Method for preparing JAK1 inhibitor

Abstract

The present application provides methods for preparing 4-[3-(cyanomethyl)-3-(3',5'-dimethyl-1H,1'H-4,4'-bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide and its phosphate salt, as well as related salt forms and intermediates thereof, which can be used as selective Janus kinase 1 (JAK1) inhibitors.

Description

[0001] Cross - reference to related applications

[0002] This application claims the priority and benefit of U.S. Provisional Patent Application No. 63 / 033,618, filed on June 2, 2020, the entire content of which is incorporated herein by reference. Technical field

[0003] This application provides methods for preparing 4 - [3 - (cyanomethyl)-3-(3',5'-dimethyl - 1H,1'H - 4,4'-bipyrazol - 1 - yl)azetidin - 1 - yl]-2,5 - difluoro - N - [(1S)-2,2,2 - trifluoro - 1 - methylethyl]benzamide and its phosphate, which can be used as a selective (Janus kinase 1) JAK1 inhibitor, as well as related salt forms and intermediates. Background art

[0004] Protein kinases (PKs) regulate a variety of biological processes, including cell growth, survival, differentiation, organ formation, morphogenesis, neovascularization, tissue repair and regeneration, etc. Protein kinases also play a special role in many human diseases, including cancer. Cytokines, low - molecular - weight polypeptides or glycoproteins regulate many pathways involved in the host's inflammatory response to sepsis. Cytokines affect cell differentiation, proliferation and activation, and can regulate pro - inflammatory and anti - inflammatory responses to enable the host to respond appropriately to pathogens. The signal transduction of a variety of cytokines involves the Janus kinase family (JAK) of protein tyrosine kinases and signal transducer and activator of transcription (STAT). There are four known mammalian JAKs: JAK1 (Janus kinase - 1), JAK2, JAK3 (also known as Janus kinase, leukocyte; JAKL; and L - JAK), and TYK2 (tyrosine - protein kinase 2).

[0005] Cytokine - stimulated immune and inflammatory responses contribute to the pathogenesis of diseases: for example, pathologies such as severe combined immunodeficiency (SCID) result from the suppression of the immune system, while overactive or inappropriate immune / inflammatory responses contribute to the pathologies of autoimmune diseases (such as asthma, systemic lupus erythematosus, thyroiditis, myocarditis) and diseases such as scleroderma and osteoarthritis (Ortmann, R.A., T. Cheng et al. (2000) Arthritis Res 2(1):16 - 32).

[0006] JAK expression defects are associated with many disease states. For example, Jak1 - / - mice are growth - retarded at birth, unable to nurse, and die perinatally (Rodig, S.J., M.A. Meraz et al. (1998) Cell 93(3):373 - 83). Due to a lack of definitive erythropoiesis, Jak2 - / - mouse embryos are anemic and die around day 12.5 post - coitum.

[0007] The JAK / STAT pathway, and in particular all four JAKs, are thought to play a role in the pathogenesis of asthma, chronic obstructive pulmonary disease, bronchitis, and other related lower respiratory inflammatory diseases. Multiple cytokines that signal through JAK are associated with inflammatory diseases / conditions of the upper respiratory tract, such as those affecting the nose and sinuses (e.g., rhinitis and sinusitis), whether or not typically allergic. The JAK / STAT pathway is also associated with inflammatory diseases / conditions of the eye and chronic allergic responses.

[0008] Activation of JAK / STAT in cancer may occur through cytokine stimulation (e.g., IL - 6 or GM - CSF) or by reducing endogenous inhibitors of JAK signaling, such as SOCS (suppressor of cytokine signaling) or PIAS (protein inhibitor of activated STAT) (Boudny, V. and Kovarik, J., Neoplasm. 49:349 - 355, 2002). Activation of STAT signaling, as well as other pathways downstream of JAK (e.g., Akt), is associated with poor prognosis in many cancer types (Bowman, T. et al. Oncogene 19:2474 - 2488, 2000). Elevated levels of circulating cytokines that signal through JAK / STAT play a causal role in cachexia and / or chronic fatigue. Thus, JAK inhibition may be beneficial for cancer patients for reasons beyond potential anti - tumor activity.

[0009] The JAK2 tyrosine kinase may be beneficial for patients with myeloproliferative disorders, such as polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis with myeloid metaplasia (MMM) (Levin et al., Cancer Cell, Volume 7, 2005:387 - 397). Inhibition of the JAK2V617F kinase reduces the proliferation of hematopoietic cells, indicating that JAK2 is a potential target for drug inhibition in PV, ET, and MMM patients.

[0010] Inhibiting JAK may benefit patients suffering from skin immune disorders (such as psoriasis) and skin allergies. The maintenance of psoriasis is thought to depend on many inflammatory cytokines plus various chemokines and growth factors (JCI, 113:1664 - 1675), many of which signal through JAK (Adv Pharmacol. 2000; 47:113 - 74).

[0011] Accordingly, there is a continuing need for new or improved agents that inhibit kinases such as JAK to develop new and more effective drugs (such as immunosuppressants for organ transplantation) that aim to enhance or inhibit immune and inflammatory pathways, as well as agents for the prevention and treatment of autoimmune diseases, diseases involving overactive inflammatory responses (such as eczema), allergies, cancers (such as prostate cancer, leukemia, multiple myeloma), and certain immune responses caused by other therapeutic agents (such as rashes or contact dermatitis or diarrhea). JAK inhibitors are currently under development. Although JAK inhibitors and methods for their preparation are in the literature, there is still a need for new methods to prepare these inhibitors that have suitable properties for manufacturing, selling, effective, and high - quality drug products. The present disclosure described herein is directed to this purpose. Summary of the Invention

[0012] The present disclosure provides a method for preparing the selective JAK1 inhibitor 4 - [3 - (cyanomethyl)-3-(3',5'-dimethyl - 1H,1'H - 4,4'-bipyrazol - 1 - yl)azetidin - 1 - yl]-2,5 - difluoro - N - [(1S)-2,2,2 - trifluoro - 1 - methylethyl]benzamide or a salt form thereof, including 4 - [3 - (cyanomethyl)-3-(3',5'-dimethyl - 1H,1'H - 4,4'-bipyrazol - 1 - yl)azetidin - 1 - yl]-2,5 - difluoro - N - [(1S)-2,2,2 - trifluoro - 1 - methylethyl]benzamide phosphate, and related intermediate compounds. Brief Description of the Drawings

[0013] Figure 1 Shows a representative differential scanning calorimetry (DSC) trace of Compound 1 phosphate prepared according to the method described in Example 1.

[0014] Figure 2 Shows a representative thermogravimetric analysis (TGA) trace of Compound 1 phosphate prepared according to the method described in Example 1.

[0015] Figure 3 Shows a representative X - ray powder diffraction (XRPD) trace of Compound 1 phosphate prepared according to the method described in Example 1, overlaid with the XRPD trace of Compound 1 phosphate prepared according to the method described in U.S. Patent No. 9,382,231. Detailed implementation mode

[0016] The present disclosure provides a method for preparing a selective JAK1 inhibitor, 4-[3-(cyanomethyl)-3-(3',5'-dimethyl-1H,1'H-4,4'-bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide (referred to as "Compound 1" hereinafter). The free base of the compound is shown hereinafter.

[0017]

[0018] The present invention also provides a method for preparing a phosphate salt of the free base of Compound 1, 4-[3-(cyanomethyl)-3-(3',5'-dimethyl-1H,1'H-4,4'-bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide phosphate (referred to as "Compound 1 phosphate (Compound1 phosphoric acid salt)", "Compound 1 phosphate (Compound 1phosphate)" or "Compound 1 phosphate (Compound 1phosphate salt)" hereinafter).

[0019]

[0020] Exemplary methods for preparing Compound 1 and its phosphate are disclosed in US2014 / 0343030, which is incorporated herein by reference in its entirety. The methods provided herein for preparing the free base of Compound 1 and its phosphate have several advantages over the methods disclosed in US2014 / 0343030, making the methods provided herein more suitable for scale-up manufacturing processes. For example, the exemplary methods described herein are convergent syntheses that provide high yields, increasing the efficacy of multi-step syntheses compared to the linear syntheses in US2014 / 0343030. On scales ranging from about 670 grams to about 2000 grams, the yields of intermediate products, such as those shown in Scheme 2 (see below), are in the range of about 93% to about 94%. Additionally, on scales ranging from 430 grams to about 5800 grams, the yields of the free base of Compound 1 and its phosphate shown in Scheme 5 (see below) are in the range of about 90% to about 97%. The overall yield of the method provided herein from the preparation of (S)-2,4,5-trifluoro-N-[1,1,1-trifluoropropan-2-yl]benzamide (Compound 1a, Scheme 2, see below) to the free base of Compound 1 is about 68% to about 70% in a five-step synthesis, while the overall yield using the method in US2014 / 0343030 is less than 5% and requires six steps from the preparation of (S)-2,4,5-trifluoro-N-[1,1,1-trifluoropropan-2-yl]benzamide to the free base of Compound 1.

[0021] The methods disclosed herein provide good product purity and high yields on a large scale. For example, in US2014 / 0343030, the Suzuki coupling reaction of 4-{3-(cyanomethyl)-3-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide with 4-bromo-3,5-dimethyl-1H-pyrazole in the presence of a palladium catalyst to produce the free base of Compound 1 resulted in a low yield (less than about 10% yield, Example 7) and required the removal of palladium contaminants from the product. In the exemplary methods provided herein, the Suzuki coupling step involving the palladium catalyst is carried out in a separate parallel synthesis to produce the bipyrazole compound (Compound 2x, Scheme 1, see below), which is then coupled with (S)-4-(3-(cyanomethylene)azetidin-1-yl)-2,5-difluoro-N-(1,1,1-trifluoropropan-2-yl)benzamide to produce the free base of Compound 1 (Scheme 5, see below). Compound 2x can be easily purified to a highly crystalline HCl salt. The crystallization process makes Compound 2x easier to purify than the complex poly-nitrogen-containing free base of Compound 1 to remove palladium impurities. This represents an advantage over existing methods that require low-yield column chromatography separations. Additionally, placing the palladium coupling step earlier in the synthesis process increases the overall yield.

[0022] In addition, the use of a bipyrazole compound (Compound 2x) in the Michael addition reaction with Compound 1x unexpectedly results in a high degree of regioselectivity. In some embodiments, the regioselectivity is about 20:1 or greater, favoring the desired regioisomer, the free base of Compound 1, rather than the undesired regioisomer (Compound R shown below). Based on electronic effects, the Compound R regioisomer is the expected product because the two electron-donating methyl groups make the 1H-NH group of Compound 2x more nucleophilic than the 1'H-NH group. Without being bound by a particular theory, it is believed that steric hindrance at the 1H-NH group results in the unexpectedly high regioselectivity.

[0023]

[0024] In some embodiments, the present disclosure relates to a method for preparing

[0025]

[0026] or a salt thereof, the method comprising reacting

[0027]

[0028] with

[0029]

[0030] to form the free base of Compound 1 or a salt thereof.

[0031] In some embodiments, the reaction of Compound 1x with Compound 2x is carried out in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and an organic solvent component. In some embodiments, the organic solvent component comprises dimethylformamide (DMF).

[0032] In some embodiments, the reaction of Compound 1x with Compound 2x is carried out at a temperature of about 40 °C to about 70 °C, about 45 °C to about 65 °C, or about 50 °C to about 60 °C. In some embodiments, the temperature is about 50 °C to about 60 °C. For example, the temperature is about 60 °C.

[0033] In some embodiments, the method for preparing the free base of Compound 1 further comprises work-up after the reaction is complete. For example, the work-up may include adding water to the reaction mixture and collecting the solid of the free base of Compound 1 by filtration, which may be washed with water.

[0034] In some embodiments, the present disclosure provides a method for preparing Compound 1 phosphate, the method comprising reacting the free base of Compound 1 prepared by the methods described herein with phosphoric acid. In some embodiments, the salt of Compound 1 is the phosphate of Compound 1, which is prepared by a method comprising reacting the free base of Compound 1 with phosphoric acid.

[0035] In some embodiments, the reaction of the free base of Compound 1 with phosphoric acid is carried out in the presence of a solvent component. In some embodiments, the solvent component comprises methanol, isopropanol, or a mixture thereof.

[0036] In some embodiments, the reaction of the free base of Compound 1 with phosphoric acid is carried out at a temperature of about 40 °C to about 70 °C or about 45 °C to about 55 °C. For example, the temperature is about 50 °C.

[0037] In some embodiments, the phosphoric acid is an aqueous solution of about 85 wt% phosphoric acid. In some embodiments, the reaction of the free base of Compound 1 with phosphoric acid further comprises adding a second solvent component to the reaction mixture. For example, the second solvent component comprises n-heptane.

[0038] The present disclosure also provides a method for preparing an intermediate compound, such as

[0039]

[0040] In some embodiments, the present disclosure provides a method for preparing Compound 1x, the method comprising:

[0041] 1a) reacting with in the presence of a base to form (Compound 1a);

[0042] 2a) reacting Compound 1a with in the presence of DBU to form (Compound 1b);

[0043] 3a) reacting Compound 1b with iodobenzene diacetate and TEMPO to form (Compound 1c); and

[0044] 4a) reacting Compound 1c with diethyl cyanomethylphosphonate in the presence of a base to form Compound 1x.

[0045] In Operation 1a, (2S)-1,1,1-trifluoropropan-2-amine can react with 2,4,5-trifluorobenzoyl chloride in the presence of a base to form Compound 1a. In some embodiments, the base is N,N-diisopropylethylamine or an aqueous sodium hydroxide solution. In some embodiments, the base is an aqueous sodium hydroxide solution. In some embodiments, the reaction is carried out in the presence of an organic solvent component (such as toluene). In some embodiments, the reaction is carried out at a temperature of from 0 °C to about 10 °C or from about 0 °C to about 5 °C. In some embodiments, a salt of (2S)-1,1,1-trifluoropropan-2-amine (such as the HCl salt) is converted to its free base prior to reaction with For example, in some embodiments, the (2S)-1,1,1-trifluoropropan-2-amine salt (such as the HCl salt) is converted in situ to its free base. In some embodiments, Operation 1a further includes work-up after the reaction is considered complete (such as according to HPLC) to obtain Compound 1a. For example, the work-up can include separating the phases of the reaction mixture and washing the organic phase with, for example, 0.5 M aqueous sodium hydroxide solution. In some embodiments, the solid of Compound 1a can be slurried in n-heptane at about 50 °C for about 1 hour. The solid can be collected by filtration and washed with n-heptane.

[0046] In Operation 2a, Compound 1a can react with azetidin-3-ol hydrochloride in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) to form Compound 1b. In some embodiments, the reaction is carried out in an organic solvent component including, for example, acetonitrile. In some embodiments, DBU can be added portionwise to the reaction mixture of Compound 1a and azetidin-3-ol hydrochloride. In some embodiments, the reaction is carried out at a temperature of from about 50 °C to about 75 °C or from about 55 °C to about 70 °C. For example, the temperature is from about 58 °C to about 68 °C. In some embodiments, Operation 2a further includes work-up after the reaction is considered complete (such as according to HPLC) to obtain Compound 1b. The work-up can include adding 1.0 M aqueous hydrochloric acid solution to the mixture of Compound 1a, azetidin-3-ol hydrochloride, and DBU, stirring the mixture with the hydrochloric acid solution at ambient temperature, adding water to the stirred mixture, and stirring the mixture to which water has been added. The work-up can further include separating the solid of Compound 1b and rinsing the solid with water.

[0047] In operation 3a, compound 1b can react with iodobenzene diacetate and 2,2,6,6-tetramethyl-1-piperidinyloxy radical (TEMPO) to form compound 1c. In some embodiments, the reaction is carried out in an organic solvent component including, for example, dichloromethane. In some embodiments, the reaction is carried out at a temperature of about 0 °C to about 20 °C or about 5 °C to about 15 °C. For example, the temperature is about 10 °C to about 12 °C. In some embodiments, operation 3a further includes work-up after the reaction is considered complete (e.g., according to HPLC) to obtain compound 1c. The work-up may include quenching the reactants with an aqueous solution of sodium thiosulfate and potassium phosphate. The two phases can be separated and the organic phase can be washed with water. The organic solution can be concentrated under reduced pressure to obtain compound 1c as a solid. The solid of compound 1c can be re-slurried in n-heptane at room temperature for about 30 minutes and washed with n-heptane.

[0048] In operation 4a, compound 1c can react with diethyl cyanomethylphosphonate in the presence of a base to form compound 1x. The base includes, for example, potassium tert-butoxide. In some embodiments, the reaction is carried out in the presence of an organic solvent component including, for example, THF, ethanol, or a mixture thereof. In some embodiments, diethyl cyanomethylphosphonate can be added to a solution of 1.0 M potassium tert-butoxide in THF at about 5 °C to about 25 °C. In some embodiments, the solution of potassium tert-butoxide in THF is about 0.95 molar equivalents relative to compound 1c. In some embodiments, the solution of potassium tert-butoxide in THF is less than about 0.95 molar equivalents relative to compound 1c (e.g., about 0.94, about 0.93, about 0.92, about 0.91, or about 0.90). In some embodiments, compound 1c can be dissolved in a mixture of organic solvent components (e.g., ethanol and tetrahydrofuran). In some embodiments, a mixture of diethyl cyanomethylphosphonate and 1.0 M potassium tert-butoxide can be added to a mixture containing compound 1c. In some embodiments, operation 4a further includes work-up after the reaction is considered complete (e.g., according to HPLC) to obtain compound 1x. The work-up may include adding water to the reaction mixture. The solid can be collected by filtration and washed with water and n-heptane. In some embodiments, the solid can be further re-slurried in methyl tert-butyl ether, collected by filtration, and washed with MTBE.

[0049] In some embodiments, the method for preparing Compound 1 free base or its salt further comprises preparing Compound 1x, wherein Compound 1x can be prepared by a method comprising reacting Compound 1c with diethyl cyanomethylphosphonate in the presence of a base. In some embodiments, the method further comprises preparing Compound 1c, wherein Compound 1c can be prepared by a method comprising reacting Compound 1b with iodobenzene diacetate and TEMPO. In some embodiments, the method further comprises preparing Compound 1b, wherein Compound 1b can be prepared by a method comprising reacting Compound 1a with azetidin-3-ol hydrochloride in the presence of DBU. In some embodiments, the method further comprises preparing Compound 1a, wherein Compound 1a can be prepared by a method comprising reacting (2S)-1,1,1-trifluoropropan-2-amine with 2,4,5-trifluorobenzoyl chloride in the presence of a base.

[0050] In some embodiments, the present disclosure provides a method for preparing Compound 2x, the method comprising:

[0051] 1b) reacting (Compound 2a) with to form (Compound 2b);

[0052] 2b) reacting Compound 2b with hydrochloric acid to form

[0053] (Compound 2x HCl); and

[0054] 3b) reacting Compound 2x HCl with a base to form Compound 2x.

[0055] In operation 1b, compound 2a can react with 4-bromo-3,5-dimethylpyrazole to form compound 2b. In some embodiments, the reaction is carried out in the presence of K2HPO4, a solvent component, and a palladium complex. For example, the solvent component includes 1-propanol, water, or a mixture thereof. In some embodiments, the palladium complex is [1,1′-bis(di-tert-butylphosphino)ferrocene]dichloropalladium(II) (Pd-118). In some embodiments, the reaction is carried out at a temperature of about 80 °C to about 100 °C or about 90 °C to about 100 °C. For example, the temperature is about 90 °C. In some embodiments, operation 1b further includes work-up to obtain compound 2a. The work-up may include cooling the reaction mixture to about 17 °C and separating the phases. The organic phase can be mixed with activated carbon, heated to about 70 °C, stirred for about 4 hours, and cooled to about 21 °C. The mixture containing compound 2a can be filtered through diatomaceous earth. In some embodiments, operation 1b further includes mixing the crude compound 2a with ethyl acetate and an aqueous NaHSO3 solution, where the resulting mixture is heated to about 65 °C to about 70 °C and maintained for about 2.5 hours. The phases can be separated and the organic phase can be mixed with an aqueous NaHSO3 solution, where the resulting mixture is heated to about 65 °C to about 70 °C and maintained for about 3.5 hours. The phases can be separated and the phase containing compound 2a can be purified by column chromatography using ethyl acetate as the eluent. In some embodiments, the purified compound 2a is further mixed with dichloromethane and Si-thiol, where the resulting mixture is filtered.

[0056] In operation 2b, compound 2b can react with hydrochloric acid to form compound 2x HCl. In some embodiments, the reaction is carried out in the presence of an organic solvent component. For example, the organic solvent component contains 2-propanol. In some embodiments, the reaction of compound 2b with hydrochloric acid is carried out at a temperature of about 50 °C to about 75 °C or about 55 °C to about 70 °C. For example, the temperature is about 60 °C to about 65 °C. In some embodiments, operation 2b further includes work-up to obtain compound 2b after the reaction is considered complete (e.g., according to HPLC). For example, the reaction mixture is cooled to room temperature and stirred for about 1 hour. The solid of compound 2b can be collected by filtration and washed with 2-propanol.

[0057] In operation 3b, compound 2x HCl can react with a base to form compound 2x. The present disclosure also relates to a method for preparing compound 2x, the method comprising reacting compound 2x HCl with a base. Exemplary bases include KOH, LiOH, K2CO3, Na2CO3, and other bases that can neutralize compound 2x HCl to its free base. In some embodiments, the base is NaOH. In some embodiments, the reaction of compound 2x HCl with the base is carried out at a temperature of about 10 °C to about 20 °C or about 15 °C to about 20 °C. For example, the temperature is about 15 °C to about 18 °C. In some embodiments, operation 3b further comprises post-treatment after the reaction is completed to obtain compound 2x. For example, the solid of compound 2x can be collected by filtration and washed with water and n-heptane.

[0058] In some embodiments, the method for preparing the free base of compound 1 or its salt further comprises preparing compound 2x, wherein compound 2x can be prepared by a method comprising reacting compound 2x HCl with a base. In some embodiments, the method further comprises preparing compound 2x HCl, wherein compound 2x HCl is prepared by a method comprising reacting compound 2b with hydrochloric acid. In some embodiments, the method further comprises preparing compound 2b, wherein compound 2b is prepared by a method comprising reacting compound 2a with 4-bromo-3,5-dimethylpyrazole.

[0059] In some embodiments, the present application further provides a method for preparing a compound of formula A:

[0060]

[0061] In some embodiments, the method for preparing a compound of formula A comprises reacting 3,5-dimethyl-1H,1'H-4,4'-bipyrazole with a compound of formula B:

[0062]

[0063] wherein Pg 1 is an amine protecting group. In some embodiments, Pg 1 is tert-butoxycarbonyl.

[0064] In some embodiments, the reaction of 3,5-dimethyl-1H,1'H-4,4'-bipyrazole with the compound of formula B is carried out in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0065] In some embodiments, less than 1 equivalent of 1,8-diazabicyclo[5.4.0]undec-7-ene is used based on 1 equivalent of the compound of formula B.

[0066] In some embodiments, about 0.2 to about 0.3 equivalents of 1,8-diazabicyclo[5.4.0]undec-7-ene are used based on 1 equivalent of the compound of formula B.

[0067] In some embodiments, greater than about 1 equivalent of 3,5-dimethyl-1H,1'H-4,4'-bipyrazole is used based on 1 equivalent of the compound of formula B.

[0068] In some embodiments, about 1.0 to about 2.0 equivalents of 3,5-dimethyl-1H,1'H-4,4'-bipyrazole are used based on 1 equivalent of the compound of formula B.

[0069] In some embodiments, about 1.0 to about 1.1 equivalents of 3,5-dimethyl-1H,1'H-4,4'-bipyrazole are used based on 1 equivalent of the compound of formula B.

[0070] In some embodiments, about 1.0 to about 1.1 equivalents of 3,5-dimethyl-1H,1'H-4,4'-bipyrazole are used based on 1 equivalent of the compound of formula B.

[0071] In some embodiments, the reaction of 3,5-dimethyl-1H,1'H-4,4'-bipyrazole with the compound of formula B is carried out at about room temperature.

[0072] In some embodiments, the reaction of 3,5-dimethyl-1H,1'H-4,4'-bipyrazole with the compound of formula B is carried out in the presence of a solvent component. In some embodiments, the solvent component comprises dimethyl sulfoxide. In some embodiments, the solvent component comprises dimethyl sulfoxide and dichloromethane.

[0073] In some embodiments, the methods provided herein further comprise deprotecting the compound of formula A to form the compound of formula C:

[0074]

[0075] or a salt thereof.

[0076] In some embodiments, deprotection of the compound of formula A comprises reacting the compound of formula A in the presence of a strong acid (such as hydrochloric acid).

[0077] In some embodiments, deprotection of the compound of formula A comprises reacting the compound of formula A in the presence of a trialkylsilyl halide.

[0078] In some embodiments, the trialkylsilyl halide is trimethylsilyl iodide.

[0079] In some embodiments, the deprotection of the compound of formula A is carried out in the presence of a solvent component. In some embodiments, the solvent component comprises dichloromethane. In some embodiments, the solvent component comprises dichloromethane and methanol.

[0080] In some embodiments, the deprotection of the compound of formula A is carried out at about room temperature.

[0081] In some embodiments, the methods provided herein further comprise reacting a compound of formula C or a salt thereof with a base to form the free base form of the compound of formula C.

[0082] In some embodiments, the methods provided herein further comprise reacting a compound of formula C or a salt thereof with an amine base to form the free base form of the compound of formula C.

[0083] In some embodiments, the base is tri(C 1-6 alkyl)amine.

[0084] In some embodiments, the base is triethylamine.

[0085] In some embodiments, the reaction of the compound of formula C or a salt thereof with an amine base is carried out in the presence of a solvent component. In some embodiments, the solvent component comprises dichloromethane.

[0086] In some embodiments, the methods provided herein further comprise reacting the free base form of the compound of formula C with compound 1a:

[0087]

[0088] To form compound 1:

[0089]

[0090] Or a salt thereof.

[0091] In some embodiments, the free base form of the compound of formula C reacts with compound 1a in the presence of a base and an alkali metal halide to form compound 1:

[0092]

[0093] Or a salt thereof.

[0094] In some embodiments, the base is a bicarbonate base.

[0095] In some embodiments, the base is sodium bicarbonate.

[0096] In some embodiments, the alkali metal halide is lithium chloride.

[0097] In some embodiments, the reaction of the free base form of the Compound C with Compound 1a is carried out at a temperature of about 80 °C to about 90 °C.

[0098] In some embodiments, the reaction of the free base form of the Compound C with Compound 1a is carried out in the presence of a solvent component. In some embodiments, the solvent component comprises dimethyl sulfoxide. In some embodiments, the solvent component comprises dimethyl sulfoxide and isopropyl acetate.

[0099] In some embodiments, the methods provided herein further comprise reacting Compound 1 with a strong acid to form the salt form of Compound 1.

[0100] In some embodiments, the methods provided herein further comprise reacting Compound 1 with hydrochloric acid to form Compound 1 hydrochloride:

[0101]

[0102] In some embodiments, greater than 1 equivalent of hydrochloric acid is used based on 1 equivalent of Compound 1.

[0103] In some embodiments, the reaction of Compound 1 with hydrochloric acid is carried out at about room temperature.

[0104] In some embodiments, the hydrochloric acid is a hydrochloric acid alcohol solution.

[0105] In some embodiments, the hydrochloric acid is an isopropyl alcohol solution of hydrochloric acid.

[0106] In some embodiments, the methods provided herein further comprise reacting Compound 1 hydrochloride with a base to form the free base form of Compound 1:

[0107]

[0108] In some embodiments, the methods provided herein further comprise reacting Compound 1 hydrochloride with a bicarbonate base to form the free base form of Compound 1:

[0109]

[0110] In some embodiments, the base is potassium bicarbonate.

[0111] In some embodiments, the potassium bicarbonate is an aqueous solution of potassium bicarbonate.

[0112] In some embodiments, the methods provided herein further comprise reacting the free base form of Compound 1 with phosphoric acid to form Compound 1 phosphate:

[0113]

[0114] The method according to embodiment 69, wherein the reaction of the free base form of compound 1 with phosphoric acid is carried out at about room temperature.

[0115] In some embodiments, the reaction of the free base form of compound 1 with phosphoric acid is carried out in the presence of a solvent component. In some embodiments, the solvent component comprises water. In some embodiments, the solvent component comprises water and isopropanol.

[0116] In some embodiments, the method provided herein further comprises separating compound 1 phosphate.

[0117] In some embodiments, compound 1 phosphate is separated by recrystallization.

[0118] In some embodiments, compound 1 phosphate is separated by recrystallization from a solvent component comprising methanol.

[0119] In some embodiments, compound 1 phosphate is separated by recrystallization from a solvent component comprising isopropanol.

[0120] In some embodiments, compound 1 phosphate is separated by recrystallization from a solvent component comprising methylcyclohexane.

[0121] In some embodiments, compound 1 phosphate is separated by recrystallization from a solvent component comprising one or more of methanol, isopropanol, and methylcyclohexane.

[0122] In some embodiments, compound 1 phosphate is separated by recrystallization from a solvent component comprising methanol, isopropanol, and methylcyclohexane.

[0123] In some embodiments, compound 1 phosphate is separated by recrystallization from a solvent component comprising methanol, isopropanol, and methylcyclohexane; and then recrystallized from a solvent component comprising methanol and isopropanol.

[0124] In some embodiments, the present application also provides a method for preparing compound 1 phosphate:

[0125]

[0126] The method comprises:

[0127] Reacting 3,5-dimethyl-1H,1'H-4,4'-bipyrazole with tert-butyl 3-(cyanomethylene)azetidine-1-carboxylate in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene to form a compound of formula A-1:

[0128]

[0129] Deprotecting the compound of formula A-1 to form a compound of formula C-1:

[0130]

[0131] or a salt thereof;

[0132] React the compound of formula C-1 with triethylamine to form the free base form of the compound of formula C-1;

[0133] React the free base form of the compound of formula C-1 with compound 1a:

[0134]

[0135] React in the presence of sodium bicarbonate and lithium chloride to form compound 1:

[0136]

[0137] React compound 1 with hydrochloric acid to form compound 1 hydrochloride:

[0138]

[0139] React compound 1 hydrochloride with potassium bicarbonate to form the free base form of compound 1; and

[0140] React the free base form of compound 1 with phosphoric acid to form the phosphate of compound 1.

[0141] In some embodiments, the present disclosure provides a compound that is 3,5-dimethyl-1H,1'H-[4,4']bipyrazolyl (compound 2x), 3,5-dimethyl-1H,1'H-4,4'-bipyrazole hydrochloride (compound 2x HCl), 1-(1-ethoxyethyl)-3',5'-dimethyl-1H,1'H-4,4'-bipyrazole (compound 2b), or 1-(1-ethoxyethyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl)-1H-pyrazole (compound 2a), or a salt of any of the foregoing.

[0142] In some embodiments, the present disclosure provides a compound that is 3,5-dimethyl-1H,1'H-[4,4']bipyrazolyl (compound 2x) or a salt thereof.

[0143] In some embodiments, the present disclosure provides a compound that is (S)-4-(3-(cyanomethylene)azetidin-1-yl)-2,5-difluoro-N-(1,1,1-trifluoropropan-2-yl)benzamide (Compound 1x), (S)-2,5-difluoro-4-(3-oxoazetidin-1-yl)-N-(1,1,1-trifluoropropan-2-yl)benzamide (Compound 1c), (S)-2,5-difluoro-4-(3-hydroxyazetidin-1-yl)-N-(1,1,1-trifluoropropan-2-yl)benzamide (Compound 1b), or (S)-2,4,5-trifluoro-N-(1,1,1-trifluoropropan-2-yl)benzamide (Compound 1a), or a salt of any of the foregoing.

[0144] As used herein, the term "about" means plus or minus 10% of a value.

[0145] As used herein, the term "reaction" is used as known in the art and generally refers to chemical reagents coming together in a manner that allows them to interact at the molecular level to effect a chemical or physical transformation. In some embodiments, a reaction involves at least two reagents. In some embodiments, the reaction steps or operations of a synthetic method may involve one or more substances in addition to the reagents, such as a solvent and / or a catalyst. The reaction steps or operations of the methods described herein may be carried out at a time and under conditions suitable for preparing the identified product. The terms "combine" and "mix" with respect to the reagents of a chemical reaction may be used interchangeably herein with the term "react". The term "couple" may also be considered interchangeable with "react", but may be used in connection with a reaction step or operation involving joining two organic moieties.

[0146] The methods described herein may be monitored according to any suitable method known in the art. For example, product formation may be monitored spectroscopically, such as by nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C), infrared spectroscopy, spectrophotometry (e.g., UV-visible), mass spectrometry; or by chromatography, such as high performance liquid chromatography (HPLC) or thin layer chromatography (TLC). Compounds obtained by a reaction may be purified by any suitable method known in the art. For example, chromatography (medium pressure) on a suitable adsorbent (such as silica gel, alumina, etc.), HPLC, or preparative thin layer chromatography; distillation; sublimation, wet milling, or recrystallization. Generally, the purity of a compound is determined by physical methods, such as measuring the melting point (in the case of a solid), obtaining an NMR spectrum, or performing an HPLC separation. A compound may be said to be purified if the melting point decreases, if unwanted signals in the NMR spectrum decrease, or if extraneous peaks in the HPLC trace are removed. In some embodiments, the compound is substantially purified.

[0147] The preparation of the compounds may involve the protection and deprotection of various chemical groups. The need for protection and deprotection and the selection of appropriate protecting groups can be readily determined by those skilled in the art. The chemical properties of protecting groups can be found, for example, in Wuts and Greene, Greene’s Protective Groups in Organic Synthesis, 4th Edition, John Wiley & Sons: New York, 2006, which is incorporated herein by reference in its entirety.

[0148] The reactions of the methods described herein can be carried out at a suitable temperature readily determined by those skilled in the art. The reaction temperature will depend on, for example, the melting and boiling points of the reagents and solvents (if any); the thermodynamics of the reaction (e.g., a highly exothermic reaction may need to be carried out at a reduced temperature); and the reaction kinetics (e.g., a high activation energy barrier may require an elevated temperature). “High temperature” refers to a temperature above room temperature (about 22 °C).

[0149] The reactions or methods described herein can be carried out in a suitable solvent, which can be readily selected by those skilled in the art of organic synthesis. A suitable solvent may not substantially react with the starting materials (reactants), intermediates, or products at the temperature at which the reaction proceeds (i.e., a temperature within the range from the freezing temperature to the boiling temperature of the solvent). A given reaction can be carried out in one solvent or a mixture of more than one solvent. Depending on the reaction step or operation, a solvent suitable for the particular reaction step or operation can be selected. Suitable solvents include water, alkanes (e.g., pentane, hexane, heptane, cyclohexane, etc., or mixtures thereof), aromatic solvents (e.g., benzene, toluene, xylene, etc.), alcohols (e.g., methanol, ethanol, isopropanol, etc.), ethers (e.g., dialkyl ethers, methyl tert-butyl ether (MTBE), tetrahydrofuran (THF), dioxane, etc.), esters (e.g., ethyl acetate, butyl acetate, etc.), halogenated hydrocarbon solvents (e.g., dichloromethane (DCM), chloroform, dichloroethane, tetrachloroethane), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetone, acetonitrile (ACN), hexamethylphosphoramide (HMPA), and N-methylpyrrolidone (NMP). Such solvents can be used in their wet or anhydrous forms.

[0150] The resolution of a racemic mixture of a compound can be carried out by any of a variety of methods known in the art. For example, the resolution of a racemic mixture can be carried out by elution on a column packed with an optically active resolving agent (e.g., dinitrobenzoyl phenylglycine). The appropriate elution solvent composition can be determined by those skilled in the art.

[0151] Method

[0152] The compounds provided herein (e.g., Compound 1 free base and Compound 1 phosphate) are JAK inhibitors, and more particularly, selective JAK1 inhibitors. A JAK1 selective inhibitor is a compound that preferentially inhibits JAK1 activity compared to other Janus kinases. For example, the compounds provided herein preferentially inhibit JAK1 relative to one or more of JAK2, JAK3, and TYK2. In some embodiments, the compound preferentially inhibits JAK1 relative to JAK2 (e.g., having a JAK2 / JAK1 IC 50 ratio). In some embodiments, the compound is about 10-fold more selective for JAK1 than for JAK2. In some embodiments, as calculated by the IC 50 measured at 1 mM ATP, the compound is about 3-fold, about 5-fold, about 10-fold, about 15-fold, or about 20-fold more selective for JAK1 than for JAK2 (e.g., see Example A).

[0153] JAK1 plays a central role in many cytokine and growth factor signaling pathways, and dysregulation of these signaling pathways can lead to or contribute to disease states. For example, elevated IL-6 levels are seen in rheumatoid arthritis, where it has been shown to have a deleterious effect (Fonesca, J.E. et al., Autoimmunity Reviews, 8:538-42, 2009). Since IL-6 signals at least in part through JAK1, inhibition of JAK1, either directly or indirectly, to antagonize IL-6 is expected to provide clinical benefit (Guschin, D., N. et al. Embo J 14:1421, 1995; Smolen, J.S. et al. Lancet 371:987, 2008). In addition, in some cancers, JAK1 mutations lead to constitutive and unwanted tumor cell growth and survival (Mullighan CG, Proc Natl Acad Sci U S A. 106:9414-8, 2009; Flex E. et al. J ExpMed. 205:751-8, 2008). In other autoimmune diseases and cancers, elevated systemic levels of inflammatory cytokines that activate JAK1 may also contribute to the disease and / or associated symptoms. Thus, patients suffering from such diseases may benefit from JAK1 inhibition. Selective inhibitors of JAK1 can be effective while avoiding the unnecessary and potentially adverse effects of inhibiting other JAK kinases.

[0154] Relative to other JAK kinases, selective inhibitors of JAK1 may have multiple therapeutic advantages over less selective inhibitors. With respect to selectivity for JAK2, many important cytokines and growth factors signal through JAK2, including, for example, erythropoietin (Epo) and thrombopoietin (Tpo) (Parganas E et al. Cell. 93:385-95, 1998). Epo is a key growth factor for erythropoiesis; thus, lack of Epo-dependent signaling results in decreased red blood cell numbers and anemia (Kaushansky K, NEJM 354:2034-45, 2006). Tpo is another example of a JAK2-dependent growth factor and plays a central role in controlling the proliferation and maturation of megakaryocytes (cells that produce platelets) (Kaushansky K, NEJ M 354:2034-45, 2006). Thus, reduced Tpo signaling reduces megakaryocyte numbers (megakaryocytopenia) and decreases circulating platelet counts (thrombocytopenia). This can lead to unwanted and / or uncontrolled bleeding. Reducing inhibition of other JAKs (such as JAK3 and Tyk2) may also be desirable because humans lacking functional forms of these kinases have been shown to suffer from a variety of diseases, such as severe combined immunodeficiency or hyperimmunoglobulin E syndrome (Minegishi, Y et al. Immunity 25:745-55, 2006; Macchi P et al. Nature. 377:65-8, 1995). Thus, JAK1 inhibitors with reduced affinity for other JAKs will have significant advantages over less selective inhibitors in reducing side effects involving immunosuppression, anemia, and thrombocytopenia.

[0155] Another aspect of the present disclosure relates to a method of treating an individual (e.g., a patient) having a JAK-related disease or disorder by administering to the individual in need of such treatment a therapeutically effective amount or dose of a compound or a pharmaceutical composition of the present disclosure. JAK-related diseases can include any disease, disorder, or condition that is directly or indirectly related to the expression or activity of JAK, including overexpression and / or abnormal activity levels. JAK-related diseases can also include any disease, disorder, or condition that can be prevented, ameliorated, or cured by modulating JAK activity.

[0156] Examples of JAK-related diseases include diseases involving the immune system, including, for example, organ transplant rejection (e.g., allograft rejection and graft-versus-host disease).

[0157] Other examples of JAK-related diseases include autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, juvenile arthritis, psoriatic arthritis, type I diabetes, lupus, psoriasis, inflammatory bowel disease, ulcerative colitis, Crohn’s disease, myasthenia gravis, immunoglobulin nephropathy, myocarditis, autoimmune thyroid disorders, chronic obstructive pulmonary disease (COPD), and the like. In some embodiments, the autoimmune disease is an autoimmune bullous skin disease such as pemphigus vulgaris (PV) or bullous pemphigoid (BP).

[0158] Other examples of JAK-related diseases include allergic conditions such as asthma, food allergy, eczematous dermatitis, contact dermatitis, atopic dermatitis (atopic eczema), and rhinitis. Other examples of JAK-related diseases include viral diseases such as Epstein Barr Virus (EBV), hepatitis B, hepatitis C, HIV, HTLV 1, varicella-zoster virus (VZV), and human papillomavirus (HPV).

[0159] Other examples of JAK-related diseases include diseases related to cartilage renewal such as gouty arthritis, suppurative or infectious arthritis, reactive arthritis, reflex sympathetic dystrophy, gouty dystrophy, Tietze syndrome, costochondropathy, endemic osteoarthritis, Mseleni disease, Handigodu disease, degeneration due to fibromyalgia, systemic lupus erythematosus, scleroderma, or ankylosing spondylitis.

[0160] Other examples of JAK-related diseases include congenital cartilage deformities including hereditary chondrolysis, chondrodysplasia, and pseudoachondroplasia (such as microtia, anotia, and metaphyseal chondrodysplasia).

[0161] Other examples of JAK-related diseases or conditions include skin disorders such as psoriasis (such as psoriasis vulgaris), atopic dermatitis, rash, skin irritation, skin sensitization (such as contact dermatitis or allergic contact dermatitis). For example, certain substances (including some drugs) can cause skin sensitization when applied topically. In some embodiments, co-administering or sequentially administering at least one JAK inhibitor of the present disclosure with an agent that causes unwanted sensitization can help treat such unwanted sensitization or dermatitis. In some embodiments, the skin disorder is treated by topically administering at least one JAK inhibitor of the present disclosure.

[0162] In additional embodiments, the JAK-related disease is cancer, including those characterized by solid tumors (e.g., prostate cancer, renal cancer, liver cancer, pancreatic cancer, gastric cancer, breast cancer, lung cancer, head and neck cancer, thyroid cancer, glioblastoma, Kaposi’s sarcoma, Castleman’s disease, uterine leiomyosarcoma, melanoma, etc.), blood cancers (e.g., lymphoma, leukemia such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), or multiple myeloma), and skin cancers such as cutaneous T-cell lymphoma (CTCL) and cutaneous B-cell lymphoma. Exemplary CTCLs include Sezary syndrome and mycosis fungoides.

[0163] In some embodiments, the JAK inhibitors described herein, or combinations with other JAK inhibitors (e.g., those reported in U.S. Publication No. 20070135461, which is incorporated herein by reference in its entirety), can be used to treat inflammation-related cancers. In some embodiments, the cancer is associated with inflammatory bowel disease. In some embodiments, the inflammatory bowel disease is ulcerative colitis. In some embodiments, the inflammatory bowel disease is Crohn's disease. In some embodiments, the inflammation-related cancer is colitis-related cancer. In some embodiments, the inflammation-related cancer is colon cancer or colorectal cancer. In some embodiments, the cancer is gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), adenocarcinoma, small intestine cancer, or rectal cancer.

[0164] JAK-related diseases can also include those characterized by the expression of: JAK2, such as those having at least one mutation in the pseudokinase domain (e.g., JAK2V617F); JAK2 mutants having at least one mutation outside the pseudokinase domain; JAK1 mutants; JAK3 mutants; erythropoietin receptor (EPOR) mutants; or dysregulated expression of CRLF2.

[0165] JAK-related diseases may also include myeloproliferative diseases (MPDs), such as polycythemia vera (PV), essential thrombocythemia (ET), myelofibrosis with myeloid metaplasia (MMM), primary myelofibrosis (PMF), chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), hypereosinophilic syndrome (HES), systemic mastocytosis (SMCD), etc. In some embodiments, the myeloproliferative disorder is myelofibrosis (e.g., primary myelofibrosis (PMF) or post-polycythemia vera / essential thrombocythemia myelofibrosis (Post-PV / ET MF)). In some embodiments, the myeloproliferative disorder is post-essential thrombocythemia myelofibrosis (Post-ET MF). In some embodiments, the myeloproliferative disorder is post-polycythemia vera myelofibrosis (Post-PV MF).

[0166] In some embodiments, the JAK inhibitors described herein may further be used to treat myelodysplastic syndromes (MDS) in patients in need thereof. In some embodiments, the patient is dependent on red blood cell transfusions.

[0167] As used herein, myelodysplastic syndromes are intended to include heterogeneous and clonal hematopoietic disorders characterized by ineffective hematopoiesis of one or more major myeloid cell lineages. Myelodysplastic syndromes are associated with bone marrow failure, peripheral cytopenias, and a propensity to progress to acute myeloid leukemia (AML). In addition, clonal cytogenetic abnormalities can be detected in approximately 50% of MDS cases. In 1997, the World Health Organization (WHO), in conjunction with the Society for Hematopathology (SH) and the European Association for Hematopathology (EAHP), proposed a new classification of hematopoietic tumors (Harris et al., J Clin Oncol 1999; 17:3835-3849; Vardiman et al., Blood 2002; 100:2292-2302). For MDS, the WHO used not only the morphological criteria from the French-American-British (FAB) classification but also incorporated existing genetic, biological, and clinical features to define subsets of MDS (Bennett et al., Br J Haematol 1982; 51:189-199). In 2008, the WHO classification of MDS (Table 1) was further refined to allow for precise and prognostically relevant subclassification of unilineage dysplasia by incorporating new clinical and scientific information (Vardiman et al., Blood 2009; 114:937-951; Swerdlow et al., WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. 4th ed Lyon France: IARC Press; 2008:88-103; Bunning and Germing, “Myelodysplastic syndromes / neoplasms” Chapter 5, Swerdlow et al. eds WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. (4th ed): Lyon, France: IARC Press; 2008:88-103).

[0168] Table 1. 2008 WHO Classification of Newly Diagnosed Myelodysplastic Syndromes

[0169]

[0170] In some embodiments, the myelodysplastic syndrome is refractory cytopenia with unilineage dysplasia (RCUD).

[0171] In some embodiments, the myelodysplastic syndrome is refractory anemia with ring sideroblasts (RARS).

[0172] In some embodiments, myelodysplastic syndrome is refractory cytopenia with multilineage dysplasia.

[0173] In some embodiments, myelodysplastic syndrome is refractory anemia with excess blasts-1 (RAEB-1).

[0174] In some embodiments, myelodysplastic syndrome is refractory anemia with excess blasts-2 (RAEB-2).

[0175] In some embodiments, myelodysplastic syndrome is myelodysplastic syndrome, unclassified (MDS-U).

[0176] In some embodiments, myelodysplastic syndrome is myelodysplastic syndrome associated with isolated del(5q).

[0177] In some embodiments, myelodysplastic syndrome is difficult to treat with erythropoiesis-stimulating agents.

[0178] The present disclosure also provides methods of treating psoriasis or other skin conditions by administering a topical formulation comprising a compound provided herein.

[0179] In some embodiments, the JAK inhibitors described herein can be used to treat pulmonary hypertension.

[0180] The present disclosure also provides a method of treating dermatological side effects of other drugs by administering a compound provided herein. For example, many agents can cause unwanted allergic reactions, which can manifest as acneiform rashes or related dermatitis. Exemplary agents with such adverse side effects include anticancer drugs such as gefitinib, cetuximab, erlotinib, etc. The compounds provided herein can be administered systemically or topically (e.g., localized near the dermatitis) in combination with (e.g., simultaneously or sequentially) an agent having adverse dermatological side effects. In some embodiments, the compounds provided herein can be administered topically with one or more other drugs, wherein the other drugs cause contact dermatitis, allergic contact sensitization, or similar skin conditions when administered topically in the absence of the compounds provided herein. Accordingly, the compositions of the present disclosure include topical formulations comprising a compound provided herein and another agent that can cause dermatitis, skin disease, or related side effects.

[0181] Additional JAK-related diseases include inflammation and inflammatory diseases. Exemplary inflammatory diseases include sarcoidosis, ocular inflammatory diseases (e.g., iritis, uveitis, scleritis, conjunctivitis or related diseases), respiratory inflammatory diseases (e.g., the upper respiratory tract including the nose and sinuses, such as rhinitis or sinusitis or the lower respiratory tract, including bronchitis, chronic obstructive pulmonary disease, etc.), inflammatory myopathies such as myocarditis, and other inflammatory diseases. In some embodiments, the inflammatory disease of the eye is blepharitis.

[0182] The JAK inhibitors described herein can also be used to treat ischemia-reperfusion injury or diseases or conditions associated with inflammatory ischemic events (e.g., stroke or cardiac arrest). The JAK inhibitors described herein can also be used to treat endotoxin-driven disease states (e.g., complications after bypass surgery or chronic endotoxin states leading to chronic heart failure). The JAK inhibitors described herein can also be used to treat anorexia, cachexia or fatigue, such as anorexia, cachexia or fatigue caused by or associated with cancer. The JAK inhibitors described herein can also be used to treat restenosis, scleroderma or fibrosis. The JAK inhibitors described herein can also be used to treat conditions associated with hypoxia or astrogliosis, such as diabetic retinopathy, cancer or neurodegeneration. See, e.g., Dudley, A.C. et al. Biochem. J. 2005, 390 (Pt 2):427-36 and Sriram, K. et al. J. Biol. Chem. 2004, 279(19):19936-47. Epub Mar 2, 2004, both of which are incorporated herein by reference in their entirety. The JAK inhibitors described herein can be used to treat Alzheimer's disease.

[0183] The JAK inhibitors described herein can also be used to treat other inflammatory diseases, such as systemic inflammatory response syndrome (SIRS) and septic shock.

[0184] The JAK inhibitors described herein can also be used to treat gout and prostate volume increase caused by, for example, benign prostatic hypertrophy or benign prostatic hyperplasia.

[0185] Other JAK-related diseases include bone resorption diseases, such as osteoporosis, osteoarthritis. Bone resorption may also be associated with other conditions, such as hormonal disorders and / or hormone therapy, autoimmune diseases (e.g., osteosarcomatosis) or cancer (e.g., myeloma). The reduction in bone resorption caused by JAK inhibitors can be about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80% or about 90%.

[0186] In some embodiments, the JAK inhibitors described herein can also be used to treat dry eye disease. As used herein, "dry eye disease" is intended to encompass the disease states summarized in a recent official report of the Dry Eye Workshop (DEWS), which defines dry eye disease as "a multifactorial disease of the tear film and ocular surface that causes symptoms such as discomfort, visual disturbances, and tear film instability, and may cause damage to the ocular surface. It is accompanied by increased osmolarity of the tear film and increased ocular surface inflammation". Lemp, "The Definition and Classification of Dry Eye Disease: Report of the Definition and Classification Subcommittee of the International Dry Eye Workshop", The Ocular Surface, 5(2), 75-92, April 2007, which is incorporated herein by reference in its entirety. In some embodiments, the dry eye disease is selected from aqueous tear-deficient dry eye (ADDE) or evaporative dry eye, or a suitable combination thereof. In some embodiments, the dry eye disease is Sjogren's syndrome dry eye (SSDE). In some embodiments, the dry eye disease is non-Sjogren's syndrome dry eye (NSSDE).

[0187] On the other hand, the present disclosure provides a method for treating a patient with conjunctivitis, uveitis (including chronic uveitis), choroiditis, retinitis, cyclitis, scleritis, episcleritis or iritis; treating inflammation or pain associated with corneal transplantation, LASIK (laser-assisted in situ keratomileusis), photorefractive keratectomy or LASEK (laser-assisted subepithelial keratomileusis); inhibiting vision loss associated with corneal transplantation, LASIK, photorefractive keratectomy or LASEK; or inhibiting transplant rejection, the method comprising administering to the patient a therapeutically effective amount of a compound provided herein or a pharmaceutically acceptable salt thereof.

[0188] In addition, the compounds provided herein, or combinations with other JAK inhibitors (such as those reported in U.S. Serial No. 11 / 637,545, which is incorporated herein by reference in its entirety) can be used to treat respiratory dysfunction or failure associated with viral infections, such as influenza and SARS.

[0189] In some embodiments, the present disclosure provides the free base of Compound 1 and Compound 1 phosphate as described in any embodiment herein, for use in a method of treating any disease or disorder described herein. In some embodiments, the present disclosure provides the use of the free base of Compound 1 and Compound 1 phosphate as described in any embodiment herein, for the preparation of a medicament for use in a method of treating any disease or disorder described herein.

[0190] In some embodiments, the present disclosure provides Compound 1 free base and Compound 1 phosphate or a pharmaceutically acceptable salt thereof as described herein for methods of modulating JAK1. In some embodiments, the present disclosure also provides uses of Compound 1 free base and Compound 1 phosphate or a pharmaceutically acceptable salt thereof as described herein for preparing an agent for methods of modulating JAK1.

[0191] As used herein, the term "contacting" means bringing together the designated moieties in an in vitro system or an in vivo system. For example, "contacting" a JAK with a compound provided herein includes administering to an individual or patient (e.g., a human) a compound of the present disclosure having a JAK, and, for example, introducing a compound provided herein into a sample containing a cell or a purified preparation containing JAK.

[0192] As used herein, the terms "individual" or "patient" used interchangeably mean any animal including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, pigs, cows, sheep, horses or primates, and most preferably humans.

[0193] As used herein, the phrase "therapeutically effective amount" means the amount of an active compound or agent that elicits a biological or medical response sought by a researcher, veterinarian, physician or other clinician in a tissue, system, animal, individual or human body. In some embodiments, the therapeutically effective amount is from about 5 mg to about 1000 mg, or from about 10 mg to about 500 mg.

[0194] As used herein, the term "treating / treatment" means one or more of the following: (1) inhibiting a disease; e.g., inhibiting a disease, condition or disorder in an individual who is experiencing or exhibiting the pathology or symptoms of the disease, condition or disorder (i.e., preventing further development of the pathology and / or symptoms); (2) alleviating a disease; e.g., alleviating a disease, condition or disorder in an individual who is experiencing or exhibiting the pathology or symptoms of the disease, condition or disorder (i.e., reversing the pathology and / or symptoms), such as reducing the severity of the disease.

[0195] As used herein, the term "preventing" or "prevention" means, for example, preventing a disease, condition or disorder in an individual who may be susceptible to the disease, condition or disorder but has not yet experienced or exhibited the pathology or symptoms of the disease.

[0196] Combination therapy

[0197] The methods described herein may further include administering one or more additional therapeutic agents. The one or more additional therapeutic agents may be administered to the patient simultaneously or sequentially.

[0198] In some embodiments, the method further comprises administering an additional therapeutic agent selected from an IMiD, an anti-IL-6 agent, an anti-TNF-α agent, a hypomethylating agent, and a biologic response modifier (BRM).

[0199] Generally, a BRM is a substance made by a living organism for treating diseases, which may occur naturally in the body or may be manufactured in the laboratory. Examples of BRMs include IL-2, interferons, various types of colony-stimulating factors (CSF, GM-CSF, G-CSF), monoclonal antibodies such as abciximab, etanercept, infliximab, rituximab, trastuzumab, and high-dose ascorbic acid.

[0200] In some embodiments, the anti-TNF-α agent is infliximab or etanercept.

[0201] In some embodiments, the demethylating agent is a DNA methyltransferase inhibitor. In some embodiments, the DNA methyltransferase inhibitor is selected from 5-azacytidine and decitabine.

[0202] Generally, an IMiD acts as an immunomodulator. In some embodiments, the IMiD is selected from thalidomide, lenalidomide, pomalidomide, CC-11006, and CC-10015.

[0203] In some embodiments, the method further comprises administering an additional therapeutic agent selected from: antithymocyte globulin, recombinant human granulocyte colony-stimulating factor (G CSF), granulocyte-monocyte CSF (GM-CSF), erythropoiesis-stimulating agent (ESA), and cyclosporine.

[0204] In some embodiments, the method further comprises administering an additional JAK inhibitor to the patient. In some embodiments, the additional JAK inhibitor is tofacitinib or ruxolitinib.

[0205] One or more additional agents, such as chemotherapeutic agents, anti-inflammatory agents, steroids, immunosuppressants, and PI3Kδ, mTor, Bcr-Abl, Flt-3, RAF, and FAK kinase inhibitors, such as those described in WO 2006 / 056399, which is incorporated herein by reference in its entirety, or other agents can be combined with the compounds described herein for treating JAK-related diseases, disorders, or conditions. The one or more additional agents can be administered to the patient simultaneously or sequentially.

[0206] Examples of chemotherapeutic agents include proteasome inhibitors (e.g., bortezomib), thalidomide, revlimid, and DNA damaging agents such as melphalan, doxorubicin, cyclophosphamide, vincristine, etoposide, carmustine, and the like.

[0207] Exemplary steroids include corticosteroids such as dexamethasone or prednisone.

[0208] Exemplary Bcr-Abl inhibitors include compounds of the genus and species disclosed in U.S. Patent No. 5,521,184, WO 04 / 005281, and U.S. Serial No. 60 / 578,491, and pharmaceutically acceptable salts thereof, which patents are hereby incorporated by reference in their entirety.

[0209] Exemplary suitable Flt-3 inhibitors include compounds and pharmaceutically acceptable salts thereof as disclosed in WO 03 / 037347, WO 03 / 099771, and WO 04 / 046120, which patents are hereby incorporated by reference in their entirety.

[0210] Exemplary suitable RAF inhibitors include compounds and pharmaceutically acceptable salts thereof as disclosed in WO 00 / 09495 and WO 05 / 028444, which patents are hereby incorporated by reference in their entirety.

[0211] Exemplary suitable FAK inhibitors include compounds and pharmaceutically acceptable salts thereof as disclosed in WO 04 / 080980, WO 04 / 056786, WO 03 / 024967, WO01 / 064655, WO 00 / 053595, and WO 01 / 014402, which patents are hereby incorporated by reference in their entirety.

[0212] In some embodiments, the compounds provided herein (e.g., Compound 1 free base and Compound 1 phosphate) can be used in combination with one or more other kinase inhibitors, including imatinib, particularly for treating patients resistant to imatinib or other kinase inhibitors.

[0213] In some embodiments, suitable chemotherapeutic agents can be selected from antimetabolites, topoisomerase 1 inhibitors, platinum analogs, taxanes, anthracyclines, and EGFR inhibitors, and combinations thereof.

[0214] In some embodiments, the antimetabolites include capecitabine, gemcitabine, and fluorouracil (5-FU).

[0215] In some embodiments, the taxanes include paclitaxel, (paclitaxel protein-bound particles for injectable suspension), and (docetaxel).

[0216] In some embodiments, the platinum analogs include oxaliplatin, cisplatin, and carboplatin.

[0217] In some embodiments, the topoisomerase 1 inhibitors include irinotecan and topotecan.

[0218] In some embodiments, the anthracyclines include doxorubicin or a liposomal formulation of doxorubicin.

[0219] In some embodiments, the chemotherapeutic agent is FOLFIRINOX (5-FU, leucovorin, irinotecan, and oxaliplatin). In some embodiments, the chemotherapeutic agent is gemcitabine and (paclitaxel protein-bound particles for injectable suspension).

[0220] In some embodiments, the compounds provided herein (e.g., Compound 1 free base and Compound 1 phosphate) can be combined with chemotherapeutic agents for the treatment of cancer, such as multiple myeloma, and can improve the treatment response without exacerbating their toxic effects as compared to the response to the chemotherapeutic agent alone. Examples of additional agents for the treatment of multiple myeloma can include, but are not limited to, melphalan, melphalan plus prednisone [MP], doxorubicin, dexamethasone, and Velcade (bortezomib). Other additional agents for the treatment of multiple myeloma include Bcr-Abl, Flt-3, RAF, and FAK kinase inhibitors. An additive or synergistic effect is the desired result of combining the JAK inhibitors of the present disclosure with another agent. In addition, after treatment with the JAK inhibitors of the present disclosure, the resistance of multiple myeloma cells to an agent such as dexamethasone can be reversible. The agent can be combined with the compounds provided herein in a single or sequential dosage form, or the agent can be administered simultaneously or sequentially as a separate dosage form.

[0221] In some embodiments, a corticosteroid, such as dexamethasone, is administered to a patient in combination with at least one JAK inhibitor, wherein dexamethasone is administered intermittently as opposed to continuously.

[0222] In some additional embodiments, the combinations of the compounds provided herein can be administered to a patient before, during, and / or after bone marrow transplantation or stem cell transplantation.

[0223] In some embodiments, the additional therapeutic agent is fluocinolone acetonide or rimexolone (AL-2178, Vexol, Alcon).

[0224] In some embodiments, the additional therapeutic agent is cyclosporine

[0225] In some embodiments, the additional therapeutic agent is a corticosteroid. In some embodiments, the corticosteroid is triamcinolone, dexamethasone, fluocinolone acetonide, cortisone, prednisolone, or fluorometholone.

[0226] In some embodiments, the additional therapeutic agent is selected from Dehydrex TM (Holles Labs), Civamide (Opko), sodium hyaluronate (Vismed, Lantibio / TRB Chemedia), cyclosporine (ST-603, Sirion Therapeutics), ARG101(T) (testosterone, Argentis), AGR1012(P) (Argentis), ecabet sodium (Senju-Ista), gefarnate (Santen), 15-(s)-hydroxyeicosatetraenoic acid (15(S)-HETE), cevimeline, doxycycline (ALTY-0501, Alacrity), minocycline, iDestrin TM(NP50301, Nascent Pharmaceuticals), Cyclosporine A (Nova22007, Novagali), Oxytetracycline (Duramycin, MOLI1901, Lantibio), CF101 ((2S,3S,4R,5R)-3,4-dihydroxy-5-[6-[(3-iodophenyl)methylamino]purin-9-yl]-N-methyl-oxolane-2-carboxamide, Can-Fite Biopharma), Voclosporin (LX212 or LX214, Lux Biosciences), ARG103 (Agentis), RX-10045 (synthetic resolvin analog, Resolvyx), DYN15 (Dyanmis Therapeutics), Rivoglitazone (DE011, Daiichi Sanko), TB4 (RegeneRx), OPH-01 (Ophtalmis Monaco), PCS101 (Pericor Science), REV1-31 (Evolutec), Lacritin (Senju), Rebamipide (Otsuka-Novartis), OT-551 (Othera), PAI-2 (University of Pennsylvania and Temple University), Pilocarpine, Tacrolimus, Pimecrolimus (AMS981, Novartis), Loteprednol Etabonate, Rituximab, Dequalinium Tetrasodium (INS365, Inspire), KLS-0611 (Kissei Pharmaceuticals), Dehydroepiandrosterone, Anakinra, Efalizumab, Mycophenolate Sodium, Etanercept Hydroxychloroquine, NGX267 (Torrey Pines Therapeutics), Actemra, Gemcitabine, Oxaliplatin, L-Asparaginase or Thalidomide.

[0227] In some embodiments, the additional therapeutic agent is an anti-angiogenic agent, a cholinergic agonist, a TRP-1 receptor modulator, a calcium channel blocker, a mucin secretagogue, a MUC1 stimulant, a calcineurin inhibitor, a corticosteroid, a P2Y2 receptor agonist, a muscarinic receptor agonist, an mTOR inhibitor, another JAK inhibitor, a Bcr-Abl kinase inhibitor, a Flt-3 kinase inhibitor, a RAF kinase inhibitor, and a FAK kinase inhibitor, such as those described in WO 2006 / 056399, which is incorporated herein by reference in its entirety. In some embodiments, the additional therapeutic agent is a tetracycline derivative (e.g., minocycline or doxycycline). In some embodiments, the additional therapeutic agent binds to FKBP12.

[0228] In some embodiments, the additional therapeutic agent is an alkylating agent or DNA cross-linking agent; an antimetabolite / demethylating agent (e.g., 5-fluorouracil, capecitabine, or azacitidine); antihormonal therapy (e.g., a hormone receptor antagonist, a SERM, or an aromatase inhibitor); a mitotic inhibitor (e.g., vincristine or paclitaxel); a topoisomerase (I or II) inhibitor (e.g., mitoxantrone and irinotecan); an apoptosis inducer (e.g., ABT-737); a nucleic acid therapy (e.g., antisense or RNAi); a nuclear receptor ligand (e.g., an agonist and / or antagonist: all-trans retinoic acid or bexarotene); an epigenetic targeting agent, such as a histone deacetylase inhibitor (e.g., vorinostat), a demethylating agent (e.g., decitabine); a regulator of protein stability, such as an Hsp90 inhibitor, ubiquitin, and / or a ubiquitin-like conjugating or deconjugating molecule; or an EGFR inhibitor (erlotinib).

[0229] In some embodiments, the additional therapeutic agent is a lubricating eye drop (also referred to as an "artificial tear"), which includes, but is not limited to, a composition containing polyvinyl alcohol, hydroxypropyl methylcellulose, glycerol, polyethylene glycol (e.g., PEG400), or carboxymethyl cellulose. Artificial tears can help treat dry eye by compensating for the reduced wetting and lubricating ability of the tear film. In some embodiments, the additional therapeutic agent is a mucolytic drug, such as N-acetyl-cysteine, which can interact with mucin and thus reduce the viscosity of the tear film.

[0230] In some embodiments, the additional therapeutic agents include antibiotics, antiviral agents, antifungal agents, anesthetics, anti-inflammatory agents (including steroidal and non-steroidal anti-inflammatory agents), and anti-allergy agents. Examples of suitable agents include aminoglycosides such as amikacin, gentamicin, tobramycin, streptomycin, netilmicin, and kanamycin; fluoroquinolones such as ciprofloxacin, norfloxacin, ofloxacin, trovafloxacin, lomefloxacin, levofloxacin, and enoxacin; nalidixic acid; sulfonamides; polymyxins; chloramphenicol; neomycin; paromomycin; colicin; bacitracin; vancomycin; tetracycline; rifampin and its derivatives (“rifampicin”); cycloserine; β-lactams; cephalosporins; amphotericin; fluconazole; flucytosine; natamycin; miconazole; ketoconazole; corticosteroids; diclofenac; flurbiprofen; ketorolac; suprofen; cromolyn; lodoxamide; levocabastine; naphazoline; antazoline; pheniramine; or azalide antibiotics.

[0231] In some embodiments, the compounds provided herein can be combined with immune checkpoint inhibitors for treating diseases such as cancer. Exemplary immune checkpoint inhibitors include inhibitors against immune checkpoint molecules such as CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3Kδ, PI3Kγ, TAM, arginase, CD137 (also known as 4-1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, PD-1, PD-L1, and PD-L2. In some embodiments, the immune checkpoint molecule is a stimulatory checkpoint molecule selected from CD27, CD28, CD40, ICOS, OX40, GITR, and CD137. In some embodiments, the immune checkpoint molecule is an inhibitory checkpoint molecule selected from A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM3, and VISTA. In some embodiments, the compounds provided herein can be used in combination with one or more agents selected from KIR inhibitors, TIGIT inhibitors, LAIR1 inhibitors, CD160 inhibitors, 2B4 inhibitors, and TGFRβ inhibitors.

[0232] In some embodiments, the inhibitor of the immune checkpoint molecule is an anti-PD1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody.

[0233] In some embodiments, the inhibitor of the immune checkpoint molecule is a PD-1 inhibitor, such as an anti-PD-1 monoclonal antibody. In some embodiments, the anti-PD-1 monoclonal antibody is nivolumab, pembrolizumab (also known as MK-3475), pidilizumab, SHR-1210, PDR001, or AMP-224. In some embodiments, the anti-PD-1 monoclonal antibody is nivolumab or pembrolizumab. In some embodiments, the anti-PD1 antibody is pembrolizumab.

[0234] In some embodiments, the inhibitor of the immune checkpoint molecule is a PD-L1 inhibitor, such as an anti-PD-L1 monoclonal antibody. In some embodiments, the anti-PD-L1 monoclonal antibody is BMS-935559, MEDI4736, MPDL3280A (also known as RG7446), or MSB0010718C. In some embodiments, the anti-PD-L1 monoclonal antibody is MPDL3280A or MEDI4736.

[0235] In some embodiments, the inhibitor of the immune checkpoint molecule is an inhibitor of CTLA-4, such as an anti-CTLA-4 antibody. In some embodiments, the anti-CTLA-4 antibody is ipilimumab.

[0236] In some embodiments, the inhibitor of the immune checkpoint molecule is an inhibitor of LAG3, such as an anti-LAG3 antibody. In some embodiments, the anti-LAG3 antibody is BMS-986016 or LAG525.

[0237] In some embodiments, the inhibitor of the immune checkpoint molecule is an inhibitor of GITR, such as an anti-GITR antibody. In some embodiments, the anti-GITR antibody is TRX518 or MK-4166.

[0238] In some embodiments, the inhibitor of the immune checkpoint molecule is an inhibitor of OX40, such as an anti-OX40 antibody or an OX40L fusion protein. In some embodiments, the anti-OX40 antibody is MEDI0562. In some embodiments, the OX40L fusion protein is MEDI6383.

[0239] The compounds of the present disclosure can be used in combination with one or more agents for treating diseases such as cancer. In some embodiments, the agent is an alkylating agent, a proteasome inhibitor, a corticosteroid, or an immunomodulator. Examples of alkylating agents include cyclophosphamide (CY), melphalan (MEL), and bendamustine. In some embodiments, the proteasome inhibitor is carfilzomib. In some embodiments, the corticosteroid is dexamethasone (DEX). In some embodiments, the immunomodulator is lenalidomide (LEN) or pomalidomide (POM).

[0240] Drug formulations and dosage forms

[0241] When used as a drug, the compounds provided herein can be administered in the form of a pharmaceutical composition. These compositions can be prepared in a manner well known in the pharmaceutical art and can be administered by a variety of routes, depending on whether local or systemic treatment is required and the area to be treated. Administration can be local (including transdermal, epidermal, ocular, and mucosal, including intranasal, vaginal, and rectal delivery), pulmonary (e.g., by inhalation or insufflation of a powder or aerosol, including by nebulizer; intratracheal or intranasal), oral, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular injection or infusion; or intracranial administration, such as intrathecal or intraventricular administration. Parenteral administration can be in the form of a single bolus dose or can be, for example, by continuous infusion pump. Pharmaceutical compositions and formulations for local administration can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickening agents, etc. may be necessary or desirable.

[0242] The present disclosure also includes pharmaceutical compositions containing, for example, the free base of Compound 1 and / or Compound 1 phosphate as the active ingredient, and one or more pharmaceutically acceptable carriers (excipients). In some embodiments, the composition is suitable for local administration. In preparing the compositions of the present disclosure, the active ingredient is usually admixed with the excipient, diluted with the excipient, or enclosed within a carrier in the form of, for example, capsules, sachets, paper, or other containers. When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material that acts as a vehicle, carrier, or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (in solid form or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.

[0243] In the preparation of formulations, the compounds provided herein (e.g., Compound 1 free base and Compound 1 phosphate) can be milled to provide a suitable particle size before being combined with other ingredients. If Compound 1 free base or Compound 1 phosphate is substantially insoluble, it can be milled to a particle size of less than 200 mesh. If Compound 1 free base and Compound 1 phosphate are substantially water-soluble, the particle size can be adjusted by milling to provide a substantially uniform distribution in the formulation, e.g., about 40 mesh.

[0244] The compounds provided herein can be milled using known milling procedures such as wet milling to obtain a particle size suitable for tablet formation and other formulation types. Fine (nanoparticle) formulations of the compounds provided herein can be prepared by methods known in the art (e.g., see International Application No. WO2002 / 000196).

[0245] Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methylcellulose. The formulation can additionally contain: lubricants such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preservatives such as methyl benzoate and propyl paraben; sweetening agents; and flavoring agents. By employing procedures known in the art, the compositions of the present disclosure can be formulated to provide rapid, sustained, or delayed release of the active ingredient after administration to a patient.

[0246] In some embodiments, the pharmaceutical composition comprises siliconized microcrystalline cellulose (SMCC) and at least one of the compounds described herein or a pharmaceutically acceptable salt thereof. In some embodiments, the siliconized microcrystalline cellulose comprises about 98% microcrystalline cellulose and about 2% silica w / w.

[0247] In some embodiments, the composition is a sustained release composition that comprises at least Compound 1 free base and / or Compound 1 phosphate, and at least one pharmaceutically acceptable carrier. In some embodiments, the composition comprises Compound 1 free base or / and Compound 1 phosphate described herein, and at least one component selected from microcrystalline cellulose, lactose monohydrate, hydroxypropyl methylcellulose, and polyethylene oxide. In some embodiments, the composition comprises Compound 1 free base and / or Compound 1 phosphate, and microcrystalline cellulose, lactose monohydrate, and hydroxypropyl methylcellulose. In some embodiments, the composition comprises Compound 1 free base and / or Compound 1 phosphate, and microcrystalline cellulose, lactose monohydrate, and polyethylene oxide. In some embodiments, the composition further comprises magnesium stearate or silica. In some embodiments, the microcrystalline cellulose is Avicel PH102TM In some embodiments, the lactose monohydrate is Fast-flo 316 TM In some embodiments, the hydroxypropyl methylcellulose is hydroxypropyl methylcellulose 2208K4M (e.g., Methocel K4 M Premier TM ) and / or hydroxypropyl methylcellulose 2208K100LV (e.g., Methocel K00LV TM ). In some embodiments, the polyethylene oxide is polyethylene oxide WSR 1105 (e.g., Polyox WSR 1105 TM ).

[0248] In some embodiments, a wet granulation process is used to produce the composition. In some embodiments, a dry granulation process is used to produce the composition.

[0249] The composition can be formulated in unit dosage form, each dose containing from about 1 to about 1,000 mg, about 1 mg to about 100 mg, 1 mg to about 50 mg, and from 1 mg to 10 mg of the active ingredient (e.g., Compound 1 free base and Compound 1 phosphate). Preferably, the dose is from about 1 mg to about 50 mg or from about 1 mg to about 10 mg of the active ingredient. In some embodiments, each dose contains about 10 mg of the active ingredient. In some embodiments, each dose contains about 50 mg of the active ingredient. In some embodiments, each dose contains about 25 mg of the active ingredient. The term "unit dosage form" refers to a physically discrete unit suitable as a unit dose for human subjects and other mammals, each unit containing a predetermined quantity of the active substance calculated to produce the desired therapeutic effect in association with a suitable pharmaceutical excipient.

[0250] In some embodiments, the composition comprises from about 1 to about 1,000 mg, about 1 mg to about 100 mg, 1 mg to about 50 mg, and from 1 mg to 10 mg of the active ingredient (e.g., Compound 1 free base and Compound 1 phosphate). Preferably, the composition comprises from about 1 mg to about 50 mg or from about 1 mg to about 10 mg of the active ingredient. Those of ordinary skill in the art will understand that this includes compounds or compositions containing from about 1 mg to about 10 mg, about 1 mg to about 20 mg, about 1 mg to about 25 mg, about 1 mg to about 50 mg of the active ingredient.

[0251] The active compound (e.g., Compound 1 free base and Compound 1 phosphate) can be effective over a wide range of doses and is generally administered in a pharmaceutically effective amount. However, it should be understood that the amount of the compound actually administered is typically determined by the physician based on relevant circumstances, including the condition to be treated, the route of administration selected, the compound actually administered, the age, weight and response of the individual patient, the severity of the patient's symptoms, etc.

[0252] To prepare solid compositions such as tablets, the primary active ingredient is mixed with pharmaceutical excipients to form a solid preformulation composition that comprises a homogeneous mixture of the compounds of the present disclosure. When these preformulation compositions are referred to as homogeneous, the active ingredient is typically uniformly dispersed throughout the composition such that the composition can be readily subdivided into equally effective unit dosage forms, such as tablets, pills, and capsules. The solid preformulation is then subdivided into unit dosage forms of the above type that contain, for example, from about 0.1 mg to about 1000 mg of the active ingredient of the present disclosure.

[0253] Tablets or pills of the present disclosure can be coated or otherwise compounded to provide dosage forms having the advantage of extended action. For example, a tablet or pill can comprise an inner dosage component and an outer dosage component, the latter being in the form of an encapsulation over the former. The two components can be separated by an enteric layer that is used to resist disintegration in the stomach and to permit the inner component to pass intact into the duodenum or to be released in a delayed manner. A variety of materials can be used for such enteric layers or coatings, including many polymeric acids and mixtures of polymeric acids with materials such as shellac, cetyl alcohol, and cellulose acetate.

[0254] Liquid forms that can be incorporated with the compounds and compositions provided herein for oral or parenteral administration include aqueous solutions, properly flavored syrups, aqueous or oil suspensions, and flavored emulsions having edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

[0255] Compositions for inhalation or insufflation include solutions and suspensions, as well as powders, in pharmaceutically acceptable aqueous or organic solvents, or mixtures thereof. The liquid or solid compositions can contain suitable pharmaceutically acceptable excipients as described above. In some embodiments, the compositions are administered by the oral or nasal respiratory route to achieve local or systemic effects. Inert gases can be used to atomize the compositions. The atomized solution can be inhaled directly from the atomizing device, or the atomizing device can be connected to a face masks tent or an intermittent positive pressure ventilator. The solution, suspension, or powder compositions can be administered orally or nasally from a device that delivers the formulation in an appropriate manner.

[0256] The topical formulation may contain one or more conventional carriers. In some embodiments, the ointment may contain water and one or more hydrophobic carriers selected from, for example, liquid paraffin, polyoxyethylene alkyl ethers, propylene glycol, white petrolatum, and the like. The carrier composition of the cream may be based on a combination of water with glycerol and one or more other components such as glycerol monostearate, PEG-glycerol monostearate, and cetearyl alcohol. The gel may be formulated using isopropyl alcohol and water, suitably in combination with other components such as glycerol, hydroxyethyl cellulose, and the like. In some embodiments, the topical formulation contains at least about 0.1, at least about 0.25, at least about 0.5, at least about 1, at least about 2, or at least about 5 weight % of the compounds provided herein. The topical formulation may be suitably packaged, for example, in a 100 g tube, which tube is optionally associated with instructions for treating a selected indication such as psoriasis or other skin conditions.

[0257] The amount of the compound or composition administered to a patient will vary depending on the substance being administered, the purpose of administration (e.g., prophylaxis or treatment), the condition of the patient, the mode of administration, and the like. In a therapeutic application, the composition is administered to a patient already suffering from a disease in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications. The effective dose will depend on the disease condition being treated and the judgment of the attending physician based on factors such as the severity of the disease, the age, weight, and general condition of the patient.

[0258] The composition administered to a patient may be in the form of the pharmaceutical compositions described above. These compositions may be sterilized by conventional sterilization techniques or may be sterile filtered. The aqueous solutions may be used as packaged or lyophilized, and the lyophilized preparation is combined with a sterile aqueous carrier prior to administration. The pH of the compound preparation is generally from 3 to 11, more preferably from 5 to 9, and most preferably from 7 to 8. It is understood that the use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of pharmaceutical salts.

[0259] The therapeutic dosage of the compounds of the present disclosure can vary depending on, for example, the particular use for treatment, the mode of administration of the compound, the health and condition of the patient, and the judgment of the prescribing physician. The proportion or concentration of Compound 1 free base or Compound 1 phosphate in the pharmaceutical composition can vary depending on many factors, including dosage, chemical properties (such as hydrophobicity), and route of administration. For example, the compounds provided herein can be provided in a physiologically buffered aqueous solution containing from about 0.1% to about 10% w / v of the compound for parenteral administration. Some typical dosage ranges are from about 1 μg / kg to about 1 g / kg body weight per day. In some embodiments, the dosage range is from about 0.01 mg / kg to about 100 mg / kg body weight per day. The dosage may depend on variables such as the type and progression of the disease or disorder, the overall health of the particular patient, the relative biological efficacy of the selected compound, the formulation of the excipient, and its route of administration. The effective dosage can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0260] The compositions of the present disclosure can also include one or more additional agents, such as chemotherapeutic agents, steroids, anti-inflammatory compounds, or immunosuppressants, examples of which are listed above.

[0261] In some embodiments, Compound 1 free base or Compound 1 phosphate is administered as an ophthalmic composition. Thus, in some embodiments, the method includes administering the compound or a pharmaceutically acceptable salt thereof and an ophthalmically acceptable carrier. In some embodiments, the ophthalmic composition is a liquid composition, a semi-solid composition, an insert, a film, microparticles, or nanoparticles.

[0262] In some embodiments, the ophthalmic composition is a liquid composition. In some embodiments, the ophthalmic composition is a semi-solid composition. In some embodiments, the ophthalmic composition is a topical composition. Topical compositions include, but are not limited to, liquid and semi-solid compositions. In some embodiments, the ophthalmic composition is a topical composition. In some embodiments, the topical composition comprises an aqueous solution, an aqueous suspension, an ointment, or a gel. In some embodiments, the ophthalmic composition is topically applied to the anterior eye segment, under the upper eyelid, on the lower eyelid, and in the cul-de-sac. In some embodiments, the ophthalmic composition is sterilized. Sterilization can be accomplished by known techniques such as sterile filtration of the solution or by heating the solution in an ampoule ready for use. The ophthalmic compositions of the present disclosure can also contain pharmaceutical excipients suitable for the preparation of ophthalmic formulations. Examples of such excipients are preservatives, buffers, chelating agents, antioxidants, and salts for adjusting the osmotic pressure.

[0263] As used herein, the term "ophthalmically acceptable carrier" refers to any material that can contain and release a compound or a pharmaceutically acceptable salt thereof and is compatible with the eye. In some embodiments, the ophthalmically acceptable carrier is water or an aqueous solution or suspension, but also includes oils such as those used to prepare ointments and polymer matrices such as those used for eye inserts. In some embodiments, the composition can be an aqueous suspension containing a compound or a pharmaceutically acceptable salt thereof. Liquid ophthalmic compositions, including ointments and suspensions, can have a viscosity suitable for the selected route of administration. In some embodiments, the ophthalmic composition has a viscosity in the range of about 1,000 to about 30,000 centipoise.

[0264] In some embodiments, the ophthalmic composition can further comprise one or more of the following: surfactants, adjuvants, buffers, antioxidants, tonicity regulators, preservatives (e.g., EDTA, BAK (benzalkonium chloride), sodium chlorite, sodium perborate, polyquaternium-1), thickening agents or viscosity regulators (e.g., carboxymethyl cellulose, hydroxymethyl cellulose, polyvinyl alcohol, polyethylene glycol, ethylene glycol 400, propylene glycol hydroxymethyl cellulose, hydroxypropyl guar gum, hyaluronic acid, and hydroxypropyl cellulose), etc. Additives in the formulation can include, but are not limited to, sodium chloride, sodium bicarbonate, sorbic acid, methylparaben, propylparaben, chlorhexidine, castor oil, and sodium perborate.

[0265] Aqueous ophthalmic compositions (solutions or suspensions) generally do not contain ingredients that are physiologically or ophthalmically harmful. In some embodiments, purified water or deionized water is used in the composition. The pH can be adjusted to a range of about 5.0 to 8.5 by adding any physiologically and ophthalmically acceptable pH-adjusting acid, base, or buffer. Examples of ophthalmically acceptable acids include acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, hydrochloric acid, etc., and examples of bases include sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, tromethamine, tris(hydroxymethyl)aminomethane, etc. Salts and buffers include citrate / glucose, sodium bicarbonate, ammonium chloride, and mixtures of the aforementioned acids and bases.

[0266] In some embodiments, the method involves forming or providing a reservoir of a therapeutic agent in contact with the outer surface of the eye. A reservoir refers to a source of a therapeutic agent that is not rapidly removed by tears or other ocular clearance mechanisms. This allows for a sustained, persistent presence of a high concentration of the therapeutic agent in the fluid on the outer surface of the eye with a single application. Without wishing to be bound by any theory, it is believed that absorption and penetration may depend on the concentration of the dissolved drug and the duration of contact of the external tissue with the drug-containing fluid. As the drug is removed by clearance through the ocular fluid and / or absorption into the ocular tissue, more drug is provided, for example, dissolved into the ocular fluid replenished from the reservoir. Thus, the use of a reservoir can more readily facilitate loading of more insoluble therapeutic agents into the ocular tissue. In some embodiments, the reservoir can be maintained for up to eight hours or longer. In some embodiments, the ophthalmic reservoir forms include, but are not limited to, aqueous polymeric suspensions, ointments, and solid inserts.

[0267] In some embodiments, the ophthalmic composition is an ointment or a gel. In some embodiments, the ophthalmic composition is an oil-based delivery vehicle. In some embodiments, the composition comprises a petrolatum or lanolin matrix with an active ingredient (usually 0.1 to 2%) and excipients added. Common bases can include, but are not limited to, mineral oil, petrolatum, and combinations thereof. In some embodiments, the ointment is applied as a strip to the lower eyelid.

[0268] In some embodiments, the ophthalmic composition is an ophthalmic insert. In some embodiments, the ophthalmic insert is bioinert, soft, bioerodible, viscoelastic, stable to sterilization after exposure to a therapeutic agent, resistant to infection by airborne bacteria, bioerodible, biocompatible, and / or viscoelastic. In some embodiments, the insert comprises an ophthalmically acceptable matrix, such as a polymeric matrix. The matrix is typically a polymer and the therapeutic agent is generally dispersed therein or bound to the polymeric matrix. In some embodiments, the therapeutic agent is slowly released from the matrix by dissolution or hydrolysis of covalent bonds. In some embodiments, the polymer is bioerodible (soluble) and its dissolution rate controls the release rate of the therapeutic agent dispersed therein. In another form, the polymeric matrix is a biodegradable polymer that decomposes, for example, by hydrolysis to release the therapeutic agent bound thereto or dispersed therein. In additional embodiments, the matrix and the therapeutic agent can be surrounded by an additional polymeric coating to further control the release. In some embodiments, the insert comprises a biodegradable polymer, such as polycaprolactone (PCL), ethylene / vinyl acetate copolymer (EVA), alkyl cyanoacrylate, polyurethane, nylon, or poly(dl-lactide-co-glycolide) (PLGA), or a copolymer of any of these. In some embodiments, the therapeutic agent is dispersed into the matrix material or dispersed in the monomer composition used to prepare the matrix material prior to polymerization. In some embodiments, the amount of the therapeutic agent is from about 0.1 to about 50%, or from about 2 to about 20%. In additional embodiments, a biodegradable or bioerodible polymeric matrix is used such that the used insert does not have to be removed. As the biodegradable or bioerodible polymer degrades or dissolves, the therapeutic agent is released.

[0269] In additional embodiments, the ophthalmic insert comprises a polymer, including but not limited to those described in “Polymers used in ocular dosage form and drug delivery systems”, Asian J. Pharm., pages 12 - 17 (January 2008), which is incorporated herein by reference in its entirety. In some embodiments, the insert comprises a polymer selected from: polyvinylpyrrolidone (PVP), acrylate or methacrylate polymers or copolymers (e.g., the series of polymers from Rohm or Degussa), hydroxyethylcellulose, polyacrylic acid, poly(amidoamine) dendrimers, poly(dimethylsiloxane), poly(ethylene oxide), poly(lactide-co-glycolide), poly(2-hydroxyethyl methacrylate), poly(vinyl alcohol), or poly(propylene fumarate). In some embodiments, the insert comprises R. In some embodiments, the insert is polyacrylic acid of a 450 kDa-cysteine conjugate.

[0270] In some embodiments, the ophthalmic composition is an ophthalmic film. Polymers suitable for such films include, but are not limited to, those described in Wagh et al. (supra). In some embodiments, the film is a soft contact lens, such as those made from a copolymer of N,N-diethylacrylamide and methacrylic acid crosslinked with ethylene glycol dimethacrylate.

[0271] In some embodiments, the ophthalmic composition comprises microspheres or nanoparticles. In some embodiments, the microspheres comprise gelatin. In some embodiments, the microspheres are injected into the posterior segment of the eye, the choroidal cavity, the sclera, the vitreous humor, or subretinally. In some embodiments, the microspheres or nanoparticles comprise polymers, including but not limited to those described in Wagh et al. (supra) which are incorporated herein by reference in their entirety. In some embodiments, the polymer is chitosan, a polycarboxylic acid such as polyacrylic acid, albumin particles, hyaluronates, polyitaconic acid, poly(butyl) cyanoacrylate, polycaprolactone, poly(isobutyl) caprolactone, poly(lactic-co-glycolic acid), or poly(lactic acid). In some embodiments, the microspheres or nanoparticles comprise solid lipid particles.

[0272] In some embodiments, the ophthalmic composition comprises an ion exchange resin. In some embodiments, the ion exchange resin is an inorganic zeolite or a synthetic organic resin. In some embodiments, the ion exchange resin includes but is not limited to those described in Wagh et al. (supra) which are incorporated herein by reference in their entirety. In some embodiments, the ion exchange resin is a partially neutralized polyacrylic acid.

[0273] In some embodiments, the ophthalmic composition is an aqueous polymeric suspension. In some embodiments, the therapeutic agent or polymeric suspending agent is suspended in an aqueous medium. In some embodiments, the aqueous polymeric suspension can be formulated such that they maintain the same or substantially the same viscosity in the eye as before being administered to the eye. In some embodiments, they can be formulated such that gelation increases upon contact with the tear fluid.

[0274] Kit

[0275] The present disclosure also includes a kit that can be used, for example, to treat or prevent JAK-related diseases or disorders (such as cancer), which includes one or more containers containing a pharmaceutical composition, the pharmaceutical composition containing a therapeutically effective amount of Compound 1 free base or Compound 1 phosphate. As will be apparent to those skilled in the art, such a kit may also include, as needed, one or more of various conventional pharmaceutical kit components, for example, containers having one or more pharmaceutically acceptable carriers, additional containers, etc. The kit may also include instructions (in the form of inserts or labels) indicating the amounts of the components to be administered, administration guidelines, and / or guidelines for mixing the components.

[0276] The present disclosure will be described in more detail by way of specific examples. The following examples are provided for illustrative purposes and are not intended to limit the present disclosure in any way. Those skilled in the art will readily identify a variety of non-critical parameters that can be changed or modified to produce substantially the same results. According to at least one of the analyses described herein, the compounds of the examples have been found to be JAK inhibitors.

[0277] Examples

[0278] Intermediate 1. 3,5-Dimethyl-4,4'-bipyrazole (Compound 2x)

[0279] Solution 1.

[0280]

[0281] Step 1. 1'-(1-Ethoxy-ethyl)-3,5-dimethyl-1H,1'H-[4,4']bipyrazolyl (Compound 2b)

[0282]

[0283] 1-Propanol (5.0 L), drinking water (6.0 L), K2HPO4 (1032 g), 4-bromo-3,5-dimethylpyrazole (1084 g), and 1-(1-ethoxyethyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (Compound 2a, 1502 g) were successively added to a 100 L glass reactor purged with nitrogen. Nitrogen was bubbled through the reaction mixture for 18 minutes, then Pd-118 (55.07 g) was charged into the reactor, and nitrogen was bubbled through the reaction mixture for an additional 18 minutes. The reaction mixture was heated to about 90 °C and stirred at about 90 °C for about 4 hours. The reaction mixture was then cooled to about 17 °C and the phases were separated. The organic phase was treated with activated carbon (1500 g), heated to about 70 °C, stirred at about 70 °C for about 4 hours, and cooled to about 21 °C. The mixture was filtered through diatomaceous earth (1500 g) and the filter cake was washed with 2-propanol (15.0 L). The combined filtrate and washings were concentrated in vacuo at about 58 °C to give the desired crude product 1'-(1-ethoxy-ethyl)-3,5-dimethyl-1H,1'H-[4,4']bipyrazolyl (2593 g), which was used for further processing.

[0284] Charge the reactor with crude 1'-(1-ethoxy-ethyl)-3,5-dimethyl-1H,1'H-[4,4']bipyrazolyl (2590 g) and ethyl acetate (EtOAc, 15.0 L). Separately, prepare an aqueous NaHSO3 solution by thoroughly mixing NaHSO3 (1500 g) and drinking water (8.0 L). Add the aqueous NaHSO3 solution to the reaction mixture, heat to 65 °C - 70 °C, and stir at 65 °C - 70 °C for about 2.5 hours. Separate the phases and retain the organic phase in the reactor. Separately, prepare an aqueous NaHSO3 solution by thoroughly mixing NaHSO3 (1500 g) and drinking water (8.0 L). Add the aqueous NaHSO3 solution to the reaction mixture, heat to 65 °C - 70 °C, and stir at 65 °C - 70 °C for about 3.5 hours. Separate the phases. Load sea sand (3000 g), ethyl acetate (EtOAc, 15.0 L), and silica gel (SiO2, 4500 g) onto the chromatography column in sequence. Mix the silica gel and the solvent, and elute the solvent onto the silica gel surface. Load sea sand (3000 g) on top of the column. Load the reaction mixture onto the column and elute with ethyl acetate (18.0 L). Combine the desired fractions and concentrate the combined solution in vacuo at about 55 °C to obtain the column-purified product (1760 g), which is then charged into the reactor together with dichloromethane (16.0 L). Charge silanethiol (160 g) into the reactor and heat the reaction mixture to 35 °C - 40 °C and stir at 35 °C - 40 °C for about 2 hours. Filter the mixture, and wash the filter cake with dichloromethane (3.5 L). Concentrate the combined filtrate and wash solution in vacuo to obtain the purified desired product 1'-(1-ethoxy-ethyl)-3,5-dimethyl-1H,1'H-[4,4']bipyrazolyl (1600 g), which contains residual solvent and is used directly in the subsequent reaction. 1 H NMR (400 MHz, DMSO-d6) δ 12.17 (s, 1H), 7.89 (s, 1H), 7.56 (s, 1H), 5.53 (q, J = 6.0 Hz, 1H), 3.41 (dq, J = 9.6, 7.0 Hz, 1H), 3.19 (dq, J = 9.6, 7.0 Hz, 1H), 2.20 (s, 6H), 2.10, 1.60 (d, J = 6.0 Hz, 3H), 1.01 (t, J = 7.0 Hz, 3H) ppm; 13 C NMR (101 MHz, DMSO-d6) δ 145.7, 137.75, 135.9, 125.48, 114.94, 108.69, 86.84, 63.57, 21.84, 15.43, 13.86 ppm.

[0285] Step 2. 3,5-Dimethyl-1H,1'H-[4,4']bipyrazolyl hydrochloride (Compound 2·x HCl)

[0286]

[0287] A 100 L glass reactor was purged with nitrogen at room temperature and charged with 1'-(1-ethoxy-ethyl)-3,5-dimethyl-1H,1'H-[4,4']bipyrazolyl (5723 g, based on the theoretical yield), 2-propanol (IPA, 13.0 L), and concentrated hydrochloric acid (HCl, 4.08 L). The resulting reaction mixture was heated to about 60 °C - 65 °C and stirred at 60 °C - 65 °C for about 2 hours. The reaction mixture was cooled to room temperature and stirred at room temperature for about 1 hour. The solid was collected by filtration, and the filter cake was washed with 2-propanol (6.5 L). The product was air-dried to give the desired product 3,5-dimethyl-1H,1'H-[4,4']bipyrazolyl hydrochloride as a white solid (3088 g, 63.6% over two steps). 1 H NMR (400 MHz, DMSO-d6) δ 7.94 (s, 2H), 2.38 (s, 6H) ppm; 13 C NMR (101 MHz, DMSO-d6) δ 141.95, 132.75, 111.78, 109.70, 10.97 ppm.

[0288] Step 3. 3,5-Dimethyl-1H,1'H-[4,4']bipyrazolyl (Compound 2·x)

[0289] A 100 L glass reactor was purged with nitrogen and charged with 3,5-dimethyl-1H,1'H-[4,4']bipyrazolyl hydrochloride (3010 g) and drinking water (24.1 L), and the reaction mixture was cooled to 0 °C - 5 °C. Separately, an aqueous NaOH solution was prepared by thoroughly mixing NaOH (1212 g) and drinking water (6.0 L). The aqueous NaOH solution was added to the reaction mixture while maintaining the temperature at about 15 °C. The reaction mixture was warmed to about 18 °C and stirred at about 18 °C for about 14 hours. The solid was collected by filtration and the filter cake was washed successively with drinking water (30.1 L) and n-heptane (13.5 L). The product was air-dried for about 16 hours and then further dried under vacuum at about 50 °C - 60 °C to give 3,5-dimethyl-1H,1'H-[4,4']bipyrazolyl as an off-white powder (2006 g, 81.6%). 1 H NMR (400 MHz, DMSO-d6) δ 7.65 (s, 2H), 2.19 (s, 6H) ppm; 1313C NMR (101 MHz, DMSO-d6) δ 140.76, 131.92, 113.44, 109.16, 12.37 ppm.

[0290] Intermediate 2. (S)-4-(3-(cyanomethylene)azetidin-1-yl)-2,5-difluoro-N-(1,1,1-trifluoropropan-2-yl)benzamide (Compound 1x)

[0291] Solution 2

[0292]

[0293] Step 1. (S)-2,4,5-Trifluoro-N-(1,1,1-trifluoropropan-2-yl)benzamide (Compound 1a)

[0294]

[0295] A mixture of (2S)-1,1,1-trifluoropropan-2-amine (520.96 g, 4.61 mol) in toluene (9.7 L) was cooled to 0 °C - 5 °C, and then a solution of 1.0 M aqueous sodium hydroxide (6.92 L, 6.92 mol, 1.5 equiv) was added at 0 °C - 8 °C. Subsequently, 2,4,5-trifluorobenzoyl chloride (995.62 g, 5.07 mol, 1.1 equiv) was added dropwise to the mixture over 20 minutes at 0 °C - 15 °C. The cooling bath was removed and the reaction mixture was warmed to room temperature and stirred for an additional 1 hour at room temperature. Subsequently, the two phases of the reaction mixture were separated. The organic phase was washed with 0.5 M aqueous sodium hydroxide (4.6 L) and concentrated under reduced pressure to give the crude product as a white solid. The solid was then slurried in n-heptane (2.3 L) at 50 °C for 1 hour, then cooled to room temperature. The solid was collected by filtration, washed with n-heptane (1 L), and dried in vacuo for 2 days to give (S)-2,4,5-trifluoro-N-(1,1,1-trifluoropropan-2-yl)benzamide as a white powder (1203.7 g, 93.2%). 1 1H NMR (300 MHz, DMSO-d6) δ 9.00 (d, J = 8.09 Hz, 1H), 7.69 (m, 2H), 4.75 (m, 1H), 1.92 (d, J = 7.00 Hz, 3H) ppm.

[0296] Step 2. (S)-2,5-Difluoro-4-(3-hydroxyazetidin-1-yl)-N-(1,1,1-trifluoropropan-2-yl)benzamide (Compound 1b)

[0297]

[0298] To a solution of (S)-2,4,5-trifluoro-N-(1,1,1-trifluoropropan-2-yl)benzamide (Compound 1a, 1807.5 g, 6.67 mol) and azetidin-3-ol hydrochloride (827.9 g, 7.56 mol, 1.13 eq) in acetonitrile (3.6 L) was added portionwise 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 2335.2 g, 15.33 mol, 2.3 eq). When the first 1000 g of DBU was charged within 25 minutes, the exothermic reaction raised the internal temperature from 12 °C to 58 °C. The remaining DBU was added at 58 °C - 68 °C within 20 minutes, and the resulting reaction mixture was stirred at 58 °C - 68 °C for 1 hour. The reaction mixture was then cooled to room temperature and treated with 1.0 M aqueous hydrochloric acid (4.34 L). The mixture was stirred at room temperature for 15 minutes and water (6 L) was added. The resulting mixture was stirred at room temperature for 1 hour. The solid was collected by filtration, washed with water (2 L), and dried in vacuo for 4 days to give (S)-2,5-difluoro-4-(3-hydroxyazetidin-1-yl)-N-(1,1,1-trifluoropropan-2-yl)benzamide (2009.8 g, 93.0%) as a white powder. 1 1H-NMR (300 MHz, DMSO-d6) δ 8.38 (d, J = 8.71 Hz, 1H), 7.26 (dd, J = 12.91 Hz, 1H), 6.38 (dd, J = 12.29 Hz, 1H), 5.70 (d, J = 6.38, 1H), 4.75 (m, 1H), 4.56 (m, 1H), 4.22 (m, 2H), 3.71 (m, 2H), 1.28 (d, J = 7.16, 3H) ppm.

[0299] Step 3. (S)-2,5-Difluoro-4-(3-oxoazetidin-1-yl)-N-(1,1,1-trifluoropropan-2-yl)benzamide (Compound 1c)

[0300]

[0301] At 10 °C - 12 °C, a solution of 2,5-difluoro-4-(3-hydroxyazetidin-1-yl)-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide (Compound 1b, 1672.6 g, 5.16 mol) and iodobenzene diacetate (1923.5 g, 5.98 mol, 1.16 eq) in dichloromethane (8.5 L) was added to 2,2,6,6-tetramethyl-1-piperidinyloxy radical (TEMPO, 20.9 g, 0.13 mol, 0.025 eq). The resulting reaction mixture was stirred at 10 °C - 12 °C, and the internal temperature reached 36 °C - 38 °C within 30 - 60 minutes. A cooling bath of IPA and dry ice was used to control the reaction temperature. Once the internal mixture temperature dropped below 25 °C, the reaction mixture was then heated to 35 °C - 38 °C and stirred at 35 °C - 38 °C for another 2 - 3 hours. The reaction mixture was then cooled to room temperature and quenched with an aqueous solution (8.0 L) of sodium thiosulfate (82.9 g, 0.52 mol) and potassium phosphate (950.0 g, 4.5 mol). The two phases were separated and the organic phase was washed with water (2 × 4 L). The organic solution was then concentrated under reduced pressure to obtain the crude desired product as a solid. The solid was slurried in n-heptane (10 L) at room temperature for 30 minutes. The solid was collected by filtration, washed with n-heptane (2 × 2 L), and dried under vacuum overnight to give (S)-2,5-difluoro-4-(3-oxoazetidin-1-yl)-N-(1,1,1-trifluoropropan-2-yl)benzamide (1552.1 g, 93.4%). 1 1H-NMR (300 MHz, DMSO-d6) δ 8.50 (d, J = 8.72 Hz, 1H), 7.35 (dd, J = 12.6 Hz, 1H), 6.62 (dd, J = 12.1 Hz, 1H), 4.81 (s, 4H), 4.56 (m, 1H), 1.30 (d, J = 7.0 Hz, 3H) ppm.

[0302] Step 4. (S)-4-(3-(Cyanomethylene)azetidin-1-yl)-2,5-difluoro-N-(1,1,1-trifluoropropan-2-yl)benzamide (Compound 1x)

[0303] Diethyl cyanomethylphosphonate (422.6 g, 2.39 mol, 0.98 eq) was added to a solution of 1.0 M potassium tert-butoxide in THF (1996.6 g, 2.27 mol, 0.94 eq) at 5 °C - 25 °C for 10 minutes under nitrogen. The resulting mixture was then warmed to room temperature and stirred for 1 hour to form a clear solution (Solution A). Under nitrogen, [2,5-difluoro-4-(3-oxoazetidin-1-yl)-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide (Compound 1c, 784.2 g, 2.43 mol) was added to a mixture of ethanol (EtOH, 0.75 L) and tetrahydrofuran (THF, 2.9 L) to form a solution (Solution B). The resulting Solution B was then cooled to -5 °C in a dry ice-IPA bath, and Solution A was added to Solution B at -5 °C - 5 °C over 30 minutes. The resulting mixture was stirred at 0 °C - 5 °C for 60 minutes. The reaction mixture was then quenched by adding water (9.4 L) over 10 minutes. The resulting mixture was stirred at room temperature for 60 minutes. The solid was then collected by filtration and washed with water (2 L) and n-heptane (2.4 L) to give a brown powder. The brown solid was slurried in methyl tert-butyl ether (MTBE, 4 L) at room temperature overnight. The solid was collected by filtration, washed with MTBE (1 L), and dried in vacuo for 3 days to give (S)-4-(3-(cyanomethylene)azetidin-1-yl)-2,5-difluoro-N-(1,1,1-trifluoropropan-2-yl)benzamide (671.1 g, 94%) as an off-white powder. 1 H-NMR (300 MHz, DMSO-d6) δ 8.50 (d, J = 9.95 Hz, 1H), 7.31 (dd, J = 12.4 Hz, 1H), 6.58 (dd, J = 12.0 Hz, 1H), 5.88 (m, 1H), 4.86–4.75 (m, 5H), 1.31 (d, J = 7.0 Hz, 3H) ppm.

[0304] Intermediate 3. tert-Butyl 3-(cyanomethylene)azetidine-1-carboxylate (Compound 1y)

[0305] Solution 3

[0306]

[0307] Step 1. 1-Diphenylmethylazetidin-3-ol hydrochloride

[0308]

[0309] Treat a solution of diphenylmethanamine (2737 g, 15.0 mol, 1.04 eq) in methanol (MeOH, 6 L) with 2-(chloromethyl)oxirane (1330 g, 14.5 mol) at ambient temperature. Stir the resulting reaction mixture at room temperature for 3 days, then heat it to reflux and maintain for 3 days. Next, cool the reaction mixture to room temperature and then cool it to 0 °C - 5 °C in an ice bath. Collect the solid by filtration and wash it with acetone (4 L) to obtain the first batch of crude desired product (1516 g). Concentrate the filtrate under reduced pressure, and dilute the resulting semi-solid with acetone (1 L). Then collect this solid by filtration to obtain the second batch of crude desired product (221 g). The crude product 1-benzhydrylazetidin-3-ol hydrochloride (1737 g, 43.4% yield) is used in the subsequent reaction without further purification. 1 HNMR (300 MHz, DMSO-d6) δ 12.28 (br.d, 1H), 7.7 (m, 5H), 7.49 (m, 5H), 6.38 (d, 1H), 4.72 (br.s, 1H), 4.46 (m, 1H), 4.12 (m, 2H), 3.85 (m, 2H) ppm; C 16 H 18 ClNO (MW 275.77; C 16 H 17 NO For the free base, MW, 239.31), LCMS (EI) m / e 240 (M + +H).

[0310] Step 2. tert-Butyl 3-hydroxyazetidine-1-carboxylate

[0311]

[0312] A suspension of 1-diphenylmethylazetidin-3-ol hydrochloride (625 g, 2.27 mol) in a 10% solution of sodium carbonate (Na2CO3, 5 L) and dichloromethane (CH2Cl2, 5 L) was stirred at room temperature until all solids dissolved. The two layers were separated, and the aqueous layer was extracted with dichloromethane (CH2Cl2, 2 L). The combined organic extracts were dried over sodium sulfate (Na2SO4) and concentrated under reduced pressure. The resulting crude 1-diphenylmethylazetidin-3-ol free base was then dissolved in THF (6 L), and the solution was placed in a Parr bomb. Di-tert-butyl dicarbonate (BOC2O, 545 g, 2.5 mol, 1.1 eq) and 20% palladium (Pd) / carbon (125 g, 50% wet) were added to the Parr bomb. The vessel was charged with hydrogen (H2) to 30 psi, and stirred at room temperature for 18 hours under a steady hydrogen atmosphere (the vessel was recharged three times to maintain the pressure at 30 psi). The reaction mixture was filtered through a Celite pad and the Celite pad was washed with THF (4 L). The filtrate was concentrated under reduced pressure to remove the solvent and the residue was loaded onto a Biotage 150 column with a minimum amount of dichloromethane (CH2Cl2). The column was eluted with heptane containing 20%-50% ethyl acetate, and the eluates containing the pure desired product tert-butyl 3-hydroxyazetidine-1-carboxylate were collected and combined. The solvent was removed under reduced pressure to give tert-butyl 3-hydroxyazetidine-1-carboxylate (357 g, 90.8% yield) as a colorless oil, which solidified after standing in vacuo at ambient temperature. 1 HNMR (300 MHz, CDCl3), δ 4.56 (m 1H), 4.13 (m, 2H), 3.81 (m, 2H), 1.43 (s, 9H) ppm.

[0313] Step 3. tert-Butyl 3-oxoazetidine-1-carboxylate

[0314]

[0315] A solution of tert-butyl 3-hydroxyazetidine-1-carboxylate (50 g, 289 mmol) in ethyl acetate (400 mL) was cooled to 0 °C. The resulting solution was then treated at 0 °C - 5 °C with a solution of solid TEMPO (0.5 g, 3.2 mmol, 0.011 equiv) and potassium bromide (KBr, 3.9 g, 33.2 mmol, 0.115 equiv) in water (60 mL). While maintaining the reaction temperature at 0 °C - 5 °C, a solution of saturated aqueous sodium bicarbonate (NaHCO3, 450 mL) and aqueous sodium hypochlorite (NaClO, 10% - 13% available chlorine, 450 mL) was added. When an additional amount of sodium hypochlorite solution was added, the color of the reaction mixture gradually faded. When the starting material was consumed, the color of the reaction mixture no longer changed. The reaction mixture was then diluted with ethyl acetate (EtOAc, 500 mL) and the two layers were separated. The organic layer was washed with water (500 mL) and saturated aqueous sodium chloride (500 mL) and dried over sodium sulfate (Na2SO4). The solvent was then removed under reduced pressure to give the crude product, tert-butyl 3-oxoazetidine-1-carboxylate (48 g, 49.47 g theoretical, 97% yield), which was used directly in the next step without further purification. 1 1H NMR (CDCl3, 300 MHz) δ 4.65 (s, 4H), 1.42 (s, 9H) ppm.

[0316] Step 4. tert-Butyl 3-(cyanomethylene)azetidine-1-carboxylate

[0317] Diethyl cyanomethylphosphonate (745 g, 4.20 mol, 1.20 eq) and anhydrous tetrahydrofuran (THF, 9 L) were added to a four-necked flask at room temperature. The solution was cooled to -14 °C with an ice-methanol bath and a solution of 1.0 M potassium tert-butoxide (t-BuOK) in anhydrous tetrahydrofuran (THF, 3.85 L, 3.85 mol, 1.1 eq) was added over 20 minutes, keeping the reaction temperature below -5 °C. The resulting reaction mixture was stirred at -10 °C for 3 h and a solution of 1-tert-butoxycarbonyl-3-azetidinone (600 g, 3.50 mol) in anhydrous tetrahydrofuran (THF, 2 L) was added over 2 h, keeping the internal temperature below -5 °C. The reaction mixture was stirred at -5 °C to -10 °C for 1 h and then slowly warmed to room temperature and stirred overnight at room temperature. The reaction mixture was then diluted with water (4.5 L) and saturated aqueous sodium chloride (NaCl, 4.5 L) and extracted with ethyl acetate (EtOAc, 2 × 9 L). The combined organic layers were washed with brine (6 L) and dried over anhydrous sodium sulfate (Na2SO4). The solvent was removed under reduced pressure and the residue was diluted with dichloromethane (CH2Cl2, 4 L) and then adsorbed onto silica gel (SiO2, 1.5 kg). The crude product adsorbed on silica gel was purified by flash column chromatography (SiO2, 3.5 kg, 0%-25% EtOAc and hexane gradient elution) to give tert-butyl 3-(cyanomethylene)azetidine-1-carboxylate (414.7 g, 61% yield) as a white solid. 1 1H NMR (300 MHz, CDCl3) δ 5.40 (m, 1H), 4.70 (m, 2H), 4.61 (m, 2H), 1.46 (s, 9H) ppm; C 10 1 14 1H + 2O2 (MW, 194.23), LCMS (EI) m / e 217 (M

[0318] Intermediate 4. Alternative synthesis of (S)-2,4,5-trifluoro-N-(1,1,1-trifluoropropan-2-yl)benzamide (Compound 1a)

[0319] Solution 4.

[0320]

[0321] Step 1. 2,4,5-Trifluorobenzoyl chloride

[0322]

[0323] Charge the 100 L reactor with SOCl2 (34.9 kg), DMF (0.34 L), and 2,4,5-trifluorobenzoic acid (32.3 kg). Heat the batch to 80 °C and stir at 80 °C - 90 °C for 9 hours. Cool the batch to 50 °C - 60 °C and vacuum distill at 60 °C until distillation ceases. Charge 14 kg of toluene into the reactor and continuously distill the batch at 60 °C to obtain crude 2,4,5-trifluorobenzoyl chloride (46.28 kg, 88% according to HPLC), which is directly used for the next reaction.

[0324] Step 2. (S)-2,4,5-Trifluoro-N-(1,1,1-trifluoropropan-2-yl)benzamide (Compound 1a)

[0325] Charge an aqueous solution (158 L) containing (S)-1,1,1-trifluoropropan-2-amine hydrochloride (35 kg) into a 1000 L reactor, and charge toluene (198 kg) into the reactor, then add K2CO3 (82 kg) portionwise. Dissolve 2,4,5-trifluorobenzoyl chloride (36.1 kg) in toluene (40 kg), and charge the toluene solution together with the toluene solution of the amine intermediate into the reactor. Stir the resulting mixture at 20 °C for 2 hours. Filter the batch and wash the filter cake with toluene (117 kg). Charge the filtrate and washings into a 1000 L reactor, and charge 1N aqueous NaOH solution (125 kg) into the reactor. Stir the mixture for 2 hours and allow phase separation. Discard the aqueous phase, wash the organic phase twice with water (135 kg) and store in a clean container (Solution 1). Treat a separate portion (Portion 2) in the same manner to obtain Solution 2. Charge Solution 1 and Solution 2 into a 1000 L reactor, and charge Na2SO4 (104 kg) into the reactor. Stir the mixture for 2 hours, filter, and wash the filter cake with toluene (90 kg). Charge the filtrate and washings into a 500 L reactor and vacuum distill the batch at 50 °C. Charge toluene (14 kg) and heptane (166 kg) into the 500 L reactor and stir the batch at 80 °C until a solution is obtained. Cool the solution to 25 °C and stir for 2 hours. Separate the product by vacuum filtration, and wash the filter cake with n-heptane (40 kg). Vacuum dry the filter cake at ≤50 °C to obtain the crude product (S)-2,4,5-trifluoro-N-(1,1,1-trifluoropropan-2-yl)benzamide (87.0 kg; 79.0 wt% by LOD; net weight: 68.7 kg; 68%; 69.4% according to HPLC; 97.1 ee% according to chiral HPLC), which is further purified from a mixture of IPA and n-heptane according to the following procedure.

[0326] Charge an IPA (30.5 kg), heptane (213 kg) and crude (S)-2,4,5-trifluoro-N-(1,1,1-trifluoropropan-2-yl)benzamide (70 kg) into a 500 L reactor. Heat the mixture to 85 °C and stir to form a clear solution. Cool the batch to 20 °C and stir for 12 hours. Filter the batch, wash the filter cake with n-heptane (48 kg), and dry in vacuo at 50 °C to obtain the purified product (S)-2,4,5-trifluoro-N-(1,1,1-trifluoropropan-2-yl)benzamide (37.5 kg, 54%; HPLC purity: 98.8%; 99.7 ee% according to chiral HPLC). 1 HNMR (300 MHz, CDCl3) δ 7.96 (m, 1H), 7.01 (m, 1H), 6.71 (m, 1H), 4.93 (m, 1H), 1.44 (d, J = 8.00 Hz, 3H) ppm.

[0327] Example 1. Synthesis of 4-[3-(cyanomethyl)-3-(3',5'-dimethyl-1H,1'H-4,4'-bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide phosphate (Compound 1 phosphate)

[0328] Solution 5.

[0329]

[0330] Step 1. 4-[3-(cyanomethyl)-3-(3',5'-dimethyl-1H,1'H-4,4'-dipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide (Compound 1 free base)

[0331]

[0332] 3,5-Dimethyl-1H,1'H-[4,4']bipyrazolyl hydrochloride (Compound 2·x HCl, 2002 g, 12.34 mol, 1.1 eq), DMF (3.9 L), and DBU (0.201 L, 204.6 g, 1.34 mol, 0.12 eq) were charged into a 50 L reactor and the reaction mixture was heated to 50 °C - 60 °C and stirred for about 30 minutes. Separately, a solution was prepared by thoroughly mixing (S)-4-(3-(cyanomethylene)azetidin-1-yl)-2,5-difluoro-N-(1,1,1-trifluoropropan-2-yl)benzamide (Compound 1·x, 3872 g, 11.21 mol) and DMF (11.6 L). Then the solution of Compound 1·x in DMF was added to the reaction mixture while maintaining the temperature at about 61 °C. The resulting reaction mixture was stirred at about 60 °C for about 3.5 hours. Then the reaction mixture was cooled to room temperature and water (77.4 L) was added to the reactor. The cooled reaction mixture was added to water while maintaining the temperature at about 21 °C. The resulting mixture was stirred at room temperature for about 1.5 hours. The solid was collected by filtration and the filter cake was washed with potable water (38.7 L). The wet filter cake was air-dried to give 4-[3-(cyanomethyl)-3-(3',5'-dimethyl-1H,1'H-4,4'-dipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide (Compound 1 free base, 5849 g).

[0333] The chromatographic column was successively loaded with ethyl acetate (9.9 L), CH2Cl2 (22.4 L), and silica gel (8000 g), mixed well, and eluted onto the silica gel surface. The crude compound 1 free base (1006 g), silica gel (4000 g), and CH2Cl2 (8.0 L) were loaded into the first rotary evaporator and rotated at about 22 °C for about 45 minutes without collecting the solvent. The crude compound 1 free base (1008 g), silica gel (4002 g), and CH2Cl2 (8.0 L) were loaded into the second rotary evaporator and rotated at about 23 °C for about 45 minutes without collecting the solvent. Then the two mixtures were concentrated under reduced pressure at about 34 °C, and the residue was loaded onto the column. Sea sand (5010 g) was loaded onto the column. The column was eluted successively with the following: the collected eluate (16 L), 30% (v / v) EtOAc-CH2Cl2 (prepared from 31.2 L of EtOAc and 72.8 L of CH2Cl2 respectively), 5% (v / v) MeOH-CH2Cl2 (prepared from 2.5 L of MeOH and 47.5 L of CH2Cl2 respectively), and 8% (v / v) MeOH-CH2Cl2 (prepared from 4.8 L of MeOH and 55.2 L of CH2Cl2 respectively). The combined eluates were concentrated under reduced pressure at about 45 °C to obtain pure compound 1 free base (1824 g). Four batches of column purification were carried out to obtain 5181 g of pure 4-[3-(cyanomethyl)-3-(3',5'-dimethyl-1H,1'H-4,4'-bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide (compound 1 free base; 91% yield). 1 H NMR (400 MHz, DMSO-d6) δ 12.22 (s, 1H), 8.50 (d, J = 8.7 Hz, 1H), 8.13 (s, 1H), 7.72 (s, 1H), 7.36 (dd, J = 12.5, 6.3 Hz, 1H), 6.62 (dd, J = 11.9, 7.3 Hz, 1H), 4.78 (m, 1H), 4.64 (d, J = 8.9 Hz, 2H), 4.40 (d, J = 9.1 Hz, 2H), 3.66 (s, 2H), 2.23 (s, 6H), 1.31 (d, J = 7.0 Hz, 3H) ppm; 1313C NMR (101 MHz, DMSO-d6) δ 162.8, 156.7 (d, J = 246.6 Hz), 146.9 (d, J = 236.9 Hz), 145.2, 141.6 (t, J = 12.3 Hz), 138.3, 135.5, 125.8 (q, J = 281.9 Hz), 125.6, 117.2, 116.4 (d, J = 26.4 Hz), 115.2, 111.3 (dd, J = 15.7, 5.8 Hz), 107.7, 102.0 (d, J = 29.1 Hz), 62.4, 57.7, 45.8 (q, J = 30.8 Hz), 27.0, 13.3, 13.3, 10.4 ppm; 19 19F NMR (282 MHz, DMSO-d6) δ -76.17 (d, J = 7.4 Hz), -116.89 (s), -139.71 (s) ppm.

[0334] Step 2. 4-[3-(Cyanomethyl)-3-(3',5'-dimethyl-1H,1'H-4,4'-bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide phosphate (crude compound 1 phosphate)

[0335]

[0336] To a clear solution of 4-[3-(cyanomethyl)-3-(3',5'-dimethyl-1H,1'H-4,4'-bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide (Compound 1 free base, 405.0 g, 798.1 mmol) in methanol (MeOH, 520.0 mL) and 2-propanol (IPA, 2550.0 mL) at 50 °C was added a solution of phosphoric acid (85 wt% aqueous solution, 119.65 g, 1037.8 mmol, 1.3 eq) in isopropyl alcohol (IPA, 120.0 mL). The resulting slurry was stirred at 50 °C for 1 h. Then n-heptane (4050.0 mL) was added over 40 min while maintaining the internal temperature between 46 °C and 53 °C. After the addition of n-heptane, the slurry was cooled to room temperature and stirred for 19 h. The solid was collected by filtration, washed with a mixture of 2-propanol / and n-heptane (3 to 10 volumes, 2 × 700 mL), then washed with n-heptane (3 × 550 mL), and dried in vacuo at room temperature to give crude 4-[3-(cyanomethyl)-3-(3',5'-dimethyl-1H,1'H-4,4'-bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide phosphate (crude Compound 1 phosphate, 434.6 g, 89.9% yield).

[0337] Step 3. 4-[3-(Cyanomethyl)-3-(3',5'-dimethyl-1H,1'H-4,4'-bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide phosphate (Compound 1 phosphate, purified)

[0338] At room temperature, 4-[3-(cyanomethyl)-3-(3',5'-dimethyl-1H,1'H-4,4'-bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide phosphate (crude compound 1 phosphate, 958.3 g, 1583 mmol) and methanol (MeOH, 9583.0 mL) were charged into a 22 L flask. The resulting slurry was heated to 50 °C to obtain a clear light orange solution. The solution was filtered through a fine filter within 70 minutes, transferred to a 22 L flask and heated to remove methanol. Then 2-propanol (IPA, 7700 mL) was added to the flask within 30 minutes while maintaining the internal temperature between 50 °C and 65 °C. Then n-heptane (14400 mL) was added portionwise within 2.5 hours while maintaining the distillation of the solvent mixture (MeOH, IPA and n-heptane). A total of 10818 g (15000 mL) of the solvent mixture was distilled off. The resulting slurry was cooled to room temperature and stirred for 17 hours. The solid was collected by filtration, washed with a mixture of 2-propanol (IPA) and n-heptane (1 to 5 volumes, 3000 mL), then washed with n-heptane (3 × 4000 mL), and dried in vacuo at room temperature to obtain 4-[3-(cyanomethyl)-3-(3',5'-dimethyl-1H,1'H-4,4'-bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide phosphate as an off-white crystalline powder (compound 1 phosphate, 925.7 g, 96.6% yield). 1 H NMR (400 MHz, DMSO-d6) δ 9.35 (br.s, 4H), 8.50 (d, J = 8.9 Hz, 1H), 8.11 (s, 1H), 7.70 (s, 1H), 7.34 (dd, J = 12.5, 6.4 Hz, 1H), 6.61 (dd, J = 12.0, 7.4 Hz, 1H), 4.86–4.69 (m, 1H), 4.61 (d, J = 8.9 Hz, 2H), 4.38 (d, J = 8.9 Hz, 2H), 3.64 (s, 2H), 2.21 (s, 6H), 1.30 (d, J = 7.1 Hz, 3H) ppm; 13 C NMR (100 MHz, DMSO-d6) δ 162.8, 156.7 (d, J CF = 246.5 Hz), 146.9 (d, J CF = 236.1 Hz), 141.6 (dd, J CF = 13.0, 11.7 Hz), 140.3, 138.3, 125.8 (q, J CF= 281.8 Hz), 125.6, 117.2, 116.4 (dd, J CF = 22.3, 4.6 Hz), 115.1, 111.3 (dd, J CF = 15.7, 5.8 Hz), 107.7, 102.0 (dd, J CF = 29.5, 4.5 Hz), 62.3, 57.7, 57.7, 45.8 (q, J CF = 30.5 Hz), 27.0, 13.3 (d, J CF = 1.7 Hz), 11.7 ppm; C 23 H 22 F5N7O (MW 507.46), LCMS (EI) m / e 508.1 (M + + H).

[0339] The phosphate ratio was measured by 1 1H NMR with 1.01 phosphoric acid: compound 1 free base. The same crystalline form of the compound 1 phosphate drug substance was prepared consistently according to the above preparation and purification procedures. This form has been confirmed by differential scanning calorimetry (DSC) as shown in Figure 1 , thermogravimetric analysis (TGA) as shown in Figure 2 and X-ray powder diffraction (XRPD) as shown in Figure 3 .

[0340] Example 2. Alternative synthesis of 4-[3-(cyanomethyl)-3-(3',5'-dimethyl-1H,1'H-4,4'-bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide phosphate (compound 1 phosphate)

[0341] Solution 6.

[0342]

[0343] Step 1. tert-Butyl 3-(cyanomethyl)-3-(3',5'-dimethyl-1H,1'H-[4,4'-bipyrazol]-1-yl)azetidine-1-carboxylate

[0344]

[0345] Charge anhydrous dimethyl sulfoxide (DMSO; 57.0 L) into a 250 L glass-lined dry reactor and heat it to 32 °C. Once the solvent reaches a certain temperature, charge tert-butyl 3-(cyanomethylene)azetidine-1-carboxylate (Compound 1y, 22.8 kg, 117.4 mol, 1.0 equiv) into the reaction vessel, and then charge 3,5-dimethyl-4,4'-bipyrazole (Compound 2x, 20.0 kg, 123.3 mol, 1.05 equiv). Cool the reaction mixture to 24 °C, charge DBU (4.4 L, 29.56 mol, 0.25 equiv) into the reaction vessel, and stir the resulting solution for at least 2 h. Then dilute the reaction mixture with dichloromethane (116 L) and pour it into an aqueous solution of 10% citric acid and 10% NaCl (97 L). Separate the lower organic layer from the biphasic mixture and extract the aqueous layer with dichloromethane (58 L). Then wash the combined organic layers twice with an aqueous solution of 10% citric acid and 10% NaCl (97 L). As part of the second wash, add additional dichloromethane (DCM) to the organic layer (58 L). After washing, charge isopropyl acetate (465 L) into the reaction mixture while performing volume-fixed distillation. A white solid forms during the distillation. Cool the resulting suspension to 20 °C, stir for at least 4 h, filter, and dry to obtain the desired product tert-butyl 3-(cyanomethyl)-3-(3',5'-dimethyl-1H,1'H-[4,4'-bipyrazol]-1-yl)azetidine-1-carboxylate (30.4 kg, 79%) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 12.19 (s, 1H), 8.06 (s, 1H), 7.70 (s, 1H), 4.41 (d, J = 9.4 Hz, 2H), 4.18 (d, J = 9.3 Hz, 2H), 3.55 (s, 2H), 2.23 (d, J = 19.5 Hz, 6H), 1.41 (s, 9H) ppm; C 18 H 24 N6O2, (MW 356.42), LCMS (EI) m / e 357.4 (M + +H).

[0346] Step 2. 2-(3-(3',5'-dimethyl-1H,1'H-[4,4'-bipyrazol]-1-yl)azetidin-3-yl)acetonitrile

[0347]

[0348] Charge a 450 L glass-lined reactor with dichloromethane (300 L) and tert-butyl 3-(cyanomethyl)-3-(3',5'-dimethyl-1H,1'H-[4,4'-bipyrazol]-1-yl)azetidine-1-carboxylate (30.0 kg, 84.17 mol, 1.000 equiv.). Add TMSI (14.4 L, 101.45 mol, 1.205 equiv.), and stir the resulting solution at 25 °C for at least 2 h. Charge methanol (4.3 L, 106.12 mol, 1.261 equiv.) to the reactor and stir the reaction mixture for an additional 30 min. Then heat the reaction mixture to remove dichloromethane (150 L) by distillation. After the distillation is complete, charge isopropyl acetate (IPAc, 150 L) to the vessel at 25 °C and stir the reaction mixture for 1 h. Filter the resulting suspension and wash with IPAc to obtain a crude mixture (68 kg) of 2-(3-(3',5'-dimethyl-1H,1'H-[4,4'-bipyrazol]-1-yl)azetidin-3-yl)acetonitrile and 2-(3-(3',5'-dimethyl-1H,1'H-[4,4'-bipyrazol]-1-yl)azetidin-3-yl)acetonitrile dihydroiodide as a yellow solid.

[0349] Next, transfer the crude solid to a 450 L glass-lined reactor charged with dichloromethane (360 L). Charge triethylamine (14 L, 100.80 mol, 1.198 equiv.) to the reactor over 30 min, and stir the resulting mixture at 25 °C for 12 h. Filter the resulting suspension, wash once with dichloromethane and three times with IPAc, filter, and dry to obtain the desired product 2-(3-(3',5'-dimethyl-1H,1'H-[4,4'-bipyrazol]-1-yl)azetidin-3-yl)acetonitrile as a white solid (16.8 kg, 78%). 1 1H NMR (600 MHz, DMSO-d6) δ 10.16 (q, J = 7.0 Hz, 1H), 9.90 (s, 1H), 8.45 (s, 1H), 7.92 (s, 1H), 4.65–4.55 (m, 2H), 4.36–4.25 (m, 2H), 3.88 (s, 2H), 2.41 (s, 6H) ppm; C 13 1 16 1 + N6, (MW 256.31), LCMS (EI) m / e 257.2 (M

[0350] Step 3. (S)-4-(3-(Cyanomethyl)-3-(3',5'-dimethyl-1H,1'H-[4,4'-bipyrazol]-1-yl)azetidin-1-yl)-2,5-difluoro-N-(1,1,1-trifluoropropan-2-yl)benzamide hydrochloride

[0351]

[0352] Charge a 250 L glass-lined reactor with 2-(3-(3',5'-dimethyl-1H,1'H-[4,4'-bipyrazol]-1-yl)azetidin-3-yl)acetonitrile (12 kg, 46.8 mol, 1.00 equiv), (S)-2,4,5-trifluoro-N-(1,1,1-trifluoropropan-2-yl)benzamide (14.6 kg, 53.8 mol, 1.15 equiv), NaHCO3 (4.1 kg, 49.1 mol, 1.05 equiv), LiCl (4.0 kg, 93.6 mol, 2.00 equiv) and DMSO (96 L, 8V). Heat the resulting reaction mixture to 85 °C and maintain for at least 7 hours, and then cool the resulting solution to room temperature. Dilute the reaction mixture with isopropyl acetate (147 L, 12V) and then with water (120 L, 10V). Separate the aqueous layer, and wash the remaining organic layer with 1 wt% aqueous citric acid solution (88 L, 7.3V) and water (88 L, 7.3V), then concentrate to approximately 133 L (11V). Next, add isopropyl acetate (147 L, 12.25V) to the mixture while performing volume-fixed distillation. Next, charge a solution of HCl in IPA (2.5 wt%, 96 L, 8V) into the reactor, and stir the resulting solution at room temperature. After 1 hour, charge methylcyclohexane (220 L, 18.1V) into the slurry, and stir the resulting suspension at room temperature for another 4 hours. Filter the resulting suspension, and wash the wet filter cake with a mixture of methylcyclohexane and isopropyl acetate (3:1, 60 L, 5V), then with methylcyclohexane (60 L, 5V). Finally, dry the wet filter cake under vacuum at 50 °C - 60 °C to obtain the crude desired product (S)-4-(3-(cyanomethyl)-3-(3',5'-dimethyl-1H,1'H-[4,4'-bipyrazol]-1-yl)azetidin-1-yl)-2,5-difluoro-N-(1,1,1-trifluoropropan-2-yl)benzamide hydrochloride (22.4 kg, 88%).

[0353] Step 4. (S)-4-(3-(Cyanomethyl)-3-(3',5'-dimethyl-1H,1'H-[4,4'-bipyrazol]-1-yl)azetidin-1-yl)-2,5-difluoro-N-(1,1,1-trifluoropropan-2-yl)benzamide phosphate

[0354]

[0355] Charge an isopropyl acetate (286 L, 10 V) and (S)-4-(3-(cyanomethyl)-3-(3',5'-dimethyl-1H,1'H-[4,4'-bipyrazole]-1-yl)azetidin-1-yl)-2,5-difluoro-N-(1,1,1-trifluoropropan-2-yl)benzamide hydrochloride (28.6 kg) into a 450 L glass-lined reactor, and then charge KHCO3 (86 L, 10 wt% in water, 3 V). Stir the suspension until a clear solution is obtained. Next, remove the aqueous layer and wash the organic matter with water (86 L (3 V)), and then filter through charcoal into a second glass-lined reactor. Concentrate the organic matter at 50 °C under a reduced pressure of 200 mbar - 400 mbar to remove 240 L (8.4 V) of solvent. Charge isopropanol (163 L, 5.7 V) into the resulting residue at 50 °C, and then cool to room temperature. Next, charge 14.9 kg (52 wt%) of IPA / water containing 48 wt% H3PO4 into the reactor over at least 2 hours, and stir the resulting solution at room temperature for at least 1 hour. Charge methylcyclohexane (172 L, 6 V) at room temperature and stir the mixture for at least 1 hour. Filter the suspension, and wash the filter cake with 1:1 IPA / methylcyclohexane (86 L, 3 V), and then with methylcyclohexane (86 L, 3 V). Then dry the wet filter cake under vacuum at 50 °C to obtain crude (S)-4-(3-(cyanomethyl)-3-(3',5'-dimethyl-1H,1'H-[4,4'-bipyrazole]-1-yl)azetidin-1-yl)-2,5-difluoro-N-(1,1,1-trifluoropropan-2-yl)benzamide phosphate (28.0 kg (88%)).

[0356] Charge a 450 L glass-lined reactor with crude phosphate (28.0 kg) and methanol (336 L (12 V)), and heat the resulting mixture to 50 °C to obtain a clear solution. Transfer the solution to a separate reactor through a polished filter. MeOH (28 L, 1 V) is used to rinse the first reactor and then transferred to the second reactor through a polished filter. Then concentrate the filtrate to 7 V by distilling 196 L (7 V) of the solvent under a reduced pressure of 300 mbar - 400 mbar at 45 °C. Next, seed the reactor with pure (S)-4-(3-(cyanomethyl)-3-(3',5'-dimethyl-1H,1'H-[4,4'-bipyrazol]-1-yl)azetidin-1-yl)-2,5-difluoro-N-(1,1,1-trifluoropropan-2-yl)benzamide phosphate (28.0 g, 0.1 wt%) and stir the mixture at 45 °C for at least 15 minutes. Charge isopropanol (196 L, 7 V) and distill 196 L (7 V) of the solvent under a reduced pressure of 100 mbar - 200 mbar at about 45 °C. Charge isopropanol (196 L, 7 V) to the reactor and remove 196 L (7 V) of the solvent by distillation. Perform IPC to confirm that the methanol in the mixture does not exceed 5%. Next, cool the mixture to room temperature and filter the resulting suspension. Wash the filter cake twice with isopropanol (56 L, 2 V), and then dry it under reduced pressure at 50 °C to obtain (S)-4-(3-(cyanomethyl)-3-(3',5'-dimethyl-1H,1'H-[4,4'-bipyrazol]-1-yl)azetidin-1-yl)-2,5-difluoro-N-(1,1,1-trifluoropropan-2-yl)benzamide phosphate (24.1 kg (86.1%) as a white solid. 1 H NMR (500 MHz, DMSO-d6) δ 8.53–8.43 (m, 1H), 8.12 (d, J = 0.7 Hz, 1H), 7.72 (s, 1H), 7.36 (dd, J = 12.5, 6.3 Hz, 1H), 6.63 (dd, J = 11.9, 7.2 Hz, 1H), 4.85–4.72 (m, J = 7.5 Hz, 1H), 4.64 (d, J = 9.0 Hz, 2H), 4.45–4.37 (m, 2H), 3.66 (s, 2H), 2.24 (s, 6H), 1.33 (d, J = 7.1 Hz, 3H) ppm; C 23 H 25 F5N7O5P (MW 605.45; C 23 H 22 F5N7O: MW 507.47), LCMS (EI) m / e 508.2 (M + +H).

[0357] Example A. In Vitro JAK Kinase Assay

[0358] The inhibitory activity of the compounds provided herein against JAK targets was tested according to the following in vitro assay described by Park et al., Analytical Biochemistry 1999, 269, 94 - 104. The catalytic domains of human JAK1 (a.a. 837 - 1142), JAK2 (a.a. 828 - 1132), and JAK3 (a.a. 781 - 1124) with an N-terminal His-tag were expressed in insect cells using baculovirus and purified. The catalytic activity of JAK1, JAK2, or JAK3 was analyzed by measuring the phosphorylation of a biotinylated peptide. The phosphorylated peptide was detected by homogeneous time-resolved fluorescence (HTRF). The IC 50 of the compound was measured for each kinase in a 40 μL reaction that contained the enzyme, ATP, and 500 nM peptide in 50 mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg / mL (0.01%) BSA. For 1 mM IC 50 measurement, the ATP concentration in the reaction was 1 mM. The reaction was carried out at room temperature for 1 hour and then terminated with 20 μL of 45 mM EDTA, 300 nM SA-APC, and 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, MA). Binding to the europium-labeled antibody occurred for 40 minutes, and the HTRF signal was measured on a Fusion plate reader (Perkin Elmer, Boston, MA). The IC 50 of compound 1 free base at 1 mM ATP was ≤300 nM, and the JAK2 / JAK1 selectivity was >10.

[0359] Example B. Cellular Assay

[0360] Cancer cell lines that are cytokine-dependent and thus JAK / STAT signal transduction-dependent for growth can be seeded at 6000 cells / well (96-well plate format) in RPMI 1640, 10% FBS, and 1 ng / mL of the appropriate cytokine. The compounds provided herein were added to the cells in DMSO / media (final concentration 0.2% DMSO) and incubated at 37 °C, 5% CO2 for 72 hours. The effect of the compounds on cell viability was evaluated using the CellTiter-Glo luminescent cell viability assay (Promega) followed by quantification on a TopCount (Perkin Elmer, Boston, MA). The potential off-target effects of the compounds were measured in parallel using a non-JAK-driven cell line with the same assay readout. All experiments were typically performed in duplicate.

[0361] The above cell lines can also be used to examine the effects of the compounds provided herein on the phosphorylation of JAK kinases or potential downstream substrates (such as STAT proteins, Akt, Shp2, or Erk). These experiments can be conducted after cytokine starvation overnight, followed by a brief pre-incubation (2 hours or less) with the compound, and cytokine stimulation for approximately 1 hour or less. Proteins are then extracted from the cells and analyzed using techniques familiar to those skilled in the art, including Western blotting or ELISA, with antibodies that can distinguish phosphorylated proteins from total proteins. These experiments can utilize normal cells or cancer cells to study the activity of the compounds on tumor cell survival biology or inflammatory disease mediators. For example, in the latter case, cytokines such as IL-6, IL-12, IL-23, or IFN can be used to stimulate JAK activation, resulting in STAT protein phosphorylation and potentially leading to the production and / or secretion of a transcriptional profile (evaluated by array or qPCR techniques) or proteins (such as IL-17). The ability of the compound to inhibit these cytokine-mediated effects can be measured using techniques commonly used by those skilled in the art.

[0362] The compounds provided herein can also be tested in cell models designed to evaluate their potency and activity against mutant JAKs, such as the JAK2V617F mutation found in myeloproliferative disorders. These experiments typically utilize cytokine-dependent cells of the blood lineage (such as BaF / 3), in which wild-type or mutant JAK kinases are ectopically expressed (James, C. et al. Nature 434:1144-1148; Staerk, J. et al. JBC 280:41893-41899). Endpoints include the effects of the compound on cell survival, proliferation, and phosphorylation of JAK, STAT, Akt, or Erk proteins.

[0363] The activity of the compounds provided herein to inhibit T cell proliferation can be evaluated. Such an analysis can be considered a second cytokine (i.e., JAK)-driven proliferation assay as well as a simplified assay of immunosuppression or inhibition of immune activation. The following is a brief overview of how such experiments can be conducted. Peripheral blood mononuclear cells (PBMCs) are prepared from human whole blood samples using the Ficoll Hypaque separation method, and T cells (fraction 2000) can be obtained from PBMCs by elutriation. Freshly isolated human T cells can be seeded at 2×10 6Maintain at a density of cells / ml in a medium (RPMI 1640, supplemented with 10% fetal bovine serum, 100 U / ml penicillin, 100 μg / ml streptomycin) for up to 2 days. For the IL-2-stimulated cell proliferation assay, T cells were first treated with phytohemagglutinin (PHA) at a final concentration of 10 μg / mL for 72 hours. After washing once with PBS, they were seeded at 6000 cells / well in a 96-well plate and treated with different concentrations of the compounds provided herein in the medium in the presence of 100 U / mL human IL-2 (ProSpec-Tany TechnoGene; Rehovot, Israel). The plates were incubated at 37 °C for 72 hours, and the proliferation index was evaluated using the CellTiter-Glo luminescent reagent according to the protocol recommended by the manufacturer (Promega; Madison, WI).

[0364] Example C. Antitumor efficacy in vivo

[0365] The compounds provided herein can be evaluated in a human tumor xenograft model of immunocompromised mice. For example, a tumorigenic variant of the INA-6 plasmacytoma cell line can be used to subcutaneously inoculate SCID mice (Burger, R. et al. Hematol J. 2:42-53, 2001). Subsequently, the tumor-bearing animals can be randomly divided into drug or vehicle treatment groups, and different doses of the compounds provided herein can be administered by any number of common routes, including orally, i.p., or continuous infusion using an implantable pump. Tumor growth can be tracked over time using calipers. In addition, tumor samples can be harvested at any time after the start of treatment for the analysis described above (Example B) to evaluate the effect of the compound on JAK activity and downstream signaling pathways. Additionally, a xenograft tumor model driven by other known kinases, such as Bcr-Abl, e.g., the K562 tumor model, can be used to evaluate the selectivity of the compound.

[0366] Example D. Mouse skin contact delayed hypersensitivity test

[0367] The efficacy (inhibiting JAK targets) of the compounds provided herein can also be tested in a T cell-driven mouse delayed hypersensitivity test model. The mouse skin contact delayed type hypersensitivity (DTH) reaction is regarded as an effective model for clinical contact dermatitis and other T lymphocyte-mediated skin immune disorders, such as psoriasis (Immunol Today. January 1998; 19(1):37-44). Mouse DTH shares many characteristics with psoriasis, including immune infiltration, increased accompanying inflammatory cytokines, and keratinocyte hyperproliferation. In addition, many classes of drugs that are clinically effective in treating psoriasis are also effective inhibitors of the mouse DTH reaction (Agents Actions. January 1993; 38(1-2):116-21).

[0368] On days 0 and 1, Balb / c mice were sensitized by topical application of the antigen 2,4-dinitro-fluorobenzene (DNFB) to their shaved abdomens. On day 5, the thickness of the ears was measured using an engineer's micrometer. This measurement was recorded and used as a baseline. Then, both ears of the animals were challenged by topical application of a total of 20 μL of DNFB at a concentration of 0.2% (10 μL on the inner ear flap and 10 μL on the outer ear flap). Forty-eight to seventy-two hours after the challenge, the ears were measured again. Treatment with the compounds provided herein was given during the entire sensitization and challenge phases (days -1 to 7) or before the challenge phase and throughout the challenge phase (usually from the afternoon of day 4 to day 7). Treatment with the test compounds (at different concentrations) was administered either systemically or topically (the treatment was topically applied to the ears). A reduction in ear swelling compared to the untreated situation indicated the efficacy of the test compounds. Test compounds that caused a 20% or greater reduction were considered effective. In some experiments, the mice were challenged but not sensitized (negative control).

[0369] The inhibitory effect of the compounds provided herein (inhibiting the activation of the JAK-STAT pathway) can be confirmed by immunohistochemical analysis. Activation of the JAK-STAT pathway leads to the formation and translocation of functional transcription factors. In addition, the influx of immune cells and an increase in keratinocyte proliferation should also provide unique expression profile changes in the ears that can be studied and quantified. Immunohistochemical analysis was performed on formalin-fixed and paraffin-embedded ear sections (harvested after the challenge phase in the DTH model) using an antibody that specifically interacts with phosphorylated STAT3 (clone 58E12, Cell Signaling Technologies). In the DTH model, the ears of mice were treated with the compounds provided herein, a vehicle, or dexamethasone (a clinically effective treatment for psoriasis), or left untreated for comparison. The test compounds and dexamethasone both produced similar transcriptional changes qualitatively and quantitatively, and both the test compounds and dexamethasone reduced the number of infiltrating cells. Both systemic and topical administration of the test compounds produced an inhibitory effect, i.e., a reduction in the number of infiltrating cells and an inhibition of transcriptional changes.

[0370] Example E. Anti-inflammatory Activity in Vivo

[0371] The compounds provided herein can be evaluated in rodent or non-rodent models designed to replicate a single or complex inflammatory response. For example, rodent models of arthritis can be used to evaluate the therapeutic potential of compounds administered prophylactically or therapeutically. These models include, but are not limited to, murine or rat collagen-induced arthritis, rat adjuvant-induced arthritis, and collagen antibody-induced arthritis. Autoimmune diseases, including, but not limited to, multiple sclerosis, type I diabetes, uveitis, thyroiditis, myasthenia gravis, immunoglobulin nephropathy, myocarditis, airway sensitization (asthma), lupus, or colitis, can also be used to evaluate the compounds provided herein. These models have been established in the research community and are familiar to those skilled in the art (Current Protocols in Immunology, Volume 3, Coligan, J.E. et al., Wiley Press.; Methods in Molecular Biology: Volume 225, Inflammation Protocols., Winyard, P.G. and Willoughby, D.A., Humana Press, 2003.).

[0372] Example F. Animal Models for Treating Dry Eye, Uveitis, and Conjunctivitis

[0373] Agents can be evaluated in one or more preclinical models of dry eye known to those skilled in the art, including, but not limited to, the rabbit concanavalin A (ConA) lacrimal gland model, the scopolamine mouse model (subcutaneous or transdermal), the botulinum toxin mouse lacrimal gland model, or any of a number of spontaneous rodent autoimmune models that result in lacrimal gland dysfunction (e.g., NOD-SCID, MRL / lpr, or NZB / NZW) (Barabino et al., Experimental Eye Research 2004, 79, 613-621 and Schrader et al., Developmental Opthalmology, Karger 2008, 41, 298-312, each of which is incorporated herein by reference in its entirety). Endpoints in these models can include histopathology of the lacrimal gland and eye (cornea, etc.), and may include the classic Schirmer test for measuring tear production or a modified version thereof (Barabino et al.). Activity can be evaluated by administering the agent via a variety of routes of administration (e.g., systemic or topical), which can be initiated before or after the presence of a measurable disease.

[0374] Agents can be evaluated in one or more preclinical models of uveitis known to those of skill in the art. These include, but are not limited to, experimental autoimmune uveitis (EAU) and endotoxin-induced uveitis (EIU) models. EAU experiments can be conducted in rabbits, rats, or mice and may involve passive or active immunization. For example, any of a variety of retinal antigens can be used to sensitize the animals to the relevant immunogen, after which the animals can be ocularly challenged with the same antigen. The EIU model is more sensitive and involves the administration of lipopolysaccharide locally or systemically at a sub-lethal dose. Endpoints for both the EIU and EAU models can include funduscopy, histopathology, etc. These models were reviewed by Smith et al. (Immunology and Cell Biology 1998, 76, 497-512, which is incorporated herein by reference in its entirety). Activity can be evaluated by administering the agent via a variety of routes (e.g., systemically or locally), which can be initiated before or after the presence of measurable disease. Some of the models listed above may also produce scleritis / sclerokeratitis, choroiditis, cyclitis, or iritis and can thus be used to study the potential activity of compounds in the therapeutic treatment of these diseases.

[0375] Agents can also be evaluated in one or more preclinical models of conjunctivitis known to those of skill in the art. These include, but are not limited to, rodent models utilizing guinea pigs, rats, or mice. Guinea pig models include those utilizing active or passive immunization and / or immunization challenge protocols using antigens such as ovalbumin or ragweed (reviewed in Gronberg, DA et al., Allergy 2003, 58, 1101-1113, which is incorporated herein by reference in its entirety). Rat and mouse models are similar in overall design to the guinea pig models (also reviewed by Groneberg). Activity can be evaluated by administering the agent via a variety of routes (e.g., systemically or locally), which can be initiated before or after the presence of measurable disease. Endpoints for such studies can include, for example, histological, immunological, biochemical, or molecular analysis of ocular tissue (e.g., the conjunctiva).

[0376] Example G. In Vivo Protection of Bone

[0377] The compounds provided herein can be evaluated in various preclinical models of osteopenia, osteoporosis, or bone resorption known to those skilled in the art. For example, ovariectomized rodents can be used to evaluate the ability of a compound to affect signs and markers of bone remodeling and / or density (W.S.S. Jee and W. Yao, J Musculoskel. Nueron. Interact., 2001, 1(3), 193-207, which is incorporated herein by reference in its entirety). Alternatively, bone density and structure can be evaluated in control or compound-treated rodents in a treatment (e.g., glucocorticoid)-induced osteopenia model (Yao et al. Arthritis and Rheumatism, 2008, 58(6), 3485-3497; and ibid. 58(11), 1674-1686, which are both incorporated herein by reference in their entirety). Additionally, the effects of the compounds provided herein on bone resorption and bone density can be evaluated in the rodent model of arthritis described above (Example E). The endpoints of all of these models may vary, but generally include histological and radiological evaluations and appropriate biochemical markers of immunohistology and bone remodeling.

[0378] Example H. S100A9 transgenic mouse model

[0379] It has been previously shown that S100A9 transgenic mice display bone marrow accumulation of MDSCs, along with the development of progressive multilineage cytopenia and cytological dysplasia similar to MDS. In addition, early forced maturation of MDSCs by all-trans retinoic acid treatment or interruption of CD33 signaling through the immunoreceptor tyrosine-based activation motif (ITAM)-bearing adaptor protein (DAP12) rescued the hematological phenotype and alleviated the disease. This system can be used to test the effect of JAK1 inhibition on MDS-like diseases in a preclinical model. J. Clin. Invest., 123(11):4595-4611 (2013). Thus, a JAK1-selective inhibitor is administered by oral gavage. Monitor the ability of the compound to reduce the cytopenia and cytological dysplasia observed in S100A9 transgenic mice.

[0380] In light of the foregoing description, various modifications of the present disclosure, in addition to those described herein, will be apparent to those skilled in the art. These modifications are also intended to fall within the scope of the appended claims. Each reference cited in this application, including all patents, patent applications, and publications, is incorporated herein by reference in its entirety.

Claims

1. A method for preparing or a salt thereof, the method comprising reacting with to form the free base of Compound 1 or a salt thereof.

2. The method according to claim 1, wherein the reaction of Compound 1x and Compound 2x is carried out in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and an organic solvent component.

3. The method according to claim 2, wherein the organic solvent component comprises dimethylformamide (DMF).

4. The method according to any one of claims 1 to 3, wherein the reaction of Compound 1x and Compound 2x is carried out at a temperature of about 50 °C to about 60 °C, wherein the term "about" means plus or minus 10% of the value.

5. The method according to claim 4, wherein the temperature is about 60 °C.

6. The method according to any one of claims 1 to 5, wherein the salt of Compound 1 is the phosphate of Compound 1, which is formed by reacting the free base of Compound 1 with phosphoric acid to form prepared by the method of 7. The method according to claim 6, wherein the reaction of the free base of Compound 1 and phosphoric acid is carried out in the presence of a solvent component.

8. The method according to claim 7, wherein the solvent component comprises methanol, isopropanol or a mixture thereof.

9. The method according to any one of claims 6 to 8, wherein the reaction of the free base of Compound 1 and phosphoric acid is carried out at a temperature of about 40 °C to about 70 °C, wherein the term "about" means plus or minus 10% of the value.

10. The method according to claim 9, wherein the temperature is about 45 °C to about 55 °C, wherein the term "about" means plus or minus 10% of the value.

11. The method according to claim 10, wherein the temperature is about 50 °C.

12. The method according to any one of claims 6 to 11, wherein the phosphoric acid is an aqueous solution of about 85 wt% phosphoric acid.

13. The method according to any one of claims 6 to 12, wherein the reaction of the free base of Compound 1 and phosphoric acid further comprises adding a second solvent component to the reaction mixture.

14. The method according to claim 13, wherein the second solvent component comprises n-heptane.

15. The method according to any one of claims 1 to 14, the method further comprising preparing Compound 2x by a method comprising reacting the following: with a base.

16. The method according to claim 15, wherein the base is NaOH.

17. The method according to claim 15 or 16, wherein the reaction of Compound 2x HCl and the base is carried out at a temperature of about 15 °C to about 18 °C, wherein the term "about" means plus or minus 10% of the value.

18. The method according to any one of claims 15 to 17, the method further comprising preparing Compound 2x HCl by a method comprising reacting the following: with hydrochloric acid.

19. The method according to claim 18, wherein the reaction of Compound 2b and hydrochloric acid is carried out in the presence of an organic solvent component.

20. The method according to claim 19, wherein the organic solvent component comprises 2-propanol.

21. The method according to any one of claims 18 to 20, wherein the reaction of compound 2b with hydrochloric acid is carried out at a temperature of about 60 °C to about 65 °C, wherein the term "about" means plus or minus 10% of the value.

22. The method according to any one of claims 18 to 21, the method further comprising preparing compound 2b by a method comprising reacting the following: with 23. The method according to claim 22, wherein the reaction of compound 2a with 4-bromo-3,5-dimethylpyrazole is carried out in the presence of K2HPO4, a solvent component and a palladium complex.

24. The method according to claim 23, wherein the solvent component comprises 1-propanol, water or a mixture thereof.

25. The method according to claim 23, wherein the palladium complex is [1,1′-bis(di-tert-butylphosphino)ferrocene]dichloropalladium(II) (Pd-118).

26. The method according to any one of claims 22 to 25, wherein the reaction of compound 2a with 4-bromo-3,5-dimethylpyrazole is carried out at a temperature of about 80 °C to about 100 °C, wherein the term "about" means plus or minus 10% of the value.

27. The method according to claim 26, wherein the temperature is about 90 °C.

28. The method according to any one of claims 1 to 14, the method further comprising preparing The preparation is carried out by a method comprising the following: Cause to react with to form Reacting compound 2b with hydrochloric acid to form and Reacting compound 2x HCl with a base to form compound 2x.

29. The method according to any one of claims 1 to 28, the method further comprising preparing wherein compound 1x is prepared by a method comprising reacting the following in the presence of a base: with diethyl cyanomethylphosphonate.

30. The method according to claim 29, wherein the reaction of compound 1c with diethyl cyanomethylphosphonate in the presence of a base is carried out in an organic solvent component.

31. The method according to claim 30, wherein the organic solvent component comprises tetrahydrofuran, ethanol or a mixture thereof.

32. The method according to any one of claims 29 to 31, the method further comprising preparing compound 1c, wherein compound 1c is prepared by a method comprising reacting the following: with iodobenzene diacetate and 2,2,6,6-tetramethyl-1-piperidinyloxy free radical (TEMPO).

33. The method according to claim 32, the method further comprising preparing compound 1b, wherein compound 1b is prepared by a method comprising reacting the following in the presence of DBU: with 34. The method according to claim 33, the method further comprising preparing compound 1a, wherein compound 1a is prepared by a method comprising reacting the following in the presence of a base:

35. The method according to any one of claims 1 to 28, the method further comprising preparing The preparation is carried out by a method comprising the following: Cause to react with in the presence of a base to form React compound 1a with in the presence of DBU to form React compound 1b with iodobenzene diacetate and TEMPO to form and Reacting compound 1c with diethyl cyanomethylphosphonate in the presence of a base to form compound 1x.

36. A method for preparing a compound of formula A: The method includes reacting 3,5-dimethyl-1H,1'H-4,4'-bipyrazole with a compound of formula B: wherein Pg 1 is an amine protecting group.

37. The method according to claim 36, wherein Pg 1 is tert-butoxycarbonyl.

38. The method according to claim 36 or 37, wherein the reaction is carried out in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene.

39. The method according to claim 38, wherein less than 1 equivalent of 1,8-diazabicyclo[5.4.0]undec-7-ene is used based on 1 equivalent of the compound of formula B.

40. The method according to claim 38, wherein about 0.2 to about 0.3 equivalent of 1,8-diazabicyclo[5.4.0]undec-7-ene is used based on 1 equivalent of the compound of formula B, wherein the term "about" means plus or minus 10% of the value.

41. The method according to any one of claims 36 to 40, wherein about 1.0 to about 1.1 equivalents of 3,5-dimethyl-1H,1'H-4,4'-bipyrazole are used based on 1 equivalent of the compound of formula B, wherein the term "about" means plus or minus 10% of the value.

42. The method according to any one of claims 36 to 41, wherein the reaction is carried out at about room temperature.

43. The method according to any one of claims 36 to 42, wherein the reaction of 3,5-dimethyl-1H,1'H-4,4'-bipyrazole with the compound of formula B is carried out in the presence of a solvent component.

44. The method according to claim 43, wherein the solvent component comprises dimethyl sulfoxide.

45. The method according to claim 43, wherein the solvent component comprises dimethyl sulfoxide and dichloromethane.

46. The method according to any one of claims 36 to 45, the method further comprising deprotecting the compound of formula A to form a compound of formula C: or a salt thereof.

47. The method according to claim 46, wherein the deprotection comprises reacting the compound of formula A in the presence of a trialkylsilyl halide.

48. The method according to claim 47, wherein the trialkylsilyl halide is trimethylsilyl iodide.

49. The method according to any one of claims 47 to 48, wherein the deprotection is carried out in the presence of a solvent component.

50. The method according to claim 49, wherein the solvent component comprises dichloromethane.

51. The method according to claim 49, wherein the solvent component comprises dichloromethane and methanol.

52. The method according to any one of claims 47 to 51, wherein the deprotection is carried out at about room temperature.

53. The method according to any one of claims 47 to 52, the method further comprising reacting the compound of formula C or a salt thereof with an amine base to form the free base form of the compound of formula C.

54. The method according to claim 53, wherein the amine base is triethylamine.

55. The method according to claim 53 or 54, wherein the reaction of the compound of formula C or a salt thereof with the amine base is carried out in the presence of a solvent component.

56. The method according to claim 55, wherein the solvent component comprises dichloromethane.

57. The method according to any one of claims 53 to 56, further comprising reacting the free base form of the compound of formula C with compound 1a: in the presence of a base and an alkali metal halide to form compound 1: or a salt thereof.

58. The method according to claim 57, wherein the base is a bicarbonate base.

59. The method according to claim 57, wherein the base is sodium bicarbonate.

60. The method according to any one of claims 57 to 59, wherein the alkali metal halide is lithium chloride.

61. The method according to any one of claims 57 to 60, wherein the reaction is carried out at a temperature of about 80 °C to about 90 °C, where the term "about" means plus or minus 10% of the value.

62. The method according to any one of claims 57 to 61, wherein the reaction of the free base form of the compound of formula C with compound 1a is carried out in the presence of a solvent component.

63. The method according to claim 62, wherein the solvent component comprises dimethyl sulfoxide.

64. The method according to claim 62, wherein the solvent component comprises dimethyl sulfoxide and isopropyl acetate.

65. The method according to any one of claims 57 to 64, further comprising reacting compound 1 with a strong acid to form the salt form of compound 1.

66. The method according to any one of claims 57 to 64, further comprising reacting compound 1 with hydrochloric acid to form compound 1 hydrochloride:

67. The method according to claim 66, further comprising reacting the compound 1 hydrochloride with a bicarbonate base to form the free base form of compound 1.

68. The method according to claim 67, wherein the bicarbonate base is potassium bicarbonate.

69. The method according to claim 67 or 68, further comprising reacting the free base form of compound 1 with phosphoric acid to form compound 1 phosphate:

70. The method according to claim 69, wherein the reaction is carried out at about room temperature.

71. The method according to claim 69 or 70, wherein the reaction of the free base form of compound 1 with phosphoric acid is carried out in the presence of a solvent component.

72. The method according to claim 71, wherein the solvent component comprises water.

73. The method according to claim 71, wherein the solvent component comprises water and isopropyl alcohol.

74. The method according to any one of claims 69 to 73, further comprising isolating the compound 1 phosphate.

75. The method according to claim 74, wherein the compound 1 phosphate is isolated by recrystallization.

76. The method according to claim 74 or 75, wherein the compound 1 phosphate is isolated by recrystallization from a mixture of methanol, isopropyl alcohol and methylcyclohexane.

77. A method for preparing compound 1 phosphate: The method comprises: reacting 3,5-dimethyl-1H,1'H-4,4'-bipyrazole with tert-butyl 3-(cyanomethylene)azetidine-1-carboxylate in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene to form a compound of formula A-1: Deprotect the compound of formula A-1 to form a compound of formula C-1: or a salt thereof; React the compound of formula C-1 with triethylamine to form the free base form of the compound of formula C-1; React the free base form of the compound of formula C-1 with compound 1a: in the presence of sodium bicarbonate and lithium chloride to form compound 1: React compound 1 with hydrochloric acid to form compound 1 hydrochloride: React compound 1 hydrochloride with potassium bicarbonate to form the free base form of compound 1; and React the free base form of compound 1 with phosphoric acid to form the phosphate of compound 1.

78. The method according to claim 77, wherein the method further comprises isolating the phosphate of compound 1.

79. The method according to claim 78, wherein the phosphate of compound 1 is isolated by recrystallization.

80. The method according to claim 78 or 79, wherein the phosphate of compound 1 is isolated by recrystallization from a mixture of methanol, isopropanol and methylcyclohexane.

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