Dihydropyrimidinethione compound and application thereof in resisting porcine epidemic diarrhea virus

Abstract

The invention discloses dihydropyrimidine (thio) ketone as shown in a general formula I or pharmaceutically acceptable salt thereof, and preparation and antiviral application of the dihydropyrimidine (thio) ketone or the pharmaceutically acceptable salt thereof. The compound has the effects of inhibiting PEDV virus replication and reducing virus titer, is used for preventing and treating PED in the pig breeding industry, and reduces the incidence rate and case fatality rate of PED. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and relates to a dihydropyrimidine (thiol) ketone compound and its preparation and antiviral use, and specifically to a dihydropyrimidine (thiol) ketone compound and its synthesis and use in anti-porcine epidemic diarrhea virus drugs. Background Art

[0002] Porcine epidemic diarrhea (PED) is a contact enteric infectious disease caused by porcine epidemic diarrhea virus (PEDV). Infected pigs often show symptoms such as severe diarrhea, vomiting, dehydration, and weight loss. There is a risk of infection with PEDV at all stages of pig farming, but the symptoms of infection vary among pigs of different ages, especially in newborn piglets, where the mortality rate can reach 100%. The high morbidity and mortality of PED have caused huge economic losses to the pig farming industry. Existing vaccines are difficult to meet the needs of PED prevention and control, so new means of preventing and controlling PEDV infection are urgently needed.

[0003] Dihydropyrimidone compounds (DHPMs) have a variety of biological activities such as antibacterial, anti-inflammatory, anti-tumor, antimalarial, and antiviral. DHPMs are often prepared by Biginelli reaction of aldehydes, β-diketones and ureas, which is a one-pot synthesis reaction. Many chemists have developed a large number of efficient catalysts for Biginelli reaction, which greatly expanded the applicable substrates of the reaction and prepared DHPMs with diverse structures. Isolated marine alkaloids, such as batzelladine A and B, inhibit the binding of HIV envelope protein gp-120 to human CD4 cells and are potential effective compounds for the treatment of AIDS. Their biological activity is attributed to the dihydropyrimidine part in their structure (Synthesis of calcium channel blocker 4-aryl-3,4-dihydropyrimidine-2(1H)-thione derivatives, Bioorganic and Medicinal Chemistry, 2006, 14, 8582-8589). Batzelladine A, a derivative of α,γ-diketobutyric acid DHPMs, has significant anti-HIV activity (Synthesis of dihydropyrimidine α,γ-diketobutyric acid derivatives targeting HIV integrase, European Medicinal Chemistry. 2015; 104: 127-138.), and its IC 50It is at the micromolar level, but cytotoxic side effects are observed. Nitractin, containing dihydropyrimidine-2(1H)-one, has a strong inhibitory effect on trachoma virus and has certain antibacterial activity. Idoxuridine containing dihydropyrimidine-2(1H)-one can be used as an antiviral drug for local treatment of herpes simplex keratitis. It can be seen that the dihydropyrimidine 2(1H)-one skeleton molecule is an antiviral advantage structure, and DHPMs obtained through rational design are expected to discover new candidate compounds with anti-PEDV activity. Summary of the invention

[0004] The object of the present invention is to provide a new dihydropyrimidinethione compound, including a preparation method and an anti-PEDV drug use thereof.

[0005] The object of the present invention is achieved through the following technical solutions:

[0006] In the first aspect, the dihydropyrimidine (thiol) ketone or its pharmaceutically acceptable salt as shown in the general formula I, including its stereoisomers or tautomers:

[0007]

[0008] Wherein: X=S or O; R1 is selected from hydrogen, or 4-halogen, phenyl, heteroaryl, N,N-diC 1-4 alkyl, or phenyl or heteroaryl at position 3, or phenyl or heteroaryl at position 2, wherein the phenyl may be substituted with one or more substituents, such as halogen, methyl, trifluoromethyl, methoxy, trifluoromethoxy, etc.; R2 is substituted or unsubstituted phenyl or heteroaryl; R3 is selected from C 1-4 Alkyl or alkoxy or C 3-5 Cycloalkyl or cycloalkoxy, preferably methyl, ethyl, isopropyl, n-butyl, cyclopropyl, cyclobutyl, cyclopentyl, or methoxy, ethoxy, isopropoxy, n-butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, said C 1-4 Alkyl or alkoxy, C 3-5 Cycloalkyl or cycloalkoxy may be substituted with one or more fluorine atoms, such as trifluoromethyl, trifluoromethylmethylene, pentafluoroethyl, trifluoromethoxy, hexafluoroisopropoxy, etc. The heteroaryl group in the present invention refers to a phenyl group in which one, two or three carbon atoms are replaced by sulfur or nitrogen atoms, and in particular refers to a phenyl group in which one or two carbon atoms are replaced by sulfur or nitrogen atoms. The dihydropyrimidine (thiol) ketone in the present invention is a brand-new compound.

[0009] As a preferred technical solution of the present invention, in the dihydropyrimidine (thio) ketone shown in the general formula I: X = S or O; R1 is selected from hydrogen, or 4-position chlorine, fluorine, phenyl, N,N-diC 1-4alkyl, or phenyl at position 3, or phenyl at position 2, the phenyl group may be further substituted by one or more substituents, such as chlorine, methyl, trifluoromethyl, methoxy, trifluoromethoxy, etc.; R2 is substituted or unsubstituted phenyl; R3 is selected from C 1-4 Straight or branched alkyl or alkoxy or C 3-5 Cycloalkyl or cycloalkoxy, preferably methyl, ethyl, isopropyl, n-butyl, cyclopropyl, cyclobutyl, cyclopentyl, or methoxy, ethoxy, isopropoxy, n-butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, said C 1-4 Straight or branched alkyl or alkoxy, C 3-5 The cycloalkyl or cycloalkoxy group may be substituted with one or more fluorine atoms, for example, trifluoromethyl, trifluoromethylmethylene, pentafluoroethyl, trifluoromethoxy, hexafluoroisopropoxy and the like.

[0010] As a preferred technical solution of the present invention, in the dihydropyrimidine (thio) ketone shown in the general formula I: X = S; R1 is selected from phenyl at the 4th, 3rd or 2nd position, and the phenyl group can be substituted by any substituent selected from chlorine, methyl, trifluoromethyl, methoxy, trifluoromethoxy; R2 is phenyl; R3 is selected from C 1-4 Straight or branched alkyl or alkoxy or C 3-5 Cycloalkyl or cycloalkoxy, the C 1-3 Straight or branched alkyl or alkoxy, C 3-4 The cycloalkyl group or cycloalkoxy group may be substituted with one or more fluorine atoms.

[0011] As a further preferred technical solution of the present invention, in the dihydropyrimidine (thio) ketone shown in general formula I: X=S; R1 is selected from phenyl at position 2, and the phenyl group can be substituted at the para position by any substituent selected from chlorine, methyl, trifluoromethyl, methoxy, and trifluoromethoxy; R2 is phenyl; R3 is selected from methyl, ethyl, isopropyl, cyclopropyl, cyclobutyl, or methoxy, ethoxy, isopropoxy, cyclopropyloxy, and cyclobutyloxy, and the hydrogen atom on R3 can be replaced by one or more fluorine atoms.

[0012] As a further preferred technical solution of the present invention, in the dihydropyrimidine (thio) ketone shown in the general formula I: X=S; R1 is selected from the phenyl group at the 2-position, and the phenyl group at the 4-position can be substituted by any substituent selected from chlorine, methyl, and methoxy; R2 is a phenyl group; R3 is selected from methyl, ethyl, isopropyl, methoxy, ethoxy, cyclopropyloxy, and cyclobutyloxy, and the hydrogen atom on R3 can be replaced by one or more fluorine atoms to form, for example, trifluoromethyl, trifluoromethylmethylene, pentafluoroethyl, and hexafluoroisopropoxy.

[0013] Among the dihydropyrimidinethione compounds described in the present invention, preferred specific compounds are: D2, D12, D22, D29, D32, D33, D34, D35, D36, D40, D41; among which further preferred compounds are: D12, D29, D32, D33, D35, D36, D40, D41; particularly preferred compounds are: D35, D40.

[0014]

[0015] In a second aspect, the present invention also relates to a compound of formula F as an intermediate, wherein X, R1, and R2 are defined the same as those of the compound of formula I.

[0016]

[0017] In the third aspect, the preparation method of the DHPMs compound of the general structural formula I of the present invention is as follows: According to the structure of the target compound, the present invention adopts different synthetic routes.

[0018] A.R3 is C 1-4 Straight chain or branched alkyl (methyl or methoxy or ethoxy as an example) compound of formula I

[0019] The preparation method is synthesized by a one-pot Biginelli reaction of substituted benzaldehyde, (thio)urea and β-diketone. Figure 1 (a), the specific operation steps are as follows: dissolve 1 mmol of substituted benzaldehyde, 1.5 times the amount of (thio)urea and 1.2 times the amount of β-diketone in 5 mL of a suitable anhydrous solvent, add 0.2 equivalents of a suitable catalyst, heat and reflux for 8-12 hours, and obtain compound D through appropriate standardized post-treatment procedures, that is, the compound represented by general formula I wherein R3 is methyl, methoxy or ethoxy.

[0020] As a preferred technical solution of the present invention, the suitable anhydrous solvent in the operation steps is glacial acetic acid, anhydrous ethanol, anhydrous methanol, anhydrous N,N-dimethylformamide and a mixture thereof, preferably glacial acetic acid and anhydrous ethanol; the suitable catalyst is glacial acetic acid or p-toluenesulfonic acid or cerium chloride heptahydrate, preferably glacial acetic acid; the appropriate standardized post-treatment procedure is recrystallization from anhydrous ethanol or recrystallization from anhydrous acetonitrile or purification by silica gel column chromatography, preferably recrystallization from anhydrous ethanol.

[0021] In some specific embodiments, the substituted benzaldehyde of the present invention needs to be prepared by Suzuki coupling reaction. The schematic diagram of the preparation route is as follows Figure 1(b), the specific operation steps are as follows: under nitrogen protection, 1 mmol of bromobenzaldehyde, 1.3 equivalents of substituted phenylboric acid and 3 equivalents of potassium carbonate are dissolved in 6 mL of a mixed solvent of toluene and water in a ratio of 5:1, 0.2 times the amount of [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride is added, and the temperature is raised and refluxed for reaction for 8 to 12 hours to obtain the substituted benzaldehyde of the present invention.

[0022] In some specific embodiments, the β-diketone of the present invention is prepared by a Claisen ester condensation reaction. The schematic diagram of the preparation route is as follows Figure 1 (c), the specific operation steps are as follows: under nitrogen protection, 1 mmol 3-acetylpyridine is dissolved in 10 mL anhydrous THF, 4 equivalents of 60% sodium hydride are slowly added under ice bath, 2.5 equivalents of ethyl acetate are added after 30 minutes, and the reaction is carried out at room temperature for 8 to 12 hours to prepare the β-diketone of the present invention.

[0023] B. Compounds of Formula I wherein R3 is hydroxyl

[0024] The compound E with R3 being ethoxy is prepared by ester hydrolysis reaction. The schematic diagram of the preparation route is shown in Figure 1 (d), the specific operation steps are as follows: dissolve 1 mmol of compound E in 5 mL of methanol, dissolve 5 times the equivalent of a suitable base in 1 mL of water, add the mixture to the reaction system, heat to 70°C and react for 20 to 24 hours to obtain compound F, which is the compound represented by the general formula II wherein R3 is a hydroxyl group as described in the present invention.

[0025] C.R3 is from C 1-4 Straight-chain or branched alkoxy compounds of formula I

[0026] It is prepared by esterification reaction of compound F wherein R3 is a hydroxyl group. The schematic diagram of the preparation route is as follows: Figure 1 (d), the specific operation steps are as follows: dissolve 1 mmol of compound F in which R3 is hydroxyl in 5 mL of anhydrous dichloromethane, add 1.5 equivalents of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.2 equivalents of 4-dimethylaminopyridine, and react at room temperature for 12 h to obtain the compound represented by the general formula I in which R3 is isopropoxy, n-butoxy, hexafluoroisopropoxy, cyclopropyloxy, cyclobutyloxy or cyclopentyloxy as described in the present invention.

[0027] As a preferred technical solution of the present invention, the suitable base in the operation steps is lithium hydroxide, sodium hydroxide, potassium hydroxide, cesium carbonate, etc., preferably lithium hydroxide.

[0028] The DHPMs synthesis raw materials of the present invention are cheap, the reaction conditions are mild, the post-treatment is simple, the environment is friendly, and the large-scale production is easy.

[0029] In the fourth aspect, the present invention relates to an anti-PEDV effect of the compound, and the mechanism of action is unique. The compound interacts with the PEDV virus in a specific way, inhibits viral replication, and reduces the virus titer. It is used for the prevention and treatment of PED in the pig farming industry, reduces the incidence and mortality of PED, thereby reducing the economic losses caused by PED and reducing the cost of breeding. In the fifth aspect, the present invention relates to a veterinary drug, which uses the dihydropyrimidine (thiol) ketone compound of the present invention as an active ingredient for the treatment of porcine epidemic diarrhea and resistance to porcine epidemic diarrhea virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The schematic diagram is a synthetic route of the compound of the present invention. DETAILED DESCRIPTION

[0031] The present invention is described in detail below with reference to the embodiments.

[0032] Example 1 Preparation of Compound D35

[0033] This example involves the synthesis of compound D29 from biphenyl-2-carboxaldehyde, thiourea and ethyl benzoylacetate via Biginelli reaction, the hydrolysis of compound D29 under alkaline conditions to synthesize compound D39, and the esterification reaction of compound D39 with isobutyl alcohol to synthesize compound D35.

[0034]

[0035] 1) Synthesis of 6-phenyl-4-(2-phenylphenyl)-2-thiolidene-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid ethyl ester (D29)

[0036] Dissolve biphenyl-2-carboxaldehyde (A12, 5mmoL, 912mg), thiourea (B1, 1.2eq, 457mg) and ethyl benzoyl acetate (C3, 1.1eq, 1058mg) in 20mL glacial acetic acid, and heat and reflux for 12 hours. After the reaction, the reaction system is cooled and concentrated under reduced pressure. The crude product is purified by silica gel column chromatography to obtain 1476mg of white solid, namely 6-phenyl-4-(2-phenylphenyl)-2-sulfylidene-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid ethyl ester (D29), with a yield of 71.2%. 1 H NMR (400MHz, DMSO-d6) δ10.33(s,1H),9.57(s,1H),7.73–7.14(m,14H),5.47(s,1H),3.53(d,J=35.9Hz,2H),0.51(s,3H). 13C NMR(101MHz,DMSO-d6)δ174.49,164.94,144.82,141.63,141.00,140.70,134.63,130.67,130.21 ,129.36,128.92,128.88,128.42,128.39,128.36,128.18,127.41,103.67,59.55,52.16,13.74.

[0037] Using different substituted benzaldehydes, (thio)urea and corresponding β-diketone compounds as raw materials, the compounds D1, D2, D3, D4, D5, D6, D7, D8, D9, D11, D12, D13, D14, D15, D16, D17, D18, D19, D20, D21, D22, D23, D30, D31, D32, D33, D34, D37, and D38 of the present invention can be synthesized in moderate to high yields according to the above method.

[0038] 1-{6-phenyl-4-[4-(pyridin-2-yl)phenyl]-2-thiolidene-1,2,3,4-tetrahydropyrimidin-5-yl}ethan-1-one (D8): 1 H NMR (400MHz, DMSO-d6) δ10.39(s,1H),9.74(s,1H),8.65(d,J=5.2Hz,1H),8.05(d,J=8.2Hz,2H),7.89(d d,J=21.4,8.4Hz,2H),7.58–7.49(m,3H),7.45(t,J=7.6Hz,2H),7.32(s,3H),5.37(s,1H),1.74(s,3H).

[0039] 1-[6-phenyl-4-(2-phenylphenyl)-2-thiolidene-1,2,3,4-tetrahydropyrimidin-5-yl]ethan-1-one (D12): 1 HNMR (400MHz, DMSO-d6) δ10.18(s,1H),9.50(s,1H),7.59(d,J=7.9Hz,1H),7.52–7.25(m,12H),7.05(d,J=7.7Hz,1H),5.48(s,1H),1.63(s,3H).

[0040] 1-{4-[4-(4-methoxyphenyl)phenyl]-6-phenyl-2-thiolidene-1,2,3,4-tetrahydropyrimidin-5-yl}ethan-1-one (D15): 1H NMR (400MHz, DMSO-d6) δ10.38(s,1H),9.71(s,1H),7.58(dd,J=8.8,2.3Hz,4H),7.56–7.51(m,3H),7.46(t, J=7.3Hz,2H),7.24(d,J=8.4Hz,2H),7.01(d,J=9.0Hz,2H),5.32(d,J=3.8Hz,1H),3.79(s,3H),1.75(s,3H).

[0041] 1-[2-Oxylidene-6-phenyl-4-(4-phenylphenyl)-1,2,3,4-tetrahydropyrimidin-5-yl]ethan-1-one (D16): 1 HNMR (400MHz, DMSO-d6) δ9.22 (s, 1H), 7.84 (s, 1H), 7.62 (t, J = 6.9Hz, 4H), 7. 46(d,J=10.6Hz,7H),7.33(dd,J=23.3,7.6Hz,3H),5.34(s,1H),1.69(s,3H).

[0042] 1-[6-methyl-4-(4-phenylphenyl)-2-thiolidene-1,2,3,4-tetrahydropyrimidin-5-yl]ethan-1-one (D19): 1 HNMR (400MHz, DMSO-d6) δ10.31(s,1H),9.79(s,1H),7.64(d,J=8.0Hz,4H),7.45(t,J=7. 5Hz,2H),7.34(dd,J=19.4,7.7Hz,3H),5.34(d,J=4.0Hz,1H),2.36(s,3H),2.20(s,3H).

[0043] 1-{6-methyl-4-[4-(methyldioxyylidene-λ6-thio)phenyl]-2-thioylidene-1,2,3,4-tetrahydropyrimidin-5-yl}ethan-1-one (D21): 1 H NMR (400MHz, DMSO-d6) δ10.40 (s, 1H), 9.83 (s, 1H), 7.91 (d, J = 8.4Hz, 2H), 7.48 (d,J=8.4Hz,2H),5.39(d,J=4.0Hz,1H),3.20(s,3H),2.36(s,3H),2.23(s,3H).

[0044] 6-phenyl-4-(4-phenylphenyl)-2-thiolidene-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid ethyl ester (D22): 1HNMR(400MHz,DMSO-d6)δ10.52(s,1H),9.80(d,J=2.2Hz,1H),7.69(dd,J=12.3,7.8Hz,4H ),7.54–7.24(m,10H),5.33(d,J=4.0Hz,1H),3.77(q,J=7.1Hz,2H),0.75(t,J=7.1Hz,3H).

[0045] 1-[6-phenyl-4-(6-phenylpyridin-3-yl)-2-thiolidene-1,2,3,4-tetrahydropyrimidin-5-yl]ethan-1-one (D23): 1 H NMR (400MHz, DMSO-d6) δ10.52(s,1H),9.80(d,J=2.2Hz,1H),7.69(dd,J=12.3,7.8Hz,4H) ,7.54–7.24(m,10H),5.33(d,J=4.0Hz,1H),3.77(q,J=7.1Hz,2H),0.75(t,J=7.1Hz,3H).

[0046] 1-{4-[2-(4-chlorophenyl)phenyl]-6-phenyl-2-thiolidene-1,2,3,4-tetrahydropyrimidin-5-yl}ethan-1-one (D32): 1 H NMR (400MHz, DMSO-d6) δ10.23(s,1H),9.58(s,1H),7.58(d,J=7.8Hz,1H),7.48(dt,J= 15.5,7.4Hz,2H),7.42–7.26(m,9H),7.05(d,J=7.7Hz,1H),5.46(s,1H),1.65(s,3H).

[0047] 1-{4-[2-(4-methylphenyl)phenyl]-6-phenyl-2-thiolidene-1,2,3,4-tetrahydropyrimidin-5-yl}ethan-1-one (D33): 1 H NMR (400MHz, DMSO-d6) δ10.19(s,1H),9.50(s,1H),7.57(d,J=7.6Hz,1H),7.49(t,J=7.3Hz,1H),7.40(d,J=8.0Hz,3H),7.3 4(t,J=7.6Hz,2H),7.27(t,J=7.4Hz,1H),7.16(s,2H),7.05(dd,J=16.6,7.9Hz,3H),5.52(s,1H),2.30(s,3H),1.65(s,3H).

[0048] 1-{4-[2-(4-methoxyphenyl)phenyl]-6-phenyl-2-thiolidene-1,2,3,4-tetrahydropyrimidin-5-yl}ethan-1-one (D34): 1 H NMR (400MHz, DMSO-d6) δ10.18(s,1H),9.49(s,1H),7.56(d,J=7.8Hz,1H),7.49(t,J=7.2Hz,1H),7.41(d,J=8.2Hz,3H),7.3 5(t,J=7.7Hz,2H),7.29–7.15(m,3H),7.02(d,J=6.4Hz,1H),6.82(d,J=8.9Hz,2H),5.52(s,1H),3.77(s,3H),1.65(s,3H).

[0049] 1-[4-(4-phenylphenyl)-6-(pyridin-3-yl)-2-thiolidene-1,2,3,4-tetrahydropyrimidin-5-yl]ethan-1-one (D37): 1 H NMR (400MHz, DMSO-d6) δ10.50(s,1H),9.83(s,1H),8.79–8.62(m,2H),7.91(dt,J=7.8,1.8Hz,1H),7.64(d,J= 8.5Hz,4H),7.52–7.41(m,3H),7.34(dd,J=17.2,7.9Hz,3H),5.33(d,J=3.7Hz,1H),1.81(s,3H),1.22(s,1H).

[0050] 1-[4-(2-phenylphenyl)-6-(pyridin-3-yl)-2-thiolidene-1,2,3,4-tetrahydropyrimidin-5-yl]ethan-1-one (D38): 1 H NMR (400MHz, DMSO-d6) δ10.28(s,1H),9.60(s,1H),8.62(d,J=5.1Hz,1H),8.51(s,1H),7.74(d,J=7.9Hz,1H),7.57(d ,J=7.8Hz,1H),7.43(t,J=7.6Hz,1H),7.31(dt,J=14.7,7.5Hz,7H),7.05(d,J=7.6Hz,1H),5.49(s,1H),1.71(s,3H).

[0051] 2) Synthesis of 6-phenyl-4-(2-phenylphenyl)-2-thiolidene-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (D39)

[0052] 6-phenyl-4-(2-phenylphenyl)-2-sulfylidene-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid ethyl ester (D29, 1 mmol, 415 mg) was dissolved in 10 mL of methanol, potassium hydroxide (5 eq, 281 mg) was dissolved in 2 mL of purified water and slowly added to the reaction system, heated to 70°C and stirred for 24 hours. After the reaction was completed, the methanol was removed by concentration under reduced pressure, the pH was adjusted to about 2 with 1 M dilute hydrochloric acid, dichloromethane (20 mL×2) was added for extraction, the organic phases were combined and dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography to obtain 38 mg of a light yellow solid, namely 6-phenyl-4-(2-phenylphenyl)-2-sulfylidene-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (D39), with a yield of 60.8%. 1 H NMR(400MHz,DMSO-d6)δ10.13(s,1H),9.41(s,1H),7.61(dd,J=24.3,7.4Hz,3 H),7.51–7.33(m,8H),7.32–7.27(m,2H),7.18(d,J=7.6Hz,1H),5.44(s,1H).

[0053] Using compound D22 as raw material, compound D25 can also be synthesized with a lower yield (15.7%) according to the above method.

[0054] 6-phenyl-4-(4-phenylphenyl)-2-thiolidene-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (D25): 1 H NMR (400MHz, DMSO-d6) δ10.37(s,1H),9.75(d,J=2.3Hz,1H),7.74–7.64(m,4H),7.47(t,J=8.5Hz,4H),7.43–7.32(m,6H),5.35(d,J=3.9Hz,1H).

[0055] 3) Synthesis of 6-phenyl-4-(2-phenylphenyl)-2-thiolidene-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid propan-2-yl ester (D35)

[0056] 6-phenyl-4-(2-phenylphenyl)-2-sulfylidene-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (D39, 50 mg, 0.13 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 1.5 eq, 38 mg) and 4-dimethylaminopyridine (DMAP, 0.2 eq, 4 mg) were dissolved in 5 mL of anhydrous dichloromethane, and isopropanol (2 eq, 20 μL) was added and stirred at room temperature for 8 hours. After the reaction was completed, the mixture was washed with water (5 mL×2), the organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 46 mg of a white solid, namely 6-phenyl-4-(2-phenylphenyl)-2-sulfylidene-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid propan-2-yl ester (D35), with a yield of 82.6%. 1 H NMR (400MHz, DMSO-d6) δ10.31(s,1H),9.49(s,1H),7.66–7.56(m,3H),7.50–7.34(m,8H),7.27(d,J=5.9H z,2H),7.19(d,J=7.6Hz,1H),5.47(d,J=2.8Hz,1H),4.40(p,J=6.2Hz,1H),0.58(dd,J=11.7,6.3Hz,6H). 13 C NMR(101MHz,DMSO-d6)δ174.36,164.75,144.23,141.21,141.08,140.63,134.80,130.75,130.28,12 9.27,128.91,128.81,128.48,128.42,128.35,128.18,127.44,104.19,67.02,52.36,21.27,21.22.

[0057] Using compound D25 or D39 and the corresponding alcohol as raw materials, compounds D27, D28, D36, D40, and D41 can also be synthesized in high yield according to the above method.

[0058] 6-phenyl-4-(4-phenylphenyl)-2-thiolidene-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid propan-2-yl ester (D27): 1 HNMR(400MHz,DMSO-d6)δ10.49(s,1H),9.79(s,1H),7.69(dd,J=12.4,7.6H z,4H),7.51–7.29(m,10H),5.75(s,1H),5.33(s,1H),0.79(t,J=6.2Hz,6H).

[0059] 6-phenyl-4-(4-phenylphenyl)-2-thiolidene-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid-1,1,1,3,3,3-hexafluoroprop-2-yl ester (D28): 1 H NMR (400MHz, DMSO-d6) δ10.99(s,1H),10.08(s,1H),7.74(d,J=8.3Hz,2H),7.68(d,J=7 .2Hz,2H),7.46(q,J=7.7Hz,7H),7.40–7.34(m,3H),5.75(s,1H),5.35(d,J=4.0Hz,1H).

[0060] 6-phenyl-4-(2-phenylphenyl)-2-thiolidene-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid-1,1,1,3,3,3-hexafluoroprop-2-yl ester (D36): 1 H NMR (400MHz, DMSO-d6) δ10.74(s,1H),9.77(s,1H),7.60(dd,J=13.2,7.3Hz,3H),7.52– 7.36(m,8H),7.24(dd,J=26.5,6.5Hz,3H),6.30(p,J=6.4Hz,1H),5.50(d,J=3.1Hz,1H).

[0061] Example 2 Synthesis of Compound D26

[0062] This embodiment relates to the synthesis of DHPMs compounds containing a pyrimidine ring. Compound S1 is synthesized into compound S2 through bromination reaction, compound S2 and compound S3 are synthesized into compound S4 through Suzuki coupling reaction, compound S4 is synthesized into an alcohol intermediate through reduction reaction and then oxidized to synthesize pyrimidine aldehyde compound S5, and compound S5, thiourea B1 and benzoylacetone C1 are synthesized into compound D26 under the catalysis of cerium chloride heptahydrate.

[0063]

[0064] 1) Synthesis of compound S2

[0065] Compound S2 was synthesized according to the method reported in the literature (Nature 2018, 561 (7722), 189–194.). Under light-shielding conditions, ethyl 2-amino-4-methylpyrimidine-5-carboxylate (S1, 10 mmol, 1812 mg) was dissolved in 30 mL of bromoform, 20 mL of isoamyl nitrite was added, and the temperature was raised to 85 °C and stirred for 4 hours. After the reaction was completed, the system was cooled and concentrated under reduced pressure to remove the solvent. The residue was dissolved in 50 mL of ethyl acetate and washed with saturated brine (50 mL × 2). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 1507 mg of white solid, which was compound S2 with a yield of 61.5%. 1 HNMR (400MHz, CDCl3) δ8.93 (s, 1H), 4.41 (q, J = 7.6Hz, 2H), 2.82 (d, J = 1.1Hz, 3H), 1.41 (t, J = 7.2Hz, 3H).

[0066] 2) Synthesis of compound S4

[0067] Compound S2 was synthesized according to the method reported in the literature (Nature 2018, 561 (7722), 189–194.). Compound S2 (5 mmol, 1226 mg), potassium carbonate (3 eq, 2073 mg), and 4-tert-butylphenylboronic acid (S3, 1.3 eq, 1158 mg) were dissolved in 15 mL of toluene, 3 mL of water was added, and [1,1'-bis(diphenylphosphino)ferrocene] palladium dichloride (0.1 eq, 365 mg) was added under nitrogen protection, and the temperature was raised to reflux for 12 hours. After the reaction was completed, the system was cooled and concentrated under reduced pressure. The residue was dissolved in 80 mL of ethyl acetate, washed with 80 mL of water and then with the same volume of saturated brine. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 1071 mg of colorless oil, which was compound S4, with a yield of 71.8%. 1 H NMR (400MHz, CD3OD) δ9.09 (s, 1H), 8.36 (d, J = 8.5Hz, 2H), 7.52 (d, J = 8.6Hz, 2H), 4. 86(s,1H),4.38(q,J=7.1Hz,2H),2.81(s,3H),1.40(t,J=7.1Hz,3H),1.36(s,9H).

[0068] Using different brominated aromatic aldehydes and corresponding substituted phenylboronic acids as raw materials, the raw materials S-D23, S-D32, S-D33, and S-D34 of compounds D23, D32, D33, and D34 can also be synthesized in high yield according to the above method.

[0069] 2-(4-Methylphenyl)benzene-1-carboxaldehyde (S-D33): 1 H NMR (400MHz, CDCl3) δ10.00 (s, 1H), 8.02 (d, J = 8.6Hz, 1H), 7.67–7.59 (m, 1H), 7.53–7.40 (m, 2H), 7.28 (s, 4H), 2.44 (s, 3H).

[0070] 2-(4-Methoxyphenyl)benzene-1-carboxaldehyde (S-D34): 1 H NMR (400MHz, CDCl3) δ10.00 (s, 1H), 8.01 (d, J = 7.7Hz, 1H), 7.62 (t, J = 7.5Hz, 1H), 7.45(q,J=7.6Hz,2H),7.31(d,J=8.8Hz,2H),7.01(d,J=8.8Hz,2H),3.87(s,3H).

[0071] 3) Synthesis of compound S5

[0072] Compound S4 (3mmoL, 895mg) was dissolved in 10mL of anhydrous tetrahydrofuran, calcium chloride (2eq, 666mg) was dissolved in 10mL of ethanol and added to the reaction system, sodium borohydride (4eq, 453mg) was slowly added under ice bath, and the mixture was stirred at room temperature for 8 hours. After the reaction, 20mL of water was added to quench the reaction, and the organic solvent was removed by concentration under reduced pressure. The residue was extracted with ethyl acetate (20mL×3), and the organic phases were combined and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude alcohol intermediate.

[0073] The obtained alcohol intermediate crude product was dissolved in 20 mL of anhydrous dichloromethane, 2,2,6,6-tetramethylpiperidinoxide (0.03 eq, 15 mg) and potassium bromide (0.1 eq, 36 mg) were added, and then 7.5% sodium hypochlorite solution (1.1 eq, 3.3 mL) was added, and the reaction was stirred at room temperature for 12 hours. After the reaction was completed, it was washed with 20 mL of water, the organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 474 mg of white solid, which was compound S5, with a yield of 62.1%. 1 H NMR (400MHz, CDCl3) δ10.26(s,1H),9.04(s,1H),8.47(d,J=8.5Hz,2H),7.54(d,J=8.5Hz,2H),2.90(s,3H),1.37(s,9H).

[0074] 4) Synthesis of Compound D26

[0075] Compound S5 (1 mmoL, 254 mg), thiourea (B1, 1.2 eq, 92 mg) and benzoyl acetone (C1, 1.1 eq, 179 mg) were dissolved in 10 mL of anhydrous ethanol, and 7-hydrate and cerium chloride (0.25 eq, 94 mg) were added, and the mixture was heated and refluxed for 12 hours. After the reaction was completed, the reaction system was cooled and concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain 242 mg of a white solid, namely 1-(4-{4-methyl-2-[4-(2-methylpropane-2-yl)phenyl]pyrimidin-5-yl}-6-phenyl-2-sulfylidene-1,2,3,4-tetrahydropyrimidin-5-yl)ethan-1-one (D26), with a yield of 52.9%. 1 H NMR (400MHz, DMSO-d6) δ10.48(s,1H),9.65(s,1H),8.61(s,1H),8.28(d,J=8.3Hz,2H), 7.52(s,5H),7.44(d,J=7.6Hz,2H),5.63(s,1H),2.59(s,3H),1.76(s,3H),1.31(s,9H). 13 C NMR(101MHz,DMSO-d6)δ194.71,174.41,164.82,162.30,156.96,154.01,143.67,140.66,134.6 1,133.00,132.46,129.17,128.41,127.99,125.94,108.98,51.17,35.04,31.45,22.16,18.87.

[0076] Using N,N-diethyl-4-aminobenzaldehyde, (thio)urea and benzoylacetone as raw materials, compounds D10 and D24 can also be synthesized in moderate yields according to the above method.

[0077] 1-{4-[4-(diethylamino)phenyl]-6-phenyl-2-thiolidene-1,2,3,4-tetrahydropyrimidin-5-yl}ethan-1-one (D10): 1 H NMR (400MHz, DMSO-d6) δ10.20(s,1H),9.50(s,1H),7.67–7.32(m,5H),6.94(d,J=8.8Hz,2H),6.57 (d,J=8.9Hz,2H),5.14(d,J=3.6Hz,1H),3.27(q,J=7.0Hz,4H),1.75(s,3H),1.03(t,J=7.0Hz,6H).

[0078] Example 3 Synthesis of Compound D38

[0079] This example involves the synthesis of DHPMs compounds wherein R2 is pyridyl. 3-Acetylpyridine and ethyl acetate are reacted via Claisen condensation to synthesize compound C5, and compound C5 is reacted with biphenyl-2-carboxaldehyde and thiourea via Biginelli reaction to synthesize compound D38.

[0080]

[0081] 1) Synthesis of compound C5

[0082] Compound C5 was synthesized according to the method reported in the literature (Bioorganic & Medicinal Chemistry 46 (2021) 116344). 3-Acetylpyridine (5mmol, 606mg) was dissolved in 20mL of anhydrous tetrahydrofuran, 60% sodium hydride (4eq, 800mg) was slowly added under an ice bath, and ethyl acetate (2.5eq, 1.3mL) was slowly added after 30 minutes, and the reaction was stirred at room temperature for 12 hours. After the reaction was completed, 20mL of saturated ammonium chloride solution was added to quench the reaction, and the mixture was extracted with ethyl acetate (40mL×2). The organic layers were combined and dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was chromatographed on a silica gel column to obtain 377mg of a light yellow solid, which was compound C5 with a yield of 46.2%. 1 H NMR (400MHz, CDCl3) δ9.04(d,J=1.5Hz,1H),8.71(dd,J=4.8,1.8Hz,1H),8.19–8.10(m,1H),7.42–7.33(m,1H),6.17(s,1H),2.20(s,3H).

[0083] 2) Synthesis of compound D38

[0084] Dissolve biphenyl-2-carboxaldehyde (A12, 1 mmoL, 183 mg), thiourea (B1, 1.2 eq, 92 mg) and compound (C5, 1.1 eq, 180 mg) in 5 mL of glacial acetic acid and heat to reflux for 12 hours. After the reaction, the reaction system was cooled and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 250 mg of a white solid, namely 1-[4-(2-phenylphenyl)-6-(pyridin-3-yl)-2-sulfylidene-1,2,3,4-tetrahydropyrimidin-5-yl]ethan-1-one (D38), with a yield of 64.9%. 1H NMR (400MHz, DMSO-d6) δ10.28(s,1H),9.60(s,1H),8.62(d,J=5.1Hz,1H),8.51(s,1H),7.74(d,J=7.9Hz,1H),7.57(d ,J=7.8Hz,1H),7.43(t,J=7.6Hz,1H),7.31(dt,J=14.7,7.5Hz,7H),7.05(d,J=7.6Hz,1H),5.49(s,1H),1.71(s,3H). 13 C NMR(101MHz,DMSO-d6)δ192.93,174.22,152.82,149.14,141.24,140.84,140.73,140.24,136.00 ,135.27,130.42,129.87,128.82,128.69,128.26,127.32,124.16,111.17,55.38,53.17,18.22.

[0085] Example 4 Study on the inhibitory effect of compounds on porcine epidemic diarrhea virus replication

[0086] Determination of the inhibitory activity of the compounds of the present invention on porcine epidemic diarrhea virus replication: Vero cells were stored in our laboratory, and the porcine epidemic diarrhea virus strain (CV777-EGFP) carrying an enhanced green fluorescent protein (EGFP) label was provided by Li Zhen, a researcher at the Shanghai Academy of Agricultural Sciences. Vero cells were cultured at a density of 1×10 5 Cells / well were cultured overnight in a 24-well cell culture dish, and then the virus (infection multiplicity MOI was 0.1) was added to infect for 1 hour, and then the virus solution was discarded, and the cells were continued to be cultured with different concentrations of the compound of the present invention. After 24 hours, the supernatant was discarded, the cells were collected and transferred to a centrifuge tube, and the fluorescence rate was detected by flow cytometry analysis, and the virus inhibition rate and EC were calculated. 50 .

[0087] The results showed that at a concentration of 10 μM, multiple compounds significantly inhibited CV777-EGFP virus replication. Compared with the control group without drug treatment, the inhibition rate of PEDV replication was >99%. The EC 50 The value is at the nanomolar level, and its antiviral activity is significantly better than the control compound remdesivir.

[0088] For compounds that can significantly inhibit PEDV replication at low concentrations, their CC 50 , and calculated the therapeutic index (SI).

[0089] EC of some compounds 50 and CC50 The data are listed in Table 1.

[0090] Table 1. Inhibitory activity against porcine epidemic diarrhea virus (CV777-EGFP) replication

[0091]

Claims

1. A dihydropyrimidinethione or a pharmaceutically acceptable salt thereof of the general formula I: in: X=S or O; R1 is selected from hydrogen, or 4-halogen, phenyl, heteroaryl, N,N-diC 1-4 alkyl, or phenyl or heteroaryl at position 3, or phenyl or heteroaryl at position 2, wherein the phenyl may be substituted with one or more substituents, such as halogen, methyl, trifluoromethyl, methoxy, trifluoromethoxy, etc.; R2 is substituted or unsubstituted phenyl or heteroaryl; R3 is selected from C 1-4 Alkyl or alkoxy or C 3-5 Cycloalkyl or cycloalkoxy, the C 1-4 Alkyl or alkoxy, C 3-5 The cycloalkyl or cycloalkoxy group may be substituted by one or more fluorine atoms; the heteroaryl group refers to a phenyl group in which one, two or three carbon atoms are substituted by sulfur or nitrogen atoms, and particularly refers to a phenyl group in which one or two carbon atoms are substituted by sulfur or nitrogen atoms.

2. The dihydropyrimidinethione according to claim 1, wherein in the general formula I: X = S or O; R1 is selected from hydrogen, or 4-position chlorine, fluorine, phenyl, N,N-diC 1-4 alkyl, or phenyl at position 3, or phenyl at position 2, the phenyl group may be further substituted by one or more substituents, such as chlorine, methyl, trifluoromethyl, methoxy, trifluoromethoxy, etc.; R2 is substituted or unsubstituted phenyl; R3 is selected from C 1-4 Straight or branched alkyl or alkoxy or C 3-5 Cycloalkyl or cycloalkoxy, the C 1-4 Straight or branched alkyl or alkoxy, C 3-5 The cycloalkyl group or cycloalkoxy group may be substituted with one or more fluorine atoms.

3. The dihydropyrimidinethione according to claim 1 or 2, wherein in the general formula I: X = S; R1 is selected from phenyl at the 4-position, 3-position or 2-position, and the phenyl group may be substituted by any substituent selected from chlorine, methyl, trifluoromethyl, methoxy, trifluoromethoxy; R2 is phenyl; R3 is selected from C 1-4 Straight or branched alkyl or alkoxy or C 3-5 Cycloalkyl or cycloalkoxy, the C 1-3 Straight or branched alkyl or alkoxy, C 3-4 The cycloalkyl group or cycloalkoxy group may be substituted with one or more fluorine atoms.

4. The dihydropyrimidinethione according to claim 3, wherein in the general formula I: X=S; R1 is selected from phenyl at position 2, and the phenyl may be substituted at the para position by any substituent selected from chlorine, methyl, trifluoromethyl, methoxy, and trifluoromethoxy; R2 is phenyl; R3 is selected from methyl, ethyl, isopropyl, cyclopropyl, cyclobutyl, or methoxy, ethoxy, isopropoxy, cyclopropyloxy, and cyclobutyloxy, and the hydrogen atom on R3 may be substituted by one or more fluorine atoms.

5. The dihydropyrimidinethione according to claim 4, wherein in the general formula I: X=S; R1 is selected from phenyl at position 2, and the phenyl at position 4 may be substituted by any substituent selected from chlorine, methyl, and methoxy; R2 is phenyl; R3 is selected from methyl, ethyl, isopropyl, methoxy, ethoxy, cyclopropyloxy, and cyclobutyloxy, and the hydrogen atom on R3 may be replaced by one or more fluorine atoms to form, for example, trifluoromethyl, trifluoromethylmethylene, pentafluoroethyl, and hexafluoroisopropoxy.

6. Any of the following dihydropyrimidinethione compounds:

7. The dihydropyrimidinethione compound according to claim 6, wherein the dihydropyrimidinethione compound is selected from: D12, D29, D32, D33, D35, D36, D40 or D41; particularly preferably D35 or D40.

8. Compounds of formula F: Wherein X, R1, and R2 are defined as the compound of general formula I in claim 1.

9. The use of the dihydropyrimidinethione compound according to any one of claims 1 to 7, characterized in that: Used to inhibit the growth of porcine epidemic diarrhea virus and / or reduce the titer of porcine epidemic diarrhea virus.

10. A veterinary drug comprising the dihydropyrimidinethione compound according to any one of claims 1 to 7.

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