Potent dual thymidylate synthase and dihydrofolate reductase inhibitors: classical and nonclassical 2-amino-4-oxo-5-arylthio-substituted-6-methylthieno[2,3-d]pyrimidine antifolates

Abstract

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.

Figure 1

Figure 2

Figure 3

Proposed binding modes of 2-amino-4-oxo-5-substituted-6-methylthieno[2,3-d]pyrimidines. The “normal” mode is defined as the binding mode of pemetrexed and raltitrexed to human TS and folic acid (a 2-amino-4-oxopyrimidine system) to human DHFR. The “flipped” mode is defined as the binding mode when pemetrexed, raltitrexed, or folic acid is rotated about the C₂-NH₂ bond by 180°.

Figure 4

Stereoview of compound 4 superimposed on pemetrexed (in green) in human TS (PDB code 1JU6).

Figure 5

Stereoview: compound 4 bound to human DHFR in “normal” mode (PDB code 1DRF), superimposed on folic acid (not shown).

Figure 6

Stereoview of compound 4 bound to human DHFR in the “flipped” mode (PDB code 1U72).

Scheme 1a

^aReagents and conditions: (a) ethylcyanoacetate, TEA, sulfur, DMF, 50 °C to room temp, 2 h; (b) chlorformamidine hydrochloride, DMSO₂, 125 °C, 30 min; (c) Br₂, AcOH, microwave 150 °C, 30 min; (d) thiols, Cu₂O, K₂CO₃, DMF, microwave 180 °C, 30 min.

Scheme 2a

^aReagents and conditions: (a) ethyl 4-mercaptobenzoate, Cu₂O, K₂CO₃, DMF, microwave 180 °C, 30 min; (b) 1 N NaOH, EtOH, room temp, 18 h; (c) L-glutamic acid diethyl ester hydrochloride, 2-chloro-4,6-dimethoxy-1,3,5-triazine, N-methylmorpholine, DMF, room temp, 5 h; (d) 1 N NaOH, EtOH, room temp, 24 h.