SYNTHESIS AND INTERACTIONS WITH THYMIDYLATE SYNTHASE OF 2,4-DITHIO ANALOGS OF DUMP AND 5-FLUORO-DUMP

The 2,4-dithio analogues of 2'-deoxyuridine and 2'-deoxy-5-fluorouridi ne have been synthesized by thiation of the previously described 2-thi o analogues, and then phosphorylated enzymatic ally or chemically to y ield 2,4-dithio-dUMP and 2,4-dithio-5-fluoro-dUMP. In striking contras t to the 2-thio and 4-thio analogues of dUMP, which are good substrate s of thymidylate synthase, 2,4-dithio-dUMP is not a substrate. But, su rprisingly, it is a competitive inhibitor, relative to dUMP, of the pu rified enzymes from both parental and FdUrd-resistant L1210 cells, wit h K-i values of 32 mu M and 55 mu M, respectively. Although 2,4-dithio -5-fluoro-dUMP behaved as a typical slow-binding inhibitor of the enzy me, its K-i value was 10(3)-10(4)-fold higher than those for the corre sponding 2-thio and 4-thio congeners. Similarly, 2,4-dithio-FdUrd was a much weaker inhibitor of tumour cell growth (IC50 approximate to 10( -5) M) than FdUrd (IC50 approximate to 10(-9) M), 2-thio-FdUrd (IC50 a pproximate to 10(-7) M) or 4-thio-FdUrd (IC50 approximate to 5 . 10(-8 ) M), while with 2,4-dithio-dUrd no influence on cell growth could be observed. Theoretical considerations, based on calculated aromaticitie s of the uracil and thiouracil rings, suggest that lack of substrate a ctivity of 2,4-dithio-dUMP may result from increased pyrimidine ring a romaticity of the latter, leading to resistance of C(6) to nucleophili c attack by the enzyme active center cysteine.