WO2022218276A1 - 含氟大环结构化合物的固体形态、制备方法和应用 - Google Patents

含氟大环结构化合物的固体形态、制备方法和应用 Download PDF

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WO2022218276A1
WO2022218276A1 PCT/CN2022/086223 CN2022086223W WO2022218276A1 WO 2022218276 A1 WO2022218276 A1 WO 2022218276A1 CN 2022086223 W CN2022086223 W CN 2022086223W WO 2022218276 A1 WO2022218276 A1 WO 2022218276A1
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compound
cancer
solvate
ray powder
diffraction pattern
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PCT/CN2022/086223
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English (en)
French (fr)
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黄浩喜
刘伟
张善军
刘川
王瑞琼
廖健宇
陈垌珲
苏忠海
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成都倍特药业股份有限公司
赛诺哈勃药业（成都）有限公司
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Priority to CN202280018582.9A priority Critical patent/CN117412970A/zh
Publication of WO2022218276A1 publication Critical patent/WO2022218276A1/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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • 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
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems

Definitions

  • the present application belongs to the technical field of drug development, and specifically relates to a polymorph or solvate of compound (I) with antitumor activity, a preparation method and application thereof.
  • the NTRK gene contains NTRK1, NTRK2 and NTRK3, which are responsible for the synthesis of the tropomyosin receptor kinase (TRK) family proteins TRKA, TRKB and TRKC, respectively.
  • Neurotrophic factors can induce receptor dimerization, phosphorylation and activation of downstream signaling cascades of PI3K, RAS/MAPK/ERK and PLC- ⁇ after binding to TRK protein.
  • TRK signaling pathway including gene fusions, protein overexpression, or single nucleotide changes, have been found to be the pathogenic cause of many tumors, especially NTRK gene fusion, which is currently the most well-defined oncogenic cause. Acting as an oncogenic driver, promoting cancer cell growth and survival, this discovery has led to the emergence of NTRK gene fusions as new targets for cancer therapy.
  • the first-generation drug LOXO-101 which can effectively treat 17 types of tumors, was launched in the United States for patients with NTRK1/NTRK2/NTRK3 fusions.
  • some cancer patients will develop resistance to the first-generation drugs.
  • Drug resistance mutations in TRK kinases are one of the main causes of drug resistance, so more effective drugs that can overcome first-generation drug resistance need to be developed.
  • the small molecule inhibitor has better biological activity, especially in vitro efficacy of mutations at NTRK1G595R, NTRK3G623R alleles, stability in human, rat and mouse liver microsomes, drug metabolism and oral bioavailability. High selectivity and security.
  • the present application studies the polymorphic forms and solvates of compound (I), these crystalline forms can improve the compound's crystal form stability, impurity removal ability, thermodynamic stability (larger onset value), and relatively better druggability, And from the perspective of preparation technology, the preparation is simpler, easy to control, and convenient for industrial production.
  • the polymorphic form of Compound (I) described in the present application is Form C, characterized in that its X-ray powder diffraction pattern includes the following peaks: 6.878 ⁇ 0.2°, 10.918 ⁇ 0.2°, 13.921 ⁇ 0.2°, 15.321 ⁇ 0.2°, 18.401 ⁇ 0.2°, 19.860 ⁇ 0.2°, 24.321 ⁇ 0.2°.
  • the X-ray powder diffraction pattern of the crystal form C of compound (I) further comprises the following peaks: 12.358 ⁇ 0.2°, 16.302 ⁇ 0.2°, 17.320 ⁇ 0.2°, 17.698 ⁇ 0.2°, 19.221 ⁇ 0.2°, 20.779 ⁇ 0.2°, 21.518 ⁇ 0.2°, 22.441 ⁇ 0.2°, 25.340 ⁇ 0.2°, 26.141 ⁇ 0.2°, 26.920 ⁇ 0.2°, 27.820 ⁇ 0.2°.
  • the X-ray powder diffraction pattern of Form C of Compound (I) is substantially as shown in FIG. 3 .
  • the crystalline form C of the compound (I) is an anhydrous form.
  • the DSC pattern of Form C of Compound (I) includes a characteristic peak at 377 ⁇ 2°C (onset).
  • the DSC spectrum of the DSC spectrum of Form C of Compound (I) includes characteristic peaks at substantially the same temperature as shown in FIG. 4 .
  • the DSC spectrum of the DSC spectrum of Form C of Compound (I) is substantially as shown in FIG. 4 .
  • the TGA profile of Form C of Compound (I) is substantially as shown in FIG. 4 .
  • the polymorphic form of Compound (I) described in the present application is Form D, characterized in that its X-ray powder diffraction pattern includes the following peaks: 9.400 ⁇ 0.2°, 10.510 ⁇ 0.2°, 12.957 ⁇ 0.2°, 13.300 ⁇ 0.2°, 17.875 ⁇ 0.2°, 18.840 ⁇ 0.2°, 20.780 ⁇ 0.2°, 22.940 ⁇ 0.2°.
  • the X-ray powder diffraction pattern of the crystal form D of compound (I) further comprises the following peaks: 10.084 ⁇ 0.2°, 16.984 ⁇ 0.2°, 19.240 ⁇ 0.2°, 21.361 ⁇ 0.2°, 22.324 ⁇ 0.2°, 24.761 ⁇ 0.2°, 29.820 ⁇ 0.2°, 30.220 ⁇ 0.2°, 33.098 ⁇ 0.2°.
  • the X-ray powder diffraction pattern of Form D of Compound (I) is substantially as shown in FIG. 5 .
  • the crystalline form D of the compound (I) is an anhydrous form.
  • the DSC pattern of Form D of Compound (I) includes a characteristic peak at 375 ⁇ 2°C (onset).
  • the DSC spectrum of the DSC spectrum of Form D of Compound (I) includes characteristic peaks at substantially the same temperature as shown in FIG. 6 .
  • the DSC spectrum of the DSC spectrum of Form D of Compound (I) is substantially as shown in FIG. 6 .
  • the TGA profile of Form D of Compound (I) is substantially as shown in FIG. 6 .
  • the polymorphic form of Compound (I) described in the present application is Form E, characterized in that its X-ray powder diffraction pattern includes the following peaks: 6.539 ⁇ 0.2°, 10.797 ⁇ 0.2°, 13.596 ⁇ 0.2°, 15.580 ⁇ 0.2°, 17.741 ⁇ 0.2°, 19.741 ⁇ 0.2°, 24.619 ⁇ 0.2°, 26.380 ⁇ 0.2°.
  • the X-ray powder diffraction pattern of the crystalline form E of compound (I) further comprises the following peaks: 10.041 ⁇ 0.2°, 13.022 ⁇ 0.2°, 16.101 ⁇ 0.2°, 16.959 ⁇ 0.2°, 18.079 ⁇ 0.2°, 18.980 ⁇ 0.2°, 20.119 ⁇ 0.2°, 21.680 ⁇ 0.2°, 23.080 ⁇ 0.2°, 25.340 ⁇ 0.2°, 26.000 ⁇ 0.2°, 28.279 ⁇ 0.2°.
  • the X-ray powder diffraction pattern of Form E of Compound (I) is substantially as shown in FIG. 7 .
  • the crystalline form E of the compound (I) is an anhydrous crystalline form.
  • the DSC pattern of Form E of Compound (I) includes a characteristic peak at 374 ⁇ 2°C (onset). In certain embodiments, the DSC pattern of the DSC pattern of Form E of Compound (I) includes characteristic peaks at substantially the same temperature as shown in FIG. 8 . In certain embodiments, the DSC spectrum of the DSC spectrum of Form E of Compound (I) is substantially as shown in FIG. 8 . In certain embodiments, the TGA profile of Form E of Compound (I) is substantially as shown in FIG. 8 .
  • the polymorphic form of Compound (I) described in the present application is Form L, characterized in that its X-ray powder diffraction pattern includes the following peaks: 5.898 ⁇ 0.2°, 9.017 ⁇ 0.2°, 11.502 ⁇ 0.2°, 13.602 ⁇ 0.2°, 15.417 ⁇ 0.2°, 16.897 ⁇ 0.2°, 19.161 ⁇ 0.2°, 21.389° ⁇ 0.2°.
  • the X-ray powder diffraction pattern of the crystal form L of compound (I) further comprises the following peaks: 16.538 ⁇ 0.2°, 18.151 ⁇ 0.2°, 19.938 ⁇ 0.2°, 20.640 ⁇ 0.2° , 22.583 ⁇ 0.2°, 23.203 ⁇ 0.2°, 23.840 ⁇ 0.2°, 24.959 ⁇ 0.2°, 27.446 ⁇ 0.2°, 29.792 ⁇ 0.2°, 30.100 ⁇ 0.2°, 30.863 ⁇ 0.2°.
  • the X-ray powder diffraction pattern of Form L of Compound (I) is substantially as shown in FIG. 9 .
  • the crystalline form L of the compound (I) is an anhydrous form.
  • the DSC pattern of the crystalline form L of Compound (I) includes a characteristic peak at 373 ⁇ 2°C (onset).
  • the DSC spectrum of the DSC spectrum of Form L of Compound (I) includes characteristic peaks at substantially the same temperature as shown in FIG. 10 .
  • the DSC spectrum of the DSC spectrum of Form L of Compound (I) is substantially as shown in FIG. 10 .
  • the TGA profile of Form L of Compound (I) is substantially as shown in FIG. 10 .
  • the polymorphic form of Compound (I) described in the present application is Form N, characterized in that its X-ray powder diffraction pattern includes the following peaks: 5.841 ⁇ 0.2°, 8.901 ⁇ 0.2°, 11.040 ⁇ 0.2°, 13.442 ⁇ 0.2°, 16.200 ⁇ 0.2°, 18.899 ⁇ 0.2°, 21.119 ⁇ 0.2°, 23.501 ⁇ 0.2°.
  • the X-ray powder diffraction pattern of the crystalline form N of compound (I) further comprises the following peaks: 7.296 ⁇ 0.2°, 9.801 ⁇ 0.2°, 12.058 ⁇ 0.2°, 17.438 ⁇ 0.2°, 20.037 ⁇ 0.2°, 20.439 ⁇ 0.2°, 22.660 ⁇ 0.2°, 25.122 ⁇ 0.2°, 27.261 ⁇ 0.2°, 28.602 ⁇ 0.2°, 29.019 ⁇ 0.2°, 29.560 ⁇ 0.2°.
  • the X-ray powder diffraction pattern of Form N of Compound (I) is substantially as shown in FIG. 11 .
  • the crystalline form N of the compound (I) is an anhydrous crystalline form.
  • the DSC pattern of the crystalline form N of compound (I) includes a characteristic peak at 370 ⁇ 2°C (onset).
  • the DSC spectrum of the DSC spectrum of Form N of Compound (I) includes characteristic peaks at substantially the same temperature as shown in FIG. 12 .
  • the DSC spectrum of the DSC spectrum of Form N of Compound (I) is substantially as shown in FIG. 12 .
  • the TGA profile of Form N of Compound (I) is substantially as shown in FIG. 12 .
  • the polymorphic form of Compound (I) described in the present application is Form A, characterized in that its X-ray powder diffraction pattern includes the following peaks: 7.383 ⁇ 0.2°, 9.762 ⁇ 0.2°, 15.157 ⁇ 0.2°, 17.660 ⁇ 0.2°, 21.002 ⁇ 0.2°, 22.539 ⁇ 0.2°, 26.300 ⁇ 0.2°; in certain embodiments, the X-ray powder diffraction pattern of the crystal form A of the compound (I) Also included are the following peaks: 10.938 ⁇ 0.2°, 16.564 ⁇ 0.2°, 16.863 ⁇ 0.2°, 19.142 ⁇ 0.2°, 19.657 ⁇ 0.2°, 19.861 ⁇ 0.2°, 23.201 ⁇ 0.2°, 23.737 ⁇ 0.2°, 24.299 ⁇ 0.2°, 28.359 ⁇ 0.2°.
  • the X-ray powder diffraction pattern of Form A of Compound (I) is substantially as shown in FIG. 1 .
  • the crystalline form A of the compound (I) is a hydrate of the compound (I), wherein the molar ratio of the compound (I) to water is 1:2.
  • the DSC pattern of Form A of Compound (I) includes characteristic peaks at 66 ⁇ 2°C (onset).
  • the DSC pattern of Form A of Compound (I) includes a characteristic peak at 370 ⁇ 2°C (onset).
  • the DSC spectrum of the DSC spectrum of Form A of Compound (I) includes characteristic peaks at substantially the same temperature as shown in FIG. 2 .
  • the DSC spectrum of the DSC spectrum of Form A of Compound (I) is substantially as shown in FIG. 2 .
  • the TGA profile of Form A of Compound (I) is substantially as shown in FIG. 2 .
  • the present application also provides a method for preparing the crystal form C of compound (I), comprising the following steps:
  • the organic solvent is: C 1 -C 6 alkyl alcohol, preferably ethanol and isopropanol.
  • the method for preparing the crystal form C of compound (I) comprises dispersing the crystal form A of compound (I) in an organic solvent and beating at a temperature of 25°C to reflux;
  • the method for preparing the crystal form C of compound (I) comprises dispersing the crystal form A of compound (I) in an organic solvent and beating at 25°C to reflux temperature for 3 to 24 hours.
  • the drying is vacuum drying.
  • the method of preparing Form C of Compound (I) comprises drying for 5-7 hours, eg, 6 hours.
  • the method for preparing crystal form C of compound (I) comprises the following steps: dispersing crystal form A in an organic solvent, beating at a certain temperature for a certain period of time, filtering, and vacuum drying for a certain period of time;
  • the organic solvent is: C1-C6 alkyl alcohol, preferably ethanol and isopropanol; wherein the temperature is 25°C to reflux; wherein the beating time is 3-24 hours; the drying time is 6 hours.
  • the present application also provides a method for preparing the crystal form D of compound (I), comprising the following steps:
  • the organic solvent is: dimethyl carbonate, methyl acetate, butyl acetate, butanone, methyl isobutyl A ketone, preferably dimethyl carbonate.
  • the method for preparing the crystal form D of compound (I) comprises dispersing the crystal form A of compound (I) in an organic solvent and beating at room temperature.
  • the method for preparing the crystal form D of compound (I) comprises dispersing the crystal form A of compound (I) in an organic solvent and beating at room temperature for 3-24 hours, preferably 24 hours.
  • the drying is vacuum drying.
  • the method of preparing Form D of Compound (I) comprises drying for 5-7 hours, eg, 6 hours.
  • the method for preparing the crystal form D of compound (I) comprises the following steps: dispersing the crystal form A in an organic solvent, beating at room temperature for a certain period of time, filtering, and vacuum drying for a certain period of time;
  • the organic solvent is: methyl acetate, butyl acetate, methyl ethyl ketone, methyl isobutyl ketone; wherein the beating time is 3-24 hours; the beating time is preferably 24 hours; the drying time is 6 hours.
  • compositions comprising a therapeutically effective amount of a polymorph or solvate of Compound (I) and a pharmaceutically acceptable carrier therefor.
  • the disease is selected from cancer.
  • the cancer is selected from neurocytoma, ovarian cancer, colorectal cancer, melanoma, cancer of the head and neck, gastric cancer, lung cancer, breast cancer, glioblastoma, neuroblastoma
  • duct cell tumor secretory breast cancer, salivary gland cancer, papillary thyroid cancer, adult myeloid leukemia, pancreatic cancer, prostate cancer, appendix cancer, cholangiocarcinoma, gastrointestinal stromal tumor, and infantile fibrosarcoma .
  • TRK-mediated disease is one or more of pain, cancer, inflammation, and neurodegenerative disease.
  • the disease is cancer.
  • the cancer is selected from neurocytoma, ovarian cancer, colorectal cancer, melanoma, cancer of the head and neck, gastric cancer, lung cancer, breast cancer, glioblastoma, neuroblastoma
  • gastric cancer lung cancer
  • breast cancer glioblastoma
  • neuroblastoma One or more of duct cell tumor, secretory breast cancer, salivary gland cancer, papillary thyroid cancer, adult myeloid leukemia, pancreatic cancer, prostate cancer, appendix cancer, cholangiocarcinoma, gastrointestinal stromal tumor, and infantile fibrosarcoma .
  • the disease is selected from cancer.
  • the cancer is selected from neurocytoma, ovarian cancer, colorectal cancer, melanoma, cancer of the head and neck, gastric cancer, lung cancer, breast cancer, glioblastoma, neuroblastoma
  • One or more of duct cell tumor, secretory breast cancer, salivary gland cancer, papillary thyroid cancer, adult myeloid leukemia, pancreatic cancer, prostate cancer, appendix cancer, cholangiocarcinoma, gastrointestinal stromal tumor, and infantile fibrosarcoma are examples of duct cell tumor, secretory breast cancer, salivary gland cancer, papillary thyroid cancer, adult myeloid leukemia, pancreatic cancer, prostate cancer, appendix cancer, cholangiocarcinoma, gastrointestinal stromal tumor, and infantile fibrosarcoma .
  • TRK-mediated disease is one or more of pain, cancer, inflammation, and neurodegenerative disease.
  • the disease is cancer.
  • the cancer is selected from neurocytoma, ovarian cancer, colorectal cancer, melanoma, cancer of the head and neck, gastric cancer, lung cancer, breast cancer, glioblastoma, neuroblastoma
  • gastric cancer lung cancer
  • breast cancer glioblastoma
  • neuroblastoma One or more of duct cell tumor, secretory breast cancer, salivary gland cancer, papillary thyroid cancer, adult myeloid leukemia, pancreatic cancer, prostate cancer, appendix cancer, cholangiocarcinoma, gastrointestinal stromal tumor, and infantile fibrosarcoma .
  • Another aspect of the application provides a method for treating pain, cancer, inflammation or neurodegenerative disease, comprising administering to a subject in need thereof a therapeutically effective amount of compound (I) described herein polymorphs or solvates or pharmaceutical compositions.
  • the disease is selected from cancer.
  • the cancer is selected from neurocytoma, ovarian cancer, colorectal cancer, melanoma, cancer of the head and neck, gastric cancer, lung cancer, breast cancer, glioblastoma, neuroblastoma
  • gastric cancer lung cancer
  • breast cancer glioblastoma
  • neuroblastoma One or more of duct cell tumor, secretory breast cancer, salivary gland cancer, papillary thyroid cancer, adult myeloid leukemia, pancreatic cancer, prostate cancer, appendix cancer, cholangiocarcinoma, gastrointestinal stromal tumor, and infantile fibrosarcoma .
  • TRK-mediated disease is one or more of pain, cancer, inflammation, and neurodegenerative disease.
  • the disease is cancer.
  • the cancer is selected from neurocytoma, ovarian cancer, colorectal cancer, melanoma, cancer of the head and neck, gastric cancer, lung cancer, breast cancer, glioblastoma, neuroblastoma
  • gastric cancer lung cancer
  • breast cancer glioblastoma
  • neuroblastoma One or more of duct cell tumor, secretory breast cancer, salivary gland cancer, papillary thyroid cancer, adult myeloid leukemia, pancreatic cancer, prostate cancer, appendix cancer, cholangiocarcinoma, gastrointestinal stromal tumor, and infantile fibrosarcoma .
  • Figure 1 shows the XRPD pattern of the compound of formula (I) Form A
  • Figure 3 shows the XRPD pattern of the anhydrous form C of the compound of formula (I);
  • FIG. 4 Shows the DSC/TGA spectrum of anhydrous Form C of the compound of formula (I).
  • Figure 5 shows the XRPD pattern of the anhydrous form D of the compound of formula (I);
  • FIG. Shows the DSC/TGA spectrum of anhydrous Form D of the compound of formula (I).
  • Figure 7 shows the XRPD pattern of the anhydrous form E of the compound of formula (I);
  • Figure 8 Shows the DSC/TGA spectrum of the anhydrous Form E of the compound of formula (I).
  • Figure 9 shows the XRPD pattern of the anhydrous form L of the compound of formula (I);
  • FIG. 10 Shows the DSC/TGA spectrum of the anhydrous form L of the compound of formula (I).
  • Figure 11 shows the XRPD pattern of the anhydrous form N of the compound of formula (I);
  • FIG. 12 Shows the DSC/TGA spectrum of the anhydrous form N of the compound of formula (I).
  • the term "pharmaceutical composition” includes a product comprising a therapeutically effective amount of a compound of the present application, as well as any product that results, directly or indirectly, from a combination of polymorphs, solvates, and solvates of a compound of the present application.
  • the pharmaceutical composition can be administered, for example, orally or parenterally.
  • the pharmaceutical composition of the present application can be prepared into various dosage forms by conventional methods in the art, including but not limited to tablets, capsules, solutions, suspensions, granules or injections, etc., for example, oral or parenteral administration.
  • the term "effective amount” refers to an amount sufficient to achieve the desired therapeutic effect, eg, an amount to achieve relief of symptoms associated with the disease to be treated.
  • treatment is intended to alleviate or eliminate the disease state or disorder targeted. If a subject has received a therapeutically effective amount of a compound according to the methods described herein, its tautomer, meso, racemate, enantiomer, diastereomer or mixture thereof , or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, the subject exhibits an observable and/or detectable reduction or improvement in one or more signs and symptoms, the subject is successfully "treated”. It should also be understood that the treatment of the disease state or disorder includes not only complete treatment, but also incomplete treatment, but the achievement of some biologically or medically relevant result.
  • the dosage and method of use of the compounds of the present application depend on many factors, including the patient's age, weight, sex, natural health, nutritional status, the active strength of the compound, the time of administration, the rate of metabolism, the severity of the condition and Subjective judgment of the treating physician.
  • the preferred dosage used is between 0.001 and 1000 mg/kg body weight/day.
  • pharmaceutically acceptable means suitable within the scope of sound medical judgment to be in contact with human and animal tissues without undue toxicity, irritation, allergic reactions or other problematic complications, with reasonable benefit/ those compounds, materials, compositions and/or dosage forms commensurate with their hazard ratios.
  • a "polymorph” or “polymorph” refers to a crystalline form having the same chemical composition, but a different spatial arrangement of the molecules, atoms and/or ions that make up the crystal.
  • polymorphs have the same chemical composition, they differ in packing and geometric arrangement and may exhibit different physical properties such as melting point, shape, color, density, hardness, deformability, stability, solubility, dissolution rate and similar properties.
  • the two polymorphs can be mono- or tautomeric. For a single-denatured system, the relative stability between the two solid phases remains unchanged when the temperature is changed.
  • subject refers to mammalian subjects, including but not limited to humans, cats, dogs, horses, cows, sheep, monkeys, etc., wherein the human subjects include male and female subjects , and includes neonatal, infant, juvenile, adolescent, adult and geriatric subjects.
  • the X-ray powder diffraction pattern (XRPD pattern) is the X-ray powder diffraction pattern expressed by Cu-K ⁇ radiation at 2 ⁇ angle.
  • two X-ray diffraction patterns or two DSC patterns are substantially identical.
  • two X-ray diffraction patterns are considered to be substantially the same when their characteristic peaks do not vary by more than ⁇ 0.3°, ⁇ 0.2°, or ⁇ 0.1° in their 2 theta angle.
  • the crystalline structures of the present application can be prepared by various methods, including crystallization or recrystallization from a suitable solvent, sublimation, growth from a melt, solid state transformation from another phase, crystallization from a supercritical fluid, and jet spraying, among others.
  • Techniques for crystallization or recrystallization of crystalline structures from solvent mixtures including solvent evaporation, lowering the temperature of the solvent mixture, seeding of supersaturated solvent mixtures of the molecule and/or salt, lyophilization of solvent mixtures, addition of antisolvents to solvent mixtures Wait.
  • Crystalline structures, including polymorphs can be prepared using high-throughput crystallization techniques.
  • Drug crystals including polymorphs, methods of preparation and characterization of drug crystals are disclosed in Solid-State Chemistry of Drugs, S.R. Byrn, R.R. Pfeiffer, and J.G. Stowell, 2nd Edition, SSCI, West Lafayette, Indiana, 1999.
  • Crystal structures equivalent to the crystal structures disclosed or claimed herein may exhibit similar, but not identical, analytical properties within reasonable error, depending on experimental conditions, purity, equipment, and other common variables known to those skilled in the art. Accordingly, it will be apparent to those skilled in the art that various modifications and variations can be made within the present invention without departing from the scope and spirit of the inventions. Other embodiments of the invention will be apparent to those skilled in the art upon consideration of the specification and practice of the invention disclosed herein. Applicants expect this specification and examples to be regarded as illustrative rather than limiting in scope.
  • room temperature or "RT” as used in this application refers to an ambient temperature of 20 to 25°C (68-77°F).
  • the term "pharmaceutically acceptable carrier” refers to a diluent, adjunct, or vehicle with which a therapeutic agent is administered and which, within the scope of sound medical judgment, is suitable for contact with human and/or other animal tissues without undue toxicity, irritation, allergic reactions or other problems or complications commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of the present application include, but are not limited to, sterile liquids such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil , sesame oil, etc. Water is an exemplary carrier when the pharmaceutical composition is administered intravenously.
  • sterile liquids such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil , sesame oil, etc.
  • Water is an exemplary carrier when the pharmaceutical composition is administered intravenously.
  • Physiological saline and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Starch glucose, lactose, sucrose, gelatin, maltose, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene glycol, water, ethanol, etc. can also be used as carriers .
  • the pharmaceutical composition may also contain a small amount of wetting agents, emulsifiers, pH buffering agents, preservatives, antioxidants, flavoring agents, fragrances, cosolvents, solubilizers, osmotic pressure regulators, colorants, etc.
  • Oral formulations may contain standard carriers such as binders, fillers, disintegrants, lubricants and the like.
  • compositions described in this application can be administered by methods known in the art, such as, but not limited to, any of the following: oral, inhalation spray, rectal, nasal, buccal, topical, parenteral Administration, such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal and intracranial injection or infusion, or via an explanted reservoir. Among them, oral, intramuscular or intravenous injection is preferred. For these routes of administration, the pharmaceutical compositions of the present application can be administered in suitable dosage forms.
  • the dosage form can be solid, semi-solid, liquid or gaseous, including but not limited to tablets, capsules, powders, granules, lozenges, hard candies, powders, sprays, creams, ointments , suppositories, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, suspensions, elixirs, syrups.
  • compositions of the present invention can be prepared by any method well known in the art, such as by mixing, dissolving, granulating, sugar coating, milling, emulsifying, lyophilizing, and the like.
  • X-ray powder diffraction (XRPD) analysis adopts Dandong Haoyuan DX-2700BH and Cu target irradiation.
  • the detection range is 3° to 40°
  • the step size is 0.02°
  • the speed is 0.2s.step -1 .
  • the simultaneous thermal analyzer (TGA/DSC) analysis instrument in the present invention is Mettler-Toledo TGA/DSC 3 + .
  • the heating rate of the apparatus was 10 K/min.
  • Step 2 Preparation of ethyl 5,7-dichloro-6-fluoropyrazolo[1,5-a]pyrimidine-3-carboxylate
  • the system was concentrated to remove phosphorus oxychloride, and then poured into saturated sodium bicarbonate solution (100 mL) to keep the solution basic, extracted with ethyl acetate three times, dried over anhydrous sodium sulfate, filtered, and concentrated.
  • the obtained crude product was filtered through the column layer. After purification, the obtained solid was washed with petroleum ether and dried to obtain 1.7 g of the title compound.
  • Step 3 Preparation of ethyl 5-chloro-6-fluoropyrazolo[1,5-a]pyrimidine-3-carboxylate
  • Step 1 Preparation of (R)-N-(((5-fluoro-2-methoxypyridin-3-yl)methylene)-2-methylpropane-2-sulfinamide
  • Step 2 Preparation of (R)-N-((R)-1-(5-fluoro-2-methoxypyridin-3-yl)ethyl)-2-methylpropane-2-sulfinamide
  • Step 1 (R)-6-Fluoro-5-((1-(5-Fluoro-2-methoxypyridin-3-yl)ethyl)amino)pyrazolo[1,5-a]pyrimidine- Preparation of ethyl 3-carboxylate
  • Step 2 (R)-6-Fluoro-5-(((1-(5-fluoro-2-methoxypyridin-3-yl)ethyl)amino)pyrazolo[1,5-a]pyrimidine - Preparation of 3-carboxylic acid
  • Step 3 (1R,3r)-3-(6-Fluoro-5-(((R)-1-(5-fluoro-2-methoxypyridin-3-yl)ethyl)amino)pyrazolo
  • Step 4 (1R,3r)-3-(6-Fluoro-5-(((R)-1-(5-fluoro-2-hydroxypyridin-3-yl)ethyl)amino)pyrazolo[1 Preparation of ,5-a]pyrimidine-3-carboxamido)-4-methylbenzenesulfonate cyclobutyl
  • Step 5 ( 31S ,33S, 63E ,64E,8R) -15,66 - difluoro- 8 -methyl-2-oxa-4,7-diaza- 6(3,5)-Pyrazolo[1,5-a]pyrimidine-1(2,3)-pyridine 3-3(1,3)-cyclobutaneocta-5-one (Compound (I) ) preparation
  • Test Examples 1-6 of this application include:
  • LOXO-195 a second-generation TRK inhibitor, has the following chemical structure:
  • Positive control 1 is the compound of Example 5 of patent WO2019210835, and its chemical structure is:
  • Positive control 2 is the compound of Example 6 of patent WO2019210835, and its chemical structure is:
  • LOXO-195 and control compounds 1-2 can be obtained commercially or synthesized according to conventional routes.
  • LOXO-195 can be obtained commercially, and positive controls 1 and 2 can be synthesized according to the method described in WO2019210835A1.
  • Compounds 1b, 2 and 4-6 can be synthesized and prepared according to conventional routes, and can also be synthesized with reference to the methods described in WO2021115401A1.
  • TRKa The inhibitory activities of the compounds of the present application on three kinases TRKa, TRKA (G595R) and TRKC (G623R) were determined.
  • Test compounds were accurately weighed and dissolved in 100% DMSO to make up a 10 mM solution.
  • Inhibition % (Inhibition) (Conversion % Maximum - Conversion % Sample) / (Conversion % Maximum - Conversion % Minimum) ⁇ 100
  • conversion rate % sample is the conversion rate reading of the sample
  • conversion rate % minimum the average value of negative control wells, representing the conversion rate readings of wells without enzymatic activity
  • conversion rate % maximum the average value of the ratio of positive control wells, representing the wells without compound inhibition Conversion rate readings.
  • Fitting the dose-response curve take the log value of the concentration as the X-axis, and the percentage inhibition rate on the Y-axis, using the analysis software GraphPad Prism 5 log (inhibitor) vs. response-variable slope (Variable slope) to fit the dose-response curve (Four-parameter model fitting) to derive the IC50 value of each compound for enzymatic activity.
  • the inhibitory effect of the compounds of the present application on the growth of three NTRK mutant cells (Ba/F3 LMNA-NTRK1-G667C, Ba/F3 EVT6-NTRK3-G623R, Ba/F3 LMNA-NTRK1-G595R) was determined.
  • Fetal bovine serum FBS (GBICO, Cat#10099-141); Luminescent Cell Viability Assay (CTG, Promega, Cat#G7573); 96-well clear flat bottom black wall plate ( Cat#165305); RPMI-1640 (Hyclone, Cat#SH30809.01)
  • the compounds to be tested were prepared with a 10-fold drug solution in a medium containing 1% DMSO, and the highest concentration was 10 ⁇ M, and were sequentially diluted 3-fold to obtain a total of 9 concentrations of drug solutions.
  • the final concentration of DMSO in the wells was 0.1%, and three replicate wells were set for each concentration of the drug.
  • Cells in medicated 96-well plates were incubated at 37° C., 5% CO 2 , and 95% humidity for an additional 72 hours before CTG analysis was performed.
  • Cell survival rate (%) (luminescence value of drug well to be tested - luminescence value of culture solution control well)/(luminescence value of cell control well - luminescence value of culture solution control well) ⁇ 100%.
  • the stability of the compounds of the present application was determined in human, rat and mouse liver microsomes.
  • Step 3.2 To each incubation system described in the aforementioned “Step 3.2", start the reaction by adding 2.5 ⁇ L of the positive control compound or the test compound solution of the present application (100 ⁇ M), the positive control is verapamil (purchased from Sigma). ), so that the final concentration of the test compound of the present application or the positive control compound is 1 ⁇ M. Incubation solutions after compound addition were batch-incubated in water at 37°C.
  • Peak areas were determined from extracted ion chromatograms.
  • the slope value k was determined by linear regression of the residual percentage of parent drug versus the natural logarithm of the incubation time curve.
  • the in vitro half-life (t 1/2 ) was calculated and determined according to the slope value, and was converted into the in vitro intrinsic clearance (CLint, expressed in ⁇ L/min/mg protein) by the mean value of the in vitro half-life.
  • the stability of compounds 1b and 2 in human, rat and mouse liver microsomes was not significantly different from that of the corresponding positive compounds, or the stability in liver microsomes of most species became worse, but the compounds of the present application (I ) in human, rat and mouse liver microsomes were significantly more stable than the positive control 2.
  • Test Example 4 In vivo pharmacokinetic studies of test compounds administered intravenously and orally to SD rats
  • Species SD rat, SPF grade.
  • Source Animals were transferred from the experimental institution animal reserve (999M-017), Shanghai Sipple-Bikai Laboratory Animal Co., Ltd. Quantity: 3 for each dosage form.
  • test product 2.1 Accurately weigh an appropriate amount of the test product, add the final volume of 5% DMSO, 10% polyethylene glycol-15 hydroxystearate, 85% normal saline, vortex or sonicate to fully mix to obtain 0.2 mg/mL dosing solution for intravenous administration.
  • test product 2.2 Accurately weigh an appropriate amount of the test product, add the final volume of 5% DMSO, 10% polyethylene glycol-15 hydroxystearate, 85% normal saline, vortex or sonicate to fully mix to obtain 0.5 mg/mL dosing solution for oral gavage administration.
  • Blood is collected through the jugular vein or other suitable methods, each sample is collected about 0.20mL, heparin sodium is anticoagulated, the blood samples are placed on ice after collection, and centrifuged within 2 hours to separate the plasma (centrifugation conditions: centrifugal force 6800g, 6 minutes, 2 -8°C).
  • the collected plasma samples were stored in a -80°C refrigerator before analysis, and the remaining plasma samples were kept in a -80°C refrigerator for temporary storage after analysis.
  • the BLQ was recorded as 0.
  • concentration before administration is calculated as 0; the BLQ before Cmax (including "No peak") is calculated as 0; the BLQ (including "No peak") after Cmax is not involved in the calculation.
  • WinNonlin to calculate the pharmacokinetic parameters, such as AUC(0-t), T 1/2 , Cmax, etc., through the plasma concentration data at different time points. The results are shown in Table A4.
  • Test Example 5 Bidirectional Permeability Study of Test Compounds on MDCK-MDR1 Cell Line
  • MDCK-MDR1 cells were purchased from the Netherlands Cancer Institute and cells between passages 10 and 20 were used.
  • the cell suspension was added to the chamber of a 96-well Transwell plate at 50 ⁇ L per well.
  • the incubator was set at 37°C, 5% CO 2 , and maintained at 95% relative humidity for 4-8 days.
  • the medium was changed 48 hours after inoculation, and the medium was cultured for 4-8 days, and the medium was changed every other day.
  • the positive control compound was prepared as a stock solution with a concentration of 10 mM in DMSO.
  • the MDCK-MDR1 Transwell culture was incubated at 37°C for 2 hours.
  • MRM Multiple Reaction Detection
  • Peak areas were calculated from ion chromatography results.
  • the apparent permeability coefficient (Papp, unit: cm/s ⁇ 10 -6 ) of the compound is calculated by the following formula:
  • VA is the volume of the receiving end solution ( Ap ⁇ Bl is 0.3mL, Bl ⁇ Ap is 0.1mL), Area is the membrane area of the Transwell-96-well plate (0.143cm 2 ); time is the incubation time (unit: s) ); [drug] is the drug concentration.
  • the compounds to be tested were dissolved in a 10 mM stock solution prepared in DMSO, and then diluted to 1 mM with DMSO.
  • the 10 compounds to be tested with a concentration gradient of 1000 ⁇ prepared were diluted 50 times with complete medium to 20 ⁇ compounds respectively, and stored in a 96-well plate (Beaver, Suzhou), with a total of 10 concentration gradients. Volume of DMSO solvent served as a negative control.
  • the logarithmic growth phase cell suspension was taken and seeded in a 96-well white cell culture plate (Corning 3917, NY, USA), and the volume of each well was 95 ⁇ l (about 2000 cells/well).
  • the culture plate was placed at room temperature for 10 minutes to stabilize the luminescence signal.
  • the luminescence signal was detected on the MD SpectraMax Paradigm plate reader.
  • Cell viability (%) (RLU Drug -RLU Min )/(RLU Max -RLU Min )*100%. Calculate the cell viability corresponding to different concentrations of compounds in EXCEL, and then use the GraphPad Prism software to draw the cell viability curve and calculate the relevant parameters, including the maximum and minimum cell viability, IC 50 value.
  • the compound (I) of the present application has a strong growth inhibitory effect on 6 BaF3 cell lines.
  • the inhibitory activity of compound 2 on 6 BaF3 cell lines was not significantly different from that of the corresponding positive compounds, but the inhibitory activity of compound (I) in the present application on 6 BaF3 cell lines was significantly higher than that of positive control 2.
  • the obtained solid is the compound crystal form A of formula (I).
  • the DSC/TGA spectrum shows ( Figure 2.), in which DSC shows endothermic peaks at 66 ⁇ 2°C (onset) and 370 ⁇ 2°C (onset); TGA shows a weight loss of 8.6% at 66 ⁇ 2°C, which is confirmed by calculation as 2 molecular hydrate, rapid weight loss after 300°C.
  • Form A includes but is not limited to the following characteristic peaks: 7.383 ⁇ 0.2°, 9.762 ⁇ 0.2°, 15.157 ⁇ 0.2°, 17.660 ⁇ 0.2°, 21.002 ⁇ 0.2°, 22.539 ⁇ 0.2°, 26.300 ⁇ 0.2°; also includes the following characteristic peaks : 10.938 ⁇ 0.2°, 16.564 ⁇ 0.2°, 16.863 ⁇ 0.2°, 19.142 ⁇ 0.2°, 19.657 ⁇ 0.2°, 19.861 ⁇ 0.2°, 23.201 ⁇ 0.2°, 23.737 ⁇ 0.2°, 24.299 ⁇ 0.2°, 28.359 ⁇ 0.2° .
  • Form C includes but is not limited to the following characteristic peaks: 6.878 ⁇ 0.2°, 10.918 ⁇ 0.2°, 13.921 ⁇ 0.2°, 15.321 ⁇ 0.2°, 18.401 ⁇ 0.2°, 19.860 ⁇ 0.2°, 24.321 ⁇ 0.2°; also includes the following characteristic peaks : 12.358 ⁇ 0.2°, 16.302 ⁇ 0.2°, 17.320 ⁇ 0.2°, 17.698 ⁇ 0.2°, 19.221 ⁇ 0.2°, 20.779 ⁇ 0.2°, 21.518 ⁇ 0.2°, 22.441 ⁇ 0.2°, 25.340 ⁇ 0.2°, 26.141 ⁇ 0.2° , 26.920 ⁇ 0.2°, 27.820 ⁇ 0.2°.
  • Form D includes but is not limited to the following characteristic peaks: 9.400 ⁇ 0.2°, 10.510 ⁇ 0.2°, 12.957 ⁇ 0.2°, 13.300 ⁇ 0.2°, 17.875 ⁇ 0.2°, 18.840 ⁇ 0.2°, 20.780 ⁇ 0.2°, 22.940 ⁇ 0.2°;
  • the organic solvent includes, but is not limited to, methyl acetate, butyl acetate, butanone, and methyl isobutyl ketone.
  • Form E includes but is not limited to the following characteristic peaks: 6.539 ⁇ 0.2°, 10.797 ⁇ 0.2°, 13.596 ⁇ 0.2°, 15.580 ⁇ 0.2°, 17.741 ⁇ 0.2°, 19.741 ⁇ 0.2°, 24.619 ⁇ 0.2°, 26.380 ⁇ 0.2°;
  • Form L includes but is not limited to the following characteristic peaks: 5.898 ⁇ 0.2°, 9.017 ⁇ 0.2°, 11.502 ⁇ 0.2°, 13.602 ⁇ 0.2°, 15.417 ⁇ 0.2°, 16.897 ⁇ 0.2°, 19.161 ⁇ 0.2°, 21.389° ⁇ 0.2° ; also includes the following characteristic peaks: 16.538 ⁇ 0.2°, 18.151 ⁇ 0.2°, 19.938 ⁇ 0.2°, 20.640 ⁇ 0.2°, 22.583 ⁇ 0.2°, 23.203 ⁇ 0.2°, 23.840 ⁇ 0.2°, 24.959 ⁇ 0.2°, 27.446 ⁇ 0.2 °, 29.792 ⁇ 0.2°, 30.100 ⁇ 0.2°, 30.863 ⁇ 0.2°.
  • Form N includes but is not limited to the following characteristic peaks: 5.841 ⁇ 0.2°, 8.901 ⁇ 0.2°, 11.040 ⁇ 0.2°, 13.442 ⁇ 0.2°, 16.200 ⁇ 0.2°, 18.899 ⁇ 0.2°, 21.119 ⁇ 0.2°, 23.501 ⁇ 0.2°;
  • pH1.2 buffer solution Measure 9.0ml of hydrochloric acid, add water to dilute to 500ml, shake well, measure 85.0ml of the solution, add water to dilute to 200ml, and shake well.
  • pH6.8 buffer solution Weigh 6.8g of potassium dihydrogen phosphate, add water to ultrasonically dissolve and dilute to 250ml, shake well, measure 62.5ml of the solution, add 29.5ml of 0.2mol/L NaOH solution, add water to dilute to 250ml, and add NaOH The solution was adjusted to pH 6.8.
  • pH7.6 buffer solution Weigh 13.6g potassium dihydrogen phosphate, add water to ultrasonically dissolve and dilute to 500ml, shake well, measure 50ml of the solution, add 42.4ml of 0.2mol/L NaOH solution, add water to dilute to 200ml, use NaOH solution Adjust pH to 7.6.
  • each crystal form has good chemical stability without obvious degradation; crystal forms C and D have better crystal form stability; crystal form C has better impurity removal ability.
  • Form C has better crystallinity, crystal form stability, anti-grinding performance, and is easier to prepare.
  • test substance solvent 50%PEG400+40%PG+10%Labrasol
  • Plasma samples were placed on ice after collection, and centrifuged within 1 hour to separate plasma (centrifugation conditions: 6800 g, 6 minutes, 2-8°C). Plasma samples were stored in a -80°C freezer prior to analysis.
  • the biological sample analysis method and the analysis of all samples are completed by the analytical laboratory of Medicipua Pharmaceutical Technology (Shanghai) Co., Ltd., and the intra-day accuracy evaluation of the quality control samples is performed at the same time as the samples are analyzed, and more than 66.7% of the quality control samples are required.
  • the accuracy is between 80-120%.
  • Form C has a larger maximum blood concentration, reaches the maximum blood concentration in a shorter time, has a larger area under the curve, and has a higher bioavailability.

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Abstract

属于药物开发技术领域，具体涉及一种具有抗肿瘤活性的化合物(I)的多晶型或溶剂化物、其制备方法和应用。

Description

含氟大环结构化合物的固体形态、制备方法和应用
本申请是以CN申请号为202110391456.5，申请日为2021年04月12日的申请，以及CN申请号为202111488763.1，申请日为2021年12月07日的申请为基础，并主张其优先权，这两件CN申请的公开内容在此作为整体引入本申请中。
技术领域
本申请属于药物开发技术领域，具体涉及一种具有抗肿瘤活性的化合物(I)的多晶型或溶剂化物、其制备方法和应用。
背景技术
NTRK基因包含NTRK1、NTRK2和NTRK3，分别负责编码原肌凝蛋白受体激酶(TRK)家庭蛋白TRKA、TRKB和TRKC的合成。神经营养因子与TRK蛋白质结合后可诱导受体二聚体化、磷酸化并激活下游PI3K、RAS/MAPK/ERK和PLC-γ的信号级联通路。
TRK信号通路的改变，包括基因融合、蛋白过度表达或单核苷酸改变，已经被发现是许多肿瘤的致病原因，特别是NTRK基因的融合，是目前其中最明确的致癌原因，NTRK融合蛋白充当致癌驱动因子，促进癌细胞生长和存活，这一发现导致了NTRK基因融合作为癌症治疗的新靶点而出现。
2018年11月，在美国新上市了可有效治疗17种肿瘤，针对NTRK1/NTRK2/NTRK3融合的患者的第一代药物LOXO-101，然而一部分癌症患者会对第一代药物产生耐药性，TRK激酶的耐药突变是产生耐药的主要原因之一，因此需要开发出更多的能够克服第一代药物耐药性的有效药物。
发明内容
成都倍特药业股份有限公司开发了一种小分子TRK或者RET抑制剂，化学名称为：(31S,33S,63E,64E,8R)-15,66-二氟-8-甲基-2-氧杂-4,7-二氮杂-6(3,5)-吡唑并[1,5-a]嘧啶-1(2,3)-吡啶3-3(1,3)-环丁环八烷-5-酮的制备)，以下简称化合物(I)，结构如式(I)所示：
Figure PCTCN2022086223-appb-000001
该小分子抑制剂在生物活性特别是在NTRK1G595R、NTRK3G623R等位点的突变的体外药效，人、大鼠和小鼠肝微粒体中的稳定性，药物代谢和口服生物利用度等方面具有更高的选择性和安全性。
本申请研究了化合物(I)的多晶型、溶剂合物，这些晶型可以改善化合物的晶型稳定性、排杂能力、热力学稳定性(onset值更大)、并且成药性相对更佳，且从制备工艺角度出发，其制备更简单，易于控制，便于工业化生产。
本申请提供一种化合物(I)((31S,33S,63E,64E,8R)-15,66-二氟-8-甲基-2-氧杂-4,7-二氮杂-6(3,5)-吡唑并[1,5-a]嘧啶-1(2,3)-吡啶3-3,(1,3)-环丁环八烷-5-酮)的多晶型物或溶剂化物：
Figure PCTCN2022086223-appb-000002
在某些实施方案中，本申请所述的化合物(I)的多晶型为晶型C，其特征在于，其X-射线粉末衍射图包括以下峰：6.878±0.2°、10.918±0.2°、13.921±0.2°、15.321±0.2°、18.401±0.2°、19.860±0.2°、24.321±0.2°。在某些实施方案中，所述的化合物(I)的晶型C的X-射线粉末衍射图还包括以下峰：12.358±0.2°、16.302±0.2°、17.320±0.2°、17.698±0.2°、19.221±0.2°、20.779±0.2°、21.518±0.2°、22.441±0.2°、25.340±0.2°、26.141±0.2°、26.920±0.2°、27.820±0.2°。在某些实施方案中，所述的化合物(I)的晶型C的X-射线粉末衍射图基本上如图3所示。在某些实施方案中，所述的化合物(I)的晶型C为无水晶型。在某些实施方案中，所述的化合物(I)的晶型C的DSC图谱包括在377±2℃(onset)处的特征峰。在某些实施方案中，化合物(I)的晶型C的DSC图谱的DSC图谱包括与图4所示基本上相同的温度处的特征峰。在某些实施方案中，化合物(I)的晶型C的DSC图谱的DSC图谱基本上如图4所示。在某些实施方案中，化合物(I)的晶型C的TGA图谱基本上如图4所示。
在某些实施方案中，本申请所述的化合物(I)的多晶型为晶型D，其特征在于，其X-射线粉末衍射图包括以下峰：9.400±0.2°、10.510±0.2°、12.957±0.2°、13.300±0.2°、17.875±0.2°、18.840±0.2°、20.780±0.2°、22.940±0.2°。在某些实施方案中，所述的化合物(I)的晶型D的X-射线粉末衍射图还包括以下峰：10.084±0.2°、16.984±0.2°、19.240±0.2°、21.361±0.2°、22.324±0.2°、24.761±0.2°、29.820±0.2°、30.220±0.2°、33.098±0.2°。在某些实施方案中，所述的化合物(I)的晶型D的X-射线粉末衍射图基本上如图5所示。在某些实施方案中，所述的化合物(I)的晶型D为无水晶型。在某些实施方案中，所述的化合物(I)的晶型D的DSC图谱包括在375±2℃(onset)处的特征峰。在某些实施方案中，化合物(I)的晶型D的DSC图谱的DSC图谱包括与图6所示基本上相同的温度处的特征峰。在某些实施方案中，化合物(I)的晶型D的DSC图谱的DSC图谱基本上如图6所示。在某些实施方案中，化合物(I)的晶型D的TGA图谱基本上如图6所示。
在某些实施方案中，本申请所述化合物(I)的多晶型为晶型E，其特征在于，其X-射线粉末衍射图包括以下峰：6.539±0.2°、10.797±0.2°、13.596±0.2°、15.580±0.2°、17.741±0.2°、19.741±0.2°、24.619±0.2°、26.380±0.2°。在某些实施方案中，所述的化合物(I)的晶型E的X-射线粉末衍射图还包括以下峰：10.041±0.2°、13.022±0.2°、16.101±0.2°、16.959±0.2°、18.079±0.2°、18.980±0.2°、20.119±0.2°、21.680±0.2°、23.080±0.2°、25.340±0.2°、26.000±0.2°、28.279±0.2°。在某些实施方案中，所述的化合物(I)的晶型E的X-射线粉末衍射图基本上如图7所示。在某些实施方案中，所述的化合物(I)的晶型E为无水晶型。在某些实施方案中，所述的化合物(I)的晶型E的DSC图谱包括在374±2℃(onset)处的特征峰。在某些实施方案中，化合物(I)的晶型E的DSC图谱的DSC图谱包括与图8所示基本上相同的温度处 的特征峰。在某些实施方案中，化合物(I)的晶型E的DSC图谱的DSC图谱基本上如图8所示。在某些实施方案中，化合物(I)的晶型E的TGA图谱基本上如图8所示。
在某些实施方案中，本申请所述的化合物(I)的多晶型为晶型L，其特征在于，其X-射线粉末衍射图包括以下峰：5.898±0.2°、9.017±0.2°、11.502±0.2°、13.602±0.2°、15.417±0.2°、16.897±0.2°、19.161±0.2°、21.389°±0.2°。在某些实施方案中，所述的化合物(I)的的晶型L的X-射线粉末衍射图还包括以下峰：16.538±0.2°、18.151±0.2°、19.938±0.2°、20.640±0.2°、22.583±0.2°、23.203±0.2°、23.840±0.2°、24.959±0.2°、27.446±0.2°、29.792±0.2°、30.100±0.2°、30.863±0.2°。在某些实施方案中，所述的化合物(I)的晶型L的X-射线粉末衍射图基本上如图9所示。在某些实施方案中，所述的化合物(I)的晶型L为无水晶型。在某些实施方案中，所述的化合物(I)的晶型L的DSC图谱包括在373±2℃(onset)处的特征峰。在某些实施方案中，化合物(I)的晶型L的DSC图谱的DSC图谱包括与图10所示基本上相同的温度处的特征峰。在某些实施方案中，化合物(I)的晶型L的DSC图谱的DSC图谱基本上如图10所示。在某些实施方案中，化合物(I)的晶型L的TGA图谱基本上如图10所示。
在某些实施方案中，本申请所述的化合物(I)的多晶型为晶型N，其特征在于，其X-射线粉末衍射图包括以下峰：5.841±0.2°、8.901±0.2°、11.040±0.2°、13.442±0.2°、16.200±0.2°、18.899±0.2°、21.119±0.2°、23.501±0.2°。在某些实施方案中，所述的化合物(I)的晶型N的X-射线粉末衍射图还包括以下峰：7.296±0.2°、9.801±0.2°、12.058±0.2°、17.438±0.2°、20.037±0.2°、20.439±0.2°、22.660±0.2°、25.122±0.2°、27.261±0.2°、28.602±0.2°、29.019±0.2°、29.560±0.2°。在某些实施方案中，所述的化合物(I)的晶型N的X-射线粉末衍射图基本上如图11所示。在某些实施方案中，所述的化合物(I)的晶型N为无水晶型。在某些实施方案中，所述的化合物(I)的晶型N的DSC图谱包括在370±2℃(onset)处的特征峰。在某些实施方案中，化合物(I)的晶型N的DSC图谱的DSC图谱包括与图12所示基本上相同的温度处的特征峰。在某些实施方案中，化合物(I)的晶型N的DSC图谱的DSC图谱基本上如图12所示。在某些实施方案中，化合物(I)的晶型N的TGA图谱基本上如图12所示。
在某些实施方案中，本申请所述的化合物(I)的多晶型为晶型A，其特征在于，其X-射线粉末衍射图包括以下峰：7.383±0.2°、9.762±0.2°、15.157±0.2°、17.660±0.2°、21.002±0.2°、22.539±0.2°、26.300±0.2°；在某些实施方案中，所述的化合物(I)的晶型A的X-射线粉末衍射图还包括以下峰：10.938±0.2°、16.564±0.2°、16.863±0.2°、19.142±0.2°、19.657±0.2°、19.861±0.2°、23.201±0.2°、23.737±0.2°、24.299±0.2°、28.359±0.2°。在某些实施方案中，所述的化合物(I)的晶型A的X-射线粉末衍射图基本上如图1所示。在某些实施方案中，所述的化合物(I)的晶型A为化合物(I)的水合物，其中所述的化合物(I)与水的摩尔比为1:2。在某些实施方案中，所述的化合物(I)的晶型A的DSC图谱包括在66±2℃(onset)处的特征峰。在某些实施方案中，所述的化合物(I)的晶型A的DSC图谱包括在370±2℃(onset)处的特征峰。在某些实施方案中，化合物(I)的晶型A的DSC图谱的DSC图谱包括与图2所示基本上相同的温度处的特征峰。在某些实施方案中，化合物(I)的晶型A的DSC图谱的DSC图谱基本上如图2所示。在某些实施方案中，化合物(I)的晶型A的TGA图谱基本上如图2所示。
本申请还提供制备化合物(I)的晶型C的方法，包括以下步骤：
将化合物(I)的晶型A分散在有机溶剂中打浆，
过滤，
干燥。
在某些实施方案中，所述的制备化合物(I)的晶型C的方法中，所述有机溶剂为：C 1-C 6烷基醇，优选乙醇、异丙醇。
在某些实施方案中，所述的制备化合物(I)的晶型C的方法包括将化合物(I)的晶型A分散在有机溶剂中在25℃～回流温度下打浆；
在某些实施方案中，所述的制备化合物(I)的晶型C的方法包括将化合物(I)的晶型A分散在有机溶剂中在25℃～回流温度下打浆3～24小时。
在某些实施方案中，所述的制备化合物(I)的晶型C的方法中，所述干燥为真空干燥。
在某些实施方案中，所述的制备化合物(I)的晶型C的方法包括干燥5～7小时，例如6小时。
在某些实施方案中，所述的制备化合物(I)的晶型C的方法包括以下步骤：将晶型A分散在有机溶剂中，在某温度下打浆一定时间，过滤、真空干燥一定时间；所述有机溶剂为：C1-C6烷基醇，优选乙醇、异丙醇；其中所述温度为25℃～回流；其中所述打浆时间为3～24小时；所述干燥时间为6小时。
本申请还提供制备化合物(I)的晶型D的方法，包括以下步骤：
将化合物(I)的晶型A分散在有机溶剂中打浆，
过滤；
干燥。
在某些实施方案中，所述的制备化合物(I)的晶型D的方法中，所述有机溶剂为：碳酸二甲酯、乙酸甲酯、乙酸丁酯、丁酮、甲基异丁基酮，优选为碳酸二甲酯。
在某些实施方案中，所述的制备化合物(I)的晶型D的方法包括将化合物(I)的晶型A分散在有机溶剂中在室温下打浆。
在某些实施方案中，所述的制备化合物(I)的晶型D的方法包括将化合物(I)的晶型A分散在有机溶剂中在室温下打浆3～24小时，优选24小时。
在某些实施方案中，所述的制备化合物(I)的晶型D的方法中，所述干燥为真空干燥。
在某些实施方案中，所述的制备化合物(I)的晶型D的方法包括干燥5～7小时，例如6小时。
在某些实施方案中，所述的制备化合物(I)的晶型D的方法包括以下步骤：将晶型A分散在有机溶剂中，在室温下打浆一定时间，过滤、真空干燥一定时间；所述有机溶剂为：乙酸甲酯、乙酸丁酯、丁酮、甲基异丁基酮；其中所述打浆时间为3～24小时；打浆时间优选24小时；所述干燥时间为6小时。
本申请另一方面提供一种药物组合物，其包括治疗有效剂量的化合物(I)的多晶型或溶剂化物及其可药用的载体。
本申请另一方面提供化合物(I)的多晶型物或溶剂化物或者药物组合物在制备治疗疼痛、癌症、炎症或神经退行性疾病中的用途。在某些实施方案中，所述疾病选自癌症。在某些实施方案中，所述癌症为选自神经细胞瘤、卵巢癌、结肠直肠癌、黑素瘤、头和颈部的癌症、胃癌、肺癌、乳腺癌、成胶质细胞瘤、成神经管细胞瘤、分泌性乳腺癌、唾液腺癌、甲状腺乳头状癌、成人髓细胞白血病、胰腺癌、前列腺癌、阑尾癌、胆管癌、胃肠道间质瘤和婴儿纤维肉瘤的一种或多种。
本申请另一方面提供化合物(I)的多晶型物或溶剂化物或者药物组合物在制备治疗由TRK介导的疾病中的用途。在某些实施方案中，所述TRK介导的疾病为疼痛、癌症、炎症和神经退行性疾病中的一种或多种。在某些实施方案中，所述疾病为癌症。在某些实施方案中，所述癌症为选自神经细胞瘤、卵巢癌、结肠直肠癌、黑素瘤、头和颈部的癌症、胃癌、肺癌、乳腺癌、成胶质细胞瘤、成神经管细胞瘤、分泌性乳腺癌、唾液腺癌、甲状腺乳头状癌、成人髓细胞白血病、胰腺癌、前列腺癌、阑尾癌、胆管癌、胃肠道间质瘤和婴儿纤维肉瘤的一种或多种。
本申请另一方面提供化合物(I)的多晶型物或溶剂化物或者药物组合物，其用于治疗疼痛、癌症、炎症或神经退行性疾病。在某些实施方案中，所述疾病选自癌症。在某些实施方案中，所述癌症为选自神经细胞瘤、卵巢癌、结肠直肠癌、黑素瘤、头和颈部的癌症、胃癌、肺癌、乳腺癌、成胶质细胞瘤、成神经管细胞瘤、分泌性乳腺癌、唾液腺癌、甲状腺乳头状癌、成人髓细胞白血病、胰腺癌、前列腺癌、阑尾癌、胆管癌、胃肠道间质瘤和婴儿纤维肉瘤的一种或多种。
本申请另一方面提供化合物(I)的多晶型物或溶剂化物或者药物组合物，其用于治疗由TRK介导的疾病。在某些实施方案中，所述TRK介导的疾病为疼痛、癌症、炎症和神经退行性疾病中的一种或多种。在某些实施方案中，所述疾病为癌症。在某些实施方案中，所述癌症为选自神经细胞瘤、卵巢癌、结肠直肠癌、黑素瘤、头和颈部的癌症、胃癌、肺癌、乳腺癌、成胶质细胞瘤、成神经管细胞瘤、分泌性乳腺癌、唾液腺癌、甲状腺乳头状癌、成人髓细胞白血病、胰腺癌、前列腺癌、阑尾癌、胆管癌、胃肠道间质瘤和婴儿纤维肉瘤的一种或多种。
本申请另一方面提供本申请另一方面提供一种治疗疼痛、癌症、炎症或神经退行性疾病的方法，包括向有需要的受试者施用治疗有效量的本申请所述的化合物(I)的多晶型物或溶剂化物或者药物组合物。在某些实施方案中，所述疾病选自癌症。在某些实施方案中，所述癌症为选自神经细胞瘤、卵巢癌、结肠直肠癌、黑素瘤、头和颈部的癌症、胃癌、肺癌、乳腺癌、成胶质细胞瘤、成神经管细胞瘤、分泌性乳腺癌、唾液腺癌、甲状腺乳头状癌、成人髓细胞白血病、胰腺癌、前列腺癌、阑尾癌、胆管癌、胃肠道间质瘤和婴儿纤维肉瘤的一种或多种。
本申请另一方面提供一种治疗由TRK介导的疾病的方法，包括向有需要的受试者施用治疗有效量的本申请所述的化合物(I)的多晶型物或溶剂化物或者药物组合物。在某些实施方案中，所述TRK介导的疾病为疼痛、癌症、炎症和神经退行性疾病中的一种或多种。在某些实施方案中，所述疾病为癌症。在某些实施方案中，所述癌症为选自神经细胞瘤、卵巢癌、结肠直肠癌、黑素瘤、头和颈部的癌症、胃癌、肺癌、乳腺癌、成胶质细胞瘤、成神经管细胞瘤、分泌性乳腺癌、唾液腺癌、甲状腺乳头状癌、成人髓细胞白血病、胰腺癌、前列腺癌、阑尾癌、胆管癌、胃肠道间质瘤和婴儿纤维肉瘤的一种或多种。
附图说明
此处所说明的附图用来提供对本发明的进一步理解，构成本申请的一部分，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：
图1.显示了式(I)化合物晶型A的XRPD图谱；
图2.显示了式(I)化合物晶型A的DSC/TGA图谱。
图3.显示了式(I)化合物无水晶型C的XRPD图谱；
图4.显示了式(I)化合物无水晶型C的DSC/TGA图谱。
图5.显示了式(I)化合物无水晶型D的XRPD图谱；
图6.显示了式(I)化合物无水晶型D的DSC/TGA图谱。
图7.显示了式(I)化合物无水晶型E的XRPD图谱；
图8.显示了式(I)化合物无水晶型E的DSC/TGA图谱。
图9.显示了式(I)化合物无水晶型L的XRPD图谱；
图10.显示了式(I)化合物无水晶型L的DSC/TGA图谱。
图11.显示了式(I)化合物无水晶型N的XRPD图谱；
图12.显示了式(I)化合物无水晶型N的DSC/TGA图谱。
具体实施方式
1.术语
本申请所用术语“药物组合物”包括包含治疗有效量的本申的化合物的产品，以及直接地或间接地由本申请化合物的多晶型、溶剂合物组合产生的任何产品。该药物组合物可通过例如口服或非肠道等途径给药。本申请的药物组合物可按本领域常规方法制备成各种剂型，包括但不限于片剂、胶囊、溶液、悬浮液、颗粒剂或注射剂等，经例如口服或非肠道等途径给药。
本申请所用术语“有效量”是指足以实现所需治疗效果的量，例如，实现减轻与待治疗疾病相关的症状的量。
本申请所用的术语“治疗”目的是减轻或消除所针对的疾病状态或病症。如果受试者按照本申请所述方法接受了治疗有效量的化合物，其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体或其混合物，或其可药用的盐，或其药物组合物，该受试者一种或多种指征和症状表现出可观察到的和/或可检测出的降低或改善，则受试者被成功地“治疗”了。还应当理解，所述的疾病状态或病症的治疗的不仅包括完全地治疗，还包括未达到完全地治疗，但实现了一些生物学或医学相关的结果。
另外需要指出，本申请化合物的使用剂量和使用方法取决于诸多因素，包括患者的年龄、体重、性别、自然健康状况、营养状况、化合物的活性强度、服用时间、代谢速率、病症的严重程度以及诊治医师的主观判断。优选的使用剂量介于0.001－1000mg/kg体重/天。
本申请所用的术语"可药用"是指在合理医疗判断的范围内适合与人类和动物的组织接触，而没有过度的毒性、刺激、过敏反应或其它问题并发症的，与合理的受益/风险比相称的那些化合物、材料、组合物和/或剂型。
本申请所用的“多晶型”或“多晶型物”是指具有相同化学组成，但构成该晶体的分子、原子和/或离子的不同空间排列的晶型。尽管多晶型物具有相同的化学组成，但它们的堆积和几何排列不同，并可能表现出不同的物理性质，如熔点、形状、颜色、密度、硬度、可形变性、稳定性、溶解度、溶出速率和类似性质。根据他们的温度-稳定性关系，两种多晶型物可以是单变性或互变性的。对于单变性体系，在温度变化时，两种固相之间的相对稳定性保持不变。相反，在互变性体系中，存在一个过渡温度，在此两种相的稳定性调换((Theory and Origin of Polymorphism in"Polymorphism in Pharmaceutical Solids"(1999)ISBN:)-8247-0237)。这种化合物以不同晶体结构存在的现象被称作药物多晶型现象。
本申请所用术语“受试者”是指哺乳动物受试者，包括但不限于人、猫、狗、马、牛、羊、猴等，其中所述人受试者包括男性和女性受试者，并且包括新生儿、婴儿、少年、青少年、成人和老人受试者。
本申请中所述的化合物，对于同一化合物而言，若名称与结构式不一致，以化合物结构式为准。
本申请中，所述的X-射线粉末衍射图(XRPD图谱)是用Cu-Kα辐射，以2θ角度表示的X-射线粉末衍射图。
本申请中，用于限定图的术语“与图X基本上一致”、“基本上如图X所示”具有相同的含义，旨在表示，考虑到本领域可接受的偏差，本领域技术人员认为所述图与参考图相同。这种偏差可能是由本领域已知的与仪器、操作条件和人为因素等有关的因素引起。在一些实施方案中，当两个图的特征峰的位置的变化不超过±5％、±4％、±3％、±2％或±1％时，则为认为这两个图基本上一致或基本上相同。例如，本领域技术人员可以理解，通过差示扫描量热法(DSC)测量的吸热起始和峰值温度可以随实验而显著变化。例如，本领域技术人员可以容易地鉴定两个X射线衍射图谱或两个DSC图谱是否基本上相同。在一些实施方案中，当两个X射线衍射图谱的特征峰的2θ角度变化不超过±0.3°、±0.2°或±0.1°时，认为所述X射线衍射图谱基本相同。
本申请的结晶结构可以通过各种方法制备，包括从合适的溶剂中结晶或重结晶、升华、从熔融体中生长、从另一相固态转化、从超临界流体中结晶和射流喷雾等。结晶结构从溶剂混合物中结晶或重结晶的技术，包括溶剂蒸发、降低溶剂混合物的温度、该分子和/或盐的过饱和溶剂混合物的引晶、冻干溶剂混合物、向溶剂混合物中加入反溶剂等。可以使用高通量的结晶技术制备结晶结构，包括多晶型物。药物晶体，包括多晶型物，药物晶体的制备方法和表征公开在Solid-State Chemistry of Drugs,S.R.Byrn,R.R.Pfeiffer,和J.G.Stowell,第2版，SSCI,West Lafayette,Indiana,1999中。
本申请公开或要求保护的晶体结构对等的晶体结构可能根据试验条件、纯度、设备和本领域技术人员已知的其它常几变量在合理误差范围内表现出类似但不完全相同的分析特性。相应地，本领域技术人员显而易见的是，可以在不背离本发明的范围和精神的情况下在本发明内作出各种修改和变动。在考虑本文公开的本发明的说明书和实践的基础上，本发明的其它实施方案是本领域技术人员显而易见的。申请人期望该说明书和实施例被视为示例性的，而非限制其范围。
本申请中所用的术语"室温"或"RT"是指20至25℃(68-77°F)的环境温度。
本申请所用术语“可药用的载体”是指与治疗剂一同给药的稀释剂、附加物或媒介物，并且其在合理的医学判断的范围内适于接触人类和/或其它动物的组织而没有过度的毒性、刺激、过敏反应或与合理的益处/风险比相应的其它问题或并发症。
在本申请的药物组合物中可使用的可药用的载体包括但不限于无菌液体，例如水和油，包括那些石油、动物、植物或合成来源的油，例如花生油、大豆油、矿物油、芝麻油等。当所述药物组合物通过静脉内给药时，水是示例性载体。还可以使用生理盐水和葡萄糖及甘油水溶液作为液体载体，特别是用于注射液。还可以使用淀粉、葡萄糖、乳糖、蔗糖、明胶、麦芽糖、白垩、硅胶、硬脂酸钠、单硬脂酸甘油酯、滑石、氯化钠、脱脂奶粉、甘油、丙二醇、水、乙醇等作为载体。所述药物组合物还可以视需要包含少量的湿润剂、乳化剂、pH缓冲剂、防腐剂、抗氧剂、矫味剂、芳香剂、助溶剂、增溶剂、渗透压调节剂、着色剂等。口服制剂可以包含标准载体，例如黏合剂、填充剂、崩解剂、润滑剂等。
本申请所述的药物组合物可以通过本领域公知的方法进行施用，例如但不限于以下面的任意方式施用：口服，喷雾吸入，直肠用药，鼻腔用药，颊部用药，局部用药，非肠道用药，如皮下，静脉，肌内，腹膜内，鞘内，心室内，胸骨内和颅内注射或输入，或借助一种外植储器用药。其中优选口服、肌内或静脉内注射给药方式。对于这些给药途径，可以适合的剂型给药本申请的药物组合物。
所述剂型可为固体制剂、半固体制剂、液体制剂或气态制剂，包括但不限于片剂、胶囊剂、散剂、颗粒剂、锭剂、硬糖剂、散剂、喷雾剂、乳膏剂、软膏剂、栓剂、凝胶剂、糊剂、洗剂、软膏剂、水性混悬剂、可注射溶液剂、混悬剂、酏剂、糖浆剂。
本发明所述的药物组合物可以通过本领域熟知的任何方法来制备，例如通过混合、溶解、制粒、糖包衣、碾磨、乳化、冻干等处理来制备。
2.分析方法
本发明中X-射线粉末衍射(XRPD)分析采用丹东浩元DX-2700BH，Cu靶照射。检测范围在3°至40°、步长为0.02°、速度为0.2s.step -1。
本发明中同步热分析仪(TGA/DSC)分析仪器为Mettler-Toledo TGA/DSC 3 +。仪器的升温速率为10K/min。
具体实施例
以下通过实施例对本发明作进一步阐述，本发明的实施例仅用于说明本发明的技术方案，并非用于限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购或得的常规产品。
制备例1：5-氯-6-氟吡唑并[1,5-a]嘧啶-3-羧酸乙酯的制备
Figure PCTCN2022086223-appb-000003
步骤1：2-氟丙二酸的制备
Figure PCTCN2022086223-appb-000004
室温下，称取2-氟丙二酸二乙酯(5.0g)及氢氧化钠(17.3g)于乙醇/水(100/100mL)中，反应过夜，LCMS显示反应完全。将体系浓缩除去乙醇，加水(50mL)，浓盐酸调节pH约为1，甲基叔丁基醚萃取四次，合并有机相，无水硫酸钠干燥，过滤，浓缩，得标题化合物3.7g，无需纯化即可用于下一步反应。
MS(ESI)m/z(M-H) +＝121.1.
步骤2：5,7-二氯-6-氟吡唑并[1,5-a]嘧啶-3-羧酸乙酯的制备
Figure PCTCN2022086223-appb-000005
室温下，称取2-氟丙二酸(2.0g)及5-氨基-1H-吡唑-4-羧酸乙酯(1.7g)于三氯氧磷(20mL)中，再将N,N-二甲基甲酰胺(2mL)和N,N-二乙基苯胺(4.9g)加入到体系中，体系升温至110℃下反应3小时，LCMS显示反应完全。将体系浓缩除去三氯氧磷，再倒入饱和碳酸氢钠溶液(100mL)中，使溶液保持为碱性，乙酸乙酯萃取三次，无水硫酸钠干燥，过滤，浓缩，所得粗品经柱层析纯化，得到的固体再用石油醚洗涤，干燥后得标题化合物1.7g。
MS(ESI)m/z(M+H) +＝278.0.
1H NMR(400MHz,DMSO-d 6)δ8.80(s,1H),4.33(q,J＝7.2Hz,2H),1.32(t,J＝7.0Hz,3H).
步骤3：5-氯-6-氟吡唑并[1,5-a]嘧啶-3-羧酸乙酯的制备
Figure PCTCN2022086223-appb-000006
称取5,7-二氯-6-氟吡唑并[1,5-a]嘧啶-3-羧酸乙酯(1.14g)及氯化铵(800mg)于乙醇/四氢呋喃/水(30/10/20mL)中，搅拌过程中加入锌粉(1.3g)，反应5分钟后将锌粉过滤，乙酸乙酯洗涤滤饼，收集滤液，无水硫酸钠干燥，过滤，浓缩，所得粗品经柱层析纯化得标题化合物800mg。
MS(ESI)m/z(M+H) +＝244.0.
1H NMR(400MHz,DMSO-d 6)δ9.93(d,J＝4.4Hz,1H),8.68(s,1H),4.31(q,J＝7.2Hz,2H),1.31(t,J＝7.2Hz,3H).
制备例2：(R)-1-(5-氟-2-甲氧基吡啶-3-基)乙-1-胺盐酸盐的制备
Figure PCTCN2022086223-appb-000007
步骤1：(R)-N-(((5-氟-2-甲氧基吡啶-3-基)亚甲基)-2-甲基丙烷-2-亚磺酰胺的制备
Figure PCTCN2022086223-appb-000008
将(R)-2-甲基丙烷-2-亚磺酰胺(12.9g)溶于四氢呋喃(100mL)中，依次加入5-氟-2-甲氧基烟碱醛(15.0g)和碳酸铯(40.9g)。体系于室温反应2小时，TLC显示原料消耗完毕。抽滤，滤饼用四氢呋喃洗涤三次，所得滤液用饱和氯化钠溶液反洗一次，无水硫酸钠干燥，过滤，浓缩。所得粗品经柱层析纯化得标题化合物23.0g。
MS(ESI)m/z(M+H) +＝259.1.
1H NMR(400MHz,DMSO-d 6)δ8.67(d,J＝2.4Hz,1H),8.42(d,J＝3.2Hz,1H),8.14(dd,J＝8.4,3.2Hz,1H),3.98(s,3H),1.18(s,9H).
步骤2：(R)-N-((R)-1-(5-氟-2-甲氧基吡啶-3-基)乙基)-2-甲基丙烷-2-亚磺酰胺的制备
Figure PCTCN2022086223-appb-000009
称取(R)-N-(((5-氟-2-甲氧基吡啶-3-基)亚甲基)-2-甲基丙烷-2-亚磺酰胺(5.0g)溶于四氢呋喃(40mL)中，体系冷却至-78℃后缓慢滴加甲基溴化镁(7.8mL,3M)，维持体系温度低于-65℃。滴毕，体系自然回温至室温，继续反应1小时，TLC显示反应完全。将反应体系倒入饱和氯化铵水溶液(1L)中，乙酸乙酯萃取，合并有机相，饱和氯化钠溶液反洗，无水硫酸钠干燥，过滤，浓缩。所得粗品经柱层析纯化得标题化合物4.5g。
MS(ESI)m/z(M+H) +＝275.2.
1H NMR(400MHz,DMSO-d 6)δ8.04(d,J＝2.8Hz,1H),7.74(dd,J＝9.2,3.2Hz,1H),5.80(d,J＝8.8Hz,1H),4.57-4.50(m,1H),3.88(s,3H),1.33(d,J＝6.8Hz,3H),1.11(s,9H).
步骤3：(R)-1-(5-氟-2-甲氧基吡啶-3-基)乙-1-胺盐酸盐的制备
Figure PCTCN2022086223-appb-000010
室温下，将(R)-N-((R)-1-(5-氟-2-甲氧基吡啶-3-基)乙基)-2-甲基丙烷-2-亚磺酰胺(4.5g)溶于氯化氢-二氧六环溶液(30mL)中，反应过夜，LCMS显示原料消耗完全。将体系浓缩得粗品3.1g，大于95％ee，无需纯化即可直接用于下一步。
MS(ESI)m/z(M+H) +＝171.2.
1H NMR(400MHz,DMSO-d 6)δ8.80-8.66(m,3H),8.18(d,J＝2.8Hz,1H),8.04-8.00(m,1H),7.09–6.60(m,1H),4.51–4.45(m,1H),3.90(s,3H),1.49(d,J＝6.4Hz,3H).
制备例3：式(I)化合物(化学名称为：(3 1S,3 3S,6 3E,6 4E,8R)-1 5,6 6-二氟-8-甲基-2-氧杂-4,7-二氮杂-6(3,5)-吡唑并[1,5-a]嘧啶-1(2,3)-吡啶3-3,(1,3)-环丁环八烷-5-酮)的制备
Figure PCTCN2022086223-appb-000011
步骤1：(R)-6-氟-5-((1-(5-氟-2-甲氧基吡啶-3-基)乙基)氨基)吡唑并[1,5-a]嘧啶-3-羧酸乙酯的制备
Figure PCTCN2022086223-appb-000012
将(R)-1-(5-氟-2-甲氧基吡啶-3-基)乙-1-胺盐酸盐(1.0g)溶于乙腈(20mL)中，依次加入N,N-二异丙基乙胺(1.9g)和5-氯-6-氟吡唑并[1,5-a]嘧啶-3-羧酸乙酯(1.2g)，体系于60℃下反应3小时，TLC显示反应完全。将体系倒入水(50mL)中，二氯甲烷萃取，合并有机相，饱和氯化钠溶液反洗，无水硫酸钠干燥，过滤，浓缩，所得粗品经柱层析纯化得标题化合物1.1g。
MS(ESI)m/z(M+H) +＝378.2.
1H NMR(400MHz,DMSO-d 6)δ9.15(d,J＝6.4Hz,1H),8.49(d,J＝8.0Hz,1H),8.18(s,1H),8.02(d,J＝3.2Hz,1H),7.67(dd,J＝9.0,3.0Hz,1H),5.60-5.52(m,1H),4.18-4.10(m,2H),3.93(s,3H),1.50(d,J＝6.8Hz,3H),1.22(t,J＝7.0Hz,3H).
步骤2：(R)-6-氟-5-(((1-(5-氟-2-甲氧基吡啶-3-基)乙基)氨基)吡唑并[1,5-a]嘧啶-3-羧酸的制备
Figure PCTCN2022086223-appb-000013
室温下，将(R)-6-氟-5-((1-(5-氟-2-甲氧基吡啶-3-基)乙基)氨基)吡唑并[1,5-a]嘧啶-3-羧酸乙酯(1.1g)溶于乙醇/水(5/15mL)中，加入氢氧化钠(584mg)，体系于50℃反应过夜，TLC显示反应完全。将体系浓缩除去乙醇，残余液倒入水(20mL)中，用氯化氢溶液(2M)调pH至5左右，二氯甲烷萃取，合并有机相，饱和氯化钠溶液反洗，无水硫酸钠干燥，过滤，浓缩得粗品800mg，无需纯化即可直接用于下一步。
MS(ESI)m/z(M+H) +＝350.1.
1H NMR(400MHz,DMSO-d 6)δ11.68(s,1H),9.13(d,J＝6.0Hz,1H),8.51(d,J＝7.6Hz,1H),8.14(s,1H),8.01(d,J＝2.8Hz,1H),7.72(dd,J＝9.0,3.0Hz,1H),5.59-5.52(m,1H),3.92(s,3H),1.50(d,J＝6.8Hz,3H).
步骤3：(1R,3r)-3-(6-氟-5-(((R)-1-(5-氟-2-甲氧基吡啶-3-基)乙基)氨基)吡唑并[1,5-a]嘧啶-3-甲酰胺基)-4-甲基苯磺酸环丁酯的制备
Figure PCTCN2022086223-appb-000014
将(R)-6-氟-5-(((1-(5-氟-2-甲氧基吡啶-3-基)乙基)氨基)吡唑并[1,5-a]嘧啶-3-羧酸(800mg)、2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(1.0g)及N,N-二异丙基乙胺(886mg)溶于干燥四氢呋喃(10mL)中，体系室温反应1小时后再加入(3-羟基环丁基)氨基甲酸叔丁酯盐酸盐(953mg)，继续反应1小时，TLC显示反应完全。将体系倒入水(30mL)中，乙酸乙酯萃取，合并有机相，饱和氯化钠溶液反洗，无水硫酸钠干燥，过滤，浓缩，所得粗品经柱层析纯化得标题化合物800mg。
MS(ESI)m/z(M+H) +＝573.2.
1H NMR(400MHz,DMSO-d 6)δ9.21(d,J＝6.0Hz,1H),8.57(d,J＝7.6Hz,1H),8.09(s,1H),8.05(d,J＝2.8Hz,1H),7.80–7.78(m,2H),7.66(dd,J＝8.8,2.8Hz,1H),7.60(d,J＝6.8Hz,1H),7.47(d,J＝8.0Hz,2H),5.44-5.37(m,1H),4.96–4.92(m,1H),4.33-4.28(m,1H),3.80(s,3H),2.47–2.38(m,5H),2.24-2.18(m,1H),2.12–2.08(m,1H),1.52(d,J＝6.8Hz,3H).
步骤4：(1R,3r)-3-(6-氟-5-(((R)-1-(5-氟-2-羟基吡啶-3-基)乙基)氨基)吡唑并[1,5-a]嘧啶-3-甲酰胺基)-4-甲基苯磺酸盐环丁基的制备
Figure PCTCN2022086223-appb-000015
将(1R,3r)-3-(6-氟-5-(((R)-1-(5-氟-2-甲氧基吡啶-3-基)乙基)氨基)吡唑并[1,5-a]嘧啶-3-甲酰胺基)-4-甲基苯磺酸环丁酯(800mg)溶于氯化氢/二氧六环(4M,10mL)中，体系于55℃条件下反应过夜，TLC显示反应完全。将体系直接浓缩以除去大部分二氧六环，得粗品600mg，无需纯化即可直接用于下一步。
MS(ESI)m/z(M+H) +＝559.2.
步骤5：(3 1S,3 3S,6 3E,6 4E,8R)-1 5,6 6-二氟-8-甲基-2-氧杂-4,7-二氮杂-6(3,5)-吡唑并[1,5-a]嘧啶-1(2,3)-吡啶3-3(1,3)-环丁环八烷-5-酮(化合物(I))的制备
Figure PCTCN2022086223-appb-000016
将(1R,3r)-3-(6-氟-5-(((R)-1-(5-氟-2-羟基吡啶-3-基)乙基)氨基)吡唑并[1,5-a]嘧啶-3-甲酰胺基)-4-甲基苯磺酸盐环丁基盐酸盐(250mg)溶于N,N-二甲基甲酰胺(6mL)中，加入碳酸钾(232mg)，体系室温反应5小时，TLC显示反应完全。将体系倒入水(10mL)中，乙酸乙酯萃取，合并有机相，饱和氯化钠溶液反洗，无水硫酸钠干燥，过滤，浓缩，所得粗品经制备薄层色谱纯化和制备高效液相色谱纯化得标题化合物58.0mg，ee值>99.5％。
MS(ESI)m/z(M+H) +＝387.1.
1H NMR(400MHz,DMSO-d6)δ9.22–9.16(m,1H),9.15(d,J＝12.0Hz,1H),8.93(d,J＝8.0Hz,1H),8.10(s,1H),8.05(d,J＝4.0Hz,1H),7.87(dd,J＝8.8,3.0Hz,1H),5.79–5.67(m,1H),5.15–5.12(m,1H),4.70–4.63(m,1H),3.10–3.04(m,1H),2.92–2.86(m,1H),2.18–2.12(m,1H),1.69–1.63(m,1H),1.54(d,J＝8.0Hz,3H).
本申请测试例1-6中所用到的化合物包括：
LOXO-195，为第二代TRK抑制剂，化学结构为：
Figure PCTCN2022086223-appb-000017
阳性对照1，为专利WO2019210835实施例5化合物，化学结构为：
Figure PCTCN2022086223-appb-000018
阳性对照2，为专利WO2019210835实施例6化合物，化学结构为：
Figure PCTCN2022086223-appb-000019
化合物1b，结构式为：
Figure PCTCN2022086223-appb-000020
化合物2，结构式为：
Figure PCTCN2022086223-appb-000021
化合物4，结构式为：
Figure PCTCN2022086223-appb-000022
化合物5，结构式为：
Figure PCTCN2022086223-appb-000023
化合物6，结构式为：
Figure PCTCN2022086223-appb-000024
上述LOXO-195和对照化合物1-2可商购获得，也可依据常规路线合成制备获得，例如，LOXO-195可商购获得，阳性对照1和2可参照WO2019210835A1记载的方法合成。化合物1b、2和4-6均可依据常规路线合成制备获得，也可参照WO2021115401A1记载的方法合成。
测试例1：激酶抑制活性
该测试例委托桑迪亚医药技术(上海)有限责任公司完成。
1.实验目的
测定本申请化合物对TRKa、TRKA(G595R)和TRKC(G623R)三种激酶的抑制活性。
2.实验材料
2.2.1试剂及耗材
试剂名称 供货商 货号 批号
TRKa Carna 08-186 13CBS-0565G
TRKA(G595R) signalchem N16-12BG-100 H2714-7
TRKC(G623R) signalchem N18-12CH-100 D2567-8
激酶底物22 GL 112393 P190329-SL112393
DMSO Sigma D8418-1L SHBG3288V
384孔板(白色) PerkinElmer 6007290 810712
2.2.2仪器
离心机(生产厂家：Eppendorf，型号：5430)；酶标仪(生产厂家：Perkin Elmer，型号：Caliper EZ ReaderII)；Echo 550(生产厂家：Labcyte，型号：Echo 550)
3.实验方法
3.1受试化合物精确称量并溶解在100％DMSO中，配制成10mM溶液。
3.2激酶反应过程
3.2.1配制1×激酶缓冲液。
3.2.2化合物浓度梯度的配制：受试化合物测试起始浓度为1000nM，在384板中稀释成100倍终浓度的100％DMSO溶液，然后3倍稀释，得到10个浓度的化合物的DMSO溶液。使用分液器Echo 550向反应板OptiPlate-384F转移250nL 100倍终浓度的化合物。
3.2.3用1×激酶缓冲液配制2.5倍终浓度的激酶溶液。
3.2.4在化合物孔和阳性对照孔分别加10μL的2.5倍终浓度的激酶溶液；在阴性对照孔中加10μL的1×激酶缓冲液。
3.2.5反应板振荡混匀后室温孵育10分钟。
3.2.6用1×激酶缓冲液配制
Figure PCTCN2022086223-appb-000025
倍终浓度的ATP和激酶底物22的混合溶液。
3.2.7加入15μL的
Figure PCTCN2022086223-appb-000026
倍终浓度的ATP和底物的混合溶液。
3.2.8将384孔板1000rpm离心30秒，振荡混匀后室温孵育相应的时间。
3.2.9终止激酶反应。
3.2.10用酶标仪Caliper EZ Reader读取转化率。
4.数据分析
计算公式
抑制率％(Inhibition)＝(转化率％最大—转化率％样品)/(转化率％最大—转化率％最小)×100
其中：转化率％样品是样品的转化率读数；转化率％最小：阴性对照孔均值，代表没有酶活孔的转化率读数；转化率％最大：阳性对照孔比值均值，代表没有化合物抑制孔的转化率读数。
拟合量效曲线：以浓度的log值作为X轴，百分比抑制率为Y轴，采用分析软件GraphPad Prism 5的log(抑制剂)vs.响应-可变斜率(Variable slope)拟合量效曲线(四参数模型拟合)，从而得出各个化合物对 酶活性的IC 50值。
结果如下表A1显示：
表A1.本申请化合物对三种激酶抑制活性的IC 50值
化合物编号 TRKa IC 50(nM) TRKA(G595R)IC 50(nM) TRKC(G623R)IC 50(nM)
化合物1b 0.62 2.50 3.70
LOXO-195 0.47 1.60 2.50
化合物2 0.42 1.20 1.80
阳性对照1 0.39 0.72 1.70
化合物(I) 0.36 0.89 1.50
阳性对照2 0.43 0.97 1.40
化合物4 320 >1000 >1000
化合物5 127 561 >1000
化合物6 0.52 2.90 8.20
结果显示，化合物(I)对TRKa、TRKA(G595R)和TRKC(G623R)三种激酶具有高抑制活性。化合物(I)与化合物4-6相比，活性有显著差异，表明不同位置的氟取代对三种激酶抑制活性的影响区别较大。
测试例2：体外细胞活性
该测试例委托合肥中科普瑞昇生物医药科技有限公司完成，其中所用的NTRK突变细胞由该公司构建。
1.实验目的
测定本申请化合物对三种NTRK突变细胞(Ba/F3 LMNA-NTRK1-G667C，Ba/F3 EVT6-NTRK3-G623R，Ba/F3 LMNA-NTRK1-G595R)生长的抑制作用。
2.试剂和耗材
细胞系：
Figure PCTCN2022086223-appb-000027
试剂：
胎牛血清FBS(GBICO,Cat#10099-141)；
Figure PCTCN2022086223-appb-000028
Luminescent Cell Viability Assay(CTG,Promega,Cat#G7573)；96孔透明平底黑壁板(
Figure PCTCN2022086223-appb-000029
Cat#165305)；RPMI-1640(Hyclone，Cat#SH30809.01)
3.实验过程
3.1细胞培养和接种：
收集处于对数生长期的细胞并采用血小板计数器进行细胞计数，调整细胞浓度至3-6×10 4cell/mL，添加90μL细胞悬液至96孔板中，将96孔板中的细胞置于37℃、5％CO 2、95％湿度条件下培养过夜。
3.2药物稀释和加药：
用含1％的DMSO的培养基将待测化合物配制10倍药物溶液，最高浓度为10μM，依次3倍稀释，共得到9个浓度的药物溶液。在接种细胞的96孔板中每孔加入10μL以上配制的药物溶液，再加入90μL含1％的DMSO的培养基，得到最高浓度为1μM的药物溶液，依次3倍稀释，共9个浓度，每孔中DMSO终浓度为0.1％，每个浓度的药物设置三个复孔。将已加药的96孔板中的细胞置于37℃、5％CO 2、95％湿度条件下继续培养72小时，之后进行CTG分析。
3.3终点读板
每孔加入等体积的CTG溶液，将96孔板放置于室温20分钟以稳定冷光信号，读取冷光值。
4.数据处理
使用GraphPad Prism 7.0软件分析数据，利用非线性S曲线回归来拟合数据得出剂量-效应曲线，并由此计算IC 50值。
细胞存活率(％)＝(待测药孔冷光值-培养液对照孔冷光值)/(细胞对照孔冷光值-培养液对照孔冷光值)×100％。
实验结果如下表A2示：
表A2.本申请化合物对三株细胞抑制活性的IC 50值
Figure PCTCN2022086223-appb-000030
结果显示，化合物(I)对三种NTRK突变细胞(Ba/F3 LMNA-NTRK1-G667C，Ba/F3 EVT6-NTRK3-G623R，Ba/F3 LMNA-NTRK1-G595R)具有强的生长抑制作用。化合物(I)和2对三种NTRK突变细胞的抑制活性与对应的阳性化合物无明显差别，但化合物(I)对三种NTRK突变细胞的抑制活性明显高于对应的阳性对照2。
测试例3：肝微粒体稳定性试验
1.实验目的
测定本申请化合物在人、大鼠和小鼠肝微粒体中的稳定性。
2.试验材料及仪器
试剂及耗材：
试剂名称 供应商 货号 批号
人肝微粒体 BiolVT X008070 SDL
大鼠肝微粒体 BiolVT M00001 TIQ
小鼠肝微粒体 Biopredic MIC255037 BQM
3.实验步骤
3.1缓冲液和肝微粒体按下表进行准备，制成孵化液：
试剂 浓度 体积
磷酸盐缓冲液 100mM 216.25μL
肝微粒体 20mg/mL 6.25μL
3.2分别进行了以下两个实验：a)添加辅酶因子NADPH的孵化体系：向孵化液(主要包含肝微粒体、磷酸盐缓冲液)中添加25μL NADPH(10mM)，使得肝微粒体和NADPH的终浓度分别为0.5mg/mL和1mM；b)不添加辅酶因子NADPH的孵化体系：向孵化液中添加25μL磷酸盐缓冲剂(100mM)，使得肝微粒体的最终浓度为0.5mg/mL。上述孵化体系分别在37℃预热10分钟。
3.3向前述“步骤3.2”中所述的各孵化体系中，分别通过添加2.5μL阳性对照化合物或本申请受试化合物溶液(100μM)开始反应，所述阳性对照为维拉帕米(购自Sigma)，使得本申请受试化合物或阳性对照化合物的最终浓度为1μM。将添加化合物后的孵化溶液在37℃的水中分批孵化。
3.4在0.5、5、15、30和45分钟时分别从反应溶液中取出30μL等分试样，通过加入5倍体积的含有200nM咖啡因和100nM甲苯磺丁脲的冷乙腈来终止反应。将等分试样在3220g重力加速度下离心40分钟，取100μL上清液与100μL超纯水混合，然后用于LC-MS/MS分析。
3.5数据分析
峰面积由提取的离子色谱图确定。斜率值k是通过母体药物的剩余百分比相对于孵育时间曲线的自然对数的线性回归来确定的。
根据斜率值分别计算确定体外半衰期(t 1/2)，通过体外半衰期平均值转换为体外固有清除率(CLint，以μL/min/mg蛋白表示)。
实验结果如下表A3所示：
表A3.本申请化合物在人、大鼠和小鼠肝微粒体中的稳定性数据
Figure PCTCN2022086223-appb-000031
结果显示，本申请化合物(I)在人、大鼠和小鼠肝微粒体中具有良好的稳定性。化合物1b和2在人、大鼠和小鼠肝微粒体中的稳定性与对应的阳性化合物无明显差别，或在多数种属的肝微粒体中的稳定性变差，但本申请化合物(I)在人、大鼠和小鼠肝微粒体中的稳定性明显优于阳性对照2。
测试例4：SD大鼠静脉和口服给予受试化合物的体内药代动力学研究
1.试验动物
种属：SD大鼠，SPF级。来源：动物转移自实验机构动物储备库(999M-017)，上海西普尔-必凯实 验动物有限公司。数量：每种剂型3只。
2.供试品配制
2.1准确称取适量的供试品，加入终体积5％DMSO、10％聚乙二醇-15羟基硬脂酸酯、85％生理盐水，涡旋或超声使充分混匀，得到0.2mg/mL的给药溶液，用于静脉注射给药。
2.2准确称取适量的供试品，加入终体积5％DMSO、10％聚乙二醇-15羟基硬脂酸酯、85％生理盐水，涡旋或超声使充分混匀，得到0.5mg/mL的给药溶液，用于口服灌胃给药。
3.实验设计
Figure PCTCN2022086223-appb-000032
4.给药方式
给药前称重，根据体重，计算给药量。通过静脉或灌胃口服给药。
5.采血时间点
给药前及给药后0.083h，0.25h，0.5h，1h，2h，4h，6h，8h，24h。
6.样品采集和处置
经颈静脉或其它合适方式采血，每个样品采集约0.20mL，肝素钠抗凝，血液样本采集后放置于冰上，并于2小时内离心分离血浆(离心条件：离心力6800g，6分钟，2-8℃)。采集的血浆样本在分析前存放于-80℃冰箱内，分析后剩余血浆样本继续存放于-80℃冰箱暂存。
7.生物分析和数据处理
检测受试物血药浓度，进行血浆药物浓度-时间曲线绘制时，BLQ均记为0。进行药代参数计算时，给药前的浓度按照0计算；C max之前的BLQ(包括“No peak”)按照0计算；Cmax之后出现的BLQ(包括“No peak”)一律不参与计算。通过不同时间点的血药浓度数据，运用WinNonlin计算药代动力学参数，如AUC(0-t)，T 1/2，Cmax等。结果见表A4。
表A4.SD大鼠静脉和口服给予受试化合物的体内药代动力学研究数据
Figure PCTCN2022086223-appb-000033
Figure PCTCN2022086223-appb-000034
结果显示，化合物2在SD大鼠体内药代动力学参数(AUC/CL/F％)与阳性化合物相比变差，化合物(I)在SD大鼠体内药代动力学参数(AUC/CL/F％)与阳性对照2相比显著提高。
测试例5：受试化合物在MDCK-MDR1细胞系上的双向渗透性研究
1.1材料
MDCK-MDR1细胞购自荷兰癌症研究所，使用10到20代之间的细胞。
1.2试验设计
1.2.1细胞培养和种板
1)细胞接种前，向Transwell上室每孔中加入50μL细胞培养基，下层培养板内加入25mL细胞培养基。将培养板置于37℃,5％CO 2培养箱内孵育1小时后可用于接种细胞。
2)使用培养基重悬MDCK-MDR1细胞，使得终浓度为1.56×10 6cells/mL。将细胞悬液以50μL每孔加入到96孔Transwell培养板上室中。培养箱设置为37℃、5％CO 2、保证相对湿度95％培养4-8天。接种后48小时开始换液，培养4-8天，隔一天换一次培养基。
1.2.2细胞单层膜完整性的评价
1)将下层培养板中的原培养基移除并在上室加入新鲜预热的培养基。
2)用电阻仪(Millipore，USA)测量单层膜电阻，记录每孔电阻。
3)测定结束后，将Transwell培养板放回培养箱。
4)电阻值的计算：测定电阻值(ohms)×膜面积(cm 2)＝TEER值(ohm·cm 2)，若TEER值<42ohms·cm 2，则该孔不能用于穿透试验。
1.2.3溶液配制
1)使用DMSO将待测化合物配置成10mM的母液，
2)阳性对照化合物用DMSO配制为浓度10mM的母液。
1.2.4药物穿透试验
1)从培养箱中取出MDCK-MDR1 Transwell培养板。使用预热的HBSS(25mM HEPES,pH 7.4) 缓冲液润洗细胞单层膜两次，37℃条件下孵育30分钟。
2)对照化合物和试验化合物的原液在DMSO中稀释得到200μM溶液，然后用HBSS(10mM HEPES，pH7.4)稀释得到1μM工作溶液。孵育体系中DMSO的最终浓度为0.5％。
3)测定化合物由顶端到基底端的转运速率。向上层小室(顶端)加入125μL工作液，然后从下层小室(基底端)立刻转移50μL样本溶液加到装有200μL含内标的乙腈的96孔板作为0分钟给药样品(A-B)进行检测，并将235μL HBSS(25mM HEPES,pH 7.4)缓冲液加入到下层小室中。内标包含了(100nM阿普唑仑，200nM咖啡因和100nM甲苯磺丁酰胺)。将上述转移的50μL样本溶液以1000rpm速度涡旋10min。
4)测定化合物由基底端到顶端的转运速率。将285μL的工作溶液添加到下层小室(基底端)，然后立刻转移50μL至上层小室(顶端)样本溶液加到装有200μL含内标的乙腈的96孔板作为0分钟给药样品(B-A)进行检测，并将75μL HBSS(25mM HEPES,pH 7.4)缓冲液加入到上层小室中。内标包含了(100nM阿普唑仑，200nM咖啡因和100nM甲苯磺丁酰胺)，将上述转移的50μL样本溶液以1000rpm速度涡旋10min。由顶端到基底端方向与基底端到顶端的实验应同时进行。
5)下室和上室分别加入缓冲液后，将MDCK-MDR1 Transwell培养置于37℃条件下孵育2小时。
6)孵育结束后，分别从给药侧(上室：Ap→Bl flux，下室：Bl→Ap)与接收侧(下室：Ap→Bl flux，上室Bl→Ap)取50μL样本溶液至新的96孔板中，向孔板中加入4倍体积的含内标物的乙醇，内标物质包含(100nM阿普唑仑，200nM咖啡因和100nM甲苯磺丁酰胺)，涡旋10分钟后，于3220g离心40分钟。吸取100μL上清液与等体积超纯水混合后进行LC-MS/MS分析。
7)用荧光黄的渗漏评价孵育2小时后细胞单层膜的完整性。用HBSS(10mM HEPES,pH 7.4)稀释荧光黄储备液至最终浓度100μM，在上层小室(顶端)每个孔中加入荧光黄溶液100μL，下层小室(基底端)基底的每个孔中加300μL HBSS(25mM HEPES,pH 7.4)。37℃下孵育30分钟后，分别从每孔上下层吸出80μL溶液至一个新的96孔板中。使用酶标仪，激发波长485nm和发射波长530nm条件下进行荧光测定。
1.2.5分析条件
LC system:Shimadzu
MS analysis:Triple Quad 5500 instrument from AB Inc with an ESI interface
1)LC参数
柱温:40℃
色谱柱:Waters XSelect HSS T3 C18,2.5μM,2.1x 50mm
流动相:0.1％甲酸溶于水(A)和0.1％甲酸溶于乙腈(B)
进样体积:5μL
洗脱速率:0.6mL/min
Time(min) 0 0.2 0.7 1.2 1.25 1.5
％B 5 5 95 95 5 5
2)MS参数
离子源:Turbo spray
电离模型:ESI
扫描类型:多反应检测(MRM)
帘气:35L/min
碰撞气:9L/min
载气:50L/min
辅助气体:50L/min
温度:500℃
离子喷雾电压:+5500V(positive)
1.3数据分析
峰面积由离子色谱结果计算得出。化合物的表观渗透系数(Papp，单位：cm/s×10 -6)用以下公式计算得出：
Figure PCTCN2022086223-appb-000035
公式中：V A为接收端溶液的体积(Ap→Bl是0.3mL，Bl→Ap是0.1mL)，Area为Transwell-96孔板膜面积(0.143cm 2)；time为孵育时间(单位：s)；[drug]为药物浓度。
实验结果如下表A5所示：
表A5.受试化合物在同批次MDCK-MDR1细胞系上的双向渗透性研究数据
化合物编号 P app(A-B)(10 -6,cm/s) P app(B-A)(10 -6,cm/s) 外排比*
化合物(I) 11.13 51.91 4.67
阳性对照2 3.31 64.41 19.42
*外排比＝P app(B-A)/P app(A-B)
结果显示，化合物(I)的渗透性(P app(A-B))优于阳性对照2，表明本申请化合物1b更容易吸收进入细胞，同时，本申请化合物(I)具有较低的外排比，表明本申请化合物1b不易被外排，从而在细胞中能维持较高的药物浓度，产生更好的药效。因此，结合SD大鼠体内药代动力学研究，更加充分说明本申请化合物(I)在体内药代动力学参数(AUC/CL/F％)与阳性对照2相比具有显著提高。
测试例6：体外细胞活性评估
该测试例委托合肥中科普瑞昇生物医药科技有限公司完成，其中所用的细胞系由该公司构建。
1.实验目的
测定本申请化合物对6株BaF3细胞系的体外抗增殖作用。
2.试剂和耗材
细胞系：
细胞系 细胞类型 细胞数量/孔 培养基
Ba/F3-LMNA-NTRK1 悬浮 2000 RPMI 1640+10％FBS+1％PS
Ba/F3-LMNA-NTRK1-V573I 悬浮 2000 RPMI 1640+10％FBS+1％PS
Ba/F3-LMNA-NTRK1-F589L 悬浮 2000 RPMI 1640+10％FBS+1％PS
Ba/F3-LMNA-NTRK1-G667S 悬浮 2000 RPMI 1640+10％FBS+1％PS
Ba/F3-TEL-NTRK2 悬浮 2000 RPMI 1640+10％FBS+1％PS
Ba/F3-TEL-NTRK3 悬浮 2000 RPMI 1640+10％FBS+1％PS
材料：
Figure PCTCN2022086223-appb-000036
3.实验过程
细胞处理及给药
细胞培养条件
6株Ba/F3细胞系采用RPMI 1640(Biological Industries，Israel)+10％胎牛血清(Biological Industries，Israel)+1％双抗(Penicillin Streptomycin solution，Coring，USA)进行培养，细胞复苏后培养两代，待测试。
1000×化合物的制备
待测化合物溶于DMSO中配成的10mM的母液，再用DMSO稀释至1mM。按照3倍稀释制备成1.0000mM、0.3333mM、0.1111mM、0.03704mM、0.01235mM、0.00412mM、0.00137mM、0.00046mM、0.00015mM、0.00005mM储存于96孔药板中(Beaver,Suzhou)，共10个浓度梯度，同时采用同等体积的DMSO溶剂作为阴性对照。
20×化合物制备
将制备好的10个浓度梯度1000×的待测化合物，分别用完全培养基稀释50倍至20×化合物，储存于96孔药板中(Beaver,Suzhou)，共10个浓度梯度，同时采用同等体积的DMSO溶剂作为阴性对照。
铺板
取对数生长期细胞悬液，接种于96孔白色细胞培养板(Corning 3917，NY，USA)，每孔体积为95μl(约2000个细胞/孔)。
取5μl 20×待测化合物分别加入上述含有95μl细胞悬液的培养板中，混匀，每个浓度梯度2个孔。待测化合物最终检测浓度分别为1.0000μM、0.3333μM、0.1111μM、0.03704μM、0.01235μM、0.00412μM、0.00137μM、0.00046μM、0.00015μM、0.00005μM。
将培养板37℃、5％CO 2培养箱中孵育72小时。
读板
以下步骤按照Promega CellTiter-Glo发光法细胞活性检测试剂盒(Promega-G7573)的说明书来进行。
(1).将CellTiter-Glo缓冲液融化并放置至室温。
(2).将CellTiter-Glo底物放置至室温。
(3).在一瓶CellTiter-Glo底物中加入CellTiter-Glo缓冲液以溶解底物，从而配制CellTiter-Glo工作液。
(4).缓慢涡旋震荡使充分溶解。
(5).取出细胞培养板放置10分钟使其平衡至室温。
(6).在每孔中加入50μl的CellTiter-Glo工作液。
(7).将培养板在轨道摇床上振摇2分钟以诱导细胞裂解。
(8).培养板在室温放置10分钟以稳定发光信号。
(9).在MD SpectraMax Paradigm读板器上检测发光信号。
4.数据分析
SpectraMax Paradigm读数得出对应的每孔荧光值RLU。细胞活力(Cell viability)数据采用下列公式来处理：
Cell viability(％)＝(RLU Drug-RLU Min)/(RLU Max-RLU Min)*100％。在EXCEL中计算不同浓度化合物对应的细胞活力，然后用GraphPad Prism软件作细胞活力曲线图并计算相关参数，参数包括细胞最大和最小活力，IC 50值。
实验结果如下表A6示：
表A6.本申请化合物对同批次6株BaF3细胞系抑制活性的IC 50值
Figure PCTCN2022086223-appb-000037
从具体实施例化合物生物活性数据来看，本申请化合物(I)对6株BaF3细胞系具有强的生长抑制作用。化合物2对6株BaF3细胞系的抑制活性与对应的阳性化合物无明显差别，但本申请化合物(I)对6株BaF3细胞系的抑制活性明显高于阳性对照2。
实施例1：化合物(I)晶型A的制备
将20g化合物(I)溶于189.6g N,N-二甲基甲酰胺中，搅拌至溶解澄清；在冰浴下加入14.84g碳酸钾，并搅拌5-10min后升温至30±2℃，反应16-24h。反应完成后，向其中快速滴加600g纯化水，继续搅拌1-2h；过滤，滤饼用100g水淋洗两次；收集固体，通过液相制备纯化，纯化液浓缩、冻干后得白色固体16.2g。对固体进行XRPD检测，图谱如图1.所示，相关数据如表1.所示，所得固体即为式(I)化合物晶型A。DSC/TGA图谱显示(图2.)，其中DSC显示在66±2℃(onset)、370±2℃(onset)出现吸热峰；TGA显示在66±2℃失重8.6％，通过计算确认为2分子水合物、300℃后存在快速失重。
表1.
Figure PCTCN2022086223-appb-000038
晶型A包括不限于以下特征峰：7.383±0.2°、9.762±0.2°、15.157±0.2°、17.660±0.2°、21.002±0.2°、22.539±0.2°、26.300±0.2°；还包括以下特征峰：10.938±0.2°、16.564±0.2°、16.863±0.2°、19.142±0.2°、19.657±0.2°、19.861±0.2°、23.201±0.2°、23.737±0.2°、24.299±0.2°、28.359±0.2°。
实施例2：化合物(I)晶型C的制备1
称取1.0g化合物(I)晶型A分散在7ml乙醇中，78℃下打浆3h，过滤，收集固体，室温下真空干燥6h。对固体进行XRPD检测，图谱如图3.所示，相关数据如表2.所示，所得固体即为晶型C。DSC/TGA图谱显示(图4)，其中DSC显示在377±2℃(onset)出现吸热峰；TGA显示在300℃内无明显失重。
表2.
Figure PCTCN2022086223-appb-000039
晶型C包括不限于以下特征峰：6.878±0.2°、10.918±0.2°、13.921±0.2°、15.321±0.2°、18.401±0.2°、19.860±0.2°、24.321±0.2°；还包括以下特征峰：12.358±0.2°、16.302±0.2°、17.320±0.2°、17.698±0.2°、19.221±0.2°、20.779±0.2°、21.518±0.2°、22.441±0.2°、25.340±0.2°、26.141±0.2°、26.920±0.2°、27.820±0.2°。
实施例2.1化合物(I)晶型C的制备2
称取1.0g化合物(I)晶型A分散在7ml有机溶剂中，某温度下打浆3～24h，过滤，收集固体，室温下真空干燥6h。对固体进行XRPD检测，结果与实施例2中获得的图3基本上一致，为晶型C。其中有机溶剂包括不限于乙醇、异丙醇；某温度指在25℃～回流。
实施例3：化合物(I)晶型D的制备1
称取1.0mg化合物(I)晶型A分散在7ml碳酸二甲酯中，室温下打浆24h，过滤，收集固体，室温下真空干燥6h。对固体进行XRPD检测，图谱如图5所示，相关数据如表3.所示，所得固体即为晶型D。DSC/TGA图谱显示(图6)，其中DSC显示在375±2℃(onset)出现吸热峰；TGA显示在300℃内无明显失重。
表3.
Figure PCTCN2022086223-appb-000040
晶型D包括不限于以下特征峰：9.400±0.2°、10.510±0.2°、12.957±0.2°、13.300±0.2°、17.875±0.2°、18.840±0.2°、20.780±0.2°、22.940±0.2°；
还包括以下特征峰：10.084±0.2°、16.984±0.2°、19.240±0.2°、21.361±0.2°、22.324±0.2°、24.761±0.2°、29.820±0.2°、30.220±0.2°、33.098±0.2°。
实施例3.1化合物(I)晶型D的制备2
称取1.0g化合物(I)晶型A分散在7ml有机溶剂中，室温度下打浆24h，过滤，收集固体，室温下真空干燥6h。对固体进行XRPD检测，结果与实施例3中获得的图5基本上一致，为晶型D。
其中有机溶剂包括不限于乙酸甲酯、乙酸丁酯、丁酮、甲基异丁基酮。
实施例4：化合物(I)晶型E的制备
称取70mg化合物(I)晶型A分散在3ml乙醚中，室温下打浆24h，过滤，收集固体，室温下真空干燥6h。对固体进行XRPD检测，图谱图7所示，相关数据如表4.所示，所得固体即为晶型E。DSC/TGA图谱显示(图8.)，其中DSC显示在374±2℃(onset)出现吸热峰；TGA显示在300℃内无明显失重。
表4.
Figure PCTCN2022086223-appb-000041
晶型E包括不限于以下特征峰：6.539±0.2°、10.797±0.2°、13.596±0.2°、15.580±0.2°、17.741±0.2°、19.741±0.2°、24.619±0.2°、26.380±0.2°；
还包括以下特征峰：10.041±0.2°、13.022±0.2°、16.101±0.2°、16.959±0.2°、18.079±0.2°、18.980±0.2°、20.119±0.2°、21.680±0.2°、23.080±0.2°、25.340±0.2°、26.000±0.2°、28.279±0.2°。
实施例5：晶型L的制备
称取70mg化合物(I)晶型A分散在3ml三氯甲烷中，室温下打浆24h，过滤，收集固体，室温下真空干燥6h。对固体进行XRPD检测，图谱如图9.所示，相关数据如表5.所示，所得固体即为晶型L。DSC/TGA图谱显示(图10.)，其中DSC显示在373±2℃(onset)出现吸热峰；TGA显示在300℃内无明显失重。
表5.
Figure PCTCN2022086223-appb-000042
晶型L包括不限于以下特征峰：5.898±0.2°、9.017±0.2°、11.502±0.2°、13.602±0.2°、15.417±0.2°、16.897±0.2°、19.161±0.2°、21.389°±0.2°；还包括以下特征峰：16.538±0.2°、18.151±0.2°、19.938±0.2°、20.640±0.2°、22.583±0.2°、23.203±0.2°、23.840±0.2°、24.959±0.2°、27.446±0.2°、29.792±0.2°、30.100±0.2°、30.863±0.2°。
实施例6：化合物(I)晶型N的制备
称取70mg化合物(I)晶型A分散在3ml乙腈中，室温下打浆24h，过滤，收集固体，室温下真空干燥6h。对固体进行XRPD检测，图谱如图11.所示，相关数据如表6.所示，所得固体即为晶型N。DSC/TGA图谱显示(图12.)，其中DSC显示在370±2℃(onset)出现吸热峰；TGA显示在300℃内无明显失重。
表6.
Figure PCTCN2022086223-appb-000043
晶型N包括不限于以下特征峰：5.841±0.2°、8.901±0.2°、11.040±0.2°、13.442±0.2°、16.200±0.2°、18.899±0.2°、21.119±0.2°、23.501±0.2°；
还包括以下特征峰：7.296±0.2°、9.801±0.2°、12.058±0.2°、17.438±0.2°、20.037±0.2°、20.439±0.2°、22.660±0.2°、25.122±0.2°、27.261±0.2°、28.602±0.2°、29.019±0.2°、29.560±0.2°。
测试例7：化合物(I)各种晶型的理化性质测试
7.1溶解度：
测试方法：
分别将各批次样品适量置于10ml离心管中，加入各pH值(1.2、6.8、7.6)的缓冲液约5ml，置于37℃的恒温振荡器中振摇，使样品处于过饱和状态，隔3h调节溶液的pH值，使溶液pH值在缓冲液pH值的±0.02范围以内，恒温振摇24h后，测定各溶液的pH值，pH值应在缓冲液pH值的±0.1范围以内，静置1h后，取上清液0.5ml与0.5ml乙腈混匀，进样检测。
pH1.2缓冲液：量取盐酸9.0ml，加水稀释至500ml，摇匀，量取该溶液85.0ml，加水稀释至200ml，摇匀。
pH6.8缓冲液：称取磷酸二氢钾6.8g，加水超声溶解并稀释至250ml，摇匀，量取该溶液62.5ml，加入0.2mol/L NaOH溶液29.5ml，加水稀释至250ml，用NaOH溶液调节pH值为6.8。
pH7.6缓冲液：称取13.6g磷酸二氢钾，加水超声溶解并稀释至500ml，摇匀，量取该溶液50ml，加入0.2mol/L NaOH溶液42.4ml，加水稀释至200ml，用NaOH溶液调节pH值为7.6。
溶解度测试结果如下：
Figure PCTCN2022086223-appb-000044
从以上结果中，可以看出，晶型C在各pH值的缓冲液(HCl-NaOH)中溶解性均优于另外两个晶型。
7.2影响因素对稳定性的影响：
Figure PCTCN2022086223-appb-000045
从表中数据可以发现，各晶型化学稳定性良好，无明显降解；晶型C、D具有更好的晶型稳定性；晶型C具有更好的排杂能力。
7.3成药性考察
Figure PCTCN2022086223-appb-000046
从上表中可以确认晶型C具有更好的结晶度、晶型稳定性、抗研磨行，且更易制备。
7.4SD大鼠药代对比
供试品信息
Figure PCTCN2022086223-appb-000047
*:配制时需要折算纯度和游离基。
供试品配制
给药当天配制。
溶媒：编号1-6的供试品溶媒：0.5％CMC-Na
编号7供试品溶媒：50％PEG400+40％PG+10％Labrasol
实验动物
种属：SD大鼠，SPF级。
来源：动物转移自实验机构动物储备库(999M-017)。上海西普尔-必凯实验动物有限公司。
数量：转移：雄性25只。实验需要：雄性21只(多余动物采空白血浆)。
动物挑选：不进行随机分组
实验设计
Figure PCTCN2022086223-appb-000048
注：*,口服给药组动物给药前禁食过夜(10-14小时)，给药4小时后给食。
给药方式
给药前称重，根据体重，计算给药量。通过灌胃口服给药。
采血时间点：
给药前，给药后0.083h，0.25h，0.5h，1h，2h，4h，6h，8h，24h。经颈静脉采血，每个样品采集约0.20mL，肝素钠抗凝，采集后放置冰上。
血浆样品处理
血液样本采集后置于冰上，并于1小时之内离心分离血浆(离心条件：6800g，6分钟，2-8℃)。血浆样本在分析前存放时则放于-80℃冰箱内。
生物样品分析方法及所有样品的分析由美迪西普亚医药科技(上海)有限公司分析实验室完成，分析样品的同时进行质控样品的日内准确度评价，并要求超过66.7％的质控样品的准确度在80-120％之间。
结果分析
通过不同时间点的血药浓度数据，运用Phoenix WinNonlin7.0计算药代动力学参数，提供AUC0-t、AUC0-∞、MRT0-∞、Cmax、Tmax、和T1/2等参数及其平均值和标准差，结果见下表。
Figure PCTCN2022086223-appb-000049
从以上药代数据中可以确认，晶型C具有更大的最大血样浓度，且在更短的时间内达到最大血样浓度，具有更大的曲线下面积、生物利用度更高。
最后应当说明的是：以上实施例仅用以说明本发明的技术方案而非对其限制；尽管参照较佳实施例对本发明进行了详细的说明，所属领域的普通技术人员应当理解：依然可以对本发明的具体实施方式进行修改或者对部分技术特征进行等同替换；而不脱离本发明技术方案的精神，其均应涵盖在本发明请求保护的技术方案范围当中。

Claims (18)

  1. 一种化合物(I)的多晶型物或溶剂化物：
    Figure PCTCN2022086223-appb-100001
  2. 根据权利要求1所述化合物(I)的多晶型或溶剂化物，其特征在于，其X-射线粉末衍射图包括以下峰：6.878±0.2°、10.918±0.2°、13.921±0.2°、15.321±0.2°、18.401±0.2°、19.860±0.2°、24.321±0.2°。
  3. 根据权利要求2所述的化合物(I)的多晶型或溶剂化物，其特征在于，其X-射线粉末衍射图还包括以下峰：12.358±0.2°、16.302±0.2°、17.320±0.2°、17.698±0.2°、19.221±0.2°、20.779±0.2°、21.518±0.2°、22.441±0.2°、25.340±0.2°、26.141±0.2°、26.920±0.2°、27.820±0.2°。
  4. 根据权利要求2所述的化合物(I)的多晶型或溶剂化物，其特征在于，其X-射线粉末衍射图基本上如图3所示。
  5. 根据权利要求1所述化合物(I)的多晶型或溶剂化物，其特征在于，其X-射线粉末衍射图包括以下峰：9.400±0.2°、10.510±0.2°、12.957±0.2°、13.300±0.2°、17.875±0.2°、18.840±0.2°、20.780±0.2°、22.940±0.2°。
  6. 根据权利要求5所述的化合物(I)的多晶型或溶剂化物,其特征在于，其X-射线粉末衍射图还包括以下峰：10.084±0.2°、16.984±0.2°、19.240±0.2°、21.361±0.2°、22.324±0.2°、24.761±0.2°、29.820±0.2°、30.220±0.2°、33.098±0.2°。
  7. 根据权利要求5所述的化合物(I)的多晶型或溶剂化物，其特征在于，其X-射线粉末衍射图基本上如图5所示。
  8. 根据权利要求1所述化合物(I)的多晶型或溶剂化物，其特征在于，其X-射线粉末衍射图包括以下峰：6.539±0.2°、10.797±0.2°、13.596±0.2°、15.580±0.2°、17.741±0.2°、19.741±0.2°、24.619±0.2°、26.380±0.2°。
  9. 根据权利要求8所述的化合物(I)的多晶型或溶剂化物其特征在于，其X-射线粉末衍射图还包括以下峰：10.041±0.2°、13.022±0.2°、16.101±0.2°、16.959±0.2°、18.079±0.2°、18.980±0.2°、20.119±0.2°、21.680±0.2°、23.080±0.2°、25.340±0.2°、26.000±0.2°、28.279±0.2°。
  10. 根据权利要求8所述的化合物(I)的多晶型或溶剂化物，其特征在于，其X-射线粉末衍射图基本上如图7所示。
  11. 根据权利要求1所述化合物(I)的多晶型或溶剂化物，其特征在于，其X-射线粉末衍射图包括以下峰：5.898±0.2°、9.017±0.2°、11.502±0.2°、13.602±0.2°、15.417±0.2°、16.897±0.2°、19.161±0.2°、 21.389±0.2°；优选地，其X-射线粉末衍射图还包括以下特征峰：16.538±0.2°、18.151±0.2°、19.938±0.2°、20.640±0.2°、22.583±0.2°、23.203±0.2°、23.840±0.2°、24.959±0.2°、27.446±0.2°、29.792±0.2°、30.100±0.2°、30.863±0.2°；更优选地，其X-射线粉末衍射图基本上如图9所示。
  12. 根据权利要求1所述化合物(I)的多晶型或溶剂化物，其特征在于，其X-射线粉末衍射图包括以下峰：5.841±0.2°、8.901±0.2°、11.040±0.2°、13.442±0.2°、16.200±0.2°、18.899±0.2°、21.119±0.2°、23.501±0.2°；优选地，其X-射线粉末衍射图还包括以下特征峰：7.296±0.2°、9.801±0.2°、12.058±0.2°、17.438±0.2°、20.037±0.2°、20.439±0.2°、22.660±0.2°、25.122±0.2°、27.261±0.2°、28.602±0.2°、29.019±0.2°、29.560±0.2°；更优选地，其X-射线粉末衍射图基本上如图11所示。
  13. 根据权利要求1所述化合物(I)的多晶型或溶剂化物，其特征在于，其X-射线粉末衍射图包括以下峰：7.383±0.2°、9.762±0.2°、15.157±0.2°、17.660±0.2°、21.002±0.2°、22.539±0.2°、26.300±0.2°；优选地，其X-射线粉末衍射图还包括以下特征峰：10.938±0.2°、16.564±0.2°、16.863±0.2°、19.142±0.2°、19.657±0.2°、19.861±0.2°、23.201±0.2°、23.737±0.2°、24.299±0.2°、28.359±0.2°；更优选地，其X-射线粉末衍射图基本上如图1所示。
  14. 一种制备权利要求2至4中任一项所述的化合物(I)的多晶型或溶剂化物的方法，包括以下步骤：
    将化合物(I)的晶型A分散在有机溶剂中打浆，
    过滤，
    干燥；
    其中所述有机溶剂为：C 1-C 6烷基醇，优选乙醇、异丙醇；
    优选地，该方法包括将化合物(I)的晶型A分散在有机溶剂中在25℃～回流温度下打浆；
    优选地，该方法包括将化合物(I)的晶型A分散在有机溶剂中在25℃～回流温度下打浆3～24小时；
    优选地，其中所述干燥为真空干燥；
    优选地，该方法包括干燥5～7小时，例如6小时。
  15. 一种制备权利要求5至7任一项所述的化合物(I)的多晶型或溶剂化物的方法，包括以下步骤：
    将化合物(I)的晶型A分散在有机溶剂中打浆，
    过滤；
    干燥；
    其中所述有机溶剂为：碳酸二甲酯、乙酸甲酯、乙酸丁酯、丁酮、甲基异丁基酮；
    优选地，该方法包括将化合物(I)的晶型A分散在有机溶剂中在室温下打浆；
    优选地，该方法包括将化合物(I)的晶型A分散在有机溶剂中在室温下打浆3～24小时，优选24小时；
    优选地，其中所述干燥为真空干燥；
    优选地，该方法包括干燥5～7小时，例如6小时。
  16. 一种药物组合物，其包括治疗有效剂量的权利要求1-13的任一项所述的化合物(I)的多晶型或溶剂化物及其可药用的载体。
  17. 权利要求1-13中任一项所述的化合物(I)的多晶型物或溶剂化物或者权利要求16所述的药物组合 物在制备治疗疼痛、癌症、炎症和神经退行性疾病中的用途；优选地，所述疾病选自癌症；优选地，所述癌症为选自神经细胞瘤、卵巢癌、结肠直肠癌、黑素瘤、头和颈部的癌症、胃癌、肺癌、乳腺癌、成胶质细胞瘤、成神经管细胞瘤、分泌性乳腺癌、唾液腺癌、甲状腺乳头状癌、成人髓细胞白血病、胰腺癌、前列腺癌、阑尾癌、胆管癌、胃肠道间质瘤和婴儿纤维肉瘤的一种或多种。
  18. 权利要求1-13中任一项所述的化合物(I)的多晶型物或溶剂化物或者权利要求16所述的药物组合物在制备治疗由TRK介导的疾病中的用途；优选地，所述TRK介导的疾病为疼痛、癌症、炎症和神经退行性疾病中的一种或多种；更为优选地，所述疾病为癌症；优选地，所述癌症为选自神经细胞瘤、卵巢癌、结肠直肠癌、黑素瘤、头和颈部的癌症、胃癌、肺癌、乳腺癌、成胶质细胞瘤、成神经管细胞瘤、分泌性乳腺癌、唾液腺癌、甲状腺乳头状癌、成人髓细胞白血病、胰腺癌、前列腺癌、阑尾癌、胆管癌、胃肠道间质瘤和婴儿纤维肉瘤的一种或多种。
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