Exploring the biological potential of N4-substituted thiosemicarbazones derived from 5-bromo salicylaldehyde: In silico and in vitro evaluation

We report the synthesis and comprehensive characterization of N⁴-substituted thiosemicarbazones derived from 5‑bromo salicylaldehyde, aimed at exploring their potential biomedical applications. Structural analyses by UV–visible, IR, NMR, and HRMS confirmed the successful formation of Schiff-base frameworks with desirable electronic and molecular features. DNA binding studies (UV–Vis and fluorescence) revealed that L₃ interact with calf thymus DNA via a mixed intercalative and groove-binding mode, showing the highest binding constant (K_b= 7.98 × 10⁴) and quenching constant (K_q= 2.4 × 10⁴, K_app= 2.5 × 10⁶). BSA fluorescence quenching studies confirmed strong binding affinity (K_b= 4.79 × 10⁴) and binding sites (n = 1), indicating efficient transport and bioavailability in serum environments. Molecular docking supported the experimental results, showing stable binding of L₃ (Vins Score = –7.0 kcal/mol) with the active site of EGFR protein. DFT calculations provided optimized geometries, HOMO–LUMO energy gaps and confirmed enhanced electronic delocalization in L₃. MEP surface analysis identified electronegative binding pockets responsible for biological recognition. Swiss ADME predictions indicated good drug-likeness, acceptable lipophilicity, and favorable pharmacokinetic profiles, with no major toxicity alerts. The strong DNA/BSA binding, favorable electronic features, and promising ADME behavior make L₃ a potential anticancer lead molecule. Overall, these combined experimental and computational results establish L₃ as a strong anticancer lead candidate (IC₅₀ values of MDA-MB-231: 26.0 ± 0.5, MCF-7: 47.2 ± 0.5, MCF-10A: 85.1 ± 0.8) meriting further biological and mechanistic investigations. The results suggest that these compounds, especially the 4-substituted analogs, may serve as promising candidates for anticancer drug development. Compound L₄ has been previously reported; however, it was resynthesized in our lab and characterized for comparison with validation purposes.