title: A potent multisubstrate analogue inhibitor of human thymidylate synthetase.
abstract: The synthesis of an 8-deazafolate analogue of the intermediate in the methylation of 2'-deoxyuridylate is described. Alkylation of diethyl 5,6,7,8-tetrahydro-8-deazafolate with 3'-O-acetyl-5-(bromomethyl)-2'-deoxyuridine 5'-[bis-(trichlorethyl) phosphate], followed by removal of the trichloroethyl groups with a Zn/Cu couple and mild saponification, gave the target inhibitor N-[4-[[[2-amino-3,4,5,6,7, 8-hexahydro-4-oxo-5-[(2'-deoxyuridin-5-yl)methyl]-pyrido[3,2-d] pyrimidin-6-yl]methyl]amino]benzoyl]-L-glutamic acid 5'-monophosphate. The free nucleoside and the 5'-(methyl phosphate) diester were similarly prepared. Each of these reactions yielded a pair of diastereoisomers about C-6 of the reduced deazafolate in approximately a 1:1 ratio. These diastereoisomeric mixtures were evaluated as inhibitors of thymidylate synthetase derived from human tumor (HeLa) cells. The 5'-monophosphate was a potent inhibitor, competitive with respect to both 2'-deoxyuridylate (Ki = 0.06 microM) and tetrahydrofolate (Ki = 0.25 microM). In contrast, the nucleoside and the nucleotide methyl ester were poorer inhibitors by more than 3 orders of magnitude, attesting to the importance of the anionic function at the nucleoside 5'-position in the affinity of an inhibitor for the enzyme active site.
mesh_terms: D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D007202:Indicators and Reagents; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008780:Methyltransferases / Q000037:antagonists & inhibitors*; D011485:Protein Binding; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors*; D014500:Uracil Nucleotides / Q000138:chemical synthesis*; D014542:Uridine Monophosphate / Q000031:analogs & derivatives / Q000138:chemical synthesis* / Q000494:pharmacology
chemical_list: D007202:Indicators and Reagents; D014500:Uracil Nucleotides; C043917:N-(4-(((2-amino-3,4,5,6,7,8-hexahydro-4-oxo-5-((2'-deoxyuridin-5-yl)methyl)pyrido(3,2-d)pyrimidin-6-yl)methyl)amino)benzoyl)glutamic acid 5'-monophosphate; D014542:Uridine Monophosphate; D008780:Methyltransferases; D013940:Thymidylate Synthase