Para-substituted benzoic acid ruthenium(ii) complexes: structural features modulating cytotoxicity

Abstract

This work describes the synthesis of six new ruthenium(II) complexes bearing para-substituted benzoic acids of general formula [Ru(L)(dppb)(bipy)]PF₆, where L = terephthalic acid (L-CO₂H), 4-(chloromethyl)benzoic acid (L-CCl), 4-(bromomethyl)benzoic acid (L-CBr), 4-(amino)benzoic acid (L-NH₂), and 4-(nitro)benzoic acid (L-NO₂), bipy = 2,2′-bipyridine and dppb = 1,4-bis(diphenylphosphino)butane. The complexes were characterized by elemental analysis, molar conductivity, NMR, cyclic voltammetry, IR spectroscopy and, for selected compounds, single-crystal X-ray diffraction. The binuclear complex RuBi exhibited a differentiated structural and spectroscopic pattern, including solvent-dependent ³¹P NMR signal duplication associated with the coexistence of closely related conformers, as supported by DFT calculations. Electrochemical investigations revealed Ru²⁺/Ru³⁺ redox couples whose E_1/2 values strongly depend on the electronic nature of the para substituent, following the trend NO₂ > COOH > CH₂Br ≈ CH₂Cl > NH₂. The strongly electron-donating –NH₂ group significantly lowers the oxidation potential and introduces an additional ligand-centered oxidation process, highlighting the pronounced electronic modulation imposed by this substituent. The in vitro cytotoxicity of the complexes, free ligands, and cisplatin was evaluated against MDA-MB-231 (breast), A549 (lung), A2780 (ovarian), A2780cis (cisplatin-resistant ovarian), and MRC-5 (non-tumor lung) cell lines using the MTT assay. All ruthenium complexes were more cytotoxic than the corresponding free ligands and cisplatin. Among them, [Ru(L-NH₂)(dppb)(bipy)]PF₆ (RuNH₂) stood out, exhibiting a submicromolar IC₅₀ value (0.5 ± 0.1 µM) against A2780 cells and the highest selectivity index (3.6) of the series. Its superior performance can be correlated with the strong donor character of the –NH₂ group, which modulates the redox properties of the metal center and enhances hydrogen-bonding capability, potentially favoring stronger biological interactions. RuNH₂ was further investigated in advanced biological assays, showing pronounced morphological alterations, significant reduction in clonogenic survival of A2780 cells, and concentration-dependent accumulation in the sub-G1 phase, consistent with induction of cell death. Finally, all complexes, except RuNH₂ due to its intrinsic fluorescence, were evaluated for interaction with human serum albumin, revealing moderate binding affinities compatible with bloodstream transport. Collectively, these findings demonstrate how subtle electronic effects govern redox behavior and cytotoxic performance, highlighting RuNH₂ as a promising candidate for ovarian cancer therapy.