As expected, chloromethylketone 12 is a potent irreversible inhibitor of SARS CoV-1 3CLpro. Compounds 22–25 possess the most electron-deficient benzoate leaving groups and display kinetics consistent with irreversible inhibition. The remaining entries appear to be reversible inhibitors in the time scale of the assay, with 28 possessing the most potent IC50. A crystal structure (PDB code 6XHN)38 of 28 in complex with SARS CoV-1 3CLpro at 2.25 Å shows a covalent adduct, which demonstrates the bimodal activity of certain acyloxy inhibitors (Figure 4). In this structure, the electron density for the 2-cyanobenzoate moiety is absent and the 3CLpro active-site cysteine (Cys145) sulfur forms a covalent bond to the methylene carbon (1.8 Å C–S bond length). The ketone carbonyl is positioned within the oxyanion hole and as such engaging in hydrogen bonds with backbone NH groups of Gly143 and Cys145. A detailed analysis of an extended hydrogen bond network from catalytic His41 reveals that the side-chain imidazole serves as a hydrogen bond donor to the interior of the protease. Specifically, the imidazole hydrogen is directed toward a lone pair electron acceptor from a structural water. We can conclude that this water must be an acceptor to His41 as one of the water hydrogens is engaged with acceptor electrons on the side chain of Asp187, and the second hydrogen engages a terminating acceptor backbone carbonyl on Asp176 through a short network that includes the side chains of an internal, neutral His164 and Thr175.