The binding of a series of amide derivatives of methotrexate to Lactobacillus casei dihydrofolate reductase has been studied by inhibition constant measurements and by 1H n.m.r. spectroscopy. Amide modification of the α‐carboxylate of methotrexate was found to prevent interaction of the γ‐carboxylate with the imidazole of His 28. Estimates of the contributions to the binding energy from the α‐carboxylate‐Arg 57 and γ‐carboxylate‐His 28 interactions have been made from a combination of inhibition and n.m.r. data.

Analogues of methotrexate (amethopterin) (1) with a-or y-monoamide functions [viz. the a-and yprimary amides (16a) and (26a); the following N-substituted amides: a-methyl-, a-ethyl-, a-and ypropyl-, a-isopropyl-, a-butyl-, a-isobutyl-, a-sec-butyl-, a-t-butyl, a-benzyl-, and a-and ycyclohexylamide (1 6b-d), (26d), (1 6e-k), (26k) respectively; and the NN-disubstituted amides: ypiperidide (261) and y-morpholide (26m)l were synthesized starting with t-butyl L-isoglutamine (1 2a), t-butyl L-glutamine (22a), or the appropriate N'-alkyl or N'N'-dialkyl analogues (1 2b-k), (22d), (22k), (221), and (22m). The corresponding N-benzyloxycarbonyl compounds (1 1) and/or (21) from which the above L-glutamic acid derivatives were obtained were generally synthesized by mixedanhydride coupling of Nbenzyloxycarbonyl-L-glutamic acid (9) with the appropriate amine, conversion into the t -butyl ester, and chromatographic separation. The resulting a-monoamide y-t-butyl ester (1 1 ) and y-monoamide a-t-butyl ester (21 ) are unambiguously distinguished by mass spectrometry and 3C n.m.r. spectroscopy. Factors which affect the y-amidelm-amide product ratio are R a RY ~~ t In some cases the separation into X -and y-monoamides could be effected by crystallization prior to ester formation.

Analoge (XI), (XV), (XVII) von Methotrexat (Amethopterin) (XVIII) werden synthetisiert: Amidierung und Veresterung der Glutaminsäure (I) führen zu Gemischen der Isomeren (IV) und (V), die getrennt werden.

The use of chemical ionization mass spectrometry for the characterization of analogues of methotrexate, N-[4-[[(2,4-diamino-6-pteridinyl)methyl]methylamino~~nzoyl~~-glutamic acid, has been studied. With methane as reactant gas, abundant (MH]+ ions were generally not produced. However, with ammonia, especially in conjunction with thermal desorption from a platinum wire, significant amounts of [MH]+ ions were formed by methotrexate, the a-, y-and diamide analogues, and series of a-and y-monoalkylamide derivatives. The 1-butyl esters of the various monoamides behaved similarly to the corresponding monoamides, except for the ready loss of isobutylene. Fragment ions from both methane and ammonia chemical ionization were formed by cleavages 'benzylic' to the pteridine ring, by bond breakage a t the amide bond between the aminobenzoyl and glutamyl moieties, and by fragmentations on both sides of this amide bond. Fragment ions from these processes, in conjunction with further disintegration of the glutamyl moiety, are diagnostic of the structures of the pteridine, aminobenzoyl and glutamyl moieties of the analogues. Examples of application to structural determination are given.