3’‐Dehydroxypurpurogallin‐4‐Carboxamides as Influenza A Endonuclease Inhibitors: Synthesis, Structure‐Activity Relationship Analysis, and Structural Characterization of Protein Complex

The influenza RNA-dependent RNA polymerase harbours an endonuclease subunit characterized by a catalytic site housing two divalent metal ions. By effectively chelating both Mg²⁺ and Mn²⁺ ions, a small-molecule inhibitor with a metal-binding pharmacophore can halt endonuclease activity. Herein, two 3'-dehydroxypurpurogallin-4-carboxamide series, namely twelve C-4' unsubstituted and twelve C-4' phenyl substituted congeners were designed and prepared to be tested as inhibitors of the metal-dependent viral enzyme. These inhibitors were accessed through the chemoenzymatic reaction of gallic acid with either pyrocatechol or phenylpyrocatechol moderated by laccase, followed by amidation. Experimental IC₅₀ values were determined using AlphaScreen technology, with the most potent inhibitors exhibiting IC₅₀ values around 0.35 μM. Using X-ray crystallography, we analyzed structure of the endonuclease in complex with one potent 3'-dehydroxypurpurogallin-carboxamide at 2.0 Å resolution, revealing the coordination of the compound's triad of oxygen atoms with the two metal ions in the influenza A endonuclease active site.