NMR spectroscopy stability studies of Ru-IM, a prodrug candidate for triple negative breast cancer.

Chemical stability and metal speciation are key determinants of the biological behavior and translational potential of metal-based chemotherapeutic agents, for which ligand exchange and chemical transformation frequently occur under physiological conditions. In these systems, pharmacological activity is defined not by a single molecular entity but by the distribution and interconversion of metal-containing species in solution and biological media. Here, we investigate the time-dependent speciation of the highly water-soluble ruthenium complex [(η6-p-cymene)Ru(κ-N,O-Ph₃P=N-CO-2-NC₅H₄)]Cl (Ru-IM), which exhibits a shelf life exceeding two years and has demonstrated robust anticancer efficacy in preclinical mechanistic and pharmacokinetic studies in triple-negative breast cancer mouse models. Using NMR spectroscopy, we characterized Ru-IM speciation in D₂O, deuterated phosphate-buffered saline, deuterated Dulbecco's Modified Eagle's Medium (DMEM), and DMEM supplemented with fetal bovine serum. Two dominant speciation pathways were identified: (A) hydrolysis with loss of O=PPh₃ from the IM ligand, which results in loss of anticancer activity, and (B) ruthenium cyclometallation of a phenyl group from the IM ligand, which preserves biological activity. The relative distribution of these species is strongly influenced by concentration, temperature, and medium composition. Notably, Ru-IM remains the predominant species in aqueous solution for at least 24 h, with stability extendable to several days under optimized storage conditions. Collectively, these results support a prodrug activation model, in which Ru-IM undergoes controlled speciation under biologically relevant conditions to generate an active cyclometalated ruthenium species.