Development of mitochondria-targeted isoquinolinium derivatives with dual fluorescence and anticancer activity against KRAS-mutant NSCLC.

Intrinsically fluorescent bioactive molecules represent an attractive strategy in drug discovery, enabling the integration of biological activity and imaging capability within a single molecular entity. In this context, isoquinolinium-based compounds have emerged as promising scaffolds owing to their tunable photophysical properties and preferential mitochondrial accumulation. Herein, we report the design, synthesis, photophysical characterization, and biological evaluation of a novel series of isoquinolinium-based derivatives as fluorescent anticancer agents for KRAS-mutant NSCLC. Structure-activity relationship studies revealed that the nature and position of the electron-donating substituent on the isoquinolinium core, together with linker architecture, strongly influenced both photophysical behavior and antiproliferative potency. This led to the identification of lead compounds IA21 and IA25, which displayed submicromolar antiproliferative activity in A549 cells (EC50 = 0.67 and 0.87 μM, respectively), low-micromolar activity across a panel of KRAS-mutant NSCLC cell lines, and high selectivity towards malignant cells over non-tumorigenic CCD-16Lu fibroblasts (SI = 10.25 and 8.15, respectively). Exploiting their intrinsic fluorescence, live-cell confocal imaging revealed efficient cellular uptake and mitochondrial accumulation. Mechanistic studies demonstrated that both compounds induced mitochondrial membrane depolarization and intracellular ROS overproduction, collectively promoting apoptotic cell death with negligible necrotic effects. Furthermore, IA21 and IA25 impaired A549 cell migration and retained antitumor activity in three-dimensional tumor spheroids. These results demonstrate the utility of isoquinolinium scaffolds for the development of intrinsically fluorescent anticancer agents and provide new leads for the treatment of KRAS-mutant NSCLC.