Chloroethylnitrosoureas (CENUs) are an important type of alkylating agent employed in the clinical treatment of cancer. However, the anticancer efficacy of CENUs is greatly decreased by a DNA repairing enzyme, O-alkylguanine-DNA alkyltransferase (AGT), by preventing the formation of interstrand cross-links (ICLs). In this study, a combi-nitrosourea prodrug, namely, -(2-chloroethyl)-'-2-(O-benzyl-9-guanine)ethyl--nitrosourea (BGCNU), which possesses an O-benzylguanine (O-BG) derivative and CENU pharmacophores simultaneously, was synthesized and evaluated for its ability to induce ICLs. The target compound is markedly more cytotoxic in human glioma cells than the clinically used CENU chemotherapies ACNU, BCNU, and their respective combinations with O-BG. In the AGT-proficient cells, significantly higher levels of DNA ICLs were observed in the groups treated by BGCNU than those by ACNU and BCNU, which indicated that the activity of AGT was effectively inhibited by the O-BG derivatives released from BGCNU.

A hypoxia-activated combi-nitrosourea prodrug, N-(2-chloroethyl)-N -2-(2-(4-nitrobenzylcarbamate)-O 6 -benzyl-9-guanine)ethyl-N-nitrosourea (NBGNU), was synthesized and evaluated for its hypoxic selectivity and anticancer activity in vitro. Results: The prodrug was designed as a tripartite molecule consisting of a chloroethylnitrosourea pharmacophore to induce DNA interstrand crosslinks (ICLs) and an O 6 -benzylguanine analog moiety masked by a 4-nitrobenzylcarbamate group to induce hypoxia-activated inhibition of O 6 -alkylguanine-DNA alkyltransferase. NBGNU was tested for hypoxic selectivity, cytotoxicity and DNA ICLs ability. The reduction product amounts, cell death rates and DNA ICL levels induced by NBGNU under hypoxic conditions were all significantly higher than those induced by NBGNU under normoxic conditions. Conclusion: The tripartite combi-nitrosourea prodrug exhibits desirable tumor-hypoxia targeting ability and abolished chemoresistance compared with the conventional chloroethylnitrosoureas.