Application of B/E bi-methoxy silybin in preparing medicaments for treating viral hepatitis B

Abstract

The invention relates to application of B / E bi-methoxy silybin in preparing medicaments for treating viral hepatitis B, in particular to application of silybin ester substituted by the methoxy on the ring B and the ring E or a pharmaceutically acceptable salt thereof in preparing medicaments for clearing away HBsAg (Hepatitis B Surface Antigen) and HBeAg (Hepatitis B e Antigen) and suppressing the HBV (Hepatitis B Virus) DNA replication. The B / E bi-methoxy silybin has strong activity on suppressing the HBsAg and the HBeAg, and in the presence of a concentration of 20 micrograms / milliliter, the intensities for clearing away the HBsAg and the HBeAg are respectively 43.9 percent and 43.7 percent which are 2.7 times and 2.6 times of that of alpha-interferon which is a positive control medicament. In the presence of the concentration, the suppression ratio on the HBV DNA is 68.6 percent, and the suppression activity is 1.8 times of that of the alpha-interferon. Accordingly, the flavone lignan or the pharmaceutically acceptable salt thereof are indicated to simultaneously have the effects of strongly suppressing the HBsAG, the HBeAg and the HBV DNA and can be expected to be used for preparing the non-nucleoside medicaments for treating HBV infection diseases.

Description

technical field

[0001] The present invention relates to the technical field of medicine, in particular, the present invention relates to a kind of silybin ester or pharmaceutically acceptable salt thereof which is substituted by ring B and ring E with dimethoxyl groups, which is used for the preparation of HBsAg and hepatitis B e Application of antigen HBeAg, inhibiting HBV DNA replication, and medicine for treating hepatitis B virus infection. The flavonoid lignans have a strong activity of inhibiting HBsAg and HBeAg, and the intensity of removing HBsAg and HBeAg at a concentration of 20 μg / ml is 43.9% and 43.7%, respectively, which are positive control drugs (10000 units / ml of α- Interferon) activity of 2.7 times and 2.6 times; at this concentration it showed 68.6% inhibition of HBVDNA, inhibitory activity is 1.8 times that of alpha-interferon. The above shows that the flavonoid lignan or its pharmaceutically acceptable salt has the effect of inhibiting HBsAg, HBeAg and HBV...

Examples

Embodiment 1

[0029] Example 1: Compound (±)-2-[2,3-dihydro-3-(4-hydroxy-3,5-dimethoxyphenyl)-2-hydroxymethyl-8-methoxy- Preparation of 1,4-benzodioxane-6-]-2,3-dihydro-3,5,7-trihydroxy-4H-1-benzopyran-4-one

[0030] 1.1 Instruments and reagents:

[0031] The ultraviolet spectrum was measured with a Shimadzu UV-240 ultraviolet spectrophotometer; the hydrogen nuclear magnetic resonance spectrum 1 H-NMR is measured by INOVA type superconducting nuclear magnetic resonance spectrometer (VARIAN INOVA-400MHz) (tetramethylsilyl ether TMS is the internal standard); (100-200, 200-300 and 300-400 mesh) and silica gel GF254 (10-40 mesh) for thin-layer chromatography are produced by Qingdao Ocean Chemical Factory; all reagents used are analytically pure, thin-layer preparative chromatography (PTLC ) uses the aluminum foil silica gel plate of Merck Company; Sephadex LH-20 used for column chromatography adopts the product of Amersham Pharmacia Biotech AB Company of Sweden; Reversed-phase silica gel RP...

Embodiment 2

[0044] Example 2: Inhibitory Effect of Compound (1) on Hepatitis B Surface Antigen (HBsAg) Secreted by HepG2.2.15 Cells

[0045] 2.1 Cell culture:

[0046] HepG2.2.15 cells were cultured in DMEM medium containing 10% inactivated fetal bovine serum, 100 U / ml penicillin and 100 U / ml streptomycin, 100 μg / ml G418 at 37°C, 5% CO 2 , cultured in an incubator with 100% relative humidity.

[0047] 2.2 The inhibitory effect of the compound of formula (1) on HepG2.2.15 cell growth was measured by MTT method:

[0048] Take the HepG2.2.15 cells in the logarithmic growth phase, and dilute the cells to 1×10 with medium 5 cells / ml, seeded in 96-well cell culture plate, 100 μl per well, at 37°C, 5% CO 2 After cultivating in an incubator with 100% relative humidity for 24 hours, add compound (1) diluted with medium, the concentration is 1000, 200, 40 and 8 μg / ml respectively, 200 μl per well, and three concentrations are set for each Duplicate wells, placed at 37°C, 5% CO 2 , cultivated...

Embodiment 3

[0058] Example 3: Inhibitory Effect of Compound (1) on Hepatitis B e Antigen (HBeAg) Secreted by HepG2.2.15 Cells

[0059]3.1 Cell culture: the method is the same as in Example 2.

[0060] 3.2 Determination of the inhibitory effect of the compound of formula (1) on the growth of HepG2.2.15 cells by MTT method: the method is the same as in Example 2.

[0061] 3.3 Determination of the inhibitory effect of the compound on hepatitis B e antigen (HBeAg): take the HepG2.2.15 cells in the logarithmic growth phase, and dilute the cells to 1 × 10 with the medium 5 / ml, seeded in 96-well cell culture plate, 100ml per well, at 37°C, 5% CO 2 After culturing in an incubator with 100% relative humidity for 24 hours, add samples diluted with culture medium at concentrations of 20 μg / ml, 4 μg / ml and 0.84 μg / ml, 200 μl per well, and set three concentrations for each Multiple wells were placed at 37°C, 5% CO 2 , cultivated in an incubator with 100% relative humidity, change the culture med...

technical field

[0001] The present invention relates to the technical field of medicine, in particular, the present invention relates to a kind of silybin ester or pharmaceutically acceptable salt thereof which is substituted by ring B and ring E with dimethoxyl groups, which is used for the preparation of HBsAg and hepatitis B e Application of antigen HBeAg, inhibiting HBV DNA replication, and medicine for treating hepatitis B virus infection. The flavonoid lignans have a strong activity of inhibiting HBsAg and HBeAg, and the intensity of removing HBsAg and HBeAg at a concentration of 20 μg / ml is 43.9% and 43.7%, respectively, which are positive control drugs (10000 units / ml of α- Interferon) activity of 2.7 times and 2.6 times; at this concentration it showed 68.6% inhibition of HBVDNA, inhibitory activity is 1.8 times that of alpha-interferon. The above shows that the flavonoid lignan or its pharmaceutically acceptable salt has the effect of inhibiting HBsAg, HBeAg and HBV...

Examples

Embodiment 1

[0029] Example 1: Compound (±)-2-[2,3-dihydro-3-(4-hydroxy-3,5-dimethoxyphenyl)-2-hydroxymethyl-8-methoxy- Preparation of 1,4-benzodioxane-6-]-2,3-dihydro-3,5,7-trihydroxy-4H-1-benzopyran-4-one

[0030] 1.1 Instruments and reagents:

[0031] The ultraviolet spectrum was measured with a Shimadzu UV-240 ultraviolet spectrophotometer; the hydrogen nuclear magnetic resonance spectrum 1 H-NMR is measured by INOVA type superconducting nuclear magnetic resonance spectrometer (VARIAN INOVA-400MHz) (tetramethylsilyl ether TMS is the internal standard); (100-200, 200-300 and 300-400 mesh) and silica gel GF254 (10-40 mesh) for thin-layer chromatography are produced by Qingdao Ocean Chemical Factory; all reagents used are analytically pure, thin-layer preparative chromatography (PTLC ) uses the aluminum foil silica gel plate of Merck Company; Sephadex LH-20 used for column chromatography adopts the product of Amersham Pharmacia Biotech AB Company of Sweden; Reversed-phase silica gel RP...

Embodiment 2

[0044] Example 2: Inhibitory Effect of Compound (1) on Hepatitis B Surface Antigen (HBsAg) Secreted by HepG2.2.15 Cells

[0045] 2.1 Cell culture:

[0046] HepG2.2.15 cells were cultured in DMEM medium containing 10% inactivated fetal bovine serum, 100 U / ml penicillin and 100 U / ml streptomycin, 100 μg / ml G418 at 37°C, 5% CO 2 , cultured in an incubator with 100% relative humidity.

[0047] 2.2 The inhibitory effect of the compound of formula (1) on HepG2.2.15 cell growth was measured by MTT method:

[0048] Take the HepG2.2.15 cells in the logarithmic growth phase, and dilute the cells to 1×10 with medium 5 cells / ml, seeded in 96-well cell culture plate, 100 μl per well, at 37°C, 5% CO 2 After cultivating in an incubator with 100% relative humidity for 24 hours, add compound (1) diluted with medium, the concentration is 1000, 200, 40 and 8 μg / ml respectively, 200 μl per well, and three concentrations are set for each Duplicate wells, placed at 37°C, 5% CO 2 , cultivated...

Embodiment 3

[0058] Example 3: Inhibitory Effect of Compound (1) on Hepatitis B e Antigen (HBeAg) Secreted by HepG2.2.15 Cells

[0059]3.1 Cell culture: the method is the same as in Example 2.

[0060] 3.2 Determination of the inhibitory effect of the compound of formula (1) on the growth of HepG2.2.15 cells by MTT method: the method is the same as in Example 2.

[0061] 3.3 Determination of the inhibitory effect of the compound on hepatitis B e antigen (HBeAg): take the HepG2.2.15 cells in the logarithmic growth phase, and dilute the cells to 1 × 10 with the medium 5 / ml, seeded in 96-well cell culture plate, 100ml per well, at 37°C, 5% CO 2 After culturing in an incubator with 100% relative humidity for 24 hours, add samples diluted with culture medium at concentrations of 20 μg / ml, 4 μg / ml and 0.84 μg / ml, 200 μl per well, and set three concentrations for each Multiple wells were placed at 37°C, 5% CO 2 , cultivated in an incubator with 100% relative humidity, change the culture med...

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