5-deazafolate analogues with a rotationally restricted glutamate or ornithine side chain: Synthesis and binding interaction with folylpolyglutamate synthetase

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 bet ween the amide nitrogen and the 6'-position of the p-aminobenzoyl moiety we re synthesized and tested as substrates for folylpolyglutamate synthetase ( FPGS), a key enzyme in folate metabolism and an important determinant of th e therapeutic potency and selectivity of classical antifolates. The corresp onding bridged analogues of 5-deazapteroyl-L-ornithine and (6R,6S)-5-deaza- 5,6,7,8-tetrahydropteroyl L-ornithine were also synthesized as potential in hibitors. Condensation of diethyl L-glutamate wi th methyl 2-bromomethyl-4- nitrobenzoate followed by catalytic reduction of the nitro group, reductive coupling with 2-acetamido-6-formylpyrido[2,3-d]pyrimid in the presence of dimethylaminoborane, and acidolysis with HBr/AcOH yielded 2-L-[5-[N-2-aceta mido-4(3H)oxopyrido[2,3-d]pyrimidin-6-yl)methylamino]-2,3-dihydro-1-oxo-2(1 H)-isoindolyl]glutaric acid (1). When acidolysis was preceded by catalytic hydrogenation, the final product was the corresponding (6R,GS)-tetrahydro d erivative 2. A similar sequence starting from methyl N-delta-benzyloxycarbo nyl-L-ornithine led to 2-L-[5-[N-(2-amino-4(3H)-oxopyrido[2,3-d]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 s ubstrates, with the reduced compound 2 giving a K-m, (0.018 CIM) lower than that of any other substrate identified to date. (6R,6S)-5-Deazatetrahydrof olate, 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 re striction of the side chain did not interfere with the initial FPGS-catalyz ed reaction and indeed seemed to facilitate it, but the ensuing gamma-gluta myl adduct was no longer an efficient substrate for the enzyme.