Breast cancer remains a leading global health challenge, driving the urgent need for innovative therapeutic strategies. This study presents the initial results of the design, synthesis, characterization, and in vitro evaluation of a novel fluorescent agent for breast cancer treatment, focusing on its subcellular localization and molecular docking. Seven novel fluorescent compounds (3a–g) were synthesized via isatin derivatives and 4-bromo 1,8-naphthalimide conjugation. The compounds were spectroscopically characterized and tested in MDA-MB-231 and MCF-7 cells using viability assays, Annexin-V and propidium iodide flow cytometry to define cytotoxic mechanisms, confocal microscopy with nuclear and mitochondrial markers for subcellular localization, and molecular docking to VEGFR2 (4AGD) and KIT (3G0E) as co-crystals. 3a (3,85 µM and 2,99 µM) and 3c (1,77 µM and 77,31 µM) emerged as two candidates with high cytotoxic potential, exhibiting the lowest IC₅₀ values in MCF-7 and MDA-MB-231 cell lines, respectively, at the 24th hour. Several conjugates, particularly 3a (87.47 %) and 3 g (96.62 %) in MCF-7, induced late apoptosis, and 3c (98.57 %) in MDA-MB-231 induced the highest early apoptosis. Across both proteins (3G0E and 4AGD), 3c consistently shows stronger predicted binding than sunitinib, with more negative binding energies and lower Ki values in each case, supporting its promise as a lead together with 3a.