1'-Cyanocytidine-5'-isobutyryl is a potent SARS-CoV-2 inhibitor in culture and infected Syrian hamsters.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and COVID-19 epidemic is relatively under control due to the rapid development and deployment of vaccines and a few drugs. However, challenges persist, as new variants and issues with vaccine durability may compromise their effectiveness. Here, we report the discovery and evaluation of 1'-cyanocytidine (CNC), a nontoxic, next-generation nucleoside analog that displays submicromolar inhibition of SARS-CoV-2 replication in various cell and 3D HAE-ALI primary culture systems. Intracellularly, CNC is metabolized to its active 5'-triphosphate form (CNC-TP), targeting the viral RNA-dependent RNA polymerase. Pre-steady-state kinetic analysis revealed CNC-TP is a reversible, competitive inhibitor. The 5'-isobutyryl ester prodrug of CNC (CNiBuC), which rapidly converts to CNC in mouse and hamster plasma, substantially reduced viral RNA levels and lung infectious virus titers in a Syrian hamster model after intraperitoneal and oral dosing. With favorable pharmacokinetics, bioavailability, and safety profiles, CNC and CNiBuC represent promising candidates for SARS-CoV-2 therapy.