N-{4-[(2-Amino-6-methyl-4-oxo-3,4-dihydrothieno[2,3- d]pyrimidin-5-yl)sulfanyl]benzoyl}-L-glutamic acid (4) and nine nonclassical analogues 5-13 were synthesized as potential dual thymidylate synthase (TS) and dihydrofolate reductase (DHFR) inhibitors. The key intermediate in the synthesis was 2-amino-6-methylthieno[2,3-d]pyrimidin-4(3 H)-one (16), which was converted to the 5-bromo-substituted compound 17 followed by an Ullmann reaction to afford 5-13. The classical analogue 4 was synthesized by coupling the benzoic acid derivative 19 with diethyl L-glutamate and saponification. Compound 4 is the most potent dual inhibitor of human TS (IC 50 = 40 nM) and human DHFR (IC 50 = 20 nM) known to date. The nonclassical analogues 5- 13 were moderately potent against human TS with IC 50 values ranging from 0.11 to 4.6 microM. The 4-nitrophenyl analogue 7 was the most potent compound in the nonclassical series, demonstrating potent dual inhibitory activities against human TS and DHFR. This study indicated that the 5-substituted 2-amino-4-oxo-6-methylthieno[2,3-d]pyrimidine scaffold is highly conducive to dual human TS-DHFR inhibitory activity.