Pharmaceutically acceptable salts and polymorphs of substituted pyrazolo[1,5-a]pyrimidine-7-amine derivatives and uses thereof

Abstract

The application relates to pharmaceutically acceptable salts and polymorphs of substituted pyrazolo[1,5-a]pyrimidin-7-amine derivatives as shown in formula (I), pharmaceutical compositions containing the same, methods of preparation, and use of the pharmaceutically acceptable salts and polymorphs in the treatment or prevention of diseases associated with or mediated by CDK9 activity.

Description

[0001] Related applications

[0002] This application claims priority to Chinese Patent Application No. 202210866135.0, filed on July 22, 2022, entitled "Pharmaceutically Acceptable Salts and Polymorphs of Substituted Pyrazolo[1,5-a]pyrimidine-7-amine Derivatives and Their Applications Thereof" and Chinese Patent Application No. 202210870337.2, entitled "Phosphates and Polymorphs of CDK9 Inhibitors and Their Applications Thereof", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure pertains to the field of pharmaceutical technology, and specifically relates to pharmaceutically acceptable salts and polymorphs of substituted pyrazolo[1,5-a]pyrimidine-7-amine derivatives, pharmaceutical compositions comprising the same, methods of their preparation, and their pharmaceutical applications. Background Technology

[0004] CDK9 is a member of the cell cycle-dependent kinase (CDK) protein family and plays a crucial role in gene transcriptional regulation. CDK9 primarily regulates gene transcriptional elongation by phosphorylating the C-terminal region of RNA complex II. CDK9 is widely and highly expressed in tumors and is an important factor in tumor cell progression and maintenance. CDK9 inhibitors promote cancer cell apoptosis by inhibiting transcriptional elongation and downregulating the expression of related oncoproteins (MYC) and the apoptosis inhibitor protein Mcl-1. CDK9 inhibitors also reactivate silenced genes by regulating the epigenetic factor BRG1, including the activation of endogenous retroviruses (ERVs) in tumor cells, promoting interferon expression, and making tumor cells more sensitive to immunotherapy.

[0005] Currently, several companies are developing CDK9 inhibitors, including Bayer's selective CDK9 inhibitor BAY1251152, AstraZeneca's selective CDK9 inhibitor AZD4573, Tolero's non-selective CDK9 inhibitor TP-1287, and Changzhou Qianhong Pharmaceutical's non-selective CDK9 inhibitor QHRD107.

[0006] The solubility and stability of active pharmaceutical ingredients have a significant impact on drug development research, and solid form is beneficial for product quality control. Therefore, it is necessary to explore the solid form of products. Summary of the Invention

[0007] Based on this, the purpose of this disclosure is to provide pharmaceutically acceptable salts and polymorphs of substituted pyrazolo[1,5-a]pyrimidine-7-amine derivatives and their applications. Specifically, the pharmaceutically acceptable salts are L-tartrate and phosphate salts of the substituted pyrazolo[1,5-a]pyrimidine-7-amine derivatives. These substituted pyrazolo[1,5-a]pyrimidine-7-amine derivatives are CDK9 inhibitors with the molecular structure (1S,3S)-N1-(5-((S)-1-cyclobutylethyl)pyrazolo[1,5-a]pyrimidine-7-yl)cyclopentane-1,3-diamine as shown in formula (I).

[0008] A first aspect of this disclosure provides a pharmaceutically acceptable salt of the compound of formula (I):

[0009]

[0010] The pharmaceutically acceptable salt is selected from phosphates and L-tartrates.

[0011] In some embodiments, the pharmaceutically acceptable salt of the compound of formula (I) is in anhydrous form, hydrate form or solvate form.

[0012] In some embodiments, a pharmaceutically acceptable salt of the compound of formula (I) is a crystal. In some embodiments, a pharmaceutically acceptable salt of the compound of formula (I) is an amorphous form.

[0013] In some embodiments, the pharmaceutically acceptable salt is a phosphate. In some embodiments, the phosphate is a first-order phosphate of a compound of formula (I), wherein the molar ratio of phosphoric acid to the compound of formula (I) is (1.8-2.4):1. In some embodiments, the molar ratio of phosphoric acid to the compound of formula (I) may be 1.8:1, 1.9:1, 2.0:1, 2.1:1, 2.2:1, 2.3:1, or 2.4:1. In some embodiments, the molar ratio of phosphoric acid to the compound of formula (I) is (1.9-2.3):1. In one embodiment, the molar ratio of phosphoric acid to the compound of formula (I) is 2:1.

[0014] In some embodiments, the phosphate is phosphate form I of compound (I), whose X-ray powder diffraction pattern has characteristic diffraction peaks at the following diffraction angles of 2θ (°): 18.234±0.2, 19.131±0.2, and 21.266±0.2. In some embodiments, the phosphate is phosphate form I of compound (I), whose X-ray powder diffraction pattern has characteristic diffraction peaks at the following diffraction angles of 2θ (°): 18.803±0.2, 18.234±0.2, 19.131±0.2, and 21.266±0.2.

[0015] In some embodiments, the X-ray powder diffraction pattern of the phosphate crystal form I, in addition to having characteristic diffraction peaks at the following diffraction angle 2θ (°) values: 18.234±0.2, 19.131±0.2, and 21.266±0.2, further includes characteristic diffraction peaks at two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc.) selected from the following group of diffraction angle 2θ (°) values: 2.121±0.2, 7.373±0.2, 9.556±0.2, 10.607±0.2, 1 1.105±0.2, 12.322±0.2, 12.917±0.2, 13.999±0.2, 14.902±0.2, 15.238±0.2, 18.803±0.2, 19.881±0.2, 20.697±0.2, 22.01±0.2, 22.464±0.2, 23.247±0.2, 24.025±0.2, 26.367±0.2, 28.513±0.2, 30.297±0.2, 31.216±0.2 and 34.017±0.2.

[0016] In some embodiments, the X-ray powder diffraction pattern of phosphate crystal form I has characteristic diffraction peaks at three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc.) or all selected from the following diffraction angle 2θ (°) values: 2.121±0.2, 7.373±0.2, 9.556±0.2, 10.607±0.2, 11.105±0.2, 12.322±0.2, 12.917±0.2, 13.999±0.2, 14.9 02±0.2, 15.238±0.2, 18.234±0.2, 18.803±0.2, 19.131±0.2, 19.881±0.2, 20.697±0.2, 21.266±0.2, 22.01±0.2, 22.464±0.2, 23.247±0.2, 24.025±0.2, 26.367±0.2, 28.513±0.2, 30.297±0.2, 31.216±0.2 and 34.017±0.2.

[0017] In some embodiments, the X-ray powder diffraction pattern of the phosphate crystal form I has characteristic diffraction peaks at the following diffraction angles 2θ (°): 13.999±0.2, 18.234±0.2, 18.803±0.2, 19.131±0.2, 21.266±0.2, 22.01±0.2, and 23.247±0.2.

[0018] In some embodiments, the X-ray powder diffraction pattern of the phosphate crystal form I has characteristic diffraction peaks at the following diffraction angles 2θ (°): 7.373±0.2, 10.607±0.2, 11.105±0.2, 12.322±0.2, 12.917±0.2, 13.999±0.2, 14.902±0.2, 15.238±0.2, 18.234±0.2, 18.803±0.2, 19.131±0.2, 20.697±0.2, 21.266±0.2, 22.01±0.2, 22.464±0.2, 23.247±0.2, 24.025±0.2, 26.367±0.2, and 28.513±0.2.

[0019] In some embodiments, the X-ray powder diffraction pattern of phosphate crystal form I has characteristic diffraction peaks at the following diffraction angles 2θ (°): 2.121±0.2, 7.373±0.2, 9.556±0.2, 10.607±0.2, 11.105±0.2, 12.322±0.2, 12.917±0.2, 13.999±0.2, 14.902±0.2, 15.238±0.2, 18.23 4±0.2, 18.803±0.2, 19.131±0.2, 19.881±0.2, 20.697±0.2, 21.266±0.2, 22.01±0.2, 22.464±0.2, 23.247±0.2, 24.025±0.2, 26.367±0.2, 28.513±0.2, 30.297±0.2, 31.216±0.2 and 34.017±0.2.

[0020] In some embodiments, the X-ray powder diffraction pattern of phosphate crystal form I of compound (I) has characteristic diffraction peaks expressed in 2θ (°) and d values ​​as shown in Table 1, and the relative intensities of each peak are shown in Table 1.

[0021] Table 1. 2θ (°), d value and relative intensity I / I0 of phosphate crystal form I

[0022]

[0023]

[0024] In some embodiments, the molar ratio of phosphoric acid to the compound of formula (I) in phosphate crystal form I is 2:1.

[0025] In some embodiments, the X-ray powder diffraction pattern of phosphate crystal form I of compound (I) is essentially as follows: Figure 1 As shown.

[0026] In some embodiments, the differential scanning calorimetry (DSC) curve of phosphate crystal form I of formula (I) exhibits an endothermic peak at 188.01℃±3℃, 188.01℃±2℃, 188.01℃±1℃, or 188.01℃±0.5℃. In some embodiments, the onset temperature of the DSC curve of phosphate crystal form I of formula (I) is 188.01℃±3℃, 188.01℃±2℃, 188.01℃±1℃, or 188.01℃±0.5℃, and the peak temperature is 193.69℃±3℃, 193.69℃±2℃, 193.69℃±1℃, or 193.69℃±0.5℃. In some embodiments, the differential scanning calorimetry (DSC) spectrum of phosphate crystal form I of formula (I) is substantially as follows: Figure 2 As shown. Figure 2 In the illustrated embodiment, the melting point of phosphate crystal form I of compound (I) is approximately 188.01 ± 0.5 °C.

[0027] In some embodiments, the thermogravimetric analysis (TGA) spectrum of phosphate crystal form I of compound (I) is basically as follows: Figure 2 As shown. In some embodiments, the TGA spectrum of phosphate crystal form I of compound (I) shows a weight loss of 1.139% at around 190 °C.

[0028] In some embodiments, the dynamic water absorption spectrum (DVS diagram) of phosphate crystal form I of formula (I) is basically as follows: Figure 3 As shown. In some embodiments, the DVS diagram of phosphate crystal form I of compound (I) shows a 9% weight gain due to moisture absorption under 80% relative humidity (RH) conditions.

[0029] In some embodiments, the microscopic image of phosphate crystal form I of compound (I) is essentially as follows: Figure 4 As shown. In some embodiments, microscopic images of phosphate crystal form I of formula (I) show that phosphate crystal form I is rod-shaped and block-shaped.

[0030] In this first aspect, in some embodiments, the pharmaceutically acceptable salt is an L-tartrate. The molar ratio of L-tartaric acid to the compound of formula (I) in the L-tartrate is (0.8-1.2):1. In some embodiments, the molar ratio of L-tartaric acid to the compound of formula (I) may be 0.8:1, 0.9:1, 1.0:1, 1.1:1, or 1.2:1. In some embodiments, the molar ratio of L-tartaric acid to the compound of formula (I) is (0.9-1.1):1. In one embodiment, the molar ratio of L-tartaric acid to the compound of formula (I) is 1:1.

[0031] In some embodiments, the L-tartrate is L-tartrate crystal form I of compound (I), whose X-ray powder diffraction pattern has characteristic diffraction peaks at the following diffraction angles 2θ (°): 13.946±0.2, 16.881±0.2, 19.405±0.2, 21.505±0.2 and 24.262±0.2.

[0032] In some embodiments, the X-ray powder diffraction pattern of the L-tartrate crystal form I, in addition to having characteristic diffraction peaks at the following diffraction angles of 2θ (°): 13.946±0.2, 16.881±0.2, 19.405±0.2, 21.505±0.2, and 24.262±0.2, further includes two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc.) characteristic diffraction peaks selected from the following diffraction angles of 2θ (°): 6.687±0.2, 7.436±0.2, 9.493±0.2, ... 10.615±0.2, 12.053±0.2, 12.776±0.2, 13.164±0.2, 14.875±0.2, 15.201±0.2, 16.013±0.2, 18.175±0.2, 19.045±0.2, 20.659±0.2, 22.434±0.2, 23.04±0.2, 25.202±0.2, 26.452±0.2, 28.105±0.2, 29.692±0.2, 31.579±0.2, 34.139±0.2 and 34.543±0.2.

[0033] In some embodiments, the X-ray powder diffraction pattern of the L-tartrate crystal form I has characteristic diffraction peaks at five or more (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc.) or all selected from the following diffraction angle 2θ (°) values: 6.687±0.2, 7.436±0.2, 9.493±0.2, 10.615±0.2, 12.053±0.2, 12.776±0.2, 13.164±0.2, 13.946±0.2, 14.875±0.2, 15.2. 01±0.2, 16.013±0.2, 16.881±0.2, 18.175±0.2, 19.045±0.2, 19.405±0.2, 20.659±0.2, 21.505±0.2, 22.434±0.2, 23.04±0.2, 24.262±0.2, 25.202±0.2, 26.452±0.2, 28.105±0.2, 29.692±0.2, 31.579±0.2, 34.139±0.2 and 34.543±0.2.

[0034] In some embodiments, the X-ray powder diffraction pattern of the L-tartrate crystal form I has characteristic diffraction peaks at the following diffraction angles 2θ (°): 7.436±0.2, 13.946±0.2, 16.013±0.2, 16.881±0.2, 18.175±0.2, 19.045±0.2, 19.405±0.2, 21.505±0.2, 24.262±0.2, 25.202±0.2, 26.452±0.2, 28.105±0.2, and 31.579±0.2.

[0035] In some embodiments, the X-ray powder diffraction pattern of the L-tartrate crystal form I has characteristic diffraction peaks at the following diffraction angles 2θ (°): 6.687±0.2, 7.436±0.2, 10.615±0.2, 12.053±0.2, 13.164±0.2, 13.946±0.2, 14.875±0.2, 15.201±0.2, 16.013±0.2, 16.881±0.2, 18. 175±0.2, 19.045±0.2, 19.405±0.2, 20.659±0.2, 21.505±0.2, 22.434±0.2, 23.04±0.2, 24.262±0.2, 25.202±0.2, 26.452±0.2, 28.105±0.2, 29.692±0.2, 31.579±0.2, 34.139±0.2 and 34.543±0.2.

[0036] In some embodiments, the X-ray powder diffraction pattern of the L-tartrate crystal form I has characteristic diffraction peaks at the following diffraction angles 2θ (°): 6.687±0.2, 7.436±0.2, 9.493±0.2, 10.615±0.2, 12.053±0.2, 12.776±0.2, 13.164±0.2, 13.946±0.2, 14.875±0.2, 15.201±0.2, 16.013±0.2, 16.8 81±0.2, 18.175±0.2, 19.045±0.2, 19.405±0.2, 20.659±0.2, 21.505±0.2, 22.434±0.2, 23.04±0.2, 24.262±0.2, 25.202±0.2, 26.452±0.2, 28.105±0.2, 29.692±0.2, 31.579±0.2, 34.139±0.2 and 34.543±0.2.

[0037] In some embodiments, the X-ray powder diffraction pattern of the L-tartrate crystal form I has characteristic diffraction peaks expressed in 2θ (°) and d values ​​as shown in Table 2, and the relative intensities of each peak are shown in Table 2.

[0038] Table 2. 2θ (°), d value and relative intensity I / I0 of L-tartrate crystal form I

[0039]

[0040] In some embodiments, the molar ratio of L-tartaric acid to the compound of formula (I) in L-tartrate crystal form I is 1:1.

[0041] In some embodiments, the X-ray powder diffraction pattern of L-tartrate crystal form I of compound (I) is essentially as follows: Figure 5 As shown.

[0042] In some embodiments, the thermogravimetric analysis spectrum of compound (I) L-tartrate crystal form I is basically as follows: Figure 6 As shown. In some embodiments, the TGA spectrum of L-tartrate crystal form I of compound (I) shows a weight loss of 1.057% near 110°C and a further weight loss of 3.482% near 178°C.

[0043] In some embodiments, the L-tartrate is L-tartrate crystal form II of compound (I), whose X-ray powder diffraction pattern has characteristic diffraction peaks at the following diffraction angles 2θ (°): 11.662±0.2 and 21.353±0.2.

[0044] In some embodiments, the X-ray powder diffraction pattern of the L-tartrate crystal form II, in addition to having characteristic diffraction peaks at the lower set of diffraction angles of 2θ (°): 11.662±0.2 and 21.353±0.2, further includes two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, etc.) characteristic diffraction peaks selected from the lower set of diffraction angles of 2θ (°): 6.985 ±0.2, 10.376±0.2, 10.884±0.2, 12.994±0.2, 14.244±0.2, 16.548±0.2, 17.481±0.2, 18.349±0.2, 18.984±0.2, 21.026±0.2, 26.925±0.2, 29.335±0.2, 30.896±0.2 and 31.784±0.2.

[0045] In some embodiments, the X-ray powder diffraction pattern of the L-tartrate crystal form II has characteristic diffraction peaks at three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc.) or all selected from the following diffraction angle 2θ (°) values: 6.985±0.2, 10.376±0.2, 10.884±0.2, 11.662± 0.2, 12.994±0.2, 14.244±0.2, 16.548±0.2, 17.481±0.2, 18.349±0.2, 18.984±0.2, 21.026±0.2, 21.353±0.2, 26.925±0.2, 29.335±0.2, 30.896±0.2 and 31.784±0.2.

[0046] In some embodiments, the X-ray powder diffraction pattern of the L-tartrate crystal form II has characteristic diffraction peaks at the following diffraction angles 2θ (°): 11.662±0.2, 14.244±0.2, 17.481±0.2, 18.349±0.2, 21.026±0.2, and 21.353±0.2.

[0047] In some embodiments, the X-ray powder diffraction pattern of the L-tartrate crystal form II has characteristic diffraction peaks at the following diffraction angles 2θ (°): 10.376±0.2, 11.662±0.2, 14.244±0.2, 16.548±0.2, 17.481±0.2, 18.349±0.2, 18.984±0.2, 21.026±0.2, 21.353±0.2, 26.925±0.2, 29.335±0.2, and 31.784±0.2.

[0048] In some embodiments, the X-ray powder diffraction pattern of the L-tartrate crystal form II has characteristic diffraction peaks at the following diffraction angles 2θ (°): 6.985±0.2, 10.376±0.2, 10.884±0.2, 11.662±0.2, 12.994±0.2, 14.244±0.2, 16.548±0.2, 17.481±0.2, 18.349±0.2, 18.984±0.2, 21.026±0.2, 21.353±0.2, 26.925±0.2, 29.335±0.2, 30.896±0.2, and 31.784±0.2.

[0049] In some embodiments, the X-ray powder diffraction pattern of the L-tartrate crystal form II has characteristic diffraction peaks expressed in 2θ (°) and d values ​​as shown in Table 3, and the relative intensities of each peak are shown in Table 3.

[0050] Table 3. 2θ (°), d value and relative intensity I / I0 of L-tartrate crystal form II

[0051]

[0052] In some embodiments, the molar ratio of L-tartaric acid to the compound of formula (I) in L-tartrate crystal form II is 1:1.

[0053] In some embodiments, the X-ray powder diffraction pattern of the L-tartrate crystal form II is substantially as follows: Figure 7 As shown.

[0054] In this first aspect, in some embodiments, the phosphate is a second type of phosphate of formula (I), wherein the molar ratio of formula (I) to phosphoric acid in the phosphate is 1:(0.8-1.2). In some embodiments, the molar ratio of formula (I) to phosphoric acid is 1:(0.9-1.1). In some embodiments, the molar ratio of formula (I) to phosphoric acid can be 1:0.8, 1:0.9, 1:1, 1:1.1, or 1:1.2. In some embodiments, the molar ratio of formula (I) to phosphoric acid in the phosphate is 1:1.

[0055] In some embodiments, the phosphate of the compound of formula (I) is in anhydrous form, hydrate form or solvate form.

[0056] In some embodiments, the phosphate of the compound of formula (I) is a crystalline powder, existing in crystalline form.

[0057] In some embodiments, the phosphate of the compound of formula (I) is phosphate crystal form II, and its X-ray powder diffraction pattern has characteristic diffraction peaks at the following diffraction angles 2θ (°): 18.230±0.2 and 21.144±0.2.

[0058] In some embodiments, the molar ratio of the phosphate crystal form II compound of formula (I) to phosphoric acid is 1:1.

[0059] In some embodiments, the X-ray powder diffraction pattern of the phosphate crystal form II, in addition to having characteristic diffraction peaks at the following diffraction angle 2θ (°) values: 18.230±0.2 and 21.144±0.2, further includes two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc.) characteristic diffraction peaks selected from the following group of diffraction angle 2θ (°) values: 10.584±0.2, 11.115±0.2, 11.813±0.2, 12.825±0.2, 13 0.969±0.2, 14.873±0.2, 15.313±0.2, 18.771±0.2, 19.85±0.2, 20.576±0.2, 22.01±0.2, 22.492±0.2, 23.153±0.2, 23.96±0.2, 24.947±0.2, 26.273±0.2, 27.837±0.2, 28.432±0.2, 30.143±0.2, 31.072±0.2, 31.88±0.2 and 33.982±0.2.

[0060] In some embodiments, the X-ray powder diffraction pattern of the phosphate crystal form II has characteristic diffraction peaks at two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) or all selected from the following diffraction angle 2θ (°) values: 10.584±0.2, 13.969±0.2, 14.873±0.2, 18.230±0.2, 20.576±0.2, 21.144±0.2, 22.01±0.2, 22.492±0.2, 23.153±0.2, and 23.96±0.2.

[0061] In some embodiments, the X-ray powder diffraction pattern of the phosphate crystal form II has characteristic diffraction peaks at the following diffraction angles 2θ (°): 10.584±0.2, 13.969±0.2, 14.873±0.2, 18.230±0.2, 20.576±0.2, 21.144±0.2, 22.01±0.2, 22.492±0.2, 23.153±0.2, and 23.96±0.2.

[0062] In some embodiments, the X-ray powder diffraction pattern of the phosphate crystal form II has characteristic diffraction peaks at two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc.) or all selected from the following diffraction angle 2θ (°) values: 10.584±0.2, 11.115±0.2, 11.813±0.2, 12.825±0.2, 13.969±0.2, 14.873±0.2, 15.313±0.2, 18.2 30±0.2, 18.771±0.2, 19.85±0.2, 20.576±0.2, 21.144±0.2, 22.01±0.2, 22.492±0.2, 23.153±0.2, 23.96±0.2, 24.947±0.2, 26.273±0.2, 27.837±0.2, 28.432±0.2, 30.143±0.2, 31.072±0.2, 31.88±0.2 and 33.982±0.2.

[0063] In some embodiments, the X-ray powder diffraction pattern of the phosphate crystal form II has characteristic diffraction peaks at the following diffraction angles 2θ (°): 10.584±0.2, 11.115±0.2, 11.813±0.2, 12.825±0.2, 13.969±0.2, 14.873±0.2, 15.313±0.2, 18.230±0.2, 18.771±0.2, 19.85±0.2. 2, 20.576±0.2, 21.144±0.2, 22.01±0.2, 22.492±0.2, 23.153±0.2, 23.96±0.2, 24.947±0.2, 26.273±0.2, 27.837±0.2, 28.432±0.2, 30.143±0.2, 31.072±0.2, 31.88±0.2 and 33.982±0.2.

[0064] In some embodiments, the X-ray powder diffraction pattern of the phosphate crystal form II has characteristic diffraction peaks expressed in 2θ (°) and d values ​​as shown in Table 4, and the relative intensities of each peak are shown in Table 4.

[0065] Table 4. 2θ (°), d value and relative intensity I / I0 of phosphate crystal form II

[0066]

[0067] In some embodiments, the X-ray powder diffraction (XRPD) pattern of the phosphate crystal form II is substantially as follows: Figure 10 As shown.

[0068] In some embodiments, the differential scanning calorimetry (DSC) curve of phosphate crystal form II has an endothermic peak at 207.48 ± 3 °C, ± 2 °C, ± 1 °C, or ± 0.5 °C. In some embodiments, the onset temperature of the DSC curve of phosphate crystal form II is 207.48 °C ± 3 °C, 207.48 °C ± 2 °C, 207.48 °C ± 1 °C, or 207.48 °C ± 0.5 °C, and the peak temperature is 214.31 °C ± 3 °C, 214.31 °C ± 2 °C, 214.31 °C ± 1 °C, or 214.31 °C ± 0.5 °C. In some embodiments, the differential scanning calorimetry (DSC) curve of phosphate crystal form II is substantially as follows: Figure 11 As shown. Figure 11 In the illustrated embodiment, the melting point of phosphate crystal form II of compound (I) is 207.48 ± 0.5 °C.

[0069] In some embodiments, the thermogravimetric analysis (TGA) curve of phosphate crystal form II of compound (I) is basically as follows: Figure 11 As shown. In some embodiments, the TGA curve of phosphate crystal form II of formula (I) shows almost no weight loss near 100°C, and begins to melt when heated to near the melting point of 207.48°C. In some embodiments, the TGA curve of phosphate crystal form II of formula (I) shows that phosphate crystal form II is anhydrous.

[0070] In some embodiments, the dynamic water absorption spectrum (DVS diagram) of phosphate crystal form II of compound (I) is basically as follows: Figure 12 As shown. In some embodiments, the DVS diagram of phosphate crystal form II of compound (I) shows a moisture gain of 0.45% under 80% relative humidity (RH) conditions.

[0071] In some embodiments, the microscopic image of phosphate crystal form II of compound (I) is essentially as follows: Figure 13 As shown. In some embodiments, a microscopic image of phosphate crystal form II of compound (I) shows that phosphate crystal form II is rod-shaped.

[0072] A second aspect of this disclosure provides a method for preparing an L-tartrate of a compound of formula (I).

[0073]

[0074] In some embodiments, the method for preparing the L-tartrate of the compound of formula (I) includes the following steps: reacting the compound of formula (I) with L-tartaric acid to form the L-tartrate of the compound of formula (I).

[0075] In some embodiments, in the method for preparing the L-tartrate, the molar ratio of L-tartaric acid to the compound of formula (I) is (0.8-1.4):1. In some embodiments, the molar ratio of L-tartaric acid to the compound of formula (I) is (0.9-1.2):1.

[0076] In some embodiments, in the method for preparing the L-tartrate, the molar ratio of L-tartaric acid to compound (I) in the formed L-tartrate of formula (I) is (0.8-1.2):1. In some embodiments, the molar ratio of L-tartaric acid to compound (I) in the formed L-tartrate of formula (I) is (0.9-1.1):1. In one embodiment, the molar ratio of L-tartaric acid to compound (I) in the formed L-tartrate of formula (I) is 1:1.

[0077] In some embodiments, a method for preparing a polymorph of the L-tartrate of formula (I) is also provided, wherein the polymorph is L-tartrate crystal form I, comprising the following steps:

[0078] The compound of formula (I) is reacted with L-tartaric acid in an organic solvent to form a salt-forming reaction solution.

[0079] The reaction solution was slowly cooled to obtain L-tartrate crystal form I.

[0080] In some embodiments, the organic solvent is selected from one or more of ethanol, acetonitrile, ethyl acetate, acetone, and methanol.

[0081] In some embodiments, in the method for preparing L-tartrate crystal form I, the molar ratio of L-tartaric acid to the compound of formula (I) is (0.8-1.4):1. In other embodiments, the molar ratio of L-tartaric acid to the compound of formula (I) is (0.9-1.2):1.

[0082] In some embodiments, in the method for preparing L-tartrate crystal form I, the molar ratio of L-tartaric acid to the compound of formula (I) in the obtained L-tartrate crystal form I is (0.8-1.2):1. In other embodiments, the molar ratio of L-tartaric acid to the compound of formula (I) in the obtained L-tartrate crystal form I is (0.9-1.1):1. In one embodiment, the molar ratio of L-tartaric acid to the compound of formula (I) in the obtained L-tartrate crystal form I is 1:1.

[0083] In some embodiments, a method for preparing L-tartrate crystal form II is also provided, comprising the following steps:

[0084] The compound of formula (I) is reacted with L-tartaric acid in an organic solvent to form a salt-forming reaction solution.

[0085] The reaction solution was slowly cooled, and an antisolvent was added to obtain the L-tartrate crystal form II.

[0086] In some embodiments, the organic solvent is selected from one or more of ethanol, acetonitrile, ethyl acetate, acetone, and methanol.

[0087] In some embodiments, the antisolvent is selected from one or more of methyl tert-butyl ether, petroleum ether, n-heptane, n-hexane, cyclohexane, isopropanol, acetone, acetonitrile, and ethyl acetate, and the antisolvent is different from the organic solvent. In other embodiments, the antisolvent is selected from one or more of methyl tert-butyl ether, n-heptane, isopropanol, and acetone.

[0088] In some embodiments, in the method for preparing L-tartrate crystal form II, the molar ratio of L-tartaric acid to the compound of formula (I) is (0.8-1.4):1. In other embodiments, the molar ratio of L-tartaric acid to the compound of formula (I) is (0.9-1.2):1.

[0089] In some embodiments, in the method for preparing L-tartrate crystal form II, the molar ratio of L-tartaric acid to the compound of formula (I) in the obtained L-tartrate crystal form II is (0.8-1.2):1. In other embodiments, the molar ratio of L-tartaric acid to the compound of formula (I) in the obtained L-tartrate crystal form II is (0.9-1.1):1. In one embodiment, the molar ratio of L-tartaric acid to the compound of formula (I) in the obtained L-tartrate crystal form II is 1:1.

[0090] In some embodiments, the temperature of the salt-forming reaction is from -10°C to 90°C. For example, the temperature of the salt-forming reaction can be any range (inclusive) selected from the extreme values ​​of -10°C, -5°C, 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, and 90°C, without any specific limitation.

[0091] In some embodiments, the reaction solution is slowly cooled to -10°C to 60°C. In some embodiments, the reaction solution is slowly cooled to 0°C to room temperature. In some embodiments, the reaction solution is slowly cooled to room temperature.

[0092] In some embodiments, the molar concentration of L-tartaric acid is 0.1-5 mol / L. For example, the molar concentration of L-tartaric acid can be 0.1 mol / L, 1 mol / L, 1.5 mol / L, 2 mol / L, 2.5 mol / L, 3 mol / L, 3.5 mol / L, 4 mol / L, 4.5 mol / L, or 5 mol / L. In other embodiments, the molar concentration of L-tartaric acid is 0.5-3 mol / L. In still other embodiments, the molar concentration of L-tartaric acid is 1-2 mol / L.

[0093] In some embodiments, the preparation method of L-tartrate crystal form I includes the following steps:

[0094] (BI-a) Dissolve the compound of formula (I) in a solvent, add an aqueous solution of L-tartaric acid, and stir to react; and

[0095] (BI-b) Cool the reaction solution obtained in step (BI-a) to precipitate a solid, separate the solid and liquid, collect the solid phase, and obtain the L-tartrate crystal form I of compound (I).

[0096] In some embodiments, the reaction temperature in step (BI-a) is from 10°C to 60°C.

[0097] In some embodiments, the molar ratio of the compound of formula (I) in step (BI-a) to the L-tartaric acid contained in the L-tartaric acid aqueous solution is 1:(0.8-1.4). In other embodiments, the molar ratio of the compound of formula (I) in step (BI-a) to the L-tartaric acid contained in the L-tartaric acid aqueous solution is 1:(0.9-1.2).

[0098] In some embodiments, the molar concentration of the L-tartaric acid aqueous solution in step (BI-a) is 0.1-5 mol / L. In other embodiments, the molar concentration of the L-tartaric acid aqueous solution in step (BI-a) is 0.5-3 mol / L. In still other embodiments, the molar concentration of the L-tartaric acid aqueous solution in step (BI-a) is 1-2 mol / L.

[0099] In some embodiments, the solvent in step (BI-a) is ethanol.

[0100] In some embodiments, after stirring at 30-60°C in step (BI-a), the mixture is then stirred at room temperature. In some embodiments, after stirring at 30-60°C for 2-5 hours in step (BI-a), the mixture is then stirred at room temperature. In some embodiments, after stirring at 30-60°C for 2-5 hours in step (BI-a), the mixture is then stirred at room temperature for 7-16 hours.

[0101] In some embodiments, in step (BI-b), the reaction solution is cooled to -10°C to 10°C. In some embodiments, the reaction solution is cooled to -5°C to 5°C. In some embodiments, the reaction solution is cooled to approximately 0°C.

[0102] In some embodiments, the reaction solution is cooled for 0.25-3 hours in step (BI-b). In some embodiments, the reaction solution is cooled for 0.5-2 hours. In some embodiments, the reaction solution is cooled for 0.5-1 hour.

[0103] In some embodiments, the separation method in step (BI-b) is selected from centrifugal separation and filtration separation.

[0104] In some embodiments, after separation in step (BI-b), the method further includes a step of evaporating the solvent or drying.

[0105] In some embodiments, the drying step following separation in step (BI-b) is performed at 25-70°C.

[0106] In some embodiments, the preparation method of L-tartrate crystal form II includes the following steps:

[0107] (BII-a) Dissolve the compound of formula (I) in a solvent, add an aqueous solution of L-tartaric acid, and stir to react;

[0108] (BII-b) Cool the reaction solution obtained in step (BII-a), add an antisolvent to crystallize, separate the solid and liquid phases, collect the solid phase, and obtain the L-tartrate crystal form II of compound (I).

[0109] In some embodiments, the reaction temperature in step (BII-a) is 10-60°C.

[0110] In some embodiments, the molar ratio of the compound of formula (I) to the L-tartaric acid contained in the L-tartaric acid aqueous solution in step (BII-a) is 1:(0.8-1.4). In other embodiments, the molar ratio of the compound of formula (I) to the L-tartaric acid contained in the L-tartaric acid aqueous solution is 1:(0.9-1.2).

[0111] In some embodiments, the molar concentration of the L-tartaric acid aqueous solution in step (BII-a) is 0.1-5 mol / L. In some embodiments, the molar concentration of the L-tartaric acid aqueous solution is 0.5-3 mol / L. In other embodiments, the molar concentration of the L-tartaric acid aqueous solution is 1-2 mol / L.

[0112] In some embodiments, the solvent in step (BII-a) is methanol.

[0113] In some embodiments, after stirring at 30-60°C in step (BII-a), the mixture is then stirred at room temperature. In some embodiments, after stirring at 30-60°C for 2-5 hours in step (BII-a), the mixture is then stirred at room temperature. In some embodiments, after stirring at 30-60°C for 2-5 hours in step (BII-a), the mixture is then stirred at room temperature for 7-16 hours.

[0114] In some embodiments, in step (BII-b), the reaction solution is cooled to -10°C to 10°C. In some embodiments, the reaction solution is cooled to -5°C to 5°C. In some embodiments, the reaction solution is cooled to approximately 0°C.

[0115] In some embodiments, the reaction solution is cooled for 0.25-3 hours in step (BII-b). In some embodiments, the reaction solution is cooled for 0.5-2 hours. In some embodiments, the reaction solution is cooled for 0.5-1 hour.

[0116] In some embodiments, the antisolvent in step (BII-b) is selected from one or more of methyl tert-butyl ether, n-heptane, n-hexane, cyclohexane, acetone, acetonitrile, and ethyl acetate, and the solvent and the antisolvent are different. In some embodiments, the antisolvent is selected from one or more of methyl tert-butyl ether, acetone, acetonitrile, and ethyl acetate. In some embodiments, the antisolvent is methyl tert-butyl ether.

[0117] In some embodiments, the separation method in step (BII-b) is selected from centrifugal separation and filtration separation.

[0118] In some embodiments, after separation in step (BII-b), a step of evaporating the solvent or drying is further included.

[0119] In some embodiments, the drying step following separation in step (BII-b) is performed at 25-70°C.

[0120] In some embodiments, the preparation method of L-tartrate amorphous products includes the following steps:

[0121] (Ca) Dissolve the compound of formula (I) in a solvent, add an aqueous solution of L-tartaric acid, and stir to react;

[0122] (Cb) Cool the reaction solution obtained in step (Ca) to precipitate a solid, separate the solid and liquid phases, collect the solid phase, and obtain the L-tartrate amorphous product.

[0123] In some embodiments, the reaction temperature in step (Ca) is 10-60°C.

[0124] In some embodiments, the molar ratio of the compound of formula (I) in step (Ca) to the L-tartaric acid contained in the L-tartaric acid aqueous solution is 1:(0.8-1.4). In some embodiments, the molar ratio of the compound of formula (I) in step (Ca) to the L-tartaric acid contained in the L-tartaric acid aqueous solution is 1:(0.9-1.2).

[0125] In some embodiments, the molar concentration of the L-tartaric acid aqueous solution in step (Ca) is 0.1-5 mol / L. In some embodiments, the molar concentration of the L-tartaric acid aqueous solution is 0.5-3 mol / L. In some embodiments, the molar concentration of the L-tartaric acid aqueous solution is 1-2 mol / L.

[0126] In some embodiments, the solvent in step (Ca) is acetone.

[0127] In some embodiments, after stirring at 30-60°C in step (Ca), the mixture is then stirred at room temperature. In some embodiments, after stirring at 30-60°C for 2-5 hours in step (Ca), the mixture is then stirred at room temperature. In some embodiments, after stirring at 30-60°C for 2-5 hours in step (Ca), the mixture is then stirred at room temperature for 7-16 hours.

[0128] In some embodiments, the reaction solution is cooled to -10°C to 10°C in step (Cb). In some embodiments, the reaction solution is cooled to -5°C to 5°C. In some embodiments, the reaction solution is cooled to approximately 0°C.

[0129] In some embodiments, the reaction solution is cooled for 0.25-3 hours in step (Cb). In some embodiments, the reaction solution is cooled for 0.5-2 hours. In some embodiments, the reaction solution is cooled for 0.5-1 hour.

[0130] In some embodiments, the separation method in step (Cb) is selected from centrifugal separation and filtration separation.

[0131] In some embodiments, after separation in step (Cb), there is also a step of evaporating the solvent or drying.

[0132] In some embodiments, the drying step following separation in step (Cb) is carried out at 25-70°C.

[0133] A third aspect of this disclosure provides a method for preparing the phosphate of the compound of formula (I).

[0134]

[0135] The method for preparing the phosphate of the compound of formula (I) includes the following steps: reacting the compound of formula (I) with phosphoric acid in the presence of an organic solvent to form a salt, thereby forming the phosphate of the compound of formula (I).

[0136] In some embodiments, the phosphate is a second type of phosphate of the compound of formula (I). The molar ratio of the compound of formula (I) to phosphoric acid in the steps is 1:(0.8-1.2). For example, the molar ratio of the compound of formula (I) to phosphoric acid can be 1:0.8, 1:0.9, 1:1.0, 1:1.1, or 1:1.2.

[0137] In some embodiments, the molar ratio of compound (I) to phosphoric acid is 1:(0.9-1.1). In some embodiments, the molar ratio of compound (I) to phosphoric acid is 1:1. In some embodiments, the molar ratio of compound (I) to phosphoric acid is 1:0.95.

[0138] In some embodiments, the molar ratio of the phosphate compound of formula (I) to phosphoric acid is 1:(0.8-1.2). In some embodiments, the molar ratio of the phosphate compound of formula (I) to phosphoric acid is 1:(0.9-1.1). In some embodiments, the molar ratio of the phosphate compound of formula (I) to phosphoric acid can be 1:0.8, 1:0.9, 1:1, 1:1.1, or 1:1.2. In some embodiments, the molar ratio of the phosphate compound of formula (I) to phosphoric acid is 1:1.

[0139] In some embodiments, the organic solvent is selected from one or more of ethanol, ethyl acetate, acetonitrile, and acetone. In some embodiments, the organic solvent is ethanol, acetone, or a mixture thereof. In some embodiments, the organic solvent is ethanol.

[0140] In some embodiments, the organic solvent is one or more selected from methanol, ethyl acetate, and acetone. In some embodiments, the organic solvent is one or more selected from methanol and ethyl acetate. In some embodiments, the organic solvent is a mixture of methanol and ethyl acetate.

[0141] In some embodiments, the molar ratio of compound (I) to phosphoric acid in the phosphate of the formed compound (I) is 1:1.

[0142] In some embodiments, a method for preparing phosphate crystal form II is also provided, comprising the following steps:

[0143] (i) reacting the compound of formula (I) with phosphoric acid in ethanol, methanol, and / or acetone to form a salt, precipitating a solid; and

[0144] (ii) Collect the solid to obtain phosphate crystal form II;

[0145] In this embodiment, the molar ratio of compound (I) to phosphoric acid is 1:(0.8-1.2), for example, it can be 1:0.8, 1:0.9, 1:1.0, 1:1.1, or 1:1.2. In some embodiments, the molar ratio of compound (I) to phosphoric acid is 1:(0.9-1.1). In one embodiment, the molar ratio of compound (I) to phosphoric acid is 1:0.95. In another embodiment, the molar ratio of compound (I) to phosphoric acid is 1:1.

[0146] In some embodiments, the salt-forming reaction is carried out in one or both of ethanol and acetone.

[0147] In some embodiments, the salt-forming reaction is carried out in ethanol. In some embodiments, the salt-forming reaction is carried out in methanol.

[0148] In some embodiments, the method for preparing phosphate crystal form II includes the following steps:

[0149] (i-1) A salt-forming reaction is carried out between an ethanol solution of the compound of formula (I) and an ethanol solution of phosphoric acid, resulting in the precipitation of a solid; and

[0150] (ii-1) Collect the solid to obtain phosphate crystal form II;

[0151] In this embodiment, the molar ratio of compound (I) to phosphoric acid is 1:(0.8-1.2), for example, it can be 1:0.8, 1:0.9, 1:1.0, 1:1.1 or 1:1.2. In some embodiments, the molar ratio of compound (I) to phosphoric acid is 1:(0.9-1.1). In one embodiment, the molar ratio of compound (I) to phosphoric acid is 1:1.

[0152] In some embodiments, the method for preparing phosphate crystal form II includes the following steps:

[0153] (i-1) A salt-forming reaction is carried out between an ethanol solution of the compound of formula (I) and an ethanol solution of phosphoric acid at reflux temperature, followed by cooling to -10°C to 60°C, resulting in the precipitation of a solid; and

[0154] (ii-1) Collect the solid to obtain phosphate crystal form II;

[0155] In this embodiment, the molar ratio of compound (I) to phosphoric acid is 1:(0.8-1.2), for example, it can be 1:0.8, 1:0.9, 1:1.0, 1:1.1 or 1:1.2. In some embodiments, the molar ratio of compound (I) to phosphoric acid is 1:(0.9-1.1). In one embodiment, the molar ratio of compound (I) to phosphoric acid is 1:1.

[0156] In some embodiments, the molar ratio of the phosphate crystal form II compound of formula (I) to phosphoric acid is 1:1.

[0157] In some embodiments, the temperature of the salt formation reaction is from -10°C to 90°C, for example, it can be any range (inclusive) consisting of the endpoints selected from -10°C, -5°C, 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C and 90°C, without any specific limitation.

[0158] In some embodiments, the collection of solids in step (ii) or (ii-1) may be carried out by centrifugation and filtration.

[0159] In some embodiments, after collecting the solids in step (ii) or (ii-1), the process further includes evaporating the solvent or drying the solids.

[0160] In some embodiments, the drying step is performed at 25°C to 70°C.

[0161] In some embodiments, the phosphate is a first-type phosphate of the compound of formula (I). The molar ratio of the compound of formula (I) to phosphoric acid in the step is 1:(1.8-2.6), for example, it can be 1:1.8, 1:1.9, 1:2.0, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5 or 1:2.6, etc.; preferably 1:(1.9-2.3), more preferably 1:(2.0-2.2).

[0162] In some embodiments, the molar ratio of phosphoric acid to the compound of formula (I) in the phosphate is (1.8-2.4):1. In some embodiments, the molar ratio of phosphoric acid to the compound of formula (I) can be 1.8:1, 1.9:1, 2.0:1, 2.1:1, 2.2:1, 2.3:1, or 2.4:1. In some embodiments, the molar ratio of phosphoric acid to the compound of formula (I) is (1.9-2.3):1. In one embodiment, the molar ratio of phosphoric acid to the compound of formula (I) is 2:1.

[0163] In some embodiments, the organic solvent is selected from one or more of ethanol, ethyl acetate, acetone, and methanol.

[0164] In some embodiments, the molar ratio of phosphoric acid to compound (I) in the phosphate of the formed compound (I) is 2:1.

[0165] In one embodiment, a method for preparing phosphate crystal form I of compound (I) includes:

[0166] (AI-a) Dissolve the compound of formula (I) in an organic solvent, add an aqueous solution of phosphoric acid, and stir to react;

[0167] (AI-b) Cool the reaction solution obtained in step (AI-a), add an antisolvent to induce crystallization, separate the solid and liquid phases, and collect the solid phase to obtain phosphate crystal form I of compound (I).

[0168] In one embodiment, the reaction temperature in step (AI-a) is 10-60°C.

[0169] In one embodiment, the molar ratio of the compound of formula (I) to the phosphoric acid contained in the aqueous phosphoric acid solution is 1:(1.8-2.6), for example, it can be 1:1.8, 1:1.9, 1:2.0, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5 or 1:2.6, etc.; preferably 1:(1.9-2.3), more preferably 1:(2.0-2.2).

[0170] In one embodiment, the molar concentration of the phosphoric acid aqueous solution is 0.1-5 mol / L, preferably 0.5-3 mol / L, and more preferably 1-2 mol / L.

[0171] In some embodiments, in step (AI-b), the reaction solution is cooled to -10°C to -10°C, preferably -5°C to -5°C; in some embodiments, the reaction solution is cooled to approximately 0°C.

[0172] In some embodiments, the reaction solution is cooled for 0.5-2 hours in step (AI-b); in some embodiments, the reaction solution is cooled for 0.5 hours.

[0173] In one embodiment, the antisolvent is selected from one or more of methyl tert-butyl ether, petroleum ether, n-heptane, n-hexane, cyclohexane, isopropanol, acetone, acetonitrile, and ethyl acetate, and the antisolvent is different from the solvent; preferably, the antisolvent is selected from one or more of methyl tert-butyl ether, n-heptane, isopropanol, and acetone.

[0174] In one embodiment, the separation method in step (AI-b) is selected from centrifugal separation and filtration separation.

[0175] In one embodiment, step (AI-b) further includes a step of evaporating the solvent or drying after separation.

[0176] In one embodiment, the drying step after separation in step (AI-b) is carried out at 25-70°C; for example, it can be 25°C, 30°C, 40°C, 50°C, 60°C or 70°C, etc., and there is no specific limitation.

[0177] In one embodiment, the reaction temperature in step (AI-a) is 10-60°C; and / or the molar ratio of the compound of formula (I) to the phosphoric acid in the aqueous phosphoric acid solution is 1:(1.8-2.4), preferably 1:(1.9-2.3), more preferably 1:(2.0-2.2); and / or the molar concentration of the aqueous phosphoric acid solution is 0.1-5 mol / L, preferably 0.5-3 mol / L, more preferably 1-2 mol / L; and / or the reaction solution in step (AI-b) is cooled to -10°C to -10°C, preferably -5°C to -5°C, more preferably about 0°C; and / or the reaction solution in step (AI-b) is cooled... The time is 0.5-2 hours, more preferably 0.5 hours; and / or the antisolvent is selected from one or more of methyl tert-butyl ether, petroleum ether, n-heptane, n-hexane, cyclohexane, isopropanol, acetone, acetonitrile, and ethyl acetate, and the antisolvent is different from the solvent; preferably, the antisolvent is selected from one or more of methyl tert-butyl ether, n-heptane, isopropanol, and acetone; and / or the separation method in step (AI-b) is selected from centrifugal separation and filtration separation; and / or the separation in step (AI-b) further includes a step of evaporating the solvent or drying; and / or the drying step in step (AI-b) is carried out at 25-70°C.

[0178] A fourth aspect of this disclosure provides a pharmaceutical composition comprising:

[0179] (a) the L-tartrate or phosphate of the compound of formula (I) as described in the first aspect of this disclosure; and (b) a pharmaceutically acceptable carrier.

[0180] In some embodiments, the pharmaceutical composition comprises (a) an L-tartrate of a compound of formula (I) as described in the first aspect of this disclosure, a first-type phosphate of a compound of formula (I) or a second-type phosphate of a compound of formula (I); and (b) a pharmaceutically acceptable carrier.

[0181] In some embodiments, the pharmaceutical composition comprises (a) an L-tartrate crystal form I, an L-tartrate crystal form II, an L-tartrate amorphous form of the compound of formula (I), a phosphate crystal form I, or a phosphate crystal form II of the compound of formula (I) as described in the first aspect of this disclosure; and (b) a pharmaceutically acceptable carrier.

[0182] A fifth aspect of this disclosure provides the use of a pharmaceutically acceptable salt of the compound of formula (I) described in the first aspect of this disclosure, or the pharmaceutical composition described in the fourth aspect of this disclosure, in the preparation of a kinase inhibitor. Specifically, the pharmaceutically acceptable salt of the compound of formula (I) includes an L-tartrate of the compound of formula (I), a first-type phosphate of the compound of formula (I), and a second-type phosphate of the compound of formula (I). In some embodiments, the pharmaceutically acceptable salt of the compound of formula (I) includes L-tartrate crystal form I, L-tartrate crystal form II, an amorphous form of L-tartrate of the compound of formula (I), a phosphate crystal form I of the compound of formula (I), and a phosphate crystal form II of the compound of formula (I). In some embodiments, the kinase inhibitor is a CDK9 inhibitor.

[0183] A sixth aspect of this disclosure provides the use of a pharmaceutically acceptable salt of a compound of formula (I) as described in the first aspect of this disclosure, or a pharmaceutical composition as described in the fourth aspect of this disclosure, in the preparation of a medicament for treating and / or preventing diseases associated with or mediated by CDK9 activity. Specifically, the pharmaceutically acceptable salt of the compound of formula (I) includes an L-tartrate of the compound of formula (I), a first-type phosphate of the compound of formula (I), and a second-type phosphate of the compound of formula (I). In some embodiments, the pharmaceutically acceptable salt of the compound of formula (I) includes L-tartrate crystal form I, L-tartrate crystal form II, an amorphous form of L-tartrate of the compound of formula (I), a phosphate crystal form I of the compound of formula (I), and a phosphate crystal form II of the compound of formula (I).

[0184] This disclosure also provides pharmaceutically acceptable salts of compounds of formula (I) described in the first aspect of this disclosure, or pharmaceutical compositions described in the fourth aspect of this disclosure, for the treatment and / or prevention of diseases associated with or mediated by CDK9 activity.

[0185] This disclosure also provides a method for inhibiting CDK9 activity, comprising administering a therapeutically effective amount of a pharmaceutically acceptable salt of a compound of formula (I) as described in the first aspect of this disclosure, or a pharmaceutical composition as described in the fourth aspect of this disclosure, to a desired subject.

[0186] This disclosure also provides a method for treating diseases associated with or mediated by CDK9 activity, the method comprising administering to a desired subject an effective amount of a pharmaceutically acceptable salt of a compound of formula (I) as described in the first aspect of this disclosure, or a pharmaceutical composition as described in the fourth aspect of this disclosure.

[0187] In some embodiments, the diseases include proliferative diseases, virus-induced infectious diseases, and cardiovascular diseases.

[0188] In some embodiments, the disease is a hyperproliferative disease. In some embodiments, the hyperproliferative disease includes angiogenesis or angioproliferative disorders, mesangial cell proliferative disorders, and solid tumors. Solid tumors include, 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. In some embodiments, the disease is selected from one or more of lymphoma, sarcoma, and leukemia. In some embodiments, the disease is cancer. Cancers include, for example, pancreatic cancer, breast cancer, ovarian cancer, cervical cancer, or leukemia. In some embodiments, the disease includes solid tumors and hematologic malignancies.

[0189] In this disclosure, diseases associated with or mediated by CDK9 activity include diseases related to or involving CDK9 activity (e.g., excessive CDK9 activity), as well as conditions accompanying these diseases. Excessive CDK9 activity refers to increased CDK9 enzyme activity compared to normal, non-disease cells, or increased CDK9 activity leading to unwanted cell proliferation, or reduced or insufficient programmed cell death (apoptosis), or mutations leading to constitutive activation of CDK9.

[0190] Excessive proliferative disorders include unintended or uncontrolled proliferative disorders involving cells, and include disorders involving reduced or insufficient programmed cell death (apoptosis). Pharmaceutically acceptable salts of compounds of formula (I) of this disclosure, or pharmaceutical compositions containing pharmaceutically acceptable salts of compounds of formula (I), can be used to prevent, inhibit, block, reduce, decrease, control, etc., cell proliferation and / or cell division, and / or induce apoptosis. Treatment and / or prevention of diseases associated with or mediated by CDK9 activity includes administering an effective treatment or prevention of said disease to a subject of need (including mammals, such as humans).

[0191] In this disclosure, the phosphate and L-tartrate salts of compound (I) exhibit higher solubility than the free base, and the L-tartrate crystal forms I, II, and amorphous forms of L-tartrate, as well as the phosphate crystal forms I and II of compound (I), all possess good physical stability, making them suitable for drug development. In particular, phosphate crystal form I of compound (I) can be converted into the more stable phosphate crystal form II under suitable conditions, such as in ethanol. Compared to phosphate crystal form I of compound (I), phosphate crystal form II of compound (I) exhibits lower hygroscopicity and better crystallinity and stability. Attached Figure Description

[0192] To more clearly illustrate the technical solutions in the embodiments or related technologies of this disclosure, the accompanying drawings used in the description of the embodiments or related technologies will be briefly introduced below. Obviously, the accompanying drawings described below are only embodiments of this application. For those skilled in the art, other drawings can be obtained based on the disclosed drawings without creative effort.

[0193] Figure 1 The X-ray powder diffraction (XRPD) pattern of phosphate crystal form I of compound (I) is shown.

[0194] Figure 2 The differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) spectra of phosphate crystal form I of compound (I).

[0195] Figure 3 The dynamic moisture absorption curve (DVS) is shown for phosphate crystal form I of compound (I).

[0196] Figure 4 This is a microscope image of phosphate crystal form I of compound (I).

[0197] Figure 5 The X-ray powder diffraction (XRPD) pattern of L-tartrate crystal form I of compound (I) is shown.

[0198] Figure 6 Thermogravimetric analysis (TGA) spectrum of L-tartrate crystal form I of compound (I).

[0199] Figure 7 The X-ray powder diffraction (XRPD) pattern of L-tartrate crystal form II of compound (I) is shown.

[0200] Figure 8 The X-ray powder diffraction (XRPD) pattern of the amorphous L-tartrate of compound (I) is shown.

[0201] Figure 9 The diagram shows the molecular stereoscopic structure ellipsoid of a single crystal of compound (II).

[0202] Figure 10 The X-ray powder diffraction (XRPD) pattern of phosphate crystal form II of compound (I) is shown.

[0203] Figure 11 The differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) spectra of phosphate crystal form II of compound (I).

[0204] Figure 12 The dynamic moisture absorption curve (DVS) is shown for phosphate crystal form II of compound (I).

[0205] Figure 13 This is a microscope image of phosphate crystal form II of compound (I).

[0206] Figure 14 Comparative X-ray powder diffraction (XRPD) patterns of phosphate crystal form I of compound (I) transformed into phosphate crystal form II of compound (I) in ethanol.

[0207] Figure 15 XRPD changes of compound (I) L-tartrate crystal form I under high temperature (60℃) and accelerated (40℃-75%RH) conditions.

[0208] Figure 16 The graph shows the XRPD changes of phosphate crystal form I of compound (I) under high temperature conditions.

[0209] Figure 17 The graph shows the changes in tumor volume after once-daily oral administration of compound D and compound (I).

[0210] Figure 18 The graph shows the changes in body weight in mice after once-daily oral administration of compound D and compound (I). Detailed Implementation

[0211] To make the above-described objects, features, and advantages of this disclosure more apparent and understandable, a detailed description of specific embodiments of this disclosure is provided below. Numerous specific details are set forth in the following description to provide a thorough understanding of this disclosure. However, this disclosure can be implemented in many other ways than those described herein, and those skilled in the art can make similar modifications without departing from the spirit of this disclosure. Therefore, this disclosure is not limited to the specific embodiments disclosed below.

[0212] The terms "crystal of this disclosure", "crystal form of this disclosure", and "polymorph of this disclosure" are used interchangeably in this disclosure.

[0213] In this disclosure, 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:

[0214]

[0215] This disclosure also includes phosphates and L-tartrates of compounds of formula (I).

[0216] Polymorphs: Solids exist either in an amorphous form or a crystalline form. In the crystalline form, molecules are located within three-dimensional lattice sites. When a compound crystallizes from a solution or slurry, it can crystallize using different spatial lattice arrangements (this property is called "polymorphism"), forming crystals with different crystalline forms, which are called "polymorphs." Different polymorphs of a given substance can differ from each other in one or more physical properties (such as solubility and dissolution rate, true specific gravity, crystal form, packing order, fluidity, and / or solid-state stability).

[0217] Crystallization: Production-scale crystallization can be achieved by manipulating the solution to exceed the solubility limit of the compound of interest. This can be accomplished by various methods, such as dissolving the compound at a relatively high temperature and then cooling the solution below the saturation limit. Alternatively, it can be done by boiling, evaporation at atmospheric pressure, vacuum drying, or other methods to reduce the liquid volume. The solubility of the compound of interest can be reduced by adding an antisolvent or a solvent in which the compound has low solubility, or a mixture of such solvents. Another alternative method is to adjust the pH value to reduce solubility. For a detailed description of crystallization, see Crystallization, 3rd Edition, JW Mullens, Butterworth-Heineman Ltd., 1993, ISBN 0750611294.

[0218] The "suspension shaking" method described in this disclosure refers to a method of obtaining crystals by mixing a compound of formula (I) with a corresponding acid or a solution of the corresponding acid in a suitable solvent to form a turbid liquid and then shaking it. A suitable solvent may be water or an organic solvent.

[0219] The "suspension centrifugation" described in this disclosure refers to a method of obtaining crystals by centrifuging a compound of formula (I) and a corresponding acid or a solution of the corresponding acid in a suitable solvent to form a turbid liquid. A suitable solvent may be water or an organic solvent.

[0220] The “slow evaporation” described in this disclosure refers to a method of slowly evaporating the solvent from a solution containing a compound of formula (I) and the corresponding acid at a certain temperature to obtain crystals.

[0221] The “antisolvent addition” or “addition of antisolvent” described in this disclosure is a method of precipitating crystals by adding another suitable solvent to a solution of a compound of formula (I).

[0222] If it is desired that salt formation and crystallization occur simultaneously, and if the salt is less soluble than the reactants in the reaction medium, then adding an appropriate acid or base can lead to the direct crystallization of the desired salt. Similarly, in a medium where the final desired form is less soluble than the reactants, the completion of the synthesis reaction can allow the final product to crystallize directly.

[0223] Optimization of crystallization may include seeding the crystallizer with the desired crystal form as a seed crystal in the crystallization medium. Additionally, many crystallization methods utilize combinations of the strategies described above. One example involves dissolving the compound of interest in a solvent at high temperature, followed by the controlled addition of an appropriate volume of antisolvent to bring the system just below saturation. At this point, seed crystals of the desired form are added (while maintaining the integrity of the seed crystals), and the system is cooled to complete crystallization. As used herein, the term "about" means ±5 from a given value.

[0224] In this article, when referring to units of data ranges, if the unit is only followed by the right endpoint, it indicates 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).

[0225] In this document, "preferred" and "better" are merely descriptions of implementation methods or embodiments with better effects, and should be understood as not constituting a limitation on the scope of protection of this disclosure. If multiple "preferred" terms appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, each "preferred" term shall be independent.

[0226] As used herein, the terms "and / or," "or / and," and "and / or" encompass any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected using 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 solutions connected by "logical AND," and also undoubtedly includes solutions connected by "logical OR."

[0227] In this article, the terms "multiple," "various types," and "multiple times" are used unless otherwise specified, referring to a quantity greater than or equal to 2. For example, "multiple types" means two or more.

[0228] In this article, the technical features described in an open-ended manner include both closed technical solutions composed of the listed features and open technical solutions that include the listed features.

[0229] As used herein, the term "polymorphs of the present disclosure" includes, but is not limited to, phosphate form I of compound (I), phosphate form II of compound (I), L-tartrate form I of compound (I), and L-tartrate form II of compound (I).

[0230] This disclosure also includes phosphates of the compound of formula (I), particularly monophosphates, and crystal form II of the phosphate of the compound of formula (I).

[0231] "Compound of Formula (I)," "Free base of Compound of Formula (I)," and "Free base" are used interchangeably.

[0232] "Polymorphs of compounds of formula (I)" and "Polymorphs of free bases of compounds of formula (I)" can be used interchangeably.

[0233] In this disclosure, certain crystal forms can be interconverted, therefore this disclosure also provides methods for interconverting some crystal forms.

[0234] Pharmaceutical Compositions and Their Applications

[0235] Typically, a pharmaceutically acceptable salt of the compound of this disclosure (I) can be used as an active ingredient to form a suitable dosage form for administration with one or more pharmaceutical carriers.

[0236] Specifically, the phosphate, L-tartrate, or polymorph thereof of the compound of formula (I) can be used as an active ingredient to form a suitable dosage form with one or more pharmaceutical carriers for administration.

[0237] "Pharmaceutically acceptable carrier" means a non-toxic, inert, solid, or semi-solid substance or liquid filling machine, diluent, encapsulation material, or excipient or any type of excipient that is compatible with the subject to which it is applied (in some embodiments, mammals, and in one embodiment, humans) and is suitable for delivering the active substance of this disclosure to the target site without terminating its activity.

[0238] The pharmaceutical compositions disclosed herein are formulated, quantified, and administered in accordance with medical practice guidelines. The "therapeutic 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 route of administration.

[0239] This disclosure provides pharmaceutically acceptable salts of compounds of formula (I) as described in the first aspect of this disclosure, and pharmaceutical compositions as described in the fourth aspect of this disclosure that can be used as medicines for treating and / or preventing diseases associated with or mediated by CDK9 activity.

[0240] This disclosure provides a method for inhibiting CDK9 activity, comprising administering to a subject a therapeutically effective amount of a pharmaceutically acceptable salt of a compound of formula (I) as described in the first aspect of this disclosure, or a pharmaceutical composition as described in the fourth aspect of this disclosure.

[0241] As used herein, "therapeutic effective amount" means the amount of a pharmaceutically acceptable salt of the compound of this disclosure (I) that will elicit a biological or medical response in an individual, such as reducing or inhibiting enzyme or protein activity or improving symptoms, alleviating symptoms, relieving or delaying disease progression, or preventing disease.

[0242] As used in this article, “object” refers to an animal, preferably a mammal, and more preferably a human. The term “mammal” refers to warm-blooded vertebrate mammals, including animals such as cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, rats, pigs, and humans.

[0243] "Treatment" refers to reducing, slowing the progression of, attenuating, preventing, or maintaining an existing disease or condition (such as cancer). Treatment also includes curing, preventing the development of, or reducing to some extent one or more symptoms of a disease or condition.

[0244] Example

[0245] The present disclosure is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the disclosure. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise defined. Unless otherwise defined, the terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this disclosure.

[0246] Reagents and Instruments

[0247] In this disclosure, the structure and purity of the compounds are determined by nuclear magnetic resonance (NMR). 1 It can be determined by 1H NMR and / or liquid chromatography-mass spectrometry (LC-MS).

[0248] 1 1H NMR: Bruker AVANCE-400 NMR spectrometer, internal standard: tetramethylsilane (TMS).

[0249] LC-MS: Agilent 1290 HPLC System / 6130 / 6150 MS liquid chromatography-mass spectrometry (manufacturer: Agilent), Waters BEH / CHS column, 50×2.1mm, 1.7μm.

[0250] HPLC analysis was performed using an Agilent 1260 Infinity HPLC system, OpenLAB CDS Chemstationworkstation, XBridge C18 4.6*250mm column, ID 5μm column, and DAD detector.

[0251] Elemental analysis was performed using an inductively coupled plasma atomic emission spectrometer, model ICP 500; power 1300W; flow rate 1mL / min.

[0252] The known starting materials can be synthesized using or according to methods known in the art, or can be purchased from companies such as ABCRGmbH & Co.KG, Acros Organics, Aldrich Chemical Company, AccelaChemBio Inc., and Darui Chemicals.

[0253] As used herein, room temperature in the following embodiments refers to approximately 20-30°C.

[0254] General Method

[0255] X-ray powder diffraction (XRPD): In this disclosure, the above-mentioned crystalline or amorphous powder X-ray diffraction patterns are obtained using an ARL Equinox 3000 X-ray powder diffractometer by methods known in the art. The XRPD test parameters are shown in Table 5 below.

[0256] Table 5

[0257]

[0258] In powder X-ray diffraction patterns, the positions of each peak are determined by the 2θ (°) value. It is understandable that different instruments and / or conditions can result in slightly different data, with variations in the positions and relative intensities of the peaks.

[0259] The intensity division of peaks merely reflects the approximate size of peaks at each position. In this disclosure, each crystal form uses its highest diffraction peak as the base peak, and its relative intensity is defined as 100%, as I0 (the peak with a 2θ (°) value of 18.803 for phosphate crystal form I is the base peak, the peak with a 2θ (°) value of 20.27 for L-tartrate crystal form I is the base peak, the peak with a 2θ (°) value of 21.353 for L-tartrate crystal form II is the base peak, and the peak with a 2θ (°) value of 18.230 for phosphate crystal form II is the base peak). The ratio of the peak height of each other peak to the peak height of the base peak is used as its relative intensity I / I0. The division of the relative intensity of each peak is defined as shown in Table 6 below.

[0260] Table 6

[0261]

[0262]

[0263] Single-crystal X-ray diffraction (SXRD): In this disclosure, the single-crystal X-ray diffraction pattern of the compound of formula (II) was obtained using a D8 Venture diffractometer by methods known in the art. The SXRD test parameters are shown in Table 7 below. After collecting the relevant data, the crystal structure was further analyzed using the direct method (SHELXT2014) to confirm the absolute configuration.

[0264] Table 7

[0265]

[0266] The phosphates and crystal forms of the compounds of formula (I) disclosed herein were determined by elemental analysis to determine the acid-base molar ratio. The L-tartrate salts and crystal forms of the compounds of formula (I) were determined by HPLC / IC or 1 H NMR was used to determine the acid-base molar ratio.

[0267] High performance liquid chromatography: In this disclosure, high performance liquid chromatography (HPLC) is performed on an Agilent 1260 HPLC.

[0268] Differential scanning calorimetry (DSC): In this disclosure, the differential scanning calorimetry spectra of the above-mentioned crystal form were obtained using a DSC25A differential scanning calorimeter by a method known in the art. The DSC test parameters are shown in Table 8 below.

[0269] Table 8

[0270]

[0271] Thermogravimetric analysis (TGA): In this disclosure, the thermogravimetric analysis spectra of the above crystal forms were obtained using a TGA550 thermogravimetric analyzer by methods known in the art. The TGA test parameters are shown in Table 9 below.

[0272] Table 9

[0273]

[0274] Dynamic moisture adsorption (DVS) curves: acquired on the DVSIntrinsic instrument of SMS (Surface Measurement Systems). Relative humidity at 25°C was corrected for the deliquescence points of LiCl, Mg(NO3)2, and KCl. Instrument testing conditions are shown in Table 10 below.

[0275] Table 10

[0276]

[0277]

[0278] The Chinese Pharmacopoeia (2020) Article 9103, the Guidelines for Hygroscopicity Testing of Drugs, specifies the description of hygroscopic characteristics and the definition of hygroscopic weight gain: (1) Deliquescence: absorbs sufficient water to form a liquid; (2) Extremely hygroscopic: hygroscopic weight gain of not less than 15%; (3) Hygroscopic: hygroscopic weight gain of less than 15% but not less than 2%; (4) Slightly hygroscopic: hygroscopic weight gain of less than 2% but not less than 0.2%; (5) No or almost no hygroscopicity: hygroscopic weight gain of less than 0.2%.

[0279] It is understood that other values ​​may be obtained when using other types of instruments that perform the same function as the instruments described above or when using test conditions different from those used in this disclosure. Therefore, the values ​​cited should not be regarded as absolute values.

[0280] Due to instrument errors or differences in operators, those skilled in the art will understand that the parameters used to characterize the physical properties of crystals may have slight differences. Therefore, the above parameters are only used to assist in characterizing the polymorphs provided in this disclosure and should not be regarded as a limitation on the polymorphs of this disclosure.

[0281] Unless otherwise specified, "phosphoric acid solution" and "L-tartaric acid solution" as used in this disclosure refer to aqueous solutions of phosphoric acid and L-tartaric acid, respectively. "Phosphate" and "L-tartarate" as used in this disclosure refer to phosphates of compounds of formula (I) and L-tartarates of compounds of formula (I).

[0282] The solvents used in this disclosure are analytical grade solvents, such as analytical grade ethanol (water content ≤0.3%).

[0283] Compound D in this application and compound E It can be prepared by referring to existing publicly available patent literature.

[0284] As used in this disclosure, MTBE refers to methyl tert-butyl ether; DMSO refers to dimethyl sulfoxide; THF refers to tetrahydrofuran; EA refers to ethyl acetate; PE refers to petroleum ether; DCM refers to dichloromethane; MeOH refers to methanol; CDI refers to N,N-carbazyldiimidazole; MOPS refers to 3-(N-morpholine)propanesulfonic acid; Tween-20 refers to Tween 20; DTT refers to dithiothreitol; EDTA refers to ethylenediaminetetraacetic acid; CDK1 refers to cell cycle-dependent kinase 1; CDK2 refers to cell cycle-dependent kinase 2; CDK9 refers to cell cycle-dependent kinase 9; K2EDTA refers to dipotassium ethylenediaminetetraacetate; IPA refers to isopropanol; and NH3 refers to ammonia.

[0285] Preparation Example 1. Preparation of Compound 6

[0286]

[0287] Step 1: Sodium hydride (18 g, 450.00 mmol, 60% purity) was dissolved in THF (300 mL) and cooled to 0 °C. Triethyl-2-phosphonopropyl ester (100 g, 419.78 mmol) was slowly added dropwise. The reaction was stirred at 0–5 °C for 1 hour. Then, cyclobutanone (25 g, 356.69 mmol) was dissolved in THF (50 mL) and slowly added dropwise. After the addition was complete, the reaction was carried out at 25 °C for 15 hours. The reaction was quenched with saturated sodium chloride solution (600 mL), and then extracted with ethyl acetate (600 mL × 2). The combined organic phases were washed with saturated sodium chloride solution (500 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by CombiFlash (120 g × 2, 0–20% EA / PE) to give compound 2 (44 g, colorless oil), yield: 80.00%. 1 H NMR (400MHz, DMSO-d6) δ4.05(q,J=7.2Hz,2H),3.01-2.93(m,2H),2.78-2.71(m,2H),2.00-1.89(m,2H),1.61-1.58(m,3H),1.18(t,J=7.2Hz,3H).

[0288] Step 2: Dissolve compound 2 (44 g, 285.33 mmol) in methanol (400 mL), and add wet palladium on carbon (4.4 g, 10% purity). Cover with a hydrogen balloon, purge with hydrogen three times, and stir at 27°C for 6–8 hours under a hydrogen atmosphere (15 psi). TLC showed the starting material spot disappeared. Filter to remove Pd / C, and remove the solvent under reduced pressure to obtain crude compound 3 (44 g, colorless oil, boiling point approximately 182°C), yield: 98.71%. Proceed directly to the next step without purification.

[0289] Step 3: Compound 3 (44 g, 281.65 mmol) was dissolved in methanol (400 mL), sodium hydroxide (45.06 g, 1.13 mol) and water (200 mL) were added, and the mixture was stirred at room temperature for 16 hours. LC-MS showed that the reaction was complete. The methanol was removed by concentration under reduced pressure, followed by extraction twice with dichloromethane. The aqueous phase was then adjusted to pH 3 with dilute hydrochloric acid (6 M), and DCM was extracted (300 mL × 3). The organic phases were combined and dried to dryness to obtain compound 4 (35 g, colorless oil, boiling point approximately 220 °C), yield: 96.96%. No purification was required before proceeding to the next step. MS m / z (ESI): 127.1 [MH] - . 1H NMR (400MHz, DMSO-d6) δ11.91 (s, 1H), 2.36-2.20 (m, 2H), 1.99-1.90 (m, 2H), 1.82-1.58 (m, 4H), 0.93 (d, J = 6.8Hz, 3H).

[0290] Step 4: In a reaction flask, compound 4 (36 g, 280.88 mmol) was dissolved in 300 mL of THF, and then CDI (68.32 g, 421.32 mmol) was added. The mixture was then reacted at room temperature for 16 hours (as solution A). In another reaction flask, potassium monomethyl malonate (143.42 g, 842.64 mmol) was added to anhydrous magnesium chloride (66.86 g, 702.20 mmol) and 900 mL of THF. The mixture was heated at 50 °C under argon protection for 16 hours (as solution B). Then, solution A was added dropwise to solution B at room temperature (approximately 10 minutes), and the mixture was stirred at 30 °C for 16 hours. The reaction was confirmed by LCMS to be complete, and the product was produced. 800 mL of water was added to the reaction mixture, and the mixture was extracted with ethyl acetate (800 mL × 3). The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, and then evaporated to dryness under reduced pressure. The residue was purified by CombiFlash (120 g × 2, 0–15% EA / PE) to give product compound 5 (42 g, pale yellow oil, boiling point approximately 240 °C), yield: 75.42%. MS m / z (ESI): 199.1 [M+H] + . 1 H 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).

[0291] Step 5: Compound 5 (42 g, 211.85 mmol) and 3-aminopyrazole (19.36 g, 233.03 mmol) were dissolved in glacial acetic acid (300 mL), and the mixture was heated to 120 °C and reacted for 16 h. LC MS showed that the reaction was complete. Acetic acid was removed by concentration under reduced pressure, and the mixture was slurried with ethyl acetate (800 mL × 4). The solid precipitated, filtered, and dried to give compound 6 (41 g, pale yellow solid), yield: 89.08%. The mixture was not purified and proceeded directly to the next step. MS m / z (ESI): 218.1 [M+H] + .

[0292] Preparation Example 2. Preparation of Compound (I)

[0293]

[0294] Step 1: Compound 6 (473.6 mg, 2.18 mmol) was dissolved in phosphorus oxychloride (6 mL) and heated to 120 °C with stirring for 3 hours. After cooling to room temperature, the solution was poured into ice water (60 g) and extracted with dichloromethane (80 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was separated by silica gel column chromatography (20 g, 0%-40% EA / DCM) to give compound 7. 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).

[0295] Step 2: Compound 7 (142.7 mg, 605.5 μmol) and ((1S,3S)-3-aminocyclopentyl)carbamate tert-butyl ester (121.27 mg, 605.52 μmol) were dissolved in acetonitrile (20 mL), and then potassium carbonate (251.0 mg, 1.81 mmol) was added. The reaction was stirred at 90 °C for 16 hours. The mixture was diluted with ethyl acetate (80 mL), washed with saturated sodium chloride solution (80 mL × 3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 8. 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).

[0296] Step 3: Compound 8 (174.7 mg, 437.38 μmol) was dissolved in 1,4-dioxane (3 mL), and hydrochloric acid solution (3.0 mL, 4 M) was added. The mixture was stirred at room temperature for 3 hours. The solvent was removed under reduced pressure, water (60 mL) was added, and 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 × 2). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Compound 9 was obtained by preparative HPLC. 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).

[0297] Step 4: Compound 9 (94.46 mg, 315.48 μmol) was chirally resolved (column type: IC-3 4.6*100 mm3 μm; co-solvent: 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 obtain compound (I) (9.70 mg, retention time 2.471 min), yield: 9.89%, purity: 96.34%. 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).

[0298] Compound (II) was prepared from compound (I), and the absolute configuration of compound (I) was determined by the absolute configuration of compound (II).

[0299]

[0300] Compound (I) (200 mg, 667.97 μmol) was dissolved in dichloromethane (10 mL), followed by the addition of 4-chlorobenzoyl chloride (175.4 mg, 1.00 mmol), and then N,N-diisopropylethylamine (0.6 mL, 3.34 mmol). The reaction was stirred at room temperature for 2 hours, and LCMS analysis confirmed complete reaction of the starting material. Water (30 mL) was added, 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, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain compound (II) (160 mg, yield: 54.69%). LCMS (ESI) m / z: 438.2 [M + H] + .

[0301] Further preparation yielded single crystals of compound (II), and the molecular stereostructure ellipsoid diagram of compound (II) is shown below. Figure 9 As shown. The Flack constant is 0.07(2), C8, C10 and C19 are S configurations, that is, the absolute configurations of the three chiral centers of compound (II) are all S configurations, and their structures are as shown in the structure of compound (II). Thus, it can be determined that the absolute configurations of the three chiral centers of compound (I) are all S configurations, and their structures are as shown in the structure of compound (I).

[0302] Example 1. Preparation of phosphate crystal form I of compound (I)

[0303] Add 500 mg of free alkali and 1 mL of methanol to a 20 mL sample vial, sonicate to dissolve, then add 1 mol / L phosphoric acid solution at an acid-base molar ratio of 2.5:1. React at 50 °C for 1 h, continue stirring at 40 °C for 1 h, and then continue stirring at 30 °C for another 1 h. Finally, turn off the heating and stir overnight. After the reaction is complete, slowly cool to 0 °C, add acetone to precipitate a solid, centrifuge to collect the solid and evaporate the solvent. The obtained solid is the phosphate crystal form I of compound (I). Elemental analysis of the obtained solid showed a phosphorus content of 12.5%, indicating that the molar ratio of compound (I) to phosphoric acid in the obtained solid is 1:2 (its theoretical phosphorus content is 12.5%). The XRPD plot of the obtained solid is basically as follows: Figure 1 As shown, the obtained solid XRPD plot has a peak at the 2θ (°) value shown in Table 1 above, and the relative intensities of each peak are shown in Table 1 above; its DSC and TGA plots are as follows. Figure 2As shown, its DVS diagram is as follows: Figure 3 As shown, its microscope image is as follows Figure 4 As shown.

[0304] Depend on Figure 2 It can be seen that there is a melting absorption peak at 188.01℃ in the DSC image, indicating good crystallinity, which means that the melting point of phosphate crystal form I of compound (I) is about 188.01℃; in the TGA image, the sample loses 1.139% of its weight when heated to around 190℃, which may be due to solvent evaporation.

[0305] Depend on Figure 3 As can be seen from the DVS diagram, under 80% RH conditions, the weight gain due to moisture absorption is 9%, indicating that the phosphate crystal form I of compound (I) has hygroscopic properties.

[0306] Depend on Figure 4 As can be seen from the polarized light microscope images, the phosphate crystal form I of compound (I) is rod-shaped and block-shaped.

[0307] Example 2. Preparation of L-tartrate crystal form I of compound (I)

[0308] Add 500 mg of free alkali and 1 mL of ethanol to a 20 mL sample vial, sonicate to dissolve, then add 1 mol / L L-tartaric acid solution at an acid-base molar ratio of 1.2:1. React at 50 °C for 1 h, stir at 40 °C for 1 h, stir at 30 °C for another 1 h, finally turn off the heating and stir at room temperature overnight. After the reaction is complete, slowly cool to 0 °C, centrifuge to collect the solid and evaporate the solvent. The obtained solid is the L-tartaric acid salt crystal form I of compound (I). Its HPLC / IC results show that the molar ratio of compound (I) to L-tartaric acid in the obtained solid is 1:1; its XRPD chromatogram is basically as follows. Figure 5 As shown, the XRPD plot of the obtained solid has a peak at the 2θ (°) value shown in Table 2 above, and the relative intensities of each peak are shown in Table 2 above; its TGA plot is shown below. Figure 6 As shown.

[0309] Depend on Figure 6 The TGA graph shows that the weight loss was 1.057% when heated to around 110°C, which may be due to water evaporation. The weight loss was 3.482% when heated to around 178°C, which is presumably due to the evaporation of organic solvents. Further heating caused melting and decomposition.

[0310] Example 3. Preparation of L-tartrate crystal form II of compound (I)

[0311] In a sample vial, add free alkali (approximately 100 mg) and methanol (0.2 mL–0.3 mL), and sonicate to dissolve. Then, add 1 mol / L L-tartaric acid solution at an acid-base molar ratio of 1.2:1. React at 50°C for 1 h, continue stirring at 40°C for 1 h, and then continue stirring at 30°C for another 1 h. After cooling to room temperature, turn off the heating and stir overnight. After the reaction is complete, slowly cool to 0°C, add MTBE to induce crystallization, and evaporate the solvent to obtain a solid. HPLC / IC analysis of the obtained solid confirmed that the molar ratio of compound (I) to L-tartaric acid in the obtained solid was 1:1. The XRPD chromatogram of the obtained solid is shown below. Figure 7 As shown, the XRPD plot of the obtained solid has a peak at the 2θ (°) value shown in Table 3 above, and the relative intensities of each peak are shown in Table 3 above. In this disclosure, it is defined as L-tartrate crystal form II of compound (I).

[0312] Example 4. Preparation of the amorphous form of L-tartrate of compound (I)

[0313] In a sample vial, add free base (approximately 100 mg) and acetone (0.2 mL–0.3 mL), and sonicate to dissolve. Then, add 1 mol / L L-tartaric acid solution at a molar ratio of 1.2:1. React at 50°C for 1 h, continue stirring at 40°C for 1 h, and then continue stirring at 30°C for another 1 h. After cooling to room temperature, turn off the heating and stir overnight. After the reaction is complete, slowly cool to 0°C to precipitate a solid. Collect the solid by centrifugation and evaporate the solvent. Perform HPLC / IC on the obtained solid to determine that the molar ratio of compound (I) to L-tartaric acid in the obtained solid is 1:1. The XRPD chromatogram of the obtained solid is shown below. Figure 8 As shown, it is defined in this application as an L-tartrate amorphous compound.

[0314] Example 5. Preparation of phosphate crystal form II of compound (I)

[0315] 100 mg of free base was dissolved in 15 mL of ethanol in a 50 mL sample vial. Then, 5 mL of an ethanol solution of phosphoric acid was added dropwise at a molar ratio of 0.95:1. The mixture was stirred at room temperature for 4 hours, filtered, washed with ethanol, and dried by rotary evaporation to obtain a white solid. Elemental analysis revealed a phosphorus content of 7.8%, indicating that the molar ratio of compound (I) to phosphoric acid in the solid was 1:1 (the theoretical phosphorus content is 7.79%). XRPD analysis of the solid yielded the following XRPD chromatogram: Figure 10 As shown, it is defined as phosphate crystal form II in this application.

[0316] The XRPD plot of the obtained solid shows a peak at the 2θ (°) value shown in Table 4 above, and the relative intensities of each peak are also shown in Table 4 above; its DSC and TGA curves are as follows. Figure 11 As shown, its DVS diagram is as follows: Figure 12 As shown, its microscope image is as follows Figure 13 As shown.

[0317] Depend on Figure 11 It can be seen that: in the DSC curve, the sample has a melting absorption peak at 207.48℃, indicating good crystallinity, which shows that the melting point of phosphate crystal form II of formula (I) is about 207.48℃; in the TGA curve, the sample loses almost no weight when heated to around 100℃, and begins to melt when heated to around 207℃, therefore phosphate crystal form II of formula (I) is anhydrous.

[0318] Depend on Figure 12 It can be seen from the DVS diagram that the weight gain due to moisture absorption is 0.45% under 80% RH conditions, indicating that the phosphate crystal form II of compound (I) has slight hygroscopicity.

[0319] Depend on Figure 13 It can be seen that the polarized light microscope shows that the phosphate crystal form II of compound (I) is rod-shaped.

[0320] Example 6. Preparation of phosphate crystal form II of compound (I)

[0321] 100 mg of free base was dissolved in 15 mL of acetone in a 50 mL sample vial. Then, 5 mL of an ethanol solution of phosphoric acid was added dropwise at a molar ratio of 0.98:1. The mixture was stirred at room temperature for 4 hours, filtered, washed with ethanol, and dried by rotary evaporation to obtain a white solid. Elemental analysis revealed a phosphorus content of 7.8%, indicating a molar ratio of compound (I) to phosphoric acid of 1:1 (the theoretical phosphorus content is 7.79%). XRPD analysis of the solid yielded a graph similar to the one shown below. Figure 10 As shown.

[0322] Example 7. Preparation of phosphate crystal form II of compound (I)

[0323] 100 mg of free base was dissolved in 10 mL of ethanol in a 50 mL sample vial. Then, 10 mL of an ethanol solution of phosphoric acid was added dropwise at a molar ratio of 1.05:1. The mixture was stirred at room temperature for 4 hours, filtered, washed with ethanol, and dried by rotary evaporation to obtain a white solid. Elemental analysis revealed a phosphorus content of 7.8%, indicating a molar ratio of compound (I) to phosphoric acid of 1:1 (the theoretical phosphorus content is 7.79%). XRPD analysis of the solid yielded a graph similar to the one shown below. Figure 10 As shown.

[0324] Example 8. Preparation of phosphate crystal form II of compound (I)

[0325] 0.5 g of free alkali was dissolved in methanol (15 ml). Phosphoric acid (commercially available) was added at a molar ratio of 1:1. After stirring for 2 hours, methanol was removed by rotary evaporation to obtain crude phosphate. Ethanol (2.5 ml) was added and the mixture was concentrated. Then, ethanol (2.5 ml) was added again and the mixture was concentrated once more. Ethanol (10 ml) was added and the mixture was heated to 60 °C and stirred for 3 hours. The mixture was then cooled to room temperature and stirred overnight. After filtration, the mixture was washed with ethanol and dried by rotary evaporation to obtain phosphate crystal form II of compound (I), with a yield of 75.8%. Elemental analysis of the obtained solid showed a phosphorus content of 7.7%, indicating that the molar ratio of compound (I) to phosphoric acid in the obtained solid was 1:1 (the theoretical value of its phosphorus content is 7.79%). XRPD analysis of the obtained solid showed that its XRPD chromatogram was basically as follows: Figure 10 As shown.

[0326] Comparative Example 1. Salt formation reaction of compound (I)

[0327] A suitable amount of compound (I) (100 mg) was weighed using the weight reduction method and placed in a transparent sample bottle. The appropriate solvent (0.2 mL–0.3 mL) was added, and the mixture was sonicated to dissolve. Then, a 1 mol / L acid solution (aqueous solution) was added at a feed acid-base molar ratio of 1.2:1. The reaction was carried out at 50°C for 1 h, then stirred at 40°C for 1 h, and then stirred again at 30°C for 1 h. After cooling to room temperature, the heating was turned off, and the mixture was stirred overnight. After the reaction was complete, the temperature was slowly lowered until solid precipitation occurred. If the solution remained clear, antisolvent addition was attempted to induce crystallization. The reaction results were sent for XRPD testing. The results showed that hydrochloric acid, sulfuric acid, oxalic acid, and other acids could not form a salt of compound (I) after cooling or the addition of an antisolvent. The types of acids and solvents are shown in Table 11 below.

[0328] Table 11

[0329]

[0330] Test Example 1. Solubility Test

[0331] At room temperature, the solubility of the phosphate and L-tartrate of the compound of formula (I) prepared in the above examples in water was tested. The solubility of the phosphate was no greater than 26.6 mg / mL, the solubility of the L-tartrate was no greater than 17.3 mg / mL, and the free base was slightly soluble in water. The salts of the compound of formula (I) significantly improve their solubility in water, and the phosphate in the resulting salts has higher solubility than the L-tartrate.

[0332] In addition, the approximate solubility (unit: mg / mL) of phosphate form I and L-tartrate form I of compound (I) in buffer solution at pH 4.5 (acetic acid-sodium acetate system) and buffer solution at pH 6.8 (sodium dihydrogen phosphate-sodium hydroxide system) was tested at room temperature, and the results are shown in Table 12. The results in Table 12 show that phosphate form I and L-tartrate form I of compound (I) have high solubility in buffer solutions at pH 4.5 and pH 6.8.

[0333] Table 12

[0334]

[0335] Weigh appropriate amounts of phosphate crystal form I or phosphate crystal form II and add them to 0.1 mol / L hydrochloric acid solution (5 mL) and pH 6.8 phosphate buffer solution (5 mL), respectively. Take samples after 24 h (placed in a 37℃ oven) to determine the solubility. The results are shown in Table 13.

[0336] Table 13

[0337]

[0338] Test Example 2. Stability Test

[0339] (1) Weigh out samples (approximately 100 mg) of phosphate crystal form I, phosphate crystal form II, L-tartrate crystal form I, and L-tartrate crystal form II of compound (I), respectively, and place them at 60℃ (high temperature) and 40℃-75%RH (accelerated) conditions. Meanwhile, another set of samples is sealed and stored at 5℃ as a control. The crystal form changes are detected at 7, 20, 30 or 60 days.

[0340] The test results show that the crystal forms of compounds (I) L-tartrate crystal form I, L-tartrate crystal form II, phosphate crystal form I, and phosphate crystal form II of formula (I) did not change significantly under the above conditions, exhibiting high stability. The XRPD images of L-tartrate crystal form I of formula (I) after storage at high temperature for 7 days and 2 months (60 days) showed no significant change. The superimposed XRPD images of L-tartrate crystal form I of formula (I) after storage under accelerated conditions for 7 days and 2 months (60 days) are shown below. Figure 15 As shown, no significant changes were observed; this indicates that L-tartrate crystal form I of compound (I) has high stability. The XRPD images of phosphate crystal form I of compound (I) stored at high temperature for 7 days and 30 days are superimposed as follows: Figure 16As shown, there is no obvious change, indicating that the phosphate crystal form I of compound (I) has high stability.

[0341] (2) Weigh approximately 40 mg of phosphate crystal form I (prepared according to Example 1 of this application) into a glass vial, add ethanol (1 mL), seal and place at 50°C and shake at 150 r / min for seven days. Then centrifuge to collect the solid and evaporate the solvent. Perform XRPD analysis on the obtained solid. The XRPD chromatogram is shown below. Figure 10 As shown. The obtained solid is phosphate crystal form II. Test results Figure 14 This indicates that phosphate crystal form I can be transformed into phosphate crystal form II, which is more thermodynamically stable.

[0342] (3) Take 25 mg of phosphate crystal form I and 25 mg of phosphate crystal form II respectively, add 5 mg of water to each, and then place them in an 80℃ oven for one week. Take samples to determine the relevant substances. The results are shown in Table 14, indicating that phosphate crystal form I and phosphate crystal form II have high chemical stability.

[0343] Table 14

[0344]

[0345] Test Example 3. Hygroscopicity Test

[0346] 3.1 Hygroscopicity test of L-tartrate

[0347] Following the procedure outlined in 9103 (Guidelines for Hygroscopicity Testing of Drugs) of the Chinese Pharmacopoeia (2020), the L-tartrate salt of compound (I) was found to be slightly hygroscopic.

[0348] 3.2 Hygroscopicity test of phosphate crystal form

[0349] DVS measurements were performed on phosphate crystal forms I and II of compound (I). The DVS diagram of phosphate crystal form I of compound (I) is shown below. Figure 3 As shown, the DVS diagram indicates that under 80% RH conditions, the hygroscopic weight gain is 9%, indicating that phosphate crystal form I of formula (I) is hygroscopic. In contrast, the DVS diagram for phosphate crystal form II shows that under 80% RH conditions, the hygroscopic weight gain is only 0.45%, indicating that phosphate crystal form II is slightly hygroscopic.

[0350] Test Example 4. Inhibition Test of CDK Family Kinase Activity

[0351] In the following LANCE Ultra assay, the kinase reagent was purchased from Carna Bioscience, the reaction substrate and assay reagent were purchased from PerkinElmer, and the remaining reagents were purchased from Thermo Scientific.

[0352] The inhibitory effects of the analytes on the activity of CDK1 / CycB (Carna bioscience, #04-102), CDK2 / CycA (Carna bioscience, #04-103), and CDK9 / CycT (Carna bioscience, #04-110) kinases were determined using the LANCE Ultra method.

[0353] The kinase activity assay uses a 10 μL system containing the following components: CDK kinase dilution buffer, substrate dilution buffer of a mixture of Ulight-Myelicbasic protein (PerkinElmer, #TRF-0109, hereinafter referred to as U-MBP) and ATP (Thermo Scientific, #PV3227), and the compound of formula (I) disclosed herein (i.e., the analyte). Each kinase assay includes three test groups: a background group (Blank), a non-inhibition group (PC), and a compound test group (Test). The components included in each test group are shown in Table 15 below.

[0354] Table 15

[0355]

[0356] The working concentrations of each component in the Test group during different kinase responses are shown in Table 16.

[0357] Compounds: The 10 mM of the test compound was dissolved at room temperature and serially diluted with DMSO, then diluted with deionized water to prepare a 4x working solution containing 2% DMSO. The highest concentration of the compound used in CDK1 and CDK2 tests was 10 μM, and that for CDK9 was 1 μM.

[0358] 1.33x reaction buffer: The components are 26.7mM MOPS, 6.67mM MgCl2 and 0.0133% Tween-20. After preparation, store in a refrigerator at 4°C protected from light. Before use, add freshly prepared DTT to a final concentration of 5.33mM.

[0359] Table 16

[0360]

[0361] The working concentration of DMSO in the reaction is 0.5%.

[0362] After mixing the above components, the mixture was placed on a shaker and incubated at room temperature in the dark for 1 hour. Subsequently, 10 μL of detection solution was added to all test groups (including Blank, PC, and Test groups).

[0363] The 10 μL test solution contains the following components: 16 mM EDTA (Thermo Scientific, #15575), 1 nM phosphorylated U-MBP protein antibody (PerkinElmer, #TRF-0201), and 1x test buffer (PerkinElmer, #CR97-100).

[0364] After adding the detection solution, place the sample on a shaker and incubate at room temperature in the dark for 1 hour. After incubation, read the signal using a PerkinElmer VictorX5 fluorescence microplate reader. The excitation wavelength was 320 nm, and the emission wavelengths were 615 nm and 665 nm. The inhibition rate was calculated as follows:

[0365] 1. Calculate the 665nm / 615nm value (hereinafter referred to as the Ratio value) for all groups, and calculate the inhibition rate based on the Ratio value of each group;

[0366] 2. Inhibition rate = (PC Ratio -Test Ratio ) / (PC Ratio -Blank Ratio )*100%;

[0367] 3. The half-maximal inhibitory concentration (IC50) of the compound was calculated using XLFIT 5.0 software (IDBS, UK), with the logarithm of the compound concentration as the X-axis and the inhibition rate as the Y-axis, using a four-parameter model. 50 The results are shown in Table 17.

[0368] Table 17

[0369]

[0370] As shown in Table 17, the compound of formula (I) disclosed in this invention has high inhibitory activity against CDK9 and high CDK9 inhibitory selectivity.

[0371] Test Example 5: In vivo pharmacokinetic test in mice

[0372] The plasma concentrations of compounds D, E, and (I) in mice were determined at different time points after intravenous injection and gavage administration using LC / MS / MS. The pharmacokinetic behavior of compounds D, E, and (I) in mice was studied, and their pharmacokinetic characteristics were evaluated.

[0373] Experimental plan:

[0374] Experimental animals: Healthy adult male ICR mice (30-40g, 12 mice; the intravenous injection group had free access to water and food, while the gavage group was fasted overnight and given free access to water and food 4 hours after administration), provided by Beijing Vital River Laboratory Animal Co.LTD;

[0375] Administration method and dosage: ICR mice were administered via tail vein (2 mg / kg, 5% DMSO, pH 4.5, 20% Captisol) and by gavage (10 mg / kg, 5% DMSO, pH 4.5, 20% Captisol).

[0376] Blood Sampling: Animals meeting experimental requirements were selected, weighed, and marked before drug administration. Before blood sampling, mice were tethered, and blood was collected from each administered mouse at predetermined time points (for intravenous administration: blood was collected at 0.083, 0.25, 0.5, 1, 2, 4, 6, 7.5, and 24 hours post-administration, for a total of 9 time points; for gavage administration: blood was collected at 0.083, 0.25, 0.5, 1, 2, 4, 6, 7.5, and 24 hours post-administration, for a total of 9 time points), approximately 100 μL of blood was collected via the orbital rim. The blood was transferred to 1.5 mL tubes pre-filled with K2EDTA, centrifuged for 4 min (8000 rpm, 4℃), and the plasma was collected. The entire process was completed within 15 min of blood collection. All samples were stored at -20℃ until analysis. Drug concentrations were determined by LC / MS / MS. The pharmacokinetic properties of compounds D, E, and (I) in mice at the same dose and administration route are shown in Table 18.

[0377] Table 18 Pharmacokinetic parameters of the compounds in mice

[0378]

[0379] Test Example 6: In vivo drug efficacy experiment

[0380] In vivo efficacy studies were conducted on BALB / c nude mice with subcutaneous implantation of xenografts (CDX) based on human tumor cell lines derived from MV4-11 acute myeloid leukemia patients.

[0381] Experimental protocol: BALB / c nude mice, female, 6-10 weeks old, weighing approximately 20-23 grams, were kept in a specific pathogen-free environment in individual ventilated cages (5 mice per cage, 2 cages per group, 10 mice in total). All cages, bedding, and water were sterilized before use. All animals had free access to standard certified commercial laboratory diets. A total of 80 mice were purchased from the Laboratory Animal Management Department of Shanghai Institute of Family Planning Science (3577 Jinke Road, Pudong, Shanghai) for research. Each mouse underwent subcutaneous implantation of tumor cells (1×10⁻⁶) in the right flank. 7 0.1 ml of Matrigel (+0.1 ml) was used to promote tumor growth. When the average tumor volume reached approximately 165 mm³, animals were randomly assigned to groups based on body weight and tumor volume, and administration began. The test compound was administered orally via gavage daily. Antitumor efficacy was determined by dividing the average tumor increase in treated animals by the average tumor increase in untreated animals.

[0382] Tumor volume was measured twice weekly using calipers, and the volume was measured in cubic millimeters. Tumor volume TV = 0.5a × b 2 Where a is the long axis of the tumor and b is the short axis of the tumor.

[0383] The relative tumor growth rate (T / C%) is the percentage of relative tumor volume (RTV) between the treatment group and the control group at a given time point. The calculation formula is as follows: T / C% = T RTV / C RTV ×100% (T) RTV : Mean RTV in the treatment group; C RTV : Average RTV of the solvent control group; RTV = V t / V0, where V0 is the tumor volume of the animal at the time of grouping, and Vt is the tumor volume of the animal after treatment. mpk refers to milligrams per kilogram of body weight.

[0384] The weight change (%) of tumor-bearing animals is calculated as follows: (weight at measurement - weight at grouping) / weight at grouping × 100.

[0385] The tumor volume changes and mouse body weight changes after once-daily oral administration of compound D and compound (I) are shown in the figures below. Figure 17 and Figure 18 As shown in Table 19, the tumor inhibition results of the compound in mice are as follows:

[0386] Table 19. Tumor inhibition results of the compounds in mice.

[0387]

[0388] All documents mentioned in this disclosure are incorporated herein by reference as if each document were individually incorporated herein by reference. Furthermore, it should be understood that after reading the foregoing teachings of this disclosure, those skilled in the art can make various alterations or modifications to this disclosure, and these equivalent forms also fall within the scope defined by the appended claims.

[0389] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above 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.

[0390] The embodiments described above are merely illustrative of several implementations of this disclosure, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this disclosure, and these all fall within the protection scope of this disclosure. Therefore, the protection scope of this patent should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A pharmaceutically acceptable salt of a compound of formula (I), ； The pharmaceutically acceptable salts are selected from phosphates and L-tartrates.

2. A pharmaceutically acceptable salt of the compound of formula (I) as described in claim 1, wherein, The pharmaceutically acceptable salt is L-tartrate; the molar ratio of L-tartrate acid to the compound of formula (I) in the L-tartrate is (0.8-1.2):

1.

3. A pharmaceutically acceptable salt of the compound of formula (I) as described in claim 2, wherein, The molar ratio of L-tartaric acid to the compound of formula (I) in the L-tartrate is (0.9-1.1):

1.

4. A pharmaceutically acceptable salt of the compound of formula (I) as described in claim 2, wherein, The molar ratio of L-tartaric acid to the compound of formula (I) in the L-tartrate salt is 1:

1.

5. A pharmaceutically acceptable salt of the compound of formula (I) as described in claim 1, wherein, The pharmaceutically acceptable salt is a phosphate; the molar ratio of phosphoric acid to the compound of formula (I) in the phosphate is (1.8-2.4):

1.

6. A pharmaceutically acceptable salt of the compound of formula (I) as described in claim 5, wherein, The molar ratio of phosphoric acid to compound of formula (I) in the phosphate is (1.9-2.3):

1.

7. A pharmaceutically acceptable salt of the compound of formula (I) as described in claim 5, wherein, The molar ratio of phosphoric acid to compound (I) in the phosphate is 2:

1.

8. A pharmaceutically acceptable salt of the compound of formula (I) as claimed in claim 1, wherein, The pharmaceutically acceptable salt is a phosphate; the molar ratio of phosphoric acid to the compound of formula (I) in the phosphate is (0.8-1.2):

1.

9. A pharmaceutically acceptable salt of the compound of formula (I) as described in claim 8, wherein, The molar ratio of phosphoric acid to compound (I) in the phosphate is 1:

1.

10. A pharmaceutically acceptable salt of the compound of formula (I) as described in claim 2, wherein, The L-tartrate is a polymorph, and the polymorph is selected from the following crystal forms: (1) L-tartrate crystal form I, its X-ray powder diffraction pattern 2θ (°) shows characteristic diffraction peaks at diffraction angles of 13.946±0.2, 16.881±0.2, 19.405±0.2, 21.505±0.2 and 24.262±0.2; and (2) L-tartrate crystal form II, its X-ray powder diffraction pattern 2θ (°) has characteristic diffraction peaks at diffraction angles of 11.662±0.2, 14.244±0.2, 17.481±0.2, 18.349±0.2 and 21.353±0.

2.

11. A pharmaceutically acceptable salt of the compound of formula (I) as described in claim 2, wherein, The L-tartrate is a polymorph, and the polymorph is selected from the following crystal forms: (1) L-tartrate crystal form I, its X-ray powder diffraction pattern 2θ (°) shows characteristic diffraction peaks at diffraction angles of 7.436±0.2, 13.946±0.2, 16.013±0.2, 16.881±0.2, 18.175±0.2, 19.045±0.2, 19.405±0.2, 21.505±0.2, 24.262±0.2, 25.202±0.2, 26.452±0.2, 28.105±0.2 and 31.579±0.2; and (2) L-tartrate crystal form II, its X-ray powder diffraction pattern 2θ (°) has characteristic diffraction peaks at diffraction angles of 11.662±0.2, 14.244±0.2, 17.481±0.2, 18.349±0.2, 21.026±0.2 and 21.353±0.

2.

12. A pharmaceutically acceptable salt of the compound of formula (I) as described in claim 2, wherein, The L-tartrate is a polymorph, and the polymorph is selected from the following crystal forms: (1) L-tartrate crystal form I, its X-ray powder diffraction pattern 2θ (°) diffraction angles are 6.687±0.2, 7.436±0.2, 10.615±0.2, 12.053±0.2, 13.164±0.2, 13.946±0.2, 14.875±0.2, 15.201±0.2, 16.013±0.2, 16.881±0.2, 18.175±0.2, Characteristic peaks are observed at values ​​of 19.045±0.2, 19.405±0.2, 20.659±0.2, 21.505±0.2, 22.434±0.2, 23.04±0.2, 24.262±0.2, 25.202±0.2, 26.452±0.2, 28.105±0.2, 29.692±0.2, 34.139±0.2, and 34.543±0.2; and (2) L-tartrate crystal form II, its X-ray powder diffraction pattern 2θ (°) has characteristic peaks at diffraction angles of 10.376±0.2, 11.662±0.2, 14.244±0.2, 16.548±0.2, 17.481±0.2, 18.349±0.2, 18.984±0.2, 21.026±0.2, 21.353±0.2, 26.925±0.2, 29.335±0.2 and 31.784±0.

2.

13. A pharmaceutically acceptable salt of the compound of formula (I) as described in claim 2, wherein, The L-tartrate is a polymorph, and the polymorph is selected from the following crystal forms: (1) L-tartrate crystal form I, which has a basic X-ray powder diffraction (XRPD) pattern as shown in Figure 5; and (2) L-tartrate crystal form II, which has a basic X-ray powder diffraction (XRPD) pattern as shown in Figure 7.

14. A pharmaceutically acceptable salt of the compound of formula (I) as described in claim 10, wherein, The L-tartrate crystal form I has a basic thermogravimetric analysis (TGA) diagram as shown in Figure 6.

15. A pharmaceutically acceptable salt of the compound of formula (I) as described in claim 2, wherein, The L-tartrate is an amorphous compound.

16. A pharmaceutically acceptable salt of the compound of formula (I) as described in claim 5, wherein, The phosphate is phosphate crystal form I, and its X-ray powder diffraction pattern 2θ (°) has characteristic diffraction peaks at diffraction angles of 18.234±0.2, 18.803±0.2, 19.131±0.2, 21.266±0.2 and 23.247±0.

2.

17. A pharmaceutically acceptable salt of the compound of formula (I) as described in claim 5, wherein, The phosphate is phosphate crystal form I, and its X-ray powder diffraction pattern 2θ (°) has characteristic diffraction peaks at diffraction angles of 13.999±0.2, 18.234±0.2, 18.803±0.2, 19.131±0.2, 21.266±0.2, 22.01±0.2 and 23.247±0.

2.

18. A pharmaceutically acceptable salt of the compound of formula (I) as described in claim 5, wherein, The phosphate is phosphate crystal form I, which has an X-ray powder diffraction (XRPD) pattern as shown in Figure 1.

19. A pharmaceutically acceptable salt of the compound of formula (I) as described in claim 16, wherein, The phosphate crystal form I has one or more characteristics selected from the following: It has a basic differential scanning calorimetry (DSC) chart as shown in Figure 2; It has a basic thermogravimetric analysis (TGA) diagram as shown in Figure 2; and It has a basic dynamic water absorption (DVS) diagram as shown in Figure 3.

20. A pharmaceutically acceptable salt of the compound of formula (I) as described in claim 8, wherein, The phosphate is phosphate crystal form II, and its X-ray powder diffraction pattern has characteristic diffraction peaks at 2θ (°) diffraction angles of 10.584±0.2, 18.230±0.2, 21.144±0.2, 23.153±0.2 and 23.96±0.

2.

21. A pharmaceutically acceptable salt of the compound of formula (I) as described in claim 8, wherein, The phosphate is phosphate crystal form II, and its X-ray powder diffraction pattern 2θ (°) shows characteristic diffraction peaks at diffraction angles of 10.584±0.2, 13.969±0.2, 14.873±0.2, 18.230±0.2, 20.576±0.2, 21.144±0.2, 22.01±0.2, 22.492±0.2, 23.153±0.2, and 23.96±0.

2.

22. A pharmaceutically acceptable salt of the compound of formula (I) as described in claim 8, wherein, The phosphate is phosphate crystal form II, which has a basic X-ray powder diffraction (XRPD) pattern as shown in Figure 10.

23. A pharmaceutically acceptable salt of the compound of formula (I) as described in claim 20, wherein, The phosphate crystal form II has one or more characteristics selected from the following: It has a melting temperature of 207.48±0.5℃; It has a basic differential scanning calorimetry (DSC) curve as shown in Figure 11; It has a basic thermogravimetric analysis (TGA) curve as shown in Figure 11; and It has a basic dynamic water absorption (DVS) diagram as shown in Figure 12.

24. A pharmaceutically acceptable salt of the compound of formula (I) as described in claim 20, wherein, The phosphate crystal form II is anhydrous.

25. A method for preparing an L-tartrate salt of a compound of formula (I), comprising the following steps: reacting a compound of formula (I) with L-tartaric acid to form an L-tartrate salt of the compound of formula (I); wherein the compound of formula (I) is... .

26. A method for preparing L-tartrate crystal form I of a compound of formula (I), comprising the following steps: The compound of formula (I) is reacted with L-tartaric acid in an organic solvent to form a salt-forming reaction solution; The reaction solution was slowly cooled to obtain L-tartrate crystal form I; The organic solvent is ethanol; the compound of formula (I) is The L-tartrate crystal form I has characteristic diffraction peaks at diffraction angles of 2θ (°) in its X-ray powder diffraction pattern at values ​​of 13.946±0.2, 16.881±0.2, 19.405±0.2, 21.505±0.2 and 24.262±0.

2.

27. A method for preparing L-tartrate crystal form II of a compound of formula (I), comprising the following steps: The compound of formula (I) is reacted with L-tartaric acid in an organic solvent to form a salt-forming reaction solution; The reaction solution was slowly cooled, and an antisolvent was added to obtain the L-tartrate crystal form II. The organic solvent mentioned above is methanol; The antisolvent is methyl tert-butyl ether; the compound of formula (I) is... The L-tartrate crystal form II has characteristic diffraction peaks at diffraction angles of 2θ (°) in its X-ray powder diffraction pattern at values ​​of 11.662±0.2, 14.244±0.2, 17.481±0.2, 18.349±0.2 and 21.353±0.

2.

28. A method for preparing the phosphate according to claim 5 or 8, comprising the following steps: reacting the compound of formula (I) with phosphoric acid in the presence of an organic solvent to form a salt-forming reaction, thereby forming a phosphate of the compound of formula (I).

29. The preparation method according to claim 28, wherein, The molar ratio of compound (I) to phosphoric acid in the phosphate forming compound (I) is 1:1 or 1:

2.

30. The preparation method according to claim 28, wherein, The organic solvent is selected from one or more of ethanol, ethyl acetate, acetonitrile, and acetone.

31. The preparation method according to claim 28, wherein, The organic solvent is selected from methanol.

32. A method for preparing phosphate crystal form II of a compound of formula (I), comprising the following steps: The compound of formula (I) is reacted with phosphoric acid in an organic solvent to form a salt, resulting in the precipitation of a solid; Collect the solid to obtain phosphate crystal form II; The molar ratio of compound (I) to phosphoric acid is 1:(0.8-1.2); the organic solvent is one or more of ethanol, methanol, and acetone; the compound (I) is... The phosphate crystal form II has characteristic diffraction peaks at 2θ (°) diffraction angles of 10.584±0.2, 18.230±0.2, 21.144±0.2, 23.153±0.2 and 23.96±0.2 in its X-ray powder diffraction pattern.

33. The preparation method according to claim 32, wherein, The organic solvent is methanol.

34. The preparation method according to claim 32, wherein, The organic solvent is ethanol.

35. The preparation method according to claim 34, wherein, The preparation method includes the following steps: An ethanol solution of the compound of formula (I) is reacted with an ethanol solution of phosphoric acid to form a salt, resulting in the precipitation of a solid. Collect the solid to obtain phosphate crystal form II; The molar ratio of compound (I) to phosphoric acid is 1:(0.8-1.2).

36. A method for preparing phosphate crystal form I of compound (I) includes: (AI-a) Dissolve the compound of formula (I) in a solvent, add an aqueous solution of phosphoric acid, and stir to react; (AI-b) The reaction solution obtained in step (AI-a) is cooled, an antisolvent is added to induce crystallization, solid-liquid separation is performed, and the solid phase is collected to obtain phosphate crystal form I of compound (I); the compound of formula (I) is The phosphate crystal form I has characteristic diffraction peaks at diffraction angles of 2θ (°) in its X-ray powder diffraction pattern at values ​​of 18.234±0.2, 18.803±0.2, 19.131±0.2, 21.266±0.2 and 23.247±0.

2.

37. A pharmaceutical composition comprising: (a) A pharmaceutically acceptable salt of the compound of formula (I) according to any one of claims 1-24; and (b) pharmaceutically acceptable carriers.

38. The use of a pharmaceutically acceptable salt of the compound of formula (I) of any one of claims 1-24, or the pharmaceutical composition of claim 37, in the preparation of a medicament for the prevention or treatment of diseases associated with or mediated by CDK9 activity.

39. The application as described in claim 38, wherein, The diseases mentioned are one or more of the following: proliferative diseases, virus-induced infectious diseases, and cardiovascular diseases.

40. A pharmaceutically acceptable salt of any one of the compounds of formula (I) of claims 1-24, or a pharmaceutical composition of claim 37, for the prevention or treatment of diseases associated with or mediated by CDK9 activity.

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