CDK inhibitor and polymorphic form of phosphate thereof

Abstract

The invention provides a polymorphic form of a compound of formula (A) (N-(1-((4-((3S, 5S)-3, 5-dimethylpiperazine-1-yl) phenyl) sulfonyl) piperidine-4-yl)-4-((S)-tetrahydrofuran-3-yl) oxy)-5-(trifluoromethyl) pyrimidine-2-amine) and a polymorphic form of a phosphate of the compound of formula (A), and provides a polymorphic form of the compound of formula (A). The crystal form provided by the invention has better chemical stability, physical stability and lower hygroscopicity, is less influenced by temperature, humidity and illumination, and is convenient to store and develop preparations.

Description

A polymorph of a CDK inhibitor and its phosphate

[0001] This application claims priority to the Chinese patent application filed with the Patent Office of China on August 5, 2022, with application number CN202210947810.2 and invention name “A polymorph of a CDK inhibitor”, the Chinese patent application filed with the Patent Office of China on August 5, 2022, with application number CN202210936709.7 and invention name “A polymorph of a CDK inhibitor phosphate”, and the Chinese patent application filed with the Patent Office of China on July 19, 2023, with application number CN202310889701.4 and invention name “A polymorph of a CDK inhibitor and its phosphate”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present application discloses a CDK inhibitor, multiple crystal forms of its phosphate salt and preparation methods thereof, as well as their use in treating cancer diseases. Background Art

[0003] The cell cycle is a fundamental process in cellular life that controls cell growth, proliferation, and differentiation. Cyclin-dependent kinases (CDKs) are an important class of cellular enzymes that, in conjunction with cyclins, play a crucial role in regulating the cell cycle. Cyclin B / CDK1, cyclin A / CDK2, cyclin E / CDK2, cyclin D / CDK4, cyclin D / CDK6, and other possible heterodimers are important regulators of different stages of the cell cycle (Harper, JW, Adams, PD, Cyclin-Dependent Kinases, Chem. Rev. 2001, 101, 2511-2526).

[0004] As a key regulatory factor in the cell cycle, CDK2 forms a kinase complex with cyclin E or A, playing a decisive role in driving the cell cycle from G1 to S phase and maintaining the S phase. Its mechanism is primarily that cyclin E and CDK2 work together to phosphorylate the retinoblastoma susceptibility gene (Rb) protein. Phosphorylation of Rb protein leads to the release of E2F (transcription factor). The released E2F binds upstream of certain genes (usually located in promoter or enhancer regions), initiating the transcriptional expression of genes related to the cell cycle and allowing cells to enter the S phase from the late G1 phase. Numerous studies have shown that abnormal expression of CDK2 is closely related to the occurrence of cancer, such as CCNE1 amplified ovarian cancer, KRAS mutant lung cancer, hormone-dependent breast cancer and prostate cancer (Tadesse S, Anshabo AT, Portman N, Lim E, Tilley W, Caldon CE, Wang S, Targeting CDK2 in cancer: challenges and opportunities for therapy, Drug Discovery Today, 2020, 25, 406-413).

[0005] With the critical role of cyclin-dependent kinases (CDKs) in cell cycle regulation established, CDK inhibitors have become a research hotspot for anti-tumor drugs. Currently, several CDK inhibitors have been approved for marketing worldwide, but most target CDK4 / 6, primarily for breast cancer. Examples include Pfizer's Palbociclib, Novartis' Ribociclib, and Eli Lilly's Abemaciclib. Multi-target inhibitors encompassing CDK2, such as fadraciclib, roscovitine, and PF-06873600, are in various stages of clinical development. Currently, no CDK2 inhibitors have been approved for marketing. Therefore, the continued development of novel CDK inhibitors, particularly those effective against CDK2, is of significant research significance.

[0006] Patent PCT / CN2022 / 074491 discloses a small molecule inhibitor targeting CDK2 / 4 / 6, particularly CDK2. Its structure is shown in Formula (A), and its chemical name is N-(1-((4-((3S,5S)-3,5-dimethylpiperazin-1-yl)phenyl)sulfonyl)piperidin-4-yl)-4-((S)-tetrahydrofuran-3-yl)oxy)-5-(trifluoromethyl)pyrimidin-2-amine. This small molecule inhibitor selectively inhibits CDK 2 / 4 / 6 kinases over CDK 1 / 7 / 9 kinases, particularly exhibiting excellent inhibitory activity against CDK 2 kinase, with kinase selectivity reaching nearly 10-fold, or even dozens or even hundreds of times higher. This small molecule inhibitor exhibits strong cell proliferation inhibition activity, and has demonstrated good tumor suppression and good tolerability in in vivo efficacy experiments, making it promising for development into a clinical drug.

[0007] Summary of the Invention

[0008] The present application discloses a polymorphic form of a CDK inhibitor, a CDK inhibitor phosphate and its polymorphic form, a preparation method of the crystal form, and their use in treating cancer diseases.

[0009] Specifically,

[0010] In a first aspect, the present application provides a crystalline form I of a compound of formula (A) (i.e., N-(1-((4-((3S,5S)-3,5-dimethylpiperazin-1-yl)phenyl)sulfonyl)piperidin-4-yl)-4-((S)-tetrahydrofuran-3-yl)oxy)-5-(trifluoromethyl)pyrimidin-2-amine), wherein the X-ray powder diffraction pattern of the crystalline form I has characteristic peaks at 2θ values ​​of 10.49°, 12.10°, 17.74°, 19.88°, and 21.66°, and the 2θ error range is ±0.2°.

[0011] In some embodiments of the present application, the X-ray powder diffraction pattern of the above-mentioned Form I has characteristic peaks at 2θ values ​​of 10.03°, 10.49°, 12.10°, 14.28°, 14.81°, 17.74°, 18.28°, 19.88°, 20.57°, 21.66°, 23.11°, 23.76°, and 26.29°, and the 2θ error range is ±0.2°.

[0012] In some embodiments of the present application, the X-ray powder diffraction pattern of the above-mentioned crystalline form I has characteristic peaks at 2θ values ​​of 10.03°, 10.49°, 11.48°, 12.10°, 13.50°, 14.28°, 14.81°, 16.16°, 16.87°, 17.74°, 18.28°, 19.88°, 20.57°, 21.10°, 21.66°, 22.13°, 22.45°, 23.11°, 23.76°, 24.67°, 25.87°, 26.29°, 30.46°, and 32.57°, and the 2θ error range is ±0.2°.

[0013] In some embodiments of the present application, the X-ray powder diffraction pattern of the above-mentioned Form I is substantially as shown in Figure 1.

[0014] In some embodiments of the present application, the DSC spectrum of the above-mentioned Form I has an endothermic characteristic peak at around 200°C.

[0015] In some embodiments of the present application, the TGA-DSC spectrum of the above-mentioned Form I is substantially as shown in Figure 2.

[0016] In some embodiments of the present application, the XRPD diffraction peak analysis data of the crystalline form I of the compound of formula (A) above are substantially as shown in Table 1.

[0017] Table 1 XRPD diffraction peak analysis data of the compound of formula (A) crystalline form I

[0018] In a second aspect, the present application also provides a crystalline form II of a compound of formula (A) (i.e., N-(1-((4-((3S,5S)-3,5-dimethylpiperazin-1-yl)phenyl)sulfonyl)piperidin-4-yl)-4-((S)-tetrahydrofuran-3-yl)oxy)-5-(trifluoromethyl)pyrimidin-2-amine), wherein the X-ray powder diffraction pattern of the crystalline form II has characteristic peaks at 2θ values ​​of 8.09°, 13.31°, 16.59°, 19.55°, 21.59°, and 25.01°, and the 2θ error range is ±0.2°.

[0019] In some embodiments of the present application, the X-ray powder diffraction pattern of the above-mentioned crystalline form II has characteristic peaks at 2θ values ​​of 6.13°, 8.09°, 11.60°, 13.31°, 14.44°, 16.59°, 17.13°, 18.27°, 19.55°, 20.93°, 21.59°, 22.54°, 25.01°, and 26.92°, and the 2θ error range is ±0.2°.

[0020] In some embodiments of the present application, the X-ray powder diffraction pattern of the above-mentioned Form II has characteristic peaks at 2θ values ​​of 6.13°, 8.09°, 9.94°, 11.60°, 13.31°, 14.44°, 16.59°, 17.13°, 17.71°, 18.27°, 19.55°, 19.98°, 20.93°, 22.07°, 21.59°, 22.54°, 25.01°, 25.35°, 25.60°, 26.92°, and 29.34°, and the 2θ error range is ±0.2°.

[0021] In some embodiments of the present application, the X-ray powder diffraction pattern of the above-mentioned Form II is substantially as shown in Figure 3.

[0022] In some embodiments of the present application, the TGA-DSC spectrum of the above-mentioned Form II is substantially as shown in Figure 4.

[0023] In some embodiments of the present application, the XRPD diffraction peak analysis data of the crystalline form II of the compound of formula (A) above are substantially as shown in Table 2.

[0024] Table 2 XRPD diffraction peak analysis data of the crystal form II of the compound of formula (A)

[0025] In a third aspect, the present application further provides a phosphate of a compound of formula (A) (i.e., N-(1-((4-((3S,5S)-3,5-dimethylpiperazin-1-yl)phenyl)sulfonyl)piperidin-4-yl)-4-((S)-tetrahydrofuran-3-yl)oxy)-5-(trifluoromethyl)pyrimidin-2-amine), wherein the molar ratio of the compound of formula (A) to phosphoric acid is 1:1.

[0026] The inventors of this application have attempted to form salts of the compound of formula (A) with various inorganic or organic acids, such as hydrochloric acid, sulfuric acid, phosphoric acid, maleic acid, fumaric acid, succinic acid, and p-toluenesulfonic acid. However, they have found that only the phosphate salt of formula (A) exhibits good crystallinity, hygroscopicity, solubility, and stability. Other acids may fail to form salts with the compound of formula (A), some may exhibit very poor crystallinity, or may result in amorphous crystals. Other acids may yield crystalline forms, but the crystalline forms may be hygroscopic or unstable.

[0027] In a fourth aspect, the present application further provides a crystalline form III of the phosphate of the compound of formula (A), wherein the X-ray powder diffraction pattern of the crystalline form III has characteristic peaks at 2θ values ​​of 5.85°, 8.94°, 14.86°, and 16.00°, and the 2θ error range is ±0.2°.

[0028] In some embodiments of the present application, the X-ray powder diffraction pattern of the above-mentioned crystalline form III has characteristic peaks at 2θ values ​​of 5.85°, 8.94°, 10.29°, 13.21°, 14.86°, 16.00°, 17.01°, 17.65°, and 19.25°, and the 2θ error range is ±0.2°.

[0029] In some embodiments of the present application, the X-ray powder diffraction pattern of the above-mentioned Form III has characteristic peaks at 2θ values ​​of 3.43°, 5.85°, 8.94°, 10.29°, 11.69°, 12.30°, 13.21°, 14.86°, 16.00°, 17.01°, 17.65°, 19.25°, 19.83°, 20.71°, 21.82°, 22.28°, and 23.86°, and the 2θ error range is ±0.2°.

[0030] In some embodiments of the present application, the X-ray powder diffraction pattern of the above-mentioned Form III is substantially as shown in Figure 8.

[0031] In some embodiments of the present application, the TGA-DSC spectrum of the above-mentioned Form III is substantially as shown in Figure 9.

[0032] In some embodiments of the present application, the XRPD diffraction peak analysis data of the above-mentioned Form III are substantially as shown in Table 3.

[0033] Table 3 XRPD diffraction peak analysis data of the phosphate crystal form III of the compound of formula (A)

[0034] In the fifth aspect of the present application, a crystalline form IV of a phosphate of a compound of formula (A) is provided, wherein the X-ray powder diffraction pattern of the crystalline form IV has characteristic peaks at 2θ values ​​of 13.07°, 15.66°, 16.11°, 16.84°, and 21.89°, and the 2θ error range is ±0.2°.

[0035] In some embodiments of the present application, the X-ray powder diffraction pattern of the above-mentioned crystalline form IV has characteristic peaks at 2θ values ​​of 7.81°, 13.07°, 15.20°, 15.66°, 16.11°, 16.84°, 19.61°, 21.89°, 22.16°, and 23.57°, and the 2θ error range is ±0.2°.

[0036] In some embodiments of the present application, the X-ray powder diffraction pattern of the above-mentioned form IV has characteristic peaks at 2θ values ​​of 7.81°, 10.97°, 13.07°, 14.20°, 15.20°, 15.66°, 16.11°, 16.84°, 19.61°, 21.89°, 22.16°, 22.93°, 23.57°, 24.70°, 25.92°, 27.42°, and 28.61°, and the 2θ error range is ±0.2°.

[0037] In some embodiments of the present application, the X-ray powder diffraction pattern of the above-mentioned Form IV is substantially as shown in Figure 10.

[0038] In some embodiments of the present application, the DSC spectrum of the above-mentioned Form IV has an endothermic characteristic peak at around 229°C.

[0039] In some embodiments of the present application, the TGA-DSC spectrum of the above-mentioned Form IV is substantially as shown in Figure 11.

[0040] In some embodiments of the present application, the XRPD diffraction peak analysis data of the above-mentioned Form IV are substantially as shown in Table 4.

[0041] Table 4 XRPD diffraction peak analysis data of the phosphate crystal form IV of the compound of formula (A)

[0042] The sixth aspect of the present application also provides a pharmaceutical composition, which comprises the crystalline form I described in the first aspect of the present application, the crystalline form II described in the second aspect of the present application, the phosphate described in the third aspect of the present application, the crystalline form III described in the fourth aspect of the present application or the crystalline form IV described in the fifth aspect of the present application, and a pharmaceutically acceptable carrier.

[0043] The seventh aspect of the present application also provides the use of the crystal form I described in the first aspect of the present application, the crystal form II described in the second aspect of the present application, the phosphate described in the third aspect of the present application, the crystal form III described in the fourth aspect of the present application, the crystal form IV described in the fifth aspect of the present application, or the pharmaceutical composition described in the sixth aspect of the present application in the preparation of drugs for treating CDK-mediated cancer.

[0044] The eighth aspect of the present application also provides the use of the crystal form I described in the first aspect of the present application, the crystal form II described in the second aspect of the present application, the phosphate described in the third aspect of the present application, the crystal form III described in the fourth aspect of the present application, the crystal form IV described in the fifth aspect of the present application, or the pharmaceutical composition described in the sixth aspect of the present application in the treatment of CDK-mediated cancer.

[0045] The ninth aspect of the present application also provides the crystal form I described in the first aspect of the present application, the crystal form II described in the second aspect of the present application, the phosphate described in the third aspect of the present application, the crystal form III described in the fourth aspect of the present application, the crystal form IV described in the fifth aspect of the present application, or the pharmaceutical composition described in the sixth aspect of the present application for use as a medicine or for treating CDK-mediated cancer.

[0046] In some embodiments of the present application, the aforementioned cancers include ovarian cancer, breast cancer, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), or small lymphocytic lymphoma (SLL).

[0047] In some embodiments of the present application, the above-mentioned cancer is breast cancer.

[0048] The tenth aspect of the present application further provides a method for preparing the crystalline form I described in the first aspect of the present application, comprising the following steps:

[0049] (a) dissolving compound A in a first solvent to obtain a solution;

[0050] (b) adding a second solvent to the solution of step (a) and stirring for a certain period of time;

[0051] (c) After the solid precipitates, it is collected by filtration and dried.

[0052] In some embodiments of the present application, in the above preparation method, the first solvent is selected from at least one of acetone, methanol, tetrahydrofuran, ethyl acetate, acetonitrile and dichloromethane.

[0053] In some embodiments of the present application, in the above preparation method, the second solvent is selected from at least one of water, n-heptane, and n-hexane.

[0054] In some embodiments of the present application, in the above preparation method, the stirring time is 1-3 hours, preferably 2 hours.

[0055] Technical Effects

[0056] The crystal form in the present application has good chemical stability, physical stability and low hygroscopicity, is less affected by temperature, humidity and light, and is convenient for storage and formulation development. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] FIG1 is an XRPD (X-ray powder diffraction) spectrum of Form I of the compound of formula (A).

[0058] FIG2 is a TGA-DSC (differential scanning calorimetry-thermogravimetric analysis) spectrum of Form I of the compound of formula (A).

[0059] FIG3 is an XRPD spectrum of Form II of the compound of formula (A).

[0060] FIG4 is a TGA-DSC spectrum of Form II of the compound of formula (A).

[0061] FIG5 is a DVS (dynamic vapor phase adsorption) spectrum of the crystalline form I of the compound of formula (A).

[0062] FIG6 is a comparison of XRPD spectra of Form I of the compound of formula (A) before and after the DVS experiment.

[0063] FIG7 is a comparative XRPD spectrum of the crystalline form I of the compound of formula (A) before and after the stability experiment.

[0064] FIG8 is an XRPD spectrum of Form III of the phosphate salt of the compound of formula (A).

[0065] FIG9 is a TGA-DSC spectrum of Form III of the phosphate salt of the compound of formula (A).

[0066] FIG10 is an XRPD spectrum of Form IV of the phosphate salt of the compound of formula (A).

[0067] FIG11 is a TGA-DSC spectrum of Form IV of the phosphate salt of the compound of formula (A).

[0068] FIG12 is a DVS spectrum of Form IV of the phosphate salt of the compound of formula (A).

[0069] FIG13 is a comparative XRPD spectrum of Form IV of the phosphate salt of the compound of formula (A) before and after the stability experiment. DETAILED DESCRIPTION

[0070] The present invention is described in detail below by way of examples, but is not intended to limit the present invention in any adverse way. The compound of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by combining the specific embodiments with other chemical synthesis methods, and equivalent replacement modes well known to those skilled in the art, and preferred embodiments include but are not limited to the examples of the present invention. It will be apparent to those skilled in the art that various changes and improvements will be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention.

[0071] Definition and Description

[0072] Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered ambiguous or unclear without a specific definition, but should be understood according to its ordinary meaning.

[0073] The term "pharmaceutically acceptable carrier" refers to a medium generally accepted in the art for delivering biologically active agents to animals, particularly mammals, and includes, for example, adjuvants, excipients, or vehicles, such as diluents, preservatives, fillers, flow regulators, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungal agents, lubricants, and dispersants, depending on the mode of administration and the nature of the dosage form. Pharmaceutically acceptable carriers are formulated within the purview of those skilled in the art based on a wide range of factors. These include, but are not limited to, the type and nature of the active agent being formulated, the subject to whom the composition containing the agent is to be administered, the intended route of administration of the composition, and the intended therapeutic indication. Pharmaceutically acceptable carriers include both aqueous and non-aqueous media, as well as a variety of solid and semisolid dosage forms. In addition to the active agent, such carriers include a variety of different ingredients and additives, and the inclusion of such additional ingredients in a formulation for various reasons (e.g., to stabilize the active agent, binders, etc.) is well known to those skilled in the art.

[0074] It is well known in the art that X-ray powder diffraction patterns have one or more measurement errors depending on slight changes in measurement conditions. The structures of the crystals, crystals or crystalline forms disclosed or claimed in this application may exhibit similar but not identical analytical characteristics within a reasonable error range depending on the experimental conditions, purity, equipment and other constant variables known to those skilled in the art. For example, the diffraction angle (2θ) in powder X-ray powder diffraction generally produces an error within the range of ±0.20°. Therefore, this application not only includes crystals with completely consistent diffraction angles in powder X-ray powder diffraction, but also includes crystals with consistent diffraction angles within the error range of ±0.20°. The crystalline form of Compound A of the present application is not limited to crystals having an X-ray powder diffraction pattern identical to the X-ray powder diffraction pattern shown in the accompanying drawings, and any crystals having an X-ray powder diffraction pattern substantially identical to that shown in the accompanying drawings are within the scope of this application.

[0075] The “X-ray powder diffraction pattern substantially identical to the X-ray powder diffraction pattern shown in the accompanying drawings” appearing herein. It should be understood that the term “substantially identical” as used in this context is also intended to indicate that the 2θ angle values ​​of the X-ray powder diffraction pattern may have slight variations due to the inherent experimental variations associated with such measurements, and that both are of the same crystalline form.

[0076] It should be understood that using different types of equipment or different test conditions may give slightly different DSC spectra and endothermic transition temperature readings. DSC data can reflect changes in the form of a substance. A strong endothermic peak can indicate that the substance has undergone dehydration or desolvation, or has undergone crystallization, or has melted, etc. When reflecting a molten state, the corresponding temperature is usually understood to be the melting point of the substance. This value will be affected by the purity of the compound, sample weight, heating rate, particle size, and calibration and maintenance of the test equipment. Those skilled in the art will understand that the temperature when a substance transforms from a solid state to a liquid state is usually a temperature range, not a fixed point value. Therefore, whether it is an onset value or a peak value or other reasonable values, it can characterize the temperature corresponding to the endothermic peak or the melting point of the substance. The maximum endothermic transition temperature of the crystal form can be within the range of ±5.0°C of the specific value disclosed above, preferably within the range of ±2.0°C.

[0077] This application also uses thermogravimetric analysis (TGA) to analyze the relationship between the degree of decomposition, sublimation, and evaporation (weight loss) of the crystal form and temperature. It should be understood that the values ​​obtained for the same crystal form are affected by sample purity, particle size, different types of equipment, different testing methods, etc., and there will be certain errors in the values ​​obtained. The temperature at which the crystal form decomposes, sublimates, or evaporates can be within the range of ±3.0°C of the specific values ​​disclosed above, for example, within the range of ±2.0°C.

[0078] The "stability" of a crystal form includes "chemical stability" and / or "physical stability." "Chemical stability" refers to the degree to which a crystal form degrades under certain conditions of temperature, humidity, and light. "Chemical stability" reflects the stability of a crystal form under storage conditions. "Physical stability" refers to the degree to which a crystal form undergoes solid-state transformation under certain conditions, such as conversion to another crystal form under conditions of high temperature, high humidity, grinding, tableting, desolvation, or solvent adsorption. Therefore, "physical stability" can, to a certain extent, reflect the stability of a crystal form during use, such as during formulation.

[0079] 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. Techniques for crystallizing or recrystallizing the crystalline structure from a solvent mixture include evaporation of the solvent, lowering the temperature of the solvent mixture, seeding of a supersaturated solvent mixture of the molecule and / or salt, freeze-drying the solvent mixture, adding an antisolvent to the solvent mixture, and the like.

[0080] The term "drying" refers to the process of removing the solvent from the obtained solid, including but not limited to natural drying at room temperature, high temperature drying, vacuum drying, etc.

[0081] The intermediate compounds of the present application can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and equivalent replacement methods well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present application.

[0082] In the examples of this application, the title compound names were derived from the compound structures using ChemDraw. In the event of inconsistencies between the compound name and the compound structure, the correct identification can be made by integrating relevant information and reaction routes. If other methods are unavailable for confirmation, the given compound structure will prevail.

[0083] The preparation methods of some compounds in this application refer to the preparation methods of the aforementioned similar compounds. Those skilled in the art should be aware that when using or referring to the preparation methods cited, the feed ratio of reactants, reaction solvent, reaction temperature, etc. can be appropriately adjusted according to the different reactants.

[0084] The compounds of the present application can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and equivalent replacement methods well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present application.

[0085] Instruments and analytical methods

[0086] 1. X-ray powder diffraction (XRPD)

[0087] Solid samples were analyzed using an X-ray powder diffractometer (X'Pert PRO). An appropriate amount of sample powder was placed in the groove of the sample holder and pressed into a flat and dense surface using a glass sheet. The XRPD measurement parameters are shown in Table 5.

[0088] Table 5 XRPD test parameters

[0089] 2. Thermogravimetric analysis (TGA)

[0090] Thermogravimetric analysis of the solids was performed using a TA Instrument thermogravimetric analyzer. Approximately 1-5 mg of sample was placed in a tared aluminum sample pan and heated according to the parameters listed in Table 6. The data was analyzed using TRIOS.

[0091] Table 6 TGA analysis method parameters

[0092] 3. Simultaneous thermal analysis (TGA-DSC)

[0093] Solids were analyzed using a Mettler Toledo simultaneous thermal analyzer (TGA) coupled with thermogravimetric and differential scanning calorimetry (DSC). An appropriate amount of the sample was placed in a crucible using a small spoon, spread evenly, and weighed. The sample was heated according to the parameters listed in Table 7, and the data was analyzed using STARe.

[0094] Table 7 TGA-DSC analysis method parameters

[0095] 4. Dynamic moisture adsorption and desorption analysis (DVS)

[0096] The hygroscopicity of the samples was measured using a DVS Intrinsic dynamic moisture sorption instrument. The samples were placed in a tared sample basket and automatically weighed. The samples were analyzed according to the parameters in Table 8.

[0097] Table 8 DVS analysis method parameters

[0098] 5. Ion chromatography analysis

[0099] Instrument: Ion chromatograph-conductivity detector;

[0100] Chromatographic column: Anion exchange chromatography column [Analytical column Ionpac TM AS11-HC (4mm×250mm), guard column Ionpac TM AG11-HC (4mm×50mm)];

[0101] Suppressor: AERS 500 4mm or equivalent;

[0102] Column temperature: 30°C;

[0103] Flow rate: 1.0 mL / min;

[0104] Injection volume: 25 μL;

[0105] Detection cell temperature: 35°C;

[0106] Mobile phase: 30 mmol / L potassium hydroxide solution;

[0107] Running time: Approximately 20 minutes.

[0108] Example 1: Preparation of compound of formula (A)

[0109] Preparation of intermediate compound 99

[0110] Step 1: Compound 1B-3 (508 mg, 1.81 mmol) was dissolved in dichloromethane (30 mL) at room temperature. Subsequently, N,N-diisopropylethylamine (702 mg, 5.43 mmol) and 4-bromo-benzenesulfonyl chloride (508 mg, 2.00 mmol) were added to the mixture under ice-water bath. The reaction mixture was stirred at room temperature for 2 hours and then quenched by addition of water (60 mL). The mixture was extracted with dichloromethane (20 mL x 3). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and finally concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography to yield 830 mg of N-(1-(1-(4-bromophenyl)sulfonyl)piperidin-4-yl)-4-chloro-5-(trifluoromethyl)pyrimidin-2-amine (99-1).

[0111] MS (ESI) M / Z: 499.0 [M+H] + .

[0112] Step 2: In a sealed jar, (S)-3-hydroxytetrahydrofuran (42 mg, 0.48 mmol) was dissolved in tetrahydrofuran (2 mL). Sodium hydride (21 mg, 0.88 mmol) was then added under an ice-water bath. After 15 minutes of reaction, compound 99-1 (200 mg, 0.40 mmol) was added. The reaction mixture was heated to 100°C for 4 hours and then quenched by the addition of water (60 mL). The mixture was extracted with dichloromethane (20 mL x 3 times). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and finally concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography to give 150 mg of (S)-N-(1-(((4-bromophenyl)sulfonyl)piperidin-4-yl)-4-((tetrahydrofuran-3-yl)oxy)-5-(trifluoromethyl)pyrimidin-2-amine (Compound 99).

[0113] MS (ESI) M / Z: 551.0 [M+H] + .

[0114] 1H NMR (400MHz, DMSO-d6) δ8.29 (s, 1H), 8.02 (d, J = 7.4Hz, 0.6H), 7.92-7.83 (m, 2.4H), 7.73-7.65 (m, 2H), 5.62-5.51 (m, 1H), 3.96- 3.86(m,1H),3.85-3.68(m,4H),3.63-3.46(m,2H),2.65-2.55(m,2H),2.28-2.13(m,1H),2.03-1.84(m,3H),1.64-1.48(m,2H).

[0115] Preparation of Compound A

[0116] Step 1: Under nitrogen, compound 99 (25 g, 45.3 mmol), tert-butyl (2S,6S)-2,6-dimethylpiperazine-1-carboxylate (11.6 g, 45.3 mmol), tris(dibenzylideneacetone)dipalladium (4.1 g, 4.53 mmol), 2-dicyclohexylphosphino-2,4,6-triisopropylbiphenyl (1.3 g, 9.02 mmol), and cesium carbonate (29.4 g, 90.6 mmol) were dissolved in 1,4-dioxane (1.25 L). The reaction mixture was heated to 100°C and stirred overnight. The reaction mixture was concentrated under reduced pressure and extracted with ethyl acetate (3 x 100 mL). The organic phases were combined, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel column chromatography to give 18.1 g of tert-butyl (2S,6S)-2,6-dimethyl-4-(4-((S)-tetrahydrofuran-3-yl)oxy)-5-(trifluoromethyl)pyrimidin-2-yl)amino)piperidin-1-yl)sulfonyl)phenyl)piperazine-1-carboxylate (Compound 168-1).

[0117] MS (ESI) M / Z: 685.2 [M+H] + .

[0118] Step 2: Compound 168-1 (18.1 g, 26.4 mmol) was dissolved in 1,4-dioxane (100 mL). A hydrochloric acid solution in dioxane (100 mL) was then added. The reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure. The resulting residue was washed with aqueous sodium bicarbonate (300 mL). The mixture was extracted with ethyl acetate (200 mL x 3), and the organic phases were combined. The organic phases were washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and finally concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography to yield 9.2 g of N-(1-((4-((3S,5S)-3,5-dimethylpiperazin-1-yl)phenyl)sulfonyl)piperidin-4-yl)-4-((S)-tetrahydrofuran-3-yl)oxy)-5-(trifluoromethyl)pyrimidin-2-amine (Compound A).

[0119] MS (ESI) M / Z: 585.2 [M+H] + .

[0120] 1H NMR (400MHz, DMSO-d6) δ8.29(s,1H),8.00(d,J=7.1Hz,0.6H),7.85(d,J=6.9Hz,0.4H),7.4 9(d,J=8.8Hz,2H),7.02(d,J=9.0Hz,2H),5.60-5.51(m,1H),3.95-3.83(m,1H),3.81-3.65( m,4H),3.57-3.44(m,2H),3.37-3.33(m,2H),3.21-3.09(m,2H),3.02-2.91(m,2H),2.46-2 .37(m,2H),2.28-2.03(m,2H),2.02-1.84(m,3H),1.66-1.47(m,2H),1.06(d,J=6.4Hz,6H).

[0121] Biological test evaluation

[0122] (1) CDK in vitro enzymatic assay

[0123] This study used the capillary migration ability assay (MSA) to test the inhibitory effect of the compounds on the kinase activity of CDK1 / CDK2 / CDK4 / CDK6 / CDK7 / CDK9, and obtained the half-maximal inhibitory concentration (IC) of the compounds on the kinase activity of CDK1 / CDK2 / CDK4 / CDK6 / CDK7 / CDK9. 50 .

[0124] 1. Experimental Materials

[0125] CDK1 / CDK2 / CDK4 / CDK6 / CDK7 / CDK9 were purchased from Carna, Carliper substrates CTD3 / substrate 18 / substrate 8 were purchased from Gill Biochemical, Dinaciclib / Palbociclib was purchased from Selleckchem, DMSO (dimethyl sulfoxide) was purchased from Sigma, and 384-well plates were purchased from Corning.

[0126] 2. Experimental Methods

[0127] (1) Prepare 1× Kinase buffer.

[0128] (2) Preparation of compound concentration gradient: Test compound concentration was 1 μM, starting at 10 μM or 30 μM, and then diluted 3-fold to 10 concentrations in duplicate. A 100% DMSO solution was diluted to 100 times the final concentration in the source 384-well plate. Using an Echo 550 dispenser, 250 nL of the compound at 100 times the final concentration was transferred to the destination 384-well plate. 250 nL of DMSO was added to the positive and negative control wells.

[0129] (3) Prepare a kinase solution with a final concentration of 2.5 times using 1× Kinase buffer.

[0130] (4) Add 10 μL of kinase solution at 2.5 times the final concentration to the compound wells and positive control wells respectively; add 10 μL of 1× Kinase buffer to the negative control wells.

[0131] (5) Centrifuge at 1000 rpm for 30 seconds, shake the reaction plate to mix, and incubate at room temperature for 10 minutes.

[0132] (6) Prepare a mixed solution of ATP and kinase substrate with a final concentration of 25 / 15 times using 1× Kinase buffer.

[0133] (7) Add 15 μL of a mixed solution of ATP and substrate at 25 / 15 times the final concentration to start the reaction.

[0134] (8) Centrifuge the 384-well plate at 1000 rpm for 30 seconds, shake to mix, and incubate at room temperature for the corresponding time.

[0135] (9) Add 30 μL of stop detection solution to stop the kinase reaction, centrifuge at 1000 rpm for 30 seconds, and shake to mix.

[0136] (10) Read the conversion rate using Caliper EZ Reader.

[0137] Calculation formula:

[0138] % inhibition rate (inhibition)=(conversion%_max-conversion%_sample) / (conversion%_max-conversion%_min)×100%

[0139] Where: Conversion%_sample is the conversion rate reading of the sample; Conversion%_min is the mean value of the negative control wells, representing the conversion rate reading of the wells without enzyme activity; Conversion%_max is the mean value of the positive control wells, representing the conversion rate reading of the wells without compound inhibition.

[0140] Fitting dose-effect curve

[0141] The log value of the concentration was used as the X-axis and the percentage inhibition rate was used as the Y-axis. The log (inhibitor) vs. response-Variable slope of the analysis software GraphPad Prism 5 was used to fit the dose-effect curve to obtain the IC of each compound on the enzyme activity. 50 (half inhibitory concentration) value.

[0142] Calculation formula: Y = Bottom + (Top - Bottom) / (1 + 10^((LogIC 50 -X)*HillSlope)).

[0143] 3. Experimental Results

[0144] The inhibitory IC of the compounds of the present application on CDK kinase activity 50 The data are shown in Table 9. Among them: IC 50 Compounds with a concentration <0.5 nM are indicated by AA, and 0.5 nM ≤ IC 50 Compounds with a concentration <2.5 nM are represented by AB, and 2.5 nM ≤ IC 50 Compounds with a concentration <10 nM are represented by AC, and 10 nM ≤ IC 50 Compounds with a concentration <50 nM are marked with B, and compounds with a concentration <50 nM are marked with B. 50 Compounds with a concentration <100 nM are marked with C, and 100 nM ≤ IC 50 Compounds with concentrations below 1000 nM are marked with D, IC 50 Compounds with concentrations >1000 nM are marked with E.

[0145] Table 9: CDK enzyme inhibition results

[0146] Conclusion: Compound A of the present application has good CDK 2 / 4 / 6 kinase inhibitory activity, especially excellent in the inhibitory activity of CDK 2 kinase; Compound A of the present application can selectively inhibit CDK 2 / 4 / 6 kinases relative to CDK 1 / 7 / 9 kinases, especially excellent in the inhibitory activity of CDK 2 kinase, and its kinase selectivity can reach nearly 10 times, or even dozens or hundreds of times.

[0147] (II) HCC1806 / NIH:OVCAR-3 cell proliferation inhibition experiment

[0148] This study used the CellTiter-Glo method to test the inhibitory effect of compounds on HCC1806 / NIH:OVCAR-3 cell proliferation and obtained the concentration of the compound that inhibited cell growth by half, IC50 (nM).

[0149] 1. Experimental Materials

[0150] HCC1806 was purchased from Tongpai (Shanghai) Biotechnology Co., Ltd.; NIH: OVCAR-3 was purchased from the ATCC cell bank in the United States.

[0151] 1640 culture medium, fetal bovine serum (FBS), Penicillin-Streptomycin, and GlutaMAX-I Supplement were purchased from GIBCO.

[0152] PF-06873600 was purchased from Selleck.

[0153] CellTiter-Glo reagent was purchased from Promega.

[0154] 2. Experimental Methods

[0155] 1) HCC1806 / NIH:OVCAR-3 cells were seeded into 96-well culture plates at a density of 600 / 1500 cells per well, with 100 μL per well.

[0156] 2) Day 0: Add 100 nL of serially diluted test compound to the cells in the culture plate using an Echo, with a final DMSO concentration of 0.5%. Incubate the plate in a cell culture incubator for 168 hours (37°C, 5 vol% CO2). For a blank control, add 30 nL of DMSO to each well.

[0157] 3) Day 7: Add 30 μL of Cell Titer-Glo reagent to each well and incubate at room temperature in the dark for 30 minutes.

[0158] 4) Chemiluminescent signals were detected using Envision microplate reader (PerkinElmer).

[0159] GraphPad Prism 6 software was used for data analysis to obtain the IC values ​​of the compounds. 50 (nM).

[0160] Experimental results and conclusions: After testing, the IC value of compound A of the present application on the cell proliferation of HCC1806 / NIH:OVCAR-3 cell line was 50 It can be less than 100nM, and has a better inhibitory effect than the reference compound PF-06873600.

[0161] (III) In vivo efficacy study in HCC1806 human breast cancer model

[0162] 1. Experimental Materials:

[0163] HCC1806 was purchased from Tongpai (Shanghai) Biotechnology Co., Ltd.; NIH: OVCAR-3 was purchased from the ATCC cell bank in the United States.

[0164] 1640 culture medium, fetal bovine serum (FBS), and Penicillin-Streptomycin were purchased from GIBCO, and PF-06873600 was purchased from MCE.

[0165] 2. Experimental methods:

[0166] Cells in the logarithmic growth phase were collected and inoculated subcutaneously on the right side of BALB / c nude mice to establish a tumor model. The day of inoculation was designated as D0, and on the fourth day after inoculation (D4), when the average tumor volume reached 150 mm 3 Around 6 weeks old, select rats with moderate tumor volumes for inclusion in each group. Start oral administration on the day of grouping. Collect body weight and tumor volume data 2-3 times a week and plot a weight and tumor growth curve. Tumor volume V = 1 / 2 × a × b 2 , where a and b represent the long and short diameters of the tumor, respectively.

[0167] Experimental results and conclusions: After testing, the tumors in the treatment group treated with compound A of the present application were effectively inhibited. Compared with the reference compound PF-06873600, the compound of the present application had a better tumor inhibitory effect, and the weight of the mice did not decrease significantly, indicating that all treatment regimens (10 mg / kg BID, 20 mg / kg BID, 30 mg / kg QD) were well tolerated.

[0168] (IV) In vivo efficacy study in the OVCAR-3 human ovarian cancer model

[0169] 1. Experimental Materials:

[0170] OVCAR-3 was purchased from the ATCC cell bank. 1640 culture medium, fetal bovine serum (FBS), and penicillin-streptomycin were purchased from GIBCO. PF-06873600 was purchased from MCE.

[0171] 2. Experimental methods:

[0172] Cells in the logarithmic growth phase were collected and inoculated subcutaneously on the right side of BALB / c nude mice to establish a tumor model. The day of inoculation was designated as D0, and 27 days after inoculation (D27), when the average tumor volume reached 180 mm 3 Around 6 weeks old, select rats with moderate tumor volumes for enrollment, with 6 rats per group. Start oral administration on the day of grouping. Continue administration for 21 days. Collect body weight and tumor volume data 2-3 times per week, and plot a weight and tumor growth curve. Tumor volume V = 1 / 2 × a × b2 , where a and b represent the long and short diameters of the tumor, respectively.

[0173] Experimental results and conclusions: After testing, the tumors in the treatment group treated with compound A of the present application were effectively inhibited. Compared with the reference compound PF-06873600, the compound of the present application had a better tumor inhibitory effect, and the weight of the mice did not decrease significantly, indicating that all treatment regimens (5 mg / kg BID, 7.5 mg / kg BID, 10 mg / kg QD, 20 mg / kg QD) were well tolerated.

[0174] Example 2 Preparation of Form I of Compound of Formula (A)

[0175] Method 1:

[0176] Approximately 10 mg of Compound A was dissolved in 1.0 mL of acetone at room temperature. Then, 4 mL of purified water was added dropwise to the solution at room temperature. After solid precipitation, the suspension was stirred at room temperature for two hours, then filtered to collect the solid and air-dried at room temperature.

[0177] The obtained solid was subjected to XRPD and TGA-DSC test characterization, and the solid was Form I.

[0178] The XRPD spectrum is shown in Figure 1.

[0179] The TGA-DSC spectrum is shown in Figure 2, which has an obvious endothermic characteristic peak at around 200°C.

[0180] Method 2:

[0181] Approximately 10 mg of Compound A was dissolved in 1.0 mL of acetone at room temperature. Then, 4 mL of n-heptane was added dropwise to the solution at room temperature. After solid precipitation, the suspension was stirred at room temperature for two hours, then filtered, the solid collected, and air-dried at room temperature. The XRPD spectrum of the resulting solid is generally shown in Figure 1.

[0182] Method 3:

[0183] Approximately 10 mg of Compound A was dissolved in 2.5 mL of ethyl acetate at room temperature. Then, 10 mL of n-heptane was added dropwise to the solution at room temperature. After solid precipitation, the suspension was stirred at room temperature for two hours, then filtered to collect the solid and air-dried at room temperature. The resulting solid had an XRPD pattern similar to that shown in Figure 1.

[0184] Method 4:

[0185] Approximately 10 mg of Compound A was dissolved in 2 mL of methanol at room temperature. Then, 8 mL of purified water was added dropwise to the solution at room temperature. After solid precipitation, the suspension was stirred at room temperature for two hours, then filtered, the solid collected, and air-dried at room temperature. The XRPD spectrum of the resulting solid is generally shown in Figure 1.

[0186] Method 5:

[0187] Approximately 10 mg of Compound A was dissolved in 0.5 mL of tetrahydrofuran at room temperature. Then, 2.0 mL of n-heptane was added dropwise to the solution at room temperature. After solid precipitation, the suspension was stirred at room temperature for two hours, then filtered to collect the solid and air-dried at room temperature. The XRPD spectrum of the resulting solid is generally shown in Figure 1.

[0188] Method 6:

[0189] Approximately 10 mg of Compound A was dissolved in 3.0 mL of acetonitrile at room temperature. Then, 15 mL of purified water was added dropwise to the solution at room temperature. After solid precipitation, the suspension was stirred at room temperature for two hours, then filtered, the solid collected, and air-dried at room temperature. The XRPD spectrum of the resulting solid is generally shown in Figure 1.

[0190] Method 7:

[0191] Approximately 10 mg of Compound A was dissolved in 0.5 mL of dichloromethane at room temperature. Then, 2.0 mL of purified water was added dropwise to the solution at room temperature. After solid precipitation, the suspension was stirred at room temperature for two hours, then filtered, the solid collected, and air-dried at room temperature. The XRPD spectrum of the resulting solid is generally shown in Figure 1.

[0192] Example 3 Preparation of Crystalline Form II of Compound of Formula (A)

[0193] About 10 mg of compound A was dissolved in 1 mL of 1,4-dioxane at room temperature, filtered, and the filtrate was placed in a ventilated place at room temperature to evaporate. After evaporation, the solid was collected and air-dried at room temperature.

[0194] The obtained solid was subjected to XRPD and TGA-DSC test characterization, and the solid was Form II.

[0195] The XRPD spectrum is shown in Figure 3.

[0196] The TGA-DSC spectrum is shown in Figure 4.

[0197] Hygroscopicity test

[0198] The moisture adsorption / desorption data of Form I were tested with reference to the "Guidelines for Hygroscopicity Tests of Drugs" in the Chinese Pharmacopoeia.

[0199] Figure 5 is a DVS curve of Form I, and Figure 6 shows XRPD spectra of Form I before and after DVS testing. DVS results indicate that Form I absorbs moisture and gains 0.14% at 80% RH and 0.16% at 90% RH, indicating that this form is virtually non-hygroscopic. XRPD analysis of the remaining solid after the DVS experiment indicates that the form has not changed.

[0200] Stability test

[0201] The stability of Form I at various temperatures and humidities was investigated with reference to the "Guidelines for Stability Testing of Drug Substances and Drug Products" in the Chinese Pharmacopoeia. Purity was tested using HPLC on days 0, 5, and 10 (denoted as D0, D5, and D10, respectively), and the crystal form was tested by XRPD. The XRPD spectra of Form I before and after the stability test are shown in Figure 7.

[0202] The experimental results are shown in Table 10. The physical properties of Form I are stable under high temperature and high humidity conditions, its crystal form does not change, and its chemical purity does not decrease significantly.

[0203] Table 10 Stability test results of Form I

[0204] Example 4 Preparation of Form III of the Phosphate Salt of the Compound of Formula (A)

[0205] 1 g of the compound of formula (A) was added to 50 ml of ethanol at room temperature, and then 125 μL of concentrated phosphoric acid was added. After the system was dissolved, a solid was quickly precipitated. The mixture was suspended and slurried at room temperature overnight, filtered, and the filter cake was dried in vacuo at 50° C. to obtain N-(1-((4-((3S,5S)-3,5-dimethylpiperazin-1-yl)phenyl)sulfonyl)piperidin-4-yl)-4-((S)-tetrahydrofuran-3-yl)oxy)-5-(trifluoromethyl)pyrimidin-2-amine phosphate.

[0206] 1 H-NMR (400MHz, DMSO-d6): δ (ppm) 8.29 (s, 1H), 8.03-7.85 (m, 1H), 7.54-7.52 ( m,2H),7.11-7.09(d,J=9.2Hz,2H),5.56-5.55(m,1H),3.94-3.86(m,1H),3.80 -3.71(m,4H),3.57-3.45(m,6H),3.25-3.22(m,2H),2.50-2.33(m,2H),2.24-2 .14(m,1H),1.98-1.91(m,3H),1.58-1.53(m,2H),1.24-1.23(d,J=6.0Hz,6H).

[0207] PO4 measured by ion chromatography3- The content is 14.9%, close to monophosphate PO4 3- The theoretical content (13.9%), combined with the above NMR results, confirmed that it was in the form of monophosphate, that is, the molar ratio of the compound of formula (A) to phosphoric acid was 1:1.

[0208] The obtained solid was subjected to XRPD and TGA-DSC test characterization, and the solid was Form III.

[0209] The XRPD spectrum is shown in Figure 8.

[0210] The TGA-DSC spectrum is shown in Figure 9.

[0211] Example 5 Preparation of Form IV of the Phosphate Salt of the Compound of Formula (A)

[0212] 1 g of the phosphate crystal form III of the compound of formula (A) was added to a high-pressure reactor, and the atmosphere was replaced with nitrogen three times. The temperature was raised to 160° C. under a nitrogen atmosphere, kept at this temperature for half an hour, and then cooled to room temperature. The nitrogen was vented to obtain a solid.

[0213] The obtained solid was subjected to XRPD and TGA-DSC test characterization, and the solid was found to be Form IV.

[0214] The XRPD spectrum is shown in Figure 10.

[0215] The TGA-DSC spectrum is shown in FIG11 . In the DSC spectrum, there is a characteristic endothermic peak at 229° C.

[0216] Hygroscopicity test

[0217] The moisture adsorption / desorption data of Form IV were tested with reference to the "Guidelines for Hygroscopicity Tests of Drugs" in the Chinese Pharmacopoeia.

[0218] FIG12 is a DVS curve of Form IV. The DVS results show that Form IV has a moisture absorption weight gain of 0.398% at 80% RH and a moisture absorption weight gain of 0.491% at 90% RH, indicating that the crystal form is slightly hygroscopic.

[0219] Stability test

[0220] The stability of Form IV at various temperatures and humidities was investigated in accordance with the "Guidelines for Stability Testing of Drug Substances and Drug Products" in the Chinese Pharmacopoeia. Purity was tested by HPLC on days 0, 5, and 10, and the crystal form was determined by XRPD. The XRPD spectra of Form IV before and after the stability test are shown in Figure 13.

[0221] The experimental results are shown in Table 11. The physical and chemical properties of Form IV are stable under high temperature and high humidity conditions, its crystal form does not change, and its chemical purity does not decrease significantly.

[0222] Table 11 Results of stability test of Form IV

[0223] The above description is only a preferred embodiment of the present application and is not intended to limit the scope of protection of the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application are included in the scope of protection of the present application.

Claims

1. A crystalline form I of a compound of formula (A), It is characterized by: The X-ray powder diffraction pattern of the crystalline form I has characteristic peaks at 2θ values ​​of 10.49°, 12.10°, 17.74°, 19.88°, and 21.66°, and the 2θ error range is ±0.2°.

2. The crystalline form I according to claim 1, wherein The X-ray powder diffraction pattern of the crystalline form I has characteristic peaks at 2θ values ​​of 10.03°, 10.49°, 12.10°, 14.28°, 14.81°, 17.74°, 18.28°, 19.88°, 20.57°, 21.66°, 23.11°, 23.76°, and 26.29°, and the 2θ error range is ±0.2°.

3. The crystalline form I according to claim 1, wherein The X-ray powder diffraction pattern of the crystalline form I has characteristic peaks at 2θ values ​​of 10.03°, 10.49°, 11.48°, 12.10°, 13.50°, 14.28°, 14.81°, 16.16°, 16.87°, 17.74°, 18.28°, 19.88°, 20.57°, 21.10°, 21.66°, 22.13°, 22.45°, 23.11°, 23.76°, 24.67°, 25.87°, 26.29°, 30.46°, and 32.57°, and the 2θ error range is ±0.2°.

4. The crystalline form I according to any one of claims 1 to 3, wherein The X-ray powder diffraction pattern of the crystalline form I is shown in FIG1 .

5. The crystalline form I according to any one of claims 1 to 4, wherein The DSC spectrum of the crystalline form I has an endothermic characteristic peak at 200°C.

6. The crystalline form I according to any one of claims 1 to 5, wherein The TGA-DSC spectrum of the crystalline form I is shown in FIG2 .

7. A crystalline form II of a compound of formula (A), It is characterized by: The X-ray powder diffraction pattern of the crystalline form II has characteristic peaks at 2θ values ​​of 8.09°, 13.31°, 16.59°, 19.55°, 21.59°, and 25.01°, and the 2θ error range is ±0.2°.

8. The crystalline form II according to claim 7, wherein The X-ray powder diffraction pattern of the crystalline form II has characteristic peaks at 2θ values ​​of 6.13°, 8.09°, 11.60°, 13.31°, 14.44°, 16.59°, 17.13°, 18.27°, 19.55°, 20.93°, 21.59°, 22.54°, 25.01°, and 26.92°, and the 2θ error range is ±0.2°.

9. The crystalline form II according to claim 8, characterized in that The X-ray powder diffraction pattern of the crystalline form II has characteristic peaks at 2θ values ​​of 6.13°, 8.09°, 9.94°, 11.60°, 13.31°, 14.44°, 16.59°, 17.13°, 17.71°, 18.27°, 19.55°, 19.98°, 20.93°, 22.07°, 21.59°, 22.54°, 25.01°, 25.35°, 25.60°, 26.92°, and 29.34°, and the 2θ error range is ±0.2°.

10. The crystalline form II according to any one of claims 7 to 9, characterized in that The X-ray powder diffraction pattern of the crystalline form II is shown in FIG3 .

11. The crystalline form II according to any one of claims 7 to 10, characterized in that The TGA-DSC spectrum of the Form II is shown in FIG4 .

12. A phosphate salt of a compound of formula (A), characterized in that The molar ratio of the compound of formula (A) to phosphoric acid is 1:1, 13. A crystalline form III of the phosphate of the compound of formula (A) according to claim 12, It is characterized by: The X-ray powder diffraction pattern of the crystalline form III has characteristic peaks at 2θ values ​​of 5.85°, 8.94°, 14.86°, and 16.00°, and the 2θ error range is ±0.2°.

14. The crystalline form III according to claim 13, characterized in that The X-ray powder diffraction pattern of the crystalline form III has characteristic peaks at 2θ values ​​of 5.85°, 8.94°, 10.29°, 13.21°, 14.86°, 16.00°, 17.01°, 17.65°, and 19.25°, and the 2θ error range is ±0.2°.

15. The crystalline form III according to claim 13, wherein The X-ray powder diffraction pattern of the crystalline form III has characteristic peaks at 2θ values ​​of 3.43°, 5.85°, 8.94°, 10.29°, 11.69°, 12.30°, 13.21°, 14.86°, 16.00°, 17.01°, 17.65°, 19.25°, 19.83°, 20.71°, 21.82°, 22.28°, and 23.86°, and the 2θ error range is ±0.2°.

16. The crystalline form III according to any one of claims 13 to 15, wherein The X-ray powder diffraction pattern of the crystalline form III is shown in FIG8 .

17. The crystalline form III according to any one of claims 13 to 16, wherein The TGA-DSC spectrum of the Form III is shown in FIG9 .

18. A crystalline form IV of the phosphate salt of the compound of formula (A) according to claim 12, It is characterized by: The X-ray powder diffraction pattern of the crystalline form IV has characteristic peaks at 2θ values ​​of 13.07°, 15.66°, 16.11°, 16.84°, and 21.89°, and the 2θ error range is ±0.2°.

19. The crystalline form IV according to claim 18, characterized in that The X-ray powder diffraction pattern of the crystalline form IV has characteristic peaks at 2θ values ​​of 7.81°, 13.07°, 15.20°, 15.66°, 16.11°, 16.84°, 19.61°, 21.89°, 22.16°, and 23.57°, and the 2θ error range is ±0.2°.

20. The crystalline form IV according to claim 19, wherein The X-ray powder diffraction pattern of the crystalline form IV has characteristic peaks at 2θ values ​​of 7.81°, 10.97°, 13.07°, 14.20°, 15.20°, 15.66°, 16.11°, 16.84°, 19.61°, 21.89°, 22.16°, 22.93°, 23.57°, 24.70°, 25.92°, 27.42°, and 28.61°, and the 2θ error range is ±0.2°.

21. The crystalline form IV according to any one of claims 18 to 20, wherein The X-ray powder diffraction pattern of the crystalline form IV is shown in FIG10 .

22. The crystalline form IV according to any one of claims 18 to 21, wherein The DSC spectrum of the crystalline form IV has an endothermic characteristic peak at 229°C.

23. The crystalline form IV according to any one of claims 18 to 22, wherein The TGA-DSC spectrum of the Form IV is shown in FIG11 .

24. A pharmaceutical composition comprising the crystalline form I according to any one of claims 1 to 6, the crystalline form II according to any one of claims 7 to 11, the phosphate salt according to claim 12, the crystalline form III according to any one of claims 13 to 17, or the crystalline form IV according to any one of claims 18 to 23, and a pharmaceutically acceptable carrier.

25. Use of the crystalline form I according to any one of claims 1-6, the crystalline form II according to any one of claims 7-11, the phosphate according to claim 12, the crystalline form III according to any one of claims 13-17, the crystalline form IV according to any one of claims 18-23, or the pharmaceutical composition according to claim 24 in the preparation of a medicament for treating CDK-mediated cancer.

26. Use of the crystalline form I according to any one of claims 1 to 6, the crystalline form II according to any one of claims 7 to 11, the phosphate according to claim 12, the crystalline form III according to any one of claims 13 to 17, the crystalline form IV according to any one of claims 18 to 23, or the pharmaceutical composition according to claim 24 in the treatment of CDK-mediated cancer.

27. The use according to claim 25 or 26, wherein the cancer comprises ovarian cancer, breast cancer, acute myeloid leukemia, chronic lymphocytic leukemia or small lymphocytic lymphoma.

28. The method for preparing the crystalline form I according to any one of claims 1 to 6, characterized in that: The following steps are involved: (a) dissolving compound A in a first solvent to obtain a solution; (b) adding a second solvent to the solution of step (a) and stirring; (c) After the solid precipitates, it is collected by filtration and dried.

29. The preparation method according to claim 28, wherein the first solvent is selected from at least one of acetone, methanol, tetrahydrofuran, ethyl acetate, acetonitrile and dichloromethane.

30. The preparation method according to claim 28, wherein the second solvent is at least one selected from water, n-heptane, n-hexane, and methyl tert-butyl ether.

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