Synthesis, Cytotoxicity, Apoptotic Activity, Oxidative Stress Inductive Capacity, and Molecular Interactions of...

Schiff bases (imines) have emerged as promising compounds with significant anticancer potential, owing to their ability to induce cytotoxicity and apoptosis in cancer cells. In this study, a series of thymol-based imines were synthesized and evaluated for their in vitro cytotoxic activity against multiple cancer cell lines. The results demonstrated that several derivatives exhibited potent cytotoxic effects, with lower IC₅₀ values compared to cisplatin and paclitaxel. Among them, compound 4f showed the highest cytotoxic activity (IC₅₀ = 4.08-9.94 µM across different cell lines), followed by 4k, 4l, and 4m, which also displayed notable potency. To explain the mode of molecular action of compounds on cells, we tested the mRNA levels of apoptotic and antiapoptotic genes such as BAX, BCL2, BCL2-xl, BiD, and caspase 3 by qRT-PCR. Moreover, we evaluated the nuclear organization of cells using DAPI staining by immunofluorescence microscopy. Additionally, cellular oxidative stress status was analyzed. Our results indicated that compounds upregulated apoptotic genes such as BAX, caspase-3, and BiD, whereas antiapoptotic BCL2 and BCL-xL genes were downregulated. DAPI results also supported these results; compounds triggered apoptotic formations. Also, compounds importantly increased the oxidative stress status in cells. The binding mode of relatively active compounds to the apoptotic and antiapoptotic targets was investigated through molecular modeling to assert and elucidate the in vitro results. The docking study implied that the active compounds could bind to BCL-xL and caspase 3 proteins. The molecular dynamics (MD) simulation study demonstrated that the complexes formed with BCL-xL were more stable. Furthermore, the MMPBSA energy study revealed that the compounds had a higher binding affinity to BCL-xL. Overall, these findings demonstrate that thymol-based Schiff bases, such as compounds 4f and 4k, exhibit potent cytotoxic activity through apoptotic stimulation and oxidative stress enhancement, suggesting their potential as novel anticancer agents targeting the BCL-xL pathway.