Solid forms of fluorine-containing macrocyclic compounds, methods of preparation and uses

Abstract

The present application relates to the technical field of drug development, and particularly relates to a polymorph or solvate of compound (I) with antitumor activity, a preparation method and application thereof.

Description

[0001] This application is based on and claims priority to CN application No. 202110391456.5, filed on April 12, 2021, and CN application No. 202111488763.1, filed on December 7, 2021. The disclosures of these two CN applications are incorporated herein by reference in their entirety. Technical Field

[0002] This application belongs to the field of drug development technology, specifically relating to a polymorph or solvate of a compound (I) with antitumor activity, its preparation method and application. Background Technology

[0003] The NTRK gene family includes NTRK1, NTRK2, and NTRK3, which are responsible for encoding the synthesis of tropomyosin receptor kinase (TRK) family proteins TRKA, TRKB, and TRKC, respectively. Neurotrophic factors binding to TRK proteins can induce receptor dimerization and phosphorylation, activating downstream PI3K, RAS / MAPK / ERK, and PLC-γ signaling cascades.

[0004] Alterations in the TRK signaling pathway, including gene fusions, protein overexpression, or single nucleotide changes, have been found to be the cause of many tumors. In particular, NTRK gene fusions are currently the most clearly defined cause of cancer. NTRK fusion proteins act as oncogenic drivers, promoting the growth and survival of cancer cells. This discovery has led to the emergence of NTRK gene fusions as a new target for cancer therapy.

[0005] In November 2018, LOXO-101, a first-generation drug that can effectively treat 17 types of tumors and targets patients with NTRK1 / NTRK2 / NTRK3 fusions, was launched in the United States. However, some cancer patients develop resistance to first-generation drugs. Resistance mutations in TRK kinases are one of the main causes of resistance. Therefore, it is necessary to develop more effective drugs that can overcome resistance to first-generation drugs. Summary of the Invention

[0006] Chengdu Better Pharmaceutical Co., Ltd. has developed a small molecule TRK or RET inhibitor, chemically named (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)-cyclobutane-octadecane-5-one, hereinafter referred to as compound (I), with the structure shown in formula (I):

[0007]

[0008] This small molecule inhibitor exhibits higher selectivity and safety in terms of bioactivity, particularly in vitro efficacy at mutation sites such as NTRK1G595R and NTRK3G623R, stability in human, rat, and mouse liver microsomes, drug metabolism, and oral bioavailability.

[0009] This application studies the polymorphs and solvates of compound (I). These polymorphs can improve the polymorph stability, impurity removal ability, thermodynamic stability (larger onset value) of the compound, and have better drug-like properties. From the perspective of preparation process, they are simpler to prepare, easier to control, and more suitable for industrial production.

[0010] This application provides a polymorph or solvate of compound (I) ((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)-cyclobutane-5-one):

[0011]

[0012] In some embodiments, the polymorph of compound (I) described in this application is crystal 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°. In some embodiments, the X-ray powder diffraction pattern of crystal form C of compound (I) further includes 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°, and 27.820±0.2°. In some embodiments, the X-ray powder diffraction pattern of crystal form C of compound (I) is substantially as follows: Figure 3 In some embodiments, the crystal form C of compound (I) is amorphous. In some embodiments, the DSC spectrum of crystal form C of compound (I) includes a characteristic peak at 377 ± 2 °C (onset). In some embodiments, the DSC spectrum of crystal form C of compound (I) includes a characteristic peak at 377 ± 2 °C (onset). Figure 4 The characteristic peaks are shown at essentially the same temperature. In some embodiments, the DSC spectrum of the crystal form C of compound (I) is essentially as follows. Figure 4As shown. In some embodiments, the TGA spectrum of crystal form C of compound (I) is essentially as follows. Figure 4 Show.

[0013] In some embodiments, the polymorph of compound (I) described in this application is crystal 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°. In some embodiments, the X-ray powder diffraction pattern of crystal form D of the compound (I) further includes 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°, and 33.098±0.2°. In some embodiments, the X-ray powder diffraction pattern of crystal form D of the compound (I) is substantially as follows: Figure 5 In some embodiments, the crystal form D of the compound (I) is amorphous. In some embodiments, the DSC spectrum of the crystal form D of the compound (I) includes a characteristic peak at 375 ± 2 °C (onset). In some embodiments, the DSC spectrum of the crystal form D of the compound (I) includes a characteristic peak at 375 ± 2 °C (onset). Figure 6 The characteristic peaks are shown at essentially the same temperature. In some embodiments, the DSC spectrum of crystal form D of compound (I) is essentially as follows: Figure 6 As shown. In some embodiments, the TGA spectrum of crystal form D of compound (I) is essentially as follows. Figure 6 Show.

[0014] In some embodiments, the polymorph of compound (I) of this application is crystal 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°, and 26.380±0.2°. In some embodiments, the X-ray powder diffraction pattern of crystal form E of the compound (I) further includes 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°, and 28.279±0.2°. In some embodiments, the X-ray powder diffraction pattern of crystal form E of the compound (I) is substantially as follows: Figure 7 In some embodiments, the crystal form E of compound (I) is amorphous. In some embodiments, the DSC spectrum of crystal form E of compound (I) includes a characteristic peak at 374 ± 2 °C (onset). In some embodiments, the DSC spectrum of crystal form E of compound (I) includes a characteristic peak at 374 ± 2 °C (onset). Figure 8 The characteristic peaks are shown at essentially the same temperature. In some embodiments, the DSC spectrum of crystal form E of compound (I) is essentially as follows. Figure 8 As shown. In some embodiments, the TGA spectrum of crystal form E of compound (I) is essentially as follows. Figure 8 Show.

[0015] In some embodiments, the polymorph of compound (I) described in this application is crystal 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°. In some embodiments, the X-ray powder diffraction pattern of crystal form L of the compound (I) further includes 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°, and 30.863±0.2°. In some embodiments, the X-ray powder diffraction pattern of crystal form L of the compound (I) is substantially as follows: Figure 9 In some embodiments, the crystal form L of compound (I) is amorphous. In some embodiments, the DSC spectrum of crystal form L of compound (I) includes a characteristic peak at 373 ± 2 °C (onset). In some embodiments, the DSC spectrum of crystal form L of compound (I) includes a characteristic peak at 373 ± 2 °C (onset). Figure 10 The characteristic peaks are shown at essentially the same temperature. In some embodiments, the DSC spectrum of crystal form L of compound (I) is essentially as follows. Figure 10 As shown. In some embodiments, the TGA spectrum of crystal form L of compound (I) is essentially as follows. Figure 10 Show.

[0016] In some embodiments, the polymorph of compound (I) described in this application is crystal 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°. In some embodiments, the X-ray powder diffraction pattern of crystal form N of compound (I) further includes 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°, and 29.560±0.2°. In some embodiments, the X-ray powder diffraction pattern of crystal form N of compound (I) is substantially as follows: Figure 11 In some embodiments, the crystal form N of compound (I) is amorphous. In some embodiments, the DSC spectrum of crystal form N of compound (I) includes a characteristic peak at 370 ± 2 °C (onset). In some embodiments, the DSC spectrum of crystal form N of compound (I) includes a characteristic peak at 370 ± 2 °C (onset). Figure 12 The characteristic peaks are shown at essentially the same temperature. In some embodiments, the DSC spectrum of the crystal form N of compound (I) is essentially as follows. Figure 12 As shown. In some embodiments, the TGA spectrum of crystal form N of compound (I) is essentially as follows. Figure 12 Show.

[0017] In some embodiments, the polymorph of compound (I) described in this application is crystal 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 some embodiments, the... The X-ray powder diffraction pattern of crystal form A of compound (I) also includes 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°, and 28.359±0.2°. In some embodiments, the X-ray powder diffraction pattern of crystal form A of compound (I) is substantially as follows: Figure 1In some embodiments, crystal form A of compound (I) is a hydrate of compound (I), wherein the molar ratio of compound (I) to water is 1:2. In some embodiments, the DSC spectrum of crystal form A of compound (I) includes a characteristic peak at 66 ± 2 °C (onset). In some embodiments, the DSC spectrum of crystal form A of compound (I) includes a characteristic peak at 370 ± 2 °C (onset). In some embodiments, the DSC spectrum of crystal form A of compound (I) includes a characteristic peak at 370 ± 2 °C (onset). Figure 2 The characteristic peaks are shown at essentially the same temperature. In some embodiments, the DSC spectrum of crystal form A of compound (I) is essentially as follows: Figure 2 As shown. In some embodiments, the TGA spectrum of crystal form A of compound (I) is essentially as follows. Figure 2 Show.

[0018] This application also provides a method for preparing crystal form C of compound (I), comprising the following steps:

[0019] The crystal form A of compound (I) was dispersed in an organic solvent and slurryed.

[0020] filter,

[0021] dry.

[0022] In some embodiments, in the method for preparing crystal form C of compound (I), the organic solvent is a C1-C6 alkyl alcohol, preferably ethanol or isopropanol.

[0023] In some embodiments, the method for preparing crystal form C of compound (I) includes dispersing crystal form A of compound (I) in an organic solvent and pulping it at 25°C to reflux temperature;

[0024] In some embodiments, the method for preparing crystal form C of compound (I) includes dispersing crystal form A of compound (I) in an organic solvent and slurrying at 25°C to reflux temperature for 3 to 24 hours.

[0025] In some embodiments, the drying in the method for preparing crystal form C of compound (I) is vacuum drying.

[0026] In some embodiments, the method for preparing crystal form C of compound (I) includes drying for 5 to 7 hours, for example, 6 hours.

[0027] In some embodiments, the method for preparing crystal form C of compound (I) includes the following steps: dispersing crystal form A in an organic solvent, pulping at a certain temperature for a certain time, filtering, and vacuum drying for a certain time; wherein the organic solvent is: C1-C6 alkyl alcohol, preferably ethanol or isopropanol; wherein the temperature is 25°C to reflux; wherein the pulping time is 3 to 24 hours; and wherein the drying time is 6 hours.

[0028] This application also provides a method for preparing crystal form D of compound (I), comprising the following steps:

[0029] The crystal form A of compound (I) was dispersed in an organic solvent and slurryed.

[0030] filter;

[0031] dry.

[0032] In some embodiments, in the method for preparing crystal form D of compound (I), the organic solvent is: dimethyl carbonate, methyl acetate, butyl acetate, butanone, methyl isobutyl ketone, preferably dimethyl carbonate.

[0033] In some embodiments, the method for preparing crystal form D of compound (I) includes dispersing crystal form A of compound (I) in an organic solvent and slurrying at room temperature.

[0034] In some embodiments, the method for preparing crystal form D of compound (I) includes dispersing crystal form A of compound (I) in an organic solvent and slurrying at room temperature for 3 to 24 hours, preferably 24 hours.

[0035] In some embodiments, the drying in the method for preparing crystal form D of compound (I) is vacuum drying.

[0036] In some embodiments, the method for preparing crystal form D of compound (I) includes drying for 5 to 7 hours, for example, 6 hours.

[0037] In some embodiments, the method for preparing crystal form D of compound (I) includes the following steps: dispersing crystal form A in an organic solvent, pulping at room temperature for a certain time, filtering, and vacuum drying for a certain time; wherein the organic solvent is: methyl acetate, butyl acetate, butanone, or methyl isobutyl ketone; wherein the pulping time is 3 to 24 hours; preferably 24 hours; and the drying time is 6 hours.

[0038] Another aspect of this application provides a pharmaceutical composition comprising a polymorph or solvate of a therapeutically effective dose of compound (I) and a pharmaceutically acceptable carrier thereof.

[0039] Another aspect of this application provides the use of a polymorph or solvate of compound (I) or a pharmaceutical composition in the preparation of a treatment for pain, cancer, inflammation, or neurodegenerative diseases. In some embodiments, the disease is selected from cancer. In some embodiments, the cancer is selected from one or more of neurocytomas, ovarian cancer, colorectal cancer, melanoma, cancers of the head and neck, gastric cancer, lung cancer, breast cancer, glioblastoma, medulloblastoma, secretory breast cancer, salivary gland cancer, papillary thyroid carcinoma, adult myeloid leukemia, pancreatic cancer, prostate cancer, appendix cancer, bile duct cancer, gastrointestinal stromal tumors, and infantile fibrosarcoma.

[0040] This application, in another aspect, provides the use of a polymorph or solvate of compound (I) or a pharmaceutical composition in the preparation of a treatment for TRK-mediated diseases. In some embodiments, the TRK-mediated disease is one or more of pain, cancer, inflammation, and neurodegenerative diseases. In some embodiments, the disease is cancer. In some embodiments, the cancer is one or more selected from neurocytoma, ovarian cancer, colorectal cancer, melanoma, cancers of the head and neck, gastric cancer, lung cancer, breast cancer, glioblastoma, medulloblastoma, secretory breast cancer, salivary gland cancer, papillary thyroid carcinoma, adult myeloid leukemia, pancreatic cancer, prostate cancer, appendix cancer, bile duct cancer, gastrointestinal stromal tumor, and infantile fibrosarcoma.

[0041] Another aspect of this application provides a polymorph or solvate of compound (I) or a pharmaceutical composition for treating pain, cancer, inflammation, or neurodegenerative diseases. In some embodiments, the disease is selected from cancer. In some embodiments, the cancer is selected from one or more of neurocytomas, ovarian cancer, colorectal cancer, melanoma, cancers of the head and neck, gastric cancer, lung cancer, breast cancer, glioblastoma, medulloblastoma, secretory breast cancer, salivary gland cancer, papillary thyroid carcinoma, adult myeloid leukemia, pancreatic cancer, prostate cancer, appendix cancer, bile duct cancer, gastrointestinal stromal tumors, and infantile fibrosarcoma.

[0042] This application, in another aspect, provides a polymorph or solvate of compound (I) or a pharmaceutical composition for treating TRK-mediated diseases. In some embodiments, the TRK-mediated disease is one or more of pain, cancer, inflammation, and neurodegenerative diseases. In some embodiments, the disease is cancer. In some embodiments, the cancer is one or more selected from neurocytoma, ovarian cancer, colorectal cancer, melanoma, head and neck cancer, gastric cancer, lung cancer, breast cancer, glioblastoma, medulloblastoma, secretory breast cancer, salivary gland cancer, papillary thyroid carcinoma, adult myeloid leukemia, pancreatic cancer, prostate cancer, appendix cancer, bile duct cancer, gastrointestinal stromal tumor, and infantile fibrosarcoma.

[0043] This application, in another aspect, provides a method for treating pain, cancer, inflammation, or neurodegenerative diseases, comprising administering a therapeutically effective amount of a polymorph or solvate of the compound (I) described in this application, or a pharmaceutical composition, to a subject in need. In some embodiments, the disease is selected from cancer. In some embodiments, the cancer is selected from one or more of neurocytomas, ovarian cancer, colorectal cancer, melanoma, head and neck cancers, gastric cancer, lung cancer, breast cancer, glioblastoma, medulloblastoma, secretory breast cancer, salivary gland cancer, papillary thyroid carcinoma, adult myeloid leukemia, pancreatic cancer, prostate cancer, appendix cancer, bile duct cancer, gastrointestinal stromal tumors, and infantile fibrosarcoma.

[0044] This application, in another aspect, provides a method for treating a TRK-mediated disease, comprising administering to a subject in need a therapeutically effective amount of a polymorph or solvate of the compound (I) described in this application, or a pharmaceutical composition. In some embodiments, the TRK-mediated disease is one or more of pain, cancer, inflammation, and neurodegenerative diseases. In some embodiments, the disease is cancer. In some embodiments, the cancer is one or more selected from neurocytoma, ovarian cancer, colorectal cancer, melanoma, head and neck cancer, gastric cancer, lung cancer, breast cancer, glioblastoma, medulloblastoma, secretory breast cancer, salivary gland cancer, papillary thyroid carcinoma, adult myeloid leukemia, pancreatic cancer, prostate cancer, appendix cancer, bile duct cancer, gastrointestinal stromal tumor, and infantile fibrosarcoma. Attached Figure Description

[0045] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, illustrate exemplary embodiments of the invention and, together with their description, serve to explain the invention and do not constitute an undue limitation thereof. In the drawings:

[0046] Figure 1. The XRPD pattern of crystal form A of compound (I) is shown;

[0047] Figure 2. The DSC / TGA spectrum of crystal form A of compound (I) is shown.

[0048] Figure 3. The XRPD spectrum of compound (I) without crystal form C is shown;

[0049] Figure 4. The DSC / TGA spectrum of the crystal-free C of compound (I) is shown.

[0050] Figure 5. The XRPD spectrum of compound (I) without crystal form D is shown;

[0051] Figure 6. The DSC / TGA spectrum of the crystal-free form D of compound (I) is shown.

[0052] Figure 7. The XRPD spectrum of compound (I) without crystal form E is shown;

[0053] Figure 8. The DSC / TGA spectrum of the crystal-free form E of compound (I) is shown.

[0054] Figure 9. The XRPD spectrum of the crystal-free L form of compound (I) is shown;

[0055] Figure 10. The DSC / TGA spectrum of the crystal-free form L of compound (I) is shown.

[0056] Figure 11. The XRPD spectrum of compound (I) without crystal N is shown;

[0057] Figure 12. The DSC / TGA spectrum of the crystal-free N of compound (I) is shown. Detailed Implementation

[0058] 1. Terminology

[0059] As used in this application, the term "pharmaceutical composition" includes products comprising a therapeutically effective amount of the compounds of this application, as well as any products derived directly or indirectly from polymorphs or solvates of the compounds of this application. The pharmaceutical composition may be administered via, for example, oral or parenteral routes. The pharmaceutical compositions of this application can be prepared into various dosage forms, including but not limited to tablets, capsules, solutions, suspensions, granules, or injections, using conventional methods in the art, for administration via, for example, oral or parenteral routes.

[0060] As used in this application, the term "effective amount" means an amount sufficient to achieve the desired therapeutic effect, such as an amount that reduces symptoms associated with the disease to be treated.

[0061] As used in this application, the term "treatment" aims to alleviate or eliminate a targeted disease state or symptom. A subject is successfully "treated" if, in accordance with the methods described in this application, a therapeutically effective amount of a compound, its tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, and the subject exhibits an observable and / or detectable reduction or improvement in one or more indications and symptoms. It should also be understood that treatment of the disease state or symptom includes not only complete treatment but also the achievement of some biological or medically relevant outcome without achieving complete treatment.

[0062] It should also be noted that the dosage and method of administration of the compounds in this application depend on many factors, including the patient's age, weight, sex, natural health condition, nutritional status, the activity intensity of the compound, the time of administration, the metabolic rate, the severity of the disease, and the subjective judgment of the treating physician. The preferred dosage is between 0.001 and 1000 mg / kg body weight / day.

[0063] As used in this application, the term "medicinal" means compounds, materials, compositions, and / or dosage forms that are suitable for contact with human and animal tissues to the extent of reasonable medical judgment, without excessive toxicity, irritation, allergic reactions, or other problematic complications, in proportion to a reasonable benefit / risk ratio.

[0064] As used in this application, "polymorph" or "polymorphic material" refers to a crystal form having the same chemical composition but different spatial arrangements of the molecules, atoms, and / or ions constituting the crystal. Although polymorphs have the same chemical composition, they differ in their 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. Based on their temperature-stability relationship, two polymorphs can be either monomorphic or tautomorphic. For monomorphic systems, the relative stability between the two solid phases remains constant with temperature changes. Conversely, in tautomorphic systems, there is a transition temperature at which the stability of the two phases is interchanged (Theory and Origin of Polymorphism in "Polymorphism in Pharmaceutical Solids" (1999) ISBN: 8247-0237). This phenomenon of compounds existing in different crystal structures is called pharmaceutical polymorphism.

[0065] As used in this application, the term "subject" refers to mammalian subjects, including but not limited to humans, cats, dogs, horses, cattle, sheep, monkeys, etc., wherein human subjects include male and female subjects, and include newborns, infants, adolescents, teenagers, adults, and elderly subjects.

[0066] In this application, if the name and structural formula of the same compound are inconsistent, the structural formula shall prevail.

[0067] In this application, the X-ray powder diffraction pattern (XRPD pattern) is an X-ray powder diffraction pattern expressed in 2θ angle using Cu-Kα radiation.

[0068] In this application, the terms "substantially identical to Figure X" and "substantially as shown in Figure X" used to define a plot have the same meaning and are intended to indicate that, given acceptable deviations in the art, those skilled in the art consider the plot to be identical to the reference plot. Such deviations may be caused by factors known in the art related to instrumentation, operating conditions, and human factors. In some embodiments, two plots are considered substantially identical or substantially the same when the positions of the characteristic peaks of the two plots vary by no more than ±5%, ±4%, ±3%, ±2%, or ±1%. For example, those skilled in the art will understand that the endothermic onset and peak temperatures measured by differential scanning calorimetry (DSC) can vary significantly with experiments. For example, those skilled in the art can readily determine whether two X-ray diffraction patterns or two DSC patterns are substantially identical. In some embodiments, two X-ray diffraction patterns are considered substantially identical when the 2θ angle of the characteristic peaks of the two X-ray diffraction patterns varies by no more than ±0.3°, ±0.2°, or ±0.1°.

[0069] The crystalline structures described in this 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. Techniques for crystallizing or recrystallizing crystalline structures from solvent mixtures include solvent evaporation, lowering the temperature of the solvent mixture, crystallization of a supersaturated solvent mixture of the molecule and / or salt, lyophilization of the solvent mixture, and adding an antisolvent to the solvent mixture. High-throughput crystallization techniques, including polymorphs, can be used to prepare crystalline structures. Methods for preparing and characterizing drug crystals, including polymorphs, are disclosed in Solid-State Chemistry of Drugs, SRByrn, RRPfeiffer, and JGStowell, 2nd edition, SSCI, West Lafayette, Indiana, 1999.

[0070] Crystal structures disclosed or claimed in this application may exhibit similar but not identical analytical properties within a reasonable margin of error, depending on experimental conditions, purity, equipment, and other constant 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 scope and spirit of this invention without departing from its scope. Other embodiments of the invention will be apparent to those skilled in the art based on consideration of the specification and practice of the invention disclosed herein. The applicant expects this specification and examples to be considered exemplary and not limiting of its scope.

[0071] As used in this application, the terms "room temperature" or "RT" refer to an ambient temperature of 20 to 25°C (68-77°F).

[0072] As used in this application, the term "pharmaceutical carrier" means a diluent, additive, or medium that is administered with a therapeutic agent and is suitable, to the extent of reasonable medical judgment, for contact with human and / or other animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0073] Pharmaceutically usable carriers that can be used in the pharmaceutical compositions of this application include, but are not limited to, sterile liquids such as water and oils, including those of petroleum, animal, plant, 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 solutions of glucose and glycerol can also be used as liquid carriers, particularly for injectable solutions. Starch, glucose, lactose, sucrose, gelatin, maltose, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, etc., can also be used as carriers. The pharmaceutical compositions may also contain small amounts of wetting agents, emulsifiers, pH buffers, preservatives, antioxidants, flavoring agents, fragrances, solubilizers, osmotic pressure regulators, colorants, etc., as needed. Oral formulations may contain standard carriers such as binders, fillers, disintegrants, lubricants, etc.

[0074] The pharmaceutical composition described in this application can be administered by methods known in the art, such as, but not limited to, any of the following: oral, spray inhalation, rectal, nasal, buccal, topical, and extracorporeal administration, such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intrasternal, and intracranial injection or infusion, or administration via an external implantation device. Oral, intramuscular, or intravenous administration is preferred. For these routes of administration, suitable dosage forms can be used to administer the pharmaceutical composition of this application.

[0075] The dosage form may be a solid dosage form, a semi-solid dosage form, a liquid dosage form, or a gaseous dosage form, 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, and syrups.

[0076] The pharmaceutical composition described in this invention can be prepared by any method known in the art, such as by mixing, dissolving, granulating, sugar coating, milling, emulsifying, lyophilizing, etc.

[0077] 2. Analytical Methods

[0078] In this invention, X-ray powder diffraction (XRPD) analysis was performed using a Dandong Haoyuan DX-2700BH Cu target irradiation. The detection range was 3° to 40°, with a step size of 0.02° and a speed of 0.2 s. -1 .

[0079] The simultaneous thermal analyzer (TGA / DSC) used in this invention is a Mettler-Toledo TGA / DSC 3. + The instrument's heating rate is 10 K / min. Specific Implementation

[0081] The present invention will be further illustrated by the following embodiments. These embodiments are only for illustrating the technical solutions of the present invention and are not intended to limit the scope of the present invention. Unless otherwise specified, specific conditions in the embodiments are performed according to conventional conditions or manufacturer's recommended conditions. Reagents or instruments used without specified manufacturers are all commercially available conventional products.

[0082] Preparation Example 1: Preparation of ethyl 5-chloro-6-flupyrazole[1,5-a]pyrimidine-3-carboxylate

[0083]

[0084] Step 1: Preparation of 2-fluoromalonic acid

[0085]

[0086] At room temperature, 5.0 g of diethyl 2-fluoromalonate and 17.3 g of sodium hydroxide were weighed into an ethanol / water mixture (100 / 100 mL). The reaction was allowed to proceed overnight, and LC-MS showed that the reaction was complete. The system was concentrated to remove the ethanol, and 50 mL of water was added. The pH was adjusted to approximately 1 with concentrated hydrochloric acid. The mixture was extracted four times with methyl tert-butyl ether, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give 3.7 g of the title compound, which could be used in the next reaction without further purification.

[0087] MS(ESI)m / z(MH) + =121.1.

[0088] Step 2: Preparation of ethyl 5,7-dichloro-6-flupyrazole[1,5-a]pyrimidine-3-carboxylate

[0089]

[0090] At room temperature, 2.0 g of 2-fluoromalonic acid and 1.7 g of ethyl 5-amino-1H-pyrazole-4-carboxylic acid were weighed into phosphorus oxychloride (20 mL). Then, 2 mL of N,N-dimethylformamide and 4.9 g of N,N-diethylaniline were added to the system. The system was heated to 110 °C and reacted for 3 hours. LC-MS showed that the reaction was complete. The system was concentrated to remove phosphorus oxychloride, and then poured into 100 mL of saturated sodium bicarbonate solution to maintain the solution alkaline. The solution was extracted three times with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography. The resulting solid was washed with petroleum ether and dried to give 1.7 g of the title compound.

[0091] MS(ESI)m / z(M+H) + =278.0.

[0092] 1 H NMR (400MHz, DMSO-d6) δ8.80 (s, 1H), 4.33 (q, J = 7.2Hz, 2H), 1.32 (t, J = 7.0Hz, 3H).

[0093] Step 3: Preparation of ethyl 5-chloro-6-flupyrazole[1,5-a]pyrimidine-3-carboxylate

[0094]

[0095] 1.14 g of ethyl 5,7-dichloro-6-flupyrazole[1,5-a]pyrimidine-3-carboxylate and 800 mg of ammonium chloride were weighed into an ethanol / tetrahydrofuran / water solution (30 / 10 / 20 mL). Zinc powder (1.3 g) was added while stirring. After reacting for 5 minutes, the zinc powder was filtered, the filter cake was washed with ethyl acetate, the filtrate was collected, dried over anhydrous sodium sulfate, filtered, concentrated, and the crude product was purified by column chromatography to obtain 800 mg of the title compound.

[0096] MS(ESI)m / z(M+H) + =244.0.

[0097] 1 H NMR (400MHz, DMSO-d6) 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).

[0098] Preparation Example 2: Preparation of (R)-1-(5-fluoro-2-methoxypyridin-3-yl)ethyl-1-amine hydrochloride

[0099]

[0100] Step 1: Preparation of (R)-N-(((5-fluoro-2-methoxypyridin-3-yl)methylene)-2-methylpropane-2-sulfinamide

[0101]

[0102] (R)-2-methylpropane-2-sulfinamide (12.9 g) was dissolved in tetrahydrofuran (100 mL), followed by the addition of 5-fluoro-2-methoxynicotinaldehyde (15.0 g) and cesium carbonate (40.9 g). The reaction was carried out at room temperature for 2 hours, and TLC showed that the starting material was completely consumed. The mixture was filtered, and the filter cake was washed three times with tetrahydrofuran. The resulting filtrate was backwashed once with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to give 23.0 g of the title compound.

[0103] MS(ESI)m / z(M+H) + =259.1.

[0104] 1 H NMR (400MHz, DMSO-d6) 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).

[0105] Step 2: Preparation of (R)-N-((R)-1-(5-fluoro-2-methoxypyridin-3-yl)ethyl)-2-methylpropane-2-sulfinamide

[0106]

[0107] (R)-N-(((5-fluoro-2-methoxypyridin-3-yl)methylene)-2-methylpropane-2-sulfinamide (5.0 g) was dissolved in tetrahydrofuran (40 mL). After cooling the system to -78 °C, methyl magnesium bromide (7.8 mL, 3 M) was slowly added dropwise, maintaining the system temperature below -65 °C. After the addition was complete, the system was allowed to return to room temperature naturally, and the reaction was continued for 1 hour. TLC showed that the reaction was complete. The reaction system was poured into a saturated ammonium chloride aqueous solution (1 L), extracted with ethyl acetate, and the organic phases were combined. The mixture was backwashed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to give 4.5 g of the title compound.

[0108] MS(ESI)m / z(M+H) + =275.2.

[0109] 1H NMR (400MHz, DMSO-d6) 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).

[0110] Step 3: Preparation of (R)-1-(5-fluoro-2-methoxypyridin-3-yl)ethyl-1-amine hydrochloride

[0111]

[0112] At room temperature, (R)-N-((R)-1-(5-fluoro-2-methoxypyridin-3-yl)ethyl)-2-methylpropane-2-sulfinamide (4.5 g) was dissolved in a hydrogen chloride-dioxane solution (30 mL) and reacted overnight. LC-MS showed complete consumption of the starting material. The system was concentrated to obtain 3.1 g of crude product with an ee greater than 95%, which could be used directly in the next step without purification.

[0113] MS(ESI)m / z(M+H) + =171.2.

[0114] 1 H NMR(400MHz,DMSO-d6)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).

[0115] Preparation Example 3: Compound of Formula (I) (chemical name: (3) 1 S,3 3 S,6 3 E,6 4 E,8R)-1 5 6 6 Preparation of -difluoro-8-methyl-2-oxa-4,7-diaza-6(3,5)-pyrazolo[1,5-a]pyrimidine-1(2,3)-pyridine 3-3,(1,3)-cyclobutane-5-one

[0116]

[0117] Step 1: Preparation of (R)-6-fluoro-5-((1-(5-fluoro-2-methoxypyridin-3-yl)ethyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid ethyl ester

[0118]

[0119] (R)-1-(5-fluoro-2-methoxypyridin-3-yl)ethyl-1-amine hydrochloride (1.0 g) was dissolved in acetonitrile (20 mL), and N,N-diisopropylethylamine (1.9 g) and ethyl 5-chloro-6-fluoropyrazolo[1,5-a]pyrimidine-3-carboxylate (1.2 g) were added sequentially. The system was reacted at 60 °C for 3 hours, and TLC showed that the reaction was complete. The system was poured into water (50 mL), extracted with dichloromethane, and the organic phases were combined. The mixture was backwashed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and the crude product was purified by column chromatography to give 1.1 g of the title compound.

[0120] MS(ESI)m / z(M+H) + =378.2.

[0121] 1 H NMR (400MHz, DMSO-d6) 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).

[0122] Step 2: Preparation of (R)-6-fluoro-5-(((1-(5-fluoro-2-methoxypyridin-3-yl)ethyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid

[0123]

[0124] At room temperature, 1.1 g of (R)-6-fluoro-5-((1-(5-fluoro-2-methoxypyridin-3-yl)ethyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid ethyl ester was dissolved in ethanol / water (5 / 15 mL), and 584 mg of sodium hydroxide was added. The system was reacted overnight at 50 °C, and TLC showed that the reaction was complete. The system was concentrated to remove ethanol, and the residue was poured into water (20 mL). The pH was adjusted to about 5 with hydrogen chloride solution (2 M), extracted with dichloromethane, and the organic phases were combined. The mixture was backwashed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 800 mg of crude product, which could be used directly in the next step without purification.

[0125] MS(ESI)m / z(M+H) + =350.1.

[0126] 1 H NMR (400MHz, DMSO-d6) 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).

[0127] Step 3: Preparation of cyclobutyl (1R,3r)-3-(6-fluoro-5-(((R)-1-(5-fluoro-2-methoxypyridin-3-yl)ethyl)amino)pyrazolo[1,5-a]pyrimidin-3-carbamate)-4-methylbenzenesulfonate

[0128]

[0129] (R)-6-fluoro-5-(((1-(5-fluoro-2-methoxypyridin-3-yl)ethyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (800 mg), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (1.0 g), and N,N-diisopropylethylamine (886 mg) were dissolved in dry tetrahydrofuran (10 mL). After reacting at room temperature for 1 hour, tert-butyl (3-hydroxycyclobutyl)carbamate hydrochloride (953 mg) was added, and the reaction was continued for another hour. TLC showed that the reaction was complete. The system was poured into water (30 mL), extracted with ethyl acetate, and the organic phases were combined. The mixture was backwashed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and the crude product was purified by column chromatography to give 800 mg of the title compound.

[0130] MS(ESI)m / z(M+H) + =573.2.

[0131] 1H NMR(400MHz,DMSO-d6)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).

[0132] Step 4: Preparation of (1R,3r)-3-(6-fluoro-5-(((R)-1-(5-fluoro-2-hydroxypyridin-3-yl)ethyl)amino)pyrazolo[1,5-a]pyrimidin-3-carboxamido)-4-methylbenzenesulfonate cyclobutyl

[0133]

[0134] (1R,3r)-3-(6-fluoro-5-(((R)-1-(5-fluoro-2-methoxypyridin-3-yl)ethyl)amino)pyrazolo[1,5-a]pyrimidin-3-carboxamido)-4-methylbenzenesulfonate cyclobutyl ester (800 mg) was dissolved in hydrogen chloride / dioxane (4 M, 10 mL), and the system was reacted overnight at 55 °C. TLC showed that the reaction was complete. The system was directly concentrated to remove most of the dioxane, giving 600 mg of crude product, which could be used directly in the next step without purification.

[0135] MS(ESI)m / z(M+H) + =559.2.

[0136] Step 5: (3) 1 S,3 3 S,6 3 E,6 4 E,8R)-1 5 6 6 Preparation of -difluoro-8-methyl-2-oxa-4,7-diaza-6(3,5)-pyrazolo[1,5-a]pyrimidine-1(2,3)-pyridine 3-3(1,3)-cyclobutane-5-one (compound (I))

[0137]

[0138] (1R,3r)-3-(6-fluoro-5-(((R)-1-(5-fluoro-2-hydroxypyridin-3-yl)ethyl)amino)pyrazolo[1,5-a]pyrimidin-3-carboxamido)-4-methylbenzenesulfonate cyclobutyl hydrochloride (250 mg) was dissolved in N,N-dimethylformamide (6 mL), and potassium carbonate (232 mg) was added. The system was reacted at room temperature for 5 hours, and TLC showed that the reaction was complete. The system was poured into water (10 mL), extracted with ethyl acetate, and the organic phases were combined. The mixture was backwashed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by preparative thin-layer chromatography and preparative high-performance liquid chromatography to obtain 58.0 mg of the title compound, with an ee value >99.5%.

[0139] MS(ESI)m / z(M+H) + =387.1.

[0140] 1 H 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).

[0141] The compounds used in Test Examples 1-6 of this application include:

[0142] LOXO-195 is a second-generation TRK inhibitor with the following chemical structure:

[0143] Positive control 1 is the compound from Example 5 of patent WO2019210835, with the following chemical structure:

[0144] Positive control 2 is the compound from Example 6 of patent WO2019210835, with the following chemical structure:

[0145] Compound 1b has the following structural formula:

[0146] Compound 2 has the following structural formula:

[0147] Compound 4 has the following structural formula:

[0148] Compound 5 has the following structural formula:

[0149] Compound 6 has the following structural formula:

[0150] LOXO-195 and control compounds 1-2 are commercially available or can be synthesized using conventional routes. For example, LOXO-195 is commercially available, and positive controls 1 and 2 can be synthesized according to the method described in WO2019210835A1. Compounds 1b, 2, and 4-6 can all be synthesized using conventional routes or according to the method described in WO2021115401A1.

[0151] Test Example 1: Kinase Inhibitory Activity

[0152] This test case was commissioned to Sandia Medical Technology (Shanghai) Co., Ltd.

[0153] 1. Experimental Objective

[0154] The inhibitory activity of the compounds in this application against three kinases, TRKa, TRKA(G595R) and TRKC(G623R), was determined.

[0155] 2. Experimental Materials

[0156] 2.2.1 Reagents and Consumables

[0157] Reagent Name Supplier Item number batch number TRKa Carna 08-186 13CBS-0565G TRKA (G595R) signalchem N16-12BG-100 H2714-7 TRKC(G623R) signalchem N18-12CH-100 D2567-8 Kinase substrate 22 GL 112393 P190329-SL112393 DMSO Sigma D8418-1L SHBG3288V 384-well plate (white) PerkinElmer 6007290 810712

[0158] 2.2.2 Instruments

[0159] Centrifuge (manufacturer: Eppendorf, model: 5430); Microplate reader (manufacturer: Perkin Elmer, model: Caliper EZ ReaderII); Echo 550 (manufacturer: Labcyte, model: Echo 550)

[0160] 3. Experimental Methods

[0161] 3.1 The test compound was accurately weighed and dissolved in 100% DMSO to prepare a 10 mM solution.

[0162] 3.2 Kinase Response Process

[0163] 3.2.1 Prepare 1×kinase buffer.

[0164] 3.2.2 Preparation of compound concentration gradients: The initial concentration of the test compound was 1000 nM. It was diluted 100 times to a final concentration of 100% DMSO in a 384 plate, and then diluted 3 times to obtain DMSO solutions of 10 concentrations of the compound. 250 nL of the 100-fold final concentration of the compound was transferred to the OptiPlate-384F reaction plate using an Echo 550 dispenser.

[0165] 3.2.3 Prepare a kinase solution with a final concentration of 2.5 times using 1× kinase buffer.

[0166] 3.2.4 Add 10 μL of kinase solution at a final concentration of 2.5 to the compound wells and the positive control wells, respectively; add 10 μL of 1× kinase buffer to the negative control wells.

[0167] 3.2.5 After shaking the reaction plate to mix, incubate at room temperature for 10 minutes.

[0168] 3.2.6 Prepare with 1× kinase buffer A mixed solution of ATP and kinase substrate 22 at a final concentration.

[0169] 3.2.7 Add 15 μL of A mixed solution of ATP and substrate at a final concentration.

[0170] 3.2.8 Centrifuge the 384-well plate at 1000 rpm for 30 seconds, shake to mix, and then incubate at room temperature for the corresponding time.

[0171] 3.2.9 Terminate the kinase response.

[0172] 3.2.10 Read the conversion rate using a Caliper EZ Reader microplate reader.

[0173] 4. Data Analysis

[0174] Calculation formula

[0175] Inhibition rate % = (Maximum conversion rate % - Sample conversion rate %) / (Maximum conversion rate % - Minimum conversion rate %) × 100

[0176] Wherein: Conversion % for sample is the conversion rate reading of the sample; Minimum conversion %: mean value of negative control wells, representing the conversion rate reading of wells without enzyme activity; Maximum conversion %: mean value of positive control wells, representing the conversion rate reading of wells without compound inhibition.

[0177] Dose-response curve fitting: With the log value of concentration as the X-axis and the percentage inhibition rate as the Y-axis, the dose-response curve was fitted using the log (inhibitor) vs. response-variable slope method (four-parameter model fitting) of GraphPad Prism 5 software to obtain the IC50 of each compound on enzyme activity. 50 value.

[0178] The results are shown in Table A1 below:

[0179] Table A1. IC50 of the compounds in this application against three kinase inhibitory activities. 50 value

[0180] Compound numbering <![CDATA[TRKa IC 50 (nM)]]> <![CDATA[TRKA(G595R)IC 50 (nM)]]> <![CDATA[TRKC(G623R)IC 50 (nM)]]> Compound 1b 0.62 2.50 3.70 LOXO-195 0.47 1.60 2.50 Compound 2 0.42 1.20 1.80 Positive control 1 0.39 0.72 1.70 Compound (I) 0.36 0.89 1.50 Positive control 2 0.43 0.97 1.40 Compound 4 320 >1000 >1000 Compound 5 127 561 >1000 Compound 6 0.52 2.90 8.20

[0181] The results showed that compound (I) exhibited high inhibitory activity against three kinases: TRKa, TRKA(G595R), and TRKC(G623R). The activity of compound (I) differed significantly from that of compounds 4-6, indicating that the fluorine substitution at different positions had a considerably different effect on the inhibitory activity of the three kinases.

[0182] Test Example 2: In vitro cell viability

[0183] The test case was commissioned to Hefei Zhongke Puruisheng Biomedical Technology Co., Ltd., and the NTRK mutant cells used were constructed by the company.

[0184] 1. Experimental Objective

[0185] The inhibitory effect of the compound of this 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.

[0186] 2. Reagents and consumables

[0187] Cell lines:

[0188]

[0189] Reagents:

[0190] Fetal bovine serum FBS (GBICO, Cat#10099-141); Luminescent Cell Viability Assay (CTG, Promega, Cat#G7573); 96-well transparent flat-bottomed black wall panel ( Cat#165305); RPMI-1640(Hyclone, Cat#SH30809.01)

[0191] 3. Experimental Procedure

[0192] 3.1 Cell Culture and Seeding:

[0193] Cells in the logarithmic growth phase were collected and counted using a platelet counter, and the cell concentration was adjusted to 3-6 × 10⁻⁶. 4 Add 90 μL of cell suspension to a 96-well plate and incubate the cells in the 96-well plate overnight at 37°C, 5% CO2, and 95% humidity.

[0194] 3.2 Drug dilution and administration:

[0195] The test compound was prepared into a 10-fold drug solution using medium containing 1% DMSO, with a maximum concentration of 10 μM. This solution was then repeatedly diluted 3-fold to obtain nine different drug concentrations. In each well of a 96-well plate inoculated with cells, at least 10 μL of the prepared drug solution was added, followed by 90 μL of medium containing 1% DMSO to obtain a drug solution with a maximum concentration of 1 μM. This was repeated 3-fold to obtain nine different concentrations, with a final DMSO concentration of 0.1% in each well. Each concentration was prepared in triplicate. Cells in the drug-treated 96-well plates were then cultured at 37°C, 5% CO2, and 95% humidity for 72 hours before CTG analysis.

[0196] 3.3 Endpoint Reading Board

[0197] Add an equal volume of CTG solution to each well, place the 96-well plate at room temperature for 20 minutes to stabilize the cold light signal, and read the cold light value.

[0198] 4. Data Processing

[0199] Data was analyzed using GraphPad Prism 7.0 software. A nonlinear S-curve regression was used to fit the data to derive the dose-response curve, from which the IC was calculated. 50 value.

[0200] Cell viability (%) = (crystal value of drug test well - crystal value of culture medium control well) / (crystal value of cell control well - crystal value of culture medium control well) × 100%.

[0201] The experimental results are shown in Table A2 below:

[0202] Table A2. IC50 values ​​of the inhibitory activity of the compounds of this application against three cell lines. 50 value

[0203]

[0204] The results showed that compound (I) had a strong growth inhibitory effect on three NTRK mutant cell lines (Ba / F3 LMNA-NTRK1-G667C, Ba / F3EVT6-NTRK3-G623R, and Ba / F3 LMNA-NTRK1-G595R). The inhibitory activities of compounds (I) and (II) on the three NTRK mutant cell lines were not significantly different from those of the corresponding positive control compounds, but the inhibitory activity of compound (I) on the three NTRK mutant cell lines was significantly higher than that of the corresponding positive control (II).

[0205] Test Example 3: Liver Microsomal Stability Test

[0206] 1. Experimental Objective

[0207] The stability of the compound of this application in human, rat and mouse liver microsomes was determined.

[0208] 2. Experimental materials and instruments

[0209] Reagents and consumables:

[0210] Reagent Name supplier Item number batch number Human liver microsomes BiolVT X008070 SDL rat liver microsomes BiolVT M00001 TIQ Mouse liver microsomes Biopredic MIC255037 BQM

[0211] 3. Experimental Procedure

[0212] 3.1 Prepare the buffer solution and liver microsomes according to the table below to make the incubation solution:

[0213] reagents concentration volume Phosphate buffer 100mM 216.25μL Liver microsomes 20mg / mL 6.25μL

[0214] 3.2 The following two experiments were conducted: a) Incubation system with added coenzyme NADPH: 25 μL of NADPH (10 mM) was added to the incubation medium (mainly containing liver microsomes and phosphate buffer) to achieve final concentrations of 0.5 mg / mL for liver microsomes and 1 mM for NADPH; b) Incubation system without added coenzyme NADPH: 25 μL of phosphate buffer (100 mM) was added to the incubation medium to achieve a final concentration of 0.5 mg / mL for liver microsomes. Both incubation systems were preheated at 37°C for 10 minutes.

[0215] 3.3 In each of the incubation systems described in "Step 3.2" above, the reaction was initiated by adding 2.5 μL of a positive control compound or a solution of the test compound of this application (100 μM), wherein the positive control was verapamil (purchased from Sigma), so that the final concentration of the test compound or positive control compound of this application was 1 μM. The incubation solution after adding the compound was incubated in batches in water at 37°C.

[0216] 3.4 At 0.5, 5, 15, 30, and 45 minutes, 30 μL aliquots were taken from the reaction solution, and the reaction was terminated by adding 5 volumes of cold acetonitrile containing 200 nM caffeine and 100 nM tolbutamide. The aliquots were centrifuged at 3220 g for 40 minutes, and 100 μL of the supernatant was mixed with 100 μL of ultrapure water for LC-MS / MS analysis.

[0217] 3.5 Data Analysis

[0218] Peak areas were determined from the extracted ion chromatograms. The slope value k was determined by linear regression of the remaining percentage of the parent drug relative to the natural logarithm of the incubation time curve.

[0219] The in vitro half-life (t) was determined by calculating the slope value. 1 / 2 The in vitro intrinsic clearance rate (CLint, expressed in μL / min / mg protein) was converted from the average in vitro half-life.

[0220] The experimental results are shown in Table A3 below:

[0221] Table A3. Stability data of the compounds of this application in human, rat, and mouse liver microsomes.

[0222]

[0223] The results showed that compound (I) of this application has good stability in human, rat, and mouse liver microsomes. The stability of compounds 1b and 2 in human, rat, and mouse liver microsomes was not significantly different from that of the corresponding positive control compounds, or the stability in liver microsomes of most species was worse. However, the stability of compound (I) of this application in human, rat, and mouse liver microsomes was significantly better than that of positive control 2.

[0224] Test Example 4: In vivo pharmacokinetic studies of the test compound administered intravenously and orally to SD rats.

[0225] 1. Laboratory animals

[0226] Species: SD rats, SPF grade. Source: Animals were transferred from the laboratory animal bank (999M-017), Shanghai Xipu-Bikai Laboratory Animal Co., Ltd. Quantity: 3 rats per dosage form.

[0227] 2. Preparation of test sample

[0228] 2.1 Accurately weigh an appropriate amount of the test sample, add 5% DMSO, 10% polyethylene glycol-15 hydroxystearate, and 85% physiological saline to a final volume, and vortex or sonicate to mix thoroughly to obtain a 0.2 mg / mL administration solution for intravenous injection.

[0229] 2.2 Accurately weigh an appropriate amount of the test sample, add 5% DMSO, 10% polyethylene glycol-15 hydroxystearate, and 85% physiological saline to the final volume, vortex or sonicate to mix thoroughly, and obtain a 0.5 mg / mL drug solution for oral gavage administration.

[0230] 3. Experimental Design

[0231]

[0232] 4. Administration method

[0233] Weigh the patient before administration and calculate the dosage based on their body weight. Administer orally via intravenous or gavage.

[0234] 5. Blood collection time point

[0235] Before administration and at 0.083h, 0.25h, 0.5h, 1h, 2h, 4h, 6h, 8h, and 24h after administration.

[0236] 6. Sample collection and processing

[0237] Blood was collected via the jugular vein or other suitable method, with approximately 0.20 mL collected per sample. Heparin sodium was used for anticoagulation. After collection, blood samples were placed on ice and centrifuged within 2 hours to separate the plasma (centrifugation conditions: 6800g, 6 minutes, 2-8℃). Collected plasma samples were stored at -80℃ before analysis, and any remaining plasma samples after analysis were also temporarily stored at -80℃.

[0238] 7. Bioanalysis and Data Processing

[0239] When measuring the plasma drug concentration of the test substance and plotting the plasma drug concentration-time curve, BLQ is recorded as 0. When calculating pharmacokinetic parameters, the concentration before administration is calculated as 0; C max Previous BLQs (including "No peak") are calculated as 0; BLQs appearing after Cmax (including "No peak") are not included in the calculation. Pharmacokinetic parameters, such as AUC(0-t) and T, are calculated using blood drug concentration data at different time points using WinNonlin. 1 / 2 Cmax, etc. The results are shown in Table A4.

[0240] Table A4. In vivo pharmacokinetic data of the test compounds administered intravenously and orally to SD rats.

[0241]

[0242]

[0243] The results showed that the pharmacokinetic parameters (AUC / CL / F%) of compound 2 in SD rats were worse than those of the positive control compound, while the pharmacokinetic parameters (AUC / CL / F%) of compound (I) in SD rats were significantly improved compared with those of the positive control 2.

[0244] Test Example 5: Study on the bidirectional permeability of the test compound in the MDCK-MDR1 cell line

[0245] 1.1 Materials

[0246] MDCK-MDR1 cells were purchased from the Dutch Cancer Institute, using cells between passage 10 and 20.

[0247] 1.2 Experimental Design

[0248] 1.2.1 Cell Culture and Seeding Plates

[0249] 1) Before cell seeding, add 50 μL of cell culture medium to each well in the upper chamber of the Transwell and 25 mL of cell culture medium to the lower culture plate. After incubating the culture plate in a 37°C, 5% CO2 incubator for 1 hour, it can be used for cell seeding.

[0250] 2) Resuspend MDCK-MDR1 cells in culture medium to a final concentration of 1.56 × 10⁻⁶. 6 cells / mL. Add 50 μL of cell suspension to each well of a 96-well Transwell plate. Incubate at 37°C, 5% CO2, and 95% relative humidity for 4-8 days. Change the medium 48 hours after inoculation and incubate for 4-8 days, changing the medium every other day.

[0251] 1.2.2 Evaluation of cell monolayer membrane integrity

[0252] 1) Remove the original culture medium from the lower culture plate and add fresh, preheated culture medium to the upper chamber.

[0253] 2) Measure the resistance of the single-layer film using a resistance meter (Millipore, USA) and record the resistance of each pore.

[0254] 3) After the measurement is completed, put the Transwell culture plate back into the incubator.

[0255] 4) Calculation of resistance value: Measure resistance value (ohms) × film area (cm²) 2 ) = TEER value (ohm·cm) 2 If the TEER value is <42 ohms·cm 2 If the hole is not suitable for a penetration test, then the hole cannot be used.

[0256] 1.2.3 Solution Preparation

[0257] 1) Prepare a 10 mM stock solution of the analyte using DMSO.

[0258] 2) The positive control compound was prepared as a stock solution with a concentration of 10 mM using DMSO.

[0259] 1.2.4 Drug penetration test

[0260] 1) Remove the MDCK-MDR1 Transwell culture plate from the incubator. Rinse the cell monolayer twice with pre-warmed HBSS (25mM HEPES, pH 7.4) buffer and incubate at 37°C for 30 minutes.

[0261] 2) The stock solutions of the control and test compounds were diluted in DMSO to obtain 200 μM solutions, and then diluted with HBSS (10 mM HEPES, pH 7.4) to obtain 1 μM working solutions. The final concentration of DMSO in the incubation system was 0.5%.

[0262] 3) Determine the transport rate of the compound from the top to the base. Add 125 μL of working solution to the upper chamber (top), then immediately transfer 50 μL of sample solution from the lower chamber (base) to a 96-well plate containing 200 μL of acetonitrile containing an internal standard as the 0-minute dosing sample (AB) for detection. Add 235 μL of HBSS (25 mM HEPES, pH 7.4) buffer to the lower chamber. The internal standard contains (100 nM alprazolam, 200 nM caffeine, and 100 nM tosylate). Vortex the transferred 50 μL of sample solution at 1000 rpm for 10 min.

[0263] 4) Determine the transport rate of the compound from the base to the top. Add 285 μL of working solution to the lower chamber (base end), then immediately transfer 50 μL to the upper chamber (top). Add the sample solution to a 96-well plate containing 200 μL of acetonitrile containing the internal standard as the 0-minute dosing sample (BA) for detection. Add 75 μL of HBSS (25 mM HEPES, pH 7.4) buffer to the upper chamber. The internal standard contains (100 nM alprazolam, 200 nM caffeine, and 100 nM tosylate). Vortex the transferred 50 μL sample solution at 1000 rpm for 10 min. The experiments from the top to the base end and from the base to the top should be performed simultaneously.

[0264] 5) After adding buffer to the lower and upper chambers respectively, incubate the MDCK-MDR1 Transwell culture at 37°C for 2 hours.

[0265] 6) After incubation, 50 μL of sample solution was taken from both the drug delivery side (upper chamber: Ap→Bl flux, lower chamber: Bl→Ap) and the receiver side (lower chamber: Ap→Bl flux, upper chamber: Bl→Ap) into new 96-well plates. Four volumes of ethanol containing internal standards (100 nM alprazolam, 200 nM caffeine, and 100 nM tosylate) were added to the plates. The plates were vortexed for 10 minutes and then centrifuged at 3220 g for 40 minutes. 100 μL of the supernatant was mixed with an equal volume of ultrapure water and analyzed by LC-MS / MS.

[0266] 7) Evaluate the integrity of the cell monolayer membrane after 2 hours of incubation using fluorescein leakage. Dilute the fluorescein stock solution to a final concentration of 100 μM with HBSS (10 mM HEPES, pH 7.4). Add 100 μL of fluorescein solution to each well in the upper compartment (top) and 300 μL of HBSS (25 mM HEPES, pH 7.4) to each well in the lower compartment (base). After incubation at 37°C for 30 minutes, aspirate 80 μL of solution from both the upper and lower layers of each well into a new 96-well plate. Perform fluorescence measurement using a microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 530 nm.

[0267] 1.2.5 Analytical Conditions

[0268] LC system: Shimadzu

[0269] MS analysis:Triple Quad 5500 instrument from AB Inc with an ESIinterface

[0270] 1) LC parameters

[0271] Column temperature: 40℃

[0272] Column: Waters XSelect HSS T3 C18, 2.5 μM, 2.1 x 50 mm

[0273] Mobile phase: 0.1% formic acid dissolved in water (A) and 0.1% formic acid dissolved in acetonitrile (B)

[0274] Injection volume: 5 μL

[0275] Elution rate: 0.6 mL / min

[0276] Time (min) 0 0.2 0.7 1.2 1.25 1.5 ％B 5 5 95 95 5 5

[0277] 2) MS parameters

[0278] Ion source: Turbo spray

[0279] Ionization model: ESI

[0280] Scan type: Multiple reaction detection (MRM)

[0281] Curtain air: 35L / min

[0282] Collision air: 9L / min

[0283] Carrier gas: 50L / min

[0284] Assist gas: 50L / min

[0285] Temperature: 500℃

[0286] Ion spray voltage: +5500V (positive)

[0287] 1.3 Data Analysis

[0288] Peak area is calculated from ion chromatography results. Apparent permeability coefficient of the compound (Papp, unit: cm / s × 10⁻⁶). -6 The following formula can be used to calculate:

[0289]

[0290] In the formula: V A The volume of the receiving solution is 0.3 mL for Ap→Bl and 0.1 mL for Bl→Ap. Area is the membrane area of ​​the Transwell-96-well plate (0.143 cm²). 2 ); time is the incubation time (unit: s); [drug] is the drug concentration.

[0291] The experimental results are shown in Table A5 below:

[0292] Table A5. Data on the bidirectional permeability of the test compounds in the same batch of MDCK-MDR1 cell lines

[0293] Compound numbering <![CDATA[P app(A-B) (10 -6 ,cm / s)]]> <![CDATA[P app(B-A) (10 -6 ,cm / s)]]> Outflow ratio* Compound (I) 11.13 51.91 4.67 Positive control 2 3.31 64.41 19.42

[0294] *Output ratio = P app(B-A) / P app(A-B)

[0295] The results showed that the permeability (P) of compound (I) was... app(A-B)The superiority of compound (I) over positive control 2 indicates that compound (I) is more easily absorbed into cells. Simultaneously, compound (I) exhibits a lower efflux ratio, suggesting that compound (I) is less likely to be effluxed, thus maintaining a higher drug concentration in cells and producing better efficacy. Therefore, combined with in vivo pharmacokinetic studies in SD rats, this further demonstrates that compound (I) exhibits significantly improved pharmacokinetic parameters (AUC / CL / F%) compared to positive control 2.

[0296] Test Example 6: In vitro cell viability assessment

[0297] The test case was commissioned to Hefei Zhongke Puruisheng Biomedical Technology Co., Ltd., and the cell line used was constructed by the company.

[0298] 1. Experimental Objective

[0299] The in vitro antiproliferative effects of the compounds in this application on six BaF3 cell lines were determined.

[0300] 2. Reagents and consumables

[0301] Cell lines:

[0302] cell lines Cell types Cell count / well culture medium Ba / F3-LMNA-NTRK1 Floating 2000 RPMI 1640 + 10% FBS + 1% PS Ba / F3-LMNA-NTRK1-V573I Floating 2000 RPMI 1640 + 10% FBS + 1% PS Ba / F3-LMNA-NTRK1-F589L Floating 2000 RPMI 1640 + 10% FBS + 1% PS

[0303] Ba / F3-LMNA-NTRK1-G667S Floating 2000 RPMI 1640 + 10% FBS + 1% PS Ba / F3-TEL-NTRK2 Floating 2000 RPMI 1640 + 10% FBS + 1% PS Ba / F3-TEL-NTRK3 Floating 2000 RPMI 1640 + 10% FBS + 1% PS

[0304] Material:

[0305]

[0306] 3. Experimental Procedure

[0307] Cell treatment and drug delivery

[0308] Cell culture conditions

[0309] Six Ba / F3 cell lines were cultured in RPMI 1640 (Biological Industries, Israel) + 10% fetal bovine serum (Biological Industries, Israel) + 1% penicillin-streptomycin solution (Coring, USA). After cell resuscitation, the cells were cultured for two generations before testing.

[0310] Preparation of 1000× compound

[0311] The test compound was dissolved in DMSO to prepare a 10 mM stock solution, which was then diluted to 1 mM with DMSO. Ten concentration gradients were prepared by three-fold dilution to 1.0000 mM, 0.3333 mM, 0.1111 mM, 0.03704 mM, 0.01235 mM, 0.00412 mM, 0.00137 mM, 0.00046 mM, 0.00015 mM, and 0.00005 mM, and stored in 96-well plates (Beaver, Suzhou). An equal volume of DMSO solvent was used as a negative control.

[0312] 20× compound preparation

[0313] The prepared 10 concentration gradients of the test compound were diluted 50 times to 20 times with complete culture medium and stored in 96-well plates (Beaver, Suzhou). A total of 10 concentration gradients were prepared, and an equal volume of DMSO solvent was used as a negative control.

[0314] planking

[0315] Take the logarithmic growth phase cell suspension and seed it into a 96-well white cell culture plate (Corning 3917, NY, USA), with a volume of 95 μl per well (approximately 2000 cells / well).

[0316] Add 5 μl of the 20× test compound to each of the above-mentioned culture plates containing 95 μl of cell suspension, mix well, and divide into two wells for each concentration gradient. The final detection concentrations of the test compounds were 1.0000 μM, 0.3333 μM, 0.1111 μM, 0.03704 μM, 0.01235 μM, 0.00412 μM, 0.00137 μM, 0.00046 μM, 0.00015 μM, and 0.00005 μM.

[0317] Incubate the culture plates at 37°C in a 5% CO2 incubator for 72 hours.

[0318] Reading board

[0319] The following steps were performed in accordance with the instructions for the Promega CellTiter-Glo luminescence assay kit (Promega-G7573).

[0320] (1). Melt the CellTiter-Glo buffer and let it come to room temperature.

[0321] (2). Place the CellTiter-Glo substrate at room temperature.

[0322] (3) Add CellTiter-Glo buffer to a bottle of CellTiter-Glo substrate to dissolve the substrate, thereby preparing CellTiter-Glo working solution.

[0323] (4) Slow vortexing to ensure complete dissolution.

[0324] (5) Remove the cell culture plate and let it stand for 10 minutes to allow it to equilibrate to room temperature.

[0325] (6) Add 50 μl of CellTiter-Glo working solution to each well.

[0326] (7) Shake the culture plate on a track shaker for 2 minutes to induce cell lysis.

[0327] (8) The culture plate was placed at room temperature for 10 minutes to stabilize the luminescence signal.

[0328] (9) Detect the light emission signal on the MD SpectraMax Paradigm reader.

[0329] 4. Data Analysis

[0330] SpectraMax Paradigm readings yielded the corresponding fluorescence value (RLU) per well. Cell viability data were processed using the following formula:

[0331] Cell viability (%) = (RLU) Drug -RLU Min ) / (RLU Max -RLU Min *100%. Calculate cell viability for different concentrations of the compound in Excel, then use GraphPad Prism software to plot cell viability curves and calculate relevant parameters, including maximum and minimum cell viability, IC50. 50 value.

[0332] The experimental results are shown in Table A6 below:

[0333] Table A6. IC50 values ​​of the inhibitory activity of the compounds of this application against six BaF3 cell lines from the same batch. 50 value

[0334]

[0335] Based on the bioactivity data of the compounds in the specific embodiments, compound (I) of this application exhibits a strong growth inhibitory effect on six BaF3 cell lines. The inhibitory activity of compound 2 on the six BaF3 cell lines is not significantly different from that of the corresponding positive control compound, but the inhibitory activity of compound (I) of this application on the six BaF3 cell lines is significantly higher than that of the positive control 2.

[0336] Example 1: Preparation of crystal form A of compound (I)

[0337] 20 g of compound (I) was dissolved in 189.6 g of N,N-dimethylformamide and stirred until dissolved and clear. 14.84 g of potassium carbonate was added under ice bath conditions, and the mixture was stirred for 5-10 min, then heated to 30 ± 2 °C and reacted for 16-24 h. After the reaction was complete, 600 g of purified water was rapidly added dropwise, and stirring was continued for 1-2 h. The mixture was filtered, and the filter cake was washed twice with 100 g of water. The solid was collected and purified by liquid chromatography. The purified solution was concentrated and lyophilized to obtain 16.2 g of a white solid. XRPD analysis of the solid yielded the following chromatogram: Figure 1. As shown in Table 1, the relevant data indicates that the obtained solid is the crystal form A of compound (I). The DSC / TGA spectrum shows ( Figure 2. The results showed that DSC showed endothermic peaks at 66±2℃ (onset) and 370±2℃ (onset); TGA showed a weight loss of 8.6% at 66±2℃, which was confirmed by calculation to be a 2-molecule hydrate, and rapid weight loss after 300℃.

[0338] Table 1.

[0339]

[0340] Crystal 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°; and 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°.

[0341] Example 2: Preparation of crystal form C of compound (I) 1

[0342] 1.0 g of compound (I) crystal form A was weighed and dispersed in 7 ml of ethanol. The mixture was stirred at 78 °C for 3 h, filtered, and the solid was collected and dried under vacuum at room temperature for 6 h. The solid was analyzed by XRPD, and the chromatogram is shown below. Figure 3.As shown in Table 2, the relevant data indicates that the obtained solid is crystal form C. The DSC / TGA spectra show (…). Figure 4) The DSC showed an endothermic peak at 377±2℃ (onset); the TGA showed no significant weight loss within 300℃.

[0343] Table 2.

[0344]

[0345] Crystal 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°; and 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°.

[0346] Example 2.1 Preparation of crystal form C of compound (I)

[0347] 1.0 g of compound (I) crystal form A was weighed and dispersed in 7 ml of organic solvent. The mixture was pulped at a certain temperature for 3–24 h, filtered, and the solid was collected and vacuum dried at room temperature for 6 h. XRPD analysis of the solid was performed, and the results were consistent with those obtained in Example 2. Figure 3 base The composition is generally consistent, and the crystal form is C. The organic solvents include, but are not limited to, ethanol and isopropanol; the specified temperature refers to reflux at 25°C.

[0348] Example 3: Preparation of crystal form D of compound (I) 1

[0349] 1.0 mg of compound (I) crystal form A was weighed and dispersed in 7 ml of dimethyl carbonate. The mixture was stirred at room temperature for 24 h, filtered, and the solid was collected and dried under vacuum at room temperature for 6 h. The solid was analyzed by XRPD, and the chromatogram is shown below. Figure 5 As shown in Table 3, the relevant data indicates that the obtained solid is crystal form D. The DSC / TGA spectra show (…). Figure 6) The DSC showed an endothermic peak at 375±2℃ (onset); the TGA showed no significant weight loss within 300℃.

[0350] Table 3.

[0351]

[0352] Crystal 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°;

[0353] It also includes the following characteristic 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°, and 33.098±0.2°.

[0354] Example 3.1 Preparation of crystal form D of compound (I) 2

[0355] 1.0 g of compound (I) crystal form A was weighed and dispersed in 7 ml of organic solvent. The mixture was pulped at room temperature for 24 h, filtered, and the solid was collected and vacuum dried at room temperature for 6 h. XRPD analysis of the solid was performed, and the results were consistent with those obtained in Example 3. Figure 5 base This is consistent with the previous description, which is crystal form D.

[0356] The organic solvents include, but are not limited to, methyl acetate, butyl acetate, butanone, and methyl isobutyl ketone.

[0357] Example 4: Preparation of crystal form E of compound (I)

[0358] 70 mg of compound (I) crystal form A was weighed and dispersed in 3 ml of diethyl ether. The mixture was stirred at room temperature for 24 h, filtered, and the solid was collected and dried under vacuum at room temperature for 6 h. The solid was then analyzed by XRPD, and the resulting chromatogram was obtained. Figure 7 As shown in Table 4, the relevant data indicates that the obtained solid is crystal form E. The DSC / TGA spectra show (…). Figure 8. The results showed that DSC showed an endothermic peak at 374±2℃ (onset), while TGA showed no significant weight loss within 300℃.

[0359] Table 4.

[0360]

[0361] Crystal 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°;

[0362] It also includes the following characteristic 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°, and 28.279±0.2°.

[0363] Example 5: Preparation of crystal form L

[0364] 70 mg of compound (I) crystal form A was weighed and dispersed in 3 ml of chloroform. The mixture was stirred at room temperature for 24 h, filtered, and the solid was collected and dried under vacuum at room temperature for 6 h. The solid was then analyzed by XRPD, and the chromatogram is shown below. Figure 9. As shown in Table 5, the relevant data indicates that the obtained solid is crystal form L. The DSC / TGA spectra show (…). Figure 10. The results showed that DSC showed an endothermic peak at 373±2℃ (onset), while TGA showed no significant weight loss within 300℃.

[0365] Table 5.

[0366]

[0367] Crystal 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°; and also includes the following characteristic peaks: 16.538±0.2°, 1 8.151±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°.

[0368] Example 6: Preparation of crystal form N of compound (I)

[0369] 70 mg of compound (I) crystal form A was weighed and dispersed in 3 ml of acetonitrile. The mixture was stirred at room temperature for 24 h, filtered, and the solid was collected and dried under vacuum at room temperature for 6 h. The solid was then analyzed by XRPD, and the resulting chromatogram is shown below. Figure 11. As shown in Table 6, the relevant data indicates that the obtained solid is crystalline N. The DSC / TGA spectra show (...). Figure 12. The results showed that DSC showed an endothermic peak at 370±2℃ (onset), while TGA showed no significant weight loss within 300℃.

[0370] Table 6.

[0371]

[0372] Crystal 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°;

[0373] It also includes the following characteristic 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°, and 29.560±0.2°.

[0374] Test Example 7: Physicochemical Properties Test of Various Crystal Forms of Compound (I)

[0375] 7.1 Solubility:

[0376] Test method:

[0377] Place appropriate amounts of each batch of samples into 10ml centrifuge tubes, add approximately 5ml of buffer solution for each pH value (1.2, 6.8, 7.6), and shake in a 37℃ constant temperature shaker to bring the samples to a supersaturated state. Adjust the pH value of the solution every 3 hours to ensure that the pH value of the solution is within ±0.02 of the buffer solution pH value. After shaking at a constant temperature for 24 hours, measure the pH value of each solution. The pH value should be within ±0.1 of the buffer solution pH value. After standing for 1 hour, take 0.5ml of the supernatant, mix it with 0.5ml of acetonitrile, and inject for analysis.

[0378] pH 1.2 buffer solution: Measure 9.0 ml of hydrochloric acid, dilute with water to 500 ml, and shake well. Measure 85.0 ml of this solution, dilute with water to 200 ml, and shake well.

[0379] pH 6.8 buffer solution: Weigh 6.8g of potassium dihydrogen phosphate, dissolve it in water by sonication and dilute it to 250ml, shake well, measure 62.5ml of this solution, add 29.5ml of 0.2mol / L NaOH solution, dilute with water to 250ml, and adjust the pH value to 6.8 with NaOH solution.

[0380] pH 7.6 buffer: Weigh 13.6g of potassium dihydrogen phosphate, dissolve it in water by sonication and dilute it to 500ml, shake well, measure 50ml of this solution, add 42.4ml of 0.2mol / L NaOH solution, dilute with water to 200ml, and adjust the pH to 7.6 with NaOH solution.

[0381] The solubility test results are as follows:

[0382]

[0383] The results above show that crystal form C has better solubility than the other two crystal forms in buffer solutions (HCl-NaOH) at all pH values.

[0384] 7.2 The impact of influencing factors on stability:

[0385]

[0386] The data in the table show that all crystal forms have good chemical stability and no obvious degradation; crystal forms C and D have better crystal form stability; and crystal form C has better impurity removal ability.

[0387] 7.3 Drugability Assessment

[0388]

[0389] The table above confirms that crystal form C has better crystallinity, crystal stability, and abrasion resistance, and is easier to prepare.

[0390] Pharmacokinetic comparison in 7.4 SD rats

[0391] Sample Information

[0392]

[0393] *: Purity and free radicals need to be calculated when preparing the formula.

[0394] Sample preparation

[0395] Prepare the medication on the day of administration.

[0396] Solvent: For test samples numbered 1-6, the solvent is 0.5% CMC-Na.

[0397] Sample No. 7: Solvent: 50% PEG400 + 40% PG + 10% Labrasol

[0398] laboratory animals

[0399] Species: SD rat, SPF grade.

[0400] Source: Animals were transferred from the laboratory animal bank (999M-017). Shanghai Xipu-Bikai Laboratory Animal Co., Ltd.

[0401] Quantity: Transferred: 25 males. Experimental requirements: 21 males (extra animals will have blank plasma collected).

[0402] Animal selection: No random grouping.

[0403] Experimental Design

[0404]

[0405] Note: * Animals in the oral administration group were fasted overnight (10-14 hours) before administration and fed 4 hours after administration.

[0406] Administration method

[0407] Weigh the patient before administration and calculate the dosage based on their body weight. Administer orally via gavage.

[0408] Blood collection time points:

[0409] Before administration, and at 0.083h, 0.25h, 0.5h, 1h, 2h, 4h, 6h, 8h, and 24h after administration. Blood was collected via the jugular vein, with approximately 0.20mL collected per sample. Heparin sodium was used for anticoagulation, and the samples were placed on ice after collection.

[0410] Plasma sample processing

[0411] Blood samples were placed on ice after collection and centrifuged within 1 hour to separate the plasma (centrifugation conditions: 6800g, 6 minutes, 2-8℃). Plasma samples were stored at -80℃ before analysis.

[0412] The biological sample analysis methods and the analysis of all samples were completed by the analytical laboratory of Medicilon Medical Technology (Shanghai) Co., Ltd. At the same time as the sample analysis, the intraday accuracy of the quality control samples was evaluated, and it was required that the accuracy of more than 66.7% of the quality control samples be between 80-120%.

[0413] Results Analysis

[0414] Using blood drug concentration data at different time points, pharmacokinetic parameters were calculated using Phoenix WinNonlin 7.0, providing parameters such as AUC0-t, AUC0-∞, MRT0-∞, Cmax, Tmax, and T1 / 2, along with their mean and standard deviation. The results are shown in the table below.

[0415]

[0416] The pharmacokinetic data above confirm that crystal 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 higher bioavailability.

[0417] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them; although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications can still be made to the specific implementation of the present invention or equivalent substitutions can be made to some technical features without departing from the spirit of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the technical solutions claimed in the present invention.

Claims

1. A crystalline form of Compound (I): ###0001### Compound (I) Form 1 which X-ray powder diffraction pattern comprises 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°. characterized in that which X-ray powder diffraction pattern 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°.

2. The morphic form of claim 1, characterized by, which X-ray powder diffraction pattern is substantially as shown in Figure 3.

3. The morphic form of claim 1, characterized by, 4. A crystalline form of Compound (I): ###0002### Compound (I) Form 4 which X-ray powder diffraction pattern comprises 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°. which X-ray powder diffraction pattern 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°. characterized in that which X-ray powder diffraction pattern is substantially as shown in Figure 5.

5. The morphic form of claim 4, characterized by, 7. A crystalline form of Compound (I): ###0003### Compound (I) Form 7 which X-ray powder diffraction pattern comprises 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°.

6. The morphic form of claim 4, characterized by an X-ray diffraction pattern substantially in accordance with Figure 30. which X-ray powder diffraction pattern 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°. which X-ray powder diffraction pattern is substantially as shown in Figure 7. characterized in that 10. A crystalline form of Compound (I): ###0004### Compound (I) Form 10 which X-ray powder diffraction pattern comprises 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°.

8. The morphic form of claim 7, characterized by an X-ray diffraction pattern substantially in accordance with Figure 30. ​ 9. The morphic form of claim 7, characterized by an X-ray diffraction pattern substantially in accordance with Figure 30, ​ ​ characterized in that ​ 11. The morphic form of claim 10, characterized by an X-ray diffraction pattern substantially in accordance with Figure 30. the X-ray powder diffraction pattern further comprising 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°。 12. The morphic form of claim 10, characterized by an X-ray diffraction pattern substantially in accordance with Figure 30. the X-ray powder diffraction pattern substantially as shown in Figure 9.

13. A crystalline form of Compound (I): characterized in that the X-ray powder diffraction pattern comprising 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°.

14. The morphic form of claim 13, characterized by an X-ray diffraction pattern substantially in accordance with Figure 30. the X-ray powder diffraction pattern further comprising the following characteristic 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°。 15. The morphic form of claim 13, characterized by an X-ray diffraction pattern substantially in accordance with Figure 30. the X-ray powder diffraction pattern substantially as shown in Figure 11.

16. A crystalline form of Compound (I): characterized in that the X-ray powder diffraction pattern comprising 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°.

17. The morphic form of claim 16, characterized by an X-ray diffraction pattern substantially in accordance with Figure 30. the X-ray powder diffraction pattern further comprising 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°。 18. The morphic form of claim 16, characterized by an X-ray diffraction pattern substantially in accordance with Figure 30. the X-ray powder diffraction pattern substantially as shown in Figure 1.

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