EFFECT OF BRIDGE REGION VARIATION ON ANTIFOLATE AND ANTITUMOR-ACTIVITY OF CLASSICAL 5-SUBSTITUTED 2,4-DIAMINOFURO[2,3-D]PYRIMIDINES

Variation of the bridge linking the heterocyclic ring and p-aminobenzo yl-L-glutamate portions of our previously descibed classical 2,4-diami nofuro[2,3-d]pyrimidines 1 and 2 are reported as inhibitors of dihydro folate reductase (DHFR) and thymidylate synthase (TS) and as antitumor agents. Specifically -CH2CH2- and -CH2NHCH2- bridged analogues, uro[2 ,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid (3) and midin-5-yl) methyl]amino]methyl]benzoyl]-L-glutamic acid (4), respectively, were s ynthesized. Compound 3 was obtained via a Wittig reaction of the tribu tylphosphonium salt of 2,4-diamino-5-(chloromethyl)furo[2,3-d]pyrimidi ne (5) and methyl 4-formylbenzoate (6) followed by reduction and coupl ing with the diethyl ester of L-glutamic acid. Compound 4 was synthesi zed by the nucleophilic displacement of 5 with diethyl N-[4-( aminomet hyl)benzoyl]-L-glutamate (15) and saponification. Both analogues were evaluated in vitro as inhibitors of DHFRs from (recombinant) human, hu man CCRF-CEM cells, and Lactobacillus casei. Compound 3 showed moderat e activity (IC50 10(-6)-10( -7) M). Compound 4 was essentially inactiv e (IC50 10(-5) M, CCRF-CEM). The compounds were also evaluated against TS from (recombinant) human and L. casei and were of low activity (IC 50 10(-6) M). The three-atom-bridged analogue 4 was somewhat more inhi bitory to human TS than methotrexate (MTX). Compound 3 inhibited the g rowth of tumor cells in culture (IC50 10(-7) M) while 4 showed a low l evel of growth inhibitory activity. The inhibition of the growth of le ukemia CCRF-CEM cells by both compounds parallels their inhibition of CCRF-CEM DHFR. Analogue 3 was a good substrate for human folylpolyglut amate synthetase (FPGS) derived from CCRF-CEM cells (K-m 8.5 mu M). Fu rther evaluation of the growth inhibitory activity of 3 against the MT X-resistant subline of CCRF-CEM cells (R30dm) with decreased FPGS indi cated that poly-gamma-glutamylation was important for its action. Prot ection studies with 3 in the FaDu squamous cell carcinoma cell line in dicated that inhibition was completely reversed by leucovorin [(6R,S-6 -formyltetrahydrofolate] or by a combination of thymidine and hypoxant hine, suggesting an antifolate effect directed at DHFR.