Arylethynyltriazole acyclonucleosides inhibit hepatitis C virus replication

Article abstract of DOI:10.1016/j.bmcl.2008.04.026

Novel acyclic triazole nucleosides with various ethynyl moieties appended on the triazole nucleobase were synthesized efficiently using a convenient one-step Sonogashira reaction in aqueous solution and under microwave irradiation. One of the compounds, 1f, inhibited HCV subgenomic replication with a 50% effective concentration (EC₅₀) of 22 μg/ml and did not inhibit proliferation of the host cell at a concentration of 50 μg/ml. A preliminary SAR study suggests that the appended phenyl ring as well as the rigid triple bond linker contributes importantly to the anti-HCV activity.

Full text of DOI:10.1016/j.bmcl.2008.04.026

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 3321–3327
Arylethynyltriazole acyclonucleosides inhibit hepatitis C
virus replication
Ruizhi Zhu,a Menghua Wang,a Yi Xia,a,b Fanqi Qu,a Johan Neytsc and Ling Penga,b,
*
aCollege of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, PR China
bDe´partement de Chimie, CNRS UPR 3118, 163 avenue de Luminy, 13288 Marseille cedex 09, France
cRega Institute for Medical Research, Minderbroedersstraat 10, B-3000 Leuven, Belgium
Received 1 April 2008; revised 9 April 2008; accepted 10 April 2008
Available online 15 April 2008
Abstract—Novel acyclic triazole nucleosides with various ethynyl moieties appended on the triazole nucleobase were synthesized
efficiently using a convenient one-step Sonogashira reaction in aqueous solution and under microwave irradiation. One of the com-
pounds, 1f, inhibited HCV subgenomic replication with a 50% effective concentration (EC50) of 22 lg/ml and did not inhibit pro-
liferation of the host cell at a concentration of 50 lg/ml. A preliminary SAR study suggests that the appended phenyl ring as well as
the rigid triple bond linker contributes importantly to the anti-HCV activity.
Ó 2008 Elsevier Ltd. All rights reserved.
Hepatitis C virus (HCV) infection constitutes a serious
public health problem: an estimated 3% of the world
population—about 170 millions people—are infected
by HCV.1 Current standard for the treatment of HCV
is pegylated interferon-a in combination with ribavirin.2
However, this therapy is only effective in about 50% of
the patients and is associated with serious side effects.2
Therefore, there is an urgent need to develop more effi-
cacious and better tolerated new antiviral candidates for
combating HCV.3
modification of the sugar moiety were identified as
inhibitors of HCV replication; the 50-triphosphate
metabolites of these drugs inhibit the viral polymerase.5
Appending aromatic systems to nucleobase may yield
nucleosides with unique properties and biological activ-
ities since the appended aromatic systems may promote
advantageous binding properties to the corresponding
nucleosides for interaction with biological targets
through stronger and more efficient binding via larger
aromatic systems. Successful examples are 6-arylpurine
nucleosides (Scheme 1), which have been shown to elicit
anti-HCV and anticancer activities.6 HEPT, an acyclo-
nucleoside analog of which the pyrimidine nucleobase
bears a phenylthio group at the 6-position (Scheme 1),
is another example that has been reported to show a po-
tent and selective anti-HIV activity.7 The appended aryl
at the 6-position in HEPT is one of the key factors con-
tributing to the anti-HIV activity. X-ray structural anal-
ysis revealed that HEPT acts as a non-nucleoside
inhibitor of the HIV reverse transcriptase and that the
aryl group at the 6-position in HEPT interacts with
the amino acid residues Tyr188 and Leu100 in the
HIV reverse transcriptase, leading to the conformational
change of this enzyme.8
Of the drugs currently marketed for the treatment of vir-
al infection, almost half of them are nucleoside analogs.
Nucleoside analogs with modified sugar and/or base
components can mimic natural nucleosides and serve
as building units or inhibitors to interfere in the nucleic
acid synthesis or to block the biological processes
involving the action of nucleos(t)ides. Ribavirin
(Scheme 1), the nucleobase of which consists of an
unnatural triazole moiety, was the first synthetic nucleo-
side to show a broad spectrum of an antiviral activity
against many RNA and DNA viruses.4 It is the only
small molecular drug available to date for treating pa-
tients infected with hepatitis C virus.2 Recently, nucleo-
side analogs with either a 20-C-methyl, a 20-F or 40-azido
In our ongoing efforts to search for potent new triazole
nucleosides with antiviral activity,9–11 we are interested
in developing acyclic triazole nucleosides with p-conju-
gated aromatic systems appended on the triazole ring.
Keywords: Triazole nucleosides; Antiviral activity; Anti-HCV; Sono-
gashira reaction; Microwave-irradiation promoted reaction.
*
Corresponding author. Tel.: +33 491 82 91 54; fax: +33 491 82 93
01; e-mail: ling.peng@univmed.fr
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.04.026

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R. Zhu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3321–3327
O
Ar
N
O
N
N
N
N
N
NH2
N
Me
S
N
HN
HO
HO
O
O
O
HO
O
OH OH
OH OH
Ribavirin
Arylpurine
HEPT
ribonucleosides
R
O
O
NH2
O
N
N
N
N
N
NH2
N
Ar
HO
N
Ar
N
R'
N
N
N N
O
HO
HO
O
O
Bitriazolyl
acyclonucleoside
Aryltriazole
1
acyclonucleosides
Scheme 1. Ribavirin, 6-arylpurine ribonucleosides, HEPT and various aryltriazole acyclonucleoside derivatives.
We expect that these appended aromatic systems will offer
the triazole advantageous binding properties to the corre-
sponding biological targets via larger aromatic systems.
Furthermore, nucleosides with unnatural triazole nucleo-
bases are generally resistant to nucleos(t)ide metabolizing
enzymes, and may lead to a better in vivo stability and effi-
ciency. Until now, very few efforts have been made on
appending aromatic systems to triazole nucleosides,
probably due to the lack of convenient and practical syn-
thetic methods. Making use of Huisgen cycloaddition and
Suzuki coupling reaction, we have recently synthesized
bitriazolyl10 and aryltriazole acyclonucleosides11
(Scheme 1), some of which displayed interesting antiviral
activity against the tobacco mosaic virus.10 Here, we re-
port the synthesis of a new family of triazole nucleosides,
5-arylethynyltriazole acyclonucleosides 1 (Scheme 1), and
the evaluation of their antiviral activity against HCV.
Sonogashira reaction12 is one of the most straightfor-
ward and convenient approach to introduce an alkynyl
functional group on aromatic systems and heterocyclic
nucleobases. Starting from bromotriazole acyclonucleo-
side (2),11a we developed herein a simple and efficient
one-step procedure to synthesize ethynyltriazole acyclo-
nucleosides (1) in an aqueous solution via a Sonogashira
reaction promoted by microwave irradiation (Tables 1
and 2).
Table 1 summarized our efforts to optimize the experi-
mental conditions for such Sonogashira reaction
between the bromotriazole derivative 2 and phenylacet-
ylene. We know from our previous experience that the
starting material 2 undergoes easily intra-molecular
cyclization under basic conditions, leading to the forma-
tion of 3.11a Our optimization effort was therefore
Table 1. Optimization of Sonogashira reaction under microwave irradiation
O
O
O
NH2
NH2
NH2
N
N
N
Br
N
N
O
N
N
N
+
N
Pd catalyst, Base, MW
HO
HO
O
O
O
2
1a
3
Entry
Solvent
Base (2 equiv)
Pd catalyst (5%)
Temperature (°C)
1a (%)
3 (%)
2 (%)
1
H2O
Li2CO3
Li2CO3
Li2CO3
Li2CO3
Li2CO3
Li2CO3
Li2CO3
Li2CO3
Li2CO3
Li2CO3
K2CO3
Et3N
Pd(PPh3)4:CuI (1:1)
Pd(PPh3)4:CuI (1:1)
Pd(PPh3)4:CuI (1:1)
Pd(PPh3)4:CuI (1:1)
Pd(PPh3)4:CuI (1:1)
100
100
100
100
100
100
100
100
100
100
100
100
80
0
30
0
0
0
0
0
0
0
0
0
9
0
0
0
2
Dioxane/H2O (1:1)
Dioxane/H2O (3:1)
CH3CN/H2O (3:1)
THF/H2O (3:1)
36
99
77
88
76
0
60
0
3
4
10
0
5
6
Dioxane/H2O (3:1)
Dioxane/H2O (3:1)
Dioxane/H2O (3:1)
Dioxane/H2O (3:1)
Dioxane/H2O (3:1)
Dioxane/H2O (3:1)
Dioxane/H2O (3:1)
Dioxane/H2O (3:1)
Pd(PPh3)2Cl2:CuI (1:1)
Pd2(dba)3:CuI (1:1)
0
7
83
66
60
50
0
8
Pd(OAc)2:CuI: BINAP (1:1:2)
Pd(PPh3)4
5
9
30
49
83
93
65
10
11
12
13
2.5% Pd(PPh3)4:CuI (1:1)
Pd(PPh3)4:CuI (1:1)
Pd(PPh3)4:CuI (1:1)
Pd(PPh3)4:CuI (1:1)
0
32
Li2CO3

R. Zhu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3321–3327
3323
Table 2. Synthesis of 1 via the Sonogashira reaction between 2 and
various terminal alkynes
by-product 3 (Table 1, entry 1). Using mixed solvent sys-
tems could efficiently suppress this side reaction (Table
1, entries 2–5). The best result was obtained with diox-
ane/H2O (3:1, v/v) which gave the desired product 1a
in almost quantitative yield (Table 1, entry 3). Among
the four common Pd-catalysts tested, Pd(PPh3)4,
Pd(PPh3)2Cl2, Pd2(dba)3, and Pd(OAc)2 (Table 1, entries
3, 6, 7, and 8), Pd(PPh3)4 was identified as the most effi-
cient. Furthermore, CuI is an important co-catalyst
since its absence led to the incomplete transformation
of the starting material 2 and significantly lower yield
of the desired product 1a (Table 1, entry 9). It is also
important to maintain the catalyst loading to 5% be-
cause lower catalyst loading led to a dramatic yield de-
crease (Table 1, entry 10). In order to avoid the
formation of the cyclization by-product 3 under the
strong basic conditions, we employed relatively weak
bases such as K2CO3, Li2CO3, and triethylamine. All
the three bases offered good to excellent yields (Table
1, entries 3, 11, and 12). However, the use of triethyl-
amine was not recommended since the triethylammo-
nium salt formed during the reaction disturbed the
purification of the desired product by chromatography
on silica gel. We have therefore chosen Li2CO3 because
of its excellent performance. Finally, we found out that
the optimal reaction temperature was 100 °C. Indeed,
higher temperatures led to the decomposition of the pal-
ladium catalyst (data not shown), while lower tempera-
ture could not ensure efficient transformation (Table 1,
entry 13). The reaction time was also finely tuned to
25 min in order to achieve maximum yields, since this
enabled the complete reaction to occur, while preventing
the decomposition of the products (data not shown).
O
N
O
N
NH2
NH2
N
N
CuI, Pd(PPh3)4, Li2CO3
Br
R
R
+
N
N
dioxane/H2O(3:1), 100°C
MW, 25 min
HO
HO
O
O
1
2
Entry
1
R
Product
Yielda (%)
99
1a
2
3
4
5
6
7
1b
1c
1d
1e
1f
91
90
92
75
86
77
MeO
H3C
F3C
F
F
1g
F
8
1h
1i
85
97
91
93
90
0
Under our optimized conditions, that is, with
Pd(PPh3)4/CuI and Li2CO3, in dioxane/H2O (3:1, v/v)
as solvents, we were able to obtain the 5-arylethynyl
and 5-alkylethynyl analogs of triazole acyclonucleo-
sides13 with good to excellent yields when the alkynes
were not directly linked with the electron-withdrawing
groups (Table 2, entries 1–4 and 9–12). The presence
of electron-withdrawing groups such as F or CF3 on
the phenylacetylene resulted in slightly lower reaction
yields (Table 2, entries 5–8), whereas alkynes directly
linked to the electron-withdrawing functionalities com-
pletely blocked the Sonogashira reaction and no prod-
uct could be identified (Table 2, entries 13 and 14).
Two factors may be responsible of these results. The first
one is that strong electron-withdrawing group will re-
duce significantly the nucleophilicity of the correspond-
ing alkyne, making the transmetalation step difficult to
occur. The second one is that the strong electron-with-
drawing group will increase electrophilicity of the corre-
sponding alkyne, leading to undesired Michael
additions.14,15 It is therefore a synthetic challenge to
introduce electrophilic alkynyl functionalities on a tria-
zole ring with high yields by using Sonogashira reaction.
OH
9
10
11
12
13
14
1j
OH
1k
1l
Cl
O
1m
1n
CH3OC
O
S
0
CH3
O
a 0.05 equiv Pd(PPh3)4, 0.05 equiv CuI, 2.0 equiv Li2CO3, dioxane/H2O
(3:1), 100 °C, microwave irradiation, 25 min.
mainly focused on preventing this cyclization side reac-
tion while promoting Sonogashira reaction. We first at-
tempted to use water as the solvent because of its
ecologic and economic advantages as well as the fact
that the starting material 2 can be well solubilized in
water. Unfortunately, the reaction in pure water did
not yield the desired product, but only the cyclization
We further obtained crystals of 1a and 1f and their X-
ray structures.16,17 It is worth to mention that two differ-
ent conformations, (I) and (II) (Fig. 1), exist for 1a in its
crystal structures, which probably results from H-bond-
ing and crystal packing. In both compounds 1a and 1f,

3324
R. Zhu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3321–3327
Figure 1. X-ray structures of compounds 1a and 1f.
the phenyl ring extended out of the triazole ring plane
via the triple bond linkage (Fig. 1), which differ from
those previously observed for the bitriazolyl10 and aryl-
triazolyl nucleosides.11
the triazole ring in 1f. Removing this rigid ethynyl triple
bond in 1f led to the inactive analog 411a (Scheme 2),
whereas replacing the rigid triple bond in 1f by the more
flexible ethylene group in 521 (Scheme 2) abolished com-
pletely the antiviral activity (data not shown). There-
fore, the triple bond linker, between the phenyl and
triazole rings, seems to be also critical for the antiviral
activity observed for 1f.
Antiviral activity of the above synthesized triazole
nucleosides (Table 3) was first evaluated in a HCV sub-
genomic RNA replicon assay using Huh-5-2 cells.18,19
All the compounds with arylethynyl functionalities on
the triazole ring 1a–1h, except 1b, exhibited anti-HCV
activity (Table 3, entries 1–8), whereas compounds bear-
ing an alkylethynyl substituent 1i–1l were devoid of anti-
HCV activity (entries 9–12 in Table 3). This may suggest
that the appended aromatic systems brought by the
arylethynyl functionality on the triazole ring in 1a–1h
are responsible for the antiviral activity, which is in line
with our initial concept for molecular design.
Finally, we compared the crystal structure of 1f with
that of HEPT.7a We noticed that the structure of 1f
resembles closely to that of HEPT (Fig. 2). HEPT is re-
ported to be a non-nucleoside inhibitor of the reverse
transcriptase of HIV, which binds to the allosteric site
and exerts its antiviral effect by inducing conformational
changes of the reverse transcriptase of HIV.8 Although
the close structural resemblance of 1f to HEPT may lead
us to speculate that 1f may target the RNA-dependent-
RNA-polymerase of HCV, it is worth to note that the
crystal structure may not necessarily represent the bio-
logically active form.
Compounds 1a and 1f inhibited HCV replication, with-
out being cytotoxic (Table 3, entries 1 and 6).20 Preli-
minary delineation on the anti-HCV activity and the
toxicity of compounds 1a–f could be obtained, based
on the different functionalities at the p-position of the
phenylacetylene moiety (Table 3, entries 1–6). Com-
pound 1b exhibited no anti-HCV activity (Table 3, entry
2), which may be due to the unfavorable electronic prop-
erties brought by the methoxyl group on the phenyl ring.
Cytotoxicity was observed with compounds 1d, 1e, and
1g (Table 3, entries 4, 5, and 7). Although both 1c and
1h had a CC50 value over 50 lg/mL, these inhibited at
50 lg/mL host cell proliferation (data not shown). Over-
all, the cytotoxicity might be the consequence of the ste-
ric congestion brought by the large and/or lipophilic
substituents such as –C6H13, –CH3, –CF3, and F.
Table 3. Antiviral activities of synthesized compounds on HCV
Subgenomic replicon replication in Huh-5-2 Cellsa
b
c
Entry
Compound
EC50 (lg/mL)
CC50 (lg/mL)
1
1a
1b
18 4d
>50
16
>50
>50
>50, Te
25
20
2
3
1c
1d
4
22
5
5
1e
6
1f
1g
22 3d
10
20
>50
29
7
8
1h
1i
>50, Te
9
>50
>50
>50
>50
>50
>50
The anti-HCV activity of 1a and 1f was further assessed
in two other HCV subgenomic RNA replicon assays
using Huh-9-13 and Huh-6 cells.18 Compound 1a had
an EC50 value over 50 lg/mL in Huh-9-13 cells, while
the activity of 1f as observed in Huh-5-2 cells was cor-
raborated by the activity noted in Huh-9-13 and Huh-
6 cells with EC50 values of 38 2 and 29 1 lg/mL,
respectively. In addition, 1f is superior to ribavirin in
selectivity, because it is much less toxic20 than ribavirin
although it is slightly less potent.
10
11
12
13
1j
1k
>50
>50
1l
Ribavirin
7
2
21 11
a Interferon-a-2b at 10,000 U/well reduced the signal in the viral RNA
(luciferase) assay to background levels; without any cytostatic activity.
b Effective concentration (EC50): concentration required to inhibit
luciferase activity in the replicon system by 50%.
c Cytotoxic concentration (CC50): concentration required to inhibit the
proliferation of exponentially growing Huh-5-2 cells by 50%.
d Average values based on six independent assays.
e
In view of these results, we further studied the impor-
tance of the triple bond bridging the phenyl ring and
T: CC50 > 50 lg/mL, however, toxicity (cell growth <80%) was
observed at sample concentration of 50 lg/mL.

R. Zhu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3321–3327
3325
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.2008.
04.026.
O
N
O
N
NH2
NH2
N
F
N
F
N
N
HO
HO
References and notes
O
O
4
5
1. Weiss, U., Eds., Nature 2005, 436, 929.
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Figure 2. Crystal structures of HEPT7a and 1f.
In conclusion, we synthesized a series of novel arylethy-
nyltriazole acyclonucleosides using a simple and conve-
nient one-step Sonogashira coupling reaction in aqueous
solution and promoted by microwave irradiation. One
of the compound 1f selectively inhibited HCV replica-
tion. Preliminary structure/activity relationship analysis
revealed that the appended aromatic phenyl ring, the
substituents at the p-position of the phenyl ring as well
as the rigid triple bond connection between the phenyl
group and the triazole ring contribute critically to the
anti-HCV activity. Analogs with large and lipophilic
substituents on the ethynylphenyl ring may have a more
pronounced inhibitory effect on proliferation of the host
cell. The arylethynyltriazole acyclonucleosides disclosed
here present simple and concise structural motifs, which
can be conveniently synthesized using the Sonogashira
reaction and easily modified for further structural opti-
mization and structural/activity relationship study.
These constitute therefore an interesting structural lead
in the search for novel antiviral compounds against
HCV, for which there is no efficient treatments and
development of new and efficacious antiviral agents is
of extreme importance and urgence.
´ ´
11. (a) Zhu, R. Z.; Qu, F. Q.; Quelever, G.; Peng, L.
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13. General: All the terminal alkynes and catalysts were
purchased from Acros or Lancaster. The microwave
assisted reactions were performed on an InitiatorTM Cre-
ator produced by Biotage. The 1H NMR spectra were
recorded at 300 or 600 MHz and the 13C NMR spectra
were recorded at 75 or 150 MHz, respectively, on Varian
Mercury-VX300 and Varian Inova-600 spectrometers. The
chemical shifts were recorded in parts per million (ppm)
with TMS as the internal reference. FAB and ESI mass
spectra were determined using ZAB-HF-3F and Finnigan
LCQ Advantage mass spectrometers, respectively. High
resolution mass spectra were obtained by Matrix-assisted
laser desorption/ionization mass spectrometry (MALDI-
MS) using an IonSpec 4.7 T Fourier Transform Mass
Spectrometer. All the compounds were purified by perform-
ing flash chromatography on silica gel (200–300 mesh).
Acknowledgments
Financial supports from the Ministry of Science and
2003CB114400,
Technology
of
China
(Nos.
2003AA2Z3506), National Science Foundation of Chi-
na (No. 20372055), Wuhan University, CNRS and the
Geconcerteerde Onderzoeksactie (KULeuven) are grate-
fully acknowledged. We thank Mrs. Katrien Geert for
´ ´
anti-HCV test, Drs. Gilles Quelever and Jessica Blanc
for manuscript reading.
Supplementary data
Procedure for preparing 1 via a microwave assisted
Sonogashira reaction: The terminal alkynes (0.24 mmol), tetra-
kis(triphenylphosphine)palladium(0) (11.6 mg, 0.01 mmol),
NMR spectra of all the new compounds are included.
This material is available free of charge via the Internet.

3326
R. Zhu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3321–3327
CuI (2.2 mg, 0.01 mmol), Li2CO3 (30.5 mg, 0.4 mmol)
and 5-bromo-1-[(2-hydroxyethoxy)methyl]-1,2,4-triazole-
NMR (300 MHz, DMSO-d6): d 8.07 (br s, 1H, –C(O)NH),
7.80–7.85 (m, 2H, ArH), 7.77 (br s, 1H, –C(O)NH), 7.39 (t,
JHH = 8.7 Hz, JFH = 8.7 Hz, 2H, ArH), 5.75 (s, 2H, H-10),
4.78(brs,1H, –OH),3.62(t, J = 4.8 Hz, 2H, H-20),3.50–3.53
(m, 2H, H-30); 13C NMR (75 MHz, DMSO-d6): d 163.9 (d,
1JCF = 249.1 Hz), 160.4, 157.6, 140.5, 135.5 (d,
3JCF = 8.6 Hz), 117.2 (d, 2JCF = 23.4 Hz), 116.6, 96.6, 78.8,
75.5, 72.1, 60.5; FAB-MS: m/z 305[M+H] +, 327 [M+Na]+;
3-carboxamide 2 (0.2 mmol) were suspended in 2.8 mL of
dioxane/H O (3/1) under argon. The vessel was sealed and
2
irradiated at 100 °C for 25 min, and then cooled to room
temperature. The reaction mixture was concentrated
under reduced pressure and the crude residue was
purified by flash chromatography on silica gel (CH2Cl2/CH
3OH, 20:1). The purified material was dried in vacuo
to afford the corresponding derivative derivatives 1a–1l.
5-Phenylethynyl- 1-[(2-hydroxyethoxy)methyl]-1,2,4-tria-
zole-3-carboxamide ( 1a ):White solid (99 %).1H NMR
(300 MHz, DMSO-d6): d 8.06 (br s, 1H, –C(O)NH), 7.73–
7.75 (m, 3H, –C(O)N H and ArH), 7.50–7.58 (m, 3H, ArH),
5.75 (s, 2H, H-10), 4.76 (br s, 1H, –OH), 3.63 (t, J = 5.1 Hz,
2H, H-20), 3.51–3.53 (m, 2H, H-30); 13C NMR (150 MHz,
DMSO-d6): d 160.4, 157.6, 140.6, 132.8, 131.5, 129.8, 120.1,
97.6, 78.8, 75.6, 72.1, 60.5; FAB-MS: m/z 287 [M+H]+, 309
þ
HRMS: Calcd. for C14H14FN4O3 305.1044. Found
305.1048; IR: 2230.09 cmꢀ1 (–C„C–).
5-[(3-Fluorophenyl)ethynyl]-1-[(2-hydroxyethoxy)methyl] -
1,2,4-triazole-3-carboxamide (1g ): White solid (77%). 1H
NMR (300 MHz, DMSO-d6): d 8.07 (br s, 1H, –C(O)N H),
7.77 (br s, 1H, –C(O)N H), 7.67 (d, J = 8.7 Hz, 1H, ArH),
7.54–7.60 (m, 2H, ArH), 7.43–7.49 (m, 1H, ArH), 5.77 (s, 2H,
H-10), 4.76 (t, J = 5.4 Hz, –OH), 3.63 (t, J = 4.8 Hz, 2H, H-
20), 3.49–3.54 (m, 2H, H-30); 13C NMR (150 MHz, DMSO-
d6):d 162.5 (d, 1JCF = 243.6 Hz), 160.3, 157.6, 140.3, 132.0(d,
[M+Na]+; HRMS: Calcd for C14H15N4O3 287.1139.
3JCF = 8.7 Hz), 129.2, 122.0 (d, JCF = 9.9 Hz), 119.4 (d,
3
þ
Found 287.1148. IR: 2232.70 cmꢀ1 (–C„C–).
2JCF = 24.2 Hz), 118.9 (d, 2JCF = 19.7 Hz), 96.0, 78.8, 76.3,
þ
5-[(4-Methoxyphenyl)ethynyl]-1-[(2-hydroxyethoxy)-
methyl]-1,2,4-triazole-3-carboxamide (1b ):White solid
(91%). 1H NMR (300 MHz, DMSO-d6): d 8.03 (br s, 1H,
–C(O)N H), 7.74 (br s, 1H, –C(O)N H), 7.68
(d,J = 8.9 Hz, 2H, ArH), 7.07 (d, J = 8.9 Hz, 2H, ArH),
5.72 (s, 2H, H-10), 4.76 (t, J = 5.6 Hz, –O H), 3.83 (s, 3H, –
OCH3), 3.62 (t, J = 4.8 Hz, 2H, H-20), 3.49–3.54 (m, 2H, H-
30); 13C NMR (75 MHz, DMSO-d6): d 161.8, 160.5, 157.5,
141.0, 134.6, 115.5, 111.8, 98.2, 78.7, 74.7, 72.1, 60.5, 56.2;
FAB-MS: m/z 317þ[M+H]+, 339 [M+Na]+; HRMS: Calcd
72.1, 60.5; HRMS: Calcd for C14H14FN4O3 305.1044.
Found 305.1043; IR: 2233.78 cmꢀ1 (–C„C–).
5-[(2-Fluorophenyl)ethynyl]-1-[(2-hydroxyethoxy)methyl]-
1,2,4-triazole-3-carboxamide (1h ): White solid (85%). 1H
NMR (300 MHz, DMSO-d6): d 8.10 (br s,1H, –C(O)NH),
7.80–7.85 (m, 1H, ArH), 7.77 (br s, 1H, –C(O)N H), 7.64–
7.66 (m, 1H, ArH), 7.35–7.48 (m, 2H, ArH), 5.74 (s, 2H, H-
10), 4.76 (t, J = 5.6 Hz, –OH), 3.63 (t, J = 5.1 Hz, 2H, H-20),
3.49–3.54 (m, 2H, H-30); 13C NMR (150 MHz, DMSO-d6): d
163.0 (d, 1JCF = 251.3 Hz), 160.3, 157.7, 140.2, 134.7, 134.0,
2
2
for C15H17N4O4
317.1244. Found 317.1248; IR:
125.9, 116.8 (d, JCF = 19.8 Hz), 108.7 (d, JCF =
2217.69 cmꢀ1 (–C„C–).
15.3 Hz), 91.0, 80.3, 78.9, 72.2, 60.4; FAB-MS: m/z 30þ5
[M+H]+, 327 [M+Na]+; HRMS: Calcd for C14H14FN4O3
305.1044. Found 305.1046; IR: 2230.12 cmꢀ1 (–C„C–).
5-[(1-Cyclopentanol)ethynyl]-1-[(2-hydroxyethoxy)methyl]-
1,2,4-triazole-3-carboxamide (1i): White solid (97%). 1H
NMR (300 MHz, DMSO-d6): d 8.00 (br s, 1H, –C(O)N H),
7.72 (br s, 1H, –C(O)N H), 5.74 (s, 1H, –O H), 5.62 (s, 2H, H-
10), 4.76 (t, J = 5.6 Hz, 1H, –O H), 3.58 (t, J = 4.4 Hz, 2H, H-
20), 3.48–3.52 (m, 2H, H-30), 1.95 (br s, 4H, –C H2–), 1.65–
1.86 (m, 4H, –C H2–); 13C NMR (75 MHz, DMSO-d6): d
160.4, 157.4, 140.7, 104.2, 78.4, 73.4, 72.1, 68.6, 60.4, 42.1,
23.7; FAB-MS: m/z 295 [M+H]+, 317 [M+Na]+; HRMS:
5-[(p-Tolyl)ethynyl]-1-[(2-hydroxyethoxy)methyl]-1,2,4-
triazole-3-carboxamide (1c): White solid (90%). H NMR
1
(300 MHz, DMSO-d6): d 8.06 (br s, 1H, –C(O)NH), 7.76 (br
s, 1H, –C(O)NH), 7.63 (d, J = 8.0 Hz, 2H, ArH), 7.34 (d,
J = 8.0 Hz, 2H, ArH), 5.73 (s, 2H, H-10), 4.77 (t, J = 5.4 Hz,
–OH), 3.62 (t, J = 4.4 Hz, 2H, H-20), 3.50–3.53 (m, 2H, H-
30), 2.38 (s, 3H, –CH3); 13C NMR (150 MHz, CDCl3): d
160.7, 156.4, 141.55, 141.47, 132.4, 129.7, 116.9, 99.1, 78.6,
73.9, 72.0, 61.6, 22.0; FAB-MS: m/z 301 [M+H]+, 323
[M+Na]+; HRMS: Calcd. for C15H17N4O3 301.1295.
þ
Found 301.1304; IR: 2229.15 cmꢀ1 (–C„C–).
þ
5-[(4-Pentylphenyl)ethynyl]-1-[(2-hydroxyethoxy)methyl]
-1,2,4-triazole-3-carboxamide (1d): White solid (92%). 1H
NMR (300 MHz, DMSO-d6 + D2O): d 7.58 (d, J = 8.0 Hz,
2H, ArH), 7.30 (d, J = 8.0 Hz, 2H, ArH), 5.68 (s, 2H, H-10),
3.58 (t, J = 5.0 Hz, 2H, H-20), 3.47 (t, J = 5.0 Hz, 2H, H-30),
2.58 (t, J = 7.7 Hz, 2H, –CH2), 1.51–1.55 (m, 2H, –CH2–),
1.22–1.26 (m, 4H, –C H2–), 0.80 (t, J = 6.6 Hz, 3H, –CH3);
13CNMR (75 MHz, DMSO-d6):d 160.5, 157.5, 146.5, 140.8,
132.7, 129.7, 117.3, 98.0, 78.7, 75.2, 72.1, 60.5, 35.8, 31.5,
30.9, 22.6, 14.6; FAB-MS: m/z 357 [M+H]+, 379 [M+Na]+;
Calcd for C13H19N4O4 295.1401. Found 295.1402; IR:
2237.00 cmꢀ1 (–C„C–).
5-[(1-Cyclohexanol)ethynyl]-1-[(2-hydroxyethoxy)methyl]-
1,2,4-triazole-3-carboxamide ( 1j ): White solid (91%). 1H
NMR (300 MHz, DMSO-d6): d 8.01 (br s, 1H, –C(O)N H),
7.71 (br s, 1H, –C(O)NH), 5.87 (s, 1H, –OH), 5.62 (s, 2H, H-
10), 4.75 (t, J = 5.1 Hz, 1H, –OH), 3.58 (t, J = 4.8 Hz, 2H, H-
20), 3.47–3.52 (m, 2H, H-30), 1.27–1.91 (m, 10H, –C H2–);
13C NMR (150 MHz, DMSO-d6): d 159.3, 156.2, 139.5,
103.0, 77.4, 70.9, 68.5, 66.7, 59.3, 38.4, 24.2, 22.0; ESI-MS:
m/z 331.1 [M+Na]+; HRMS: Calcd for C14H21 N4O4
þ
þ
HRMS: Calcd for C19H25N4O3
357.1921. Found
357.1929; IR: 2230.07 cmꢀ1 (–C„C–).
5-[(4-Trifluoromethyl-phenyl)ethynyl]-1-[(2-hydroxyeth-
oxy)methyl]-1,2,4-triazole-3-carboxamide (1e): White solid
309.1557.
Found
309.1560;
IR:
2243.43 cmꢀ1
(–C„C–).
5-[(5-Chloropentyl)ethynyl]-1-[(2-hydroxyethoxy)methyl]-
1,2,4-triazole-3-carboxamide ( 1k): White solid (93%). 1H
NMR (300 MHz, DMSO-d6): d7.98 (br s, 1H, –C(O)NH),
7.70 (br s, 1H, –C(O)N H), 5.61 (s, 2H, H-10), 4.73 (t,
J = 5.1 Hz, 1H, –OH), 3.78 (t, J = 6.2 Hz, 2H, –CH2–), 3.56
(t, J = 4.4 Hz, 2H, H-20), 3.46–3.51 (m, 2H, H-30), 2.74 (t,
J = 6.9 Hz, 2H, –C H2–), 2.01–2.10 (m, 2H, –CH2–); 13C
NMR (75 MHz, CDCl3): d 160.8, 156.2, 141.1, 99.4, 78.5,
71.9, 67.6, 61.6, 43.5, 30.5, 17.1; FAB-MS: m/z 287 [M+H]+þ,
309 [M+Na]+; HRMS: Calcd for C11H15ClN4 NaO3
309.0725. Found 309.0727; IR: 2248.32 cmꢀ1 (–C„C–).
5-[(1-Cyclohexene)ethynyl]-1-[(2-hydroxyethoxy)methyl]-
1,2,4-triazole-3-carboxamide (1l ): Colorless oil (90%). 1H
1
(75%). H NMR (300 MHz, DMSO-d6): d 8.08 (br s, 1H,
–C(O)N H), 7.98 (d, J = 8.4 Hz, 2H, ArH), 7.90 (d,
J = 8.4 Hz, 2H, ArH), 7.78 (br s, 1H, –C(O)NH), 5.78 (s,
2H, H-10), 4.76 (t, J = 5.3 Hz, –O H), 3.63 (t, J = 5.1 Hz, 2H,
H-20), 3.49–3.54 (m, 2H, H-30); 13C NMR (75 MHz,
DMSO-d6):
d 160.5, 157.3, 140.3, 133.6, 131.2 (q,
1
2JCF = 32.0 Hz), 126.6, 124.3 (q, JCF = 270.4 Hz), 124.2,
96.0, 78.9, 77.3, 72.0, 60.3; FAB-MS: m/z 355 [M+H]+, 377
[M+Na]+; HRMS: Calcd for C15H14F3N4O3 355.1013.
þ
Found 355.1013; IR: 2233.78 cmꢀ1 (–C„C–).
5-[(4-Fluorophenyl)ethynyl]-1-[(2-hydroxyethoxy)methyl]-
1,2,4-triazole-3-carboxamide ( 1f ): White solid (86%). 1H

R. Zhu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3321–3327
8.02 (br s, 1H,
3327
NMR (300 MHz, DMSO-d6):
d
Anti-HCV Assay in Huh-9-13 cells. Huh-9-13 cells were
seeded at a density of 5000 cells per well in 96-well cell
culture plates in complete DMEM supplemented with
1,000 lg/mL G418. After 24 h incubation at 37 °C, cell
culture medium was removed and threefold serial dilutions
of the test compounds in complete DMEM without G418
were added in a total volume of 100 lL. After 4 days
incubation at 37 °C, cell culture fluid was removed and
monolayers were washed once with phosphate-buffered
saline. Cells were lysed in 350 lL RLT buffer (Qiagen,
Venlo, The Netherlands) according to the manufacturer’s
instruction. Lysates were used to determine the amount of
HCV replicon RNA by means of quantitative real-time
PCR. The values of EC50 were calculated as the concen-
tration of compound that caused a 50% reduction in HCV
RNA levels compared to that of the untreated control.
Anti-HCV Assay in Huh-6 cells. Huh-6 cells were seeded at
a density of 15,000 cells per well in 96-well cell culture
plates as described for Huh 9-13. After a 3-day incubation
period at 37 °C, cells were lysed in cells-to-cDNA lysis
buffer (Ambion, Cambridgeshire, United Kingdom), and
lysates were used to determine the amount of HCV
replicon RNA by means of quantitative real-time PCR.
The values of EC50 were calculated as the concentration of
compound that caused a 50% reduction in HCV RNA
levels compared to that of the untreated control.
–C(O)N H), 7.71 (br s, 1H, –C(O)N H), 6.52 (s, 1H, –C
H@C–), 5.62 (s, 2H, H-10), 4.75 (t, J = 5.1 Hz, 1H, –O H),
3.57 (t, J = 4.7 Hz, 2H, H-20), 3.48–3.52 (m, 2H, H-30), 2.17–
2.22 (m, 4H, –C H2–), 1.58–1.65 (m, 4H, –C H2–); 13C NMR
(75 MHz, CDCl3): d 161.0, 156.3, 141.7, 141.4, 118.8, 100.7,
78.4, 72.0, 71.9, 61.5, 28.4, 26.1, 22.1, 21.3; ESI-MS: m/z291.0
[M+H]+; HRMS: Calcd for C14H19N4Oþ3 291.1452. Found
291.1457; IR: 2214.48 cmꢀ1 (–C„C–).
14. Negishi, E. I.; Anastasia, L. Chem. Rev. 2003, 103,
1979.
15. Anastasia, L.; Negishi, E. I. Org. Lett. 2001, 3, 3111.
16. Single crystals of product 1a suitable for X-ray crystal-
lographic analysis were obtained via slow evaporation of
CH2Cl2–CH3OH solution. Crystallographic data of 1a:
colorless, monoclinic space group P 21/c, Z = 8, a = 17.9247
˚
(9), b = 9.1151 (5), c = 18.0680 (9) A, a = 90.00, b = 106.6130
3
2
2
˚
(10), c = 90.00, V = 2828.8 (3) A , R (F > 2rF ) = 0.0553
and wR = 0.1269 (w = 1/[r2(Fo ) + (0.0637P) 2 + 0.4295P],
2
P = (Fo2 + 2Fc )/3). The detailed X-ray structure data have
2
been deposited in the Cambridge Crystallographic Data
Center with deposition No. CCDC 680416. Copy of the
data can be obtained, free of charge, on application to
the CCDC, 12 Union Road, Cambridge CB2 1EZ UK
(Email: deposit@ccdc.cam.ac.uk).
17. Single crystals of product 1f suitable for X-ray crystallo-
graphic analysis were obtained via slow evaporation of
CH2Cl2–CH 3OH solution. Crystallographic data of 1f:
colorless, triclinic space group P ꢀ 1, Z = 2, a = 5.1149 (6),
20. Cytotoxicity of 1a and 1f was further evaluated on the
MiaPaCa, Capan-2 and PC-3 cells using MTT assay. No
significant toxicity was observed at concentrations even up
to 200 lM (Xia, Y. et al., unpublished results).
21. Synthesis of 5: 1f (31.8 mg, 0.105 mmol) and 10% Pd/C
(11 mg) were suspended in 5 mL of CH3OH and the
obtained mixture was stirred under atmospheric hydrogen
at room temperature for 24 h. The reaction mixture was
filtered through Celite. The filtrate was concentrated under
reduced pressure and the residue was dried in vacuo to
afford product.
˚
b = 8.4420 (9), c = 17.0589 (18) A, a= 93.615 (2), b = 90.062
(2), c = 101.108 (2), V = 721.30 (14) A , R (F 2 > 2rF2) =
3
˚
2
0.0531 and wR = 0.1479 (w = 1/[r2(Fo ) + (0.1018P) 2
+
0.0000P], P = (Fo2 + 2Fc2)/3). The detailed X-ray structure
data have been deposited in the Cambridge Crystallo-
graphic Data Center with deposition No. CCDC 680417.
Copy of the data can be obtained, free of charge, on
application to the CCDC, 12 Union Road, Cambridge CB2
1EZ UK (Email: deposit@ccdc.cam.ac.uk).
Compound 5: White solid (30.8 mg, 95%). 1H NMR
(300 MHz, CDCl3): d 7.09–7.14 (m, 2H, ArH), 6.94–7.00
(m, 3H, ArH and –C(O)NH), 5.75 (br s, 1H,
–C(O)NH), 5.37 (s, 2H, H-10), 3.66–3.69 (m, 2H, H-20),
3.59–3.62 (m, 2H, H-30), 3.12 (s, 4H, –C H2–), 1.80 (t,
J = 5.6 Hz, –OH); 13C NMR (75 MHz, CDCl3): d 161.9 (d,
1JCF = 243.8 Hz), 161.3, 157.7, 155.3, 135.7, 130.1(d,
18. (a) Paeshuyse, J.; Kaul, A.; De Clercq, E.; Rosenwirth, B.;
Dumont, J. M.; Scalfaro, P.; Bartenschlager, R.; Neyts, J.
Hepatology 2006, 43, 761; (b) Coelmont, L.; Paeshuyse, J.;
Windisch, M. P.; De Clercq, E.; Bartenschlager, R.; Neyts,
J. Antimicrob. Agents Chemother. 2006, 50, 3444.
19. Anti-HCV Assay in Huh-5-2 cells. Huh-5-2 cells were
seeded at a density of 5000 per well in a tissue culture-
treated white 96-well view plate (Packard, Canberra,
Canada) in complete Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 250 lg/mL G418. After
incubation for 24 h at 37 °C (5% CO2) medium was
removed and threefold serial dilutions in complete
DMEM (without G418) of the test compounds were
added in a total volume of 100 lL. After 4 days of
incubation at 37 °C, cell culture medium was removed and
luciferase activity was determined using the Steady-Glo
luciferase assay system (Promega, Leiden, The Nether-
lands); the luciferase signal was measured using a Lumi-
noskan ascent (Thermo, Vantaa, Finland).
2
3JCF = 7.4 Hz), 115.7 (d, JCF = 21.3 Hz), 78.0, 71.3,
þ
61.6, 33.0, 28.3; HRMS: Calcd for C14H18FN4O3
309.1357. Found 309.1353.
O
N
O
N
NH2
NH2
F
N
N
F
N
N
H2, Pd/C
HO
HO
O
O
1f
5