Crystalline forms of pyrrolopyrimidine compounds and their preparation methods

Abstract

The present invention relates to crystalline forms of the compound of formula (I) and processes for its preparation, and further includes the use of said crystalline forms in the preparation of medicaments for treating related diseases. [Formula 1] JPEG2024518790000027.jpg4966

Description

[Technical field]

[0001] This invention claims priority to CN202110518999.9, filed on May 12, 2021.

[0002] The present invention relates to crystalline forms of the compound of formula (I) and processes for its preparation and its use in the preparation of medicaments for treating related diseases. [Background technology]

[0003] Kinases control the transfer of phosphate groups from a phosphate donor (e.g., ATP) to a specific substrate. Protein kinases are a large subset of kinases that play a central role in regulating many cell signals and processes, and BTK is one of the protein kinases.

[0004] BTK is a member of the TEC cytoplasmic tyrosine kinase family (TEC family kinases, TFKs), which has five other members in addition to BTK: ITK, TEC, BMX, and TXK. TFKs are a very ancient kinase family with an evolutionary history of over 600 million years that functions primarily in the hematopoietic system.

[0005] BTK is involved in the transduction and amplification of various intracellular and extracellular signals primarily in B lymphocytes and is essential for B cell maturation. Inactivation of BTK function in XLA patients results in peripheral B cell and immunoglobulin deficiency. Signaling receptors upstream of BTK include growth factor and cytokine receptors, G protein-coupled receptors such as chemokine receptors, antigen receptors (especially B cell receptor [BCR]), and integrins. BTK activates many major downstream signaling pathways, including the phosphoinositide-3 kinase (PI3K)-AKT pathway, phospholipase-C (PLC), protein kinase C, and nuclear factor kappa B (NF-κB). BTK's role in BCR signaling and cell migration is well established, and these functions also appear to be primary targets of BTK inhibitors. Increased BTK activity is detected in hematological cancer cells, such as B cell chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). Because aberrant activation of BTK function often leads to B cell malignancies or autoimmune diseases, BTK has become a popular target for research and development. Summary of the Invention

[0006] The present invention provides crystalline form A of the compound represented by formula (I), characterized in that the X-ray powder diffraction pattern of said crystalline form A has characteristic diffraction peaks at the following 2θ angles: 16.16±0.20°, 16.60±0.20°, 22.02±0.20°, and 23.08±0.20°. [ka]

[0007] In some embodiments of the present invention, the X-ray powder diffraction pattern of crystalline form A of the compound of formula (I) comprises at least 5, 6, 7, or 8 diffraction peaks selected from the group consisting of 7.51±0.20°, 10.63±0.20°, 15.06±0.20°, 16.16±0.20°, 16.60±0.20°, 21.28±0.20°, 22.02±0.20°, and 23.08±0.20°, expressed in terms of 2θ angles.

[0008] In some embodiments of the invention, the X-ray powder diffraction pattern of crystalline form A has characteristic diffraction peaks at the following 2θ angles: 7.51±0.20°, 10.63±0.20°, 15.06±0.20°, 16.16±0.20°, 16.60±0.20°, 21.28±0.20°, 22.02±0.20°, and 23.08±0.20°.

[0009] In some embodiments of the present invention, the X-ray powder diffraction pattern of crystalline form A of the compound of formula (I) comprises at least 9, 10, 11, or 12 diffraction peaks selected from the group consisting of 7.51±0.20°, 10.63±0.20°, 12.00±0.20°, 13.17±0.20°, 15.06±0.20°, 16.16±0.20°, 16.60±0.20°, 18.61±0.20°, 21.28±0.20°, 22.02±0.20°, 22.49±0.20°, and 23.08±0.20°, expressed in terms of 2θ angles.

[0010] In some embodiments of the invention, the X-ray powder diffraction pattern of crystalline form A has characteristic diffraction peaks at the following 2θ angles: 7.51±0.20°, 10.63±0.20°, 12.00±0.20°, 13.17±0.20°, 15.06±0.20°, 16.16±0.20°, 16.60±0.20°, 18.61±0.20°, 21.28±0.20°, 22.02±0.20°, 22.49±0.20°, and 23.08±0.20°.

[0011] In some embodiments of the present invention, the X-ray powder diffraction pattern of the crystalline form A of the compound of formula (I) above is at the following 2θ angles: 7.51±0.20°, 10.04±0.20°, 10.63±0.20°, 12.00±0.20°, 13.17±0.20°, 14.25±0.20°, 15.06±0.20°, 15.51±0.20°, 16.06±0.20°, 17.06±0.20°, 18.06±0.20°, 19.06±0.20°, 20.06±0.20°, 21.06±0.20°, 22.06±0.20°, 23.06±0.20°, 24.06±0.20°, 25.06±0.20°, 26.06±0.20°, 27.06±0.20°, 28.06±0.20°, 29.06±0.20°, 30.06±0.20°, 31.06±0.20°, 32.06±0.20°, 33.06±0.20°, 34.06±0.20°, 35.06±0.20°, 36.06±0.20°, 37.06±0.20°, 38.06±0.20°, 39.06±0.20°, 39.06±0.20°, 39.06±0.20°, 40.06±0.20°, 41.06±0.20°, 42.0 The diffraction peaks are at 16.16±0.20°, 16.60±0.20°, 17.31±0.20°, 18.61±0.20°, 19.53±0.20°, 20.07±0.20°, 20.60±0.20°, 21.28±0.20°, 21.78±0.20°, 22.49±0.20°, and 23.08±0.20°.

[0012] In some embodiments of the invention, the X-ray powder diffraction pattern of crystalline form A has characteristic diffraction peaks at the following 2θ angles: 7.51°, 9.45°, 10.04°, 10.63°, 12.00°, 13.17°, 14.25°, 15.06°, 15.51°, 16.16°, 16.60°, 17.31°, 17.58°, 18.61°, 18.95°, 19.53°, 20.07°, 20.60°, 21.28°, 21.78°, 22.02°, 22.49°, and 23.08°.

[0013] In some embodiments of the present invention, the crystalline form A has the XRPD pattern shown in FIG.

[0014] In some embodiments of the present invention, analytical data of the XRPD pattern of crystalline form A is shown in Table 1.

[0015] [Table 1]

[0016] In some embodiments of the present invention, the differential scanning calorimetry curve for crystalline form A has endothermic peak onsets at 145.7° C.±3.0° C., 208.9° C.±3.0° C., and 218.7° C.±3.0° C., respectively.

[0017] In some embodiments of the present invention, the crystalline form A has a DSC pattern as shown in FIG.

[0018] In some embodiments of the present invention, the thermogravimetric analysis curve for the crystalline form A shows a weight loss of 7.66% at 200.0°C±3.0°C.

[0019] In some embodiments of the present invention, the crystalline form A has a TGA pattern as shown in FIG.

[0020] The present invention further provides crystalline form B of compound of formula (I), characterized in that the X-ray powder diffraction pattern of said crystalline form B has characteristic diffraction peaks at the following 2θ angles: 7.47±0.20°, 10.41±0.20°, 16.04±0.20°, 16.73±0.20°. [ka]

[0021] In some embodiments of the present invention, the X-ray powder diffraction pattern of crystalline form B of the compound of formula (I) comprises at least 5, 6, 7, or 8 diffraction peaks selected from the group consisting of 7.47±0.20°, 10.41±0.20°, 15.23±0.20°, 16.04±0.20°, 16.73±0.20°, 20.87±0.20°, 21.46±0.20°, and 21.91±0.20°, expressed in terms of 2θ angles.

[0022] In some embodiments of the invention, the X-ray powder diffraction pattern of crystalline form B has characteristic diffraction peaks at the following 2θ angles: 7.47±0.20°, 10.41±0.20°, 15.23±0.20°, 16.04±0.20°, 16.73±0.20°, 20.87±0.20°, 21.46±0.20°, and 21.91±0.20°.

[0023] In some embodiments of the present invention, the X-ray powder diffraction pattern of crystalline form B of compound of formula (I) comprises at least 9, 10, 11, or 12 diffraction peaks selected from the group consisting of 7.47±0.20°, 9.53±0.20°, 10.41±0.20°, 11.91±0.20°, 13.91±0.20°, 15.23±0.20°, 16.04±0.20°, 16.73±0.20°, 18.82±0.20°, 20.87±0.20°, 21.46±0.20°, and 21.91±0.20°, expressed in terms of 2θ angles.

[0024] In some embodiments of the invention, the X-ray powder diffraction pattern of crystalline form B has characteristic diffraction peaks at the following 2θ angles: 7.47±0.20°, 9.53±0.20°, 10.41±0.20°, 11.91±0.20°, 13.91±0.20°, 15.23±0.20°, 16.04±0.20°, 16.73±0.20°, 18.82±0.20°, 20.87±0.20°, 21.46±0.20°, and 21.91±0.20°.

[0025] In some embodiments of the invention, the X-ray powder diffraction pattern of crystalline form B has characteristic diffraction peaks at the following 2θ angles: 7.47°, 9.53°, 9.78°, 10.41°, 11.45°, 11.91°, 12.40°, 13.22°, 13.91°, 14.87°, 15.23°, 15.44°, 16.04°, 16.73°, 17.33°, 17.88°, 18.82°, 19.61°, 19.88°, 20.25°, 20.87°, 21.46°, and 21.91°.

[0026] In some embodiments of the present invention, the crystalline form B has the XRPD pattern shown in FIG.

[0027] In some embodiments of the present invention, the analytical data of the XRPD pattern of crystalline form B is shown in Table 2.

[0028] [Table 2]

[0029] In some embodiments of the present invention, the differential scanning calorimetry curve for crystalline form B has an endothermic peak onset at 214.3°C ± 3.0°C.

[0030] In some embodiments of the present invention, the crystalline form B has the DSC pattern shown in FIG.

[0031] In some embodiments of the present invention, the thermogravimetric analysis curve for crystalline form B exhibits a weight loss of 0.64% at 200.0°C±3.0°C.

[0032] In some embodiments of the present invention, the crystalline form B has a TGA pattern as shown in FIG.

[0033] The present invention further provides crystalline form C of compound of formula (I), characterized in that the X-ray powder diffraction pattern of said crystalline form C has characteristic diffraction peaks at the following 2θ angles: 5.28±0.20°, 8.58±0.20°, 20.56±0.20°, 24.55±0.20°. [ka]

[0034] In some embodiments of the present invention, the X-ray powder diffraction pattern of crystalline form C of compound of formula (I) comprises at least 5, 6, 7, or 8 diffraction peaks selected from the group consisting of 5.28±0.20°, 8.58±0.20°, 11.00±0.20°, 11.52±0.20°, 15.72±0.20°, 20.56±0.20°, 21.84±0.20°, and 24.55±0.20°, expressed in terms of 2θ angles.

[0035] In some embodiments of the invention, the X-ray powder diffraction pattern of crystalline form C has characteristic diffraction peaks at the following 2θ angles: 5.28±0.20°, 8.58±0.20°, 11.00±0.20°, 11.52±0.20°, 15.72±0.20°, 20.56±0.20°, 21.84±0.20°, and 24.55±0.20°.

[0036] In some embodiments of the present invention, the X-ray powder diffraction pattern of crystalline form C of compound of formula (I) comprises at least 9, 10, 11, or 12 diffraction peaks selected from the group consisting of 5.28±0.20°, 8.58±0.20°, 11.00±0.20°, 11.52±0.20°, 15.72±020°, 16.69±020°, 17.16±020°, 18.62±020°, 19.76±020°, 20.56±020°, 21.84±020°, and 24.55±020°, expressed in terms of 2θ angles.

[0037] In some embodiments of the invention, the X-ray powder diffraction pattern of crystalline form C has characteristic diffraction peaks at the following 2θ angles: 5.28±0.20°, 8.58±0.20°, 11.00±0.20°, 11.52±0.20°, 15.72±0.20°, 16.69±0.20°, 17.16±0.20°, 18.62±0.20°, 19.76±0.20°, 20.56±0.20°, 21.84±0.20°, and 24.55±0.20°.

[0038] In some embodiments of the present invention, the X-ray powder diffraction pattern of crystalline form C of compound of formula (I) has diffraction peaks at the following 2θ angles: 5.28±0.20°, 8.58±0.20°, 11.00±0.20°, 11.52±0.20°, 14.08±0.20°, 15.08±0.20°, 15.72±0.20°, 16.69±0.20°, 17.16±0.20°, 18.62±0.20°, 19.76±0.20°, 20.56±0.20°, 21.84±0.20°, 23.14±0.20°, 23.89±0.20°, 24.55±0.20°, 25.40±0.20°, 29.21±0.20°.

[0039] In some embodiments of the invention, the X-ray powder diffraction pattern of crystalline form C has characteristic diffraction peaks at the following 2θ angles: 5.28°, 8.58°, 11.00°, 11.52°, 14.08°, 15.08°, 15.72°, 16.69°, 17.16°, 18.62°, 19.76°, 20.56°, 20.77°, 21.84°, 23.14°, 23.89°, 24.55°, 25.40°, 28.24°, 29.21°, 31.36°.

[0040] In some embodiments of the present invention, the crystalline form C has the XRPD pattern shown in FIG.

[0041] In some embodiments of the present invention, the analytical data of the XRPD pattern of crystalline form C is shown in Table 3.

[0042] [Table 3]

[0043] In some embodiments of the present invention, the differential scanning calorimetry curve for crystalline form C has an endothermic peak onset at 218.8°C ± 3.0°C.

[0044] In some embodiments of the present invention, the crystalline form C has the DSC pattern shown in FIG.

[0045] In some embodiments of the present invention, the thermogravimetric analysis curve for crystalline form C exhibits a weight loss of 1.45% at 200.0°C ± 3.0°C.

[0046] In some embodiments of the present invention, the crystalline form C has a TGA pattern as shown in FIG.

[0047] The present invention further provides the use of the above crystalline form A, or crystalline form B, or crystalline form C in the preparation of a medicament for treating a disease associated with the BTK protein kinase.

[0048] In some embodiments of the present invention, the disease associated with the BTK protein kinase is a hematological neoplasm.

[0049] Effect of the Invention The crystalline form of the compound of the present invention has good stability, is easily druggable, and has a remarkable tumor growth inhibiting effect.

[0050] Definitions and Explanations The following terms and phrases as used herein are intended to include the following meanings unless otherwise specified. A particular phrase or term, unless specifically defined, should not be considered indefinite or unclear, but should be understood according to its general meaning. When a trade name appears herein, it is intended to refer to the corresponding trade name or its active ingredient.

[0051] References throughout this specification to "one embodiment" or "an embodiment" or "another embodiment" or "in an embodiment" are meant to include the relevant specific reference elements, structures, or features described in the embodiment in at least one embodiment. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" or "in another embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, particular elements, structures, or features may be combined in any suitable manner in one or more embodiments.

[0052] The article "a" (corresponding to the English "a", "an" and "the"), as used in the specification and the appended claims, should be understood to include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a reaction including a "catalyst" includes one catalyst or two or more catalysts. It should also be understood that the term "or" is generally used in the sense of including "and / or" unless the context clearly dictates otherwise.

[0053] The intermediate compounds of the present invention can be prepared by various synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments in combination with other chemical synthetic methods, and equivalent methods known to those skilled in the art, and preferred embodiments include, but are not limited to, the examples of the present invention.

[0054] The chemical reactions described in the detailed description of the present invention are carried out in suitable solvents suitable for the chemical transformations of the present invention and the reagents and materials required therefor. To obtain the compounds of the present invention, those skilled in the art may need to modify or select synthetic steps or reaction schemes based on existing embodiments.

[0055] The structure of the compound of the present invention can be confirmed by conventional methods known to those skilled in the art, and when the present invention relates to the absolute configuration of a compound, the absolute configuration can be confirmed by conventional techniques in the art. For example, single crystal X-ray diffraction (SXRD) is performed by collecting diffraction intensity data from a cultured single crystal using a Bruker D8 venture diffractometer, the light source is CuKα radiation, and the scanning method is φ / ω scanning. After collecting relevant data, the crystal structure can be further analyzed by a direct method (Shelxs97) to confirm the absolute configuration.

[0056] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[0057] All solvents used in this invention are commercially available and are used without further purification.

[0058] Compounds are named manually or with ChemDraw® software; commercially available compounds use the supplier's catalogue name.

[0059] The X-ray powder diffractometer (XRPD) method of the present invention Model number of the device: PANalytical X'pert3 X-ray diffractometer Measurement method: Approximately 10 to 20 mg of sample is used for XRPD measurement.

[0060] Detailed XRPD parameters are as follows: Tube:Cu,kα1,λ=1.54060Å,kα2,λ=1.54443Å Tube voltage: 45kV, tube current: 40mA Divergence slit: 0.60mm Detector slit: 10.50mm Anti-scatter slit: 7.10mm Scanning range: 3-40deg Step size: 0.0263deg Scan Step Time: 46.665s Sample pan rotation speed: 15 rpm

[0061] Differential Scanning Calorimetry (DSC) Method of the Present Invention Model number: TA 2500 Differential Scanning Calorimeter Measurement method: Place the sample (approximately 1 mg) in a DSC aluminum pan, close the lid, and measure. 2 Under these conditions, the sample is heated from 25°C to 350°C at a heating rate of 10°C / min.

[0062] The Thermogravimetric Analysis (TGA) Method of the Present Invention Model number of the instrument: TA 5500 Thermogravimetric Analyzer Measurement method: Place the sample (2-5 mg) in a TGA aluminum pan, open the lid, and perform the measurement. 2 Under these conditions, the sample is heated from room temperature to 350° C. at a heating rate of 10° C. / min.

[0063] The Dynamic Vapor Sorption (DVS) Method of the Present Invention Model number: SMS DVS Advantage Dynamic Vapor Sorption Apparatus Measurement conditions: Place the sample (10 to 15 mg) in a DVS sample pan and measure.

[0064] The detailed DVS parameters are as follows: Temperature: 25℃ Equilibrium: dm / dt=0.01% / min (minimum: 10min, maximum: 180min) Drying: 0% RH for 120 min Measurement step of RH(%): 10% RH(%) measurement stage range: 0%~90%~0%

[0065] The evaluation classification of moisture absorption is as shown in Table 4.

[0066] [Table 4] [Brief description of the drawings]

[0067] [Figure 1] FIG. 1 is a Cu-Kα XRPD pattern of crystalline form A of the compound of formula (I). [Diagram 2] FIG. 2 is a DSC pattern of crystalline form A of the compound of formula (I). [Diagram 3] FIG. 3 is a TGA pattern of crystalline form A of the compound of formula (I). [Figure 4] FIG. 4 is a Cu-Kα XRPD pattern of crystalline form B of compound of formula (I). [Diagram 5] FIG. 5 is a DSC pattern of crystalline form B of compound of formula (I). [Figure 6] FIG. 6 is a TGA pattern of crystalline form B of compound of formula (I). [Figure 7] FIG. 7 is a Cu-Kα XRPD pattern of crystalline form C of the compound of formula (I). [Figure 8] FIG. 8 is a DSC pattern of crystalline form C of compound of formula (I). [Figure 9] FIG. 9 is a TGA pattern of crystalline form C of compound of formula (I). [Figure 10]FIG. 10 is a DVS pattern of crystalline form C of the compound of formula (I). [Figure 11] FIG. 11 shows the tumor volume growth curves with drug effects. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0068] In order to better understand the present invention, the present invention will be further described below with reference to specific examples, but the specific embodiments do not limit the present invention.

[0069] Reference example [ka]

[0070] The reference example was prepared according to the method described in Patent WO2017111787 Compound (I).

[0071] Example 1: Preparation of compounds of formula (I) [ka]

[0072] Synthesis Route: [ka] [ka]

[0073] Step 1: Synthesis of compound A2 Phenol (7.49 g, 79.54 mmol, 7.00 mL, 1.5 eq), compound A1 (10 g, 53.03 mmol, 1 eq), and cesium carbonate (25.92 g, 79.54 mmol, 1.5 eq) were dissolved in N,N-dimethylformamide (20 mL) and reacted at 80° C. for 3 hours. After the reaction was completed, the reaction solution was filtered, water was added, and the mixture was extracted with ethyl acetate (100 mL×3), and the organic phases were combined and dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation, and the mixture was purified by column chromatography (petroleum ether: ethyl acetate = 10:1) to obtain compound A2. LCMS: (ESI) m / z: 263.1 [M+1] + .

[0074] Step 2: Synthesis of compound A Compound B (6 g, 25.81 mmol, 1 eq) was added to tetrahydrofuran (180 mL), cooled to -78 ° C (suspension, partially dissolved), and n-butyl lithium (2.5 M, 21.68 mL, 2.1 eq) was added dropwise. After the addition was completed, the mixture was stirred at -78 ° C for 1 hour to react, and then a solution of compound A2 (7.12 g, 27.10 mmol, 1.05 eq) in tetrahydrofuran (10 mL) was added dropwise thereto, and the mixture was stirred for 1 hour to react. After the reaction was completed, the reaction was quenched with saturated ammonium chloride and stirred for 5 minutes, the organic phase was separated, and the aqueous phase was extracted with ethyl acetate (100 mL × 3). The organic phases were combined and concentrated to obtain crude intermediate A. LCMS: (ESI) m / z: 384.2 [M + 1] + .

[0075] Step 3: Synthesis of Compound 1-2 Compound 1-1 (10 g, 29.11 mmol, 1 eq) was dissolved in dichloromethane (100 mL), m-chloroperbenzoic acid (7.53 g, 43.66 mmol, 1.5 eq) was added at 0° C., and the mixture was reacted at 20° C. for 15 hours. The reaction mixture was quenched by adding saturated sodium sulfite solution, and then extracted with ethyl acetate (50 mL×3), washed with saturated saline, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was purified by column chromatography (petroleum ether:ethyl acetate=10:1) to obtain compound 1-2. LCMS: (ESI) m / z: 304.2 [M- t Bu+1].

[0076] Step 4: Synthesis of compounds 1-3 Compound 1-2 (5 g, 13.91 mmol, 1 eq) was dissolved in tetrahydrofuran (10 mL), and lithium aluminum hydride (2.5 M, 8.34 mL, 1.5 eq) was added at -20°C. After the addition was completed, the reaction was allowed to proceed for 3 hours at 20°C. After the reaction was completed, sodium hydroxide solution was added to the reaction solution to quench the reaction, filtered, the filter cake was washed with ethyl acetate, and the filtrate was combined and concentrated to obtain the crude product. The crude product was purified by column chromatography (petroleum ether: ethyl acetate = 3:1) to obtain compound 1-3. LCMS: (ESI) m / z: 306.1 [M- t Bu+1].

[0077] Step 5: Synthesis of Compounds 1-4 Compound 1-3 (0.117 g, 323.61 μmol, 1 eq) was dissolved in acetonitrile (2 mL). Under a nitrogen gas atmosphere, silver oxide (224.97 mg, 970.83 μmol, 3 eq) and iodomethane (459.33 mg, 3.24 mmol, 201.46 μL, 10 eq) were added in sequence at 20° C. Then, the mixture was heated to 80° C. and reacted for 12 hours. After cooling, the reaction solution was filtered, and the filtrate was removed by rotary evaporation to obtain compound 1-4. Compound 1-4 was directly put into the next step without purification. LCMS: (ESI) m / z: 319.85 [M- t Bu+H].

[0078] Step 6: Synthesis of Compounds 1-5 Compound 1-4 (121.54 mg, 323.61 μmol, 1 eq) was dissolved in dichloromethane (2 mL), trifluoroacetic acid (30.80 mg, 270.12 μmol, 0.02 mL, 0.84 eq) was added, and the mixture was reacted at 20° C. for 1 hour after the addition. The reaction solution was directly rotary evaporated to give compound 1-5. Compound 1-5 was used directly in the next reaction without purification. LCMS: (ESI) m / z: 276.20 [M+H] + .

[0079] Step 7: Synthesis of Compounds of Formula (I) Compound A (99.47 mg, 258.89 μmol, 0.8 eq) and compound 1-5 (89.14 mg, 323.61 μmol, 1 eq) were dissolved in isopropanol (2 mL), and N,N-diisopropylethylamine (104.56 mg, 809.03 μmol, 140.91 μL, 2.5 eq) was added. The reaction was then carried out at 130°C for 1 hour in a microwave oven. The reaction solution was removed by rotary evaporation to obtain the crude product. The crude product was purified by preparative chromatography (column: Phenomenex Gemini-NX 80 mm*40 mm*3 μm; mobile phase: [water (0.05% NH 3 H 2 O + 10 mM NH 4 HCO 3 )-acetonitrile]; B (acetonitrile) %: 37%-57%, 8 min) to give the compound of formula (I). LCMS: (ESI) m / z: 508.9 [M]. 1 H NMR (400 MHz, CDCl 3 )δ9.25(d,J=8.3Hz,1H),8.35(s,1H),7.50-7.39(m,3H),7.39-7.32(m,1H) ,7.28-7.22(m,1H),7.14-7.08(m,3H),6.98(dd,J=2.3,8.3Hz,1H),4.57(br d,J=5.5Hz,1H),4.08(dd,J=5.4,10.2Hz,1H),3.93-3.82(m,2H),3.81-3.69(m,2H),3.64-3.53(m,4H),2.03(br d,J=13.1Hz,1H),1.70(br s,1H).

[0080] Example 2: Preparation of crystalline form A of compound of formula (I) Compound A (3.01 g, 7.81 mmol, 1.0 eq) and compound 1-5 (1.62 g, 8.20 mmol, 1.05 eq) were dissolved in isopropanol (15 mL), N,N-diisopropylethylamine (3.03 g, 23.4 mmol, 3.0 eq) was further added, and the mixture was reacted at 90° C. for 20 hours. The reaction was detected to be complete by LCMS, and 50 mL of water was added, and the mixture was extracted with 100 mL of ethyl acetate. The organic phase was separated, dried with anhydrous sodium sulfate, and the organic solvent was removed under reduced pressure. The mixture was slurried with ethyl acetate at room temperature to obtain a solid product, i.e., crystalline form A of the compound of formula (I).

[0081] Example 3: Preparation of crystalline form B of compound of formula (I) Approximately 100 mg of crystalline form A of the compound was placed in different 2.0 mL glass vials, and 1 mL of solvent or solvent mixture was added to obtain a suspension, and the suspension sample was placed in a hot magnetic stirrer and stirred at 25° C. for 16 hours, and the suspension was centrifuged and dried in vacuum at 40° C. for 60 hours until the weight remained unchanged to obtain crystalline form B of the compound of formula (I) (shown in Table 5).

[0082] [Table 5]

[0083] Example 4: Preparation of crystalline form C of compound of formula (I) 1.2 g of the compound of crystalline form A was dispersed in 12 mL of methyl tert-butyl ether to form a suspension, and the system was stirred at 25° C., 40° C., or 60° C. for 16 hours, filtered, and the filter cake was dried in vacuum at 40° C. for 60 hours until the weight remained constant to obtain crystalline form C of the compound of formula (I).

[0084] [Table 6]

[0085] Example 5: Competition experiment between crystalline form B and crystalline form C 30 mg of the compound of crystalline form B and 30 mg of the compound of crystalline form C were taken in three groups, each of which was added to a different 2.0 mL glass vial, and 0.6 mL of methyl tert-butyl ether was added to obtain a suspension. The suspension samples were placed in a hot magnetic stirrer and stirred at 20°C, 40°C, and 60°C for 16 hours, and the suspension was centrifuged to obtain a white solid, which was vacuum dried until the weight remained unchanged to obtain crystalline form C of the compound of formula (I).

[0086] [Table 7]

[0087] Conclusion: Crystal form C is more stable than crystal form B.

[0088] Example 6 Hygroscopicity study of crystalline form C of compound of formula (I) Test materials: SMS DVS Advantage Dynamic Vapor Sorption Apparatus Testing Method: 10-15 mg of crystalline form C of compound of formula (I) was measured in a DVS sample pan.

[0089] Test Results: The DVS pattern of crystalline form C of compound of formula (I) is shown in FIG. 10 and had a ΔW=0.262%.

[0090] Test conclusion: The hygroscopic weight gain of crystalline form C of compound of formula (I) at 25° C. and 80% RH was 0.262%, making it slightly hygroscopic.

[0091] Example 7: Solid-state stability study of crystalline form C of compound of formula (I) In accordance with the "Guideline for Stability Testing of Drug Substances and Preparations" (Chinese Pharmacopoeia 2015, Part 4 General Principles 9001), the stability of crystalline form C of compound of formula (I) was examined under conditions of high temperature (60°C, open), high humidity (a. room temperature / relative humidity 92.5%, open; b. 40°C / relative humidity 75%, open; c. 60°C / relative humidity 75%, open) and strong light exposure (50001x, closed).

[0092] Form C of compound of formula (I) (15 mg) was weighed and placed at the bottom of a glass sample bottle and spread into a thin layer. The bottle mouth of the sample left under high temperature and high humidity conditions was sealed with aluminum foil paper, and several small holes were made in the aluminum foil paper to allow the sample to fully contact with the surrounding air. The sample left under strong light conditions was sealed with a screw cap. The samples left under different conditions were sampled and detected (XRPD) after 5 days, 10 days, and 1 month, and the detection results were compared with the initial detection results on day 0, and the test results are shown in Table 8 below.

[0093] [Table 8]

[0094] Conclusion: The crystalline form C of compound of formula (I) had good stability under the conditions of high temperature, high humidity and strong light.

[0095] Biometric Data: Experimental example 1: BTK enzyme activity measurement The specific experimental process for measuring BTK enzyme activity was as follows:

[0096] Buffer: 20 mM hydroxyethylpiperazineethanesulfonic acid (Hepes) (pH 7.5), 10 mM magnesium chloride, 1 mM ethylene glycol bisaminoethyl ether tetraacetic acid (EGTA), 0.02% polyoxyethylene lauryl ether (Brij35), 0.02 mg / mL BSA, 0.1 mM sodium vanadate (Na 3 VO 4), 2 mM dithiothreitol (DTT), 1% DMSO, and 200 μM adenosine triphosphate (ATP).

[0097] 1. The substrate was placed in freshly prepared reaction buffer. 2. The necessary cofactors were added to the above substrate solution. 3. Kinase BTK C481S was added to the above substrate solution and mixed uniformly. 4. Compounds dissolved in DMSO were added to the kinase reaction mixture via Echo550 (Acoustic technology; nanoliter range) and incubated at room temperature for 20 minutes. 5. 33 P-ATP (specific activity 10 μCi / μL) was added to the reaction mixture to initiate the reaction. 6. Incubate at room temperature for 2 hours. 7. Radioactivity was detected by the filtration-binding method. 8. Kinase activity data was expressed as the percentage of remaining kinase activity in the measurement sample compared to the solvent (dimethyl sulfoxide) reaction. IC 50 Values ​​and fitting curves were obtained using Prism (GraphPad software) and the results are shown in Table 9.

[0098] [Table 9]

[0099] Conclusion: Compounds of formula (I) inhibit BTK C481S It had a strong inhibitory effect on mutations.

[0100] Experimental Example 2: Pharmacokinetic evaluation of compounds of formula (I) in mice 2.1 Experimental Objective: To determine the in vivo pharmacokinetics (intravenous) of the compound in CD-1 mice.

[0101] The compound of formula (I) and the Reference Examples were dissolved in a solvent at 0.10 mg / mL in 10% NMP / 60% PEG400 / 30% H 2The mixture was mixed with 020, vortexed, and sonicated to prepare a clear solution of 0.1 mg / mL. Male CD-1 mice aged 7 to 10 weeks were selected and intravenously administered with the solution of the candidate compound at a dose of 0.21 mg / kg.

[0102] Whole blood was collected at certain times, plasma was prepared, and the drug concentrations were analyzed by LC-MS / MS method, and drug parameters were calculated by Phoenix WinNonlin software (Pharsight, USA), and the results were as shown in Table 10.

[0103] [Table 10]

[0104] Conclusion: The compound of formula (I) has a higher free drug concentration in mouse plasma than the reference example.

[0105] 2.2 Experimental Objective: To determine the in vivo pharmacokinetics (oral) of the compound in CD-1 mice.

[0106] The compounds of formula (I) and the Reference Examples were dissolved in a solvent of 10% NMP / 60% PEG400 / 30% H 2 The mixture was mixed with 0, vortexed and sonicated to prepare a clear solution of 0.6 mg / mL. Male CD-1 mice aged 7 to 10 weeks were selected and the solution of the candidate compound was administered orally at a dose of 3.1 mg / kg by force. Whole blood was collected at a certain time point, plasma was prepared, and the drug concentration was analyzed by LC-MS / MS method, and drug parameters were calculated by Phoenix WinNonlin software (Pharsight, USA), and the results were as shown in Table 11.

[0107] [Table 11]

[0108] Conclusion: The compound of formula (I) has a higher free drug concentration in mouse plasma than the reference example.

[0109] Experimental Example 3: Pharmacokinetic evaluation of compounds in rats 3.1 Experimental Objective: To determine the in vivo pharmacokinetics (intravenous) of the compound in SD rats.

[0110] The compound of formula (I) and the Reference Examples were dissolved in a solvent at 0.10 mg / mL in 10% NMP / 60% PEG400 / 30% H 2 The mixture was mixed with 0, vortexed and sonicated to prepare a clear solution of 0.5 mg / mL. SD rats aged 7 to 10 weeks were selected and intravenously injected with the solution of the candidate compound. Whole blood was collected at a certain time, plasma was prepared, and the drug concentration was analyzed by LC-MS / MS method. The drug parameters were calculated by Phoenix WinNonlin software (Pharsight, USA), and the results were as shown in Table 12.

[0111] [Table 12]

[0112] Conclusion: The compound of formula (I) has a higher free drug concentration in rat plasma than the reference example.

[0113] 3.2 Experimental Objective: To determine the in vivo pharmacokinetics (oral) of the compound in SD rats.

[0114] The compounds of formula (I) and the Reference Examples were dissolved in a solvent of 10% NMP / 60% PEG400 / 30% H 2 The solution was mixed with O, vortexed, and sonicated to prepare a clear solution of 2 mg / mL. SD rats aged 7 to 10 weeks were selected and the candidate compound solution was orally administered by force.

[0115] Whole blood was collected at certain times, plasma was prepared, and drug concentrations were analyzed by LC-MS / MS method, and drug parameters were calculated by Phoenix WinNonlin software (Pharsight, USA), and the results were as shown in Table 13.

[0116] [Table 13]

[0117] Conclusion: The compound of formula (I) has a higher free drug concentration in rat plasma than the reference example.

[0118] Experimental Example 4: In vivo studies OCI-LY10 SCID mouse xenograft tumor model Experimental method: A human B-cell lymphoma subcutaneous tumor model was prepared. Tumor cells in the logarithmic growth phase were harvested and resuspended in IMDM basal medium, and Matrigel was added at a 1:1 ratio to a cell concentration of 4 × 10 7 Under sterile conditions, 0.1 mL of the cell suspension was diluted with 4 × 10 6 The tumors were inoculated subcutaneously into the right dorsal region of SCID mice at a concentration of 0.1 mL / mouse. 3 When the tumor volume reached the threshold, the tumor-bearing mice were randomly divided into 5 groups, each with 8 mice, according to the size of the tumor volume. On the day of the experiment, the animals were administered the corresponding drugs according to the group.

[0119] [Table 14]

[0120] During the experiment, the body weight and tumor size of the animals were measured three times a week, and the clinical symptoms of the animals were observed and recorded daily. For each administration, the body weight of the animals was referred to as the last weighed body weight.

[0121] The tumor was measured by measuring the length (a) and width (b) with a digital caliper, and the tumor volume (TV) was calculated as TV=a×b. 2 / 2.

[0122] Test Results: The antitumor effects of the test compounds evaluated in a human B cell lymphoma OCI-LY10 cell subcutaneous xenograft tumor model are shown in Table 15.

[0123] The tumor volume growth curve under drug action is shown in FIG.

[0124] [Table 15]

[0125] The mean tumor volume was calculated based on the tumor volume of the animals at each measurement. Data points represent the mean tumor volume change within a group, and error bars represent the standard error of the mean (SEM).

[0126] Conclusion: In the in vivo study of human B-cell lymphoma OCI-LY10 cell subcutaneous xenograft tumor model, crystalline form C could significantly inhibit tumor growth, and the tumor inhibition effect was positively correlated with the dose. At the same dose, the tumor inhibition effect of crystalline form C was significantly superior to that of the reference.

Claims

1. A crystal of crystalline form A of compound of formula (I), The X-ray powder diffraction pattern of the crystalline form A is characterized by having characteristic diffraction peaks at the following 2θ angles: 16.16±0.20°, 16.60±0.20°, 22.02±0.20°, and 23.08±0.20°. 【Chemistry 1】

2. 2. The crystal of claim 1, wherein the X-ray powder diffraction pattern of crystalline form A has characteristic diffraction peaks at the following 2θ angles: 7.51±0.20°, 10.63±0.20°, 15.06±0.20°, 16.16±0.20°, 16.60±0.20°, 21.28±0.20°, 22.02±0.20°, and 23.08±0.20°.

3. 3. The crystal of claim 2, wherein the X-ray powder diffraction pattern of crystalline form A has characteristic diffraction peaks at the following 2θ angles: 7.51±0.20°, 10.63±0.20°, 12.00±0.20°, 13.17±0.20°, 15.06±0.20°, 16.16±0.20°, 16.60±0.20°, 18.61±0.20°, 21.28±0.20°, 22.02±0.20°, 22.49±0.20°, and 23.08±0.20°.

4. 4. The crystal of claim 3, wherein the X-ray powder diffraction pattern of crystalline form A has characteristic diffraction peaks at the following 2θ angles: 7.51°, 9.45°, 10.04°, 10.63°, 12.00°, 13.17°, 14.25°, 15.06°, 15.51°, 16.16°, 16.60°, 17.31°, 17.58°, 18.61°, 18.95°, 19.53°, 20.07°, 20.60°, 21.28°, 21.78°, 22.02°, 22.49°, and 23.08°.

5. The crystal according to claim 4, wherein the crystalline form A has an X-ray powder diffraction pattern as shown in FIG. [Figure 1]

6. The crystal according to any one of claims 1 to 5, wherein the differential scanning calorimetry curve of the crystalline form A has endothermic peak onset values ​​at 145.7°C ± 3.0°C, 208.9°C ± 3.0°C, and 218.7°C ± 3.0°C, respectively.

7. The crystal of claim 6, wherein the crystalline form A has a DSC pattern as shown in FIG. [Figure 2]

8. The crystal according to any one of claims 1 to 5, wherein the thermogravimetric analysis curve of the crystalline form A shows a weight loss of 7.66% at 200.0°C ± 3.0°C.

9. The crystal according to claim 8, wherein the crystalline form A has a TGA pattern as shown in Figure 3 below. [Figure 3]

10. A crystal of crystalline form B of compound of formula (I), The X-ray powder diffraction pattern of the crystalline form B is characterized by having characteristic diffraction peaks at the following 2θ angles: 7.47±0.20°, 10.41±0.20°, 16.04±0.20°, and 16.73±0.20°. 【Chemistry 2】

11. 11. The crystal of claim 10, wherein the X-ray powder diffraction pattern of crystalline form B has characteristic diffraction peaks at the following 2θ angles: 7.47±0.20°, 10.41±0.20°, 15.23±0.20°, 16.04±0.20°, 16.73±0.20°, 20.87±0.20°, 21.46±0.20°, and 21.91±0.20°.

12. 12. The crystal of claim 11, wherein the X-ray powder diffraction pattern of crystalline form B has characteristic diffraction peaks at the following 2θ angles: 7.47±0.20°, 9.53±0.20°, 10.41±0.20°, 11.91±0.20°, 13.91±0.20°, 15.23±0.20°, 16.04±0.20°, 16.73±0.20°, 18.82±0.20°, 20.87±0.20°, 21.46±0.20°, and 21.91±0.20°.

13. 13. The crystal of claim 12, wherein the X-ray powder diffraction pattern of crystalline form B has characteristic diffraction peaks at the following 2θ angles: 7.47°, 9.53°, 9.78°, 10.41°, 11.45°, 11.91°, 12.40°, 13.22°, 13.91°, 14.87°, 15.23°, 15.44°, 16.04°, 16.73°, 17.33°, 17.88°, 18.82°, 19.61°, 19.88°, 20.25°, 20.87°, 21.46°, and 21.91°.

14. The crystal according to claim 13, wherein the crystalline form B has an X-ray powder diffraction pattern as shown in FIG. [Figure 4]

15. The crystal according to any one of claims 10 to 14, wherein the differential scanning calorimetry curve of the crystalline form B has an onset value of an endothermic peak at 214.3°C ± 3.0°C.

16. 16. The crystal of claim 15, wherein the crystalline form B has a DSC pattern as shown in FIG. [Figure 5]

17. The crystal according to any one of claims 10 to 14, wherein the thermogravimetric analysis curve of the crystalline form B shows a weight loss of 0.64% at 200.0°C ± 3.0°C.

18. 18. The crystal of claim 17, wherein the crystalline form B has a TGA pattern as shown in FIG. [Figure 6]

19. A crystal of crystalline form C of compound of formula (I), The X-ray powder diffraction pattern of the crystalline form C is characterized by having characteristic diffraction peaks at the following 2θ angles: 5.28±0.20°, 8.58±0.20°, 20.56±0.20°, and 24.55±0.20°. 【Chemistry 3】

20. 20. The crystal of claim 19, wherein the X-ray powder diffraction pattern of crystalline form C has characteristic diffraction peaks at the following 2θ angles: 5.28±0.20°, 8.58±0.20°, 11.00±0.20°, 11.52±0.20°, 15.72±0.20°, 20.56±0.20°, 21.84±0.20°, and 24.55±0.20°.

21. 21. The crystal of claim 20, wherein the X-ray powder diffraction pattern of crystalline form C has characteristic diffraction peaks at the following 2θ angles: 5.28±0.20°, 8.58±0.20°, 11.00±0.20°, 11.52±0.20°, 15.72±0.20°, 16.69±0.20°, 17.16±0.20°, 18.62±0.20°, 19.76±0.20°, 20.56±0.20°, 21.84±0.20°, and 24.55±0.20°.

22. 22. The crystal of claim 21, wherein the X-ray powder diffraction pattern of crystalline form C has characteristic diffraction peaks at the following 2θ angles: 5.28°, 8.58°, 11.00°, 11.52°, 14.08°, 15.08°, 15.72°, 16.69°, 17.16°, 18.62°, 19.76°, 20.56°, 20.77°, 21.84°, 23.14°, 23.89°, 24.55°, 25.40°, 28.24°, 29.21°, 31.36°.

23. The crystal according to claim 22, wherein the crystalline form C has an X-ray powder diffraction pattern as shown in FIG. [Figure 7]

24. The crystal according to any one of claims 19 to 23, wherein the differential scanning calorimetry curve of the crystalline form C has an onset value of an endothermic peak at 218.8 ° C. ± 3.0 ° C.

25. 25. The crystal of claim 24, wherein the crystalline form C has a DSC pattern as shown in FIG. [Figure 8]

26. The crystal according to any one of claims 19 to 23, wherein the thermogravimetric analysis curve of the crystalline form C shows a weight loss of 1.45% at 200.0°C ± 3.0°C.

27. 27. The crystal of claim 26, wherein the crystalline form C has a TGA pattern as shown in FIG. [Figure 9]

28. 27. Use of the crystal of any one of claims 1 to 5, 10 to 14, or 19 to 23 in the preparation of a medicament for treating a disease associated with BTK protein kinase.

29. 29. The use of claim 28, wherein the disease associated with the BTK protein kinase is a hematological tumor.

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