Design and synthesis of multi-substituted pyrazole derivatives as potential allosteric inhibitors of 3CL^pro

The COVID-19 pandemic severely threatened global public health, and the SARS-CoV-2 main protease (3CL^pro) is a vital target for anti-COVID-19 drug development. Nevertheless, most reported 3CL^pro orthosteric inhibitors readily trigger viral drug resistance, limiting their long-term clinical application. Herein, we identified a novel 3CL^pro allosteric site AS1 via AlloSite and AlloReverse tools. Through virtual screening and structural optimization, a series of potent 3CL^pro allosteric derivatives were designed and synthesized. FRET assays confirmed that derivatives E42 (IC₅₀ = 59.85 ± 4.10 μM) and E60 (IC₅₀ = 7.64 ± 0.25 μM) possessed superior inhibitory potency to the lead compound E0. Structure-activity relationship and molecular docking analyses revealed that the p-trifluoromethylphenethylamine fragment, trimethylene linker, amide bond, methyl group and 4-nitropyrazole ring are key active structural motifs, enabling E42 to specifically bind the AS1 allosteric site. The antiviral activity and cytotoxicity of E42 and E60 were evaluated using a SARS-CoV-2 trans-complementation cell culture system (containing mNG/Fluc) and the CCK-8 assay, respectively. The luciferase reporter assay revealed a consistent trend in antiviral activity among nirmatrelvir, E42, and E60, with E42 exhibiting moderate potency (EC₅₀ = 13.79 ± 1.28 μM). Molecular dynamics simulations indicated that E42 inhibits 3CL^pro activity by stabilizing its inactive conformation and disrupting protein allosteric equilibrium. Compared with conventional orthosteric inhibitors prone to drug resistance and poorly active, unsafe reported allosteric inhibitors, the optimized compounds in this study combine favorable potency and biosafety. This work provides novel lead structures and mechanistic insights for developing anti-COVID-19 3CL^pro allosteric inhibitors.