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-glutamic 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 restriction at the C8-C9 bridge of N-[4-[2-(2,4-diaminofuro[2, 3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid (1) with respect to dihydrofolate reductase (DHFR) inhibitory activity as well as antitumor activity. The compounds were 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 biological results indicated that the addition of a 9-methyl group to the C8-C9 bridge, as in 5, increased recombinant human (rh) DHFR inhibitory potency (IC(50) = 0.42 microM) as well as the potency against the growth inhibition of tumor cells in culture (CCRF-CEM EC(50) = 29 nM, A253 EC(50) = 28.5 nM, and FaDu EC(50) = 17.5 nM) compared with the 9-desmethyl analogue 1. However, the conformationally 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 three 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)-resistant CCRF-CEM cell sublines suggested that polyglutamylation was crucial for their mechanism of action. Metabolite protection studies of 5 implicated DHFR as the primary intracellular target. Compound 5 showed GI(50) values in 10(-9)-10(-7) M range against more than 30 tumor cell lines in culture.
Gangjee
A
A
Division of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne University, Pittsburgh, Pennsylvania 15282, USA. gangjee@duq.edu
Zeng
Y
Y
McGuire
J J
JJ
Kisliuk
R L
RL
D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D019008:Drug Resistance, Neoplasm; D004354:Drug Screening Assays, Antitumor; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D004926:Escherichia coli / Q000737:chemistry; D005492:Folic Acid / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D005493:Folic Acid Antagonists / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D007780:Lacticaseibacillus casei / Q000737:chemistry; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000378:metabolism; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry; D014122:Toxoplasma / Q000737:chemistry; D014407:Tumor Cells, Cultured
D000964:Antimetabolites, Antineoplastic; D004791:Enzyme Inhibitors; D005493:Folic Acid Antagonists; C095762:N-(4-((N-((2,4-diaminofuro(2,3-d)pyrimidin-5-yl)methyl)amino)methyl)benzoyl)glutamic acid; D011994:Recombinant Proteins; D005492:Folic Acid; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase