Synthesis and pharmacological exploitation of clioquinol-derived copper-binding apoptosis inducers triggering reactive oxygen species generation and MAPK pathway activation

Article abstract of DOI:10.1016/j.bmc.2009.08.054

In the present study, we carried out Mannich-type reaction to synthesize clioquinol-derived 7-methyl-arylsulfonylpiperazine analogs with improved growth-inhibitory effects. 11 bearing 5-nitro group on the quinoline ring exhibited 26-fold more potent than that of clioquinol against HeLa cells with a GI₅₀ value of 0.71 μM. In addition, 11 revealed synergistic effects on the growth inhibition of HeLa cells with GI₅₀ values of 0.65, 0.25, and 0.06 μM in the presence of 1, 10, and 50 μM copper, respectively. Consistent to the clioquinol-mediated apoptosis, mechanistic study indicates that 9- and 11-induced growth inhibition is attributed to caspase-dependent pathway. Detection of reactive oxygen species in response to clioquinol, 9 and 11 confirmed that ROS was dramatically stimulated in the presence of copper and partially abolished upon treatment of 1 mM tempol. Further study indicated that 9- and 11-mediated induction of oxidative stress by ROS generation resulted in the activation MAPK pathway.

Full text of DOI:10.1016/j.bmc.2009.08.054

Bioorganic & Medicinal Chemistry 17 (2009) 7239–7247
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and pharmacological exploitation of clioquinol-derived
copper-binding apoptosis inducers triggering reactive oxygen species
generation and MAPK pathway activation
Hui-Ling Chen a, Chun-Yi Chang b, Hsun-Tzu Lee b, Hua-Hsuan Lin b, Pei-Jung Lu a, Chia-Ning Yang c,
Chung-Wai Shiau d, Arthur Y. Shaw b,
*
a Institute of Clinical Medicine, National Cheng Kung University, Tainan 701, Taiwan
b Department of Chemistry, Tamkang University, Tamsui 251, Taiwan
c Institute of Biotechnology, National University of Kaohsiung, Kaohsiung 700, Taiwan
d Institute of Biopharmaceutical Sciences, National Yang-Ming University, Taipei 112, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present study, we carried out Mannich-type reaction to synthesize clioquinol-derived 7-methyl-
arylsulfonylpiperazine analogs with improved growth-inhibitory effects. 11 bearing 5-nitro group on
the quinoline ring exhibited 26-fold more potent than that of clioquinol against HeLa cells with a GI50
Received 10 July 2009
Revised 26 August 2009
Accepted 26 August 2009
Available online 1 September 2009
value of 0.71
l
M. In addition, 11 revealed synergistic effects on the growth inhibition of HeLa cells with
M in the presence of 1, 10, and 50 M copper, respectively. Consistent
GI50 values of 0.65, 0.25, and 0.06
l
l
to the clioquinol-mediated apoptosis, mechanistic study indicates that 9- and 11-induced growth inhibi-
tion is attributed to caspase-dependent pathway. Detection of reactive oxygen species in response to cli-
oquinol, 9 and 11 confirmed that ROS was dramatically stimulated in the presence of copper and partially
abolished upon treatment of 1 mM tempol. Further study indicated that 9- and 11-mediated induction of
oxidative stress by ROS generation resulted in the activation MAPK pathway.
Keywords:
Clioquinol
Mannich-type reaction
Metal-binding property
Reactive oxygen species (ROS)
MAPK pathway activation
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
oxide, are mitogenic to promote cell proliferation, while medium
levels result in either transient or permanent growth arrest.
Reactive oxygen species (ROS) are highly reactive molecules
presenting radical and non-radical structure: superoxide anion,
hydrogen peroxide, hydroxyl radical, and singlet oxygen. In the
biological system, ROS are generated by enzymatic and non-enzy-
matic reactions which play a central role as second messengers in a
number of signal transduction pathways, such as mitogen-acti-
vated protein kinases (MAPK), protein phosphatases, and tran-
Redundant ROS eventually cause cell death through apoptotic or
necrotic mechanisms.1,2
Clioquinol (5-chloro-7-iodo-8-hydoxyquinoline) was widely
used as an antibiotic for the treatment of diarrhea and skin infec-
tion (Fig. 1). The metal-binding properties of clioquinol led to its
application in a mouse model of Alzheimer’s disease via the reduc-
tion or prevention of amyloid plaque accumulation in the brain. It
was then moved to clinical trials for patients with Alzheimer’s dis-
ease.3,4 On the other hand, clioquinol also exhibited growth-inhib-
itory effect against a panel of cancer cell lines. The mechanistic
study of clioquinol-induced apoptosis revealed that clioquinol
served as an ionophore that elevated levels of intracellular zinc
for cytotoxic potentiation. The apoptosis induced by clioquinol
was attributed to the activation of caspase-dependent pathways
and inhibition of proteasome activity.5–7
In the present study we sought to synthesize clioquinol-derived
apoptosis inducers by means of Mannich-type reaction with aim to
examine their growth-inhibitory effects against carcinoma cell
lines. We further provided evidence that apoptosis induced by cli-
oquinol and its analogs was potentiated by copper ion through
reactive oxygen species generation.
scription factors. The oxidative stress generally describes
a
condition in which cellular antioxidant defense mechanisms are
insufficient to deactivate ROS, or excessive ROS are generated, or
both. The maintenance of cellular redox balance is regulated by a
powerful antioxidant system that inactivates ROS. It comprises of
SOD, catalase, the glutathione system (glutathione, glutathione
reductase, peroxidase, and transferase), the thioredoxin system
(thioredoxins, thioredoxin peroxidase, and peroxiredoxins), vita-
min E and C. The influence of cellular response to redox modula-
tion is extensive and includes both proliferative and apoptotic
pathways. Generally, low levels of ROS, particularly hydrogen per-
* Corresponding author. Tel.: +886 2 2621 5656x2842; fax: +886 2 2620 9924.
E-mail address: shaw299@mail.tku.edu.tw (A.Y. Shaw).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.08.054

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H.-L. Chen et al. / Bioorg. Med. Chem. 17 (2009) 7239–7247
Table 1
Cl
Growth inhibition of clioquinol (CQ) and Mannich bases 7–11 against carcinoma cell
lines
a
Entry
GI50
(
lM)
N
I
HeLA
BT483
SKHep
CE81T
OH
Clioquinol
CQ
7
8
9
10
11
18.6 2.4
14.5 1.7
25.5 2.7
6.8 1.1
38.6 4.0
0.71 0.15
10.2 0.6
26.9 3.2
NDb
10.7 2.6
ND
9.5 0.6
16.6 2.5
ND
14.6 3.6
ND
23.4 2.5
16.7 0.8
ND
5.6 1.8
ND
Figure 1. Chemical structure of clioquinol.
1.9 0.1
14.6 0.6
5.9 0.5
2. Results and discussion
a
GI50 values are presented as the mean SEM (standard error of the mean) from
four to six separated experiments.
2.1. Synthesis of Mannich bases
b
ND indicated that no appreciable growth inhibition was observed upon treat-
ment of maximal concentration at 40 lM.
Herein, we take advantage of the three-component one-pot
synthesis of Mannich-type reaction as shown in Scheme 1. In gen-
eral, the mixture of 8-hydroxyquinoline (1), 1-naphthol (2), or 5-
nitro-8-hydroxyquinoline (3), along with formaldehyde and corre-
sponding secondary amines was stirred in ethanol at reflux for 16 h
to afford Mannich bases 7–11.8–10
inhibition against HeLa cells with GI50 values of 25.5 and
38.6 M, respectively. Moreover, as high as 40 M treatment of 8
l
l
and 10, no appreciable growth-inhibitory effect was observed on
the rest of BT483, SKHep, and CE81T cell lines, indicating that 8-
hydroxyquinoline skeleton plays a crucial role for growth-inhibi-
tory activity.
2.2. Growth inhibition of clioquinol and Mannich bases against
carcinoma cell lines
2.3. Enhancement of growth inhibition of clioquinol and
Mannich bases against HeLa cells in the presence of metal ion
The evaluation of growth-inhibitory activity of clioquinol and
Mannich bases 7–11 was examined on a panel of carcinoma cell
lines, including HeLa (cervical epithelioid carcinoma cell), BT483
(mammary gland adenocarcinoma cell), SKHep (hepatocellular car-
cinoma cell), and CE81T (esophageal carcinoma cell). The MTT (3-
[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) as-
say was employed for growth inhibition studies and the GI50 values
are summarized in Table 1. The tested compound concentration
causing a 50% cell growth inhibition (GI50) was determined by
interpolation from dose-response curves.
Among four carcinoma cell lines, HeLa cells were most sensitive
in response to tested compounds on growth inhibition. As shown,
exposure of HeLa cells to clioquinol and Mannich bases 7–11 in
10% FBS-supplemented DMEM medium for 48 h resulted in the
growth inhibition with a range of GI50 values between 0.7 and
Similar to the results demonstrated in the literature, the metal-
binding properties of clioquinol enhanced its growth-inhibitory ef-
fect on HeLa cells in the presence of both copper and zinc. As
shown in Table 2, both copper and zinc potentiated clioquinol-in-
duced growth inhibition toward HeLa cells in a dose-dependent
manner. Meanwhile, 7, 9, and 11 exhibited significantly synergistic
effects on growth inhibition in the presence of 50
GI50 values of 0.35, 0.27, and 0.06 M, respectively, all of which
exhibited more potent than that of clioquinol with a GI50 of
4.4 M. However, the synergistic effect with zinc was not as obvi-
lM copper with
l
l
ous as that of copper. This discrepancy might be attributed to the
distinct redox sensitivity of specific metal ions that counts for
the stability of intracellular redox balance. On the other hand,
the counterparts 8 and 10 did not show significant growth inhibi-
tion in the presence of both copper and zinc. These findings clearly
confirmed the metal-binding property of 8-hydroxyquinoline skel-
eton in clioquinol, 7, 9, and 11.
25.5
growth-inhibitory effect on HeLa cells with a GI50 value of
18.6 M. An appreciable growth inhibition on HeLa cells was ob-
served while introducing 7-methyl-piperidine (7, GI50, 14.5 M)
and 7-methyl-arylsulfonylpiperazine moieties (9, GI50, 6.8 M) on
lM. Clioquinol as a positive control showed a moderate
l
l
l
the 8-hydroxyquinoline scaffold. In particular, 11 bearing 5-nitro
2.4. Mannich bases induce apoptosis through caspase-
dependent pathways
group on the quinoline ring exhibited 26-, 5-, and 4-fold more po-
tent than that of clioquinol against HeLa (GI50, 0.7 vs 18.6
lM),
BT483 (GI50, 1.9 vs 10.2 M) and CE81T (GI50, 5.9 vs 23.4 M) cells,
l
l
To determine whether induction of apoptosis mediated by clio-
quinol-derived Mannich bases is similar to that of clioquinol in the
literature,5 analysis of HeLa cells was examined after treatment of
9 for 24 and 48 h. According to the flow cytometric analysis shown
respectively. Together, these results indicate that structural opti-
mization could be achieved by the appropriate elongation of 8-
hydroxyquinoline and substitution on quinoline ring. On the con-
trary, 1-naphthol-bearing 8 and 10 exhibited decreased growth
Y
Y
EtOH
reflux
Z
Z
+
HCHO
+
HN
N
X
X
OH
OH
1 X = N, Y = H
2 X = CH, Y = H
3 X = N, Y = NO2
4 Z = CH2
5 Z = NSO2Ph
6 Z = NSO2C6H4(4-CH3)
7
8
9
X = N, Y = H, Z = CH2
X = CH, Y = H, Z = CH2
X = N, Y = H, Z = NSO2Ph
10 X = CH, Y = H, Z = NSO2Ph
11 X = N, Y= NO2, Z = NSO2C6H4(4-CH3)
Scheme 1.

H.-L. Chen et al. / Bioorg. Med. Chem. 17 (2009) 7239–7247
7241
Table 2
Growth inhibition of clioquinol (CQ) and Mannich bases 7–11 against HeLa cells in the presence of metal ion
a
Entry
GI50
(l
M)
CuCl2
(lM)
ZnCl2 (lM)
0
1
10
5.3 1.2
0.58 0.09
22.8 4.2
0.31 0.07
28.4 3.4
0.25 0.07
50
4.4 0.9
0.35 0.08
48.8 4.5
0.27 0.06
22.6 2.4
0.06 0.05
50
5.6 0.8
8.5 1.9
34.0 5.9
7.5 1.6
100
2.1 0.8
12.9 1.6
44.5 4.8
7.3 2.4
CQ
7
8
9
10
11
18.6 2.4
14.5 1.7
25.5 2.7
6.8 1.1
38.6 4.0
0.71 0.15
6.1 1.8
3.0 0.9
30.7 3.8
2.5 0.8
40.1 5.2
0.65 0.12
29.7 4.8
0.48 0.09
20.1 4.3
0.26 0.05
a
GI50 values are presented as the mean SEM (standard error of the mean) from four to six separated experiments.
in Figure 2A, HeLa cells exposed to 9 for 24 h treatment was found
to exhibit sub-G1 phase arrest in a dose-dependent manner, sug-
gesting that growth inhibition mediated by 8-hydroxyquinoline
analogs resulted in DNA fragmentation. Moreover, western blot
of cytosolic extracts prepared from 9-treated cells revealed cleav-
age of the 116-kDa protein poly(ADP-ribose) polymerase (PARP)
and generation of the 89-kDa fragment. The appearance of a 85-
kDa fragment coincided with the activation of caspase-3, as shown
by the reduced level of the 32-kDa proenzyme and the increased
level of the 17/19-kDa cleaved caspase-3 (Fig. 2B). The activation
of the upstream caspase-9 was also observed with the reduced
level of the 45-kDa proenzyme, indicating the mitochondrial apop-
totic pathway was involved. Taken together, the 9-mediated
apoptotic effects are in agreement with the findings in clio-
quinol-mediated apoptosis through the caspase-dependent path-
way. In light of growth-inhibitory activity of Mannich bases
enhanced through metal-binding property, we demonstrated the
synergistic effect of apoptosis in the presence of copper.
Three major MAPK subfamilies have been identified: extracellular
signal-regulated kinase (ERK), p38 MAPK, and c-Jun N-terminal ki-
nase (JNK). In addition, activation of MAPK subfamilies has been
shown to be important in the oxidative stress-mediated cell death.
Therefore, as indicated in Figure 5A, we showed that upon expo-
sure of HeLa cells to 3 lM 9 in the presence of 10 lM copper,
JNK, p38 and ERK were dramatically activated in a time-dependent
manner in response to oxidative stress induced by 9. In addition,
the activation of MAPK pathway was reversed upon treatment of
tempol and that without copper treatment. Moreover, the activa-
tion of MAPK pathways as well as caspase-3 and PARP were also
observed upon the long-term treatment of HeLa cells with 11 at
1 lM for 24–48 h in the presence of 10 lM copper as shown in
Figure 5B.
3. Conclusions
In summary, the present study we take advantage of the practi-
cal Mannich-type reaction to synthesize a new class of clioquinol-
derived 7-methyl-arylsulfonylpiperazine analogs with improved
growth-inhibitory effects. In particular, analog 11 bearing 5-nitro
group on the quinoline ring exhibited 26-fold more potent than
the parent compound clioquinol against HeLa cells. In the presence
As shown in Figure 3, exposure of HeLa cells to 9 at 3
lM and 11
at 1 M in the presence of 10 M copper enhanced sub-G1 phase
l
l
arrest rising from 7.9% and 6.8% to 29.8% and 17.5%, respectively.
The sub-G1 phase arrest was reduced upon treatment of caspase
inhibitor z-VAD at 10 lM (Fig. 3L). Moreover, accumulation of
sub-G1 phase cells diminished upon treatment of 1 mM tempol
(Fig. 3K), a cell membrane-permeable radical scavenger, which
prompted us to examine reactive oxygen species (ROS) formation.
of 50
lM copper ion, 11 revealed significantly synergistic effects on
the growth inhibition of HeLa cells with a GI50 value of 0.06
lM.
These results suggest that clioquinol containing 8-hydroxyquino-
line scaffold is amenable for structural modifications with im-
proved growth-inhibitory activity independent of metal-binding
property. The mechanistic study on the growth inhibition of HeLa
cell mediated by Mannich bases agreed with clioquinol-induced
caspase-dependent apoptotic pathway. The well-known redox
property of copper ion enables its cytotoxic when present in excess
of cellular requirements. For example, the intracellular cytotoxic
effects associated with copper exposure are consistent with oxida-
tive damage of lipids, proteins, and nucleic acids. Current study
shows that clioquinol and Mannich bases trigger potential stimula-
tion of reactive oxygen species upon co-treatment of copper ion. In
addition, Western blot analysis reveals that upon treatment of 11
in the presence of copper ion phosphorylated histone H2AX in a
time-dependent manner (data not shown), indicating the response
of DNA damage to 11-Cu-induced oxidative stress. Detection of
reactive oxygen species in response to clioquinol, 7, 9, and 11 also
confirmed that ROS could be dramatically stimulated in the pres-
ence of copper ion. Upon treatment of 1 mM tempol, ROS produc-
tion was partially abolished which was corresponding to the
minimized sub-G1 phase arrest in the flow cytometric analysis.
We conclude that consecutive activation of MAPK pathway was
accompanied by ROS production that leads to the oxidative-
stressed microenvironment and eventually executed programmed
cell death.
2.5. Clioquinol and Mannich bases trigger reactive oxygen
species (ROS) generation
Clioquinol is a metal-binding agent that binds metal ions extra-
cellularly and transports them into the cells. In addition, copper ion
is a redox-active metal that may be more redox active as com-
plexed with some metal-binding agents, which may result in the
generation of reactive oxygen species and exhibit cytotoxicty.11,12
As shown in Figure 4, the production of ROS was dramatically stim-
ulated upon treatment of CQ at 10
lM, 7 and 9 at 3 lM, and 11 at
1
l
M in the presence of indicated concentrations of copper ion.
Moreover, ROS generation was partially abolished by the addition
of 1 mM tempol. Consequently, modulation of ROS production is
in accordance with our flow cytometric data in which the sub-G1
phase arrest is reduced upon co-treatment of 1 mM tempol. Taken
together, we conclude that the apoptosis-inducing effect mediated
by clioquinol and Mannich bases is, in part, through the generation
of reactive oxygen species.
2.6. 8-Hydroxyquinoline-induced activation of mitogen-
activated protein kinase (MAPK) pathway
MAPK is one of the most common signaling pathways activated
in response to various cellular stimuli such as oxidative stress.

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H.-L. Chen et al. / Bioorg. Med. Chem. 17 (2009) 7239–7247
Figure 2. Induction of apoptosis in HeLa cells by 9. (A) Untreated HeLa cells were taken as blank group. Cells were treated with 0.5% DMSO as the control. Cells were
harvested after 24 h exposure to 3 and 10 M of 9 and followed by fixation, propidium iodide staining prior to the flow cytometric analysis. Sub-G1 phase indicated the DNA
l
fragmentation and cell death. (B) Induction of poly(ADP-ribose) polymerase (PARP) cleavage by 9 at the indicated concentrations after 24 and 48 h of treatment. PARP
proteolysis to the apoptosis-specific 85-kDa fragment was monitored by western blotting. The appearance of a 89-kDa fragment coincided with the activation of caspase-3, as
shown by the reduced level of the 32-kDa proenzyme and the increased level of the 17/19-kDa cleaved caspase-3. The activation of the upstream caspase-9 was also observed
with the reduced level of the 45-kDa proenzyme, indicating the mitochondrial apoptotic pathway was involved. CPT as the positive control.
the TMS peak. Mass spectra were obtained by FAB on a Jeol JMS-
700 instrument. Flash column chromatography was performed
with silica gel (230–400 mesh). Elemental Analysis was carried
out on a Heraeus VarioEL-III C, H, N analyzer.
4. Materials and methods
4.1. Synthesis
Chemical reagents and organic solvents were purchased from
TCI and Alfa Aesar unless otherwise mentioned. Melting points
were determined by Fargo MP-2D. Nuclear magnetic resonance
spectra (1H and 13C NMR) were measured on a Bruker AC-300
instrument. Chemical shifts (d) are reported in ppm relative to
4.1.1. The general experimental procedure
To a solution of 8-hydroxyquinoline or 1-naphthnol (1.05 mmol)
and paraformaldehyde (36 mg, 1.26 mmol) in dry ethanol (8 mL)
was added the appropriate secondary amine (1.26 mmol) at room

H.-L. Chen et al. / Bioorg. Med. Chem. 17 (2009) 7239–7247
7243
Figure 3. Flow cytometric analysis of HeLa cells. Cells were harvested after 24 h treatment and followed by fixation, propidium iodide staining prior to the flow cytometric
analysis. Sub-G1 phase indicated the DNA fragmentation and cell death. Exposure of HeLa cells to 9 at 3 M and 11 at 1 M in the presence of 10 M copper enhanced sub-G1
phase arrest increasing from 7.9% and 6.8% to 29.8% and 17.5%, respectively. The sub-G1 phase arrest was reduced upon treatment of tempol (Fig. 3K) and z-VAD (Fig. 3L). (A)
Untreated HeLa cells were taken as blank group; (B) cells were treated with 0.5% DMSO as the control; (C) CuCl2 10 M; (D) tempol 1 mM; (E) treatment of 9 at 3 M; (F)
treatment of 9 at 3 M in the presence of CuCl2 10 M; (G) treatment of 9 at 3 M in the presence of CuCl2 at 10 M and temopl at 1 mM; (H) treatment of 11 at 1 M; (I)
treatment of 11 at 1 M in the presence of CuCl2 at 10 M; (J) treatment of 11 at 2 M; (K) treatment of 11 at 1 M in the presence of CuCl2 at 10 M and temopl at 1 mM; (L)
treatment of 11 at 1 M in the presence of CuCl2 at 10 M and z-VAD at 10 M.
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
temperature. The resultingmixture washeated at reflux for 18–22 h.
The solvent was removed in vacuo and the crude product was puri-
fied by flash chromatography and/or recrystallization.
4.1.2. 7-(Piperidin-1-ylmethyl)quinolin-8-ol (7)
Mp 115–117 °C. (lit.8 114–117 °C). 1H NMR (300 MHz, CDCl3) d
1.45 (d, J = 4.1 Hz, 2H), 1.58–1.65 (m, 4H), 2.52 (br s, 4H), 3.78 (d,

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H.-L. Chen et al. / Bioorg. Med. Chem. 17 (2009) 7239–7247
Figure 4. Reactive oxygen species detection. The production of ROS in HeLa cells was observed for 1 h treatment with CQ, 7, 9, and 11 at indicated concentrations. Generation
of ROS could be partially abolished by the treatment of 1 mM tempol. (A) Untreated HeLa cells were taken as blank group; (B) Cells were treated with 0.5% DMSO as the
control; (C) treatment of CQ at 10
presence of CuCl2 10 M and tempol 1 mM; (G) treatment at 7 at 3
CuCl2 M and tempol 1 mM; (J) treatment at 9 at 3
l
M; (D) treatment of CQ at 10
l
l
M; (E) treatment of CQ at 10
M; (H) treatment of 7 at 3 M in the presence of CuCl2
M in the presence of CuCl2 M; (L) treatment of 9 at 3
M; (O) treatment of 11 at 3 M in the presence of CuCl2 5 l
l
M in the presence of CuCl2 10
M; (I) treatment of 7 at 3
M in the presence of CuCl2
M and
l
M; (F) treatment of CQ at 10
lM in the
l
l
5
l
lM in the presence of
5
l
lM; (K) treatment of 9 at 3
l
5
l
l
5 lM
and tempol 1 mM; (M) treatment at 11 at 1
lM; (N) treatment of 11 at 1
l
M in the presence of CuCl2
5
l
l
tempol 1 mM.
J = 4.1 Hz, 2H), 7.10 (d, J = 8.3 Hz, 1H), 7.16 (d, J = 8.3 Hz, 1H), 7.26–
7.30 (m, 1H), 7.97–8.01 (dd, J = 10.0 Hz, J = 6.6 Hz, 1H), 8.82–8.83
(dd, J = 5.8 Hz, J = 2.5 Hz, 1H) ppm. 13C NMR (75 MHz, CDCl3) d
24.0, 25.9, 54.1, 61.8, 117.1, 118.0, 121.1, 127.3, 128.5, 135.6,

H.-L. Chen et al. / Bioorg. Med. Chem. 17 (2009) 7239–7247
7245
Figure 5. The activation of mitogen-activated protein kinase (MAPK) pathway mediated by Mannich bases 9 and 11. (A) The phosphorylation of ERK, p38 and JNK was
observed upon exposure of HeLa cells to 9 at 3 M in the presence of CuCl2 (10 M) within 2 h treatment. In addition, the activation of MAPK pathway was reversed upon
treatment of tempol and without copper treatment. (B) Cleavage of pro-caspase-3, poly(ADP-ribose) polymerase (PARP) and the phosphorylation of ERK, p38, and JNK were
l
l
observed upon exposure of HeLa cells to 11 at 1
lM in the presence of CuCl2 (10
l
M) after 24 and 48 h treatment.
139.5, 149.0, 153.9 ppm. HRMS (M+1)+ calcd for C15H19N2O
243.1497; found 243.1500. Anal. Calcd for C15H19N2O: C, 74.35;
H, 7.49; N, 11.56. Found: C, 74.10; H, 7.41; N, 11.46.
4.1.3. 2-(Piperidin-1-ylmethyl)naphthalen-1-ol (8)
Mp 133–135 °C. (lit.10 133.5–134.5 °C). 1H NMR (300 MHz,
CDCl3) d 1.53 (br s, 2H), 1.62–1.72 (m, 4H), 2.59 (br s, 4H), 3.82

7246
H.-L. Chen et al. / Bioorg. Med. Chem. 17 (2009) 7239–7247
(s, 2H), 7.07 (d, J = 8.3 Hz, 1H), 7.28 (d, J = 8.3 Hz, 1H), 7.40–7.48 (m,
2H), 7.73–7.78 (m, 1H), 8.23–8.28 (m, 1H) ppm. 13C NMR (75 MHz,
CDCl3) d 24.2, 26.0, 54.1, 62.4, 114.0, 118.2, 122.2, 124.9, 125.1,
126.0, 126.7, 127.5, 134.0, 153.9 ppm. HRMS (M)+ calcd for
C16H19NO 241.1467; found 241.1465. Anal. Calcd for C16H19NO:
C, 79.63; H, 7.94; N, 5.80. Found: C, 79.15; H, 7.91; N, 5.76.
on the reduction of MTT by mitochondrial dehydrogenases of via-
ble cell to a blue formazan product, which come be measured spec-
trophotometrically. Tumor cells were incubated in each well with
serial dilutions of the tested compounds. After 2 days of incubation
(37 °C, 5% CO2 in a humid atmosphere) 100
PBS) was added to each well and the plate was incubated for a fur-
ther 2 h (37 °C). The resulting formazan was dissolved in 100
lL of MTT (2 mg/mL in
lL
DMSO and read at 570 nm. The percentage of growth inhibition
was calculated by the following equation: percentage growth inhi-
bition = (1 À At/Ac) Â 100, where At and Ac represent the absor-
bance in treated and control cultures, respectively. The drug
concentration causing a 50% cell growth inhibition (GI50) was
determined by interpolation from dose-response curves. All deter-
minations were carried out in four to six separated experiments.
4.1.4. 7-((4-(Phenylsulfonyl)piperazin-1-yl)methyl)quinolin-8-
ol (9)
Mp 201–203 °C. 1H NMR (300 MHz, CDCl3) d 2.69 (t, J = 4.9 Hz,
4H), 3.09 (br s, 4H), 3.85 (s, 2H), 7.24 (d, J = 5.4 Hz, 2H), 7.37 (dd,
J = 10.5, 5.9 Hz, 1H), 7.50–7.56 (m, 2H), 7.59–7.64 (m, 1H), 7.72–
7.75 (m, 2H), 8.07 (dd, J = 10.5 Hz, 1H), 8.79 (dd, J = 5.9 Hz, 1H)
ppm. 13C NMR (75 MHz, CDCl3) d 46.3, 52.0, 59.0, 117.6, 117.8,
121.7, 127.9, 128.4, 129.3, 133.3, 135.4, 135.9, 139.0, 148.9,
152.2 ppm. HRMS (M)+ calcd for C20H21N3O3S 383.1304; found
383.1304. Anal. Calcd for C20H21N3O3S: C, 62.64; H, 5.52; N,
10.96. Found: C, 62.56; H, 5.53; N, 10.85.
4.4. Determination of apoptosis by flow cytometry
Apoptosis and cell cycle profile were assessed by DNA fluores-
cence flow cytometry. HeLa cells treated with DMSO or tested
compounds at indicated concentrations for 24 and 48 h were har-
vested, rinsed in PBS, resuspended and fixed in 80% ethanol, and
stored at À20 °C in fixation buffer until ready for analysis. Then
the pellets were suspended in 1 mL of fluorochromic solution
(0.08 mg/mL PI (propidium iodide), 0.1% TritonX-100 and 0.2 mg/
ml RNase A in 1Â PBS) at room temperature in the dark for
30 min. The DNA content was analyzed by FACScan flow cytometer
(Becton Dickinson, Mountain View, CA) and CELLQUEST software (Bec-
ton Dickinson). The population of apoptotic nuclei (subdiploid DNA
peak in the DNA fluorescence histogram) was expressed as the per-
centage in the entire population.
4.1.5. 2-((4-(Phenylsulfonyl)piperazin-1-yl)methyl)naphthalen-
1-ol (10)
Mp 125.5–126.5 °C. 1H NMR (300 MHz, CDCl3) d 2.71 (br s, 4H),
3.11 (br s, 4H), 3.85 (s, 2H), 7.05 (d, J = 8.4 Hz, 1H), 7.29 (d,
J = 8.3 Hz, 1H), 7.43 (t, J = 3.8 Hz, 2H), 7.57 (d, J =7.9 Hz, 2H), 7.65
(d, J = 7.0 Hz, 1H), 7.73–7.77 (m, 3H), 8.11 (t, J = 6.9 Hz, 1H) ppm.
13C NMR (75 MHz, CDCl3) d 46.1, 51.9, 61.3, 113.0, 119.0, 122.4,
125.1, 126.4, 126.7, 127.6, 127.7, 127.9, 129.5, 133.4, 134.2,
135.7, 153.0 ppm. HRMS (M)+ calcd for C21H22N2O3S 382.1351;
found 382.1350. Anal. Calcd for C21H22N2O3S: C, 65.95; H, 5.80;
N, 7.32. Found: C, 65.56; H, 5.63; N, 7.81.
4.5. Protein extraction and Western blotting
4.1.6. 5-Nitro-7-((4-tosylpiperazin-1-yl)methyl)quinolin-8-ol
(11)
After the treatment of cells with vehicle (1% DMSO) or tested
compounds for indicated time treatment, the cells were washed
twice with PBS and reaction was terminated by the addition of
Mp 135.5–136.5 °C. 1H NMR (300 MHz, CDCl3) d 2.46 (s, 3H),
2.72 (t, J = 4.8 Hz, 4H), 3.08 (t, J = 4.8 Hz, 4H), 3.89 (s, 2H), 7.35
(d, J = 8.2 Hz, 2H), 7.63 (d, J = 8.2 Hz, 2H), 7.67 (m, 1H), 8.45 (s,
1H), 8.91 (m, 1H), 9.25 (m, 1H) ppm. 13C NMR (75 MHz, CDCl3) d
21.7, 46.1, 52.0, 57.4, 116.6, 122.2, 124.9, 127.9, 129.0, 130.0,
132.0, 133.2, 135.9, 137.8, 144.2, 149.6, 158.5 ppm. HRMS (M)+
calcd for C21H22N4O5S 442.1311; found 442.1335. Anal. Calcd for
C21H22N4O5S: C, 57.00; H, 5.01; N, 12.66. Found: C, 56.45; H,
5.28; N, 12.55.
100
lL lysis buffer. For Western blot analysis, the amount of pro-
teins (50
l
g) were separated by electrophoresis in a 15% SDS–PAGE
and transferred to a nitrocellulose membrane. After an overnight
incubation at 4 °C in TBST/5% non-fat milk, the membrane was
washed with TBST three times and immuno-reacted with the
monocolonal primary antibodies, anti-poly-ADP-ribose polymer-
ase(PARP) (1:500), anti-pro-caspase-3 (1:1000), anti-pro-caspase-
9 (1:1000), anti-cleaved caspase-3 (1:1000), anti-phospho-ERK1/
2
(1:1000), anti-ERK1/2 (1:1000), anti-phospho-p38 (1:1000),
4.2. Cell culture
anti-p38 (1:1000), anti-phospho-c-Jun N-terminal kinase (1:500),
anti-c-Jun N-terminal kinase (1:500), and anti-b-actin (1:1000)
from Cell Signaling Technology (Beverly, MA) were used. After four
washings with TBST, the anti-mouse or anti-rabbit IgG (dilute
1:10,000) was applied to the membranes for 1 h at room tempera-
ture. The membranes were washed with TBST for 1 h and the
detection of signal was performed with an enhanced chemilumi-
nescence (ECL) detection reagents.
Cancer cells were purchased from Bioresource Collection and
Research Center in Taiwan. Each cell line was maintained in the
standard medium and grown as a monolayer in Dulbecco’s Modi-
fied Eagle Medium (DMEM) containing 10% fetal bovine serum,
2 mM glutamine, 100 units/ml penicillin, and 100 g/ml streptomy-
cin. Cultures were maintained at 37 °C with 5% CO2 in a humidified
atmosphere.
4.6. Reactive oxygen species detection
4.3. MTT assay for cell viability
Cells were assessed for the production of ROS using the dye,
dihydroethidium (DHE).13 DHE is a nonfluorescent, reduced form
of ethidium that can passively cross plasma membranes of live
Cells were plated in 96-well microtiter plates at a density of
5 Â 103/well and incubated for 24 h. After that, cells were treated
with vehicle alone (control) or compounds (drugs were dissolved
in DMSO previously) at the concentrations indicated. Treated cells
were further incubated for 48 h. Cell survival is expressed as per-
centage of control cell growth. The 3-[4,5-dimethylthiazol-2-yl]-
2,5-diphenyltetrazolium bromide (MTT, 2 mg/mL) dye reduction
assay in 96-well microplates was used. The assay is dependent
cells. Cells were incubated with 5 lmol/L (Invitrogen, Carlsbad,
CA) for 30 min, washed in PBS and visualized by light microscopy,
using a Zeiss microscope with epifluorescence optics. Investiga-
tions were performed on four separate cultures, with replicates
of two to six coverslips per culture analyzed.

H.-L. Chen et al. / Bioorg. Med. Chem. 17 (2009) 7239–7247
7247
4. Ritchie, C. W.; Bush, A. I.; Mackinnon, A.; Macfarlane, S.; Mastwyk, M.;
MacGregor, L.; Kiers, L.; Cherny, R.; Li, Q. X.; Tammer, A.; Carrington, D.;
Mavros, C.; Volitakis, I.; Xilinas, M.; Ames, D.; Davis, S.; Beyreuther, K.; Tanzi, R.
E.; Masters, C. L. Arch. Neurol. 2003, 60, 1685.
5. Ding, W.-Q.; Liu, B.; Vaught, J. L.; Yamauchi, H.; Lind, S. E. Cancer Res. 2005, 65,
3389.
6. Ding, W.-Q.; Yu, H.-J.; Lind, S. E. Cancer Lett. 2008, 271, 251.
7. Chen, D.; Cui, Q. C.; Yang, H.; Barrea, R. A.; Sarkar, F. H.; Sheng, S.; Yan, B.;
Reddy, G. P. V.; Dou, Q. P. Cancer Res. 2007, 67, 1636.
8. Burckhalter, J. H.; Leib, R. I. J. Org. Chem. 1961, 26, 4078.
9. Negm, N. A.; Morsy, S. M. I.; Said, M. M. Bioorg. Med. Chem. 2005, 13, 5921.
10. Shriner, R. L.; Grillot, G. F.; Teeters, W. O. J. Am. Chem. Soc. 1946, 68, 946.
11. Goodman, V. L.; Brewer, G. J.; Merajver, S. D. Curr. Cancer Drug Targets 2005, 5,
543.
Acknowledgments
Financial support from the National Science Council of the
Republic of China (NSC 96-2113-M-032-007-MY2) and Tamkang
University to A. Y. Shaw is gratefully acknowledged.
References and notes
1. Martindale, J. L.; Holbrook, N. J. J. Cell Physiol. 2002, 192, 1.
2. Manda, G.; Nechifor, M. T.; Neagu, T.-M. Curr. Chem. Biol. 2009, 3, 22.
3. Cherny, R. A.; Atwood, C. S.; Xilinas, M. E.; Gray, D. N.; Jones, W. D.; McLean, C.
A.; Barnham, K. J.; Volitakis, I.; Fraser, F. W.; Kim, Y.; Huang, X.; Goldstein, L. E.;
Moir, R. D.; Lim, J. T.; Beyreuther, K.; Zheng, H.; Tanzi, R. E.; Masters, C. L.; Bush,
A. I. Neuron 2001, 30, 665.
12. Mattie, M. D.; McElwee, M. K.; Freedman, J. H. J. Mol. Biol. 2008, 383, 1008.
13. King, A.; Gottlieb, E.; Brooks, D. G.; Murphy, M. P.; Dunaief, J. L. Photochem.
Photobiol. 2004, 79, 470.