Application of a 6,8-dihydroxypurine compound in the preparation of anti-hepatitis B virus drugs

Abstract

The invention provides an application of a 6,8-xanthine compound in preparing anti-HBV (Hepatitis B Virus) drugs. The 6,8-xanthine compound according to the invention is used for preparing the anti-HBV drugs, has a function of inhibiting HBV core protein assembly, is capable of fundamentally inhibiting HBV, has no toxic side effect to cells and has a wide application prospect at the aspect of HBVtreatment.

Description

technical field

[0001] The invention belongs to the application technical field of ethanol compounds, and relates to the application of a 6,8-dihydroxypurine compound in the preparation of anti-hepatitis B virus drugs. Background technique

[0002] Hepatitis B virus (HBV) infection is an important public health problem worldwide. After acute hepatitis B virus infection, about 8% still develop into chronic hepatitis B infection, and persistent HBV infection will lead to liver cirrhosis and even liver cancer. my country is a big country with hepatitis B, and there are nearly 130 million hepatitis B virus carriers, accounting for about 9% of the total population. Although the new hepatitis B infection rate has been effectively controlled with the widespread popularization of hepatitis B vaccine, the population base of hepatitis B carriers is large, and the prevention and treatment of hepatitis B has become the top priority of public health problems in my country. Hepatitis B ...

Examples

Embodiment 1

[0027] Study on the Effect of 6,8-Dihydroxypurine on Inhibiting the Reciprocal Binding of HBV c Antigen

[0028] Experimental method: Take well-grown human adrenal cell line 239t cells and inoculate them in a 96-well transparent flat-bottomed plate, with 5×104 cells per well. The medium used is complete medium: high-glucose DMEM, 10% fetal bovine serum and 1% double antibody, the culture condition is 5% carbon dioxide, 37°C; C and pcDNA3.1-HBVcAg-VFP-N two plasmids. For transfection, liposome-encapsulated transfection was used, lipo2000 was used as the reagent, and 20 μl of transfection solution was used. After 4 hours of transfection, compound A to be screened was added, 2 μl per well, with a final concentration of 50 μM. After culturing for 48 hours, the expression of green fluorescent protein VFP was detected. If there is a decrease in the expression of green fluorescent protein VFP, the compound may become an antiviral drug candidate. Experimental results such as figu...

Embodiment 2

[0030] 6,8-Dihydroxypurine inhibits viral replication of wild-type HBV

[0031] Experimental method: Take the well-grown cell line HepG2.2.15 that can produce wild-type HBV virus, the amount of cells is 2×104 / well, add compound A after 24 hours of laying on a 96-well plate, and compound 2 μl per hole (final concentration 250 μM, 50 μM , 10 μM, 2 μM, 0.4 μM, 0.08 μM, 0.016 μM, 0 μM); use 2% Triton X-100 to treat the collected supernatant, harvest the cultured cell supernatant for 8 days, and then detect the HBV content in the cell culture supernatant DNA content. Experimental results such as figure 2 shown. It can be seen from the experimental results that 6,8-dihydroxypurine has a good effect of inhibiting the replication of wild-type HBV virus, and its IC50 is 1.891 μM.

Embodiment 3

[0033] Cytotoxicity of 6,8-dihydroxypurine in 293t cells

[0034] Experimental method: use DMEM culture medium containing 10% fetal calf serum to prepare 293t into a single cell suspension, inoculate 1000 cells per well into a 96-well plate with a volume of 200ul per well; 2 μl, the final concentration is 50 μM, 5 μM, 0.5 μM, 0.05 μM, 0.005 μM, 0.0005 μM, 0 μM; after 48 hours of culture, add 20 μl of MTS solution to each well, and continue to incubate in the incubator for 2 to 4 hours; The light absorption value of each well was measured on an enzyme-linked immunosorbent monitor, and the cytotoxicity of the compound to 293t cells was observed. Experimental results such as image 3 shown. It can be seen from the experimental results that 6,8-dihydroxypurine has low toxicity, and it shows no cytotoxicity in 293t cells.

technical field

[0001] The invention belongs to the application technical field of ethanol compounds, and relates to the application of a 6,8-dihydroxypurine compound in the preparation of anti-hepatitis B virus drugs. Background technique

[0002] Hepatitis B virus (HBV) infection is an important public health problem worldwide. After acute hepatitis B virus infection, about 8% still develop into chronic hepatitis B infection, and persistent HBV infection will lead to liver cirrhosis and even liver cancer. my country is a big country with hepatitis B, and there are nearly 130 million hepatitis B virus carriers, accounting for about 9% of the total population. Although the new hepatitis B infection rate has been effectively controlled with the widespread popularization of hepatitis B vaccine, the population base of hepatitis B carriers is large, and the prevention and treatment of hepatitis B has become the top priority of public health problems in my country. Hepatitis B ...

Examples

Embodiment 1

[0027] Study on the Effect of 6,8-Dihydroxypurine on Inhibiting the Reciprocal Binding of HBV c Antigen

[0028] Experimental method: Take well-grown human adrenal cell line 239t cells and inoculate them in a 96-well transparent flat-bottomed plate, with 5×104 cells per well. The medium used is complete medium: high-glucose DMEM, 10% fetal bovine serum and 1% double antibody, the culture condition is 5% carbon dioxide, 37°C; C and pcDNA3.1-HBVcAg-VFP-N two plasmids. For transfection, liposome-encapsulated transfection was used, lipo2000 was used as the reagent, and 20 μl of transfection solution was used. After 4 hours of transfection, compound A to be screened was added, 2 μl per well, with a final concentration of 50 μM. After culturing for 48 hours, the expression of green fluorescent protein VFP was detected. If there is a decrease in the expression of green fluorescent protein VFP, the compound may become an antiviral drug candidate. Experimental results such as figu...

Embodiment 2

[0030] 6,8-Dihydroxypurine inhibits viral replication of wild-type HBV

[0031] Experimental method: Take the well-grown cell line HepG2.2.15 that can produce wild-type HBV virus, the amount of cells is 2×104 / well, add compound A after 24 hours of laying on a 96-well plate, and compound 2 μl per hole (final concentration 250 μM, 50 μM , 10 μM, 2 μM, 0.4 μM, 0.08 μM, 0.016 μM, 0 μM); use 2% Triton X-100 to treat the collected supernatant, harvest the cultured cell supernatant for 8 days, and then detect the HBV content in the cell culture supernatant DNA content. Experimental results such as figure 2 shown. It can be seen from the experimental results that 6,8-dihydroxypurine has a good effect of inhibiting the replication of wild-type HBV virus, and its IC50 is 1.891 μM.

Embodiment 3

[0033] Cytotoxicity of 6,8-dihydroxypurine in 293t cells

[0034] Experimental method: use DMEM culture medium containing 10% fetal calf serum to prepare 293t into a single cell suspension, inoculate 1000 cells per well into a 96-well plate with a volume of 200ul per well; 2 μl, the final concentration is 50 μM, 5 μM, 0.5 μM, 0.05 μM, 0.005 μM, 0.0005 μM, 0 μM; after 48 hours of culture, add 20 μl of MTS solution to each well, and continue to incubate in the incubator for 2 to 4 hours; The light absorption value of each well was measured on an enzyme-linked immunosorbent monitor, and the cytotoxicity of the compound to 293t cells was observed. Experimental results such as image 3 shown. It can be seen from the experimental results that 6,8-dihydroxypurine has low toxicity, and it shows no cytotoxicity in 293t cells.

Images