Felczak K, Gołos B, Dzik J M, Rode W, Bretner M, Shugar D, Kulikowski T
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw.
Acta Biochim Pol. 1998;45(1):75-82.
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.
1-[(2-羟基乙氧基)甲基]-5-氟尿嘧啶（HEMFU）和1-[(1,3-二羟基-2-丙氧基)甲基]-5-氟尿嘧啶（DHPFU）通过5-氟尿嘧啶的二-O-三甲基硅烷基衍生物的烷基化反应制备，并利用小麦芽磷酸转移酶系统将其磷酸化为单磷酸盐，即HEMFUMP和DHPFUMP。1-(2-膦酰甲氧基乙基)-5-氟尿嘧啶（PMEFU）通过2-氯乙氧基甲基膦酸二乙酯与5-氟尿嘧啶缩合，并用三甲基溴硅烷裂解烷基磷酸酯得到。研究了核苷酸类似物DHPFUMP、PMEFU和HEMFUMP对小鼠肿瘤艾氏腹水癌和白血病L1210细胞中高度纯化的胸苷酸合成酶的抑制作用，以及核苷（HEMFU）类似物对L5178Y小鼠白血病细胞生长的抑制作用。结果表明，DHPFUMP是最强的抑制剂，对dUMP呈非竞争性抑制，与酶和N5,N10-亚甲基四氢叶酸（CH2H4PteGlu）的非时间依赖性相互作用的K(i)app为2.8 microM。在CH2H4PteGlu存在下，DHPFUMP表现出对酶的时间依赖性失活，失活速率曲线呈双相，表明其Ki值在 microM范围内（比5-氟-dUMP高10(3)倍）。HEMFUMP和PMEFU对该酶的抑制作用较弱，K(i)app值分别为0.26 mM（对dUMP呈非竞争性抑制）和30 mM（对dUMP呈非竞争性抑制）。尽管HEMFU的核苷酸类似物与酶的相互作用较弱，但它被证明是一种强力的细胞（L5178Y）生长抑制剂，IC50在10(-5) M范围内。
