Integrated synthetic, biological, and molecular simulation exploration of halogenated benzohydrazide-based acyl hydrazones: Assessing stability and binding affinity toward COX isoforms and MCF7 cells

Fifteen 2,5-dichloro-N′-(substituted benzylidene)benzohydrazide derivatives (3a-o) were synthesized and evaluated for their biological activities. Structures were confirmed via1H-NMR,13C-NMR, FT-IR spectra, and elemental analysis. In vitro MTT assays revealed that derivatives 3a, 3n and 3o showed the highest anti-proliferative effects against the MCF7 breast adenocarcinoma cell line, with IC50 values ranging from 30.19 to 33.98 μM. These compounds displayed lower toxicity toward non-cancerous HEK293 cells. Enzyme inhibition screens indicated a general preference for the COX-1 isoform, where the 4-methylsulfanyl derivative (3g) emerged as the most active inhibitor, reducing COX-1 activity by 60.74% at 20 μM. A divergence was observed between enzymatic COX-1 inhibition and cellular cytotoxicity; the most potent COX-1 inhibitor (3g) lacked corresponding anti-proliferative activity, suggesting the cytotoxicity of 3a, 3n and 3o likely involves multi-target mechanisms rather than direct COX-1 blockade. In silico ADME predictions supported the high membrane permeability and oral bioavailability of the series. To elucidate the active site dynamics, molecular docking and 300 ns molecular dynamics simulations were performed for compound 3n. The simulations demonstrated a stable binding mode within the COX-1 pocket, maintained primarily by a persistent hydrogen bond with Arg120. These findings present the acyl hydrazone scaffold as a viable starting point for targeted agent development, while highlighting the need for further pathway-specific studies.