UC Santa Barbara

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 (t_1/2 = 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.