A Series of Oleanolic Acid Derivatives as Anti-Hepatitis B Virus Agents: Design, Synthesis, and in Vitro and in Vivo Biological Evaluation

Abstract

A series of oleanolic acid derivatives were synthesized by diverse reactions, including the introduction of conjugated alkadiene and epoxy ring moieties formed by means of photosensitized oxidation. Eosin Y was used as photosensitizer during this process. Next the cytotoxicity of the products was evaluated on HepG2.2.15 cells to determine the appropriate treatment concentration for the subsequent experiments. Most of the OA derivatives exhibited anti-HBV antigens secretion activity in HepG2.2.15 cells. Among the tested compounds, OA-4 (3.13 µg/mL) showed significant activity against the secretion of HBsAg, HBeAg, and HBV DNA replication with inhibitory ratios of 90.52% ± 1.78%, 31.55% ± 3.65%, and 94.57% ± 3.11% after 6 days, respectively. Besides, OA-4 was further investigated in a duck model with DHBV infection. When OA-4 was administered at a dosage of 500 mg/kg, the results revealed a significant inhibitory effects of DHBV at 19.94% ± 2.87%, 28.80% ± 3.62% and 29.25% ± 2.65% at days 5, 10, and 3 after the cessation of OA-4 treatment, respectively. It's worth noting that OA-4 is superior to lamivudine in the inhibition of rebound of viral replication rate. The structure-activity relationships of OA derivatives had been preliminary discussed, which should be useful to explore further novel anti-HBV agents.

Keywords: anti-hepatitis B virus; eosin Y; epoxy ring; oleanolic-acid derivatives; virus recrudescence.

Figure 1

Structure of oleanolic acid.

Scheme 1

Synthesis route to OA-n derivatives. Reagents and Conditions: (a) NBS, CCl₄, light, reflux, 4 h; (b) chromic acid solution, acetone, 0 °C, 1 h; (c) Eosin Y, dichloromethane, light, 10 h; (d) chromic acid solution, acetone, 0 °C, 1 h; (e) chromic acid solution, acetone, 0 °C, 1 h.

Figure 2

Cytotoxic effect of OA-n on HepG2.2.15 cell line. Data were expressed as inhibitory ratio ± SD based on three independent experiments.

Figure 3

The structure of lamivudine (3TC).

Figure 4

The activity against HBV antigen secretion of OA-4 in HepG2.2.15 cells. Data are expressed as OD value ± SD based on three independent experiments. ** p < 0.01, * p < 0.05 compared with the control group.

Figure 5

Inhibitory effect of OA-4 and 3TC on HBV DNA expression in HepG2.2.15 cells. HBV DNA levels were quantified by fluorescence quantitative PCR. Data are expressed as HBV DNA copies ± SD based on three independent experiments. ** p < 0.01, * p < 0.05 compared with the control group.

Figure 6

Dot blots of DHBV DNA in duck serum in the OA-4 treatment study. (a) 125 mg/kg OA-4 treatment; (b) 250 mg/kg OA-4 treatment; (c) 500 mg/kg OA-4 treatment; (d) 50 mg/kg 3TC treatment.

Figure 7

Inhibitory effect of OA-4 on DHBV DNA expression in duck serum at days 5, 10, and 3 after the cessation of OA-4 treatment. Data are expressed as DHBV DNA inhibitory ratio ± SD based on the independent experiments.