Fumarate crystal form of JAK tyrosine kinase inhibitor as well as preparation method and application of fumarate crystal form

Abstract

The invention discloses a polymorphic form of a fumarate of a 2-(3-(3-amino-4-(7H-pyrrolo [2, 3-d] pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl) isonicotinoyl) piperidine-4-yl) azetidin-3-yl) acetonitrile compound, and the crystal form A of the polymorphic form comprises the following characteristic peaks measured by reflection angles of 2 theta in an X-ray powder diffraction pattern: 8.69, 9.82, 11.58, 14.79, 15.54, 16.05, 17.82, 18.02, 18.55, 19.68 and 20.39. The invention further discloses a preparation method of the polymorphic form. The crystal form is good in stability and high in solubility. The invention further provides a preparation method of the fumarate crystal form and application of the fumarate crystal form in preparation of a JAK1 inhibitor.

Description

A fumarate crystal form of a JAK tyrosine kinase inhibitor, and its preparation method and application Technical Field

[0001] The present disclosure relates to a fumarate salt of a 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinoyl)piperidin-4-yl)azetidin-3-yl)acetonitrile compound and a crystalline form thereof, as well as a preparation method and application of the crystalline form. Background Art

[0002] 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinyl)piperidin-4-yl)azetidin-3-yl)acetonitrile belongs to the JAK class of small molecule non-receptor tyrosine kinase (PTK) inhibitors. Its JAK-STAT signaling pathway is closely related to inflammatory cytokines and tumors, and is widely involved in cell proliferation, differentiation, metastasis, apoptosis, and immune regulation in human health and disease. The compound 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinyl)piperidin-4-yl)azetidin-3-yl)acetonitrile has great application potential because it maintains excellent JAK1 enzyme inhibition while avoiding potential side effects caused by inhibiting other JAK kinases (JAK2 and / or JAK3). The structural formula is as follows:

[0003] The synthesis method of this compound is disclosed in invention patent CN109867676A, but the salt form of the compound is not involved. No other literature has reported the salt form of 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinyl)piperidin-4-yl)azetidin-3-yl)acetonitrile. The salt form of a drug is of great significance to the physical properties, bioavailability, quality and process of the drug.

[0004] Summary of the Invention

[0005] The present disclosure studies various salts of 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinyl)piperidin-4-yl)azetidin-3-yl)acetonitrile and their crystalline forms. Based on a comprehensive analysis of some properties, such as dissolution rate, solubility in simulated gastrointestinal fluid, stability, hygroscopicity, and pharmacokinetic properties, it is shown that Form A of the fumarate salt (Formula I) of the compound has obvious advantages and better drugability than other salts and their crystalline forms.

[0006] According to one aspect, the present disclosure provides Form A of a fumarate salt of 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinoyl)piperidin-4-yl)azetidin-3-yl)acetonitrile compound (Formula I), wherein the Form A has an X-ray powder diffraction pattern having characteristic peaks at the following positions represented by 2θ: 8.69, 9.82, 11.58, 14.79, 15.54, 16.05, 17.82, 18.02, 18.55, 19.68, and 20.39.

[0007] According to some embodiments, the X-ray powder diffraction pattern of Form A of Formula I is shown in FIG1 .

[0008] According to some embodiments, Form A of Formula I melts at 193 ± 3 °C.

[0009] According to another aspect, the present disclosure also provides a method for preparing the crystalline form A, comprising the step of recrystallizing 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinoyl)piperidin-4-yl)azetidin-3-yl)acetonitrile fumarate (Formula I) in a mixed solvent of one or more of ethanol, tetrahydrofuran, and n-heptane.

[0010] According to some embodiments, the preparation method further comprises the step of reacting 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinoyl)piperidin-4-yl)azetidin-3-yl)acetonitrile with fumaric acid. In some embodiments, the molar ratio of 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinoyl)piperidin-4-yl)azetidin-3-yl)acetonitrile to fumaric acid can be 1.0:0.5 to 2.0, for example, 0.5 to 1.0.

[0011] According to another aspect, the present disclosure provides a pharmaceutical composition comprising the crystalline form A of the fumarate salt and a pharmaceutically acceptable carrier.

[0012] According to another aspect, the present disclosure provides use of the crystalline form A of the fumarate salt in the preparation of a JAK1 inhibitor.

[0013] According to some embodiments, the JAK1 inhibitor can be used to treat autoimmune-related diseases, including psoriasis, atopic dermatitis, vitiligo, pruritus, scleroderma, alopecia areata, alopecia totalis, alopecia universalis, androgenic alopecia, ankylosing spondylitis, psoriatic arthritis, rheumatoid arthritis, Crohn's disease, ulcerative colitis, systemic lupus erythematosus, and graft-versus-host disease.

[0014] The fumarate or hemifumarate salts mentioned in the present disclosure all refer to the compound of formula I. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG1 is an X-ray powder diffraction (XRD) pattern of the fumarate salt form A.

[0016] FIG2 is a differential scanning calorimetry (DSC) diagram of fumarate salt form A.

[0017] FIG3 is an X-ray powder diffraction (XRD) pattern of the fumarate salt form B.

[0018] FIG4 is a differential scanning calorimetry (DSC) diagram of fumarate salt Form B.

[0019] FIG5 is a thermogravimetric analysis (TGA) diagram of the fumarate salt form A.

[0020] Figure 6 is the NMR of the fumarate salt form A ( 1 HNMR) diagram.

[0021] FIG7 is an X-ray powder diffraction (XRD) pattern of succinate salt form A.

[0022] FIG8 is a differential scanning calorimetry (DSC) diagram of succinate salt form A.

[0023] Figure 9 is the NMR of succinate form A ( 1 HNMR) diagram.

[0024] FIG10 is an X-ray powder diffraction (XRD) pattern of malonate salt form A.

[0025] FIG11 is a differential scanning calorimetry (DSC) diagram of malonate salt form A.

[0026] FIG12 is a three-dimensional structure diagram of a fumarate single crystal.

[0027] FIG13 is a unit cell diagram of a fumarate single crystal.

[0028] Figure 14 shows the dynamic vapor sorption (DVS) results of different salt types.

[0029] Detailed Description of the Invention

[0030] The following describes in detail the studies conducted on various salts of 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinoyl)piperidin-4-yl)azetidin-3-yl)acetonitrile and their crystalline forms.

[0031] In the salt screening, different salts were prepared by mixing the free base with the acid in a 1:1 molar ratio in acetone, acetonitrile, ethanol, isopropanol, and tetrahydrofuran. Table 1 shows the physicochemical properties of the different salts.

[0032] The present disclosure studies various crystalline forms of each salt form, for example, by measuring X-ray diffraction patterns, differential scanning calorimetry patterns, and thermogravimetric analysis patterns. Form A of 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinyl)piperidin-4-yl)azetidin-3-yl)acetonitrile fumarate has an X-ray powder diffraction pattern as shown in Figure 1, with a measurement error of ±0.2 degrees in 2θ, and contains multiple absorption peaks between 0 and 50 degrees, as shown in Table 2.

[0033] Table 2: d-values ​​and 2θ angles of Form A

[0034] Form B of 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinoyl)piperidin-4-yl)azetidin-3-yl)acetonitrile fumarate has an X-ray powder diffraction pattern as shown in Figure 3, with a measurement error of ±0.2 degrees in 2θ, and contains multiple absorption peaks between 0 and 50 degrees as shown in Table 3.

[0035] Table 3: d-values ​​and 2θ angles of Form B

[0036] Form A of 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinyl)piperidin-4-yl)azetidin-3-yl)acetonitrile succinate has an X-ray powder diffraction pattern as shown in Figure 7, with a measurement error of ±0.2 degrees in 2θ, and contains multiple absorption peaks between 0 and 50 degrees, as shown in Table 4.

[0037] Table 4: d-values ​​and 2θ angles of Form A

[0038] 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinoyl)piperidin-4-yl)azetidin-3-yl)acetonitrile malonate Form A has an X-ray powder diffraction pattern as shown in Figure 10, with a measurement error of ±0.2 degrees in 2θ, and contains multiple absorption peaks between 0 and 50 degrees as shown in Table 5.

[0039] Table 5: d-values ​​and 2θ angles of Form A

[0040] In the five tables above, the relative strength expressed by numbers is defined as follows:

[0041] In the present disclosure, the X-ray diffraction patterns were measured using the following method: Instrument: PANalytical Empyrean X-ray diffractometer: Method: Target: Cu: K-Alpha; Wavelength Tube voltage: 45kV; tube current: 40mA; scanning range: 3~40°; scanning speed: 0.2 seconds per step, 0.013° per step.

[0042] In the present disclosure, differential scanning calorimetry (DSC) analysis is measured using the following method: Instrument: Discovery DSC (TA Instruments, US) differential scanning calorimeter; Method: Take a sample (~5 mg) and place it in a DSC aluminum pan for testing, the method is 30°C-300°C, and the heating rate is 10°C / min.

[0043] It should be noted that in X-ray diffraction spectroscopy, the diffraction pattern obtained from a crystalline compound is often characteristic of a specific crystal form. The relative intensities of the bands (especially at low angles) may vary due to preferential orientation effects caused by differences in crystallization conditions, particle size, and other measurement conditions. Therefore, the relative intensities of the diffraction peaks are not characteristic of the intended crystal form. When determining whether a crystal form is identical to a known crystal form, the relative positions of the peaks, rather than their relative intensities, should be considered. Furthermore, for any given crystal form, the positions of the peaks may vary slightly, as is well known in the art of crystallography. For example, peak positions can shift due to temperature fluctuations, sample movement, or instrument calibration during sample analysis, resulting in a measurement error of approximately ±0.2° in 2θ values. Therefore, this error should be taken into account when determining the structure of each crystal form. In XRD patterns, peak positions are typically expressed in 2θ angles or interplanar distances d. The two are converted to a simple formula: d = λ / 2sinθ, where d represents the interplanar distance, λ represents the wavelength of the incident X-ray, and θ is the diffraction angle. For the same compound and the same crystal form, the peak positions of their XRD spectra are generally similar, but the relative intensity errors may be large. It should also be noted that in the identification of mixtures, some diffraction lines may be missing due to factors such as a decrease in content. In such cases, it is necessary to rely on the full spectrum of bands observed in the high-purity sample. Sometimes, even a single band may be characteristic for a given crystal.

[0044] It should be noted that DSC measures the transition temperature when a crystal absorbs or releases heat due to changes in its crystal structure or melting. For the same crystalline form of the same compound, the error in thermal transition temperatures and melting points in consecutive analyses is typically within approximately 5°C. When a compound is said to have a given DSC peak or melting point, this refers to the DSC peak or melting point ±5°C. DSC provides an auxiliary method for distinguishing different crystalline forms. Different crystal forms can be identified based on their distinct transition temperature characteristics. DETAILED DESCRIPTION

[0045] The present invention discloses the synthesis of a 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinoyl)piperidin-4-yl)azetidin-3-yl)acetonitrile compound, and the preparation of fumarate salt crystal forms A and B, succinate salt, and malonate salt.

[0046] The following examples provide further non-limiting details of the disclosed technical solution. These examples should not be considered as limiting the scope of the present invention, but are merely exemplary and typical representations of the present invention.

[0047] Example 1 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinyl)piperidin-4-yl)azetidin-3-yl)acetonitrile (I)

[0048] Step A 2-{4-[7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl]-1H-pyrazol-3-yl}isoindole-1,3-dione

[0049] To a 3 L reaction flask, add 1.5 L of toluene, 165 g (0.50 mol, 1.0 eq) of 4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-3-amine, and 81.5 g (0.55 mol, 1.1 eq) of phthalic anhydride with stirring at room temperature. Install a water separator and heat to reflux for 8 h. Then, stop heating, cool, and filter. The resulting solid was dried at 55-60°C to constant weight. The product weighed 191 g, for a yield of 83.1%.

[0050] 1 H-NMR (400MHz, DMSO-d6): δ13.85(s,1H),8.97(d,1H,J=1.6Hz),8.25(s,1H),8.14–7.91(m,4H),7.80(d,1H,J=3.7H z),7.12(d,1H,J=3.7Hz),5.61(s,2H),3.52(t,2H,J=8.0Hz),0.82(t,2H,J=8.0Hz),-0.09(s,9H); m / z=461.57[M+H] + .

[0051] Step B: Benzyl 4-{3-(cyanomethyl)-3-[3-(1,3-phthaloyl-2-yl)-4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}piperidine-1-carboxylate

[0052] In a 2L reaction flask, 138 g (0.30 mol, 1.0 eq) of 2-{4-[7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl]-1H-pyrazol-3-yl}isoindole-1,3-dione, 9.13 g (0.06 mol, 0.2 eq) of 1,8-diazabicyclo[5.4.0]undec-7-ene, and N,N-dimethylformamide were added. 450 mL of amide and 102.7 g (0.33 mol, 1.1 eq) of benzyl 4-(3-(cyanomethyl)azetidin-1-yl)piperidine-1-carboxylate were heated to 30-40°C and stirred for 3 h. The reaction was stopped and the reaction solution was extracted with 5 L of water and 1.5 L of ethyl acetate. After ethyl acetate was concentrated, 600 mL of isopropanol was added, stirred for crystallization, and filtered. The resulting solid was dried at 55-60°C to obtain an off-white solid. The weight was 200.8 g, with a yield of 86.9%.

[0053] 1 H-NMR (400MHz, DMSO-d6): δ9.08(s,1H),8.28(s,1H),8.08–7.97(m,4H),7.87(d,1H,J =3.7Hz),7.47–7.31(m,5H),7.20(d,1H,J=3.7Hz),5.63(s,2H),5.10(s,2H),3.87(t,4 H,J=13.2Hz),3.65(d,4H,J=8.0Hz),3.52(t,2H,J=8.0Hz),3.35(s,3H),3.07(s,2H),1 .73(d,2H,J=10.3Hz,),1.21(dd,2H),-0.09(s,9H,J=24.3,14.7Hz); m / z=772.95[M+H] + .

[0054] Step C: Benzyl 4-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(cyanomethyl)azetidin-1-yl)piperidine-1-carboxylate

[0055] To a 2 L reaction flask, add 154 g (0.20 mol, 1.0 eq) of benzyl 4-{3-(cyanomethyl)-3-[3-(1,3-phthaloyl-2-yl)-4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}piperidine-1-carboxylate and 700 mL of acetonitrile. Stir at room temperature, then dropwise add 85.2 g (0.60 mol, 3.0 eq) of boron trifluoride in ether. After addition, raise the temperature to 40-50°C and stir for 5 h. Stop the reaction, concentrate the reaction solution, adjust the pH to 9-10 with saturated sodium carbonate solution, extract with 1 L of ethyl acetate, and concentrate to obtain an oily product.

[0056] Dissolve the oily product in 500 mL of ethanol, add 100 g (1.2 mol, 6.0 eq) of hydrazine hydrate (60% content), and heat to 70-80°C with stirring for 5 hours. Stop the reaction, add 2 L of water, cool, stir, and crystallize. Filter, wash the filter cake with 300 mL of water, and dry at 55-60°C to obtain a light yellow solid. Weighing 92.1 g, yield 90.0%.

[0057] 1 H-NMR (400MHz, DMSO-d6): δ12.08(s,1H),8.69(s,1H),8.53(s,1H),7.57(d,1H,J=3 .5Hz),7.30-7.46(m,5H),7.09(d,1H,J=3.6Hz),6.34(d,2H,J=11.4Hz),5.10(s,2H ),3.76-3.90(m,2H),3.70(d,2H,J=8.0Hz),3.44-3.56(m,4H),3.06(s,2H),2.44(d d,1H,J=10.3,6.9Hz),1.69(d,2H,J=10.2Hz),1.12-1.24(m,2H); m / z=512.59[M+H] + .

[0058] Step D: Preparation of 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(piperidin-4-yl)azacycl-3-yl)acetonitrile.

[0059] To a 5 L reaction flask, add 76.7 g (0.15 mol, 1.0 eq) of benzyl 4-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(cyanomethyl)azetidin-1-yl)piperidine-1-carboxylate and 760 mL of methanol / tetrahydrofuran. Heat to 50-60°C and stir to dissolve. Add 7.6 g of Pd / C (0.015 mol, 0.1 eq) and allow reduction to proceed for 8 h. The reaction is then stopped, cooled, filtered, and concentrated. 500 mL of isopropyl ether is added, stirred to allow crystallization, filtered, and the filter cake is dried at 55-60°C to obtain a light yellow solid. The solid weighs 52.1 g, with a yield of 92.0%.

[0060] 1 H-NMR (400MHz, DMSO-d6): δ8.69 (s, 1H), 8.53 (s, 1H), 7.57 (d, 1H, J = 3.5Hz), 7.09 (d, 1H, J = 3.6Hz), 6.34 (d, 2H, J = 11.2Hz), 3.66 (d, 2H, J = 8.1Hz), 3. 41-3.53(m,6H),2.94(d,2H,J=11.9Hz),2.44(t,2H,J=11.1Hz),2.24(t,1 H, J=9.6Hz), 1.65 (d, 2H, J=10.1Hz), 0.74-1.15 (m, 2H); m / z=378.46[M+H] + .

[0061] Step E: Preparation of 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinoyl)piperidin-4-yl)azetidin-3-yl)acetonitrile (I).

[0062] In a 2L reaction flask, 50.0 g (0.13 mol, 1.0 eq) of 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(piperidin-4-yl)azacyclyl)acetonitrile, 250 ml of N,N-dimethylformamide / 250 ml of acetonitrile, 30.7 g (0.16 mol, 1.2 eq) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(piperidin-4-yl)azacyclyl)acetonitrile were added. 18.4 g (0.16 mol, 1.2 eq) of succinimide, 39.5 g (0.39 mol, 3.0 eq) of triethylamine, and 29.3 g (0.14 mol, 1.1 eq) of 3-fluoro-2-trifluoromethylisonicotinic acid were stirred and dissolved. The mixture was heated to 30-40°C and reacted for 4 h. The reaction was stopped, and 2.5 L of water and 1.5 L of ethyl acetate were added for extraction and concentration. 200 mL of ethanol was added for crystallization, and the mixture was filtered. The filter cake was dried at 55-60°C to obtain a light yellow solid weighing 65.7 g, with a yield of 87.25%.

[0063] 1 H NMR (400MHz, DMSO-d6): δ12.08(s,1H),8.69(q,2H,J=4.8Hz),8.54(s,1H),7.94(t,1H, J=4.7Hz),7.52-7.65(m,1H),7.09(dd,1H,J=3.4,1.5Hz),6.35(s,2H),3.72(d,2H,J=7. 6Hz),3.42-3.58(m,6H),3.26-3.33(m,1H),3.12(t,1H,J=10.0Hz),2.58(s,1H),1.81(d ,1H,J=10.2Hz),1.68(d,1H,J=10.4Hz),1.29(dd,2H,J=31.2,8.9Hz); m / z=569.54[M+H] + .

[0064] Example 2 Free Base Form I

[0065] Take 2.0 g of the compound obtained in Example 1, add 6.0 ml of acetonitrile and stir to dissolve, stir and crystallize at room temperature 20-30°C for 16 h, filter, rinse the filter cake with 2 ml of acetonitrile, and dry under reduced pressure at 50°C to obtain 1.62 g of the product.

[0066] Example 3 Free Base Form II

[0067] Take 2.0 g of the compound obtained in Example 1, add 10 ml of acetonitrile and 10 ml of isopropyl acetate mixed solvent, heat to 70 ° C, dissolve and filter, cool to crystallize, filter, rinse the filter cake with 2 ml of mixed solvent, and dry under reduced pressure at 50 ° C to obtain 1.03 g of product.

[0068] Example 4 Free Base Form III

[0069] 1.8 g of Form I obtained in Example 2 was suspended in 7 ml of ethanol and stirred at room temperature overnight. 3 ml of methyl tert-butyl ether was then added as an antisolvent and stirred for several hours. The solid was collected by filtration and dried under vacuum at 40°C for 4 hours to obtain 1.4 g of compound of Formula I.

[0070] Example 5 Free Base Form IV

[0071] 2.0 g of the compound obtained in Example 2 was added to 20 ml of water, heated to 60-70° C., stirred and crystallized for 6 hours, filtered, the filter cake was rinsed with 10 ml of water, and dried under reduced pressure at 50° C. to obtain 1.80 g of the product.

[0072] Example 6 Fumarate Crystal Form A

[0073] Take 2.0 g of the compound obtained in Example 5 and 0.41 g of fumaric acid, add 18 ml of tetrahydrofuran and 3 ml of ethanol, stir evenly, heat to 60-70 ° C, stir to dissolve and clarify, add 18 ml of n-heptane after 0.5 h, slowly cool and crystallize for 12 hours, filter, and dry under reduced pressure at 50 ° C to obtain the product.

[0074] NMR analysis of the fumarate salt form A showed an acid-base ratio of 0.5 / 1, indicating a hemi-fumarate salt. Form A had a weight loss of approximately 0.8% before 80°C on the TGA spectrum, and two endothermic peaks were shown on the DSC spectrum. The first broad peak was presumably caused by dehydration / solvent, the second sharp endothermic peak was caused by melting, and the partially overlapping exothermic peaks were caused by decomposition. After drying at 80°C on the DSC, the fumarate salt form A still had a certain weight loss on the TGA test, but it was easy to quickly absorb moisture from the air, indicating that the stable state of the fumarate salt form A may be a channel hydrate, and the single crystal will be further characterized later.

[0075] The XRD pattern of fumarate salt form A is shown in FIG1 .

[0076] The DSC spectrum of fumarate form A is shown in FIG2 .

[0077] The TGA spectrum of fumarate crystal form A is shown in Figure 5

[0078] The NMR spectrum of the fumarate salt form A is shown in FIG6 .

[0079] Example 6 Fumarate Crystal Form B

[0080] Take 2.0 g of the compound obtained in Example 4 and 0.41 g of fumaric acid, add them to 18.0 ml of acetonitrile and stir evenly, heat to 60-70 ° C and stir to crystallize, cool to 15-25 ° C after 0.5 h, crystallize for 12 hours, filter, rinse the filter cake with 2 ml of acetonitrile, and dry under reduced pressure at 50 ° C to obtain the product.

[0081] A typical XRD pattern of fumarate form B is shown in FIG3 .

[0082] A typical DSC spectrum of fumarate form B is shown in FIG4 .

[0083] Example 7 Preparation of succinate

[0084] 100 mg of the compound obtained in Example 4 and 20.79 mg of succinic acid were added to 1.0 ml of ethanol and stirred evenly. The mixture was stirred at room temperature (25-35°C) for 12 h to crystallize. The product was filtered and dried under reduced pressure at 50°C to obtain the product. NMR analysis revealed a ratio of free base to succinic acid of 1:0.5, indicating a hemisuccinate.

[0085] A typical XRD pattern of succinate form A is shown in FIG7 .

[0086] A typical DSC spectrum of succinate salt form A is shown in FIG8 .

[0087] The NMR spectrum of succinate salt Form A is shown in FIG9 .

[0088] Example 8 Preparation of Malonate

[0089] 100 mg of the compound obtained in Example 4 and 18.32 mg of malonic acid were added to 1.0 ml of ethanol and stirred evenly. The mixture was stirred at room temperature (25-35° C.) for 12 h to crystallize. The mixture was filtered and dried under reduced pressure at 50° C. to obtain the product.

[0090] A typical XRD pattern of malonate form A is shown in FIG10 .

[0091] A typical DSC spectrum of malonate form A is shown in FIG11 .

[0092] Example 9 Single Crystal Study of Fumarate

[0093] 20 mg of the compound obtained in Example 1 was dissolved in 3 ml of methanol, 4.1 mg of fumaric acid was added, and the mixture was evaporated for 6 hours and then diffused into about 1 ml of ether.

[0094] Select 0.15×0.12×0.1mm from the above slurry 3 The selected crystals were fixed to a thin glass fiber with a small amount of non-sticky grease and mounted on a Bruker Apex II single crystal diffractometer equipped with a rotating copper anode at room temperature.

[0095] The fumarate salt single crystals were characterized by unit cell parameters approximately equal to those listed in Table 6. The unit cell parameters were measured at a temperature of approximately 25°C.

[0096] Table 6 Unit cell parameters

[0097] Structural solution and modification Monoclinic space group P21 / with four formula units in the unit cell C This structure contains a 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isoamido)piperidin-4-yl)azetidin-3-yl)acetonitrile cation, a fumarate anion, and water in a ratio of 1:0.5:1.5, indicating that the free base and fumaric acid in Salt Form A form a 1:0.5 salt. The atomic coordinates of the compound calculated at 25°C for a single crystal are shown in Table 7.

[0098] Table 7 Single crystal atomic coordinates

[0099] The spatial three-dimensional structure diagram of the fumarate single crystal is shown in Figure 12.

[0100] The unit cell diagram of the fumarate single crystal is shown in FIG13 .

[0101] Example 10 Determination of solubility

[0102] 1. Determination of the dissolution rate of different types of salt

[0103] Approximately 2.50 g of each of the succinate Form A, malonate Form A, and fumarate Form A were weighed and added to pH 4.5 and 6.8 buffer solutions to prepare 5 mg / mL suspensions. The resulting suspensions were stirred at 37°C at 75 rpm, and samples were collected and analyzed at 5, 30, 60, and 240°C. Approximately 1 ml of the suspension was aspirated with a syringe and filtered through a membrane. After dilution several times, the filtrate was subjected to HPLC analysis for solubility using the area normalization method.

[0104] Table 8 Tests of different crystal forms at different pH values

[0105] The results showed that the solubility of the fumarate salt form A at pH = 4.5 and pH = 6.8 was 5 to 10 times that of the free base, and the solubility of the fumarate salt was better than that of the succinate salt and the malonate salt.

[0106] 2. Determination of Solubility of Fumarate, Succinate and Malonate in Simulated Intestinal Fluid

[0107] About 15 mg of solid sample was weighed and added to 5 mL of FaSSIF and FeSSIF solutions respectively to prepare suspensions. The suspensions were shaken at 100 rpm in a PLS200Plus water bath at 37°C. After a certain period of time, samples were taken for HPLC, pH, and XRPD testing (0.5 h, 2 h, and 24 h).

[0108] Table 9 Solubility of different salts in simulated intestinal fluid

[0109] From the above data, it can be seen that the solubility after a meal is greater than the solubility before a meal, and both are greater than 0.16 mg / mL. The solubility of the three salts in FaSSIF and FeSSIF media is relatively good, and the fumarate salt has the best solubility.

[0110] 3. Stability investigation

[0111] Appropriate amounts of succinate Form A, fumarate Form A, and malonate Form A were weighed into a sample bottle and placed in an open container at 60°C and 40°C / 75% RH for 7 days. Purity changes after 7 days were measured.

[0112] Table 10 Stability test results of different salt types

[0113] The results showed that the succinate salt form and the fumarate salt form A were relatively stable at both 60°C and 40°C / 75% RH for 7 days, while the malonate salt form was significantly degraded and had poor stability.

[0114] 4. Hygroscopicity investigation

[0115] 100 mg samples of succinate Form A, fumarate Form A, and malonate Form A were weighed and tested for dynamic vapor sorption (DVS) simultaneously. After the test, the solids were tested for XPRD. As shown in Figure 20, malonate Form A absorbed approximately 3.2% moisture at 70% RH during a slow rise in humidity. It rapidly absorbed moisture when the humidity was above 70% RH (14% water content at 90% RH). It contained 11% water at 50% RH during a drop in humidity. It rapidly dehydrated when the humidity was below 50% RH. Combining the stability results and the XPRD patterns before and after DVS, it can be inferred that malonate crystal transformation may have occurred during the DVS process. Both the hemisuccinate and fumarate forms absorbed 1.7% moisture at 80% RH, indicating slight hygroscopicity.

[0116] The dynamic vapor sorption (DVS) results of the above three crystal forms are shown in FIG14 .

[0117] 5. Pharmacokinetic (PK) Determination in Adult Male / Female SD Rats

[0118] Healthy adult female SD rats were obtained from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. Three groups were divided into this study and administered by oral gavage at a dose of 10 mg / kg. Animals receiving oral gavage were fasted overnight before the experiment and from 10 hours before to 4 hours after dosing. Blood samples were collected 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, and 24 hours after gavage. After isoflurane anesthesia using a small animal anesthesia machine, 0.4 ml of whole blood was collected from the retinal venous plexus. The sample was centrifuged at 4000 rpm for 20 minutes at 4°C. The separated plasma was placed in labeled EP tubes. Plasma samples should be immediately stored in an ultra-low temperature freezer until analysis. Plasma samples should be stored at -70°C until analysis. Plasma samples were extracted using protein precipitation, and the extracts were analyzed by LC / MS / MS.

[0119] It can be seen from the above PK data that the fumarate crystal form A has better pharmacokinetic properties than the hemisuccinate crystal form and the malonate crystal form.

[0120] Comprehensive analysis shows that the dissolution rate, solubility in simulated gastrointestinal fluid, stability, hygroscopicity and pharmacokinetic properties of fumarate salt form A all have obvious advantages, and have better drugability than other salts.

Claims

1. Form A of the fumarate salt of 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinyl)piperidin-4-yl)azetidin-3-yl)acetonitrile, It is characterized in that The structure of the fumarate is shown in Formula I The X-ray powder diffraction pattern of the crystalline form A has characteristic peaks at the following positions represented by 2θ: 8.69, 9.82, 11.58, 14.79, 15.54, 16.05, 17.82, 18.02, 18.55, 19.68 and 20.

39.

2. The crystalline form A of the fumarate according to claim 1, It is characterized in that Its X-ray powder diffraction pattern is shown in FIG1 .

3. The crystalline form A of the fumarate according to claim 1 or 2, It is characterized in that It melts at 193±3°C.

4. A method for preparing the crystalline form A of the fumarate salt according to claim 1, 2 or 3, It is characterized in that The compound of formula I is recrystallized in one or more mixed solvents of ethanol, tetrahydrofuran and n-heptane to obtain Form A.

5. The method according to claim 5 or 6, It is characterized in that The method also includes forming compound I by reacting 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinyl)piperidin-4-yl)azetidin-3-yl)acetonitrile with fumaric acid, wherein the molar ratio of the two is 1.0:0.5-2.

0.

6. A pharmaceutical composition, It is characterized in that It comprises the crystalline form A of the fumarate according to any one of claims 1 to 3.

7. Use of the crystalline form A of the fumarate according to any one of claims 1 to 3 in the preparation of a JAK1 inhibitor.

8. The use according to claim 7, It is characterized in that The JAK1 inhibitor can be used to treat autoimmune-related diseases selected from psoriasis, atopic dermatitis, vitiligo, pruritus, scleroderma, alopecia areata, alopecia totalis, alopecia universalis, androgenic alopecia, ankylosing spondylitis, psoriatic arthritis, rheumatoid arthritis, Crohn's disease, ulcerative colitis, systemic lupus erythematosus or graft-versus-host disease.

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