A potent multisubstrate analog inhibitor of human thymidylate synthetase

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Abstract
The synthesis of an 8-deazafolate analog of the intermediate in the methylation of 2''-deoxyuridylate is described. Alkaylation 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 [potential antitumor agents] 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 .mu.M) and tetrahydrofolate (Ki = 0.25 .mu.M). 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.