The Synthesis Of Seco-A-Pentacyclic Triterpene Derivatives And Their Anti-HBV Activities In Vitro

Hepatitis B virus(HBV)represents a major global health problem with an estimated 2 billion carriers in the worldwide.This virus causes the disease hepatitis B,which can cause both acute and chronic infections.Most of those with chronic disease have no symptoms;however,cirrhosis and liver cancer may eventually develop.These complications result in the death of 15 to 25%of those with chronic disease.Furthermore,the low curing rate,serious side effects,severe drug resistance and rebound phenomena of the approved drugs restrict the drugs' clinical application.Natural products with various skeletons and diverse biological activities are considered as important sources in drug discovery.1.ObjectiveIn this paper,a series of pentacyclic triterpenoids from Chinese herbs with anti-virus and protecting liver activities were modified via ring A expansion and opening to find anti-HBV lead compounds.2.Methods2.1 C-3 hydroxyl groups of pentacyclic triterpenoids,such as oleanolic acid(OA),ursolic acid(UA)and glycyrrhetinic acid(GA)were first oxidized to C-3 ketones by Jones reagent respectively.And then Baeyer-Villiger reaction was successfully performed using 3-chloroperbenzoid acid(m-CPBA)to yield seco-A-3,4-lactones,which were subsequently converted to the ring A cleaved 3,28-dioic acids by the hydrolysis of lactones.The structures of the compounds were fully verified by 1H NMR,13C NMR and HRMS.2.2 The cytotoxicity of the compounds was measured on HepG2.2.15 cell line by MTT assay.2.3 The inhibitory effects of the compounds on the secretion of HBsAg and HBeAg were determined by both ELISA and flow cytometry.2.4 The anti-HBV DNA and cccDNA replication activities were investigated by the fluorogenic quantitative PCR(FQ-PCR).3.Results3.1 Seven seco-A-triterpene lactones,OA-2-1BV,OA-2-2BV,OA-Br-1-1,OA-4,GA-2,UA-2 and F-1 were synthesized,and three of hydrolysis products,OA-3,GA-3 and UA-3 were obtained.Compounds GA-3,OA-2-2BV,OA-Br-1 and OA-Br-1-1 were new compounds.3.2 The CC50 values of all compounds were above 400 ?M except compound 8a(100.4?M).3.3 Seven seco-A-3,4-triterpene lactones showed significantly inhibitory effects against the secretion of both HBsAg and HBeAg compared to raw compounds,while the hydrolysis products slightly decreased compared to their lactones.Compounds OA-2-2BV and OA-4 displayed the most promising inhibition against the secretion of HBeAg with IC50 values of 0.14 ?M and 0.86 ?M,and inhibition ratio of 33.8%and 34.0%at the concentration of 25 ?M via flow cytometry.The inhibitory effects of the compounds on HBeAg secretion were stronger than on HBsAg secretion.The C12/13 double bond and the oleanane type played an important role on the antigen inhibition.3.4 Seven seco-A-3,4-triterpene lactones showed significantly inhibitory effects against HBV DNA replication compared to the parent compounds.It was exciting that the inhibitory rate of compounds OA-2-2BV,OA-4 and GA-3 were 83.32%,82.36%and 91.58%respectively at the concentration of 25 ?M,while the inhibitory effect of GA-3 had no statistic difference with the positive control 3TC(93.01%).The inhibitory activities of OA-2-2BV,OA-4,and GA-3 on HBV cccDNA were further investigated.The level of HBV cccDNA in cell line was decreased after treatment of tested compounds,the inhibitory strength attained to the micromole level with IC50 of 32.7?M,33.5?M,and 12.3 p,M respectively.The C12/13 double bond and the oleanane type played an important role on the DNA relpication.4.ConclusionsThe synthesized seco-A-triterpenoid-3,4-lactones and hydrolysis products exhibited significantly inhibitory effects on HBsAg,HBeAg secretion and HBV DNA,HBV cccDNA replication compared to the parent compounds.Compound GA-3 had the possibility to be a potential novel non-nucleoside anti-HBV lead compound.The C12/13 double bond of the synthesized compounds played an important role on the anti-HBV activities.The inhibitory effects of the compounds on HBeAg secretion were stronger than on HBsAg secretion.The oleanane type derivatives exhibited more obvious function on anti-HBV activities.