WO2024017365A1 - 取代的吡唑并[1,5-a]嘧啶-7-胺衍生物的药学上可接受的盐和多晶型物及其应用 - Google Patents

取代的吡唑并[1,5-a]嘧啶-7-胺衍生物的药学上可接受的盐和多晶型物及其应用 Download PDF

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WO2024017365A1
WO2024017365A1 PCT/CN2023/108554 CN2023108554W WO2024017365A1 WO 2024017365 A1 WO2024017365 A1 WO 2024017365A1 CN 2023108554 W CN2023108554 W CN 2023108554W WO 2024017365 A1 WO2024017365 A1 WO 2024017365A1
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compound
formula
crystal form
phosphate
pharmaceutically acceptable
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French (fr)
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但招陵
江涛涛
王吉标
薛原
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上海海雁医药科技有限公司
扬子江药业集团有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems

Definitions

  • the present disclosure belongs to the field of pharmaceutical technology, and specifically relates to pharmaceutically acceptable salts and polymorphs of substituted pyrazolo[1,5-a]pyrimidin-7-amine derivatives, pharmaceutical compositions containing the same, and pharmaceutical compositions thereof. Preparation methods, and pharmaceutical applications.
  • CDK9 is a member of the cell cycle-dependent kinase (CDK) protein family and plays an important role in the transcriptional regulation of genes.
  • CDK9 mainly regulates gene transcription elongation by phosphorylating the carbon-terminal region of RNA complexase II.
  • CDK9 is generally highly expressed in tumors and is also an important factor in the progression and maintenance of tumor cells.
  • CDK9 inhibitors downregulate the expression of related oncoprotein (MYC) and the expression of apoptosis inhibitor protein Mcl-1 by inhibiting the extension of gene transcription, thereby promoting the apoptosis of cancer cells.
  • CDK9 inhibitors reactivate silenced genes by regulating the epigenetic factor BRG1, including the activation of endogenous retroviruses (ERVs) in tumor cells, promote the expression of interferon, and make tumor cells more sensitive to immunotherapy.
  • EMVs endogenous retroviruses
  • CDK9 inhibitors including the selective CDK9 inhibitor BAY1251152 developed by Bayer, the selective CDK9 inhibitor AZD4573 developed by AstraZeneca, the non-selective CDK9 inhibitor TP-1287 developed by Tolero, and The non-selective CDK9 inhibitor QHRD107 developed by Changzhou Qianhong Pharmaceutical Company, etc.
  • the purpose of the present disclosure is to provide pharmaceutically acceptable salts and polymorphs of substituted pyrazolo[1,5-a]pyrimidin-7-amine derivatives and their applications.
  • the pharmaceutically acceptable salts are the L-tartrate and phosphate salts of substituted pyrazolo[1,5-a]pyrimidin-7-amine derivatives.
  • the substituted pyrazolo[1,5-a]pyrimidin-7-amine derivative is a CDK9 inhibitor, and its molecular structure is (1S,3S)-N1-(5-((S) represented by formula (I) )-1-cyclobutylethyl)pyrazolo[1,5-a]pyrimidin-7-yl)cyclopentane-1,3-diamine.
  • a first aspect of the present disclosure provides pharmaceutically acceptable salts of compounds of formula (I):
  • the pharmaceutically acceptable salt is selected from: phosphate and L-tartrate.
  • the pharmaceutically acceptable salt of the compound of Formula (I) is an anhydrous form, a hydrate form, or a solvate form.
  • the pharmaceutically acceptable salt of the compound of Formula (I) is a crystal. In some embodiments, the pharmaceutically acceptable salt of the compound of Formula (I) is amorphous.
  • the pharmaceutically acceptable salt is a phosphate salt.
  • the phosphate salt is a compound of formula (I) The first type of phosphate, wherein the molar ratio of phosphoric acid to the compound of formula (I) is (1.8-2.4):1.
  • the molar ratio of the 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.
  • the molar ratio of the phosphoric acid to the compound of formula (I) is (1.9-2.3):1.
  • the molar ratio of the phosphoric acid to the compound of formula (I) is 2:1.
  • the phosphate is the phosphate crystal form I of the compound of formula (I), and its X-ray powder diffraction pattern has characteristic diffraction peaks at the following diffraction angle 2 ⁇ (°) values: 18.234 ⁇ 0.2, 19.131 ⁇ 0.2 and 21.266 ⁇ 0.2.
  • the phosphate is the phosphate crystal form I of the compound of formula (I), and its X-ray powder diffraction pattern has characteristic diffraction peaks at the following diffraction angle 2 ⁇ (°) values: 18.803 ⁇ 0.2, 18.234 ⁇ 0.2 , 19.131 ⁇ 0.2 and 21.266 ⁇ 0.2.
  • the X-ray powder diffraction pattern of the phosphate crystal form I further has Including 2 or more (such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc.) diffraction angle 2 ⁇ (°) values selected from the following group Characteristic diffraction peaks at: 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, 18.803 ⁇ 0. 2.
  • the X-ray powder diffraction pattern of the phosphate crystal form I is in 3 or more (such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc.) or all selected from the following diffraction angle 2 ⁇ (°) values with characteristic diffraction peaks: 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, 18.234 ⁇ 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.
  • the X-ray powder diffraction pattern of the phosphate crystal form I has characteristic diffraction peaks at the following diffraction angle 2 ⁇ (°) values: 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.
  • the X-ray powder diffraction pattern of the phosphate crystal form I has characteristic diffraction peaks at the following diffraction angle 2 ⁇ (°) values: 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.
  • the X-ray powder diffraction pattern of the phosphate crystal form I has characteristic diffraction peaks at the following diffraction angle 2 ⁇ (°) 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, 14.902 ⁇ 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.
  • the X-ray powder diffraction pattern of the phosphate crystal form I of the compound of formula (I) has the characteristic diffraction peaks expressed in 2 ⁇ (°) values and d values as shown in Table 1, and the relative intensity of each peak is as shown in Table 1 shown.
  • the molar ratio of phosphoric acid to the compound of formula (I) in the phosphate crystal form I of the compound of formula (I) is 2:1.
  • the X-ray powder diffraction pattern of the phosphate crystal form I of the compound of formula (I) is substantially as shown in Figure 1.
  • the differential scanning calorimetry curve of the phosphate crystal form I of the compound of formula (I) has an absorption at 188.01°C ⁇ 3°C, 188.01°C ⁇ 2°C, 188.01°C ⁇ 1°C, or 188.01°C ⁇ 0.5°C. Hot peak.
  • the starting temperature in the differential scanning calorimetry curve of the phosphate crystal form I of the compound of formula (I) is 188.01°C ⁇ 3°C, 188.01°C ⁇ 2°C, 188.01°C ⁇ 1°C, or 188.01°C °C°C ⁇ 0.5°C
  • the peak temperature is 193.69°C ⁇ 3°C, 193.69°C ⁇ 2°C, 193.69°C ⁇ 1°C or 193.69°C ⁇ 0.5°C.
  • the differential scanning calorimetry spectrum (DSC spectrum) of the phosphate crystal form I of the compound of formula (I) is substantially as shown in Figure 2. In the embodiment shown in Figure 2, the melting point of the phosphate crystal form I of the compound of formula (I) is approximately 188.01 ⁇ 0.5°C.
  • thermogravimetric analysis pattern (TGA pattern) of the phosphate crystal form I of the compound of formula (I) is substantially as shown in Figure 2.
  • TGA spectrum of the phosphate crystal form I of the compound of formula (I) shows a weight loss of 1.139% near 190°C.
  • the dynamic moisture absorption spectrum (DVS chart) of the phosphate crystal form I of the compound of formula (I) is substantially as shown in Figure 3.
  • the DVS plot of the phosphate crystal form I of the compound of formula (I) shows a hygroscopic weight gain of 9% under 80% relative humidity (RH) conditions.
  • a micrograph of the phosphate crystal form I of the compound of formula (I) is substantially as shown in Figure 4.
  • microscopic images of the phosphate crystal form I of the compound of formula (I) show that the phosphate crystal form I is in the form of rods and blocks.
  • the pharmaceutically acceptable salt is 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.
  • the molar ratio of the 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.
  • the molar ratio of the L-tartaric acid to the compound of formula (I) is (0.9-1.1):1.
  • the molar ratio of L-tartaric acid to the compound of formula (I) is 1:1.
  • the L-tartrate is the L-tartrate crystal form I of the compound of formula (I), and its X-ray powder diffraction pattern has a characteristic diffraction peak at the following set of diffraction angle 2 ⁇ (°) values: 13.946 ⁇ 0.2 , 16.881 ⁇ 0.2, 19.405 ⁇ 0.2, 21.505 ⁇ 0.2 and 24.262 ⁇ 0.2.
  • the X-ray powder diffraction pattern of the L-tartrate crystal form I has characteristic diffraction peaks at the following set of diffraction angle 2 ⁇ (°) values: 13.946 ⁇ 0.2, 16.881 ⁇ 0.2, 19.405 ⁇ 0.2, 21.505 In addition to ⁇ 0.2 and 24.262 ⁇ 0.2, it further includes 2 or more (such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc.) selected from The characteristic diffraction peaks at the lower set of diffraction angle 2 ⁇ (°) 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, 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 ⁇
  • the X-ray powder diffraction pattern of the L-tartrate crystal form I is in 5 or more (such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 etc.) or all selected from the following diffraction angle 2 ⁇ (°) values with characteristic diffraction peaks: 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, 16.881 ⁇ 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.
  • the X-ray powder diffraction pattern of the L-tartrate crystal form I has characteristic diffraction peaks at the following diffraction angle 2 ⁇ (°) values: 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.
  • the X-ray powder diffraction pattern of the L-tartrate crystal form I has characteristic diffraction peaks at the following diffraction angle 2 ⁇ (°) values: 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.
  • the X-ray powder diffraction pattern of the L-tartrate crystal form I has characteristic diffraction peaks at the following diffraction angle 2 ⁇ (°) 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, 15.201 ⁇ 0.2, 16.013 ⁇ 0.2, 16.881 ⁇ 0.2, 18.175 ⁇ 0.2, 19.045 ⁇ 0.2, 19.405 ⁇ 0.
  • the X-ray powder diffraction pattern of the L-tartrate crystal form I has the characteristic diffraction peaks expressed in 2 ⁇ (°) values and d values as shown in Table 2, and the relative intensity of each peak is as shown in Table 2 Show.
  • the molar ratio of L-tartaric acid to the compound of formula (I) in the L-tartrate crystal form I is 1:1.
  • the X-ray powder diffraction pattern of compound L-tartrate crystal form I of formula (I) is substantially as shown in Figure 5.
  • thermogravimetric analysis spectrum of compound L-tartrate crystal form I of formula (I) is substantially as shown in Figure 6.
  • the TGA spectrum of compound L-tartrate crystal form I of formula (I) shows a weight loss of 1.057% near 110°C and a continued weight loss of 3.482% near 178°C.
  • the L-tartrate is the L-tartrate crystal form II of the compound of formula (I), and its X-ray powder diffraction pattern has a characteristic diffraction peak at the following set of diffraction angle 2 ⁇ (°) values: 11.662 ⁇ 0.2 and 21.353 ⁇ 0.2.
  • the X-ray powder diffraction pattern of the L-tartrate crystal form II further includes: 2 or more (such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, etc.) selected from the following group of characteristic diffraction peaks at the diffraction angle 2 ⁇ (°) value : 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.
  • the X-ray powder diffraction pattern of the L-tartrate crystal form II is in 3 or more (such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc.) or all selected from the following diffraction angle 2 ⁇ (°) values with characteristic diffraction peaks: 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.
  • the X-ray powder diffraction pattern of the L-tartrate crystal form II has characteristic diffraction peaks at the following diffraction angle 2 ⁇ (°) values: 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.
  • the X-ray powder diffraction pattern of the L-tartrate crystal form II has characteristic diffraction peaks at the following diffraction angle 2 ⁇ (°) values: 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.
  • the X-ray powder diffraction pattern of the L-tartrate crystal form II has characteristic diffraction peaks at the following diffraction angle 2 ⁇ (°) 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.
  • the X-ray powder diffraction pattern of the L-tartrate crystal form II has the characteristic diffraction peaks expressed in 2 ⁇ (°) values and d values as shown in Table 3, and the relative intensity of each peak is as shown in Table 3 Show.
  • the molar ratio of L-tartaric acid to the compound of formula (I) in the L-tartrate crystal form II is 1:1.
  • the X-ray powder diffraction pattern of the L-tartrate crystal form II is substantially as shown in Figure 7.
  • the phosphate is a second type of phosphate of the compound of formula (I), wherein the molar ratio of the compound of formula (I) to phosphoric acid in the phosphate is 1: (0.8-1.2) .
  • the molar ratio of the compound of formula (I) to phosphoric acid is 1: (0.9-1.1).
  • the molar ratio of the compound of formula (I) to phosphoric acid may be 1:0.8, 1:0.9, 1:1, 1:1.1 or 1:1.2.
  • the molar ratio of the compound of formula (I) to phosphoric acid in the phosphate is 1:1.
  • the phosphate salt of the compound of Formula (I) is in an anhydrous form, a hydrate form, or a solvate form.
  • the phosphate salt of the compound of formula (I) is a crystalline powder and exists in crystal form.
  • the phosphate salt 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 angle 2 ⁇ (°) values: 18.230 ⁇ 0.2 and 21.144 ⁇ 0.2.
  • the molar ratio of the compound of formula (I) to phosphoric acid in the phosphate crystal form II is 1:1.
  • the X-ray powder diffraction pattern of the phosphate crystal form II further includes 2 Or more than 2 (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc.) selected from the following set of features at the diffraction angle 2 ⁇ (°) value Diffraction peaks: 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, 18.771 ⁇ 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.
  • the X-ray powder diffraction pattern of the phosphate crystal form II is selected from 2 or more (such as 2, 3, 4, 5, 6, 7, 8, 9, 10) or all There are characteristic diffraction peaks at the following diffraction angle 2 ⁇ (°) 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.
  • the X-ray powder diffraction pattern of the phosphate crystal form II has characteristic diffraction peaks at the following diffraction angle 2 ⁇ (°) 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.
  • the X-ray powder diffraction pattern of the phosphate crystal form II is in 2 or more (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc.) or all have characteristic diffraction peaks at the diffraction angle 2 ⁇ (°) values selected from the following: 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, 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, 26.2 73 ⁇ 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.
  • the X-ray powder diffraction pattern of the phosphate crystal form II has characteristic diffraction peaks at the following diffraction angle 2 ⁇ (°) 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, 18.230 ⁇ 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, 30.14 3 ⁇ 0.2, 31.072 ⁇ 0.2, 31.88 ⁇ 0.2 and 33.982 ⁇ 0.2.
  • the X-ray powder diffraction pattern of the phosphate crystal form II has the characteristic diffraction peaks expressed in 2 ⁇ (°) values and d values as shown in Table 4, and the relative intensity of each peak is as shown in Table 4.
  • the X-ray powder diffraction pattern (XRPD pattern) of the phosphate crystal form II is substantially as shown in Figure 10.
  • the differential scanning calorimetry curve of the phosphate crystal form II has an endothermic peak at 207.48 ⁇ 3°C, ⁇ 2°C, ⁇ 1°C, or ⁇ 0.5°C.
  • the starting temperature in the differential scanning calorimetry curve of the 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
  • 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.
  • the differential scanning calorimetry curve (DSC curve) of the phosphate crystal form II is substantially as shown in Figure 11.
  • the melting point of the phosphate crystal form II of the compound of formula (I) is 207.48 ⁇ 0.5°C.
  • thermogravimetric analysis curve (TGA curve) of the phosphate crystal form II of the compound of formula (I) is substantially as shown in Figure 11.
  • the TGA curve of the phosphate crystal form II of the compound of formula (I) shows almost no weight loss near 100°C, and begins to melt when heated to a melting point of 207.48°C.
  • the TGA curve of the phosphate crystalline form II of the compound of formula (I) shows that the phosphate crystalline form II is anhydrous.
  • the dynamic moisture absorption spectrum (DVS chart) of the phosphate crystal form II of the compound of formula (I) is substantially as shown in Figure 12.
  • the DVS plot of the phosphate crystal form II of the compound of formula (I) shows a hygroscopic weight gain of 0.45% under 80% relative humidity (RH) conditions.
  • a micrograph of the phosphate crystal form II of the compound of Formula (I) is substantially as shown in Figure 13.
  • a microscopic image of the phosphate form II of the compound of formula (I) shows that the phosphate form II is rod-shaped.
  • a second aspect of the present disclosure provides a method for preparing L-tartrate salt of the compound of formula (I).
  • the preparation method of the L-tartrate salt of the compound of formula (I) includes the following steps: carrying out a salt-forming reaction of the compound of formula (I) and L-tartaric acid to form the L-tartrate salt of the compound of formula (I). -Tartrates.
  • 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.
  • the molar ratio of L-tartaric acid in the L-tartrate of the compound of formula (I) formed to the compound of formula (I) is (0.8-1.2): 1. In some embodiments, the molar ratio of L-tartaric acid to the compound of formula (I) in the formed L-tartrate salt of the compound of formula (I) is (0.9-1.1):1. In one embodiment, the molar ratio of L-tartaric acid and the compound of formula (I) in the formed L-tartrate salt of the compound of formula (I) is 1:1.
  • a method for preparing the polymorph of L-tartrate of the compound of formula (I) is also provided, wherein the polymorph is L-tartrate crystal form I, including the following steps:
  • the organic solvent is selected from one or more of ethanol, acetonitrile, ethyl acetate, acetone and methanol.
  • 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.
  • 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.
  • a method for preparing L-tartrate crystal form II including the following steps:
  • reaction solution was slowly cooled down, and an antisolvent was added to obtain the L-tartrate crystal form II.
  • the organic solvent is selected from one or more of ethanol, acetonitrile, ethyl acetate, acetone and methanol.
  • the antisolvent is selected from one or more of methyl tert-butyl ether, petroleum ether, n-heptane, n-hexane, cyclohexane, isopropyl alcohol, 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, isopropyl alcohol and acetone.
  • 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.
  • the molar ratio of L-tartaric acid and 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 salt 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.
  • the temperature of the salt-forming reaction is -10°C to 90°C.
  • the temperature of the salt-forming reaction can be 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 Any range (including the end values) consisting of the end values of °C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C and 90°C, without specific limitations.
  • 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.
  • the molar concentration of L-tartaric acid is 0.1-5 mol/L.
  • the molar concentration of L-tartaric acid can be 0.1mol/L, 1mol/L, 1.5mol/L, 2mol/L, 2.5mol/L, 3mol/L, 3.5mol/L, 4mol/L, 4.5mol /L or 5mol/L.
  • the molar concentration of L-tartaric acid is 0.5-3 mol/L.
  • the molar concentration of L-tartaric acid is 1-2 mol/L.
  • the preparation method of L-tartrate crystal form I includes the following steps:
  • step (BI-b) Cool the reaction solution obtained in step (BI-a) to precipitate a solid, separate the solid and liquid, and collect the solid phase to obtain the L-tartrate crystal form I of the compound of formula (I).
  • the temperature of the reaction in step (BI-a) is 10°C to 60°C.
  • the molar ratio of the compound of formula (I) to the L-tartaric acid contained in the L-tartaric acid aqueous solution in step (BI-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 in step (BI-a) is 1: (0.9-1.2).
  • 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 other embodiments, the molar concentration of the L-tartaric acid aqueous solution in step (BI-a) is 1-2 mol/L.
  • the solvent in step (BI-a) is ethanol.
  • step (BI-a) after stirring at 30-60°C in step (BI-a), the mixture is stirred at room temperature. In some embodiments, in step (BI-a), after stirring at 30-60°C for 2-5 hours, the mixture is stirred at room temperature. In some embodiments, in step (BI-a), after stirring at 30-60°C for 2-5 hours, the mixture is stirred at room temperature for 7-16 hours.
  • the reaction solution is cooled to -10°C to 10°C in step (BI-b). In some embodiments, the reaction solution is cooled to -5°C to 5°C. In some embodiments, the reaction solution is cooled to about 0°C.
  • the cooling time of the reaction solution in step (BI-b) is 0.25-3h. In some embodiments, the cooling time of the reaction solution is 0.5-2 h. In some embodiments, the cooling time of the reaction solution is 0.5-1 h.
  • the separation method in step (BI-b) is selected from centrifugal separation and filtration separation.
  • the method further includes a step of evaporating the solvent or drying.
  • the drying step after separation in step (BI-b) is performed at 25-70°C.
  • the preparation method of L-tartrate crystal form II includes the following steps:
  • step (BII-b) Cool the reaction solution obtained in step (BII-a), add an antisolvent for crystallization, separate the solid and liquid, and collect the solid phase to obtain the L-tartrate crystal form II of the compound of formula (I).
  • the reaction temperature in step (BII-a) is 10-60°C.
  • 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).
  • 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.
  • the solvent in step (BII-a) is methanol.
  • step (BII-a) after stirring at 30-60°C in step (BII-a), the mixture is stirred at room temperature. In some embodiments, in step (BII-a), after stirring at 30-60°C for 2-5 hours, the mixture is stirred at room temperature. In some embodiments, in step (BII-a), after stirring at 30-60°C for 2-5 hours, the mixture is stirred at room temperature for 7-16 hours.
  • the reaction solution is cooled to -10°C to 10°C in step (BII-b). In some embodiments, the reaction solution is cooled to -5°C to 5°C. In some embodiments, the reaction solution is cooled to about 0°C.
  • the cooling time of the reaction solution in step (BII-b) is 0.25-3h. In some embodiments, the cooling time of the reaction solution is 0.5-2 h. In some embodiments, the cooling time of the reaction solution is 0.5-1 h.
  • 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.
  • the antisolvent is selected from one or more of methyl tert-butyl ether, acetone, acetonitrile and ethyl acetate.
  • the antisolvent is methyl tert-butyl ether.
  • the separation method in step (BII-b) is selected from centrifugal separation and filtration separation.
  • a step of evaporating the solvent or drying is further included.
  • drying step after separation in step (BII-b) is performed at 25-70°C.
  • the preparation method of L-tartrate amorphous substance includes the following steps:
  • step (C-b) Cool the reaction liquid obtained in step (C-a) to precipitate a solid, separate the solid from the liquid, and collect the solid phase to obtain amorphous L-tartrate.
  • the temperature of the reaction in step (C-a) is 10-60°C.
  • the molar ratio of the compound of formula (I) to the L-tartaric acid contained in the L-tartaric acid aqueous solution in step (C-a) is 1: (0.8-1.4). 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 (C-a) is 1: (0.9-1.2).
  • the molar concentration of the L-tartaric acid aqueous solution in step (C-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 some embodiments, the molar concentration of the L-tartaric acid aqueous solution is 1-2 mol/L.
  • the solvent in step (C-a) is acetone.
  • step (C-a) after stirring at 30-60°C in step (C-a), the mixture is stirred at room temperature. In some embodiments, in step (C-a), after stirring at 30-60°C for 2-5 hours, the mixture is stirred at room temperature. In some embodiments, in step (C-a), after stirring at 30-60°C for 2-5 hours, the mixture is stirred at room temperature for 7-16 hours.
  • the reaction solution is cooled to -10°C to 10°C in step (C-b). In some embodiments, the reaction solution is cooled to -5°C to 5°C. In some embodiments, the reaction solution is cooled to about 0°C.
  • the cooling time of the reaction solution in step (C-b) is 0.25-3h. In some embodiments, the cooling time of the reaction solution is 0.5-2 h. In some embodiments, the cooling time of the reaction solution is 0.5-1 h.
  • the separation method in step (C-b) is selected from centrifugal separation and filtration separation.
  • a step of evaporating the solvent or drying is further included.
  • drying step after separation in step (C-b) is performed at 25-70°C.
  • a third aspect of the present disclosure provides a method for preparing a phosphate salt of a compound of formula (I).
  • the method for preparing the phosphate salt of the compound of formula (I) includes the following steps: carrying out a salt-forming reaction between the compound of formula (I) and phosphoric acid in the presence of an organic solvent to form the phosphate salt of the compound of formula (I).
  • the phosphate salt is a second type of phosphate salt of a compound of Formula (I).
  • the molar ratio of the compound of formula (I) to phosphoric acid is 1: (0.8-1.2).
  • 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.
  • the molar ratio of the compound of formula (I) to phosphoric acid is 1: (0.9-1.1). In some embodiments, the molar ratio of the compound of formula (I) to phosphoric acid is 1:1. In some embodiments, the molar ratio of the compound of formula (I) to phosphoric acid is 1:0.95.
  • the molar ratio of the compound of formula (I) to phosphoric acid in the phosphate is 1: (0.8-1.2). In some embodiments, the molar ratio of the compound of formula (I) to phosphoric acid in the phosphate salt is 1: (0.9-1.1). In some embodiments, the molar ratio of the compound of formula (I) to phosphoric acid in the phosphate salt may be 1:0.8, 1:0.9, 1:1, 1:1.1 or 1:1.2. In some embodiments, the molar ratio of the compound of formula (I) to phosphoric acid in the phosphate salt is 1:1.
  • 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.
  • the organic solvent is one or more of methanol, ethyl acetate, and acetone. In some embodiments, the organic solvent is one or more of methanol and ethyl acetate. In some embodiments, the organic solvent is a mixture of methanol and ethyl acetate.
  • the molar ratio of the compound of formula (I) to phosphoric acid in the formed phosphate salt of the compound of formula (I) is 1:1.
  • a method for preparing phosphate crystal form II including the following steps:
  • the feeding molar ratio of the compound of formula (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 the compound of formula (I) to phosphoric acid is 1: (0.9-1.1). In one embodiment, the molar ratio of the compound of formula (I) to phosphoric acid is 1:0.95. In another embodiment, the molar ratio of the compound of formula (I) to phosphoric acid is 1:1.
  • the salt-forming reaction is performed in one or both of ethanol and acetone.
  • the salt-forming reaction is performed in ethanol. In some embodiments, the salt-forming reaction is performed in methanol.
  • a method for preparing phosphate crystal form II includes the following steps:
  • the feeding molar ratio of the compound of formula (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 the compound of formula (I) to phosphoric acid is 1: (0.9-1.1). In one embodiment, the molar ratio of the compound of formula (I) to phosphoric acid is 1:1.
  • a method for preparing phosphate crystal form II includes the following steps:
  • the feeding molar ratio of the compound of formula (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 the compound of formula (I) to phosphoric acid is 1: (0.9-1.1). In one embodiment, the molar ratio of the compound of formula (I) to phosphoric acid is 1:1.
  • the molar ratio of the compound of formula (I) to phosphoric acid in the phosphate crystal form II is 1:1.
  • the temperature of the salt-forming reaction is -10°C to 90°C, for example, it can be selected from -10°C, -5°C, 0°C, 5°C, 10°C, 15°C, 20°C, 25°C Any range (including the end values) consisting of the end values of °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. value), there is no specific limit.
  • centrifugal separation and filtration separation may be used to collect the solid in step (ii) or (ii-1).
  • a step of evaporating the solvent or drying is further included.
  • the drying step is performed at 25°C to 70°C.
  • the phosphate salt is a first type of phosphate salt of a compound of Formula (I).
  • the molar ratio of the compound of formula (I) to phosphoric acid described 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).
  • the molar ratio of phosphoric acid to the compound of formula (I) in the phosphate salt is (1.8-2.4):1. In some embodiments, the molar ratio of the 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 the phosphoric acid to the compound of formula (I) is (1.9-2.3):1. In one embodiment, the molar ratio of the phosphoric acid to the compound of formula (I) is 2:1.
  • the organic solvent is selected from one or more of ethanol, ethyl acetate, acetone and methanol.
  • the molar ratio of phosphoric acid to the compound of formula (I) in the formed phosphate salt of the compound of formula (I) is 2:1.
  • a method for preparing the phosphate crystal form I of the compound of formula (I) includes:
  • step (AI-b) Cool the reaction solution obtained in step (AI-a), add an antisolvent to crystallize, separate the solid and liquid, and collect the solid phase to obtain the compound of formula (I) Phosphate crystal form I.
  • reaction temperature in step (AI-a) is 10-60°C.
  • 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).
  • 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.
  • the reaction liquid in step (AI-b), is cooled to -10°C-10°C, preferably -5°C-5°C; in some embodiments, the reaction liquid is cooled to about 0°C °C.
  • reaction liquid cooling time in step (AI-b) is 0.5-2 h; in some embodiments, the reaction liquid cooling time is 0.5 h.
  • the antisolvent is selected from one or more of methyl tert-butyl ether, petroleum ether, n-heptane, n-hexane, cyclohexane, isopropyl alcohol, acetone, acetonitrile and ethyl acetate.
  • the antisolvent is different from the solvent; preferably, the antisolvent is selected from one or more of methyl tert-butyl ether, n-heptane, isopropyl alcohol and acetone.
  • the separation method in step (AI-b) is selected from centrifugal separation and filtration separation.
  • the separation in step (AI-b) further includes a step of evaporating the solvent or drying.
  • the drying step after separation in step (AI-b) is performed at 25-70°C; for example, it can be 25°C, 30°C, 40°C, 50°C, 60°C or 70°C, etc., specifically No restrictions.
  • 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 contained 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 phosphoric acid aqueous solution is 0.1-5mol/L, preferably 0.5-3mol/L, more preferably 1-2mol/L ; and/or the reaction solution is cooled to -10°C-10°C in step (AI-b), preferably -5°C-5°C, more preferably about 0°C; and/or the reaction in step (AI-b)
  • the liquid cooling time is 0.5-2h, more preferably 0.5h; and/or the anti-solvent is selected from methyl tert-butyl ether, petroleum ether, n-heptane, n-hexane, cyclohexane, isopropyl alcohol
  • One or more; 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) also includes the step of evaporating the solvent or drying; and/or Or the drying step after separation in step (AI-b) is performed at 25-70°C.
  • a fourth aspect of the present disclosure provides a pharmaceutical composition comprising:
  • the pharmaceutical composition includes (a) an L-tartrate salt of a compound of formula (I), a first type of phosphate salt of a compound of formula (I), or a compound of formula (I) described in the first aspect of the present disclosure. a second type of phosphate salt of the compound; and (b) a pharmaceutically acceptable carrier.
  • the pharmaceutical composition includes (a) L-tartrate crystal form I of the compound of formula (I) described in the first aspect of the present disclosure, L-tartrate crystal form II of the compound of formula (I) , L-tartrate amorphous form of the compound of formula (I), phosphate crystal form I of the compound of formula (I), or phosphate crystal form II of the compound of formula (I); and (b) a pharmaceutically acceptable carrier .
  • the fifth aspect of the present disclosure provides a pharmaceutically acceptable salt of the compound of formula (I) described in the first aspect of the present disclosure, or the use of the pharmaceutical composition described in the fourth aspect of the present disclosure in the preparation of a kinase inhibitor.
  • the pharmaceutically acceptable salts of the compound of formula (I) include the L-tartrate salt of the compound of formula (I), the first type of phosphate salt of the compound of formula (I) and the second type of the compound of formula (I). Phosphate.
  • the pharmaceutically acceptable salts of the compound of formula (I) include L-tartrate crystal form I of the compound of formula (I), L-tartrate crystal form II of the compound of formula (I), The L-tartrate amorphous form of compound (I), the phosphate crystalline form I of the compound of formula (I), and the phosphate crystalline form II of the compound of formula (I).
  • the kinase inhibitor is a CDK9 inhibitor.
  • the sixth aspect of the present disclosure provides a pharmaceutically acceptable salt of the compound of formula (I) described in the first aspect of the present disclosure, or the pharmaceutical composition described in the fourth aspect of the present disclosure for the preparation of treatment and/or prevention and Applications in medicines for diseases related to or mediated by CDK9 activity.
  • the pharmaceutically acceptable salts of the compound of formula (I) include the L-tartrate salt of the compound of formula (I), the first type of phosphate salt of the compound of formula (I) and the second type of the compound of formula (I). Phosphate.
  • the pharmaceutically acceptable salts of the compound of formula (I) include L-tartrate crystal form I of the compound of formula (I), L-tartrate crystal form II of the compound of formula (I), (I) L- of the compound Tartrate amorphous form, phosphate crystal form I of the compound of formula (I), and phosphate crystal form II of the compound of formula (I).
  • the present disclosure also provides pharmaceutically acceptable salts of the compounds of formula (I) described in the first aspect of the present disclosure for the treatment and/or prevention of diseases related to or mediated by CDK9 activity, or the present disclosure.
  • the pharmaceutical composition according to the fourth aspect is provided.
  • the present disclosure also provides a method for inhibiting CDK9 activity, which includes administering to a desired subject a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) described in the first aspect of the present disclosure, or a pharmaceutically acceptable salt of the compound of formula (I) described in the fourth aspect of the present disclosure.
  • a method for inhibiting CDK9 activity which includes administering to a desired subject a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) described in the first aspect of the present disclosure, or a pharmaceutically acceptable salt of the compound of formula (I) described in the fourth aspect of the present disclosure.
  • the pharmaceutical composition described above includes administering to a desired subject a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) described in the first aspect of the present disclosure, or a pharmaceutically acceptable salt of the compound of formula (I) described in the fourth aspect of the present disclosure.
  • the present disclosure also provides a method for treating diseases related to or mediated by CDK9 activity, the method comprising administering to a desired subject an effective amount of a pharmaceutical compound of formula (I) described in the first aspect of the present disclosure.
  • a pharmaceutical compound of formula (I) described in the first aspect of the present disclosure comprising administering to a desired subject an effective amount of a pharmaceutical compound of formula (I) described in the first aspect of the present disclosure.
  • the above acceptable salt, or the pharmaceutical composition according to the fourth aspect of the present disclosure are examples of a pharmaceutical compound of formula (I) described in the first aspect of the present disclosure.
  • the diseases include hyperproliferative diseases, virus-induced infectious diseases, and cardiovascular diseases.
  • the disease is a hyperproliferative disease.
  • the hyperproliferative diseases include angiogenic or vasoproliferative disorders, mesangial cell proliferative disorders, and solid tumors.
  • the solid tumors are, for example, cancers of the breast, respiratory tract, brain, reproductive organs, digestive tract, urinary tract, eyes, liver, skin, head and neck, thyroid or parathyroid glands, and their distant metastases.
  • the disease is selected from one or more of lymphoma, sarcoma, and leukemia.
  • the disease is cancer.
  • the cancer is, for example, pancreatic cancer, breast cancer, ovarian cancer, cervical cancer or leukemia.
  • the disease includes solid tumors and hematological tumors.
  • diseases related to or mediated by CDK9 activity include diseases related to or involving CDK9 activity (eg, excessive activity of CDK9), as well as conditions accompanying these diseases.
  • Hyperactivity of CDK9 refers to increased CDK9 enzymatic activity compared to normal, non-disease cells, or to increased CDK9 leading to unwanted cell proliferation, or reduced or insufficient programmed cell death (apoptosis) activity, or refers to mutations that lead to constitutive activation of CDK9.
  • Hyperproliferative diseases include diseases involving undesired or uncontrolled proliferation of cells, and it includes diseases involving reduced or insufficient programmed cell death (apoptosis).
  • the pharmaceutically acceptable salt of the compound of formula (I) of the present disclosure, or the pharmaceutical composition containing the pharmaceutically acceptable salt of the compound of formula (I) can be used to prevent, inhibit, and inhibit cell proliferation and/or cell division. interrupt, reduce, reduce, control, etc., and/or produce apoptosis.
  • Treating and/or preventing a disease associated with or mediated by CDK9 activity includes administering to a subject in need thereof (including a mammal, such as a human) an amount of Formula (I) of the present disclosure effective to treat or prevent the disease.
  • the phosphate and L-tartrate salts of the compound of formula (I) have higher solubility than the free base, and the compound of formula (I) L-tartrate crystal form I, the compound of formula (I) L-tartaric acid Salt crystal form II, compound of formula (I) L-tartrate amorphous substance, compound of formula (I) phosphate crystal form I and compound of formula (I) phosphate crystal form II all have good physical stability and are suitable for Drug development.
  • the phosphate crystal form I of the compound of formula (I) can be converted into the more stable phosphate crystal form II under suitable conditions, such as in ethanol.
  • the phosphate crystal form II of the compound of formula (I) has lower hygroscopicity and better crystallinity and stability.
  • Figure 1 is an X-ray powder diffraction (XRPD) pattern of the phosphate crystal form I of the compound of formula (I).
  • Figure 2 is a differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) spectrum of the phosphate crystal form I of the compound of formula (I).
  • Figure 3 is the dynamic moisture absorption curve (DVS) of the phosphate crystal form I of the compound of formula (I).
  • Figure 4 is a microscopic picture of the phosphate crystal form I of the compound of formula (I).
  • Figure 5 is an X-ray powder diffraction (XRPD) pattern of compound L-tartrate crystal form I of formula (I).
  • FIG. 6 is a thermogravimetric analysis (TGA) spectrum of compound L-tartrate crystal form I of formula (I).
  • Figure 7 is an X-ray powder diffraction (XRPD) pattern of compound L-tartrate crystal form II of formula (I).
  • Figure 8 is an X-ray powder diffraction (XRPD) pattern of the amorphous L-tartrate salt of compound (I).
  • Figure 9 is an ellipsoid diagram of the molecular three-dimensional structure of a single crystal of the compound of formula (II).
  • Figure 10 is an X-ray powder diffraction (XRPD) pattern of the phosphate crystal form II of the compound of formula (I).
  • Figure 11 is a differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) spectrum of the phosphate crystal form II of the compound of formula (I).
  • Figure 12 is the dynamic moisture absorption curve (DVS) of the phosphate crystal form II of the compound of formula (I).
  • Figure 13 is a microscope image of the phosphate crystal form II of the compound of formula (I).
  • Figure 14 is a comparative X-ray powder diffraction (XRPD) pattern of the phosphate crystal form I of the compound of formula (I) transformed into the phosphate crystal form II of the compound of formula (I) in ethanol.
  • XRPD X-ray powder diffraction
  • Figure 15 is an XRPD change diagram of compound L-tartrate crystal form I of formula (I) under high temperature (60°C) and accelerated (40°C-75% RH) conditions.
  • Figure 16 is an XRPD change diagram of the phosphate crystal form I of the compound of formula (I) under high temperature conditions.
  • Figure 17 is a graph showing changes in tumor volume after once-daily oral administration of compound D and compound of formula (I).
  • Figure 18 is a graph showing changes in body weight of mice after oral administration of compound D and compound of formula (I) once a day.
  • Crystals of the present disclosure may be used interchangeably.
  • the compound of formula (I) is (1S, 3S)-N1-(5-((S)-1-cyclobutylethyl)pyrazolo[1,5-a]pyrimidin-7-yl) Cyclopentane-1,3-diamine has the following structure:
  • Polymorph A solid that exists in either amorphous or crystalline form. In the case of the crystalline form, the molecules are positioned within a three-dimensional lattice site. When a compound crystallizes from a solution or slurry, it can crystallize in different spatial lattice arrangements (a property known as "polymorphism"), forming crystals with different crystalline forms, which are known as Called "polymorph”. Different polymorphs of a given substance may differ from one another in one or more physical properties, such as solubility and dissolution rate, true specific gravity, crystalline form, packing pattern, flowability, and/or solid state stability.
  • Production-scale crystallization can be accomplished by manipulating the solution so that the solubility limit of the compound of interest is exceeded. This can be accomplished by a variety of methods, such as dissolving the compound at a relatively high temperature and then cooling the solution below the saturation limit. Or reduce the liquid volume by boiling, normal pressure evaporation, vacuum drying or other methods.
  • the solubility of a compound of interest can be reduced by adding an antisolvent or a solvent or a mixture of such solvents in which the compound has low solubility. Another option is to adjust the pH to reduce solubility.
  • the "suspension shaking” mentioned in this disclosure refers to a method of mixing the compound of formula (I) and the corresponding acid or a solution of the corresponding acid in a suitable solvent to form a turbid liquid and then shaking to obtain crystals.
  • suitable solvents can be water or organic solvents.
  • “Suspension centrifugation” as described in the present disclosure refers to mixing the compound of formula (I) and the corresponding acid or a solution of the corresponding acid in a suitable solvent to form a turbid A method of centrifuging the turbid liquid to obtain crystals.
  • suitable solvents can be water or organic solvents.
  • the “slow volatilization” mentioned in this disclosure refers to a method in which a solution containing a compound of formula (I) and the corresponding acid is placed at a certain temperature to slowly evaporate the solvent to obtain crystals.
  • antisolvent addition or “antisolvent addition” described in this disclosure refers to a method of adding another suitable solvent to a solution of the compound of formula (I) and then precipitating to obtain crystals.
  • optimization of crystallization may include seeding the crystallization medium with crystals of the desired form. Additionally, many crystallization methods use a combination of the above strategies. One embodiment involves dissolving the compound of interest in a solvent at elevated temperatures and subsequently adding an appropriate volume of antisolvent in a controlled manner to bring the system just below saturation levels. At this point, the desired form of seed crystal can be added (and the integrity of the seed crystal maintained), and the system is cooled to complete crystallization. As used herein, the term "about” means ⁇ 5 based on a given numerical value.
  • polymorphs of the present disclosure includes, but is not limited to, Compound of Formula (I) phosphate crystalline Form I, Compound of Formula (I) phosphate crystalline Form II, Compound of Formula (I) L-tartrate crystalline salt Form I, and the compound of formula (I) L-tartrate salt form II.
  • phosphate salts of compounds of formula (I) particularly monophosphate salts, and Form II of the phosphate salts of compounds of formula (I).
  • a pharmaceutically acceptable salt of a compound of Formula (I) of the present disclosure as an active ingredient can be administered in a suitable dosage form with one or more pharmaceutical carriers.
  • the phosphate salt, L-tartrate salt, or polymorphic form of the compound of formula (I) of the present disclosure as the active ingredient can be formed into a suitable dosage form with one or more pharmaceutical carriers for administration.
  • “Pharmaceutically acceptable carrier” refers to a non-toxic, inert, solid, semi-solid substance or liquid filling machine, diluent, packaging material or auxiliary preparation or any type of excipient, which is in contact with the subject to be administered (in some embodiments (e.g., mammals, in one embodiment human), which are suitable for delivering the active substances of the present disclosure to the intended target site without terminating their activity.
  • compositions of the present disclosure are formulated, dosed, and administered in a manner consistent with good medical practice.
  • the "therapeutically effective amount" of an 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 method of administration.
  • the present disclosure provides pharmaceutically acceptable salts of the compounds of formula (I) described in the first aspect of the present disclosure, and the pharmaceutical compositions described in the fourth aspect of the present disclosure can be used to treat and/or prevent CDK9 activity-related or Drugs for diseases mediated by CDK9 activity.
  • the present disclosure provides a method for inhibiting CDK9 activity, which includes administering to a subject a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) described in the first aspect of the present disclosure, or a drug described in the fourth aspect of the present disclosure. combination.
  • a "therapeutically effective amount” refers to a formula (I) of the present disclosure that will cause a biological or medical response in an individual, such as reducing or inhibiting enzymatic protein activity or improving symptoms, alleviating disease, alleviating or delaying disease progression, or preventing disease, etc. ) amount of a pharmaceutically acceptable salt of a compound.
  • subject refers to an animal, preferably a mammal, and more preferably a human.
  • mammal refers to warm-blooded vertebrate mammals, including, for example, cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, rats, pigs, and humans.
  • Treatment means to alleviate, delay the progression, attenuate, prevent, or maintain an existing disease or condition (eg, cancer). Treatment also includes curing, preventing the progression of, or alleviating to some degree one or more symptoms of a disease or condition.
  • the structure and purity of compounds are determined by nuclear magnetic resonance ( 1H NMR) and/or liquid mass spectrometry (LC-MS).
  • LC-MS Agilent 1290 HPLC System/6130/6150 MS liquid mass spectrometer (manufacturer: Agilent), column Waters BEH/CHS, 50 ⁇ 2.1mm, 1.7 ⁇ m.
  • HPLC analysis uses Agilent 1260 Infinity HPLC, OpenLAB CDS Chemstation workstation, column XBridge C18 4.6*250mm, ID 5 ⁇ m column, and detector DAD.
  • Elemental analysis was performed using an inductively coupled plasma optical emission spectrometer, model icp 500; power 1300w; flow rate 1mL/min.
  • Known starting materials can be synthesized by methods known in the art, or can be purchased from ABCR GmbH&Co.KG, Acros Organics, Aldrich Chemical Company, Shaoyuan Chemical Technology (Accela ChemBio Inc) and Dari Chemicals, etc. company.
  • room temperature in the following examples refers to about 20-30°C.
  • X-ray powder diffraction (XRPD): In this disclosure, the above-mentioned crystalline or amorphous powder X-ray diffraction pattern is obtained by known methods in the art, using the ARL Equinox3000 X-ray powder diffraction analyzer. The XRPD test parameters are as follows in Table 5 shown.
  • the position of each peak is determined by the 2 ⁇ (°) value. It is understood that different instruments and/or conditions may result in slightly different data, with variations in the position and relative intensity of individual peaks.
  • the intensity division of the peaks only reflects the approximate size of the peaks at each location.
  • the diffraction peak with the highest peak height of each crystal form is used as the base peak, and its relative intensity is defined as 100%.
  • the peak of I 0 phosphate crystal form I with a 2 ⁇ (°) value of 18.803
  • the peak with a 2 ⁇ (°) value of 20.27 for L-tartrate crystal form I is the base peak
  • the peak with a 2 ⁇ (°) value of 21.353 for L-tartrate crystal form II is the base peak
  • the peak with a 2 ⁇ (°) value of 21.353 for L-tartrate crystal form II is the base peak.
  • the peak with a 2 ⁇ (°) value of 18.230 is the base peak), and the ratio of the peak height of each other peak to the peak height of the base peak is regarded as its relative intensity I/I 0.
  • the division and definition of the relative intensity of each peak is as shown in Table 6 below.
  • Single crystal X-ray diffraction SXRD:
  • SXRD Single crystal X-ray diffraction
  • the phosphate of the compound of formula (I) and its crystal form of the present disclosure are determined by elemental analysis to determine the acid-base molar ratio.
  • the L-tartrate of the compound of formula (I) and its crystal form and its amorphous form are determined by HPLC/IC or 1 H NMR. Determine the acid-base molar ratio.
  • High Performance Liquid Chromatography In this disclosure, high performance liquid chromatography (HPLC) was collected on an Agilent 1260 HPLC.
  • DSC Differential Scanning Calorimetry
  • thermogravimetric analysis In this disclosure, the thermogravimetric analysis spectrum of the above crystalline form is obtained by a method known in the art, using a TGA550 thermogravimetric analyzer. The TGA test parameters are shown in Table 9 below.
  • Dynamic moisture adsorption (DVS) curve collected on the DVS Intrinsic of SMS (Surface Measurement Systems). The relative humidity at 25°C is corrected for the deliquescent points of LiCl, Mg(NO 3 ) 2 and KCl. Instrument test conditions are shown in Table 10 below.
  • phosphoric acid solution and “L-tartaric acid solution” used in this disclosure refer to phosphoric acid aqueous solution and L-tartaric acid aqueous solution.
  • Phosphate and “L-tartrate” used in this disclosure refer to the phosphate of the compound of formula (I) and the L-tartrate of the compound of formula (I).
  • the solvent used in this disclosure is an analytically pure solvent, such as ethanol, which is analytically pure ethanol (moisture content ⁇ 0.3%).
  • MTBE used in this disclosure 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 methyl alcohol; CDI refers to N , N-carbonyldiimidazole; 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 iso
  • Step 1 Dissolve sodium hydride (18g, 450.00mmol, purity 60%) in THF (300mL), cool to 0°C, and add triethyl 2-phosphonopropyl ester (100g, 419.78mmol) slowly dropwise. The reaction was stirred at 0-5°C for 1 hour, and then cyclobutanone (25g, 356.69mmol) was dissolved in THF (50mL) and slowly added dropwise. After the dropwise addition was completed, the reaction was carried out at 25°C for 15 hours. The reaction was quenched with saturated sodium chloride solution (600mL), and then extracted with ethyl acetate (600mL ⁇ 2).
  • Step 2 Dissolve compound 2 (44g, 285.33mmol) in methanol (400mL), and add wet palladium on carbon (4.4g, purity 10%). Cover with a hydrogen balloon, replace the hydrogen three times, and stir under a hydrogen atmosphere (15 psi) at 27°C for 6 to 8 hours. TLC showed that the raw material spot disappeared. Pd/C was filtered off, and the solvent was removed under reduced pressure to obtain crude compound 3 (44 g, colorless oil, boiling point approximately 182°C), yield: 98.71%, proceed to the next step without purification.
  • Step 3 Dissolve compound 3 (44g, 281.65mmol) in methanol (400mL), add sodium hydroxide (45.06g, 1.13mol) and water (200mL), and then stir at room temperature for 16 hours. LCMS showed the reaction was complete. Concentrate under reduced pressure to remove methanol, then extract twice with dichloromethane, adjust the pH value to 3 with dilute hydrochloric acid (6M) in the aqueous phase, extract DCM (300mL ⁇ 3), combine the organic phases, dry and spin to obtain compound 4 (35g, Colorless oil, boiling point is about 220°C), yield: 96.96%, go to the next step without purification. MS m/z(ESI):127.1[MH] - .
  • Step 4 Dissolve compound 4 (36g, 280.88mmol) in THF (300mL) in a reaction bottle, then add CDI (68.32g, 421.32mmol), and then react at room temperature for 16 hours (as solution A).
  • solution A potassium monomethyl malonate
  • anhydrous magnesium chloride 66.86g, 702.20mmol
  • THF 900mL
  • solution B solution B
  • solution A was added dropwise to solution B at room temperature (about 10 minutes), and then the mixed solution was stirred at 30°C for 16 hours.
  • LCMS detects that the reaction is complete and the product is produced.
  • Step 5 Dissolve compound 5 (42g, 211.85mmol) and 3-aminopyrazole (19.36g, 233.03mmol) in glacial acetic acid (300mL), raise the temperature to 120°C and react for 16h. LCMS showed the reaction was complete. Concentrate under reduced pressure to remove acetic acid, and slurry with ethyl acetate (800 mL ⁇ 4). The solid is precipitated, filtered, and dried to obtain compound 6 (41 g, light yellow solid), yield: 89.08%. Go to the next step without purification. MS m/z(ESI):218.1[M+H] + .
  • Step 1 Dissolve compound 6 (473.6 mg, 2.18 mmol) in phosphorus oxychloride (6 mL) and heat to 120°C and stir for 3 hours. Cool to room temperature, pour into ice water (60g), extract with dichloromethane (80mL), dry the organic phase over anhydrous sodium sulfate, filter, and concentrate under reduced pressure. The residue was separated by silica gel column chromatography (20 g, 0%-40% EA/DCM) to obtain compound 7.
  • Step 2 Dissolve compound 7 (142.7 mg, 605.5 ⁇ mol) and ((1S,3S)-3-aminocyclopentyl)carbamic acid tert-butyl ester (121.27 mg, 605.52 ⁇ mol) 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. Add ethyl acetate (80 mL) to dilute, wash with saturated sodium chloride solution (80 mL ⁇ 3), dry the organic phase over anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain compound 8.
  • Step 3 Dissolve compound 8 (174.7 mg, 437.38 ⁇ mol) in 1,4-dioxane (3 mL), add hydrochloric acid solution (3.0 mL, 4 M), and stir at room temperature for 3 hours.
  • the solvent was evaporated under reduced pressure, water (60mL) was added, and extracted with ethyl acetate (50mL).
  • the organic phase was extracted with ethyl acetate (60mL ⁇ 2). Dry over sodium sulfate, filter, and concentrate under reduced pressure.
  • Compound 9 was isolated by preparative HPLC chromatography.
  • Step 4 Chiral separation of compound 9 (94.46mg, 315.48 ⁇ mol) (column type: IC-3 4.6*100mm 3um; co-solvent: IPA [1% NH 3 (7M in MeOH)]; injection volume: 5.00uL; wavelength: 220.0nm; running time: 6.0 minutes; flow rate: 3.0mL/min; pressure: 2000psi; column temperature: 40°C) to obtain the compound of formula (I) (9.70mg, retention time 2.471min), yield: 9.89%, purity: 96.34%. MS m/z(ESI):300.2[M+H] + .
  • the compound of formula (II) is prepared from the compound of formula (I), and the absolute configuration of the compound of formula (I) is determined by the absolute configuration of the compound of formula (II).
  • a single crystal of the compound of formula (II) was further prepared.
  • the ellipsoid diagram of the molecular three-dimensional structure of the compound of formula (II) is shown in Figure 9.
  • the Flack constant is 0.07(2), and C8, C10 and C19 are S configuration, that is, the absolute configurations of the three chiral centers of the compound of formula (II) are all S configuration, and their structure is the same as that of the compound of formula (II). shown. Therefore, it can be determined that the absolute configurations of the three chiral centers of the compound of formula (I) are all S configurations, and their structures are as shown in the structure of the compound of formula (I).
  • the obtained solid obtained a phosphorus content of 12.5% through elemental analysis, indicating that the molar ratio of the compound of formula (I) to phosphoric acid in the obtained solid was 1:2 (the theoretical value of the phosphorus content was 12.5%);
  • the XRPD pattern of the obtained solid was basically as follows As shown in Figure 1, the XRPD pattern of the obtained solid has a peak at the 2 ⁇ (°) value shown in the aforementioned Table 1, and the relative intensity of each peak is as shown in the aforementioned Table 1; its DSC and TGA patterns are shown in Figure 2.
  • the DVS diagram is shown in Figure 3, and its microscopic picture is shown in Figure 4.
  • the polarized light microscope picture shows that the phosphate crystal form I of the compound of formula (I) is in the shape of rods and blocks.
  • HPLC/IC results show that the molar ratio of the compound of formula (I) to L-tartaric acid in the obtained solid is 1:1; its XRPD pattern is basically as shown in Figure 5, and the XRPD pattern of the obtained solid is as shown in the aforementioned Table 2 There are peaks at the 2 ⁇ (°) value, and the relative intensity of each peak is shown in the aforementioned Table 2; its TGA chart is shown in Figure 6.
  • the TGA chart shows that the weight loss is 1.057% when heated to around 110°C, which may be due to the volatilization of water.
  • the weight loss continues to be 3.482% when heated to around 178°C, which is speculated to be due to the volatilization of organic solvents, and melting and decomposition occur with continued heating.
  • the obtained solid was subjected to HPLC/IC detection, and it was determined that the molar ratio of the compound of formula (I) to L-tartaric acid in the obtained solid was 1:1; the XRPD pattern of the obtained solid was shown in Figure 7, and the XRPD pattern of the obtained solid was in the aforementioned table There is a peak at the 2 ⁇ (°) value shown in 3, and the relative intensity of each peak is as shown in the aforementioned Table 3.
  • Compound L-tartrate of formula (I) is defined in this disclosure as Form II.
  • the obtained solid was subjected to HPLC/IC, and it was determined that the molar ratio of the compound of formula (I) to L-tartaric acid in the obtained solid was 1:1; the XRPD pattern of the obtained solid was shown in Figure 8, and was defined as L-tartrate in this application. Amorphous.
  • the XRPD pattern of the obtained solid has a peak at the 2 ⁇ (°) value shown in the aforementioned Table 4, and the relative intensity of each peak is as shown in the aforementioned Table 4; its DSC and TGA curves are shown in Figure 11, and its DVS pattern is shown in Figure 12 shows, and its microscopic picture is shown in Figure 13.
  • the obtained solid obtained a phosphorus content of 7.7% through elemental analysis, indicating that the molar ratio of the compound of formula (I) to phosphoric acid in the obtained solid was 1:1 (the theoretical value of the phosphorus content was 7.79%); the obtained solid was subjected to XRPD detection, and the The XRPD diagram is basically shown in Figure 10.
  • the solubility of the phosphate and L-tartrate of the compound of formula (I) prepared in the above example in water was tested.
  • the solubility of the phosphate was no more than 26.6 mg/mL, and the solubility of the L-tartrate was no more than 26.6 mg/mL. Greater than 17.3mg/mL, while the free base is slightly soluble in water.
  • the salt of the compound of formula (I) greatly improves its solubility in water, and the phosphate in the resulting salt has higher solubility than L-tartrate.
  • the test results show that: the compound of formula (I) L-tartrate crystal form I, the compound of formula (I) L-tartrate crystal form II, the compound of formula (I) phosphate crystal form I, the compound of formula (I) phosphate crystal form
  • the crystal form of II has no obvious changes under the above conditions and has high stability.
  • the XRPD pattern of the crystal form I of compound L-tartrate of formula (I) was stored under high temperature conditions for 7 days and 2 months (60 days) and showed no obvious change; the crystal form of L-tartrate of compound formula (I) was The overlay of the XRPD pattern of the crystal form of I stored under accelerated conditions for 7 days and 2 months (60 days) is shown in Figure 15, showing no obvious change; this shows that the compound of formula (I) L-tartrate crystal form I Has high stability.
  • the phosphate crystal form I of the compound of formula (I) was stored under high temperature conditions for 7 days and 30 days.
  • the overlay of the XRPD pattern of its crystal form is shown in Figure 16, showing no obvious change, indicating that the phosphate crystal of the compound of formula (I) Type I has higher stability.
  • the hygroscopicity test was carried out in accordance with the operation of 9103 (Guiding Principles for Drug Hygroscopicity Test) in the "Chinese Pharmacopoeia (2020)", and it was found that the L-tartrate salt of the compound of formula (I) was slightly hygroscopic.
  • the DVS diagram of the phosphate crystal form I of the compound of formula (I) is shown in Figure 3.
  • the DVS diagram is shown in Under the condition of 80% RH, the hygroscopic weight gain was 9%, indicating that the phosphate crystal form I of the compound of formula (I) has hygroscopicity.
  • the DVS chart of the phosphate crystal form II shows that under 80% RH conditions, the moisture absorption only increases by 0.45%, indicating that the phosphate crystal form II is slightly hygroscopic.
  • Test Example 4 Activity inhibition test on CDK family kinases
  • the kinase reagent was purchased from Carna Bioscience, the reaction substrate and detection reagent were purchased from PerkinElmer, and the remaining reagents were purchased from Thermo scientific.
  • the kinase activity test uses a 10 ⁇ L system, which contains the following components: CDK kinase diluent, Ulight-Myelic basic protein (PerkinElmer, #TRF-0109, hereinafter referred to as U-MBP) and ATP (Thermo scientific, #PV3227) mixed base diluent, and the compound of formula (I) of the present disclosure (i.e., the test substance).
  • U-MBP Ulight-Myelic basic protein
  • ATP Thermo scientific, #PV3227
  • Each kinase in the test includes three test groups: background group (Blank), non-inhibitory group (PC) and compound test group (Test). The components included in each set of tests are shown in Table 15 below.
  • Compound Dissolve 10mM of the compound to be tested at room temperature and perform gradient dilution with DMSO, and then dilute it with deionized water to make a 4x compound working solution.
  • the DMSO content is 2%.
  • the highest concentrations of compounds used in testing were 10 ⁇ M for CDK1 and CDK2 and 1 ⁇ M for CDK9.
  • 1.33x reaction buffer The ingredients are 26.7mM MOPS, 6.67mM MgCl2 and 0.0133% Tween-20. After preparation, store it in a 4°C refrigerator in the dark. Add freshly prepared DTT to a final concentration of 5.33mM before use.
  • the DMSO working concentration in the reaction was 0.5%.
  • test groups including Blank, PC, and Test groups.
  • 10 ⁇ L detection solution The components of 10 ⁇ L detection solution are: 16mM EDTA (Thermo scientific, #15575), 1nM phosphorylated U-MBP protein antibody (PerkinElmer, #TRF-0201) and 1x detection buffer (PerkinElmer, #CR97-100).
  • Ratio value 665nm/615nm value
  • the LC/MS/MS method was used to determine the drug concentrations in the plasma of mice at different times after intravenous injection and intragastric administration of compound D, compound E and compound of formula (I).
  • Compound D, compound E and compound of formula (I) were studied. Pharmacokinetic behavior of the compound in mice and evaluation of its pharmacokinetic characteristics.
  • mice healthy adult male ICR mice (weight 30-40g, 12 mice, mice in the intravenous injection group were allowed to drink water and eat freely, mice in the intragastric administration group were fasted overnight, and were allowed to drink water and eat freely 4 hours after administration), provided by Beijing Provided by Vital River Laboratory Animal Co.LTD;
  • Tail vein administration (2 mg/kg, 5% DMSO, pH 4.5 20% Captisol) and intragastric administration (10 mg/kg, 5% DMSO, pH 4.5 20% Captisol) in ICR mice.
  • Blood sample collection Select animals that meet the experimental requirements before administration, and weigh and mark them. Before collecting blood samples, mice were bound, and each administered mouse was collected at predetermined blood collection time points (intravenous administration: 0.083, 0.25, 0.5, 1, 2, 4, 6, 7.5, respectively, after administration). Collect blood at 24 hours, a total of 9 time points; intragastric administration: Collect blood at 0.083, 0.25, 0.5, 1, 2, 4, 6, 7.5, 24 hours after administration, a total of 9 time points), and collect blood through the orbit for about 100 ⁇ L. Transfer the blood to a 1.5mL test tube pre-added with K 2 EDTA, centrifuge for 4 minutes (8000rpm, 4°C), and remove the plasma. The entire process is completed within 15 minutes after blood collection.
  • mice Female, 6-10 weeks old, weighing approximately 20-23 grams, maintained in a special pathogen-free environment in individual ventilated cages (5 mice per cage , 2 cages (10 animals per group). All cages, bedding and water are disinfected before use. All animals had free access to a standard certified commercial laboratory diet.
  • tumor volume When the average tumor volume reaches approximately 165 cubic millimeters, patients will be randomly divided into groups according to body weight and tumor volume, and drug administration will begin. Test compounds were administered orally by gavage every day. Antitumor efficacy was determined by dividing the mean tumor volume increase in compound-treated animals by the mean tumor volume increase in untreated animals.
  • Tumor volume was measured twice weekly using a two-dimensional caliper, and the volume was measured in cubic millimeters.
  • Tumor volume TV 0.5a ⁇ b 2 .
  • a is the long diameter of the tumor and b is the short diameter of the tumor.
  • the relative tumor proliferation rate T/C is the percentage value of the relative tumor volume (RTV) of the treatment group and the control group at a certain time point.
  • mpk refers to milligrams per kilogram of body weight.
  • the body weight change (%) of tumor-bearing animals was calculated as follows: (body weight at measurement - body weight at grouping)/body weight at grouping ⁇ 100.

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Abstract

涉及一种如式(I)所示的取代的吡唑并[1,5-a]嘧啶-7-胺衍生物的药学上可接受的盐和多晶型物，包含其的药物组合物，其制备方法，和该药学上可接受的盐和多晶型物在治疗或预防与CDK9活性相关的或由CDK9活性介导的疾病中的应用。

Description

取代的吡唑并[1,5-a]嘧啶-7-胺衍生物的药学上可接受的盐和多晶型物及其应用
相关申请
本申请要求2022年07月22日提交中国专利局，申请号为No.202210866135.0，名称为“取代的吡唑并[1,5-a]嘧啶-7-胺衍生物的药学上可接受的盐和多晶型物及其应用”以及申请号为No.202210870337.2，名称为“CDK9抑制剂的磷酸盐和多晶型物及其应用”的中国专利申请的优先权，在此将其全文引入作为参考。
技术领域
本公开属于医药技术领域，具体地涉及取代的吡唑并[1,5-a]嘧啶-7-胺衍生物的药学上可接受的盐和多晶型物、包含其的药物组合物、其制备方法，及其医药应用。
背景技术
CDK9是细胞周期依赖性激酶(CDK)蛋白家族的成员之一，在基因的转录调节中具有重要作用。CDK9主要通过磷酸化RNA复合酶II的碳端区域来调控基因的转录延长。CDK9在肿瘤中普遍高表达，也是肿瘤细胞进展和维持的重要因素。CDK9抑制剂通过抑制基因的转录延长下调相关癌蛋白(MYC)的表达，及凋亡抑制剂蛋白Mcl-1的表达，从而促进癌细胞的凋亡。CDK9抑制剂通过调节表观遗传因子BRG1，使沉默的基因重新被激活，包括肿瘤细胞中内源性逆转录病毒(ERVs)的激活，促进干扰素表达，使肿瘤细胞对免疫疗法更敏感。
目前已有多家企业进行CDK9抑制剂的开发，包括Bayer公司开发的选择性CDK9抑制剂BAY1251152，AstraZeneca公司开发的选择性CDK9抑制剂AZD4573，Tolero公司开发的非选择性CDK9抑制剂TP-1287以及常州千红制药公司开发的非选择性CDK9抑制剂QHRD107等。
药品活性成分的溶解度、稳定性对于成药性研究具有重要的影响，并且固体形式有利于产品的质量控制，因此，有必要对产品的固体形式进行探索。
发明内容
基于此，本公开的目的在于提供取代的吡唑并[1,5-a]嘧啶-7-胺衍生物的药学上可接受的盐和多晶型物及其应用。具体地，所述药学上可接受的盐为取代的吡唑并[1,5-a]嘧啶-7-胺衍生物的L-酒石酸盐和磷酸盐。该取代的吡唑并[1,5-a]嘧啶-7-胺衍生物是CDK9抑制剂，其分子结构为式(I)所示的(1S,3S)-N1-(5-((S)-1-环丁基乙基)吡唑并[1,5-a]嘧啶-7-基)环戊烷-1,3-二胺。
本公开的第一方面，提供了式(I)化合物的药学上可接受的盐：
其中，所述药学上可接受的盐选自：磷酸盐和L-酒石酸盐。
在一些实施例中，所述式(I)化合物的药学上可接受的盐为无水形式、水合物形式或溶剂合物形式。
在一些实施例中，所述式(I)化合物的药学上可接受的盐为晶体。在一些实施例中，所述式(I)化合物的药学上可接受的盐为无定型物。
在一些实施例中，所述药学上可接受的盐为磷酸盐。在一些实施例中，所述磷酸盐为式(I)化合物的 第一类磷酸盐，其中磷酸与所述式(I)化合物的摩尔比为(1.8-2.4)：1。在一些实施例中，所述磷酸与所述式(I)化合物的摩尔比可以为1.8:1、1.9:1、2.0:1、2.1:1、2.2:1、2.3:1或2.4:1。在一些实施例中，所述磷酸与所述式(I)化合物的摩尔比为(1.9-2.3)：1。在一个实施例中，所述磷酸与所述式(I)化合物的摩尔比为2:1。
在一些实施例中，所述磷酸盐为式(I)化合物磷酸盐晶型I，其X射线粉末衍射图在以下衍射角2θ(°)值处具有特征衍射峰：18.234±0.2、19.131±0.2和21.266±0.2。在一些实施例中，所述磷酸盐为式(I)化合物磷酸盐晶型I，其X射线粉末衍射图在以下衍射角2θ(°)值处具有特征衍射峰：18.803±0.2、18.234±0.2、19.131±0.2和21.266±0.2。
在一些实施例中，所述磷酸盐晶型I的X射线粉末衍射图除在以下衍射角2θ(°)值处具有特征衍射峰：18.234±0.2、19.131±0.2和21.266±0.2以外，进一步还包括在2个或2个以上(如3、4、5、6、7、8、9、10、11、12、13、14、15个等)选自下组的衍射角2θ(°)值处的特征衍射峰：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、18.803±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和34.017±0.2。
在一些实施例中，所述磷酸盐晶型I的X射线粉末衍射图在3个或更多个(如3、4、5、6、7、8、9、10、11、12、13、14、15个等)或全部选自以下衍射角2θ(°)值处具有特征衍射峰：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、18.234±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和34.017±0.2。
在一些实施例中，所述磷酸盐晶型I的X射线粉末衍射图在以下衍射角2θ(°)值处具有特征衍射峰：13.999±0.2、18.234±0.2、18.803±0.2、19.131±0.2、21.266±0.2、22.01±0.2和23.247±0.2。
在一些实施例中，所述磷酸盐晶型I的X射线粉末衍射图在以下衍射角2θ(°)值处具有特征衍射峰：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和28.513±0.2。
在一些实施例中，所述磷酸盐晶型I的X射线粉末衍射图在以下衍射角2θ(°)值处具有特征衍射峰：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、18.234±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和34.017±0.2。
在一些实施例中，式(I)化合物磷酸盐晶型I的X射线粉末衍射图具有表1所示的以2θ(°)值和d值表达的特征衍射峰，各峰相对强度如表1所示。
表1.磷酸盐晶型I的2θ(°)、d值及其相对强度I/I0

在一些实施例中，所述式(I)化合物磷酸盐晶型I中磷酸与所述式(I)化合物的摩尔比为2:1。
在一些实施例中，式(I)化合物磷酸盐晶型I的X射线粉末衍射图基本如图1所示。
在一些实施例中，式(I)化合物磷酸盐晶型I的差示扫描量热曲线在188.01℃±3℃、188.01℃±2℃、188.01℃±1℃或188.01℃±0.5℃处具有吸热峰。在一些实施例中，式(I)化合物磷酸盐晶型I的差示扫描量热曲线中的起始温度为188.01℃±3℃、188.01℃℃±2℃、188.01℃℃±1℃或188.01℃℃±0.5℃，峰值温度为193.69℃±3℃、193.69℃±2℃、193.69℃±1℃或193.69℃±0.5℃。在一些实施例中，式(I)化合物磷酸盐晶型I的差示扫描量热分析谱图(DSC谱图)基本如图2所示。图2所示实施例中，式(I)化合物磷酸盐晶型I的熔点约为188.01±0.5℃。
在一些实施例中，式(I)化合物磷酸盐晶型I的热重分析图谱(TGA图)基本如图2所示。在一些实施例中，式(I)化合物磷酸盐晶型I的TGA图谱显示在190℃附近失重1.139％。
在一些实施例中，式(I)化合物磷酸盐晶型I的动态水分吸收图谱(DVS图)基本如图3所示。在一些实施例中，式(I)化合物磷酸盐晶型I的DVS图显示，在80％相对湿度(RH)条件下，吸湿增重9％。
在一些实施例中，式(I)化合物磷酸盐晶型I的显微镜图基本如图4所示。在一些实施例中，式(I)化合物磷酸盐晶型I的显微镜图显示磷酸盐晶型I呈棒状和块状。
在该第一方面，在一些实施例中，所述药学上可接受的盐为L-酒石酸盐。所述L-酒石酸盐中L-酒石酸与所述式(I)化合物的摩尔比为(0.8-1.2)：1。在一些实施例中，所述L-酒石酸与所述式(I)化合物的摩尔比可以为0.8:1、0.9:1、1.0:1、1.1:1或1.2:1。在一些实施例中，所述L-酒石酸与所述式(I)化合物的摩尔比为(0.9-1.1)：1。在一个实施例中，L-酒石酸与所述式(I)化合物的摩尔比为1:1。
在一些实施例中，所述L-酒石酸盐为式(I)化合物L-酒石酸盐晶型I，其X射线粉末衍射图在下组衍射角2θ(°)值处具有特征衍射峰：13.946±0.2、16.881±0.2、19.405±0.2、21.505±0.2和24.262±0.2。
在一些实施例中，所述L-酒石酸盐晶型I的X射线粉末衍射图除在下组衍射角2θ(°)值处具有特征衍射峰：13.946±0.2、16.881±0.2、19.405±0.2、21.505±0.2和24.262±0.2以外，进一步还包括在2个或2个以上(如3、4、5、6、7、8、9、10、11、12、13、14、15个等)选自下组衍射角2θ(°)值处的特征衍射峰：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和34.543±0.2。
在一些实施例中，所述L-酒石酸盐晶型I的X射线粉末衍射图在5个或更多个(如5、6、7、8、9、10、11、12、13、14、15个等)或全部选自以下衍射角2θ(°)值处具有特征衍射峰：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、16.881±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和34.543±0.2。
在一些实施例中，所述L-酒石酸盐晶型I的X射线粉末衍射图在以下衍射角2θ(°)值处具有特征衍射峰：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和31.579±0.2。
在一些实施例中，所述L-酒石酸盐晶型I的X射线粉末衍射图在以下衍射角2θ(°)值处具有特征衍射峰：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和34.543±0.2。
在一些实施例中，所述L-酒石酸盐晶型I的X射线粉末衍射图在以下衍射角2θ(°)值处具有特征衍射峰：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、16.881±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 和34.543±0.2。
在一些实施例中，所述L-酒石酸盐晶型I的X射线粉末衍射图具有表2所示的以2θ(°)值和d值表达的特征衍射峰，各峰相对强度如表2所示。
表2.L-酒石酸盐晶型I的2θ(°)、d值及其相对强度I/I0
在一些实施例中，所述L-酒石酸盐晶型I中L-酒石酸与所述式(I)化合物的摩尔比为1:1。
在一些实施例中，式(I)化合物L-酒石酸盐晶型I的X射线粉末衍射图基本如图5所示。
在一些实施例中，式(I)化合物L-酒石酸盐晶型I的热重分析图谱基本如图6所示。在一些实施例中，式(I)化合物L-酒石酸盐晶型I的TGA图谱显示在110℃附近失重1.057％，在178℃附近继续失重3.482％。
在一些实施例中，所述L-酒石酸盐为式(I)化合物L-酒石酸盐晶型II，其X射线粉末衍射图在下组衍射角2θ(°)值处具有特征衍射峰：11.662±0.2和21.353±0.2。
在一些实施例中，所述L-酒石酸盐晶型II的X射线粉末衍射图除在下组衍射角2θ(°)值处具有特征衍射峰：11.662±0.2和21.353±0.2以外，进一步还包括在2个或2个以上(如3、4、5、6、7、8、9、10、11、12、13、14个等)选自下组衍射角2θ(°)值处的特征衍射峰：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和31.784±0.2。
在一些实施例中，所述L-酒石酸盐晶型II的X射线粉末衍射图在3个或更多个(如3、4、5、6、7、8、9、10、11、12、13、14、15个等)或全部选自以下衍射角2θ(°)值处具有特征衍射峰：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和31.784±0.2。
在一些实施例中，所述L-酒石酸盐晶型II的X射线粉末衍射图在以下衍射角2θ(°)值处具有特征衍射峰：11.662±0.2、14.244±0.2、17.481±0.2、18.349±0.2、21.026±0.2和21.353±0.2。
在一些实施例中，所述L-酒石酸盐晶型II的X射线粉末衍射图在以下衍射角2θ(°)值处具有特征衍射峰：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和31.784±0.2。
在一些实施例中，所述L-酒石酸盐晶型II的X射线粉末衍射图在以下衍射角2θ(°)值处具有特征衍射峰：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和31.784±0.2。
在一些实施例中，所述L-酒石酸盐晶型II的X射线粉末衍射图具有表3所示的以2θ(°)值和d值表达的特征衍射峰，各峰相对强度如表3所示。
表3.L-酒石酸盐晶型II的2θ(°)、d值及其相对强度I/I0
在一些实施例中，所述L-酒石酸盐晶型II中L-酒石酸与所述式(I)化合物的摩尔比为1:1。
在一些实施例中，所述L-酒石酸盐晶型II的X射线粉末衍射图基本如图7所示。
在该第一方面，在一些实施例中，所述磷酸盐为式(I)化合物的第二类磷酸盐，其中磷酸盐中式(I)化合物与磷酸的摩尔比为1：(0.8-1.2)。在一些实施例中，式(I)化合物与磷酸的摩尔比为1：(0.9-1.1)。在一些实施例中，式(I)化合物与磷酸的摩尔比可以为1：0.8，1：0.9，1：1，1：1.1或1：1.2。在一些实施例中，所述磷酸盐中式(I)化合物与磷酸的摩尔比为1：1。
在一些实施例中，所述式(I)化合物的磷酸盐为无水形式、水合物形式或溶剂合物形式。
在一些实施例中，所述式(I)化合物的磷酸盐为结晶性粉末，以晶体形式存在。
在一些实施例中，所述式(I)化合物的磷酸盐为磷酸盐晶型II，其X射线粉末衍射图在以下衍射角2θ(°)值处具有特征衍射峰：18.230±0.2和21.144±0.2。
在一些实施例中，所述磷酸盐晶型II中式(I)化合物与磷酸的摩尔比为1：1。
在一些实施例中，所述磷酸盐晶型II的X射线粉末衍射图除在以下衍射角2θ(°)值处具有特征衍射峰：18.230±0.2和21.144±0.2以外，进一步还包括在2个或2个以上(如2、3、4、5、6、7、8、9、10、11、12、13、14、15个等)选自下组衍射角2θ(°)值处的特征衍射峰：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、18.771±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和33.982±0.2。
在一些实施例中，所述磷酸盐晶型II的X射线粉末衍射图在2个或更多个(如2、3、4、5、6、7、8、9、10个)或全部选自以下的衍射角2θ(°)值处具有特征衍射峰：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和23.96±0.2。
在一些实施例中，所述磷酸盐晶型II的X射线粉末衍射图在以下衍射角2θ(°)值处具有特征衍射峰：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和23.96±0.2。
在一些实施例中，所述磷酸盐晶型II的X射线粉末衍射图在2个或更多个(如2、3、4、5、6、7、8、9、10、11、12、13、14、15个等)或全部选自以下的衍射角2θ(°)值处具有特征衍射峰：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、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和33.982±0.2。
在一些实施例中，所述磷酸盐晶型II的X射线粉末衍射图在以下衍射角2θ(°)值处具有特征衍射峰：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、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和33.982±0.2。
在一些实施例中，所述磷酸盐晶型II的X射线粉末衍射图具有表4所示的以2θ(°)值和d值表达的特征衍射峰，各峰相对强度如表4所示。
表4.磷酸盐晶型II的2θ(°)、d值及其相对强度I/I0
在一些实施例中，所述磷酸盐晶型II的X射线粉末衍射图(XRPD图)基本如图10所示。
在一些实施例中，所述磷酸盐晶型II的差示扫描量热曲线在207.48±3℃、±2℃、±1℃或±0.5℃处具有吸热峰。在一些实施例中，所述磷酸盐晶型II的差示扫描量热曲线中的起始温度为207.48℃℃±3℃、207.48℃±2℃、207.48℃±1℃或207.48℃±0.5℃，峰值温度为214.31℃±3℃、214.31℃±2℃、214.31℃±1℃或214.31℃±0.5℃。在一些实施例中，所述磷酸盐晶型II的差示扫描量热分析曲线(DSC曲线)基本如图11所示。图11所示实施例中，式(I)化合物磷酸盐晶型II的熔点为207.48±0.5℃。
在一些实施例中，式(I)化合物磷酸盐晶型II的热重分析曲线(TGA曲线)基本如图11所示。在一些实施例中，式(I)化合物磷酸盐晶型II的TGA曲线显示在100℃附近几乎没有失重，加热到熔点207.48℃附近开始熔化。在一些实施例中，式(I)化合物磷酸盐晶型II的TGA曲线显示磷酸盐晶型II为无水物。
在一些实施例中，式(I)化合物磷酸盐晶型II的动态水分吸收图谱(DVS图)基本如图12所示。在一些实施例中，式(I)化合物磷酸盐晶型II的DVS图显示，在80％相对湿度(RH)条件下，吸湿增重0.45％。
在一些实施例中，式(I)化合物磷酸盐晶型II的显微镜图基本如图13所示。在一些实施例中，式(I)化合物磷酸盐晶型II的显微镜图显示磷酸盐晶型II呈杆状。
本公开的第二方面，提供了一种式(I)化合物的L-酒石酸盐的制备方法。
在一些实施例中，式(I)化合物的L-酒石酸盐的制备方法包括如下步骤：将所述式(I)化合物与L-酒石酸进行成盐反应，形成所述式(I)化合物的L-酒石酸盐。
在一些实施例中，所述L-酒石酸盐的制备方法中，L-酒石酸与式(I)化合物的投料摩尔比为(0.8-1.4)：1。在一些实施例中，L-酒石酸与式(I)化合物的投料摩尔比为(0.9-1.2)：1。
在一些实施例中，所述L-酒石酸盐的制备方法中，所形成的式(I)化合物L-酒石酸盐中的L-酒石酸与式(I)化合物的摩尔比为(0.8-1.2)：1。在一些实施例中，所形成的式(I)化合物L-酒石酸盐中的L-酒石酸与式(I)化合物的摩尔比为(0.9-1.1)：1。在一个实施例中，所形成的式(I)化合物L-酒石酸盐中的L-酒石酸与式(I)化合物的摩尔比为1:1。
在一些实施例中，还提供了一种式(I)化合物的L-酒石酸盐的多晶型物的制备方法，其中所述多晶型物为L-酒石酸盐晶型I，包括如下步骤：
将式(I)化合物与L-酒石酸在有机溶剂中进行成盐反应，形成反应液溶液；
将所述反应液溶液缓慢降温，得到所述L-酒石酸盐晶型I。
在一些实施例中，所述有机溶剂选自乙醇、乙腈、乙酸乙酯、丙酮和甲醇中的一种或多种。
在一些实施例中，所述L-酒石酸盐晶型I的制备方法中，L-酒石酸与式(I)化合物的投料摩尔比为(0.8-1.4)：1。在另一些实施例中，L-酒石酸与式(I)化合物的投料摩尔比为(0.9-1.2)：1。
在一些实施例中，所述L-酒石酸盐晶型I的制备方法中，所得的L-酒石酸盐晶型I中L-酒石酸与式(I)化合物的摩尔比为(0.8-1.2)：1。在另一些实施例中，所得的L-酒石酸盐晶型I中L-酒石酸与式(I)化合物的摩尔比为(0.9-1.1)：1。在一个实施例中，所得的L-酒石酸盐晶型I中L-酒石酸与式(I)化合物的摩尔比为1:1。
在一些实施例中，还提供了L-酒石酸盐晶型II的制备方法，包括如下步骤：
将式(I)化合物与L-酒石酸在有机溶剂中进行成盐反应，形成反应液溶液；
将所述反应液溶液缓慢降温，添加反溶剂，得到所述L-酒石酸盐晶型II。
在一些实施例中，所述有机溶剂选自乙醇、乙腈、乙酸乙酯、丙酮和甲醇中的一种或多种。
在一些实施例中，所述反溶剂选自甲基叔丁醚、石油醚、正庚烷、正己烷、环己烷、异丙醇、丙酮、乙腈和乙酸乙酯中的一种或多种，且所述反溶剂与所述有机溶剂不同。在另一些实施例中，所述反溶剂选自甲基叔丁醚、正庚烷、异丙醇和丙酮中的一种或多种。
在一些实施例中，所述L-酒石酸盐晶型II的制备方法中，L-酒石酸与式(I)化合物的投料摩尔比为(0.8-1.4)：1。在另一些实施例中，L-酒石酸与式(I)化合物的投料摩尔比为(0.9-1.2)：1。
在一些实施例中，所述L-酒石酸盐晶型II的制备方法中，所得的L-酒石酸盐晶型II中的L-酒石酸与式(I)化合物的摩尔比为(0.8-1.2)：1。在另一些实施例中，所得的L-酒石酸盐晶型II中的L-酒石酸与式(I)化合物的摩尔比为(0.9-1.1)：1。在一个实施例中，所得的L-酒石酸盐晶型II中的L-酒石酸与式(I)化合物的摩尔比为1:1。
在一些实施例中，成盐反应的温度为-10℃至90℃。例如，成盐反应的温度可以为选自-10℃、-5℃、0℃、5℃、10℃、15℃、20℃、25℃、30℃、35℃、40℃、45℃、50℃、55℃、60℃、65℃、70℃、75℃、80℃、85℃和90℃的端值组成的任意范围(包含端值)，具体不做限定。
在一些实施例中，将所述反应液溶液缓慢降温至-10℃至60℃。在一些实施例中，将所述反应液溶液缓慢降温至0℃至室温。在一些实施例中，将所述反应液溶液缓慢降温至室温。
在一些实施例中，所述L-酒石酸的摩尔浓度为0.1-5mol/L。例如，所述L-酒石酸的摩尔浓度可以为0.1mol/L、1mol/L、1.5mol/L、2mol/L、2.5mol/L、3mol/L、3.5mol/L、4mol/L、4.5mol/L或5mol/L。在另一些实施例中，所述L-酒石酸的摩尔浓度为0.5-3mol/L。在另一些实施例中，所述L-酒石酸的摩尔浓度为1-2mol/L。
在一些实施例中，L-酒石酸盐晶型I的制备方法包括如下步骤：
(BI-a)将式(I)化合物溶于溶剂中，加入L-酒石酸水溶液，搅拌反应；和
(BI-b)将步骤(BI-a)得到的反应液冷却，析出固体，固液分离，收集固相，得到式(I)化合物L-酒石酸盐晶型I。
在一些实施例中，步骤(BI-a)中反应的温度为10℃至60℃。
在一些实施例中，步骤(BI-a)中式(I)化合物与L-酒石酸水溶液中含有的L-酒石酸的摩尔比为1：(0.8-1.4)。在另一些实施例中，步骤(BI-a)中式(I)化合物与L-酒石酸水溶液中含有的L-酒石酸的摩尔比为1：(0.9-1.2)。
在一些实施例中，步骤(BI-a)中L-酒石酸水溶液的摩尔浓度为0.1-5mol/L。在另一些实施例中，步骤(BI-a)中L-酒石酸水溶液的摩尔浓度为0.5-3mol/L。在另一些实施例中，步骤(BI-a)中L-酒石酸水溶液的摩尔浓度为1-2mol/L。
在一些实施例中，步骤(BI-a)中溶剂为乙醇。
在一些实施例中，步骤(BI-a)中在30-60℃搅拌后，再在室温下搅拌。在一些实施例中，步骤(BI-a)中在30-60℃搅拌2-5h后，再在室温下搅拌。在一些实施例中，步骤(BI-a)中在30-60℃搅拌2-5h后，再在室温下搅拌7-16h。
在一些实施例中，步骤(BI-b)中将反应液冷却至-10℃至10℃。在一些实施例中，将反应液冷却至-5℃至5℃。在一些实施例中，将反应液冷却至约为0℃。
在一些实施例中，步骤(BI-b)中反应液冷却的时间为0.25-3h。在一些实施例中，反应液冷却的时间为0.5-2h。在一些实施例中，反应液冷却的时间为0.5-1h。
在一些实施例中，步骤(BI-b)中分离方式选自离心分离和过滤分离。
在一些实施例中，步骤(BI-b)中的分离之后，所述方法还包括挥干溶剂或干燥的步骤。
在一些实施例中，步骤(BI-b)中分离之后的干燥步骤在25-70℃下进行。
在一些实施例中，L-酒石酸盐晶型II的制备方法包括如下步骤：
(BII-a)将式(I)化合物溶于溶剂中，加入L-酒石酸水溶液，搅拌反应；
(BII-b)将步骤(BII-a)得到的反应液冷却，加入反溶剂析晶，固液分离，收集固相，得到式(I)化合物的L-酒石酸盐晶型II。
在一些实施例中，步骤(BII-a)中反应的温度为10-60℃。
在一些实施例中，步骤(BII-a)中式(I)化合物与L-酒石酸水溶液中含有的L-酒石酸的摩尔比为1：(0.8-1.4)。在另一些实施例中，式(I)化合物与L-酒石酸水溶液中含有的L-酒石酸的摩尔比为1：(0.9-1.2)。
在一些实施例中，步骤(BII-a)中L-酒石酸水溶液的摩尔浓度为0.1-5mol/L。在一些实施例中，L-酒石酸水溶液的摩尔浓度为0.5-3mol/L。在另一些实施例中，L-酒石酸水溶液的摩尔浓度为1-2mol/L。
在一些实施例中，步骤(BII-a)中溶剂为甲醇。
在一些实施例中，步骤(BII-a)中在30-60℃搅拌后，再在室温下搅拌。在一些实施例中，步骤(BII-a)中在30-60℃搅拌2-5h后，再在室温下搅拌。在一些实施例中，步骤(BII-a)中在30-60℃搅拌2-5h后，再在室温下搅拌7-16h。
在一些实施例中，步骤(BII-b)中将反应液冷却至-10℃至10℃。在一些实施例中，将反应液冷却至-5℃至5℃。在一些实施例中，将反应液冷却至约为0℃。
在一些实施例中，步骤(BII-b)中反应液冷却的时间为0.25-3h。在一些实施例中，反应液冷却的时间为0.5-2h。在一些实施例中，反应液冷却的时间为0.5-1h。
在一些实施例中，步骤(BII-b)中反溶剂选自甲基叔丁醚、正庚烷、正己烷、环己烷、丙酮、乙腈和乙酸乙酯中的一种或多种，且所述溶剂和所述反溶剂不同。在一些实施例中，所述反溶剂选自甲基叔丁基醚、丙酮、乙腈和乙酸乙酯中的一种或多种。在一些实施例中，所述反溶剂为甲基叔丁基醚。
在一些实施例中，步骤(BII-b)中分离方式选自离心分离和过滤分离。
在一些实施例中，步骤(BII-b)中的分离之后，还包括挥干溶剂或干燥的步骤。
在一些实施例中，步骤(BII-b)中分离之后的干燥步骤在25-70℃下进行。
在一些实施例中，L-酒石酸盐无定型物的制备方法包括如下步骤：
(C-a)将式(I)化合物溶于溶剂中，加入L-酒石酸水溶液，搅拌反应；
(C-b)将步骤(C-a)得到的反应液冷却，析出固体，固液分离，收集固相，得到L-酒石酸盐无定型物。
在一些实施例中，步骤(C-a)中反应的温度为10-60℃。
在一些实施例中，步骤(C-a)中式(I)化合物与L-酒石酸水溶液中含有的L-酒石酸的摩尔比为1：(0.8-1.4)。在一些实施例中，步骤(C-a)中式(I)化合物与L-酒石酸水溶液中含有的L-酒石酸的摩尔比为1：(0.9-1.2)。
在一些实施例中，步骤(C-a)中L-酒石酸水溶液的摩尔浓度为0.1-5mol/L。在一些实施例中，L-酒石酸水溶液的摩尔浓度为0.5-3mol/L。在一些实施例中，L-酒石酸水溶液的摩尔浓度为1-2mol/L。
在一些实施例中，步骤(C-a)中溶剂为丙酮。
在一些实施例中，步骤(C-a)中在30-60℃搅拌后，再在室温下搅拌。在一些实施例中，步骤(C-a)中在30-60℃搅拌2-5h后，再在室温下搅拌。在一些实施例中，步骤(C-a)中在30-60℃搅拌2-5h后，再在室温下搅拌7-16h。
在一些实施例中，步骤(C-b)中将反应液冷却至-10℃至10℃。在一些实施例中，将反应液冷却至-5℃至5℃。在一些实施例中，将反应液冷却至约为0℃。
在一些实施例中，步骤(C-b)中反应液冷却的时间为0.25-3h。在一些实施例中，反应液冷却的时间为0.5-2h。在一些实施例中，反应液冷却的时间为0.5-1h。
在一些实施例中，步骤(C-b)中分离方式选自离心分离和过滤分离。
在一些实施例中，步骤(C-b)中的分离之后，还包括挥干溶剂或干燥的步骤。
在一些实施例中，步骤(C-b)中分离之后的干燥步骤在25-70℃下进行。
本公开的第三方面，提供了式(I)化合物的磷酸盐的制备方法。
所述式(I)化合物的磷酸盐的制备方法包括如下步骤：将所述式(I)化合物与磷酸在有机溶剂存在下进行成盐反应，形成所述式(I)化合物的磷酸盐。
在一些实施例中，所述磷酸盐为式(I)化合物的第二类磷酸盐。步骤中式(I)化合物与磷酸的投料摩尔比为1：(0.8-1.2)。例如，式(I)化合物与磷酸的投料摩尔比可以为1:0.8、1:0.9、1:1.0、1:1.1或1:1.2。
在一些实施例中，式(I)化合物与磷酸的投料摩尔比为1：(0.9-1.1)。在一些实施例中，式(I)化合物与磷酸的投料摩尔比为1：1。在一些实施例中，式(I)化合物与磷酸的投料摩尔比为1：0.95。
在一些实施例中，所述磷酸盐中式(I)化合物与磷酸的摩尔比为1：(0.8-1.2)。在一些实施方案中，所述磷酸盐中式(I)化合物与磷酸的摩尔比为1：(0.9-1.1)。在一些实施方案中，所述磷酸盐中式(I)化合物与磷酸的摩尔比可以为1：0.8，1：0.9，1：1，1：1.1或1：1.2。在一些实施方案中，所述磷酸盐中式(I)化合物与磷酸的摩尔比为1：1。
在一些实施例中，所述有机溶剂选自乙醇、乙酸乙酯、乙腈和丙酮中的一种或多种。在一些实施例中， 所述有机溶剂为乙醇、丙酮或其混合。在一些实施例中，所述有机溶剂为乙醇。
在一些实施例中，所述有机溶剂为甲醇、乙酸乙酯、丙酮中的一种或多种。在一些实施例中，所述有机溶剂为甲醇、乙酸乙酯中的一种或多种。在一些实施例中，所述有机溶剂为甲醇和乙酸乙酯的混合。
在一些实施例中，形成的所述式(I)化合物的磷酸盐中式(I)化合物与磷酸的摩尔比为1：1。
在一些实施例中，还提供了磷酸盐晶型II的制备方法，包括如下步骤：
(i)将所述式(I)化合物与磷酸在乙醇、甲醇和/或丙酮中进行成盐反应，析出固体；和
(ii)收集固体，得到磷酸盐晶型II；
其中，式(I)化合物与磷酸的投料摩尔比为1：(0.8-1.2)，例如，可以为1:0.8、1:0.9、1:1.0、1:1.1或1:1.2。在一些实施例中，式(I)化合物与磷酸的投料摩尔比为1：(0.9-1.1)。在一个实施例中，式(I)化合物与磷酸的投料摩尔比为1：0.95。在另一个实施例中，式(I)化合物与磷酸的投料摩尔比为1：1。
在一些实施例中，所述成盐反应是在乙醇和丙酮的一种或二者中进行的。
在一些实施例中，所述成盐反应是在乙醇中进行的。在一些实施例中，所述成盐反应是在甲醇中进行的。
在一些实施例中，磷酸盐晶型II的制备方法，包括如下步骤：
(i-1)将所述式(I)化合物的乙醇溶液与磷酸的乙醇溶液进行成盐反应，析出固体；和
(ii-1)收集固体，得到磷酸盐晶型II；
其中，式(I)化合物与磷酸的投料摩尔比为1：(0.8-1.2)，例如，可以为1:0.8、1:0.9、1:1.0、1:1.1或1:1.2。在一些实施例中，式(I)化合物与磷酸的投料摩尔比为1：(0.9-1.1)。在一个实施例中，式(I)化合物与磷酸的投料摩尔比为1：1。
在一些实施例中，磷酸盐晶型II的制备方法，包括如下步骤：
(i-1)将所述式(I)化合物的乙醇溶液与磷酸的乙醇溶液在回流温度下进行成盐反应，降温至-10℃至60℃，析出固体；和
(ii-1)收集固体，得到磷酸盐晶型II；
其中，式(I)化合物与磷酸的投料摩尔比为1：(0.8-1.2)，例如，可以为1:0.8、1:0.9、1:1.0、1:1.1或1:1.2。在一些实施例中，式(I)化合物与磷酸的投料摩尔比为1：(0.9-1.1)。在一个实施例中，式(I)化合物与磷酸的投料摩尔比为1：1。
在一些实施例中，磷酸盐晶型II中式(I)化合物与磷酸的摩尔比为1：1。
在一些实施例中，成盐反应的温度为-10℃至90℃，例如，可以为由选自-10℃、-5℃、0℃、5℃、10℃、15℃、20℃、25℃、30℃、35℃、40℃、45℃、50℃、55℃、60℃、65℃、70℃、75℃、80℃、85℃和90℃的端值组成的任意范围(包括端值)，具体不做限定。
在一些实施例中，步骤(ii)或(ii-1)中收集固体可采取离心分离和过滤分离的方式。
在一些实施例中，步骤(ii)或(ii-1)中收集固体之后，还包括挥干溶剂或干燥的步骤。
在一些实施例中，所述干燥步骤在25℃至70℃下进行。
在一些实施例中，所述磷酸盐为式(I)化合物的第一类磷酸盐。步骤中所述式(I)化合物与磷酸的投料摩尔比为1：(1.8-2.6)，例如，可以为1:1.8、1:1.9、1:2.0、1:2.1、1:2.2、1:2.3、1:2.4、1:2.5或1:2.6等；优选为1：(1.9-2.3)，更优选为1：(2.0-2.2)。
在一些实施例中，所述磷酸盐中磷酸与所述式(I)化合物的摩尔比为(1.8-2.4)：1。在一些实施例中，所述磷酸与所述式(I)化合物的摩尔比可以为1.8:1、1.9:1、2.0:1、2.1:1、2.2:1、2.3:1或2.4:1。在一些实施例中，所述磷酸与所述式(I)化合物的摩尔比为(1.9-2.3)：1。在一个实施例中，所述磷酸与所述式(I)化合物的摩尔比为2:1。
在一些实施例中，所述有机溶剂选自乙醇、乙酸乙酯、丙酮和甲醇中的一种或多种。
在一些实施例中，形成的所述式(I)化合物的磷酸盐中磷酸与式(I)化合物的摩尔比为2：1。
在一个实施例中，制备式(I)化合物磷酸盐晶型I的方法包括：
(AI-a)将式(I)化合物溶于有机溶剂中，加入磷酸水溶液，搅拌反应；
(AI-b)将步骤(AI-a)得到的反应液冷却，加入反溶剂析晶，固液分离，收集固相得到式(I)化合 物磷酸盐晶型I。
在一个实施例中，步骤(AI-a)中反应温度为10-60℃。
在一个实施例中，式(I)化合物与磷酸水溶液中含有的磷酸的摩尔比为1：(1.8-2.6)，例如，可以为1:1.8、1:1.9、1:2.0、1:2.1、1:2.2、1:2.3、1:2.4、1:2.5或1:2.6等；优选为1：(1.9-2.3)，更优选为1：(2.0-2.2)。
在一个实施例中，所述磷酸水溶液的摩尔浓度为0.1-5mol/L，优选0.5-3mol/L，更优选1-2mol/L。
在一些实施例中，步骤(AI-b)中将反应液冷却至-10℃-10℃，优选为-5℃-5℃；在其中的一些实施例中，将反应液冷却至约为0℃。
在一些实施例中，步骤(AI-b)中反应液冷却的时间为0.5-2h；在其中的一些实施例中，反应液冷却的时间为0.5h。
在一个实施例中，所述反溶剂选自甲基叔丁醚、石油醚、正庚烷、正己烷、环己烷、异丙醇、丙酮、乙腈和乙酸乙酯中的一种或多种，且所述反溶剂与所述溶剂不同；优选地，所述反溶剂选自甲基叔丁醚、正庚烷、异丙醇和丙酮中的一种或多种。
在一个实施例中，步骤(AI-b)中的分离方式选自离心分离和过滤分离。
在一个实施例中，步骤(AI-b)中分离之后还包括挥干溶剂或干燥的步骤。
在一个实施例中，步骤(AI-b)中分离之后的干燥步骤在25-70℃下进行；例如，可以为25℃、30℃、40℃、50℃、60℃或70℃等，具体不做限定。
在一个实施例中，步骤(AI-a)中反应温度为10-60℃；和/或式(I)化合物与磷酸水溶液中含有的磷酸的摩尔比为1：(1.8-2.4)，优选为1：(1.9-2.3)，更优选为1：(2.0-2.2)；和/或所述磷酸水溶液的摩尔浓度为0.1-5mol/L，优选0.5-3mol/L，更优选1-2mol/L；和/或步骤(AI-b)中将反应液冷却至-10℃-10℃，优选为-5℃-5℃，更优选约为0℃；和/或步骤(AI-b)中反应液冷却的时间为0.5-2h，更优选为0.5h；和/或所述反溶剂选自甲基叔丁醚、石油醚、正庚烷、正己烷、环己烷、异丙醇、丙酮、乙腈和乙酸乙酯中的一种或多种，且所述反溶剂与所述溶剂不同；优选地，所述反溶剂选自甲基叔丁醚、正庚烷、异丙醇和丙酮中的一种或多种；和/或步骤(AI-b)中的分离方式选自离心分离和过滤分离；和/或步骤(AI-b)中分离之后还包括挥干溶剂或干燥的步骤；和/或步骤(AI-b)中分离之后的干燥步骤在25-70℃下进行。
本公开的第四方面，提供了一种药物组合物，所述药物组合物包括：
(a)本公开第一方面所述的式(I)化合物的L-酒石酸盐或磷酸盐；以及(b)药学上可接受的载体。
在一些实施例中，所述药物组合物包括(a)本公开第一方面所述的式(I)化合物的L-酒石酸盐、式(I)化合物的第一类磷酸盐或式(I)化合物的第二类磷酸盐；以及(b)药学上可接受的载体。
在一些实施例中，所述药物组合物包括(a)本公开第一方面所述的式(I)化合物的L-酒石酸盐晶型I、式(I)化合物的L-酒石酸盐晶型II、式(I)化合物的L-酒石酸盐无定型物、式(I)化合物的磷酸盐晶型I、或式(I)化合物的磷酸盐晶型II；以及(b)药学上可接受的载体。
本公开的第五方面，提供了本公开第一方面所述的式(I)化合物的药学上可接受的盐、或本公开第四方面所述的药物组合物在制备激酶抑制剂中的应用。具体地，所述式(I)化合物的药学上可接受的盐包括式(I)化合物的L-酒石酸盐、式(I)化合物的第一类磷酸盐和式(I)化合物的第二类磷酸盐。在一些实施例中，所述式(I)化合物的药学上可接受的盐包括式(I)化合物的L-酒石酸盐晶型I、式(I)化合物的L-酒石酸盐晶型II、式(I)化合物的L-酒石酸盐无定型物、式(I)化合物的磷酸盐晶型I、和式(I)化合物的磷酸盐晶型II。在一些实施例中，所述激酶抑制剂为CDK9抑制剂。
本公开的第六方面，提供了本公开第一方面所述的式(I)化合物的药学上可接受的盐、或本公开第四方面所述的药物组合物在制备治疗和/或预防与CDK9活性相关的或由CDK9活性介导的疾病的药物中的应用。具体地，所述式(I)化合物的药学上可接受的盐包括式(I)化合物的L-酒石酸盐、式(I)化合物的第一类磷酸盐和式(I)化合物的第二类磷酸盐。在一些实施例中，所述式(I)化合物的药学上可接受的盐包括式(I)化合物的L-酒石酸盐晶型I、式(I)化合物的L-酒石酸盐晶型II、式(I)化合物的L- 酒石酸盐无定型物、式(I)化合物的磷酸盐晶型I、和式(I)化合物的磷酸盐晶型II。
本公开还提供了用于治疗和/或预防与CDK9活性相关的或由CDK9活性介导的疾病的本公开第一方面所述的式(I)化合物的药学上可接受的盐、或本公开第四方面所述的药物组合物。
本公开还提供了一种抑制CDK9活性的方法，包括给予所需对象治疗有效量的本公开第一方面所述的式(I)化合物的药学上可接受的盐、或本公开第四方面所述的药物组合物。
本公开还提供了一种治疗与CDK9活性相关的或由CDK9活性介导的疾病的方法，所述方法包括给予所需对象有效量的本公开第一方面所述的式(I)化合物的药学上可接受的盐、或本公开第四方面所述的药物组合物。
在一些实施例中，所述疾病包括过度增殖性疾病、病毒诱导的感染性疾病和心血管疾病。
在一些实施例中，所述疾病为过度增殖性疾病。在一些实施例中，所述过度增殖性疾病包括血管生成或血管增殖性病症、系膜细胞增殖性疾病和实体瘤。所述实体瘤例如为乳腺、呼吸道、脑、生殖器官、消化道、泌尿道、眼、肝脏、皮肤、头和颈、甲状腺或甲状旁腺的癌症以及它们的远端转移等。在一些实施例中，所述疾病选自淋巴瘤、肉瘤和白血病中的一种或多种。在一些实施例中，所述疾病为癌症。所述癌症例如为胰腺癌、乳腺癌、卵巢癌、子宫颈癌或白血病等。在一些实施例中，所述疾病包括实体瘤和血液瘤。
本公开中，与CDK9活性相关的或由CDK9活性介导的疾病包括与CDK9活性(例如CDK9的过度活性)相关或涉及CDK9活性的疾病，以及伴随这些疾病的病况。CDK9的过度活性是指与正常的非疾病细胞相比的增加的CDK9酶活性，或者是指导致不需要的细胞增殖、或者降低的或不足的程序性细胞死亡(细胞凋亡)的增加的CDK9活性，或者是指导致CDK9的组成性激活的突变。
过度增殖性疾病包括涉及细胞的不期望的或不受控制的增殖性疾病，并且其包括涉及降低的或不足的程序性细胞死亡(细胞凋亡)的疾病。本公开的式(I)化合物的药学上可接受的盐、或含有式(I)化合物的药学上可接受的盐的药物组合物可用于对细胞增殖和/或细胞分裂进行预防、抑制、阻断、减少、降低、控制等，和/或产生细胞凋亡。治疗和或预防与CDK9活性相关的或由CDK9活性介导的疾病包括对有需要的对象(包含哺乳动物，例如人)给予有效治疗或预防所述疾病的一定量的本公开的式(I)化合物的药学上可接受的盐、或含有式(I)化合物的药学上可接受的盐的药物组合物。
本公开中，式(I)化合物的磷酸盐和L-酒石酸盐相比于游离碱具有更高的溶解度，并且式(I)化合物L-酒石酸盐晶型I、式(I)化合物L-酒石酸盐晶型II、式(I)化合物L-酒石酸盐无定型物、式(I)化合物磷酸盐晶型I和式(I)化合物磷酸盐晶型II都具有较好的物理稳定性，适用于药物开发。特别地，式(I)化合物磷酸盐晶型I在合适的条件下，如在乙醇中可转变成更稳定的磷酸盐晶型II。与式(I)化合物磷酸盐晶型I相比，式(I)化合物磷酸盐晶型II的引湿性更低以及结晶度、稳定性更好。
附图说明
为了更清楚地说明本公开的实施例或相关技术中的技术方案，下面将对实施例或相关技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本申请的实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据公开的附图获得其他的附图。
图1为式(I)化合物磷酸盐晶型I的X射线粉末衍射(XRPD)图谱。
图2为式(I)化合物磷酸盐晶型I的差示扫描量热(DSC)和热重分析(TGA)的图谱。
图3为式(I)化合物磷酸盐晶型I的动态水分吸收曲线(DVS)。
图4为式(I)化合物磷酸盐晶型I的显微镜图。
图5为式(I)化合物L-酒石酸盐晶型I的X射线粉末衍射(XRPD)图谱。
图6为式(I)化合物L-酒石酸盐晶型I的热重分析(TGA)图谱。
图7为式(I)化合物L-酒石酸盐晶型II的X射线粉末衍射(XRPD)图谱。
图8为式(I)化合物L-酒石酸盐无定型物的X射线粉末衍射(XRPD)图谱。
图9为式(II)化合物的单晶的分子立体结构椭球图。
图10为式(I)化合物磷酸盐晶型II的X射线粉末衍射(XRPD)图谱。
图11为式(I)化合物磷酸盐晶型II的差示扫描量热(DSC)和热重分析(TGA)的图谱。
图12为式(I)化合物磷酸盐晶型II的动态水分吸收曲线(DVS)。
图13为式(I)化合物磷酸盐晶型II的显微镜图。
图14为式(I)化合物磷酸盐晶型I在乙醇中转变为式(I)化合物磷酸盐晶型II的X射线粉末衍射(XRPD)的对比图谱。
图15为式(I)化合物L-酒石酸盐晶型I在高温(60℃)和加速(40℃-75％RH)条件下XRPD变化图。
图16为式(I)化合物磷酸盐晶型I在高温条件下XRPD变化图。
图17为每日一次口服给药化合物D与式(I)化合物后的肿瘤体积变化图。
图18为每日一次口服给药化合物D与式(I)化合物后的小鼠体重变化图。
具体实施方式
为使本公开的上述目的、特征和优点能够更加明显易懂，下面对本公开的具体实施方式做详细的说明。在下面的描述中阐述了很多具体细节以便于充分理解本公开。但是本公开能够以很多不同于在此描述的其它方式来实施，本领域技术人员可以在不违背本公开内涵的情况下做类似改进，因此本公开不受下面公开的具体实施例的限制。
本公开所用的“本公开的晶体”、“本公开的晶型”、“本公开的多晶型物”等可互换使用。
本公开中，式(I)化合物为(1S，3S)-N1-(5-((S)-1-环丁基乙基)吡唑并[1,5-a]嘧啶-7-基)环戊烷-1,3-二胺，其结构如下所示：
本公开中还包括式(I)化合物的磷酸盐和L-酒石酸盐。
多晶型物：固体不是以无定型的形式就是以结晶的形式存在。在结晶形式的情况下，分子定位于三维晶格格位内。当化合物从溶液或浆液中结晶出来时，它可以不同的空间点阵排列结晶(这种性质被称作“多晶型现象”)，形成具有不同的结晶形式的晶体，这各种结晶形式被称作“多晶型物”。给定物质的不同多晶型物可在一个或多个物理属性方面(如溶解度和溶解速率、真比重、晶形、堆积方式、流动性和/或固态稳定性)彼此不同。
结晶：可以通过操作溶液，使得感兴趣化合物的溶解度极限被超过，从而完成生产规模的结晶。这可以通过多种方法来完成，例如，在相对高的温度下溶解化合物，然后冷却溶液至饱和极限以下。或者通过沸腾、常压蒸发、真空干燥或通过其它的一些方法来减小液体体积。可通过加入抗溶剂或化合物在其中具有低的溶解度的溶剂或这样的溶剂的混合物，来降低感兴趣化合物的溶解度。另一种可选方法是调节pH值以降低溶解度。有关结晶方面的详细描述请参见Crystallization，第三版，J W Mullens，Butterworth-Heineman Ltd.，1993，ISBN 0750611294。
本公开所述的“悬浮振摇”是指将式(I)化合物和相应的酸或相应酸的溶液在合适的溶剂中混合形成浑浊液后振荡得到晶体的一种方法。合适的溶剂可以为水或有机溶剂。
本公开所述的“悬浮离心”是指将式(I)化合物和相应的酸或相应酸的溶液在合适的溶剂中混合形成浑 浊液后离心得到晶体的一种方法。合适的溶剂可以为水或有机溶剂。
本公开中所述的“缓慢挥发”是指将含式(I)化合物和相应的酸的溶液置于一定温度下缓慢挥发掉溶剂得到晶体的一种方法。
本公开中所述的“反溶剂添加”或“添加反溶剂”是指向式(I)化合物的一种溶液中加入另一种合适溶剂后析出得到晶体的一种方法。
假如期望盐的形成与结晶同时发生，如果盐在反应介质中比原料溶解度小，那么加入适当的酸或碱可导致所需盐的直接结晶。同样，在最终想要的形式比反应物溶解度小的介质中，合成反应的完成可使最终产物直接结晶。
结晶的优化可包括用所需形式的晶体作为晶种接种于结晶介质中。另外，许多结晶方法使用上述策略的组合。一个实施例式在高温下将感兴趣的化合物溶解在溶剂中，随后通过受控方式加入适当体积的抗溶剂，以使体系正好在饱和水平之下。此时，可加入所需形式的晶种(并保持晶种的完整性)，将体系冷却以完成结晶。如本文所用，术语“约”，是指在给定数值的基础上有±5。
在本文中，涉及数据范围的单位，如果仅在右端点后带有单位，则表示左端点和右端点的单位是相同的。比如，3-5h表示左端点“3”和右端点“5”的单位都是h(小时)。
本文中，“优选”、“更好”仅为描述效果更好的实施方式或实施例，应当理解，并不构成对本公开保护范围的限制。如果一个技术方案中出现多处“优选”，如无特别说明，且无矛盾之处或相互制约关系，则每项“优选”各自独立。
本文所使用的术语“和/或”、“或/和”、“及/或”的选择范围包括两个或两个以上相关所列项目中任一个项目，也包括相关所列项目的任意的和所有的组合，所述任意的和所有的组合包括任意的两个相关所列项目、任意的更多个相关所列项目、或者全部相关所列项目的组合。需要说明的是，当用至少两个选自“和/或”、“或/和”、“及/或”的连词组合连接至少三个项目时，应当理解，在本申请中，该技术方案毫无疑问地包括均用“逻辑与”连接的技术方案，还毫无疑问地包括均用“逻辑或”连接的技术方案。
本文中涉及“多个”、“多种”、“多次”等，如无特别限定，指在数量上大于2或等于2。例如，“多种”表示大于或等于两种。
本文中，以开放式描述的技术特征中，包括所列举特征组成的封闭式技术方案，也包括包含所列举特征的开放式技术方案。
如本文所用，术语“本公开的多晶型物”包括但不限于式(I)化合物磷酸盐晶型I、式(I)化合物磷酸盐晶型II、式(I)化合物L-酒石酸盐晶型I、和式(I)化合物L-酒石酸盐晶型II。
本公开中还包括式(I)化合物的磷酸盐，特别是单磷酸盐以及式(I)化合物磷酸盐的晶型II。
“式(I)化合物”、“式(I)化合物游离碱”与“游离碱”可互换使用。
“式(I)化合物的多晶型物”与“式(I)化合物游离碱的多晶型物”可互换使用。
本公开中，某些晶型可以相互转化，因此本公开还提供了部分晶型相互转化的方法。
药物组合物及其应用
通常，本公开式(I)化合物的药学上可接受的盐作为活性成分可以与一种或多种药用载体形成合适的剂型施用。
具体地，本公开式(I)化合物的磷酸盐、L-酒石酸盐、或其多晶型物作为活性成分可以与一种或多种药用载体形成合适的剂型施用。
“药学上可接受的载体”是指无毒、惰性、固态、半固态的物质或液体灌装机、稀释剂、封装材料或辅助制剂或任何类型辅料，其与被施用的对象(在一些实施例中为哺乳动物，在一个实施例中为人)相兼容，其适合将本公开的活性物质输送到目标靶点而不终止其活性。
本公开的药物组合物以符合医学实践规范的方式配制、定量和给药。给予活性成分的“治疗有效量”由要治疗的具体病症、治疗的个体、病症的起因、药物的靶点以及给药方式等因素决定。
本公开提供了本公开第一方面所述的式(I)化合物的药学上可接受的盐、本公开第四方面所述的药物组合物可用作治疗和/或预防与CDK9活性相关的或由CDK9活性介导的疾病的药物。
本公开提供了一种抑制CDK9活性的方法，包括给予对象治疗有效量的本公开第一方面所述的式(I)化合物的药学上可接受的盐、或本公开第四方面所述的药物组合物。
如本文所用，“治疗有效量”指将引起个体的生物学或医学响应，例如降低或抑制酶活蛋白质活性或改善症状、缓解病症、缓解或延迟疾病进程或预防疾病等的本公开式(I)化合物的药物学上可接受的盐的量。
如本文所用，“对象”是指一种动物，最好为哺乳动物，更好的为人。术语“哺乳动物”是指温血脊椎类哺乳动物，包括如猫、狗、兔、熊、狐狸、狼、猴子、鹿、鼠、猪和人类。
“治疗”是指减轻、延缓进展、衰减、预防，或维持现有疾病或病症(例如癌症)。治疗还包括将疾病或病症的一个或多个症状治愈、预防其发展或减轻到某种程度。
实施例
下面结合具体实施例，进一步阐述本公开。应理解，这些实施例仅用于说明本公开而不用于限制本公开的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。除非另行定义，本文所用的术语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或同等的方法及材料皆可应用于本公开中。
试剂与仪器
本公开中，化合物的结构和纯度通过核磁共振(1H NMR)和/或液质联用质谱(LC-MS)来确定。
1H NMR：Bruker AVANCE-400核磁仪，内标为四甲基硅烷(TMS)。
LC-MS：Agilent 1290 HPLC System/6130/6150 MS液质联用质谱仪(生产商：安捷伦)，柱子Waters BEH/CHS，50×2.1mm，1.7μm。
HPLC分析采用Agilent 1260 Infinity HPLC,OpenLAB CDS Chemstation workstation，色谱柱XBridge C18 4.6*250mm,ID 5μm column，检测器DAD。
元素分析采用电感耦合等离子体发射光谱仪，型号icp 500；功率1300w；流速1mL/min。
已知的起始原料可以采用或按照本领域已知的方法来合成，或可以购自ABCR GmbH&Co.KG，Acros Organics，Aldrich Chemical Company，韶远化学科技(Accela ChemBio Inc)和达瑞化学品等公司。
如本文所用，下述各实施例中室温是指约为20-30℃。
通用方法
X射线粉末衍射(XRPD)：本公开中，上述晶型或无定型的粉末X射线衍射图谱是通过本领域的已知方法，使用ARL Equinox3000 X射线粉末衍射分析仪获得，XRPD测试参数如下表5所示。
表5
在粉末X射线衍射图中，各峰的位置由2θ(°)值确定。可以理解，不同的仪器和/或条件可导致产生的数据会略有不同，各峰的位置和相对强度会有变化。
峰的强度划分仅仅反映了各位置上峰的近似大小。在本公开中，各晶型均以其峰高最高的衍射峰作为基峰，定义其相对强度为100％，作为I0(磷酸盐晶型I的2θ(°)值为18.803的峰为基峰，L-酒石酸盐晶型I的2θ(°)值为20.27的峰为基峰，L-酒石酸盐晶型II的2θ(°)值为21.353的峰为基峰，磷酸盐晶型II的2θ(°)值为18.230的峰为基峰)，其它各峰以其峰高与基峰峰高的比值作为其相对强度I/I0，各峰相对强度的划分定义如下表6所示。
表6

单晶X射线衍射(SXRD)：本公开中，式(II)化合物的单晶X射线衍射图谱是通过本领域的已知方法，使用D8 Venture衍射仪获得，SXRD测试参数如下表7所示。收集相关数据后，进一步采用直接法(SHELXT2014)解析晶体结构，便可以确证绝对构型。
表7
本公开的式(I)化合物的磷酸盐及其晶型通过元素分析确定酸碱摩尔比，式(I)化合物的L-酒石酸盐及其晶型及其无定型通过HPLC/IC或1H NMR确定酸碱摩尔比。
高效液相色谱：本公开中，高效液相色谱(HPLC)在Agilent1260 HPLC上采集。
差示扫描量热(DSC)：本公开中，上述晶型的差示扫描量热图谱是通过本领域的已知方法，使用DSC25A差示扫描量热仪获得，DSC测试参数如下表8所示。
表8
热重分析(TGA)：本公开中，上述晶型的热重分析图谱是通过本领域的已知方法，使用TGA550热重分析仪获得，TGA测试参数如下表9所示。
表9
动态水分吸附(DVS)曲线：在SMS(Surface Measurement Systems)的DVS Intrinsic上采集。在25℃时的相对湿度用LiCl，Mg(NO3)2和KCl的潮解点校正。仪器测试条件如下表10所示。
表10

《中国药典》(2020)第9103药物引湿性试验指导原则中规定了引湿性特征描述与引湿性增重的界定：(1)潮解：吸收足量水分形成液体；(2)极具引湿性：引湿增重不小于15％；(3)有引湿性：引湿增重小于15％但不小于2％；(4)略有引湿性：引湿增重小于2％但不小于0.2％；(5)无或几乎无引湿性：引湿增重小于0.2％。
可以理解的是，使用与上述仪器作用相同的其他类型的仪器或使用不同于本公开中使用的测试条件时，可能会得到另外的数值，因此，所引用的数值不应视为绝对的数值。
由于仪器的误差或操作人员的区别，本领域技术人员能理解，以上用于表征晶体的物理性质的参数可能有微小差别，所以上述的参数仅用于辅助表征本公开提供的多晶型物，而不能视为是对本公开的多晶型物的限制。
若无特别说明，本公开所用的“磷酸溶液”、“L-酒石酸溶液”指磷酸水溶液、L-酒石酸水溶液。本公开所用的“磷酸盐”、“L-酒石酸盐”指式(I)化合物的磷酸盐、式(I)化合物的L-酒石酸盐。
本公开所用的溶剂为分析纯的溶剂，如乙醇为分析纯乙醇(含水量≤0.3％)。
本申请中的化合物D和化合物E可参照现有公开专利文献进行制备得到。
本公开所使用的MTBE指甲基叔丁基醚；DMSO指二甲基亚砜；THF指四氢呋喃；EA指乙酸乙酯；PE指石油醚；DCM指二氯甲烷；MeOH指甲醇；CDI指N,N-碳酰二咪唑；MOPS指3-(N-吗啉)丙磺酸；Tween-20指吐温20；DTT指二硫苏糖醇；EDTA指乙二胺四乙酸；CDK1指细胞周期依赖性激酶1；CDK2指细胞周期依赖性激酶2；CDK9指细胞周期依赖性激酶9；K2EDTA指乙二胺四乙酸二钾；IPA指异丙醇；NH3指氨。
制备例1.化合物6的制备
步骤1：将氢化钠(18g,450.00mmol,纯度60％)溶于THF(300mL)中，冷却至0℃，将三乙基2-膦酰基丙酯(100g,419.78mmol)缓慢滴加入其中。该反应在0-5℃条件下搅拌1小时,然后将环丁酮(25g,356.69mmol)溶于THF(50mL)缓慢滴加入其中，滴加完毕后，25℃反应15小时。用饱和氯化钠溶液(600mL)淬灭反应，然后用乙酸乙酯萃取(600mL×2)，合并有机相用饱和氯化钠溶液(500mL)洗涤，无水硫酸钠干燥，过滤，减压浓缩。残留物经CombiFlash分离纯化(120g×2，0～20％EA/PE)得到化合物2(44g,无色油状物)，收率：80.00％。1H 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).
步骤2：将化合物2(44g,285.33mmol)溶于甲醇(400mL)中，加入湿钯碳(4.4g，纯度10％)。罩上氢气球，置换氢气三次，在氢气氛围下(15psi)27℃搅拌6～8小时。TLC显示原料点消失。过滤掉Pd/C，减压除去溶剂得到粗品化合物3(44g,无色油状物，沸点约为182℃),收率：98.71％，不纯化直接下一步。
步骤3：将化合物3(44g,281.65mmol)溶于甲醇(400mL)中，加入氢氧化钠(45.06g,1.13mol)和水(200mL)，然后室温搅拌16小时。LCMS显示反应完成。减压浓缩除去甲醇，然后用二氯甲烷萃取两次，水相再用稀盐酸(6M)调pH值到3，萃取DCM(300mL×3),有机相合并干燥旋干得到化合物4(35g,无色油状物，沸点为约220℃)，收率：96.96％，不纯化直接下一步。MS m/z(ESI):127.1[M-H]-。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).
步骤4：在反应瓶中将化合物4(36g,280.88mmol)溶于THF(300mL)中，然后将CDI(68.32g,421.32mmol)加入其中，然后室温下反应16小时(作为A溶液)。在另一个反应瓶中将丙二酸单甲酯钾盐(143.42g,842.64mmol)加入无水氯化镁(66.86g,702.20mmol)和THF(900mL)中，氩气保护下加热50℃反应16小时(作为B溶液)。然后室温下将A溶液滴加到B溶液中(约10分钟)，然后混合溶液30℃下搅拌16小时。LCMS检测反应完成，产物产生。反应液中加入800mL水，用乙酸乙酯萃取(800mL×3)。有机相合并后用食盐水洗涤，无水硫酸钠干燥，再减压旋干。残留物经CombiFlash分离纯化(120g×2，0～15％EA/PE)得到产物化合物5(42g,浅黄色油状物，沸点约240℃),收率：75.42％。MS m/z(ESI):199.1[M+H]+。1H NMR(400MHz,DMSO-d6)δ4.13-4.00(m,2H),3.55(d,J＝0.8Hz,2H),2.63-2.54(m,1H),2.42-2.28(m,1H),1.97-1.85(m,2H),1.83-1.74(m,1H),1.73-1.61(m,3H),1.16(t,J＝7.2Hz,3H),0.91(d,J＝6.8Hz,3H).
步骤5：将化合物5(42g,211.85mmol)和3-胺基吡唑(19.36g,233.03mmol)溶于冰乙酸(300mL)中，升温至120℃反应16h。LCMS显示反应完成。减压浓缩除去乙酸，用乙酸乙酯打浆(800mL×4)，固体析出，过滤，干燥，得到化合物6(41g,淡黄色固体)，收率：89.08％。不纯化直接下一步。MS m/z(ESI):218.1[M+H]+。
制备例2.式(I)化合物的制备
步骤1：将化合物6(473.6mg,2.18mmol)溶于三氯氧磷(6mL)中加热至120℃搅拌3小时。冷却至室温，将其倒入冰水(60g)中，用二氯甲烷(80mL)萃取，有机相用无水硫酸钠干燥，过滤，减压浓缩。残留物经硅胶柱色谱分离(20g，0％-40％EA/DCM)得到化合物7。MS m/z(ESI):236.1[M+H]+；1H 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).
步骤2：将化合物7(142.7mg,605.5μmol)和((1S,3S)-3-氨基环戊基)氨基甲酸叔丁酯(121.27mg,605.52μmol)溶于乙腈(20mL)中，然后将碳酸钾(251.0mg,1.81mmol)加入其中。该反应在90℃条件下搅拌16小时。加入乙酸乙酯(80mL)稀释,用饱和氯化钠溶液(80mL×3)洗涤，有机相用无水硫酸钠干燥，过滤，减压浓缩得到化合物8。MS m/z(ESI):400.3[M+H]+；1H 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),4.02–3.93(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).
步骤3：将化合物8(174.7mg,437.38μmol)溶于1,4-二氧六环(3mL)中，加入的盐酸溶液(3.0mL，4M)，在室温条件下搅拌3小时。减压蒸除溶剂，加入水(60mL)，用乙酸乙酯(50mL)萃取，水相用饱和碳酸钠溶液调pH＝9-10,用乙酸乙酯(60mL×2)萃取，有机相用无水硫酸钠干燥，过滤，减压浓缩。用制备HPLC色谱法分离得到化合物9。MS m/z(ESI):300.2[M+H]+；1H 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).
步骤4：将化合物9(94.46mg,315.48μmol)进行手性拆分(柱型:IC-3 4.6*100mm 3um；助溶剂:IPA[1％NH3(7M in MeOH)]；进样体积:5.00uL；波长:220.0nm；运行时间:6.0分钟；流速:3.0mL/min；压力:2000psi；柱温:40℃)得式(I)化合物(9.70mg,保留时间2.471min)，收率：9.89％,纯度：96.34％。MS m/z(ESI):300.2[M+H]+。1H 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).
通过式(I)化合物制备得到了式(II)化合物，以通过式(II)化合物的绝对构型确定式(I)化合物的绝对构型。
将式(I)化合物(200mg,667.97μmol)溶于二氯甲烷(10mL)中，然后将4-氯苯甲酰氯(175.4mg,1.00mmol)加入其中，随后加入N,N-二异丙基乙胺(0.6mL,3.34mmol)，该反应在室温条件下搅拌2小时，LCMS检测原料反应完全。加入水(30mL)，用乙酸乙酯(30mL)萃取，有机相用饱和氯化钠溶液(30mL×3)洗涤，无水硫酸钠干燥，过滤，减压浓缩得到粗品。粗品经硅胶柱色谱分离纯化，得到式(II)化合物(160mg,收率：54.69％)。LCMS(ESI)m/z:438.2[M+H]+。
进一步制备得到了式(II)化合物的单晶，式(II)化合物的分子立体结构椭球图如图9所示。Flack常数为0.07(2)，C8和C10和C19为S构型，即式(II)化合物的3个手性中心的绝对构型均为S构型，其结构如式(II)化合物的结构所示。从而可以确定式(I)化合物的3个手性中心的绝对构型均为S构型，其结构如式(I)化合物的结构所示。
实施例1.式(I)化合物磷酸盐晶型I的制备
20mL样品瓶中加入游离碱(500mg)、甲醇(1mL)，超声使溶解，后按照投料酸碱摩尔比＝2.5:1的比例加入1mol/L的磷酸溶液，50℃反应1h，40℃继续搅拌1h，30℃再继续搅拌1h，最后关闭加热，搅拌过夜。反应结束后缓慢降温至0℃，加入丙酮使析出固体，离心收集固体并挥干溶剂，所得固体即为式(I)化合物磷酸盐晶型I。所得固体通过元素分析得到磷元素含量为12.5％，表明所得固体中式(I)化合物与磷酸的摩尔比为1:2(其磷元素含量的理论值为12.5％)；所得固体的XRPD图基本如图1所示，且所得固体的XRPD图在前述表1所示的2θ(°)值处具有峰，各峰相对强度如前述表1所示；其DSC和TGA图如图2所示，其DVS图如图3所示，其显微镜图如图4所示。
由图2可知，DSC图中，在188.01℃有一个熔化吸收峰，结晶度较好，表明式(I)化合物磷酸盐晶型I的熔点约为188.01℃；在TGA图中，样品加热到190℃附近失重1.139％，可能是溶剂挥发。
由图3可知，DVS图中，在80％RH条件下，吸湿增重9％，表明式(I)化合物磷酸盐晶型I具有引湿性。
由图4可知，偏光显微镜图显示式(I)化合物磷酸盐晶型I呈棒状和块状。
实施例2.式(I)化合物L-酒石酸盐晶型I的制备
20mL样品瓶中加入游离碱(500mg)、乙醇(1mL)，超声使溶解，后按照投料酸碱摩尔比＝1.2:1的比例加入1mol/L的L-酒石酸溶液，50℃反应1h，40℃继续搅拌1h，30℃再继续搅拌1h，最后关闭加热，室温搅拌过夜。反应结束后，缓慢降温至0℃，离心收集固体并挥干溶剂。所得固体即为式(I)化合物 L-酒石酸盐晶型I。其HPLC/IC结果表明所得固体中的式(I)化合物与L-酒石酸的摩尔比为1:1；其XRPD图基本如图5所示，且所得固体的XRPD图在前述表2所示的2θ(°)值处具有峰，各峰相对强度如前述表2所示；其TGA图如图6所示。
由图6可知，TGA图表明加热到110℃附近失重1.057％，可能为水分挥发，加热到178℃附近继续失重3.482％，推测为有机溶剂挥发，继续加热发生熔化分解。
实施例3.式(I)化合物L-酒石酸盐晶型II的制备
样品瓶中，加入游离碱(约100mg)、甲醇(0.2mL～0.3mL)，超声使溶解，后按照投料酸碱摩尔比＝1.2:1的比例加入1mol/L的L-酒石酸溶液，50℃反应1h，40℃继续搅拌1h，30℃再继续搅拌1h，降至室温后关闭加热，搅拌过夜。反应结束后缓慢降温至0℃，加入MTBE使析晶，挥干溶剂得到固体。所得固体进行HPLC/IC检测，确定所得固体中的式(I)化合物与L-酒石酸的摩尔比为1:1；所得固体的XRPD图如图7所示，且所得固体的XRPD图在前述表3所示的2θ(°)值处具有峰，各峰相对强度如前述表3所示。在本公开中定义为式(I)化合物L-酒石酸盐晶型II。
实施例4.式(I)化合物L-酒石酸盐无定型物的制备
样品瓶中，加入游离碱(约100mg)、丙酮(0.2mL～0.3mL)，超声使溶解，后按照投料酸碱摩尔比＝1.2:1的比例加入1mol/L的L-酒石酸溶液，50℃反应1h，40℃继续搅拌1h，30℃再继续搅拌1h，降至室温后关闭加热，搅拌过夜。反应结束后缓慢降温至0℃使析出固体，离心收集固体并挥干溶剂。所得固体进行HPLC/IC，确定所得固体中的式(I)化合物与L-酒石酸的摩尔比为1:1；所得固体的XRPD图如图8所示，在本申请中定义为L-酒石酸盐无定型物。
实施例5.式(I)化合物磷酸盐晶型II的制备
50mL样品瓶中游离碱(100mg)溶于乙醇(15mL)，后按照投料酸碱摩尔比＝0.95:1的比例滴加磷酸的乙醇溶液(5ml)，室温搅拌4h，过滤并用乙醇淋洗，旋蒸干燥得到白色固体。所得固体通过元素分析得到磷元素含量为7.8％，表明所得固体中式(I)化合物与磷酸的摩尔比为1:1(其磷元素含量的理论值为7.79％)；所得固体进行XRPD检测，其XRPD图如图10所示，在本申请中定义为磷酸盐晶型II。
所得固体的XRPD图在前述表4所示的2θ(°)值处具有峰，各峰相对强度如前述表4所示；其DSC和TGA曲线如图11所示，其DVS图如图12所示，其显微镜图如图13所示。
由图11可知：DSC曲线中，样品在207.48℃有一个熔化吸收峰，结晶度好，可见式(I)化合物磷酸盐晶型II的熔点约为207.48℃；TGA曲线中，样品加热到100℃附近几乎没有失重，加热到207℃附近开始熔化，因此式(I)化合物磷酸盐晶型II是无水物。
由图12可知：DVS图中，在80％RH条件下，吸湿增重0.45％，表明式(I)化合物磷酸盐晶型II略有引湿性。
由图13可知：偏光显微镜显示式(I)化合物磷酸盐晶型II呈杆状。
实施例6.式(I)化合物磷酸盐晶型II的制备
50mL样品瓶中游离碱(100mg)溶于丙酮(15mL)，后按照投料酸碱摩尔比＝0.98:1的比例滴加磷酸的乙醇溶液(5ml)，室温搅拌4h，过滤并用乙醇淋洗，旋蒸干燥得到白色固体。所得固体通过元素分析得到磷元素含量为7.8％，表明所得固体中式(I)化合物与磷酸的摩尔比为1:1(其磷元素含量的理论值为7.79％)；所得固体进行XRPD检测，其XRPD图基本如图10所示。
实施例7.式(I)化合物磷酸盐晶型II的制备
50mL样品瓶中游离碱(100mg)溶于乙醇(10mL)，后按照投料酸碱摩尔比＝1.05:1的比例滴加磷酸的乙醇溶液(10ml)，室温搅拌4h，过滤并用乙醇淋洗，旋蒸干燥得到白色固体。所得固体通过元素分析得到磷元素含量为7.8％，表明所得固体中式(I)化合物与磷酸的摩尔比为1:1(其磷元素含量的理论值为7.79％)；所得固体进行XRPD检测，其XRPD图基本如图10所示。
实施例8.式(I)化合物磷酸盐晶型II的制备
将0.5g游离碱溶解在甲醇(15ml)中，按照投料酸碱摩尔比＝1:1的比例加入磷酸(市售)，搅拌2h后旋蒸除去甲醇，得到磷酸盐粗品，加入乙醇(2.5ml)然后浓缩，再加入乙醇(2.5ml)然后再次浓缩，加入乙醇(10ml)加热至60℃，搅拌3h后降至室温搅拌过夜，过滤，乙醇淋洗，旋蒸干燥，得到式(I) 化合物磷酸盐晶型II，收率75.8％。所得固体通过元素分析得到磷元素含量为7.7％，表明所得固体中式(I)化合物与磷酸的摩尔比为1:1(其磷元素含量的理论值为7.79％)；所得固体进行XRPD检测，其XRPD图基本如图10所示。
对比例1.式(I)化合物的成盐反应
采用减重法称取适量的式(I)化合物(100mg)，置于透明样品瓶中，加入相应溶剂(0.2mL～0.3mL)，超声使溶解后按照投料酸碱摩尔比＝1.2:1的比例加入1mol/L的酸溶液(水溶液)，50℃反应1h，40℃继续搅拌1h，30℃再继续搅拌1h，降至室温后关闭加热，搅拌过夜。反应结束后缓慢降温待固体析出，若仍为澄清溶液尝试用反溶剂添加的方法诱导析晶。将反应结果送XRPD测试，结果显示，盐酸、硫酸、乙二酸等酸在降温后和添加反溶剂后均不能使式(I)化合物成盐；其中，酸和溶剂的种类分别如下表11所示。
表11
测试例1.溶解度测试
室温条件下，对上述实施例制备的式(I)化合物的磷酸盐、L-酒石酸盐在水中的溶解度进行测试，磷酸盐的溶解度为不大于26.6mg/mL，L-酒石酸盐的溶解度为不大于17.3mg/mL，而游离碱在水中微溶。式(I)化合物的盐大大提高其在水中的溶解度，所成的盐中磷酸盐较L-酒石酸盐具有更高的溶解度。
另外，在室温下，对式(I)化合物磷酸盐晶型I、L-酒石酸盐晶型I在pH4.5缓冲液(醋酸-醋酸钠体系)、pH6.8的缓冲液(磷酸二氢钠-氢氧化钠体系)中的粗略溶解度(单位：mg/mL)进行测试，结果如表12所示。由表12的结果可知，式(I)化合物磷酸盐晶型I、L-酒石酸盐晶型I在pH4.5、pH6.8的缓冲液中均具有较高的溶解度。
表12
称取适量磷酸盐晶型I或磷酸盐晶型II分别加入0.1mo/L盐酸溶液(5mL)与pH6.8磷酸盐缓冲液(5mL)，于24h(置于37℃烘箱内)取样测定溶解度，结果如表13所示。
表13
测试例2.稳定性测试
(1)分别称取式(I)化合物磷酸盐晶型I、式(I)化合物磷酸盐晶型II和式(I)化合物L-酒石酸盐晶型I以及式(I)化合物L-酒石酸盐晶型II的样品(约100mg)，在60℃(高温)条件下以及40℃-75％RH(加速)条件下放置，同时将另一组样品在5℃条件下密封保存作为对照，于7天、20天、30天或60天分别检测晶型变化。
检测结果表明：式(I)化合物L-酒石酸盐晶型I、式(I)化合物L-酒石酸盐晶型II、式(I)化合物磷酸盐晶型I、式(I)化合物磷酸盐晶型II的晶型在上述条件下均无明显变化，具有较高的稳定性。式(I)化合物L-酒石酸盐晶型I在高温条件下保存7天、2个月(60天)，其晶型的XRPD图显示无明显变化；式(I)化合物L-酒石酸盐晶型I在加速条件下保存7天、2个月(60天)其晶型的XRPD图的叠加图如图15所示，显示无明显变化；这表明式(I)化合物L-酒石酸盐晶型I具有较高的稳定性。式(I)化合物磷酸盐晶型I在高温条件下保存7天、30天，其晶型的XRPD图的叠加图如图16所示，显示无明显变化，表明式(I)化合物磷酸盐晶型I具有较高的稳定性。
(2)称取约40mg的磷酸盐晶型I(参照本申请实施例1制备得到)至玻璃小瓶中，加入乙醇(1mL)，密封并置于50℃，150r/min条件下振摇七天，后离心收集固体并挥干溶剂，将所得固体进行XRPD检测，其XRPD图如图10所示。该所得固体为磷酸盐晶型II。测试结果图14表明磷酸盐晶型I可以转变为热力学更加稳定的磷酸盐晶型II。
(3)分别取磷酸盐晶型I与磷酸盐晶型II各25mg，分别加入5mg水，然后置于80℃烘箱内一周，取样测定有关物质，测定结果如表14所示，表明磷酸盐晶型I和磷酸盐晶型II具有较高的化学稳定性。
表14
测试例3.吸湿性测试
3.1 L-酒石酸盐吸湿性测试
按照《中国药典(2020)》中9103(药物引湿性试验指导原则)的操作进行引湿性试验，发现式(I)化合物的L-酒石酸盐略有引湿性。
3.2磷酸盐晶型的吸湿性测试
对式(I)化合物磷酸盐晶型I、式(I)化合物磷酸盐晶型II进行了DVS测定，式(I)化合物磷酸盐晶型I的DVS图如图3所示，DVS图显示在80％RH条件下，吸湿增重9％，表明式(I)化合物磷酸盐晶型I具有引湿性。而磷酸盐晶型II的DVS图显示在80％RH条件下，吸湿仅增重0.45％，表明磷酸盐晶型II略有引湿性。
测试例4.对CDK家族激酶的活性抑制测试
以下LANCE Ultra测试方法中，激酶试剂购自Carna Bioscience，反应底物和检测试剂购自PerkinElmer，其余试剂购自Thermo scientific。
利用LANCE Ultra方法测定待测物对CDK1/CycB(Carna bioscience，#04-102)、CDK2/CycA(Carna bioscience，#04-103)、CDK9/CycT(Carna bioscience，#04-110)激酶活性的抑制作用。
激酶活性测试采用10μL的体系，包含的组分为：CDK激酶稀释液，Ulight-Myelic basic protein(PerkinElmer，#TRF-0109，下称U-MBP)和ATP(Thermo scientific，#PV3227)混合的底物稀释液，以及本公开的式(I)化合物(即待测物)。测试中每一种激酶包括三个测试组：背景组(Blank)，无抑制组(PC)和化合物测试组(Test)。各组测试中包含的组分如下表15所示。
表15
不同激酶反应中Test组的各组分工作浓度如表16所示：
化合物：室温溶解10mM的待测化合物以DMSO进行梯度稀释，随后用去离子水稀释为4x化合物工作液，DMSO的含量为2％。CDK1和CDK2测试中使用的化合物最高浓度为10μM，CDK9为1μM。
1.33x反应缓冲液：成分为26.7mM MOPS，6.67mM MgCl2和0.0133％Tween-20，配置后放于4℃冰箱内避光保存，使用前加入新鲜配置的DTT至终浓度为5.33mM。
表16
反应中的DMSO工作浓度为0.5％。
上述组分混合后，置于摇床上，室温下避光孵育1小时。随后在所有测试组(包括Blank、PC和Test组)中加入10μL检测液。
10μL检测液包含的组分为：16mM EDTA(Thermo scientific，#15575)，1nM磷酸化U-MBP蛋白抗体(PerkinElmer，#TRF-0201)和1x检测缓冲液(PerkinElmer，#CR97-100)。
加入检测液后，置于摇床上，室温下继续避光孵育1小时。孵育结束后，使用PerkinElmer公司的VictorX5荧光酶标仪读取信号，激发光波长为320nm，发射光波长为615nm和665nm，计算抑制率的方法如下：
1.所有的组别计算665nm/615nm的值(下称Ratio值)，抑制率以各组的Ratio值进行计算；
2.抑制率＝(PCRatio-TestRatio)/(PCRatio-BlankRatio)*100％；
3.用XLFIT5.0软件(英国IDBS公司)进行拟合，以化合物浓度的对数值作为X轴，以抑制率作为Y轴，使用四参数模型计算化合物的半数抑制浓度IC50，结果如表17所示。
表17
由表17中结果可知，本公开的式(I)化合物对CDK9具有较高的抑制活性，并且具有较高的CDK9抑制选择性。
测试例5：小鼠体内药代试验
应用LC/MS/MS法测定了小鼠分别静脉注射和灌胃给药化合物D、化合物E和式(I)化合物后不同时刻血浆中的药物浓度，研究化合物D、化合物E和式(I)化合物在小鼠体内的药代动力学行为，评价其药动学特征。
实验方案：
试验动物：健康成年雄性ICR小鼠(体重30-40g，12只，静脉注射组小鼠自由饮水和饮食，灌胃给药组禁食整晚，给药4h后自由饮水和饮食)，由Beijing Vital River Laboratory Animal Co.LTD提供；
给药方式与剂量：ICR小鼠尾静脉给药(2mg/kg,5％DMSO,pH 4.5 20％Captisol)和灌胃给药(10mg/kg,5％DMSO,pH 4.5 20％Captisol)。
血样采集：给药前挑选符合实验要求的动物，称重标记。采集血样前，绑定小鼠，每一只给药的小鼠在预定的采血时间点(静脉给药：分别于给药后的0.083,0.25,0.5,1,2,4,6,7.5,24h采血,共9个时间点；灌胃给药：分别于给药后的0.083,0.25,0.5,1,2,4,6,7.5,24h采血,共9个时间点)，通过眼眶采血约100μL。血液转移至预先加入K2EDTA的1.5mL试管中，离心4min(8000rpm,4℃)，取出血浆，整个过程在采血后15min内完成。所有的样品都需要存放于-20℃冰箱直至样品分析。应用LC/MS/MS法测定药物浓度，化合物D、化合物E和式(I)化合物在相同剂量和给药方式下，小鼠体内的药代动力学性质参数如表18所示。
表18化合物在小鼠体内药代动力学参数
测试例6：体内药效实验
在皮下植入MV4-11急性髓系白血病患者来源的基于人源肿瘤细胞系的异种移植(CDX)BALB/c裸小鼠上进行体内药效实验。
实验方案：BALB/c裸鼠，雌性，6-10周，体重约20-23克，将小鼠保持在一个特殊的无病原体的环境中，且在单个通风笼中(5只小鼠每笼，每组2笼10只)。所有的笼子，铺垫和水在使用前进行消毒。所有的动物都可以自由获取标准认证的商业实验室饮食。共有80只购于上海市计划生育科学研究所实验动物经营部(上海浦东金科路3577号)的小鼠用于研究。每只小鼠在右肋腹皮下植入肿瘤细胞(1×1070.1ml+Matrigel 0.1ml)，用于肿瘤的生长。当平均肿瘤体积达到约165立方毫米时，按照体重、瘤体积随机分组，并开始给药。将试验化合物每天口服灌胃给药。抗肿瘤药效通过用化合物处理过的动物的平均肿瘤增加体积除以未处理过动物的平均肿瘤增加体积来确定。
肿瘤体积每周2次用二维卡尺测量，体积以立方毫米计量。肿瘤体积TV＝0.5a×b2。其中a是肿瘤的长径，b是肿瘤的短径。
相对肿瘤增值率T/C(％)，即在某一时间点，治疗组和对照组的相对肿瘤体积(RTV)的百分比值。计算公式如下：T/C％＝TRTV/CRTV×100％(TRTV：治疗组平均RTV；CRTV：溶媒对照组平均RTV；RTV＝Vt/V0，V0为分组时该动物的瘤体积，Vt为治疗后该动物的瘤体积)。mpk是指每千克体重的毫克数。
荷瘤动物的体重变化(％)计算如下：(测量时体重-分组时体重)/分组时体重×100。
每日一次口服给药化合物D与式(I)化合物后的肿瘤体积变化图和小鼠体重变化图分别如图17和图18所示，化合物在小鼠体内的肿瘤抑制结果如表19所示：
表19化合物在小鼠体内的肿瘤抑制结果
在本公开提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本公开的上述讲授内容之后，本领域技术人员可以对本公开作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本公开的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本公开构思的前提下，还可以做出若干变形和改进，这些都属于本公开的保护范围。因此，本公开专利的保护范围应以所附权利要求为准，说明书及附图可以用于解释权利要求的内容。

Claims (36)

  1. 一种式(I)化合物的药学上可接受的盐，
    所述药学上可接受的盐选自：磷酸盐和L-酒石酸盐。
  2. 如权利要求1所述的式(I)化合物的药学上可接受的盐，其中，所述药学上可接受的盐为L-酒石酸盐；所述L-酒石酸盐中L-酒石酸与式(I)化合物的摩尔比为(0.8-1.2)：1，优选为(0.9-1.1)：1，更优选为1:1。
  3. 如权利要求1所述的式(I)化合物的药学上可接受的盐，其中，所述药学上可接受的盐为磷酸盐；所述磷酸盐中磷酸与式(I)化合物的摩尔比为(1.8-2.4)：1，优选为(1.9-2.3)：1，更优选为2:1。
  4. 如权利要求1所述的式(I)化合物的药学上可接受的盐，其中，所述药学上可接受的盐为磷酸盐；所述磷酸盐中磷酸与式(I)化合物的摩尔比为(0.8-1.2)：1，优选为1：1。
  5. 如权利要求2所述的式(I)化合物的药学上可接受的盐，其中，所述L-酒石酸盐为多晶型物，所述多晶型物是选自以下的晶型：
    (1)L-酒石酸盐晶型I，其X射线粉末衍射图2θ(°)衍射角在13.946±0.2、16.881±0.2、19.405±0.2、21.505±0.2和24.262±0.2值处具有特征衍射峰；和
    (2)L-酒石酸盐晶型II，其X射线粉末衍射图2θ(°)衍射角在11.662±0.2和21.353±0.2值处具有特征衍射峰。
  6. 如权利要求2所述的式(I)化合物的药学上可接受的盐，其中，所述L-酒石酸盐为多晶型物，所述多晶型物是选自以下的晶型：
    (1)L-酒石酸盐晶型I，其X射线粉末衍射图2θ(°)衍射角在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和31.579±0.2值处具有特征衍射峰；和
    (2)L-酒石酸盐晶型II，其X射线粉末衍射图2θ(°)衍射角在11.662±0.2、14.244±0.2、17.481±0.2、18.349±0.2、21.026±0.2和21.353±0.2值处具有特征衍射峰。
  7. 如权利要求2所述的式(I)化合物的药学上可接受的盐，其中，所述L-酒石酸盐为多晶型物，所述多晶型物是选自以下的晶型：
    (1)L-酒石酸盐晶型I，其X射线粉末衍射图2θ(°)衍射角在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、34.139±0.2和34.543±0.2值处具有特征峰；和
    (2)L-酒石酸盐晶型II，其X射线粉末衍射图2θ(°)衍射角在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和31.784±0.2值处具有特征峰。
  8. 如权利要求2所述的式(I)化合物的药学上可接受的盐，其中，所述L-酒石酸盐为多晶型物，所述多晶型物是选自以下的晶型：
    (1)L-酒石酸盐晶型I，其具有基本如图5所示的X射线粉末衍射(XRPD)图；和
    (2)L-酒石酸盐晶型II，其具有基本如图7所示的X射线粉末衍射(XRPD)图。
  9. 如权利要求5所述的式(I)化合物的药学上可接受的盐，其中，所述L-酒石酸盐晶型I具有基本如图6所示的热重分析(TGA)图。
  10. 如权利要求2所述的式(I)化合物的药学上可接受的盐，其中，所述L-酒石酸盐为无定型物。
  11. 如权利要求3所述的式(I)化合物的药学上可接受的盐，其中，所述磷酸盐为磷酸盐晶型I，其X射线粉末衍射图2θ(°)衍射角在18.234±0.2、19.131±0.2和21.266±0.2值处具有特征衍射峰。
  12. 如权利要求3所述的式(I)化合物的药学上可接受的盐，其中，所述磷酸盐为磷酸盐晶型I，其X射线粉末衍射图2θ(°)衍射角在13.999±0.2、18.234±0.2、18.803±0.2、19.131±0.2、21.266±0.2、22.01±0.2和23.247±0.2值处具有特征衍射峰。
  13. 如权利要求3所述的式(I)化合物的药学上可接受的盐，其中，所述磷酸盐为磷酸盐晶型I，其具有基本如图1所示的X射线粉末衍射(XRPD)图。
  14. 如权利要求11所述的式(I)化合物的药学上可接受的盐，其中，所述磷酸盐晶型I具有选自以下的一个或多个特征：
    具有基本如图2所述的差示扫描量热(DSC)图；
    具有基本如图2所示的热重分析(TGA)图；和
    具有基本如图3所示的动态水分吸收(DVS)图。
  15. 如权利要求4所述的式(I)化合物的药学上可接受的盐，其中，所述磷酸盐为磷酸盐晶型II，其X射线粉末衍射图的2θ(°)衍射角在18.230±0.2和21.144±0.2值处具有特征衍射峰。
  16. 如权利要求4所述的式(I)化合物的药学上可接受的盐，其中，所述磷酸盐为磷酸盐晶型II，其X射线粉末衍射图2θ(°)衍射角在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和23.96±0.2值处具有特征衍射峰。
  17. 如权利要求4所述的式(I)化合物的药学上可接受的盐，其中，所述磷酸盐为磷酸盐晶型II，其具有基本如图10所示的X射线粉末衍射(XRPD)图。
  18. 如权利要求15至17中任一项所述的式(I)化合物的药学上可接受的盐，其中，所述磷酸盐晶型II具有选自以下的一个或多个特征：
    具有207.48±0.5℃的熔融温度；
    具有基本如图11所示的差示扫描量热(DSC)曲线；
    具有基本如图11所示的热重分析(TGA)曲线；和
    具有基本如图12所示的动态水分吸收(DVS)图。
  19. 如权利要求15至17中任一项所述的式(I)化合物的药学上可接受的盐，其中，所述磷酸盐晶型II是无水物。
  20. 一种式(I)化合物的L-酒石酸盐的制备方法，包括如下步骤：将式(I)化合物与L-酒石酸进行成盐反应，形成所述式(I)化合物的L-酒石酸盐。
  21. 一种式(I)化合物的L-酒石酸盐晶型I的制备方法，包括如下步骤：
    将式(I)化合物与L-酒石酸在有机溶剂中进行成盐反应，形成反应液溶液；
    将所述反应液溶液缓慢降温，得到所述L-酒石酸盐晶型I；
    其中所述有机溶剂选自乙醇、乙腈、乙酸乙酯、丙酮和甲醇中的一种或多种。
  22. 一种式(I)化合物的L-酒石酸盐晶型II的制备方法，包括如下步骤：
    将式(I)化合物与L-酒石酸在有机溶剂中进行成盐反应，形成反应液溶液；
    将所述反应液溶液缓慢降温，添加反溶剂，得到所述L-酒石酸盐晶型II；
    其中所述有机溶剂选自乙醇、乙腈、乙酸乙酯、丙酮和甲醇中的一种或多种；
    所述反溶剂选自甲基叔丁醚、石油醚、正庚烷、正己烷、环己烷、异丙醇、丙酮、乙腈和乙酸乙酯中的一种或多种，且所述反溶剂与所述有机溶剂不同。
  23. 如权利要求22所述的制备方法，其中，所述反溶剂选自甲基叔丁醚、正庚烷、异丙醇和丙酮中的一种或多种，且所述反溶剂与所述有机溶剂不同。
  24. 一种权利要求3或4所述的磷酸盐的制备方法，包括如下步骤：将所述式(I)化合物与磷酸在有机溶剂存在下进行成盐反应，形成所述式(I)化合物的磷酸盐。
  25. 如权利要求24所述的制备方法，其中，形成所述式(I)化合物的磷酸盐中式(I)化合物与磷酸的摩尔比为1：1或者1:2。
  26. 如权利要求24所述的制备方法，其中，所述有机溶剂选自乙醇、乙酸乙酯、乙腈和丙酮中的一种或多种。
  27. 如权利要求24所述的制备方法，其中，所述有机溶剂选自甲醇。
  28. 一种式(I)化合物的磷酸盐晶型II的制备方法，包括如下步骤：
    将所述式(I)化合物与磷酸在有机溶剂中进行成盐反应，析出固体；
    收集固体，得到磷酸盐晶型II；
    其中式(I)化合物与磷酸的投料摩尔比为1：(0.8-1.2)；所述有机溶剂为乙醇、甲醇和丙酮中的一种或多种；优选地，所述有机溶剂为乙醇和丙酮中的一种或二者；优选地，所述有机溶剂为甲醇。
  29. 如权利要求28所述的制备方法，其中，所述有机溶剂为乙醇。
  30. 如权利要求29所述的制备方法，其中，所述制备方法包括如下步骤：
    将所述式(I)化合物的乙醇溶液与磷酸的乙醇溶液进行成盐反应，析出固体；
    收集固体，得到磷酸盐晶型II；
    其中式(I)化合物与磷酸的投料摩尔比为1：(0.8-1.2)。
  31. 一种式(I)化合物磷酸盐晶型I的制备方法包括：
    (AI-a)将式(I)化合物溶于溶剂中，加入磷酸水溶液，搅拌反应；
    (AI-b)将步骤(AI-a)得到的反应液冷却，加入反溶剂析晶，固液分离，收集固相得到式(I)化合物磷酸盐晶型I。
  32. 一种药物组合物，包括：
    (a)权利要求1-19中任一项所述的式(I)化合物的药学上可接受的盐；以及(b)药学上可接受的载体。
  33. 权利要求1-19中任一项所述的式(I)化合物的药学上可接受的盐、或权利要求32所述的药物组合物在制备预防或治疗与CDK9活性相关的或由CDK9活性介导的疾病的药物中的应用。
  34. 如权利要求33所述的应用，其中，所述的疾病为过度增殖性疾病、病毒诱导的感染性疾病和心血管疾病中的一种或多种。
  35. 一种治疗与CDK9活性相关的或由CDK9活性介导的疾病的方法，所述方法包括给予对象有效量的权利要求1-19中任一项所述的式(I)化合物的药学上可接受的盐、或权利要求32所述的药物组合物。
  36. 用于预防或治疗与CDK9活性相关的或由CDK9活性介导的疾病的权利要求1-19中任一项所述的式(I)化合物的药学上可接受的盐、或权利要求32所述的药物组合物。
PCT/CN2023/108554 2022-07-22 2023-07-21 取代的吡唑并[1,5-a]嘧啶-7-胺衍生物的药学上可接受的盐和多晶型物及其应用 WO2024017365A1 (zh)

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