Anticancer potential of 1H-naphth[2,3-d]imidazolium salts through structural, DNA binding, and tumor necrosis factor alpha (TNF-α) modulation studies

Abstract

A structurally diverse series of 1H-naphth[2,3-d]imidazolium salts was prepared by N-alkylation/quaternization and isolated in high yields. Structural assignment by ¹H NMR spectroscopy confirmed successful quaternization, while single-crystal x-ray diffraction analyses of BIM, CIM, and CIC revealed that the fused naphthimidazolium core remains essentially planar and that peripheral substituents, counterions and solvent molecules govern torsion and supramolecular packing. Interaction of the salts with calf thymus DNA was investigated by UV–vis absorption titration and agarose gel electrophoresis. All compounds showed measurable but substituent-dependent DNA affinity, with intrinsic binding constants in the range 0.08 × 10⁶–1.37 × 10⁶ M⁻¹, consistent with mixed binding involving π-stacking together with groove and/or electrostatic contributions. In vitro anticancer evaluation against MCF-7 and A549 cells revealed a pronounced structure–activity relationship: sulfonate-containing analogues were inactive, whereas bromobenzyl- and carbazolyl-containing halide salts displayed low-micromolar cytotoxicity, with BIC and CIC among the most active members of the series. Because cytotoxic potency did not correlate directly with DNA binding strength, TNF-α was examined by docking, 300 ns molecular dynamics simulations, and cellular ELISA. Docking/MD suggested favorable interactions of the active salts within the Tyr-rich hydrophobic cavity of TNF-α, while ELISA assays showed that BIB and CIC suppressed LPS-induced TNF-α production in HeLa cells by up to 47.2% and 52.8%, respectively, at 2 μM. Overall, hydrophobic and π-extended substitution around the naphthimidazolium core is a major determinant of cytotoxic activity and TNF-α modulation, but further optimization is required to improve selectivity and establish direct target engagement.