Crystal form of benzamide of pyrazolyl-amino-pyrimidinyl derivative, preparation method therefor and use thereof

Abstract

Provided are a crystal form of benzamide of a pyrazolyl-amino-pyrimidinyl derivative, a preparation method therefor and the use thereof. Specifically, provided are a crystal form I of a compound shown as formula 3, the crystal form satisfying one or more of the following effect advantages: good physical and chemical properties, solid stability, good solubility, low hygroscopicity and good processibility in formulation processes.

Description

Crystal form, preparation method and application of benzamide of a pyrazolyl-amino-pyrimidinyl derivative

[0001] This application claims priority to Chinese patent application No. 202410116896.3, filed on January 26, 2024, and priority to Chinese patent application No. 202411961767.0, filed on December 27, 2024. This application incorporates the entirety of the aforementioned Chinese patent application. Technical Field

[0002] The present application relates to a crystalline form, preparation method and application of a benzamide of a pyrazolyl-amino-pyrimidinyl derivative. Background Art

[0003] The JAK kinase family is a class of non-receptor tyrosine protein kinases with four members: JAK1, JAK2, JAK3, and tyrosine kinase 2 (TYK2). With the exception of JAK3, which is expressed only in the bone marrow and lymphatic system, the other three are widely present in various tissues and cells. Gain-of-function expression or mutation analysis has revealed that JAK1, JAK3, and TYK2 are closely associated with immune regulation, while JAK2 is directly involved in the production of red blood cells and platelets. Loss-of-function analysis has shown that loss of function in JAK1 and JAK2 causes embryonic lethality in mice, while loss of function in JAK3 leads to severe immunodeficiency, and loss of function in TYK2 results in moderate immunodeficiency. No diseases associated with loss of function in JAK1 or JAK2 have been identified in humans, indirectly demonstrating the importance of the physiological functions of JAK1 and JAK2.

[0004] The JAK / STAT signaling pathway is a crucial intracellular signal transduction pathway involved in regulating the survival, proliferation, differentiation, activation, apoptosis, and function of various cells. When the extracellular domain of a receptor binds to its corresponding ligand (e.g., IL-2, IL-4, IL-6, IL-7, IL-9, IL-15, GM-CSF, etc.), the intracellular domain of the receptor undergoes structural changes, bringing the two regions closer together. This brings the bound JAKs closer together, leading to activation through mutual phosphorylation or autophosphorylation. Activated JAKs phosphorylate receptor tyrosine residues, causing further structural changes and creating space for binding to signaling molecules called STATs. This also provides JAKs with the opportunity to phosphorylate STATs. Phosphorylated STATs then form homodimers or heterodimers, translocate to the cell nucleus, and bind to specific regulatory sequences on DNA, regulating the transcription of related genes.

[0005] Preclinical research data show that the compound shown in Formula 3 can inhibit JAK kinase activity in vitro and block the cytokine-JAK-STAT signaling pathway involved in JAK in intestinal epithelial cells and immune cells. Polymorphism is common in compounds, and general drugs may exist in two or more different crystalline states. The existence form and quantity of polymorphic compounds are unpredictable. Different crystalline forms of the same drug have significant differences in solubility, melting point, density, stability, etc., which affect the stability, uniformity, bioavailability, efficacy and safety of the drug to varying degrees. Therefore, in the process of new drug research and development, it is necessary to conduct comprehensive polymorph screening of compounds, and selecting a crystalline form suitable for drug formulation development has important clinical significance. Summary of the Invention

[0006] The present invention provides a benzamide crystal form of a pyrazolyl-amino-pyrimidinyl derivative, a preparation method, and an application thereof. The crystal form satisfies one or more of the following advantages: good physical and chemical properties, solid-state stability, good solubility, low hygroscopicity, and good processability in formulation processes.

[0007] The present invention provides a crystalline form I of a compound as shown in Formula 3;

[0008] The crystalline form I uses Cu-Kα radiation, and the X-ray powder diffraction pattern expressed in 2θ has diffraction peaks at the following positions: 12.600°±0.2°, 16.899°±0.2°, 18.640°±0.2°, 20.860°±0.2°, 23.381°±0.2°, and 27.021°±0.2°.

[0009] In one embodiment, the X-ray powder diffraction pattern of the crystalline form I expressed in 2θ angles further has diffraction peaks at one or more of the following positions: 6.020°±0.2°, 7.183°±0.2°, 8.300°±0.2°, 10.418°±0.2°, 17.999°±0.2°, 21.815°±0.2°, 24.420°±0.2°, 24.998°±0.2°, and 28.560°±0.2°.

[0010] In a certain embodiment, the X-ray powder diffraction pattern of the crystalline form I expressed in 2θ angles further has diffraction peaks at one or more of the following positions: 14.437°±0.2°, 29.001°±0.2°, 30.800°±0.2°, 31.058°±0.2°, 31.498°±0.2°, 33.895°±0.2°, 36.140°±0.2°, 36.900°±0.2°, 38.522°±0.2°, 39.619°±0.2°, 42.609°±0.2°, 45.115°±0.2°, and 51.622°±0.2°.

[0011] In one embodiment, the X-ray powder diffraction pattern of the crystalline form I expressed in 2θ angles has the diffraction peaks and diffraction peak properties shown in the following table:

[0012] In one embodiment, the X-ray powder diffraction pattern of the crystalline form I expressed in 2θ angles has the properties shown in the following table:

[0013] In one embodiment, the X-ray powder diffraction pattern of the crystalline form I expressed in 2θ angles is substantially as shown in FIG1 .

[0014] In one embodiment, the differential scanning calorimetry diagram of the crystalline form I has an endothermic peak at 269°C±2°C.

[0015] In one embodiment, the differential scanning calorimetry diagram of the crystalline form I has an endothermic peak at 269°C±2°C, and the heat of melting is 407 J / g.

[0016] In one embodiment, the differential scanning calorimetry diagram of the crystalline form I is substantially as shown in FIG2 .

[0017] In one embodiment, the thermogravimetric analysis of the Form I shows a weight loss of 0.03±0.01% when heated to 120°C.

[0018] In one embodiment, the thermogravimetric analysis diagram of the crystalline form I is substantially as shown in FIG3 .

[0019] In one embodiment, the PLM diagram of the crystalline form I is substantially as shown in FIG4 .

[0020] In one embodiment, the DVS diagram of the crystal form I is substantially as shown in FIG5 .

[0021] The present invention also provides a method for preparing the above-mentioned crystal form I, which is Scheme 1 or Scheme 2;

[0022] Scheme 1 comprises the following steps: the compound represented by Formula 3 is mixed with a solvent to prepare a suspension, and the suspension is slurried, and the solid is collected to obtain the crystalline form I, wherein the solvent is THF:water = 1:1.

[0023] Scheme 2 comprises the following steps: adding acetonitrile to a DMSO solution of the compound represented by Formula 3 to precipitate Form I.

[0024] In one embodiment, in embodiment 1, during the beating, the beating temperature is 40-60°C, preferably 50°C.

[0025] In one embodiment, in embodiment 1, the beating time is related to the scale of the reaction. Generally, the end point of the reaction is when the solids no longer increase. The beating time is generally 2-4 days, preferably 3 days.

[0026] In one embodiment, in embodiment 1, during the beating, the concentration of the suspension is 20-30 g / L, preferably 25 g / L.

[0027] In one embodiment, the first embodiment comprises the following steps: preparing a suspension of the compound of Formula 3, stirring at 50° C. for three days, and collecting the wet solid by centrifugation at 12,000 rpm for 5 minutes; if no solid precipitates or little solid precipitates, placing the suspension in a fume hood to evaporate the solvent naturally, and then drying the suspension under reduced pressure and vacuum at room temperature.

[0028] In one embodiment, in embodiment 2, the concentration of the DMSO solution of the compound represented by Formula 3 is 95-105 g / L, preferably 100 g / L.

[0029] In one embodiment, in embodiment 2, the volume ratio of the DMSO to the acetonitrile is 1:3-1:5, preferably 1:4.

[0030] In one embodiment, the second embodiment comprises the following steps: preparing a DMSO solution of the compound represented by Formula 3, adding acetonitrile dropwise until a large amount of solid precipitates, and absorbing moisture from the solid with absorbent paper.

[0031] The present invention also provides a pharmaceutical composition comprising the above-mentioned crystal form I and pharmaceutical excipients.

[0032] The present invention also provides a use of the above-mentioned crystal form I or the above-mentioned pharmaceutical composition in the preparation of a drug for treating and / or preventing diseases associated with JAK kinase.

[0033] In one embodiment, the disease associated with JAK kinase is inflammatory bowel disease, psoriasis, vitiligo, atopic dermatitis, systemic lupus erythematosus, asthma, diabetic nephropathy, chronic myeloid leukemia (CML), essential thrombocythemia (ET), polycythemia vera (PV), myelofibrosis (MF), breast cancer or ovarian cancer.

[0034] The term “substantially” means that the positions of the peaks in the graph may vary slightly with slight variations in the measuring equipment, measuring conditions, and batches of the product to be measured, and are not to be regarded as absolute values.

[0035] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention.

[0036] The reagents and raw materials used in the present invention are commercially available.

[0037] The positive progress of the present invention is that: Form I has good physical and chemical properties, good high temperature (e.g., 60°C) and high humidity (75% RH) stability, good solubility (especially under acidic conditions), low hygroscopicity, uniform particle size distribution, good solid form and formulation processability, and good development prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 is the XRPD spectrum of Form I.

[0039] Figure 2 is the DSC spectrum of Form I.

[0040] FIG3 is a TGA spectrum of Form I.

[0041] Figure 4 is a PLM spectrum of Form I.

[0042] Figure 5 is a DVS spectrum of Form I. DETAILED DESCRIPTION

[0043] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0044] 1. Experimental Equipment

[0045] 2. Experimental instrument parameter setting

[0046] 2.1 XRPD analysis method

[0047] The equipment is Shimadzu XRD-6000, and the sample is scanned according to the following parameters:

[0048] The radiation source is Cu~Kα target

[0049] The minimum operating voltage and current of the fluorescent tube are 40kV and 30mA respectively.

[0050] The sample scanning range was 2-Theta value from 5° to 50° and the scanning speed was 5 deg / min.

[0051] 2.2 Thermogravimetric analysis (TGA)

[0052] Weigh approximately 5 mg of sample into a crucible, protect with nitrogen, heat from 30°C to 350°C at a rate of 20°C / min, and maintain at 350°C for 1 min.

[0053] 2.3 Differential Scanning Calorimetry (DSC)

[0054] Weigh approximately 1 mg of powder sample and place it in a sealed aluminum crucible with a pinhole pierced in the crucible lid. Under nitrogen, perform a differential calorimetric scan from 30°C to 300°C, holding at 300°C for 1 minute. The heating rate is 20°C / min.

[0055] 2.4 Dynamic Water Sorption (DVS)

[0056] Dynamic moisture adsorption experiments consist of adsorption and desorption. It is generally believed that at a set relative humidity, when the sample weight dm / dt ≤ 0.01%, the sample is considered to have reached equilibrium in terms of moisture adsorption or desorption at that relative humidity.

[0057] Sample test temperature: T = 25 ° C;

[0058] Equilibrium time: dm / dt: 0.01% / min;

[0059] Relative humidity change range: 0% ~ 95% ~ 0%; RH (%) test humidity change per step: 5%.

[0060] 2.5 Polarized Light Microscope (PLM)

[0061] The sample was dispersed in a medium (silicone oil), and the sample was observed using a 10X eyepiece and a 10X objective lens, and the image was recorded using a camera computer system.

[0062] Example 1 Preparation and Characterization of Form I

[0063] To a liquid phase vial was added the compound of Formula 3 (49.15 mg) (prepared with reference to Example 4 of compound patent CN113227074A), 1 ml of tetrahydrofuran, and 1 ml of water to form a suspension. The suspension was stirred at 50°C for three days. The resulting suspension was centrifuged (12,000 rpm, 5 min), but solids could not be separated. The solvent was naturally evaporated in a fume hood. The collected wet solid was dried under reduced pressure in vacuo at room temperature for 24 hours. The above operation was repeated twice to obtain Form I with a purity of 99.13%.

[0064] Characterization of Form I

[0065] The XRPD pattern of Form I using Cu-Kα radiation, expressed in 2θ angles, is shown in Figure 1, and the specific data are provided in the table below. The DSC analysis results of Form I are shown in Figure 2, which show an endothermic peak at 268.69°C with an enthalpy of 166.97 J / g. The TGA analysis results are shown in Figure 3, which show that Form I exhibits no weight loss before 120°C, indicating that Form I is a solvent-free crystalline form. A polarizing light microscopy (PLM) pattern of Form I is preferably substantially as shown in Figure 4.

[0066] Example 2 Preparation of Form I

[0067] The compound represented by Formula 3 (47.88 mg) (prepared with reference to Example 4 of compound patent CN113227074A) was placed in a glass vial and completely dissolved in 0.5 ml of DMSO. Acetonitrile (2 ml) was added dropwise until a large amount of solid precipitated. The wet solid was separated by centrifugation (12000 rpm, 5 min), and the residual solvent was dried with absorbent paper to obtain Form I.

[0068] Example 3 Stability Experiment of Form I

[0069] The stability of Form I was investigated under the following conditions.

[0070] High temperature: 60℃, open;

[0071] High temperature and high humidity: 40℃ / 75%RH, open.

[0072] 40 mg of Form I was weighed into a glass bottle, opened, and placed in a stability chamber. Samples were taken after 1 and 2 weeks for stability testing. The stability test included purity and crystal form.

[0073] Crystal form I can remain stable for a long time (more than two months) under high temperature and high humidity, and has good stability.

[0074] Example 4 Hygroscopicity Experiment of Form I

[0075] Testing using Dynamic Water Sorption (DVS)

[0076] Sample test temperature: T = 25 ° C;

[0077] Equilibrium time: dm / dt: 0.01% / min;

[0078] Relative humidity change range: 0% ~ 95% ~ 0%; RH (%) test humidity change per step: 5%.

[0079] The test results are shown in FIG5 . DVS shows that the compound absorbs moisture at 80% RH and increases in weight by 0.45%, which is slightly hygroscopic. *At 25±1°C and 80±2% RH (Ph. Eur. 6.0).

[0080] Example 5 Solubility Experiment of Form I

[0081] Medium preparation:

[0082] SGF: Measure 0.2g NaCl and 0.7mL hydrochloric acid in a 100mL volumetric flask, dissolve and dilute with water to the mark, and shake well.

[0083] FaSSIF: Dissolve 0.042 g of NaOH, 0.343 g of anhydrous NaH2PO4, and 0.619 g of NaCl in 90 mL of water by ultrasonication. Adjust the pH to 6.50 with 1 N NaOH and make up to 100 mL with 10 mL of water. Add 0.224 g of FaSSIF powder to 50 mL of buffer medium by ultrasonication. Dissolve the mixture by ultrasonication. Add the remaining 50 mL of medium and mix thoroughly.

[0084] FeSSIF: Dissolve 0.404 g of NaOH, 0.8865 g of glacial acetic acid, and 0.619 g of NaCl in 90 mL of water by ultrasonication. Adjust the pH to 5.00 with 1 N NaOH and make up to 100 mL with 10 mL of water. Add 1.12 g of FaSSIF powder to 50 mL of buffer by ultrasonication. Dissolve the mixture by ultrasonication. Add the remaining 50 mL of buffer and mix thoroughly.

[0085] FaSSIF / FeSSIF / FaSSGF were purchased from Bio-Relevant

[0086] Other buffer media were prepared according to USP.

[0087] Experimental process:

[0088] Weigh approximately 8 mg of the drug substance of the compound represented by Formula 3, add 4.0 mL of various media (target concentration 2 mg / mL), add a stir bar, and stir on a 37°C constant-temperature magnetic stirrer for 24 hours. Measure and record the final pH. Transfer 1.0 mL of the suspension to a 1.5 mL centrifuge tube and centrifuge at 12,000 rpm for 5 minutes. Dilute the supernatant appropriately with 50% THF and determine its concentration by HPLC.

[0089] Control solution and standard curve:

[0090] Use the starting API as a reference substance for solubility testing. Accurately weigh 10 mg of the API into a 50 mL volumetric flask. Dissolve in 50% THF and dilute to volume. Shake well. Prepare two replicates.

[0091] Take control 1, dilute it 2-fold, 4-fold, 20-fold, 200-fold, and 400-fold with 50% THF, inject 10 μL, and draw a standard curve.

[0092] The solubility test results of Form I are as follows:

[0093] The results showed that the solubility of Form I was higher under strong acid conditions (pH < 2), which was better than that under neutral and alkaline conditions.

[0094] Example 6 Stability Experiment of Form I

[0095] Form I was ground in an agate mortar for 10 min and 30 min, respectively, and then XRPD analysis of the ground samples was performed.

[0096] Approximately 10 mg of Form I was weighed and heated to 240° C. and maintained at this temperature for 20 minutes. The XRPD of the heated solid was then measured.

[0097] According to XRPD results, the crystalline form of Form I did not change after grinding and heating.

Claims

1. Crystal form I of a compound represented by Formula 3, characterized in that: For the crystalline form I, using Cu-Kα radiation, the X-ray powder diffraction pattern expressed in 2θ has diffraction peaks at the following positions: 12.600° ± 0.2°, 16.899° ± 0.2°, 18.640° ± 0.2°, 20.860° ± 0.2°, 23.381° ± 0.2°, 27.021° ± 0.2°; 2. The crystalline form I according to claim 1, characterized in that, The X-ray powder diffraction pattern of the said Form I using Cu-Kα radiation and expressed in terms of 2θ also has diffraction peaks at the following positions: 6.020° ± 0.2°, 7.183° ± 0.2°, 8.300° ± 0.2°, 10.418° ± 0.2°, 17.999° ± 0.2°, 21.815° ± 0.2°, 24.420° ± 0.2°, 24.998° ± 0.2°, 28.560° ± 0.2°.

3. The crystalline form I according to claim 1, wherein The X-ray powder diffraction pattern of the said Form I using Cu-Kα radiation and expressed in terms of 2θ also has diffraction peaks at the following positions: 14.437° ± 0.2°, 29.001° ± 0.2°, 30.800° ± 0.2°, 31.058° ± 0.2°, 31.498° ± 0.2°, 33.895° ± 0.2°, 36.140° ± 0.2°, 36.900° ± 0.2°, 38.522° ± 0.2°, 39.619° ± 0.2°, 42.609° ± 0.2°, 45.115° ± 0.2°, 51.622° ± 0.2°.

4. The crystalline form I according to claim 1, wherein The X-ray powder diffraction pattern of the crystalline form I in terms of 2θ angle has diffraction peaks and relative peak heights as shown in the following table:

5. The crystalline form I according to claim 1, characterized in that, It satisfies one or more of the following conditions: (1) The differential scanning calorimetry chart of the said Form I has an endothermic peak at 269°C ± 2°C; (2) The thermogravimetric analysis chart of the said Form I loses 0.03% ± 0.01% of its weight when heated to 120°C.

6. The crystalline form I according to claim 5, wherein, The differential scanning calorimetry chart of the said Form I has an endothermic peak at 269°C ± 2°C, and the heat of fusion is 407 J / g.

7. The crystalline form I according to claim 1, characterized in that, It satisfies one or more of the following conditions: (1) The X-ray powder diffraction pattern of the said Form I expressed in terms of 2θ angle is basically as shown in Figure 1; (2) The differential scanning calorimetry chart of the said Form I is basically as shown in Figure 2; (3) The thermogravimetric analysis chart of the said Form I is basically as shown in Figure 3; (4) The polarized light microscopy (PLM) chart of the said Form I is basically as shown in Figure 4; (5) The dynamic vapor sorption (DVS) chart of the said Form I is basically as shown in Figure 5.

8. A method for preparing polymorphic form I of the compound shown in Formula 3 as described in any one of claims 1-7, characterized in that, It is Scheme 1 or Scheme 2; Scheme 1: The compound shown in Formula 3 is slurried with a solvent to obtain the said Form I, and the solvent is THF: water volume ratio = 1:1; Scheme 2: Acetonitrile is added to the DMSO solution of the compound shown in Formula 3 to precipitate Form I.

9. The preparation method according to claim 8, characterized in that, It satisfies one or more of the following conditions: (1) In Scheme 1, during the said slurrying, the temperature of the slurrying is 40 - 60°C, preferably 50°C; (2) In Scheme 1, the time of the slurrying is 2 - 4 days, preferably 3 days; (3) In Scheme 1, during the said slurrying, the mass-to-volume ratio of the compound shown in Formula 3 and the solvent is 20 - 30 g / L, preferably 25 g / L; (4) In Scheme 2, the concentration of the DMSO solution of the compound shown in Formula 3 is 95 - 105 g / L, preferably 100 g / L; (5) In Scheme 2, the volume ratio of DMSO and acetonitrile is 1:3 - 1:5, preferably 1:4; Preferably, the first scheme includes the following operations: preparing a suspension of the compound shown in Formula 3, stirring at 50 °C for three days, centrifuging at 12,000 rpm for 5 minutes to collect the wet solid; if no solid precipitates or only a small amount of solid precipitates, the solvent is allowed to evaporate naturally and dried under reduced pressure at room temperature. The second scheme includes the following operations: preparing a DMSO solution of the compound shown in Formula 3, and adding acetonitrile dropwise until a large amount of solid precipitates.

10. Crystal form I of a compound represented by Formula 3, characterized in that, It is prepared by the preparation method described in any one of claims 8-9.

11. A pharmaceutical composition comprising the polymorphic form I described in any one of claims 1-8 and a pharmaceutical excipient.

12. Use of the polymorphic form I described in any one of claims 1-7 or claim 10 or the pharmaceutical composition described in claim 11 in the preparation of a drug for treating and / or preventing a disease related to JAK kinase; preferably, the disease related to JAK kinase is autoinflammatory bowel disease, psoriasis, vitiligo, atopic dermatitis, systemic lupus erythematosus, asthma, diabetic nephropathy, chronic myeloid leukemia, essential thrombocythemia, polycythemia vera, myelofibrosis, breast cancer or ovarian cancer.

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