The mitochondrial calcium uniporter (MCU) is a transmembrane protein that resides on the inner membrane of the mitochondria and mediates calcium uptake into this organelle. Given the critical role of mitochondrial calcium trafficking in cellular function, inhibitors of this channel have arisen as tools for studying the biological relevance of this process and as potential therapeutic agents. In this study, four new analogues of the previously reported Ru-based MCU inhibitor [ClRu(NH 3 ) 4 (μ-N)Ru(NH 3 ) 4 Cl]Cl 3 (Ru265) are reported. These compounds, which bear axial carboxylate ligands, are of the general formula [(RCO 2 )Ru(NH 3 ) 4 (μ-N)Ru(NH 3 ) 4 (O 2 CR)]X 3 , where X = NO 3 − or CF 3 SO 3 − and R = H (1), CH 3 (2), CH 2 CH 3 (3), and (CH 2 ) 2 CH 3 (4). These complexes were fully characterized by IR spectroscopy, NMR spectroscopy, and elemental analysis. X-ray crystal structures of 1 and 3 were obtained, revealing the expected presence of both the linear Ru(μ-N)Ru core and axial formate and propionate ligands. The axial carboxylate ligands of complexes 1− 4 are displaced by water in buffered aqueous solution to give the aquated compound Ru265′. The kinetics of these processes were measured by 1 H NMR spectroscopy, revealing half-lives that span 5.9−9.9 h at 37 °C. Complex 1 with axial formate ligands underwent aquation approximately twice as fast as the other compounds. In vitro cytotoxicity and mitochondrial membrane potential measurements carried out in HeLa and HEK293T cells demonstrated that none of these four complexes negatively affects cell viability or mitochondrial function. The abilities of 1−4 to inhibit mitochondrial calcium uptake in permeabilized HEK293T cells were assessed and compared to that of Ru265. Fresh solutions of 1−4 are approximately 2-fold less potent than Ru265 with IC 50 values in the range of 14.7−19.1 nM. Preincubating 1−4 in aqueous buffers for longer time periods to allow for the aquation reactions to proceed increases their potency of mitochondrial uptake inhibition to match that of Ru265. This result indicates that 1− 4 are aquation-activated prodrugs of Ru265′. Finally, 1−4 were shown to inhibit mitochondrial calcium uptake in intact, nonpermeabilized cells, revealing their value as tools and potential therapeutic agents for mitochondrial calcium-related disorders.

The synthesis and characterization of the 15 N-labeled analogue of the mitochondrial calcium uptake inhibitor [Cl(NH 3 ) 4 Ru(μ-N)Ru(NH 3 ) 4 Cl] 3 + (Ru265) bearing [ 15 N]NH 3 ligands is reported. Using [ 1 H, 15 N] HSQC NMR spectroscopy, the rate constants for the axial chlorido ligand aquation of [ 15 N]Ru265 in pH 7.4 buffer at 25 °C were found to be k 1 = (3.43 � 0.03) × 10 À 4 s À 1 and k 2 = (4.03 � 0.09) × 10 À 3 s À 1 . The reactivity of [ 15 N]Ru265 towards biologically relevant small molecules was also assessed via this method, revealing that this complex can form coordination bonds to anionic oxygen and sulfur donors. Time-based studies on these ligand-binding reactions reveal this process to be slow relative to the time required for the complex to inhibit mitochondrial calcium uptake, suggesting that hydrogen-bonding interactions, rather than the formation of coordination bonds, may play a more significant role in mediating the inhibitory properties of this complex.

An analogue of the mitochondrial calcium uniporter (MCU) inhibitor Ru265 containing axial ferrocenecarboxylate ligands is reported. This new complex exhibits enhanced cellular uptake compared to the parent compound Ru265.