Design,Synthesis And Bioactivity Evaluation Of Novel Polyphenols As HIV-1 RNase H Inhibitors

Acquired immunodeficiency syndrome(AIDS)mainly caused by human immunodeficiency virus type 1(HIV-1),is still an important global health concern.Currently,highly active antiretroviral therapies(HAART)are the most effective treatments to prevent the development of AIDS,which dramatically decreased the mortality resulted from HIV-1 infection.However,the efficacy of HAART can be reduced due to the emergence of HIV resistant mutants.Therefore,there is an urgent need to develop novel antiviral drugs with new structures and mechanisms of action,via targeting non-validated and underexplored processes crucial in HIV-1 replication.HIV-1 reverse transcriptase(RT)is a well-established target,which plays a critical role in HIV-1 replication by converting the viral single-stranded RNA into double-stranded DNA.RT possesses two distinct enzymatic functions:an DNA polymerase activity that synthesizes the proviral DNA,and an ribonuclease H(RNase H)activity that selectively degrades the RNA strand in DNA/RNA heteroduplex replication intermediates.Both activities play a critical role in viral replication since they are responsible for the conversion of the viral(+)-strand RNA into double stranded DNA.Currently approved antiretroviral drugs target the DNA polymerase activity of the RT and are classified in two groups:nucleos(t)ide RT inhibitors(N(t)RTIs)and non-nucleoside RT inhibitors(NNRTIs).No inhibitor of HIV-1 RNase H has been introduced to the market.Due to its important role in viral replication and highly conserved amino acid residues in the RNase H active site,the RNase H is a potentially important and underexploited target for novel anti-HIV drug design.According to their mechanism of action,the newly reported HIV-1 RNase H inhibitors can be classified into two groups:active-site inhibitors and allosteric inhibitors.Notably,seeking RNase H active-site inhibitors becomes an important topic in antiviral drug research field.The RNase H active-site inhibitors act mainly by chelating the two divalent metal ions which are essential in the active site.Although they belong to a wide range of structurally diverse scaffolds,the RNase H active-site inhibitors contain many ubiquitous fragments in their structures and possess a common pharmacophore model,including Divalent metal ion chelating group,Linker and Hydrophobic group.It should be noted that,despite extensive medchem campaigns over the last decade,no RNase H inhibitors have reached the clinic,let alone the market,underscoring the challenges to find a safe and efficacious drug candidate for the treatment of HIV infection.One of the main challenges is to adjust physicochemical properties,such as low membrane permeability,to improve cell-based antiviral activity.Therefore,it is of great significance to develop novel,highly effective and cell-active HIV-1 RNase H inhibitors.Fortunately,the structural diversity of RNase H active-site inhibitors provided a wide space for novel lead discovery,and the pharmacophore similarity of them gave valuable hints for lead discovery and optimization.Natural products have become one of the most important resources of novel lead compounds including antiviral agents.Notably,polyphenols have a variety of physiological activities,such as anti-oxidation,anti-tumor,anti-cardiovascular disease and antivirus activities.The ortho-phenolic hydroxyl groups of polyphenols can be considered as a multidentate ligand chelating metal ions to inhibit the corresponding metal enzyme activity.Compound 1,3,4,5-tetragalloylapiitol,was isolated from the plant Hylodendrongabunensis and was found to be a potent inhibitor with selective HIV-1 RNase H enzymatic activity(no inhibition against human RNase H and E.coli RNase H)and lower cytotoxicity.Therefore,1,3,4,5-tetragalloylapiitol was taken as the lead compound for further optimization in this thesis.Based on structural simplification and structure-based drug design strategy,we chose the key pharmacophore element(galloyl group)as the chelating skeleton,diversely substituted non-aromatic aliphatic rings(piperidine and piperazine)as the peripheric substituent groups.Finally,two kinds of polyphenols were designed and synthesized via simple and convenient procedures with aim to explore the structure-activity relationship,and to improve their inhibitory activity and physicochemical properties.Initially,these compounds were tested for their ability to inhibit HIV-1 RNase H activity by denaturing polyacrylamide gel electrophoresis.Fortunately,most of these compounds showed remarkable HIV-1 RNase H inhibitory activity.Compared with the piperidine series,the piperazine series exhibited ten times higher HIV-1 RNase H inhibitory activity,and most of them were found to be more active than the positive control(?-thujaplicinol).Totally,14 compounds of series II displayed potent inhibitory activity against RNase H in the sub-micromolar level.Especially,compound ?-25 was the most promising one(IC50 = 0.72 ± 0.07 ?M),about 2.8 times as potent as ?-thujaplicinol(IC50 = 1.98 ± 0.22 ?M).Furthermore,some compounds were also tested for RT(polymerase)inhibitory activity using ELISA.Among them,only four compounds showed weak inhibitory activity(<100 ?M)against HIV-1 RT,indicating that these compounds are selective inhibitor of RNase H enzymatic activity.Moreover,these compounds were evaluated for their anti-HIV replication activity against HIV-1(?B),HIV-2(ROD)and wild-type HIV-1/NL4-3.However,all of these compounds showed no anti-HIV potency but low cytotoxicity in MT-4 cell line.Then we predict the physicochemical properties of compounds ?-5 and ?-25 with "Molinspiration Cheminformatics Software".The results showed that the two compounds were extremely highly polar(LogP was 0.04 and-0.48,respectively),far below the appropriate range of oral drugs(LogP = 2-3).Therefore,the low antiviral activity may be due to the poor physico-chemical properties(probably,membrane permeability).In order to acquire more information about the binding modes of these compounds,a further investigation was performed with a particular focus on the four "hits" compounds,namely,?-5,?-6,?-25 and ?-26,via comparing their binding modes with that of?-thujaplicinol,using the Glide module of the Schrodinger suit(PDB:3QIO).The results showed that the binding modes of these compounds were similar to that of?-thujaplicinol,and some of them developed additional interactions with other important amino acid residues such as His539 and Lys540,which providing a rational explanation of the improved activity.In summary,taking 1,3,4,5-tetragalloylapiitol as lead compound,two series of novel polyphenols(series I and II,totally 51 compounds)were designed,synthesized and evaluated as selective HIV-1 RNase H inhibitors via structural simplification strategy and structure-based drug design strategy.Though these compounds showed no anti-HIV potency in the tested concentration,they represent novel leads to yield more potent antiviral agents in further optimization.