]-4-nitro-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazole (171 g, 408 mmol), ethanol (2000 mL) and water (200 mL). The reaction mixture was stirred and refluxed for 3 h under nitrogen and cooled to room temperature. The solid was filtered out. The filtrate was concentrated under vacuum. The residue was dissolved in 3000 mL of ethyl acetate and washed with 1x500 mL of brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under vacuum. 148 g of 5-(2-(difluoromethoxy)-5-(methylsulfanyl)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-4-amine was obtained as a yellow oil. LC / MS (method F, ESI): [M+H]+=402.1, R T =0.93min.

[0611] 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

[0612]

[0613] DMA (1500 mL), 5-(2-(difluoromethoxy)-5-(methylthio)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-amine (148 g), pyrazolo[l,5-a]pyrimidine-3-carboxylic acid (102 g), HATU (325 g), 4-dimethylaminopyridine (4.5 g), DIPEA (142 g) were placed in a 3000 mL 3-necked round-bottom flask. 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 and eluted with ethyl acetate / petroleum ether (4:1) to give 200 g of N-(5-(2-(difluoromethoxy)-5-(methylthio)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide as a light yellow solid. LC / MS (Method A, ESI): [M+H]+=547.2, RT=1.10 min; 1H NMR (300MHz, CDC13) 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) ,5.46–5.38(m,2H),3.70–3.59(m,2H),2.52(s,3H),0.92-0.85(m,2H),0.03(s,9H).

[0614] Step 4: Synthesis of N-(3-(2-difluoromethoxy)-5-(methylthio)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0615]

[0616] To a solution of N-(5-(2-(difluoromethoxy)-5-(methylthio)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-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 of the solution was adjusted to 8 with saturated sodium bicarbonate. The product was collected by filtration and dried to give 30 g (66%) of N-(3-(2-(difluoromethoxy)-5-(methylthio)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide as a light yellow solid. LC / MS (method G, ESI): [M+H]+=417.0, R T =0.80min; 1 H NMR(300MHz, DMSO-d6)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).

[0617] Intermediate 8

[0618]

[0619] 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

[0620] Step 1: Synthesis of 4-bromo-5-chloro-2-iodophenol

[0621]

[0622] Under stirring, 70 ℃, add CuBr2 (264g, 1.18mol) several times to the solution of 5-chloro-2-iodophenol (100g, 393mmol) in acetonitrile (1000mL). The resulting mixture is stirred at 70 ℃ for 4h, cooled to room temperature and concentrated under vacuum. The residue is then quenched by adding 3000mL water / ice, and the organic layers are merged by extracting with 3x2000mL ethyl acetate. The extract is washed with 1x1000mL brine, dried over sodium sulfate, and concentrated under vacuum. The residue is purified by flash chromatography on silica gel, eluted with ethyl acetate / petroleum ether (1:30). Collect appropriate fractions and concentrate under vacuum. The reaction is repeated more than once. 140g (53%) of 4-bromo-5-chloro-2-iodophenol as a white solid is obtained. 1 H NMR (400MHz, CDCl3): δ (ppm) 7.89 (s, 1H), 7.14 (s, 1H), 5.32 (s, 1H).

[0623] Step 2: Synthesis of 1-bromo-2-chloro-4-(difluoromethoxy)-5-iodobenzene

[0624]

[0625] 2-Chloro-2,2-difluoroacetic acid sodium (95.8g, 628mmol), cesium carbonate (274g, 840mmol) were added to a solution of 4-bromo-5-chloro-2-iodophenol (140g, 420mmol) in DMF (1200mL). The reaction mixture was stirred in an oil bath at 120 ° C for 2h, cooled to room temperature, and then quenched by adding 2500mL water / ice. The resulting solution was extracted with 3x2000mL ethyl acetate, and the organic layers were merged. The extract was washed with 1x1000mL brine, dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was purified by flash chromatography on silica gel, eluted with ethyl acetate / petroleum ether (1 / 30). Appropriate fractions were combined and concentrated under vacuum to obtain 130g (81%) of light yellow solid l-bromo-2-chloro-4-(difluoromethoxy)-5-iodobenzene.

[0626] Step 3: Synthesis of 5-(5-bromo-4-chloro-2-(difluoromethoxy)phenyl)-4-nitro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole

[0627]

[0628] LiHMDS (340 mL, 1 M THF) was added dropwise to a solution of 4-nitro-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazole (67.0 g, 275 mmol) in tetrahydrofuran (1000 mL) under stirring at -70°C under nitrogen. The resulting solution was stirred at -70°C for 1 h. ZnCl2 (400 ml, 0.7 M THF solution) was added dropwise to the solution under stirring at -70°C. The resulting solution was stirred under nitrogen for 1 h at -70°C. 1-Bromo-2-chloro-4-(difluoromethoxy)-5-iodobenzene (105 g, 274 mmol) and Pd(PPh3)4 (16.0 g, 13.9 mmol) were added to the mixture under nitrogen. The resulting solution was stirred at 90°C overnight, cooled 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). The appropriate fractions were collected and concentrated under vacuum. 115 g (84%) of 5-[5-bromo-4-chloro-2-(difluoromethoxy)phenyl]-4-nitro-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazole was obtained as a light yellow solid. LC / MS (Method B, ESI): [M+H]+=498.0 & 500.0, Rt=1.27 min.

[0629] Step 4: Synthesis of 5-[5-bromo-4-chloro-2-(difluoromethoxy)phenyl]-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazol-4-amine

[0630]

[0631] To a solution of 5-(5-bromo-4-chloro-2-(difluoromethoxy)phenyl)-4-nitro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole (102g, 205mmol) in ethanol (1000mL) and water (100mL) was added iron powder (102g, 1.82mol) and ammonium chloride (53g, 1.00mol). At 100°C, the reaction mixture was stirred in an oil bath for 3h. The solid was filtered off. The filtrate was concentrated under vacuum. The residue was dissolved in 2000mL ethyl acetate. The organic solution was washed with 1x500mL brine, dried over anhydrous sodium sulfate and concentrated under vacuum. 102g (crude) light yellow oily 5-[5-bromo-4-chloro-2-(difluoromethoxy)phenyl]-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazole-4-amine was obtained. LC / MS (method E, ESI): [M+H]+=467.9 & 469.9, R T =1.29min.

[0632] 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

[0633]

[0634] To a solution of 5-[5-bromo-4-chloro-2-(difluoromethoxy)phenyl]-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazole-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 in an oil bath at 60 °C. The reactants were then quenched by adding 2000 mL of water / ice. The resulting solution was extracted with 3x1500 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. 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 was obtained as an off-white solid. LC / MS (Method A, ESI): [M+H]+=613.2&615.2, RT=2.29 min. 1 H NMR (400MHz, CDCl3): δ (ppm) 9.56 (s, 1H), 8.81 (dd, J = 6.8, 1.5Hz, 1H), 8.73 (s, 1H), 8.53 (d, J = 4.0Hz, 1H), 8.33 (s, 1H), 7.92 (s, 1H), 7.54 (s, 1H), 7.0 3(dd,J=6.8Hz,4.0Hz,1H),6.45(t,J=72.2Hz,1H),5.43(d,J=11.2Hz,1H), 5.35(d,J=11.2Hz,1H),3.68-3.56(m,2H),0.94-0.84(m,2H),0.00(s,9H).

[0635] Intermediate 9

[0636]

[0637] 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

[0638] Step 1: Synthesis 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

[0639]

[0640] To a solution of intermediate 8 (200 mg, 0.326 mmol) in toluene (10 mL) under nitrogen was added 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). The resulting solution was stirred at 60 ° C for 4 h and concentrated under vacuum. The residue was purified by flash chromatography on silica gel 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.

[0641] 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

[0642]

[0643] 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) under nitrogen was added BrettPhos Palladacycle Gen.3 (CAS1470372-59-8, supplier 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). The resulting solution was stirred at 110 °C for 20 h, 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 / 1). The 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.

[0644] 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

[0645]

[0646] 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 aqueous solution) (4.0 mL). The resulting solution was stirred at 25 °C for 4 h and concentrated under vacuum. The crude product (50.0 mg) was purified by Prep-HPLC under the following conditions: column, XBridge Shield RP18OBD column, 19*150 mm, 5 um; mobile phase, 10 mM NH4HCO3 aqueous solution and acetonitrile (10.0% acetonitrile up to 38.0% in 10 min); detector, UV 254 nm to obtain 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 HNMR (400MHz, CD3OD): δ (ppm) 8.98 (d, J = 6.8 Hz, 1H), 8.57-8.51 (m, 2H), 8.12 (s, 1H), 7.10 (dd, J = 7.0 ,4.2Hz,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).

[0647] Intermediate 10

[0648]

[0649] N-(1-((2H-tetrazol-5-yl)methyl)-3-(2,5-bis(difluoromethoxy)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0650] Step 1: Synthesis of red oily 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. (Mixture of positional isomers)

[0651]

[0652] To 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-tetrazolyl (11.6 g, 57.3 mmol) at room temperature. The reaction mixture was stirred for 1.5 h, after which it was washed with brine (300 mL) and extracted with ethyl acetate (3x300 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 flash chromatography on silica gel eluting with DCM (1% TEA) / EA (1 / 2) to give 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. LC / MS (Method I, ESI): [M+H]+=603.25, Rt=1.02 min

[0653] 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

[0654]

[0655] 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 overnight at room temperature. 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, yield: 69.2%) as a white solid. LC / MS (Method I, ESI): [M+H]+=519.3, Rt=0.71 min.

[0656] Intermediate 11

[0657]

[0658] N-(1-((2H-tetrazol-5-yl)methyl)-3-(2-(difluoromethoxy)-5-(methylthio)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0659] 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 tetrazolylated isomers)

[0660]

[0661] A 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-tetrazolyl (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 along with tetrazolylated regioisomers as a white solid (3.51 g, 6.02 mmol). LC / MS (Method M, ESI): [M+H]+=583.1, Rt=0.66 min.

[0662] 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

[0663]

[0664] 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. 1,4-Dioxane (80 mL) (4M HCl) was then 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

[0665] Intermediate 12

[0666]

[0667] N-(1-((2H-tetrazol-5-yl)methyl)-3-(5-chloro-2-(difluoromethoxy)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0668] 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 tetrazolylated isomers)

[0669]

[0670] To 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-tetrazolyl (4.05 g, 20.0 mmol) at room temperature. The resulting solution was stirred at room temperature for 2 hours. 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 layer was concentrated under vacuum. The residue was purified by flash chromatography on silica gel eluting with EA / DCM (24%) to give 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, yield: 67.3%) as a yellow oil together with a mixture of tetrazolylated regioisomers. LC / MS (Method M, ESI): [M+H]+=471.1, RT=0.66 min

[0671] 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

[0672]

[0673] 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 4M 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 flash chromatography on C18 gel eluting with ACN / H2O(TFA) (35%) to give 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, yield: 74.4%) as a yellow solid. LC / MS (Method H, ESI): [M+H]+=487.1, RT=1.12 min.

[0674] Intermediate 13

[0675]

[0676] N-(1-((2H-tetrazol-5-yl)methyl)-3-(5-cyclopropyl-2-(difluoromethoxy)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0677] 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

[0678]

[0679] Step 1: Synthesis of N-[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

[0680] 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-tetrazolyl (3.75 g, 18.5 mmol) at room temperature. The resulting solution was stirred at room temperature for 1.5 hours. The residue was filtered 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, eluted 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 tetrazolylated regioisomers (3.26 g, 5.65 mmol, yield: 76.8%) as a yellow oil. LC / MS (Method G, ESI): [M+H]+ = 577.2, RT = 0.98 min.

[0681] Step 2: Synthesis of N-[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

[0682]

[0683] 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 4M HCl / methanol (20 mL) was stirred at room temperature for 2 h. The resulting solution was concentrated under vacuum. The residue was purified by reverse phase chromatography (acetonitrile 0-45 / water 0.1% TFA in water) to give a pale yellow solid 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, yield: 66.5%). LC / MS (Method H, ESI): [M+H]+=493.2, RT=1.16 min.

[0684] Examples

[0685] Example 1

[0686]

[0687] N-(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

[0688] N-[3-[5-chloro-2-(difluoromethoxy)phenyl]-1-(2H-1,2,3,4-tetrazolyl-5-ylmethyl)-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (Intermediate 12, 200 mg, 0.412 mmol), 1,3-dioxolane-2-one (122 mg, 1.38 mmol, 3.35 equiv), sodium hydroxide (44 mg, 1.10 mmol, 2.67 equiv), N,N-dimethylformamide (30 mL) were placed in a 100 mL round-bottom flask. The resulting solution was stirred in an oil bath at 120 ° C for 4 h. The resulting mixture was concentrated under vacuum. The residue was applied to a silica gel column using dichloromethane / petroleum ether (11.5:1). The crude product was purified by chiral Prep-HPLC (Prep-HPLC-009) under the following conditions: column, Phenomenex Lux 5u Cellulose-4XIAPacked, 2.12*25cm, 5um; mobile phase, Hex and ethanol (maintained at 60.0% ethanol in 32min); detector, UV 220 / 254nm. 19.6mg (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 was obtained as a white solid. LC / MS (Method N, ESI): [M+H]+=531.2, RT=1.33min. 1 H NMR (300MHz, DMSO-d6): 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,4H),5.78(s,2H),5.07(t,J=5.6Hz,1H),4.72(t,J=5.1Hz,2H),3.94–3.88(m,2H).

[0689] Example 21

[0690] 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

[0691]

[0692] 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

[0693]

[0694] 1-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 cooled to room temperature and heated to 40 ° C for 1 h. Filter. Dilute the filtrate with brine (120 mL). Extract the resulting mixture with EA (3 x 50 mL) and combine the organic layers. Wash the organic layer with brine (2 x 50 mL). The residue was purified by flash chromatography on silica gel 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 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.

[0695] 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% NH4HCO3 aqueous 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, yield: 41.2%) as a white solid. LC / MS (Method C, ESI): [M+H]+=674.2 RT=2.63 min.

[0696] 330 mg of the mixture of diastereomers was purified by flash chromatography on silica gel 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, yield: 5.8%) as a yellow solid. LC / MS (Method G, ESI): [M+H]+ = 674.2 RT = 0.97 min.

[0697] 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

[0698]

[0699] A solution 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) in 2,2,2-trifluoroacetic acid (3 mL) / dichloromethane (12 mL) was stirred at room temperature for 2 h. The reaction mixture was concentrated under vacuum and the resulting product was used without further purification. LC / MS (Method R, ESI): [M+H]+=574.2 RT=1.58 min.

[0700] 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

[0701]

[0702] To 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) was added potassium carbonate (593 mg, 4.29 mmol) 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 purified by flash chromatography on a C18 column eluting with 42% ACN / NH4HCO3 (0.05%) to give 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, yield: 69.8%) as a yellow oil. LC / MS (Method M, ESI): [M+H]+=717.4, RT=0.67 min.

[0703] 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

[0704]

[0705] 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 under vacuum. The residue was purified by flash chromatography on a C18 column eluting with 45% ACN / water (0.05% HCl) to afford 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, yield: 99.3%) as a yellow oil. LC / MS (Method M, ESI): [M+H]+=617.4, RT=0.54 min.

[0706] 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

[0707]

[0708] To a 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) was added HCHO / H2O (256 mg, 3.15 mmol) at room temperature. The resulting solution was stirred at 25°C for 2 h. NaBH(AcO)3 (846 mg, 3.99 mmol) was added and the reaction mixture was stirred at 25°C for 3 h before it was concentrated under vacuum. The residue was purified by flash chromatography on silica gel eluting with (0.05% NH4HCO3). A slurry of 560 mg of the product in ethanol (4 mL) was slowly stirred at room temperature for 2 days to give N-[3-[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, yield: 66.4%) as a white solid. LC / MS (Method X, ESI): [M+H]+=645.3, RT=3.38 min. 1 H NMR (400MHz, DMSO-d6): 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,5H),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).

[0709] Example 10

[0710]

[0711] N-(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

[0712] A solution of N-[1-[[2-(azetidine-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) and HCHO / H2O (113 mg, 1.4 mmol) in methanol (10 mL) was stirred at 25 ° C for 2 h. NaBH(CH3COO)3 (301 mg, 1.42 mmol) was then added and stirred overnight at 25 ° C. 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 layer was dried over anhydrous sodium sulfate and concentrated under vacuum. The product was purified by Prep-HPLC under the following conditions: column: XBridge Prep OBD C18 column 30x; 150mm 5um; mobile phase A: water (10mmol / L NH4HCO3), mobile phase B: ACN; flow rate: 60mL / min; gradient: 27% B to 37% B in 10min; 254 / 220nm; Rt: 13min to obtain 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 (507mg, 0.858mmol, yield: 73.4%) as a white solid. LC / MS (Method E, ESI): [M+H]+=588.2, RT=2.28min. 1 HNMR (400MHz, DMSO-d6): 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) ),7.50–7.00(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).

[0713] Example 12

[0714]

[0715] N-(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

[0716] 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 flash chromatography on silica gel, eluted with H2O (0.1% TFA) / ACN (69%) to give yellow oily N-[1-[[1-(azetidine-3-yl)tetrazol-5-yl]methyl]-3-[2,5-bis(difluoromethoxy)phenyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (184 mg). LC / MS (Method H, ESI): [M+H]+=574.2, RT=0.97 min. 1 H NMR (300MHz, DMSO-d6): 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).

[0717] Example 78

[0718]

[0719] N-(3-(2,5-bis(difluoromethoxy)phenyl)-1-((2-(1'-methyl-[1,3'-diazetidine]-3-yl)-2H-tetrazol-5-yl)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0720] 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'-aziridine]-1'-carboxylate

[0721]

[0722] 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 (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 for 2 h at 25 ° C. NaBH(OAc) 3 (1.1 g, 5.19 mmol) was then added and the mixture was stirred for 3 h at room temperature. After 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% NH4HCO3), 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-carboxamido)-1H-pyrazol-1-yl)methyl)-2H-tetrazol-2-yl)-[1,3'-aziridine]-1'-carboxylate (650 mg, 0.890 mmol, yield: 50.1%) as a pale yellow solid. LC / MS (Method P, ESI): [M+H]+=729.15, RT=0.85 min.

[0723] Step 2: Synthesis of N-(1-((2-([1,3'-azidetidine]-3-yl)-2H-tetrazol-5-yl)methyl)-3-(2,5-bis(difluoromethoxy)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0724]

[0725] 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]azetidine-1-yl]azetidine-1-carboxylate (650 mg, 0.890 mmol) in dichloromethane (6 mL) and 2,2,2-trifluoroacetic acid (2 mL) was stirred for 3 h at room temperature. After the reaction was complete, the solvent was removed under vacuum to give 600 mg of the crude product as a yellow solid. LC / MS (Method G, ESI): [M+H]+=629.2, RT=0.92 min.

[0726] Step 3: Synthesis of N-(3-(2,5-bis(difluoromethoxy)phenyl)-1-((2-(1'-methyl-[1,3'-aziridine]-3-yl)-2H-tetrazol-5-yl)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0727]

[0728] A solution of N-(1-((2-([1,3'-azididine]-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) and formaldehyde / water (2 mL) in a mixture of acetic acid (1 mL) and methanol (10 mL) was stirred at room temperature for 1 h. NaBH(OAc)3 (1.0 g, 4.72 mmol) was then added and the mixture was stirred for 3 h. After 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% NH4HCO3), 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'-aziridine]-3-yl)-2H-tetrazol-5-yl)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (400 mg, 0.603 mmol, yield: 63.3%) as a pale yellow solid. LC / MS (Method O, ESI): [M+H]+=643.3, RT=1.42 min. 1 H NMR (300MHz, DMSO-d6): 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),7.53–6.96(m,6H),5.82(s,2H),5.64–5.59(m,1H),3.87–3.82(m,2H),3.71–3.35(m,10H).

[0729] Example 51

[0730]

[0731] 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

[0732] 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

[0733]

[0734] At room temperature N-[1-[[2-(azetidine-3-yl)tetrazol-5-yl]methyl]-3-[2,5-bis(difluoromethoxy)phenyl]pyrazole-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (500mg, 0.870mmol), acetic acid (104mg, 1.74mmol) and N-(3-oxopropyl)carbamic acid tert-butyl ester (302mg, 1.74mmol) in methanol (8mL) solution. The resulting solution was stirred at room temperature for 2 hours. Then NaBH(AcO)3(555mg, 2.62mmol) was added and stirred at room temperature for 3h. The reaction mixture was concentrated under vacuum. The residue was purified by flash chromatography on silica gel eluting with ACN / water (0.05% NH4HCO3) (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, yield: 73.8%) as a yellow oil. LC / MS (Method Q, ESI): [M+H]+=731.3, RT=1.35 min.

[0735] 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

[0736]

[0737] 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) was added at room temperature. The resulting solution was stirred at room temperature for 2 hours. The reaction mixture was concentrated under vacuum. The residue was purified by flash chromatography on a C18 column gel, 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, yield: 61.1%) as a yellow oil. LC / MS (Method M, ESI): [M+H]+=631.4, RT=0.55 min.

[0738] 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

[0739]

[0740] A solution of HCHO / H2O (39.4 mg, 0.490 mmol) and 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) in methanol (5 mL) was added at room temperature. The resulting solution was stirred at 25 °C for 2 h. NaBH(AcO)3 (114 mg, 0.540 mmol) was then added and stirred at 25 °C for 3 h. NaBH3CN (14 mg, 0.23 mmol) was then added and the mixture was stirred for 1 h. The reaction mixture was concentrated under vacuum. The residue was purified by HPLC under the following conditions: column: XBridge Prep OBD C18 column, 19*250mm, 5um; mobile phase A: water (10mmol / L H4HCO3), mobile phase B: ACN; flow rate: 25mL / min; gradient: 20B to 50B in 7min; 254nm; RT1: 5.88; to obtain 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 (25.4mg, 0.038mmol, yield: 27.9%) as a yellow solid. LC / MS (method A, ESI): [M+H]+=659.3, RT=1.17min, 1 H NMR (300MHz, DMSO-d6): δ (ppm) 9.78 (s, 1H), 9.36 (dd, J = 9.4, 2.2Hz, 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).

[0741] Example 80

[0742]

[0743] 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

[0744]

[0745] 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. NaBH(OAc)3 (148 mg, 0.700 mmol) was then added and the mixture was stirred at room temperature for another 1 h. NaBH3CN (21.9 mg, 0.350 mmol) was then added and the mixture was stirred at room temperature for another 1 h. After the reaction was complete, 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, yield: 51.5%) as a pale yellow solid. LC / MS (Method H, ESI): [M+H]+=757.3, RT=1.10 min.

[0746] Step 2: 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

[0747]

[0748] A solution of tert-butyl N-[1-[2-[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]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 h. 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.

[0749] Step 3: 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

[0750]

[0751] 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 h. NaBH(OAc)3 (154 mg, 0.730 mmol) was then added and stirred at 35°C for 3 h. Finally, NaBH3CN (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 products were purified by HPLC: YMC-Actus Triart C18, 30*250, 5um; Mobile phase A: water (10mmol / L NH4HCO3), Mobile phase B: ACN; Flow rate: 60mL / min; Gradient: 38B to 44B in 7min; 220nm to afford 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 (15mg, 0.022mmol, Yield: 6%). LC / MS (Method H, ESI): [M+H]+=685.3, RT=0.94min.1 H NMR (400MHz, DMSO-d6): 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), 5.8 0(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).

[0752] Example 83

[0753]

[0754] 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

[0755] 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

[0756]

[0757] 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. NaBH(OAc)3 (333 mg, 1.57 mmol) was then added and the mixture was stirred for an additional 2 h. After the reaction was complete, the organic solvent was removed under vacuum. The residue was purified by reverse phase separation column [mobile phase A: water (0.1% NH4HCO3), 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, yield: 74%) as a pale yellow solid. LC / MS (Method Q, ESI): [M+H]+=749.25, RT=1.98 min.

[0758] 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

[0759]

[0760] 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. After completion of the reaction, the solvent was concentrated under vacuum. The residue was purified by Prep Chiral HPLC [column: hiralpak 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; number of runs: 12] to obtain two stereoisomers (65 mg (peak 1) and 52 mg (peak 2)). LC / MS (method I, ESI): [M+H]+=649.3, RT=0.96 min.

[0761] 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

[0762]

[0763] A 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 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. NaBH3CN (5.0 mg, 0.08 mmol) was added and the mixture was stirred at room temperature for another 1 h. The solvent was removed under vacuum. By Prep-HPLC [column: Xselect CSH OBD column 30*150mm 5um; mobile phase A: water (10mmol / L NH4HCO3), mobile phase B: ACN:MeOH=4:1; flow rate: 60mL / min; gradient: 27B to 49B in 11min; 220nm; RT1: 10.08, 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 (10.6mg, 0.016mmol, yield: 19.3%) was obtained as a light yellow solid. LC / MS (Method A, ESI): [M+H]+=677.2, RT=1.24 min. 1 H NMR (400MHz, CDCl3): δ (ppm) 9.87 (s, 1H), 8.80 (dd, J = 7.2, 1.6Hz, 1H), – 9.38 (m, 1H), 8.72 (s, 1H),8.54–8.52(m,2H),7.47(d,J=2.8Hz,1H),7.36–7.33(m,1H),7.25–7.22(m,1H),7.03–7.0 0(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).

[0764] Determination

[0765] Test reagent

[0766] The test agent samples were provided as 10 mM dimethyl sulfoxide (DMSO) solutions and stored in the dark at room temperature until use.

[0767] Biochemical assays of JAK1 and JAK2

[0768] In vitro biochemical assays to quantify JAK-catalyzed phosphorylation of synthetic peptides, such as using EZ ReaderII microfluidic flow transfer instrument (PerkinElmer; Waltham, MA) detected. Substrate peptide Y-1B has the sequence 5-FAM-VALVDGYFRLTT-NH2. 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. The substrate peptide stock solution was prepared at 5mM with DMSO. The purified recombinant human JAK1 kinase domain protein (residues 854-1154) was expressed in insect cells and obtained from Proteros Biostructures GmbH (Martinsried, Germany). The recombinant human JAK2 kinase domain protein (residues 812-1132) was expressed in insect cells and purified at Genentech, Inc. (South San Francisco, CA).

[0769] The kinase reaction mixture contained 100 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 7.2), 10 mM magnesium chloride, 0.015% 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 of test compound in DMSO at a final concentration of 2% (volume to volume [v / v]). In each titration experiment, the test compound was tested in duplicate at each of the twelve concentrations. Blank reactions contained ATP, peptide, and DMSO, but no JAK or test compound, while uncontrolled control reactions contained ATP, peptide, JAK, and DMSO, but no test compound.

[0770] The peptide and ATP mixture (24 μL) was added to the test compound (or DMSO alone) in 1 μL DMSO. The reaction was initiated by adding 25 μL of the JAK enzyme to the inhibitor / peptide / ATP mixture and then mixing the resulting solution thoroughly. The reaction was incubated in a final volume of 50 μL per well of a 384-well plate at room temperature (22°C-23°C). After incubation for 30 minutes, the reaction was terminated 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.

[0771] In each reaction, the residual Y-1B substrate and the generated phospho-peptide product were separated using an EZ Reader II instrument. The electrophoretic separation of the product molecule and the substrate molecule was achieved using -500V and -2600V downstream and upstream voltages respectively under a -1.3psi operating pressure. The 5-FAM group present in the substrate and product peptide was excited at 488nm, and fluorescence was detected at 530nm, and the peak height was reported.

[0772] Data analysis

[0773] The degree (or percentage) of conversion of substrate to product was calculated from the corresponding peak height in the electropherogram obtained using HTS Well Analyzer software version 5.2 (PerkinElmer) and the following equation (Equation 1):

[0774] Equation 1

[0775] % conversion = [P ÷ (S + P)] × 100

[0776] where S and P represent the peak heights of substrate and product, respectively. After subtracting any baseline signal from blank wells without JAK from the signals of all test wells, the % conversion data were converted to fractional activity as shown in Equation 2, where v i and v o The % conversion observed in the uninhibited control reaction containing JAK and DMSO vehicle but no test compound is defined as having a fractional activity = 1 (in the absence of inhibitor, v i =v o , and blank wells without JAK were defined as having fractional activity = 0. Fractional activity was plotted against the concentration of test compound, and the data were fitted to the tight binding apparent inhibition constant (K) using XLfit software (IDBS; Guildford, United Kingdom). i 表观) quadratic equation (see Equation 2) (Williams JW, Morrison JF. The kinetics of reversible tight-binding inhibition. Methods Enzymol 1979; 63: 437-67.), which is used to calculate the fractional activity and K i 表观

[0777] Equation 2

[0778]

[0779] Where [E] T and[I] T are the total concentrations of active enzyme (initial estimates were 0.15 nM for JAK1 and 0.048 nM for JAK2) and inhibitor (variable parameter), respectively. Finally, K i By applying the competitive inhibition relationship (Equation 3) from K i 表观 Calculated.

[0780] Equation 3

[0781] K i =K i 表观 / (1+[ATP] / K m 表观 )

[0782] Where [ATP] is the concentration of ATP = 25 μM, K m 表观 is the ATP Michaelis constant, which for JAK1 is 32.1 μM, and for JAK2, K m 表观 = 11.7 μM. By applying the tight binding equation 2 that takes into account any consumption of the inhibitor and the competitive inhibition relationship equation 3, the sensitivity of the assay can be extended to at least the K calculated for JAK1. i 0.008nM and 0.0015nM for JAK2.

[0783] Kinase selectivity

[0784] The in vitro kinase selectivity of the test agents at 1 μM concentration was evaluated in a panel of recombinant human kinase activity and binding assays, including cytoplasmic and receptor tyrosine kinases, serine / threonine kinases, and lipid kinases ( Kinase Profiling Services, ThermoFisher Scientific, Madison, WI). Kinase activity assays measure peptide phosphorylation (Z'- ) or ADP production The binding assay monitors the displacement of the ATP site binding probe The ATP concentration used in activity assays is usually determined experimentally based on the apparent Michaelis constant (K) for each kinase. m 表观 ) value, while the competitive binding trace concentration used in binding assays is usually within 2 times of the experimentally determined dissociation constant (K d ) value. Inhibitors were tested in duplicate for each kinase and the average of the % inhibition values ​​was reported. For kinases that were inhibited by nearly or greater than 50% at the initial 1-μM test concentration, a 10-point inhibitor titration was performed using the same assay to determine the inhibitor that caused 50% inhibition (IC 50 ). The total JAK1 concentration used in this assay set was 75 nM. If 100% of the 75 nM JAK1 protein was catalytically active, the JAK1 inhibitor sensitivity limit from the donor JAK1 assay would theoretically be an IC of 37.5 nM. 50 value (half of the total enzyme concentration). However, JAK1 assay generates JAK1IC of several inhibitors 50 values, which are much lower than 37.5 nM and consistent with the internally determined values. The concentration of active JAK1 enzyme in the assay must be much lower than the total nominal JAK1 protein concentration of 75 nM used in the assay, and the observed sensitivity of this assay is much higher than the theoretical sensitivity IC 50 Limit 37.5nM.

[0785] Data analysis

[0786] For the data fitting in the concentration-kinase inhibition curve, The kinase analysis service uses XLfit software (IDBS), model number 205 (sigmoidal concentration-response model), which is a four-parameter logistic fitting model described by Equation 4

[0787] Equation 4

[0788]

[001] y=A+{(B-A)÷[1+(C÷x) D ]}

[0789] where x is the inhibitor concentration, y is the observed % inhibition, A is the minimum y-value, B is the maximum y-value, and C is the IC 50The value is , and D is the Hill slope. In some cases, a three-parameter logistic fit is used. For example, if the plateau of the curve at infinitely low inhibitor concentration does not fit between -20% and 20% inhibition, the lower plateau limit is set to 0% inhibition, and if the plateau of the curve at infinite inhibitor concentration does not fit between 70% and 130% inhibition, the upper plateau limit is set to 100% inhibition.

[0790] Phospho-STAT JAK1 and JAK2 pathway selectivity assay in TF-1 cell line

[0791] TF-1 human erythroleukemia cells ( Manassas, VA; Catalog No. CRL-2003 TM ) 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. One day before the assay, the cultures were transferred to Opti-MEM TM , 1×NEAA, 1 mM sodium pyruvate, and 0.5% charcoal-stripped FBS (starvation medium). The inhibitor stock solution (5 mM DMSO solution) was serially diluted 1:2 with DMSO to generate a 10-point concentration titration (at 500× test concentration), which was further diluted by 50-fold dilution in assay medium (RPMI containing 1×NEAA and 1 mM sodium pyruvate) to generate a 10× concentration titration (2% DMSO). Cells (300,000 cells / well in 35 μL assay medium) were seeded in 384-well Greiner plates. Inhibitors diluted at 10× concentration (5 μL) were added to the cells and the plates were incubated at 37°C in a humidified incubator for 30 minutes. The corresponding EC 90Cells were stimulated with human recombinant cytokines at concentrations of 10 μL. For the determination of phosphorylated signal transduction and transcription activator 6 (P-STAT6) TF-1 + interleukin-13 (IL-13), 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 determination of P-STAT5 TF-1 + erythropoietin (EPO), 10 μL of 110 IU / mL EPO (Gibco Life Technologies, catalog number PHC2054) was added to the cells, which were then incubated at 37°C for 30 min. For both determinations, after incubation, 5 μL of ice-cold 10× cell lysis buffer (Cell Signaling Technologies; Danvers, MA; catalog number 9803S) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) was added to the cells. The assay plate was frozen at -80°C for at least 1 hour. In the IL-13 assay, P-STAT6 was measured by coating goat anti-rabbit (GAR) plates (Meso Scale Discovery [MSD]; Rockville, MD; Catalog No. MSD L21RA-1) with rabbit anti-human total STAT6 antibody (Cell Signaling Technologies; Catalog No. 9362S), incubating cell lysates in the coated plates overnight at 4°C, and then detecting mouse anti-P-STAT6 (Tyr641) clone 16E12 antibody ((MilliporeSigma; Burlington, MA; Catalog No. 05-590, custom labeled by MSD with SULFO-tag) using standard MSD plate processing, washing, and detection protocols. In the EPO assay, P-STAT5 was detected using the phospho-STAT5a,b whole cell lysate kit (MSD; Catalog No. K150IGD-1). The electrochemiluminescence (ECL) signal of the wells was read on a MESO SECTOR S600 (MSD) reader.

[0792] Data analysis

[0793] Data analysis was performed by subtracting the mean ECL value of the negative control (cells stimulated with cytokines and treated with 20 μM control inhibitor) from the ECL value of all wells, determining the control percentage of the ECL value of the test compound wells relative to the mean ECL value of the positive control (cells stimulated with cytokines and treated with DMSO), and determining the IC of the test compound using a four-parameter logistic fit model as shown in Equation 4. 50 .

[0794] P-STAT6 BEAS-2B+IL-13 cell assay

[0795] To investigate the effects of JAK1 inhibitors in cell lines relevant to human asthma cell biology, an Il-13-stimulated STAT6 phosphorylation assay of the human lung bronchial epithelial BEAS-2B cell line was developed.

[0796] BEAS-2B cells ( CRL-9609 TM ) were grown in bronchial epithelial growth medium (BEGM) (Lonza catalog number CC-3170; Walkersville, MD; or PromoCell catalog number C-21060; Heidelberg, Germany). The test compound stock solution (0.5mM DMSO solution) was diluted serially 1:2 with DMSO to generate a 10-point concentration curve (at 500× test concentration), which was further diluted by 50-fold dilution steps BEGM to generate a 10× concentration curve (2% DMSO solution). The cells were plated in 200 μL BEGM in a 96-well plate at 100,000 cells / well and incubated in a humidified incubator at 37°C for 48 hours. The culture medium was removed from the cells and replaced with 70 μL fresh BEGM. The diluted test compound (10 μL; or 2% DMSO in the assay medium) was added to the cells, and the plate was incubated in a humidified incubator at 37°C for 1 hour. Twenty μL of 250 ng / mL human recombinant IL-13 (Bio Techne catalog number 213-ILB) was then added to the cells and incubated at 37°C for 15 minutes. The culture medium was aspirated from the cells and 60 μL of ice-cold 1× cell lysis buffer (Cell Signaling Technologies; catalog number 9803S) containing 1 mM PMSF was added to the cells. The assay plate was incubated at -80°C for at least 1 hour. P-STAT was measured by the following method: GAR plates (MSD; catalog number L45RA-1) were coated with rabbit anti-human total STAT6 antibody (Cell Signaling Technologies; catalog number 9362S), cell lysates in the coated plates were incubated overnight at 4°C, and then mouse anti-phospho-STAT6 (Tyr641) homologous 16E12 antibodies (Millipore; catalog number 05-590, custom labeled by MSD with SULFO-tag) were detected using standard MSD plate processing, washing and detection protocols. Plates were read on a MESO SECTOR S600.

[0797] Data analysis

[0798] Data analysis was performed by subtracting the negative control values ​​from all wells and using the positive control values ​​to determine the percentage of control; using a four-parameter logistic fit model as shown in Equation 4 to determine the IC50 .

[0799] P-STAT6 BEAS-2B+IL-13 cell assay using inhibitor washout (WO)

[0800] To assess the ability of JAK1 inhibitors to inhibit Il-13-stimulated STAT6 phosphorylation after cell washing to remove free unbound inhibitors, an inhibitor washout (WO) assay was developed for the human lung bronchial epithelial BEAS-2B cell line. Retention of inhibitory activity after inhibitor washout is consistent with persistent binding of the inhibitor to the JAK1 protein and / or retention of inhibitor molecules within the cell after washout.

[0801] As in the standard BEAS-2B cell assay (see above), BEAS-2B cells were grown in bronchial epithelial growth medium (BEGM). The test compound stock solution (0.5mM DMSO solution) was diluted 1:2 with DMSO to generate a 10-point concentration curve (at 500× test concentration), which was further diluted by 50-fold dilution steps BEGM to generate a 10× concentration curve (2% DMSO solution). The cells were plated in 200 μL BEGM in a 96-well plate at 100,000 cells / well and incubated in a humidified incubator for 48 hours at 37°C. The culture medium was withdrawn from the cells and replaced with 70 μL fresh BEGM. The diluted test compound (10 μL; or 2% DMSO in the assay culture medium) was added to the cells, and the plate was incubated in a humidified incubator for 1 hour at 37°C. The culture medium is removed from the cells and replaced with 80 μL of fresh BEGM to flush the inhibitors from the cells, and then the cell plate is incubated in a humidified incubator at 37°C for 10 minutes. This flushing procedure is repeated twice more. After the third washing step, the cell plate is returned to a 37°C humidified incubator and incubated for 1 hour. Then twenty μL of 250 ng / mL IL-13 is added to the cells and incubated at 37°C for 15 minutes. The culture medium is extracted from the cells and 60 μL of ice-cold 1× cell lysis buffer (Cell Signaling Technologies; Catalog No. 9803S) containing 1 mM PMSF is added to the cells. The assay plate is incubated at -80°C for at least 1 hour. P-STAT was measured by coating GAR plates (MSD; catalog number L45RA-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 mouse anti-phospho-STAT6 (Tyr641) cloned 16E12 antibody (Millipore; catalog number 05-590, custom labeled by MSD with SULFO-tag) using standard MSD plate handling, washing, and detection protocols. Plates were read on a MESO SECTOR S600.

[0802] Data analysis

[0803] Data analysis was performed by subtracting the negative control values ​​from all wells and using the positive control values ​​to determine the percentage of control; using a four-parameter logistic fit model as shown in Equation 4 to determine the IC 50 .

[0804] Cytotoxicity assay

[0805] A549 ( CCL-185 TM )、Jurkat pure line E6-1( TIB-152 TM ) and HEK-293T( CRL-1573 TM ) cells. Cells in the exponential growth phase were plated (450 cells in 45 μL of medium) in Greiner 384-well black / clear tissue culture treated plates (Greiner catalog number 781091). After dispensing the cells, the plates were allowed to equilibrate at room temperature for 30 minutes and then placed in a humidity controlled incubator at 37°C and CO2 overnight. The next day, cells were treated with test agents diluted in 100% DMSO (0.5% final DMSO concentration on cells) at a 10-point titration and a maximum concentration of 50 μM. The cells and compounds were then incubated in a humidity controlled incubator at 37°C and CO2 for 72 hours and then incubated with CellTiter- (Promega G7572) reagent was added to all wells to measure cell viability. The plates were incubated at room temperature for 20 minutes and then the wells were read for luminescence on an EnVision plate reader (Perkin Elmer Life Sciences).

[0806] The above JAK1 and JAK2 assay data of the compounds in Table 1 are shown in Table 2 below.

[0807] Table 2:

[0808]

[0809]

[0810]

[0811] As shown in Table 2, compounds of the invention have well-balanced affinities for both JAK1 and JAK2, and many are active in cell-based assays.

[0812] Animal models

[0813] Mouse House Dust Mite (HDM) Model

[0814] Seven to eight week old female C57BL / 6J mice were purchased from Jackson West. Mice were immunized by intraperitoneal injection of a mixture of house dust mites (HDM, D. Pteronyssinus, purchased from Greer Laboratories, normalized to 0.918ug DerP1 per mouse) and 2mg alum (Thermo Scientific) diluted in sterile PBS on days 0 & 14. Mice were challenged with HDM in PBS (normalized again to 0.918ug DerP1) and administered by intratracheal inhalation on days 21 & 24. Prior to each inhalation HDM challenge (in a subset of groups, on days 22 & 23), animals received test compounds via nasal inhalation only (using a dry powder inhalation device from Electro-Medical Measurement Systems (EMMS), including a Wright powder feeder and a 4-layer / 24-port or 2-layer / 12-port, directional flow, nasal inhalation tower only), ending 1 hour before challenge. Control animals received nasal air inhalation only. 24 hours after the final treatment, mice were bled retro-orbitally for plasma PK and then euthanized by CO2 inhalation. After euthanasia, BAL fluid was collected for total (by FACS, using known amounts of added reference beads) and differentiation (by Wright Giemsa-stained cytospin) cell counts. Lungs and spleens were collected, weighed, and frozen for PK. There were 5 or 6 animals per group.

[0815] In addition, to verify the pulmonary delivery dose, the test compound was administered to a PK satellite group of 3 naive animals via nasal inhalation only on a single day or four consecutive days. After the final inhalation dose, blood was drawn retro-orbitally from the PK satellite animals for plasma PK and then euthanized by CO2. Lungs and spleens were collected and weighed for PK analysis.

[0816] Rat OVA model

[0817] Six-week-old male Brown Norway rats were from Charles River-Kingston. Rats were immunized on day 0 by intraperitoneal injection of a mixture of 150ug OVA (Sigma) and 40mg alum (Thermo Scientific) diluted in sterile PBS. On day 28 after sensitization, rats were stimulated for 30 minutes with 2% OVA in PBS aerosolized by a nebulizer for three consecutive days. Before each OVA stimulation, animals received JAK1 / JAK2 test compounds via nasal inhalation only (using a dry powder inhalation device from an electronic medical measurement system (EMMS), including a Wright powder feeder and a 4-layer, 24-mouth, directional flow, nasal inhalation tower only), ending 1 hour before stimulation. Control animals received oral MCT buffer or only nasal air inhalation. 24 hours after the final treatment, animals were euthanized by CO2 inhalation. Blood was drawn from the abdominal aorta of the animals for plasma PK and whole blood FACS analysis. After euthanasia, BAL fluid was collected for total (by FACS, using known amount of added reference beads) and differentiation (by Wright Giemsa-stained cytospin) cell counts. Lungs were collected, weighed, and frozen for PK. Spleens were weighed and cut in half for PK and FACS analysis. Blood and spleen samples were analyzed by FACS for total cell counts and % NK cells (CD161a positive). There were 6 animals in each group except the pure control group which contained 5 animals.

[0818] In addition, to verify the pulmonary delivery dose, the JAK1 / JAK2 test compound was administered to a PK satellite group of 3 naive animals via nasal inhalation only on a single or three days. After the final inhalation dose, the PK satellite animals were euthanized by CO2 inhalation. Blood was drawn from the abdominal aorta of the animals for plasma PK. The lungs and spleen were collected and weighed for PK analysis.

[0819] The plasma and lung levels of the test compound and their ratios were determined in the following manner. BALB / c mice from Charles River Laboratories were used in this assay. The test compound was prepared separately with 0.2% Tween 80 saline solution, and the dosing solution was introduced into the mouse trachea by oral inhalation. At different time points after administration (usually 0.167, 2, 6, 24h), blood samples were removed via cardiac puncture and complete lungs were cut from the mice. The blood samples were centrifuged at approximately 12,000rpm and 4°C for 4 minutes (Eppendorf centrifuge, 5804R) to collect plasma. The lung pad was dried, weighed and homogenized by dilution with sterile water at 1:3. The plasma and lung levels of the test compound were determined by LC-MS analysis for the analytical standards constructed into the standard curve of the test matrix. The lung to plasma ratio was determined as the ratio of the lung AUC in μg h / g to the plasma AUC in μg h / mL, where AUC is generally defined as the area of ​​the curve of the test compound concentration relative to time.

[0820] Pharmacokinetics in mouse plasma and lung

[0821] The plasma and lung levels of the test compound and their ratios were determined in the following manner. BALB / c mice from Charles River Laboratories were used in this assay. The test compound was prepared separately at a concentration of 0.2 mg / mL with 20% propylene glycol in a citrate buffer at pH 4 and 50uL of the dosing solution was introduced into the mouse trachea by oral inhalation. At different time points after administration (usually 0.167, 2, 6, 24h), blood samples were removed via cardiac puncture and complete lungs were cut from the mice. The blood samples were centrifuged at approximately 12,000rpm and 4°C for 4 minutes (Eppendorf centrifuge, 5804R) to collect plasma. The lung pads were dried, weighed and homogenized with a 1:3 dilution with sterile water. The plasma and lung levels of the test compound were determined by LC-MS analysis for the analytical standards constructed into the standard curve of the test matrix. The lung to plasma ratio was determined as the ratio of lung AUC in μg h / g to plasma AUC in μg h / mL, where AUC is generally defined as the area of ​​the test compound concentration versus time curve.

[0822] Pharmacokinetics in mouse plasma and lung

[0823] The pharmacokinetics of the compound in female Balb / c mice were determined after a single intranasal (IN) bolus injection of the solution / suspension at a target dose of 0.3 mg / kg in 0.2% Tween 80 saline. Female Balb / c mice aged 7-8 weeks were purchased from Charles River. Mice were housed under specific pathogen-free conditions until used in the study.

[0824] Animals were not fasted before administration. Blood samples were obtained from 3 animals via cardiac puncture under anesthesia (intraperitoneal injection of pentobarbital) at each time point of 0.083, 2, 7 and 24 hours after administration, and placed in microcontainers coated with EDTA. Blood samples were centrifuged (1500g, 10min at 4°C) to separate plasma. Plasma samples were frozen at approximately -80°C. After intranasal administration, before lung perfusion, the spleen was removed, weighed, and flash-frozen. After confirming death, the lungs of the administered animals were perfused with frozen PBS to remove residual blood from the pulmonary vessels. The lungs were then cut and weighed (recording total weight). All tissue samples were immersed in liquid nitrogen and frozen. Tissue samples were stored frozen (approximately -80°C) until used for analysis.

[0825] Prior to PK analysis, thawed tissue samples (spleen and lung) were weighed and homogenized at 4°C using an Omni-Prep ball mill (Omni Inc., Kennesaw, GA) after adding 4 mL of HPLC-grade water per gram of tissue. Plasma and tissue homogenate samples were extracted using protein precipitation with four volumes of acetonitrile containing tulubitamide (200 ng / mL) and labetalol (100 ng / mL) as internal standards. The samples were mixed and centrifuged at 3200 g and 4°C for 30 minutes to remove precipitated proteins, and the supernatant was appropriately diluted with HPLC-grade water in a 96-well plate (e.g., 1:1, v:v). Compound concentrations of representative aliquots of plasma, spleen, and lung were determined by LC-MS / MS in positive ion mode using Waters Xevo TQ-S (Waters, Elstree, UK) for matrix-matched calibration curves and quality control standards. Standards were prepared by spiking control plasma, spleen, and lung tissue homogenates with compounds and extracted for experimental samples. The detection limits determined in all matrices ranged from 0.168 mg / mL to 4000 ng / mL.

[0826] The concentration below the lower limit of quantitation (LLOQ) was taken as zero for calculating the mean and SD. The mean concentration measured in the sample was used to construct a semi-logarithmic concentration-time curve feature. Pharmacokinetic (PK) analysis was performed using the non-compartmental method in Biobook (E-Workbook IDBS).

[0827] A murine model of eosinophilic inflammation induced by Alternaria alternata in the lung

[0828] Airway eosinophilia is a hallmark of human asthma. Alternaria alternata is a fungal aeroallergen that can exacerbate human asthma and induce eosinophilic inflammation in the lungs of mice (Havaux et al. Clin Exp Immunol. 2005, 139(2): 179-88. In mice, Alternaria alternata has been shown to indirectly activate type 2 tissue-resident innate lymphocytes in the lungs, which respond to (e.g., IL-2 and 11-7) and release JAK-dependent cytokines (e.g., IL-5 and 11-13) and regulate eosinophilic inflammation (Bartemes et al. J Immunol. 2012, 188(3): 1503-13).

[0829] The study used seven to nine week old male C57 mice from Taconic. On the day of the study, the animals were lightly anesthetized with isoflurane and injected with vehicle or test compound via oropharyngeal aspiration. After administration, the animals were placed in a side-lying state and returned to their home cages after monitoring that the animals had fully recovered from anesthesia. One hour later, the animals were briefly anesthetized again and stimulated with vehicle or alternating septal ...

[0830] Compound activity in the model is demonstrated by reduced levels of eosinophils present in the BALF of treated animals forty-eight hours compared to vehicle-treated, Alternaria-challenged control animals. Data are expressed as percent inhibition of the vehicle-treated, Alternaria-challenged BALF eosinophil response. To calculate percent inhibition, the number of BALF eosinophils for each condition was converted to the percentage of the mean vehicle-treated, Alternaria-challenged BALF eosinophils and subtracted from 100%.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof: in: R 1 Selected from: and R 2 is: chloro; difluoromethoxy; methylthio; or cyclopropyl; R 3 Yes: Hydrogen; R 4 Yes: Hydrogen; R 5 Yes: Hydrogen; R 6 Yes: Hydrogen.

2. A compound or a pharmaceutically acceptable salt thereof, wherein the compound is selected from: and 3. Use of a compound according to any one of claims 1 to 2 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing, treating or alleviating the severity of a disease or condition responsive to inhibition of Janus kinase activity in a patient.

4. The use according to claim 3, wherein the disease or disorder is an allergic disease or an inflammatory disease.

5. The use according to claim 3, wherein the disease or disorder is psoriasis, rheumatoid arthritis, inflammatory bowel disease or asthma.

6. A pharmaceutical composition comprising a compound according to any one of claims 1 to 2 or a pharmaceutically acceptable salt thereof, wherein the pharmaceutical composition comprises microparticles of the compound suitable for inhalation delivery.

7. The pharmaceutical composition according to claim 6, wherein the microparticles are prepared by spray drying, freeze drying or micronization.

8. A kit, comprising: (a) a first pharmaceutical composition, comprising a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 2; and (b) Instructions for use.

9. The kit according to claim 8, further comprising a second pharmaceutical composition comprising an agent for treating an inflammatory disease, or a chemotherapeutic agent.

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