Iron Complexes of an Antiproliferative Aroyl Hydrazone: Characterization of Three Protonation States by Electron Paramagnetic Resonance Methods

Tridentate aroyl hydrazones are effective metal chelators in biological settings, and their activity has been investigated extensively for medicinal applications in metal overload, cancer, and neurodegenerative diseases. The aroyl hydrazone motif is found in the recently reported prochelator (AH1-S)(2), which has shown antiproliferative proapoptotic activity in mammalian cancer cell lines. Intracellular reduction of this disulfide prochelator leads to the formation of mercaptobenzaldehyde benzoylhydrazone chelator AH1 and to iron sequestration, which in turn impacts cell growth. Herein, we investigate the iron coordination chemistry of AH1 to determine the structural and spectroscopic properties of the iron complexes in the solid state and in solution. A neutral iron(III) complex of 2:1 ligand-to-metal stoichiometry was isolated and characterized fully to reveal two different binding modes for the tridentate AH1 ligand. Specifically, one ligand binds in the monoanionic keto form, whereas the other ligand coordinates as a dianionic enolate. Continuous-wave electron paramagnetic resonance experiments in frozen solutions indicated that this neutral complex is one of three low-spin iron(III) complexes observed depending on the pH of the solution. Electron spin echo envelope modulation (ESEEM) experiments allowed assignment of the three species to different protonation states of the coordinated ligands. Our ESEEM analysis provides a method to distinguish the coordination of aroyl hydrazones in the keto and enolate forms, which influences both the ligand field and overall charge of the complex. As such, this type of analysis could provide valuable information in a variety of studies of iron complexes of aroyl hydrazones, ranging from the investigation of spin-crossover behavior to tracking of their distribution in biological samples.