An N-hydroxythienopyrimidinedione derivative, and a preparation method and application thereof

Abstract

The application discloses an N-hydroxythienopyrimidinedione derivative, a preparation method and application thereof. The compound has a structure shown in general formula I or II. The application further relates to a pharmaceutical composition containing the compound with the structure of formula I or formula II. Activity screening experiments show that the compound has good anti-positive poxvirus activity, and therefore the application further provides application of the compound in preparation of an anti-positive poxvirus drug.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of organic compound synthesis and medical application, and particularly relates to an N-hydroxy thienopyrimidine diketone derivative, a preparation method thereof and application thereof as an anti-orthopoxvirus drug. BACKGROUND

[0002] Poxvirus, belonging to the family of poxviridae, is a virus that can cause a wide range of pox diseases in humans and other animals. Currently, there are four genera of viruses that can infect humans, including orthopoxvirus, parapoxvirus, yatapoxvirus and molluscipoxvirus. Among them, smallpox virus, monkeypox virus, cowpox virus and vaccinia virus belonging to orthopoxvirus are well known in the history of human epidemics. With the eradication of smallpox virus in 1980 and the discontinuation of smallpox vaccination, monkeypox virus has become the most important orthopoxvirus to infect humans. On August 14, 2024, the monkeypox outbreak occurred in multiple regions around the world, and the World Health Organization declared the monkeypox outbreak as a global public health emergency, which sounded the alarm for the prevention and treatment of global orthopoxvirus.

[0003] Currently, there are three antiviral drugs approved for emergency treatment of smallpox outbreaks, namely cidofovir, brincidofovir and tecovirimat. Among them, cidofovir and brincidofovir are limited in clinical use due to kidney toxicity and damage to the gastrointestinal tract. In addition, for the emerging outbreak of monkeypox, there is no specific drug available, and only tecovirimat was urgently approved by the European Medicines Agency in 2022 for the treatment of monkeypox. Therefore, in view of the crisis of monkeypox virus outbreak and the clinical demand for anti-orthopoxvirus drugs, it is urgent to discover new anti-orthopoxvirus drugs.

[0004] SUMMARY

[0005] The present application provides a kind of N-hydroxy thienopyrimidine diketone derivative and preparation method thereof to solve the technical problems of prior art;The present application also provides the application of N-hydroxy thienopyrimidine diketone derivative as orthopoxvirus inhibitor.

[0006] The technical scheme of the present application is as follows:

[0007] 1.N-hydroxy thienopyrimidine diketone derivative

[0008] An N-hydroxy thienopyrimidine diketone derivative, or a pharmaceutically acceptable salt, ester or prodrug thereof, has the structure shown in general formula I or II:

[0009]

[0010] n=0, 1, 2 or 3;

[0011] R1is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

[0012] R2is C1-C6alkyl, OC1-C6alkyl, C2-C6alkenyl, C3-C6cycloalkyl, OC3-C6cycloalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted phenyl ring, substituted or unsubstituted six-membered heterocycle, substituted or unsubstituted five-membered heterocycle, substituted or unsubstituted six-membered fused five-membered heterocycle, substituted or unsubstituted six-membered fused six-membered heterocycle, substituted or unsubstituted five-membered fused five-membered heterocycle, substituted or unsubstituted benzofused five-membered heterocycle, substituted or unsubstituted benzofused six-membered heterocycle;

[0013] The substituents in R1, R2are selected from alkyl, cycloalkyl, methoxy, nitro, cyano, amino, trifluoromethyl, ester, halogen, 2,6-diF, 2,5-diF, 3,4-diF, 2,4-diF, 2-F,4-CF3, 3-F,4-CF3, CONH2, SO2NH2, SO2CH3, NHMe, NHCOMe, NHSO2Me or NHSO2CF3.

[0014] According to the present application, preferably,

[0015] R1is methyl, ethyl, isopropyl, methylenecyclopropyl or methylenecyclobutyl;

[0016] n = 1,

[0017] R2is a substituted phenyl ring having the general formula a;

[0018]

[0019] wherein R3is F, Cl, Br, Me, OMe, CN, NO2, NH2, 2,6-diF, 2,5-diF, 3,4-diF, 2,4-diF, 2-F,4-CF3, 3-F,4-CF3, CONH2, SO2NH2, SO2CH3, NHMe, NHCOMe, NHSO2Me or NHSO2CF3.

[0020] According to the present application, preferably, the pharmaceutically acceptable salt of the compound is a sodium salt, a hydrochloride salt, a sulfate salt, a tartrate salt or a citrate salt.

[0021] According to the present application, further preferably, the N-hydroxythienopyrimidine dione derivative of general formula I or II is one of the following compounds:

[0022]

[0023] 2. Preparation method of N-hydroxythiophenepyrimidine dione derivatives

[0024] The preparation method of N-hydroxythiophene-pyrimidine dione derivatives includes the following steps: Methyl 2-aminothiophene-3-carboxylate is used as the starting material and reacts with carbonyl diimidazole and o-benzyl hydroxylamine to generate intermediate compound 1; intermediate compound 1 undergoes an iodination reaction with N-iodosuccinimide to generate intermediate compound 2 (or methyl 3-amino-5-iodothiophene-2-carboxylate directly reacts with carbonyl diimidazole and o-benzyl hydroxylamine to generate intermediate compound 2); intermediate compound 2 undergoes a nucleophilic substitution reaction in the presence of potassium carbonate to generate intermediate compound 3. Intermediate compound 3 undergoes a spur-coupling reaction with cuprous iodide and bis(triphenylphosphine)palladium dichloride as catalysts and triethylamine as a base to yield key intermediate compound 4; intermediate compound 4 is detrimethylsilylated in the presence of potassium carbonate to yield intermediate 5; intermediate compound 5 and different azide fragments undergo an azide-alkynyl Husigen-Click cycloaddition reaction with the participation of sodium ascorbate and copper sulfate pentahydrate to generate intermediate compound 6; intermediate compound 6 is refluxed in a hydrobromic acid and acetic acid system to remove benzyl protection to generate target product I or II;

[0025] The synthesis route is as follows:

[0026]

[0027] Reagents and conditions: (i) a) carbonyl diimidazole, O-benzylhydroxylamine, tetrahydrofuran, 80℃, 5-8h; b) sodium hydroxide, ethanol, 85℃, 1-3h; c) acetic acid, rt, 0.5h; (ii) N,N-dimethylformamide, dichloromethane, N-iodosuccinimide, room temperature, 1-3h; (iii) N,N-dimethylformamide, iodoalkane or iodoaromatic hydrocarbon, carbonic acid Potassium, 20-60℃, 4-8h; (iv) Tetrahydrofuran, cuprous iodide, bis(triphenylphosphine)palladium dichloride, triethylamine, trimethylsilylacetylene, nitrogen, room temperature, 5-8h; (v) Methanol, potassium carbonate, room temperature, 1-3h; (vi) Sodium ascorbate, copper sulfate pentahydrate, water, tetrahydrofuran, azide, room temperature, 4-5h; (vii) Hydrobromic acid, acetic acid, 120℃, 1-2h;

[0028] R1, R2, and n are the same as those shown in general formulas I and II above.

[0029] The azides are various substituted benzene ring azides, various substituted naphthyl ring azides, various substituted six-membered heterocyclic azides, various substituted five-membered heterocyclic azides, various substituted six-membered pentane-heterocyclic azides, various substituted six-membered pentane-six-membered heterocyclic azides, various substituted five-membered pentane-heterocyclic azides, various substituted benzo[a]-pentane-heterocyclic azides, or various substituted benzo[a]-six-membered heterocyclic azides. The substituents are selected from alkyl, cycloalkyl, methoxy, nitro, cyano, amino, trifluoromethyl, ester, halogen, 2,6-diF, 2,5-diF, 3,4-diF, 2,4-diF, 2-F,4-CF3, 3-F,4-CF3, CONH2, SO2NH2, SO2CH3, NHMe, NHCOMe, NHSO2Me, or NHSO2CF3.

[0030] 3. Applications of N-hydroxythiophenepyrimidine dione derivatives

[0031] This invention discloses the screening results of the anti-orchiopeptidosis virus activity of N-hydroxythiophene-pyrimidine dione derivatives and their first application as orchiopeptidosis virus inhibitors. Experiments demonstrate that the N-hydroxythiophene-pyrimidine dione derivatives of this invention can be used as orchiopeptidosis virus inhibitors in the preparation of anti-orchiopeptidosis virus drugs.

[0032] Anti-pox virus activity and toxicity experiments of the target compound

[0033] The N-hydroxythiophene-pyrimidine dione derivatives synthesized according to the above method were screened for anti-orthopoxvirus (monkeypoxvirus, vaccinia virus, cowpoxvirus, and camelpoxvirus). Activity and toxicity data are listed in Table 1, with cidofovir, brincidofovir, and tecoviril as positive controls. As shown in Table 1, among the four newly synthesized N-hydroxythiophene-pyrimidine dione compounds, Z1 and Z2 exhibited significant antiviral activity against monkeypoxvirus and camelpoxvirus, superior to the positive control drug cidofovir. Simultaneously, both compounds showed low cytotoxicity (MCC). Z1 ≥100μM, MCC Z2 (≥100μM), which has further research and development value.

[0034] The N-hydroxythiophene-pyrimidine dione derivatives of the present invention are a series of novel orthopoxvirus inhibitors that can be used as lead compounds against orthopoxvirus.

[0035] The N-hydroxythiophene-pyrimidine dione derivatives of the present invention can be used as inhibitors of orthopoxvirus. Specifically, they can be used as orthopoxvirus inhibitors in the preparation of anti-orthopoxvirus drugs.

[0036] An anti-vaccinia virus pharmaceutical composition comprising an N-hydroxythiophene-pyrimidine dione derivative of the present invention and one or more pharmaceutically acceptable carriers or excipients. Detailed Implementation

[0037] The following examples help to understand the present invention, but they are not intended to limit the scope of the invention.

[0038] The synthetic routes involved in the embodiments are as follows:

[0039]

[0040] Example 1: Preparation of the key intermediate 3-(benzyloxy)-1-(cyclobutylmethyl)-6-ethynylthiophenopyrimidine-2,4(1H,3H)-dione (5-1,5-2)

[0041] 2-Aminothiophene-3-carboxylic acid methyl ester (3.14 g, 20.00 mmol), carbonyl diimidazole (8.11 g, 50.00 mmol), and o-benzylhydroxylamine (3.07 g, 25.00 mmol) were weighed and mixed in 30 mL of tetrahydrofuran. The mixture was heated to 75 °C and refluxed for 5 h. After the reaction was completed by TLC, the tetrahydrofuran was evaporated to dryness, and sodium hydroxide (1.60 g, 40.00 mmol) and 30 mL of ethanol were added. The mixture was refluxed at 80 °C for 1 h. After the reaction was completed by TLC, the reaction solution was cooled to room temperature and acidified with glacial acetic acid to produce a large amount of white precipitate. The precipitate was filtered, washed successively with ethanol and water, and dried to obtain the crude intermediate 3-(benzyloxy)thieno[2,3-d]pyrimidine-2,4(1H,3H)-dione (1), a pale yellow solid, with a yield of 62%. 1 H NMR(400MHz,DMSO-d6)δ12.50(s,1H,NH),7.58–7.55(m,2H,Ph-H),7.43–7.40(m,2H,Ph-H),7.21(d,J=5.6Hz,1H, C4-thienopyrimidinone-H),7.17(d,J=5.6Hz,1H,C3-thienopyrimidinone-H),5.06(s,2H,O-CH2).ESI-MS:m / z 571.25[2M+Na] + C 13 H 10 N2O3S(274.04).

[0042] The crude intermediate 1 obtained above (4.11 g, 15 mmol) was dissolved in 20 mL of N,N-dimethylformamide, and N-iodosuccinimide (3.71 g, 16.5 mmol) was added in portions. The mixture was stirred at room temperature for 1 h. After the reaction was completed as detected by TLC, 80 mL of saturated sodium chloride was added to the reaction solution, producing a large amount of white precipitate. The precipitate was filtered off, dried, and used as a raw material to obtain the crude intermediate 3-(benzyloxy)-6-iodothieno[2,3-d]pyrimidine-2,4-(1H,3H)-dione (2-1), a white solid, yield: 75%. ESI-MS: m / z 423.25 [M+Na] + C 13 H9IN2O3S(399.94).

[0043] 5.66 g (20.00 mmol) of methyl 3-amino-5-iodothiophene-2-carboxylate, 8.11 g (50.00 mmol) of carbonyl diimidazole, and 3.07 g (20.00 mmol) of o-benzylhydroxylamine were weighed and mixed in 30 mL of tetrahydrofuran. The mixture was heated to 75 °C and refluxed for 5 h. After the reaction was completed by TLC, the solvent was evaporated to dryness, and 30 mL of ethanol was added to the residue to dissolve it. Sodium hydroxide (1.60 g, 40.00 mmol) was added, and the mixture was refluxed for another 1 h. After the reaction was completed by TLC, the mixture was cooled to room temperature and acidified with glacial acetic acid to produce a large amount of white precipitate. The precipitate was filtered, washed successively with ethanol and water, and dried to obtain crude 3-(benzyloxy)-6-iodothiophene[3,2-d]pyrimidine-2,4-(1H,3H)-dione (2-2), a pale yellow solid, yield: 59%. ESI-MS: m / z 822.49 [2M+Na] + C 13 H9IN2O3S(399.94).

[0044] Intermediate 2-1 (or 2-2) (6.00 g, 15.00 mmol) was added to 20 mL of N,N-dimethylformamide, followed by the addition of potassium carbonate (4.15 g, 30.00 mmol) and bromomethylcyclobutane (3.35 g, 22.50 mmol). The mixture was stirred at room temperature for 4 h. After the reaction was completed by TLC, 100 mL of ice water was added to the mixture, resulting in a large amount of white precipitate. The precipitate was filtered and washed with water to obtain 3-(benzyloxy)-1-(cyclobutylmethyl)-6-ethynyl-3-(benzyloxy)thiophenepyrimidine-2,4(1H,3H)-dione (3-1, 3-2).

[0045] 3-(benzyloxy)-1-(cyclobutylmethyl)-thieno[2,3-d]pyrimidine-2,4(1H,3H)-dione (3-1): white solid, yield: 59%. 1H NMR(400MHz,DMSO-d6)δ7.56(s,1H,C3-thienopyrimidinone-H),7.55–7.52(m,2H,Ph-H),7.41(dd,J=5.0,2.1Hz,3H,Ph-H),5.05(s,2H, O-CH2),3.93(d,J=7.2Hz,2H,CH2),2.76(p,J=7.4Hz,1H,CH),2.00–1.95(m,2H,CH2),1.84–1.79(m,4H,2×CH2).ESI-MS:m / z468.97[M+H] + C 18 H 17 IN2O3S (468.00).

[0046] 3-(benzyloxy)-1-(cyclobutylmethyl)-6-ethynyl-3-(benzyloxy)thiopheno[3,2-d]pyrimidine-2,4(1H,3H)-dione (3-2): white solid, yield: 63%. 1 H NMR(400MHz,DMSO-d6)δ7.78(s,1H,C3-thienopyrimidinone-H),7.55–7.52(m,2H,Ph-H) ,7.41–7.39(m,3H,Ph-H),5.06(s,2H,O-CH2),4.05(d,J=7.3Hz,2H,CH2),2.67(p,J=7.3Hz 1H,CH),1.96–1.89(m,2H,CH2),1.82–1.75(m,4H,2×CH2).ESI-MS:m / z 500.62[M+Na] + C 18 H 17 IN2O3S (468.00).

[0047] To a 40 mL anhydrous tetrahydrofuran solution containing 4.68 g (10 mmol) of 3-(benzyloxy)-1-(cyclobutylmethyl)-thienopyrimidine-2,4(1H,3H)-dione (3-1,3-2) , bis(triphenylphosphine)palladium dichloride (0.35 g, 0.5 mmol), cuprous iodide (0.38 g, 2 mmol), and triethylamine (5.56 mL, 40 mmol) were added sequentially. Under nitrogen protection, trimethylsilylacetylene (3.11 mL, 22 mmol) was injected, and the mixture was stirred at room temperature for 8 h. After the reaction was complete as detected by TLC, the reaction solution was filtered through diatomaceous earth, concentrated, and separated by Flash column chromatography to obtain the intermediate 3-benzyloxy-1-(cyclobutylmethyl)-6-((trimethylsilyl)ethynyl)-thienopyrimidine-2,4(1H,3H)-dione (4-1,4-2). The obtained intermediate (4-1, 4-2) (2.19 g, 5 mmol) was dissolved in 20 mL of methanol solution, and potassium carbonate (1.38 g, 10 mmol) was added. The mixture was stirred at room temperature for 1 h. After the reaction was completed by TLC, the methanol was evaporated to dryness, and 50 mL of aqueous solution was added to the reaction solution. The mixture was extracted with ethyl acetate (30 mL × 3). The organic phase was dried over anhydrous sodium sulfate and concentrated by filtration. Finally, it was recrystallized in methanol to give the key intermediate 3-benzyloxy-1-(cyclobutylmethyl)-6-ethynyl-thienopyrimidine-2,4(1H,3H)-dione (5-1, 5-2).

[0048] 3-Benzyloxy-1-(cyclobutylmethyl)-6-ethynyl-thieno[2,3-d]pyrimidine-2,4(1H,3H)-dione (5-1), white solid, yield: 65%, melting point: 150-152℃. 1 H NMR(400MHz,DMSO-d6)δ7.57(s,1H,C3-thienopyrimidinone-H),7.56–7.53(m,2H,Ph-H),7.45–7.39(m,3H,Ph-H),5.06(s,2H,O-CH2 ),4.77(s,1H,C≡CH),3.96(d,J=7.1Hz,2H,CH2),2.82–2.73(m,1H,CH),2.03–1.95(m,2H,CH2),1.87–1.79(m,4H,2×CH2).ESI-MS:m / z 405.21[M+K] + C 20 H 18 N2O3S(366.10).

[0049] 3-Benzyloxy-1-(cyclobutylmethyl)-6-ethynyl-thieno[3,2-d]pyrimidine-2,4(1H,3H)-dione (5-2), pale yellow solid, yield: 56%, melting point: 156-158℃.1 H NMR(400MHz,DMSO-d6)δ7.77(s,1H,C3-thienopyrimidinone-H),7.56–7.53(m,2H,Ph-H),7.45–7.38(m,3H,Ph-H),5.12(s,1H,C≡CH) ,5.08(s,2H,O-CH2),4.07(d,J=7.2Hz,2H,CH2),2.72–2.65(m,1H,CH),1.96–1.90(m,2H,CH2),1.82–1.76(m,4H,2×CH2).ESI-MS:m / z 405.45[M+K] + C 20 H 18 N2O3S (366.10). Example 2: Preparation of target products Z1-Z4

[0050] The key intermediate 3-benzyloxy-1-(cyclobutylmethyl)-6-ethynyl-thiophenopyrimidine-2,4(1H,3H)-dione (5-1,5-2) (0.70 mmol) and the azide prepared from substituted benzyl halides (1.05 mmol) were added to a mixed solvent of tetrahydrofuran and water (v / v = 4:1, 15 mL). Then, 1 M freshly prepared aqueous solution of sodium ascorbate (0.195 mmol) and an aqueous solution of 7.5% copper sulfate pentahydrate (0.065 mmol) were added to this solution, and the mixture was stirred at room temperature for 5 h. After the reaction was complete as determined by TLC, 20 mL of water was added to the reaction solution, resulting in a large precipitate. The precipitate was filtered, washed with water, and then recrystallized in methanol to obtain intermediates Z1a-Z4a of the target product. 2 mL of 48% hydrobromic acid and 2 mL of glacial acetic acid were added to the target product intermediate Z1a-Z4a (0.6 mmol), and the mixture was heated under reflux at 120 °C for 2 h. After the reaction was completed by TLC, the reaction solution was cooled to room temperature and concentrated under reduced pressure to obtain the crude target product. The crude product was then recrystallized in methanol to obtain the target product Z1-Z4.

[0051]

[0052] The procedure is as described above, using 1-(azidomethyl)-3-fluoro-4-(trifluoromethyl)benzene. Z1 is a white solid with a yield of 60% and a melting point of 232–234 °C. 1 H NMR (400MHz, DMSO-d6) δ10.77(s,1H,OH),8.73(s,1H,C3-thienopyrimidinone-H),7.85(t,J=7.9Hz,2H,Ph-H),7.66(s,1H,C 15-triazole-H),7.55(d,J=11.5Hz,1H,Ph-H),7.34(d,J=8.1Hz,1H,Ph-H),5.82(s,2H,CH2),4.03( d,J=7.3Hz,2H,CH2),2.84(p,J=7.5Hz,1H,CH),2.04–1.98(m,2H,CH2),1.87–1.81(m,4H,2×CH2). 13 C NMR (100MHz, DMSO-d6) δ 159.33 (d, J = 253.1Hz), 155.82, 151.05, 149.58, 143.86 (d, J = 7.4Hz), 141.40, 128.48 (d, J = 4.9Hz), 125.02 (d, J=4.9Hz),124.35(d,J=4.3Hz),122.27,119.87,117.34,117.12,115.30,54.15,52.51,33.70,26.19,18.39.ESI-MS:m / z496.22[M+H] + C 21 H 17 F4N5O3S(495.10).

[0053]

[0054] The procedure is as described above, using 1-(azidomethyl)-2-fluoro-4-(trifluoromethyl)benzene. Z2 is a white solid with a yield of 56% and a melting point of 197–199 °C. 1 H NMR(400MHz,DMSO-d6)δ10.73(s,1H,-OH),8.72(s,1H,C3-thienopyrimidinone-H),7.84–7.78(m,1H,Ph-H),7.69-7.66(m,2H,C 15 -triazole-H,Ph-H),7.62(t,J=7.6Hz,1H,Ph-H),5.84(s,2H,CH2),4.02(d,J=7.3Hz, 2H,CH2),2.84(p,J=7.6Hz,1H,CH),2.03-1.97(m,2H,CH2),1.89–1.81(m,4H,2×CH2). 13C NMR(100MHz,DMSO-d6)δ160.42(d,J=249.8Hz),155.79,151.06,149.56,141.30,132.50(d,J=3.7Hz),127.66(d,J=14.9Hz),124.35,1 22.34(d,J=3.8Hz),122.17,119.92,115.31,113.93(d,J=4.0Hz),113.66(d,J=3.9Hz),54.13,47.53,33.70,26.19,18.38.ESI-MS:m / z 496.23[M+H] + C 21 H 17 F4N5O3S(495.10).

[0055]

[0056] The procedure is as described above, using 1-(azidomethyl)-3-fluoro-4-(trifluoromethyl)benzene. Z3 is a white solid with a yield of 64% and a melting point of 186–188 °C. 1 H NMR (400MHz, DMSO-d6) δ10.77(s,1H,-OH),8.83(s,1H,C3-thienopyrimidinone-H),7.86(t,J=7.9Hz,1H,Ph-H),7.77(s,1H,C 15 -triazole-H,),7.57(d,J=11.5Hz,1H,Ph-H),7.36(d,J=8.1Hz,1H,Ph-H),5.87(s,2H,CH2),4.09(d,J=7.3Hz,2H CH2),2.75(p,J=7.3Hz,1H,CH),1.98–1.92(m,2H,CH2),1.87–1.80(m,4H,2×CH2). 13 C NMR (100MHz, DMSO-d6) δ159.35(d,J=252.4Hz),155.48,150.58,145.17,143.79(d,J=7.8Hz),141.36,140.99,128.54(d, J=4.8Hz),125.04(d,J=3.4Hz),123.83,117.37,117.16,114.62,109.86,52.60,50.83,34.29,25.82,18.31.ESI-MS:m / z 496.08[M+H] + C 21 H 17F4N5O3S(495.10).

[0057]

[0058] The procedure is as described above, using 1-(azidomethyl)-2-fluoro-4-(trifluoromethyl)benzene. Z4 is a white solid with a yield of 58% and a melting point of 240–242 °C. 1 H NMR (400MHz, DMSO-d6) δ10.75(s,1H,-OH),8.82(s,1H,C3-thienopyrimidinone-H),7.82(d,J=10.0Hz,1H,Ph-H),7.79(s,1H,C 15 -triazole-H,),7.72–7.62(m,2H,Ph-H),5.90(s,2H,CH2),4.09(d,J=7.4Hz,2H, CH2),2.75(p,J=7.3Hz,1H,CH),1.99–1.94(m,2H,CH2),1.87–1.81(m,4H,2×CH2). 13 C NMR (100MHz, DMSO-d6) δ160.45(d,J=249.9Hz),155.47,150.57,145.19,141.27,140.99,132.54(d,J=3.7Hz),127.60(d,J=15.1 Hz),123.69,122.42,114.63,113.96(d,J=3.8Hz),113.72(d,J=4.0Hz),109.84,50.82,47.67,34.28,25.81,18.31.ESI-MS:m / z 496.14[M+H] + C 21 H 17 F4N5O3S(495.10).

[0059] Example 3: In vitro anti-vaccinia virus activity assay of the target compound (HEL cells)

[0060] Test Principles

[0061] After a virus infects a cell, due to the limitation of the solid medium, the released virus can only spread from the initially infected cell to the surrounding area. After several proliferation cycles, a localized lesion cell area is formed, which is called a viral plaque. Theoretically, a plaque is formed from a single viral particle that initially infected the cell; therefore, this technique is often used for viral particle counting and the isolation of viral clones.

[0062] Test method:

[0063] Viral plaque assay (PFU)

[0064] HEL cells were digested, centrifuged, counted, and seeded into 96-well plates. After 24 hours of incubation at 37°C and 5% CO2, 100 CClD50 of virus was added to infect the cells. After 1 hour, residual virus was removed, and different concentrations of compounds were added. Incubation continued for another hour, followed by covering the cells with 1.6% carboxymethyl cellulose diluted in DMEM. After 3 days of culture, the monolayer cells were fixed and stained with 3.7% formaldehyde, 0.1% crystal violet, and 1.5% methanol. Plaques were counted under a microscope, and the concentration of the compound that reduced the number of plaques by 50% was calculated as the antiviral activity (EC50) of the target compound. 50 ).

[0065] Cytotoxicity testing is based on the inhibitory effect of the test compound on cell growth. First, a quantitative amount of the test compound at 5 × 10⁻⁶ mg / well is seeded into each well of a 96-well microtiter plate. 3 HEL cells were proliferated at a certain rate, then serially diluted test compounds were added and incubated for 3 days. Cells in each well of HEL were then counted using a Coulter counter. The minimum concentration of the compound required to produce normal cell morphological changes visible under a microscope was defined as the MCC.

[0066] Table 1. Antiviral activity and toxicity of N-hydroxythiophenepyrimidine dione derivatives (HEL cells)

[0067]

[0068] a EC 50 The concentration of the compound that inhibits the formation of 50% of viral plaques. 0 <A<10μM,10μM<B<100μM,C> 100μM.

[0069] b MCC: The minimum concentration of a compound required to cause a change in the morphology of normal cells visible under a microscope.

[0070] Experimental conclusions and analysis: Among the four newly synthesized N-hydroxythiophenepyrimidine dione compounds, Z1 and Z2 exhibited significant antiviral activity against monkeypox virus and camelpox virus, superior to the positive control drug cidofovir. Simultaneously, both compounds showed low cytotoxicity (MCC). Z1 ≥100μM, MCC Z2 (≥100μM), which has further research and development value.

Claims

1. An N-hydroxythiophenepyrimidine dione derivative, characterized in that... is one of the following compounds:

2. The process for the preparation of N-hydroxythienopyrimidine dione derivatives according to claim 1, characterized in that The steps are as follows: 2-aminothiophene-3-carboxylic acid methyl ester as starting material, with carbonyl diimidazole, O-benzyl hydroxylamine cyclization to generate intermediate compound 1; intermediate compound 1 and N-iodosuccinimide iodination reaction under the generation of intermediate compound 2; The intermediate compound 2 is subjected to nucleophilic substitution reaction with iodoalkane under the action of potassium carbonate to generate intermediate compound 3; the intermediate compound 3 is subjected to Sonogashira coupling reaction under the condition that cuprous iodide and bis(triphenylphosphine)palladium dichloride are used as catalysts and triethylamine is used as base to obtain key intermediate compound 4; the intermediate compound 4 is subjected to trimethylsilyl removal under the action of potassium carbonate to obtain intermediate compound 5; the intermediate compound 5 and different azides are subjected to azide-alkynyl Husigen-Click cycloaddition reaction under the participation of sodium ascorbate and copper sulfate pentahydrate to generate intermediate compound 6; the intermediate compound 6 is subjected to benzyl protection removal in hydrobromic acid and acetic acid system under reflux to generate the target product I; The synthetic route is as follows: Reagents and conditions: (i) a) carbonyl diimidazole, O-benzyl hydroxylamine, tetrahydrofuran, 80°C, 5h; b) sodium hydroxide, ethanol, 85°C, 1h; c) acetic acid, r.t., 0.5h; (ii) N,N-dimethylformamide, dichloromethane, N-iodosuccinimide, room temperature, 3h; (iii) N,N-dimethylformamide, iodoalkane, potassium carbonate, 60°C, 8h; (iv) tetrahydrofuran, cuprous iodide, bis(triphenylphosphine)palladium dichloride, triethylamine, trimethylsilylacetylene, nitrogen, room temperature, 8h; (v) methanol, potassium carbonate, room temperature, 1h; (vi) sodium ascorbate, copper sulfate pentahydrate, water, tetrahydrofuran, azide, room temperature, 5h; (vii) hydrobromic acid, acetic acid, 120°C, 1h; Wherein, R1 is methylenecyclobutyl, R2 is 3-fluoro-4-(trifluoromethyl)phenyl or 2-fluoro-4-(trifluoromethyl)phenyl; The iodoalkane is (iodomethyl)cyclobutane, and the azide is 4-(azidomethyl)-2-fluoro-1-(trifluoromethyl)benzene or 1-(azidomethyl)-2-fluoro-4-(trifluoromethyl)benzene.

3. Use of the compound of claim 1 in the preparation of a medicament for the treatment of monkeypox virus and camelpox virus.

4. A pharmaceutical composition comprising the compound of claim 1 and one or more pharmaceutically acceptable carriers.

5. A pharmaceutical composition comprising the compound of claim 1 and one or more pharmaceutically acceptable excipients.

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