5-Substituted N⁴-Hydroxy-2‘-deoxycytidines and Their 5‘-Monophosphates:  Synthesis, Conformation, Interaction with Tumor Thymidylate Synthase, and in Vitro Antitumor Activity

Abstract

Convenient procedures are described for the synthesis of 5-substituted N(4)-hydroxy-2'-deoxycytidines 5a,b,d-h via transformation of the respective 5-substituted 3', 5'-di-O-acetyl-2'-deoxyuridines 1a-c,e-h. These procedures involved site-specific triazolation or N-methylimidazolation at position C(4), followed by hydroxylamination and deblocking with MeOH-NH(3). Nucleosides 5a,b,d-h were selectively converted to the corresponding 5'-monophosphates 6a,b,d-h with the aid of the wheat shoot phosphotransferase system. Conformation of each nucleoside in D(2)O solution, deduced from (1)H 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 N(4)-hydroxy-dCMP (6a) and FdUMP, were also N(5), N(10)-methylenetetrahydrofolate-dependent, hence mechanism-based, slow-binding inhibitors. 5-Chloro-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-(3)H]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.

Ribonucleoside Analogue That Blocks Replication of Bovine Viral Diarrhea and Hepatitis C Viruses in Culture

“…In this hypothesis, NHC metabolites act with a cellular enzyme involved in the de novo synthesis of purine (most likely not GTP, as the activity was not reversed by purines) or pyrimidine triphosphates, resulting in the specific depletion of such natural NTPs. For comparison, a molecule closely related to NHC, the 2Ј-deoxy analogue of NHC in its 5Ј-monophosphate form (dNHC-TP), was reported to be a strong inhibitor of thymidylate synthase (TS) (EC 2.1.1.45) (15). When this compound was tested in the replicon system together with the TS inhibitors ␤-D-2Ј-deoxy-5-fluorouridine and ␤-D-5-fluorouridine, all three compounds were found inactive (⌬⌬Ct at day 4; EC 90 Ͼ 100 M) (data not shown), suggesting that TS inhibition does not influence HCV replicon levels.…”

Prodrug Activation by Cryptosporidium Thymidine Kinase

“…KP1308 and KP1309 were synthesized as previously described (17). The syntheses of KP330, KP332-4, and KP1283 are described in Ref.…”

X-ray Crystal and Ab Initio Structures of 3′,5′-di-O-Acetyl-N(4)-Hydroxy-2′-Deoxycytidine and Its 5-Fluoro Analogue: Models of the N(4)-OH-dCMP and N(4)-OH-FdCMP Molecules Interacting with Thymidylate Synthase

“…were synthesized according to the protocol described in Felczak et al [18]. Crystals of the both compounds were grown at room temperature by slow evaporation of the solvent containing saturated methanol and ethanol mixed at the molar ratio close to 1:2.…”

“…In addition, a series of various 5-substituted 1-methyl-N (4)-hydroxycytosines, investigated using molecular mechanics, exhibited invariantly very low populations of the trans form, with the hydroxyl pointed toward C(5), relatively to much higher populations of the cis conformer [18]. The population of the trans form at 298 K was estimated to be 2 × bond formation.…”

“…However, the subsequent ab initio studies proved that the intramolecular hydrogen bond would be too weak to considerably improve the internal stability of N(4)-hydroxy-5-fluorocytosine [17]. Furthermore, recent results of the molecular mechanics calculations indicated very similar populations of the trans conformer in 1-methyl-N (4)-hydroxycytosine and its C(5)-fluoro derivative [18]. Thus, although some interplay between the C(4)-and C(5) substituents may exist, the true reasons for a stronger inhibitory potency of the 5-fluoro congener of N (4)-OHdCMP against TS may likely lie elsewhere.…”