Design, synthesis and in vitro evaluations of new cyclotriphosphazenes as safe drug candidates

Although it is possible to discover new drug candidate molecules using in silico approaches, chemical synthesis followed by screening of their functions is still at the center of bioactive molecule discovery. While determining the potential effects of compounds on target signaling molecules or pathways, assessing their effects on the circadian rhythm is also very important for determining the efficacy of drug candidates because they control most of the signaling pathways. Herein, new members of the biocompatible cyclotriphosphazene family were prepared, and their in vitro biological activities and effects on circadian rhythm were evaluated for the first time. In particular, new cyclotriphosphazene derivatives carrying morpholine, thiomorpholine and triazole groups were designed and synthesized, and their chemical structures were characterized using appropriate spectroscopic methods. Cellular toxicity analyses of the compounds were performed using different biological methods, such as determination of IC₅₀ values, calculation of population doubling times, and colony formation patterns. Subsequently, the effects of the compounds on the cell cycle were analyzed using the flow cytometry technique. Finally, the effects of the synthesized compounds on circadian rhythm were determined using a real-time bioluminescence approach. Based on these studies, it was determined that some compounds demonstrated varying degrees of antiproliferative activity, with the most potent compounds causing G₂/M phase arrest. Additionally, most derivatives had no adverse effects on the circadian rhythm, indicating their potential for safe therapeutic application in targeting cell proliferation. Furthermore, an important pharmacological characteristic of the drug candidate molecules, namely, membrane permeability in terms of log P values, was assessed. In conclusion, these novel cyclotriphosphazene-based compounds are a class of circadian rhythm-safe drug candidate compounds.