N-Aryltriazole ribonucleosides with potent antiproliferative activity against drug-resistant pancreatic cancer

Article abstract of DOI:10.1016/j.bmcl.2010.02.104

Novel N-aryltriazole nucleosides were synthesized via Cu-mediated C-N cross-coupling reaction starting with 3-aminotriazole ribonucleoside and various boronic acids. Two of them exhibited potent apoptosis-related antiproliferative activity against the drug-resistant pancreatic cancer cell line MiaPaCa-2, with an increased potency compared to gemcitabine, the reference treatment for pancreatic cancer. A preliminary SAR study suggests that the appended N-aryl moiety and the substituent at its para-position, as well as the ribose sugar component, contribute considerably to the observed antiproliferative activity.

Full text of DOI:10.1016/j.bmcl.2010.02.104

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2503–2507
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
N-Aryltriazole ribonucleosides with potent antiproliferative activity against
drug-resistant pancreatic cancer
Yang Liu a, Yi Xia b, Yuting Fan a, Alain Maggiani b, 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 N-aryltriazole nucleosides were synthesized via Cu-mediated C–N cross-coupling reaction starting
with 3-aminotriazole ribonucleoside and various boronic acids. Two of them exhibited potent apoptosis-
related antiproliferative activity against the drug-resistant pancreatic cancer cell line MiaPaCa-2, with an
increased potency compared to gemcitabine, the reference treatment for pancreatic cancer. A preliminary
SAR study suggests that the appended N-aryl moiety and the substituent at its para-position, as well as
the ribose sugar component, contribute considerably to the observed antiproliferative activity.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 29 January 2010
Revised 24 February 2010
Accepted 26 February 2010
Available online 3 March 2010
Keywords:
Nucleosides
N-Arylated triazole nucleoside
Triazole nucleosides
Pancreatic cancer
N-Arylation
Synthetic nucleoside analogs are of considerable importance in
the search for new structural leads with biologically interesting
activity.1 Over the last few years, our research program has aimed
to develop novel triazole nucleoside analogs bearing aromatic sys-
tems on the nucleobase,2–9 with a view to identifying new drug
candidates against various cancers, particularly drug-resistant
pancreatic cancer.2,3 Pancreatic cancer represents one of the most
lethal forms of human cancer, and drug resistance develops extre-
mely fast.10 The current first-line treatment for pancreatic cancer is
based on gemcitabine (Fig. 1), a nucleoside drug which is only
moderately effective yielding a mere 12% response rate, with a
median survival period of five months and a five-year survival rate
as low as 3%.11 Consequently, there is an urgent need to develop
new and more efficacious drug candidates to treat pancreatic
cancer.12
We have previously developed a series of triazole nucleosides
with appended aromatic moieties on the triazole ring, such as
arylethynyltriazolyl,2–4 bitriazolyl,5,6 aryltriazolyl,7,8 and N-aryl
nucleosides,9 with the rationale to combine the special properties
of triazole heterocycles13 with aromatic systems as enlarged nucle-
obases. Such triazole nucleoside analogs may exhibit favorable
binding properties with the corresponding biological targets via
greater and stronger interactions14 and in turn offer novel mecha-
nisms of action. Some of these triazole nucleosides (I and II in
Fig. 1) indeed demonstrated potent anticancer activity against
drug-resistant pancreatic cancer in vitro and in vivo,2,3 with en-
tirely new mechanisms of action responsible for the down-regula-
tion of the heat shock protein Hsp27,2 a novel target for anticancer
therapy which plays an important role in the drug-resistance ob-
served in certain cancers.15–17
In our continuing efforts to develop triazole nucleosides as no-
vel anticancer drug candidates, we became interested in N-aryl-
triazole ribonucleosides (A and
B in Fig. 1). The frequent
occurrence of the N-aryl motif in many natural products and syn-
thetic drugs18 justifies the interest in their presence within the
nucleobases of nucleoside analogs with respect to designing new
nucleoside mimics. Additionally, we postulated that the relatively
flexible amine linkage between the triazole ring and aryl moiety
may confer an adaptive binding flexibility to the corresponding
biological targets. This would be an advantage over our previously
identified lead compounds I and II (Fig. 1), which have rather rigid
triple bond bridge between the aryl moiety and triazole ring of the
nucleobase.2,3
We have previously established the synthesis of N-aryltriazole
acyclonucleoside analogs (A in Fig. 1) using Chan-Lam modified
Cu-mediated cross-coupling reaction.9 However, none of the syn-
thesized acyclonucleoside analogs demonstrated any notable
anticancer activity. Here, we report the synthesis of novel N-aryl-
triazole ribonucleoside analogs (B in Fig. 1) using the Chan-Lam
cross-coupling reaction and the evaluation of their antiprolifera-
tive activity against pancreatic cancer cells. Two of the novel
* 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.2010.02.104

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Y. Liu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2503–2507
C5H11
NH2
N
O
N
OCH3
N
N
O
F3C
N
N
N
N
O
NH2
HO
AcO
O
HO
F
F
O
O
OH
OAc OAc
OH OH
II
I
Gemcitabine
Ar
HN
Ar
HN
N
N
R'
N
N
RO
O
N
R'
O
N
RO
O
O
OR OR
A
B
Figure 1. Gemcitabine, identified anticancer triazole nucleoside leads I and II, as well as N-aryltriazole acyclonucleosides (A) and ribonucleosides (B).
N-aryltriazole ribonucleoside analogs exhibited potent antiprolif-
erative activity against the drug-resistant pancreatic cancer cell
line MiaPaCa-2, presumably via a mechanism of caspase-depen-
dent apoptosis induction.
The synthesis of N-aryltriazole ribonucleosides A, specifically 2
and 3 in Scheme 1, was achieved using our previously developed
protocol of Cu-mediated C–N cross coupling (Scheme 1, Table 1).9
The starting material 3-aminotriazole ribonucleoside 1 was ob-
tained via reductive hydrogenation of the corresponding 3-azido-
triazole ribonucleoside.19,20 The N-arylation was performed with
various boronic acids in the presence of Cu(OAc)2 and pyridine
with freshly prepared molecular sieves in CH2Cl2 at room temper-
ature and open air for 3 days.21 It should be noted that in many
cases we were unable to obtain the corresponding products in rea-
sonably good yields (Table 1). In particular, the Cu-mediated N-
arylation reaction was not at all effective with sterically demand-
ing arylboronic reagents (Table 1, entries 4 and 13). This could
mainly be ascribed to the steric congestion created by the substitu-
ent at the ortho-position, which may in turn strongly impede C–N
coupling. In addition, no clear-cut electronic effect was observed
for the products bearing either electron-donating (Table 1, entries
7 and 8) or electron-withdrawing substituents (Table 1, entries 9–
12) on the phenylamine. After further treatment in NH3/MeOH at
room temperature, the nucleosides 2 were transformed to the cor-
responding deprotected form 3 with good to excellent yields
(Table 1).22
N3
H2N
N
N
O
O
N
N
N
N
OCH3
OCH3
AcO
AcO
O
H2, Pd/C
O
OAc OAc
OAc OAc
1
Ar B(OH)2
Ar
Ar
HN
N
HN
N
O
O
N
N
N
N
NH2
OCH3
HO
AcO
NH3
O
O
OH OH
OAc OAc
3
2
Scheme 1. Synthesis of N-aryltriazole ribonucleosides 2 and 3 starting from aminotriazole nucleoside 1 via Cu-mediated N-arylation followed by subsequent ammonolysis.

Y. Liu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2503–2507
2505
Table 1
Table 2
N-Aryltriazole ribonucleosides 2 and 3 synthesized via Cu-mediated N-arylation and
subsequent ammonolysis
The proliferation of human pancreatic cancer MiaPaCa-2 and Capan-2 cells as well as
human prostate cancer PC-3 cells was assessed using MTT assay after treatment with
50
lM compound 2 or 3, in comparison with gemcitabine and taxol, the clinical drugs
Entry Ar
1
Product 2 Yield (%) Product 3 Yield (%)
for pancreatic cancer and prostate cancer, respectively
Entry
Compound
Cell proliferation (%)
Capan-2
2a
2b
44
44
3a
3b
98
98
MiaPaCa-2
PC-3
2
1
2
3
2a
2b
2c
2d
137.7 5.6
58.6 1.7
99.9 0.04
—
96.2 2.2
47.1 1.4
84.9 6.0
—
91.0 5.2
68.6 4.8
94.2 2.0
—
H3C
H3C
4
3
4
2c
60
0
3c
81
—
5
6
7
8
2e
2f
2g
2h
2i
2j
2k
2l
2m
3a
3b
3c
3d
3e
3f
3g
99.0 1.8
102.8 1.9
96.4 1.0
85.4 2.5
98.6 3.6
80.2 6.4
43.8 1.6
72.4 6.4
—
68.0 1.5
85.6 4.0
100.1 3.3
—
99.8 3.7
103.1 2.8
126.4 1.0
98.6 2.8
100.3 0.4
68.1 4.7
95.9 1.5
88.7 1.5
—
90.0 5.2
95.9 6.5
72.0 2.4
88.1 4.8
64.4 4.1
79.8 3.6
59.6 2.2
65.8 3.5
—
46.6 2.0
63.9 3.0
76.3 2.8
—
66.0 3.1
71.5 1.4
126.2 7.2
71.1 2.9
121.9 2.3
62.9 3.1
72.8 3.5
66.2 1.5
—
96.1 1.3
90.2 2.9
85.6 4.5
105.6 1.9
88.4 2.3
84.7 2.4
60.2 1.2
69.8 4.3
—
73.9 3.8
107.5 4.3
96.2 1.8
—
91.4 0.7
90.3 1.5
102.2 2.3
92.1 2.7
89.4 3.9
67.0 5.4
70.2 4.2
77.4 1.8
—
CH3
2d
3d
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
5
6
2e
2f
43
46
34
29
21
50
42
3e
3f
94
82
95
86
96
94
98
7
2g
2h
2i
3g
3h
3i
H3CO
H3CS
F3C
F
8
3h
3i
3j
3k
9
10
11
2j
3j
3l
3m
Gemcitabine
Taxol
70.4 3.4
—
32.7 1.0
—
—
2k
3k
Cl
Cl
41.3 0.2
12
13
2l
40
0
3l
90
—
Dose-dependent assay on MiaPaCa-2 cells
140
A
Cl
2m
3m
100
2b
2k
gemcitabine
60
20
We then assessed the anticancer activity of the synthesized N-
aryltriazole ribonucleosides 2 and 3 against the drug-resistant hu-
man pancreatic cancer cell line MiaPaCa-2, drug-sensitive human
pancreatic cancer cell line Capan-2 and hormone-refractory human
prostate cancer cell line PC-3.23 Cells were treated with 50
lM
compound for 48 h in comparison with non-treatment control.
The number of viable cells remaining after treatment was deter-
mined by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide, MTT) colorimetric assay (Table 2). To our great delight,
two compounds 2b and 2k displayed interesting dose–responsive
inhibitory activity on cell growth of MiaPaCa-2 cells, with a much
better potency than the control reference drug gemcitabine
(Fig. 2A), and an equivalent activity compared to the previously
identified two triazole nucleoside leads I and II (Fig. 2B). In addi-
tion, the inhibitory effect of 2b and 2k on cell growth seems to
be general, because both of them elicited also some inhibition on
the drug-sensitive pancreatic cancer Capan-2 cells and hormone-
refractory prostate cancer PC-3 cells (Table 2). However, their
inhibitory effects on Capan-2 and PC-3 cells are much less effective
than the corresponding clinical references, gemcitabine and taxol,
respectively. We thus focused our attention only on their activity
on drug-resistant pancreatic cancer MiaPaCa-2 cells.
MTT assay on MiaPaCa-2 cell lines
B
120
100
80
60
40
20
0
Figure 2. Inhibitory effect of compounds 2b and 2k on the pancreatic cancer
MiaPaCa-2 cell growth, assayed by MTT test. (A) Dose–responsive effect of 2b and
2k on the growth of MiaPaCa-2 cells compared to gemcitabine. (B) Antiproliferative
activity of 2b and 2k on the MiaPaCa-2 cells in comparison with non-treatment
control, gemcitabine control and the previously identified two triazole nucleoside
leads I and II.
A preliminary delineation of the structural features behind the
antiproliferative activity shown by compounds 2b and 2k on Mia-
PaCa-2 cells was obtained, based on comparing the analogs with
different substituents on the phenyl ring. Compounds bearing
strong electron-donating (2g and 2h) and electron-withdrawing
groups (2i) on the phenyl ring elicited no considerable inhibitory
effect on cell proliferation. This may be due to their unfavorable
electronic properties. In addition, replacing the methyl functional-

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Y. Liu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2503–2507
ity and the Cl atom on the phenyl moiety in 2b and 2k with either
smaller atoms such as H and F (2a and 2j) or larger groups such as
ethyl and propyl groups (2e and 2f), led to a significant reduction
in activity. Further analogs harboring a substitution at the meta-
position (2c and 2l) displayed considerably decreased activity.
Consequently, the size and the position, as well as the electronic
properties of the substituent on the phenyl ring all contribute to
the observed antiproliferative activity of 2b and 2k.
compared to non-treatment control (Fig. 3A), indicating that the
induction of apoptosis represented the main mode of activity.
Interestingly, both 2b and 2k elicited similar apoptotic cell popula-
tion compared to lead I, whereas much more important apoptosis
than lead II at 50
lM. It is to note that lead II showed very signif-
icant apoptosis induction only at an elevated concentration of
100 l
M.2
We next examined the importance of the sugar component
based on the structural analogs of 2b. Removing the protecting
groups at the ribose sugar led to an inactive analog 3b. The reason
that the acetyl protected analog 2b elicited activity may be as-
cribed to the increased lipophilicity brought by the acetyl protec-
tion, which may facilitate the cell uptake and result in effective
biological activity; whereas the unprotected analog is more polar,
which may lead to an inefficient cell uptake and thus no activity.
Further substituting the ribose sugar by an acyclic sugar compo-
nent (4 and 5 in Scheme 2)9,24 significantly decreased the activity.
These findings indicate the importance of the acetyl protection of
ribose sugar. It is worthy to mention that 2b and 2k may have com-
pleted different mechanisms of action from gemcitabine, which
may require detailed investigation in this direction.
5
4
3
2
1
0
A
control
I
II
2b
2k
3
2
1
0
B
We also assessed the role of the amine bridge between the aryl
group and the triazole ring in 2b (Scheme 2). Either removing the
amine linkage (6 in Scheme 2)25 or replacing the amine functional-
ity in 2b with a rigid triple bond connection (7 in Scheme 2)2 or
triazolyl ring (8 in Scheme 2)5a led to a considerable reduction in
activity (data not shown). We therefore concluded that both the
flexible structure and the balanced polarity of amine linkage may
contribute collectively to the observed antiproliferative activity.
We further studied the apoptosis-inducing activity of 2b and 2k
in MiaPaCa-2 cells using fluorescence-activated cell sorting (FACS)
control
I
II
2b
2k
flow cytometry.26 Cells were treated with 50
lM of either 2b or 2k,
using non-treatment as control. After 48 h of treatment, the cells
were labeled with propidium iodide and immediately analyzed.
An increase in the fraction of cells undergoing apoptosis (sub-G1/
G0 fraction) was revealed following treatment with 2b and 2k
Figure 3. Apoptosis-induction in MiaPaCa-2 cells after treatment with the
compounds I, II, 2b and 2k, using non-treatment as control. (A) Flow cytometry
was used to quantify the percentage of cells undergoing apoptosis (cells in sub-G1/
G0). (B) Caspase-3/7 activity was measured using Caspase-Glo luminescent assay.
CH3
CH3
HN
HN
N
O
N
O
N
N
OCH3
N
OCH3
N
AcO
BzO
O
O
4
5
H3C
H3C
H3C
AcO
N
N
O
N
N
N
N
N
O
O
O
N
N
N
N
N
OCH3
OCH3
OCH3
AcO
AcO
O
O
OAc OAc
OAc OAc
OAc OAc
7
8
6
Scheme 2. Structural analogs for SAR analysis of 2b.

Y. Liu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2503–2507
2507
9. Li, W.; Fan, Y. T.; Xia, Y.; Rocchi, P.; Zhu, R. Z.; Qu, F. Q.; Neyts, J.; Iovanna, J.;
Peng, L. Helv. Chim. Acta 2009, 92, 1503.
To further confirm the apoptosis-inducing activity of 2b and 2k
in MiaPaCa-2 cells, we determined caspase-3/7 activity using a
luminescent assay.27 MiaPaCa-2 cells were treated with the test
10. Li, D.; Xie, K.; Wolff, R.; Abbruzzese, J. L. Lancet 2004, 363, 1049.
11. (a) Rivera, F.; López-Tarruella, S.; Vega-Villegas, M. E.; Salcedo, M. Cancer Treat.
Rev. 2009, 35, 335; (b) Pierantoni, C.; Pagliacci, A.; Scartozzi, M.; Berardi, R.;
Bianconi, M.; Cascinu, S. Crit. Rev. Oncol. Hematol. 2008, 67, 27; (c) Mini, E.;
Nobili, S.; Caciagli, B.; Landini, I.; Mazzei, T. Ann. Oncol. 2006, 17(Suppl 5), v7.
12. Wong, H. H.; Lemoine, N. R. Nat. Rev. Gastroenterol. Hepatol. 2009, 6, 412.
13. Loakes, D. Nucleic Acids Res. 2001, 29, 2437.
14. Xia, Y.; Wan, J. Q.; Qu, F. Q.; Peng, L. Synthesis of Nucleoside Analogues with
Aromatic Systems Appended on the Triazole Nucleosides. In Chemistry of
Nucleic Acid Components, Hocek, M.; Eds.; Collection Symposium Series;
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the
Czech Republic: Prague 2008; Vol. 10, pp 224–228.
15. (a) Arrigo, A.-P.; Simon, S.; Gibert, B.; Kretz-Remy, C.; Nivon, M.; Czekalla, A.;
Guillet, D.; Moulin, M.; Diaz-Latoud, C.; Vicart, P. FEBS Lett. 2007, 581, 3665; (b)
Concannon, C. G.; Gorman, A. M.; Samali, A. Apoptosis 2003, 8, 61.
16. (a) Mori-Iwamoto, S.; Kuramitsu, Y.; Ryozawa, S.; Mikuria, K.; Fujimoto, M.;
Maehara, S.-I.; Maehara, Y.; Okita, K.; Nakamura, K.; Sakaida, I. Int. Journal
Oncol. 2007, 31, 1345; (b) Mori-Iwamoto, S.; Taba, K.; Kuramitsu, Y.; Ryozawa,
S.; Tanaka, T.; Maehara, S.-I.; Maehara, Y.; Okita, K.; Nakamura, K.; Sakaida, I.
Pancreas 2009, 38, 224.
17. (a) Rocchi, P.; So, A.; Kojima, S.; Signaevsky, M.; Beraldi, E.; Fazli, L.; Hurtado-
coll, A.; Yamanaka, K.; Gleave, M. Cancer Res. 2004, 64, 6595; (b) Rocchi, P.;
Beraldi, E.; Ettinger, S.; Fazli, L.; Vessella, R. L.; Nelson, C.; Gleave, M. Cancer Res.
2005, 65, 11083.
compound at 50 lM and the caspase-3/7 activity was measured
by the cleavage of the luminogenic substrate containing the tetra-
peptide sequence DEVD according to the instructions.27 The ob-
tained results revealed
a similarly increase in caspase-3/7
activation following treatment with 2b and 2k compared to non-
treatment control and positive control with previous leads I and
II (Fig. 3B). Altogether, these findings suggest a caspase-3/7-depen-
dent mechanism of apoptosis-induction.
In conclusion, we have synthesized a novel family of N-aryl-
triazole ribonucleoside analogs via Cu-mediated C–N cross-cou-
pling reaction. Although the N-arylation reaction could not
deliver the corresponding products in good yields, the yield was
sufficient to allow a preliminary in vitro assay to investigate their
antiproliferative activity against drug-resistant human pancreatic
cancer MiaPaCa-2 cells, drug-sensitive human pancreatic cancer
Capan-2 cells and hormone-refractory human prostate cancer PC-
3 cells. Two compounds elicited potent anticancer activity against
the drug-resistant human pancreatic cancer cell line MiaPaCa-2,
with a better potency than gemcitabine, the reference treatment
for pancreatic cancer. This finding suggests that these two N-aryl-
triazole nucleosides may represent novel structural leads in the
search for new anticancer candidate drugs. We are currently pur-
suing the chemistry of N-arylation on triazole nucleosides with a
view to significantly improving the reaction yield and broadening
the substrate scope. Not only would we then have the necessary
quantity of products for further in vivo and mechanistic studies
but also have access to a large diversity of molecular structures
for future structure/activity relationship analysis, essential to the
search for novel anticancer drug candidates.
18. Evano, G.; Blanchard, N.; Toumi, M. Chem. Rev. 2008, 108, 3054.
19. (a) Wu, Q. Y.; Qu, F. Q.; Wan, J. Q.; Zhu, X.; Xia, Y.; Peng, L. Helv. Chim. Acta 2004,
87, 811; (b) Wu, Q. Y.; Qu, F. Q.; Wan, J. Q.; Xia, Y.; Peng, L. Nucleosides,
Nucleotides Nucleic Acids 2005, 999.
20. Preparation of 3-aminotriazole ribonucleoside 1: To a solution of 1.00 g of 3-
azido-1-[2,3,5-tri-O-acetyl-b-
D-ribofuranosyl]-1,2,4-triazole-5-carboxylate
(2.3 mmol) in 15 mL CH2Cl2, 0.10 g Pd/C catalyst (5 wt % of Pd, 25.6 mmol) was
added. The reaction was then carried out with H2 at a pressure of 1 atm. After
the complete consumption of the starting material (detected with TLC), the
mixture was filtered over Celite, the filtrate was concentrated under reduced
pressure, giving 0.92 g of 1 as white powder in the yield of 98%.
21. General procedure for preparing
2 via copper-mediated N-arylation with
arylboronic acid: To mixture of 3-amino-1-[2,3,5-tri-O-acetyl-b-D-
a
ribofuranosyl]-1,2,4-triazole-5-carboxylate (100.0 mg, 0.25 mmol), 6 equiv
arylboronic acid (1.50 mmol) and 68.2 mg anhydrous copper acetate
(0.38 mmol) was added 5 mL dichloromethane (distilled freshly over calcium
hydride). Then, 40.2 lL freshly distilled pyridine (0.50 mmol) and ca. 20 mg
powder of 4 Å molecular sieve (activated at 500 °C for 5 h) were added rapidly.
The mixture was stirred at room temperature for 3 days, and then filtered over
Celite. The filtration was concentrated under reduced pressure, and the
obtained residue was purified on silica gel with petroleum ether/ethyl
acetate (1:1, v/v), giving the corresponding product 2 as powder.
Acknowledgments
Financial supports from the Ministry of Science and Technology
of China (Nos. 2003CB114400, 2003AA2Z3506), National Science
Foundation of China (Nos. 20372055, 20473112), Wuhan Univer-
sity, CNRS and INSERM are gratefully acknowledged. We thank
Mr. Joël Tardivel-Lacombe for assistance in cell culture, and Mrs.
Emily Witty for English correction of manuscript. Dr. Yi Xia is sup-
ported by a post-doctoral fellowship from la Fondation pour la
Recherche Médicale.
22. General procedure for preparing 3: The corresponding 2 was dissolved in 12 mL
of a saturated NH3/MeOH solution and stirred at room temperature for 2 days.
Then the solvent was removed and the residue was washed three times with
CH2Cl2. The washed residue was dried in vacuo to afford the corresponding
product 3.
23. Anti-cancer assay in MiaPaCa-2: Pancreatic cancer 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 well in 96-well
View Plate™ (Packard) in 250 lL of medium containing the same components
as described above. Cells were allowed to attached 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 with 50 lM compound was determined by (3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide, MTT) colorimetric assay.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.02.104.
24. Compound 4 was synthesized following the protocol described in Ref. 9.
25. Compound 6 was synthesized following the protocol described in Ref. 8.
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
References and notes
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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-ethanol 70% overnight at 4 °C. After a wash
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). Each sample was performed in triplicate.
27. Caspase-3/7 cleavage assay: Caspase-3/7 activity was measured using the
Caspase-Glo 3/7 Assay Kit (Promega). MiaPaCa-2 cells were initially seeded at
15,000 cells/well on 96-well plates. Twenty-four hours later, cells were treated
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Iovanna, J. L.; Peng, L. J. Med. Chem. 2009, 52, 1144.
4. Zhu, R. Z.; Wang, M. H.; Xia, Y.; Qu, F. Q.; Neyts, J.; Peng, L. Bioorg. Med. Chem.
Lett. 2008, 18, 3321.
5. (a) Xia, Y.; Li, W.; Qu, F. Q.; Fan, Z. J.; Liu, X. F.; Berro, C.; Rauzy, E.; Peng, L. Org.
Biomol. Chem. 2007, 5, 1695; (b) Xia, Y.; Fan, Z. J.; Yao, J. H.; Liao, Q.; Li, W.; Qu,
F. Q.; Peng, L. Bioorg. Med. Chem. Lett. 2006, 16, 2693; (c) Xia, Y.; Qu, F. Q.; Li, W.;
Wu, Q. Y.; Peng, L. Heterocycles 2005, 65, 345.
with the test compound at 50
Assay Reagent was added to each well of a white 96-well plate containing
100 L of blank, control or cells in culture. The caspase-3/7 activity was
lM for 24 h. Next 100 lL of Caspase-Glo 3/7
l
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.
6. Li, W.; Xia, Y.; Fan, Z. J.; Qu, F. Q.; Wu, Q. Y.; Peng, L. Tetrahedron Lett. 2008, 49,
2804.
7. Zhu, R. Z.; Qu, F. Q.; Quéléver, G.; Peng, L. Tetrahedron Lett. 2007, 48, 2389.
8. Wan, J. Q.; Zhu, R. Z.; Xia, Y.; Qu, F. Q.; Wu, Q. Y.; Yang, G. F.; Neyts, J.; Peng, L.
Tetrahedron Lett. 2006, 47, 6727.