Electrophilic warhead engagement and structure-activity relationship of Benzothiazole-based putative covalent inhibitors targeting SARS-CoV-2 Main protease.

SARS-CoV-2 has precipitated a global health crisis. The Main Protease (Mpro) represents a validated pharmacological target. A series of benzothiazole-based derivatives was designed, synthesized, and evaluated as Mpro inhibitors. The synthesized 5-nitro benzothiazole intermediates (26-29) displayed moderate Mpro inhibition with low to moderate cytotoxicity (CC₅₀ = 16-83 μM in HEK-293 cells). These compounds were subsequently optimized through SAR studies guided by binding site analyses. Lead optimization afforded highly potent derivatives, notably compound 35 (IC50 = 0.026 μM), exhibiting superior potency compared to GC376 and an exceptionally high selectivity index (10,653.8), indicating a favorable potency and safety profile. Acute oral toxicity studies classified compound 35 (LD50 = 947.6 mg/kg BW) and 48 (LD50 = 274.6 mg/kg BW) as Category IV and III toxicants, indicating low acute oral toxicity. Histopathological analysis revealed that the intermediate compound 28 induced hepatic alterations, whereas the final SAR-optimized compound 35 exhibited preserved liver architecture with no apparent pathological changes. Molecular Docking analyses revealed that these benzothiazole derivatives effectively occupied the Mpro active site sub-pocket through stable H-bonding and hydrophobic interactions, thereby facilitating catalytic dyad engagement and pocket adaptability. Covalent docking of compound 35 indicated favorable binding and putative covalent interaction with Cys145. MD simulations confirmed that compound 35 stabilized the protein structure with enhanced compactness, stable interactions, and reduced conformational fluctuations compared to the apo protein. Collectively, these findings highlight benzothiazole-based scaffolds as promising SARS-CoV-2 Mpro inhibitors.