Design,Synthesis And Biological Evaluation Of Novel Oseltamivir Derivatives Targeting 150-cavity Of Influenza Virus Neuraminidase

Influenza virus(IFV),as a kind of respiratory pathogen,always causes severe public health crisis because of its seasonal epidemicity,high contagiosity,various reservoirs,high morbidity and mortality.According to the data from WHO,seasonal influenza leads to about 3,000,000-5,000,000 cases of severe illness and about 290,000-650,000 respiratory deaths per year around the world.Thus,the prophylaxis and treatment of influenza virus is a important and urgent task for all the countries.Neuraminidase(NA),as a surface glycoprotein,plays a crucial role in the release stage of IFV life cycle.The currently listed NA inhibitors have contributed to prophylaxis and treatment of influenza virus.However,due to the extensive application,drug-resistant strains against NA inhibitors have emerged around the world.Therefore,the development of potent NA inhibitors against drug resistance have been a hotspot in anti-influenza research.According to the structural biology research,there is an naturally opened or induced flexible cavity(volume:10(?)×5(?)×5(?)),named 150-cavity,near the catalytic site of NAs.which inspires us to develop dual-site binding NA inhibitors.On the basis of the research described above and multiple medicinal chemistry strategies,my research,which focused on the drug resistance of Oseltamivir,was divided into two parts:"Optimization of the pharmacokinetic properties of existed potent Oseltamivir derivatives" and "Development of novel dual-site binding Oseltamivir derivatives".1.Design,Synthesis and Pharmacokinetic study of I-3b Prodrug targeting Influenza Virus NeuraminidaseTargeting 150-cavity,neuraminidase inhibitor I-3b was developed by our group with higher anti-influenza activities than Oseltamivir.However,low bioavailability of I-3b hindered its further development.Thus,with the method of prodrug and salification,I designed and synthesized P-13(pivaloyloxymethyl ester of I-3b)and P-13Y(P-13 phosphate)in order to improve the bioavailability of I-3b.Unfortunately,pharmacokinetic study on rats indicated that T1/2,Cmax,Tmax,MRT0-∞,AUC0-t and AUC0-∞ of P-13 and P-13Y were much lower than those of I-3b.Espeically,P-13 and P-13Y exhibited lower bioavailability(0.3%and 0.2%,respectively)than I-3b(3.7%).Therefore,pivaloyloxymethyl ester prodrug is unsuitable for the optimization of I-3b because of gastrointestinal stability or first pass effect problem.Although the research in this part didn’t meet the expected goals,the experimental results also accumulated valuable experience for the development of Ⅰ-3b.2.Design,Synthesis and Biological Evaluation of Ferrocene-based Oseltamivir Derivatives Targeting 150-cavity of NeuraminidaseBased on the binding model of NA active site and 150-cavity,with multiple-sites binding strategy,I designed and synthesized 19 N-substituted ferrocene-based oseltamivir derivatives targeting 150-cavity.In vitro NA inhibitory assay indicated that most of compounds exhibited moderate activities compared with OSC.However,compound 18 possessed prominent inhibitory potencies(IC50=2.80 μM,0.44 μM,1.22 μM,0.92 μM and 562.40 μM for H1N1,H3N2,H5N1,H5N6 and H5N1-H274Y,respectively),which also means the activity of compound 18 was 3.6-7.9 times lower than that of OSC(IC50=0.45 μM,0.12 μM,0.16 μM,0.16 μM and 132.70 μM for H1N1,H3N2,H5N1,H5N6 and H5N1-H274Y,respectively).As for anti-influenza assay in chicken embryo fibroblasts(CEFs),compound 18(EC50=2.01 μM and 20.68 μM for H5N1 and H5N6,respectively)exhibited moderate activities with low toxicity(CC50>1000 μM),compared with OSC(EC50=1.17 μM and 3.45 μM for H5N1 and H5N6,respectively).The molecular modeling result indicated that,compound 18 successfully binded to not only the active site but also the 150-cavity.The carboxy group of compound 18 formed hydrogen bonds with Arg292,Tyr347 and Arg371.Besides,the carbanyl group formed one hydrogen bond with Argl52.Notably,the elongated modification side chain well occupied 150-cavity and the nitro group interacted with Val116 and formed one hydrogen bond,which accounts for the higher activity of compound 18 compared to the other compound in this series.Although the activities of these compounds exhibited lower activities than OSC on enzyme and CEFs,the experimental results also accumulated valuable experience for the development of Oseltamivir derivatives targeting 150-cavity of neuraminidase.Taken together,for the problem of drug resistance of Oseltamivir in clinical,based on the structural biology research and multiple medicinal chemistry strategies,the research concentrated on two parts:"Design,Synthesis and Pharmacokinetic study of I-3b Prodrug targeting Influenza Virus Neuraminidase" and "Design,Synthesis and Biological Evaluation of Ferrocene-based Oseltamivir Derivatives Targeting 150-cavity of Neuraminidase".which provided valuable information for the development of anti-influenza drugs.