Ruthenium(II)-p-Cymene/Ferrocenyl Complexes as Multifunctional Therapeutic Candidates: Integrated Biological Evaluation, Anticancer Mechanisms, Antimicrobial Activity, and Biomolecular Interactions

Background/Objectives: Two novel heteronuclear Ru(II)-p-cymene complexes bearing ferrocenyl thioether ligands were synthesized and evaluated for their anticancer and antimicrobial potential. Methods/Results: Both complexes exhibited pronounced antiproliferative activity against human cancer cell lines, with IC50 values ranging from 8.42 to 24.62 μM after 72 h, showing activity corresponding to the level of activity of standard cisplatin treatment. Mechanistic studies in HeLa cells revealed concentration-dependent cell cycle disruption accompanied by an increased sub-G1 population and only a slight elevation in intracellular reactive oxygen species, without the potential to induce apoptosis, indicating that growth inhibition is predominantly mediated through cytostatic rather than apoptotic mechanisms. In contrast, both compounds displayed substantially lower activity in three-dimensional multicellular tumor spheroids, emphasizing the need to improve tumor penetration and efficacy in physiologically relevant models. The complexes inhibited all microorganisms included in the tested panel, with MIC values ranging from 0.16 to 10 mg/mL; Staphylococcus aureus was the most susceptible strain (MIC = 0.16 mg/mL). Biophysical studies revealed weak-to-moderate DNA binding of both complexes, consistent with a predominantly groove-associated and external binding mode rather than classical intercalation, as supported by fluorescence displacement, viscosity, and molecular docking analyses. Both complexes exhibited moderate-to-high affinity for human serum albumin, while the single-methylene spacer difference resulted in distinct albumin binding-site preferences, highlighting the sensitivity of protein recognition to subtle structural modifications without substantially affecting overall binding affinity. Conclusions: Overall, these Ru(II)-p-cymene/ferrocenyl complexes combine promising antiproliferative and antimicrobial activities with a mechanism of action distinct from cisplatin, representing attractive candidates for further structural optimization aimed at improving selectivity and efficacy in three-dimensional tumor models.