<i>In Vitro</i> and <i>In Vivo</i> Biological Evaluation of Novel 1,4‐Naphthoquinone Derivatives as Potential Anticancer Agents

A novel library of naphthoquinone derivatives (3-5 aa) was synthesized and evaluated for their anticancer properties. Specifically, compounds 5 i, 5 l, 5 o, 5 q, 5 r, 5 s, 5 t, and 5 v demonstrated superior cytotoxic activity against the cancer cell lines that were studied. All the studied compounds exhibited a higher selectivity index (SI) and a favourable safety profile than the standard drug doxorubicin. Notably, compound 5 v displayed a greater cytotoxic effect on MCF-7 cells (IC₅₀=1.2 μM, and 0.9 μM at 24 h and 48 h, respectively) compared to the standard drug doxorubicin (IC₅₀=2.4 μM, and 2.1 μM at 24 h and 48 h, respectively). To further investigate the mechanism of cytotoxic effect, additional anticancer studies were conducted with 5 v in MCF-7 cells. The studies are including morphological changes, AO/EB (acridine orange/ethidium bromide) double staining, apoptosis analysis, cell colony assay, SDS-PAGE and Western blotting, cell cycle analysis, and detecting reactive oxygen species (ROS) assay. The findings showed that 5 v triggered cytotoxic effects in MCF-7 cells through the initiation of cell cycle arrest at the G1/S phase and necrosis. In vivo ecotoxicity studies indicated that 5 v had lower toxicity towards zebrafish larvae (LC₅₀=50.15 μM) and had an insignificant impact on cardiac functions. In vivo xenotransplantation of MCF-7 cells in zebrafish larvae demonstrated a significant reduction in tumour volume in the xenograft. Approximately 95 % of the zebrafish larvae with 5 v xenografts survived after 10 days of the treatment. Finally, a computational modelling study was conducted on four protein receptors, namely ER, EFGR, BRCA1, and VEFGR2. The findings highlight the importance of the aminonaphthoquinone moiety, amide linkage, and propyl thio moiety in enhancing the anticancer properties. 5 v exhibited superior drug-likeness features and docking scores (-9.1, -7.1, -8.9, and -10.9 kcal/mol) compared to doxorubicin (-7.2, -6.1, -6.9, and -7.3 kcal/mol) against ER, EFGR, BRCA1, and VEGFR2 receptors, respectively. Therefore, the notable antitumor effects of naphthoquinone derivatives (3-5 aa) suggest that these molecular frameworks may play a role in the development of promising anticancer agents for cancer treatment.