Mitochondrial calcium uniporter (MCU) is a Ca2+ transporter located in the inner membrane of mitochondria that plays a crucial role in Ca2+ homeostasis. Mitochondrial Ca2+ overload has implications in many pathological conditions such as neurodegenerative disorders, ischemia-reperfusion injury (IRI), diabetes and cancer. Therefore, the development of MCU inhibitors to reduce mitochondrial Ca2+ overload has therapeutic potential for improving these conditions. The known MCU inhibitor Ru265, which aquates rapidly (t1/2 = 2.3 min) at axial positions under physiological conditions to form the active species Ru265′ to interact with MCU, has limitations in kinetic stability and cell permeability. To address these issues, two new Ru265 derivatives are developed by attaching phosphinate and phosphonate ligands at the axial positions. The first Ru265 derivative featuring diphenylphosphinate ligands has enhanced cell permeability due to increased lipophilicity. It also exhibits improved kinetic stability with an aquation half-life within hour regime. The second Ru265 derivative incorporating methylphosphonate ligands exhibits pH-dependent thermodynamic and kinetic stability, where the compound undergoes a higher level of aquation (i.e. activation) at a faster rate at acidic pH whereas remains mostly intact at physiological pH. It indicates that this compound can be selectively activated in diseased cells with acidic microenvironments, like cancer cells and cells undergoing IRI. Furthermore, formation of stable asymmetric intermediates with different axial ligands (one aqua and the other phosphonate) is captured during aquation. This observation suggests the potential for developing MCU probes using aqua, phosphonate-capped Ru265 derivatives, where the aqua end can dock into MCU opening pore for interaction whereas the phosphonate end can carry a fluorophore or click-chemistry handle to function as a reporter. In conclusion, the two Ru265 derivatives developed in this work are able to aquate to form active species Ru265' for MCU inhibition. Besides, compared with the parental compound, these derivatives demonstrate better prodrug properties and potential for broader applications.