A Potent Multisubstrate Analogue Inhibitor of Human Thymidylate Synthetase

Article abstract of DOI:10.1021/jm00378a031

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'-, 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 similary prepared.Each of these reactions yielded a pair of diastereoisomers about C-6 of the reduced deazafolate in approximately a 1:1 ratio.These diastereomeric 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 (K_i = 0.06 μM) and tetrahydrofolate (K_i = 0.25 μM).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.