Red Fluorescent Aminoferrocene (Pro)Drugs for in Cellulo and in Vivo Imaging

Red fluorescent dyes are usually charged, lipophilic molecules with relatively high molecular weight, which tend to localize in specific intracellular locations, e. g., a cyanine dye Cy5 is biased towards mitochondria. They are often used as markers of biomolecules including nucleic acids and proteins. Since the molecular weight of the dyes is much smaller than that of the biomolecules, the labelling has a negligible effect on the properties of the biomolecules. In contrast, conjugation of the dyes to low molecular weight (pro)drugs can dramatically alter their properties. For example, conjugates of Cy5 with lysosome-targeting aminoferrocenes accumulate in mitochondria and exhibit no intracellular effects characteristic for the parent (pro)drugs. Herein we tested several neutral and negatively charged dyes for labelling lysosome-targeting aminoferrocenes 7 and 8 as well as a non-targeted control 3. We found that a BODIPY derivative BDP-TR exhibits the desired unbiased properties: the conjugation does not disturb the intracellular localization of the (pro)drugs, their mode of action, and cancer cell specificity. We used the conjugates to clarify the mechanism of action of the aminoferrocenes. In particular, we identified new intermediates, explained why lysosome-targeting aminoferrocenes are more potent than their non-targeted counterparts, and evaluated their distribution in vivo.