N-Benzyl Indole-Benzoxazole Hybrids as Multi-Target Anticancer Agents Modulating mTOR, Apoptosis, and Lipid Dynamics in Breast Cancer

Objectives: Indole-benzoxazole hybrids are structurally versatile scaffolds with significant promise in anticancer drug development. This study aimed to synthesize N-benzyl-substituted indole-benzoxazole derivatives (IBB1-5) and to evaluate their antiproliferative potential and mechanistic effects in human breast cancer models. Materials and Methods: IBB1-5 were synthesized and screened for cytotoxic activity against MDA-MB-231 and MCF-7 cells using MTT assays. The most active compound, IBB-4, was further investigated in MDA-MB-231 cells through clonogenic assays and morphological analysis. Immunofluorescence staining was performed to assess mTOR, LC3, caspase-3, and β-tubulin expression and distribution. Lipid droplet dynamics were evaluated using Oil Red O and Nile Red staining, combined with three-dimensional surface-plot analysis. Results: values of 12.2 μM (MDA-MB-231) and 12.8 μM (MCF-7). Clonogenic assays revealed significant dose-dependent suppression of long-term proliferative capacity. Morphological alterations included cytoplasmic vacuolization and structural disorganization. Immunofluorescence analyses showed a marked downregulation of mTOR and a spatial redistribution of LC3, without significant changes in total fluorescence intensity. Increased caspase-3 immunoreactivity and pronounced disruption of β-tubulin indicated activation of apoptotic pathways and cytoskeletal destabilization. Lipid staining, supported by three-dimensional surface analysis, demonstrated prominent clustering and redistribution of intracellular lipid droplets. Conclusion: IBB-4 exerts multi-layered anticancer effects through coordinated modulation of proliferative signaling, apoptotic activation, cytoskeletal integrity, and lipid compartmentalization. These findings identify IBB-4 as a promising lead candidate for further molecular and preclinical investigations, particularly in triple-negative breast cancer.