Polymorphic substance of JAK tyrosine kinase inhibitor and application thereof

Abstract

The present application relates to a polymorphic form 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 of formula (I) and a pharmaceutical use thereof.

Description

A polymorph of a JAK tyrosine kinase inhibitor and its use Technical Field

[0001] The present application relates to a polymorph 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)isonicotinyl)piperidin-4-yl)azetidin-3-yl)acetonitrile compound, a preparation method thereof, and pharmaceutical uses thereof. 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 crystal form of the compound is not involved. There is no other literature reporting the crystal 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, so the crystal form needs to be studied.

[0004] Summary of the Invention

[0005] The present application provides crystalline forms I, II, III, IV and V 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 methods for their preparation.

[0006] According to one aspect of the present application, the present application provides a crystalline form I 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, whose X-ray powder diffraction pattern has characteristic peaks at the following positions represented by 2θ: 5.17, 5.55, 6.30, 8.67, 10.34, 15.96, 19.06, 19.75, 20.39, and 21.42.

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

[0008] According to another aspect of the present application, the present application provides a crystalline form II 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, whose X-ray powder diffraction pattern has characteristic peaks at the following positions represented by 2θ: 5.55, 6.85, 7.89, 9.78, 13.78, 15.89, 16.38, 16.88, 18.05, 20.42, and 24.00.

[0009] According to some embodiments, the X-ray powder diffraction pattern of Form II is shown in FIG2 .

[0010] According to another aspect of the present application, the present application provides a crystalline form III 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, whose X-ray powder diffraction pattern has characteristic peaks at the following positions represented by 2θ: 8.76, 9.53, 10.17, 15.11, 16.70, 19.18, 20.49, 20.79, 22.33, and 25.18.

[0011] According to some embodiments, the X-ray powder diffraction pattern of Form III is shown in FIG4 .

[0012] According to another aspect of the present application, the present application provides Form IV 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, whose X-ray powder diffraction pattern has characteristic peaks at the following positions represented by 2θ: 7.95, 11.57, 12.08, 14.66, 16.04, 16.60, 18.12, 18.53, 18.96, 19.73, 23.32, and 25.24.

[0013] According to some embodiments, the X-ray powder diffraction pattern of Form IV is shown in FIG6 .

[0014] According to some embodiments, Form IV melts at 224±3° C., and its DSC graph is shown in FIG7 .

[0015] According to another aspect of the present application, the present application provides a crystalline form V 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, whose X-ray powder diffraction pattern has characteristic peaks at the following positions represented by 2θ: 9.73, 11.11, 13.03, 14.20, 14.66, 15.74, 17.10, 17.64, 18.35, 18.85, 20.09, and 23.33.

[0016] According to some embodiments, the X-ray powder diffraction pattern of Form V is shown in FIG8 .

[0017] According to some embodiments, Form V melts at 219±3° C., and its DSC graph is shown in FIG9 .

[0018] According to another aspect of the present application, the present application provides a pharmaceutical composition comprising any of the aforementioned crystal forms and a pharmaceutically acceptable carrier.

[0019] According to another aspect of the present application, the present application provides a use of any of the aforementioned crystal forms in the preparation of a JAK1 inhibitor. According to some embodiments, the JAK1 inhibitor is 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG1 is an X-ray powder diffraction (XRD) pattern of Form I.

[0021] FIG2 is an X-ray powder diffraction (XRD) pattern of Form II.

[0022] FIG3 is a differential scanning calorimetry (DSC) diagram of Form II.

[0023] FIG4 is an X-ray powder diffraction (XRD) pattern of Form III.

[0024] FIG5 is a differential scanning calorimetry (DSC) diagram of Form III.

[0025] FIG6 is an X-ray powder diffraction (XRD) pattern of Form IV.

[0026] FIG7 is a differential scanning calorimetry (DSC) diagram of Form IV.

[0027] FIG8 is an X-ray powder diffraction (XRD) pattern of Form V.

[0028] FIG9 is a differential scanning calorimetry (DSC) diagram of Form V.

[0029] Figure 10 is the XRD pattern of Form II / III competitive beating.

[0030] Figure 11 is the XRD pattern of competitive beating of Form III / IV / V.

[0031] FIG12 is a graph showing the stability of Form III at 25° C. / 60% RH for 7 days.

[0032] FIG13 is a graph showing the stability of Form IV at 25° C. / 60% RH for 7 days.

[0033] FIG14 is a graph showing the stability of Form V at 25° C. / 60% RH for 7 days.

[0034] Figure 15 is a graph showing the crystalline stability of Form III / IV / V under accelerated and long-term conditions for 7 days.

[0035] Detailed Description of the Invention

[0036] Form I 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 has an X-ray powder diffraction pattern as shown in Figure 1, a measurement error of 2θ of ±0.2 degrees, and contains multiple absorption peaks between 0 and 50 degrees, as shown in Table 1.

[0037] Table 1: d-values ​​and 2θ angles of Form I

[0038] Form II 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 has an X-ray powder diffraction pattern as shown in Figure 2, 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.

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

[0040] Form III 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 has an X-ray powder diffraction pattern as shown in Figure 4, 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.

[0041] Table 3: d-values ​​and 2θ angles of Form III

[0042] Form IV 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 has an X-ray powder diffraction pattern as shown in Figure 6, 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.

[0043] Table 4: d-values ​​and 2θ angles of Form IV

[0044] Form V 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 has an X-ray powder diffraction pattern as shown in Figure 8, 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.

[0045] Table 5: d-values ​​and 2θ angles of Form V

[0046] In the five tables above, the relative strength expressed by numbers is defined as follows:

[0047] In this application, the X-ray diffraction patterns were measured using the following method: Instrument: Bruker D2 PHASER X-ray diffractometer: Method: Target: Cu: K-Alpha; Wavelength Tube voltage: 30kV; tube current: 10mA; scanning range: 3~40°; scanning speed: 0.2 seconds per step, 0.02° per step.

[0048] In this application, differential scanning calorimetry (DSC) analysis was performed using the following method: instrument: Mettler DSC-1 differential scanning calorimeter; method: a sample (~5 mg) was placed in a DSC aluminum pan for testing, the method was 30°C-300°C, and the heating rate was 10°C / min.

[0049] 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 may 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 this case, it is not necessary to rely on all the bands observed in a high-purity sample; sometimes, even a single band may be characteristic for a given crystal.

[0050] 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.

[0051] The present application also relates to the synthesis 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 compounds and the preparation of polymorphs I, II, III, IV, and V. At the same time, the preparation method of polymorph IV of the compound of formula I involved in the present application is simple, the solvent is cheap and readily available, the crystallization conditions are mild, and it is suitable for industrial production.

[0052] The following examples provide further non-limiting details of the technical solution of this application. They should not be considered as limiting the scope of the present invention, but are merely exemplary and typical representations of the present invention.

[0053] 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)

[0054] 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

[0055] 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%.

[0056] 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] + .

[0057] 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

[0058] 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%.

[0059] 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] + .

[0060] 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

[0061] 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.

[0062] 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%.

[0063] 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] + .

[0064] 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.

[0065] 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%.

[0066] 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] + .

[0067] 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).

[0068] 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%.

[0069] 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] + .

[0070] Example 2: Crystalline Form I of Compound of Formula I

[0071] 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.

[0072] The XRD pattern of Form I of the compound of Formula I is shown in FIG1 .

[0073] Example 3: Crystalline Form II of the Compound of Formula I

[0074] Method 1

[0075] 5 mg of the crystal form I obtained in Example 2 was taken for variable temperature XRD, and it was found that the crystal form I was transformed into the crystal form II after desolvation.

[0076] Method 2

[0077] 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.

[0078] The XRD pattern of Form II of the compound of Formula I is shown in FIG2 .

[0079] The DSC spectrum of Form II of the compound of Formula I is shown in FIG3 .

[0080] Example 4: Crystalline Form III of the Compound of Formula I

[0081] 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 yield 1.4 g of product.

[0082] The XRD pattern of Form III of the compound of Formula I is shown in FIG4 .

[0083] The DSC spectrum of Form III of the compound of Formula I is shown in FIG5 .

[0084] Example 5: Crystalline Form IV of the Compound of Formula I

[0085] 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.

[0086] The XRD pattern of Form IV of the compound of Formula I is shown in FIG6 .

[0087] The DSC spectrum of Form IV of the compound of Formula I is shown in FIG7 .

[0088] Example 6: Crystalline Form V of the Compound of Formula I

[0089] 2.0 g of the compound obtained in Example 1 was added to 6 ml of ethanol at room temperature (20-30° C.), stirred for crystallization for 16 hours, and filtered. The filter cake was rinsed with 2 ml of solvent and dried under reduced pressure at 50° C. to obtain 1.46 g of the product.

[0090] The XRD pattern of Form V of the compound of Formula I is shown in FIG8 .

[0091] The DSC spectrum of Form V of the compound of Formula I is shown in FIG9 .

[0092] Example 7 Crystal Form Evaluation

[0093] Each crystalline form of the compound of formula I was evaluated, including competitive beating, stability and solubility studies.

[0094] 1. Competitive beating experiment

[0095] The relative stability of Form II and Form III was investigated by slurrying. Approximately 5 mg of Form II and Form III were weighed, mixed, and then added to methanol / n-heptane (1 / 1), ethanol, and ethyl acetate, respectively, to prepare suspensions. The suspensions were stirred at room temperature for several hours, then centrifuged and the solids were analyzed by XRD. The results showed that the solids crystallized into Form III, indicating that Form III is more stable than Form II.

[0096] The competitive beating XRD patterns of Form II and Form III are shown in Figure 10.

[0097] Competitive beating studies were conducted to investigate the relative stability of Forms III, IV, and V. 5 mg of Forms III, IV, and V were weighed and uniformly mixed. Suspensions were then prepared in ethanol (80°C), ethanol / water (1 / 1) (80°C), water (60°C), water (25°C), acetonitrile (70°C), and acetonitrile / water (1 / 1) (70°C). The suspensions were stirred for 12 hours, and the solids were centrifuged and analyzed by XRD.

[0098] Table 6: Competitive beating results of Forms III, IV and V

[0099] The results showed that Form III and Form V were converted to Form IV in the ethanol / water (1 / 1) (80°C), acetonitrile (70°C), and acetonitrile / water (70°C) systems. Therefore, Form IV is the most stable of the five forms.

[0100] The competitive beating XRD patterns of Forms III, IV, and V are shown in Figure 11 .

[0101] 2. Stability investigation

[0102] The analysis method is as follows:

[0103] Octadecylsilane bonded silica gel was used as filler (Kromasil C 18 The column was mounted on a 4.6 mm × 250 mm column, with a flow rate of 1.0 ml / min. The mobile phase A was 0.01 mol / L potassium dihydrogen phosphate solution (pH adjusted to 3.0 with phosphoric acid), and the mobile phase B was methanol. Gradient elution was performed according to the table below. The detection wavelength was 220 nm. The column temperature was 40°C. The injection volume was 10 μl. The HPLC purity was calculated by the area normalization method.

[0104] Appropriate amounts of Form III, IV, and Form V were weighed into sample bottles and stored uncovered at 80°C, 40°C / 75% RH, and 25°C / 60% RH for 7 days. Samples were collected at 0, 1, 3, and 7 days for HPLC and XRD analysis.

[0105] Table 7 Stability test results of Form III, IV and Form V

[0106] The results showed that the chemical stability of Form III, Form IV and Form V was relatively stable under three conditions of 80°C, 40°C / 75% RH and 25°C / 60% RH for 7 days, and the crystal form did not change.

[0107] The chemical stability profiles of Forms III, IV, and V are shown in Figures 12, 13, and 14.

[0108] The crystal stability profiles of Forms III, IV, and V are shown in Figure 15 .

[0109] 3. Solubility Determination

[0110] Appropriate amounts of Forms III, IV, and V were weighed and added to pH 4.5 and 6.8 buffer solutions to prepare a 5 mg / mL suspension. The resulting suspension was stirred at 37°C, and samples were collected and analyzed at 5, 30, 60, and 240 minutes. A syringe was drawn up, ~1 mL of the suspension was filtered through a membrane, and after dilution several times, the filtrate was subjected to HPLC analysis for solubility using the area normalization method.

[0111] Table 8 Tests of different crystal forms at different pH values

[0112] The results show that in pH = 4.5 (postprandial gastric juice), the solubility of Form IV at 240 min is comparable to that of Form III, which is better than the solubility of Form V and is twice the solubility of Form V; in pH = 6.8 (simulated intestinal fluid), the solubility of Form IV at 240 min is comparable to that of Form III and is twice the solubility of Form V. Therefore, Form IV exhibits good solubility characteristics.

[0113] Example 8 Biological Activity Test

[0114] 1. Compound enzymatic activity (IC50) test

[0115] The JAK1 / 2 / 3 kinase activity detection platform was established using the Lance Ultra principle to measure the activity of the compounds, while the known Itacitinib was also measured as a control. In the detection plate, the enzyme, Ulight-labeled peptide substrate, ATP, and the detection compound were mixed and incubated. After the reaction, EDTA was added to terminate the reaction, and Eu-labeled antibodies were added for detection. The detection plate was analyzed using PE's Envision, and the analysis mode was TR-FRET. The data were represented by the readings of the fluorescence signal at 665nm and 615nm, respectively. A high ratio of 665nm / 615nm indicates high enzyme activity, while a low ratio of 665nm / 615nm indicates that the enzyme activity is inhibited.

[0116] Reagents: kinases (JAK1 / 2 / 3), substrates (ULight-JAK-1 peptide and ATP), detection reagents (Eu-W1024 Anti-phosphotyrosine and EDTA). Instruments: Echo, Envision.

[0117] Dissolve the test compound into a 10mM DMSO solution and place it in a nitrogen cabinet for long-term storage. Take 10μL of 10mM test compound solution and dilute it to a 1mM working solution. Dilute it 3 times with Echo for a total of 11 concentrations. The concentration of the compound in the final reaction system is 10μM to 0.17nM. Use an electric pipette to add 5μL of enzyme and polypeptide substrate mixture to the detection plate, centrifuge the detection plate, and incubate the detection plate at room temperature (23°C) for 15 minutes. Use an electric pipette to add 5μL of kinase buffer containing ATP to the detection plate, centrifuge the detection plate, and seal the plate with aluminum foil. Incubate the detection plate at room temperature (23°C) for 90 minutes. Terminate the reaction, add detection reagent to the detection plate with an electric pipette, centrifuge the detection plate, and seal the plate with aluminum foil. Incubate the detection plate at room temperature (23°C) for half an hour, and use the Envision instrument to detect the signal value of the reaction plate. The results show that the IC50 values ​​of the compound of formula I (amorphous form) and the control Itacitinib or Barcitinib are all less than 10 nM.

[0118] 2. Determination of Bioavailability in Adult SD Rats

[0119] Healthy adult female Sprague-Dawley rats were obtained from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. The study was conducted in two phases: first, animals received a single intravenous injection of 1 mg / kg; second, one week later, the same group received a single oral gavage of the suspension at a dose of 10 mg / kg. Animals receiving oral gavage were fasted overnight and from 10 hours before to 4 hours after dosing. Animals in the intravenous group were not restricted to food. Blood samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after dosing for the intravenous group and at 0.25, 0.5, 1, 2, 4, 8, and 24 hours after dosing for the oral group. 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. Plasma was separated and placed in labeled EP tubes. Plasma samples were immediately stored in an ultra-low temperature freezer until analysis. Plasma samples should be stored in a refrigerator at -70°C before testing. Plasma samples were extracted using protein precipitation, and the extracts were analyzed by LC / MS / MS.

[0120] It can be seen from the above PK (IV and PO) data that the PO bioavailability of the compound of Formula I (Form IV) reaches 17%.

[0121] 3. Pharmacokinetic (PK) Determination in Adult Beagle Dogs

[0122] Healthy adult male Beagle dogs were obtained from Beijing Mas Biotechnology Co., Ltd. Three groups were divided into the study and administered via oral gavage at a dose of 19.5 mg / kg. Animals receiving oral gavage were fasted overnight and from 10 hours before to 4 hours after dosing. Blood samples were collected 15 minutes, 30 minutes, 45 minutes, 1 hour, 1.5 hours, 3 hours, 4 hours, 6 hours, 8 hours, and 24 hours after gavage. Blood samples were collected from the forelimb vein or other suitable venous sampling method. One mL of blood was placed in a labeled EDTA-K2 anticoagulant tube. After gentle inversion to thoroughly mix the anticoagulant with the blood, the tube was immediately placed on wet ice. Plasma was separated by centrifugation within 1 hour of blood collection at 4°C, 2200g, and 10 minutes. The separated plasma was placed in labeled EP tubes. Plasma samples were immediately stored in an ultra-low temperature freezer until analysis. Plasma samples were stored at -70°C until analysis. Plasma samples were extracted using protein precipitation, and the extracts were analyzed by LC / MS / MS.

[0123] Through comprehensive analysis of the PK data and other data of this application, it can be seen that the crystalline form IV of the compound of Formula I exhibits better pharmacokinetic properties after PO administration.

[0124] From the analysis of cell activity, bioavailability, stability, solubility and pharmacokinetic properties, it can be seen that Form IV has better drugability.

Claims

1. Form I 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, characterized in that: Its X-ray powder diffraction pattern has characteristic peaks at the following positions represented by 2θ: 5.17, 5.55, 6.30, 8.67, 10.34, 15.96, 19.06, 19.75, 20.39, and 21.

42.

2. According to the crystalline form I of claim 1, it is characterized in that Its X-ray powder diffraction pattern is shown in FIG1 .

3. Form II 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, characterized in that: Its X-ray powder diffraction pattern has characteristic peaks at the following positions represented by 2θ: 5.55, 6.85, 7.89, 9.78, 13.78, 15.89, 16.38, 16.88, 18.05, 20.42, and 24.

00.

4. The crystal form II according to claim 3, characterized in that Its X-ray powder diffraction pattern is shown in FIG2 .

5. Crystalline Form II I 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, characterized in that: Its X-ray powder diffraction pattern has characteristic peaks at the following positions represented by 2θ: 8.76, 9.53, 10.17, 15.11, 16.70, 19.18, 20.49, 20.79, 22.33, and 25.

18.

6. The crystal form III according to claim 5, characterized in that Its X-ray powder diffraction pattern is shown in FIG4 .

7. Form IV 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, characterized in that Its X-ray powder diffraction pattern has characteristic peaks at the following positions represented by 2θ: 7.95, 11.57, 12.08, 14.66, 16.04, 16.60, 18.12, 18.53, 18.96, 19.73, 23.32, and 25.

24.

8. The crystalline form IV according to claim 7, characterized in that Its X-ray powder diffraction pattern is shown in FIG6 .

9. The crystalline form IV according to claim 7 or 8, characterized in that It melts at 224±3°C, as shown in FIG7 .

10. Form V 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, characterized in that Its X-ray powder diffraction pattern has characteristic peaks at the following positions represented by 2θ: 9.73, 11.11, 13.03, 14.20, 14.66, 15.74, 17.10, 17.64, 18.35, 18.85, 20.09, and 23.

33.

11. The crystal form V according to claim 10, characterized in that: Its X-ray powder diffraction pattern is shown in FIG8 .

12. The crystalline form V according to claim 10 or 11, characterized in that: It melts at 219±3°C, as shown in FIG9 .

13. A pharmaceutical composition comprising any of the aforementioned crystal forms.

14. Use of any of the aforementioned crystal forms in the preparation of JAK1 inhibitors.

15. The use according to claim 14, characterized in that The JAK1 inhibitor is 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.

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