1-[(2-Hydroxyethoxy)methyl]-
5-fluorouracil (
HEMFU) and 1-[(1,3-dihydroxy-2-propoxy)methyl]-
5-fluorouracil (
DHPFU) were prepared by alkylation of the di-O-TMS derivative of
5-fluorouracil and phosphorylated with the use of the wheat shoot
phosphotransferase system to their monophosphates, HEMFUMP and DHPFUMP. 1-(2-Phosphonylmethoxyethyl)-5-fluorouracil (PMEFU) was obtained by condensation of diethyl-2-chloroethoxymethanephosphonate with
5-fluorouracil and cleavage of the alkylphosphoester with
trimethylbromosilane. Inhibition of highly purified
thymidylate synthase from mouse tumour Ehrlich
carcinoma and
leukemia L1210 cells by each of the
nucleotide analogues, DHPFUMP, PMEFU and HEMFUMP, and of L5178Y mouse
leukemia cell growth by the
nucleoside (
HEMFU) analogue, were studied. DHPFUMP proved to be the strongest inhibitor, non-competitive vs dUMP, with K(i)app 2.8 microM for time-independent interaction with the
enzyme and N5,N10-methylenetetrahydrofolate (CH2H4PteGlu). In the presence of CH2H4PteGlu, DHPFUMP exhibited time-dependent inactivation of the
enzyme, the inactivation rate plots being biphasic and pointing to Ki values in the microM range (10(3)-fold higher than for 5-fluoro-dUMP). HEMFUMP and PMEFU were much weaker inhibitors of the
enzyme, with K(i)app values of 0.26 mM (non-competitive vs dUMP) and 30 mM (non-competitive vs dUMP), respectively.
HEMFU, despite the weak interaction of its
nucleotide analogue with the
enzyme, proved to be a strong cell (L5178Y)
growth inhibitor, with IC50 in the range 10(-5) M.