Study On Construction And Antitumor Activity Of Novel Adamantyl Nitroxide Derivatives For Human Hepatocellular Carcinnoma

[Objectives]To design and synthesis a series of novel adamantyl nitroxide derivatives, and determine the structures of target compounds; To evaluate the anti-hepatoma activity of target compounds by in vitro and in vivo assay; To explore the anti-hepatoma mechanism of target compounds, and provide a theoretical basis and experimental data for the development of new drugs against hepatocellular carcinoma with high efficiency and low toxicity.[Methods]1. Synthesis of adamantyl nitroxide derivatives: In this paper, we will use the principle of dual-target drug design to build up a serise adamantyl nitroxide derivatives,which contained an adamantyl moiety and a nitroxyl nitroxide radicals.2. In vitro and in vivo of anti-hepatoma activity evaluate for adamantyl nitroxide derivatives: In this paper, we investigated the synthesis of adamantyl nitroxide derivatives inhibitory rates for Hep G2, MHCC-97 H, SMMC-7721 and Bel-7404 on human hepatocellular carcinoma and toxicity for L-02 normal cells by MTT assay in vitro, and screened the advantages compounds of anti-hepatoma activity; Established a Bel-7404 xenograft models mice to investigate the effects of the tumor growth inhibition for compound 2 in vivo; TUNEL assay was used to observe the effect of compound 2 on induced Bel-7404 cells apoptosis;HE staining was used to study the influence on the histopathology; Immunohistochemical method was used to study microvessel density effects of compound 2 for Bel-7404 tumor mice tissue tumor.3. Study on the anti-hepatoma mechanism of the adamantyl nitroxide derivatives:Flow cytometry to detect compounds 2 and 5 on Bel-7404 cell cycle, cell apoptosis and ROS level; Transwell assay to detect compound 2 on Bel-7404 cell migration and invasion;TEM to observe the ultrastructure of Bel-7404 cells; Western blot to detect Bax, Bcl-2,caspase-9 and caspase-3 protein levels of expression.[Results]1. We synthesized five novel adamantyl nitroxide derivatives, and characterized by IR, ESI-MS, elemental and X-ray crystal analysis.2. In vitro, we selected two compounds 2 and 5, which have significant inhibitory effect on Bel-7404 hepatoma cells and less toxic to L-02 normal cells. The results showed that the inhibitory effect of the compound 2 on tumor mice was significant, and there was no obvious toxicity in vivo. At the same time, compound 2 group could induce tumor cell apoptosis in xenograft models mice in different degree, cause the tumor tissue necrosis, decrease the tumor tissue MVD and significantly inhibit the formation of tumor angiogenesis.3. The anti-hepatoma mechanism results showed that there was a significant G2/M phase arrest after compound 2 treatment, and the compound 5 exhibits a cell cycle arrest in G0/G1 and G2/M phase;Compounds 2 and 5 induced cell apoptosis in a dose-and timedependent manner;With increasing drug concentration,compound 2 significantly inhibited migration and invasion of Bel-7404 cells;TEM showed that compound 2 could damage the cellular ultrastructure and inhibite the cell growth; Compound 2 could notablely increase the ROS level in Bel-7404 cells; Compound 2 decreased expression levels of Bcl-2 and increased expression level of Bax,caspase-9 and caspase-3.[Conclusions]1. Using the principle of dual-target drug design, we synthesize five novel adamantyl nitroxide derivatives and the structure of compounds was confirmed.2. In vitro and in vivo anti-hepatoma activity experiments showed that the compound 2 could significantly inhibit the proliferation and metastasis of human hepatocellular carcinoma Bel-7404 cells, promote tumor tissue necrosis, and induce apoptosis;3. Compound 2 inhibit cell proliferation and metastasis by arresting the cell cycle and inhibiting Bel-7404 cell migration and invasion, and induce the apoptosis of Bel-7404 cells through activation of the mitochondrial apoptosis pathway. Thus, compound 2 may become a promising anti-hepatoma drug candidate.