Heterocyclic P2X3 receptor inhibitor

Abstract

The invention provides a heterocyclic compound as a P2X3 receptor inhibitor. The heterocyclic compound is a compound as shown in a formula (I), an isotope variant, a tautomer or a stereoisomer. The invention also provides a pharmaceutical composition containing the compound, and application of the pharmaceutical composition in treatment of diseases related to P2X3.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and particularly relates to P2X3 receptor inhibitors. BACKGROUND

[0002] P2X purinoreceptors are ATP-gated ion channel receptors, the family of receptors includes seven homologous receptors: P2X1, P2X2, P2X3, P2X4, P2X5, P2X6 and P2X7, and three heterologous receptors: P2X2 / 3, P2X4 / 6, P2X1 / 5. Among them, P2X3 receptors are selectively expressed in the dorsal root ganglion, spinal cord and brain neurons of nerve endings, i.e. in small and medium diameter primary sensory neurons. P2X3 can also form P2X2 / 3 heterodimers with P2X2, and P2X2 / 3 is highly expressed on the terminals of sensory neurons (central and peripheral).

[0003] Studies have shown that ATP released by damaged or inflamed tissues in the airway directly acts on the P2X3 receptors of primary neurons, thereby triggering depolarization and action potentials, and the transmission of these potentials will cause a cough impulse, which in turn triggers cough. Preclinical and clinical studies have shown that P2X3 receptors play an important role in the hypersensitivity of the cough reflex, thereby leading to chronic cough. Therefore, P2X3 receptor inhibitors can be used for the treatment of patients with chronic cough. In addition, it has also been reported that P2X3 receptors play a key role in mediating the primary sensory effects of ATP, including the sensation of itching, pain, urinary tract disease and other related diseases, so P2X3 inhibitors also have the potential to be used for the treatment of related diseases.

[0004] Globally, 5-10% of adults suffer from chronic cough, which has a large clinical demand. Currently, the P2X3 inhibitor gefapixant (MK-7264) has been approved for marketing in Japan for the treatment of refractory chronic cough (RCC) or unexplained chronic cough (UCC). However, this drug has very strong inhibition on P2X2 / 3, and has very obvious taste disorder side effects in clinical practice, which will greatly affect the use and efficacy of the drug.

[0005] Therefore, the development of P2X3 inhibitors with high activity, high selectivity and safety is of great significance for the treatment of cough and other related diseases. SUMMARY

[0006] In the present application, we have found a series of small molecule inhibitors with very strong inhibitory activity on P2X3 receptors, and particularly high selectivity for P2X2 / 3.

[0007] In one aspect, the present application provides a compound, or an isotopic variant, tautomer or stereoisomer thereof:

[0008] In another aspect, the present application provides a pharmaceutical composition comprising a compound of the present application, and optionally a pharmaceutically acceptable excipient.

[0009] In another aspect, the present application provides a pharmaceutical composition comprising a compound of the present application and a pharmaceutically acceptable excipient, further comprising another therapeutic agent.

[0010] In another aspect, the present application provides the use of a compound of the present application for the manufacture of a medicament for the treatment of a disease associated with P2X3 receptor activity.

[0011] In another aspect, the present application provides a method of treating a disease associated with P2X3 receptor activity in a subject, comprising administering to said subject a compound of the present application or a composition of the present application.

[0012] In another aspect, the present application provides a compound of the present application or a composition of the present application for use in the treatment of a disease associated with P2X3 receptor activity.

[0013] Other objects and advantages of the present application will be readily apparent to those skilled in the art from the following detailed description of the application, wherein specific embodiments of the application are shown and described, together with the accompanying drawings. DETAILED DESCRIPTION

[0014] The reagents employed in the present application are either commercially available or synthesized by methods well known in the art.

[0015] Common Abbreviation Notes: Abbreviations: PE = petroleum ether; EA = ethyl acetate; MeOH = methanol; DCM = dichloromethane; DCE = dichloroethane; MeCN = acetonitrile; 1,4-dioxane = 1,4-dioxane; DMSO = dimethylsulfoxide; HFIP = hexafluoroisopropanol; DMF = N,N-dimethylformamide; THF = tetrahydrofuran; Hex = n-hexane; IPA = isopropanol; NMP = N-methylpyrrolidinone; NMO = N-methylmorpholine-N-oxide; TEA = triethylamine; DIEA = diisopropylethylamine; CuI = copper iodide; CuCN = copper cyanide; triphosgene = triphosgene; p-TsOH = p-toluenesulfonic acid; T3P = 1-propylphosphonic anhydride; TsN3 = p-toluenesulfonyl azide; PPA = polyphosphoric acid; SEM-Cl = 2-(trimethylsilyl)ethoxymethyl chloride; DMA = N,N-dimethylacetamide.

[0016] Example 1 Preparation of key intermediates

[0017] ​​ First step: Dissolve int 1-1 (5 g, 0.05 mol) in CH3CN:MeOH (3:1) 100 mL, add triethylamine (8.48 mL, 0.06 mol), then place in an ice ethanol bath (-20 °C), add chloroacetyl chloride (5.68 mL, 0.071 mol) dropwise via a dropping funnel, approximately two hours to add, naturally raise to room temperature, stir for 15 h. TLC test reaction complete (EA:MeOH = 10:1, KMnO4 coloration), stop reaction. The reaction solution is concentrated under reduced pressure to remove a large amount of solvent, and the crude product is purified by silica gel column chromatography (0-10% MeOH / EA) to obtain compound int 1-2 (6 g) as a yellowish oil, yield: 65%.

[0018] Second step: Dissolve potassium tert-butoxide (10 g, 0.09 mol) in tert-butanol (70 mL), and add a solution of compound int 1-2 (6 g, 0.036 mol) in tert-butanol (70 mL) dropwise via a dropping funnel. After approximately 2 h of dropwise addition, react at room temperature for 15 h, and TLC test reaction complete (EA:MeOH = 5:1, KMnO4 coloration), stop reaction. Filter the reaction solution, and concentrate the filtrate to obtain a crude product, which is purified by silica gel column chromatography (0-5% MeOH / EA) to obtain compound int 1-3 (3 g) as a white solid, yield: 64%.

[0019] Third step: Dissolve compound int 1-3 (3 g, 0.023 mol) in DCM (30 mL), add triethylamine (3.8 mL, 0.027 mol), and then add benzoyl chloride (2.9 mL, 0.025 mol) dropwise, and react at room temperature for 2 h. LC-MS test reaction complete (DCM:MeOH = 10:1), stop reaction. Wash the reaction solution with water, extract with DCM, wash with saturated brine, dry over anhydrous sodium sulfate, filter, and rotary evaporate to obtain a crude product. Purify the crude product by silica gel column chromatography (0-10% MeOH / DCM) to obtain compound int 1-4 (3.5 g) as a white solid, yield: 65%.

[0020] Fourth step: Dissolve int 1-4 (3.5 g, 0.149 mol) in DCM (50 mL), add trimethyloxonium tetrafluoroborate (2.6 g, 0.018 mol, CAS:420-37-1), and react at room temperature for 6 h. After LC-MS test raw material disappears, add methyl hydrazinecarboxylate (1.6 g, 0.018 mol, CAS:6294-89-9), and then react at room temperature for 15 h. LC-MS test reaction complete, stop reaction. Concentrate the reaction solution under reduced pressure to obtain crude product int 1-5.

[0021] Step 5: The crude int 1-5 was dissolved in DMF (20 mL), and the reaction was completed after 1 h of microwave reaction at 170 °C, which was detected by LC-MS. The reaction was stopped. The reaction mixture was diluted with water and extracted with EA. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product. The crude product was purified by silica gel column chromatography (0-10% MeOH / DCM) to give compound int 1-6 (2 g) in 49% yield.

[0022] Step 6: Compound int 1-6 (2 g, 0.007 mol) was dissolved in DMF (10 mL), and potassium carbonate (3 g, 0.022 mol) and iodomethane (1.4 mL, 0.022 mol) were added successively. The reaction was completed after 2 h of reaction at room temperature, which was detected by LC-MS or TLC (DCM:MeOH=10:1). The reaction was stopped. The reaction mixture was washed with water and concentrated to give the crude product. The crude product was purified by silica gel column chromatography (0-10% MeOH / DCM) to give compound int 1-7 (1 g) in 48% yield.

[0023] Step 7: Int 1-7 (1 g, 0.003 mol) was dissolved in THF:MeOH:H2O (10:1:1), and the reaction was completed after 2 h of reaction at room temperature, which was detected by LC-MS or TLC (DCM:MeOH=10:1). The reaction was stopped. The reaction mixture was concentrated to give the crude product. The crude product was purified by silica gel column chromatography (0-10% MeOH / DCM) to give compound int 1-8 (600 mg) in 90% yield.

[0024] Step 8: Compound int 1-8 (600 mg, 0.003 mol) was dissolved in DCM (20 mL), and DIPEA (1.4 ml, 0.008 mol) was added. Methanesulfonyl chloride (0.35 ml, 0.005 mol) was added dropwise. The reaction was completed after 2 h of reaction at room temperature, which was detected by TLC (DCM:MeOH=15:1, KMnO4coloration). The reaction was stopped. The organic layer was washed with water, dried, and concentrated to give the crude product. The crude product was purified by silica gel column chromatography (0-15% MeOH / DCM) to give compound int 1-9 (400 mg) in 47% yield.

[0025] Ninth step: Compound int 1-9 (400 mg, 1.5 mmol) was dissolved in DMF (10 mL), sodium azide (200 mg, 3.0 mmol) was added in batches at room temperature, and then the reaction was carried out at 80 °C for 15 h. LC-MS or TLC detection reaction complete (DCM:MeOH=10:1), after the reaction was complete, concentrated to obtain the crude compound int 1-10 (200 mg).

[0026] Tenth step: Compound int 1-10 (200 mg, 0.95 mmol) was dissolved in EtOH (5 mL), Pd / C (8 mg, 4wt%) was added under hydrogen atmosphere, and the reaction was carried out at room temperature for 15 h. LC-MS or TLC detection reaction complete (DCM:MeOH=10:1), stop reaction. The reaction solution was concentrated under reduced pressure to obtain int 1 (100 mg), yield: 36%.

[0027] Synthesis of intermediate int 2 First step: Compound int 2-1 (2 g, 10.5 mmol) was dissolved in super dry acetonitrile (20 mL), and after nitrogen replacement, benzyl mercaptan (1.56 g, 12.6 mmol), triethylamine (3.18 g, 31.5 mmol) were added, and the reaction was carried out at room temperature for 15 h. LC-MS detection reaction complete, stop reaction. Diluted with water, EA extraction, saturated brine washing, anhydrous sodium sulfate drying, filtration, rotary evaporation to obtain the crude product. The crude product was purified by silica gel column chromatography (0-20% EA / PE) to obtain compound int 2-2 (1.2 g), yield: 38.7%. Recovered raw material (660 mg).

[0028] Second step: int 2-2 (500 mg, 1.7 mmol) was dissolved in dichloromethane (5 mL), acetic acid (510 mg, 8.5 mmol), chlorosulfonic acid (850 mg, 6.8 mmol) were added at 0 °C, and after the reaction was no longer vigorous, the ice bath was removed, and the reaction was carried out at room temperature for 4 h. TLC detection reaction complete, stop reaction. The reaction solution of compound int 2-3 was obtained, and the reaction solution was directly used for the next step.

[0029] Third step: Under ice-bath, add excess amount of ammonia water to the reaction solution of int 2-3, a large amount of white smoke appears, remove the ice-bath after intense heat, react at room temperature for 2 h. LC-MS detection shows that the reaction is complete, stop the reaction. Extract with DCM, wash with saturated brine, dry over anhydrous sodium sulfate, filter, and rotary evaporation to obtain the crude product. The crude product is purified by silica gel column chromatography (0-20% MeOH / DCM) to obtain compound int 2-4 (240 mg), yield: 56.3%.

[0030] Fourth step: Compound int 2-4 (50 mg, 0.20 mmol) is dissolved in DMF (2 mL), NaH (18 mg, 0.46 mmol) is added at 0°C, and the reaction is carried out at room temperature for 1 h. Then PMBCl (78 mg, 0.50 mmol) is added and the reaction is carried out at room temperature for 2 h. LC-MS detection shows that the reaction is complete, stop the reaction. Extract with DCM, wash with saturated brine, dry over anhydrous sodium sulfate, filter, and rotary evaporation to obtain the crude product. The crude product is purified by silica gel column chromatography (0-20% MeOH / DCM) to obtain compound int 2-5 (40 mg), yield: 41.2%.

[0031] Fifth step: Compound int 2-5 (40 mg, 0.08 mmol) is dissolved in THF / H2O=1:1 mixed solvent, LiOH·H2O (13 mg, 0.33 mmol) is added, and the reaction is carried out at room temperature for 2 h. LC-MS detection shows that the reaction is complete, stop the reaction. The pH is adjusted to be acidic after treatment, extracted with DCM, dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain the crude product. The crude product is purified by silica gel column chromatography (0-10% MeOH / DCM) to obtain compound int 2 (35 mg), yield: 92.1%.

[0032] Example 2 Preparation of target compound

[0033] First step: Compound int 1 (78 mg, 0.43 mmol) and P 1-1 (73 mg, 0.47 mmol) are dissolved in DMF (3 mL), K2CO3 (107 mg, 0.77 mmol) is added, and the reaction is carried out at 80°C for 15 h. LC-MS monitoring shows that the reaction is complete, stop the reaction. The reaction solution is quenched with water, extracted with DCM, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain the crude product. The crude product is purified by silica gel column chromatography (0-35% EA / PE) to obtain compound P 1-2 (35 mg), yield: 25.9%.

[0034] Second Step: Dissolve P 1-2 (35 mg, 0.11 mmol) in ethanol (3 mL), add palladium-carbon according to 20% of raw material mass fraction, replace with hydrogen three times, and react at room temperature for 2 h. Monitor the reaction completion by LC-MS, and stop the reaction. Filter the reaction solution, and distill the filtrate under reduced pressure to obtain the crude compound P 1-3 (30 mg), yield: 93.7%. LC-MS: ESI-MS (m / z): [M+H] + = 290.

[0035] Third Step: Dissolve P 1-3 (30 mg, 0.10 mmol) and int 2 (54 mg, 0.11 mmol) in DMF (3 mL), add HATU (49 mg, 0.13 mmol) and TEA (30 mg, 0.30 mmol), and stir at room temperature for 15 h. Monitor the reaction completion by LC-MS, and stop the reaction. Quench the reaction solution with water, extract with DCM, wash with saturated brine, dry over anhydrous sodium sulfate, filter, and rotary evaporate to obtain the crude product. Purify the crude product by silica gel column chromatography (0-10% MeOH / DCM) to obtain compound P 1-4 (30 mg), yield: 33.3%. LC-MS: ESI-MS (m / z): [M+H] + = 749.

[0036] Fourth Step: Dissolve compound P 1-4 (30 mg, 0.04 mmol) in DCE (2 mL), add TFA (0.5 mL) and anhydrous magnesium sulfate (7 mg, 0.06 mmol), and microwave at 130°C for 40 min. Monitor the reaction completion by LC-MS, and stop the reaction. Dilute the reaction solution with water, extract with DCM, dry over anhydrous sodium sulfate, filter, and rotary evaporate to obtain the crude product. Purify the crude product by reverse phase preparation to obtain compound P 1 (6.0 mg), yield: 31.5%. LC-MS: ESI-MS (m / z): [M+H] + = 491.

[0037] 1 H NMR (600 MHz, DMSO- d 6) δ 7.81 (s, 2H), 7.74 (d, J = 8.4 Hz, 1H),7.71 (d, J = 7.2 Hz, 2H), 7.55 (s, 1H), 7.23 (dd, J = 8.4, 1.8 Hz, 1H), 4.62(dd, J= 15.6, 2.4 Hz, 1H), 4.49 (d, J = 15.6 Hz, 1H), 4.30 (d, J = 15.6 Hz, 1H), 4.21 (dd, J = 15.6, 8.4 Hz, 1H), 4.05 (tdd, J = 12.6, 7.8, 2.4 Hz, 1H),3.84 (dd, J = 12.6, 3.6 Hz, 1H), 3.24 (s, 3H), 3.20 (dd, J = 12.6, 10.8 Hz,1H), 2.46 (s, 3H)。

[0038] Synthesis was carried out according to the preparation of P 1 with the replacement of intermediate P 1-1 by P 2-1.

[0039] 1 H NMR (600 MHz, Chloroform- d ) δ 7.75 (s, 1H), 7.36 (d, J = 8.4 Hz,1H), 7.30 – 7.27 (m, 1H), 7.23 (dd, J = 8.4, 1.8 Hz, 1H), 4.64 (d, J = 15.6Hz, 1H), 4.35 – 4.29 (m, 2H), 4.25 (dd, J = 15.6, 7.8 Hz, 1H), 3.97 (ddt, J =11.4, 7.2, 3.0 Hz, 1H), 3.78 (dd, J = 12.0, 3.6 Hz, 1H), 3.39 (s, 3H), 3.29(t, J = 10.8 Hz, 1H), 3.27 – 3.23 (m, 2H), 2.53 (s, 3H), 1.45 (t, J = 7.8 Hz,3H)。

[0040] Evaluation of compounds of example 3 for inhibitory activity at the human P2X3 receptor The antagonist properties of the compounds of the present application are determined by the FLIPR (fluorometric imaging plate reader) method as inhibitors of intracellular calcium elevation induced by activation of hP2X3 (human purinergic P2X receptor subtype 3, Accession No. NM_002559.5) expressed in HEK293 cells (human embryonic kidney 293 cell line, ATCC).

[0041] HEK293 cells stably expressing hP2X3 are maintained in a cell culture incubator at 37 °C, 5% humidity, in DMEM high glucose medium containing 10% FBS (fetal bovine serum, Gibco, 10569-010), 1% penicillin-streptomycin (Invitrogen, 15140), and 350 μg / mL G418 (Invitrogen, 11811031). 18-24 hours before the FLIPR experiment, cells are seeded at a density of 666666 cells / mL (20000 cells / well) in 384-well plates and incubated overnight in the cell culture incubator. On the day of the experiment, the medium is discarded. Probenecid (Sigma, P8761) is dissolved in ddH2O to 500 mM (NaOH is added to dissolve). The reaction buffer (HBSS (Invitrogen, 14025) containing 20 mM HEPES (Invitrogen, 15630), pH 7.4) is prepared by adding 10 mL buffer to the compound A dye solution prepared as 20x (stored at -20 °C for 6 months). 2x loading buffer is prepared according to the Calcium 6 kit (Molecular Device, R8191) instructions: i. dilute the dye with reaction buffer; ii. add the probenecid stock solution to a final concentration of 5 mM; iii. adjust the pH to 7.4 using a vortex.

[0042] 10 μL of buffer is added per well, 10 μL of 2x dye solution is added per well, and the cell plate is incubated in a 37 °C carbon dioxide incubator for 2 hours and then in a 25 °C incubator for 15 minutes. 10 μL of test compound (dissolved in DMSO at a concentration of 10 mM and serially diluted with buffer) or vehicle is then added to each well and allowed to equilibrate at room temperature for 30 min. The cell plate is then placed in the FLIPR, and baseline fluorescence measurements are taken (excitation wavelength 475-495 nm, emission wavelength 515-575 nm). 10 μL / well of agonist (2.5 μM final concentration of αβ-me ATP (Sigma, M6517) or vehicle (ultrapure water) is then added, and fluorescence values are measured at 1 second intervals for 2 minutes, and the output fluorescence counts are finally analyzed. The results of a specific experiment are shown in Table 1.

[0043] The selectivity of the compounds of the present application for P2X2 / 3 receptors is determined by the FLIPR (fluorometric imaging plate reader) assay, which measures the inhibition of intracellular calcium elevation induced by activation of hP2X2 / 3 (heterodimeric receptor formed by human purinergic PX receptor subtypes 2 and 3, accession number NM_170683.4 for P2X2 and accession number NM_002559.5 for P2X3) expressed in CHO-K1 cells (Chinese hamster ovary cells, ATCC).

[0044] The CHO-K1 cells stably expressing hP2X2 / 3 are maintained in a cell incubator at 37°C, 5% humidity, in F12 medium containing 10% FBS (fetal bovine serum, Gibco, 10569-010), 1% penicillin-streptomycin (Invitrogen, 15140), and 600 μg / mL G418 (Invitrogen, 11811031) and 300 μg / mL Zeocin (Invitrogen, R25005). 18-24 hours before the FLIPR experiment, the cells are seeded at a density of 400000 cells / mL (20000 cells / well) in 384-well plates and incubated overnight in the cell incubator. On the day of the experiment, the medium is discarded. Probenecid (Sigma, P8761) is dissolved in ddH2O to 500 mM (NaOH is added to dissolve). The reaction buffer (HBSS (Invitrogen, 14025) containing 20 mM HEPES (Invitrogen, 15630), pH 7.4) is prepared by adding 10 mL buffer to the compound A dye solution prepared at 20x (stored at -20°C for 6 months). The 2X loading buffer is prepared according to the Calcium 6 kit (Molecular Device, R8191) instructions: i. Dilute the dye with the reaction buffer ii. Add the probenecid stock solution to a final concentration of 5 mM; iii. Adjust the pH to 7.4 using a shaker for 1-2 minutes.

[0045] Each well was added with 10 μL of buffer, 10 μL of 2x dye solution was added to each well, and the cell plate was incubated in a 37 °C carbon dioxide incubator for 2 hours, and then placed in a 25 °C incubator for 15 minutes. Subsequently, 10 μL of test compound (dissolved in DMSO at a concentration of 10 mM and serially diluted with buffer) or vehicle was added to each well, and allowed to equilibrate at room temperature for 30 min. Then the cell plate was placed in the FLIPR, and the baseline fluorescence was measured (excitation wavelength 475-495 nm, emission wavelength 515-575 nm). Subsequently, 10 μL / well of agonist (10 μM final concentration of αβ-me ATP (Sigma, M6517) or vehicle (ultrapure water) was added, and the fluorescence value was measured at 1 second intervals for 2 minutes, and finally the output fluorescence count was analyzed. The specific experimental results are shown in Table 2.

[0046] Table 1. Inhibitory activity of compounds on human P2X3 Compound hP2X3 IC 50 (nM) <!-- 6 -->]]> P1 67 P2 46 BLU-5937 48 QR052107B 41 Table 2. Inhibitory activity of compounds on human P2X2 / 3 Compound hP2X2 / 3 inhibition (inhibition @ 50 μΜ) hP2X2 / 3 inhibition (inhibition @ 100 μΜ) hP2X2 / 3 IC 50 (nM) P1 -1.8% 23% / P2 2.6% -0.8% / BLU-5937 / / QR052107B 35% 59% / # “ / ” = no data. The structures of the comparative compounds BLU-5937 and QR052107B are as follows: , .

[0047] The above test results show that the molecules of the present application have excellent inhibitory effect on hP2X3 receptors, and have comparable activity to the control compounds. At the same time, the inhibitory activity of the molecules of the present application on hP2X2 / 3 is very weak, and the inhibitory effect of the compounds on hP2X2 / 3 under high concentration test conditions is obviously weaker than that of the control compounds. The comprehensive data prove that the compounds of the present patent are high-activity and high-selectivity hP2X3 receptor inhibitors, and in the future they will have better safety.

Claims

1. A compound, or an isotopic variant, tautomer, or stereoisomer thereof, wherein the compound is selected from: 、 2. A pharmaceutical composition comprising a compound of claim 1, or a pharmaceutically acceptable salt, isotopic variant, tautomer, stereoisomer thereof, and a pharmaceutically acceptable carrier, adjuvant, or vehicle, optionally other therapeutic agents.

3. Use of a compound of claim 1, or a pharmaceutically acceptable salt, isotopic variant, tautomer, stereoisomer thereof, in the manufacture of a medicament for the treatment and / or prevention of a disease associated with P2X3 receptors.

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