Novel triazole ribonucleoside down-regulates heat shock protein 27 and induces potent anticancer activity on drug-resistant pancreatic cancer

Article abstract of DOI:10.1021/jm900960v

A series of novel 3-arylethynyltriazolyl ribonucleosides were synthesized and assessed for their anticancer activity on the drug-resistant pancreatic cancer cell line MiaPaCa-2. Among them, one compound exhibited potent apoptosis-inducing properties and a

Full text of DOI:10.1021/jm900960v

J. Med. Chem. 2009, 52, 6083–6096 6083
DOI: 10.1021/jm900960v
Novel Triazole Ribonucleoside Down-Regulates Heat Shock Protein 27 and Induces Potent Anticancer
Activity on Drug-Resistant Pancreatic Cancer
Yi Xia,†,‡ Yang Liu,† Jinqiao Wan,† Menghua Wang,† Palma Rocchi,§ Fanqi Qu,† Juan L. Iovanna,§ and Ling Peng*,†,‡
†State Key Laboratory of Virology, College of Chemistry and Molecular Sciences, Wuhan University, Wuhan, 430072, P. R. China,
‡
ꢀ
Departement de Chimie, Centre Interdisciplinaire de Nanoscience de Marseille, CNRS UPR 3118, 163, Avenue de Luminy, 13288 Marseille,
France, and INSERM U624, 163, Avenue de Luminy, 13288 Marseille, France
§
Received June 30, 2009
A series of novel 3-arylethynyltriazolyl ribonucleosides were synthesized and assessed for their anti-
cancer activity on the drug-resistant pancreatic cancer cell line MiaPaCa-2. Among them, one com-
pound exhibited potent apoptosis-inducing properties and anticancer activity against the pancreatic
cancer model MiaPaCa-2 both in vitro and in vivo with no adverse effects. This compound did not
inhibit DNA synthesis and therefore does not resemble the clinical drug gemcitabine. It did, however,
significantly down-regulate the expression of heat shock protein 27 (Hsp27), a small molecular
chaperone playing an important role in drug resistance and highly expressed in drug-resistant cancer
forms, and thus represents the first small molecular anticancer lead with such a mode of action.
Introduction
Pancreatic cancer is one of the most lethal and devastating
forms of human cancer, ranking as the fifth most common
cause of death by cancer in the Western world and with a
5-year survival rate as low as 3%.1 Conventional cancer
treatments have little impact on this extremely aggressive
form of cancer, and drug resistance develops very quickly.
The current first-line treatment is based on gemcitabine
(Figure 1), a nucleoside drug that exerts its anticancer activity
mainly via inhibiting DNA synthesis alongside other possible
modes of action.2 Unfortunately, gemcitabine is only moder-
ately effective, yielding a mere 12% response rate and a
median survival period of 5 months.1,2 There is therefore an
urgent need to explore new drug candidates for combating
pancreatic cancer, particularly drug-resistant pancreatic cancer.
The resistance of pancreatic cancer to apoptosis is an
important factor contributing to its drug resistance. The
investigation of several proteins such as p8,3 Bcl2, Bcl2-like
proteins,4 and their respective signaling pathways has pro-
Figure 1. Gemcitabine, 5-arylethynyltriazole ribonucleoside 1, and
vided evidence to this effect. Of particular interest is the heat
3-ethynyltriazole ribonucleosides 2 and 3 described in this study.
shock protein 27 (Hsp27a), which plays an important role in
the resistance shown by patients with pancreatic cancer to
apoptotic properties.6 Increased levels of Hsp27 expression
gemcitabine.5 Hsp27 is an ATP-independent small molecular
render tumors more resistant to many chemotherapeutic
chaperone functioning to protect against drug toxicity, which
agents. Inhibiting Hsp27 function can increase cell sensitivity
displays enhanced synthesis in response to stress and anti-
to drugs, thus overcoming the problem of drug resistance.
Antisense oligonucleotides and siRNA molecules, developed
ꢀ
*To whom correspondence should be addressed. Address: Departe-
to down-regulate Hsp27, have potent caspase-dependent
apoptotic anticancer activity on prostate cancer7,8 and are
currently in clinical trials for use in the treatment of several
human cancers, such as lung, breast, prostate, bladder, and
ovarian cancers.9 One recent study has shown that down-
regulation of Hsp27 via interferon-γ could also produce effec-
tive anticancer activity in drug-resistant pancreatic cancer
cells, further demonstrating the general utility and importance
ofdown-regulatingHsp27in treatingdrug-resistantcancers.10
ment de Chimie, 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.
a Abbreviations: Hsp27, heat shock protein 27; DMEM, Dulbecco’s
modified Eagle medium; FBS, fetal bovine serum; MTT, 3-(4,5-di-
methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; FACS, fluores-
cence activated cell sorting; ELISA, enzyme linked immunosorbent
assay; LDH, lactate dehydrogenase; 7-AAD, 7-aminoactinomycin D;
qRT-PCR, quantitative real-time polymerase chain reaction; DAPI,
4,6-diamidino-2-phenylindole; ip, intraperitoneal; O/N, overnight; SE,
standard error.
r
2009 American Chemical Society
Published on Web 09/16/2009
pubs.acs.org/jmc

6084 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Xia et al.
Table 1. Synthesis of 3-Arylethylynyltriazole Nucleosides 2 and 3 via the Sonogashira Reaction and the Subsequent Ammonolysis
However, as yet, no small molecular weight compound able to
down-regulate Hsp27 has been identified and the related drug
discovery process is difficult due to the lack of structural
information on Hsp27.

Article
We recently discovered a triazole ribonucleoside 1 (Figure 1),
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 6085
which showed potent anticancer activity against the drug-
resistant pancreatic cancer cell line MiaPaCa-2 both in vitro
and in vivo while having no adverse effect.11 1 belongs to the
family of arylethylnyltriazolyl ribonucleosides, the chemistry
and biological activity of which have never been described
before.12 This family of triazole nucleosides may thus provide
a new structural lead in the search for drug candidates with
novel mode of action to treat drug-resistant pancreatic cancer.
In addition, arylethylnyltriazolyl nucleosides also exhibit anti-
viral activity against HCV.11,13 Consequently, we feel that
arylethylnyltriazolyl nucleosides may represent an important
structural motif in the search of bioactive compounds.
In our continuing efforts to identify novel triazole nucleo-
sides as potential anticancer drug candidates and to provide
molecular diversity of triazole nucleosides for further struc-
ture-activity relationship analysis, we are particularly
interested in 3-arylethynyltriazolyl ribonucleosides 2 and 3
(Figure 1), the structural isomers of the corresponding
5-arylethynyltriazolyl ribonucleoside 1 previously developed
in our laboratory.11 We wished to investigate their potential
anticancer activity and more particularly their ability to
down-regulate Hsp27, with the aim of developing small
molecular weight compounds having novel mechanisms of
action. This represents part of our ongoing program of
developing structurally novel and diverse triazole nucleosides
by appending π-conjugated systems onto the triazole nucleo-
base.11-18 These compounds are expected to offer advanta-
geous binding properties to their corresponding biological
targets via more efficient and stronger interactions provided
by their larger aromatic systems.14 In addition, they may lead
to better in vivo stability and efficiency due to the unnatural
triazole heterocyclic system. Here we report on the chemical
synthesis of the 3-arylethynyltriazolyl ribonucleosides 2 and 3
(Figure 1), the biological assessment of their anticancer
activity against drug-resistant pancreatic cancer MiaPaCa-2
cell line, and the results of our preliminary investigation on
their mechanism of action. One of the newly synthesized
3-arylethynyltriazolyl ribonucleosides showed effective antic-
ancer activity on the MiaPaCa-2 cell line both in vitro and in
vivo, probably via a mechanism involving the down-regula-
tion of Hsp27.
Figure 2. Structural analogues for SAR analysis of 3e.
relationship analysis. Compounds 4, 7, and 8 were prepared
using the synthetic procedures previously developed by our
group (Scheme 1).11,13,15 Molecule 5 was obtained by Pd/
C-catalyzed hydrogenation of 3e, and analogue 6 was ob-
tained via the Huisgen reaction using 3-azidotriazole nucleo-
side19 and 1-ethynyl-4-pentylbenzene followed by ammono-
lysis in NH3/MeOH (Scheme 1).16a,17
2. Biology. In Vitro Antiproliferative Activity Assay. We
used a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay to evaluate the anticancer activity of
all the synthesized nucleosides on the MiaPaCa-2 cells. One
of the compounds, 3e, led to a significant reduction in
MiaPaCa-2 cell proliferation and/or viability by 80% 2 days
following treatment (Figure 3). Under the same conditions,
gemcitabine only moderately inhibited cell proliferation by
less than 30% (Figure 3).
Since 3e bears the alky chain in its structure, we further
examined its analogues 3c and 3d (Figure 2), each bearing
different alkyl chain lengths. These compounds were found
to be inactive against MiaPaCa-2 cell proliferation. More-
over, we checked the activity of the analogue 3r, which
corresponds to 3e with a phenyl group removed. Compound
3r failed to significantly inhibit the growth of MiaPaCa-2
cells. Therefore, both the alkyl chain C5H11 and the phenyl
ring seem crucial for 3e to retain its activity.
In addition, we found that removing the triple bond from
3e or replacing it with either a flexible -CH2CH2- linker or
a rigid triazole ring led to the inactive analogues 4, 5, and 6,
respectively (Figure 2). These data reflect the importance of
the triple bond connection between the phenyl group and the
triazole ring for the activity of 3e. Moreover, neither the
acyclic nucleoside analogue 7 nor the 5-isomeric analogue 8
(Figure 2) exhibited any considerable inhibition on cell
Results and Discussion
1. Chemistry. The synthesis of 3-ethynyltriazole ribonu-
cleosides was carried out via a microwave-assisted Sonoga-
shira coupling reaction, using a strategy similar to that used
to synthesize 5-ethynyltriazole ribonucleosides (Table 1).11
The Sonogashira coupling reaction between the 3-bromo-
triazole nucleoside and various alkynes led to the nucleosides
2, which were then subjected to ammonolysis to give the
deprotected nucleosides 3 in good yields (Table 1). Similar to
that observed previously with 5-ethynyltriazole ribonucleo-
sides, there was no important difference in the yields of the
Sonogashira reaction between using alkynes with electron-
donating and those with electron-withdrawing groups ap-
pended on phenylacetylene. In addition, very good yields
were obtained with both heterocyclic arylacetylenes (Table 1,
entries 13-15) and alkylacetylenes (Table 1, entries 16-20)
bearing various functionalities including a chloride, a double
bond, and a hydroxyl group etc (Table 1, entries 16-20).
We further synthesized several structural analogues of 3e
(Figure 2 and Scheme 1) for the relevant structure-activity

6086 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Xia et al.
Scheme 1. Synthesis of Compounds 4-8 the Structural Analogues of 3e
growth. It is interesting to note that 2e, the corresponding
protected form of 3e, was devoid of any activity on MiaPa-
Ca-2 cells. This may have resulted from the reduced H-bond-
ing possibility and increased steric hindrance related to the
acetyl protected ribose sugar moiety in 2e. Furthermore,
both 2j, the structural isomer of our previously identified
anticancer lead 1, and 3j, the deprotected form of 2j, failed to
yield any notable activity. All of these observations led us to
conclude that the ribose component and the 3-isomeric
structure contribute largely to the activity of 3e. In our
previous study with triazole ribonucleosides, only protected
arylethynyltriazole nucleosides showed anticancer activity.11
The identification of the active compound 3e in this work
revealed that the sugar-deprotected alkylnyltriazole ribonu-
cleosides 3 may also constitute a new structural lead with
anticancer activity.
Caspase-Dependent Apoptosis-Inducing Properties and An-
ticancer Activity. Our previous molecular lead 1 was shown
to display caspase-dependent apoptosis-inducing properties
and anticancer activity.11 As for lead 1, we studied the
apoptosis-inducing activity of 3e in MiaPaCa-2 cells using
fluorescence-activated cell sorting (FACS) flow cytometry,20
ELISA for DNA/histone release,21and a caspase-3/7 activity
assay20 (Figure 4). A significant 14-fold increase in the
fraction of cells undergoing apoptosis (sub-G1/G0 fraction)
was revealed by FACS flow analysis following treatment

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 6087
Figure 3. Inhibitory effect of compound 3e on the pancreatic
cancer MiaPaCa-2 cell growth. MiaPaCa-2 cells were incubated
with compound 3e for 48 h and then assayed by MTT, using no
treatment and gemcitabine as controls. The values are the mean (
SE of three independent experiments.
Figure 5. Necrosis induced by 3e assessed by (A) LDH assay at
different concentration. Negative control determines the LDH
activity released from the untreated normal cells (=spontaneous
LDH release), and positive control determines the maximum relea-
sable LDH activity in the cells (=maximum LDH release). (B) Flow
cytometry: the necrotic cells were determined by staining with
7-aminoactinomycin D (7AAD).
12-fold increase compared to gemcitabine control (Figure 4C),
suggesting a caspase-3/7-dependent mechanism of apoptosis
induction by 3e.
Necrosis Effect. Because of the presence of the long
hydrophobic alkyl chain C5H11, we suspected that 3e might
execute its antiproliferative effect on MiaPaCa-2 cells addi-
tionally via necrosis. Necrosis is a passive, nonprogrammed
form of cell death mainly occurring following the induction
of cell membrane damage/leakage and the consequent rup-
ture of the cell and its organelles. We therefore performed a
lactate dehydrogenase (LDH) cytotoxicity assay to measure
membrane damage and LDH release. Compound 3e in
DMSO of different concentrations was tested. Even at
concentrations up to 300 μM, no significant induction of
LDH release (<2%) was observed (Figure 5A). We further
used flow cytometry to assess necrosis by staining the cells
treated by 3e with 7-aminoactinomycin (7-AAD), a mem-
brane-impermeable dye that binds to DNA. As shown in
Figure 5B, no significant fraction of necrosis cells (<2%)
was observed following up to 12 h of treatment with 3e.
Collectively, these results demonstrate that 3e does not act as
a membrane destabilizing/disrupting agent and is devoid of
any toxicity leading to cell membrane damage.
Effect on DNA Synthesis. Since 3e is a nucleoside analogue
and may mimic natural nucleosides, one of its possible
mechanisms of action could therefore be to interfere in the
pathways of DNA synthesis and, consequently, inhibit DNA
synthesis and cell growth. Gemcitabine, the current first-line
drug in the treatment of pancreatic cancer, is able to inhibit
DNA replication and subsequently induce apoptosis.22 In
order to examine whether 3e also exerts its antiproliferative
activity by inhibiting DNA synthesis, the action of 3e on the
metabolism of nucleic acids was evaluated in MiaPaCa-2
Figure 4. Apoptosis-induction in MiaPaCa-2 cells after 48 h of
treatment with 3e using gemcitabine and no treatment as control:
(A) flow cytometry was used to quantify the percentage of cells
undergoing apoptosis (cells in sub-G1/G0); (B) ELISA test for
detection of DNA/histone nucleosome release; (C) caspase-3/7
activity measurement. All the experiments were done in triplicate.
with 3e compared to nontreatment control (Figure 4A),
indicating that induction of apoptosis represented the main
mode of activity for 3e. Further results from ELISA showed
a spectacular 40-fold increase in DNA/histone nucleosome
release following treatment with 3e compared to nontreat-
ment control (Figure 4B), in line with those obtained from
FACS flow (Figure 4A), thus confirming the apoptosis-
induced antiproliferative activity of 3e. Moreover, using
caspase-3/7 colorimetric assay, we revealed a similarly im-
pressive 30-fold increase in caspase-3/7 activation following
treatment with 3e compared to nontreatment control and a

6088 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Xia et al.
Figure 6. Inhibition of 3e on DNA synthesis. Cells in exponential
growth phase were incubated with gemcitabine and 3e, respectively,
for 4 h and then labeled with [3H]thymidine for 6 h. The incorpora-
tion of [3H]dTMP was determined as described in Experimental
Section.
Figure 8. Immunofluorescence staining for Hsp27 protein in Mia-
PaCa-2 cells treated with 3e (A-C) compared to untreated cells
(D-F). Hsp27 protein was revealed by the antibody labeled with
green fluorescent Alexa fluor 488 dye, and the nuclei were visualized
by being stained with DAPI. All photomicrographs were taken with
the same magnification: green channel image of cells treated with 3e
(A) and untreated cells (D); blue channel image of the nuclei of cells
treated with 3e (B) and untreated cells (E); (C) merged fluorescent
images of (A) and (B); (F) merged fluorescent images of (D) and (E).
90% DNA inhibition was achieved with gemcitabine under
the same conditions. This finding suggests different mechan-
isms of anticancer action between 3e and gemcitabine.
Effective Down-Regulation of Heat Shock Protein 27. Since
the identified active compound 3e inhibits cell growth of
drug-resistant pancreatic cancer MiaPaCa-2 cells, we rea-
soned that it may specifically target some apoptotic path-
ways related to drug resistance. As Hsp27 plays an important
role in gemcitabine resistance in patients with pancreatic
cancer,5 we wondered whether 3e exerted its anticancer
activity by down-regulating Hsp27 expression. We then
focused our attention on investigating the effect of 3e on
Hsp27 using quantitative real-time PCR and Western blot-
ting analysis to assess the mRNA level and the protein level
of Hsp27, respectively. Indeed, treatment with 3e signifi-
cantly down-regulated the level of Hsp27 mRNA expression
by 80% after 24 h (Figure 7A) and similarly decreased the
Hsp27 protein expression after 48 h (Figures 7B and
Figure 7C). The efficient down-regulation of Hsp27 by 3e
was further revealed using immunofluorescence. As demon-
strated in Figure 8, effective inhibition on Hsp27 expression
after treatment with 3e was revealed noticeably by the
corresponding antibody labeled with green fluorescent Alexa
fluor 488 (Figure 8A), compared to the nontreatment control
(Figure 8D), and the resulting cell death was observed with
cells treated with 3e (Figures 8A-C) compared with un-
treated cells (Figures 8D-F). Overall, these results demon-
strate the ability of 3e to effectively inhibit the expression of
Hsp27 and provide insight on the anticancer activity path-
way of 3e on drug-resistant pancreatic cancer MiaPaCa-2
cells. To our knowledge, 3e is the first small molecule known
to down-regulate Hsp27, leading to potent anticancer activ-
ity. It may thus constitute a new lead in the development of
novel anticancer candidates, although the detailed mechan-
isms underlying the biological activity of 3e require further
investigation.
Figure 7. Effects of 3e on Hsp27 mRNA (A) and protein (B) ex-
pression in MiaPaCa-2 cells. (A) MiaPaCa-2 cells were treated with
3e for 24 h. Purified RNA was then analyzed by quantitative RT-
PCR to monitor Hsp27 mRNA expression. Quantitative analysis of
Hsp27 mRNA levels was normalized to 18S ribosomal mRNA
levels in MiaPaCa-2 cells. (B) MiaPaCa-2 cells were treated with 3e.
At 48 h after the treatment, proteins were extracted from cultured
cells and Hsp27 protein levels were analyzed by Western blotting
using vinculin as reference. (C) Quantification of Western blotting
analysis of Hsp27 protein expression with treatment of 3e compared
to no treatment as control.
In Vivo Tumor Growth Inhibition Assay. We finally eval-
uated the antitumor effect of 3e in nude mice bearing
MiaPaCa-2-xenografed tumors (Figure 9). Each mouse was
inoculated with 107 human pancreatic cancer MiaPaCa-2
cells on the shoulder and was allowed to grow for 7-10 days
cells by measuring the incorporation of [3H]thymidine into
the DNA. As shown in Figure 6, no significant inhibition on
DNA synthesis was observed with 3e, whereas more than

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 6089
bilizing activity or necrosis was observed for 3e. We further
confirmed that the caspase-dependent apoptosis induction
represents the main mode of action for the anticancer effect of
3e. Preliminary investigations on the mechanism of action
revealed that 3e did not inhibit DNA synthesis but signifi-
cantly down-regulated Hsp27 expression. These observations
collectively suggest that 3e differs from gemcitabine and may
have a novel mode of action involving the down-regulation of
Hsp27 and consequent anticancer effect. It therefore is a novel
anticancer lead, opening the door to new possibilities in the
discovery of drugs targeting Hsp27
To our knowledge, the arylethynyltriazole ribonucleoside
3e represents the first small molecule found to down-regulate
Hsp27 and induce potent caspase-dependent apoptosis with a
resulting anticancer activity. This compound bears a simple
and concise structural motif, which can be easily modu-
lated for further structural optimization and the study of
structure-activity relationships. It therefore emerges as a
promising structural lead in the search for new anticancer
candidates with novel mechanisms of action against pancrea-
tic cancer. Future work is directed toward lead optimization
with other types of triazole nucleosides and comprehensive
investigations on structure-activity relationships as well as
on the precise mechansims of action related to Hsp27 down-
regulation or other possible pathways.
Figure 9. (A) Effect of 3e treatment on drug-resistant MiaPaCa-2
tumor growth in vivo. (B) Mice weight recording throughout the in
vivo experiments. Nude mice bearing MiaPaCa-2 tumors of 100 mm3
were randomly selected for treatment with 3e and no treatment as
control. 3e was injected intraperitoneally (150 mg/kg/mice) every
3 days for 5 weeks, and tumor volume was measured once a week.
All of the results were expressed as the mean ( SE (n = 8): points are
the means values of analysis; bars are the standard errors.
Experimental Section
General. All the terminal alkynes and catalysts were pur-
chased from Acros or Lancaster. The microwave assisted reac-
tions were performed on an Initiator Creator produced by
Biotage. The 1H 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. The
chemical shifts were recorded in parts per million (ppm) with
TMS as the internal reference. ESI mass spectra were deter-
mined 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 to be >95% 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 of CH3CN/H2O, 90/10 (method 2).
General Procedure for Preparing 2 via a Microwave Assisted
Sonogashira Reaction. 3-Bromo-1-[2,3,5-tri-O-acetyl-β-D-ribo-
furanosyl]-1,2,4-triazole-5-carboxylate (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 irra-
diated at 100 °C for 30 min and then cooled to room tempera-
ture. The reaction mixture was concentrated under reduced
pressure, and the crude residue was 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
products 2a-t.
to reach an average tumor volume of 100 mm3. The mice
were then randomly selected for treatment with 3e versus no
treatment as a control. Compound 3e was administrated
intraperitoneally (ip) once every 3 days for 5 consecutive
weeks. Tumor growth was measured every week and sys-
temic toxicity evaluated by monitoring the weight loss of the
mice throughout the in vivo experiments. Figure 9A shows
that treatment with 3e significantly reduced MiaPaCa-2
tumor volume by 50% after 3 weeks of treatment. In addi-
tion, no toxicity was observed, as demonstrated by the
absence of weight loss (Figure 9B) and by the normal growth
and behavior of all mice tested. These results clearly demon-
strate the potent and efficacious antitumor activity of com-
pound 3e and the lack of adverse effects. This compound
may therefore constitute a novel lead in the search for
anticancer drug candidates with new mechanisms of action.
Conclusion
On the basis of our earlier anticancer lead compound 1, we
further developed 3-ethynyltriazole ribonucleosides 2 and 3,
the structural isomers of 5-ethynyltriazole ribonucleosides
previously synthesized in our group. The 3-ethynyltriazole
ribonucleosides were prepared using a simple and efficient
Sonogashira coupling reaction. Among the newly synthesized
ribonucleosides, we have identified the lead compound 3e,
which displayed potent and effective anticancer activity in
vitro and in vivo in the drug-resistant pancreatic cancer model
MiaPaCa-2 with no adverse effects.
The preliminary structure-activity relationship study of 3e
demonstrated that both alkyl chain C5H11 and the triple bond
connection between the phenyl group and the triazole ring are
critical for the anticancer activity. Despite the presence of a
relative hydrophobic alkyl chain C5H11 attached to the phenyl
ring on the triazole nucleobase, no obvious membrane-desta-
2a. An amount of 74.7 mg (81%) of product was obtained,
isolated as a colorless oil. HPLC: t = 6.62 min, purity >99%
(method 1); t = 6.65 min, purity 98.8% (method 2). H NMR
1
(300 MHz, CDCl3): δ 7.58-7.61 (m, 2H, phenyl-H), 7.37-
7.42 (m, 3H, phenyl-H), 6.96 (d, 1H, J = 2.1 Hz, H-10),
5.86-5.89 (m, 1H, H-20), 5.77-5.81 (m, 1H, H-30), 4.45-4.49

6090 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Xia et al.
(m, 2H, H-40 þ H-50), 4.17-4.22 (m, 1H, H-50), 4.03 (s, 3H,
-OCH3), 2.15 (s, 3H, -C(O)CH3), 2.13 (s, 3H, -C(O)CH3),
2.10 (s, 3H, -C(O)CH3). 13C NMR (75 MHz, CDCl3): δ 169.5,
168.5, 168.3, 156.4, 146.8, 143.9, 131.1, 128.6, 127.4, 120.0, 89.8,
88.5, 79.7, 78.0, 73.3, 69.7, 61.7, 52.5, 19.6, 19.4. Maldi-MS: m/z
508.1 [M þ Na]þ. HRMS: calcd for C23H23N3O9Naþ 508.1326,
(d, 2H, J = 8.1 Hz, phenyl-H), 6.97 (d, 1H, J = 2.1 Hz, H-10),
5.84-5.87 (m, 1H, H-20), 5.77-5.81 (m, 1H, H-30), 4.44-4.48
(m, 2H, H-40 þ H-50), 4.15-4.21 (m, 1H, H-50), 4.04 (s, 3H,
-OCH3), 2.16 (s, 3H, -C(O)CH3), 2.14 (s, 3H, -C(O)CH3),
2.09 (s, 3H, -C(O)CH3). 13C NMR (75 MHz, CDCl3): δ 170.8,
169.8, 169.7, 157.7, 147.9, 145.2, 136.1, 133.6, 129.1, 119.8, 89.9,
89.8, 80.9, 80.2, 74.6, 71.0, 63.1, 53.8, 20.9, 20.8. Maldi-MS:
m/z 542.1 [M þ Na]þ. HRMS: calcd for C23H22N3O9ClNaþ
found 508.1323. IR: 2230.8 cm-1
.
2b. An amount of 77.2 mg (75%) of product was obtained,
isolated as a colorless oil. HPLC: t = 6.62 min, purity >99%
(method 1); t = 6.51 min, purity >99% (method 2). 1H NMR
(300 MHz, CDCl3): δ 7.54 (d, 2H, J=8.7 Hz, phenyl-H), 6.96 (d,
1H, J = 2.1 Hz, H-10), 6.90 (d, 2H, J = 8.7 Hz, phenyl-H),
5.86-5.88 (m, 1H, H-20), 5.78-5.82 (m, 1H, H-30), 4.46-4.50
(m, 2H, H-40 þ H-50), 4.15-4.21 (m, 1H, H-50), 4.03 (s, 3H,
-OCH3), 3.84 (s, 3H, -OCH3), 2.16 (s, 3H, -C(O)CH3), 2.13
(s, 3H, -C(O)CH3), 2.10 (s, 3H, -C(O)CH3). 13C NMR (75
MHz, CDCl3): δ 171.2, 170.1, 170.0, 161.1, 158.1, 148.7, 145.3,
134.3, 114.7, 113.6, 91.7, 90.0, 81.2, 78.6, 74.9, 71.3, 63.3, 55.9,
54.1, 21.3, 21.1. Maldi-MS: m/z 576.1 [M þ Na]þ. HRMS: calcd
for C24H22N3O9F3Naþ 576.1200, found 576.1188. IR: 2232.2
542.0937, found 542.0924. IR 2230.6 cm-1
.
2g. An amount of 73.4 mg (73%) of product was obtained,
isolated as a colorless oil. HPLC: t = 6.61 min, purity >99%
(method 1); t = 6.55 min, purity 98.6% (method 2). H NMR
1
(300 MHz, CDCl3): δ 7.57-7.61 (m, 2H, phenyl-H), 7.08 (dd,
2H, JHH = JHF = 8.7 Hz, phenyl-H), 6.96 (d, 1H, J = 2.1 Hz,
H-10), 5.85-5.87 (m, 1H, H-20), 5.77-5.80 (m, 1H, H-30),
4.44-4.49 (m, 2H, H-40 þ H-50), 4.15-4.21 (m, 1H, H-50),
4.03 (s, 3H, -OCH3), 2.15 (s, 3H, -C(O)CH3), 2.13 (s, 3H,
-C(O)CH3), 2.09 (s, 3H, -C(O)CH3). 13C NMR (75 MHz,
CDCl3): δ 170.8, 169.8, 169.6, 163.5 (d, 1JCF=250.5 Hz), 157.7,
3
2
148.0, 145.2, 134.4 (d, JCF = 9.0 Hz), 117.4, 116.2 (d, JCF
21.4 Hz), 90.0, 89.8, 80.9, 79.1, 74.6, 71.0, 63.0, 53.8, 20.9, 20.7.
Maldi-MS: m/z 526.1 [M
Na]þ. HRMS: calcd for
C23H22N3O9FNaþ 526.1232, found 526.1222. IR: 2239.7 cm-1
=
cm-1
.
þ
2c. An amount of 62.9 mg (63%) of product was obtained,
isolated as a colorless oil. HPLC: t = 6.72 min, purity >99%
(method 1); t = 6.62 min, purity >99% (method 2). 1H NMR
(300 MHz, CDCl3): δ 7.49 (d, 2H, J=8.1 Hz, phenyl-H), 7.18 (d,
2H, J = 8.1 Hz, phenyl-H), 6.95 (d, 1H, J = 2.1 Hz, H-10),
5.86-5.88 (m, 1H, H-20), 5.77-5.81 (m, 1H, H-30), 4.45-4.48
(m, 2H, H-40 þ H-50), 4.15-4.21 (m, 1H, H-50), 4.03 (s, 3H,
-OCH3), 2.38 (s, 3H, -CH3), 2.15 (s, 3H, -C(O)CH3), 2.13 (s,
3H, -C(O)CH3), 2.09 (s, 3H, -C(O)CH3). 13C NMR (150
MHz, CDCl3): δ 170.8, 169.7, 169.6, 157.7, 148.3, 145.1,
140.2, 132.3, 129.4, 118.2, 91.4, 89.7, 81.0, 78.8, 74.6, 71.0,
62.9, 53.7, 21.8, 20.9, 20.7. Maldi-MS: m/z 522.1 [M þ Na]þ.
HRMS: calcd for C24H25N3O9Naþ 522.1483, found 522.1481.
.
2h:. An amount of 64.4 mg (64%) of product was obtained,
isolated as a colorless oil. HPLC: t = 6.60 min, purity 99.0%
(method 1); t = 6.54 min, purity 95.8% (method 2). H NMR
1
(300 MHz, CDCl3): δ 7.30-7.40 (m, 3H, phenyl-H), 7.09-7.15
(m, 1H, phenyl-H), 6.96 (s, 1H, H-10), 5.85-5.87 (m, 1H, H-20),
5.76-5.80 (m, 1H, H-30), 4.44-4.49 (m, 2H, H-40 þ H-50),
4.15-4.21 (m, 1H, H-50), 4.04 (s, 3H, -OCH3), 2.15 (s, 3H,
-C(O)CH3), 2.13 (s, 3H, -C(O)CH3), 2.09 (s, 3H, -C(O)CH3).
13C NMR (75 MHz, CDCl3): δ 170.8, 169.8, 169.6, 162.5 (d,
1JCF =246.0 Hz), 157.7, 147.8, 145.3, 130.4 (d, 3JCF =7.8 Hz),
128.3, 123.2, 119.1 (d, 2JCF =23.6 Hz), 117.3 (d, 2JCF =20.2 Hz),
89.8, 89.6, 81.0, 80.0, 74.6, 71.0, 63.0, 53.8, 20.9, 20.7. Maldi-
MS: m/z 526.1 [M þ Na]þ. HRMS: calcd for C23H22N3O9FNaþ
IR: 2234.1 cm-1
.
2d. An amount of 73.2 mg (69%) of product was obtained,
isolated as a white solid. HPLC: t = 7.00 min, purity >98.3%
(method 1); t=6.88 min, purity >95.2% (method 2). 1H NMR
(300 MHz, CDCl3): δ 7.51 (d, 2H, J=8.1 Hz, phenyl-H), 7.19 (d,
2H, J = 8.1 Hz, phenyl-H), 6.96 (d, 1H, J = 1.5 Hz, H-10),
5.86-5.89 (m, 1H, H-20), 5.78-5.82 (m, 1H, H-30), 4.46-4.49
(m, 2H, H-40 þ H-50), 4.16-4.21 (m, 1H, H-50), 4.03 (s, 3H,
-OCH3), 2.61 (t, 2H, J = 7.8 Hz, -CH2-), 2.16 (s, 3H,
-C(O)CH3), 2.13 (s, 3H, -C(O)CH3), 2.10 (s, 3H, -C(O)CH3),
526.1232, found 526.1223. IR: 2240.8 cm-1
.
2i. An amount of 64.4 mg (64%) of product was obtained,
isolated as a pink solid. HPLC: t = 6.63 min, purity >99%
(method 1); t = 6.61 min, purity 95.6% (method 2). H NMR
1
(300 MHz, CDCl3): δ 7.58 (dd, 1H, JHH=JHF=7.8 Hz, phenyl-
H), 7.39-7.41 (m, 1H, phenyl-H), 7.09-7.18 (m, 2H, phenyl-H),
6.96 (d, 1H, J = 2.4 Hz, H-10), 5.87-5.89 (m, 1H, H-20),
5.76-5.80 (m, 1H, H-30), 4.46-4.50 (m, 2H, H-40 þ H-50),
4.15-4.21 (m, 1H, H-50), 4.04 (s, 3H, -OCH3), 2.15 (s, 3H,
-C(O)CH3), 2.13 (s, 3H, -C(O)CH3), 2.11 (s, 3H, -C(O)CH3).
1.61-1.69 (m, 2H, -CH2-), 0.95 (t, 3H, J=7.2 Hz, -CH3). 13
C
NMR (150 MHz, CDCl3): δ 170.6, 169.5, 169.4, 166.3, 157.5,
148.0, 144.8, 144.7, 132.0, 128.6, 118.2, 91.2, 89.4, 80.7, 78.5,
74.4, 70.7, 62.7, 53.5, 38.0, 24.2, 20.7, 20.5, 20.4, 13.7. Maldi-
MS: m/z 550.2 [M þ Na]þ. HRMS: calcd for C26H29N3O9Naþ
13C NMR (75 MHz, CDCl3): δ 170.9, 169.7, 169.6, 163.3 (d,
3
1JCF = 252.8 Hz), 157.7, 147.8, 145.3, 134.2, 131.8 (d, JCF
=
7.9 Hz), 124.4 (d, 4JCF=3.4 Hz), 116.0 (d, 2JCF=20.3 Hz), 110.1
(d, 2JCF =14.6 Hz), 89.8, 84.6, 84.0, 81.1, 74.6, 71.0, 62.7, 53.8,
20.9, 20.7. Maldi-MS: m/z 526.1 [M þ Na]þ. HRMS: calcd for
550.1796, found 550.1808. IR: 2229.4 cm-1
.
2e. An amount of 81.0 mg (73%) of product was obtained,
isolated as a colorless oil. HPLC: t = 7.49 min, purity 97.9%
(method 1); t = 7.16 min, purity 96.5% (method 2). H NMR
C23H22N3O9FNaþ 526.1232, found 526.1227. IR: 2237.8 cm-1
.
1
2j. An amount of 64.1 mg (58%) of product was obtained,
isolated as a colorless oil. HPLC: t = 6.75 min, purity >99%
(method 1); t = 6.63 min, purity 96.9% (method 2). H NMR
(300 MHz, CDCl3): δ 7.51 (d, 2H, J=8.1 Hz, phenyl-H), 7.18 (d,
2H, J = 8.1 Hz, phenyl-H), 6.96 (d, 1H, J = 2.1 Hz, H-10),
5.86-5.89 (m, 1H, H-20), 5.78-5.81 (m, 1H, H-30), 4.45-4.50
(m, 2H, H-40 þ H-50), 4.15-4.21 (m, 1H, H-50), 4.03 (s, 3H,
-OCH3), 2.62 (t, 2H, J = 7.7 Hz, -CH2-), 2.15 (s, 3H,
-C(O)CH3), 2.13 (s, 3H, -C(O)CH3), 2.10 (s, 3H, -C(O)CH3),
1.60-1.67 (m, 2H, -CH2-), 1.31-1.38 (m, 4H, -CH2-), 0.90
(t, 3H, J = 6.6 Hz, -CH3). 13C NMR (150 MHz, CDCl3): δ
170.8, 169.7, 169.6, 157.8, 148.3, 145.2, 145.1, 132.3, 128.8,
118.4, 91.5, 89.7, 81.0, 78.7, 74.6, 71.0, 62.9, 53.7, 36.2, 31.6,
31.0, 22.7, 20.9, 20.7, 14.2. Maldi-MS: m/z 578.2 [M þ Na]þ.
HRMS: calcd for C28H33N3O9Naþ 578.2109, found 578.2101.
1
(300 MHz, CDCl3): δ 7.63-7.73 (m, 4H, phenyl-H), 6.97 (s, 1H,
H-10), 5.85-5.86 (m, 1H, H-20), 5.76-5.80 (m, 1H, H-30),
4.45-4.48 (m, 2H, H-40 þ H-50), 4.15-4.21 (m, 1H, H-50),
4.04 (s, 3H, -OCH3), 2.15 (s, 3H, -C(O)CH3), 2.13 (s, 3H,
-C(O)CH3), 2.08 (s, 3H, -C(O)CH3). 13C NMR (75 MHz,
CDCl3): δ 170.7, 169.8, 169.6, 157.6, 147.6, 145.3, 132.6, 131.2,
1
125.7, 125.1, 121.4 (q, JCF = 272 Hz), 89.9, 89.3, 81.4, 81.0,
74.6, 71.0, 63.0, 53.8, 20.9, 20.7. Maldi-MS: m/z 576.1 [M þ
Na]þ. HRMS: calcd for C24H22N3O9F3Naþ 576.1200, found
576.1188. IR: 2235.8 cm-1
.
IR: 2234.4 cm-1
.
2k. An amount of 70.8 mg (64%) of product was obtained,
isolated as a colorless oil. HPLC: t = 6.75 min, purity 96.0%
(method 1); t = 6.62 min, purity 95.3% (method 2). H NMR
2f. An amount of 50.9 mg (49%) of product was obtained,
isolated as a yellow solid. HPLC: t = 6.81 min, purity 97.2%
(method 1); t = 6.54 min, purity 95.2% (method 2). H NMR
1
1
(300 MHz, CDCl3): δ 7.88 (s, 1H, phenyl-H), 7.76-7.79 (m, 1H,
phenyl-H), 7.66-7.68 (m, 1H, phenyl-H), 7.49-7.55 (m, 1H,
(300 MHz, CDCl3): δ 7.53 (d, 2H, J = 8.1 Hz, phenyl-H), 7.36

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 6091
phenyl-H), 6.97 (d, 1H, J = 1.5 Hz, H-10), 5.85-5.87 (m, 1H,
H-20), 5.77-5.81 (m, 1H, H-30), 4.44-4.49 (m, 2H, H-40 þ H-50),
4.16-4.22 (m, 1H, H-50), 4.04 (s, 3H, -OCH3), 2.16 (s, 3H,
-C(O)CH3), 2.14 (s, 3H, -C(O)CH3), 2.09 (s, 3H, -C(O)CH3).
13C NMR (150 MHz, CDCl3): δ 170.7, 169.8, 169.6, 157.6,
4.01 (s, 3H, -OCH3), 2.05-2.21 (m, 13H, cyclohexane-
Hþ -C(O)CH3), 1.60-1.67 (m, 4H, cyclohexane-H). 13C NMR
(150 MHz, CDCl3): δ 170.6, 169.5, 169.3, 157.6, 148.4, 144.8,
138.8, 119.4, 92.9, 89.4, 80.8, 74.5, 70.9, 62.8, 53.4, 28.4, 25.8,
22.1, 20.7, 20.5, 20.4. Maldi-MS: m/z 512.2 [M þ Na]þ. HRMS:
calcd for C23H27N3O9Naþ 512.1639, found 512.1631. IR:
2
147.6, 145.3, 135.4, 131.4 (q, JCF = 32.9 Hz), 129.3, 129.1,
126.4, 123.7 (q, JCF = 271 Hz), 122.3, 89.8, 89.2, 80.9, 80.6,
1
2219.9 cm-1
.
74.6, 70.9. 63.0, 53.8, 20.9, 20.7. Maldi-MS: m/z 576.1 [M þ
2q. An amount of 72.7 mg (75%) of product was obtained,
isolated as a colorless oil. HPLC: t = 6.58 min, purity 97.1%
(method 1); t = 6.51 min, purity >99% (method 2). 1H NMR
(300 MHz, CDCl3): δ 6.91 (s, 1H, H-10), 5.80-5.82 (m, 1H,
H-20), 5.72-5.76 (m, 1H, H-30), 4.43-4.46 (m, 2H, H-40 þ H-50),
4.13-4.19 (m, 1H, H-50), 4.01 (s, 3H, -OCH3), 3.70 (t, 2H, J=
6.3 Hz, -CH2-), 2.64 (t, 2H, J=6.5 Hz, -CH2-), 2.14 (s, 3H,
-C(O)CH3), 2.12 (s, 3H, -C(O)CH3), 2.07 (s, 3H, -C(O)CH3).
13C NMR (75 MHz, CDCl3): δ 170.7, 169.7, 169.5, 157.6, 147.9,
144.9, 91.1. 89.6, 80.8, 74.5, 71.8, 70.9, 63.0, 53.7, 43.6, 30.8,
20.8, 20.6, 16.7. Maldi-MS: m/z 508.1 [M þ Na]þ. HRMS:
calcd for C20H24N3O9ClNaþ 508.1093, found 508.1088. IR:
Na]þ. HRMS: calcd for C24H22N3O9F3Naþ 576.1200, found
576.1187. IR: 2235.7 cm-1
.
2l. An amount of 60.8 mg (55%) of product was obtained,
isolated as a colorless oil. HPLC: t = 6.70 min, purity >99%
(method 1); t = 6.60 min, purity 96.9% (method 2). H NMR
1
(300 MHz, CDCl3): δ 7.70-7.78 (m, 2H, phenyl-H), 7.51-7.58
(m, 2H, phenyl-H), 6.97 (d, 1H, J=2.1 Hz, H-10), 5.88-5.91 (m,
1H, H-20), 5.75-5.79 (m, 1H, H-30), 4.46-4.51 (m, 2H, H-40 þ
H-50), 4.16-4.21 (m, 1H, H-50), 4.04 (s, 3H, -OCH3), 2.15 (s,
3H, -C(O)CH3), 2.14 (s, 3H, -C(O)CH3), 2.10 (s, 3H, -C-
(O)CH3). 13C NMR (150 MHz, CDCl3): δ 170.8, 169.7, 169.6,
2
157.6, 147.6, 145.4, 135.0, 132.1 (q, JCF = 30.9 Hz), 131.8,
2227.8 cm-1
.
1
129.7, 126.2, 123.4 (q, JCF = 272 Hz), 119.4, 89.7, 86.4, 84.2,
2r. An amount of 62.6 mg (65%) of product was obtained,
isolated as a colorless oil. HPLC: t = 6.81 min, purity >99%
(method 1); t=6.70 min, purity 95.6% (method 2). 1H NMR (300
MHz, CDCl3): δ 6.91 (s, 1H, H-10), 5.82-5.83 (m, 1H,
H-20), 5.74-5.78 (m, 1H, H-30), 4.43-4.46 (m, 2H, H-40 þ H-50),
4.12-4.18 (m, 1H, H-50), 4.01 (s, 3H, -OCH3), 2.41 (t, 2H, J=7.1
Hz, -CH2-), 2.14 (s, 3H, -C(O)CH3), 2.12 (s, 3H, -C(O)CH3),
2.08 (s, 3H, -C(O)CH3), 1.57-1.67 (m, 4H, -CH2-), 1.30-1.47
(m, 4H, -CH2-), 0.91 (t, 3H, J=7.1 Hz, -CH3). 13C NMR (150
MHz, CDCl3): δ 170.5, 169.4, 169.2, 157.4, 147.9, 144.5, 93.1, 89.2,
80.5, 74.2, 70.6, 62.6, 53.3, 30.9, 27.5, 22.0, 20.6, 20.4, 20.3, 18.9,
13.8. Maldi-MS: m/z 512.2 [M þ Na]þ. HRMS: calcd for
81.1, 74.5, 70.8, 62.5, 53.8, 20.8, 20.6. Maldi-MS: m/z 576.1 [M þ
Na]þ. HRMS: calcd for C24H22N3O9F3Naþ 576.1200, found
576.1190. IR: 2233.8 cm-1
.
2m. An amount of 71.7 mg (73%) of product was obtained,
isolated as a colorless oil. HPLC: t = 6.66 min, purity >99%
(method 1); t = 6.52 min, purity 96.3% (method 2). H NMR
1
(300 MHz, CDCl3): δ 7.38-7.43 (m, 2H, thiophenyl-H),
7.03-7.06 (m, 1H, thiophenyl-H), 6.96 (d, 1H, J = 2.1 Hz,
H-10), 5.85-5.87 (m, 1H, H-20), 5.76-5.80 (m, 1H, H-30),
4.45-4.48 (m, 2H, H-40 þ H-50), 4.15-4.21 (m, 1H, H-50),
4.03 (s, 3H, -OCH3), 2.16 (s, 3H, -C(O)CH3), 2.13 (s, 3H,
-C(O)CH3), 2.10 (s, 3H, -C(O)CH3). 13C NMR (150 MHz,
CDCl3): δ 170.9, 169.8, 169.6, 157.7, 148.0, 145.2, 134.4, 129.4,
127.5, 121.1, 89.8, 84.8, 83.0, 81.0, 74.6, 71.0, 63.0, 53.8, 20.9,
20.7. Maldi-MS: m/z 514.1 [M þ Na]þ. HRMS: calcd for
C22H29N3O9Naþ 502.1796, found 502.1809. IR: 2249.8 cm-1
.
2s. An amount of 77.9 mg (79%) of product was obtained,
isolated as a colorless oil. HPLC: t = 6.61 min, purity >99%
(method 1); t = 6.55 min, purity >99% (method 2). 1H NMR
(300 MHz, CDCl3): δ 6.92 (d, 1H, J=2.4 Hz, H-10), 5.80-5.83
(m, 1H, H-20), 5.72-5.76 (m, 1H, H-30), 4.43-4.46 (m, 2H,
H-40 þ H-50), 4.12-4.19 (m, 1H, H-50), 4.01 (s, 3H, -OCH3),
2.47 (br, 1H, -OH), 2.14 (s, 3H, -C(O)CH3), 2.12 (s, 3H,
-C(O)CH3), 2.08 (s, 3H, -C(O)CH3), 1.99-2.05 (m, 4H, cyclo-
pentanyl-H), 1.75-1.87 (m, 4H, cyclopentanyl-H). 13C NMR (75
MHz, CDCl3): δ 170.9, 169.8, 169.6, 157.6, 147.7, 145.1, 95.5,
89.7, 80.9, 74.6, 74.4, 73.2, 71.0, 63.0, 53.8, 42.3, 23.7, 20.9, 20.7.
C21H21N3O9SNaþ 514.0890, found 514.0889. IR: 2228.5 cm-1
.
2n. An amount of 51.1 mg (52%) of product was obtained,
isolated as a colorless oil. HPLC: t = 6.61 min, purity >99%
(method 1); t = 6.53 min, purity >99% (method 2). 1H NMR
(300 MHz, CDCl3): δ 7.67-7.68 (m, 1H, thiophenyl-H),
7.32-7.34 (m, 1H, thiophenyl-H), 7.24-7.27 (m, 1H, thiophe-
nyl-H), 6.96 (d, 1H, J=1.5 Hz, H-10), 5.85-5.87 (m, 1H, H-20),
5.77-5.81 (m, 1H, H-30), 4.45-4.49 (m, 2H, H-40 þ H-50),
4.14-4.21 (m, 1H, H-50), 4.03 (s, 3H, -OCH3), 2.16 (s, 3H,
-C(O)CH3), 2.13 (s, 3H, -C(O)CH3), 2.09 (s, 3H, -C(O)CH3).
13C NMR (75 MHz, CDCl3): δ 170.8, 169.8, 169.6, 157.7, 148.1,
145.1, 131.5, 130.1, 126.0, 120.5, 89.7, 86.5, 80.9, 79.0, 74.6, 71.0,
63.0, 53.8, 20.9, 20.7. Maldi-MS: m/z 514.1 [M þ Na]þ. HRMS:
calcd for C21H21N3O9SNaþ 514.0890, found 514.0876. IR:
Maldi-MS: m/z 516.2 [M
þ
Na]þ. HRMS: calcd for
C22H27N3O10Naþ 516.1589, found 516.1581. IR: 2242.8 cm-1
.
2t. An amount of 80.1 mg (79%) of product was obtained,
isolated as a colorless oil. HPLC: t = 6.62 min, purity >99%
1
(method 1); t = 6.39 min, purity 95.3% (method 2). H NMR
(300 MHz, CDCl3): δ 6.93 (d, 1H, J=1.5 Hz, H-10), 5.80-5.83
(m, 1H, H-20), 5.74-5.77 (m, 1H, H-30), 4.43-4.47 (m, 2H,
H-40 þ H-50), 4.13-4.19 (m, 1H, H-50), 4.02 (s, 3H, -OCH3),
2.24 (br, 1H, -OH), 2.14 (s, 3H, -C(O)CH3), 2.12 (s, 3H,
-C(O)CH3), 2.08 (s, 3H, -C(O)CH3), 2.02-2.05 (m, 2H,
cyclohexanyl-H), 1.65-1.78 (m, 8H, cyclohexane-H). 13C
NMR (75 MHz, CDCl3): δ 170.9, 169.8, 169.6, 157.6, 147.7,
145.0, 95.4, 89.6, 80.9, 74.5, 71.0, 68.8, 63.0, 62.9, 53.8, 42.3,
39.5, 25.2, 23.7, 23.2, 20.9, 20.7. Maldi-MS: m/z 530.2 [M þ
Na]þ. HRMS: calcd for C23H29N3O10Naþ 530.1745, found
2228.8 cm-1
.
2o. An amount of 59.6 mg (61%) of product was obtained,
isolated as a colorless oil. HPLC: t = 7.60 min, purity 98.0%
(method 1); t = 7.15 min, purity 98.1% (method 2). H NMR
1
(300 MHz, CDCl3): δ 7.53 (s, 1H, imidazole-H), 7.49 (s, 1H,
imidazole-H), 6.97 (d, 1H, J=1.2 Hz, H-10), 5.84-5.86 (m, 1H,
H-20), 5.74-5.78 (m, 1H, H-30), 4.44-4.49 (m, 2H, H-40 þ H-50),
4.15-4.21 (m, 1H, H-50), 4.04 (s, 3H, -OCH3), 3.77 (s, 3H,
-NCH3), 2.16 (s, 3H, -C(O)CH3), 2.14 (s, 3H, -C(O)CH3),
2.08 (s, 3H, -C(O)CH3). 13C NMR (150 MHz, CDCl3): δ 170.7,
169.8, 169.6, 157.6, 147.7, 145.3, 139.4, 137.2, 114.7, 89.8, 86.7,
80.9, 79.3, 74.6, 70.9, 63.0, 53.8, 32.6, 20.9, 20.71, 20.67. Maldi-
530.1740. IR: 2236.7 cm-1
.
General Procedure for Preparing 3. 2 was dissolved in 10 mL
of saturated NH3/MeOH and stirred at room temperature for 2
days. Then the solvent was removed and the residue was washed
with CH2Cl2. The washed residuel was dried in vacuo to afford
the corresponding products 3a-t.
3a. An amount of 87.8 mg (0.18 mmol) of 2a was used for the
reaction. An amount of 50.4 mg (81%) of product was obtained,
isolated as a white solid. HPLC: t = 6.57 min, purity 97.2%
(method 1); t = 6.21 min, purity >99% (method 2). 1H NMR
(300 MHz, DMSO-d6): δ 8.51 (br s, 1H, -C(O)NH), 8.18 (br s,
þ
MS: m/z 490.2 [M þ H]þ. HRMS: calcd for C21H24N5O9
490.1567, found 490.1548. IR: 2228.4 cm-1
.
2p. An amount of 60.6 mg (62%) of product was obtained,
isolated as a colorless oil. HPLC: t = 6.80 min, purity 96.5%
1
(method 1); t = 6.54 min, purity 98.3% (method 2). H NMR
(300 MHz, CDCl3): δ 6.92 (s, 1H, H-10), 6.37 (s, 1H, cyclohex-
enyl-H), 5.82-5.85 (m, 1H, H-20), 5.74-5.78 (m, 1H, H-30),
4.43-4.47 (m, 2H, H-40 þ H-50), 4.14-4.18 (m, 1H, H-50),

6092 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Xia et al.
1H, -C(O)NH), 7.64-7.67 (m, 2H, phenyl-H), 7.48-7.53 (m,
3H, phenyl-H), 6.77 (d, 1H, J=3.0 Hz, H-10), 5.51 (d, 1H, J=
5.1 Hz, -OH), 5.20 (d, 1H, J=6.0 Hz, -OH), 4.78 (t, 1H, J=
6.0 Hz, -OH), 4.40-4.44 (m, 1H, H-20), 4.19-4.23 (m, 1H,
H-30), 3.90-3.92 (m, 1H, H-40), 3.41-3.57 (m, 2H, H-50). 13C
NMR (150 MHz, DMSO-d6): δ 161.3, 158.5, 149.0, 145.8, 132.5,
130.8, 129.7, 120.9, 91.4, 90.1, 86.1, 80.7, 74.9, 71.3, 62.7. Maldi-
MS: m/z 367.1 [M þ Na]þ. HRMS: calcd for C16H16N4O5Naþ
calcd for C21H26N4O5Naþ 437.1795, found 437.1804. IR:
2228.0 cm-1
.
3f. An amount of 60.0 mg (0.12 mmol) of 2f was used for the
reaction. An amount of 33.6 mg (77%) of product was obtained,
isolated as a yellow solid. HPLC: t = 6.81 min, purity 97.1%
1
(method 1); t = 6.32 min, purity 98.4% (method 2). H NMR
(300 MHz, DMSO-d6): δ 8.48 (br s, 1H, -C(O)NH), 8.17 (br s,
1H, -C(O)NH), 7.68 (d, 2H, J=8.1 Hz, phenyl-H), 7.55 (d, 2H,
J = 8.1 Hz, phenyl-H), 6.76 (d, 1H, J = 3.6 Hz, H-10), 5.52 (d,
1H, J=6.0 Hz, OH), 5.19 (d, 1H, J=6.0 Hz, OH), 4.78 (t, 1H,
J = 5.9 Hz, OH), 4.40-4.45 (m, 1H, H-20), 4.19-4.23 (m, 1H,
H-30), 3.90-3.92 (m, 1H, H-40), 3.40-3.59 (m, 2H, H-50). 13C
NMR (75 MHz, DMSO-d6): δ 163.3, 153.8, 150.4, 140.4, 139.0,
134.6, 124.6, 96.2, 93.7, 90.9, 86.4, 79.7, 76.0, 67.5. Maldi-MS:
m/z 401.1 [M þ Na]þ. HRMS: calcd for C16H15N4O5ClNaþ
367.1013, found 367.1017. IR: 2228.4 cm-1
.
3b. An amount of 93.8 mg (0.18 mmol) of 2b was used for the
reaction. An amount of 48.3 mg (72%) of product was obtained,
isolated as a white solid. HPLC: t = 6.68 min, purity 98.6%
1
(method 1); t = 6.27 min, purity 96.3% (method 2). H NMR
(300 MHz, DMSO-d6): δ 8.44 (br s, 1H, -C(O)NH), 8.12 (br s,
1H, -C(O)NH), 7.57 (d, 2H, J=8.4 Hz, phenyl-H), 7.01 (d, 2H,
J = 9.0 Hz, phenyl-H), 6.74 (d, 1H, J = 2.7 Hz, H-10), 5.47 (d,
1H, J=5.7 Hz, -OH), 5.15 (d, 1H, J=6.3 Hz, -OH), 4.74 (t,
1H, J=5.7 Hz, -OH), 4.37-4.42 (m, 1H, H-20), 4.19-4.22 (m,
1H, H-30), 3.88-3.91 (m, 1H, H-40), 3.80 (s, 3H, -OCH3),
3.37-3.57 (m, 2H, H-50). 13C NMR (150 MHz, DMSO-d6): δ
163.3, 160.9, 150.7, 148.4, 136.4, 117.5, 114.5, 93.5, 93.1, 87.8,
81.2, 76.9, 73.2, 64.6, 58.1. Maldi-MS: m/z 397.1 [M þ Na]þ.
HRMS: calcd for C17H18N4O6Naþ 397.1119, found 397.1130.
401.0623, found 401.0625. IR: 2234.9 cm-1
.
3g. An amount of 86.4 mg (0.17 mmol) of 2g was used for the
reaction. An amount of 49.0 mg (80%) of product was obtained,
isolated as a yellow solid. HPLC: t = 6.66 min, purity 98.6%
1
(method 1); t = 6.16 min, purity 98.2% (method 2). H NMR
(300 MHz, DMSO-d6): δ 8.48 (br s, 1H, -C(O)NH), 8.16 (br s,
1H, -C(O)NH), 7.69-7.74 (m, 2H, phenyl-H), 7.32 (dd, 2H,
J
HH =JHF =9.2 Hz, phenyl-H), 6.74 (d, 1H, J=3.0 Hz, H-10),
IR: 2233.5 cm-1
.
5.51 (d, 1H, J=5.1 Hz, -OH), 5.19 (d, 1H, J=5.7 Hz, -OH),
4.77 (t, 1H, J = 5.4 Hz, -OH), 4.38-4.42 (m, 1H, H-20),
4.17-4.22 (m, 1H, H-30), 3.86-3.91 (m, 1H, H-40), 3.38-3.57
(m, 2H, H-50). 13C NMR (150 MHz, DMSO-d6): δ 163.5 (d,
1JCF = 255 Hz), 158.5, 149.0, 145.8, 135.1 (d, 3JCF = 8.55 Hz),
117.3, 117.1 (d, 2JCF=21.3 Hz), 91.4, 89.1, 86.1, 80.4, 74.9, 71.3,
62.7. Maldi-MS: m/z 385.1 [M þ Na]þ. HRMS: calcd for
3c. An amount of 75.7 mg (0.15 mmol) of 2c was used for the
reaction. An amount of 42.4 mg (78%) of product was obtained,
isolated as a white solid. HPLC: t = 6.82 min, purity 98.5%
(method 1); t = 6.31 min, purity >99% (method 2). 1H NMR
(300 MHz, DMSO-d6): δ 8.49 (br s, 1H, -C(O)NH), 8.16 (br s,
1H, -C(O)NH), 7.54 (d, 2H, J=7.8 Hz, phenyl-H), 7.29 (d, 2H,
J = 7.8 Hz, phenyl-H), 6.75 (d, 1H, J = 3.0 Hz, H-10), 5.51 (d,
1H, J=5.4 Hz, -OH), 5.19 (d, 1H, J=5.4 Hz, -OH), 4.77 (t,
1H, J=5.4 Hz, -OH), 4.41-4.42 (m, 1H, H-20), 4.19-4.22 (m,
1H, H-30), 3.90-3.91 (m, 1H, H-40), 3.41-3.57 (m, 2H, H-50),
2.36 (s, 3H, -OCH3). 13C NMR (150 MHz, DMSO-d6): δ 158.6,
148.9, 146.0, 140.8, 132.4, 130.3, 117.9, 91.4, 90.3, 86.1, 80.2,
74.9, 71.3, 62.7, 21.8. Maldi-MS: m/z 381.1 [M þ Na]þ. HRMS:
calcd for C17H18N4O5Naþ 381.1169, found 381.1177. IR:
C16H15N4O5FNaþ 385.0919, found 385.0923. IR: 2233.1 cm-1
.
3h. An amount of 66.1 mg (0.14 mmol) of 2h was used for the
reaction. An amount of 36.3 mg (77%) of product was obtained,
isolated as a yellow solid. HPLC: t = 6.64 min, purity 96.6%
1
(method 1); t = 6.19 min, purity 96.5% (method 2). H NMR
(300 MHz, DMSO-d6): δ 8.49 (br s, 1H, -C(O)NH), 8.17 (br s,
1H, -C(O)NH), 7.49-7.55 (m, 3H, phenyl-H), 7.35-7.40 (m,
1H, phenyl-H), 6.74 (d, 1H, J=3.6 Hz, H-10), 5.52 (d, 1H, J=
5.1 Hz, -OH), 5.20 (d, 1H, J=5.1 Hz, -OH), 4.79 (t, 1H, J=
5.6 Hz, -OH), 4.38-4.42 (m, 1H, H-20), 4.17-4.22 (m, 1H, H-
30), 3.87-3.92 (m, 1H, H-40), 3.38-3.58 (m, 2H, H-50). 13C
NMR (150 MHz, DMSO-d6): δ 158.5, 149.1, 145.5, 131.8,
2230.6 cm-1
.
3d. An amount of 62.7 mg (0.12 mmol) of 2d was used for the
reaction. An amount of 38.2 mg (83%) of product was obtained,
isolated as a white solid. HPLC: t = 7.04 min, purity 96.4%
2
2
1
129.0, 122.8, 119.1 (d, JCF = 22.7 Hz), 118.1 (d, JCF = 20.4
Hz), 91.5, 88.6, 86.2, 81.5, 74.9, 71.3, 62.7. Maldi-MS: m/z 385.1
[M þ Na]þ. HRMS: calcd for C16H15N4O5FNaþ 385.0919,
(method 1); t = 6.51 min, purity 97.7% (method 2). H NMR
(300 MHz, DMSO-d6): δ 8.51 (br s, 1H, -C(O)NH), 8.18 (br s,
1H, -C(O)NH), 7.56 (d, 2H, J=7.8 Hz, phenyl-H), 7.31 (d, 2H,
J = 7.8 Hz, phenyl-H), 6.78 (d, 1H, J = 3.9 Hz, H-10), 5.56 (d,
1H, J=5.7 Hz, -OH), 5.24 (d, 1H, J=4.8 Hz, -OH), 4.84 (t,
1H, J=5.4 Hz, -OH), 4.42-4.46 (m, 1H, H-20), 4.21-4.26 (m,
1H, H-30), 3.90-3.95 (m, 1H, H-40), 3.55-3.63 (m, 2H, H-50),
2.61 (t, 2H, J=7.4 Hz, -CH2-), 1.57-1.64 (m, 2H, -CH2-),
0.90 (t, 3H, J =7.4 Hz, -CH3). 13C NMR (150 MHz, DMSO-
d6): δ 157.3, 147.7, 144.8, 144.1, 131.2, 131.2, 128.5, 128.4, 117.0,
90.2, 89.1, 85.0, 84.9, 79.0, 73.7, 73.6, 70.1, 61.5, 36.6, 23.2,
13.05, 12.96. Maldi-MS: m/z 409.1 [M þ Na]þ. HRMS: calcd for
found 385.0920. IR: 2235.5 cm-1
.
3i. An amount of 69.8 mg (0.14 mmol) of 2i was used for the
reaction. An amount of 38.0 mg (77%) of product was obtained,
isolated as a yellow solid. HPLC: t = 6.63 min, purity 98.3%
1
(method 1); t = 6.19 min, purity 97.9% (method 2). H NMR
(300 MHz, DMSO-d6): δ 8.53 (br s, 1H, -C(O)NH), 8.19 (br s,
1H, -C(O)NH), 7.74 (dd, 1H, JHH =JHF =7.2 Hz, phenyl-H),
7.55-7.60 (m, 1H, phenyl-H), 7.30-7.44 (m, 2H, phenyl-H),
6.77 (d, 1H, J =3.0 Hz, H-10), 5.53 (d, 1H, J = 5.1 Hz, -OH),
5.20 (d, 1H, J=6.0 Hz, -OH), 4.78 (t, 1H, J=5.9 Hz, -OH),
4.42-4.43 (m, 1H, H-20), 4.19-4.24 (m, 1H, H-30), 3.88-3.93
(m, 1H, H-40), 3.41-3.59 (m, 2H, H-50). 13C NMR (150 MHz,
DMSO-d6): δ 158.5, 149.1, 145.5, 134.5, 133.2, 125.8, 116.7 (d,
2JCF = 19.4 Hz), 109.4, 91.5, 86.2, 85.5, 83.5, 74.9, 71.3, 62.7.
C19H22N4O5Naþ 409.1482, found 409.1485. IR: 2231.0 cm-1
.
3e. An amount of 110.1 mg (0.20 mmol) of 2e was used for the
reaction. An amount of 58.3 mg (71%) of product was obtained,
isolated as a yellow solid. HPLC: t = 7.20 min, purity >99%
(method 1); t = 6.83 min, purity >99% (method 2). 1H NMR
(300 MHz, DMSO-d6): δ 8.50 (br s, 1H, -C(O)NH), 8.17 (br s,
1H, -C(O)NH), 7.55 (d, 1H, J=7.8 Hz, phenyl-H), 7.30 (d, 1H.
J = 8.1 Hz, phenyl-H), 6.76 (d, 1H, J = 3.0 Hz, H-10), 5.51 (d,
1H, J=5.1 Hz, -OH), 5.19 (d, 1H, J=5.7 Hz, -OH), 4.78 (t,
1H, J=5.6 Hz, -OH), 4.40-4.45 (m, 1H, H-20), 4.19-4.24 (m,
1H, H-30), 3.89-3.94 (m, 1H, H-40), 3.42-3.59 (m, 2H, H-50),
2.62 (t, 2H, J=7.2 Hz, -CH2-), 1.56-1.61 (m, 2H, -CH2-),
Maldi-MS: m/z 385.1 [M
þ
Na]þ. HRMS: calcd for
C16H15N4O5FNaþ 385.0919, found 385.0917. IR: 2235.2 cm-1
.
3j. An amount of 120.8 mg (0.22 mmol) of 2j was used for the
reaction. An amount of 67.4 mg (74%) of product was obtained,
isolated as a yellow solid. HPLC: t = 6.65 min, purity 98.5%
1
(method 1); t = 6.16 min, purity 98.8% (method 2). H NMR
(300 MHz, DMSO-d6): δ 8.66 (br s, 1H, -C(O)NH), 8.34 (br s,
1H, -C(O)NH), 7.96-8.04 (m, 4H, phenyl-H), 6.77 (d, 1H, J=
3.6 Hz, H-10), 5.69 (d, 1H, J = 5.4 Hz, -OH), 5.36 (d, 1H, J =
5.7 Hz, -OH), 4.93 (t, 1H, J=5.7 Hz, -OH), 4.40-4.45 (m, 1H,
H-20), 4.20-4.25 (m, 1H, H-30), 4.04-4.05 (m, 1H, H-40),
1.25-1.35 (m, 4H, -CH2-), 0.86 (t, 3H, J=6.9 Hz, -CH3). 13
C
NMR (75 MHz, DMSO-d6): δ 158.6, 148.9, 146.0, 145.6, 132.5,
129.6, 118.2, 91.4, 90.3, 86.1, 80.2, 74.9, 71.3, 62.7, 35.7,
31.5, 31.0, 22.6, 14.6. Maldi-MS: m/z 437.2 [M þ Na]þ. HRMS:

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 6093
3.40-3.59 (m, 2H, H-50). 13C NMR (75 MHz, DMSO-d6): δ
158.5, 149.1, 145.4, 133.4, 130.5 (q, 2JCF =31 Hz), 126.5, 125.2,
123.9 (q, 1JCF =192 Hz), 91.5, 88.5, 86.2, 82.8, 74.9, 71.4, 62.7.
Maldi-MS: m/z 435.1 [M þ Na]þ. HRMS: calcd for C17H15-
1H, -C(O)NH), 7.87 (br s, 1H, imidazole-H), 7.53 (br s, 1H,
imidazole-H), 6.76 (d, 1H, J=3.6 Hz, H-10), 5.51 (d, 1H, J=5.1
Hz, -OH), 5.19 (d, 1H, J=5.7 Hz, -OH), 4.77 (t, 1H, J=5.9
Hz, -OH), 4.40-4.45 (m, 1H, H-20), 4.19-4.24 (m, 1H, H-30),
3.89-3.94 (m, 1H, H-40), 3.72 (s, 3H, -NCH3), 3.42-3.61 (m,
2H, H-50). 13C NMR (150 MHz, DMSO-d6): δ 158.5, 149.1,
145.7, 141.1, 137.0, 114.2, 91.5, 87.6, 86.1, 79.0, 74.9, 71.3, 62.7,
32.6. Maldi-MS: m/z 349.1 [M þ H]þ. HRMS: calcd for
N4O5F3Naþ 435.0887, found 435.0892. IR: 2237.0 cm-1
.
3k. An amount of 62.2 mg (0.11 mmol) of 2k was used for the
reaction. An amount of 35.3 mg (76%) of product was obtained,
isolated as a white solid. HPLC: t = 6.66 min, purity 98.8%
(method 1); t = 6.25 min, purity 97.7% (method 2). H NMR
C14H17N6O5þ 349.1255, found 349.1251. IR: 2235.1 cm-1
.
1
(300 MHz, DMSO-d6): δ 8.47 (br s, 1H, -C(O)NH), 8.19 (br s,
1H, -C(O)NH), 8.02 (s, 1H, phenyl-H), 7.97 (d, 1H, J=7.8 Hz,
phenyl-H), 7.89 (d, 1H, J = 7.8 Hz, phenyl-H), 7.73-7.76 (m,
1H, phenyl-H), 6.77 (d, 1H, J=3.0 Hz, H-10), 5.52 (d, 1H, J=
4.8 Hz, -OH), 5.20 (d, 1H, J=5.7 Hz, -OH), 4.78 (t, 1H, J=
5.9 Hz, -OH), 4.41-4.43 (m, 1H, H-20), 4.21-4.23 (m, 1H, H-
30), 3.91-3.93 (m, 1H, H-40), 3.44-3.57 (m, 2H, H-50). 13C
NMR (75 MHz, DMSO-d6): δ 158.5, 149.1, 145.5, 136.5,
131.0, 128.9, 127.3, 122.1, 91.6, 88.3, 86.2, 82.0, 74.9, 71.3,
62.7. Maldi-MS: m/z 435.1 [M þ Na]þ. HRMS: calcd for
3p. An amount of 66.1 mg (0.14 mmol) of 2p was used for the
reaction. An amount of 36.2 mg (74%) of product was obtained,
isolated as a white solid. HPLC: t = 6.65 min, purity 98.5%
1
(method 1); t = 6.53 min, purity 96.5% (method 2). H NMR
(300 MHz, DMSO-d6): δ 8.45 (br s, 1H, -C(O)NH), 8.12 (br s,
1H, -C(O)NH), 6.22 (d, 1H, J = 3.3 Hz, H-10), 6.37 (s, 1H,
-CdCH), 5.48 (d, 1H, J=5.4 Hz, -OH), 5.17 (d, 1H, J=5.7
Hz, -OH), 4.76 (t, 1H, J = 5.9 Hz, -OH), 4.37-4.39 (m, 1H,
H-20), 4.17-4.19 (m, 1H, H-30), 3.87-3.89 (m, 1H, H-40),
3.40-3.54 (m, 2H, H-50), 2.14-2.17 (m, 4H, cyclohexenylethy-
nyl-H), 1.56-1.61 (m, 4H, cyclohexenylethynyl-H). 13C NMR
(150 MHz, DMSO-d6): δ 158.6, 148.8, 146.2, 139.3, 119.3, 92.1,
91.3, 86.0, 78.3, 74.8, 71.3, 62.7, 28.7, 26.0, 22.3, 21.4. Maldi-
MS: m/z 371.1 [M þ Na]þ. HRMS: calcd for C16H20N4O5Naþ
C17H15N4O5F3Naþ 435.0886, found 435.0890. IR: 2238.4 cm-1
.
3l. An amount of 50.9 mg (0.10 mmol) of 2l was used for the
reaction. An amount of 30.1 mg (73%) of product was obtained,
isolated as a white solid. HPLC: t = 6.66 min, purity 98.4%
371.1326, found 371.1335. IR: 2220.0 cm-1
.
1
(method 1); t = 6.23 min, purity 98.6% (method 2). H NMR
3q. An amount of 88.4 mg (0.18 mmol) of 2q was used for the
reaction. An amount of 46.0 mg (74%) of product was obtained,
isolated as a white solid. HPLC: t = 6.58 min, purity 98.2%
(300 MHz, DMSO-d6): δ 8.50 (br s, 1H, -C(O)NH), 8.19 (br s,
1H, -C(O)NH), 7.89-7.95 (m, 2H, phenyl-H), 7.70-7.82 (m,
2H, phenyl-H), 6.77 (d, 1H, J=3.6 Hz, H-10), 5.52 (d, 1H, J=
5.7 Hz, -OH), 5.20 (d, 1H, J=6.0 Hz, -OH), 4.78 (t, 1H, J=
5.6 Hz, -OH), 4.42-4.47 (m, 1H, H-20), 4.20-4.25 (m, 1H,
H-30), 3.91-3.96 (m, 1H, H-40), 3.42-3.62 (m, 2H, H-50). 13C
NMR (150 MHz, DMSO-d6): δ 158.5, 149.2, 145.4, 135.5, 133.6,
131.2, 130.9 (q, 2JCF = 30.3 Hz), 127.0, 124.1 (q, 1JCF = 271.7
Hz), 118.8, 91.5, 86.2, 85.6, 85.4, 74.9, 71.3, 62.7. Maldi-MS: m/z
435.1 [M þ Na]þ. HRMS: calcd for C17H15N4O5F3Naþ
1
(method 1); t = 6.08 min, purity 97.5% (method 2). H NMR
(300 MHz, DMSO-d6): δ 8.41 (br s, 1H, -C(O)NH), 8.12 (br s,
1H, -C(O)NH), 6.71 (d, 1H, J=3.3 Hz, H-10), 5.47 (d, 1H, J=
5.4 Hz, -OH), 5.16 (d, 1H, J=5.7 Hz, -OH), 4.75 (t, 1H, J=
5.7 Hz, -OH), 4.34-4.38 (m, 1H, H-20), 4.15-4.19 (m, 1H, H-
20), 3.86-3.90 (m, 1H, H-30), 4.74 (t, 2H, J=6.5 Hz, -CH2-),
3.40-3.60 (m, 2H, H-50), 2.63 (t, 2H, J = 7.1 Hz, -CH2-),
1.99-2.03 (m, 2H, -CH2-). 13C NMR (150 MHz, DMSO-d6):
δ 158.6, 148.6, 146.0, 91.23, 91.16, 86.0, 74.8, 72.8, 71.3, 62.7,
44.7, 31.0, 16.5. Maldi-MS: m/z 367.1 [M þ Na]þ. HRMS: calcd
for C13H17N4O5ClNaþ 367.0780, found 367.0782. IR: 2251.4
435.0886, found 435.0877. IR: 2234.6 cm-1
.
3m. An amount of 64.9 mg (0.13 mmol) of 2m was used for the
reaction. An amount of 37.3 mg (81%) of product was obtained,
isolated as a white solid. HPLC: t = 6.60 min, purity 98.9%
cm-1
.
1
(method 1); t = 6.13 min, purity 95.5% (method 2). H NMR
3r. An amount of 51.4 mg (0.11 mmol) of 2r was used for the
reaction. An amount of 28.7 mg (79%) of product was obtained,
isolated as a white solid. HPLC: t = 6.72 min, purity 98.7%
(300 MHz, DMSO-d6): δ 8.46 (br s, 1H, -C(O)NH), 8.16 (br s,
1H, -C(O)NH), 7.80 (d, 1H, J = 5.1 Hz, thiophenyl-H),
7.61-7.62 (m, 1H, thiophenyl-H), 7.18-7.21 (m, 1H, thiophe-
nyl-H), 6.77 (d, 1H, J =3.6 Hz, H-10), 5.50 (d, 1H, J=5.1 Hz,
-OH), 5.18 (d, 1H, J=5.7 Hz, -OH), 4.77 (t, 1H, J=6.0 Hz,
-OH), 4.40-4.44 (m, 1H, H-20), 4.20-4.25 (m, 1H, H-30),
3.89-3.94 (m, 1H, H-40), 3.42-3.61 (m, 2H, H-50). 13C NMR
(150 MHz, DMSO-d6): δ 158.5, 149.0, 145.7, 135.4, 131.4, 128.8,
120.2, 91.5, 86.1, 84.3, 83.8, 74.9, 71.3, 62.7. Maldi-MS: m/z
373.1 [M þ Na]þ. HRMS: calcd for C14H14N4O5SNaþ
1
(method 1); t = 6.32 min, purity 97.8% (method 2). H NMR
(600 MHz, DMSO-d6): δ 8.38 (br s, 1H, -C(O)NH), 8.08 (br s,
1H, -C(O)NH), 6.69 (d, 1H, J=3.0 Hz, H-10), 5.46 (d, 1H, J=
4.8 Hz, -OH), 5.15 (d, 1H, J=6.0 Hz, -OH), 4.74 (t, 1H, J=
5.9 Hz, -OH), 4.32-4.35 (m, 1H, H-20), 4.14-4.16 (m, 1H, H-
30), 3.84-3.87 (m, 1H, H-40), 3.39-3.53 (m, 2H, H-50), 2.44 (t,
2H, J = 6.9 Hz, -CH2-), 1.50-1.54 (m, 2H, -CH2-),
1.27-1.36 (m, 4H, -CH2-), 0.86 (t, 3H, J = 7.5 Hz, -CH3).
13C NMR (150 MHz, DMSO-d6): δ 158.7, 148.6, 146.2, 92.8,
91.2, 86.0, 74.9, 72.4, 71.3, 62.8, 31.2, 27.9, 22.3, 18.8, 14.6.
373.0577, found 373.0580. IR: 2221.8 cm-1
.
3n. An amount of 90.1 mg (0.18 mmol) of 2n was used for the
reaction. An amount of 40.5 mg (76%) of product was obtained,
isolated as a white solid. HPLC: t = 6.62 min, purity 98.3%
Maldi-MS: m/z 361.1 [M
þ
Na]þ. HRMS: calcd for
1
(method 1); t = 6.05 min, purity 97.5% (method 2). H NMR
C19H22N4O5Naþ 361.1482, found 361.1490. IR: 2250.0 cm-1
.
(300 MHz, DMSO-d6): δ 8.48 (br s, 1H, -C(O)NH), 8.17 (br s,
1H, -C(O)NH), 8.13-8.14 (m, 1H, thiophenyl-H), 7.72-7.74
(m, 1H, thiophenyl-H), 7.38 (d, 1H, J=4.8 Hz, thiophenyl-H),
6.77 (d, 1H, J = 3.0 Hz, H-10), 5.51 (d, 1H, J = 5.1 Hz, -OH),
5.19 (d, 1H, J=6.0 Hz, -OH), 4.78 (t, 1H, J=5.4 Hz, -OH),
4.41-4.45 (m, 1H, H-20), 4.21-4.26 (m, 1H, H-30), 3.90-3.95
(m, 1H, H-40), 3.43-3.63 (m, 2H, H-50). 13C NMR (75 MHz,
DMSO-d6): δ 158.6, 148.9, 146.0, 133.1, 130.3, 128.2, 119.8,
91.4, 86.1, 85.9, 80.2, 74.9, 71.3, 62.7. Maldi-MS: m/z 373.1 [M þ
Na]þ. HRMS: calcd for C14H14N4O5SNaþ 373.0577, found
3s. An amount of 90.1 mg (0.18 mmol) of 2s was used for the
reaction. An amount of 40.5 mg (63%) of product was obtained,
isolated as a white solid. HPLC: t = 6.57 min, purity 98.6%
1
(method 1); t = 5.99 min, purity 98.4% (method 2). H NMR
(300 MHz, DMSO-d6): δ 8.43 (br s, 1H, -C(O)NH), 8.13 (br s,
1H, -C(O)NH), 6.73 (d, 1H, J = 3.0 Hz, H-10), 5.56 (s, 1H,
-OH), 5.49 (d, 1H, J=5.1 Hz, -OH), 5.17 (d, 1H, J=5.1 Hz,
-OH), 4.76 (t, 1H, J=5.4 Hz, -OH), 4.35-4.40 (m, 1H, H-20),
4.16-4.21 (m, 1H, H-30), 3.86-3.91 (m, 1H, H-40), 3.38-3.57
(m, 2H, H-50), 1.80-1.96 (m, 4H, cyclopentanyl-H), 1.68-1.76
(m, 4H, cyclopentanyl-H). 13C NMR (150 MHz, DMSO-d6): δ
158.6, 148.7, 146.0, 96.2, 91.3, 86.0, 74.9, 73.4, 73.2, 71.3, 62.7,
42.3, 23.7. Maldi-MS: m/z 375.1 [M þ Na]þ. HRMS: calcd for
373.0587. IR: 2234.0 cm-1
.
3o. An amount of 54.8 mg (0.11 mmol) of 2o was used for the
reaction. An amount of 33.5 mg (86%) of product was obtained,
isolated as a white solid. HPLC: t = 7.18 min, purity 98.7%
C15H20N4O6Naþ 375.1275, found 375.1275. IR: 2243.9 cm-1
.
1
(method 1); t = 6.07 min, purity 97.2% (method 2). H NMR
(300 MHz, DMSO-d6): δ 8.48 (br s, 1H, -C(O)NH), 8.16 (br s,
3t. An amount of 76.9 mg (0.15 mmol) of 2t was used for the
reaction. An amount of 32.9 mg (60%) of product was obtained,

6094 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Xia et al.
isolated as a white solid. HPLC: t = 6.58 min, purity 98.6%
(method 1); t = 6.04 min, purity 97.6% (method 2). H NMR
yield of 95%. HPLC: t=7.43 min, purity >99% (method 1); t=
6.93 min, purity 95.4% (method 2). 1H NMR (300 MHz,
DMSO-d6): δ 8.51 (br s, 1H, -C(O)NH), 8.14 (br s, 1H,
-C(O)NH), 7.54 (d, 2H, J = 7.8 Hz, phenyl-H), 7.30 (d, 2H,
J=7.8 Hz, phenyl-H), 5.93 (s, 2H, H-10), 4.73 (t, 1H, J=5.4 Hz,
-OH), 3.58 (t, 2H, J=5.0 Hz, H-20), 3.44-3.49 (m, 2H, H-30),
2.62 (t, 2H, J=7.4 Hz, -CH2-), 1.56-1.60 (m, 2H, -CH2-),
1
(300 MHz, DMSO-d6): δ 8.55 (br s, 1H, -C(O)NH), 8.23 (br s,
1H, -C(O)NH), 6.82 (d, 1H, J = 3.6 Hz, H-10), 5.82 (s, 1H,
-OH), 5.61 (d, 1H, J=5.1 Hz, -OH), 5.31 (d, 1H, J=5.1 Hz,
-OH), 4.89 (t, 1H, J=5.1 Hz, -OH), 4.46-4.49 (m, 1H, H-20),
4.25-4.31 (m, 1H, H-30), 3.96-4.01 (m, 1H, H-40), 3.46-3.67
(m, 2H, H-50), 1.53-2.01 (m, 10H, cyclohexanyl-H). 13C NMR
(150 MHz, DMSO-d6): δ 158.6, 148.7, 145.9, 96.2, 91.2, 86.0,
74.8, 73.4, 73.2, 71.2, 67.5, 62.7, 42.2, 25.3, 23.7, 23.6, 23.1.
1.21-1.28 (m, 4H, -CH2-), 0.87 (t, 3H, J=6.8 Hz, -CH3). 13
C
NMR (150 MHz, CDCl3): δ 157.8, 146.8, 146.4, 145.1, 132.1,
128.6, 118.1, 91.1, 79.5, 78.3, 71.7, 61.5, 36.0, 31.4, 30.8,
22.5, 14.0. FAB-MS: m/z 357 [M þ H]þ, 379 [M þ Na]þ.
Maldi-MS: m/z 389.1 [M
þ
Na]þ. HRMS: calcd for
þ
HRMS: calcd for C19H25N4O3 357.1921, found 357.1911.
C16H22N4O6Naþ 389.1432, found 389.1439. IR: 2249.9 cm-1
.
4. Compounds 4 was synthesized according to the procedures
described in ref 15a. HPLC: t=7.08 min, purity >99% (method
1); t=6.55 min, purity 98.7% (method 2). 1H NMR (600 MHz,
DMSO-d6): δ 8.31 (br, 1H, -C(O)NH), 8.14 (br, 1H, -C-
(O)NH), 7.95 (d, 2H, J = 8.4 Hz, phenyl-H), 7.34 (d, 2H, J =
8.4 Hz, phenyl-H), 6.81 (d, 1H, J = 2.4 Hz, H-10), 5.52 (d, 1H,
J=5.4 Hz, -OH), 5.18 (d, 1H, J=4.8 Hz, -OH), 4.80 (t, 1H,
J=5.4 Hz, -OH), 4.47-4.78 (m, 1H, H-20), 4.36-4.37 (m, 1H,
H-30), 3.95-3.98 (m, 1H, H-40), 3.51-3.67 (m, 2H, H-50), 2.62 (t,
2H, J = 7.5 Hz, -CH2-), 1.58-1.63 (m, 2H, -CH2-),
1.27-1.33 (m, 4H, -CH2-), 0.87 (t, 3H, J = 6.9 Hz, -CH3).
13C NMR (150 MHz, DMSO-d6): δ 160.3, 159.3, 149.2, 145.0,
129.4, 128.1, 126.7, 90.9, 86.1, 75.2, 71.6, 63.0, 35.6, 31.5, 31.1,
22.6, 14.6. Maldi-MS: m/z 413.2 [M þ Na]þ. HRMS: calcd for
C19H26N4O5Naþ 413.1801, found 413.1820.
IR: 2227.35 cm-1
.
8. The protected precursor of 8 was synthesized according to
the procedures described in ref 11. The obtained protected
compound was dissolved in 10 mL of saturated NH3/MeOH
and stirred at room temperature for 2 days. Then the solvent was
removed and the residue was washed with CH2Cl2. The washed
residue was dried in vacuo to afford the corresponding product 8
as white solid (71%). HPLC: t = 7.28 min, purity >99%
1
(method 1); t = 6.55 min, purity 95.4% (method 2). H NMR
(300 MHz, DMSO-d6): δ 8.03 (br s, 1H, -C(O)NH), 7.76 (br s,
1H, -C(O)NH), 7.62 (d, 2H, J=7.8 Hz, phenyl-H), 7.35 (d, 2H,
J = 8.1 Hz, phenyl-H), 5.99 (d, 1H, J = 4.2 Hz, H-10), 5.63 (d,
1H, J=5.7 Hz, -OH), 5.28 (d, 1H, J=6.0 Hz, -OH), 4.80 (t,
1H, J=6.0 Hz, -OH), 4.49-4.54 (m, 1H, H-20), 4.21-4.26 (m,
1H, H-30), 3.95-4.00 (m, 1H, H-40), 3.33-3.62 (m, 2H, H-50),
2.64 (t, 2H, J=7.5 Hz, -CH2-), 1.54-1.64 (m, 2H, -CH2-),
5. An amount of 40.9 mg (0.099 mmol) of 3e 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 over-
night. The insoluble residue was filtered by Celite and the
solvent removed by reduced pressure. The residue was dried in
vacuo to afford 40.7 mg (98.6%) of product 10 as a white solid.
HPLC: t = 7.14 min, purity >99% (method 1); t = 6.49 min,
1.25-1.35 (m, 4H, -CH2-), 0.86 (t, 3H, J=6.9 Hz, -CH3). 13
C
NMR (150 MHz, DMSO-d6): δ 160.4, 157.7, 146.6, 140.8, 132.7,
129.8, 117.1, 98.2, 91.1, 86.9, 74.9, 74.7, 71.2, 62.7, 35.7, 31.5,
30.9, 22.6, 14.6. Maldi-MS: m/z 437 [M þ Na]þ. HRMS: calcd
for C21H26N4O5Naþ 437.1795, found 437.1798. IR: 2226.2
cm-1
.
1
purity 95.1% (method 2). H NMR (300 MHz, DMSO-d6): δ
In Vitro Cell Growth Inhibition Assay. Pancreatic cancer
chemoresistant MiaPaCa-2 cells were cultured in DMEM med-
ium (Gibco) supplemented with 10% fetal bovine serum (FBS).
Cells were seeded at a densitiy of 15 000 cells per well in 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 medium 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 determined
by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) colorimetric assay.
8.21 (br s, 1H, -C(O)NH), 8.00 (br s, 1H, -C(O)NH), 7.14 (d,
2H, J=7.8 Hz, phenyl-H), 7.09 (d, 2H, J=7.8 Hz, phenyl-H),
6.69 (d, 1H, J = 2.7 Hz, H-10), 5.40 (d, 1H, J = 6.0 Hz, -OH),
5.10 (d, 1H, J=6.0 Hz, -OH), 4.73 (t, 1H, J=6.0 Hz, -OH),
4.32-3.35 (m, 1H, H-20), 4.17-4.23 (m, 1H, H-30), 3.85-3.88
(m, 1H, H-40), 3.43-3.63 (m, 2H, H-50), 2.94 (br, 4H, -CH2-),
2.51-2.54 (m, 2H, -CH2-), 1.51-1.59 (m, 2H, -CH2-),
1.23-1.28 (m, 4H, -CH2-), 0.86 (t, 3H, J = 6.9 Hz, -CH3).
13C NMR (150 MHz, DMSO-d6): δ 162.4, 159.3, 148.5, 140.6,
138.7, 128.9, 128.8, 90.9, 85.5, 75.9, 71.5, 63.1, 35.4, 33.6, 31.6,
31.3, 30.3, 22.6, 14.6. Maldi-MS: 441 [M þ Na]þ. HRMS: calcd
for C21H30N4O5Naþ 441.2108, found 441.2099.
FACS Flow Cytometry. Cells were seeded in 10 cm dishes at
the density of 106 cells/dish and allowed to adhere and prolifer-
ate 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 ethanol, 70%, 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.
ELISA Assay for DNA/Histone Release. 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 cytoplasmic nucleosomes produced during apoptosis
(Cell Death Detection ELISAPlus, Roche). The 96-well plates
were centrifuged (200g) for 10 min, the supernatant was dis-
carded, and lysis buffer was added. After lysis, the samples were
centrifuged and an amount of 20 μL of the supernatant was
6. The protected precursor of 6 was synthesized according to
the procedures described in ref 16a. The obtained protected
compound was dissolved in 10 mL of saturated NH3/MeOH
and stirred at room temperature for 2 days. Then the solvent was
removed and the residue was was purified by flash chromatog-
raphy on silica gel (CH2Cl2/MeOH, 6:1). The purified product
was dried in vacuo to afford 6 as a white solid (85.4%). HPLC:
t = 7.11 min, purity >99% (method 1); t = 6.42 min, purity
>99% (method 2). 1H NMR (300 MHz, DMSO-d6): δ 9.15 (s,
1H, triazole-H), 8.52 (br s, 1H, -C(O)NH), 8.31 (br s, 1H,
-C(O)NH), 7.90 (d, 2H, J=7.8 Hz, phenyl-H), 7.30 (d, 2H, J=
8.1 Hz, phenyl-H), 6.80 (d, 1H, J = 3.3 Hz, H-10), 5.59 (d, 1H,
J=5.4 Hz, -OH), 5.18 (d, 1H, J=6.0 Hz, -OH), 4.75 (t, 1H,
J=5.7 Hz, -OH), 4.44-4.47 (m, 1H, H-20), 4.25-4.31 (m, 1H,
H-30), 3.93-3.94 (m, 1H, H-40), 3.46-3.60 (m, 2H, H-50), 2.60 (t,
J = 7.5 Hz, 2H, -CH2-), 1.56-1.61 (m, 2H, -CH2-),
1.28-1.29 (m, 4H, -CH2-), 0.85 (t, 3H, J = 6.8 Hz, -CH3).
13C NMR (150 MHz, DMSO-d6): δ 158.3, 153.5, 149.4, 147.8,
143.7, 129.6, 127.6, 126.3, 120.8, 91.9, 86.4, 75.0, 71.3, 62.8, 35.5,
31.5, 31.2, 22.6, 14.6. ESI-MS: m/z 479.9 [M þ Na]þ. HRMS:
calcd for C21H27N7O5Naþ 480.1971, found 480.1985.
7. Compounds 7 was synthesized according to the procedures
described in ref 13. The obtained product is a white solid with a

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 6095
transferred to a streptavidin-coated microtiter plate. Biotin-
labeled anti-histone 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 incuba-
tion, the plates were read at 405 nm in a microplate reader. The
enrichment in histone-DNA fragments is expressed as a fold
increase in absorbance compared with control.
Caspase-3/7 Cleavage Assay. Caspase-3/7 activity was mea-
sured 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 fluoro-
metric substrate Z-DEVD-R110 according to the instructions of
the manufacturer (Promega). Next 100 μL of Apo-ONE homo-
geneous 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 measuring the fluorescence of
each well.
Lactate Dehydrogenase (LDH) Assay. MiaPaCa-2 cells were
seeded 15 000 cells per well in 96-well plates. Twenty-four
hours later, cells were treated with the test compound for 1 h.
Then the LDH concentration was measured by using commer-
cial LDH kit (cytotoxicity detection kit, Roche). The LDH
reaction mixture was freshly prepared according to the man-
ufacturer’s protocol (Roche Diagnostics), 100 μL added to
each well of a 96-well plate containing 100 μL of blank, control,
or cells in culture, and the plate incubated for 10 min at 25 °C.
Control was performed with lysis buffer and medium and
set as 100% and 0% LDH release, respectively. The relative
LDH release is defined by the ratio of LDH released over
total LDH in the intact cells. All samples were performed in
triplicate.
Necrosis Assay on Flow Cytometry. MiaPaCa-2 cells were
seeded in 6 cm dishes at a density of 500 000 cells/dish and
allowed to adhere and proliferate O/N. Culture medium was
then removed, and fresh medium containing the proper con-
centration of compound was added. No treatment was done as
negative controls. After 3, 6, and 12 h of treatment, the cells were
trypsinized and the collected cell pellet was washed with cold
PBS twice. The samples were pelleted through centrifugation at
3000 rpm for 5 min and resuspended in 100 μL of binding buffer
(10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4). The
nuclear stain 7-AAD (7-aminoactinomycin D) was added to the
samples, which were then incubated for 15 min at 25 °C.
Necrotic cells, as well as those cells in the advanced stages of
apoptosis, have permeablized membranes and allow 7-AAD to
stain the cell nucleus. Immediately after staining, flow cytometry
was performed on fluorescence activated cell sorting (FACS
Calibur, Becton Dickinson, Le Pont-De-Claix, France). Each
sample was performed in triplicate.
dNTP mix, 4.8 μL of 25 mM MgCl2, 4 μL of 5ꢀRT buffer) in a
final volume of 20 μL. The thermal cycling protocol employed
included two steps (step 1 consisting of 70 °C for 5 min, 4 °C for
5 min; step 2 consisting of 25 °C for 5 min, 42 °C for 1 h, 70 °C for
15 min, and 4 °C forever). The real time PCR was conducted
using the LightCycler 2.0 instrument (Roche Diagnostics
GmbH, Mannheim, Germany). Reaction mixtures contained a
total volume of 20 μL consisting of 5 μL of each diluted cDNA
(1:10), 10 μL of SYBR Premix Ex Taq (2ꢀ) (TaKaRa Bio. Inc.,
Japan), 0.4 μL of primers forward and primers reverse (10 μM)
of target gene Hsp27 and internal control 18S, and 4.2 μL of
H2O. The sequences of primers are as follows: Hsp27 primer F,
50-TCCCTGGATGTCAACCACTTC-30, and Hsp27 primer R,
50-TCTCCACCACGCCATCCT-30; 18S primer F, 50-CTAC-
CACATCCAAGGAAGGC-30, and 18S primer R, 50-TTTTC-
GTCACTACCTCCCCG-3 (Eurogentec S. A., Seraing, Belgium).
The PCR conditions were as follows: an initial denaturation step
at 95 °C for 10 s, then 45 cycles of 95 °C for 5 s, 57 °C for 6 s, and
72 °C for 12 s. A melting curve was carried out after the
amplification program by heating at temperatures from 65 to
95 °C in 1.5 min. And a final cooling step at 40 °C for 1 min was
performed. Each sample was analyzed in triplicate in the PCR
reaction to estimate the reproducibility of data. The data were
acquired by using Roche Molecular Biochemicals LightCycler
software, version 3.5, and statistical analysis was done with
RelQuant (Roche).
Western Blotting Analysis. Samples containing equal
amounts of protein (15 μg) from lysates of cultured MiaPaCa-
2 cells were analyzed by Western blot analysis as described
previously7,8 with 1:5000 diluted antihuman Hsp27 rabbit poly-
clonal antibody (Stressgen Assay Designs Inc., MI) and 1:2000
diluted antihuman vinculin mouse monoclonal antibody (Sigma
Chemical Co., St. Louis, MO). Specific proteins were detected
using an enhanced chemiluminescence Western blotting analysis
system (Amersham Life Science, Arlington Heights, IL).
Immunofluorescence Detection of Hsp27 Protein Expression.
For immunofluorescence detection of Hsp27 protein in MiaPa-
Ca-2 cells, the cells after different treatments were fixed with
paraformaldehyde (Sigma-Aldrich, Lyon, France) (4% aqu-
eous solution) and permeabilized with Triton 100ꢀ 1%. Then
the cells were incubated with 1:500 diluted antimouse Hsp27
monoclonal antibody (Stressgen Assay Designs Inc., MI) at 4 °C
overnight. After saturation in BSA/PBS, the cells were incu-
bated with 1:750 diluted secondary green-fluorescent labeled
antimouse antibody (Invitrogen Ltd., Paisley, U.K.). Then the
cells were mounted with Prolong Gold antifade reagent contain-
ing DAPI (4, 6-diamidino-2-phenylindole) (Invitrogen Ltd.,
Paisley, U.K.). The samples were visualized using a Nikon
Eclipse 90i microscope equipped with a Nikon digital sight
DS-1QM/H digital camera (Nikon Instruments Inc., Japan).
Images were acquired using NIS-Eliments AR 2.30 software
(Nikon Instruments Inc.)
Assessment of in Vivo Tumor Growth. Institutional guidelines
for the proper and humane use of animals in research were
followed. Approximately 1 ꢀ 107 MiaPaCa-2 cells were inocu-
lated subcutaneously with 0.1 mL of Matrigel (BD Biosciences
Discovery Labware) to 5-week-old male xenografed nude mice.
When MiaPaCa-2 tumors reached 100 mm3, mice were ran-
domly selected for treatment with test compound and no treated
mice were used as control. Each experimental group consisted of
eight mice. After randomization, 150 mg/kg test compound was
injected twice a week by intraperitoneal (ip) injection for 5
weeks. Tumor volume measurements were performed once
weekly and calculated by the formula length ꢀ width ꢀ depth ꢀ
0.5236.
Determination of DNA Synthesis in Whole Cells. MiaPaCa-2
Cells were seeded at a densitiy 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 labeled with
[3H]thymidine (10 μCi/mL) for 6 h at 37 °C and 5% CO2. Then
the cells were harvested and DNA synthesis activity was deter-
mined according to the radioactivity by using liquid scintillation
counting.
Quantitative Real-Time PCR (qRT-PCR). The expression of
the Hsp27 mRNAs was analyzed by quantitative real-time PCR
amplification analysis. Total RNAs were isolated using TRIzol
method (Invitrogen). Then 1 μg of RNAs was reverse-tran-
scribed into cDNA by using the ImProm-II reverse transcription
system (Promega) (0.5 μg of oligo dT primer, 1 μL of ImProm-II
reverse transcriptase, 0.5 μL of RNase inhibitor, 1 μL of 10 mM
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).

6096 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Xia et al.
Acknowledgment. Financial support from the CNRS, IN-
SERM, Wuhan University, National Natural Science Foun-
dation of China (Grants 20572081 and 20025311), La Ligue
Contre le Cancer, and Ministere des Affaires Etrangeres de la
France is gratefully acknowledged. Y.X. is supported by a
postdoctoral fellowship from the Fondation pour la Re-
(11) Wan, J. Q.; Xia, Y.; Liu, Y.; Wang, M. H.; Rocchi, P.; Yao, J. H.;
Qu, F. Q.; Neyts, J.; Iovanna, J. L.; Peng, L. Discovery of novel
arylethynyltriazole ribonucleosides with selective and effective
antiviral and antiproliferative activity. J. Med. Chem. 2009, 52,
1144–1155.
(12) Peng, L. Xia, Y.; Wan, J. Q.; Rocchi, P.; Qu, F. Q.; Iovana, J. L.
Novel Triazole Nucleoside Derivatives, Their Preparation and
Their Application in Therapeutics. Patent PCT/EP2009/055213,
2009.
(13) Zhu, R. Z.; Wang, M. H.; Xia, Y.; Qu, F. Q.; Neyts, J.; Peng, L.
Arylethynyltriazole acyclonucleosides inhibit hepatitis C virus
replication. Bioorg. Med. Chem. Lett. 2008, 18, 3321–3327.
(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., , Ed.; Collection Symposium Series Vol 10; Institute of Organic
Chemistry and Biochemistry, Academy of Sciences of the Czech
Republic: Prague, Czech Republic, 2008; pp224-228.
ꢀ
€
cherche Medicale. We thank Joel Tardivel-Lacombe for his
assistance with cell culture, Drs. Sandrine Henri and Gene-
vieve Victorero for their help with radioactivity experiment,
Prof. Jingui Qin for allowing us to use his microwave reactor,
and Emily Witty for her careful reading of the manuscript.
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