Synthesis And Activity Evaluation Of Hypoxia-activated O~6-benzylguanine Derivatives

Chloroethylnitrosoureas(CENUs) are an family of anticancer bifunctional alkylating agents widely used in the clinical treatments of malignancies, including brain tumor, myeloma, neuroglioma, malignant melanoma and so on. At present, nimustine(ACNU), carmustine(BCNU), lomustine(CCNU) and semustine(Me CCNU) are the most common alkylating agents used in clinical practice. Anti cancer effect of CENUs is mediated mainly by inducing DNA interstrand cross-links(ICLs) within GC base pairs to form d G-d C cross-links. However, O6-alkylguanineDNA alkyltransferase(AGT) in tunor cells could repair the DNA lesions caused by CENUs and lead to drug resistance. O6-benzylguanine(O6-BG) is one of the most effective inhibitors of AGT, which can react with AGT and form S-benzylcysteine in the active site of protein to deplete AGT in tumor cells. Since, O6-BG was not a specific inhibitor for AGT in tumor cells, which can deplete the AGT in normal cells and increase the sensitivity of both tumor and host cells to CENUs. In this study, the inhibition mechanisms of AGT inhibitors were analyzed by molecular simulation methods. Three AGT inhibitors targeted to deplete AGT in hypoxic tumor cells were synthesized and the AGT inhibitory activity of those three agents were evaluated by vitro cytotoxicity assay.Molecular docking combined with molecular dynamics simulation and quantum mechanical was performed to investigate the inhibition mechanisms of O6-methylguanine(O6-Me G), O6-benzylguanine(O6-BG) and O6-(4-bromothenyl)guanine(4-BTG) to AGT. Three stable substrates and AGT(1T38) models were constructed, including AGT-O6-Me G, AGT-O6-BG and AGT-4-BTG. After that, we picked out the essential residues and substrate molecules of these three stable conformations and explored the inhibition mechanisms of O6-Me G, O6-BG and 4-BTG by ONIOM computations at M062x/6-31+G(d,p):AM1 theoretical level. The results showed that the energy barrier of 4-bromothenyl transferring step in 4-BTG was lower than that of methyl transferring step in O6-Me G and benzyl transferring step O6-BG. This is in agreement with the higher AGT inhibitory activity of 4-BTG than O6-Me G and O6-BG.Due to the rapid growth of solid tumors, the tumor tissue oxygen supply is reduced and malignant tumor is often in hypoxic condition We have synthesized three kind of hypoxia activated O6-BG derivatives(HA-BG) according to the characteristics of hypoxia microenvironment in tumor cells, including N2-((((4-nitrobenzyl)oxy) carbonyl)amino)-N9-chloroethyl-O6-benzyl-guanine(HA-BG-1), N2-((((4-nitrobenzyl) oxy)carbonyl)amino)-N9-bromomethyl-O6-benzyl-guanine(HA-BG-2) and N2-((((4-nitrobenzyl)oxy)carbonyl)amino)-N9-(2-amino)ethyl)-O6-benzyl-guanine(HA-BG-3). These three compounds can be decomposed to release N9-chloroethyl-O6-benzylguanine(HA-BG-1A), N9-bromomethyl-O6-benzyl-guanine(HA-BG-2A) and N9-(2-amino)ethyl)-O6-benzyl-guanine(HA-BG-3A) in hypoxia conditions respectively. The structures of those three agents were characterized by 1H NMR,IR, UV and ESI-MS.The AGT inhibitory activity and the effect of hypoxia activation of HA-BG-1, HA-BG-2 and HA-BG-3 were evaluated in this study. The cell survival rate was conducted by CCK-8 method after exposured to ACNU, ACNU combination with O6-BG and ACNU combination with three kind of HA-BG under condition of normoxic and hypoxia. The results showed that the cell survival rates of ACNU combination with three kind of HA-BG under the condition of hypoxia are significantly lower than that of normoxic groups and HA-BG-2 had a better AGT inhibitory activity than HA-BG-1 and HA-BG-3. This proved that HA-BG-1 HA-BG-2 and HA-BG-3 can be activated under hypoxic conditions and release O6-BG analogs to selectively deplete AGT in hypoxia tumor cells. HA-BG-1, HA-BG-2, HA-BG-3 and N9-chloroethyl-O6-benzyl-guanine(HA-BG-1A), N9-bromomethyl-O6-benzyl-guanine(HA-BG-2A), N9-(2-amino)ethyl)-O6-benzylguanine(HA-BG-3A), which were hypoxia-actived by HA-BG-1, HA-BG-2 and HA-BG-3 were docked into the active site of AGT. The results showed that HA-BG-1, HA-BG-2 and HA-BG-3 can’t enter into the active site of AGT, however HA-BG-1A, HA-BG-2A and HA-BG-3A can bind with AGT, which agreed with the cytotoxicity experimental results.In this study, the inhibition mechanisms of O6-alkylguanine to AGT were clarified. Based on that, three hypoxia-activated AGT inhibitors with 4-nitrobenzyl were synthesized and the AGT inhibitory activity of those three agents were evaluated by vitro cytotoxicity assay. The result show that this three N2-((((4-nitrobenzyl)oxy) carbonyl)amino)-O6-benzyl-guanine can targeted to deplete AGT in hypoxic tumor cells and may become a new kind of targeted AGT inhibitors applied combined with nitrosourea alkylating agent. This work provided a new breach for the further designing and synthesis anticancer nitrosoureas with anti-drug-resistant and remarkable magnetic target to tumor.