The　drug　resistance　of　CENUs　induced　by　O-6-alkylguanine-DNA　alkyltransferase　(AGT),　which　repairs　the　O-6-alkylated　guanine　and　subsequently　inhibits　the　formation　of　dG-dC　cross-links,　hinders　the　application　of　CENU　chemotherapies.　Therefore,　the　discovery　of　CENU　analogs　with　AGT　inhibiting　activity　is　a　promising　approach　leading　to　novel　CENU　chemotherapies　with　high　therapeutic　index.　In　this　study,　a　new　combi-nitrosourea　prodrug　3-(3-(((2-amino-9H-purin-6-yl)oxy)methyl)　benzyl)-1-(2-chloroethyl)-1-nitrosourea　(6),　designed　to　release　a　DNA　cross-linking　agent　and　an　inhibitor　of　AGT,　was　synthesized　and　evaluated　for　its　antitumor　activity　and　ability　to　induce　DNA　interstrand　cross-links　(ICLs).　The　results　indicated　that　6　exhibited　higher　cytotoxicity　against　mer(+)　glioma　cells　compared　with　ACNU,　BCNU,　and　their　respective　combinations　with　O-6-benzylguanine　(O-6-BG).　Quantifications　of　dG-dC　cross-links　induced　by　6　were　performed　using　HPLC-ESI-MS/MS.　Higher　levels　of　dG-dC　cross-link　were　observed　in　6-treated　human　glioma　SF763　cells　(mer(+)),　whereas　lower　levels　of　dG-dC　cross-link　were　observed　in　6-treated　calf　thymus　DNA,　when　compared　with　the　groups　treated　with　BCNU　and　ACNU.　The　results　suggested　that　the　superiority　of　6　might　result　from　the　AGT　inhibitory　moiety,　which　specifically　functions　in　cells　with　AGT　activity.　Molecular　docking　studies　indicated　that　five　hydrogen　bonds　were　formed　between　the　O-6-BG　analogs　released　from　6　and　the　five　residues　in　the　active　pocket　of　AGT,　which　provided　a　reasonable　explanation　for　the　higher　AGT-inhibitory　activity　of　6　than　O-6-BG.　(C)　2016　Elsevier　Ltd.　All　rights　reserved.