Synthesis and biological evaluation of dimeric cinnamaldehydes as potent antitumor agents

Article abstract of DOI:10.1016/j.bmc.2005.11.028

It has been reported that 2-hydroxycinnamaldehyde and 2-benzoyl- oxycinnamaldehyde inhibited the activity of farnesyl protein transferase, angiogenesis, cell-cell adhesion, and tumor growth in vivo model. In order to improve its anti-tumor activity, dimer

Full text of DOI:10.1016/j.bmc.2005.11.028

Bioorganic & Medicinal Chemistry 14 (2006) 2498–2506
Synthesis and biological evaluation of dimeric cinnamaldehydes
as potent antitumor agents
Dae-Seop Shin,a,b, Jong-Han Kim,a, Su-Kyung Lee,a Dong Cho Han,a Kwang-Hee Son,a
Hwan-Mook Kim,a Hyae-Gyeong Cheon,c Kwang-Rok Kim,c Nack-Do Sung,d
Seung Jae Lee,d Sung Kwon Kangd,* and Byoung-Mog Kwona,b,
*
aKorea Research Institute of Bioscience and Biotechnology, 52 Uendong Yoosung, Taejeon 305-600, Republic of Korea
bBiomolecular Science, University of Science and Technology in Korea, Taejeon 305-600, Republic of Korea
cKorea Research Institute of Chemical Technology, Taejon 305-600, Republic of Korea
dChungnam National University, Taejon 305-764, Republic of Korea
Received 30 September 2005; revised 14 November 2005; accepted 15 November 2005
Available online 15 December 2005
Abstract—It has been reported that 2-hydroxycinnamaldehyde and 2-benzoyl-oxycinnamaldehyde inhibited the activity of farnesyl
protein transferase, angiogenesis, cell–cell adhesion, and tumor growth in vivo model. In order to improve its anti-tumor activity,
dimeric cinnamaldehydes have been synthesized based on 2-hydroxycinnamaldehyde. The synthesized compounds strongly inhibited
the growth of human colon tumor cells with GI50 values of 0.6–10 lM. Especially, 2-piperazine derivative blocked in vivo growth of
human colon tumor xenograft in nude mice at 10 mg/kg. It was found that their anti-tumor effects induce apoptosis and cell cycle
arrest at G2/M phase by the compounds. It was confirmed by detection of apoptosis markers such as activated caspase-3 and cleaved
PARP, and cell cycle analysis. The dimeric compounds also inhibited Cdc25B phosphatase which is essential for preinitiating G2/M
transition and S phase progression.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
reported that HCA inhibited farnesyl protein transferase
activity,7 angiogenesis,8 and CDK4/cyclinD1 kinase.9
HCA and BCA also have been shown to inhibit the
growth of human cancer cell lines including breast, leuke-
mia, ovarian, lung, and colon tumor cells.3,6 Also, CB403,
one of cinnamaldehyde derivatives, was reported to inhib-
it tumor growth through the cell cycle arrest at G2/M
phase.10
Cinnamomum cassia Blume (Lauraceae) has been used as
a spice and tea in the whole world and considered to
have pharmacological properties.1 Oil extracted from
Cinnamomum cassia had been shown to inhibit the growth
of molds2 and exhibit cytotoxicity against human cancer
cells.3 This antitumor effect has been attributed to cinn-
amaldehydes such as 2-methoxycinnamaldehyde and
2-hydroxycinnamaldehyde (HCA). Various essential oils
of C. cassia were identified by the GC–MS analysis and
their biological properties studied.4 Also, it was reported
that cinnamaldehyde induces apoptosis by reactive oxy-
gen species (ROS)-mediated mitochondrial permeability
transition in human promyelocytic leukemia HL-60
cells.5 In our previous study, we found that the antitumor
effect by 2-benzoyloxycinnamaldehyde (BCA) was due to
induction of reactive oxygen species.6 Previously, we
Cdc25B phosphatase has been thought as a potential
human oncogene, because it is essential protein for
the G2/M phase transition in human cells.11,12 In
human cells, Cdc25 phosphatases are encoded by a
multigene family consisting of Cdc25A, Cdc25B, and
Cdc25C.13–15 Each Cdc25 phosphatase stimulates pro-
gression through the cell cycle by activating cyclin
and CDK complexes. Cdc25C dephosphorylates and
activates the mitotic kinase Cdc2/cyclin B, which is re-
quired for entry into mitosis.16 Cdc25A is important
for entry into S phase,17 whereas Cdc25B is essential
for preinitiating G2/M transition and S phase progres-
sion.12 Cdc25B is thought to function as a mitotic
starter by dephosphorylating and activating Cdk2/
cyclin A and Cdk1/cyclin B.18 Because of these reasons,
Keywords: Cinnamaldehyde; Dimeric cinnamaldehyde; Apoptosis;
Cell cycle; Cdc25B phosphatase.
*
Corresponding authors. Tel.: +82 42 860 4557; fax: +82 42 861
2675; e-mail addresses: skkang@cnu.ac.kr; kwonbm@kribb.re.kr
These authors contributed equally to this work.
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.11.028

D.-S. Shin et al. / Bioorg. Med. Chem. 14 (2006) 2498–2506
2499
identification of Cdc25B inhibitors becomes an active
area for the development of antitumor agents.
could not obtain the dimeric compound. As shown in
Scheme 1, the 2-hydroxyl group reacted with the aldehyde
carbonyl group to give a cyclic compound 22.
In this study, we evaluated the biological activity of
dimeric cinnamaldehyde as a potent anti-tumor agent.
Inhibitory activity of Cdc25B of the synthesized com-
pounds was also examined, because the compounds
arrested the cell cycle at the G2/M phase in tumor cells.
Especially, we focused on two types of dimeric cinna-
maldehyde derivatives such as 2-(4-Br-benzyl)-dimer
(11) and 2-(N-CH3-piperazine)-dimer (21).
2.1.1. X-ray structure of cinnamaldehyde dimers and a
cyclic product. The crystallized compounds 3, 17, and 22
were obtained from acetone–hexane to confirm the
structure and to see three-dimensional shape of the syn-
thesized compounds. X-ray analysis of the crystallized
compounds 3 and 17 confirmed the proposed structure
with a linear dimeric system. The X-ray structures of 3
and 17 show that the aldehyde groups are on the same
plane in the solid state (Fig. 1). X-ray analysis of the
crystallized compound 22 obtained from acetone–hex-
ane confirmed the proposed structure with a unique
fused ring system.
2. Results and discussion
2.1. Chemistry
As shown in Scheme 1, the cinnamaldehyde derivatives
are prepared by linear dimerization of benzaldehyde
substituted by R1, R2, and R3 in the presence of 2,5-di-
methoxytetrahydrofuran, potassium acetate, acetic acid,
and water (wherein, R1, R2, and R3 are described in
Table 1).19,20 The benzaldehydes substituted with R1,
R2 and, R3 are used as commercially available formula-
tion. Also, the benzaldehydes substituted with R1, R2,
and R3 are prepared by reacting alkylating agents to
benzaldehyde substituted with activating substituent
such as halogen or hydroxy group in the presence of
base. For example, 2-n-propyloxybenzaldehyde was pre-
pared by reacting 1-iodopropane (alkylating agent) to 2-
hydroxybenzaldehyde substituted with hydroxyl group
in the presence of potassium carbonate. To improve the
solubility in water, 2-(N-CH3-piperazine)-dimer (21)
was synthesized by the described method. The solubility
of compound 21 is 0.2 mg/mL in water and it is freely sol-
uble in acidic water (pH 3). We also tried to synthesize of
2-hydroxy dimeric cinnamaldehyde, unfortunately, we
2.2. Inhibition of Cdc25B activity by dimeric
cinnamaldehydes
Cdc25B isanessentialprotein for the G2/M phase transition
invarioushumancancercelllines.11,12 Therefore,inhibition
of Cdc25B causes cell cycle arrest leading to suppression of
tumor growth. In our previous study, it was shown that
cinnamaldehyde derivatives, CB403, induced cell cycle
arrest at G2/M phase.10 Therefore, we primarily examined
the inhibitory activity of Cdc25B by dimeric cinnamaldehy-
des. The dimeric cinnamaldehydes such as 4, 9, 10, and
11 strongly and selectively inhibited Cdc25B activity with
IC50 values of less than 10 lM (Table 2).
2.3. Inhibition of human colon tumor cell growth by
dimeric cinnamaldehydes
In order to investigate whether the dimeric cinnamalde-
hydes inhibit cancer cell proliferation, HCT116 and
SW620 (human colon tumor cells) were treated at
O
H
R3
H
CHO
R3
R1
CH3COOH/CH3COOK
H2O (1mL)/Reflux
R2
R2
CHO
H
R1
+
R3
R1
O
R2
H3CO
OCH3
Compound 1-21
O
H
OH
H
O
O
CH3COOH/CH3COOK
H2O (1mL)/Reflux
+
O
H3CO
OCH3
22
O
H
Scheme 1. Synthesis of dimeric cinnamaldehydes 1–21 and compound 22.19

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D.-S. Shin et al. / Bioorg. Med. Chem. 14 (2006) 2498–2506
Table 1. Synthesized dimeric cinnamaldehydes
Compound
R1
R2
R3
Mp (ꢁC)
1
2
–H
–F
–H
–H
–H
–H
150–151
141–142
177–178
194–195
163–164
109–110
117–118
109–110
120–121
186–187
210–211
116–117
107–108
93–94
3
–Cl
–Br
–H
–H
–H
–H
4
5
–OCH3
–OCH2CH2CH3
–H
–H
–H
–H
6
7
–OCH2CHCH2
–OCH(CH3)2
–H
–H
–H
–H
8
9
–OCH2C6H5
–OCH2C6H4–4-Cl
–H
–H
–H
–H
10
11
12
13
14
15
16
17
18
19
20
21
–OCH2C6H4-4-Br
–OCH2C6H4–4-NO2
–H
–H
–H
–H
–H
–H
–Cl
–H
–H
–OCH2CH2CH3
–OCH(CH3)2
–OCH2C6H5
–OCH3
–OCH3
–OCH3
–OCH3
–H
–H
–H
–H
–H
116–117
126–127
177–178
149–150
95–96
–H
–H
–OH
–OCH3
–OCH2CH2CH3
–OCH(CH3)2
–H
–H
–H
119–120
118–220
–Piperazine-N-CH3
different concentrations from 0.1 to 100 lM for 24 h
(Table 3). Both these tumor cells were inhibited by the
compounds with a GI50 value of 0.6–10 lM.
As shown in Figure 2, 2-(4-Br-benzyl)-dimer (11) and 2-
(N-CH3-piperazine)-dimer (21) exhibited a dose-depen-
dent inhibition of tumor cell growth in a broad range of
concentrations and the GI50 value of 21 for in vitro
growth inhibition was approximately 2.8 lM for
MDA-MB-231 (human breast cancer cell) and 2.1 lM
for SW620 cells. It was also found that morphology of
two cell lines was changed at the concentration of 5 lM
Table 2. Inhibitory activity of dimeric cinnamaldehydes against
phosphatase
Compound
Cdc25B
% inhibition
(at 10 lM)
% inhibition
(at 10 lM)
IC50 (lM)
Cdc25A
PTP-1B
1
2
6.70
5.99
3
39.07
56.21
1.10
11.80
25.70
2.63
5.63
4
7.79
5
6
29.95
6.26
7.97
7
8
9
60.49
89.56
95.00
1.31
7.08
4.77
1.94
8.85
6.74
1.80
2.87
7.49
10
11
12
13
14
15
16
17
18
19
20
21
19.24
28.41
22.80
2.25
55.16
5.60
9.34
14.66
11.61
1.86
9.84
3.08
Figure 1. ORTEP drawing of the X-ray structure of compounds 3, 17,
and 22.
5.33

D.-S. Shin et al. / Bioorg. Med. Chem. 14 (2006) 2498–2506
Table 3. The effects of dimeric compounds on tumor cell growth
2501
Western blot analysis of PARP and cleavage caspase-3
was performed (Fig. 5). The PARP and cleavage cas-
pase-3 was enhanced by 10 lM of 2-(N-CH3-pipera-
zine)-dimer (21) in MDA-MB-231 cells, although we
could not detect any significant increase of PARP or
caspase-3 over 10 lM (Fig. 5A left panel). However,
remarkable increase of PARP and cleavage caspase-3
was shown by 5 lM of 2-(4-Br-benzyl)-dimer (11)
(Fig. 5A right panel). Although, a trace amount of
PARP degradation and cleavage caspase-3 was observed
in 2-(N-CH3-piperazine)-dimer (21)-treated cells, these
results state that the dimeric compounds inhibited can-
cer cell growth through apoptosis.
No.
Compound
HCT116
GI50 (lM)
SW620
GI50 (lM)
1
2
H
2-F
8.39
4.02
0.91
1.05
3.72
5.28
5.28
5.34
1.41
0.84
2.63
5.19
3.62
2.74
4.23
7.59
7.87
5.49
0.62
0.71
3.88
1.52
10.73
0.91
2.14
1.55
2.11
2.64
5.34
0.63
0.54
4.64
7.09
9.21
6.74
0.61
3.58
3.53
10.46
2.28
0.91
6.22
3
2-Cl
2-Br
4
5
2-OCH3
6
2-n-Propyl
2-i-Propyl
2-Allyl
7
8
9
2-Benzyl
10
11
12
13
14
15
16
17
18
19
20
21
2-(4-Cl-benzyl)
2-(4-Br-benzyl)
2-(4-NO2-benzyl)
3-Cl
2.6. In vivo effect of 2-(N-CH3-piperazine)-dimer 21 on
tumor cell growth
3-n-Propyl
3-i-Propyl
3-Benzyl
SW620 tumor xenograft model of nude mice was used to
investigate the inhibitory activity of the compound 21
on tumor growth. SW620 cells were implanted subcuta-
neously into the right flank of nude mice on day 0 and
compound was intraperitoneously administered at a
concentration of 10 mg/kg per day for 20 days. To deter-
mine the toxicity of the compound, the body weight of
tumor-bearing animals was measured. The mice were
sacrificed and the tumors were weighed on day 20.
Tumor volume was decreased by 45.6% compared to
control mice. Twenty days after implantation (Fig. 6),
tumor weights were also decreased by 50.2% compared
to control mice in SW620 cells. Body weight loss was
not observed in mice implanted with SW620 cells (data
not shown).
4-OH, 5-OCH3
3,4-OCH3
3-OCH3, 4-n-propyl
3-OCH3, 4-i-propyl
2-Piperazine-N-CH3
of 2-(4-Br-benzyl)-dimer (11) and 2-(N-CH3-piperazine)-
dimer (21) (Fig. 3). Unfortunately, new cyclic compound
22 did not inhibit the growth of human tumor cells.
2.4. Effects of 2-(N-CH3-piperazine)-dimer 21 on the cell
cycle of cancer cells
To determine the effects of 2-(N-CH3-piperazine)-dimer
(21) on the cell cycle, after treatment of the compound
(5 lM) in SW620 cells, the cells were harvested and ana-
lyzed with a FACScalibur. As shown in Figure 4. 2-(N-
CH3-piperazine)-dimer (21) arrested the progression of
the cells at G2/M phase.
3. Conclusion
Cinnamaldehydes have been known to have antitumor
activity.5,6 However, the mechanism is still in debate.
In our previous study, we reported that cinnamaldehyde
derivatives have an inhibitory activity against FPTase7
and induce reactive oxygen species6 that may (at least
in part) explain the mechanism by which cinnamaldehy-
des exhibit their antitumor activity. In this study, we
showed that growth inhibition of tumor cells was in-
2.5. 2-(4-Br-benzyl)-dimer (11) and 2-(N-CH3-pipera-
zine)-dimer (21) induce apoptosis in MDA-MB-231
and SW620 cells
To determine whether the growth inhibition by two
dimeric cinnamaldehydes was associated with apoptosis,
B
A
120
100
80
60
40
20
0
120
Compound 21
Compound 11
Compound 21
Compound 11
100
80
60
40
20
0
0.1
1
10
100
0.1
1
10
100
Conc. (μM)
Conc. (μM)
Figure 2. Growth inhibition of cancer cells by 2-(4-Br-benzyl)-dimer (11) and 2-(N-CH3-piperazine)-dimer (21). Compounds were treated in MDA-
MB-231 (A) and SW620 (B) cell line with different concentrations of the compounds. Viability was determined by WST-1 at 24 h after compound
treatment.

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D.-S. Shin et al. / Bioorg. Med. Chem. 14 (2006) 2498–2506
5 uM
10 uM
20 uM
A
Compound 21
Compound 11
Con
5 uM
10 uM
20 uM
5 uM
5 uM
10 uM
10 uM
20 uM
20 uM
B
Compound 21
Compound 11
Con
Figure 3. Morphology change by compound 11 and 21. MDA-MB-231 (A) and SW620 (B) cells were imaged by Nikon fluorescence microscope after
24 h from compound treatment.
Control (DMSO)
2-(N-CH3-piperazine)-dimer
Figure 4. Effects of 2-(N-CH3-piperazine)-dimer (21) on cell cycle. SW620 cells were treated with 2-(N-CH3-piperazine)-dimer (21). Cells were
harvested after 24 h and were subjected to FACScalibur analysis to determine the distribution of cells through the G1, S, and G2/M phases.
Experiments were performed at least three times with consistent and repeatable results.
duced by the dimeric cinnamaldehydes through PARP
degradation via caspase-3 pathway or cell cycle arrest in
G2/M phase. A SW620 tumor xenograft model of nude
mice was used to investigate the inhibitory activity on
tumor growth by 21, which was intraperitoneously
administered at concentrations of 10 mg/kg from day 1
to day 20. As shown in Fig. 6, compound 21 inhibited
the growth of tumors and also significantly reduced the
weight of tumors. A promising aspect of 21 is apparent
lack of obvious toxicity in tumor-bearing nude mice.
Therefore, the dimeric cinnamaldehyde derivative exerts
antitumor effects by inducing G2/M phase arrest in cancer
cells and also apoptosis of tumor cells. The growth
inhibitory activity of the compounds in cells and animal
studies supports their promise as anti-tumor compounds.
4. Experimental
4.1. Materials and methods
NMR spectra were measured at 300 and 400 MHz
(1H and 13C), and chemical shifts are reported rela-
tive to internal Me4Si. Melting points are reported
in degrees Celsius and are uncorrected. Thin-layer
chromatography was effected on silica gel 60 F254
(layer thickness 0.2 mm), and components were locat-
ed by observation under UV light and/or by treating
the plates with a p-anisaldehyde reagent followed by
heating. Flash chromatography was performed on
Merck silica gel, 60 and 230–400 mesh by StillÕs
methods.20

D.-S. Shin et al. / Bioorg. Med. Chem. 14 (2006) 2498–2506
2503
A
2-(N-CH3-piperazine)-dimer
Con 10 20
2-(4-Br-benzyl)-dimer
Con 10 20 M
1
2
5
M
1
2
5
116 KDa
89 KDa
PARP
cleavage
19 KDa
17 KDa
-caspase3
-actin
B
2-(N-CH3-piperazine)-dimer
Con 10 20
2-(4-Br-benzyl)-dimer
Con 10 20 M
1
2
5
M
1
2
5
116 KDa
89 KDa
PARP
cleavage
-caspase3
19 KDa
17 KDa
-actin
Figure 5. Induction of PARP and cleavage caspase-3 by dimeric cinnamaldehydes. 2-(4-Br-benzyl)-dimer (11) and 2-(N-CH3-piperazine)-dimer (21)
were treated in MDA-MB-231 and SW620 cells. Cell lysates were prepared with RIPA buffer after 24 h and 40 lg of lysates was resolved by
SDS–PAGE. Western blot analysis of PARP and cleaved caspase-3 was carried with each MDA-MB-231 (A) or SW620 cell lysate (B).
flux for 12 h. After cooling, the mixture is poured into
Vehicle (water
)
250
200
150
100
50
water. The mixture is extracted with chloroform, the ex-
tract is washed with water, dried (MgSO4), and evapo-
rated, which was purified by column chromatography.
compound 21
1
Yield = 13%, H NMR (CDCl3/TMS): d 7.26–7.53 (m,
10H), 7.71 (s, 2H), 9.67 (s, 2H).
4.2.2. 2,3-Bis-(2-fluorobenzylidene)succinaldehyde (2).
1
Yield = 10%, H NMR (CDCl3/TMS): d 7.0 (m, 4H),
7.29 (m, 4H), 7.88 (s, 2H), 9.69 (s, 2H).
4.2.3. 2,3-Bis-(2-chlorobenzylidene)succinaldehyde (3).
1
Yield = 14%, H NMR (CDCl3/TMS): d 7.13–7.28 (m,
8H), 7.83 (s, 2H), 9.73 (s, 2H).
0
4.2.4. 2,3-Bis-(2-bromobenzylidene)-succinaldehyde (4).
Yield = 10%, H NMR (CDCl3/TMS) d 7.12–7.46 (m,
8H), 7.73 (s, 2H), 9.74 (s, 2H).
1
0
4
6
8
10 12
14
16
18
20
Days after implantation
Figure 6. Tumor volume change of SW620 xenografted nude mice
treated with compound 21. For the evaluation of in vivo antitumor
activity of compound 21, SW620 (0.3 mL of 3 · 107 cells/mL) was
implanted subcutaneously into the right flank of nude mice on day 0.
Tumor volumes were estimated by the formula length (mm) · width
(mm) · height (mm)/2. Compounds were dissolved in water and
intraperitoneally administered at the concentration of 10 mg/kg per
day from day 1 to 20. Error bar is standard deviation.
4.2.5. 2,3-Bis-(2-methoxybenzylidene)succinaldehyde (5).
1
Yield = 15%, H NMR (CDCl3/TMS): d 3.85 (s, 6H),
6.76 (m, 4H), 7.5 (m, 2H), 7.4 (dd, 2H, J = 1.2,
7.5 Hz), 8.07 (s, 2H), 9.65 (s, 2H).
4.2.6. 2,3-Bis-(2-n-propyloxybenzylidene)succinaldehyde
(6). A solution of 6.1 g (50 mmol) of 2-hydroxybenzalde-
hyde in 50 mL of acetonitrile was stirred with K2CO3
(60 mmol, 8.28 g) and 1-iodopropane (55 mmol, 9.35 g)
under reflux, and the progress of the reaction was mon-
itored by TLC. When TLC indicated the disappearance
of starting material, the reaction was stopped. The reac-
tion mixture was then cooled to room temperature,
quenched with water, and extracted with ethyl acetate.
The organic layer was washed with water, dried over
4.2. Synthesis of dimeric cinnamaldehydes
4.2.1. 2,3-Bis-benzylidenesuccinaldehyde (1). A mixture
of 2,5-dimethoxytetrahydrofuran (2 mL, 15 mmol),
benzaldehyde (30 mmol), acetic acid (1 mL), water
(1 mL), and potassium acetate (2 g) was heated under re-

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D.-S. Shin et al. / Bioorg. Med. Chem. 14 (2006) 2498–2506
anhydrous MgSO4, filtered, and concentrated. Purifica-
tion by column chromatography over silica gel afforded
the corresponding products 6. Yield = 10%, H NMR
(CDCl3/TMS): d 1.08 (t, 6H, J = 7.5 Hz), 1.86 (m,
4H), 3.96 (m. 4H), 6.76 (m, 2H), 6.83 (d, 2H,
J = 7.5 Hz), 7.26 (m, 2H), 7.42 (dd, 2H, J = 1.2,
7.5 Hz), 8.12 (s, 2H), 9.66 (s, 2H).
4.2.17. 2,3-Bis-(4-hydroxy-3-methoxybenzylidene)succin-
aldehyde (17). Yield = 10%, 1H NMR (CDCl3/TMS)
(ppm); d 3.75 (s, 6H), 5.92 (s, 2H), 7.10 (m, 6H), 7.63
(s, 2H), 9.63 (s, 2H).
1
4.2.18. 2,3-Bis-(3,4-dimethoxybenzylidene)succinaldehyde
1
(18). Yield = 15%, H NMR (CDCl3/TMS): d 6.73 (s,
6H), 3.88 (s, 6H), 6.82 (d, 2H, J = 8.1 Hz), 7.11 (d, 2H,
J = 1.8 Hz), 7.19 (dd, J = 1.8, 8.1 Hz), 7.65 (s, 2H), 9.65
(s, 2H).
4.2.7. 2,3-Bis-(2-allyloxybenzylidene)succinaldehyde (7).
Yield = 10%, H NMR (CDCl3/TMS): d 4.57 (m, 4H),
1
5.36 (m, 4H), 6.07 (m, 2H), 6.7 (m, 4H), 7.25 (dt, 2H,
J = 1.5, 8.1 Hz), 7.41 (dd, 2H, J = 1.5, 8.1 Hz), 8.11 (s,
2H), 9.67 (s, 2H).
4.2.19. 2,3-Bis-(3-methoxy-4-n-propyloxybenzylidene)suc-
cinaldehyde (19). Yield = 75%, H NMR (CDCl3/TMS):
1
d 1.01 (t, 6H, J = 7.2 Hz), 1.84 (m, 4H), 3.70 (s, 6H), 3.97
(t, 4H, J = 6.6 Hz), 6.80 (d, 2H, J = 8.4 Hz), 7.11 (d, 2H,
J = 1.8 Hz), 7.19 (dd, J = 1.8, 8.4 Hz), 7.63 (s, 2H), 9.64
(s, 2H).
4.2.8. 2,3-Bis-(2-isopropyloxybenzylidene)succinaldehyde
(8). Yield = 8%, 1H NMR (CDCl3/TMS): d 1.38 (t, 12H,
J = 6.0 Hz), 4.58 (m. 2H), 6.75 (t, 2H, J = 7.8 Hz), 6.86
(d, 2H, J = 7.8 Hz), 7.26 (dt, 2H, J = 1.8, 7.8 Hz), 7.43
(dd, 2H, J = 1.8, 7.8 Hz), 8.11 (s, 2H), 9.66 (s, 2H).
4.2.20. 2,3-Bis-(3-methoxy-4-isopropyloxybenzylidene)suc-
cinaldehyde (20). Yield = 80%, 1H NMR (CDCl3/TMS): d
1.36 (d, 12H, J = 6.0 Hz), 3.69 (s, 6H), 4.57 (m, 2H), 6.82
(d, 2H, J = 8.4 Hz), 7.11 (d, 2H, J = 2.1 Hz), 7.15 (dd,
2H, J = 2.1, 8.4 Hz), 7.63 (s, 2H), 9.64 (s, 2H).
4.2.9. 2,3-Bis-(2-benzyloxybenzylidene)succinaldehyde (9).
1
Yield = 10%, H NMR (CDCl3/TMS): d 5.09 (dd, 4H,
J = 1.2, 9.0 Hz), 6.78 (t, 2H, J = 7.8 Hz), 6.88 (d, 2H,
J = 7.8 Hz), 7.26 (dt, 2H, J = 1.8, 7.8 Hz), 7.26 (m, 12H),
8.13(s, 2H), 9.65 (s, 2H).
4.2.21.
2,3-Bis-(2-(4-N-methyl)-piperazine)succinalde-
hyde (21). 2-Fluorobenzaldehyde (42.16 mL, 400 mmol)
was added to the 250 mL round-bottomed flask, 100 mL
of dimethylformamide (DMF) was further added there-
in. Thereafter, 8.28 g of potassium carbonate (60 mmol)
and 44.4 mL of N-methylpiperazine (400 mmol) were
added therein. The reaction mixture was refluxed at
150 ꢁC for 10 hr. After the reaction was terminated,
the reaction mixture was cooled to room temperature.
Thereafter, 400 mL of water was added to the reaction
mixture. The reaction mixture was extracted with
200 mL of ethyl acetate three times. The ethyl acetate
layers were collected, washed with water three times,
and dried with anhydrous magnesium sulfate. The ethyl
acetate was removed under reduced pressure. The result-
ing residue was purified with column chromatography,
to give 2-(4-N-methylpiperazine)benzaldehyde.
4.2.10. 2,3-Bis-{2-(4-chlorobenzyloxy)benzylidene}succin-
aldehyde (10). Yield = 8%, H NMR (CDCl3/TMS); d
5.02 (dd, 4H, J = 1.2, 9.0 Hz), 6.73 (t, 2H, J = 7.8 Hz),
6.78 (d, 2H, J = 7.8 Hz), 7.32 (m, 14H), 8.01 (s, 2H),
9.56 (s, 2H).
1
4.2.11. 2,3-Bis-{2-(4-bromobenzyloxy)benzylidene}succin-
1
aldehyde (11). Yield = 15%, H NMR (CDCl3/TMS): d
5.07 (dd, 4H, J = 1.2, 9.0 Hz), 6.82 (m, 4H), 7.32
(m, 8H), 7.41 (dd, J = 1.8, 7.8 Hz), 8.08 (s, 2H), 9.63
(s, 2H).
4.2.12. 2,3-bis-{2-(4-nitrobenzyloxy)benzylidene}succinal-
dehyde (12). Yield = 9%, 1H NMR (CDCl3/TMS): d 5.31
(s, 4H), 7.01 (d, 2H, 7.5 J = 7.5 Hz), 7.10 (t, 2H,
J = 7.5 Hz), 7.55 (dt, 2H, J = 1.5, 7.5 Hz), 7.63 (d, 4H,
7.7 Hz), 7.88 (dd, 2H, J = 1.5, 7.5 Hz), 8.27 (d, 4H,
7.7 Hz), 10.56 (s, 2H).
2-(N-CH3-piperazine)benzaldehyde (4.5 g, 30 mmol)
prepared in the above step was dissolved in 250 mL of
round-bottomed flask. Thereafter, 2 mL of 2,5-dim-
ethoxytetrahydrofuran (15 mmol), 2 g of potassium ace-
tate (20 mmol), 1 mL of acetic acid (16 mmol), and 1 mL
water were added to the flask. The reaction mixture was
refluxed at 110 ꢁC for 12 h. After the reaction was termi-
nated, the reaction mixture was cooled to room temper-
ature. Thereafter, water was added to the reaction
mixture. The reaction mixture was extracted with
100 mL chloroform for three times. The chloroform lay-
ers were collected, washed with water for three times,
and dried with anhydrous magnesium sulfate. The chlo-
roform was removed under reduced pressure. The result-
ing residue was purified with column chromatography,
to give the title compound as a yellow crystal (4.5 g,
yield: 65%). 1H NMR (CDCl3/TMS): d 2.25 (s, 6H,
OCH3), 2.40–2.85 (br d 16H), 6.94 (dt, 2H, J = 1.2,
7.5 Hz), 7.04 (dd, 2H, 2H, J = 1.2, 7.5 Hz), 7.22 (dd,
2H, J = 1.2, 7.8 Hz), 7.32 (dt, 2H, J = 1.2, 7.5 Hz),
7.92 (s, 2H), 9.70 (s, 2H).
4.2.13. 2,3-Bis-(3-chlorobenzylidene)succinaldehyde (13).
Yield = 15%, 1H NMR (CDCl3/TMS): d 7.24 ꢀ 7.42 (m,
8H), 7.64 (s, 2H), 9.67 (s, 2H).
4.2.14. 2,3-Bis-(3-n-propyloxybenzylidene)succinaldehyde
(14). Yield = 10%, 1H NMR (CDCl3/TMS): d 0.99 (t, 6H
J = 7.2 Hz), 1.76 (m, 4H), 3.81 (dt, 4H, J = 1.2, 6.6 Hz),
7.22 (m, 8H), 7.67 (s, 2H), 9.65 (s, 2H).
4.2.15. 2,3-Bis-(3-isopropyloxybenzylidene)succinaldehyde
1
(15). Yield = 10%, H NMR (CDCl3/TMS): d 1.27 (dd,
12H, J = 5.7, 15.4 Hz), 4.39 (m, 2H), 7.16 (m, 8H), 7.66
(s, 2H), 9.65 (s, 2H).
4.2.16.
2,3-Bis-(3-benzyloxybenzylidene)succinaldehyde
1
(16). Yield = 10%, H NMR (CDCl3/TMS): d 4.97 (s,
4H), 7.01 (m, 6H), 7.25 (m, 12H), 7.51 (s, 2H), 9.48 (s,
2H).

D.-S. Shin et al. / Bioorg. Med. Chem. 14 (2006) 2498–2506
2505
4.2.22. Cyclic compound 22. Yield = 20%, 1H NMR
(CDCl3/TMS): d 5.63 (s, 1H), 7.1–7.4 (m, 8H), 7.51
(dd, 1H, J = 1.2, 6.0 Hz), 7.75 (s, 1H), 9.79 (s, 1H).
10 lg/mL leupeptin, 100 lg/mL AEBSF, and 10 mM
DTT. The fusion protein was eluted with three succes-
sive washes using 10 mM glutathione in 2· reaction
buffer. Active fractions were pooled and supplemented
with 20% glycerol prior to storage at À80 ꢁC (Brisson
et al., 2004).22
4.3. X-ray structure
The data for X-ray structure determination were collect-
ed on Bruker P4 and CAD-4 diffractometers equipped
with graphite monochromated Mo Ka radiation
4.6. Dual-specific Cdc25B and phosphatase assay22
˚
(k = 0.71073 A) at 295 K. The unit cell dimensions were
The activity of the GST fusion Cdc25B or A phospha-
tase was measured with FDP (Molecular Probes, Inc.,
Eugene, OR), which is readily metabolized to the fluo-
rescent fluorescein monophosphate, as a substrate in a
96-well microtiter plate. The final incubation mixture
(150 lL) comprised of 30 mM Tris (pH 8.5), 75 mM
NaCl, 0.67 mM EDTA, 0.033% bovine serum albumin,
1 mM DTT, and 20 lM FDP for Cdc25B or A phos-
phatase. Inhibitors were resuspended in DMSO, and
all reactions including controls were performed at a
final concentration of 7% DMSO. Reactions were
initiated by adding ꢀ0.25 lg of fusion protein and
incubated at ambient temperature for 1 h for Cdc25B
phosphatase. Fluorescence emission from the product
was measured with a multiwell plate reader (Perseptive
Biosystems Cytofluor II, Framingham, MA; excitation
filter, 485/20; emission filter, 530/30).
determined on the basis of 36 reflections in the range of
5.15ꢁ < h < 12.56ꢁ for compound 3, 25 reflections in the
range of 9.86ꢁ < h < 13.97ꢁ for compound 17, and 23
reflections in the range of 11.41ꢁ < h < 12.67ꢁ for com-
pound 22. The data were collected by the x/2h scan
mode. The standard direct method was used to position
the heavy atoms. The remaining non-hydrogen atoms
were located from the subsequent difference Fourier syn-
thesis. All non-hydrogen atoms were refined anisotropi-
cally. All hydrogen atoms were calculated in ideal
positions and were riding on their respective carbon
atoms (Biso = 1.2Beq and Biso = 1.5Beq). The structure
was refined in a full matrix least-squares calculation
on F2. Programs used to solve and refine the structure
were: SHELXS97 and SHELXL97. Molecular graphics;
Ortep-3 for windows.21
Crystallographic data for the structures reported here
have been deposited with the Cambridge Crystallo-
graphic Data Centre [Deposition Nos. CCDC-280899
for 2-Cl-dimer (3), CCDC-280901 for 3-MeO-4-OH di-
mer (17), and CCDC-280900 for 22]. The data can be
obtained free of charge via www.ccdc.cam.ac.uk/perl/
catreq/catreq.cgi (or from the CCDC, 12 Union Road,
Cambridge CB2 1EZ, UK; Fax: +44-1223 336033;
E-mail: deposit@ccdc.cam.ac.uk).
4.7. PTP-1B enzyme assay
In order to carry out PTP-1B counterscreenig, the
cDNA encoding the catalytic domain of PTP-1B (resi-
dues 1–322) was cloned into the NdeI site of pET14b
and expressed in Escherichia coli BL21. After overnight
culture with 0.1 mM IPTG for induction, the histidine-
tagged PTP-1B fusion protein was purified from bacte-
rial lysates by using a nickel-chelated affinity column.
The enzyme assay for PTP-1B was carried out on 96-
well plates. To each well (final volume: 200 mL) were
added 20 AM FDP and 0.1 Ag PTP-1B in a buffer con-
taining 30 mM Tris (pH 8.0), 75 mM NaCl, 0.67 mM
EDTA and, 1 mM dithiothreitol with or without test
compounds. Following incubation at room temperature
for 1 h, the fluorescence released by enzyme catalysis
was measured at 485 nm (excitation) and 538 nm (emis-
sion) by using a fluorometer (Synergy HT, BioTek).
4.4. Cell culture
MDA-MB-231 (a human breast cancer) and SW620 (a
human colon cancer) cell line were originally purchased
from ATCC and maintained in RPMI 1640 (Gibco/
BRL) containing 10% heat-inactivated fetal bovine ser-
um (hyclone) and 25 mM HEPES. Cells were grown in
37 ꢁC incubator maintained 5% CO2 under humidified
condition and subcultured into a new cell culture dishes
per 72 h after detaching the cells with trypsin-EDTA
(Gibco/BRL).
4.8. Cell proliferation assays
Cells (5000 cells/well) were seeded into 96-well plates in
RPMI 1640 containing 10% FBS. After 24 h, each com-
pound diluted with DMSO was treated to each well. Cell
proliferation reagent, WST-1 (Roche Applied Science),
was added after 24 h. The plate was incubated in
37 ꢁC incubator for 2 h and absorbance was measured
at 450 nm using an ELISA Reader (Bio-Rad).
4.5. Purification of GST fusion proteins
The bacterial pellet was disrupted by sonication at 4 ꢁC
in lysis buffer containing 10 lg/mL aprotinin, 10 lg/mL
leupeptin, 100 lg/mL AEBSF, and 10 mM DTT. The
homogenate was then centrifuged for 10 min at 4 ꢁC
at 10,000g. The resulting supernatant fraction was
immediately mixed and rotated with glutathione beads
(equilibrated with lysis buffer) for 1 h at 4 ꢁC (5 vol of
supernatant/1 vol of 50% bead slurry). The glutathione
beads were washed two times with 10 vol of lysis buffer
and then twice with 10 vol of 2· reaction buffer
(60 mM Tris, pH 8.5, 150 mM NaCl, 1.34 mM EDTA,
and 0.066% BSA) containing 10 lg/mL aprotinin,
4.9. Cell cycle analysis
Cells were trypsinized at the time after compound treat-
ment and gathered by centrifugation at 300g for 5 min at
room temperature. Supernatant was discarded and
precipitated cells were washed with phosphate-buffered
saline. Cells were fixed with ice-cold 70% ethanol

2506
D.-S. Shin et al. / Bioorg. Med. Chem. 14 (2006) 2498–2506
overnight. Fixed cells were centrifuged at 300g for 5 min
at room temperature and washed twice with phosphate-
buffered saline. Cells were diluted with PBS solution
(1 · 105 cells/100 lL) and treated with 100 lg/mL
RNase A at 37 ꢁC for 30 min. For DNA staining,
50 lg/mL of propidium iodide was added to the cells.
Analysis was carried by 20,000 cells with FACScalibur
(Becton Dickinson). Cell cycle distribution was analyzed
by ModifitÕs program (Becton Dickinson).
(CBM1-B500-001-1-00) of the 21st Century Frontier
Research Program.
References and notes
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K.; Kwon, B. M.; Kim, H. M. Planta Med. 1999, 65,
263–266.
4.10. Western blotting
4. Choi, J.; Lee, K. T.; Ka, H.; Jung, W. T.; Jung, H. J.;
Park, H. J. Arch. Pharm. Res. 2001, 24, 418–423.
5. Ka, H.; Park, H. J.; Jung, H. J.; Choi, J. W.; Cho, K. S.;
Ha, J.; Lee, K. T. Cancer Lett. 2003, 196, 143–152.
6. Han, D. C.; Lee, M. Y.; Shin, K. D.; Jeon, S. B.; Kim, J.
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Cell lysates treated with chemicals were prepared by
RIPA lysis buffer (50 mM Tris, pH 7.0, 150 mM NaCl,
5 mM EDTA, 1% Triton X-100, 1% deoxycholic acid,
0.1% SDS, 30 mM Na2HPO4, 50 mM NaF, and 1 mM
NaVO4) containing protease inhibitor cocktail (Roche
Applied Science). Proteins (40 lg) were resolved by 7.5
or 10% SDS–PAGE and transferred to PVDF mem-
brane (Roche Applied Science). The membrane were
blocked with 5% nonfat dried milk in TBS-T (50 mM
Tris–HCl, pH 7.6, 150 mM NaCl, and 0.1% Tween 20)
and probed with primary antibodies (Cell Signaling
Technology) for 2–2.5 h. The blot was washed, exposed
to HRP-conjugated anti-rabbit IgG for 1.5 h, and exam-
ined by chemiluminescence POD reagents (Roche
Applied Science).
4.11. Nude-mouse xenograft assay
For the evaluation of compound 21 for antitumor
activity in vivo, SW620 human colon adenocarcinoma
cells (3 · 107 cells/mL) were implanted subcutaneously
into the right flank of nude mice on day 0. Compound
was dissolved in acidic water (pH 3) and was intra-
peritoneously administered at
a concentration of
10 mg/kg per day for 20 days. Tumor volumes were
estimated as: length (mm) · width (mm) · height
(mm). To determine the toxicity of the compound,
the body weight of tumor-bearing animals was mea-
sured. The mice were sacrificed and the tumors were
weighed at day 20.
17. Jinno, S.; Suto, J.; Nagata, A.; Igarashi, M.; Kanaoka, Y.;
Nojima, H.; Okayama, H. EMBO J. 1994, 13, 1549–1556.
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20. El Gendy, A. M.; Mallouli, A.; Lepage, Y. Synthesis 1980,
898–899.
Acknowledgments
21. Sheldrick, G. M. SHELXS97 and SHELXL97, Program
for the Refinement of Crystal Structures, University of
Go¨ttingen, Germany, 1997.
22. Brisson, M.; Nguyen, T.; Vogt, A.; Yalowich, J.; Giorgi-
anni, A.; Tobi, D.; Bahr, I.; Stephenson, C. R. J.; Wipf, P.;
Lazo, J. S. Mol. Pharm. 2004, 66, 824–833.
This research was financially supported by grants from
Plant Diversity Research Center of 21st Century Fron-
tier Research Program, KRIBB Research Initiative
Program, the National Chemical Genomics Research
Program, and Center for Biological Modulators