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Journal ArticleDOI

3'-[4-Aryl-(1,2,3-triazol-1-yl)]-3'-deoxythymidine Analogues as Potent and Selective Inhibitors of Human Mitochondrial Thymidine Kinase

10 Mar 2010-Journal of Medicinal Chemistry (American Chemical Society)-Vol. 53, Iss: 7, pp 2902-2912

TL;DR: The synthesis of new thymidine analogues containing a 4- or 5-substituted 1,2,3-triazol-1-yl substituent at the 3'-position of the 2'-deoxyribofuranosyl ring is described.

AbstractIn an effort to increase the potency and selectivity of earlier identified substrate-based inhibitors of mitochondrial thymidine kinase 2 (TK-2), we now describe the synthesis of new thymidine analogues containing a 4- or 5-substituted 1,2,3-triazol-1-yl substituent at the 3′-position of the 2′-deoxyribofuranosyl ring. These analogues were prepared by Cu- and Ru-catalyzed cycloadditions of 3′-azido-3′-deoxythymidine and the appropriate alkynes, which produced the 1,4- and 1,5-triazoles, respectively. Selected analogues showed nanomolar inhibitory activity for TK-2, while virtually not affecting the TK-1 counterpart. Enzyme kinetics indicated a competitive and uncompetitive inhibition profile against thymidine and the cosubstrate ATP, respectively. This behavior is rationalized by suggesting that the inhibitors occupy the substrate-binding site in a TK-2−ATP complex that maintains the enzyme’s active site in a closed conformation through the stabilization of a small lid domain.

Topics: Thymidine kinase (62%), Uncompetitive inhibitor (56%), Nucleoside (54%), Thymidine (54%), Active site (52%)

Summary (1 min read)

Introduction of a -CH

  • Of an electron-withdrawing Cl in the para position of the phenyl (8f) significantly improved the inhibitory activity.
  • A similar effect had previously been observed in the thiourea series.
  • This revealed that 14b inhibited the enzyme in a purely competitive fashion and had Ki values as low as 0.012 µM.
  • Its Ki/Km ratios were markedly lower than 1 (0.011), pointing to an affinity for the enzyme that largely exceeds the affinity of the natural substrate.
  • Combination of the 3'-modification and the 5'-O-trityl substituent led, as expected, to an analogue that was completely devoid of affinity for TK-2.

Molecular modeling and str uctur e-activity r elationship

  • To gain insight into the mode of binding of this new class of inhibitors, docking experiments were undertaken using the previously reported homology-based model of TK-2 as the target.
  • This "lid loop" appears as a disordered region in many dNK crystal structures apparently due to its high mobility, and side-chain orientation varies depending on the nature of the molecules binding in the long active-site cleft and on the presence of an additional negative charge (e.g. sulfate ions) on the enzyme surface.
  • -the 3'-OH of the thymidine substrate can be held in place with the aid of two direct hydrogen bonds, one with the carboxylate of Glu201 and another one with the phenol of Tyr99.
  • O -rotation of the triazole ring relative to the sugar would lead to steric clash of the attached phenyl ring with the lid loop, and also that this model does not favor substitution at position 5 of the triazole ring, in good agreement with the much lower inhibitory activity measured for the 1,5-substituted derivatives 8i and 8j.
  • To reveal whether the TK-2 inhibitors are able to be taken up by the cells, the most active compound 14b was chosen for further studies.

Conclusions

  • Cycloaddition of organic azides and alkynes is the most direct route to 1,2,3-triazoles.
  • The authors used two different catalysts to achieve this reaction: the Cu(I) catalyst, which provided the 1,4- ] catalyst, which has recently been described for regioselective synthesis of 1,5-disubstituted 1,2,3-triazole systems.

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This item is the archived peer-reviewed author-version of:
Title: 3’-[4-Aryl-(1,2,3-triazol-1-yl)]-3’-deoxythymidine Analogues as Potent and Selective Inhibitors
of Human Mitochondrial Thymidine Kinase.
Authors: Van Poecke, Sara; Negri, Ana ; Gago, Federico ; Van Daele, Ineke ; Solaroli, Nicola ;
Karlsson, Anna ; Balzarini, Jan and Van Calenbergh, Serge
In: JOURNAL OF MEDICINAL CHEMISTRY, 53(7), 2902 2912 (2010), DOI 10.1021/jm901532h

1
3’-[4-Aryl-(1,2,3-triazol-1-yl)]-3’-deoxythymidine
Analogues as Potent and Selective Inhibitors of Human
Mitochondrial Thymidine Kinase.
Sara Van Poecke,
a
Ana Negri,
b
Federico Gago,
b
Ineke Van Daele,
a
Nicola Solaroli,
c
Anna Karlsson,
c
Jan Balzarini
d
and Serge Van Calenbergh
a,*
a
Laboratory for Medicinal Chemistry (FFW), Ghent University, 9000 Gent, Belgium
b
Departamento de Farmacologia, Universidad de Alcalá, E-28871 Alcalá de Henares, Madrid, Spain
c
Karolinska Institute, S-14157 Stockholm, Sweden
d
serge.vancalenbergh@ugent.be
Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required
according to the journal that you are submitting your paper to)
*Corresponding author: Laboratory for Medicinal Chemistry (FFW), UGent, Harelbekestraat 72, 9000
Ghent, Belgium. E-mail: Serge.VanCalenbergh@Ugent.be, Phone: +32 9 264 81 24. Fax + 32 9 264 81
46.
Abbreviations: TK: thymidine kinase; HSV: herpes simplex virus; VZV, varicella zoster virus; Dm:
Drosophila melanogaster; dNK: deoxynucleoside kinase; dThd: thymidine; AZT: azidothymidine.

2
Abstract In an effort to increase the potency and selectivity of earlier identified substrate-based
inhibitors of mitochondrial thymidine kinase 2 (TK-2), we now describe the synthesis of new thymidine
analogues containing a 4- or 5-substituted 1,2,3-triazol-1-yl substituent at the 3’-position of the 2’-
deoxyribofuranosyl ring. These analogues were prepared by Cu- and Ru-catalysed cycloadditions of 3'-
azido-3’-deoxythymidine and the appropriate alkynes, which produced the 1,4- and 1,5-triazoles,
respectively. Selected analogues showed nanomolar inhibitory activity for TK-2, while virtually not
affecting the TK-1 counterpart. Enzyme kinetics indicated a competitive and uncompetitive inhibition
profile against thymidine and the co-substrate ATP, respectively. This behavior is rationalized by
suggesting that the inhibitors occupy the substrate-binding site in a TK-2–ATP complex that maintains
the enzyme’s active site in a closed conformation through the stabilization of a small lid domain.
KEYWORDS: thymidine kinase 2; thymidine analogues; click chemistry.

3
Introduction
In mammalian cells, four different deoxynucleoside kinases can be found: thymidine (dThd) kinase 1
(TK-1), thymidine kinase 2 (TK-2), deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK). The
main role of these kinases is to convert deoxynucleosides to their monophosphates by γ-phosphoryl
transfer of ATP, an essential step in the biosynthesis of the DNA-building blocks. A second fundamental
role lies in the activation of nucleoside analogues with pharmacological (anticancer and antiviral)
properties.
Among these mammalian deoxynucleoside kinases, two enzymes phosphorylate thymidine (dThd),
TK-1 and TK-2. The main differences between these two kinases with respect to amino acid sequences,
substrate specificities, localization and levels of expression during the different cell cycle phases are
summarized in Table 1.
1, 2
Mitochondrial DNA (mtDNA) replication takes place throughout the whole
cell cycle, thus constantly requiring deoxynucleoside triphosphates for mtDNA synthesis. Being active
in non-proliferating tissues, TK-2 provides the nucleotides for mtDNA synthesis. Consequently, TK-2
deficiency leads to mitochondrial disorders, designated as mtDNA depletion syndromes, mostly
affecting skeletal muscles.
3
Besides the mitochondrial disorders linked to TK-2 deficiency, severe mitochondrial toxicity is also
associated to long-term treatment with antiviral nucleoside analogues such as AZT.
4,5
Although the
mechanism by which these nucleoside analogues exert their mitochondrial toxicity is not fully
understood, it has been suggested that after phosphorylation of the nucleoside analogues by TK-2, their
triphosphates accumulate in the mitochondria. In the case of AZT, phosphorylation in non-replicating
cells by TK-2 is significant, despite the fact that it is not an ideal substrate for TK-2. The accumulation
of AZT-TP is suggested to affect DNA-polymerase-γ, resulting in mtDNA depletion.
Likewise, mitochondrial toxicity is a major concern in the development of new nucleoside drugs as
exemplified back in 1993 by the halting of a clinical trial of fialuridine (FIAU) because patients
developed serious liver and kidney toxicity, later found to originate from incorporation of the drug into
mitochondrial DNA.

4
TK-2 inhibitors can be a valuable tool to answer the many open questions regarding the real
contribution of TK-2 in the maintenance and homeostasis of mitochondrial dNTP pools and to clarify
the role of this enzyme in the mitochondrial toxicity of a variety of antiviral and anticancer drugs.
Despite the lack of a crystal structure of TK-2 for structure-based inhibitor design, several TK-2
inhibitors have been identified in the past (Chart 1). A noteworthy example is the ribonucleoside 5-(
E
)-
(2-bromovinyl)uridine (1;
K
i
= 10.4 µM), whose 2’-deoxy congener is an alternative substrate for the
enzyme.
6
Another study describes nucleosides modified at the sugar moiety, including 3’-
O
-alkyl
analogues and 3’-hexanoylamino-3’-deoxythymidine 2, a very potent inhibitor of TK-2 (
K
i
= 0.15 µM).
7
While 1-β-D-arabinofuranosylthymine (Ara-T) and (
E
)-5-(2-bromovinyl)-1-β-D-arabinofuranosyluracil
(BVaraU) represent good substrates for TK-2, the introduction of long chain acyl substituents at the 2’-
OH (as in 3; IC
50
= 6.3 µM) turned these substrates into potent inhibitors. Unfortunately, these 2’-
O
-
acyl derivatives cannot be used as tools to study TK-2 in intact cells because they are unstable in cell
culture and readily converted to the parent nucleoside.
After the identification of 5’-
O
-trityl-thymidine as a moderately active inhibitor of TK-2 (IC
8
50
= 33
µM), Pérez-Peréz et al. replaced the sugar moiety of this nucleoside by acyclic spacers to tether the
thymine base to a distal triphenylmethoxy moiety.
9
Elaborate optimization of the TK-2 inhibitory
activity of 1-[(
Z
)-4-(triphenylmethoxy)-2-butenyl]thymine 4 (IC
50
= 1.5 µM) yielded the acyclic
analogue 5 with an IC
50
-value of 0.4 µM.
Recently, we evaluated two series of thymidine analogues, which had been originally designed as
M.
tuberculosis
thymidylate kinase inhibitors, for their inhibitory activity against a panel of other
nucleoside kinases (TK-1, TK-2, HSV-1 and VZV TK).
10
11
Several substituted 3’-thiourea derivatives of
β-dThd proved highly inhibitory to and selective for human mitochondrial TK-2 compared to the other
enzymes. Compound 6, which emerged as the most potent analogue of this series, inhibited TK-2 at
concentrations 2,100-fold lower than those required to inhibit cytosolic TK-1 (IC
50
: TK-1: 316 µM; TK-
2: 0.15 µM). Kinetic experiments indicated that this inhibitor specifically binds to the enzyme-ATP

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