In this work, a new fused pyrimidine derivatives {5-(4-chlorophenyl)-4-oxo-3,5-dihydro-4H-chromeno [2,3-d]pyrimidin-8-yl formate)} was prepared from chromene compound {(2-amino-4-(4-(dimethylamino)phenyl)-5-oxo-4,5-dihydropyran[3,2-c]chromene-3-carbonitrile} with formic acid in presence of POCl3 and identified by FT-IR and 1 H NMR spectra. The pyrimidine compounds have medicinal and biological activities. Therefore the effect of these compounds was studied on human serum acetylthiocholine esterase activity. The results concluded that the greater inhibition percent was found at concentrations (10 -2 ) M for each compounds chromene and pyrimidine respectively and indicated that Km varied from higher, lesser or the same in the presence of chromene and pyrimidine compared with non-inhibiting system. The maximum and minimum inhibitor concentrations of chromene appeared mix inhibition during enzymatic reaction, in contrast pyrimidine does not compute with substrate on the active site of enzyme (noncompetitive and uncompetitive inhibition). The Vmax value for control sample was higher than in inhibited samples, The biochemical tests revealed that Ki (the binding affinity of the inhibitor) for chromene and pyrimidine compounds are higher at 10 -2 M (1.5 × 10 -3 , 1.11 × 10 -3 ).