Checkpoint kinase 1 (CHK1) inhibitors can potentiate the effectiveness of deoxyribonucleic acid (DNA) damaging agents in the treatment of cancer. A novel series of 2,6-disubstituted-9H-purine (3a-p, 5a and 5b), 2,4-disubstituted-thieno[3,2-d]pyrimidine (8a-c) and 2,4-disbustituted-7H-pyrrolo[2,3-d]pyrimidine (11a-c) analogues were designed and synthesized as potent CHK1 inhibitors. Compounds (3a, 3d, 3f and 3j-l) with 9H-purine core displayed more potent inhibition against CHK1. The most potent compound (3l) also exhibited low anti-proliferative effects towards HT29 and Hek293 cell lines. In addition, 3l showed strong potentiating effect (7-fold) on the anti-proliferative activity of gemcitabine towards HT29 cells. The results of cell cycle assay indicated that 3l could strikingly affect the cell cycle distribution of the gemcitabine-treated HT29 cells and induce a significant S phase accumulation. The kinase selectivity profile of 3l displayed acceptable selectivity against other kinases. These results rendered 3l a potent lead compound of CHK1 inhibitor for further investigation.