O-6-alkylguanine-DNA　alkyltransferase　(AGT)　is　the　main　cause　of　tumor　cell　resistance　to　DNA-alkylating　agents,　so　it　is　valuable　to　design　tumor-targeted　AGT　inhibitors　with　hypoxia　activation.　Based　on　the　existing　benchmark　inhibitor　O-6-benzylguanine　(O-6-BG),　four　derivatives　with　hypoxia-reduced　potential　and　their　corresponding　reduction　products　were　synthesized.　A　reductase　system　consisting　of　glucose/glucose　oxidase,　xanthine/xanthine　oxidase,　and　catalase　were　constructed,　and　the　reduction　products　of　the　hypoxia-activated　prodrugs　under　normoxic　and　hypoxic　conditions　were　determined　by　high-performance　liquid　chromatography　electrospray　ionization　tandem　mass　spectrometry　(HPLC-ESI-MS/MS).　The　results　showed　that　the　reduction　products　produced　under　hypoxic　conditions　were　significantly　higher　than　that　under　normoxic　condition.　The　amount　of　the　reduction　product　yielded　from　ANBP　(2-nitro-6-(3-amino)　benzyloxypurine)　under　hypoxic　conditions　was　the　highest,　followed　by　AMNBP　(2-nitro-6-(3-aminomethyl)benzyloxypurine),　2-NBP　(2-nitro-6-benzyloxypurine),　and　3-NBG　(O6-(3-nitro)benzylguanine).　It　should　be　noted　that　although　the　levels　of　the　reduction　products　of　2-NBP　and　3-NBG　were　lower　than　those　of　ANBP　and　AMNBP,　their　maximal　hypoxic/normoxic　ratios　were　higher　than　those　of　the　other　two　prodrugs.　Meanwhile,　we　also　investigated　the　single　electron　reduction　mechanism　of　the　hypoxia-activated　prodrugs　using　density　functional　theory　(DFT)　calculations.　As　a　result,　the　reduction　of　the　nitro　group　to　the　nitroso　was　proven　to　be　a　rate-limiting　step.　Moreover,　the　2-nitro　group　of　purine　ring　was　more　ready　to　be　reduced　than　the　3-nitro　group　of　benzyl.　The　energy　barriers　of　the　rate-limiting　steps　were　34-37　kcal/mol.　The　interactions　between　these　prodrugs　and　nitroreductase　were　explored　via　molecular　docking　study,　and　ANBP　was　observed　to　have　the　highest　affinity　to　nitroreductase,　followed　by　AMNBP,　2-NBP,　and　3-NBG.　Interestingly,　the　theoretical　results　were　generally　in　a　good　agreement　with　the　experimental　results.　Finally,　molecular　docking　and　molecular　dynamics　simulations　were　performed　to　predict　the　AGT-inhibitory　activity　of　the　four　prodrugs　and　their　reduction　products.　In　summary,　simultaneous　consideration　of　reduction　potential　and　hypoxic　selectivity　is　necessary　to　ensure　that　such　prodrugs　have　good　hypoxic　tumor　targeting.　This　study　provides　insights　into　the　hypoxia-activated　mechanism　of　nitro-substituted　prodrugs　as　AGT　inhibitors,　which　may　contribute　to　reasonable　design　and　development　of　novel　tumor-targeted　AGT　inhibitors.