Singh, VipinVipinSinghGupta, PrabalPrabalGuptaHaribabu, JebitiJebitiHaribabuArulraj, ArunachalamArunachalamArulrajMoraga, DanielDanielMoragaSreekanth, AnandaramAnandaramSreekanth2026-07-072026-07-072026JOURNAL OF MOLECULAR STRUCTURE, 1353, 144676 (2026). https://doi.org/10.1016/j.molstruc.2025.1446760022-28601872-8014https://hdl.handle.net/20.500.12740/24806We 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 (Kb= 7.98 x 104) and quenching constant (Kq= 2.4 x 104, Kapp= 2.5 x 106). BSA fluorescence quenching studies confirmed strong binding affinity (Kb= 4.79 x 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."info:eu-repo/semantics/openAccessThiosemicarbazone5-Bromo salicylaldehydeBSA bindingDNA interactionMolecular dockingCytotoxicityArticulohttps://doi.org/10.1016/j.molstruc.2025.144676