Dual prodrugs of N⁴-hydroxycytidine with ProTide and ester modifications as anti-SARS-CoV-2 agents

Molnupiravir, an oral prodrug of β-D-N⁴-hydroxycytidine (NHC), is a broad-spectrum antiviral agent. However, its clinical efficacy is hampered by suboptimal plasma stability and inefficient intracellular monophosphorylation. To circumvent these pharmacological limitations, a novel series of NHC dual-prodrug derivatives was designed and synthesized by integrating 5'-ProTide and N⁴-ester modification strategies. Structure-activity relationship (SAR) analysis revealed that derivatives functionalized with short-chain N⁴-esters achieved a more favorable balance between antiviral potency and cytotoxicity. Specifically, 5b exhibited favorable anti-SARS-CoV-2 activity with low cytotoxicity, displaying anti-SARS-CoV-2 activity (EC₅₀ = 3.22 μM) with negligible cytotoxicity. Subsequent mechanistic studies supported that the ProTide moiety mediates rapid enzymatic activation, effectively bypassing the rate-limiting first phosphorylation step to release the nucleoside monophosphate. Furthermore, pharmacokinetic evaluations revealed a tunable profile: 4 demonstrated significantly enhanced metabolic stability in human plasma, suggesting its potential as a long-circulating systemic reservoir, whereas 5b displayed favorable in vitro antiviral activity, likely associated with its balanced physicochemical properties and rapid esterase-mediated conversion to the more plasma-stable ProTide intermediate 4. Consequently, these findings demonstrate prodrugs 4 and 5b represent promising antiviral agents with favorable metabolic stability and high potency.