Synthetic 2′,5′-dimethoxychalcones as G2/M arrest-mediated apoptosis-inducing agents and inhibitors of nitric oxide production in rat macrophages

Article abstract of DOI:10.1016/j.ejmech.2006.12.009

In an effort to develop novel antitumor or chemopreventive agents, a series of 2′,5′-dimethoxychalcone derivatives were prepared by Claisen-Schmidt condensation of appropriate acetophenones with suitable aromatic aldehyde. In vitro screening revealed low micromolar activity (IC₅₀) against several human cancer lines. Activity in MCF-7 cells correlated with the ability to induce G₂/M arrest-mediated apoptosis following drug treatment by the most potent agent, 8, an observation further reinforced by fluorescence microscopy. Compounds 3, 8, and 10 showed potent inhibitory effect on NO production in lipopolysaccharide (LPS)-activated RAW 264.7 macrophage-like cells. The present results demonstrated that 3, 8, and 10 are potential anti-inflammatory and cancer chemopreventive agents.

Full text of DOI:10.1016/j.ejmech.2006.12.009

European Journal of Medicinal Chemistry 42 (2007) 660e668
http://www.elsevier.com/locate/ejmech
Original article
Synthetic 20,50-dimethoxychalcones as G2/M arrest-mediated
apoptosis-inducing agents and inhibitors of nitric oxide
production in rat macrophages
Bai-Luh Wei a, Chi-Huang Teng b, Jih-Pyang Wang c, Shen-Jeu Won d, Chun-Nan Lin b,
*
a Institute of Life Science, National Taitung University, Taitung 950, Taiwan
b School of Pharmacy, Kaohsiung Medical University, 100 Shih-Chuan 1st Rd, Kaohsiung 807, Taiwan
c Department of Education and Research, Taichung Veterans General Hospital, Taichung 407, Taiwan
d Department of Microbiology and Immunology, National Cheng Kung University, Tainan 701, Taiwan
Received 16 August 2006; received in revised form 28 November 2006; accepted 5 December 2006
Available online 9 January 2007
Abstract
In an effort to develop novel antitumor or chemopreventive agents, a series of 20,50-dimethoxychalcone derivatives were prepared by
ClaiseneSchmidt condensation of appropriate acetophenones with suitable aromatic aldehyde. In vitro screening revealed low micromolar
activity (IC50) against several human cancer lines. Activity in MCF-7 cells correlated with the ability to induce G2/M arrest-mediated apoptosis
following drug treatment by the most potent agent, 8, an observation further reinforced by fluorescence microscopy. Compounds 3, 8, and 10
showed potent inhibitory effect on NO production in lipopolysaccharide (LPS)-activated RAW 264.7 macrophage-like cells. The present results
demonstrated that 3, 8, and 10 are potential anti-inflammatory and cancer chemopreventive agents.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: 20,50-Dimethoxychalcones; G2/M arrest; Apoptosis; NO; Anti-inflammatory effect
1. Introduction
chalcone, 20,50-dimethoxy-4-hydroxychalcone (2), showed po-
tent inhibitory effect on NO accumulation from RAW 264.7
cells, and manifested potent selective cytotoxicity against hu-
man MCF-7 cells and caused cell death by apoptosis [5].
These findings suggested that some chalcones might be prom-
ising antitumor or cancer chemopreventive agents and have
potential in the therapy of tumor. This report described the
chemistry of further synthesized series of 20,50-dimethoxychal-
cone derivatives, cytotoxic activity, anti-inflammatory activity
and the structureecytotoxic or anti-inflammatory activity rela-
tionships of these series of novel antitumor or cancer chemo-
preventive agents.
Chalcones have displayed a variety of biological activities,
among which anti-malaria, anti-protozoal, anti-inflammatory,
immunomodulatory, nitric oxide inhibition, tyrosinase inhibi-
tion, cytotoxic, anticancer, and antileishmanial activities
have been cited in the literature [1e3]. A series of synthetic
chalcones were reported as antimitotic agents [4]. One of
these,
(E )-1-(2,5-dimethoxyphenyl)-3-[4-(dimethylamino)-
phenyl]-2-methyl-2-propen-1-one) (1), was reported to be an
effective antimitotic agent at a concentration of 4 mM in an
in vitro Hela cell test system [4]. Our recent report has dem-
onstrated that some synthetic chalcone derivatives showed se-
lective cytotoxicity against MCF-7 cells [4]. One of the
2. Chemistry
As depicted in Scheme 1 for the specific synthesis of new
chalcone 8, we have prepared a number of new 20,50-dimethox-
ychalcones (3e10) using ClaiseneSchmidt condensation of
* Corresponding author. Tel.: þ886 7 3121101-9x2163; fax: þ886 7
5562365.
E-mail address: lincna@cc.kmu.edu.tw (C.-N. Lin).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.12.009

B.-L. Wei et al. / European Journal of Medicinal Chemistry 42 (2007) 660e668
661
OMe
OMe
a
S
+
CH3
CH3
OHC
S
OMe
O
O
OMe
8a
8b
8
Scheme 1. (a) Reagents and condition: BaOH, 8H2O, 40 ꢁC, HCl.
20,50-dimethoxyacetophenone (8a) with appropriate aromatic
aldehyde (8b) [5]. For the synthesis of new chalcones 12e
14 using compound 2 reacted with appropriate cinnamoyl
chloride. New chalcone 11 was prepared as depicted in
Scheme 2. These reactions afforded various 20,50-dimethoxy-
chalcones in a good yield except for compounds 11 and 14
(Table 1).
group at C-4 of 2 by cinnamyl chloride or cinnamoyl chloride
did not enhance the cytotoxicity against the cell lines used in
Table 2.
It has been recognized that apoptotic cells reduced DNA
stainability with a variety of fluorochromes [7]. The appear-
ance of cells with low DNA stainability forms a ‘‘sub-G1
peak’’, which has been considered to be the hallmark of all
death by apoptosis [8]. In this study, various concentrations
of 8 were added to MCF-7 cells for different time periods.
As shown in Fig. 1, a sub-G1 peak was detected in the DNA
histograms of 8 at various concentrations for different time pe-
riods. The shift of G0/G1 and S phase cell cycles to the G2/M
phase is decreased dose dependently in the MCF-7 cell treated
by 8 for different time periods, while a significant G2/M
accumulation was observed at 24 h and 48 h time periods,
respectively, following the administration of 8. However,
a maximum of 63.79 ꢀ 5.1% apoptosis cells were detected
at 72 h. It seems that treatment with 8 was associated with ac-
cumulation of cells at G2/M which in part appeared to be due
to mitotic arrest prior to metaphase.
Within various time periods, Hoechst 33258 staining of cells
exposed to various concentrations of 8 demonstrated typical
morphological changes of apoptosis (Fig. 2). The cell shrunk,
turned round, and had a relatively smaller volume than control
cells. Chromatin DNA was extensively condensed and DNA
was fragmented. The typical morphological and biochemical
features clearly revealed that 8 causes cell death by apoptosis.
Previously we report compound 2 revealed the shift of G0/G1
and G2/M cell cycles to the sub-G1 phase and caused cell death
by apoptosis [5]. The B ring of 2 replaced by a 2-(5-methyl)-
thienyl moiety, such as 8, may induce G2/M phase arrest and
caused cell death by apoptosis. Further experiments are needed
to elucidate the mechanism of action of 8. The anti-inflamma-
tory activities of randomly selected compounds 3, 5, 7, 8, 10,
3. Biological results and discussion
Evaluation of the growth-inhibitory properties of 20,50-di-
methoxychalcones was undertaken in four human cancer cell
lines, A549 (lung), Hep 3B (liver), HT-29 (colorectal), and
MCF-7 (breast), using a MTT microassay for cytotoxicity
and following the method described previously [6]. The results
are listed in Table 2. A number of compounds indicated signif-
icant inhibitory activities against several cancer cell types. All
compounds in Table 2 showed selective cytotoxicity against
MCF-7 cells except for compounds 6 and 8 which showed sig-
nificant activity against all the cell lines used in Table 2. Com-
pounds with a thienyl moiety revealed significant cytotoxic
activity and showed stronger cytotoxic activity than those of
compounds with a furfuryl moiety except for compound 9.
Compound with a 3-thienyl moiety, such as 6, indicated stron-
ger cytotoxic activity than that of compound with a 2-thienyl
moiety such as 5. A methyl group substituted at C-5 of com-
pound with a 2-thienyl moiety, such as 8, significantly en-
hanced the cytotoxicity while a methyl group substituted at
C-3 of compound with same skeleton, such as 9, did not en-
hance the cytotoxicity. The B ring of 2 replaced with 3-thieny
moiety or 2-(5-methylthienyl) moiety, such as 6 or 8, signifi-
cantly enhanced the cytotoxicity against Hep 3B and HT-29
cell lines while it did not enhance the cytotoxicity against
MCF-7 cells. The etherification or esterification of phenolic
O
OMe
a
Cl
2
+
O
OMe
11a
11
Scheme 2. (a) Reagents and condition: KOH, MeOH, reflux 1 h.

662
B.-L. Wei et al. / European Journal of Medicinal Chemistry 42 (2007) 660e668
Table 1
Structure and analytical data of 20,50-dimethoxychalcones
R'
R'
4
5
4
OMe
A
OMe
OMe
OMe
3
B
5
X
R
3
2
X
2
R
OMe
O
OMe O
O
4,6
3,5,7-10
1 ( R = CH3, R' = N(CH3)2)
2 ( R = H, R' = OH)
5''
5''
6''
O
R"
6''
3''
3''
4''
9''
1''
3
6
4''
1''
2''
O
O
7''
8''
2''
R'
2
1
OMe
OMe
OMe
5'
7''
4
9''
8''
R
5
4'
3'
6'
1'
OMe
2'
O
O
11
12-14
Compound
R
H
R0
H
R00
X
Formula
Mp ꢁC
Yield %
Analysisa
3
O
O
S
C15H14O4
C15H14O4
C15H14O3S
Oil
Oil
41
33
50b
42
58b
56b
31
C, H
C, H
4
5
H
H
70e72
Oil
C, H, S
C, H, S
C, H
6
S
C15H14O3S
7
Me
Me
H
H
H
O
S
C16H16O4
C16H16O3S
C16H16O3S
64e65
74e75
Oil
8
C, H, S
C, H, S
C, H
9
Me
H
S
10
11
12
13
14
a
Br
O
C
15H13BrO4
C26H24O4
27H24O5
C27H24O6
29H28O8
Powder
118e120
116e119
140e143
153e155
30
23b
41b
40b
11b
C, H
C, H
H
Me
H
H
C
H
OMe
OMe
OMe
C, H
C, H
OMe
C
C, H and S analyses were within ꢀ0.4% of the theoretical values.
b
Solvents used for recrystallization: CHCl3.
and 11 were studied in vitro for their inhibitory effects on
chemical mediators released from mast cells, neutrophils and
macrophages. Compound 3 had significant and concentration-
dependent inhibitory effect on the release of b-glucuronidase
from rat mast cell stimulated with compound 48/80 (Table 3).
It indicated that the B ring of 20,50-dimethoxychalcones
replaced by an unsubstituted 2-furan ring such as 3 revealed
significant inhibition of mast cell degradation. Formyl-Met-
Table 2
Cytotoxicity of 3e14 (IC50 values in mM)a
Compound
Cell line
A549
Hep 3B
HT-29
MCF-7
3
27.9 ꢀ 1.5
22.1 ꢀ 0.9
11.0 ꢀ 1.1
3.8 ꢀ 0.3
19.0 ꢀ 1.2
12.0 ꢀ 0.7
8.4 ꢀ 0.9
24.8 ꢀ 1.4
21.3 ꢀ 1.5
13.3 ꢀ 1.3
5.3 ꢀ 0.2
10.5 ꢀ 0.9
18.2 ꢀ 0.7
3.8 ꢀ 0.08
3.7 ꢀ 0.1
10.3 ꢀ 0.4
2.6 ꢀ 0.08
7.6 ꢀ 0.3
11.0 ꢀ 0.9
19.0 ꢀ 1.5
12.6 ꢀ 1.1
4.1 ꢀ 0.1
6.4 ꢀ 0.5
1.5 ꢀ 0.1
4
5
Leu-Phe (FMLP) (1 mM)/cytochalasin B (CB) (5 mg mlꢂ1
)
6
5.7 ꢀ 0.6
7
51.5 ꢀ 2.1
4.5 ꢀ 0.09
22.2 ꢀ 1.1
14.9 ꢀ 1.3
39.8 ꢀ 2.7
29.7 ꢀ 2.2
60.4 ꢀ 4.1
36.7 ꢀ 3.6
3.1 ꢀ 0.2
26.5 ꢀ 1.2
3.4 ꢀ 0.01
16.7 ꢀ 0.7
11.0 ꢀ 1.7
17.3 ꢀ 1.9
29.0 ꢀ 2.5
42.6 ꢀ 3.8
23.2 ꢀ 2.1
0.6 ꢀ 0.3
43.0 ꢀ 2.3
1.8 ꢀ 0.03
19.4 ꢀ 1.4
15.2 ꢀ 2.1
26.3 ꢀ 2.4
16.4 ꢀ 1.5
23.0 ꢀ 2.0
23.8 ꢀ 2.5
0.6 ꢀ 0.1
stimulated the release of b-glucuronidase and lysozyme
from rat neutrophils. Compounds 3 and 7 had potent and
concentration-dependent inhibitory effects on the release of
b-glucuronidase and lysozyme from rat neutrophils stimulated
with fMLP/CB, respectively (Table 4), while compound 5
showed significant inhibitory effect on the release of lysozyme
from rat neutrophils stimulated with fMLP/CB. Based on
the data appeared on the Table 4, it clearly indicates that
the 20,50-dimethoxychalcones with an unsubstituted or a
5-methyl-2-furan ring enhances the inhibition of rat neutrophil
degranulation.
8
9
10
11
12
13
14
5-Fu
a
Data are presented as mean ꢀ SD (n ¼ 3). Compounds 3e14 or 5-Fu dis-
solved in DMSO, were diluted with culture medium containing 0.1% DMSO
respectively. The control cells were treated with culture medium containing
0.1% DMSO. 5-Fu (5-fluorouracil) was used as a positive control.

B.-L. Wei et al. / European Journal of Medicinal Chemistry 42 (2007) 660e668
663
Fig. 1. Flow cytometry analysis of 8-treated MCF-7 cells. MCF-7 (1 ꢃ 104 cells/mL) was treated with various concentrations of 8 for different time periods. At the
times indicated, cells were stained with propidium iodide (PI), and DNA contents were analyzed by flow cytometry, apoptosis was measured by the accumulation
of sub-G1 DNA contents in cells. The control cells were treated with medium. Results were representative of three independent experiments.
FMLP (0.3 mM)/CB (5 mg mlꢂ1) or phorbol myristate
(PMA; 3 nM) stimulated superoxide anion generation in rat neu-
trophils. As shown in Table 5, compounds 3 and 10 had signif-
icant and concentration-dependent inhibitory effects on fMLP/
CB-induced superoxide anion generation. Based on the data
shown on Table 5, it indicates that 20,50-dimethoxychalcones
with an unsubstituted or a 5-methyl-2-furan ring enhances the
inhibitory effect on fMLP/CB-induced superoxide anion gener-
ation in rat neutrophils while it does not enhance the inhibitory
effect on PMA-induced superoxide anion generation.
As shown in Table 5, compounds 3, 5, 7, 8, 10, and 11
showed different inhibitory effects on superoxide anion forma-
tion from rat neutrophils caused by fMLP/CB and PMA. It fur-
ther established that fMLP and PMA elicit the superoxide
anion formation from neutrophils through a different transduc-
tion mechanism and are regulated differently [9].
NO may contribute to the pathophysiology of septic shock
[11]. The excessive production of NO also can destroy func-
tional normal tissue during acute and chronic inflammation
[12]. The inhibition of NO production by 8 in macrophages
and significant cytotoxic activity by 8 against the cell lines
listed in Table 2 suggest that 8 may be used as cancer chemo-
preventive drugs.
Effect on the generation of tumor necrosis factor-a (TNF-a)
was determined in RAW 264.7 cells activated by LPS [13e15].
As shown in Table 6, compound 3 strongly inhibited TNF-
a generation in LPS-stimulated RAW 264.7 cells. Further ex-
periments are needed to elucidate the mechanism of actions
of cytotoxic and anti-inflammatory effects of these compounds.
4. Conclusions
Treatment of RAW 264.7 macrophage-like cells with lipo-
polysaccharide (LPS; 1 mg mlꢂ1) for 24 h induced NO pro-
duction as assessed by measuring the accumulation of
nitrite, a stable metabolite of NO in the media, based on Gri-
ess [10]. As shown in Table 6, compounds 3, 8, and 10
showed potent and concentration-dependent inhibitory effects
on NO accumulation from RAW 264.7 cells. NO plays a cen-
tral role in macrophage-induced cytotoxicity and expressed
In this study we have synthesized a novel family of 20,50-di-
methoxychalcones and studied their ability to inhibit the
growth in human tumor cell lines. This series of compounds
revealed potent and selective cytotoxicity against human
MCF-7 cells. The most potent compound 8 showed G2/M ar-
rest-mediated apoptosis in human MCF-7 cell lines. This com-
pound has the potential to develop as a new class of antimitotic
agent. The present study also suggested that the inhibition of

664
B.-L. Wei et al. / European Journal of Medicinal Chemistry 42 (2007) 660e668
Fig. 2. Fluorescence microscopy observation of chromatin condensation in MCF-7 cells induced by 8. Co-staining of Hoechst 33258 and PI showed shrunken
nuclei and nuclear fragmentation in MCF-7 cells treated with 8 (8.7, 34.7, and 52 mM). The control cells (C) were treated with the medium.
NO production by 3, 8, and 10 in macrophages may be used as
cancer chemopreventive drugs.
determined with an ABBE refractomer, Model 1T, ATAGO.
IR spectra were determined with a PerkineElmer system
1
2000 FTIR spectrophotometer. H (400 MHz) and 13C NMR
(100 MHz) spectra were recorded on a Varian UNITY-400
spectrometer, and mass spectra were obtained on a JMX-HX
100 mass spectrometer. Elemental analyses were within
ꢀ0.4% of the theoretical values, unless otherwise noted.
5. Experimental protocols
Melting points (uncorrected) were determined with a Yanco
micro melting point apparatus. Refractive indexes were
Table 3
The inhibitory effects of dimethoxychalcones on the release of b-glucuroni-
dase and histamine from rat mast cells stimulated with compound 48/80
Table 4
The inhibitory effects of dimethoxychalcones on the release of b-glucuroni-
dase and lysozyme from rat neutrophils stimulated with fMLP (1 mM)/CB
(5 mg mlꢂ1
)
(10 mg mlꢂ1
)
Compound
IC50 (mM)a
Compound
IC50 (mM)a
b-Glucuronidase
Histamine
b-Glucuronidase
Lysozyme
3
18.4 ꢀ 5.4
>30 (ꢂ4.6 ꢀ 12.4)
>30 (12.7 ꢀ 12.0)
>30 (ꢂ2.7 ꢀ 8.8)
>30 (ꢂ13.3 ꢀ 7.9)
>30 (ꢂ11.2 ꢀ 12.5)
2.2 ꢀ 0.7
>30 (38.5 ꢀ 6.6)
>30 (0.8 ꢀ 2.5)
>30 (17.5 ꢀ 12.4)
>30 (43.5 ꢀ 1.3)
>30 (6.3 ꢀ 3.9)
>30 (0.3 ꢀ 3.3)
2.0 ꢀ 0.4
3
6.2 ꢀ 0.8
>30 (38.5 ꢀ 9.1)
22.2 ꢀ 5.1
8.3 ꢀ 4.4
5
5
14.6 ꢀ 13.1
17.8 ꢀ 7.2
7
7
8
8
>30 (45.6 ꢀ 4.7)
>30 (36.9 ꢀ 12.2)
>30 (18.8 ꢀ 7.6)
13.2 ꢀ 1.2
>30 (37.4 ꢀ 5.4)
>30 (33.4 ꢀ 15.7)
>30 (33.1 ꢀ 13.1)
12.7 ꢀ 3.5
10
11
10
11
TFP
Acetylshikonin
Data are presented as mean ꢀ SD (n ¼ 3). Acetylshikonin was used as a posi-
Data are presented as mean ꢀ SD (n ¼ 3). Trifluoperazine (TFP) was used as
tive control.
a
a positive control.
a
When 50% inhibition could not be reached at the highest concentration,
the percentage of inhibition is given in parentheses.
When 50% inhibition could not be reached at the highest concentration,
the percentage of inhibition is given in parentheses.

B.-L. Wei et al. / European Journal of Medicinal Chemistry 42 (2007) 660e668
665
Table 5
acid (3.2 g, 25 mmol) in anhydrous C6H6 (60 ml) was treated
with 5.0 ml of oxalyl chloride under an Ar atmosphere and thor-
oughly stirred at room temperature. After 2 h of stirring, the sol-
vent and the excess reagent were removed under reduced
pressure. The residue was dissolved in anhydrous pyridine
(100 ml) and reacted with 2 (25 mmol). The mixture was re-
fluxed for 1 h and then evaporated in vacuo. The residue was
chromatographed over a silica gel column and eluted with ap-
propriate solvent to yielded 12e14.
The inhibitory effects of dimethoxychalcones on superoxide anion generation
(nmol) in rat neutrophils stimulated with fMLP (0.3 mM)/CB (5 mg mlꢂ1) or
PMA (3 nM)
Compound
IC50 (mM)a
FMLP
PMA
3
3.0 ꢀ 1.8
>30 (49.1 ꢀ 4.1)
5
>30 (47.2 ꢀ 8.2)
>30 (48.1 ꢀ 1.1)
>30 (49.1 ꢀ 8.6)
19.7 ꢀ 4.6
>30 (7.9 ꢀ 5.3)
>30 (39.0 ꢀ 13.7)
>30 (ꢂ11.9 ꢀ 10.2)
>30 (ꢂ2.2 ꢀ 14.7)
>10 (4.1 ꢀ 2.9)
3.3 ꢀ 0.9
7
8
10
11
DPI
5.1.1. 20,50-Dimethoxy-2-furfurylchalcone (3)
>10 (ꢂ71.9 ꢀ 15.1)
5.8 ꢀ 2.8
Furan-2-carbaldehyde (2.4 g, 25 mmol), 20,50-dimethoxya-
cetophenone (4.5 g, 25 mmol) and barium hydroxide octahy-
drate (7.89 g, 25 mmol) were treated as in general method
for the synthesis of chalcone to give 3 as yellow oil (2.64 g,
10.25 mmol, 41%), n2D7 1.356. IR (KBr) 1654, 1593, 1493,
Data are presented as mean ꢀ SD (n ¼ 3). Diphenylene iodonium chloride
(DPI) was used as a positive control.
a
When 50% inhibition could not be reached at the highest concentration,
the percentage of inhibition is given in parentheses.
1280, 1223, 1035, 1017 cmꢂ1 1H NMR (CDCl3): d 3.78
;
(3H, s, OCH3), 3.85 (3H, s, OCH3), 6.58 (1H, dd, J ¼ 3.2,
2.0 Hz, H-4), 6.88 (1H, d, J ¼ 3.2, 0.9 Hz, H-3), 7.04 (1H, d,
J ¼ 9.2 Hz, H-30), 7.06 (1H, dd, J ¼ 9.2, 2.0 Hz, H-40), 7.16
(1H, dd, J ¼ 2.0, 0.9 Hz, H-5), 7.33 (1H, d, J ¼ 15.2 Hz, H-
a), 7.45 (1H, d, J ¼ 15.2 Hz, H-b), 7.70 (1H, d, J ¼ 2.0 Hz,
H-60). 13C NMR (CDCl3): d 56.6 (OCH3), 57.3 (OCH3),
114.1 (C-60), 114.9 (C-30), 115.9 (C-3), 117.3 (C-4), 119.9
(C-a), 125.7 (C-40), 130.2 (C-5), 131.1 (C-10), 146.7 (C-b),
153.1 (C-2), 153.9 (C-50), 155.1 (C-20), 192.1 (C]O); MS
(EI) m/z 258 (Mþ, 100). Anal Calcd for C15H14O4: C, 69.80;
H, 5.50. Found: C, 69.35; H, 5.50.
Chromatography was performed using a flash-column tech-
nique on silica gel 60 supplied by E. Merck.
5.1. General procedure for obtaining chalcones 3e14
Appropriate aldehyde (25 mmol), barium hydroxide octahy-
drate (25 mmol) and 20,50-dimethoxyacetophenone (25 mmol)
were dissolved in MeOH (100 ml). The mixture was stirred
for 12 h at 40 ꢁC and then evaporated in vacuo, water
(100 ml) was added and the mixture was neutralized with
HCl (1 M, 35 ml) and extracted with EtOAc. The organic layer
was separated, washed with water, dried, and evaporated in va-
cuo. The residue was chromatographed over a silica gel column
and eluted with appropriate solvent to yield compounds 3e10.
Compound 2 (25 mmol) was dissolved in MeOH (100 ml) and
KOH (25 mmol) and cinnamyl chloride (25 mmol) were added
and refluxed for 1 h. The mixture was evaporated in vacuo, neu-
tralized with 1 M HCl, and extracted with EtOAc. The organic
layer was separated and evaporated in vacuo. The residue was
chromatographed over a silica gel column and eluted with ap-
propriate solvent to yield 11. Various substituted cinnamic
5.1.2. 20,50-Dimethoxy-3-furfurylchalcone (4)
Furan-3-carbaldehyde (2.4 g, 25 mmol), 20,50-dimethoxya-
cetophenone (4.5 g, 25 mmol) and barium hydroxide octahy-
drate (7.89 g, 25 mmol) were treated as in general method
for the synthesis of chalcone to give 4 as yellow oil (2.13 g,
8.25 mmol, 33%), n2D6 1.335. IR (KBr) 1665, 1594, 1493,
1
1227, 1223 cmꢂ1; H NMR (CDCl3): d 3.70 (3H, s, OCH3),
3.74 (3H, s, OCH3), 6.57 (1H, m, H-4), 6.83 (1H, d,
J ¼ 8.8 Hz, H-30), 6.90 (1H, dd, J ¼ 9.2, 3.2 Hz, H-40), 7.05
(1H, d, J ¼ 15.6 Hz, H-a), 7.08 (1H, d, J ¼ 3.2 Hz, H-5),
7.34 (1H, m, H-2), 7.46 (1H, d, J ¼ 15.6 Hz, H-b), 7.61 (1H,
s, H-60). 13C NMR (CDCl3): d 55.4 (OCH3), 56.0 (OCH3),
107.3 (C-4), 113.0 (C-60), 114.1 (C-30), 118.4 (C-a), 123.0
(C-3), 126.6 (C-40), 129.2 (C-10), 133.1 (C-2), 144.1 (C-b),
144.9 (C-5), 152.1 (C-50), 153.2 (C-20), 191.9 (C]O); MS
(EI) m/z 258 (Mþ, 100). Anal Calcd for C15H14O4: C, 69.80;
H, 5.50. Found: C, 69.55; H, 5.58.
Table 6
The inhibitory effects of dimethoxychalcones on the accumulation of NOꢂ2 and
TNF-a formation in the culture media of RAW 264.7 cells in response to LPS
(1 mg mlꢂ1
)
Compound
IC50 (mM)a
NOꢂ2
TNF-a
3
5.7 ꢀ 2.0
4.6 ꢀ 0.1
5
>10 (49.8 ꢀ 9.0)
>10 (29.5 ꢀ 3.5)
6.7 ꢀ 1.2
>30 (5.9 ꢀ 2.0)
>10 (ꢂ10.5 ꢀ 4.2)
>30 (46.0 ꢀ 15.0)
>30 (35.6 ꢀ 12.2)
>30 (15.0 ꢀ 13.1)
18.1 ꢀ 2.5
5.1.3. 20,50-Dimethoxy-2-thienylchalcone (5)
7
2-Thiophenecarboxaldehyde (2.8 g, 25 mmol), 20,50-dime-
thoxyacetophenone (4.5 g, 25 mmol) and barium hydroxide
octahydrate (7.89 g, 25 mmol) were treated as in general
method for the synthesis of chalcone to give 5 as yellow nee-
dles (3.42 g, 12.5 mmol, 50%). IR (KBr) 1650, 1581, 1493,
8
10
12.8 ꢀ 4.7
11
Positive control
>30 (27.9 ꢀ 4.2)
5.2 ꢀ 1.1
Data are presented as mean ꢀ SD (n ¼ 3). N-(3-Aminomethyl) benzylacetami-
dine (1400w) and genistein were used as a positive control for the accumula-
tion of NOꢂ2 and TNF-a formation in the culture media of RAW 264.7 cells in
response to LPS, respectively.
1413, 1280, 1221, 1035 cmꢂ1 1H NMR (CDCl3): d 3.78
;
(3H, s, OCH3), 3.84 (3H, s, OCH3), 6.92 (1H, d, J ¼ 8.8 Hz,
H-30), 6.99 (1H, dd, J ¼ 8.8, 3.2 Hz, H-40), 7.03 (1H, dd,
J ¼ 4.8, 3.6 Hz, H-4), 7.18 (1H, d, J ¼ 3.2 Hz, H-60), 7.24
a
When 50% inhibition could not be reached at the highest concentration,
the percentage of inhibition is given in parentheses.

666
B.-L. Wei et al. / European Journal of Medicinal Chemistry 42 (2007) 660e668
(1H, d, J ¼ 15.6 Hz, H-a), 7.29 (1H, d, J ¼ 3.0, 0.8 Hz, H-3),
7.37 (1H, d, J ¼ 4.8 Hz, H-5), 7.76 (1H, d, J ¼ 15.6 Hz, H-b).
13C NMR (CDCl3): d 55.7 (OCH3), 56.3 (OCH3), 113.3 (C-60),
114.3 (C-30), 119.1 (C-a), 125.8 (C-40), 128.2 (C-3), 128.4 (C-
4), 129.3 (C-10), 131.4 (C-5), 135.5 (C-b), 140.5 (C-2),þ152.5
(C-50), 153.5 (C-20), 191.6 (C]O); MS (EI) m/z 274 (M , 42).
Anal Calcd for C15H14O3S: C, 65.70; H, 5.10; S, 11.70.
Found: C, 65.70; H, 5.02; S, 11.67.
J ¼ 3.6 Hz, H-4), 7.08 (1H, d, J ¼ 15.6 Hz, H-a), 7.15 (1H,
d, J ¼ 3.2 Hz, H-60), 7.65 (1H, d, J ¼ 15.6 Hz, H-b). 13C
NMR (CDCl3): d 15.6 (CH3), 55.5 (OCH3), 56.1 (OCH3),
113.1 (C-60), 114.2 (C-30), 118.6 (C-a), 124.4 (C-3), 126.6
(C-40), 129.3 (C-10), 132.2 (C-4), 136.0 (C-b), 138.4 (C-2),
144.0 (C-5), 152.2 (C-50), 153.3 (C-20), 191.4 (C]O); MS
(EI) m/z 288 (Mþ, 28). Anal Calcd for C16H16O3S: C,
66.60; H, 5.60; S, 11.10. Found: C, 66.66; H, 5.47; S, 10.92.
5.1.4. 20,50-Dimethoxy-3-thienylchalcone (6)
5.1.7. 20,50-Dimethoxy-2-(3-methylthienyl)chalcone (9)
3-Thiophenecarboxaldehyde (2.8 g, 25 mmol), 20,50-dime-
thoxyacetophenone (4.5 g, 25 mmol) and barium hydroxide oc-
tahydrate (7.89 g, 25 mmol) were treated as in general method
for the synthesis of chalcone to give 6 as yellow oil (2.88 g,
10.5 mmol, 42%), n2D6 1.341. IR (KBr) 1653, 1586, 1493,
3-Methyl-2-thiophenecarboxaldehyde (3.15 g, 25 mmol),
20,50-dimethoxyacetophenone (4.5 g, 25 mmol) and barium
hydroxide octahydrate (7.89 g, 25 mmol) were treated as in
general method for the synthesis of chalcone to give 9 as yel-
low oil (2.23 g, 7.75 mmol, 31%), n2D6 1.360. IR (KBr) 1648,
1
1282, 1222, 1035 cmꢂ1; H NMR (CDCl3): d 3.80 (3H, s,
1575, 1493, 1281, 1222, 1038 cmꢂ1 1H NMR (CDCl3):
;
OCH3), 3.84 (3H, s, OCH3), 6.93 (1H, d, J ¼ 8.8 Hz, H-30),
6.94 (1H, dd, J ¼ 8.8, 2.8 Hz, H-40), 7.16 (1H, d, J ¼ 2.8 Hz,
H-60), 7.22 (1H, d, J ¼ 15.6 Hz, H-a), 7.32 (1H, ddd, J ¼ 5.2,
2.8, 0.4 Hz, H-4), 7.37 (1H, dd, J ¼ 5.2, 1.6 Hz, H-5), 7.53
(1H, dd, J ¼ 2.8, 1.6 Hz, H-2), 7.62 (1H, d, J ¼ 15.6 Hz, H-
b). 13C NMR (CDCl3): d 55.8 (OCH3), 56.4 (OCH3), 113.3
(C-60), 114.3 (C-30), 118.9 (C-a), 125.3 (C-5), 126.7 (C-40),
126.8 (C-2), 128.7 (C-4), 129.6 (C-10), 136.9 (C-b), 138.3 (C-
2), 152.4 (C-50), 153.5 (C-20), 192.7 (C]O); MS (EI) m/z
274 (Mþ, 59). Anal Calcd for C15H14O3S: C, 65.70; H, 5.10;
S, 11.70. Found: C, 65.26; H, 5.06; S, 12.10.
d 2.35 (3H, s, CH3), 3.80 (3H, s, OCH3), 3.85 (3H, s,
OCH3), 6.87 (1H, d, J ¼ 4.8 Hz, H-4), 6.92 (1H, d,
J ¼ 8.8 Hz, H-30), 6.99 (1H, dd, J ¼ 9.2, 3.2 Hz, H-40), 7.20
(1H, d, J ¼ 4.8 Hz, H-50), 7.21 (1H, d, J ¼ 3.2 Hz, H-60),
7.25 (1H, d, J ¼ 15.6 Hz, H-a), 7.86 (1H, d, J ¼ 15.6 Hz, H-
b). 13C NMR (CDCl3): d 14.2 (CH3), 55.8 (OCH3), 56.4
(OCH3), 113.3 (C-60), 114.3 (C-30), 119.1 (C-a), 124.9 (C-
40), 127.0 (C-5), 129.6 (C-10), 131.3 (C-4), 133.8 (C-b),
142.1 (C-2), 152.6 (C-50), 153.5 (C-20), 191.4 (C]O); MS
(EI) m/z 288 (Mþ, 59). Anal Calcd for C16H16O3S: C,
66.60; H, 5.60; S, 11.10. Found: C, 66.30; H, 5.56; S, 11.02.
5.1.5. 20,50-Dimethoxy-2-(5-methylfurfuryl)chalcone (7)
5-Methyl-2-furaldehyde (2.75 g, 25 mmol), 20,50-dimethox-
yacetophenone (4.5 g, 25 mmol) and barium hydroxide octa-
hydrate (7.89 g, 25 mmol) were treated as in general method
for the synthesis of chalcone to give 7 as yellow needles
(3.94 g, 14.5 mmol, 58%). IR (KBr) 1653, 1599, 1568, 1493,
5.1.8. 20,50-Dimethoxy-2-(5-bromofurfuryl)chalcone (10)
5-Bromo-2-furaldehyde (4.37 g, 25 mmol), 20,50-dimethox-
yacetophenone (4.5 g, 25 mmol) and barium hydroxide octahy-
drate (7.89 g, 25 mmol) were treated as in general method for
the synthesis of chalcone to give 10 as brown powder (2.04 g,
7.5 mmol, 30%). IR (KBr) 1655, 1594, 1493, 1279,
1
1276, 1223, 1037, 1021 cmꢂ1
;
1H NMR (CDCl3): d 2.30
1223 cmꢂ1; H NMR (CDCl3): d 3.80 (3H, s, OCH3), 3.86
(3H, s, CH3), 3.74 (3H, s, OCH3), 3.79 (3H, s, OCH3), 6.04
(1H, dd, J ¼ 3.2, 0.8 Hz, H-3), 6.53 (1H, d, J ¼ 3.2 Hz, H-
4), 6.86 (1H, d, J ¼ 9.2 Hz, H-30), 6.93 (1H, dd, J ¼ 8.8,
2.8 Hz, H-40), 7.12 (1H, d, J ¼ 2.8 Hz, H-60), 7.16 (1H, d,
J ¼ 15.6 Hz, H-a), 7.32 (1H, d, J ¼ 15.6 Hz, H-b). 13C NMR
(CDCl3): d 13.9 (CH3), 55.5 (OCH3), 56.2 (OCH3), 109.1
(C-a), 113.1 (C-60), 114.2 (C-30), 117.5 (C-3), 118.4 (C-4),
122.4 (C-40), 129.6 (C-10), 129.7 (C-b), 150.1 (C-5), 152.2
(C-2), 153.3 (C-50), 155.5 (C-20), 191.8 (C]O); MS (EI) m/
z 272 (Mþ, 100). Anal Calcd for C16H14O4: C, 70.60; H,
5.90. Found: C, 70.50; H, 5.91.
(3H, s, OCH3), 6.41 (1H, d, J ¼ 3.6 Hz, H-4), 6.60 (1H, d,
J ¼ 3.6 Hz, H-3), 6.92 (1H, d, J ¼ 9.2 Hz, H-30), 7.00 (1H, dd,
J ¼ 8.8, 3.2 Hz, H-40), 7.18 (1H, d, J ¼ 3.2 Hz, H-60), 7.28
(1H, d, J ¼ 15.6 Hz, H-a), 7.33 (1H, d, J ¼ 15.6 Hz, H-b). 13
C
NMR (CDCl3): d 55.8 (OCH3), 56.4 (OCH3), 113.3 (C-30),
114.3 (C-60), 114.4 (C-4), 117.5 (C-3), 119.4 (C-40), 124.6 (C-
a), 125.4 (C-2), 127.9 (C-b), 129.4 (C-10), 152.7 (C-20), 153.5
(C-50), 153.7 (C-5), 191.4 (C]O); MS (EI) m/z 336 (Mþ, 3).
Anal Calcd for C15H13BrO4: C, 53.40; H, 3.90. Found: C,
53.66; H, 3.94. The structure was supported by 2D spectra.
5.1.9. 20,50-Dimethoxy-4-cinnamyloxychalcone (11)
5.1.6. 20,50-Dimethoxy-2-(5-methylthienyl)chalcone (8)
Cinnamyl chloride (3.4 g, 25 mmol), compound 2 (7.1 g,
25 mmol) and KOH (1.4 g, 25 mmol) were dissolved in
MeOH (100 ml). The reaction mixture was treated as in general
method for the synthesis of chalcone to give 11 as yellow nee-
dles (2.3 g, 5.7 mmol, 23%). IR (KBr) 1648, 1586, 1508, 1492,
5-Methyl-2-thiophenecarboxaldehyde (3.15 g, 25 mmol),
20,50-dimethoxyacetophenone (4.5 g, 25 mmol) and barium
hydroxide octahydrate (7.89 g, 25 mmol) were treated as in
general method for the synthesis of chalcone to give 8 as yel-
low needles (4.03 g, 14 mmol, 56%). IR (KBr) 1648, 1578,
1
1249, 1222, 1171 cmꢂ1; H NMR (CDCl3): d 3.81 (3H, s,
1493, 1280, 1221, 1035 cmꢂ1
;
1H NMR (CDCl3): d 2.44
OCH3), 3.86 (3H, s, OCH3), 4.73 (2H, dd, J ¼ 6.0, 1.6 Hz,
OCH2), 6.41 (H, dt, J ¼ 15.6, 6.0 Hz, H-200), 6.75 (1H, d,
J ¼ 16 Hz, H-a), 6.94 (1H, d, J ¼ 9.2 Hz, H-30), 6.96 (2H, dd,
J ¼ 8.8, 2.8 Hz, H-3 and H-5), 7.00 (1H, dd, J ¼ 8.8, 2.8 Hz,
(3H, s, CH3), 3.74 (3H, s, OCH3), 3.80 (3H, s, OCH3), 6.67
(1H, dd, J ¼ 3.6, 1.2 Hz, H-3), 6.87 (1H, d, J ¼ 8.8 Hz, H-
30), 6.95 (1H, dd, J ¼ 8.8, 3.2 Hz, H-40), 7.05 (1H, d,

B.-L. Wei et al. / European Journal of Medicinal Chemistry 42 (2007) 660e668
667
H-40), 7.17 (1H, d, J ¼ 2.8 Hz, H-60), 7.25e7.40 (6H, m, H-300e
5.1.12. 20,50-Dimethoxy-4-(3,4,5-trimethyoxy cinnamoyloxy)-
chalcone (14)
H-900), 7.55 (2H, dd, J ¼ 8.8, 2.8 Hz, H-2 and H-6), 7.61 (1H, d,
J ¼ 16 Hz, H-b). 13C NMR (CDCl3): d 55.8 (OCH3), 56.5
(OCH3), 68.7 (C-100), 113.4 (C-60), 114.3 (C-30), 115.1 (C-2
and C-6), 118.8 (C-a), 123.8 (C-200), 124.8 (C-40), 126.6 (C-
500 and C-900), 128.0 (C-100 and C-700), 128.6 (C-600 and C-800),
130.0 (C-1), 130.2 (C-2 and C-6), 133.4 (C-300), 136.2 (C-400),
143.4 (C-b), 152.4 (C-50), 154.6 (C-20), 160.5 (C-4), 192.6
(C]O); MS (EI) m/z 400 (Mþ, 1). Anal Calcd for C26H24O4:
C, 78.00; H, 6.00. Found: C, 77.89; H, 6.1.
3,4,5-Trimethoxycinnamic acid (6.0 g, 25 mmol) in anhy-
drous C6H6 (60 ml) was treated as in general method for the
synthesis of chalcone to give 14 as yellow needles (3.4 g,
25 mmol, 11%). IR (KBr) 1726, 1635, 1581, 1503, 1273,
1
1210, 1166, 1127 cmꢂ1; H NMR (CDCl3): d 3.79 (3H, s,
OCH3), 3.84 (3H, s, OCH3), 3.89 (3H, s, OCH3), 3.89 (6H, s,
OCH3), 6.53 (1H, d, J ¼ 15.6 Hz, H-a), 6.81 (2H, s, H-500 and
H-900), 6.92 (1H, d, J ¼ 9.2 Hz, H-30), 7.00 (1H, dd, J ¼ 9.2,
3.2 Hz, H-40), 7.18 (1H, d, J ¼ 3.2 Hz, H-60), 7.18 (1H, d,
J ¼ 3.2 Hz, H-60), 7.20 (2H, dd, J ¼ 9.2, 3.2 Hz, H-3 and H-
5), 7.38 (2H, d, J ¼ 16.0 Hz, H-200), 7.62 (2H, dd, J ¼ 9.2,
3.2 Hz, H-2 and H-6), 7.63 (1H, d, J ¼ 16.0 Hz, H-300), 7.78
(1H, d, J ¼ 15.6 Hz, H-b). 13C NMR (CDCl3): d 55.7
(OCH3), 56.1 (OCH3), 56.4 (OCH3), 60.9 (OCH3), 105.4 (C-
500 and C-900), 113.3 (C-60), 114.3 (C-30), 116.0 (C-200), 119.2
(C-a), 122.0 (C-3 and C-5), 126.9 (C-40), 129.4 (C-10, C-6
and C-400), 132.7 (C-1), 141.9 (C-b), 146.9 (C-300), 152.1 (C-
4), 152.5 (C-50), 153.4 (C-20, C-600 and C-800), 165.0 (C-100),
192.0 (C]O); MS (EI) m/z 504 (Mþ, 1). Anal Calcd for
C29H28O8: C, 69.00; H, 5.60. Found: C, 68.81; H, 5.63.
5.1.10. 20,50-Dimethoxy-4-(4-methylcinnamoyloxy)chalcone (12)
4-Methylcinnamic acid (3.2 g, 25 mmol) in anhydrous
C6H6 (60 ml) was treated as in general method for the synthe-
sis of chalcone to give 12 as yellow needles (4.38 g,
10.25 mmol, 41%). IR (KBr) 1733, 1630, 1594, 1578, 1491,
1
1212, 1165, 1132 cmꢂ1; H NMR (CDCl3): d 2.39 (3H, s,
CH3), 3.80 (3H, s, OCH3), 3.86 (3H, s, OCH3), 6.58 (1H, d,
J ¼ 16.0 Hz, H-a), 6.94 (1H, d, J ¼ 8.8 Hz, H-30), 7.02 (1H,
dd, J ¼ 9.2, 2.8 Hz, H-40), 7.22 (5H, m, H-60 and H-2e6),
7.40 (1H, d, J ¼ 15.6 Hz, H-200), 7.48 (2H, d, J ¼ 8.0 Hz, H-
600 and H-800), 7.61 (2H, dd, J ¼ 8.0, 2.0 Hz, H-500 and H-900),
7.65 (1H, d, J ¼ 15.6 Hz, H-300), 7.86 (1H, d, J ¼ 16.0 Hz,
H-b). 13C NMR (CDCl3): d 21.4 (CH3), 55.7 (OCH3), 56.4
(OCH3), 113.3 (C-60), 114.3 (C-30), 115.6 (C-200), 119.2 (C-
a), 122.1 (C-3 and C-5), 126.8 (C-40), 128.3 (C-600 and C-
800), 129.4 (C-10, C-2 and C-6), 129.7 (C-500 and C-900),
131.2 (C-1), 132.7 (C-400), 141.3 (C-700), 142.0 (C-b), 147.0
(C-300), 152.2 (C-4), 152.5 (C-40), 153.5 (C-20), 165.2 (C-100),
192.1 (C]O); MS (EI) m/z 428 (Mþ, 1). Anal Calcd for
C27H24O5: C, 75.70; H, 5.60. Found: C, 75.61; H, 5.71.
5.2. Tumor cell growth inhibition assays
The cytotoxicity of compounds 3e14 against human lung
cancer cell A549, human hepatomacellular carcinoma Hep
3B, human colerectal adenocarcinoma HT-29 and human
breast adenocarcinoma MCF-7 cells obtained from American
Type Culture Collection (ATCC, Rockville, MD), were per-
formed by the method as described in Ref. [5].
5.3. Flow cytometry
5.1.11. 20,50-Dimethoxy-4-(4-methoxycinnamoyloxy)-
chalcone (13)
Compounds were added to cells (1 ꢃ 107). At various time
intervals, the reactions were terminated by washing with PBS.
The cells were fixed with 4% paraformadehyde/PBS (pH 7.4)
at room temperature for 30 min. After centrifugation at
1000 rpm for 10 min, the cells were permeabilized with
0.1% Triton-X-100/0.1% sodium citrate at 4 ꢁC for 2 min. Pro-
pidium iodide (Sigma) in PBS (10 g/mL) was added to stain
the cells at 37 ꢁC for 30 min. The intensity of fluorescence
was measured with a FACScan flow cytometer (Becton Dick-
inson, Mountain View, CA). A minimum of 5000 cell counts
were collected for the analysis by LYSIS II Software.
4-Methoxycinnamic acid (4.5 g, 25 mmol) in anhydrous
C6H6 (60 ml) was treated as in general method for the synthe-
sis of chalcone to give 13 as yellow needles (4.45 g,
10.03 mmol, 40.1%). IR (KBr) 1718, 1654, 1629, 1601,
1
1508, 1166, 1131 cmꢂ1; H NMR (CDCl3): d 3.81 (3H, s,
OCH3), 3.86 (3H, s, OCH3), 3.86 (3H, s, OCH3), 6.49 (1H,
d, J ¼ 15.6 Hz, H-a), 6.94 (1H, d, J ¼ 8.8 Hz, H-30), 6.95
(2H, dd, J ¼ 8.8, 2.8 Hz, H-3 and H-5), 7.02 (1H, dd,
J ¼ 8.8, 2.8 Hz, H-40), 7.20 (1H, d, J ¼ 2.8 Hz, H-60), 7.22
(2H, dd, J ¼ 8.8, 2.8 Hz, H-2 and H-6), 7.39 (1H, d,
J ¼ 16.0 Hz, H-300), 7.53 (2H, dd, J ¼ 8.8, 2.0 Hz, H-600 and
H-800), 7.61 (2H, dd, J ¼ 8.8, 2.0 Hz, H-500 and H-900), 7.64
(1H, d, J ¼ 16.0 Hz, H-200), 7.82 (1H, d, J ¼ 15.6 Hz, H-b).
13C NMR (CDCl3): d 55.4 (OCH3), 55.8 (OCH3), 56.5
(OCH3), 113.4 (C-60), 114.2 (C-30), 114.4 (C-200, C-600 and
C-800), 119.3 (C-a), 122.2 (C-3 and C-5), 126.8 (C-400),
126.9 (C-40), 129.5 (C-10), 129.6 (C-2 and C-6), 130.1 (C-500
and C-900), 132.7 (C-1), 142.1 (C-b), 146.7 (C-300), 152.4 (C-
4), 152.6 (C-40), 153.6 (C-20), 161.8 (C-700), 165.4 (C-100),
192.2 (C]O); MS (EI) m/z 444 (Mþ, 1). Anal Calcd for
C27H24O6: C, 73.00; H, 5.40. Found: C, 72.90; H, 5.45.
5.4. Microscopic observation of morphology and nuclear
fragmentation
Morphological changes of the cells were observed directly
in culture vehicles. To observe chromatin condensation and
plasma membrane permeability, cell were resuspended in fresh
culture medium after collection by centrifugation, co-stained
propidium iodide (PI, 0.05 g/L) and Hoechst 33258 (0.01
g/L) at 37 ꢁC for 30 min, then centrifuged to remove most

668
B.-L. Wei et al. / European Journal of Medicinal Chemistry 42 (2007) 660e668
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of the supernatant. The pellet was suspended in the remaining
culture medium and smeared for fluorescence microscope.
~
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Acknowledgements
This work was supported by a grant from the National
Science Council of the Republic of China (NSC 94-2320-B-
037-033).
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