Objective：To establish a new approach to synthesis of diethyl N-［4-［(2,4-diaminopyrido［3,2-d］pyrimidin-6-yl)methylamino］benzoyl］-L-glutamate. Methods:Target compound (5) was synthesized by the use of (2,4-dioxo-tetrahydropyridopyrimidin-6-yl)methyl acetate (1) as starting material via hydrolysis, chlorination, condensation with diethyl (paminobenzoyl)glutamate and aminolysis. Results: A new approach to synthesis of diethyl N［4［(2,4diaminopyrido［3,2-d］pyrimidin-6-yl)methylamino］benzoyl］-L-glutamatewas established. This synthetic route has hydrolysis reaction, chlorination, diethyl N-(p-aminobenzoyl)-L-glutamate condensation reaction and ammonolysis reaction. The total yield is 36.7%.The structures of those compounds have identified by 1H nuclear magnetic resonance, 13C nuclear magnetic resonance and mass spectrometry. This synthetic route avoid the unstable brominated re-action product and improves the harsh condition of ammonolysis reaction. Conclusion:The new synthetic route has improved the reaction condition and the stability of the intermediate, and increased the extent of the derivative compounds, which has great significance to anti-folic acid of anti-tumor inhibitor synthesis.