Crystal Forms of Heterocyclic Compounds, Preparation Methods Thereof and Applications Thereof

Abstract

The present invention discloses a crystal form of a heterocyclic compound, a preparation method thereof and an application thereof. The present invention provides a crystal form of a compound represented by formula A: the crystal form is 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, and discloses its preparation method, composition and its application in the preparation of P2X3 receptor antagonists, or in the preparation of drugs for preventing and / or treating pain, urinary tract diseases or respiratory diseases. It has high P2X3 antagonistic activity, good selectivity, low toxicity, good metabolic stability and little taste impact.

Description

Technical Field

[0001] The present invention relates to the field of medicinal chemistry, and particularly to a crystal form of a heterocyclic compound, a preparation method thereof, and an application thereof. Background Art

[0002] 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, P2X2 / 3, P2X4 / 6, P2X1 / 5. It has been found that P2X3 receptors are specifically expressed in the primary afferent nerve fibers of "hollow viscera", such as the lower urinary tract and the respiratory tract.

[0003] Cough is the main symptom manifestation of respiratory diseases. In the outpatient department of respiratory medicine, 70% - 80% of patients have cough symptoms. With the gradually increasing prevalence of COPD, IPF, etc., and cough being the main manifestation symptom of most airway diseases, the demand also increases accordingly. As a defensive nerve reflex of the body, cough is beneficial to clearing respiratory tract secretions and harmful factors, but frequent and severe cough will have a serious impact on the patient's work, life, and social activities.

[0004] There are not many varieties of P2X3 antagonists specifically developed for cough indications. Currently, the project with relatively fast progress is Roche's AF-219 project, which has good efficacy for refractory cough in the latest completed Phase II clinical trial, but has serious taste disorder problems.

[0005] Currently, there are no drugs on the market for treating many diseases including chronic cough through the P2X3 inhibition pathway. Therefore, the development of new compounds that can inhibit P2X3 activity has positive significance for the treatment of diseases.

[0006] Patent application CN201911379293.8 involves a P2X3 antagonist as shown below, which has high P2X3 antagonistic activity, good selectivity, low toxicity, good metabolic stability, and little taste impact. It has good prospects for drug development. However, it does not involve the crystal form of the above compound.

[0007]

[0008] Compounds generally exhibit polymorphism, and a general drug may exist in two or more different crystalline forms. The existence form and quantity of polymorphic compounds are unpredictable. Different crystalline forms of the same drug have significant differences in solubility, melting point, density, stability, etc., thus affecting to varying degrees the temperature type, uniformity, bioavailability, efficacy, and safety of the drug. 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 crystalline form suitable for drug formulation development. Summary of the Invention

[0009] The present invention provides a crystalline form of a heterocyclic compound, a preparation method thereof, and an application thereof. The crystalline form of the present invention has good stability and is of great value for the optimization and development of drugs.

[0010] The present invention provides a crystalline form of a compound represented by formula A or a solvate thereof:

[0011]

[0012] It is selected from crystalline form I, crystalline form II, crystalline form III, crystalline form IV, crystalline form V, crystalline form VI, crystalline form VII, crystalline form VIII, and crystalline form IX.

[0013] The present invention provides crystalline form III of the compound of formula A, and its X-ray powder diffraction pattern (XRPD) has characteristic peaks at 2θ values of 12.91° ± 0.20°, 16.77 ± 0.20°, 19.27° ± 0.20°, and 22.80° ± 0.20°.

[0014] In some preferred embodiments of the present invention, the XRPD of the crystalline form III represented by the 2θ angle further has characteristic peaks at 13.75° ± 0.20°, 14.46° ± 0.20°, and 20.86° ± 0.20°; further, it has characteristic peaks at 21.08° ± 0.20°, 23.75° ± 0.20°, and 24.05° ± 0.20°.

[0015] In some preferred embodiments of the present invention, the XRPD pattern of the crystalline form III represented by the 2θ angle is substantially as Figure 1 shown. In the thermogravimetric analysis spectrum (TGA) of the crystalline form III, the weight loss gradient in the range of room temperature (RT) - 100 °C is 1.5%, and the "%" is weight percentage. In the differential scanning calorimetry spectrum (DSC) of the crystalline form III, the first endothermic peak is for the removal of 0.4 water molecules, and the second endothermic peak is attributed to the melting endothermic peak of the sample after dehydration. Its TGA and DSC spectra are preferably as Figure 2 shown. After the crystalline form III is dehydrated, it quickly absorbs moisture again under ambient humidity and returns to crystalline form III. The XRPD patterns before and after heating to dehydration are preferably asFigure 3 As shown. In the dynamic vapor sorption (DVS) profile of Form III, the sample shows a certain hygroscopicity, and the water content changes little within a wide humidity range. Its DVS profile is preferably as shown in Figure 4 As shown. In the X-ray powder diffraction (XRPD) profiles of Form III before and after DVS testing, there are no obvious changes in XRPD before and after DVS testing. Its XRPD profiles before and after DVS testing are preferably as shown in Figure 5 As shown. In the polarized light microscopy (PLM) image of Form III, the crystal form is irregular crystals of about 2 μm, aggregated into 20 - 50 μm. Its PLM image is preferably substantially as shown in Figure 6 As shown. The purity of Form III is generally above 90%, preferably above 95%.

[0016] The present invention provides Form V of the compound of formula A, whose XRPD has characteristic peaks at 2θ values of 8.38° ± 0.20°, 9.15° ± 0.20°, 13.52° ± 0.20° and 18.44 ± 0.20°.

[0017] In certain preferred embodiments of the present invention, the XRPD pattern of Form V expressed in 2θ angle also has characteristic peaks at 16.26° ± 0.20°, 16.89° ± 0.20° and 17.86° ± 0.20°; further, it has characteristic peaks at 22.35° ± 0.20°, 23.56° ± 0.20°, 24.74° ± 0.20°.

[0018] In certain preferred embodiments of the present invention, the XRPD pattern of Form V expressed in 2θ angle is substantially as shown in Figure 7 As shown. In the thermogravimetric analysis (TGA) profile of Form V, there is no weight loss in the temperature range of RT - 230°C. In the differential scanning calorimetry (DSC) profile of Form V, there is an endothermic peak at 166°C ± 2°C, and the melting enthalpy is 70 ± 2 J / g. Its TGA and DSC profiles are preferably as shown in Figure 8 As shown. Combining the DSC and TGA diagrams, it can be known that this product is a non - hydrous crystal form. In the DVS profile of Form V, the sample shows a certain hygroscopicity (0.7%, 80% RH). Its DVS profile is preferably as shown in Figure 10 As shown. In the XRPD profiles of Form V before and after DVS testing, there are no obvious changes in XRPD before and after DVS testing. Its XRPD profiles before and after DVS testing are preferably as shown in Figure 9 As shown. In the PLM of Form V, Form V is irregular crystals of about 5 μm. Its PLM image is preferably substantially as shown in Figure 11 As shown. The purity of Form V is generally above 90%, preferably above 95%.

[0019] The present invention provides polymorphic form I of a compound of formula A, whose XRPD pattern has characteristic peaks at 2θ values of 8.56° ± 0.20°, 12.48 ± 0.20° and 22.13° ± 0.20°.

[0020] In certain preferred embodiments of the present invention, for the polymorphic form I, its X-ray powder diffraction pattern further has characteristic peaks at 2θ values of 13.53° ± 0.20°, 14.25 ± 0.20°, 25.18° ± 0.20° and 26.07° ± 0.20°; further, it has characteristic peaks at 22.32° ± 0.20°, 23.23° ± 0.20° and 23.42° ± 0.20°.

[0021] In certain preferred embodiments of the present invention, the XRPD pattern of the polymorphic form I expressed in 2θ angle is substantially as Figure 12 shown. In the DSC pattern of the polymorphic form I, there is an endothermic peak at 152°C ± 2°C, and the enthalpy of fusion is 44 ± 2 J / g. In the TGA pattern of the polymorphic form I, there is no weight loss in the temperature range of RT - 230°C. Its TGA and DSC spectra are preferably substantially as Figure 13 shown. Combining the DSC and TGA patterns, it can be known that this product is an anhydrous polymorphic form. The DVS pattern of the polymorphic form I is as Figure 14 shown. When the relative humidity is greater than 40%, the weight of the polymorphic form I increases sharply. When the relative humidity drops to 40%, all the absorbed moisture is discharged. In the XRPD pattern of the polymorphic form I after moisture absorption, the polymorphic form I undergoes crystal transformation to a hydrate polymorphic form IV in a high-humidity environment, and after drying it in a vacuum drying oven at 30°C, it transforms back to the initial polymorphic form I. The XRPD patterns before and after moisture absorption are preferably substantially as Figure 15 shown. It can be known that, combining the DVS test results ( Figure 14 ), when the environmental relative humidity is higher than 40%, the polymorphic form I rapidly absorbs moisture and transforms into a hydrate; when the relative humidity is lower than 50%, the adsorbed water rapidly desorbs and transforms into the polymorphic form I. That is, the transformation between the polymorphic form I and IV is reversible. The polymorphic form I shows hygroscopicity (6.8%, 80% RH), and the polymorphic form I remains unchanged in crystal form after DVS testing. The XRPD patterns before and after DVS testing are preferably as Figure 16 shown. In the PLM of the polymorphic form I, the polymorphic form I is irregular crystals of about 5 μm, and its PLM is preferably substantially as Figure 17 shown. The purity of the polymorphic form I is generally above 90%, preferably above 95%.

[0022] The present invention provides polymorphic form II of a MTBE solvate of a compound of formula A, whose XRPD pattern has characteristic peaks at 2θ values of 8.42° ± 0.20°, 12.09° ± 0.20°, 13.68° ± 0.20° and 20.87° ± 0.20°.

[0023] In certain preferred embodiments of the present invention, the XRPD of the crystal form II expressed in 2θ angle further has characteristic peaks at 16.17°±0.20°, 16.93°±0.20°, 17.55°±0.20° and 21.20°±0.20°; further, there are characteristic peaks at 22.60°±0.20°, 23.23°±0.20° and 24.40°±0.20°.

[0024] In certain preferred embodiments of the present invention, the XRPD of the crystal form II expressed in 2θ angle is substantially as Figure 18 shown. In the 1H NMR spectrum of the crystal form II, there are residual signals of MTBE at chemical shifts of 1.11 and 3.08, and the 1H NMR spectrum of the residual MTBE is as Figure 19 shown. In the TGA spectrum of the crystal form II, the weight loss is 3.5% in the temperature range of 100 - 160 °C and 2.9% in the range of 160 - 200 °C. In the DSC spectrum of the crystal form II, there are two adjacent endothermic peaks, and the TGA and DSC spectra are preferably as Figure 20 shown. Compared with the residual MTBE hydrogen spectrum as Figure 19 shown, it can be known that this product is an MTBE solvate. In the PLM of the crystal form II, the crystal form is about 2 μm irregular crystals, and its PLM is preferably substantially as Figure 21 shown.

[0025] The present invention provides crystal form IV of the hydrate of the compound of formula A, and its XRPD pattern has characteristic peaks at 2θ values of 8.65°±0.20°, 12.69°±0.20° and 22.56°±0.20°.

[0026] In certain preferred embodiments of the present invention, the XRPD of the crystal form IV expressed in 2θ angle further 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°.

[0027] In certain preferred embodiments of the present invention, the XRPD of the crystal form IV expressed in 2θ angle is substantially as Figure 22 shown. In the TGA spectrum of the crystal form IV, the weight loss is 1.2% in the temperature range of RT - 60 °C. In the DSC spectrum of the crystal form IV, there are two endothermic peaks. The first broad endothermic peak is presumably caused by dehydration, and the subsequent endothermic peak is a melting peak. The TGA and DSC spectra are preferably as Figure 23As shown. The crystalline form IV is only stable in a high-humidity environment. After dehydration, it quickly absorbs moisture in the ambient humidity and reverts to crystalline form I. The XRPD patterns before and after heating to dehydration are preferably as Figure 24 shown. In the PLM of the crystalline form IV, the crystal form is irregular crystals of about 5 μm, and its PLM diagram is preferably substantially as Figure 25 shown. The purity of the crystalline form IV is generally above 90%, preferably above 95%.

[0028] The present invention provides a crystalline form VI of a hydrate of a compound of formula A, whose X-ray powder diffraction pattern has characteristic peaks at 2θ values of 8.62° ± 0.20°, 12.69° ± 0.20°, and 22.59° ± 0.02°;

[0029] In certain preferred embodiments of the present invention, the XRPD of the crystalline form VI expressed in 2θ angles further has characteristic peaks at 13.46° ± 0.20°, 17.41° ± 0.20°, 26.51° ± 0.02°, 25.62° ± 0.02°, and 25.24° ± 0.20°; further, it has characteristic peaks at 23.64° ± 0.20°, 21.00° ± 0.20°, and 27.85° ± 0.20°.

[0030] In certain preferred embodiments of the present invention, the XRPD of the crystalline form VI expressed in 2θ angles is substantially as Figure 26 shown. In the superimposed XRPD pattern of the crystalline form VI, the crystal form sample converts to crystalline form I after being placed at ambient humidity (35% RH) for a few minutes. Its superimposed XRPD pattern is as Figure 27 shown. This indicates that the crystalline form VI may be a very unstable hydrate. The purity of the crystalline form VI is generally above 90%, preferably above 95%.

[0031] The present invention provides a crystalline form VII of a glycol solvate of a compound of formula A, whose X-ray powder diffraction pattern has characteristic peaks at 2θ values of 8.36° ± 0.20°, 12.13° ± 0.20°, 12.45° ± 0.20°, 16.84° ± 0.20°, and 21.66° ± 0.20°.

[0032] In certain preferred embodiments of the present invention, the XRPD of the crystalline form VII further has characteristic peaks at the following 2θ values: 21.07° ± 0.20°, 24.82° ± 0.20°; further, at 13.61° ± 0.20°, 23.22° ± 0.20°, and 24.57° ± 0.20°.

[0033] In certain preferred embodiments of the present invention, the XRPD of the crystalline form VII expressed in 2θ angles is substantially as Figure 28 shown. The1 In the HNMR spectrum, residual ethylene glycol solvent is shown at chemical shifts δ of 3.39 and 4.44, and the residual ethylene glycol 1 The HNMR spectrum is preferably as Figure 29 shown. In the TGA spectrum of the crystal form VII, there is a weight loss of 25.7% in the temperature range of RT - 120°C. In the DSC spectrum of the crystal form VII, there are two broad endothermic peaks. The first endothermic peak is presumably caused by desolvation, and its TGA and DSC spectra are preferably as Figure 30 shown. Combining the DSC and TGA diagrams, it can be known that the product is a solvate of ethylene glycol. The purity of the crystal form VII is generally above 90%, preferably above 95%.

[0034] The present invention provides crystal form VIII of the THF solvate of the compound of formula A, and its X-ray powder diffraction pattern has characteristic peaks at 2θ values of 8.53° ± 0.20°, 12.38° ± 0.20°, 13.66° ± 0.20° and 21.49° ± 0.20°.

[0035] In some preferred embodiments of the present invention, the X-ray powder diffraction pattern of the crystal form VIII expressed in 2θ angle also has characteristic peaks at 20.99° ± 0.20°, 24.94° ± 0.20° and 25.31° ± 0.20°; further, it has characteristic peaks at 17.14° ± 0.20°, 21.72° ± 0.20° and 23.00° ± 0.20°.

[0036] In some preferred embodiments of the present invention, the X-ray powder diffraction pattern of the crystal form VIII expressed in 2θ angle is substantially as Figure 31 shown. In the TGA spectrum of the crystal form VIII, there is a weight loss of 5.7% in the temperature range of RT - 160°C. In the DSC spectrum of the crystal form VIII, there is only one endothermic peak, which is the melting peak after desolvation of the sample. Therefore, the crystal form VIII is a solvate containing THF, and its TGA and DSC spectra are preferably as Figure 32 shown. The 1 In the HNMR spectrum of the crystal form VIII, residual THF solvent is shown at chemical shifts δ of 1.76 and 3.60, and its 1 The HNMR spectrum is preferably as Figure 33 shown. The crystal form VIII is unstable and undergoes crystal transformation after desolvation (drying in vacuum at 40°C for 3 hours), turning into crystal form I, and its XRPD spectra before and after drying are preferably as Figure 34 shown. The purity of the crystal form VIII is generally above 90%, preferably above 95%.

[0037] The present invention provides polymorphic form IX of the DMSO solvate of the compound of formula A, whose XRPD pattern has characteristic peaks at 2θ values of 8.55° ± 0.20°, 12.43° ± 0.20°, 21.75° ± 0.20° and 25.07° ± 0.20°.

[0038] In certain preferred embodiments of the present invention, the XRPD pattern of the polymorphic form IX expressed in 2θ angle further has peaks 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°.

[0039] In certain preferred embodiments of the present invention, the XRPD pattern of the polymorphic form IX expressed in 2θ angle is substantially as Figure 35 shown. In the TGA pattern of the polymorphic form IX, the weight loss is 18.23% in the temperature range of RT - 160°C, and there is a corresponding endothermic peak in the DSC pattern corresponding to the weight loss in TGA. Its TGA and DSC patterns are preferably as Figure 36 shown. In the 1 1H NMR spectrum of the polymorphic form IX, the chemical shift δ shows the residue of DMSO solvent at 2.68. The 1 1H NMR spectrum of the residual DMSO is preferably as Figure 60 shown. Combining the DSC and TGA patterns, it can be known that this product is a DMSO solvate. The purity of the polymorphic form IX is generally above 90%, preferably above 95%.

[0040] In the present invention, the ray used in the XRPD is Kα ray.

[0041] In the present invention, the target type used in the X-ray powder diffraction is Cu target.

[0042] The present invention also provides a preparation method of polymorphic form III of a substance A, which is Scheme 1, Scheme 2 or Scheme 3;

[0043] Scheme 1 includes the following steps: subjecting the suspension of the amorphous form of the compound of formula A and a solvent to polymorph conversion to obtain polymorphic form III of substance A; the solvent is water or an alcohol solvent;

[0044] Scheme 2 includes the following steps: adding an anti-solvent to the solution of the compound of formula A and a solvent for crystallization to obtain polymorphic form III of substance A; the solvent is one or more of alcohols, furans or DMSO; the anti-solvent is water;

[0045] Scheme III, which comprises the following steps: adding a solution of compound A and a solvent to aqueous solution A, followed by crystallization to obtain crystalline form III of substance A. The aqueous solution A is a suspension of crystalline form III of the substance A and water; the solvent is DMSO.

[0046] In the method for preparing crystalline form III, when using Scheme I, the solvent is preferably water or methanol.

[0047] In the method for preparing crystalline form III, when using Scheme I, the crystallization temperature is 20 - 50 °C, preferably 40 °C or 50 °C.

[0048] In the method for preparing crystalline form III, when using Scheme I, the mass - volume ratio of the amorphous form of compound A to the solvent is 50 mg / mL.

[0049] In the method for preparing crystalline form III, when using Scheme II, the solvent is preferably one or more of methanol, tetrahydrofuran, or DMSO.

[0050] In the method for preparing crystalline form III, when using Scheme II, the volume ratio of the solvent to water is 3:1 - 1:1 (such as 1:1 or 3:1).

[0051] In the method for preparing crystalline form III, when using Scheme III, the volume ratio of the solvent to water is 1:1 - 1:4 (such as 1:1, 2:3, 1:2, or 1:4).

[0052] The method for preparing crystalline form III, when using Scheme I, preferably comprises the following steps: subjecting a suspension of the amorphous form of compound A and a solvent to polymorph conversion; the solvent is water or methanol. The stirring temperature is 20 - 50 °C, preferably 40 °C. The mass - volume ratio of the amorphous form of compound A to the solvent is 50 mg / mL.

[0053] The method for preparing crystalline form III, when using Scheme II, preferably comprises the following steps: mixing compound A and a solvent, and then adding the mixture drop - wise to an anti - solvent; 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 (such as 3:1 or 1:1).

[0054] For the preparation method of the crystal form III, when adopting the third scheme, it preferably includes the following steps: adding a solution of compound A and a solvent to aqueous solution A for crystallization. The aqueous solution A is a suspension of crystal seeds of the crystal form III of substance A and water; the solvent is DMSO; the volume ratio of DMSO to water is 1:1 - 1:4 (such as 1:1, 2:3, 1:2 or 1:4).

[0055] The present invention also provides a preparation method of crystal form V of compound A, which is Scheme A or Scheme B;

[0056] Scheme A includes the following steps: subjecting the amorphous form of the compound shown by formula A to polymorphic transformation with a suspension of a solvent at 20 - 50 °C to obtain crystal form V of compound A; the solvent is water or a nitrile solvent.

[0057] Scheme B includes the following steps: evaporating the solvent in the solution of the compound shown by formula A and a solvent to obtain crystal form V of compound A; the solvent is an alcohol solvent.

[0058] In Scheme A, the mass - volume ratio of the amorphous form of compound A to the solvent is preferably 3.0 mg / mL or 50 mg / mL.

[0059] In Scheme A, the solvent is preferably water or acetonitrile.

[0060] In Scheme A, the temperature of the polymorphic transformation is preferably 50 °C.

[0061] In Scheme B, the solvent is preferably methanol.

[0062] In a certain scheme of Scheme A, it includes the following steps: subjecting the amorphous form of compound A to polymorphic transformation with a suspension of a solvent at 20 - 50 °C to obtain 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 compound A to the solvent is 50 mg / mL or 3.0 mg / mL.

[0063] In a certain scheme of Scheme B, it includes the following steps: evaporating the solvent in the solution of compound A and a solvent to obtain crystal form V of compound A; the solvent is methanol, and the temperature is 50 °C.

[0064] In the present invention, the amorphous form of the compound shown by formula A is prepared by referring to the method in patent application CN201911379293.8 (see the specific examples for details).

[0065] A pharmaceutical composition comprising a crystal form of a compound of formula A as described above or a solvate thereof (e.g., one or more 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) and / or crystal form III of substance A as described above, and a pharmaceutical excipient. Wherein, the dose of the crystal form can be a therapeutically effective amount.

[0066] The pharmaceutical excipients mentioned above can be those widely used in the field of drug production. Excipients are mainly used to provide a safe, stable, and functional pharmaceutical composition, and can also provide methods to enable the active ingredient to dissolve at a desired rate after the subject receives the administration, or to promote the effective absorption of the active ingredient after the subject receives the composition. The pharmaceutical excipients can be inert fillers, or provide certain functions, such as stabilizing the overall pH value of the composition or preventing the degradation of the active ingredient of the composition. The pharmaceutical excipients can include one or more of the following excipients: binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, adhesives, disintegrants, lubricants, anti-adhesion agents, glidants, wetting agents, gelling agents, absorption retardants, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.

[0067] 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, buffering substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures 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, polyvinylpyrrolidone, polyacrylates, waxes, polyethylene - polypropylene - block polymers, lanolin, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; gum powder; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; diol compounds such as propylene glycol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic salts; Ringer's solution; ethanol, phosphate buffer solution, and other non-toxic suitable lubricants such as sodium lauryl sulfate and magnesium stearate, colorants, release agents, coating materials, sweeteners, flavoring agents, and fragrances, preservatives, and antioxidants.

[0068] The pharmaceutical composition of the present invention can be prepared by any method known to those skilled in the art according to the disclosed content. For example, conventional mixing, dissolving, granulating, emulsifying, grinding, encapsulating, embedding, or lyophilization processes.

[0069] The present invention provides the use of a crystal form of a compound of formula A or a solvate thereof as described above (for example, one or more 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), and crystal form III of substance A as described above in the preparation of a P2X3 inhibitor. In the said use, the P2X3 inhibitor can be used in mammalian organisms; it can also be used in vitro, mainly for experimental purposes, for example: provided for comparison as a standard sample or a control sample, or made into a kit according to conventional methods in the art to provide a rapid detection of the inhibitory effect of P2X3.

[0070] The present invention also provides the use of a crystal form of a compound of formula A or a solvate thereof as described above (for example, one or more 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), and crystal form III of substance A as described above in the preparation of a drug; the drug is a drug for protecting, treating, or alleviating at least partially P2X3-mediated or activity-related diseases in animals; or, the drug is a drug for treating pain, urinary tract diseases, or respiratory diseases.

[0071] The present invention provides the use of a crystal form of a compound of formula A or a solvate thereof as described above (for example, one or more 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), crystal form III of substance A as described above, or the drug composition as described above in protecting, treating, or alleviating at least partially P2X3-mediated or activity-related diseases in animals (such as humans). The said diseases include but are not limited to respiratory diseases, cough, chronic cough, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, pain, urinary incontinence, autoimmune diseases, overactive bladder, dysuria, inflammation, Alzheimer's disease, Parkinson's disease, sleep disorders, epilepsy, mental diseases, arthritis, neurodegeneration, traumatic brain injury, myocardial infarction, rheumatoid arthritis, stroke, thrombosis, atherosclerosis, colon syndrome, inflammatory bowel disease, digestive tract diseases; gastrointestinal dysfunction, respiratory failure, sexual dysfunction, cardiovascular diseases, heart failure, hypertension, urinary incontinence, cystitis, arthritis, endometriosis, blood diseases, musculoskeletal and connective tissue developmental disorders, systemic disorders.

[0072] In some embodiments, the said diseases include pain; the pain includes but is not limited to: inflammatory pain, surgical pain, visceral pain, toothache, premenstrual pain, central pain, pain caused by burns, migraine, or cluster headache.

[0073] In some embodiments, the disease includes urinary system diseases; the urinary tract diseases include: urinary incontinence, overactive bladder, dysuria, cystitis;

[0074] In some embodiments, the disease includes respiratory system diseases, and the respiratory system diseases include but are not limited to: respiratory disorders, including idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, bronchospasm, or chronic cough.

[0075] The above-mentioned pharmaceutical composition is characterized in that by administering the pharmaceutical composition, the side effect of taste disorder related to treatment is reduced.

[0076] On the basis of conforming to the common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

[0077] The reagents and raw materials used in the present invention are all commercially available.

[0078] The positive and progressive effects of the present invention are as follows:

[0079] 1. In the prior art, there is no report on the crystal forms of this heterocyclic compound. The present application discovers various new crystal forms of this compound for the first time. Through a large number of experiments and screenings, the present invention prepares 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 uses them as candidate objects.

[0080] 2. Some of the 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, avoid changes in bioavailability and drug efficacy, and can be developed into dosage forms suitable for clinical use, having strong economic value.

[0081] 3. The present invention also provides a preparation method for the crystal forms of 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 at the same time. The operation is simple and has high reproducibility. The solvent is not easy to remain, is environmentally friendly, and is suitable for different large-scale productions. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] Figure 1 is the XRPD pattern of crystal form III;

[0083] Figure 2 is the superimposed TGA / DSC pattern of crystal form III;

[0084] Figure 3 is the superimposed XRPD pattern of crystal form III before and after heating to dehydration;

[0085] Figure 4 is the DVS pattern of crystal form III;

[0086] Figure 5 XRPD overlay diagrams before and after DVS testing for polymorph III;

[0087] Figure 6 PLM diagram for polymorph III;

[0088] Figure 7 XRPD diagram for polymorph V;

[0089] Figure 8 TGA / DSC overlay diagram for polymorph V;

[0090] Figure 9 XRPD overlay diagrams before and after DVS testing for polymorph V;

[0091] Figure 10 DVS diagram for polymorph V;

[0092] Figure 11 PLM diagram for polymorph V;

[0093] Figure 12 XRPD diagram for polymorph I;

[0094] Figure 13 TGA / DSC overlay diagram for polymorph I;

[0095] Figure 14 DVS diagram for polymorph I;

[0096] Figure 15 XPRD diagram for hygroscopic polymorph I;

[0097] Figure 16 XRPD overlay diagrams before and after DVS testing for polymorph I;

[0098] Figure 17 PLM diagram for polymorph I;

[0099] Figure 18 XRPD diagram for polymorph II;

[0100] Figure 19 HNMR spectrum of residual MTBE for polymorph II;

[0101] Figure 20 TGA / DSC overlay diagram for polymorph II;

[0102] Figure 21 PLM diagram for polymorph II;

[0103] Figure 22 XRPD diagram for polymorph IV;

[0104] Figure 23 TGA / DSC overlay diagram for polymorph IV;

[0105] Figure 24 XRPD overlay diagrams before and after dehydration of polymorph IV;

[0106] Figure 25 PLM diagram of polymorph IV;

[0107] Figure 26 XRPD diagram of polymorph VI;

[0108] Figure 27 XRPD overlay diagram of polymorph VI;

[0109] Figure 28 XRPD diagram of polymorph VII;

[0110] Figure 29 Residual ethylene glycol HNMR spectrum of polymorph VII;

[0111] Figure 30 TGA / DSC overlay diagram of polymorph VII;

[0112] Figure 31 XRPD diagram of polymorph VIII;

[0113] Figure 32 TGA / DSC overlay diagram of polymorph VIII;

[0114] Figure 33 HNMR spectra of polymorph VIII and the raw material;

[0115] Figure 34 XRPD overlay diagram of polymorph VIII and the sample before drying;

[0116] Figure 35 XRPD diagram of polymorph IX;

[0117] Figure 36 TGA / DSC overlay diagram of polymorph IX;

[0118] Figure 37 XRPD overlay diagram of polymorph III obtained from the amorphous sample;

[0119] Figure 38 DSC&TGA overlay diagram of polymorph III obtained from the amorphous sample;

[0120] Figure 39 XRPD overlay diagram of polymorph III slurried in mixed solvents with different volume ratios;

[0121] Figure 40 XRPD overlay diagram of 200mg-scale polymorph III;

[0122] Figure 41 PLM diagram of polymorph III at the 200 mg level;

[0123] Figure 42 XRPD overlay diagram of polymorph III (3 g);

[0124] Figure 43 XRPD overlay diagram of polymorph III before and after slurrying in water at room temperature;

[0125] Figure 44 Water activity experimental result diagram of polymorph I;

[0126] Figure 45 Water activity experimental result diagram of polymorph V;

[0127] Figure 46 XRPD overlay diagram of the competitive slurrying experiment of polymorph I and polymorph V;

[0128] Figure 47 Stability data result diagram of polymorph I and V in the solid state;

[0129] Figure 48 XRPD overlay diagram of the stability test sample of polymorph I;

[0130] Figure 49 XRPD overlay diagram of the stability test sample of polymorph III;

[0131] Figure 50 XRPD overlay diagram of polymorph I before and after grinding test;

[0132] Figure 51 XRPD overlay diagram of polymorph I before and after pressure test;

[0133] Figure 52 XRPD overlay diagram of polymorph III before and after grinding test;

[0134] Figure 53 XRPD overlay diagram of polymorph III before and after pressure test;

[0135] Figure 54 Solubility data diagram of polymorph I, III and V;

[0136] Figure 55 XRPD overlay diagram of the remaining solid in the solubility test of polymorph I;

[0137] Figure 56 XRPD overlay diagram of the remaining solid in the solubility test of polymorph V;

[0138] Figure 57 XRPD overlay diagram of the remaining solid in the solubility test of polymorph III;

[0139] Figure 58 PLM diagram of the amorphous sample;

[0140] Figure 59 DSC & TGA superimposed diagram of the amorphous sample;

[0141] Figure 60 For polymorph IX 1 HNMR spectrum;

[0142] Figure 61 XRPD superimposed diagram of the sample for the stability test of polymorph V. Detailed implementation manners

[0143] The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the described examples. For the experimental methods without specific conditions in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0144] The following further describes the present invention in detail with specific examples. It should be understood that these examples are used to illustrate the basic principles, main features and advantages of the present invention, and the present invention is not limited by the following examples. The implementation conditions adopted in the examples can be further adjusted according to specific requirements, and the implementation conditions not specified are usually the conditions in conventional experiments.

[0145] In the following examples, the experimental methods are generally 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 are obtained by commercially available methods and the purity reaches 99%.

[0146] The abbreviations used in the present invention are explained as follows:

[0147] XPRD - X-ray powder diffraction

[0148] TGA - Thermogravimetric analysis

[0149] DSC - Differential scanning calorimetry

[0150] DVS - Dynamic vapor sorption analysis

[0151] PLM - Polarizing microscopy analysis

[0152] The test conditions are as follows:

[0153] XRPD

[0154] The solid is characterized using an X-ray powder diffractometer (Bruker D8 advance or D2 Phase).

[0155] Scanning angle: 3°(2θ) - 40°(2θ).

[0156] Step size: 0.02° (2θ).

[0157] Scanning speed: 0.3 sec / step (D8), 0.2 sec / step (D2).

[0158] Tube voltage: 40 KV (D8), 30 KV (D2).

[0159] Tube current: 40 mA (D8), 10 mA (D2).

[0160] Rotation: On.

[0161] Sample pan: Zero-background sample pan.

[0162] TGA

[0163] Thermogravimetric analysis of solid samples was performed using a TA Instrument thermogravimetric analyzer Q500 or Discovery TGA55. After balancing the sample pan, the sample was suspended on a hanging wire and lifted into the furnace. After stabilization, the sample was heated to different end temperatures at a rate of 10 °C / min.

[0164] DSC

[0165] DSC analysis of solid samples was performed using a TA Instrument differential scanning calorimeter Q200 and Discovery DSC 250. The sample was weighed and the value was recorded, and then the sample was placed in the sample chamber. The sample was heated from 25 °C to different end temperatures at a rate of 10 °C / min.

[0166] DVS

[0167] DVS analysis of solids was performed using an IGAsorp dynamic water sorption instrument.

[0168] Temperature: 25 °C.

[0169] Gas flow: 250 mL / min.

[0170] Scanning cycle: 2.

[0171] Shortest test time: 30 min.

[0172] Longest test time: 2 h.

[0173] Waiting for equilibrium: 98%.

[0174] PLM

[0175] The sample was observed using a Nikon Eclipse LV100N POL polarizing microscope.

[0176] Example 1: Preparation of Compound of Formula A

[0177]

[0178] Step 1. Preparation of tert-butyl (S)-2-((2-(4-bromo-2,6-difluorophenyl)-7-chloroimidazo[1,2-a]pyridin-3-yl)methyl)morpholine-4-carboxylate

[0179]

[0180] In a 100 mL round-bottom flask, successively add tert-butyl (S)-2-ethynylmorpholine-4-carboxylate (3.1 g, 1.0 eq, Intermediate 1-4), 4-bromo-2,6-difluorobenzaldehyde (2.76 g, 1.0 eq, Compound 172-1), 4-chloropyridin-2-amine (1.61 g, 1.0 eq, Compound 172-2), CuCl (0.37 g, 0.3 eq), Cu(OTf) 2 (1.36 g, 0.3 eq), isopropanol (50 mL), displace with nitrogen three times, heat in an oil bath at 80 °C overnight, and detect by TLC until the raw material Compound 172-2 disappears. Rotavaporize the isopropanol, extract successively with EA and ammonia water, take the EA phase, wash successively with saturated brine and citric acid, dry over anhydrous sodium sulfate, rotavaporize and column chromatograph to obtain Intermediate 172-3, white solid (3.0 g, purity 78%). LC-MS: [M+H] + = 542.2.

[0181] Step 2. Preparation of (S)-2-((2-(4-bromo-2,6-difluorophenyl)-7-chloroimidazo[1,2-a]pyridin-3-yl)methyl)morpholine

[0182]

[0183] Dissolve Intermediate 172-3 (2.67 g) in dichloromethane (24 mL), then add dioxane hydrochloride (24 mL), stir at room temperature for 1.0 h, and detect by LC-MS until the reaction is complete. Rotavaporize the reaction solution, add water (15 mL) and dichloromethane (15 mL) to the reaction solution, extract and discard the aqueous phase after extraction, adjust the pH of the aqueous phase to weakly alkaline (pH = 8 - 9) with aqueous sodium bicarbonate solution. Separate the dichloromethane phase, and extract the aqueous phase with dichloromethane (10 mL × 2). Combine the dichloromethane phases, wash with saturated brine, and rotavaporize to obtain Intermediate 172-4, white solid (1.70 g, purity 88.6%). LC-MS: [M+H] + = 442.1.

[0184] Step 3. Preparation of methyl (S)-2-((2-(4-bromo-2,6-difluorophenyl)-7-chloroimidazo[1,2-a]pyridin-3-yl)methyl)morpholine-4-carboxylate

[0185]

[0186] Dissolve intermediate 172-4 (1.4 g, 1.0 eq) in dichloromethane (10 mL), add triethylamine (480 mg, 1.5 eq), and dropwise add methyl chloroacetate (388 mg, 1.3 eq). After reacting for 1.0 h, LC-MS shows the formation of the product. After the reaction, add water (10 mL), stir for 30 min, then separate the layers to obtain the dichloromethane phase. The aqueous phase is extracted with dichloromethane (10 mL × 2). Combine the dichloromethane phases, wash with saturated sodium chloride, dry over anhydrous sodium sulfate, evaporate to dryness, and purify by column chromatography to obtain intermediate 172-5, a white solid (1.01 g, purity 93.02%). LC-MS: [M+H] + = 499.8.

[0187] Step 4 Preparation of methyl (S)-2-((2-(4-(benzylthio)-2,6-difluorophenyl)-7-chloroimidazo[1,2-a]pyridin-3-yl)methyl)morpholine-4-carboxylate

[0188]

[0189] Dissolve intermediate 172-5 (0.73 g, 1.0 eq) in dioxane (4 mL), add BnSH (0.24 g, 1.3 eq), Pd2(dba)3 (0.04 g, 0.03 eq), Xantphos (0.04 g, 0.05 eq), DIEA (0.60 g, 3.0 eq), and displace N 2 three times. React overnight at 80 °C. Monitor by LCMS until the raw materials disappear completely. Add dichloromethane (10 mL) and water (10 mL) to the reaction solution, separate the layers to obtain the dichloromethane phase. The aqueous phase is extracted with dichloromethane (10 mL × 2). Combine the dichloromethane phases, wash with saturated sodium chloride, dry over anhydrous sodium sulfate, evaporate to dryness, and purify by column chromatography to obtain intermediate 172-6, a white solid (0.82 g, purity 91.53%). LC-MS: [M+H] + = 544.2.

[0190] Step 5 Preparation of methyl (S)-2-((7-chloro-2-(4-(chlorosulfonyl)-2,6-difluorophenyl)imidazo[1,2-a]pyridin-3-yl)methyl)morpholine-4-carboxylate

[0191]

[0192] Add intermediate 172-6 (510 mg) to a reaction flask, dissolve it in acetonitrile (3 mL), then add glacial acetic acid (281 mg, 5.0 eq), and dropwise add SO2Cl 2(506 mg, 4.0 eq). The reaction was carried out at 0 °C for 1 h. LCMS showed that the raw material disappeared and intermediate 172-7 was formed. This reaction was not treated, and the reaction solution was directly used for the next step.

[0193] Step 6 Preparation of Methyl (S)-2-((7-chloro-2-(2,6-difluoro-4-sulfamoylphenyl)imidazo[1,2-a]pyridin-3-yl)methyl)morpholine-4-carboxylate

[0194]

[0195] At 0 °C, ammonia water (2 mL) diluted with acetonitrile (1 mL) was added dropwise to the above reaction solution, and the reaction was carried out at room temperature for 0.5 h. LCMS showed that the raw material disappeared completely and the target product was formed. The reaction solution was extracted twice with water and ethyl acetate, washed with brine solution, dried over anhydrous sodium sulfate, concentrated, and separated and purified using a C18 chromatographic column (water / acetonitrile, RRt = 22.5 min). Amorphous compound A, a white solid, was obtained. (185 mg, purity 99.74%). LC-MS: [M+H] + = 501.1.

[0196] 1 H NMR (400 MHz, 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).

[0197] Example 2: Post-treatment of Raw Materials

[0198] A total of 11.1 g of compound A was prepared with reference to Example 1, 20 mL of acetone was added, and the mixture was refluxed at 65 °C (under nitrogen protection) for 2.0 h. The acetone was directly evaporated to dryness, and vacuum dried at 40 °C for 12 h. NMR showed that about 1% of acetone remained. It was vacuum dried again at 80 °C for 12 h, and NMR still showed acetone residue. 5.3 g of it was taken and vacuum dried again at 80 °C for 12 h, and NMR still showed acetone residue. This batch of products was added with acetonitrile (16 mL), refluxed at 85 °C (under nitrogen protection) for 2.0 h, the acetonitrile was directly evaporated to dryness, and then vacuum dried at 80 °C for 12 h. NMR showed that it was qualified without solvent residue, and 5.2 g was put into storage. The product purity was 99.29%, and it was in the form of a white powder.

[0199] The PLM diagram and XRPD results show that the raw material is irregularly shaped crystals with a size of 10 - 50 μm, having a general crystallinity and being in an amorphous state. As Figure 37 , the DSC diagram shows that the raw material has two connected endothermic peaks at around 150 - 180 °C, with the peak temperatures being 164 ± 2 °C and 173 °C ± 2 °C respectively. As Figure 38 , the TGA diagram shows that the sample has basically no weight loss before 230 °C.

[0200] Example 3: Preparation and Characterization of Amorphous Form of Compound A

[0201] 3.1 Dissolve Compound A in a certain amount of THF and concentrate it to dryness under reduced pressure to obtain an amorphous sample. For XRPD characterization, see the appendix Figure 37 .

[0202] Example 4: Preparation and Characterization of Crystal Form I of Compound A

[0203] 4.1 Select 9 solvents: EtOH, IPA, NBA, MEK, ACN, acetone, EA, IPAc, Hept, and conduct suspension milling at room temperature with a milling concentration of 60 mg / mL to obtain Crystal Form I;

[0204] 4.2 Select 6 solvents: IPA, NBA, MEK, acetone, Tol, EA, and conduct suspension milling at 50 °C with a milling concentration of 100 mg / mL to obtain Crystal Form I;

[0205] 4.3 Select the solvent: IPAc, conduct suspension milling at 50 °C with a milling concentration of 50 mg / mL to obtain Crystal Form I;

[0206] 4.4 Slowly cool and crystallize in methanol and ethanol, with the cooling temperature ranging from 50 °C to RT, and Crystal Form I can be obtained;

[0207] 4.5 Use tetrahydrofuran as a good solvent to dissolve the sample to form a sample solution with a certain concentration, and slowly add Tol, Hept, and water as anti-solvents to the sample solution respectively, with a volume ratio of 1:10 for all, to increase the supersaturation and thus precipitate solids to obtain Crystal Form I;

[0208] 4.6 Select EtOH as the solvent for evaporation crystallization experiment to obtain Crystal Form I.

[0209] The XRPD of Crystal Form I expressed in 2θ angle is as Figure 12 shown. In the DSC diagram of the said Crystal Form I, there is an endothermic peak at 152 °C, and the melting enthalpy is 44 ± 2 J / g. In the TGA diagram of the said Crystal Form I, there is no weight loss in the temperature range of RT - 230 °C. Its TGA and DSC diagrams are as Figure 13As shown. Combining the DSC and TGA diagrams, it can be known that the product is a non-crystalline form. The DVS diagram of the crystalline form I is as Figure 14 shown. When the relative humidity is greater than 40%, the weight of the crystalline form I increases sharply. When the relative humidity drops to 40%, all the adsorbed water is discharged. In the XRPD diagram of the hygroscopic crystalline form I, the crystalline form I undergoes a crystal transformation upon moisture absorption in a high-humidity environment and transforms into the hydrate crystalline form IV. After drying it in a vacuum drying oven at 30°C, it transforms back into the initial crystalline form I. Its XRPD diagram after moisture absorption is as Figure 15 shown. It can be seen that, combining the DVS test results ( Figure 14 ), when the environmental relative humidity is higher than 40%, the crystalline form I rapidly absorbs moisture and transforms into the hydrate; when the relative humidity is lower than 50%, the adsorbed water is rapidly removed and transforms into the crystalline form I. That is, the transformation between the crystalline form I and IV is reversible. The said crystalline form I exhibits hygroscopicity (6.8%, 80% RH). After the DVS test of the crystalline form I, the crystal form remains unchanged. Its XRPD diagrams before and after the DVS test are as Figure 16 shown. In the PLM diagram of the crystalline form I, the crystal form is irregular crystals of about 5 μm. Its PLM is as Figure 17 shown.

[0210] Example 5: Preparation and Characterization of Crystal Form II of Compound A

[0211] 5.1 Suspension and pulping in MTBE at room temperature to obtain crystal form II:

[0212] 5.2 Select the solvent: MTBE, suspension and pulping at 50°C, and the pulping concentration is 100 mg / mL to obtain crystal form II;

[0213] 5.3 Use tetrahydrofuran as a good solvent to dissolve the sample to form a sample solution of a certain concentration, and slowly add MTBE as an anti-solvent to the sample solution, with a volume ratio of 1:10, to increase the supersaturation, so that the solid precipitates to obtain crystal form II.

[0214] The X-ray powder diffraction pattern of crystal form II expressed in 2θ angle is as Figure 18 shown. In the nuclear magnetic resonance hydrogen spectrum of the crystal form II, there are solvent residue signals of MTBE at chemical shifts of 1.10 and 3.08; their molar ratio is 0.39. Its HNMR spectrum is as Figure 19 shown. In the TGA diagram of the crystal form II, the weight loss is 3.5% in the temperature range of 100 - 160°C and 2.9% in the range of 160 - 200°C. In the DSC diagram of the crystal form II, there are two adjacent endothermic peaks. Its TGA and DSC diagrams are as Figure 20 shown. Compared with Figure 19The residual MTBE proton NMR spectrum shown is equivalent to approximately 6.0%, indicating that the product is an MTBE solvate. In the PLM of the crystalline form II, the crystal form is irregular crystals of approximately 2 μm, and its polarized light micrograph is as shown in Figure 21 shown.

[0215] Example 6: Preparation and Characterization of Crystalline Form III of Compound A

[0216] 6.1 Select the solvent: water, suspend and grind at 50 °C, and the grinding concentration is 50 mg / mL to obtain crystalline form III;

[0217] 6.2 Prepared by suspending and grinding the amorphous sample in water at 40 °C to obtain crystalline form III; specifically: dissolve the sample in methanol, filter to obtain the sample solution, and then rotary evaporate to obtain 250 mg of the amorphous sample. Crystalline form III was obtained after suspending and grinding in water for 20 hours. Through XRPD, DSC, and TGA characterization tests, it was determined that pure crystalline form III was successfully prepared and used as the seed crystal for subsequent scale-up experiments.

[0218] 6.3 Prepare crystalline form III by solvent-antisolvent precipitation crystallization and adding seed crystals to induce nucleation. Specifically: first dissolve the sample in the mixed solvent in a specific ratio shown in Table 1, filter to obtain a saturated solution, add crystalline form III as the seed crystal to the above saturated solution, and then slowly add water as the anti-solvent to precipitate solids and induce crystallization. XRPD tests were performed on the solid samples obtained by filtration. The experimental results are shown in Table 1, and the results show that crystalline form III is unstable in the above system and transforms into a solvate or an unstable hydrate. Considering from the perspectives of solubility and solvent residue limit, DMSO is a third-class solvent and its solubility is higher than 100 mg / mL, so DMSO was selected for the next step of research.

[0219] Table 1 Experimental Results of Solvent-Antisolvent Precipitation Crystallization

[0220]

[0221] 6.4 As can be seen from the experimental results in 6.3 above, it is difficult to obtain the target crystalline form III by the crystallization method of adding the anti-solvent to the sample solution of the good solvent. Therefore, it is considered to adopt the method of preparing the target crystal form by adding the sample dissolved in the organic solvent dropwise to the aqueous solution dispersed with seed crystals for the crystallization of compound A. The specific operation method is: first dissolve the sample in DMSO and filter to obtain the DMSO solution, and then add it dropwise to the aqueous solution dispersed with seed crystals in a slow manner. The precipitated solid is transformed into the target crystalline form III under the induction of the seed crystal. As shown in Table 2 and Figure 1As shown, the stability of polymorph III in DMSO / water mixed solvent systems with different ratios was investigated separately to determine the appropriate solvent / antisolvent ratio to ensure the stable existence of polymorph III in this system. The experimental results show that polymorph III can stably exist in DMSO / water mixed solvents with a water content higher than 60% (volume percentage) at room temperature without crystal transformation. When the water content is lower than 60%, polymorph III transforms into the solvate of DMSO (polymorph IX).

[0222] Table 2 Stability results of polymorph III in mixed solvents with different volume ratios

[0223]

[0224] From the perspective of the yield of the target product, the theoretical yields of polymorph III at the above ratios were calculated separately 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 polymorph III in these three ratios of solvents is relatively small, the theoretical yields are not very different, and the yields are all above 99%.

[0225] Table 3 Theoretical yields of polymorph III in DMSO / water mixed solvents with different ratios

[0226] Solvent Solubility (mg / mL, RT) Theoretical yield (%) DMSO >200 / DMSO / Water 1:2 ～0.5 99.3％ DMSO / Water 1:3 ～0.28 99.4％ DMSO / Water 1:4 <0.25 99.4％

[0227] 6.5 Scale-up of polymorph III (200 mg)

[0228] At room temperature, the sample was dissolved in DMSO and filtered to obtain a DMSO solution, which was slowly added dropwise to water containing polymorph III seeds at a rate of 25 μL / min. As the amount of the DMSO solution added increased, the precipitation of solids gradually increased (DMSO / water 1:2, volume ratio). Confirmed by XRPD ( Figure 2 ), the freshly precipitated solid sample was almost amorphous. With the extension of the reaction time and the induction of seeds, after magnetic stirring at room temperature for 16 hours, it was all transformed into polymorph III. It was experimentally observed that after the reaction ended, the stirring was stopped, and the solids quickly settled to the bottom, and the upper liquid was basically transparent. Observed by PLM ( Figure 6 ), it was found that the obtained polymorph III sample itself was crystals about 2 μm in size, but easily agglomerated into large particles of 20 - 50 μm. Therefore, once the stirring was stopped, the solids settled due to agglomeration. Thus, this polymorph was easy to filter. In summary, a 200 mg sample of polymorph III was successfully prepared by the anti-solvent precipitation crystallization method, with a yield of 93.0%, and 1 HNMR showed that the DMSO residue was 0.1% wt, meeting the residue limit (<0.5% wt).

[0229] 6.6 g-Scale Preparation of Polymorph III

[0230] Reducing the volume of DMSO, increasing the reaction temperature, and allowing the temperature to drop to room temperature after the polymorph conversion and appropriately prolonging the slurrying experiment are beneficial to reducing the residual solvent and increasing the yield. Based on the above considerations, when preparing 3 g of polymorph III on a larger scale, a 1:4 DMSO / water system at 40 °C was used, the amount of seed added was 2% wt, and the dropping rate of the DMSO (5V) solution was controlled at 25 μL / min. To simulate scale-up production, an anchor agitator paddle was used at a rotation speed of 150 rpm. The reaction system was slurried at 40 °C for 22 h, and the XRPD results showed that the solid had been converted to polymorph III. To further increase the yield, the reaction temperature was lowered to room temperature, and the slurry was continued at room temperature. After 22 h, the solid sample was filtered and slurried in water to remove the DMSO residual solvent. As Figure 42 shown, 3 g of polymorph III was successfully prepared on a larger scale, with a yield of 96.7%. As Figure 43 shown, 0.7% of the DMSO residual solvent could be removed by slurrying at room temperature in water, and the polymorph remained unchanged.

[0231] Considering issues such as the production of this compound, the amount of solvent used, the residual solvent, and the product yield, the crystallization process temperature of polymorph III was finally 40 °C, the reaction system was 1:4 DMSO / water, and the solvent volume was 25V. In summary, polymorph III can be prepared on a gram scale with a yield as high as 97%.

[0232] The XRPD pattern of polymorph III expressed in 2θ angle is as Figure 1 shown. In the TGA pattern of the described polymorph III, the weight loss gradient in the RT-100 °C range is 1.5%, and the "%" is weight percentage. In the DSC pattern of the described polymorph III, the first endothermic peak is for the removal of 0.4 water molecules, and the second endothermic peak is attributed to the endothermic peak of melting after the sample is dehydrated. Its TGA and DSC patterns are as Figure 2 shown. After the dehydration of the described polymorph III, it quickly absorbs moisture in the ambient humidity and reverts to polymorph III again. The XRPD patterns before and after heating to dehydration are as Figure 3 shown. The DVS pattern of the described polymorph III shows that the sample has a certain hygroscopicity, and the water content changes little in a relatively wide humidity range. Its DVS pattern is as Figure 4 shown. In the XPRD patterns of the described polymorph III before and after DVS testing, there is no obvious change in XRPD before and after DVS testing. Its XPRD pattern is as Figure 5 shown. In the PLM of the described polymorph III, the polymorph is irregular crystals of about 2 μm, aggregated into 20-50 μm. Its PLM pattern is as Figure 6 shown.

[0233] Example 7: Preparation and Characterization of Crystal Form IV of Compound A

[0234] 7.1 Absorb moisture from the crystal form I product under high humidity conditions to obtain crystal form IV;

[0235] 7.2 Pulp the crystal form I product in 50 - 95% water / acetone (V / V) to obtain crystal form IV.

[0236] The XRPD pattern of IV expressed in 2θ angle is basically as Figure 22 shown. In the TGA pattern of the crystal form IV, the weight loss is 1.2% in the temperature range of RT - 60°C. In the DSC pattern of the crystal form IV, there are two endothermic peaks. The first broad endothermic peak is presumably caused by dehydration, and the subsequent endothermic peak is the melting peak. Its TGA and DSC patterns are as Figure 23 shown. Combining the DSC and TGA diagrams, it can be known that this product is a hydrate crystal form with about 0.34 water molecules. The crystal form IV is only stable in a high humidity environment. After dehydration, it quickly absorbs moisture in the ambient humidity and reverts to crystal form I again. The XRPD patterns before and after heating to dehydration are as Figure 24 shown. In the PLM of the crystal form IV, the crystal form is about 5μm irregular crystals, and its PLM diagram is as Figure 25 shown.

[0237] Example 8: Preparation and Characterization of Crystal Form V of Compound A

[0238] 8.1 Select the solvent: water, suspend and pulp at 50°C, and the pulping concentration is 50mg / mL to obtain crystal form V;

[0239] 8.2 Select the solvent: ACN, suspend and pulp at 50°C, and the pulping concentration is 3.0mg / mL to obtain crystal form V;

[0240] 8.3 Select MeOH as the solvent for evaporation crystallization test to obtain crystal form V.

[0241] The XRPD pattern of crystal form V expressed in 2θ angle is as Figure 7 shown. In the TGA pattern of the crystal form V, there is no weight loss in the temperature range of RT - 230°C. In the DSC pattern of the crystal form II, there is an endothermic peak at 166°C ± 2°C, and the melting enthalpy is 70 ± 2J / g. Its TGA and DSC patterns are as Figure 8 shown. Combining the DSC and TGA diagrams, it can be known that this product is an anhydrous crystal form. The DVS pattern of the crystal form V shows that the sample has a certain hygroscopicity (0.7%, 80% RH), and its DVS pattern is as Figure 10 shown. In the XPRD patterns of the crystal form V before and after DVS testing, there is no obvious change in XRPD before and after DVS testing, and its XPRD pattern is as Figure 9As shown. In the PLM of polymorph V, the polymorph is about 5 μm irregular crystals, and its PLM diagram is as Figure 11 shown.

[0242] Example 9: Preparation and Characterization of Polymorph VI of Compound of Formula A

[0243] 9.1 Polymorph VI was obtained by slurrying polymorph I or polymorph V in a water / acetone mixed solvent with a water content of 10% (volume ratio) at 60 °C for 60 °C.

[0244] The X-ray powder diffraction pattern of polymorph VI expressed in 2θ angle is as Figure 26 shown. In the superimposed XRPD pattern of polymorph VI, the polymorph sample transformed into polymorph I after being placed at ambient humidity (35% RH) for a few minutes. Its superimposed XRPD pattern is as Figure 27 shown. This indicates that polymorph VI may be a very unstable hydrate.

[0245] Example 10: Preparation and Characterization of Polymorph VII of Compound of Formula A

[0246] 10.1 Solvent selected: ethylene glycol, slurried at 90 °C, and the slurrying concentration was 320 mg / mL to obtain polymorph VII;

[0247] 10.2 Slowly cooled and crystallized in ethylene glycol, and the cooling temperature was from 50 °C to RT to obtain polymorph VII.

[0248] The XRPD of polymorph VII expressed in 2θ angle is basically as Figure 28 shown. In the 1 1H NMR spectrum of polymorph VII, there are residual solvents of ethylene glycol at chemical shifts δ of 3.39 and 4.44, and its 1 1H NMR spectrum is as Figure 29 shown. In the TGA spectrum of polymorph VII, there is a weight loss of 25.7% in the range of RT - 120 °C. In the DSC spectrum of polymorph VII, there are two broad endothermic peaks. The first endothermic peak is presumably caused by desolvation. Its TGA and DSC spectra are as Figure 30 shown. Combining the DSC and TGA diagrams, it can be seen that this product is a solvate containing 2.79 molecules of ethylene glycol.

[0249] Example 11: Preparation and Characterization of Polymorph VIII of Compound of Formula A

[0250] 11.1 Using polymorph III as a seed crystal, water was added dropwise as an anti-solvent to a saturated 50% THF / aqueous solution at 40 °C, and an oiliness occurred. After cooling to room temperature, it was further slurried to obtain polymorph VIII (wet filter cake)

[0251] The XRPD diagram of polymorph VIII expressed in 2θ angle is basically asFigure 31 As shown. In the TGA spectrum of the crystal form VIII, the weight loss is 5.7% in the temperature range of RT-160°C. In 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, the crystal form VIII is a solvate, and its TGA and DSC spectra are as follows Figure 32 As shown. The crystal form VIII 1 In the HNMR spectrum, the chemical shifts δ at 1.76 and 3.60 show THF residues. 1 HNMR spectrum Figure 33 As shown. The crystal form VIII contains 0.42 molecules of THF. The crystal form VIII is unstable and transforms into crystal form I after dehydration (vacuum drying at 40°C for 3 hours). Its XRPD spectrum before and after heating is as follows Figure 34 shown.

[0252] Example 12: Preparation and characterization of Form IX of the compound of formula A

[0253] 12.1 Form IX is prepared by slurrying Form III in a 50% DMSO / water saturated solution at 40°C.

[0254] In the TGA spectrum of the crystal form IX, the weight loss in the temperature range of RT-160°C is 18.23%. In the DSC spectrum of the crystal form IX, there is a corresponding endothermic peak corresponding to the TGA weight loss on the DSC spectrum. The TGA and DSC spectra are as follows: Figure 36 As shown. The crystal form IX 1 In the HNMR spectrum, the chemical shift δ at 2.68 shows that there is residual DMSO solvent. 1 HNMR spectrum Figure 60 As shown. Combining the DSC and TGA graphs, it can be seen that the product is a DMSO solvate. Combining the DSC and TGA graphs, it can be seen that the product is a DMSO solvate.

[0255] Example 13: Crystal form interconversion study

[0256] A total of nine different crystalline forms were obtained through screening experiments, including two anhydrous crystalline forms (Form I and Form V), three solvates (Form II, Form VII, Form IX, Form VIII), and four hydrates (Form III, Form IV, Form VI, Form VIII).

[0257] 13.1 Transformation between Form I and Form IV

[0258] Polymorph I can be repeatedly obtained by different crystallization methods. However, Polymorph I is a hygroscopic form and converts to the hydrate Polymorph IV when the relative humidity is higher than 40% RH. Therefore, there is a risk of polymorph conversion during the production and scale-up of APIs in Polymorph I.

[0259] 13.2 Conversion between Other Hydrate Polymorphs and Polymorph I

[0260] Except for Polymorph III, the other hydrates are unstable and convert to Polymorph I after dehydration.

[0261] Polymorph III (a hemihydrate) has a low risk of polymorph conversion.

[0262] Polymorph IV (a hydrate) converts to Polymorph I after dehydration;

[0263] Polymorph VI (a hydrate) is extremely unstable and quickly converts to Polymorph I under ambient humidity.

[0264] 13.3 Study on the Conversion between Anhydrous Polymorphs and Hydrates

[0265] Perform water activity experiments. Pulp Polymorph I and V (both anhydrous polymorphs) in acetone / water mixed solvents with different water contents at room temperature and 60 °C for three days. After three days, filter to obtain solid samples and test their XRPD. The experimental results are shown in Table 4. As Figure 44 and 45 shown, whether at room temperature or high temperature, Polymorph I and V both convert to the hydrate Polymorph IV in the acetone / water system with a water content of 50 - 90%. When the water content is 10%, Polymorph I or V both convert to another hydrate (Polymorph VI) at high temperature. It is found that this hydrate is extremely unstable under ambient humidity (35% RH) and converts to Polymorph I after being placed for a few minutes. At room temperature, Polymorph V converts to Polymorph I in this solvent system.

[0266] Table 4 Results of Water Activity Experiments for Polymorph I and Polymorph V

[0267]

[0268] N / A indicates: Unable to obtain

[0269] 13.4 Competitive Pulping Experiments between Anhydrous Polymorphs

[0270] Perform competitive pulping experiments: Weigh equal amounts of Polymorph I and V, mix them evenly, and test their XRPD. The result is used as T 0 in the competitive pulping experiment. Pulp the mixture in acetone, ethanol, and water respectively, and the reaction temperature is room temperature and 60 °C. After three days, filter to obtain solid samples and test their XRPD. As Figure 46As shown, in an organic solvent system (acetone or ethanol), Form I is obtained at room temperature or high temperature; when Form I and Form V are slurried competitively in water, due to their extremely low solubility at room temperature, no crystal transformation occurs and it remains a mixed crystal of the two, while at 60 °C, it transforms into Form V. Combining the results of the water activity experiment, it can be inferred that Form I is more stable in an organic solvent system or in a mixed solvent with low water content (≤10%), while Form V is stable at high temperature (60 °C) in water.

[0271] 13.5 Transformation between anhydrous crystal forms

[0272] After gently grinding an equal amount of Form I and Form V to make the mixture homogeneous, the mixture was placed in an oven at 60 °C and a desiccator at room temperature (to avoid the moisture absorption and crystal transformation of Form I) respectively, and after one week, XRPD was used to test whether there was any mutual transformation of the crystal forms. As Figure 47 shown, after being placed under the above conditions for one week, there is no tendency for Form I and Form V to transform into each other, indicating that both Form I and Form V are stable in the solid state.

[0273] Example 14: Crystal form stability study

[0274] Compared with other crystal forms, Form I, III, and V exhibit good solid-state properties, so their stability tests were carried out respectively.

[0275] 14.1 Physical and chemical stability study

[0276] Approximately 10 mg of Form I, III, and V were respectively placed in a stability chamber at 40 °C / 75% RH (open) and 60 °C (covered) for seven days. Part of the samples were taken out at 0 day and 7 days to test XRPD and HPLC respectively to investigate their physical and chemical stability. The results are shown in Tables 5, Figure 48 , 49 and 61. After seven days under the above test conditions, the crystal forms of Form III and Form V did not change, and at the same time, the chemical purity did not decrease significantly. This indicates that the physical and chemical stability of Form III and Form V is good. For Form I, under the condition of 40 °C / 75% RH (open), it absorbs moisture and transforms into the hydrate crystal form IV. This result is consistent with the DVS result. After being placed at 60 °C for one week, the XRPD pattern of Form I shows changes at 22 - 28° (2θ). According to the TGA result, there is a 1.5% weight loss in the temperature range from room temperature to 60 °C, presumably due to water absorption leading to the change in the diffraction peak. However, the chemical stability of Form I is good.

[0277] Table 5 Results of stability evaluation

[0278]

[0279] 14.2 Mechanical stability test

[0280] The mechanical stabilities of polymorphs I and III were investigated by grinding and tableting, respectively. The grinding test method was as follows: the samples to be tested were placed in a mortar and ground for 2 minutes and 5 minutes respectively, and then XRPD was tested to observe the changes in polymorph and crystallinity. The pressure test method was as follows: the samples were placed in the mold of a tablet press, kept at 40 MPa for one minute, and then taken out for XRPD testing.

[0281] The stability results of polymorph I and polymorph III after grinding (2 minutes and 5 minutes) and tableting (40 MPa, 1 minute) are as Figure 50 and 51 shown. After grinding, the crystallinity of polymorph I decreased significantly, but the polymorph remained unchanged. For polymorph III, neither the polymorph nor the crystallinity changed significantly after grinding ( Figure 52 ), however, the crystallinity decreased significantly after tableting ( Figure 53 ).

[0282] Example 15: Solubility study

[0283] Compared with other polymorphs, polymorphs I, III and V showed good solid-state properties, so their solubility tests were carried out respectively. The solubility test temperatures of polymorphs I, III and V in SGF, FaSSIF and FeSSIF simulated human body temperature of 37 °C, and the test duration was 24 hours.

[0284] Weigh 10 mg of the sample, then add 2.0 mL of biological media buffer (i.e., the target concentration is 5 mg / mL) to form a suspension. Place it in a thermostatic water bath shaker (37 °C, 100 rpm), and take 500 μL of samples at 0.5, 2 and 24 hours respectively. After filtration, test the pH value and HPLC concentration of the filtrate respectively, and at the same time test XRPD of the solid sample obtained by filtration.

[0285] The solubility test results of polymorphs I, III and V in biological media of SGF, FaSSIF and FeSSIF are shown in Table 6 and Figure 54 shown. The solubilities of the above three polymorphs in different biological media were not very different, and their solubilities in SGF were all higher than 5 mg / mL. The solubility in FeSSIF was about 3 times that in FaSSIF, indicating that food may help the absorption of the drug. During the test, the pH value of the biological media buffer did not change significantly.

[0286] As Figure 55 shown, polymorph I transformed into hydrate polymorph IV in FaSSIF and FeSSIF buffers at 0.5 hour. While the polymorphs of polymorph III and V did not change during the test, and the results are shown in Figure 56 and Figure 57 .

[0287] Table 6 Solubility test results

[0288]

[0289] "Dissolution Clear": It means that through naked-eye observation, the solubility of the sample in the medium is greater than 5 mg / mL.

[0290] "N / A": It means that there is no solid sample for testing XRPD.

[0291] Example 16: Pharmacokinetic Study

[0292] After beagle dogs (ordinary grade, Beijing Mas Biotechnology Co., Ltd.) were given compound A by single oral gavage, the pharmacokinetic status of the compound in plasma was studied. The test drug (at a dose of 100 mg / kg and a dosing volume of 5 mL / kg) was administered by gavage. Samples were collected at 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 24 hours, and 48 hours after dosing: Peripheral venous puncture was performed, and approximately 1.0 mL of whole blood was collected from each animal at each time point. The blood samples were collected into K 2 EDTA blood collection tubes, inverted several times to mix well, and placed at room temperature. Within 60 minutes after blood collection, 0.2 mL of whole blood was separated into cryopreservation tubes, deionized water was added at a ratio of 1:1, mixed well by inverting up and down, and then frozen. The remaining samples were centrifuged at 2000 g for 10 minutes at 2 - 8 °C to obtain plasma samples. The hemolysis situation of each sample was recorded to evaluate the impact on detection, and the plasma color was observed and recorded on the blood collection form ("-" for no hemolysis, "+" for pink supernatant, "++" for red supernatant).

[0293] The concentration of compound A in plasma samples was determined by LC-MS / MS method (Non-GLP). Pharmacokinetic parameters were calculated using WinNonlin (PhoenixTM, version 8.1) or other similar software. If applicable plasma drug concentration-time data were available, the following pharmacokinetic parameters were calculated: CL (clearance rate), Vss (steady-state apparent volume of distribution), T1 / 2 (elimination half-life), Cmax (peak concentration), Tmax (time to peak concentration), 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 using Microsoft Excel 2007 or 2010.

[0294] 16.1 Comparison of PK Property Differences between Polymorph I and Polymorph V

[0295] The comparison between polymorph I and polymorph V was for the same 3 dogs (male), 10 mg / kg po, fasted, administered (suspended) after stirring with 0.5% CMC-Na for 3 h. The data obtained are shown in Table 7.

[0296] Table 7 PK Comparison between Crystal Form I and Crystal Form V

[0297]

[0298] Calculated AUC (Crystal Form I): AUC (Crystal Form V) = 300.53%, and it was found that the exposure level and absolute bioavailability of Crystal Form V were less than those of Crystal Form I.

[0299] 16.2 Pharmacokinetic Study of Crystal Form III

[0300] Comparing the differences in female and fasting status for oral administration of Crystal Form III, 100 mg / kg, po, fasting and non-fasting, administered after stirring with 0.5% CMC-Na for 8 h, suspended, and the obtained data are shown in Table 8.

[0301] Table 8 Pharmacokinetic Data of Crystal Form III

[0302]

[0303]

[0304] N / A: Unable to obtain

[0305] It was found that: under fasting conditions, there were certain differences in plasma and whole blood drug concentrations between male and female dogs. In plasma, the concentration in males was about 6 times higher than that in females, and in whole blood, the concentration in males was 1.9 times higher than that in females. However, under non-fasting conditions, there were no significant differences in plasma and whole blood drug concentrations between male and female dogs. Under fasting conditions, there were differences in the erythrocyte distribution of the drug in male and female dogs (male blood: plasma = 1452% Vs 521%), but under non-fasting conditions, there were no significant differences in the erythrocyte distribution of the drug in male and female dogs (male blood: plasma = 114% Vs 116%). Regardless of gender, the drug concentrations in plasma and whole blood under non-fasting conditions were higher than those under fasting conditions. Under non-fasting conditions, the increase in plasma drug concentration was higher than that in whole blood, and the plasma drug concentration in female dogs was most affected by whether fasting or not. The increase in whole blood drug concentration may be due to the increased absorption of the drug under non-fasting conditions, resulting in an increase in the overall exposure level in the body. It is speculated that diet helps the absorption of Compound A. However, the increase in plasma drug concentration being higher than that in whole blood may be due to: 1. The drug concentration in red blood cells is saturated, resulting in a sudden increase in plasma drug concentration (presumably the most likely); 2. After fasting administration on the first day, the drug continuously accumulates in red blood cells, and the 7-day washout period is not sufficient to completely metabolize the drug distributed in red blood cells, so the plasma drug concentration increases significantly during the second non-fasting administration; 3. After fasting administration on the first day, it affects red blood cells, resulting in a decrease in the ability of red blood cells to bind to the drug during the second non-fasting administration, leading to a significant increase in plasma drug concentration.

[0306] Biological test

[0307] In vitro biological activity evaluation of Example A

[0308] The antagonist properties of the compounds in the present invention were determined by the FLIPR (fluorescence imaging plate reader) method. The compounds are inhibitors of the intracellular calcium elevation induced by the activation of hP2X3 (human purinergic P2X receptor subtype 3, accession number NM_002559.4) expressed in HEK293 cells (human renal epithelial cell line, ATCC).

[0309] HEK293 cells stably expressing hP2X3 were placed in a cell culture incubator at 37 °C and 5% humidity and cultured in DMEM high-glucose medium containing 10% FBS (fetal bovine serum, Gibco, 10099-141), 1% penicillin-streptomycin (Gibco, 15140-122), and 1 mg / mL G418 (Invitrogen, 10131027). 18-24 hours before the FLIPR experiment, the cells were seeded into 384-well plates at a density of 400000 cells / mL (10000 cells / well) and incubated overnight in the cell culture incubator. On the day of the experiment, the medium was discarded and the cells were washed in FLIPR buffer (containing 0.3 mL probenecid (Thermo, P36400), 0.6 mL 1 M HEPES (Invitrogen, 15630080), and 29.1 mL HBSS (Invitrogen, 14065056) in every 30 mL of buffer). 20 μL of 0.5× Calcium 6 fluorescent dye (Molecular Devices, R8190) was added to each well and incubated at 37 °C for 1.5 hours for dye loading. Subsequently, 10 μL of the test compound (dissolved in DMSO at a concentration of 10 mM and serially diluted with buffer) or the vehicle was added to each well and allowed to equilibrate at room temperature for 30 min. Then the cell plate was placed into the FLIPR for baseline fluorescence measurement (excitation wavelength: 485 nm, emission wavelength: 525-535 nm). Subsequently, 10 μL / well of the agonist (BZ-ATP (Sigma, B6396) at a final concentration of 2.5 μM) or the vehicle (ultrapure water) was added, and the fluorescence values were measured at 1-second intervals for 2 minutes. Finally, the output fluorescence counts were analyzed.

[0310] The IC 50 obtained using the above method is shown in the following table.

[0311] Table 9 IC data using the above method 50 Data

[0312] Compound <![CDATA[P2X3 IC 50 (μM)]]> Compound 172 0.0007 Positive compound 1 0.0115 Positive compound 2 0.0522

[0313] Positive Compound 1:

[0314] Positive Compound 2:

[0315] In vitro P2X2 / 3 receptor selectivity evaluation of Example B

[0316] The selectivity of the compounds in the present invention for the P2X2 / 3 receptor was determined by the FLIPR (fluorescence imaging plate reader) method. The compounds are inhibitors of the intracellular calcium elevation induced by the activation of hP2X2 / 3 (heterodimeric receptor formed by human purinergic P 2 X receptor subtype 2 and subtype 3) expressed in HEK293 cells (human renal epithelial cell line, ATCC). The accession number of P2X2 is NM_170682.3, and the accession number of P2X3 is NM_002559.4).

[0317] HEK293 cells stably expressing hP2X2 / 3 were placed in a cell culture incubator at 37 °C and 5% humidity, and cultured in DMEM high-glucose medium containing 10% FBS (fetal bovine serum, Gibco, 10099-141), 1% penicillin-streptomycin (Gibco, 15140-122), and 1 mg / mL G418 (Invitrogen, 10131027). 18-24 hours before the FLIPR experiment, the cells were seeded into 96 wells at a density of 250,000 cells / mL (25,000 cells / well) and incubated overnight in the cell culture incubator. On the day of the experiment, the medium was discarded, and the cells were washed in FLIPR buffer (containing 0.3 mL probenecid (Thermo, P36400), 0.6 mL 1 M HEPES (Invitrogen, 15630080), and 29.1 mL HBSS (Invitrogen, 14065056) in every 30 mL of buffer). 75 μL of 1 mM Fluo-4 AM fluorescent dye (Thermo, F14202) was added to each well, and the dye loading was incubated at 37 °C for 1.0 hour. Subsequently, the 96-well plate was washed once with buffer, 50 μL of buffer containing the test compound or solvent was added to each well, and incubated at room temperature for 30 min. Then the cell plate was placed into the FLIPR, and the baseline fluorescence measurement was performed (excitation wavelength: 485 nm, emission wavelength: 525-535 nm). Subsequently, the agonist (BZ-ATP (Sigma, B6396) at a final concentration of 5 μM) or solvent (ultrapure water) was added at 50 μL / well, and the fluorescence values were measured at 1-second intervals for 2 minutes. Finally, the output fluorescence counts were analyzed.

[0318] ICs obtained using the above method 50 are shown in the following table.

[0319] Table 10 ICs using the above method 50 Data

[0320] Compound <![CDATA[IC 50 > Selectivity multiple Compound 172 >60 >85714 Positive compound 1 >60 >5217 Positive compound 2 0.36 6.9

[0321] Activity test of simple citric acid cough model in Example C

[0322] Male Dunkin Hartley guinea pigs (300 - 350 g) were placed in an animal atomization chamber. The door of the atomization chamber was closed, and at the same time, an ultrasonic atomizer (Guangdong Yuehua) was turned on. 17.5% citric acid gas was filled into the atomization chamber at the maximum atomization rate (about 2 mL / min) for 20 s, and timing started from the beginning of atomization. The coughing performance of the animals was continuously observed for 10 min. During the 10 - min observation period, manual counting of coughs was required. The number of coughs was judged based on the coughing postures of the guinea pigs such as abdominal twitching, mouth opening, head lowering, etc. and the coughing sound. The number of coughs in the first 5 min and the number of coughs in 10 min were recorded. At the same time, the cough latency of the guinea pigs was recorded, that is, the time from the start of citric acid induction to the first cough.

[0323] Table 11 Animal experiment

[0324]

[0325]

[0326] Compound 172 has the effect of reducing the number of coughs and increasing the cough latency, and is comparable to the efficacy of the positive compound.

[0327] Activity test of ATP - citric acid cough model in Example D

[0328] Male Dunkin Hartley guinea pigs (300 - 400 g) were placed in a whole - body volume scanning chamber and allowed to adapt for 3 - 5 mins. Then, ATP atomization was carried out for 2 min, with an interval of 3 min, and then citric acid atomization was given for 5 min. All atomization rates were about 300 μL / min. Starting from the start of citric acid atomization, the number of coughs and the cough latency of the animals within 10 mins were recorded. During the 10 - min observation period, manual counting of coughs was required. The number of coughs was judged based on the coughing postures of the guinea pigs such as abdominal twitching, mouth opening, head lowering, etc. and the coughing sound. The number of coughs in 10 min was recorded. At the same time, the cough latency of the guinea pigs was recorded.

[0329] Animals were divided into a vehicle group, a positive group of dextromethorphan (63 mg / kg), a positive group of AF-219 (30 mg / kg), and administration groups of the compound of Example 172 (3, 10, 30 mg / kg). All compounds were administered orally. Except for dextromethorphan which was administered 40 min before ATP-citrate exposure, the other compounds were administered 2 h before ATP-citrate exposure.

[0330] Table 12 Quinine / water drinking ratios obtained for the compounds

[0331]

[0332] As shown in Table 5, after treatment with all compounds, there was a tendency for the number of coughs to decrease compared with the vehicle group. Among them, both the positive group of dextromethorphan and compound 172 could significantly reduce the number of coughs, and compound 172 had a dose-dependent inhibitory effect. Compared with the positive compound 2, the cough inhibitory effect of compound 172 was stronger.

[0333] Example E In vitro cytotoxicity test

[0334] The in vitro cytotoxicity test of the compounds in the present invention was determined by the CCK-8 method in HepG2 cells. Logarithmic-phase HepG2 cells (Beijing Na Biotechnology) were collected, the cell suspension concentration was adjusted, and plated in a 96-well cell culture plate at 50000 cells / well. The cells were incubated overnight in a cell culture incubator at 5%, 37 °C. After the cell confluence in the plate reached 80 - 90%, the medium was changed and the test compounds or vehicle (DMSO) at each concentration gradient was added, and incubated in a cell culture incubator at 5%, 37 °C for 48 hours. After the treatment was completed, the medium in the plate was discarded, washed twice with PBS, 100 μL of CCK-8 working solution (Beyotime Biotechnology) was added to each well, incubated at 37 °C in the dark for 1.5 hours, and the absorbance value at OD 450nm at each well was measured, and the CC of each compound was analyzed and calculated 50 .

[0335] Table 13 IC of each compound 50 value

[0336] Compound <![CDATA[HepG2 CC 50 (μM)]]> Compound 172 57.05 Positive compound 1 128.4 Positive compound 2 >200

[0337] Example F In vitro metabolic stability test

[0338] The in vitro metabolic stability of the compounds in the present invention was determined by the incubation method with liver microsomes of various species. In the liver microsome reaction system (1 mg / mL liver microsome protein, 25 U / mL glucose-6-phosphate dehydrogenase, 1 mM NADP, 6 mM D-glucose-6-phosphate, 5 mM MgCl 2)Add an appropriate amount of the test compound, place it in a water bath at 37 °C for incubation to initiate the reaction. At each time point, take 100 μL of the reaction solution and add it to a centrifuge tube containing 400 μL of internal standard working solution (acetonitrile solution containing 200 ng / mL of dexamethasone, diclofenac, tolbutamide, and labetalol) pre-cooled at 0 °C to terminate the reaction. Centrifuge at 10000 g for 10 min in a 4 °C centrifuge. Take the supernatant and perform LC-MS analysis and detection to obtain the in vitro metabolic half-life of the test compound in various species of liver microsomes.

[0339] Table 14 In vitro metabolic stability test

[0340]

[0341] Example G Two-bottle method for taste disorder test

[0342] After the SPF-grade male SD rats (6 - 8 weeks old) are put into storage, they immediately receive 3 days of adaptive drinking water training. The specific training content is that the animals are housed individually in cages, and two bottles of water (both are ordinary drinking water) are placed in each cage. During the adaptive training period, water treatment is prohibited throughout the night (remove the water bottles), and drinking water is re-given from 8:30 am to 5:30 pm. This cycle lasts for 3 days, and the positions of the two bottles of water are changed around every day. The animals can eat freely throughout the adaptive training period. Twenty hours before the formal experiment, remove all the animals' water bottles and prohibit water until the experiment starts. During the formal experiment, all the animals are randomly grouped. Before re-supplying water, the test compound or the vehicle is given by single intraperitoneal injection. The administration time is determined according to the T max of the test compound. Then place the animals individually in cages and give two bottles of drinking water, one is ordinary drinking water, and the other is 0.3 mM quinine water. Observe the water intake of the animals within 15 min, and perform statistical analysis with the quinine water intake / ordinary drinking water intake. Use positive compound 2 as the positive control for taste disorder.

[0343] Table 15 Animal water intake test

[0344]

[0345] The results show that: compared with the blank vehicle group, compound 172 at 5, 10, and 20 mg / kg has no obvious effect on the ratio of quinine / water consumption in rats, indicating that compound 172 has little effect on the taste of animals; compared with positive compound 2, the ratio of quinine / water consumption is significantly lower than that of positive compound 2, indicating that the risk of compound 172 causing taste disorder is far better than that of positive compound 2, and the safety is better.

Claims

1. A crystal form of a compound of formula A or its solvate: It is selected from 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; Wherein, Crystal form III of the hydrate of the compound of formula A has characteristic peaks in the X-ray powder diffraction pattern at 2θ values of 12.91° ± 0.20°, 16.77 ± 0.20°, 19.27° ± 0.20°, and 22.80° ± 0.20°; Crystal form V of the compound of formula A has characteristic peaks in the X-ray powder diffraction pattern at 2θ values of 8.38° ± 0.20°, 9.15° ± 0.20°, 13.52° ± 0.20°, and 18.44 ± 0.20°; Crystal form I of the compound of formula A has characteristic peaks in the X-ray powder diffraction pattern at 2θ values of 8.56° ± 0.20°, 12.48 ± 0.20°, 22.13° ± 0.20°, 13.53° ± 0.20°, 14.25 ± 0.20°, 25.18° ± 0.20°, and 26.07° ± 0.20°; Crystal form II of the MTBE solvate of the compound of formula A has characteristic peaks in the X-ray powder diffraction pattern at 2θ values of 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°, and 21.20° ± 0.20°; Crystal form IV of the hydrate of the compound of formula A has characteristic peaks in the X-ray powder diffraction pattern at 2θ values of 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°, and 26.52° ± 0.20°; Crystal form VI of the hydrate of the compound of formula A has characteristic peaks in the X-ray powder diffraction pattern at 2θ values of 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°, and 25.24° ± 0.20°; Crystal form VII of the ethylene glycol solvate of the compound of formula A has characteristic peaks in the X-ray powder diffraction pattern at 2θ values of 8.36° ± 0.20°, 12.13° ± 0.20°, 12.45° ± 0.20°, 16.84° ± 0.20°, 21.66° ± 0.20°, 21.07° ± 0.20°, and 24.82° ± 0.20°; Polymorph VIII of the THF solvate of the compound of formula A, the X-ray powder diffraction pattern of which has characteristic peaks at 2θ values of 8.53° ± 0.20°, 12.38° ± 0.20°, 13.66° ± 0.20°, 21.49° ± 0.20°, 20.99° ± 0.20°, 24.94° ± 0.20° and 25.31° ± 0.20°; Polymorph IX of the DMSO solvate of the compound of formula A, the X-ray powder diffraction pattern of which has characteristic peaks at 2θ values of 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° and 25.57° ± 0.20°.

2. The crystal form of compound A or its solvate according to claim 1, wherein, for the said polymorph III, the X-ray powder diffraction pattern thereof has characteristic peaks at 2θ values of 12.91° ± 0.20°, 16.77 ± 0.20°, 19.27° ± 0.20°, 22.80° ± 0.20°, 13.75° ± 0.20°, 14.46° ± 0.20° and 20.86° ± 0.20°; and / or, for the said polymorph V, the X-ray powder diffraction thereof has characteristic peaks at 2θ values of 8.38° ± 0.20°, 9.15° ± 0.20°, 13.52° ± 0.20°, 18.44 ± 0.20°, 16.26° ± 0.20°, 16.89° ± 0.20° and 17.86° ± 0.20°.

3. The crystal form of compound A or its solvate according to claim 2, wherein, for the said polymorph III, the X-ray powder diffraction pattern thereof has characteristic peaks at 2θ values of 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° and 24.05° ± 0.20°; and / or, in the thermogravimetric analysis spectrum of the said polymorph III, the weight loss gradient in the room temperature - 100°C range is 1.5%; and / or, in the differential scanning calorimetry spectrum of the said polymorph III, the first endothermic peak is for the removal of 0.4 water molecules; and / or, for the said polymorph V, the X-ray powder diffraction thereof has characteristic peaks at 2θ values of 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°. And / or, the differential scanning calorimetry (DSC) pattern of Form V has an endothermic peak at 166 °C with a melting enthalpy of 70 ± 2 J / g; And / or, for Form I, its X-ray powder diffraction pattern has characteristic peaks at 2θ values of 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° and 23.42° ± 0.20°; And / or, the DSC pattern of Form I has an endothermic peak at 152 °C with a melting enthalpy of 44 ± 2 J / g; And / or, for Form II, its X-ray powder diffraction pattern has characteristic peaks at 2θ values of 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° and 24.40° ± 0.20°; And / or, in the thermogravimetric analysis (TGA) pattern of Form II, there is a weight loss of 3.5% in the temperature range of 100 - 160 °C and a weight loss of 2.9% in the range of 160 - 200 °C; And / or, in the TGA pattern of Form IV, there is a weight loss of 1.2% in the temperature range of RT - 60 °C; And / or, for Form VII, its X-ray powder diffraction pattern has characteristic peaks at 2θ values of 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° and 24.57° ± 0.20°; And / or, in the TGA pattern of Form VII, there is a weight loss of 25.7% in the temperature range from room temperature to 120 °C; And / or, for Form VIII, its X-ray powder diffraction pattern has characteristic peaks at 2θ values of 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° and 23.00° ± 0.20°; And / or, in the TGA pattern of Form VIII, there is a weight loss of 5.7% in the temperature range from room temperature to 160 °C; And / or, for the crystalline form IX, its X-ray powder diffraction pattern has characteristic peaks at 2θ values of 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° and 23.12° ± 0.20°; And / or, in the thermogravimetric analysis spectrum of the crystalline form IX, there is a weight loss of 18.23% in the temperature range from room temperature to 160°C.

4. The crystalline form of the compound A or its solvate according to claim 3, wherein, the X-ray powder diffraction pattern of the crystalline form III expressed in 2θ angle is substantially as shown in Figure 1; And / or, the thermogravimetric analysis spectrum and differential scanning calorimetry spectrum of the crystalline form III are substantially as shown in Figure 2; And / or, the dynamic water adsorption spectrum of the crystalline form III is as shown in Figure 4; And / or, the polarized light microscope spectrum of the crystalline form III is substantially as shown in Figure 6; And / or, the crystalline form III contains 0.4 equivalents of water; And / or, the X-ray powder diffraction pattern of the crystalline form V expressed in 2θ angle is substantially as shown in Figure 7; And / or, the dynamic water adsorption spectrum of the crystalline form V is as shown in Figure 10; And / or, the thermogravimetric analysis spectrum and differential scanning calorimetry spectrum of the crystalline form V are substantially as shown in Figure 8; And / or, the polarized light microscope spectrum of the crystalline form V is substantially as shown in Figure 11; And / or, the X-ray powder diffraction pattern of the crystalline form I expressed in 2θ angle is substantially as shown in Figure 12; And / or, the thermogravimetric analysis spectrum and differential scanning calorimetry spectrum of the crystalline form I are substantially as shown in Figure 13; And / or, the dynamic water adsorption spectrum of the crystalline form I is as shown in Figure 14; And / or, the polarized light microscope spectrum of the crystalline form I is substantially as shown in Figure 17; And / or, the nuclear magnetic resonance hydrogen spectrum of the crystalline form II is as shown in Figure 19; And / or, the X-ray powder diffraction pattern of the crystalline form II expressed in 2θ angle is substantially as shown in Figure 18; And / or, the polarized light microscope spectrum of the crystalline form II is substantially as shown in Figure 21; And / or, the thermogravimetric analysis spectrum and differential scanning calorimetry spectrum of the crystalline form II are substantially as shown in Figure 20; And / or, the X-ray powder diffraction pattern of the crystalline form IV expressed in 2θ angle is substantially as shown in Figure 22; And / or, the thermogravimetric analysis spectrum and differential scanning calorimetry spectrum of the crystalline form IV are as shown in Figure 23; And / or, the polarized light microscope spectrum of the crystalline form IV is substantially as shown in Figure 25; And / or, the X-ray powder diffraction pattern of the crystalline form VI expressed in 2θ angle is substantially as shown in Figure 26; And / or, the X-ray powder diffraction pattern of the crystalline form VII expressed in 2θ angle is substantially as shown in Figure 28; And / or, the nuclear magnetic resonance hydrogen spectrum of the crystalline form VII is as shown in Figure 29; And / or, the thermogravimetric analysis spectrum and differential scanning calorimetry spectrum of the crystalline form VII are substantially as shown in Figure 30; And / or, the X-ray powder diffraction pattern of the crystalline form VIII expressed in terms of 2θ angle is substantially as shown in Figure 31; And / or, the thermogravimetric analysis spectrum and differential scanning calorimetry spectrum of the crystalline form VIII are substantially as shown in Figure 32; And / or, the nuclear magnetic resonance hydrogen spectrum of the crystalline form VIII is as shown in Figure 33; And / or, the X-ray powder diffraction pattern of the crystalline form IX expressed in terms of 2θ angle is substantially as shown in Figure 35; And / or, the thermogravimetric analysis spectrum and differential scanning calorimetry spectrum of the crystalline form IX are substantially as shown in Figure 36.

5. A method for preparing crystalline form III of a substance A, characterized in that, it comprises the following steps: adding a solution of the compound shown by formula A and a solvent to aqueous solution A, crystallizing to obtain crystalline form III of substance A, wherein the aqueous solution A is a suspension of the crystalline seed of crystalline form III of the substance A and water; the solvent is DMSO; the crystalline form III of the substance A is the hydrate crystalline form III of compound A as described in any one of claims 1-4.

6. The preparation method according to claim 5, characterized in that, the volume ratio of DMSO to water is 1:1 - 1:

4.

7. A crystalline form III of a substance A, characterized in that, the crystalline form III of the substance A is prepared according to the preparation method of claim 5 or 6.

8. A method for preparing crystalline form V of a compound shown by formula A, characterized in that, it comprises the following steps: subjecting the amorphous form of the compound shown by formula A to polymorphic transformation in a suspension with a solvent at 50°C to obtain crystalline form V of compound A; the solvent is acetonitrile; the compound shown by formula A and crystalline form V are as defined in any one of claims 1-4.

9. The preparation method according to claim 8, characterized in that, the mass-volume ratio of the amorphous form of compound A to the solvent is 3 mg / mL.

10. A pharmaceutical composition, which comprises the crystalline form of the compound shown by formula A as described in any one of claims 1-4 or its solvate and / or the crystalline form III of the substance A as described in claim 7, and pharmaceutical excipients.

11. Use of the crystalline form of the compound shown by formula A as described in any one of claims 1-4 or its solvate, the crystalline form III of the substance A as described in claim 7 or the pharmaceutical composition as described in claim 10 in the preparation of a P2X3 receptor antagonist or a drug; the drug is a drug for protecting, treating, or alleviating at least partially P2X3-mediated or activity-related diseases in animals.

12. The use according to claim 11, characterized in that, the disease is pain, urinary tract disease or respiratory system disease.

13. The use according to claim 12, characterized in that, the pain is selected from: inflammatory pain, surgical pain, visceral pain, toothache, premenstrual pain, central pain, pain caused by burns, migraine or cluster headache; the urinary tract diseases are selected from: urinary incontinence, overactive bladder, dysuria, cystitis; the respiratory system disease is respiratory disorder.

14. The use according to claim 12, characterized in that, The respiratory disease is idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, bronchospasm or chronic cough.

15. The use according to claim 11, wherein, by administering the pharmaceutical composition, the side effect of taste disorder related to the treatment is reduced.

Images