Synthesis And Antitumor Activities Of O~6-Benzylguanine Cholroethylnitrosoureas

Cholroethylnitrosoureas(CENUs) are an important type of DNA alkylating agents for clinical cancer treatment, including brain tumor, malignant lymphoma, leukemia, neuroglioma, melanoma and so on. Studies indicated that good antitumor effect of CENUs is attributed to their ability to induce the performance of DNA interstrand crosslinks(ICLs), as well as their carcinogenesis. However, it was reported that the formation of dG-dC ICLs could be prevented by O~6-Alkylguanine-DNA alkyltransferase which could remove the alkyl group at the O~6-positon of guanine from DNA and lead to the cell resistance. Therefore, a novel combi-nitrosourea, which possesses the O~6-benzylguanine(O~6-BG) and the CENU pharmacophores simultaneously, was designed and synthesized. This compound not only can inhibit AGT, but also can target to tumor tissue to improve the activity of anticancer. This work is also significant for the design of new anticancer nitrosoueas with high activity and low toxicity.A novel combi-nitrosourea named N-(2-chloroethyl)-N’-2-(O~6-benzyl-9-guanine)-ethyl-N-nitrosourea(BGCNU) was synthesized using O~6-BG as a starting compound. The synthesis procedure mainly includes the following steps. Firstly, O~6-benzyl-guanine(O~6-BG, a) reacted with dibromoethane to prepare N9-bromomethyl-O~6-benzyl-guanine(b), N9-(2-amino)ethyl-O~6-benzylguanine(d) was obtained through a Gabriel reaction. Then the target compound was synthesized by isocyanate substitution of compound d followed by a nitrosation reaction. A pharmacokinetic analysis was performed using BGCNU in phosphate buffer saline(PBS) and in minimum essential medium with Earle’s balanced salts(MEM-EBSS) supplemented with antibiotics(100 U/mL penicillin and 100 μg/m L streptomycin). The half-lives(t1/2) of BGCNU in PBS and MEM-EBSS were approximately 20 min and 30 min. The structures of the target compound and all the intermediate products were confirmed by UV, IR, 1H NMR, 13 C NMR and MS.The antitumor activities of BGCNU was evaluated by CCK-8 method and the data was compared with Nimustine(ACNU), ACNU plus O~6-BG, Carmustine(BCNU), BCNU plus O~6-BG in three types of glioma cells(SF763, SF767, SF126) with different AGT expression levels. The results showed BGCNU expressed high activities in glioma cells, the IC50 of BGCNU is much lower than that of ACNU and ACNU plus O~6-BG. The IC50 of BCNU and BCNU plus O~6-BG are low, which may due to the release of the highly cytotoxic 2-chloroethyl isocyanate from the decomposition of BCNU. A molecular docking study of O~6-BG and O~6-BG analogs which is generated by the decomposition of BGCNU to AGT(from PDB code 1T39) was performed. The results showed that two compounds are able to enter the AGT active site to form hydrogen bond that the number of 3 and 4, respectively. The ChemScore of O~6-BG analogs is higher than O~6-BG, it’s indicated that O~6-BG analogs showed greater activity as AGT inhibitors than O~6-BG.We quantitatively analysed the DNA ICLs(l-[N-(2’-deoxycytidyl)]-2-[N-(2’-deoxyguanosyl)]ethan) induced by ACNU, BCNU and BGCNU in glioma cell lines(SF763, SF767 and SF126) through high-performance liquid chromatography-electrospray ion tandem mass spectrometry(HPLC-ESI-MS/MS). The interstrand crosslinking levels in glioma cells were compared after exposure to drugs for 24 h with different concentrations. The results indicated that BGCNU showed a more superior activity in glioma cells than ACNU and BCNU. The results suggested that BGCNU which have strong crosslinking ability can be decomposed to generate the O~6-BG derivatives to inhibit the activities of AGT in cells, so the dG-dC level in human glioma cell line SF763 was highly improved. The combi-nitrosourea is expected to be developed as a novel chemotherapy with high efficiency, low toxicity and eliminated drug resistance.In this study, a novel combi-nitrosourea was synthesized, the structure of BGCNU was characterized by UV, IR, 1H NMR, 13 C NMR and MS. And its antitumor activities were evaluated by CCK-8 method, then the DNA ICLs induced by BGCNU in glioma cell lines were quantitatively analysed. The results suggested that BGCNU could possess the O~6-BG and the CENU pharmacophores simultaneously, so the activity of CENUs can be improved. This work has important implications for clarifying the antitumor mechanism and developing high-efficient nitrosoureas antitumor drugs with low cytotoxicity.