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We herein report the synthesis, and antiproliferative evaluation of a novel series of N(4)-substituted 5,7-dibromoisatin thiosemicarbazones (TSCs). Structure and activity based approach was used to synthesize derivatives: N(4)-pyrrolidinyl (L1), N(4)-piperidinyl (L2), N(4)-morpholinyl (L3), N(4)-methyl (L4), and N(4)-ethyl (L5). The compounds were characterized by elemental analysis, FTIR, ¹H NMR, ¹³C NMR, UV-Vis spectroscopy, HRMS and single crystal X-ray analysis. The antiproliferative activity of the synthesized TSCs was evaluated in a dose-dependent manner against breast (MCF-7, MDA-MB-231), skin (A431), lung (A549, NCI-H460), and prostate (PC3) cancer cell lines. L3 and L5 exhibited enhanced cytotoxicity in the low micromolar range of IC₅₀; 1.16-2.47 µM. Notably, L5 showed superior potency in MCF-7 cells with IC₅₀; 1.16 µM compared to the FDA-approved thiosemicarbazone Triapine with IC₅₀; 4.27 µM, while displaying minimal toxicity toward non-tumorigenic MCF-10a breast epithelial cells with selectivity index > 86.20, consistent with ADMET predictions. Molecular docking and molecular dynamics simulations demonstrated stronger binding affinity and greater complex stability of L5 with PTOV1 compared to the FDA approved drug Lenalidomide, supporting L5 drug likeness and therapeutic potential. Mechanistic investigations through functional assays like crystal violet assays, flow cytometry, immunoblotting, and microscopy revealed that L5 induces G0/G1 cell-cycle arrest, suppresses cell migration, invasion, colony formation, 3D spheroid growth, and promotes apoptotic cell death in MCF-7. Western blot analysis provided direct mechanistic evidence that L5 downregulates PTOV1 expression, leading to reduced phosphorylation of AKT1/2/3 and c-Jun, reduced β-catenin nuclear translocation, and decreased MMP-2 expression. L5 enhanced H2AX phosphorylation, suppressed PARP and BCL-XL levels, and increased active caspase-3 driving L5 induced apoptosis. This study identifies L5 as a potent anticancer agent in breast cancer, acting through modulation of the PTOV1-AKT-β-catenin signaling axis, and highlights PTOV1 as a promising therapeutic target.