Patent Application
20240391904
This application claims priority to a Chinese patent application No. 202111137108.1 filed to the Chinese Patent Office on Sep. 27, 2021 under the title Dihydropyrimidine based Compound, Preparation Method and Use Thereof, the entire contents of which are incorporated herein by reference.
The present disclosure pertains to the technical field of pharmaceutical chemistry, and particularly to a dihydropyrimidine-based compound and the preparation method and use of the same.
It is estimated that 5-10% of adults worldwide suffer from chronic cough. Some of these patients who have refractory chronic cough (RCC) and unexplained chronic cough (UCC) are more sensitive to a variety of triggers that do not normally cause coughing in healthy subjects, including daily activities (e.g., talking and laughing), changes in temperature, exposure to aerosols, or food volatile. To date, treatment options for these patients have been extremely limited, and many have often gone years without relief. There are no approved medications for chronic cough. Commonly used clinical medications such as codeine and dextromethorphan, which are central cough suppressants, frequently lead to adverse effects such as constipation and drowsiness. Statistics shows that chronic cough with a duration of >8 weeks accounts for more than ⅓ of respiratory outpatient cases, with a prevalence of 2-10%.
P2X3 receptors, a member of the purine receptor family, are non-selective ligand-gated ion channels that play an important role in the generation and transmission of injurious information. Recent studies have found that overactivation of P2X3 receptors is associated with hyper-sensitization of sensory neurons. Neuronal hypersensitivity in the airways and lungs induced by injury or infection can lead to excessive, persistent and frequent coughing. P2X3 receptors have been implicated in a variety of disorders, including chronic cough, neuralgia, and sleep apnea, all of which have high prevalence rates and large unmet clinical needs. A New Drug Application (NDA) has been filed with the FDA for Merck & Co's new cough suppressant gefapixant (MK-7264), an oral, selective P2X3 receptor antagonist for the treatment of adult patients with refractory chronic cough (RCC) or unexplained chronic cough (UCC). Two phase III clinical trials have demonstrated a statistically significant reduction in 24-hour cough frequency (the number of coughs per hour measured objectively using 24-hour recording) at Week 12 (COUGH-1 study) and Week 24 (COUGH-2 study) in the treatment group with a 45 mg dose of gefapixant twice a day compared to the placebo group. In these two studies, the primary efficacy endpoint was not met in the treatment group with a 15 mg dose of gefapixant twice a day. The clinical endpoint was met in the 45 mg group, but there was a higher frequency of discontinuation due to adverse events and a higher incidence of taste-related adverse events with 45 mg. Therefore, there is a clinical need for a safer and more effective P2X3 receptor antagonist.
In view of the above defects or shortages in the prior art, one object of the present disclosure is to provide a dihydropyrimidine-based compound, which has improved P2X3 receptor antagonism and improved safety.
It is another object of the present disclosure to provide a method for preparing the compound.
It is yet another object of the present disclosure to provide the use of the compound.
In order to achieve the above objects, the technical solutions employed in the present disclosure are as follows.
The present disclosure provides a compound having a structure represented by Formula I, or a salt, a solvate, an isomer, a metabolite, a nitrogen oxide, or a prodrug thereof:
In some embodiments of the present disclosure, R4 is selected from substituted or unsubstituted phenyl, substituted or unsubstituted pyridyl, substituted or unsubstituted pyrimidinyl, substituted or unsubstituted pyrazinyl, substituted or unsubstituted pyridazinyl, substituted or unsubstituted benzothiazolyl, substituted or unsubstituted benzisoxazolyl, substituted or unsubstituted imidazopyridazinyl.
In some embodiments of the present disclosure, R1 is selected from hydroxyl, substituted or unsubstituted alkoxy, substituted or unsubstituted sulfonamide group; and/or R4 is selected from a substituted or unsubstituted phenyl, a substituted or unsubstituted pyridyl, a substituted or unsubstituted pyrimidinyl, a substituted or unsubstituted pyrazinyl, a substituted or unsubstituted pyridazinyl, a substituted or unsubstituted benzothiazolyl, a substituted or unsubstituted benzisoxazolyl, and a substituted or unsubstituted imidazopyridazinyl, wherein the substituent of R4 is selected from 1 to 5 of hydrogen, deuterium, amino, cyano, halogen, a substituted or unsubstituted alkyl, and a substituted or unsubstituted alkoxy;
In some embodiments of the present disclosure, R2 and R3 are independently selected from hydrogen, deuterium, substituted or unsubstituted methyl, substituted or unsubstituted ethyl, substituted or unsubstituted propyl, and substituted or unsubstituted cyclopropyl;
In some embodiments of the present disclosure,
In some embodiments of the present disclosure, R1 is selected from hydroxyl, a C1-C16 alkoxy, a C1-C16 alkylsulfonamide group, a C1-C6 alkylaminosulfonamide group, and a C1-C6 cycloalkylaminosulfonamide group.
In some embodiments of the present disclosure, R4 is selected from 4-phenyl, 2-pyridinyl, 2-pyrimidinyl, 2-pyrazinyl, 3-pyridazinyl, 3-fluoropyridin-2-yl, (5-trifluoromethoxy)pyridin-2-yl, (5-difluoromethoxy)pyridin-2-yl, 5-chloro-pyridin-2-yl, 3-pyridinyl, 4-pyridinyl, 6-fluoro-pyridin-2-yl, 2-cyanopyrimidin-5-yl, 3-methylpyrazin-2-yl, 2-chloro-pyridin-4-yl, 6-methoxypyrimidin-3-yl, 5-chloropyridin-3-yl, 2-methoxypyrimidin-4-yl, 2-cyanopyridin-4-yl, 3-chloropyrazin-2-yl, 6-chloropyrimidin-4-yl, 5-methylpyridin-2-yl, 3-chloropyridin-2-yl, 5-methoxypyridin-2-yl, 5-chloropyridin-2-yl, benzo[D]thiazol-2-yl, imidazo[1,2-B]pyridazin-6-yl, benzo[D]isoxazol-3-yl, 3,6-difluoropyridin-2-yl, and 3-chloro-6-fluoropyridin-2-yl.
In some embodiments of the present disclosure, the compound is selected from the compounds shown in Table 2 below or a pharmaceutically acceptable salt thereof.
In some embodiments of the present disclosure, the hydrogen in the above compounds may be substituted by one or more deuterium.
The method for preparing the compound or a salt, a solvate, an isomer, a metabolite, a nitrogen oxide, or a prodrug thereof provided by the present disclosure comprises the following steps:
Step I: subjecting compound a and compound k under an alkaline condition to a substitution reaction to produce compound b;
Step II: subjecting intermediate compounds c and d in the presence of an organic alkaline or an inorganic alkaline to a substitution reaction to produce e;
Step III: subjecting intermediate compounds e and f to a Mitsunobu reaction to produce an intermediate g;
Step IV: subjecting intermediate compounds g and b to a coupling reaction to produce an intermediate h;
Provided is the use of the compound or a salt, a solvate, an isomer, a metabolite, a nitrogen oxide, or a prodrug thereof according to the present disclosure in the manufacture of a medicament for treating or preventing a disease associated with a P2X3 and/or P2X2/3 receptor, preferably for treating or preventing a respiratory disease, and more preferably for treating or preventing cough, asthma, pain, or sleep apnea.
The present disclosure has the following beneficial effects over the prior art.
The dihydropyrimidine-based compound of the present disclosure has good P2X3 receptor antagonism and improved safety. Tests have shown that the cough suppressant effect of the present disclosure is stronger than that of the positive drugs and has a longer duration of action. The inhibitory effect on the P2X3 receptor is good and, and it has almost no impact on the sense of taste in mice.
The dihydropyrimidine-based compound of the present disclosure is useful for treating or preventing P2X3 and/or/P2X2/3 receptor-related diseases with improved efficacy and safety.
Hereinafter the present disclosure will be described in further details in connection with the examples and test examples. The examples and test examples of the present disclosure are only used to illustrate the technical solutions of the present disclosure, and not to limit the present disclosure, and any equivalent substitutions in the related field in accordance with the disclosure of the present disclosure are within the protection scope of the present disclosure.
Compound Structures were Determined by 1H NMR or LC-MS.
The LC-MS instrument is an Agilent G6120B (in connection with an Agilent 1260); the 1H NMR instrument is a Bruker AVANCE-400 or a Bruker AVANCE-800, and the 1H NMR shift (6) is given in parts per million (ppm), with DMSO as the test solvent, and tetramethylsilane (TMS) as the internal standard. Chemical shifts are given in a unit of 10−6 (ppm).
The term “room temperature” in the present disclosure refers to a temperature of 10-25° C.
2-Fluoropyrazine (50.0 g, 0.52 mol) and p-aminophenol (53.5 g, 0.49 mol) were dissolved in dimethyl sulfoxide (360 ml), into which cesium carbonate (320 g, 0.98 mol) was added to obtain a reaction mixture. The reaction mixture was stirred at a constant speed under mechanical stirring. Then, the temperature of the reaction system was raised to 80° C. for a reaction for 2-3 h. The reaction progress was monitored by TLC (thin-layer chromatography), and after the reaction was complete, the reaction mixture was added into three times volume of water (about 1 L) while stirring was maintained. Thereafter, the resultant was extracted three times with ethyl acetate, and a crude product was combined after it has been concentrated after drying the ethyl acetate. The crude product was prepared into a slurry with 500 ml water for 1 h, filtrated, and dried in an air-drying oven to obtain 4-(pyrazin-2-oxyl)aniline (92.8 g, brown granular solid), yield: 96.8%.
ESI-MS: m/z=187.1 (M+H)+.
6-Chlorouracil (36.8 g, 0.25 mol) and 4-chlorobenzyl bromide (52.5 g, 0.256 mol) were mixed and dissolved in 300 ml DMF, and DIPEA (96.9 g, 0.75 mol) was added dropwise thereto to conduct a reaction for 3-5 h while maintained at 30° C. The reaction progress was monitored by TLC, and after the reaction was complete, the reaction mixture was added into three times volume of water. The precipitated solid was filtered after wash and dried, and the filter cake was prepared into a slurry with 300 ml ethyl acetate. The resulting solid upon filtration was dried in an air-drying oven to obtain 6-chloro-1-(4-chlorobenzyl)pyrimidine-2,4 (1H,3H)-dione (Compound e-1) (51.9 g, white solid), yield: 76.6%, purity: 99.26%.
ESI-MS: m/z=271.0 (M+H)+.
Compound e-1 (27.1 g, 0.1 mol), methyl (S)-(+)-3-hydroxy-2-methylpropionate (11.8 g, 0.1 mol) and triphenylphosphine (52.4 g, 0.2 mol) was dissolved in 300 ml anhydrous tetrahydrofuran until it was clear, and after replacing the air in the reaction system with argon, the reaction system was cooled in an iced water bath. Diisopropyl azodicarboxylate (40.4 g, 0.2 mol) was slowly added dropwise at a constant speed while stirring was maintained, and after the addition was completed in 30 min, the reaction was conducted at rt. 2-3 hours later the reaction progress was monitored by TLC, and after the reaction was complete, the reaction solution was quenched in 500 ml water, extracted three times with 30 ml ethyl acetate, and an oily crude product was obtained after drying and concentrating the organic solution. The oily crude product was dispersed in a mixed solvent of 100 ml ethyl acetate and 500 ml petroleum ether, and a large amount of triphenylphosphine was precipitated. The triphenylphosphine was removed upon filtration, and the mother liquid was concentration and purified by chromatography to obtain methyl (S)-3-(4-chloro-3-(4-chlorobenzyl)-2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl)-2-methylpropionate (32.1 g, white solid), yield: 86.6%, purity: 98.71%.
ESI-MS: m/z=371.1 (M+H)+.
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) and potassium phosphate (4.24 g, 0.02 mol) were mixed and then dissolved in 30 ml dioxane, and the air in the reaction flask was replaced with argon. The reaction was conducted under argon protection, and the reaction mixture was heated to 80° C. in an oil bath for a reaction for 1-2 h. The reaction was monitored by TLC until Compound g-1 was completely depleted, and the reaction mixture was evaporated under reduced pressure to remove dioxane and then extracted three times with 100 ml ethyl acetate and 100 ml water. The ethyl acetate phase was concentrated and purified by column chromatography to obtain methyl (S)-3-(3-(4-chlorobenzyl)-2,6-dioxo-4-(4-(pyrazin-2-oxyl)phenyl)amino)-3,6-dihydropyrimidin-1(2H)-yl)-2-methylpropionate (Compound 1) 4.56 g (tawny foamy solid), yield: 87.3%, purity: 96.82%.
ESI-MS: m/z=522.2 (M+H)+.
1H NMR (400 MHz, DMSO-d6) δ 8.65 (s, 1H), δ 8.17 (s, 1H), 7.55-7.48 (m, 1H), 7.46-7.37 (m, 1H), 7.35-7.26 (m, 2H), 7.16 (m, 4H), 7.14-7.11 (m, 1H), 7.04 (d, J=8.3 Hz, 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).
Methyl (S)-3-(3-(4-chlorobenzyl)-2,6-dioxo-4-(4-(pyrazin-2-oxyl)phenyl)-amino)-3,6-dihydropyrimidin-1(2H)-yl)-2-methylpropionate (522 mg, 1.0 mmol) prepared according to the method in Example 1 was dissolved in a mixed solvent of methanol (3 ml) and tetrahydrofuran (3 ml), and while the temperature was maintained at around 10° C., an aqueous solution (3 ml) of lithium hydroxide (168 mg, 4 mmol) was added thereto to obtain a reaction mixture. The reaction mixture was reacted at RT overnight. The reaction progress was monitored by TLC, and after the reaction was complete, the reaction mixture was purified by column chromatography to obtain S-3-(3-(4-chlorobenzyl)-2,6-dioxo-4-(4-(pyrazin-2-oxyl)phenyl)amino)-3,6-dihydropyrimidin-1(2H)-yl)-2-methylpropionic acid (388 mg, off-white solid), yield: 76.5%, purity: 98.62%.
ESI-MS: m/z=508.1 (M+H)+.
1H NMR (400 MHz, DMSO-d6) δ 12.11 (s, 1H), δ 8.85 (s, 1H), δ 8.18 (s, 1H), 7.56-7.49 (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=8.3 Hz, 1H), 5.30 (s, 2H), 4.62 (s, 1H), 4.06-3.79 (m, 2H), 2.75 (m, 1H), 0.98 (m, 3H).
S-3-(3-(4-chlorobenzyl)-2,6-dioxo-4-(4-(pyrazin-2-oxyl)phenyl)amino)-3,6-dihydropyrimidin-1(2H)-yl)-2-methylpropionic acid (Compound 2) (288 mg, 0.57 mmol) prepared according to the method in Example 2, DIPEA (183 mg, 1.42 mmol) and EDCI (130.8 mg, 0.68 mmol) were dissolved in dichloromethane (5 ml) and stirred for 15 min, and methanesulfonamide group (64.7 mg, 0.68 mmol) and DMAP (83 mg, 0.68 mmol) were then added and a reaction was conducted at room temperature for 2-3 h. The reaction progress was monitored by TLC, and after the reaction was complete, the reaction mixture was washed with water, extracted three times with dichloromethane, and combined, dried, concentrated and purified by column chromatography to obtain (S)-3-(3-(4-chlorobenzyl)-2,6-dioxo-4-(4-(pyrazin-2-oxyl)phenyl)amino)-3,6-dihydropyrimidin-1(2H)-yl)-2-methyl-N-(methylsulfonyl)propionamide group (198 mg) yield: 59.6%, purity: 99.87%.
ESI-MS: m/z=585.1 (M+H)+.
1H NMR (400 MHz, DMSO-d6) δ 11.31 (s, 1H), δ 8.65 (s, 1H), δ 8.19 (s, 1H), 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=8.3 Hz, 1H), 5.42-5.15 (s, 2H), 4.62 (s, 1H), 3.88 (m, 2H), 3.01 (s, 3H), 2.69 (m, 1H), 0.99 (m, 3H).
The preparation method of this example was conducted under the same conditions as those in Example 1, with the exception that compound f-1: methyl (S)-3-hydroxy-2-methylpropionate in Step III was replaced with an equimolar of methyl 3-hydroxy-2,2-dimethylpropionate, to obtain a white solid-like methyl 3-(3-(4-chlorobenzyl)-2,6-dioxo-4-(4-(pyrazin-2-oxyl)phenyl)amino)-3,6-dihydropyrimidin-1(2H)-yl)-2,2-dimethylpropionate (Compound 4), yield: 76.5%, purity: 98.78%.
ESI-MS: m/z=536.2 (M+H)+.
1H NMR (400 MHz, DMSO-d6) δ 8.65 (s, 1H), 8.18 (s, 1H), 7.84 (d, J=8.1 Hz, 1H), 7.42 (d, J=8.3 Hz, 1H), 7.29 (d, J=8.4 Hz, 2H), 7.16 (m, 4H), 7.11 (d, J=6.6 Hz, 1H), 7.03 (d, J=8.4 Hz, 1H), 5.26 (s, 2H), 4.59 (s, 1H), 3.93 (s, 2H), 3.44 (s, 3H), 1.06 (s, 6H).
The preparation method of this example was conducted under the same conditions as those in Example 2, with the exception that compound f-1: methyl (S)-3-hydroxy-2-methylpropionate in Step III was replaced with an equimolar of methyl 3-hydroxy-2,2-dimethylpropionate, to obtain a white solid-like 3-(3-(4-chlorobenzyl)-2,6-dioxo-4-(4-(pyrazin-2-oxyl)phenyl)amino)-3,6-dihydropyrimidin-1(2H)-yl)-2,2-dimethylpropionic acid (Compound 5), yield: 82.2%, purity: 98.51%.
ESI-MS: m/z=522.2 (M+H)+.
1H NMR (400 MHz, DMSO-d6) δ 12.07 (s, 1H), 8.58 (s, 1H), 8.20 (s, 1H), 7.89-7.79 (m, 1H), 7.46-7.37 (m, 1H), 7.28 (d, J=8.2 Hz, 2H), 7.13 (m, 4H), 7.15-7.10 (m, 1H), 7.02 (d, J=8.3 Hz, 1H), 5.28 (s, 2H), 4.60 (s, 1H), 3.99 (s, 2H), 1.03 (s, 6H).
The preparation method of this example was conducted under the same conditions as those in Example 3, with the exception that compound f-1: methyl (S)-3-hydroxy-2-methylpropionate in Step III was replaced with an equimolar of methyl 3-hydroxy-2,2-dimethylpropionate, to obtain a white solid-like 3-(3-(4-chlorobenzyl)-2,6-dioxo-4-(4-(pyrazin-2-oxyl)phenyl)amino)-3,6-dihydropyrimidin-1(2H)-yl)-2,2-dimethyl-N-(methylsulfonyl)propionamide group (Compound 6), yield: 78.6%, purity: 97.68%.
ESI-MS: m/z=599.1 (M+H)+.
1H NMR (400 MHz, DMSO-d6) δ 11.30 (s, 1H), 8.64 (s, 1H), 8.18 (s, 1H), 7.84 (d, J=8.1 Hz, 1H), 7.42 (d, J=8.3 Hz, 1H), 7.29 (d, J=8.4 Hz, 2H), 7.16 (m, 4H), 7.11 (d, J=6.6 Hz, 1H), 7.03 (d, J=8.4 Hz, 1H), 5.25 (s, 2H), 4.60 (d, J=1.6 Hz, 1H), 4.03 (s, 2H), 3.02 (s, 3H), 1.05 (s, 6H).
The preparation method of this example was conducted under the same conditions as those in Example 2, with the exception that 2-fluoropyrazine in Step I was replaced with an equimolar of 2-fluoropyridine, to obtain a white solid-like (S)-3-(3-(4-chlorobenzyl)-2,6-dioxo-4-(4-(pyridin-2-oxyl)phenyl)amino)-3,6-dihydropyrimidin-1(2H)-yl)-2-methylpropionic acid (Compound 7), yield: 78.2%, purity: 98.68%.
ESI-MS: m/z=507.1 (M+H)+.
1H NMR (400 MHz, DMSO-d6) δ 8.16 (dd, J=5.1, 2.0 Hz, 1H), 7.94-7.80 (m, 1H), 7.48-7.39 (m, 2H), 7.35-7.26 (m, 2H), 7.16 (m, 4H), 7.14-7.11 (m, 1H), 7.04 (d, J=8.3 Hz, 1H), 5.30 (s, 2H), 4.62 (s, 1H), 4.06-3.79 (m, 2H), 2.75 (m, 1H), 0.98 (m, 3H).
The preparation method of this example was conducted under the same conditions as those in Example 1, with the exception that 2-fluoropyrazine in Step I was replaced with an equimolar of 2-fluoropyridine, to obtain a white solid-like methyl 3-(3-(4-chlorobenzyl)-2,6-dioxo-4-(4-(pyridin-2-oxyl)phenyl)amino)-3,6-dihydropyrimidin-1(2H)-yl)-2-methylpropionate (Compound 8), yield: 72.8%, purity: 98.56%.
ESI-MS: m/z=521.2 (M+H)+.
1H NMR (400 MHz, DMSO-d6) δ 8.63 (s, 1H), 8.20-8.11 (m, 1H), 7.90-7.79 (m, 1H), 7.48-7.38 (m, 2H), 7.29 (d, J=8.3 Hz, 2H), 7.21-7.14 (m, 4H), 7.14-7.10 (m, 1H), 7.03 (d, J=8.3 Hz, 1H), 5.28 (s, 2H), 4.61 (s, 1H), 3.89 (m, 2H), 3.47 (s, 3H), 2.77 (m, 1H), 1.00 (m, 3H).
The preparation method of this example was conducted under the same conditions as those in Example 3, with the exception that 2-fluoropyrazine in Step I was replaced with an equimolar of 2-fluoropyridine, to obtain a white solid-like (S)-3-(3-(4-chlorobenzyl)-2,6-dioxo-4-(4-(pyridin-2-oxyl)phenyl)amino)-3,6-dihydropyrimidin-1(2H)-yl)-2-methyl-N-(methylsulfonyl)acrylamide group (Compound 9), yield: 85.2%, purity: 99.21%.
ESI-MS: m/z=584.2 (M+H)+.
1H NMR (400 MHz, DMSO-d6) δ 11.69 (s, 1H), 8.60 (s, 1H), 8.15 (dd, J=5.0, 2.0 Hz, 1H), 7.90-7.79 (m, 1H), 7.45-7.37 (m, 2H), 7.30 (d, J=8.4 Hz, 2H), 7.15 (m, 4H), 7.14-7.09 (m, 1H), 7.03 (d, J=8.3 Hz, 1H), 5.42-5.15 (m, 2H), 4.62 (s, 1H), 3.88 (m, 2H), 3.01 (s, 3H), 2.69 (m, 1H), 0.98 (m, 3H).
The preparation method of this example was conducted under the same conditions as those in Example 2, with the exception that 2-fluoropyrazine in Step I was replaced with an equimolar of 2-fluoropyridine and that methyl (S)-3-hydroxy-2-methylpropionate in Step III was replaced with an equimolar of methyl 3-hydroxy-2,2-dimethylpropionate, to obtain a white solid-like 3-(3-(4-chlorobenzyl)-2,6-dioxo-4-(4-(pyridin-2-oxyl)phenyl)amino)-3,6-dihydropyrimidin-1(2H)-yl)-2,2-dimethylpropionic acid (Compound 10), yield: 79.6%, purity: 98.26%.
ESI-MS: m/z=521.2 (M+H)+.
1H NMR (400 MHz, DMSO-d6) δ 12.08 (s, 1H), 8.59 (s, 1H), 8.14 (dd, J=4.8, 2.1 Hz, 1H), 7.89-7.79 (m, 1H), 7.46-7.37 (m, 2H), 7.29 (d, J=8.2 Hz, 2H), 7.15 (m, 4H), 7.14-7.10 (m, 1H), 7.03 (d, J=8.3 Hz, 1H), 5.27 (s, 2H), 4.60 (s, 1H), 3.98 (s, 2H), 1.02 (s, 6H).
The preparation method of this example was conducted under the same conditions as those in Example 1, with the exception that 2-fluoropyrazine in Step I was replaced with an equimolar of 2-fluoropyridine and that methyl (S)-3-hydroxy-2-methylpropionate in Step III was replaced with an equimolar of methyl 3-hydroxy-2,2-dimethylpropionate, to obtain a white solid-like methyl 3-(3-(4-chlorobenzyl)-2,6-dioxo-4-(4-(pyridin-2-oxyl)phenyl)amino)-3,6-dihydropyrimidin-1(2H)-yl)-2,2-dimethylpropionate (Compound 11), yield: 75.2%, purity: 97.58%.
ESI-MS: m/z=535.2 (M+H)+.
1H NMR (400 MHz, DMSO-d6) δ 8.62 (s, 1H), 8.15 (d, J=4.7 Hz, 1H), 7.84 (t, J=8.1 Hz, 1H), 7.42 (d, J=8.3 Hz, 2H), 7.29 (d, J=8.4 Hz, 2H), 7.16 (m, 4H), 7.11 (d, J=6.6 Hz, 1H), 7.03 (d, J=8.4 Hz, 1H), 5.26 (s, 2H), 4.59 (s, 1H), 3.93 (s, 2H), 3.44 (s, 3H), 1.06 (s, 6H).
The preparation method of this example was conducted under the same conditions as those in Example 3, with the exception that 2-fluoropyrazine in Step I was replaced with an equimolar of 2-fluoropyridine and that methyl (S)-3-hydroxy-2-methylpropionate in Step III was replaced with an equimolar of methyl 3-hydroxy-2,2-dimethylpropionate, to obtain a white solid-like 3-(3-(4-chlorobenzyl)-2,6-dioxo-4-(4-(pyridin-2-oxyl)phenyl)amino)-3,6-dihydropyrimidin-1(2H)-yl)-2,2-dimethyl-N-(methylsulfonyl)propionamide group (Compound 12), yield: 77.8%, purity: 98.98%.
ESI-MS: m/z=598.1 (M+H)+.
1H NMR (400 MHz, DMSO-d6) δ 11.30 (s, 1H), 8.64 (s, 1H), 8.20-8.09 (m, 1H), 7.84 (m, 1H), 7.42 (d, J=8.6 Hz, 2H), 7.31-7.25 (m, 2H), 7.20-7.13 (m, 4H), 7.13-7.09 (m, 1H), 7.03 (d, J=8.3 Hz, 1H), 5.25 (s, 2H), 4.60 (s, 1H), 4.03 (s, 2H), 3.02 (s, 3H), 1.05 (s, 6H).
The preparation method of this example was conducted under the same conditions as those in Example 2, with the exception that 2-fluoropyrazine in Step I was replaced with an equimolar of 2-fluoropyridine and that methyl (S)-3-hydroxy-2-methylpropionate in Step III was replaced with an equimolar of methyl 1-(hydroxymethyl)cyclopropane-1-carboxylate, to obtain a white solid-like 1-(3-(4-chlorobenzyl)-2,6-dioxo-4-(4-(pyridin-2-oxyl)phenyl)amino)-3,6-dihydropyrimidinyl-1(2H)-methyl)-cyclopropane-1-carboxylic acid (Compound 13), yield: 76.6%, purity: 98.53%.
ESI-MS: m/z=519.2 (M+H)+.
1H NMR (400 MHz, DMSO-d6) δ 8.14 (dd, J=4.9, 2.1 Hz, 1H), 7.83 (m, 1H), 7.45-7.37 (m, 2H), 7.29 (d, J=8.3 Hz, 2H), 7.11 (m, 5H), 7.01 (d, J=8.3 Hz, 1H), 5.26 (s, 2H), 4.54 (s, 1H), 4.22 (s, 2H), 0.91 (m, 2H), 0.61 (m, 4H).
The preparation method of this example was conducted under the same conditions as those in Example 1, with the exception that 2-fluoropyrazine in Step I was replaced with an equimolar of 2-fluoropyridine and that methyl (S)-3-hydroxy-2-methylpropionate in Step III was replaced with an equimolar of methyl 1-(hydroxymethyl)cyclopropane-1-carboxylate, to obtain a white solid-like 1-(3-(4-chlorobenzyl)-2,6-dioxo-4-(4-(pyridin-2-oxyl)phenyl)amino)-3,6-dihydropyrimidinyl-1(2H)-methyl)-cyclopropane-1-carboxylate (Compound 14), yield: 79.2%, purity: 98.26%.
ESI-MS: m/z=533.2 (M+H)+.
1H NMR (400 MHz, DMSO-d6) δ 8.68 (s, 1H), 8.13 (d, J=4.7 Hz, 1H), 7.83 (t, J=8.1 Hz, 1H), 7.45-7.38 (m, 2H), 7.29 (d, J=8.4 Hz, 2H), 7.16 (m, 4H), 7.12 (d, J=6.6 Hz, 1H), 7.03 (d, J=8.4 Hz, 1H), 5.26 (s, 2H), 4.54 (s, 1H), 4.03 (s, 2H), 3.42 (s, 3H), 1.07-0.92 (m, 4H).
The preparation method of this example was conducted under the same conditions as those in Example 3, with the exception that 2-fluoropyrazine in Step I was replaced with an equimolar of 2-fluoropyridine and that methyl (S)-3-hydroxy-2-methylpropionate in Step III was replaced with an equimolar of methyl 1-(hydroxymethyl)cyclopropane-1-carboxylate, to obtain a white solid-like 1-(3-(4-chlorobenzyl)-2,6-dioxo-4-(4-(pyridin-2-oxyl)phenyl)amino)-3,6-dihydropyrimidin-1(2H)yl)methyl)-N-(methylsulfonyl)cyclopropane-1-carboxamide group (Compound 15), yield: 75.8%, purity: 98.31%.
ESI-MS: m/z=596.1 (M+H)+.
1H NMR (400 MHz, DMSO-d6) δ 11.30 (s, 1H), 8.64 (s, 1H), 8.15 (dd, J=5.2, 2.0 Hz, 1H), 7.85 (m, 1H), 7.46-7.38 (m, 2H), 7.31 (d, J=8.4 Hz, 2H), 7.23-7.15 (m, 4H), 7.15-7.09 (m, 1H), 7.04 (d, J=8.3 Hz, 1H), 5.28 (s, 2H), 4.65 (s, 1H), 4.13 (s, 2H), 3.12 (s, 3H), 1.07-0.91 (m, 4H).
The preparation method of this example was conducted under the same conditions as those in Example 3, with the exception that 2-fluoropyrazine in Step I was replaced with an equimolar of 2,2-difluoropyridine, to obtain a white solid-like (S)-3-(3-(4-chlorobenzyl)-4-(4-(3-(3-fluoropyridin-2-yl)oxyphenyl)amino)-2,6-dioxo-3,6-dihydropyrimidin-1(2H)yl)-2-methyl-N-(methylsulfonyl)propionamide group (Compound 16), yield: 78.5%, purity: 98.89%.
ESI-MS: m/z=602.1 (M+H)+.
1H NMR (400 MHz, DMSO-d6) δ 11.30 (s, 1H), 8.65 (s, 1H), 8.03 (q, J=8.2 Hz, 1H), 7.44 (d, J=8.2 Hz, 2H), 7.32 (d, J=8.2 Hz, 2H), 7.22 (m, 4H), 6.95 (dd, J=8.0, 1.7 Hz, 1H), 6.90 (dd, J=7.9, 2.5 Hz, 1H), 5.31 (s, 2H), 4.66 (s, 1H), 4.08-3.80 (m, 2H), 3.01 (s, 3H), 2.73 (m, 1H), 0.99 (m, 3H).
The preparation method of this example was conducted under the same conditions as those in Example 3, with the exception that 2-fluoropyrazine in Step I was replaced with an equimolar of 2,3-difluoropyridine and that 1-(bromomethyl)-4-chlorobenzene in Step II was replaced with an equimolar of 1-(bromomethyl)-4-fluorobenzene, to obtain a white solid-like (S)-3-(3-(4-fluorobenzyl)-4-(4-(3-fluoropyridin-2-yl)phenyl)amino)-2,6-dioxo-3,6-dihydropyrimidin-1(2H)yl)-2-methyl-N-(methylsulfonyl)propionamide group (Compound 17), yield: 78.9%, purity: 99.12%.
ESI-MS: m/z=586.2 (M+H)+.
1H NMR (400 MHz, DMSO-d6) δ 11.30 (s, 1H), 8.65 (s, 1H), 8.03 (q, J=8.2 Hz, 1H), 7.44 (d, J=8.2 Hz, 2H), 7.39 (m, 2H), 7.32 (d, J=8.2 Hz, 2H), 7.08 (m, 2H), 6.95 (dd, J=8.0, 1.7 Hz, 1H), 6.90 (dd, J=7.9, 2.5 Hz, 1H), 5.31 (s, 2H), 4.66 (s, 1H), 4.08-3.80 (m, 2H), 3.01 (s, 3H), 2.73 (m, 1H), 0.99 (m, 3H).
The preparation method of this example was conducted under the same conditions as those in Example 3, with the exception that 2-fluoropyrazine in Step I was replaced with an equimolar of 2,3-difluoropyridine and that 1-(bromomethyl)-4-chlorobenzene in Step II was replaced with an equimolar of 1-(bromomethyl)-4-trifluoromethylbenzene, to obtain a white solid-like (S)-3-(2,6-dioxo-4-(4-(pyridin-2-oxyl)phenyl)amino)-3-(4-(trifluoromethyl)benzyl)-3,6-dihydropyrimidin-1(2H)yl)-2-methyl-N-(methylsulfonyl)propionamide group (Compound 18), yield: 88.2%, purity: 98.23%.
ESI-MS: m/z=617.2 (M+H)+.
1H NMR (400 MHz, DMSO-d6) δ 11.69 (s, 1H), 8.60 (s, 1H), 8.13 (dd, J=5.0, 2.0 Hz, 1H), 7.90-7.79 (m, 1H), 7.58-7.50 (m, 2H), 7.45-7.37 (m, 2H), 7.30 (d, J=8.4 Hz, 2H), 7.18-7.10 (m, 2H), 7.14-7.09 (m, 1H), 7.03 (d, J=8.3 Hz, 1H), 5.42-5.15 (m, 2H), 4.63 (s, 1H), 3.86 (m, 2H), 3.02 (s, 3H), 2.68 (m, 1H), 0.99 (m, 3H).
The preparation method of this example was conducted under the same conditions as those in Example 3, with the exception that 2-fluoropyrazine in Step I was replaced with an equimolar of 2-fluoro-5-(trifluoromethoxy)pyridine, to obtain a white solid-like (S)-3-(3-(4-chlorobenzyl)-2,6-dioxo-4-(4-(5-((5-trifluoromethoxy)pyridin-2-yl)oxyphenyl)amino)-3,6-dihydropyrimidin-1(2H)-yl)-2-methyl-N-(sulfonyl)propionamide group (Compound 19), yield: 82.1%, purity: 98.68%.
ESI-MS: m/z=668.1 (M+H)+.
1H NMR (400 MHz, DMSO-d6) δ 11.68 (s, 1H), 8.59 (s, 1H), 8.16 (s, 1H), 7.90-7.79 (m, 1H), 7.45-7.37 (m, 2H), 7.30 (d, J=8.4 Hz, 2H), 7.15 (m, 4H), 7.14-7.09 (m, 1H), 5.42-5.15 (m, 2H), 4.62 (s, 1H), 3.86 (m, 2H), 3.01 (s, 3H), 2.68 (m, 1H), 0.98 (m, 3H).
The preparation method of this example was conducted under the same conditions as those in Example 3, with the exception that 2-fluoropyrazine in Step I was replaced with an equimolar of 2-fluoro-5-(trifluoromethoxy)pyridine, to obtain a white solid-like (S)-3-(3-(4-chlorobenzyl)-2,6-dioxo-4-(4-(5-((5-difluoromethoxy)pyridin-2-yl)oxyphenyl)amino)-3,6-dihydropyrimidin-1(2H)-yl)-2-methyl-N-(sulfonyl)propionamide group (Compound 20), yield: 79.1%, purity: 98.18%.
ESI-MS: m/z=650.1 (M+H)+.
1H NMR (400 MHz, DMSO-d6) δ 11.68 (s, 1H), 8.59 (s, 1H), 8.02 (s, 1H), 7.82-7.73 (m, 1H), 7.52 (s, 1H), 7.45-7.37 (m, 2H), 7.30 (d, J=8.4 Hz, 2H), 7.15 (m, 4H), 7.11-7.06 (m, 1H), 5.42-5.15 (m, 2H), 4.62 (s, 1H), 3.86 (m, 2H), 3.01 (s, 3H), 2.68 m, 1H), 0.98 (m, 3H).
The preparation method of this example was conducted under the same conditions as those in Example 3, with the exception that 2-fluoropyrazine in Step I was replaced with an equimolar of 2-fluoro-5-chloropyridine, to obtain a white solid-like (S)-3-(3-(4-chlorobenzyl)-4-(4-(5-chloropyridin-2-oxyl)phenyl)amino)-2,6-dioxo-3,6-dihydropyrimidin-1(2H)yl)-2-methyl-N-(methylsulfonyl)propionamide group (Compound 21), yield: 85.9%, purity: 98.56%.
ESI-MS: m/z=618.1 (M+H)+.
1H NMR (400 MHz, DMSO-d6) δ 11.66 (s, 1H), 8.58 (s, 1H), 8.18 (s, 1H), 7.90-7.79 (m, 1H), 7.45-7.37 (m, 2H), 7.30 (d, J=8.4 Hz, 2H), 7.22 (d, J=8.3 Hz, 1H), 7.13 (m, 4H), 5.42-5.15 (s, 2H), 4.62 (s, 1H), 3.88 (m, 2H), 3.01 (s, 3H), 2.69 (m, 1H), 0.98 (m, 3H).
The preparation method of this example was conducted under the same conditions as those in Example 3, with the exception that 2-fluoropyrazine in Step I was replaced with an equimolar of 2-fluoropyrimidine, to obtain a white solid-like (S)-3-(3-(4-chlorobenzyl)-2,6-dioxo-4-(4-(pyrimidin-2-oxyl)phenyl)amino)-3,6-dihydropyrimidin-1(2H)-yl)-2-methyl-N-(methylsulfonyl)propionamide group (Compound 22), yield: 81.5%, purity: 99.74%.
ESI-MS: m/z=585.1 (M+H)+.
1H NMR (400 MHz, DMSO-d6) δ 11.69 (s, 1H), 8.60 (s, 1H), 8.28 (dd, J=5.0, 2.0 Hz, 2H), 7.45-7.37 (m, 2H), 7.30 (d, J=8.4 Hz, 2H), 7.15 (m, 4H), 7.08-7.01 (m, 1H), 5.42-5.15 (m, 2H), 4.62 (s, 1H), 3.88 (m, 2H), 3.01 (s, 3H), 2.69 (m, 1H), 0.98 (m, 3H).
The preparation method of this example was conducted under the same conditions as those in Example 3, with the exception that 2-fluoropyrazine in Step I was replaced with an equimolar of 2-fluorobenzene, to obtain a white solid-like (S)-3-(3-(4-chlorobenzyl)-2,6-dioxo-4-(4-phenoxyphenyl)amino)-3,6-dihydropyrimidin-1(2H)-yl)-2-methyl-N-(methylsulfonyl)propionamide group (Compound 23), yield: 78.3%, purity: 98.11%.
ESI-MS: m/z=583.1 (M+H)+.
1H NMR (400 MHz, DMSO-d6) δ 11.69 (s, 1H), 8.60 (s, 1H), 8.03 (m, 2H), 7.61-7.53 (m, 2H), 7.30 (d, J=8.4 Hz, 2H), 7.15 (m, 4H), 7.24-7.19 (m, 1H), 7.03 (d, J=8.3 Hz, 2H), 5.42-5.15 (s, 2H), 4.62 (s, 1H), 3.88 (m, 2H), 3.03 (s, 3H), 2.69 (m, 1H), 0.98 (m, 3H).
The preparation method of this example was conducted under the same conditions as those in Example 3, with the exception that 2-fluoropyrazine in Step I was replaced with an equimolar of 2,6-difluorobenzene, to obtain a white solid-like (S)-3-(3-(4-chlorobenzyl)-4-(4-(6-fluoropyridin-2-oxyl)phenyl)amino)-2,6-dioxo-3,6-dihydropyrimidin-1(2H)yl)-2-methyl-N-(methylsulfonyl)propionamide group (Compound 24), yield: 78.2%, purity: 98.22%.
ESI-MS: m/z=602.1 (M+H)+.
1H NMR (400 MHz, DMSO-d6) δ 11.69 (s, 1H), 8.60 (s, 1H), 8.15 (dd, J=5.0, 2.0 Hz, 1H), 7.62-7.53 (m, 1H), 7.45-7.37 (m, 2H), 7.30 (d, J=8.4 Hz, 2H), 7.15 (m, 4H), 7.14-7.09 (m, 1H), 5.42-5.15 (s, 2H), 4.62 (s, 1H), 3.88 (m, 2H), 3.01 (s, 3H), 2.69 (m, 1H), 0.98 (m, 3H).
In addition, the present inventors synthesized the exemplary compounds as in Table 3 below by using the aforementioned methods.
The preparation method of this comparative example was conducted under the same conditions as those in Example 2, with the exception that 2-fluoropyrazine in Step I was replaced with an equimolar of 2,3-difluoropyridine, to obtain a white solid-like compound of Comparative Example 1, yield: 81.6%, purity: 99.21%.
ESI-MS: m/z=525.1 (M+H)+.
1H NMR (400 MHz, DMSO-d6) δ 12.09 (s, 1H), 8.65 (s, 1H), 8.03 (q, J=8.2 Hz, 1H), 7.44 (d, J=8.2 Hz, 2H), 7.32 (d, J=8.2 Hz, 2H), 7.22 (brs, 4H), 6.95 (dd, J=8.0, 1.7 Hz, 1H), 6.90 (dd, J=7.9, 2.5 Hz, 1H), 5.31 (s, 2H), 4.66 (s, 1H), 4.08-3.80 (m, 2H), 2.77 (m, 1H), 0.99 (m, 3H).
Compounds of Example 1-24 (synthesized in the inventor's laboratory), positive control drug (gefapixant, batch No: 01030-210326-2-1, purchased from Holder Pharmaceuticals), the compound of Comparative Example 1 (synthesized in the inventor's laboratory).
Physiological saline, ammonia.
Healthy adult KM mice, half of them are male and half of them are female, 6 per group, weighing about 28-30 g.
Most of the animal coughing models currently reported in the literatures use mechanical, chemical and electrical stimulations to stimulate the nerves and receptors of the animals to trigger coughing. According to the characteristics of the candidate compounds and the existing similar target compounds for reference, the method of concentrated ammonia induction was initially chosen to establish the cough modeling test in mice.
Preparation of 50% ammonia solution: 2.5 ml of ammonia was dissolved in 5 ml of a 0.9% sodium chloride solution for injection and mixed evenly.
Preparation of a solution of the compound of Comparative Example 1: 9 mg of the compound of Comparative Example 1 was weighed and dissolved in 3 ml of a 0.5% CMC-Na solution, and mixed evenly to prepare a 3 mg/ml solution.
Preparation of a solution of the example compounds: 9 mg of the example compound was weighed and dissolved in 3 ml of a 0.5% CMC-Na solution, and mixed evenly to prepare a 3 mg/ml solution.
Grouping: a positive control group, a Comparative Example 1 group, Example 1 group-Example 24 group, and a model group were set up. Six KM mice were taken from each group and administered by gavage; here, the Comparative Example 1 group was given the compound of Comparative Example 1, the positive control group was given the positive control drug (gefapixant, commercially purchased), the Example groups were given the compounds prepared in the corresponding Examples, with each group given one of three dosages: 30 mg/kg, 10 ml/kg, and 3 mg/kg; and the model group was given the same volume of 0.5% CMC-Na solution.
30 min, 60 min or 120 min after administration, the mice were placed in a 500 ml beaker with a cotton ball (weight 100±5 mg) containing 0.3 ml of 50% ammonia. The number of typical coughing of the mice (typical coughing action: contraction of the abdominal muscles or chest contraction, with mouth wide opening and a coughing sound) was observed in 3 min.
(1) Judgment criteria of coughing: coughing was manifest in contraction of abdominal muscles or chest contraction, with mouth wide opening and a coughing sound.
(2) The number of coughs (times) within 3 min was recorded with a stopwatch, and the data were statistically analyzed by software, with the data in each group shown in mean±standard deviation. A one-way analysis of variance among the groups was conducted, and P<0.05 was considered a difference having a statistical significance.
4.2.1 the Number of Coughs in Mice 30 Min after Administration of the Example Compounds at 30 mg/kg
As seen from the above table, the number of coughs in mice induced by ammonia 30 min after the administration was significantly reduced in the positive control group compared with the model group, with a statistical difference (P23.01), indicating that the modeling was successful. The number of coughs was significantly reduced in several example compound groups compared with the model group, with a statistical difference (P<0.01, P<0.05); and the cough suppressing effect of the inventive compounds of Examples 1, 3, 4, 6, 7, 9, 10, 12, 15, 19, 20 and 24 was stronger than that of the positive compound.
4.2.2 Number of Coughs in Mice 60 Min after Administration of Different Doses
ΔP < 0.05.
As shown in Table 5, with ammonia-induced coughing in mice 60 min after the administration, the number of coughs was significantly reduced in the 30 mg/kg positive control group compared with the model group, with a statistical difference (P<0.01); whereas the reduction in the number of coughs in the 10 mg/kg positive control group was not statistically significant when compared with the model group, suggesting that the 10 mg/kg positive control drug did not have a significant effect in reducing coughs, and that the 10 mg/kg positive control drug did not have any significant cough-reducing effect. However, at this dose (10 mg/kg), the number of coughs in groups having the compounds of Examples 3, 7, 9, 12, 15, 16, and 24 were statistically significantly reduced in t compared with the positive control group (° P<0.05), and also were statistically significantly reduced in compared with the model group (**P<0.01), suggesting that these example compounds had improved cough suppressing efficacy than the positive control drug; the compounds of Examples 3, 7, 9, 15, 16, and 24 even showed statistically significant (P<0.01) reduction in the number of coughs when the dosage was lowered to 3 mg/kg. In addition, in the Example 10 group, the cough suppressing effect was obvious at 30 min, but no cough suppressing effect at 60 min, indicating that the compound in the Example 10 group had a short duration of action.
4.2.3 Number of Coughs 60 and 120 Min after Administration of 30 mg/kg of Test Samples
As shown in Table 6, with administration of the test drug at 30 mg/kg, the number of coughs of mice in the Comparative Example 1 group compared with the model group was significantly reduced 60 min after the administration, which was statistically different (P<0.01); and the reduction in the number of coughs in the Comparative Example 1 group was not statistically significant compared with the model group 120 min after the administration, indicating that the compound in the Comparative Example 1 group did not have a significant cough suppressing effect at 120 min. As for the compounds of Examples 3, 9, 12, 15, 16 and 24, the number of coughs was significantly reduced both 60 min and 120 min after the administration, which were statistically different from that of the model group (P<0.01), and statistically different from that of the compound of Comparative Example 1 at 120 min (P<0.05), suggesting that the duration of the cough suppression action of the compounds of Example 3, 9, 12, 15, 16 and 24 was significantly prolonged compared with that of the compound of Comparative Example 1.
The reagents, consumables and instruments used in this test example were commercially available.
The HEK 293 cell line stably transfected with human P2X3 receptor was used.
Cell culture medium: DMEM high glucose; 10% FBS; 1% PenStrep.
Cell lines are usually passaged twice a week at a dilution of 1:3 to 1:4 (a 1:3 passaging ratio was more commonly used), and the cells after passaging needed 2-3 days before they grew to 85% confluence.
Antagonist mode
The IC50 inhibition of the Example compounds to the P2X3 receptor is shown in the table below, where A indicates less than 10 nM, B indicates 10.1-50 nM, and C indicates 50.1-100 nM.
The results show that the compounds of the present disclosure have improved inhibitory effect to the P2X3 receptor in vitro than the positive control drug and the compound of Comparative Example 1.
Compounds of Examples 1, 9, 12, 15, 16, 24 (synthesized in the laboratory of the present inventor), the positive control drug (gefapixant, batch No: 01030-210326-2-1, purchased from Holder Pharmaceuticals).
0.9% sodium chloride solution for injection, quinine hydrochloride (Quinie, batch No: C12476271)
Healthy adult SD rats, all males, weighing about 280-300 g.
Preparation of 0.3 mM quinine solution: 119.20 mg of quinine hydrochloride was weighed and dissolved in 1000 ml of tap water, and mixed evenly.
Preparation of the test sample solution: 40 mg of a test sample was dissolved in DMSO and a solution of the solubilizer HS-15 was added, mixed evenly, and 16 ml of saline was added to formulate a 2.5 mg/ml solution.
Animal and grouping: male SD rats weighed about 160-180 g each, with 10 of a similar average weight in each group, were housed in seperate cages.
Drinking habit training: each group was given normal drinking water for 30 minutes at 8:30 am and 16:30 pm every day, and no access of water for the rest of the day for 5 days, and two bottles of water were changed every day at the left and right positions.
Administration: with no access to water the night before the experiment, in the following morning the test group was given 4 mL/kg (10 mg/kg) of the test sample by tail intravenous injection, and the model group was given 4 mL/kg (10 mg/kg) of 0.5% HS-15 by intravenous injection.
Measurement of water intake: after the injection, the animals were put back to their previous cages, and the measurement time of each group was within the Tmax interval of each drug (the measurement time was 0 min-15 min after the administration), and one bottle of normal drinking water and one bottle of drinking water containing 0.3 mM quinine hydrochloride (Quinie) were both placed in each cage, with the left and right positions of the two bottles of water being the same in all the animal cages. The animals were allowed to drink freely for 15 min, and the amount of water consumed from the two bottles was measured to the nearest 0.1 ml.
(2) Statistical analysis of the data: the drinking volume of the quinine bitter water and tap water were recorded separately, as well as the percentage of quinine water to the tap water, and the significance of the difference between the groups was determined by ANOVA.
ΔP<0.01.
As seen from Table 8, the ratio of quinine/tap water consumed by mice in the positive control group was statistically different from that in the vehicle group (P<0.01), indicating that the compound gefapixant in the positive control group had a significant effect on the taste sense of mice. In contrast, in the groups of Example compounds 1, 9, 12, 15, 16 and 24, the ratio of quinine/tap water consumed by the mice was not statistically different from that in the vehicle group, indicating that the compounds of the present disclosure had little or no effect on the taste sense of the mice when administered intravenously at 10 mg/kg, which was a statistically significant difference from that of the positive control group.
The above examples are only preferred embodiments of the present disclosure, and should not be construed as limitation to the protection scope of the present disclosure. Any non-substantive alteration or modification made in within the principal idea and spirit of the present disclosure, which solve a technical problem consistent with that of the present disclosure, should be encompassed within the protection scope of the present disclosure.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202111137108.1 | Sep 2021 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2022/121194 | 9/26/2022 | WO |
