Novel pyrido[2,3-d]pyrimidines for bioimaging: Effect of water content on enhanced fluorescence and the mechanism of aggregation-induced blue-shifted emission.

Compounds containing the pyridopyrimidine core have attracted significant interest due to their promising biological properties and potential for medical applications. However, they represent an unexplored area in cellular imaging. Herein, a new class of aggregation-induced emission (AIE) fluorophores based on the pyrido[2,3-d]pyrimidine scaffold has been developed. We synthesized a small series of derivatives, which were evaluated for their spectroscopic properties in various solvents, considering fluorescence quantum yields, fluorescence lifetimes, and their decay components. Importantly, the physicochemical parameters were also evaluated in a MeOH/HO environment, suggesting the possible behavior of the derivatives in the cellular environment. Our analyses revealed the excellent photophysical properties of the ET06 compound, which exhibited enhanced fluorescence as a result of increasing water content, retaining significant Stokes shifts. An in-depth characterization of the molecular mechanism of fluorescence formation was linked to an aggregation-induced blue-shifted emission effect (AIBSE). TD-DFT calculations showed the existence of different spatial conformational states of ET06 in the single molecule state and aggregated states in at sandwich arrangement. Molecular conformational and energy changes in the ground and excited states, which had a significant effect on fluorescence emission in water-containing media, were also discussed. Finally, biological studies revealed negligible cytotoxicity of ET06 against cancer and normal cells. High fluorescence intensity in cells and a possible affinity and tendency to accumulate in mitochondria and lysosomes were also confirmed. These findings provide valuable insights into the molecular behavior and properties of pyrido[2,3-d]pyrimidines, boosting the landscape for designing new fluorescent probes for bioimaging.