5-ARYLTHIO-SUBSTITUTED 2-AMINO-4-OXO-6-METHYLPYRROLO[2,3-D]PYRIMIDINEANTIFOLATES AS THYMIDYLATE SYNTHASE INHIBITORS AND ANTITUMOR AGENTS

Classical antifolate inhibitors of thymidylate synthase (TS) often req uire the reduced folate uptake system in order to exert their antitumo r effects. In addition, these analogues are polyglutamylated via the e nzyme folylpoly-gamma-glutamate synthetase (FPGS), which prevents anal ogue efflux from the cell and usually-increases their inhibitory poten cy against TS. Impaired function of the reduced folate uptake system a nd that of FPGS are potential sources of resistance to such antifolate s. We designed and synthesized a classical 6-5 ring-fused analogue hyd ro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)thio]- benzoyl]-L-glutamic ac id (5) and a nonclassical 6-5 ring-fused analogue hio)-3,4-dihydro-4-o xo-7H-pyrrolo[2,3-d]pyrimidine (6) as TS inhibitors and antitumor agen ts. The syntheses of analogues 5 and 6 were acheived via the oxidative addition of the sodium salt of ethyl 4-mercaptobenzoate or 4-mercapto pyridine to thyl-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidine (17) in the presence of iodine. For the synthesis of 5 the ester obtained fro m the reaction was deprotected and coupled with diethyl L-glutamate fo llowed by saponification. Compound 5 was a potent inhibitor of human a nd bacterial TS with IC50 values of 42 and 21 nM, respectively. Compou nd 6 was 10-fold less potent than 5 against human TS but more than 470 0-fold less potent than 5 against Lactobacillus casei TS. The classica l analogue 5 was neither a substrate nor an inhibitor of human FPGS de rived from CCRF-CEM cells. Compound 5 was cytotoxic to CCRF-CEM and Fa Du tumor cell lines as well as to an FPGS-deficient subline of CCRF-CE M. Thymidine protection studies established that TS was the primary ta rget of 5.