Syntheses of .alpha.- and .gamma.-substituted amides, peptides, and esters of methotrexate and their evaluation as inhibitors of folate metabolism

Abstract

N-[4-[[(Benzyloxy)carbonyl]methylamino]benzoyl]-L-glutamic acid alpha-benzyl ester (2) and gamma-benzyl ester (6) served as key intermediates in syntheses of precursors to amides and peptides of methotrexate (MTX) involving both the alpha- and gamma-carboxyl groupings of the glutamate moiety. Coupling of 2 and 6 at the open carboxyl grouping with amino compounds was affected by the mixed anhydride method (using isobutyl chloroformate); carboxyl groupings of amino acids coupled with 2 and 6 were protected as benzyl esters. N-[4-[[(Benzyloxy)carbonyl]methylamino]benzoyl]-L-glutamic acid gamma-methyl ester (5), a precursor to MTX gamma-methyl ester, was prepared from L-glutamic acid gamma-methyl ester and 4-[[(benzyloxy)carbonyl]methylamino]benzoyl chloride (1) in a manner similar to that used to prepare 2 and 6. The precursor to MTX alpha-methyl ester was prepared from gamma-benzyl ester 6 by treatment with MeI in DMF containing (i-Pr)2NEt. Benzyl and (benzyloxy)carbonyl protective groupings were removed by hydrogenolysis, and the deprotected side-chain precursors were converted to alpha- and gamma-substituted amides, peptides, and esters of MTX by alkylation with 6-(bromomethyl)-2,4-pteridinediamine hydrobromide (12). Biochemical-pharmacological studies on the prepared compounds aided in establishing that the alpha-carboxyl grouping of the glutamate moiety contributes to the binding of MTX to dihydrofolate reductase while the gamma-carboxyl does not. Other studies on the peptide MTX-gamma-Glu (13h) are concerned with the contribution toward antifolate activity of this metabolite of MTX. The compounds prepared were also evaluated and compared with MTX with respect to cytotoxicity toward H.Ep.-2 cells and effect on L1210 murine leukemia.

The structure of mouse L1210 dihydrofolate reductase‐drug complexes and the construction of a model of human enzyme

“…Arg-57ec) is observed. This is somewhat surprising, given that a-substitution of MTX increases the Ki by about 105 [18], consistent with an ionic interaction of the a,-carboxylate with, for example, Arg-70. As the MTX complex was prepared by the soaking of pre-grown DHFR/NADPH/TMP crystals in the inhibitor, it is possible that packing constraints within the crystal are preventing either rearrangement of protein side-chains (improbably, given the presence of a large solvent-filled cavity in this region) or a more substantial conformational change.…”

A 1H n.m.r. study of the role of the glutamate moiety in the binding of methotrexate to Lactobacillus casei dihydrofolate reductase

“…and methotrexate y-monoamide (III) (Piper et al, 1982), were synthesized from N -benzyloxycarbonyl -Lglutamic acid and N -benzyloxy -carbonyl -Lglutamine respectively, as described by Antonjuk et al (1983).…”

Antibody-targeted liposomes: increase in specific toxicity of methotrexate-gamma-aspartate.

“…Methotrexate was obtained from Bristol Myers, and methotrexate-,y-aspartate was synthesized as described (12). Monoclonal antibodies used in these experiments were anti-H2Kk IgG2A secreted by the clone 11-4.1 (13), antisheep erythrocyte IgG2A (hybridoma S-S. 1), and anti-Thy 1.1 IgG2A secreted by the hybridoma 19VE12 (14).…”

“…Methotrexate has been used successfully in targeted liposomes by Leserman et al (11) but it readily penetrates cells via the folate transport system. Piper et aL (12) have examined the effects of structural alterations of methotrexate on cytotoxicity, influx kinetics, and ability to inhibit dihydrofolate reductase. Methotrexate-y-aspartate proved to be virtually indistinguishable from methotrexate in its ability to inhibit dihydrofolate reductase.…”