A series of pyrimido[5,4-c]quinoline-4-(3H)-one derivatives variously substituted at positions 2 and 3 were synthesized and evaluated for their in vitro antiproliferative activities against a panel of six human cancer cell lines. Biological evaluation revealed that the vast majority of derivatives exhibited moderate tumor growth inhibitory activities. In particular, compound 7e showed effective anti-tumor activity with broad-spectrum toward numerous cell lines and the most active member in this study. This derivative displaying significant activity against KB (IC(50): 4.9 μM), CNE2 (IC(50): 13.8 μM), MGC-803 (IC(50): 4.8 μM), GLC-82 (IC(50): 7.88 μM), MDA-MB-453 (IC(50): 18.2 μM) and MCF-7 (IC(50): 10.1 μM) cell lines could be considered as the most promising and useful template for future development to obtain more potent anti-tumor agent(s).