Annulated cationic tetramethyl indo(di)carbocyanine dyes exhibit potent dark cytotoxicity in cancer cells

We report the synthesis, characterization, and biological evaluation of a new series of chain-substituted pentamethine cyanine dyes, including annulated cationic derivatives. Palladium-catalyzed Sonogashira coupling of γ-bromo-substituted pentamethine cyanine (Cy5) dyes with electron-neutral or weakly donating arylalkynes yielded the expected linear products, whereas strongly electron-donating arylalkynes produced annulated dyes with blue-shifted absorption and pH-responsive “turn-on” fluorescence with pKa values between 4.9 and 6.3. Anionic sulfonated dyes were biocompatible, while cationic N -methyl derivatives selectively localized to mitochondria and exhibited potent dark cytotoxicity in multiple cancer cell lines, with submicromolar IC 50 values and some cases selectivity over a noncancerous cell-line. Fluorescence microscopy confirmed mitochondrial targeting, and reactive oxygen species (ROS) imaging demonstrated dose-dependent induction of oxidative stress. Seahorse Mito-Stress and Glyco-Stress assays revealed mild mitochondrial uncoupling by the cationic annulated dyes, leading to increased basal and maximal respiration and compensatory glycolytic activity. Collectively, these findings highlight the potential of these new annulated cationic cyanine dyes as mitochondria-targeting cytotoxic agents with pH-responsive fluorescence properties. • Turn-on deep-red fluorescence upon protonation with pKa 4.9 – 6.3 • Potent dark cytotoxicity in multiple cancer cell lines, partial selectivity over noncancer cells • Mitochondrial targeting uncoupling by the cationic annulated dyes. • Mitochondrial uncoupling increases basal and maximal respiration and compensatory glycolytic activity.