Synthesis and inhibitory activity of thymidine analogues targeting Mycobacterium tuberculosis thymidine monophosphate kinase

Article abstract of DOI:10.1016/j.bmc.2011.10.021

We report on Mycobacterium tuberculosis thymidine monophosphate kinase (TMPKmt) inhibitory activities of a series of new 3′- and 5′-modified thymidine analogues including α- and β-derivatives. In addition, several analogues were synthesized in which the 4

Full text of DOI:10.1016/j.bmc.2011.10.021

Bioorganic & Medicinal Chemistry 19 (2011) 7603–7611
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and inhibitory activity of thymidine analogues targeting
Mycobacterium tuberculosis thymidine monophosphate kinase
Sara Van Poecke a, Hélène Munier-Lehmann b,c, Olivier Helynck b,c, Matheus Froeyen d,
Serge Van Calenbergh a,
⇑
a Laboratory for Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium
b Institut Pasteur, Unité de Chimie et Biocatalyse, 28 rue du Dr Roux, 75724 Paris Cedex 15, France
c CNRS URA2128, 28 rue du Dr Roux, France
d Rega Institute for Medical Research, Minderbroedersstraat 10, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
We report on Mycobacterium tuberculosis thymidine monophosphate kinase (TMPKmt) inhibitory activities
of a series of new 30- and 50-modified thymidine analogues including
a- and b-derivatives. In addition,
Received 13 August 2011
Revised 6 October 2011
Accepted 7 October 2011
Available online 18 October 2011
several analogues were synthesized in which the 4-oxygen was replaced by a more lipophilic sulfur atom
to probe the influence of this modification on TMPKmt inhibitory activity. Several compounds showed an
inhibitory potency in the low micromolar range, with the 50-arylthiourea 4-thio-
a-thymidine analogue
being the most active one (Ki = 0.17
l
M). This compound was capable of inhibiting mycobacteria growth
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
TMPKmt
Thymidine analogues
at a concentration of 25 g/mL.
l
a- and b-Nucleosides
Spectrophotometric binding assay
Inhibitory activity
1. Introduction
replication of bacteria. TMPK acts at the junction of the de novo
and salvage pathways for the synthesis of deoxythymidine triphos-
phate (dTTP), which is indispensable for growth and survival. There-
fore, TMPK represents a promising target for developing new TB
drugs. Experiments with TMPK-deficient mutant of Saccharomyces
cerevisiae underscore the criticality of this enzyme for DNA replica-
tion and cellular growth.5
Although the global folding of TMPKmt is similar to that of
other TMPKs, the configuration of its active site is unique. Com-
pared to the human isozyme TMPKmt is peculiar in that it is com-
Worldwide, tuberculosis (TB) remains one of the leading causes
of death from infectious diseases. About one third of the world’s
population is infected with Mycobacterium tuberculosis that causes
TB. On average 5–10% of these carriers become sick or infectious at
some time during their life. Annually, more than 9 million new
cases are reported and TB claims almost 2 million lives each year.1
TB forms a lethal combination with HIV, each speeding the
other’s progress. TB is a leading cause of HIV-related deaths world-
wide. In 2008, there were an estimated 1.4 million new cases of TB
among persons with HIV infection and TB accounted for 23% of
AIDS-related deaths. The global resurgence of TB due to HIV infec-
tion and the rapid emergence of multidrug-resistant (MDR) and
extensively drug-resistant (XDR) strains of TB bacilli underscore
the importance of developing new antimycobacterial drugs against
TB.2
petitively inhibited by AZT-MP (Ki = 10 lM), making the latter an
attractive starting point for the design of selective inhibitors.3,6
On the basis of the structure of a dinucleoside 1 (Chart 1), dis-
covered by chance to produce significant inhibition of TMPKmt
(Ki = 37
atives of b-
(Ki = 5.0
M).8 Modeling experiments suggested a binding mode
l
M),7 we have prepared a series of 30-C-arylthiourea deriv-
D-thymidine, which led to the arylthiourea analogue 2
l
Recently, thymidine monophosphate kinase of M. tuberculosis
(TMPKmt)3 was put forward as an attractive target for new antitu-
berculosis agents.4 TMPK catalyzes the conversion of dTMP to dTDP
using ATP as phosphate donor and is crucial for maintaining the
thymidine triphosphate pools required for DNA synthesis and
for these 30-C-arylthiourea analogues that differs from that of the
natural substrate in that the sugar ring of the thymidine moiety
is tilted over 180° compared to that of dTMP, thereby positioning
the aromatic 30-substituent into the phosphoryl donor binding area
and the nucleobase below the sugar plane (Fig. 1).
This unusual binding mode led us to explore if an alternative
sugar scaffold could be used to impose a similar relative orienta-
tion of the thymine and the phenylthiourea moieties for TMPKmt
⇑
Corresponding author. Tel.: +32 09 264 81 24; fax: +32 09 264 81 46.
E-mail address: serge.vancalenbergh@ugent.be (S. Van Calenbergh).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.10.021

7604
S. Van Poecke et al. / Bioorg. Med. Chem. 19 (2011) 7603–7611
O
O
O
N
NH
NH
NH
N
O
HO
O
N
O
CF3
O
HO
S
O
O
OH
O
Cl
Cl
S
β
α
HN
N
O
O
N
N
N
N
H
H
F3C
N
N
O
H
H
H
H
OH
1: Ki (TMPKmt) = 37 μM
2: Ki (TMPKmt) = 5.0 μM
3: Ki (TMPKmt) = 0.6 μM
Chart 1.
H
O
N
O
F3C
Cl
F3C
Cl
5'
5'
O
O
3'
N
H
H
N
NH
HO
N
NH
HO
T
T
O
3'
NH
5'
3'
S
S
H
N
=
Cl
F3C
HO
α-nucleoside 3
5'-area
S
β-nucleoside 2
Figure 1. Suggested inverse sugar binding of 30-C-arylthiourea-modified b-thymidine
50-deoxy-50-arylthiourea modified
-thymidine 3.
2 and anticipated similar relative orientation of the colored moieties in
a
inhibition. It was hypothesized that an
a
-nucleoside in which the
(e.g., 7, 10, 18 and 19) to probe the influence of this modification
on TMPKmt inhibitory activity.
50-position served as the thiourea anchor might fulfill this crite-
rion. From a small library of easily accessible 50-N-arylthiourea
derivatives of
TMPKmt inhibitors to date with a Ki of 0.6
(vs TMPKh) of 600, and good inhibitory activity on the growing
Mycobacterium bovis (MIC99 20 g/mL) and M. tuberculosis (39%
inhibition at 6.25
g/mL) strains.8 Next to the relative orientation
between the aryl moiety and the nucleobase, structural exploration
a
-thymidine, 3 emerged as one of the most potent
2. Results and discussion
2.1. Chemistry
lM, a selectivity index
l
l
With the exception of compounds 7,10, 14–16, 18–20 and 22, the
chemical synthesis of all other final compounds has been reported
before.9–11 For the preparation of 4-thio-AZT (7), 50-O-acetylated
AZT 2313 was treated with Lawesson’s reagent to generate the cor-
responding 4-thio pyrimidine 24, followed by hydrolysis of the
acetate ester (Scheme 1). A CuAAC reaction14,15 between 23 and
1-chloro-4-ethynylbenzene, followed by thionation of the result-
ing 1,4-disubstituted 1,2,3-triazole 25 gave analogue 10 after final
deprotection.
of the
a-thymidine derivatives revealed the importance for
aromatic residues at the 50-position and the positive impact of
electronic-withdrawing and lipophilic substituents on the aryl
moiety for optimal inhibition of TMPKmt.
In this contribution we report on the TMPKmt inhibitory activ-
ities of a series of new thymidine analogues. Analogues 4 and 5
represent close analogues of 30-C-arylthiourea 2, in which the
methylene group between C-30 and the (thio)urea group has been
omitted.9 Analogues 8–11, derived from AZT (6), were selected to
investigate if a 1,4-disubstituted 1,2,3-triazole motif can act as a
bioisostere for the 30-C-thiourea linker of 2 as previously found
to be the case for TK-2 inhibition.10 The aminotetrazole isomers
12 and 13 were recently synthesized in the context of TK-2 inhibi-
tion11 and are also characterized by the presence of a heterocyclic
linker to connect the aromatic moiety to position 3 of the 20-deoxy-
ribofuranose ring.
Coupling of amine 2716 with 4-chloro-3-(trifluoromethyl)phenyl
isothiocyanate gave thiourea derivative 28 which was deprotected
using TBAF in THF. A mercury(II)-promoted reaction of 28 with
NaN3 and Et3N gave access to aminotetrazole 15 after final desilyla-
tion (Scheme 2).11
The synthesis of the 50-substituted b-thymidine analogue 16
started from 50-azido-50-deoxy-b-thymidine17 (Scheme 3). ‘Click
chemistry’ followed by HPLC purification allowed to isolate enough
pure material of 16 for testing.
For the synthesis of compounds 18 and 19, 30,50-O-diacetyl-
a-D-
To assess the inhibitory activity of anomeric variants of 3, its
b-anomer 14, as well as two heterocyclic analogues 15 and 16
are included in this study. Compounds 18–20 are derived from
thymidine 3118 was first thionated using Lawesson’s reagent fol-
lowed by deprotection and conversion to the monomesylate ester
34 (Scheme 4). Upon treatment with NaN3, 34 was converted into
azide 35, which was reduced to afford the 50-amino-50-deoxy-4-
50-azido-50-deoxy-
a-D-thymidine (17) and synthesized in an effort
to improve the activity of compound 3. To further investigate the
influence of the relative orientation between the aryl moiety and
thio-a-D-thymidine 36. Final treatment of this amine with the
the nucleobases, compound 22, which is the
a-analogue of 9,
appropriate isothiocyanate analogues afforded derivatives 18 and
was synthesized and evaluated.
19.
Starting from 50-azido-50-deoxy- -thymidine 17,8 compound
a-D
Based on earlier reports of 4-thiothymidine analogues showing
promising antimycobacterial potency against M. bovis and M.
tuberculosis in vitro and thus capable of entering the bacillus,12 sev-
eral analogues were synthesized in which the 4-oxygen of the
thymine moiety was replaced by a more lipophilic sulfur atom
20 was synthesized using the same method as described for tria-
zole 16 (Scheme 5).
The synthesis of 30-modified
a
-thymidine analogue 21 started
with the anomerisation of 50-O-acetylated AZT 36.13 Deprotection

S. Van Poecke et al. / Bioorg. Med. Chem. 19 (2011) 7603–7611
7605
O
N
S
S
N
O
N
O
NH
NH
NH
NH
NH
O
N
O
O
N
O
N
N
N
O
AcO
HO
AcO
Cl
N3
O
O
O
a
b
O
O
a
N3
N3
N3
OH
16
OH
30
23
24
7
c
Scheme 3. Reagents and conditions: (a) 1-chloro-4-ethynylbenzene, CuSO4ꢀ5H2O,
sodium ascorbate, H2O/t-BuOH 1:2, rt, 7 d, 2%.
O
N
S
S
NH
NH
NH
O
N
O
N
O
while this trend was less pronounced with the thiourea analogue
5. In the 1,4-substituted 1,2,3-triazole series, the anti-TMPKmt
activity was clearly influenced by the nature of the substituent at
C-4 of the triazole. The click product of AZT and phenylacetylene
(8) proved to be more potent than AZT itself. p-Chloro-substitution
of the phenyl ring of 8 or introduction of a methylene between the
triazole and the phenyl caused a moderate increase in activity (11).
In this series of 30-modified thymidine analogues, replacement of
the oxygen at position 4 of the thymine moiety by a sulfur typically
led to a significant drop in affinity for the target enzyme (compare
couples 6/7 and 9/10). Compounds 12 and 13, both containing a
1,5-disubstituted tetrazole, significantly differed in their capacity
to inhibit TMPKmt. The aminotetrazole analogue 13, in which the
tetrazole ring is directly attached to the sugar ring, showed a sig-
nificantly better activity compared to analogue 12 in which the
tetrazole ring is connected to C-30 via a NH-bridge. Remarkably,
an opposite trend was observed for these tetrazole analogues on
mitochondrial thymidine kinase 2.11
AcO
AcO
HO
d
e
O
O
O
N
N
N
N
N
N
N
N
N
Cl
Cl
Cl
25
26
10
Scheme 1. Reagents and conditions: (a) Lawesson’s reagent, toluene, 80 °C,
overnight, 20%; (b) 7 N NH3 in MeOH, rt, 6 h, 42%, (c) 1-chloro-4-ethynylbenzene,
CuSO4ꢀ5H2O, sodium ascorbate, H2O/t-BuOH 2:1, rt, 24 h, 41%; (d) Lawesson’s
reagent, toluene, 80 °C, overnight, 49%; (e) 7 N NH3 in MeOH, rt, 6 h, 66%.
of 37 followed by CuAAC with 1-chloro-4-ethynylbenzene afforded
triazole 22 in moderate yield (Scheme 6).
The inhibitory activity of a series of 50-modified b-thymidine ana-
logues appeared to be weak. Introduction of a 3-CF3-4-Cl-phenyl-
thiourea substituent (14) gave micromolar inhibition, while
replacement of the thiourea by a 1-(3-CF3-4-Cl-phenyl)-tetrazol-
5-amine (15) or a 4-(p-chlorophenyl)-triazol-1-yl (16) caused a 5
and 14-fold drop in Ki value, respectively. Comparison of the ano-
2.2. Biological evaluation
All compounds have been evaluated for TMPKmt inhibition as
described in the Section 4 and results are summarized in Table 1.
Replacement of the 30-azido group of AZT (6) by a 3-CF3-4-Cl-phe-
nylurea substituent (4) resulted in a 10-fold increased activity,
meric couples 3/14 and 16/20 demonstrate that the
a-anomers,
O
O
NH
NH
S
NH NH
N
O
N
O
Cl
H2N
O
O
a
F3C
OTBDMS
OTBDMS
28
27
c
b
O
O
N
O
N
NH
NH
NH
O
N
O
O
N
N
N
N
H
N
H
N
N
S
N
N
N
O
O
Cl
NH NH
b
O
OTBDMS
OH
F3C
OH
CF3
CF3
Cl
Cl
14
29
15
Scheme 2. Reagents and conditions: (a) 4-chloro-3-(trifluoromethyl)phenyl isothiocyanate, DMF, 0 °C?rt, 1 h, 76%; (b) 1 M TBAF in THF, THF, rt, 1 h, 53–60%; (c) NaN3, HgCl2,
Et3N, DMF, 0 °C?rt, overnight, 83%.

7606
AcO
S. Van Poecke et al. / Bioorg. Med. Chem. 19 (2011) 7603–7611
AcO
HO
O
N
O
O
O
AcO
NH
a
b
O
N
O
N
O
N
O
OAc
OAc
OH
O
N
O
a
NH
NH
NH
N3
AcO
b
O
NH
O
S
S
N3
36
O
31
32
33
37
N3
MsO
HO
HO
O
O
O
O
e
c
d
N
O
N
O
N
O
OH
OH
N
O
N
N
N
N3
c
NH
NH
NH
NH
S
S
O
O
34
35
22
21
Cl
H2N
Scheme 6. Reagents and conditions: (a) acetic anhydride, H2SO4, CH2Cl2, rt, 2 h, 20%
(b) 7 N NH3 in MeOH, rt, overnight, 72%, (c) 1-chloro-4-ethynylbenzene,
CuSO4ꢀ5H2O, sodium ascorbate, H2O/t-BuOH 1:3, rt, 24 h, 47%.
R1
NH NH
S
O
O
R2
f
N
O
OH
N
S
O
OH
NH
NH
S
In addition,
a-analogue 22, which was synthesized to further
assess the influence of the relative orientation between the aryl
moiety and the nucleobases, showed poor inhibitory activity
against TMPKmt, indicating that the preferred trans-orientation
of the base and the aromatic substituent also holds for the 30-mod-
ified analogues.
36
18: R1 = H, R2 = H
19: R1 = Cl, R2 = CF3
Scheme 4. Reagents and conditions: (a) Lawesson’s reagent, anhydrous 1,4-
dioxane, reflux, 4 h; (b) 7 N NH3 in MeOH, rt, 4 h, 37% over 2 steps; (c) MsCl,
pyridine, ꢁ78 °C?0 °C, 1 h, 70%; (d) NaN3, DMF, 60 °C, overnight, 89%; (e) PPh3,
THF, H2O, rt, 1 d, 89%; (f) appropriate isothiocyanate, DMF, 0 °C?rt, 3 h, 42–69%.
Compound 19 was evaluated for its in vitro inhibitory activity
against M. bovis BCG. It showed 100% inhibition of bacterial growth
at a concentration of 25 lg/mL.
In an effort to rationalize the threefold increase in affinity upon
replacement of the 4-O of the original hit 3 by a 4-S (19), both com-
pound were docked into the substrate binding site of the TMPKmt
enzyme (Fig. 2).8 The interactions of the base moiety of inhibitor 3
with the surrounding residues are similar to those observed in the
dTMP-TMPKmt complex6: a stacking with Phe70, H-bond of base
atom N3 with Asn100 and base atom O4 hydrogen bonding with
Arg74. An additional hydrogen bond between O30 and Tyr39 may
explain the better activity of 3 compared to its 30-deoxygenated
analogue.8 In compound 19 where the C(4)@O is replaced by a
C(4)@S, the hydrogen bonds to Arg74 is lost. However, due to the
bigger size of the sulfur atom, a better van der Waals interaction
is seen with surrounding residues Phe70, Arg74 and Asn100 which
may explain a higher affinity for this inhibitor.
N3
N
N
N
O
Cl
O
N
O
a
OH
N
O
OH
NH
NH
O
O
17
20
Scheme 5. Reagents and conditions: (a) 1-chloro-4-ethynylbenzene, CuSO4ꢀ5H2O,
sodium ascorbate, H2O/t-BuOH 2:1, rt, 4 d, 31%.
which feature a trans orientation of the nucleobase and the 50-sub-
stituent, exhibit superior TMPKmt inhibition compared to their b-
epimers (factor 22–24).
3. Conclusions
The 50-modified
a-thymidine analogues (3, 17–20) demon-
On the basis of the structures of nucleosides 2 and 3, which
were identified earlier as potent TMPKmt inhibitors, this paper
describes the synthesis and biological evaluation of a series of
strated moderate to excellent inhibitory activity for TMPKmt. Also
in this small series, the activity is influenced by the nature of the
substituent at the 50-position. Derivative 17, containing an azide
function, gave moderate inhibition with a strikingly comparable
new thymidine analogues, including a- and b-derivatives. In both
the 30- and the 50-derivatised analogues, the anomer that places
the thymine base trans to the aromatic substituent showed the
best TMPKmt inhibition. In addition, several analogues were syn-
thesized in which the 4-oxygen was replaced by a more lipophilic
sulfur atom to probe the influence of this modification on TMPKmt
inhibitory activity. Remarkably, a 4-thio modification of the pyrim-
Ki value (26.5 lM) as its AZT counterpart (28 lM). As observed in
the 30-modified b-series, conversion of the 50-azide moiety by a
4-(p-chlorophenyl)-1,2,3-triazol-1-yl substituent improved the
inhibitory activity, although to a lesser extent. Most interestingly
and in contrast to what was observed in the 30-modified b-series,
substitution of the 4-O of the original hit 3 by a 4-S, increased
the activity by a factor 3, ranking compound 19 amongst the most
potent TMPKmt inhibitors together with the (Z)-butenylthymines
with a naphtholactam or naphthosultam moiety at position 4 (Ki
idine base was favorable for the 50-modified
a-analogues, while it
caused an opposite effect the 30-modified b-analogues. Several
compounds showed an inhibitory potency in the low micromolar
range, with the 50-arylthiourea 4-thio-
a-thymidine analogue 19
values of 0.42 and 0.27 l
M, respectively).19
being the most active one (Ki = 0.17
lM). This compound is capable

S. Van Poecke et al. / Bioorg. Med. Chem. 19 (2011) 7603–7611
7607
Table 1
Kinetic parameters of TMPKmt with compounds 3–22
O
X
NH
R
NH
HO
O
O
N
O
N
O
R
HO
N
O
O
N
X
O
R
OH
O
O
NH
NH
OH
14-16
R
3, 17-20
4-13
21-22
Compound
X
R
Ki (l
M) TMPKmt
Ki (
lM) TMPKh
SI (Ki TMPKh/Ki TMPKmt)
MIC99 M. bovis (lg/mL)
3
4
5
O
O
O
O
S
O
O
S
O
O
O
O
O
O
O
S
3-CF3-4-Cl-Phenylthiourea
3-CF3-4-Cl-Phenylurea
3-CF3-4-Cl-Phenylthiourea
N3
0.6
2.8
9.9
—
95
—
—
—
—
—
—
—
34
—
—
—
—
—
—
>100
—
>100
—
—
—
—
—
>100
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
6 (AZT)
7
8
28
P100
4.2
2.1
15
2.7
45
N3
4-(Phenyl)-triazol-1-yl
4-(p-Chlorophenyl)-triazol-1-yl
4-(p-Chlorophenyl)-triazol-1-yl
4-(Benzyl)-triazol-1-yl
1-(3-CF3-4-Cl-Phenyl)-tetrazol-5-amine
5-(Aminobenzyl)-tetrazol-1-yl
3-CF3-4-Cl-Phenylthiourea
1-(3-CF3-4-Cl-Phenyl)-tetrazol-5-amine
4-(p-Chlorophenyl)-triazol-1-yl
N3
9
10
11
12
13
14
15
16
17
18
19
20
21
22
N.Ib
—
2.3
14.5
73
N.Ib
—
—
—
—
201
26.5
N. Ia
0.17
9
6
35
Phenylthiourea
—
>>100
25
—
—
—
S
3-CF3-4-Cl-Phenylthiourea
4-(p-Chlorophenyl)-triazol-1-yl
N3
N.Ia
—
O
O
O
—
—
4-(p-Chlorophenyl)-triazol-1-yl
N.I.: no inhibition detected at a final concentration of (a) 0.05 mM and (b) 1 mM.
Figure 2. Compound 3 (a) and compound 19 (b) docked in the substrate binding site of TMPKmt. The carbon atoms of both inhibitors are colored green. The enzyme contact
surface is colored cyan, and the contact surface with the 4S atom is colored magenta.
of inhibiting M. bovis at a concentration of 25
TMPKmt as an attractive target for further inhibitor design.
l
g/mL, promoting
ECOM 6122 photometer and a wavelength of 334 nm. The reaction
medium (0.5 mL final volume) contained 50 mM Tris–HCl, pH 7.4,
50 mM KCl, 2 mM MgCl2, 0.2 mM NADH, 1 mM phosphoenol pyru-
vate, and 2 units each of lactate dehydrogenase, pyruvate kinase,
and nucleoside diphosphate kinase. The concentrations of ATP
and dTMP were kept constant at 0.5 and 0.05 mM, respectively,
whereas the concentrations of analogues varied between 0.003
and 1.5 mM. Eq. 1 was used to calculate the Ki values using
Eqs. 2 and 3 (classical competitive inhibition model following the
Lineweaver–Burk representation):
4. Experimental section
4.1. Spectrophotometric binding assay
TMPKmt activities were determined using the coupled spectro-
photometric assay described by Blondin et al.20 using an Eppendorf

7608
S. Van Poecke et al. / Bioorg. Med. Chem. 19 (2011) 7603–7611
J = 5.7 Hz, J = 7.2 Hz, H-10), 7.58 (1H, s, H-6), 12.75 (1H, s, 3-NH).
Km½Iꢂ
Ki ¼
ð1Þ
ð
ꢁ 1ÞðKm þ ½SꢂÞ
13C NMR (75 MHz, DMSO-d6): d 16.78 (5-CH3), 20.58 (OAc), 35.96
(C-20), 59.81 (C-30), 63.09 (C-50), 81.10 (C-40), 84.64 (C-10), 117.95
(C-5), 133.32 (C-6), 147.67 (C-2), 171.92 (OAc), 190.95 (C-4). Exact
mass (ESI-MS) for C12H16N5O4S [M+H]+ found, 326.0941; calcd
326.0918.
v
vi
Vm½Sꢂ
v
¼
ð2Þ
ð3Þ
½Sꢂ þ Km
Vm½Sꢂ
vi
¼
½Sꢂ þ Kmð1 þ K½IꢂÞ
i
4.3.2. 30-Azido-30-deoxy-4-thio-b-
D-thymidine (7)
where
absence and in the presence of the analogue at a concentration va-
lue [I]; Km is the Km for dTMP (4.5 M for TMPKmt and 5 M for
TMPKh); [S] is the concentration of dTMP (50 M). For each com-
v and vi are the reaction velocities, respectively, in the
Compound 24 (16 mg, 0.050 mmol) was dissolved in a 7 N NH3
in MeOH solution (1 mL) and stirred at room temperature for 6 h.
The reaction mixture was concentrated in vacuo and the residue
was purified on a silica gel column using CH2Cl2/MeOH (95:5) as
the eluent to afford compound 7 as a yellow powder (6.0 mg,
42%). 1H NMR (300 MHz, DMSO-d6): d 1.97 (3H, d, J = 0.6 Hz,
5-CH3), 2.29–2.38 (1H, m, H-20a), 2.41–2.48 (1H, m, H-20b), 3.59–
3.71 (2H, m, H-50a and H-50b), 3.83–3.87 (1H, m, H-40), 4.40 (1H,
app dd, J = 6.0 Hz, J = 12.6 Hz, H-30), 5.28 (1H, br s, 50-OH), 6.04
(1H, app t, J = 5.7 Hz, H-10), 7.86 (1H, s, H-6). 13C NMR (75 MHz,
DMSO-d6): d 16.90 (5-CH3), 36.64 (C-20), 59.44 (C-30), 60.35 (C-
50), 84.32 and 84.44 (C-40 and C-10), 117.63 (C-5), 133.34 (C-6),
147.71 (C-2), 190.76 (C-4). Exact mass (ESI-MS) for C10H14N5O3S
[M+H]+ found, 326.284.0813; calcd 284.0812. Spectroscopic data
of 7 were in accordance with literature data.22
l
l
l
pound, vi determinations were performed at least at two different
concentration values [I].
4.2. Biological assays on M. bovis (BCG)
Compounds 18 and 19 were assayed for their inhibitory potency
on M. bovis var. BCG growth in vitro.21 A micro-method of culture
was performed in 7H9 Middlebrook broth medium containing 0.2%
glycerol and 0.5% Tween-80. Serial twofold dilutions of each com-
pound were prepared directly in 96-well plates. The bacterial inoc-
ulum was prepared previously at a concentration in the range of
107 bacteria (M. bovis BCG 1173P2) in 7H9 medium and stored at
ꢁ80 °C until used. The bacteria, adjusted at 105/mL, were delivered
4.3.3. 50-O-Acetyl-30-(4-chlorophenyl-1,2,3-triazol-1-yl)-30-deoxy-
in 100
and incubated at 37 °C in plastic boxes containing a humidified
normal atmosphere. At day 8 of incubation, 30 L of a resazurin
lL per well. The covered plates were sealed with parafilm
b-D-thymidine (25)
Compound 23 (146 mg, 0.47 mmol), sodium ascorbate (5 mg,
l
0.024 mmol) and CuSO45H2O (5 mg, 0.019 mmol) were suspended
in 9 mL of H2O/t-BuOH (2:1). 1-Chloro-4-ethynylbenzene
(129 mg, 0.94 mmol) was added after 15 min and the mixture
was stirred at room temperature for 24 h. The reaction mixture
was extracted with EtOAc and the combined organic phases were
dried over anhydrous MgSO4 and evaporated to anhydrousness.
The crude product was purified column chromatography
(CH2Cl2/MeOH 95:5) affording 25 (87 mg, 41%) as a white pow-
der. 1H NMR (300 MHz, DMSO-d6): d 1.84 (3H, s, 5-CH3), 2.04
(3H, s, OAc), 2.73–2.94 (2H, m, H-20a and H-20b), 4.27–4.37 (2H,
m, H-50a and H-50b), 4.43–4.49 (1H, m, H-40), 5.47–5.54 (1H, m,
H-30), 6.45 (1H, t, J = 7.2 Hz, H-10), 7.52–7.57 (2H, m, subs Ph),
7.63 (1H, d, J = 1.2 Hz, H-6), 7.85–7.90 (2H, m, subs Ph), 8.86
(1H, s, H-500), 11.41 (1H, s, 3-NH). 13C NMR (75 MHz, DMSO-d6):
d 12.14 (5-CH3), 20.53 (OAc), 36.36 (C-20), 59.50 and 63.29 (C-50
and C-30), 80.85 (C-40), 84.15 (C-10), 110.00 (C-5), 121.34 (C-500),
126.86, 129.07, 129.42 and 132.48 (subs Ph), 136.39 (C-6),
145.54 (C-400), 150.44 (C-2), 163.74 (C-4), 170.08 (OAc). Exact
mass (ESI-MS) for C20H21ClN5O5 [M+H]+ found, 446.1238; calcd
446.1226.
(Sigma) solution at 0.01% (wt/vol) in water was added to each well.
After an overnight incubation at 37 °C, the plates were assessed for
color development using the optical density difference at 570 and
630 nm on a microplate reader. The change from blue to pink indi-
cates reduction of resazurin and therefore bacterial growth. The
lowest compound concentration that prevented the color change
determined the MIC for the assayed compound.
4.3. Synthesis
General: All reagents were from standard commercial sources
and of analytical grade. Precoated Merck silica gel F254 plates were
used for TLC, spots were examined under ultraviolet light at
254 nm and further visualized by sulfuric acid-anisaldehyde spray.
Column chromatography was performed on silica gel (63–200 lm,
60 Å, Biosolve, Valkenswaard, The Netherlands). NMR spectra were
determined using a Varian Mercury 300 MHz spectrometer. Chem-
ical shifts are given in ppm (d) relative to the residual solvent peak:
in the case of DMSO-d6, it is 2.54 ppm for 1H and 40.5 ppm for 13C;
in the case of CDCl3, it is 7.26 ppm for 1H and 77.4 ppm for 13C.
Structural assignment was confirmed with COSY and DEPT. All sig-
nals assigned to hydroxyl groups were exchangeable with D2O.
Exact mass measurements were performed on a Waters LCT Pre-
mier XETM Time of flight (TOF) mass spectrometer equipped with
a standard electrospray ionization (ESI) and modular LockSpray TM
interface. Samples were infused in a CH3CN/water (1:1) mixture at
4.3.4. 50-O-Acetyl-30-(4-chlorophenyl-1,2,3-triazol-1-yl)-30-deo-
xy-4-thio-b-D-thymidine (26)
Lawesson’s reagent (158 mg, 0.39 mmol) was added to a solu-
tion of compound 25 (87 mg, 0.20 mmol) in 10 mL anhydrous tol-
uene. The mixture was refluxed overnight and the solvent was
removed in vacuo. The residue was purified by column chromatog-
raphy (CH2Cl2/MeOH 95:5) to give compound 26 as a brown–yel-
low solid (44 mg, 49%). 1H NMR (300 MHz, CDCl3): d 2.00 (3H, s,
5-CH3), 2.05 (3H, s, OAc), 2.86–2.94 (1H, m, H-20a), 3.17–3.26
(1H, m, H-20b), 4.35 (2H, d, J = 3.3 Hz, H-50a and H-50b), 4.59–4.65
(1H, m, H-40), 5.49 (1H, app dd, J = 7.5 Hz, J = 15.3 Hz, H-30),6.01–
6.04 (1H, m, H-10), 7.24 (1H, s, H-6), 7.32 (2H, d, J = 8.4 Hz, subs
Ph), 7.68 (2H, d, J = 8.4 Hz, subs Ph), 7.93 (1H, s, H-500), 11.10 (1H,
s, 3-NH). 13C NMR (75 MHz, CDCl3): d 17.41 (5-CH3), 21.00 (OAc),
38.16 (C-20), 60.31 and 63.45 (C-50 and C-30), 82.99 (C-40), 89.57
(C-10), 120.21 and 120.71 (C-5 and C-500), 127.23, 128.77, 129.39,
134.01 and 134.47 (C-6 and subs Ph), 147.20 and 148.49 (C-400
10
lL/min.
4.3.1. 50-O-Acetyl-30-azido-30-deoxy-4-thio-b-
D
-thymidine (24)
Lawesson’s reagent (154 mg, 0.38 mmol) was added to a solu-
tion of compound 23 (111 mg, 0.36 mmol) in 10 mL anhydrous tol-
uene. The mixture was refluxed overnight and the solvent was
removed in vacuo. The residue was purified by column chromatog-
raphy (CH2Cl2/MeOH 95:5) to give compound 24 as a brown–
yellow solid (23 mg, 20%). 1H NMR (300 MHz, DMSO-d6): d 1.99
(3H, d, J = 0.9 Hz, 5-CH3), 2.06 (3H, s, OAc), 2.34–2.43 (1H, m, H-
20a), 2.53–2.57 (1H, m, H-20b), 3.99–4.04 (1H, m, H-40), 4.21–4.32
(2H,m, H-50a an H-50b), 4.44–4.50 (1H, m, H-30), 6.07 (1H, dd,

S. Van Poecke et al. / Bioorg. Med. Chem. 19 (2011) 7603–7611
7609
and C-2), 170.66 (OAc), 190.91 (C-4). Exact mass (ESI-MS) for
20H21ClN5O4S [M+H]+ found, 462.1042; calcd 462.0997.
4.3.8. 5-(50-Amino-50-deoxy-30-O-tert-butyldimethylsilyl-b-
D-
C
thymidin-50N-yl)-1-(4-chloro-3-trifluoro-methylphenyl)-
tetrazole (29)
To a suspension of compound 28 (504 mg, 0.85 mmol), sodium
azide (166 mg, 2.55 mmol) and HgCl2 (253 mg, 0.93 mmol) in anhy-
drous DMF (3.3 mL) was added Et3N (0.36 mL, 2.55 mmol) under N2
atmosphere. The resulting black suspension was stirred overnight
at room temperature. The mixture was filtered through a pad of
Celite, washing with CH2Cl2. The filtrate was diluted with water
and extracted with CH2Cl2. The combined organic layers were dried
over MgSO4, filtered and concentrated under reduced pressure. The
resulting residue was purified by silica gel chromatography
(CH2Cl2/MeOH 98:2) affording compound 29 (424 mg, 83%) as a
colorless solid. 1H NMR (300 MHz, DMSO-d6): d 0.029–0.042 (6H,
m, TBDMS), 0.84–0.86 (9H, m, TBDMS), 1.78 (3H, s, 5-CH3), 2.00–
2.07 (1H, m, H-20a), 2.23–2.33 (1H, m, H-20b), 3.48–3.62 (2H, m,
H-50a and H-50b), 3.94–3.99 (1H, m, H-40), 4.42–4.45 (1H, m, H-
30), 6.10–6.14 (1H, m, H-10), 7.41 (1H, t, J = 6.0 Hz, 50-NH), 7.53
(1H, d, J = 1.2 Hz, H-6), 7.88–8.03 (3H, m, subs Ph), 11.31 (1H, s,
3-NH). 13C NMR (75 MHz, DMSO-d6): d ꢁ5.06 and ꢁ4.91 (TBDMS),
11.92 (5-CH3), 17.56 (TBDMS), 25.56 (TBDMS), C-20 (under solvent
peak), 45.82 (C-50), 72.90 (C-30), 84.07 and 84.58 (C-10 and C-40),
109.61 (C-5), 128.00–136.27 (subs Ph, CF3 and C-6), 150.35 (C-2),
155.28 (C@N), 163.62 (C-4). Exact mass (ESI-MS) for
4.3.5. 30-(4-Chlorophenyl-1,2,3-triazol-1-yl)-30-deoxy-4-thio-b-
D
-thymidine (10)
Compound 26 (42 mg, 0.090 mmol) was dissolved in a 7 N NH3
in MeOH solution (1 mL) and stirred at room temperature for 6 h.
The reaction mixture was concentrated in vacuo and the residue
was purified on a silica gel column using CH2Cl2/MeOH (95:5) as
the eluent to afford compound 10 as a yellow powder (25.2 mg,
66%). 1H NMR (300 MHz, DMSO-d6): d 1.75 (3H, d, J = 0.9 Hz, 5-
CH3), 1.96 (1H, app dt, J = 3.6 Hz, J = 14.1 Hz, H-20a), 2.45–2.55
(1H, m, H-20b), 4.28 (1H, app s, H-30), 4.47–4.64 (3H, m, H-40, H-
50a and H-50b), 5.63 (1H, s, 50-OH), 6.18 (1H, dd, J = 3.9 Hz,
J = 7.5 Hz, H-10), 7.49–7.54 (2H, m, subs Ph), 7.72 (1H, d,
J = 1.2 Hz, H-6), 7.86–7.91 (2H, m, subs Ph), 8.62 (1H, s, H-500). 13C
NMR (75 MHz, DMSO-d6): d 16.94 (5-CH3), 37.51 (C-20), 59.02 (C-
30), 60.41 (C-50), 84.80 and 84.90 (C-40 and C-10), 117.81 (C-5),
121.40 (C-500), 126.87, 129.03, 129.47, 132.44 and 133.50 (C-6
and subs Ph), 145.49 and 147.79 (C-400 and C-2), 190.90 (C-4). Exact
mass (ESI-MS) for C18H19ClN5O3S [M+H]+ found, 420.0915; calcd
420.0892.
C
24H32ClF3N7O4Si [M+H]+ found, 602.1910; calcd 602.1920.
4.3.6. N-(50-Deoxy-30-O-tert-butyldimethylsilyl-b- -thymidin-50-
D
yl)-N0-(4-chloro-3-trifluoromethyl-phenyl)-thiourea (28)
To a solution of compound 27 (403 mg, 1.13 mmol) in DMF
(4 mL) was added a solution of 4-chloro-3-(trifluoromethyl)phenyl-
isothiocyanate (0.18 mL, 1.13 mmol) in DMF (2 mL) at 0 °C. The
reaction mixture was stirred for 1 h. The solvents were evaporated
to anhydrousness and the residue was purified by column chroma-
tography (CH2Cl2/MeOH 98:2) affording compound 28 as a color-
less solid (510 mg, 76%). 1H NMR (300 MHz, DMSO-d6): d 0.098
(6H, s, TBDMS), 0.87–0.88 (9H, m, TBDMS), 1.80 (3H, s, 5-CH3),
2.02–2.10 (1H, m, H-20a), 2.24–2.34 (1H, m, H-20b), 3.55–3.65 (1H,
m, H-40), 3.98–3.99 (2H, m, H-50a and H-50b), 4.45–4.47 (1H, m,
H-30), 6.15–6.19 (1H, m, H-10), 7.52 (1H, s, subs Ph), 7.62–7.72
(2H, m, subs Ph and H-6), 7.96 (1H, subs Ph), 8.12 (1H, s, 50-NH),
9.93 (1H, s, N0H), 11.33 (1H, s, 3-NH). 13C NMR (75 MHz, DMSO-
d6): d ꢁ4.99 and ꢁ4.93 (TBDMS), 11.95 (5-CH3), 17.57 (TBDMS),
25.57 (TBDMS), C-20 (under solvent peak), 45.74 (C-50), 72.80 (C-
30), 83.98 and 84.19 (C-10 and C-40), 109.74 (C-5), 124.50–139.00
(subs Ph, CF3 and C-6), 150.36 (C-2), 163.56 (C-4), 180.77 (C@S). Ex-
act mass (ESI-MS) for C24H33ClF3N4O4Si [M+H]+ found, 593.1643;
calcd 593.1627.
4.3.9. 5-(50-Amino-50-deoxy-b- -thymidin-50N-yl)-1-(4-chloro-
3-trifluoromethylphenyl)-tetrazole (15)
D
Compound 29 (241 mg, 0.400 mmol) was dissolved in THF
(2.5 mL). A solution of 1 M tetra-n-butylammoniumfluoride in
THF (0.88 mL) was added. After 1 h at room temperature the
reaction was completed. The solvent was evaporated and the
anhydrous residue was purified by column chromatography
(CH2Cl2/MeOH 95:5) to give pure compound 15 (104 mg, white
solid) in 53% yield. 1H NMR (300 MHz, DMSO-d6): d 1.76 (3H, d,
J = 0.9 Hz, 5-CH3), 2.04–2.11 (1H, m, H-20a), 2.14–2.24 (1H, m,
H-20b), 3.49–3.65 (2H, m, H-50a and H-50b), 3.93–3.98 (1H, m, H-
40), 4.22–4.27 (1H, m, H-30), 5.32 (1H, d, J = 4.5 Hz, 30-OH), 6.12–
6.17 (1H, m, H-10), 7.39 (1H, t, J = 5.7 Hz, 50-NH), 7.50 (1H, d,
J = 1.5 Hz, H-6), 7.90–8.07 (3H, m, subs Ph), 11.29 (1H, s, 3-NH).
13C NMR (75 MHz, DMSO-d6): d 12.02 (5-CH3), C-20 (under solvent
peak), 46.16 (C-50), 71.24 (C-30), 83.91 (C-10), 84.28 (C-40), 109.73
(C-5), 124.95–136.26 (subs Ph, CF3 and C-6), 150.47 (C-2), 155.42
(C@N), 163.76 (C-4). Exact mass (ESI-MS) for C18H18ClF3N7O4
[M+H]+ found, 488.1052; calcd 488.1055.
4.3.10. 50-(4-Chlorophenyl-1,2,3-triazol-1-yl)-50-deoxy-b-
D-thy-
midine (16)
4.3.7. N-(50-Deoxy-b- -thymidin-50-yl)-N0-(4-chloro-3-trifluo-
D
romethylphenyl)-thiourea (14)
Compound 30 (56 mg, 0.21 mmol), sodium ascorbate (cat.
amount) and CuSO4ꢀ5H2O (cat. amount) were suspended in
3 mL of H2O/t-BuOH (1:2). 1-Chloro-4-ethynylbenzene (57 mg,
0.42 mmol) was added after 15 min and the mixture was stirred
at room temperature for 7 days. The reaction mixture was ex-
tracted with EtOAc and the combined organic phases were dried
over anhydrous MgSO4 and evaporated to anhydrousness. Purifi-
cation of the crude using RP-HPLC (Phenomenex Luna C-18,
H2O/0.1% HCOOH in CH3CN, 90:10?0:100 in 23 min, flow
17.5 mL/min) afforded compound 16 (2.0 mg, 2%) as a white
powder. 1H NMR (300 MHz, DMSO-d6): d 1.68 (3H, s, 5-CH3),
2.02–2.24 (2H, m, H-20a and H-20b), 4.09–4.14 (1H, m, H-40),
4.28–4.31 (1H, m, H-30), 4.64–4.80 (2H, m, H-50a and H-50b),
5.59 (1H, br s, 30-OH), 6.18 (1H, app t, J = 6.9 Hz, H-10), 7.23
(1H, d, J = 1.2 Hz, H-6), 7.49–7.53 (2H, m, subs Ph), 7.85–7.90
(2H, m, subs Ph), 8.62 (1H, s, H-500), 11.30 (1H, s, 3-NH). 13C
NMR (75 MHz, DMSO-d6): d 11.96 (5-CH3), 37.84 (C-20), 51.13
(C-50), 70.47 (C-30), 83.66 (C-40), 83.77 (C-10), 109.79 (C-5),
Compound 28 (84 mg, 0.14 mmol) was dissolved in THF
(0.9 mL). A solution of 1 M tetra-n-butylammoniumfluoride in
THF (0.31 mL) was added. After 1 h at room temperature the reac-
tion was completed. The solvent was evaporated and the anhy-
drous residue was purified by column chromatography (CH2Cl2/
MeOH 95:5) to give pure compound 14 (40 mg, white solid) in
60% yield. 1H NMR (300 MHz, DMSO-d6): d 1.79 (3H, s, 5-CH3),
2.05–2.13 (1H, m, H-20a), 2.17–2.27 (1H, m, H-20b), 3.59–3.66
(1H, m, H-40), 3.87–3.96 (2H, m, H-50a and H-50b), 4.23–4.24 (1H,
m, H-30), 5.38 (1H, d, J = 4.2 Hz, 30-OH), 6.18–6.22 (1H, m, H-10),
7.51–7.7.75 (3H, m, subs Ph and H-6), 8.17–8.22 (2H, m, subs Ph
and 50-NH), 9.99 (1H, s, N0H), 11.32 (1H, s, 3-NH). 13C NMR
(75 MHz, DMSO-d6): d 12.73 (5-CH3), C-20 (under solvent peak),
46.95 (C-50), 71.97 (C-30), 84.56 and 84.69 (C-10 and C-40), 110.54
(C-5), 121.59–139.91 (subs Ph, CF3 and C-6), 151.18 (C-2), 164.38
(C-4), 181.44 (C@S). Exact mass (ESI-MS) for C18H19ClF3N4O4
[M+H]+ found, 479.0778; calcd 479.0762.

7610
S. Van Poecke et al. / Bioorg. Med. Chem. 19 (2011) 7603–7611
122.53–132.25 (subs Ph and C-500), 135.96 (C-6), 145.26 (C-400),
150.37 (C-2), 163.56 (C-4). Exact mass (ESI-MS) for C18H19ClN5O4
[M+H]+ found, 404.1125; calcd 404.1120.
4.3.15. N-(50-Deoxy-4-thio-
phenylthiourea (18)
a-D
-thymidin-50-yl)-N0-
For the synthesis of compound 18, amine 36 (26 mg, 0.10 mmol)
was dissolved in DMF (1 mL). At 0 °C, phenyl isothiocyanate (16 mg,
0.12 mmol) was added and the reaction mixture was allowed to stir
at room temperature during 3 h. After completion of the reaction,
the reaction mixture was evaporated to anhydrousness and the res-
idue was purified by column chromatography (CH2Cl2/MeOH 95:5)
to obtain the pure final compound 18 (27.0 mg, 69%) as a yellow
powder. 1H NMR (300 MHz, DMSO-d6): d 1.97 (3H, d, J = 0.9 Hz, 5-
CH3), 2.01–2.03 (1H, m, H-20a), 2.54–2.63 (1H, m, H-20b), 3.53–
3.70 (2H, m, H-50a and H-50b), 4.21–4.24 (1H, m, H-30), 4.42–4.46
(1H, m, H-40), 5.43 (1H, d, J = 2.7 Hz, 30-OH), 6.12 (1H, dd,
J = 2.7 Hz, J = 7.5 Hz, H-10), 7.08–7.13 (1H, m, Ph), 7.29–7.34 (2H,
m, Ph), 7.43–7.47 (2H, m, Ph), 7.80 (1H, s, H-6). 13C NMR (75 MHz,
DMSO-d6): d 17.03 (5-CH3), under DMSO (C-20), 45.49 (C-50),
70.79 (C-30), 86.08 (C-10), 86.78 (C-40), 117.22 (C-5), 123.22,
124.30, 128.63 and 134.25 (Ph), 139.13 (C-6), 147.87 (C-2),
180.74 (C@S), 190.56 (C-4). Exact mass (ESI-MS) for C17H21N4O3S2
[M+H]+ found, 393.1053; calcd 393.1050.
4.3.11. 4-Thio-
Lawesson’s reagent (777 mg, 1.92 mmol) was added to a solu-
tion of compound 30-50-di-O-acetyl-
-thymidine (31) (519 mg,
a-D-thymidine (33)
a-D
1.59 mmol) in 15 mL anhydrous 1,4-dioxane. The mixture was re-
fluxed for 4 h. After the reaction mixture had been cooled, the sol-
vent was removed in vacuo. The crude product thus obtained was
treated with 8 mL of a 7 N NH3 in MeOH solution and stirred at
room temperature for 4 h. The reaction mixture was concentrated
in vacuo and the residue was purified on a silica gel column using
CH2Cl2/MeOH (94:6) as the eluent to afford compound 33 as a yel-
low foam (150 mg, 37%). 1H NMR (300 MHz, DMSO-d6): d 1.75 (3H,
s, 5-CH3), 1.94–1.98 (1H, m, H-20a), 2.51–2.57 (1H, m, H-20b), 3.40
(2H, t, J = 5.2 Hz, H-50a and H-50b), 4.23–4.25 (2H, m, H-30 and H-
40), 4.86 (1H, t, J = 5.7 Hz, 50-OH), 5.26 (1H, d, J = 2.7 Hz, 30-OH),
6.04 (1H, dd, J = 2.7 Hz, J = 7.5 Hz, H-10), 7.81 (1H, d, J = 0.9 Hz, H-
6), 12.65 (1H, s, 3-NH). Exact mass (ESI-MS) for C10H15N2O4S
[M+H]+ found, 259.0753; calcd 259.0747.
4.3.16. N-(50-Deoxy-4-thio- D-thymidin-50-yl)-N0-(3-
a-D
trifluoromethyl-4-chlorophenyl)thiourea (19)
4.3.12. 50-O-Methanesulfonyl-4-thio-
To a solution of 4-thio- -thymidine 33 (146 mg, 0.57 mmol) in
pyridine (5 mL) at ꢁ78 °C, methanesulfonylchloride (42 L,0.54
a-D-thymidine (34)
Compound 19 was synthesized from amine 36 (52 mg,
0.20 mmol) and 4-chloro-3-trifluoromethylphenyl isothiocyanate
(57 mg, 0.24 mmol) using the same procedure as described for
the synthesis of compound 18. After purification by column chro-
matography (CH2Cl2/MeOH 95:5), compound 19 (41.7 mg, 42%)
was obtained as a yellow powder. 1H NMR (300 MHz, DMSO-d6):
d 1.98 (3H, d, J = 0.6 Hz, 5-CH3), 2.06 (1H, t, J = 2.1 Hz, H-20a),
2.57–2.66 (1H, m, H-20b), 3.56–3.59 (1H, m, H-50a), 3.67–3.72
(1H, m, H-50b), 4.25–4.26 (1H, m, H-30), 4.44–4.48 (1H, m, H-40),
5.47 (1H, d, J = 3.0 Hz, 30-OH), 6.14 (1H, dd, J = 2.7 Hz, J = 7.5 Hz,
H-10), 7.64 (2H, d, J = 8.7 Hz, subs Ph), 7.74 (2H, dd; J = 2.1 Hz,
J = 8.4 Hz, subs Ph), 7.83 (1H, d, J = 0.9 Hz, H-6). 13C NMR
(75 MHz, DMSO-d6): d 17,11 (5-CH3), under DMSO (C-20), 45.62
(C-50), 70.95 (C-30), 86.18 and 86.55 (C-40 and C-10), 117.22 (C-5),
124.61, 127.50, 131.77 and 134.28 (CF3 and subs Ph), 139.23 (C-
6), 148.00 (C-2), 180.95 (C@S), 190.70 (C-4). Exact mass (ESI-MS)
for C18H19ClF3N4O3S2 [M+H]+ found, 495.0508; calcd 495.0534.
a-D
l
mmol) was added. The reaction mixture was stirred for 1 h at 0 °C.
The reaction was quenched with saturated aqueous NaHCO3-solu-
tion and extracted with CH2Cl2 three times, dried over MgSO4 and
evaporated. The residue was purified by column chromatography
(CH2Cl2/MeOH 95:5) to give mesylated compound 34 as a yellow
foam (133 mg, 70%). 1H NMR (300 MHz, DMSO-d6): d 1.97 (3H, d,
J = 0.6 Hz, 5-CH3), 1.99–2.04 (1H, m, H-20a), 2.53–2.30 (1H, m, H-
20b), 3.22 (3H, s, SO2CH3), 4.08–4.10 (2H, m, H-50a and H-50b),
4.16–4.30 (1H, m, H-30), 4.40–4.46 (1H, m, H-40), 5.55 (1H, br s, 30-
OH), 6.09 (1H, dd, J = 3.6 Hz, J = 7.5 Hz, H-10), 7.69 (1H, s, H-6). Exact
mass (ESI-MS) for C11H17N2O6S2 [M+H]+ found, 337.0533; calcd
337.0523.
4.3.13. 50-Azido-50-deoxy-4-thio-
A solution of 50-mesylated 4-thio-
a-
D
-thymidine (35)
a-D-thymidine 34 (129 mg,
4.3.17. 50-(4-Chlorophenyl-1,2,3-triazol-1-yl)-50-deoxy-
thymidine (20)
a-D-
0.38 mmol) and NaN3 (250 mg, 3.86 mmol) in DMF (7 mL) was
heated to 60 °C overnight. The reaction mixture was evaporated
in vacuo. The residue was resolved in CH2Cl2 and washed with
brine. The organic layer was dried over MgSO4, evaporated and
purified by column chromatography (CH2Cl2/MeOH 95:5) to afford
compound 35 (97 mg, 89%) as a yellow oil. 1H NMR (300 MHz,
DMSO-d6): d 1.98 (3H, d, J = 0.9 Hz, 5-CH3), 2.04 (1H, t, J = 3.3 Hz,
H-20a), 2.56–2.65 (1H, m, H-20b), 3.40–3.44 (2H, m, H-50a and H-
50b), 4.14–4.17 (1H, m, H-30), 4.34–4.39 (1H, m, H-40), 5.47 (1H,
br s, 30-OH), 6.09 (1H, dd, J = 3.6 Hz, J = 7.5 Hz, H-10), 7.78 (1H, s,
Compound 17 (85 mg, 0.32 mmol), sodium ascorbate (3 mg,
0.016 mmol) and CuSO4ꢀ5H2O (3 mg, 0.013 mmol) were suspended
in 3 mL of H2O/t-BuOH (2:1). 1-Chloro-4-ethynylbenzene (87 mg,
0.64 mmol) was added after 15 min and the mixture was stirred
at room temperature for 4 days. Water was added and the triazole
product precipitated. Filtration of the mixture afforded pure com-
pound 20 (40.0 mg, 31%) as a white powder. 1H NMR (300 MHz,
DMSO-d6): d 1.75 (3H, d, J = 1.2 Hz, 5-CH3), 1.94 (1H, app t,
J = 3.9 Hz, H-20a), 1.99 (1H, app t, J = 3.9 Hz, H-20b), 4.22–4.32
(1H, m, H-40), 4.43–4.66 (3H, m, H-30, H-50a and H-50b), 5.65 (1H,
br s, 30-OH), 6.18 (1H, dd, J = 4.2 Hz, J = 7.5 Hz, H-10), 7.49–7.54
(2H, m, subs Ph), 7.72 (1H, d, J = 1.2 Hz, H-6), 7.86–7.91 (2H, m,
subs Ph), 8.62 (1H, s, H-500), 11.26 (1H, s, 3-NH). 13C NMR
(75 MHz, DMSO-d6): d 12.15 (5-CH3), 51.20 (C-50), 70.53 (C-40),
84.72 (C-10), 85.37 (C-30), 108.81 (C-5), 122.41 (C-500), 126.73,
128.83, 129.45 and 132.15 (subs Ph), 136.64 (C-6), 145.12 (C-400),
150.29 (C-2), 163.65 (C-4). Exact mass (ESI-MS) for C18H19ClN5O4
[M+H]+ found, 404.1129; calcd 404.1120.
H-6). Exact mass (ESI-MS) for
284.0813; calcd 284.0812.
C
10H14N5O3S [M+H]+ found,
4.3.14. 50-Amino-50-deoxy-4-thio-
a
-D
-thymidine (36)
Compound 35 (97 mg, 0.34 mmol) and PPh3 (187 mg,
0.71 mmol) were dissolved in THF (6 mL). After stirring for
10 min, H2O was added (883 lL) and the mixture was stirred for
1 day. The mixture was extracted with CH2Cl2 and the water phase
lyophilized to give amine 36 (78 mg, 89%). 1H NMR (300 MHz,
DMSO-d6): d 1.96 (3H, d, J = 0.6 Hz, 5-CH3), 2.00 (1H, t, J = 3.6 Hz,
H-20a), 2.58–2.68 (1H, m, H-20b), 2.76–2.87 (1H, m, H-50a), 2.96–
3.04 (1H, m, H-50b), 4.18–4.20 (1H, m, H-30), 4.32 (1H,dt,
J = 3.3 Hz, J = 9.6 Hz, H-40), 5.57 (1H, br s, 30-OH), 6.15 (1H, dd,
J = 3.3 Hz, J = 7.5 Hz, H-10), 7.77 (1H, s, H-6). Exact mass (ESI-MS)
for C10H16N3O3S [M+H]+ found, 258.0907; calcd 258.0907.
4.3.18. 50-O-Acetyl-30-azido-30-deoxy-
a-D-thymidine (38)
To a solution of compound 37 (642 mg, 2.08 mmol) in 1 mL anhy-
drous CH2Cl2, was added a freshly prepared solution, containing
34 lL H2SO4 and 140 lL acetic acid anhydride in 1 mL anhydrous

S. Van Poecke et al. / Bioorg. Med. Chem. 19 (2011) 7603–7611
7611
CH2Cl2. After 2 h, the mixture was quenched with saturated NaH-
CO3-solution and extracted three times with EtOAc. Purification of
the crude on a silica gel column (EtOAc/hexane 9:1) yielded
compound 38 as a white foam (126 mg, 20%). 1H NMR (300 MHz,
DMSO-d6): d 1.80 (3H, d, J = 0.9 Hz, 5-CH3), 2.06 (3H, s, OAc),
2.15–2.22 (1H, m, H-20a), 2.70–2.75 (1H, m, H-20b), 4.11–4.14 (2H,
m, H-50a and H-50b), 4.34–4.39 (1H, m, H-30), 4.42–4.45 (1H, m,
H-40), 6.07 (1H, dd, J = 6.0 Hz, J= 6.9 Hz, H-10), 7.59 (1H, d,
J = 1.2 Hz, H-6), 11.32 (1H, s, 3-NH). 13C NMR (75 MHz, DMSO-d6):
d 12.15 (5-CH3), 20.62 (OAc), 36.23 (C-20), 60.43 (C-3), 63.68 (C-50),
81.46 (C-40), 84.93 (C-10), 109.38 (C-5), 136.25 (C-6), 150.36 (C-2),
163.80 (C-4), 170.15 (OAc). Exact mass (ESI-MS) for C12H16N5O5
[M+H]+ found, 310.1164; calcd 310.1146. Spectroscopic data of 38
were in accordance with literature data.13
(C-10), 109.58 (C-5), 121.38 (C-500), 126.88–132.47 (subs Ph),
136.07 (C-6), 145.49 (C-400), 150.46 (C-2), 163.78 (C-4). Exact mass
(ESI-MS) for
404.1120.
C
18H19ClN5O4 [M+H]+ found, 404.1156; calcd
Acknowledgments
We thank the UGent Research Fund (BOF, Ghent University), the
Fund for Scientific Research-Flanders (F.W.O.-Vlaanderen) for
funding and the Institute for the Promotion of Innovation by Sci-
ence and Technology in Flanders (IWT) for providing a scholarship
to S.V.P. We also thank the Institut Pasteur (GPH Tuberculose, DAR-
RI), the CNRS and INSERM for funding.
Supplementary data
4.3.19. 30-Azido-30-deoxy-
a-D-thymidine (21)
Compound 38 (146 mg, 0.47 mmol) was dissolved in a 7 N NH3
in MeOH solution (2.6 mL) and stirred at room temperature for 6 h.
The reaction mixture was concentrated in vacuo and the residue
was purified on a silica gel column using EtOAc/hexane (8:2) as
the eluent to afford compound 21 as a colorless solid (91 mg,
72%). 1H NMR (300 MHz, CDCl3): d 1.95 (3H, d, J = 0.9 Hz, 5-CH3),
2.12–2.17 (1H, m, H-20a), 2.82–2.91 (1H, m, H-20b), 3.08 (1H, t,
J = 5.4 Hz, 50-OH), 3.65–3.72 (1H, m, H-50a), 3.81–3.85 (1H, m, H-
50b), 4.27–4.35 (2H, m, H-30 and H-40), 6.29 (1H, dd, J = 4.2 Hz,
J = 6.9 Hz, H-10), 7.32 (1H, d, J = 1.5 Hz, H-6), 9.48 (1H, s, 3-NH).
13C NMR (75 MHz, CDCl3): d 12.67 (5-CH3), 38.22 (C-20), 60.83 (C-
3), 62.62 (C-50), 85.93 and 86.02 (C-40 and C-10), 111.24 (C-5),
135.35 (C-6), 150.72 (C-2), 164.03 (C-4). Exact mass (ESI-MS) for
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.10.021.
References and notes
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Compound 21 (72 mg, 0.27 mmol), sodium ascorbate (3 mg,
0.024 mmol) and CuSO4ꢀ5H2O (3 mg, 0.011 mmol) were suspended
in 3 mL of H2O/t-BuOH (1:2). 1-Chloro-4-ethynylbenzene (74 mg,
0.54 mmol) was added after 15 min and the mixture was stirred
at room temperature for 24 h. The reaction mixture was extracted
with EtOAc and the combined organic phases were dried over anhy-
drous MgSO4 and evaporated to anhydrousness. The crude product
was purified column chromatography (EtOAc/hexane 8:2) affording
22 (50.8 mg, 47%) as a white powder. 1H NMR (300 MHz, DMSO-d6):
d 1.75 (3H, s, 5-CH3), 2.71–2.80 (1H, m, H-20a), 2.98–3.07 (1H, m, H-
20b), 3.54–3.70 (2H, m, H-50a and H-50b), 4.65–4.70 (1H, m, H-40),
5.12 (1H, t, J = 5.4 Hz, 50-OH), 5.29–5.36 (1H, m, H-30), 6.26 (1H,
app t, J = 6.6 Hz, H-10), 7.50–7.54 (2H, m, subs Ph), 7.63 (1H, d,
J = 1.2 Hz, H-6), 7.84–7.87 (2H, m, subs Ph), 8.82 (1H, s, H-500),
11.29 (1H, s, 3-NH). 13C NMR (75 MHz, DMSO-d6): d 12.18 (5-
CH3), 37.12 (C-20), 59.23 (C-3), 61.12 (C-50), 83.89 (C-40), 84.43
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