WO2022001820A1 - 杂环类化合物的晶型及其制备方法和应用 - Google Patents

杂环类化合物的晶型及其制备方法和应用 Download PDF

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WO2022001820A1
WO2022001820A1 PCT/CN2021/102068 CN2021102068W WO2022001820A1 WO 2022001820 A1 WO2022001820 A1 WO 2022001820A1 CN 2021102068 W CN2021102068 W CN 2021102068W WO 2022001820 A1 WO2022001820 A1 WO 2022001820A1
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crystal form
solvent
compound
ray powder
powder diffraction
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PCT/CN2021/102068
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English (en)
French (fr)
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王亮
娄军
洪华云
郭晓丹
钱丽娜
张轶涵
陈永凯
王朝东
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武汉朗来科技发展有限公司
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Priority to EP21831769.1A priority Critical patent/EP4169921A1/en
Priority to CA3184482A priority patent/CA3184482A1/en
Priority to JP2022581651A priority patent/JP7478858B2/ja
Priority to AU2021302056A priority patent/AU2021302056B2/en
Priority to US18/013,620 priority patent/US20230406850A1/en
Publication of WO2022001820A1 publication Critical patent/WO2022001820A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/10—Drugs for disorders of the urinary system of the bladder
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/08—Antiepileptics; Anticonvulsants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/20—Hypnotics; Sedatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/12—Antidiuretics, e.g. drugs for diabetes insipidus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/13—Crystalline forms, e.g. polymorphs

Definitions

  • the invention relates to the field of medicinal chemistry, in particular to a crystal form of a heterocyclic compound and a preparation method and application thereof.
  • ATP receptors are classified into two main families, P2Y- and P2X-purinoceptors, based on molecular structure, transduction mechanism, and pharmacological properties.
  • P2X-purinoceptors are a family of ATP-gated cation channels, and several subtypes have been cloned, including: six homomeric receptors, P2X1; P2X2; P2X3; P2X4; P2X5; and P2X7; and three heteromeric receptors Receptors P2X2/3, P2X4/6, P2X1/5.
  • P2X3 receptors are specifically expressed on primary afferent nerve fibers in "hollow guts," such as the lower urinary tract and respiratory tract.
  • Cough is the main symptom of respiratory diseases. In respiratory outpatient clinics, 70% to 80% of patients have cough symptoms. With the increasing prevalence of COPD, IPF, etc., and cough as the main symptom of most respiratory diseases, the demand also increases. As the body's defensive nerve reflex, coughing is beneficial to clear respiratory secretions and harmful factors, but frequent and severe coughing will seriously affect the patient's work, life and social activities.
  • Patent application CN201911379293.8 relates to a P2X3 antagonist as shown below, which has high P2X3 antagonistic activity, better selectivity, lower toxicity, better metabolic stability and less impact on taste. It has good prospects for drug development. However, it does not refer to the crystalline form of the above-mentioned compound.
  • Compounds generally have polymorphism, and general drugs may exist in two or more different crystalline states.
  • the existence form and quantity of polymorphic compounds are unpredictable, and different crystal forms of the same drug have significant differences in solubility, melting point, density, stability, etc., which affect the temperature, homogeneity, biological properties of the drug to varying degrees Availability, efficacy and safety. Therefore, in the process of new drug research and development, it is necessary to conduct a comprehensive polymorph screening of compounds, and it is of great clinical significance to select a crystal form suitable for the development of pharmaceutical preparations.
  • the invention provides a crystal form of a heterocyclic compound and a preparation method and application thereof.
  • the crystal form of the invention has good stability and has important value for optimization and development of medicines.
  • the present invention provides a crystal form of the compound represented by formula A or its solvate:
  • Form I It is selected from Form I, Form II, Form III, Form IV, Form V, Form VI, Form VII, Form VIII and Form IX.
  • the present invention provides a crystalline form III of the compound of formula A, whose X-ray powder diffraction pattern (XRPD) has characteristic peaks at 2 ⁇ values of 12.91° ⁇ 0.20°, 16.77° ⁇ 0.20°, 19.27° ⁇ 0.20° and 22.80° ⁇ 0.20° .
  • XRPD X-ray powder diffraction pattern
  • the XRPD of the crystalline form III expressed at 2 ⁇ angle also has characteristic peaks at 13.75° ⁇ 0.20°, 14.46° ⁇ 0.20° and 20.86° ⁇ 0.20°; further, in There are characteristic peaks at 21.08° ⁇ 0.20°, 23.75° ⁇ 0.20° and 24.05° ⁇ 0.20°.
  • the XRPD pattern of the crystalline form III represented by the 2 ⁇ angle is substantially as shown in FIG. 1 .
  • TGA thermogravimetric analysis spectrum
  • RT room temperature
  • % a weight percentage
  • DSC differential scanning calorimetry
  • the XRPD pattern before and after heating to dehydration is preferably as shown in FIG. 3 .
  • the dynamic moisture adsorption spectrum (DVS) spectrum of the crystal form III shows that the sample has a certain hygroscopicity, and the water content changes little in a wide humidity range.
  • the DVS spectrum is preferably as shown in FIG. 4 .
  • the XPRD pattern of the crystal form III before and after the DVS test there is no significant change in the XRPD before and after the DVS test, and the XPRD pattern before and after the DVS test is preferably as shown in FIG. 5 .
  • the crystal form is an irregular crystal of about 2 ⁇ m, and the agglomeration is 20-50 ⁇ m, and the PLM image is preferably substantially as shown in FIG. 6 .
  • the purity of the crystal form III is generally above 90%, preferably above 95%.
  • the present invention provides the crystalline form V of the compound of formula A, whose XRPD has characteristic peaks at 2 ⁇ values of 8.38° ⁇ 0.20°, 9.15° ⁇ 0.20°, 13.52° ⁇ 0.20° and 18.44 ⁇ 0.20°.
  • the XRPD pattern of the crystalline form V represented by 2 ⁇ angle also has characteristic peaks at 16.26° ⁇ 0.20°, 16.89° ⁇ 0.20° and 17.86° ⁇ 0.20°; further, in There are characteristic peaks at 22.35° ⁇ 0.20°, 23.56° ⁇ 0.20°, and 24.74° ⁇ 0.20°.
  • the XRPD pattern of the crystalline form V expressed in 2 ⁇ angle is substantially as shown in FIG. 7 .
  • TGA spectrum of the crystal form V there is no weight loss in the temperature range of RT-230°C.
  • DSC spectrum of the crystal form V there is an endothermic peak at 166°C ⁇ 2°C, and the melting enthalpy is 70 ⁇ 2J/g, and its TGA and DSC spectrums are preferably as shown in FIG. 8 .
  • the product is an anhydrous crystal.
  • the DVS spectrum of the crystal form V shows that the sample has a certain hygroscopicity (0.7%, 80% RH), and its DVS spectrum is preferably as shown in FIG. 9 .
  • the XPRD pattern of the crystal form V before and after the DVS test there is no significant change in the XRPD before and after the DVS test, and the XPRD pattern before and after the DVS test is preferably as shown in FIG. 10 .
  • the crystal form is an irregular crystal of about 5 ⁇ m, and its PLM diagram is preferably substantially as shown in FIG. 11 .
  • the purity of the crystal form V is generally above 90%, preferably above 95%.
  • the present invention provides the crystalline form I of the compound of formula A, whose XRPD pattern has characteristic peaks at 2 ⁇ values of 8.56° ⁇ 0.20°, 12.48° ⁇ 0.20° and 22.13° ⁇ 0.20°.
  • the X-ray powder diffraction pattern of the crystal form I also has 2 ⁇ values of 13.53° ⁇ 0.20°, 14.25° ⁇ 0.20°, 25.18° ⁇ 0.20° and 26.07° ⁇ 0.20° There are characteristic peaks at; further, there are characteristic peaks at 22.32° ⁇ 0.20°, 23.23° ⁇ 0.20° and 23.42° ⁇ 0.20°.
  • the XRPD pattern of the crystalline form I represented by the 2 ⁇ angle is substantially as shown in FIG. 12 .
  • the DSC chart of the crystal form I there is an endothermic peak at 152°C ⁇ 2°C, and the melting enthalpy is 44 ⁇ 2 J/g.
  • the TGA diagram of the crystal form I there is no weight loss in the temperature range of RT-230°C. Its TGA and DSC spectra are preferably substantially as shown in FIG. 13 . Combined with the DSC and TGA diagrams, it can be seen that the product is an anhydrous crystal.
  • the DVS diagram of the crystal form I is shown in FIG.
  • the weight of the crystal form I sharply increases. When the relative humidity drops to 40%, all the absorbed moisture is discharged.
  • the crystal form I absorbs moisture and transforms into a hydrate crystal form IV under a high humidity environment.
  • the XRPD patterns before and after hygroscopicity are preferably substantially as shown in FIG. 15 . It can be seen that, combined with the DVS test results (Fig. 14), when the relative humidity of the environment is higher than 40%, the crystal form I rapidly absorbs moisture and transforms into hydrate; when the relative humidity is lower than 50%, the adsorbed water is quickly removed and transformed into crystals. Type I.
  • the crystal form I exhibits hygroscopicity (6.8%, 80% RH), the crystal form of the crystal form I remains unchanged after the DVS test, and the preferred XRPD diagrams before and after the DVS test are shown in FIG. 16 .
  • the crystal form is an irregular crystal of about 5 ⁇ m, and its PLM is preferably substantially as shown in FIG. 17 .
  • the purity of the crystal form I is generally above 90%, preferably above 95%.
  • the present invention provides the crystalline form II of the MTBE solvate of the compound of formula A, whose XRPD pattern has characteristic peaks at 2 ⁇ values of 8.42° ⁇ 0.20°, 12.09° ⁇ 0.20°, 13.68° ⁇ 0.20° and 20.87° ⁇ 0.20°.
  • said Form II XRPD in 2 ⁇ angles is also characterized at 16.17° ⁇ 0.20°, 16.93° ⁇ 0.20°, 17.55° ⁇ 0.20° and 21.20° ⁇ 0.20° peaks; further, there were characteristic peaks at 22.60° ⁇ 0.20°, 23.23° ⁇ 0.20° and 24.40° ⁇ 0.20°.
  • the XRPD of the crystalline form II expressed by 2 ⁇ angle is substantially as shown in FIG. 18 .
  • HNMR hydrogen nuclear magnetic resonance
  • the HNMR spectrum of the residual MTBE is shown in FIG. 19 .
  • the weight loss is 3.5% in the temperature range of 100-160°C, and the weight loss is 2.9% in the temperature range of 160-200°C.
  • the DSC pattern of the crystal form II there are two adjacent endothermic peaks, and the TGA and DSC patterns are preferably as shown in FIG. 20 .
  • the product is a MTBE solvate.
  • the crystal form is an irregular crystal of about 2 ⁇ m, and its PLM is preferably substantially as shown in FIG. 21 .
  • the present invention provides the hydrated crystal form IV of the compound of formula A, whose XRPD pattern has characteristic peaks at 2 ⁇ values of 8.65° ⁇ 0.20°, 12.69° ⁇ 0.20° and 22.56° ⁇ 0.20°.
  • the XRPD of the crystalline form IV in 2 ⁇ angle also has characteristic peaks at 13.48° ⁇ 0.20°, 17.39° ⁇ 0.20°, 21.04° ⁇ 0.20° and 23.63° ⁇ 0.20° ; Further, there are characteristic peaks at 14.39° ⁇ 0.20°, 25.60° ⁇ 0.20° and 26.52° ⁇ 0.20°.
  • the XRPD of the crystalline form IV in 2 ⁇ angle is substantially as shown in FIG. 22 .
  • the weight loss was 1.2% in the temperature range of RT-60°C.
  • the DSC spectrum of the crystal form IV there are two endothermic peaks, the first broad endothermic peak is presumed to be caused by dehydration, and the latter endothermic peak is a melting peak, and its TGA and DSC spectra are preferably as shown in Figure 23.
  • the crystal form IV is only stable in a high-humidity environment. After dehydration, it quickly absorbs moisture and becomes crystal form I again under ambient humidity.
  • the XRPD pattern before and after heating to dehydration is preferably as shown in FIG. 24 .
  • the crystal form is an irregular crystal of about 5 ⁇ m, and its PLM diagram is preferably substantially as shown in FIG. 25 .
  • the purity of the crystal form IV is generally above 90%, preferably above 95%.
  • the present invention provides the hydrated crystal form VI of the compound of formula A, and its X-ray powder diffraction pattern has characteristic peaks at 2 ⁇ values of 8.62° ⁇ 0.20°, 12.69° ⁇ 0.20° and 22.59° ⁇ 0.02°;
  • the XRPD of the crystalline form VI expressed in 2 ⁇ angles is also 13.46° ⁇ 0.20°, 17.41° ⁇ 0.20°, 26.51° ⁇ 0.02°, 25.62° ⁇ 0.02° and 25.24° ⁇ 0.02° There are characteristic peaks at 0.20°; further, there are characteristic peaks at 23.64° ⁇ 0.20°, 21.00° ⁇ 0.20° and 27.85° ⁇ 0.20°.
  • the XRPD of the crystalline form VI expressed as a 2 ⁇ angle is substantially as shown in FIG. 26 .
  • the crystal form sample is transformed into the crystal form I after being placed under ambient humidity (35% RH) for a few minutes. Its XRPD pattern overlay is shown in Figure 27. This suggests that Form VI may be an extremely unstable hydrate.
  • the purity of the crystal form VI is generally above 90%, preferably above 95%.
  • the present invention provides the crystalline form VII of the ethylene glycol solvate of the compound of formula A, whose X-ray powder diffraction pattern has 2 ⁇ values of 8.36° ⁇ 0.20°, 12.13° ⁇ 0.20°, 12.45° ⁇ 0.20°, 16.84° ⁇ 0.20° and characteristic peaks at 21.66° ⁇ 0.20°.
  • the XRPD of the crystalline form VII also has characteristic peaks at the following 2 ⁇ values: 21.07° ⁇ 0.20°, 24.82° ⁇ 0.20°; further, at 13.61° ⁇ 0.20°, 23.22 ° ⁇ 0.20° and 24.57° ⁇ 0.20°.
  • the XRPD of the crystalline Form VII in 2 ⁇ angle is substantially as shown in FIG. 28 .
  • the chemical shifts ⁇ at 3.39 and 4.44 show residual ethylene glycol solvent
  • the 1 HNMR spectrum of the residual ethylene glycol is preferably as shown in FIG. 29 .
  • the TGA spectrum of the crystal form VII there is a weight loss of 25.7% in the range of rt-120°C.
  • the DSC spectrum of the crystal form VII there are two broad endothermic peaks, the first endothermic peak is presumed to be caused by desolvation, and its TGA and DSC spectrum are preferably as shown in FIG. 30 .
  • the product is a solvate of ethylene glycol.
  • the purity of the crystal form VII is generally above 90%, preferably above 95%.
  • the present invention provides a crystalline form VIII of the THF solvate of the compound of formula A, whose X-ray powder diffraction pattern has 2 ⁇ values at 8.53° ⁇ 0.20°, 12.38° ⁇ 0.20°, 13.66° ⁇ 0.20° and 21.49° ⁇ 0.20° has characteristic peaks.
  • the X-ray powder diffraction pattern of the crystalline form VIII at 2 ⁇ angle also has characteristic peaks at 20.99° ⁇ 0.20°, 24.94° ⁇ 0.20° and 25.31° ⁇ 0.20°; further There are characteristic peaks at 17.14° ⁇ 0.20°, 21.72° ⁇ 0.20° and 23.00° ⁇ 0.20°.
  • the X-ray powder diffraction pattern of the crystalline form VIII at 2 ⁇ angle is substantially as shown in FIG. 31 .
  • the weight loss is 5.7% in the temperature range of rt-160°C.
  • the DSC spectrum of the crystal form VIII there is only one endothermic peak, which is the melting peak after the sample is desolvated. Therefore, Form VIII is a solvate containing THF, and its TGA and DSC spectra are preferably as shown in FIG. 32 .
  • the chemical shifts ⁇ at 1.76 and 3.60 show that THF solvent remains, and its 1 HNMR spectrum is preferably as shown in FIG. 33 .
  • the crystal form VIII is unstable, and after desolventizing (vacuum drying at 40° C. for 3 hours), it is transformed into crystal form I, and its XRPD pattern before and after drying is preferably as shown in FIG. 34 .
  • the purity of the crystal form VIII is generally above 90%, preferably above 95%.
  • the present invention provides the crystalline form IX of the DMSO solvate of the compound of formula A, whose XRPD pattern has characteristic peaks at 2 ⁇ values of 8.55° ⁇ 0.20°, 12.43° ⁇ 0.20°, 21.75° ⁇ 0.20° and 25.07° ⁇ 0.20°.
  • the XRPD pattern of the crystalline Form IX expressed at 2 ⁇ angles is also at 13.57° ⁇ 0.20°, 17.18° ⁇ 0.20°, 20.94° ⁇ 0.20° and 25.57° ⁇ 0.20°; further , at 21.37° ⁇ 0.20° and 23.12° ⁇ 0.20°.
  • the XRPD pattern of the crystalline Form IX expressed in 2 ⁇ angle is substantially as shown in FIG. 35 .
  • the weight loss in the temperature range of rt-160°C is 18.23%, and there is a corresponding endothermic peak corresponding to the TGA weight loss on the DSC spectrum.
  • the chemical shift ⁇ at 2.68 shows that DMSO solvent remains, and the 1 HNMR spectrum of the residual DMSO is preferably as shown in FIG. 60 .
  • the product is a DMSO solvate.
  • the purity of the crystal form IX is generally above 90%, preferably above 95%.
  • the rays used in the XRPD are K ⁇ rays.
  • the target type used in the X-ray powder diffraction is a Cu target.
  • the present invention also provides a method for preparing the crystal form III of substance A, which is scheme one, scheme two or scheme three;
  • Scheme 1 it comprises the following steps: crystallize the suspension of the amorphous compound represented by the formula A and the solvent to obtain the crystal form III of the substance A;
  • the solvent is water or an alcohol solvent;
  • Scheme 2 which includes the following steps: adding an anti-solvent to a solution of a compound represented by formula A and a solvent, and performing crystallization to obtain the crystal form III of substance A;
  • the solvent is alcohols, furans or DMSO One or more in; Described anti-solvent is water;
  • Scheme 3 which includes the following steps: adding a solution of compound A and a solvent into an aqueous solution A, crystallization, to obtain the crystal form III of the substance A, and the aqueous solution A is the crystal form III of the substance A.
  • the suspension of seed crystal and water; the solvent is DMSO.
  • the solvent is preferably water or methanol.
  • the crystallization temperature is 20-50°C, preferably 40°C or 50°C.
  • the mass-volume ratio of the amorphous form of the compound A to the solvent is 50 mg/mL.
  • the solvent is preferably one or more of methanol, tetrahydrofuran or DMSO.
  • the volume ratio of the solvent to the water is 3:1-1:1 (for example, 1:1 or 3:1).
  • the volume ratio of the solvent to the water of the solvent is 1:1-1:4 (for example, 1:1, 2:3, 1 :2 or 1:4).
  • the preparation method of the crystal form III when the scheme 1 is adopted, preferably includes the following steps: transcrystallization of the suspension of the amorphous form of compound A and the solvent; the solvent water or methanol.
  • the stirring temperature is 20-50°C, preferably 40°C.
  • the mass-volume ratio of the amorphous form of the compound A to the solvent is 50 mg/mL.
  • the preparation method of the crystal form III when the scheme 2 is adopted, preferably includes the following steps: mixing compound A with a solvent, and then adding it to the anti-solvent in a slow dropwise manner;
  • the solvent is One or more of methanol, tetrahydrofuran or DMSO;
  • the anti-solvent is water;
  • the volume ratio of the solvent to water is 3:1-1:1 (for example, 3:1 or 1:1).
  • the preparation method of the crystal form III when the scheme 3 is adopted, preferably includes the following steps: adding the solution of compound A and the solvent into the aqueous solution A, and crystallization, the aqueous solution A is the The suspension of the seed crystal of the crystal form III of substance A and water; the solvent is DMSO; the volume ratio of DMSO and water is 1:1-1:4 (for example 1:1, 2:3, 1:2 or 1:4).
  • the present invention also provides a method for preparing the crystal form V of compound A, which is scheme A or scheme B;
  • Scheme A which includes the following steps: crystallizing the amorphous form of the compound represented by the formula A with a solvent suspension at 20-50° C. to obtain the crystal form V of the compound A; the solvent is water or Nitrile solvents.
  • Scheme B which includes the following steps: volatilizing the solvent in the solution of the compound represented by formula A and the solvent to obtain the crystal form V of compound A; the solvent is an alcohol solvent.
  • the mass-volume ratio of the amorphous form of the compound A to the solvent is preferably 3.0 mg/mL or 50 mg/mL.
  • the solvent is preferably water or acetonitrile.
  • the temperature of the crystal transformation is preferably 50°C.
  • the solvent is preferably methanol.
  • the scheme A includes the following steps: transforming the amorphous form of compound A with a suspension of a solvent at 20-50° C. to obtain a crystal form V of the compound; the solvent is: Water or acetonitrile; the crystallization temperature is preferably 50°C, and the mass-volume ratio of the amorphous form of the compound A to the solvent is 50 mg/mL or 3.0 mg/mL.
  • the scheme B includes the following steps: volatilizing the solvent in the solution of the compound A and the solvent to obtain the crystal form V of the compound A; for the solvent methanol, the temperature is 50°C.
  • the amorphous form of the compound represented by the formula A is prepared by referring to the method in the patent application CN201911379293.8 (see the examples for details).
  • a pharmaceutical composition comprising the above-mentioned crystalline form of the compound represented by formula A or its solvate (such as crystalline form I, crystalline form II, crystalline form III, crystalline form IV, crystalline form V, crystalline form VI , one or more of Form VII, Form VIII and Form IX) and/or Form III of Substance A as described above, and pharmaceutical excipients.
  • the dosage of the crystal form can be a therapeutically effective amount.
  • the pharmaceutical excipients can be those widely used in the field of pharmaceutical production. Excipients are mainly used to provide a safe, stable and functional pharmaceutical composition, and can also provide a method to enable the subject to dissolve the active ingredient at a desired rate after administration, or to promote the activity of the subject after the composition is administered. The ingredients are effectively absorbed.
  • the pharmaceutical excipients can be inert fillers, or provide some function, such as stabilizing the overall pH of the composition or preventing degradation of the active ingredients of the composition.
  • Described pharmaceutical adjuvants may include one or more of the following adjuvants: binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, adhesives, disintegrating agents, lubricants, anti-sticking agents Agents, glidants, wetting agents, gelling agents, absorption delaying agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavors and sweeteners.
  • adjuvants may include one or more of the following adjuvants: binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, adhesives, disintegrating agents, lubricants, anti-sticking agents Agents, glidants, wetting agents, gelling agents, absorption delaying agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavors and sweeten
  • Substances that can be used as pharmaceutically acceptable excipients include, but are not limited to, ion exchangers, aluminum, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphate, glycine, sorbic acid, sorbic acid Potassium acid, partial glyceride mixture of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silicon, magnesium trisilicate, polyethylene Pyrrolidones, polyacrylates, waxes, polyethylene-polyoxypropylene-blocking polymers, lanolin, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as carboxymethyl Sodium cellulose, ethyl cellulose and cellulose acetate; gum powder; malt; gelatin; talc; adjuvants
  • compositions of the present invention can be prepared in light of the disclosure using any method known to those skilled in the art. For example, conventional mixing, dissolving, granulating, emulsifying, attenuating, encapsulating, entrapping or lyophilizing processes.
  • the present invention provides a crystalline form of the above-mentioned compound represented by formula A or its solvate (for example, crystal form I, crystal form II, crystal form III, crystal form IV, crystal form V, crystal form VI, The use of one or more of crystal form VII, crystal form VIII and crystal form IX), crystal form III of substance A as described above in the preparation of P2X3 inhibitor.
  • the P2X3 inhibitor can be used in mammalian organisms; it can also be used in vitro, mainly for experimental purposes, such as: providing comparison as a standard sample or a control sample, or preparing according to conventional methods in the art into a kit to provide rapid detection of the inhibitory effect of P2X3.
  • the present invention also provides a crystal form of the compound represented by formula A or its solvate (for example, crystal form I, crystal form II, crystal form III, crystal form IV, crystal form V, crystal form VI, crystal form VII, one or more of crystal form VIII and crystal form IX), the use of the crystal form III of substance A as described above in the preparation of medicaments; the medicament is used for the protection, treatment, treatment or alleviation of animals A drug for a disease at least partially mediated or activity-related by P2X3; alternatively, the drug is a drug for the treatment of pain, urinary tract disease or respiratory disease.
  • a crystal form of the compound represented by formula A or its solvate for example, crystal form I, crystal form II, crystal form III, crystal form IV, crystal form V, crystal form VI, crystal form VII, one or more of crystal form VIII and crystal form IX
  • the drug is a drug for the treatment of pain, urinary tract disease or respiratory disease.
  • the present invention provides a crystal form of the above-mentioned compound represented by formula A or its solvate (for example, crystal form I, crystal form II, crystal form III, crystal form IV, crystal form V, crystal form VI, crystal form VII, one or more of Form VIII and Form IX), Form III of Substance A as described above, or a pharmaceutical composition as described above in use in the protection, treatment, treatment or amelioration of animals (eg, humans). ) in diseases that are at least partially mediated or activity-related by P2X3.
  • a crystal form of the above-mentioned compound represented by formula A or its solvate for example, crystal form I, crystal form II, crystal form III, crystal form IV, crystal form V, crystal form VI, crystal form VII, one or more of Form VIII and Form IX
  • Form III of Substance A as described above
  • a pharmaceutical composition as described above in use in the protection, treatment, treatment or amelioration of animals (eg, humans).
  • the diseases include, but are not limited to, respiratory diseases, cough, chronic cough, idiopathic pulmonary fibrosis, chronic pulmonary obstruction, asthma, pain, urinary incontinence, autoimmune diseases, overactive bladder, dysuria, inflammation, Alzheimer's disease, Parkinson's, sleep disorders, epilepsy, mental illness, arthritis, neurodegeneration, traumatic brain injury, myocardial infarction, rheumatoid arthritis, stroke, thrombosis, atherosclerosis, colon syndrome Symptoms, inflammatory bowel disease, digestive tract diseases; gastrointestinal disorders, respiratory failure, sexual dysfunction, cardiovascular system diseases, heart failure, hypertension, urinary incontinence, cystitis, arthritis, endometriosis, blood diseases, musculoskeletal and connective tissue developmental disorders, systemic disorders.
  • the disorder includes pain; the pain includes, but is not limited to: inflammatory pain, surgical pain, visceral pain, dental pain, premenstrual pain, central pain, pain due to burns, migraine, or Cluster headaches.
  • the diseases include urinary system diseases; the urinary tract diseases include: urinary incontinence, overactive bladder, dysuria, cystitis;
  • the disease comprises a respiratory disease including, but not limited to, a breathing disorder including idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, bronchospasm, or chronic cough.
  • a respiratory disease including, but not limited to, a breathing disorder including idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, bronchospasm, or chronic cough.
  • the present invention provides a crystal form of the above-mentioned compound represented by formula A or its solvate (for example, crystal form I, crystal form II, crystal form III, crystal form IV, crystal form V, crystal form VI, crystal form VII, one or more of Form VIII and Form IX), Form III of Substance A as described above, or a pharmaceutical composition as described above to protect, treat, treat or ameliorate at least an animal (eg, a human) Methods for diseases mediated in part or activity-related by P2X3.
  • a crystal form of the above-mentioned compound represented by formula A or its solvate for example, crystal form I, crystal form II, crystal form III, crystal form IV, crystal form V, crystal form VI, crystal form VII, one or more of Form VIII and Form IX
  • Form III of Substance A as described above
  • a pharmaceutical composition as described above to protect, treat, treat or ameliorate at least an animal (eg, a human) Methods for diseases mediated in part or activity-related by P2X3.
  • the diseases include, but are not limited to, respiratory diseases, cough, chronic cough, idiopathic pulmonary fibrosis, chronic pulmonary obstruction, asthma, pain, urinary incontinence, autoimmune diseases, overactive bladder, dysuria, inflammation, Alzheimer's disease, Parkinson's, sleep disorders, epilepsy, mental illness, arthritis, neurodegeneration, traumatic brain injury, myocardial infarction, rheumatoid arthritis, stroke, thrombosis, atherosclerosis, colon syndrome Symptoms, inflammatory bowel disease, digestive tract diseases; gastrointestinal disorders, respiratory failure, sexual dysfunction, cardiovascular system diseases, heart failure, hypertension, urinary incontinence, cystitis, arthritis, endometriosis, blood diseases, musculoskeletal and connective tissue developmental disorders, systemic disorders.
  • the disorder includes pain; the pain includes, but is not limited to: inflammatory pain, surgical pain, visceral pain, dental pain, premenstrual pain, central pain, pain due to burns, migraine, or Cluster headaches.
  • the diseases include urinary system diseases; the urinary tract diseases include: urinary incontinence, overactive bladder, dysuria, cystitis;
  • the disease comprises a respiratory disease including, but not limited to, a breathing disorder including idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, bronchospasm, or chronic cough.
  • a respiratory disease including, but not limited to, a breathing disorder including idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, bronchospasm, or chronic cough.
  • the above pharmaceutical composition characterized in that, by administering the pharmaceutical composition, the side effects of dysgeusia associated with the treatment are reduced.
  • the reagents and raw materials used in the present invention are all commercially available.
  • the crystal form of the heterocyclic compound has not been reported in the prior art, and this application is the first time to discover a variety of new crystal forms of the compound.
  • the present invention has prepared crystal form I, crystal form II, crystal form III, crystal form IV, crystal form V, crystal form VI, crystal form VII, crystal form VIII or crystal form IX for the first time, and the as a candidate.
  • the partial crystal forms prepared by the present invention have good stability, are convenient for storage, can avoid the risk of crystal transformation during drug development or production process, avoid changes in bioavailability and drug efficacy, and can be developed into suitable for clinical use.
  • the dosage form has strong economic value.
  • the present invention also provides salts or solvates of crystal form I, crystal form II, crystal form III, crystal form IV, crystal form V, crystal form VI, crystal form VII, crystal form VIII and crystal form IX.
  • the preparation method of the crystal form has the advantages of simple operation and high reproducibility, the solvent is not easy to remain, is environmentally friendly, and is suitable for different large-scale production.
  • Fig. 1 is the XRPD pattern of crystal form III
  • Figure 2 is a TGA/DSC overlay of Form III
  • Fig. 3 is the XRPD overlay of crystal form III before and after heating to dehydration
  • Fig. 4 is the DVS diagram of crystal form III
  • Fig. 5 is the XRPD overlay before and after the DVS test of crystal form III;
  • Fig. 6 is the PLM diagram of crystal form III
  • Fig. 7 is the XRPD pattern of crystal form V
  • Figure 8 is a TGA/DSC overlay of Form V
  • Fig. 9 is the XRPD overlay diagram before and after the DVS test of crystal form V;
  • Figure 10 is a DVS diagram of Form V
  • Fig. 11 is the PLM diagram of crystal form V
  • Figure 12 is the XRPD pattern of Form I
  • Figure 13 is a TGA/DSC overlay of Form I
  • Figure 14 is a DVS diagram of Form I
  • Fig. 15 is XPRD figure after crystal form I absorbs moisture
  • Figure 16 is the XRPD overlay before and after the DVS test of Form I;
  • Figure 17 is a PLM diagram of Form I
  • Figure 18 is the XRPD pattern of Form II
  • Figure 19 is the HNMR spectrum of the residual MTBE of crystal form II.
  • Figure 20 is a TGA/DSC overlay of Form II
  • Figure 21 is the PLM diagram of crystal form II
  • Figure 22 is an XRPD pattern of Form IV
  • Figure 23 is a TGA/DSC overlay of Form IV
  • Figure 24 is the XRPD overlay before and after dehydration of crystal form IV;
  • Figure 25 is a PLM diagram of Form IV
  • Figure 26 is the XRPD pattern of crystal form VI
  • Figure 27 is an XRPD overlay of Form VI
  • Figure 28 is the XRPD pattern of crystal form VII
  • Figure 29 is the residual ethylene glycol HNMR spectrum of crystal form VII.
  • Figure 30 is a TGA/DSC overlay of Form VII
  • Figure 31 is the XRPD pattern of crystal form VIII
  • Figure 32 is a TGA/DSC overlay of Form VIII
  • Figure 33 is the HNMR spectrum of crystal form VIII and starting material
  • Figure 34 is the XRPD overlay of Form VIII and the sample before drying
  • Figure 35 is an XRPD pattern of Form IX
  • Figure 36 is a TGA/DSC overlay of Form IX
  • Figure 37 is an XRPD overlay of Form III obtained from an amorphous sample
  • Figure 38 is a DSC & TGA overlay of Form III obtained from an amorphous sample
  • Figure 39 is the XRPD overlay of the crystal form III slurried in mixed solvents of different volume ratios;
  • Figure 40 is the XRPD overlay of the 200mg grade crystal form III;
  • Figure 41 is the PLM image of 200mg grade crystal form III
  • Figure 42 is an XRPD overlay of Form III (3g).
  • Figure 43 is the XRPD overlay of Form III before and after beating in water at room temperature
  • Fig. 44 is the water activity experiment result figure of crystal form I
  • Fig. 45 is the water activity experiment result graph of crystal form V
  • Figure 46 is the XRPD overlay of the competitive beating experiment of crystal form I and crystal form V;
  • Fig. 47 is the stability data result graph under the solid state of crystal form I and V;
  • Figure 48 is an XRPD overlay of the Form I stability test sample
  • Figure 49 is the XRPD overlay of the Form III stability test sample
  • Figure 50 is the XRPD overlay diagram before and after the grinding test of crystal form I;
  • Figure 51 is the XRPD overlay diagram before and after the crystal form I stress test
  • Figure 52 is the XRPD overlay before and after the grinding test of crystal form III
  • Figure 53 is the XRPD overlay diagram before and after the pressure test of crystal form III;
  • Figure 54 is a graph of solubility data for Forms I, III and V;
  • Figure 55 is an XRPD overlay of the remaining solids from the Form I solubility test
  • Figure 56 is an XRPD overlay of the remaining solids from the Form V solubility test
  • Figure 57 is an XRPD overlay of the remaining solids from the Form III solubility test.
  • Figure 58 is a PLM image of an amorphous sample
  • Figure 59 is a DSC&TGA overlay of an amorphous sample
  • Figure 60 is the 1 HNMR spectrum of crystal form IX
  • Figure 61 is an XRPD overlay of the Form V stability test sample.
  • the described experimental methods are usually completed under conventional conditions or conventional test conditions, and the compounds can be obtained by organic synthesis or by commercially available methods.
  • the compounds used in the following examples were obtained by commercially available methods with a purity of 99%.
  • test conditions are as follows:
  • the solids were characterized using an X-ray powder diffractometer (Bruker D8 advance or D2 Phase).
  • Step size 0.02° (2 ⁇ ).
  • Light tube voltage 40KV (D8), 30KV (D2).
  • Light tube current 40mA (D8), 10mA (D2).
  • Thermogravimetric analysis of solid samples was performed using a TA Instruments Thermogravimetric Analysis Q500 or Discovery TGA 55. After equilibrating the sample pan, the sample was suspended on the hanging wire and raised to the furnace. After stabilization, the samples were heated to different endpoint temperatures at a rate of 10°C/min.
  • DSC analysis of solid samples was performed using a TA Instruments Differential Scanning Calorimeter Q200 and a Discovery DSC 250. Weigh the sample and record the value, then place the sample in the sample chamber. The samples were heated from 25°C to various endpoint temperatures at a rate of 10°C/min.
  • Airflow 250 mL/min.
  • the shortest test time 30min.
  • the samples were observed using a Nikon Eclipse LV100N POL type polarizing microscope.
  • a total of 11.1 g of compound A was prepared with reference to Example 1, and 20 mL of acetone was added, refluxed at 65 °C (nitrogen protection) for 2.0 h, directly spin-dried acetone, and vacuum dried at 40 °C for 12 h. NMR showed that there was about 1% acetone residue. It was vacuum-dried again at 80°C for 12h, and NMR showed that there was still acetone residue. Take 5.3 g of it and vacuum dry it again at 80 °C for 12 h, and NMR shows that there is still acetone residue.
  • the batch of product was added to acetonitrile (16 mL), refluxed at 85 °C (nitrogen protection) for 2.0 h, directly spin-dried acetonitrile, and then vacuum-dried at 80 °C for 12 h.
  • the product has a purity of 99.29% and is in the form of white powder.
  • the results of PLM and XRPD show that the raw material is an irregular shape crystal of 10-50 ⁇ m, the crystallinity is average, and it is an amorphous state type.
  • the DSC spectrum shows that the raw material has two connected endothermic peaks between about 150-180 °C, and the peak temperatures are 164 ⁇ 2 °C and 173 °C ⁇ 2 °C, respectively, as shown in Figure 38, the TGA spectrum shows that the sample is at There is basically no weight loss before 230°C.
  • Example 3 Amorphous preparation and characterization of the compound of formula A
  • the XRPD of Form I in 2 ⁇ angles is shown in Figure 12.
  • the DSC chart of the crystal form I there is an endothermic peak at 152° C., and the melting enthalpy is 44 ⁇ 2 J/g.
  • the TGA spectrum of the crystal form I there is no weight loss in the temperature range of RT-230°C. Its TGA and DSC spectra are shown in Figure 13. Combined with the DSC and TGA diagrams, it can be seen that the product is an anhydrous crystal.
  • the DVS spectrum of the crystal form I is shown in FIG. 14 , when the relative humidity is greater than 40%, the weight of the crystal form I increases sharply. When the relative humidity drops to 40%, all the absorbed moisture is discharged.
  • the crystal form I absorbs moisture and transforms into a hydrate crystal form IV under a high humidity environment.
  • the XRPD pattern of the initial crystal form I after moisture absorption is shown in Figure 15. It can be seen that, combined with the DVS test results (Fig. 14), when the relative humidity of the environment is higher than 40%, the crystal form I rapidly absorbs moisture and transforms into hydrate; when the relative humidity is lower than 50%, the adsorbed water is quickly removed and transformed into crystals.
  • Type I That is, the interconversion between Forms I and IV is reversible.
  • the crystal form I showed hygroscopicity (6.8%, 80% RH), the crystal form remained unchanged after the IDVS test, and the XRPD patterns before and after the DVS test were shown in FIG. 16 .
  • the crystal form is an irregular crystal of about 5 ⁇ m, and its PLM is shown in FIG. 17 .
  • the X-ray powder diffraction pattern of Form II in 2 ⁇ angle is shown in FIG. 18 .
  • the H NMR spectrum of the crystal form II the residual solvent signals of MTBE at chemical shifts of 1.10 and 3.08; the molar ratio thereof is 0.39
  • the H NMR spectrum is shown in FIG. 19 .
  • the weight loss is 3.5% in the temperature range of 100-160°C, and the weight loss is 2.9% in the temperature range of 160-200°C.
  • the DSC pattern of the crystal form II there are two adjacent endothermic peaks, and the TGA and DSC patterns are shown in FIG. 20 .
  • the product is an MTBE solvate.
  • the crystal form is an irregular crystal of about 2 ⁇ m, and its polarizing microscope image is shown in FIG. 21 .
  • Crystal form III Prepare by suspending and beating the amorphous sample in water at 40 °C to obtain crystal form III; specifically: dissolve the sample in methanol, filter to obtain the sample solution, and then rotate to obtain 250 mg of amorphous sample. Form III was obtained after 20 hours of suspension and beating in water. Through XRPD, DSC, and TGA characterization tests, it was determined that pure crystal form III was successfully prepared and used as a seed crystal for subsequent scale-up experiments.
  • the crystal form III was prepared by the method of solvent-antisolvent precipitation and nucleation induced by adding seed crystals. Specifically: pre-dissolve the sample in a mixed solvent of a specific ratio shown in Table 1, filter to obtain a saturated solution, add crystal form III as a seed crystal to the above saturated solution, and then slowly add water as an anti-solvent to precipitate solids and induce crystallization. XRPD testing was performed on the filtered solid samples. The experimental results are shown in Table 1. The results show that the crystal form III is unstable in the above system, and is transformed into a solvate or an unstable hydrate. From the perspective of solubility and solvent residue limit, DMSO is the third type of solvent and its solubility is higher than 100 mg/mL, so DMSO is selected for further research.
  • the target crystalline form was prepared by crystallization of Compound A in an aqueous solution of .
  • the specific operation method is as follows: firstly, the sample is dissolved in DMSO and filtered to obtain a DMSO solution, and then it is slowly added dropwise to the aqueous solution in which the seeds are dispersed. The precipitated solid was transformed into the target crystal form III under the induction of seed crystals.
  • crystal form III As shown in Table 2 and Figure 1, the stability of crystal form III in different proportions of DMSO/water mixed solvent systems was investigated respectively to determine the appropriate solvent/anti-solvent ratio to ensure that crystal form III can be stable in this system. exist.
  • the experimental results show that the crystal form III can exist stably in a DMSO/water mixed solvent with a water content higher than 60% (volume percent) at room temperature without transcrystallization. When the water content is less than 60%, the crystal form III is transformed into a solvate of DMSO (crystal form IX).
  • the theoretical yield of the crystal form III under the above ratios was calculated to further determine the ratio of the mixed solvent. As shown in Table 3, for DMSO/water 1:2, 1:3, and 1:4, since the solubility of crystal form III in these three ratios of solvents is relatively small, the theoretical yields are not much different. The rate is above 99%.
  • the reaction temperature was lowered to room temperature, and the slurry was continued to be suspended at room temperature. After 22 hours, a solid sample was obtained by filtration, which was slurried in water to remove DMSO residues. As shown in Figure 42, 3 g of crystal form III was successfully amplified and prepared, and the yield was 96.7%. As shown in Figure 43, 0.7% of DMSO dissolved residue can be removed by beating in water at room temperature, and the crystal form remains unchanged.
  • the crystallization temperature of the final crystal form III was 40°C
  • the reaction system was 1:4 DMSO/water
  • the solvent volume was 25V.
  • the crystal form III can be prepared by gram scale scale up, and the yield is as high as 97%.
  • the XRPD pattern of Form III in 2 ⁇ angle is shown in Figure 1.
  • the weight loss gradient in the range of RT-100° C. is 1.5%
  • the “%” is the weight percentage.
  • the first endothermic peak is the dehydration of 0.4 water
  • the second endothermic peak is attributed to the melting endothermic peak after dehydration of the sample.
  • the TGA and DSC spectrums are shown in Figure 2. After the described crystal form III is dehydrated, it quickly absorbs moisture and becomes crystal form III again under ambient humidity, and its XRPD pattern before and after dehydration is shown in FIG. 3 .
  • the DVS spectrum of the crystal form III shows that the sample has a certain degree of hygroscopicity, and the water content does not change much in a wide humidity range.
  • the DVS spectrum is shown in FIG. 4 .
  • the XRPD did not change significantly before and after the DVS test, and the XPRD pattern thereof is shown in FIG. 5 .
  • the crystal form is an irregular crystal of about 2 ⁇ m, and the agglomeration is 20-50 ⁇ m, and its PLM diagram is shown in FIG. 6 .
  • the XRPD pattern of IV in 2 ⁇ angles is basically as shown in Figure 22.
  • the weight loss was 1.2% in the temperature range of RT-60°C.
  • the DSC spectrum of the crystal form IV there are two endothermic peaks. The first broad endothermic peak is presumed to be caused by dehydration, and the latter endothermic peak is a melting peak.
  • the TGA and DSC spectra are shown in Figure 23. Combining the DSC and TGA images, it can be known that the product is a hydrate crystal form with 0.34 water molecules per month.
  • the crystal form IV is only stable in a high-humidity environment. After dehydration, it quickly absorbs moisture and becomes crystal form I again under ambient humidity.
  • the XRPD pattern before and after heating to dehydration is shown in Figure 24.
  • the crystal form is an irregular crystal of about 5 ⁇ m, and its PLM diagram is shown in FIG. 25 .
  • the XRPD pattern of Form V in 2 ⁇ angle is shown in FIG. 7 .
  • TGA spectrum of the crystal form V there is no weight loss in the temperature range of RT-230°C.
  • DSC spectrum of the crystal form II there is an endothermic peak at 166°C ⁇ 2°C, and the melting enthalpy is 70 ⁇ 2J/g, and the TGA and DSC spectrums are shown in FIG. 8 .
  • the product is an anhydrous crystal.
  • the DVS spectrum of the crystal form V shows that the sample has a certain hygroscopicity (0.7%, 80% RH), and its DVS spectrum is shown in FIG. 9 .
  • the XPRD pattern of the crystal form V before and after the DVS test the XRPD did not change significantly before and after the DVS test, and its XPRD pattern is shown in FIG. 10 .
  • the crystal form In the PLM of the crystal form V, the crystal form is an irregular crystal of about 5 ⁇ m, and its PLM diagram is shown in FIG. 11 .
  • Crystal form VI is obtained by beating crystal form I or crystal form V in a water/acetone mixed solvent with a water content of 10% (volume ratio) at 60°C at 60°C.
  • the X-ray powder diffraction pattern of Form VI expressed at 2 ⁇ angle is shown in FIG. 26 .
  • the crystal form sample is transformed into the crystal form I after being placed under ambient humidity (35% RH) for a few minutes. Its XRPD pattern overlay is shown in Figure 27. This suggests that Form VI may be an extremely unstable hydrate.
  • the XRPD of Form VII in 2 ⁇ angles is substantially as shown in Figure 28 .
  • the chemical shifts ⁇ at 3.39 and 4.44 have ethylene glycol residues
  • the 1 HNMR spectrum is shown in FIG. 29 .
  • the TGA spectrum of the crystal form VII there is a weight loss of 25.7% in the range of rt-120°C.
  • the DSC spectrum of the crystal form VII there are two broad endothermic peaks, the first endothermic peak is presumed to be caused by desolvation, and the TGA and DSC spectrums are shown in FIG. 30 . Combined with DSC and TGA, it can be known that the product is a solvate containing 2.79 molecules of ethylene glycol.
  • Example 11 Preparation and characterization of the compound of formula A crystal form VIII.
  • Crystal form III was used as a seed crystal, and water was added dropwise to a saturated 50% THF/water solution at 40°C as an anti-solvent, resulting in an oily state. After cooling to room temperature, continue beating to obtain crystal form VIII (wet filter cake)
  • the XRPD pattern of Form VIII in 2 ⁇ angles is substantially as shown in Figure 31 .
  • the weight loss is 5.7% in the temperature range of Rt-160°C.
  • the DSC spectrum of the crystal form VIII there is only one endothermic peak, which is the melting peak after the sample is desolvated. Therefore, Form VIII is a solvate whose TGA and DSC spectra are shown in Figure 32.
  • the chemical shift ⁇ is at 1.76, and there is a soluble residue of THF at 3.60, and its 1 HNMR spectrum is shown in FIG. 33 .
  • the crystal form VIII contains 0.42 molecules of THF.
  • the crystal form VIII is unstable, and is transformed into crystal form I after dehydration (vacuum drying at 40° C. for 3 hours), and the XRPD patterns before and after heating are shown in FIG. 34 .
  • Form IX is prepared by beating form III in 50% DMSO/water saturated solution at 40°C
  • the weight loss in the temperature range of rt-160°C is 18.23%.
  • the DSC spectrum of the crystal form IX there is a corresponding endothermic peak corresponding to the TGA weight loss on the DSC spectrum, and its TGA and DSC spectrums As shown in Figure 36.
  • the chemical shift ⁇ at 2.68 shows that DMSO solvent remains, and the 1 HNMR spectrum of the residual DMSO is shown in FIG. 60 .
  • crystal form I anhydrous crystal forms
  • crystal form V four solvates
  • crystal form II crystal form VII
  • crystal form IX crystal form IX
  • crystal form VIII three hydrates
  • Form III Form IV, Form VI
  • the crystal form I can be repeatedly obtained by different crystallization methods, however, the crystal form I exhibits a hygroscopic form and is hygroscopically transformed into a hydrate crystal form IV when the relative humidity is higher than 40% RH. Therefore, Form I has the risk of transcrystallization during API production and scale-up.
  • Form III (as a hemihydrate) with low risk of transcrystallization.
  • Crystal form IV (as a hydrate), transformed into crystal form I after dehydration;
  • the crystal form VI (as a hydrate) is extremely unstable, and it is quickly transformed into the crystal form I under ambient humidity.
  • Crystal forms I and V (both are anhydrous crystal forms) were beaten for three days at room temperature and 60 ° C in acetone/water mixed solvents with different water contents, respectively. After three days, the solid samples were filtered to test XRPD. The experimental results are shown in Table 4. Show. As shown in Figures 44 and 45, regardless of room temperature or high temperature, crystal forms I and V were transformed into hydrate crystal form IV in acetone/water system with a water content of 50-90%. When the water content is 10%, both crystal forms I or V are transformed into another hydrate (crystal form VI) at high temperature. It was found that the hydrate was extremely unstable under ambient humidity (35% RH), and transformed into crystal form I after being placed for a few minutes. At room temperature, the crystal form V was transformed into the crystal form I in the solvent system.
  • N/A means: not available
  • crystal forms I, III and V Compared with other crystal forms, crystal forms I, III and V exhibited good solid-state properties, so their stability tests were carried out respectively.
  • Forms I, III and V were placed in stability chambers at 40°C/75% RH (open) and 60°C (covered) for seven days, and some samples were taken at 0 and 7 days for XRPD and HPLC, respectively, to investigate its physical and chemical stability.
  • Table 5 Figures 48, 49 and 61, the crystal forms of Form III and Form V did not change after seven days under the above test conditions, and the chemical purity did not decrease significantly. This indicates that Form III and Form V have good physical and chemical stability.
  • the hydrated crystal form IV was transformed into the hydrate crystal form IV by hygroscopic transformation under the condition of 40°C/75%RH (open). This result is consistent with the DVS result.
  • the mechanical stability of Forms I and III was investigated by grinding and tableting, respectively.
  • the grinding test method is as follows: the sample to be tested is ground in a mortar for 2 minutes and 5 minutes, respectively, and then XRPD is tested, and the changes of crystal form and crystallinity are observed.
  • the pressure test method is as follows: the sample is placed in the mold of the tablet press, kept at 40 MPa for one minute, and then taken out to test the XRPD.
  • crystal forms I, III and V showed good solid state properties, so their solubility tests were carried out respectively.
  • a 10 mg sample was weighed and 2.0 mL of biological vehicle buffer (ie, a target concentration of 5 mg/mL) was added to form a suspension. It was placed in a constant temperature water bath shaker (37°C, 100 rpm), and 500 mL were sampled at 0.5, 2 and 24 hours, respectively. After filtration, the pH value and HPLC concentration of the filtrate were tested respectively, and the solid sample obtained by filtration was tested for XRPD.
  • biological vehicle buffer ie, a target concentration of 5 mg/mL
  • Form I was transformed to hydrate form IV at 0.5 hours in FaSSIF and FeSSIF buffers.
  • the crystal forms of Forms III and V did not change during the testing process, and the results are shown in Figure 56 and Figure 57 .
  • Dissolved clear indicates that the solubility of the sample in the medium is greater than 5 mg/mL by visual observation
  • N/A Indicates no solid sample to test for XRPD.
  • the pharmacokinetics of the compound in plasma were studied after single oral gavage administration of compound A to beagle dogs (general grade, Beijing Mars Biotechnology Co., Ltd.).
  • the test drug (100mg/kg dose, 5mL/kg administration volume) was administered by gavage, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 24 hours after administration samples were taken and 48 hours: peripherally venipuncture, each animal was collected at each time point ⁇ 1.0mL whole blood, the blood samples were collected into K 2 EDTA blood collection tubes, inverted several times up and down thoroughly mixed, left at room temperature.
  • the concentration of Compound A in plasma samples was determined by LC-MS/MS method (Non-GLP). Pharmacokinetic parameter calculations are performed with WinNonlin (PhoenixTM, version 8.1) or other similar software. The following pharmacokinetic parameters will be calculated if available plasma drug concentration-time data: CL (clearance), Vss (apparent volume of distribution at steady state), T1/2 (elimination half-life), Cmax (peak concentration) ), Tmax (time to peak), AUC (area under the plasma concentration-time curve), MRT (mean residence time). Pharmacokinetic data were described by descriptive statistics such as mean, standard deviation and sample size. Calculations were performed with Microsoft Excel 2007 or 2010.
  • the drug concentration in erythrocytes is saturated, resulting in a similar sudden increase in plasma drug concentration (presumably the highest possibility); 2.
  • the drug continues to accumulate in erythrocytes after the first day of fasting administration, and the 7-day washout period is not enough to make distribution
  • the drug is completely metabolized in red blood cells, so the plasma drug concentration is significantly increased during the second non-fasting administration; 3. After the first day of fasting administration, it will affect the red blood cells, so that the second non-fasting administration
  • the ability of red blood cells to bind to the drug is reduced, resulting in a significant increase in plasma drug concentrations.
  • the antagonistic properties of the compounds of the present invention were determined by the FLIPR (fluorescence imaging plate reader) method. Type 3, accession number NM_002559.4) inhibitor of activation-induced increase in intracellular calcium.
  • HEK293 cells stably expressing hP2X3 were placed in a 37°C, 5% humidity cell incubator containing 10% FBS (fetal bovine serum, Gibco, 10099-141), 1% penicillin-streptomycin (Gibco, 15140- 122), and cultured in DMEM high glucose medium with 1 mg/mL G418 (Invitrogen, 10131027). 18-24 hours before FLIPR experiments, cells were seeded into 384 wells (10,000 cells/well) at a density of 400,000 cells/mL and incubated overnight in a cell incubator.
  • FBS fetal bovine serum
  • Gibco fetal bovine serum
  • 1% penicillin-streptomycin Gibco, 15140- 122
  • DMEM high glucose medium 1 mg/mL G418
  • test compound 10 [mu]L of test compound (dissolved in DMSO at a concentration of 10 mM and serially diluted with buffer) or vehicle was then added to each well and allowed to equilibrate for 30 min at room temperature.
  • the cell plate was then placed in the FLIPR for baseline fluorescence measurements (excitation at 485 nm, emission at 525-535 nm).
  • agonist BZ-ATP (Sigma, B6396) at a final concentration of 2.5 ⁇ M) or vehicle (ultrapure water) was added at 10 ⁇ L/well, the fluorescence value was measured at 1 second intervals for 2 minutes, and finally the output fluorescence was counted. analyze.
  • the selectivity of the compounds of the present invention to P2X2/3 receptors was determined by FLIPR (fluorescence imaging plate reader) method, and the compounds were hP2X2/3 ( man purinergic receptor subtypes P 2 X 2 and heterodimeric receptor subtype 3 is formed, P2X2 accession numbers NM_002559.4, P2X3 the accession number NM_002559.4) induced by the activation of intracellular calcium l high inhibitor.
  • HEK293 cells stably expressing hP2X2/3 were placed in a 37°C, 5% humidity cell incubator containing 10% FBS (fetal bovine serum, Gibco, 10099-141), 1% penicillin-streptomycin (Gibco, 15140-122), and 1 mg/mL G418 (Invitrogen, 10131027) in DMEM high glucose medium. 18-24 hours before the FLIPR experiment, cells were seeded into 96 wells (25000 cells/well) at a density of 250000 cells/mL and incubated overnight in a cell culture incubator.
  • FBS fetal bovine serum
  • Gibco fetal bovine serum
  • Gibco penicillin-streptomycin
  • G418 Invitrogen, 10131027
  • the 96-well plate was then washed once with buffer and 50 [mu]L of buffer containing test compound or vehicle was added to each well and allowed to incubate for 30 min at room temperature.
  • the cell plate was then placed in the FLIPR for baseline fluorescence measurements (excitation at 485 nm, emission at 525-535 nm).
  • 50 ⁇ L/well of agonist BZ-ATP (Sigma, B6396) at a final concentration of 5 ⁇ M
  • vehicle ultrapure water
  • Compound 172 has the effect of reducing the number of coughs and increasing the cough latency, and the efficacy is comparable to that of the positive compound.
  • the male Dunkin Hartley guinea pigs (300-400g) were placed in the whole body volume scanning box for 3-5 minutes, and then nebulized with ATP for 2 minutes, with an interval of 3 minutes, and then given 5 minutes of citric acid atomization. All atomization rates were 300ul/min. about. From the start of citric acid nebulization, the number of coughs and the cough latency of the animals were recorded within 10 mins.
  • In vitro cytotoxicity assays for compounds of the present invention were determined in HepG2 cells using the CCK-8 method. Collect HepG2 cells in log phase (Beina Biosciences), adjust the concentration of cell suspension, plate them in a 96-well cell culture plate at 50,000 cells/well, and incubate the cells in a 5%, 37°C cell culture incubator overnight. After the cells in the plate reached 80-90% confluence, the test compound or vehicle (DMSO) of each concentration gradient was added to the medium, and incubated for 48 hours in a cell culture incubator at 5%, 37°C.
  • DMSO test compound or vehicle
  • the in vitro metabolic stability of the compounds of the present invention was determined using the body temperature incubation method of various hepatic microparticles.
  • liver microsomal reaction system (1 mg/mL liver microsomal protein, 25 U/mL 6-phosphate glucose dehydrogenase, 1 mM NADP, 6 mM D-6-phosphate glucose, 5 mM MgCl 2 )
  • an appropriate amount of the test compound was added and put into Incubate the reaction in a 37°C water bath to start the reaction, and at each time point, take 100 ⁇ L of the reaction solution and add it to 400 ⁇ L of 0°C pre-cooled internal standard working solution (containing 200 ng/mL dexamethasone, diclofenac, tolbutamide, and acetaminophen).
  • the specific training content was that the animals were reared in a single cage, and two bottles of water (both ordinary drinking water) were placed in each cage. During the adaptive training period The water was forbidden to be treated overnight (removing the drinking bottle) every day, and drinking water was given again from 8:30am to 5:30pm. This cycle was repeated for 3 days. The placement of two bottles of water was changed every day, and the animals could eat freely during the entire adaptive training period. All animal drinking bottles were removed 20 hours before the formal experiment, and water was deprived until the experiment began.

Abstract

提供了一种如式A所示的化合物的晶型：所述晶型是晶型I、晶型II、晶型III、晶型IV、晶型V、晶型VI、晶型VII、晶型VIII或晶型IX，及其制备方法、组合物以及其在制备P2X3受体拮抗剂中的应用，或者在制备用于预防和/或治疗疼痛、泌尿道疾病或呼吸***疾病的药物中的应用。所述化合物具有高的P2X3拮抗活性，且具有较好的选择性，毒性较低、代谢稳定性较好、味觉影响较小。

Description

杂环类化合物的晶型及其制备方法和应用
本申请要求申请日为2020/6/29的中国专利申请2020106100526的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明涉及药物化学领域，具体涉及一种杂环类化合物的晶型及其制备方法和应用。
背景技术
ATP受体基于分子结构、转导机理和药理学特性被分类成两个主要家族，P2Y-和P2X-嘌呤受体。P2X-嘌呤受体是ATP-门控的阳离子通道的家族，已克隆数种亚型，包括：六种同聚受体，P2X1；P2X2；P2X3；P2X4；P2X5；和P2X7；和三种杂聚受体P2X2/3，P2X4/6，P2X1/5。研究发现，P2X3受体特别表达于“中空内脏”的初级传入神经纤维，例如下尿路和呼吸道。
咳嗽是呼吸***疾病的主要症状表现，呼吸科门诊中，70％～80％的患者都具有咳嗽症状。随着COPD、IPF等患病率逐渐升高，而咳嗽作为大多数呼气道疾病的主要表现症状，需求也随之增大。作为机体的防御性神经反射，咳嗽有利于清除呼吸道分泌物和有害因子，但频繁剧烈的咳嗽会对患者的工作、生活和社会活动造成严重影响。
P2X3拮抗剂明确针对咳嗽适应症进行开发的品种并不多，目前进展较快的项目为罗氏的AF-219项目，其在已最新完成的II期临床试验针对难治性咳嗽疗效较好，但味觉障碍问题严重。
目前尚无P2X3抑制途径治疗包括慢性咳嗽在内的众多病症的药物上市。因此，开发新的可抑制P2X3活性的化合物对于疾病的治疗具有积极意义。
专利申请CN201911379293.8中涉及一种如下所示的P2X3拮抗剂，具有高的P2X3拮抗活性，且具有较好的选择性，毒性较低、代谢稳定性较好、味觉影响较小。具有良好的药物开发前景。但是其并未涉及上述化合物的晶型。
Figure PCTCN2021102068-appb-000001
化合物普遍存在多晶现象，一般药物可能存在两种或是两种以上的不同晶型物质状态。多晶型化合物的存在形态和数量是不可预期的，同一药物的不同晶型在溶解度、熔点、密度、稳定性等方面有显著的差异，从而不同程度地影响药物的温度型、均一性、生 物利用度、疗效和安全性。因此，在新药研发过程中需要对化合物进行全面的多晶型筛选，选择适合药物制剂开发的晶型具有重要的临床意义。
发明内容
本发明提供了一种杂环类化合物的晶型及其制备方法和应用。本发明的晶型具有良好的稳定性，对药物的优化和开发具有重要的价值。
本发明提供了一种式A所示化合物或其溶剂化物的晶型：
Figure PCTCN2021102068-appb-000002
其选自晶型I、晶型II、晶型III、晶型IV、晶型V、晶型VI、晶型VII、晶型VIII和晶型IX。
本发明提供式A化合物的晶型III，其X射线粉末衍射图(XRPD)在2θ值为12.91°±0.20°、16.77±0.20°、19.27°±0.20°和22.80°±0.20°处具有特征峰。
在本发明某些优选实施方案中，所述的晶型III以2θ角表示的XRPD还在13.75°±0.20°、14.46°±0.20°和20.86°±0.20°处具有特征峰；进一步地，在21.08°±0.20°、23.75°±0.20°和24.05°±0.20°处具有特征峰。
在本发明某些优选实施方案中，所述的晶型III以2θ角表示的XRPD图基本如图1所示。所述的晶型III的热重分析图谱(TGA)中，在室温(RT)-100℃区间重量损失梯度为1.5％，所述“％”为重量百分比。所述的晶型III的示差扫描量热图谱(DSC)中第一处吸热峰为脱去0.4个水，第二处吸热峰归结于样品脱水后的熔融吸热峰，其TGA和DSC图谱优选如图2所示。所述的晶型III脱水后，在环境湿度下又很快吸湿重新变为晶型III，其加热至脱水前后XRPD图谱优选如图3所示。所述的晶型III的动态水分吸附图谱(DVS)图谱中显示样品具有一定的有吸湿性，在较宽的湿度范围内水含量变化不大，其DVS图谱优选如图4所示。所述的晶型III在DVS测试前后的XPRD图谱中，DVS测试前后XRPD没有明显变化，其DVS测试前后的XPRD图谱优选如图5所示。所述的晶型III的偏光显微镜图谱(PLM)中，晶型是约2μm不规则晶体，团聚为20-50μm，其PLM图优选基本如图6所示。所述的晶型III的纯度一般为90％以上，优选95％以上。
本发明提供式A化合物的晶型V，其XRPD在2θ值为8.38°±0.20°、9.15°±0.20°、13.52°±0.20°和18.44±0.20°处具有特征峰。
在本发明某些优选实施方案中，所述晶型V以2θ角表示的XRPD图还在16.26°±0.20°、16.89°±0.20°和17.86°±0.20°处有特征峰；进一步地，在22.35°±0.20°、23.56°±0.20°、24.74°±0.20°处具有特征峰。
在本发明某些优选实施方案中，所述晶型V以2θ角表示的XRPD图基本如图7所示。所述晶型V的TGA图谱中，RT-230℃温度区间内没有失重。所述的晶型V的DSC图谱中，在166℃±2℃具有吸热峰，熔化焓为70±2J/g，其TGA和DSC图谱优选如图8所示。结合DSC和TGA图可知该产品为无水晶型。所述的晶型V的DVS图谱中显示样品具有一定的有吸湿性(0.7％，80％RH)，其DVS图谱优选如图9所示。所述的晶型V在DVS测试前后的XPRD图谱中，DVS测试前后XRPD没有明显变化，其DVS测试前后XPRD图谱优选如图10所示。所述的晶型V的PLM中，晶型是约5μm不规则晶体，其PLM图优选基本如图11所示。所述的晶型V的纯度一般为90％以上，优选95％以上。
本发明提供式A化合物的晶型I，其XRPD图在2θ值为8.56°±0.20°、12.48±0.20°和22.13°±0.20°处具有特征峰。
在本发明某些优选实施方案中，所述的晶型I，其X射线粉末衍射图还在2θ值为13.53°±0.20°、14.25±0.20°、25.18°±0.20°和26.07°±0.20°处有特征峰；进一步地，在22.32°±0.20°、23.23°±0.20°和23.42°±0.20°处有特征峰。
在本发明某些优选实施方案中，所述的晶型I以2θ角表示的XRPD图基本如图12所示。所述的晶型I的DSC图中，在152℃±2℃具有吸热峰，熔化焓为44±2J/g。所述的晶型I的TGA图中，在RT-230℃温度区间内没有失重。其TGA和DSC图谱优选基本如图13所示。结合DSC和TGA图可知该产品为无水晶型。所述晶型I的DVS图如图14所示，当相对湿度大于40％时，晶型I的重量急剧增加。当相对湿度降至40％时，吸收的水分全部排出。所述的晶型I的吸湿后的XRPD图中，晶型I在高湿度环境下吸湿转晶，转变为水合物晶型IV，将其置于30℃真空干燥箱中干燥后又转晶为初始晶型I，其吸湿前后的XRPD图优选基本如图15所示。可知，结合DVS测试结果(图14)，当环境相对湿度高于40％时，晶型I快速吸湿转晶为水合物；在相对湿度低于50％时，吸附的水快速脱去转变为晶型I。即，晶型I和IV之间相互转变时可逆的。所述的晶型I表现为吸湿性(6.8％，80％RH)，所述的晶型I的DVS测试后晶型不变，其DVS测试前后的XRPD优选图如图16所示。所述的晶型I的PLM中，晶型是约5μm不规则晶体，其PLM优选基本如图17所示。所述的晶型I的纯度一般为90％以上，优选95％以上。
本发明提供式A化合物的MTBE溶剂化物的晶型II，其XRPD图在2θ值为8.42°±0.20°、12.09°±0.20°、13.68°±0.20°和20.87°±0.20°处具有特征峰。
在本发明某些优选实施方案中，所述的晶型Ⅱ以2θ角表示的XRPD还在16.17°±0.20°、16.93°±0.20°、17.55°±0.20°和21.20°±0.20°处有特征峰；进一步地，在22.60°±0.20°、23.23°±0.20°和24.40°±0.20°处有特征峰。
在本发明某些优选实施方案中，所述的晶型Ⅱ以2θ角表示的XRPD基本如图18所示。所述晶型II的核磁共振氢谱(HNMR)图谱中，化学位移在1.11和3.08处有MTBE的残留信号，其残留MTBE的HNMR谱图如图19所示。所述晶型II的TGA图谱中，在100-160℃温度区间内失重3.5％，在160-200℃区间内失重2.9％。所述的晶型II的DSC图谱中，有两个相邻的吸热峰，其TGA和DSC图谱优选如图20所示。与图19所示的残留MTBE氢谱，可知该产品为一种MTBE溶剂化物。所述的晶型II的PLM中，晶型是约2μm不规则晶体，其PLM优选基本如图21所示。
本发明提供式A化合物的水合物的晶型IV，其XRPD图在2θ值为8.65°±0.20°、12.69°±0.20°和22.56°±0.20°处具有特征峰。
在本发明某些优选实施方案中，所述晶型IV以2θ角表示的XRPD还在13.48°±0.20°、17.39°±0.20°、21.04°±0.20°和23.63°±0.20°处具有特征峰；进一步地，在14.39°±0.20°、25.60°±0.20°和26.52°±0.20°处具有特征峰。
在本发明某些优选实施方案中，所述晶型IV以2θ角表示的XRPD基本如图22所示。所述晶型IV的TGA图谱中，RT-60℃温度区间内失重1.2％。所述的晶型IV的DSC图谱中，有两个吸热峰，第一个宽吸热峰推测为脱水导致，后面的吸热峰为熔融峰，其TGA和DSC图谱优选如图23所示。所述的晶型IV只在高湿环境下稳定，其脱水后，在环境湿度下又很快吸湿重新变为晶型I，其加热至脱水前后XRPD图谱优选如图24所示。所述的晶型IV的PLM中，晶型是约5μm不规则晶体，其PLM图优选基本如图25所示。所述的晶型IV的纯度一般为90％以上，优选95％以上。
本发明提供式A化合物的水合物的晶型VI，其X射线粉末衍射图在2θ值为8.62°±0.20°、12.69°±0.20°和22.59°±0.02°处具有特征峰；
在本发明某些优选实施方案中，所述晶型VI以2θ角表示的XRPD还在13.46°±0.20°、17.41°±0.20°、26.51°±0.02°、25.62°±0.02°和25.24°±0.20°处具有特征峰；进一步地，在23.64°±0.20°、21.00°±0.20°和27.85°±0.20°处具有特征峰。
在本发明某些优选实施方案中，所述晶型VI以2θ角表示的XRPD基本如图26所示。所述晶型VI的XRPD图谱叠加图中，该晶型样品在环境湿度(35％RH)下放置几分钟后转晶为晶型I。其XRPD图谱叠加图如图27所示。这表明晶型VI可能是一种极不稳定的水合物。所述的晶型VI的纯度一般为90％以上，优选95％以上。
本发明提供式A化合物的乙二醇溶剂化物的晶型VII，其X射线粉末衍射图在2θ值为8.36°±0.20°、12.13°±0.20°、12.45°±0.20°、16.84°±0.20°和21.66°±0.20°处具有特征峰。
在本发明某些优选实施方案中，所述晶型VII的XRPD还在下列2θ值处具有特征峰：21.07°±0.20°、24.82°±0.20°；进一步地，在13.61°±0.20°、23.22°±0.20°和24.57°±0.20°。
在本发明某些优选实施方案中，所述晶型VII以2θ角表示的XRPD基本如图28所示。所述晶型VII的 1HNMR图谱中，化学位移δ在3.39和4.44处显示有乙二醇溶剂残 留，其残留乙二醇的 1HNMR图谱优选如图29所示。所述晶型VII的TGA图谱中，在rt-120℃区间内有25.7％的失重。所述的晶型VII的DSC图谱中，有两个宽的吸热峰，第一处的吸热峰推测为脱溶剂导致，其TGA和DSC图谱优选如图30所示。结合DSC和TGA图可知该产品为一种乙二醇的溶剂化物。所述的晶型VII的纯度一般为90％以上，优选95％以上。
本发明提供式A化合物的THF溶剂化物的晶型VIII，其X射线粉末衍射图在2θ值为在8.53°±0.20°、12.38°±0.20°、13.66°±0.20°和21.49°±0.20°处具有特征峰。
在本发明某些优选实施方案中，所述晶型VIII以2θ角表示的X射线粉末衍射图还在20.99°±0.20°、24.94°±0.20°和25.31°±0.20°处具有特征峰；进一步地，在17.14°±0.20°、21.72°±0.20°和23.00°±0.20°处具有特征峰。
在本发明某些优选实施方案中，所述晶型VIII以2θ角表示的X射线粉末衍射图基本如图31所示。所述晶型VIII的TGA图谱中，rt-160℃温度区间内失重5.7％。所述的晶型VIII的DSC图谱中，只有一个吸热峰，为样品脱溶剂后的熔融峰。所以，晶型VIII是含有THF的溶剂化物，其TGA和DSC图谱优选如图32所示。所述晶型VIII的 1HNMR图谱中，化学位移δ在1.76,和3.60处显示有THF溶剂残留，其 1HNMR图谱优选如图33所示。所述的晶型VIII不稳定，在脱溶剂(40℃真空干燥3小时)后转晶，转变为晶型I，其干燥前后XRPD图谱优选如图34所示。所述的晶型VIII的纯度一般为90％以上，优选95％以上。
本发明提供式A化合物的DMSO溶剂化物的晶型IX，其XRPD图在2θ值为8.55°±0.20°、12.43°±0.20°、21.75°±0.20°和25.07°±0.20°处具有特征峰。
在本发明某些优选实施方案中，所述晶型IX以2θ角表示的XRPD图还在13.57°±0.20°、17.18°±0.20°、20.94°±0.20°和25.57°±0.20°；进一步地，在21.37°±0.20°和23.12°±0.20°。
在本发明某些优选实施方案中，所述晶型IX以2θ角表示的XRPD图基本如图35所示。所述晶型IX的TGA图谱中，rt-160℃温度区间失重18.23％，DSC谱图上对应TGA失重有相应的吸热峰，其TGA和DSC图谱优选如图36所示。所述晶型IX的 1HNMR图谱中，化学位移δ在2.68处显示有DMSO溶剂残留，其残留DMSO的 1HNMR图谱优选如图60所示。结合DSC和TGA图可知该产品为一种DMSO溶剂化物。所述的晶型IX的纯度一般为90％以上，优选95％以上。
本发明中，所述的XRPD中使用的射线为Kα射线。
本发明中，所述的X射线粉末衍射中使用的靶型为Cu靶。
本发明还提供了一种物质A的晶型Ⅲ的制备方法，其为方案一、方案二或方案三；
方案一，其包括如下步骤：将式A所示化合物的无定型与溶剂的混悬液进行转晶，得到物质A的晶型III即可；所述的溶剂为水或醇类溶剂；
方案二，其包括如下步骤：将反溶剂加入到式A所示化合物与溶剂的溶液中，进行析晶，得到物质A的晶型III即可；所述的溶剂为醇类、呋喃类或DMSO中的一种或多种；所述的反溶剂为水；
方案三，其包括如下步骤：将化合物A与溶剂的溶液加入到水溶液A中，析晶，得到物质A的晶型III即可，所述的水溶液A为所述的物质A的晶型III的晶种与水的混悬液；所述的溶剂为DMSO。
所述的晶型III的制备方法中，当采用所述的方案一时，所述的溶剂优选水或甲醇。
所述的晶型III的制备方法中，当采用所述的方案一时，所述的析晶温度为20-50℃，优选地40℃或50℃。
所述的晶型III的制备方法中，当采用所述的方案一时，所述的化合物A的无定型与溶剂的质量体积比为50mg/mL。
所述的晶型III的制备方法中，当采用所述的方案二时，所述的溶剂优选甲醇、四氢呋喃或DMSO中的一种或多种。
所述的晶型III的制备方法中，当采用所述的方案二时，所述的溶剂的溶剂与水的体积比为3:1-1:1(例如1:1或3:1)。
所述的晶型III的制备方法中，当采用所述的方案三时，所述的溶剂的溶剂与水的体积比为1:1-1:4(例如1:1、2:3、1:2或1:4)。
所述的晶型III的制备方法，当采用所述的方案一时，其优选包括以下步骤：将化合物A的无定型与溶剂的混悬液进行转晶；所述的溶剂水或甲醇。所述的搅拌温度为20-50℃，优选40℃。所述的所述的化合物A的无定型与溶剂的质量体积比为50mg/mL。
所述的晶型III的制备方法，当采用所述的方案二时，其优选包括以下步骤：将化合物A与溶剂混合，然后以缓慢滴加的方式加入到反溶剂中；所述的溶剂为甲醇、四氢呋喃或DMSO中的一种或多种；所述的反溶剂为水；所述的溶剂与水的体积比为3:1-1:1(例如3:1或1:1)。
所述的晶型III的制备方法，当采用所述的方案三时，其优选包括以下步骤：将化合物A与溶剂的溶液加入到水溶液A中，析晶，所述的水溶液A为所述的物质A的晶型III的晶种与水的混悬液；所述的溶剂为DMSO；所述的DMSO与水的体积比为1:1-1:4(例如1:1、2:3、1:2或1:4)。
本发明还提供了一种化合物A的晶型Ⅴ的制备方法，其为方案A或方案B；
方案A，其包括如下步骤：将式A所示化合物的无定型在20-50℃下与溶剂的混悬液进行转晶，得到化合物A的晶型V即可；所述的溶剂为水或腈类溶剂。
方案B，其包括如下步骤：将式A所示化合物与溶剂的溶液中的溶剂挥发，得到化合物A的晶型V即可；所述的溶剂为醇类溶剂。
方案A中，所述的化合物A的无定型与溶剂的质量体积比优选3.0mg/mL或50mg/mL。
方案A中，所述的溶剂优选水或乙腈。
方案A中，所述的转晶的温度优选50℃。
方案B中，所述的溶剂优选甲醇。
所述的方案A的某一方案中，其包括以下步骤：将化合物A的无定型在20-50℃下与溶剂的混悬液进行转晶，得到化合物的晶型V；所述的溶剂为水或乙腈；所述的结晶温度优选50℃，所述的化合物A的无定型与溶剂的质量体积比为50mg/mL或3.0mg/mL。
所述的方案B的某一方案中，其包括以下步骤：将化合物A与溶剂的溶液中的溶剂挥发，得到化合物A的晶型V；所述的溶剂甲醇，所述的温度为50℃。
本发明中，所述式A所示化合物的无定型参照专利申请CN201911379293.8中的方法制备获得(具体见实施例)。
一种药物组合物，其包括如上所述的如式A所示化合物或其溶剂化物的晶型(例如晶型I、晶型II、晶型III、晶型Ⅳ、晶型Ⅴ、晶型Ⅵ、晶型Ⅶ、晶型Ⅷ和晶型Ⅸ中的一种或多种)和/或如上所述的物质A的晶型III，和药用辅料。其中，所述的晶型的剂量可为治疗有效量。
所述的药用辅料可为药物生产领域中广泛采用的那些辅料。辅料主要用于提供一个安全、稳定和功能性的药物组合物，还可以提供方法，使受试者接受给药后活性成分以所期望速率溶出，或促进受试者接受组合物给药后活性成分得到有效吸收。所述的药用辅料可以是惰性填充剂，或者提供某种功能，例如稳定该组合物的整体pH值或防止组合物活性成分的降解。所述的药用辅料可以包括下列辅料中的一种或多种：粘合剂、助悬剂、乳化剂、稀释剂、填充剂、成粒剂、胶粘剂、崩解剂、润滑剂、抗粘着剂、助流剂、润湿剂、胶凝剂、吸收延迟剂、溶解抑制剂、增强剂、吸附剂、缓冲剂、螯合剂、防腐剂、着色剂、矫味剂和甜味剂。
可作为药学上可接受辅料的物质包括，但并不限于，离子交换剂，铝，硬脂酸铝，卵磷脂，血清蛋白，如人血清蛋白，缓冲物质如磷酸盐，甘氨酸，山梨酸，山梨酸钾，饱和植物脂肪酸的部分甘油酯混合物，水，盐或电解质，如硫酸鱼精蛋白，磷酸氢二钠，磷酸氢钾，氯化钠，锌盐，胶体硅，三硅酸镁，聚乙烯吡咯烷酮，聚丙烯酸脂，蜡，聚乙烯-聚氧丙烯-阻断聚合体，羊毛脂，糖，如乳糖，葡萄糖和蔗糖；淀粉如玉米淀粉和土豆淀粉；纤维素和它的衍生物如羧甲基纤维素钠，乙基纤维素和乙酸纤维素；树胶粉；麦芽；明胶；滑石粉；辅料如可可豆脂和栓剂蜡状物；油如花生油，棉子油，红花油，麻油，橄榄油，玉米油和豆油；二醇类化合物，如丙二醇和聚乙二醇；酯类如乙基油酸酯和乙基月桂酸酯；琼脂；缓冲剂如氢氧化镁和氢氧化铝；海藻酸；无热原的水；等渗盐；林格(氏)溶液；乙醇，磷酸缓冲溶液，和其他无毒的合适的润滑剂如月桂硫酸钠和硬脂酸镁，着色剂，释放剂，包衣衣料，甜味剂，调味剂和香料，防腐剂和抗氧化剂。
本发明的药物组合物可根据公开的内容使用本领域技术人员已知的任何方法来制备。 例如，常规混合、溶解、造粒、乳化、磨细、包封、包埋或冻干工艺。
本发明的提供了一种如上所述的如式A所示化合物或其溶剂化物的晶型(例如晶型I、晶型II、晶型III、晶型Ⅳ、晶型Ⅴ、晶型Ⅵ、晶型Ⅶ、晶型Ⅷ和晶型Ⅸ中的一种或多种)、如上所述的物质A的晶型III在制备P2X3抑制剂中的应用。在所述的应用中，所述的P2X3抑制剂可用于哺乳动物生物体内；也可用于生物体外，主要作为实验用途，例如：作为标准样或对照样提供比对，或按照本领域常规方法制成试剂盒，为P2X3的抑制效果提供快速检测。
本发明还提供了一种上述的如式A所示化合物或其溶剂化物的晶型(例如晶型I、晶型II、晶型III、晶型Ⅳ、晶型Ⅴ、晶型Ⅵ、晶型Ⅶ、晶型Ⅷ和晶型Ⅸ中的一种或多种)、如上所述的物质A的晶型III在制备药物中的应用；所述的药物为用于防护、处理、治疗或减轻动物的至少部分由P2X3介导的或活性相关的疾病的药物；或者，所述的药物为用于治疗疼痛、泌尿道疾病或呼吸***疾病的药物。
本发明的提供了一种上述的如式A所示化合物或其溶剂化物的晶型(例如晶型I、晶型II、晶型III、晶型Ⅳ、晶型Ⅴ、晶型Ⅵ、晶型Ⅶ、晶型Ⅷ和晶型Ⅸ中的一种或多种)、如上所述的物质A的晶型III或如上所述的药物组合物在用于防护、处理、治疗或减轻动物(例如人类)的至少部分由P2X3介导的或活性相关的疾病中的应用。所述的疾病包括但不限于，呼吸***疾病、咳嗽、慢性咳嗽、特发性肺纤维化、慢性肺阻塞、哮喘、疼痛、尿失禁、自身免疫病、膀胱过度活动症、排尿困难、炎症、老年痴呆症、帕金森、睡眠障碍、癫痫、精神疾病、关节炎、神经退行性变、外伤性脑损伤、心肌梗死、类风湿性关节炎、脑卒中、血栓症、动脉粥样硬化、结肠综合症、炎性肠病、消化道疾病；胃肠功能紊乱、呼吸衰竭、性功能障碍、心血管***疾病、心衰、高血压、尿失禁、膀胱炎、关节炎、子宫内膜异位、血液病、肌肉骨骼和***发育障碍、***性障碍疾病。
在一些实施方案中，所述疾病包括疼痛；所述疼痛包括但不限于：炎性疼痛、手术疼痛、内脏疼痛、牙痛、经前期疼痛、中枢性疼痛、由灼伤所致疼痛、偏头痛、或簇性头痛。
在一些实施方案中，所述疾病包括泌尿***疾病；所述的泌尿道疾病包括：尿失禁、膀胱过度活动症、排尿困难、膀胱炎；
在一些实施方案中，所述疾病包括呼吸***疾病，所述呼吸***疾病包括但不限于：呼吸障碍，包括特发性肺纤维化、慢性阻塞性肺病、哮喘、支气管痉挛、或慢性咳嗽。
本发明的提供了一种上述的如式A所示化合物或其溶剂化物的晶型(例如晶型I、晶型II、晶型III、晶型Ⅳ、晶型Ⅴ、晶型Ⅵ、晶型Ⅶ、晶型Ⅷ和晶型Ⅸ中的一种或多种)、如上所述的物质A的晶型III或如上所述的药物组合物防护、处理、治疗或减轻动物(例如人类)的至少部分由P2X3介导的或活性相关的疾病的方法。所述的疾病包括但不限于，呼吸***疾病、咳嗽、慢性咳嗽、特发性肺纤维化、慢性肺阻塞、哮喘、疼痛、尿失禁、自身免疫病、膀胱过度活动症、排尿困难、炎症、老年痴呆症、帕金森、睡眠障 碍、癫痫、精神疾病、关节炎、神经退行性变、外伤性脑损伤、心肌梗死、类风湿性关节炎、脑卒中、血栓症、动脉粥样硬化、结肠综合症、炎性肠病、消化道疾病；胃肠功能紊乱、呼吸衰竭、性功能障碍、心血管***疾病、心衰、高血压、尿失禁、膀胱炎、关节炎、子宫内膜异位、血液病、肌肉骨骼和***发育障碍、***性障碍疾病。
在一些实施方案中，所述疾病包括疼痛；所述疼痛包括但不限于：炎性疼痛、手术疼痛、内脏疼痛、牙痛、经前期疼痛、中枢性疼痛、由灼伤所致疼痛、偏头痛、或簇性头痛。
在一些实施方案中，所述疾病包括泌尿***疾病；所述的泌尿道疾病包括：尿失禁、膀胱过度活动症、排尿困难、膀胱炎；
在一些实施方案中，所述疾病包括呼吸***疾病，所述呼吸***疾病包括但不限于：呼吸障碍，包括特发性肺纤维化、慢性阻塞性肺病、哮喘、支气管痉挛、或慢性咳嗽。
上述的药物组合物，其特征在于，通过给药所述药物组合物，减少了与治疗相关的味觉障碍的副作用。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：
1、现有技术中并没有报道该杂环类化合物的晶型，本申请首次发现该化合物多种新晶型。通过大量的实验与筛选，本发明首次制备得到晶型I、晶型II、晶型III、晶型Ⅳ、晶型Ⅴ、晶型Ⅵ、晶型Ⅶ、晶型Ⅷ或晶型Ⅸ，并将其作为候选对象。
2、本发明制备得到的部分晶型的稳定性好，方便保存，能够避免药物开发或是生产过程中发生转晶的风险，避免生物利用度以及药效发生改变，可以开发成适合临床使用的剂型，具有很强的经济价值。
3、本发明还同时提供了晶型I、晶型II、晶型III、晶型Ⅳ、晶型Ⅴ、晶型Ⅵ、晶型Ⅶ、晶型Ⅷ、晶型Ⅸ的盐或溶剂合物的晶型的制备方法，操作简便且重现性高，溶剂不易残留，对环境友好，适合不同规模化生产。
附图说明
图1为晶型III的XRPD图；
图2为晶型III的TGA/DSC叠加图；
图3为加热至脱水前后晶型III的XRPD叠加图；
图4为晶型III的DVS图；
图5为晶型III的DVS测试前后的XRPD叠加图；
图6为晶型III的PLM图；
图7为晶型V的XRPD图；
图8为晶型V的TGA/DSC叠加图；
图9为晶型V的DVS测试前后的XRPD叠加图；
图10为晶型V的DVS图；
图11为晶型V的PLM图；
图12为晶型I的XRPD图；
图13为晶型I的TGA/DSC叠加图；
图14为晶型I的DVS图；
图15为晶型I吸湿后XPRD图；
图16为晶型I的DVS测试前后的XRPD叠加图；
图17为晶型I的PLM图；
图18为晶型II的XRPD图；
图19为晶型II的残留MTBE的HNMR谱；
图20为晶型II的TGA/DSC叠加图；
图21为晶型II的PLM图；
图22为晶型IV的XRPD图；
图23为晶型IV的TGA/DSC叠加图；
图24为晶型IV脱水前后的XRPD叠加图；
图25为晶型IV的PLM图；
图26为晶型VI的XRPD图；
图27为晶型VI的XRPD叠加图；
图28为晶型VII的XRPD图；
图29为晶型VII的残留乙二醇HNMR谱；
图30为晶型VII的TGA/DSC叠加图；
图31为晶型VIII的XRPD图；
图32为晶型VIII的TGA/DSC叠加图；
图33为晶型VIII和原料的HNMR谱；
图34为晶型VIII及干燥后前样品的XRPD叠加图；
图35为晶型IX的XRPD图；
图36为晶型IX的TGA/DSC叠加图；
图37为由无定型样品得到的晶型III的XRPD叠加图；
图38为由无定型样品得到的晶型III的DSC&TGA叠加图；
图39为晶型III在不同体积比的混合溶剂中打浆的XRPD叠加图；图40为200mg级晶型III的XRPD叠加图；
图41为200mg级晶型III的PLM图；
图42为晶型III(3g)的XRPD叠加图；
图43为晶型III在水中室温打浆前后的XRPD叠加图；
图44为晶型I的水活度实验结果图；
图45为晶型V的水活度实验结果图；
图46为晶型I、晶型V竞争打浆实验的XRPD叠加图；
图47为晶型I与V固态下稳定性数据结果图；
图48为晶型I稳定性测试样品的XRPD叠加图；
图49为晶型III稳定性测试样品的XRPD叠加图；
图50为晶型I研磨测试前后的XRPD叠加图；
图51为晶型I压力测试前后XRPD叠加图；
图52为晶型III研磨测试前后的XRPD叠加图；
图53为晶型III压力测试前后XRPD叠加图；
图54为晶型I、III和V的溶解度数据图；
图55为晶型I溶解度测试剩余固体的XRPD叠加图；
图56为晶型V溶解度测试剩余固体的XRPD叠加图；
图57为晶型III溶解度测试剩余固体的XRPD叠加图；
图58为无定型样品的PLM图；
图59为无定型样品的DSC&TGA叠加图；
图60为晶型IX的 1HNMR图谱；
图61为晶型V稳定性测试样品的XRPD叠加图。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
以下结合具体实施例对本发明做进一步详细说明。应理解，这些实施例是用于说明本发明的基本原理、主要特征和优点，而本发明不受以下实施例的限制。实施例中采用的实施条件可以根据具体要求做进一步调整，未注明的实施条件通常为常规实验中的条件。
下列实施例中，所述的实验方法通常按照常规条件或是常规测试条件完成，所述化合物可以通过有机合成或是通过市售的方法获得。以下实施例所用的化合物通过市售的方法获得，纯度达到99％。
本发明中所使用的缩写解释如下：
XPRD—X射线粉末衍射
TGA—热重分析
DSC—差示扫描量热分析
DVS—动态水分吸脱附分析
PLM—偏光显微镜分析
其测试条件如下：
XRPD
使用X射线粉末衍射仪(布鲁克D8 advance或D2 Phase)对固体进行表征。
扫描角度：3°(2θ)-40°(2θ)。
步长：0.02°(2θ)。
扫描速度：0.3sec/step(D8)，0.2sec/step(D2)。
光管电压：40KV(D8)，30KV(D2)。
光管电流：40mA(D8)，10mA(D2)。
旋转：开。
样品盘：零背景样品盘。
TGA
使用TA Instrument热重分析Q500或Discovery TGA 55对固体样品进行热重分析。平衡样品盘后，将样品悬挂于挂丝上并升上炉子。待稳定后，以10℃/min的速率加热样品至不同终点温度。
DSC
使用TA Instrument差示扫描量热仪Q200和Discovery DSC 250对固体样品进行DSC分析。称量样品并记录数值，然后将样品置于样品室中。以10℃/min的速率将样品从25℃加热至不同终点温度。
DVS
使用IGAsorp动态水吸附仪对固体进行DVS分析。
温度：25℃。
气流：250mL/min。
扫描循环：2。
最短测试时间：30min。
最长测试时间：2h。
等待平衡：98％。
PLM
使用尼康Eclipse LV100N POL型偏光显微镜对样品进行观察。
实施例1：式A化合物的制备
Figure PCTCN2021102068-appb-000003
步骤1 (S)-2-((2-(4-溴-2,6-二氟苯基)-7-氯咪唑并[1,2-a]吡啶-3-基)甲基)吗啉-4-甲酸叔丁酯的制备
Figure PCTCN2021102068-appb-000004
在100mL圆底瓶中依次加入(S)-2-乙炔基吗啉-4-甲酸叔丁酯(3.1g，1.0eq，中间体1-4)，4-溴-2,6-二氟苯甲醛(2.76g，1.0eq，化合物172-1)，4-氯吡啶-2-胺(1.61g，1.0eq，化合物172-2)，CuCl(0.37g，0.3eq)，Cu(OTf) 2(1.36g，0.3eq)，异丙醇(50mL)，氮气置换3次，80℃油浴加热过夜，TLC检测原料化合物172-2消失。旋干异丙醇，依次用EA和氨水萃取，取EA相，依次用饱和食盐水，柠檬酸洗涤，无水硫酸钠干燥，旋干过柱得中间体172-3，白色固体(3.0g，纯度为78％)。LC-MS:[M+H] +＝542.2。
步骤2 (S)-2-((2-(4-溴-2,6-二氟苯基)-7-氯咪唑并[1,2-a]吡啶-3-基)甲基)吗啉的制备
Figure PCTCN2021102068-appb-000005
将中间体172-3(2.67g)溶于二氯甲烷(24mL)，再加入盐酸二氧六环(24mL)，室温搅拌1.0h，LC-MS检测反应完。将反应液旋干，往反应液中加入水(15mL)和二氯甲烷(15mL)，萃取完去水相，用碳酸氢钠水溶液调pH至水相呈弱碱性(pH＝8～9)。分液取二氯甲烷相，水相再用二氯甲烷萃取(10mL×2)。合并二氯甲烷相，用饱和食盐水洗涤，旋干得中间体172-4，白色固体(1.70g，纯度88.6％)。LC-MS:[M+H] +＝442.1。
步骤3.(S)-2-((2-(4-溴-2,6-二氟苯基)-7-氯咪唑并[1,2-a]吡啶-3-基)甲基)吗啉-4-甲酸甲酯的制备
Figure PCTCN2021102068-appb-000006
将中间体172-4(1.4g，1.0eq)溶于二氯甲烷(10mL)中，加入三乙胺(480mg，1.5eq)，滴加氯乙酸甲酯(388mg，1.3eq)。反应1.0h后LC-MS显示产物生成。反应完后加水(10mL)搅拌30min后分液取二氯甲烷相，水相再用二氯甲烷萃取(10mL×2)。合并二氯甲烷相，用饱和氯化钠洗涤，无水硫酸钠干燥旋干，过柱得中间体172-5，白色固体(1.01g，纯度93.02％)。LC-MS:[M+H] +＝499.8。
步骤4 (S)-2-((2-(4-(苯甲硫基)-2,6-二氟苯基)-7-氯咪唑并[1,2-a]吡啶-3-基)甲基)吗啉-4-甲酸甲酯的制备
Figure PCTCN2021102068-appb-000007
将中间体172-5(0.73g，1.0eq)溶于二氧六环(4mL)中，加入BnSH(0.24g，1.3eq)，Pd2(dba)3(0.04g，0.03eq)，Xantphos(0.04g，0.05eq)，DIEA(0.60g，3.0eq)， 并置换N 2三次，在80℃下反应过夜。LCMS监测原料消失完全。往反应液加入二氯甲烷(10mL)和水(10mL)，分液取二氯甲烷相，水相再用二氯甲烷萃取(10mL×2)。合并二氯甲烷相，用饱和氯化钠洗涤，无水硫酸钠干燥旋干，过柱得中间体172-6，白色固体(0.82g，纯度91.53％)。LC-MS:[M+H] +＝544.2。
步骤5 (S)-2-((7-氯-2-(4-(氯磺酰)-2,6-二氟苯基)咪唑并[1,2-a]吡啶-3-基)甲基)吗啉-4-甲酸甲酯的制备
Figure PCTCN2021102068-appb-000008
将中间体172-6(510mg)加入到反应瓶中，加乙腈(3mL)溶解，再加入冰醋酸(281mg，5.0eq)，在冰浴下滴加SO2Cl 2(506mg，4.0eq)。并在0℃反应1h。LCMS显示原料消失，有中间体172-7生成。该反应未加处理，反应液直接用于下一步。
步骤6 (S)-2-((7-氯-2-(2,6-二氟-4-氨磺酰苯基)咪唑并[1,2-a]吡啶-3-基)甲基)吗啉-4-甲酸甲酯的制备
Figure PCTCN2021102068-appb-000009
在0℃下，将氨水(2mL)用乙腈(1mL)稀释滴加到上述反应液中，并在室温下反应0.5h。LCMS显示原料消失完全，有目标产物生成。将反应液用水和乙酸乙酯萃取2次，食盐水溶液洗涤，无水硫酸钠干燥，浓缩，利用C18色谱柱(水/乙腈，RRt＝22.5min)分离纯化。得到无定型状态化合物A，白色固体。(185mg，纯度99.74％)。LC-MS:[M+H] +＝501.1。
1H NMR(400MHz,DMSO-d 6)δ＝8.11(d,J＝7.4,1H),7.29(d,J＝1.6,1H),7.22(s,2H),7.14(d,J＝6.6,2H),6.60(dd,J＝7.4,2.1,1H),3.33(d,J＝12.8,1H),3.13(d,J＝11.3,2H),3.07(s,3H),2.97(d,J＝7.8,1H),2.77–2.69(m,1H),2.69–2.61(m,1H),2.53(dd,J＝15.5,8.3,1H).
实施例2：原料后处理
参照实施例1制备获得化合物A共11.1g，加入20mL丙酮，65℃(氮气保护)回流2.0h，直接旋干丙酮，40℃真空干燥12h，核磁显示有1％左右丙酮残余。80℃再次真空干燥12h，核磁显示仍有丙酮残余。取其中5.3g再次用80℃真空干燥12h，核磁 显示仍有丙酮残余。将该批次产物加入乙腈(16mL)，85℃(氮气保护)回流2.0h，直接旋干乙腈，再用80℃真空干燥12h，核磁显示合格无溶残，入库5.2g。产品纯度99.29％，呈白色粉末状。
PLM图和XRPD结果显示，原料为10-50μm的不规则形状晶体，结晶度一般，为无定型状态型。如图37，DSC图谱显示原料在约150-180℃之间处有两个相连的吸热峰，峰温分别为164±2℃和173℃±2℃，如图38，TGA图谱显示样品在230℃前基本无失重。
实施例3：式A化合物无定型的制备与表征
3.1 将化合物A溶解在一定量的THF中，减压浓缩至干，得到无定型样品。XRPD表征参见附图37。
实施例4：式A化合物晶型I的制备与表征
4.1 分别选用9种溶剂：EtOH、IPA、NBA、MEK、ACN、丙酮、EA、IPAc、Hept，室温下混悬打浆，打浆浓度为60mg/mL，得到晶型I；
4.2 分别选用6种溶剂：IPA、NBA、MEK、丙酮、Tol、EA，50℃下混悬打浆，打浆浓度为100mg/mL，得到晶型I；
4.3 选用溶剂：IPAc，50℃下混悬打浆，打浆浓度为50mg/mL，得到晶型I；
4.4 在甲醇和乙醇中缓慢冷却结晶，冷却温度从50℃至RT，均可得到晶型I；
4.5 将四氢呋喃作为良溶剂用于溶解样品形成一定浓度的样品溶液，分别将Tol、Hept、水作为反溶剂缓慢滴加到样品溶液中，体积比均为1：10，以提高过饱和度，从而使固体析出，得到晶型I；
4.6 选择EtOH为溶剂进行挥发结晶试验，得到晶型I。
晶型I以2θ角表示的XRPD如图12所示。所述的晶型I的DSC图中，在152℃具有吸热峰，熔化焓为44±2J/g。所述的晶型I的TGA图谱中，在RT-230℃温度区间内没有失重。其TGA和DSC图谱如图13所示。结合DSC和TGA图可知该产品为无水晶型。所述晶型I的DVS图谱如图14所示，当相对湿度大于40％时，晶型I的重量急剧增加。当相对湿度降至40％时，吸收的水分全部排出。所述的晶型I的吸湿后的XRPD图中，晶型I在高湿度环境下吸湿转晶，转变为水合物晶型IV，将其置于30℃真空干燥箱中干燥后又转晶为初始晶型I，其吸湿后的XRPD图如图15所示。可知，结合DVS测试结果(图14)，当环境相对湿度高于40％时，晶型I快速吸湿转晶为水合物；在相对湿度低于50％时，吸附的水快速脱去转变为晶型I。即，晶型I和IV之间相互转变时可逆的。所述的晶型I表现为吸湿性(6.8％，80％RH)，所述的晶型IDVS测试后晶型不变，其DVS测试前后的XRPD图如图16所示。所述的晶型I的PLM图中，晶型是约5μm不规则晶体，其PLM如图17所示。
实施例5：式A化合物晶型II的制备与表征
5.1 在MTBE中，室温下混悬打浆，得到晶型II：
5.2 选用溶剂：MTBE，50℃下混悬打浆，打浆浓度为100mg/mL，得到晶型II；
5.3 将四氢呋喃作为良溶剂用于溶解样品形成一定浓度的样品溶液，MTBE作为反溶剂缓慢滴加到样品溶液中，体积比为1：10，提高过饱和度，从而使固体析出，得到晶型II。
晶型Ⅱ以2θ角表示的X射线粉末衍射图如图18所示。所述晶型II的核磁共振氢谱图谱中，化学位移1.10和3.08处MTBE的溶剂残留信号；其摩尔比为0.39，其HNMR谱图如图19所示。所述晶型II的TGA图谱中，在100-160℃温度区间内失重3.5％，在160-200℃区间内失重2.9％。所述的晶型II的DSC图谱中，有两个相邻的吸热峰，其TGA和DSC图谱如图20所示。与图19所示的残留MTBE氢谱约6.0％相当，可知该产品为一种MTBE溶剂化物。所述的晶型II的PLM中，晶型是约2μm不规则晶体，其偏光显微镜图如图21所示。
实施例6：式A化合物晶型III的制备与表征
6.1 选用溶剂：水，50℃下混悬打浆，打浆浓度为50mg/mL，得到晶型III；
6.2 通过无定型样品在水中40℃混悬打浆制备，得到晶型III；具体为：将样品溶解在甲醇中，过滤得到样品溶液，然后旋蒸得到250mg无定型样品。在水中混悬打浆20小时后得到晶型III。通过XRPD、DSC、TGA表征测试，确定成功制备出纯的晶型III并作为后续放大实验的晶种。
6.3 通过溶剂-反溶剂沉淀结晶，并加入晶种诱导成核的方法制备晶型III。具体为：预先将样品溶解在表1所示的特定比例的混合溶剂中，过滤得到饱和溶液，晶型III作为晶种加入到上述饱和溶液中，然后缓慢加入水作为反溶剂使析出固体并诱导结晶。对过滤得到固体样品进行XRPD测试。实验结果如表1所示，结果表明晶型III在上述体系中不稳定，转变为溶剂化物或不稳定的水合物。从溶解度和溶剂残留限度角度考虑，DMSO作为第三类溶剂且溶解度高于100mg/mL，因此选择DMSO进行下一步的研究。
表1 溶剂-反溶剂沉淀结晶实验结果
Figure PCTCN2021102068-appb-000010
Figure PCTCN2021102068-appb-000011
6.4 从上述6.3的实验结果可以看出，将反溶剂加入到良溶剂的样品溶液中的结晶方式难以得到目标晶型III，所以考虑采用通过滴加溶解在有机溶剂中的样品于分散有晶种的水溶液中对化合物A进行结晶制备的方式制备目标晶型。具体的操作方法为：首先将样品溶解在DMSO中过滤得到DMSO溶液，然后以缓慢滴加的方式加入到分散有晶种的水溶液中。析出的固体在晶种的诱导下转晶为目标晶型III。如表2和图1所示，分别考察了晶型III在不同比例的DMSO/水混合溶剂体系中的稳定性，以确定出合适的溶剂/反溶剂比例保证晶型III在该体系中能够稳定存在。实验结果表明，室温下晶型III能够在水含量高于60％(体积百分比)的DMSO/水混合溶剂中稳定存在而不发生转晶。而当水含量低于60％时，晶型III转晶为DMSO的溶剂化物(晶型IX)。
表2 晶型III在不同体积比的混合溶剂中的稳定性结果
Figure PCTCN2021102068-appb-000012
从目标产物收率角度考虑，分别计算了上述比例下晶型III的理论收率以进一步确定混合溶剂的比例。如表3所示，对于DMSO/水1:2、1:3、1:4而言，由于晶型III在这三种比例的溶剂中溶解度均比较小，所以理论收率相差不大，收率均在99％以上。
表3 晶型III在不同比例DMSO/水混合溶剂中的理论收率
溶剂 溶解度(mg/mL,RT) 理论收率(％)
DMSO >200 /
DMSO/水 1:2 ～0.5 99.3％
DMSO/水 1:3 ～0.28 99.4％
DMSO/水 1:4 <0.25 99.4％
6.5 晶型III的放大(200mg)
室温下，样品溶解在DMSO中过滤得到DMSO溶液中，以25μL/min的速度缓慢滴加到分散有晶种晶型III的水中，随着DMSO溶液滴加量的增大，固体析出逐渐增多(DMSO/水1:2，体积比)。经XRPD确认(图2)，刚析出的固体样品几乎为无定型，随着反应时间的延长以及晶种的诱导，室温下磁力搅拌16小时后，全部转晶为晶型III。实验观察到，反应结束后，停止搅拌，固体很快沉降在底部，上层液体基本呈透明状。经过PLM(图6)观察，发现得到的晶型III样品本身为2μm左右的晶体，但容易团聚为20-50μm的大颗粒，所以一旦停止搅拌，固体因为团聚而沉降。因而，该晶型容易过滤。综上，通过反反溶剂沉淀结晶的方式成功制备得到了200mg的晶型III样品，收率为93.0％，且 1HNMR显示DMSO溶残为0.1％wt，符合溶残限度(<0.5％wt)。
6.6 克级晶型III的放大制备
降低DMSO的体积、升高反应温度以及转晶结束后降至室温并适当延长打浆实验有利于降低溶残并提高收率。基于上述考虑，放大制备3g的晶型III时采用40℃下1:4DMSO/水的体系，晶种的加入量为2％wt，DMSO(5V)溶液的滴加速度控制在25μL/min。为模拟生产放大，采用锚式搅拌桨，转速为150rpm。反应体系在40℃下混悬打浆22小时，XRPD结果显示固体已转晶为晶型III。为进一步提高收率，将反应温度降至室温，并继续在室温下混悬打浆，22小时后过滤得到固体样品，并对其在水中打浆以除去DMSO溶残。如图42所示，成功放大制备得到了3g的晶型III，收率为96.7％。如图43所示，0.7％的DMSO溶残可以通过在水中室温打浆除去，且晶型不变。
考虑到该化合物生产、溶剂使用量、溶残以及产品收率等问题，最终晶型III的结晶工艺温度为40℃，反应体系为1:4DMSO/水，溶剂体积25V。综上所述，晶型III可进行克级放大制备，且收率高达97％。
晶型III以2θ角表示的XRPD图如图1所示。所述的晶型III的TGA图谱中，在RT-100℃区间重量损失梯度为1.5％，所述“％”为重量百分比。所述的晶型III的DSC图谱中第一处吸热峰为脱0.4个水，第二处吸热峰归结于样品脱水后的熔融吸热峰，其TGA和DSC图谱如图2所示。所述的晶型III脱水后，在环境湿度下又很快吸湿重新变为晶型III，其加热至脱水前后XRPD图谱如图3所示。所述的晶型III的DVS图谱中显示样品具有一定的有吸湿性，在较宽的湿度范围内水含量变化不大，其DVS图谱如图4所示。所述的晶型III在DVS测试前后的XPRD图谱中，DVS测试前后XRPD没有明显变化，其XPRD图谱如图5所示。所述的晶型III的PLM中，晶型是约2μm不规则晶体，团聚为20-50μm，其PLM图如图6所示。
实施例7：式A化合物晶型IV的制备与表征
7.1 将晶型I产品在高湿条件下吸湿得到晶型IV；
7.2 将晶型I产品料浆在50～95％的水/丙酮(V/V)中打浆得到晶型IV。
IV以2θ角表示的XRPD图基本如图22所示。所述晶型IV的TGA图谱中，RT-60℃温度区间内失重1.2％。所述的晶型IV的DSC图谱中，有两个吸热峰，第一个宽吸热峰推测为脱水导致，后面的吸热峰为熔融峰，其TGA和DSC图谱如图23所示。结合DSC和TGA图可以知该产品为水合物晶型，月0.34个水分子。所述的晶型IV只在高湿环境下稳定，其脱水后，在环境湿度下又很快吸湿重新变为晶型I，其加热至脱水前后XRPD图谱如图24所示。所述的晶型IV的PLM中，晶型是约5μm不规则晶体，其PLM图如图25所示。
实施例8：式A化合物晶型V的制备与表征
8.1 选用溶剂：水，50℃下混悬打浆，打浆浓度为50mg/mL，得到晶型V；
8.2 选用溶剂：ACN，50℃下混悬打浆，打浆浓度为3.0mg/mL，得到晶型V；
8.3 选择MeOH为溶剂进行挥发结晶试验，得到晶型V。
晶型V以2θ角表示的XRPD图如图7所示。所述晶型V的TGA图谱中，RT-230℃温度区间内没有失重。所述的晶型II的DSC图谱中，在166℃±2℃具有吸热峰，熔化焓为70±2J/g，其TGA和DSC图谱如图8所示。结合DSC和TGA图可知该产品为无水晶型。所述的晶型V的DVS图谱中显示样品具有一定的有吸湿性(0.7％，80％RH)，其DVS图谱如图9所示。所述的晶型V在DVS测试前后的XPRD图谱中，DVS测试前后XRPD没有明显变化，其XPRD图谱如图10所示。所述的晶型V的PLM中，晶型是约5μm不规则晶体，其PLM图如图11所示。
实施例9：式A化合物晶型VI的制备与表征
9.1 晶型VI是60℃下晶型I或晶型V在含水量10％(体积比)的水/丙酮混合溶剂中在60℃下打浆得到。
晶型VI以2θ角表示的X射线粉末衍射图如图26所示。所述晶型VI的XRPD图谱叠加图中，该晶型样品在环境湿度(35％RH)下放置几分钟后转晶为晶型I。其XRPD图谱叠加图如图27所示。这表明晶型VI可能是一种极不稳定的水合物。
实施例10：式A化合物晶型VII的制备与表征
10.1 选用溶剂：乙二醇，90℃下混悬打浆，打浆浓度为320mg/mL，得到晶型VII；
10.2 在乙二醇中缓慢冷却结晶，冷却温度从50℃至RT，得到晶型VII。
晶型VII以2θ角表示的XRPD基本如图28所示。所述晶型VII的 1HNMR图谱中化学位移δ在3.39和4.44处有乙二醇的溶残，其 1HNMR图谱如图29所示。所述晶型VII的TGA图谱中，在rt-120℃区间内有25.7％的失重。所述的晶型VII的DSC图谱中，有两个宽的吸热峰，第一处的吸热峰推测为脱溶剂导致，其TGA和DSC图谱如图30所示。结合DSC和TGA图可知该产品为一种含有2.79个分子乙二醇的溶剂化物。实施例11：式A化合物晶型VIII的制备与表征。
实施例11：式A化合物晶型Ⅷ的制备与表征
11.1 晶型III作为晶种，40℃向饱和的50％THF/水溶液中滴加水作为反溶剂，发生油状，降温至室温后继续打浆得到晶型VIII(湿滤饼)
晶型VIII以2θ角表示的XRPD图基本如图31所示。所述晶型VIII的TGA图谱中，Rt-160℃温度区间内失重5.7％。所述的晶型VIII的DSC图谱中，只有一个吸热峰，为样品脱溶剂后的熔融峰。所以，晶型VIII是溶剂化物，其TGA和DSC图谱如图32所示。所述晶型VIII的 1HNMR图谱中，化学位移δ在1.76,和3.60处有THF的溶残，其 1HNMR图谱如图33所示。所述的晶型VIII中，含有0.42个分子THF。所述的晶型VIII不稳定，在脱水(40℃真空干燥3小时)后转晶，转变为晶型I，其加热前后XRPD图谱如图34所示。
实施例12：式A化合物晶型IX的制备与表征
12.1 晶型IX是晶型III在40℃的50％DMSO/水饱和溶液中打浆制备
所述晶型IX的TGA图谱中，rt-160℃温度区间失重18.23％，所述的晶型IX的DSC图谱中，DSC谱图上对应TGA失重有相应的吸热峰，其TGA和DSC图谱如图36所示。所述晶型IX的 1HNMR图谱中，化学位移δ在2.68处显示有DMSO溶剂残留，其残留DMSO的 1HNMR图谱如图60所示。结合DSC和TGA图可知该产品为一种DMSO溶剂化物。结合DSC和TGA图可知该产品为一种DMSO溶剂化物。
实施例13：晶型相互转化研究
通过筛选实验共得到九种不同的晶型，其中包括两种无水晶型(晶型I、晶型V)、四种溶剂化物(晶型II、晶型VII、晶型IX、晶型VIII)、以及三种水合物(晶型III、晶型IV、晶型VI)。
13.1 晶型I与晶型IV之间的转换
晶型I可以通过不同的结晶方法重复得到，然而晶型I表现为吸湿型在相对湿度高于40％RH时吸湿转晶为水合物晶型IV。因而晶型I在API生产和放大时存在转晶的风险。
13.2 其它水合物晶型与晶型I之间的转换
除晶型III外，其余水合物均不稳定，脱水后转变为晶型I。
晶型III(为半水合物)，转晶风险低。
晶型IV(为水合物)，脱水后转变为晶型I；
晶型VI(为水合物)，极不稳定，环境湿度下很快转晶为晶型I。
13.3 无水晶型与水合物之间的转化研究
开展水活度实验。将晶型I和V(均为无水晶型)分别在不同水含量的丙酮/水混合溶剂中室温和60℃下打浆三天，三天后，过滤得到固体样品测试XRPD，实验结果如表4所示。如图44和45所示，无论室温还是高温，晶型I和V在含水量50-90％的丙酮/水体系中均转变为水合物晶型IV。当水含量为10％，高温下晶型I或V均转晶为另外一种水合物(晶型VI)。研究发现该水合物在环境湿度(35％RH)下极不稳定，放置几分钟 后转晶为晶型I。室温下，晶型V在该溶剂体系下转晶为晶型I。
表4 晶型I和晶型V的水活度实验结果
Figure PCTCN2021102068-appb-000013
N/A表示：无法得到
13.4 无水晶型间的竞争打浆实验
开展竞争打浆实验：称取等量的晶型I和V混合均匀后，测试XRPD其结果作为竞争打浆实验的T 0。将混合物分别在丙酮、乙醇、水中打浆，反应温度为室温和60℃。三天后，过滤得到固体样品，测试XRPD。如图46所示，在有机溶剂体系(丙酮或乙醇)，室温或高温得到的均为晶型I；晶型I和V在水中竞争打浆，室温下由于溶解度过低没有转晶仍为两者的混晶，而在60℃下转变为晶型V。结合水活度实验结果，可以推断晶型I在有机溶剂体系或者在水含量低(≤10％)的混合溶剂中是更为稳定，而晶型V在水中高温(60℃)稳定。
13.5 无水晶型之间的转化
等量的晶型I和晶型V轻微研磨使其混合均匀后，将混合物分别放置于60℃烘箱和室温干燥器(避免晶型I吸湿转晶)中，一周后测试XRPD研究其晶型有无相互转变。如图47所示，在上述条件下放置一周后，晶型I和晶型V没有相互转变的趋势，表明晶型I与V在固态下均稳定。
实施例14：晶型稳定性研究
相比于其它晶型，晶型I、III和V表现良好的固态性质，所以分别对其进行了稳定性测试。
14.1 物理和化学稳定性研究
约10mg的晶型I、III和V分别置于40℃/75％RH(敞口)和60℃(加盖)的稳定性箱中七天，分别于0天和7天时取出部分样品测试XRPD和HPLC，分别考察其物理和化学稳定性。结果如表5、图48、49和61所示，晶型III和晶型V在上述测试条件下七天后晶型未发生改变，同时化学纯度无明显降低。这表明晶型III和晶型V的物理和化学稳定性良好。而对于晶型I，在40℃/75％RH(敞口)条件下吸湿转晶，转变为水合物晶型IV。这一结果与DVS结果是一致的。在60℃下放置一周后，晶型I的XRPD谱图显示在22-28°(2θ)有变化，根据TGA结果显示在室温至60℃区间内有1.5％的失重，推测为吸水导致衍射峰的变化。但晶型I的化学稳定性良好。
表5 稳定性评价结果
Figure PCTCN2021102068-appb-000014
14.2 机械稳定性测试
通过研磨和压片，分别考察了晶型I和III的机械稳定性。研磨测试方法为：将待测样品置于研钵中分别研磨2分钟和5分钟后测试XRPD，观察晶型和结晶度的变化情况。压力测试方法为：将样品置于压片机的模具中，在40MPa下保持一分钟后，取出测试XRPD。
晶型I、晶型III在研磨(2分钟和5分钟)和压片(40MPa，1分钟)后的稳定性结果如图50和51所示，研磨后晶型I结晶度明显下降明显，但晶型未变。对于晶型III，研磨后晶型和结晶度未见明显变化(图52)，然而压片后结晶度下降明显(图53)。
实施例15：溶解度研究
相比于其它晶型，晶型I、III和V表现良好的固态性质，所以分别对其进行了溶解度测试。晶型I、III和V分别在SGF、FaSSIF、FeSSIF中的溶解度测试温度模拟的人体体温37℃，测试时长为24小时。
称取10mg的样品，然后加入2.0mL的生物媒介缓冲液(即目标浓度为5mg/mL)形成混悬液。将其置于恒温水浴摇床中(37℃，100rpm)，分别在0.5、2和24小时时取样500mL。过滤后分别测试滤液的pH值和HPLC浓度，同时对过滤得到的固体样品测试XRPD。
晶型I、III和V在SGF、FaSSIF、FeSSIF中的生物媒介中的溶解度测试结果如表6和图54所示。上述三种晶型在不同生物媒介中的溶解度相差不大，在SGF中的溶解度均高于5mg/mL。在FeSSIF中的溶解度约是FaSSIF中的3倍，表明食物可能有助于药物的吸收。测试过程中，生物媒介缓冲液的pH值无明显变化。
如图55所示，晶型I在FaSSIF和FeSSIF缓冲液中0.5小时时转晶为水合物晶型IV。而晶型III和V在测试过程中晶型未发生改变，结果见图56和图57。
表6 溶解度测试结果
Figure PCTCN2021102068-appb-000015
“溶清”：表示通过肉眼观察，样品在媒介中的溶解度大于5mg/mL；
“N/A”：表示无固体样品以测试XRPD.
实施例16：药代动力学研究
比格犬(普通级，北京玛斯生物技术有限公司)经单次经口灌胃给予化合物A后，研究该化合物在血浆内的药代动力学状况。将受试药物(以100mg/kg剂量，5mL/kg给药体积)灌胃，给药后10分钟、30分钟、1小时、2小时、4小时、6小时、8小时、10小时、24小时和48小时采集样品：进行外周静脉穿刺，每只动物每个时间点采集～1.0mL全血，将血样采集到K 2EDTA采血管中，上下颠倒数次充分混匀，置于室温。并于采血后60分钟内，分离出0.2mL全血于冻存管内，1:1加入去离子水，上下颠倒混匀后冻存。剩余样品在2-8℃下2000g，离心10分钟，得到血浆样品，记录每个样品的溶血情况，评估对检测的影响，观察血浆颜色并记录在采血表上(“-”为无溶血”，“+”为粉红色上清， “++”为红色上清)。
血浆样品中化合物A的浓度用LC-MS/MS方法(Non-GLP)测定。用WinNonlin(PhoenixTM，8.1版本)或其它类似软件进行药代动力学参数计算。如有适用的血浆的药物浓度-时间数据，将计算以下药代动力学参数：CL(清除率)、Vss(稳态表观分布容积)、T1/2(消除半衰期)、Cmax(达峰浓度)、Tmax(达峰时间)、AUC(血药浓度-时间曲线下面积)、MRT(平均滞留时间)。药代动力学数据通过描述性统计学如平均值、标准偏差和样本量进行描述。用Microsoft Excel 2007或2010进行计算。
16.1 对比晶型I和晶型V的PK性质差异
晶型I与晶型V的比较为相同的3只犬(雄性)，10mg/kg po，禁食，0.5％CMC-Na搅拌3h后给药(混悬)，获得的数据示于表7中。
表7 晶型I与晶型V的PK对比
Figure PCTCN2021102068-appb-000016
计算AUC(晶型I)：AUC(晶型V)＝300.53％，结果发现晶型V的暴露水平及绝对生物利用度小于晶型I。
16.2 晶型III的药代动力学研究
对比晶型III口服给药雌性差异&禁食与否差异，100mg/kg，po，禁食&非禁食，0.5％CMC-Na搅拌8h后给药，混悬，获得的数据示于表8中。
表8 晶型III的药代动力学数据
Figure PCTCN2021102068-appb-000017
Figure PCTCN2021102068-appb-000018
N/A：无法得到
结果发现：禁食条件下，雌雄犬血浆及全血药物浓度均存在一定差异，血浆中雄性比雌雄高6倍左右，全血中雄性比雌雄高1.9倍；但在非禁食条件下，雌雄犬血浆及全血药物浓度均无明显差异；禁食条件下，药物在雌雄犬中的红细胞分布存在差异(雌雄blood：plasma＝1452％Vs 521％)，但非禁食条件下，药物在雌雄犬中的红细胞分布无显著差异差异(雌雄blood：plasma＝114％Vs 116％)；无论雌雄，非禁食条件下血浆和全血中药物的浓度均高于禁食条件下，非禁食条件下，血浆药物浓度升高水平高于全血，且雌性犬血浆药物浓度受是否禁食影响程度最大。全血药物浓度升高可能是由于非禁食条件下药物吸收增加导致体内整体暴露水平升高，推测饮食有助于化合物A吸收；但血浆药物浓度升高水平高于全血可能是由于：1、红细胞中药物浓度饱和，导致血浆药物浓度类似突跃性升高(推测可能性最高)；2、第一天禁食给药后药物持续蓄积于红细胞中，7天的清洗期不足以使分布于红细胞中的药物代谢完全，所以第二次非禁食给药时导致血浆药物浓度显著升高；3、第一天禁食给药后，对红细胞造成影响，使第二次非禁食给药时红细胞与药物结合的能力下降，导致血浆药物浓度显著升高。
生物试验
实施例A体外生物活性评价
对本发明中化合物的拮抗剂特性利用FLIPR(荧光成像读板仪)法进行测定，所述化合物是由HEK293细胞(人肾上皮细胞系，ATCC)中所表达的hP2X3(人嘌呤能P2X受体亚型3，登录号NM_002559.4)激活所诱导的细胞内钙升高的抑制剂。
将稳定表达hP2X3的HEK293细胞置于37℃，湿度5％的细胞培养箱中，以含有10％FBS(胎牛血清，Gibco，10099-141)，1％青霉素-链霉素(Gibco，15140-122)，和1mg/mL G418(Invitrogen，10131027)的DMEM高糖培养基进行培养。在FLIPR实验前18-24小时，将细胞以400000cells/mL的密度接种到384孔中(10000cells/well)，在细胞培养箱中温育过夜。实验当天，弃去培养基，将细胞在FLIPR缓冲液(每30mL缓冲液中含有0.3mL丙磺舒(Thermo，P36400)，0.6mL 1M HEPES(Invitrogen，15630080)和29.1mL HBSS(Invitrogen，14065056))中进行洗涤。每孔加入20μL 0.5×Calcium 6荧光染料(Molecular Devices，R8190)，在37℃下染料荷载温育1.5小时。随后将10μL供试化合物(以10 mM的浓度溶解于DMSO中并用缓冲液进行系列稀释)或溶媒加入各孔，并使其在室温下平衡30min。然后将细胞板放入FLIPR中，进行基线荧光测量(激发波长为485nm，发射波长为525-535nm)。随后以10μL/孔加入激动剂(终浓度2.5μM的BZ-ATP(Sigma，B6396))或溶媒(超纯水)，以1秒的时间间隔测量荧光值2分钟，最后对输出的荧光计数进行分析。
使用上述方法获得的IC 50示于下表中。
表9 使用上述方法IC 50数据
化合物 P2X3 IC 50(μM)
化合物172 0.0007
阳性化合物1 0.0115
阳性化合物2 0.0522
阳性化合物1：
Figure PCTCN2021102068-appb-000019
阳性化合物2：
Figure PCTCN2021102068-appb-000020
实施例B体外P2X2/3受体选择性评价
对本发明中化合物对P2X2/3受体的选择性利用FLIPR(荧光成像读板仪)法进行测定，所述化合物是由HEK293细胞(人肾上皮细胞系，ATCC)中所表达的hP2X2/3(人嘌呤能P 2X受体亚型2和亚型3形成的异源二聚受体，P2X2的登录号为NM_002559.4，P2X3的登录号为NM_002559.4)激活所诱导的细胞内钙升高的抑制剂。
将稳定表达hP2X2/3的HEK293细胞置于37℃，湿度5％的细胞培养箱中，以含有10％FBS(胎牛血清，Gibco，10099-141)，1％青霉素-链霉素(Gibco，15140-122)，和1mg/mL G418(Invitrogen，10131027)的DMEM高糖培养基进行培养。在FLIPR实验前18-24小时，将细胞以250000cells/mL的密度接种到96孔中(25000cells/well)，在细胞培养箱中温育过夜。实验当天，弃去培养基，将细胞在FLIPR缓冲液(每30mL缓冲液中含有0.3mL丙磺舒(Thermo，P36400)，0.6mL 1M HEPES(Invitrogen，15630080)和29.1mL HBSS(Invitrogen，14065056))中进行洗涤。每孔加入75μL 1mM Fluo-4 AM荧光染料(Thermo，F14202)，在37℃下染料荷载温育1.0小时。随后用缓冲液清洗96孔板一次，将50μL含有供试化合物或溶媒的缓冲液加入各孔，并使其在室温下孵育30min。 然后将细胞板放入FLIPR中，进行基线荧光测量(激发波长为485nm，发射波长为525-535nm)。随后以50μL/孔加入激动剂(终浓度5μM的BZ-ATP(Sigma，B6396))或溶媒(超纯水)，以1秒的时间间隔测量荧光值2分钟，最后对输出的荧光计数进行分析。
使用上述方法获得的IC 50示于下表中。
表10 使用上述方法IC 50数据
化合物 IC 50 选择性倍数
化合物172 >60 >85714
阳性化合物1 >60 >5217
阳性化合物2 0.36 6.9
实施例C单纯柠檬酸咳嗽模型活性测试
将雄性Dunkin Hartley豚鼠(300-350g)置于动物雾化箱中，关闭雾化箱门，同时开启超声雾化器(广东粤华)，以最大雾化量(约2mL/min)往雾化箱中充17.5％的柠檬酸气体，持续雾化20s，并从雾化开始时计时，持续观察动物在10min内的咳嗽表现。10min观察期间需要进行咳嗽人工计数，根据豚鼠咳嗽姿势如腹部抽动、嘴巴张开、头部下勾等以及咳嗽声音判断咳嗽次数，记录前5min咳嗽次数、10min咳嗽次数，同时记录豚鼠的咳嗽潜伏期，即从柠檬酸诱导开始至第1次咳嗽出现的时间。
表11 动物实验
Figure PCTCN2021102068-appb-000021
化合物172具有减少咳嗽次数，提高咳嗽潜伏期的作用，且与阳性化合物效能相 当。
实施例D ATP-柠檬酸咳嗽模型活性测试
将雄性Dunkin Hartley豚鼠(300-400g)置于放入全身体积扫描箱适应3～5mins后，进行2min ATP雾化，间隔3min，再给予5min的柠檬酸雾化，所有雾化速率为300ul/min左右。从柠檬酸雾化开始，记录10mins内动物的咳嗽次数和咳嗽潜伏期。10min观察期间需要进行咳嗽人工计数，根据豚鼠咳嗽姿势如腹部抽动、嘴巴张开、头部下勾等以及咳嗽声音判断咳嗽次数，记录10min咳嗽次数，同时记录豚鼠的咳嗽潜伏期。
动物分组为溶媒组、右美沙芬(63mg/kg)阳性组、AF-219(30mg/kg)阳性组、实施例172化合物(3、10、30mg/kg)给药组，所有化合物口服给药，除右美沙芬为ATP-柠檬酸暴露前40min给药，其余化合物为ATP-柠檬酸暴露前2h给药。
表12 对于化合物所获得的奎宁/水饮用比例
Figure PCTCN2021102068-appb-000022
如表5所示，所有化合物处理后与溶媒组相比均具有咳嗽次数下降的趋势，其中右美沙芬阳性组和化合物172均可获得显著性降低咳嗽次数的作用，且化合物172具有剂量依赖的抑制作用。相较于阳性化合物2，化合物172的咳嗽抑制作用更强。
实施例E体外细胞毒性测试
对本发明中化合物的体外细胞毒性测试在HepG2细胞中利用CCK-8法进行测定。收集对数期的HepG2细胞(北纳生物)，调整细胞悬液浓度，以50000cells/well在96孔细胞培养板中铺板，将细胞置于5％，37℃的细胞培养箱中孵育过夜，待板中细胞融合度达到80-90％后，换液加入各浓度梯度的供试化合物或溶媒(DMSO)，在5％，37℃的细胞培养箱中孵育48小时。处理结束后，弃去板内培养基，用PBS洗涤2遍，每孔加入100μL CCK-8工作液(碧云天生物技术)，37℃避光孵育1.5小时，酶标仪上检测OD 450nm处各孔的吸光值，分析计算各化合物的CC 50。
表13 各化合物的IC 50值
化合物 HepG2 CC 50(μM)
化合物172 57.05
阳性化合物1 128.4
阳性化合物2 >200
实施例F体外代谢稳定性试验
对本发明中化合物的体外代谢稳定性利用各种属肝微粒体温孵法进行测定。在肝微粒体反应体系中(1mg/mL肝微粒体蛋白，25U/mL 6-磷酸葡萄糖脱氢酶，1mM NADP，6mM D-6-磷酸葡萄糖，5mM MgCl 2)加入适量供试化合物，放入37℃水浴锅温孵启动反应，于各时间点取100μL反应液加入至含400μL 0℃预冷的内标工作液(含200ng/mL***、双氯酚酸、甲苯磺丁脲、拉贝洛尔的乙腈溶液)离心管中，终止反应，4℃离心机10000g离心10min，取上清液进LC-MS进行分析检测，获得供试化合物在各种属肝微粒体中的体外代谢半衰期。
表14 体外代谢稳定性测试
Figure PCTCN2021102068-appb-000023
实施例G 双瓶法味觉障碍测试
SPF级雄性SD大鼠(6-8周)入库后随即接受3天适应性饮水训练，具体训练内容为动物单笼饲养，每笼放置两瓶水(均为普通饮水)，适应性训练期间每天彻夜禁水处理(撤去饮水瓶)，8:30am～5:30pm重新给予饮水，如此循环3天，每天更换两瓶水左右摆放位置，整个适应性训练期间动物均可自由进食。正式实验开展前20小时撤去所有动物饮水瓶，禁水至试验开展时。正式实验时所有动物随机分组，在重新供水前给予供试化合物或溶媒，采用单次腹腔注射方式给药，给药时间根据供试化合物的T max确定。随后将动物单笼放置，给予两瓶饮水，一瓶为普通饮水，一瓶为0.3mM的奎宁水，观察15min内动物的饮水量，以奎宁饮水量/普通饮水量进行统计分析，以阳性化合物2为味觉障碍阳性对照。
表15 动物饮水量测试
Figure PCTCN2021102068-appb-000024
Figure PCTCN2021102068-appb-000025
结果显示：与空白溶媒组组相比，5、10、20mg/kg的化合物172均对大鼠饮用奎宁/水的比例无明显影响，说明化合物172对动物味觉影响较小；与阳性化合物2相比，奎宁/水的饮用比例均显著低于阳性化合物2，说明化合物172引起味觉障碍的风险远远优于阳性化合物2，安全性更佳。

Claims (14)

  1. 一种如式A所示化合物或其溶剂化物的晶型：
    Figure PCTCN2021102068-appb-100001
    其选自晶型I、晶型II、晶型III、晶型IV、晶型V、晶型VI、晶型VII、晶型VIII和晶型IX；
    其中，式A化合物的水合物的晶型III，其X射线粉末衍射图在2θ值为12.91°±0.20°、16.77±0.20°、19.27°±0.20°和22.80°±0.20°处具有特征峰；
    式A化合物的晶型V，其X射线粉末衍射图在2θ值为8.38°±0.20°、9.15°±0.20°、13.52°±0.20°和18.44±0.20°处具有特征峰；
    式A化合物的晶型I，其X射线粉末衍射图在2θ值为8.56°±0.20°、12.48±0.20°和22.13°±0.20°处具有特征峰；
    式A化合物的MTBE溶剂化物的晶型II，其X射线粉末衍射图在2θ值为8.42°±0.20°、12.09°±0.20°、13.68°±0.20°和20.87°±0.20°处具有特征峰；
    式A化合物的水合物的晶型IV，其X射线粉末衍射图在2θ值为8.65°±0.20°、12.69°±0.20°和22.56°±0.20°处具有特征峰；
    式A化合物的水合物的晶型VI，其X射线粉末衍射图在2θ值为8.62°±0.20°、12.69°±0.20°和22.59°±0.02°处具有特征峰；
    式A化合物的乙二醇溶剂化物的晶型VII，其X射线粉末衍射图在2θ值为8.36°±0.20°、12.13°±0.20°、12.45°±0.20°、16.84°±0.20°和21.66°±0.20°处具有特征峰；
    式A化合物的THF溶剂化物的晶型VIII，其X射线粉末衍射图在2θ值为8.53°±0.20°、12.38°±0.20°、13.66°±0.20°和21.49°±0.20°处具有特征峰；
    式A化合物的DMSO溶剂化物的晶型IX，其X射线粉末衍射图在2θ值为8.55°±0.20°、12.43°±0.20°、21.75°±0.20°和25.07°±0.20°处具有特征峰。
  2. 如权利要求1所述的化合物A或其溶剂化物的晶型，其特征在于，
    所述的晶型III，其X射线粉末衍射图在2θ值为12.91°±0.20°、16.77±0.20°、19.27°±0.20°、22.80°±0.20°、13.75°±0.20°、14.46°±0.20°和20.86°±0.20°处具有特征峰；
    和/或，所述的晶型V，其X射线粉末衍射在2θ值为8.38°±0.20°、9.15°±0.20°、13.52°±0.20°、18.44±0.20°、16.26°±0.20°、16.89°±0.20°和17.86°±0.20°处具有特征峰；
    和/或，所述的晶型I，其X射线粉末衍射图在2θ值为8.56°±0.20°、12.48±0.20°、22.13°±0.20°、13.53°±0.20°、14.25±0.20°、25.18°±0.20°和26.07°±0.20°处有特征峰；
    和/或，所述的晶型II，其X射线粉末衍射图在2θ值为8.42°±0.20°、12.09°±0.20°、13.68°±0.20°、20.87°±0.20°、16.17°±0.20°、16.93°±0.20°、17.55°±0.20°和21.20°±0.20°处有特征峰；
    和/或，所述的晶型IV，其X射线粉末衍射图在2θ值为8.65°±0.20°、12.69°±0.20°、22.56°±0.20°、13.48°±0.20°、17.39°±0.20°、21.04°±0.20°和23.63°±0.20°处具有特征峰；
    和/或，所述的晶型VI，其X射线粉末衍射图在2θ值为8.62°±0.20°、12.69°±0.20°、22.59°±0.02°、13.46°±0.20°、17.41°±0.20°、26.51°±0.02°、25.62°±0.02°和25.24°±0.20°处具有特征峰；
    和/或，所述的晶型VII，其X射线粉末衍射图在2θ值为8.36°±0.20°、12.13°±0.20°、12.45°±0.20°、16.84°±0.20°、21.66°±0.20°、21.07°±0.20°和24.82°±0.20°处具有特征峰；
    和/或，式A化合物的THF溶剂化物的晶型VIII，其X射线粉末衍射图在2θ值为8.53°±0.20°、12.38°±0.20°、13.66°±0.20°、21.49°±0.20°、20.99°±0.20°、24.94°±0.20°和25.31°±0.20°处具有特征峰；
    和/或，所述的晶型IX，其X射线粉末衍射图在2θ值为8.55°±0.20°、12.43°±0.20°、21.75°±0.20°、25.07°±0.20°、13.57°±0.20°、17.18°±0.20°、20.94°±0.20°和25.57°±0.20°处具有特征峰。
  3. 如权利要求2所述的化合物A或其溶剂化物的晶型，其特征在于，
    所述的晶型III，其X射线粉末衍射图在2θ值为12.91°±0.20°、16.77±0.20°、19.27°±0.20°、22.80°±0.20°、13.75°±0.20°、14.46°±0.20°、20.86°±0.20°、21.08°±0.20°、23.75°±0.20°和24.05°±0.20°处具有特征峰；
    和/或，所述的晶型III的热重分析图谱中，在室温-100℃区间重量损失梯度为1.5％；
    和/或，所述的晶型III的示差扫描量热图谱中第一处吸热峰为脱去0.4个水；
    和/或，所述的晶型V，其X射线粉末衍射在2θ值为8.38°±0.20°、9.15°±0.20°、13.52°±0.20°、18.44±0.20°、16.26°±0.20°、16.89°±0.20°、17.86°±0.20°、22.35°±0.20°、23.56°±0.20°、24.74°±0.20°处具有特征峰；
    和/或，所述的晶型Ⅴ的示差扫描量热图谱在166℃具有吸热峰，熔化焓为70±2J/g；
    和/或，所述的晶型I，其X射线粉末衍射图在2θ值为8.56°±0.20°、12.48±0.20°、22.13°±0.20°、13.53°±0.20°、14.25±0.20°、25.18°±0.20°、26.07°±0.20°、22.32°±0.20°、23.23°±0.20°和23.42°±0.20°处有特征峰；
    和/或，所述的晶型I的示差扫描量热图谱中在152℃具有吸热峰，熔化焓为44±2J/g；
    和/或，所述的晶型II，其X射线粉末衍射图在2θ值为8.42°±0.20°、12.09°±0.20°、13.68°±0.20°、20.87°±0.20°、16.17°±0.20°、16.93°±0.20°、17.55°±0.20°、21.20°±0.20°、22.60°±0.20°、23.23°±0.20°和24.40°±0.20°处有特征峰；
    和/或，所述的晶型II的热重分析图谱中，在100-160℃温度区间内失重3.5％，在160- 200℃区间内失重2.9％；
    和/或，所述的晶型IV，其X射线粉末衍射图在2θ值为8.65°±0.20°、12.69°±0.20°、22.56°±0.20°、13.48°±0.20°、17.39°±0.20°、21.04°±0.20°、23.63°±0.20°、14.39°±0.20°、25.60°±0.20°和26.52°±0.20°处具有特征峰；
    和/或，所述的晶型IV的TGA图谱中，RT-60℃温度区间内失重1.2％；
    和/或，所述的晶型VII，其X射线粉末衍射图在2θ值为8.36°±0.20°、12.13°±0.20°、12.45°±0.20°、16.84°±0.20°、21.66°±0.20°、21.07°±0.20°、24.82°±0.20°、13.61°±0.20°、23.22°±0.20°和24.57°±0.20°处具有特征峰；
    和/或，所述的晶型VII的热重分析图谱中，在室温-120℃区间内有25.7％的失重；
    和/或，所述的晶型VIII，其X射线粉末衍射图在2θ值为8.53°±0.20°、12.38°±0.20°、13.66°±0.20°、21.49°±0.20°、20.99°±0.20°、24.94°±0.20°、25.31°±0.20°、17.14°±0.20°、21.72°±0.20°和23.00°±0.20°处具有特征峰；
    和/或，所述的晶型VIII的热重分析图谱中，在室温-160℃温度区间内失重5.7％；
    和/或，所述的晶型IX，其X射线粉末衍射图在2θ值为8.55°±0.20°、12.43°±0.20°、21.75°±0.20°、25.07°±0.20°、13.57°±0.20°、17.18°±0.20°、20.94°±0.20°、25.57°±0.20°、21.37°±0.20°和23.12°±0.20°具有特征峰；
    和/或，所述的晶型IX的热重分析图谱中，在室温-60℃温度区间失重18.23％。
  4. 如权利要求3所述的化合物A或其溶剂化物的晶型，其特征在于，
    所述的晶型III以2θ角表示的X射线粉末衍射图基本如图1所示；
    和/或，所述的晶型III的热重分析图谱和示差扫描量热图谱基本为如图2所示；
    和/或，所述的晶型III的动态水分吸附图谱如图4所示；
    和/或，所述的晶型III偏光显微镜图谱基本如图6所示；
    和/或，所述的晶型III中含0.4当量的水；
    和/或，所述的晶型Ⅴ以2θ角表示的X射线粉末衍射图基本如图7所示；
    和/或，所述的晶型Ⅴ的动态水分吸附图谱如图9所示；
    和/或，所述的晶型Ⅴ的热重分析图谱和示差扫描量热图谱为基本如图8所示；
    和/或，所述的晶型Ⅴ偏光显微镜图谱基本如图11所示；
    和/或，所述的晶型I以2θ角表示的X射线粉末衍射图基本如图12所示；
    和/或，所述的晶型I的热重分析图谱和示差扫描量热图谱基本如图13所示；
    和/或，所述的晶型I的动态水分吸附图谱如图14所示；
    和/或，所述的晶型I偏光显微镜图谱基本如图17所示；
    和/或，所述晶型II的核磁共振氢谱图谱图如图19所示；
    和/或，所述的晶型II以2θ角表示的X射线粉末粉末衍射图基本如图18所示；
    和/或，所述的晶型II偏光显微镜图谱基本如图21所示；
    和/或，所述的晶型II的热重分析图谱和示差扫描量热图谱基本如图20所示；
    和/或，所述的晶型IV的以2θ角表示的X射线粉末衍射图基本如图22所示；
    和/或，所述的晶型IV的热重分析图谱和示差扫描量热图谱如图23所示；
    和/或，所述的晶型IV的偏光显微镜图谱基本如图25所示；
    和/或，所述的晶型VI以2θ角表示的X射线粉末衍射图基本如图26所示；
    和/或，所述的晶型VII以2θ角表示的X射线粉末衍射图基本如图28所示；
    和/或，所述的晶型VII的核磁共振氢图谱为如图29所示；
    和/或，所述的晶型VII的热重分析图谱和示差扫描量热图谱为基本如图30所示；
    和/或，所述的晶型VIII以2θ角表示的X射线粉末衍射图基本如图31所示；
    和/或，所述的晶型VIII的热重分析图谱和示差扫描量热图谱基本如图32所示；
    和/或，所述的晶型VIII的核磁共振氢谱谱图如图33所示；
    和/或，所述的晶型IX以2θ角表示的X射线粉末衍射图基本如图35所示；
    和/或，所述的晶型IX的热重分析图谱和示差扫描量热图谱基本如图36所示。
  5. 一种物质A的晶型III的制备方法，其特征在于，其为方案一、方案二或方案三；其中，
    方案一，其包括如下步骤：将式A所示化合物的无定型与溶剂的混悬液进行转晶，得到物质A的晶型III即可；所述的溶剂为水或醇类溶剂；
    方案二，其包括如下步骤：将反溶剂加入到式A所示化合物与溶剂的溶液中，进行析晶，得到物质A的晶型III即可；所述的溶剂为醇类、呋喃类或DMSO中的一种或多种；所述的反溶剂为水；
    方案三，其包括如下步骤：将式A所示化合物与溶剂的溶液加入到水溶液A中，析晶，得到物质A的晶型III即可，所述的水溶液A为所述的物质A的晶型III的晶种与水的混悬液；所述的溶剂为DMSO。
  6. 如权利要求5所述的制备方法，其特征在于，所述的方案一中，所述的溶剂为水或甲醇；
    和/或，所述的方案一中，所述的析晶温度为20-50℃，优选地40℃或50℃；
    和/或，所述的方案一中，所述的化合物A的无定型与溶剂的质量体积比为50mg/mL；
    和/或，所述的方案二中，所述的溶剂为甲醇、四氢呋喃或DMSO中的一种或多种；
    和/或，所述的方案三中，所述的溶剂与水的体积比为3:1-1:1；
    和/或，所述的方案三中，所述的DMSO与水的体积比为1:1-1:4；
    和/或，当采用所述的方案一时，其包括以下步骤：将化合物A的无定型样品与溶剂混合，混悬进行转晶；所述的溶剂为水或甲醇；所述的搅拌温度为20-50℃，优选40℃；所述的化合物A的无定型与溶剂的质量体积比为50mg/mL；
    和/或，当采用所述的方案二时，其包括以下步骤：将化合物A与溶剂混合，滴加到反溶剂中；所述的溶剂为甲醇、四氢呋喃或DMSO中的一种或多种；所述的反溶剂为水；所述的溶剂与水的体积比为3:1-1:1；
    和/或，当采用所述的方案三时，其包括以下步骤：将化合物A与溶剂的溶液加入到水溶液A中，析晶，所述的水溶液A为所述的物质A的晶型III的晶种与水的混悬液；所述的溶剂为DMSO；所述的DMSO与水的体积比为1:1-1:4。
  7. 一种物质A的晶型III，其特征在于，所述的物质A的晶型III按照如权利要求5或6的制备方法制备得到。
  8. 如权利要求7所述的物质A的晶型III，其特征在于，所述的物质A的晶型III为如权利要求1-4中任一项所述的化合物A的水合物晶型III。
  9. 一种式A所示化合物的晶型Ⅴ的制备方法，其特征在于，其为方案A或方案B；
    方案A，其包括如下步骤：将式A所示化合物的无定型在20-50℃下与溶剂的混悬液进行转晶，得到化合物A的晶型V即可；所述的溶剂为水或腈类溶剂；所述的结晶的温度优选为50℃；
    方案B，其包括如下步骤：将式A所示化合物与溶剂的溶液中的溶剂挥发，得到化合物A的晶型V即可；所述的溶剂为醇类溶剂。
  10. 如权利要求9所述的制备方法，其特征在于，
    方案A中，所述的溶剂为水或乙腈；
    和/或，方案A中，所述化合物A的无定型与所述的溶剂的质量体积比为3.0mg/mL或50mg/mL；
    和/或，方案A中的转晶温度为50℃；
    和/或，方案B中，所述的溶剂为甲醇；
    和/或，方案B中的析晶温度为50℃；
    和/或，当采用所述的方案A时，其包括以下步骤：将化合物A的无定型在20-50℃下与溶剂的混悬液进行转晶，得到化合物的晶型V；所述的转晶温度50℃；所述的溶剂为水或乙腈；所述的化合物A的无定型与溶剂的质量体积比为50mg/mL或3.0mg/mL；
    和/或，当采用所述的方案B时，其包括以下步骤：将化合物A与溶剂的溶液中的溶剂挥发析晶，得到化合物A的晶型V；所述的溶剂甲醇，所述的析晶温度为50℃。
  11. 一种药物组合物，其包括如权利要求1～4中任一项所述的式A所示化合物或其溶剂化物的晶型和/或如权利要求7或8所述的物质A的晶型III，和药用辅料。
  12. 一种如权利要求1～4中任一项所述的式A所示化合物或其溶剂化物的晶型、如权利要求7或8所述的物质A的晶型III在制备P2X3受体拮抗剂或药物中的应用；所述的药物可为用于防护、处理、治疗或减轻动物的至少部分由P2X3介导的或活性相关的疾病的药物；或者，所述的药物可为用于治疗疼痛、泌尿道疾病或呼吸***疾病的药物。
  13. 如权利要求12所述的应用，其特征在于，所述疾病包括疼痛、泌尿道疾病或呼吸***疾病；所述疼痛较佳地包括：炎性疼痛、手术疼痛、内脏疼痛、牙痛、经前期疼痛、中枢性疼痛、由灼伤所致疼痛、偏头痛或簇性头痛；所述的泌尿道疾病包括：尿失禁、膀胱过度活动症、排尿困难、膀胱炎；所述呼吸***疾病较佳地包括：呼吸障碍，包括特发性肺纤维化、慢性阻塞性肺病、哮喘、支气管痉挛或慢性咳嗽。
  14. 如权利要求13所述的药物组合物，其特征在于，通过给药所述药物组合物，减少了与治疗相关的味觉障碍的副作用。
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