Abstract
Although the coronavirus disease 2019 (COVID-19) crisis has passed, there remains a necessity for continuous efforts toward developing more targeted drugs and preparing for potential future virus attacks. Currently, most of the drugs received authorization for the treatment of COVID-19 have exhibited several limitations, such as poor metabolic stability, formidable preparation, and uncertain effectiveness. It is still significant to develop novel, structurally diverse small-molecule antiviral drugs targeting SARS-CoV-2 3-chymotrypsin-like protease (3CL(pro)). Herein, we report a class of alkynylamide-based nonpeptidic 3CL(pro) inhibitors that can be prepared conveniently by our previously developed one-pot synthetic method. The structure-activity relationships of alkynylamides as SARS-CoV-2 3CL(pro) inhibitors have been carefully investigated and discussed in this study. The two stereoisomers of the resulting molecules exhibit stereoselective interaction with 3CL(pro), and the optimized compound (S,R)-4y inhibits 3CL(pro) with high potency (IC(50) = 0.43 μM), low cytotoxicity, and acceptable cell permeability. Compound (S,R)-4y presents as a noncovalent inhibitor of 3CL(pro) against SARS-CoV-2 by the time-dependent inhibition assay (TDI) and mass spectrometry analysis. The Lineweaver-Burk plots, binding energy, surface plasmon resonance, and molecular docking studies suggest that (S,R)-4y specifically binds to an allosteric pocket of the SARS-CoV-2 3CL(pro). These findings provide a novel class of nonpeptidic alkynylamide-based allosteric inhibitors with high selectivity against SARS-CoV-2 3CL(pro) featured by a simplified one-pot synthesis at room temperature in air.