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5H-benzo[h]thiazolo[2,3-b]quinazoline scaffold is known to have an antitumor effect on certain types of malignancies; however, its effect on hepatocellular carcinoma (HCC) remains unclear. Previously, we reported p-toluenesulfonic acid-promoted syntheses, molecular modeling and in vitro antitumor activity of 5H-benzo[h]thiazolo[2,3-b]quinazoline against human hepatoma (Hep-G2) cells where compounds 4A and 6A were found to be potent inhibitors among the series. In continuation to our previous effort to develop novel therapeutic strategies for HCC treatment, here we investigated the in vivo antitumor activity and the mechanism underlying the effects of 4A and 6A in N-nitrosodiethylamine (NDEA)-induced HCC using male Wistar rats. NDEA was administered weekly intraperitoneally at a dose of 100 mg/kg for 6 weeks. Various physiological and morphological changes, oxidative parameters, liver marker enzymes and cytokines were assessed to evaluate the antitumor effect of 4A and 6A. In addition, proton nuclear magnetic resonance-based serum metabolomics were performed to analyze the effects of 4A and 6A against HCC-induced metabolic alterations. Significant tumor incidences with an imbalance in carcinogen metabolizing enzymes and cellular redox status were observed in carcinogenic rats. Tumor inhibitory effects of 4A and 6A were noted by histopathology and biochemical profiles in NDEA-induced hepatic cancer. Compounds 4A and 6A had a potential role in normalizing the elevated levels of inflammatory mediators such as interleukin-1β (IL-1β), IL-2, IL-6 and IL-10. At molecular level, the real-time quantitative reverse-transcribed polymerase chain reaction analysis revealed that 4A and 6A attenuated the IL-6 gene overexpression in hepatic cancer. Further, orthogonal partial least squares discriminant analysis scores plot demonstrated a significant separation of 4A and 6A-treated groups from carcinogen control group. Both the compounds have potential to restore the imbalanced metabolites due to HCC, signifying promising hepatoprotective activities. All these findings suggested that 4A and 6A could be potential drug candidates to treat HCC.
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