A pharmaceutically acceptable salt of a p2x3 receptor antagonist and a process for its preparation

Abstract

The present disclosure relates to a pharmaceutically acceptable salt of a P2X3 receptor antagonist and a method of preparing the same. Specifically, the present disclosure provides a pharmaceutically acceptable salt of 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 a method of preparing the same.

Description

[0001] This application claims priority to Chinese patent application 202210127699.2, filed on February 11, 2022. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This disclosure belongs to the field of pharmaceutical technology and relates to a pharmaceutically acceptable salt of a P2X3 receptor antagonist and its preparation method. Background Technology

[0003] The P2X3 receptor has four ATP-binding sites on a single subunit, consisting of two transmembrane domains, an intracellular N-terminus and a C-terminus, and a conserved sequence in an extracellular loop. High expression of the P2X3 receptor has been found in small- to medium-diameter neurons specifically associated with nociceptive information. Simultaneously, the P2X3 receptor is also involved in the transmission of some non-nociceptive sensations. It has been confirmed that the P2X3 receptor is involved in bladder sensory function, serving as a key receptor-mediated bladder sensory signal, and is expressed in bladder mucosa tissue rich in sensory nerve fibers. P2X3 is also expressed in sensory nerve fibers of the pharyngeal mucosa, and it is involved in the transmission and formation of taste.

[0004] When the body is injured or nerves are damaged, a large amount of ATP is released, activating the presynaptic membrane P2X3 receptors and causing a surge in calcium. 2+ Influx of calcium into the cell increases intracellular calcium concentration, activating protein kinase A (PKA) and protein kinase C (PKC), leading to phosphorylation of PKA and PKC. This also promotes glutamate release, further activating NMDA receptors and resulting in excitatory postsynaptic currents, causing central sensitization. Many studies have shown that upregulation of P2X3 receptor expression can lead to pain hypersensitivity and participate in pain signal transmission.

[0005] 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.

[0006] (S)-1-(4-(1-((4-acetylmorpholin-2-yl)methyl)-5-methyl-1H-benzo[d]imidazol-2-yl)-3-chloro-5-fluorophenyl)pyrrolidone-2-one (as shown below, WO2022033567) is a novel P2X3 antagonist. In vitro experiments have shown that it has excellent inhibitory activity against P2X3, and also exhibits excellent in vivo metabolic performance.

[0007] Summary of the Invention

[0008] This disclosure provides a pharmaceutically acceptable salt of the compound (S)-1-(4-(1-((4-acetylmorpholin-2-yl)methyl)-5-methyl-1H-benzo[d]imidazol-2-yl)-3-chloro-5-fluorophenyl)pyrrolidone, wherein the pharmaceutically acceptable salt is selected from hydrochloride, sulfate, methanesulfonate, phosphate, acetate, fumarate, maleate, tartrate, succinate, and citrate.

[0009]

[0010] In some embodiments, the chemical ratio of the compound represented by Formula I to the acid molecule is selected from 1:0.5 to 1:3. In some embodiments, the chemical ratio of the compound represented by Formula I to the acid molecule is selected from 1:0.5, 1:1, 1:2, or 1:3. In some embodiments, the chemical ratio of the compound represented by Formula I to the acid molecule is selected from 1:1.

[0011] In some embodiments, the pharmaceutically acceptable salt of the compound represented by Formula I is a hydrochloride salt. In other embodiments, the pharmaceutically acceptable salt of the compound represented by Formula I is a hydrochloride salt.

[0012] This disclosure also provides a method for preparing the aforementioned pharmaceutically acceptable salt, comprising the step of reacting (S)-1-(4-(1-((4-acetylmorpholin-2-yl)methyl)-5-methyl-1H-benzo[d]imidazol-2-yl)-3-chloro-5-fluorophenyl)pyrrolidone-2-one with an acid selected from hydrochloric acid, sulfuric acid, methanesulfonic acid, phosphoric acid, acetic acid, fumaric acid, maleic acid, tartaric acid, succinic acid, and citric acid.

[0013] In some embodiments, the solvent used for the salt formation reaction is selected from at least one of methanol, isopropyl acetate, ethanol, isopropanol, tetrahydrofuran, acetone, acetonitrile, and water.

[0014] In some embodiments, the volume (μl) of the solvent used in the salt-forming reaction described in this disclosure can be 1-200 times the mass (mg) of the compound shown in formula (I), and in non-limiting embodiments, it can be 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 of these values.

[0015] This disclosure also provides the I crystal form of the hydrochloride salt of compound (S)-1-(4-(1-((4-acetylmorpholin-2-yl)methyl)-5-methyl-1H-benzo[d]imidazol-2-yl)-3-chloro-5-fluorophenyl)pyrrolidine-2-one.

[0016] In some embodiments, the I crystal form, in its X-ray powder diffraction pattern expressed as a diffraction angle 2θ, has characteristic peaks at 6.784, 12.078, 20.759, 23.920, 24.193, and 26.185.

[0017] In some embodiments, the X-ray powder diffraction pattern of the I crystal form, expressed as a diffraction angle 2θ, has characteristic peaks at 6.784, 7.867, 10.489, 12.078, 14.256, 20.759, 21.816, 23.920, 24.193, 24.872, and 26.185.

[0018] In some embodiments, the X-ray powder diffraction pattern of the I crystal form, expressed as a diffraction angle 2θ, has characteristic peaks at 6.784, 7.090, 7.867, 10.489, 12.078, 13.674, 14.256, 18.328, 18.438, 20.045, 20.759, 21.816, 23.920, 24.193, 24.872, and 26.185.

[0019] In some embodiments, the X-ray powder diffraction pattern of the I crystal form, expressed as a diffraction angle 2θ, has characteristic peaks at 5.978, 6.784, 7.090, 7.867, 9.070, 10.489, 12.078, 13.226, 13.674, 14.256, 16.934, 18.328, 18.438, 20.045, 20.759, 21.816, 23.452, 23.920, 24.193, 24.872, 26.185, 26.784, 27.675, 30.413, and 31.227.

[0020] In other embodiments, the X-ray powder diffraction pattern of the I crystal form, expressed in terms of a diffraction angle 2θ, is as follows: Figure 1 As shown.

[0021] In other embodiments, the 2θ angle error range is ±0.20.

[0022] This disclosure also provides a method for preparing the aforementioned crystal form I, the method comprising:

[0023] Method 1:

[0024] (a) The compound (S)-1-(4-(1-((4-acetylmorpholin-2-yl)methyl)-5-methyl-1H-benzo[d]imidazol-2-yl)-3-chloro-5-fluorophenyl)pyrrolidone-2-one is mixed with solvent (I), and hydrochloric acid solution is added, wherein solvent (I) is preferably ethanol.

[0025] (b) Crystallization;

[0026] Method 2:

[0027] (a) The compound (S)-1-(4-(1-((4-acetylmorpholin-2-yl)methyl)-5-methyl-1H-benzo[d]imidazol-2-yl)-3-chloro-5-fluorophenyl)pyrrolidone-2-one is mixed with solvent (II), a hydrochloric acid solution is added, followed by the addition of solvent (III), wherein solvent (II) is preferably one or more of methanol, ethanol, tetrahydrofuran, and 10% water / acetone, and solvent (III) is preferably one or more of acetone, isopropyl acetate, and tetrahydrofuran.

[0028] (b) Crystallization.

[0029] In other embodiments, the volume (μl) of solvents (I), (II), and (III) used in this disclosure may be 1-200 times the mass (mg) of the compound represented by formula (I), and in non-limiting embodiments may be 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 of these values. In some embodiments, the preparation method described in this disclosure further includes centrifugation (filtration), washing, or drying steps.

[0030] The crystallization methods disclosed herein include, but are not limited to, stirred crystallization, static crystallization, or evaporative crystallization. In some embodiments, the crystallization is stirred crystallization. In some embodiments, the crystallization is static crystallization.

[0031] In other embodiments, the method for preparing the aforementioned compound further includes one or more steps of filtration, washing, or drying, wherein the drying is preferably vacuum drying at a pressure < -0.08 MPa.

[0032] This disclosure also provides a pharmaceutical composition comprising the aforementioned pharmaceutically acceptable salt, crystal form I or crystal form I prepared by the aforementioned methods, and pharmaceutical excipients optionally selected from pharmaceutically acceptable excipients.

[0033] This disclosure also provides a pharmaceutical composition prepared from the aforementioned pharmaceutically acceptable salt, crystal form I, or crystal form I prepared by the aforementioned methods, and optionally a pharmaceutically acceptable excipient.

[0034] This disclosure also provides a method for preparing a pharmaceutical composition, comprising the step of mixing the aforementioned pharmaceutically acceptable salt, I-form, or I-form prepared by the aforementioned method with a pharmaceutically acceptable excipient.

[0035] This disclosure also provides the use of the aforementioned pharmaceutically acceptable salt, I-form, or I-form prepared by the aforementioned method, or the aforementioned composition in the preparation of a medicament for treating or preventing diseases related to P2X3 activity.

[0036] This disclosure also provides the use of the aforementioned pharmaceutically acceptable salt, I-form, or I-form prepared by the aforementioned method, or the aforementioned composition in the preparation of a medicament for the treatment or prevention of pain, urinary tract diseases, or cough.

[0037] This disclosure also provides an I-type crystal for the treatment or prevention of pain, urinary tract diseases or cough, with an X-ray powder diffraction pattern expressed in diffraction angle 2θ, showing characteristic peaks at 6.784, 12.078, 20.759, 23.920, 24.193 and 26.185.

[0038] The "2θ or 2θ angle" mentioned in this disclosure refers to the diffraction angle, where θ is the Bragg angle, and the unit is ° or degree; the error range of 2θ for each characteristic peak is ±0.20 (including the case where the number has more than one decimal place 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.

[0039] The “excipients” described in this disclosure include, but are not limited to, any adjuvants, carriers, flow aids, sweeteners, diluents, preservatives, dyes / colorants, flavoring agents, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, or emulsifiers that have been approved by the U.S. Food and Drug Administration for use in humans or livestock.

[0040] The "pulping" described in this disclosure refers to a purification method that utilizes the characteristic that substances have poor solubility in solvents, but impurities have good solubility in solvents. Pulping purification can remove color, change crystal form, or remove a small amount of impurities.

[0041] The starting material used in the crystal form preparation method disclosed herein can be any form of compound shown in Formula I, including but not limited to: amorphous, arbitrary crystal form, hydrate, solvate, etc.

[0042] The “differential scanning calorimetry or DSC” described in this disclosure refers to measuring the temperature difference and heat flow difference between the sample and the reference material during the sample heating or isothermal process, in order to characterize all physical and chemical changes related to thermal effects and obtain phase transition information of the sample.

[0043] The drying temperature described in this disclosure is generally 25℃~100℃, preferably 40℃~70℃, more preferably 45℃~55℃. It can be dried under normal pressure or reduced pressure, with a pressure <-0.08MPa, and the drying time is 5~10h, preferably 7~8h.

[0044] The numerical values ​​in this disclosure, such as those relating to the content of certain substances, are calculated data and inevitably contain a certain degree of error. Generally, ±10% is within the reasonable error range. The error may vary to some extent depending on the context in which it is used, but this variation shall not exceed ±10%, and may be ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1%, preferably ±5%.

[0045] Compound A: (S)-1-(4-(1-((4-acetylmorpholin-2-yl)methyl)-5-methyl-1H-benzo[d]imidazol-2-yl)-3-chloro-5-fluorophenyl)pyrrolidone-2-one. Its preparation method is based on the method in WO2022033567, and relevant content is cited in this text for illustration.

[0046] The testing conditions of the instruments used in the experiments in this disclosure are as follows:

[0047] 1. Differential Scanning Calorimeter (DSC)

[0048] Instrument Model: Mettler Toledo DSC 3+STARe System

[0049] Purging gas: nitrogen; Nitrogen purging rate: 50 mL / min

[0050] Heating rate: 10.0℃ / min

[0051] Temperature range: 25-250℃

[0052] 2. X-ray Powder Diffraction (XRPD)

[0053] Instrument Model: BRUKER D8 Discover X-ray Powder Diffractometer

[0054] Rays: Monochromatic Cu-Kα rays (λ = 1.5406)

[0055] Scanning mode: θ / 2θ, scanning range (2θ range): 3~50°

[0056] Voltage: 40kV, Current: 40mA

[0057] 3. Thermogravimetric Analysis (TGA)

[0058] Instrument model: Mettler Toledo TGA2

[0059] Purging gas: nitrogen; Nitrogen purging rate: 50 mL / min

[0060] Heating rate: 10.0℃ / min

[0061] Temperature range: 30-350℃

[0062] 4. DVS is a dynamic moisture adsorption method.

[0063] The test was performed using Surface Measurement Systems advantage 2 at 25°C with humidity ranging from 50% to 95% to 0% to 95% to 50% RH in 10% increments. The judgment criterion was that the mass change dM / dT for each gradient was less than 0.002%, with a TMAX of 360 min and two cycles.

[0064] 5. The structure of the compound was determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). NMR shifts (δ) are given in units of 10⁻⁶ (ppm).

[0065] The NMR measurements were performed using a Bruker AVANCE NEO 500M spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD), with tetramethylsilane (TMS) as the internal standard.

[0066] MS measurements were performed using an Agilent 1200 / 1290 DAD-6110 / 6120 Quadrupole MS LC-MS system (manufacturer: Agilent, MS model: 6110 / 6120 Quadrupole MS). Other instruments used included: a Waters ACQuity UPLC-QD / SQD system (manufacturer: Waters, MS model: Waters ACQuity Qda Detector / Waters SQ Detector); and a THERMOUltimate 3000-Q Exactive system (manufacturer: THERMO, MS model: THERMO Q Exactive).

[0067] 6. The known starting materials can be synthesized using or according to methods known in the art, or can be purchased from companies such as ABCRGmbH & Co.KG, Acros Organics, Aldrich Chemical Company, AccelaChemBio Inc, and Darui Chemicals.

[0068] 7. HPLC determination was performed using an Agilent 1260DAD high-performance liquid chromatograph (ACE Excel C18 150×4.6mm column) and a Thermo Dionex Ultimate 3000 high-performance liquid chromatograph (Waters Xbridge C18 150×4.6mm column). Attached Figure Description

[0069] Figure 1 XRPD pattern of the I crystal form of the hydrochloride salt of the compound shown in Formula I. Detailed Implementation

[0070] 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 are not intended to limit the substance and scope of the present disclosure.

[0071] Example 1

[0072]

[0073] Step 1: (R)-2-(((methanesulfonyl)oxy)methyl)morpholine-4-carboxylic acid tert-butyl ester (1b)

[0074] Under nitrogen protection at 0 °C, triethylamine (96 mL, 690.41 mmol) and methanesulfonyl chloride (49.63 mL, 635.71 mmol) were added dropwise to a toluene (1000 mL) solution of (2R)-2-(hydroxymethyl)morpholine-4-carboxylic acid tert-butyl ester (100 g, 460.28 mmol). The reaction mixture was then stirred at 30 °C for 12 hours. TLC (petroleum ether / ethyl acetate = 1 / 1) showed that the starting material reacted completely. The reaction mixture was filtered, and the filtrate was washed with water (500 mL x 3) and brine (500 mL x 3), dried over anhydrous sodium sulfate, filtered, and concentrated to give title compound 1b (135 g, yield: 99%).

[0075] 1 H NMR (400MHz, CDCl3) δ4.24(d,J=4.8Hz,2H),3.92(d,J=11.2Hz,3H),3.74-3.65(m,1H),3.55(dt ,J=2.4,11.6Hz,1H),3.08-3.06(m,3H),3.04-2.91(m,1H),2.79(d,J=14.4Hz,1H),1.47(s,9H).

[0076] Step 2: (S)-2-((1,3-dioxoisoindoline-2-yl)methyl)morpholine-4-carboxylic acid tert-butyl ester (1c)

[0077] Potassium phthalimide (93.12 g, 502.78 mmol) and tetrabutylammonium bromide (14.73 g, 45.71 mmol) were added to a solution of compound 1b (135 g, 457.07 mmol) in N,N-dimethylacetamide (1350 mmol). The reaction mixture was stirred at 80 °C for 6 h. TLC (petroleum ether / ethyl acetate = 1 / 1) showed that the starting material was completely consumed. The reaction mixture was filtered, the filtrate was poured into water (3 L), filtered again, and the filter cake was stirred at 25 °C for 12 h with N-methylpyrrolidone-water (1 / 5, 2.4 L), then filtered, and the filter cake was dried under reduced pressure to give title compound 1c (120 g, yield: 73%).

[0078] MS(ESI)m / z = 291.1[M-56+H] + .

[0079] 1H NMR (400MHz, CDCl3) δ7.86 (dd, J = 3.2, 5.2Hz, 2H), 7.76-7.69 (m, 2H), 4.04-3.83 (m, 3H), 3.82-3.71 (m, 2H), 3.67(dd,J=4.8,13.5Hz,1H),3.44(dt,J=2.8,11.2Hz,1H),3.05-2.92(m,1H),2.76(br.s,1H),1.45(s,9H).

[0080] Step 3: (S)-2-(aminomethyl)morpholine-4-carboxylic acid tert-butyl ester (1d)

[0081] Hydrazine hydrate (10.40 mL, 181.88 mmol) was added to a solution of compound 1c (100 g, 288.70 mmol) in 2-methyltetrahydrofuran (1000 mmol). The reaction mixture was stirred at 80 °C for 3 h, 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 title compound 1d (68 g, yield: 87%).

[0082] MS(ESI)m / z = 217.1[M+H] + .

[0083] 1 H NMR (400MHz, CDCl3) δ3.88-3.84(m,3H),3.59-3.45(m,1H),3.37-3.25(m,1H),2.97-2. 82(m,1H),2.73(d,J=1.2Hz,1H),2.71(d,J=3.6Hz,1H),2.61-2.56(m,1H),1.45(s,9H).

[0084] Step 4: (S)-2-((2-(4-bromo-2-chloro-6-fluorophenyl)-5-methyl-1H-benzo[d]imidazol-1-yl)methyl)morpholine-4-carboxylic acid tert-butyl ester (1f)

[0085] Calcium carbonate (27 g, 270.74 mmol) and 1-fluoro-4-methyl-2-nitrobenzene (35 g, 225.62 mmol) were added to a 350 mL solution of compound 1d (67 g, 248.18 mmol) in dimethyl sulfoxide. The reaction mixture was stirred at 110 °C for 12 h. TLC (petroleum ether / ethyl acetate = 1 / 2) showed that the starting material was completely consumed. 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 h. The reaction mixture was poured into water (2 L), extracted with ethyl acetate (800 mL x 3), the organic layer was concentrated, the residue was dissolved in ethyl acetate (1.5 L), washed with water (1 L x 3) and brine (800 mL x 2), dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give title compound 1f (113 g, yield: 93%).

[0086] MS(ESI)m / z = 538.1 [M+H] + .

[0087] 1 H NMR (400MHz, CDCl3) δ7.64 (s, 1H), 7.56-7.52 (m, 1H), 7.40-7.32 (m, 2H), 7.20 (d, J = 8.4Hz, 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=10.4Hz,1H),2.51(s,3H),2.50-2.46(m,1H),1.42(s,9H).

[0088] Step 5: (R)-1-(3-chloro-5-fluoro-4-(5-methyl-1-(morpholin-2-ylmethyl)-1H-benzo[d]imidazol-2-yl)phenyl)pyrrolidone-2-one (1h)

[0089] To a solution of compound 1f (113 g, 152.92 mmol) in dioxane (1000 mL), pyrrolidone-2-one (47 mL, 611.67 mmol), cesium carbonate (100 g, 305.84 mmol), bis(diphenylacetone)palladium (8.79 g, 15.29 mmol), and 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene (17.70 g, 30.58 mmol) were added. The reaction mixture was purged three times with nitrogen, and stirred at 100 °C for 2 hours under nitrogen. Then, p-toluenesulfonic acid (29 g, 1529.18 mmol) was added at 25 °C, and the mixture was stirred at 100 °C for 1 hour. The reaction mixture was poured into water (1000 mL), extracted with ethyl acetate (800 mL x 6), and the aqueous phase was adjusted to pH 9 with 1 mol / L sodium hydroxide solution. The aqueous phase was then extracted with dichloromethane (800 mL x 5), the organic phase was dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the title compound 1h (113 g, yield: 93%).

[0090] MS(ESI)m / z = 443.1 [M+H] + .

[0091] 1 H NMR (400MHz, CDCl3) δ7.74-7.65(m,1H),7.61(br.s,2H),7.38-7.36(m,1H),7.16( d,J=8.4Hz,1H),4.15-3.91(m,2H),3.87(t,J=7.2Hz,2H),3.75-3.64(m,2H),3.49- 3.34(m,2H),2.75-2.69(m,2H),2.66(t,J=8.0Hz,2H),2.50(s,3H),2.45-2.33(m,2 H),2.31-2.25(m,1H),2.21(t,J=7.6Hz,2H),2.15-2.09(m,1H),2.15-2.08(m,1H).

[0092] Step Six: (S)-1-(4-(1-((4-acetylmorpholin-2-yl)methyl)-5-methyl-1H-benzi[d]imidazol-2-yl)-3-chloro-5-fluorophenyl)pyrrolidone-2-one (I)

[0093] To a 1-hour (480 mg, 1.08 mmol) solution of the compound in tetrahydrofuran (2.5 mL), a saturated sodium bicarbonate solution (2.5 mL) and acetic anhydride (221.3 mg, 2.17 mmol) were added at 0 °C. 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 overnight in n-hexane / ethyl acetate (1 / 1, 4 mL). The precipitate was filtered and dried to give the title product (400.0 mg, yield: 76%). X-ray powder diffraction analysis showed that the product was amorphous.

[0094] MS(ESI)m / z = 485.4 [M+H] + .

[0095] 1 H NMR (400MHz, DMSO-d6) δ7.88(br s,1H),7.79(br d,J=11.6Hz,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=15.0Hz,1H),3.97-3.87(m,3H),3.78(br d,J=17.6Hz,1H),3.68-3.55(m,1H),3.12(br d,J=11.0Hz,1H),2.99(br d,J=11.6Hz,1H),2.81-2.70(m,2H),2.58(br d,J=7.9Hz,2H),2.44(br s,3H),2.14-2.06(m,2H),1.93(s,2H).

[0096] Test Example 1: In vitro bioactivity evaluation

[0097] FLIPR assay was used to screen compounds for their antagonistic activity against hP2X3 and hP2X2 / 3 receptors (the effect of compounds on ion channels was expressed as changes in calcium flow signal).

[0098] 1. Experimental instruments and materials

[0099]

[0100]

[0101] 2. Experimental Procedure

[0102] Stable 1321N1 cells (adherent cells) transfected with hP2X3 and hP2X2 / 3 receptors were digested, centrifuged, resuspended in plating medium (DMEM + 10% DFBS), and counted. The cell volume was adjusted to 2*10⁻⁶ cells / cells. 5 Cells / mL were seeded at 50 μL per well in a 384-well Assay Plate and incubated for 16–24 hours in a 5% CO2, 37°C incubator. 500 nmol of the desired concentration of the test compound (20 mM DMSO stock solution) was prepared at 180-fold concentration and added to each well of the 384-well Compound Plate, followed by 30 μL of LLIPR buffer (containing 1.26 mM Ca). 2+ Mix 1*HBSS + 2mM CaCl2 + 20mM HEPES, shake for 20-40 min to homogenize. Prepare 3 times the required concentration of α,β-meATP agonist using FLIPR buffer (500nM for hP2X3 cells, 1000nM for hP2X2 / 3 cells), and add 35μL of agonist to each well into another 384-well Compound Plate. Remove the cell culture plate after 16-24 hours of culture, aspirate the cell supernatant, and add 30μL of Dye to each well. Incubate with Calcium 4 Assay Kit (diluted with FLIPR buffer) for 1 hour. Add 15 μL of the compound to each well (using a FLIPR instrument). After 15 minutes, add 22.5 μL of the agonist to each well and detect the fluorescence signal (excitation wavelength 470 nm-495 nm, emission wavelength 515 nm-575 nm). Use the difference between the signal peak and trough as the baseline data. Use the highest concentration of the positive control as the 100% inhibition rate and DMSO as the 0% inhibition rate. Fit the inhibition effect curve of the compound to Graphpad Prism 6 software and calculate the IC50. 50 value.

[0103] Table 1. Half-maximal inhibitory concentrations (IC50) of compounds against hP2X3 and hP2X2 / 3 receptors 50 )

[0104]

[0105] Test Example 2: CYP Inhibition Experiment

[0106] The representative substrate metabolism responses of the five major human CYP subtypes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 / 5) were evaluated using a mixture of 150 donor human liver microsomes (purchased from Corning, catalog number 452117). The effects of different concentrations of the analytes on the metabolic responses of phenacetin (CYP1A2), diclofenac sodium (CYP2C9), S-metphenytoin (CYP2C19), bufrolol hydrochloride (CYP2D6), and midazolam (CYP3A4 / 5) were determined by liquid chromatography-tandem mass spectrometry (LC / MS / MS).

[0107] A 200 μL reaction mixture (100 mmol / L phosphate buffer, pH 7.4, containing 0.3% DMSO, 0.6% acetonitrile, and 0.1% methanol, by volume) of 30 μM phenacetin, 10 μM diclofenac sodium, 35 μM S-mphenanthridine, 5 μM bromide hydrochloride, 3 μM midazolam, 1 mM NADPH, the test compound (concentrations of 0.1, 0.3, 1, 3, 10, and 30 μmol / L, respectively), a positive compound, or a blank control, and mixed human liver microsomes (0.2 mg / mL) was incubated at 37 °C for 5 min. Then, 200 μL of acetonitrile solution containing 3% formic acid and 40 nM internal standard verapamil was added, and the mixture was centrifuged at 4000 rpm for 50 min. The mixture was cooled on ice for 20 min, and then centrifuged at 4000 rpm for 20 min to precipitate the protein. 200 μL of the supernatant was analyzed by LC / MS / MS.

[0108] Peak area is calculated from the chromatogram. Residual activity percentage (%) is calculated using the following formula:

[0109] Peak area ratio = Metabolite peak area / Internal standard peak area

[0110] Residual activity percentage (%) = Peak area percentage of the test compound group / Peak area percentage of the blank group

[0111] CYP half-maximal inhibitory concentration (IC50) 50 This was calculated using Excel XLfit 5.3.1.3.

[0112] The measured CYP half-maximal inhibitory concentration (IC50) 50 The values ​​are shown in Table 2.

[0113] Table 2. Half-maximal inhibitory concentrations (IC50) of compounds against CYP 50 )

[0114]

[0115] Test Example 3: Detection of In Vitro Metabolic Stability of Human Hepatocytes

[0116] The concentration of compounds in the reaction system was determined by LC / MS / MS to calculate the intrinsic clearance rate of the analyte and to assess its in vitro metabolic stability in human hepatocytes.

[0117] 247.5 μL 1×10 6 A mixture of human hepatocytes / mL (purchased from Bioreclamation IVT, catalog number S01205) and 2.5 μL of 100 μM of the test compound or positive control was added to the incubation plate to initiate the reaction. Incubation was performed at 37 °C and 600 rpm. 20 μL of the incubation mixture was transferred to the stop plate at 0.5, 5, 15, 30, 45, 60, 80, 100, and 120 minutes, respectively. The mixture was then vortexed for 2 minutes. The stop plate was centrifuged at 4000 rpm for 20 minutes. 40 μL of the supernatant from each compound was transferred to a 96-well plate, followed by dilution with 160 μL of purified water.

[0118] The obtained samples were quantified by ion chromatogram. The residual rate was calculated based on the peak area of ​​the analyte or positive control. The slope k was determined using Microsoft Excel by linear regression of the natural logarithm of the residual rate against incubation time.

[0119] Intrinsic clearance rate (in vitro CL) int μL / min / 10 6 (The cell) is calculated from the slope value according to the following equation:

[0120] in vitro CL int =kV / N

[0121] V = incubation volume (0.25 mL);

[0122] N = Number of cells per well (0.25 × 10⁻⁶) 6 cell)

[0123] The measured intrinsic clearance rate of human hepatocytes is shown in Table 3.

[0124] Table 3. Intrinsic clearance rate of compounds from human hepatocytes

[0125]

[0126] Example 2

[0127] Approximately 7 mg of compound A was weighed and added to 70 μl of ethanol. The mixture was stirred until dissolved, then 11 μl of 2M hydrochloric acid aqueous solution was added. The mixture was heated at 50-10℃ for 3 days, resulting in the precipitation of a solid. The solid was centrifuged and dried under vacuum. X-ray powder diffraction analysis showed that the product was of crystal form I. The XRPD spectrum is shown below. Figure 1As shown in Table 4, the positions of its characteristic peaks are as follows. The DSC spectrum shows that the endothermic peaks have peak values ​​of 124.80℃ and 153.79℃. The TGA spectrum shows a weight loss of 11.60% between 30-160℃ and 2.91% between 160-275℃.

[0128] Table 4

[0129]

[0130]

[0131] Example 3

[0132] Approximately 5 mg of compound A was weighed and added to 50 μl of methanol. The mixture was stirred until dissolved, then 22 μl of 2M hydrochloric acid aqueous solution was added and stirred. The solution was then added dropwise to 0.5 mL of isopropyl acetate and stirred overnight. A solid precipitated out, which was then centrifuged and dried under vacuum to obtain the solid. X-ray powder diffraction analysis showed that the X-ray diffraction pattern was consistent with that of Example 2, indicating that the product was in crystal form I.

[0133] Example 4

[0134] Approximately 5 mg of compound A was weighed and added to 50 μl of ethanol. The mixture was stirred until dissolved, then 22 μl of 2M hydrochloric acid aqueous solution was added and stirred. The solution was then added dropwise to 0.5 ml of isopropyl acetate and stirred overnight. A solid precipitated out, which was then centrifuged and dried under vacuum to obtain the solid. X-ray powder diffraction analysis showed that the X-ray diffraction pattern was consistent with that of Example 2, indicating that the product was in crystal form I.

[0135] Example 5

[0136] Approximately 5 mg of compound A was weighed and added to 50 μl of methanol. The mixture was stirred until dissolved, then 22 μl of 2M hydrochloric acid aqueous solution was added and stirred. The solution was then added dropwise to 0.5 mL of tetrahydrofuran and stirred overnight. A solid precipitated out, which was then centrifuged and dried under vacuum to obtain the solid. X-ray powder diffraction analysis showed that the X-ray diffraction pattern was consistent with that of Example 2, indicating that the product was in crystal form I.

[0137] Example 6

[0138] Approximately 200 mg of compound A was weighed, added to 1 ml of ethanol, and stirred until dissolved. 39 μl of hydrochloric acid was added, and the mixture was stirred. 10 ml of isopropyl acetate was added, and the mixture was stirred overnight. A solid precipitated, which was then centrifuged and dried under vacuum to obtain the solid. X-ray powder diffraction analysis showed that the X-ray diffraction pattern was consistent with that of Example 2, indicating that the product was in crystal form I.

[0139] Example 7

[0140] Using Surface Measurement Systems advantage 2, at 25°C and with humidity starting at 50%, the humidity range was investigated from 0% to 95% in 10% increments. The criterion was that the mass change dM / dT for each gradient was less than 0.002%, and the running time TMAX for each humidity gradient was 360 min, with two cycles.

[0141]

[0142] Example 8

[0143] The hydrochloride I crystal form of compound A was laid out in an open container, and the stability of the samples was investigated under light (4500 Lux), high temperature (40℃, 60℃), and high humidity (RH 75%, RH 92.5%) conditions. The sampling period was 30 days.

[0144] Table 5

[0145]

[0146] Conclusion: Except for a crystal form transformation under prolonged exposure to high humidity, the drug exhibits good physical stability under other conditions. Its chemical stability is slightly lower under high humidity (92.5% RH) and light exposure for 30 days, but good chemical stability under other conditions, meeting the basic requirements for drug development.

[0147] Example 9

[0148] The stability of the hydrochloride I crystal form of the compound shown in formula (I) was investigated under conditions of -20℃, 4℃, and 25℃ / 60%RH.

[0149] Table 6

[0150]

[0151] Conclusion: Long-term accelerated experiments show that the I-type hydrochloride exhibits good physical and chemical stability under conditions of -20℃, 4℃, and 25℃ / 60RH for 6 months, meeting the basic requirements for drug development.

Claims

1. A compound of formula I ( S Pharmaceutically acceptable salts of 1-(4-(1-((4-acetylmorpholin-2-yl)methyl)-5-methyl-1H-benzo[d]imidazol-2-yl)-3-chloro-5-fluorophenyl)pyrrolidine-2-one, wherein the pharmaceutically acceptable salt is selected from hydrochloride, sulfate, methanesulfonate, phosphate, acetate, fumarate, maleate, tartrate, succinate, and citrate. 。 2. The pharmaceutically usable salt according to claim 1, wherein the chemical ratio of the compound of formula I to the acid molecule is selected from 1:0.5 to 1:

3.

3. The pharmaceutically usable salt according to claim 2, wherein the chemical ratio of the compound represented by formula I to the acid molecule is selected from 1:0.5, 1:1, 1:2 or 1:

3.

4. The pharmaceutically acceptable salt according to claim 3, wherein the chemical ratio of the compound represented by formula I to the acid molecule is selected from 1:

1.

5. The pharmaceutically usable salt according to claim 1, wherein it is a hydrochloride salt.

6. The pharmaceutically usable salt according to claim 5, wherein the hydrochloride is a hydrochloride salt.

7. A method for preparing the pharmaceutically acceptable salt according to any one of claims 1-6, comprising ( S The step of reacting 1-(4-(1-((4-acetylmorpholin-2-yl)methyl)-5-methyl-1H-benzo[d]imidazol-2-yl)-3-chloro-5-fluorophenyl)pyrrolidine-2-one with an acid selected from hydrochloric acid, sulfuric acid, methanesulfonic acid, phosphoric acid, acetic acid, fumaric acid, maleic acid, tartaric acid, succinic acid and citric acid.

8. A hydrochloride salt, crystal form I, of 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-one, characterized in that, The X-ray powder diffraction pattern, expressed as a diffraction angle 2θ, shows characteristic peaks at 6.784, 12.078, 20.759, 23.920, 24.193, and 26.

185.

9. The I-type as described in claim 8, characterized in that, The X-ray powder diffraction pattern, expressed as a diffraction angle 2θ, shows characteristic peaks at 6.784, 7.867, 10.489, 12.078, 14.256, 20.759, 21.816, 23.920, 24.193, 24.872, and 26.

185.

10. The I-type as described in claim 8, characterized in that, The X-ray powder diffraction pattern, expressed as a diffraction angle 2θ, shows characteristic peaks at 6.784, 7.090, 7.867, 10.489, 12.078, 13.674, 14.256, 18.328, 18.438, 20.045, 20.759, 21.816, 23.920, 24.193, 24.872, and 26.

185.

11. The I-type as described in claim 8, characterized in that, The X-ray powder diffraction pattern, expressed as a diffraction angle 2θ, shows characteristic peaks at 5.978, 6.784, 7.090, 7.867, 9.070, 10.489, 12.078, 13.226, 13.674, 14.256, 16.934, 18.328, 18.438, 20.045, 20.759, 21.816, 23.452, 23.920, 24.193, 24.872, 26.185, 26.784, 27.675, 30.413, and 31.

227.

12. The I-type as described in claim 8, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern expressed in terms of angle is shown in Figure 1.

13. The I-type as described in any one of claims 8-12, characterized in that, The 2 θ The angular error range is ±0.

20.

14. A method for preparing the I-type crystal form according to any one of claims 8-12, 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)pyrrolidone-2-one is mixed with solvent (I), and hydrochloric acid solution is added, wherein solvent (I) is selected from ethanol. (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)pyrrolidone-2-one is mixed with solvent (II), and hydrochloric acid solution is added, followed by solvent (III), wherein solvent (II) is selected from one or more of methanol, ethanol, tetrahydrofuran, and 10% water / acetone, and solvent (III) is selected from one or more of acetone, isopropyl acetate, and tetrahydrofuran. (b) Crystallization.

15. A pharmaceutical composition comprising a pharmaceutically acceptable salt according to any one of claims 1-6, a crystal form according to any one of claims 8-12 or a crystal form prepared by the method of claim 14, and optionally a pharmaceutically acceptable excipient.

16. A method for preparing a pharmaceutical composition, comprising the step of mixing a pharmaceutically acceptable salt according to any one of claims 1-6, a crystal form according to any one of claims 8-12, or a crystal form prepared by the method of claim 14 with a pharmaceutically acceptable excipient.

17. Use of the pharmaceutically acceptable salt of any one of claims 1-6, the I-crystal form of any one of claims 8-12, or the I-crystal form prepared by the method of claim 14, or the composition of claim 15 in the preparation of a medicament for the treatment or prevention of diseases related to P2X3 activity.

18. Use of the pharmaceutically acceptable salt according to any one of claims 1-6, the I-crystal form according to any one of claims 8-12, or the I-crystal form prepared by the method of claim 14, or the composition according to claim 15, in the preparation of a medicament for the treatment or prevention of pain, urinary tract diseases, or cough.

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