Comprehensive Investigation of a Novel Schiff Base: Synthesis, Anticancer Efficacy, Gene Expression Profiling, and Computational Analyses

(1) <b>Background:</b> This study evaluates the anticancer potential of a newly synthesized azomethine-based compound, 6,6',5,8-Dioxa-2,11-diazadodeca-1,11-diene-1,12-diyl)bis(4-bromo-2-methoxyphenol) (B-134-0), against osteosarcoma (SAOS-2) cells, focusing on its effects on apoptosis and DNA-damage-related gene expression. (2) <b>Methods:</b> B-134-0 was synthesized via condensation and tested at eight concentrations (0.5-100 μg/mL) for 24, 48, and 72 h. Cytotoxicity was assessed through MTT assay, and gene expression levels of <i>TP53</i>, <i>RAD51</i>, <i>BRCA2</i>, <i>CASP2</i>, <i>MYC, MDM2</i>, <i>CDKN1A</i>, <i>ERCC1</i>, <i>ATR</i>, and <i>PRKDC</i> were quantified through qPCR using the ΔΔ_Ct method. Molecular docking and DFT analyses were performed to explore structural stability and protein interactions. (3) <b>Results:</b> B-134-0 exhibited strong time-dependent cytotoxicity (IC₅₀: 71.58, 54.36, and 12.59 μg/mL at 24, 48, and 72 h, respectively) and significantly modulated the expression of cell cycle and DNA-repair-associated genes. The compound notably downregulated <i>TP53</i>, <i>RAD51</i>, <i>CASP2</i>, <i>MYC</i>, and <i>MDM2</i>, while <i>CDKN1A</i> and <i>BRCA2</i> showed relative upregulation, indicating activation of the DNA damage response. Docking results revealed strong binding affinity with <i>BRCA2</i> and <i>CDKN1A</i>, consistent with experimental findings. (4) <b>Conclusions:</b> These results indicate that B-134-0 exhibits potent anticancer activity by modulating DDR and apoptosis pathways, with strong molecular stability, suggesting its promise as a therapeutic candidate for osteosarcoma.