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

Krzysztof Felczak , Agnieszka Miazga , Jarosław Poznański , Maria Bretner , Tadeusz Kulikowski , Jolanta M. Dzik , Barbara Gołos , Zbigniew Zieliński , Joanna Cieśla , Wojciech Rode

Abstract

Convenient procedures are described for the synthesis of 5-substituted N4-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−NH3. 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 D2O solution, deduced from 1H NMR spectra and confirmed by molecular mechanics calculations, showed the pentose ring to exist predominantly in the conformation S (C-2‘-endo) and the N4-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-N4-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 N4-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 N5,N10-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 [5-3H]dUMP. 5-Bromo-dUMP and 5-iodo-dUMP showed substrate activity independent of N5,N10-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.
Author Krzysztof Felczak (PEC)
Krzysztof Felczak,,
- The Phisical Education Centre
, Agnieszka Miazga - [Institute of Biochemistry and Biophysics of the Polish Academy of Sciences]
Agnieszka Miazga,,
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, Jarosław Poznański - Department of Microbial Biochemistry, Institute of Biochemistry and Biophysics of the Polish Academy of Sciences, Poland [Polish Academy of Sciences (PAN)]
Jarosław Poznański,,
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, Maria Bretner (FC / IBC / DDTB)
Maria Bretner,,
- Department Of Drug Technology And Biotechnology
, Tadeusz Kulikowski - [Institute of Biochemistry and Biophysics of the Polish Academy of Sciences]
Tadeusz Kulikowski,,
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, Jolanta M. Dzik - [Nencki Institute of Experimental Biology of the Polish Academy of Sciences]
Jolanta M. Dzik,,
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, Barbara Gołos - [Nencki Institute of Experimental Biology of the Polish Academy of Sciences]
Barbara Gołos,,
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, Zbigniew Zieliński - [Nencki Institute of Experimental Biology of the Polish Academy of Sciences]
Zbigniew Zieliński,,
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, Joanna Cieśla (FC / IBC / DDTB)
Joanna Cieśla,,
- Department Of Drug Technology And Biotechnology
, Wojciech Rode - [Nencki Institute of Experimental Biology of the Polish Academy of Sciences]
Wojciech Rode,,
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Journal seriesJournal of Medicinal Chemistry, ISSN 0022-2623, [1520-4804]
Issue year2000
Vol43
No24
Pages4647-4656
Publication size in sheets0.5
ASJC Classification1313 Molecular Medicine; 3002 Drug Discovery
DOIDOI:10.1021/jm000975u
URL http://pubs.acs.org/doi/abs/10.1021/jm000975u
Languageen angielski
Score (nominal)45
Score sourcejournalList
Publication indicators Scopus Citations = 20; WoS Citations = 18; Scopus SNIP (Source Normalised Impact per Paper): 2000 = 1.752; WoS Impact Factor: 2006 = 5.115 (2) - 2007=5.054 (5)
Citation count*29 (2018-06-25)
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