Multitarget Anticancer Activity of Pyrazoline‐Based Hybrids: Insights into Apoptosis, Bovine Serum Albumin/DNA Interactions, and Caspase Targeting

A series of novel pyrazoline-based hybrids, bearing a pyrrole or indole scaffold, is designed, synthesized, and evaluated for anticancer potential. MTT assay reveals 4'a, 4'c, and 4'e as promising cytotoxic agents, where 4'e possess good pharmacological profile exhibiting prominent potency toward tumor cell lines HeLa, K562, and HL-60 (IC₅₀ = 3.7-5.1 µM), and selectivity toward normal MRC-5 cell line (IC₅₀ > 50 µM). Apoptosis induction is confirmed for all three compounds, with 4'e activating both caspase-3 and caspase-9 demonstrating a dual apoptotic mechanism. Molecular docking supports these findings, revealing a strong binding affinity of 4'e toward both caspases. Interaction with bovine serum albumin (BSA) is confirmed by fluorescence quenching and docking, indicating specific site binding and potential impact on pharmacokinetics. In addition, Förster resonance energy transfer (FRET) analysis provides valuable insight into the binding interaction between the investigated complexes and BSA. DNA-binding studies demonstrate that 4'e preferentially binds within the minor groove, while docking also suggests intercalative potential, which is further supported by viscosity measurements, confirming its ability to modulate DNA-dependent apoptotic signaling via intercalative binding. These results highlight compound 4'e as a promising anticancer candidate with selective cytotoxicity and multitarget engagement.