Synthesis of 5-haloethynyl- and 5-(1,2-dihalo)vinyluracil nucleosides: Antiviral activity and cellular toxicity

Article abstract of DOI:10.1016/j.bmc.2005.06.021

In this article, we report the synthesis of hitherto unknown 5-haloethynyl and 5-(1,2-dihalo)vinyluracil nucleosides in the 2′-deoxy, 3′-deoxy- and ribosyl series, and we discuss their in vitro anti-HIV and anti-HCV activities and cellular toxicitites. As a result, on the basis of their selectivity index (SI) obtained with the HCV replicon system, but also on their cytotoxicity on peripheral blood mononuclear, CEM and VERO cell lines, the best compounds were the 5-bromoethynyluridine (SI = 3.2) and the 5-(1-chloro-2-iodo) vinyluridine (SI > 2.8).

Full text of DOI:10.1016/j.bmc.2005.06.021

Bioorganic & Medicinal Chemistry 13 (2005) 6015–6024
Synthesis of 5-haloethynyl- and 5-(1,2-dihalo)vinyluracil
nucleosides: Antiviral activity and cellular toxicity
Vanessa Escuret,b Vincent Aucagne,a Nicolas Joubert,a David Durantel,b
Kimberly L. Rapp,c Raymond F. Schinazi,c Fabien Zoulimb and Luigi A. Agrofoglioa,*
a
´
Institut de Chimie Organique et Analytique, ICOA UMR 6005, UFR Sciences, BP 6759, 45067 Orleans Cedex 2, France
bInserm U271, 151 Cours Albert Thomas, 69003 Lyon, France
cVeterans Affairs Medical Center and Laboratory of Biochemical Pharmacology, Department of Pediatrics,
Emory University School of Medicine, Atlanta, GA 30033, USA
Received 18 April 2005; revised 6 June 2005; accepted 10 June 2005
Available online 14 July 2005
Abstract—In this article, we report the synthesis of hitherto unknown 5-haloethynyl and 5-(1,2-dihalo)vinyluracil nucleosides in the
20-deoxy, 30-deoxy- and ribosyl series, and we discuss their in vitro anti-HIV and anti-HCV activities and cellular toxicitites. As a
result, on the basis of their selectivity index (SI) obtained with the HCV replicon system, but also on their cytotoxicity on peripheral
blood mononuclear, CEM and VERO cell lines, the best compounds were the 5-bromoethynyluridine (SI = 3.2) and the 5-(1-chloro-
2-iodo)vinyluridine (SI > 2.8).
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
HCV infection remains a main public health problem
with 175 million people infected in the world. Cur-
rently, the best treatment available consists in the
combination of pegylated interferon alpha 2b
(IFNa-2b) and ribavirin [1, 1-b-D-ribofuranosyl-1,2,4-
triazole-3-carboxamide, Fig. 1]. Twenty percent of
patients infected with HCV genotype 2 or 3 and
50% of patients infected with HCV genotype 1 are
non-responders to this treatment.1–3 Moreover, IFN
and ribavirin are responsible for numerous adverse ef-
fects. Therefore, a new treatment regimen based on
more potent and specific inhibitors of HCV replica-
tion is required to improve the current antiviral strat-
egies against chronic HCV infection. The inhibition of
NS5B bearing the RNA-dependent RNA polymerase
activity has been intensively studied4 because of the
central role of this protein in viral replication. Thus,
Figure 1. Some nucleoside inhibitors of NS5B.
specific inhibitors of this domain may represent po-
tent antiviral agents.5,6 The pharmaceutical impor-
tance of nucleoside analogues against the HIV,
HBV or HSV DNA polymerases has prompted the
Keywords: Nucleosides; HCV replicon; 5-Haloethynyl nucleosides;
5-(1,2-Dihalo)ethynyl nucleosides.
*
design and the synthesis of nucleoside analogues
against viral RNA polymerase.
Corresponding author. Tel.: +33 2 3849 4582; fax: +33 2 3841
7281; e-mail: luigi.agrofoglio@univ-orleans.fr
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.06.021

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V. Escuret et al. / Bioorg. Med. Chem. 13 (2005) 6015–6024
While numerous non-nucleoside inhibitors of NS5B-
mediated HCV RNA replication have been reported,7
only a few nucleoside inhibitors have been identified
so far,8 (Fig. 1). Among nucleoside inhibitors,9b the 20-
C-methylarabinoguanosine (2a) and the cytosine ana-
logue (2b)10a are phosphorylated in cultured uninfected
cells and are orally bioavailable in primates; meanwhile,
the triazolo analogue (2c) exhibited an effective concen-
tration (EC50) of 19.7 lM on HCV replicon without any
toxicity on peripheral blood mononuclear (PBM) and
VERO cells.10b The use of these compounds effectively
eliminates wild-type viruses and those with mutations
in the polymerase gene survive. These selected mutants
are therefore resistant to the drugs.9,10 Therefore, the
discovery of new inhibitors of HCV is mandatory in
the perspective of the development of combination
therapy.
Thus, as a part of our drug discovery programme, we
report here the synthesis and evaluation of 18, hither-
to unknown, 5-haloethynyl- and 5-(1,2-dihalo)vinyl-
uracil-(20-deoxy-, ribo- and 30-deoxy)-nucleosides.
The anti-HCV activity and the cellular toxicity were
evaluated for these new 5-halosubstituted uridine
derivatives using a cell line harbouring a genotype-
1b HCV subgenomic replicon,11 and for some com-
pounds, against the bovine viral diarrhoea virus
(BVDV) strain NADL. The molecules were also test-
ed on HIV-1 using different cell lines and their cyto-
toxicities evaluated in lymphocytic CEM, African
Green monkey (VERO) and activated human PBM
cells.
Scheme 1. Synthesis of 5-ethynyluracil acetylated nucleosides 5a–c.
We then turned our attention to the synthesis of 5-haloe-
thynyluracil nucleosides 12a–c and 13a–c, for which
only the 5-bromoethynyl-20-deoxyuridine has been al-
ready reported by Eger et al.15 to exhibit a certain selec-
tivity [similar to the E-5-(bromovinyl)-20-deoxyuridine]
towards HSV-1. The halogenation of alkynes 5a–c was
realized using either the iodonium di-syn-collidine per-
chlorate (IDCP, for iodination) or bromonium di-syn-
collidine perchlorate (Br(coll)2ClO4, for bromination)
in anhydrous CH3CN (Scheme 2). Both reagents are
known to act as very reactive electrophile (I+ and Br+,
respectively). The 5-iodoethynyluracil nucleosides 6a–c
were obtained with a good yield ranging from 68 to
93%, whereas the bromination yielded the desired 5-bro-
moethynyluracil derivatives 7a–c in lower yields (from
21 to 58%). The final deacylation of nucleosides 6a–c
and 7a–c was performed under smooth basic conditions
(1 M NaOH in a mixture of H2O/EtOH/pyridine), and
the 5-haloethynyluracil nucleosides 12a–c (for X = I)
and 13a–c (for X = Br) were obtained, respectively, in
good-to-moderate yields.
2. Chemistry
Halogenated pyrimidines 4a–c can be prepared by
direct reaction with halogens following the procedure
recently reported by Asakura and Robins12 via a ceri-
um(IV)-mediated halogenation at the C-5 of uracil
derivatives (Scheme 1). Thus, the treatment of known
acetylated nucleosides 3a–c with elemental iodine and
ceric ammonium nitrate (CAN) at 80 ꢁC gave the cor-
responding protected 5-iodouracil nucleosides 4a–c
with excellent yields. This CAN-mediated halogena-
tion has advantages over other available methods as
mild reaction conditions and short reaction times are
employed. The introduction of a C-5-alkynyl group
was performed by a Pd(0)-mediated reaction, using
the Sonogashira conditions.13 Thus, reaction of the
resulting pure acetylated 5-iodouracil 4a–c with trim-
ethylsilylacetylene (TMS) in the presence of Et3N
(base), CuI (cocatalyst) and PdCl2(PPh3)2 (catalyst)
in anhydrous dimethylsufoxide (DMSO) at room tem-
perature, followed by the removal of the trimethylsilyl
group with n-Bu4NF yielded the corresponding 5-ethy-
nyluracil nucleosides 5a–c with a high efficiency (80–
99%). In most cases, it has been observed that slight
elevations in the reaction temperature led to an in-
creased rate of coupling. In all cases, a by-product,
furanopyrimidine derivatives,14 has been either isolat-
ed with a <7% yield or just detected by thin-layer
chromatography (TLC).
Finally, we worked on the dihalogenation of 5-ethynyl
nucleosides to reach the hitherto unknown 5-(1,2-
dihalogenated)vinyluracil nucleosides 14a–c to 17a–c
(Scheme 3). Several halogenating reagents (i.e., I2, IBr,
ICl and Br2) were used to obtain the corresponding
1,2-dihaloderivatives. In all cases of halo-iodination,
the formation of vicinal halo-iodoalkenes (8a–c to
10a–c) occurred with high anti-stereospecificity, impli-
cating the intermediary of an iodonium ion in the reac-
tion sequence, and a high and unique regioselectivity,
implicating a predominantly Markovnikov addition.
The formation of 11a–c using Br2 occurs also with an
E-stereochemistry.

V. Escuret et al. / Bioorg. Med. Chem. 13 (2005) 6015–6024
6017
in Table 1. Human PBM cells were used to assay their
ability to inhibit HIV-1.16 Among the 20-deoxyribosyl
nucleosides, 12a–17a exhibited moderate anti-HIV
activity with a median EC50 ranging from 0.9 to
73.3 lM; the best anti-HIV activity was exhibited
by the 5-(1-bromo-2-iodo)vinyl-20-deoxyuridine (15a)
(EC50 = 0.9 lM, SI = 14.8). Nevertheless, all those com-
pounds also showed toxicity against PBM, CEM or
VERO cells, probably by inhibiting cellular DNA syn-
thesis. Among the C-5-substituted uridines, 12b–17b,
the best (but moderate) anti-HIV activity was achieved
for compound 12b (5-iodoethynyluridine) with an
EC50 of 20.8 lM (SI > 4.8); this nucleoside analogue
did not exhibited any toxicity in PBM, CEM or VERO
cells. In the 30-deoxyribosyl series, no or very weak anti-
HIV activity was noted for compounds 12c–17c that
were also not toxic.
The HCV subgenomic replicon system was used to
assess the inhibitory activity of the synthesized
compounds on the HCV replication. Among the 20-de-
oxyuridine analogues (12a–17a), the 5-iodoethynyl-20-
deoxyuridine (12a) and the 5-(1-chloro-2-iodo)-20-deox-
yuridine (16a) have the best selectivity index (SI) on
HCV replication, respectively, of 3.2 and 2.7. It is worth
noting that ribavirin in the same system had an SI < 1,
although its mechanism of action on HCV replication
remains debated. However, in this series, all iodinated
analogues were also the most toxic ones in Huh7 cells
and also in PBM, CEM and VERO cells, whereas the
bromo derivative compounds (13a and 17a) were less
toxic, but also unable to inhibit HCV significantly.
The important cell toxicity exhibited by the 20-deoxyur-
idine derivatives is also in favour of a non-specific inhi-
bition of viral genomic replication by those compounds.
Indeed, in the replicon system, it was shown that HCV
replication is dependent on cell cycle progression. Drugs
that have a cytostatic effect may therefore exhibit HCV
replication by an indirect mechanism. Efficient com-
pounds for inhibiting HCV replication were also found
among the uridine analogues. The 5-bromoethynyluri-
dine (13b), the 5-(1,2-diiodo)vinyluridine (14b) and the
5-(1-chloro,2-iodo)vinyluridine (16b) exhibited an EC50
of 58, 162 and 75 lM, respectively, against HCV repli-
con, with a CC50 > 200 lM for 14b and 16b. The selec-
tivity index against HCV, for 13b, 14b and 16b, was
found to be equal to 3.2, 1.5 and 2.8, respectively.
Finally, the 30-deoxyribosyl nucleosides (12c–17c) did
not show any antiviral activity against neither HCV
or BVDV (Table 2).
Scheme 2. Synthesis of 5-haloethynyluracil nucleosides 12a–c, 13a–c.
Ribavirin, which shows activity in vivo in chronically
infected patients together with IFNa, is inactive against
HCV replication in the replicon system, but inhibits
viral replication in the BVDV system in combination
with IFNa.17 It is well known that ribavirin when used
alone has no anti-HCV activity in humans infected with
HCV. On the basis of observation of possibly toxic com-
pounds (12b, 14a and 15a) that exhibited only modest
antiviral activity (SI < 1), this may therefore indicate
for 13b and 16b a specific anti-HCV effect, as these com-
pounds were not active against the replication of BVDV,
a closely related virus member of the Flaviviridae family
Scheme 3. Synthesis of 5-(1,2-dihalo)vinyluracil nucleosides from 14a–
c to 17a–c.
3. Biological assay
All synthesized compounds, the 5-haloethynyluracil
nucleosides (12a–c and 13a–c) and the 5-(1,2-
dihalo)vinyluracil nucleosides (14a–c to 17a–c) along
with the known antiviral compounds (ribavirin and
IFNa-2b for HCV, and AZT for HIV), were tested for
their antiviral activities in vitro, and the results are given

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V. Escuret et al. / Bioorg. Med. Chem. 13 (2005) 6015–6024
Table 1. Evaluation of the antiviral activity and cell viability (obtained with neutral red test) against HCV replication using cell line harbouring HCV
subgenomic replicon, against HIV-1 in PBM cells and of the cytotoxicity against PBM, CEM and VERO cells in vitro
Compound
Anti-HCV activity and cytotoxicity
on replicon system
Anti-HIV activity
Cytotoxicity (CC50)
CC50
EC50
EC90
SI
EC50
EC90
SI
PBM
CEM
VERO
IFNa-2b
Ribavirin
MPA
AZT
12a
>1000
110
7
19
760
>512
52
170
40
<1
<1
0.016
9.2
>6250
<1
>100
7.5
14.0
15.8
>100
19.5
0.13
14.8
43.9
29.0
>100
>100
19.5
6.6
20-Deoxyribosyl moiety
>182
>200
77
57
>239
>200
>263
>100
>207
>200
>3.2
61
<1
34.8
>100
39.1
6.9
13a
14a
>200
>161
>100
80
73.3
10.3
0.9
>1.4
1.4
>100
14.7
13.3
3.2
15a
16a
43
213
<1
14.8
1.2
2.7
61
2.6
43.2
20.3
86.9
1.4
>100
17a
>200
<200
>2.3
>100
Ribosyl moiety
12b
13b
14b
15b
16b
17b
148
188
>200
58
>200
>200
>250
>100
>250
>200
<1
3.2
20.8
95.2
>100
62.4
90.8
60.1
>100
>100
>100
>100
>100
>100
>4.8
1
>100
>100
100
>100
>100
41.7
>100
>100
49.5
>100
100
>250
>100
>200
>200
>162
>100
75
>1.5
1
61
>1.6
1.1
>100
>100
>100
93.6
>100
>100
>2.8
6 1
>200
>1.7
59.8
30-Deoxyribosyl moiety
12c
13c
14c
15c
16c
17c
>200
>300
>200
>200
>200
>200
>200
>300
>200
>200
>200
=200
>200
>300
>200
>200
>200
>200
6 1
6 1
6 1
6 1
6 1
6 1
88.7
>100
49.4
44.5
47.8
81.1
>100
>100
>100
90.7
>1.1
6 1
1.8
>100
>100
87.2
>100
>100
>100
79.3
3.2
6.4
42.6
>100
15.3
7.8
1.6
71.5
94.4
>100
>100
2
>1.2
66.6
79.3
>100
Results are expressed in lM, except for IFN which is expressed as international units/ml. Selectivity index (SI) is the ratio between CC50 and EC50
.
Table 2. Neutral red testing and antiviral assay on BVDV strain
NADL expressed in lM
anti-HCV activity and cytotoxicity, the order of potency
for these anti-HCV nucleoside analogues was found to
be: ribosyl- > 20-deoxyribosyl- > 30-deoxyribosyl substi-
tuted nucleoside analogues. Our results are also in
accordance with other observations of the general inca-
pacity of 30-deoxy purine and pyrimidine ribonucleo-
sides to inhibit the HCV RNA replication in cell
assays, despite their efficient inhibition in in vitro
NS5B assays.8d
Compound
CC50
EC50 CPE
SI
(Neutral red) inhibitiona
(3rd day pi)b
Ribavirin >100
12a–c, 13b, 14a–c, 15c, 16a–c >100
36
>100
>2.8
<1
a Cytopathic effect.
b Post inoculation.
It will be interesting to compare their anti-HCV activity
to that of IFNa and to study whether their combination
with IFNa may lead to a synergistic antiviral effect.
(Table 2). This may suggest that the MDBK-cpBVDV
assay is not always a reliable model for predicting
anti-HCV activity. Therefore, the active and non-toxic
13b and 16b warrant further evaluation in combination
with IFNa both in in vitro and in animal models.22
4. Experimental
4.1. Chemistry
In summary, the substituted 20-deoxyuridines 12a and
16a, and the substituted uridines 13b and 16b exhibited
anti-HCV activity in the HCV replicon system. Never-
theless, the best compounds, based on their SI obtained
with the HCV replicon system, but also on their cytotox-
icity on PBM, CEM and VERO cell lines, are bearing
the 5-(2-bromoethynyl)-uridine (13b) and the (E)-5-(1-
chloro-2-iodovinyl)uridine (16b). Finally, of significance
was the finding that all active compounds have a 30-hy-
droxyl group (either in the ribosyl or 20-deoxyribosyl
series) while all 30-deoxyuridine analogues did not pos-
sess any antiviral activity neither against HCV nor
against HIV-1. As all of these active compounds possess
a 30-hydroxyl group on the ribose, our results confirm
the crucial role of the 30-hydroxyl group for inhibition
of the HCV polymerase.18 Thus, based on the
4.1.1. General methods. Commercially available chemi-
cals and solvents were of reagent grade and used
as received. Dry tetrahydrofuran, pyridine and dichloro-
methane were obtained from distillation over CaH2 or
Na, N,N-dimethylformamide over BaO. The reactions
were monitored by TLC analysis using silica gel plates
(Kieselgel 60 F254; E. Merck). Compounds were visual-
ized by UV irradiation and/or spraying with 20%
H2SO4 in EtOH, followed by charring at 150 ꢁC. Col-
umn chromatography was performed on Silica Gel 60
M (0.040–0.063 mm; E. Merck). Melting points were
recorded on a Buchi (Dr. Tottoli) and were uncorrected.
¨
Optical rotations were measured at 20–25 ꢁC with a
Perkin-Elmer Model 141 polarimeter. 1H and 13C NMR

V. Escuret et al. / Bioorg. Med. Chem. 13 (2005) 6015–6024
6019
spectra were recorded on a Bruker AVANCE DPX 250
Fourier Transform spectrometer at 250 MHz for 1H and
62.9 MHz for 13C, respectively, using tetramethylsilane
as the internal standard; signals are reported as s (sin-
glet), d (doublet), t (triplet), q (quartet), and m (multi-
plet). Mass spectra were recorded on a Perkin-Elmer
SCIEX API-300 (heated nebullizer) spectrometer.
High-resolution mass analyses (HRMS) were performed
by the CRMPO, University of Rennes 1–Fr using the
fast atom bombardment or electron spray ionization
mode. The nomenclature of the obtained compounds
is in accordance with the IUPAC rules and was checked
with Autonome. The numbering and assignment of the
chemical shifts for all described compounds are related
to the corresponding ribose derivatives. Evidence of
purity has been done from a proton-decoupled 13C
NMR spectrum with a signal-to-noise ratio sufficient
to permit seeing peak with 5% of the intensity of the
strongest peak.
(5· 40 mL) and brine (40 mL). The organic layer was
dried over MgSO4 and the solvents were evaporated un-
der reduced pressure to a dark oil. A first purification
using
a
short path flash chromatography (elu-
ent:CH2Cl2, then MeOH/CH2Cl2 95:5) afforded the de-
sired compound contaminated with coloured reaction
coproducts. Pure silylated alkynes (2 mmol), obtained
after a second flash chromatography (eluent: hexanes/
AcOEt, 7:3 then 1:1), were dissolved in dry CH3CN
(20 mL). TBAF monohydrate (583 mg, 1.05 equiv) was
added and the resulting solution was stirred at rt until
completion (typically 30 min to 2 h, checked by TLC).
Solvents were evaporated under reduced pressure at rt
and the oily residue was submitted to a flash column
chromatography (eluent: hexanes/AcOEt 1:1 then
AcOEt then MeOH/AcOEt 98:2) to afford pure 5-ethy-
nyl nucleosides 5a–c.
4.1.7. 30,50-Di-O-acetyl-20-deoxy-5-ethynyluridine (5a).
The physicochemical data of this compound are fully
related with those previously published.20
4.1.2. General procedure for iodination of acetylated
nucleosides. A solution of acetylated nucleoside 3a–c
(15 mmol) in dry CH3CN (150 mL), CAN (4.94 g,
0.6 equiv) and I2 (2.28 g, 0.6 equiv) was refluxed until
completion (typically 1 h, checked by TLC). After cool-
ing to rt solvents were evaporated under reduced pres-
sure, and the dark oily residue was dissolved in AcOEt
(300 mL) and H2O (50 mL). The biphasic mixture was
cooled in an ice bath, and a saturated Na2S2O3 solution
was smoothly added until complete decolouration. The
organic layer was washed with water (2· 50 mL) and
brine (50 mL), dried over MgSO4 and concentrated un-
der reduced pressure. The white foam was triturated
with pentane (50 mL), filtered and dried under reduced
pressure to afford pure iodinated compounds 4a–c,
respectively.
4.1.8. 20,30,50-Tri-O-acetyl-5-ethynyluridine (5b). The
physicochemical data of this compound are fully related
with those previously published.21
4.1.9. 20,50-Di-O-acetyl-30-deoxy-5-ethynyluridine (5c).
20
D
1
75% yield; ½aꢀ ꢁ42 (c 1.0, CHCl3); H NMR (CDCl3)
d 9.8 (br s, 1H, NH), 7.88 (s, 1H, H-6), 5.78 (s, 1H,
H-10), 5.31 (d, J = 7.5 Hz, 1H, H-20), 4.48 (m, 1H, H-
40), 4.35 (dd, J = 12.5, 1.8 Hz, 1H, H-50a), 4.25 (dd,
J = 12.5, 3.4 Hz, 1H, H-50b), 3.1 (s, 1H, H-Csp), 2.31–
1.95 (m, 2H, H-30), 2.12 (s, 3H, OAc), 2.05 (s, 3H,
OAc). HRMS m/z calcd for C15H16N2O7Na, 360.2968;
found, 360.2971.
4.1.10. General procedure for monohalogenation of 5-
ethynyl nucleosides. 5-Ethynyl nucleoside 5a–c
(0.5 mmol) was dissolved in 5 mL dry CH3CN, and
the mixture was cooled in an ice bath. 0.7 mmol
(1.4 equiv) of the halogenating reagent (i.e., IDCP or
Br(coll)2ClO4) and 11 mg Ag(coll)2ClO4 (0.05 equiv)
were added and the mixture was stirred in the dark at
rt (typically 2–20 h, checked by TLC). The reaction mix-
ture was cooled to 0 ꢁC then quenched by a saturated
Na2S2O3 solution (2 mL) and extracted with AcOEt
(4· 10 mL). The organic layer was washed with water
(10 mL), a 1 M HCl solution (3· 5 mL), water (2·
5 mL) and brine (10 mL) and then dried over MgSO4
and concentrated in vacuo. The solid residue was puri-
fied by flash chromatography (eluent: hexanes/AcOEt,
1:1, v/v) to leave the desired monohalogenated com-
pound (6a–c and 7a–c).
4.1.3. 30,50-Di-O-acetyl-20-deoxy-5-iodouridine (4a). The
physicochemical data of this compound are in accor-
dance with those previously published.12,19
4.1.4. 20,30,50-Tri-O-acetyl-5-iodouridine (4b). The physi-
cochemical data of this compound are in accordance
with those previously published.12,19
4.1.5. 20,50-Di-O-acetyl-30-deoxy-5-iodouridine (4c). 89%
1
yield; H NMR (CDCl3) d 9.53 (br s, 1H, NH), 7.93
(s, 1H, H-6), 5.82 (d, J = 1.6 Hz, 1H, H-10), 5.30 (m,
1H, H-20), 4.55 (m, 1H, H-40), 4.41 (dd, J = 12.8,
2.8 Hz, 1H, H-50a), 4.33 (dd, J = 12.8, 3.8 Hz, 1H, H-
50b), 2.31–1.95 (m, 2H, H-30), 2.23 (s, 3H, OAc), 2.13
(s, 3H, OAc).
4.1.6. General procedure for Sonogashira cross-coupling
and subsequent desilylation. Iodinated nucleoside 4a–c
(5 mmol) was dissolved in a mixture of dry DMF
(15 mL), dry Et3N (2.06 mL, 3 equiv) and TMS
(2.06 mL, 3 equiv). CuI (190 mg, 0.2 equiv) and
PdCl2(PPh3)2 (350 mg, 0.1 equiv) were then added and
the reaction mixture was stirred at rt until completion
(typically 5–20 h, checked by TLC). Solvents were evap-
orated under reduced pressure. The oily residue was dis-
solved in AcOEt (250 mL) and then washed with water
4.1.11. 30,50-Di-O-acetyl-5-(2-iodoethynyl)-20-deoxyuri-
20
dine (6a). 93% yield; mp 175–177 (dec) ꢁC; ½aꢀ ꢁ32 (c
D
0.5, CHCl3); 1H NMR (CDCl3) d 11.68 (br s, 1H,
NH), 8.02 (s, 1H, H-6), 6.12 (t, J = 7.1 Hz, 1H, H-10),
5.20 (m, 1H, H-30), 4.33–4.15 (m, 3H, H-40,50), 2.55
(m, 1H, H-20a), 2.31 (m, 1H, H-20b), 2.07 (s, 3H,
OAc), 2.05 (s, 3H, OAc). HRMS m/z calcd for C15H15I-
N2O7Na, 486.1982; found, 486.1986.

6020
V. Escuret et al. / Bioorg. Med. Chem. 13 (2005) 6015–6024
4.1.12.
20,30,50-Tri-O-acetyl-5-(2-iodoethynyl)-uridine
7.67 (s, 1H, H-6), 7.45 (s, 1H, @CHI), 6.36 (dd,
20
(6b). 68% yield; mp 100–102 ꢁC; ½aꢀ ꢁ71 (c 1.0,
J = 8.1, 5.6 Hz, 1H, H-10), 5.25 (m, 1H, H-30), 4.50–
4.28 (m, 3H, H-40,50), 2.60 (m, 1H, H-20a), 2.30 (m,
1H, H-20b), 2.17 (s, 3H, OAc), 2.08 (s, 3H, OAc).
HRMS m/z calcd for C15H16I2N2O7Na, 614.1106;
found, 614.1110.
D
CHCl3); 1H NMR (CDCl3) d 9.41 (br s, 1H, NH),
7.91 (s, 1H, H-6), 6.07 (d, J = 4.6 Hz, 1H, H-10), 5.35
(m, 2H, H-20,30), 4.38 (m, 3H, H-40,50), 2.25 (s, 3H,
OAc), 2.12 (s, 2 · 3H, 2 · OAc). HRMS m/z calcd for
C17H17IN2O9Na, 544.2352; found 544.2349.
4.1.19.
(E)-20,30,50-Tri-O-acetyl-5-(1,2-diiodovinyl)uri-
20
4.1.13. 20,50-Di-O-acetyl-5-(2-iodoethynyl)-30-deoxyuri-
dine (8b). 98% yield; mp 83–85 ꢁC; ½aꢀ ꢁ41 (c 1.0,
D
20
D
dine (6c). 76% yield as an oil; ½aꢀ ꢁ49 (c 0.8, CHCl3);
CHCl3); 1H NMR (CDCl3) d 9.56 (br s, 1H, NH),
7.58 (s, 1H, H-6), 7.40 (s, 1H, @CHI), 6.15 (d,
J = 4.6 Hz, 1H, H-10), 5.36 (m, 2H, H-20,30), 4.38 (br s,
3H, H-40,50), 2.19 (s, 3H, OAc), 2.14 (s, 3H, OAc),
2.11 (s, 3H, OAc). HRMS m/z calcd for C17H18I2N2O9-
Na, 672.1476; found, 672.1480.
1H NMR (CDCl3) d 11.68 (br s, 1H, NH), 7.98 (s, 1H,
H-6), 5.74 (s, 1H, H-10), 5.30 (d, J = 7.5 Hz, 1H, H-20),
4.43 (m, 1H, H-40), 4.28 (br s, 2H, H-50), 2.31–1.95
(m, 2H, H-30), 2.09 (s, 3H, OAc), 2.05 (s, 3H, OAc).
HRMS m/z calcd for C15H15IN2O7Na, 486.1982; found,
486.1986.
4.1.20. (E)-20,50-Di-O-acetyl-5-(1,2-diiodovinyl)-30-deoxy-
20
4.1.14. 30,50-Di-O-acetyl-5-(2-bromoethynyl)-20-deoxyur-
uridine (8c). 87% yield; mp 75–77 ꢁC; ½aꢀ ꢁ23 (c 0.3,
D
20
D
idine (7a). 21% yield as an oil; ½aꢀ ꢁ68 (c 1.0, CHCl3);
CHCl3); 1H NMR (CDCl3) d 9.38 (br s, 1H, NH),
7.64 (s, 1H, H-6), 7.39 (s, 1H, @CHI), 5.92 (d,
J = 1.6 Hz, 1H, H-10), 5.38 (m, 1H, H-40), 4.58 (m,
1H, H-20), 4.44 (dd, J = 12.5, 4.4 Hz, 1H, H-50a), 4.33
(dd, 1H, H-50b), 2.31–1.95 (m, 2H, H-30), 2.14 (s, 3H,
OAc), 2.12 (s, 3H, OAc). HRMS m/z calcd for C15H16I2-
N2O7Na, 614.1106; found, m/z 614.1101.
1H NMR (CDCl3) d 8.88 (br s, 1H, NH), 7.95 (s, 1H, H-
6), 6.31 (dd, J = 7.3, 6.4 Hz, 1H, H-10), 5.25 (m, 1H, H-
30), 4.38–4.30 (m, 3H, H-40,50), 2.55 (m, 1H, H-20a), 2.31
(m, 1H, H-20b), 2.18 (s, 3H, OAc), 2.12 (s, 3H, OAc).
HRMS m/z calcd for C15H15BrN2O7Na, 439.1978;
found, 439.1981.
4.1.15. 20,30,50-Tri-O-acetyl-5-(2-bromoethynyl)-uridine
4.1.21. (E)-30,50-Di-O-acetyl-5-(1-bromo-2-iodovinyl)-20-
20
20
D
(7b). 58% yield as a colourless oil; ½aꢀ ꢁ59 (c 0.4,
deoxyuridine (9a). 96% yield; mp 82–84 ꢁC; ½aꢀ ꢁ14 (c
D
CHCl3); 1H NMR (CDCl3) d 9.75 (br s, 1H, NH),
7.92 (s, 1H, H-6), 6.07 (d, J = 4.4 Hz, 1H, H-10), 5.35
(m, 2H, H-20,30), 4.37 (m, 3H, H-40,50), 2.22 (s, 3H,
OAc), 2.12 (s, 2 · 3H, 2 · OAc). HRMS m/z calcd for
C17H17BrN2O9Na, 497.2348; found 497.2351.
4.0, CHCl3); H NMR (CDCl3) d 9.78 (br s, 1H, NH),
7.71 (s, 1H, H-6), 7.08 (s, 1H, @CHI), 6.25 (dd,
J = 8.1, 5.6 Hz, 1H, H-10), 5.28 (m, 1H, H-30), 4.50–
4.05 (m, 3H, H40, 50), 2.58 (m, 1H, H-20a), 2.20 (m,
1H, H-20b), 2.12 (s, 3H, OAc), 1.96 (s, 3H, OAc).
HRMS m/z calcd for C15H16BrIN2O7Na, 567.1102;
found, 567.1107.
1
4.1.16. 20,50-Di-O-acetyl-5-(2-bromoethynyl)-30-deoxyur-
20
idine (7c). 25% yield as an oil; ½aꢀ ꢁ52 (c 0.4, CHCl3);
D
1H NMR (DMSO-d6) d 11.75 (br s, 1H, NH), 8.07 (s,
1H, H-6), 5.85 (s, 1H, H-10), 5.31 (d, J = 7.5 Hz, 1H,
H-20), 4.43 (m, 1H, H-40), 4.28 (br s, 2H, H-50), 2.31-
1.95 (m, 2H, H-30), 2.08 (s, 3H, OAc), 2.06 (s, 3H,
OAc). HRMS m/z calcd for C15H15BrN2O7Na,
439.1978; found 439.1975.
4.1.22. (E)-20,30,50-Tri-O-acetyl-5-(1-bromo-2-iodovinyl)-
20
uridine (9b). 92% yield; mp 86–88 ꢁC; ½aꢀ ꢁ39 (c 2.0,
D
CHCl3); 1H NMR (CDCl3) d 9.57 (br s, 1H, NH),
7.66 (s, 1H, H-6), 7.15 (s, 1H, @CHI), 6.15 (d,
J = 4.7 Hz, 1H, H-10), 5.38 (m, 2H, H-20,30), 4.38 (m,
3H, H-40,50), 2.18 (s, 3H, OAc), 2.15 (s, 3H, OAc),
2.13 (s, 3H, OAc). HRMS m/z calcd for C17H18BrI-
N2O9Na, 625.1469; found, m/z 625.1473.
4.1.17. General procedure for dihalogenation of 5-ethynyl
nucleosides. 5-Ethynyl nucleoside 5a–c (0.5 mmol) was
dissolved in 5 mL dry CH3CN, and the mixture was
cooled in an ice bath. 0.6 mmol (1.2 equiv) of a solution
of the halogenating reagent (i.e., I2, IBr, ICl or Br2) in
1 mL dry CH3CN was added dropwise and the mixture
was stirred at 0 ꢁC until completion (typically 15 min to
2 h, checked by TLC). The reaction mixture was
quenched at 0 ꢁC by a saturated Na2S2O3 solution
(2 mL) and then extracted with AcOEt (3· 10 mL).
The organic layer was washed with water (2· 5 mL)
and brine (10 mL) then dried over MgSO4 and concen-
trated in vacuo to let the desired dihalogenated com-
pound (8a–c to 11a–c). Pure analytical samples were
obtained using flash chromatography (eluent: hexanes/
AcOEt, 1:1).
4.1.23. (E)-20,50-Di-O-acetyl-5-(1-bromo-2-iodovinyl)-30-
20
deoxyuridine (9c). 89% yield as an oil; ½aꢀ ꢁ19 (c 0.5,
D
CHCl3); 1H NMR (CDCl3) d 9.63 (br s, 1H, NH),
7.61 (s, 1H, H-6), 7.13 (s, 1H, @CHI), 5.92 (s, 1H, H-
10), 5.38 (m, 1H, H-40), 4.58 (m, 1H, H-20), 4.44 (dd,
J = 12.4, 2.6 Hz, 1H, H-50a), 4.33 (dd, J = 12.4,
4.4 Hz, 1H, H-50b), 2.31–1.95 (m, 2H, H-30), 2.16 (s,
3H, OAc), 2.14 (s, 3H, OAc). HRMS m/z calcd for
C15H16BrIN2O7Na, 567.1102; found, m/z 567.1105.
4.1.24. (E)-30,50-Di-O-acetyl-5-(1-chloro-2-iodovinyl)-20-
20
deoxyuridine (10a). 91% yield; mp 81–83 ꢁC; ½aꢀ ꢁ14
D
(c 0.7, CHCl3); 1H NMR (CDCl3) d 9.28 (br s, 1H,
NH), 7.75 (s, 1H, H-6), 6.91 (s, 1H, @CHI), 6.32 (m,
1H, H-10), 5.24 (m, 1H, H-30), 4.50–4.30 (m, 3H, H-
40,50), 2.75 (m, 1H, H-20a), 2.18 (m, 1H, H-20b), 2.13
(s, 2 · 3H, 2 · OAc). HRMS m/z calcd for C15H16ClI-
N2O7Na, 522.6592; found, 522.6597.
4.1.18. (E)-30,50-Di-O-acetyl-5-(1,2-diiodovinyl)-20-deoxy-
20
D
uridine (8a). 94% yield; mp 76–78 ꢁC; ½aꢀ ꢁ19 (c 0.3,
CHCl3); 1H NMR (CDCl3) d 9.72 (br s, 1H, NH),

V. Escuret et al. / Bioorg. Med. Chem. 13 (2005) 6015–6024
6021
4.1.25.
(E)-20,30,50-Tri-O-acetyl-5-(1-chloro-2-iodovi-
4.1.31. 5-(2-Iodoethynyl)-20-deoxyuridine (12a). 68%
20
D
20
yield; mp 135–137 ꢁC; ½aꢀ +14 (c 0.8, MeOH); 1H
nyl)uridine (10b). 97% yield; mp 85–87 ꢁC; ½aꢀ ꢁ27 (c
D
1
0.8, CHCl3); H NMR (CDCl3) d 9.43 (br s, 1H, NH),
7.68 (s, 1H, H-6), 6.92 (s, 1H, @CHI), 6.15 (br s, 1H,
H-10), 5.36 (m, 2H, H-20,30), 4.38 (m, 3H, H-40,50),
2.17 (s, 3H, OAc), 2.14 (s, 3H, OAc), 2.12 (s, 3H,
OAc). HRMS m/z calcd for C17H18ClIN2O9Na,
580.6962; found, 580.6958.
NMR (CD3OD) d 8.32 (s, 1H, H-6), 6.22 (t,
J = 6.6 Hz, 1H, H-10), 4.41 (m, 1H, H-30), 3.95 (m,
1H, H-40), 3.83 (dd, J = 12.1, 3.0 Hz, 1H, H-50a), 3.73
(dd, J = 12.1, 3.4 Hz, 1H, H-50b), 2.40–2.12 (m, 2H,
H-20). HRMS m/z calcd for C11H11IN2O5Na,
402.1229; found, 402.1231.
4.1.26. (E)-20,50-Di-O-acetyl-5-(1-chloro-2-iodovinyl)-30-
4.1.32. 5-(2-Iodoethynyl)-uridine (12b). 70% yield as an
20
20
D
1
deoxyuridine (10c). 83% yield; mp 87–89 ꢁC; ½aꢀ ꢁ13
oil; ½aꢀ ꢁ12 (c 0.2, MeOH); H NMR (DMSO-d6) d
D
(c 1.0, CHCl3); 1H NMR (CDCl3) d 9.75 (br s, 1H,
NH), 7.74 (s, 1H, H-6), 6.90 (s, 1H, @CHI), 5.91
(d, J = 1.7 Hz, 1H, H-10), 5.38 (m, 1H, H-40), 4.58
(m, 1H, H-20), 4.43 (dd, J = 12.5, 2.8 Hz, 1H, H-
50a), 4.33 (dd, J = 12.5, 4.1 Hz, 1H, H-50b), 2.35-1.95
(m, 2H, H-30), 2.14 (s, 2 · 3H, 2 · OAc). HRMS
m/z calcd for C15H16ClIN2O7Na, 522.6592; found,
522.6593.
11.65 (br s, 1H, NH), 8.29 (s, 1H, H-6), 5.72 (m, 1H,
H-10), 5.05–5.12 (3 · br s, 3 · OH), 4.05–3.95 (m, 2H,
H-20,30), 3.85 (m, 1H, H-40), 3.70–355 (m, 2H, H-50).
HRMS m/z calcd for C11H11IN2O6Na, 418.1223; found,
418.1219.
4.1.33. 5-(2-Iodoethynyl)-30-deoxyuridine (12c). 66%
20
D
yield as an oil; ½aꢀ +6 (c 1.0, MeOH); 1H NMR
(DMSO-d6) d 11.65(s, 1H, NH), 8.48 (s, 1H, H-6),
5.59 (s, 1H, H-10), 5.54 (d, J = 4.1 Hz, 1H, OH), 5.23
(t, J = 5.1 Hz, 1H, OH), 4.32 (m, 1H, H-40), 4.21 (m,
1H, H-20), 3.75 (m, 1H, H-50a), 3.52 (m, 1H, H-50b),
1.98 (m, 1H, H-30a), 1.76 (m, 1H, H-30b). HRMS m/z
calcd for C11H11IN2O5Na, 402.1229; found, 402.1233.
4.1.27. 30,50-Di-O-acetyl-5-(1,2-dibromovinyl)-20-deoxy-
20
uridine (11a). 97% yield; mp 79–81 ꢁC; ½aꢀ ꢁ15 (c 1.0,
D
CHCl3); 1H NMR (CDCl3) d 9.85 (br s, 1H, NH),
7.75 (s, 1H, H-6), 6.89 (s, 1H, @CHBr), 6.33 (dd,
J = 8.1, 5.6 Hz, 1H, H-10), 5.24 (m, 1H, H-30), 4.45–
4.25 (m, 3H, H-40,50), 2.65 (m, 1H, H-20a), 2.18 (m,
1H, H-20b), 2.14 (s, 2 · 3H, 2 · OAc). HRMS m/z calcd
for C15H16Br2N2O7Na, 520.1098; found, 520.1099.
4.1.34. 5-(2-Bromoethynyl)-20-deoxyuridine (13a). 80%
20
D
yield as an oil; ½aꢀ +3 (c 0.3, MeOH); 1H NMR
(CD3OD) d 8.30 (s, 1H, H-6), 6.05 (t, J = 6.3 Hz, 1H,
H-10), 4.21 (m, 1H, H-30), 3.78 (m, 1H, H-40), 3.65–
3.50 (m, 2H, H-50), 2.15 (dd, J = 5.6, 5.0 Hz, 2H, H-
20). HRMS m/z calcd for C11H11BrN2O5Na, 355.1225;
found, 355.1227.
4.1.28. (E)-20,30,50-Tri-O-acetyl-5-(1,2-dibromo-vinyl)uri-
20
dine (11b). 92% yield; mp 83–85 ꢁC; ½aꢀ ꢁ43 (c 0.5,
D
CHCl3); 1H NMR (CDCl3) d 9.38 (br s, 1H, NH),
7.66 (s, 1H, H-6), 6.90 (s, 1H, @CHBr), 6.12 (d,
J = 4.8 Hz, 1H, H-10), 5.36 (m, 2H, H-20,30), 4.38 (m,
3H, H-40,50), 2.17 (s, 3H, OAc), 2.14 (s, 3H, OAc),
2.12 (s, 3H, OAc). HRMS m/z calcd for
C17H18Br2N2O9Na, 578.1468; found, 578.1473.
4.1.35. 5-(2-Bromoethynyl)-uridine (13b). 74% yield as an
20
D
1
oil; ½aꢀ ꢁ23 (c 0.5, MeOH); H NMR (DMSO-d6) d
11.69 (br s, 1H, NH), 8.39 (s, 1H, H-6), 5.73 (d,
J = 4.7 Hz, 1H, H-10), 5.41 (d, J = 5.3 Hz, OH), 5.24
(t, J = 4.7 Hz, OH), 5.08 (d, J = 5.3 Hz, OH), 4.10–
3.91 (m, 2H, H-20,30), 3.85 (m, 1H, H-40), 3.75–350 (m,
2H, H-50). HRMS m/z calcd for C11H11BrN2O6Na,
371.1219; found, 371.1224.
4.1.29. 20,50-Di-O-acetyl-5-(1,2-dibromovinyl)-30-deoxy-
20
uridine (11c). 82% yield; mp 78–80 ꢁC; ½aꢀ ꢁ16 (c 0.8,
D
CHCl3); 1H NMR (CDCl3) d 9.61 (br s, 1H, NH),
7.72 (s, 1H, H-6), 6.88 (s, 1H, @CHI), 5.88 (d,
J = 1.5 Hz, 1H, H-10), 5.38 (m, 1H, H-40), 4.58 (m,
1H, H-20), 4.43 (dd, J = 12.5, 2.8 Hz, 1H, H-50a), 4.33
(dd, J = 12.5, 4.1 Hz, 1H, H-50b), 2.35–1.95 (m, 2H,
H-30), 2.15 (s, 3H, OAc), 2.14 (s, 3H, OAc). HRMS
m/z calcd for C15H16Br2N2O7Na, 520.1098; found,
520.1103.
4.1.36. 5-(2-Bromoethynyl)-30-deoxyuridine (13c). 72%
20
D
yield as an oil; ½aꢀ ꢁ29 (c 1.0, MeOH); 1H NMR
(DMSO-d6) d 11.63(s, 1H, NH), 8.53 (s, 1H, H-6),
5.59 (s, 1H, H-10), 5.56 (d, J = 4.1 Hz, 1H, OH), 5.26
(t, J = 5.1 Hz, 1H, OH), 4.32 (m, 1H, H-40), 4.25 (m,
1H, H-20), 3.81 (m, 1H, H-50a), 3.61 (m, 1H, H-50b),
1.95 (m, 1H, H-30a), 1.72 (m, 1H, H-30b). HRMS m/z
calcd for C11H11BrN2O5Na, 355.1225; found, 355.1223.
4.1.30. General procedure for deacetylation. Acetylated
nucleoside analogue (6a–c to 11a–c) (1 mmol) was dis-
solved in 10 mL pyridine and 5 mL EtOH. The reac-
tion mixture was cooled to ꢁ10 ꢁC and 5 mL of a
1 M NaOH aqueous solution was added. The resulting
solution was stirred at this temperature until comple-
tion (typically 1–4 h, checked by TLC). The reaction
mixture was neutralized with Dowex 50X2-200 then
filtered through a fritted glass funnel. Solvents were
evaporated in vacuo, and the oily residue was submit-
ted to a flash column chromatography using an
appropriate eluent (typically hexanes/EtOAc 25:75
then EtOAc then MeOH/AcOEt 99:1) to let pure
12a–c to 17a–c.
4.1.37. (E)-5-(1,2-Diiodovinyl)-20-deoxyuridine (14a).
20
72% yield; mp 103–105 ꢁC; ½aꢀ +28 (c 0.3, MeOH);
D
1H NMR (CD3OD) d 8.27 (s, 1H, H-6), 7.52 (s, 1H,
@CHI), 6.28 (t, J = 6.3 Hz, 1H, H-10), 4.43 (m, 1H, H-
30), 3.97 (m, 1H, H-40), 3.83 (dd, J = 11.9, 3.2 Hz, 1H,
H-50a), 3.73 (m, J = 11.9, 3.4 Hz, 1H, H-50b), 2.43–
2.12 (m, 2H, H-20). HRMS m/z calcd for C11H12I2N2O5-
Na, 530.0353; found, 530.0558.
4.1.38. (E)-5-(1,2-Diiodovinyl)uridine (14b). 82% yield;
20
mp 107–109 ꢁC; ½aꢀ +5 (c 1.0, MeOH); 1H NMR
D

6022
V. Escuret et al. / Bioorg. Med. Chem. 13 (2005) 6015–6024
(DMSO-d6) d 11.65 (br s, 1H, NH), 8.05 (s, 1H, H-6),
7.05 (s, IH, @CHI), 5.80 (d, J = 4.7 Hz, 1H, H-10),
5.50–5.10 (3 · br s, 3 · OH), 4.12–3.95 (m, 2H, H-
20,30), 3.87 (m, 1H, H-40), 3.75–355 (m, 2H, H-50).
HRMS m/z calcd for C11H12I2N2O6Na, 546.0347;
found, 546.0351.
NMR (DMSO-d6 + D2O) d 8.38 (s, 1H, H-6), 7.18 (s,
1H, @CHI), 5.65 (d, J = 1.2 Hz, 1H, H-10), 4.32 (m,
1H, H-40), 4.25 (m, 1H, H-20), 3.76 (dd, J = 12.3,
2.6 Hz, 1H, H-50a), 3.52 (dd, J = 12.3, 2.8 Hz, 1H, H-
50b), 2.01 (m, 1H, H-30a), 1.75 (m, 1H, H-30b). HRMS
m/z calcd for C11H12ClIN2O5Na, 438.5839; found, m/z
438.5837.
4.1.39. (E)-5-(1,2-Diiodovinyl)-30-deoxyuridine (14c).
20
D
76% yield as an oil; ½aꢀ +1.0 (c 0.8, MeOH); 1H
4.1.46. (E)-5-(1,2-Dibromovinyl)-20-deoxyuridine (17a).
20
D
1
NMR (DMSO-d6 + D2O) d 8.21 (s, 1H, H-6), 7.55 (s,
1H, @CHI), 5.61 (s, 1H, H-10), 4.32 (m, 1H, H-40),
4.25 (m, 1H, H-20), 3.81 (m, 1H, H-50a), 3.52 (m, 1H,
H-50b), 1.97 (m, 1H, H-30a), 1.74 (m, 1H, H-30b).
HRMS m/z calcd for C11H12I2N2O5Na, 530.0553;
found, 530.0554.
85% yield as an oil; ½aꢀ +26 (c 0.5, MeOH); H NMR
(CD3OD) d 8.39 (s, 1H, H-6), 7.06 (s, 1H, @CHBr),
6.28 (t, J = 6.5 Hz, 1H, H-10), 4.41 (m, 1H, H-30), 3.95
(m, 1H, H-40), 3.82 (dd, J = 11.9, 2.9 Hz, 1H, H-50a),
3.73 (dd, J = 11.9, 3.4 Hz, 1H, H-50b), 2.45–2.10 (m,
2H, H-20). HRMS m/z calcd for C11H12Br2N2O5Na,
436.0345; found, m/z 436.0348.
4.1.40.
(E)-5-(1-Bromo-2-iodovinyl)-20-deoxyuridine
20
1
(15a). 78% yield as an oil; ½aꢀ +26 (c 2.0, MeOH); H
4.1.47. (E)-5-(1,2-Dibromovinyl)uridine (17b). 72% yield
D
20
D
1
NMR (CD3OD) d 8.35 (s, 1H, H-6), 7.39 (s, 1H, @CHI),
6.25 (t, J = 6.5 Hz, 1H, H-10), 4.45 (m, 1H, H-30), 3.94
(m, 1H, H-40), 3.86 (dd, J = 11.9, 3.2 Hz, 1H, H-50a),
3.72 (dd, J = 11.9, 3.4 Hz, 1H, H-50b), 2.45–2.15 (m,
2H, H-20). HRMS m/z calcd for C11H12BrIN2O5Na,
483.0349; found, 483.0352.
as an oil; ½aꢀ ꢁ14 (c 0.5, MeOH); H NMR (D2O) d
8.45 (s, 1H, H-6), 7.07 (s, IH, @CHBr), 5.93 (d,
J = 5.2 Hz, 1H, H-10), 4.18 (m, 2H, H-20,30), 4.05 (m,
1H, H-40), 3.88 (dd, J = 12.5, 2.5 Hz, 1H, H-50a), 3.74
(dd, J = 12.5, 2.8 Hz, 1H, H-50b). HRMS m/z calcd for
C11H12Br2N2O6Na, 452.0339; found, 452.0342.
4.1.41. (E)-5-(1-Bromo-2-iodovinyl)uridine (15b). 76%
4.1.48. (E)-5-(1,2-Dibromovinyl)-30-deoxyuridine (17c).
20
D
20
D
1
yield as an oil; ½aꢀ ꢁ1 (c 0.3, MeOH); 1H NMR
74% yield as an oil; ½aꢀ +5 (c 1.0, MeOH); H NMR
(D2O) d 8.27 (s, 1H, H-6), 7.45 (s, IH, @CHI), 5.79 (d,
J = 4.7 Hz, 1H, H-10), 4.15–3.92 (m, 2H, H-20,30), 3.88
(m, 1H, H-40), 3.67 (dd, J = 11.8, 2.5 Hz, 1H, H-50a),
3.56 (dd, 1H, H-50b). HRMS m/z calcd for C11H12BrI-
N2O6Na, 499.0343; found, m/z 499.0348.
(DMSO-d6 + D2O) d 8.42 (s, 1H, H-6), 7.25 (s, 1H,
@CHBr), 5.64 (br s, 1H, H-10), 4.35 (m, 1H, H-40),
4.25 (m, 1H, H-20), 3.78 (m, H-50a), 3.54 (m, H-50b),
1.95 (m, 1H, H-30a), 1.75 (m, 1H, H-30b). HRMS m/z
calcd for C11H12Br2N2O5Na, 436.0345; found, 436.0340.
4.1.42.
(E)-5-(1-Bromo-2-iodovinyl)-30-deoxyuridine
4.2. Biological evaluation
20
D
1
(15c). 68% yield as an oil; ½aꢀ +3 (c 1.0, MeOH); H
NMR (DMSO-d 6 + D2O) d 8.35 (s, 1H, H-6), 7.39 (s,
1H, @CHI), 5.61 (d, J = 1.3 Hz, 1H, H-10), 4.32 (m,
1H, H-40), 4.25 (m, 1H, H-20), 3.76 (dd, J = 12.2,
2.4 Hz, 1H, H-50a), 3.52 (dd, J = 12.2, 2.8 Hz, 1H, H-
50b), 1.97 (m, 1H, H-30a), 1.75 (m, 1H, H-30b). HRMS
m/z calcd for C11H12BrIN2O5Na, 483.0349; found, m/z
483.0344.
4.2.1. Antiviral and Cytotoxicity assays for HCV. Huh7
cells harbouring the subgenomic HCV replicon BM4-
5, kindly provided by Seeger,11 were used in this study.
Cells were maintained in DulbeccoÕs modified EagleÕs
medium high glucose 4.5 g/L (LifeTechnologies) supple-
mented with 10% fetal bovine serum, 1% L-glutamine,
1% penicillin and streptomycin, 1% L-pyruvate and
500 lg/mL of geneticin (G418; Invitrogen). Geneticin
was used to select cells permitting the HCV RNA repli-
cation. Cells were passaged every 4 days with a 1:4 ratio.
4.1.43.
(E)-5-(1-Chloro-2-iodovinyl)-20-deoxyuridine
20
(16a). 71% yield; mp 109–111 ꢁC; ½aꢀ +34 (c 0.5,
D
1
MeOH); H NMR (CD3OD) d 8.38 (s, 1H, H-6), 7.06
(s, 1H, @CHI), 6.30 (t, J = 6.5 Hz, 1H, H-10), 4.43
(ddd, J = 6.5, 3.8, 3.6 Hz, 1H, H-30), 3.96 (ddd,
J = 3.6, 3.4, 3.0 Hz, 1H, H-40), 3.83 (dd, J = 11.9,
3.1 Hz, 1H, H-50a), 3.73 (dd, J = 11.9, 3.4 Hz, 1H, H-
50b), 2.45–2.10 (m, 2H, H-20). HRMS m/z calcd for
C11H12ClIN2O5Na, 438.5839; found, m/z 438.5843.
Cells were seeded in 6-well plates at a density of
2.5 · 105 cells per well, 16 h before the beginning of
treatment. Cells were treated with the molecules admin-
istered at different concentrations in complete medium
that did not contain geneticin. The administration of
each drug was renewed every day for three consecutive
days. Ribavirin (ICN Pharmaceuticals), mycophenolic
acid (Sigma) and IFNa-2b(IntronAꢂ) were used in the
same conditions as positive controls. The concentrations
used for these drugs were, 0, 0.5, 1, 2, 4, 8, 16, 32, 64,
128, 256, and 512 lM for ribavirin: 0, 2.5, 5, 10, 20,
and 40 lM for mycophenolic acid, and 0, 0.1, 1, 10,
100 and 1000 UI/mL for IFNa-2b. Total RNA was
extracted at the end of treatment (24 h after the last
day of treatment) with the ÔExtract allÕ reagent (Euro-
bio), which is a mix of phenol and guanidinium thiocy-
anate. Northern blot analysis was then performed using
4.1.44. (E)-5-(1-Chloro-2-iodovinyl)uridine (16b). 78%
20
D
yield; mp 109–111 ꢁC; ½aꢀ +5 (c 0.4, MeOH); 1H
NMR (D2O) d 8.45 (s, 1H, H-6), 7.05 (s, IH, @CHI),
5.89 (br s, 1H, H-10), 4.30 (br s, 2H, H-20,30), 4.10 (m,
1H, H-40), 3.85 (dd, J = 11.5, 2.2 Hz, 1H, H-50a), 3.75
(dd, 1H, H-50b). HRMS m/z calcd for C11H12IClN2O6-
Na, 454.5833; found, m/z 454.5835.
4.1.45.
(E)-5-(1-Chloro-2-iodovinyl)-30-deoxyuridine
20
1
(16c). 70% yield as an oil; ½aꢀ +2 (c 2.0, MeOH); H
D

V. Escuret et al. / Bioorg. Med. Chem. 13 (2005) 6015–6024
6023
the NorthernMaxTM-Gly kit (Ambion), following man-
ufacturerÕs instructions. Five micrograms of total RNA
were denaturated in glyoxal buffer at 50 ꢁC for 30 min,
separated by 1.1% agarose gel electrophoresis and then
transferred for 12 h onto a charged nylon membrane
(HybondN+; Amersham). Hybridization was carried
out with three different [32P]CTP-labelled riboprobes
obtained by in vitro transcription (Riboprobe in vitro
transcription system; Promega). Two probes were com-
plementary to the NS5A region of the HCV genome of
negative polarity and positive polarity. A third probe
was complementary to the beta-actin mRNA and ob-
tained by in vitro transcription from a specific plasmid
(pTRI beta actin human, reference 7424; Ambion).
First, the blot was hybridized with the riboprobes direct-
ed against the negative strand of HCV RNA and beta-
actin mRNA. After one night of hybridization at
68 ꢁC, the membrane was washed, and then exposed to
X-ray film and a phosphor screen (phosphorimager).
This screen was then scanned and quantitative analysis
was achieved using ImageQuant software. The amount
of beta-actin mRNA was used as an internal loading
control to standardize the amount of HCV RNA detect-
ed. The same membrane was subsequently hybridized
with the negative sense riboprobe to determine the level
of positive strand HCV RNA, using the same approach.
postinoculation to evaluate the EC50 on BVDV (strain
NADL), according to the different conditions of treat-
ment. By definition, the EC50 is the concentration of
drug that inhibits 50% of the CPE in comparison to cells
inoculated but untreated presenting 100% of CPE.
Uninfected MDBK cells were grown in the absence or
presence of varying concentrations of drug tested. After
3 days, toxicity was evaluated by neutral red colouration
as described previously.
4.2.3. Cell culture assays for HIV-1. Antiviral and cyto-
toxicity assays were conducted as described recently by
Stuyver et al.16 HIV-1 antiviral assays were performed
in activated primary human PBM.
Acknowledgments
We thank the CNRS and MENRT for financial support.
´
N.J. thanks the CNRS and la Region Centre for a PhD
fellowship (BDI). R.F.S. was supported by EmoryÕs Cen-
ter for AIDS Research NIH Grant 2P30-AI-50409 and
by the Department of Veterans Affairs. Part of this work
was supported by a grant (LSHM-CT-2004-503359,
VIRGIL European Network of Excellence on Antiviral
Drug Resistance) from the Priority 1 ÔLife Sciences,
Genomics and Biotechnology for HealthÕ programme
in the 6th Framework Programme of the EU.
For cell viability assays, cells were seeded in 96-well
plates at a density of 12,500 cells per well. They were
treated by the different molecules with the same concen-
trations and conditions than those used for the antiviral
assays. Then, cell viability was measured by neutral red
assay. Neutral red which specifically colours lysosomes
and its accumulation depends on cellular membrane
integrity. The yield of neutral red incorporated in cells
is proportional to the number of living cells. At the
end of treatment, culture medium was removed; cells
were washed by PBS and then coloured with neutral
red at 0.005% for 3 h at 37 ꢁC. Cells were then fixed
1 min by formol calcium and lysed by a treatment with
a v/v mixture of acetic acid and ethanol. After 15 min
incubation, absorbance was read at 490 nm.
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