Methotrexate analogues. 30. Dihydrofolate reductase inhibition and in vitro tumor cell growth inhibition by N epsilon-(haloacetyl)-L-lysine and N delta-(haloacetyl)-L-ornithine analogues and an acivicin analogue of methotrexate.
Analogues of methotrexate (MTX) with strong alkylating activity were prepared by replacing the L-glutamate side chain with N omega-haloacetyl derivatives of L-lysine and L-ornithine. Haloacetylation was accomplished in 30-40% yield by reaction of the preformed L-lysine and L-ornithine analogues of MTX with p-nitrophenyl bromoacetate or chloroacetate in aqueous sodium bicarbonate at room temperature. All four haloacetamides were potent inhibitors in spectrophotometric assays measuring noncovalent binding to purified dihydrofolate reductase (DHFR) from L1210 cells. In experiments designed to measure time-dependent inactivation of DHFR from L1210 cells and Candida albicans, the N epsilon-(bromoacetyl)-L-lysine and N delta-(bromoacetyl)-L-ornithine analogues gave results consistent with covalent binding, whereas N epsilon- and N delta-chloroacetyl analogues did not. The N delta-(bromoacetyl)-L-ornithine analogue appeared to be the more reactive one toward both enzymes. Amino acid analysis of acid hydrolysates of the L1210 enzyme following incubation with the bromoacetamides failed to demonstrate the presence of a carboxymethylated residue, suggesting that alkylation had perhaps formed an acid-labile bond. In growth inhibition assays with L1210 cultured murine leukemia cells, the four haloacetamides were all more potent than their nonacylated precursors but less potent than MTX. The greater than 40,000-fold MTX-resistant mutant cell line L1210/R81 was only partly cross-resistant to the haloacetamides. An analogue of MTX with acivicin replacing glutamate was a potent inhibitor of DHFR from chicken liver and L1210 cells but was 200 times less potent than MTX against L1210 cells in culture.
D000085:Acetates / Q000138:chemical synthesis / Q000494:pharmacology / Q000627:therapeutic use; D000818:Animals; D002176:Candida albicans / Q000201:enzymology; D055598:Chemical Phenomena; D002621:Chemistry; D005493:Folic Acid Antagonists; D007555:Isoxazoles / Q000138:chemical synthesis / Q000494:pharmacology / Q000627:therapeutic use; D007939:Leukemia L1210 / Q000188:drug therapy* / Q000201:enzymology; D008239:Lysine / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000494:pharmacology / Q000627:therapeutic use; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000494:pharmacology / Q000627:therapeutic use; D051379:Mice; D009249:NADP / Q000494:pharmacology; D009952:Ornithine / Q000138:chemical synthesis / Q000494:pharmacology / Q000627:therapeutic use; D011487:Protein Conformation / Q000187:drug effects; D013053:Spectrophotometry
D000085:Acetates; D005493:Folic Acid Antagonists; D007555:Isoxazoles; C053402:(N-(4-amino-4-deoxy-N(10)-methylpteroyl)amino)-3-chloro-4,5-dihydro-5-isoxazoleacetic acid; D009249:NADP; D009952:Ornithine; D008239:Lysine; D008727:Methotrexate