Design Synthesis and X-ray Crystal Structure of Classical and Nonclassical 2-Amino-4-oxo-5-substituted-6-ethyl-thieno23-dpyrimidines as Dual Thymidylate Synthase and Dihydrofolate Reductase Inhibitors and as Potential Antitumor Agents

摘要

N-{4-[(2-amino-6-ethyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-5-yl)thio]benzoyl}-L-glutamic acid >2 and thirteen nonclassical analogues >2a–2m were synthesized as potential dual thymidylate synthase (TS) and dihydrofolate reductase (DHFR) inhibitors and as antitumor agents. The key intermediate in the synthesis was 2-amino-6-ethyl-5-iodothieno[2,3-d]pyrimidin-4(3H)-one, >7, to which various aryl thiols were attached at the 5-position. Coupling >8 with L-glutamic acid diethyl ester and saponification afforded >2. X-ray crystal structure of >2 and >1 (the 6-methyl analogue of >2), DHFR and NADPH showed for the first time that the thieno[2,3-d]pyrimidine ring binds in a “folate” mode. Compound >2 was an excellent dual inhibitor of human TS (IC50 = 54 nM) and human DHFR (IC50 = 19 nM), and afforded nanomolar GI50 values against tumor cells in culture. The 6-ethyl substitution in >2 increases both the potency (by two- to three-orders of magnitude) as well as the spectrum of tumor inhibition in vitro compared to the 6-methyl analogue >1. Some of the nonclassical analogues were potent and selective inhibitors of DHFR from Toxoplasma gondii.