Synthesis, structure activity relationship and in vitro anti-influenza virus activity of novel polyphenol-pentacyclic triterpene conjugates.

It is urgently necessary to develop more effective anti-influenza agents due to the continuous emergence of drug-resistant strains of influenza virus. Our earlier studies have identified that certain pentacyclic triterpene derivatives are effective inhibitors of influenza virus infection. In the present study, a series of C-28 modified pentacyclic triterpene derivatives via conjugation with a series of polyphenols were synthesized, and their antiviral activities against influenza A/WSN/33 (H1N1) virus in MDCK (Madin-Darby canine kidney) cells were evaluated. Four compounds 23m, 23o, 23q and 23s displayed robust anti-influenza potency with averaged IC50 values at the low-micromole level, surpassing the potency of oseltamivir. In addition, the in vitro cytotoxic activity of the four conjugates against MDCK cells showed no toxicity at 100 µM. Further mechanism studies of compound 23s, one of the best representative conjugates with IC50 value of 5.80 µM and a selective index (SI) value of over 17.2, by hemagglutination inhibition (HI), surface plasmon resonance and molecular modeling indicated that this conjugate bound tightly to the viral envelope hemagglutinin (KD = 15.6 µM), thus blocking the invasion of influenza viruses into host cells.

Design, synthesis and biological evaluation of novel L-ascorbic acid-conjugated pentacyclic triterpene derivatives as potential influenza virus entry inhibitors.

Since the influenza viruses can rapidly evolve, it is urgently required to develop novel anti-influenza agents possessing a novel mechanism of action. In our previous study, two pentacyclic triterpene derivatives (Q8 and Y3) have been found to have anti-influenza virus entry activities. Keeping the potential synergy of biological activity of pentacyclic triterpenes and l-ascorbic acid in mind, we synthesized a series of novel l-ascorbic acid-conjugated pentacyclic triterpene derivatives (18-26, 29-31, 35-40 and 42-43). Moreover, we evaluated these novel compounds for their anti-influenza activities against A/WSN/33 virus in MDCK cells. Among all evaluated compounds, the 2,3-O,O-dibenzyl-6-deoxy-l-ascorbic acid-betulinic acid conjugate (30) showed the most significant anti-influenza activity with an EC50 of 8.7 µM, and no cytotoxic effects on MDCK cells were observed. Time-of-addition assay indicated that compound 30 acted at an early stage of the influenza life cycle. Further analyses revealed that influenza virus-induced hemagglutination of chicken red blood cells was inhibited by treatment of compound 30, and the interaction between the influenza hemagglutinin (HA) and compound 30 was determined by surface plasmon resonance (SPR) with a dissociation constant of KD = 3.76 µM. Finally, silico docking studies indicated that compound 30 and its derivative 31 were able to occupy the binding pocket of HA for sialic acid receptor. Collectively, these results suggested that l-ascorbic acid-conjugated pentacyclic triterpenes were promising anti-influenza entry inhibitors, and HA protein associated with viral entry was a promising drug target.

Pentacyclic triterpenes grafted on CD cores to interfere with influenza virus entry: A dramatic multivalent effect.

Multivalent effect plays an important role in biological processes, particularly in the specific recognition of virus with its host cell during the first step of infection. Here we report the synthesis of multivalent pentacyclic triterpene grafted on cyclodextrin core and potency of against influenza entry activity. Nine star-shaped compounds containing six, seven and eight pentacyclic triterpene pharmacophore on cyclodextrin scaffold were prepared by way of copper-catalyzed azide-alkyl cycloaddition reaction under microwave activation. Some of the multimers exhibited much potent antiviral activity against H1N1 virus (A/WSN/33), even equivalent or superior to oseltamivir. The most active compound 31, a heptavalent oleanolic acid-ß-cyclodextrin conjugate, shows an up to 125-fold potency enhancement by its IC50 value over the corresponding monovalent conjugate and oleanolic acid, disclosing a clear multivalent effect. Further studies show that three compounds 31-33 exhibited broad spectrum inhibitory activity against other two human influenza A/JX/312 (H3N2) and A/HN/1222 (H3N2) viruses with the IC50 values at 2.47-14.90 µM. Most importantly, we found that compound 31, one of the best representative conjugate, binds tightly to the viral envelope hemagglutinin with a dissociation constant of KD = 2.08 µM, disrupting the interaction of hemagglutinin with the sialic acid receptor and thus the attachment of viruses to host cells. Our study might establish a strategy for the design of new pharmaceutical agents based on multivalency so as to block influenza virus entry into host cells.

Synthesis and biological evaluation of ring A and/or C expansion and opening echinocystic acid derivatives for anti-HCV entry inhibitors.

Echinocystic acid (EA), a naturally occurring oleanane-type triterpene isolated from Dipsacus asperoides, was found to have anti-HCV entry activity in our previous study. Expansion of triterpene structural diversity, including the ring A and/or C expansion and opening, was performed. To elucidate the pharmacophore of EA, seven lactones (8, 16, 17, 24, 26, 35 and 41), three 3,28-dioic acids (9, 36 and 42) and two pentols (10 and 27) were synthesized. The anti-HCV entry activities of those derivatives, along with their parental compound EA and analogs α,ß-unsaturated ketone (18), were evaluated. All the products showed no improvement but detrimental effect on potency of EA. The results demonstrated that ring A and C of EA are highly conserved, indicating the steric effects of the rigid skeleton have a profound effect on the potency.