N-hydroxy-isocytidine: A direct-acting broad-Spectrum antiviral agent against respiratory viruses with potent...

A series of C2-modified pyrimidine nucleosides and phosphoramidate derivatives were designed, synthesized, and evaluated against a panel of respiratory viruses. Among these, N-hydroxy-isocytidine (5) exhibited broad-spectrum antiviral activity against SARS-CoV, SARS-CoV-2, HCoV, RSV, and influenza viruses, with potent activity against MERS-CoV (EC₅₀ = 1.27 μM, SI₅₀ > 300). The Nucleoside 5 showed an approximately tenfold higher selectivity index than remdesivir under identical conditions. The phosphoramidate derivative 6 displayed enhanced potency against influenza A (H1N1; A/California/07/2009), with an EC₅₀ of 0.49 μM and SI₅₀ > 380. Structure-activity relationship studies revealed that antiviral activity is highly sensitive to both nucleobase and sugar modifications, as alterations such as C2-oxime substitution, 4-oxo modification, 2'-C-methylation, or inversion of the 2'-stereochemistry reduced or abolished activity. Density functional theory analysis indicated that C2-oxime formation stabilizes a purine-like tautomer, providing a structural basis for activity. The enhanced activity of 6 supports efficient intracellular conversion of 5 to its active triphosphate, which inhibits viral RNA elongation upon incorporation. These findings identify N-hydroxy-isocytidine as a promising direct-acting antiviral agent and highlight the importance of nucleobase and sugar modifications in modulating antiviral activity.