CN113620888B - Dihydropyrimidine compound and preparation method and application thereof - Google Patents
Dihydropyrimidine compound and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a dihydropyrimidine compound, a preparation method and application thereof, belongs to the technical field of pharmaceutical chemistry, and solves the problems of poor effect and large adverse reaction of a P2X3 receptor inhibitor in the prior art. The structure of the dihydropyrimidine compound is shown as a formula I. The invention also provides a preparation method of the compound shown in the formula I and application of the compound in preparation of medicines for treating or preventing P2X3 and/or P2X2/3 receptor related diseases. The dihydropyrimidine compound provided by the invention has good affinity with P2X3, and has stronger antagonism to P2X3 receptor, thereby being safe and effective.
Description
Technical Field
The invention relates to the field of pharmaceutical chemistry, in particular to dihydropyrimidine compounds or salts, solvates, allosteric structures, metabolites, nitrogen oxides and prodrugs thereof, a preparation method thereof and application thereof in preparing medicaments for treating and preventing diseases related to P2X3 and/or P2X2/3 receptors, in particular to application in preparing medicaments for treating and preventing respiratory diseases.
Background
Chronic cough is one of the common clinical symptoms of the respiratory system. Chronic cough is cough with cough time lasting over 8 weeks and no obvious lung disease evidence in X-ray chest, and is often the only diagnosis of the patient. There are various causes of chronic cough. Patients with chronic cough are more sensitive to various causes that do not normally cause cough in healthy subjects. No approved drug specifically for chronic cough is available in the prior art. Clinically common central antitussive drugs such as codeine, dextromethorphan and the like are easy to cause adverse reactions such as constipation, somnolence and the like.
In recent years, purine receptors P2X3 have been found to be closely related to a variety of diseases, including chronic cough, neuralgia, sleep apnea and the like. gefasixant (MK-7264) is a novel antitussive developed by the company moesadong (Merck & Co), which is the first P2X3 blocker to publish clinical data in phase III, and has been applied for FDA New Drugs (NDA). Two clinical phase III trials showed a statistically significant reduction in the frequency of COUGH at week 12 (COUGH-1 study) and 24 (COUGH-2 study) 24 hours (measured objectively as the number of COUGH per hour using 24 hour recordings) in the 45mg dose gefasixant treatment group compared to the placebo group. In 2 studies, the 15mg dose of gecapixant 2 times daily treatment group did not reach the primary efficacy endpoint. The 45mg group reached the clinical endpoint, but the frequency of discontinuation of 45mg due to adverse events was higher and the incidence of taste-related adverse events was higher. Thus, providing a safer, more potent P2X3 receptor antagonist would be a major challenge to those skilled in the art.
Disclosure of Invention
The invention aims to provide a dihydropyrimidine compound with a structure shown as a formula I or salts, solvates, allosteric structures, metabolites, nitrogen oxides and prodrugs thereof, which has good affinity with a P2X3 receptor, good antagonism to the P2X3 receptor and small adverse reaction.
The second object of the present invention is to provide a process for producing the compound.
The invention also aims to provide the application of the compound in preparing a medicine for treating or preventing P2X3 and/or P2X2/3 receptor related diseases.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the invention provides a compound with a structure shown as a formula I, or salts, solvates, allosteric structures, metabolites, nitrogen oxides and prodrugs thereof,
wherein R is 1 Selected from hydrogen, alkyl, cycloalkyl, pyrrolyl or piperidinyl;
R 2 、R 3 independently selected from hydrogen, deuterium, substituted or unsubstituted C 1-12 Alkyl, or R 2 、R 3 A substituted or unsubstituted 3 to 15 membered cycloalkyl group formed by ligation;
R 4 selected from the group consisting of a substituted or unsubstituted heterocyclic group, a substituted or unsubstituted aryl group;
R 5 selected from 1 to 5 hydrogens, deuterium, substituted and unsubstituted alkyl groups, substituted or unsubstituted alkoxy groups, substituted or unsubstituted alkylthio groups, substituted or unsubstituted amino groups or halogens.
In some embodiments of the invention, R 2 、R 3 Independently selected from hydrogen, deuterium, substituted or unsubstituted methyl, substituted or unsubstituted ethyl, substituted or unsubstituted propyl, substituted or unsubstituted cyclopropyl; wherein R is 2 、R 3 Each substituent of (a) is independently selected from one or more of deuterium, halogen, hydroxy, amino, methyl, ethyl, cyclopropyl, t-butyl, methoxy, ethoxy, cyclopropoxy, t-butoxy, methylthio, ethylthio, cyclopropylthio, t-butylthio, amino, methylamino, ethylamino, cyclopropylamino, t-butylamino, carboxamide, acetamido, cyclopropylamido, t-butyrylamino, NO 2 CN or CF 3 ；
Or R is 2 、R 3 A substituted or unsubstituted cyclopropyl group formed by ligation; wherein the substituents for the cyclopropyl group are selected from one or more ofA plurality of deuterium, halogen, hydroxyl, amino, methyl, ethyl, cyclopropyl, t-butyl, methoxy, ethoxy, cyclopropoxy, t-butoxy, methylthio, ethylthio, cyclopropylthio, t-butylthio, amino, methylamino, ethylamino, cyclopropylamino, t-butylamino, carboxamide, acetamido, cyclopropylamido, t-butyrylamino, NO 2 CN or CF 3 The method comprises the steps of carrying out a first treatment on the surface of the Or/and R 4 Selected from the group consisting of substituted or unsubstituted phenyl, substituted or unsubstituted pyridinyl, substituted or unsubstituted pyrimidinyl, substituted or unsubstituted pyrazinyl, substituted or unsubstituted pyridazinyl, substituted or unsubstituted benzothiazolyl, substituted or unsubstituted benzisoxazolyl, substituted or unsubstituted imidazopyridazinyl, and R 4 The substituent of (2) is selected from 1-5 deuterium, amino, cyano, methyl, methoxy, halogen, trifluoromethoxy or difluoromethoxy;
or/and R 4 Selected from the group consisting of substituted or unsubstituted phenyl, substituted or unsubstituted pyridinyl, substituted or unsubstituted pyrimidinyl, substituted or unsubstituted pyrazinyl, substituted or unsubstituted pyridazinyl, substituted or unsubstituted benzothiazolyl, substituted or unsubstituted benzisoxazolyl, substituted or unsubstituted imidazopyridazinyl, and R 4 The substituent of (2) is selected from 1-5 deuterium, amino, cyano, methyl, methoxy, halogen, trifluoromethoxy or difluoromethoxy;
or/and R 5 Selected from 1 to 5 hydrogen, deuterium, amino, methyl, halogen, trifluoromethyl and difluoromethyl.
In some embodiments of the invention, R 2 、R 3 Independently selected from hydrogen, deuterium, methyl, ethyl, propyl or cyclopropyl;
or R is 2 、R 3 The connection forms cyclopropyl.
In some embodiments of the invention, the compound selected from the following compounds, or pharmaceutically acceptable salts or prodrugs thereof:
TABLE 1
In some embodiments of the invention, the hydrogen in the compounds of formula I may be replaced by one or more deuterium.
The preparation method provided by the invention comprises the following steps:
step 1: the compound a and the compound k undergo substitution reaction under the catalysis of alkali to generate a compound b;
step 2: the compounds c and d undergo substitution reaction under alkaline conditions to obtain e;
step 3: carrying out a photo-delay reaction on the compounds e and f to obtain an intermediate g;
step 4: the coupling reaction of the compounds g and b is carried out in the presence of a catalyst to obtain a compound h;
step 5: the compound h undergoes hydrolysis reaction under inorganic base catalysis or acid catalysis to obtain a compound i;
step 6: the compound I and the compound j undergo condensation or esterification reaction to obtain a compound of formula (I);
preferably, the catalyst used in step 4 is a palladium-based catalyst.
The invention provides an application of a compound shown in a formula I or a salt, a solvate, an allosteric structure, a metabolite, a nitrogen oxide and a prodrug thereof in preparing medicines for treating or preventing P2X3 and/or P2X2/3 receptor related diseases.
In some embodiments of the invention, the disease is a respiratory disease.
In some embodiments of the invention, the compounds or salts, solvates, allosteric structures, metabolites, nitroxides and prodrugs thereof are used for the preparation of a medicament for the treatment or prophylaxis of cough, asthma, pain, sleep apnea.
The compound name corresponding to the English abbreviation is:
DMF: n, N-dimethylformamide
DIPEA: 1-Hydroxybenzotriazole (HOBT) N, N-diisopropylethylamine
xant-phos:4, 5-bis (diphenylphosphine) -9, 9-dimethylxanthene
HATU:2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate
DMSO: dimethyl sulfoxide
Compared with the prior art, the invention has the following beneficial effects:
the dihydropyrimidine compound provided by the invention has good affinity with P2X3, and has stronger antagonism to P2X3 receptor, thereby being safe and effective. The cough test of mice shows that the compound has good antitussive effect, and the in vitro biological activity evaluation shows that the compound has stronger inhibition effect on P2X3 receptor, and the taste disorder test shows that the taste of mice has little influence and has obvious statistical difference with a positive control group. The invention is useful for the treatment or prevention of P2X3 and/or P2X2/3 receptor-related diseases.
The preparation method of the invention is simple, is easy to operate and is easy to industrialize.
Detailed Description
The present invention will be described in further detail with reference to the following examples and experimental examples, which are only for illustrating the technical scheme of the present invention, but not for limiting the present invention, and any equivalent substitution in the art according to the disclosure of the present invention shall fall within the scope of the present invention.
The structure of the compound in the embodiment of the invention adopts nuclear magnetic resonance 1 H NMR) or liquid mass spectrometry (LC-MS).
The liquid chromatography-mass spectrometry (LC-MS) in the embodiment of the invention is Agilent G6120B (matched with liquid Agilent 1260); nuclear magnetic resonance apparatus 1 H NMR) of Bruker AVANCE-400 or Bruker AVANCE-800, nuclear magnetic resonance 1 H NMR) shift (δ) given in parts per million (ppm), solvent of measurement DMSO, internal standard Tetramethylsilane (TMS), chemical shift of 10 -6 (ppm) is given as a unit.
As used herein, "room temperature" means 10 to 25 ℃.
Example 1: preparation of (S) -3- (3- (4-chlorobenzyl) -2, 6-dioxo-4- (4- (pyrazin-2-yloxy) phenyl) amino) -3, 6-dihydropyrimidin-1 (2H) -yl) -2-methylpropanamide (Compound 1)
Step 1: preparation of 4- (pyrazin-2-yloxy) aniline (Compound b-1)
2-Fluoropyrazine (50.0 g,0.52 mol) and p-aminophenol (53.5 g,0.49 mol) were dissolved in dimethyl sulfoxide (360 ml), cesium carbonate (320 g,0.98 mol) was added to obtain a reaction mixture, and the reaction mixture was stirred with mechanical stirring at a constant speed. Then the internal temperature of the reaction system is raised to 80 ℃ for reaction for 2 hours. Thin layer chromatography followed the progress of the reaction and after completion of the reaction mixture was added to three volumes (about 1L) of water and stirring was maintained. The product was then extracted three times with ethyl acetate, the ethyl acetate was combined and dried, and concentrated to give crude product, which was slurried with 500ml of water for 1h, filtered, and air-dried in an air-drying oven to give 4- (pyrazin-2-yloxy) aniline (92.8 g, brown granular solid) in 96.8% yield.
ESI-MS:m/z＝187.1(M+H) + 。
Step 2: preparation of 6-chloro-1- (4-chlorobenzyl) pyrimidine-2, 4 (1H, 3H) -dione (Compound e-1)
6-chlorouracil (36.8 g,0.25 mol) and 4-chlorobenzyl bromide (52.5 g,0.256 mol) were mixed, dissolved in 300ml DMF, DIPEA (96.9 g,0.75 mol) was added dropwise and reacted at 30℃for 3 hours, after completion of the reaction by thin layer chromatography, the reaction mixture was added to 3 volumes of water, the washed solid was filtered, the cake was slurried with 300ml ethyl acetate after drying, the solid was obtained by filtration, and after drying with a forced air dryer, 6-chloro-1- (4-chlorobenzyl) pyrimidine-2, 4 (1H, 3H) -dione compound (51.9 g, white solid) was obtained in a yield of 76.6% and a purity of 99.26%
ESI-MS:m/z＝271.0(M+H) + 。
Step 3: preparation of methyl (S) -3- (4-chloro-3- (4-chlorobenzyl) -2, 6-dioxo-3, 6-dihydropyrimidin-1 (2H) -yl) -2-methylpropionate (Compound g-1)
Compound e-1 (27.1 g,0.1 mol), (S) - (+) -3-hydroxy-2-methylpropanoic acid methyl ester (11.8 g,0.1 mol) and triphenylphosphine (52.4 g,0.2 mol) were dissolved and clarified with 300ml of anhydrous tetrahydrofuran, the reaction system was cooled in an ice-water bath after replacing the air in the reaction system with argon, diisopropyl azodicarboxylate (40.4 g,0.2 mol) was slowly and uniformly added dropwise with stirring, after the dropwise addition was completed within 30min, the reaction was kept at room temperature, the progress of the reaction was followed by thin layer chromatography, after the completion of the reaction, the reaction solution was quenched with 500ml of water, extracted three times with 30ml of ethyl acetate, and the organic solution was dried and concentrated to an oily crude product. The crude oil was dispersed with a mixed solvent of 100ml of ethyl acetate and 500ml of petroleum ether, a large amount of triphenylphosphine oxide solid was precipitated, triphenylphosphine oxide was removed by filtration, and after concentration of the mother liquor, this was purified by chromatography to give methyl (S) -3- (4-chloro-3- (4-chlorobenzyl) -2, 6-dioxo-3, 6-dihydropyrimidin-1 (2H) -yl) -2-methylpropionate (32.1 g, white solid) in 86.6% yield and 98.71% purity.
ESI-MS:m/z＝371.1(M+H) + 。
Step 4: preparation of methyl (S) -3- (3- (4-chlorobenzyl) -2, 6-dioxo-4- (4- (pyrazin-2-yloxy) phenyl) amino) -3, 6-dihydropyrimidin-1 (2H) -yl) -2-methylpropionate (Compound H-1)
Compound g-1 (3.71 g,0.01 mol), compound b-1 (1.87 g,0.01 mol), xant-phos (868 mg,1.5 mmol), palladium acetate (337 mg,1.5 mmol), potassium phosphate (4.24 g,0.02 mol) were mixed and dissolved with 30ml dioxane, air in the reaction flask was replaced with argon, and the reaction mixture was heated to 80 ℃ in an oil bath for 1H under argon protection, the reaction was checked until the compound g-1 was consumed completely by thin layer chromatography, the reaction mixture was distilled off under reduced pressure to remove dioxane, and extracted three times with 100ml of ethyl acetate and 100ml of water, and after ethyl acetate was concentrated by drying, column chromatography was purified to give methyl (S) -3- (3- (4-chlorobenzyl) -2, 6-dioxo-4- (4- (pyrazin-2-oxy) phenyl) amino) -3, 6-dihydropyrimidin-1 (2H) -yl) -2-methylpropionate (4.56 g, 87g, as a yellow brown foam solid with a purity of 96.82%.
ESI-MS:m/z＝522.2(M+H) + 。
1 H NMR(400MHz,DMSO-d6)δ8.65(s,1H),δ8.17(s,1H),7.55–7.48(m,1H),7.46–7.37(m,1H),7.35–7.26(m,2H),7.16(s,4H),7.14–7.11(m,1H),7.04(d,J＝8.3Hz,1H),5.28(s,2H),4.61(s,1H),3.88(m,2H),3.46(s,3H),2.76(m,1H),0.99(m,3H)。
Preparation of step 5,S-3- (3- (4-chlorobenzyl) -2, 6-dioxo-4- (4- (pyrazin-2-yloxy) phenyl) amino) -3, 6-dihydropyrimidin-1 (2H) -yl) -2-methylpropanoic acid (Compound i-1)
Compound h-1 (522 mg,1.0 mmol) was dissolved in a mixed solvent of methanol (3 ml) and tetrahydrofuran (3 ml), the temperature was kept around 10℃and a solution of lithium hydroxide (168 mg,4 mmol) in water (3 ml) was added to obtain a reaction mixture, and the reaction mixture was allowed to react overnight at room temperature. The progress of the reaction was followed by thin layer chromatography, and after completion of the reaction, the compound i-1 (388 mg, off-white solid) was purified by column chromatography to give the yield: 76.5% and purity of 98.62%.
ESI-MS:m/z＝508.1(M+H) + 。
1 HNMR(400MHz,DMSO-d6)δ12.11(s,1H)，δ8.85(s,1H)，δ8.18(s,1H),7.56–7.49(m,1H),7.48–7.39(m,1H),7.35–7.26(m,2H),7.16(s,4H),7.14–7.11(m,1H),7.04(d,J＝8.3Hz,1H),5.30(s,2H),4.62(s,1H),4.06–3.79(m,2H),2.79-2.68(m,1H),0.99-0.95(m,3H)。
Step 6 preparation of (S) -3- (3- (4-chlorobenzyl) -2, 6-dioxo-4- (4- (pyrazin-2-yloxy) phenyl) amino) -3, 6-dihydropyrimidin-1 (2H) -yl) -2-methylpropanamide (Compound 1)
Compound j-1 (288 mg,0.57 mmol), HATU (433 mg,1.14 mmol), HOBT (38 mg,0.29 mmol), NH 4 Cl (46 mg,0.86 mmol), DIPEA (183 mg,1.42 mmol) was dissolved in DMF (5 ml), the reaction was carried out at room temperature for 16 hours, thin layer chromatography followed by ice water quenching after completion of the reaction, extraction with dichloromethane three times, combination, drying and concentration followed by purification by column chromatography to give compound 1 (198 mg) in a yield of 60.6% and a purity of 99.87%.
ESI-MS:m/z＝585.1(M+H) + 。
1 HNMR(400MHz,DMSO-d6)δ8.65(s,1H)，δ8.19(s,1H)，7.91(s，2H),7.86–7.76(m,1H),7.48–7.39(m,1H),7.35–7.26(m,2H),7.16(m,4H),7.14–7.11(m,1H),7.04(d,J＝8.3Hz,1H),5.42–5.15(s,2H),4.62(s,1H),3.92-3.86(m,2H),2.73-2.67(m,1H),0.98-0.92(m,3H)。
Example 2: preparation of 3- (3- (4-chlorobenzyl) -2, 6-dioxo-4- (4- (pyrazin-2-yloxy) phenyl) amino) -3, 6-dihydropyrimidin-1 (2H) -yl) -N-ethyl-2, 2-dimethylpropionamide (Compound 2)
The preparation method of this example compared to example 1, replaced methyl (S) -3-hydroxy-2-methylpropionate in step 3 with equimolar methyl 3-hydroxy-2, 2-dimethylpropionate, and replaced ammonium chloride in step 6 with equimolar ethylamine, all the other conditions being the same; compound 2 was obtained as a white solid in yield: 66.5% and purity of 99.78%.
ESI-MS:m/z＝549.2(M+H) + 。
1 HNMR(400MHz,DMSO-d6)δ9.52(s,1H)，δ8.66(s,1H)，δ8.16(s,1H),7.85–7.76(m,1H),7.48–7.38(m,1H),7.35–7.26(m,2H),7.16(m,4H),7.14–7.11(m,1H),7.04(d,J＝8.3Hz,1H),5.42–5.15(s,2H),4.62(s,1H),3.92(s,2H),3.22-3.16(m,2H),1.09(s,6H),1.02-0.96(m,3H)。
Example 3: preparation of (S) -3- (3- (4-chlorobenzyl) -4- (4- (2-cyanopyrimidin-5-yloxy) phenyl) -amino) -2, 6-dioxo-3, 6-dihydropyrimidin-1 (2H) -yl) -2-methylpropanamide (Compound 3)
The preparation method of this example was compared with example 1, and the 2-fluoropyrazine in step 1 was replaced with equimolar 5-fluoro-2-cyanopyrimidine, with the remaining conditions being identical. Compound 3 was obtained in yield: 67.3% and a purity of 99.76%.
ESI-MS:m/z＝532.2(M+H) + 。
1 HNMR(400MHz,DMSO-d6)δ8.63(s,1H)，δ8.11(s,2H),7.95(s，2H),7.86–7.76(m,1H),7.35–7.26(m,2H),7.18-7.09(m,4H),7.14–7.11(m,1H),5.32(s,2H),4.62(s,1H),3.88-3.79(m,2H),2.72-2.68(m,1H),1.01-0.96(m,3H)。
Example 4: preparation of (S) -3- (3- (4-chlorobenzyl) -4- (4- (3- (3-methylpyrazin-2-oxo) phenyl) amino) -2, 6-dioxo-3, 6-dihydropyrimidin-1 (2H) -yl) -2-methylpropanamide (Compound 4)
The preparation method of this example was compared with example 1, except that 2-fluoropyrazine in step 1 was replaced with equimolar 2-fluoro-3-methylpyrazine, and the other conditions were identical. Compound 4 was obtained as a white solid in yield: 66.5% and purity 98.54%.
ESI-MS:m/z＝521.2(M+H) + 。
1 H NMR(400MHz,DMSO-d6)δ8.66(s,1H),7.96(s，2H),7.84(d,J＝8.1Hz,1H),7.42(d,J＝8.3Hz,1H),7.29(d,J＝8.4Hz,2H),7.16(d,J＝2.5Hz,4H),7.11(d,J＝6.6Hz,1H),7.03(d,J＝8.4Hz,1H),5.26(s,2H),4.59(s,1H),3.95-3.89(m,2H),2.98(s,3H),2.71-2.63(m,1H),1.02-0.98(m,3H)。
Example 5: preparation of (S) -3- (3- (4-chlorobenzyl) -2, 6-dioxo-4- (4-phenoxyphenyl) amino) -3, 6-dihydropyrimidin-1 (2H) -yl) -2-methylpropanamide (Compound 5)
In the preparation method of this example, compared with example 2, 2-fluoropyrazine in step 1 was replaced with equimolar fluorobenzene, and the other conditions were identical to each other, to obtain compound 5 as a white solid, yield: 62.2% and purity of 99.56%.
ESI-MS:m/z＝505.2(M+H) + 。
1 H NMR(400MHz,DMSO-d6)δ8.65(s,1H),7.99(s，2H)，7.92(m,2H),7.58(m,2H),7.29(d,J＝8.4Hz,2H),7.22(m,1H),7.16(d,J＝2.5Hz,4H),7.11(d,J＝6.6Hz,1H),7.03(d,J＝8.4Hz,1H),5.26(s,2H),4.59(s,1H),3.98-3.90(m,2H),2.72-2.68(m,1H),1.03-0.98(m,3H)。
Example 6: preparation of (S) -3- (3- (4-chlorobenzyl) -4- (4- (2-methoxypyrimidin-4-yloxy) phenyl) -amino) -2, 6-dioxo-3, 6-dihydropyrimidin-1 (2H) -yl) -2-methylpropanamide (Compound 6)
In the preparation method of this example, compared with example 3, the 2-fluoropyrazine in step 1 was replaced with equimolar 4-fluoro-2-methoxypyrimidine, and the other conditions were identical, to obtain compound 6 as a white solid, yield: 68.6% and a purity of 98.68%.
ESI-MS:m/z＝537.2(M+H) + 。
1 H NMR(400MHz,DMSO-d6)δ8.66(s,1H),7.99(s,2H),7.82(d,J＝8.4Hz,1H),7.56(d,J＝8.3Hz,1H),7.29(d,J＝8.4Hz,2H),7.16(d,J＝2.5Hz,4H),7.11(d,J＝6.6Hz,1H),7.03(d,J＝8.4Hz,1H),5.26(s,2H),4.59(s,1H),3.98-3.90(m,2H),2.72-2.66(m,1H),1.02-0.95(m,3H)。
Example 7: preparation of (S) -3- (3- (4-chlorobenzyl) -2, 6-dioxo-4- (4- (pyridin-2-yloxy) phenyl) amino) -3, 6-dihydropyrimidin-1 (2H) -yl) -2-methylpropanamide (Compound 7)
The preparation method of this example was compared with example 1, except that 2-fluoropyrazine in step 1 was replaced with equimolar 2-fluoropyridine, and the other conditions were identical. Compound 7 was obtained as a white solid in yield: 68.2% and a purity of 98.64%.
ESI-MS:m/z＝506.2(M+H) + 。
1 H NMR(400MHz,DMSO-d6)δ8.65(s,1H),7.99(s,2H),8.16-8.05(m,1H),7.94–7.80(m,1H),7.48–7.39(m,2H),7.35–7.26(m,2H),7.16(s,4H),7.14–7.11(m,1H),7.04(d,J＝8.3Hz,1H),5.30(s,2H),4.62(s,1H),4.06–3.79(m,2H),2.75-2.68(m,1H),1.02-0.96(m,3H)。
Example 8: preparation of (S) -3- (4- (4- ((5-chloropyridin-2-yl) phenyl) amino) -3- (4-methylbenzyl) -2, 6-dioxo-3, 6-dihydropyrimidin-1 (2H) yl) -2-methylpropanamide (Compound 8)
The preparation method of this example was compared with example 1, in which 2-fluoropyrazine in step 1 was replaced with equimolar 2-fluoro-5-chloro-pyridine, and in which 1- (bromomethyl) -4-chlorobenzene in step 2 was replaced with equimolar 1- (bromomethyl) -4-toluene, and the other conditions were identical. Compound 8 was obtained as a white solid in yield: 62.8% purity 98.51%.
ESI-MS:m/z＝520.2(M+H) + 。
1 H NMR(400MHz,DMSO-d6)δ8.63(s,1H),8.20–8.11(m,1H),7.97(s,2H),7.90–7.79(m,1H),7.48–7.38(m,1H),7.29(d,J＝8.3Hz,2H),7.21–7.14(m,4H),7.14–7.10(m,1H),7.03(d,J＝8.3Hz,1H),5.28(s,2H),4.61(s,1H),3.92-3.85(m,2H),2.81-2.75(m,1H),1.02-0.98(m,3H)。
Example 9: preparation of 1- (3- (4-chlorobenzyl) -2, 6-dioxo-4- (4- (pyridin-2-yloxy) phenyl) amino) -3, 6-dihydropyrimidine-1 (2H) -methyl) -n-hexylcyclopropane-1-carboxamide (Compound 9)
In the preparation method of this example, compared with example 3, 2-fluoropyrazine in step 1 was replaced with equimolar 2-fluoropyridine, and ammonium chloride in step 6 was replaced with equimolar n-hexane amine, and the other conditions were identical. Compound 9 was obtained as a white solid in yield: 65.2% and a purity of 99.27%.
ESI-MS:m/z＝602.3(M+H) + 。
1 H NMR(400MHz,DMSO-d6)δ8.91(s,1H),8.60(s,1H),8.15(dd,J＝5.0,2.0Hz,1H),7.90–7.79(m,1H),7.45–7.37(m,2H),7.30(d,J＝8.4Hz,2H),7.15(s,4H),7.14–7.09(m,1H),7.03(d,J＝8.3Hz,1H),5.28(s,2H),4.62(s,1H),4.03(s,2H),3.12-3.03(m,2H),1.38-1.26(m,8H),1.07–0.92(m,4H),0.91-0.88(m,3H)。
Example 10: preparation of 1- (3- (4-chlorobenzyl) -2, 6-dioxo-4- (4- (pyridin-2-yloxy) phenyl) amino) -3, 6-dihydropyrimidine-1 (2H) -methyl) -N-cyclohexylcyclopropane-1-carboxamide (Compound 10)
The preparation method of this example was comparable to that of example 2, wherein the 2-fluoropyrazine in step 1 was replaced with equimolar 2-fluoropyridine and the ammonium chloride in step 6 was replaced with equimolar cyclohexane amine, with the remaining conditions being identical. Compound 10 was obtained as a white solid in yield: 69.6% and a purity of 98.66%.
ESI-MS:m/z＝600.2(M+H) + 。
1 H NMR(400MHz,DMSO-d6)δ8.91(s,1H),8.60(s,1H),8.15(dd,J＝5.0,2.0Hz,1H),7.90–7.79(m,1H),7.45–7.37(m,2H),7.30(d,J＝8.4Hz,2H),7.15(s,4H),7.14–7.09(m,1H),7.03(d,J＝8.3Hz,1H),5.28(s,2H),4.62(s,1H),4.03(s,2H),1.52-1.39(m,6H),1.28-1.19(m,4H),1.08–0.92(m,4H)。
Example 11: preparation of (S) -3- (3- (4-fluorobenzyl) -4- (4- (3- (3-fluoropyridin-2-yl) oxyphenyl) amino) -2, 6-dioxo-3, 6-dihydropyrimidin-1 (2H) yl) -2-methyl-N- (piperidin-4-yl) propanamide (Compound 11)
The preparation method of this example was compared with example 1, wherein 2-fluoropyrazine in step 1 was replaced with equimolar 2-fluoropyridine, 1- (bromomethyl) -4-chlorobenzene in step 2 was replaced with equimolar 1- (bromomethyl) -4-fluoro-benzene, and ammonium chloride in step 6 was replaced with equimolar 4-piperidylamine, and the other conditions were identical. Compound 11 was obtained as a white solid in yield: 65.2% and 97.57% purity.
ESI-MS:m/z＝591.3(M+H) + 。
Example 12: preparation of (S) -3- (3- (4-chlorobenzyl) -4- (4- (3- (3-fluoropyridin-2-yl) oxyphenyl) amino) -2, 6-dioxo-3, 6-dihydropyrimidin-1 (2H) yl) -2-methylpropanamide (Compound 12)
In the preparation method of this example, compared with example 3, the 2-fluoropyrazine in step 1 was replaced with equimolar 2-fluoropyridine, and the other conditions were identical. Compound 12 was obtained as a white solid in yield: 67.8% and purity 98.48%.
ESI-MS:m/z＝538.2(M+H) + 。
Example 13: preparation of (2S) -3- (2, 6-dioxo-4- (4- (pyridin-2-yloxy) phenyl) amino) -3- (4- (trifluoromethyl) benzyl) -3, 6-dihydropyrimidin-1 (2H) yl) -2-methyl-N- (pyrrolidin-3-yl) propanamide (Compound 13)
The preparation method of this example was compared with example 2, wherein 2-fluoropyrazine in step 1 was replaced with equimolar 2-fluoropyridine, 1- (bromomethyl) -4-chlorobenzene in step 2 was replaced with equimolar 1- (bromomethyl) -4-trifluoromethylbenzene, and ammonium chloride in step 6 was replaced with equimolar 3-pyrrolidinamine, and the other conditions were identical. Compound 13 was obtained as a white solid in yield: 66.6% and purity 98.53%.
ESI-MS:m/z＝609.3(M+H) + 。
Example 14: preparation of (S) -3- (3- (4-chlorobenzyl) -2, 6-dioxo-4- (4- (pyrimidin-2-yloxy) phenyl) amino) -3, 6-dihydropyrimidin-1 (2H) -yl) -2-methylpropanamide (Compound 14)
The preparation method of this example was compared with example 1, except that 2-fluoropyrazine in step 1 was replaced with equimolar 2-fluoropyridine, and the other conditions were identical. Compound 14 was obtained as a white solid in yield: 79.2% and a purity of 98.96%.
ESI-MS:m/z＝507.2(M+H) + 。
Example 15: preparation of (S) -3- (3- (4-chlorobenzyl) -4- (4- (5-chloropyridin-2-yloxy) phenyl) amino) -2, 6-dioxo-3, 6-dihydropyrimidin-1 (2H) -yl) -2-methylpropanamide (Compound 15)
In the preparation method of this example, compared with example 3, the 2-fluoropyrazine in step 1 was replaced with equimolar 2-fluoropyridine, and the other conditions were identical. Compound 15 was obtained as a white solid in yield: 65.8% and a purity of 98.51%.
ESI-MS:m/z＝540.1(M+H) + 。
Example 16: preparation of (S) -3- (4- (4- (benzo [ D ] isoxazol-3-yloxy) phenyl) amino) -3- (4-methylbenzyl) -2, 6-dioxo-3, 6-dihydropyrimidin-1 (2H) -yl) -2-methylpropanamide (Compound 16)
The preparation method of this example was compared with example 3, in which 2-fluoropyrazine in step 1 was replaced with equimolar 2, 3-difluoropyridine, and in which 1- (bromomethyl) -4-chlorobenzene in step 2 was replaced with equimolar 1- (bromomethyl) -4-toluene, and the other conditions were identical. Compound 16 was obtained as a white solid in yield: 68.5% and a purity of 98.89%.
ESI-MS:m/z＝526.2(M+H) + 。
Example 17: preparation of (S) -3- (3- (4-chlorobenzyl) -4- (4- (6-methoxypyridazin-3-yloxy) phenyl) -amino) -2, 6-dioxo-3, 6-dihydropyrimidin-1 (2H) -yl) -2-methylpropanamide (Compound 17)
Preparation procedure following the preparation of example 3 substituting 2-fluoropyrazine in step 1 with equimolar 3-fluoro-6-methoxy-pyridazine, the title compound was obtained as a white solid in yield: 68.9% and a purity of 99.19%.
ESI-MS:m/z＝537.2(M+H) + 。
Example 18: preparation of (S) -3- (3- (4-chlorobenzyl) -4- (4- (6-fluoropyridin-2-yl) phenyl) -amino) -2, 6-dioxo-3, 6-dihydropyrimidin-1 (2H) -yl) -2-methylpropanamide (Compound 18)
The preparation method of this example was compared with example 3, in which 2-fluoropyrazine in step 1 was replaced with equimolar 2, 6-difluoropyridine, and the other conditions were identical. Compound 18 was obtained as a white solid in yield: 68.2% and purity 98.29%.
ESI-MS:m/z＝524.1(M+H) + 。
Example 19: preparation of (S) -3- (3- (4-chlorobenzyl) -2, 6-dioxo-4- (4- (5- ((trifluoromethoxy) pyridin-2-yl) oxyphenyl) amino) -3, 6-dihydropyrimidin-1 (2H) -yl) -2-methylpropanamide (Compound 19)
The preparation method of this example was compared with example 3, and the 2-fluoropyrazine in step 1 was replaced with equimolar 2-fluoro-5- (trifluoromethoxy) pyridine, and the other conditions were identical. Compound 19 was obtained as a white solid in yield: 62.1% purity 98.28%.
ESI-MS:m/z＝590.1(M+H) + 。
Example 20: preparation of (S) -3- (3- (4-chlorobenzyl) -4- (4- ((5- (difluoromethoxy) pyridin-2-yl) oxyphenyl) -amino) -2, 6-dioxo-3, 6-dihydropyrimidin-1 (2H) -yl) -2-methylpropanamide (Compound 20)
The preparation method of this example was compared with example 3, and the 2-fluoropyrazine in step 1 was replaced with equimolar 2-fluoro-5- (difluoromethoxy) pyridine, with the remaining conditions being identical. Compound 20 was obtained as a white solid in yield: 69.1% and the purity is 98.17%.
ESI-MS:m/z＝572.2(M+H) + 。
Example 21: preparation of (S) -3- (4- (4- (benzo [ D ] thiazol-2-yloxy) phenyl) amino) -3- (4-chlorobenzyl) -2, 6-dioxo-3, 6-dihydropyrimidin-1 (2H) -yl) -2-methylpropanamide (Compound 21)
The preparation method of this example is comparable to that of example 3, except that the 2-fluoropyrazine in step 1 is replaced with equimolar 2-fluoro-4-benzo [ D ] thiazole, all under the same conditions. Compound 21 was obtained as a white solid in yield: 65.6% and a purity of 98.59%.
ESI-MS:m/z＝562.1(M+H) + 。
Example 22: preparation of (S) -3- (3- (4-chlorobenzyl) -4- (4- (imidazo [1,2-B ] pyridin-6-yloxy) phenyl) amino) -2, 6-dioxo-3, 6-dihydropyrimidin-1 (2H) -yl) -2-methylpropanamide (Compound 22)
The preparation method of this example is compared with example 3, wherein the 2-fluoropyrazine in step 1 is replaced by equimolar 6-fluoro-4-imidazo [1,2-B ] pyridine, and the other conditions are identical. Compound 22 was obtained as a white solid in yield: 61.1% and a purity of 99.84%.
ESI-MS:m/z＝546.2(M+H) + 。
Example 23: preparation of (S) -3- (3- (4-chlorobenzyl) -4- (4- (5-chloropyridin-2-yl) phenyl) amino) -2, 6-dioxa-3, 6-dihydropyrimidin-1 (2H) yl) -N-hexadecyl-2-methylpropanamine (Compound 23)
In the preparation method of this example, compared with example 3, 2-fluoropyrazine in step 1 was replaced with equimolar 2-fluoro-5-chloropyridine, and ammonium chloride in step 6 was replaced with equimolar hexadecylamine, and the other conditions were identical. Compound 23 was obtained as a white solid in yield: 78.3% and purity 98.19%.
ESI-MS:m/z＝764.4(M+H) + 。
Example 24: preparation of (S) -3- (3- (4-chlorobenzyl) -4- (4- (6- (6-fluoropyridin-2-yl) oxyphenyl) amino) -2, 6-dioxo-3, 6-dihydropyrimidin-1 (2H) yl) -N-hexadecyl-2-methylpropanamide (Compound 24)
In the preparation method of this example, compared with example 3, the 2-fluoropyrazine in step 1 was replaced with equimolar 2, 6-difluoropyridine, and the ammonium chloride in step 6 was replaced with equimolar hexadecylamine to obtain compound 24 as a white solid, yield: 68.6% and a purity of 98.82%.
ESI-MS:m/z＝748.4(M+H) + 。
Comparative example 1: preparation of (S) -3- (3- (4-chlorobenzyl) -4- (4- (3-fluoropyridin-2-yloxy) phenyl) amino) -2, 6-dioxa-3, 6-dihydropyrimidin-1 (2H) yl) -2-methylpropanoic acid
The preparation method of this comparative example was identical to that of the compound i-1 of example 1 except that 2-fluoropyrazine in step 1 was replaced with equimolar 2, 3-difluoropyridine. The compound of comparative example 1 was obtained as a white solid in yield: 61.4% and a purity of 99.27%.
ESI-MS:m/z＝525.1(M+H) + 。
1 H NMR(400MHz,DMSO-d6)δ12.09(s,1H),8.65(s,1H),8.03(q,J＝8.2Hz,1H),7.44(d,J＝8.2Hz,2H),7.32(d,J＝8.2Hz,2H),7.22(brs,4H),6.95(dd,J＝8.0,1.7Hz,1H),6.90(dd,J＝7.9,2.5Hz,1H),5.31(s,2H),4.66(s,1H),4.08–3.80(m,2H),2.77(m,1H),0.99(m,3H)。
Test example 1: cough test in mice
1 test materials
1.1 basic information of test article: the compounds of examples 1-22 (laboratory synthesis of the present invention), the positive control (gefasixant, lot number: 01030-210326-2-1, commercially available from the palm), and the compound of comparative example 1 (laboratory synthesis of the present invention).
1.2 test reagents: physiological saline and ammonia water.
2 experimental animals: healthy adult KM mice, male and female, each group of 6 mice, and the weight of the mice is about 28 g to 30 g.
3 test method
3.1 dose design and amount of test sample to be used
The animal cough model reported in the literature mostly adopts methods such as mechanical, chemical, electrical stimulation and the like to stimulate nerves and receptors of animals so as to cause cough. And (3) establishing a mouse cough modeling test by initially selecting a strong ammonia water induction method according to the characteristics of the candidate compound and the existing similar target compound as references.
3.2 preparation method of sample
The preparation method of 50% ammonia water solution comprises the following steps: 2.5ml of ammonia water is measured and dissolved in 5ml of 0.9% sodium chloride injection, and the mixture is fully and uniformly mixed.
Positive control drug and comparative example 1 solution formulation method: 9mg of the positive control and the compound of comparative example 1 were each weighed and dissolved in 3ml of 0.5% CMC-Na solution, and mixed well to prepare a 3mg/ml solution.
Example solution formulation method: 9mg of the compound of the example was weighed and dissolved in 3ml of 0.5% CMC-Na solution, and the mixture was thoroughly mixed to prepare a 3mg/ml solution.
3.3 Experimental methods of operation
Grouping: the test sample was divided into a model group, a positive control group, a comparative example 1 group, and example 1 to example 22 groups; 6 KM mice are taken from each group and are subjected to gastric lavage administration; wherein, the positive control group is given a positive control drug (purchased) and the comparative example 1 group is given a compound of comparative example 1; examples groups the compounds obtained were prepared by giving the corresponding examples; three doses for each group: 30mg/kg, 10ml/kg; the model group was given an equal volume of 0.5% cmc-Na solution. After administration for 60min or 120min, the mice were placed in 500ml beakers, and 1 cotton ball (weight of 100.+ -. 5 mg) containing 0.3ml of 50% ammonia water was placed in each beaker. Mice were observed for the number of typical coughs that occurred within 3min (typical coughing actions: abdominal muscle contraction or chest constriction, with simultaneous large mouth opening, with coughing).
Results and discussion 4
4.1 result judgment criteria
(1) Cough criterion:
cough manifests as: the abdominal muscles contract or contract the chest, and simultaneously open the large mouth with cough.
(2) The number of coughs (times) in 3min of the mice was recorded by stopwatch, statistical analysis was performed by software, each group of data was statistically described by mean ± standard deviation, multi-group single factor analysis of variance was performed, and P <0.05 was statistically significant.
4.2 discussion of results
TABLE 2
Remarks: comparison to model set: * P<0.01，*P<0.05. Comparison with comparative example 1 group: △ P<0.05。
as can be seen from Table 2, at a dose of 30mg/kg, the number of times of typical cough occurring in the compound mice of the positive control group, the comparative example 1 group and the plurality of example groups within 3min was significantly different from that of the model group at the time of administration for 60min (P)<0.01，*P<0.05 However, the mice of comparative example 1 showed no statistical difference in the number of typical coughs occurring within 3min from the model group when tested for 120 min; while mice of examples 1,2, 3, 7, 8, 9, 18, 22 showed significant differences (×p) in the number of typical coughs occurring within 3min compared to the model and comparative example 1 at 120min<0.01， △ P<0.05)。
At the dose of 10mg/kg, the mice in the positive control group and the mice in the comparative example 1 showed no obvious difference in the number of times of typical cough in 3min compared with the model group. However, mice in example 1, 7, 8 had significant differences in the number of typical coughs occurring within 3min (P < 0.01) compared to the model group.
Test example 2: in vitro biological Activity evaluation
1. The reagents, consumables and instruments used in this test example are all commercially available.
2. Cell lines
HEK293 cell lines stably transfected with human P2X3 receptor were used.
3. Cell culture
Growth medium: DMEM high glucose;10% fbs;1% PenStrep.
4. Cell culture process:
a) Resuscitating cells
1) Immersing the cell cryopreservation tube in a water bath at 37 ℃ and continuously shaking to dissolve the cell cryopreservation tube as soon as possible;
2) Slowly blowing the cells up and down to suspension by using a 1mL pipetting gun, dripping the cells into a 15mL centrifuge tube containing 10mL of fresh pre-warmed growth medium, and centrifuging the cells for 5 minutes at 1000 rpm/min;
3) The supernatant was discarded and the cells were resuspended in 5mL fresh growth medium. The cell suspension was transferred to a petri dish and placed in 5% CO 2 Is subjected to stationary culture at 37 ℃ in an incubator;
4) After 24 hours, the medium was slowly removed (taking care not to disrupt the cell monolayer) and incubated with fresh growth medium.
b) Subculture
Cell lines are generally expressed in terms of 1:3 to 1:4 (more commonly used in a 1:3 passaging ratio) twice a week, and the cells after passaging need 2-3 days to grow to reach 85% confluency;
1) After the cells reached >85% saturation in a 10cm dish, the cells in the dish were aspirated by digestion with 0.25% Trypsin-EDTA solution for about 1 min;
2) Cells were transferred to dishes containing complete growth medium according to dilution ratio. Note that: to maintain logarithmic growth of cells, cell monolayer culture should be maintained;
3) According to the cell line cell doubling time (HEK 293-P2X3:24 hours), cells were passaged using 0.25% trypsin solution.
c) Cryopreserved cells
1) Taking the culture dish out of the incubator, placing the culture dish in an ultra-clean workbench, digesting the culture dish with 0.25% Trypsin-EDTA solution for about 1min, collecting and counting cells, and centrifuging at 1000rpm/min for 5min;
2) The supernatant was aspirated and the cells resuspended in cryopreservation (90% FBS and 10% DMSO) at a density of 2X 10 6 1mL of cell suspension is added into each freezing tube;
3) Placing the cell cryopreservation tube into a cryopreservation box, and then transferring the cell cryopreservation tube to-80 ℃ overnight;
4) The frozen tube was transferred to a liquid nitrogen tank (-196 ℃).
5. Experimental procedure
Step 1: cellsPreparation of the laboratory plate
1) When the cells in the 15cm culture dish grow to 80% and fuse, removing the supernatant, adding 5mL of DPBS to clean the cells and suck out, adding 2.5mL 0.25%Trypsin-EDTA solution into the culture dish, placing the culture dish into an incubator for 1-3 minutes or until the cells digest, adding 3mL of complete culture medium to terminate digestion, and detecting the cell density by a cell counter;
2) After centrifugation at 1000rpm/min for 5min, the cells were resuspended in growth medium and the volume of the suspension was adjusted to a cell density of 4X 10 5 cells/mL(1×10 4 cells/25μL)；
3) To a black microplate, 10. Mu.L of 5 Xmatrigel was added, and after placing the microplate in an incubator for 15 minutes, the microplate was taken out, centrifuged at 300g/min for 30 seconds, and 5 Xmatrigel was removed. The prepared cell suspension was then added to 384 black microwell plates, 25 μl per well;
4) Placing microplates into 5% CO 2 Is incubated overnight at 37℃until the next day the cells grow to confluent state.
Step 2: preparation of the Compounds
Antagonist mode
1) Test compound mother liquor concentration: 20mM;
2) The compounds on 384-LDV plates were 12-point diluted with Bravo, starting concentration of compound 10 μm, dilution fold 3-fold;
3) HPE (high potency control): a single dose of positive control compound; FAC (final concentration): 40. Mu.M; ZPE (zero effect control): 100% dmso;
4) Compounds on 384-LDV plates and HPE, ZPE were transferred to 384 well plates (PE 6008590) using ECHO as compound plates;
5) Compound plates were stored at-20 ℃.
Step 3: screening assays were performed
1) Taking out the cell plates grown to a fusion state from the incubator;
2) Preparing detection buffer solution: 30mL of buffer containing 0.3mL 250mM probenecid, 0.6mL of 1M HEPES and 29.1mL of HBSS, the actual amount of detection buffer will depend on the number of cell plates;
3) Preparing C6 dyne, wherein the stock solution of the C6 dyne is 10×, and diluting the C6 dyne to 1×witha buffer solution;
4) The medium was discarded by inverted centrifugation using the genetle spin mode of Bluewanser.
5) C6 dyne, 20. Mu.L/well was added to the cell plate using a pipette gun;
6) After centrifugation of the cell plates at 300rpm/min for 30s, incubation was performed in an incubator for 1.5h;
7) mu.L of assay buffer was added to each well on pre-prepared compound plates using a Dragonfly automated applicator, 10. Mu.L of compound was transferred to the cell plates with Bravo according to the assay plate layout, and the test compound was tested for the highest concentration of FAC: 10. Mu.M, 3-fold dilution, 12 concentration spots.
8) Centrifuging the cell plate at 300rpm/min for 30s, and placing the cell plate into an incubator for incubation for 30min;
9) 25. Mu.L of 4 XBZATP (final concentration 3.5. Mu.M) agonist was prepared on an agonist plate (PE 6008590) and acted on P2X3 cells.
10 Placing the cell plate, FLIPR gun head and agonist plate at room temperature for 15min;
11 Transfer 10 μl of BZATP agonist into the cell plate with FLIPR and read.
Step 4: data analysis with Excel and Xlfit6. Experimental results and analysis
Inhibition of P2X3 receptor by Compounds of examples IC 50 As shown in the following Table, wherein A represents less than 10nM, B represents 10.1-50 nM, and C represents greater than 50.1nM.
TABLE 3 Table 3
| Test sample | P2X3 IC 50 (nM) |
| Positive control medicine | C |
| Comparative example 1 | B |
| Compound 1 | A |
| Compound 2 | B |
| Compound 3 | A |
| Compound 4 | C |
| Compound 5 | C |
| Compound 7 | A |
| Compound 8 | A |
| Compound 11 | C |
| Compound 12 | A |
| Compound 13 | C |
| Compound 14 | B |
| Compound 18 | A |
| Compound 21 | A |
| Compound 22 | A |
From Table 3, it is clear that compounds 1, 3, 7, 8, 12, 18, 21, 22 inhibit the P2X3 receptor IC 50 Is superior to the positive control and the compound of comparative example 1.
Test example 3: gustatory disorder test
1 test materials
1.1 basic information of test sample
The compounds of examples 3, 8 and 21 (synthesized in the laboratory of the present invention) and a positive control drug (gefasixant, lot number: 01030-210326-2-1, obtained by the purchase of palm medicine).
1.2 test reagents
0.9% sodium chloride injection, quinine hydrochloride (Quinie, batch number: C12476271)
2 laboratory animals
Healthy adult SD rats, all male, weigh around 280-300 g.
3 test method
3.1 preparation method of sample
Preparation method of quinine solution with 0.3 mM: weighing 119.20mg quinine hydrochloride, dissolving in 1000ml tap water, and mixing thoroughly.
The preparation method of the test sample solution comprises the following steps: 40mg of test sample is weighed, a proper amount of DMSO is added for dissolution, then HS-15 solution is added, the mixture is fully and uniformly mixed, and 16ml of physiological saline is added to prepare 2.5mg/ml of solution.
3.2 Experimental methods of operation
Animals and groupings: about 160-180 g/each male SD rat, 10 rats in each group, the average weight of each group is similar, and the rats are bred in a single cage.
Training drinking habit: the animals of each group drink water normally for 30 minutes at 8:30 a.m. and 16:30 a.m. each day, water is forbidden for the rest of the time, the time lasts for 5 days, and the placement positions of two bottles of water are changed each day.
Administration: the water was stopped in the evening the day before the experiment, and the following morning test group was given 4mL/kg (10 mg/kg) of test sample by tail intravenous injection and the model group was given 4mL/kg (10 mg/kg) of 0.5% HS-15 by intravenous injection.
Results and discussion 4
4.1 result judgment criteria
(1) Measuring water intake: after injection, animals are put back into the original cages, the measurement time of each group is respectively in the Tmax interval of various medicines (the measurement time is 0min-15min after administration), each cage is simultaneously put into one bottle of normal drinking water, one bottle of drinking water containing 0.3mM quinine hydrochloride (Quinie), and the left and right positions of two bottles of water in all animal feeding cages are consistent. The animals were allowed to drink water freely for 15min, and the water intake of two bottles of water was measured separately, to an accuracy of 0.1ml.
(2) Data statistical analysis: the amounts of quinine bitter water and tap water were counted separately, and the percentage of quinine water in tap water was compared with variance analysis to determine whether the differences between the groups were significant.
4.2 discussion of results
TABLE 4 Table 4
| Group of | Quinine/tap water (%) |
| Solvent group | 38.16％ |
| Positive control group | 79.01％* |
| Example 3 group (Compound 3) | 44.21％ △ |
| Example 8 group (Compound 8) | 36.16％ △ |
| Example 21 group (Compound 21) | 39.22％ △ |
Remarks: comparison to vehicle group: * P (P)<0.01; comparison to the positive control group: △ P<0.01。
from the above table, the ratio of quinine/tap water consumed by mice in the positive control group versus the vehicle group was statistically different (P < 0.01), indicating that the positive control group compounds had a significant effect on the taste of mice, whereas the ratios of quinine/tap water consumed by mice in the examples 3, 8 and 21 were not statistically significant, so that the compounds in the above examples had little effect on the taste of mice at 10mg/kg intravenous administration, and were statistically significantly different from the positive control group.
The above embodiment is only one of the preferred embodiments of the present invention, and should not be used to limit the scope of the present invention, but all the insubstantial modifications or color changes made in the main design concept and spirit of the present invention are still consistent with the present invention, and all the technical problems to be solved are included in the scope of the present invention.
Claims (9)
1. A compound with a structure shown as a formula I or a salt thereof,
wherein R is 1 Selected from hydrogen, alkyl;
R 2 、R 3 independently selected from hydrogen, deuterium, C 1-12 Alkyl, or R 2 、R 3 A cyclopropyl group is formed by connection;
R 4 selected from the group consisting of substituted or unsubstituted pyridyl, substituted or unsubstituted pyrimidinyl, substituted or unsubstituted benzothiazolyl, substituted or unsubstituted imidazopyridazinyl; wherein R is 4 The substituent of (2) is selected from 1-5 deuterium, amino, cyano, methyl, methoxy, halogen, trifluoromethoxy or difluoromethoxy;
R 5 selected from 1 to 5 hydrogens, deuterium, alkyl groups, or halogens.
2. A compound or salt thereof according to claim 1, wherein R 2 、R 3 Independently selected from hydrogen, deuterium, methyl, ethyl, propyl;
or R is 2 、R 3 A cyclopropyl group is formed by connection;
or/and R 4 Selected from the group consisting of substituted or unsubstituted pyridyl, substituted or unsubstituted pyrimidinyl, substituted or unsubstituted benzothiazolyl, or substituted or unsubstituted imidazopyridazinyl, wherein R 4 Is selected from cyano, methyl, methoxy, halogen, trifluoromethoxy or difluoromethoxy;
or/and R 5 Selected from 1 to 5 hydrogen, deuterium, methyl, or halogen.
3. A compound or salt thereof according to claim 2, wherein R 2 、R 3 Independently selected from hydrogen, deuterium, methyl, ethyl or propyl;
or R is 2 、R 3 The connection forms cyclopropyl.
4. A compound or salt thereof according to claim 1, selected from the following compounds or pharmaceutically acceptable salts thereof:
5. the compound or salt thereof according to any one of claims 1 to 4, wherein the hydrogen in the compound of formula I is replaced by one or more deuterium.
6. A process for the preparation of a compound or salt thereof according to any one of claims 1 to 5, comprising the steps of:
step 1: the compound a and the compound k undergo substitution reaction under the catalysis of alkali to generate a compound b;
step 2: the compounds c and d undergo substitution reaction under alkaline conditions to obtain e;
step 3: carrying out a photo-delay reaction on the compounds e and f to obtain an intermediate g;
step 4: the coupling reaction of the compounds g and b is carried out in the presence of a catalyst to obtain a compound h;
step 5: the compound h undergoes hydrolysis reaction under inorganic base catalysis or acid catalysis to obtain a compound i;
step 6: the compound I and the compound j undergo condensation or esterification reaction to obtain a compound of the formula I;
wherein R is 1 、R 2 、R 3 、R 4 、R 5 Is as defined in claim 1; x is fluorine.
7. Use of a compound according to any one of claims 1 to 5 or a salt thereof for the manufacture of a medicament for the treatment or prophylaxis of P2X3 and/or P2X2/3 receptor-related disorders.
8. The use according to claim 7, wherein the disease is a respiratory system disease.
9. The use according to claim 7 or 8, wherein the compound or salt thereof is for the manufacture of a medicament for the treatment or prophylaxis of cough, asthma, pain, sleep apnea.
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