Discovery and Crystallographic Studies of Trisubstituted Piperazine Derivatives as Non-Covalent SARS-CoV-2 Main Protease Inhibitors with High Target Specificity and Low Toxicity

The continuous spread of SARS-CoV-2 calls for more direct-acting Antiviral agents to combat the highly infectious variants. The main protease (M^pro) is an promising target for anti-SARS-CoV-2 drug design. Here, we report the discovery of potent non-covalent non-peptide M^pro inhibitors featuring a 1,2,4-trisubstituted piperazine scaffold. We systematically modified the non-covalent hit MCULE-5948770040 by structure-based rational design combined with multi-site binding and privileged structure assembly strategies. The optimized compound GC-14 inhibits M^pro with high potency (IC₅₀ = 0.40 μM) and displays excellent Antiviral activity (EC₅₀ = 1.1 μM), being more potent than Remdesivir. Notably, GC-14 exhibits low cytotoxicity (CC₅₀ > 100 μM) and excellent target selectivity for SARS-CoV-2 M^pro (IC₅₀ > 50 μM for cathepsins B, F, K, L, and Caspase 3). X-ray co-crystal structures prove that the inhibitors occupy multiple subpockets by critical non-covalent interactions. These studies may provide a basis for developing a more efficient and safer therapy for COVID-19.