5-substituted N4-hydroxy-2′-deoxycytidines and their 5′-monophosphates:: Synthesis, conformation, interaction with tumor thymidylate synthase, and in vitro antitumor activity

Convenient procedures are described for the synthesis of 5-substituted N-4-hydroxy-2'-deoxycytidines Ba,lb,d-h via transformation of the respective 5-substituted 3',5'-di-O-acetyl-2'-deoxyuridines la-c,e-h. These procedures involved site-specific triazolation or N-methylimidazolation at position C(4), followed by hydroxylamination and deblocking with MeOH-NH3. Nucleosides Ba,b,d-h were selectively converted to the corresponding 5'-monophosphates Ga,b,d-h with the aid of the wheat shoot phosphotransferase system. Conformation of each nucleoside in D2O solution, deduced from H-1 NMR spectra and confirmed by molecular mechanics calculations, showed the pentose ring to exist predominantly in the conformation S (C-2'-endo) and the N-4-OH group as the cis rotamer; Cell growth inhibition was studied with two L5178Y murine leukemia cell lines, parental and 5-fluoro-2'-deoxyuridine (FdUrd);resistant, the latter 70-fold less sensitive toward FdUrd than the former, With FdUrd-resistant L5178Y cells, 5-fluoro-N-4-hydroxy-2'-deoxycytidine (5e) caused almost 3-fold stronger growth inhibition than FdUrd; 5e was only some 3-fold weaker growth inhibitor of the resistant cells than of the parental cells. Thymidylate synthase inhibition was studied with two forms of the enzyme differing in sensitivities toward 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP), isolated from parental and FdUrd-resistant L1210 cell lines; All N-4-hydroxy-dCMP (6a,b,d-h) and dUMP analogues studied were competitive vs dUMP inhibitors of the enzyme. Analogues 6b,d-h and 5-hydroxymethyl-dUMP, similar to N4-hydroxy-dCMP (6a) and FdUMP, were also N-5,N-10-methylenetetrahydrofolate-dependent mechanism-based, slow-binding inhibitors. 6-Chlolo-dUMP, 5-bromo-dUMP, and 5-iodo-dUMP, similar to dTMP, did not cause a time-dependent inactivation of the enzyme. Instead, they behaved as classic inhibitors of tritium release from [5-H-3]dUMP. 5-Bromo-dUMP and 5-iodo-dUMP showed substrate activity independent of N-5,N-10-methylenetetrahydrofolate in the thymidylate synthase-catalyzed dehalogenation reaction. The =N-OH substituent of the pyrimidine C(4) prevented the enzyme-catalyzed release from the C(5) of Br- and I- (the same shown previously for H+). While FdUMP and 6a showed a higher affinity and greater inactivation power with the parental cell than FdUrd-resistant cell enzyme, an opposite relationship could be seen with 5-hydroxymethyl-dUMP.