title: 5-deazafolate analogues with a rotationally restricted glutamate or ornithine side chain: synthesis and binding interaction with folylpolyglutamate synthetase.
abstract: Rotationally restricted analogues of 5-deazapteroyl-L-glutamate and (6R,6S)-5-deaza-5,6,7,8-tetrahydropteroyl-L-glutamate with a one-carbon bridge between the amide nitrogen and the 6'-position of the p-aminobenzoyl moiety were synthesized and tested as substrates for folylpolyglutamate synthetase (FPGS), a key enzyme in folate metabolism and an important determinant of the therapeutic potency and selectivity of classical antifolates. The corresponding bridged analogues of 5-deazapteroyl-L-ornithine and (6R,6S)-5-deaza-5,6,7, 8-tetrahydropteroyl-L-ornithine were also synthesized as potential inhibitors. Condensation of diethyl L-glutamate with methyl 2-bromomethyl-4-nitrobenzoate followed by catalytic reduction of the nitro group, reductive coupling with 2-acetamido-6-formylpyrido[2, 3-d]pyrimidin-4(3H)-one in the presence of dimethylaminoborane, and acidolysis with HBr/AcOH yielded 2-L-[5-[N-(2-acetamido-4(3H)-oxopyrido[2, 3-d]pyrimidin-6-yl)methylamino]-2, 3-dihydro-1-oxo-2(1H)-isoindolyl]glutaric acid (1). When acidolysis was preceded by catalytic hydrogenation, the final product was the corresponding (6R,6S)-tetrahydro derivative 2. A similar sequence starting from methyl N(delta)-benzyloxycarbonyl-L-ornithine led to 2-L-[5-[N-(2-amino-4(3H)-oxopyrido[2, 3-d]pyrimidin-6-yl)methylamino]-2, 3-dihydro-1-oxo-2(1H)-isoindolyl]-5-aminopentanoic acid (3) and the (6R,6S)-tetrahydro derivative 4. Compounds 3 and 4 were powerful inhibitors of recombinant human FPGS, whereas 1 and 2 were exceptionally efficient FPGS substrates, with the reduced compound 2 giving a K(m) (0.018 microM) lower than that of any other substrate identified to date. (6R,6S)-5-Deazatetrahydrofolate, in which the side chain is free to rotate, was rapidly converted to long-chain polyglutamates. In contrast, the reaction of 1 and 2 was limited to the addition of a single molecule of glutamic acid. Hence rotational restriction of the side chain did not interfere with the initial FPGS-catalyzed reaction and indeed seemed to facilitate it, but the ensuing gamma-glutamyl adduct was no longer an efficient substrate for the enzyme.
authors.affiliation: Dana-Farber Cancer Institute and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: A
authors.initials: A
authors.lastname: Rosowsky
authors.forename: R A
authors.initials: RA
authors.lastname: Forsch
authors.forename: A
authors.initials: A
authors.lastname: Null
authors.forename: R G
authors.initials: RG
authors.lastname: Moran
mesh_terms: D002455:Cell Division / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000737:chemistry*; D005492:Folic Acid / Q000031:analogs & derivatives* / Q000737:chemistry; D005493:Folic Acid Antagonists / Q000138:chemical synthesis / Q000494:pharmacology; D018698:Glutamic Acid / Q000737:chemistry; D006801:Humans; D009952:Ornithine / Q000737:chemistry; D010453:Peptide Synthases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011994:Recombinant Proteins
chemical_list: D004791:Enzyme Inhibitors; D005493:Folic Acid Antagonists; D011994:Recombinant Proteins; D018698:Glutamic Acid; C040505:5-deazafolic acid; D005492:Folic Acid; D009952:Ornithine; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase