Discovery, Synthesis and Mechanism Study of 2,3,5-Substituted [1,2,4]-Thiadiazoles as Covalent Inhibitors Targeting 3C-Like Protease of SARS-CoV-2

3C-like protease (3CLpro) is indispensable for the replication and transcription of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), rendering it to be a promising druggable target for treating Corona Virus Disease 2019 (COVID-19). In this study, a series of 2,3,5-substituted [1,2,4]-thiadiazole analogs were discovered to inhibit 3CLpro as non-peptidomimetic covalent binders at sub-micromolar levels, with IC50 values ranging from 0.118 to 0.582 µM. The antiviral abilities of these compounds were also evaluated in cell-based assays and the compound 6g exhibited potent antiviral activity with an EC50 value of 7.249 μM. These compounds were proposed to covalently bind with the catalytic cysteine 145 via a ring-opening metathesis reaction mechanism. We chose compound 6a, one of the identified hit compounds, as the representation to study the reaction mechanism in great depth through the combination of Mass Spectroscopic (MS) analysis, docking study and Density Function Theory (DFT) calculations. The covalent-binding reaction between 6a and 3CLpro was proved by the MS assays. The binding modes of the compound against 3CLpro before and after the reaction were predicted by non-covalent and covalent docking simulations, respectively. The process of ring-opening metathesis was theoretically studied by the transition state search using Gaussian. The electrophilicity of the 2,3,5-substituted [1,2,4]-thiadiazole group was demonstrated by the Frontier Molecular Orbitals (FMOs) and Molecular Electrostatic Potentials (MEPs). The DFT calculations indicated that the 2,3,5-substituted [1,2,4]-thiadiazole group could covalently modify the catalytic cysteine in the binding pocket of 3CLpro as a potential warhead.