Design, synthesis, and X-ray crystal structure of classical and nonclassical 2-amino-4-oxo-5-substituted-6-ethylthieno[2,3-d]pyrimidines as dual thymidylate synthase and dihydrofolate reductase inhibitors and as potential antitumor agents

Abstract

N-{4-[(2-Amino-6-ethyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-5-yl)thio]benzoyl}-L-glutamic acid 2 and 13 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 arylthiols were attached at the 5-position. Coupling 8 with L-glutamic acid diethyl ester and saponification afforded 2. X-ray crystal structures 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 2-3 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.

Figure 1

Representative antifolates.

Figure 2

6-Methyl and Target 6-Ethyl–2-amino-4-oxo-5-substituted thieno[2,3-d]pyrimidines.

Figure 3

Difference electron density map (2Fo-Fc, 1σ blue, Fo-Fc 3σ green) for the ternary complex of NADPH and 1 in human DHFR

Figure 4

Stereoview of superposition of active site for human DHFR-Q35K/N64F double mutant ternary complex with the inhibitor 2 and NADPH (green), human DHFR-Q35S/N64S double mutant ternary complex with the inhibitor 2 and NADPH (cyan), human DHFR-Q35K single mutant ternary complex with 1 (violet) and human DHFR ternary complex with 1 (yellow). The figure was prepared by PyMol.

Figure 5

Stereoview of the superposition of folate (cyan) from hDHFR (PDB1drf) on the structure of the hDHFR NADPH double mutant protein Q35K/N64F with 2 (green).

Figure 6

Stereoview of active site for human DHFR-Q35S/N64S double mutant ternary complex with the inhibitor 2 and NADPH. The figure was prepared by SYBY 8.0.

Scheme 1^a

^a Reagents: (a) Ethylcyanoacetate, Et₃N, Sulfur, DMF, 55 °C, 3h; (b) carbamimidic chloride hydrochloride, DMSO₂, 120 °C, 1 h; (c) (1) Hg(AcO)₂, AcOH, 100 °C, 3 h; (2) I₂, CH₂Cl₂, rt, 5 h; (d) thiols, Pd₂(dba)₃, Xantphos, i-Pr₂NEt, DMF, microwave 190 °C, 30 min; (e) 1 N NaOH, MeOH; (f) Lglutamate hydrochloride, 2-chloro-4,6-dimethoxy-1,3,5-triazine, N-methylmorpholine, DMF, rt, 5h; (g) 1 N NaOH, EtOH.