N-Dialkylaminoferrocene-Prodrugs Targeting Lysosomes in Cancer Cells (LysoCene)

• Applicant: Professor Dr. Andriy Mokhir
• Subject Area: Biological and Biomimetic Chemistry
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 573739262

In the context of previous projects, we developed N-alkylaminoferrocene-based (NAAF) prodrugs that are selectively activated in cancer cells due to their elevated levels of reactive oxygen species (ROS). Upon activation, these prodrugs release NAAF drugs, which further amplify ROS production—specifically superoxide anions and hydroxyl radicals—ultimately leading to cancer cell death. In contrast, normal cells exhibit significantly lower ROS levels and therefore do not activate the prodrugs, rendering them non-toxic to healthy tissue. We markedly improved the efficacy of these prodrugs by targeting them to ROS-enriched organelles such as mitochondria, the endoplasmic reticulum, and lysosomes. Among these, lysosome-targeting prodrugs demonstrated the most favorable therapeutic profile and were selected for further optimization. To mitigate off-target oxidation, we tuned the redox potential of the lead compound, developed a clinically viable formulation, and elucidated its unique mechanism of action. However, a key remaining limitation of our most promising candidate, ND21, is the instability of the active drug released within cancer cells. This instability restricts the number of catalytic ROS-generating cycles to only three. Preliminary studies identified the cause of this instability as oxidation of the (cyclopentadienyl ligand)C–NHR moiety, leading to the formation of unstable imine intermediates ((Cp)C=NR). To overcome this inherent drawback of NAAF, we propose the development of N,N-dialkylaminoferrocene (DAFc) analogues. These compounds lack the critical N–H bond necessary for imine formation, thereby providing significantly improved oxidative stability. We propose two complementary synthetic strategies to generate these prodrugs—classified as type A and type B—which, upon activation in cancer cells, form stable DAFc drugs via intra- or intermolecular alkylation reactions, respectively. These synthetic routes, validated in model systems, are modular and adaptable, allowing for systematic exploration of structure–activity relationship (SAR). The biological properties of the DAFc prodrugs will be evaluated in vitro using cancer cell lines and in vivo by assessing prodrug biodistribution, tumor accumulation, and antitumor efficacy in a fibrosarcoma mouse model. Our objective is to develop prodrugs whose active drugs, generated specifically within cancer cells, can catalytically induce the formation of more than 100 equivalents of reactive oxygen species (ROS) from molecular oxygen. We aim to achieve IC₅₀ values in the nanomolar range (below 100 nM) against selected cancer cell types that are particularly dependent on lysosomal function. These include models of acute myeloid leukemia, fibrosarcoma, lung cancer, triple-negative breast cancer, and pancreatic cancer. Furthermore, we aim to demonstrate in vivo anticancer efficacy at doses below 4 mg/kg.

• DFG Programme: Research Grants