Preparation process of JAK1 inhibitors

Abstract

The present application provides processes 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 salts, as well as related salt forms and intermediates, which are useful as selective (Janus kinase 1) JAK1 inhibitors. TIFF2023528868000099.tif52164

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 033,618, filed June 2, 2020, the entire contents of which are incorporated herein by reference.

[0002] The present application provides processes 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 salts, as well as related salt forms and intermediates, which are useful as selective (Janus kinase 1) JAK1 inhibitors. [Background technology]

[0003] Protein kinases (PKs) regulate diverse biological processes, including cell proliferation, survival, differentiation, organogenesis, morphogenesis, angiogenesis, tissue repair, and regeneration, among others. Protein kinases also play specialized roles in the host in numerous human diseases, including cancer. Cytokines, small polypeptides or glycoproteins, regulate many pathways involved in the host inflammatory response to sepsis. Cytokines affect cell differentiation, proliferation, and activation, and can regulate both pro- and anti-inflammatory responses, enabling the host to respond appropriately to pathogens. A wide range of cytokine signaling pathways involve the Janus kinase (JAK) family of protein tyrosine kinases and signal transducers and activators of transcription (STATs). There are four known mammalian JAKs: JAK1 (Janus kinase-1), JAK2, JAK3 (also known as leukocyte Janus kinase, JAKL, and L-JAK), and TYK2 (protein tyrosine kinase 2).

[0004] Cytokine-stimulated immune and inflammatory responses contribute to disease pathogenesis in the following ways: conditions such as severe combined immunodeficiency (SCID) result from a suppressed immune system, while overactive or inappropriate immune / inflammatory responses contribute to the pathology of autoimmune diseases (e.g., 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).

[0005] Defective JAK expression is associated with many disease states. For example, Jak1- / - mice are stunted at birth, fail to lactate, and die perinatally (Rodig, SJ, MAMeraz, et al. (1998) Cell 93(3):373-83). Jak2- / - mouse embryos are anemic and die approximately 12.5 days postcoitum due to the absence of significant erythropoiesis.

[0006] The JAK / STAT pathway, particularly all four JAKs, is thought to play a role in the pathogenesis of asthma responses, chronic obstructive pulmonary disease, bronchitis, and other related lower airway inflammatory diseases. Multiple cytokines that signal through JAKs have been implicated in upper airway inflammatory diseases / pathologies, such as those affecting the nose and paranasal sinuses (e.g., rhinitis and sinusitis), both classical and non-allergic. The JAK / STAT pathway has also been implicated in ocular inflammatory diseases / pathologies and chronic allergic responses.

[0007] Activation of JAK / STAT in cancer can occur through cytokine stimulation (e.g., IL-6 or GM-CSF) or through the reduction of endogenous inhibitors of JAK signaling, such as SOCS (suppressor of cytokine signaling) or PIAS (protein inhibitors of activated STATs) (Boudny, V., and Kovarik, J., Neoplasm. 49:349-355, 2002). In addition to STAT signaling, activation of other pathways downstream of JAK (e.g., Akt) correlates 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 may play a causal role in cachexia and / or chronic fatigue. Therefore, JAK inhibition may be beneficial to cancer patients for reasons beyond its potential antitumor activity.

[0008] JAK2 tyrosine kinase may be beneficial for patients with myeloproliferative syndromes, such as polycythemia vera (PV), essential thrombocythemia (ET), and myeloid metaplasia with myelofibrosis (MMM) (Levin, et al., Cancer Cell, vol. 7, 2005: 387-397). Inhibition of JAK2V617F kinase reduces hematopoietic cell proliferation, suggesting JAK2 as a potential target for pharmacological inhibition in patients with PV, ET, and MMM.

[0009] Inhibition of JAK may benefit patients suffering from skin immune disorders and skin sensitization, such as psoriasis. The persistence of psoriasis is thought to depend on several inflammatory cytokines in addition to various chemokines and growth factors (JCI, 113:1664-1675), many of which signal through JAK (Adv Pharmacol. 2000; 47:113-74).

[0010] Thus, new or improved agents that inhibit kinases such as JAKs are continually needed to develop new, more effective pharmaceuticals aimed at enhancing or suppressing immune and inflammatory pathways (e.g., immunosuppressants for organ transplants), as well as drugs for the prevention and treatment of autoimmune diseases, diseases involving an overactive inflammatory response (e.g., eczema), allergies, cancers (e.g., prostate, leukemia, multiple myeloma), and some immune reactions caused by other therapies (e.g., rashes, contact dermatitis, diarrhea). Inhibitors of JAKs are currently under development. While JAK inhibitors and processes for their preparation exist in the literature, there remains a need for new processes for preparing these inhibitors with suitable properties useful for the manufacture of marketable, effective, high-quality pharmaceuticals. The disclosure described herein is directed to this end. Summary of the Invention

[0011] The present disclosure provides processes 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 salt forms 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 intermediate compounds related thereto. [Brief explanation of the drawings]

[0012] [Figure 1] 1 shows a representative differential scanning calorimetry (DSC) trace of Compound 1 phosphate prepared according to the process described in Example 1. [Figure 2] 1 shows a representative thermogravimetric analysis (TGA) trace of Compound 1 phosphate prepared according to the process described in Example 1. [Figure 3]FIG. 1 shows a representative X-ray powder diffraction (XRPD) trace of Compound 1 phosphate prepared according to the process described in Example 1 overlaid with an XRPD trace of Compound 1 phosphate prepared according to the process described in U.S. Pat. No. 9,382,231. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present disclosure provides a process 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 (see below), referred to herein as "Compound 1." The free base of the compound is shown below. [ka]

[0014] The present disclosure also provides a process for preparing the phosphate salt of Compound 1 free base (see below), 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]benzamidophosphate, referred to herein as “Compound 1 phosphoric acid salt,” “Compound 1 phosphate,” or “Compound 1 phosphate salt.” [ka]

[0015] Exemplary processes for preparing Compound 1 and its phosphate salts are disclosed in US 2014 / 0343030, which is incorporated herein by reference in its entirety. The processes for preparing Compound 1 free base and its phosphate salts provided herein have several advantages over the processes disclosed in US 2014 / 0343030, making them more suitable for scale-up manufacturing processes. For example, the exemplary processes described herein are convergent syntheses that provide high yields, increasing the efficiency of multi-step synthesis compared to the linear synthesis in US 2014 / 0343030. The yield of intermediate products, as shown in Scheme 2 (see below), ranges from about 93% to about 94% at scales ranging from about 670 grams to about 2000 grams. Furthermore, the yield of Compound 1 free base and its phosphate salts, as shown in Scheme 5 (see below), ranges from about 90% to about 97% at scales ranging from 430 grams to about 5800 grams. The overall yield of the process provided herein, which begins with the preparation of (S)-2,4,5-trifluoro-N-[1,1,1-trifluoropropan-2-yl]benzamide (compound 1a, Scheme 2, see below) to compound 1 free base, is about 68% to about 70% over a five-step synthesis, whereas the overall yield using the process of US2014 / 0343030, which requires six steps beginning with the preparation of (S)-2,4,5-trifluoro-N-[1,1,1-trifluoropropan-2-yl]benzamide to compound 1 free base, is less than 5%.

[0016] The process disclosed herein provides 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 Compound 1 free base resulted in low yields (less than about 10% yield, Example 7) and required removal of palladium contaminants from the product. In the exemplary process provided herein, a palladium-catalyzed Suzuki coupling step is performed in a separate, parallel synthesis to generate a 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 generate compound 1 free base (Scheme 5, see below). Compound 2x can be easily purified as a highly crystalline HCl salt. The crystallization process allows compound 2x to be more easily purified to remove palladium impurities than the complex, multi-nitrogen-containing compound 1 free base. This represents an advantage over previous processes that required low-yield column chromatographic separations. Furthermore, placing the palladium coupling step early in the synthetic process improved overall yields.

[0017] Furthermore, when the bipyrazole compound (compound 2x) was used in the Michael addition reaction with compound 1x, an unexpectedly high degree of regioselectivity was obtained. In some embodiments, the regioselectivity was approximately 20:1, favoring the desired regioisomer, compound 1 free base, over the undesired regioisomer (compound R shown below). Based on electronic effects, the two electron-donating methyl groups make the 1H-NH group of compound 2X more nucleophilic than the 1'H-NH group, and thus the compound R regioisomer was the expected product. Without being limited to a particular theory, it is believed that steric hindrance at the 1H-NH group leads to the unexpectedly high degree of regioselectivity. [ka]

[0018] In some embodiments, the present disclosure provides: [ka] or a salt thereof, the process comprising: [ka] of [ka] to form Compound 1 free base or a salt thereof.

[0019] 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).

[0020] 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.

[0021] In some embodiments, the process for preparing Compound 1 free base further comprises workup after the reaction is complete. For example, the workup can include adding water to the reaction mixture and recovering the solid Compound 1 free base by filtration, which can be washed with water.

[0022] In some embodiments, the present disclosure provides a process for preparing Compound 1 phosphate, comprising reacting Compound 1 free base, prepared by a process described herein, with phosphoric acid. In some embodiments, the salt of Compound 1 is Compound 1 phosphate, prepared by a process comprising reacting Compound 1 free base with phosphoric acid.

[0023] In some embodiments, the reaction of Compound 1 free base 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.

[0024] In some embodiments, the reaction of Compound 1 free base 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.

[0025] In some embodiments, the phosphoric acid is an aqueous solution of about 85% by weight of phosphoric acid. In some embodiments, the reaction of Compound 1 free base with phosphoric acid further comprises adding a second solvent component to the reaction mixture. For example, the second solvent component comprises n-heptane. The present disclosure also provides intermediate compounds, e.g., [ka] and [ka] The present invention includes a process for preparing the above compound.

[0026] In some embodiments, the present disclosure provides a process for preparing compound 1x, the process comprising: [ka] of, [ka] and, React in the presence of a base [ka] and forming 2a) Compound 1a, [ka] and reacted in the presence of DBU [ka] and forming 3a) Reacting compound 1b with iodobenzene diacetate and TEMPO to give [ka] and forming 4a) reacting compound 1c with diethyl cyanomethylphosphonate in the presence of a base to form compound 1x.

[0027] In operation 1a, (2S)-1,1,1-trifluoropropan-2-amine can be reacted 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 aqueous sodium hydroxide. In some embodiments, the base is aqueous sodium hydroxide. In some embodiments, the reaction is carried out in the presence of an organic solvent component (e.g., toluene). In some embodiments, the reaction is carried out at a temperature of about 0°C to about 10°C or about 0°C to about 5°C. In some embodiments, a salt of (2S)-1,1,1-trifluoropropan-2-amine (e.g., an HCl salt) can be prepared from [ka] The compound 1a is converted to its free base prior to reaction with HCl. For example, in some embodiments, a (2S)-1,1,1-trifluoropropan-2-amine salt (e.g., an HCl salt) is converted to its free base in situ. In some embodiments, procedure 1a further comprises a workup to obtain compound 1a after the reaction is deemed complete, for example, by HPLC. For example, the workup 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.

[0028] In operation 2a, compound 1a can be reacted 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, for example, acetonitrile. In some embodiments, DBU can be added in portions to the reaction mixture of compound 1a and azetidin-3-ol hydrochloride. In some embodiments, the reaction 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 58°C to about 68°C. In some embodiments, operation 2a further includes a workup to obtain compound 1b after the reaction is deemed complete, for example, by HPLC. The workup can include adding 1.0 M aqueous hydrochloric acid to a 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 with the added water. The workup can further include isolating the compound 1b solid and rinsing the solid with water.

[0029] In operation 3a, compound 1b can be reacted with iodobenzene diacetate and 2,2,6,6-tetramethyl-1-piperidinyloxy free radical (TEMPO) to form compound 1c. In some embodiments, the reaction is carried out in an organic solvent, for example, containing methylene chloride. 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 a workup to obtain compound 1c after the reaction is deemed complete, for example, by HPLC. The workup can include quenching the reaction 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 solvent can be concentrated under reduced pressure to provide compound 1c as a solid. The solid compound 1c can be reslurried in n-heptane at room temperature for about 30 minutes and washed with n-heptane.

[0030] In operation 4a, compound 1c can be reacted with diethyl cyanomethylphosphonate in the presence of a base to form compound 1x. The base can include, for example, potassium tert-butoxide. In some embodiments, the reaction is carried out in the presence of an organic solvent component, such as THF, ethanol, or a mixture thereof. In some embodiments, diethyl cyanomethylphosphonate can be added to a 1.0 M solution of potassium tert-butoxide in THF at about 5° C. to about 25° C. In some embodiments, the molar equivalent of the potassium tert-butoxide solution in THF relative to compound 1c is about 0.95. In some embodiments, the molar equivalent of the potassium tert-butoxide solution in THF relative to compound 1c is less than about 0.95 (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 the mixture containing compound 1c. In some embodiments, procedure 4a further comprises workup to obtain compound 1x after the reaction is deemed complete, for example, by HPLC. The HPLC can 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 reslurried in methyl tert-butyl ether, collected by filtration, and washed with MTBE.

[0031] In some embodiments, the process for preparing compound 1 free base or a salt thereof further comprises preparing compound 1x, which can be prepared by a process comprising reacting compound 1c with diethyl cyanomethylphosphonate in the presence of a base. In some embodiments, the process further comprises preparing compound 1c, which can be prepared by a process comprising reacting compound 1b with iodobenzene diacetate and TEMPO. In some embodiments, the process further comprises preparing compound 1b, which can be prepared by a process comprising reacting compound 1a with azetidin-3-ol hydrochloride in the presence of DBU. In some embodiments, the process further comprises preparing compound 1a, which can be prepared by a process comprising reacting (2S)-1,1,1-trifluoropropan-2-amine with 2,4,5-trifluorobenzoyl chloride in the presence of a base.

[0032] In some embodiments, the present disclosure provides a process for preparing compound 2x, the process comprising: [ka] of, [ka] React with [ka] and forming 2b) reacting compound 2b with hydrochloric acid, [ka] and forming 3b) reacting compound 2xHCl with a base to form compound 2x.

[0033] In operation 1b, compound 2a can be reacted with 4-bromo-3,5-dimethylpyrazole to form compound 2b. In some embodiments, the reaction is carried out in the presence of KHPO, 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 a workup to obtain compound 2a. The workup can 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 Celite. In some embodiments, operation 1b further comprises combining crude compound 2a with ethyl acetate and aqueous NaHSO3 and heating the resulting mixture to about 65°C to about 70°C for about 2.5 hours. The phases can be separated, and the organic phase can be combined with aqueous NaHSO3 and heated to about 65°C to about 70°C 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 an eluent. In some embodiments, purified compound 2a is further combined with methylene chloride and Si-thiol, and the resulting mixture is filtered.

[0034] In operation 2b, compound 2b can be reacted with hydrochloric acid to form compound 2xHCl. In some embodiments, the reaction is carried out in the presence of an organic solvent component. For example, the organic solvent component includes 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 workup to obtain compound 2b after the reaction is deemed complete, for example, by HPLC. For example, the reaction mixture is cooled to room temperature and stirred for about 1 hour. The solid compound 2b can be collected by filtration and washed with 2-propanol.

[0035] In operation 3b, compound 2xHCl can be reacted with a base to form compound 2x. The present disclosure also relates to a process for preparing compound 2x, comprising reacting compound 2xHCl with a base. Exemplary bases include KOH, LiOH, K2CO3, Na2CO3, and other bases capable of neutralizing compound 2xHCl to its free base. In some embodiments, the base is NaOH. In some embodiments, the reaction of compound 2xHCl with a 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 workup to obtain compound 2x after the reaction is complete. For example, the solid of compound 2x can be collected by filtration and washed with water and n-heptane.

[0036] In some embodiments, the process for preparing compound 1 free base or a salt thereof further comprises preparing compound 2x, which can be prepared by a process comprising reacting compound 2xHCl with a base. In some embodiments, the process further comprises preparing compound 2xHCl, which is prepared by a process comprising reacting compound 2b with hydrochloric acid. In some embodiments, the process further comprises preparing compound 2b, which is prepared by a process comprising reacting compound 2a with 4-bromo-3,5-dimethylpyrazole.

[0037] In some embodiments, the present application further provides a process for preparing a compound of formula A: [ka]

[0038] In some embodiments, the process for preparing a compound of formula A includes reacting 3,5-dimethyl-1H,1′H-4,4′-bipyrazole with a compound of formula B: [ka] In the formula, Pg 1 is an amino protecting group. In some embodiments, Pg 1 is tert-butoxycarbonyl.

[0039] In some embodiments, the reaction of 3,5-dimethyl-1H,1'H-4,4'-bipyrazole with a compound of formula B is carried out in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] In some embodiments, the reaction of 3,5-dimethyl-1H,1'H-4,4'-bipyrazole with a compound of Formula B is carried out at about room temperature.

[0047] In some embodiments, the reaction of 3,5-dimethyl-1H,1'H-4,4'-bipyrazole with a 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 methylene chloride.

[0048] In some embodiments, the processes provided herein involve deprotecting a compound of formula A to form a compound of formula C: [ka] or a salt thereof.

[0049] In some embodiments, deprotecting the compound of formula A comprises reacting the compound of formula A in the presence of a strong acid (eg, hydrochloric acid).

[0050] In some embodiments, deprotecting the compound of formula A comprises reacting the compound of formula A in the presence of a trialkylsilyl halide.

[0051] In some embodiments, the trialkylsilyl halide is trimethylsilyl iodide.

[0052] 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 comprises methylene chloride. In some embodiments, the solvent comprises methylene chloride and methanol.

[0053] In some embodiments, the deprotection of the compound of formula A is carried out at about room temperature.

[0054] In some embodiments, the processes provided herein further include reacting the compound of formula C, or a salt thereof, with a base to form the free base form of the compound of formula C.

[0055] In some embodiments, the processes provided herein further include 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.

[0056] In some embodiments, the base is tri(C 1-6 alkyl)amine.

[0057] In some embodiments, the base is triethylamine.

[0058] 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 methylene chloride.

[0059] In some embodiments, the processes provided herein involve converting the free base form of a compound of formula C to compound 1a: [ka] to give compound 1: [ka] , or a salt thereof.

[0060] In some embodiments, the free base form of the compound of formula C is reacted with compound 1a in the presence of a base and an alkali metal halide to produce compound 1: [ka] , or a salt thereof.

[0061] In some embodiments, the base is a bicarbonate base.

[0062] In some embodiments, the base is sodium bicarbonate.

[0063] In some embodiments, the alkali metal halide is lithium chloride.

[0064] In some embodiments, the reaction of the compound of Formula C with the free base form of compound 1a is carried out at a temperature of about 80°C to about 90°C.

[0065] In some embodiments, 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. In some embodiments, the solvent component comprises dimethyl sulfoxide. In some embodiments, the solvent component comprises dimethyl sulfoxide and isopropyl acetate.

[0066] In some embodiments, the processes provided herein further include reacting Compound 1 with a strong acid to form a salt form of Compound 1.

[0067] In some embodiments, the processes provided herein further include reacting Compound 1 with hydrochloric acid to form Compound 1 hydrochloride: [ka]

[0068] In some embodiments, more than 1 equivalent of hydrochloric acid based on 1 equivalent of compound 1 is used.

[0069] In some embodiments, the reaction of compound 1 with hydrochloric acid is carried out at about room temperature.

[0070] In some embodiments, the hydrochloric acid is an alcoholic hydrochloric acid solution.

[0071] In some embodiments, the hydrochloric acid is a solution of hydrochloric acid in isopropanol.

[0072] In some embodiments, the processes provided herein further include reacting Compound 1 hydrochloride with a base to form Compound 1 free base form: [ka]

[0073] In some embodiments, the processes provided herein further include reacting Compound 1 hydrochloride with a bicarbonate base to form Compound 1 free base form: [ka]

[0074] In some embodiments, the base is potassium bicarbonate.

[0075] In some embodiments, the potassium bicarbonate is an aqueous potassium bicarbonate solution.

[0076] In some embodiments, the processes provided herein further include reacting Compound 1 free base form with phosphoric acid to form Compound 1 phosphate: [ka]

[0077] 70. The process of claim 69, wherein the reaction of Compound 1 free base form with phosphoric acid is carried out at about room temperature.

[0078] In some embodiments, the reaction of Compound 1 free base form 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 isopropyl alcohol.

[0079] In some embodiments, the processes provided herein further include isolating compound monophosphate.

[0080] In some embodiments, compound monophosphate is isolated by recrystallization.

[0081] In some embodiments, compound monophosphate is isolated by recrystallization from a solvent component comprising methanol.

[0082] In some embodiments, compound monophosphate is isolated by recrystallization from a solvent component comprising isopropanol.

[0083] In some embodiments, compound monophosphate is isolated by recrystallization from a solvent component comprising methylcyclohexane.

[0084] In some embodiments, compound monophosphate is isolated by recrystallization from a solvent component comprising one or more of methanol, isopropanol, and methylcyclohexane.

[0085] In some embodiments, compound monophosphate is isolated by recrystallization from a solvent mixture comprising methanol, isopropanol, and methylcyclohexane.

[0086] In some embodiments, compound monophosphate is isolated by recrystallization from a solvent component comprising methanol, isopropanol, and methylcyclohexane, followed by recrystallization from a solvent component comprising methanol and isopropanol.

[0087] In some embodiments, the present application provides compound monophosphate: [ka] The present invention further provides a process for preparing 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; [ka] , Deprotecting the compound of formula A-1 to give a compound of formula C-1, [ka] or a salt thereof; and reacting the compound of formula C-1 with triethylamine to form the free base form of the compound of formula C-1; The free base form of the compound of formula C-1 is prepared as compound 1a: [ka] in the presence of sodium bicarbonate and lithium chloride to give Compound 1: [ka] and forming Compound 1 is reacted with hydrochloric acid to give Compound 1 hydrochloride: [ka] and forming reacting Compound 1 hydrochloride with potassium bicarbonate to form Compound 1 free base form; reacting Compound 1 free base form with phosphoric acid to form Compound 1 phosphate.

[0088] 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 2xHCl), 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-dioxaborolan-2-yl)-1H-pyrazole (compound 2a), or a salt of any of the foregoing.

[0089] 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.

[0090] 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.

[0091] In some embodiments, the term "about" refers to ±10% of a value.

[0092] As used herein, the term "reacting" is used as known in the art and generally refers to bringing together chemical reagents in a manner that allows their interaction at a molecular level to achieve a chemical or physical change. In some embodiments, a reaction involves at least two reagents. In some embodiments, a reaction step or operation of a synthetic process 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 processes described herein may be carried out for a time and under conditions suitable for the preparation of a specified product. The terms "combining" and "mixing" with respect to reagents of a chemical reaction are used interchangeably with the term "reacting" herein. The term "coupling" may also be considered interchangeable with "reacting," but may be used in conjunction with reaction steps and operations involving the joining of two organic fragments.

[0093] The processes described herein can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 1H or 13The purity of a compound can be monitored by spectroscopy, infrared spectroscopy, spectrophotometry (e.g., UV-visible light), or mass spectrometry; or chromatography, for example, high-performance liquid chromatography (HPLC) or thin-layer chromatography. The compound obtained by the reaction can be purified by any suitable method known in the art. For example, chromatography (medium pressure) on a suitable adsorbent (e.g., silica gel, alumina, etc.), HPLC, or preparative thin-layer chromatography; distillation; sublimation; titration; or recrystallization. The purity of a compound is generally determined by physical methods, such as measuring the melting point (if solid), obtaining an NMR spectrum, or performing HPLC separation. A compound can be said to be purified if the melting point is reduced, if undesired signals in the NMR spectrum are reduced, or if extraneous peaks in the HPLC trace are eliminated. In some embodiments, the compound is substantially purified.

[0094] Preparation of compounds can involve the protection and deprotection of various chemical groups. The chemistry of protecting groups is described, for example, in Wuts and Greene, Greene's Protective Groups in Organic Synthesis, 4 th Ed., John Wiley & Sons: New York, 2006, which is incorporated herein by reference in its entirety.

[0095] The reactions of the processes described herein can be carried out at any suitable temperature, which can be readily determined by one skilled in the art. The reaction temperature will depend, for example, on the melting and boiling points of the reagents and solvent (if present), the thermodynamics of the reaction (e.g., a highly exothermic reaction may need to be carried out at a reduced temperature), and the kinetics of the reaction (e.g., a high activation energy barrier may require an elevated temperature). "Elevated temperature" refers to a temperature greater than room temperature (about 22°C).

[0096] The reactions of the processes described herein can be carried out in a suitable solvent that can be easily selected by one skilled in the art of organic synthesis. A suitable solvent can be substantially non-reactive with the starting materials (reactants), intermediates, or products at the temperature at which the reaction is carried out, i.e., a temperature that can range from the freezing temperature of the solvent to the boiling temperature of the solvent. A given reaction can be carried out in one solvent or a mixture of two or more solvents. Depending on the reaction step or operation, a suitable solvent(s) for that particular reaction step and 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 (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 either their wet or anhydrous form.

[0097] The resolution of a racemic mixture of compounds can be carried out by many 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., dinitrobenzoylphenylglycine). The appropriate elution solvent composition can be determined by one skilled in the art.

[0098] method The compounds provided herein (e.g., Compound 1 free base and Compound 1 phosphate) are JAK inhibitors, more specifically, selective JAK1 inhibitors. A JAK1 selective inhibitor is a compound that preferentially inhibits JAK1 activity over other Janus kinases. For example, the compounds described herein preferentially inhibit JAK1 over one or more of JAK2, JAK3, and TYK2. In some embodiments, the compounds preferentially inhibit JAK1 over JAK2 (e.g., JAK2 / JAK1IC). 50 In some embodiments, the compound is about 10-fold selective for JAK1 over JAK2. In some embodiments, the compound has an IC50 at 1 mM AP. 50 The antibody is about 3-fold, about 5-fold, about 10-fold, about 15-fold, or about 20-fold selective for JAK1 over JAK2, as calculated by measuring the β-glucan bond (β-glucan bond) (see, eg, Example A).

[0099] JAK1 plays a central role in many cytokine and growth factor signaling pathways, which, when dysregulated, can lead to or contribute to disease states. For example, IL-6 levels are elevated in rheumatoid arthritis, and it has been suggested that IL-6 has a detrimental effect in this disease (Fonesca, JE et al., Autoimmunity Reviews, 8:538-42, 2009). Because IL-6 signals at least in part through JAK1, antagonizing IL-6 directly or indirectly through JAK1 inhibition is expected to provide clinical benefit (Guschin, D., N., et al., Embo J14:1421, 1995; Smolen, JS, et al., Lancet 371:987, 2008). Furthermore, in some cancers, JAK1 is mutated, resulting in the constitutive proliferation and survival of unwanted tumor cells (Mullighan CG, ProcNatl Acad Sci USA. 106:9414-8, 2009; Flex E., et al. J Exp Med. 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. Therefore, patients with such diseases may benefit from inhibition of JAK1. A selective inhibitor of JAK1 could be beneficial while avoiding the unnecessary and potentially undesirable effects of inhibiting other JAK kinases.

[0100] Substances that selectively inhibit JAK1 over other JAK kinases may have several therapeutic advantages over less selective inhibitors. Regarding selectivity for JAK2, several important cytokines and growth factors, including erythropoietin (Epo) and thrombopoietin (Tpo), signal through JAK2 (Parganas E, et al. Cell. 93:385-95, 1998). Epo is a key growth factor for erythropoiesis; therefore, deficiency of Epo-dependent signaling can lead to reduced red blood cell counts and anemia (Kaushansky K, NEJM 354:2034-45, 2006). Tpo, another example of a JAK2-dependent growth factor, plays a central role in regulating the proliferation and maturation of megakaryocytes, the cells from which platelets are produced (Kaushansky K, NEJM 354:2034-45, 2006). Therefore, reducing Tpo signaling would result in a decrease in the number of megakaryocytes (megakaryocytopenia) and a decrease in the number of circulating platelets (thrombocytopenia). This can lead to undesirable and / or uncontrollable bleeding. Reducing the inhibition of other JAKs (such as JAK3 and Tyk2) may also be desirable, since humans lacking functional versions of these kinases have been shown to suffer from a number of diseases, including 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). Therefore, JAK1 inhibitors with reduced affinity for other JAKs would have significant advantages over less selective inhibitors in terms of reducing side effects related to immunosuppression, anemia, and thrombocytopenia.

[0101] Another aspect of the present disclosure relates to a method for treating a JAK-associated disease or disorder in an individual (e.g., a patient) by administering to the individual in need thereof a therapeutically effective amount or dose of one or more compounds of the present disclosure or a pharmaceutical composition thereof. A JAK-associated disease can include any disease, disorder, or condition that is directly or indirectly associated with JAK expression or activity, including overexpression and / or abnormal activity levels. A JAK-associated disease can also include any disease, disorder, or condition that can be prevented, ameliorated, or cured by modulating JAK activity.

[0102] Examples of JAK-associated diseases include diseases involving the immune system, including, for example, transplant organ rejection (eg, allograft rejection and graft-versus-host disease).

[0103] Further examples of JAK-associated 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 disease, chronic obstructive pulmonary disease (COPD), and the like. In some embodiments, the autoimmune disease is an autoimmune bullous skin disorder (such as pemphigus vulgaris (PV) or bullous pemphigoid (BP)).

[0104] Further examples of JAK-associated diseases include allergic conditions such as asthma, food allergies, eczematous dermatitis, contact dermatitis, atopic dermatitis (atopic eczema), and rhinitis. Further examples of JAK-associated 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).

[0105] Further examples of JAK-associated diseases include diseases associated with cartilage turnover, such as gouty arthritis, septic or infectious arthritis, reactive arthritis, reflex sympathetic dystrophy, painful dystrophy, Tietze's syndrome, costoarthropathy, endemic osteoarthritis, Mseleni's disease, Handigodu's disease, degeneration resulting from fibromyalgia, systemic lupus erythematosus, scleroderma, or ankylosing spondylitis.

[0106] Further examples of JAK-associated disorders include congenital chondrodysplasia, including hereditary chondrolysis, achondroplasia, and pseudoachondroplasia (e.g., microtia, anotia, and metaphyseal achondroplasia).

[0107] Further examples of JAK-related diseases or conditions include skin disorders such as psoriasis (e.g., plaque psoriasis), atopic dermatitis, skin rash, skin irritation, and skin sensitization (e.g., contact dermatitis or allergic contact dermatitis). For example, certain substances, including some pharmaceuticals, can cause skin sensitization when applied topically. In some embodiments, co-administration or sequential administration of at least one JAK inhibitor of the present disclosure together with an agent that causes unwanted sensitization can be useful in treating such unwanted sensitization or dermatitis. In some embodiments, the skin disorder is treated by topical administration of at least one JAK inhibitor of the present disclosure.

[0108] In further embodiments, the JAK-associated 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.), hematological 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.

[0109] In some embodiments, the JAK inhibitors described herein, or in combination with other JAK inhibitors such as those reported in U.S. Publication No. 20070135461, the entire contents of which are incorporated herein by reference, can be used to treat inflammation-associated 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-associated cancer is colitis-associated cancer. In some embodiments, the inflammation-associated 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.

[0110] JAK-associated diseases may further include those characterized by the expression of JAK2 mutations, such as those with at least one mutation in the pseudokinase domain (e.g., JAK2V617F), the expression of JAK2 mutations with at least one mutation outside the pseudokinase domain, the expression of JAK1 mutations, the expression of JAK3 mutations, the expression of erythropoietin receptor (EPOR) mutations, or the deregulated expression of CRLF2.

[0111] JAK-associated diseases may further include myeloproliferative syndromes (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), and the like. In some embodiments, the myeloproliferative disorder is myelofibrosis (e.g., primary myelofibrosis (PMF) or post-polycythemia vera / post-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).

[0112] In some embodiments, the JAK inhibitors described herein can further be used to treat myelodysplastic syndrome (MDS) in a patient in need thereof, in some embodiments, the patient is red blood cell transfusion dependent.

[0113] As used herein, myelodysplastic syndrome is intended to encompass heterogeneous and clonal hematopoietic disorders characterized by ineffective hematopoiesis of one or more major myeloid cell lineages. Myelodysplastic syndrome is associated with bone marrow failure, peripheral blood cytopenia, and a tendency to progress to acute myeloid leukemia (AML). Furthermore, clonal cytogenetic abnormalities can be detected in approximately 50% of MDS cases. In 1997, the World Health Organization (WHO), in collaboration with the Society of Hematology (SH) and the European Association of Hematology and Pathology (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). Regarding MDS, the WHO has defined subsets of MDS incorporating not only the morphological criteria of the French-American-British (FAB) classification but also available genetic, biological, and clinical features (Bennett, et al., Br J Haematol 1982;51:189-199). In 2008, the WHO classification of MDS (Table 1) was further refined to incorporate new clinical and scientific information, allowing for accurate and prognostically relevant subclassification of unilineage dysplasias (Vardiman, et al., Blood 2009;114:937-951; Swerdlow, et al., WHO Classification of Tumors of Haematopoietic and Lymphoid Tissues. 4th Edition. Lyon, France: IARC Press;2008:88-103; Bunning and Germing, "Myelodysplastic syndromes / neoplasms" in Chapter 5, Swerdlow, et al., eds. WHO Classification of Tumors of Haematopoietic and Lymphoid Tissues. (ed. 4th Edition): Lyon, France: IARC Press;2008:88-103). [Table 1]

[0114] In some embodiments, the myelodysplastic syndrome is refractory cytopenia with single lineage dysplasia (RCUD).

[0115] In some embodiments, the myelodysplastic syndrome is refractory anemia with ringed sideroblasts (RARS).

[0116] In some embodiments, the myelodysplastic syndrome is refractory cytopenia with multilineage dysplasia.

[0117] In some embodiments, the myelodysplastic syndrome is refractory anemia with excess blasts-1 (RAEB-1).

[0118] In some embodiments, the myelodysplastic syndrome is refractory anemia with excess blasts-2 (RAEB-2).

[0119] In some embodiments, the myelodysplastic syndrome is myelodysplastic syndrome, unclassifiable (MDS-U).

[0120] In some embodiments, the myelodysplastic syndrome is myelodysplastic syndrome associated with isolated del(5q) chromosomal abnormality.

[0121] In some embodiments, the myelodysplastic syndrome is refractory to an erythropoiesis-stimulating agent.

[0122] The present disclosure further provides methods of treating psoriasis or other skin disorders by administering a topical formulation containing a compound provided herein.

[0123] In some embodiments, the JAK inhibitors described herein can be used to treat pulmonary arterial hypertension.

[0124] The present disclosure further provides methods for treating dermatological side effects of other pharmaceuticals by administering the compounds provided herein. For example, many pharmaceutical agents cause unwanted allergic reactions that can manifest as acneiform skin eruptions or related dermatitis. Exemplary pharmaceutical agents with such unwanted side effects include anticancer drugs such as gefitinib, cetuximab, erlotinib, and the like. The compounds provided herein can be administered systemically or locally (e.g., localized near dermatitis) in combination (e.g., simultaneously or sequentially) with pharmaceutical agents that have unwanted dermatological side effects. In some embodiments, the compounds provided herein can be administered topically with one or more other pharmaceutical agents where the other pharmaceutical agents, when applied topically in the absence of the compounds provided herein, cause contact dermatitis, allergic contact sensitization, or similar skin disorders. Thus, compositions of the present disclosure include topical formulations containing the compounds provided herein and additional pharmaceutical agents that may cause dermatitis, skin disorders, or related side effects.

[0125] Further JAK-associated diseases include inflammation and inflammatory diseases. Exemplary inflammatory diseases include sarcoidosis, inflammatory diseases of the eye (e.g., iritis, uveitis, scleritis, conjunctivitis, or related diseases), inflammatory diseases of the respiratory tract (e.g., upper respiratory tract including the nose and sinuses, such as rhinitis or sinusitis, or lower respiratory tract including bronchitis, chronic obstructive pulmonary disease, and the like), inflammatory myopathies such as myocarditis, and other inflammatory diseases. In some embodiments, the inflammatory disease of the eye is blepharitis.

[0126] The JAK inhibitors described herein can also be used to treat diseases or conditions associated with ischemia-reperfusion injury or inflammatory ischemic events, such as 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 contributing to chronic heart failure). The JAK inhibitors described herein can also be used to treat anorexia, cachexia, or fatigue, such as those 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(Pt2):427-36 and Sriram, K. et al. J. Biol. Chem. 2004, 279(19):19936-47. Epub 2004 Mar 2, both of which are incorporated by reference in their entireties. The JAK inhibitors described herein can be used to treat Alzheimer's disease.

[0127] The JAK inhibitors described herein can further be used to treat other inflammatory diseases such as systemic inflammatory response syndrome (SIRS) and septic shock.

[0128] The JAK inhibitors described herein can further be used to treat gout and increased prostate size due to, for example, benign prostatic hyperplasia or benign prostatic hyperplasia.

[0129] Further JAK-associated diseases include bone resorption diseases such as osteoporosis and osteoarthritis. Bone resorption may be associated with other conditions, such as hormonal imbalance and / or hormone therapy, autoimmune diseases (e.g., skeletal sarcoidosis), or cancer (e.g., myeloma). Reduction of bone resorption by JAK inhibitors can be about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%.

[0130] In some embodiments, the JAK inhibitors described herein can further be used to treat dry eye disorders. As used herein, "dry eye disorder" is intended to encompass the disease state summarized in a recent official report from the Dry Eye Workshop (DEWS), which defined dry eye as "a multifactorial disorder of the tears and ocular surface resulting in symptoms of discomfort, visual disturbance, and tear film instability with potential damage to the ocular surface. It is accompanied by increased tear film osmolality and 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, the entire contents of which are incorporated herein by reference). In some embodiments, the dry eye disorder is selected from aqueous tear-deficient dry eye (ADDE) or evaporative dry eye disorder, or an appropriate combination thereof. In some embodiments, the dry eye disorder is Sjögren's syndrome dry eye (SSDE). In some embodiments, the dry eye disorder is non-Sjogren's syndrome dry eye (NSSDE).

[0131] In further aspects, the present disclosure provides methods for treating conjunctivitis, uveitis (including chronic uveitis), choroiditis, retinitis, cardiitis, scleroveciitis, episcleritis, or iritis; treating inflammation or pain associated with corneal transplantation, LASIK (laser in situ keratomileusis), photorefractive keratectomy, or LASEK (laser epithelial cell keratomileusis); or inhibiting visual loss associated with corneal transplantation, LASIK, photorefractive keratectomy; or inhibiting graft rejection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound provided herein, or a pharmaceutically acceptable salt thereof.

[0132] Additionally, the compounds provided herein, or in combination with other JAK inhibitors, such as those reported in US Ser. 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.

[0133] In some embodiments, the present disclosure provides a compound 1 free base and compound 1 phosphate salt described in any of the embodiments herein for use in a method of treating any of the diseases or disorders described herein. In some embodiments, the present disclosure provides a compound 1 free base and compound 1 phosphate salt described in any of the embodiments herein for the preparation of a medicament for use in a method of treating any of the diseases or disorders described herein.

[0134] In some embodiments, the present disclosure provides compound 1 free base and compound 1 phosphate, or pharmaceutically acceptable salts thereof, as described herein, for use in methods of modulating JAK1. In some embodiments, the present disclosure provides use of compound 1 free base and compound 1 phosphate, as described in any of the embodiments herein, for the preparation of a medicament for use in methods of modulating JAK1.

[0135] As used herein, the term "contacting" refers to bringing the indicated moieties together in an in vitro system or in vivo system. For example, "contacting" a JAK with a compound described herein includes administering a compound of the present disclosure to an individual or patient, such as a human, having a JAK, as well as introducing a compound provided herein into a sample, including, for example, a cell preparation or purified preparation containing a JAK.

[0136] As used herein, the terms "individual" or "patient," used interchangeably, refer to any animal, including a mammal, preferably a mouse, rat, other rodent, rabbit, dog, cat, pig, cow, sheep, horse, or primate, most preferably a human.

[0137] As used herein, the phrase "therapeutically effective amount" refers to an amount of an active compound or pharmaceutical agent that elicits the biological or medicinal response that a researcher, veterinarian, physician, or other clinician is seeking in a tissue, system, animal, individual, or human. 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.

[0138] As used herein, the term "treating" or "treatment" refers to one or more of: (1) inhibiting a disease, e.g., inhibiting a disease, condition, or disorder (i.e., halting further progression of the symptoms and / or symptomology) in an individual experiencing or exhibiting the symptoms or symptomology of the disease, condition, or disorder; and (2) ameliorating a disease, e.g., improving a disease, condition, or disorder (i.e., reversing the symptoms and / or symptomology) in an individual experiencing or exhibiting the symptoms or symptomology of the disease, condition, or disorder, such as reducing the severity of the disease.

[0139] As used herein, the terms "prevent" or "prevention" refer to preventing a disease, condition or disorder, for example, in an individual who may be predisposed to the disease, condition or disorder but who has not yet experienced or exhibited the pathology or symptomology of the disease.

[0140] Combination therapy The methods described herein can further comprise administering one or more additional therapeutic agents. The one or more additional therapeutic agents can be administered to the patient simultaneously or sequentially.

[0141] 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 biological response modifier (BRM).

[0142] Generally, BRMs are substances derived from living organisms to treat disease, and may occur naturally in the body or be created in a 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, and trastuzumab, and high-dose ascorbate.

[0143] In some embodiments, the anti-TNF-α agent is infliximab or etanercept.

[0144] In some embodiments, the hypomethylating agent is a DNA methyltransferase inhibitor. In some embodiments, the DNA methyltransferase inhibitor is selected from 5-azacytidine and decitabine.

[0145] Generally, the IMiD is an immunomodulatory agent, hi some embodiments, the IMiD is selected from thalidomide, lenalidomide, pomalidomide, CC-11006, and CC-10015.

[0146] In some embodiments, the method further comprises administering an additional therapeutic agent selected from antithymocyte globulin, recombinant human granulocyte colony-stimulating factor (GCSF), granulocyte-monocyte CSF (GM-CSF), an erythropoiesis-stimulating agent (ESA), and cyclosporine.

[0147] In some embodiments, the method further comprises administering to the patient an additional JAK inhibitor, hi some embodiments, the additional JAK inhibitor is tofacitinib or ruxolitinib.

[0148] For example, one or more additional pharmaceutical 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 used in combination with the compounds described herein for the treatment of a JAK-associated disease, disorder, or condition. The one or more additional pharmaceutical agents can be administered to the patient simultaneously or sequentially.

[0149] Examples of chemotherapeutic agents include proteosome inhibitors (eg, bortezomib), thalidomide, revlimid, and DNA damaging agents such as melphalan, doxorubicin, cyclophosphamide, vincristine, etoposide, carmustine, and the like.

[0150] Exemplary steroids include corticosteroids such as dexamethasone or prednisone.

[0151] Examples of Bcr-Abl inhibitors are provided in US Pat. No. 5,521,184, WO 04 / 005281, and US Ser. No. 60 / 578,491, all of which are incorporated herein by reference in their entireties.

[0152] Exemplary suitable Flt-3 inhibitors include compounds, and pharmaceutically acceptable salts thereof, as disclosed in WO03 / 037347, WO03 / 099771, and WO04 / 046120, all of which are incorporated herein by reference in their entireties.

[0153] Exemplary suitable RAF inhibitors include compounds such as those disclosed in WO00 / 09495 and WO05 / 028444, and pharmaceutically acceptable salts thereof, all of which are incorporated herein by reference in their entireties.

[0154] Examples of suitable FAK inhibitors include compounds and pharmaceutically acceptable salts thereof, such as those disclosed in WO04 / 080980, WO04 / 056786, WO03 / 024967, WO01 / 064655, WO00 / 053595, and WO01 / 014402, all of which are incorporated herein by reference in their entirety.

[0155] In some embodiments, the compounds provided herein (e.g., Compound 1 free base and Compound 1 phosphate) may be used in combination with one or more other kinase inhibitors, including imatinib, particularly to treat patients who are resistant to imatinib or other kinase inhibitors.

[0156] In some embodiments, suitable chemotherapeutic agents may be selected from antimetabolites, topoisomerase 1 inhibitors, platinum analogs, taxanes, anthracyclines, and EGFR inhibitors, and combinations thereof.

[0157] In some embodiments, antimetabolites include capecitabine, gemcitabine, and fluorouracil (5-FU).

[0158] In some embodiments, taxanes include paclitaxel, Abraxane® (paclitaxel protein-bound particles for injectable suspension), and Taxotere® (docetaxel).

[0159] In some embodiments, platinum analogs include oxaliplatin, cisplatin, and carboplatin.

[0160] In some embodiments, topoisomerase 1 inhibitors include irinotecan and topotecan.

[0161] In some embodiments, the anthracycline includes doxorubicin or a liposomal formulation of doxorubicin.

[0162] In some embodiments, the chemotherapy agent is FOLFIRINOX (5-FU, Recovorin, Irinotecan, and Oxaliplatin). In some embodiments, the chemotherapy agent is Gemcitabine and Abraxane® (Paclitaxel Protein-Bound Particles for Injectable Suspension).

[0163] In some embodiments, the compounds provided herein (e.g., Compound 1 free base and Compound 1 phosphate) can be used in combination with chemotherapeutic agents in the treatment of cancer (such as multiple myeloma) and may improve the therapeutic response compared to the response to the chemotherapeutic agent alone without exacerbating its toxic effects. Examples of additional pharmaceutical agents used in the treatment of multiple myeloma can include, but are not limited to, melphalan, melphalan and prednisone [MP], doxorubicin, dexamethasone, and Velcade (bortezomib). Further additional agents used in the treatment of multiple myeloma include Bcr-Abl, Flt-3, RAF, and FAK kinase inhibitors. Additive or synergistic effects are desirable results when a JAK inhibitor of the present disclosure is combined with an additional agent. Furthermore, resistance of multiple myeloma cells to agents such as dexamethasone can be reversible upon treatment with a JAK inhibitor of the present disclosure. The agents can be combined with the compounds provided herein in a single or sequential dosage form, or the agents can be administered simultaneously or sequentially as separate dosage forms.

[0164] In some embodiments, a corticosteroid such as dexamethasone is administered to a patient in combination with at least one JAK inhibitor, and the dexamethasone is administered intermittently rather than continuously.

[0165] In some further embodiments, combinations of compounds provided herein with other therapeutic agents can be administered to patients before, during, and / or after bone marrow or stem cell transplantation.

[0166] In some embodiments, the additional therapeutic agent is fluocinolone acetonide (Retisert®) or rimexolone (AL-2178, Vexol, Alcon).

[0167] In some embodiments, the additional therapeutic agent is cyclosporine (Restasis®).

[0168] In some embodiments, the additional therapeutic agent is a corticosteroid, hi some embodiments, the corticosteroid is triamcinolone, dexamethasone, fluocinolone, cortisone, prednisone, prednisolone, or flumetholone.

[0169] In some embodiments, the additional therapeutic agent is Dehydrex™ (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), sevillemine, doxycycline (ALTY-0501, Alacrity), minocycline, iDestrin™ (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-carbamyl, 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 and Temple University), pilocarpine, tacrolimus, pimecrolimus (AMS981, Novartis), loteprednol etabonate, rituximab, diquafosol tetrasodium (INS365, Inspire), KLS-0611 (Kissei Pharmaceuticals), dehydroepiandrosterone, anakinra, efalizumab, mycophenolate sodium, etanercept (Embrel®), hydroxychloroquine, NGX267 (TorreyPines Therapeutics), Actemra, gemcitabine, oxaliplatin, L-asparaginase, or thalidomide.

[0170] In some embodiments, the additional therapeutic agent is an antiangiogenic agent, a cholinergic agonist, a TRP-1 receptor modulator, a calcium channel blocker, a mucin secretagogue, a MUC1 stimulator, a calcineurin inhibitor, a corticosteroid, a P2Y2 receptor agonist, a muscarinic receptor agonist, an mTOR inhibitor, another JAK inhibitor, a Bcr-Abl kinase inhibitor, an Flt-3 kinase inhibitor, a RAF kinase inhibitor, and a FAK kinase inhibitor, such as those described in WO 2006 / 056399, the entire contents of which are incorporated herein by reference. 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.

[0171] In some embodiments, the additional therapeutic agent is an alkylating or DNA cross-linking agent; antimetabolite / demethylating agent (e.g., 5-fluorouracil, capecitabine, or azacitidine); anti-hormonal therapy (e.g., hormone receptor antagonist, SERM, aromatase inhibitor); mitotic inhibitor (e.g., vincristine or paclitaxel); topoisomerase (I or II) inhibitor (e.g., mitoxantrone and irinotecan); apoptosis inducers (e.g., ABT-737); nucleic acid therapy nuclear receptor ligands (e.g., agonists and / or antagonists: all-trans retinoic acid or bexarotene); epigenetic targeting agents such as histone deacetylase inhibitors (e.g., vorinostat), hypomethylating agents (e.g., decitabine); modulators of protein stability such as Hsp90 inhibitors, ubiquitin and / or ubiquitin-like binding or debinding molecules; or EGFR inhibitors (erlotinib).

[0172] In some embodiments, the additional therapeutic agent is a demulcent eye drop (also known as "artificial tears"), including, but not limited to, compositions containing polyvinyl alcohol, hydroxypropyl methylcellulose, glycerin, polyethylene glycol (e.g., PEG 400), or carboxymethylcellulose. Artificial tears help treat dry eye by compensating for the reduced moistening 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 mucosal proteins and thus reduce the viscosity of the tear film.

[0173] In some embodiments, the additional therapeutic agent includes antibiotics, antivirals, antifungals, anesthetics, anti-inflammatory agents, including steroidal and non-steroidal anti-inflammatory drugs, and anti-allergy agents. Examples of suitable pharmaceutical agents include aminoglycosides such as amikacin, gentamicin, tobramycin, streptomycin, netilmicin, and kanamycin; fluoroquinolones such as ciprofloxacin, norfloxacin, ofloxacin, trovafloxacin, lomefloxacin, levofloxacin, and enoxacin; naphthyridines; sulfonamides; polymyxins; chloramphenicol; neomycin; paramomycin; colistimethate; bacitracin; bacitracin; Examples of antibiotics include cyclosporin; tetracyclines; rifampin and its derivatives ("rifampin"); cycloserine; beta-lactams; cephalosporins; amphotericin; fluconazole; flucytosine; natamycin; miconazole; ketoconazole; corticosteroids; diclofenac; flurbiprofen; ketorolac; suprofen; cromolyn; lodoxamide; levocabastine; naphazoline; antazoline; pheniramine; or azalide antibiotics.

[0174] In some embodiments, the compounds provided herein can be used in combination with one or more immune checkpoint inhibitors for the treatment of cancers described herein. Exemplary immune checkpoint inhibitors include inhibitors of 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 a KIR inhibitor, a TIGIT inhibitor, a LAIR1 inhibitor, a CD160 inhibitor, a 2B4 inhibitor, and a TGFRbeta inhibitor.

[0175] In some embodiments, the inhibitor of an immune checkpoint molecule is an anti-PD1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody.

[0176] In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of PD-1, 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.

[0177] In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of PD-L1, 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.

[0178] In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of CTLA-4, e.g., an anti-CTLA-4 antibody. In some embodiments, the anti-CTLA-4 antibody is ipilimumab.

[0179] In some embodiments, the inhibitor of an 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.

[0180] In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of GITR, e.g., an anti-GITR antibody. In some embodiments, the anti-GITR antibody is TRX518 or MK-4166.

[0181] In some embodiments, the inhibitor of an 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.

[0182] The compounds of the present disclosure can be used in combination with one or more drugs for the treatment of diseases such as cancer. In some embodiments, the drug is an alkylating agent, a proteasome inhibitor, a corticosteroid, or an immunomodulatory agent. 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 immunomodulatory agent is lenalidomide (LEN) or pomalidomide (POM).

[0183] Pharmaceutical Preparations and Dosage Forms When used as a pharmaceutical, the compounds provided herein can be administered in the form of pharmaceutical compositions. These compositions can be prepared in a manner well known in the pharmaceutical arts and can be administered by various routes, depending on whether local or systemic treatment is desired and the area to be treated. Administration can be topical (including transdermal, epidermal, ophthalmic, and mucosal delivery, including intranasal, vaginal, and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal or intranasal), oral, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion, or intracranial, e.g., intrathecal or intraventricular, administration. Parenteral administration can be in the form of a single bolus dose or, for example, by a continuous perfusion pump. Pharmaceutical compositions and formulations for topical administration can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

[0184] The present disclosure also includes pharmaceutical compositions containing, for example, Compound 1 free base and / or Compound 1 phosphate as an active ingredient in combination with one or more pharmaceutically acceptable carriers (excipients). In some embodiments, the compositions are suitable for topical administration. When preparing these compositions, the active ingredient is typically mixed with an excipient, diluted by the excipient, or enclosed in such a carrier, for example, in the form of a capsule, sachet, paper, or other container. 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 (as solid or liquid media), for example, ointments containing up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.

[0185] When preparing a formulation, the compounds provided herein (Compound 1 free base and / or Compound 1 phosphate) can be milled to provide an appropriate particle size before combining with other ingredients. If Compound 1 free base and / 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 / or Compound 1 phosphate is substantially water-soluble, the particle size can be adjusted by milling to provide a substantially uniform distribution in the formulation, for example, about 40 mesh.

[0186] The compounds provided herein can be milled using known milling procedures, such as wet milling, to obtain particle sizes suitable for tableting and other formulation types. Finely divided (nanoparticulate) preparations of the compounds provided herein can be prepared by processes known in the art, see, for example, International Application No. WO2002 / 000196.

[0187] Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methylcellulose.The formulation may further include lubricants such as talc, magnesium stearate, and mineral oil, wetting agents, emulsifying and suspending agents, preservatives such as methylbenzoate and propylhydroxybenzoate, sweeteners, and flavoring agents.The compositions of the present disclosure can be formulated to provide quick, sustained, or delayed release of the active ingredient after administration to a patient by using procedures known in the art.

[0188] In some embodiments, the pharmaceutical composition comprises silicified microcrystalline cellulose (SMCC) and at least one compound described herein, or a pharmaceutically acceptable salt thereof. In some embodiments, the silicified microcrystalline cellulose comprises about 98% microcrystalline cellulose and about 2% silicon dioxide w / w.

[0189] In some embodiments, the composition is a sustained-release composition comprising 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 and / or Compound 1 phosphate as 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 silicon dioxide. In some embodiments, the microcrystalline cellulose is Avicel PH102™. In some embodiments, the lactose monohydrate is Fast-flo 316™. In some embodiments, the hydroxypropyl methylcellulose is hydroxypropyl methylcellulose 2208K4M (e.g., Methocel K4M Premier™) and / or hydroxypropyl methylcellulose 2208K100LV (e.g., Methocel K00LV™). In some embodiments, the polyethylene oxide is polyethylene oxide WSR 1105 (e.g., Polyox WSR 1105™).

[0190] In some embodiments, a wet granulation process is used to produce the composition. In some embodiments, a dry granulation process is used to manufacture the composition.

[0191] The compositions can be formulated in unit dosage form, with each dosage containing about 1 to about 1,000 mg, about 1 mg to about 100 mg, 1 mg to about 50 mg, and about 1 mg to 10 mg of active ingredient (e.g., Compound 1 free base and Compound 1 phosphate). Preferably, the dosage is about 1 mg to about 50 mg or about 1 mg to about 10 mg of active ingredient. In some embodiments, each dosage contains about 10 mg of active ingredient. In some embodiments, each dosage contains about 50 mg of active ingredient. In some embodiments, each dosage contains about 25 mg of active ingredient. The term "unit dosage form" refers to a physically discrete unit suitable as a unitary dosage for human subjects and other mammals, each unit containing a predetermined quantity of active ingredient calculated to produce a desired therapeutic effect in association with a suitable pharmaceutical excipient.

[0192] In some embodiments, the composition contains about 1 to about 1,000 mg, about 1 mg to about 100 mg, 1 mg to about 50 mg, and about 1 mg to 10 mg of active ingredient (e.g., Compound 1 free base and Compound 1 phosphate). Preferably, the composition contains about 1 mg to about 50 mg or about 1 mg to about 10 mg of active ingredient. One of skill in the art will appreciate that this embodies compounds or compositions containing about 1 mg to about 10 mg, about 1 mg to about 20 mg, about 1 mg to about 25 mg, or about 1 mg to about 50 mg of active ingredient.

[0193] Active compounds (e.g., Compound 1 free base and Compound 1 phosphate) can be effective over a wide dosage range and are therefore generally administered in a pharmaceutically effective amount. However, it will be understood that the amount of compound actually administered will usually be determined by a physician according to the relevant circumstances, including the condition being treated, the selected route of administration, the compound actually administered, the age, weight, and response of the individual patient, the severity of the patient's condition, etc.

[0194] To prepare solid compositions such as tablets, the primary active ingredient is mixed with pharmaceutical excipients to form a solid preformulation composition containing a homogeneous mixture of the compounds of the present disclosure. When these preformulation compositions are referred to as homogeneous, the active ingredient is typically dispersed evenly throughout the composition so that the composition can be readily subdivided into equally effective unit dosage forms such as tablets, pills, and capsules. This solid preformulation is then subdivided into unit dosage forms of the type described above, containing, for example, from about 0.1 to about 1000 mg of the active ingredient of the present disclosure.

[0195] The tablets or pills of the present disclosure can be coated or otherwise compounded to provide a dosage form that provides the advantage of prolonged action.For example, the tablets or pills can comprise an inner dose and an outer dose component, the latter being in the form of an envelope over the former.The two components can be separated by an enteric layer that resists disintegration in the stomach and allows the inner component to pass intact into the duodenum or be delayed in release.A variety of materials can be used as such enteric layers or coatings, including numerous polymeric acids and mixtures of polymeric acids with materials such as shellac, cetyl alcohol, and cellulose acetate.

[0196] Liquid forms into which the compounds and compositions provided herein may be incorporated for oral or injectable administration include aqueous solutions, suitably flavored syrups, aqueous or oily suspensions, and emulsions flavored with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

[0197] Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents, or mixtures thereof, as well as powders. Liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described above. In some embodiments, compositions are administered by the oral or nasal respiratory route for local or systemic effect. Compositions may be nebulized by use of inert gases. Nebulized solutions may be inhaled directly from the nebulizing device, or the nebulizing device may be attached to a face mask, tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions may be administered orally or nasally from devices that deliver the formulation in an appropriate manner.

[0198] Topical formulations may contain one or more conventional carriers. In some embodiments, ointments may contain water and one or more hydrophobic carriers selected from, for example, liquid paraffin, polyoxyethylene alkyl ether, propylene glycol, white Vaseline® (petrolatum), etc. Cream carrier compositions may be based on a combination of water with glycerol and one or more other components, such as glycerol monostearate, PEG-glycerol monostearate, and cetylstearyl alcohol. Gels may be formulated using isopropyl alcohol and water, preferably in combination with other components such as glycerol and hydroxyethylcellulose. In some embodiments, topical formulations contain 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% by weight of a compound provided herein. Topical formulations may be suitably packaged in 100g tubes, optionally accompanied by instructions for treating a selected indication, such as psoriasis or other skin conditions.

[0199] The amount of compound or composition administered to a patient will vary depending on what is being administered, the purpose of the administration, such as prophylaxis or treatment, the condition of the patient, the method of administration, etc. In therapeutic applications, compositions can be 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 state 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.

[0200] The compositions administered to patients may be in the form of pharmaceutical compositions described above. These compositions may be sterilized by conventional sterilization techniques or may be sterile filtered. Aqueous solutions may be packaged for immediate use or lyophilized, with the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the compound preparation will typically be 3-11, more preferably 5-9, and most preferably 7-8. It will be appreciated that the use of certain of the aforementioned excipients, carriers, or stabilizers may result in the formulation of pharmaceutical salts.

[0201] Therapeutic amounts of the compounds of the present disclosure can vary according to, for example, the particular application for which the treatment is being administered, the method 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 and / or Compound 1 phosphate in a pharmaceutical composition can vary depending on several factors, including dosage, chemical properties (e.g., hydrophobicity), and route of administration. For example, for parenteral administration, the compounds provided herein can be provided in an aqueous physiological buffer solution containing about 0.1 to about 10% w / v of the compound. Some typical dosage ranges are about 1 μg / kg to about 1 g / kg of body weight per day. In some embodiments, the dosage range is about 0.01 mg / kg to about 100 mg / kg of body weight per day. The dosage can depend on variables such as the type and progression of the disease or disorder, the overall health of the particular patient, the relative bioefficacy of the selected compound, the formulation of excipients, and its route of administration. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0202] The compositions of the present disclosure can further comprise one or more additional pharmaceutical agents, such as chemotherapeutic agents, steroids, anti-inflammatory compounds, or immunosuppressants, examples of which are listed above.

[0203] In some embodiments, Compound 1 free base or Compound 1 phosphate is administered as an ophthalmic composition. Thus, in some embodiments, the method comprises administering the compound or a pharmaceutically acceptable salt thereof and an ophthalmologically acceptable carrier. In some embodiments, the ophthalmic composition is a liquid composition, a semi-solid composition, an injectate, a film, a microparticle, or a nanoparticle.

[0204] 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 applied topically to the anterior part of the eye, under the upper eyelid, above the lower eyelid, and in the cul-de-sac. In some embodiments, the ophthalmic composition is sterilized. Sterilization can be achieved by known techniques such as sterilizing by filtering the solution or by heating the solution in a ready-to-use ampoule. The ophthalmic compositions of the present disclosure can further comprise pharmaceutical excipients suitable for the preparation of ophthalmic formulations. Examples of such excipients are preservatives, buffers, chelating agents, antioxidants, and salts to adjust osmolality.

[0205] As used herein, the term "ophthalmically acceptable carrier" refers to any substance that can contain and release a compound or a pharmaceutically acceptable salt thereof and is compatible with the eye. In some embodiments, ophthalmically acceptable carriers are water or aqueous solutions or suspensions, but also include oils, such as those used in the manufacture of ointments, and polymer matrices, such as those used in ocular inserts. In some embodiments, the composition can be an aqueous suspension containing the compound or a pharmaceutically acceptable salt thereof. Liquid ophthalmic compositions, including both ointments and suspensions, can have a viscosity appropriate for the selected route of administration. In some embodiments, the ophthalmic composition has a viscosity ranging from about 1,000 to about 30,000 centipoise.

[0206] In some embodiments, the ophthalmic composition may include one or more surfactants, adjuvants, buffers, antioxidants, tonicity adjusters, preservatives (e.g., EDTA, BAK (benzalkonium chloride), sodium chlorite, sodium perborate, polyquaternium-1), thickeners or viscosity adjusters (e.g., carboxymethylcellulose, hydroxymethylcellulose, polyvinyl alcohol, polyethylene glycol, glycol 400, propylene glycol hydroxymethylcellulose, hydroxpropyl-guar, hyaluronic acid, and hydroxypropylcellulose). Additives in the formulation may include, but are not limited to, sodium chloride, sodium bicarbonate, sorbic acid, methylparaben, propylparaben, chlorhexidine, castor oil, and sodium perborate.

[0207] Aqueous ophthalmic compositions (solutions or suspensions) generally do not contain physiologically or ophthalmically harmful components. In some embodiments, purified water or deionized water is used in the composition. The pH can be adjusted by adding any physiologically and ophthalmically acceptable pH-adjusting acid, base, or buffer within the range of about 5.0 to 8.5. Examples of ophthalmologically 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, trishydroxymethylaminomethane, etc. Salts and buffers include citrate / dextrose, sodium bicarbonate, ammonium chloride, and mixtures of the aforementioned acids and bases.

[0208] In some embodiments, the method includes forming or providing a depot of therapeutic agent that contacts the external surface of the eye. A depot refers to a source of therapeutic agent that is not rapidly removed by tears or other ocular clearance mechanisms. This allows for a continuous, sustained high concentration of therapeutic agent in the fluid on the external 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 both the dissolved drug concentration and the contact time between the external tissue and the drug-containing fluid. As the drug is removed by ocular fluid clearance and / or absorption into the ocular tissue, more drug is provided, for example, dissolved in replenished ocular fluid from the depot. Thus, the use of a depot can facilitate the loading of more insoluble therapeutic agents into ocular tissues. In some embodiments, the depot can remain in place for up to 8 hours or longer. In some embodiments, ophthalmic depot forms include, but are not limited to, aqueous polymer suspensions, ointments, and solid inserts.

[0209] In some embodiments, the ophthalmic composition is an ointment or gel. In some embodiments, the ophthalmic composition is an oily delivery vehicle. In some embodiments, the composition comprises a petroleum or lanolin base, typically containing 0.1-2% of the active ingredient and excipients. Common bases include, but are not limited to, mineral oil, petrolatum, and combinations thereof. In some embodiments, the ointment is applied to the lower eyelid as a ribbon.

[0210] In some embodiments, the ophthalmic composition is an ophthalmic insert. In some embodiments, the ophthalmic insert is biologically inert, soft, bioerodible, viscoelastic, stable to sterilization after exposure to a therapeutic agent, resistant to infection from airborne bacteria, bioerodible, biocompatible, and / or viscoelastic. In some embodiments, the insert comprises an ophthalmologically 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 can be slowly released from the matrix by dissolution or hydrolysis of covalent bonds. In some embodiments, the polymer is bioerodible (soluble), and its dissolution rate can control the release rate of the therapeutic agent dispersed therein. In another embodiment, the polymeric matrix is ​​a biodegradable polymer that degrades, such as by hydrolysis, thereby releasing the bound or dispersed therapeutic agent. In further embodiments, the matrix and therapeutic agent can be surrounded by an additional polymeric coating to further control release. In some embodiments, the insert is a biodegradable polymer such as polycaprolactone (PCL), ethylene / vinyl acetate copolymer (EVA), polyalkyl 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 in the matrix material or in the monomer composition used to make the matrix material prior to polymerization. In some embodiments, the amount of therapeutic agent is about 0.1 to about 50%, or about 2 to about 20%. In further embodiments, a biodegradable or bioerodible polymer matrix is ​​used to eliminate the need to remove the insert after use. The therapeutic agent is released as the biodegradable or bioerodible polymer degrades or dissolves.

[0211] In further embodiments, the ophthalmic insert is described in Wagh, et al., "Polymers used in ocular dosage forms and drug delivery systems," Asian J. Pharm., pages 12-17 (Jan. 2008), which is incorporated herein by reference in its entirety. In some embodiments, the insert is polyvinylpyrrolidone (PVP), an acrylate or methacrylate polymer or copolymer (e.g., the Eudragit® family of polymers from Rohm or Degussa), hydroxymethylcellulose, polyacrylic acid, poly(amidoamine) dendrimer, poly(dimethylsiloxane), polyethylene oxide, poly(lactide-co-glycolide), poly(2-hydroxyethyl methacrylate), poly(vinyl alcohol), or poly(propylene fumarate). In some embodiments, the insert comprises Gelfoam®. In some embodiments, the insert is a 450 kDa polyacrylic acid-cysteine ​​conjugate.

[0212] In some embodiments, the ophthalmic composition is an ophthalmic film. Suitable polymers for such films include, but are not limited to, those described in Wagh, et al. (ibid.). In some embodiments, the film is a soft contact lens, such as one made from a copolymer of N,N-diethylacrylamide and ethylene glycol dimethacrylate crosslinked with methacrylic acid.

[0213] 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, chronically intraluminally, intrasclerally, intravitreally, or subretinaly. In some embodiments, the microspheres or nanoparticles comprise a polymer, including but not limited to those described in Wagh, et al. (ibid.), 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, hyaluronic acid esters, 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.

[0214] 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. (ibid.), which are incorporated herein by reference in their entireties. In some embodiments, the ion exchange resin is a partially neutralized polyacrylic acid.

[0215] In some embodiments, the ophthalmic composition is an aqueous polymer suspension. In some embodiments, the therapeutic agent or polymer suspending agent is suspended in an aqueous medium. In some embodiments, the aqueous polymer suspensions can be formulated to retain the same or substantially the same viscosity in the eye as they had before administration to the eye. In some embodiments, they can be formulated to increase gelation upon contact with tear fluid.

[0216] kit The present disclosure also includes pharmaceutical kits useful in the treatment or prevention of JAK-associated diseases or disorders, such as cancer, which include one or more containers housing a pharmaceutical composition comprising a therapeutically effective amount of Compound 1 free base or Compound 1 phosphate. As will be apparent to those skilled in the art, such kits can optionally further include one or more of a variety of conventional pharmaceutical kit components, such as, for example, a container with one or more pharmaceutically acceptable carriers, additional containers, etc. Instructions, either as an insert or label, indicating the amounts of components to be administered, administration guidelines, and / or guidelines for mixing the components, can also be included in the kit.

[0217] The present disclosure will be described in more detail by 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 easily recognize various non-critical parameters that can be changed or modified to achieve essentially the same results.The compound of the examples has been found to be a JAK inhibitor by at least one assay described herein. [Example]

[0218] Intermediate 1. 3,5-Dimethyl-4,4'-bipyrazole (Compound 2x) Scheme 1. [ka] Step 1. 1'-(1-ethoxyethyl)-3,5-dimethyl-1H,1'H-[4,4']bipyrazolyl (compound 2b) [ka] A 100 L glass reactor purged with nitrogen was charged with 1-propanol (5.0 L), drinking water (6.0 L), KHPO (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). Nitrogen gas was bubbled through the reaction mixture for 18 minutes, and then Pd-118 (55.07 g) was charged to the reactor, and nitrogen gas was bubbled through the reaction mixture for an additional 18 minutes. The reaction mixture was heated to approximately 90°C and stirred at approximately 90°C for approximately 4 hours. The reaction mixture was then cooled to approximately 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 Celite (1500 g), and the filter cake was washed with 2-propanol (15.0 L). The combined filtrate and washings were concentrated under vacuum at about 58° C. to give the desired crude product, 1′-(1-ethoxyethyl)-3,5-dimethyl-1H,1′H-[4,4′]bipyrazolyl (2593 g), which was used in further processing.

[0219] Crude 1'-(1-ethoxyethyl)-3,5-dimethyl-1H,1'H-[4,4']bipyrazolyl (2590 g) and ethyl acetate (EtOAc, 15.0 L) were charged into a reactor. Separately, an aqueous NaHSO3 solution was prepared by thoroughly mixing NaHSO3 (1500 g) with drinking water (8.0 L). The reaction mixture was then heated to 65-70°C and stirred at 65-70°C for approximately 2.5 hours. The phases were separated, and the organic phase was retained in the reactor. Separately, an aqueous NaHSO3 solution was prepared by thoroughly mixing NaHSO3 (1500 g) with drinking water (8.0 L). The reaction mixture was then heated to 65-70°C and stirred at 65-70°C for approximately 3.5 hours. The phases were separated. A chromatography column was sequentially packed with sea sand (3000 g), ethyl acetate (EtOAc, 15.0 L), and silica gel (SiO2, 4500 g). The silica gel and solvent were mixed, and the solvent was eluted onto the surface of the silica gel. Sea sand (3000 g) was loaded onto the top of the column. The reaction mixture was loaded onto the column and eluted with ethyl acetate (18.0 L). The desired fractions were combined, and the combined solution was concentrated under vacuum at approximately 55 °C to obtain the column-purified product (1760 g), which was then placed in a reactor together with methylene chloride (16.0 L). Si-thiol (160 g) was added to the reactor, and the reaction mixture was heated to 35 °C-40 °C and stirred at 35 °C-40 °C for approximately 2 hours. The mixture was filtered, and the filter cake was washed with methylene chloride (3.5 mL). The combined filtrate and washings were concentrated under vacuum to give the purified desired product, 1′-(1-ethoxy-ethyl)-3,5-dimethyl-1H,1′H-[4,4′]pyrazolyl (1600 g), which contained residual solvent and was used directly in the next reaction. 1 H NMR(400MHz,DMSO-d6)δ12.17(s,1H),7.89(s,1H),7.56(s,1H),5.53(q,J=6.0Hz,1H),3.41(dq,J=9.6,7. 0Hz,1H),3.19(dq,J=9.6,7.0Hz,1H),2.20(s,6H),2.10,1.60(d,J=6.0Hz,3H),1.01(t,J=7.0Hz,3H)ppm; 13C NMR(101MHz,DMSO-d6)δ145.7,137.75,135.9,125.48,114.94,108.69,86.84,63.57,21.84,15.43,13.86ppm.

[0220] Step 2. 3,5-Dimethyl-1H,1'H-[4,4']bipyrazolyl hydrochloride (compound 2xHCl) [ka] A 100 L glass reactor was purged with nitrogen and charged with 1'-(1-ethoxyethyl)-3,5-dimethyl-1H,1'H-[4,4']bipyrazolyl (5723 g, based on theoretical yield), 2-propanol (IPA, 13.0 L), and concentrated hydrochloric acid (HCl, 4.08 L) at room temperature. The resulting reaction mixture was heated to 60 °C and stirred for 4 h. The reaction mixture was cooled to room temperature and stirred at room temperature for approximately 1 h. The solid was collected by filtration, and the filter cake was washed with 2-propanol (6.5 L). The product was air-dried to afford the desired product, 3,5-dimethyl-1H,1'H-[4,4']bipyrazolyl hydrochloride (3088 g, 63.6% over two steps), as a white solid. 1 H NMR(400MHz,DMSO-d6)δ7.94(s,2H),2.38(s,6H)ppm; 13 C NMR(101MHz,DMSO-d6)δ141.95,132.75,111.78,109.70,10.97ppm.

[0221] Step 3. 3,5-Dimethyl-1H,1'H-[4,4']bipyrazolyl (Compound 2x) 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). The reaction mixture was cooled to 0-5°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 approximately 15°C. The reaction mixture was warmed to approximately 18°C ​​and stirred at approximately 18°C ​​for approximately 14 hours. The solids were collected by filtration, and the filter cake was washed sequentially 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 (2006 g, 81.6%) as an off-white powder. 1 H NMR(400MHz,DMSO-d6)δ7.65(s,2H),2.19(s,6H)ppm; 13 C NMR(101MHz,DMSO-d6)δ140.76,131.92,113.44,109.16,12.37ppm.

[0222] Intermediate 2. (S)-4-(3-(cyanomethylene)azetidin-1-yl)-2,5-difluoro-N-(1,1,1-trifluoropropan-2-yl)benzamide (Compound 1x) Scheme 2 [ka]

[0223] Step 1. (S)-2,4,5-trifluoro-N-(1,1,1-trifluoropropan-2-yl)benzamide (compound 1a) [ka] 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. Next, 2,4,5-trifluorobenzoyl chloride (995.62 g, 5.07 mol, 1.1 equiv.) was added dropwise to the mixture over 20 min at 0°C–15°C. The cooling bath was removed, and the reaction mixture was allowed to warm to room temperature and stirred for an additional 1 h at room temperature. The two phases of the reaction mixture were then 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 h and then cooled to room temperature. The solid was collected by filtration, washed with n-heptane (1 L), and dried under vacuum for 2 days to give (S)-2,4,5-trifluoro-N-(1,1,1-trifluoropropan-2-yl)benzamide (1203.7 g, 93.2%) as a white powder. 1 H NMR(300MHz,DMSO-d6)δ9.00(d,J=8.09Hz,1H),7.69(m,2H),4.75(m,1H),1.92(d,J=7.00Hz,3H)ppm.

[0224] Step 2. (S)-2,5-Difluoro-4-(3-hydroxyazetidin-1-yl)-N-(1,1,1-trifluoropropan-2-yl)benzamide (Compound 1b) [ka] To (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 equiv.) in acetonitrile (3.6 L), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 2335.2 g, 15.33 mol, 2.3 equiv.) was added portionwise. The first 1000 g of DBU was charged over 25 min, causing an exothermic reaction that raised the internal temperature from 12 °C to 58 °C. The remaining DBU was added over 20 min at 58 °C–68 °C, and the resulting reaction mixture was stirred at 58 °C–68 °C for 1 h. 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 under vacuum 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 H-NMR (300MHz,DMSO-d6)δ8.38(d,J=8.71Hz,1H),7.26(dd,J=12.91Hz,1H),6.38(dd,J=12.29Hz,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.

[0225] Step 3. (S)-2,5-Difluoro-4-(3-hydroxyazetidin-1-yl)-N-(1,1,1-trifluoropropan-2-yl)benzamide (Compound 1c) [ka] To 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 equiv.) in methylene chloride (8.5 L), 2,2,6,6-tetramethyl-1-piperidinyloxy free radical (TEMPO, 20.9 g, 0.13 mol, 0.025 equiv.) was added at 10°C to 12°C. The resulting reaction mixture was stirred at 10°C to 12°C, and the internal temperature was allowed to reach 36°C to 38°C over 30 to 60 min. A cooling bath of IPA and dry ice was used to control the reaction temperature. Once the temperature of the internal mixture had dropped below 25°C, the reaction mixture was heated to 35-38°C and stirred at 35-38°C for an additional 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 x 4 L). The organic solvent was then concentrated under reduced pressure to give the desired crude 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 x 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%) as a tan powder. 1 H-NMR(300MHz,DMSO-d6)δ8.50(d,J=8.72Hz,1H),7.35(dd,J=12.6Hz,1H),6.62(dd,J=12.1Hz,1H),4.81(s,4H),4.56(m,1H),1.30(d,J=7.0Hz,3H)ppm.

[0226] Step 4. (S)-4-(3-(cyanomethylene)azetidin-1-yl)-2,5-difluoro-N-(1,1,1-trifluoropropan-2-yl)benzamide (Compound 1x) Diethyl cyanomethylphosphonate (422.6 g, 2.39 mol, 0.98 equiv.) was added to a 1.0 M solution of potassium tert-butoxide in THF (1996.6 g, 2.27 mol, 0.94 equiv.) under nitrogen over 10 min at 5°C to 25°C. The resulting mixture was then warmed to room temperature and stirred for 1 h to form a clear solid (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 over 30 minutes at -5°C to 5°C. The resulting mixture was stirred at 0°C to 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 collected by filtration and washed with water (2 L) and n-heptane (2.4 L) to give a brown solid. 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 under vacuum 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(300MHz,DMSO-d6)δ8.50(d,J=9.95Hz,1H),7.31(dd,J=12.4Hz,1H),6.5 8(dd,J=12.0Hz,1H),5.88(m,1H),4.86-4.75(m,5H),1.31(d,J=7.0Hz,3H)ppm.

[0227] Intermediate 3. tert-Butyl 3-(cyanomethylene)azetidine-1-carboxylate (Compound 1y) Scheme 3 [ka]

[0228] Step 1. 1-Benzhydrylazetidin-3-ol hydrochloride [ka] A solution of diphenylmethanamine (2737 g, 15.0 mol, 1.04 equiv.) in methanol (MeOH, 6 L) was treated with 2-(chloromethyl)oxirane (1330 g, 14.5 mol) at ambient temperature. The resulting reaction mixture was stirred at room temperature for 3 days and then warmed to reflux for an additional 3 days. The reaction mixture was then cooled to room temperature and then cooled to 0-5°C in an ice bath. The solid was collected by filtration and washed with acetone (4 L) to give a first crop of the desired crude product (1516 g). The filtrate was concentrated under reduced pressure, and the resulting semi-solid was diluted with acetone (1 L). The solid was then collected by filtration to give a second crop of the desired crude product (221 g). The crude product, 1-benzhydrylazetidin-3-ol hydrochloride (1737 g, 43.4% yield), was used in subsequent reactions without further purification. 1 C 16 H 18 ClNO (molecular weight 275.77; C for the free base) 16 H 17 NO, molecular weight, 239.31), LCMS (EI) m / e 240 (M + +H).

[0229] Step 2. tert-Butyl 3-hydroxyazetidine-1-carboxylate [ka] A suspension of 1-benzhydrylazetidin-3-ol hydrochloride (625 g, 2.27 mol) in 10% aqueous sodium carbonate (NaCO, 5 L) and dichloromethane (CHCl, 5 L) was stirred at room temperature until all solids dissolved. The two layers were separated, and the aqueous layer was extracted with dichloromethane (CHCl, 2 L). The combined organic extracts were dried over sodium sulfate (NaSO) and concentrated under reduced pressure. The resulting crude 1-benzhydrylazetidin-3-ol free base was then dissolved in THF (6 L), and the solution was placed in a large Parr bomb. Di-tert-butyl dicarbonate (BOCO, 545 g, 2.5 mol, 1.1 equiv.) and 20% palladium on carbon (Pd) (125 g, 50% moisture) were added to the Parr bomb. The vessel was charged with hydrogen gas (H2) to 30 psi and stirred at room temperature for 18 hours under a constant hydrogen atmosphere (the vessel was recharged three times to maintain 30 psi pressure). The reaction mixture was filtered through a Celite pad, which 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 minimal amount of dichloromethane (CHCl2). The column was eluted with 20–50% ethyl acetate in n-heptane, and fractions 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 upon standing in vacuo at ambient temperature. 1 H NMR (300MHz, CDCl3), δ4.56(m1H), 4.13(m,2H), 3.81(m,2H), 1.43(s,9H)ppm.

[0230] Step 3. tert-Butyl 3-oxoazetidine-1-carboxylate [ka] 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 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) at 0–5 °C. Saturated aqueous sodium bicarbonate (NaHCO3, 450 mL) and aqueous sodium hypochlorite (NaClO, 10–13% available chlorine, 450 mL) were added while maintaining the reaction temperature between 0 and 5 °C. The color of the reaction mixture gradually faded as additional sodium hypochlorite solution was added. When all of 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 solution (500 mL) and dried over sodium sulfate (NaSO). The solvent was then removed under reduced pressure to give the crude product (tert-butyl 3-oxoazetidine-1-carboxylate (48 g, theoretical 49.47 g, 97% yield)), which was used in the subsequent step without further purification. 1 H NMR(CDCl3,300MHz)δ4.65(s,4H),1.42(s,9H)ppm.

[0231] Step 4. tert-Butyl 3-(cyanomethylene)azetidine-1-carboxylate Diethyl cyanomethylphosphate (745 g, 4.20 mol, 1.20 equiv.) and anhydrous tetrahydrofuran (THF, 9 L) were added to a four-necked flask at room temperature. The solution was cooled to −14° C. in an ice-methanol bath, and a solution of 1.0 M potassium tert-butoxide (t-BuOK) (3.85 mol, 1.1 equiv.) in anhydrous tetrahydrofuran (THF, 3.85 L) was added over 20 minutes, maintaining the reaction temperature below −5° C. The resulting reaction mixture was stirred at −10° C. for 3 hours, and a solution of 1-tert-butoxycarbonyl-3-azetidinone (600 g, 3.50 mol) in anhydrous tetrahydrofuran (THF, 2 L) was added over 2 hours, maintaining the internal temperature below −5° C. The reaction mixture was stirred at −5 to −10° C. for 1 hour, 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 (NaSO). The solvent was removed under reduced pressure, and the residue was diluted with dichloromethane (CHCl, 4 L) and then absorbed onto silica gel (SiO, 1.5 kg). The crude product absorbed onto silica gel was purified by flash column chromatography (SiO, 3.5 kg, 0–25% EtOAc / hexane gradient elution) to afford tert-butyl 3-(cyanomethylene)azetidine-1-carboxylate (414.7 g, 61% yield) as a white solid. 1 H NMR(300MHz,CDCl3)δ5.40(m,1H),4.70(m,2H),4.61(m,2H),1.46(s,9H)ppm;C 10 H 14 N2O2 (molecular weight, 194.23), LCMS (EI) m / e 217 (M + +Na).

[0232] Intermediate 4. Alternative synthesis of (S)-2,4,5-trifluoro-N-(1,1,1-trifluoropropan-2-yl)benzamide (compound 1a) Scheme 4. [ka]

[0233] Step 1. 2,4,5-Trifluorobenzoyl Chloride [ka] A 100 L reactor was charged with SOCl2 (34.9 kg), DMF (0.34 L), and 2,4,5-trifluorobenzoic acid (32.3 kg). The batch was heated to 80°C and stirred at 80-90°C for 9 hours. The batch was cooled to 50-60°C and distilled under vacuum at 60°C until distillation stopped. 14 kg of toluene was charged to the reactor, and the batch was continuously distilled at 60°C to give crude 2,4,5-trifluorobenzoyl chloride (46.28 kg, 88% by HPLC).

[0234] Step 2. (S)-2,4,5-trifluoro-N-(1,1,1-trifluoropropan-2-yl)benzamide (Compound 1a) An aqueous solution (158 L) containing (S)-1,1,1-trifluoropropan-2-amine hydrochloride (35 kg) was charged to a 1000 L reactor, and toluene (198 kg) was added to the reactor, followed by the portionwise addition of K2CO3 (82 kg). 2,4,5-Trifluorobenzoyl chloride (36.1 kg) was dissolved in toluene (40 kg), and the toluene solution was charged to the reactor along with the toluene solution of the amine intermediate. The resulting mixture was stirred at 20 °C for 2 hours. The batch was filtered, and the filter cake was washed with toluene (117 kg). The filtrate and washings were charged to a 1000 L reactor, and 1 N aqueous NaOH solution (125 kg) was charged to the reactor. The mixture was stirred for 2 hours, and the phases were allowed to separate. The aqueous phase was discarded, and the organic phase was washed twice with water (135 kg) and stored in a clean container (Solution 1). Another portion (portion 2) was treated in the same manner to obtain solution 2. Solutions 1 and 2 were charged to a 1000 L reactor, and Na2SO4 (104 kg) was charged to the reactor. The mixture was stirred for 2 hours, filtered, and the filter cake was washed with toluene (90 kg). The filtrate and washes were charged to a 500 L reactor, and the batch was distilled under vacuum at 50°C. Toluene (14 kg) and heptane (166 kg) were charged to a 500 L reactor, and the batch was stirred at 80°C until a solution was obtained. The solution was cooled to 25°C and stirred for 2 hours. The product was isolated by vacuum filtration, and the filter cake was washed with n-heptane (40 kg). The filter cake was dried under vacuum at ≦50° C. to give crude product (S)-2,4,5-trifluoro-N-(1,1,1-trifluoropropan-2-yl)benzamide (87.0 kg; LOD 79.0 wt %; Net weight: 68.7 kg; 68% by HPLC; 69.4% by HPLC; 97.1 ee% by chiral HPLC), which was further purified from a mixture of IPA and n-heptane according to the following procedure.

[0235] A 500 L reactor was charged with IPA (30.5 kg), heptane (213 kg), and crude (S)-2,4,5-trifluoro-N-(1,1,1-trifluoropropan-2-yl)benzamide (70 kg). The mixture was heated to 85°C and stirred to obtain a clear solution. The batch was cooled to 20°C and stirred for 12 hours. The batch was filtered, and the filter cake was washed with n-heptane (48 kg) and dried under vacuum at 50°C to give 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% by chiral HPLC). 1 H NMR(300MHz, CDCl3) δ7.96(m,1H),7.01(m,1H),6.71(m,1H),4.93(m,1H),1.44(d,J=8.00Hz,3H)ppm.

[0236] 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]benzamidophosphate (Compound 1 Phosphate) Scheme 5. [ka]

[0237] Step 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 (Compound 1 free base) [ka] 3,5-Dimethyl-1H,1'H-[4,4']bipyrazolyl hydrochloride (Compound 2xHCl, 2002 g, 12.34 mol, 1.1 equiv.), DMF (3.9 L), and DBU (0.201 L, 204.6 g, 1.34 mol, 0.12 equiv.) were placed in a 50 L reactor. The reaction mixture was heated to 50-60 °C and stirred for approximately 30 min. Separately, (S)-4-(3-(cyanomethylene)azetidin-1-yl)-2,5-difluoro-N-(1,1,1-trifluoropropan-2-yl)benzamide (Compound 1x, 3872 g, 11.21 mol) was thoroughly mixed with DMF (11.6 L) to prepare a solution. Then, a solution of Compound 1x in DMF was added to the reaction mixture while maintaining the temperature at approximately 61 °C. The resulting reaction mixture was stirred at about 60°C for about 3.5 hours. The reaction mixture was then cooled to room temperature, and water (77.4 L) was added to the reactor. The cooled reaction mixture was added to the 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 cake was air-dried to give 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 (Compound 1 free base, 5849 g).

[0238] A chromatography column was sequentially packed with ethyl acetate (9.9 L), CHCl (22.4 L), and silica gel (8000 g), mixed thoroughly, and eluted onto the surface of the silica gel. Crude Compound 1 free base (1006 g), silica gel (4000 g), and CHCl (8.0 L) were loaded onto a first rotary evaporator and rotated at about 22 °C for about 45 minutes without solvent recovery. Crude Compound 1 free base (1008 g), silica gel (4002 g), and CHCl (8.0 L) were loaded onto a second rotary evaporator and rotated at about 23 °C for about 45 minutes without solvent recovery. Both mixtures were then 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 sequentially eluted with the collected eluate (16 L), 30% (v / v) EtOAc-CHCl (prepared separately from 31.2 L of EtOAc and 72.8 L of CHCl), 5% (v / v) MeOH-CHCl (prepared separately from 2.5 L of MeOH and 47.5 L of CHCl), and 8% (v / v) MeOH-CHCl (prepared separately from 4.8 L of MeOH and 55.2 L of CHCl). The combined fractions were concentrated under reduced pressure at approximately 45 °C to give pure Compound 1 free base (1824 g). Four batches of column purification were performed 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(400MHz,DMSO-d6)δ12.22(s,1H),8.50(d,J=8.7Hz,1H),8.13(s,1H),7.72(s,1H),7.36(dd,J=12.5,6.3Hz,1H),6.62(dd,J= 11.9,7.3Hz,1H),4.78(m,1H),4.64(d,J=8.9Hz,2H),4.40(d,J=9.1Hz,2H),3.66(s,2H),2.23(s,6H),1.31(d,J=7.0Hz,3H)ppm; 13C NMR(101MHz,DMSO-d6)δ162.8,156.7(d,J=246.6Hz),146.9(d,J=236.9Hz) ,145.2,141.6(t,J=12.3Hz),138.3,135.5,125.8(q,J=281.9Hz),125.6,1 17.2,116.4(d,J=26.4Hz),115.2,111.3(dd,J=15.7,5.8Hz),107.7,102.0 (d,J=29.1Hz),62.4,57.7,45.8(q,J=30.8Hz),27.0,13.3,13.3,10.4ppm; 19 F NMR(282MHz,DMSO-d6)δ-76.17(d,J=7.4Hz),-116.89(s),-139.71(s)ppm.

[0239] 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]benzamidophosphate (crude compound 1 phosphate) [ka] 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, 119.65 g, 1037.8 mmol, 1.3 equiv.) in isopropyl alcohol (IPA, 120.0 mL) over 18 minutes. The resulting slurry was stirred at 50° C. for 1 hour. n-Heptane (4050.0 mL) was then added over 40 minutes, maintaining the internal temperature at 46-53°C. After the addition of n-heptane, the slurry was cooled to room temperature and stirred for 19 hours. The solid was collected by filtration, washed with a mixture of 2-propanol and n-heptane (3-10 volumes, 2 x 700 mL), followed by n-heptane (3 x 550 mL), and dried under vacuum 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]benzamidophosphate (crude Compound 1 phosphate, 434.6 g, 89.9% yield).

[0240] 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]benzamidophosphate (compound 1 phosphate, purified)

[0241] 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]benzamidophosphate (crude Compound 1 phosphate, 958.3 g, 1583 mmol) and methanol (MeOH, 9583.0 mL) were charged to a 22 L flask at room temperature. The resulting slurry was heated to 50 °C, yielding a clear, pale orange solution. The solution was polish filtered, transferred to a 22 L flask, and heated to remove the methanol over 70 minutes. 2-Propanol (IPA, 7700 mL) was then added to the flask over 30 minutes, while maintaining the internal temperature between 50 °C and 65 °C. Then, n-heptane (14,400 mL) was added in portions over 2.5 hours while maintaining the distillation of the solvent mixture (MeOH, IPA, and n-heptane). A total of 10,818 g (15,000 mL) of the solvent mixture was distilled. 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 (1-5 volumes, 3000 mL), followed by n-heptane (3 x 4000 mL), and dried under vacuum at room temperature to give 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]benzamidophosphate (compound phosphate, 925.7 g, 96.6% yield) as an off-white crystalline powder. 1 H NMR(400MHz,DMSO-d6)δ9.35(br.s,4H),8.50(d,J=8.9Hz,1H),8.11(s,1H),7.70(s,1H),7.34(dd,J=12.5,6.4Hz,1H),6.61(dd,J=1 2.0,7.4Hz,1H),4.86-4.69(m,1H),4.61(d,J=8.9Hz,2H),4.38(d,J=8.9Hz,2H),3.64(s,2H),2.21(s,6H),1.30(d,J=7.1Hz,3H)ppm; 13 C NMR(100MHz,DMSO-d6)δ162.8,156.7(d,J CF=246.5Hz), 146.9(d,J CF =236.1Hz), 141.6(dd,J CF =13.0,11.7Hz),140.3,138.3,125.8(q,J CF =281.8Hz),125.6,117.2,116.4(dd,J CF =22.3,4.6Hz),115.1,111.3(dd,J CF =15.7,5.8Hz),107.7,102.0(dd,J CF =29.5,4.5Hz),62.3,57.7,57.7,45.8(q,J CF =30.5Hz), 27.0, 13.3(d,J CF =1.7Hz), 11.7ppm;C 23 H 22 F5N7O (molecular weight 507.46), LCMS (EI) m / e 508.1 (M + +H).

[0242] The phosphate ratio was 1.01 phosphate to compound 1 free base. 1 The crystal structure was determined by HNMR. The same crystalline form of Compound 1 phosphate drug substance has been consistently prepared 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.

[0243] 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]benzamidophosphate (Compound 1 Phosphate) Scheme 6. [ka]

[0244] Step 1. tert-Butyl 3-(cyanomethyl)-3-(3',5'-dimethyl-1H,1'H-[4,4'-bipyrazol]-1-yl)azetidine-1-carboxylic acid [ka] A 250 L glass-lined dry reactor was charged with anhydrous dimethyl sulfoxide (DMSO; 57.0 L) and heated to 32 °C. Once the solvent was at temperature, tert-butyl 3-(cyanomethylene)azetidine-1-carboxylate (compound 1y, 22.8 kg, 117.4 mol, 1.0 equiv.) was charged to the reactor, followed by 3,5-dimethyl-4,4'-bipyrazole (compound 2x, 20.0 kg, 123.3 mol, 1.05 equiv.). The reaction mixture was cooled to 24 °C, and DBU (4.4 L, 29.56 mol, 0.25 equiv.) was charged to the reactor. The resulting solution was stirred for at least 2 hours. The reaction mixture was then diluted with methylene chloride (116 L) and poured into a solution of 10% citric acid and 10% NaCl in water (97 L). The lower organic layer was separated from the biphasic mixture, and the aqueous layer was extracted with methylene chloride (58 L). The combined organic layers were then washed twice with an aqueous solution of 10% citric acid and 10% NaCl (97 L). As part of the second wash, additional methylene chloride (DCM) was added to the organic layer (58 L). After washing, isopropyl acetate (465 L) was charged to the reaction mixture while performing a constant volume distillation. A white solid formed during the distillation. The resulting suspension was cooled to 20 °C, stirred for at least 4 hours, filtered, and dried to give the desired 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(400MHz,DMSO-d6)δ12.19(s,1H),8.06(s,1H),7.70(s,1H),4.41(d,J=9.4Hz, 2H),4.18(d,J=9.3Hz,2H),3.55(s,2H),2.23(d,J=19.5Hz,6H),1.41(s,9H)ppm;C 18 H 24N6O2, (molecular weight 356.42), LCMS (EI) m / e 357.4 (M + +H).

[0245] Step 2. 2-(3-(3',5'-dimethyl-1H,1'H-4,4'-bipyrazol]-1-yl)azetidin-3-yl)acetonitrile [ka] A 450 L glass-lined reactor was charged with methylene chloride (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.). TMSI (14.4 L, 101.45 mol, 1.205 equiv.) was added, and the resulting solution was stirred at 25°C for at least 2 hours. Methanol (4.3 L, 106.12 mol, 1.261 equiv.) was charged to the reactor, and the reaction mixture was stirred for an additional 30 minutes. The reaction mixture was then heated to remove methylene chloride (150 L) by distillation. After distillation was complete, isopropyl acetate (IPAc, 150 L) was charged to the vessel at 25°C, and the reaction mixture was stirred for 1 hour. The resulting suspension was filtered and washed with IPAc to give a crude mixture 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 (68 kg).

[0246] The crude solid was then transferred to a 450 L glass-lined reactor charged with methylene chloride (360 L). Triethylamine (14 L, 100.80 mol, 1.198 equiv.) was charged to the reactor over 30 minutes, and the resulting mixture was stirred at 25° C. for 12 hours. The resulting suspension was filtered, washed once with methylene chloride and three times with IPAc, filtered, and dried to give the desired product, 2-(3-(3',5'-dimethyl-1H,1'H-[4,4'-bipyrazol]-1-yl)azetidin-3-yl)acetonitrile (16.8 kg, 78%) as a white solid. 1 H NMR(600MHz,DMSO-d6)δ10.16(q,J=7.0Hz,1H),9.90(s,1H),8.45(s,1H),7.9 2(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 H 16 N6, (molecular weight 256.31), LCMS (EI) m / e 257.2 (M + +H).

[0247] 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 [ka] A 250 L glass-lined reactor was charged 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.), NaHCO (4.1 kg, 49.1 mol, 1.05 equiv.), LiCl (4.0 kg, 93.6 mol, 2.00 equiv.), and DMSO (96 L, 8 V). The resulting reaction mixture was heated to 85°C for at least 7 hours, and then the resulting solution was cooled to room temperature. The reaction mixture was diluted with isopropyl acetate (147 L, 12 V) and then with water (120 L, 10 V). The aqueous layer was separated, and the remaining organic layer was washed with 1 wt% aqueous citric acid (88 L, 7.3 V) and water (88 L, 7.3 V) before being concentrated to approximately 133 L (11 V). Isopropyl acetate (147 L, 12.25 V) was then added to the mixture while performing constant volume distillation. Next, a solution of HCl in IPA (2.5 wt%, 96 L, 8 V) was charged to the reactor, and the resulting solution was stirred at room temperature. After 1 hour, methylcyclohexane (220 L, 18.1 V) was charged to the slurry, and the resulting suspension was stirred at room temperature for an additional 4 hours. The resulting suspension was filtered, and the wet cake was washed with a mixture of methylcyclohexane and isopropyl acetate (3:1, 60 L, 5 V), followed by methylcyclohexane (60 L, 5 V). Finally, the wet cake was dried under vacuum at 50 °C–60 °C to give 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%).

[0248] 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)benzamidophosphate [ka] A 450 L glass-lined reactor was charged with isopropyl acetate (286 L, 10 V) and (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 (28.6 kg), followed by KHCO (86 L, 10 wt % aqueous solution in water, 3 V). The suspension was stirred until a clear solution was obtained. The aqueous layer was then removed, and the organics were washed with water (86 L (3 V)) before being filtered through charcoal and transferred to a second glass-lined reactor. The organics were concentrated to remove 240 L (8.4 V) of solvent under reduced pressure of 200 mbar to 400 mbar at 50 °C. The resulting residue was charged with isopropanol (163 L, 5.7 V) at 50°C, followed by cooling to room temperature. Next, 14.9 kg (52 wt%) of 48 wt% H3PO4 in IPA / water was charged to the reactor over at least 2 hours, and the resulting solution was stirred at room temperature for at least 1 hour. Methylcyclohexane (172 L, 6 V) was charged at room temperature, and the mixture was stirred for at least 1 hour. The suspension was filtered, and the cake was washed with 1:1 IPA / methylcyclohexane (86 L, 3 V), followed by methylcyclohexane (86 L, 3 V). The wet cake was then dried under vacuum at 50° C. to give crude (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)benzamidophosphate (28.0 kg (88%)).

[0249] A 450 L glass-lined reactor was charged with crude phosphate (28.0 kg) and methanol (336 L (12 V)), and the resulting mixture was heated to 50 °C to obtain a clear solution. The solution was transferred to another reactor through a polish filter. MeOH (28 L, 1 V) was used to rinse the first reactor and then transferred to the second reactor through a polish filter. The filtrate was then concentrated to 7 V by distilling 196 L (7 V) of solvent at 45 °C under a reduced pressure of 300 mbar to 400 mbar. Next, seeds of 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)benzamidophosphate (28.0 g, 0.1 wt%) were charged to the reactor, and the mixture was stirred at 45 °C for at least 15 min. Isopropanol (196 L, 7 V) was charged, and 196 L (7 V) of the solvent was distilled off at approximately 45 °C under a reduced pressure of 100 mbar to 200 mbar. Isopropanol (196 L, 7 V) was charged to the reactor, and 196 L (7 V) of the solvent was removed by distillation. An IPC was performed to confirm that the methanol content in the mixture was 5% or less. Next, the mixture was cooled to room temperature, and the resulting suspension was filtered. The cake was washed twice with isopropanol (56 L, 2 V) and then dried under reduced pressure at 50°C to give (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)benzamidophosphate (24.1 kg (86.1%)) as a white solid. 1 H NMR(500MHz,DMSO-d6)δ8.53-8.43(m,1H),8.12(d,J=0.7Hz,1H),7.72(s,1H),7.36(dd,J=12.5,6.3Hz,1H),6.63(dd,J=11.9,7.2H C23 H 25 F5N7O5P(molecular weight 605.45;C 23 H 22 F5N7O: Molecular weight 507.47), LCMS (EI) m / e 508.2 (M + +H).

[0250] Example A. In vitro JAK kinase assay

[0251] The compounds provided herein were tested for inhibitory activity against JAK targets according to the following in vitro assay described in Parket et al., Analytical Biochemistry 1999, 269, 94-104. The catalytic domains of human JAK1 (amino acids 837-1142), JAK2 (amino acids 828-1132), and JAK3 (amino acids 781-1124) with N-terminal His tags were expressed in insect cells using baculovirus and purified. The catalytic activity of JAK1, JAK2, or JAK3 was assayed by measuring the phosphorylation of a biotinylated peptide. The phosphorylated peptide was detected by homogeneous time-resolved fluorescence (HTRF). The IC of the compound for each kinase was measured in a 40 μL reaction containing enzyme, ATP, and 500 nM peptide in 50 mM Tris (pH 7.8) buffer containing 100 mM NaCl, 5 mM DTT, and 0.1 mg / mL (0.01%) BSA. 50 The IC of 1 mM was measured. 50 For measurements, the ATP concentration in the reaction was 1 mM. The reaction was carried out at room temperature for 1 hour and then stopped with 20 μL of 45 mM EDTA, 300 nM SA-APC, and 6 nM Eu-Py20 assay buffer (Perkin Elmer, Boston, MA). Binding to the europium-labeled antibody was carried out for 40 minutes, and the HTRF signal was measured using a Fusion plate reader (Perkin Elmer, Boston, MA). Compound 1 free base exhibited an IC of ≦300 nM with JAK2 / JAK1 selectivity of >10 at 1 mM ATP. 50 had.

[0252] Example B: Cellular Assay Cancer cell lines that depend on cytokines and thus JAK / STAT signal transduction for growth can be plated at 6000 cells per well (96-well plate format) in RPMI1640, 10% FBS, and 1 nG / mL of the appropriate cytokine. Compounds provided herein are added to the cells in DMSO / medium (final DMSO concentration of 0.2%) and incubated at 37°C, 5% CO2 for 72 hours. The effect of the compound on cell viability is assessed using CellTiter-GloLuminescent CellViability Assay (Promega) followed by quantification with TopCount (Perkin Elmer, Boston, MA). Potential off-target effects of the compound are measured in parallel using non-JAK-driven cell lines with the same assay readout. All experiments are typically performed in duplicate.

[0253] The cell lines described above can be used to examine the effects of 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 performed after overnight cytokine starvation, followed by a short preincubation (up to 2 hours) with the compound and cytokine stimulation for approximately 1 hour or less. Proteins are then extracted from the cells and analyzed by techniques familiar to those skilled in the art, including Western blot or ELISA, using antibodies capable of distinguishing between phosphorylated and total proteins. These experiments can utilize normal or cancer cells to investigate the activity of compounds on tumor cell survival biology or inflammatory disease mediators. For example, with regard to the latter, cytokines such as IL-6, IL-12, IL-23, or IFN can be used to stimulate JAK activation, resulting in the phosphorylation of STAT protein(s) and, potentially, the transcriptional profile (assessed by array or qPCR techniques) or the production and / or secretion of proteins such as IL-17. The ability of compounds to inhibit these cytokine-mediated effects can be measured using techniques common to those skilled in the art.

[0254] The compounds provided herein can also be tested in cell models designed to evaluate their efficacy and activity against mutant JAKs, such as the JAK2V617F mutation found in myeloproliferative disorders. These experiments frequently utilize cytokine-dependent cells of the blood lineage (e.g., 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. Nature 434:1144-1148; Staerk, J., et al. JBC 280:41893-41899). Endpoints include the effects of compounds on cell survival, proliferation, and phosphorylated JAK, STAT, Akt, or Erk proteins.

[0255] Compounds provided herein can be evaluated for their activity in inhibiting T cell proliferation. Such assays can be considered as secondary cytokine (i.e., JAK)-driven proliferation assays and simplified assays for the inhibition of immune suppression or immune activation. The following is a brief outline of how such experiments can be performed. Peripheral blood mononuclear cells (PBMCs) can be prepared from human whole blood samples using FicollHypaque separation, and T cells (fraction 2000) can be obtained from the PBMCs by elution. Freshly isolated human T cells can be cultured at 37°C at a concentration of 2 x 10 6T cells can be maintained at a density of 1000 cells / ml in culture medium (RPMI 1640 supplemented with 10% fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin) for up to 2 days. For IL-2-stimulated cell proliferation analysis, T cells are first treated with phytohemagglutinin (PHA) at a final concentration of 10 μg / ml for 72 hours. After washing once with PBS, 6000 cells / well are plated in a 96-well plate and treated with different concentrations of the compounds provided herein in culture medium in the presence of 100 U / ml human IL-2 (ProSpec-TanyTechnoGene; Rehovot, Israel). The plate is incubated at 37°C for 72 hours, and proliferation indicators are assessed using CellTiter-Glo Luminescent reagent according to the manufacturer's suggested protocol (Promega; Madison, WI).

[0256] Example C. In vivo antitumor efficacy The compounds provided herein can be evaluated in human tumor xenograft models in immunodeficient 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). Tumor-bearing animals can then be randomized into drug-treated or vehicle-treated groups, and different doses of the compounds provided herein can be administered by several common routes, including orally, intraperitoneally, or by continuous infusion using an implantable pump. Tumor growth is tracked over time using calipers. Furthermore, tumor samples can be harvested at any time after the start of treatment for analysis as described above (Example B) to evaluate the compound's effect on JAK activity and downstream signaling pathways. In addition, the selectivity of the compound can be assessed using xenograft tumor models driven by other known kinases (e.g., Bcr-Abl), such as the K562 tumor model.

[0257] Example D. Testing of Cutaneous Contact Delayed-Type Hypersensitivity Responses in Mice The compounds provided herein can also be tested for their efficacy (inhibition of JAK targets) in a T cell-driven mouse delayed-type hypersensitivity test model. Mouse cutaneous contact delayed-type hypersensitivity (DTH) response is considered an effective model of clinical contact dermatitis and other T lymphocyte-mediated skin immune disorders, such as psoriasis (Immunol Today. 1998 January; 19(1): 37-44). Mouse DTH shares several characteristics with psoriasis, including immune infiltration, a concomitant increase in inflammatory cytokines, and keratinocyte hyperproliferation. Furthermore, many classes of drugs that are effective in treating psoriasis in the clinic are also effective inhibitors of DTH response in mice (Agents Actions. 1993 January; 38(1-2): 116-21).

[0258] Balb / c mice are sensitized on days 0 and 1 by topical application of the antigen 2,4,dinitro-fluorobenzene (DNFB) to the shaved abdomen. On day 5, ear thickness is measured using an engineer's micrometer. This measurement is recorded and used as a baseline. Both ears of the animals are then challenged with a topical application of DNFB (10 μL on the inner pinna and 10 μL on the outer pinna) at a concentration of 0.2% in a total of 20 μL. 24-72 hours after challenge, the ears are measured again. Treatment with a compound provided herein is given throughout the sensitization and challenge phases (days -1 to 7), or before and throughout the challenge phase (usually the afternoon of day 4 to day 7). Treatment with a test compound (at different concentrations) is administered either systemically or locally (topical application of the treatment to the ear). The efficacy of the test compound is indicated by a reduction in ear swelling compared to the no-treatment condition. Test compounds that caused a reduction of 20% or more were considered effective. In some experiments, mice were challenged but not sensitized (negative control).

[0259] The inhibitory effect of the compounds provided herein (inhibition of JAK-STAT pathway activation) can be confirmed by immunohistochemical analysis. Activation of the JAK-STAT pathway(s) leads to the formation and translocation of functional transcription factors. Furthermore, immune cell influx and increased keratinocyte proliferation should also provide specific expression profile changes in the ear that can be investigated and quantified. Formalin-fixed, paraffin-embedded ear sections (harvested after the challenge phase in the DTH model) are subjected to immunohistochemical analysis using an antibody (clone 58E12, Cell Signaling Technology) that specifically interacts with phosphorylated STAT3. For comparison, mouse ears are treated with the compounds provided herein, vehicle, or dexamethasone (a clinically effective treatment for psoriasis) or no treatment in the DTH model. The test compounds and dexamethasone can produce similar transcriptional changes, both qualitatively and quantitatively, and both the test compounds and dexamethasone can reduce the number of infiltrating cells. Both systemic and local administration of test compounds can produce inhibitory efficacy, ie, reduction in the number of infiltrating cells and inhibition of transcriptional changes.

[0260] Example E. In vivo anti-inflammatory effects The compounds provided herein can be evaluated in rodent or non-rodent models designed to reproduce simple or complex inflammatory responses.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, collagen-induced arthritis in mice or rats, adjuvant-induced arthritis in rats, and collagen antibody-induced arthritis.The therapeutic potential of the compounds provided herein can be evaluated using autoimmune diseases, including, but not limited to, multiple sclerosis, type 1 diabetes, uveoretinitis, thyroiditis, myasthenia gravis, immunoglobulin A nephropathy, myocarditis, airway sensitization (asthma), lupus, or colitis. These models are well established in the research community and familiar to those skilled in the art (Current Protocols in Immunology, Vol. 3., Coligan, JE et al., Wiley Press., Methods in Molecular Biology: Vol. 225, Inflammation Protocols., Winyard, PG and Willoughby, DA, Humana Press, 2003.).

[0261] Example F. Animal Models for Treatment of Dry Eye, Uveitis, and Conjunctivitis Drugs 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 mouse scopolamine model (subcutaneous or transdermal), the mouse Botulinum lacrimal gland model, or a number of naturally occurring rodent autoimmune models resulting in ocular 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 Ophthalmology, 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 ocular glands and eyes (such as the cornea), and, in some cases, the classic Schirmer test, which measures tear production, or a modified version thereof (Barabino et al.). Activity can be assessed by dosing via multiple routes of administration (eg, systemic or local), which can begin before or after measurable disease is present.

[0262] Drugs can be evaluated in one or more preclinical models of uveitis known to those skilled in the art. These include, but are not limited to, experimental autoimmune uveitis (EAU) and endotoxin-induced uveitis (EIU) models. EAU experiments can be performed in rabbits, rats, or mice and can involve passive or active immunization. For example, animals can be sensitized to a relevant immunogen using any of several retinal antigens, and then the animals can be challenged ocularly with the same antigen. The EIU model is more acute and involves local or systemic administration of sublethal doses of lipopolysaccharide. Endpoints for both the EIU and EAU models can include fundus examination, particularly histopathology. These models are reviewed by Smith et al. (Immunology and Cell Biology 1998, 76, 497-512, the entire contents of which are incorporated herein by reference). Activity is assessed by dosing via multiple routes of administration (e.g., systemic or topical), which may be initiated before or after measurable disease is present. Some of the models listed above may also develop scleritis / episcreitis, choroiditis, cyclitis, or iritis, and are therefore useful in investigating the potential activity of compounds for the therapeutic treatment of these diseases.

[0263] Agents can also be evaluated in one or more preclinical models of conjunctivitis known to those skilled 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 immune challenge protocols with antigens such as ovalbumin or ragweed (reviewed in Groneberg, DA, et al., Allergy 2003, 58, 1101-1113, incorporated herein by reference in its entirety). Rat and mouse models are similar in overall design to those of guinea pigs (also reviewed by Groneberg). Activity can be assessed by dosing via multiple routes of administration (e.g., systemic or topical), which can begin before or after the presence of measurable disease. Endpoints for such studies may include, for example, histological, immunological, biochemical, and molecular analysis of ocular tissues (e.g., conjunctiva).

[0264] Example G. In vivo bone protection 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 compounds to affect the 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, the entire contents of which are incorporated herein by reference). Alternatively, bone density and structure can be evaluated in control or compound-treated rodents in models of treatment (e.g., glucocorticoid-induced) osteopenia (Yao, et al., Arthritis and Rheumatism, 2008, 58(6), 3485-3497, and ibid., 58(11), 1674-1686, the entire contents of which are incorporated herein by reference). Additionally, the effects of compounds provided herein on bone resorption and density can be evaluated in the rodent models of arthritis discussed above (Example E). Endpoints for all of these models can vary but often include immunohistology and appropriate biochemical markers of bone remodeling, in addition to histological and radiological assessments.

[0265] Example H. S100A9 Transgenic Mouse Model S100A9 transgenic mice were previously shown to exhibit bone marrow accumulation of MDSCs, accompanied by the development of progressive multilineage cytopenias and cytological dysplasia similar to MDS. Furthermore, early forced maturation of MDSCs by either all-trans retinoic acid treatment or disruption of CD33 signaling by the activating immunoreceptor tyrosine-based activation motif-bearing (ITAM-bearing) adaptor protein (DAP12) rescued the hematological phenotype and reduced disease. This system may be useful for testing the effects of JAK1 inhibition on MDS-like disease in preclinical models. J. Clin. Invest., 123(11):4595-4611 (2013). Therefore, JAK1-selective inhibitors are administered by oral gavage. The ability of compounds to reduce the cytopenias and cytological dysplasia observed in S100A9 transgenic mice was monitored.

[0266] Various modifications of the present disclosure, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims. Each reference, including all patents, patent applications, and publications cited in this application, is hereby incorporated by reference in its entirety. The present application also includes the following aspects. [Aspect 1] [ka] 、 or a salt thereof, said process comprising: [ka] of [ka] to form Compound 1 free base, or a salt thereof. [Aspect 2] 2. The process of embodiment 1, wherein reacting 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. [Aspect 3] 3. The process of embodiment 2, wherein the organic solvent component comprises dimethylformamide (DMF). [Aspect 4] The process of any one of aspects 1 to 3, wherein reacting compound 1x with compound 2x is carried out at a temperature of about 50°C to about 60°C. [Aspect 5] 5. The process of embodiment 4, wherein the temperature is about 60°C. [Aspect 6] The salt of Compound 1 can be prepared by reacting Compound 1 free base with phosphoric acid,

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Claims

【Request Item 1】 【Chemistry 1】 or a salt thereof, comprising: 【Chemistry 2】 of 【Transformation 3】 to form Compound 1 free base, or a salt thereof. The process.

2. 2. The process of claim 1, wherein reacting 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.

3. 3. The process of claim 2, wherein the organic solvent component comprises dimethylformamide (DMF).

4. 4. The process of any one of claims 1 to 3, wherein the reacting of compound 1x with compound 2x is carried out at a temperature of 50°C (±10%) to 60°C (±10%).

5. 5. The process of claim 4, wherein the temperature is 60°C (±10%).

6. The salt of Compound 1 can be prepared by reacting Compound 1 free base with phosphoric acid, 【Chemistry 4】 6. The process of any one of claims 1 to 5, wherein the phosphate salt of Compound 1 is prepared by a process comprising forming

7. 7. The process of claim 6, wherein reacting Compound 1 free base with phosphoric acid is carried out in the presence of a solvent component.

8. 8. The process of claim 7, wherein the solvent component comprises methanol, isopropanol, or a mixture thereof.

9. 9. The process of any one of claims 6 to 8, wherein reacting Compound 1 free base with phosphoric acid is carried out at a temperature of 40°C (±10%) to 70°C (±10%).

10. 10. The process of claim 9, wherein the temperature is between 45°C (±10%) and 55°C (±10%).

11. 11. The process of claim 10, wherein the temperature is 50°C (±10%).

12. 12. The process of any one of claims 6 to 11, wherein the phosphoric acid is an aqueous solution of 85% by weight (±10%) phosphoric acid.

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

14. The process of claim 13, wherein the second solvent component comprises n-heptane. 【Request Item 15】 【Chemistry 5】 15. The process of any one of claims 1 to 14, further comprising preparing compound 2x by a process comprising reacting

16. 16. The process of claim 15, wherein the base is NaOH.

17. 17. The process of claim 15 or 16, wherein the reacting of the compound 2xHCl with a base is carried out at a temperature of 15°C (±10%) to 18°C ​​(±10%). 【Request Item 18】 【Chemistry 6】 18. The process of any one of claims 15 to 17, further comprising preparing the compound 2xHCl by a process comprising reacting

19. 20. The process of claim 18, wherein reacting compound 2b with hydrochloric acid is carried out in the presence of an organic solvent component.

20. 20. The process of claim 19, wherein the organic solvent component comprises 2-propanol.

21. 21. The process of any one of claims 18 to 20, wherein reacting compound 2b with hydrochloric acid is carried out at a temperature of 60°C (±10%) to 65°C (±10%). 【Request Item 22】 【Chemistry 7】 of 【Transformation 8】 22. The process of any one of claims 18 to 21, further comprising preparing compound 2b by a process comprising reacting

23. Reacting compound 2a with 4-bromo-3,5-dimethylpyrazole is ２ HPO ４ 23. The process of claim 22, carried out in the presence of a solvent component and a palladium complex.

24. 24. The process of claim 23, wherein the solvent component comprises 1-propanol, water, or a mixture thereof.

25. 24. The process of claim 23, wherein the palladium complex is [1,1'-bis(di-tert-butylphosphino)ferrocene]dichloropalladium(II) (Pd-118).

26. 26. The process of any one of claims 22 to 25, wherein reacting compound 2a with 4-bromo-3,5-dimethylpyrazole is carried out at a temperature of from 80°C (±10%) to 100°C (±10%).

27. 27. The process of claim 26, wherein the temperature is 90°C (±10%). 【Request Item 28】 【Chemistry 9】 of 【Chemistry 10】 React with 【Chemistry 11】 and forming Compound 2b is reacted with hydrochloric acid to give 【Chemistry 12】 and forming The compound 2xHCl is reacted with a base to give 【Chemistry 13】 and forming 15. The process of any one of claims 1 to 14, further comprising preparing compound 2x by a process comprising: 【Request Item 29】 【Chemistry 14】 and further comprising preparing wherein compound 1x is 【Chemistry 15】 with diethyl cyanomethylphosphonate in the presence of a base, The process of any one of claims 1 to 28.

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

31. 31. The process of claim 30, wherein the organic solvent component comprises tetrahydrofuran, ethanol, or a mixture thereof.

32. further comprising preparing compound 1c, wherein compound 1c is 【Chemistry 16】 with iodobenzene diacetate and 2,2,6,6-tetramethyl-1-piperidinyloxy free radical (TEMPO), The process of any one of claims 29 to 31.

33. further comprising preparing compound 1b, wherein compound 1b is 【Chemistry 17】 of [Chemistry 18] in the presence of DBU, 33. The process of claim 32.

34. further comprising preparing compound 1a, wherein compound 1a is 【Chemistry 19】 of 【Chemistry 20】 in the presence of a base, 34. The process of claim 33. 【Request Item 35】 【Chemistry 21】 of 【Chemistry 22】 reacted with in the presence of a base 【Chemistry 23】 and forming Compound 1a 【Chemistry 24】 and reacted in the presence of DBU 【Chemistry 25】 and forming Compound 1b was reacted with iodobenzene diacetate and TEMPO to give 【Chemistry 26】 and forming Compound 1c is reacted with diethyl cyanomethylphosphonate in the presence of a base to give 【Chemistry 27】 and forming 29. The process of any one of claims 1 to 28, further comprising preparing compound 1x by a process comprising:

36. Formula A: 【Chemistry 28】 1. A process for preparing a compound of formula (I), comprising: 3,5-dimethyl-1H,1′H-4,4′-bipyrazole of formula B: 【Chemistry 29】 with a compound of In the formula, Pg １ is an amine protecting group, The process.

37. Pg １ 37. The process of claim 36, wherein is tert-butoxycarbonyl.

38. 38. The process of claim 36 or 37, wherein said reacting is carried out in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene.

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

40. 39. The process of claim 38, wherein 0.2 (±10%) to 0.3 (±10%) equivalents of the 1,8-diazabicyclo[5.4.0]undec-7-ene are used, based on 1 equivalent of the compound of Formula B.

41. 41. The process of any one of claims 36 to 40, wherein 1.0 (±10%) to 1.1 (±10%) equivalents of the 3,5-dimethyl-1H,1'H-4,4'-bipyrazole are used, based on 1 equivalent of the compound of formula B.

42. 42. The process of any one of claims 36 to 41, wherein the reacting is carried out at room temperature (±10%).

43. 43. The process of any one of claims 36 to 42, wherein the reacting 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. 44. The process of claim 43, wherein the solvent component comprises dimethyl sulfoxide.

45. 44. The process of claim 43, wherein the solvent component comprises dimethyl sulfoxide and methylene chloride.

46. The compound of formula A is deprotected to give a compound of formula C: 【Transformation 30】 46. ​​The process of any one of claims 36 to 45, further comprising forming a compound of formula (I) or a salt thereof.

47. 47. The process of claim 46, wherein said deprotecting comprises reacting said compound of formula A in the presence of a trialkylsilyl halide.

48. 48. The process of claim 47, wherein the trialkylsilyl halide is trimethylsilyl iodide.

49. 49. The process of claim 47 or 48, wherein the deprotection is carried out in the presence of a solvent component.

50. 50. The process of claim 49, wherein the solvent component comprises methylene chloride.

51. 50. The process of claim 49, wherein the solvent component comprises methylene chloride and methanol.

52. 52. The process of any one of claims 47 to 51, wherein the deprotecting is carried out at room temperature (±10%).

53. 53. The process of any one of claims 47 to 52, 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. 54. The process of claim 53, wherein the amine base is triethylamine.

55. 55. The process of claim 53 or 54, wherein 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.

56. 56. The process of claim 55, wherein the solvent component comprises methylene chloride.

57. The free base form of the compound of formula C may be prepared as compound 1a: 【Chemistry 31】 in the presence of a base and an alkali metal halide to give Compound 1: 【Chemistry 32】 57. The process of any one of claims 53 to 56, further comprising forming 2-(2-methyl-2-propanol)-3-hydroxybenzoate, or a salt thereof.

58. 58. The process of claim 57, wherein the base is a bicarbonate base.

59. 58. The process of claim 57, wherein the base is sodium bicarbonate.

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

61. 61. The process of any one of claims 57 to 60, wherein the reacting is carried out at a temperature of from 80°C (±10%) to 90°C (±10%).

62. 62. The process of any one of claims 57 to 61, wherein the reacting 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. 63. The process of claim 62, wherein the solvent component comprises dimethyl sulfoxide.

64. 63. The process of claim 62, wherein the solvent component comprises dimethyl sulfoxide and isopropyl acetate.

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

66. Compound 1 was reacted with hydrochloric acid to give Compound 1 hydrochloride: 【Transformation 33】 65. The process of any one of claims 57 to 64, further comprising forming

67. 67. The process of claim 66, further comprising reacting the Compound 1 hydrochloride salt with a bicarbonate base to form the free base form of Compound 1.

68. 68. The process of claim 67, wherein the bicarbonate base is potassium bicarbonate.

69. The free base form of Compound 1 is reacted with phosphoric acid to form Compound 1 phosphate: 【Transformation 34】 69. The process of claim 67 or 68, further comprising forming

70. 70. The process of claim 69, wherein said reacting is carried out at room temperature (±10%).

71. 71. The process of 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. 72. The process of claim 71 , wherein the solvent component comprises water.

73. 72. The process of claim 71, wherein the solvent component comprises water and isopropyl alcohol.

74. 74. The process of any one of claims 69 to 73, further comprising isolating the compound monophosphate.

75. 75. The process of claim 74, wherein the compound monophosphate is isolated by recrystallization.

76. 76. The process of claim 74 or 75, wherein the compound monophosphate is isolated by recrystallization from a mixture of methanol, isopropanol, and methylcyclohexane.

77. Compound 1 Phosphate: 【Chemistry 35】 A process for preparing 3,5-Dimethyl-1H,1′H-4,4′-bipyrazole is reacted with tert-butyl 3-(cyanomethylene)azetidine-1-carboxylate in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene to give the compound of formula A-1: 【Transformation 36】 and forming a compound of The compound of formula A-1 is deprotected to give a compound of formula C-1: 【Chemistry 37】 or a salt thereof; reacting the compound of formula C-1 with triethylamine to form the free base form of the compound of formula C-1; The free base form of the compound of formula C-1 is reacted with compound 1a: 【Transformation 38】 in the presence of sodium bicarbonate and lithium chloride to give Compound 1: 【Chemistry 39】 and forming Compound 1 is reacted with hydrochloric acid to give Compound 1 hydrochloride: 【Chemistry 40】 and forming reacting Compound 1 hydrochloride with potassium bicarbonate to form the free base form of said Compound 1; reacting the free base form of Compound 1 with phosphoric acid to form Compound 1 phosphate; The process comprising:

78. 78. The process of claim 77, further comprising isolating the compound monophosphate.

79. 79. The process of claim 78, wherein the compound monophosphate is isolated by recrystallization.

80. 80. The process of claim 78 or 79, wherein the compound monophosphate is isolated by recrystallization from a mixture of methanol, isopropanol, and methylcyclohexane.

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