Based on our previous work, a series of novel 2-amino-7,8-dihydropteridin-6(5H)-one derivatives were designed and synthesized via a ring-closing strategy. Biological evaluation with four human cancer cell lines (BT549, T47D, MDA-MB-468, and MDA-MB-231) showed that most of these compounds possessed moderate to potent antiproliferative activities. The most promising compound 8-benzyl-2-(phenethylamino)-7,8-dihydropteridin-6(5H)-one (6q) possessing IC50 values of 7.75, 6.37, and 10.73μM against MDA-MB-468, T47D, and BT549, respectively, which were 49, 11, and 8 folds more active than the positive control fluorouracil. Moreover, fluorescence-activated cell sorting analysis revealed that compound 6q displayed a significant effect on G1 cell-cycle arrest in a concentration-dependent manner in T47D cells. The initial structure-activity relationship studies indicated that linker-length of amine chain in C-2 position of pyrimidine ring played a crucial role in modulating the antitumor activity, which could be of help in the rational design of dihydropteridin-6(5H)-ones as novel anticancer drugs.