title: Methotrexate analogues. 26. Inhibition of dihydrofolate reductase and folylpolyglutamate synthetase activity and in vitro tumor cell growth by methotrexate and aminopterin analogues containing a basic amino acid side chain.
abstract: Analogues of the antitumor antifolate methotrexate (MTX) were synthesized in which the glutamate (Glu) moiety was replaced by ornithine (Orn), 2,4-diaminobutyric acid (Dab), or 2,3-diaminopropionic acid (Dap). An aminopterin (AMT) analogue with Orn in place of Glu was also synthesized. The MTX analogues were obtained by reaction of 4-amino-4-deoxy-N10-methylpteroic acid (mAPA) and N omega-Boc-alpha,omega-diaminoalkanoic acids in the presence of diethyl phosphorocyanidate, followed by deprotection with trifluoroacetic acid (TFA) or by reaction of p-nitrophenyl-mAPA and N omega-Boc-alpha,omega-diaminoalkanoic acids and subsequent treatment with TFA. The AMT analogue (APA-Orn) was synthesized by reaction of p-nitrophenyl 4-amino-4-deoxy-N10-formylpteroate with silylated N delta-Boc-L-ornithine in DMF at 55 degrees C for 3 days (45% yield), saponification (83%), and TFA cleavage (89%). APA-Orn was a potent inhibitor of both dihydrofolate reductase (DHFR) from L1210 mouse leukemia (IC50 = 0.072 microM) and partly purified folylpolyglutamate synthetase (FPGS) from mouse liver (Ki = 0.15 +/- 0.06 microM). The MTX analogue (mAPA-Orn) was likewise active against both enzymes, with an IC50 of 0.160 microM for DHFR and a Ki of 20.4 +/- 7.7 microM for FPGS inhibition. The other MTX analogues and the previously reported lysine derivative (mAPA-Lys) showed DHFR affinity similar to that of mAPA-Orn but lacked activity as FPGS inhibitors. The positively charged amino group appears to be detrimental to cellular uptake, as evidenced by the low cytotoxicity of these compounds (IC50 = 0.40-2.4 microM) in comparison with MTX and AMT (IC50 = 0.002 microM) against wild-type L1210 cells. On the other hand, mAPA-Orn and APA-Orn were both more potent than the corresponding Glu derivatives MTX and AMT against L1210/R81 cells, suggesting that in these MTX-resistant cells there may occur a "self-potentiation" process involving enhanced antifolate activity via interference with the polyglutamylation of reduced folates. APA-Orn is the most potent dual inhibitor of DHFR and FPGS discovered to date, but its effectiveness as a therapeutic agent may require some form of prodrug modification to neutralize the terminal amino group of the side chain.
authors.forename: A
authors.initials: A
authors.lastname: Rosowsky
authors.forename: J H
authors.initials: JH
authors.lastname: Freisheim
authors.forename: R G
authors.initials: RG
authors.lastname: Moran
authors.forename: V C
authors.initials: VC
authors.lastname: Solan
authors.forename: H
authors.initials: H
authors.lastname: Bader
authors.forename: J E
authors.initials: JE
authors.lastname: Wright
authors.forename: M
authors.initials: M
authors.lastname: Radike-Smith
mesh_terms: D000613:Aminobutyrates; D000630:Aminopterin / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D002455:Cell Division / Q000187:drug effects; D005493:Folic Acid Antagonists; D005971:Glutamates; D018698:Glutamic Acid; D007700:Kinetics; D007939:Leukemia L1210 / Q000201:enzymology; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000494:pharmacology; D051379:Mice; D008970:Molecular Weight; D009952:Ornithine; D010453:Peptide Synthases / Q000037:antagonists & inhibitors*; D015091:beta-Alanine / Q000031:analogs & derivatives
chemical_list: D000613:Aminobutyrates; D005493:Folic Acid Antagonists; D005971:Glutamates; D015091:beta-Alanine; C005959:2,4-diaminobutyric acid; D018698:Glutamic Acid; C011757:2,3-diaminopropionic acid; D009952:Ornithine; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase; D000630:Aminopterin; D008727:Methotrexate