Design,Synthesis And Activity Evaluation Of Oseltamivir And Peramivir Derivatives As Influenza Neuraminidase Inhibitors

Influenza is an acute respiratory infectious disease caused by influenza virus,with high morbidity,high variability and high mortality,which poses a serious threat to human health and life.With the continuous emergence of COVID-19 and highly pathogenic avian influenza,SARS-CoV-2 co-infection with influenza viruses leads to the morbidity and mortality,which makes people more concerned about a possible influenza pandemic.Neuraminidase inhibitors(NAIs)are first-line drugs for both influenza prophylaxis and treatment in many countries due to their broad-spectrum antiviral potencies and low side effects.However,their drug resistance and oral bioavailability still need further improvement.Neuraminidase(NA)subtypes are structurally categorized into two groups:Group-1 NAs and Group-2 NAs.The opening of 150-cavity near the active sites of Group-1 NAs have been considered as new opportunities for the design of specific Group-1 NA inhibitors.The design of these inhibitors is mainly to extend the structure of existing inhibitors by introducing substituents with spatial adaptability to fill 150cavity.In this work,by modifying the oseltamivir(C-5 amino group)and peramivir(C4 guanidine group),and two types of "two-site binding" NAIs through targeting both the 150-cavity and active center of NA were designed and synthesized.Design,synthesis and activity evaluation of oseltamivir amino derivatives.Although compound 17 showed strong NA inhibition and drug resistance,but the side chain of 17 failed to occupy the "entrance channel" of 150-cavity.To find more resistant oseltamivir derivatives,we introduced substituents on the linker benzene ring of 17,hoping to further occupy the 150-cavity.For NA inhibition screening,approximate half of compounds were demonstrated to be potent inhibitors against both wild-type and oseltamivir-resistant group-1 NAs,which were much better than OSC.In cellular assays,compounds Ⅱ-4a,Ⅱ-4d,Ⅱ-4e,Ⅱ-7a,Ⅱ-7e and Ⅱ-7i also showed more potent or equipotent antiviral activity against H1N1,H5N1 and H5N8 strains compared to OSC,with no obvious cytotoxicity(CC50>250 μM).Furthermore,based on molecular docking studies,key hydrogen bonds or hydrophobic interactions with amino acids(Arg118 and Glu119)at the entrance channel were formed by introduced substituents(-Cl,-CH3 and-OCH3 etc.),which might be the reason for the increased activity of these compounds.More importantly,Ⅱ-4e and Ⅱ-7e exhibited the high metabolic stability in the human liver microsomes,low inhibitory effect on some CYP enzymes and good pharmacokinetic characteristics.Therefore,Ⅱ-4e and Ⅱ-7e possess great potential to serve as anti-influenza candidates and deserve further investigation.Design,synthesis and activity evaluation of peramivir amino derivatives.The guanidine group of peramivir makes the greatest contribution to its polarity,which is the main reason for its low bioavailability.The amino-peramivir was obtained by replacing the guanidine group with amino group,which could be modified toward NA 150-cavity.Therefore,according to the structural characteristics of 150-cavity,a series of peramivir amino derivatives were designed and synthesized to improve the oral bioavailability.It is expected to enhance bioavailability by increasing the hydrophobic interaction between the compound and NA.In NA inhibition assay,compound Ⅲ-6j(IC50=0.68 μM)with the best inhibitory activity on H5N8 NA was still significantly lower than that of peramivir(IC50=0.012 μM).In cellular assays,compound Ⅲ-6j showed no anti-influenza effect at the maximum test concentration(100 μM).Furthermore,molecular docking studies showed that the 2-furanacrylamide side chain of Ⅲ-6j did not fully penetrate into the 150-cavity,so it was unable to form more hydrophobic interactions with amino acids in the 150-cavity to compensate for the loss of binding energy.It’s an important reason for the poor activity of these compounds.In summary,based on the analysis of cocrystal structure of NA and its inhibitor,in this thesis,two series of compounds were designed and synthesized by using the "twosite binding" strategy.Among them,oseltamivir amino derivatives Ⅱ-4e and Ⅱ-7e not only showed excellent inhibitory effect at the enzyme and cellular levels,but also achieved good results in preliminary druggability evaluation,which can be used as candidates for further research.