Methotrexate 5-aminoallyl-2'-deoxyuridine 5'-monophosphate: a potential bifunctional inhibitor of thymidylate synthase.
Mercuration of 2'-deoxyuridine 5'-phosphate (dUMP) followed by alkylation with allylamine in the presence of K2PdCl4 afforded the 5-aminoallyl deoxynucleotide, which was isolated by sequential Dowex 50 H+ and DEAE-Sephadex chromatography. Further reaction of the product with the N-hydroxysuccinimide ester of methotrexate (MTX) in dry dimethyl sulfoxide gave an MTX-aminoallyl-dUMP covalent complex separable by DEAE-Sephadex chromatography. Reprecipitation with acid from basic solution offered further purification and the structure was confirmed by elemental analysis, NMR and absorbance spectra. The product was an inhibitor of rat liver dihydrofolate reductase (I50 approximately 250 nM, cf. MTX I50 approximately 60 nM) and Lactobacillus casei thymidylate synthase. With the latter enzyme, inhibition was competitive with both nucleotide and folate substrates (Ki = 2.6 and 3.5 microM, respectively) and partial enzyme-inhibitor binary complex could be detected by gel electrophoresis. Large fluorescence changes were observed on titration of the synthase with MTX-aminoallyl-dUMP and alterations in the UV difference spectra similar to those seen on titration of the enzyme with MTX were also noted. The compound was a poor growth inhibitor for cultured murine L1210 and human CCRF-CEM cell lines, which probably reflects low cellular uptake.
Stuhmiller
L M
LM
Department of Basic and Clinical Research, Research Institute of Scripps Clinic, La Jolla, CA 92037.
Nazarbaghi
R
R
Webber
S
S
Whiteley
J M
JM
D000818:Animals; D001692:Biological Transport; D002455:Cell Division / Q000187:drug effects; D003856:Deoxyuracil Nucleotides / Q000138:chemical synthesis / Q000493:pharmacokinetics / Q000494:pharmacology*; D005493:Folic Acid Antagonists; D006801:Humans; D007780:Lacticaseibacillus casei / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000493:pharmacokinetics / Q000494:pharmacology; D051381:Rats; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors*; D014407:Tumor Cells, Cultured / Q000187:drug effects
D003856:Deoxyuracil Nucleotides; D005493:Folic Acid Antagonists; C063734:methotrexate 5-aminoallyl-2'-deoxyuridine 5'-monophosphate; D013940:Thymidylate Synthase; D008727:Methotrexate