Synthesis And Antitumor Activities Of A Hypoxia-activated AGT Inhibitor

Chloroethylnitrosoureas(CENUs) compounds are chemotherapeutic agents employed for the clinical treatment of glioma tumors and other malignant tumors by inducing DNA interstrand cross-links(ICLs) within GC base pairs to form dG-dC cross-links to inhibit cell replication. However, the cell resistance showed in the clinical application of CENUs blocked its further development. It was reported that O6-Alkylguanine-DNA alkyltransferase(AGT) could repair the DNA ICLs by removing the alkyl group at the O6 positon of guanine to restore the DNA damage and lead to drug resistance. In recent years, many kinds of AGT inhibitors were developed and used in combination with CENUs. The most potent one used in preclinical treatment was O6-Benzylguanine. Since it was not a specific inactivator for alkyltransferase in tumor cells, AGT levels were also depleted by O6-BG in normal cells, increased the DNA damage of host tissues, and resulting in the failure of chemotherapy. Therefore, it has become an urgent problem to develop the AGT inhibitor targeted to tumor cells.This thesis is mainly about the synthesis of a kind of AGT inhibitor, 4-nitrobenzyl(6-(benzyloxy)-9H- purin-2-yl)carbamate, which could target to tumor cells based on the characteristics of hypoxia microenvironment in tumor tissue. The reaction used O6-BG as the starting material and reacted with chloromethyl pivalate to prepare(2-amino-6-(benzyloxy)-9H-purin-9-yl)methyl pivalate. After reacting with phosgene and 4-nitrobenzyl alcohol, targeted compound was produced and the structure was characterized by UV, IR, 1H NMR, 13 C NMR and ESI-MS.The cell models applied to the AGT inhibitor cytotoxicity assay were constructed in order to lay the foundation for the evaluation of target compound biology activities. Western Blot was carried out to investigate the AGT level within the three glioma cell lines SF126, SF763 and SF767. The survival rate of glioma cell lines after exposured to Nimustine(ACNU) 24 h was measured by CCK-8 method in order to evaluate the relationship between AGT level and cell cytotoxicity. The results showed that SF763 cells had the highest level of AGT with the lowest motality rate while SF126 cells had the lowest level of AGT with the highest motality rate, which indicated that AGT level was negatively correlated with cell death rate and the three glioma cell lines were appropriate for the cell model used in AGT inhibitor cytotoxicity assay.The antitumor activities of compound 4 combination with CENUs were evaluated by cytotoxicity assay. The assay was conducted by CCK-8 method to measure the cell survival rate after exposured to ACNU, ACNU combination with O6-BG and ACNU combination with compound 4 under condition of normoxic and oxygen deficient. The results showed that there were no differences between ACNU combination with compound 4 experimental group and ACNU experimental group while the cell survival rate of ACNU combination with O6-BG decreased significantly under condition of normoxic, which illustrated that compound 4 did not have AGT inhibitory activities under condition of normoxic. And the cell survival rate of ACNU combination with compound 4 under condition of oxygen deficient was significantly lower than that of the three experimental groups under the condition of normoxic, which illustrated that compound 4 could selectively deplete AGT in tumor cells relative to normal cells. In summary, compound 4 could be specifically activated and selectively inhibit AGT activities under condition of oxygen deficient.In this study, a AGT inhibitor targeted to tumor cells under hypoxia microenvironment was synthesized, a series of cell models applied to AGT inhibitor cytotoxicity assay were constructed and its antitumor activities were evaluated by CCK-8. This work not only provided a new idea for the designing of targeted AGT inhibitor, but also laid the foundation for synthesis novel nitrosoureas with AGT inhibitory activity.