5-Deazafolate Analogues with a Rotationally Restricted Glutamate or Ornithine Side Chain: Synthesis and Binding Interaction with Folylpolyglutamate Synthetase

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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 Nδ-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 Km (0.018 μM) 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 γ-glutamyl adduct was no longer an efficient substrate for the enzyme.

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