Miniaturized Modular Click Chemistry‐enabled Rapid Discovery of Unique SARS‐CoV‐2 Mpro Inhibitors With Robust Potency and Drug‐like Profile

Abstract The COVID‐19 pandemic has required an expeditious advancement of innovative antiviral drugs. In this study, focused compound libraries are synthesized in 96‐ well plates utilizing modular click chemistry to rapidly discover potent inhibitors targeting the main protease (M pro ) of SARS‐CoV‐2. Subsequent direct biological screening identifies novel 1,2,3‐triazole derivatives as robust M pro inhibitors with high anti‐SARS‐CoV‐2 activity. Notably, C5N17B demonstrates sub‐micromolar M pro inhibitory potency (IC 50 = 0.12 µM) and excellent antiviral activity in Calu‐3 cells determined in an immunofluorescence‐based antiviral assay (EC 50 = 0.078 µM, no cytotoxicity: CC 50 > 100 µM). C5N17B shows superior potency to nirmatrelvir (EC 50 = 1.95 µM) and similar efficacy to ensitrelvir (EC 50 = 0.11 µM). Importantly, this compound displays high antiviral activities against several SARS‐CoV‐2 variants (Gamma, Delta, and Omicron, EC 50 = 0.13 – 0.26 µM) and HCoV‐OC43, indicating its broad‐spectrum antiviral activity. It is worthy that C5N17B retains antiviral activity against nirmatrelvir‐resistant strains with T21I/E166V and L50F/E166V mutations in M pro (EC 50 = 0.26 and 0.15 µM, respectively). Furthermore, C5N17B displays favorable pharmacokinetic properties. Crystallography studies reveal a unique, non‐covalent multi‐site binding mode. In conclusion, these findings substantiate the potential of C5N17B as an up‐and‐coming drug candidate targeting SARS‐CoV‐2 M pro for clinical therapy.