Small-Molecule Thioesters as SARS-CoV-2 Main Protease Inhibitors: Enzyme Inhibition, Structure–Activity Relationships, Antiviral Activity, and X-ray Structure Determination

Abstract

The main protease (M^pro, 3CL^pro) of SARS-CoV-2 is an attractive target in coronaviruses because of its crucial involvement in viral replication and transcription. Here, we report on the design, synthesis, and structure-activity relationships of novel small-molecule thioesters as SARS-CoV-2 M^pro inhibitors. Compounds <b>3w</b> and <b>3x</b> exhibited excellent SARS-CoV-2 M^pro inhibition with <i>k</i>_inac/<i>K</i>_i of 58,700 M^-1 s^-1 (<i>K_i</i> = 0.0141 μM) and 27,200 M^-1 s^-1 (<i>K</i>_i = 0.0332 μM), respectively. In Calu-3 and Vero76 cells, compounds <b>3h</b>, <b>3i, 3l</b>, <b>3r</b>, <b>3v</b>, <b>3w</b>, and <b>3x</b> displayed antiviral activity in the nanomolar range without host cell toxicity. Co-crystallization of <b>3w</b> and <b>3af</b> with SARS-CoV-2 M^pro was accomplished, and the X-ray structures showed covalent binding with the catalytic Cys145 residue of the protease. The potent SARS-CoV-2 Mpro inhibitors also inhibited the M^pro of other beta-coronaviruses, including SARS-CoV-1 and MERS-CoV, indicating that they might be useful to treat a broader range of coronaviral infections.