Application of piperazinone and hydroxypyridone compounds in the preparation of drugs for treating herpes simplex virus

Abstract

The present invention discloses the use of piperazinone hydroxypyridone compounds in the preparation of anti-herpes simplex virus drugs. Experiments have shown that the piperazinone hydroxypyridone compounds of the present invention can effectively inhibit the replication of herpes simplex virus in Vero cell lines, particularly having an inhibitory effect on acyclovir-resistant herpes simplex virus. The present invention also discloses the antiviral mechanism of the piperazinone hydroxypyridone compounds, which is different from the clinical first-line drug acyclovir. The general structural formula of the piperazinone hydroxypyridone compounds is shown as (I):

Description

Technical Field

[0001] The present invention belongs to the field of medicinal chemistry, and in particular relates to the application of piperazinone and hydroxypyridone compounds in the preparation of anti-herpes simplex virus drugs. Background Art

[0002] Herpes simplex virus (HSV) is an enveloped, linear, double-stranded DNA virus. HSV is a common pathogen that causes skin diseases in humans, and humans are the sole natural reservoir for HSV infection. HSV is divided into type I (HSV-1) and type II (HSV-2) based on serotype. HSV primarily infects the skin and mucous membranes of the face and genitals, causing diseases such as herpes labialis, oral mucositis, keratitis, genital herpes, and even herpes encephalitis. Currently, only a few approved drugs are available for the general treatment of herpes simplex virus infections, primarily nucleoside analogs, including acyclovir (ACV) and its derivatives. While these nucleoside analogs can specifically block viral DNA replication, they also suffer from limited structural types, low bioavailability, and short half-lives. Furthermore, HSV has developed resistance to current therapies. Therefore, the search for anti-HSV compounds that act on novel targets is crucial. Summary of the Invention

[0003] The object of the present invention is to provide a piperazinone and hydroxypyridone compound, which is a small molecule compound that is resistant to herpes simplex virus, and the general formula is shown as (I):

[0004]

[0005] Wherein R1 is an aromatic group or an aliphatic hydrocarbon group, specifically:

[0006] The aryl group is selected from naphthyl, phenyl, substituted phenyl, benzyl, aryl-substituted benzyl, pyridin-3-ylmethyl, (pyridine-1-oxide)-2-ylmethyl; the substituted phenyl group is selected from o-, m-, and p-substituted phenyl groups; the substituents on the substituted phenyl group are selected from halogen, trihalogen-substituted methyl, and 4-acetylpiperazinyl, and the halogen is selected from fluorine, chlorine, and bromine; the aryl substituents of the aryl-substituted benzyl group are selected from halogen, and the halogen is selected from fluorine, chlorine, and bromine.

[0007] The aliphatic hydrocarbon group is selected from methyl, ethyl, propyl, butyl, methoxycarbonylmethyl, 2-morpholinylethyl, 2,2-dimethoxyethyl, 2-[(4-methylphenyl)sulfonamide]ethyl, 4-[(tert-butoxycarbonyl)amino]butyl, and cyclopropylmethyl.

[0008] R2 is selected from tert-butyl, cyclohexyl, benzyl, naphthyl, 2,6-dimethylphenyl, 4-methylphenylsulfonylmethyl.

[0009] The piperazinone and hydroxypyridone compound is selected from any one of the following compounds:

[0010] 2-n-Butyl-3-tert-butylaminocarbonyl-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid

[0011] 2-Methoxycarbonylmethyl-3-tert-butylaminocarbonyl-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid

[0012] 2-(2-Morpholinylethyl)-3-cyclohexylaminocarbonyl-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid

[0013] 2-(2,2-Dimethoxyethyl)-3-cyclohexylaminocarbonyl-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid

[0014] 2-{2-[(4-methylphenyl)sulfonamido]ethyl}-3-cyclohexylaminocarbonyl-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid

[0015] 2-{4-[(tert-Butyloxycarbonyl)amino]butyl}-3-cyclohexylaminocarbonyl-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid

[0016] 2-Benzyl-3-benzylaminocarbonyl-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid

[0017] 2-Benzyl-3-(naphthalen-2-ylaminocarbonyl)-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid

[0018] 2-Benzyl-3-((2,6-dimethylphenyl)aminocarbonyl)-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid

[0019] 2-(2,6-dimethylphenyl)aminocarbonyl-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid

[0020] 2-(2-morpholinoethyl)-3-((2,6-dimethylphenyl)aminocarbonyl)-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid

[0021] 2-(2,4-Difluorobenzyl)-3-((2,6-dimethylphenyl)aminocarbonyl)-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid

[0022] 2-(Naphth-1-yl)-3-((2,6-dimethylphenyl)aminocarbonyl)-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid

[0023] 2-(2-Fluoro-3-chlorophenyl)-3-((2,6-dimethylphenyl)aminocarbonyl)-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid

[0024] 2-(3,5-difluorophenyl)-3-((2,6-dimethylphenyl)aminocarbonyl)-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid

[0025] 2-(Pyridin-3-ylmethyl)-3-((2,6-dimethylphenyl)aminocarbonyl)-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid

[0026] 2-(3,5-Bistrifluoromethylphenyl)-3-((2,6-dimethylphenyl)aminocarbonyl)-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid

[0027] 2-(4-(4-acetylpiperazin-1-yl)phenyl)-3-((2,6-dimethylphenyl)aminocarbonyl)-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid

[0028] 2-((Pyridine-1-oxide)-2-ylmethyl)-3-((2,6-dimethylphenyl)aminocarbonyl)-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid

[0029] 2-(2,4-Difluorophenyl)-3-((2,6-dimethylphenyl)aminocarbonyl)-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid

[0030] 2-(2,2-dimethoxyethyl)-3-((2,6-dimethylphenyl)aminocarbonyl)-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid

[0031] 2-(Cyclopropylmethyl)-3-((4-methylphenylsulfonylmethyl)aminocarbonyl)-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid.

[0032] Another object of the present invention is to provide the use of the piperazinone and hydroxypyridone compounds in the preparation of anti-herpes simplex virus drugs.

[0033] The present invention discloses the application of general formula I in inhibiting herpes simplex virus replication in Vero cells

[0034] The present invention discloses the inhibitory effect of a compound of general formula I on acyclovir-resistant herpes simplex virus.

[0035] The invention discloses the mechanism of action of the compound of general formula I against herpes simplex virus.

[0036] Compared with the prior art, the present invention has the following advantages:

[0037] The piperazino-hydroxypyridone compounds described herein can effectively inhibit the replication of herpes simplex virus in Vero cell lines, with inhibitory activity comparable to that of ACV. In particular, the compounds described herein have a strong inhibitory effect against acyclovir-resistant herpes simplex virus. The present invention also discloses the antiviral mechanism of the piperazino-hydroxypyridone compounds, which differs from acyclovir, a first-line clinical drug. The novel target-based anti-herpes simplex virus compounds disclosed herein provide more options for the treatment of herpes simplex virus infections and have promising application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is the toxicity of piperazinone and hydroxypyridone compounds and acyclovir to the test cells.

[0039] Figure 2 The experiments of Examples 9 and 12 were respectively incubated with HSV-1 virus.

[0040] Figure 3 The adsorption experiments of Examples 9 and 12 on HSV-1 virus are shown.

[0041] Figure 4 Examples 9 and 12 are HSV-1 virus entry experiments.

[0042] Figure 5 The effects of Examples 9 and 12 on HSV-1 viral gene expression.

[0043] Figure 6 The effects of Examples 9 and 12 on HSV-1 viral protein translation are shown. DETAILED DESCRIPTION

[0044] The present invention will be further described in detail below with reference to the embodiments, but the embodiments of the present invention are not limited thereto. Without departing from the above-mentioned technical concept of the present invention, various substitutions and changes can be made according to common technical knowledge and customary means in the field, and all of these should be included within the scope of the present invention.

[0045] Example 1. 2-n-Butyl-3-tert-butylaminocarbonyl-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid (1)

[0046] 1-(2,2-dihydroxyethyl)-3-hydroxy-4-oxo-1,4-dihydropyridine-2,5-dicarboxylic acid (259 mg, 1 mmol) and n-butylamine (99 μL, 1 mmol) were added to a reaction flask, followed by 5 mL of methanol. The mixture was stirred at room temperature for 30 minutes. Tert-butyl isocyanide (105 μL, 1 mmol) was then added to the reaction flask and allowed to react at room temperature for 2 hours. After completion of the reaction as monitored by TLC, the crude white product was filtered. The crude product was slurried once with methanol, filtered, and dried to obtain the product in a yield of 68%.

[0047] White solid, mp>250℃. 1 H NMR (500MHz, DMSO-d6) δ = 15.51 (s, 1H), 12.68 (s, 1H), 8.66 (s, 1H), 8.14 (s, 1H), 4.76 (dd, J = 14.0, 1.5Hz, 1H), 4.63 (dd, J = 14.0, 4.5Hz, 1H), 4.48(dd,J=4.5,2.0Hz,1H),3.59-3.53(m,1H),3.33-3.27(m,1H),1.56-1.42(m,2H),1.34-1.27(m,2H),1.21(s,9H),0.90(t,J=7.5Hz,3H). 13C NMR (125MHz, DMSO-d6)δ=172.2,166.1,165.6,163.3,152.4,140.4,119.9,112.7,56.5,52.6,50.8,46.3,28.8,28.0,19.5,13.7.HRMS(ESI):m / zcalcd for C 18 H 26 N3O6 + [M+H] + :380.1816,found 380.1815.

[0048] Example 2. 2-methoxycarbonylmethyl-3-tert-butylaminocarbonyl-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid (2)

[0049] Glycine methyl ester was used instead of n-butylamine. Other steps were the same as in Example 1. The yield was 59%.

[0050] White solid, mp>250℃. 1 H NMR (500MHz, DMSO-d6) δ = 15.35 (s, 1H), 12.00 (s, 1H), 8.71 (s, 1H), 8.08 (s, 1H), 4.84 ( d,J=13.0Hz,1H),4.62-4.58(m,3H),4.08(d,J=17.0Hz,1H),3.70(s,3H),1.19(s,9H). 13 C NMR (125MHz, DMSO-d6)δ=172.1,168.4,165.5,165.4,163.1,152.4,141.0,119.4,112.7,57.6,53.1,52.4,50.8,47.6,28.1.HRMS(ESI):m / zcalcd for C 17 H 22 N3O8 + [M+H] + :396.1401,found 396.1408.

[0051] Example 3. 2-(2-morpholinoethyl)-3-cyclohexylaminocarbonyl-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid (3)

[0052] 2-morpholinoethylamine was used instead of n-butylamine. Other steps were the same as in Example 1. The yield was 65%.

[0053] White solid, mp 160.4-161.3℃. 1 H NMR (500MHz, DMSO-d6) δ = 15.49 (s, 1H), 12.35 (s, 1H), 8.65 (s, 1H), 8.36 (d, J = 7.5Hz, 1H), 4.74 (dd, J = 13.5, 1.5Hz, 1H), 4.65-4.59 (m, 2H), 3.79-3. 73(m,1H),3.57(t,J=4.5Hz,4H),3.46-3.33(m,4H),2.47-2.42(m,1H),2. 40-2.33(m,3H),1.73-1.62(m,4H),1.54-1.50(m,1H),1.25-1.11(m,5H). 13 C NMR (125MHz, DMSO-d6)δ=172.2,165.8,165.6,163.2,152.4,140.5,119.8,112.7,66 .2,57.1,55.3,53.2,52.9,48.2,43.4,32.0,31.9,25.1,24.3,24.2.HRMS(ESI):m / z calcd for C 22 H 31 N4O7 + [M+H] + :463.2187,found463.2190.

[0054] Example 4. 2-(2,2-dimethoxyethyl)-3-cyclohexylaminocarbonyl-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid (4)

[0055] 2,2-dimethoxyethylamine was used instead of n-butylamine, cyclohexyl isocyanide was used instead of tert-butyl isocyanide, and the rest of the process was the same as in Example 1. The yield was 74%.

[0056] White solid, mp 142.4-143.3℃. 1H NMR (500MHz, DMSO-d6) δ=15.43(s,1H),12.38(s,1H),8.66(s,1H),8.29(d,J= 7.5Hz,1H),4.72(dd,J=15.0,4.0Hz,1H),4.61-4.57(m,2H),4.44(t,J=5.0Hz, 1H),3.68(dd,J=14.0,6.0Hz,1H),3.46-3.38(m,2H),3.35(s,3H),3.24(s,3H ),1.75-1.72(m,1H),1.68-1.62(m,3H),1.53-1.50(m,1H),1.28-1.09(m,5H). 13 CNMR(125MHz,DMSO-d6)δ=172.2,165.6,165.5,163.6,152.4,140.6,119.8,112.8,10 1.9,57.7,54.4,54.0,53.2,48.4,48.1,32.1,32.0,25.1,24.2,24.1.HRMS(ESI):m / z calcdfor C 20 H 28 N3O8 + [M+H] + :438.1871,found 438.1870.

[0057] Example 5. 2-{2-[(4-methylphenyl)sulfonamide]ethyl}-3-cyclohexylaminocarbonyl-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid (5)

[0058] N-(2-aminoethyl)-4-methylbenzenesulfonamide was used instead of n-butylamine, and cyclohexyl isocyanide was used instead of tert-butyl isocyanide. The rest of the reaction was the same as in Example 1. The yield was 69%.

[0059] White solid, mp>250℃. 1H NMR (500MHz, DMSO-d6) δ = 15.40 (s, 1H), 12.41 (s, 1H), 8.63 (s, 1H), 8.40 (d, J = 7.5Hz, 1H), 7.81 (t, J=6.0Hz,1H),7.68(d,J=8.0Hz,2H),7.40(d,J=8.5Hz,2H),4.74(dd,J=14.0,2.0Hz,1H),4.61(dd ,J=13.5,4.5Hz,1H),4.55(dd,J=4.5,2.0Hz,1H),3.82-3.76(m,1H),3.43-3.38(m,1H),3.14-3.0 9(m,1H),3.02-2.95(m,1H),2.92-2.86(m,1H),2.36(s,3H),1.66-1.50(m,5H),1.25-1.06(m,5H). 13 C NMR (125MHz, DMSO-d6)δ=172.2,165.6,165.5,163.6,152.5,142.9,140.7,137.3,129.8,126 .5,119.5,112.8,56.9,52.5,48.2,46.4,31.9,31.9,25.1,24.2,24.2,21.0.HRMS(ESI):m / z calcdfor C 25 H 31 N4O8S + [M+H] + :547.1857,found 547.1851.

[0060] Example 6. 2-{4-[(tert-Butoxycarbonyl)amino]butyl}-3-cyclohexylaminocarbonyl-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid (6)

[0061] Boc-1,4-butanediamine hydrochloride was used instead of n-butylamine, and cyclohexyl isocyanide was used instead of tert-butyl isocyanide. The rest of the process was the same as in Example 1, and the yield was 77%.

[0062] White solid, mp 150.1-150.9℃. 1H NMR (500MHz, DMSO-d6) δ = 15.49 (s, 1H), 12.58 (s, 1H), 8.62 (s, 1H), 8.38 (d, J = 7.5Hz, 1 H),6.83(t,J=5.5Hz,1H),4.75(d,J=13.5Hz,1H),4.64(dd,J=13.5,3.5Hz,1H),4.50(d ,J=2.5Hz,1H),3.62-3.56(m,1H),3.46-3.41(m,1H),3.29-3.23(m,1H),2.93(q,J=6. 5Hz,2H),1.74-1.63(m,4H),1.53-1.45(m,3H),1.41-1.36(m,10H),1.27-1.12(m,6H). 13 C NMR (125MHz, DMSO-d6)δ=172.2,166.0,165.6,163.2,155.6,152.4,140.5,119.8,112.7 ,77.4,56.3,52.4,48.2,46.4,31.9,28.3,26.7,25.1,24.5,24.2,24.1.HRMS(ESI):m / z calcd for C 25 H 37 N4O8 + [M+H] + :521.2606,found521.2603.

[0063] Example 7. 2-Benzyl-3-benzylaminocarbonyl-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid (7)

[0064] Benzylamine was used instead of n-butylamine, and benzyl isocyanide was used instead of tert-butyl isocyanide. The rest of the process was the same as in Example 1. The yield was 89%.

[0065] Off-white solid, mp 256.5-257.2℃. 1H NMR (500MHz, DMSO-d6) δ = 15.39 (s, 1H), 12.41 (s, 1H), 8.86 (t, J = 5.5Hz, 1H), 8.66 (s, 1H),7.40-7.32(m,5H),7.28-7.20(m,3H),7.09-7.07(m,2H),5.12(d,J=15.5Hz,1H), 4.86(dd,J=14.0,1.5Hz,1H), 4.68(dd,J=14.0,4.0Hz,1H), 4.59(dd,J=4.0,1.5Hz,1H ), 4.34(d,J=15.5Hz,1H), 4.25(dd,J=15.5,5.5Hz,1H), 4.19(dd,J=15.0,5.5Hz,1H). 13 C NMR (125MHz, DMSO-d6)δ=172.2,166.6,165.4,163.4,152.5,140.7,138.3,135.1,128.6,12 8.3,128.2,127.8,127.0,127.0,119.8,112.8,56.3,52.8,49.4,42.7.HRMS(ESI):m / zcalcd for C 24 H 22 N3O6 + [M+H] + :448.1503,found 448.1499.

[0066] Example 8. 2-Benzyl-3-(naphthalen-2-ylaminocarbonyl)-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid (8)

[0067] Benzylamine was used instead of n-butylamine, 2-isocyanonaphthalene was used instead of tert-butyl isocyanide, and the rest of the process was the same as in Example 1. The yield was 71%.

[0068] Off-white solid, mp>250℃. 1 H NMR (500MHz, DMSO-d6) δ=15.37(s,1H),12.54(s,1H),10.61(s,1H),8.65(s,1H),8.15(d,J=2.0Hz,1H),7.87-7.79(m,3H),7.48-7.40(m,5 H),7.39-7.35(m,2H),7.31-7.28(m,1H),5.16(d,J=15.0Hz,1H),5.04(dd,J=14.0,1.5Hz,1H),4.78-4.71(m,2H),4.47(d,J=15.0Hz,1H).13 C NMR (125MHz, DMSO-d6)δ=172.3,165.4,165.3,163.7,152.7,141.0,135.5,135.0,133.2,130.1,128.7,128 .5,128.3,127.9,127.5,127.4,126.6,125.0,119.8,119.7,115.9,112.8,56.6,52.2,49.1.HRMS(ESI):m / z calcd for C 27 H 22 N3O6 + [M+H] + :484.1503,found 484.1502.

[0069] Example 9. 2-Benzyl-3-((2,6-dimethylphenyl)aminocarbonyl)-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid (9)

[0070] Benzylamine replaced n-butylamine, and 2,6-dimethylphenyl isocyanide replaced tert-butyl isocyanide. The remaining conditions were the same as in Example 1. Yield: 70%. White solid, mp 265.3-266.1°C. 1 H NMR (500MHz, DMSO-d6)δ=15.36(s,1H),12.27(s,1H),9.81(s,1H),8.82(s,1H),7.45-7.38(m,4H),7.38-7.34(m,1H),7.07 -7.01(m,3H),5.18(d,J=15.0Hz,1H),5.02(dd,J=18.0,5.0Hz,1H),4.83-4.78(m,2H),4.39(d,J=15.0Hz,1H),1.95(s,6H). 13 C NMR (125MHz, DMSO-d6)δ=172.2,165.4,165.0,163.6,152.6,140.7,135.0,134.7,133.7,12 8.8,128.3,128.0,127.8,127.0,119.9,112.8,56.3,53.2,49.5,17.8.HRMS(ESI):m / zcalcd for C 25 H 24 N3O6 + [M+H] + :462.1660,found 462.1659.

[0071] Example 10. 2-(2,6-dimethylphenyl)aminocarbonyl)-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid (10)

[0072] m-Trifluoromethylaniline was used instead of n-butylamine, 2,6-dimethylphenyl isocyanide was used instead of tert-butyl isocyanide, and the rest of the process was the same as in Example 1. The yield was 75%.

[0073] White solid, mp>250℃. 1 H NMR(500MHz,DMSO-d6)15.26(s,1H),11.92(s,1H),10.02(s,1H),8.91(s,1H),7.87(s,1H), 7.85-7.78(m,3H),7.10-7.03(m,3H),5.27(t,J=3.0Hz,1H),5.17-5.10(m,2H),1.94(s,6H). 13 C NMR (125MHz, DMSO-d6)172.2,165.4,165.3,162.9,153.1,141.0,140.1,134.7,133.6,130.8,130.7,130.4(q,J=32.5Hz),127 .9,127.2,127.0(q,J=271.2Hz),125.1(q,J=8.7Hz),123.2(q,J=8.7Hz),119.9,112.8,60.1,53.3,17.7.HRMS(ESI):m / zcalcd for C25H21F3N3O6+[M+H]+:516.1377, found 516.1378.

[0074] Example 11. 2-(2-morpholinoethyl)-3-((2,6-dimethylphenyl)aminocarbonyl)-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid (11)

[0075] 2-morpholinoethylamine was used instead of n-butylamine, 2,6-dimethylphenyl isocyanide was used instead of tert-butyl isocyanide, and the rest was the same as in Example 1. The yield was 67%.

[0076] Off-white solid, mp>250℃. 1H NMR(500MHz,DMSO-d6)δ15.38(s,1H),12.32(s,1H),10.04(s,1H),8.80(s,1H),7.08–7.05( m,3H),5.06–5.00(m,3H),3.59(d,J=33.3Hz,8H),2.19(dd,J=37.1,5.0Hz,4H),2.00(s,6H). 13 C NMR(125MHz,DMSO-d6)δ167.8,166.8,165.4,160.6,159.6,157.2,148.9,147.1,14 3.2,140.8,134.8,128.0,66.0,62.1,58.3,57.5,55.0,53.1,17.9.HRMS(ESI):m / z calcd for C24H29N4O7+[M+H]+:485.2031,found485.2036.

[0077] Example 12. 2-(2,4-difluorobenzyl)-3-((2,6-dimethylphenyl)aminocarbonyl)-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid (12)

[0078] 2,4-difluorobenzylamine was used instead of n-butylamine, 2,6-dimethylphenyl isocyanide was used instead of tert-butyl isocyanide, and the rest of the process was the same as in Example 1. The yield was 85%.

[0079] White solid, mp 236.4-236.7℃. 1 H NMR(500MHz, Acetonitrile-d3)δ15.07(s,1H),12.39(s,1H),8.39(s,1H),8.29(s,1H),7.56–7.52(m,1H),7.09(dd,J=8.2,6.6Hz,1H),7.04(s,1H), 7.02(d,J=11.2Hz,3H),5.13(d,J=15.1Hz,1H),4.74(d,J=3.9Hz,1H),4.71 (s,1H),4.66(dd,J=13.9,3.8Hz,1H),4.51(d,J=15.1Hz,1H),1.99(s,6H). 13C NMR(125MHz, Acetonitrile-d3)δ174.0,166.5,165.9,165.2,155.0,141.4,136.3,134.3,133.9(dd,J=10Hz,6.3Hz),129.0 ,128.5,120.2,114.8,112.8(d,J=3.8Hz),112.6(d,J=3.8Hz),105.1,104.9,104.7,58.1,54.6,44.7,18.3.HRMS(ESI):m / z calcd forC25H22F2N3O6+[M+H]+:498.1471, found 498.1481.

[0080] Example 13. 2-(Naphth-1-yl)-3-((2,6-dimethylphenyl)aminocarbonyl)-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid (13)

[0081] 2-naphthylamine was used instead of n-butylamine, 2,6-dimethylphenyl isocyanide was used instead of tert-butyl isocyanide, and the rest of the process was the same as in Example 1. The yield was 70%.

[0082] Brown solid, mp>250℃. 1 H NMR (500MHz, DMSO-d6) δ15.35(s,1H),12.16(s,1H),9.86(s,1H),9.00(s,1H),8.25(d,J=7.9Hz,1H),8.13–8.08(m,2H),7.73–7.67(m,2H),7.6 2(d,J=7.8Hz,1H),7.44(d,J=7.2Hz,1H),7.07(q,J=5.6Hz,3H),5.43(dd,J=14.0,3.7Hz,1H),5.19(d,J=13.8Hz,1H),5.03(s,1H),2.00(s,6H). 13 C NMR (125MHz, DMSO-d6) δ172.3,165.8,165.4,164.0,152.8,140.9,135.7,134.7,134.3,129.5,128.9,128. 7,128.0,127.8,127.7,127.1,127.0,125.8,124.9,122.5,120.5,113.0,59.9,53.3,17.9.HRMS(ESI):m / z calcd for C28H24N3O6+[M+H]+:498.1660,found 498.1660.

[0083] Example 14. 2-(2-Fluoro-3-chlorophenyl)-3-((2,6-dimethylphenyl)aminocarbonyl)-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid (14)

[0084] 2-Fluoro-3-chloroaniline was used instead of n-butylamine, 2,6-dimethylphenyl isocyanide was used instead of tert-butyl isocyanide, and the rest of the process was the same as in Example 1. The yield was 75%.

[0085] White solid, mp>250℃. 1 H NMR (500MHz, DMSO-d6) δ15.23(s,1H),11.75(s,1H),9.99(s,1H),8.91(s,1H),7.75–7.71(m,1H),7.46(t,J=6 .6Hz,1H),7.41(t,J=8.0Hz,1H),7.09–7.03(m,3H),5.21(t,J=3.1Hz,1H),5.10(d,J=2.8Hz,2H),1.97(s,6H). 13 C NMR (125MHz, DMSO-d6) δ172.2,165.3,165.0,162.1,153.8,153.0,151.8,141.2,134.7,133.6,130.8,128.1(d,J=12.5H z),127.9,127.1(d,J=3.8Hz),125.8(d,J=3.8Hz),120.8(d,J=16.3Hz),119.8,112.7,59.5,53.4,17.8.HRMS(ESI):m / z calcd for C24H20ClFN3O6+[M+H]+:500.1019, found 500.1010.

[0086] Example 15. 2-(3,5-difluorophenyl)-3-((2,6-dimethylphenyl)aminocarbonyl)-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid (15)

[0087] 3,5-difluoroaniline was used instead of n-butylamine, 2,6-dimethylphenyl isocyanide was used instead of tert-butyl isocyanide, and the rest of the process was the same as in Example 1. The yield was 90%.

[0088] White solid, mp>250℃. 1H NMR (500MHz, DMSO-d6) δ15.22(s,1H),11.83(s,1H),10.01(s,1H),8.90(s,1H),7.41(ddd,J=9.3,7.0,2.2 Hz,1H),7.28(dd,J=8.0,2.1Hz,2H),7.09–7.04(m,3H),5.28–5.26(m,1H),5.14–5.07(m,2H),1.94(s,6H). 13 C NMR (125MHz, DMSO-d6) δ172.2,165.3,165.2,163.2(d,J=15Hz),162.4,161.3(d,J=15Hz),153.1,141.1,134.7,133 .6,128.0,127.2,119.9,112.7,110.1(dd,J=21.2Hz,7.5Hz),103.9(t,J=25Hz),,60.0,53.2,17.7.HRMS(ESI):m / z calcd for C24H20F2N3O6+[M+H]+:484.1315, found 484.1307.

[0089] Example 16. 2-(Pyridin-3-ylmethyl)-3-((2,6-dimethylphenyl)aminocarbonyl)-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid (16)

[0090] 3-aminomethylpyridine was used instead of n-butylamine, 2,6-dimethylphenyl isocyanide was used instead of tert-butyl isocyanide, and the rest of the process was the same as in Example 1. The yield was 85%.

[0091] Off-white solid, mp 215.3-215.7℃. 1 H NMR(500MHz,DMSO-d6)δ15.34(s,1H),12.26(s,1H),9.83(s,1H),8.82(s,1H),8.68 (d,J=1.7Hz,1H),8.56(dd,J=4.8,1.4Hz,1H),7.90(d,J=7.9Hz,1H),7.45(dd,J=7.8 ,4.9Hz,1H),7.02(t,J=7.6Hz,3H),5.05(d,J=15.3Hz,1H),5.00(d,J=13.0Hz,1H),4 .93–4.91(m,1H),4.87(dd,J=13.7,3.9Hz,1H),4.61(d,J=15.3Hz,1H),1.92(s,6H). 13C NMR(125MHz,DMSO-d6)δ172.2,165.3,165.0,163.6,152.5,149.1,148.7,140.8,136.7,134 .7,133.7,131.2,127.8,127.0,123.8,120.0,112.8,56.9,53.3,47.6,17.8.HRMS(ESI):m / z calcd for C24H23N4O6+[M+H]+:463.1612, found 463.1613.

[0092] Example 17. 2-(3,5-ditrifluoromethylphenyl)-3-((2,6-dimethylphenyl)aminocarbonyl)-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid (17)

[0093] 3,5-ditrifluoromethylaniline was used instead of n-butylamine, 2,6-dimethylphenyl isocyanide was used instead of tert-butyl isocyanide, and the rest of the process was the same as in Example 1. The yield was 90%.

[0094] White solid, mp>250℃. 1 H NMR(500MHz,DMSO-d6)δ15.17(s,1H),11.63(s,1H),10.06(s,1H),8.90(s,1H),8.26(s, 1H),8.23(s,2H),7.06(q,J=5.9Hz,3H),5.42(t,J=3.0Hz,1H),5.15(s,2H),1.91(s,6H). 13 C NMR(125MHz, DMSO-d6)δ172.2,165.2(d,J=10Hz),162.2,153.1,141.1(d,J=3.8Hz),134.7,133.6,131.4,131.1, 127.9,127.3(d,J=21.3Hz),124.0,121.8(m),119.8,112.7,99.5,60.0,53.3,45.6,17.50.HRMS(ESI):m / zcalcd for C26H20F6N3O6+[M+H]+:584.1251,found 584.1258.

[0095] Example 18. 2-(4-(4-acetylpiperazin-1-yl)phenyl)-3-((2,6-dimethylphenyl)aminocarbonyl)-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid (18)

[0096] 1-acetyl-4-(4-aminophenyl)piperazine was used instead of n-butylamine, 2,6-dimethylphenyl isocyanide was used instead of tert-butyl isocyanide, and the rest of the process was the same as in Example 1. The yield was 65%.

[0097] Yellow solid, mp>250℃. 1 H NMR (500MHz, DMSO-d6) δ15.39(s,1H),12.37(s,1H),9.93(s,1H),8.90(s,1H),7.29(d,J=9.0Hz,2H),7.08–7.06(m,3H),7.04(d,J=6.0Hz,2H), 5.12–5.08(m,1H),5.07(s,1H),5.05(dd,J=3.9,2.1Hz,1H),3.60–3.57 (m,4H),3.24–3.22(m,2H),3.18–3.15(m,2H),2.05(s,3H),1.96(s,6H). 13 C NMR (125MHz, DMSO-d6) δ172.2, 168.3, 165.5 (d, J = 18Hz), 165.4, 163.3, 152.9, 150.4, 140.8, 134.8, 133.7, 13 0.5,127.9,126.9,120.1,115.8,112.9,60.4,53.2,48.3,47.9,45.3,40.5,21.2,17.9.HRMS(ESI):m / zcalcd for C30H32N5O7+[M+H]+:574.2296, found 574.2295.

[0098] Example 19. 2-((Pyridine-1-oxide)-2-ylmethyl)-3-((2,6-dimethylphenyl)aminocarbonyl)-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid (19)

[0099] 2-aminomethylpyridine oxide was used instead of n-butylamine, 2,6-dimethylphenyl isocyanide was used instead of tert-butyl isocyanide, and the rest of the process was the same as in Example 1. The yield was 72%.

[0100] Off-white solid, mp>250℃. 1H NMR (500MHz, DMSO-d6) δ15.31(s,1H),12.10(s,1H),9.96(s,1H),8.83(s,1H),8.34(d,J=6.0Hz,1H),7.72–7.69(m,1H),7.41(p,J=7.3,6.5 Hz,2H),7.05(q,J=5.5Hz,3H),5.38(s,1H),5.06(dd,J=14.5,6.0Hz,2H),4.88(dd,J=13.8,3.6Hz,1H),4.55(d,J=15.6Hz,1H),2.01(s,6H). 13 C NMR(125MHz,DMSO-d6)δ172.1,165.4,165.3,163.8,152.4,145.0,140.9,139.2,134.8,133 .9,127.8,127.0,126.9,125.8,125.4,119.9,112.8,59.1,53.5,47.2,17.8.HRMS(ESI):m / z calcd forC24H23N4O7+[M+H]+:479.1561, found 479.1563.

[0101] Example 20. 2-(2,4-difluorophenyl)-3-((2,6-dimethylphenyl)aminocarbonyl)-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid (20)

[0102] 2,4-difluoroaniline was used instead of n-butylamine, 2,6-dimethylphenyl isocyanide was used instead of tert-butyl isocyanide, and the rest of the process was the same as in Example 1. The yield was 87%.

[0103] White solid, mp>250℃. 1 H NMR (500MHz, DMSO-d6) δ15.24(s,1H),11.83(s,1H),9.94(s,1H),8.90(s,1H),7.58–7.53(m,2H),7.29(td,J=8.2 ,2.4Hz,1H),7.09–7.06(m,1H),7.04(d,J=6.0Hz,2H),5.14(t,J=3.2Hz,1H),5.09(d,J=3.1Hz,2H),1.97(s,6H). 13C NMR (125MHz, DMSO-d6) δ172.2,165.3,165.0,162.5,160.9(d,J=11.3Hz),158.4(d,J=13.8Hz),156.3(d,J=12.5Hz),152.9,141.2(d,J=3 1.3Hz),134.7,133.6,129.9(d,J=8.8Hz),127.9,127.1,123.2(m),119.9,112.8,112.4(d,J=22.5Hz),59.6,53.4,17.8.HRMS(ESI):m / z calcd for C24H20F2N3O6+[M+H]+:484.1315,found 484.1325.

[0104] Example 21. 2-(2,2-dimethoxyethyl)-3-((2,6-dimethylphenyl)aminocarbonyl)-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid (21)

[0105] 2,2-dimethoxyethylamine was used instead of n-butylamine, 2,6-dimethylphenyl isocyanide was used instead of tert-butyl isocyanide, and the rest of the process was the same as in Example 1. The yield was 82%.

[0106] White solid, mp 190.2-190.9℃. 1 H NMR(500MHz,DMSO-d6)δ15.34(s,1H),12.25(s,1H),9.87(s,1H),8.82(s,1H),7 .06(d,J=5.1Hz,1H),7.03(d,J=5.5Hz,2H),5.03–5.00(m,1H),4.96(dd,J=3.7, 1.8Hz, 1H), 4.76 (dd, J=13.7, 3.9Hz, 1H), 4.56 (t, J=5.0Hz, 1H), 3.98 (dd, J=14. 0,4.8Hz,1H),3.40(s,3H),3.3(dd,J=14,5.5Hz,1H),3.29(s,3H),2.01(s,6H). 13CNMR(125MHz,DMSO-d6)δ172.2,165.4,165.0,163.4,152.5,134.6,13.9,127.9,12 7.9,119.8,127.8,101.9,100.5,95.6,58.0,54.8,48.2,17.9.HRMS(ESI):m / zcalcd for C22H26N3O8+[M+H]+:460.1714, found 460.1724.

[0107] Example 22. 2-(Cyclopropylmethyl)-3-((4-methylphenylsulfonylmethyl)aminocarbonyl)-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid (22)

[0108] The yield was 75% except that 2-cyclopropylmethylamine was used instead of n-butylamine and p-toluenesulfonylmethyl isocyanide was used instead of tert-butyl isocyanide. The rest of the reaction was the same as in Example 1.

[0109] Off-white solid, mp 172.5-173.2℃. 1 H NMR(500MHz,DMSO-d6)δ15.38(s,1H),11.47(s,1H),9.63(s,1H),8.64(s, 1H),7.71(d,J=8.0Hz,2H),7.37(d,J=7.9Hz,2H),4.76(s,1H),4.74–4.71( m,2H),4.66(s,2H),3.28(dd,J=14.1,7.4Hz,1H),3.15(dd,J=14.1,6.7Hz, 1H),2.30(s,3H),0.82–0.77(m,1H),0.47–0.42(m,2H),0.23–0.20(m,2H). 13 C NMR(125MHz,DMSO-d6)δ172.2,167.5,165.6,163.0,152.6,144.7,140.9,134.5,129 .8,128.4,119.3,112.7,60.1,55.5,52.1,50.2,20.9,8.3,3.6,3.4.HRMS(ESI):m / z calcd forC22H24N3O8S+[M+H]+:490.1279, found 490.1282.

[0110] Example 23. Antiviral effect of the compounds disclosed in the present invention in HSV-1 infected Vero cells

[0111] 3×10 4Vero cells were seeded into 96-well plates, and 100 μL of cell suspension was added to the plates. The plates were pre-incubated for 24 hours (at 37°C, 5% CO₂). Test substances were added at 20 μM and 2 μM concentrations, with three replicates for each drug. A DMSO negative control group and an ACV positive control group were also established. Vero cells were infected with HSV-1-luciferase virus at an MOI of 0.01. Luciferase activity in infected cells was measured using a Firefly Luciferase Assay Kit (Yeasen Biotech 11401ES) 36 hours after infection (at 37°C, 5% CO₂). Luminescence intensity was measured using a Thermo Scientific multi-wavelength scanning reader. Data were analyzed using GraphPad Prism 9.0.0 software. Relative inhibition (%) = fluorescence intensity of drug-treated group / fluorescence intensity of negative control group × 100.

[0112] The inhibition rates of Examples 1-22 and acyclovir are shown in Table 1. The results show that piperazinone and hydroxypyridone compounds can effectively inhibit HSV-1 in Vero cells.

[0113] Table 1. Inhibition rates of piperazinone and hydroxypyridone compounds and acyclovir

[0114]

[0115]

[0116] Example 24. Inhibitory activity against acyclovir-sensitive strain (HSV-1KOS)

[0117] Drug treatment and virus infection: 1.5×10 5 Vero cells were inoculated into 24-well plates. 500 μL of cell suspension was prepared in the 24-well plate. The culture plate was pre-cultured in an incubator for 24 hours (at 37°C, 5% CO2). The drug to be tested was diluted in a gradient, at least 5 concentration gradients to be tested were set, and 3 parallel replicates were set for each drug concentration. Different concentrations of the test substance were added to the culture plate, and the Vero cells were infected with HSV-1KOS virus at an MOI of 0.01. After 24 hours of infection (at 37°C, 5% CO2), the cells were scraped, the cells and supernatant were collected, and three freeze-thaw cycles were performed.

[0118] Determination of sample virus titer:

[0119] Vero cells were plated one day in advance in 24-well plates, 1x10 5Cells / well. Perform a 10-fold serial dilution of the frozen-thawed sample. Aspirate the culture medium from the plate and add 200 μL of dilution to each well. Incubate at 37°C, 5% CO2 for 1 hour (shake every 15 minutes) to allow for viral adsorption. After 1 hour of incubation, add 0.5 mL of methylcellulose culture medium to each well and incubate at 37°C for 2-3 days. After 2-3 days, plaques should be observed under a microscope. Add a 1:1 (v / v) formaldehyde-acetic acid fixative to the plate and let it sit for at least 0.5 hour. Rinse the fixed cell monolayer with water and then stain with crystal violet stain. Let it sit for at least 0.5 hour, rinse with water, and air dry. Count the fixed and stained plaques using a stereomicroscope to calculate the viral titer (PFU / mL, plaque-forming units): PFU / mL = number of viral plaques / 5 × dilution factor. The number of plaques at different concentrations was used to generate a dose-dependent curve using GraphPad Prism 9.0.0, and the EC was determined by fitting the dose-dependent curve using Prism. 50 .

[0120] Some Examples and EC of Acyclovir 50 The results are shown in Table 2. The results show that the piperazinone and hydroxypyridone compounds can effectively inhibit HSV-1 in Vero cells, and Examples 9 and 12 have an inhibitory activity comparable to that of ACV.

[0121] Table 2. EC values ​​of piperazino- and hydroxypyridino- nes and acyclovir against HSV-1 50 value

[0122] Compound <![CDATA[EC 50 (μM)]]> Compound <![CDATA[EC 50 (μM)]]> 9 4.9 20 12.2 10 10.7 ACV 0.7 12 3.3

[0123] Example 25. Cytotoxicity of the compounds disclosed in the present invention

[0124] 3×10 4 Vero cells were seeded into 96-well plates. 100 μL of cell suspension was prepared in the 96-well plate. The plate was pre-incubated in an incubator for 24 hours (at 37°C, 5% CO2). The test drug was diluted to 20 μM and 2 μM in the plate. Each well was incubated in the incubator for an appropriate period of time in DMEM medium containing 10% fetal bovine serum and 1% (v / v) penicillin-streptomycin solution. Subsequently, 10 μL of CCK-8 (Solarbio CA1210) solution was added to each well and incubated in the dark at 37°C for an additional hour. Cell viability was determined by measuring absorbance at 450 nm using a Thermo Scientific full-wavelength scanning multi-function reader.

[0125] Cell viability (%) = [A(drug added) - A(blank)] / [A(0 drug added) - A(blank)] × 100

[0126] A (drug added): absorbance of the wells containing cells, CCK-8 solution, and drug solution

[0127] A (blank): absorbance of the well with culture medium and CCK-8 solution but no cells

[0128] A(0 drug addition): absorbance of the well containing cells, CCK-8 solution but no drug solution

[0129] Data analysis was performed using GraphPad Prism 9.0.0 software. The cytotoxicity results of Examples 9, 12, 20 and ACV are shown in Figure 1 The results showed that Examples 9, 12, and 20 had no obvious toxicity at 20 μM and 2 μM.

[0130] Example 26. Inhibitory activity against acyclovir-resistant strain (HSV-1tkLTRZ1)

[0131] Drug treatment and viral infection:

[0132] 1.5×10 5 Vero cells were inoculated into 24-well plates. 500 μL of cell suspension was prepared in the 24-well plate. The culture plate was pre-cultured in an incubator for 24 hours (at 37°C, 5% CO2). The drug to be tested was diluted gradiently, at least 5 concentration gradients to be tested were set, and 3 parallel replicates were set for each drug concentration. Different concentrations of the test substance were added to the culture plate, and a TK-HSV-1 virus strain tkLTRZ1 was used to infect Vero cells at an MOI of 0.01. After 24 hours of infection (at 37°C, 5% CO2), the cells were scraped, the cells and supernatant were collected, and frozen and thawed three times.

[0133] Determination of sample virus titer:

[0134] Vero cells were plated one day in advance in 24-well plates, 1x10 5Cells / well. Perform a 10-fold serial dilution of the frozen-thawed sample. Aspirate the culture medium from the plate and add 200 μL of dilution to each well. Incubate at 37°C, 5% CO2 for 1 hour (shake every 15 minutes) to allow for viral adsorption. After 1 hour of incubation, add 0.5 mL of methylcellulose culture medium to each well and incubate at 37°C for 2-3 days. After 2-3 days, plaques should be observed under a microscope. Add a 1:1 (v / v) formaldehyde-acetic acid fixative to the plate and let it sit for at least 0.5 hour. Rinse the fixed cell monolayer with water and then stain with crystal violet stain. Let it sit for at least 0.5 hour, rinse with water, and air dry. Count the fixed and stained plaques using a stereomicroscope to calculate the viral titer (PFU / mL, plaque-forming units): PFU / mL = number of viral plaques / 5 × dilution factor. The number of plaques at different concentrations was used to generate a dose-dependent curve using GraphPad Prism 9.0.0, and the EC was determined by fitting the dose-dependent curve using Prism. 50 .

[0135] Some Examples and EC of Acyclovir 50 The results are shown in Table 3. The results show that the inhibitory activity of acyclovir against acyclovir-resistant strain (HSV-1tkLTRZ1) decreased significantly, and Examples 9, 12, and 20 still retained the same EC level as the sensitive strain against the resistant strain. 50 The results show that the compounds disclosed in the present invention can solve the drug resistance problem of acyclovir, a first-line drug.

[0136] Table 3. EC of piperazinone and hydroxypyridone compounds against ACV-resistant HSV-1 50 value

[0137] Compound <![CDATA[EC 50 (μM)]]> Compound <![CDATA[EC 50 (μM)]]> 9 7.6 20 9.4 12 4.8 ACV 17.3

[0138] Example 27. In vitro co-incubation of the compounds disclosed in Examples 9 and 12 with HSV-1 virus. Treatment of drugs and incubation of viruses:

[0139] The compounds were diluted to a final concentration of 20 μM using cell culture medium and incubated with the virus dilution solution at 37°C for 2 h. Three replicates were set for each drug. 5 Vero cells were seeded into a 24-well plate. 500 μL of cell suspension was prepared in the 24-well plate. The culture plate was pre-incubated in an incubator for 24 hours (at 37° C., 5% CO 2 ).

[0140] Determination of sample virus titer:

[0141] Perform serial dilutions of at least 100-fold from the co-incubated sample to ensure that the drug is diluted to a non-working concentration. Aspirate the medium from the plate and add 200 μL of dilution to each well. Incubate at 37°C, 5% CO₂ for 1 hour (shaking every 15 minutes) to allow for viral adsorption. After 1 hour of incubation, add 0.5 mL of methylcellulose medium to each well and incubate at 37°C for 2-3 days. After 2-3 days, plaques should be observed under a microscope. Add a 1:1 (v / v) formaldehyde-acetic acid fixative to the plate and let it sit for at least 0.5 hour. The fixed cell monolayer should then be rinsed with water. Crystal violet stain should then be added for staining, let it sit for at least 0.5 hour, rinsed with water, and air-dried. Count the fixed and stained plaques using a stereomicroscope to calculate the viral titer (PFU / mL, plaque formation units): PFU / mL = number of viral plaques / 5 × dilution factor.

[0142] GraphPad Prism 9.0.0 was used for statistical analysis, and Bonferroni's multiple comparison test was used to compare multiple groups of data. Data are expressed as mean ± standard deviation (SDs).

[0143] The experimental results are as follows Figure 2 As shown, the results showed that there was no significant difference between the drug-treated group and the DMSO group, indicating that piperazinone and hydroxypyridone compounds cannot directly kill viruses and need to rely on infected hosts to exert their effects.

[0144] Example 28. Effects of Examples 9 and 12 on Adsorption and Entry of HSV-1 Virus

[0145] Adsorption experiment: the day before, 1.5×10 5 Vero cells were inoculated into a 24-well plate. 500 μL of cell suspension was prepared in a 24-well plate. The culture plate was pre-cultured in an incubator for 24 hours (at 37°C, 5% CO2). The cells were pre-cooled at 4°C for 30 minutes, then the tray was filled with ice, and the well plate was placed on ice and transferred to a biosafety cabinet. The drug to be tested was added to dilute the drug to 20 μM, and 3 parallel replicates were set for each drug. Virus infection was performed according to MOI = 1 (the virus solution was pre-cooled), placed at 4°C for one hour, and shaken every 15 minutes. After one hour, the non-adsorbed virus was removed by washing three times with pre-cooled PBS. Finally, the cells were scraped to complete the sample collection for subsequent experiments.

[0146] Enter the experiment:

[0147] The initial steps are the same as for the adsorption experiment. After one hour of adsorption at 4°C, remove the viral fluid and wash with PBS. Warm culture medium is then added, and the cells are incubated at 37°C. After 2 hours, wash the cells once with PBS (a control group should be included, i.e., centrifugation is performed directly after adsorption at 4°C without incubation at 37°C to indicate possible background). The cells are then scraped with 1 mL of PBS (containing 0.5 mg / mL proteinase K) into a 1.5 mL EP tube and centrifuged at 3500 rpm for 3 minutes. Remove the supernatant and repeat the above steps several times. After the final centrifugation, resuspend the cells in 1 mL of pre-chilled hypotonic buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT) and incubate on ice. The cells are then transferred to a 1 mL homogenizer and triturated up and down 15 times using the included pestle. Finally, the cells were transferred to another EP tube and centrifuged at 5000 rpm for 5 minutes at 4°C. The supernatant was discarded, and the cell pellet was retained. Genomic DNA was extracted using a cell DNA extraction kit, and viral genome levels were detected using quantitative PCR. Statistical analysis was performed using GraphPad Prism 9.0.0 (GraphPad Software), and multiple data sets were compared using one-way ANOVA with Bonferroni multiple comparison tests. Data are expressed as mean ± standard deviation (SD).

[0148] The results of HSV-1 virus adsorption and entry experiments are as follows Figure 3 、 Figure 4 As shown, the results show that the piperazinone and hydroxypyridone compounds disclosed in the present invention have no significant effect on the adsorption and entry process of the virus, indicating that the inhibitory effect of the drug on the virus is not through affecting the adsorption and entry process of the virus.

[0149] Example 29. Effects of the compounds disclosed in Examples 9 and 12 on HSV-1 viral gene expression

[0150] The day before, 1.5×10 5Vero cells were inoculated into 24-well plates. 500 μL of cell suspension was prepared in the 24-well plate. The culture plate was pre-cultured in an incubator for 24 hours (at 37°C, 5% CO2). The drug to be tested was added to dilute the drug to 20 μM in a gradient manner, and 3 parallel replicates were set for each drug. The cells were scraped and sampled using RNA lysis buffer at 2 hours and 5 hours after infection. RNA extraction and reverse transcription experiments were performed using an RNA extraction kit and a reverse transcription kit, and the mRNA expression levels of viral ICP27, TK, and gC were detected using quantitative PCR. GraphPad Prism 9.0.0 (GraphPad Software) was used for statistical analysis, and Bonferroni multiple comparison tests of one-way ANOVA and two-way ANOVA were used to compare multiple groups of data. The data are expressed as mean ± standard deviation (SDs). The experimental results are shown in Figure 2. Figure 5 shown.

[0151] The results showed that 2h and 5h after HSV-1 infected Vero cells, the transcript levels of viral ICP27, TK, and gC in the drug-treated group were significantly lower than those in the DMSO group, indicating that piperazinone and hydroxypyridone compounds significantly inhibited the expression of HSV-1 viral genes.

[0152] Example 30. Effects of the compounds disclosed in Examples 9 and 12 on HSV-1 viral protein translation

[0153] The day before, 1.5×10 5 Vero cells were inoculated into a 24-well plate. 500 μL of cell suspension was prepared in the 24-well plate. The culture plate was pre-cultured in an incubator for 24 hours (at 37°C, 5% CO2). The drug to be tested was added to make a gradient dilution of the drug to be tested to 20 μM. After drug treatment, HSV-1 MOI=5 was used to infect Vero cells. After aspirating the supernatant 3h, 6h and 9h after infection, an appropriate amount of SDS loading buffer was added, the cells were scraped, and denatured in a 95°C metal bath for 5 minutes. Western Blot experiments were performed to detect the protein expression of viral ICP4, gC and TK. The experimental results are shown in the figure. Figure 6 shown.

[0154] The results showed that at 6h and 9h after infection, the viral ICP4, gC, and TK protein levels were significantly lower than those in the DMSO group, indicating that the piperazinone and hydroxypyridone compounds disclosed in the present invention can significantly inhibit the expression of HSV-1 proteins.

[0155] Preliminary studies on the mechanism of action of piperazino-hydroxypyridone compounds against HSV-1 have demonstrated that piperazino-hydroxypyridone compounds exhibit potent inhibitory effects against both HSV-1 KOS strains and ACV-resistant strains, suggesting a novel mechanism of action distinct from that of ACV in inhibiting HSV-1. Piperazino-hydroxypyridone compounds significantly inhibit HSV-1 protein and transcript levels at relatively early post-infection time points, but do not affect viral adsorption or entry. Furthermore, the inhibitory effects of piperazino-hydroxypyridone compounds on HSV-1 are dependent on host cell function, and they lack a direct antiviral effect on HSV-1. This suggests that piperazino-hydroxypyridone compounds may inhibit viral replication by directly affecting viral transcription or indirectly by influencing host antiviral immunity.

Claims

1. The use of a piperazinone and hydroxypyridone compound in the preparation of an anti-herpes simplex virus drug, characterized in that: Any one of the following compounds: 2-n-Butyl-3-tert-butylaminocarbonyl-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid 2-Methoxycarbonylmethyl-3-tert-butylaminocarbonyl-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid 2-(2-Morpholinylethyl)-3-cyclohexylaminocarbonyl-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid 2-(2,2-Dimethoxyethyl)-3-cyclohexylaminocarbonyl-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid 2-{2-[(4-methylphenyl)sulfonamido]ethyl}-3-cyclohexylaminocarbonyl-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid 2-{4-[(tert-Butyloxycarbonyl)amino]butyl}-3-cyclohexylaminocarbonyl-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid 2-Benzyl-3-benzylaminocarbonyl-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid 2-Benzyl-3-(naphthalen-2-ylaminocarbonyl)-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid 2-Benzyl-3-((2,6-dimethylphenyl)aminocarbonyl)-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid 2-(2,6-dimethylphenyl)aminocarbonyl-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid 2-(2-morpholinoethyl)-3-((2,6-dimethylphenyl)aminocarbonyl)-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid 2-(2,4-Difluorobenzyl)-3-((2,6-dimethylphenyl)aminocarbonyl)-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid 2-(Naphth-1-yl)-3-((2,6-dimethylphenyl)aminocarbonyl)-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid 2-(2-Fluoro-3-chlorophenyl)-3-((2,6-dimethylphenyl)aminocarbonyl)-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid 2-(3,5-difluorophenyl)-3-((2,6-dimethylphenyl)aminocarbonyl)-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid 2-(Pyridin-3-ylmethyl)-3-((2,6-dimethylphenyl)aminocarbonyl)-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid 2-(3,5-Bistrifluoromethylphenyl)-3-((2,6-dimethylphenyl)aminocarbonyl)-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid 2-(4-(4-acetylpiperazin-1-yl)phenyl)-3-((2,6-dimethylphenyl)aminocarbonyl)-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid 2-((Pyridine-1-oxide)-2-ylmethyl)-3-((2,6-dimethylphenyl)aminocarbonyl)-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid 2-(2,4-difluorophenyl)-3-((2,6-dimethylphenyl)aminocarbonyl)-9-hydroxy-1,8-dioxo-1,3,4,8-tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylic acid.

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