Phenyl Versus Pyridyl Substituents as Molecular Switches for Cellular Toxicity and Cellular Localization in Tetraarylvinylthiophenes

Subtle structural modifications in organic fluorophores greatly influence their biological profiles. Herein, we report a structure-activity relationship study of 2,5‑bis(vinyl)thiophene derivatives bearing phenyl (PhT) or pyridyl substituents (2PyT, 3PyT, and 4PyT) to assess their suitability for live‑cell fluorescence imaging. PhT exhibits excellent biocompatibility across five human cell lines (HEK293T, HeLa, MCF‑7, HEPG2, A549), showing no cytotoxicity at concentrations up to 100 µM over 72 h, and localizes predominantly in the cytoplasm. In contrast, with cytoplasmic localization, 2PyT and 3PyT display significant cytotoxicity and weak fluorescence. Notably, 4PyT, featuring a weakly donating 4‑pyridyl substituent, is non‑toxic, highly emissive, and selectively accumulates in the nuclei of cancer cells. Mechanistic studies reveal that PhT enters cells via passive, energy‑independent uptake, remains chemically inert intracellularly, and neither generates reactive oxygen species nor disrupts the cell cycle. These findings demonstrate that careful choice of substituent position and electronic properties can tune both biocompatibility and subcellular targeting. PhT and 4PyT thus emerge as promising scaffolds for developing live‑cell imaging agents and organelle‑specific fluorescent probes.