The　negative　hyperconjugation　(NHC)　of　sp2　and　sp3　hybrid　nitrogen　atoms　were　studied　based　on　the　calculation　of　quinoxaline　derivatives　containing　piperazine　moiety.　The　energy　level,　electronic　density　and　spatial　position　of　acceptor　orbital　are　all　important　factors　of　NHC.　NHC　of　nitrogen　atom　does　not　limit　in　the　case　that　the　sigma*-acceptor　orbital　is　in　the　anti-staggered　positon　of　donor　orbital,　but　it　will　switch　off　when　nitrogen　atom　loses　its　lone　pair　electron.　These　results　are　proved　by　the　crystal　structure　and　the　calculation　of　vibration　frequencies.　At　last,　two　quinoxaline　derivatives　synthesized　in　this　work　show　good　anticancer　activities　against　leukemia　cell　lines.