Esterase‐responsive Self‐immolative Prodrugs for the Sustained Delivery of the Anticancer Drug 5‐Fluorouracil with Turn‐on Fluorescence

Stimuli‐responsive prodrugs of anticancer drugs are advantageous for the selective delivery of drugs to cancer cells with minimized off‐target side effects. In the present study, esterase‐activatable fluorogenic prodrugs of the chemotherapeutic drug 5‐fluorouracil (5‐FU) have been rationally designed and synthesized using multi‐step organic synthesis. While 5‐FU was connected directly with the fluorophore via a C‐N bond in the prodrug BJ‐50, an additional self‐immolative benzylic spacer with a carbonate linker was incorporated in the prodrug BJ‐92. Although absorption and emission spectroscopic studies revealed the activation of both the prodrugs by porcine liver esterase (PLE), reverse‐phase HPLC studies confirmed the inability of BJ‐50 to release the active drug 5‐FU. In contrast, a sustained release of 5‐FU and Cou‐OH was observed from BJ‐92 in the presence of PLE. The endogenous esterase‐mediated activation of the prodrug BJ‐92 was validated by the turn‐on fluorescence in HeLa cells and the anti‐proliferative activities in A549, HeLa, and HEK‐293 cells. Modulation of the expression of a few cancer marker proteins by BJ‐92 and 5‐FU was studied to evaluate their anticancer activities. As esterases are overexpressed in cancer cells, the prodrug in the present study would be helpful in selectively delivering 5‐FU to cancer cells with reduced off‐target side‐effects.