Discovery of novel arylethynyltriazole ribonucleosides with selective and effective antiviral and antiproliferative activity

Article abstract of DOI:10.1021/jm800927r

Novel ethynyltriazole ribonucleosides were synthesized using a simple and efficient two-step procedure involving Sonogashira coupling and subsequent ammonolysis. Compounds 2f and 3o inhibited hepatitis C virus (HCV) replication efficiently, whereas compou

Full text of DOI:10.1021/jm800927r

1144
J. Med. Chem. 2009, 52, 1144–1155
Discovery of Novel Arylethynyltriazole Ribonucleosides with Selective and Effective Antiviral
and Antiproliferative Activity
Jinqiao Wan,†,# Yi Xia,‡,# Yang Liu,† Menghua Wang,† Palma Rocchi,§ Jianhua Yao,| Fanqi Qu,† Johan Neyts,
Juan L. Iovanna,§ and Ling Peng*,†,‡
State Key Laboratory of Virology, College of Chemistry and Molecular Sciences, Wuhan UniVersity, Wuhan, 430072, P. R. China, De´partement
de Chimie, UniVersite´ de la Méditerranée, CNRS UMR 3118, CINaM, 163, AVenue de Luminy, 13288 Marseille, France, INSERM U624, 163,
AVenue de Luminy, 13288 Marseille, France, Department of Computer Chemistry and Cheminformatics, Shanghai Institute of Organic
Chemistry, Chinese Academy of Sciences, Shanghai, 200032, P. R. China, and Rega Institute for Medical Research, Minderbroedersstraat 10,
B-3000 LeuVen, Belgium
ReceiVed July 28, 2008
Novel ethynyltriazole ribonucleosides were synthesized using a simple and efficient two-step procedure
involving Sonogashira coupling and subsequent ammonolysis. Compounds 2f and 3o inhibited hepatitis C
virus (HCV) replication efficiently, whereas compound 3f demonstrated potent apoptosis-induced antipro-
liferative activity against pancreatic cancer MiaPaCa-2 cells both in vitro and in vivo. Most interestingly,
the notable selective antiviral and antiproliferative activities were achieved respectively for 2f and 3f by
modulating the ribose sugar moiety into deprotected and protected forms while retaining a similar
trifluoromethylphenylethynyltriazole as the nucleobase. Preliminary structure-activity relationship study
revealed that not only the ribose moiety but also the CF3 group at the p-position of the phenyl ring and the
rigid triple bond functionality contributed critically to the observed antiviral activity of 2f against HCV and
antiproliferative activity of 3f against pancreatic cancer. These two compounds constitute therefore promising
leads in the search for new antiviral and anticancer candidates.
Introduction
1), with aromatic systems appended on the 6-position of adenine,
have been shown to elicit interesting anti-HCVa and anticancer
activity.3 Another example includes HEPT (Figure 1), an
acyclonucleoside analogue with the pyrimidine nucleobase
bearing a phenylthio group at the 6-position, which has been
reported to show potent and selective anti-HIV activity.4 The
appended aryl at the 6-position is one of the key factors
contributing to this anti-HIV activity because of interactions
with the amino acid residues Tyr188 and Leu100 in the enzyme
HIV reverse transcriptase, leading to its conformational change.5
Synthetic nucleoside analogues with modified nucleobase and/
or sugar moieties are of considerable importance in the search
for new structural leads endowed with antiviral and anticancer
activity.1 These nucleoside analogues can mimic natural nucleo-
sides and serve as building units or inhibitors that interfere in
nucleic acid synthesis or block nucleos(t)ide-dependent biologi-
cal processes. Some well-known synthetic nucleoside drugs
include the antiviral drugs ribavirin, acyclovir, and zidovudine,
as well as the anticancer drugs gemcitabine and cladribine
(Figure 1).
Introducing various functionalities to the natural nucleobase
is the most frequently used method of obtaining novel nucleoside
mimics with base modifications. Such nucleosides can be
imparted with not only novel biologically interesting activities
but also particular resistance to nucleos(t)ide metabolizing
enzymes, thus leading to better in vivo stability and efficiency.
A successful example is the clinical anticancer drug cladribine
(Figure 1) with a chlorine atom attached to the 2-position of
adenine nucleobase.2 Recently, 6-arylpurine nucleosides (Figure
Using unnatural heterocycles as nucleobases is another
approach to develop novel nucleoside analogues. Ribavirin was
the first synthetic nucleoside with an unnatural triazole hetero-
cycle as the nucleobase and exhibits a broad spectrum of
antiviral activity against a variety of RNA and DNA viruses.6
Used in combination with pegylated interferon-R to treat patients
infected with hepatitis C virus (HCV),7 this therapy with
ribavirin is unfortunately only effective in 40-60% of the
patients (depending on the genotype) and is often associated
with serious side effects.7 An estimated 3% of the world
population, about 170 millon people, are infected by HCV and
at increased risk of developing liver cirrhosis and primary
hepatocellular carcinoma. The development of new drugs with
improved efficacity and tolerance therefore represents an urgent
need to treat HCV infection.8 Moreover, it is also of paramount
importance to explore new drug candidates for the treatment of
pancreatic cancer, one of the most lethal forms of human
cancers, on which conventional cancer therapy has little impact
because of both the aggressivity of this cancer and the rapid
development of drug resistance.9 The current first-line treatment
is based on gemcitabine, which is moderately effective, and
results in a mere 12% response and 3% overall survival rate.9
* To whom correspondence should be addressed. Address: De´partement
de Chimie, Universite´ de la Méditerranée, CNRS UPR 3118, CINaM, 13288
Marseille Cedex 09, France. Phone: 00 33 4 91 82 91 54. Fax: 00 33 4 91
82 93 01. E-mail: ling.peng@univmed.fr.
†
Wuhan University.
These authors contributed equally to this work.
Universite´ de la Méditerranée.
INSERM U624.
#
‡
§
|
Shanghai Institute of Organic Chemistry.
Rega Institute for Medical Research.
a Abbreviations: HCV, hepatitis C virus; EC50, 50% effective concentra-
tion; CC50, 50% cytostatic concentration; MEM, minimum essential medium;
DMEM, Dulbecco’s modified Eagle media; FCS, fetal calf serum; FBS,
fetal bovine serum; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide; FACS, fluorescence activated cell sorting; ELISA, enzyme
linked immunosorbent assay; ip, intraperitoneal; O/N, overnight; SE,
standard error.
Although ribavirin was discovered over 30 years ago, the
search for triazole nucleoside analogues with improved antiviral
activity and selectivity has achieved little success. One reason
10.1021/jm800927r CCC: $40.75
2009 American Chemical Society
Published on Web 01/26/2009

Arylethynyltriazole Ribonucleosides
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1145
Figure 1. Ribavirin, acyclovir, zidovudine, gemcitabine, cladribine, 6-arylpurine ribonucleoside, HEPT, arylethynyltriazole acyclonucleoside 1,
and proposed ethynyltriazole ribonucleosides 2 and 3.
for this may be the limited synthetic methods available to
conveniently obtain structurally diverse triazole nucleosides.
Over the past few years, our laboratories have been actively
engaged in elaborating different synthetic strategies for the
development of novel structural motifs of triazole nucleosides
in an attempt to identify new antiviral and anticancer drug
candidates.10-16 We have mainly focused our efforts on app-
ending π-conjugated systems onto the triazole nucleobase.11-16
Triazole nucleosides may possess unique and intrinsic properties
due to the triazole heterocycle which is a universal base17 for
base-pairing and has broad scope for H-bonding and special
geometry between purine and pyrimidine, as well as a high
polarity. We also expect the appended π-conjugated systems
to offer advantageous binding properties with respect to the
corresponding biological targets via the stronger interactions
afforded by a larger aromatic binding surface and better shape-
complementary conjugated system. Furthermore, we would
expect nucleosides with these unnatural triazole nucleobases to
be generally resistant to nucleos(t)ide metabolizing enzymes that
may result in increased in vivo stability and efficiency.
and another revealed potent antiproliferative activity on Mia-
PaCa-2 cells, a drug-resistant pancreatic cancer cell line.
Results and Discussion
1. Chemistry. Similar to the synthesis of ethynyltriazole
acyclonucleosides,15 we employed the Sonogashira coupling
reaction to prepare the corresponding ethynyltriazole ribo-
nucleosides 2 and 3 (Scheme 1). However, contrary to our
previous one-pot synthetic protocol for acyclonucleosides, here
we used a two-step procedure with a view to obtaining both
sugar-protected and -deprotected nucleosides 3 and 2 (Scheme
1). The synthesis was achieved via Sonogashira coupling
reaction between the bromotriazole nucleoside 411 and various
alkynes to obtain first the protected nucleosides 3 (Scheme 1).
These were then subjected to subsequent ammonolysis to yield
the deprotected nucleosides 2 (Scheme 1).
On the basis of our previous experience synthesizing aryl-
triazole nucleosides,11,15 we attempted to optimize the Sono-
gashira coupling reaction between the bromotriazole nucleoside
4 and phenylacetylene under microwave irradiation by varying
the catalysts (Pd(PPh3)4, Pd(PPh3)2Cl2, Pd2dba3, Pd(OAc)2,
PdCl2, etc.), bases (K2CO3, Li2CO3, triethylamine, TBAF, etc.),
solvents (CH3CN, THF, toluene, etc.), reaction temperature, and
microwave irradiation duration (data not shown). The best results
for synthesizing 3 were achieved with the cocatalyst system of
Pd(PPh3)4/CuI (1/2) in the presence of triethylamine in CH3CN
at 100 °C under microwave irradiation for 30 min.
It is worth noting that the presence of the electron-donating
or electron-withdrawing groups on phenylacetylene did not
significantly affect the Sonogashira coupling yields (Scheme
1, entries 1-6). Neither was any noteworthy steric effect
observed (Scheme 1, entries 6-9). Furthermore, both hetero-
cyclic arylacetylenes (Scheme 1, entries 10-12) and alkylacety-
lenes (Scheme 1, entries 14-16) gave good to excellent yields.
The reaction was also tolerated for a large range of alkyne
substrates having various functionalities including a halide,
double bond, and hydroxyl group, etc. (Scheme 1, entries
Using Suzuki coupling, Huisgen cycloaddition, Sonogashira
reaction, and N-arylation, we have previously synthesized
aryltriazolyl,11,12 bitriazolyl,13,14 ethynyltriazolyl,15 and N-
aryltriazolyl nucleosides,16 respectively. Some of these triazole
nucleoside analogues displayed promising antiviral activity
against TMV (tobacco mosaic virus)13,14 and HCV.15 Of
particular interest is the arylethynyltriazolyl acyclonucleoside15
(1 in Figure 1) discovered in our laboratories, which showed
interesting anti-HCV activity and a greatly reduced toxicity
compared to the current clinical drug in use, ribavirin. In our
continuing efforts to develop novel triazole nucleosides as
potential drug candidates, we became interested in synthesizing
ethynyltriazole ribonucleosides (2 and 3 in Figure 1). Here, we
report on the synthesis and characterization of these triazole
nucleosides as well as the assessment of their biological activity.
Two of the newly synthesized ethynyltriazole ribonucleosides
in this work elicited effective antiviral activity against HCV,

1146 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Wan et al.
Scheme 1. Synthesis of 3 and 2 via the Sonogashira Reaction and the Subsequent Ammonolysis
13-16). However, no coupling product could be obtained when
alkynes were directly linked to the electron-withdrawing func-
tionalities (Scheme 1, entries 17-18), similar to that observed
previously with ethynyltriazole acyclonucleosides.15 This is
mainly due to the significantly reduced nucleophilicity and
increased electrophilicity of the corresponding alkyne induced
by the strong electron-withdrawing group, making transmeta-
lation difficult and at the same time leading to undesired Michael
additions.18,19
ture-activity relationship studies. Compounds 5, 6, 9, and 11
were prepared according to our previous synthetic procedure.11,15
Compound 7 was obtained by selective acetylation of 615 using
acetic anhydride in the presence of triethylamine at room
temperature, while 8 was synthesized by direct Sonogashira
coupling between the corresponding bromotriazole acyclo-
nucleoside12 and the p-trifluoromethylphenylacetylene. Finally,
10 and 12 were achieved via Pd-catalyzed hydrogenation of 2f
and 3f, respectively, with almost quantitative yields.
Treatment of 3 in NH3/MeOH at room temperature resulted
in the deprotection of the sugar moiety and amination of the
carboxylester group, yielding the corresponding deprotected
nucleoside 2 in good to excellent yields (Scheme 1).
We next synthesized several structural analogues of 2f and
3f (Figure 2) with a view to undertaking the relevant struc-
We further succeeded in obtaining crystal structures of 2a
(Figure 3). Two different conformational structures were
observed in the same unit cell: one with the syn conformation
and the other the anti. This finding suggests that the glycosidic
bond in 2a is relatively flexible allowing different rotational
conformations. In both structures, the phenyl ring in 2a extended

Arylethynyltriazole Ribonucleosides
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1147
but unfortunately also an increased toxicity (Table 1). Changing
the acyclic sugar part in 6 to the cyclic ribose sugar moiety
gave the ribonucleoside 2f (Figure 4), which has an antiviral
potential at a level similar to 6 though markedly reduced
cytotoxicity (Table 1). These data demonstrate the importance
of the cyclic ribose sugar component in compound 2f in the
view to both maintain anti-HCV activity and reduce the
associated toxicity.
Furthermore, the effect of the CF3 group at the p-position of
the phenyl ring in 2f was also studied using 2g and 2h containing
this group at the m- and o-position, respectively (Figure 4).
Neither 2g nor 2h displayed favorable anti-HCV activity.
Therefore, the CF3 group at the p-position of the phenyl ring
seems to be critical for the observed anti-HCV activity.
We next studied the importance of the triple bond bridging
the trifluoromethylphenyl ring and the triazole ring in 2f.
Removing the rigid ethynyl triple bond in 2f resulted in an
inactive analogue 9 (Figure 4),11 whereas replacing it with a
more flexible ethylene group (compound 10 in Figure 4)
completely abolished the antiviral activity (data not shown). The
triple bond linker is therefore an important requirement for the
observed antiviral activity of 2f, which is in line with results
obtained earlier with acyclonucleoside 1.15
The alkylnyltriazole ribonucleoside 3o also inhibited HCV
replication. In a previous study with triazole acyclonucleosides,
only arylethynyltriazole nucleosides exhibited antiviral activity.15
Alkylnyltriazole ribonucleoside 3o may also constitue a new
structural anti-HCV hit. It is worth noting that compound 2o,
the corresponding deprotected analogue of 3o, was devoid of
anti-HCV activity.
Figure 2. Structural analogues of 2f and 3f.
Antiproliferative Activity on Pancreatic Cancer Mia-
PaCa-2 Cells. We next evaluated the antiproliferative activity
on MiaPaCa-2 cells, a drug-resistant pancreatic cancer cell line.
Identification of active compounds against cell proliferation of
MiaPaCa-2 may lead to an anticancer candidate to treat
pancreatic cancer. We first used a MTT assay to evaluate the
antiproliferative activity of all the synthesized nucleosides. Our
results reveal a significant reduction in MiaPaCa-2 cell prolif-
eration and/or viability by 59% after 2 days post-treatment with
compound 3f. Under the same conditions, gemcitabine only
moderately inhibited (29%) cell proliferation.
It is interesting to note that 2f, the corresponding deprotected
form of 3f, which elicited effective anti-HCV activity, displayed
no antiproliferative activity on MiaPaCa-2 cells. This is in
agreement with the low toxicity of 2f observed during anti-
HCV assay in different Huh cells and in L1210, HepG2, Hela,
and PC-3 cells. In addition, an acyclic nucleoside analogue of
2f, 6 (Figure 5), which displayed important toxicity on Huh
cells (Table 1), exhibited no antiproliferative activity on
MiaPaCa-2 cells nor did the acetyl-protected acylconucleoside
analogues 7 and 8 (Figure 5). Therefore, the acetyl-protected
ribose component represents a crucial factor contributing to the
antiproliferative activity of 3f. Modulation of the protection/
deprotection state of the ribose sugar moiety is able to generate
distinct and selective antiviral/antiproliferative activity. It is not
clear at this stage whether 3f exerts its antiproliferative activity
per se or through hydrolysis of its acetyl protecting groups in
vivo. Also, the mechanism of action of 2f remains the subject
of further study. It can be hypothesized that 2f may (i) akin to
ribavirin be incorporated into RNA or (ii) be a non-nucleoside
inhibitor of DNA/RNA polymerase.
Figure 3. X-ray structure of 2a.
out of the triazole ring plane via the triple bond linkage, similar
to that observed for its acyclic nucleoside homologue 515 but
different from the corresponding bitriazolyl ribonucleosides.13
2. Biology. Antiviral Activity against Hepatitis C Virus.
We first evaluated the anti-HCV activity of the above synthe-
sized nucleosides using a HCV subgenomic RNA replicon assay
in Huh-5-2 cells (Table 1).20 Compounds with interesting
antiviral activity with no or little effect on the proliferation of
the host cells were further evaluated on HCV subgenomic RNA
replicon assays using Huh-9-13 and Huh-6 cells.20 Two
compounds 2f and 3o resulted in reproducible anti-HCV activity
in all three replicon assays and had limited or no adverse effects
on the host cell. Neither was any noteworthy toxicity observed
on various cell lines such as L1210, HepG2, Hela, MiaPaCa-2,
and PC-3 cells, even with concentrations up to 200 µM (data
not shown).
Some interesting observations were made during the struc-
ture-activity relationship studies with the acyclo- and ribo-
nucleoside analogues of 2f (Figure 4). We previously noted that
the acyclonucleoside 5 (Figure 4), with a simple phenyl ring
connected to the triple bond, had elusive anti-HCV activity
(Table 1).15 By introduction of one F atom at the p-position,
the corresponding analogue 1 (Figure 4) resulted in reproducible
anti-HCV activity (Table 1).15 Replacing this F with CF3 in 1
led to analogue 6 (Figure 4) and an improved anti-HCV activity
The preliminary structure-activity relationship was further
studied using various analogues of 3f (Figure 5). Two isomers
of 3f, 3h and 3g, bearing the CF3 group at the o- and m-position,

1148 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Wan et al.
Figure 4. Structural analogues of 2f for the structure-activity relationship analysis related to its anti-HCV activity.
Figure 5. Structural analogues of 3f for the structure-activity relationship analysis related to its antiproliferative activity on MiaPaCa-2 cells.
Table 1. Antiviral Activities of Ethynyltriazole Nucleoside Analogues on HCV Subgenomic Replicon Replication in Huh-5-2, Huh-9-13 and Huh-6
Cellsa
Huh-5-2
Huh-9-13
Huh-6
entry
EC50
CC50
EC50
>164
125 ( 6.6
36.7 ( 23
19.4 ( 7.0
54.3 ( 18
84.0 ( 15
CC50
>170
>160
79.0 ( 19
>120
>105
229
EC50
CC50
>170
132 ( 18
87.5 ( 14
>120
5
1
6
62.9 ( 14
72.3 ( 9.9
14.1 ( 3.7
17.7 ( 1.9
52.3 ( 3.1
28.7 ( 8.2
>170
>160
112 ( 1.7
95.3 ( 3.3
50.8 ( 21
43.7 ( 20
25.1 ( 4.8
33.0
56.5 ( 14
82.5 ( 9.7
>105
2f
3o
ribavirin
>105
>100
86.0 ( 45
a EC50: 50% effective concentration or the concentration (µM) required to inhibit replicon replication by 50%. CC50: 50% cytostatic concentration or the
concentration (µM) required to inhibit Huh cell proliferation by 50%. Values are expressed as the mean ( SE (n g 3).
respectively, showed no notable inhibition on MiaPaCa-2 cell
proliferation. Moreover, removing the triple bond and replacing
it with the flexible -CH2CH2- connection in 3f led to inactive
analogues 11 and 12, respectively (Figure 5). These data suggest

Arylethynyltriazole Ribonucleosides
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1149
Figure 7. Effect of 3f treatment on chemoresistant MiaPaCa-2 tumor
growth in vivo. Nude mice bearing MiaPaCa-2 tumors of 100 mm3
were randomly selected for treatment with 3f and no treatment as
control. 3f was injected intraperitoneally (150 (mg/kg)/mice) every 3
days for 4 weeks and tumor volume measured once a week. All of the
results were expressed as the mean ( SE (n ) 5): points, mean values
of analysis; bars, standard errors; *, differing from control (p e 0.05)
by Student’s t test.
(Figure 6C), suggesting a caspase-3/7-dependent mechanism of
apoptosis-induction.
We next evaluated the antitumor effect of 3f in nude mice
bearing MiaPaCa-2-xenografed tumors (Figure 7).23 Mice with
mean tumor volume of 100 mm3 were randomly selected for
treatment with 3f versus no treatment as a control. Compound
3f was administrated intraperitoneally (ip) once every 3 days
for 4 consecutive weeks. Figure 7 shows that treatment with 3f
significantly reduced MiaPaCa-2 tumor volume by 60% after
just two weeks of treatment (*, p e 0.05). Under the
experimental conditions used, no adverse effects were observed.
Altogether, our results reveal that compound 3f has potent
antiproliferative activity and may constitute a promising novel
anticancer lead.
Figure 6. Apoptosis-induction in MiaPaCa-2 cells after 48 h of
treatment with 3f or gemcitabine as well as no treatment as control:
(A) flow cytometry analysis to quantify the percentage of cells
undergoing apoptosis (cells in sub-G1/G0); (B) ELISA test for detection
of DNA/histone release; (C) caspase-3/7 activity measurements. All
the experiments were done in triplicate. All of the results were expressed
as the mean ( SE (n ) 3): points, mean values of triplicate analysis;
bars, standard errors; *, **, and ***, differing from control (p e 0.05,
p e 0.01, and p e 0.001, respectively) by Student’s t test.
Conclusion
We succeeded in developing novel ethynyltriazole ribo-
nucleosides based on our earlier anti-HCV leads of triazole
acyclonucleosides. Their synthesis was achieved using a simple
and efficient Sonogashira coupling with or without subsequent
ammonolysis. Among the new ethynyltriazole ribonucleosides
synthesized here, two compounds 2f and 3o exhibited effective
inhibition of HCV replication in vitro and another compound
3f demonstrated potent antiproliferative activity in vitro and in
vivo in chemoresistant pancreatic cancer MiaPaCa-2 cells.
Compared to the acyclic nucleoside homologue, the ribo-
nucleoside 2f developed in this work had considerably reduced
toxicity, while retaining in vitro anti-HCV activity. Furthermore,
replacing the acyclic sugar component with the ribose resulted
in a new anti-HCV lead, alkylethynyltriazole ribonucleoside 3o.
This finding differs from our previous results obtained with
acyclonucleosides, although 3o may have potential carcinogenic
toxicity.
Most interestingly, we have also identified the lead compound
3f, which displayed potent and effective apoptosis-induced
antitumor activity in vitro and in vivo in the chemoresistant
pancreatic cancer MiaPaCa-2 model with no adverse effects. It
is mentioned here that 2f and 3f have a similar triazole
nucleobase and 3f differs from 2f by having the ribose sugar
moiety protected. Surprisingly, simple modulation of the protec-
tion form of the ribose sugar component resulted in distinct
antiviral (2f) and anticancer (3f) activities. This may provide
new direction for searching of novel antiviral and anticancer
nucleoside candidates.
that both the CF3- functionality at the p-position of the phenyl
ring and the triple bond connection between the phenyl group
and the triazole ring are critical for the antiproliferative activity
of 3f.
We further studied the apoptosis-inducing activity of 3f in
MiaPaCa-2 cells using fluorescence-activated cell sorting (FACS)
flow cytometry,21 ELISA test for DNA/histone release22 and
caspase-3/7 activity assay21 (Figure 6). FACS flow analysis
revealed a 3-fold increase in the fraction of cells undergoing
apoptosis (sub-G1/G0 fraction) after treatment with 3f compared
to nontreatment control (Figure 6A). This suggests that for 3f,
the induction of apoptosis represents the main mode of
antiproliferative activity. Importantly, DNA fragmentation and
the release of DNA/histone nucleosomes into the cytoplasm
represent early events in apoptosis. Results from ELISA test
show a 3-fold increase in DNA/histone nucleosome release after
treatment with 3f compared to nontreatment control (Figure 6B),
in good agreement with those obtained from FACS flow (Figure
6A) and further confirming the apoptosis-induced antiprolif-
erative activity of 3f. Caspase-3/7 activation, another hallmark
of apoptosis, was also demonstrated with 3f using caspase-3/7
colorimetric assay. Our results demonstrate a 6.8-fold increase
in caspase-3/7 activation with 3f treatment compared to non-
treatment and a 2-fold increase compared to gemcitabine control

1150 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Wan et al.
1H, H-2′), 5.72-5.76 (m, 1H, H-3′), 4.44-4.54 (m, 2H, H-4′ +
H-5′), 4.16-4.21 (m, 1H, H-5′), 3.99 (s, 3H, -OCH3), 3.86 (s,
3H, -OCH3), 2.15 (s, 9H, -C(O)CH3). 13C NMR (75 MHz,
CDCl3): δ 170.9, 169.6, 169.4, 161.7, 159.7, 155.1, 142.3, 134.3,
114.6, 112.0, 99.6, 89.0, 81.4, 74.4, 73.3, 71.2, 63.1, 55.7, 53.0,
20.9, 20.7. Maldi-MS: m/z 538.1 [M + Na]+. HRMS: calcd for
C24H25N3O10Na+ 538.1432, found 538.1430. IR: 2221.6 cm-1
(-Ct C-).
3c. A total of 81.8 mg (82%) of product was obtained, isolated
as a colorless oil. HPLC: t ) 6.69 min, purity >99% (method 1);
t ) 6.39 min, purity >99% (method 2). 1H NMR (300 MHz,
CDCl3): δ 7.51 (d, 2H, J ) 5.1 Hz, phenyl-H), 7.24 (d, 2H, J )
5.1 Hz, phenyl-H), 6.28 (d, 1H, J ) 3.6 Hz, H-1′), 5.85-5.88 (m,
1H, H-2′), 5.72-5.76 (m, 1H, H-3′), 4.46-4.53 (m, 2H, H-4′ +
H-5′), 4.16-4.22 (m, 1H, H-5′), 3.99 (s, 3H, -OCH3), 2.41 (s,
3H, -CH3), 2.14 (s, 9H, -C(O)CH3). 13C NMR (150 MHz, CDCl3):
δ 170.8, 169.6, 169.4, 159.6, 155.0, 142.1, 141.5, 132.4, 129.7,
116.9, 99.5, 88.9, 81.4, 74.4, 73.6, 71.1, 63.0, 52.9, 21.9, 20.6.
Maldi-MS: m/z 522.1 [M + Na]+. HRMS: calcd for C24H25N3O9Na+
522.1483, found 522.1480. IR: 2224.0 cm-1 (-Ct C-).
The preliminary structure-activity relationship study of 2f
and 3f revealed also that the CF3 group at the p-position of the
phenyl ring and the rigid triple bond connection between the
phenyl group and the triazole ring contribute importantly to both
the antiviral (2f) and antiproliferative (3f) activity against our
HCV and pancreatic cancer MiaPaCa-2 cells. The mechanism
of action and the pharmacological properties of these compounds
remain the subject for further study.
The two arylethynyltriazole ribonucleosides 2f and 3f dis-
covered in this work present simple and concise structural
motifs, which can be conveniently synthesized using the
Sonogashira reaction and modified for further structural opti-
mization and structure-activity relationship study. Altogether,
both compounds constitute promising structural leads in the
search for novel antiviral and antiproliferative candidates against
HCV and pancreatic cancer, respectively. A lead-optimization
process will be initiated, and we are actively pursuing this angle
in current work.
3d. A total of 64.4 mg (62%) of product was obtained, isolated
as a white solid. HPLC: t ) 6.70 min, purity 97.6% (method 1); t
) 6.40 min, purity 97.3% (method 2). 1H NMR (300 MHz, CDCl3):
δ 7.55 (d, 2H, J ) 8.1 Hz, phenyl-H), 7.41 (d, 2H, J ) 8.1 Hz,
phenyl-H), 6.26 (d, 1H, J ) 3.0 Hz, H-1′), 5.84-5.87 (m, 1H, H-2′),
5.71-5.75 (m, 1H, H-3′), 4.49-4.54 (m, 2H, H-4′ + H-5′),
4.16-4.21 (m, 1H, H-5′), 4.00 (s, 3H, OCH3), 2.14 (s, 9H,
C(O)CH3). 13C NMR (150 MHz, CDCl3): δ 169.7, 168.4, 168.2,
158.3, 153.9, 140.4, 136.0, 132.5, 128.2, 117.3, 96.6, 87.8, 80.2,
73.7, 73.2, 69.8, 61.8, 51.9, 19.6, 19.5. Maldi-MS: m/z 542.1 [M
+ Na]+. HRMS: calcd for C23H23N3O9Cl+ 520.1117, found
520.1106. IR: 2228.6 cm-1 (-Ct C-).
Experimental Section
General. All the terminal alkynes and catalysts were purchased
from Acros or Lancaster. The microwave assisted reactions were
performed on an Initiator Creator produced by Biotage. The H
1
NMR spectra were recorded at 300 or 600 MHz and the 13C NMR
spectra recorded at 75 or 150 MHz on Varian Mercury-VX300 and
Varian Inova-600 spectrometers, respectively. The chemical shifts
were recorded in parts per million (ppm) with TMS as the internal
reference. ESI mass spectra were determined using Finnigan LCQ
Advantage mass spectrometers. MALDI mass spectra and 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, with 2,5-
dihydroxybenzoic acid (DHB) being the matrix. All compounds
were purified by performing flash chromatography on silica gel
(200-300 mesh). The purity of compound was further verified by
HPLC analysis using an analytical column of Hypersil C18 (ELITE)
(4.6 mm × 250 mm) with an isocratic elution of CH3CN/H2O 90/
10 (method 1) and an analytical column of Nucleosil 120-5C8
(HICHROM) (4.6 mm × 250 mm) with an isocratic elution with
CH3CN/H2O 90/10 (method 2).
3e. A total of 77.5 mg (82%) of product was obtained, isolated
as a colorless oil. HPLC: t ) 6.56 min, purity >99% (method 1);
t ) 6.55 min, purity 96.7% (method 2). 1H NMR (300 MHz,
CDCl3): δ 7.60-7.64 (m, 2H, phenyl-H), 7.13 (dd, 2H, 3JHF ) 9.0
3
Hz, JHH ) 8.7 Hz, phenyl-H), 6.26 (d, 1H, J ) 3.3 Hz, H-1′),
5.85-5.88 (m, 1H, H-2′), 5.73-5.76 (m, 1H, H-3′), 4.46-4.52 (m,
2H, H-4′ + H-5′), 4.18-4.21 (m, 1H, H-5′), 3.99 (s, 3H, -OCH3),
2.15 (s, 3H, -C(O)CH3), 2.14 (s, 3H, -C(O)CH3), 2.13 (s, 3H,
-C(O)CH3). 13C NMR (75 MHz, CDCl3): δ 170.8, 169.6, 169.4,
1
3
164.0 (d, JCF ) 252.4 Hz), 159.5, 155.1, 141.7, 134.7 (d, JCF
)
General Procedure for Preparing 3 via a Microwave As-
sisted Sonogashira Reaction. 5-Bromo-1-[2,3,5-tri-O-acetyl-ꢀ-D-
ribofuranosyl]-1,2,4-triazole-3-carboxylate 4 (92.8 mg, 0.2 mmol),
the terminal alkynes (0.4 mmol), tetrakis(triphenylphosphine)-
palladium(0) (11.6 mg, 0.01 mmol), CuI (3.8 mg, 0.02 mmol), and
triethylamine (0.4 mL) were suspended in 3 mL of fresh distilled
MeCN under argon. The vessel was sealed and irradiated at 100
°C for 30 min and then cooled to room temperature. The reaction
mixture was concentrated under reduced pressure and the crude
residue purified by flash chromatography on silica gel (petroleum
ether/ethyl acetate, 2:1). The purified material was dried in vacuo
to afford the corresponding product 3a-p.
3a. A total of 78.6 mg (81%) of product was obtained, isolated
as a colorless oil. HPLC: t ) 6.58 min, purity >99% (method 1);
t ) 6.64 min, purity 96.2% (method 2). 1H NMR (300 MHz,
CDCl3): δ 7.61-7.63 (m, 2H, phenyl-H), 7.42-7.49 (m, 3H,
phenyl-H), 6.28 (d, 1H, J ) 3.6 Hz, H-1′), 5.86-5.89 (m, 1H, H-2′),
5.74-5.75 (m, 1H, H-3′), 4.47-4.52 (m, 2H, H-4′ + H-5′),
4.18-4.21 (m, 1H, H-5′), 3.99 (s, 3H, -OCH3), 2.14 (s, 9H,
-C(O)CH3). 13C NMR (75 MHz, CDCl3): δ 170.9, 169.7, 169.4,
159.6, 155.1, 142.0, 132.5, 130.9, 128.9, 120.1, 99.1, 89.0, 81.5,
74.4, 74.1, 71.1, 63.1, 53.1, 20.9, 20.9, 20.8, 20.7. ESI-MS: m/z
486.0 [M + H]+. HRMS: calcd for C23H23N3O9Na+ 508.1327, found
508.1322. IR: 2228.8 cm-1 (-Ct C-).
2
8.6 Hz), 116.4 (d, JCF ) 22.4 Hz), 97.9, 89.0, 81.4, 74.4, 74.0,
71.1, 62.9, 53.0, 20.8, 20.7, 20.6. Maldi-MS: m/z 526.1 [M + Na]+.
HRMS: calcd for C23H22N3O9FNa+ 526.1232, found 526.1242. IR:
2228.5 cm-1 (-Ct C-).
3f. A total of 70.9 mg (64%) of product was obtained, isolated
as a colorless oil. HPLC: t ) 7.26 min, purity >99% (method 1);
t ) 6.45 min, purity 98.9% (method 2). 1H NMR (300 MHz,
CDCl3): δ 7.68-7.76 (m, 4H, phenyl-H), 6.27 (d, 1H, J ) 3.0 Hz,
H-1′) 5.85-5.88 (m, 1H, H-2′), 5.72-5.75 (m, 1H, H-3′), 4.46-4.54
(m, 2H, H-4′ + H-5′), 4.17-4.23 (m, 1H, H-5′), 4.00 (s, 3H,
-OCH3), 2.15 (s, 6H, -C(O)CH3), 2.13 (s, 3H, -C(O)CH3). 13C
NMR (150 MHz, CDCl3): δ 170.8, 169.9, 169.5, 159.4, 155.3,
2
1
141.3, 132.8, 132.6 (q, JCF ) 33.5 Hz), 123.8, 123.7 (q, JCF
)
271.4 Hz), 97.0, 89.2, 81.5, 76.0, 74.5, 71.1, 63.0, 53.1, 20.8, 20.71,
20.66. Maldi-MS: m/z 576.1 [M + Na]+. HRMS: calcd for
C24H22N3O9F3Na+ 576.1200, found 576.1204. IR: 2233.6 cm-1
(-Ct C-).
3g. A total of 78.3 mg (71%) of product was obtained, isolated
as a colorless oil. HPLC: t ) 6.86 min, purity >99% (method 1);
t ) 6.83 min, purity 96.9% (method 2). 1H NMR (300 MHz,
CDCl3): δ 7.88 (s, 1H, phenyl-H), 7.73-7.81 (m, 2H, phenyl-H),
7.55-7.61 (m, 1H, phenyl-H), 6.28 (d, 1H, J ) 3.9 Hz, H-1′),
5.84-5.87 (m, 1H, H-2′), 5.73 (t, 1H, J ) 5.4 Hz, H-3′), 4.48-4.54
(m, 2H, H-4′ + H-5′), 4.19-4.23 (m, 1H, H-5′), 4.00 (s, 3H,
-OCH3), 2.15 (s, 9H, -C(O)CH3). 13C NMR (75 MHz, CDCl3):
3b. A total of 80.3 mg (78%) of product was obtained, isolated
as a colorless oil. HPLC: t ) 6.57 min, purity >99% (method 1);
t ) 6.51 min, purity 96.2% (method 2). 1H NMR (300 MHz,
CDCl3): δ 7.56 (d, 2H, J ) 8.1 Hz, phenyl-H), 6.93 (d, 2H, J )
8.1 Hz, phenyl-H), 6.28 (d, 1H, J ) 3.9 Hz, H-1′), 5.85-5.88 (m,
δ 170.9, 169.7, 169.5, 159.4, 155.3, 141.4, 135.6, 131.6 (q, 2JCF
)
1
33.3 Hz), 129.7, 129.2, 127.4, 123.6 (q, JCF ) 271.7 Hz), 121.1,
97.0, 89.1, 81.5, 75.4, 74.5, 71.1, 63.0, 53.1, 20.8, 20.71, 20.66.

Arylethynyltriazole Ribonucleosides
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1151
Maldi-MS: m/z 576.1 [M
+
Na]+. HRMS: calcd for
H-1′), 5.80-5.83 (m, 1H, H-2′), 5.70-5.74 (m, 1H, H-3′),
4.41-4.52 (m, 2H, H-4′ + H-5′), 4.15-4.20 (m, 1H, H-5′), 3.97
(s, 3H, -OCH3), 2.18-2.22 (m, 4H, -CH2-), 2.13 (s, 9H,
-C(O)CH3), 1.63-1.70 (m, 4H, -CH2-). 13C NMR (150 MHz,
CDCl3): δ 170.9, 169.6, 169.4, 160.4, 159.7, 154.9, 142.3, 119.0,
101.2, 88.8, 81.3, 74.3, 71.8, 71.1, 63.0, 52.9, 28.3, 26.2, 22.1, 21.3,
20.7. Maldi-MS: m/z 512.2 [M + Na]+. HRMS: calcd for
C24H22N3O9F3Na+ 576.1200, found 576.1195. IR: 2235.3 cm-1
(-Ct C-).
3h. A total of 76.3 mg (69%) of product was obtained, isolated
as a colorless oil. HPLC: t ) 6.64 min, purity >99% (method 1);
t ) 6.87 min, purity 97.8% (method 2). 1H NMR (300 MHz,
CDCl3): δ 7.75-7.79 (m, 2H, phenyl-H), 7.59-7.66 (m, 2H,
phenyl-H), 6.35 (d, 1H, J ) 5.1 Hz, H-1′), 5.81-5.84 (m, 1H, H-2′),
5.67-5.71 (m, 1H, H-3′), 4.46-4.58 (m, 2H, H-4′ + H-5′),
4.17-4.23 (m, 1H, H-5′), 4.00 (s, 3H, -OCH3), 2.18 (s, 3H,
-C(O)CH3), 2.15 (s, 3H, -C(O)CH3), 2.10 (s, 3H, -C(O)CH3).
13C NMR (75 MHz, CDCl3): δ 170.9, 169.8, 169.4, 159.5, 155.4,
-1
C23H27N3O9Na+ 512.1639, Found 512.1637. IR: 2214.5 cm
(-Ct C-).
3n. A total of 77.6 mg (80%) of product was obtained, isolated
as a colorless oil. HPLC: t ) 6.52 min, purity 98.6% (method 1);
t ) 6.45 min, purity 98.2% (method 2). 1H NMR (300 MHz,
CDCl3): δ 6.19 (d, 1H, J ) 3.6 Hz, H-1′), 5.79-5.82 (m, 1H, H-2′),
5.68-5.72 (m, 1H, H-3′), 4.46-4.53 (m, 2H, H-4′ + H-5′),
4.14-4.20 (m, 1H, H-5′), 3.97 (s, 3H, -OCH3), 3.71 (t, 2H, J )
6.2 Hz, -CH2-), 2.74 (t, 2H, J ) 6.9 Hz, -CH2-), 2.09-2.14
(m, 11H, -C(O)CH3 and -CH2-). 13C NMR (75 MHz, CDCl3):
δ 170.8, 169.7, 169.5, 159.6, 154.8, 141.7, 100.0, 88.8, 81.3, 74.3,
71.1, 67.3, 62.9, 53.0, 43.5, 30.4, 20.8, 20.7, 20.6, 17.0. Maldi-
1
141.5, 135.0, 132.3, 131.8, 130.7, 126.5, 123.6 (q, JCF ) 271.7
Hz), 118.3, 94.4, 88.7, 81.5, 79.0, 74.2, 71.3, 63.0, 53.1, 20.9, 20.6,
20.4. Maldi-MS: m/z 576.1 [M + Na]+. HRMS: calcd for
C24H22N3O9F3Na+ 576.1200, found 576.1196. IR: 2233.8 cm-1
(-Ct C-).
3i. A total of 79.1 mg (65%) of product was obtained, isolated
as a yellow solid. HPLC: t ) 8.14 min, purity 96.5% (method 1);
t ) 8.73 min, purity 96.2% (method 2). 1H NMR (300 MHz,
CDCl3): δ 8.60 (d, 1H, J ) 8.7 Hz, pyrene-H), 8.07-8.31 (m, 8H,
pyrene-H), 6.51 (d, 1H, J ) 3.6 Hz, H-1′), 5.94-5.97 (m, 1H, H-2′),
5.77-5.81 (m, 1H, H-3′), 4.54-4.59 (m, 2H, H-4′ + H-5′),
4.23-4.27 (m, 1H, H-5′), 4.04 (s, 3H, -OCH3), 2.16 (s, 9H,
-C(O)CH3). 13C NMR (75 MHz, CDCl3): δ 170.9, 169.7, 169.5,
159.7, 155.3, 142.3, 133.1, 131.2, 131.0, 130.3, 129.7, 129.6, 127.3,
126.9, 126.6, 125.0, 124.7, 124.4, 124.1, 113.8, 98.9, 89.1, 81.6,
79.3, 74.6, 71.3, 63.1, 53.1, 20.9, 20.8, 20.7. Maldi-MS: m/z 632.2
MS: m/z 508.1 [M + Na]+. HRMS: calcd for C20H24N3O9ClNa+
-1
508.1093, Found 508.1087. IR: 2217.8 cm
(-Ct C-).
3o. A total of 87.7 mg (89%) of product was obtained, isolated
as a colorless oil. HPLC: t ) 6.51 min, purity >99% (method 1);
t ) 6.38 min, purity 98.6% (method 2). 1H NMR (300 MHz,
CDCl3): δ 6.20 (d, 1H, J ) 2.4 Hz, H-1′), 5.74-5.77 (m, 1H, H-2′),
5.66-5.70 (m, 1H, H-3′), 4.43-4.54 (m, 2H, H-4′ + H-5′),
4.19-4.24 (m, 1H, H-5′), 3.97 (s, 3H, -OCH3), 3.00 (br s, 1H,
-OH), 2.14 (s, 9H, -C(O)CH3), 2.06-2.08 (m, 4H, cyclopentanyl-
H), 1.77-1.93 (m, 4H, cyclopentanyl-H). 13C NMR (75 MHz,
CDCl3): δ 170.9, 169.8, 159.4, 154.6, 141.4, 104.6, 89.1, 81.0, 74.4,
70.8, 68.1, 62.9, 52.9, 42.2, 23.7, 20.7, 20.6. Maldi-MS: m/z 516.2
[M + Na]+. HRMS: calcd for C22H27N3O10Na+ 516.1589, found
516.1586. IR: 2240.8 cm-1 (-Ct C-).
[M + Na]+. HRMS: calcd for C33H28N3O9 610.1820, found
+
610.1824. IR: 2217.9 cm-1 (-Ct C-).
3j. A total of 75.6 mg (77%) of product was obtained, isolated
as a colorless oil. HPLC: t ) 6.62 min, purity >99% (method 1);
t ) 6.49 min, purity 96.6% (method 2). 1H NMR (300 MHz,
CDCl3): δ 7.49-7.50 (m, 2H, thiphenyl-H), 7.08-7.11 (m, 1H,
thiophenyl-H), 6.25 (d, 1H, J ) 3.6 Hz, H-1′), 5.85-5.88 (m, 1H,
H-2′), 5.71-5.74 (m, 1H, H-3′), 4.48-4.53 (m, 2H, H-4′ + H-5′),
4.16-4.21 (m, 1H, H-5′), 3.99 (s, 3H, -OCH3), 2.15 (s, 9H,
-C(O)CH3). 13C NMR (150 MHz, CDCl3): δ 170.9, 169.7, 169.4,
159.5, 155.2, 141.8, 135.6, 130.9, 127.9, 119.7, 92.7, 89.0, 81.5,
77.8, 74.4, 71.1, 63.0, 53.1, 20.7. Maldi-MS: m/z 514.1 [M + Na]+.
HRMS: calcd for C21H22N3O9S+ 492.1071, found 492.1062. IR:
2223.7 cm-1 (-Ct C-).
3p. A total of 97.3 mg (96%) of product was obtained, isolated
as a colorless oil. HPLC: t ) 6.56 min, purity 98.9% (method 1);
t ) 6.47 min, purity 97.8% (method 2). 1H NMR (300 MHz,
CDCl3): δ 6.21 (d, 1H, J ) 2.7 Hz, H-1′), 5.72-5.75 (m, 1H, H-2′),
5.65-5.69 (m, 1H, H-3′), 4.45-4.53 (m, 2H, H-4′ + H-5′),
4.18-4.24 (m, 1H, H-5′), 3.98 (s, 3H, -OCH3), 3.42 (br s, 1H,
-OH), 2.13 (s, 9H, -C(O)CH3), 2.05-2.06 (m, 2H, cyclohexanyl-
H), 1.61-1.74 (m, 4H, cyclohexanyl-H), 1.56-1.59 (m, 4H,
cyclohexanyl-H). 13C NMR (75 MHz, CDCl3): δ 170.9, 169.7,
169.6, 159.4, 154.6, 141.3, 104.5, 89.0, 81.0, 74.4, 70.8, 69.1, 68.8,
62.9, 52.9, 39.2, 25.1, 23.1, 20.7, 20.6. Maldi-MS: m/z 530.2 [M
+ Na]+. HRMS: calcd for C23H29N3O10Na+ 530.1745, found
530.1740. IR: 2234.7 cm-1 (-Ct C-).
General Procedure for Preparing 2. 3 was dissolved in 10 mL
of saturated NH3/MeOH and stirred at room temperature for 2 days.
Then the solvent was removed, the residue was dissolved in MeOH
and crystallized by precipitation in CH2Cl2. Then the crystalline
state precipitation was washed with CH2Cl2 and dried in vacuo to
afford the corresponding product 2a-p.
2a. An amount of 64.4 mg (0.133 mmol) of 3a was used for the
reaction. A total of 38.4 mg (84%) of product was obtained, isolated
as a white solid. HPLC: t ) 8.04 min, purity 96.0% (method 1); t
) 6.12 min, purity 98.7% (method 2). 1H NMR (300 MHz, DMSO-
d6): δ 8.05 (br, 1H, -C(O)NH2), 7.77 (br, 1H, -C(O)NH2),
7.71-7.73 (m, 2H, phenyl-H), 7.51-7.62 (m, 3H, phenyl-H), 6.01
(d, 1H, J ) 5.2 Hz, H-1′), 5.65 (d, 1H, J ) 6.0 Hz, -OH), 5.29 (d,
1H, J ) 5.1 Hz, -OH), 4.82 (t, 1H, J ) 5.6 Hz, -OH), 4.51-4.54
(m, 1H, H-2′), 4.24-4.27 (m, 1H, H-3′), 3.98-4.00 (m, 1H, H-4′),
3.42-3.63 (m, 2H, H-5′). 13C NMR (75 MHz, DMSO-d6): δ 157.7,
140.7, 132.7, 131.6, 129.8, 119.9, 97.9, 91.3, 87.0, 75.4, 74.8, 71.3,
62.7. Maldi-MS: m/z 367.1 [M + Na]+. HRMS: calcd for
C16H16N4O5Na+ 367.1013, found 367.1012. IR: 2226.7 cm-1
(-Ct C-).
3k. A total of 77.6 mg (89%) of product was obtained, isolated
as a colorless oil. HPLC: t ) 6.84 min, purity 99.0% (method 1);
t ) 6.29 min, purity 98.5% (method 2). 1H NMR (300 MHz,
CDCl3): δ 7.78 (d, 1H, J ) 1.5 Hz, thiphenyl-H), 7.37-7.40 (m,
1H, thiophenyl-H), 7.28 (s, 1H, thiophenyl-H), 6.27 (d, 1H, J )
3.6 Hz, H-1′), 5.85-5.88 (m, 1H, H-2′), 5.72-5.75 (m, 1H, H-3′),
4.48-4.54 (m, 2H, H-4′ + H-5′), 4.11-4.22 (m, 1H, H-5′), 3.99
(s, 3H, -OCH3), 2.14 (s, 9H, -C(O)CH3). 13C NMR (150 MHz,
CDCl3): δ 170.9, 169.7, 169.4, 159.5, 155.1, 141.9, 132.9, 129.9,
126.6, 119.2, 94.5, 89.0, 81.4, 74.4, 73.9, 71.1, 63.0, 53.0, 20.8,
20.7. Maldi-MS: m/z 514.1 [M + Na]+. HRMS: calcd for
C21H22N3O9S+ 492.1071, found 492.1062. IR: 2225.1 cm-1
(-Ct C-).
3l. A total of 85.1 mg (87%) of product was obtained, isolated
as a pink solid. HPLC: t ) 7.19 min, purity >99% (method 1); t
) 6.35 min, purity 97.2% (method 2). 1H NMR (300 MHz, CDCl3):
δ 7.58 (s, 1H, imidazole-H), 7.56 (s, 1H, imidazole-H), 6.23 (d,
1H, J ) 3.0 Hz, H-1′), 5.84-5.86 (m, 1H, H-2′), 5.71-5.74 (m,
1H, H-3′), 4.49-4.54 (m, 2H, H-4′ + H-5′), 4.17-4.22 (m, 1H,
H-5′), 4.00 (s, 3H, -OCH3), 3.78 (s, 3H, -NCH3), 2.15 (s, 9H,
-C(O)CH3). 13C NMR (150 MHz, CDCl3): δ 170.9, 169.7, 169.5,
159.5, 155.2, 141.5, 140.3, 138.2, 113.8, 89.1, 87.4, 81.7, 81.4,
74.5, 71.0, 62.9, 53.2, 32.7, 20.9, 20.7. Maldi-MS: m/z 490.2 [M
+ H]+. HRMS: calcd for C21H24N5O9+ 490.1569, found 490.1548.
IR: 2226.1 cm-1 (-Ct C-).
A single crystal of product 2a suitable for X-ray crystallographic
analysis was obtained via slow evaporation of H2O solution.
Crystallographic data of 2a: colorless, triclinic space group P1, Z
) 2, a ) 7.5538 (11) Å, b ) 11.114 (17) Å, c ) 11.5618 (17) Å,
R ) 66.563° (2), ꢀ ) 89.504° (3), γ ) 78.045° (3), V ) 868.3 (2)
3m. A total of 86.5 mg (86%) of product was obtained, isolated
as a colorless oil. HPLC: t ) 7.10 min, purity 98.9% (method 1);
t ) 6.44 min, purity 96.7% (method 2). 1H NMR (300 MHz,
CDCl3): δ 6.45-6.47 (m, 1H, alkene-H), 6.18 (d, 1H, J ) 3.0 Hz,

1152 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Wan et al.
Å3, R (F2 > 2σF2) ) 0.0505 and wR ) 0.0894 (w ) 1/[σ2(Fo ) +
435.1 [M + Na]+. HRMS: calcd for C17H15N4O5F3Na+ 435.0887,
found 435.0885. IR: 2232.8 cm-1 (-Ct C-).
2
(0.0426P)2 + 0.0000P], P ) (Fo + 2Fc2)/3.
2
2b. An amount of 54.1 mg (0.105 mmol) of 3b was used for the
reaction. A total of 30.6 mg (78%) of product was obtained, isolated
as a white solid. HPLC: t ) 7.95 min, purity >99% (method 1); t
) 6.17 min, purity 97.3% (method 2). 1H NMR (300 MHz, DMSO-
d6): δ 8.03 (br, 1H, -C(O)NH2), 7.76 (br, 1H, -C(O)NH2), 7.66
(d, 2H, J ) 8.7 Hz, phenyl-H), 7.08 (d, 2H, J ) 8.1 Hz, phenyl-
H), 5.99 (d, 1H, J ) 3.6 Hz, H-1′), 5.64 (d, 1H, J ) 5.7 Hz, -OH),
5.29 (d, 1H, J ) 6.0 Hz, -OH), 4.78-4.85 (m, 1H, -OH),
4.51-4.52 (m, 1H, H-2′), 4.23-4.24 (m, 1H, H-3′), 3.97-3.99 (m,
1H, H-4′), 3.84 (s, 3H, -OCH3), 3.42-3.58 (m, 2H, H-5′). 13C
NMR (75 MHz, DMSO-d6): δ 161.8, 160.4, 157.6, 141.0, 134.6,
115.5, 111.6, 98.4, 91.1, 86.9, 74.8, 74.4, 71.2, 62.7, 56.2. Maldi-
MS: m/z 397.1 [M + Na]+. HRMS: calcd for C17H18N4O6Na+
397.1119, found 397.1119. IR: 2222.1 cm-1 (-Ct C-).
2c. An amount of 65.9 mg (0.105 mmol) of 3c was used for the
reaction. A total of 38.8 mg (82%) of product was obtained, isolated
as a white solid. HPLC: t ) 7.92 min, purity >99% (method 1); t
) 6.17 min, purity >99% (method 2). 1H NMR (300 MHz, DMSO-
d6): δ 8.04 (br, 1H, -C(O)NH2), 7.76 (br, 1H, -C(O)NH2), 7.60
(d, 2H, J ) 7.5 Hz, phenyl-H), 7.34 (d, 2H, J ) 8.1 Hz, phenyl-
H), 5.99 (d, 1H, J ) 3.6 Hz, H-1′), 5.64 (d, 1H, J ) 5.7 Hz, -OH),
5.29 (d, 1H, J ) 5.4 Hz, -OH), 4.81 (t, 1H, J ) 5.1 Hz, -OH),
4.50-4.52 (m, 1H, H-2′), 4.22-4.24 (m, 1H, H-3′), 3.97-3.98 (m,
1H, H-4′), 3.43-3.58 (m, 2H, H-5′), 2.39 (s, 3H, -CH3). 13C NMR
(150 MHz, DMSO-d6): δ 160.4, 157.7, 141.8, 140.8, 132.7, 130.4,
116.9, 98.2, 91.2, 86.9, 74.9, 74.8, 71.3, 62.7, 21.9. Maldi-MS:
m/z 381.1 [M + Na]+. HRMS: calcd for C17H18N4O5Na+ 381.1169,
found 381.1169. IR: 2223.9 cm-1 (-Ct C-).
2d. An amount of 48.9 mg (0.094 mmol) of 3d was used for the
reaction. A total of 27.1 mg (76%) of product was obtained, isolated
as a white solid. HPLC: t ) 7.94 min, purity 97.1% (method 1); t
) 6.18 min, purity 98.1% (method 2). 1H NMR (300 MHz, DMSO-
d6): δ 8.03 (br s, 1H, -C(O)NH2), 7.74-7.76 (m, 3H, C(O)NH2 +
phenyl-H), 7.61 (d, 2H, J ) 8.7 Hz, phenyl-H), 6.00 (d, 1H, J )
4.5 Hz, H-1′), 5.63 (d, 1H, J ) 5.7 Hz, -OH), 5.28 (d, 1H, J )
5.7 Hz, -OH), 4.80 (t, 1H, J ) 6.0 Hz, -OH), 4.50-4.52 (m, 1H,
H-2′), 4.22-4.24 (m, 1H, H-3′), 3.97-3.99 (m, 1H, H-4′),
3.43-3.58 (m, 2H, H-5′). 13C NMR (150 MHz, DMSO-d6): δ 160.3,
157.8, 140.5, 136.5, 134.5, 130.0, 118.8, 96.6, 91.3, 87.0, 76.2,
74.8, 71.2, 62.7. Maldi-MS: m/z 401.1 [M + Na]+. HRMS: calcd
for C16H16N4O5Cl+ 379.0804, found 379.0804. IR: 2230.7 cm-1
(-Ct C-).
2g. An amount of 55.7 mg (0.101 mmol) of 3g was used for the
reaction. A total of 31.1 mg (78%) of product was obtained, isolated
as a white solid. HPLC: t ) 7.08 min, purity >99% (method 1); t
) 6.12 min, purity >99% (method 2). 1H NMR (300 MHz, DMSO-
d6): δ 8.13 (s, 1H, phenyl-H), 8.03-8.05 (m, 2H, -C(O)NH2 +
phenyl-H), 7.96 (d, 1H, J ) 8.1 Hz, phenyl-H), 7.76-7.81 (m,
2H, phenyl-H), 6.08 (d, 1H, J ) 3.6 Hz, H-1′), 5.65 (d, 1H, J )
5.7 Hz, -OH), 5.30 (d, 1H, J ) 5.7 Hz, -OH), 4.82 (t, 1H, J )
5.9 Hz, -OH), 4.50-4.55 (m, 1H, H-2′), 4.24-4.29 (m, 1H, H-3′),
4.00-4.01 (m, 1H, H-4′), 3.46-3.62 (m, 2H, H-5′). 13C NMR (150
MHz, DMSO-d6): δ 160.3, 157.8, 140.2, 136.8, 131.1, 130.6 (d,
1
2JCF ) 31.8 Hz), 129.3, 128.1, 124.2 (d, JCF ) 270.6 Hz), 121.2,
95.9, 91.3, 86.9, 76.5, 74.9, 71.2, 62.7. ESI-MS: m/z 435.1 [M +
Na]+. HRMS: calcd for C17H15N4O5F3Na+ 435.0887, found 435.0882.
IR: 2234.2 cm-1 (-Ct C-).
2h. An amount of 59.0 mg (0.107 mmol) of 3h was used for the
reaction. A total of 30.8 mg (75%) of product was obtained, isolated
as a white solid. HPLC: t ) 7.04 min, purity >99% (method 1); t
) 6.14 min, purity >99% (method 2). 1H NMR (300 MHz, DMSO-
d6): δ 8.06 (br s, 1H, -C(O)NH2), 7.94-8.01 (m, 2H, phenyl-H),
7.82-7.85 (m, 2H, phenyl-H), 7.78 (br s, 1H, -C(O)NH2), 5.99
(d, 1H, J ) 4.2 Hz, H-1′), 5.62 (d, 1H, J ) 6.0 Hz, -OH), 5.25 (d,
1H, J ) 6.0 Hz, -OH), 4.80 (t, 1H, J ) 5.6 Hz, -OH), 4.49-4.52
(m, 1H, H-2′), 4.24-4.28 (m, 1H, H-3′), 3.96-4.00 (m, 1H, H-4′),
3.41-3.58 (m, 2H, H-5′). 13C NMR (150 MHz, DMSO-d6): δ 160.3,
157.9, 140.1, 135.7, 133.7, 132.0, 130.9 (q, 2JCF ) 30.2 Hz), 127.2,
1
123.9 (q, JCF ) 272.4 Hz), 117.6, 93.2, 91.3, 86.9, 79.9, 74.8,
71.1, 62.6. ESI-MS: m/z 435.1 [M + Na]+. HRMS: calcd for
C17H15N4O5F3Na+ 435.0887, found 435.0878. IR: 2232.9 cm-1
(-Ct C-).
2i. An amount of 65.9 mg (0.108 mmol) of 3i was used for the
reaction. A total of 41.6 mg (82%) of product was obtained, isolated
as a yellow solid. HPLC: t ) 8.23 min, purity 98.8% (method 1);
t ) 7.02 min, purity 97.3% (method 2). 1H NMR (300 MHz,
DMSO-d6): δ 8.14-8.55 (m, 11H, pyrene-H and br, 1H,
-C(O)NH2), 7.83 (br s, 1H, -C(O)NH2), 6.25 (d, 1H, J ) 3.6 Hz,
H-1′), 5.75 (d, 1H, J ) 5.7 Hz, -OH), 5.36 (d, 1H, J ) 6.0 Hz,
-OH), 4.87 (t, 1H, J ) 5.7 Hz, -OH), 4.62-4.67 (m, 1H, H-2′),
4.29-4.34 (m, 1H, H-3′), 4.07-4.11 (m, 1H, H-4′), 3.51-3.67 (m,
2H, H-5′). 13C NMR (150 MHz, DMSO-d6): δ 160.5, 157.8, 141.0,
133.1, 132.6, 131.3, 130.93, 130.86, 130.5, 130.2, 127.84, 127.76,
127.4, 127.3, 125.7, 124.6, 124.1, 123.7, 113.7, 97.2, 91.4, 87.1,
80.7, 74.8, 71.3, 62.7. Maldi-MS: 491.1 [M + Na]+. HRMS: calcd
for C26H20N4O5Na+ 491.1326, found 491.1314. IR: 2218.7 cm-1
(-Ct C-).
2j. An amount of 51.1 mg (0.104 mmol) of 3j was used for the
reaction. A total of 31.0 mg (85%) of product was obtained, isolated
as a yellow solid. HPLC: t ) 7.11 min, purity >99% (method 1);
t ) 6.05 min, purity >99% (method 2). 1H NMR (300 MHz,
DMSO-d6): δ 8.04 (br s, 1H, -C(O)NH2), 7.91-7.92 (m, 1H,
thiophene-H), 7.77 (br s, 1H, -C(O)NH2), 7.72-7.73 (m, 1H,
thiophene-H), 7.24-7.26 (m, 1H, thiophene-H), 5.94 (d, 1H, J )
3.9 Hz, H-1′), 5.65 (d, 1H, J ) 5.4 Hz, -OH), 5.30 (d, 1H, J )
5.4 Hz, -OH), 4.75-4.85 (m, 1H, -OH), 4.51-4.53 (m, 1H, H-2′),
4.23-4.25 (m, 1H, H-3′), 3.97-3.99 (m, 1H, H-4′), 3.44-3.60 (m,
2H, H-5′). 13C NMR (150 MHz, DMSO-d6): δ 160.4, 157.7, 140.6,
136.7, 132.9, 129.0, 119.0, 91.6, 91.2, 86.9, 78.8, 74.7, 71.2, 62.6.
ESI-MS: m/z 373.1 [M + Na]+. HRMS: calcd for C14H14N4O5SNa+
373.0577, found 373.0585. IR: 2216.9 cm-1(-Ct C-).
2e. An amount of 75.5 mg (0.15 mmol) of 3e was used for the
reaction. A total of 41.8 mg (77%) of product was obtained, isolated
as a white solid. HPLC: t ) 7.77 min, purity >99% (method 1); t
) 6.07 min, purity 98.3% (method 2). 1H NMR (300 MHz, DMSO-
d6): δ 8.03 (br s, 1H, -C(O)NH2), 7.77-7.82 (m, 3H, -C(O)NH2
+ phenyl-H), 7.39 (dd, 2H, 3JHF ) 8.7 Hz, 3JHH ) 9.0 Hz, phenyl-
H), 6.00 (d, 1H, J ) 3.9 Hz, H-1′), 5.63 (d, 1H, J ) 5.7 Hz, -OH),
5.28 (d, 1H, J ) 5.7 Hz, -OH), 4.80 (t, 1H, J ) 5.0 Hz, -OH),
4.50-4.52 (m, 1H, H-2′), 4.23-4.24 (m, 1H, H-3′), 3.97-3.99 (m,
1H, H-4′), 3.44-3.60 (m, 2H, H-5′). 13C NMR (150 MHz, DMSO-
3
d6): δ 162.2, 160.3, 157.7, 140.6, 135.5 (d, JCF ) 9.6 Hz), 117.2
(d, 2JCF ) 22.4 Hz), 116.4, 96.9, 91.3, 87.0, 75.2, 74.8, 71.2, 62.7.
Maldi-MS: m/z 385.1 [M
+
Na]+. HRMS: calcd for
C16H15N4O5FNa+ 385.0919, found 385.0917. IR: 2229.4 cm-1
(-Ct C-).
2f. An amount of 87.7 mg (0.159 mmol) of 3f was used for the
reaction. A total of 47.7 mg (73%) of product was obtained, isolated
as a yellow solid. HPLC: t ) 8.26 min, purity >99% (method 1);
t ) 6.11 min, purity >99% (method 2). 1H NMR (300 MHz,
DMSO-d6): δ 8.07 (br s, 1H, -C(O)NH2), 7.89-7.98 (m, 4H,
phenyl-H), 7.80 (br s, 1H, -C(O)NH2), 6.05 (d, 1H, J ) 3.6 Hz,
H-1′), 5.66 (d, 1H, J ) 5.1 Hz, -OH), 5.31 (d, 1H, J ) 5.7 Hz,
-OH), 4.83 (t, 1H, J ) 5.6 Hz, -OH), 4.51-4.56 (m, 1H, H-2′),
4.24-4.29 (m, 1H, H-3′), 3.99-4.01 (m, 1H, H-4′), 3.45-3.62 (m,
2H, H-5′). 13C NMR (150 MHz, DMSO-d6): δ 160.3, 157.8, 140.2,
2k. An amount of 68.7 mg (0.14 mmol) of 3k was used for the
reaction. A total of 37.2 mg (76%) of product was obtained, isolated
as a yellow solid. HPLC: t ) 7.08 min, purity >99% (method 1);
t ) 6.05 min, purity >99% (method 2). 1H NMR (300 MHz,
DMSO-d6): δ 8.24 (d, 1H, J ) 1.5 Hz, thiophenyl-H), 8.03 (br s,
1H, -C(O)NH2), 7.76-7.78 (m, 2H, thiophenyl-H and -C(O)NH2),
7.42 (d, 1H, J ) 4.5 Hz, thiophenyl-H), 5.98 (d, 1H, J ) 4.5 Hz,
H-1′), 5.65 (d, 1H, J ) 5.7 Hz, -OH), 5.30 (d, 1H, J ) 5.7 Hz,
-OH), 4.84 (t, 1H, J ) 5.6 Hz, -OH), 4.49-4.54 (m, 1H, H-2′),
4.22-4.27 (m, 1H, H-3′), 3.96-3.99 (m, 1H, H-4′), 3.46-3.61 (m,
2
1
133.6, 131.2 (q, JCF ) 31.5 Hz), 126.7, 124.4 (q, JCF ) 271.2
Hz), 124.2, 96.0, 91.4, 87.0, 77.3, 74.9, 71.2, 62.6. Maldi-MS: m/z

Arylethynyltriazole Ribonucleosides
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1153
2H, H-5′). 13C NMR (150 MHz, DMSO-d6): δ 160.4, 157.7, 140.8,
134.6, 130.3, 128.7, 118.8, 93.6, 91.1, 86.9, 74.9, 74.8, 71.2, 62.7.
Maldi-MS: m/z 373.1 [M + Na]+. HRMS: calcd for C14H15N4O5S+
351.0758, found 351.0760. IR: 2223.6 cm-1(-Ct C-).
d6): δ 160.4, 157.5, 140.7, 104.5, 91.0, 86.7, 74.7, 71.2, 69.4, 67.9,
62.7, 25.3, 23.2. Maldi-MS: m/z 389.1 [M + Na]+. HRMS: calcd
for C16H22N4O6Na+ 389.1432, found 389.1438. IR: 2231.8 cm-1
(-Ct C-).
2l. An amount of 60.0 mg (0.123 mmol) of 3l was used for the
reaction. A total of 38.8 mg (91%) of product was obtained, isolated
as a yellow solid. HPLC: t ) 9.29 min, purity 98.5% (method 1);
t ) 6.18 min, purity 96.0% (method 2). 1H NMR (300 MHz,
DMSO-d6): δ 8.04 (br s, 1H, -C(O)NH2), 7.94 (s, 1H, H-
imidazole), 7.77 (br s, 1H, -C(O)NH2), 7.62 (s, 1H, H-imidazole),
5.96 (d, 1H, J ) 4.5 Hz, H-1′), 5.64 (d, 1H, J ) 5.4 Hz, -OH),
5.29 (d, 1H, J ) 5.1 Hz, -OH), 4.81 (t, 1H, J ) 4.5 Hz, -OH),
4.51-4.53 (m, 1H, H-2′), 4.21-4.26 (m, 1H, H-3′), 3.97-3.98 (m,
1H, H-4′), 3.76 (s, 3H, CH3), 3.45-3.62 (m, 2H, H-5′). 13C NMR
(150 MHz, DMSO-d6): δ 160.4, 157.7, 141.9, 140.6, 137.9, 113.4,
91.3, 87.1, 86.9, 82.5, 74.7, 71.2, 62.6, 32.7. Maldi-MS: m/z 349.1
7. An amount of 35.9 mg (0.1 mmol) of 6 was dissolved in 3
mL acetic anhydride and stirred at 75 °C until TLC indicated the
complete consumption of 6. Then the solvent was removed and
the residue purified by flash chromatography on silica gel (CH2Cl2/
MeOH, 40:1). The purified material was dried in vacuo to afford
37.8 mg (94%) of product 7 as a white solid. HPLC: t ) 6.91 min,
purity >99% (method 1); t ) 6.35 min, purity 97.8% (method 2).
1H NMR (300 MHz, CDCl3): δ 7.69-7.77 (m, 4H, phenyl-H), 7.07
(br s, 1H, -C(O)NH2), 6.01 (br s, 1H, -C(O)NH2), 5.75 (s, 2H,
-CH2-), 4.23 (t, 2H, J ) 4.4 Hz, -CH2-), 3.91 (t, 2H, J ) 4.4
Hz, -CH2-), 2.06 (s, 3H, -C(O)CH3). 13C NMR (150 MHz,
CDCl3): δ 171.1, 160.5, 156.5, 140.7, 132.8, 132.4 (q, 2JCF ) 31.8
[M + H]+. HRMS: calcd for C14H17N6O5 349.1255, found
Hz), 126.0 (d, JCF ) 3.2 Hz), 123.7, 123.7 (q, JCF ) 271.7 Hz),
+
3
1
349.1247. IR: 2223.9 cm-1(-Ct C-).
96.7, 78.6, 76.2, 68.6, 62.9, 21.1. Maldi-MS: 419.1 [M + Na]+.
HRMS: calcd for C17H16F3N4O4 397.1118, found 397.1123. IR:
2237.8 cm (-Ct C-).
+
2m. An amount of 76.6 mg (0.157 mmol) of 3m was used for
the reaction. A total of 45.2 mg (86%) of product was obtained,
isolated as a yellow solid. HPLC: t ) 7.38 min, purity >99%
(method 1); t ) 6.23 min, purity >99% (method 2). 1H NMR (300
MHz, DMSO-d6): δ 8.01 (br s, 1H, -C(O)NH2), 7.73 (br s, 1H,
-C(O)NH2), 6.51 (s, 1H, alkene-H), 5.85 (d, 1H, J ) 4.2 Hz, H-1′),
5.61 (d, 1H, J ) 5.1 Hz, -OH), 5.28 (d, 1H, J ) 5.1 Hz, -OH),
4.80 (t, 1H, J ) 5.1 Hz, -OH), 4.46-4.48 (m, 1H, H-2′),
4.19-4.21 (m, 1H, H-3′), 3.93-3.95 (m, 1H, H-4′), 3.40-3.60 (m,
2H, H-5′), 2.19-2.20 (m, 4H, -CH2-), 1.58-1.65 (m, 4H,
-CH2-). 13C NMR (150 MHz, DMSO-d6): δ 160.4, 157.6, 141.6,
141.1, 118.8, 99.9, 91.0, 86.9, 74.6, 73.1, 71.2, 62.7, 28.4, 26.1,
22.1, 21.3. Maldi-MS: m/z 371.1 [M + Na]+. HRMS: calcd for
C16H20N4O5Na+ 371.1326, found 371.1324. IR: 2214.1 cm-1
(-Ct C-).
-1
8. 5-Bromo-1-[(2-acetyl)methyl]-1,2,4-triazole-3-carboxylate (63.5
mg, 0.2 mmol), 4-trifluoromethylphenylacetylene 39 µL (0.24
mmol), tetrakis(triphenylphosphine)palladium(0) (11.2 mg, 0.01
mmol), CuI (1.8 mg, 0.01 mmol), and triethylamine (0.3 mL) were
suspended in 3 mL of fresh distilled dioxane under argon. The vessel
was sealed and irradiated at 100 °C for 30 min and then cooled to
room temperature. The reaction mixture was concentrated under
reduced pressure, and the crude residue purified by flash chroma-
tography on silica gel (petroleum ether/ethyl acetate, 2:1). The
purified material was dried in vacuo to afford the corresponding
product 8. A total of 48.6 mg (60%) of product was obtained,
isolated as a white oil. HPLC: t ) 6.76 min, purity >99% (method
1
1); t ) 6.43 min, purity >99% (method 2). H NMR (300 MHz,
2n. An amount of 80.1 mg (0.165 mmol) of 3n was used for the
reaction. A total of 40.3 mg (71%) of product was obtained, isolated
as a white solid. HPLC: t ) 7.29 min, purity >99% (method 1); t
) 5.99 min, purity >99% (method 2). 1H NMR (300 MHz, DMSO-
d6): δ 7.96 (br s, 1H, -C(O)NH2), 7.71 (br s, 1H, -C(O)NH2),
5.87 (d, 1H, J ) 4.5 Hz, H-1′), 5.59 (d, 1H, J ) 6.0 Hz, -OH),
5.25 (d, 1H, J ) 5.1 Hz, -OH), 4.79 (t, 1H, J ) 5.6 Hz, -OH),
4.44-4.49 (m, 1H, H-2′), 4.18-4.23 (m, 1H, H-3′), 3.92-3.97 (m,
1H, H-4′), 3.77 (t, 2H, J ) 6.3 Hz, -CH2-), 3.43-3.59 (m, 2H,
H-5′), 2.76 (t, 2H, J ) 6.9 Hz, -CH2-), 2.01-2.10 (m, 2H,
-CH2-). 13C NMR (150 MHz, DMSO-d6): δ 160.4, 157.3, 140.8,
99.9, 90.8, 86.8, 74.6, 71.2, 67.8, 62.7, 44.6, 30.8, 16.9. Maldi-
MS: m/z 367.1 [M + Na]+. HRMS: calcd for C13H17N4O5ClNa+
367.0780, found 367.0784. IR: 2246.1 cm-1 (-Ct C-).
2o. An amount of 77.0 mg (0.156 mmol) of 3o was used for the
reaction. A total of 46.7 mg (85%) of product was obtained, isolated
as a white solid. HPLC: t ) 7.48 min, purity >99% (method 1); t
) 5.94 min, purity 97.7% (method 2). 1H NMR (300 MHz, DMSO-
d6): δ 7.98 (br s, 1H, -C(O)NH2), 7.72 (br s, 1H, -C(O)NH2),
5.88 (d, 1H, J ) 3.9 Hz, H-1′), 5.77 (s, 1H, -OH), 5.60 (d, 1H, J
) 6.0 Hz, -OH), 5.26 (d, 1H, J ) 5.1 Hz, -OH), 4.79 (t, 1H, J
) 5.4 Hz, -OH), 4.44-4.49 (m, 1H, H-2′), 4.20-4.25 (m, 1H,
H-3′), 3.94-3.95 (m, 1H, H-4′), 3.39-3.60 (m, 2H, H-5′),
1.94-1.96 (m, 4H, cyclopentane-H), 1.73-1.75 (m, 4H, cyclo-
pentane-H). 13C NMR (150 MHz, DMSO-d6): δ 160.4, 157.4, 140.8,
104.5, 91.0, 86.7, 74.7, 73.4, 71.2, 68.3, 62.7, 42.3, 23.7. Maldi-
MS: m/z 375.1 [M + Na]+. HRMS: calcd for C15H20N4O6Na+
375.1275, found 375.1274. IR: 2241.5 cm-1 (-Ct C-).
CDCl3): δ 7.64-7.75 (m, 4H, phenyl-H), 5.75 (s, 2H, -CH2-),
4.23 (t, 2H, J ) 4.8 Hz, -CH2-), 4.04 (s, 3H, -OCH3), 3.89 (t,
2H, J ) 4.7 Hz, -CH2-), 2.04 (s, 3H, -C(O)CH3). 13C NMR
(150 MHz, CDCl3): δ 171.0, 159.7, 154.8, 141.4, 132.8, 126.0,
123.9, 96.7, 78.7, 76.3, 68.5, 62.8, 53.3, 21.0. Maldi-MS: 434.1
[M + Na]+. HRMS: calcd for C18H17F3N3O5 412.1115, found
+
412.1123. IR: 2234.0 cm-1 (-Ct C-).
10. An amount of 37.2 mg (0.09 mmol) of 2f was dissolved in
5 mL of MeOH and 4.0 mg of 10% Pd/C added. H2 was flushed
in, and the reaction mixture was stirred at room temperature
overnight. The insoluble residue was filtered by Celite and the
solvent removed by reduced pressure. The residue was dried in
vacuo to afford 37.0 mg (98.5%) of product 10 as a white solid.
HPLC: t ) 7.44 min, purity 98.8% (method 1); t ) 6.03 min, purity
1
98.1% (method 2). H NMR (300 MHz, DMSO-d6): δ 7.74 (br s,
1H, -C(O)NH2), 7.66 (d, 2H, J ) 8.1 Hz, phenyl-H), 7.61 (br s,
1H, -C(O)NH2), 7.53 (d, 2H, J ) 8.1 Hz, phenyl-H), 5.83 (d, 1H,
J ) 4.2 Hz, H-1′), 5.46 (d, 1H, J ) 5.7 Hz, -OH), 5.22 (d, 1H, J
) 5.1 Hz, -OH), 4.78 (t, 1H, J ) 5.6 Hz, -OH), 4.45-4.48 (m,
1H, H-2′), 4.19-4.21 (m, 1H, H-3′), 3.93-3.94 (m, 1H, H-4′),
3.42-3.58 (m, 2H, H-5′), 3.15-3.22 (m, 4H, -CH2-). 13C NMR
(75 MHz, DMSO-d6): δ 161.2, 157.8, 156.3, 145.8, 129.9, 127.7
(q, 2JCF ) 32.3 Hz), 125.8, 125.1 (d, 1JCF ) 270.5 Hz), 90.1, 86.5,
74.7, 71.2, 62.7, 33.2, 26.9. Maldi-MS: 439.1 [M + Na]+. HRMS:
calcd for C17H19F3N4O5Na+ 439.1200, found 439.1200.
12. An amount of 39.0 mg (0.071 mmol) of 3f was dissolved in
5 mL of CH2Cl2 and 4.0 mg of 10% Pd/C added. H2 was flushed
in, and the reactants were stirred at room temperature overnight.
The insoluble residue was filtered by Celite and the solvent removed
by reduced pressure. The residue was dried in vacuo to afford 39.0
mg (99.3%) of product 12 as colorless oil. HPLC: t ) 6.76 min,
purity >99% (method 1); t ) 6.21 min, purity >99% (method 2).
1H NMR (300 MHz, CDCl3): δ 7.56 (d, 2H, J ) 8.1 Hz, phenyl-
H), 7.32 (d, 2H, J ) 8.1 Hz, phenyl-H), 5.81 (br, 2H, H-1′ and
H-2′), 5.68 (br, 1H, H-3′), 4.40-4.47 (m, 2H, H-5′), 4.11-4.16
(m, 1H, H-4′), 3.98 (s, 3H, -OCH3), 3.21-3.23 (m, 2H, -CH2-),
3.11-3.17 (m, 2H, -CH2-), 2.13 (s, 3H, -C(O)CH3), 2.11 (s,
3H, -C(O)CH3), 2.07 (s, 3H, -C(O)CH3). 13C NMR (150 MHz,
2p. An amount of 69.0 mg (0.136 mmol) of 3p was used for the
reaction. A total of 43.8 mg (88%) of product was obtained, isolated
as a white solid. HPLC: t ) 7.48 min, purity 98.5% (method 1); t
) 5.97 min, purity 98.6% (method 2). 1H NMR (300 MHz, DMSO-
d6): δ 8.01 (br s, 1H, -C(O)NH2), 7.74 (br s, 1H, -C(O)NH2),
5.91 (s, 1H, -OH), 5.89 (d, 1H, J ) 4.2 Hz, H-1′), 5.62 (d, 1H, J
) 5.1 Hz, -OH), 5.27 (d, 1H, J ) 5.1 Hz, -OH), 4.80 (t, 1H, J
) 5.4 Hz, -OH), 4.43-4.48 (m, 1H, H-2′), 4.20-4.25 (m, 1H,
H-3′), 3.91-3.94 (m, 1H, H-4′), 3.40-3.58 (m, 2H, H-5′),
1.44-1.88 (m, 10H, cyclohexane-H). 13C NMR (150 MHz, DMSO-

1154 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Wan et al.
CDCl3): δ 170.7, 169.6, 160.2, 157.9, 154.2, 144.0, 129.2 (q, 2JCF
cytoplasmic nucleosomes produced during apoptosis (Cell Death
Detection ELISA plus, Roche). The 96-well plates were centrifuged
(200g) for 10 min, the supernatant was discarded, and lysis buffer
was added. After lysis, the samples were centrifuged and an amount
20 µL of the supernatant was transferred to a streptavidin-coated
microtiter plate. Biotin-labeled antihistone antibodies and peroxidase
conjugated anti-DNA antibodies were added to each well, and the
plate was incubated at room temperature for 2 h. After three washes
with buffer, the peroxidase substrate was added to each well to
quantitate the captured nucleosomes. After 20 min of incubation,
the plates were read at 405/490 nm in a microplate reader. The
enrichment in histone-DNA fragments is expressed as a fold
increase in absorbance compared with control.
Apoptosis Assay by Caspase-3/7 Cleavage Assay. Caspase-
3/7 activity was measured using the Apo-ONE homogeneous
caspase-3/7 assay fluorometric kit (Promega). MiaPaCa-2 cells were
initially seeded at 15 000 cells/well on 96-well plates. Twenty-four
hours later, cells were treated with the test compound for 48 h and
caspase-3 activity was measured by the cleavage of the fluorometric
substrate Z-DEVD-R110 according to the instructions of the
manufacturer (Promega). Next 100 µL of Apo-ONE homogeneous
caspase-3/7 reagent was added to each well of a black 96-well plate
containing 100 µL of blank, control, or cells in culture. Each
experiment was performed in triplicate. The plate was covered with
a plate sealer and incubated at room temperature for 30 min before
the fluorescence of each well was measured.
1
) 32.6 Hz), 128.9, 125.8, 124.3 (q, JCF ) 270.2 Hz), 88.2, 81.3,
74.5, 71.1, 62.9, 52.9, 33.5, 27.7, 20.72, 20.65, 20.57. Maldi-MS:
580.1 [M + Na]+. HRMS: calcd for C24H26N3O9F3Na+ 580.1513,
found 580.1498.
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 Dulbec-
co’s modified Eagle medium (DMEM) supplemented with 2% fetal
calf serum (FCS) and 250 µg/mL G418. After incubation for 24 h
at 37 °C (5% CO2) the medium was replaced by 3-fold serial
dilutions in complete DMEM (without G418) of the test compounds
in a total volume of 100 µL. After 4 days of incubation at 37 °C,
cell culture medium was removed and luciferase activity determined
using the Steady-Glo luciferase assay system (Promega, Leiden,
The Netherlands) and was measured using a Luminoskan ascent
(Thermo, Vantaa, Finland).
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 2% fetal calf serum
(FCS) and 1000 µg/mL G418. After 24 h of incubation at 37 °C,
cell culture medium was replaced by 3-fold serial dilutions of the
test compounds in complete DMEM without G418 in a total volume
of 100 µL. After 4 days of incubation at 37 °C, cell culture fluid
was removed and monolayers were washed once with phosphate-
buffered saline. Cells were lysed in 350 µL of 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 concentration of compound causing a 50%
reduction in HCV RNA levels compared to 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, Cam-
bridgeshire, U.K.), and the amount of HCV replicon RNA was
analyzed by quantitative real-time PCR. The values of EC50 were
calculated as the concentration of compound causing a 50%
reduction in HCV RNA levels compared to that of the untreated
control.
Anticancer Assay in MiaPaCa-2 Cells. In Vitro Cell Growth
Inhibition. Pancreatic cancer chemoresistant MiaPaCa-2 cells were
cultured in DMEM medium (Gibco) supplemented with 10% fetal
bovine serum (FBS). Cells were seeded at a densitiy of 15 000 cells
per well in a 96-well view plate (Packard) in 250 µL of medium
containing the same components as described above. Cells were
allowed to attach overnight (O/N), and then culture medium was
removed and replaced with fresh media alone as control or
containing different concentrations of compounds. Plates were
further incubated at 37 °C and 5% CO2 for 48 h. The number of
viable cells remaining after the appropriate treatment was deter-
mined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) colorimetric assay.
Apoptosis Assay by Flow Cytometry (FACS). Cells were
seeded in 10 cm dishes at a density of 106 cells/dish and allowed
to adhere and proliferate O/N. Culture medium was then removed,
and fresh medium containing the proper concentration of compound
was added. No treatment was done as negative controls. After 48 h
of treatment, the cells were trypsinized and the collected cell pellet
was washed with PBS and fixed in cold 70% ethanol overnight at
4 °C. After a wash with phosphate-citrate buffer, cells were treated
with 200 µL of RNase (500 µg/mL), labeled with 1 mL of
propidium iodide (50 µg/mL), and immediately analyzed by
fluorescence activated cell sorting (FACS Calibur, Becton Dick-
inson, Le Pont-De-Claix, France). Cell death analysis was done on
106 cells, evaluating the sub-G1/G0 ratio. Each sample was
performed in triplicate.
Assessment of in Vivo Tumor Growth. Institutional guidelines
for the proper and human use of animals in research were followed.
Approximately 1 × 107 MiaPaCa-2 cells were inoculated subcu-
taneously with 0.1 mL of Matrigel (BD Biosciences Discovery
Labware) to 6-week-old male xenografed nude mice. When
MiaPaCa-2 tumors reached 100 mm3, mice were randomly selected
for treatment with test compound, and no treated mice were used
as control. Each experimental group consisted of five mice. After
randomization, 150 mg/kg test compound was injected every 3 days
by ip injection for 4 weeks. Tumor volume measurements were
performed once weekly and calculated by the formula length ×
width × depth × 0.5236.
Statistical Analysis. All of the results were expressed as the
mean ( standard error (SE). Statistical analysis was performed by
a one way ANOVA followed by Fisher’s protected least significant
difference (PLSD) test (Statview 512, Brain Power Inc., Calabases,
CA). p e 0.05 was considered significant (*); p e 0.01 (**); p e
0.001 (***).
Cytotoxicity Assay. MiaPaCa-2, PC-3, and Hela cells were
cultured in DMEM medium (Gibco) supplemented with 10% fetal
bovine serum (FBS). HepG2 cells were cultured in MEM medium
supplemented with 10% FBS. Cells were seeded at a density of
15 000 cells per well in a 96-well view plate (Packard) in 250 µL
of medium containing the same components as described above.
L1210 cells were cultured in RPMI 1640 medium supplemented
with 5% FBS and were seeded at a density of 10 000 cells per
well in a 96-well view plate (Packard) in 50 µL of medium. Cells
were allowed to attach O/N, and then culture medium was removed
and replaced with fresh media alone as control or containing serial
dilutions of the test compounds from 10 nM to 200 µM. Plates
were further incubated at 37 °C and 5% CO2 for 48 h. The number
of viable cells remaining after the appropriate treatment was
determined by MTT colorimetric assay. The cell viability of L1210
cells were determined by CellTiter 96 AQueous One solution cell
proliferation assay (Promega).
Acknowledgment. Financial support from the Ministry of
Science and Technology of China (Grant Nos. 2003CB114400,
2003AA2Z3506, and 2006AA02Z339), National Natural Sci-
ence Foundation of China (Grant Nos. 20572081 and 20025311),
Ministry of Environmental Protection of China (Grant No.
200709046), Wuhan University, CNRS, INSERM, Ministere des
Affaires Etrangeres de la France, and the Geconcerteerde
Onderzoeksactie (KULeuven) are gratefully acknowledged. Y.X.
Apoptosis Assay by ELISA. Cells were seeded for 24 h in 96-
well plates (15 000 cells/well), and cells were treated by test
compounds or not as negative control. After 48 h, apoptosis was
assessed by an enzyme linked immunoassay (ELISA) that quantifies

Arylethynyltriazole Ribonucleosides
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1
Supporting Information Available: H and 13C NMR spectra
and HPLC analysis results for all new compounds described in the
work. This material is available free of charge via the Internet at
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