Cafeic acid benzylidene hydrazides as novel procaspase-3 activators: Rational design, synthesis, and mechanistic evaluation of antitumor efficacy

Caspase-3 is an essential intracellular protein mediating cell Apoptosis and a key target in the Apoptosis pathway of Cancer cell. Activating its existing inactive proenzyme, Procaspase-3, induces Apoptosis in various tumor cells and has emerged as a promising Cancer therapy strategy. In this study, a novel series of hybrid caffeic acid benzylidene hydrazide derivatives were rationally designed and evaluated as potent procaspase-3 activators. Caffeic acid was first identified as a privileged scaffold for procaspase-3 activation through structure-based pharmacophore modeling. By hybridizing this natural product scaffold with optimized structural elements from PAC-1, a reference procaspase-3 activator, 28 target compounds (K-01 ∼ K-28) were successfully synthesized. Molecular modeling studies revealed that these hybrid compounds possess enhanced zinc chelation capacity, a crucial mechanism for procaspase-3 activation. The antiproliferative activity of these compounds was systematically evaluated against A549 (non-small cell lung Cancer) and A375 (melanoma) cell lines. Among the synthesized derivatives, 23 compounds (82.1 %) demonstrated superior potency compared to PAC-1 (positive control). Notably, compounds K-15 and K-22 exhibited significant activity against A549 cells (IC₅₀ = 3.96 ± 1.07 μM and 5.60 ± 2.20 μM, respectively) while maintaining minimal cytotoxicity toward human lung epithelial cells (BEAS-2B), as evidenced by high cell viability rates of 91.99 % and 95.37 %. Direct procaspase-3 activation by these lead compounds was further confirmed through mechanistic studies. In silico ADMET profiling demonstrated that the lead compound K-15 exhibits optimal pharmacokinetic properties, including moderate aqueous solubility (Level 2), negligible blood-brain barrier penetration (Level 4), absence of hepatotoxicity risk, and favorable intestinal absorption (Level 1). All compounds were confirmed to be non-inhibitors of CYP2D6, indicating minimal potential for drug-drug interactions. These results demonstrate that the hybrid molecular design strategy effectively combines the pharmacophoric advantages of both natural products and synthetic activators. The developed procaspase-3 activators exhibit improved therapeutic potential and represent promising candidates for further development as targeted Anticancer agents modulating the Caspase-3 activation pathway.

Apoptosis induction; Caffeic acid derivatives; In silico ADMET; Pharmacophore modeling; Procaspase-3 activator; Targeted therapy.