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 (Mpro) is an promising target for anti-SARS-CoV-2
drug design. Here, we report the discovery of potent non-covalent
non-peptide Mpro 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 Mpro 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 Mpro (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.