Design, Synthesis and Evaluation of Inhibitors of the SARS-CoV2 nsp3 Macrodomain

A series of amino acid based 7H -pyrrolo[2,3- d ]pyrimidines were designed and synthesized to discern the structure activity relationships against the SARS-CoV-2 nsp3 macrodomain (Mac1), an ADP-ribosylhydrolase that is critical for coronavirus replication and pathogenesis. Structure activity studies identified compound 15c as a low-micromolar inhibitor of Mac1 in two ADP-ribose binding assays. This compound also demonstrated inhibition in an enzymatic assay of Mac1 and displayed a thermal shift comparable to ADPr in the melting temperature of Mac1 supporting binding to the target protein. A structural model reproducibly predicted a binding mode where the pyrrolo pyrimidine forms a hydrogen bonding network with Asp ²² and the amide backbone NH of Ile ²³ in the adenosine binding pocket and the carboxylate forms hydrogen bonds to the amide backbone of Phe ¹⁵⁷ and Asp ¹⁵⁶ , part of the oxyanion subsite of Mac1. Compound 15c also demonstrated notable selectivity for coronavirus macrodomains when tested against a panel of ADP-ribose binding proteins. Together, this study identified several low MW, low μM Mac1 inhibitors to use as small molecule chemical probes for this potential anti-viral target and offers starting points for further optimization.