Design and Synthesis of PAN Endonuclease Inhibitors through Spirocyclization Strategy against Influenza A Virus

The PA N endonuclease inhibitor baloxavir marked a significant advancement in influenza treatment. Herein, we reported the development of PA N inhibitors through structural simplification of baloxavir̀s tricyclic system coupled with strategic incorporation of a spirocyclic architecture, which aims to construct a new metal-binding pharmacophore with distinct three-dimensional features. Lead 39-( S ) was identified and exhibited nanomolar potency against influenza virus polymerase complexes (IC 50 = 17.4 nM), which was mechanistically attributed to PA N endonuclease inhibition. Furthermore, 39-( S ) demonstrated robust antiviral activities against multiple current and different influenza virus strains while showing minimal cytotoxicity in MDCK cells, ensuring an exceptional selectivity index of more than 2.4 × 10 5 during antiviral treatment. Additionally, both 39-( S ) and its prodrug 41 significantly suppressed viral replication in an A/WSN/33 infected mouse model with efficacy profiles remarkably comparable to those of baloxavir. Collectively, 39-( S ) emerges as a potent PA N inhibitor with the potential for further development.