Conventional chemotherapy remains limited by systemic toxicity and emerging drug resistance, requiring therapeutic strategies that specifically activate cytotoxic agents within cancer cells. Reactive oxygen species (ROS)-responsive prodrugs offer a promising approach by exploiting higher oxidative stress in many cancer cells compared with most normal tissues. However, most existing ROS-responsive systems rely on elevated bulk ROS levels without accounting for the subcellular compartmentalization of oxidative stress. Recent evidence indicates that ROS is concentrated in discrete intracellular microenvironments rather than being uniformly increased throughout the cell. Directing prodrug activation to such ROS-rich compartments therefore offers promising potential to improve therapeutic specificity and efficacy. This dissertation addresses this gap through the development of two complementary H2O2-responsive prodrugs based on the N-alkylaminoferrocene (NAAF) scaffold. Prodrug A targets mitochondria, a major source of ROS production in cancer cells. It also integrates an arylboronic pinacol ester as an H2O2-sensitive trigger and a self-immolative linker connecting the topoisomerase I inhibitor CPT-OH. This design achieves significant improvements over a previously reported mitochondria-targeting NAAF-based prodrug from our group. The H2O2-dependent release efficiency is enhanced by 12.5-fold, and anticancer potency is improved (IC50 values of 142 ± 68 nM in A2780 and 479 ± 32 nM in THP-1 cells, compared with 573 ± 171 nM and > 3 μM for the previously published system. Critically, Prodrug A demonstrated cancer selectivity, showing no detectable toxicity toward healthy cells in vitro within the tested concentration range and being well tolerated in primary cells in vivo under the applied dosing regimen. Prodrug B represents an expansion of this strategy towards the actin cytoskeleton, an underexplored target for anticancer therapy despite actin’s central role in cancer progression and metastasis. The design integrates an actin-binding fluorescent probe for subcellular targeting with a ROS-responsive boronic diethylamine ester trigger that confers cancer cell-specific activation. In the intact prodrug, the ferrocene unit efficiently quenches the fluorophore. However, upon ROS-dependent cleavage, separation from the ferrocene is expected to restore fluorescence, suggesting the potential for real-time imaging of drug release and actin dynamics. Together, these prodrugs establish a general framework for a compartment-specific, H2O2-responsive prodrug design and provide a foundation for advancing next-generation anticancer therapeutics.
Michael Raucheisen (Thu,) studied this question.