Abstract: A series of 4-(N-aryl)amino-6-alkoxyl pteridine derivatives was designed and synthesized via the bioisostere principle using anti-cancer drugs with quinazoline cores as lead compounds. The proliferative inhibitory activities of the synthesized compounds against tumor cells A549, KG1a and HGC-27 were also performed by MTT assay. Using methyl 3-aminopyrazine-2-carboxylate as the starting material, 12 target compounds( 7a - 7l )were synthesized through six-step reaction, and characterized by ¹H NMR, ¹³C NMR and MS. It was showed that antitumor activity of pteridines with 2-chloro-5-nitro-anilino substituted in position 4 was more potent than that of others. The activity of compound 7b on A549 cells(IC₅₀=11. 55 μmol/L)was closely approximate to that of positive control gefitinib(IC₅₀=5. 95 μmol/L). IC₅₀ values of compound 7k on three cell lines were all close to those of gefitinib. A 2-chloro-5-nitro-anilino fragment was contained in preferred compounds which might be modified for further investigation.