Effect of C9-methyl substitution and C8-C9 conformational restriction on antifolate and antitumor activity of classical 5-substituted 2,4-diaminofuro[2,3-d] pyrimidines

N-[4-[1-Methyl-2-(2,4-diaminofuro[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glu tamic acid(5) and its C8-C9 conformationally restricted E- and Z-isomers (6 and 7) were designed and synthesized in order to investigate the effect of incorporating a methyl group at the C9 position and of conformational rest riction at the C8-C9 bridge of N-[4-[2-(2,4-diaminofuro[2,3-d]pyrimidin-5-y l)ethyl]benzoyl]-L-glutamic acid (1) with respect to dihydrofolate reductas e (DHFR) inhibitory activity as well as antitumor activity. The compounds w ere synthesized by a Wittig reaction of 2,4-diamino-5-(chloromethyl)furo[2, 3-d]pyrimidine with ethyl 4-acetylbenzoate followed by catalytic reduction, hydrolysis, and standard peptide coupling with diethyl L-glutamate. The bi ological results indicated that the addition of a 9-methyl group to the C8- C9 bridge, as in 5, increased recombinant human (rh) DHFR inhibitory potenc y (IC50 = 0.42 CIM) as well as the potency against the growth inhibition of tumor cells in culture (CCRF-CEM EC50 = 29 nM, A253 EC50 = 28.5 nM, and Fa Du EC50 = 17.5 nM) compared with the g-desmethyl analogue 1. However, the c onformationally restricted 4:1 Z/E mixture of 7 and 6 was less potent than 5 in both assays, and the pure E-isomer 6 was essentially inactive. These t hree classical analogues were also evaluated as inhibitors of Lactobacillus casei, Escherichia coli, and rat and rh thymidylate synthase (TS) and were found to be weak inhibitors. All three analogues 5-7 were good substrates for human folylpolyglutamate synthetase (FPGS). These data suggested that; FPGS is relatively tolerant to different conformations in the bridge region . Further evaluation of the cytotoxicity of 5 and 7 in methotrexate (MTX)-r esistant CCRF-CEM cell sublines suggested that polyglutamylation was crucia l. for their mechanism of action. Metabolite protection studies of 5 implic ated DHFR as the primary intracelluar target. Compound 5 showed GI(50) valu es in 10(-9)-10(-7) M range against more than 30 tumor cell lines in cultur e.