WO2024027825A1 - Cdk inhibitor and polymorph of phosphate thereof - Google Patents

Abstract

The present application provides a polymorph of a compound of formula (A), i.e., N-(1-((4-((3S,5S)-3,5-dimethylpiperazine-1-yl)phenyl)sulfonyl)piperidin-4-yl)-4-((S)-tetrahydrofuran-3-yl)oxy)-5(trifluoromethyl)pyrimidine-2-amine, and a polymorph of a phosphate of the compound of formula (A). A crystalline form provided by the present application has good chemical stability and physical stability, and low hygroscopicity, is less affected by temperature, humidity, and illumination, and facilitates storage and preparation development.

Description

A polymorphic form of a CDK inhibitor and its phosphate

This application is required to be submitted to the China Patent Office on August 5, 2022, with the application number CN202210947810.2 and the invention title "A polymorphic form of a CDK inhibitor". It is submitted to the China Patent Office on August 5, 2022. , the Chinese patent application with the application number CN202210936709.7 and the invention name "A polymorphic form of a CDK inhibitor phosphate" was submitted to the China Patent Office on July 19, 2023. The application number is CN202310889701.4 and the invention name is "a polymorphic form of a CDK inhibitor phosphate". The priority of the Chinese patent application "A CDK inhibitor and polymorphic form of its phosphate", the entire content of which is incorporated by reference in this application.

Technical field

This application discloses a CDK inhibitor, multiple crystal forms of its phosphate, preparation methods thereof, and their application in treating cancer diseases.

Background technique

The cell cycle is the basic process of cell life activities, controlling the growth, proliferation and differentiation of cells. Cyclin-dependent kinases (CDKs) are an important class of cellular enzymes that cooperate with cyclins and play an important role in the regulation of the cell cycle. Cyclin B/CDK1, cyclin A/CDK2, cyclin E/CDK2, cyclin D/CDK4, cyclin D/CDK6 and possibly other heterodimers are important regulators of different stages of the cell cycle Factor (Harper, J.W., Adams, P.D., Cyclin-Dependent Kinases, Chem. Rev. 2001, 101, 2511-2526).

As an important regulatory factor in the cell cycle, CDK2 forms a kinase complex with cyclin E or A and plays a decisive role in driving the cell cycle from the G1 phase to the S phase and maintaining the S phase. The main mechanism is that Cyclin E and CDK2 work together to phosphorylate the retinoblastoma susceptibility gene (Rb) protein. The phosphorylation of the Rb protein leads to the release of E2F (transcription factor). The released E2F binds to the upstream of some genes ( Usually located in the promoter or enhancer region), it initiates the transcriptional expression of those genes related to the cell cycle, causing cells to enter the S phase from the end of 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).

With the identification of the important role of cyclin-dependent kinases (CDKs) in cell cycle regulation, CDK inhibitors have become a current research focus on anti-tumor drugs. Currently, multiple CDK inhibitors have been approved for marketing around the world, but most of them act on the CDK4/6 target, mainly for breast cancer, such as Pfizer's Palbociclib, Novartis' Ribociclib and Eli Lilly's abemaciclib. Molecules including CDK2 multi-target inhibitors such as fadraciclib, Roscovitine and PF-06873600 are in different clinical stages. Currently, no CDK2 inhibitors have been approved for marketing. Therefore, new CDK inhibitors continue to be developed, especially those that are effective against CDK2 targets. inhibitors, which are of great research significance.

Patent PCT/CN2022/074491 discloses a small molecule inhibitor targeting CDK2/4/6, especially the CDK2 target. 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 methyl)-5-(trifluoromethyl)pyrimidin-2-amine. This small molecule inhibitor can selectively inhibit CDK 2/4/6 kinases compared to CDK 1/7/9 kinases. It is especially excellent in the inhibitory activity of CDK 2 kinases. Its kinase selectivity can reach nearly 10 times, even Dozens or hundreds of times more, this small molecule inhibitor has good cell proliferation inhibitory activity, and has shown good tumor inhibitory activity and good tolerance in in vivo efficacy experiments, and is expected to be developed into clinical drugs.

Contents of the invention

The present application discloses a polymorphic form of a CDK inhibitor, a CDK inhibitor phosphate, a polymorphic form, a preparation method of the crystal form, and their application in the treatment of cancer diseases.

specific,

The first aspect of the application provides a compound of formula (A) (i.e., N-(1-((4-((3S,5S)-3,5-dimethylpiperazin-1-yl)phenyl)sulfonate) Crystalline Form I of acyl)piperidin-4-yl)-4-((S)-tetrahydrofuran-3-yl)oxy)-5-(trifluoromethyl)pyrimidin-2-amine), wherein, The X-ray powder diffraction pattern of 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°.

In some embodiments of the present application, the X-ray powder diffraction pattern of the above-mentioned crystalline form I has a 2θ value of 10.03°, 10.49°, 12.10°, There are characteristic peaks at 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°.

In some embodiments of the present application, the X-ray powder diffraction pattern of the above-mentioned crystalline form I has a 2θ value of 10.03°, 10.49°, 11.48°, 12.10°, 13.50°, 14.28°, 14.81°, 16.16°, 16.87° There are characteristic peaks at , 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°, 32.57°, 2θ error range is ±0.2°.

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.

In some embodiments of the present application, the DSC spectrum of the above-mentioned crystalline form I has an endothermic characteristic peak at about 200°C.

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

In some embodiments of the present application, the XRPD pattern diffraction peak analysis data of the crystal form I of the compound of formula (A) is basically as shown in Table 1.

Table 1 XRPD diffraction peak analysis data of crystal form I of the compound of formula (A)

The second aspect of the application also provides a compound of formula (A) (i.e. N-(1-((4-((3S,5S)-3,5-dimethylpiperazin-1-yl)phenyl)) Form II of sulfonyl)piperidin-4-yl)-4-((S)-tetrahydrofuran-3-yl)oxy)-5-(trifluoromethyl)pyrimidin-2-amine), the crystal The X-ray powder diffraction pattern of Type 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°.

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

In some embodiments of the present application, the X-ray powder diffraction pattern of the above-mentioned crystalline form II has a 2θ value of 6.13°, 8.09°, 9.94°, 11.60°, 13.31°, 14.44°, 16.59°, 17.13°, 17.71° There are characteristic peaks at , 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°.

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.

In some embodiments of the present application, the TGA-DSC spectrum of the above-mentioned crystal form II is substantially as shown in Figure 4.

In some embodiments of the present application, the XRPD pattern diffraction peak analysis data of the crystal form II of the compound of formula (A) is basically as shown in Table 2.

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

The third aspect of the application also provides 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) phosphate, wherein formula (A ) compound and phosphoric acid molar ratio is 1:1,

In the research, the inventor of the present application has tried to form salts of the compound of formula (A) with various inorganic acids or organic acids, such as hydrochloric acid, sulfuric acid, phosphoric acid, maleic acid, fumaric acid, succinic acid and p-toluenesulfonic acid. etc., but only the phosphate of formula (A) was found to have better crystallinity, hygroscopicity, solubility and stability. Some other acids cannot form salts with the compound of formula (A), some have poor crystallinity, or are mostly amorphous, and some can obtain crystal forms, but the crystal forms are prone to moisture or have poor stability.

The fourth aspect of the present application also provides a crystal form III of the phosphate salt of the compound of formula (A). The X-ray powder diffraction pattern of the crystal form III has a 2θ value of 5.85°, 8.94°, 14.86°, and 16.00°. There is a characteristic peak at 2θ, and the 2θ error range is ±0.2°.

In some embodiments of the present application, the X-ray powder diffraction pattern of the above-mentioned crystal form III has a 2θ value of 5.85°, 8.94°, 10.29°, 13.21°, 14.86°, 16.00°, 17.01°, 17.65°, 19.25° There is a characteristic peak at 2θ, and the 2θ error range is ±0.2°.

In some embodiments of the present application, the X-ray powder diffraction pattern of the above-mentioned crystal form III has a 2θ value of 3.43°, 5.85°, 8.94°, 10.29°, 11.69°, 12.30°, 13.21°, 14.86°, 16.00° There are characteristic peaks at , 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°.

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

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

In some embodiments of the present application, the XRPD pattern diffraction peak analysis data of the above-mentioned crystal form III is basically as shown in Table 3.

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

The fifth aspect of the present application also provides a crystalline form IV of the phosphate of the compound of formula (A). The X-ray powder diffraction pattern of the crystalline form IV has a 2θ value of 13.07°, 15.66°, 16.11°, and 16.84°. , there is a characteristic peak at 21.89°, and the 2θ error range is ±0.2°.

In some embodiments of the present application, the X-ray powder diffraction pattern of the above crystalline form IV has a 2θ value of 7.81°, 13.07°, 15.20°, 15.66°, 16.11°, 16.84°, 19.61°, 21.89°, 22.16° , there is a characteristic peak at 23.57°, and the 2θ error range is ±0.2°.

In some embodiments of the present application, the X-ray powder diffraction pattern of the above crystalline form IV has a 2θ value of 7.81°, 10.97°, 13.07°, 14.20°, 15.20°, 15.66°, 16.11°, 16.84°, 19.61° There are characteristic peaks at , 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°.

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

In some embodiments of the present application, the DSC spectrum of the above crystalline form IV has an endothermic characteristic peak at about 229°C.

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

In some embodiments of the present application, the XRPD pattern diffraction peak analysis data of the above-mentioned crystal form IV is basically as shown in Table 4.

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

The sixth aspect of the application also provides a pharmaceutical composition, which includes the crystal form I described in the first aspect of the application, the crystal form II described in the second aspect of the application, and the phosphate salt described in the third aspect of the application. , the crystal form III described in the fourth aspect of this application or the crystal form IV described in the fifth aspect of this application, and a pharmaceutically acceptable carrier.

The seventh aspect of the application also provides the crystal form I described in the first aspect of the application, the crystal form II described in the second aspect of the application, the phosphate described in the third aspect of the application, and the crystal form II described in the fourth aspect of the application. The use of the crystal form III, 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.

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

The ninth aspect of the present application also provides 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, and the third aspect of the present application for use as medicine or for treating CDK-mediated cancer. The phosphate, the crystal form III described in the fourth aspect of the application, the crystal form IV described in the fifth aspect of the application or the pharmaceutical composition described in the sixth aspect of the application.

In some embodiments of the present application, the cancer includes ovarian cancer, breast cancer, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), or small lymphocytic lymphoma (SLL).

In some embodiments of the present application, the cancer is breast cancer.

The tenth aspect of this application also provides a preparation method for the crystal form I described in the first aspect of this application, including the following steps:

(a) Dissolve compound A in the first solvent to obtain a solution;

(b) Add a second solvent to the solution in step (a) and stir for a certain period of time;

(c) After the solid is precipitated, filter, collect, and dry the solid.

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

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

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

Technical effect

The crystal form in this 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.

Description of the drawings

Figure 1 is an XRPD (X-ray powder diffraction) spectrum of crystal form I of the compound of formula (A).

Figure 2 is a TGA-DSC (differential scanning calorimetry-thermogravimetric analysis) spectrum of crystal form I of the compound of formula (A).

Figure 3 is the XRPD spectrum of crystal form II of the compound of formula (A).

Figure 4 is a TGA-DSC spectrum of crystal form II of the compound of formula (A).

Figure 5 is a DVS (dynamic vapor phase adsorption) spectrum of crystal form I of the compound of formula (A).

Figure 6 is a comparative XRPD spectrum of crystal form I of the compound of formula (A) before and after the DVS experiment.

Figure 7 is a comparative XRPD spectrum before and after the stability experiment of the crystal form I of the compound of formula (A).

Figure 8 is an XRPD spectrum of Form III of the phosphate salt of the compound of formula (A).

Figure 9 is a TGA-DSC spectrum of Form III of the phosphate salt of the compound of formula (A).

Figure 10 is an XRPD spectrum of Form IV of the phosphate salt of the compound of formula (A).

Figure 11 is a TGA-DSC spectrum of Form IV of the phosphate salt of the compound of formula (A).

Figure 12 is a DVS spectrum of Form IV of the phosphate salt of the compound of formula (A).

Figure 13 is a comparative XRPD spectrum before and after the stability experiment of the phosphate form IV of the compound of formula (A).

Detailed ways

The present application is described in detail through examples below, but does not mean any adverse limitations to the present application. 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 methods well known to those skilled in the art. Equivalent alternatives and preferred implementations 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 improvements can be made to the specific embodiments of the present application without departing from the spirit and scope of the present application.

Definition and Description

Unless otherwise stated, the following terms and phrases used herein are intended to have the following meanings. a specific term or phrase in no particular Defined situations should not be considered uncertain or unclear, but should be understood in their ordinary meaning.

The term "pharmaceutically acceptable carrier" refers to a medium generally accepted in the art for delivering biologically active agents to animals, especially mammals, including, for example, adjuvants, excipients, or Excipients such as diluents, preservatives, fillers, flow regulators, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavorings, aromatics, antibacterial agents, antifungal agents, Lubricants and dispersants. The formulation of pharmaceutically acceptable carriers depends on a number of factors within the purview of one of ordinary skill in the art. This includes, but is not limited to: the type and nature of the formulated active agent, the subjects 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 and a variety of solid and semi-solid dosage forms. Such carriers include many different ingredients and additives in addition to the active agent, and such additional ingredients are well known to those of ordinary skill in the art to be included in the formulation for a variety of reasons (e.g., to stabilize the active agent, binders, etc.) .

It is known in the art that X-ray powder diffraction patterns have one or more measurement errors depending on slight changes in measurement conditions. The structure of the crystals, crystals or crystal forms disclosed or claimed in this application may vary depending on test conditions, purity, equipment It exhibits similar but not identical analytical properties within a reasonable error range as other constant variables known to those skilled in the art. For example, the diffraction angle (2θ) in powder X-ray powder diffraction usually 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 an error range of ±0.20°. The crystalline form of Compound A of the present application is not limited to crystals having the same X-ray powder diffraction pattern as shown in the accompanying drawings, and has substantially the same X-ray powder diffraction pattern as shown in the accompanying drawings. Any crystal with a diffraction pattern falls within the scope of this application.

The occurrence of "substantially the same X-ray powder diffraction pattern as the X-ray powder diffraction pattern shown in the accompanying drawings." It will be understood that the term "substantially the same" used in this context is also intended to indicate X-ray powder diffraction The 2θ angle values of the spectrum may vary slightly due to the inherent experimental variations that accompany these measurements, both for the same crystal form.

It should be understood that using different types of equipment or using 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. Strong endothermic peaks can indicate that the substance has dehydrated or desolvated, or has undergone crystallization, or has melted. When reflecting the melting state, the corresponding temperature is usually understood as the substance. melting point. This value will be affected by compound purity, sample weight, heating rate, particle size, and calibration and maintenance of the test equipment. Those skilled in the art can understand that the temperature when a substance transforms from a solid state to a liquid state is usually a temperature range rather than a fixed point value. Therefore, the temperature corresponding to the endothermic peak can be characterized by the onset value or peak value or other reasonable values. or the melting point of a substance. The maximum endothermic transition temperature of the crystal form may be within the range of the above-disclosed specific value ±5.0°C, preferably within the range of ±2.0°C.

This application also uses thermogravimetric analysis (TGA) to analyze the relationship between the degree of decomposition, sublimation, and evaporation (loss of weight) of the crystal form and temperature. It should be understood that the same crystal form is 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 numerical value disclosed above, for example, within the range of ±2.0°C.

"Stability" of a crystalline form includes "chemical stability" and/or "physical stability". "Chemical stability" refers to the degree of degradation reaction of the crystal form under certain temperature, humidity, and light conditions. "Chemical stability" reflects the stability of the crystal form under storage conditions. "Physical stability" refers to the degree to which the crystal form is converted into a solid form under certain specific conditions, such as high temperature, high humidity, grinding, tableting, desolvation, and adsorption of solvents, into another crystal form. Therefore, "physical stability" can reflect to a certain extent the stability of the crystal form during the use of preparations and other processes.

The crystalline structures of the present application can be prepared by a variety of methods, including crystallization or recrystallization from a suitable solvent, sublimation, growth from a melt, solid state conversion from another phase, crystallization from a supercritical fluid, and jet spray. Techniques for crystallizing or recrystallizing a crystalline structure from a solvent mixture, including solvent evaporation, lowering the temperature of the solvent mixture, seeding of a supersaturated solvent mixture of the molecule and/or salt, lyophilizing the solvent mixture, adding an antisolvent to the solvent mixture wait.

The term "drying" refers to the process of removing solvent from the obtained solid, including but not limited to natural drying at room temperature, high temperature drying, vacuum drying and other methods.

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 those skilled in the art. Well-known equivalents and preferred embodiments include but are not limited to the embodiments of the present application.

In the examples of this application, the naming of the title compound was converted from the compound structure with the help of Chemdraw. If there is any inconsistency between the compound name and the compound structure, it can be determined by comprehensively integrating relevant information and reaction routes; if it cannot be confirmed through other methods, the given compound structural formula shall prevail.

The preparation methods of some compounds in this application refer to the preparation methods of similar compounds mentioned above. Persons in the art should know that when using or referring to the preparation methods cited therein, the feed ratio of the reactants, the reaction solvent, the reaction temperature, etc. can be appropriately adjusted according to the different reactants.

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 methods well known to those skilled in the art. Equivalent alternatives and preferred implementations include but are not limited to the embodiments of this application.

Instruments and analytical methods

1. X-ray powder diffraction (XRPD)

The solid sample is analyzed with an X-ray powder diffractometer (X'Pert PRO). Take an appropriate amount of fine powder of the test sample, place it in the groove of the sample holder, and press it into a flat and dense plane with a glass piece. The XRPD measurement parameters are shown in Table 5. .

Table 5 XRPD test parameters

2. Thermogravimetric analysis (TGA)

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

Table 6 TGA analysis method parameters

3. Simultaneous thermal analysis (TGA-DSC)

Thermogravimetry-differential scanning calorimetry analysis of solids was performed using a simultaneous thermal analyzer from Mettler Toledo. Use a small spoon to take an appropriate amount of the test sample and place it in the crucible, spread it evenly, weigh it, heat the sample according to the parameters listed in Table 7, and use STARe to analyze the data.

Table 7 TGA-DSC analysis method parameters

4. Dynamic moisture adsorption and desorption analysis (DVS)

The hygroscopicity of the samples was measured using the DVS Intrinsic dynamic moisture adsorption instrument. Place the sample into the tared sample basket, the instrument automatically weighs, and analyzes the sample according to the parameters in Table 8.

Table 8 DVS analysis method parameters

5. Ion chromatography analysis

Instrument: ion chromatograph-conductivity detector;

Chromatographic column: Use an anion exchange chromatographic column [analytical column Ionpac ^TM AS11-HC (4mm × 250mm), guard column Ionpac ^TM AG11-HC (4mm×50mm)];

Suppressor: AERS 500 4mm or equivalent suppressor;

Column temperature: 30℃;

Flow rate: 1.0mL/min;

Injection volume: 25μL;

Detection pool temperature: 35℃;

Mobile phase: 30mmol/L potassium hydroxide solution;

Running time: approximately 20 minutes.

Example 1: Preparation of compounds of formula (A)

Preparation of intermediate compound 99

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 thereto under an ice-water bath. After the reaction solution was stirred at room temperature for 2 hours, water (60 ml) was added to quench the mixture. 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 obtained residue was purified by silica gel column chromatography to obtain 830 mg of N-(1-(1-(4-bromophenyl)sulfonyl)piperidin-4-yl)-4-chloro-5-(trifluoromethyl) Pyrimidine-2-amine (99-1).

MS(ESI)M/Z:499.0[M+H] ⁺ .

Step 2: In a sealed jar, dissolve (S)-3-hydroxytetrahydrofuran (42 mg, 0.48 mmol) in tetrahydrofuran (2 mL). Subsequently, sodium hydride (21 mg, 0.88 mmol) was added thereto under an ice-water bath. After reacting for 15 minutes, compound 99-1 (200 mg, 0.40 mmol) was added, and the reaction solution was heated to 100°C. After reaction for 4 hours, water (60 ml) was added to the reaction solution to quench the reaction solution. 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 obtained residue was purified by silica gel column chromatography to obtain 150 mg of (S)-N-(1-(((4-bromophenyl)sulfonyl)piperidin-4-yl)-4-((tetrahydrofuran-3-yl) )oxy)-5-(trifluoromethyl)pyrimidin-2-amine (compound 99).

MS(ESI)M/Z:551.0[M+H] ⁺ .

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).

Preparation of Compound A

Step 1: Under nitrogen protection conditions, compound 99 (25g, 45.3 mmol), (2S, 6S)-2,6-dimethylpiperazine-1-carboxylic acid tert-butyl ester (11.6 g, 45.3 mmol) ), tris(dibenzylideneacetone)dipalladium (4.1 g, 4.53 mmol), 2-dicyclohexylphosphon-2,4,6-triisopropylbiphenyl (1.3 g, 9.02 mmol) and carbonic acid Cesium (29.4 g, 90.6 mmol) was dissolved in 1,4-dioxane (1.25 L). The reaction solution was heated to 100°C and stirred overnight. The reaction solution 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 obtained residue was purified by silica gel column chromatography to obtain 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).

MS(ESI)M/Z:685.2[M+H] ⁺ .

Step 2: Compound 168-1 (18.1 g, 26.4 mmol) was dissolved in 1,4dioxane (100 mL). Subsequently, dioxane hydrochloride solution (100 ml) was added thereto. The reaction was stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure. The obtained residue was washed with sodium bicarbonate aqueous solution (300 ml), the mixture was extracted with ethyl acetate (200 ml × 3 times), and the organic phases were combined. The organic phase Wash with saturated brine (100 ml), dry over anhydrous sodium sulfate, filter, and finally concentrate under reduced pressure. The obtained residue was purified by silica gel column chromatography to obtain 9.2 g of N-(1-((4-((3S,5S)-3,5-dimethylpiperazin-1-yl)phenyl)sulfonyl)piperidine -4-yl)-4-((S)-tetrahydrofuran-3-yl)oxy)-5-(trifluoromethyl)pyrimidin-2-amine (Compound A).

MS(ESI)M/Z:585.2[M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.29 (s, 1H), 8.00 (d, J = 7.1Hz, 0.6H), 7.85 (d, J = 6.9Hz, 0.4H), 7.49 (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).

Biological test evaluation

(1) CDK in vitro enzymology experiment

This experiment uses the capillary migration ability change assay (MSA) method to test the inhibitory effect of the compound on CDK1/CDK2/CDK4/CDK6/CDK7/CDK9 kinase activity, and concludes that the compound’s inhibitory effect on CDK1/CDK2/CDK4/CDK6/CDK7/CDK9 kinase The half inhibitory concentration IC ₅₀ of activity.

1. Experimental materials

CDK1/CDK2/CDK4/CDK6/CDK7/CDK9 were purchased from Carna Company, Carliper substrate CTD3/substrate 18/substrate 8 were purchased from Gill Biochemical Company, Dinaciclib/Palbociclib were purchased from Selleckchem Company, DMSO (dimethyl sulfoxide) It was purchased from Sigma Company, and the 384-well plate was purchased from Corning Company.

2. Experimental methods

(1) Prepare 1×Kinase buffer.

(2) Preparation of compound concentration gradient: test compound test concentration is 1 μM, 10 μM or 30 μM is the starting concentration, dilute 3 times to 10 concentrations, and detect in duplicate. Dilute to a 100x final concentration of 100% DMSO solution in a 384-well source plate. Use the Dispenser Echo 550 to transfer 250 nL of compound at 100x the final concentration to the destination 384-well plate. Add 250nL DMSO to the positive and negative control wells.

(3) Use 1×Kinase buffer to prepare a kinase solution with 2.5 times the final concentration.

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

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

(6) Use 1×Kinase buffer to prepare a mixed solution of ATP and Kinase substrate at 25/15 times the final concentration.

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

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

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

(10) Use Caliper EZ Reader to read the conversion rate.

Calculation formula:

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

Among them: 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 the wells without enzyme activity; Conversion%_max: the mean value of the positive control wells, representing the conversion rate reading of the wells without compound inhibition.

Fitted dose-response curve

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

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

3.Experimental results

The inhibitory IC ₅₀ data of the compounds of the present application on CDK kinase activity are shown in Table 9. Among them: compounds with IC ₅₀ <0.5nM are represented by AA, compounds with 0.5nM≤IC ₅₀ <2.5nM are represented by AB, compounds with 2.5nM≤IC ₅₀ <10nM are represented by AC, and compounds with 10nM≤IC ₅₀ <50nM are represented by B. To identify, compounds with 50nM≤IC ₅₀ <100nM are identified with C, compounds with 100nM≤IC ₅₀ <1000nM are identified with D, and compounds with IC ₅₀ >1000nM are identified with E.

Table 9: CDK enzymatic inhibition results

Conclusion: Compound A of this application has good CDK 2/4/6 kinase inhibitory activity, especially excellent in the inhibitory activity of CDK 2 kinase; Compound A of this application can be selected compared to CDK 1/7/9 kinase It specifically inhibits CDK 2/4/6 kinases, especially in the inhibitory activity of CDK 2 kinase. Its kinase selectivity can reach nearly 10 times, even dozens or hundreds of times.

(2) HCC1806/NIH:OVCAR-3 cell proliferation inhibition experiment

This experiment uses the CellTiter-Glo method to test the inhibitory effect of compounds on HCC1806/NIH:OVCAR-3 cell proliferation, and Find the concentration IC ₅₀ (nM) at which the compound inhibits cell growth by half.

1. Experimental materials

HCC1806 was purchased from Tongpai (Shanghai) Biotechnology Co., Ltd.; NIH:OVCAR-3 was purchased from the ATCC Cell Bank of the United States.

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

PF-06873600 was purchased from Selleck.

CellTiter-Glo reagent was purchased from Promega Company.

2. Experimental methods

1) Seed HCC1806/NIH:OVCAR-3 cells into a 96-well culture plate at a density of 600/1500 cells per well, 100 μL per well.

2) Day 0: Use Echo to add 100nL gradient dilution of the test compound to the culture plate cells. The final concentration of DMSO is 0.5%. Place the culture plate in a cell culture incubator and incubate for 168 hours (37°C, 5vol %CO ₂ ). For blank control, add 30 nL DMSO to each well.

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

4) Envision microplate reader (PerkinElmer) detects chemiluminescence signal.

Data analysis was performed using GraphPad Prism 6 software to obtain the IC ₅₀ (nM) of the compound.

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

(3) In vivo drug efficacy experiment of HCC1806 human breast cancer model

1. Experimental materials:

HCC1806 was purchased from Tongpai (Shanghai) Biotechnology Co., Ltd.; NIH:OVCAR-3 was purchased from the ATCC Cell Bank of the United States.

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

2.Experimental method:

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 named D0. On the fourth day after inoculation (D4), when the average tumor volume reached about 150 mm ³ , those with moderate tumor volume were selected and included in each group, with 6 animals in each group. Administration by intragastric administration was started on the day of grouping. Collect weight data and tumor volume data 2-3 times a week, and draw weight and tumor growth curves. Tumor volume V=1/2×a×b ² , where a and b represent the long and short diameters of the tumor respectively.

Experimental results and conclusions: After testing, the tumors in the treatment group administered with the 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 was not significantly reduced. , showing that each treatment regimen (10mg/kg BID, 20mg/kg BID, 30mg/kg QD) can be well tolerated.

(4) In vivo efficacy experiment of OVCAR-3 human ovarian cancer model

1. Experimental materials:

OVCAR-3 was purchased from ATCC cell bank in the United States. 1640 medium, fetal bovine serum (FBS), and Penicillin-Streptomycin were purchased from GIBCO. PF-06873600 was purchased from MCE Company.

2.Experimental method:

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 named D0. On the 27th day after inoculation (D27), when the average tumor volume reached about 180 mm ³ , those with moderate tumor volume were selected and included in each group, with 6 animals in each group. Administration by intragastric administration was started on the day of grouping. Administration was continued for 21 days. Collect weight data and tumor volume data 2-3 times a week, and draw weight and tumor growth curves. Tumor volume V=1/2×a×b ² , where a and b represent the long and short diameters of the tumor respectively.

Experimental results and conclusions: After testing, the tumors in the treatment group administered with the 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 was not significantly reduced. , showing that each treatment regimen (5mg/kg BID, 7.5mg/kg BID, 10mg/kg QD, 20mg/kg QD) can be well tolerated.

Example 2 Preparation of Crystal Form I of Compound of Formula (A)

method 1:

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 medicinal solution at room temperature. After the solid precipitated, the suspension was stirred at room temperature for two hours, then filtered, and the solid was collected and dried at room temperature.

The obtained solid was subjected to XRPD and TGA-DSC testing and characterization. The solid was crystalline form I.

The XRPD spectrum is shown in Figure 1.

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

Method 2:

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 medicinal solution at room temperature. After the solid precipitated, the suspension was stirred at room temperature for two hours, then filtered, and the solid was collected and dried at room temperature. The XRPD spectrum of the obtained solid is basically as shown in Figure 1.

Method 3:

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 medicinal solution at room temperature. After the solid precipitated, the suspension was stirred at room temperature for two hours, then filtered, and the solid was collected and dried at room temperature. The XRPD spectrum of the obtained solid is basically as shown in Figure 1.

Method 4:

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 medicinal solution at room temperature. After the solid precipitated, the suspension was stirred at room temperature for two hours, then filtered, and the solid was collected and dried at room temperature. The XRPD spectrum of the obtained solid is basically as shown in Figure 1.

Method 5:

About 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 medicinal solution at room temperature. After the solid precipitated, the suspension was stirred at room temperature for two hours, then filtered, and the solid was collected and dried at room temperature. The XRPD spectrum of the obtained solid is basically as shown in Figure 1.

Method 6:

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 medicinal solution at room temperature. After the solid precipitated, the suspension was stirred at room temperature for two hours, then filtered, and the solid was collected and dried at room temperature. The XRPD spectrum of the obtained solid is basically as shown in Figure 1.

Method 7:

Approximately 10 mg of Compound A was dissolved in 0.5 mL of methylene chloride at room temperature. Then, 2.0 mL of purified water was added dropwise to the medicinal solution at room temperature. After the solid precipitated, the suspension was stirred at room temperature for two hours, then filtered, and the solid was collected and dried at room temperature. The XRPD spectrum of the obtained solid is basically as shown in Figure 1.

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

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

The obtained solid was subjected to XRPD and TGA-DSC testing and characterization, and the solid was crystalline form II.

The XRPD spectrum is shown in Figure 3.

The TGA-DSC spectrum is shown in Figure 4.

Hygroscopicity test

Refer to the "Guiding Principles for Drug Hygroscopicity Testing" in the Chinese Pharmacopoeia to test the moisture adsorption/desorption data of crystalline form I.

Figure 5 is the DVS curve of Form I, and Figure 6 is the XRPD spectrum of Form I before and after the DVS test. The DVS results show that the crystalline form I absorbs moisture and gains weight by 0.14% at 80% RH and 0.16% at 90%RH, indicating that the crystalline form has almost no hygroscopicity. The XRPD of the remaining solid after the DVS experiment was tested, and the results showed that The crystal form has not changed.

stability test

Referring to the "Guiding Principles for Stability Testing of Raw Materials and Preparations" in the Chinese Pharmacopoeia, the stability of Form I at different temperatures and humidity was investigated. HPLC was used to test the purity on days 0, 5 and 10 (recorded as D0, D5 and D10 respectively), and XRPD was used to test the crystal form. The XRPD comparison spectra of Form I before and after the stability experiment are shown in Figure 7.

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

Table 10 Form I stability experimental results

Example 4 Preparation of Form III of the Phosphate of the Compound of Formula (A)

Add 1g of the compound of formula (A) to 50 ml of ethanol at room temperature, and then add 125 μL of concentrated phosphoric acid. After the system is dissolved, a solid precipitates out quickly. The mixture is suspended and beaten at room temperature overnight, filtered, and the filter cake is vacuum dried 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.

¹ H-NMR (400MHz, DMSO-d ₆ ): 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) .

The PO ₄ ^3- content measured by ion chromatography is 14.9%, which is close to the theoretical content of monophosphate PO ₄ ^3- (13.9%). Combined with the above nuclear magnetic results, it is confirmed that it is in the form of monophosphate, that is, the compound of formula (A) and phosphoric acid The molar ratio is 1:1.

The obtained solid was subjected to XRPD and TGA-DSC testing and characterization, and the solid was crystalline form III.

The XRPD spectrum is shown in Figure 8.

The TGA-DSC spectrum is shown in Figure 9.

Example 5 Preparation of Form IV of the Phosphate of the Compound of Formula (A)

Add 1 g of the phosphate crystal form III of the compound of formula (A) into a high-pressure reaction kettle, replace it with nitrogen three times, raise the temperature to 160°C in a nitrogen atmosphere, keep it for half an hour and then lower it to room temperature. Evacuate the nitrogen to obtain a solid.

The obtained solid was characterized by XRPD and TGA-DSC tests and found that the solid was crystal form IV.

The XRPD spectrum is shown in Figure 10.

The TGA-DSC spectrum is shown in Figure 11. In the DSC spectrum, there is a characteristic endothermic peak at 229°C.

Hygroscopicity test

Refer to the "Guiding Principles for Drug Hygroscopicity Testing" in the Chinese Pharmacopoeia to test the moisture adsorption/desorption data of crystalline form IV.

Figure 12 is the DVS curve of the crystal form IV. The DVS results show that the hygroscopic weight gain of the crystal form IV is 0.398% at 80% RH and 0.491% at 90% RH, indicating that the crystal form is slightly hygroscopic.

stability test

Refer to the "Guiding Principles for Stability Testing of Raw Materials and Preparations" in the Chinese Pharmacopoeia to examine the stability of Form IV at different temperatures and humidity. HPLC was used to test purity on days 0, 5 and 10, and XRPD was used to test crystal form. The XRPD comparison spectra of Form IV before and after the stability experiment are shown in Figure 13.

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

Table 11 Form IV stability experimental results

The above descriptions are only preferred embodiments of the present application and are not intended to limit the protection scope of the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of this application are included in the protection scope of this application.

Claims (30)

1. Form I of a compound of formula (A),
   

   It is characterized in that the X-ray powder diffraction pattern of the crystal 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 crystal form I of claim 1, wherein the X-ray powder diffraction pattern of the crystal form I has a 2θ value of 10.03°, 10.49°, 12.10°, 14.28°, 14.81°, 17.74°, 18.28 There are characteristic peaks at 19.88°, 20.57°, 21.66°, 23.11°, 23.76°, and 26.29°, and the 2θ error range is ±0.2°.
3. The crystal form I of claim 1, wherein the X-ray powder diffraction pattern of the crystal form I has a 2θ value 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°, There is a characteristic peak at 32.57°, and the 2θ error range is ±0.2°.
4. The crystalline form I according to any one of claims 1 to 3, characterized in that the X-ray powder diffraction pattern of the crystalline form I is as shown in Figure 1.
5. The crystal form I according to any one of claims 1 to 4, characterized in that the DSC spectrum of the crystal form I has an endothermic characteristic peak at 200°C.
6. The crystal form I according to any one of claims 1 to 5, characterized in that the TGA-DSC spectrum of the crystal form I is as shown in Figure 2.
7. Form II of a compound of formula (A),
   

   It is characterized in that 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°, 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 a 2θ value of 6.13°, 8.09°, 11.60°, 13.31°, 14.44°, 16.59°, 17.13 There are characteristic peaks at 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 crystal form II of claim 8, wherein the X-ray powder diffraction pattern of the crystal form II has a 2θ value of 6.13°, 8.09°, 9.94°, 11.60°, 13.31°, 14.44°, and 16.59 There are characteristic peaks at °, 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°, 29.34°, 2θ error The range is ±0.2°.
10. The crystal form II according to any one of claims 7 to 9, characterized in that the X-ray powder diffraction pattern of the crystal form II is as shown in Figure 3.
11. The crystal form II according to any one of claims 7 to 10, characterized in that the TGA-DSC spectrum of the crystal form II is as shown in Figure 4.
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 salt of the compound of formula (A) according to claim 12,
    

    It is characterized in that 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°.
14. The crystal form III of claim 13, wherein the X-ray powder diffraction pattern of the crystal form III has a 2θ value of 5.85°, 8.94°, 10.29°, 13.21°, 14.86°, 16.00°, and 17.01 There are characteristic peaks at 17.65° and 19.25°, and the 2θ error range is ±0.2°.
15. The crystal form III of claim 13, wherein the X-ray powder diffraction pattern of the crystal form III has a 2θ value of 3.43°, 5.85°, 8.94°, 10.29°, 11.69°, 12.30°, and 13.21 There are characteristic peaks at 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 crystal form III according to any one of claims 13 to 15, wherein the X-ray powder diffraction pattern of the crystal form III is as shown in Figure 8.
17. The crystal form III according to any one of claims 13 to 16, characterized in that the TGA-DSC spectrum of the crystal form III is as shown in Figure 9.
18. A crystalline form IV of the phosphate salt of the compound of formula (A) according to claim 12,
    

    It is characterized in that the X-ray powder diffraction pattern of the crystal 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 crystal form IV of claim 18, wherein the X-ray powder diffraction pattern of the crystal form IV has a 2θ value of 7.81°, 13.07°, 15.20°, 15.66°, 16.11°, 16.84°, and 19.61 There are characteristic peaks at 21.89°, 22.16°, and 23.57°, and the 2θ error range is ±0.2°.
20. The crystal form IV of claim 19, wherein the X-ray powder diffraction pattern of the crystal form IV has a 2θ value of 7.81°, 10.97°, 13.07°, 14.20°, 15.20°, 15.66°, 16.11 There are characteristic peaks at °, 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 crystal form IV according to any one of claims 18 to 20, wherein the X-ray powder diffraction pattern of the crystal form IV is as shown in Figure 10.
22. The crystal form IV according to any one of claims 18 to 21, wherein the DSC spectrum of the crystal form IV has an endothermic characteristic peak at 229°C.
23. The crystal form IV according to any one of claims 18 to 22, characterized in that the TGA-DSC spectrum of the crystal form IV is as shown in Figure 11.
24. A pharmaceutical composition comprising the crystal form I described in any one of claims 1-6, the crystal form II described in any one of claims 7-11, the phosphate salt described in claim 12, The crystal form III according to any one of claims 13-17 or the crystal form IV according to any one of claims 18-23, and a pharmaceutically acceptable carrier.
25. Crystal form I according to any one of claims 1 to 6, crystal form II according to any one of claims 7 to 11, phosphate according to claim 12, any one of claims 13 to 17 The use of the crystal form III, the crystal form IV according to any one of claims 18-23 or the pharmaceutical composition according to claim 24 in the preparation of drugs for the treatment of CDK-mediated cancer.
26. Crystal form I according to any one of claims 1 to 6, crystal form II according to any one of claims 7 to 11, phosphate according to claim 12, any one of claims 13 to 17 The use of the crystal form III, the crystal form IV according to any one of claims 18-23 or the pharmaceutical composition according to claim 24 in the treatment of CDK-mediated cancer.
27. The use of claim 25 or 26, wherein the cancer includes ovarian cancer, breast cancer, acute myeloid leukemia, chronic lymphocytic leukemia or small lymphocytic lymphoma.
28. The preparation method of crystal form I according to any one of claims 1-6, characterized in that it includes the following steps:

    (a) Dissolve compound A in the first solvent to obtain a solution;

    (b) Add the second solvent to the solution in step (a) and stir;

    (c) After the solid is precipitated, filter, collect, and dry the solid.
29. The preparation method according to claim 28, wherein the first solvent is selected from at least one selected from the group consisting of acetone, methanol, tetrahydrofuran, ethyl acetate, acetonitrile and dichloromethane.
30. The preparation method according to claim 28, wherein the second solvent is selected from at least one of water, n-heptane, n-hexane, and methyl tert-butyl ether.