Synthesis and in vitro study of novel neuraminidase inhibitors against avian influenza virus

Evidences of oseltamivir resistant influenza patients raised the need of novel neuraminidase inhibitors. In this study, five oseltamivir analogs PMC-31-PMC-36, synthesized according to the outcomes of a rational design anal. aimed to investigate the effects of substitution at the 5-amino and 4-amido groups of oseltamivir on its antiviral activity, were screened for their inhibition against neuraminidase N1 and N3. The enzymes used as models were from the avian influenza A H7N1 and H7N3 viruses. The neuraminidase inhibition assay was carried out by using recombinant species obtained from a baculovirus expression system and the fluorogenic substrate MUNANA. The assay was validated by using oseltamivir carboxylate as a ref. inhibitor. Among the tested compds., PMC-36 (I) showed the highest inhibition on N1 with an IC50 of 14.6 ± 3.0 nM (oseltamivir 25 ± 4 nM), while PMC-35 showed a significant inhibitory effect on N3 with an IC50 of 0.1 ± 0.03 nM (oseltamivir 0.2 ± 0.02 nM). The anal. of the inhibitory properties of this panel of compds. allowed a preliminary assessment of a structure-activity relationship for the modification of the 4-amido and 5-amino groups of oseltamivir carboxylate. The substitution of the acetamido group in the oseltamivir structure with a 2-butenylamido moiety reduced the obsd. activity, while the introduction of a propenylamido group was well tolerated. Substitution of the free 5-amino group of oseltamivir carboxylate with an azide, decreased the activity against both N1 and N3. When these structural changes were both introduced, a dramatic redn. of activity was obsd. for both N1 and N3. The alkylation of the free 5-amino group in oseltamivir carboxylate introducing an iso-Pr group seemed to increase the inhibitory effect for both N1 and N3 neuraminidases, displaying a more pronounced effect against N1.