Benzoyl Ring Halogenated Classical 2-Amino-6-methyl-3,4-dihydro-4-oxo-5-substituted Thiobenzoyl-7H-pyrrolo[2,3-d]pyrimidine Antifolates as Inhibitors of Thymidylate Synthase and as Antitumor Agents

Abstract

In an attempt to circumvent resistance to and toxicity of clinically used folate-based thymidylate synthase (TS) inhibitors that require folylpoly-gamma-glutamate synthetase (FPGS) for their antitumor activity, we designed and synthesized two classical 6-5 ring-fused analogues, N-[4-[(2-amino-6-methyl-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)thio]-2'-fluorobenzoyl]-l-glutamic acid (4) and N-[4-[(2-amino-6-methyl-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)thio]-2'-chlorobenzoyl]-l-glutamic acid (5), as TS inhibitors and antitumor agents. The key intermediates in the synthesis of these classical analogues were the mercaptans 10 and 11, which were obtained from the corresponding nitro compounds 6 and 7 respectively, by reduction of the nitro groups followed by diazotization of the amines. The syntheses of analogues 4 and 5 were achieved via the oxidative addition of the sodium salt of ethyl 2-halo-substituted-4-mercaptobenzoate (16 or 17) to 2-amino-6-methyl-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidine (18) in the presence of iodine. The esters obtained from the reaction were deprotected and coupled with diethyl-l-glutamate followed by saponification. Compounds 4 and 5 were both more potent inhibitors of human TS (IC(50) values of 54 and 51 nM, respectively) than were PDDF and the clinically used ZD1694 and LY231514. Compounds 4 and 5 were not substrates for human FPGS up to 250 muM. In addition, 4 and 5 were growth inhibitory against CCRF-CEM cells as well as a number of other tumor cell lines in culture, and protection studies established TS as the principal target of these analogues.