Synthesis of monoamides of methotrexate from L-glutamic acid monoamide t-butyl esters

Analogues of methotrexate (amethopterin)(1) with α- or γ-monoamide functions [viz. the α- and γ-primary amides (16a) and (26a); the following N-substituted amides: α-methyl-, α-ethyl-, α- and γ-propyl-, α-isopropyl-, α-butyl-, α-isobutyl-, α-sec-butyl-, α-t-butyl, α-benzyl-, and α- and γ-cyclohexylamide (16b–d), (26d), (16e–k), (26k) respectively; and the NN-disubstituted amides: γ-piperidide (26l) and γ-morpholide (26m)] were synthesized starting with t-butyl L-isoglutamine (12a), t-butyl L-glutamine (22a), or the appropriateN′-alkyl or N′N′-dialkyl analogues (12b–k), (22d), (22k), (22l), and (22m). The corresponding N-benzyloxycarbonyl compounds (11) and/or (21) from which the above L-glutamic acid derivatives were obtained were generally synthesized by mixed-anhydride coupling of N-benzyloxycarbonyl-L-glutamic acid (9) with the appropriate amine, conversion into the t-butyl ester, and chromatographic separation. The resulting α-monoamide γ-t-butyl ester (11) and γ-monoamide α-t-butyl ester (21) are unambiguously distinguished by mass spectrometry and 13C n.m.r. spectroscopy. Factors which affect the γ-amide/α-amide product ratio are discussed. The N-deprotected L-glutamic acid monoamide t-butyl esters (12) or (22) were individually coupled to N-trifluoracetyl-p-methylaminobenzoic acid, and the resulting α- or γ-monoamide t-butyl esters (13) or (23) of N-(p-methyl (trifluoroacetyl)aminobenzoyl)-L-glutamic acid was hydrolysed. The N-deprotected product, viz. t-butyl N-(p-methylaminobenzoyl)-L-glutamate α- or γ-monoamide (14) or (24) was converted into the appropriate methotrexate-monoamide t-butyl ester (15) or (25), and thence the desired methotrexate-monoamide (16) or (26), by reaction with 2,4-diamino-6-bromomethylpteridine (17) or by the Taylor procedure. Features of the mass and 13C n.m.r. spectra of the intermediates are discussed.