Design and synthesis of hydrolyzable coumarin-naphthalimide dual probes for NIR fluorescence detection and selective photodynamic activity in cancer cells

Developing multifunctional agents capable of combining diagnostic and therapeutic functions remains a major challenge in cancer research. Herein, we report the design, synthesis, and photophysical characterization of a family of dual probes (C-NAF) based on coumarin-naphthalimide hybrids linked through an imine bond, combining near-infrared (NIR) fluorescence with light-induced cytotoxicity. C-NAF derivatives exhibit broad NIR emission and pronounced sensitivity to microenvironmental polarity, arising from locally excited (LE) and intramolecular charge-transfer (ICT) states modulated by solvent polarity. In their intact form, the C-NAF derivatives show negligible photodynamic activity. However, under polar protic conditions, imine hydrolysis generates photoactive naphthalimide aldehydes (NAF). Intracellular ROS detection and selective scavenger experiments indicate that the photodynamic response proceeds predominantly through a type I pathway involving radical-mediated ROS generation. Functionalization with butyl, morpholine, and biotin moieties was introduced to modulate physicochemical and molecular-recognition properties. In vitro studies in MCF-7 breast cancer cells showed that C-NAF derivatives are non-toxic in the dark and exhibit negligible phototoxicity under 540 nm irradiation, whereas significant phototoxicity occurs under 460 nm irradiation, consistent with photoactivity of the NAF hydrolysis products. C-NAF-B7 exhibited the strongest phototoxic response in MCF-7 cells, suggesting a possible contribution of the biotin moiety to cellular recognition or uptake. Conversely, negligible phototoxicity was observed in HEK-293 cells. Overall, these results identify C-NAF derivatives as promising dual probes combining NIR fluorescence with hydrolysis-activated, predominantly type I photodynamic activity and preferential photoactivity toward cancer cells under investigated conditions.