A New Class of Anionic Near-Infrared Fluorescent Dyes with 1,6-Diheterophenalene-2,5-dione End Groups

We describe the synthesis and evaluation of oxonol-based near-infrared (NIR) dyes designed for in vivo optical imaging applications. These small-molecule anionic dyes, dubbed FROGs (Fiercely Radiant Oxonol Green), exhibit strong NIR absorption and emission, high molar absorptivity, and good photostability. In vitro studies revealed distinct cellular uptake profiles: whereas compound FROG-1 displayed minimal cellular internalization, FROG-2 showed robust accumulation in cancer cells. Fluorescence lifetime imaging microscopy (FLIM) further revealed that FROG-2 produced a uniform and prolonged lifetime signal within cancer cells, indicative of stable intracellular localization. Both representative compounds were administered to nude mice and monitored using multispectral fluorescence imaging across a broadband excitation/emission channel combinations between 500 and 840 nm. Principal component analysis of the imaging data revealed distinct temporal and spatial distribution patterns: FROG-1 demonstrated renal clearance with minimal off-target accumulation, while FROG-2 showed mixed renal and hepatic clearance and spectral heterogeneity at early time points, suggesting differential stability or interaction with tissue microenvironments. Ex vivo organ imaging at 24 h confirmed these clearance routes. These findings highlight the potential of tailored oxonol dyes as versatile NIR probes and provide insight into the structure–biodistribution relationship critical for biomedical imaging probe development.