CN107501257B - Dihydropyrimidine-triazole derivative and preparation method and application thereof - Google Patents
Dihydropyrimidine-triazole derivative and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a dihydropyrimidine-triazole derivative and a preparation method and application thereof. The compound has a structure shown in formula I. The invention also relates to a preparation method of the compound containing the structure shown in the formula I, a pharmaceutical composition and application of the compound in preparing anti-HBV drugs.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a dihydropyrimidine-triazole derivative, and a preparation method and pharmaceutical application thereof.
Background
Viral Hepatitis B (Hepatitis B), abbreviated as Hepatitis B, is a serious infectious disease caused by Hepatitis B Virus (HBV), and can lead to acute and chronic viral Hepatitis, severe Hepatitis, cirrhosis and primary hepatocellular carcinoma (HCC) after long-term development. According to the World Health Organization (WHO), nearly 20 million people worldwide have been infected with HBV, about 2.4 million people are chronic HBV infected people, and on average, about 78 million people die each year from acute and chronic hepatitis and related complications caused by HBV infection. The current drugs for preventing and treating Chronic Hepatitis B (CHB) are mainly vaccines, interferons, immunomodulators and DNA polymerase inhibitors. However, due to the defects of drug resistance, side effect, rebound after drug withdrawal, incomplete hepatitis B virus elimination and the like, the discovery and research of a novel safe, high-efficiency, low-toxicity and drug-resistance non-nucleoside hepatitis B virus inhibitor are very important.
The core protein is the main structural protein composed of HBV nucleocapsid, and is relatively conserved in the virus evolution process, and the assembly of the core protein plays an important role in the life cycle of hepatitis B virus. However, no relevant target drugs are currently on the market. Aiming at the defects of strong hepatotoxicity, poor water solubility and poor metabolic stability of the existing clinical candidate drugs, a reasonable drug design based on a target point is carried out through a crystal compound structure of a core protein and a ligand, and a novel dihydropyrimidine-triazole compound is designed and synthesized, and the compound is not reported in the prior art.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a dihydropyrimidine-triazole derivative and a preparation method thereof, and also provides an activity screening result of the compound as a non-nucleoside HBV inhibitor and application thereof.
The technical scheme of the invention is as follows:
mono-or dihydropyrimidine-triazole derivatives
The dihydropyrimidine-triazole derivative has a structure shown in the following general formula I:
wherein the content of the first and second substances,
r is hydrogen, alkyl with different substitution, benzene ring with or without substituent, heterocycle with or without substituent;
according to the invention, in the formula I, R is hydrogen, benzene ring, 2-amino substituted benzene ring, 3-amino substituted benzene ring, 4-amino substituted benzene ring, pyridine ring, thiophene ring, 1-hydroxypentane group, p-methylbenzamide methyl group and mesitylene sulfonamide methyl group.
Further preferably, the dihydropyrimidine-triazole derivative is one of the compounds having the following structure:
TABLE 1 structural formula of dihydropyrimidine-triazole derivatives
Preparation method of di-or dihydropyrimidine-triazole derivatives
Firstly, taking a compound 2-thiazolecarboxamidine hydrochloride, 2-bromo-4-fluorobenzaldehyde and ethyl acetoacetate as starting raw materials, cyclizing by a 'one-pot method' to obtain a key intermediate 2, carrying out bromination reaction on the intermediate 2 and N-monobromo succinimide in a carbon tetrachloride solution to obtain an important intermediate 3, carrying out substitution reaction with sodium azide to obtain an important intermediate 4, and finally, carrying out cycloaddition on the intermediate 4 and alkyne containing different substituents to obtain a target compound I;
the synthetic route is as follows:
the reagent and the conditions are (i) 2-bromo-4-fluorobenzaldehyde, ethyl acetoacetate, sodium acetate and ethanol, and 80 ℃; (ii) n-bromosuccinimide, carbon tetrachloride, 50 ℃; (iii) sodium azide, acetone, 25 ℃; (iv) copper sulfate pentahydrate, sodium ascorbate, water, tetrahydrofuran, various substituted alkynes, 25 ℃;
wherein R is as described in the general formula I;
the alkyne ring containing different substituents is 2-aminophenylacetylene, 3-aminophenylacetylene, 2-ethynylpyridine, 2-ethynylthiophene, phenylacetylene, 4-aminophenylacetylene, propiolic acid, 1-heptyne-3-ol, N-propargyl- (4 methyl) benzamide and 2,4, 6-trimethyl-N (2-propynyl) benzenesulfonamide.
The preparation method of the dihydropyrimidine-triazole derivative comprises the following specific steps:
(1) dissolving 12.22mmol of 2-thiazole formamidine hydrochloride in 250mL of absolute ethanol, sequentially adding 18.42mmol of 2-bromo-4-fluorobenzaldehyde, 12.22mmol of ethyl acetoacetate and 12.22mmol of sodium acetate, and carrying out reflux reaction at 80 ℃ for 6 h; after the reaction is finished, cooling to room temperature, removing absolute ethyl alcohol by rotary evaporation, adding water, extracting for three times by ethyl acetate, combining organic phases, washing with saturated salt water for three times, and drying with anhydrous sodium sulfate; concentrating, loading by a dry method, separating by a rapid preparative chromatographic silica gel column, and recrystallizing to obtain a compound 2;
(2) dissolving the intermediate 24.71 mmol in 200mL carbon tetrachloride, slowly adding N-bromosuccinimide 4.94mmol, and refluxing at 50 ℃ for 10 h. After the reaction is finished, cooling to room temperature, removing carbon tetrachloride by rotary evaporation, adding water, extracting with ethyl acetate for three times, combining organic phases, washing with saturated salt water for three times, and drying with anhydrous sodium sulfate; concentrating, loading by a dry method, separating by a rapid preparative chromatographic silica gel column, and recrystallizing to obtain a compound 3;
(3) intermediate 3 was dissolved in 45mL acetone and NaN was added33.54mmol, the reaction was stirred at room temperature overnight. After the reaction is finished, cooling to room temperature, removing carbon tetrachloride by rotary evaporation, adding water, extracting with ethyl acetate for three times, combining organic phases, washing with saturated salt water for three times, and drying with anhydrous sodium sulfate; concentrating and recrystallizing to obtain a compound 4;
(4) 40.43 mmol of intermediate is dissolved in 6mL tetrahydrofuran, 0.043mmol of copper sulfate pentahydrate, 0.13mmol of sodium ascorbate and 0.86mmol of different substituted alkynes are added in sequence, and the mixture is stirred at room temperature and reacts overnight. After the reaction is finished, cooling to room temperature, adding water, extracting for three times by ethyl acetate, combining organic phases, washing for three times by saturated salt water, and drying by anhydrous sodium sulfate; concentrating, loading by a dry method, separating by a rapid preparative chromatographic silica gel column, and recrystallizing to obtain the target compound I.
The room temperature of the invention is 15-25 ℃.
Application of tri-dihydropyrimidine-triazole derivatives
The invention discloses an anti-HBV activity screening result of dihydropyrimidine-triazole derivatives and application thereof as an anti-HBV inhibitor. Experiments prove that the dihydropyrimidine-triazole derivative can be used as a classical HBV non-nucleoside inhibitor.
As shown in Table 2, the synthesized object compound I (5a-5j) was evaluated for anti-HBV activity in vitro, and the cell mortality was measured by CCK-8 method at drug concentrations of 20. mu.M and 5. mu.M; meanwhile, the activity of inhibiting HBV DNA replication under the drug concentration of 20 mu M and 5 mu M is determined by a PCR method, a lead compound GLS4 and a marketed drug lamivudine are selected as positive controls, and 5a and 5g of the positive controls show better activity of inhibiting HBV DNA replication.
As shown in Table 3, based on the results of the primary screening, the cytotoxicity of the drug at different concentrations was determined by the CCK-8 method for further evaluation of the anti-HBV activity in vitro of the primary screened target compounds 5a and 5 g; the inhibition activity of the drug on the HBV DNA replication under different concentrations is determined by a PCR method; simultaneously, by enzyme linked immunosorbent assayThe secretion inhibiting activity of the medicine on HBsAg and HBeAg antigen under different concentrations is determined. Lead compound GLS4 and marketed drug lamivudine were selected as positive controls, each compound was set to five concentration gradients (50. mu.M, 5. mu.M, 0.5. mu.M, 0.05. mu.M and 0.005. mu.M), and half inhibitory concentrations CC were calculated respectively50、IC50And a selectivity coefficient SI.
The dihydropyrimidine-triazole derivatives are non-nucleoside HBV inhibitors with novel structures, and can be used as anti-HBV lead compounds.
The dihydropyrimidine-triazole derivative can be used as a non-nucleoside HBV inhibitor. In particular to the application of the derivative as an HBV inhibitor in preparing anti-hepatitis B medicines.
An anti-HBV pharmaceutical composition comprises the dihydropyrimidine-triazole derivative and one or more pharmaceutically acceptable carriers or excipients.
The invention discloses a dihydropyrimidine-triazole derivative, a preparation method thereof, an anti-HBV activity screening result and a first application of the dihydropyrimidine-triazole derivative as an anti-HBV inhibitor. Experiments prove that the dihydropyrimidine-triazole derivative can be used as an HBV inhibitor for preparing anti-hepatitis B drugs.
Detailed Description
The present invention will be understood by reference to the following examples, in which all the numbers of the objective compounds are the same as those in Table 1, but the contents of the present invention are not limited thereto.
The synthetic route is as follows:
the reagent and the conditions are (i) 2-bromo-4-fluorobenzaldehyde, ethyl acetoacetate, sodium acetate and ethanol, and 80 ℃; (ii) n-bromosuccinimide, carbon tetrachloride, 50 ℃; (iii) sodium azide, acetone, 25 ℃; (iv) copper sulfate pentahydrate, sodium ascorbate, water, tetrahydrofuran, various substituted alkynes, 25 ℃;
EXAMPLE 1 preparation of Compound 2
A500 mL round-bottom flask was taken, 2-thiazolecarboxamidine hydrochloride (2.00g,12.22mmol) was dissolved in 250mL absolute ethanol, and 2-bromo-4-fluorobenzaldehyde (3.74g,18.42mmol), ethyl acetoacetate (1559. mu.L, 12.22mmol), sodium acetate (1.66g,12.22mmol) and the mixture was added sequentially at room temperature and reacted at 80 ℃ for 6h under reflux. After the reaction is finished, cooling to room temperature, removing anhydrous ethanol by rotary evaporation, adding water (60mL), extracting three times (25mL x3) by ethyl acetate, combining organic phases, washing three times (30mL x3) by saturated salt water, and drying by anhydrous sodium sulfate; concentrating, loading by a dry method, separating by a rapid preparative chromatography silica gel column, and recrystallizing with a dichloromethane-n-hexane mixed solvent to obtain 3.98g of yellow solid with a yield of 76%; melting point 153-156 ℃.
Compound 2 spectral data:1H NMR(400MHz,CDCl3)δ7.81(d,J＝2.8Hz,1H),7.46(s,1H),7.38–7.28(m,2H),6.97(t,J＝8.2Hz,1H),6.15(s,1H),4.05(q,J＝7.1Hz,2H),2.53(s,3H),1.13(t,J＝7.1Hz,3H)；EI-MS:424.3[M+H]+.
EXAMPLE 2 preparation of Compound 3
A500 mL round bottom flask was taken, intermediate 2(2.00g, 4.71mmol) was dissolved in 200mL carbon tetrachloride, NBS (0.88g, 4.94mmol) was added slowly, and the reaction was refluxed at 50 ℃ for 10 h. After the reaction is finished, cooling to room temperature, removing carbon tetrachloride by rotary evaporation, adding water (50mL), extracting with ethyl acetate for three times (20mL x3), combining organic phases, washing with saturated salt water for three times (25mL x3), and drying with anhydrous sodium sulfate; concentrating, loading by a dry method, separating by a rapid preparative chromatography silica gel column, and recrystallizing by a dichloromethane-n-hexane mixed solvent to obtain 1.21g of yellow solid with the yield of 51%; melting point 123-128 ℃.
Spectroscopic data for compound 1:1H NMR(400MHz,CDCl3)δ7.84(d,J＝3.1Hz,1H),7.52(s,2H),7.44–7.35(m,1H),7.32(dd,J＝8.1,2.6Hz,1H),7.02(t,J＝8.0Hz,1H),6.09(s,1H),4.94(d,J＝8.9Hz,1H),4.61(s,1H),4.09(d,J＝7.0Hz,2H),1.16(t,J＝7.1Hz,3H)；EI-MS:502.2[M+H]+.
EXAMPLE 3 preparation of Compound 4
A100 mL round-bottom flask was taken, intermediate X-3(0.89g, 1.77mmol) was dissolved in 45mL acetone, and NaN was added3(0.23g, 3.54mmol), and stirred at room temperature overnight. After the reaction is finished, cooling to room temperature, removing carbon tetrachloride by rotary evaporation, adding water (50mL), extracting with ethyl acetate for three times (20mL x3), combining organic phases, washing with saturated salt water for three times (25mL x3), and drying with anhydrous sodium sulfate; concentrating, loading by a dry method, separating by a rapid preparative chromatography silica gel column, and recrystallizing with a dichloromethane-n-hexane mixed solvent to obtain 0.85g of yellow solid with a yield of 91%; melting point 123-126 ℃.
Compound 4 spectroscopic data:1H NMR(400MHz,CDCl3)δ8.64(s,1H),7.85(d,J＝3.1Hz,1H),7.55(d,J＝3.1Hz,1H),7.48–7.37(m,1H),7.35–7.29(m,1H),7.10–6.92(m,1H),6.29–6.02(m,1H),4.97(s,1H),4.60(d,J＝2.6Hz,1H),4.17–4.00(m,2H),1.13(t,J＝7.1Hz,3H)；13C NMR(100MHz,CDCl3)δ165.78,165.03,163.31,162.71,162.36,162.09,160.80,160.22,154.83,150.00,143.92,143.54,143.10,142.75,139.59,137.74(d,J＝3.5Hz),130.80,130.72,130.61,124.92,123.38,122.07(d,J＝9.7Hz),120.23(dd,J＝24.4,17.0Hz),115.83,115.62,115.18,114.97,106.27,98.60,77.37,77.06,76.74,60.70,60.32,58.37,51.91(d,J＝2.0Hz),49.79,14.07(d,J＝5.7Hz)；EI-MS:465.4[M+H]+.
EXAMPLE 4 preparation of Compound 5a
A25 mL round-bottomed flask was taken, intermediate 4(200mg,0.43mmol) was dissolved in 6mL tetrahydrofuran, and copper sulfate pentahydrate (10.8mg,0.043mmol), sodium ascorbate (25.6mg,0.13mmol), and o-aminophenylacetylene (101mg,0.86mmol) were added in this order at room temperature, and stirred at room temperature overnight. After the reaction is finished, cooling to room temperature, adding water (40mL), extracting with ethyl acetate for three times (20mL x3), combining organic phases, washing with saturated salt water for three times (25mL x3), and drying with anhydrous sodium sulfate; concentrating, loading by a dry method, separating by a rapid preparative chromatography silica gel column, and recrystallizing by a dichloromethane-n-hexane mixed solvent to obtain a yellow solid with the yield: 73%, melting point: 184-187 ℃.
Compound 5a spectral data:1H NMR(400MHz,CDCl3)δ8.08(s,1H),7.79(d,J＝3.1Hz,1H),7.52(s,1H),7.47(d,J＝3.1Hz,1H),7.43(dd,J＝7.7,1.3Hz,1H),7.34(ddd,J＝9.3,8.4,4.2Hz,2H),7.16–7.07(m,1H),7.03(td,J＝8.3,2.5Hz,1H),6.77(dd,J＝6.6,5.9Hz,1H),6.72(dd,J＝11.6,4.3Hz,1H),6.17(t,J＝15.1Hz,1H),5.94(dd,J＝39.1,15.3Hz,2H),5.54(s,2H),4.14(q,J＝7.2Hz,2H),1.17(t,J＝7.1Hz,3H)；13C NMR(100MHz,CDCl3)δ165.02,162.10(d,J＝252.8Hz),161.77,152.47,150.09,147.97,145.20,143.95,137.62,130.77(d,J＝8.8Hz),128.78,127.74,125.06,122.04(d,J＝9.7Hz),121.84,120.37(d,J＝24.7Hz),117.24,116.67,115.85(d,J＝21.1Hz),114.17,106.48,60.92,52.12,52.00,14.08；EI-MS:582.3[M+H]+.
EXAMPLE 5 preparation of Compound 5b
The procedure is as in example 4, except that o-aminophenylacetylene is replaced by 2-aminophenylacetylene. Yellow solid, yield: 75%, melting point: 148-151 ℃.
Compound 5b spectral data:1H NMR(400MHz,CDCl3)δ8.02(s,1H),7.78(d,J＝3.1Hz,1H),7.54(s,1H),7.45(d,J＝3.1Hz,1H),7.41–7.28(m,3H),7.23–7.15(m,2H),7.03(td,J＝8.3,2.5Hz,1H),6.73–6.58(m,1H),6.17(d,J＝32.5Hz,1H),5.92(q,J＝15.4Hz,2H),4.13(q,J＝7.1Hz,2H),3.77(s,2H),1.16(t,J＝7.1Hz,3H)；13C NMR(100MHz,CDCl3)δ165.04,162.08(d,J＝252.8Hz),161.82,152.60,150.06,147.41,146.88,143.93,137.66(d,J＝3.4Hz),132.04,130.80(d,J＝8.8Hz),129.71,125.07,123.28,122.03(d,J＝9.7Hz),121.70,120.34(d,J＝24.7Hz),116.10,115.82(d,J＝21.1Hz),114.69,112.34,106.33,60.90,52.12,52.00,14.08；EI-MS:582.3[M+H]+.
EXAMPLE 6 preparation of Compound 5c
The procedure is as in example 4, except that o-aminophenylacetylene is replaced by 2-ethynylpyridine. Orange solid, yield: 74% and a melting point of 194-197 ℃.
Compound 5c spectral data:1H NMR(400MHz,CDCl3)δ8.59(d,J＝4.8Hz,1H),8.39(s,1H),8.22(d,J＝7.9Hz,1H),7.84–7.71(m,2H),7.53(s,1H),7.43(d,J＝3.1Hz,1H),7.38(dd,J＝8.6,5.9Hz,1H),7.32(dd,J＝6.6,4.0Hz,1H),7.22(ddd,J＝7.5,4.9,1.1Hz,1H),7.04(td,J＝8.3,2.5Hz,1H),6.13(d,J＝2.4Hz,1H),5.95(dd,J＝31.5,15.3Hz,2H),4.14(q,J＝7.1Hz,2H),1.16(t,J＝7.1Hz,3H)；13C NMR(100MHz,CDCl3)δ164.97,162.08(d,J＝252.7Hz),161.83,152.44,150.77,150.12,149.41,148.02,143.87,137.69(d,J＝3.5Hz),136.81,130.81(d,J＝8.8Hz),125.04,123.71,122.61,122.02(d,J＝9.7Hz),120.45,120.27,120.20,115.85(d,J＝21.1Hz),106.58,60.91,52.31,51.99,14.07；EI-MS:568.4[M+H]+.
EXAMPLE 7 preparation of Compound 5d
The procedure is as in example 4, except that o-aminophenylacetylene is replaced by 2-ethynylthiophene. Orange solid, yield: 76%, melting point: 108-110 ℃.
Compound 5d spectroscopic data:1H NMR(400MHz,CDCl3)δ7.99(s,1H),7.79(d,J＝3.1Hz,1H),7.52(s,1H),7.47(d,J＝3.1Hz,1H),7.44–7.39(m,1H),7.39–7.27(m,3H),7.08(dd,J＝5.0,3.6Hz,1H),7.04(td,J＝8.3,2.6Hz,1H),6.13(s,1H),5.91(q,J＝15.5Hz,2H),4.21–4.07(m,2H),1.17(t,J＝8.3Hz,3H)；13C NMR(100MHz,CDCl3)δ165.01,162.11(d,J＝252.8Hz),161.77,152.38,150.07,143.96,142.38,137.61(d,J＝3.6Hz),133.53,130.78(d,J＝8.8Hz),127.64(d,J＝10.3Hz),125.04,124.72,123.91,122.04(d,J＝9.7Hz),121.28,120.37(d,J＝24.6Hz),115.84(d,J＝21.0Hz),106.41,60.93,52.14,52.00,14.07；EI-MS:573.4[M+H]+.
EXAMPLE 8 preparation of Compound 5e
The procedure is as in example 4, except that the o-aminophenylacetylene is replaced by phenylacetylene. Orange solid, yield: 71%, melting point: 109-112 ℃.
Compound 5e spectral data:1H NMR(400MHz,CDCl3)δ8.07(s,1H),7.90(d,J＝1.3Hz,1H),7.88(s,1H),7.78(d,J＝3.1Hz,1H),7.54(s,1H),7.48–7.29(m,6H),7.03(td,J＝8.3,2.6Hz,1H),6.13(s,1H),5.93(dd,J＝36.0,15.4Hz,2H),4.14(q,J＝7.1Hz,2H),1.17(t,J＝7.1Hz,3H)；13C NMR(101MHz,CDCl3)δ165.04,162.09(d,J＝252.8Hz),161.81,152.55,150.06,147.33,137.65(d,J＝3.5Hz),131.14,130.80(d,J＝8.8Hz),128.91,128.81,127.89,125.84,125.75,125.01,122.05(d,J＝9.7Hz),121.66,120.36(d,J＝24.6Hz),115.82(d,J＝21.1Hz),106.41,60.91,52.13,52.01,14.08；EI-MS:567.4[M+H]+.
example 9 preparation of Compound 5f
The procedure is as in example 4, except that o-aminophenylacetylene is replaced by 4-aminophenylacetylene. Orange solid, yield: 52%, melting point: 123-127 ℃.
Compound 5f spectral data:1H NMR(400MHz,CDCl3)δ7.93(s,1H),7.78(d,J＝3.0Hz,1H),7.68(d,J＝8.2Hz,2H),7.52(s,1H),7.45(d,J＝2.8Hz,1H),7.40–7.34(m,1H),7.32(dd,J＝8.1,2.5Hz,1H),7.02(t,J＝9.1Hz,1H),6.74(d,J＝8.5Hz,2H),6.17(d,J＝29.7Hz,1H),5.90(q,J＝15.2Hz,2H),4.13(q,J＝7.1Hz,2H),3.78(s,2H),1.17(t,J＝7.1Hz,3H)；13CNMR(100MHz,CDCl3)δ165.05,162.08(d,J＝252.6Hz),161.88,152.71,150.04,147.66(s),146.26,143.91,137.64,130.81(d,J＝8.9Hz),126.96,125.01,121.98,121.74,120.35,120.33(d,J＝24.6Hz),115.81(d,J＝21.1Hz),115.27,106.37,60.89,52.08,51.99,14.08；EI-MS:582.3[M+H]+.
EXAMPLE 10 preparation of 5g Compound
The procedure is as in example 4, except that o-aminophenylacetylene is replaced by propiolic acid. Orange solid, yield: 72%, melting point: 115-119 ℃.
Compound 5g spectral data:1H NMR(400MHz,CDCl3)δ7.85(d,J＝0.7Hz,1H),7.80(d,J＝3.1Hz,1H),7.75(s,1H),7.52(s,1H),7.49(d,J＝3.1Hz,1H),7.39–7.28(m,2H),7.03(td,J＝8.3,2.6Hz,1H),6.13(d,J＝2.4Hz,1H),5.95(s,1H),5.90(s,1H),4.13(q,J＝7.1Hz,2H),1.15(t,J＝7.1Hz,3H)；13C NMR(100MHz,CDCl3)δ165.00,162.08(d,J＝252.8Hz),161.81,152.58,150.03,143.97,137.64(d,J＝3.5Hz),133.35,130.76(d,J＝8.8Hz),125.23,124.90,122.03(d,J＝9.6Hz),120.34(d,J＝24.6Hz),115.80(d,J＝21.1Hz),106.44,60.88,51.96,51.85,14.05；EI-MS:491.4[M+H]+.
EXAMPLE 11 preparation of Compound 5h
The procedure is as in example 4, except that o-aminophenylacetylene is replaced by 1-heptyn-3-ol. Orange solid, yield: 61%, melting point: 79 to 83 ℃.
Compound 5h spectral data:1H NMR(400MHz,CDCl3)δ7.78(d,J＝3.1Hz,1H),7.76(s,1H),7.46(d,J＝2.9Hz,1H),7.39–7.25(m,2H),7.02(td,J＝8.3,2.3Hz,1H),6.13(s,1H),5.93(dd,J＝15.4,7.7Hz,1H),5.82(dd,J＝15.4,7.5Hz,1H),4.93(t,J＝6.5Hz,1H),4.11(q,J＝7.1Hz,2H),2.60(s,1H),1.99–1.84(m,2H),1.63–1.28(m,4H),1.15(t,J＝7.1Hz,3H),0.88(t,J＝7.1Hz,3H)；EI-MS:577.5[M+H]+.
EXAMPLE 12 preparation of Compound 5i
The procedure is as in example 4, except that o-aminophenylacetylene is replaced by N-propargyl- (4-methyl) benzamide. Orange solid, yield: 47%, melting point: 85-89 ℃.
Compound 5i spectral data:1H NMR(400MHz,CDCl3)δ7.93(s,1H),7.72(d,J＝8.1Hz,2H),7.70(d,J＝3.1Hz,1H),7.30(ddd,J＝15.0,7.8,5.2Hz,3H),7.20(d,J＝7.9Hz,2H),7.15(s,1H),7.00(td,J＝8.3,2.5Hz,1H),6.11(s,1H),5.89(dd,J＝40.6,15.7Hz,2H),4.76(d,J＝5.2Hz,2H),4.11(q,J＝7.1Hz,2H),2.37(s,3H),1.14(t,J＝7.1Hz,3H)；EI-MS:638.3[M+H]+.
example 13 preparation of Compound 5j1
The procedure is as in example 4, except that o-aminophenylacetylene is replaced by 2,4, 6-trimethyl-N (2-propynyl) benzenesulfonamide. Orange solid, yield: 61%, melting point: 96-99 ℃.
Compound 5j spectral data:1H NMR(400MHz,CDCl3)δ7.81(d,J＝3.1Hz,1H),7.69(s,1H),7.51(d,J＝3.1Hz,2H),7.36–7.29(m,2H),7.04(td,J＝8.3,2.5Hz,1H),6.95(s,2H),6.12(d,J＝2.5Hz,1H),5.05(t,J＝6.0Hz,1H),4.25(d,J＝6.1Hz,2H),4.12(d,J＝7.1Hz,3H),2.65(s,6H),2.31(s,3H),1.15(t,J＝7.1Hz,3H)；13C NMR(100MHz,CDCl3)δ164.94,162.09(d,J＝252.9Hz),161.70,152.26,150.06,144.00,142.94,142.34,139.11,137.63(d,J＝3.5Hz),133.50,132.01,130.77(d,J＝8.8Hz),125.08,124.14,122.02(d,J＝9.7Hz),120.35(d,J＝24.7Hz),115.85(d,J＝21.1Hz),106.36,60.89,58.31,51.97,38.34,22.98,20.95,14.05；EI-MS:702.4[M+H]+.
example 14 in vitro anti-HBV cell Activity screening assay for Compounds of interest
Principle of testing
The HBV transfected hepatoma cell HepG2.2.15 cell strain can secrete HBV virus particles (comprising HBsAg, HBeAg and DNA) when being cultured. Under the intervention of anti-HBV target compounds, the content of HBsAg and HBeAg secreted by cells and the generated DNA are changed, so that the content of HBsAg and HBeAg secreted by cells into culture supernatant and the generated HBV DNA are detected, and the antiviral activity of a sample medicament can be reflected by referring to the content of an unformed control group. Using lamivudine as positive control drug, and detecting the concentration value of the sample drug reaching 50% of the secretion of HBsAg and HBeAg for inhibiting virus by enzyme-linked immunosorbent assay (ELISA) to be IC50(ii) a Polymerase Chain Reaction (PCR) method for detecting concentration value IC of drug for inhibiting 50% of HBV DNA replication50(ii) a The numerical concentration of the drug causing 50% cytotoxic death in the sample tested using CCK-8 was CC50A value; and calculating the 'selection coefficient' (selectivity index) of the compound to be detected, and calculating the formula: SI ═ CC50/IC50。
Test method
(1) Cytotoxicity test
The stock concentration (100 mu mol/L) of samples required by the experiment is prepared, 2 dilution concentrations (20 mu mol/L and 5 mu mol/L) of each sample are prepared by HepG2.2.15 cell culture solution for primary activity screening, a blank control is set, and lamivudine is used as a positive control drug. Adding 96-well plate cell culture plate, repeating the wells at a concentration of 3 times, changing the liquid medicine with the same concentration every 4 days, setting a drug-free cell control group, and culturing for 9 days. The cell survival rate is detected by a CCK-8 method, and the toxicity of the drug to HepG2.2.15 cells is determined. For the active compound, 5 dilutions (50. mu. mol/L and 5. mu. mol/L, 0.5. mu. mol/L, 0.05. mu. mol/L, 0.005. mu. mol/L) were prepared in HepG2.2.15 cell culture medium, and a blank control was set up and lamivudine was used as a positive control. Adding 96-well plate cell culture plate, repeating the wells at a concentration of 3 times, changing the liquid medicine with the same concentration every 4 days, setting a drug-free cell control group, and culturing for 9 days. The cell survival rate is detected by a CCK-8 method, and the toxicity of the drug to HepG2.2.15 cells is determined.
(2) Experiment for inhibiting HBeAg and HBsAg antigen secretion
After the HepG22.2.15 cells were cultured in a 96-well cell culture plate for 24 hours, the prepared drug-containing culture solutions of different concentrations were added, the culture was continued for 8 days (the solution was changed every 4 days), and the supernatant was collected and HBsAg and HBeAg were detected using HBsAg and HBeAg diagnostic kits (ELISA).
(3) Experiment for inhibiting HBV DNA Synthesis (PCR method)
HepG22.2.15 cells were cultured in a 96-well cell culture plate for 24 hours, then the prepared drug-containing culture medium of 20. mu. mol/L and 5. mu. mol/L was added thereto, and the cells were cultured for another 8 days (changing the medium every 4 days), and the supernatant was collected and subjected to PCR detection by the probe method.
TABLE 2 preliminary evaluation of inhibition of HBV DNA replication and cytotoxicity by Compounds of interest
As shown in Table 2, 13 of the synthesized compounds were evaluated for anti-HBV activity in vitro, and cell mortality was measured by CCK-8 at drug concentrations of 20. mu.M and 5. mu.M; meanwhile, the inhibitory activity of HBV DNA replication was determined by PCR method at drug concentrations of 20. mu.M and 5. mu.M.
The primary activity screening results show that the compounds 3, 4, 5b, 5c, 5f, 5h and 5i show larger cytotoxicity at the concentration of 20 mu M, and the inhibition rate of most compounds for inhibiting the HBV DNA replication activity is more than 50 percent and is equivalent to the activity of lamivudine, so the primary activity screening at the concentration of 5 mu M is carried out. The experimental results show that at 5 μ M concentration, all compounds show lower cell death rate and are close to or lower than the cytotoxicity of the lead compound GLS4 and the positive drug lamivudine; in addition, the target compounds 5a and 5g show relatively good inhibitory activity against HBV DNA replication, the inhibitory rates are 59.9 +/-2.0 and 68.2 +/-6.8 respectively, which are equivalent to and superior to the inhibitory activity against HBV DNA replication of the positive drug lamivudine (59.4 +/-1.8), but are weaker than the inhibitory activity against HBV DNA replication of the lead compound GLS4 (83.7 +/-1.6), and further activity study can be carried out.
TABLE 3 anti-HBV activity of active compound, lead compound GLS4 and marketed drug lamivudine
As shown in Table 3, based on the results of the primary screening, the cytotoxicity of the drug at different concentrations was determined by the CCK-8 method for further evaluation of the anti-HBV activity in vitro of the primary screened target compounds 5a and 5 g; the inhibition activity of the drug on the HBV DNA replication under different concentrations is determined by a PCR method; meanwhile, the secretion inhibition activity of the medicament to HBsAg and HBeAg antigens under different concentrations is measured by an enzyme-linked immunosorbent assay. Lead compound GLS4 and marketed drug lamivudine were selected as positive controls, each compound was set to five concentration gradients (50. mu.M, 5. mu.M, 0.5. mu.M, 0.05. mu.M and 0.005. mu.M), and half inhibitory concentrations CC were calculated respectively50、IC50And a selectivity coefficient SI.
The activity results show that the target compound 5a shows less cytotoxicity, the CC of which50Greater than 50 mu M, obviously superior to GLS4(22.4 +/-2.1 mu M); in addition, it also shows better HBV DNA replication inhibition activity, IC thereof500.35 +/-0.04 mu M, which is superior to the marketed drug lamivudine (0.54 +/-0.18 mu M) and is weaker than GLS4(0.13 +/-0.05 mu M); 5a the selectivity coefficient (SI) for inhibiting HBV DNA replication is more than 143, is better than or similar to that of a lead compound GLS4(22.4 +/-2.1 mu M) and a marketed drug lamivudine (Lamivudine) (II)>93) However, the inhibitory activity against HBsAg and HBeAg secretion was not exhibited. 5g of the compound showed a certain cytotoxicity, its CC5020.9 +/-1.2 mu M, slightly higher than the precursorCompound GLS4 and lamivudine; in addition, certain inhibitory activity of HBV DNA replication, IC thereof, was also shown500.86 +/-0.32 mu M, which is the same order of magnitude as GLS4 and lamivudine; it may show weak HBsAg and HBeAg secretion inhibiting activity due to its cytotoxicity, and its IC5016.5. + -. 0.42. mu.M and 14.2. + -. 0.85. mu.M, respectively, and therefore further modification studies were required.
Claims (5)
1. Dihydropyrimidine-triazole derivatives characterized by being one of the compounds having the following structure:
2. the process for preparing dihydropyrimidine-triazole derivatives as claimed in claim 1, which comprises the following steps:
the reagent and the conditions are (i) 2-bromo-4-fluorobenzaldehyde, ethyl acetoacetate, sodium acetate and ethanol, and 80 ℃; (ii) n-bromosuccinimide, carbon tetrachloride, 50 ℃; (iii) sodium azide, acetone, 25 ℃; (iv) copper sulfate pentahydrate, sodium ascorbate, tetrahydrofuran, various substituted alkynes, 25 ℃;
wherein R is 2-amino substituted benzene ring, 3-amino substituted benzene ring, 2-substituted pyridine ring, 4-amino substituted benzene ring, 1-hydroxypentanyl and p-methylbenzamide methyl;
the alkyne ring containing different substituents is 2-aminophenylacetylene, 3-aminophenylacetylene, 2-ethynylpyridine, 4-aminophenylacetylene, 1-heptyne-3-ol and N-propargyl- (4 methyl) benzamide.
3. The process for producing a dihydropyrimidine-triazole derivative according to claim 2, which comprises the steps of:
(1) dissolving 12.22mmol of 2-thiazole formamidine hydrochloride in 250mL of absolute ethanol, sequentially adding 18.42mmol of 2-bromo-4-fluorobenzaldehyde, 12.22mmol of ethyl acetoacetate and 12.22mmol of sodium acetate, and carrying out reflux reaction at 80 ℃ for 6 h; after the reaction is finished, cooling to room temperature, removing absolute ethyl alcohol by rotary evaporation, adding water, extracting for three times by ethyl acetate, combining organic phases, washing with saturated salt water for three times, and drying with anhydrous sodium sulfate; concentrating, loading by a dry method, separating by a rapid preparative chromatographic silica gel column, and recrystallizing to obtain a compound 2;
(2) dissolving the intermediate 24.71 mmol in 200mL of carbon tetrachloride, slowly adding N-bromosuccinimide 4.94mmol, and carrying out reflux reaction at 50 ℃ for 10 h; after the reaction is finished, cooling to room temperature, removing carbon tetrachloride by rotary evaporation, adding water, extracting with ethyl acetate for three times, combining organic phases, washing with saturated salt water for three times, and drying with anhydrous sodium sulfate; concentrating, loading by a dry method, separating by a rapid preparative chromatographic silica gel column, and recrystallizing to obtain a compound 3;
(3) intermediate 3 was dissolved in 45mL acetone and NaN was added33.54mmol, stirring and reacting at 25 ℃ overnight; after the reaction is finished, cooling to room temperature, removing carbon tetrachloride by rotary evaporation, adding water, extracting with ethyl acetate for three times, combining organic phases, washing with saturated salt water for three times, and drying with anhydrous sodium sulfate; concentrating and recrystallizing to obtain a compound 4;
(4) dissolving 40.43 mmol of intermediate in 6mL of tetrahydrofuran, sequentially adding 0.043mmol of copper sulfate pentahydrate, 0.13mmol of sodium ascorbate and 0.86mmol of different substituted alkynes, and stirring at 25 ℃ for reaction overnight; after the reaction is finished, cooling to room temperature, adding water, extracting for three times by ethyl acetate, combining organic phases, washing for three times by saturated salt water, and drying by anhydrous sodium sulfate; concentrating, loading by a dry method, separating by a rapid preparative chromatographic silica gel column, and recrystallizing to obtain a target compound; the different substituted alkynes are as defined in claim 2.
4. Use of a compound according to claim 1 for the manufacture of a medicament against HBV.
5. An anti-HBV pharmaceutical composition comprising a compound of claim 1 and one or more pharmaceutically acceptable carriers or excipients.
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