Employing phosphonate and phosphinate ligands for prodrug development of the mitochondrial calcium uniporter inhibitor Ru265.

Ru265, [Ru2(μ-N)(NH3)8Cl2]Cl3, is a potent nanomolar inhibitor of the mitochondrial calcium uniporter (MCU), the transporter that mediates Ca2+ uptake into the mitochondria. This compound and related MCU inhibitors are promising therapeutic agents and chemical biology tools for studying intracellular Ca2+ dynamics. Axial ligand modification of these Ru-based complexes provides a facile way to tune their properties and make prodrugs. In this study, the use of axial phosphonate and phosphinate ligands was explored within this compound class, yielding [Ru2(μ-N)(NH3)8(OPO(OH)Me)2](CF3SO3)3 (1) and [Ru2(μ-N)(NH3)8(OPOPh2)2](CF3SO3)3 (2). Both complexes were characterized by multinuclear NMR spectroscopy, revealing downfield shifts of the 31P resonances of the coordinated ligands. The crystal structure of 1 was also obtained, which confirmed coordination of the methylphosphonate ligands to the axial sites of the Ru core. The aquation of 1 and 2 were studied by NMR and UV-vis spectroscopy. Unexpectedly, compound 1 undergoes partial aquation, arresting at a final product in which only one axial methylphosphonate is displaced by water. By contrast, 2 loses both diphenylphosphinates via aquation (t1/2 = 1.7 h) under physiological conditions. The in vitro biological investigations of 1 and 2 in HeLa cells showed that neither demonstrate high cytotoxicity nor depolarize the mitochondrial membrane potential. Both compounds exhibit nanomolar inhibitory activity against mitochondrial Ca2+ uptake in permeabilized HEK293T cells and modest inhibitory activity against this process in intact HeLa cells. Notably, 1 shows pH-dependent activity for MCU inhibition, with greater inhibition in more acidic conditions, while 2 shows improvement in cellular uptake efficiency.