Polymorphs of a CDK inhibitor and its phosphate

Abstract

The application provides a polymorph of a compound of formula (A) (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) and a polymorph of a phosphate salt of the compound of formula (A). The crystal form provided by the application has good chemical stability, physical stability and low hygroscopicity, is less affected by temperature, humidity and light, and is convenient to store and develop into a preparation.

Description

[0001] This application claims priority to Chinese Patent Application No. CN202210947810.2, filed on August 5, 2022, entitled "A Polymorph of a CDK Inhibitor"; Chinese Patent Application No. CN202210936709.7, filed on August 5, 2022, entitled "A Polymorph of a CDK Inhibitor Phosphate"; and Chinese Patent Application No. CN202310889701.4, filed on July 19, 2023, entitled "A Polymorph of a CDK Inhibitor and Its Phosphate", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This application discloses a CDK inhibitor, multiple crystal forms of its phosphate, methods for preparing the inhibitor, and their applications in the treatment of cancer. Background Technology

[0003] The cell cycle is a fundamental process of cellular life, controlling cell growth, proliferation, and differentiation. Cyclin-dependent kinases (CDKs) are an important class of cellular enzymes that work in conjunction with cyclins to play a crucial role in the regulation of 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] CDK2, an important regulator of the cell cycle, forms a kinase complex with cyclin E or A, playing a decisive role in driving the cell cycle from G1 phase to S phase and maintaining S phase. The mechanism primarily involves the combined action of Cyclin E and CDK2 to phosphorylate the retinoblastoma susceptibility gene (Rb) protein. Rb phosphorylation leads to the release of E2F (transcription factor), which binds to the upstream regions of some genes (usually located in promoter or enhancer regions), initiating the transcriptional expression of those cell cycle-related genes and enabling the cell to transition from late G1 phase to S phase. Numerous studies have shown that abnormal expression of CDK2 is closely related to the occurrence of cancer, such as ovarian cancer with CCNE1 amplification, 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 established crucial role of cyclin-dependent kinases (CDKs) in cell cycle regulation, CDK inhibitors have become a hot research topic in anti-tumor drugs. Currently, several CDK inhibitors have been approved for marketing globally, but most target CDK4 / 6, primarily for breast cancer, such as Pfizer's Palbociclib, Novartis' Ribociclib, and Eli Lilly's abemaciclib. Multi-target inhibitors containing CDK2, such as fadraciclib, Roscovitine, and PF-06873600, are in different clinical stages. No CDK2 inhibitors have yet been approved for marketing; therefore, continued development of novel CDK inhibitors, especially those effective against CDK2, is of significant research importance.

[0006] Patent PCT / CN2022 / 074491 discloses a small molecule inhibitor targeting CDK2 / 4 / 6, especially CDK2, with the structure shown in formula (A) and the chemical name 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 CDK2 / 4 / 6 kinases relative to CDK1 / 7 / 9 kinases, exhibiting particularly outstanding inhibitory activity against CDK2 kinase, with a kinase selectivity reaching nearly 10 times, or even tens or hundreds of times higher. This small molecule inhibitor demonstrates good cell proliferation inhibitory activity and good tumor suppression activity and tolerability in in vivo pharmacodynamic experiments, showing promise for development into a clinical drug.

[0007] Summary of the Invention

[0008] This application discloses a polymorph of a CDK inhibitor, CDK inhibitor phosphate and its polymorphs, a method for preparing the polymorph, and their applications in the treatment of cancer.

[0009] Specifically,

[0010] The first aspect of this 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)pyrimidine-2-amine), wherein the X-ray powder diffraction pattern of crystalline form I has characteristic peaks at 2θ values ​​of 10.49°, 12.10°, 17.74°, 19.88°, and 21.66°, with a 2θ error range of ±0.2°.

[0011] In some embodiments of this application, the X-ray powder diffraction pattern of the crystal 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°, with a 2θ error range of ±0.2°.

[0012] In some embodiments of this application, the X-ray powder diffraction pattern of the crystal 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°, with a 2θ error range of ±0.2°.

[0013] In some embodiments of this application, the X-ray powder diffraction pattern of crystal form I is substantially as follows: Figure 1 As shown.

[0014] In some embodiments of this application, the DSC spectrum of the crystal form I has an endothermic characteristic peak at around 200°C.

[0015] In some embodiments of this application, the TGA-DSC spectrum of crystal form I is essentially as follows: Figure 2 As shown.

[0016] In some embodiments of this application, the XRPD diffraction peak resolution data of crystal form I of the compound of formula (A) above are basically as shown in Table 1.

[0017] Table 1 shows the XRPD diffraction peak analysis data for crystal form I of compound (A).

[0018]

[0019]

[0020] The second aspect of this application also provides a crystal form II of the 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)pyrimidine-2-amine), wherein the X-ray powder diffraction pattern of the crystal form II has characteristic peaks at 2θ values ​​of 8.09°, 13.31°, 16.59°, 19.55°, 21.59°, and 25.01°, with a 2θ error range of ±0.2°.

[0021] In some embodiments of this application, the X-ray powder diffraction pattern of the crystal 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°, with a 2θ error range of ±0.2°.

[0022] In some embodiments of this application, the X-ray powder diffraction pattern of the crystal 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°, with a 2θ error range of ±0.2°.

[0023] In some embodiments of this application, the X-ray powder diffraction pattern of the crystal form II described above is substantially as follows: Figure 3 As shown.

[0024] In some embodiments of this application, the TGA-DSC spectrum of the above-mentioned crystal form II is essentially as follows: Figure 4 As shown.

[0025] In some embodiments of this application, the XRPD diffraction peak resolution data of crystal form II of the compound of formula (A) above are basically as shown in Table 2.

[0026] Table 2 shows the XRPD diffraction peak analysis data for crystal form II of compound (A).

[0027]

[0028]

[0029] A third aspect of this application also provides a phosphate of compound (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 compound (A) to phosphoric acid is 1:1.

[0030]

[0031] The inventors of this application attempted to form salts of compound (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, only the phosphate of formula (A) was found to have good crystallinity, hygroscopicity, solubility, and stability. Some other acids could not form salts with compound (A), some had very poor crystallinity, or were mostly amorphous. Some could form crystalline forms, but the crystalline forms were hygroscopic or had poor stability.

[0032] The fourth aspect of this application also provides a phosphate crystal form III of the compound of formula (A), wherein the X-ray powder diffraction pattern of the crystal 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°.

[0033] In some embodiments of this application, the X-ray powder diffraction pattern of the crystal 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°, with a 2θ error range of ±0.2°.

[0034] In some embodiments of this application, the X-ray powder diffraction pattern of the crystal 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°, with a 2θ error range of ±0.2°.

[0035] In some embodiments of this application, the X-ray powder diffraction pattern of the above-mentioned crystal form III is substantially as follows: Figure 8 As shown.

[0036] In some embodiments of this application, the TGA-DSC spectrum of the above-mentioned crystal form III is essentially as follows: Figure 9 As shown.

[0037] In some embodiments of this application, the XRPD diffraction peak resolution data of the crystal form III described above are basically as shown in Table 3.

[0038] Table 3 shows the XRPD diffraction peak analysis data for phosphate crystal form III of compound (A).

[0039]

[0040]

[0041] The fifth aspect of this application also provides a crystalline form IV of the phosphate of compound (A), wherein the X-ray powder diffraction pattern of 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°.

[0042] In some embodiments of this application, the X-ray powder diffraction pattern of the above-mentioned crystal 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°, with a 2θ error range of ±0.2°.

[0043] In some embodiments of this application, the X-ray powder diffraction pattern of the above-mentioned crystal 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°, with a 2θ error range of ±0.2°.

[0044] In some embodiments of this application, the X-ray powder diffraction pattern of the above-mentioned crystal form IV is substantially as follows: Figure 10 As shown.

[0045] In some embodiments of this application, the DSC spectrum of the above-mentioned crystal form IV has an endothermic characteristic peak at around 229°C.

[0046] In some embodiments of this application, the TGA-DSC spectrum of the above-mentioned crystal form IV is essentially as follows: Figure 11 As shown.

[0047] In some embodiments of this application, the XRPD diffraction peak resolution data of the above-mentioned crystal form IV are basically as shown in Table 4.

[0048] Table 4 shows the XRPD diffraction peak analysis data for phosphate crystal form IV of compound (A).

[0049]

[0050]

[0051] A sixth aspect of this application also provides a pharmaceutical composition comprising crystal form I as described in the first aspect of this application, crystal form II as described in the second aspect of this application, phosphate as described in the third aspect of this application, crystal form III as described in the fourth aspect of this application, or crystal form IV as described in the fifth aspect of this application, and a pharmaceutically acceptable carrier.

[0052] The seventh aspect of this application also provides the use of the crystal form I described in the first aspect of this application, the crystal form II described in the second aspect of this application, the phosphate described in the third aspect of this application, the crystal form III described in the fourth aspect of this application, the crystal form IV described in the fifth aspect of this application, or the pharmaceutical composition described in the sixth aspect of this application in the preparation of a medicament for treating CDK-mediated cancer.

[0053] The eighth aspect of this application also provides the use of the crystal form I described in the first aspect of this application, the crystal form II described in the second aspect of this application, the phosphate described in the third aspect of this application, the crystal form III described in the fourth aspect of this application, the crystal form IV described in the fifth aspect of this application, or the pharmaceutical composition described in the sixth aspect of this application in the treatment of CDK-mediated cancer.

[0054] The ninth aspect of this application also provides crystal form I, crystal form II, phosphate, crystal form III, crystal form IV, or pharmaceutical composition as described in the first aspect of this application, for use as a drug or for treating CDK-mediated cancers.

[0055] In some embodiments of this application, the aforementioned cancers include ovarian cancer, breast cancer, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), or small lymphocytic lymphoma (SLL).

[0056] In some embodiments of this application, the aforementioned cancer is breast cancer.

[0057] The tenth aspect of this application also provides a method for preparing crystal form I as described in the first aspect of this application, comprising the following steps:

[0058] (a) Compound A is dissolved in a first solvent to obtain a solution;

[0059] (b) Add a second solvent to the solution from step (a) and stir for a certain period of time;

[0060] (c) After the solid precipitates, filter, collect, and dry the solid.

[0061] In some embodiments of this application, the first solvent in the above preparation method is selected from at least one of acetone, methanol, tetrahydrofuran, ethyl acetate, acetonitrile, and dichloromethane.

[0062] In some embodiments of this application, the second solvent in the above preparation method is selected from at least one of water, n-heptane, and n-hexane.

[0063] In some embodiments of this application, the stirring time in the above preparation method is 1-3 hours, preferably 2 hours.

[0064] Technical effect

[0065] The crystal form in this application has good chemical stability, physical stability, and low hygroscopicity. It is less affected by temperature, humidity, and light, making it easy to store and develop formulations. Attached Figure Description

[0066] Figure 1 The image shows the XRPD (X-ray powder diffraction) pattern of crystal form I of compound (A).

[0067] Figure 2 The TGA-DSC (differential scanning calorimetry-thermogravimetric analysis) spectrum of crystal form I of compound (A) is shown.

[0068] Figure 3 The image shows the XRPD spectrum of crystal form II of compound (A).

[0069] Figure 4 The TGA-DSC spectrum of crystal form II of compound (A) is shown.

[0070] Figure 5 The image shows the DVS (Dynamic Gas-Phase Adsorption) spectrum of crystal form I of compound (A).

[0071] Figure 6 The XRPD comparison spectra of crystal form I of compound (A) before and after the DVS experiment are shown.

[0072] Figure 7 The XRPD comparison spectra of crystal form I of compound (A) before and after the stability experiment are shown.

[0073] Figure 8 The XRPD spectrum of phosphate of compound (A) in crystal form III is shown.

[0074] Figure 9 The TGA-DSC spectrum of the phosphate of compound (A) in crystal form III is shown.

[0075] Figure 10 The image shows the XRPD spectrum of phosphate form IV of compound (A).

[0076] Figure 11 The TGA-DSC spectrum of phosphate form IV of compound (A) is shown.

[0077] Figure 12 The image shows the DVS spectrum of crystal form IV of the phosphate of compound (A).

[0078] Figure 13 XRPD comparison spectra before and after the stability experiment of crystal form IV of the phosphate of compound (A). Detailed Implementation

[0079] The present application is described in detail below through examples, but this does not imply any adverse limitation on the present application. The compounds of this application can be prepared by various synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include, but are not limited to, the embodiments of this application. It will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of this application without departing from the spirit and scope of this application.

[0080] Definitions and Explanations

[0081] Unless otherwise stated, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered uncertain or unclear unless specifically defined, but should be understood in its ordinary sense.

[0082] The term "pharmaceutical-grade carrier" refers to a medium generally acceptable in the art for delivering a bioactive pharmaceutical agent to animals, particularly mammals. Depending on the route of administration and dosage form, this includes, for example, adjuvants, excipients, or excipients such as diluents, preservatives, fillers, flow modifiers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, aromatizers, antibacterial agents, antifungal agents, lubricants, and dispersants. Pharmaceutically acceptable carriers are formulated based on a multitude of factors, within the scope of those skilled in the art. These include, but are not limited to, the type and nature of the active pharmaceutical agent being formulated, the recipient to whom the composition containing the pharmaceutical agent is to be administered, the intended route of administration of the composition, and the target therapeutic indication. Pharmaceutically acceptable carriers include both aqueous and non-aqueous media, as well as various solid and semi-solid dosage forms. In addition to the active pharmaceutical agent, such carriers include many different components and additives, and the inclusion of such additional components in the formulation for various reasons (e.g., stabilizing active pharmaceutical agents, binders, etc.) is well known to those skilled in the art.

[0083] As is known in the art, X-ray powder diffraction patterns have one or more measurement errors due to minute variations in measurement conditions. The structures of the crystals, crystal forms, or crystalline forms disclosed or claimed in this application may exhibit similar but not identical analytical characteristics within a reasonable error range, depending on 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 typically produces an error within ±0.20°. Therefore, this application includes not only crystals with completely consistent diffraction angles in powder X-ray powder diffraction, but also crystals with consistent diffraction angles within an error range of ±0.20°. The crystalline form of compound A in this application is not limited to crystals having the same X-ray powder diffraction pattern as shown in the accompanying drawings; any crystal having a substantially identical X-ray powder diffraction pattern as shown in the accompanying drawings falls within the scope of this application.

[0084] The text refers to "X-ray powder diffraction patterns that are substantially the same as those shown in the accompanying figures." It should be understood that the term "substantially the same" used in this context also indicates that the 2θ angle values ​​of the X-ray powder diffraction patterns may vary slightly due to inherent experimental variations accompanying these measurements, and both are of the same crystalline form.

[0085] It should be understood that different types of equipment or different testing conditions may yield slightly different DSC spectra and endothermic transition temperature readings. DSC data can reflect changes in the state of matter; strong endothermic peaks may indicate that the substance has undergone dehydration or desolvation, or crystal transformation, or melting, etc.; when reflecting the molten state, the corresponding temperature is generally understood as the melting point of the substance. This value will be affected by compound purity, sample weight, heating rate, particle size, and calibration and maintenance of the testing equipment. Those skilled in the art will understand that the temperature at which a substance changes from a solid to a liquid state is usually a temperature range, not a fixed point value; therefore, the temperature corresponding to the endothermic peak or the melting point of the substance can be characterized by either the onset value, the peak value, or other reasonable values. The maximum endothermic transition temperature of the crystal form can be within the range of ±5.0℃, preferably ±2.0℃, of the specific values ​​disclosed above.

[0086] This application also uses thermogravimetric analysis (TGA) to analyze the relationship between the degree of decomposition, sublimation, or evaporation of the crystal form (weight loss) and temperature. It should be understood that the values ​​obtained for the same crystal form may have some error due to factors such as sample purity, particle size, different types of equipment, and different testing methods. The temperature at which the crystal form decomposes, sublimates, or evaporates can be within ±3.0℃ of the specific values ​​disclosed above, for example, within ±2.0℃.

[0087] The "stability" of a crystal form includes "chemical stability" and / or "physical stability." "Chemical stability" refers to the degree to which the crystal form undergoes degradation reactions under certain temperature, humidity, and light conditions; it reflects the stability of the crystal form under storage conditions. "Physical stability" refers to the degree to which the crystal form undergoes a solid-state transformation under certain specific conditions, such as under conditions of high temperature, high humidity, grinding, tableting, solvent removal, or solvent adsorption, transforming into another crystal form. Therefore, "physical stability" can, to some extent, reflect the stability of the crystal form during its use in formulations and other processes.

[0088] The crystalline structure of 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 the crystalline structure from a solvent mixture include solvent evaporation, lowering the temperature of the solvent mixture, crystallization of a supersaturated solvent mixture of the molecule and / or salt, lyophilizing the solvent mixture, and adding an antisolvent to the solvent mixture.

[0089] The term "drying" refers to the process of removing solvent from the resulting solid, including but not limited to air drying at room temperature, high-temperature drying, and vacuum drying.

[0090] The intermediate compounds of this application can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitutions known to those skilled in the art. Preferred embodiments include, but are not limited to, the embodiments of this application.

[0091] In the embodiments of this application, the title compound was named using Chemdraw to convert the compound structure. If there is a discrepancy between the compound name and the compound structure, it can be determined by combining relevant information and reaction routes; if it cannot be confirmed by other means, the given compound structure shall prevail.

[0092] The preparation methods for some compounds in this application reference the preparation methods for the aforementioned similar compounds. Those skilled in the art should understand that when using or referring to the referenced preparation methods, the reactant ratios, reaction solvents, reaction temperatures, etc., can be appropriately adjusted according to the different reactants.

[0093] The compounds of this application can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitutions known to those skilled in the art. Preferred embodiments include, but are not limited to, the embodiments of this application.

[0094] Instruments and Analytical Methods

[0095] 1. X-ray powder diffraction (XRPD)

[0096] Solid samples were analyzed using an X-ray powder diffractometer (X'Pert PRO). An appropriate amount of fine powder was placed in the groove of the sample holder and pressed into a flat and dense plane using a glass slide. The XRPD measurement parameters are shown in Table 5.

[0097] Table 5 XRPD Test Parameters

[0098]

[0099] 2. Thermogravimetric analysis (TGA)

[0100] Thermogravimetric analysis (TGA) of solids was performed using a TA Instrument thermogravimetric analyzer. Approximately 1-5 mg of sample was placed in a peeled aluminum sample pan, heated according to the parameters listed in Table 6, and the data were analyzed using TRIOS.

[0101] Table 6 TGA Analysis Method Parameters

[0102]

[0103] 3. Simultaneous Thermal Analysis (TGA-DSC)

[0104] Thermogravimetric-differential scanning calorimetry (TGC) analysis of solids was performed using a Mettler Toledo simultaneous thermal analyzer. A suitable amount of the sample was placed in a crucible using a small spoon, ensuring even distribution, and weighed. The sample was heated according to the parameters listed in Table 7, and the data were analyzed using STARE.

[0105] Table 7 Parameters of TGA-DSC Analysis Method

[0106]

[0107] 4. Dynamic moisture adsorption-desorption analysis (DVS)

[0108] The hygroscopicity of the samples was determined using a DVS Intrinsic dynamic moisture adsorption analyzer. The samples were placed in a pre-peeled sample basket, and the instrument automatically weighed them. The samples were then analyzed according to the parameters in Table 8.

[0109] Table 8 Parameters of DVS Analysis Method

[0110]

[0111] 5. Ion chromatography analysis

[0112] Instrument: Ion chromatograph - conductivity detector;

[0113] Chromatographic column: Anion exchange column [analytical column Ionpac] TM AS11-HC (4mm×250mm), protective post Ionpac TM AG11-HC (4mm×50mm)];

[0114] Suppressor: AERS 500 4mm or equivalent suppressor;

[0115] Column temperature: 30℃;

[0116] Flow rate: 1.0 mL / min;

[0117] Injection volume: 25 μL;

[0118] Detection pool temperature: 35℃;

[0119] Mobile phase: 30 mmol / L potassium hydroxide solution;

[0120] Running time: Approximately 20 minutes.

[0121] Example 1: Preparation of compound (A)

[0122] Preparation of intermediate compound 99

[0123]

[0124] Step 1: Compound 1B-3 (508 mg, 1.81 mmol) was dissolved in dichloromethane (30 mL) at room temperature. Then, N,N-diisopropylethylamine (702 mg, 5.43 mmol) and 4-bromo-benzenesulfonyl chloride (508 mg, 2.00 mmol) were added to the solution under ice-water bath conditions. The reaction mixture was stirred at room temperature for 2 hours, then quenched with water (60 mL). The mixture was extracted with dichloromethane (20 mL × 3 times), and the organic phases were combined. The organic phase was washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and finally concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give 830 mg of N-(1-(1-(4-bromophenyl)sulfonyl)piperidin-4-yl)-4-chloro-5-(trifluoromethyl)pyrimidin-2-amine (99-1).

[0125] MS(ESI)M / Z:499.0[M+H] + .

[0126] Step 2: In a sealed container, (S)-3-hydroxytetrahydrofuran (42 mg, 0.48 mmol) was dissolved in tetrahydrofuran (2 mL). Then, sodium hydride (21 mg, 0.88 mmol) was added under ice-water bath conditions. After reacting for 15 minutes, compound 99-1 (200 mg, 0.40 mmol) was added. The reaction mixture was heated to 100°C and reacted for 4 hours. Water (60 mL) was then added to quench the reaction. The mixture was extracted with dichloromethane (20 mL × 3 times), and the organic phases were combined. The organic phase was first washed with saturated brine (30 mL), then dried over anhydrous sodium sulfate, filtered, and finally concentrated under reduced pressure. The 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).

[0127] MS(ESI)M / Z:551.0[M+H] + .

[0128] 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).

[0129] Preparation of compound A

[0130]

[0131] Step 1: Under nitrogen protection, compound 99 (25 g, 45.3 mmol), (2S,6S)-2,6-dimethylpiperazine-1-carboxylic acid tert-butyl ester (11.6 g, 45.3 mmol), tris(dibenzylacetone)dipalladium (4.1 g, 4.53 mmol), 2-dicyclohexylphosphine-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 × 100 mL). The organic phases were combined, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The 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-carboxylic acid tert-butyl ester (compound 168-1).

[0132] MS(ESI)M / Z:685.2[M+H] + .

[0133] Step 2: Compound 168-1 (18.1 g, 26.4 mmol) was dissolved in 1,4-dioxane (100 mL). Then, a solution of dioxane hydrochloride (100 mL) was added. The reaction mixture was stirred overnight at room temperature. The reaction mixture was concentrated under reduced pressure. The residue was washed with an aqueous sodium bicarbonate solution (300 mL), and the mixture was extracted with ethyl acetate (200 mL × 3 times). The organic phases were combined. The organic phase was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and finally concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give 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).

[0134] MS(ESI)M / Z:585.2[M+H] + .

[0135] 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).

[0136] Biological testing evaluation

[0137] (I) CDK in vitro enzyme experiments

[0138] This experiment used the capillary migration capacity variation assay (MSA) to test the inhibitory effect of the compound on the activity of CDK1 / CDK2 / CDK4 / CDK6 / CDK7 / CDK9 kinases, and determined the half-maximal inhibitory concentration (IC50) of the compound on the activity of CDK1 / CDK2 / CDK4 / CDK6 / CDK7 / CDK9 kinases. 50 .

[0139] 1. Experimental Materials

[0140] CDK1 / CDK2 / CDK4 / CDK6 / CDK7 / CDK9 were purchased from Carna, Carliper substrates CTD3 / 18 / 8 were purchased from Gill Biochemical, Dinaciclib / Palbociclib were purchased from Selleckchem, DMSO (dimethyl sulfoxide) was purchased from Sigma, and 384-well plates were purchased from Corning.

[0141] 2. Experimental Methods

[0142] (1) Prepare 1×kinase buffer.

[0143] (2) Preparation of compound concentration gradients: The test concentration of the test compound is 1 μM, 10 μM, or 30 μM as the starting concentration. Ten concentrations are obtained by 3-fold dilution and replicate detection is performed. Dilute to a final concentration of 100% DMSO in the source 384-well plate. Use an Echo 550 dispenser to transfer 250 nL of the 100-fold final concentration of the compound to the target 384-well plate. Add 250 nL of DMSO to the positive and negative control wells.

[0144] (3) Prepare a kinase solution with a final concentration of 2.5 times using 1×Kinase buffer.

[0145] (4) Add 10 μL of kinase solution at 2.5 times the final concentration to the compound wells and the positive control wells respectively; add 10 μL of 1×Kinase buffer to the negative control wells.

[0146] (5) Centrifuge at 1000 rpm for 30 seconds, shake the reaction plate to mix, and incubate at room temperature for 10 minutes.

[0147] (6) Prepare a 25 / 15 times final concentration of ATP and kinase substrate using 1×Kinase buffer.

[0148] (7) Add 15 μL of a mixture of ATP and substrate at a final concentration of 25 / 15 to initiate the reaction.

[0149] (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.

[0150] (9) Add 30 μL of the stop detection solution to stop the kinase reaction, centrifuge at 1000 rpm for 30 seconds, and shake to mix.

[0151] (10) Read the conversion rate using Caliper EZ Reader.

[0152] Calculation formula:

[0153] % inhibition rate (inhibition)=(conversion%_max-conversion%_sample) / (conversion%_max-conversion%_min)×100%

[0154] Where: Conversion%_sample is the conversion rate reading of the sample; Conversion%_min: the mean value of the negative control wells, representing the conversion rate reading of wells without enzyme activity; Conversion%_max: the mean value of the positive control wells, representing the conversion rate reading of wells without compound inhibition.

[0155] Fitting dose-response curve

[0156] Plotting the logarithmic value of concentration on the X-axis and the percentage inhibition rate on the Y-axis, dose-response curves were fitted using the log(inhibitor) vs. response-variable slope method in GraphPad Prism 5 to derive the IC50 values ​​of each compound on enzyme activity. 50 (Half-inhibitory concentration) value.

[0157] Calculation formula: Y = Bottom + (Top - Bottom) / (1 + 10^(LogIC)) 50 -X)*HillSlope)).

[0158] 3. Experimental Results

[0159] The compound in this application inhibits CDK kinase activity by IC50. 50 The data is shown in Table 9. Among them: IC 50 Compounds <0.5 nM are denoted by AA, and 0.5 nM ≤ IC 50 Compounds with a molecular weight <2.5 nM are designated as AB, and 2.5 nM ≤ IC. 50 Compounds <10 nM are denoted by AC, and 10 nM ≤ IC. 50 Compounds with a molecular weight <50 nM are identified by B, and compounds with a molecular weight ≤50 nM are identified by IC. 50 Compounds with a molecular weight <100 nM are identified by C, and 100 nM ≤ IC 50 Compounds with a molecular weight <1000 nM are identified by D, IC 50 Compounds with a molecular weight greater than 1000 nM are identified by E.

[0160] Table 9: Results of CDK Enzyme Inhibition

[0161]

[0162] Conclusion: Compound A of this application has good CDK 2 / 4 / 6 kinase inhibitory activity, especially showing excellent inhibitory activity on CDK 2 kinase; compared with CDK 1 / 7 / 9 kinases, compound A of this application can selectively inhibit CDK 2 / 4 / 6 kinases, especially showing excellent inhibitory activity on CDK 2 kinase, with kinase selectivity reaching nearly 10 times, or even tens or hundreds of times.

[0163] (II) HCC1806 / NIH:OVCAR-3 Cell Proliferation Inhibition Experiment

[0164] This experiment used the CellTiter-Glo method to test the inhibitory effect of the compound on the proliferation of HCC1806 / NIH:OVCAR-3 cells, and determined the half-maximal concentration (IC50) of the compound that inhibits cell growth. 50 (nM).

[0165] 1. Experimental Materials

[0166] HCC1806 was purchased from Tongpai (Shanghai) Biotechnology Co., Ltd.; NIH: OVCAR-3 was purchased from the American ATCC cell bank.

[0167] 1640 culture medium, fetal bovine serum (FBS), penicillin-streptomycin, and GlutaMAX-ISupplement were purchased from GIBCO.

[0168] PF-06873600 was purchased from Selleck.

[0169] CellTiter-Glo reagents were purchased from Promega.

[0170] 2. Experimental Methods

[0171] 1) HCC1806 / NIH:OVCAR-3 cells were seeded into 96-well culture plates at a density of 600 / 1500 cells per well, 100 μL per well.

[0172] 2) Day 0: Using Echo, add 100 nL of serially diluted test compound to the cells in the culture plate, with a final DMSO concentration of 0.5%. Incubate the culture plate in a cell culture incubator for 168 hours (37°C, 5 vol% CO2). Add 30 nL of DMSO to each well for the blank control.

[0173] 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.

[0174] 4) Envision microplate reader (PerkinElmer) detects chemiluminescence signals.

[0175] Data analysis was performed using GraphPad Prism 6 software to determine the IC50 of the compound. 50 (nM).

[0176] Experimental Results and Conclusions: The IC50 of compound A in this application on the proliferation of the HCC1806 / NIH:OVCAR-3 cell line was tested and found to be effective. 50 It can be less than 100 nM, and has a better inhibitory effect than the reference compound PF-06873600.

[0177] (III) In vivo efficacy experiment of HCC1806 human breast cancer model

[0178] 1. Experimental materials:

[0179] HCC1806 was purchased from Tongpai (Shanghai) Biotechnology Co., Ltd.; NIH: OVCAR-3 was purchased from the American ATCC cell bank.

[0180] 1640 medium, fetal bovine serum (FBS), and penicillin-streptomycin were purchased from GIBCO. PF-06873600 was purchased from MCE.

[0181] 2. Experimental Methods:

[0182] Cells in the logarithmic growth phase were collected and subcutaneously injected into BALB / c nude mice on the right side to establish a tumor model. The cells on the day of injection were designated D0. On day four (D4), when the average tumor volume reached 150 mm², the tumor model was established. 3 Around the time of grouping, six animals with tumors of moderate size were selected for inclusion in the group. Administering medication via gavage began on the day of grouping. Body weight and tumor volume data were recorded 2-3 times per week, and a body weight vs. tumor growth curve was plotted. Tumor volume V = 1 / 2 × a × b 2 , where a and b represent the long and short diameters of the tumor, respectively.

[0183] Experimental results and conclusions: The tumors in the treatment group treated with compound A of this application were effectively inhibited. Compared with the reference compound PF-06873600, the compound of this application had better tumor inhibition effect, and the body weight of mice did not decrease significantly, showing that all treatment regimens (10 mg / kg BID, 20 mg / kg BID, 30 mg / kg QD) were well tolerated.

[0184] (IV) In vivo efficacy experiment of OVCAR-3 human ovarian cancer model

[0185] 1. Experimental materials:

[0186] OVCAR-3 was purchased from the American College of Cell Bank (ATCC). 1640 medium, fetal bovine serum (FBS), and penicillin-streptomycin were purchased from GIBCO. PF-06873600 was purchased from MCE.

[0187] 2. Experimental Methods:

[0188] Cells in the logarithmic growth phase were collected and subcutaneously injected into BALB / c nude mice on the right side to establish a tumor model. The cells on the day of injection were named D0. On day 27 (D27), when the average tumor volume reached 180 mmHg, the tumor model was established. 3Around the same time, six mice with moderate tumor volume were selected for the group. Gavage administration began on the day of grouping. Administration continued for 21 days. Body weight and tumor volume data were recorded 2-3 times per week to plot a body weight vs. 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.

[0189] Experimental results and conclusions: The tumors in the treatment group treated with compound A of this application were effectively inhibited. Compared with the reference compound PF-06873600, the compound of this application had better tumor inhibition effect, and the body weight of mice did not decrease significantly, showing that all treatment regimens (5 mg / kg BID, 7.5 mg / kg BID, 10 mg / kg QD, 20 mg / kg QD) were well tolerated.

[0190] Example 2 Preparation of crystal form I of compound (A)

[0191] Method 1:

[0192] 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 the solid precipitated, the suspension was stirred at room temperature for two hours, then filtered, the solid was collected, and air-dried at room temperature.

[0193] The obtained solid was characterized by XRPD and TGA-DSC tests, and the solid was identified as crystal form I.

[0194] XRPD spectrum as shown Figure 1 As shown.

[0195] TGA-DSC spectrum as follows Figure 2 As shown, it has a distinct endothermic characteristic peak at around 200℃.

[0196] Method 2:

[0197] 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 the solid precipitated, the suspension was stirred at room temperature for two hours, then filtered, the solid was collected, and air-dried at room temperature. The XRPD spectrum of the obtained solid is basically as follows. Figure 1 As shown.

[0198] Method 3:

[0199] 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 the solid precipitated, the suspension was stirred at room temperature for two hours, then filtered, the solid was collected, and dried at room temperature. The XRPD spectrum of the obtained solid is basically as shown below. Figure 1 As shown.

[0200] Method 4:

[0201] 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 the solid precipitated, the suspension was stirred at room temperature for two hours, then filtered, the solid was collected, and air-dried at room temperature. The XRPD spectrum of the obtained solid is basically as follows. Figure 1 As shown.

[0202] Method 5:

[0203] 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 the solid precipitated, the suspension was stirred at room temperature for two hours, then filtered, the solid was collected, and air-dried at room temperature. The XRPD spectrum of the obtained solid is basically as shown below. Figure 1 As shown.

[0204] Method 6:

[0205] 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 the solid precipitated, the suspension was stirred at room temperature for two hours, then filtered, the solid was collected, and air-dried at room temperature. The XRPD spectrum of the obtained solid is basically as shown below. Figure 1 As shown.

[0206] Method 7:

[0207] 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 the solid precipitated, the suspension was stirred at room temperature for two hours, then filtered, the solid was collected, and air-dried at room temperature. The XRPD spectrum of the obtained solid is basically as shown below. Figure 1 As shown.

[0208] Example 3 Preparation of crystal form II of compound (A)

[0209] Dissolve approximately 10 mg of compound A in 1 mL of 1,4-dioxane at room temperature, filter, and allow the filtrate to evaporate in a ventilated place at room temperature. After evaporation, collect the solid and air dry it at room temperature.

[0210] The obtained solid was characterized by XRPD and TGA-DSC tests, and the solid was found to be crystal form II.

[0211] XRPD spectrum as shown Figure 3 As shown.

[0212] TGA-DSC spectrum as follows Figure 4 As shown.

[0213] Hygroscopicity test

[0214] Referencing the "Guidelines for Hygroscopicity Testing of Drugs" in the Chinese Pharmacopoeia, the water adsorption / desorption data of crystal form I were tested.

[0215] Figure 5 This is the DVS curve for crystal form I. Figure 6 The XRPD spectra of crystal form I before and after DVS testing are shown. The DVS results show that crystal form I has a moisture absorption weight gain of 0.14% at 80% RH and a moisture absorption weight gain of 0.16% at 90% RH, indicating that this crystal form has almost no hygroscopicity. The XRPD of the remaining solid after the DVS experiment shows that the crystal form has not changed.

[0216] Stability test

[0217] Following the "Guidelines for Stability Testing of Drug Substances and Preparations" in the Chinese Pharmacopoeia, the stability of crystal form I under different temperatures and humidity conditions was investigated. Purity was determined by HPLC on days 0, 5, and 10 (denoted as D0, D5, and D10, respectively), and crystal form was determined by XRPD. The XRPD comparison spectra of crystal form I before and after the stability test are shown below. Figure 7 As shown.

[0218] The experimental results are shown in Table 10. Crystal form I is physically stable under high temperature and high humidity conditions, its crystal form has not changed, and its chemical purity has not decreased significantly.

[0219] Table 10. Experimental results of crystal form I stability

[0220]

[0221] Example 4: Preparation of crystal form III of the phosphate of compound (A)

[0222] 1 g of compound (A) was added to 50 ml of ethanol at room temperature, followed by 125 μL of concentrated phosphoric acid. After the system was dissolved, a solid quickly precipitated out. The solid was suspended and stirred overnight at room temperature, filtered, and the filter cake was dried under vacuum 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)pyrimidine-2-amine phosphate.

[0223] 1H-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).

[0224] PO4 measured by ion chromatography 3- The content is 14.9%, which is close to that of monophosphate PO4. 3- The theoretical content (13.9%), combined with the above NMR results, confirms that it is in the form of monophosphate, that is, the molar ratio of compound (A) to phosphoric acid is 1:1.

[0225] The obtained solid was characterized by XRPD and TGA-DSC tests, and the solid was found to be crystal form III.

[0226] XRPD spectrum as shown Figure 8 As shown.

[0227] TGA-DSC spectrum as follows Figure 9 As shown.

[0228] Example 5: Preparation of crystal form IV of the phosphate of compound (A)

[0229] 1g of compound (A) phosphate crystal form III was added to a high-pressure reactor, purged with nitrogen three times, heated to 160°C under a nitrogen atmosphere, held at that temperature for half an hour, cooled to room temperature, and the nitrogen was purged to obtain a solid.

[0230] The obtained solid was characterized by XRPD and TGA-DSC tests, and the solid was found to be crystal form IV.

[0231] XRPD spectrum as shown Figure 10 As shown.

[0232] TGA-DSC spectrum as follows Figure 11 As shown, its DSC spectrum has a characteristic endothermic peak at 229℃.

[0233] Hygroscopicity test

[0234] Referencing the "Guidelines for Hygroscopicity Testing of Drugs" in the Chinese Pharmacopoeia, we tested the water adsorption / desorption data for crystal form IV.

[0235] Figure 12 The DVS curve of crystal form IV is shown. The DVS results show that crystal 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 this crystal form has slight hygroscopicity.

[0236] Stability test

[0237] Following the "Guidelines for Stability Testing of Drug Substances and Preparations" in the Chinese Pharmacopoeia, the stability of crystal form IV under different temperatures and humidity conditions was investigated. Purity was determined by HPLC on days 0, 5, and 10, and crystal form was determined by XRPD. The XRPD comparison spectra of crystal form IV before and after the stability test are shown below. Figure 13 As shown.

[0238] The experimental results are shown in Table 11. Crystal form IV is stable in physical and chemical properties under high temperature and high humidity conditions, its crystal form has not changed, and its chemical purity has not decreased significantly.

[0239] Table 11 Results of Crystal Form IV Stability Experiments

[0240]

[0241] The above description is merely a preferred embodiment of this application and is not intended to limit the scope of protection of this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application are included within the scope of protection of this application.

Claims

1. A crystal form I of a compound of formula (A), Its features are, The X-ray powder diffraction pattern of crystal form I has characteristic peaks at 2θ values ​​of 10.49°, 12.10°, 17.74°, 19.88°, and 21.66°, with a 2θ error range of ±0.2°.

2. The crystal form I as described in claim 1, characterized in that, The X-ray powder diffraction pattern of crystal 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°, with a 2θ error range of ±0.2°.

3. The crystal form I as described in claim 1, characterized in that, The X-ray powder diffraction pattern of crystal 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°, with a 2θ error range of ±0.2°.

4. Crystal form I as described in any one of claims 1-3, characterized in that, The X-ray powder diffraction pattern of crystal form I is shown in Figure 1.

5. Crystal form I as described in any one of claims 1-3, characterized in that, The DSC spectrum of crystal form I shows an endothermic characteristic peak at 200°C.

6. Crystal form I as described in any one of claims 1-3, characterized in that, The TGA-DSC spectrum of crystal form I is shown in Figure 2.

7. A crystal form II of a compound of formula (A), Its features are, The X-ray powder diffraction pattern of crystal form II has characteristic peaks at 2θ values ​​of 8.09°, 13.31°, 16.59°, 19.55°, 21.59°, and 25.01°, with a 2θ error range of ±0.2°.

8. The crystal form II as described in claim 7, characterized in that, The X-ray powder diffraction pattern of crystal 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°, with a 2θ error range of ±0.2°.

9. The crystal form II as described in claim 8, characterized in that, The X-ray powder diffraction pattern of crystal 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°, with a 2θ error range of ±0.2°.

10. Crystal form II as described in any one of claims 7-9, characterized in that, The X-ray powder diffraction pattern of crystal form II is shown in Figure 3.

11. Crystal form II as described in any one of claims 7-9, characterized in that, The TGA-DSC spectrum of crystal form II is shown in Figure 4.

12. A phosphate of a compound of formula (A), characterized in that, The molar ratio of compound (A) to phosphoric acid is 1:

1.

13. A phosphate of the compound of formula (A) according to claim 12, crystal form III, Its features are, The X-ray powder diffraction pattern of crystal form III has characteristic peaks at 2θ values ​​of 5.85°, 8.94°, 13.21°, 14.86°, 16.00°, and 19.25°, with a 2θ error range of ±0.2°.

14. The crystal form III as described in claim 13, characterized in that, The X-ray powder diffraction pattern of crystal 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°, with a 2θ error range of ±0.2°.

15. Crystal form III as described in claim 13, characterized in that, The X-ray powder diffraction pattern of crystal 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°, with a 2θ error range of ±0.2°.

16. Crystal form III as described in any one of claims 13-15, characterized in that, The X-ray powder diffraction pattern of crystal form III is shown in Figure 8.

17. Crystal form III as described in any one of claims 13-15, characterized in that, The TGA-DSC spectrum of crystal form III is shown in Figure 9.

18. A phosphate crystal form IV of the compound of formula (A) according to claim 12, Its features are, The X-ray powder diffraction pattern of crystal form IV has characteristic peaks at 2θ values ​​of 13.07°, 15.66°, 16.11°, 16.84°, and 21.89°, with a 2θ error range of ±0.2°.

19. Crystal form IV as described in claim 18, characterized in that, The X-ray powder diffraction pattern of crystal 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°, with a 2θ error range of ±0.2°.

20. Crystal form IV as described in claim 19, characterized in that, The X-ray powder diffraction pattern of crystal 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°, with a 2θ error range of ±0.2°.

21. Crystal form IV as described in any one of claims 18-20, characterized in that, The X-ray powder diffraction pattern of crystal form IV is shown in Figure 10.

22. Crystal form IV as described in any one of claims 18-20, characterized in that, The DSC spectrum of crystal form IV shows an endothermic characteristic peak at 229 °C.

23. Crystal form IV as described in any one of claims 18-20, characterized in that, The TGA-DSC spectrum of crystal form IV is shown in Figure 11.

24. A pharmaceutical composition comprising crystal form I as described in any one of claims 1-6, crystal form II as described in any one of claims 7-11, the phosphate as described in claim 12, crystal form III as described in any one of claims 13-17 or crystal form IV as described in any one of claims 18-23, and a pharmaceutically acceptable carrier.

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

26. The use as described in claim 25, wherein the cancer includes ovarian cancer, breast cancer, acute myeloid leukemia, chronic lymphocytic leukemia, or small lymphocytic lymphoma.

27. The method for preparing crystal form I according to any one of claims 1-6, characterized in that, Includes the following steps: (a) Compound A is dissolved in a first solvent to obtain a solution; (b) Add the second solvent to the solution from step (a) and stir; (c) After the solid precipitates, filter, collect, and dry the solid; The first solvent is selected from at least one of acetone, methanol, tetrahydrofuran, ethyl acetate, acetonitrile, and dichloromethane; The second solvent is selected from at least one of water, n-heptane, n-hexane, and methyl tert-butyl ether.

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