Crystal form of N-formamido pyrazoline compound and preparation method and application thereof

Abstract

The invention discloses a crystal form of an N-formamido pyrazoline compound as well as a preparation method and application of the crystal form. The invention provides a crystal form of a compound as shown in a formula A. The crystal form is a crystal form I, a crystal form II, a crystal form III, a crystal form IV, a crystal form V, a crystal form VI or a crystal form VII, and discloses a preparation method, a composition and application of the crystal form in preparation of a P2X3 receptor antagonist. The invention also relates to application of the compound in preparation of drugs for treating respiratory system diseases, pains caused by various reasons, and skin diseases. The crystal form stability is good, the hygroscopicity is small, and the patent medicine prospect is good. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the field of medical technology and relates to a crystal form of an N-formamidopyrazoline compound and a preparation method and application thereof. Background Art

[0002] P2X3 is a ligand-gated non-selective cation channel receptor subtype, belonging to the ionotropic P2X receptor in the purinergic receptor P2 class. It was first cloned in mammals in 1995 (Chen C et al., Nature, 1995, 428; Lewis, C et al., Nature, 1995, 432). To date, there are 7 P2X receptor subtypes cloned in mammals (P2X1 to P2X7). Each P2X receptor molecule consists of an intracellular N-terminus and a C-terminus and two transmembrane structural regions. Although there are subtypes and species differences in P2X receptors, there is no obvious difference in the basic structure of P2X receptors. They are all trimers composed of three homologous or heterologous subunits, such as P2X3 is a homotrimer, and P2X1 / 2 and P2X2 / 3 are heterotrimers (Jacobson et al., Neuropharmacology, 2016, 31). P2X3 receptors are widely distributed in the body, mainly expressed in peripheral sensory neurons related to noxious information, and play an important mediating role in the generation and transmission of noxious information. When the body is injured or nerves are damaged, a large amount of ATP is released, which binds to the P2X3 receptors and causes conformational changes in the transmembrane region of the receptors, thereby activating the P2X3 receptors and causing a large amount of Ca 2+ Influx of calcium and increase in intracellular calcium concentration activate the phosphorylation of protein kinase A, protein kinase C, etc., promote the release of glutamate and further activation of NMDA receptors, etc., ultimately leading to sensitization of the central nervous system.

[0003] P2X3 is involved in a variety of physiological and pathological reactions, including pathological pain such as inflammatory pain, neuropathic pain, cancer pain, cough, hypertension, bladder urination, etc. For example, upregulation of P2X3 receptor expression can lead to the formation of pain sensitivity and participate in the signal transmission of pain. After P2X3 receptor knockout, pain-related behaviors in mice can be alleviated (Cockayne DA et al., Nature, 2000, 1011). The inhibition of P2X3 receptors can improve the reflex function of spontaneous cardiac pressure receptors, help inhibit the sympathetic nerves of rats, and thus play an antihypertensive effect (Pijacks W et al., Nature Medicine, 2016, 1151). P2X3 receptors are correlated with the urination frequency of mice. The loss of P2X3 receptors will also reduce the urination frequency of mice (Gao XF et al., Nature Communication, 2015, 7650). The ATP / P2X3 signaling pathway in human skin is associated with chronic pruritus, so P2X3 receptor inhibitors can also be used to treat chronic pruritus (Chauret, N. et al., 49th Annual ESDR Meeting, 2019).

[0004] Other studies have shown that P2X3 is expressed in the vagus nerve of guinea pig C-fibres. After being activated by its agonist ATP, it can stimulate the sensory nerves in the respiratory tract and induce cough (Abdulqawi R et al., Eur Respir J, 2013). Studies have found that Cough Hypersensitivity Syndrome, that is, chronic cough, especially refractory chronic cough, is one of the most common diseases of the respiratory system. The causes include a variety of respiratory diseases, environment, smoking and drug allergies. This type of disease seriously affects the health and quality of life of patients, and is increasing year by year due to factors such as environmental deterioration and population aging, but there are few treatment options for existing drugs. Therefore, it is necessary to study the cough mechanism to uncover new therapeutic targets and develop new treatments to improve the quality of life of patients. Clinical data in recent years have shown that the P2X3 receptor inhibitor AF-219 is effective in reducing the frequency and severity of cough in patients with chronic cough (Abdulqawi R et al., Eur Respir J, 2013, 42; Abdulqawi R et al., Lancet, 2015, 1198; Garceau D et al., Pulmonary Pharmacology & Therapeutics, 2019, 56; Muccino D et al., Pulmonary Pharmacology & Therapeutics, 2019, 75). However, clinical trials have also shown that AF-219 can cause taste disorders in the treated subjects by affecting the taste of the tongue (Abdulqawi et al., Lancet 2015).

[0005] Chinese patent ZL202110695453.0 discloses a class of N-formamidopyrazoline compounds that can be used as P2X3 receptor antagonists, and specifically discloses a preparation method of a P2X3 antagonist compound as shown below and its use, but does not involve the crystal form of the compound. The inventors have found through research that the compound has a selective P2X3 receptor antagonist effect and has little effect on taste. It can be used to treat, prevent or alleviate patients' chronic obstructive pulmonary disease, asthma, acute, subacute and chronic cough, bronchospasm, surgical pain, inflammatory pain, cancer pain, bladder pain, endometriosis pain, diabetic neuropathy, traumatic pain, toothache, migraine and pain related to irritable bowel syndrome, chronic itching and other diseases or symptoms.

[0006] Summary of the invention

[0007] The present invention provides a crystalline form of (R)-4-(2-oxopyrrolidin-1-yl)-3-(4-methylphenyl)-N-((R)-1-(2-(trifluoromethyl)pyrimidin-5-yl)ethyl)-4,5-dihydro-1H-pyrazole-1-carboxamide (compound shown in formula A), a preparation method and application thereof.

[0008] The present invention provides a crystalline form of a compound represented by formula A,

[0009]

[0010] It is selected from the group consisting of Form I, Form II, Form III, Form IV, Form V, Form VI and Form VII.

[0011] In some embodiments of the present invention, the crystalline form of the compound represented by formula A is crystalline form IV.

[0012] In some embodiments of the present invention, the crystalline form IV is characterized in that the X-ray powder diffraction pattern of the crystalline form IV has diffraction peaks at the following 2θ angles: 9.68±0.2°, 11.96±0.2°, 19.00±0.2°, and 19.98±0.2°.

[0013] In some embodiments of the present invention, the crystalline form IV is characterized in that the X-ray powder diffraction pattern of the crystalline form IV has diffraction peaks at the following 2θ angles: 9.68±0.2°, 11.96±0.2°, 17.76±0.2°, 19.00±0.2°, 19.98±0.2°, 21.50±0.2°, and 22.48±0.2°.

[0014] In some embodiments of the present invention, the crystalline form IV is characterized in that the X-ray powder diffraction pattern of the crystalline form IV has diffraction peaks at the following 2θ angles: 8.18±0.2°, 9.68±0.2°, 11.96±0.2°, 14.30±0.2°, 17.76±0.2°, 19.00±0.2°, 19.98±0.2°, 21.50±0.2°, 22.48±0.2°, and 24.68±0.2°.

[0015] In some embodiments of the present invention, the crystalline form IV is characterized in that the X-ray powder diffraction pattern of the crystalline form IV is as follows Fig.10 shown.

[0016] In some embodiments of the present invention, the crystal form IV is characterized in that the X-ray powder diffraction pattern analysis data of the crystal form IV is shown in Table 4.

[0017] In some embodiments of the present invention, the crystalline form IV is characterized in that the differential scanning calorimetry diagram of the crystalline form IV comprises an endothermic peak at 170.95±3°C.

[0018] In some embodiments of the present invention, the crystalline form IV is characterized in that the differential scanning calorimetry diagram of the crystalline form IV is as follows Fig.11 shown.

[0019] In some embodiments of the present invention, the crystalline form IV is characterized in that when the crystalline form IV is heated to about 150° C., the weight loss is about 0.02%.

[0020] In some embodiments of the present invention, the crystalline form IV is characterized in that the thermogravimetric analysis diagram of the crystalline form IV is as follows Fig.12 shown.

[0021] According to one aspect of the invention, a method for preparing the crystalline form IV of the compound of formula A is provided, comprising: adding the compound of formula A to a solvent and stirring at 20 to 35°C; or adding the compound of formula A to a solvent, heating and dissolving it, and then stirring; solid-liquid separation; or cooling to 20 to 35°C, adding an insoluble solvent, continuing to stir, and then cooling to 0 to 10°C and then solid-liquid separation; drying to obtain the crystalline form IV.

[0022] As a further preferred embodiment, it is Scheme 1 or Scheme 2;

[0023] In some embodiments of the present invention, the scheme 1 comprises the following steps: adding a suitable volume of solvent to the compound of formula A, stirring magnetically at room temperature for 2 to 3 days, collecting the solid by centrifugation, and drying under reduced pressure and vacuum.

[0024] In some embodiments of the present invention, in the scheme 1, the solvent is an alcohol solvent, preferably one or more of isopropanol, n-butanol or isobutanol. Preferably, the mass volume ratio of the compound of formula A to the solvent is 40 to 80 mg / mL. Further preferably, the solvent is isopropanol.

[0025] In some embodiments of the present invention, in the scheme 1, the centrifugation conditions are 10000-14000 rpm, centrifugation for 3-6 min; preferably 12000 rpm, centrifugation for 5 min.

[0026] In some embodiments of the present invention, in the scheme 1, the reduced pressure vacuum drying temperature is 30-50°C, preferably 40°C.

[0027] In some embodiments of the present invention, the second scheme comprises the following steps:

[0028] Add a proper volume of solvent to the compound of formula A, heat and stir for 1 to 2 hours, cool naturally to 20 to 35°C (such as room temperature), add a proper volume of poor solvent (such as n-heptane) and continue stirring for 8 to 12 hours, cool to 0 to 10°C, filter and collect the solid, and dry under reduced pressure.

[0029] In some embodiments of the present invention, in the second scheme, the solvent is an alcohol solvent, preferably one or more of isopropanol, n-butanol or isobutanol. Preferably, the mass volume ratio of the compound of formula A to the solvent is 40 to 80 mg / mL. Further preferably, the solvent is isopropanol. The poor solvent is one or more of petroleum ether, n-hexane, cyclohexane, and n-heptane. Further preferably, the poor solvent is n-heptane.

[0030] In some embodiments of the present invention, in the second scheme, the heating and stirring temperature is 60-80°C, preferably 70°C.

[0031] In some embodiments of the present invention, in the second scheme, the reduced pressure vacuum drying temperature is 30-50°C, preferably 40°C.

[0032] In some embodiments of the present invention, the crystalline form of the compound represented by formula A is crystalline form I.

[0033] In some embodiments of the present invention, the crystalline form I has an X-ray powder diffraction pattern having diffraction peaks at the following 2θ angles: 7.24°±0.2°, 11.46°±0.2°, 18.58°±0.2°, and 19.16°±0.2°.

[0034] In some embodiments of the present invention, the crystalline form I, the X-ray powder diffraction pattern of the crystalline form I has diffraction peaks at the following 2θ angles: 7.24°±0.2°, 9.22°±0.2°, 11.46°±0.2°, 13.58°±0.2°, 15.60°±0.2°, 18.58°±0.2°, 19.16°±0.2°, 21.92°±0.2°, and 27.52°±0.2°.

[0035] In some embodiments of the present invention, the crystalline form I, the X-ray powder diffraction pattern of the crystalline form I has diffraction peaks at the following 2θ angles: 7.24°±0.2°, 9.22°±0.2°, 11.46°±0.2°, 13.58°±0.2°, 15.60°±0.2°, 18.58°±0.2°, 19.16°±0.2°, 19.92°±0.2°, 21.92°±0.2°, 22.62°±0.2°, 24.20°±0.2°, and 27.52°±0.2°.

[0036] In some embodiments of the present invention, the crystalline form I has an X-ray powder diffraction pattern as shown in the attached Figure 1 shown.

[0037] In some embodiments of the present invention, the crystal form I, the X-ray powder diffraction pattern analysis data of the crystal form I is shown in Table 1.

[0038] In some embodiments of the present invention, the crystalline form I, the differential scanning calorimetry diagram of the crystalline form I comprises an endothermic peak at 169.46±3°C.

[0039] In some embodiments of the present invention, the crystalline form I is characterized in that the differential scanning calorimetry diagram of the crystalline form I is as follows Figure 2 shown.

[0040] In some embodiments of the present invention, the crystalline form I is characterized in that when the crystalline form I is heated to about 90° C., the weight loss is about 5.6%.

[0041] In some embodiments of the present invention, the crystal form I is characterized in that the thermogravimetric analysis diagram of the crystal form I is as follows Figure 3 shown.

[0042] According to one aspect of the invention, a method for preparing the crystalline form I of the compound of formula A is provided, the method comprising: adding an appropriate volume of solvent to the compound of formula A, magnetically stirring at 20 to 30°C for 2 to 3 days, collecting the solid by centrifugation (10,000 to 14,000 rpm, centrifugation for 3 to 6 min), and drying under reduced pressure and vacuum at 30 to 50°C.

[0043] In some embodiments of the present invention, in the method for preparing the crystalline form I of the compound of formula A, the solvent in the method comprises one or more of ethanol, acetone, and methyl ethyl ketone.

[0044] In some embodiments of the present invention, the crystalline form of the compound represented by formula A is crystalline form II.

[0045] In some embodiments of the present invention, the crystal form II has an X-ray powder diffraction pattern having diffraction peaks at the following 2θ angles: 9.80±0.2°, 24.90±0.2°.

[0046] In some embodiments of the present invention, the crystalline form II has an X-ray powder diffraction pattern having diffraction peaks at the following 2θ angles: 9.80±0.2°, 18.06±0.2°, 22.84±0.2°, and 24.90±0.2°.

[0047] In some embodiments of the present invention, the crystalline form II has an X-ray powder diffraction pattern having diffraction peaks at the following 2θ angles: 9.80±0.2°, 12.76±0.2°, 14.78±0.2°, 18.06±0.2°, 19.12±0.2°, 22.84±0.2°, 24.90±0.2°, 27.12±0.2°, and 29.38±0.2°.

[0048] In some embodiments of the present invention, the crystalline form II has an X-ray powder diffraction pattern as shown in Figure 4 shown.

[0049] In some embodiments of the present invention, the crystal form II, the X-ray powder diffraction pattern analysis data of the crystal form II is shown in Table 2.

[0050] In some embodiments of the present invention, the crystalline form II has a differential scanning calorimetry diagram comprising endothermic peaks at 81.47±3°C and 132.62±3°C.

[0051] In some embodiments of the present invention, the crystalline form II has a differential scanning calorimetry diagram as shown in Figure 5 shown.

[0052] In some embodiments of the present invention, the crystalline form II loses about 3.2% of its weight when heated to about 90°C.

[0053] In some embodiments of the present invention, the crystal form II, the thermogravimetric analysis diagram of the crystal form II is as follows Figure 6 shown.

[0054] According to one aspect of the invention, a method for preparing the crystalline form II of the compound of formula A is provided, the method comprising: adding an appropriate volume of solvent to the compound of formula A, heating to 40-60°C to dissolve, cooling to room temperature, adding an appropriate amount of water, stirring for 1-2 hours, collecting the solid by centrifugation (10000-14000 rpm, centrifugation for 3-6 min), and drying under reduced pressure and vacuum at 30-50°C.

[0055] In some embodiments of the present invention, in the method for preparing the crystalline form II of the compound of formula A, the solvent in the method comprises one or more of ethanol, acetone, methyl ethyl ketone, and acetonitrile.

[0056] In some embodiments of the present invention, the crystalline form of the compound represented by formula A is crystalline form III.

[0057] In some embodiments of the present invention, the crystalline form III has an X-ray powder diffraction pattern having diffraction peaks at the following 2θ angles: 6.86±0.2°, 11.76±0.2°, and 18.16±0.2°.

[0058] In some embodiments of the present invention, the crystalline form III has an X-ray powder diffraction pattern having diffraction peaks at the following 2θ angles: 6.86±0.2°, 11.76±0.2°, 18.16±0.2°, 20.54±0.2°, and 22.22±0.2°.

[0059] In some embodiments of the present invention, the crystalline form III has an X-ray powder diffraction pattern having diffraction peaks at the following 2θ angles: 6.86±0.2°, 11.76±0.2°, 18.16±0.2°, 19.08±0.2°, 20.54±0.2°, 22.22±0.2°, and 26.52±0.2°.

[0060] In some embodiments of the present invention, the crystalline form III has an X-ray powder diffraction pattern as shown in Figure 7 shown.

[0061] In some embodiments of the present invention, the crystal form III, the X-ray powder diffraction pattern analysis data of the crystal form III is shown in Table 3.

[0062] In some embodiments of the present invention, the crystalline form III is characterized in that the differential scanning calorimetry diagram of the crystalline form III comprises endothermic peaks at 155.41±3°C and 173.55±3°C, and an exothermic peak at 160.48±3°C.

[0063] In some embodiments of the present invention, the crystalline form III is characterized in that the differential scanning calorimetry diagram of the crystalline form III is as follows Figure 8 shown.

[0064] In some embodiments of the present invention, the crystalline form III is characterized in that when the crystalline form III is heated to about 150° C., the weight loss is about 2.2%.

[0065] In some embodiments of the present invention, the crystalline form III is characterized in that the thermogravimetric analysis diagram of the crystalline form III is as follows Fig. 9 shown.

[0066] According to one aspect of the invention, a method for preparing the crystalline form III of the compound of formula A is provided, the method comprising: adding an appropriate volume of solvent to the compound of formula A, magnetically stirring at 20 to 30° C. for 2 to 3 days, collecting the solid by centrifugation (10,000 to 14,000 rpm, centrifugation for 3 to 6 min), and drying under reduced pressure and vacuum at 30 to 50° C.

[0067] In some embodiments of the present invention, in the method for preparing the crystalline form III of the compound of formula A, the solvent in the method comprises methyl tert-butyl ether.

[0068] In some embodiments of the present invention, the crystalline form of the compound represented by formula A is crystalline form V.

[0069] In some embodiments of the present invention, the crystalline form V has an X-ray powder diffraction pattern having diffraction peaks at the following 2θ angles: 6.64±0.2°, 17.56±0.2°, and 20.22±0.2°.

[0070] In some embodiments of the present invention, the crystalline form V has an X-ray powder diffraction pattern having diffraction peaks at the following 2θ angles: 6.64±0.2°, 12.14±0.2°, 17.56±0.2°, 19.56±0.2°, and 20.22±0.2°.

[0071] In some embodiments of the present invention, the crystalline form V has an X-ray powder diffraction pattern having diffraction peaks at the following 2θ angles: 6.64±0.2°, 12.14±0.2°, 17.56±0.2°, 19.56±0.2°, 20.22±0.2°, 22.06±0.2°, and 25.80±0.2°.

[0072] In some embodiments of the present invention, the crystalline form V has an X-ray powder diffraction pattern as shown in Fig.13 shown.

[0073] In some embodiments of the present invention, the crystal form V, the X-ray powder diffraction pattern analysis data of the crystal form V is shown in Table 5.

[0074] In some embodiments of the present invention, the crystalline form V, the differential scanning calorimetry diagram of the crystalline form V comprises endothermic peaks at 154.03±3°C and 172.16±3°C, and an endothermic peak at 159.54±3°C.

[0075] In some embodiments of the present invention, the crystalline form V has a differential scanning calorimetry diagram as shown in Fig.14 shown.

[0076] In some embodiments of the present invention, the crystalline form V loses about 7.9% of its weight when heated to about 145°C.

[0077] In some embodiments of the present invention, the crystal form V is characterized in that the thermogravimetric analysis diagram of the crystal form V is as follows Fig.15 shown.

[0078] According to one aspect of the invention, a method for preparing the crystalline form V of the compound of formula A is provided, the method comprising: adding an appropriate volume of solvent to the compound of formula A, magnetically stirring at 20 to 30° C. for 2 to 3 days, collecting the solid by centrifugation (10,000 to 14,000 rpm, centrifugation for 3 to 6 minutes), and naturally drying the solid in a fume hood.

[0079] In some embodiments of the present invention, in the method for preparing the crystalline form V of the compound of formula A, the solvent in the method comprises methyl tert-butyl ether.

[0080] In some embodiments of the present invention, the crystalline form of the compound represented by formula A is crystalline form VI.

[0081] In some embodiments of the present invention, the crystal form VI has an X-ray powder diffraction pattern having diffraction peaks at the following 2θ angles: 6.60±0.2°, 12.18±0.2°, and 20.28±0.2°.

[0082] In some embodiments of the present invention, the crystalline form VI has an X-ray powder diffraction pattern having diffraction peaks at the following 2θ angles: 6.60±0.2°, 9.92±0.2°, 12.18±0.2°, 18.08±0.2°, and 20.28±0.2°.

[0083] In some embodiments of the present invention, the crystalline form VI has an X-ray powder diffraction pattern having diffraction peaks at the following 2θ angles: 6.60±0.2°, 9.92±0.2°, 12.18±0.2°, 18.08±0.2°, 19.16±0.2°, 20.28±0.2°, and 25.00±0.2°.

[0084] In some embodiments of the present invention, the crystalline form VI has an X-ray powder diffraction pattern as shown in Fig.16 shown.

[0085] In some embodiments of the present invention, the crystal form VI, the X-ray powder diffraction pattern analysis data of the crystal form VI is shown in Table 6.

[0086] In some embodiments of the present invention, the crystalline form VI, the differential scanning calorimetry diagram of the crystalline form VI comprises an endothermic peak at 171.30±3°C.

[0087] In some embodiments of the present invention, the crystalline form VI has a differential scanning calorimetry diagram as shown in Fig.17 shown.

[0088] In some embodiments of the present invention, the crystalline form VI loses about 1.4% of its weight when heated to about 80°C.

[0089] In some embodiments of the present invention, the crystal form VI, the thermogravimetric analysis diagram of the crystal form VI is as follows Fig.18 shown.

[0090] According to one aspect of the invention, a method for preparing the crystalline form VI of the compound of formula A is provided, the method comprising: adding an appropriate volume of solvent to the compound of formula A, magnetically stirring at 40 to 60° C. for 2 to 3 days, collecting the solid by centrifugation (10,000 to 14,000 rpm, centrifugation for 3 to 6 min), and drying under reduced pressure and vacuum at 30 to 50° C.

[0091] In some embodiments of the present invention, in the method for preparing the crystalline form VI of the compound of formula A, the solvent in the method comprises isopropyl acetate.

[0092] In some embodiments of the present invention, the crystalline form of the compound represented by formula A is crystalline form VII.

[0093] In some embodiments of the present invention, the crystalline form VII has an X-ray powder diffraction pattern having diffraction peaks at the following 2θ angles: 17.74±0.2°, 21.30±0.2°.

[0094] In some embodiments of the present invention, the crystalline form VII, the X-ray powder diffraction pattern of the crystalline form VII has diffraction peaks at the following 2θ angles: 11.78±0.2°, 17.74±0.2°, 18.40±0.2°, 19.14±0.2°, 21.30±0.2°, 22.20±0.2°, 24.26±0.2°.

[0095] In some embodiments of the present invention, the crystalline form VII, the X-ray powder diffraction pattern of the crystalline form VII has diffraction peaks at the following 2θ angles: 5.82±0.2°, 11.78±0.2°, 17.74±0.2°, 18.40±0.2°, 19.14±0.2°, 20.68±0.2°, 21.30±0.2°, 22.20±0.2°, 24.26±0.2°, 25.88±0.2°.

[0096] In some embodiments of the present invention, the crystalline form VII has an X-ray powder diffraction pattern as shown in Fig.19 shown.

[0097] In some embodiments of the present invention, the crystal form VII, the X-ray powder diffraction pattern analysis data of the crystal form VII is shown in Table 7.

[0098] In some embodiments of the present invention, the crystalline form VII, the differential scanning calorimetry diagram of the crystalline form VII comprises endothermic peaks at 87.69±3°C and 172.33±3°C.

[0099] In some embodiments of the present invention, the crystalline form VII has a differential scanning calorimetry diagram as shown in Fig. 20 shown.

[0100] In some embodiments of the present invention, the crystalline form VII loses about 14.2% of its weight when heated to about 110°C, and loses about 1.8% of its weight when further heated to about 145°C.

[0101] In some embodiments of the present invention, the crystalline form VII, the thermogravimetric analysis diagram of the crystalline form VII is as follows Fig.21 shown.

[0102] According to one aspect of the invention, a method for preparing the crystalline form VII of the compound of formula A is provided, the method comprising: adding an appropriate volume of solvent to the compound of formula A, magnetically stirring at 20 to 30° C. for 2 to 3 days, and then collecting the solid by centrifugation (10,000 to 14,000 rpm, centrifugation for 3 to 6 min).

[0103] In some embodiments of the present invention, in the method for preparing the crystalline form VII of the compound of formula A, the solvent in the method comprises isopropyl acetate.

[0104] According to one aspect of the present invention, there is provided a pharmaceutical composition, comprising one or more of the above-mentioned Form I, Form II, Form III, Form IV, Form V, Form VI or Form VII and an optional pharmaceutically acceptable carrier, excipient, diluent, adjuvant or a combination thereof.

[0105] In some embodiments of the present invention, the pharmaceutical composition is used to treat diseases mediated by P2X3.

[0106] According to one aspect of the present invention, provided is a use of the above-mentioned Form I, Form II, Form III, Form IV, Form V, Form VI or Form VII in the preparation of a medicament for treating a disease mediated by P2X3.

[0107] According to one aspect of the present invention, there is provided a method for treating a disease mediated by P2X3, the method comprising administering a therapeutically effective amount of a compound of formula A, a crystalline form I of a compound of formula A, a pharmaceutical composition comprising crystalline form I of a compound of formula A, a crystalline form II of a compound of formula A, a pharmaceutical composition comprising crystalline form II of a compound of formula A, a crystalline form III of a compound of formula A, a pharmaceutical composition comprising crystalline form III of a compound of formula A, a crystalline form IV of a compound of formula A, a pharmaceutical composition comprising crystalline form IV of a compound of formula A, a crystalline form V of a compound of formula A, a pharmaceutical composition comprising crystalline form V of a compound of formula A, a crystalline form VI of a compound of formula A, a pharmaceutical composition comprising crystalline form VI of a compound of formula (I), a crystalline form VII of a compound of formula A, and a pharmaceutical composition comprising crystalline form VII of a compound of formula A.

[0108] In some embodiments of the present invention, the diseases include respiratory diseases, pain caused by various reasons, and skin diseases; the respiratory diseases include chronic obstructive pulmonary disease, asthma, acute, subacute and chronic cough, and bronchospasm; the pain includes surgical pain, inflammatory pain, cancer pain, bladder pain, endometriosis pain, diabetic neuropathy, traumatic pain, toothache, migraine, and pain associated with irritable bowel syndrome; the skin diseases include chronic itching.

[0109] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

[0110] Advantages of the present invention:

[0111] 1. The present invention prepares multiple new crystal forms of the compound of formula A for the first time through a large number of experiments and screenings, including crystal form I, crystal form II, crystal form III, crystal form IV, crystal form V, crystal form VI and crystal form VII;

[0112] 2. The partial crystal form prepared by the present invention has good stability, low hygroscopicity, and is easy to store. It can avoid the risk of crystal transformation during drug development or production, avoid changes in bioavailability and efficacy, and can be developed into a dosage form suitable for clinical use, which has strong economic value;

[0113] 3. The present invention also provides a method for preparing Form I, Form II, Form IV, Form VI and Form VII, which is simple to operate and has high reproducibility, does not easily leave solvent residues, is environmentally friendly, and has excellent potential for industrial implementation;

[0114] 4. The crystal form of the present invention has high in vitro inhibitory activity and subtype selectivity, good pharmacokinetic properties and significant efficacy, and has good prospects for drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0115] Figure 1 It is the X-ray powder diffraction (XRPD) pattern of Form I of the compound represented by Formula A.

[0116] Figure 2 It is a differential scanning calorimetry (DSC) diagram of Form I of the compound represented by Formula A.

[0117] Figure 3 This is a thermogravimetric analysis (TGA) diagram of Form I of the compound represented by Formula A.

[0118] Figure 4 It is the X-ray powder diffraction (XRPD) pattern of Form II of the compound represented by Formula A.

[0119] Figure 5 It is a differential scanning calorimetry (DSC) diagram of Form II of the compound represented by Formula A.

[0120] Figure 6 This is a thermogravimetric analysis (TGA) diagram of Form II of the compound represented by Formula A.

[0121] Figure 7 It is the X-ray powder diffraction (XRPD) pattern of Form III of the compound represented by Formula A.

[0122] Figure 8 It is a differential scanning calorimetry (DSC) diagram of Form III of the compound represented by Formula A.

[0123] Fig. 9 This is a thermogravimetric analysis (TGA) diagram of Form III of the compound represented by Formula A.

[0124] Fig.10 It is the X-ray powder diffraction (XRPD) pattern of Form IV of the compound represented by Formula A.

[0125] Fig.11 It is a differential scanning calorimetry (DSC) diagram of Form IV of the compound represented by Formula A.

[0126] Fig.12 This is a thermogravimetric analysis (TGA) diagram of Form IV of the compound represented by Formula A.

[0127] Fig.13 It is the X-ray powder diffraction (XRPD) pattern of Form V of the compound represented by Formula A.

[0128] Fig.14 It is a differential scanning calorimetry (DSC) diagram of Form V of the compound represented by Formula A.

[0129] Fig.15 This is a thermogravimetric analysis (TGA) diagram of Form V of the compound represented by Formula A.

[0130] Fig.16 It is the X-ray powder diffraction (XRPD) pattern of Form VI of the compound represented by Formula A.

[0131] Fig.17 It is a differential scanning calorimetry (DSC) diagram of Form VI of the compound represented by Formula A.

[0132] Fig.18 This is a thermogravimetric analysis (TGA) diagram of Form VI of the compound represented by Formula A.

[0133] Fig.19 It is the X-ray powder diffraction (XRPD) pattern of Form VII of the compound represented by Formula A.

[0134] Fig. 20 It is a differential scanning calorimetry (DSC) diagram of Form VII of the compound represented by Formula A.

[0135] Fig.21 This is a thermogravimetric analysis (TGA) diagram of Form VII of the compound represented by Formula A.

[0136] Fig. 22 The one-week and two-week stability XRPD results of Form I of the compound represented by Formula A.

[0137] Fig.23 The one-week and two-week stability XRPD results of Form II of the compound represented by Formula A.

[0138] Fig.24 The one-week and two-week stability XRPD results of Form IV of the compound represented by Formula A.

[0139] Fig.25 This is the DVS result of Form I of the compound represented by Formula A.

[0140] Fig.26 This is the DVS result of Form IV of the compound represented by Formula A.

[0141] Fig. 27 This is the XRPD result of the grinding experiment of Form I of the compound represented by Formula A.

[0142] Fig.28 This is the XRPD result of the grinding experiment of Form IV of the compound represented by Formula A. DETAILED DESCRIPTION

[0143] Definitions and general terms

[0144] Unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those of ordinary skill in the art to which the present invention belongs. All patent documents and publications to which the present invention relates are incorporated herein by reference in their entirety. Although any methods and materials similar or identical to those described in the present invention may be used in the practice or testing of the present invention, preferred methods, apparatus and materials are described in the present invention.

[0145] "Crystal form" or "crystalline form" refers to a solid having a highly regular chemical structure, including, but not limited to, single-component or multi-component crystals, and / or polymorphs, solvates, hydrates, inclusion compounds, co-crystals, salts, solvates of salts, hydrates of salts of compounds. The crystalline form of a substance can be obtained by many methods known in the art. Such methods include, but are not limited to, melt crystallization, melt cooling, solvent crystallization, crystallization in a confined space, for example, in a nanopore or capillary, crystallization on a surface or template, for example, on a polymer, crystallization in the presence of an additive such as a co-crystallization countermolecule, desolvation, dehydration, rapid evaporation, rapid cooling, slow cooling, vapor diffusion, sublimation, reactive crystallization, anti-solvent addition, grinding and solvent drop grinding, etc.

[0146] "Solvent" refers to a substance (typically a liquid) that is capable of completely or partially dissolving another substance (typically a solid). Solvents useful in the practice of the present invention include, but are not limited to, water, acetic acid, acetone, acetonitrile, benzene, chloroform, carbon tetrachloride, dichloromethane, dimethyl sulfoxide, 1,4-dioxane, ethanol, ethyl acetate, butanol, tert-butanol, N,N-dimethylacetamide, N,N-dimethylformamide, formamide, formic acid, heptane, hexane, isopropanol, methanol, methyl ethyl ketone, mesitylene, nitromethane, polyethylene glycol, propanol, pyridine, tetrahydrofuran, toluene, xylene, mixtures thereof, and the like.

[0147] "Solvate" refers to a compound having a solvent on the surface, in the crystal lattice, or on the surface and in the crystal lattice, and the solvent may be water, acetic acid, acetone, acetonitrile, benzene, chloroform, carbon tetrachloride, dichloromethane, dimethyl sulfoxide, 1,4-dioxane, ethanol, ethyl acetate, butanol, tert-butanol, N,N-dimethylacetamide, N,N-dimethylformamide, formamide, formic acid, heptane, hexane, isopropanol, methanol, methyl ethyl ketone, methyl pyrrolidone, mesitylene, nitromethane, polyethylene glycol, propanol, pyridine, tetrahydrofuran, toluene, xylene, and mixtures thereof, etc. A specific example of a solvate is a hydrate, in which the solvent on the surface, in the crystal lattice, or on the surface and in the crystal lattice is water. Hydrates may or may not have other solvents other than water on the surface, in the crystal lattice, or on the surface and in the crystal lattice of a substance.

[0148] The crystal form can be identified by a variety of technical means, such as X-ray powder diffraction (XRPD), infrared absorption spectroscopy (IR), melting point method, differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), nuclear magnetic resonance, Raman spectroscopy, X-ray single crystal diffraction, solution calorimetry, scanning electron microscopy (SEM), quantitative analysis, solubility and dissolution rate, etc.

[0149] X-ray powder diffraction (XRPD) can detect information such as changes in crystal forms, crystallinity, and crystal structure states, and is a common means of identifying crystal forms. The peak position of the XRPD spectrum depends mainly on the structure of the crystal form, is relatively insensitive to experimental details, and its relative peak height depends on many factors related to sample preparation and instrument geometry. Therefore, in some embodiments, the crystal form of the present invention is characterized by an XRPD pattern with certain peak positions, which is substantially as shown in the XRPD pattern provided in the accompanying drawings of the present invention. At the same time, the measurement of 2θ of the XRPD spectrum may have experimental errors, and the measurement of 2θ of the XRPD spectrum may be slightly different between different instruments and different samples, so the value of the 2θ cannot be regarded as absolute. According to the instrument conditions used in this experiment, there is an error tolerance of ±0.2° for the diffraction peak.

[0150] Differential scanning calorimetry (DSC) is a technique that measures the energy difference between a sample and an inert reference (usually α-Al2O3) as it changes with temperature by continuously heating or cooling under program control. The endothermic peak height of the DSC curve depends on many factors related to sample preparation and instrument geometry, while the peak position is relatively insensitive to experimental details. Therefore, in some embodiments, the crystal form of the present invention is characterized by a DSC graph with a characteristic peak position, which is substantially as shown in the DSC graph provided in the accompanying drawings of the present invention. At the same time, the DSC spectrum may have experimental errors, and the peak position and peak value of the DSC spectrum may be slightly different between different instruments and different samples, so the peak position or peak value of the DSC endothermic peak cannot be regarded as absolute. According to the instrument conditions used in this experiment, there is an error tolerance of ±3°C for the endothermic peak.

[0151] Thermogravimetric analysis (TGA) is a technique for measuring the mass change of a substance with temperature under program control. It is suitable for checking the loss of solvent in crystals or the process of sample sublimation and decomposition, and can infer the presence of water or crystallization solvent in the crystals. The mass change shown by the TGA curve depends on many factors such as sample preparation and instrumentation; the mass change detected by TGA varies slightly between different instruments and different samples. According to the instrument conditions used in the test of the present invention, the mass change has an error tolerance of ±0.1%.

[0152] In the context of the present invention, 2θ values ​​in X-ray powder diffraction patterns are given in degrees (°).

[0153] When referring to a spectrum and / or data appearing in a graph, a "peak" refers to a feature that can be identified by one skilled in the art and which cannot be attributed to background noise.

[0154] The present invention relates to the crystalline forms of the compound represented by formula A, for example, crystalline forms I to crystalline forms VII, which exist in substantially pure crystalline forms.

[0155] "Substantially pure" means that one crystalline form is substantially free of one or more other crystalline forms, that is, the purity of the crystalline form is at least 80%, or at least 85%, or at least 90%, or at least 93%, or at least 95%, or at least 98%, or at least 99%, or at least 99.5%, or at least 99.6%, or at least 99.7%, or at least 99.8%, or at least 99.9%, or the crystalline form contains other crystalline forms, and the percentage of the other crystalline forms in the total volume or total weight of the crystalline form is less than 20%, or less than 10%, or less than 5%, or less than 3%, or less than 1%, or less than 0.5%, or less than 0.1%, or less than 0.01%.

[0156] "Substantially free" means that the percentage of one or more other crystalline forms in the total volume or total weight of the crystalline form is less than 20%, or less than 10%, or less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1%, or less than 0.5%, or less than 0.1%, or less than 0.01%.

[0157] The "relative intensity" (or "relative peak height") in the XRPD pattern refers to the ratio of the intensity of other peaks to the intensity of the first strongest peak among all diffraction peaks in the X-ray powder diffraction pattern (XRPD) when the intensity of the first strongest peak is 100%.

[0158] In the context of the present invention, when or whether the words "about" or "approximately" are used, it means within 10%, suitably within 5%, and especially within 1% of a given value or range. Alternatively, for those of ordinary skill in the art, the term "about" or "approximately" means within an acceptable standard error range of the mean. Whenever a number having a value of N is disclosed, any number having a value within N+ / -1%, N+ / -2%, N+ / -3%, N+ / -5%, N+ / -7%, N+ / -8% or N+ / -10% will be explicitly disclosed, where "+ / -" means plus or minus.

[0159] Test method:

[0160] X-ray powder diffraction (XRPD) method of the present invention

[0161] The equipment is Shimadzu XRD-6000, and the samples are scanned according to the following parameters:

[0162] The radiation source is Cu~Kα target

[0163] The minimum operating voltage and current of the fluorescent tube are 40kV and 30mA respectively.

[0164] The sample scanning range was 2-Theta value from 5° to 50° and the scanning speed was 5 deg / min.

[0165] Thermogravimetric Analyzer (TGA) Method of the Present Invention

[0166] About 5 mg of sample was weighed into a crucible, and the temperature was raised from 30°C to 300°C under nitrogen protection at a rate of 20°C / min, and maintained at 300°C for 1 min.

[0167] Differential Scanning Calorimetry (DSC) Method of the Present Invention

[0168] Weigh about 1-5 mg of powder sample and place it in a closed aluminum crucible with a pinhole on the crucible cover. Under nitrogen protection, heat from 30°C to 300°C for differential thermal scanning, and keep at 300°C for 1 minute. The heating rate is 20°C / min.

[0169] Parameters of the HPLC method of the present invention

[0170]

[0171]

[0172] Specific preparation method

[0173] The present invention provides a preparation example of a compound of formula A and a crystal form thereof. Those skilled in the art can refer to the content of the present invention and appropriately improve the process parameters to achieve the above. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are deemed to be included in the present invention.

[0174] The method of the present invention has been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0175] In order to further understand the present invention, the present invention is described in detail below in conjunction with embodiments.

[0176] Example

[0177] Example 1 Synthesis of the compound represented by formula A (R)-4-(2-oxopyrrolidin-1-yl)-3-(4-methylphenyl)-N-((R)-1-(2-(trifluoromethyl)pyrimidin-5-yl)ethyl)-4,5-dihydro-1H-pyrazole-1-carboxamide

[0178] Step 1 Synthesis of intermediate 1-6:

[0179]

[0180] Step 1.1 Synthesis of intermediate 1-2:

[0181] 2-Pyrrolidone (1.0 g, 11.76 mmol) and dimethyl sulfate (1.11 mL, 11.76 mmol) were added to a 100 mL three-necked flask and stirred at 60 ° C for 16 h. TLC showed that the raw material disappeared (phosphomolybdic acid color development). The temperature was lowered to -5-0 ° C, and saturated potassium carbonate solution (20 mL) was added dropwise to the reaction flask at this temperature. After the addition was completed, the mixture was stirred at room temperature for 0.5 h, methyl tert-butyl ether was added for extraction (3*20 mL), the organic phases were combined, and dried over anhydrous sodium sulfate. The organic phase was concentrated to dryness under reduced pressure at 20 ° C to obtain the crude intermediate 1-2 (2-methoxy-1-pyrrolidine), a colorless oil, 0.61 g, yield = 52%, which was directly used in the next step.

[0182] Step 1.2 Synthesis of intermediate 1-3:

[0183] 1-2 (2-methoxy-1-pyrrolidine) (0.61 g, 6.16 mmol) and 2-bromo-4'-methylacetophenone (1.43 g, 6.77 mmol) were added to DMF (4 mL), and the mixture was stirred at 50 °C overnight under N2 protection. TLC was monitored until there was no starting material. The temperature was cooled to room temperature, and water (40 mL) was added. The mixture was stirred and extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness under reduced pressure. Intermediate 1-3 was separated by column chromatography (petroleum ether: ethyl acetate = 4:1 to 1:1) as a light yellow solid, 0.68 g, yield = 51%.

[0184] Step 1.3 Synthesis of intermediate 1-4:

[0185] 1-3 (0.68 g, 3.13 mmol), 37% formaldehyde solution (0.70 mL, 9.39 mmol) and piperidine (0.14 mL, 1.56 mmol) were added to dioxane (10 mL), stirred at 105 ° C for 6 h, and the mixture was cooled to room temperature after TLC monitoring until there was no raw material. The dioxane was evaporated under reduced pressure. 15 mL of ethyl acetate and 30 mL of saturated brine were added to the system, stirred for 5 min, and allowed to stand for stratification. The aqueous phase was extracted once with ethyl acetate (15 mL), and the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure to obtain the crude intermediate 1-4, a yellow oil, 1.35 g, which was directly used in the next step.

[0186] Step 1.4 Synthesis of intermediate 1-5:

[0187] 1-4 (1.35 g, 5.89 mmol) and hydrazine hydrate (0.45 mL, 9.42 mmol) were added to EtOH (5 mL), stirred at 80 ° C for 3 h under N2 protection, monitored by TLC until there was no starting material, cooled to room temperature, and ethanol in the system was evaporated under reduced pressure. Water was added, stirred, and extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure to obtain a crude intermediate 1-5 as a yellow oil, 1.63 g, which was directly used in the next step.

[0188] Step 1.5 Synthesis of intermediate 1-6:

[0189] Triphosgene (0.99 g, 3.35 mmol) and DCM (15 mL) were added to a three-necked flask, and the temperature of N2 protection was lowered to -10 to -5 ° C. 1-5 (1.63 g, 6.70 mmol) and pyridine (1.61 mL, 20.1 mmol) were added to DCM (15 mL), and added dropwise to the three-necked flask at -10 to -5 ° C. After the addition was completed, the mixture was stirred at room temperature for 10 min, and dilute hydrochloric acid was added to quench, and the mixture was extracted twice with DCM, and the organic phases were combined. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate, concentrated to dryness under reduced pressure, and separated by column chromatography (petroleum ether: ethyl acetate = 4:1 to 1:2) to obtain intermediate 1-6 as a light yellow solid, 0.14 g, and the total yield of three steps = 14%.

[0190] Step 2 Synthesis of intermediate 2-4:

[0191]

[0192] Step 2.1 Synthesis of intermediate 2-2:

[0193] 2-1 (5.0 g, 28.4 mmol), S-tert-butylsulfenamide (3.79 g, 31.3 mmol), anhydrous copper sulfate (9.06 g, 56.8 mmol) were added to 1,2-dichloroethane (70 mL), stirred at 50 ° C for 18 to 20 h, monitored by TLC until there was no raw material, cooled to room temperature, and filtered. The filtrate was concentrated by vacuum distillation and column chromatography (petroleum ether: ethyl acetate = 5:1 to 1:1) to obtain a white solid 2-2, 5.5 g, with a yield of 70%.

[0194] Step 2.2 Synthesis of intermediate 2-3:

[0195] 2-2 (4.0 g, 14.3 mmol) and toluene (80 mL) were added to a three-necked flask, and the temperature was lowered to -70 °C under N2 protection. Methyl lithium (1.6 M, 10.2 mL, 16.3 mmol) was slowly added dropwise to the three-necked flask at -70 °C and stirred at -70 °C for 1 h. Saturated ammonium chloride (50 mL) was added to quench and stirred at room temperature for 10 min. The mixture was allowed to stand and separated into layers. The aqueous phase was extracted three times with DCM. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure. 2-3 was obtained as a white solid by column chromatography (petroleum ether: ethyl acetate = 3:1 to 1:3). The yield was 30%.

[0196] Step 2.3 Synthesis of intermediate 2-4:

[0197] 2-3 (1.2 g, 4.06 mmol) and methanol (20 mL) were added to a 100 mL reaction bottle, and HCl (4.0 Min dioxane; 5 mL, 20 mmol) was added dropwise to the reaction bottle at room temperature, and stirred at room temperature for 1 h. The raw material disappeared after TLC detection. The reaction solution was spin-dried, 10 ml of potassium carbonate aqueous solution was added, and after stirring for 10 min, it was extracted four times with DCM: MeOH (10: 1), and the organic phase was spin-dried to obtain a light yellow oily intermediate 2-4, 0.72 g, with a yield of 80%.

[0198] Step 3: Synthesis of compound of formula A:

[0199]

[0200] 1-6 (1.04 g, 3.4 mmol) and 2-4 (0.62 g, 3.25 mmol) were added to DCM (10 mL), and DIPEA (1.6 mL, 9.75 mmol) was added, and the mixture was stirred at room temperature overnight, and hydrochloric acid (0.5 N, 10 mL) was added to quench the mixture and stirred for 10 min. The mixture was allowed to stand, separated, and the aqueous phase was extracted three times with DCM. The organic phases were combined, dried over anhydrous sodium sulfate, concentrated to dryness under reduced pressure, and separated by gradient chromatography on a silica gel column (petroleum ether: ethyl acetate = 2:1 to 1:3) to obtain 0.73 g of the compound of formula A (R)-4-(2-oxopyrrolidin-1-yl)-3-(4-methylphenyl)-N-((R)-1-(2-(trifluoromethyl)pyrimidin-5-yl)ethyl)-4,5-dihydro-1H-pyrazole-1-carboxamide, with a yield of 49%. 1H NMR (500MHz, CDCl3) δ8.94(s,2H),7.62(d,J=8.2Hz,2H),7.21(d,J=8.0Hz,2H),6. 36(d,J=6.7Hz,1H),6.07(dd,J=10.8,3.8Hz,1H),5.15(m,1H),4.00(dd,J=12.6,1 0.9Hz,1H),3.87(dd,J=12.6,3.8Hz,1H),3.23(m,1H),2.89(m,1H),2.37(d,J=4.5 Hz,3H),2.29(m,1H),1.98–1.91(m,1H),1.90–1.78(m,2H),1.66(d,J=7.1Hz,3H).

[0201] Example 2 Preparation methods of various crystalline forms of the compound of formula A

[0202] Weigh approximately 100 mg of the compound of formula A into a glass vial, add 0.6 mL of ethanol, stir the sample magnetically at 20-30°C for 2-3 days, and collect the solid by centrifugation (12000 rpm, centrifugation for 3-6 min). Dry the collected solid under reduced pressure and vacuum at 30-50°C overnight. The dried solid was characterized by XRPD (e.g. Figure 1 As shown), it was determined to be the crystalline form I of the compound of formula A, and DSC was detected at the same time (as Figure 2 as shown) and TGA (as Figure 3 shown).

[0203] Table 1: XRPD pattern analysis data of Form I of Formula A compound

[0204] Peak number 2θ(±0.2°) d value strength% Peak number 2θ(±0.2°) d value strength% 1 7.24 12.20 61.7 17 24.64 3.61 9.6 2 9.22 9.58 37.0 18 26.72 3.33 13.7 3 11.46 7.72 100.0 19 27.52 3.24 38.1 4 13.58 6.52 36.0 20 28.06 3.18 4.8 5 14.30 6.19 5.8 21 29.38 3.04 5.4 6 14.62 6.05 5.4 22 29.82 2.99 9.3 7 15.60 5.68 30.7 23 30.96 2.89 4.3 8 17.18 5.16 8.6 24 31.56 2.83 3.4 9 17.70 5.01 12.5 25 32.42 2.76 5.2 10 18.58 4.77 70.4 26 34.82 2.57 4.7 11 19.16 4.63 75.1 27 36.22 2.48 3.0 12 19.92 4.45 20.8 28 37.28 2.41 2.9 13 21.92 4.05 41.2 29 41.66 2.17 3.8 14 22.62 3.93 18.4 30 44.38 2.04 5.1 15 23.74 3.75 7.0 31 45.02 2.01 3.2 16 24.20 3.67 18.8 32 48.18 1.89 2.6

[0205] Weigh about 50 mg of the compound of formula A into a glass vial, add 0.6 mL of ethanol, heat to 40-60°C to dissolve, cool to 20-30°C, add 1.5 mL of water, stir for 1-2 hours, and collect the solid by centrifugation (12000 rpm, centrifugation for 3-6 minutes). Dry the collected solid under reduced pressure and vacuum at 30-50°C overnight. The dried solid was characterized by XRPD (such as Figure 4 As shown), it was determined to be the crystalline form II of the compound of formula A, and DSC was detected at the same time (as shown Figure 5 as shown) and TGA (as Figure 6 shown).

[0206] Table 2: XRPD pattern analysis data of Form II of Formula A compound

[0207] Peak number 2θ(±0.2°) d value strength% Peak number 2θ(±0.2°) d value strength% 1 9.80 9.02 70.0 12 24.90 3.57 100.0 2 12.76 6.93 15.2 13 25.90 3.44 7.5 3 14.78 5.99 13.3 14 27.12 3.29 15.0 4 15.78 5.61 12.7 15 29.38 3.04 13.8 5 18.06 4.91 35.5 16 30.00 2.98 5.7 6 19.12 4.64 14.5 17 30.42 2.94 5.5 7 19.90 4.46 5.2 18 31.28 2.86 5.4 8 21.08 4.21 12.7 19 34.08 2.63 4.2 9 22.22 4.00 10.2 20 38.89 2.31 3.7 10 22.84 3.89 42.3 21 46.76 1.94 3.7 11 23.28 3.82 8.1

[0208] Weigh about 50 mg of the compound of formula A into a glass vial, add 0.4 mL of methyl tert-butyl ether, stir the sample magnetically at 20-30°C for 2-3 days, and then collect the solid by centrifugation (10000-14000 rpm, centrifugation for 3-6 min). Dry the collected solid under reduced pressure and vacuum at 30-50°C overnight. The dried solid was characterized by XRPD (e.g. Figure 7 As shown), it was determined to be the crystalline form III of the compound of formula A, and DSC was detected at the same time (as Figure 8 as shown) and TGA (as Fig. 9 shown).

[0209] Table 3: XRPD pattern analysis data of Form III of Formula A compound

[0210] Peak number 2θ(±0.2°) d value strength% Peak number 2θ(±0.2°) d value strength% 1 6.86 12.88 62.6 10 20.54 4.32 27.1 2 9.34 9.46 8.3 11 22.22 4.00 31.3 3 11.76 7.52 100.0 12 23.28 3.82 6.7 4 13.04 6.78 5.4 13 26.52 3.36 17.1 5 13.62 6.50 5.3 14 28.76 3.10 6.0 6 14.55 6.08 4.3 15 29.44 3.03 6.1 7 15.94 5.56 9.9 16 32.82 2.73 4.7 8 18.16 4.88 51.8 17 34.20 2.62 4.6 9 19.08 4.65 23.9

[0211] Weigh approximately 50 mg of the compound of formula A into a glass vial, add 0.8 mL of isopropanol, stir the sample magnetically at 20-30°C for 2-3 days, and collect the solid by centrifugation (12000 rpm, centrifugation for 3-6 min). Dry the collected solid under reduced pressure and vacuum at 30-50°C overnight. The dried solid was characterized by XRPD (e.g. Fig.10 As shown), it was determined to be the crystalline form IV of the compound of formula A, and DSC was detected at the same time (as shown Fig.11 as shown) and TGA (as Fig.12 shown).

[0212] Table 4: XRPD pattern analysis data of Form IV of the compound of Formula A

[0213] Peak number 2θ(±0.2°) d value strength% Peak number 2θ(±0.2°) d value strength% 1 4.84 18.25 6.8 12 21.50 4.13 30.1 2 7.58 11.65 8.1 13 22.06 4.03 12.8 3 8.18 10.80 26.2 14 22.48 3.95 31.1 4 9.68 9.13 73.2 15 23.16 3.84 14.3 5 11.96 7.39 92.7 16 24.68 3.60 23.2 6 14.30 6.19 20.0 17 26.24 3.39 17.3 7 15.30 5.79 19.1 18 26.80 3.32 14.1 8 16.80 5.27 15.5 19 27.64 3.22 18.4 9 17.76 4.99 38.7 20 28.10 3.17 17.5 10 19.00 4.67 50.9 21 28.72 3.11 9.0 11 19.98 4.44 100.0 22 30.98 2.88 8.5

[0214] Weigh about 50 mg of the compound of formula A into a glass vial, add 0.8 mL of isopropanol, heat to 60-80°C to dissolve, cool to 20-30°C, add 2.4 mL of n-heptane and continue stirring for 8-12 hours, finally cool to 0-10°C, filter and collect the solid. Dry the collected solid under reduced pressure and vacuum at 30-50°C overnight. XRPD characterization of the dried solid is shown. Fig.10 The characteristics were consistent and it was determined to be the crystalline form IV of the compound of formula A. At the same time, DSC (with Fig.11 Characterization consistent) and TGA (with Fig.12 consistent representation).

[0215] Weigh approximately 50 mg of the compound of formula A into a glass vial, add 0.4 mL of methyl tert-butyl ether, stir the sample magnetically at 20-30°C for 2-3 days, and then collect the solid by centrifugation (10000-14000 rpm, centrifugation for 3-6 min). The solid was naturally evaporated in a fume hood. XRPD characterization of the solid (such as Fig.13 As shown), it was determined to be the crystalline form V of the compound of formula A, and DSC was detected at the same time (as shown Fig.14 as shown) and TGA (as Fig.15 shown).

[0216] Table 5: XRPD pattern analysis data of Form V of the compound of Formula A

[0217] Peak number 2θ(±0.2°) d value strength% Peak number 2θ(±0.2°) d value strength% 1 6.64 13.30 46.1 11 22.06 4.03 29.5 2 12.14 7.28 38.6 12 22.98 3.87 7.0 3 13.02 6.79 7.5 13 24.20 3.67 16.0 4 13.40 6.60 15.1 14 25.80 3.45 18.5 5 14.84 5.97 5.2 15 27.12 3.29 5.5 6 15.92 5.56 7.6 16 27.64 3.22 11.6 7 16.44 5.39 8.1 17 29.00 3.08 6.8 8 17.56 5.05 54.3 18 29.86 2.99 5.6 9 19.56 4.54 30.6 19 40.64 2.22 2.9 10 20.22 4.39 100.0

[0218] Weigh about 60 mg of the compound of formula A into a glass vial, add 0.4 mL of isopropyl acetate, dissolve the sample at 40-60°C, stir magnetically for 2-3 days, and collect the solid by centrifugation (12000 rpm, centrifugation for 3-6 min). Dry the collected solid under reduced pressure and vacuum at 30-50°C overnight. The dried solid was characterized by XRPD (e.g. Fig.16 As shown), it was determined to be the crystalline form VI of the compound of formula A, and DSC was detected at the same time (as shown Fig.17 as shown) and TGA (as Fig.18 shown).

[0219] Table 6: XRPD pattern analysis data of Form VI of the compound of Formula A

[0220] Peak number 2θ(±0.2°) d value strength% Peak number 2θ(±0.2°) d value strength% 1 5.02 17.59 6.0 11 20.28 4.38 94.7 2 6.60 13.38 100.0 12 20.74 4.28 16.8 3 8.32 10.62 17.1 13 21.64 4.10 15.3 4 9.92 8.91 66.0 14 22.16 4.01 11.3 5 12.18 7.26 92.8 15 22.64 3.92 28.7 6 13.24 6.68 24.7 16 25.00 3.56 37.2 7 14.46 6.12 9.8 17 26.26 3.39 10.7 8 16.06 5.51 8.7 18 27.78 3.21 18.6 9 18.08 4.90 58.3 19 28.18 3.16 10.0 10 19.16 4.63 37.9 20 30.72 2.91 10.6

[0221] Weigh approximately 50 mg of the compound of formula A into a glass vial, add 0.3 mL of isopropyl acetate, stir the sample magnetically at 20-30°C for 2-3 days, and collect the solid by centrifugation (12000 rpm, centrifugation for 3-6 min). The solid was naturally evaporated in a fume hood. The dried solid was characterized by XRPD (e.g. Fig.19 As shown), it was determined to be the crystalline form VII of the compound of formula A, and DSC was detected at the same time (as shown Fig. 20 as shown) and TGA (as Fig.21 shown).

[0222] Table 7: XRPD pattern analysis data of Form VII of the compound of Formula A

[0223] Peak number 2θ(±0.2°) d value strength% Peak number 2θ(±0.2°) d value strength% 1 5.82 15.18 30.0 16 22.20 4.00 45.5 2 7.94 11.13 10.9 17 23.36 3.81 6.8 3 9.02 9.80 11.7 18 23.82 3.73 6.5 4 11.78 7.51 40.9 19 24.26 3.67 42.8 5 13.52 6.54 9.9 20 25.88 3.44 33.5 6 14.38 6.15 20.6 21 26.86 3.32 14.2 7 15.76 5.62 10.0 22 27.74 3.21 23.9 8 16.24 5.45 24.8 23 28.24 3.16 9.2 9 17.28 5.13 11.2 24 29.24 3.05 9.1 10 17.74 5.00 100.0 25 29.68 3.01 13.3 11 18.40 4.82 39.7 26 30.96 2.89 5.7 12 19.14 4.63 39.4 27 32.88 2.72 8.1 13 20.22 4.39 20.4 28 33.42 2.68 5.8 14 20.68 4.29 30.2 29 39.94 2.26 4.9 15 21.30 4.17 78.4 30 44.84 2.02 4.8

[0224] Example 3 Stability Study of Crystalline Form

[0225] Weigh appropriate amounts of Form I, Form II and Form IV into 4 mL glass vials, cover the vials with pierced aluminum foil, and place them in an environment of 60°C and 40°C 75% RH. Samples were taken for HPLC testing after 1 week and 2 weeks. The test results are shown in Table 8. The XRPD comparison of stability is shown in Table 8. Fig. 22(From bottom to top in the figure: Form I placed for 0 days, Form I placed at 40°C 75% RH for 7 days, Form I placed at 40°C 75% RH for 14 days, Form I placed at 60°C for 7 days, Form I placed at 60°C for 14 days), Fig.23 (From bottom to top in the figure: Form II placed for 0 days, Form II placed at 40°C 75% RH for 7 days, Form II placed at 40°C 75% RH for 14 days, Form II placed at 60°C for 7 days, Form II placed at 60°C for 14 days), Fig.24 (From bottom to top in the figure, they are: Form IV placed at 0 days, Form IV placed at 40°C and 75% RH for 7 days, Form IV placed at 40°C and 75% RH for 14 days, Form IV placed at 60°C for 7 days, and Form IV placed at 60°C for 14 days); the experimental results are shown in Table 8.

[0226] Table 8 Stability test of crystal form

[0227]

[0228] *: Place in an open air (humidity greater than or equal to 75%) at 60°C.

[0229] The test results show that both the crystal form II and the crystal form IV of the present invention have high stability under the conditions of 40°C, 75% RH and 60°C.

[0230] Example 4 Hygroscopicity study of crystal form

[0231] Appropriate amounts of Form I and Form IV were weighed respectively for dynamic moisture sorption (DVS) test, and the experimental results are shown in Table 9. Among them, the description of hygroscopic characteristics and the definition of hygroscopic weight gain (Guidelines for hygroscopicity test of drugs in Appendix 9103 of the 2020 edition of the Chinese Pharmacopoeia, experimental conditions: 25°C ± 1°C, 80% ± 2% relative humidity) are as follows:

[0232] Deliquesce: Absorbs enough water to form a liquid;

[0233] Highly hygroscopic: weight gain due to moisture absorption is not less than 15%;

[0234] Hygroscopic: weight gain due to moisture absorption is less than 15% but not less than 2%;

[0235] Slightly hygroscopic: weight gain due to moisture absorption is less than 2% but not less than 0.2%;

[0236] No or almost no hygroscopicity: weight gain due to moisture is less than 0.2%.

[0237] From the experimental results in Table 9 below, it can be seen that the crystal form I and the crystal form IV of the present invention are slightly hygroscopic and are not easily deliquesced by high humidity. Fig.25 and Fig.26 The experimental results are shown in Table 9.

[0238] Table 9 Hygroscopicity test of crystal form

[0239]

[0240] Example 5 Grinding experiment of crystal form

[0241] The crystal form I was dry-milled for 30 seconds and 1 minute respectively to test XRPD. The crystal form I was wet-milled with water for 30 seconds and then tested for XRPD. The XRPD results are shown in Figure 2. Fig. 27 As shown (from bottom to top: initial crystal form I, crystal form I wet grinding for 30s, crystal form I dry grinding for 1min, initial crystal form II). The results show that the crystal form did not change after dry grinding for 30s and 1min, and the crystallinity decreased; after wet grinding for 30s, the crystal form became crystal form I + crystal form II.

[0242] Form IV was dry-milled for 30 seconds and wet-milled for 30 seconds, and then XRPD was tested. Fig.28 (From bottom to top: initial crystal form IV, crystal form IV dry grinding for 30s, crystal form IV wet grinding for 30s). The results show that the crystal form does not change after dry grinding for 30s and wet grinding for 30s.

[0243] In summary, the crystalline form IV of the compound represented by formula A of the present invention has certain advantages in terms of stability and hygroscopicity, and has good drugability.

[0244] Example 6 Pharmacokinetic Study

[0245] Test process:

[0246] Beagle dogs were given a single oral dose of compound A in different crystalline forms to study the pharmacokinetics of the compound in plasma. The test drug was divided into three dose groups: low (5 mg / kg), medium (10 mg / kg) and high (20 mg / kg). Blood was collected from the single-dose group before and 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 24 h, and 48 h after administration; about 1.0 mL of blood was collected from each animal at each time point, placed in a heparin anticoagulation tube, centrifuged at 1000 g for 10 min (4 ° C), and plasma was separated within 2 h and stored in a refrigerator at -80 to -75 ° C for testing. The concentration of compound A in plasma was detected by a validated LC-MS / MS analysis method, with a linear range of 2.00 to 2000 ng / mL and a lower limit of quantification of 2.00 ng / mL. The non-compartmental model of WinNonlin 8.3.5.340 software was used to calculate the pharmacokinetic parameters of dogs after administration, and the statistical moment method was used to calculate the AUC last , C max And other main pharmacokinetic parameters.

[0247] 6.1 Comparison of PK properties between Form I and Form II of the compound of Formula A

[0248] The comparison of Form I and Form II was conducted on the same 3 dogs (male), 5 mg / kg po, and the data obtained are shown in Table 10.

[0249] Table 10 PK comparison between Form I and Form II

[0250]

[0251] The exposure level and bioavailability of Form I are slightly lower than those of Form II, but there is no significant difference between the two.

[0252] 6.2 Pharmacokinetic study of the crystalline form IV of the compound of formula A. The results are shown in Table 11:

[0253] Table 11 Plasma pharmacokinetic data of dogs after single oral administration of different doses of Form IV

[0254]

[0255] After a single oral administration of 5, 10 and 20 mg / kg of Form IV, there was no significant gender difference in the exposure of Form IV in male and female dogs, and the average plasma elimination half-life was 3.7 to 4.7 h. The plasma C max and AUC last All of them increase with the increase of dose, showing a linear kinetic trend. last It is calculated that the bioavailability of the crystal form IV of the compound of formula A after oral administration to dogs is 79% to 88%, and the bioavailability of this crystal form is significantly better than that of the crystal form I and the crystal form II.

[0256] The following Examples A to D are biological evaluations of Form IV of the compound of Formula A:

[0257] Example A In vitro biological activity evaluation

[0258] Electrophysiological determination of the inhibitory activity of the crystalline form IV of the compound of formula A on P2X3

[0259] 1) Cell culture

[0260] HEK293 cell line was used as the cell line. HEK293 cells were cultured in a 37°C incubator containing 5% CO2. When the cells grew to 80%-90% density, they were passaged. The culture medium in the original 60mm culture dish was discarded, and the cells were washed 3 times with sterilized PBS solution and then the solution was aspirated. 1mL of 0.25% trypsin (Gibco) was added to digest for 10s, and after the trypsin was aspirated, 3mL of cell culture medium was added to resuspend the cells. 0.5mL of the resuspended solution was added to a new culture dish, and the total culture solution was supplemented to 5mL. The culture dish was gently shaken to evenly distribute the cells, and then placed in a 37°C incubator containing 5% CO2 for culture.

[0261] 2) Plasmid transfection

[0262] First, place the treated slides in a 35mm small culture dish, add 100μL of resuspended cells, and then add the total amount of culture medium to 2mL. Use calcium transfer to transfer the target gene into the HEK293 cell line. For the small dish to be transfected, change the medium 1h before transfection and add 2mL of fresh cell culture medium. Take a 1.5mL EP tube, add 200μL of 0.25M CaCl2 solution, add 3μg of the required transfection plasmid, and mix evenly to form a DNA-CaCl2 solution. Then slowly add this mixed solution to an equal volume of 2×HBS solution (NaCl 140mM, Na2HPO4 1.5mM, HEPES 50mM, adjust pH to 6.96), and use the pipette tip to mix while adding. After all are added, gently blow 8-10 times. After standing for 5min, add it to the small dish, shake the small dish slightly to mix the solution evenly, and then place it in the cell culture incubator. Change the culture medium after 8-10h. Electrophysiological experiments were performed 24-48 h after transfection.

[0263] 3) Electrophysiological experiments

[0264] Electrophysiological recording electrodes were obtained by two-step pulling using an electrode puller (PP-10, Narishige). After the internal solution was injected into the electrode, the water resistance was 3–5 MΩ. During the experiment, a high potassium salt internal solution containing nystatin (K2SO4 75mM, KCl120mM, MgSO4 5mM, HEPES10mM, pH adjusted to 7.2 using Tris-base, filtered and used) was used as the electrode internal solution. After the electrode was sealed with the cell, there was no need to further absorb and break the cell membrane. Instead, nystatin was used to perforate the cell membrane, causing it to drill holes in the cell membrane to form channels. This experiment used an Axon 200B amplifier and a Digidata 1440A digital-to-analog converter, and Clampfit was used to record data in voltage clamp mode. The clamping voltage was -60mv, the sampling frequency was 10kHz, and filtering was performed at 2kHz. The concentration of P2X3 receptor agonist ATP was 1 μM, the interval between two ATP administrations was 10 min, and all test compounds including the control AF-219 were pre-administered for 2 min before co-administration with ATP. The experimental results are shown in Table 12:

[0265] Table 12 Inhibitory activity of the crystalline form IV of the compound of formula A on human P2X3 (electrophysiological method)

[0266] Compound No. <![CDATA[hP2X3(IC 50 ,μM)]]> Form IV 0.082 AF-219 0.333

[0267] Compared with the positive compound AF-219, the crystalline form IV of the compound of formula A has stronger inhibitory activity against P2X3.

[0268] Example B In vitro P2X2 / 3 receptor selectivity evaluation

[0269] Using the same method as in Example A, the inhibitory activity of the obtained compounds on human P2X2 / 3 is shown in Table 13:

[0270] Table 13 Inhibitory activity of the crystalline form IV of the compound of formula A on human P2X2 / 3 (electrophysiological method)

[0271] Compound No. <![CDATA[P2X2 / 3(IC 50 ,μM)]]> Form IV 2.23±0.13

[0272] IC of Form IV against heteromeric hP2X2 / 3 receptors in human embryonic kidney 293 (HEK293) cells exogenously expressing heteromeric hP2X2 / 3 receptors at 1 μM ATP concentration 50 The result showed that the selectivity of Form IV for homomeric hP2X3 was about 20 times higher than that for heteromeric hP2X2 / 3.

[0273] Example C ATP+citrate cough model activity test

[0274] Guinea pigs were randomly divided into groups according to gender and cough frequency: (1) model control group; (2) AF-219 30 mg / kg group; (3) Formula A compound crystal IV 3 mg / kg group; (4) Formula A compound crystal IV 10 mg / kg group; (5) Formula A compound crystal IV 30 mg / kg group. Each group was given intragastric administration, and the guinea pigs were placed in a recording box for 1 minute, and the ATP solution was continuously atomized into the box with a compressed nebulizer inhaler for 2 minutes (spray flow rate 0.6 mL / min). Then, citric acid solution was atomized for 10 minutes, and then the number of coughs of the animals within 10 minutes and the time of the first cough (cough latency) were recorded using a multi-channel physiological signal acquisition and processing system. The results are shown in Table 14.

[0275] Table 14 Effect of Form IV of Formula A Compound on ATP+citrate-induced cough in guinea pigs

[0276] Group Dosage mg / kg Number of coughs (times) Inhibition rate (%) Model Group / 17.9 - AF-219 30 6.9 61.45 Form IV 3 9.4 47.49 Form IV 10 7.4 58.66 Form IV 30 5.7 68.16

[0277] As shown in the table, all compounds showed a trend of decreasing the number of coughs compared with the model group after treatment, among which the AF-219 positive group and crystal form IV could significantly reduce the number of coughs, and crystal form IV had a dose-dependent inhibitory effect. In the same dose group, compared with the positive AF-219, the cough inhibitory effect of crystal form IV of Formula A compound was stronger.

[0278] Example D In vitro metabolic stability test

[0279] The in vitro metabolic stability of the compounds of the present invention was determined using different liver microsome incubation methods. Take out the frozen liver microsomes, thaw on ice, and shake gently. Pipette the liver microsomes and add them to 100mM phosphate buffer, and then add the working solution of the administration group of the crystal form IV of the compound of formula A to make the microsome concentration 0.625mg / mL and the crystal form IV concentration of the compound of formula A to be 1.25μM. Pipette 80μL of the above solution, pre-incubate in a 37°C water bath for 5min, add 20μL of 5mM NADPH solution, and incubate in a 37°C water bath. The total volume of each metabolic incubation system sample is 100μL, and the incubation system includes liver microsomal protein with a final concentration of 0.5mg / mL, 1μM crystal form IV of the compound of formula A, and 1mM NADPH. After 0, 10, 30, 60, and 90 minutes of reaction, the reaction was terminated with 4 times the volume of acetonitrile (containing internal standard loratadine 1 ng / mL), vortexed and mixed, centrifuged at 4°C, 20000 rcf for 10 minutes, and the supernatant was taken for LC-MS / MS detection of the prototype drug content. The in vitro metabolic half-life of the test compound in various liver microsomes was obtained. The results are shown in Table 15.

[0280] Table 15 Half-life and intrinsic clearance of Form IV of Formula A in liver microsomes of various species

[0281]

[0282] The above contents are only basic descriptions of the concept of the present invention, and any equivalent changes made according to the technical solution of the present invention shall fall within the protection scope of the present invention.

[0283] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms are not necessarily directed to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0284] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A crystalline form IV of a compound as shown in formula A: It is characterized in that The X-ray powder diffraction pattern of the crystalline form IV of the compound of formula A has diffraction peaks at the following 2θ angles: 9.68±0.2°, 11.96±0.2°, 19.00±0.2°, and 19.98±0.2°.

2. The crystalline form IV according to claim 1, characterized in that The X-ray powder diffraction pattern of the crystalline form IV has diffraction peaks at the following 2θ angles: 9.68±0.2°, 11.96±0.2°, 17.76±0.2°, 19.00±0.2°, 19.98±0.2°, 21.50±0.2°, and 22.48±0.2°.

3. The crystalline form IV according to claim 1, characterized in that The X-ray powder diffraction pattern of the crystalline form IV has diffraction peaks at the following 2θ angles: 8.18±0.2°, 9.68±0.2°, 11.96±0.2°, 14.30±0.2°, 17.76±0.2°, 19.00±0.2°, 19.98±0.2°, 21.50±0.2°, 22.48±0.2°, and 24.68±0.2°.

4. The crystalline form IV according to any one of claims 1 to 3, characterized in that: The X-ray powder diffraction pattern of the crystalline form IV is shown in FIG10 .

5. The crystalline form IV according to any one of claims 1 to 3, characterized in that: The differential scanning calorimetry diagram of the Form IV comprises an endothermic peak at 170.95±3°C.

6. The crystalline form IV according to any one of claims 1 to 3, characterized in that: When the crystal form IV is analyzed by a thermogravimetric analyzer, the weight loss is about 0.02% when heated to about 150°C.

7. A method for preparing the crystalline form IV of the compound of formula A according to any one of claims 1 to 6, characterized in that: include: Add the compound of formula A to a solvent and stir at 20-35°C; or add the compound of formula A to a solvent, heat and dissolve it, and then stir; solid-liquid separation; or after the solution is cooled to 20-35°C, a poor solvent is added, stirring is continued, and then the temperature is cooled to 0-10°C for solid-liquid separation; drying is performed to obtain the crystal form IV.

8. The method of Form IV according to claim 7, characterized in that: The solvent is an alcohol solvent, preferably one or more of isopropanol, n-butanol and isobutanol.

9. The method of Form IV according to claim 7, characterized in that: The solid-liquid separation is achieved by centrifugation, wherein the centrifugation conditions are 10000-14000 rpm, centrifugation for 3-6 minutes; drying is performed by reduced pressure vacuum drying at a temperature of 30-50°C; the stirring time at 20-35°C is 2-12 hours; and the heating stirring temperature is 40-60°C.

10. A crystalline form I of the compound represented by formula A as claimed in claim 1, characterized in that: The X-ray powder diffraction pattern of the crystalline form I has diffraction peaks at the following 2θ angles: 7.24°±0.2°, 9.22°±0.2°, 11.46°±0.2°, 13.58°±0.2°, 15.60°±0.2°, 18.58°±0.2°, 19.16°±0.2°, 21.92°±0.2°, 27.52°±0.2°; or, the differential scanning calorimetry diagram of the crystalline form I includes an endothermic peak of 169.46±3°C; or, when the crystalline form I is analyzed using a thermogravimetric analyzer, when heated to about 90°C, the weight loss is about 5.6%.

11. A crystalline form II of the compound represented by formula A as claimed in claim 1, characterized in that: The X-ray powder diffraction pattern of the crystalline form II has diffraction peaks at the following 2θ angles: 9.80±0.2°, 18.06±0.2°, 22.84±0.2°, 24.90±0.2°; or, the differential scanning calorimetry diagram of the crystalline form II contains endothermic peaks at 81.47±3°C and 132.62±3°C; or, when the crystalline form II is analyzed using a thermogravimetric analyzer, when heated to about 90°C, the weight loss is about 3.2%.

12. A crystalline form III of the compound represented by formula A as claimed in claim 1, characterized in that: The X-ray powder diffraction pattern of the crystalline form III has diffraction peaks at the following 2θ angles: 6.86±0.2°, 11.76±0.2°, 18.16±0.2°, 20.54±0.2°, 22.22±0.2°; or, the differential scanning calorimetry diagram of the crystalline form III includes endothermic peaks at 155.41±3°C and 173.55±3°C, and an exothermic peak at 160.48±3°C; or, when the crystalline form III is analyzed using a thermogravimetric analyzer, the weight loss is about 2.2% when heated to about 150°C.

13. A crystalline form V of the compound represented by formula A as claimed in claim 1, characterized in that: The X-ray powder diffraction pattern of the crystalline form V has diffraction peaks at the following 2θ angles: 6.64±0.2°, 12.14±0.2°, 17.56±0.2°, 19.56±0.2°, 20.22±0.2°; or, the differential scanning calorimetry diagram of the crystalline form V comprises endothermic peaks at 154.03±3°C and 172.16±3°C, and an endothermic peak at 159.54±3°C; or, when the crystalline form V is analyzed using a thermogravimetric analyzer, the weight loss is approximately 7.9% when heated to about 145°C.

14. A crystalline form VI of the compound represented by formula A as claimed in claim 1, characterized in that: The X-ray powder diffraction pattern of the crystalline form VI has diffraction peaks at the following 2θ angles: 6.60±0.2°, 9.92±0.2°, 12.18±0.2°, 18.08±0.2°, 20.28±0.2°; the differential scanning calorimetry diagram of the crystalline form VI includes an endothermic peak at 171.30±3°C; when the crystalline form VI is analyzed using a thermogravimetric analyzer, the weight loss is approximately 1.4% when heated to about 80°C.

15. A crystalline form VII of the compound represented by formula A as claimed in claim 1, characterized in that: The X-ray powder diffraction pattern of the crystalline form VII has diffraction peaks at the following 2θ angles: 11.78±0.2°, 17.74±0.2°, 18.40±0.2°, 19.14±0.2°, 21.30±0.2°, 22.20±0.2°, 24.26±0.2°; or the differential scanning calorimetry diagram of the crystalline form VII contains endothermic peaks at 87.69±3°C and 172.33±3°C; or when analyzed by a thermogravimetric analyzer, the crystalline form VII loses about 14.2% of its weight when heated to about 110°C, and loses about 1.8% of its weight when further heated to about 145°C.

16. A pharmaceutical composition, characterized in that It includes one or more of the crystalline form IV according to any one of claims 1 to 6, the crystalline form I according to claim 10, the crystalline form II according to claim 11, the crystalline form III according to claim 12, the crystalline form V according to claim 13, the crystalline form VI according to claim 14, and the crystalline form VII according to claim 15, and optionally a pharmaceutically acceptable carrier, excipient, diluent, adjuvant or a combination thereof.

17. Use of the crystalline form IV according to any one of claims 1 to 6, the crystalline form I according to claim 10, the crystalline form II according to claim 11, the crystalline form III according to claim 12, the crystalline form V according to claim 13, the crystalline form VI according to claim 14, or the crystalline form VII according to claim 15 in the preparation of a P2X3 receptor antagonist or a drug; the drug may be a drug for protecting, treating, curing or alleviating a disease at least partially mediated by or activity-related to P2X3; or, the drug may be a drug for treating respiratory diseases, pain caused by various reasons, or skin diseases.

18. The use according to claim 17, characterized in that The respiratory diseases include chronic obstructive pulmonary disease, asthma, acute, subacute and chronic cough, bronchospasm; the pain includes surgical pain, inflammatory pain, cancer pain, bladder pain, endometriosis pain, diabetic neuropathy, traumatic pain, toothache, migraine, and pain associated with irritable bowel syndrome; the skin diseases include chronic pruritus.

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