Pyrazolopyrimidine aryl ether inhibitors of JAK kinase and uses thereof

Abstract

Compounds and salts thereof useful as JAK kinase inhibitors are described herein. Pharmaceutical compositions comprising such JAK inhibitors and pharmaceutically acceptable carriers, adjuvants or vehicles, as well as methods for treating or alleviating the severity of a disease or condition responsive to inhibition of Janus kinase activity in a patient are also provided. #imgabs0#

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to International Application No. PCT / CN2019 / 091710 filed on June 18, 2019 and U.S. Provisional Application No. 63 / 036,577 filed on June 9, 2020, the disclosures of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to Janus kinase (such as JAK1 and JAK2) inhibitor compounds, and compositions comprising these compounds, and methods of use including but not limited to diagnosing or treating patients suffering from disorders responsive to JAK kinase inhibition. 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 associated with type I and type II cytokine receptors and regulate cytokine signal transduction. The binding of cytokines to cognate receptors triggers the activation of receptor-associated JAKs, and this results in JAK-mediated tyrosine phosphorylation of signal transduction and transcription activator (STAT) proteins and ultimately leads to transcriptional activation of specific gene sets (Schindler et al., 2007, J.Biol.Chem.282:20059-63). JAK1, JAK2 and TYK2 show a wide range of gene expression patterns, while JAK3 expression is limited to leukocytes. Cytokine receptors usually play a role as heterodimers, and therefore usually more than one type of JAK kinase is associated with cytokine receptor complexes. In many cases, the specific JAK associated with different cytokine receptor complexes has been determined by genetic studies and has been 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 is functionally and physically associated 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 perinatally due to defects in LIF receptor signaling (Kisseleva et al., 2002, Gene 285: 1-24; O'Shea et al., 2002, Cell, 109 (Suppl): S121-S131). Characterization of tissues derived from JAK1 knockout mice demonstrated a key role for this kinase in the IFN, IL-10, IL-2 / IL-4, and IL-6 pathways. A humanized monoclonal antibody (tocilizumab) targeting the IL-6 pathway 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). IL-4 and IL-13 induce increased mucus production, recruitment of eosinophils to the lungs, and increased 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). The IL-4R chain activates JAK1 and binds to IL-4 or IL-13 when α binds to the common γ chain or IL-13Rα1 chain, respectively (Pernis et al., 2002, J. Clin. Invest. 109(10):1279-1283). The common γ chain can also bind to 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 signaling 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 reduce asthma symptoms in preclinical lung inflammation models (Mathew et al., 2001, J. Exp. Med. 193(9):1087-1096; Kudlacz et al., 2008, Eur. J. Pharmacol. 582(1-3):154-161).

[0007] Biochemical and genetic studies have shown an association between JAK2 and the 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, JAK2 knockout mice die of anemia (O'Shea et al., 2002, Cell, 109 (Suppl.): S121-S131). JAK2 kinase activating mutations (e.g., JAK2 V617F) are associated with human myeloproliferative disorders. In addition, JAK2 is associated with cytokine receptors such as IL-5 and thymic stromal lymphopoietin (TSLP). IL-5 is a key cytokine responsible for eosinophil differentiation, growth, activation, survival, and recruitment to the airways (Pelaia et al., 2019, Front. Physiol., 10:1514; Stirling et al., 2001, Am. J. Respir. Crit. Care Med., 164:1403–9; Fulkerson and Rothenberg, 2013, Nat. Rev. Drug Discov., 12:117–9.; Varricchi and Canonica, 2016, Expert. Rev. Clin. Immunol., 12:903–5). Three monoclonal antibody drugs targeting IL-5 (mepolizumab, retinizumab) or its receptor α chain (benralizumab) have been approved for the treatment of eosinophilic phenotype asthma. TSLP is an epithelial cell-derived cytokine that plays an important role in regulating type II immunity and acts as an alarm upstream of TH2 cytokine production (Kitajima et al., 2011, Eur J Immunol., 41: 1862-71). Tezepelumab is an antagonist antibody to TSLP. Results from a phase 2 trial showed that it successfully reduced asthma exacerbations in patients with and without type 2 high features (Corren et al., 2017, 377: 936-46).

[0008] JAK3 is only associated 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 (supplement): 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 is associated 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 and O'Shea, JJ, 2006, Immunity 25: 695-697). Consistent with this, primary cells derived from TYK2-deficient humans are defective in type I interferon, IL-6, IL-10, IL-12, and IL-23 signaling. A fully human monoclonal antibody (ustekinumab) targeting the 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 tested in clinical trials 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 reportedly useful as inhibitors of one or more Janus kinases. Data for certain specific compounds showing inhibition of JAK1 as well as JAK2, JAK3, and / or TYK2 kinases are provided.

[0011] At present, other compounds as Janus kinase inhibitors are still needed. For example, it is necessary to have a useful effect as an inhibitor 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 selectivity for one Janus kinase over selectivity for other kinases (e.g., selectivity for JAK1 and / or JAK2 over selectivity for other kinases such as leucine-rich repeat kinase 2 (LRRK2)). Effective compounds that show selectivity for one Janus kinase over selectivity for other Janus kinases (e.g., selectivity for JAK1 and / or JAK2 over selectivity for JAK3 and / or TYK2) are also needed. Compounds that show selectivity for both JAK1 and JAK2 over selectivity for JAK3 and TYK2 can provide therapeutic benefits under conditions that respond to JAK1 inhibition. In addition, there is currently a need for effective JAK1 inhibitors with other properties (e.g., melting point, pK, solubility, etc.) necessary for formulation and inhalation administration. 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.

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

[0014]

[0015] or a pharmaceutically acceptable salt thereof, wherein:

[0016] Ar is: phenyl; 1,2,3,4-tetrahydroisoquinolyl; pyrazolyl; pyridinyl; or pyridazinyl:

[0017] R 1 is: hydrogen; C1-C6 alkyl; halo-C1-C6 alkyl; hydroxy-C1-C6 alkyl; -(CHR a ) h -

[0018] het 1 ;-(CHR a ) k -NR a -het 1 ; or -(CHR a )m -C 3-6 Cycloalkyl, wherein the cycloalkyl portion may be unsubstituted or substituted by R d Replace once or twice;

[0019] Each R 2 are independently: C1-C6 alkyl; hydroxy-C1-C6 alkyl; halo-C1-C6 alkyl; C1-

[0020] C6 alkoxy; C1-C6 alkoxy-C1-C6 alkyl; halo-C1-C6 alkoxy; halo-

[0021] C1-C6 alkoxy-C1-C6 alkyl; C1-C6 alkyl-SO2-C1-C6 alkyl; hydroxy; cyano; cyano-C1-C6 alkyl; halo; acetyl; -(CHR a ) p -het 2 ;-(CHR a ) q -

[0022] NR b R c ;-(CHR a ) r -C(O)-NR b R c ;-(CHR a ) s -NR a -(CHR a ) s -C(O)-

[0023] NR b R c ; or -(CHR a ) t -C 3-6 Cycloalkyl, wherein the cycloalkyl portion may be unsubstituted or substituted by R e Replace once or twice;

[0024] R 3 , R 4 and R 5 Each independently is: hydrogen; or C1-C6-alkyl;

[0025] Each R a are independently: hydrogen; or C 1-6 alkyl;

[0026] Each R b are independently: hydrogen; C 1-6 Alkyl; or hydroxy-C1-C6 alkyl;

[0027] Each Rc are independently: hydrogen; C 1-6 Alkyl; Hydroxy-C1-C6 alkyl; Cyano-C1-C6 alkyl;

[0028] C1-C6 alkoxy-C1-C6 alkyl; oxetanyl; 2-morpholinoethyl; 1-methyl-azetidin-3-yl; 2-(N,N-dimethylamino)-ethyl; hydroxycyclobutyl; or 3-

[0029] (N,N-dimethylamino)-pyrrolidin-1-yl; (CHR a ) u -C 3-6 Cycloalkyl, wherein cycloalkyl

[0030] The moiety may be unsubstituted or replaced by R e Replace once or twice;

[0031] or R b and R c Together with the nitrogen atom to which they are attached they can form het 3 ;

[0032] Each R d Independently: C1-C6 alkyl, hydroxyl or halo;

[0033] Each R e Independently: C 1-6 Alkyl; Hydroxyl; Cyano-C1-C6 Alkyl; Hydroxyl-C1-C6 Alkyl

[0034] methyl; morpholinyl; or -(CHR a ) v -NR g R h , where R g and R h Each independently is hydrogen

[0035] or C 1-6 alkyl;

[0036] h is 0 to 2;

[0037] k is 0 to 2;

[0038] m is 0 to 2;

[0039] n is 0 to 2;

[0040] p is 0 to 2;

[0041] q is 0 to 2;

[0042] r is 0 to 2;

[0043] s is 0 to 2;

[0044] t is 0 to 2;

[0045] u is 0 to 2;

[0046] v is 0 to 2;

[0047] het 1 is: oxetane; tetrahydrofuran; tetrahydropyran; or pyrrolidinyl;

[0048] Each of may be unsubstituted or replaced by R d Replace once or twice;

[0049] het 2 are: azetidinyl; pyrrolidinyl; oxetanyl; piperidinyl; morpholinyl; piperazinyl; azepine quinuclidine; or pyrazolyl; each of which may be unsubstituted or replaced by R e replaced once or twice; and

[0050] het 3 is: azetidinyl; pyrrolidinyl; piperidinyl; morpholinyl; piperazinyl; or aza each of which may be unsubstituted or replaced by R e Replace once or twice;

[0051] 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.

[0052] Also provided is the use of the JAK inhibitors described herein or their pharmaceutically acceptable salts 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 their pharmaceutically acceptable salts in the preparation of a medicament for the treatment of 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 its pharmaceutically acceptable salt.

[0053] 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 both JAK1 and JAK2. Some of these compounds optionally have an optimally balanced co-activity against both JAK1 and JAK2, or have a slightly higher affinity for JAK1 than JAK2, rather than having much greater activity against one of these kinases than the other. The subject compounds also have good selectivity for off-target kinases associated with pulmonary toxicity, such as LRRK2.

[0054] Although many compounds may show high affinity for both JAK1 and JAK2 in simple biochemical assays, not all of these compounds are effective in mediating the asthma-related 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 in cell-based assays to be effective in mediating the asthma-related cytokines associated with JAK1 and JAK2.

[0055] The compounds of the present invention also show favorable pharmacokinetic (PK) properties in lung tissue and can be used for inhalation therapy. When administered by inhalation route using a technology such as dry powder inhalation (DPI) or intranasal (IN) delivery, some compounds unexpectedly show sustained retention in lung tissue, while the concentration in systemic circulation is much lower. This improved PK property can advantageously lead to smaller doses and less frequent dosing requirements for effective treatment. Some compounds show unexpectedly improved solubility, again providing improved efficacy in the lungs. Compared with other JAK inhibitors, some compounds of the present invention also show unexpectedly reduced cytotoxicity. DETAILED DESCRIPTION

[0056] definition

[0057] "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.

[0058] The term "alkyl" refers to a saturated straight or branched monovalent hydrocarbon group, wherein the alkyl group may be optionally substituted. In one example, an alkyl group is one to eighteen 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)CH 2CH3), 2-methyl-2-propyl (t-Bu, tert-butyl, -C(CH3)3), 1-pentyl (n-pentyl, -CH2CH2CH2CH2CH3), 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 (-CH2 1-Hexyl (-CH2CH2CH2CH2CH2CH3), 2-Hexyl (-CH(CH3)CH2CH2CH2CH3), 3-Hexyl (-CH(CH2CH3)(CH2CH2CH3)), 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.

[0059] 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 two to eighteen 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-ene, but-1-ene, but-2-enyl, but-3-enyl, but-1,3-dienyl, 2-methylbut-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.

[0060] 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 (-C≡CH), 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.

[0061] "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 one to eighteen 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-1,2-yl (-C(CH3)2CH2-), 1,4-butyl (-CH2CH2CH2CH2-), and the like.

[0062] 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 one to five 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.

[0063] "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 alkyl, cycloalkyl, aryl, and heterocyclyl groups are as defined herein. In many such embodiments, R is a C1-C6 alkyl. 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.

[0064] "Aryl" refers to a carbocyclic aromatic group having a specified number of carbon atoms, or if no number is specified, up to 14 carbon atoms, whether or not fused to one or more groups. One example includes an aryl group having 6-14 carbon atoms. Another example includes an aryl group having 6-10 carbon atoms. Examples of aryl groups include phenyl, naphthyl, biphenyl, phenanthrenyl, naphthacenyl, 1,2,3,4-tetrahydronaphthyl, 1H-indenyl, 2,3-dihydro-1H-indenyl, etc. (see, e.g., Lang's Handbook of Chemistry (Dean, JA, ed.) 13 thed.Table 7-2

[1985] ). A specific aryl group 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 the alkyl, phenyl and heterocyclic portions thereof may be optionally substituted, such as substituted with one to four substituents selected from the 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). 0-4 CONR'R", wherein R' and R" each independently refer to a group including, for example, hydrogen; unsubstituted C1-C6 alkyl; C1-C6 alkyl substituted with halogen, OH, CN, unsubstituted C1-C6 alkyl, unsubstituted C1-C6 alkoxy, oxo or NR'R"; unsubstituted C1-C6 heteroalkyl; C1-C6 heteroalkyl substituted with halogen, OH, CN, unsubstituted C1-C6 alkyl, unsubstituted C1-C6 alkoxy, oxo or NR'R"; unsubstituted C6-C6 heteroalkyl; 10 Aryl; C6-C6 substituted by halogen, OH, CN, unsubstituted C1-C6 alkyl, unsubstituted C1-C6 alkoxy or NR'R" 10 aryl; unsubstituted 3-11 membered heterocyclic group (for example, 5-6 membered heteroaryl containing 1 to 4 heteroatoms selected from O, N and S or 4-11 membered heterocyclic alkyl containing 1 to 4 heteroatoms selected from O, N and S); and 3-11 membered heterocyclic group (for example, 5-6 membered heteroaryl 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 " atom or a 4-11 membered heterocycloalkyl containing 1 to 4 heteroatoms selected from O, N and S); 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 C1-C6 alkyl, unsubstituted C1-C6 alkoxy, oxo or NR'R".

[0065] "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 examples, the cycloalkyl group is C3-C8, C3-C 10 or C5-C10 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 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 C1-C6 alkyl; C1-C6 alkyl substituted with halogen, OH, CN, unsubstituted C1-C6 alkyl, unsubstituted C1-C6 alkoxy, oxo or NR'R"; unsubstituted C1-C6 heteroalkyl; C1-C6 heteroalkyl substituted with halogen, OH, CN, unsubstituted C1-C6 alkyl, unsubstituted C1-C6 alkoxy, oxo or NR'R"; unsubstituted C6-C6 heteroalkyl; 10 Aryl; C6-C6 substituted by halogen, OH, CN, unsubstituted C1-C6 alkyl, unsubstituted C1-C6 alkoxy or NR'R" 10aryl; unsubstituted 3-11 membered heterocyclic group (for example, 5-6 membered heteroaryl containing 1 to 4 heteroatoms selected from O, N and S or 4-11 membered heterocyclic alkyl containing 1 to 4 heteroatoms selected from O, N and S); and 3-11 membered heterocyclic group (for example, 5-6 membered heteroaryl 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 " atom or a 4-11 membered heterocycloalkyl containing 1 to 4 heteroatoms selected from O, N and S); 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 C1-C6 alkyl, unsubstituted C1-C6 alkoxy, oxo or NR'R".

[0066] "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 system, 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 of the ring system is a heteroatom, 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 includes 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 having 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 having 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 heterocyclic alkyl group includes at least one nitrogen. In one example, the heterocyclic group includes 0 to 3 double bonds. Any nitrogen or sulfur heteroatom may be optionally oxidized (e.g., NO, SO, SO2), and any nitrogen heteroatom may be optionally quaternized (e.g., [NR4] + Cl - 、[NR4]+ OH -Examples of 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, isoquinolyl, tetrahydroisoquinolyl, morpholinyl, thiomorpholinyl, 1,1-dihydro-thiomorpholinyl, dihydropyranyl, tetrahydropyranyl, hexahydrothiopyranyl, hexahydropyrimidinyl, oxazinyl, thiazinyl, thioxanyl, homopiperazinyl, homopiperidinyl, piperidinyl), azepanyl, oxepanyl, thiepanyl, oxazepinyl, oxazepanyl, diazepanyl, 1,4-diazepanyl, diazepine, triazepine, thiazepanyl, tetrahydrothiopyranyl, oxazolidinyl, thiazolidinyl, isothiazolidinyl, 1,1-dioxoisothiazolinyl, oxazolidinediyl, 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, oxatriazinyl, dithiadiazinyl, imidazolinyl, dihydropyrimidinyl, tetrahydropyrimidinyl, 1-pyrrolidinyl, 2-pyrrolidinyl, 3-pyrrolinyl, indolyl, thiopyranyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolyl, pyrazolidinyl, dithienyl, dithiolanyl, pyrimidinone, pyrimidinedione, pyrimidine-2,4-dicarboxyl, piperazinone, piperazinedione, pyrazolidiniminoimidazolinyl, 3- azabicyclo[3.1.0]hexanyl, 3,6-diazabicyclo[3.1.1]heptanyl, 6-azabicyclo[3.1.1]heptanyl, 3-azabicyclo[3.1.1]heptanyl, 3-azabicyclo[4.1.0]heptanyl, azabicyclo[2.2.2]hexanyl, 2-azabicyclo[3.2.1]octanyl, 8-azabicyclo[3.2.1]octanyl, 2-azabicyclo[2.2.2]octanyl, 8-azabicyclo[2.2.2]octanyl, 7-oxabicyclo[2.2.1]heptanyl, azaspiro[3.5]nonanyl, azaspiro[2.5]octanyl, azaspiro[4.5]decan-2-yl, 1-azaspiro[4.5]decan-2-yl, azaspiro[5.5] undecyl, tetrahydroindolyl, octahydroindolyl, tetrahydroisoindolyl, tetrahydroindolyl, 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, for example 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 benzooxazol-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, such as 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 pyridyl, pyrimidine-2-yl, pyrimidine-4-yl, pyridazinyl and 1,3,4-triazine-2-yl groups are other exemplary heterocyclic groups.Heterocycle can be optionally substituted.For example, the substituent for "optionally substituted heterocycle" includes 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, piperidyl, piperazinyl and pyrimidyl one to four examples, 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, a substituent of a heterocyclic group (such as a heteroaryl or heterocycloalkyl) includes an amide. For example, a heterocyclic (e.g., heteroaryl or heterocycloalkyl) substituent can be -(CH2). 0-4 CONR'R", wherein R' and R" each independently refer to a group including, for example, hydrogen; unsubstituted C1-C6 alkyl; C1-C6 alkyl substituted with halogen, OH, CN, unsubstituted C1-C6 alkyl, unsubstituted C1-C6 alkoxy, oxo or NR'R"; unsubstituted C1-C6 heteroalkyl; C1-C6 heteroalkyl substituted with halogen, OH, CN, unsubstituted C1-C6 alkyl, unsubstituted C1-C6 alkoxy, oxo or NR'R"; unsubstituted C6-C6 heteroalkyl; 10Aryl; C6-C6 substituted by halogen, OH, CN, unsubstituted C1-C6 alkyl, unsubstituted C1-C6 alkoxy or NR'R" 10 aryl; unsubstituted 3-11 membered heterocyclic group (for example, 5-6 membered heteroaryl containing 1 to 4 heteroatoms selected from O, N and S or 4-11 membered heterocyclic alkyl containing 1 to 4 heteroatoms selected from O, N and S); and 3-11 membered heterocyclic group (for example, 5-6 membered heteroaryl 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 " atom or a 4-11 membered heterocycloalkyl containing 1 to 4 heteroatoms selected from O, N and S); 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 C1-C6 alkyl, unsubstituted C1-C6 alkoxy, oxo or NR'R".

[0067] "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, Lang's Handbook of Chemistry (Dean, JA, ed.) 13 thed.Table 7-2

[1985] . This definition includes any bicyclic group in which any of the above heteroaryl rings is fused to an aryl ring, wherein the aryl ring or the heteroaryl ring is attached to the rest of the molecule. In one embodiment, heteroaryl includes a 5-6 membered monocyclic aromatic group in which one or more of the ring atoms is 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 C1-C6 alkyl; C1-C6 alkyl substituted with halogen, OH, CN, unsubstituted C1-C6 alkyl, unsubstituted C1-C6 alkoxy, oxo or NR'R"; unsubstituted C1-C6 heteroalkyl; C1-C6 heteroalkyl substituted with halogen, OH, CN, unsubstituted C1-C6 alkyl, unsubstituted C1-C6 alkoxy, oxo or NR'R"; unsubstituted C6-C6 heteroalkyl; 10 Aryl; C6-C6 substituted by halogen, OH, CN, unsubstituted C1-C6 alkyl, unsubstituted C1-C6 alkoxy or NR'R" 10aryl; unsubstituted 3-11 membered heterocyclic group (for example, 5-6 membered heteroaryl containing 1 to 4 heteroatoms selected from O, N and S or 4-11 membered heterocyclic alkyl containing 1 to 4 heteroatoms selected from O, N and S); and 3-11 membered heterocyclic group (for example, 5-6 membered heteroaryl 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 " atom or a 4-11 membered heterocycloalkyl containing 1 to 4 heteroatoms selected from O, N and S); 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 C1-C6 alkyl, unsubstituted C1-C6 alkoxy, oxo or NR'R".

[0068] In certain embodiments, the heterocyclyl group is attached at a carbon atom of the heterocyclyl group. By way of example, carbon-bonded heterocyclyl groups include the following bonding arrangements: at the 2, 3, 4, 5, or 6 position of a pyridine ring, at the 3, 4, 5, or 6 position of a pyridazine ring, at the 2, 4, 5, or 6 position of a pyrimidine ring, at the 2, 3, 5, or 6 position of a pyrazine ring, at the 2, 3, 4, or 5 position of a furan, tetrahydrofuran, thiophene, thiophene, pyrrole, or tetrahydropyrrole ring, at the 2, 4, or 5 position of an oxazole, imidazole, or thiazole ring, at the 3, 4, or 5 position of an isoxazole, pyrazole, or isothiazole ring, at the 2 or 3 position of an aziridine ring, at the 2, 3, or 4 position of an azetidine ring, at the 2, 3, 4, 5, 6, 7, or 8 position of a quinoline ring, or at the 1, 3, 4, 5, 6, 7, or 8 position of an isoquinoline ring.

[0069] In certain embodiments, the heterocyclyl group is N-linked. By way of example, nitrogen-bonded heterocyclyl or heteroaryl groups include the following bonding arrangements: at the 1 position of aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, 1H-indazole, at the 2 position of isoindole or isoindoline, at the 4 position of morpholine, and at the 9 position of carbazole or β-carboline.

[0070] 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.

[0071] "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., pyridyl).

[0072] Unless otherwise indicated, "optionally substituted" means that the group can be unsubstituted or substituted with one or more (e.g., 0, 1, 2, 3, 4, or 5 or more, or any range derivable therein) of the substituents listed for the group, wherein the substituents can be the same or different. In an 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.

[0073] The alkyl radical, alone or as part of another substituent (e.g., alkoxy), and the optional substituents for the alkylene, alkenyl, alkynyl, heteroalkyl, heterocycloalkyl, and cycloalkyl radicals, 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-C1-C4 alkoxy; unsubstituted C3-C7 cycloalkyl; C3-C7 cycloalkyl substituted with 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 " -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 substituents ranges from zero to (2m'+1), where m' is the total number of carbon atoms in such radicals. R', R'' and R''' each independently refer to a group including, for example, hydrogen; unsubstituted C1-C6 alkyl; C1-C6 alkyl substituted with halogen, OH, CN, unsubstituted C1-C6 alkyl, unsubstituted C1-C6 alkoxy, oxo or NR'R''; unsubstituted C1-C6 heteroalkyl; C1-C6 heteroalkyl substituted with halogen, OH, CN, unsubstituted C1-C6 alkyl, unsubstituted C1-C6 alkoxy, oxo or NR'R''; unsubstituted C6-C6 heteroalkyl; 10 Aryl; 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); and 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 "substituted O, N and S heteroatoms). 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 intended to include 1-pyrrolidinyl and 4-morpholinyl.

[0074] Similarly, there are a wide variety of optional substituents for aryl and heteroaryl groups. In some embodiments, the substituents for aryl and heteroaryl groups are selected from the group consisting of: halogen; CN; NO; N3; -OR'; perfluoro-C1-C4 alkoxy; unsubstituted C3-C7 cycloalkyl; C3-C7 cycloalkyl substituted with 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'' 10 aryl; 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 " -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- 4CONR'R", or a combination thereof, the amount of the optional substituents is in the range of zero to (2m'+1), wherein m' is the total number of carbon atoms in such groups. R', R'' and R''' each independently refer to groups including, for example, hydrogen; unsubstituted C1-C6 alkyl; C1-C6 alkyl substituted with halogen, OH, CN, unsubstituted C1-C6 alkyl, unsubstituted C1-C6 alkoxy, oxo or NR'R''; unsubstituted C1-C6 heteroalkyl; C1-C6 heteroalkyl substituted with halogen, OH, CN, unsubstituted C1-C6 alkyl, unsubstituted C1-C6 alkoxy, oxo or NR'R''; unsubstituted C6-C6 heteroalkyl; 10 Aryl; C6-C6 substituted by halogen, OH, CN, unsubstituted C1-C6 alkyl, unsubstituted C1-C6 alkoxy or NR'R" 10 aryl; 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); and 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 "substituted O, N and S heteroatoms). 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 intended to include 1-pyrrolidinyl and 4-morpholinyl.

[0075] The term "oxo" refers to =0 or (=0)2.

[0076] As used herein, a wavy line intersecting a bond in a chemical structure Indicates the point of connection between an atom connected to a wavy bond in a chemical structure and the rest of the molecule or the rest of a molecular fragment. In some embodiments, an arrow and an asterisk are used together in a wavy line to indicate a point of connection.

[0077] In certain embodiments, a divalent group is generally described as having no specific bonding configuration. It should be understood that unless otherwise specified, the general description is intended to include both bonding structures. 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.

[0078] 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.

[0079] 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.

[0080] 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 and base addition salts. "Pharmaceutically acceptable acid addition salts" refer to salts that retain the biological effectiveness and properties of free alkalis formed with inorganic acids (such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, carbonic acid, phosphoric acid, etc.) and organic acids 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.

[0081] "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, methylglucosamine, 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.

[0082] 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).

[0083] A "sterile" preparation is sterile or free from all living microorganisms and their spores.

[0084] "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.

[0085] "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.

[0086] "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, for example, melting points, boiling points, spectral properties or biological activities. Mixtures of diastereomers can be separated under high resolution analytical procedures such as electrophoresis and chromatography such as HPLC.

[0087] "Enantiomers" refers to two stereoisomers of a compound that are non-superimposable mirror images of one another.

[0088] The stereochemical definitions and conventions used herein generally follow those of 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. In describing optically active compounds, the prefixes D and L or R and S are used to indicate the absolute configuration of the molecule about its chiral center. The prefixes d and l or (+) and (-) are used to indicate the sign of the rotation of the compound with respect 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.

[0089] 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.

[0090] 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. Generally, all physical forms are intended to be within the scope of the invention. The term "hydrate" refers to a complex in which the solvent molecule is water.

[0091] "Metabolite" refers to a product produced by the metabolism of a particular compound or its salt in vivo. Such products can be produced, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, deesterification, enzymatic cleavage, etc. of the administered compound.

[0092] Metabolites are usually prepared by (e.g. 14 C or 3 H) The compounds of the invention are identified with radiolabeled isotopes and administered to animals (such as rats, mice, guinea pigs, monkeys or humans) at a detectable dose (e.g., greater than about 0.5 mg / kg), allowing sufficient time for metabolism (usually about 30 seconds to 30 hours), and their conversion products are isolated 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 metabolites 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 assays for therapeutic administration of the compounds of the invention as long as they are not found in vivo.

[0093] A "subject," "individual," or "patient" is a vertebrate. In certain embodiments, the vertebrate is a mammal. Mammals include, but are not limited to, livestock (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.

[0094] The term "Janus kinase" refers to JAK1, JAK2, JAK3 and TYK2 protein kinases. In some embodiments, the Janus kinase can be further defined as one of JAK1, JAK2, JAK3 or TYK2. In any embodiment, any one of JAK1, JAK2, JAK3 and TYK2 can be specifically excluded from the Janus kinases. In some embodiments, the Janus kinase is JAK1. In some embodiments, the Janus kinase is a combination of JAK1 and JAK2.

[0095] The terms "inhibit" and "reduce", or any variation of these terms, include any measurable reduction or complete inhibition to achieve the desired result. For example, the activity (e.g., JAK1 activity) can be reduced by about, up to about, or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more compared to normal, or any range of activity (e.g., JAK1 activity) that can be derived therefrom.

[0096] "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., to some extent, slow down and preferably prevent) cancer cell infiltration into surrounding organs, inhibit (i.e., to some extent, slow down and preferably prevent) tumor metastasis, inhibit tumor growth to some extent, or alleviate one or more symptoms associated with cancer to some extent. To some extent, the drug can prevent growth or kill existing cancer cells, it can inhibit cell growth or have cytotoxicity. For cancer therapy, for example, efficacy can be measured by assessing the time to disease progression (TTP) or determining the effective rate (RR).

[0097] "Treatment" (and variations thereof such as "treat" or "treating") refers to clinical intervention that attempts to alter the natural course of the treated individual or cell, 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, alleviating any direct or indirect pathological consequences of the disease, stabilizing (i.e., not worsening) the disease state, reducing the rate of disease progression, improving or alleviating the disease state, if the survival period is prolonged compared to the expected survival period without treatment and alleviation or improved prognosis. In some embodiments, the compounds of the present invention or salts thereof (e.g., pharmaceutically acceptable salts thereof) are used to delay the development of a disease or condition or to slow the progression of a disease or condition. Those in need of treatment include those already suffering from a condition or condition and those susceptible to a condition or condition (e.g., by genetic mutation) or those of a condition or condition to be prevented.

[0098] "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.

[0099] "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 responses of specific defense systems as well as responses of non-specific defense systems.

[0100] "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.

[0101] 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.

[0102] "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.

[0103] 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.

[0104] "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, perpetuation or exacerbation of inflammatory conditions.

[0105] 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.

[0106] The terms "cancer" and "cancerous," "neoplasm" and "tumor," 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 cancers include carcinomas, blastomas, sarcomas, seminoma, 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 disorders, such as polycythemia vera, essential thrombocythemia, myelofibrosis, such as primary myelofibrosis, and chronic myeloid leukemia (CML).

[0107] A "chemotherapeutic agent" is a formulation useful for treating a given condition (e.g., cancer or an inflammatory condition). Examples of chemotherapeutic agents are well known in the art, and include examples such as those disclosed in U.S. Publication 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.

[0108] "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.

[0109] 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 or substrate tissue distribution assays. 3 H) and carbon-14 (i.e., 14 C) isotopes are useful because of their ease of preparation and detectability. In addition, heavier isotopes such as deuterium (i.e., 2 H) substitution may provide certain therapeutic advantages due to 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 18 F) 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.

[0110] It is particularly 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 or salt thereof (e.g., pharmaceutically acceptable salt thereof) or composition of the present invention 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 or salt thereof (e.g., pharmaceutically acceptable salt thereof) or composition of the present invention.

[0111] Although the present disclosure supports definitions referring to optional options only and "and / or", use of the term "or" is intended to mean "and / or" unless explicitly stated to refer to optional options only or the optional options are mutually exclusive.

[0112] Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0113] As used herein, "a" or "an" means one or more, unless expressly stated otherwise. As used herein, "another" means at least a second or more.

[0114] The headings used herein are for editorial purposes only.

[0115] Inhibitors of JANUS kinases

[0116] One embodiment provides a compound of formula (I):

[0117]

[0118] or a pharmaceutically acceptable salt thereof, wherein:

[0119] Ar is: phenyl; 1,2,3,4-tetrahydroisoquinolyl; pyrazolyl; pyridinyl; or pyridazinyl:

[0120] R 1 is: hydrogen; C1-C6 alkyl; halo-C1-C6 alkyl; hydroxy-C1-C6 alkyl; -(CHR a ) h -

[0121] het 1 ;-(CHR a ) k -NR a -het 1 ; or -(CHR a ) m -C 3-6 Cycloalkyl, wherein the cycloalkyl portion may be unsubstituted or substituted by R d Replace once or twice;

[0122] Each R 2 are independently: C1-C6 alkyl; hydroxy-C1-C6 alkyl; halo-C1-C6 alkyl; C1-

[0123] C6 alkoxy; C1-C6 alkoxy-C1-C6 alkyl; halo-C1-C6 alkoxy; halo-

[0124] C1-C6 alkoxy-C1-C6 alkyl; C1-C6 alkyl-SO2-C1-C6 alkyl; hydroxy; cyano; cyano-C1-C6 alkyl; halo; acetyl; -(CHR a ) p -het 2 ;-(CHR a ) q -

[0125] NR b R c ;-(CHR a ) r -C(O)-NR b R c ;-(CHR a ) s -NR a -(CHR a ) s -C(O)-

[0126] NR b R c ; or -(CHR a ) t -C 3-6 Cycloalkyl, wherein the cycloalkyl portion may be unsubstituted or substituted by R e Replace once or twice;

[0127] R 3 , R 4 and R 5 Each independently is: hydrogen; or C1-C6-alkyl;

[0128] Each R a are independently: hydrogen; or C 1-6 alkyl;

[0129] Each R b are independently: hydrogen; C 1-6 Alkyl; or hydroxy-C1-C6 alkyl;

[0130] Each R c are independently: hydrogen; C 1-6 Alkyl; Hydroxy-C1-C6 alkyl; Cyano-C1-C6 alkyl;

[0131] C1-C6 alkoxy-C1-C6 alkyl; oxetanyl; 2-morpholinoethyl; 1-methyl-azetidin-3-yl; 2-(N,N-dimethylamino)-ethyl; hydroxycyclobutyl; or 3-

[0132] (N,N-dimethylamino)-pyrrolidin-1-yl; (CHR a ) u -C3-6 Cycloalkyl, wherein cycloalkyl

[0133] The moiety may be unsubstituted or replaced by R e Replace once or twice;

[0134] or R b and R c Together with the nitrogen atom to which they are attached they can form het 3 ;

[0135] Each R d Independently: C1-C6 alkyl, hydroxyl or halo;

[0136] Each R e Independently: C 1-6 Alkyl; Hydroxyl; Cyano-C1-C6 Alkyl; Hydroxyl-C1-C6 Alkyl

[0137] methyl; morpholinyl; or -(CHR a ) v -NR g R h , where R g and R h Each independently is hydrogen

[0138] or C 1-6 alkyl;

[0139] h is 0 to 2;

[0140] k is 0 to 2;

[0141] m is 0 to 2;

[0142] n is 0 to 2;

[0143] p is 0 to 2;

[0144] q is 0 to 2;

[0145] r is 0 to 2;

[0146] s is 0 to 2;

[0147] t is 0 to 2;

[0148] u is 0 to 2;

[0149] v is 0 to 2;

[0150] het 1 is: oxetane; tetrahydrofuranyl; tetrahydropyranyl; or pyrrolidinyl; each of which may be unsubstituted or replaced by R d Replace once or twice;

[0151] het 2are: azetidinyl; pyrrolidinyl; oxetanyl; piperidinyl; morpholinyl; piperazinyl; azepine quinuclidine; or pyrazolyl; each of which may be unsubstituted or replaced by R e replaced once or twice; and

[0152] het 3 is: azetidinyl; pyrrolidinyl; piperidinyl; morpholinyl; piperazinyl; or aza each of which may be unsubstituted or replaced by R e Replace once or twice.

[0153] In certain embodiments, Ar is phenyl; or pyrazolyl.

[0154] In certain embodiments, Ar is phenyl.

[0155] In certain embodiments, Ar is pyrazolyl.

[0156] In certain embodiments, R 1 It is hydrogen or C1-C6 alkyl.

[0157] In certain embodiments, R 1 For hydrogen.

[0158] In certain embodiments, R 1 It is a C1-C6 alkyl group.

[0159] In certain embodiments, R 1 It is methyl.

[0160] In certain embodiments, R 3 For hydrogen.

[0161] In certain embodiments, R 4 For hydrogen.

[0162] In certain embodiments, R 5 For hydrogen.

[0163] In certain embodiments, h is zero.

[0164] In certain embodiments, h is 1.

[0165] In some embodiments, h is 2.

[0166] In some embodiments, k is zero.

[0167] In some embodiments, k is 1.

[0168] In some embodiments, k is 2.

[0169] In certain embodiments, m is 0.

[0170] In certain embodiments, m is 1.

[0171] In some embodiments, m is 2.

[0172] In certain embodiments, n is 0.

[0173] In certain embodiments, n is 1.

[0174] In certain embodiments, n is 2.

[0175] In certain embodiments, p is 0.

[0176] In certain embodiments, p is 1.

[0177] In some embodiments, p is 2.

[0178] In some embodiments, q is zero.

[0179] In certain embodiments, q is 1.

[0180] In some embodiments, q is 2.

[0181] In certain embodiments, r is zero.

[0182] In certain embodiments, r is 1.

[0183] In certain embodiments, r is 2.

[0184] In some embodiments, s is 0.

[0185] In certain embodiments, s is 1.

[0186] In some embodiments, s is 2.

[0187] In certain embodiments, t is 0.

[0188] In certain embodiments, t is 1.

[0189] In certain embodiments, t is 2.

[0190] In some embodiments, u is zero.

[0191] In some embodiments, u is 1.

[0192] In some embodiments, u is 2.

[0193] In some embodiments, v is zero.

[0194] In some embodiments, v is 1.

[0195] In some embodiments, v is 2.

[0196] In some embodiments, each R 2 Independently selected from:

[0197]

[0198]

[0199] In some embodiments, each R 2 Independently selected from:

[0200]

[0201]

[0202] In some embodiments, each R 2 Independently selected from:

[0203]

[0204]

[0205] In some embodiments, each R 2 Independently selected from:

[0206]

[0207]

[0208] In some embodiments, n is 2, R 2 One is selected from: -CH2NH2; -OCF3; -CH2CN; -F; -CF3; -CHF2; -CH2CN; -OCF3; and And other R 2 Selected from:

[0209]

[0210]

[0211] In some embodiments, wherein n is 1, R 2 Selected from;

[0212]

[0213]

[0214] In some embodiments, wherein n is 1, R 2 Selected from;

[0215]

[0216]

[0217] In certain embodiments, 3 Selected from: morpholinyl; azetidinyl: and piperazinyl.

[0218] In certain embodiments, the subject compounds have Formula (II):

[0219]

[0220] Where R 1 , R 2 and n are as defined herein.

[0221] In certain embodiments, the subject compounds have Formula (III):

[0222]

[0223] Where R 1 and R 2 As defined herein.

[0224] In some embodiments, a compound or salt (eg, a pharmaceutically acceptable salt) or a stereoisomer thereof selected from the following Table 1 is provided:

[0225] 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.

[0226] Also provided is the use of the JAK inhibitors described herein or their pharmaceutically acceptable salts 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 their pharmaceutically acceptable salts in the preparation of a medicament for the treatment of 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 its pharmaceutically acceptable salt.

[0227] 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.

[0228] 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.

[0229] In one embodiment, a composition is provided that is formulated for administration by inhalation.

[0230] 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.

[0231] In one embodiment, a JAK inhibitor described herein, or a pharmaceutically acceptable salt thereof, is at least five times more potent as a JAK1 inhibitor than as a LRRK2 inhibitor.

[0232] 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.

[0233] In one embodiment, a method for treating hair loss in a mammal is provided, the method comprising administering a JAK inhibitor described herein or a pharmaceutically acceptable salt thereof to the mammal.

[0234] In one embodiment, provided is a use of a JAK inhibitor described herein or a pharmaceutically acceptable salt thereof for the treatment of hair loss.

[0235] 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.

[0236] The compounds of the 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 the compounds may use racemates, diastereomers or enantiomers as starting materials or intermediates. A mixture of specific diastereomeric compounds may be separated or enriched in one or more specific diastereomers by chromatography or crystallization. Similarly, the same techniques or other techniques known in the art may be used to separate or enrich enantiomeric mixtures. Asymmetric carbon or nitrogen atoms may each be in R or S configuration, and these configurations are within the scope of the present invention.

[0237] 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. When stereochemistry is specified by a solid wedge or dashed line representing a specific configuration, then that stereoisomer is specified and defined. Unless otherwise indicated, relative stereochemistry is intended when a solid wedge or dashed line is used.

[0238] 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.

[0239] 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 is capable of being activated or converted to a more active parent form by enzymes or hydrolysis. See, for example, Wilman, "Prodrugs in Cancer Chemotherapy" Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) and Stella et al., "Prodrugs: A Chemical Approach to Targeted Drug Delivery," Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267, Humana Press (1985). Prodrugs include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, β-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, and 5-fluorocytosine and 5-fluorouridine prodrugs.

[0240] A particular class of prodrugs is a compound 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 compounds containing a leaving group bonded to the carbonyl group, and include, for example, acyl halides, acyl amines, acyl pyridinium salts, acyl alcohol salts, acyl phenoxides, such as p-nitrophenoxy acyl, dinitrophenoxy acyl, fluorophenoxy acyl, and difluorophenoxy acyl. The reaction is usually 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.).

[0241] 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 groups such as (C1-C6) alkanoyloxymethyl, 1-((C1-C6) alkanoyloxy)ethyl, 1-methyl-1-((C1-C6) alkanoyloxy)ethyl, (C1-C6) alkoxycarbonyloxymethyl, N-(C1-C6) alkoxycarbonylaminomethyl, succinyl, (C1-C6) alkanoyl, α-amino(C1-C4) alkanoyl, aryl and α-aminoacyl or α-aminoacyl-α-aminoacyl, wherein each α-aminoacyl group is independently selected from natural L-amino acids, P(O)(OH)2, -P(O)(O(C1-C6) alkyl)2 or glycosyl (free radicals generated by removal of hydroxyl groups in the hemiacetal form of carbohydrates).

[0242] "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 (trifluoromethanesulfonate)) and arylsulfonyloxy (e.g., p-toluenesulfonyloxy (toluenesulfonate) and p-nitrosulfonyloxy (nonanoate)).

[0243] Synthesis of Janus kinase inhibitor compounds

[0244] Compounds can be synthesized by synthetic routes 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.

[0245] Compounds can be prepared individually or as a compound library 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 compound library comprising at least 2 compounds of the present invention is provided.

[0246] For the purpose of illustration, the reaction scheme described below provides the approach to synthesizing the compounds of the present invention and key intermediates. For detailed description of each reaction step, see the following example chapters. It will be appreciated by those skilled in the art that other synthetic routes can be used. Although certain specific starting materials and reagents are described in the flow, and discussed below, other starting materials and reagents can be substituted to provide various derivatives or reaction conditions. In addition, according to the present disclosure, conventional chemical methods well known to those skilled in the art can be used to further modify many compounds prepared by the following methods.

[0247] 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.

[0248] 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:

[0249] (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.

[0250] (2) The reaction of a primary or secondary amine with an aryl halide or quasi-halide (e.g., a triflate) can be accomplished using a variety of catalysts, ligands, and bases, commonly referred to as the "Buchwald-

[0251] Hartwig cross-coupling". A review of these methods is provided in Comprehensive Organic Name Reactions and Reagents, 2010, 575-581.

[0252] (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.

[0253] (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 aqueous base, such as lithium hydroxide, sodium hydroxide or potassium hydroxide, or a strong aqueous mineral acid, such as HCl; for tert-butyl esters, an acid (e.g., a solution of HCl in dioxane or a solution of trifluoroacetic acid (TFA) in dichloromethane (DCM)) is used for hydrolysis.

[0254] Reaction Scheme 1

[0255]

[0256] Reaction Scheme 1 illustrates the synthesis of compounds of Formula I. Nitropyrazole compound 1 can be arylated with 4-bromo-1-(difluoromethoxy)-2-iodobenzene under palladium catalysis to produce compound 2. The nitro group of compound 2 can be reduced using conditions such as iron and ammonium chloride to produce aminoaniline 3. The amide bond is coupled with commercially available pyrazolo[1,5-a]pyrimidine-3-carboxylic acid in the presence of a coupling agent such as, but not limited to, PyAOP and an organic base such as, but not limited to, DIPEA and DMAP in an organic solvent such as, but not limited to, DMF to provide compound 4. Compounds of Formula 6 can be synthesized by treating compound 4 with an appropriately substituted hydroxyaryl compound 5 such as phenol, pyrazole or pyridinol and a base such as, but not limited to, cesium carbonate in a solvent such as, but not limited to toluene under Pd catalyzed coupling conditions. The SEM protecting group of compound 6 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 provide compound 7. Compound 7 can then be reacted with a group R 1 -X undergoes N-alkylation, wherein X is halo (eg bromo) to provide compound 8.

[0257] 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.

[0258] 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 a suitable solvent such as dichloromethane in the presence of a base such as triethylamine, 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 a suitable solvent such as dichloromethane in the presence of a suitable base such as triethylamine. 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 a suitable solvent such as dichloromethane in the presence of a suitable base such as triethylamine.

[0259] 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).

[0260] 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.

[0261] 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).

[0262] 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.

[0263] 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.

[0264] 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.

[0265] 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.

[0266] The carboxylic acid group (-CO2H) can be converted to an amide (CONHR' or -CONR'R") by reaction with an appropriate amine in a suitable coupling reagent such as HATU in an appropriate solvent such as dichloromethane.

[0267] 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 the Arndt-Eistert synthesis.

[0268] 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.

[0269] 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.

[0270] 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).

[0271] Aromatic halogen substituents in compounds can be treated with a base, for example, a lithium base (such as n-butyl lithium or tert-butyl lithium), optionally at low temperatures (e.g., about -78°C), in a solvent (such as tetrahydrofuran) for halogen metal exchange, 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.

[0272] 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.

[0273] Separation methods

[0274] In each exemplary scheme, it may be advantageous to separate the reaction products from each other or from the starting 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.

[0275] 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.

[0276] 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.

[0277] 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.

[0278] A single stereoisomer, such as an enantiomer substantially free of its stereoisomer, can be obtained by resolving a racemic mixture using an optically active resolving agent using a method such as diastereoisomer formation (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)). The racemic mixture of the chiral compounds of the present 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) directly separating 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).

[0279] 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.

[0280] 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.

[0281] 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.

[0282] Pharmaceutical compositions and administration

[0283] 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.

[0284] Therefore, another embodiment provides a pharmaceutical composition or medicament 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 medicament using the compound of the present invention.

[0285] 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 nontoxic to the recipient at the dosage and concentration used) under an appropriate pH value and desired purity at ambient temperature. The pH 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.

[0286] 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.

[0287] 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, time of administration, route of administration, 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. Typically, the daily dosage range for administration will be in the following range: about 0.001 mg to about 100 mg per kg of human body weight, typically 0.01 mg to about 50 mg per kg of human body weight, for example 0.1 to 10 mg per kg of human body weight, in a single dose or divided dose. Typically, 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.

[0288] The compounds of the present invention or their pharmaceutically acceptable salts can be administered in any suitable manner, 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 administration is adopted.

[0289] 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.

[0290] Suitable carriers and excipients are well known to those skilled in the art and are described in detail in, for example, Ansel, Howard C. et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems. Philadelphia: Lippincott, Williams and 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 the case where any conventional carrier is incompatible with the active ingredient, the use of the carrier in treatment or pharmaceutical compositions is predictable. Exemplary excipients include dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate or a combination thereof. Pharmaceutical compositions may include 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 routes of administration.

[0291] 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.

[0292] 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.

[0293] 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.

[0294] 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).

[0295] 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.

[0296] 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.

[0297] In the case of aerosol-based formulations, examples are:

[0298] Compound of the present invention*24 mg / can

[0299] Lecithin, NF liquid concentration 1.2mg / can

[0300] Trichlorofluoromethane, NF 4.025g / can

[0301] Dichlorodifluoromethane, NF 12.15g / can.

[0302] * or its pharmaceutically acceptable salt

[0303] 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 compound, the administration form may also contain excipients as described above, or for example propellants (e.g. Frigen in the case of a metered aerosol), surfactants, emulsifiers, stabilizers, preservatives, flavoring agents, fillers (e.g. lactose in the case of a powder inhaler) or, if appropriate, further active compounds.

[0304] 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.

[0305] 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 a vehicle. Advantageously, an adjuvant such as a local anesthetic, a preservative or a buffer can be dissolved in the vehicle.

[0306] Targeted inhaled drug delivery

[0307] The compounds of the invention may be used for targeted inhalation delivery. The optimization of drugs delivered to the lung by topical (inhalation) administration has been recently reviewed (Cooper, AE et al. Curr. Drug Metab. 2012, 13, 457-473).

[0308] Due to the limitations of the delivery device, the dose of inhaled drugs may be limited in the human body, which requires efficient molecules with good lung pharmacokinetic properties. Due to factors such as the limited amount of drug that can be delivered from an inhaler at one time, and safety issues related to high lung spray loads (e.g., cough or inflammation), high efficacy for targets of interest 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 assay based on JAK1-dependent cells as described herein is about 20nM or less. In other embodiments, the estimated human dose of the compound of the present invention or its pharmaceutically acceptable salt is at least twice less than the estimated human dose of the compound known in the art. Therefore, in some embodiments, the compound described herein (or its pharmaceutically acceptable salt) demonstrates such efficacy values. The following procedure is used to evaluate the potential use of the subject compound as an inhaled drug.

[0309] IL13 signaling.IL13 signaling is closely related to the onset of asthma. IL13 is a cytokine that requires active JAK1 for signaling. Therefore, inhibiting JAK1 will also inhibit IL13 signaling, which will benefit asthma patients. Inhibition of IL13 signaling in animal models (e.g., mouse models) can predict future benefits for human asthma patients. Therefore, inhaled JAK1 inhibitors may be beneficial in showing IL13 signaling inhibition in animal models. Methods for measuring such 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 effects on pSTAT6 levels, the compounds of the present invention were co-administered intranasally with IL13 to female Balb / c mice. The compound was formulated in 0.2% (v:v) Tween 80 in saline and mixed with IL13 immediately before administration at a ratio of 1:1 (v:v). Intranasal doses were 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 hours 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 analysis. After adding 2 mL of HPLC grade water per gram of tissue, the defrosted lung samples were weighed and homogenized using Omni-Prep Bead Ruptor at 4 ° C. Plasma and lung samples were extracted by protein precipitation, wherein three volumes of acetonitrile containing tolbutamide (50 ng / mL) and labetalol (25 ng / mL) were used as analytical internal standards. After vortex mixing and centrifugation at 3200g 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 hours).

[0310] In order 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 protease (Sigma Aldrich, catalog number P8340) and phosphatase (Sigma Aldrich, catalog number P5726 and P0044) inhibitor mixtures in the buffer. The sample was centrifuged at 4 ° C for 4 minutes with 16060 x g to remove tissue debris, and the protein concentration of the homogenate was determined using Pierce BCA protein assay kit (catalog number 23225). It is 5 mg / ml for the sample to be diluted to a protein concentration in ice-cold distilled water, and pSTAT6 levels are determined by Meso Scale Discovery electrochemiluminescent immunoassay. Briefly, 5 μl / well of 150 μg / 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 μl / well of 30 mg / ml Meso Scale Discovery blocking agent A (catalog number R93BA-4) and cultured on a microplate oscillator at room temperature for 2 hours. The blocked plate was washed 4 times with Meso Scale Discovery TRIS wash buffer (catalog number R61TX-1), after which 50 μl / well of lung homogenate was transferred to reach a protein load of 250 μg / well. The assay plate was incubated overnight at 4°C and washed 4 times with TRIS wash buffer, followed by addition of 25 μl / well 2.5 μg / ml thio-tagged pSTAT6 detection antibody (BD Pharmingen, catalog number 558241) 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.

[0311] JAK and JAK2 inhibitionCompounds that inhibit JAK1 and JAK2 can potentially be used to treat different types of asthma. The selectivity between JAK1 and JAK2 is also important for inhaled JAK1 inhibitors. For example, GMCSF (granulocyte-macrophage colony stimulating factor) is a cytokine that signals only through JAK2. Neutralization of GMCSF activity is associated with pulmonary alveolar proteinosis (PAP) in the lungs. However, submaximal JAK2 inhibition does not seem to be related to PAP. Therefore, even moderate JAK1 and JAK2 selectivity, or approximately equivalent inhibition of JAK1 and JAK2, helps to avoid complete inhibition of the GMCSF pathway and avoid PAP. For example, in certain embodiments, compounds that are equivalent to JAK1 and JAK2 are needed. In other embodiments, compounds that are about 2-5 times more selective for JAK1 than for JAK2 can be beneficial for inhaled JAK1 inhibitors. Therefore, in some embodiments, the compounds described herein (or pharmaceutically acceptable salts thereof) demonstrate such selectivity. Methods for measuring JAK1 and JAK2 selectivity are known in the art, and information can also be found in the examples herein.

[0312] 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 compared to a group of non-JAK kinases. Therefore, in some embodiments, the compounds described herein (or pharmaceutically acceptable salts thereof) demonstrate such selectivity.

[0313] Cytotoxicity assay.Hepatotoxicity, general cytotoxicity or cytotoxicity of unknown mechanisms are undesirable features 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 evaluating cytotoxicity include primary cells (e.g., human hepatocytes) and cell lines (e.g., Jurkat and HEK-293) established by proliferation. Therefore, in some embodiments, the compounds described herein (or pharmaceutically acceptable salts thereof) demonstrate these values. Methods for measuring cytotoxicity are known in the art. In some embodiments, the compounds described herein are tested as follows:

[0314] (a) 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, 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 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);

[0315] (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) TMThe cells were plated 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 plated with 1% Torpedo TM InVitroGro with antibiotic cocktail and 1% DMSO (serum-free conditions) TM Hepatocytes were treated with compounds diluted in HI culture 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, 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 measurements 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 Estimates with top and bottom limits constrained to constant values ​​of 100 and 0, respectively (GraphPad Prism TM , GraphPad Software, La Jolla, CA, USA).

[0316] 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 minimized hERG inhibition under the following conditions:

[0317] (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 μM. 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 of approximately 10-90% 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

[0318] (b) In the example of WO 2014 / 074775, in “Effect on Cloned hERG Potassium Channels Expressed in Mammalian Cells”, ChanTest by Charles River Company TM Those described under the scheme, the following changes were made: the cells stably expressing hERG were maintained at -80mV. Use a pulse mode of fixed amplitude (adjustment pre-pulse: +20mV lasts 1s; repeat repolarization test ramepto-90mV (-0.5V / s) at intervals of 5s). Each record ends with the final application of the super-large concentration of reference substance 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.

[0319] CYP (cytochrome P450) inhibition test. 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 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. The CYP inhibition of the compounds described herein was evaluated over a concentration range of 0.16-10 μM using pooled (n=150) human liver microsomes (Corning, Tewksbury, MA) using a previously reported method (Halladay et al., Drug Metab. Lett. 2011, 5, 220-230). The incubation duration and protein concentration depended on the CYP isoform and the 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 minutes, 0.03 mg / ml protein; CYP2C9, warfarin / 7-hydroxywarfarin, 30 minutes, 0.2 mg / ml protein; CYP2C19, mephenytoin / 4-hydroxymephenytoin, 40 minutes, 0.2 mg / ml protein; CYP2D6, dextromethorphan / dextrorphan, 10 minutes, 0.03 mg / ml protein; CYP3A4, midazolam / 1-hydroxymidazolam, 10 minutes, 0.03 mg / ml protein and CYP3A4 testosterone / 6-hydroxytestosterone, 10 minutes, 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 (with appropriate stable labeled internal standards). Samples were analyzed by LC-MS / MS.

[0320] Mouse lung tissue binding A high fraction or percentage of JAK1 / JAK2 inhibitor binding to lung tissue may be undesirable because it would reduce the amount of free drug available to inhibit JAK1 or JAK2.

[0321] (a) Tissue binding experiments were performed in triplicate (n=3) using single-use REDs according to standard protocols. Initially, individual drugs were added to tissue homogenate (pH 7.4) to a final concentration of 1 μM, and then 300 μL of the drug-tissue homogenate mixture was transferred to the donor well of the RED preloaded with 500 μL of phosphate-buffered saline (133 mM) on the receiving well. The RED plate was sealed with a gas-permeable membrane and placed in a shaking incubator (450 rpm, VWR Symphony TM ) and cultured for 6 hours at 37°C and 5% CO2. At the end of the culture, a 30 μL aliquot of the sample was taken from the RED device and the matrix was equilibrated with an equal volume of tissue homogenate or buffer, and then the resulting sample was immediately quenched with ice-cold acetonitrile (sample: acetonitrile 1:4) containing propranolol or labetalol as internal standard. After shaking at 500 rpm for 15 min on a Thermo Scientific Compact Digital MicroPlate Shaker, 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.

[0322] (b) In an alternative 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) apparatus (Fisher Scientific 89811 & 89809). A 10 mM solution of the compound in DMSO was prepared and diluted to 1 mM with DMSO. An aliquot of this 1 mM (4 μL) was added to lung homogenate (dilution factor 1:9, lung tissue: potassium phosphate buffer (0.05 M, pH 7.4)) to give a final compound incubation concentration of 5 μM, with solvent accounting for 0.5% (v / v) of the final incubation volume.

[0323] For each assay, the percentage of lung tissue bound was determined in triplicate. Lung homogenate (200 μL) was loaded into one side of the RED device insert in triplicate, and 350 μL of potassium phosphate buffer was loaded into the other side. The RED device was sealed and incubated at ea 37°C on an orbital shaker (approximately 150 rpm) for 4 hours.

[0324] After incubation, a portion of lung homogenate (8 μL) and a portion of dialysate (72 μL) were matrix matched before analysis (lung homogenate with 72 μL phosphate buffer, dialysate with 8 μL lung homogenate). 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 aliquots of lung homogenate sampled at the beginning of the experiment (t = 0 min sample) to assess mass balance. The quenched samples were centrifuged (4000 rpm, 30 min, 4 ° C), the resulting supernatant was diluted with water (3: 1 (v / v), supernatant: water), and the samples or parent compounds were analyzed by liquid chromatography mass spectrometry.

[0325] The unbound fraction (fu) in lung homogenate was determined from the ratio of dialysate to homogenate peak areas, corrected to account for lung homogenate dilution (D), allowing estimation of binding in whole lung tissue using the following equation:

[0326] Undiluted fu = (1 / D) / [((1 / surface fu)-1)+(1 / D)]

[0327] Corrected binding fraction (%) = (1-undiluted FU) * 100

[0328] Kinetic solubility. In order to reduce the amount of undissolved particulate matter in the lung, it may be desirable for the JAK1 / JAK2 inhibitors delivered by inhalation to have good aqueous solubility. In one procedure to measure kinetic solubility, 4 μL of a 10 mM DMSO test compound stock solution was added to a Millipore A test concentration of 200 μM with 2% residual DMSO was obtained in 196 μL of pH 7.4 phosphate buffered saline solution in a 96-well filter plate. The filter plate was sealed with an aluminum sealing film and shaken at room temperature for 24 hours, and then the mixture was vacuum filtered into a clean 96-well plate. The filtrate sample was diluted two-fold using pH 7.4 phosphate buffered saline solution, and 5 μL of the resulting solution was analyzed at a wavelength of 254 nm by ultra-high performance liquid chromatography (UHPLC) with chemiluminescent nitrogen detection (CLND) and ultraviolet (UV) detection. The sample concentration is usually quantified by the CLND intensity, which is related to the number of nitrogen in the compound. UV detection is mainly used to confirm sample purity, except in rare cases where the test compound does not contain nitrogen. In those cases, a compound-specific calibration curve is collected based on UV absorbance. This curve is then used to determine the sample concentration.

[0329] Lipophilic.Lipophilicity is often correlated with the solubility, absorption, tissue penetration, protein binding, distribution, and ADME and PK properties of a potential drug. The calculated logP (cLogP), the logarithm of the partition coefficient of a compound between n-octanol and water (i.e., concentration of compound in n-octanol / concentration of compound in water), can therefore be an important consideration for inhaled-delivered JAK1 / JAK inhibitors.

[0330] Hepatic microsomal stability. In order to minimize the systemic exposure of inhaled JAK1 / JAK2 inhibitors, it may be beneficial to optimize the rapid metabolism in the liver. The liver microsomal stability assay was performed on a BioCel 1200 liquid handling workstation (gilent Technologies, Santa Clara, CA). The compound (1.0 μM) was incubated at 37 ° C for 5 min in a 100 μL 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), a 20 μL aliquot of the reaction mixture was taken out and mixed with 4 volumes of acetonitrile (ACN) containing 0.1 μM propranolol as an internal standard to stop 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 and analyzed 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 remaining percentage (T = 0 min) was calculated using the peak area ratio of the test compound to the internal standard relative to the control 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.

[0331] Solid state properties. For compounds intended to be delivered by dry powder inhalation, it is also desirable 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 less than 5 micrometers (μm) are generally defined as respirable. 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 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 lung, a particle size of 1-5 μm can be beneficial. Typical methods for measuring particle size include laser diffraction and cascade impaction. Typical values ​​used to define particle size include:

[0332] • 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.

[0333] • 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.

[0334] • Geometric Standard Deviation (GSD). GSD is a measure of the dispersion value or aerodynamic particle size distribution spread according to MMAD.

[0335] A common formulation for inhaled drugs is a dry powder formulation comprising an active pharmaceutical ingredient (API) blended with a carrier (such as lactose) and (or without) additional additives (such as magnesium stearate). For this formulation and other formulations, it may be beneficial for the API itself to have a property that allows it to be ground into a respirable particle size of 1-5 μm. Agglomeration of particles should be avoided, and the agglomeration 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 pharmaceutically acceptable salts thereof) can be prepared to have such a respirable particle size with little or no agglomeration.

[0336] 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.

[0337] 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.

[0338] 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 a short lung half-life, so it is necessary to attenuate the lung absorption rate by changing one or more characteristics of the compound: minimizing membrane permeability, optimizing pKa, cLogP, solubility, dissolution rate, or introducing a degree of alkalinity 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.

[0339] 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 medications rather than inhaled medications. For example, compounds that are fast to absorb are generally difficult to retain in the lungs when inhaled.

[0340] Methods of treatment and uses using JANUS kinase inhibitors

[0341] The compound of the present invention or its pharmaceutically acceptable salt inhibits the activity of Janus kinases such as JAK1 kinases. For example, the compound or its pharmaceutically acceptable salt inhibits the phosphorylation of signal transduction and transcription activator (STAT) by JAK1 kinase and STAT-mediated cytokine production. The compound 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 cells with a compound of the present invention or its pharmaceutically acceptable salt to inhibit Janus kinase activity (e.g., JAK1 activity) in cells.

[0342] 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.

[0343] Thus, one embodiment includes a compound of the present invention or a pharmaceutically acceptable salt thereof for use in therapy.

[0344] In some embodiments, the use of a compound of the present invention or a pharmaceutically acceptable salt thereof in the treatment of an inflammatory disease is provided. Further provided is the use of a compound of the present invention or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating an inflammatory disease such as asthma. Also provided is a compound of the present invention or a pharmaceutically acceptable salt thereof for treating an inflammatory disease such as asthma.

[0345] 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 kinases, such as JAK1 kinases, is asthma.

[0346] 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 disorders, inflammation, nervous system disorders, hormone-related diseases, disorders related to organ transplantation (e.g., transplant rejection), immunodeficiency disorders, destructive bone diseases, proliferative disorders, infectious diseases, disorders related to cell death, thrombin-induced platelet aggregation, liver disease, pathological immune disorders involving T cell activation, CNS disorders, or myeloproliferative disorders.

[0347] 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.

[0348] In another embodiment, the compound of the present invention or its pharmaceutically acceptable salt can be used for treating lung disease, such as fibrotic lung disease or interstitial lung disease (e.g., 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 sclerotic interstitial lung disease (SSc-ILD), nonspecific interstitial pneumonia (NSIP), rheumatoid arthritis related interstitial lung disease (RA-ILD), sarcoidosis, hypersensitivity pneumonitis or the ILD (e.g., polymyositis, dermatomyositis, rheumatoid arthritis, systemic lupus erythematosus (SLE) or mixed connective tissue disease) secondary to connective tissue disease other than scleroderma.

[0349] 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.

[0350] In one embodiment, the disease is a myeloproliferative disorder. In one embodiment, the myeloproliferative disorder is polycythemia vera, essential thrombocythemia, myelofibrosis, or chronic myeloid leukemia (CML).

[0351] 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.

[0352] Combination therapy

[0353] The compound can be used alone or in combination with other agents for treatment. The second compound or other compounds (e.g., the third) of the pharmaceutical composition or dosage regimen generally have 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 effective amount combination 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.

[0354] For example, other compounds can be used in combination with the compounds of the present invention or their pharmaceutically acceptable salts 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; 2-adrenergic receptor agonists; CCR1 antagonists; chemokine antagonists (not CCR1); corticosteroids; CRTh2 antagonists; DP1 antagonists; formyl peptide receptor antagonists; histone deacetylase activators; chloride channel hCLCA1 blockers; epithelial sodium channel blockers (ENAC blockers; intercellular adhesion molecule 1 blockers (ICAM blockers); IKK2 inhibitors; JNK inhibitors; transient receptor potential anchor protein 1 (TRPA1) inhibitors; Bruton's tyrosine kinase (BTK) inhibitors (such as fenebrutinib); spleen tyrosine kinase (SYK) inhibitors; tryptase β antibodies; ST2 receptor antibodies (such as AMG 282); cyclooxygenase inhibitors (COX inhibitors); lipoxygenase inhibitors; leukotriene receptor antagonists; dual 2-adrenergic receptor agonists / M3 receptor antagonists (MABA compounds); MEK-1 inhibitors; myeloperoxidase inhibitors (MPO inhibitors); muscarinic antagonists; p38MAPK inhibitors; phosphodiesterase PDE4 inhibitors; phosphatidylinositol 3-kinase delta inhibitors (PI3-kinase delta inhibitors); phosphatidylinositol 3-kinase gamma inhibitors (PI3-kinase inhibitors); peroxisome proliferator-activated receptor agonists (PPAR agonists); protease inhibitors; retinoic acid receptor modulators (RAR modulators); statins; thromboxane antagonists; TLR7 receptor agonists; or vasodilators.

[0355] In addition, the compounds of the present invention or their pharmaceutically acceptable salts may be combined with the following substances: (1) corticosteroids such as aclometasone dipropionate, amelometasone, beclomethasone dipropionate, budesonide, buticotinide propionate, Biclesonide, clobetasol propionate, desisobutyrylciclesonide, dexamethasone, dtiprednol dicloacetate, fluocinolone, 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, tritoquinol, tulobuterol, and long-acting β2-adrenergic receptor agonists such as metaproterenol, isoproterenol, isoprenaline, salmeterol, indacaterol, formoterol (including formoterol fumarate), arformoterol, carmoterol, abediterol, vilanterol trifluoperate, and dapoxetine. trifenate), olodaterol; (3) corticosteroid / long-acting β2-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 (LAS-34273), glycopyrrolate, or umeclidinium bromide; (5) M3-anticholinergic / β2-adrenergic receptor agonist combination products, such as vilanterol / umeclidinium Olodaterol / tiotropium, glycopyrrolate / indacaterol Sales), Fenoterol Hydrobromide / Ipratropium Bromide Salbutamol sulfate / ipratropium bromide Formoterol fumarate / glycopyrrolate or aclidinium / formoterol; (6) dual pharmacology M3-anticholinergic / β2-adrenergic receptor agonist, such as Batefenterol 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 (inhaled 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, loratadine or astemizole, or dual H1 / H3 receptor antagonists such as GSK 835726 or GSK 1004723; (10) antitussives, such as codeine or dextramorphan; (11) mucolytics, such as N-acetylcysteine ​​or fudostein; (12) expectorants / viscoelastic modulators, such as ambroxol, hypertonic solutions (e.g., saline or mannitol), or surfactants; (13) mucolytic peptides, such as 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, such as 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 WO 2005 / 026124, WO 2003 / 053930 and WO 2006 / 082412, each of which is incorporated herein by reference; (21) A2b antagonists, such as those described in WO 2002 / 42298, which is incorporated herein by reference; (22) Modulators of chemokine receptor function, such as antagonists of CCR3 and CCR8; (23) Compounds that modulate the action of other prostanoid receptors, such as thromboxane A2 antagonists; DP1 antagonists, such as laropiprant or asapiprant; CRTH2 antagonists, such as OC000459, fevipiprant, ADC 3680 or ARRY 502; (24) PPAR 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 / beclomethasone 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 anakinra 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 (glycopyrrolate); (33) triple combination products, such as TRELEGY ELLIPTA (fluticasone furoate, umeclidinium bromide, and vilanterol); or (34) small molecule inhibitors of TRPA1, BTK, or SYK.

[0356] In some embodiments, the compounds of the present invention or their pharmaceutically acceptable salts 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 their pharmaceutically acceptable salts can also be used in combination with radiotherapy or surgery.

[0357] Combinations of any of the foregoing with a compound of the invention or a pharmaceutically acceptable salt thereof are specifically contemplated.

[0358] Products

[0359] 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:

[0360] (a) a first pharmaceutical composition comprising a compound of the present invention or a pharmaceutically acceptable salt thereof; and

[0361] (b) Instructions for use.

[0362] In another embodiment, the kit further comprises:

[0363] (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.

[0364] 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.

[0365] 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.

[0366] 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 effectively treating 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 pierced 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 condition. In addition, the label or package insert may indicate that the patient to be treated is a patient with a condition 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 conditions.

[0367] 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.

[0368] 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.

[0369] Examples

[0370] General Experimental 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 (50 x 3 mm Shim-Pack XR-ODS, 2.2 μm particle size) and eluted with solvent A: water + 0.05% trifluoroacetic acid; solvent B: acetonitrile + 0.05% trifluoroacetic acid. Gradient:

[0375]

[0376]

[0377] Detection - UV (220 and 254 nm) and ELSD

[0378] Method B

[0379] The experiment was performed on a SHIMADZU LCMS-2020 using a C18 reverse phase column (50 x 3 mm Shim-Pack XR-ODS, 2.2 μm particle size) and eluted with solvent A: water + 0.05% trifluoroacetic acid; solvent B: acetonitrile + 0.05% trifluoroacetic acid. Gradient:

[0380]

[0381] Detection - UV (220 and 254 nm) and ELSD

[0382] Method C

[0383] The experiment was performed on a SHIMADZU LCMS-2020 using a C18 reverse phase column (50 x 3 mm Shim-Pack XR-ODS, 2.2 μm particle size) and eluted with solvent A: water + 0.05% trifluoroacetic acid; solvent B: acetonitrile + 0.05% trifluoroacetic acid. Gradient:

[0384]

[0385] Detection - UV (220 and 254 nm) and ELSD

[0386] Method D

[0387] The experiment was performed on a SHIMADZU LCMS-2020 using a C18 reverse phase column (50 x 3 mm Shim-Pack XR-ODS, 2.2 μm particle size) and eluted with solvent A: water + 0.05% trifluoroacetic acid; solvent B: acetonitrile + 0.05% trifluoroacetic acid. Gradient:

[0388]

[0389]

[0390] Detection - UV (220 and 254 nm) and ELSD

[0391] Method E

[0392] The experiment was performed on a SHIMADZU LCMS-2020 using a C18 reverse phase column (50 x 3 mm Shim-Pack XR-ODS, 2.2 μm particle size) and eluted with solvent A: water + 0.05% trifluoroacetic acid; solvent B: acetonitrile + 0.05% trifluoroacetic acid. Gradient:

[0393]

[0394] Detection - UV (220 and 254 nm) and ELSD

[0395] Method F

[0396] The experiment was performed on a SHIMADZU LCMS-2020 using a C18 reverse phase column (50 x 3 mm Shim-Pack XR-ODS, 2.2 μm particle size) and eluted with solvent A: water + 0.05% trifluoroacetic acid; solvent B: acetonitrile + 0.05% trifluoroacetic acid. Gradient:

[0397]

[0398]

[0399] Detection - UV (220 and 254 nm) and ELSD

[0400] Method G

[0401] The experiment was conducted on a SHIMADZU 20A HPLC using a C18 reverse phase column (50 x 2.1 mm Ascentis Express C18, 2.7 μm particle size) and the eluents were: solvent A: water + 0.05% trifluoroacetic acid; solvent B: acetonitrile + 0.05% trifluoroacetic acid. Gradient:

[0402]

[0403] Detection - UV (220 and 254 nm) and ELSD

[0404] Method H

[0405] The experiment was performed on a SHIMADZU LCMS-2020 using a C18 reverse phase column (50 x 3 mm Shim-Pack XR-ODS, 2.2 μm particle size) and eluted with solvent A: water + 0.05% trifluoroacetic acid; solvent B: acetonitrile + 0.05% trifluoroacetic acid. Gradient:

[0406]

[0407] Detection - UV (220 and 254 nm) and ELSD

[0408] Method I

[0409] The experiments were performed on a SHIMADZU 20A HPLC using a Poroshell HPH-C 18 Chromatographic column (50x 3mm, 2.7μm particle size), using: solvent A: water / 5mM NH4HCO3; solvent B: acetonitrile elution. Gradient:

[0410]

[0411]

[0412] Detection - UV (220 and 254 nm) and ELSD

[0413] Method J

[0414] The experiment was performed on a SHIMADZU LCMS-2020 using a C18 reverse phase column (50x 3mm Kinetex XB-C 18 , 2.6 μm particle size), eluted with solvent A: water + 0.05% trifluoroacetic acid; solvent B: acetonitrile + 0.05% trifluoroacetic acid. Gradient:

[0415]

[0416] Detection - UV (220 and 254 nm) and ELSD

[0417] Method K

[0418] The experiment was performed on a SHIMADZU LCMS-2020 using a C18 reverse phase column (50 x 3 mm Shim-Pack XR-ODS, 2.2 μm particle size) and eluted with solvent A: water + 0.05% trifluoroacetic acid; solvent B: acetonitrile + 0.05% trifluoroacetic acid. Gradient:

[0419]

[0420] Detection - UV (220 and 254 nm) and ELSD

[0421] Method L

[0422] The experiment was performed on a SHIMADZU LCMS-2020 using a C18 reverse phase column (50 x 2.1 mm Kinetex XB-C 18 100A, 2.6 μm particle size), eluted with solvent A: water + 0.05% trifluoroacetic acid; solvent B: acetonitrile + 0.05% trifluoroacetic acid. Gradient:

[0423]

[0424] Detection - UV (220 and 254 nm) and ELSD

[0425] Method M

[0426] The experiment was performed on a SHIMADZU LCMS-2020 using a C18 reverse phase column (30 x 2.1 mm Kinetex C18-100A, 1.7 μm particle size) and eluted with solvent A: water + 0.05% trifluoroacetic acid; solvent B: acetonitrile + 0.05% trifluoroacetic acid. Gradient:

[0427]

[0428] Detection - UV (220 and 254 nm) and ELSD

[0429] Method N

[0430] The experiment was performed on a SHIMADZU LCMS-2020 using a C18 reverse phase column (50 x 3.0 mm Poroshell HPH-C18, 2.7 μm particle size) and eluted with solvent A: water + 5 mM ammonium bicarbonate; solvent B: acetonitrile. Gradient:

[0431]

[0432] Detection - UV (220 and 254 nm) and ELSD

[0433] Method O

[0434] The experiment was performed on a SHIMADZU LCMS-2020 using a C18 reverse phase column (50 x 3.0 mm Titank C18, 3.0 μm particle size) and eluted with solvent A: water + 5 mM ammonium bicarbonate; solvent B: acetonitrile. Gradient:

[0435]

[0436] Detection - UV (220 and 254 nm) and ELSD

[0437] Method P

[0438] The experiment was performed on a SHIMADZU LCMS-2020 using a C18 reverse phase column (30 x 2.1 mm Halo C18, 2.0 μm particle size) and eluted with solvent A: water + 0.05% trifluoroacetic acid; solvent B: acetonitrile + 0.05% trifluoroacetic acid. Gradient:

[0439]

[0440] Detection - UV (220 and 254 nm) and ELSD

[0441] Method Q

[0442] The experiment was performed on a SHIMADZU LCMS-2020 using a C18 reverse phase column (50 x 3.0 mm YMC-Triart C18, 2.5 μm particle size) and eluted with solvent A: water + 0.1% formic acid; solvent B: acetonitrile + 0.1% formic acid. Gradient:

[0443]

[0444]

[0445] Detection - UV (220 and 254 nm) and ELSD

[0446] Method R

[0447] The experiment was performed on a SHIMADZU LCMS-2020 using a C18 reverse phase column (50 x 3 mm Shim-Pack XR-ODS, 2.2 μm particle size) and eluted with solvent A: water + 0.05% trifluoroacetic acid; solvent B: acetonitrile + 0.05% trifluoroacetic acid. Gradient:

[0448]

[0449] Detection - UV (220 and 254 nm) and ELSD

[0450] Method S

[0451] The experiment was performed on a SHIMADZU LCMS-2020 using a C18 reverse phase column (50 x 3.0 mm Poroshell HPH-C18, 2.7 μm particle size) and eluted with solvent A: water + 5 mM ammonium bicarbonate; solvent B: acetonitrile. Gradient:

[0452]

[0453] Detection - UV (220 and 254 nm) and ELSD

[0454] Method T

[0455] The experiment was conducted on a SHIMADZU 20A HPLC using a C18 reverse phase column (50 x 2.1 mm Ascentis Express C18, 2.7 μm particle size) and the eluents were: solvent A: water + 0.05% trifluoroacetic acid; solvent B: acetonitrile + 0.05% trifluoroacetic acid. Gradient:

[0456]

[0457]

[0458] Detection - UV (220 and 254 nm) and ELSD

[0459] Method U

[0460] The experiment was performed on a SHIMADZU LCMS-2020 using a C18 reverse phase column (50 x 3 mm Shim-Pack XR-ODS, 2.2 μm particle size) and eluted with solvent A: water + 0.05% trifluoroacetic acid; solvent B: acetonitrile + 0.05% trifluoroacetic acid. Gradient:

[0461]

[0462] Detection - UV (220 and 254 nm) and ELSD

[0463] Method V

[0464] The experiment was performed on a SHIMADZU LCMS-2020 using a C18 reverse phase column (50 x 3.0 mm Poroshell HPH-C18, 2.7 μm particle size) and eluted with solvent A: water + 5 mM ammonium bicarbonate; solvent B: acetonitrile. Gradient:

[0465]

[0466] Detection - UV (220 and 254 nm) and ELSD

[0467] Method W

[0468] The experiment was performed on a SHIMADZU LCMS-2020 using a C18 reverse phase column (50 x 2.1 mm Waters AcquityBEH, 1.7 μm particle size) and eluted with solvent A: water + 0.1% formic acid; solvent B: acetonitrile + 0.1% formic acid. Gradient:

[0469]

[0470] Detection - UV (220 and 254 nm) and ELSD

[0471] Method X

[0472] 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, 1.7 μm, 50×2.1 mm column was used to separate the LC at a flow rate of 0.4 ml / min. Mobile phase A was water containing 0.1% formic acid, and mobile phase B was acetonitrile containing 0.1% formic acid. The gradient started with 2% B and ended with 98% B for 7 min, and maintained at 98% B for 1.5 min, then equilibrated for 1.5 min. The LC column temperature was 40°C. UV absorbance at 220 nm and 254 nm was collected, and all experiments used a full mass spectrometry scan.

[0473] Method Y

[0474] The experiment was performed on a SHIMADZU LCMS-2020 using a C18 reverse phase column (50x 3.0 mm Gemini-NX, 3.0 μm particle size) and eluted with solvent A: water + 5 mM NH4HCO3; solvent B: acetonitrile + 5 mM NH4HCO3. Gradient:

[0475]

[0476] Detection - UV (220 and 254 nm) and ELSD

[0477] Method Z

[0478] The experiment was performed on a SHIMADZU LCMS-2020 using a C18 reverse phase column (50x 3.0 mm Gemini-NX, 3.0 μm particle size) and eluted with solvent A: water + 5 mM NH4HCO3; solvent B: acetonitrile + 5 mM NH4HCO3. Gradient:

[0479]

[0480] Detection - UV (220 and 254 nm) and ELSD

[0481] List of common abbreviations

[0482] ACN Acetonitrile

[0483] Brine Saturated sodium chloride solution

[0484] CH3OD Deuterated methanol

[0485] CDCl3 deuterated chloroform

[0486] DCM Dichloromethane

[0487] DIEA or DIPEA Diisopropylethylamine

[0488] DMA Dimethylacetamide

[0489] DMAP 4-dimethylaminopyridine

[0490] DMF Dimethylformamide

[0491] DMSO Dimethyl sulfoxide

[0492] DMSO-d6 Deuterated dimethyl sulfoxide

[0493] EDC or EDCI 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

[0494] EtOAc Ethyl acetate

[0495] EtOH

[0496] FA Formic acid

[0497] HOAc Acetic acid

[0498] g

[0499] h hour

[0500] HATU (O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate)

[0501] HCl

[0502] HOBt Hydroxybenzotriazole

[0503] HPLC High Performance Liquid Chromatography

[0504] IMS Industrial Methylated Spirits

[0505] L Liter

[0506] LCMS liquid chromatography-mass spectrometry

[0507] LiHMDS or LHMDS Lithium Hexamethyldisilazide

[0508] MDAP mass spectrometry-guided automated purification

[0509] MeCN Acetonitrile

[0510] MeOH Methanol

[0511] min

[0512] mg milligram

[0513] mL milliliters

[0514] NMR Nuclear Magnetic Resonance Spectroscopy

[0515] Pd2(dba)3.CHCl3 tris(dibenzylideneacetone)dipalladium(0)-chloroform adduct

[0516] PE Petroleum Ether

[0517] Prep-HPLC Preparative High Performance Liquid Chromatography

[0518] SCX-2 Strong Cation Exchange

[0519] TBAF Tetra-n-butylammonium fluoride

[0520] THF Tetrahydrofuran

[0521] TFA Trifluoroacetic acid

[0522] Xantphos 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene

[0523] The following representative compounds of Table 1 were prepared using procedures analogous to those described in the Schemes and Examples herein. The absolute stereochemistry of each of the following compounds may not be depicted: therefore, a structure may appear more than once, each representing one stereoisomer.

[0524] Table 1

[0525]

[0526]

[0527]

[0528]

[0529]

[0530]

[0531]

[0532]

[0533]

[0534]

[0535]

[0536]

[0537]

[0538]

[0539]

[0540]

[0541]

[0542]

[0543]

[0544]

[0545]

[0546]

[0547]

[0548]

[0549]

[0550]

[0551]

[0552]

[0553]

[0554]

[0555]

[0556]

[0557]

[0558]

[0559]

[0560]

[0561]

[0562]

[0563]

[0564]

[0565]

[0566]

[0567]

[0568]

[0569]

[0570]

[0571]

[0572]

[0573]

[0574]

[0575]

[0576]

[0577]

[0578]

[0579]

[0580]

[0581] Table 1 Intermediate 1

[0582]

[0583] N-(5-(5-bromo-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0584] Step 1: Synthesis of 4-bromo-1-(difluoromethoxy)-2-iodobenzene

[0585]

[0586] 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 releasing CO2. The resulting mixture is stirred overnight at 120 °, cooled to room temperature, and poured into ice water (3000mL). The resulting solution is extracted with ethyl acetate (3x1500 mL), and the organic layer is mixed. 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), and 300g (91%) of 4-bromo-1-(difluoromethoxy)-2-iodobenzene is obtained as a yellow oil. 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).

[0587] Step 2: Synthesis of 5-[5-bromo-2-(difluoromethoxy)phenyl]-4-nitro-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazole

[0588]

[0589] A solution of 4-nitro-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazole (100 g, 411 mmol) in anhydrous THF (1000 mL) was added dropwise to a solution of LiHMDS (490 mL, 1.0 mol / L in THF) and stirred 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 in THF) was added dropwise at -70°C. The resulting solution was heated to room temperature and stirred at room temperature for 1 h. 4-bromo-1-(difluoromethoxy)-2-iodobenzene (150 g, 860 mmol) and 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 was repeated once more at this scale and the crude product from the two runs was combined for purification. The residue was purified by flash chromatography on silica gel, eluting with ethyl acetate / petroleum ether (1 / 20). The appropriate fractions were combined and concentrated under reduced pressure. This resulted in 300 g (79%) of 5-[5-bromo-2-(difluoromethoxy)phenyl]-4-nitro-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazole as a pale 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).

[0590] Step 3: Synthesis of 5-(5-bromo-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-amine

[0591]

[0592] 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) was added iron powder (60.1 g, 1.07 mol) and NH4Cl (28.0 g, 0.523 mol). The reaction mixture was stirred for 3 h at reflux temperature under nitrogen. The solid was filtered out and washed with ethanol (100 mL). The filtrate was concentrated under reduced pressure. The residue was dissolved in 3000 mL ethyl acetate. The ethyl acetate solution was washed with 1x500 mL brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 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.

[0593] 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

[0594]

[0595] 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 in an oil bath at 60 °C for 3 h and then cooled to room temperature. The reaction mixture was then partitioned between water / ice (2000 mL) and ethyl acetate (2000 mL). The aqueous phase was extracted with ethyl acetate (2x). The organic layers were combined, washed with brine (1000 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel, eluting with ethyl acetate / petroleum ether (4:1). The appropriate fractions were combined and concentrated under reduced pressure. Water (150 mL) was added to the residue, and the mixture was stirred in water for 1 h at room temperature. The solid was collected by filtration and air-dried to give 60.1 g (91%) of N-(5-(5-bromo-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide as a 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).

[0596] Intermediate 2

[0597]

[0598] N-[3-[5-Bromo-2-(difluoromethoxy)phenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0599] N-[5-[5-bromo-2-(difluoromethoxy)phenyl]-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (Intermediate 2, 5.00 g, 8.63 mmol) was treated with HCl / dioxane (150 mL, 4 M) at room temperature overnight. The resulting mixture was concentrated under reduced pressure. This gave 3.80 g of N-[3-[5-bromo-2-(difluoromethoxy)phenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide as a yellow solid. The 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 H NMR(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).

[0600] Intermediate 3

[0601]

[0602] N-[3-[5-bromo-2-(difluoromethoxy)phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-

[0603] Formamide

[0604] 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 1, 10.1 g, 17.3 mmol) in dichloromethane (200 mL) was added Me3OBF4 (2.81 g, 18.9 mmol) at room temperature. The resulting solution was stirred at room temperature for 2 hours. 10 mL of EtOH was then added to the reaction mixture, and the reaction mixture was stirred for 1 h, 5.0 mL of HCl (concentrated) was added to the solution and stirred for 1 h. The resulting mixture was concentrated under vacuum. The pH value of the solution was adjusted to 8 with sodium bicarbonate (20%). The resulting solution was extracted with 3x300 mL of ethyl acetate, the organic layers were combined, dried over anhydrous sodium sulfate, and then concentrated under vacuum. The residue was applied to a silica gel column and eluted with ethyl acetate / petroleum ether (80%) to give 5.5 g (69%) of N-[3-[5-bromo-2-(difluoromethoxy)phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[l,5-a]pyrimidine-3-carboxamide as a light yellow solid. 1 H NMR (400MHz, CDC13): δ (ppm) 9.86 (s, 1H), 8.80 (dd, J = 7.0, 1.6Hz, 1H), 8.74 (s, 1H), 8.60 (dd, J = 4.2, 1.6Hz, 1H), 8.32 (s, 1H), 7.85 (d ,J=2.4Hz,1H),7.58(dd,J=8.4,2.4Hz,1H),7.24(d,J=8.8Hz,1H),7.02(dd,J=7.0,4.2Hz,1H),6.49(t,J=74.0Hz,1H),4.01(s,3H).

[0605] Intermediate 4

[0606]

[0607] N-(3-(2-(difluoromethoxy)-5-hydroxyphenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-

[0608] 3-Formamide

[0609] Step 1: Synthesis of N-[3-[2-(difluoromethoxy)-5-(tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0610]

[0611] N-[3-[5-bromo-2-(difluoromethoxy)phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (Intermediate 3, 1000 mg, 2.16 mmol), 4,4,5,5-tetramethyl-2-(tetramethyl-1,3,2-dioxaborolane-2-yl)-1,3,2-dioxaborolane (824 mg, 3.25 mmol, 1.50 equiv), Pd(dppf)Cl 2- CHCl (176 mg, 0.216 mmol, 0.100 equiv), DPPF (119 mg, 0.215 mmol, 0.100 equiv), potassium acetate (636 mg, 6.48 mmol, 3.00 equiv) and dioxane (18 mL) were placed in a 30-mL sealed tube, which was purged and maintained under an inert atmosphere of nitrogen. The resulting mixture was stirred overnight at 110 ° C and then concentrated under vacuum. The residue was purified by silica gel column and eluted with ethyl acetate / petroleum ether (60:40). This gave 965 mg (88%) of N-[3-[2-(difluoromethoxyy)-5-(tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide as a light yellow solid. LC / MS (method H, ESI): [M+H] + =511.2,R T =1.31min.

[0612] Step 2: Synthesis of N-(3-(2-(difluoromethoxy)-5-hydroxyphenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0613]

[0614] In a 100 mL round bottom bottle, N-[3-[2-(difluoromethoxy)-5-(tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (965 mg, 1.89 mmol), tetrahydrofuran (10 mL), H2O2 (0.5 mL, 21.5 mmol) were placed. The resulting solution was stirred at room temperature overnight and then concentrated under vacuum. This gave 680 mg (90%) of N-[3-[2-(difluoromethoxy)-5-hydroxyphenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide as a gray solid. LC / MS (Method H, ESI): [M+H] + =401.1,R T =1.04min.

[0615] Examples

[0616] Example 1

[0617]

[0618] N-(3-(2-(difluoromethoxy)-5-phenoxyphenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0619] Step 1: Synthesis of N-(5-(2-(difluoromethoxy)-5-phenoxyphenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0620]

[0621] N-(5-(5-bromo-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (Intermediate 1, 100 mg, 0.173 mmol), phenol (32.5 mg, 0.345 mmol, 2.00 equiv), [PdCl(allyl)]2 (6.31 mg, 0.017 mmol, 0.10 equiv), RockPhos (16.2 mg, 0.035 mmol, 0.20 equiv), cesium carbonate (84.3 mg, 0.26 mmol, 1.50 equiv), toluene (2 mL) were placed in a 10-mL round bottom flask, which was purged and maintained under an inert atmosphere of nitrogen. The resulting solution was stirred in an oil bath at 100 °C for 14 h. The resulting mixture was concentrated under vacuum. The residue was applied to a silica gel column and eluted with ethyl acetate / petroleum ether (1:1). This gave 100 mg (98%) of N-(5-(2-(difluoromethoxy)-5-phenoxyphenyl)-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] + =593.3,R T =1.35min.

[0622] Step 2: Synthesis of N-(3-(2-(difluoromethoxy)-5-phenoxyphenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0623]

[0624] N-(5-(2-(difluoromethoxy)-5-phenoxyphenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (100 mg, 0.169 mmol), trifluoroacetic acid (2 mL) and dichloromethane (8 mL) were placed in a 25 mL round bottom bottle. The resulting solution was stirred at room temperature for 30 min and concentrated under vacuum. The crude product was purified by Flash-Prep-HPLC under the following conditions (IntelFlash-1): chromatographic column, C18 silica gel; mobile phase, increasing from CH3CN:H2O=30:70 to CH3CN:H2O=70:30 in 14 min; detector, UV 254 nm. This gave 28.5 mg (37%) of N-(3-(2-(difluoromethoxy)-5-phenoxyphenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide as a light yellow solid. LC / MS (Method G, ESI): [M+H] + =463.2,R T =0.86min. 1 H NMR (300MHz, CD3OD) δ9.14–9.12(m,1H),8.69–8.65(m,2H),8.28–8.26(m,1H),7.45–7.42(m,1H),7.33–7.04(m,8H),6.72(t,J=73.7Hz,1H).

[0625] Example 133

[0626]

[0627] N-(3-(2-(difluoromethoxy)-5-((5-((dimethylamino)methyl)pyridin-3-yl)oxy)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0628] N-[3-[5-bromo-2-(difluoromethoxy)phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (Intermediate 3, 300 mg, 0.648 mmol), 5-[(dimethylamino)methyl]pyridin-3-ol (197 mg, 1.30 mmol), [PdCl(allyl)]2 (9.48 mg, 0.026 mmol), RockPhos (30.4 mg, 0.065 mmol), cesium carbonate (422 mg, 1.30 mmol) and toluene (13 mL) were added to a 100 mL round-bottom flask and degassed with nitrogen for 5 min. The reaction mixture was stirred at 100 ° C overnight and then concentrated under vacuum. The crude product was purified by reverse phase HPLC to give the title compound (39.0 mg, 0.071 mmol, 10.9% yield) as a white solid. LC / MS (Method W, ESI): [M+H] + =535.2,R T =1.04min. 1 H NMR(400MHz, DMSO-d6)δ9.73(s,1H),9.35(dd,J=7.0,1.6Hz,1H),8.80–8.51(m,2H),8.41–8 .09(m,3H),7.47(d,J=8.9Hz,1H),7.40–6.96(m,5H),3.89(s,3H),3.39(s,2H),2.10(s,6H).

[0629] Example 157

[0630]

[0631] N-(3-(2-(difluoromethoxy)-5-((1,2,3,4-tetrahydroisoquinolin-7-yl)oxy)phenyl)-1-(oxetane

[0632] -3-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0633] Step 1: Synthesis of N-(3-(5-bromo-2-(difluoromethoxy)phenyl)-1-(oxetan-3-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0634]

[0635] N-[3-[5-bromo-2-(difluoromethoxy)phenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (Intermediate 2, 200 mg, 0.445 mmol), N,N-dimethylformamide (8 mL), cesium carbonate (403 mg, 1.24 mmol, 3.0 equivalents), 3-iodooxane (97.3 mg, 0.529 mmol, 1.2 equivalents) were placed in a 25 mL round-bottom flask. The resulting solution was stirred at 60 ° C for 4 h. The reaction mixture was concentrated in vacuo. The residue was applied to a silica gel column and eluted with dichloromethane / methanol (10:1). This gave 187 mg (83%) of N-(3-(5-bromo-2-(difluoromethoxy)phenyl)-1-(oxetan-3-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide as a yellow solid. LC / MS (Method J, ESI): [M+H] + =505.1&507.1,R T =1.03min.

[0636] Step 2. Synthesis of tert-butyl 7-(4-(difluoromethoxy)-3-(1-(oxetan-3-yl)-4-(pyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-3-yl)phenoxy)-3,4-dihydroisoquinoline-2(1H)-carboxylate

[0637]

[0638] N-[3-[5-bromo-2-(difluoromethoxy)phenyl]-1-(oxetane-3-yl)-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (187 mg, 0.37 mmol), toluene (10 mL), Pd2(allyl)2Cl2 (6.8 mg, 0.019 mmol, 0.050 equiv), t-BuBrettPhos (17.9 mg, 0.037 mmol, 0.10 equiv), cesium carbonate (145 mg, 0.45 mmol, 1.2 equiv), tert-butyl 7-hydroxy-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (111 mg, 0.45 mmol, 1.2 equiv) were placed in a 30-mL sealed tube. The resulting solution was stirred at 100° C. overnight. The resulting mixture was concentrated under vacuum. The residue was applied to a silica gel column and eluted with ethyl acetate / petroleum ether (1:1). This gave 125 mg (50%) of tert-butyl 7-(4-(difluoromethoxy)-3-(1-(oxetan-3-yl)-4-(pyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-3-yl)phenoxy)-3,4-dihydroisoquinoline-2(1H)-carboxylate as a yellow solid. LC / MS (Method H, ESI): [M+H]+ =674.4,R T =1.40min.

[0639] Step 3. Synthesis of N-(3-(2-(difluoromethoxy)-5-((1,2,3,4-tetrahydroisoquinolin-7-yl)oxy)phenyl)-1-(oxetan-3-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0640]

[0641] Tert-butyl 7-(4-(difluoromethoxy)-3-(1-(oxetan-3-yl)-4-(pyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-3-yl)phenoxy)-3,4-dihydroisoquinoline-2(1H)-carboxylate (150 mg, 0.223 mmol), hydrogen chloride (4M in dioxane, 1 mL) and 1,4-dioxane (4 mL) were placed in a 25 mL round bottom flask. The resulting solution was stirred at room temperature for 1 hour. The resulting mixture was concentrated under vacuum. This gave 120 mg (94%) of N-(3-(2-(difluoromethoxy)-5-((1,2,3,4-tetrahydroisoquinolin-7-yl)oxy)phenyl)-1-(oxetan-3-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide as a yellow solid. LC / MS (Method A, ESI): [M+H] + =574.3,R T =1.30min. 1 H NMR(300MHz,CD3OD)δ9.11(dd,J=7.2,1.8Hz,1H),8.67–8.64(m,2H),8.41(s,1H),7.40(d,J=8.8Hz,1H),7.25–7.21(m,2H),7 .17–7.14(m,1H),7.08–6.51(m,3H),5.62–5.57(m,1H),5.12–5.08(m,4H),3.89(s,2H),3.10–3.06(m,2H),2.82–2.80(m,2H).

[0642] Example 128

[0643]

[0644] N-(3-(2-(difluoromethoxy)-5-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)oxy)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0645] Step 1: N-(3-(2-(difluoromethoxy)-5-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)oxy)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0646]

[0647] N-(3-(5-bromo-2-(difluoromethoxy)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide was added at 25°C under N2. 1 To a solution of (Intermediate 3, 56 g, 121 mmol), 1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-ol (35.0 g, 163 mmol), di-tert-butyl-[6-methoxy-3-methyl-2-(2,4,6-triisopropylphenyl)phenyl]phosphine (5.78 g, 12.3 mmol) and allyl(chloro)palladium (2.30 g, 6.29 mmol) in toluene (1.40 L) was added K2CO3 (50.0 g, 361 mmol). The resulting solution was stirred at 100 °C for 3 h. The resulting mixture was concentrated under vacuum and diluted with water (500 mL). The resulting solution was extracted with ethyl acetate (3 x 400 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 ethyl acetate / petroleum ether (75%) to give N-(3-(2-(difluoromethoxy)-5-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)oxy)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (150 g, crude) as a green solid. LC / MS (Method G, ESI): [M+H] + =597.2,R T =1.19min.

[0648] Step 2: N-(3-(5-((1H-pyrazol-4-yl)oxy)-2-(difluoromethoxy)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0649]

[0650] To a solution of N-(3-(2-(difluoromethoxy)-5-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)oxy)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (45 g, 75.4 mmol) in methanol (450 mL) was added HCl (230 g, 6.29 mol, 225 mL). The reaction mixture was heated to 60 °C and stirred for 30 min. This sequence was repeated two more times on the same scale and the three reaction mixtures were combined for workup. The reaction mixture was cooled to 25 °C and concentrated under vacuum. The crude product was dissolved in THF (1.50 L) and the solution was adjusted to pH 8 with sodium bicarbonate. The resulting solid was filtered from the solution and dissolved in THF. Ethyl acetate was added and the resulting precipitate was isolated by vacuum filtration (76.5 g). The filtrate was concentrated in vacuo, dissolved in THF, and precipitated with EtOAc. Vacuum filtration gave a second portion (4.95 g) of the desired product. The filtrate was concentrated and purified by prep HPLC (column: Phenomenex lunac18 250 mm*100 mm*10 μm; mobile phase: [water (0.1% TFA)-ACN]; B%: 20%-50%, 23 min) to give a third portion (13.8 g) of the desired product. A total of N-(3-(5-((1H-pyrazol-4-yl)oxy)-2-(difluoromethoxy)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (95.3 g, 65.6%) was obtained as an off-white solid. 1 H NMR: DMSO 400MHzδ12.83(s,1H),9.74(s,1H),9.35-9.33(m,1H),8.68-8.66(m,2H),8.27-7.81(s,1H),7.48-7.41(s,1H),7.38-7.31(s,1H),7.29-7.28(m,1H),7.17-6.90(m,4H).LC / MS(Method I,ESI):[M+H] + =467.3,R T =0.91min.

[0651] Step 3: tert-Butyl 3-((4-(4-(difluoromethoxy)-3-(1-methyl-4-(pyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-3-yl)phenoxy)-1H-pyrazol-1-yl)methyl)-3-hydroxyazetidine-1-carboxylate

[0652]

[0653] To a solution of N-(3-(5-((1H-pyrazol-4-yl)oxy)-2-(difluoromethoxy)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (850 mg, 1.82 mmol), tert-butyl 1-oxa-5-azaspiro[2.3]hexane-5-carboxylate (675 mg, 3.65 mmol) in methanol (12 mL) was added DIEA (706 mg, 5.47 mmol) at room temperature. The resulting solution was stirred at 80° C. overnight. The resulting residue was purified by reverse phase chromatography (acetonitrile 0-50 / 0.05% NH4HCO3 in water) to give tert-butyl 3-((4-(4-(difluoromethoxy)-3-(1-methyl-4-(pyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-3-yl)phenoxy)-1H-pyrazol-1-yl)methyl)-3-hydroxyazetidine-1-carboxylate (837 mg, 1.28 mmol, 70.5% yield) as a yellow solid. LC / MS (Method H, ESI): [M+H] + =652.3,R T =1.20min.

[0654] Step 4: N-(3-(2-(difluoromethoxy)-5-((1-((3-hydroxyazetidin-3-yl)methyl)-1H-pyrazol-4-yl)oxy)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0655]

[0656] To a solution of tert-butyl 3-((4-(4-(difluoromethoxy)-3-(1-methyl-4-(pyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-3-yl)phenoxy)-1H-pyrazol-1-yl)methyl)-3-hydroxyazetidine-1-carboxylate (737 mg, 1.13 mmol) at room temperature was added trifluoroacetic acid (84 mg, 0.73 mmol) in 2-hexylfluoropropanol (5.0 mL, 1.13 mmol). The resulting solution was stirred at 30° C. for two days. The resulting residue was purified by reverse phase chromatography (acetonitrile 0-40 / 0.05% NH4HCO3 in water) to give N-(3-(2-(difluoromethoxy)-5-((1-((3-hydroxyazetidin-3-yl)methyl)-1H-pyrazol-4-yl)oxy)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (1.11 g) as a white solid. LC / MS (Method N, ESI): [M+H] + =552.3,R T =1.11min.1 H NMR (300MHz, DMSO-d6) δ9.75 (s, 1H), 9.35 (dd, J = 6.9, 1.5Hz, 1H), 8.67–8.65 (m, 2H), 8. 27(s,1H),7.70(s,1H),7.41–6.84(m,6H),5.67(s,1H),3.91(s,3H),3.34–3.17(m,5H).

[0657] Step 5: N-(3-(2-(difluoromethoxy)-5-((1-((3-hydroxy-1methylazetidin-3-yl)methyl)-1H-pyrazol-4-yl)oxy)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0658]

[0659] To a solution of N-(3-(2-(difluoromethoxy)-5-((1-((3-hydroxyazetidin-3-yl)methyl)-1H-pyrazol-4-yl)oxy)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (950 mg, 1.72 mmol) was added HCHO / HO (1.79 g, 1.72 mmol) in methanol (24 mL) at room temperature. The resulting solution was stirred for 2 h. Sodium triacetoxyborohydride (5.04 g, 1.72 mmol) was then added. The reaction mixture was stirred at room temperature for 2 h and concentrated in vacuo. The resulting crude product was purified by reverse phase chromatography (acetonitrile 0-45 / 0.1% NH4HCO3 in water) to give the desired product as a white solid (864.1 mg). After stirring at 55 ° C for 2 h, and then at room temperature for 3 days, the solid was recrystallized from cyclohexane / isopropanol = 3 / 1 (60 mL). After filtration, N-(3-(2-(difluoromethoxy)-5-((1-((3-hydroxy-1-methylazetidin-3-yl)methyl)-1H-pyrazol-4-yl)oxy)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (836.6 mg, 1.4793 mmol, 85.9% yield) was obtained as a white solid. LC / MS (Method A, ESI): [M+H] + =566.3,R T =1.21min. 1H NMR(400MHz,DMSO-d6)δ9.74(s,1H),9.34(dd,J=7.2,1.6Hz,1H),8.67–8.65(m,2H),8.27(s,1H),7.70(s,1H) ,7.41–6.90(m,6H),5.60(s,1H),4.22(s,2H),3.90(s,3H),3.37–3.31(m,2H),2.75–2.73(m,2H),2.20(s,3H).

[0660] Examples 32 and 33

[0661]

[0662] (N-[3-[2-(difluoromethoxy)-5-[3-[(3R)-morpholin-3-yl]phenoxy]phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide and (N-[3-[2-(difluoromethoxy)-5-[3-[(3S)-morpholin-3-yl]phenoxy]phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0663] [phenoxy]phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0664] Step 1. Synthesis of tert-butyl 3-(3-(4-(difluoromethoxy)-3-(1-methyl-4-(pyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-3-yl)phenoxy)phenyl)morpholine-4-carboxylate

[0665]

[0666] Toluene (10 mL), N-[3-[2-(difluoromethoxy)-5-hydroxyphenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (intermediate 4, 400 mg, 1.00 mmol), tert-butyl 3-(3-bromophenyl)morpholine-4-carboxylate (410 mg, 1.2 mmol, 1.20 equivalents), [PdCl(allyl)]2 (7.31 mg, 0.020 mmol, 0.02 equivalents), RockPhos (18.7 mg, 0.040 mmol, 0.040 equivalents), cesium carbonate (651 mg, 2.0 mmol, 2.0 equivalents) were placed in a 30-mL sealed tube, which was purged and maintained under an inert atmosphere of nitrogen. The resulting solution was stirred in an oil bath at 90 ° C for 14 h. The resulting mixture was concentrated under vacuum. The residue was applied to a silica gel column and eluted with dichloromethane / methanol (10:1). This gave 200 mg (30%) of tert-butyl 3-[3-[4-(difluoromethoxy)-3-(1-methyl-4-[pyrazolo[1,5-a]pyrimidin-3-amino]-1H-pyrazol-3-yl)phenoxy]phenyl]morpholine-4-carboxylate as a light yellow solid.

[0667] Step 2. Synthesis of (N-[3-[2-(difluoromethoxy)-5-[3-[(3R)-morpholin-3-yl]phenoxy]phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide and (N-[3-[2-(difluoromethoxy)-5-[3-[(3S)-morpholin-3-yl]phenoxy]phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0668]

[0669] Hydrogen chloride / dioxane (4M, 3mL), tert-butyl 3-[3-[4-(difluoromethoxy)-3-(1-methyl-4-[pyrazolo[1,5-a]pyrimidine-3-amino]-1H-pyrazol-3-yl)phenoxy]phenyl]morpholine-4-carboxylate (200mg, 0.302mmol) were placed in a 25mL round-bottom bottle. The resulting solution was stirred at room temperature for 2 hours. The resulting mixture was concentrated under vacuum. The crude product was purified by Prep-HPLC under the following conditions: (2#-AnalyseHPLC-SHIMADZU (HPLC-10)): chromatographic column, XBridge Prep C18 OBD chromatographic column, 19*150mm 5μm 13nm; mobile phase, 10mmol NH4HCO3 aqueous solution and ACN (24.0% ACN, increased to up to 42.0% in 8min); detector, UV 220nm. The racemic product was purified by chiral preparative HPLC under the following conditions (Prep-HPLC-009): column, CHIRALPAK-AD-H-SL001, 20*250 mm; mobile phase, Hex and IPA (maintaining 50.0% IPA within 50 min); detector, UV 254 / 220 nm. This afforded (N-[3-[2-(difluoromethoxy)-5-[3-[(3R)-morpholin-3-yl]phenoxy]phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (peak 1, 11.4 mg, 7% yield) as a white solid and (N-[3-[2-(difluoromethoxy)-5-[3-[(3S)-morpholin-3-yl]phenoxy]phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (peak 2, 8.4 mg, 5% yield) as a white solid with arbitrary stereochemical assignments. 1 H NMR (300 MHz, DMSO-d6) δ 9.74 (s, 1H), 9.35 (dd, J = 6.9, 1.5 Hz, 1H), 8.68–8.66 (m, 2H), 8.26 (s, 1H), 7.45–6.90 (m, 9H), 3.89 (s, 3H), 3.76–3.68 (m, 3H), 3.32 (m, 1H), 3.09 (m, 1H), 2.82–2.80 (m, 2H), 1.24 (m, 2H), 0.88–0.83 (m, 1H). LC / MS (Method E, ESI): [M+H] + =562.3,R T =2.73min.

[0670] Example 73

[0671]

[0672] N-(3-(2-(difluoromethoxy)-5-((1-(2-(dimethylamino)ethyl)-1H-pyrazol-4-yl)oxy)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0673] Step 1: Synthesis of N-(3-(5-((1-(2-bromoethyl)-1H-pyrazol-4-yl)oxy)-2-(difluoromethoxy)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0674]

[0675] N-(3-(5-((1H-pyrazol-4-yl)oxy)-2-(difluoromethoxy)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (200 mg, 0.429 mmol), N,N-dimethylformamide (15 mL), cesium carbonate (700 mg, 2.15 mmol, 5.00 equiv), 1,2-dibromoethane (1.6 g, 8.52 mmol, 20.000 equiv) were placed in a 30-mL sealed tube. The resulting solution was stirred at 60 ° C in an oil bath for 3 h. The resulting mixture was concentrated under vacuum. The residue was applied to a silica gel column and eluted with ethyl acetate / petroleum ether (100% EA). This gave 180 mg (73%) of N-(3-(5-((1-(2-bromoethyl)-1H-pyrazol-4-yl)oxy)-2-(difluoromethoxy)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide as a yellow oil. LC / MS (Method J, ESI): [M+H] + =573.2&575.2,R T =1.00min.

[0676] Step 2. Synthesis of N-(3-(2-(difluoromethoxy)-5-((1-(2-(dimethylamino)ethyl)-1H-pyrazol-4-yl)oxy)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0677] N-[3-(5-[[1-(2-bromoethyl)-1H-pyrazol-4-yl]oxy]-2-(difluoromethoxy)phenyl)-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (180 mg, 0.314 mmol, 1.000 equiv), CH3CN (14 mL, 266 mmol), DIEA (203 mg, 1.57 mmol, 5.00 equiv), dimethylamine hydrochloride (76.5 mg, 0.94 mmol, 3.00 equiv) were placed in a 30-mL sealed tube. The resulting solution was stirred at 70 ° C in an oil bath for 3 h. The resulting mixture was concentrated under vacuum. The residue was applied to a silica gel column and eluted with dichloromethane / methanol (85:15). The crude product (120 mg) was purified by Flash-Prep-HPLC under the following conditions (IntelFlash-1): silica gel column; mobile phase, ACN / H2O (10 mmol NH4HCO3) = 15%, increasing to 37% in 8 min; detector, UV 254 nm. This gave 34.9 mg (21%) of N-(3-(2-(difluoromethoxy)-5-((1-(2-(dimethylamino)ethyl)-1H-pyrazol-4-yl)oxy)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide as a light yellow solid. LC / MS (Method T, ESI): [M+H] + =538.3,R T =0.94min. 1 H NMR(300MHz,DMSO-d6)δ9.74(s,1H),9.35(dd,J=6.9,1.5Hz,1H),8.67–8.66(m,2H),8.27(s,1H),7.82(s, 1H),7.41–7.38(m,2H),7.33–6.84(m,4H),4.14–4.10(m,2H),3.90(s,3H),2.63–2.59(m,2H),2.13(s,6H).

[0678] Example 124

[0679]

[0680] N-(3-(2-(difluoromethoxy)-5-((1-(2-hydroxy-2-methylpropyl)-1H-pyrazol-4-yl)oxy)phenyl)-1-

[0681] Methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0682] N-[3-[2-(difluoromethoxy)-5-(1H-pyrazol-4-yloxy)phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (80 mg, 0.172 mmol), DIEA (90 mg, 0.70 mmol, 4.06 equiv), methanol (5 mL), 2,2-dimethyloxirane (25 mg, 0.35 mmol, 2.02 equiv) were placed in an 8-mL sealed tube. The resulting solution was stirred at 80 ° C for 20 h in an oil bath. The resulting mixture was concentrated under vacuum. The residue was applied to a silica gel column and eluted with dichloromethane / methanol (5% MeOH). This gave 33.3 mg (36%) of N-(3-(2-(difluoromethoxy)-5-((1-(2-hydroxy-2-methylpropyl)-1H-pyrazol-4-yl)oxy)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide as a white solid. LC / MS (Method A, ESI): [M+H] + =539.3,R T =1.70min. 1 H NMR(400MHz, DMSO-d6)δ9.75(s,1H),9.35(dd,J=7.2,1.6Hz,1H),8.67–8.66(m,2H),8.28(s,1H),7.71(s,1H) ,7.41–7.39(m,2H),7.30–7.27(m,1H),7.18–6.90(m,3H),4.68(s,1H),3.96(s,2H),3.90(s,3H),1.05(s,6H).

[0683] Example 117

[0684]

[0685] N-(3-(5-((1-(3-(cyanomethyl)-1-methyl-azetidin-3-yl)-1H-pyrazol-4-yl)oxy)-2-

[0686] (difluoromethoxy)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0687] N-[3-[5-bromo-2-(difluoromethoxy)phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (Intermediate 3, 100 mg, 0.216 mmol), 2-[3-(4-hydroxy-1H-pyrazol-1-yl)-1-methylazetidin-3-yl]acetonitrile (40 mg, 0.21 mmol, 0.96 equiv), Pd2(allyl)2Cl2 (3 mg, 0.008 mmol, 0.038 equiv), t-BuBrettPhos (8 mg, 0.017 mmol, 0.076 equiv), cesium carbonate (60 mg, 0.18 mmol, 0.85 equiv), toluene (5 mL) were placed in a 10-mL round-bottom flask, which was purged and maintained under an inert atmosphere of nitrogen. The resulting solution was stirred at 80° C. overnight. The resulting mixture was concentrated in vacuo. The residue was applied to a silica gel column and eluted with dichloromethane / methanol (90 / 10). The collected fractions were combined and concentrated under vacuum. The crude product was purified by Flash-Prep-HPLC under the following conditions (IntelFlash-1): silica gel column; mobile phase, 2O(NH4HCO3) / CH3CN=90 / 10, increasing to 50 / 50 within 10 min; detector, UV 254nm. This gave 16.4 mg (13%) of N-(3-(5-((1-(3-(cyanomethyl)-1-methyl-azetidin-3-yl)-1H-pyrazol-4-yl)oxy)-2-(difluoromethoxy)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide as an off-white solid. LC / MS (Method A, ESI): [M+H] + =575.3,R T =1.28min. 1 H NMR(400MHz, DMSO-d6)δ9.76(s,1H),9.36(dd,J=6.8,1.6Hz,1H),8.68–8.67(m,2H),8.28(s,1H),8.16(s,1H),7. 60(s,1H),7.41(d,J=Hz,1H),7.31–6.93(m,4H),3.92(s,3H),3.58–3.56(m,2H),3.49–3.47(m,2H),3.43(s,2H).

[0688] Example 203

[0689]

[0690] N-(3-(2-(difluoromethoxy)-5-((1-(1-methylpiperidin-4-yl)-1H-pyrazol-4-yl)oxy)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0691] Step 1. Synthesis of tert-butyl 4-(4-(4-(difluoromethoxy)-3-(1-methyl-4-(pyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-3-yl)phenoxy)-1H-pyrazol-1-yl)piperidine-1-carboxylate

[0692]

[0693] Under nitrogen, a solution of N-[3-[2-(difluoromethoxy)-5-(1H-pyrazol-4-yloxy)phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (100, 0.210 mmol), tert-butyl 4-iodopiperidine-1-carboxylate (334 mg, 1.07 mmol), cesium carbonate (209 mg, 0.640 mmol) and N,N-dimethylformamide (10 mL) was added at room temperature. The resulting solution was stirred at 120° C. overnight. The crude reaction mixture was passed through celite. Filter. Dilute the organic layer with water (100 mL). Extract the resulting solution with EA (100*3 mL) and combine the organic layers. The organic layer is dried over anhydrous sodium sulfate and concentrated in vacuo. The resulting residue is purified by reverse phase chromatography (acetonitrile 0-56 / 0.05% NH4HCO3 in water) to give tert-butyl 4-(4-(4-(difluoromethoxy)-3-(1-methyl-4-(pyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-3-yl)phenoxy)-1H-pyrazol-1-yl)piperidine-1-carboxylate (96.4 mg) as a white solid. LC / MS (Method H, ESI): [M+H] + =467.2,R T =1.09min.

[0694] Step 2. Synthesis of N-(3-(2-(difluoromethoxy)-5-((1-(piperidin-4-yl)-1H-pyrazol-4-yl)oxy)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0695]

[0696] To a solution of tert-butyl 4-[4-[4-(difluoromethoxy)-3-[1-methyl-4-(pyrazolo[1,5-a]pyrimidine-3-carboxamido)pyrazol-3-yl]phenoxy]pyrazol-1-yl]piperidine-1-carboxylate (96.4 mg, 0.150 mmol) in dichloromethane (5 mL) at room temperature, trifluoroacetic acid (1.0 mL, 0.150 mmol) was added. The resulting solution was stirred at room temperature for 4 h and concentrated under vacuum. The crude product was used directly without further purification.

[0697] Step 3. Synthesis of N-(3-(2-(difluoromethoxy)-5-((1-(1-methylpiperidin-4-yl)-1H-pyrazol-4-yl)oxy)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0698]

[0699] A solution of N-[3-[2-(difluoromethoxy)-5-[1-(4-piperidinyl)pyrazol-4-yl]oxy-phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (150 mg, 0.270 mmol), HCHO / HO (360 mg, 12.0 mmol) in methanol (6 mL) was added at room temperature. The resulting solution was stirred at rt for 2 h. Sodium triacetoxyborohydride (1.1 mg, 0.01 mmol) was then added and stirred at room temperature for 2 h. The reaction mixture was concentrated under vacuum. The resulting residue was purified by reverse phase chromatography; column: XBridge Prep OBD C18 column, 19*250mm, 5μm; mobile phase A: water (10mmol / LNH4HCO3), mobile phase B: EtOH; flow rate: 25mL / min; gradient: 40B to 64B in 10min to give N-(3-(2-(difluoromethoxy)-5-((1-(1-methylpiperidin-4-yl)-1H-pyrazol-4-yl)oxy)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (47.2mg, 0.084mmol, 30.6% yield) as a white solid. LC / MS (Method A, ESI): [M+H] + =564.3,R T =1.23min. 1H NMR(400MHz, DMSO-d6)δ9.73(s,1H),9.34(dd,J=7.2,1.6Hz,1H),8.66–8.65(m,2H),8.27(s,1H),7.87(s,1H),7.41–7.40(m,2H), 7.28(dd,J=7.2,4.4Hz,1H),7.17–6.89(m,3H),4.04–3.98(m,1H),3.90(s,3H),2.83–2.80(m,2H),2.18(s,3H),2.03–1.87(m,6H).

[0700] Example 79

[0701]

[0702] N-(3-(2-(difluoromethoxy)-5-(3-(3-hydroxy-1-methylazetidin-3-yl)phenoxy)phenyl)-1-

[0703] Methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0704] Step 1: Synthesis of tert-butyl 3-(3-(4-(difluoromethoxy)-3-(1-methyl-4-(pyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-3-yl)phenoxy)phenyl)-3-hydroxyazetidine-1-carboxylate

[0705]

[0706] N-[3-[5-bromo-2-(difluoromethoxy)phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (Intermediate 3, 462 mg, 1.00 mmol), tert-butyl 3-hydroxy-3-(3-hydroxyphenyl)azetidine-1-carboxylate (549 mg, 2.07 mmol, 2.07 equiv), [PdCl(allyl)]2 (41.1 mg, 0.112 mmol, 0.113 equiv), t-BuBrettPhos (98.1 mg, 0.202 mmol, 0.203 equiv), cesium carbonate (395 mg, 1.21 mmol, 1.22 equiv) and toluene (15 mL) were placed in a 30-mL sealed tube, which was purged and maintained under an inert atmosphere of nitrogen. The resulting solution was stirred in an oil bath at 100° C. for 12 h. The reaction mixture was brought to room temperature and the solid was removed by filtration. The filtrate was concentrated under vacuum. The residue was applied to a silica gel column and eluted with ethyl acetate / petroleum ether (10: 1). This gave 3-(3-(4-(difluoromethoxy)-3-(1-methyl-4-(pyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-3-yl)phenoxy)phenyl)-3-hydroxyazetidine-1-carboxylic acid tert-butyl ester as a yellow-green solid. LC / MS (Method I, ESI): [M+H] + =648.3,R T =1.18min.

[0707] Step 2: Synthesis of N-(3-(2-(difluoromethoxy)-5-(3-(3-hydroxyazetidin-3-yl)phenoxy)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0708]

[0709] 3-[3-[4-(difluoromethoxy)-3-(1-methyl-4-[pyrazolo[1,5-a]pyrimidine-3-amino]-1H-pyrazol-3-yl)phenoxy]phenyl]-3-hydroxyazetidine-1-carboxylic acid tert-butyl ester (1.15 g, 1.78 mmol), dichloromethane (20 mL) and trifluoroacetic acid (4 mL) were placed in a 50 mL round-bottom flask. The resulting solution was stirred at room temperature for 2 hours and concentrated under vacuum. This gave 1.21 g (crude) N-[3-[2-(difluoromethoxy)-5-[3-(3-hydroxyazetidine-3-yl)phenoxy]phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide as a solid which was used without further purification. LC / MS (Method G, ESI): [M+H] + =548.3,R T=0.69min.

[0710] Step 3: Synthesis of N-(3-(2-(difluoromethoxy)-5-(3-(3-hydroxy-1-methylazetidin-3-yl)phenoxy)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0711]

[0712] A solution of N-[3-[2-(difluoromethoxy)-5-[3-(3-hydroxyazetidine-3-yl)phenoxy]phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (1.21 g, 2.21 mmol, 1.00 equiv) in dichloromethane (20 mL), formaldehyde (0.75 mL, 30.2 mmol, 13.7 equiv), NaBH(OAc)3 (937 mg, 4.42 mmol, 2.00 equiv) were placed in a 50 mL round bottom flask. The resulting solution was stirred at room temperature for 18 hours. The resulting mixture was concentrated under vacuum. The residue was applied to a silica gel column and eluted with tetrahydrofuran / MeOH (10 / 1). This gave 0.68 g (55%) of N-(3-(2-(difluoromethoxy)-5-(3-(3-hydroxy-1-methylazetidin-3-yl)phenoxy)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide as a white solid. LC / MS (Method H, ESI): [M+H] + =562.3,R T =0.69min. 1 H NMR (400MHz, CD3OD) δ9.13–9.11(m,1H),8.67–8.65(m,2H),8.25(s,1H),7.44–7.36(m,3H),7.28–7.20 (m,4H),7.02–6.97(m,1H),6.73(t,J=74Hz,1H),3.98(s,3H),3.73(d,J=9.2Hz,2H),3.51–3.50(m,2H).

[0713] The liquid chromatography-mass spectrometry (LCMS) method, retention time and m / z of each compound in Table 1 are shown in Table 2.

[0714] Table 2

[0715]

[0716]

[0717]

[0718]

[0719]

[0720]

[0721] Table 2

[0722] Determination

[0723] Test Agent

[0724] Test agent samples were provided as dimethyl sulfoxide (DMSO) solutions at a concentration of 10 mM and stored in the dark at room temperature prior to use.

[0725] Biochemical analysis of JAK1 and JAK2

[0726] In vitro biochemical assays quantify JAK-catalyzed phosphorylation of synthetic peptides, such as using EZ Reader II microfluidic flow displacement instrument (PerkinElmer; Waltham, MA). The 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 tyrosine residue (Y) that can be phosphorylated by JAK activity. Substrate peptide stocks were prepared in DMSO at 5 mM. Purified recombinant human JAK1 kinase domain protein (residues 854-1154) was expressed in insect cells and purchased from Proteros Biostructures GmbH (Martinsried, Germany). Recombinant human JAK2 kinase domain protein (residues 812-1132) was expressed in insect cells and purified at Genentech (South San Francisco, CA). 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, 4mM dithiothreitol, 1.5μM Y-1B peptide substrate, 25μM adenosine triphosphate (ATP), 1nM total JAK1 or 0.2nM total JAK2, and up to 1000nM of the test compound at a final concentration of 2% (volume ratio [v / v]) DMSO. In each titration experiment, the test compound was tested in duplicate at each of the twelve concentrations. The blank reaction contained ATP, peptide and DMSO, but no JAK or test compound, while the non-inhibition control reaction contained ATP, peptide, JAK and DMSO, but no test compound.

[0727] The peptide plus ATP mixture (24 μL) was added to the test compound in 1 μL DMSO (or DMSO alone). The reaction was started by adding 25 μL of JAK enzyme to the inhibitor / peptide / ATP mixture before the resulting solution was thoroughly mixed. The reaction was incubated at room temperature (22°C-23°C) in a final volume of 50 μL per well in a 384-well plate. After 30 minutes of incubation, the reaction was stopped by adding 25 μL of 150 mM ethylenediaminetetraacetic acid (pH 7.2) to 100 mM HEPES buffer (containing 0.015% Brij35) to each well.

[0728] 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 product molecules from substrate molecules was achieved using downstream and upstream voltages of -500 and -2600 V, respectively, at an operating pressure of -1.3 psi. The 5-FAM group present on the substrate and product peptide was excited at 488 nm, fluorescence was detected at 530 nm, and the peak height was reported.

[0729] Data analysis

[0730] The extent (or percentage) of substrate conversion to product was calculated from the corresponding peak heights in the electropherogram using HTS Well Analyzer software version 5.2 (PerkinElmer) and the following equation (Equation 1):

[0731] % conversion rate = [P ÷ (S + P)] × 100

[0732] 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 non-inhibitory control reaction containing JAK and DMSO vehicle but no test compound was defined as having a fractional activity = 1 (no inhibitor, v i =v o ), while blank wells without JAK were defined as having fractional activity = 0. Fractional activity was plotted against test compound concentration, and the data were fitted to the tight-binding apparent inhibition constant (K) using XLfit software (IDBS; Guildford, United Kingdom). i app) quadratic equation (see Equation 2) (Williams JW, Morrison JF. The kinetics of reversible tight-binding inhibition. Methods Enzymol 1979; 63: 437-67.), which was used to calculate the fractional activity and K i app

[0733] Reaction 2

[0734]

[0735] Where [E] T and[I] T is the total concentration of active enzyme (initial estimates are 0.15 nM for JAK1 and 0.048 nM for JAK2) and inhibitor (variable parameters). Finally, by applying the competitive inhibition relationship (Equation 3), from K i app Calculate K i

[0736] Reaction 3

[0737] K i =K i app / (1+[ATP] / K m app )

[0738] Where [ATP] is the concentration of ATP, = 25 μM, K m app is the apparent ATP Michaelis constant of JAK1, = 32.1 μM, K m app = is the apparent ATP Michaelis constant for JAK2, 11.7 μM. By applying the tight-binding equation 2 to account for any depletion of inhibitor, and the competition-inhibition relationship equation 3, the sensitivity of the assay can be extended to at least the calculated K for JAK1. i is 0.008nM, while that of JAK2 is 0.0015nM.

[0739] Kinase selectivity

[0740] In vitro kinase selectivity of test drugs was evaluated at 1 μM 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 measuring peptide phosphorylation or ADP generation The binding assay monitors the displacement of the probe bound to the ATP site The ATP concentration used in activity assays is usually within the experimentally determined apparent Michaelis constant (K) of each kinase. m app ) value, while the competitive binding tracer concentration used in binding assays is usually within 2-fold of the experimentally determined dissociation constant (K d ) values. Inhibitors were tested in duplicate for each kinase and the average % inhibition values ​​are 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 concentration 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 is catalytically active, the JAK1 inhibitor sensitivity limit derived from the supplier's JAK1 assay is theoretically an IC of 37.5 nM. 50 value (half of the total enzyme concentration). However, JAK1 IC of several inhibitors generated by JAK1 analysis 50 The value is much lower than 37.5 nM, which is consistent with our internal assay results. The active JAK1 enzyme concentration 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 the assay is much higher than the theoretical sensitivity IC of 37.5 nM. 50 Limit value.

[0741] Data analysis

[0742] To fit the data in the concentration-kinase inhibition plot, SelectScreen The service used XLfit software (IDBS), model 205 (sigmoidal concentration-response model), which is a four-parameter logistic fit model as described in Equation 4

[0743] Reaction 4

[0744]

[001] y=A+{(BA)÷[1+(C÷x) D ]}

[0745] where x is the inhibitor concentration, y is the observed inhibition percentage, A is the minimum y-value, B is the maximum y-value, and C is the IC 50= value, D is the (Hill) slope. In some cases, a three-parameter logistic fit was used. For example, if the curve plateau at infinitely low inhibitor concentrations did not fit between -20% and 20% inhibition, the lower plateau was set to 0% inhibition, and if the curve plateau at infinitely low inhibitor concentrations did not fit between 70% and 130% inhibition, the upper plateau was set to 100% inhibition.

[0746] Phosphorylation of STAT JAK1 and JAK2 pathways in TF-1 cell line

[0747] 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. The day before the assay, the cultures were transferred to Opti-MEM TM , 1× NEAA, 1 mM sodium pyruvate, and 0.5% charcoal-treated FBS (starvation medium). Inhibitor stock solutions (5 mM in DMSO) were serially diluted 1:2 in DMSO to generate 10-point concentration titrations (at 500× test concentrations) and then further diluted by 50-fold dilutions in assay medium (RPMI containing 1× NEAA and 1 mM sodium pyruvate) to generate 10× concentration titrations (in 2% DMSO). Cells (300,000 cells / well in 35 μL assay medium) were seeded in 384-well Greiner plates. The diluted inhibitors were added to the cells at 10× concentration (5 μL) and plated in a humidified incubator at 37°C for 30 min. Human recombinant cytokines were then used at their corresponding EC 90The cells were stimulated with 10 μL of ... In the IL-13 assay, P-STAT6 was measured by coating goat anti-rabbit (GAR) plates (Meso Scale Discovery [MSD]; Rockville, MD; catalog number MSD L21RA-1) with rabbit anti-human STAT6 total antibody (Cell Signaling Technologies; catalog number 9362S), and cell lysates were incubated in the coated plates at 4°C overnight, followed by detection using mouse anti-P-STAT6 (Tyr641) Clone 16E12 antibody (MilliporeSigma; Burlington, MA; catalog number 05-590, by MSD using SULFO-labeling). In the EPO assay, P-STAT5 was detected using a phosphorylated-STAT5a,b whole cell lysate kit (MSD; catalog number K150IGD-1). The electrochemiluminescence (ECL) signal of the wells was read on a MESO SECTOR S600 (MSD) plate reader.

[0748] Data analysis

[0749] Determine the control percentage of the ECL value of the test compound wells relative to the mean ECL value of the positive control (cytokine stimulated and DMSO treated cells) by subtracting the mean ECL value of the negative control (cytokine stimulated and 20 μM control inhibitor treated cells) from the ECL value of all wells and determine the IC of the test compound using a four-parameter logistic fit model 50 , as shown in Equation 4.

[0750] P-STAT6 BEAS-2B+IL-13 cell assay

[0751] To investigate the effects of JAK1 inhibitors in cell lines relevant to human asthma cell biology, an IL-13-stimulated STAT6 phosphorylation assay in the human lung bronchial epithelial BEAS-2B cell line was developed.

[0752] 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). Test compound stock solutions (0.5 mM in DMSO) were serially diluted in DMSO at a ratio of 1:2 to generate a 10-point concentration curve (at 500× test concentration), and then further diluted in BEGM by 50-fold dilution steps to generate a 10× concentration curve (in 2% DMSO). Cells were seeded in 200 μL BEGM in 96-well plates at 100,000 cells / well and incubated in a humidified incubator at 37°C for 48 hours. The medium was aspirated from the cells and replaced with 70 μL fresh BEGM. Diluted test compounds (10 μL; or 2% DMSO in assay medium) were added to the cells and incubated in a humidified incubator at 37°C for 1 hour. Then twenty μL of 250ng / mL human recombinant IL-13 (Bio-Techne catalog number 213-ILB) was 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 1mM PMSF was added to the cells. The assay plate was incubated at -80°C for at least 1 hour. P-STAT6 was measured by coating GAR plates (MSD; catalog number MSD L45RA-1) with rabbit anti-human STAT6 total antibody (Cell Signaling Technologies; catalog number 9362S), and cell lysates were cultured overnight at 4°C in the coated plates, followed by standard MSD plate processing, washing and detection procedures, using mouse anti-phosphorylated-STAT6 (Tyr641) Clone 16E12 antibody (Millipore; catalog number 05-590, by MSD using SULFO-labeling) for detection. Plates were read on a MESO SECTOR S600.

[0753] Data analysis

[0754] Data analysis was performed by subtracting the negative control value from all wells and using the positive control value to determine the percentage of control; IC 50Determined by a four-parameter logistic fit model, as shown in Equation 4. P- STAT6BEAS-2B+IL-13 cell assay

[0755] To evaluate the ability of JAK1 inhibitors to retain their ability to inhibit IL-13-stimulated STAT6 phosphorylation after cell washing to remove free unbound inhibitor, an inhibitor washout (WO) assay in the human lung bronchial epithelial BEAS-2B cell line was developed. Retention of inhibitory activity after inhibitor washout is consistent with persistent binding of the inhibitor to the JAK1 protein and / or retention of the inhibitor molecule within the cell after washout.

[0756] As with the standard BEAS-2B cell assay (see above), BEAS-2B cells are grown in bronchial epithelial growth medium (BEGM). Test compound stock solutions (0.5mM in DMSO) are serially diluted in DMSO at a ratio of 1:2 to generate a 10-point concentration curve (at 500× test concentration), and then further diluted in BEGM by a 50-fold dilution step to generate a 10× concentration curve (in 2% DMSO). Cells are seeded 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 is aspirated from the cells and replaced with 70 μL fresh BEGM. Diluted test compounds (10 μL; or 2% DMSO in the assay culture medium) are added to the cells and incubated in a humidified incubator at 37°C for 1 hour. The culture medium was drawn from the cells and replaced with 80 μL of fresh BEGM to wash away the inhibitors in the cells, and then the cell plates were incubated in a 37°C humidified incubator for 10 minutes. The flushing procedure was repeated twice. After the third washing step, the cell plates were returned to a 37°C humidified incubator and incubated for 1 hour. Then twenty μL of 250ng / mL IL-13 were added to the cells and incubated for 15 minutes at 37°C. The culture medium was drawn from the cells, and 60 μL of ice-cold 1× cell lysis buffer (Cell Signaling Technologies; Catalog No. 9803S) containing 1mM PMSF was added to the cells. The assay plate was incubated at -80°C for at least 1 hour. P-STAT6 was measured by coating GAR plates (MSD; catalog number MSD L45RA-1) with rabbit anti-human STAT6 total antibody (Cell Signaling Technologies; catalog number 9362S), incubating cell lysates in coated plates at 4°C overnight, then using standard MSD plate handling, washing and detection procedures, using mouse anti-phosphorylated-STAT6 (Tyr641) Clone 16E12 antibody (Millipore; catalog number 05-590, by MSD using SULFO-label). Plates were read on MESO SECTOR S600.

[0757] Data analysis

[0758] Data analysis was performed by subtracting the negative control value from all wells and using the positive control value to determine the percentage of control; IC 50 Determined by a four-parameter logistic fit model, as shown in Equation 4.

[0759] Cytotoxicity assay

[0760] A549 cells ( CCL-185 TM ), Jurkat clone E6-1 ( TIB-152 TM ) and HEK-293T( CRL-1573 TM ) cells. Cells in exponential growth phase (450 cells in 45 μL of medium) were plated 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 37°C CO2 and humidity controlled incubator overnight. The next day, cells were treated with test agents (10 point titration, highest concentration was 50 μM) diluted in 100% DMSO (0.5% final dimethyl sulfoxide concentration on cells). The cells and compounds were then incubated in a 37°C CO2 and humidity controlled incubator for 72 hours and then incubated by adding Cell viability was measured by adding 50 μL of ELISA (Promega G7572) reagent to all wells. The plates were incubated at room temperature for 20 minutes and then the luminescence of the wells was read on an EnVision plate reader (Perkin Elmer LifeSciences).

[0761] The enzymatic assay data of the compounds in Table 1 are shown in Table 3.

[0762] Table 3

[0763]

[0764]

[0765]

[0766]

[0767]

[0768]

[0769]

[0770]

[0771]

[0772] Table 3

[0773] Animal models

[0774] House dust mite (HDM) model

[0775] Seven to eight week old female C57BL / 6J mice purchased from Jackson West. On the 0th and 14th days, the mice were immunized by intraperitoneal administration of 2mg alum (Thermo Scientific) mixed with house dust mites (HDM, D. Pteronyssinus, purchased from Greer Laboratories, standardized to 0.918 μg DerP1 content per mouse). On the 21st and 24th days, the mice were challenged with HDM in PBS (standardized to 0.918 μg DerP1 content again) and administered by intratracheal inhalation. Before each inhalation of HDM stimulation (and in the subgroups on the 22nd and 23rd days), the animals received the test compound by nasal inhalation only (using a dry powder inhalation device from an electronic medical measurement system (EMMS), including a Wright dust feeder and 4 layers / 24 ports or 2 layers / 12 ports, directional flow, nasal inhalation tower only), which ended 1 hour before the attack. The control group animals only received air nasal inhalation. 24 hours after the final treatment, mice were retro-orbitally bled for plasma PK and then euthanized by CO2 inhalation. After euthanasia, BAL fluid was collected for total (by FACS, using known peaks in reference beads) and differential (by Wright Giemsa-stained cell smears) cell counts. Lungs and spleens were harvested, weighed and frozen for PK. There were 5 or 6 animals per group.

[0776] In addition, to verify the lung dose, PK satellite groups (3 naive animals per group) were administered test compounds by nasal inhalation only for one or four consecutive days. Directly after the final inhalation dose, PK satellite animals were bled retro-orbitally for plasma PK and then euthanized by CO2 inhalation. Lungs and spleens were collected and weighed for PK analysis.

[0777] Rat OVA Model

[0778] Six-week-old male brown Norway rats from Charles River-Kingston. On day 0, rats were immunized by intraperitoneal injection of 150 μg OVA (Sigma) and 40 mg alum (Thermo Scientific) diluted in sterile PBS. 28 days after sensitization, rats were challenged for three consecutive days with 2% OVA in PBS nebulized by a nebulizer for 30 minutes. Before each OVA challenge, animals received JAK1 / JAK2 test compounds by nasal inhalation only (using a dry powder inhalation device from an electronic medical measurement system (EMMS), including a Wright dust feeder and a 4-layer, 24-port, directional flow, nasal inhalation tower only), ending 1 hour before the attack. Control animals received MCT buffer orally, or only nasally inhaled air. After the final treatment for 24 hours, rats were euthanized by inhalation of CO2. Blood was drawn from the abdominal aorta for plasma PK and whole blood FACS analysis. After euthanasia, BAL fluid was collected for total (by FACS, using the peak of known amount in reference beads) and differential (by Wright Giemsa-stained cell smear) cell counts. Lungs were collected, weighed and frozen for PK. Spleen was weighed and cut in half for PK and FACS analysis. Total cell counts and NK cell percentages (CD161a positive) of blood and spleen samples were analyzed by FACS. There were 6 animals in each group, except for the natural control group, which contained 5 animals.

[0779] In addition, to verify the lung dose, the PK satellite group (3 immature animals per group) was administered JAK1 / JAK2 test compound by nasal inhalation only for one or three days. Directly after the final inhalation dose, the PK satellite animals were euthanized by CO2 inhalation. Blood was drawn from the abdominal aorta for plasma PK. Lungs and spleens were collected and weighed for PK analysis.

[0780] The plasma and lung levels of the test compound and their ratios were determined in the following manner. BALB / c mice from Charles River Laboratory were used in the assay. The test compound was prepared separately in 0.2% Tween 80 in saline, and the dosing solution was introduced into the mouse trachea by inhalation. At different time points after administration (usually 0.167, 2, 6, 24 hours), blood samples were taken by cardiac puncture, and complete lungs were removed from mice. The blood samples were centrifuged at about 12,000 rpm (Eppendorf centrifuge, 5804R) for 4 minutes at 4°C to collect plasma. The lungs were filled dry, weighed, and homogenized in sterile water at a dilution of 1:3. The plasma and lung levels of the test compound were determined by LC-MS analysis according to the analytical standards in the standard curve in the test matrix. The lung-plasma ratio is determined as the ratio of lung AUC (unit: μg hr / g) to plasma AUC (unit: μg hr / mL), where AUC is generally defined as the area under the test compound concentration and time curve.

[0781] Pharmacokinetics in mouse plasma and lung

[0782] The pharmacokinetics of the compound were determined in female Balb / c mice after administration of a target dose of 0.3 mg / kg formulated in 0.2% Tween 80 in saline via a single intranasal (IN) instillation of the solution / suspension. Female Balb / c mice, 7-8 weeks old, were purchased from Charles River. Mice were housed under specific pathogen-free conditions prior to use in the study.

[0783] Animals were not fasted before dosing. At 0.083, 2, 7 and 24 hours after dosing, blood samples were collected from 3 animals at each time point under anesthesia (intraperitoneal injection of pentobarbital) and placed into EDTA-coated microcontainers by cardiac puncture. Blood samples were centrifuged (1500g, 10min at 4°C) to separate plasma. Plasma samples were frozen at approximately -80°. After nasal administration, the spleen was removed, weighed and quickly frozen before lung perfusion. After confirmation of death, the lungs of the administered animals were perfused with frozen PBS to remove residual blood from the pulmonary vessels. The lungs were then removed and weighed (all weights were recorded). All tissue samples were frozen by immersion in liquid nitrogen. Tissue samples were stored frozen (approximately -80°C) until analysis.

[0784] Prior to PK analysis, 4 mL of HPLC grade water was added per gram of tissue, and the defrosted tissue samples (spleen and lung) were weighed and homogenized at 4 ° C using Omni-Prep BeadRuptor (Omni Inc., Kennesaw, GA). Plasma and tissue homogenate samples were extracted by protein precipitation, using four volumes of acetonitrile containing tolbutamide (200 ng / mL) or labetalol (100 ng / mL) as internal standards. Samples were mixed and centrifuged at 3200 g and 4 ° C for 30 minutes to remove precipitated proteins, and the supernatant was appropriately diluted (e.g., 1: 1, v / v) with HPLC grade water in a 96-well plate. Representative aliquots of plasma, spleen, and lung samples were analyzed for compound concentrations by LC-MS / MS for matrix matching calibration curves and quality control standards using Waters Xevo TQ-S (Waters, Elstree, UK) in positive ion mode. Standards were prepared by spiking aliquots of compound into control plasma, spleen and lung tissue homogenates and extractions were performed as described for experimental samples. Limits of detection for all matrices ranged from 0.168 mg / mL to 4000 ng / mL.

[0785] When calculating the mean and SD, concentrations below the lower limit of quantification (LLOQ) were considered zero. The mean concentration measured in the samples was used to construct a semi-logarithmic concentration-time curve. Pharmacokinetic (PK) analysis was performed using the non-divisional method in Biobook (E-Workbook IDBS).

[0786] Mouse model of eosinophilic pulmonary inflammation induced by Alternaria alternata

[0787] Airway eosinophilia is a characteristic of human asthma. Alternaria is a fungal aeroallergen that can aggravate 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 has been shown to indirectly activate tissue-resident type 2 innate lymphocytes in the lungs, which respond to (such as IL-2 and IL-7), release JAK-dependent cytokines (such as IL-5 and IL-13), and coordinate eosinophilic inflammation (Bartemes et al. J Immunol. 2012, 188 (3): 1503-13).

[0788] Seven to nine week old male C57 mice from Taconic were used in the study. On the day of the study, the animals were lightly anesthetized with isoflurane and the vehicle or test compound was administered by oropharyngeal aspiration. After dosing, the animals were placed in lateral recumbency and monitored for complete recovery from anesthesia before being returned to their home cages. One hour later, the animals were briefly anesthetized again and challenged with vehicle or Alternaria extract by oropharyngeal aspiration, then monitored for recovery from anesthesia and returned to their home cages. Forty-eight hours after Alternaria administration, bronchoalveolar lavage fluid (BALF) was collected and eosinophils in the BALF were counted using the Advia 120 hematology system (Siemens).

[0789] Compared to vehicle-treated, Alternaria-challenged control animals, at 48 hours, the levels of eosinophils present in the BALF of treated animals were reduced, demonstrating compound activity in the model. Data are expressed as percent inhibition of vehicle-treated, Alternaria-challenged BALF eosinophil responses. To calculate percent inhibition, the number of BALF eosinophils in each case was converted to the percentage of average vehicle-treated, Alternaria-challenged BALF eosinophils and subtracted from 100 percent.

Claims

1. A compound of formula (I): or a pharmaceutically acceptable salt thereof, wherein: Ar is: phenyl; 1,2,3,4-tetrahydroisoquinolyl; pyrazolyl; pyridinyl; or pyridazinyl: R 1 is: hydrogen; C1-C6 alkyl; Each R 2 are independently: C1-C6 alkyl; hydroxy-C1-C6 alkyl; halo-C1-C6 alkyl; C1-C6 alkoxy; C1-C6 alkoxy-C1-C6 alkyl; halo-C1-C6 alkoxy; halo-C1-C6 alkoxy-C1-C6 alkyl; C1-C6 alkyl-SO2-C1-C6 alkyl; hydroxy; cyano; cyano-C1-C6 alkyl; halo; acetyl; -(CHR a ) p -het 2 ;-(CHR a ) q -NR b R c ;-(CHR a ) r -C(O)-NR b R c ;-(CHR a ) s -NR a -(CHR a ) s -C(O)-NR b R c ; or -(CHR a ) t -C 3-6 Cycloalkyl, wherein the cycloalkyl portion may be unsubstituted or substituted by R e Replace once or twice; R 3 , R 4 and R 5 are each independently: hydrogen; Each R a are independently: hydrogen; or C 1-6 alkyl; Each R b are independently: hydrogen; C 1-6 Alkyl; or hydroxy-C1-C6 alkyl; Each R c are independently: hydrogen; C 1-6 Alkyl; hydroxy-C1-C6 alkyl; cyano-C1-C6 alkyl; C1-C6 alkoxy-C1-C6 alkyl; oxetanyl; 2-morpholinoethyl; 1-methyl-azetidin-3-yl; 2-(N,N-dimethylamino)-ethyl; hydroxycyclobutyl; or 3-(N,N-dimethylamino)-pyrrolidin-1-yl; (CHR a ) u -C 3-6 Cycloalkyl, wherein the cycloalkyl portion may be unsubstituted or substituted by R e Replace once or twice; or R b and R c Together with the nitrogen atom to which they are attached they can form het 3 ; Each R e Independently: C 1-6 alkyl; hydroxy; cyano-C1-C6 alkyl; hydroxy-C1-C6 alkyl; morpholinyl; or -(CHR a ) v -NR g R h , where R g and R h are independently hydrogen or C 1-6 alkyl; n is 0 to 2; p is 0 to 2; q is 0 to 2; r is 0 to 2; s is 0 to 2; t is 0 to 2; u is 0 to 2; v is 0 to 2; het 2 are: azetidinyl; pyrrolidinyl; oxetanyl; piperidinyl; morpholinyl; piperazinyl; azepine quinuclidine; or pyrazolyl; each of which may be unsubstituted or replaced by R e replaced once or twice; and het 3 are: azetidinyl; pyrrolidinyl; piperidinyl; morpholinyl; piperazinyl; or Aza each of which may be unsubstituted or replaced by R e Replace once or twice.

2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein Ar is: phenyl; or pyrazolyl.

3. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein Ar is phenyl.

4. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein Ar is pyrazolyl.

5. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R 1 It is methyl.

6. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein n is 0.

7. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein n is 1.

8. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein n is 2.

9. The compound according to claim 1, wherein each R 2 Independently selected from:

10. The compound according to claim 1, wherein each R 2 Independently selected from:

11. The compound according to claim 1, wherein n is 1, and R 2 Selected from: Salt.

12. The compound according to claim 1, wherein the compound is a compound of formula (II): or pharmaceutically acceptable salt.

13. The compound according to claim 1, wherein the compound is a compound of formula (III): or pharmaceutically acceptable salt.

14. A compound selected from: or pharmaceutically acceptable salt.

15. Use of a compound or a pharmaceutically acceptable salt for preparing a medicament for preventing, treating or alleviating the severity of a disease or condition responsive to inhibition of Janus kinase activity in a patient, comprising administering to the patient a therapeutically effective amount of a compound or a pharmaceutically acceptable salt according to any one of claims 1 to 14.

16. The use according to claim 15, wherein the disease or disorder is cancer, stroke, diabetes, cardiovascular disease, cystic fibrosis, viral disease, autoimmune disease, allergic disease, inflammation, nervous system disease, hormone-related disease, organ transplant rejection, immunodeficiency disease, destructive bone disease, proliferative disease, infectious disease, disorder associated with cell death, thrombin-induced platelet aggregation, liver disease or pathological immune disorder involving T cell activation.

17. A pharmaceutical composition comprising a compound according to any one of claims 1 to 14, or a pharmaceutically acceptable salt thereof, wherein the pharmaceutical composition comprises microparticles of the compound suitable for delivery by inhalation.

18. The pharmaceutical composition according to claim 17, wherein the microparticles are prepared by spray drying, freeze drying or micronization.

19. A kit comprising: (a) a first pharmaceutical composition comprising a compound according to any one of claims 1 to 14, or a pharmaceutically acceptable salt thereof; and (b) Instructions for use.

20. The kit of claim 19, further comprising a second pharmaceutical composition comprising an agent for treating an inflammatory disease, or a chemotherapeutic agent.

21. The use according to claim 15, wherein the disease or disorder is an inflammatory disease, wherein the inflammatory disease is asthma.

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