Many pyridopyrimidine moieties linked to the coumarin ring were synthesized by the reaction of malononitrile with 7-hydroxy-4-methyl-2-oxo-2 H-chromene-8-carbaldehyde (2) directly in the presence of ammonium acetate and different ketones and studied the effect of other basic catalysis, ratio of reactants and the effect of the solvent. The newly synthesized compounds were evaluated for their cytotoxic activity against four cell lines namely HepG2, WI-38, VERO, and MCF-7. The cytotoxic activity showed that compounds 8, 9, 10, and 7 have the highest activity against the studied cell lines. Focusing on the binding affinity and interactions between the five synthesized derivatives with the highest anticancer activity and specific amino acids of 4HJO residues over the molecular docking analysis. Derivative 10 recorded the highest energy score with good RMSD compared to the rest of the derivatives.