Development of a Folate Receptor (FR)-Targeted Indenoisoquinoline Using a pH-Sensitive N-Ethoxybenzylimidazole (NEBI) Bifunctional Cross-Linker

[Image: see text] This Communication describes the synthesis and evaluation of a folate-conjugated drug delivery system (DDS) that incorporates an acid-sensitive N-ethoxybenzylimidazole (NEBI) bifunctional linker and a novel imidazole-containing indenoisoquinoline. Indenoisoquinolines are a class of TOP1 inhibitors that exhibit broad anticancer activity. Here, we examined whether a DDS that comprised an indenoisoquinoline attached to a folate moiety could help target activity to cancer cells that naturally overexpress the folate receptor (FR), thereby increasing the specificity of these compounds. Evaluation of the DDS revealed an 11-fold increased toxicity in folate receptor (FR)-overexpressing cells compared to in FR-knockdown cancer cells. Microscopy studies demonstrate enhanced internalization and localization of the DDS in acidic lysosomal compartments of FR-overexpressing cells, supporting a receptor-mediated mechanism for uptake and activation. Together with control experiments, the results support that the cytotoxic activity of this DDS is dependent on both the presence of the folate group as well as the presence of the acid-sensitive hydrolyzable group. This work represents the first example of a cell receptor-targeted indenoisoquinoline, which could help pave the way for the use of this class of compounds in anticancer therapy.