Discovery of a spirocyclic influenza RNA-dependent RNA polymerase inhibitor.

The cap-dependent endonuclease (CEN) inhibitor baloxavir marboxil (1) was approved by the FDA as a significant advancement in influenza treatment in nearly two decades. By expanding the tricyclic scaffold of baloxavir and introducing a spirocyclic architecture, a range of innovative compounds were designed and synthesized. Through iterative cycles of compound design and structure-activity relationship (SAR) analysis, guided by the cytopathic effect (CPE) assay, influenza polymerase inhibitory activity assay and molecular docking results, we hypothesized that the newly designed spirocyclic compounds could form a novel hydrogen-bonding interaction with the Tyr24 amino acid residue within the receptor binding pocket. Lead compound (S,S)-5a exhibited potent anti-influenza virus activity (EC50 = 3.76 nM), relatively low cytotoxicity (CC50 = 29.91 μM), and favorable liver microsomal stability. Moreover, both (S,S)-5a and its prodrug 6 effectively suppressed viral replication in a mouse model infected with A/WSN/33, demonstrating promising efficacy profiles. Both compound (S,S)-5a and its unique binding mode with the receptor warrant further investigation.