Expanding the π-system of Fatty Acid-Anion Transporter Conjugates Modulates Their Mechanism of Proton Transport and Mitochondrial Uncoupling Activity

Mitochondrial uncoupling by small molecule protonophores is a promising strategy for developing novel anticancer agents. Recently, aryl urea substituted fatty acids (aryl ureas) were identified as a new class of protonophoric anticancer agents. To mediate proton transport these molecules self-assemble into membrane-permeable anionic dimers in which intermolecular hydrogen bonds between the carboxylate and aryl-urea anion receptor delocalise the negative charge across the aromatic pi-system. In this work, we extend the aromatic pi-system by introducing a second phenyl substituent to the aryl urea scaffold and compare the proton transport mechanisms and mitochondrial uncoupling actions of these compounds to their monoaryl analogues. It was found that incorporation of meta-linked phenyl substituents into the aryl urea scaffold enhanced proton transport in vesicles and demonstrated superior capacity to depolarise mitochondria, inhibit ATP production and reduce the viability of MDA-MB-231 breast cancer cells. In contrast, diphenyl ureas linked through a 1,4-distribution across the phenyl ring displayed diminished proton transport activity, despite both diphenyl urea isomers possessing similar binding affinities for carboxylates. Mechanistic studies suggest that inclusion of a second aryl ring changes the proton transport mechanism, presumably due to steric factors that impose higher energy penalties for dimer formation. In this paper we introduce a second phenyl substituent to the aryl urea scaffold and evaluate the proton transport mechanisms and mitochondrial uncoupling actions of these compounds against their monoarylated analogues. Notably, incorporation of meta- but not para-linked proximal rings enhanced proton transport and mitochondrial uncoupling actions in MDA-MB-231 cells, consistent with improved charge delocalisation by the urea anion binding group to produce membrane-permeable complexes. image