Synthesis and structural characterization of novel paraben-substituted cyclotriphosphazene derivatives with preliminary cytotoxic and genotoxic assessment

A novel series of paraben-substituted cyclotriphosphazene derivatives (C1–C5) were synthesized and structurally characterized by ³¹P, ¹H, and ¹³C NMR spectroscopy, mass spectrometry, and single-crystal X-ray crystallography for compounds C2 and C3. The crystal structures represent the first crystallographically characterized examples of this paraben–phosphazene hybrid scaffold. Preliminary biological evaluation was performed through cytotoxicity, gene expression, protein-level, and genotoxicity assays using both in vitro and in vivo models. Cytotoxic activity, assessed by the sulforhodamine B (SRB) assay in Caco-2 colorectal cancer cells, demonstrated concentration-dependent reductions in cell viability (1.56–100 µM), with estimated IC₅₀ values exceeding 100 µM for all compounds. Comparatively lower toxicity was observed in CCD-18Co normal colon fibroblast cells; however, additional studies are required to determine whether this observation reflects true cancer-cell selectivity. RT-qPCR analyses revealed marked upregulation of CASP3 together with differential modulation of BCL-2, BAX, SOD1, SOD2, CAT, and GSS. ELISA-based protein analyses further demonstrated compound-dependent glutathione depletion and altered caspase-3 protein responses, indicating involvement of redox-associated apoptotic processes. Genotoxic potential was evaluated using the standard Drosophila melanogaster somatic mutation and recombination test (SMART) together with larval toxicity analysis, where no statistically significant genotoxic effects were detected at the tested sublethal concentrations. RT-qPCR profiling in D. melanogaster further indicated modulation of apoptotic (dronc, drice) and detoxification-related (gstD) genes. Collectively, these findings indicate that these newly synthesized phosphazene derivatives exhibit moderate biological activity under the experimental conditions employed and provide a useful structural framework for future structural optimization and mechanistic investigations.