Design and Synthesis of PA_N 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 <b>39-(</b><i><b>S</b></i><b>)</b> was identified and exhibited nanomolar potency against influenza virus polymerase complexes (IC₅₀ = 17.4 nM), which was mechanistically attributed to PA_N endonuclease inhibition. Furthermore, <b>39-(</b><i><b>S</b></i><b>)</b> 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 × 10⁵ during antiviral treatment. Additionally, both <b>39-(</b><i><b>S</b></i><b>)</b> and its prodrug <b>41</b> significantly suppressed viral replication in an A/WSN/33 infected mouse model with efficacy profiles remarkably comparable to those of baloxavir. Collectively, <b>39-(</b><i><b>S</b></i><b>)</b> emerges as a potent PA_N inhibitor with the potential for further development.