Aminoferrocene‐based prodrugs represent a promising class of redox‐active anticancer agents that can be selectively activated in oxidative cellular environments. However, activation of these systems typically relies on reactive oxygen species (ROS) and is therefore inefficient in intracellular compartments with low ROS levels, such as the cytoplasm. In this study, we explored strategies to enhance activation of organelle‐non‐biased aminoferrocene prodrugs through peroxynitrite (ONOO − ). Using a previously reported aminoferrocene–coumarin fluorogenic probe ( 11a ) and a set of aminoferrocene‐based prodrugs 13 and 20a and ROS‐insensitive controls 20b and 20c earlier developed in our group, we demonstrated that ONOO ‐ generated from the donor 3‐morpholinosydnonimine hydrochloride (SIN‐1) efficiently triggers cleavage of arylboronic acid groups and formation of the active aminoferrocenium species both in cell‐free systems and in cancer cells. Consistent with this mechanism, the anticancer activity of these previously described aminoferrocene‐based prodrugs was significantly enhanced in the presence of SIN‐1 across multiple cancer cell lines. To exploit this effect in a self‐contained system, we designed and synthesized a hybrid aminoferrocene prodrug bearing an organic nitrate ester moiety capable of releasing nitric oxide, which, upon reaction with endogenous superoxide anion radical, should form ONOO − . The resulting compound 7 and several control derivatives were prepared and characterized. Although the design was intended to promote intracellular ONOO − formation and ROS‐mediated activation, biological studies revealed that the nitrate ester functionality itself is the dominant determinant of cytotoxicity. Mechanistic investigations showed that prodrug 7 is efficiently internalized by cancer cells and induces moderate mitochondrial ROS formation but strongly increases intracellular aldehyde levels, likely via intramolecular nitrate‐mediated oxidation at the benzylic position. These findings highlight the complex interplay between redox chemistry, NO‐donor reactivity, and aminoferrocene activation pathways. Overall, this work demonstrates that peroxynitrite can efficiently activate aminoferrocene prodrugs in cytoplasmic environments and provides new insights into the design of redox‐active organometallic therapeutics.
Özkan et al. (Wed,) studied this question.