WO2022048551A1 - 布鲁顿酪氨酸激酶抑制剂的多晶型物及其制备方法和应用 - Google Patents

布鲁顿酪氨酸激酶抑制剂的多晶型物及其制备方法和应用 Download PDF

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WO2022048551A1
WO2022048551A1 PCT/CN2021/115856 CN2021115856W WO2022048551A1 WO 2022048551 A1 WO2022048551 A1 WO 2022048551A1 CN 2021115856 W CN2021115856 W CN 2021115856W WO 2022048551 A1 WO2022048551 A1 WO 2022048551A1
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crystal form
compound
preparation
formula
ray powder
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PCT/CN2021/115856
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French (fr)
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李金晶
江涛涛
曾振亚
杨菡
陈曦
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上海海雁医药科技有限公司
扬子江药业集团有限公司
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Priority to CN202180053967.4A priority Critical patent/CN116096369A/zh
Publication of WO2022048551A1 publication Critical patent/WO2022048551A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00

Definitions

  • the invention belongs to the technical field of medicine, in particular, the invention relates to a polymorphic form of a BTK inhibitor, a preparation method and application thereof, and the inhibitor is (R)-6-((1-acryloylpiperidine- 3-yl)amino)-7-fluoro-4-((2-fluoro-4-morpholinophenyl)amino)-1,2-dihydro-3H-pyrrolo[3,4-c]pyridine- 3-keto.
  • Bruton's tyrosine kinase (BTK) kinase is a non-receptor tyrosine kinase in the TEC kinase family. It is a key regulator of the BCR signaling pathway and plays an important role in the maturation, proliferation and survival of B cells.
  • BTK is overexpressed in a variety of B-cell lymphomas and is currently the only clinically validated target for drug development in the TEC kinase family. Inhibition of BTK inhibits the proliferation of a range of B-cell lymphomas.
  • BCR B-cell antigen receptor
  • BTK inhibitors act on chronic lymphocytic leukemia (CLL) cells to induce cytotoxicity and inhibit the proliferation of CLL cells. It inhibits the proliferation of primary B cells activated by BCR, and inhibits the secretion of TNF ⁇ , IL-1 ⁇ and IL-6 in primary monocytes. In a collagen-induced arthritis model, BTK inhibitors significantly reduce clinical arthritis symptoms such as foot swelling and joint inflammation by inhibiting B cell activity.
  • CLL chronic lymphocytic leukemia
  • Patent WO2019062329A1 discloses the structure of a class of compounds with better 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]pyridin-3-one, the compound has high inhibitory activity on enzymes and cells.
  • 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 the later drug development.
  • the present invention further researches the R-configuration compound, and completes the present application based on the research.
  • the present invention provides two kinds of (R)-6-((1-acryloylpiperidin-3-yl)amino)-7-fluoro-4-((2-fluoro-4)
  • the new crystal form provided by the present invention has improved solubility and more favorable in vivo pharmacokinetic characteristics, which is helpful for further drug development.
  • the Form IV is an anhydrous form or a hydrate form.
  • the Form IV is an anhydrous form.
  • the X-ray powder diffraction pattern of the crystal form IV detected using Cu-K ⁇ radiation has diffraction peaks at the diffraction angle 2 ⁇ (°) 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.
  • the X-ray powder diffraction pattern of Form IV further comprises diffraction peaks at 1, 2, or more or all of the diffraction angle 2 ⁇ (°) values selected from the following Group IV-2: 10.41 ⁇ 10.41 ⁇ 0.2, 12.19 ⁇ 0.2, 18.80 ⁇ 0.2, 21.49 ⁇ 0.2 and 25.44 ⁇ 0.2.
  • the X-ray powder diffraction pattern of Form IV further includes diffraction peaks at 8.91 ⁇ 0.2, 12.49 ⁇ 0.2, and 12.76 ⁇ 0.2 diffraction angle 2 ⁇ (°) values.
  • the X-ray powder diffraction pattern of Form IV further comprises diffraction angle 2 ⁇ (°) values at 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 diffraction peaks at.
  • the X-ray powder diffraction pattern of Form IV further comprises diffraction peaks at 1, 2, 3, or more or all of the diffraction angle 2 ⁇ (°) 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.
  • the X-ray powder diffraction pattern of the crystal form IV is selected from 6 or more or all of the group IV-1, IV-2 and IV-3 (eg 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc.) with peaks at 2 ⁇ (°) values.
  • the X-ray powder diffraction pattern of the crystal form IV has diffraction peaks at the 2 ⁇ (°) values shown in Table 1, and the relative intensities of each peak are shown in Table 1:
  • the X-ray powder diffraction pattern of the Form IV is substantially as characterized in FIG. 1 .
  • the differential scanning calorimetry curve of the crystal form IV has an endothermic peak at 167.89°C ⁇ 3°C, and the heat of fusion is about 55.84 J/g.
  • the differential scanning calorimetry curve of Form IV has an endothermic peak at 167.89°C ⁇ 1°C.
  • the Form IV has a differential scanning calorimetry profile substantially as shown in FIG. 2 .
  • the crystalline form IV has a weight loss of about 0.29% as detected by thermogravimetric analysis at 30°C to 150°C.
  • the Form IV has a thermogravimetric analysis pattern substantially as shown in FIG. 3 .
  • the dynamic moisture adsorption of the crystal form IV has a weight gain of about 0.7% in a relative humidity range of 0% to 80%, and the percentage is a percentage by weight.
  • the Form IV has a DVS pattern substantially as shown in FIG. 4 .
  • the preparation method is simple, the obtained crystal form has high purity, and is suitable for industrial production.
  • the preparation method of the crystal form IV comprises the following steps:
  • the mixture in step (S102) is cooled to room temperature.
  • the mixture in step (S102) is cooled to 20°C to 25°C.
  • the solvent in step (S101) is selected from one or more of water, ethanol, n-propanol and isopropanol.
  • the solvent in step (S101) is isopropanol.
  • the heating time is 0.5-12 hours; preferably, the heating time is 1-2 hours.
  • the mixture in the step (S102), after the mixture is cooled to room temperature, the mixture is kept for 0-12 hours, preferably 1-2 hours.
  • the Form V is an anhydrous form or a hydrate form.
  • the Form V is an anhydrous form.
  • the X-ray powder diffraction pattern of Form V detected using Cu-K ⁇ radiation has diffraction peaks at the following values of diffraction angle 2 ⁇ (°) of set V-1: 7.33 ⁇ 0.2, 9.91 ⁇ 0.2 and 17.53 ⁇ 0.2.
  • the X-ray powder diffraction pattern of Form V further includes diffraction peaks at 13.67 ⁇ 0.2 diffraction angle 2 ⁇ (°) values.
  • the X-ray powder diffraction pattern of Form V further includes diffraction peaks at diffraction angle 2 ⁇ (°) values of 15.69 ⁇ 0.2 and 16.20 ⁇ 0.2.
  • the X-ray powder diffraction pattern of the Form V further includes diffraction peaks at diffraction angle 2 ⁇ (°) values of 12.49 ⁇ 0.2.
  • the X-ray powder diffraction pattern of Form V further includes diffraction peaks at diffraction angle 2 ⁇ (°) values of 18.95 ⁇ 0.2.
  • the X-ray powder diffraction pattern of Form V further includes diffraction peaks at diffraction angle 2 ⁇ (°) values of 20.19 ⁇ 0.2 and 21.76 ⁇ 0.2.
  • the X-ray powder diffraction pattern of Form V further includes diffraction peaks at the following values of diffraction angle 2 ⁇ (°) of Group V-2: 12.49 ⁇ 0.2, 15.69 ⁇ 0.2, 20.19 ⁇ 0.2, and 23.02 ⁇ 0.2.
  • the X-ray powder diffraction pattern of Form V further includes diffraction peaks at diffraction angle 2 ⁇ (°) values of 13.67 ⁇ 0.2 and 16.20 ⁇ 0.2.
  • the X-ray powder diffraction pattern of the Form V further includes diffraction peaks at diffraction angle 2 ⁇ (°) values of 13.67 ⁇ 0.2 and 18.95 ⁇ 0.2.
  • the X-ray powder diffraction pattern of Form V further includes diffraction peaks at diffraction angle 2 ⁇ (°) values of 16.20 ⁇ 0.2 and 18.95 ⁇ 0.2.
  • the X-ray powder diffraction pattern of Form V further includes diffraction peaks at diffraction angle 2 ⁇ (°) values of 13.67 ⁇ 0.2, 16.20 ⁇ 0.2, and 18.95 ⁇ 0.2.
  • the X-ray powder diffraction pattern of Form V further includes diffraction peaks at diffraction angle 2 ⁇ (°) values of 21.76 ⁇ 0.2 and 30.21 ⁇ 0.2.
  • the X-ray powder diffraction pattern of Form V further includes diffraction peaks at diffraction angle 2 ⁇ (°) values of 24.80 ⁇ 0.2 and 25.53 ⁇ 0.2.
  • the X-ray powder diffraction pattern of Form V further comprises diffraction peaks at 2 or more or all of the diffraction angle 2 ⁇ (°) values selected from the following Group V-3: 13.67 ⁇ 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.
  • the X-ray powder diffraction pattern of the Form V is at 6 or more or all selected from Groups V-1, V-2, and V-3 (eg, 6, 7, 8, 9, 10, 11, 12, 13, 14, etc.) have diffraction peaks at 2 ⁇ (°) values.
  • the X-ray powder diffraction pattern of the crystal form V has diffraction peaks at the 2 ⁇ (°) values shown in Table 2, and the relative intensities of the diffraction peaks are shown in Table 2:
  • the X-ray powder diffraction pattern of the Form V is substantially as characterized in FIG. 5 .
  • the differential scanning calorimetry curve of the crystal form V has an endothermic peak at 197.31°C ⁇ 3°C, and the heat of fusion is about 71.30 J/g.
  • the differential scanning calorimetry curve of the Form V has an endothermic peak at 197.31°C ⁇ 1°C.
  • the Form V has a differential scanning calorimetry profile substantially as shown in FIG. 6 .
  • the crystal form V has a weight loss of about 0.50% as detected by thermogravimetric analysis at 25°C to 150°C; and a weight loss of about 0.29% detected by thermogravimetric analysis between 150°C and 225°C.
  • the Form V has a thermogravimetric analysis pattern substantially as shown in FIG. 7 .
  • the dynamic moisture adsorption of the crystal form V has a weight gain of less than 0.2% in a relative humidity range of 0% to 80%, and the percentage is a percentage by weight.
  • the Form V has a DVS pattern substantially as shown in FIG. 8 .
  • a preparation method of the crystal form V of the compound of formula X has good reproducibility, easy process control, stable process method, high purity of the obtained crystal form, and is suitable for industrial production.
  • the preparation method of the crystal form V comprises the following steps:
  • step (S202) the mixture is cooled to room temperature.
  • step (S202) the mixture is cooled to 20°C to 25°C.
  • the solvent is selected from water, C 2-4 alkyl alcohol, acetonitrile, toluene, xylene, 1,4-dioxane, 1,2-dichloroethane One or more of alkane and ethyl acetate.
  • the C 2-4 alkyl alcohol is one or more of ethanol, n-propanol, isopropanol, and ethylene glycol, and the preferred solvent is isopropanol.
  • the ratio (m/v) of the mass (m) of the compound of formula X to the solvent volume (v) is 50g/L ⁇ 400g/L (preferably 100g/L ⁇ 200g/L) ).
  • 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, more preferably 70-80°C.
  • the heating time is 0.5-48 hours, preferably 0.5-12 hours, more preferably 1-5 hours, and more preferably 1-2 hours.
  • a step (S2021) is further included: after cooling the mixture to 40-65°C (preferably 55-65°C), keeping the mixture for a period of time.
  • the holding time is 1-12 hours, and further, preferably, the holding time is 8-10 hours.
  • step (S2021) the mixture is cooled to 40-65°C (preferably 55-65°C) at a cooling rate of 4°C/hour-6°C/hour (preferably 5°C/hour), and kept for 8 ⁇ 10 hours.
  • step (S2021) the mixture is naturally cooled to 40-65°C (preferably 55-65°C), and kept for 8-9 hours.
  • step (S202) after the mixture is cooled to room temperature, the mixture is kept for 1-2 hours.
  • the preparation method of the crystal form V comprises the following steps:
  • step (S301) the compound of formula X and isopropanol are mixed and heated to reflux or 70-80° C., and kept for 0.5 to 48 hours; further, for 0.5 to 12 hours; further, for 0.5 to 12 hours 1 to 5 hours; further, keep the temperature for 1 to 2 hours.
  • step (S302) after cooling the mixture to 40-65° C., the mixture is kept for 1-12 hours; further, the mixture is kept for 8-10 hours.
  • step (S303) after cooling the mixture to room temperature, the temperature is maintained for 1-12 hours; further, preferably, the mixture is maintained for 1-2 hours.
  • the sixth aspect of the present invention provides a crystal form V of the compound of formula X prepared by the preparation method of the fifth aspect.
  • the seventh aspect of the present invention provides the crystal form IV of the first aspect, the crystal form IV of the third aspect, the crystal form V of the fourth aspect, or the crystal form V of the sixth aspect. Use in the preparation of BTK inhibitors.
  • the eighth aspect of the present invention provides the crystal form IV of the first aspect, the crystal form IV of the third aspect, the crystal form V of the fourth aspect, or the crystal form V of the sixth aspect. Use in the preparation of a medicament for the treatment and/or prevention of diseases mediated by B cells.
  • a ninth aspect of the present invention provides a method for treating a disease mediated by B cells, comprising administering to a patient in need a therapeutically effective amount of the crystal form IV described in the first aspect, the crystal form IV described in the third aspect, The crystal form V of the fourth aspect or the crystal form V of the sixth aspect.
  • the disease mediated by B cells is selected from the group consisting of: neoplastic disease, proliferative disease, allergic disease, autoimmune disease and inflammatory disease.
  • 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, psoriasis Arthritis, osteoarthritis, systemic lupus erythematosus, psoriasis, rheumatoid spondylitis and gouty arthritis.
  • the disease mediated by B cells is a solid tumor.
  • the solid tumor is at least one selected from the group consisting of lymphoma, soft tissue sarcoma, lymphocytic lymphoma, mantle cell lymphoma, melanoma and multiple myeloma.
  • Fig. 2 Differential scanning calorimetry (DSC) spectrum of crystal form IV (the abscissa is temperature (°C), and the ordinate is heat flow rate (mW));
  • Fig. 6 Differential scanning calorimetry (DSC) spectrum of crystal form V (the abscissa is temperature (°C), and the ordinate is heat flow rate (mW));
  • Fig. 7 Thermogravimetric analysis (TGA) pattern of crystal form V (the abscissa is temperature (°C), and the ordinate is weight percentage (%));
  • Fig. 9 Comparison of XRPD patterns of Form IV placed at 60°C for 1 month, 40°C/75%RH for 1 month and the initial sample (the abscissa is the angle 2 ⁇ (°), the ordinate is the intensity) ;
  • Fig. 10 Comparison of XRPD patterns of crystal form V placed at 60°C for 1 month, 40°C/75%RH for 1 month and the initial sample (the abscissa is 2 ⁇ (°); the ordinate is the intensity);
  • Figure 11 The amorphous X-ray powder diffraction (XRPD) pattern of the free base of the compound of formula X (using Cu-K ⁇ radiation, the abscissa is 2 ⁇ (°); the ordinate is the intensity);
  • 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 crystal forms IV and V have improved solubility and more favorable in vivo pharmacokinetics It is not easy to absorb moisture and has good chemical and physical stability. In addition, it was found that the compound of formula X has better inhibitory activity on BTK WT kinase than its racemate compound.
  • the crystal form IV and crystal form V of the compound of formula X provided by the present invention can be used for preparing BTK inhibitors or preparing medicines for treating BTK-related diseases.
  • the BTK-related disease is cancer, abnormal cell proliferative disease, infection, inflammatory disorder, autoimmune disease, cardiovascular disease, neurodegenerative disease, hematopoietic toxicity disease caused by radiation, or a combination thereof.
  • 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.
  • the breast cancer is HR-positive, HER2-negative advanced breast cancer.
  • a “therapeutically effective amount” refers to an amount that produces function or activity in humans and/or animals and is acceptable to humans and/or animals.
  • patient refers to an animal, preferably a mammal, more preferably a human.
  • mammal refers to warm-blooded vertebrate mammals including, for example, cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, mice, pigs and humans.
  • treating refers to alleviating, delaying progression, attenuating, preventing, or maintaining an existing disease or disorder (eg, cancer). Treatment also includes curing, preventing the development or alleviating to some extent one or more symptoms of a disease or disorder.
  • "using Cu-K ⁇ radiation” means that the corresponding spectrum is obtained by detecting the K ⁇ radiation of the Cu target.
  • the diffraction peaks may have deviations within the acceptable range in the art, which should not be interpreted as Limitations of the present invention.
  • Solids exist in either amorphous or crystalline form. In the case of crystalline forms, the molecules are localized within three-dimensional lattice sites. When a compound crystallizes out of a solution or slurry, it can crystallize in different spatial lattice arrangements (a property known as "polymorphism"), forming crystals with different crystalline forms that 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 form, packing pattern, flowability and/or solid state stability.
  • Production scale crystallization can be accomplished by manipulating the solution such that the solubility limit of the compound of interest is exceeded. This can be accomplished by a variety of methods, for example, dissolving the compound at a relatively high temperature and then cooling the solution below the saturation limit. Or by boiling, atmospheric evaporation, vacuum drying or by some other method to reduce the liquid volume.
  • the solubility of the compound of interest can be reduced by adding an antisolvent or a solvent in which the compound has low solubility, or a mixture of such solvents. Another alternative is to adjust the pH to reduce solubility.
  • crystallisation see Crystallization, Third Edition, JW Mullens, Butterworth-Heineman Ltd., 1993, ISBN 0750611294.
  • room temperature generally refers to 4-30°C, preferably 20 ⁇ 5°C.
  • XRPD can detect the change of crystal form, crystallinity, crystal structure and other information, and is a common method to identify crystal form.
  • the peak position of the XRPD pattern mainly depends on the structure of the crystal form, and the measurement of the 2 ⁇ of the XRPD pattern may be slightly different between different instruments, so the value of the 2 ⁇ cannot be regarded as absolute. According to the condition of the instrument used in the experiment of the present invention, there is an error of ⁇ 0.2° in the diffraction peak.
  • the crystal form of the compound of formula X of the present invention has a specific crystal form and has a specific characteristic peak in the XRPD pattern.
  • DSC scanning analysis is a technique for measuring the relationship between the energy difference between the measured substance and the reference substance and the temperature 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 qualitatively used to identify the substance. This method is commonly used in the art to detect various parameters such as phase transition temperature, glass transition temperature, and reaction heat of substances.
  • the peak positions of the DSC spectra may vary slightly between different instruments, so the numerical values of the peak positions of the DSC endothermic peaks cannot be regarded as absolute.
  • the value of 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.
  • TGA is a technique for measuring the change of the mass of a substance with temperature under program control. It is suitable for checking the loss of solvent in the crystal or the process of sublimation and decomposition of the sample. It can be speculated that the crystal contains water of crystallization or crystallization solvent.
  • the mass change shown by the TGA curve depends on many factors such as sample preparation and instrument; the mass change detected by TGA varies slightly between different instruments. Depending on the condition of the instrument used for the experiments of the present invention, there is an error of ⁇ 0.1% in mass variation.
  • the crystal form IV and crystal form V of the compound of the formula X of the present invention have good crystallinity, high stability, not easy to absorb moisture, and have better solubility and more favorable pharmacokinetic characteristics compared with the amorphous form. , including relatively high plasma exposure and long half-life, contribute to the enhanced bioavailability of the amorphous compound of formula X.
  • the preparation method of the crystal form IV is simple, the obtained crystal form has high purity, and is suitable for industrial production.
  • the preparation method of crystal form V has good reproducibility, easy process control and stable process method, and is suitable for industrial production. Therefore, Form IV and Form V have the possibility of being further developed into medicines.
  • LC-MS liquid mass spectrometry
  • 1 H NMR nuclear magnetic resonance
  • LC-MS liquid mass spectrometry
  • 1 H NMR Bruker AVANCE-400 nuclear magnetic instrument, the internal standard is tetramethylsilane (TMS).
  • LC-MS Agilent 1200HPLC System, 6140MS LC/MS mass spectrometer (purchased from Agilent), column WatersX-Bridge, 150 ⁇ 4.6 mm, 3.5 ⁇ m.
  • Preparative high performance liquid chromatography Pre-HPLC: Waters PHW007, column XBridge C18, 4.6*150mm, 3.5um.
  • DCM dichloromethane
  • DMF dimethylformamide
  • DMSO dimethyl sulfoxide
  • THF tetrahydrofuran
  • EA ethyl acetate
  • DIPA diisopropylamine
  • DIPEA N,N-diisopropylamine Propylethylamine
  • n-BuLi n-butyllithium
  • NaBH(OAc) 3 sodium triacetoxyborohydride
  • Xantphos means 4,5-bis(diphenylphosphine)-9,9-dimethyloxy Xanthene
  • TFA for trifluoroacetic acid
  • Pd 2 (dba) 3 for tris(dibenzylideneacetone)dipalladium
  • Xphos for 2-dicyclohexylphosphorus-2,4,6-triisopropylbiphenyl
  • NMP represents N-methylpyrrolidone
  • Et 3 SiH represents triethyls
  • the powder X-ray diffraction pattern of the crystal form is obtained by a known method in the art, using an Equinox 3000S/N X-ray powder diffraction analyzer.
  • the test conditions of the instrument are shown in Table 3 below:
  • the position of each peak is determined by 2 ⁇ (°). It will be appreciated that different instruments and/or conditions may result in slightly different data, with variations in peak position and relative intensities.
  • the intensity division of the peaks only reflects the approximate size of the peaks at each position.
  • 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 % .
  • the peak with the 2 ⁇ (°) value of V 7.33 is the base peak), and the ratio of the peak height to the base peak height of other peaks is taken as the relative intensity I/I 0 .
  • the definition of the relative intensity of each peak is shown in the following table. :
  • High performance liquid chromatography In the present invention, high performance liquid chromatography (HPLC) was collected on Agilent 1260 HPLC.
  • the TGA spectrum was measured by a TA Q500/5000 thermogravimetric analyzer. Measurement conditions: protective gas: nitrogen (40 mL/min); temperature range: room temperature -350 °C; scanning rate: 10.0 °C/min.
  • the DSC spectrum was measured by a METTLER DSC3 differential scanning calorimeter. Measurement conditions: protective gas: nitrogen (50 mL/min); temperature range: -30-300 °C; scanning rate: 10.0 °C/min.
  • the dynamic moisture adsorption (DVS) curve was measured with a TA Q5000SA dynamic moisture adsorption instrument. Measurement conditions: temperature: 25°C; relative humidity range: 0%-80%.
  • Step 1 A solution of compound 1a-1 (6.0 g, 30.0 mmol) in THF (80 mL) was added at -78 °C with n-BuLi (27 mL, 66 mmol) and DIPA (6.6 g, 66 mmol), the mixture was stirred for 1 h and then DMF ( 10 mL) and warmed to room temperature to continue stirring for 2 h.
  • LC-MS was followed to complete the reaction.
  • HCl (2N) was added to the system to adjust pH to 5-6, extracted with ethyl acetate, the organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain 6.8 g of compound 1a-2.
  • Step 2 Compound 1a-2 (6.8 g, 30.0 mmol) 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) and the mixture was stirred at 50 °C overnight. LC-MS was followed to complete the reaction. The reaction solution was evaporated to dryness under reduced pressure, washed with saturated brine, extracted with DCM, the organic layer was dried, concentrated, and purified by column chromatography to obtain 4.8 g of compound 1a. MS m/z (ESI): 371 [M+H] + .
  • Step 1 Compound 1b-1 (1.5 g, 5.17 mmol), morpholine (470 mg, 5.39 mmol), Pd 2 (dba) 3 (210 mg, 0.23 mmol), Xphos (240 mg, 0.503 mmol) were added to a 100 mL three-necked flask ), a solution of cesium carbonate (3.38 g, 10.37 mmol) in 1,4-dioxane (20 mL) was reacted at 110 °C for 3 h. LC-MS was followed to complete the reaction.
  • Step 2 Compound 1b-2 (1.38 g, 4.657 mmol), methanol (20 mL) and HCl/1,4-dioxane (4M, 10 mL) were added to a 100 mL flask, and the mixture was stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure to remove the solvent, the residue was washed with saturated sodium bicarbonate, extracted with dichloromethane, and the organic layer was dried and concentrated to obtain 900 mg of compound 1b as a yellow solid. MS m/z (ESI): 197.2 [M+H] + .
  • Step 1 Compound 1a (740 mg, 2 mmol) in NMP (10 mL) was added with (R)-3-aminopiperidine-1-carboxylic acid tert-butyl ester, namely compound 2.1 (600 mg, 3 mmol) and DIPEA (780 mg, 6 mmol), The mixture was microwaved at 180°C for 30 min under argon atmosphere, followed by LC-MS until the reaction was complete. The reaction solution was cooled to room temperature, diluted with DCM, washed with water and saturated brine respectively, the organic layer was dried, concentrated, and purified by column chromatography to obtain 300 mg of compound X-1. MS m/z (ESI): 535 [M+H] + .
  • Step 2 Compound X-1 (250 mg, 0.5 mmol), Compound 1b (118 mg, 0.6 mmol), Pd 2 (dba) 3 (45 mg, 0.05 mmol), Xantphos (54 mg, 0.1 mmol), Cesium Carbonate (326 mg, 1 mmol) ) solution in 1,4-dioxane (15 mL) was microwaved at 160 °C for 50 min under argon atmosphere. LC-MS was followed to complete the reaction. The reaction solution was cooled to room temperature, diluted with EA, 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] + .
  • 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 followed to complete the reaction. Most of the TFA was removed under reduced pressure, and saturated sodium bicarbonate solution was added to adjust the pH to 7-8, extracted with DCM, the organic layers were combined, dried and concentrated to obtain compound X-3, which was directly used in the next reaction. MS m/z (ESI): 595.2 [M+H] + .
  • Step 4 A solution of compound X-3 (100 mg) in DCM (10 mL) was added under argon atmosphere acryloyl chloride (15.4 mg, 0.17 mmol) and DIPEA (66 mg, 0.51 mmol), respectively. The mixture was stirred at room temperature for 2 h. LC-MS was followed to complete the reaction. The reaction solution was washed with saturated brine, extracted with DCM, the organic layer was dried and concentrated to obtain a crude product, which was purified by column chromatography to obtain 86 mg of compound X-4. MS m/z (ESI): 649 [M+H] + .
  • the obtained solid compound X was sent to XRPD for detection, and its powder X-ray diffraction pattern showed no characteristic peaks, and the powder X-ray diffraction pattern was shown in FIG. 11 , and it was in an amorphous form.
  • Method 1 Weigh 3.5 g of the compound of formula X (amorphous) into a container, add 35 mL of isopropanol, heat up to reflux, and reflux for 1.5 h. Then, the temperature was lowered at a cooling rate of 5 °C/h. When the temperature was lowered to 63 °C, a large amount of solid was precipitated, and the temperature was kept and stirred for 10 h. Then naturally cooled to room temperature, and then kept stirring for 1.5 h; filtered, and the filter cake was dried under reduced pressure at 45°C to constant weight, and the obtained solid was sent to DSC to detect that it was a single crystal form, as shown in Figure 6.
  • Method 2 Weigh 1.54 kg of the compound of formula X (amorphous) into a container, add to 7.7 L of isopropanol, heat up at 70-80° C., and keep stirring for 1.5 h. Cool down naturally to 55-65°C, keep stirring for 8-9h. Naturally and slowly cool down to 20-25°C, keep stirring for 2h. Filter, rinse with isopropanol, and dry under reduced pressure at 40-50°C to constant weight to obtain 1.223 kg of solid.
  • thermogravimetric analyzer (3) Analysis and identification by TA Q500/5000 thermogravimetric analyzer:
  • the TGA patterns of the solids obtained by the first and second methods are basically as shown in Figure 7, and the crystal form V has a slow weight loss of about 0.50% before 150 °C, There is a slow weight loss of about 0.29% between 150 ° C and 225 ° C, and the stability is good at high temperature.
  • the purity of the initial sample was determined by HPLC prior to the start of the accelerated stability test.
  • the ratio of the purity of the sample in the accelerated stability test to the purity of the initial sample was used as the criterion for evaluating the stability of the sample. If the ratio of the purity was less than 95%, the chemical property of the sample was considered unstable.
  • the specific experimental results are shown in Table 4.
  • 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.
  • pH 6.8 medium Weigh 6.8 g of potassium dihydrogen phosphate and 0.94 g of sodium hydroxide, add water and dilute to 1L.
  • 0.2% SLS/pH6.8 medium Weigh 7.8g of sodium dihydrogen phosphate dihydrate and 0.9g of sodium hydroxide into 1L of water, adjust the pH to 6.8 with 2N sodium hydroxide, mix well and add 2g of SLS, mix well That is, 0.2% SLS/pH6.8 medium is obtained.
  • solubility of crystal form IV and crystal form V in 0.2% SLS/pH6.8 medium is better than that in water, pH4.5 and pH6.8 medium.
  • the solubility of the crystal form IV and the crystal form V is significantly improved, which improves the solubility of the drug and is beneficial to improve the bioavailability.
  • Administration and blood sample collection Oral administration (PO) by gavage, the dose is 1 capsule/rat (5mg/rat), after administration 0.083, 0.25, 0.5, 1, 2, 4, 6, 8 and Blood samples were collected at 9 time points over 24 hours. The animals were manually controlled at the time of collection, and approximately 150 ⁇ L of blood was collected through the fundus venous plexus of the rat into a tube containing K 2 EDTA. Blood samples were placed on wet ice and plasma samples were obtained by centrifugation (8000 rpm, 4 minutes, 4°C) within 15 minutes and stored at -80°C until analysis. Compounds were prepared on the day of the experiment.
  • the plasma samples were analyzed by liquid chromatography-tandem mass spectrometry (model: Triple Quad TM 4000), and the chromatographic column was: Waters XBridge-C18 (2.1 ⁇ 50 mm, 5 ⁇ m).
  • Preparation of standard curve prepare a standard curve with a linear range of 1.00-3000ng/mL for the compounds to be tested in blank Wistar rat plasma matrix, and at the same time prepare quality control samples with concentrations of 3,500, 2400ng/mL at low, medium and high concentrations.
  • Processing of biological samples Place the frozen plasma samples on ice to thaw, and after the samples are thawed, place them on a vortexer and vortex for 5 minutes. Take 20 ⁇ L of plasma samples, standard curve and quality control reference samples and add them to a 96-well plate, and then add 200 ⁇ L of acetonitrile-precipitated protein containing internal standard dexamethasone (brand: NIFDC, batch number: 6TUC-T4C2, preparation concentration 2000ng/mL).
  • both the crystalline form IV and the crystalline form V have significantly improved half-life and plasma exposure levels.
  • the plasma exposure level AUC of the crystal form IV administration group was about 1.5 times that of the amorphous form
  • the half-life T 1/2 of the crystal form IV was about 1.3 times that of the amorphous form
  • the plasma exposure level of the crystal form IV was about 1.3 times.
  • AUC and half-life T 1/2 are better than amorphous.
  • the plasma exposure level AUC of the crystal form V administration group is about 2 times that of amorphous, the half-life T 1/2 of crystal form V is about 3 times that of amorphous, and the plasma exposure level AUC and half-life T 1/2 of crystal form V Significantly better than amorphous.
  • Embodiment 8 Lantha screening kinase reaction experimental method
  • the blank well replaced the kinase with reaction buffer, and the kinase well (Enzyme) did not add any drug.
  • After 60 minutes of reaction in the dark on a shaker at 25°C. Add 10 ⁇ l Detection Solution (mixture of Invitrogen PV3528 and EDTA, dilute with TR-FRET dilution buffer, EDTA working concentration is 5 mM, Lanthascreening Tb PY20 antibody working concentration is 0.2 nM), and shake for 30 minutes at room temperature. Plates were read on a Victor X5 fluorescence microplate reader (PerkinElmer) and absorbance was measured at excitation wavelength 340 nm, emission wavelengths 500 nm and 520 nm.
  • the calculation method of the inhibition rate (refer to the specification of Invitrogen, PV3363) is as follows:
  • Emission rate(ER) Coumarin Emission(520nm)/Fluorescein Emission(500nm)
  • Inhibition rate (ER kinase -ER test compound )/(ER kinase -ER blank ) ⁇ 100%.
  • the median inhibitory concentration IC50 was calculated by fitting with XLFIT 5.0 software (IDBS, UK). The results are shown in Table 8:

Abstract

提供一种布鲁顿酪氨酸激酶抑制剂的多晶型物及其制备方法和应用，具体地提供了(R)-6-((1-丙烯酰基哌啶-3-基)氨基)-7-氟-4-((2-氟-4-吗啉代苯基)氨基)-1,2-二氢-3H-吡咯并[3，4-c]吡啶-3-酮的两种晶型、其制备方法和应用，所述两种晶型具有较好的稳定性、不易吸湿、改善的溶解性和药代动力学特征，且制备方法稳定，能够进行大规模生产。

Description

布鲁顿酪氨酸激酶抑制剂的多晶型物及其制备方法和应用 技术领域
本发明属于医药技术领域，具体地说，本发明涉及一种BTK抑制剂的多晶型物及其制备方法和应用，该抑制剂为(R)-6-((1-丙烯酰基哌啶-3-基)氨基)-7-氟-4-((2-氟-4-吗啉代苯基)氨基)-1,2-二氢-3H-吡咯并[3，4-c]吡啶-3-酮。
背景技术
布鲁顿酪氨酸激酶(BTK)激酶是TEC激酶家族中的一种非受体络氨酸激酶，是BCR信号通路的关键调节因子，对于B细胞成熟、增殖、存活具有重要的作用。在多种B细胞淋巴瘤中BTK都有过度表达，是目前TEC激酶家族中唯一经过临床验证的有效的药物开发的靶点。抑制BTK能够抑制一系列B细胞淋巴瘤的增殖。
B细胞抗原受体(BCR)信号通道的活化对诱发和维持B细胞恶性肿瘤及自免疫疾病有重要作用。Bruton's酪氨酸激酶(Btk)在造血细胞BCR信号通道中起着关键作用，是淋巴瘤新疗法研究中非常良好的靶点。BTK抑制剂作用于BCR通路,抑制Btk自磷酸化，Btk's生理底物PLCγ磷酸化和下游激酶ERK的磷酸化。
BTK抑制剂作用于慢性淋巴细胞白血病(CLL)细胞，诱导细胞毒性，抑制CLL细胞增殖能力。抑制BCR激活的原代B细胞增殖，且抑制原代单核细胞中TNFα,IL-1β和IL-6等分泌。BTK抑制剂作用于胶原诱导的关节炎模型，通过抑制B细胞活性，显著降低足肿胀和关节发炎等临床关节炎症状。
专利WO2019062329A1公开了一类具有较好BTK抑制活性的化合物结构，并具体公开了化合物6-((1-丙烯酰基哌啶-3-基)氨基)-7-氟-4-((2-氟-4-吗啉代苯基)氨基)-1,2-二氢-3H-吡咯并[3,4-c]吡啶-3-酮，该化合物对酶和细胞具有较高的抑制活性。然而对该化合物进一步开发时发现该化合物的R构型以无定形态存在时其溶解性较差，这样的结果可能会对后期的药物开发产生影响。为了更好、更有效地开发研究该化合物并能够潜在地应用于临床，本发明对该R构型化合物作了进一步研究，并基于该研究而完成本申请。
发明内容
为解决现有技术存在的问题，本发明提供了两种(R)-6-((1-丙烯酰基哌啶-3-基)氨基)-7-氟-4-((2-氟-4-吗啉代苯基)氨基)-1,2-二氢-3H-吡咯并[3，4-c]吡啶-3-酮(式(X)化合物)的新晶型及其制备方法和应用。本发明提供的新晶型具有改善的溶解性以及更有利的体内药代动力学特征，有助于进一步的药物开发。
在本发明的第一方面，提供了一种式X化合物的晶型Ⅳ：
Figure PCTCN2021115856-appb-000001
在一些实施例中，所述晶型Ⅳ为无水形式或水合物形式。
在一些实施例中，所述晶型Ⅳ为无水形式。
在一些实施例中，所述晶型Ⅳ的使用Cu-Kα辐射检测的X射线粉末衍射图在下组Ⅳ-1的衍射角2θ(°)值处具有衍射峰：5.90±0.2、14.91±0.2、17.51±0.2、24.90±0.2和26.37±0.2。
在一些实施例中，晶型Ⅳ的X射线粉末衍射图还包括在1、2个或2个以上或全部选自下组Ⅳ-2的衍射角2θ(°)值处的衍射峰：10.41±0.2、12.19±0.2、18.80±0.2、21.49±0.2和25.44±0.2。
在一些实施例中，所述晶型Ⅳ的X射线粉末衍射图还包括在8.91±0.2、12.49±0.2和12.76±0.2衍射角2θ(°)值处的衍射峰。
在一些实施例中，所述晶型Ⅳ的X射线粉末衍射图还包括在15.48±0.2、16.05±0.2、18.29±0.2、19.76±0.2、22.45±0.2和23.57±0.2衍射角2θ(°)值处的衍射峰。
在一些实施例中，晶型Ⅳ的X射线粉末衍射图还包括在1、2、3个或3个以上或全部选自下组Ⅳ-3的衍射角2θ(°)值处的衍射峰：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和35.37±0.2。
在一些实施例中，所述晶型Ⅳ的X射线粉末衍射图在选自组Ⅳ-1、Ⅳ-2和Ⅳ-3中的6个或更多个或全部(如6、7、8、9、10、11、12、13、14、15等)的2θ(°)值处具有峰。
在一些实施例中，所述晶型Ⅳ的X射线粉末衍射图在表1所示的2θ(°)值处具有衍射峰，各峰相对强度如表1所示：
表1
2θ(°) I/I 0 2θ(°) I/I 0 2θ(°) I/I 0
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    
在一些实施例中，所述晶型Ⅳ的X射线粉末衍射图基本如图1所表征。
在一些实施例中，所述晶型Ⅳ的差示扫描量热曲线在167.89℃±3℃处具有吸热峰，熔化热约为55.84J/g。
在一些实施例中，所述晶型Ⅳ的差示扫描量热曲线在167.89℃±1℃处具有吸热峰。
在一些实施例中，所述晶型Ⅳ具有基本上如图2所示的差示扫描量热曲线。
在一些实施例中，所述晶型Ⅳ在30℃～150℃下，热重分析检测其失重约为0.29％。
在一些实施例中，所述晶型Ⅳ具有基本上如图3所示的热重分析图谱。
在一些实施例中，所述晶型Ⅳ的动态水分吸附在0％～80％相对湿度范围内增重约为0.7％，所述百分比为重量百分数比。
在一些实施例中，所述晶型Ⅳ具有基本上如图4所示的DVS图谱。
在本发明的第二方面，提供了式X化合物晶型Ⅳ的制备方法，该制备方法简单，制得的晶型纯度高，适合工业化生产。
所述晶型Ⅳ的制备方法包括以下步骤：
(S101)将式X化合物和溶剂的混合物加热至55～65℃(优选60～65℃)；及
(S102)将混合物冷却，分离析出的固体，得到所述晶型Ⅳ。
在一些实施例中，步骤(S102)中的混合物冷却至室温。
在一些实施例中，步骤(S102)中的混合物冷却至20℃～25℃。
在一些实施例中，步骤(S101)中的溶剂选自水、乙醇、正丙醇和异丙醇中的一种或多种。
在一些实施例中，步骤(S101)中的溶剂为异丙醇。
在一些实施例中，所述步骤(S101)中，加热时间为0.5-12小时；优选加热时间为1-2小时。
在一些实施例中，所述步骤(S102)中，混合物冷却至室温后，保温0-12小时，优选保温1-2小时。
在本发明的第三方面，提供一种根据第二方面的制备方法制备得到的式X化合物晶型Ⅳ。
在本发明的第四方面，提供了另一种式X化合物的晶型Ⅴ，其中式X化合物具有以下结构：
Figure PCTCN2021115856-appb-000002
在一些实施例中，所述晶型Ⅴ为无水形式或水合物形式。
在一些实施例中，所述晶型Ⅴ为无水形式。
在一些实施例中，所述晶型Ⅴ的使用Cu-Kα辐射检测的X射线粉末衍射图在下组Ⅴ-1的衍射角2θ(°)值处具有衍射峰：7.33±0.2、9.91±0.2和17.53±0.2。
在一些实施例中，所述晶型Ⅴ的X射线粉末衍射图还包括在13.67±0.2衍射角2θ(°)值处的衍射峰。
在一些实施例中，所述晶型Ⅴ的X射线粉末衍射图还包括在15.69±0.2和16.20±0.2的衍射角2θ(°)值处的衍射峰。
在一些实施例中，所述晶型Ⅴ的X射线粉末衍射图还包括在12.49±0.2的衍射角2θ(°)值处的衍射峰。
在一些实施例中，所述晶型Ⅴ的X射线粉末衍射图还包括在18.95±0.2的衍射角2θ(°)值处的衍射峰。
在一些实施例中，所述晶型Ⅴ的X射线粉末衍射图还包括在20.19±0.2和21.76±0.2的衍射角2θ(°)值处的衍射峰。
在一些实施例中，所述晶型Ⅴ的X射线粉末衍射图还包括在下组Ⅴ-2的衍射角2θ(°)值处的衍射峰：12.49±0.2、15.69±0.2、20.19±0.2和23.02±0.2。
在一些实施例中，所述晶型Ⅴ的X射线粉末衍射图还包括在13.67±0.2和16.20±0.2的衍射角2θ(°)值处的衍射峰。
在一些实施例中，所述晶型Ⅴ的X射线粉末衍射图还包括在13.67±0.2和18.95±0.2的衍射角2θ(°)值处的衍射峰。
在一些实施例中，所述晶型Ⅴ的X射线粉末衍射图还包括在16.20±0.2和18.95±0.2的衍射角2θ(°)值处的衍射峰。
在一些实施例中，所述晶型Ⅴ的X射线粉末衍射图还包括在13.67±0.2、16.20±0.2和18.95±0.2的衍射角2θ(°)值处的衍射峰。
在一些实施例中，所述晶型Ⅴ的X射线粉末衍射图还包括在21.76±0.2和30.21±0.2的衍射角2θ(°)值处的衍射峰。
在一些实施例中，所述晶型Ⅴ的X射线粉末衍射图还包括在24.80±0.2和25.53±0.2的衍射角2θ(°)值处的衍射峰。
在一些实施例中，所述晶型Ⅴ的X射线粉末衍射图还包括在2个或2个以上或全部选自下组Ⅴ-3的衍射角2θ(°)值处的衍射峰：13.67±0.2、16.20±0.2、18.95±0.2、21.76±0.2、24.80±0.2、25.53±0.2和30.21±0.2。
在一些实施例中，所述晶型Ⅴ的X射线粉末衍射图在选自组Ⅴ-1、Ⅴ-2和Ⅴ-3中的6个或更多个或全部(如6、7、8、9、10、11、12、13、14等)的2θ(°)值处具有衍射峰。
在一些实施例中，所述晶型Ⅴ的X射线粉末衍射图在表2所示的2θ(°)值处具有衍射峰，各衍射峰相对强度如表2所示：
表2
2θ(°) I/I 0 2θ(°) I/I 0 2θ(°) I/I 0
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    
在一些实施例中，所述晶型Ⅴ的X射线粉末衍射图基本如图5所表征。
在一些实施例中，所述晶型Ⅴ的差示扫描量热曲线在197.31℃±3℃处具有吸热峰，熔化热约为71.30J/g。
在一些实施例中，所述晶型Ⅴ的差示扫描量热曲线在197.31℃±1℃处具有吸热峰。
在一些实施例中，所述晶型Ⅴ具有基本上如图6所示的差示扫描量热曲线。
在一些实施例中，所述晶型Ⅴ在25℃～150℃下，热重分析检测其失重约为0.50％；在150℃～225℃之间，热重分析检测其失重约为0.29％。
在一些实施例中，所述晶型Ⅴ具有基本上如图7所示的热重分析图谱。
在一些实施例中，所述晶型Ⅴ的动态水分吸附在0％～80％相对湿度范围内增重为小于0.2％，所述百分比为重量百分数比。
在一些实施例中，所述晶型Ⅴ具有基本上如图8所示的DVS图谱。
在本发明的第五方面，提供了式X化合物晶型Ⅴ的制备方法，该制备方法重现性好，过程易于控制，工艺方法稳定，制得的晶型纯度高，适合工业化生产。
所述晶型Ⅴ的制备方法包括以下步骤：
(S201)将式X化合物和溶剂的混合物加热到70℃或70℃以上；及
(S202)将混合物冷却，分离析出的固体，得到所述晶型Ⅴ。
在一些实施例中，步骤(S202)中，将混合物冷却至室温。
在一些实施例中，步骤(S202)中，将混合物冷却至20℃～25℃。
在一些实施例中，步骤(S201)中，所述溶剂选自水、C 2-4烷基醇、乙腈、甲苯、二甲苯、1,4-二氧六环、1，2-二氯乙烷和乙酸乙酯中的一种或多种。
在一些实施例中，所述C 2-4烷基醇为乙醇、正丙醇、异丙醇、乙二醇中的一种或多种，优选溶剂为异丙醇。
在一些实施例中，步骤(S201)中，式X化合物的质量(m)与溶剂体积(v)的比值(m/v)为50g/L～400g/L(优选100g/L～200g/L)。
在一些实施例中，步骤(S201)中，将式X化合物和溶剂的混合物加热到70～150℃，优选70～120℃，更优选70～90℃，更优选70～80℃。
在一些实施例中，步骤(S201)中，加热时间为0.5～48小时，优选0.5～12小时，更优选为1～5小时，更优选为1～2小时。
在一些实施例中，在步骤(S202)之前还包括步骤(S2021)：将混合物冷却至40～65℃(优选55～65℃)后，保温一段时间。
在一些实施例中，步骤(S2021)中，保温时间为1～12小时，进一步地，优选保温时间为8～10小时。
在一些实施例中，步骤(S2021)中，混合物以4℃/小时-6℃/小时(优选5℃/小时)的降温速率冷却至40～65℃(优选55～65℃)，保温8～10小时。
在一些实施例中，步骤(S2021)中，混合物自然降温至40～65℃(优选55～65℃)，保温8-9小时。
在一些实施例中，步骤(S202)中，混合物冷却至室温后，保温1～2小时。
在一些实施例中，所述晶型Ⅴ的制备方法包括以下步骤：
(S301)将式X化合物和异丙醇混合后加热至回流或70-80℃；
(S302)将混合物冷却至40～65℃；及
(S303)将混合物冷却至室温，分离析出的固体，得到所述晶型Ⅴ。
在一些实施例中，步骤(S301)中，式X化合物和异丙醇混合后加热至回流或70-80℃后，保温0.5～48小时；进一步地，保温0.5～12小时；进一步地，保温1～5小时；进一步地，保温1～2小时。
在一些实施例中，步骤(S302)中，将混合物冷却至40～65℃后，保温1～12小时；进一步地，保温8～10小时。
在一些实施例中，步骤(S303)中，将混合物冷却至室温后，保温度1～12小时；进一步地，优选保温1～2小时。
本发明第六方面，提供了一种根据第五方面的制备方法制备得到的式X化合物晶型Ⅴ。
本发明第七方面，提供了第一方面所述的晶型Ⅳ、第三方面所述的晶型Ⅳ、第四方面所述的晶型Ⅴ、或第六方面的所述的晶型Ⅴ在制备BTK抑制剂中的应用。
本发明第八方面，提供了第一方面所述的晶型Ⅳ、第三方面所述的晶型Ⅳ、第四方面所述的晶型Ⅴ、或第六方面的所述的晶型Ⅴ在制备治疗和/或预防由B细胞介导的疾病的药物中的应用。
本发明第九方面，提供了一种治疗由B细胞介导的疾病的方法，包括给予所需患者治疗有效量的第一方面所述的晶型Ⅳ、第三方面所述的晶型Ⅳ、第四方面所述的晶型Ⅴ或第六方面的所述的晶型Ⅴ。
在另一优选实施例中，由B细胞介导的疾病选自：肿瘤疾病、增殖性疾病、***反应性疾病、自身免疫性疾病和炎症性疾病。
在另一优选实施例中，由B细胞介导的疾病选自：实体瘤、急性淋巴细胞白血病、慢性淋巴细胞白血病、急性骨髓性白血病、慢性骨髓性白血病、类风湿性关节炎、银屑病关节炎、骨关节炎、***性红斑狼疮、牛皮癣、类风湿性脊椎炎和痛风性关节炎。
在另一优选实施例中，由B细胞介导的疾病为实体瘤。
在另一优选实施例中，所述实体瘤为选自淋巴瘤、软组织肉瘤、淋巴细胞性淋巴瘤、套细胞淋巴瘤、黑色素瘤和多发性骨髓瘤中的至少一种。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1晶型Ⅳ的X射线粉末衍射(XRPD)图谱(使用Cu-Kα辐射，横坐标为角度2θ(°)，纵坐标为强度)；
图2晶型Ⅳ的差示扫描量热分析(DSC)图谱(横坐标为温度(℃)，纵坐标为热流率(mW))；
图3晶型Ⅳ的热重分析(TGA)图谱(横坐标为温度(℃)，纵坐标为重量百分比(％))；
图4晶型Ⅳ的动态水分吸附(DVS)图谱(横坐标为相对湿度(％)，纵坐标为样品重量变化(％))；
图5晶型Ⅴ的X射线粉末衍射(XRPD)图谱(使用Cu-Kα辐射，横坐标为角度2θ(°)，纵坐标为强度)；
图6晶型Ⅴ的差示扫描量热分析(DSC)图谱(横坐标为温度(℃)，纵坐标为热流率(mW))；
图7晶型Ⅴ的热重分析(TGA)图谱(横坐标为温度(℃)，纵坐标为重量百分比(％))；
图8晶型Ⅴ的动态水分吸附(DVS)图谱(横坐标为相对湿度(％)，纵坐标为样品重量变化(％))；
图9晶型Ⅳ分别在60℃条件下放置1个月、40℃/75％RH条件下放置1个月和初始样品的XRPD图谱对比(横坐标为角度2θ(°)，纵坐标为强度)；
图10晶型Ⅴ分别在60℃条件下放置1个月、40℃/75％RH条件下放置1个月和初始样品的XRPD图谱对比(横坐标为2θ(°)；纵坐标为强度)；
图11式X化合物游离碱无定形的X射线粉末衍射(XRPD)图谱(使用Cu-Kα辐射，横坐标为2θ(°)；纵坐标为强度)；
图12晶型Ⅰ的X射线粉末衍射(XRPD)图谱(使用Cu-Kα辐射，横坐标为2θ(°)；纵坐标为强度)。
具体实施方式
式X化合物
在本发明中，式X化合物为(R)-6-((1-丙烯酰基哌啶-3-基)氨基)-7-氟-4-((2-氟-4-吗啉代苯基)氨基)-1,2-二氢-3H-吡咯并[3，4-c]吡啶-3-酮，其晶型Ⅳ和晶型Ⅴ具有改善的溶解性以及更有利的体内药代动力学特征，不易吸湿，具有很好的化学和物理稳定性。此外研究发现式X化合物相比于其消旋体化合物对BTK WT激酶具有更好的抑制活性。
本发明提供的式X化合物的晶型Ⅳ和晶型Ⅴ可用于制备BTK抑制剂或制备治疗BTK相关疾病的药物。作为优选，所述BTK相关疾病为癌症、异常细胞增殖性疾病、感染、炎性病症、自身免疫性疾病、心血管疾病、神经变性疾病、由辐射引起的造血毒性疾病，或其组合。优选地，所述癌症为乳腺癌、卵巢癌、***癌、黑色素瘤、脑瘤、食管癌、胃癌、肝癌、胰腺癌、结肠直肠癌、肺癌、肾癌、皮肤癌、成胶质细胞瘤、神经母细胞 瘤、肉瘤、脂肪肉瘤、骨软骨瘤、骨瘤、骨肉瘤、***瘤、睾丸肿瘤、子宫癌、头颈肿瘤、多发性骨髓瘤、恶性淋巴瘤、真性红细胞增多症、白血病、甲状腺肿瘤、输尿管肿瘤、***、胆囊癌、胆管癌、绒毛膜上皮癌或儿科肿瘤，或它们的任何组合。优选地，所述乳腺癌是HR-阳性、HER2-阴性晚期乳腺癌。
如本文所用，“治疗有效量”是指可对人和/或动物产生功能或活性的且可被人和/或动物所接受的量。
如本文所用，“患者”是指一种动物，优选为哺乳动物，更优选为人。术语“哺乳动物”是指温血脊椎类哺乳动物，包括如猫、狗、兔、熊、狐狸、狼、猴子、鹿、鼠、猪和人类。
如本文所用，“治疗”是指减轻、延缓进展、衰减、预防，或维持现有疾病或病症(例如癌症)。治疗还包括将疾病或病症的一个或多个症状治愈、预防其发展或减轻到某种程度。
本发明中，“使用Cu-Kα辐射”指相应的谱图使用Cu靶的Kα射线检测获得，当采用其他方法检测时，各衍射峰可以存在本领域可接受范围内的偏差，不应理解为对本发明的限制。
多晶型物
固体不是以无定形的形式就是以结晶的形式存在。在结晶形式的情况下，分子定位于三维晶格格位内。当化合物从溶液或浆液中结晶出来时，它可以不同的空间点阵排列结晶(这种性质被称作“多晶型现象”)，形成具有不同的结晶形式的晶体，这各种结晶形式被称作“多晶型物”或“晶型”。给定物质的不同多晶型物可在一个或多个物理属性方面(如溶解度和溶解速率、真比重、晶形、堆积方式、流动性和/或固态稳定性)彼此不同。
结晶
可以通过操作溶液，使得感兴趣化合物的溶解度极限被超过，从而完成生产规模的结晶。这可以通过多种方法来完成，例如，在相对高的温度下溶解化合物，然后冷却溶液至饱和极限以下。或者通过沸腾、常压蒸发、真空干燥或通过其它的一些方法来减小液体体积。可通过加入抗溶剂或化合物在其中具有低的溶解度的溶剂或这样的溶剂的混合物，来降低感兴趣化合物的溶解度。另一种可选方法是调节pH值以降低溶解度。有关结晶方面的详细描述请参见Crystallization,第三版,J W Mullens,Butterworth-Heineman Ltd.，1993,ISBN 0750611294。
如本文所用，术语“室温”一般指4-30℃，较佳地指20±5℃。
晶型的鉴定和性质
本发明在制备式X化合物的晶型后，采用如下多种方式和仪器对其性质进行了研究。X射线粉末衍射(XRPD)
测定晶型的X射线粉末衍射的方法在本领域中是已知的。XRPD可检测晶型的变化、结晶度、晶构状态等信息，是鉴别晶型的常用手段。XRPD图谱的峰位置主要取决于晶型 的结构，不同仪器之间，XRPD图谱的2θ的量度可能会略有差别，因此所述2θ的数值不能视为绝对的。根据本发明试验所用仪器状况，衍射峰存在±0.2°的误差。本发明的式X化合物的晶型，具有特定的晶型形态，在XRPD图中具有特定的特征峰。
示差扫描量热分析(DSC)
又称“差示量热扫描分析”，是在加热过程中，测量被测物质与参比物之间的能量差与温度之间关系的一种技术。DSC图谱上的峰位置、形状和峰数目与物质的性质有关，故可以定性地用来鉴定物质。本领域常用该方法来检测物质的相变温度、玻璃化转变温度、反应热等多种参数。不同仪器之间，DSC图谱的峰位置可能会略有差别，因此所述DSC吸热峰的峰位置的数值不能视为绝对的。根据本发明试验所用仪器状况，实验误差或差别的数值可能小于等于5℃，或小于等于4℃，或小于等于3℃，或小于等于2℃，或小于等于1℃。
热重分析(TGA)
TGA是在程序控制下，测定物质的质量随温度变化的一种技术，适用于检查晶体中溶剂的丧失或样品升华、分解的过程，可推测晶体中含结晶水或结晶溶剂的情况。TGA曲线显示的质量变化取决于样品制备和仪器等许多因素；不同仪器之间TGA检测的质量变化略有差别。根据本发明试验所用的仪器状况，质量变化存在±0.1％的误差。
本发明的主要优点在于：本发明式X化合物的晶型Ⅳ和晶型Ⅴ结晶性好，稳定性高，不易吸湿，相对于无定形具有更好的溶解性和更有利的药代动力学特征，包括相对较高的血浆暴露量和较长的半衰期，有助于提高无定形的式X化合物的生物利用度。此外，晶型Ⅳ的制备方法简单，制得的晶型纯度高，适合工业化生产。晶型Ⅴ的制备方法重现性好，过程易于控制，工艺方法稳定，适合工业化生产。因此，晶型Ⅳ和晶型Ⅴ具有进一步开发成为药物的可能。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
试剂与仪器
本发明中，化合物的结构和纯度通过核磁共振( 1H NMR)和/或液质联用质谱(LC-MS)来确定。 1H NMR：BrukerAVANCE-400核磁仪，内标为四甲基硅烷(TMS)。LC-MS：Agilent1200HPLC System、6140MS液质联用质谱仪(购自安捷伦)，柱子WatersX-Bridge,150×4.6mm,3.5μm。制备高效液相色谱(pre-HPLC)：用Waters PHW007，柱子XBridge C18,4.6*150mm,3.5um。
如本文所用，DCM表示二氯甲烷，DMF表示二甲基甲酰胺，DMSO表示二甲基亚砜，THF表示四氢呋喃，EA表示乙酸乙酯，DIPA表示二异丙胺，DIPEA表示N,N-二异丙基乙胺，n-BuLi表示正丁基锂，NaBH(OAc) 3表示三乙酰氧基硼氢化钠，Xantphos表示4,5-双(二苯基膦)-9,9-二甲基氧杂蒽，TFA表示三氟乙酸，Pd 2(dba) 3表示三(二亚苄基 丙酮)二钯，Xphos表示2-二环己基磷-2,4,6-三异丙基联苯，NMP表示N-甲基吡咯烷酮，Et 3SiH表示三乙基硅烷，SLS表示月桂基硫酸钠，羧甲基淀粉钠(品牌：安徽山河，批号SSG3017006)。
通用方法
X射线粉末衍射：本发明中，晶型的粉末X衍射图谱是通过本领域的已知方法，使用Equinox 3000S/N X射线粉末衍射分析仪获得。仪器测试条件如下表3所示：
表3
Figure PCTCN2021115856-appb-000003
在粉末X射线粉末衍射图中，各峰的位置由2θ(°)确定。可以理解，不同的仪器和/或条件可导致产生的数据会略有不同，各峰的位置和相对强度会有变化。峰的强度划分仅仅反映了各位置上峰的近似大小。在本发明中，晶型以其峰高最高的衍射峰作为基峰，定义其相对强度为100％，作为I 0(晶型Ⅳ的2θ(°)值为14.91的峰为基峰，晶型Ⅴ的2θ(°)值为7.33的峰为基峰)，其它各峰以其峰高与基峰峰高的比值作为其相对强度I/I 0，各峰相对强度的划分定义如下表所示：
相对强度I/I 0(％) 定义
50～100 VS(很强)
25～50 S(强)
10～25 M(中等)
1～10 W(弱)
高效液相色谱：本发明中，高效液相色谱(HPLC)在Agilent1260HPLC上采集。
TGA图谱采用TA Q500/5000热重分析仪测得，测定条件：保护气体：氮气(40mL/min)；温度范围：室温-350℃；扫描速率：10.0℃/min。
DSC图谱采用METTLER DSC3差示扫描量热仪测得，测定条件：保护气体：氮气(50mL/min)；温度范围：-30-300℃；扫描速率：10.0℃/min。
动态水分吸附(DVS)曲线采用TA Q5000SA动态水分吸附仪测得，测定条件：温度： 25℃；相对湿度范围：0％-80％。
可以理解的是，使用与上述仪器作用相同的其他类型的仪器或使用不同与本发明中使用的测试条件时，可能会得到另外的数值，因此，所引用的数值不应视为绝对的数值。由于仪器的误差或操作人员的区别，本领域技术人员能理解，以上用于表征晶体的物理性质的参数可能有微小的差别，所以上述的参数仅用于辅助表征本发明提供的晶型，而不能视为是对本发明的晶型的限制。
具体实施例
实施例1无定形的式X化合物游离碱的制备
中间体1a的制备
Figure PCTCN2021115856-appb-000004
步骤1：化合物1a-1(6.0g,30.0mmol)的THF(80mL)溶液在-78℃下加入n-BuLi(27mL,66mmol)和DIPA(6.6g,66mmol)，混合物搅拌1h后加入DMF(10mL)并升至室温继续搅拌2h。LC-MS跟踪至反应完全。向体系中加HCl(2N)调pH至5-6，乙酸乙酯萃取，有机层经饱和食盐水洗涤，无水硫酸钠干燥，浓缩，柱层析纯化得6.8g化合物1a-2。MS m/z(ESI):238[M+H] +。
步骤2：化合物1a-2(6.8g,30.0mmol)的1,4-二氧六环(80mL)加入化合物1a.1(15g,90.0mmol)、醋酸(2mL)和NaBH(OAc) 3(18.9g,90.0mmol)，混合物于50℃搅拌过夜。LC-MS跟踪至反应完全。反应液减压蒸干，加饱和食盐水洗涤，经DCM萃取，有机层干燥、浓缩，柱层析纯化得4.8g化合物1a。MS m/z(ESI):371[M+H] +。
中间体1b的制备
Figure PCTCN2021115856-appb-000005
步骤1：100mL的三颈烧瓶中加入化合物1b-1(1.5g,5.17mmol)、吗啉(470mg,5.39mmol)、Pd 2(dba) 3(210mg,0.23mmol)、Xphos(240mg,0.503mmol)、碳酸铯(3.38g,10.37mmol)的1,4-二氧六环(20mL)溶液于110℃反应3h。LC-MS跟踪至反应完全。反应液冷却至室温，浓缩后经柱层析纯化(含0-20％EA的正己烷)得灰色固体化合物1b-2(1.38g,产率为90.1％)。MS m/z(ESI):297.2[M+H] +。
步骤2：100mL的烧瓶中加入化合物1b-2(1.38g,4.657mmol)、甲醇(20mL)和HCl/1,4-二氧六环(4M,10mL)，混合物室温搅拌过夜。反应液减压浓缩去除溶剂，残留物加饱和碳酸氢钠洗涤，二氯甲烷萃取，有机层干燥后浓缩得900mg黄色固体化合物1b。MS  m/z(ESI):197.2[M+H] +。
式X化合物的制备
Figure PCTCN2021115856-appb-000006
步骤1：化合物1a(740mg,2mmol)的NMP(10mL)溶液加入(R)-3-氨基哌啶-1-羧酸叔丁酯即化合物2.1(600mg,3mmol)和DIPEA(780mg,6mmol)，混合物在氩气氛围下于180℃微波反应30min，LC-MS跟踪至反应完全。反应液冷却至室温，加DCM稀释，分别经水与饱和食盐水洗涤，有机层干燥、浓缩后经柱层析纯化得300mg化合物X-1。MS m/z(ESI):535[M+H] +。
步骤2：化合物X-1(250mg,0.5mmol)、化合物1b(118mg,0.6mmol)、Pd 2(dba) 3(45mg,0.05mmol)、Xantphos(54mg,0.1mmol)、碳酸铯(326mg,1mmol)的1,4-二氧六环(15mL)溶液在氩气氛围下于160℃微波反应50min。LC-MS跟踪至反应完全。反应液冷却至室温，加EA稀释，分别经水与饱和食盐水洗涤，有机层干燥、浓缩后经柱层析纯化得185mg化合物X-2。MS m/z(ESI):695.3[M+H] +。
步骤3：化合物X-2(185mg,0.27mmol)的DCM(12mL)溶液中加入TFA(4.5mL)。混合物室温搅拌1h。LC-MS跟踪至反应完全。减压去除大部分TFA，加饱和碳酸氢钠溶液，调pH至7-8，DCM萃取，有机层合并后干燥、浓缩得化合物X-3，直接用于下一步反应。MS m/z(ESI):595.2[M+H] +。
步骤4：化合物X-3(100mg)的DCM(10mL)溶液在氩气氛围下分别加入丙烯酰氯(15.4mg,0.17mmol)和DIPEA(66mg,0.51mmol)。混合物室温搅拌2h。LC-MS跟踪至反应完全。反应液经饱和食盐水洗涤，DCM萃取，有机层干燥、浓缩得粗品，柱层析纯化得86mg化合物X-4。MS m/z(ESI):649[M+H] +。
步骤5：化合物X-4(86mg,0.13mmol)的TFA(3mL)溶液加入Et 3SiH(0.2mL)，混合物加热至80℃搅拌2h。LC-MS跟踪至反应完全。减压去除大部分TFA，加饱和碳酸氢钠溶液调pH至7-8，乙酸乙酯萃取，合并有机层，干燥、浓缩后经Prep-HPLC(洗脱剂DCM:MeOH＝100:3)纯化得10mg固体化合物X。MS m/z(ESI):499.3[M+H] +。 1H NMR(400MHz,DMSO-d 6)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＝12.4Hz,0.5H),4.37(s,2H),4.17(d,J＝12.6Hz,0.5H),3.99(s,1H),3.89(s,1H),3.71(t,J＝4.6Hz,4H),3.03-2.98(m,5H),2.66-2.85(m,1H),2.00(s,1H),1.79(d,J＝13.5Hz,1H),1.65(d,J＝12.5Hz,1H),1.43(s,1H)。将所得固体化合物X送XRPD检测，其粉末X射线衍射图显示无特征峰，粉末X射线衍射图如图11所示，为无定形形式。
实施例2晶型Ⅳ的制备
1.式X化合物晶型Ⅳ的制备方法
称取2.0g式X化合物(无定形)置于容器中，加入18mL异丙醇，升温至60～65℃，保温1～2小时；然后自然降温到室温，保温1～2小时；过滤，滤饼在45℃减压干燥至恒重，将所得固体送DSC检测显示为单一晶型，如图2所示。
2.式X化合物晶型Ⅳ的表征
(1)通过Equinox 3000S/N X射线粉末衍射分析仪分析鉴定：所得固体的X射线粉末衍射基本上如图1所示。在本申请中定义为晶型Ⅳ。
(2)通过TA Q200/2000差示扫描量热仪分析鉴定：晶型Ⅳ在167.89℃处具有吸热峰；熔化热为55.84J/g，其DSC图谱基本上如图2所示。
(3)通过TA Q500/5000热重分析仪分析鉴定：晶型Ⅳ在150℃之前有约为0.29％的缓慢失重，高温下稳定性良好，其TGA图谱基本上如图3所示。
(4)通过TA Q5000SA动态水分吸附仪分析鉴定：晶型Ⅳ在0％～80％相对湿度范围内吸湿增重约为0.7％，略有吸湿性。其DVS图谱如图4所示。
实施例3晶型Ⅴ的制备
1.式X化合物晶型Ⅴ的制备方法
方法一：称取3.5g式X化合物(无定形)置于容器中，加入35mL异丙醇，升温至回流，回流1.5h。然后以5℃/h的降温速率降温，降温至63℃时，有大量固体析出，保温搅拌10h。然后自然降温至室温，再保温搅拌1.5h；过滤，滤饼在45℃减压干燥至恒重，将所得固体送DSC检测显示为单一晶型，如图6所示。
方法二：称取1.54kg的式X化合物(无定形)置于容器中，加入至7.7L异丙醇，升温70-80℃，保温搅拌1.5h。自然降温至55～65℃，保温搅拌8-9h。自然缓慢降温至20～25℃，保温搅拌2h。过滤，用异丙醇淋洗，40～50℃减压干燥至恒重，得到固体1.223kg。
2.式X化合物晶型Ⅴ的表征
(1)通过Equinox 3000S/N X射线粉末衍射(XRPD)分析仪分析鉴定：方法一和方法二所得固体的X射线粉末衍射均基本上如图5所示，在本申请中定义为晶型Ⅴ。
(2)通过TA Q200/2000差示扫描量热仪分析鉴定：方法一和方法二所得固体的DSC图谱均基本如图6所示，晶型Ⅴ在197.31℃处具有吸热峰，熔化热为71.30J/g。
(3)通过TA Q500/5000热重分析仪分析鉴定：方法一和方法二所得固体的TGA图谱均基本上如图7所示，晶型Ⅴ在150℃之前有约为0.50％的缓慢失重，在150℃～225℃之间有约为0.29％的缓慢失重，高温下稳定性良好。
(4)通过TA Q5000SA动态水分吸附仪分析鉴定：方法一和方法二所得固体的DVS图谱均基本如图8所示，晶型Ⅴ在0％～80％相对湿度范围内吸湿增重小于0.2％，几乎无吸湿性。
实施例4晶型Ⅰ的制备
称取10.17mg式X化合物游离碱(无定形)于样品瓶中，室温下边搅拌边逐步往瓶中加入3ml水，得到式X化合物的混悬液，将混悬液离心干燥得到固体，将固体送X射线粉末衍射检测。所得固体的X射线粉末衍射图如图12所示，在本申请中定义为晶型Ⅰ。本发明中，晶型Ⅰ的粉末X衍射图谱是使用D8ADVANCE X射线粉末衍射分析仪获得。仪器测试条件如下表所示：
Figure PCTCN2021115856-appb-000007
实施例5稳定性实验
1.加速稳定性试验
将晶型Ⅳ、晶型Ⅴ分别置于40℃/75％RH加速稳定性箱和60℃烘箱内，放置1个月后取出，称取10mg用甲醇溶解，用HPLC测试其纯度，用XRPD测试其晶型变化。
加速稳定性试验开始前，初始样品用HPLC测定样品的纯度。用加速稳定性试验样品纯度与初始样品纯度的比值，作为样品稳定性评判标准，若纯度比值小于95％，则认为样品化学性质不稳定。具体实验结果如表4所示。
表4
Figure PCTCN2021115856-appb-000008
Figure PCTCN2021115856-appb-000009
由表4以及图9、图10可知，晶型Ⅳ和晶型Ⅴ在40℃/75％RH条件和60℃条件下放置1个月，杂质物质未明显增加，XRPD图谱显示无特征峰变化，表明晶型Ⅳ和晶型Ⅴ的化学稳定性和物理稳定性均良好。
2.混悬竞争试验
为进一步确认晶型Ⅴ和晶型Ⅳ在有溶剂条件下的相互转变关系，分别在室温及50℃下设置了混悬竞争试验。准备12份等量混合的晶型Ⅴ和晶型Ⅳ样品，分别加入乙醇、异丙醇、丙酮和乙酸乙酯四种溶剂，密封后置于相应振荡器和摇床中，分别在室温下混悬振摇1天和7天，在50℃混悬振摇1天，离心干燥后进行XRPD检测，查看晶型变化。实验结果如表5所示：
表5
Figure PCTCN2021115856-appb-000010
实施例6溶解性实验
溶出介质：
pH 4.5介质：称取乙酸钠18g，加入冰醋酸9.8ml，加水稀释至1L。
pH 6.8介质：称取磷酸二氢钾6.8g，氢氧化钠0.94g，加水稀释至1L。
0.2％SLS/pH6.8介质：称取7.8g的二水合磷酸二氢钠和0.9g氢氧化钠加入至1L水中，用2N氢氧化钠调节pH至6.8，混匀后加入2g SLS，混合均匀即得0.2％SLS/pH6.8介质。
分别称取5mg左右晶型Ⅳ、晶型Ⅴ、式X化合物(无定形)与晶型Ⅰ于1ml相应介质中，超声振摇使其充分分散，振摇2h，离心取上清液，稀释一定倍数，采用HPLC分析其中式X化合物的含量，计算溶解度。实验结果如表6所示：
表6
Figure PCTCN2021115856-appb-000011
从表6可知，晶型Ⅳ和晶型Ⅴ在0.2％SLS/pH6.8介质中的溶解性比在水、pH4.5和pH6.8介质中的溶解性好。晶型Ⅳ和晶型Ⅴ的溶解度分别与式X化合物(无定形)、晶型Ⅰ相比均有显著提高，改善了药物的溶解性，有利于提高生物利用度。
实施例7动物血浆药代动力学实验
1.动物处方胶囊制备
1)分别取一定量无定形、晶型Ⅳ和晶型Ⅴ，研磨5分钟后取少量于显微镜观察，确保粒径尽量相似；
2)定灌装量，分别称取5mg的无定形、晶型Ⅳ和晶型Ⅴ，灌装于对应的动物胶囊(成分：羧甲基淀粉钠)中，制备结果如下表。
2.动物血浆药代动力学实验
实验动物：Wistar大鼠，体重180-200g，雌性，动物数n＝4/晶型，从Beijing Vital River Laboratory Animal Co.LTD购买。于给药前一晚禁食，给药后4小时自由饮食。
给药及血样采集：通过灌胃口服给药(PO)，剂量为1粒/大鼠(5mg/大鼠)，于给药后0.083，0.25，0.5，1，2，4，6，8以及24小时采集9个时间点的血样。采集的时候手动控制好动物，通过大鼠眼底静脉丛采集大约150μL的血液到含K 2EDTA的管中。血液样本放在湿冰上，在15分钟内，通过离心(8000rpm，4分钟，4℃)，获得血浆样品，将血浆样品储存在-80℃直至分析。于实验当天配制好化合物。
分析方法：采用液相色谱-串联质谱法(型号：Triple Quad TM 4000)分析血浆样品，色谱柱为：Waters XBridge-C18(2.1×50mm,5μm)。
标准曲线的配制：配制待测化合物在空白Wistar大鼠血浆基质中线性范围为1.00-3000ng/mL的标准曲线，同时配制浓度为3，500，2400ng/mL的低中高浓度的质控样品。
生物样品的处理：将冻存的血浆样品放置冰上解冻，待样品解冻后，放置涡旋仪上涡旋5分钟。取20μL血浆样品、标准曲线及质控指控样品加至96孔板中，再加入200μL含内标***(品牌：NIFDC，批号:6TUC-T4C2，配制浓度2000ng/mL)的乙腈沉淀蛋白，涡旋混合5分钟，然后在3700rpm，4℃离心15分钟，提取上清同样条件下二次离心，最后进2μL上清溶液进行LC-MS/MS分析。大鼠的药代动力学参数数据如表7所示。
表7
Figure PCTCN2021115856-appb-000012
从表7可知，与无定形相比，晶型Ⅳ和晶型Ⅴ在半衰期和血浆暴露水平方面均有明显的提高。同等剂量下给药后，晶型Ⅳ给药组的血浆暴露水平AUC是无定形的1.5倍左右，晶型Ⅳ的半衰期T 1/2是无定形的1.3倍左右，晶型Ⅳ的血浆暴露水平AUC和半衰期T 1/2优于无定形。晶型Ⅴ给药组的血浆暴露水平AUC是无定形的2倍左右，晶型Ⅴ的半衰期T 1/2是无定形的3倍左右，晶型Ⅴ的血浆暴露水平AUC和半衰期T 1/2明显优于无定形。
实施例8Lantha screening激酶反应实验方法
化合物预先溶解在100％DMSO中。室温溶解10mM的药物储存液，经8vol％DMSO溶液梯度稀释至终浓度10-0.005μM。384孔板(Corning 3676)每孔中加入2.5μl的待测物溶液以及2.5μl经反应缓冲液稀释的激酶(Invitrogen PV3363)，再加入5μl的反应缓冲液稀释Fluososcei-PolyGT(Invitrogen PV3610)底物与ATP(Invitrogen PV3227)的混合物启动反应。其中空白孔(Blank)用反应缓冲液代替激酶，激酶孔(Enzyme)不加入任何药物。在25℃摇床避光反应60分钟后。加入10μl Detection Solution(Invitrogen PV3528与EDTA的混合液，用TR-FRET稀释缓冲液进行稀释，EDTA工作浓度为5mM，Lanthascreening Tb PY20 antibody工作浓度为0.2nM)，于室温摇床反应30分钟。在VictorX5荧光酶标仪(PerkinElmer)上读板，测定激发波长为340nm、发射波长为500nm和520nm的光吸收。
抑制率计算方法(参照Invitrogen，PV3363的说明书)如下：
1.Emission rate(ER):Coumarin Emission(520nm)/Fluorescein Emission(500nm)
2.抑制率(IR):(ER kinase-ER test compound)/(ER kinase-ER blank)×100％。用XLFIT 5.0软件(英国IDBS公司)拟合计算半数抑制浓度IC 50。结果如表8所示：
表8化合物对BTK WT的抑制活性
Figure PCTCN2021115856-appb-000013
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (27)

  1. 一种式X化合物的晶型Ⅳ，其特征在于，所述式X化合物具有以下结构：
    Figure PCTCN2021115856-appb-100001
    其中，所述晶型Ⅳ的使用Cu-Kα辐射检测的X射线粉末衍射图在以下衍射角2θ(°)值处具有衍射峰：5.90±0.2、14.91±0.2、17.51±0.2、24.90±0.2和26.37±0.2。
  2. 如权利要求1所述的晶型Ⅳ，其特征在于，所述晶型Ⅳ的X射线粉末衍射图在以下衍射角2θ(°)值处具有衍射峰：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和26.37±0.2。
  3. 如权利要求1所述的晶型Ⅳ，其特征在于，所述晶型Ⅳ的X射线粉末衍射图在以下衍射角2θ(°)值处具有衍射峰：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、16.05±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和35.37±0.2。
  4. 如权利要求1所述的晶型Ⅳ，其特征在于，所述晶型Ⅳ的X射线粉末衍射图基本如图1所表征。
  5. 如权利要求1所述的晶型Ⅳ，其特征在于，所述晶型Ⅳ的差示扫描量热曲线在167.89℃±3℃处具有吸热峰。
  6. 如权利要求1所述的晶型Ⅳ，其特征在于，所述晶型Ⅳ具有基本如图2所示的差示扫描量热曲线。
  7. 如权利要求1所述的晶型Ⅳ，其特征在于，所述晶型Ⅳ具有基本如图3所示的热重分析图谱。
  8. 如权利要求1所述的晶型Ⅳ，其特征在于，所述晶型Ⅳ具有基本如图4所示的DVS图谱。
  9. 一种权利要求1所述的式X化合物的晶型Ⅳ的制备方法，其特征在于，所述方法包括以下步骤：
    (S101)将式X化合物和溶剂的混合物加热至55℃～65℃；及
    (S102)将混合物冷却，分离析出的固体，得到所述晶型Ⅳ。
  10. 如权利要求9所述的制备方法，其特征在于，所述步骤(S101)中的溶剂选自水、乙醇、正丙醇和异丙醇中的一种或多种。
  11. 如权利要求9所述的制备方法，其特征在于，所述步骤(S102)中的混合物冷却至 20℃～25℃。
  12. 一种式X化合物的晶型Ⅴ，其特征在于，所述式X化合物具有以下结构：
    Figure PCTCN2021115856-appb-100002
    其中，所述晶型Ⅴ的使用Cu-Kα辐射检测的X射线粉末衍射图在以下衍射角2θ(°)值处具有衍射峰：7.33±0.2、9.91±0.2和17.53±0.2。
  13. 如权利要求12所述的晶型Ⅴ，其特征在于，所述晶型Ⅴ的X射线粉末衍射图在以下衍射角2θ(°)值处具有衍射峰：7.33±0.2、9.91±0.2、12.49±0.2、15.69±0.2、17.53±0.2、20.19±0.2和23.02±0.2。
  14. 如权利要求12所述的晶型Ⅴ，其特征在于，所述晶型Ⅴ的X射线粉末衍射图在以下衍射角2θ(°)值处具有衍射峰：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和30.21±0.2。
  15. 如权利要求12所述的晶型Ⅴ，其特征在于，所述晶型Ⅴ的X射线粉末衍射图基本如图5所表征。
  16. 如权利要求12所述的晶型Ⅴ，其特征在于，所述晶型Ⅴ的差示扫描量热曲线在197.31℃±3℃处具有吸热峰。
  17. 如权利要求12所述的晶型Ⅴ，其特征在于，所述晶型Ⅴ具有基本如图6所示的差示扫描量热曲线。
  18. 如权利要求12所述的晶型Ⅴ，其特征在于，所述晶型Ⅴ具有基本如图7所示的热重分析图谱。
  19. 如权利要求12所述的晶型Ⅴ，其特征在于，所述晶型Ⅴ具有基本如图8所示的DVS图谱。
  20. 一种权利要求12所述的式X化合物晶型Ⅴ的制备方法，其特征在于，所述方法包括以下步骤：
    (S201)将式X化合物和溶剂的混合物加热到70℃以上；及
    (S202)将混合物冷却，分离析出的固体，得到所述晶型Ⅴ。
  21. 如权利要求20所述的制备方法，其特征在于，所述步骤(S201)中的溶剂选自：水、C 2-4烷基醇、乙腈、甲苯、二甲苯、1,4-二氧六环、1，2-二氯乙烷和乙酸乙酯中的一种或多种。
  22. 如权利要求21所述的制备方法，其特征在于，所述C 2-4烷基醇为乙醇、正丙醇、异丙醇和乙二醇中的一种或多种。
  23. 如权利要求20所述的制备方法，其特征在于，所述步骤(S201)中的式X化合物的质量与溶剂体积的比值为50g/L～400g/L。
  24. 如权利要求20所述的制备方法，其特征在于，所述步骤(S201)中的式X化合物和溶剂的混合物加热到70℃-80℃；所述步骤(S202)中混合物冷却至20℃～25℃。
  25. 如权利要求20所述的制备方法，其特征在于，在所述步骤(S202)之前还包括以下步骤：将所述混合物冷却至40℃～65℃，保温1-12小时。
  26. 权利要求1-8中任一项所述的晶型Ⅳ、或权利要求9-11任一项所述的制备方法制备得到的晶型Ⅳ在制备治疗和/或预防肿瘤、癌症、增殖性疾病、***反应性疾病、自身免疫性疾病或炎症性疾病的药物中的用途。
  27. 权利要求12-19中任一项所述的晶型Ⅴ、或权利要求20-25任一项所述的制备方法制备得到的晶型Ⅴ在制备治疗和/或预防肿瘤、癌症、增殖性疾病、***反应性疾病、自身免疫性疾病或炎症性疾病的药物中的用途。
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CN102753548A (zh) * 2009-12-23 2012-10-24 武田药品工业株式会社 作为syk抑制剂的稠合的杂芳族吡咯烷酮
WO2017128917A1 (zh) * 2016-01-29 2017-08-03 北京诺诚健华医药科技有限公司 吡唑稠环类衍生物、其制备方法及其在治疗癌症，炎症和免疫性疾病上的应用
WO2019062329A1 (zh) * 2017-09-28 2019-04-04 上海海雁医药科技有限公司 4,6,7-三取代1,2-二氢吡咯并[3,4-c]吡啶/嘧啶-3-酮衍生物及用途
WO2020187267A1 (zh) * 2019-03-18 2020-09-24 上海海雁医药科技有限公司 Btk抑制剂及其药学上可接受的盐和多晶型物及其应用

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Publication number Priority date Publication date Assignee Title
CN102753548A (zh) * 2009-12-23 2012-10-24 武田药品工业株式会社 作为syk抑制剂的稠合的杂芳族吡咯烷酮
WO2017128917A1 (zh) * 2016-01-29 2017-08-03 北京诺诚健华医药科技有限公司 吡唑稠环类衍生物、其制备方法及其在治疗癌症，炎症和免疫性疾病上的应用
WO2019062329A1 (zh) * 2017-09-28 2019-04-04 上海海雁医药科技有限公司 4,6,7-三取代1,2-二氢吡咯并[3,4-c]吡啶/嘧啶-3-酮衍生物及用途
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