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 synthesis of 5-methyl-5,6,7,8-tetrahydropteroly tri-, pena-, and heptaglutamate has been accomplished by reductive methylation of the tetrahydropteroyl oligoglutamate with formaldehyde, followed by purification on DEAE-Sephadex. The corresponding [5-14-C]methyltetrahydropteroyl oligoglutamates were prepared from 14-CH-2-0, and tested as substrates for methionine synthetase (EC 2.1.1.13) ISOLATED FROM BOVINE BRAIN. In all cases, the polyglutamate conjugates were better substrates (lower Km, higher Vmax) than the corresponding monoglutamate forms. In addition, the nonradioactive methyltetrahydropteroyl oligoglutamates inhibited the methylation of homocysteine by methyltetrahydrofolate. This indicates that the monoglutamate and polyglutamates compete for the same enzyme, and established a role for the ubiquitous methyltetrahydropteroyl oligoglutamates in mammalian methionine biosynthesis.
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