Synthesis of Triphenylphosphonium-Linked Derivative of 3,5-Di tert -butyl-4-hydroxybenzylidene-malononitrile (SF6847) via Knoevenagel Reaction Yields an Effective Mitochondria-Targeted Protonophoric Uncoupler

Mitochondrial uncouplers are actively sought as potential therapeutics. Here, we report the first successful synthesis of mitochondria-targeted derivatives of the highly potent uncoupler 3,5-di -butyl-4-hydroxybenzylidene-malononitrile (SF6847), bearing a cationic alkyl(triphenyl)phosphonium (TPP) group. As a key step of the synthesis, we used condensation of a ketophenol with malononitrile via the Knoevenagel reaction. SF-C -TPP with a pentamethylene linker between SF6847 and TPP, stimulating respiration and collapsing membrane potential of rat liver mitochondria at submicromolar concentrations, proved to be the most effective uncoupler of the series. SF-C -TPP showed pronounced protonophoric activity on a model planar bilayer lipid membrane. Importantly, SF-C -TPP exhibited rather low toxicity in fibroblast cell culture, causing mitochondrial depolarization in cells at concentrations that only slightly affected cell viability. SF-C -TPP was more effective in decreasing the mitochondrial membrane potential in the cell culture than SF6847, in contrast to the case of isolated mitochondria. Like other zwitterionic uncouplers, SF-C -TPP inhibited the growth of in the micromolar concentration range.