A novel arylethynyltriazole acyclonucleoside inhibits proliferation of drug-resistant pancreatic cancer cells

Article abstract of DOI:10.1016/j.bmcl.2010.08.093

Novel arylethynyltriazole acyclonucleosides were synthesized and assessed for their anticancer activity on drug-resistant pancreatic cancer MiaPaCa-2 cells. One lead compound was found to have much more potent apoptosis-related antiproliferative effects than gemcitabine, the current first-line treatment for pancreatic cancer. Further investigations showed that this active compound did not inhibit DNA synthesis, which means that it does not resemble gemcitabine and may involve a different mechanism of action.

Full text of DOI:10.1016/j.bmcl.2010.08.093

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5979–5983
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A novel arylethynyltriazole acyclonucleoside inhibits proliferation of
drug-resistant pancreatic cancer cells
Menghua Wang a, Yi Xia b, Yuting Fan a, Palma Rocchi c, Fanqi Qu a, Juan L. Iovanna c, Ling Peng b,
⇑
a State Key Laboratory of Virology, College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, PR China
b Département de Chimie, CNRS UPR 3118 CINaM, 163, avenue de Luminy, 13288 Marseille, France
c INSERM U624, 163, avenue de Luminy, 13288 Marseille, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel arylethynyltriazole acyclonucleosides were synthesized and assessed for their anticancer activity
on drug-resistant pancreatic cancer MiaPaCa-2 cells. One lead compound was found to have much more
potent apoptosis-related antiproliferative effects than gemcitabine, the current first-line treatment for
pancreatic cancer. Further investigations showed that this active compound did not inhibit DNA synthe-
sis, which means that it does not resemble gemcitabine and may involve a different mechanism of action.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 13 July 2010
Revised 17 August 2010
Accepted 17 August 2010
Available online 21 August 2010
Keywords:
Acyclic nucleosides
Triazole nucleosides
Pancreatic cancer
Anticancer activity
Sonogashira reaction
Pancreatic cancer is one of the most devastating forms of human
cancer: it is associated with a 5-year survival rate of only 3%.1 The
current first-line treatment is based on gemcitabine (Fig. 1), a nucle-
oside drug which acts mainly by inhibiting DNA synthesis along
with other possible modes of action.2 However, gemcitabine is only
moderately effective, since the response rate in humans is only 12%.
In addition, drug resistance develops very fast, resulting in a median
survival period of only five months.1,2 Consequently, there is an ur-
gent need to develop new and more efficacious drug candidates for
combating pancreatic cancer, especially the drug-resistant form.3
We recently discovered several novel aryltriazole ribonucleo-
sides with promising inhibitory effects on the proliferation of
drug-resistant pancreatic cancer cells (1–4 in Fig. 1).4–6 With the
special triazole heterocycle as nucleobase, the corresponding
nucleosides may be endowed with novel mechanisms of action,
different from the traditional nucleoside analogues with natural
nucleobases.7 Ribavirin, the first synthetic triazole nucleoside anti-
viral drug discovered 40 years ago,8a constitutes one of the best
examples in this regard, because it exerts its antiviral activities
by several modes of action including the inhibition of viral RNA
polymerases and viral capping enzymes, the lethal mutagenesis
of viral RNA genomes, the interference of host inosine monophos-
phate dehydrogenase (IMPDH) and the modulation of the host
immune responses.8 Recently, there has been renewed interest in
ribavirin and its apoptosis-related anticancer effect.9a,b The anti-
cancer mechanisms of ribavirin are also complex and pleiotropic:
ribavirin may inhibit the activity of an oncogene, the eukaryotic
translation initiation factor eIF4E, or modulate the immune system
through eIF4E and/or IMPDH.9b Ribavirin treatment led to signifi-
cant clinical improvement in patients with poor-prognosis acute
myeloid leukemia (AML),9c with its clinical benefit likely being
far beyond AML.9d Furthermore, the aromatic moieties appended
on the triazole ring of these ribonucleosides are expected to yield
enlarged aromatic systems serving as nucleobases.7 These triazole
nucleoside analogs may bind to the corresponding biological tar-
gets via greater and stronger interactions and thus provide novel
mechanisms of action for combating cancer. It was established
for example that triazole ribonucleoside 2 (Fig. 1) has potent anti-
cancer effects involving entirely new mechanisms,4,7 namely
down-regulation of the heat shock protein Hsp27, a small molecu-
lar chaperone which contributes importantly to the drug resistance
observed in many forms of cancer and provides a novel target for
developing new means of combating drug resistance in pancreatic
cancer.10
In our ongoing efforts to develop novel triazole nucleosides11–18
capable of anticancer activity, we have been focusing on acyclic
aryltriazole nucleoside analogs. Replacing ribose sugar with an
acyclic sugar component is known to yield nucleoside analogs with
further interesting biological properties. Acyclovir (Fig. 1) is one of
the most successful examples of acyclic nucleoside drugs showing
antiviral activity.19 Although acyclic ribavirin analogues were first
⇑
Corresponding author. Tel.: +33 491 82 91 54; fax: +33 491 82 93 01.
E-mail address: ling.peng@univmed.fr (L. Peng).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.093

5980
M. Wang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5979–5983
NH2
N
O
N
N
NH2
O
N
N
N
O
N
N
NH
NH2
HO
HO
F
F
O
O
HO
O
OH
OH OH
Gemcitabine
Ribavirin
Acyclovir
CH3
Cl
C5H11
O
HN
O
HN
N
N
N
O
N
OCH3
F3C
N
N
OMe
OMe
N
N
N
N
N
N
NH2
HO
AcO
AcO
AcO
O
O
O
O
O
OH OH
2
OAc OAc
3
OAc OAc
OAc OAc
1
4
O
O
N
N
N
N
NH2
NH2
R
F
N
N
HO
HO
O
O
A
5
Figure 1. Gemcitabine, ribavirin, acyclovir, the identified anticancer triazole ribonucleoside leads 1–4 and antiviral triazole acyclonucleoside 5, as well as the proposed
arylethynyltriazole acyclonucleosides (A).
synthesized in 1985 by Beauchamp et al., none of them showed
interesting biological activity.20 We have recently identified an
arylethynyl acyclonucleoside 5 (Fig. 1),13 which has significant
antiviral effects on the hepatitis C virus (HCV). All these findings
led us to study more closely the presence of the acyclic sugar com-
ponent in aryltriazole nucleoside analogs, with a view to designing
new nucleoside analogs with antitumoral properties. Here we
report on our recent studies, in which novel arylethynyltriazole
acyclonucleoside analogs were synthesized by performing Sono-
gashira cross-coupling reactions and the anticancer effects were
assessed on cancer cells. One of these novel acyclonucleoside ana-
logs was found to have particularly noteworthy antiproliferative
effects on drug-resistant pancreatic cancer MiaPaCa-2 cells, pre-
sumably via caspase-dependent apoptosis induction processes. In
addition, the potency of this analog is greater than that of gemcit-
abine, the reference drug to treat pancreatic cancer.
Arylethynyltriazole acyclonucleoside A was synthesized in a
single step, using our previously developed Pd-catalyzed Sono-
gashira cross-coupling method in aqueous media under microwave
irradiation (Table 1).13,21 In line with our previous findings, good to
excellent yields were obtained with both alkylacetylene (Table 1,
entry 1) and heterocyclic arylacetylenes (Table 1, entries 7 and 8)
in comparison with the reference n-butylphenylacetylene (Table
1, entry 2). Electron-withdrawing groups such as the Cl, Br, and
CN groups located at the para-position of the phenyl ring in phen-
ylacetylene resulted in lower yields (Table 1, entries 3–5), possibly
because the reduced nucleophilicity and increased electrophilicity
of the alkyne induced by the electron-withdrawing group impede
the transmetallation process and, in addition, induce undesirable
Michael additions.22 It is worth mentioning that the yield was
much lower in the case of A4, and that considerable amounts of
side products could be observed on the TLC plate. This may be
mainly due to the presence of the Br atom in A4, which may further
react with the remaining excess alkyne. For reasons so far not fully
understand, no reaction occurred with 4-ethynylaniline, and the
starting materials were recovered with a yield of up to 80%.
We then assessed the anticancer activity of the above synthe-
sized compounds and that of the previously synthesized arylethy-
nyltriazole acyclonucleosides13 by performing MTT assays to
determine the inhibition on the proliferation of drug-resistant
human pancreatic cancer MiaPaCa-2 cells.23 Among all the acyclic
nucleosides tested, A4 turned out to be the most promising lead,
since it was found to significantly inhibit the growth of MiaPaCa-
2 cells in a dose-dependent manner (Fig. 2). In addition, it is much
more potent than the control reference, gemcitabine, which is cur-
rently the first-line drug used to treat pancreatic cancer. In our pre-
vious study on aryltriazole nucleosides, only nucleoside analogs
bearing a ribose sugar component showed effective anticancer
activity (1–4 in Fig. 1).4–7 The results obtained in the present study
on the active compound A4 show that the acyclic analog A4 may
also constitute a promising new structural lead with potent anti-
cancer effects. To our knowledge, A4 is the first acyclic aryltriazole
nucleoside analog reported to inhibit the proliferation of drug-
resistant pancreatic cancer MiaPaCa-2 cells more efficiently than
gemcitabine.
We were particularly interested in the role of the acyclic sugar
component relating to the anticancer activity of A4. We therefore
synthesized two analogues of A4, 6 and 7,24,25 which have different
sugar components from A4. Compound 6 is a ribose analog of A4,
whereas 7 has the acyclic sugar part being protected by acetyl
group (Fig. 3). No anticancer activity was observed with 6, neither
did 7 elicit any significant activity (data not shown). Therefore, the

M. Wang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5979–5983
5981
Table 1
O
Synthesis of arylethynyltriazole acyclonucleosides
coupling in aqueous media using microwave irradiation, and their inhibition on the
cell growth of drug-resistant human pancreatic cancer MiaPaCa-2 cells
A
via the Sonogashira cross-
N
N
NH2
O
Br
N
N
N
NH2
HO
Br
N
O
O
O
AcO
N
N
N
N
NH2
NH2
O
Br
R
CuI, Pd(PPh3)4, Li2CO3
OH OH
N
N
dioxane/H2O(3:1), 100 ºC
MW, 25 min
HO
HO
7
6
O
O
Figure 3. Structural analogs of A4.
A
Entry
1
R
Products
A1
Yieldsa (%)
80
Inhibition (%)
NA
cells. After 48 h of treatment, the cells were labeled with propidi-
um iodide and immediately analyzed. An increase in the fraction
of cells undergoing apoptosis (sub-G1/G0 fraction) was observed
in cells treated with A4 in comparison with non-treated control
cells (Fig. 4A), which indicates that A4 acts mainly by inducing
apoptosis. To further confirm this point, caspase-3/7 activity was
determined by performing a colorimetric assay and measuring
the cleavage of the luminogenic substrate containing the tetrapep-
tide sequence DEVD.27 The results obtained showed the occurrence
of a twofold increase in caspase-3/7 activation in cells treated with
A4 as compared with non-treated control cells (Fig. 4B). Collec-
tively, these findings suggest that a caspase-3/7-dependent process
of apoptosis induction was involved.
We then examined whether A4 exerted its antiproliferative
effects by inhibiting DNA synthesis, because the anticancer nucle-
oside drug gemcitabine is known to inhibit DNA replication and
subsequently induce apoptosis, resulting in effective anticancer
effects.28 The action of A4 on the metabolism of nucleic acids
was assessed in MiaPaCa-2 cells by measuring the incorporation
of [3H]-thymidine into the DNA.29 As shown in Figure 5, no signif-
icant inhibitory effects on DNA synthesis were observed with A4,
whereas more than 80% DNA inhibition was achieved with
gemcitabine under the same conditions. This finding suggests that
A4 may have a different mechanism of action from that of
2
3
4
5
6
A2
A3
A4
A5
A6
90
67
21
22
69
19
—
Cl
Br
b
48
NC
53
0(80)c
H2N
7
8
A7
A8
75
70
NA
NA
S
S
CF3
9
A9
72
72
13
F3C
10
11
A10
21
30
Gemcitabine
NA: no activity.
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.
b
Flow Cytometry Analysis
By-products were observed by TLC.
No product and about 80% of starting material was recovered.
A
c
5
4
3
2
1
0
Dose-dependent assay on MiaPaCa-2 cells
120
gemcitabine
A4
100
80
60
40
20
0
control
A4
Caspase-3/7 assay on MiaPaCa-2 cells
B
2
1.5
1
Figure 2. Inhibitory effect of A4, in comparison with gemcitabine, on the drug-
resistant pancreatic cancer MiaPaCa-2 cell growth, assessed by MTT test.
0.5
0
control
A4
deprotected acyclic sugar component appears to be essential and
contributed crucially to the observed anticancer activity of A4.
We further studied the apoptosis-inducing anticancer effects of
A4 on MiaPaCa-2 cells, using fluorescence-activated cell sorting
(FACS) flow cytometry,26 in comparison with non-treated control
Figure 4. Apoptosis-induction in MiaPaCa-2 cells after treatment with A4, using
non treatment as control. (A) Flow cytometry was used to quantify the percentage
of cells undergoing apoptosis (cells in sub-G1/G0);26 (B) Caspase-3/7 activity was
measured using Caspase-Glo luminescent assay.27

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Figure 5. Inhibition of A4 on DNA synthesis by measuring the incorporation of
[3H]-dTMP in MiaPaCa-2 cells, in comparison with gemcitabine.29
gemcitabine. Whether A4 resembles ribavirin in anticancer
mechanisms is an open question and requires further insightful
investigation, which is not in the scope of the present work. As
the modes for ribavirin action in cancer therapy are complex,
pleiotropic, and not yet fully understood, a comparison of A4 with
ribavirin may be helpful to underpin their anticancer mecha-
nisms. This will constitute the research program of our future
investigation.
In conclusion, a series of novel arylethynyltriazole acyclonucle-
oside analogs was synthesized by performing Pd-catalyzed Sono-
gashira cross-coupling reactions using a previously described
simple, efficient one-step procedure. The antiproliferative effects
of these analogs were assessed on drug-resistant human pancreatic
cancer MiaPaCa-2 cells. One lead compound was found to have
superior antiproliferative effects to and different mechanism from
gemcitabine, the current reference treatment for pancreatic cancer.
This finding suggests that this acyclonucleoside could be a promis-
ing candidate in the search for new anticancer drugs with novel
mechanisms of action. We are currently engaged on detailed study
on its mode of action and further structure/activity relationship
analyses, the results of which are essential to our ongoing search
for potent novel anticancer drug candidates.
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17. Wu, Q. Y.; Qu, F. Q.; Wan, J. Q.; Zhu, X.; Xia, Y.; Peng, L. Helv. Chim. Acta 2004, 87,
811.
18. Wu, Q. Y.; Qu, F. Q.; Wan, J. Q.; Xia, Y.; Peng, L. Nucleosides Nucleotides Nucleic
Acids 2005, 999.
19. De Clercq, E.; Holy´, A. Nat. Rev. Drug Disc. 2005, 4, 928.
20. Beauchamp, L. M.; Dolmatch, B. L.; Schaeffer, H. J.; Collins, P.; Bauer, D. J.;
Keller, P. M.; Fyfe, J. A. J. Med. Chem. 1985, 28, 982.
21. General procedure for preparing A: The terminal alkynes (0.24 mmol),
tetrakis(triphenylphosphine)palladium(0) (11.6 mg, 0.01 mmol), CuI (1.9 mg,
Acknowledgments
0.01 mmol),
Li2CO3
(29.6 mg,
0.4 mmol)
and
(0.2 mmol)
5-bromo-1-[(2-
were
Financial supports from the Ministry of Science and Technology
of China (N°2003CB114400, N°2003AA2Z3506), National Science
Foundation of China (N°20372055, N°20473112), Wuhan Univer-
sity, CNRS and INSERM are gratefully acknowledged. We thank
Mr. Joël Tardivel-Lacombe for assistance in cell culture, and Mrs.
Jessica Blanc for English correction of manuscript. Dr. Yi Xia is sup-
ported by a post-doctoral fellowship from la Fondation pour la
Recherche Médicale.
hydroxyethoxy)methyl]-1,2,4-triazole-3-carboxamide
suspended in 2.8 mL of dioxane/H2O (3:1) under argon. The vessel was
sealed and 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/CH3OH, 20:1). The purified material was dried in vacuo to afford the
corresponding products.
22. (a) Agrofoglio, L. A.; Gillaizeau, I.; Saito, Y. Chem. Rev. 2003, 103, 1875; (b)
Hocek, M. Eur. J. Org. Chem. 2003, 245; (c) Negishi, E. I.; Anastasia, L. Chem. Rev.
2003, 103, 1979; (d) Anastasia, L.; Negishi, E. I. Org. Lett. 2001, 3, 3111.
23. In vitro cell growth inhibition assay: Pancreatic cancer chemo-resistant
MiaPaCa-2 cells were cultured in DMEM medium (Gibco) supplemented with
10% fetal bovine serum (FBS). Cells were seeded at a density of 15,000 cells per
Supplementary data
well in 96 well View Plate (Packard) in 250 lL of medium containing the same
Supplementary data (1H and 13C NMR spectra and MS analysis
of all new compounds are included) associated with this article
can be found, in the online version, at doi:10.1016/j.bmcl.2010.
08.093.
components as described above. Cells were allowed to attach overnight and
then culture medium was removed and replace with fresh media alone as
control or containing different 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 determined by (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide, MTT) colorimetric assay.
24. Compound 6 was synthesized following the protocol described in Ref. 5: 1H
NMR (250 MHz, DMSO-d6): d 8.06 (br s, 1H, –C(O)NH), 7.79 (br s, 1H, –C(O)NH),
7.76 (d, 2H, J = 8.8 Hz, phenyl-H), 7.68 (d, 2H, J = 8.5 Hz, phenyl-H), 6.01 (d, 1H,
J = 4.0 Hz, H-10), 5.65 (br s, 1H, –OH), 5.33 (br s, 1H, –OH), 4.82 (t, 1H, J = 5.6 Hz,
–OH), 4.53–4.50 (m, 1H, H-20), 4.26–4.23 (m, 1H, H-30), 4.02–3.96 (m, 1H, H-40),
3.63–3.41 (m, 2H, H-50); 13C NMR (62.5 MHz, DMSO-d6): d 159.6, 157.1, 139.7,
133.9, 132.2, 124.6, 118.4, 96.0, 90.5, 86.2, 75.6, 74.1, 70.5, 62.0; ESI-HRMS:
References and notes
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calcd. for C16H16BrN4O 423.0299, found 423.0299; IR: 2231 cmꢀ1 (–C„C–).
þ
25. Preparation of 7: 40.0 5mg (0.11 mmol) of A4 was dissolved in 6 mL acetic
anhydride and stirred under the catalyst 4-DMAP (0.01 mmol, 1.2 mg) until
TLC indicated the complete consumption of A4. Then the solvent was removed
and the residue purified by flash chromatography on silica gel (CH2Cl2/MeOH,
4. Xia, Y.; Liu, Y.; Wan, J. Q.; Wang, M. H.; Rocchi, P.; Qu, F. Q.; Neyts, J.; Iovanna, J.
L.; Peng, L. J. Med. Chem. 2009, 52, 6083.

M. Wang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5979–5983
5983
40:1). The purified material was dried in vacuo to afford 35.7 mg (80%) product
7 as a white solid: 1H NMR (250 MHz, CDCl3): d 7.56 (d, 2H, J = 8.5 Hz, phenyl-
H), 7.47 (d, 2H, J = 8.5 Hz, phenyl-H), 7.09 (br s, 1H, –C(O)NH), 6.38 (br s,1H, –
C(O)NH), 5.71 (s, 1H, –NCH2O–), 4.20 (t, 2H, J = 4.6 Hz, –CH2CH2OAc), 3.89 (t,
2H, J = 4.6 Hz, –CH2CH2OAc), 2.03 (s, 3H, –C(O)CH3); 13C NMR (62.5 MHz,
CDCl3): d 170.8, 160.3, 156.2, 140.8, 133.6, 132.2, 125.5, 118.7, 97.3, 78.3, 75.1,
68.3, 62.7, 20.9; ESI-HRMS: calcd for C16H16N4O4Br+ 407.0349, found 407.0345.
26. FACS flow cytometry: Cells were seeded in 10 cm dishes at the density of 106
cells/dish and allowed to adhere and proliferate overnight. Culture medium
was then removed and fresh media 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
27. Caspase-3/7 cleavage assay: 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. Next 100
lL of Caspase-Glo 3/7 Assay Reagent
(Promega) was added to each well of a white 96-well plate containing 100
lL
of blank, negative control cells or treated cells in culture medium. The caspase-
3/7 activity was measured by the cleavage of the luminogenic substrate
containing the tetrapeptide sequence DEVD according to the instructions of the
manufacturer (Promega). The plate was incubated at room temperature for 1 h
before measuring the luminescence of each well. Each experiment was
performed in triplicate.
28. Huang, P.; Chubb, S.; Hertel, L. W.; Grindey, G. B.; Plunkett, W. Cancer Res. 1991,
51, 6110.
29. Determination of DNA synthesis: MiaPaCa-2 Cells were seeded at a density of
15,000 cells per well in 96 well View Plate. Then the cells in exponential
growth phase were treated with the compounds for 4 h and then labelled with
with phosphate–citrate buffer, cells were treated with 200
lL RNase (500 lg/
mL), labeled with 1 mL propidium iodide (50 g/mL), and immediately
l
analyzed by Fluorescence Activated Cell Sorting (FACS Calibur, Becton
Dickinson, 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.
[3H]-thymidine (10
lCi/mL) for 4 h at 37 °C and 5% CO2. Then the cells were
harvested and DNA synthesis activity was determined according to the
radioactivity by using liquid scintillation counting.