In this work the corresponding 4-amino-6-pyridyl-2-phenyl-5-carbethoxypyrimidine (3) was synthesized through a one-pot, three-component reaction of pyridine-3-carbaldehyde, ethyl cyanoacetate and benzamidine hydrochloride in the presence of TEA in refluxing ethanol-DMF mixture. The synthesized pyrimidine (3) was directly transformed to medicinally important pyrimido[4,5-d]pyrimidine derivatives. All the synthesized products were tested and evaluated as antimicrobial agents. Keywords: Pyrimidine-5-ethyl carboxylate derivative, Pyrimido[4,5-d]pyrimidine, Chloroacetyl chlorid