Synthetic chalcones as potential anti-inflammatory and cancer chemopreventive agents

Article abstract of DOI:10.1016/j.ejmech.2004.09.006

In an effort to develop potent anti-inflammatory and cancer chemopreventive agents, a series of chalcones were prepared by Claisen-Schmidt condensation of appropriate acetophenones with suitable aromatic aldehyde or prepared with appropriate dihydrochalco

Full text of DOI:10.1016/j.ejmech.2004.09.006

European Journal of Medicinal Chemistry 40 (2005) 103–112
www.elsevier.com/locate/ejmech
Short communication
Synthetic chalcones as potential anti-inflammatory
and cancer chemopreventive agents
Shen-Jeu Won a, Cheng-Tsung Liu b, Lo-Ti Tsao c, Jing-Ru Weng b, Horng-Huey Ko b,
Jih-Pyang Wang c, Chun-Nan Lin b,*
a Department of Microbiology and Immunology, National Cheng Kung University, Tainan 701, Taiwan
b School of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan
c Department of Education and Research, Taichung Veterans General Hospital, Taichung 407, Taiwan
Received 1 March 2004; received in revised form 1 September 2004; accepted 6 September 2004
Available online 25 November 2004
Abstract
In an effort to develop potent anti-inflammatory and cancer chemopreventive agents, a series of chalcones were prepared by Claisen–
Schmidt condensation of appropriate acetophenones with suitable aromatic aldehyde or prepared with appropriate dihydrochalcone reacted
with appropriate alkyl bromide or prepared in one-pot procedure involving acetophenone and convenient aromatic aldehyde using ultrasonic
agitation on basic alumina. The synthesized products were tested for their inhibitory effects on the activation of mast cells, neutrophils,
macrophages, and microglial cells. The potent inhibitors of NO production in macrophages and microglial cells were further evaluated for
their in vitro cytotoxic effects against several human cancer cell lines. 2′-Hydroxychalcones 1–3, and 2′,5′-dihydroxychalcone 7 exhibited
potent inhibitory effects on the release of b-glucuronidase or lysozyme from rat neutrophils stimulated with formyl-Met-Leu-Phe
(fMLP)/cytochalasin B (CB). Two 2′-hydroxychalcones (1 and 3) showed potent inhibitory effects on superoxide anion generation in rat
neutrophils in response to fMLP/CB. The previously reported chalcone, 5, 6, and 12, exhibited potent inhibitory effect on NO production in
lipopolysaccharide (LPS)/interferon-c (IFN-c)-activated N9 microglial cells or in LPS-activated RAW 264.7 macrophage-like cells. The
potent inhibitors 5, 6, and 12 of NO production in macrophages or microglial cells revealed significant or marginal cytotoxic effects against
several human cancer lines. Compound 12 manifested potent selective cytotoxicity against human MCF-7 cells and caused cell death by
apoptosis. The present results demonstrated that 1–3, and 7 have anti-inflammatory effects and 5, 6, and 12 are potential anti-inflammatory
and cancer chemopreventive agents.
© 2004 Elsevier SAS. All rights reserved.
Keywords: Chalcone; Anti-inflammatory; Cancer chemopreventive agent
1. Introduction
macrophage-induced cytotoxicity and expressed NO may con-
tribute to the pathophysiology of septic shock [1]. The exces-
sive production of NO also can destroy functional normal tis-
sues during acute and chronic inflammation [2]. This
phenomenon is also closely related mechanistically to car-
cinogenesis [2]. Thus, inhibitors of NO production in mac-
rophages are potential anti-inflammatory and cancer chemo-
preventive drugs.
It is conceivable that mast cells, neutrophils, and macroph-
ages are important players in inflammatory disorders. Acti-
vation of microglial cells also plays a crucial role in inflam-
matory diseases of the CNS. Thus, inhibition of the activation
of these inflammatory cells appears to be an important thera-
peutic target for small molecule drug design for the treatment
of inflammatory diseases. NO plays a central role in
Our recent reports have demonstrated that some synthetic
chalcones inhibited the release of chemical mediators from
mast cells, neutrophils, macrophages, and microglial cells in
vitro, and suppressed the oedematous response in vivo [3–6].
We also reported that broussochalcone A, a natural chalcone
* Corresponding author. Tel.: +886 7 312 1101 9x2163;
fax: +886 7 556 2365.
E-mail address: lincna@cc.kmu.edu.tw (C.-N. Lin).
0223-5234/$ - see front matter © 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2004.09.006

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S.-J. Won et al. / European Journal of Medicinal Chemistry 40 (2005) 103–112
isolated from Broussonetia papyrifea (Moraceae), exerts a
potent antioxidant activity and inhibits the respiratory burst
in neutrophils and the inducible nitric oxide synthase (iNOS)
expression in macrophages [7,8]. Moreover, two synthetic
products, 2′,5′-dihydroxy-4-chloro-dihydrochalcone and
2′,5′-dihydroxydihydrochalcone inhibit the iNOS protein ex-
pression and the former also inhibits cyclooxygenase-2
(COX-2) activity in RAW 264.7 cells [6,9].
dihydroxydihydrochalcone (10b) and anhydrous K2CO3
were stirred with 3-bromo-1-propene in N,N-dimethyl-
formamide (DMF) at room temperature to give the novel
dihydrochalcone 10. Compound 10b was also refluxed with
3-bromo-1-butene (Scheme 3). In this latter case, the subse-
quent Claisen rearrangement gave 11′. The occurrence of
secondary Claisen rearrangement give the new dihydrochal-
cone, 11 [26]. The 1H-NMR and 13C-NMR spectra (Section
5) supported the structure of 11. One-pot synthesis involving
the appropriate acetophenone and the suitable aromatic alde-
hyde by ultrasonic agitation on basic alumina provided the
novel chalcone 6 (Scheme 2). This is the first report dealing
with a convenient one-pot synthesis of chalcones by ultra-
sonic agitation on basic alumina [27].
Chalcones constitute an important group of natural prod-
ucts and some of them posses antitumor activity [10]. These
findings suggested that some chalcones may be promising
anti-inflammatory and cancer chemopreventive agents and
have potential in the therapy of septic shock. This report
described the chemistry of further synthesized chalcones,
biological activity, and the structure–activity relationships of
these series of anti-inflammatory and cancer chemopreven-
tive agents.
3. Biological results and discussion
The anti-inflammatory activities of 1–11 were studied in
vitro for their inhibitory effects on chemical mediators re-
2. Chemistry
As depicted in Scheme 1 for the specific synthesis of
compound 5, in addition to the new chalcones 3, 5–7, we
have prepared compounds 1 [3,11–17], 2 [18–22], 4
[19,20,23,24], the 3,4-dichloro-2′,5′-dimethoxychalcone
(8a), 3,4-dichloro-2′,5′-dihydroxychalcone (9a) [4,6] and
the 4-chloro-2′,5′-dihydroxychalcone (10a) [4,26] by carry-
ing out Claisen–Schmidt condensations starting from appro-
priate tetrahydropyran-2-yloxyacetophenones and requisite
heterocyclic aldehydes or aromatic aldehydes (protected as
tetrahydropyran-2-yl ethers if necessary) [4]. This procedure
afforded various chalcones in good yield (Table 1). A solu-
tion 8a, 9a, or 10a was hydrogenated in the presence of Pd/C
at room temperature to give the new dihydrochalcones 8 and
9 [5], respectively. The reduced product: 4-chloro-2′,5′-
Scheme 2. Reagents: (iv) Al2O3, 40 °C, 12 h.
Scheme 1. Reagents : (i) pyridium p-toluenesulfonate, 3,4-dihydro-a-pyran,
r.t., 4 h ; (ii) BaOH·8H2O, 40 °C, HCl ; (iii) P-toluenesulfonic acid, r.t., 4 h,
Scheme 3
. Reagents: (i) EtOAc, H2, 5% Pd/C, r.t.; (ii) 3-bromo-1-propene,
5 % NaHCO3.
K2CO3, DMF, r.t., 18 h; (iii) 3-bromo-1-butene, K2CO3, DMF, reflux 18 h.

S.-J. Won et al. / European Journal of Medicinal Chemistry 40 (2005) 103–112
105
Table 1
Structure and analytical data of chalcone derivatives
leased from mast cells, neutrophils, macrophages, and mi-
croglial cells. Compounds 1–11 did not show significant
inhibition of mast cell degranulation (data not shown). Pre-
viously, we reported that 2′,5′-dihydroxy-3,4-dichloro-
chalcone showed inhibition of mast cell degranulation [6],
while 5 and 9 did not indicate significant inhibition of mast
cell degranulation. It shows that the OH-5′ and enone moi-
eties of 2′,5′-dihydroxy-3,4-dichlorochalcone appear to be
required for the inhibition of mast cell degranulation.
Formyl-Met-Leu-Phe (fMLP) (1 µM)/cytochalasin B (CB)
(5 µg ml–1) stimulated the release of b-glucuronidase and
lysozyme from rat neutrophils. Compounds 2, 3, and 7, and
1–3 had potent and concentration-dependent inhibitory ef-
fects on the release of b-glucuronidase and lysozyme from
rat neutrophils, respectively (Table 2). Based on the data
appeared on the Table 2, it clearly indicates that the OH-5′
enhances the inhibitory effects on the release of b-glucuro-
nidase and lysozyme from rat neutrophils in the series of
2′-hydroxy-3,4-dichloro or 2-furfuryl or 2- or 3-thienyl-
chalcone [4,6]. In addition, the essential role of the enone
moiety of chalcones in the inhibition of fMLP/CB-stimulated
neutrophil degranulation reconcile our earlier observation
[5]. The B ring of 2′,5′-dihydroxychalcone substituted by a
indole ring such as 7 (Table 2) showed potent and
concentration-dependent inhibition effect on the release of
b-glucurnidase from rat neutrophils. Trifluoperazine was
used in this study as a positive control.
FMLP (0.3 µM)/CB (5 µg ml–1) or phorbol myristate
(PMA; 3 nM) stimulated superoxide anion generation in rat
neutrophils. As shown in Table 3, compounds 1 and 3 had
potent and concentration-dependent inhibitory effects on
fMLP/CB-induced superoxide anion generation. Based on
the data shown on Table 3, it indicates that a hydroxyl group
substituted at C-5′ of 2 or 3 does not enhance the inhibitory
effect on fMLP/CB-induced superoxide anion generation in

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S.-J. Won et al. / European Journal of Medicinal Chemistry 40 (2005) 103–112
Table 2
response suggest the involvement of PMA-independent sig-
naling pathway. In addition, the essential role of the enone
moiety of chalcones in the inhibition of fMLP/CB- or PMA-
stimulated superoxide anion generation also reconcil on ear-
lier observation [6].
The inhibitory effects of chalcone derivatives on the release of
b-glucuronidase and lysozyme from rat neutrophils stimulated with
fMLP/CB
Compound
IC50 (µM) a
b-glucuronidase
b
Lysozyme
Treatment of RAW 264.7 macrophage-like cells with li-
popolysaccharide (LPS; 1 µg ml–1) for 24 h induced NO
production as assessed by measuring the accumulation of
nitrite, a stable metabolite of NO, in the media based on
Griess reaction. As shown in Table 4, 5, 6, and 2′,5′-
dimethoxy-4-hydroxychalcone (12), a previously reported
compound [6], showed potent and concentration-dependent
inhibitory effects on NO accumulation from RAW
264.7 cells. The parallel inhibition of NO production in
N9 microglial cells as well as in RAW 264.7 cells by 5 and
12. The dihydrochalcones, 8–11 (Table 4) did not show
significant inhibitory effects on NO accumulation from RAW
264.7 stimulated with LPS and N9 microglial cells stimu-
lated with LPS/interferon-c (IFN-c), while 2′,5′-dihydroxy-
dihydrochalcone (17) showed potent inhibitory effect on NO
production from RAW 264.7 cells [5]. It suggests that 5, 12,
and 17 may be the potential leading compounds for the
development of more potent drugs to inhibit the NO produc-
tion in macrophages or microglial cells.
1
7.5 0.3
2
3
4
5
6
7
8
9
10
11
13*
14*
15*
16*
23.5 1.4
27.4 0.7
8.7 0.7
13.4 2.5
>30 (35.1 2.8)
>30 (21.8 3.6)
>3 (33.7 4.7)
17.1 4.2
>30 (17.7 12.2)
>30 (15.0 0.9)
>3 (14.8 2.0)
>30 (45.7 6.5)
>30 (−3.6 2.4)
>30 (22.6 0.5)
>30 (2.0 3.3)
>3 (−5.5 3.1)
19.7 5.0 c
>30 (9.4 3.5)
>30 (41.5 2.8)
>30 (9.2 0.5)
>3 (2.7 5.4)
17.3 4.1 c
9.7 1.9 c
23.6 4.9 c
4.6 0.4 d
1.3 0.1 d
6.7 0.3 d
1.2 0.1 d
Trifluoperazine
12.2 0.3
13.2 0.7
13*: 2′,5′-Dihydroxy-2-furfurylchalcone. 14*: 2′,5′-Dihydroxy-2-thienyl-
chalcone. 15*: 2′,5′-Dihydroxy-3-thienylchalcone. 16*: 2′,5′-Dihydroxy-
3,4-dichlorochalcone. Trifluoperazine was used as a positive control.
a When 50% inhibition could not be reached at the highest concentration,
the % of inhibition is given in parentheses.
b Not determined. Data are presented as mean S.E.M. (n = 3–5).
c Data cited from Ref. [4].
Cytotoxic activities of compounds 3, 5, 6, and 12 with
potent inhibition of NO production from RAW 264.7 cells
activated with LPS except that 3 did not show significant
inhibition of NO production, were studied against a number
of cancer cell types. The results were listed in Table 5. All
compounds in Table 5 showed selective cytotoxicity against
MCF-7 cells. The lipophilicity of A or B ring did affect the
d Data cited from Ref. [6].
Table 3
The inhibitory effects of chalcone derivatives on superoxide anion genera-
tion in rat neutrophils stimulated with fMLP/CB or PMA
Compound
IC50 (µM) a
fMLP
PMA
Table 4
1
2
3
4
5
6
7
8
9
10
11
14
6.4 0.3
>30 (35.5 2.5)
>30 (8.4 4.6)
>30 (32.0 6.9)
>30 (17.6 1.5)
>30 (24.7 6.0)
>1 (12.7 9.5)
>1 (−3.1 4.2)
>30 (6.9 4.2)
>30 (−12.6 3.4)
>30 (−19.8 2.2)
>10 (−74.7 8.2)
24.3 4.5 b
–
The inhibitory effects of chalcone derivatives on the accumulation of NO2
in the culture media of RAW 264.7 cells in response to LPS and N9 cells in
response to LPS/IFN-c
>30 (41.8 9.8)
8.2 0.3
>30 (39.2 12.2)
>30 (46.1 7.4)
>1 (0.1 7.6)
>1 (16.1 1.9)
>30 (−42.4 4.6)
>30 (−6.0 5.1)
>30 (−7.0 1.9)
>10 (−1.1 5.7)
>100 (−10.8 8.5) b
>1 (43.4 6.1) c
8.3 0.8
Compound
IC50 (µM) a
N9 cells
RAW 264.7 cells
>30 (49.5 1.9)
>30 (13.5 2.1)
>30 (−4.8 0.4)
>30 (23.2 2.7)
23.8 1.0
1
2
3
4
5
6
7
8
>10 (7.3 0.8)
>30 (12.7 2.3)
>30 (20.4 1.2)
>30 (18.8 2.3)
11.8 0.2
23.3 2.6
>10 (42.3 1.6)
>10 (15.8 1.1)
>10 (28.7 2.2)
>3 (43.7 1.4)
>30 (12.5 1.2)
>10 (1.3 8.1)
17.8 0.2 b
15
>1 (23.5 10.4) c
6.1 0.4
>10 (33.6 0.6)
>30 (3.3 1.7)
>1 (27.0 0.1)
>30 (−1.0 0.4)
>30 (30.3 2.9)
14.6 0.1 c
Trifluoperazine
9
10
11
12
17*
1400 W
Data are presented as mean S.E.M. (n = 3–5). Trifluoperazine was used as
a positive control.
a When 50% inhibition could not be reached at the highest concentration,
the % of inhibition is given in parentheses.
b Data cited from Ref. [4].
11.8 0.9 c
>3 (48.8 3.3) c
4.5 0.2
c Data cited from Ref. [6].
6.1 0.4
Data are presented as mean S.E.M. (n = 3–5). 1400 W [N-(3-amino-
methyl)benzyl-acetamidine] was used as a positive control. 17*: 2′,5′-
Dihydroxydihydrochalcone.
rat neutrophils while a hydroxyl group substituted at C-5′ of
2 does enhance the inhibitory effect on PMA-induced super-
oxide anion generation [4,6]. Because fMLP and PMA acti-
vate NADPH oxidase to produce superoxide anion through
different cellular signaling mechanisms [28]. The observa-
tions of 1–11 had no appreciable effect on PMA-induced
a When 50% inhibition could not be reached at the highest concentration,
the % of inhibition is given in parentheses.
b Data cited from Ref. [6].
c Data cited from Ref. [5].

S.-J. Won et al. / European Journal of Medicinal Chemistry 40 (2005) 103–112
107
Table 5
cycles to the sub-G1 phase is increased dose-dependently in
the MCF-7 cells treated by 12 for different time periods.
However a maximum 65.88% apoptotic cells was detected at
72 h. In addition, DNA fragmentation is generally used to
characterize cell death by apoptosis [32,33]. Thus, apoptosis
of the MCF-7 cells after 12 treatment was also studied by
DNA fragmentation. DNA fragmentation in MCF-7 cells
was significantly observed after 48 and 72 h incubation with
12 (1.0 and 2.0 µg ml–1; Fig. 2). Further experiments are
needed to elucidate its mechanism of action.
Cytotocixity of chalcone derivatives against different cell lines a
Compound
ED50 (µg ml–1
)
T-24
>8
MCF-7
2.10
Hep 3B
>8
HT-29
>8
3
5
6
12
2.98
7.40
2.12
3.00
7.10
2.00
4.00
9.00
4.60
3.60
>4
b
0.16
b
5-Fu
0.072
0.074
b
b
b
Mitomycin C
0.042
a For significant activity of the pure compounds, an ED50 ≤ 4.0 µg ml–1 is
required.
b Not determined. 5-Fu (5-fluorouracil) was used as a positive control.
4. Conclusions
cytotoxicity of chalcones against the cell lines used in
Table 5.
This study verifies that 2′-hydroxychalcones 1–3, 2′,5′-
dihydroxychalcone 7 exert potent inhibitory effects on the
release of chemical mediators from inflammatory cells. NO
plays a central role in macrophage-induced cytotoxicity and
has been demonstrated to implicate in the pathology of cen-
tral neurologic diseases and also in the peripheral tissue
damage associated with acute and chronic inflammation
[6,34,35], and septic shock [1]. The present study suggests
that the inhibition of NO production by 5 and 6 in macroph-
It has been recognized that apoptotic cells have reduced
DNA stainability with a variety of fluorochromes [29,30].
The appearance of cells with low DNA stainability forms a
“sub-G1 peak”, which has been considered to be the hallmark
of cell death by apoptosis [31]. In the study, various concen-
trations of 12 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 12 at various concentrations for
different time periods. The shift of G0/G1 and G2/M cell
Fig. 1.
Flow cytometry analysis of 12 treated MCF-7 cells. MCF-7 (1 × 104 cells ml–1) were treated with various concentrations of 12 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.

108
S.-J. Won et al. / European Journal of Medicinal Chemistry 40 (2005) 103–112
Fig. 2
.
Compound 12 induces DNA fragmentation in MCF-7 cells. MCF-7 (1 × 104 cells ml–1) cells were treated with various concentrations of 12 for different
time periods. At the times indicated, the cells were lyzed and DNA was prepared. DNA fragmentation was analyzed by 1% agarose gel electrophoresis. The
compound 12 was diluted with culture medium. The control cells were treated with medium. M, DNA molecular ladder marker. Results were representative of
three independent experiments.
ages and 5 in microglial cells may have value in the therapeu-
tic treatment or prevention of certain central as well as
peripheral inflammatory diseases associated with the in-
crease of NO production. The inhibition of NO production by
5, 6, and 12 in macrophages and significant cytotoxic activi-
ties by 5, 6, and 12 against the cell lines used in Table 5
suggest that 5, 6, and 12 may be used as cancer chemopre-
ventive drugs [2]. Thus, compound such 12 has the potential
to develop as cancer chemopreventive drug for treatment or
prevention of MCF-7 cell type cancer.
dihydroxychalcone (9a), and 4-chloro-2′,5′-dihydroxy-
chalcone (10a) [4].
5.1. 2′,4-Dihydroxychalcone (1)
2-Hydroxyacetophenone (3.4 g, 25 mmol) or 4-hydroxy-
benzaldehyde (3.05 g, 25 mmol) and pyridinium p-toluene-
sulfonate (0.15 g, 0.6 mmol) were treated as in the synthesis
of 2′,3-dihydroxychalone to give 2-(tetra-hydropyran-2-
yloxy)acetophenone (1a) or 4-(tetrahydroxypyran-2-
yloxy)benzyl-aldehyde (1b) [4]. The 1a or 1b was identified
with various spectral data and compared with those of au-
thentic samples, respectively [4,6]. Compound 1a (5.5 g,
25 mmol), 1b (5.15 g, 25 mmol), and barium hydroxide
octahydrate (4.29 g, 25 mmol) were treated as in the synthe-
sis of 2′,3-dihydroxychalcone to give 1 (2.47 g, 10.3 mmol,
5. Experimental protocols
Melting points (uncorrected) were determined with a
Yanaco Micro-Melting Point apparatus. IR spectra were de-
termined with a Perkin–Elmer system 2000 FTIR spectro-
photometer. 1H (400 MHz) and 13C (100 MHz) NMR spectra
were recorded on aVarian Unity-400 spectrometer, and Mass
were obtained on a JMS-HX 100 mass spectrometer. El-
emental analyses were within 0.4% of the theoretical val-
ues, unless otherwise noted. Chromatography was performed
using a flash-column technique on silica gel 60 supplied by
E. Merck.
1
41%): IR (KBr) 3347, 1634, 1610, 1580 cm–1. H-NMR
(CDCl3): d 6.89 (2H, dd, J = 8.2, 1.4 Hz, H-3 and 5), 6.96
(1H, dd, J = 8.2, 2.0 Hz, H-3′), 7.03 (1H, td, J = 8.2, 2.0 Hz,
H-5′), 7.50 (2H, dd, J = 8.2, 1.4 Hz, H-2 and 6), 7.54 (1H, d,
J = 15.2 Hz, H-a), 7.59 (1H, td, J = 8.2, 2.0 Hz, H-4′), 7.89
(1H, d, J = 15.2 Hz, H-b), 7.92 (1H, dd, J = 8.2, 2.0 Hz, H-6′),
12.94 (1H, s, OH-2′). 13C-NMR (CDCl3): d 118 (C-3 and 5),
119.8 (C-3′), 120.5 (C-a), 129.2 (C-2 and 6), 129.5 (C-5′),
129.7 (C-1′), 133.0 (C-6′), 136.4 (C-4′), 136.8 (C-1′), 143.8
(C-b), 163.5 (C-2′ and 4), 193.3 (C=O). EIMS (70 eV) m/z
(% rel. int.): 240 (75) [M]+.
Chalcone 12 has been synthesized as previously reported
[6].
General procedure for obtaining chalcones 1–5, 7, 3,4-
dichloro-2′,5′-dimethoxy-chalcone (8a), 3,4-dichloro-2′,5′-

S.-J. Won et al. / European Journal of Medicinal Chemistry 40 (2005) 103–112
109
5.2. 2′-Hydroxy-2-thienylchalcone (2)
sis of 1 to give 5 (4.24 g, 14.5 mmol, 58%): IR (KBr) 3460,
1643, 1570 cm–1. 1H-NMR (CDCl3): d 6.95 (1H, td, J = 8.1,
1.6 Hz, H-5′), 7.03 (1H, dd, J = 8.1, 1.6 Hz, H-3′), 7.43–7.56
(3H, m, H-4′, 5 and 6), 7.60 (1H, d, J = 15.5 Hz, H-a), 7.73
(1H, d, J = 1.7 Hz, H-2), 7.78 (1H, d, J = 15.5 Hz, H-b), 7.89
(1H, dd, J = 8.1, 1.6 Hz, H-6′), 12.66 (1H, s, OH-2′). 13C-
NMR (CDCl3): d 118.6 (C-2), 118.9 (C-3′), 119.7 (C-1′),
121.6 (C-5), 127.6 (C-a), 129.5 (C-6), 129.8 (C-5′), 130.9
(C-4′), 133.3 (C-1), 134.5 (C-3), 134.7 (C-4), 136.6 (C-6′),
142.4 (C-b), 163.6 (C-2′), 193.0 (C=O). EIMS (70 eV) m/z
(% rel. int.): 392 (22) [M]+.
Compound 1a (5.5 g, 25 mmol), 2-thiophen-aldehyde
(2.8 g, 25 mmol), and barium hydroxide octahydrate (4.29 g,
25 mmol) were treated as in the synthesis of 1 to give 2
(3.06 g, 13.3 mmol, 53%): IR (KBr) 3484, 1636, 1560 cm–1.
1H-NMR (CDCl3): d 6.95 (1H, td, J = 8.2, 1.0 Hz, H-5′), 7.03
(1H, dd, J = 8.2, 1.4 Hz, H-3′), 7.39 (1H, d, J = 3.6 Hz, H-3),
7.43 (1H, dd, J = 4.3, 3.6 Hz, H-4), 7.51 (1H, td, J = 8.2,
1.4 Hz, H-4′), 7.61 (1H, d, J = 4.3 Hz, H-5), 7.62 (1H, d,
J = 15.4 Hz, H-a), 7.86 (1H, d, J = 15.4 Hz, H-b), 7.91 (1H,
dd, J = 8.2, 1.4 Hz, H-6′), 12.70 (1H, s, OH-2′). 13C-NMR
(CDCl3): d 118.5 (C-3′), 118.7 (C-a), 119.8 (C-1′), 128.4
(C-5′), 129.4 (C-3 and 6′), 132.6 (C-4), 136.2 (C-4′ and 5),
137.7 (C-b), 140.0 (C-2), 193.0 (C=O). EIMS (70 eV) m/z
(% rel. int.): 230 (74) [M]+.
5.6. 2′,5′-Dihydroxy-indol-3-yl-chalcone (7)
2′,5′-Bis(tetrahydropyran-2-yloxy)acetophenone
(7a)
was synthesis as previously reported [6]. Compound 7a
(8.00 g, 25 mmol), indole-3-aldehyde (3.62 g, 25 mmol) and
barium hydroxide octahydrate (4.29 g, 25 mmol) were
treated as in the synthesis of 1 to give 7 (1.59 g, 5.73 mmol,
23%): IR (KBr) 3380, 1637, 1558 cm–1. 1H-NMR (acetone-
d6): d 6.83 (1H, d, J = 8.8 Hz, H-3′), 7.09 (1H, dd, J = 8.8,
2.8 Hz, H-4′), 7.26–7.31 (2H, m, H-5, 6), 7.54–7.58 (1H, m,
H-7), 7.60 (1H, d, J = 2.8 Hz, H-6′), 7.74 (1H, d, J = 15.2 Hz,
H-a), 8.08 (1H, s, H-2), 8.10–8.14 (1H, m, H-4), 8.25 (1H, d,
J = 15.2 Hz, H-b), 12.70 (1H, s, OH-2′). 13C-NMR (acetone-
d6): d 114.0 (C-7), 115.2 (C-3), 115.6 (C-6′), 115.8 (C-a),
120.1 (C-3′), 121.5 (C-1′), 121.9 (C-4), 123.1 (C-5), 124.6
(C-6), 125.6 (C-4′), 127.0 (C-3a), 134.8 (C-2), 139.6 (C-7a),
141.6 (C-b), 150.7 (C-2′), 158.4 (C-5′), 194.9 (C=O). EIMS
(70 eV) m/z (% rel. int.): 279 (6.8) [M]+.
5.3. 2′-Hydroxy-3-thienylchalcone (3)
Compound 1a (5.5 g, 25 mmol), 3-thiophen-aldehyde
(2.8 g, 25 mmol), and barium hydroxide octahydrate (4.29 g,
25 mmol) were treated as in the synthesis of 1 to give 3
(2.94 g, 12.8 mmol, 51%): IR (KBr) 3498, 1639, 1560 cm–1.
1H-NMR (CDCl3): d 6.91 (1H, td, J = 8.4, 1.5 Hz, H-5′), 7.01
(1H, dd, J = 8.4, 1.5 Hz, H-3′), 7.35 (1H, dd, J = 5.1, 2.7 Hz,
H-5), 7.41 (1H, dd, J = 5.1, 1.4 Hz, H-4), 7.43 (1H, d,
J = 15.1 Hz, H-a), 7.48 (1H, dd, J = 8.4, 1.5 Hz, H-4′), 7.60
(1H, dd, J = 2.7, 1.4 Hz, H-2), 7.86 (1H, dd, J = 8.4, 1.5 Hz,
H-6′), 7.88 (1H, d, J = 15.1 Hz, H-b), 12.90 (1H, s, OH-2).
13C-NMR (CDCl3): d 118.4 (C-2), 118.6 (C-3′), 119.5 (C-a),
119.8 (C-1′), 125.0 (C-5′), 127.0 (C-4), 129.4 (C-4′), 129.8
(C-5), 136.1 (C-6′), 137.7 (C-3), 138.6 (C-b), 163.3 (C-2′),
193.7 (C=O). EIMS (70 eV) m/z (% rel. int.): 230 (100) [M]+.
5.7. 3,4-Dichloro-2′,5′-dimethoxychalcone (8a)
2,5-Dimethoxyacetophenone (4.50 g, 25 mmol), 3,4-
dichlorobenzaldehyde (4.38 g, 25 mmol), and barium hy-
droxide octahydrate (4.29 g, 25 mmol) were treated as in the
synthesis of 1 to give 8a (4.27 g, 12.6 mmol, 50.4%). Com-
pound 8a was identified with various spectral data compared
with those of authentic sample [6].
5.4. 2′-Hydroxy-2-furfurylchalcone (4)
Compound 1a (5.5 g, 25 mmol), 2-furaldehyde (2.4 g,
25 mmol), and barium hydroxide octahydrate (4.29 g,
25 mmol) were treated as in the synthesis of 1 to give 4
(2.18 g, 10.2 mmol, 41%): IR (KBr) 3425, 1639, 1582 cm–1.
1H-NMR (CDCl3): d 6.52 (1H, dd, J = 3.4, 1.8 Hz, H-4), 6.75
(1H, d, J = 3.4 Hz, H-3), 6.92 (1H, td, J = 8.5, 1.6 Hz, H-5′),
7.01 (1H, dd, J = 8.5, 1.6 Hz, H-3′), 7.48 (1H, td, J = 8.5,
1.6 Hz, H-4′), 7.53 (1H, d, J = 15.0 Hz, H-a), 7.55 (1H, d,
J = 1.8 Hz, H-5), 7.57 (1H, d, J = 15.0 Hz, H-b), 7.90 (1H, dd,
J = 8.5, 1.6 Hz, H-6′), 12.90 (1H, s, OH-2′). 13C-NMR
(CDCl3): d 112.8 (C-3), 117.0 (C-4), 117.5 (C-3′), 118.4
(C-5′), 118.7 (C-a), 119.9 (C-1′), 129.5 (C-4′), 131.0 (C-6′),
136.2 (C-5), 145.3 (C-b), 151.4 (C-2), 163.4 (C-2′), 193.2
(C=O). EIMS (70 eV) m/z (% rel. int.): 214 (53) [M]+.
5.8. 3,4-Dichloro-2′,5′-dihydroxychalcone (9a)
2,5-Dihydroxyacetophenone (3.80 g, 25 mmol) was
treated as in synthesis of 2,5-bis(tetrahydropyran-2-yloxy)-
acetophenone (7a) [6] to give 7a. Compound 7a (8.0 g,
25 mmol), 3,4-dichlorobenzaldehyde (4.38 g, 25 mmol) and
barium hydroxide octahydrate (4.29 g, 25 mmol) were
treated as in the synthesis of 1 to give 9a (5.02 g, 16.3 mmol,
65%). The 9a was identified with various spectral data and
compared with those of authentic sample [6].
5.5. 2′-Hydroxy-3,4-dichlorochalcone (5)
5.9. 4-Chloro-2′,5′-dihydroxychalcone (10a)
Compound 1a (5.5 g, 25 mmol), 3,4-di-chloro-
benzaldehyde (4.38 g, 25 mmol), and barium hydroxide
octahydrate (4.29 g, 25 mmol) were treated as in the synthe-
2,5-Dihydroxyacetophenone (3.80 g, 25 mmol) was
treated as in the synthesis of 9a to give 7a. Compound 7a
(8.0 g, 25 mmol), 4-chloro-benzylaldehyde (3.50 g,

110
S.-J. Won et al. / European Journal of Medicinal Chemistry 40 (2005) 103–112
25 mmol) and barium hydroxide octahydrate (4.29 g,
25 mmol) were treated as in the synthesis of 1 to give 10a.
The 10a was identified with various spectral data and com-
pared with those of authentic sample [4].
(3.30 g, 10.8 mmol, 43.2%): IR (KBr) 3335, 1642,
1514 cm–1. 1H-NMR (acetone-d6): d 3.04 (2H, t, J = 14.8 Hz,
–COCH2CH2–), 3.44 (2H, t, J = 14.8 Hz, –COCH2CH2–),
6.81 (1H, d, J = 8.8 Hz, H-3), 7.09 (1H, dd, J = 8.8, 3.0 Hz,
H-4′), 7.31 (1H, d, J = 8.3, 2.1 Hz, H-6), 7.34 (1H, t,
J = 3.0 Hz, H-6′), 7.46 (1H, d, J = 8.3 Hz, H-5), 7.55 (1H, d,
J = 2.1 Hz, H-2), 11.65 (1H, s, OH-2′). 13C-NMR (acetone-
d6): d 30.0 (–COCH2CH2–), 40.5 (–COCH2CH2–), 116.1
(C-6′), 120.1 (C-3′), 120.5 (C-1′), 126.3 (C-4′), 130.2 (C-6),
130.8 (C-3), 131.8 (C-5), 132.1 (C-2), 133.0 (C-4), 143.9
(C-1), 150.8 (C-5′), 157.1 (C-2′), 206.9 (CO). EIMS (70 eV)
m/z (% rel. int.): 310 (12) [M]+.
5.10. 3,5-Di-tert-butyl-2′,4,5′-trihydroxychalcone (6)
2,5-Dihydroxyacetophenone (3.80 g, 25 mmol) and 3,5-
di-tert-butyl-4-hydroxybenzaldehyde (5.80 g, 25 mmol) dis-
solved in 50 ml of acetone. Al2O3 (12 g) was added to the
above mixture of acetone solution at room temperature and
stirred for 1 min, and the solvent was removed under reduced
pressure. The reaction mixture was then subjected to sonic
agitation (Prothermo Shaker NTS-211) at 40 °C for 12 h [28].
The product was eluted through a alumina column with
n-hexane/CH2Cl2 (1:9) to give 6 (0.96 g, 2.61 mmol, 10.4%):
IR (KBr) 3387, 1639, 1563 cm–1. 1H-NMR (CDCl3): d 1.48
(18H, s, C(CH3)3 × 2), 5.63 (1H, s, OH-5′), 6.92 (1H, d,
J = 8.7 Hz, H-3′), 7.04 (1H, dd, J = 8.7, 2.9 Hz, H-4′), 7.39
(1H, d, J = 2.9 Hz, H-6), 7.40 (1H, d, J = 15.3 Hz, H-a), 7.50
(2H, s, H-2 and 6), 7.91 (1H, d, J = 15.3 Hz, H-b), 12.55 (1H,
s, OH-2′), 13C-NMR (CDCl3): d 30.1 (–CH3), 34.3
(–C(CH3)3), 114.6 (C-6′), 116.5 (C-3′), 119.2 (C-a), 119.9
(C-1′), 124.4 (C-4′), 125.9 (C-3 and 5), 126.3 (C-2 and 6),
136.6 (C-1), 147.3 (C-b), 147.4 (C-4), 157.0 (C-5′), 157.6
(C-2′), 193.3 (C=O). EIMS (70 eV) m/z (% rel. int.): 368 (31)
[M]+. HREIMS m/z [M]+ 368.1988 (calcd. for C23H28O4,
368.1987).
5.13. 5′-Allyloxy-4-chloro-2′-dihydroxydihydrochalcone
(10)
A solution of 10a (5.48 g, 20 mmol) and 5% Pd/C
(100 mg) in ethyl acetate were treated as in the synthesis of 8
to give 4-chloro-2′,5′-dihydroxydihydrochalcone (10b)
(2.76 g, 10 mmol). Compound 10b was identified with vari-
ous spectral data and compared with those of authentic
sample [4]. 3-Bromo-1-propene (1.20 g, 10 mmol) was
added to a solution of 10b (2.76 g, 10 mmol) and anhydrous
K2CO3 (6.00 g, 10 mmol) in DMF (50 ml) and stirred at room
temperature for 18 h. The mixture was diluted with water and
washed three times with water. The organic phase was dried
over sodium sulfate, filtered and concentrated in vacuo to
give the product. The product was purified by chromatogra-
phy over a silica gel column using a n-hexane/ethyl acetate
(2:1) mixture as eluant to afford 10 (0.35 g, 1.12 mmol,
11.2%): IR (KBr) 3387, 1646, 1617 cm–1. 1H-NMR
(CDCl3): d 3.03 (2H, t, J = 14.6 Hz, –COCH2CH2–), 3.27
(2H, t, J = 14.6 Hz, –COCH2CH2–), 4.48 (2H, d, J = 5.2 Hz,
CH2=CHCH2–), 5.30 (1H, d, JE = 11.8 Hz, H2C=CHCH2–),
5.40 (1H, d, JZ = 17.2 Hz, H2C=CHCH2–), 5.94–6.13 (1H,
m, H2C=CH–CH2–), 6.90 (1H, d, J = 8.9 Hz, H-3′), 7.31–
7.09 (6H, m, H-2, 3, 4′, 5, 6, 6′), 11.84 (1H, s, OH-2′).
General Procedure for obtaining dihydrochalcones 8–11.
5.11. 3,4-Dichloro-2′,5′-dimethoxydihydrochalcone (8)
A solution of 8a (8.50 g, 25 mmol) in ethyl acetate (50 ml)
was hydrogenated in an autoclave for 3 h at an initial pressure
60 kg cm–3 in the presence of 5% Pd/C (100 mg) at room
temperature. The catalyst was removed by filtration and the
filtrate was evaporated in vacuo and purified by chromatog-
raphy over a silica gel column using a n-hexane/ethyl acetate
(5:1) mixture as eluant to afford the crude product. The crude
product was recrystallized from CHCl3 to give 8 (4.27 g,
13C-NMR (CDCl3):
d
29.2 (–COCH2CH2–), 39.7
(–COCH2CH2–), 69.8 (–OCH2–), 114.0 (H2C=CHCH2–),
117.9 (C-3′), 118.7 (C-1′), 119.3 (C-6′), 124.9 (C-4′), 128.6
(C-3 and 5), 129.7 (C-2 and 6), 132.0 (C-4), 133.0
(H2C=CHCH2–), 139.1 (C-1), 150.6 (C-5′), 156.9 (C-2′),
204.3 (CO). EIMS (70 eV) m/z (% rel. int.): 316 (1) [M]+.
1
12.6 mmol, 50.4%): IR (KBr) 1662, 1596 cm–1. H-NMR
(CDCl3): d 2.97 (2H, t, J = 14.6 Hz, –COCH2CH2–), 3.29
(2H, t, J = 14.6 Hz, –COCH2CH2–), 3.78 (3H, s, OMe), 3.85
(3H, s, OMe), 6.89 (1H, d, J = 8.9 Hz, H-3′), 7.02 (1H, dd,
J = 8.9, 3.0 Hz, H-4′), 7.09 (1H, d, J = 1.7 Hz, H-2), 7.25 (1H,
d, J = 3.0 Hz, H-6′), 7.32 (1H, dd, J = 8.1, 1.7 Hz, H-6), 7.32
(1H, d, J = 8.1 Hz, H-5). 13C-NMR (CDCl3): d 29.4
(–COCH2CH2–), 44.7 (–COCH2CH2–), 55.7 (OMe), 55.9
(OMe), 112.9 (C-6′), 113.8 (C-3′), 120.1 (C-4′), 127.8 (C-
1′), 127.9 (C-5), 129.6 (C-1), 130.1 (C-2), 130.3 (C-6), 132.2
(C-4), 141.9 (C-3), 153.0 (C-5′), 153.3 (C-2′), 200.1 (CO).
EIMS (70 eV) m/z (% rel. int.): 338 (9) [M]+.
5.14. 4-Chloro-2′,5′-dihydroxy-3′-(1-methylallyl)dihydro-
chalcone (11)
3-Bromo-1-butene (1.35 g, 10 mmol) was added to a
solution of 10b (2.76 g, 10 mmol) and anhydrous K2CO3
(6.00 g, 10 mmol) in DMF (50 ml) and refluxed for 18 h. The
mixture was treated as in the synthesis of 10 to give 11
(0.207 g, 0.63 mmol, 7.3%): IR (KBr) 3421, 1637,
1
1591 cm–1. H-NMR (CDCl3): d 1.29 (3H, d, J = 7.0 Hz,
5.12. 3,4-Dichloro-2′,5′-dihydroxydihydrochalcone (9)
CH3), 2.99 (2H, t, J = 14.0 Hz, –COCH2CH2–), 3.20 (2H, t,
J = 14.0 Hz, –COCH2CH2–), 3.95 (1H, m, –CH(CH3)–), 5.06
(1H, m, JE = 10.3 Hz, H2C=CH–), 5.08 (1H, m, JZ = 17.2 Hz,
A solution of 9a (7.70 g, 25 mmol) and 5% Pd/C (100 mg)
in ethyl acetate were treated as in the synthesis of 8 to give 9

S.-J. Won et al. / European Journal of Medicinal Chemistry 40 (2005) 103–112
111
H2C=CH–), 5.70 (1H, s, OH-5′), 5.90–6.07 (1H, m,
H2C=CH–), 6.95 (1H, d, J = 2.9 Hz, H-6′), 7.05 (1H, d,
J = 2.9 Hz, H-4′), 7.13 (2H, dd, J = 8.2, 2.1 Hz, H-3 and 5),
7.24 (2H, dd, J = 8.2, 2.1 Hz, H-2 and 6), 12.3 (1H, s, OH-2′).
13C-NMR (CDCl3): d 18.8 (CH3), 29.2 (–CO–CH2CH2–),
34.9 (=CH–CH(CH3)–), 39.8 (–COCH2–), 112.2 (C-6′),
113.7 (H2C=CH–), 118.4 (C-1′), 123.0 (C-4′), 128.6 (C-
3 and 5), 129.7 (C-2 and 6), 132.0 (C-3′), 136.3 (C-1), 139.0
(C-4), 141.5 (H2C=CH–), 147.0 (C-5′), 154.3 (C-2′), 204.7
(CO). EIMS (70 eV) m/z (% rel. int.): 330 (29) [M]+.
After the addition of 100 µl lysis buffer [1% of NP-40
(Sigma) in 20 mM EDTA, 50 mM Tris–HCl, pH 7.5] and
mixing; the cell lysates were then centrifuged and the super-
natants were collected. The supernatants were incubated with
50 µl of RNase A (20 mg ml–1) and 20 µl of SDS (10%) at
56 °C for 2 h. Then, 35 µl of proteinase K (20 mg ml–1) was
added and incubated at 37 °C overnight. DNA fragments
were precipitated after the addition of 150 µl of 10 M
NH4OAc and 1.2 ml of 100% ethanol at −20 °C overnight.
After centrifuging and drying, the DNA pellets were sus-
pended in 15 µl Tris–EDTA buffer and electrophoresed on a
1% (W/V) agarose gel in TBE buffer at 50 V for 1 h, DNA
laddering was observed after staining with ethidium bromide
solution and exposing to the UV light [39].
5.15. Tumor cell growth inhibition assays
A microassay for cytotoxicity was performed using MTT
[36,37]. Briefly, 1–3 × 103 cells/100 µl were seeded in 96-
well microplates (Nunk, Roskidle, Denmark) and preincu-
bated for 6 h to allow attachment. The cells were incubated
with each drug for 6 days and then pulsed 10 µl of MTT
(5 mg ml–1; Sigma, St. Louis, MO) and incubated for an
additional 4 h at 37 °C. The microplates were read 550 nm on
a Multiskan Photometer (MRX II; Dynatech, McLean, VA)
after lysis of cells with 100 µl of 10% sodium dodecyl sulfate
(SDS) in 0.01 M HCl. Control wells contained medium and
cells (total absorbance) or medium alone (background absor-
bance). Cell death was calculated as the percentage of MTT
inhibition.
Human hepatomacellular carcinoma Hep 3B, T-24, hu-
man colorectal adenocarcinoma HT-29, and human breast
adenocarcinoma MCF-7 cells were obtained from American
Type Culture Collection (ATCC; Rockville, MO) and grown
in DMEM [37,38], containing 10% FBS, 2 mM L-glutamine,
100 units ml–1 of penicillin, and 100 µg ml–1 of streptomycin.
For the microassay, the growth medium was supplemented
with 10 mM HEPES (4-(2-hydroxyethyl)piperazine-1-
ethanesulfonic acid) buffer pH 7.3 and incubated at 37 °C in
a CO2 incubator.
Acknowledgments
This work was supported by a grant from the National
Science Council of the Republic of China (NSC 91-2320-
B037-031).
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