Design,Synthesis And Antiviral Activity Evaluation Of Novel "Multiple-Sites Binding"-typed Influenza Virus Neuraminidase Inhibitors

Annually,influenza viruses(IFV)have a major impact on global health,causing 3-5 million severe cases of disease and 1 million deaths.Human infections caused by avian influenza virus(AIV)have continually brought great panic and death threats to people all over the world.During the life cycle of IFVs,influenza virus neuraminidase(NA)is an important surface antigenic glycoprotein,which facilitates viral shedding by cleaving glucosidic bonds between another essential surface antigenic glycoproteins hemagglutinin(HA)and sialic acid on the surface of the host cell,thus enables continued infection to other cells.Therefore,NA is an attractive target for anti-influenza drug design.Oseltamivir phosphate,a cyclohexene NA inhibitor(NAI),is the only oral NAI with the advantages of high efficiency,low toxicity and high bioavailability.However,due to the inherent high mutability of IFVs,various oseltamivir resistant mutants have emerged,such as the most frequently encountered H5N1-H274Y and H3N2-E119V,which have severely limited the therapeutic efficacy of oseltamivir.Therefore,the occurrence of highly pathogenic oseltamivir resistant strains(including H274Y)highlights the urgent need for new classes of antiviral drugs.On the basis of the phylogenetic tree,the N1-N9 subtypes can be divided into two groups:group-1 NAs(N1,N4,N5,and N8 subtypes),and group-2 NAs(N2,N3,N6,N7,and N9 subtypes).X-ray crystallography of NAs shows that a flexible 150-loop in group-1 NAs usually adopts an open conformation,whereas in 09N1 and group-2 NAs,this loop is always closed.The open 150-loop can form in an open large cavity(150-cavity)adjacent to the active site in group-1 NAs,but this cavity does not present in 09N1 and group-2 NAs.However,recent studies have shown that the open 150-loop and 150-cavity may also exist in 09N1 and group-2 NAs under certain conditions.In addition,the active site in NAs of both groups can be linked to another binding site(430-cavity)through 430-loop.These two cavities are directly connected with active site and have large volumes,providing important opportunity for developing new NAIs with high activity and favorable anti-drug resistance profile.In order to overcome the shortcomings of the existing neuraminidase inhibitors and find new and highly effective anti-influenza virus candidate drugs,in this thesis,inspired by the structural characteristics of oseltamivir-NAs complex,several series of C-5-amino and C-1-carboxyl derivatives of oseltamivir,targeting the 150-and 430-cavity adjacented to the active site of influenza A viral NAs,were designed and synthesized by multi-sites binding strategy.The research work is divided into the following aspects:Discovery of oseltamivir derivatives targeting both the active site and 150-cavity of group-1 and-2 NAs.Based on our previously discovered N1 selective inhibitors(as exemplified by compounds 58 and 59 in Chapter ?)and other reported N2 selective inhibitors,novel oseltamivir derivatives were designed and synthesized by exploiting the active site and 150-cavity of NAs.Among them,I-3b and I-3c were exceptionally active against both group-1 and-2 NAs.Especially for 09N1,N2,N6,and N9 subtypes,they showed 6.80-12.47 and 1.20-3.94 times greater activity than oseltamivir carboxylate(OSC),respectively.Besides,these two compounds also showed greater inhibitory activity than OSC toward N1-H274Y and N2-E119V variant,and their ability to inhibit influenza B virus NA were comparable to that of OSC.Furthermore,in cellular assays,they both exhibited greater potency than OSC toward H5N1,H5N2,H5N6,and H5N8 viruses while I-3b also had comparable antiviral activity to OSC for H1N1 and H3N2.It is noteworthy that the anti-AIV(H5N2)activity of I-3b in vivo(chicken embryo)was superior to that of OSC as well.More importantly,I-3b demonstrated high metabolic stability,low cytotoxicity in vitro,and low acute toxicity in mice.Molecular dynamics simulation provided insights into the role of the R group of I-3b in improving potency toward group-1 and-2 NAs.To the best of our knowledge,the hypothesis,that the 150-loop and 150-cavity of the 09N1 and group-2 NAs can be induced to open by small molecular binders was first verified by this study.Discovery of group-1 NAs selective and anti-N1-H274Y drug-resistant oseltamivir derivatives using structure-based drug design.In this part,we aimed to improve the activity profile of oseltamivir derivatives towards drug-resistant viral strains by further exploiting the 150-cavity adjacent to the active sites of viral NAs.Taking 58 and 59 as lead compounds,five sub-series of N-substituted oseltamivir derivatives were designed and synthesized based on bioisosterism,molecular hybridization and other drug design strategies,and evaluated their bioactivity in cell-based and enzymatic assays.Among them,compound II-15h exhibited antiviral activities similar to or better than OSC against H5N1,H5N2,H5N6,and H5N8.Besides,II-15h was 5-to 86-fold more potent than OSC toward N1,N8,and N1-H274Y in the inhibitory assays.The high potency of II-15h against 09N1 again supported the idea that 09N1 favors an open form of the 150-loop.The anti-AIV activity of II-15h in vivo(chicken embryo)was better than that of OSC.Computational studies provided a plausible rationale for the high potency of II-15h against group-1 and N1-H274Y NAs.In addition,II-15h demonstrated acceptable oral bioavailability,low acute toxicity in vivo,and high metabolic stability.Overall,the above excellent profiles make II-15h a promising drug candidate for the treatment of influenza virus infection.Discovery of other "two-site binding" oseltamivir derivatives targeting the active site and 150-cavity of IFV group-1 NAs.Inspired by the outcomes of Chapter 2 and Chapter 3,in Chapter 4,more OSC derivatives were obtained by further modifying the C-5-amino groups of OSC.The anti-IFV activity results demonstrated that compounds containing strong electron-withdrawing groups at C-5-position amino were not suitable for binding to 150-cavity.Most of the C-5-amino-bi-aromatic heterocyclic derivatives of OSC exhibited selective inhibition of AIVs and their corresponding NAs.The slender allyl benzene group bearing conjugated ? system at C-5-amino position was favorable for binding to 150-cavity,and therefore some of these compounds have robust potency inhibiting the replication of group-1 NAs subtype AIV and the function of NA,even comparable to OSC.The activity of C-5-position amides and sulfonamides derivatives displayed significantly reduced antiviral activity toward H5N1,H5N2,H5N6 and H5N8.Overall,the inhibitory enzyme activities of the compounds in this chapter was consistent with the trend of anti-AIV activities in the cellular level,which was explained by the detailed analysis of the molecular docking of ?-9a and ?-lle with N1 and N8.Design,synthesis and activity evaluation of "three-site binding"-typed oseltamivir derivatives targeting active site,150-cavity and 430-cavity of IFV NAs.Further,a novel series of "three-site binding"-typed oseltamivir derivatives were designed and synthesized by modifying the C-1-carboxyl and C-5-amino groups of OSC simultaneously,based on molecular hybridization principle.Biological evaluations demonstrated that these oseltamivir derivatives(e.g.?-9e,which was modified simultaneously by C-1-carboxyl and C-5-amino groups)showed a significant decrease in the inhibition of AIV.As a comparison,compound ?-11e,the most potent N1-selective inhibitor targeting active site and 150-cavity,showed 1.5 and 1.8 times greater activity than OSC against N1 and N1-H274Y.In cellular assay,?-11e also exhibited greater potency than OSC against H5N1.In addition,?-11e demonstrated low cytotoxicity in vitro and low acute toxicity in mice.Although the antiviral profile of "three-site binding"-typed NAIs were less than encouraging,this study represents the first attempt to rationally target three binding sites of NA and may have potential implications for further design.In conclusion,to discover novel and highly effective anti-influenza virus agents,in this thesis,inspired by the co-crystal structural information of oseltamivir with NAs and the structural characteristics of NAs active site,150-cavity,and 430-cavity,several series of "two-site binding"-typed and "three-site binding"-typed oseltamivir derivatives were designed and synthesized,via structure-based drug design and"multiple-sites binding" concept,and by modifying the C-1-carboxyl group and/or C-5-amino group of oseltamivir.Finally,the medicinal chemistry optimization and extensive bioassay culminated in the identification of novel lead compounds with high activity,selectivity and anti-drug resistance profiles.Notably,on the basis of these excellent in vitro and in vivo results,we consider that ?-3b and ?-15h are promising new drug candidates to treat influenza and may have the potential to prevent or ameliorate the next pandemic.Besides,to the best of our knowledge,this study also verified for the first time the hypothesis that the 150-loop and 150-cavity of 09N1 and group-2 NAs can be induced to open by small molecular compounds.Furthermore,in this paper,the feasibility of designing "three-sites binding"-typed inhibitors is discussed for the first time,providing valuable information for further study.All in all,we envisioned the 150-cavity and 430-cavity of influenza virus neuraminidases(NAs)could be further exploited to yield more potent oseltamivir derivatives.