TW202342468A - A crystalline form of p2x3 receptor antagonist and preparation method thereof - Google Patents

Abstract

The invention relates to a crystalline form of P2X3 receptor antagonist and preparation method thereof. In particular, the present disclosure provides the crystal form of compound (S) - 1 - (4 - (1 - ((4-acetylmorpholine-2-yl) methyl) - 5 - methyl- 1H - benzo [d] imidazol - 2- yl) - 3 - chloro - 5 - fluorophenyl) pyrrolidin - 2 - one and preparation method thereof.

Description

Crystal form of P2X3 receptor antagonist and preparation method thereof

The present disclosure belongs to the field of medical technology and relates to a crystalline form of a P2X3 receptor antagonist and a preparation method thereof.

This application claims priority from Chinese patent application 202210127962.8 with a filing date of 2022/2/11. This application cites the full text of the above-mentioned Chinese patent application.

The P2X3 receptor has four ATP-binding sites on a single subunit and is composed of two transmembrane domains, the N-terminus and C-terminus located in the cell, and a conserved sequence located in the extracellular loop structure. High expression of P2X3 receptors was found in small and medium-diameter neurons specifically related to injury information. At the same time, P2X3 receptors are also involved in the transmission of some non-nociceptive sensations. It has been confirmed that P2X3 receptor is involved in bladder sensory function and is a key receptor that mediates bladder sensory signals and is expressed in bladder mucosal tissue containing abundant sensory nerve fibers. P2X3 is also expressed in the sensory nerve fibers of the pharyngeal mucosa, which is related to the conduction and formation of taste.

When the organism is injured or nerve damaged, a large amount of ATP is released, activating the presynaptic membrane P2X3 receptor, causing a large amount of Ca ²⁺ influx, and the intracellular calcium concentration increases to activate protein kinase A (PKA), protein kinase C ( protein kinase C, PKC), phosphorylates PKA and PKC, and promotes the release of glutamate, further activating NMDA receptors, resulting in the generation of excitatory postsynaptic currents and central sensitization. Many studies have shown that upregulation of P2X3 receptor expression can lead to the formation of pain sensitivity and participate in pain signal transmission.

MK-7264 (Gefapixant) is a sulfonamide drug that acts as an antagonist of the purine P2X receptor P2RX3 and can be used to treat chronic cough.

( S )-1-(4-(1-((4-ethylmorpholin-2-yl)methyl)-5-methyl-1H-benzo[d]imidazol-2-yl)-3 -Chloro-5-fluorophenyl)pyrrolidin-2-one (shown below, WO2022033567) is a new P2X3 antagonist. In vitro experiments show that it has excellent inhibitory activity on P2X3 and has excellent in vivo metabolic properties. .

The crystal structure of the active ingredient of a drug often affects the physical and chemical stability of the drug. Different crystallization conditions and storage conditions may lead to changes in the crystal structure of the compound, sometimes accompanied by the production of other crystal forms. Generally speaking, amorphous pharmaceutical products do not have regular crystal structures, and are often accompanied by defects such as poor product stability, fine crystallization, difficulty in filtration, easy agglomeration, and poor fluidity. In view of the importance of solid drug crystal forms and their stability in clinical treatment, an in-depth study of compound ( S )-1-(4-(1-((4-ethylmorpholin-2-yl)methyl)- The solid form of 5-methyl-1H-benzo[d]imidazol-2-yl)-3-chloro-5-fluorophenyl)pyrrolidin-2-one is useful for the development of drugs suitable for industrial production and with good biological activity of great significance.

The present disclosure provides compound (S)-1-(4-(1-((4-acetylmorpholin-2-yl)methyl)-5-methyl-1H-benzo[d]imidazole-2 X-ray powder diffraction pattern expressed in diffraction angle 2θ of crystal form A of -3-chloro-5-fluorophenyl)pyrrolidin-2-one, at 11.750, 13.120, 19.224, 20.324 and There is a characteristic peak at 23.625.

In some embodiments, the Form A crystalline form has an There are characteristic peaks everywhere.

In some embodiments, the Form A crystalline form has an X-ray powder diffraction pattern expressed as a diffraction angle 2θ at 7.135, 9.212, 9.946, 11.750, 12.656, 13.120, 14.216, 14.471, 17.170, 18.615, 19.224 There are characteristic peaks at , 20.324, 20.905, 22.081, 23.625, 25.216 and 27.718.

In some embodiments, the Form A crystalline form has an X-ray powder diffraction pattern expressed as a diffraction angle 2θ at 7.135, 9.212, 9.946, 11.082, 11.750, 12.656, 13.120, 14.216, 14.471, 16.558, 17.170 There are characteristic peaks at , 18.615, 19.224, 20.324, 20.905, 21.637, 22.081, 23.312, 23.625, 24.343, 25.216, 25.719, 26.058, 27.718, 29.504 and 30.269.

In other embodiments, the X-ray powder diffraction pattern of Form A expressed as a diffraction angle 2θ is as shown in Figure 2.

In other embodiments, the 2θ angle error range is ±0.20.

Another aspect of the present disclosure also provides a method for preparing the aforementioned crystal form A, the method comprising: method one: (a) Compound (S)-1-(4-(1-((4-acetylmorpholin-2-yl)methyl)-5-methyl-1H-benzo[d]imidazole-2 -(yl)-3-chloro-5-fluorophenyl)pyrrolidin-2-one is mixed with the solvent (I), stirred to dissolve or heated to dissolve, the solvent (I) is selected from alcohol solvents, ketone solvents, esters One or more solvents, (b) Crystallization; Method Two: (a) Compound (S)-1-(4-(1-((4-acetylmorpholin-2-yl)methyl)-5-methyl-1H-benzo[d]imidazole-2 -3-Chloro-5-fluorophenyl)pyrrolidin-2-one is mixed with the solvent (II), stirred to dissolve or heated to dissolve, the solvent (II) is selected from one of ethanol, acetonitrile and tetrahydrofuran or Many kinds, (b) Add solvent (III) and crystallize. The solvent (III) is selected from one or more of methyl tertiary butyl ether, n-heptane, and isopropyl ether; Method three: (a) Compound (S)-1-(4-(1-((4-acetylmorpholin-2-yl)methyl)-5-methyl-1H-benzo[d]imidazole-2 -3-chloro-5-fluorophenyl)pyrrolidin-2-one is mixed with solvent (IV), stirred and beaten, and the solvent (IV) is selected from one of MTBE, cyclohexane and isopropyl ether. or more; Method four: (a) Compound (S)-1-(4-(1-((4-acetylmorpholin-2-yl)methyl)-5-methyl-1H-benzo[d]imidazole-2 -(yl)-3-chloro-5-fluorophenyl)pyrrolidin-2-one is mixed with the solvent (V), and evaporates and crystallizes. The solvent (V) is selected from DMSO.

In some embodiments, the solvent (I) is selected from one or more of ethanol, isopropyl alcohol, acetone, methyl isobutyl ketone, 2-butanone, ethyl acetate and isopropyl acetate.

On the other hand, in some embodiments, the volume (ml) of solvent (I), solvent (II), solvent (IV), and solvent (V) is compound ( S )-1-(4-(1-((4 -Acetylmorpholin-2-yl)methyl)-5-methyl-1H-benzo[d]imidazol-2-yl)-3-chloro-5-fluorophenyl)pyrrolidin-2-one 1-200 times the weight, specifically 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 200 times or any two numbers in between.

The crystallization methods of the present disclosure include but are not limited to stirring crystallization, static crystallization or volatilization crystallization. In some embodiments, the crystallization is stirred crystallization. In some embodiments, the crystallization is static crystallization.

The preparation methods described in the present disclosure also include centrifugation (filtration), washing or drying steps.

In other embodiments, the method for preparing the aforementioned compounds also includes one or more steps of filtration, washing or drying. The drying is preferably drying under reduced pressure with a pressure of <-0.08MPa.

The present disclosure also provides a pharmaceutical composition, which contains the aforementioned A crystal form or the A crystal form prepared by the aforementioned method, and pharmaceutical excipients optionally selected from pharmaceutically acceptable excipients.

The present disclosure also provides a pharmaceutical composition prepared from the aforementioned crystal form A and optional pharmaceutically acceptable excipients.

The present disclosure also provides a method for preparing a pharmaceutical composition, which includes the step of mixing the aforementioned crystal form A or the crystal form A prepared by the aforementioned method and a pharmaceutically acceptable excipient.

The present disclosure also provides the use of the aforementioned A crystal form or the A crystal form prepared by the aforementioned method or the aforementioned composition in preparing a medicament for treating or preventing diseases related to P2X3 activity.

The present disclosure also provides the use of the aforementioned A crystal form or the A crystal form prepared by the aforementioned method or the aforementioned composition in the preparation of medicaments for treating or preventing pain, urinary tract diseases or cough.

The present disclosure also provides X-ray powder diffraction patterns expressed in diffraction angles 2 theta at 11.750, 13.120, 19.224, 20.324 and 23.625 for Form A for the treatment or prevention of pain, urinary tract disease or cough. There are characteristic peaks.

According to the description of hygroscopic characteristics and the definition of hygroscopic weight gain in the "9103 Guidelines for Hygroscopicity of Drugs" in the 2015 edition of the Chinese Pharmacopoeia, Part Four, Deliquescence: Absorbing enough water to form a liquid; Extremely hygroscopic: weight gain by moisture absorption is not less than 15%; Hygroscopic: weight gain due to moisture absorption is less than 15% but not less than 2%; Slightly hygroscopic: hygroscopic weight gain is less than 2% but not less than 0.2%; No or almost no hygroscopicity: moisture absorption weight gain is less than 0.2%.

In some embodiments, Form A of the present disclosure is slightly hygroscopic.

The "2 θ or 2 θ angle" mentioned in this disclosure refers to the diffraction angle, θ is the Bragg angle, and the unit is ° or degree; the error range of each characteristic peak 2 θ is ±0.20 (including more than 1 decimal place) The figures after rounding), specifically -0.20, -0.19, -0.18, -0.17, -0.16, -0.15, -0.14, -0.13, -0.12, -0.11, -0.10, -0.09, -0.08, - 0.07, -0.06, -0.05, -0.04, -0.03, -0.02, -0.01, 0.00, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20.

"Excipients" as used in this disclosure include, but are not limited to, any auxiliary, carrier, glidant, sweetener, diluent that has been approved by the U.S. Food and Drug Administration as acceptable for use in humans or livestock animals. , preservatives, dyes/colorants, flavoring agents, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents or emulsifiers.

The "beating" mentioned in this disclosure refers to a method of purification that utilizes the characteristics of substances with poor solubility in solvents but good solubility of impurities in solvents. Beating and purification can remove color, change crystal form, or remove a small amount of impurities.

The starting material used in the method for preparing the crystal form of the present disclosure can be any form of the compound represented by Formula I. Specific forms include but are not limited to: amorphous, any crystal form, hydrate, solvate, etc.

"Differential scanning calorimetry or DSC" as described in this disclosure refers to measuring the temperature difference and heat flow difference between the sample and the reference during the process of heating or constant temperature of the sample to characterize all physical changes related to thermal effects and Chemical changes to obtain phase change information of the sample.

The drying temperature described in this disclosure is generally 25°C to 100°C, preferably 40°C to 70°C, and more preferably 45°C to 55°C. It can be dried under normal pressure or under reduced pressure. The pressure is <-0.08MPa, and the drying time is 5 ~10h, preferably 7~8h.

The numerical values in this disclosure, such as the content of relevant substances, are measured and calculated data, and there is inevitably a certain degree of error. Generally speaking, ±10% is within a reasonable error range. There is a certain degree of error change depending on the context where it is used. The error change does not exceed ±10% and can be ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2% or ±1%, preferably ±5%.

Compound A: ( S )-1-(4-(1-((4-acetylmorpholin-2-yl)methyl)-5-methyl-1H-benzo[d]imidazol-2-yl )-3-chloro-5-fluorophenyl)pyrrolidin-2-one. The preparation method refers to the method in WO2022033567, and the relevant content is quoted from this text for explanation.

Test conditions for the instruments used in the experiments in this disclosure:

1. Differential Scanning Calorimeter (DSC) Instrument model: Mettler Toledo DSC 3+STARe System Purge gas: nitrogen; nitrogen purge speed: 50 mL/min Heating rate: 10.0 ℃/min Temperature range: 25-250℃

2. X-ray Powder Diffraction (XRPD) Instrument model: BRUKER D8 Discover X-ray powder diffractometer Ray: Monochromatic Cu-Kα ray (λ=1.5406) Scanning method: θ/2θ, scanning range (2θ range): 3~50° Voltage: 40kV, current: 40mA

3. Thermogravimetric Analysis (TGA) Instrument model: Mettler Toledo TGA2 Purge gas: nitrogen; nitrogen purge speed: 50 mL/min Heating rate: 10.0℃/min Temperature range: 30-350℃

4. DVS is dynamic moisture adsorption The test uses Surface Measurement Systems advantage 2. At 25°C, the humidity ranges from 50% -95%-0%-95%-50%RH in steps of 10%. The judgment standard is that the change in dM/dT of each gradient is less than 0.002%. , TMAX 360min, two laps.

5. The structure of the compound is determined by nuclear magnetic resonance (NMR) or/and mass spectrometry (MS). NMR shifts (δ) are given in units of 10-6 (ppm). NMR was measured using Bruker AVANCE NEO 500M. The measurement solvents were deuterated dimethyl styrene (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD). The internal standard was tetramethylsilane (TMS). . MS was measured using Agilent 1200/1290 DAD-6110/6120 Quadrupole MS liquid mass spectrometer (manufacturer: Agilent, MS model: 6110/6120 Quadrupole MS). waters ACQuity UPLC-QD/SQD (manufacturer: waters, MS model: waters ACQuity Qda Detector/waters SQ Detector). THERMO Ultimate 3000-Q Exactive (Manufacturer: THERMO, MS model: THERMO Q Exactive)

6. Known starting materials can be synthesized by methods known in the art, or can be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc, and Da Rui Chemical and other companies.

7. HPLC determination uses Agilent 1260DAD high performance liquid chromatograph (ACE Excel C18 150×4.6mm column) and Thermo Dionex Ultimate 3000 high pressure liquid chromatograph (Waters Xbridge C18 150×4.6mm column).

The present disclosure will be explained in more detail below with reference to embodiments or experimental examples. The embodiments or experimental examples in the present disclosure are only used to illustrate the technical solutions in the present disclosure and do not limit the essence and scope of the present disclosure.

Example 1

Step 1: (R)-2-(((methanesulfonyl)oxy)methyl)morpholine-4-carboxylic acid tertiary butyl ester (1b) at 0°C under nitrogen protection, add to (2R)-2- To a solution of (hydroxymethyl)morpholine-4-carboxylic acid tertiary butyl ester (100 g, 460.28 mmol) in toluene (1000 mL), triethylamine (96 mL, 690.41 mmol) and methanesulfonyl chloride (49.63 mL, 635.71 mmol), and the reaction mixture was stirred at 30°C for 12 hours. TLC (petroleum ether/ethyl acetate = 1/1) showed that the raw material reaction was complete. The reaction solution was filtered. The filtrate was washed with water (500 mL x 3) and brine (500 mL x 3), dried over anhydrous sodium sulfate, filtered, and concentrated. The title compound 1b was obtained (135 g, yield: 99%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 4.24 (d, J = 4.8 Hz, 2H), 3.92 (d, J = 11.2 Hz, 3H), 3.74-3.65 (m, 1H), 3.55 (dt, J = 2.4, 11.6 Hz, 1H), 3.08-3.06 (m, 3H), 3.04-2.91 (m, 1H), 2.79 (d, J = 14.4 Hz, 1H), 1.47 (s, 9H).

Step 2: (S)-2-((1,3-dioxoisoindolin-2-yl)methyl)morpholine-4-carboxylic acid tertiary butyl ester (1c) was added to compound 1b (135 g , 457.07 mmol) of N,N-dimethylacetamide (1350 mmol) was added to a solution of phthalimide potassium salt (93.12 g, 502.78 mmol) and tetrabutylammonium bromide (14.73 g, 45.71 mmol). The reaction mixture was stirred at 80°C for 6 hours. TLC (petroleum ether/ethyl acetate = 1/1) showed complete consumption of starting material. Filter the reaction mixture, pour the filtrate into water (3L), filter, and stir the filter cake with N-methylpyrrolidone-water (1/5, 2.4 L) at 25°C for 12 hours, then filter and dry under reduced pressure. Filter the cake to obtain the title compound 1c (120 g, yield: 73%). MS (ESI) m/z =291.1 [M-56+H] ⁺ . ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.86 (dd, J = 3.2, 5.2 Hz, 2H), 7.76-7.69 (m, 2H ), 4.04-3.83 (m, 3H), 3.82-3.71 (m, 2H), 3.67 (dd, J = 4.8, 13.5 Hz, 1H), 3.44 (dt, J = 2.8, 11.2 Hz, 1H), 3.05- 2.92 (m, 1H), 2.76 (br. s, 1H), 1.45 (s, 9H).

Step 3: (S)-2-(Aminomethyl)morpholine-4-carboxylic acid tertiary butyl ester (1d) was added to a solution of compound 1c (100 g, 288.70 mmol) in 2-methyltetrahydrofuran (1000 mmol). Add hydrazine (10.40 mL, 181.88 mmol). The reaction mixture was stirred at 80°C for 3 hours, filtered, and the filtrate was concentrated. The residue was stirred in ethyl acetate (200 mmol) for 30 min, filtered, and concentrated under reduced pressure to give the title compound 1d (68 g, yield: 87%). MS (ESI) m/z =217.1 [M+H] ⁺ . ¹ H NMR (400 MHz, CDCl ₃ ) δ 3.88-3.84 (m, 3H), 3.59-3.45 (m, 1H), 3.37-3.25 (m , 1H), 2.97-2.82 (m, 1H), 2.73 (d, J = 1.2 Hz, 1H), 2.71 (d, J = 3.6 Hz, 1H), 2.61-2.56 (m, 1H), 1.45 (s, 9H).

Step 4: (S)-2-((2-(4-bromo-2-chloro-6-fluorophenyl)-5-methyl-1H-benzo[d]imidazol-1-yl)methyl) Morpholine-4-carboxylic acid tertiary butyl ester (1f) To a solution of compound 1d (67 g, 248.18 mmol) in dimethylstyrene (350 mL) was added calcium carbonate (27 g, 270.74 mmol) and 1-fluoro -4-Methyl-2-nitrobenzene (35 g, 225.62 mmol). The reaction mixture was stirred at 110°C for 12 hours. TLC (petroleum ether/ethyl acetate=1/2) showed complete consumption of starting material. The reaction mixture was cooled to 47 °C and EtOH (350 mL), sodium dithionite (157 g, 902.47 mmol) and 4-bromo-2-chloro-6-fluorobenzaldehyde (43 g, 180.49 mmol) were added. The reaction mixture was stirred at 90°C for 16 hours. Pour the reaction mixture into water (2 L), extract with ethyl acetate (800 mL x 3), concentrate the organic layer, dissolve the residue in ethyl acetate (1.5 L), add water (1 L x 3) and Wash with brine (800 mL x 2), dry over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain the title compound 1f (113 g, yield: 93%). MS (ESI) m/z = 538.1 [M+H] ⁺ . ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.64 (s, 1H), 7.56-7.52 (m, 1H), 7.40-7.32 (m, 2H ), 7.20 (d, J = 8.4 Hz, 1H), 4.11-3.96 (m, 3H), 3.76-3.67 (m, 2H), 3.61-5.57 (m, 1H), 3.33-3.29 (m, 1H), 2.79 (t, J = 10.4 Hz, 1H), 2.51 (s, 3H), 2.50-2.46 (m, 1H), 1.42 (s, 9H).

Step 5: (R)-1-(3-chloro-5-fluoro-4-(5-methyl-1-(morpholin-2-ylmethyl)-1H-benzo[d]imidazole-2- (113 g, 152.92 mmol) in dihexane (1000 mL), add pyrrolidin-2-one (47 mL, 611.67 mmol) , cesium carbonate (100 g, 305.84 mmol), bis(dibenzylideneacetone)palladium (8.79 g, 15.29 mmol), 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene (17.70 g, 30.58 mmol). The reaction system was replaced with nitrogen three times, and the reaction mixture was stirred at 100°C for 2 hours under nitrogen. Then p-toluenesulfonic acid (29 g, 1529.18 mmol) was added at 25°C and stirred at 100°C for 1 hour. Pour the reaction mixture into water (1000 mL), extract with ethyl acetate (800 mLx6), and adjust the aqueous phase to pH=9 with 1 mol/L sodium hydroxide solution. Then the aqueous phase was extracted with dichloromethane (800 mL x 5), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the title compound 1h (113 g, yield: 93%). MS (ESI) m/z = 443.1 [M+H] ⁺ . ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.74-7.65 (m, 1H), 7.61 (br. s, 2H), 7.38-7.36 (m , 1H), 7.16 (d, J = 8.4 Hz, 1H), 4.15-3.91 (m, 2H), 3.87 (t, J = 7.2 Hz, 2H), 3.75-3.64 (m, 2H), 3.49-3.34 ( m, 2H), 2.75-2.69 (m, 2H), 2.66 (t, J = 8.0 Hz, 2H), 2.50 (s, 3H), 2.45-2.33 (m, 2H), 2.31-2.25 (m, 1H) , 2.21 (t, J = 7.6 Hz, 2H), 2.15-2.09 (m, 1H), 2.15-2.08 (m, 1H).

Step 6: (S)-1-(4-(1-((4-acetylmorpholin-2-yl)methyl)-5-methyl-1H-benzo[d]imidazol-2-yl )-3-Chloro-5-fluorophenyl)pyrrolidin-2-one (I) To a solution of compound 1h (480 mg, 1.08 mmol) in tetrahydrofuran (2.5 mL) was added saturated sodium bicarbonate solution ( 2.5 mL) and acetic anhydride (221.3 mg, 2.17 mmol). The mixture was stirred at room temperature for 0.5 hours, then diluted with ethyl acetate and separated. The aqueous phase was extracted with ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate, filtered and concentrated. The crude product (500 mg) was stirred in n-hexane/ethyl acetate (1/1, 4 mL) overnight. The precipitate was filtered and dried to give the title product (400.0 mg, yield: 76%). After X-ray powder diffraction detection, the product was amorphous, and the XRPD is shown in Figure 1. MS (ESI) m/z = 485.4 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 7.88 (br s, 1H), 7.79 (br d, J = 11.6 Hz, 1H), 7.70 - 7.60 (m, 1H), 7.49 (br s, 1H), 7.20 - 7.12 (m, 1H), 4.33 - 4.16 (m, 2H), 4.03 (br d, J = 15.0 Hz, 1H), 3.97 - 3.87 (m, 3H), 3.78 (br d, J = 17.6 Hz, 1H), 3.68 - 3.55 (m, 1H), 3.12 (br d, J = 11.0 Hz, 1H), 2.99 (br d, J = 11.6 Hz, 1H), 2.81 - 2.70 (m, 2H), 2.58 (br d, J = 7.9 Hz, 2H), 2.44 (br s, 3H), 2.14 - 2.06 (m, 2H), 1.93 (s, 2H) .

Test example 1, in vitro biological activity evaluation

The FLIPR assay is used to screen the antagonistic activity of compounds on hP2X3 and hP2X2/3 receptors (the change in calcium flux signal represents the effect of compounds on ion channels).

1. Experimental instruments and materials Instrument name Equipment manufacturer Model FLIPR Molecular Devices TETRA _CO2 incubator Thermo Fisher 3111 Reagents and consumables names Source Item number FLIPR® Calcium 4 Assay Kit Molecular Devices R8141 FLIPR pipette Tips Molecular Devices 9000-0764 Matrix DARTs Tips-30ul Thermo 5416 FBS Gibco 10099-141 DMEM Gibco 11965 Hygromycin B Invitrogen 10687010 G418 DISULFATE SALT SIGMA G5013 384-well Assay Plate Corning CC3712 384-well Compound Plate Corning CC3657 Echo qualified, 384-well polypropylene microplate LABCYTE P-05525 Probenecid Sigma P8761-25G 1xHBSS Invitrogen 14025 ATP hydrolytic enzyme Sigma A7646 HEPES Invitrogen 15630-080 Versene Gibco 15040066 αβ-meATP Sigma M6517 Stable cell line Chempartner 1321N1/hP2X3, 1321N1/hP2X2/3

2. Experimental steps: Digest the 1321N1 cells (adherent cells) stably transfected with hP2X3 and hP2X2/3 receptors, centrifuge, resuspend and count in plating medium (DMEM + 10% DFBS), and adjust the cells to 2*10 ⁵ cells/mL, spread 50 μL cells in each well of the 384-well Assay Plate, and place in a 5% CO ₂ , 37°C incubator for 16-24 hours. Use DMSO to prepare 180 times the required concentration of the test compound (20 mM DMSO stock solution), add 500 nL from each well to the 384-well Compound Plate, and supplement 30 μL FLIPR buffer (1* HBSS + containing 1.26 mM Ca ²⁺ 2 mM CaCl ₂ + 20 mM HEPES), shake for 20-40 min to mix. Use FLIPR buffer to prepare 3 times the required concentration of agonist α, β-meATP (hP2X3 cells require a final concentration of 500 nM, hP2X2/3 cells require a final concentration of 1000 nM), and add 35 μL of agonist to each well to another 384- well Compound Plate. Take out the cell plate that has been cultured for 16-24 hours, aspirate the cell supernatant, add 30 μL Dye (FLIPR® Calcium 4 Assay Kit, diluted in FLIPR buffer) to each well, and incubate for 1 hour. Add 15 μL of compound to each well of cells (FLIPR instrument). After 15 minutes, add 22.5 μL of agonist to each well and detect the fluorescence signal (excitation light wavelength 470 nm-495 nm, emission light wavelength 515 nm-575 nm). Take the difference between the peak value and the valley value of the signal as the basic data, use the highest concentration data of the positive drug as the 100% inhibition rate, and the DMSO data as the 0% inhibition rate. Fit the inhibitory effect curve of the compound on the software Graphpad Prism 6 and calculate the IC ₅₀ value. Table 1. Half inhibitory concentration (IC ₅₀ ) of the compounds of the present invention on hP2X3 and hP2X2/3 receptors No. hP2X3 (IC ₅₀ , nM) hP2X2/3 (IC ₅₀ , nM) I 31.6 1927

Test example 2, CYP inhibition experiment Representative substrate metabolic responses of the five major human CYP isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4/5) were evaluated using 150 donor pooled human liver microsomes (purchased from Corning, Cat. No. 452117). Liquid chromatography tandem mass spectrometry (LC/MS/MS) was used to determine the response of test compounds at different concentrations to phenacetin (CYP1A2), diclofenac sodium (CYP2C9), S-mephenytoin (CYP2C19), and bufuralol hydrochloride. Effects of salt (CYP2D6) and midazolam (CYP3A4/5) metabolic reactions.

30 μM phenacetin, 10 μM diclofenac sodium, 35 μM S-mephenytoin, 5 μM bufurolol hydrochloride, 3 μM midazolam, 1 mM NADPH, and the test compound (the concentrations are respectively 0.1, 0.3, 1, 3, 10, 30 μmol/L) or the reaction system of positive compound or blank control and mixed human liver microsomes (0.2 mg/mL) 200 μL (100 mmol/L phosphate buffer, pH 7.4 , containing 0.3% DMSO, 0.6% acetonitrile, and 0.1% methanol by volume) and incubated at 37 °C for 5 minutes. Then add 200 μL of acetonitrile solution containing 3% formic acid and 40 nM internal standard verapamil, and centrifuge at 4000 rpm for 50 minutes. Place on ice to cool for 20 minutes, then centrifuge at 4000 rpm for 20 minutes to precipitate protein. Take 200 μL of supernatant for LC/MS/MS analysis.

Peak areas were calculated from the chromatograms. The residual activity ratio (%) is calculated using the following formula: Peak area ratio = metabolite peak area / internal standard peak area

Residual activity ratio (%) = peak area ratio of the compound group to be tested / peak area ratio of the blank group

The CYP half inhibitory concentration (IC ₅₀ ) was calculated by Excel XLfit 5.3.1.3. The measured CYP half inhibitory concentration (IC ₅₀ ) values are shown in Table 2. Table 2. 50% inhibitory concentrations (IC ₅₀ ) of compounds on CYP No. CYP 1A2 (μM) CYP 2C9 (μM) CYP 2C19 (μM) CYP 2D6 (μM) CYP 3A4/5 (μM) I >30 >30 >30 >30 >30

Test Example 3: In vitro metabolic stability test of human liver cells. Use LC/MS/MS to measure the concentration of the compound in the reaction system to calculate the intrinsic clearance rate of the compound to be tested and evaluate the in vitro metabolic stability of human hepatocytes. . Add 247.5 μL of 1×10 ⁶ cells/mL human hepatocyte (purchased from BioreclamationIVT, Cat. No. S01205) mixture and 2.5 μL of 100 μM test compound or positive control to the incubation plate to start the reaction. Incubate at 37 ^° C and 600 rpm. Transfer 20 μL of the incubation system to the stop plate at 0.5, 5, 15, 30, 45, 60, 80, 100 and 120 minutes respectively. Then mix by vortexing for 2 minutes. Centrifuge the stop plate at 4000 rpm for 20 minutes. Transfer 40 μL of the supernatant of each compound to a 96-well injection plate, and then add 160 μL of pure water to dilute the sample.

The resulting samples were quantified from ion chromatograms. Calculate the residual rate based on the peak area of the test compound or positive control. The slope k was determined from a linear regression of the natural logarithm of the residual rate versus incubation time using Microsoft Excel.

Intrinsic clearance (in vitro CL _int , μL/min/ ¹⁰ cells) was calculated from the slope value according to the following equation: in vitro CL _int = kV/N V = incubation volume (0.25 mL); N = number of cells per well (0.25×10 ⁶ cells) The measured intrinsic clearance rate values of human hepatocytes are shown in Table 3. Table 3. Intrinsic clearance rates of compounds in human hepatocytes No. Intrinsic clearance rate (μL/min/10 ⁶ cells) I <1

Example 2 Weigh about 8 mg of compound A, add 0.04 mL of isopropyl alcohol to dissolve, stir for crystallization, centrifuge, and vacuum dry to obtain a solid. After X-ray powder diffraction detection, the product is crystal form A. The XRPD spectrum is shown in Figure 2, and its characteristic peak positions are shown in Table 4. The DSC spectrum shows that the endothermic peak peak is 198.77°C. The TGA spectrum shows that the weight loss is 0.27% at 30-210℃. Table 4 Peak number 2θ value [° or degree] d[Å] Relative strength % 1 7.135 12.37901 34.4 2 9.212 9.59269 33.7 3 9.946 8.88639 32.6 4 11.082 7.97763 12.0 5 11.750 7.52533 56.7 6 12.656 6.98862 32.9 7 13.120 6.74280 89.3 8 14.216 6.22526 30.9 9 14.471 6.11603 47.6 10 16.558 5.34959 11.1 11 17.170 5.16036 27.5 12 18.615 4.76278 36.3 13 19.224 4.61337 86.6 14 20.324 4.36603 100.0 15 20.905 4.24603 32.4 16 21.637 4.10384 21.9 17 22.081 4.02240 25.7 18 23.312 3.81263 25.7 19 23.625 3.76293 66.1 20 24.343 3.65352 10.5 twenty one 25.216 3.52895 49.9 twenty two 25.719 3.46106 16.9 twenty three 26.058 3.41678 13.0 twenty four 26.751 3.32985 5.3 25 27.718 3.21580 25.3 26 28.367 3.14368 6.8 27 29.504 3.02514 14.8 28 30.269 2.95036 16.4 29 31.402 2.84644 5.1 30 31.666 2.82331 5.0 31 33.019 2.71069 4.5 32 33.975 2.63652 6.0 33 34.998 2.56179 8.1

Example 3 Weigh about 8 mg of compound A, add 0.04 ml of acetone to dissolve, the solid precipitates after 5 minutes, centrifuge, and vacuum dry to obtain a solid. After X-ray powder diffraction detection, the X-ray diffraction pattern was consistent with Example 2, and the product was crystal form A.

Example 4 Weigh about 8 mg of compound A, add 0.04 mL of ethyl acetate to dissolve, and solid precipitate after 5 min. Centrifuge and dry under vacuum to obtain a solid. After X-ray powder diffraction detection, the X-ray diffraction pattern was consistent with Example 2, and the product was crystal form A.

Example 5 Weigh about 8 mg of compound A, add 0.8 mL of MTBE, beat at room temperature, centrifuge, and vacuum dry to obtain a solid. After X-ray powder diffraction detection, the X-ray diffraction pattern was consistent with Example 2, and the product was crystal form A.

Example 6 Weigh about 8 mg of compound A, add 0.8 mL of cyclohexane, beat at room temperature, centrifuge, and vacuum dry to obtain a solid. After X-ray powder diffraction detection, the X-ray diffraction pattern was consistent with Example 2, and the product was crystal form A.

Example 7 Weigh about 8 mg of compound A, add 0.04 ml of DMSO, stir and dissolve at room temperature, and slowly evaporate the solution sample at room temperature to obtain a solid. After X-ray powder diffraction detection, the X-ray diffraction pattern was consistent with Example 2, and the product was crystal form A.

Example 8 Weigh about 7 mg of compound A, add 0.07 ml of ethanol, stir and dissolve at room temperature, add 0.7 ml of MTBE, stir, and the solid will precipitate, centrifuge, and dry under vacuum to obtain a solid. After X-ray powder diffraction detection, the X-ray diffraction pattern was consistent with Example 2, and the product was crystal form A.

Example 9 Weigh about 7 mg of compound A, add 0.07 ml of acetonitrile, stir and dissolve at room temperature, add 0.7 ml of isopropyl ether, stir, and the solid will precipitate, centrifuge, and dry under vacuum to obtain a solid. After X-ray powder diffraction detection, the X-ray diffraction pattern was consistent with Example 2, and the product was crystal form A.

Example 10 Weigh about 7 mg of compound A, add 0.07 ml of tetrahydrofuran, stir and dissolve at room temperature, add 0.7 ml of isopropyl ether, stir, and the solid precipitates, centrifuge, and dry under vacuum to obtain a solid. After X-ray powder diffraction detection, the X-ray diffraction pattern was consistent with Example 2, and the product was crystal form A.

Example 11 Using Surface Measurement Systems advantage 2, at 25°C, the humidity starts from 50%, the humidity range is 0%-95%, the step is 10%, the judgment standard is that the change of each gradient quality dM/dT is less than 0.002 %, the execution time TMAX of each humidity gradient is 360min, and there are two loops. test article 0.0%RH-95.0%RH 0%RH-80.0%RH Crystal form Form A 1.92% 1.49% (slightly hygroscopic) Not converted

Example 12 The crystal form A of the compound represented by formula (I) was placed flat in the open, and the effects under light (4500 Lux), high temperature (40°C, 60°C), and high humidity (RH 75%, RH 92.5%) were investigated. ) conditions, the sampling inspection period is 30 days. table 5 condition time (days) Crystal form A of the compound represented by formula (I) color, properties Purity % Crystal form start 0 Off-white solid 99.5 Form A 40℃ 5 Off-white solid 99.4 Not converted 10 Off-white solid 99.5 Not converted 30 Off-white solid 99.3 Not converted 60℃ 5 Off-white solid 99.2 Not converted 10 Off-white solid 99.4 Not converted 30 Off-white solid 99.3 Not converted 75%RH 5 Off-white solid 99.3 Not converted 10 Off-white solid 99.4 Not converted 30 Off-white solid 99.4 Not converted 92.5% RH 5 Off-white solid 99.3 Not converted 10 Off-white solid 99.3 Not converted 30 Off-white solid 99.4 Not converted 4500 Lux 5 Off-white solid 99.3 Not converted 10 Off-white solid 99.3 Not converted 30 Off-white solid 99.4 Not converted Conclusion: Experiments on influencing factors show that the physical and chemical stability of the crystalline form A of the compound represented by formula (I) is good under the conditions of light, high temperatures of 40°C and 60°C, and high humidity of 75% and 92.5% for 30 days.

Example 13 The crystal form A of the compound represented by formula (I) was placed under the conditions of -20°C, 4°C, 25°C/60%RH and 40°C/75%RH respectively to investigate the stability. Table 6 sample Placement conditions Purity % Purity % Purity % Purity % Purity % Crystal form start 1 month 2 months 3 months 6 months Form A -20℃ 99.5 99.5 99.3 99.4 99.4 Not converted 4℃ 99.5 99.3 99.3 99.3 99.3 Not converted 25℃/60%RH 99.5 99.3 99.4 99.5 99.4 Not converted 40℃/75%RH 99.5 99.3 99.4 99.4 99.4 Not converted Conclusion: Long-term accelerated experiments show that the physical and chemical stability of crystalline form A of the compound represented by formula (I) is good under the conditions of -20℃, 4℃, 25℃/60RH and 40℃/75RH for 6 months.

without

Figure 1: Amorphous XRPD pattern of the compound. Figure 2: XRPD pattern of compound Form A.

Claims (10)

A compound (S)-1-(4-(1-((4-ethylmorpholin-2-yl)methyl)-5-methyl-1H-benzo[d]imidazol-2-yl) -Crystalline Form A of 3-chloro-5-fluorophenyl)pyrrolidin-2-one, in which the X-ray powder diffraction pattern expressed as a diffraction angle 2θ is at 11.750, 13.120, 19.224, 20.324 and 23.625 There are characteristic peaks at 7.135, 9.212, 9.946, 11.750, 13.120, 14.471, 18.615, 19.224, 20.324, 23.625 and 25.216, and more preferably at 7.135, 9.212, 9.946, 11.750, 12.656, 1 3.120, 14.216, There are characteristic peaks at 14.471, 17.170, 18.615, 19.224, 20.324, 20.905, 22.081, 23.625, 25.216 and 27.718. Crystal form A as described in claim 1, wherein the X-ray powder diffraction pattern expressed in the diffraction angle 2θ is at 7.135, 9.212, 9.946, 11.082, 11.750, 12.656, 13.120, 14.216, 14.471, 16.558, There are characteristic peaks at 17.170, 18.615, 19.224, 20.324, 20.905, 21.637, 22.081, 23.312, 23.625, 24.343, 25.216, 25.719, 26.058, 27.718, 29.504 and 30.269. The A crystal form according to claim 1 or 2, wherein the X-ray powder diffraction pattern represented by the diffraction angle 2 θ is as shown in Figure 2. The A crystal form according to any one of claims 1 to 3, wherein the 2θ angle error range is ±0.20. A method for preparing the A crystal form described in any one of claims 1-4, selected from any of the following methods, Method 1: (a) Compound ( S )-1-(4-(1-((4- Acetylmorpholin-2-yl)methyl)-5-methyl-1H-benzo[d]imidazol-2-yl)-3-chloro-5-fluorophenyl)pyrrolidin-2-one and The solvent (I) is mixed, stirred and dissolved or heated to dissolve. The solvent (I) is selected from one or more alcohol solvents, ketone solvents, and ester solvents, preferably ethanol, isopropyl alcohol, acetone, and methyl isobutyl. One or more of base ketone, 2-butanone, ethyl acetate and isopropyl acetate, (b) crystallization; Method 2: (a) Compound ( S )-1-(4-(1-((( 4-acetylmorpholin-2-yl)methyl)-5-methyl-1H-benzo[d]imidazol-2-yl)-3-chloro-5-fluorophenyl)pyrrolidine-2- The ketone is mixed with the solvent (II) and stirred to dissolve or heated to dissolve. The solvent (II) is selected from one or more of ethanol, acetonitrile and tetrahydrofuran. (b) Add the solvent (III) and crystallize. The solvent (III) ) is selected from one or more of methyl tertiary butyl ether, n-heptane, and isopropyl ether; Method three: (a) Compound ( S )-1-(4-(1-((4-acetyl ether) (morpholin-2-yl)methyl)-5-methyl-1H-benzo[d]imidazol-2-yl)-3-chloro-5-fluorophenyl)pyrrolidin-2-one and solvent ( IV) Mixing, stirring and beating, the solvent (IV) is selected from one or more of MTBE, cyclohexane and isopropyl ether; Method 4: (a) Compound ( S )-1-(4-(1- ((4-acetylmorpholin-2-yl)methyl)-5-methyl-1H-benzo[d]imidazol-2-yl)-3-chloro-5-fluorophenyl)pyrrolidine- The 2-ketone is mixed with the solvent (V), and the solvent (V) is selected from DMSO to evaporate and crystallize. A pharmaceutical composition prepared from the crystal form A described in any one of claims 1 to 4. A pharmaceutical composition containing the A crystal form described in any one of claims 1 to 4 or the A crystal form prepared by the method described in claim 5, and optionally selected from pharmaceutically acceptable excipients. A method for preparing a pharmaceutical composition, including the step of mixing the crystal form described in any one of claims 1 to 4 or the crystal form prepared by the method described in claim 5 and a pharmaceutically acceptable excipient. The A crystal form described in any one of claims 1 to 4 or the A crystal form prepared by the method described in claim 5 or the composition described in claim 6 or 7 is used for the treatment or prevention of diseases related to P2X3 activity. uses in medicines. The A crystal form described in any one of claims 1 to 4 or the A crystal form prepared by the method described in claim 5 or the composition described in claim 6 or 7 is used for the treatment or prevention of pain and urinary tract diseases. or use in cough medicines.