title: Effect of bridge region variation on antifolate and antitumor activity of classical 5-substituted 2,4-diaminofuro[2,3-d]pyrimidines.
abstract: Variation of the bridge linking the heterocyclic ring and p-aminobenzoyl-L-glutamate portions of our previously described classical 2,4-diaminofuro[2,3-d]pyrimidines 1 and 2 are reported as inhibitors of dihydrofolate reductase (DHFR) and thymidylate synthase (TS) and as antitumor agents. Specifically -CH2CH2- and -CH2NHCH2- bridged analogues, N-[4-[2-(2,4-diaminofuro[2,3-d]pyrimidin-5-yl) ethyl]benzoyl]-L-glutamic acid (3) and N-[4-[[N-[(2,4-diaminofuro[2,3-d]pyrimidin-5-yl) methyl]amino]methyl]benzoyl]-L-glutamic acid (4), respectively, were synthesized. Compound 3 was obtained via a Wittig reaction of the tributylphosphonium salt of 2,4-diamino-5-(chloromethyl)furo[2,3-d]pyrimidine (5) and methyl 4-formylbenzoate (6) followed by reduction and coupling with the diethyl ester of L-glutamic acid. Compound 4 was synthesized by the nucleophilic displacement of 5 with diethyl N-[4-(aminomethyl)benzoyl]-L-glutamate (15) and saponification. Both analogues were evaluated in vitro as inhibitors of DHFRs from (recombinant) human, human CCRF-CEM cells, and Lactobacillus casei. Compound 3 showed moderate activity (IC50 10(-6)-10(-7) M). Compound 4 was essentially inactive (IC50 10(-5) M, CCRF-CEM). The compounds were also evaluated against TS from (recombinant) human and L. casei and were of low activity (IC50 10(-5) M). The three-atom-bridged analogue 4 was somewhat more inhibitory to human TS than methotrexate (MTX). Compound 3 inhibited the growth of tumor cells in culture (IC50 10(-7) M) while 4 showed a low level of growth inhibitory activity. The inhibition of the growth of leukemia CCRF-CEM cells by both compounds parallels their inhibition of CCRF-CEM DHFR. Analogue 3 was a good substrate for human folylpolyglutamate synthetase (FPGS) derived from CCRF-CEM cells (Km 8.5 microM). Further evaluation of the growth inhibitory activity of 3 against the MTX-resistant subline of CCRF-CEM cells (R30dm) with decreased FPGS indicated that poly-gamma-glutamylation was important for its action. Protection studies with 3 in the FaDu squamous cell carcinoma cell line indicated that inhibition was completely reversed by leucovorin [(6R,S-5-formyltetrahydrofolate] or by a combination of thymidine and hypoxanthine, suggesting an antifolate effect directed at DHFR.
authors.affiliation: Division of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne University, Pittsburgh, Pennsylvania 15282, USA.
authors.forename: A
authors.initials: A
authors.lastname: Gangjee
authors.forename: R
authors.initials: R
authors.lastname: Devraj
authors.forename: J J
authors.initials: JJ
authors.lastname: McGuire
authors.forename: R L
authors.initials: RL
authors.lastname: Kisliuk
mesh_terms: D000970:Antineoplastic Agents / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology*; D002455:Cell Division / Q000187:drug effects; 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; D019271:Hypoxanthine; D007042:Hypoxanthines / Q000494:pharmacology; D007700:Kinetics; D007780:Lacticaseibacillus casei / Q000201:enzymology; D002955:Leucovorin / Q000494:pharmacology; D008727:Methotrexate / Q000494:pharmacology; D015394:Molecular Structure; D010453:Peptide Synthases / Q000037:antagonists & inhibitors / Q000378:metabolism; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma; D011743:Pyrimidines / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D013329:Structure-Activity Relationship; D013936:Thymidine / Q000494:pharmacology; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D000970:Antineoplastic Agents; D005493:Folic Acid Antagonists; D007042:Hypoxanthines; C095762:N-(4-((N-((2,4-diaminofuro(2,3-d)pyrimidin-5-yl)methyl)amino)methyl)benzoyl)glutamic acid; C095761:N-(4-(2-(2,4-diaminofuro(2,3-d)pyrimidin-5-yl)ethyl)benzoyl)glutamic acid; D011743:Pyrimidines; D011994:Recombinant Proteins; D019271:Hypoxanthine; D005492:Folic Acid; D013940:Thymidylate Synthase; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase; D002955:Leucovorin; D013936:Thymidine; D008727:Methotrexate