Design,Synthesis And Activity Evaluation Of Novel AGT Inhibitors

Alkylating agents, such as TMZ, ACNU and BCNU, are an important type of antitumor drugs widely used in clinic leading to the generation of a series of alkylating lesions. A large amount of cellular biological processes would be affected, such as DNA replication and transcription, which result in replication arrest and cell death. However, the O~6-alkylguanine-DNA alkyltransferase(AGT) expressed in tumor cells can repair the O~6-postion lesions of guanine residues in DNA which induced by alkylating agents, leading to drug resistance and reduced chemotherapeutic efficacy. Therefore, a variety of AGT inhibitors were used in clinical to improve the curative effect of chemotherapy drugs. AGT inhibitors without tumor cells targeting will also decrease AGT level in normal cells, result in an increase in the sensitivity of normal cells to drugs and their death. This study has developed an AGT inhibitor with tumor cell targeting property to inhibit the activity of AGT in tumor cells specifically, result in improving the chemotherapy drug efficacy.We synthesized two AGT inhibitors, O~6-(3-nitrobenzyl)-guanine(O~6-3-NBG, Compound d), which could be selectively activated to inhibit the AGT activity in solid tumor tissues based on the hypoxia microenvironment, and, O~6-(3-aminobenzyl)-guanine(O~6-3-ABG, Compound h). 2-amino-6-chloropurine were used as the starting material, and reacted with N-methylpyrrolidine to prepare 1-(2-aminopurin-6-yl)-1-methyl-pyrrolidinium chloride. After reacting with 3-nitrobenzyl alcohol, targeted compound was produced. 3-aminobenzyl alcohol were used as the starting material, and reacted with trifluoroaceticanhydride to prepare 2,2,2-trifluoroN-(3-hydroxy-benzyl)-acetamide. After reacting with compound 2 and deamination, targeted compound was produced. The final products were characterized by UV, IR, 1H NMR and ESI-MS.Reduction of O~6-3-NBG in the system of Zn/EDTA(p H 7.4) in vitro has been measured. The initial concentration of O~6-3-NBG was set to 50, 75 or 100 μM and the solution was mixed with zinc powder for 1, 5, 10, 20 and 30 min, respectively. A high performance liquid chromatography(HPLC) method was used for quantitative analysis of the composition in Zn/EDTA system. And the rationality of the quantitative analysis method was confirmed. The intra-day and inter-day RSD of the method for the assay of concentration of main components were 0.12-2.85% and 1.11-8.23%, these satisfied the requirement of quantitative analysis. The results showed that O~6-3-NBG can be reduced to generate O~6-3-ABG in Zn/EDTA system, made it generate O~6-3-ABG under oxygen-deficient condition and be a tumor targeting AGT inhibitor.While O~6-3-NBG can be reduced to generate O~6-3-ABG, we performed the activity evaluation of O~6-3-NBG or O~6-3-ABG combined with CENUs. The cell survival rates(SF763) of ACNU group and three treatment groups, ACNU plus O~6-BG(1), O~6-3-NBG(2) or O~6-3-ABG(3) were measured under normoxia or hypoxia by CCK-8 method. The results showed that there were no significant differences of survival rate between ACNU group and treatment group 2, indicated that O~6-3-NBG did not have AGT inhibitory activity under normoxia. While the cell survival rates of treatment groups 1 and 3 were significantly lower than ACNU group under normoxia, which suggested that O~6-BG and O~6-3-ABG have AGT inhibitory activity. Furthermore, the cell survival rates of treatment 3 under hypoxia was almost like the corresponding group under normoxia, indicated that O~6-3-ABG has AGT inhibitory activity under both conditions. The cell survival rate of treatment group 2 under hypoxia was the lowest and significantly lower than the corresponding group under normoxia, which indicated that O~6-3-NBG could be selectively activated to inhibit AGT activity under hypoxia. A molecular docking study of O~6-BG, O~6-3-NBG and O~6-3-ABG to AGT(from PDB code 1T39) were performed. The results showed that three compounds were able to enter the AGT active site to form hydrogen bond with the number of 4, 2 and 5, respectively. It is indicated that O~6-3-ABG showed the greatest activity followed by O~6-BG and O~6-3-NBG.In summary, in this work, we synthesized two AGT inhibitors O~6-3-NBG and O~6-3-ABG, demonstrated that O~6-3-NBG can be reduced to generate O~6-3-ABG by HPLC. And the activities of the compounds were evaluated by CCK-8 method under normoxia or hypoxia. O~6-3-ABG showed well AGT inhibitory activity under both conditions while O~6-3-NBG could barely decrease the activity of AGT under normoxia. But O~6-3-NBG could inhibit AGT activity significantly under hypoxia. This work provided a new idea for designing targeted AGT inhibitors, and the experimental support of the combination of AGT inhibitors and alkylating agents.