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

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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 effected 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, a precursor to MTX .gamma.-methyl ester, was prepared from L-glutamic acid .gamma.-methyl ester and 4-[[(benzyloxy)carbonyl]methylamino]benzoyl chloride 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; the deprotected side-chian precursors were converted to .alpha.- and .gamma.-substituted amides, peptide, and esters of MTX by alkylation with 6-(bromomethyl)-2,4-pteridinediamine hydrobromide. 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 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 [human laryngeal carcinoma] H.Ep.-2 cells and effect on L1210 murine leukemia cells.