Novel Alkynylamide-Based\nNonpeptidic Allosteric Inhibitors\nfor SARS-CoV‑2 3‑Chymotrypsin-like Protease

Although\nthe coronavirus disease 2019 (COVID-19) crisis\nhas passed,\nthere remains a necessity for continuous efforts toward developing\nmore targeted drugs and preparing for potential future virus attacks.\nCurrently, most of the drugs received authorization for the treatment\nof COVID-19 have exhibited several limitations, such as poor metabolic\nstability, formidable preparation, and uncertain effectiveness. It\nis still significant to develop novel, structurally diverse small-molecule\nantiviral drugs targeting SARS-CoV-2 3-chymotrypsin-like protease\n(3CL<sup>pro</sup>). Herein, we report a class of alkynylamide-based\nnonpeptidic 3CL<sup>pro</sup> inhibitors that can be prepared conveniently\nby our previously developed one-pot synthetic method. The structure–activity\nrelationships of alkynylamides as SARS-CoV-2 3CL<sup>pro</sup> inhibitors\nhave been carefully investigated and discussed in this study. The\ntwo stereoisomers of the resulting molecules exhibit stereoselective\ninteraction with 3CL<sup>pro</sup>, and the optimized compound (<i>S</i>,<i>R</i>)-<b>4y</b> inhibits 3CL<sup>pro</sup> with high potency (IC<sub>50</sub> = 0.43 μM), low cytotoxicity,\nand acceptable cell permeability. Compound (<i>S</i>,<i>R</i>)-<b>4y</b> presents as a noncovalent inhibitor of\n3CL<sup>pro</sup> against SARS-CoV-2 by the time-dependent inhibition\nassay (TDI) and mass spectrometry analysis. The Lineweaver–Burk\nplots, binding energy, surface plasmon resonance, and molecular docking\nstudies suggest that (<i>S</i>,<i>R</i>)-<b>4y</b> specifically binds to an allosteric pocket of the SARS-CoV-2\n3CL<sup>pro</sup>. These findings provide a novel class of nonpeptidic\nalkynylamide-based allosteric inhibitors with high selectivity against\nSARS-CoV-2 3CL<sup>pro</sup> featured by a simplified one-pot synthesis\nat room temperature in air.