New antifolates, characterized by a 6-5 fused ring system, a pyrrolo[2,3-d]pyrimidine ring, and a trimethylene bridge at position 5 (12a,b and 13a,b) were designed and efficiently synthesized. The synthetic method included (1) construction of the key intermediary acyclic skeleton, 5-[4-(tert-butoxycarbonyl)phenyl]- 2-(dicyanomethyl)pentanoates (6a,b), (2) cyclization with guanidine, followed by reduction to the pyrrolo[2,3-d]pyrimidine derivatives (8a,b and 9a,b), and (3) subsequent glutamate coupling and saponification. These antifolates were more growth-inhibitory by about 1 order of magnitude than methotrexate (MTX) against KB human epidermoid carcinoma cells and A549 human nonsmall cell lung carcinoma cells in in vitro culture. Growth inhibitory IC50 values for N-[4-[3-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5- yl)propyl]benzoyl]-L-glutamic acid (12a) against KB and A549 were 0.27 and 4.5 ng/mL, while those for MTX were 5.0 and 35 ng/mL, respectively. Other members of this class of antifolates, 12b and 13a,b, showed good activities nearly equal to that of 12a.