A series of 2,6-diamine-9H-purine derivatives substituted with phenyl groups at the 8-position through three carbon bridges were synthesized as nonclassical antifolates. The anti-proliferative activities of these compounds against HL60, HeLa and A549 cells were tested. The inhibitory activities against rhDHFR and behavior towards thymine synthase (TS) and aminoimidazole carbonamide ribonucleotide transformylase (AICARFT) of our target compounds were determined. Compound 4e displayed the best inhibitory activity against HL-60 and HeLa cells. Flow cytometry studies indicated that HL-60 cells treated with 4e displayed S-phase arrest and induction of apoptosis. The effect of 4e on lysosomes and mitochondria were confirmed which indicated that the induction of apoptosis of 4e was acting through a lysosome-nonmitochondrial pathway. The results suggested that compound 4e, containing an m-methoxyphenyl side chain substituent linked by an α,β-unsaturated carbonyl group as a three-carbon bridge, is worth to be further investigated as a novel potent anticancer agent.