CN113666914B - 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 and a preparation method and application thereof, belonging to the technical field of pharmaceutical chemistry. The structure of the dihydropyrimidine compound provided by the invention is shown as a formula I. The invention also discloses a preparation method of the dihydropyrimidine compound. The invention provides an application of a compound shown in a formula I or a salt, a solvate, a allosteric isomer, a metabolite, a nitrogen oxide and a prodrug thereof in preparing a medicament for treating or preventing P2X3 and/or P2X2/3 receptor related diseases. The cough-relieving action time of the compound is obviously prolonged compared with that of the compound in the comparative example 1; the inhibition activity on P2X3 is better than that of the compound in the comparative example 1 and the gefapixant serving as the positive control drug, the taste of the mice is hardly influenced under the condition of intravenous administration of 10mg/kg, and the statistical difference with the positive control drug gefapixant is significant.
Description
Technical Field
The invention relates to the technical field of medicinal chemistry, in particular to dihydropyrimidine compounds or salts, solvates, allosteric isomers, 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, especially application thereof in preparing medicaments for treating and preventing respiratory diseases.
Background
It is estimated that chronic cough with a duration of >8 weeks, which is more than 1/3 at the outpatient clinic in the respiratory department, occurs at a rate of 2-10%. Patients with chronic cough are more susceptible to various causes that do not normally cause cough in healthy subjects, such as daily activities (talking and laughing), changes in air temperature, exposure to aerosols or food odors. Chronic cough not only affects the quality of life of the patient, but also creates a serious psychological burden. In the prior art, the treatment means for chronic cough is limited. The clinically commonly used drugs include: glucocorticoid, beta 2 receptor agonist, antihistaminic, antireflux, antibiotics and the like, and no approved medicine specially aiming at chronic cough exists at present.
The P2X3 receptor is a member of the purine receptor family, is a non-selective ligand-gated ion channel, and plays an important role in the generation and transmission of nociceptive information. Recent studies have found that over-activation of the P2X3 receptor is associated with hypersensitization (hyper-sensitization) of sensory neurons. Hypersensitivity of airway and pulmonary neurons caused by injury or infection can cause excessive, persistent and frequent coughing. Thus, P2X3 receptor antagonists that inhibit neuronal hypersensitivity have been used in the prior art for the treatment of chronic cough.
gefapixant (MK-7264) is an oral, selective P2X3 receptor antagonist for the treatment of chronic cough (RCC) of difficult or unknown cause (UCC) in adult patients. Gefapixant was developed by Merck & Co, Inc. of Moshadong, and has currently filed for New Drug Application (NDA) with the FDA. Two clinical phase III trials of this drug showed a statistically significant reduction in 24 hour COUGH frequency (measured objectively with 24 hour recordings for hourly COUGHs) at week 12 (COUGH-1 study) and week 24 (COUGH-2 study) in the 45mg dose gefapixant treatment group 2 times daily compared to the placebo group. In 2 studies, the 15mg dose gefapixant treatment group did not reach the primary efficacy endpoint 2 times daily. Although the 45mg group reached the clinical endpoint, 45mg was discontinued more frequently due to adverse events and the incidence of taste-related adverse events was higher. There is therefore a clinical need for safer, more potent P2X3 receptor antagonists.
Disclosure of Invention
The invention aims to provide a dihydropyrimidine compound with the structure shown in the formula I, which has good affinity to a P2X3 receptor, strong antagonism and good safety.
The second object of the present invention is to provide a process for producing the compound.
The invention also aims to provide application of the compound.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the invention provides a compound with a structure shown in formula I, or salt, solvate, allosteric isomer, metabolite, nitrogen oxide and prodrug thereof,
wherein R is1Selected from substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted C1-C12Alkylamino, substituted or unsubstituted ring C4-C8An alkylamino group;
R2、R3independently selected from hydrogen, deuterium, substituted or unsubstituted C1-12An alkyl group; or R2、R3Linked to form a substituted or unsubstituted 3-to 15-membered cycloalkyl group;
R4selected from substituted or unsubstituted heterocyclic groups, substituted or unsubstituted aryl groups;
R5selected from 1-5 hydrogen, deuterium, substituted and unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted alkylthio, substituted or unsubstituted amino or halogen.
In some embodiments of the invention, R2、R3Independently selected from hydrogen, deuterium, substituted or unsubstituted methyl, substituted or unsubstituted ethyl, substituted or unsubstituted propyl, substituted or unsubstituted cyclopropyl; wherein R is2Or R3The substituents of (a) are independently selected from one or more of deuterium, halogen, hydroxy, amino, methyl, ethyl, cyclopropyl, tert-butyl, methoxy, ethoxy, cyclopropoxy, tert-butoxy, methylthio, ethylthio, cyclopropylthio, tert-butylthio, amino, methylamino, ethylamino, cyclopropylamino, tert-butylamino, carboxamido, acetamido, cyclopropylamino, tert-butylamino, NO2、CN、CF3；
Or R2、R3Linked to form a substituted or unsubstituted cyclopropyl group, wherein the substituents of the substituted cyclopropyl group are 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, carboxamido, acetamido, cyclopropylamino, t-butylamino, NO2、CN、CF3；
Or/and R1Selected from substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted C1-C12Alkylamino, substituted or unsubstituted cyclic C4-C8An alkylamino group; wherein R is1The substituent(s) is selected from one or more of deuterium, halogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, amido, NO2、CN、CF3；
Or/and R4Selected from the group consisting of substituted or unsubstituted phenyl, substituted or unsubstituted pyridyl, substituted or unsubstituted pyrimidyl, substituted or unsubstituted pyrazinyl, substituted or unsubstituted pyridazinyl, substituted or unsubstituted pyridazylOr unsubstituted benzothiazolyl, substituted or unsubstituted benzisoxazolyl, substituted or unsubstituted imidazopyridazinyl, wherein R is4The substituent is selected from 1-5 deuterium, amino, cyano, methyl, methoxy, halogen, trifluoromethoxy or difluoromethoxy;
or/and R5Selected from 1-5 hydrogen, deuterium, amino, methyl, halogen, trifluoromethyl and difluoromethyl.
In some embodiments of the invention, R2、R3Independently selected from hydrogen, deuterium, methyl, ethyl, propyl or cyclopropyl;
or R2、R3Linked to form a cyclopropyl group;
or/and R1Selected from hydroxy, C1-C16Alkyl or C1-C12Cycloalkyl, substituted or unsubstituted C1-C6An alkyl amine group;
or/and R4Selected from the group consisting of 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, wherein R is4The substituent is selected from 1-5 of deuterium, amino, cyano, methyl, methoxy, halogen, trifluoromethoxy and difluoromethoxy;
or/and R5Selected from 1-5 hydrogen, deuterium, amino, methyl, halogen, trifluoromethyl and difluoromethyl.
In some embodiments of the invention, the compound is selected from the group consisting of:
TABLE 1
In some embodiments of the invention, the hydrogen in the compound is substituted with one or more deuterium.
The invention provides a preparation method of a compound of formula I or salt, solvate, allosteric isomer, metabolite, nitrogen oxide and prodrug thereof, which comprises the following steps:
step 1: carrying out substitution reaction on the compound a and the compound k under the catalysis of alkali to generate a compound b;
step 2: carrying out substitution reaction on the compound c and the compound d under an alkaline condition to obtain e;
and step 3: carrying out mitsunobu reaction on the compound e and the compound f to obtain a compound g;
and 4, step 4: carrying out coupling reaction on the compound g and the compound b in the presence of a catalyst to generate a compound h;
and 5: the body compound h is subjected to hydrolysis reaction under the catalysis of inorganic base or acid to obtain a compound j;
step 6: carrying out condensation or esterification reaction on the compound j and the compound I to obtain a compound shown in the formula I;
the invention provides a pharmaceutical preparation, which comprises the compound and a pharmaceutically acceptable carrier.
The invention provides an application of a compound shown in formula I or a salt, a solvate, an allosteric isomer, a metabolite, a nitrogen oxide and a prodrug thereof in preparing a medicament for treating or preventing P2X3 and/or P2X2/3 receptor related diseases.
In some embodiments of the invention, the use of a compound of formula (I) for the manufacture of a medicament for the treatment or prevention of a respiratory disorder.
In some embodiments of the invention, the use for the manufacture of a medicament for the treatment or prevention of cough, asthma, pain, sleep apnea.
In the present invention, the "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic agent is administered, and which is, within the scope of sound medical judgment, suitable for contact with the tissues of humans and/or other animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio.
Compared with the prior art, the invention has the following beneficial effects:
the dihydropyrimidine compound shown in the formula I has good affinity to a P2X3 receptor, strong antagonistic action and good safety. Tests show that the cough relieving effect time of the compound is obviously prolonged compared with that of the compound in the comparative example 1; the inhibition activity on P2X3 is better than that of the compound in the comparative example 1 and the gefapixant serving as the positive control drug, the taste of the mice is hardly influenced under the condition of intravenous administration of 10mg/kg, and the statistical difference with the positive control drug gefapixant is significant.
The preparation method of the compound of the formula I provided by the invention has the advantages of easily available raw materials, simple and convenient operation and easy industrialization.
Detailed Description
The present invention will be described in further detail with reference to examples and experimental examples, which are provided for illustration of the technical solution of the present invention and are not intended to limit the present invention, and any equivalent replacement in the field made in accordance with the disclosure of the present invention is within the scope of the present invention.
The structure of the compound is nuclear magnetic resonance (1H NMR) or liquid mass spectrometry (LC-MS).
The liquid mass spectrometer (LC-MS) is Agilent G6120B (used with liquid Agilent 1260); nuclear magnetic resonance apparatus (1H NMR is Bruker AVANCE-400 or Bruker AVANCE-800, nuclear magnetic resonance: (1HNMR) displacement (δ) is given in parts per million (ppm) and the solvent is determined as DMSO, internal standard Tetramethylsilane (TMS), chemical shift is 10-6(ppm) is given as a unit.
The term "room temperature" in the present invention means a temperature of 10 to 25 ℃.
Example 1: preparation of (S) -3- (3- (4-chlorobenzyl) -2, 6-dioxa-4- (4- (pyrazin-2-ylsulfanyl) phenyl) amino) -3, 6-dihydropyrimidin-1 (2H) yl) -2-methyl-N- (methylsulfonyl) propionamide (compound 1):
step 1: preparation of 4- (pyrazin-2-thio) aniline (Compound b-1)
2-Fluopyrazine (51.0g, 0.52mol) and p-aminophenol (61.3g, 0.49mol) were dissolved in dimethyl sulfoxide (360ml), cesium carbonate (320g, 0.98mol) was added to obtain a reaction mixture, and the reaction mixture was stirred at a uniform speed with mechanical stirring. Then the temperature of the reaction system is increased to 80 ℃ for reaction for 2 hours. The progress of the reaction was followed by thin layer chromatography and, after completion of the reaction, the reaction mixture was added to three volumes (about 1L) of water and kept under stirring. Then, the product was extracted with ethyl acetate three times, and the ethyl acetate was combined and dried, and then concentrated to obtain a crude product, which was slurried with 500ml of water for 1 hour, filtered, and dried in a forced air drying oven to obtain 4- (pyrazin-2-oxy) aniline (95.6g, brown granular solid) with a yield of 96.0%.
ESI-MS:m/z＝204.1(M+H)+。
Step 2: preparation of 6-chloro-1- (4-chlorobenzyl) pyrimidine-2, 4(1H,3H) -dione (Compound e-1)
6-chlorouracil (36.6g, 0.25mol) and 4-chlorobenzyl bromide (53.4g, 0.26mol) are mixed and dissolved by 300ml DMF, DIPEA (96.9g, 0.75mol) is added dropwise, then the reaction is kept at 30 ℃ for 3H, the reaction process is tracked by thin layer chromatography, after the reaction is completed, the reaction mixture is added into 3 times volume of water, the washed solid is filtered, after the drying, the filter cake is pulped by 300ml ethyl acetate, the solid is obtained by the filtration, and after the drying by a forced air drier, the 6-chloro-1- (4-chlorobenzyl) pyrimidine-2, 4(1H,3H) -diketone compound (52.25g, white solid) is obtained, the yield is 77.1%, and the purity is 99.28%.
ESI-MS:m/z＝271.0(M+H)+。
And step 3: preparation of methyl (S) -3- (4-chloro-3- (4-chlorobenzyl) -2, 6-dioxy-3, 6-dihydropyrimidin-1 (2H) -yl) -2-methylpropionate (Compound g-1)
Dissolving and clarifying a compound e-1(27.1g, 0.1mol), (S) - (+) -3-hydroxy-2-methyl propionate (11.8g, 0.1mol) and triphenylphosphine (52.4g, 0.2mol) by using 300ml of anhydrous tetrahydrofuran, replacing air in a reaction system by using argon, cooling the reaction system by using an ice water bath, slowly and uniformly dropwise adding diisopropyl azodicarboxylate (40.4g, 0.2mol) under stirring, keeping the reaction at room temperature after dropwise adding is finished within 30min, tracking the reaction progress by using a thin layer chromatography after 2 hours, quenching a reaction liquid by using 500ml of water after the reaction is finished, extracting three times by using 30ml of ethyl acetate, drying an organic solution, and concentrating to obtain an oily crude product. The crude oil was dispersed in a mixed solvent of 100ml of ethyl acetate and 500ml of petroleum ether to precipitate a large amount of triphenylphosphine oxide solid, the triphenylphosphine oxide was removed by filtration, and the mother liquor was concentrated and then purified by chromatography to give methyl (S) -3- (4-chloro-3- (4-chlorobenzyl) -2, 6-dioxo-3, 6-dihydropyrimidin-1 (2H) -yl) -2-methylpropionate (32.1g, white solid) in a yield of 86.5% and a purity of 98.71%.
ESI-MS:m/z＝371.1(M+H)+。
And 4, step 4: preparation of methyl (S) -3- (3- (4-chlorobenzyl) -2, 6-dioxo-4- (4- (pyrazin-2-sulfanyl) phenyl) amino) -3, 6-dihydropyrimidin-1 (2H) -yl) -2-methylpropionate (Compound H-1)
Mixing compound g-1(3.71g, 0.01mol), compound b-1(2.03g, 0.01mol), xant-phos (868mg, 1.5mmol), palladium acetate (337mg, 1.5mmol), potassium phosphate (4.24g, 0.02mol), dissolving with 30ml dioxane, replacing air in a reaction bottle with argon, reacting under protection of argon, heating the reaction mixture in an oil bath to 80 ℃ for 1h, detecting by thin layer chromatography until compound g-1 is completely consumed, distilling the reaction mixture under reduced pressure to remove dioxane, separating and extracting with 100ml ethyl acetate and 100ml water for three times, drying and concentrating the ethyl acetate phase, purifying by column chromatography to obtain methyl (S) -3- (3- (4-chlorobenzyl) -2, 6-dioxo-4- (4- (pyrazine-2-thio) phenyl) amino) -3, 6-dihydropyrimidin-1 (2H) -yl) -2-methylpropionate (4.58g, tan foamy solid), yield 85.1%, purity 98.89%.
ESI-MS:m/z＝538.2(M+H)+。
1H 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),7.21(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)。
Step 5, preparation of S-3- (3- (4-chlorobenzyl) -2, 6-dioxo-4- (4- (pyrazin-2-sulfanyl) phenyl) amino) -3, 6-dihydropyrimidin-1 (2H) -yl) -2-methylpropionic acid (Compound j-1)
Compound h-1(538mg, 1.0mmol) was dissolved in a mixed solvent of methanol (3ml) and tetrahydrofuran (3ml), the temperature was maintained at about 10 ℃, a solution of lithium hydroxide (168mg, 4mmol) in water (3ml) was added to obtain a reaction mixture, and the reaction mixture was allowed to react at room temperature overnight. The reaction progress is tracked by thin layer chromatography, and after the reaction is completed, the compound j-1(397mg, white-like solid) is obtained by column chromatography purification, and the yield is as follows: 75.8 percent and the purity is 99.35 percent.
ESI-MS:m/z＝524.1(M+H)+。
1HNMR(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), 2.75(m,1H),0.98(m,3H)。
Step 6 preparation of (S) -3- (3- (4-chlorobenzyl) -2, 6-dioxa-4- (4- (pyrazin-2-ylsulfanyl) phenyl) amino) -3, 6-dihydropyrimidin-1 (2H) yl) -2-methyl-N- (methylsulfonyl) propanamide (Compound 1)
Dissolving the compound j-1(299mg, 0.57mmol), DIPEA (183mg,1.42mmol) and EDCI (130.8mg, 0.68mmol) in dichloromethane (5ml), stirring for 15min, adding methylsulfonamide (64.7mg, 0.68mmol) and DMAP (83mg, 0.68mmol), reacting at room temperature for 2-3 h, following the reaction progress by thin layer chromatography, washing the reaction mixture after completion of the reaction with water, extracting with dichloromethane three times, combining, drying, concentrating, and purifying by column chromatography to obtain the compound 1(215.5mg), with a yield of 62.9% and a purity of 99.39%.
ESI-MS:m/z＝601.1(M+H)+。
1HNMR(400MHz,DMSO-d6)δ11.41(s,1H)，δ8.83(s,1H)，δ8.79(s,1H), 7.96–7.86(m,1H),7.48–7.39(m,1H),7.35–7.26(m,2H),7.20(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),3.88(m,2H), 3.01(s,3H),2.69(m,1H),0.99(m,3H)。
Example 2: preparation of (S) -3- (3- (4-chlorobenzyl) -2, 6-dioxa-4- (4- (pyridin-2-ylsulfanyl) phenyl) amino) -3, 6-dihydropyrimidin-1 (2H) yl) -2-methyl-N- (methylsulfonyl) propanamide (Compound 2)
The preparation method of this example compared to example 1, replacing 2-fluoropyrazine with equimolar 2-fluoropyridine in step 1, and the same conditions were followed to obtain compound 2 as a white solid in yield: 76.0 percent and the purity of 99.05 percent.
ESI-MS:m/z＝600.1(M+H)+。
1H NMR(400MHz,DMSO-d6)δ11.70(s,1H),8.80(s,1H),8.31(dd,J＝5.0, 2.0Hz,1H),7.99–7.89(m,1H),7.45–7.37(m,2H),7.30(d,J＝8.4Hz,2H),7.25 (m,4H),7.14–7.09(m,1H),7.03(d,J＝8.3Hz,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)。
Example 3: preparation of 1- (2, 6-dioxo-4- (4- (pyridine-2-thiophenyl) amino) -3- (4- (trifluoromethyl) benzyl) -3, 6-dihydropyrimidin-1 (2H) ylmethyl) -N- (methylsulfonyl) cyclopropane-1-carboxamide (Compound 3)
The same conditions were used for the preparation of this example compared to example 1 by replacing 2-fluoro-pyrazine with equimolar 2-fluoropyridine in step 1 and replacing methyl (S) -3-hydroxy-2-methylpropionate with equimolar methyl 1- (hydroxymethyl) cyclopropane-1-carboxylate in step 3, to give compound 3 as a white solid in yield: 76.1 percent and the purity is 99.05 percent.
ESI-MS:m/z＝646.1(M+H)+。
1H NMR(400MHz,DMSO-d6)δ11.40(s,1H),8.89(s,1H),8.35(dd,J＝5.2, 2.0Hz,1H),7.85(m,1H),7.46–7.38(m,2H),7.35(d,J＝8.4Hz,2H),7.23–7.15 (m,4H),7.15–7.09(m,1H),7.04(d,J＝8.3Hz,1H),5.28(s,2H),4.65(s,1H),4.13 (s,2H),3.12(s,3H),1.07–0.92(m,4H).
Example 4: preparation of (S) -3- (3- (4-chlorobenzyl) -2, 6-dioxo-4- (4- (pyrimidin-2-ylthio) phenyl) amino) -3, 6-dihydropyrimidin-1 (2H) -yl) -2-methyl-N- (methylsulfonyl) propanamide (Compound 4)
In the preparation method of this example, compared to example 1, the 2-fluoropyrazine in step 1 was replaced with equimolar 2, 6-difluoropyridine, and the same conditions were applied to obtain compound 4 as a white solid in yield: 79.1 percent and the purity is 98.98 percent.
ESI-MS:m/z＝634.2(M+H)+。
1H NMR(400MHz,DMSO-d6)δ11.30(s,1H),8.79(s,1H),8.33(q,J＝8.2Hz, 1H),7.44(d,J＝8.2Hz,2H),7.36(d,J＝8.2Hz,2H),7.22(s,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),3.01(s,3H),2.73(m,1H),0.98(m,3H).
Example 5: preparation of (S) -3- (3- (4-chlorobenzyl) -2, 6-dioxo-4- (4- (5- ((5-trifluoromethoxy) pyridin-2-yl) thio) phenyl) -3, 6-dihydropyrimidin-1 (2H) -yl) -2-methyl-N- (sulfonyl) propanamide (Compound 5)
In comparison with example 1, the preparation method of this example was carried out under the same conditions except that 2-fluoropyrazine in step 1 was replaced with equimolar 2-fluoro-5-trifluoromethoxy pyridine, so as to obtain compound 5 as a white solid in the yield: 80.1 percent and the purity is 99.50 percent.
ESI-MS:m/z＝684.2(M+H)+。
1H NMR(400MHz,DMSO-d6)δ11.70(s,1H),8.89(s,1H),8.36(s,1H),7.90– 7.79(m,1H),7.45–7.37(m,2H),7.36(d,J＝8.4Hz,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)。
Example 6: preparation of (S) -3- (3- (4-chlorobenzyl) -2, 6-dioxo-4- (4- (pyrimidin-2-ylthio) phenyl) amino) -3, 6-dihydropyrimidin-1 (2H) -yl) -2-methyl-N- (methylsulfonyl) propanamide (Compound 6)
The preparation method of this example compared to example 1, replacing 2-fluoropyrazine with equimolar 2-fluoropyrimidine in step 1, and the same conditions were followed to give compound 6 as a white solid in yield: 78.9% and the purity is 99.61%.
ESI-MS:m/z＝599.1(M+H)+。
1H NMR(400MHz,DMSO-d6)δ11.69(s,1H),8.80(s,1H),8.38(dd,J＝5.0, 2.0Hz,2H),7.45–7.37(m,2H),7.30(d,J＝8.4Hz,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), 0.99(m,3H)。
Example 7: preparation of (S) -3- (3- (4-chlorobenzyl) -4- (4- (2-cyanopyrimidin-5-ylthio) phenyl) -amino) -2, 6-dioxido-3, 6-dihydropyrimidin-1 (2H) -yl) -2-methyl-N- (methylsulfonyl) propanamide (Compound 7)
The procedure of this example was followed in comparison with example 1, except that 2-fluoropyrazine was replaced with 5-fluoro 2-cyanopyrimidine in an equimolar amount in step 1 and the conditions were the same, to obtain compound 7 as a white solid in a yield: 79.1 percent and the purity is 98.99 percent.
ESI-MS:m/z＝626.1(M+H)+。
1H NMR(400MHz,DMSO-d6)δ11.68(s,1H),8.79(s,1H),8.38(s,1H),7.90– 7.79(m,1H),7.46–7.38(m,2H),7.34(d,J＝8.4Hz,2H),7.15(s,4H),7.14–7.09 (m,1H),5.42–5.15(m,2H),3.86(m,2H),3.01(s,3H),2.68(m,1H),0.98(m,3H)。
Example 8: preparation of (S) -3- (3- (4-chlorobenzyl) -4- (4- (6-methoxypyridazin-3-yl) thio) phenyl) -2, 6-dioxo-3, 6-dihydropyrimidin-1 (2H) -yl) -2-methyl-N- (methylsulfonyl) propanamide (Compound 8)
The procedure of this example was followed in the same manner as in example 1 except that 2-fluoropyrazine in step 1 was replaced with equimolar 3-fluoro-6-methoxypyridazine to give compound 8 as a white solid in yield: 77.8 percent and the purity of 98.96 percent.
ESI-MS:m/z＝631.2(M+H)+。
1H NMR(400MHz,DMSO-d6)δ11.70(s,1H),8.69(s,1H),8.34(s,1H),7.90– 7.79(m,1H),7.46–7.38(m,2H),7.34(d,J＝8.4Hz,2H),7.15(s,4H),7.14–7.09 (m,1H),5.42–5.15(m,2H),4.06(m,2H),3.76(s,3H),3.01(s,3H),2.68(m,1H), 0.98(m,3H)。
Example 9: preparation of 3- (3- (4-chlorobenzyl) -2, 6-dioxo-4- (4- (phenylthio) phenyl) amino) -3, 6-dihydropyrimidin-1 (2H) -yl) -2, 2-dimethyl-N- (methylsulphonyl) propanamide (Compound 9)
The procedure of this example was followed in the same manner as in example 1 except that 2-fluoropyrazine was replaced with an equimolar amount of fluorobenzene in step 1 and methyl (S) -3-hydroxy-2-methylpropionate was replaced with an equimolar amount of methyl 3-hydroxy-2, 2-dimethylpropionate in step 3, to give compound 9 as a white solid in a yield: 75.1% and the purity is 99.26%.
ESI-MS:m/z＝613.1(M+H)+。
1H NMR(400MHz,DMSO-d6)δ9.74(s,1H),9.16(s,1H),8.60(s,1H),8.03 (m,1H),7.61–7.53(m,2H),7.30(d,J＝8.4Hz,2H),7.15(m,4H),7.24–7.19(m, 2H),7.03(d,J＝8.3Hz,2H),5.42–5.15(s,2H),4.06(m,2H),3.88(m,3H),0.99 (s,6H)。
Example 10: preparation of (S) -3- (3- (4-chlorobenzyl) -4- (4- (6-fluoropyridin-2-ylthio) phenyl) -2, 6-dioxo-3, 6-dihydropyrimidin-1 (2H) -yl) -2-methyl-N- (methylsulfonyl) propanamide (Compound 10)
In the preparation method of this example, compared to example 1, the 2-fluoropyrazine in step 1 was replaced with equimolar 2, 6-difluoropyridine, and the same conditions were applied to obtain compound 10 as a white solid in yield: 75.1% and the purity is 99.11%.
ESI-MS:m/z＝618.2(M+H)+。
1H NMR(400MHz,DMSO-d6)δ11.60(s,1H),8.95(s,1H),8.43(q,J＝8.2Hz, 1H),7.44(d,J＝8.2Hz,2H),7.35(d,J＝8.2Hz,2H),7.22(s,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),3.01(s,3H),2.73(m,1H),0.99(m,3H).
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-methylpropionic acid
In comparison with example 1, the preparation method of this example was carried out under the same conditions as those of example 1 except that p-aminophenol in step 1 was replaced with equimolar p-aminophenol and 2-fluoropyrazine in step 1 was replaced with equimolar 2, 3-difluoropyridine, and the other steps 2 to 5 were carried out without the condensation reaction of step 6 to obtain the compound of comparative example 1 as a white solid in yield: 81.6% and the purity is 99.21%.
ESI-MS:m/z＝525.1(M+H)+。
1H 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 Material
1.1 basic information of the test sample
Examples 1-10 (synthesized in the laboratory of the inventors), a positive control (gefapixant, lot: 01030-.
1.2 test reagents: normal saline, ammonia water.
2, experimental animals: healthy adult KM mice, half male and female, 6 mice per group, weight 28-30 g.
3 test method
3.1 dose design and sample usage
The animal cough model reported in the literature at present mostly adopts methods such as mechanical, chemical and electrical stimulation to stimulate nerves and receptors of animals to cause cough. According to the characteristics of the candidate compound and the existing similar target compounds as references, a mouse cough modeling test is established by primarily selecting a strong ammonia water induction method.
3.2 preparation method of test article
The preparation method of the 50% ammonia water solution comprises the following steps: 2.5ml of ammonia water is measured and dissolved in 5ml of 0.9 percent sodium chloride injection, and the mixture is fully and evenly mixed.
Comparative example 1 solution preparation method: 9mg of comparative example 1 was weighed out and dissolved in 3ml of 0.5% CMC-Na solution, and the solution was mixed well to prepare a 3mg/ml solution.
Examples solution formulation methods: 9mg of the example was dissolved in 3ml of 0.5% CMC-Na solution and mixed well to prepare a 3mg/ml solution.
3.3 Experimental operating methods
Grouping: the test pieces were divided into a positive control group, a comparative example 1 group, an example 1 group to an example 10 group, and a model group. Each group was prepared from 6 KM mice, and the administration was by intragastric administration; wherein the comparative example 1 group was administered with the compound of comparative example 1; the positive control group was given a positive control drug (gefapixant, purchased), and the example group was given the compound prepared in the corresponding example; each group corresponded to three doses: 30mg/kg, 10 ml/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, respectively, into which 1 cotton ball (weight 100. + -.5 mg) containing 0.3ml of 50% ammonia water was placed. The number of typical coughs occurring within 3min was observed in the mice (typical coughing action: contraction of abdominal muscles or chest contraction while mouth widening with coughing sound).
4 results and discussion
4.1 criteria for determination of results
Judging a cough standard:
the manifestations of cough are: the abdominal muscles contract or contract the chest while the mouth is enlarged, with a cough.
Secondly, a stopwatch is used for timing, the number of coughing of the mice within 3min is recorded, statistical analysis is carried out by software, all groups of data are statistically described by means of mean values plus or minus standard deviations, single-factor variance analysis among multiple groups is carried out, and the difference P <0.05 has statistical significance.
4.2 discussion of results
The number of coughs after 60, 120min administration of the test samples is shown in the following table:
TABLE 2
Remarking: comparison with model groups: p<0.01，*P<0.05. Comparison with comparative example 1 group:△P<0.05。
as shown in the table, when the test of 30mg/kg is used, the cough frequency of mice is obviously reduced after 60min administration and has statistical difference (P is less than 0.01) when the positive control group, the comparative example 1 group and the model group are compared; after 120min of administration, the comparative example 1 group had a reduced number of coughs compared to the model group, but had no statistical significance, indicating that the compound of comparative example 1 did not have a significant antitussive effect at 120 min.
After the compound of the invention is administrated for 60min and 120min, the number of cough times is still obviously reduced compared with the model group, and the compound has statistical difference (P < 0.01); furthermore, there was a statistical difference (P <0.05) from the compound of comparative example 1 after 120min of administration. Illustrating that the compounds of the examples of the present invention have longer time to relieve cough than the compounds of comparative example 1.
Test example 2: evaluation of biological Activity in vitro
1. Reagents, consumables and instruments: the reagents, consumables and instruments used in this test example were commercially available.
2. Cell line (b): HEK293 cell line stably transfected with human P2X3 receptor was used.
3. Cell culture
Growth medium: DMEM high glucose; 10% FBS; 1% PenStrep.
4. The cell culture process comprises the following steps:
a) reviving cells
1) Immersing the cell freezing tube in water bath at 37 ℃, and continuously shaking to dissolve the cell freezing tube as soon as possible;
2) slowly blowing the cells up and down by using a 1mL pipette to suspend the cells, dripping the cells into a 15mL centrifuge tube containing 10mL fresh pre-warming growth medium, and centrifuging the cells for 5 minutes at 1000 rpm/min;
3) the supernatant was discarded and 5mL of fresh green tea was usedThe long medium resuspended the cells. Transfer the cell suspension to a petri dish and place in 5% CO2The incubator (2) is subjected to static culture at 37 ℃;
4) after 24 hours, the medium was slowly removed (taking care not to disrupt the cell monolayer) and cultured with fresh growth medium.
b) Subculturing
Cell lines are typically expressed as 1: 3 to 1: 4, twice a week (the passage ratio of 1: 3 is more common), and the cells after passage can grow to reach 85 percent of confluency in 2-3 days;
1) when the cells reach more than 85% saturation in a 10cm culture dish, digesting the cells for about 1min by using 0.25% Trypsin-EDTA solution, and sucking out the cells in the culture dish;
2) cells were transferred to petri dishes containing complete growth medium according to the dilution ratio. Note that: to maintain logarithmic cell growth, cell monolayer culture should be maintained;
3) cell doubling time according to cell line (HEK293-P2X 3: 24 hours), cells were passaged using 0.25% trypsin solution.
c) Cryopreserved cells
1) Taking out the culture dish from the incubator, placing the culture dish in an ultra-clean workbench, digesting the culture dish for about 1min by using 0.25% Trypsin-EDTA solution, collecting and counting cells, and centrifuging the cells for 5min at 1000 rpm/min;
2) the supernatant was aspirated and the cells were resuspended in a cryopreservative (90% FBS and 10% DMSO) at a density of 2X 106cells/mL, 1mL of cell suspension is added into each cryopreservation tube;
3) putting the cell freezing tube into a freezing box, and then transferring the cell freezing tube to-80 ℃ for overnight;
4) the vial was transferred to a liquid nitrogen tank (-196 ℃ C.).
5. Experimental procedures
Step 1: preparation of cell assay plates
1) When the cells in a 15cm culture dish grow to 80% and are fused, removing supernatant, adding 5mL of DPBS to clean the cells and sucking out, adding 2.5mL of 0.25% Trypsin-EDTA solution into the culture dish, putting the culture dish into an incubator for 1-3 minutes or until the cells are digested, adding 3mL of complete culture medium to stop digestion, and detecting the cell density by using a cell counter;
2) after centrifugation at 1000rpm/min for 5min, the cells were resuspended in growth medium and the suspension volume was adjusted to achieve a cell density of 4X 105cells/mL(1×104cells/25μL)；
3) Adding 10 mu L of 5 Xmatrigel into a black microplate, placing the microplate in an incubator for 15 minutes, taking out the microplate, inverting the microplate, centrifuging the microplate at 300g/min for 30s, and removing the 5 Xmatrigel. Then adding the prepared cell suspension into a 384 black micro-porous plate, wherein each hole is 25 mu L;
4) placing the microporous plate in 5% CO2The incubator of (1) was incubated overnight at 37 ℃ until the next day the cells grew to confluency.
Step 2: preparation of the Compounds
Antagonist modes
1) Test compound mother liquor concentration: 20 mM;
2) compounds on 384-LDV plates were diluted 12-point using Bravo, starting at 10 μ M concentration, at a 3-fold dilution;
3) HPE (high potency control): a single dose of a 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) as compound plates using ECHO;
5) the compound plate was stored at-20 ℃.
And 3, step 3: performing a screening test
1) Taking the cell plate growing to the fusion state out of the incubator;
2) preparation of detection buffer: 30mL of buffer containing 0.3mL of 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 a stock solution of C6 dye, C6 dye as 10 ×, and diluting C6 dye to 1 ×, with a buffer solution;
4) the medium was discarded by inverted centrifugation using the genetle spin model of Bluewashbher.
5) Add C6 dye, 20 μ L/well, onto the cell plate with a pipetting gun;
6) centrifuging the cell plate at 300rpm/min for 30s, and incubating for 1.5h in an incubator;
7) mu.L of assay buffer was added to each well using a Dragonfly autosampler on a pre-prepared compound plate, 10. mu.L of compound was transferred to the cell plate with Bravo according to the assay plate layout, test compound finally detected the highest concentration dose of FAC: 10 μ M, 3 fold dilution, 12 concentration points.
8) Centrifuging the cell plate at 300rpm/min for 30s, and putting the cell plate into an incubator to incubate for 30 min;
9) 25 μ L of 4 XBZATP (final concentration 3.5uM) agonist was prepared on an agonist plate (PE 6008590) to act on P2X3 cells.
10) Placing the cell plate, FLIPR tip and agonist plate at room temperature for 15 min;
11) transfer 10 μ L of BZATP agonist to cell plates with FLIPR and read.
And 4, step 4: data analysis with Excel and Xlfit
6. Results and analysis of the experiments
Inhibition of the P2X3 receptor by the Compounds of the examples IC50As shown in the following table, wherein A represents less than 10nM, B represents 10.1-50 nM, and C represents 50.1-100 nM.
TABLE 3
| Test sample | P2X3 IC50(nM) |
| Positive control drug | C |
| Comparative example 1 | B |
| Compound 1 | A |
| Compound 2 | A |
| Compound 3 | B |
| Compound 4 | B |
| Compound 5 | A |
| Compound 6 | C |
| Compound 7 | A |
| Compound 8 | B |
| Compound 9 | C |
| Compound 10 | A |
As can be seen from table 3, the compounds of examples 1, 2, 5, 7, and 10 had inhibitory activity against P2X3, and were superior to those of comparative example 1 and the positive control.
Test example 3: taste disorder test
1 test Material
1.1 basic information of the test sample
Examples 1, 2 and 10 (synthesized in the laboratory of the present inventors), and a positive control (gefapixant, lot # 01030-210326-2-1, purchased from palmtop medicine).
1.2 test reagents
0.9% sodium chloride injection, quinine hydrochloride (Quinie, batch No.: C12476271)
2, experimental animals: healthy adult SD rats, full male, weighing about 280-300 g.
3 test method
3.1 preparation method of test article
The preparation method of the 0.3mM quinine solution comprises the following steps: weighing 119.20mg quinine hydrochloride, dissolving in 1000ml tap water, and mixing well.
The preparation method of the test sample solution comprises the following steps: weighing 40mg of test sample, adding a proper amount of DMSO, dissolving, adding the HS-15 solution, fully and uniformly mixing, adding 16ml of physiological saline, and preparing into a solution of 2.5 mg/ml.
3.2 Experimental operating methods
Animals and groups: 160-180 g/male SD rat, 10 rats in each group, similar average body weight of each group, and single cage breeding.
Training drinking habits: the animals in each group are respectively fed with normal water for 30 minutes at 8:30 am and 16:30 pm every day, the water is forbidden in the rest time for 5 days, and the left and right placement positions of two bottles of water are changed every day.
Administration: the evening before the experiment was deprived of water, the test group was administered 4mL/kg (10mg/kg) of the test sample by tail vein injection at the following dose in the morning of the next day, and the model group was administered 4mL/kg (10mg/kg) of 0.5% HS-15 by intravenous injection.
4 results and discussion
4.1 criteria for determination of results
Measuring water intake: the animals are placed back into the original cages after injection, the measurement time of each group is respectively within the interval of Tmax of various medicines (the measurement time is 0min-15min after administration), one bottle of normal drinking water and one bottle of drinking water containing 0.3mM quinine hydrochloride (Quinie) are simultaneously placed into each cage, and the left and right positions of the two bottles of water in all animal raising cages are consistent. Animals were allowed to drink water freely for 15min, and the water consumption of two bottles of water was measured separately to 0.1 ml.
And (2) statistical analysis of data: the drinking amount of the quinine bitter water and tap water and the percentage of quinine water in tap water are counted respectively, and the difference between the groups is compared by analysis of variance to determine whether the difference is significant.
4.2 discussion of results
TABLE 4
| Test sample | Quinine/tap water (%) |
| Solvent group | 38.16％ |
| Positive control group | 79.01％** |
| EXAMPLE 1 group (Compound 1) | 40.34％△ |
| EXAMPLE 2 group (Compound 2) | 41.54％△ |
| EXAMPLE 10 group (Compound 10) | 39.43％△ |
Remarking: comparison with vehicle group: p<0.01; comparison with Positive control group：△P<0.01。
As can be seen from the above table, the ratio of quinine/tap water consumed by the mice in the positive control group relative to the vehicle group was statistically different (P <0.01), indicating that the compounds in the positive control group have a significant effect on the taste of the mice. On the other hand, the ratio of quinine drinking to tap water in the mice in the groups of example 1, 2 and 10 was not statistically significant compared to the vehicle group, so that the above compounds 1, 2 and 10 had little effect on the taste of the mice when administered intravenously at 10mg/kg, and were statistically significantly different from the positive control group.
The above-mentioned 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 changes made in the spirit and the concept of the main body of the present invention, which still solve the technical problems consistent with the present invention, should be included in the scope of the present invention.
Claims (10)
1. A compound or its salt with a structure shown in formula I,
wherein R is1Is C1-C16 alkyl;
R2、R3independently selected from hydrogen, methyl, ethyl or propyl; or R2、R3Linked to form an unsubstituted cyclopropane;
R4selected from the group consisting of substituted or unsubstituted phenyl, substituted or unsubstituted pyridyl, substituted or unsubstituted pyrimidinyl, substituted or unsubstituted pyrazinyl, substituted or unsubstituted pyridazinyl; r4The substituent(s) is selected from halogen, methoxy, cyano, trifluoromethoxy or difluoromethoxy;
R5selected from halogen, trifluoromethyl or difluoromethyl.
2. A compound or salt thereof according to claim 1, wherein in formula I:
R1is methyl;
R2、R3independently selected from hydrogen, methyl, ethyl or propyl; or R2、R3Linked to form an unsubstituted cyclopropane;
R4selected from the group consisting of substituted or unsubstituted phenyl, substituted or unsubstituted pyridyl, substituted or unsubstituted pyrimidinyl, substituted or unsubstituted pyrazinyl, substituted or unsubstituted pyridazinyl; r4The substituent(s) is selected from halogen, methoxy, cyano, trifluoromethoxy or difluoromethoxy;
R5selected from halogen, trifluoromethyl or difluoromethyl.
3. A compound or salt thereof according to claim 1, wherein in formula I:
R1is methyl;
R2、R3independently selected from hydrogen or methyl; or R2、R3Linked to form an unsubstituted cyclopropane;
R4selected from the group consisting of substituted or unsubstituted phenyl, substituted or unsubstituted pyridyl, substituted or unsubstituted pyrimidinyl, substituted or unsubstituted pyrazinyl, substituted or unsubstituted pyridazinyl; r4The substituent(s) is selected from fluoro, methoxy, cyano or trifluoromethoxy;
R5selected from chloro, trifluoromethyl or difluoromethyl.
4. The compound or salt thereof according to claim 1, selected from the following compounds or pharmaceutically acceptable salts thereof:
5. a compound or a salt thereof according to any one of claims 1 to 4, wherein the hydrogen in the compound is substituted with one or more deuterium.
6. The process for producing a compound or a salt thereof according to claim 4, comprising the steps of:
step 1: carrying out substitution reaction on the compound a and the compound k under the catalysis of alkali to generate a compound b;
step 2: carrying out substitution reaction on the compound c and the compound d under an alkaline condition to obtain e;
and 3, step 3: carrying out mitsunobu reaction on the compound e and the compound f to obtain a compound g;
and 4, step 4: carrying out coupling reaction on the compound g and the compound b in the presence of a catalyst to generate a compound h;
and 5: the body compound h is subjected to hydrolysis reaction under the catalysis of inorganic base or acid to obtain a compound j;
step 6: carrying out condensation or esterification reaction on the compound j and the compound I to obtain a compound shown in the formula I;
wherein R is1、R2、R3、R4、R5Is as defined in claim 4.
7. A pharmaceutical formulation comprising a compound according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier.
8. Use of a compound according to any one of claims 1 to 4 or a salt thereof for the manufacture of a medicament for the treatment or prevention of a P2X3 and/or P2X2/3 receptor related disease.
9. The use according to claim 8, in the manufacture of a medicament for the treatment or prevention of a respiratory disorder.
10. Use according to claim 8, in the manufacture of a medicament for the treatment or prevention of cough, asthma, pain or sleep apnea.
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| CN103140221A (en) * | 2010-08-10 | 2013-06-05 | 盐野义制药株式会社 | Novel heterocyclic derivatives and pharmaceutical composition containing same |
| WO2013118855A1 (en) * | 2012-02-09 | 2013-08-15 | 塩野義製薬株式会社 | Heterocyclic ring and carbocyclic derivative |
| CN107709299A (en) * | 2015-04-24 | 2018-02-16 | 盐野义制药株式会社 | 6 membered heterocycle derivatives and the pharmaceutical composition containing it |
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| CN103140221A (en) * | 2010-08-10 | 2013-06-05 | 盐野义制药株式会社 | Novel heterocyclic derivatives and pharmaceutical composition containing same |
| WO2013118855A1 (en) * | 2012-02-09 | 2013-08-15 | 塩野義製薬株式会社 | Heterocyclic ring and carbocyclic derivative |
| CN107709299A (en) * | 2015-04-24 | 2018-02-16 | 盐野义制药株式会社 | 6 membered heterocycle derivatives and the pharmaceutical composition containing it |
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