Polymorph of phosphate salt of pyrazolopyrimidine derivative and use thereof

Abstract

The present invention relates to a polymorph of phosphate salt of substituted pyrazolo[1,5-a]pyrimidin-7-amine derivative and the use thereof. Specifically, provided in the present invention are a crystal form III of phosphate salt of a compound (1S,3S)-N1-(5-((S)-1-cyclobutylethyl)pyrazolo[1,5-a]pyrimidin-7-yl)cyclopentane-1,3-diamine and the use thereof.

Description

A phosphate polymorph of a pyrazolopyrimidine derivative and its application

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on January 19, 2024, with application number CN202410081846.6 and invention name “A phosphate polymorph of a pyrazolopyrimidine derivative and its application”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention relates to the field of medical technology, and specifically to a phosphate crystal form III of the compound (1S,3S)-N1-(5-((S)-1-cyclobutylethyl)pyrazolo[1,5-a]pyrimidin-7-yl)cyclopentane-1,3-diamine and its application. Background Art

[0003] CDK9 is a member of the cyclin-dependent kinase (CDK) protein family and plays an important role in gene transcription regulation. CDK9 primarily regulates gene transcriptional elongation by phosphorylating the carbon-terminal region of RNA complex II. CDK9 is widely overexpressed in tumors and is an important factor in the progression and maintenance of tumor cells. CDK9 inhibitors have demonstrated excellent therapeutic effects in tumor treatment. Currently, several companies are developing CDK9 inhibitors, including the selective CDK9 inhibitor BAY1251152 developed by Bayer, the selective CDK9 inhibitor AZD4573 developed by AstraZeneca, the non-selective CDK9 inhibitor TP-1287 developed by Tolero, and the non-selective CDK9 inhibitor QHRD107 developed by Changzhou Qianhong Pharmaceutical.

[0004] Since the stability of active ingredients of pharmaceuticals has an important impact on drugability research, and the solid form is beneficial to product quality control, it is necessary to further explore the solid form of the product. Summary of the Invention

[0005] Based on this, the object of the present invention is to provide a phosphate crystal form III of the compound (1S,3S)-N1-(5-((S)-1-cyclobutylethyl)pyrazolo[1,5-a]pyrimidin-7-yl)cyclopentane-1,3-diamine (i.e., a compound of formula (I)) and its application, which has good solubility and stability.

[0006] In the first aspect of the present invention, there is provided a phosphate crystal form III of a compound of formula (I):

[0007] Wherein, the molar ratio of the compound of formula (I) to phosphoric acid in the phosphate is 1:1.

[0008] In some embodiments, the phosphate crystal form III has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following diffraction angle 2θ (°) values: 14.8±0.2, 18.12±0.2 and 22.26±0.2.

[0009] In some embodiments, the X-ray powder diffraction pattern of the phosphate crystal form III has characteristic diffraction peaks at the following diffraction angle 2θ (°) values: 14.8 ± 0.2, 18.12 ± 0.2, and 22.26 ± 0.2, and further includes one or more (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22) selected from the following group of characteristic diffraction peaks at diffraction angle 2θ (°) values: 7.42 ± 0.2, 10.58 ± 0.2, 11 .08±0.2, 11.8±0.2, 12.76±0.2, 13.94±0.2, 15.22±0.2, 16.34±0.2, 18.54±0.2, 20.96±0.2, 21.28±0.2, 21.78±0.2, 23.04±0.2, 23.6±0.2, 23.84±0.2, 24.62±0.2, 26.04±0.2, 28.08±0.2, 29.84±0.2, 30.82±0.2, 31.68±0.2 and 33.52±0.2. That is, the X-ray powder diffraction pattern of the phosphate crystal form III has characteristic diffraction peaks at diffraction angle 2θ (°) values ​​of 14.8±0.2, 18.12±0.2, and 22.26±0.2, and also has characteristic diffraction peaks at diffraction angle 2θ values ​​of 7.42±0.2, 10.58±0.2, 11.08±0.2, 11.8±0.2, 12.76±0.2, 13.94±0.2, 15.22±0.2, 16.34±0 .2, 18.54±0.2, 20.96±0.2, 21.28±0.2, 21.78±0.2, 23.04±0.2, 23.6±0.2, 23.84±0.2, 24.62±0.2, 26.04±0.2, 28.08±0.2, 29.84±0.2, 30.82±0.2, 31.68±0.2 and 33.52±0.2 have a characteristic diffraction peak.

[0010] In some embodiments, the X-ray powder diffraction pattern of the phosphate crystalline form III has characteristic diffraction peaks at 3 or more (such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24) or all of the following diffraction angle 2θ (°) values: 7.42 ± 0.2, 10.58 ± 0.2, 11.08 ± 0.2, 11.8 ± 0.2, 12.76 ± 0.2, 13.94 ± 0.2, 14.8 ±0.2, 15.22±0.2, 16.34±0.2, 18.12±0.2, 18.54±0.2, 20.96±0.2, 21.28±0.2, 21.78±0.2, 22.26±0.2, 23.04±0.2, 23.6±0.2, 23.84±0.2, 24.62±0.2, 26.04±0.2, 28.08±0.2, 29.84±0.2, 30.82±0.2, 31.68±0.2 and 33.52±0.2. That is, the X-ray powder diffraction pattern of the phosphate crystal form III has a diffraction angle 2θ (°) value of 7.42±0.2, 10.58±0.2, 11.08±0.2, 11.8±0.2, 12.76±0.2, 13.94±0.2, 14.8±0.2, 15.22±0.2, 16.34±0.2, 18.12±0.2, 18.54±0.2, 20.96±0 .2, 21.28±0.2, 21.78±0.2, 22.26±0.2, 23.04±0.2, 23.6±0.2, 23.84±0.2, 24.62±0.2, 26.04±0.2, 28.08±0.2, 29.84±0.2, 30.82±0.2, 31.68±0.2 and 33.52±0.2 have characteristic diffraction peaks.

[0011] In some embodiments, the X-ray powder diffraction pattern of the phosphate crystal form III has characteristic diffraction peaks at the following diffraction angle 2θ (°) values: 14.8±0.2, 18.12±0.2, 21.28±0.2 and 22.26±0.2.

[0012] In some embodiments, the X-ray powder diffraction pattern of the phosphate crystal form III has characteristic diffraction peaks at the following diffraction angle 2θ (°) values: 7.42±0.2, 10.58±0.2, 11.08±0.2, 13.94±0.2, 14.8±0.2, 18.12±0.2, 20.96±0.2, 21.28±0.2, 22.26±0.2, 26.04±0.2, and 29.84±0.2.

[0013] In some embodiments, the X-ray powder diffraction pattern of the phosphate crystal form III has characteristic diffraction peaks at the following diffraction angle 2θ (°) values: 7.42±0.2, 10.58±0.2, 11.08±0.2, 11.8±0.2, 12.76±0.2, 13.94±0.2, 14.8±0.2, 15.22±0.2, 16.34±0.2, 18.12±0.2, 1 8.54±0.2, 20.96±0.2, 21.28±0.2, 21.78±0.2, 22.26±0.2, 23.04±0.2, 23.6±0.2, 23.84±0.2, 24.62±0.2, 26.04±0.2, 28.08±0.2, 29.84±0.2, 30.82±0.2, 31.68±0.2 and 33.52±0.2.

[0014] In some embodiments, the X-ray powder diffraction pattern of the phosphate crystal form III expressed in 2θ (°) value and d value has the characteristic diffraction peaks shown in Table 1, and the relative intensity of each peak is shown in Table 1:

[0015] Table 1 2θ(°), d value and relative intensity I / I0 of phosphate crystal form III

[0016] In some embodiments, the X-ray powder diffraction pattern (XRPD pattern) of the phosphate salt Form III is substantially as shown in FIG1 .

[0017] In some embodiments, the peak temperature of the differential scanning calorimetry curve of the phosphate salt form III is 216.25±3°C, 216.25±2°C, 216.25±1°C or 216.25±0.5°C.

[0018] In some embodiments, the differential scanning calorimetry curve of the phosphate salt form III has an onset temperature of 212.90±3°C, 212.90±2°C, 212.90±1°C or 212.90±0.5°C.

[0019] In some embodiments, the differential scanning calorimetry curve (DSC curve) of the phosphate salt crystal form III is substantially as shown in Figure 2. In the embodiment shown in Figure 2, the melting point of the phosphate salt crystal form III of the compound of formula (I) is 212.90±0.5°C.

[0020] In some embodiments, the thermogravimetric analysis curve (TGA curve) of the phosphate crystal form III of the compound of formula (I) is substantially as shown in FIG3 .

[0021] In some embodiments, the TGA curve of Form III of the phosphate salt of the compound of Formula (I) shows almost no weight loss near 120°C.

[0022] In some embodiments, the TGA curve of the phosphate salt Form III of the compound of formula (I) shows that the phosphate salt Form III is an anhydrate.

[0023] The second aspect of the present invention provides a method for preparing the phosphate crystal form III of the compound of formula (I) according to the first aspect of the present invention.

[0024] The method comprises the following steps: forming a salt with the compound of formula (I) and phosphoric acid in a solvent, and performing crystallization treatment to obtain the phosphate crystal form III of the compound of formula (I).

[0025] In some embodiments, the temperature of the reaction system for salt formation between the compound of formula (I) and phosphoric acid in a solvent is 40°C to 70°C, preferably 50°C to 70°C, and more preferably 50°C to 65°C.

[0026] In some embodiments, the molar ratio of the compound of formula (I) to phosphoric acid is 1:(0.85-1.25).

[0027] In some embodiments, the molar ratio of the compound of formula (I) to phosphoric acid is 1:(0.8-1.2), for example, 1:0.8, 1:0.85, 1:0.9, 1:0.95, 1:1.0, 1:1.05, 1:1.1, 1:1.15, or 1:1.2. In some embodiments, the molar ratio of the compound of formula (I) to phosphoric acid is 1:(0.9-1.1). In some embodiments, the molar ratio of the compound of formula (I) to phosphoric acid is 1:0.95. In some embodiments, the molar ratio of the compound of formula (I) to phosphoric acid is 1:1.

[0028] In some embodiments, the solvent is a mixture of an organic solvent and water. Further, the organic solvent is selected from one or more of ethanol, methanol, and acetone. Further, the organic solvent is selected from one or more of ethanol and acetone.

[0029] In some embodiments, the solvent is an organic solvent. Further, the organic solvent is selected from ethanol, acetone, or a combination thereof. Further, the organic solvent is ethanol. Further, the organic solvent is acetone.

[0030] In some embodiments, the preparation method of the phosphate crystal form III of the compound of formula (I) comprises the following steps: dispersing the compound of formula (I) in a solvent, adding a phosphoric acid ethanol solution to form a salt, and performing crystallization treatment to obtain the phosphate crystal form III of the compound of formula (I); wherein the molar ratio of the compound of formula (I) to phosphoric acid is 1: (0.85-1.25).

[0031] In some embodiments, the compound of formula (I) is dispersed in a solvent and a phosphoric acid ethanol solution is added to form a salt. The reaction system temperature is 40°C to 70°C, preferably 50°C to 70°C, and more preferably 50°C to 65°C.

[0032] In some embodiments, the crystallization treatment is one or more of seed-induced crystallization, cooling crystallization, anti-solvent addition crystallization, suspension shaking, suspension centrifugation, slow evaporation, or stirring crystallization.

[0033] In some embodiments, the crystallization treatment is temperature-lowering crystallization.

[0034] In some embodiments, the crystallization treatment is to lower the temperature of the reaction system to -20°C-35°C for crystallization. For example, the temperature for crystallization can be any range (including the end values) selected from the group consisting of -20°C, -15°C, -10°C, -5°C, 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, and 35°C, without specific limitation.

[0035] In some embodiments, the crystallization process is seed-induced crystallization.

[0036] In some embodiments, the seed crystal induced crystallization is to add seed crystals at 0-80° C. to induce crystallization. For example, the temperature for adding seed crystals can be any range consisting of (including end values) end values ​​selected from 0° C., 5° C., 10° C., 15° C., 20° C., 25° C., 30° C., 35° C., 40° C., 45° C., 50° C., 60° C., 65° C., 70° C., 75° C., and 80° C., and is not specifically limited.

[0037] In some embodiments, the seed crystal induced crystallization is performed by adding seed crystals at 20°C-80°C to induce crystallization. Preferably, the seed crystals are added at 20°C-35°C. Preferably, the seed crystals are added at 50°C-65°C. More preferably, the seed crystals are added at 55°C-65°C.

[0038] In some embodiments, the seed-induced crystallization is performed by adding seed crystals at 55-65°C, maintaining the temperature, and cooling the solution to crystallize. Further, the temperature is lowered to 0-40°C, preferably 15-35°C, and more preferably 20-30°C.

[0039] In some embodiments, the amount of the seed crystals is 0.1-1 wt% of the amount of free alkali. In some embodiments, the amount of the seed crystals is 0.2-0.8 wt% of the amount of free alkali. In some embodiments, the amount of the seed crystals is 0.3-0.8 wt% of the amount of free alkali. In other embodiments, the amount of the seed crystals is 0.3-0.5 wt% of the amount of free alkali.

[0040] In some embodiments, the crystallization process further includes separation and / or drying steps.

[0041] In some embodiments, the separation step is one or more of filtration, suction filtration, and centrifugation. Further, the separation step is suction filtration. Further, the separation step is centrifugation.

[0042] In some embodiments, the drying step is one or more of vacuum oven drying, drying in a drying oven (non-vacuum), and vacuum drying on a rotary evaporator. Preferably, the drying step is vacuum oven drying. Preferably, the drying step is vacuum drying on a rotary evaporator.

[0043] The third aspect of the present invention provides a phosphate crystal form III of the compound of formula (I) prepared by the preparation method of the second aspect of the present invention.

[0044] A fourth aspect of the present invention provides a pharmaceutical composition comprising:

[0045] (a) the phosphate salt form III of the compound of formula (I) according to the first aspect of the present invention or the phosphate salt form III of the compound of formula (I) according to the third aspect of the present invention; and (b) a pharmaceutically acceptable carrier.

[0046] The fifth aspect of the present invention provides the use of the phosphate crystal form III of the compound of formula (I) according to the first aspect of the present invention, the phosphate crystal form III of the compound of formula (I) according to the third aspect of the present invention, or the pharmaceutical composition according to the fourth aspect of the present invention in the preparation of kinase inhibitors.

[0047] In some embodiments, the kinase inhibitor is a CDK9 inhibitor.

[0048] In the sixth aspect of the present invention, there is provided the use of the phosphate crystal form III of the compound of formula (I) according to the first aspect of the present invention, the phosphate crystal form III of the compound of formula (I) according to the third aspect of the present invention, or the pharmaceutical composition according to the fourth aspect of the present invention in the preparation of a drug for treating and / or preventing diseases related to or mediated by CDK9 activity.

[0049] In some embodiments, the disease includes a hyperproliferative disease, a virus-induced infectious disease, and a cardiovascular disease.

[0050] In some embodiments, the disease is a hyperproliferative disease.

[0051] In some embodiments, the hyperproliferative diseases include angiogenic or angioproliferative disorders, mesangial cell proliferative diseases, and solid tumors; for example, cancers of the breast, respiratory tract, brain, reproductive organs, digestive tract, urinary tract, eye, liver, skin, head and neck, thyroid or parathyroid glands, and their distant metastases, etc.

[0052] In some embodiments, the disease is selected from one or more of lymphoma, sarcoma, and leukemia.

[0053] In some embodiments, the disease is cancer; for example, pancreatic cancer, breast cancer, ovarian cancer, cervical cancer, or leukemia, among others.

[0054] In some embodiments, the disease includes solid tumors and hematological tumors.

[0055] In the present invention, diseases associated with CDK9 activity or mediated by CDK9 activity include diseases associated with CDK9 activity (e.g., overactivity of CDK9) or involving CDK9 activity, as well as conditions associated with these diseases. Overactivity of CDK9 refers to increased CDK9 enzymatic activity compared to normal, non-disease cells, or results in unwanted cell proliferation, or decreased or insufficient programmed cell death (apoptosis) leading to increased CDK9 activity, or mutations that result in constitutive activation of CDK9.

[0056] Hyperproliferative diseases include undesirable or uncontrolled proliferative diseases involving cells, and include diseases involving reduced or insufficient programmed cell death (apoptosis). The phosphate crystal form III of the compound of formula (I) of the present invention or a pharmaceutical composition containing the phosphate crystal form III of the compound of formula (I) can be used to prevent, inhibit, block, reduce, reduce, control, etc. cell proliferation and / or cell division, and / or produce apoptosis. The method includes giving a certain amount of the phosphate crystal form III of the compound of formula (I) of the present invention or a pharmaceutical composition containing the phosphate crystal form III of the compound of formula (I) to a subject in need (including mammals, including humans) for effectively treating or preventing the disease.

[0057] The present invention also provides a method for inhibiting CDK9 activity, comprising administering to a patient in need thereof a therapeutically effective amount of the phosphate crystal form III of the compound of formula (I) according to the first aspect of the present invention, the phosphate crystal form III of the compound of formula (I) according to the third aspect of the present invention, or the pharmaceutical composition according to the fourth aspect of the present invention.

[0058] The present invention also provides a method for treating a disease associated with or mediated by CDK9 activity, comprising administering to a subject an effective amount of the phosphate crystal form III of the compound of formula (I) according to the first aspect of the present invention, the phosphate crystal form III of the compound of formula (I) according to the third aspect of the present invention, or the pharmaceutical composition according to the fourth aspect of the present invention.

[0059] The purity of the compound of formula (I) obtained by the experiment is not high, and impurities will be produced during long-term storage, and the product color will change significantly, affecting the product quality. The phosphate crystal form III of the present invention has excellent chemical stability, and the impurity content does not change significantly in the long-term stability test. And it has excellent physical stability, and the transformation of crystal form will not occur in formulation development, thereby ensuring the quality uniformity of the formulation product. In addition, the phosphate crystal form III of the compound of formula (I) of the present invention has lower hygroscopicity while having excellent solubility compared with the free alkali. The phosphate crystal form III of the compound of formula (I) of the present invention has lower hygroscopicity while having excellent solubility compared with the phosphate crystal form I of the compound of formula (I), and is more suitable for the development of pharmaceutical preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] FIG1 is an X-ray powder diffraction (XRPD) pattern of the phosphate crystal form III of the compound of formula (I);

[0061] FIG2 is a differential scanning calorimetry (DSC) spectrum of the phosphate crystal form III of the compound of formula (I);

[0062] FIG3 is a thermogravimetric analysis (TGA) graph of the phosphate crystal form III of the compound of formula (I);

[0063] FIG4 is a single crystal structure diagram of the compound of formula (II);

[0064] FIG5 is an X-ray powder diffraction (XRPD) pattern of the phosphate crystal form I of the compound of formula (I);

[0065] FIG6 is a graph of differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) of the phosphate salt of the compound of formula (I) in Form I;

[0066] FIG7 is a dynamic water absorption curve (DVS) diagram of the phosphate salt crystal form I of the compound of formula (I);

[0067] FIG8 is an XRPD overlay of Form III of the phosphate salt of the compound of formula (I) under high temperature conditions;

[0068] FIG9 is a polarized light microscope image of the crystalline compound obtained in Example 2;

[0069] FIG10 is a polarized light microscope image of the crystalline compound obtained in Example 3;

[0070] FIG11 is a polarized light microscope image of the crystalline compound obtained in Example 4. DETAILED DESCRIPTION

[0071] To make the above-mentioned objects, features, and advantages of the present invention more readily apparent, specific embodiments of the present invention are described in detail below. The following description sets forth numerous specific details to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art may make similar modifications without departing from the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0072] The "crystal of the present invention", "crystal form of the present invention", "polymorph of the present invention" and the like used in the present invention are used interchangeably and refer to the phosphate polymorph of the compound of formula (I), particularly to the monophosphate polymorph of the compound of formula (I), and more particularly to the phosphate crystal form III.

[0073] In the present invention, the compound of formula (I) is (1S,3S)-N1-(5-((S)-1-cyclobutylethyl)pyrazolo[1,5-a]pyrimidin-7-yl)cyclopentane-1,3-diamine, and its structure is shown below:

[0074] The present invention also includes phosphates of the compound of formula (I), in particular monophosphates and the crystal form III of the phosphate of the compound of formula (I).

[0075] In the present invention, "compound of formula (I)", "free base of compound of formula (I)" and "free base" can be used interchangeably.

[0076] The "phosphate crystal form III" used in the present invention and the "phosphate crystal form III of the compound of formula (I)" can be used interchangeably.

[0077] Polymorphs: Solids exist in either amorphous or crystalline forms. In the crystalline form, the molecules are arranged in a three-dimensional lattice. When a compound crystallizes from a solution or slurry, it can crystallize in different spatial arrangements (a property known as "polymorphism"), forming crystals with different crystalline forms, which are called "polymorphs." Different polymorphs of a given substance can differ from one another in one or more physical properties, such as solubility and dissolution rate, true specific gravity, crystal shape, packing pattern, flowability, and / or solid-state stability.

[0078] Crystallization: Production-scale crystallization can be accomplished by manipulating the solution so that the solubility limit of the compound of interest is exceeded. This can be accomplished in a variety of ways, for example, by dissolving the compound at a relatively high temperature and then cooling the solution to below the saturation limit. Alternatively, the liquid volume can be reduced by boiling, atmospheric evaporation, vacuum drying, or by some other method. The solubility of the compound of interest can be reduced by adding an antisolvent or a solvent in which the compound has a low solubility, or a mixture of such solvents. Another alternative is to adjust the pH to reduce the solubility. For a detailed description of crystallization, see Crystallization, 3rd edition, J.W. Mullens, Butterworth-Heineman Ltd., 1993, ISBN 0750611294, the entire contents of which are incorporated herein by reference.

[0079] The "suspension shaking" method of the present invention refers to a method of mixing the compound of formula (I) and the corresponding acid or a solution of the corresponding acid in a suitable solvent to form a turbid solution and then shaking the solution to obtain crystals. The suitable solvent may be water or an organic solvent.

[0080] The "suspension centrifugation" of the present invention refers to a method in which the compound of formula (I) and the corresponding acid or a solution of the corresponding acid are mixed in a suitable solvent to form a turbid solution, followed by centrifugation to obtain crystals. The suitable solvent may be water or an organic solvent.

[0081] The "slow volatilization" mentioned in the present invention refers to a method in which a solution containing the compound of formula (I) and the corresponding acid is placed at a certain temperature to slowly volatilize the solvent to obtain crystals.

[0082] The "anti-solvent addition crystallization" mentioned in the present invention refers to a method of adding another suitable solvent to a solution of the compound of formula (I) to precipitate crystals.

[0083] If it is desired that salt formation and crystallization occur simultaneously, then if the salt is less soluble in the reaction medium than the starting materials, then the addition of an appropriate acid or base can result in direct crystallization of the desired salt. Similarly, completion of the synthesis reaction in a medium in which the final desired form is less soluble than the reactants can result in direct crystallization of the final product.

[0084] Optimization of crystallization can include seeding the crystallization medium with crystals of the desired form. In addition, many crystallization methods use a combination of the above strategies. One embodiment is to dissolve the compound of interest in a solvent at an elevated temperature, followed by the controlled addition of an appropriate volume of antisolvent to bring the system just below saturation. At this point, seeds of the desired form can be added (while maintaining the integrity of the seeds) and the system cooled to complete crystallization. As used herein, the term "about" refers to fluctuations within a range of ±5 on the basis of a given value.

[0085] In this article, when referring to a range of units, if the unit is only after the right endpoint, it means that the units of the left and right endpoints are the same. For example, 3~5h means that the units of the left endpoint "3" and the right endpoint "5" are both h (hours).

[0086] Herein, the terms "preferred," "better," "more preferred," and "suitable" are merely used to describe preferred implementations or examples and should not be construed as limiting the scope of protection of the present invention. If multiple "preferred" terms appear in a technical solution, each "preferred" term is considered independent unless otherwise specified and there are no contradictions or mutual constraints.

[0087] The terms "and / or", "or / and", and "and / or" used herein include any one of two or more related listed items, and also include any and all combinations of the related listed items, wherein the any and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND" and technical solutions connected by "logical OR".

[0088] As used herein, "plurality," "multiple," "multiple times," etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "multiple" means greater than or equal to two.

[0089] In this article, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0090] Pharmaceutical composition and its application

[0091] Generally, the phosphate crystal form III of the compound of formula (I) of the present invention as an active ingredient can be formed into a suitable dosage form for administration with one or more pharmaceutically acceptable carriers.

[0092] "Pharmaceutically acceptable carrier" refers to a non-toxic, inert, solid, semi-solid substance or liquid filling machine, diluent, encapsulating material or auxiliary formulation or any type of excipient, which is compatible with the subject to be administered (preferably a mammal, more preferably a human), and is suitable for delivering the active substance of the present invention to the target site without terminating its activity.

[0093] The pharmaceutical compositions of the present invention are formulated, dosed, and administered in a manner consistent with standard medical practice. The "therapeutically effective amount" of the active ingredient administered is determined by factors such as the specific condition to be treated, the individual being treated, the cause of the condition, the target of the drug, and the mode of administration.

[0094] The phosphate crystal form III of the compound of formula (I) according to the first aspect of the present invention, the phosphate crystal form III of the compound of formula (I) according to the third aspect of the present invention, or the pharmaceutical composition according to the fourth aspect of the present invention can be used to prepare drugs for treating and / or preventing diseases related to CDK9 activity or mediated by CDK9 activity.

[0095] The present invention provides a method for inhibiting CDK9 activity, comprising administering to a subject a therapeutically effective amount of the phosphate crystal form III of the compound of formula (I) according to the first aspect of the present invention, the phosphate crystal form III of the compound of formula (I) according to the third aspect of the present invention, or the pharmaceutical composition according to the fourth aspect of the present invention.

[0096] As used herein, "therapeutically effective amount" refers to the phosphate salt form III of the compound of formula (I) of the present invention that will cause a biological or medical response in a subject, such as reducing or inhibiting the activity of an enzyme or protein, or improving symptoms, alleviating symptoms, relieving or delaying disease progression, or preventing disease.

[0097] As used herein, "subject" refers to an animal, preferably a mammal, more preferably a human. The term "mammal" refers to warm-blooded vertebrate mammals, including cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, mice, pigs and humans.

[0098] "Treatment" refers to alleviating, slowing the progression of, attenuating, preventing, or maintaining an existing disease or condition (eg, cancer). Treatment also includes curing, preventing the development of, or alleviating to some extent, one or more symptoms of a disease or condition.

[0099] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight. Unless otherwise defined, the terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described contents may be applied to the present invention.

[0100] Reagents and instruments

[0101] In the present invention, the structure and purity of the compound were determined by nuclear magnetic resonance ( 1 H NMR) and / or liquid chromatography-mass spectrometry (LC-MS).

[0102] 1 H NMR: Bruker AVANCE-400 nuclear magnetic spectrometer, internal standard is tetramethylsilane (TMS).

[0103] LC-MS: Agilent 1290 HPLC System / 6130 / 6150MS liquid chromatography-mass spectrometer (manufacturer: Agilent), column: Waters BEH / CHS, 50×2.1 mm, 1.7 μm.

[0104] HPLC analysis was performed using an Agilent 1260 Infinity HPLC, OpenLAB CDS Chemstation workstation, an XBridge C18 4.6*250 mm column, ID 5 μm column, and a DAD detector.

[0105] Elemental analysis was performed using an inductively coupled plasma optical emission spectrometer (ICP 500) with a power of 1300 W and a flow rate of 1 mL / min.

[0106] Known starting materials can be synthesized by methods known in the art, or can be purchased from companies such as ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc, and Darui Chemicals.

[0107] As used herein, room temperature in the following examples refers to about 20-30°C.

[0108] General Methods

[0109] X-ray powder diffraction (XRPD): In the present invention, the X-ray diffraction pattern of the above-mentioned crystalline or amorphous powder is obtained by a method known in the art using an ARL Equinox 3000 X-ray powder diffraction analyzer. The XRPD test parameters are shown in Table 2 below:

[0110] Table 2

[0111] In the powder X-ray diffraction pattern, the position of each peak is determined by the 2θ (°) value. It will be appreciated that different instruments and / or conditions may result in slightly different data being generated, and the position and relative intensity of each peak may vary. The intensity division of the peaks merely reflects the approximate size of the peaks at each position. In the present invention, the diffraction peak with the highest peak height of the crystal form is taken as the base peak, and its relative intensity is defined as 100%, as I0 (the peak with a 2θ (°) value of 18.12 for the phosphate crystal form III is the base peak), and the ratio of the peak height to the base peak height of the other peaks is taken as their relative intensity I / I0. The division definition of the relative intensity of each peak is shown in Table 3 below:

[0112] Table 3

[0113] Single Crystal X-ray Diffraction (SXRD): In the present invention, single crystal X-ray diffraction patterns of the compound of Formula (II) were obtained using a D8 Venture diffractometer using methods known in the art. The SXRD test parameters are shown in Table 4 below. After collecting relevant data, the crystal structure was further analyzed using a direct method (SHELXT2014) to confirm the absolute configuration.

[0114] Table 4

[0115] The acid-base molar ratio of the phosphate of the compound of formula (I) and its crystal form is determined by elemental analysis.

[0116] High performance liquid chromatography (HPLC) was performed on an Agilent 1260 HPLC.

[0117] Differential Scanning Calorimetry (DSC): In the present invention, the differential scanning calorimetry spectrum of the above-mentioned crystal form is obtained by a method known in the art using a DSC25A differential scanning calorimeter. The DSC test parameters are shown in Table 5 below:

[0118] Table 5

[0119] Thermogravimetric analysis (TGA): In the present invention, the thermogravimetric analysis spectrum of the above-mentioned crystal form is obtained by a method known in the art using a TGA550 thermogravimetric analyzer. The TGA test parameters are shown in Table 6 below:

[0120] Table 6

[0121] Dynamic moisture sorption (DVS) curves were collected on an SMS (Surface Measurement Systems) DVS Intrinsic instrument. Relative humidity at 25°C was calibrated using the deliquescent points of LiCl, Mg(NO₃)₂, and KCl. Instrument test conditions are shown in Table 7.

[0122] Table 7

[0123] The "Guidelines for Hygroscopicity Tests of Drugs" No. 9103 of the "Chinese Pharmacopoeia" (2020) stipulates the description of hygroscopic characteristics and the definition of hygroscopic weight gain: (1) Deliquescent: absorbing sufficient water to form a liquid; (2) Extremely hygroscopic: hygroscopic weight gain is not less than 15%; (3) Hygroscopic: hygroscopic weight gain is less than 15% but not less than 2%; (4) Slightly hygroscopic: hygroscopic weight gain is less than 2% but not less than 0.2%; (5) No or almost no hygroscopic: hygroscopic weight gain is less than 0.2%.

[0124] The General Provisions section of Part II of the Chinese Pharmacopoeia (2020) defines solubility as a physical property of a drug. The selected solvents and their solubility in each solvent are provided for reference when refining or preparing solutions. When conducting quality control on solubility in unspecified solvents, specific regulations are provided under the inspection section for that drug. The approximate solubility of a drug is expressed using the following terms: Very soluble: 1 g (mL) of solute can be dissolved in less than 1 mL of solvent; Freely soluble: 1 g (mL) of solute can be dissolved in 1 to less than 10 mL of solvent; Soluble: 1 g (mL) of solute can be dissolved in 10 to less than 30 mL of solvent; Slightly soluble: 1 g (mL) of solute can be dissolved in 30 to less than 100 mL of solvent; Slightly soluble: 1 g (mL) of solute can be dissolved in 100 to less than 1000 mL of solvent; Very slightly soluble: 1 g (mL) of solute can be dissolved in 1000 to less than 10000 mL of solvent; Almost insoluble or insoluble: 1 g (mL) of solute can be dissolved in 10000 mL of solvent. Test method: Unless otherwise specified, weigh the powdered test sample or measure the liquid test sample and place it in a certain volume of solvent at 25℃±2℃. Shake vigorously for 30 seconds every 5 minutes. Observe the dissolution within 30 seconds. If no solute particles or droplets are visible, it is considered to be completely dissolved.

[0125] It is understood that other values ​​may be obtained when using other types of instruments with the same function as the above-mentioned instruments or using test conditions different from those used in the present invention. Therefore, the quoted values ​​should not be regarded as absolute values.

[0126] Due to instrument errors or differences in operators, those skilled in the art will understand that the above parameters used to characterize the physical properties of the crystals may vary slightly. Therefore, the above parameters are only used to assist in characterizing the polymorphs provided by the present invention and cannot be regarded as limitations on the polymorphs of the present invention.

[0127] Unless otherwise specified, "phosphoric acid solution" as used herein refers to aqueous phosphoric acid. "Phosphate" as used herein refers to the phosphate of the compound of formula (I). The wt% of seed crystals used is (amount of seed crystals used / amount of raw material free base used) x 100%.

[0128] As used in the present invention, CDK1 refers to cyclin-dependent kinase 1; CDK2 refers to cyclin-dependent kinase 2; CDK9 refers to cyclin-dependent kinase 9; THF: tetrahydrofuran; CDI: N,N'-carbonyldiimidazole; EA: ethyl acetate; PE: petroleum ether; DCM: dichloromethane; DMSO: dimethyl sulfoxide; NH3: ammonia; IPA: isopropanol; MeOH: methanol.

[0129] The compound of formula (I) of the present invention can be prepared with reference to the contents disclosed in WO2022156779A1. Specifically, take the following Preparation Example 1 as an example:

[0130] Preparation Example 1 Preparation of the compound of formula (I)

[0131] Step 1: In a reaction flask, compound 1 (36 g, 280.88 mmol) was dissolved in THF (300 mL). CDI (68.32 g, 421.32 mmol) was then added and the mixture was allowed to react at room temperature for 16 hours (Solution A). In a separate reaction flask, potassium monomethyl malonate (143.42 g, 842.64 mmol) was added to anhydrous magnesium chloride (66.86 g, 702.20 mmol) and THF (900 mL). The mixture was heated at 50°C under argon for 16 hours (Solution B). Solution A was then added dropwise to Solution B at room temperature (approximately 10 minutes). The mixture was then stirred at 30°C for 16 hours. LCMS confirmed the reaction was complete and the product was produced. Water (800 mL) was added to the reaction solution, which was extracted with ethyl acetate (800 mL x 3). The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, concentrated, and purified using a CombiFlash (120 g × 2, 0-15% EA / PE) to afford compound 2 (42 g, light yellow oil) in a yield of 75.42%. MS m / z (ESI): 199.1 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ4.13-4.00(m,2H),3.55(d,J=0.8Hz,2H),2.63-2.54(m,1H),2.42-2.28(m,1H) ,1.97-1.85(m,2H),1.83-1.74(m,1H),1.73-1.61(m,3H),1.16(t,J=7.2Hz,3H),0.91(d,J=6.8Hz,3H).

[0132] Step 2: Dissolve compound 2 (42 g, 211.85 mmol) and 3-aminopyrazole (19.36 g, 233.03 mmol) in glacial acetic acid (300 mL) and heat to 120°C for 16 h. LCMS indicated the reaction was complete. Concentrate under reduced pressure to remove the acetic acid, then slurry with ethyl acetate (800 mL x 4). The solid precipitated, was filtered, and dried to afford compound 3 (41 g, pale yellow solid) in a yield of 89.08%. The product was carried on to the next step without purification. MS m / z (ESI): 218.1 [M+H] + .

[0133] Step 3: Dissolve compound 3 (473.6 mg, 2.18 mmol) in phosphorus oxychloride (6 mL) and heat to 120°C with stirring for 3 h. Cool to room temperature, pour into ice water (60 g), and extract with DCM (80 mL). The organic phase is dried over anhydrous sodium sulfate, concentrated, and separated by silica gel column chromatography (20 g, 0%-40% EA / DCM) to provide compound 4. MS m / z (ESI): 236.1 [M+H] + ; 1 H NMR (400MHz, DMSO-d6) δ8.25(d,J=2.4Hz,1H),7.35(s,1H),6.76(d,J=2.4Hz,1H),2.893-2 .84(m,1H),2.61-2.51(m,1H),2.12-2.01(m,1H),1.80-1.59(m,5H),1.14(d,J=6.8Hz,3H).

[0134] Step 4: Dissolve compound 4 (142.7 mg, 605.5 μmol) and tert-butyl ((1S,3S)-3-aminocyclopentyl)carbamate (121.27 mg, 605.52 μmol) in acetonitrile (20 mL), add potassium carbonate (251.0 mg, 1.81 mmol), and stir at 90°C for 16 h. Dilute with ethyl acetate (80 mL), wash with saturated sodium chloride solution (80 mL x 3), dry the combined organic phases over anhydrous sodium sulfate, and concentrate to yield compound 5. MS m / z (ESI): 400.3 [M+H] +; 1 H NMR (400MHz, DMSO-d6) δ8.00(d,J=2.0Hz,1H),7.58(d,J=7.6Hz,1H),6.97(d,J= 7.6Hz,1H),6.30(d,J=2.0Hz,1H),6.00(s,1H),4.19(q,J=7.2Hz,1H),4.02-3.9 3(m,1H),2.77-2.66(m,1H),2.61-2.53(m,1H),2.21-2.01(m,3H),1.97-1.88(m ,2H),1.80-1.63(m,6H),1.56-1.42(m,1H),1.40(s,9H),1.13(d,J=6.8Hz,3H).

[0135] Step 5: Compound 5 (174.7 mg, 437.38 μmol) was dissolved in 1,4-dioxane (3 mL), and dilute hydrochloric acid (3.0 mL, 4 mol / L) was added. The mixture was stirred at room temperature for 3 h. The solvent was evaporated under reduced pressure, and water (60 mL) was added. The mixture was extracted with ethyl acetate (50 mL). The aqueous phase was adjusted to pH 9-10 with saturated sodium carbonate solution and extracted with ethyl acetate (60 mL x 2). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by preparative HPLC to obtain compound 6. MS m / z (ESI): 300.2 [M+H] + ; 1 H NMR(400MHz,DMSO-d6)δ7.97(s,1H),7.54(s,1H),6.28(s,1H),5.99(s,1H),4.13-4.22(m,1H),3.90-3.99(m,1H),2.75-2.6.3 (m,1H),2.51-2.57(m,1H),2.20-1.96(m,3H),1.85-1.94(m,2H),1.60-1.78(m,6H),1.40-1.49(m,1H),1.11(d,J=6.8Hz,3H).

[0136] Step 6: Compound 6 (94.46 mg, 315.48 μmol) was subjected to chiral separation (IC-3 4.6*100 mm 3 μm column; cosolvent: IPA [1% NH3 (7 M in MeOH)]; injection volume: 5.00 μL; wavelength: 220.0 nm; run time: 6.0 min; flow rate: 3.0 mL / min; pressure: 2000 psi; column temperature: 40°C) to afford compound (I) (9.70 mg, retention time 2.471 min) in 9.89% yield and 96.34% purity. MS m / z (ESI): 300.2 [M+H]+ . 1 H NMR(400MHz,DMSO-d6)δ7.96(d,J=2.0Hz,1H),7.41(d,J=7.6Hz,1H),6.27(d ,J=2.0Hz,1H),5.96(s,1H),4.21(q,J=7.2Hz,1H),3.42(q,J=6.0Hz,1H),2. 65-2.74(m,1H),2.58-2.50(m,1H),2.16-2.24(m,1H),2.03-2.09(m,1H),1. 83-1.95(m,2H),1.80-1.61(m,7H),1.35-1.25(m,1H),1.11(d,J=6.8Hz,3H).

[0137] In order to determine the absolute configuration of the compound of formula (I), the present application prepared the compound of formula (II) from the compound of formula (I).

[0138] The compound of formula (I) obtained in Preparation Example 1 (200 mg, 667.97 μmol) was dissolved in DCM (10 mL), and 4-chlorobenzoyl chloride (175.4 mg, 1.00 mmol) was added, followed by N,N-diisopropylethylamine (0.6 mL, 3.34 mmol). The mixture was stirred at room temperature for 2 hours. LC-MS analysis confirmed the complete reaction. Water (30 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (30 mL). The organic phase was washed with saturated sodium chloride solution (30 mL × 3), dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography to obtain the compound of formula (II) (160 mg, 54.69% yield). LC-MS (ESI) m / z: 438.2 [M+H] + .

[0139] Further, a single crystal of the compound of formula (II) was prepared, and the molecular structure ellipsoid diagram of the compound of formula (II) is shown in Figure 4. The Flack constant is 0.07(2), and C8, C10, and C19 are in the S configuration, that is, the absolute configuration of the three chiral centers of the compound of formula (II) is all S. Therefore, it can be determined that the absolute configuration of the three chiral centers of the compound of formula (I) is all S, and its structure is shown in the structure of the compound of formula (I).

[0140] Example 1. Preparation of Phosphate Form III of the Compound of Formula (I)

[0141] The free base (5 g) (prepared according to Preparation Example 1) was dissolved in ethanol (75 mL) and heated to 60°C. An ethanolic solution of phosphoric acid was added dropwise at a molar ratio of 0.95:1 between the acid and base. After addition, the mixture was stirred for 30 minutes, cooled to 20°C-25°C, stirred for 4 hours, filtered, rinsed with ethanol, and dried in a drying oven at 60°C. Elemental analysis of the resulting solid revealed a phosphorus content of 7.7%, indicating that the molar ratio of the compound of formula (I) to phosphoric acid in the resulting solid was 1:1 (the theoretical value of its phosphorus content is 7.79%). The resulting solid was subjected to XRPD analysis, and its XRPD pattern is shown in Figure 1. It is defined as phosphate crystal form III in this application. The XRPD pattern of the resulting solid has peaks at the 2θ (°) values ​​shown in Table 1 above, and the relative intensities of each peak are shown in Table 1 above. Its DSC curve is shown in Figure 2, and its TGA curve is shown in Figure 3. As shown in Figure 2, in the DSC curve, the starting temperature is 212.90°C and the peak temperature is 216.25°C, indicating that the melting point of the phosphate crystal form III of the compound of formula (I) is approximately 212.90°C. As shown in Figure 3, in the TGA curve, there is almost no weight loss when the sample is heated to around 120°C, indicating that the phosphate crystal form III of the compound of formula (I) is anhydrous.

[0142] Example 2-Example 4. Preparation of Phosphate Form III of Compound of Formula (I)

[0143] The free base (1.38 kg) (prepared with reference to Preparation Example 1) was transferred to a 50 L reactor with 12 kg of ethanol. The temperature was raised and maintained at 55-70°C. An ethanolic solution of phosphoric acid (448 g of phosphoric acid (85% content), 1.58 kg of ethanol) was added dropwise. The salt-forming reaction was carried out for 0.5 to 1 hour, and then crystallized (according to the crystallization operation in Table 8, the effects of different crystallization temperatures, crystallization conditions, and seed crystal addition amounts on the residual solvent were investigated). The solid was filtered and vacuum dried in a 20 L rotary evaporator for 12 hours to obtain the phosphate salt form III of the compound of formula (I). The obtained solid was subjected to XRPD analysis, and its XRPD pattern is generally shown in Figure 1.

[0144] Table 8 Crystallization conditions

[0145] Example 5. Preparation of Phosphate Crystalline Form I of the Compound of Formula (I)

[0146] A 20mL sample vial was charged with free base (500mg) and methanol (1mL) and sonicated to dissolve. A 1mol / L phosphoric acid solution was then added at a molar ratio of 2.5:1 between the acid and base. The reaction was allowed to proceed at 50°C for 1h, followed by stirring at 40°C for 1h, and then at 30°C for another 1h. The heat was then turned off and the mixture was stirred overnight. After the reaction, the temperature was slowly lowered to 0°C, and acetone was added to precipitate a solid. The solid was collected by centrifugation and the solvent was evaporated to obtain the phosphate salt Form I of the compound of formula (I). Elemental analysis of the resulting solid revealed a phosphorus content of 12.5%, indicating a molar ratio of the compound of formula (I) to phosphoric acid of 1:2 (the theoretical phosphorus content is 12.5%). The XRPD pattern of the resulting solid is shown in Figure 5 ; its DSC and TGA patterns are shown in Figure 6 ; and its DVS pattern is shown in Figure 7 .

[0147] Comparative Example 1. Salt-forming reaction of the compound of formula (I)

[0148] An appropriate amount of the compound of formula (I) (about 100 mg) was weighed by the weight loss method and placed in a transparent sample bottle. 0.2 mL to 0.3 mL of the corresponding solvent was added and dissolved by ultrasound. Then, a 1 mol / L aqueous solution of the acid was added according to an acid-base molar ratio of 1.2:1. The reaction was continued at 50 ° C for 1 hour, and stirring was continued at 40 ° C for 1 hour. Stirring was continued at 30 ° C for another 1 hour. After cooling to room temperature, the heating was turned off and stirred overnight. After the reaction was completed, the temperature was slowly lowered to precipitate the solid. If it was still a clear solution, the anti-solvent addition method was tried to induce crystallization. The reaction results showed that the compound of formula (I) did not form salts with acids such as hydrochloric acid, sulfuric acid, and oxalic acid after cooling and adding anti-solvents; wherein, the types and results of the acid and solvent are shown in Table 9 below.

[0149] Table 9 Salt Screening Results

[0150] Test Example 1. Solubility Test

[0151] (1) Solubility test of phosphate crystal form III

[0152] Referring to the test method for solubility in the general provisions of Part II of the "Chinese Pharmacopoeia" (2020), the obtained Form III (prepared with reference to Example 4) was ground into fine powder, added to a certain volume of solvent at 25°C±2°C, and shaken vigorously for 30 seconds every 5 minutes. The dissolution within 30 minutes was observed. If no solute particles were visible to the naked eye, it was considered to be completely dissolved.

[0153] Table 10-1 Solubility test results of Form III

[0154] As shown in Table 10-1, Form III is nearly insoluble in dichloromethane and ethanol, but readily soluble in dimethyl sulfoxide and water. Test results for the free base show that it is slightly soluble in water, indicating that Form III has better solubility in water than the free base, which is more conducive to improving the drug's bioavailability in the body.

[0155] (2) Comparison of the solubility of the phosphate crystal form I and the phosphate crystal form III of the compound of formula (I)

[0156] Prepare 0.1 mol / L hydrochloric acid solution and pH 6.8 phosphate buffer, weigh 200 mg of phosphate crystal form I (prepared according to Example 5) and phosphate crystal form III (prepared according to Example 4), add 5 mL of 0.1 mol / L hydrochloric acid solution and pH 6.8 phosphate buffer (sodium dihydrogen phosphate-sodium hydroxide system), respectively, and take samples at 0 h and 24 h (placed in a 37°C oven) to measure solubility. The experimental results are shown in the following table:

[0157] Table 10-2 Solubility comparison

[0158] It can be seen from the experimental results in Table 10-2 that the phosphate crystal form III has a higher solubility than the phosphate crystal form I.

[0159] Test Example 2. Stability Test of Phosphate Crystalline Form III

[0160] (1) Chemical stability

[0161] With reference to the provisions of the long-term test in the "Guidelines for Stability Tests of Raw Materials and Preparations" of the "Chinese Pharmacopoeia" (2020) 9001, a long-term stability test was conducted on Form III (prepared with reference to Example 4). The test method was as follows: the test sample was placed at a temperature of 25°C ± 2°C and a relative humidity of 60% ± 5% for 12 months. Samples were taken every 3 months, and samples were taken at 0 months, 3 months, 6 months, 9 months, and 12 months for testing according to the key stability inspection items. The test results are shown in Table 11:

[0162] Table 11 Long-term stability test results of Form III

[0163] The stability test results in Table 11 show that Form III has excellent stability in long-term stability tests and is more suitable for further development of pharmaceutical formulations. However, the free base of the compound of formula (I) turns from light color to black after being left at room temperature for a period of time, indicating severe deterioration, indicating that the phosphate Form III has better chemical stability.

[0164] (2) Physical stability

[0165] A sample (about 100 mg) of the phosphate crystal form III (prepared as described in Example 4) was weighed and placed under 60°C (high temperature) and 40°C-75% RH (accelerated) conditions. At the same time, another group of samples was sealed and stored at 5°C as a control. The crystal form changes were detected at 20 days and 30 days.

[0166] The test results show that the XRPD pattern of the phosphate crystal form III at 60°C does not change significantly. Its XRPD overlay is shown in Figure 8, indicating that this crystal form has good stability under high temperature conditions. The XRPD pattern of the phosphate crystal form III does not change significantly under 40°C-75% RH conditions, indicating that this crystal form has good stability under accelerated conditions.

[0167] Test Example 3. Hygroscopicity test

[0168] The free base of the compound of formula (I) (prepared as described in Preparation Example 1) and the phosphate crystal form III of the compound of formula (I) (prepared as described in Example 4) were subjected to a hygroscopicity test according to the procedure of 9103 (Guidelines for Hygroscopicity Test of Drugs) in the Chinese Pharmacopoeia (2020). The test method is as follows: (1) Take a dry stoppered glass weighing bottle (outer diameter 50 mm, height 15 mm), place it in a suitable 25℃±1℃ constant temperature desiccator (with ammonium chloride or ammonium sulfate saturated solution placed at the bottom) or an artificial climate box (set temperature of 25℃±1℃, relative humidity of 80%±2%) the day before the test, and accurately weigh the weight (m1). (2) Take an appropriate amount of the test sample and spread it flat in the above-mentioned weighing bottle. The thickness of the test sample is generally about 1 mm, and accurately weigh the weight (m2). (3) Open the weighing bottle and place it under the above-mentioned constant temperature and humidity conditions with the bottle cap for 24 hours. (4) Cover the weighing bottle cap and accurately weigh the weight (m3). (5) Calculate the weight gain percentage: The test results show that the free base of the compound of formula (I) has a moisture absorption weight increase of 3.95% under the test conditions, and is hygroscopic; the phosphate crystal form III of the compound of formula (I) has a moisture absorption weight increase of <0.5% under the test conditions, and is slightly hygroscopic.

[0169] The DVS test was performed on the phosphate crystal form I of the compound of formula (I). The DVS graph is shown in FIG7 . The DVS graph shows that under 80% RH conditions, the weight gain after moisture absorption is 9%, indicating that the compound has hygroscopic properties.

[0170] The above hygroscopicity results show that the hygroscopicity of the free base of the compound of formula (I) is greatly enhanced after conversion to the phosphate salt crystal form I (the weight gain due to hygroscopicity increases from 3.95% to 9%), while the hygroscopicity is greatly reduced after conversion from the free base to the phosphate salt crystal form III (the weight gain due to hygroscopicity decreases from 3.95% to <0.5%). It can be seen that the phosphate salt crystal form III has unexpectedly low hygroscopicity.

[0171] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

[0172] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0173] The above-described embodiments merely represent several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art may make several modifications and improvements without departing from the concept of the present invention, and these modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be based on the appended claims, and the description and drawings may be used to interpret the content of the claims.

Claims

1. A phosphate crystal form III of a compound of formula (I) Among them, The molar ratio of the compound of formula (I) to phosphoric acid in the phosphate is 1:

1.

2. The phosphate crystal form III according to claim 1, characterized in that, Its X-ray powder diffraction pattern has characteristic diffraction peaks at the following diffraction angle 2θ (°) values: 14.8 ± 0.2, 18.12 ± 0.2, and 22.26 ± 0.

2.

3. The phosphate crystal form III according to claim 1, wherein Its X-ray powder diffraction pattern, in addition to having characteristic diffraction peaks at the following diffraction angle 2θ (°) values: 14.8 ± 0.2, 18.12 ± 0.2, 22.26 ± 0.2, further includes characteristic diffraction peaks at 1 or more diffraction angle 2θ (°) values selected from the following group: 7.42 ± 0.2, 10.58 ± 0.2, 11.08 ± 0.2, 11.8 ± 0.2, 12.76 ± 0.2, 13.94 ± 0.2, 15.22 ± 0.2, 16.34 ± 0.2, 18.54 ± 0.2, 20.96 ± 0.2, 21.28 ± 0.2, 21.78 ± 0.2, 23.04 ± 0.2, 23.6 ± 0.2, 23.84 ± 0.2, 24.62 ± 0.2, 26.04 ± 0.2, 28.08 ± 0.2, 29.84 ± 0.2, 30.82 ± 0.2, 31.68 ± 0.2, and 33.52 ± 0.

2.

4. The phosphate crystal form III according to claim 1, characterized in that, Its X-ray powder diffraction pattern has characteristic diffraction peaks at 3 or more or all of the diffraction angle 2θ (°) values selected from the following: 7.42 ± 0.2, 10.58 ± 0.2, 11.08 ± 0.2, 11.8 ± 0.2, 12.76 ± 0.2, 13.94 ± 0.2, 14.8 ± 0.2, 15.22 ± 0.2, 16.34 ± 0.2, 18.12 ± 0.2, 18.54 ± 0.2, 20.96 ± 0.2, 21.28 ± 0.2, 21.78 ± 0.2, 22.26 ± 0.2, 23.04 ± 0.2, 23.6 ± 0.2, 23.84 ± 0.2, 24.62 ± 0.2, 26.04 ± 0.2, 28.08 ± 0.2, 29.84 ± 0.2, 30.82 ± 0.2, 31.68 ± 0.2, and 33.52 ± 0.

2.

5. The phosphate crystal form III according to claim 1, wherein It has one or more of the following characteristics: (1) It has a melting point of 212.90 ± 0.5 °C; (2) It is an anhydrous substance.

6. The phosphate crystal form III according to any one of claims 1-5, characterized in that, It has one or more of the following characteristics: (1) It has an XRPD pattern substantially as shown in Figure 1; (2) It has a DSC curve substantially as shown in Figure 2; (3) It has a TGA curve substantially as shown in Figure 3.

7. A process for preparing the phosphate crystal form III of the compound of formula (I) according to any one of claims 1-6, comprising: The compound of formula (I) is salted with phosphoric acid in a solvent and subjected to crystallization treatment to obtain crystalline form III of the compound of formula (I) phosphate.

8. The preparation method according to claim 7, characterized in that: The crystallization treatment is seeding-induced crystallization.

9. The preparation method according to claim 8, characterized in that: The seeding-induced crystallization is: adding seeds under the condition of 20 °C - 80 °C to induce crystallization; preferably, adding seeds at 20 °C - 35 °C; preferably, adding seeds at 50 °C - 65 °C; more preferably, adding seeds at 55 °C - 65 °C.

10. The preparation method according to claim 8, characterized in that: The dosage of the seed crystal is 0.1-1 wt% of the dosage of the free base; preferably, the dosage of the seed crystal is 0.2-0.8 wt% of the dosage of the free base; preferably, the dosage of the seed crystal is 0.3-0.8 wt% of the dosage of the free base; preferably, the dosage of the seed crystal is 0.3-0.5 wt% of the dosage of the free base.

11. The preparation method according to claim 7, characterized in that: The crystallization treatment is cooling crystallization.

12. The preparation method according to claim 11, characterized in that: The crystallization treatment is to carry out crystallization by reducing the temperature of the reaction system to -20°C to 35°C.

13. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: (a) The phosphate crystal form III of the compound of formula (I) according to any one of claims 1-6, and (b) A pharmaceutically acceptable carrier.

14. Use of the phosphate crystal form III of the compound of formula (I) according to any one of claims 1-6 or the pharmaceutical composition according to claim 13 in the preparation of a medicament for treating and / or preventing a disease related to or mediated by CDK9 activity.

15. The application according to claim 14, characterized in that The disease is one or more of hyperproliferative diseases, virus-induced infectious diseases or cardiovascular diseases.

16. A method for treating a disease related to or mediated by CDK9 activity, the method comprising administering to a subject an effective amount of the phosphate crystal form III of the compound of formula (I) according to any one of claims 1-6 or the pharmaceutical composition according to claim 13.

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