Polymorphs of Bruton's tyrosine kinase inhibitors and their preparation methods and applications

Abstract

The present invention provides polymorphs of Bruton's tyrosine kinase inhibitors, their preparation methods, and uses. Specifically, two crystalline forms of (R)-6-((1-acryloylpiperidin-3-yl)amino)-7-fluoro-4-((2-fluoro-4-morpholinophenyl)amino)-1,2-dihydro-3H-pyrrolo[3,4-c]pyridin-3-one, their preparation methods, and uses are provided. The two crystalline forms provided by the present invention have good stability, are not hygroscopic, have improved solubility and pharmacokinetic characteristics, and the preparation method is stable and capable of large-scale production.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a polymorph of a Bruton's tyrosine kinase inhibitor, a preparation method, and an application thereof. The inhibitor is (R)-6-((1-acryloylpiperidin-3-yl)amino)-7-fluoro-4-((2-fluoro-4-morpholinophenyl)amino)-1,2-dihydro-3H-pyrrolo[3,4-c]pyridin-3-one. Background Art

[0002] Bruton's tyrosine kinase (BTK), a non-receptor tyrosine kinase in the TEC kinase family, is a key regulator of the BCR signaling pathway and plays a crucial role in B cell maturation, proliferation, and survival. BTK is overexpressed in various B cell lymphomas and is currently the only clinically validated target for effective drug development in the TEC kinase family. Inhibiting BTK can suppress the proliferation of a range of B cell lymphomas.

[0003] Activation of the B cell antigen receptor (BCR) signaling pathway plays a crucial role in the induction and maintenance of B cell malignancies and autoimmune diseases. Bruton's tyrosine kinase (Btk) plays a key role in BCR signaling in hematopoietic cells and is a promising target for novel lymphoma therapies. BTK inhibitors act within the BCR pathway to inhibit Btk autophosphorylation, phosphorylation of Btk's physiological substrate, PLCγ, and phosphorylation of the downstream kinase, ERK.

[0004] BTK inhibitors induce cytotoxicity and inhibit CLL cell proliferation in chronic lymphocytic leukemia (CLL) cells. They also inhibit the proliferation of BCR-activated primary B cells and suppress the secretion of TNFα, IL-1β, and IL-6 in primary monocytes. In a collagen-induced arthritis model, BTK inhibitors significantly reduce clinical arthritis symptoms such as paw swelling and joint inflammation by suppressing B cell activity.

[0005] Patent WO2019062329A1 discloses a class of compound structures with good BTK inhibitory activity, and specifically discloses the compound 6-((1-acryloylpiperidin-3-yl)amino)-7-fluoro-4-((2-fluoro-4-morpholinophenyl)amino)-1,2-dihydro-3H-pyrrolo[3,4-c]pyridine-3-one, which has high inhibitory activity against enzymes and cells. However, when the compound was further developed, it was found that the R configuration of the compound had poor solubility when it existed in an amorphous state, and such a result may have an impact on later drug development. In order to better and more effectively develop and study the compound and to be able to potentially apply it in clinical practice, the present invention further studied the R-configuration compound and completed the present invention based on this research. Summary of the Invention

[0006] Based on this, the present invention provides two new crystalline forms of (R)-6-((1-acryloylpiperidin-3-yl)amino)-7-fluoro-4-((2-fluoro-4-morpholinophenyl)amino)-1,2-dihydro-3H-pyrrolo[3,4-c]pyridin-3-one (compound of formula (X)), as well as preparation methods and uses thereof. The new crystalline forms provided by the present invention have improved solubility and more favorable in vivo pharmacokinetic characteristics, facilitating further drug development.

[0007] The present invention is achieved through the following technical solutions.

[0008] A crystalline form IV of a compound of formula X, wherein the compound of formula X has the following structure:

[0009]

[0010] The X-ray powder diffraction pattern of the crystalline form IV detected using Cu-Kα radiation has diffraction peaks at the following diffraction angle 2θ (°) values: 5.90±0.2, 14.91±0.2, 17.51±0.2, 24.90±0.2 and 26.37±0.2.

[0011] In one embodiment, the X-ray powder diffraction pattern of the crystalline form IV has diffraction peaks at the following diffraction angle 2θ (°) values: 5.90±0.2, 10.41±0.2, 12.19±0.2, 14.91±0.2, 17.51±0.2, 18.80±0.2, 21.49±0.2, 24.90±0.2, 25.44±0.2 and 26.37±0.2.

[0012] In one embodiment, the X-ray powder diffraction pattern of the crystalline form IV has diffraction peaks at the following diffraction angle 2θ (°) values: 5.90±0.2, 8.91±0.2, 10.41±0.2, 12.19±0.2, 12.49±0.2, 12.76±0.2, 13.94±0.2, 14.91±0.2, 15.48±0.2, 16.0 5±0.2, 17.51±0.2, 18.29±0.2, 18.80±0.2, 19.76±0.2, 21.49±0.2, 22.45±0.2, 23.57±0.2, 24.90±0.2, 25.44±0.2, 26.37±0.2, 27.73±0.2, 28.67±0.2 and 35.37±0.2.

[0013] In one embodiment, the X-ray powder diffraction pattern of the crystalline form IV is substantially as follows Figure 1 Represented.

[0014] In one embodiment, the differential scanning calorimetry curve of the crystalline form IV has an endothermic peak at 167.89°C±3°C.

[0015] In one embodiment, the crystalline form IV has substantially Figure 2 The differential scanning calorimetry curve is shown.

[0016] In one embodiment, the crystalline form IV has substantially Figure 3 Thermogravimetric analysis diagram shown.

[0017] In one embodiment, the crystalline form IV has substantially Figure 4 DVS map shown.

[0018] The present invention also provides a method for preparing the crystalline form IV of the compound of formula X as described above, the method comprising the following steps:

[0019] S101, heating a mixture of the compound of formula X and a solvent to 55° C. to 65° C.; and

[0020] S102, cooling the mixture, separating the precipitated solid, and obtaining the crystalline form IV.

[0021] In one embodiment, the solvent in step S101 is selected from one or more of water, ethanol, n-propanol and isopropanol.

[0022] In one embodiment, the mixture in step S102 is cooled to 20°C to 25°C.

[0023] The present invention also provides a crystalline form V of a compound of formula X, wherein the compound of formula X has the following structure:

[0024]

[0025] The X-ray powder diffraction pattern of the crystalline form V detected using Cu-Kα radiation has diffraction peaks at the following diffraction angle 2θ (°) values: 7.33±0.2, 9.91±0.2 and 17.53±0.2.

[0026] In one embodiment, the X-ray powder diffraction pattern of the crystalline form V has diffraction peaks at the following diffraction angle 2θ (°) values: 7.33±0.2, 9.91±0.2, 12.49±0.2, 15.69±0.2, 17.53±0.2, 20.19±0.2 and 23.02±0.2.

[0027] In one embodiment, the X-ray powder diffraction pattern of the crystalline form V has diffraction peaks at the following diffraction angle 2θ (°) values: 7.33±0.2, 9.91±0.2, 12.49±0.2, 13.67±0.2, 15.69±0.2, 16.20±0.2, 17.53±0.2, 18.95±0.2, 20.19±0.2, 21.76±0.2, 23.02±0.2, 24.80±0.2, 25.53±0.2 and 30.21±0.2.

[0028] In one embodiment, the X-ray powder diffraction pattern of the crystalline form V is substantially as follows Figure 5 Represented.

[0029] In one embodiment, the differential scanning calorimetry curve of the crystalline form V has an endothermic peak at 197.31°C±3°C.

[0030] In one embodiment, the crystalline form V has substantially Figure 6 The differential scanning calorimetry curve is shown.

[0031] In one embodiment, the crystalline form V has substantially Figure 7 Thermogravimetric analysis spectrum shown.

[0032] In one embodiment, the crystalline form V has substantially Figure 8 DVS spectrum shown.

[0033] The present invention also provides a method for preparing the crystalline form V of the compound of formula X as described above, the method comprising the following steps:

[0034] S201, heating a mixture of the compound of formula X and a solvent to above 70°C; and

[0035] S202, cooling the mixture, separating the precipitated solid, and obtaining the crystalline form V.

[0036] In one embodiment, the solvent in step S201 is selected from: water, C 2-4 One or more of alkyl alcohol, acetonitrile, toluene, xylene, 1,4-dioxane, 1,2-dichloroethane and ethyl acetate.

[0037] In one embodiment, the C 2-4 The alkyl alcohol is one or more of ethanol, n-propanol, isopropanol and ethylene glycol.

[0038] In one embodiment, the ratio of the mass of the compound of formula X to the volume of the solvent in step S201 is 50 g / L to 400 g / L.

[0039] In one embodiment, the mixture of the compound of formula X and the solvent in step S201 is heated to 70° C. to 80° C.; and the mixture in step S202 is cooled to 20° C. to 25° C.

[0040] In one embodiment, the process further includes the following steps before step S202: cooling the mixture to 40° C. to 65° C. and keeping the mixture warm for 1 to 12 hours.

[0041] The present invention also provides use of the above-described crystalline Form IV, or the crystalline Form IV prepared by the above-described preparation method, in the preparation of a medicament for treating and / or preventing tumors, cancers, proliferative diseases, allergic diseases, autoimmune diseases or inflammatory diseases.

[0042] The present invention also provides use of the above-described Form V, or the Form V prepared by the above-described preparation method, in the preparation of drugs for treating and / or preventing tumors, cancers, proliferative diseases, allergic diseases, autoimmune diseases or inflammatory diseases.

[0043] Compared with the prior art, the new crystal form of the present invention has the following beneficial effects:

[0044] The crystalline Forms IV and V of the compound of Formula X described herein exhibit excellent crystallinity, high stability, and low hygroscopicity. Compared to the amorphous form, they possess improved solubility and more favorable pharmacokinetic characteristics, including relatively high plasma exposure and a longer half-life, which contribute to improved bioavailability of the amorphous form of the compound of Formula X. Furthermore, the preparation method of Form IV of the compound of Formula X is simple, resulting in a high-purity crystal suitable for industrial production. The preparation method of Form V is highly reproducible, easily controllable, and stable, making it suitable for industrial production.

[0045] In summary, the crystalline form IV and crystalline form V of the compound of formula X have the potential to be further developed into drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 is the X-ray powder diffraction (XRPD) pattern of Form IV (using Cu-Kα radiation, the abscissa is the angle 2θ (°), and the ordinate is the intensity);

[0047] Figure 2 is the differential scanning calorimetry (DSC) spectrum of Form IV (the abscissa is temperature (°C), the ordinate is heat flow rate (mW));

[0048] Figure 3 The thermogravimetric analysis (TGA) spectrum of Form IV (the abscissa is temperature (°C), the ordinate is weight percentage (%));

[0049] Figure 4The dynamic moisture sorption (DVS) spectrum of Form IV (the abscissa is relative humidity (%), and the ordinate is the sample weight change (%));

[0050] Figure 5 is the X-ray powder diffraction (XRPD) pattern of Form V (using Cu-Kα radiation, the abscissa is the angle 2θ (°), and the ordinate is the intensity);

[0051] Figure 6 is the differential scanning calorimetry (DSC) spectrum of Form V (the abscissa is temperature (°C), the ordinate is heat flow rate (mW));

[0052] Figure 7 Thermogravimetric analysis (TGA) spectrum of Form V (the abscissa is temperature (°C), the ordinate is weight percentage (%));

[0053] Figure 8 The dynamic moisture sorption (DVS) spectrum of Form V (the horizontal axis is relative humidity (%), and the vertical axis is the sample weight change (%));

[0054] Figure 9 Comparison of XRPD patterns of Form IV after being stored at 60°C for 1 month, 40°C / 75% RH for 1 month, and the initial sample (the horizontal axis is the angle 2θ (°), and the vertical axis is the intensity);

[0055] Figure 10 Comparison of XRPD patterns of Form V after storage at 60°C for 1 month, 40°C / 75% RH for 1 month, and the initial sample (abscissa: 2θ(°); ordinate: intensity);

[0056] Figure 11 is the X-ray powder diffraction (XRPD) pattern of the amorphous free base of the compound of formula X (using Cu-Kα radiation, the abscissa is 2θ (°); the ordinate is intensity);

[0057] Figure 12 The X-ray powder diffraction (XRPD) pattern of Form I (using Cu-Kα radiation, the abscissa is 2θ (°); the ordinate is intensity). DETAILED DESCRIPTION

[0058] To facilitate understanding of the present invention, the present invention will be described more fully below with reference to the accompanying drawings. The accompanying drawings illustrate preferred embodiments of the present invention. However, the present invention may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the present disclosure.

[0059] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe a feature or characteristic, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein are understood to include any and all subranges subsumed therein.

[0060] Unless otherwise indicated, all percentages, fractions, and ratios are calculated based on the total weight of the compositions of the present invention. Unless otherwise indicated, all masses relating to listed ingredients are given as active ingredients and therefore do not include solvents or byproducts that may be present in commercially available materials. The term "mass percentage content" may be expressed herein with the symbol "%." All molecular weights herein are weight-average molecular weights expressed in Daltons, unless otherwise indicated. All formulations and testing herein took place at 25°C, unless otherwise indicated. The terms "comprise," "include," "contain," "have," "have," and other variations herein are intended to encompass non-exclusive inclusions, and do not distinguish between these terms. The term "comprising" means that additional steps and ingredients that do not affect the end result may be added. The compositions and methods / processes of the present invention comprise, consist of, and consist essentially of the essential elements and limitations described herein, as well as any additional or optional ingredients, components, steps, or limitations described herein. The terms "efficacy," "performance," "effect," and "efficacy" are not used herein to distinguish between.

[0061] Compound of formula X

[0062] In the present invention, the compound of formula X is (R)-6-((1-acryloylpiperidin-3-yl)amino)-7-fluoro-4-((2-fluoro-4-morpholinophenyl)amino)-1,2-dihydro-3H-pyrrolo[3,4-c]pyridin-3-one. Its crystalline forms IV and V have improved solubility and more favorable in vivo pharmacokinetic characteristics, are non-hygroscopic, and have excellent chemical and physical stability. Furthermore, the compound of formula X exhibits superior inhibitory activity against BTK WT kinase compared to its racemic compound.

[0063] The crystalline form IV and crystalline form V of the compound of formula X provided by the present invention can be used to prepare BTK inhibitors or prepare drugs for treating BTK-related diseases. Preferably, the BTK-related disease is cancer, abnormal cell proliferation disease, infection, inflammatory disease, autoimmune disease, cardiovascular disease, neurodegenerative disease, hematopoietic toxicity disease caused by radiation, or a combination thereof. Preferably, the cancer is breast cancer, ovarian cancer, prostate cancer, melanoma, brain tumor, esophageal cancer, gastric cancer, liver cancer, pancreatic cancer, colorectal cancer, lung cancer, kidney cancer, skin cancer, glioblastoma, neuroblastoma, sarcoma, liposarcoma, osteochondroma, osteoma, osteosarcoma, seminoma, testicular tumor, uterine cancer, head and neck tumor, multiple myeloma, malignant lymphoma, polycythemia vera, leukemia, thyroid tumor, ureteral tumor, bladder tumor, gallbladder cancer, bile duct cancer, choriocarcinoma or pediatric tumor, or any combination thereof. Preferably, the breast cancer is HR-positive, HER2-negative advanced breast cancer.

[0064] As used herein, "therapeutically effective amount" refers to an amount that can produce a function or activity on humans and / or animals and can be accepted by humans and / or animals.

[0065] As used herein, "patient" refers to an animal, preferably a mammal, more preferably a human. The term "mammal" refers to warm-blooded vertebrate mammals, including cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, mice, pigs and humans.

[0066] As used herein, "treat" refers to alleviating, slowing the progression, attenuating, preventing, or maintaining an existing disease or condition (eg, cancer). Treatment also includes curing, preventing the development of, or alleviating to some extent, one or more symptoms of a disease or condition.

[0067] In the present invention, "using Cu-Kα radiation" means that the corresponding spectrum is obtained using Kα ray detection of a Cu target. When other detection methods are used, the diffraction peaks may have deviations within the acceptable range in the art, which should not be construed as limiting the present invention.

[0068] polymorphs

[0069] Solids exist in either amorphous or crystalline form. In the case of crystalline forms, the molecules are positioned within a three-dimensional lattice. When a compound crystallizes from a solution or slurry, it can crystallize in different spatial arrangements (a property known as "polymorphism"), forming crystals with different crystalline forms, which are referred to as "polymorphs" or "crystal forms." Different polymorphs of a given substance may differ from one another in one or more physical properties, such as solubility and dissolution rate, true specific gravity, crystal shape, packing pattern, flowability, and / or solid-state stability.

[0070] crystallization

[0071] The solubility limit of the compound of interest can be exceeded by operating the solution, thereby completing production-scale crystallization. This can be accomplished by a variety of methods, for example, dissolving the compound at a relatively high temperature and then cooling the solution to below the saturation limit. Alternatively, the liquid volume can be reduced by boiling, atmospheric evaporation, vacuum drying, or other methods. The solubility of the compound of interest can be reduced by adding an antisolvent or a solvent or a mixture of such solvents in which the compound has low solubility. Another alternative is to adjust the pH value to reduce solubility. For a detailed description of the relevant crystallization aspects, see Crystallization, 3rd edition, JW Mullens, Butterworth-Heineman Ltd., 1993, ISBN 0750611294.

[0072] As used herein, the term "room temperature" generally refers to 4-30°C, preferably 20±5°C.

[0073] Identification and properties of crystal forms

[0074] After preparing the crystalline form of the compound of formula X, the present invention studied its properties using the following methods and instruments.

[0075] X-ray powder diffraction (XRPD)

[0076] Methods for determining crystal forms by X-ray powder diffraction are known in the art. XRPD can detect changes in crystal form, crystallinity, crystalline state, and other information, and is a common method for identifying crystal forms. The peak position of an XRPD pattern depends primarily on the structure of the crystal form. The 2θ measurement of an XRPD pattern may vary slightly between different instruments, so the 2θ value cannot be considered absolute. Depending on the instrument conditions used in the present invention's experiments, the diffraction peaks may have an error of ±0.2°. The crystal form of the compound of Formula X of the present invention has a specific crystal morphology and exhibits specific characteristic peaks in the XRPD pattern.

[0077] Differential Scanning Calorimetry (DSC)

[0078] Also known as "differential scanning calorimetry", it is a technique that measures the relationship between the energy difference and temperature between the substance being tested and the reference substance during the heating process. The peak position, shape and number of peaks on the DSC spectrum are related to the properties of the substance, so they can be used to qualitatively identify the substance. This method is commonly used in this field to detect various parameters such as the phase transition temperature, glass transition temperature, reaction heat, etc. of a substance. The peak position of the DSC spectrum may vary slightly between different instruments, so the numerical value of the peak position of the DSC endothermic peak cannot be regarded as absolute. According to the condition of the instrument used in the test of the present invention, the numerical value of the experimental error or difference may be less than or equal to 5°C, or less than or equal to 4°C, or less than or equal to 3°C, or less than or equal to 2°C, or less than or equal to 1°C.

[0079] Thermogravimetric analysis (TGA)

[0080] TGA is a technique that measures the mass change of a substance with temperature under program control. It is suitable for examining the loss of solvent from crystals or the sublimation or decomposition of a sample, and can infer the presence of water of crystallization or solvent in the crystals. The mass change shown by the TGA curve depends on many factors, including sample preparation and instrumentation; the mass change detected by TGA varies slightly between different instruments. Depending on the instrument used in the present test, the mass change has an error of ±0.1%.

[0081] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. The terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0082] In the first aspect of the present invention, there is provided a crystalline form IV of a compound of formula X:

[0083]

[0084] In one embodiment, Form IV is in an anhydrous form or a hydrate form.

[0085] In one embodiment, Form IV is in an anhydrous form.

[0086] In a specific example, the X-ray powder diffraction pattern of Form IV detected using Cu-Kα radiation has diffraction peaks at diffraction angle 2θ (°) values ​​of the following group IV-1: 5.90±0.2, 14.91±0.2, 17.51±0.2, 24.90±0.2 and 26.37±0.2.

[0087] In a specific example, the X-ray powder diffraction pattern of Form IV further includes diffraction peaks at 1, 2, or more than 2 or all of the diffraction angle 2θ (°) values ​​selected from the following group IV-2: 10.41±0.2, 12.19±0.2, 18.80±0.2, 21.49±0.2 and 25.44±0.2.

[0088] In a specific example, the X-ray powder diffraction pattern of Form IV further includes diffraction peaks at diffraction angle 2θ (°) values ​​of 8.91±0.2, 12.49±0.2, and 12.76±0.2.

[0089] In a specific example, the X-ray powder diffraction pattern of Form IV further includes diffraction peaks at diffraction angle 2θ (°) values ​​of 15.48±0.2, 16.05±0.2, 18.29±0.2, 19.76±0.2, 22.45±0.2 and 23.57±0.2.

[0090] In a specific example, the X-ray powder diffraction pattern of Form IV further includes diffraction peaks at 1, 2, 3 or more or all of the diffraction angle 2θ (°) values ​​selected from the following group IV-3: 8.91±0.2, 12.49±0.2, 12.76±0.2, 13.94±0.2, 15.48±0.2, 16.05±0.2, 18.29±0.2, 19.76±0.2, 22.45±0.2, 23.57±0.2, 27.73±0.2, 28.67±0.2 and 35.37±0.2.

[0091] In a specific example, the X-ray powder diffraction pattern of Form IV has peaks at 2θ (°) values ​​of 6 or more or all (such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc.) selected from Group IV-1, IV-2 and IV-3.

[0092] In a specific example, the X-ray powder diffraction pattern of Form IV has diffraction peaks at the 2θ (°) values ​​shown in Table 1, and the relative intensities of the peaks are shown in Table 1:

[0093] Table 1

[0094] 2θ(°) <![CDATA[I / I0]]> 2θ(°) <![CDATA[I / I0]]> 2θ(°) <![CDATA[I / I0]]> 5.90 S 15.48 W 23.57 W 8.91 W 16.05 W 24.90 S 10.41 M 17.51 S 25.44 M 12.19 M 18.29 W 26.37 S 12.49 W 18.80 M 27.73 W 12.76 W 19.76 W 28.67 W 13.94 W 21.49 M 35.37 W 14.91 VS 22.45 W

[0095] In a specific example, the X-ray powder diffraction pattern of Form IV is substantially as follows Figure 1 Represented.

[0096] In a specific example, the differential scanning calorimetry curve of Form IV has an endothermic peak at 167.89°C±3°C, and the heat of melting is about 55.84 J / g.

[0097] In a specific example, the differential scanning calorimetry curve of Form IV has an endothermic peak at 167.89°C±1°C.

[0098] In one specific example, Form IV has substantially Figure 2 The differential scanning calorimetry curve is shown.

[0099] In a specific example, the weight loss of Form IV at 30° C. to 150° C. was determined by thermogravimetric analysis to be approximately 0.29%.

[0100] In one specific example, Form IV has substantially Figure 3 Thermogravimetric analysis diagram shown.

[0101] In a specific example, the dynamic moisture adsorption of Form IV increases weight by about 0.7% in the relative humidity range of 0% to 80%, where the percentage is weight percentage.

[0102] In one specific example, Form IV has substantially Figure 4 DVS map shown.

[0103] In a second aspect of the present invention, a method for preparing the crystalline form IV of the compound of formula X is provided. The preparation method is simple, the obtained crystalline form has high purity, and is suitable for industrial production.

[0104] The preparation method of the crystalline form IV of the compound of formula X comprises the following steps:

[0105] S101, heating a mixture of the compound of formula X and a solvent to 55° C. to 65° C. (preferably 60 to 65° C.); and

[0106] S102. Cool the mixture, separate the precipitated solid, and obtain Form IV.

[0107] In a specific example, the mixture in step S102 is cooled to room temperature.

[0108] In a specific example, the mixture in step S102 is cooled to 20°C to 25°C.

[0109] In a specific example, the solvent in step S101 is selected from one or more of water, ethanol, n-propanol and isopropanol.

[0110] In a specific example, the solvent in step S101 is isopropyl alcohol.

[0111] In a specific example, in step S101, the heating time is 0.5-12 hours; preferably, the heating time is 1-2 hours.

[0112] In a specific example, in step S102, after the mixture is cooled to room temperature, it is kept warm for 0-12 hours, preferably for 1-2 hours.

[0113] In the third aspect of the present invention, a crystalline form IV of the compound of formula X prepared according to the preparation method of the second aspect is provided.

[0114] In a fourth aspect of the present invention, another crystalline form V of a compound of formula X is provided, wherein the compound of formula X has the following structure:

[0115]

[0116] In one embodiment, Form V is in an anhydrous form or a hydrated form.

[0117] In one embodiment, Form V is in an anhydrous form.

[0118] In a specific example, the X-ray powder diffraction pattern of Form V detected using Cu-Kα radiation has diffraction peaks at diffraction angle 2θ (°) values ​​of the following group V-1: 7.33±0.2, 9.91±0.2 and 17.53±0.2.

[0119] In a specific example, the X-ray powder diffraction pattern of Form V further includes a diffraction peak at a diffraction angle 2θ (°) value of 13.67±0.2.

[0120] In a specific example, the X-ray powder diffraction pattern of Form V further includes diffraction peaks at diffraction angle 2θ (°) values ​​of 15.69±0.2 and 16.20±0.2.

[0121] In a specific example, the X-ray powder diffraction pattern of Form V further includes a diffraction peak at a diffraction angle 2θ (°) value of 12.49±0.2.

[0122] In a specific example, the X-ray powder diffraction pattern of Form V further includes a diffraction peak at a diffraction angle 2θ (°) value of 18.95±0.2.

[0123] In a specific example, the X-ray powder diffraction pattern of Form V further includes diffraction peaks at diffraction angle 2θ (°) values ​​of 20.19±0.2 and 21.76±0.2.

[0124] In a specific example, the X-ray powder diffraction pattern of Form V further includes diffraction peaks at diffraction angle 2θ (°) values ​​of the following group V-2: 12.49±0.2, 15.69±0.2, 20.19±0.2 and 23.02±0.2.

[0125] In a specific example, the X-ray powder diffraction pattern of Form V further includes diffraction peaks at diffraction angle 2θ (°) values ​​of 13.67±0.2 and 16.20±0.2.

[0126] In a specific example, the X-ray powder diffraction pattern of Form V further includes diffraction peaks at diffraction angle 2θ (°) values ​​of 13.67±0.2 and 18.95±0.2.

[0127] In a specific example, the X-ray powder diffraction pattern of Form V further includes diffraction peaks at diffraction angle 2θ (°) values ​​of 16.20±0.2 and 18.95±0.2.

[0128] In a specific example, the X-ray powder diffraction pattern of Form V further includes diffraction peaks at diffraction angle 2θ (°) values ​​of 13.67±0.2, 16.20±0.2, and 18.95±0.2.

[0129] In a specific example, the X-ray powder diffraction pattern of Form V further includes diffraction peaks at diffraction angle 2θ (°) values ​​of 21.76±0.2 and 30.21±0.2.

[0130] In a specific example, the X-ray powder diffraction pattern of Form V further includes diffraction peaks at diffraction angle 2θ (°) values ​​of 24.80±0.2 and 25.53±0.2.

[0131] In a specific example, the X-ray powder diffraction pattern of Form V also includes diffraction peaks at 2 or more or all of the diffraction angle 2θ (°) values ​​selected from the following group V-3: 13.67±0.2, 16.20±0.2, 18.95±0.2, 21.76±0.2, 24.80±0.2, 25.53±0.2 and 30.21±0.2.

[0132] In a specific example, the X-ray powder diffraction pattern of Form V has diffraction peaks at 2θ (°) values ​​of 6 or more or all (such as 6, 7, 8, 9, 10, 11, 12, 13, 14, etc.) selected from Groups V-1, V-2 and V-3.

[0133] In a specific example, the X-ray powder diffraction pattern of Form V has diffraction peaks at the 2θ (°) values ​​shown in Table 2, and the relative intensities of the diffraction peaks are shown in Table 2:

[0134] Table 2

[0135] 2θ(°) <![CDATA[I / I0]]> 2θ(°) <![CDATA[I / I0]]> 2θ(°) <![CDATA[I / I0]]> 7.33 VS 16.20 W 23.02 W 9.91 M 17.53 M 24.80 W 12.49 W 18.95 W 25.53 W 13.67 W 20.19 W 30.21 W 15.69 W 21.76 W

[0136] In a specific example, the X-ray powder diffraction pattern of Form V is substantially as follows Figure 5 Represented.

[0137] In a specific example, the differential scanning calorimetry curve of Form V has an endothermic peak at 197.31°C±3°C, and the heat of melting is about 71.30 J / g.

[0138] In a specific example, the differential scanning calorimetry curve of Form V has an endothermic peak at 197.31°C±1°C.

[0139] In one specific example, Form V has substantially Figure 6 The differential scanning calorimetry curve is shown.

[0140] In a specific example, the weight loss of Form V at 25°C to 150°C was determined by thermogravimetric analysis to be approximately 0.50%; and the weight loss at 150°C to 225°C was determined by thermogravimetric analysis to be approximately 0.29%.

[0141] In one specific example, Form V has substantially Figure 7 Thermogravimetric analysis spectrum shown.

[0142] In a specific example, the dynamic moisture adsorption of Form V has a weight gain of less than 0.2% in the relative humidity range of 0% to 80%, where the percentage is weight percentage.

[0143] In one specific example, Form V has substantially Figure 8 DVS spectrum shown.

[0144] In a fifth aspect of the present invention, a method for preparing the crystalline form V of the compound of formula X is provided. The preparation method has good reproducibility, easy process control, stable process, high purity of the obtained crystalline form, and is suitable for industrial production.

[0145] The preparation method of Form V comprises the following steps:

[0146] S201, heating a mixture of the compound of formula X and a solvent to 70° C. or above; and

[0147] S202. Cool the mixture, separate the precipitated solid, and obtain Form V.

[0148] In a specific example, in step S202 , the mixture is cooled to room temperature.

[0149] In a specific example, in step S202, the mixture is cooled to 20°C to 25°C.

[0150] In a specific example, in step S201, the solvent is selected from water, C 2-4 One or more of alkyl alcohol, acetonitrile, toluene, xylene, 1,4-dioxane, 1,2-dichloroethane and ethyl acetate.

[0151] In a specific example, C 2-4 The alkyl alcohol is one or more of ethanol, n-propanol, isopropanol, and ethylene glycol, and the preferred solvent is isopropanol.

[0152] In a specific example, in step S201, the ratio (m / v) of the mass (m) of the compound of formula X to the volume (v) of the solvent is 50 g / L to 400 g / L (preferably 100 g / L to 200 g / L).

[0153] In a specific example, in step S201, the mixture of the compound of formula X and the solvent is heated to 70-150°C, preferably 70-120°C, more preferably 70-90°C, and even more preferably 70-80°C.

[0154] In a specific example, in step S201 , the heating time is 0.5 to 48 hours, preferably 0.5 to 12 hours, more preferably 1 to 5 hours, and even more preferably 1 to 2 hours.

[0155] In a specific example, before step S202 , the method further includes step S2021 : cooling the mixture to 40-65° C. (preferably 55-65° C.) and then keeping the mixture warm for a period of time.

[0156] In a specific example, in step S2021, the insulation time is 1 to 12 hours, and further, preferably the insulation time is 8 to 10 hours.

[0157] In a specific example, in step S2021, the mixture is cooled to 40°C to 65°C (preferably 55°C to 65°C) at a cooling rate of 4°C / hour to 6°C / hour (preferably 5°C / hour), and kept warm for 8 hours to 10 hours.

[0158] In a specific example, in step S2021, the mixture is naturally cooled to 40°C to 65°C (preferably 55°C to 65°C) and kept warm for 8 to 9 hours.

[0159] In a specific example, in step S202 , the mixture is cooled to room temperature and then kept warm for 1 to 2 hours.

[0160] In a specific example, the preparation method of Form V comprises the following steps:

[0161] S301, mixing the compound of formula X and isopropyl alcohol and heating to reflux or 70-80° C.;

[0162] S302, cooling the mixture to 40-65°C; and

[0163] S303. Cool the mixture to room temperature, separate the precipitated solid, and obtain Form V.

[0164] In a specific example, in step S301, the compound of formula X and isopropyl alcohol are mixed and heated to reflux or 70° C. to 80° C., and then kept warm for 0.5 to 48 hours; further, kept warm for 0.5 to 12 hours; further, kept warm for 1 to 5 hours; further, kept warm for 1 to 2 hours.

[0165] In a specific example, in step S302, the mixture is cooled to 40°C to 65°C and then kept warm for 1 hour to 12 hours; further, kept warm for 8 hours to 10 hours.

[0166] In a specific example, in step S303, after the mixture is cooled to room temperature, it is kept warm for 1 hour to 12 hours; further, preferably, it is kept warm for 1 hour to 2 hours.

[0167] In a sixth aspect, the present invention provides a crystalline form V of the compound of formula X prepared according to the preparation method of the fifth aspect.

[0168] In a seventh aspect, the present invention provides use of the crystalline form IV described in the first aspect, the crystalline form IV described in the third aspect, the crystalline form V described in the fourth aspect, or the crystalline form V described in the sixth aspect in the preparation of a BTK inhibitor.

[0169] In an eighth aspect, the present invention provides the use of the crystalline form IV described in the first aspect, the crystalline form IV described in the third aspect, the crystalline form V described in the fourth aspect, or the crystalline form V described in the sixth aspect in the preparation of a medicament for treating and / or preventing diseases mediated by B cells.

[0170] In the ninth aspect, the present invention provides a method for treating a disease mediated by B cells, comprising administering to a patient in need thereof a therapeutically effective amount of the crystalline form IV described in the first aspect, the crystalline form IV described in the third aspect, the crystalline form V described in the fourth aspect, or the crystalline form V described in the sixth aspect.

[0171] In a preferred example, the disease mediated by B cells is selected from the group consisting of: tumor diseases, proliferative diseases, allergic diseases, autoimmune diseases and inflammatory diseases.

[0172] In a preferred example, the disease mediated by B cells is selected from the group consisting of solid tumors, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, rheumatoid arthritis, psoriatic arthritis, osteoarthritis, systemic lupus erythematosus, psoriasis, rheumatoid spondylitis and gouty arthritis.

[0173] In a preferred embodiment, the disease mediated by B cells is a solid tumor.

[0174] In a preferred example, the solid tumor is one or more selected from lymphoma, soft tissue sarcoma, lymphocytic lymphoma, mantle cell lymphoma, melanoma and multiple myeloma.

[0175] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one.

[0176] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0177] Reagents and instruments

[0178] In the present invention, the structure and purity of the compound were determined by nuclear magnetic resonance ( 1 H NMR) and / or liquid chromatography-mass spectrometry (LC-MS). 1 H NMR: Bruker AVANCE-400 NMR, internal standard: tetramethylsilane (TMS). LC-MS: Agilent 1200 HPLC System, 6140MS LC / MS / MS (Agilent), Waters X-Bridge column, 150 × 4.6 mm, 3.5 μm. Preparative HPLC (pre-HPLC): Waters PHW007, XBridge C18 column, 4.6 × 150 mm, 3.5 μm.

[0179] As used herein, DCM represents dichloromethane, DMF represents dimethylformamide, DMSO represents dimethyl sulfoxide, THF represents tetrahydrofuran, EA represents ethyl acetate, DIPA represents diisopropylamine, DIPEA represents N,N-diisopropylethylamine, n-BuLi represents n-butyllithium, NaBH(OAc)3 represents sodium triacetoxyborohydride, Xantphos represents 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene, TFA represents trifluoroacetic acid, Pd2(dba)3 represents tris(dibenzylideneacetone)dipalladium, Xphos represents 2-dicyclohexylphosphino-2,4,6-triisopropylbiphenyl, NMP represents N-methylpyrrolidone, Et3SiH represents triethylsilane, SLS represents sodium lauryl sulfate, sodium carboxymethyl starch (brand: Anhui Shanhe, batch number SSG3017006).

[0180] General Methods

[0181] X-ray powder diffraction: In the present invention, the powder X-ray diffraction pattern of the crystalline form is obtained by methods known in the art using an Equinox 3000S / NX X-ray powder diffraction analyzer. The instrument test conditions are shown in Table 3 below:

[0182] Table 3

[0183]

[0184] In the powder X-ray diffraction pattern, the position of each peak is determined by 2θ (°). It is understood that different instruments and / or conditions may result in slightly different data, and the position and relative intensity of each peak may vary. The intensity division of the peak only reflects the approximate size of the peak at each position. In the present invention, the diffraction peak with the highest peak height of the crystal form is used as the base peak, and its relative intensity is defined as 100%, as I0 (the peak with a 2θ (°) value of 14.91 for crystal form IV is the base peak, and the peak with a 2θ (°) value of 7.33 for crystal form V is the base peak). The ratio of the peak height to the base peak height of each other peak is used as its relative intensity I / I0. The division definition of the relative intensity of each peak is shown in Table 4 below:

[0185] Table 4

[0186] <![CDATA[Relative intensity I / I0 (%)]]> definition 50～100 VS (very strong) 25～50 S (strong) 10～25 M (medium) 1～10 W(weak)

[0187] High Performance Liquid Chromatography: In the present invention, high performance liquid chromatography (HPLC) was acquired on an Agilent 1260 HPLC.

[0188] The TGA spectrum was measured using a TA Q500 / 5000 thermogravimetric analyzer. The measurement conditions were as follows: protective gas: nitrogen (40 mL / min); temperature range: room temperature-350°C; scanning rate: 10.0°C / min.

[0189] The DSC spectrum was measured using a METTLER DSC3 differential scanning calorimeter. The measurement conditions were as follows: protective gas: nitrogen (50 mL / min); temperature range: -30-300°C; scanning rate: 10.0°C / min.

[0190] The dynamic moisture sorption (DVS) curve was measured using a TA Q5000SA dynamic moisture sorption instrument under the following conditions: temperature: 25°C; relative humidity range: 0%-80%.

[0191] It is understood that when using other types of instruments with the same function as the above-mentioned instruments or using different test conditions from those used in the present invention, different values ​​may be obtained. Therefore, the quoted values ​​should not be regarded as absolute values. Due to instrument errors or operator differences, those skilled in the art will understand that the parameters used to characterize the physical properties of the crystals may vary slightly. Therefore, the above parameters are only used to assist in characterizing the crystal forms provided by the present invention and cannot be regarded as limitations on the crystal forms of the present invention.

[0192] Example 1

[0193] This example provides a preparation of an amorphous free base of the compound of formula X, as follows:

[0194] 1. Preparation of intermediate 1a

[0195]

[0196] Step 1: To a solution of compound 1a-1 (6.0 g, 30.0 mmol) in THF (80 mL) at -78°C was added n-BuLi (27 mL, 66 mmol) and DIPA (6.6 g, 66 mmol). The mixture was stirred for 1 hour, then DMF (10 mL) was added and the mixture was allowed to warm to room temperature and stirred for another 2 hours. LC-MS was used to monitor the reaction until completion. HCl (2N) was added to adjust the pH to 5-6, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to yield 6.8 g of compound 1a-2. MS m / z (ESI): 238 [M+H] + .

[0197] Step 2: Compound 1a-2 (6.8 g, 30.0 mmol) dissolved in 1,4-dioxane (80 mL) was added to compound 1a.1 (15 g, 90.0 mmol), acetic acid (2 mL), and NaBH(OAc)3 (18.9 g, 90.0 mmol). The mixture was stirred at 50°C overnight. LC-MS was used to monitor the reaction until completion. The reaction solution was evaporated to dryness under reduced pressure, washed with saturated brine, extracted with DCM, and the organic layer was dried, concentrated, and purified by column chromatography to yield 4.8 g of compound 1a. MS m / z (ESI): 371 [M+H] + .

[0198] 2. Preparation of intermediate 1b

[0199]

[0200] Step 1: A 100 mL three-necked flask was charged with a solution of compound 1b-1 (1.5 g, 5.17 mmol), morpholine (470 mg, 5.39 mmol), Pd2(dba)3 (210 mg, 0.23 mmol), Xphos (240 mg, 0.503 mmol), and cesium carbonate (3.38 g, 10.37 mmol) in 1,4-dioxane (20 mL). The mixture was reacted at 110°C for 3 h. LC-MS was used to monitor the reaction until completion. The reaction mixture was cooled to room temperature, concentrated, and purified by column chromatography (0-20% EA in n-hexane) to afford compound 1b-2 (1.38 g, 90.1% yield) as a gray solid. MS m / z (ESI): 297.2 [M+H] + .

[0201] Step 2: Compound 1b-2 (1.38 g, 4.657 mmol), methanol (20 mL), and HCl / 1,4-dioxane (4 M, 10 mL) were added to a 100 mL flask and stirred overnight at room temperature. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was washed with saturated sodium bicarbonate and extracted with dichloromethane. The organic layer was dried and concentrated to yield 900 mg of compound 1b as a yellow solid. MS m / z (ESI): 197.2 [M+H] + .

[0202] 3. Preparation of Compound X

[0203]

[0204] Step 1: To a solution of compound 1a (740 mg, 2 mmol) in NMP (10 mL) was added (R)-tert-butyl 3-aminopiperidine-1-carboxylate, compound 2.1 (600 mg, 3 mmol), and DIPEA (780 mg, 6 mmol). The mixture was microwave-treated at 180°C under an argon atmosphere for 30 min. LC-MS was used to monitor the reaction until completion. The reaction mixture was cooled to room temperature, diluted with DCM, and washed with water and saturated brine. The organic layer was dried, concentrated, and purified by column chromatography to yield 300 mg of compound X-1. MS m / z (ESI): 535 [M+H]. + .

[0205] Step 2: A solution of compound X-1 (250 mg, 0.5 mmol), compound 1b (118 mg, 0.6 mmol), Pd2(dba)3 (45 mg, 0.05 mmol), Xantphos (54 mg, 0.1 mmol), and cesium carbonate (326 mg, 1 mmol) in 1,4-dioxane (15 mL) was microwave-treated at 160°C for 50 min under an argon atmosphere. LC-MS was used to monitor the reaction until completion. The reaction solution was cooled to room temperature, diluted with EA, and washed with water and saturated brine, respectively. The organic layer was dried, concentrated, and purified by column chromatography to obtain 185 mg of compound X-2. MS m / z (ESI): 695.3 [M+H] + .

[0206] Step 3: To a solution of compound X-2 (185 mg, 0.27 mmol) in DCM (12 mL) was added TFA (4.5 mL). The mixture was stirred at room temperature for 1 h. LC-MS was used to monitor the reaction until completion. Most of the TFA was removed under reduced pressure, and saturated sodium bicarbonate solution was added. The pH was adjusted to 7-8, and the mixture was extracted with DCM. The organic layers were combined, dried, and concentrated to afford compound X-3, which was used directly in the next reaction. MS m / z (ESI): 595.2 [M+H] + .

[0207] Step 4: To a solution of compound X-3 (100 mg) in DCM (10 mL) under argon atmosphere, acryloyl chloride (15.4 mg, 0.17 mmol) and DIPEA (66 mg, 0.51 mmol) were added. The mixture was stirred at room temperature for 2 h. LC-MS was used to monitor the reaction until completion. The reaction solution was washed with saturated brine and extracted with DCM. The organic layer was dried and concentrated to obtain the crude product, which was then purified by column chromatography to yield 86 mg of compound X-4. MS m / z (ESI): 649 [M+H] + .

[0208] Step 5: Et3SiH (0.2 mL) was added to a solution of compound X-4 (86 mg, 0.13 mmol) in TFA (3 mL). The mixture was heated to 80°C and stirred for 2 h. LC-MS was used to monitor the reaction until completion. Most of the TFA was removed under reduced pressure, and the pH was adjusted to 7-8 with saturated sodium bicarbonate solution. The mixture was extracted with ethyl acetate. The organic layers were combined, dried, concentrated, and purified by Prep-HPLC (eluent: DCM:MeOH = 100:3) to obtain 10 mg of solid compound X. MS m / z (ESI): 499.3 [M+H] + . 1H NMR(400MHz, DMSO-d6)δ8.65(d,J=2.6Hz,1H),8.28-8.16(m,2H),7.08–6.99(m,1H),6.87(d,J=14.3Hz,1.5H ),6.58(d,J=9.1Hz,1.5H),6.18-5.98(m,1H),5.72(d,J=10.4Hz,0.5H),5.42(d,J=10.6Hz,0.5H),4.55(d,J =12.4 Hz, 0.5H), 4.37(s, 2H), 4.17(d, J=12.6 Hz, 0.5H), 3.99(s, 1H), 3.89(s, 1H), 3.71(t, J=4.6 Hz, 4H), 3.03-2.98(m, 5H), 2.66-2.85(m, 1H), 2.00(s, 1H), 1.79(d, J=13.5 Hz, 1H), 1.65(d, J=12.5 Hz, 1H), 1.43(s, 1H). The obtained solid compound X was sent to XRPD for detection, and its powder X-ray diffraction pattern showed no characteristic peaks. The powder X-ray diffraction pattern was as follows Figure 11 Shown is an amorphous form.

[0209] Example 2

[0210] This embodiment provides a preparation method of Form IV, as follows:

[0211] 1. Preparation method of crystal form IV of compound of formula X

[0212] Weigh 2.0 g of the compound of formula X (amorphous form) into a container, add 18 mL of isopropanol, raise the temperature to 60°C to 65°C, and keep warm for 1 to 2 hours; then naturally cool to room temperature and keep warm for 1 to 2 hours; filter, and dry the filter cake at 45°C under reduced pressure to constant weight. The resulting solid is sent to DSC for analysis, which shows a single crystalline form, such as Figure 2 shown.

[0213] 2. Characterization of Form IV of Compound X

[0214] (1) The X-ray powder diffraction of the obtained solid was analyzed and identified by Equinox 3000S / NX X-ray powder diffraction analyzer. Figure 1 In the present invention, it is defined as crystal form IV.

[0215] (2) Analysis and identification by TA Q200 / 2000 differential scanning calorimeter: Form IV has an endothermic peak at 167.89°C; the heat of fusion is 55.84 J / g, and its DSC spectrum is basically as follows Figure 2 shown.

[0216] (3) Analysis and identification by TA Q500 / 5000 thermogravimetric analyzer: Form IV has a slow weight loss of about 0.29% before 150°C, and has good stability at high temperatures. Its TGA spectrum is basically as follows Figure 3 shown.

[0217] (4) Analysis and identification by TA Q5000SA dynamic moisture adsorption instrument: Crystal form IV has a moisture absorption weight increase of about 0.7% in the relative humidity range of 0% to 80%, indicating slight hygroscopicity. Its DVS spectrum is as follows Figure 4 shown.

[0218] Example 3

[0219] This embodiment provides a preparation of Form V, as follows:

[0220] 1. Preparation method of crystal form V of compound of formula X

[0221] Method 1: Weigh 3.5 g of the compound of formula X (amorphous form) into a container, add 35 mL of isopropanol, raise the temperature to reflux, and reflux for 1.5 hours. Then cool at a cooling rate of 5°C / h. When the temperature drops to 63°C, a large amount of solid precipitates. Maintain the temperature and stir for 10 hours. Then cool naturally to room temperature and maintain the temperature and stir for another 1.5 hours. Filter, and dry the filter cake under reduced pressure at 45°C to constant weight. The resulting solid is sent to DSC for detection, which shows a single crystalline form, such as Figure 6 shown.

[0222] Method 2: Weigh 1.54 kg of compound X (amorphous form) into a container, add 7.7 L of isopropanol, raise the temperature to 70-80°C, and stir at this temperature for 1.5 hours. Naturally cool to 55-65°C, and stir at this temperature for 8-9 hours. Naturally cool slowly to 20-25°C, and stir at this temperature for 2 hours. Filter, rinse with isopropanol, and dry under reduced pressure at 40-50°C to constant weight to yield 1.223 kg of a solid.

[0223] 2. Characterization of Form V of Compound X

[0224] (1) The X-ray powder diffraction (XRPD) analysis of the solids obtained by the first and second methods was basically as follows: Figure 5 As shown, it is defined as crystal form V in the present invention.

[0225] (2) Analyzed and identified by TA Q200 / 2000 differential scanning calorimeter: The DSC spectra of the solids obtained by method 1 and method 2 were basically as follows Figure 6 As shown, Form V has an endothermic peak at 197.31°C and a melting heat of 71.30 J / g.

[0226] (3) Analyzed and identified by TA Q500 / 5000 thermogravimetric analyzer: The TGA spectra of the solids obtained by method 1 and method 2 were basically as follows Figure 7 As shown, Form V has a slow weight loss of about 0.50% before 150°C and a slow weight loss of about 0.29% between 150°C and 225°C, and has good stability at high temperatures.

[0227] (4) The DVS spectra of the solids obtained by method 1 and method 2 were basically the same as Figure 8 As shown, the weight gain of crystal form V due to moisture absorption is less than 0.2% in the relative humidity range of 0% to 80%, and it is almost non-hygroscopic.

[0228] Example 4

[0229] This embodiment provides a preparation of Form I, as follows:

[0230] 10.17 mg of the free base of the compound of formula X (amorphous form) was weighed into a sample bottle. 3 ml of water was gradually added to the bottle while stirring at room temperature to obtain a suspension of the compound of formula X. The suspension was centrifuged and dried to obtain a solid, which was then subjected to X-ray powder diffraction analysis. The X-ray powder diffraction pattern of the obtained solid is shown below. Figure 12 As shown, it is defined as Form I in the present invention. In the present invention, the powder X-ray diffraction pattern of Form I was obtained using a D8 ADVANCE X-ray powder diffraction analyzer. The instrument test conditions are shown in Table 5 below:

[0231] Table 5

[0232]

[0233]

[0234] Example 5 Stability Test

[0235] 1. Accelerated stability test

[0236] The crystal form IV and the crystal form V were placed in a 40°C / 75% RH accelerated stability box and a 60°C oven, respectively. After 1 month, they were taken out, 10 mg was weighed and dissolved in methanol, and the purity was tested by HPLC, and the crystal form change was tested by XRPD.

[0237] Before the accelerated stability test began, the initial sample was tested for purity using HPLC. The ratio of the accelerated stability test sample's purity to the initial sample's purity was used as the sample stability criterion. If the purity ratio was less than 95%, the sample was considered chemically unstable. The specific experimental results are shown in Table 6.

[0238] Table 6

[0239]

[0240] From Table 6 and Figure 9 、 Figure 10 It can be seen that when Form IV and Form V are placed under 40°C / 75% RH conditions and 60°C conditions for one month, there is no obvious increase in impurities, and the XRPD spectrum shows no change in characteristic peaks, indicating that Form IV and Form V have good chemical and physical stability.

[0241] 2. Suspension competition test

[0242] To further confirm the interconversion relationship between Form V and Form IV in the presence of solvents, suspension competition experiments were conducted at room temperature and 50°C. Twelve equal volumes of Form V and Form IV samples were prepared, and four solvents, ethanol, isopropanol, acetone, and ethyl acetate, were added. After sealing, the mixture was placed in a corresponding oscillator and shaker. The suspension was shaken at room temperature for 1 day and 7 days, and at 50°C for 1 day. After centrifugation and drying, XRPD analysis was performed to examine the changes in the crystal form. The experimental results are shown in Table 7.

[0243] Table 7

[0244]

[0245] Example 6 Solubility Experiment

[0246] Dissolution medium:

[0247] pH 4.5 medium: Weigh 18 g of sodium acetate, add 9.8 ml of glacial acetic acid, and dilute to 1 L with water.

[0248] pH 6.8 medium: Weigh 6.8 g of potassium dihydrogen phosphate and 0.94 g of sodium hydroxide, and dilute to 1 L with water.

[0249] 0.2% SLS / pH 6.8 medium: Weigh 7.8 g of sodium dihydrogen phosphate dihydrate and 0.9 g of sodium hydroxide into 1 L of water. Adjust the pH to 6.8 with 2N sodium hydroxide. Mix thoroughly, then add 2 g of SLS and mix thoroughly to obtain the 0.2% SLS / pH 6.8 medium.

[0250] Approximately 5 mg of each of Form IV, Form V, Compound X (amorphous form), and Form I were weighed into 1 ml of the corresponding medium and thoroughly dispersed by ultrasonic shaking for 2 hours. The supernatant was centrifuged, diluted to a predetermined multiple, and analyzed by HPLC to determine the content of Compound X and calculate the solubility. The experimental results are shown in Table 8.

[0251] Table 8

[0252]

[0253] As shown in Table 8, Form IV and Form V exhibited better solubility in 0.2% SLS / pH 6.8 than in water, pH 4.5, and pH 6.8. The solubility of Form IV and Form V was significantly improved compared to the compound of Formula X (amorphous form) and Form I, respectively, improving drug solubility and enhancing bioavailability.

[0254] Example 7 Animal Plasma Pharmacokinetic Experiment

[0255] 1. Preparation of animal prescription capsules

[0256] 1) Take a certain amount of amorphous, Form IV, and Form V respectively, grind for 5 minutes, and then take a small amount to observe under a microscope to ensure that the particle sizes are as similar as possible;

[0257] 2) Determine the filling volume: weigh 5 mg of amorphous form, crystalline form IV, and crystalline form V, respectively, and fill them into corresponding animal capsules (ingredients: sodium carboxymethyl starch).

[0258] 2. Animal plasma pharmacokinetic experiments

[0259] Experimental animals: Wistar rats, weighing 180-200 g, female, n = 4 / crystal form, purchased from Beijing Vital River Laboratory Animal Co. LTD. They were fasted the night before administration and allowed to eat freely 4 hours after administration.

[0260] Dosing and Blood Sampling: 1 capsule / rat (5 mg / rat) was administered orally (PO). Blood samples were collected at nine time points: 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after dosing. Animals were manually restrained and approximately 150 μL of blood was collected from the retinal venous plexus into tubes containing K2EDTA. Blood samples were placed on wet ice and centrifuged within 15 minutes (8000 rpm, 4 minutes, 4°C) to obtain plasma samples, which were stored at -80°C until analysis. Compounds were prepared on the day of the experiment.

[0261] Analytical method: Liquid chromatography-tandem mass spectrometry (Triple Quad TM Plasma samples were analyzed using a 4000 column (Waters XBridge-C18, 2.1 × 50 mm, 5 μm).

[0262] Preparation of standard curve: Prepare a standard curve with a linear range of 1.00-3000 ng / mL for the test compound in blank Wistar rat plasma matrix, and simultaneously prepare low, medium and high concentration quality control samples with concentrations of 3,500 and 2400 ng / mL.

[0263] Processing of biological samples: Thaw frozen plasma samples on ice. Once thawed, vortex for 5 minutes. Add 20 μL of plasma sample, standard curve, and quality control sample to a 96-well plate. Add 200 μL of acetonitrile containing the internal standard dexamethasone (brand: NIFDC, lot number: 6TUC-T4C2, prepared at a concentration of 2000 ng / mL) to precipitate protein. Vortex mix for 5 minutes, then centrifuge at 3700 rpm and 4°C for 15 minutes. Extract the supernatant and centrifuge again under the same conditions. Finally, 2 μL of the supernatant was used for LC-MS / MS analysis. Pharmacokinetic parameters for rats are shown in Table 9.

[0264] Table 9

[0265]

[0266]

[0267] As shown in Table 9, compared with the amorphous form, both the crystal form IV and the crystal form V have significantly improved half-life and plasma exposure levels. After administration at the same dose, the plasma exposure level AUC of the crystal form IV group is about 1.5 times that of the amorphous form, and the half-life T 1 / 2 It is about 1.3 times that of the amorphous form. The plasma exposure level AUC and half-life T 1 / 2 The plasma exposure level AUC of the group treated with crystal form V was about 2 times that of the amorphous form. The half-life of crystal form V was T 1 / 2 The plasma exposure level AUC and half-life T of crystal form V are about 3 times that of the amorphous form. 1 / 2 Significantly better than amorphous.

[0268] Example 8 Lantha screening kinase reaction assay method

[0269] Compounds were pre-dissolved in 100% DMSO. A 10 mM stock solution of drug was dissolved at room temperature and serially diluted with 8 vol% DMSO to a final concentration of 10-0.005 μM. To each well of a 384-well plate (Corning 3676), 2.5 μl of the test solution and 2.5 μl of kinase (Invitrogen PV3363) diluted in reaction buffer were added. The reaction was initiated by adding 5 μl of a mixture of Fluososcei-PolyGT (Invitrogen PV3610) substrate and ATP (Invitrogen PV3227) diluted in reaction buffer. Blank wells were treated with reaction buffer instead of kinase, while no drug was added to the kinase wells. The reaction was incubated at 25°C in a shaker protected from light for 60 minutes. Add 10 μl of detection solution (a mixture of Invitrogen PV3528 and EDTA, diluted in TR-FRET dilution buffer, with a working concentration of 5 mM EDTA and 0.2 nM Lanthascreening Tb PY20 antibody) and incubate on a shaker at room temperature for 30 minutes. Read the plate on a Victor X5 fluorescence microplate reader (PerkinElmer), measuring absorbance at excitation wavelengths of 340 nm and emission wavelengths of 500 and 520 nm.

[0270] The inhibition rate calculation method (refer to the instructions of Invitrogen, PV3363) is as follows:

[0271] 1. Emission rate (ER): Coumarin Emission (520nm) / Fluorescein Emission (500nm);

[0272] 2. Inhibition rate (IR): (ER kinase -ER test compound ) / (ER kinase -ER blank ) × 100%. XLFIT 5.0 software (IDBS, UK) was used to calculate the half-maximal inhibitory concentration (IC) 50 The results are shown in Table 10:

[0273] Table 10 Inhibitory activity of compounds against BTK WT

[0274]

[0275] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0276] The embodiments described above only express several implementation methods of the present invention, which are convenient for understanding the technical solutions of the present invention in a specific and detailed manner, but they cannot be understood as limiting the scope of protection of the invention patent. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present invention, several variations and improvements can be made, which all fall within the scope of protection of the present invention. It should be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided by the present invention are all within the scope of protection of the claims attached to the present invention. Therefore, the scope of protection of the patent of the present invention shall be based on the content of the attached claims, and the description and drawings can be used to interpret the content of the claims.

Claims

1. A crystalline form V of a compound of formula X, characterized in that: The compound of formula X has the following structure: Among them, the X-ray powder diffraction pattern of the crystalline form V detected using Cu-Kα radiation has diffraction peaks at the following diffraction angle 2θ (°) values: 7.33±0.2, 9.91±0.2, 12.49±0.2, 13.67±0.2, 15.69±0.2, 16.20±0.2, 17.53±0.2, 18.95±0.2, 20.19±0.2, 21.76±0.2, 23.02±0.2, 24.80±0.2, 25.53±0.2 and 30.21±0.

2.

2. The crystalline form V according to claim 1, characterized in that The X-ray powder diffraction pattern of the crystalline form V is basically represented by FIG5 .

3. The crystalline form V according to claim 1, characterized in that The differential scanning calorimetry curve of the crystalline form V has an endothermic peak at 197.31°C±3°C.

4. The crystalline form V according to claim 1, characterized in that The crystalline form V has a differential scanning calorimetry curve substantially as shown in FIG6 .

5. The crystalline form V according to claim 1, characterized in that The crystalline form V has a thermogravimetric analysis spectrum substantially as shown in FIG7 .

6. The crystalline form V according to claim 1, characterized in that The crystalline form V has a DVS pattern substantially as shown in FIG8 .

7. A method for preparing the crystalline form V of the compound of formula X according to any one of claims 1 to 6, characterized in that: The method comprises the following steps: S201, heating a mixture of the compound of formula X and a solvent to above 70°C; and S202, cooling the mixture, separating the precipitated solid, and obtaining the crystalline form V.

8. The preparation method according to claim 7, wherein The solvent in step S201 is selected from: water, C 2-4 One or more of alkyl alcohol, acetonitrile, toluene, xylene, 1,4-dioxane, 1,2-dichloroethane and ethyl acetate.

9. The preparation method according to claim 8, wherein The C 2-4 The alkyl alcohol is one or more of ethanol, n-propanol, isopropanol and ethylene glycol.

10. The preparation method according to claim 7, wherein The ratio of the mass of the compound of formula X to the volume of the solvent in step S201 is 50 g / L to 400 g / L.

11. The preparation method according to claim 7, wherein In step S201, the mixture of the compound of formula X and the solvent is heated to 70° C. to 80° C.; in step S202, the mixture is cooled to 20° C. to 25° C.

12. The preparation method according to claim 7, wherein Before step S202, the method further includes the following steps: cooling the mixture to 40° C. to 65° C. and keeping the mixture warm for 1 hour to 12 hours.

13. Use of the crystalline form V according to any one of claims 1 to 6, or the crystalline form V prepared by the preparation method according to any one of claims 7 to 12, in the preparation of a medicament for treating and / or preventing a proliferative disease, an allergic disease, an autoimmune disease or an inflammatory disease.

14. Use of the crystalline form V according to any one of claims 1 to 6, or the crystalline form V prepared by the preparation method according to any one of claims 7 to 12, in the preparation of a drug for treating and / or preventing tumors.

15. Use of the crystalline form V according to any one of claims 1 to 6, or the crystalline form V prepared by the preparation method according to any one of claims 7 to 12, in the preparation of a drug for treating and / or preventing cancer.

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