AbstractA series of amino acid based7H-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 compound15cas 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 Asp22and the amide backbone NH of Ile23in the adenosine binding pocket and the carboxylate forms hydrogen bonds to the amide backbone of Phe157and Asp156, part of the oxyanion subsite of Mac1. Compound15calso 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.Graphical Abstract