Synthesis and cytotoxic, anti-inflammatory, and anti-oxidant activities of 2′,5′-dialkoxylchalcones as cancer chemopreventive agents

Article abstract of DOI:10.1016/j.bmc.2008.06.031

In an effort to develop novel anti-tumor, or cancer chemopreventive agents, a series of 2′,5′-dialkoxylchalcones were prepared by Claisen-Schmidt condensation of appropriate acetophenones with suitable aromatic aldehyde. In vitro screening revealed low mi

Full text of DOI:10.1016/j.bmc.2008.06.031

Bioorganic & Medicinal Chemistry 16 (2008) 7270–7276
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and cytotoxic, anti-inflammatory, and anti-oxidant activities
of 20,50-dialkoxylchalcones as cancer chemopreventive agents
c
Jen-Hao Cheng a, , Chi-Feng Hung b, , Shyh-Chyun Yang a, , Jih-Pyang Wang ,
*
Shen-Jeu Won d, Chun-Nan Lin a,e,f,
*
a School of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan
b School of Medicine, Fu-Jen Catholic University, Taipei County 242, Taiwan
c Department of Education and Research, Taichung Veterans General Hospital, Taichung 407, Taiwan
d Departmant of Microbiology and Immunology, National Cheng Kung University, Tainan 701, Taiwan
e Institute of Life Science, National Taitung University, Taitung 950, Taiwan
f Center of Excellence for Environmental Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan
a r t i c l e i n f o
a b s t r a c t
In an effort to develop novel anti-tumor, or cancer chemopreventive agents, a series of 20,50-dialkoxyl-
chalcones were prepared by Claisen–Schmidt condensation of appropriate acetophenones with suitable
aromatic aldehyde. In vitro screening revealed low micromolar activity (IC50) against several human can-
cer cell lines. Selective compound 10 induced an accumulation of A549 cells in the G2/M phase arrest
which was well correlated with inhibitory activity against tubulin polymerization. Cytotoxic compounds
3 and 12 showed significant inhibitory effects on NO production in lipopolysaccharide (LPS)-activated
RAW 264.7 macrophage-like cells while cytotoxic compound 10 revealed potent inhibitory effect on
Article history:
Received 4 March 2008
Revised 17 June 2008
Accepted 18 June 2008
Available online 21 June 2008
Keywords:
Dialkoxylchalcones
Synthesis
Cytotoxicity
Anti-inflammatory activity
Anti-oxidant activity
TNF-
a formation in RAW 264.7 cells in response to LPS. Compounds 3 and 10 also showed significant
inhibitory effects on xanthine oxidase. The present results suggested that compounds 3 and 10 were
potential to be served as cancer chemopreventive agents.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
20,50-Dimethoxy-2-(5-methylthienyl)chalcone was also suggested
to be a significant G2/M arrest-mediated apoptosis-inducing agent
Chalcones are characterized with diverse biological activities,
among which anti-inflammatory, anti-malaria, anti-protozoal,
anti-bacterial, nitric oxide inhibition, tyrosinase inhibition, cyto-
toxic, anticancer, and anti-leishmanial activities have been re-
ported in the past decade.1–5 In our previous papers, we have
demonstrated that various synthetic chalcones were potential can-
cer chemopreventive agents.6,7
Mast cells, neutrophils and macrophages play important roles in
inflammatory disorders. Nitric oxide (NO) accumulation from
macrophages induced cytotoxicity and expressed NO may lead to
pathophysiology of septic shock.8 The excess production of NO also
can destroy functional normal tissues during acute and chronic
inflammation related mechanistically to carcinogenesis.9 Among
the synthetic chalcone derivatives in our previous paper, 20,50-dime-
thoxy-4-hydroxychalcone showed potent inhibitory effect on NO
accumulation from RAW 264.7 cells and potent selective cytotoxic-
ity against human MCF-7 cells and caused cell death by apoptosis.7
and inhibitor of NO production in rat macrophages.7
Reactive oxygen species (ROS) is implicated in numerous
pathological events including inflammation, metabolic disorders,
cellular aging, reperfusion damage, artherosclerosis, and carcino-
genesis.10 The oxidative damage of DNA induced by ROS lead to
certain cancers. Xanthine oxidase (XO) is a key enzyme that cata-
lyzes the oxidation of xanthine and hypoxanthine into uric acid
and plays a vital role in causing hyperuricemia and gout.11 In the
investigation on anti-oxidant agent, we found that synthetic chal-
cones were significantly inhibited oxidative damage of DNA and
XO activity. For continual discovery of potential cancer chemopre-
ventive agents with cytotoxic, anti-inflammatory and anti-oxidant
activities, we reported the synthesis, cytotoxicity, anti-inflamma-
tory activity and anti-oxidant activity of a series of 20,50-dialkoxyl
and related chalcones in the present paper.
2. Results and discussion
2.1. Chemistry
* Corresponding authors. Tel.: +886 7 3121101; fax: +886 7 5562365.
E-mail addresses: scyang@kmu.edu.tw (S.-C. Yang), lincna@cc.kmu.edu.tw (C.-N.
Lin).
As shown in Scheme 1, new chalcones, 1, 9, 13, and 14 were pre-
pared by using Claisen–Schmidt condensation of appropriate
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.06.031

J.-H. Cheng et al. / Bioorg. Med. Chem. 16 (2008) 7270–7276
7271
O
O
O
OH
OH
i
ii
+
CH3
CH3
CH3
OHC
S
C
O
C
O
O
1a
1b
O
O
O
4
3
2
OR'
OH
OH
5
S
5'
β
6'
S
4'
3'
S
iv
iii
α
1'
O
O
2'
OR
O
O
1c
1
2-12
Scheme 1. Reagents and conditions: (i) Pyridinium p-toluenesulfonate, 3,4-dihydro-
5% NaHCO3; (iv) alkyl halides, K2CO3, acetone, rt.
a
-pyran, CH2Cl2, rt, 4 h; (ii) BaOH ꢀ 8H2O, MeOH, 40 °C; (iii) p-toluenesulfonic acid, rt, 4 h,
hydroxyacetophenones, protected as tetrahydropyranyl ether,
with appropriate aromatic aldehyde.12 A mixture of 1, appropriate
alkyl halide, and potassium carbonate in appropriate solvent were
stirred at room temperature to afford new chalcones, 2–8 and 10–
12. These procedures afforded various chalcone derivatives in a
good yield (Table 1).
2.2. Biological results and discussion
The MTT microassay was used for the evaluation of cytotoxic
properties of the synthetic compounds in this research. The
growth-inhibitory effects was undertaken in four human cell lines,
including MCF-7 (breast), A549 (lung), Hep3B (liver) and HT-29
Table 1
Structure and analytical data of chalcones
OR'
R''O
HO
S
S
OH
O
OR
O
1-12
13,14
Compound
R
R0
H
R00
Formula
Mp (°C)
Yield%
Analysisa
1
2
3
H
H
C14H12O3S
C16H16O3S
C18H20O3S
184–185
99–100
68–69
58
89
77
C, H, S
C, H, S
C, H, S
CH2CH3
CH2CH3
CH2CH3
4
H
C17H18O3S
83–84
85
C, H, S
5
6
H
H
C17H18O3S
C18H20O3S
100–101
86–87
79
75
C, H, S
C, H, S
7
8
H
H
C18H20O3S
C19H22O3S
99–100
82–83
64
68
C, H, S
C, H, S
9
10
H
CH3
CH3
C15H14O3S
C17H18O3S
78–79
70–71
55
71
C, H, S
C, H, S
CH2CH3
11
CH3
CH3
C18H20O3S
Oil
65
C, H, S
12
C19H22O3S
C15H14O4S
Oil
53
C, H, S
13
14
CH3
H
131–132
182–183
48
46
C, H, S
C, H, S
C
14H12O4S
a
C, H and S analyses were within 0.4% of the theoretical values.

7272
J.-H. Cheng et al. / Bioorg. Med. Chem. 16 (2008) 7270–7276
(colorectal) followed the method described previously.13 The re-
sults are listed in Table 2. All compounds in Table 2 showed selec-
tive cytotoxicity against MCF-7 cells except for compound 10. In
our previous report, we have definitely indicated that 5-methylthi-
ophene substituted at B ring of 20,50-dimethoxychalcone signifi-
cantly enhanced the cytotoxicity.7 In this study, we founded that
the different alkoxyl groups and hydroxy groups substituted at
both C-20 and C-50 leaded to different cytotoxic effects. Compounds
2 and 4–9 with hydroxyl group at C-20 and various alkoxyl groups
at C-50 showed different cytotoxic effect against several human
cancer cell lines used in Table 2. The increasing length of alkoxyl
branched side chains at C-50 significantly enhanced cytotoxic activ-
ities against MCF-7 and HT-29 cells, while both C-20 and C-50 were
substituted with ethoxy groups, such as compound 3, revealed
more stronger cytotoxic effects than that of 2, especially for cyto-
toxicity against MCF-7 cells. Compound 10, a 20-ethoxy-50-meth-
oxy chalcone derivative revealed potent cytotoxicity against the
four human cancer cell lines used in Table 2. We previously re-
ported 20,50-dimethoxy-2-(5-methylthienyl)chalcone, 15, showed
potent cytotoxicity against A 549, Hep3B, HT-29 and MCH-7 cells.7
When the methoxyl group substituted at C-20 of 15 replaced by
ethoxyl group, such as 10, significantly enhanced the cytotoxicity
against MCF-7, Hep3B, and HT-29 cells.
Whereas cell proliferation and differentiation are specifically
controlled in the G1 phase and the G1-S transition in the cell
cycle, oncogenic progress exert their greatest effect by targeting
particular regulators of G1 phase progression.14 To determine
the phase of the cell cycle of human A549 cells at which selective
compound 10 exerts its growth-inhibitory effect, cells were trea-
ted with different concentration of 10 for 24 h and analyzed by
flow cytometry (Fig. 1 and Table 3). In the cell cycle of 10, G2/M
Table 2
Cytotoxicity of 2–14 (IC50 values in l
M)a
Compound
Cell line
MCF-7
A549
Hep3B
HT-29
2
3
4
5
6
7
8
9
10
11
12
13
14
5-Fu
23.2 2.1
1.4 1.9
21.5 2.1
15.5 2.1
38.9 1.2
13.0 2.2
48.4 2.2
34.6 2.1
2.3 2.1
0.8 0.3
13.9 1.6
20.7 1.5
—
—
90.2 2.0
31.6 2.3
23.2 2.3
81.7 2.0
91.7 1.2
54.4 0.5
112.0 1.2
87.5 0.9
0.2 0.1
7.0 0.1
10.7 1.5
10.7 2.1
—
43.4 1.2
3.2 2.0
phase arrest was apparent after treatment with 10 lM of 10. This
4.4 2.6
—
—
—
—
—
—
6.3 2.4
—
—
—
125.7 2.0
30.2 0.5
71.4 0.9
30.7 0.6
86.9 2.1
71.5 1.5
0.1 0.3
86.6 0.3
61.1 2.3
53.4 2.3
35.9 0.5
0.6 0.1
indicated that the G2/M phase arrest of 10 is probably induced by
inhibition of tubulin polymerization, which is also correlated with
cell growth inhibition.15 These results demonstrated that this ser-
ies may be a anti-mitotic agent inhibiting tubulin polymerization
with anti-proliferative activity.
Selective compounds 3, 10, 11, 12, and 13 with significant cyto-
toxicity against several human cancer cells were studied in vitro
for their inhibitory effects on chemical mediators released from
mast cells, neutrophils and macrophages. These compounds had
no significant inhibitory effects on the release of b-glucuronidase
from rat mast cells stimulated with compound 48/80 (data not
shown), the release of b-glucuronidase and lysozyme from neutro-
phils stimulated with fromyl-Met-Leu-Phe (fMLP)/cytochalasin B
(CB) (data not shown) and superoxide anion generation in rat neu-
—
1.5 0.1
3.1 0.2
0.6 0.3
a
Data are presented as means SEM (n = 6). Compounds 2–14 or 5-Fu dissolved
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. —, not determined.
Figure 1. Flow cytometric analysis of the DNA histograms of propidium iodide (PI)-stained A549 cells. The cells were incubated with various concentrations of 10 for 24 h.
After incubation, the cells were fixed and incubated with PI and RNase before reading red fluorescence exited by blue light. The control (C) cells were treated without 10.

J.-H. Cheng et al. / Bioorg. Med. Chem. 16 (2008) 7270–7276
7273
Table 3
Cell populations of cell cycle of A549 cells treated with various concentration of 10
100
G1
%
S %
G2/M %
80
60
40
20
0
C
67.857
70.207
65.919
64.102
30.683
27.69
28.384
24.271
1.46
2.104
5.698
11.627
10 (0.03 lM)
10 (0.1
10 (10
l
M)
l
M)
The cell populations of cell cycle were analyzed by LYSIS II software. The control (C)
cells were treated with medium.
trophils stimulated with fMLP/CB or phorbol myristate (PMA) (data
not shown). Treatment of RAW 264.7 macrophage-like cells with
LPS 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.16 As shown in Figure 2, compounds 3 and 12
showed potent and concentration-dependent inhibitory effects
on NO accumulation from 264.7 cells with IC50 values of
10
Genistein
0
5
10
15
20
25
30
35
Concentration (μM)
Figure 3. The inhibitory effects of 10 on the accumulation of TNF-
the culture media of RAW 264.7 cells in response to LPS (1
presented as mean SD (n = 3). Genistein was used as a positive control.
a
formation in
16.5 1.5 and 28.0 4.2 lM, respectively. NO plays a central role
l
g/mL). Data are
in macrophage-induced cytotoxicity and expressed NO may con-
tribute to the pathophysiology of septic shock.8 NO with excessive
production also lead to destruction of functional normal tissues
during acute and chronic inflammation.9 Effect on the generation
itory effect on XO. For determination of the anti-oxidant activity of
3 and 10, the radical scavenging activity and XO inhibitory activity
of 3 and 10 were analyzed. As shown at Figure 5, compound 3 and
allopurinol (positive control) significantly inhibit the XO activity in
a concentration-dependent manner with IC50 values of 21.3 15.3
of tumor necrosis factor-
a (TNF-a) was determined in RAW
264.7 cells activated by LPS.17–19 As shown in Figure 3, compound
10 showed significantly inhibitory effects on the generation of
TNF-
0.4 0.2
ation of TNF-
oxy and 50-methoxy groups substituted on the A ring revealed
significant inhibition of TNF- in RAW 264.7 cells. The strong inhi-
bition of TNF- and potent cytotoxic activity against the cell lines
a
in RAW 264.7 cells activated by LPS with an IC50 value of
M. It indicated a stronger inhibitory effect on the gener-
than that of positive control (genistein) and 20-eth-
l
and 2.0 0.7
lM, respectively, while compound 10 weakly inhibit
a
a
a
used in Table 2 of 10 suggested that 10 may be a potential cancer
chemopreventive agent.
The anti-oxidant efficiency of free radical scavenging activities,
oxidase inhibition and inhibition of oxidative DNA damage were
studied in vitro by agarose gel electrophoresis.20 As shown in Fig-
ure 4, selective compounds, 3 and 10, showed protective effects of
ꢁÅ
oxidative DNA damage caused by O2 generated by xanthine (XA)/
xanthine oxidase (XO). Compounds 3 and 10 each at 300 lM
showed protective effect on oxidative DNA damage caused by
O2ꢁÅ. The above results clearly indicated that 3 and 10 possessed
1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity or inhib-
ꢁÅ
Figure 4. Inhibition of DNA strand breaks induced by O2 (generated by XA/XO) in
the presence of 3 and 10 studied by gel electrophoresis. Supercoiled plasmid
pBR322 DNA (500 ng) in phosphate buffer (pH 7.4) solution was incubated for
20 min with XA/XO acting as the control. Lane 1, DNA (without XA/XO); lane 2,
control; lane 3, control + SOD (300
serving as positive control; lane 5, control + 3 (300
(300 M).
l
M); lane 4, control + quercetin (300
lM)
l
M); lane 6, control + 10
l
100
100
80
60
40
20
0
3
80
60
40
20
0
10
11
12
13
1400W
allopurinol
-20
3
10
0
10
20
30
40
50
Concentration (μM)
0
50
100
150
200
ꢁ
Concentration (μM)
Figure 2. The inhibitory effects of 3 and 10–13 on the accumulation of NO2
formation in the culture media of RAW 264.7 cells in response to LPS (1 g/mL).
l
Data are presented as mean SD (n = 3). N-(3-Aminomethyl)benzylacetamidine
(1400 W) was used as a positive control.
Figure 5. Dose-dependent inhibition of XO by 3, 10, and allopurinol. Data are
presented as means SEM, n = 3–6.

7274
J.-H. Cheng et al. / Bioorg. Med. Chem. 16 (2008) 7270–7276
the XO activity also in a concentration-dependent manner with an
chromatographed over a silica gel column and eluted with appro-
IC50 value of 131.0 10.9
lM. Compounds 3 and 10 did not show
priate solvent to yield compounds 2–8, 10–12.
DPPH scavenging activity.
4.2.3. 50-Ethoxy-20-hydroxy-2-(5-methylthienyl)chalcone (2)
Compound
1
(2.95 g, 10.2 mmol), ethyl iodide (1.59 g,
3. Conclusion
10.2 mmol) and potassium carbonate (1.41 g, 10.2 mmol) were
treated as in general method for the synthesis of chalcone to afford
2 (2.62 g, 9.1 mmol, 89%) as orange crystals, mp 99–100 °C. IR (KBr)
In this study, we have synthesized a series of new compounds of
20,50-dialkoxy-2-(5-methylthienyl)chalcone and studied on their
biological activities of cytotoxicity, anti-inflammation, anti-oxida-
tion, and xanthine oxidase inhibitory effect. This series of com-
pounds revealed cytotoxicity against human cancer cells. Among
them, compounds 3 and 10 showed potent cytotoxicity against a
several human cancer cells, potent activity on the inhibition of
oxidative DNA damage, potent anti-inflammatory activity, and
anti-oxidant effect. The present research suggested that com-
pounds 3 and 10 may be used as cancer chemopreventive agents.
3446, 1646, 1570 cmꢁ1 1H NMR (CDCl3): d 1.44 (3H, t, J = 6.8 Hz,
;
CH3), 2.55 (3H, s, CH3), 4.05 (2H, q, J = 7.2 Hz, OCH2), 6.78 (1H,
dd, J = 3.6, 1.2 Hz, H-3), 6.95 (1H, d, J = 8.8 Hz, H-30), 7.12 (1H, dd,
J = 8.8, 2.8 Hz, H-40), 7.21 (1H, d, J = 3.6 Hz, H-4), 7.23 (1H, d,
J = 15.2 Hz, H-a
), 7.32 (1H, d, J = 2.8 Hz, H-60), 7.97 (1H, d,
J = 15.2 Hz, H-b), 12.47 (1H, s, OH). 13C NMR (CDCl3): d 14.9
(CH3), 16.0 (CH3), 64.5 (OCH2), 114.0 (C-60), 117.4 (C-30), 119.1
(C-a
), 119.7 (C-10), 124.0 (C-3), 127.1 (C-40), 133.8 (C-4), 138.2
(C-2), 138.4 (C-b), 145.6 (C-5), 150.9 (C-20), 157.7 (C-50), 192.8
(C@O). EIMS (70 eV) m/z (% rel. int.): 288 (39) [M]+.
4. Experimental
4.1. Chemistry
4.2.4. 20,50-Diethoxy-2-(5-methylthienyl)chalcone (3)
Compound
1
(2.95 g, 10.2 mmol), ethyl iodide (3.18 g,
20.4 mmol) and potassium carbonate (1.41 g, 10.2 mmol) were
treated as in general method for the synthesis of chalcone to afford
3 (2.50 g, 7.9 mmol, 77%) as yellow crystals, mp 68–69 °C. IR (KBr)
Melting points (uncorrected) were determined with a Yanco
micro melting point apparatus. IR spectra were determined with
a
Perkin-Elmer system 2000 FTIR spectrophotometer. 1H
1641, 1565 cmꢁ1 1H NMR (CDCl3): d 1.39 (3H, t, J = 7.2 Hz, CH3),
;
(400 MHz) and 13C (100 MHz) NMR 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.
Chromatography was performed using a flash-column technique
on silica gel 60 supplied by E. Merck.
1.42 (3H, t, J = 6.8 Hz, CH3), 2.51 (3H, s, CH3), 4.02 (2H, q,
J = 7.2 Hz, OCH2), 4.07 (2H, q, J = 7.2 Hz, OCH2), 6.72 (1H, dd,
J = 3.6, 1.2 Hz, H-3), 6.90 (1H, d, J = 8.8 Hz, H-30), 7.00 (1H, dd,
J = 8.8, 3.2 Hz, H-40), 7.10 (1H, d, J = 3.6 Hz, H-4), 7.23 (1H, d,
J = 3.2 Hz, H-60), 7.25 (1H, d, J = 15.6 Hz, H-
a), 7.71 (1H, d,
J = 15.2 Hz, H-b). 13C NMR (CDCl3): d 14.9 (CH3), 15.0 (CH3), 15.9
(CH3), 64.1 (OCH2), 65.1 (OCH2), 114.6 (C-60), 115.0 (C-30), 120.2
(C-a
), 124.9 (C-3), 126.7 (C-40), 129.6 (C-10), 132.4 (C-4), 135.7
4.2. Synthesis of chalcones
(C-b), 138.9 (C-2), 144.0 (C-5), 152.1 (C-20), 152.9 (C-50), 191.4
(C@O). EIMS (70 eV) m/z (% rel. int.): 316 (57) [M]+.
4.2.1. 20,50-Dihydroxy-2-(5-methylthienyl)chalcone (1)
20,50-Dihydroxyacetophenone (3.8 g, 25 mmol), 3,4-dihydro-
a-
4.2.5. 20-Hydroxy-50-propoxy-2-(5-methylthienyl)chalcone (4)
Compound 1 (2.95 g, 10.2 mmol), normal propyl iodide (1.73 g,
10.2 mmol) and potassium carbonate (1.41 g, 10.2 mmol) were
treated as in general method for the synthesis of chalcone to afford
4 (2.63 g, 8.7 mmol, 85%) as orange crystals, mp 83–84 °C. IR (KBr)
pyran (13 mL) and pyridinium p-toluenesulfonate (0.15 g,
0.6 mmol) were reacted in CH2Cl2 (100 mL) to give 20,50-di(tetra-
hydropyran-2-yloxy)acetophenone (1a). Compound 1a (8.0 g,
25 mmol), 5-methylthiophene-2-aldehyde (3.2 g, 25 mmol) and
barium hydroxide octahydrate (4.29 g, 25 mmol) were refluxed in
methanol (100 mL) and then the solvent was evaporated under re-
duced pressure. The residue and p-toluenesulfonic acid (0.1 g,
0.6 mmol) in methanol (100 mL) was stirred for 4 h at room tem-
perature, and then evaporated in vacuo. Water was added to the
mixture, neutralized with 5% NaHCO3 (50 mL) and extracted with
EtOAc. The organic layer was separated, washed with H2O, dried
and evaporated in vacuo. The residue was eluted through a silica
3446, 1641, 1570 cmꢁ1 1H NMR (CDCl3): d 1.07 (3H, t, J = 7.2 Hz,
;
CH3), 1.83 (2H, m, CH2), 2.55 (3H, s, CH3), 3.93 (2H, t, J = 6.4 Hz,
OCH2), 6.78 (1H, dd, J = 3.6, 1.2 Hz, H-3), 6.95 (1H, d, J = 9.2 Hz,
H-30), 7.12 (1H, dd, J = 9.2, 3.2 Hz, H-40), 7.21 (1H, d, J = 3.6 Hz, H-
4), 7.25 (1H, d, J = 15.2 Hz, H-a
), 7.31 (1H, d, J = 3.2 Hz, H-60), 7.97
(1H, d, J = 15.2 Hz, H-b), 12.47 (1H, s, OH). 13C NMR (CDCl3): d
10.5 (CH3), 16.0 (CH3), 22.7 (CH2), 70.6 (OCH2), 113.9 (C-60),
117.4 (C-30), 119.1 (C- ), 119.7 (C-10), 124.1 (C-3), 127.1 (C-40),
a
gel column with n-hexane/EtOAc, 4:1 to afford
1 (3.77 g,
133.7 (C-4), 138.2 (C-2), 138.4 (C-b), 145.6 (C-5), 151.1 (C-20),
157.7 (C-50), 192.8 (C@O). EIMS (70 eV) m/z (% rel. int.): 302 (70)
[M]+.
14.5 mmol, 58%) as red crystals, mp 184–185 °C. IR (KBr) 3442,
1635, 1569 cmꢁ1 1H NMR (CDCl3): d 2.60 (3H, s, CH3), 6.78 (1H,
;
dd, J = 3.6, 0.8 Hz, H-3), 6.92 (1H, d, J = 8.8 Hz, H-30), 7.04 (1H, dd,
J = 8.8, 2.8 Hz, H-40), 7.21 (1H, d, J = 3.6 Hz, H-4), 7.21 (1H, d,
J = 14.8 Hz, H-
J = 14.8 Hz, H-b), 12.46 (1H, s, OH). 13C NMR (CDCl3): d 16.0
a
), 7.32 (1H, d, J = 2.8 Hz, H-60), 7.96 (1H, d,
4.2.6. 20-Hydroxy-50-iso-propoxy-2-(5-methylthienyl)chalcone
(5)
(CH3), 114.4 (C-30), 117.3 (C-40), 119.3 (C- ), 119.8 (C-10), 124.6
a
Compound 1 (2.95 g, 10.2 mmol), isopropyl iodide (1.73 g,
10.2 mmol) and potassium carbonate (1.41 g, 10.2 mmol) were
treated as in general method for the synthesis of chalcone to afford
5 (2.44 g, 8.1 mmol, 79%) as orange crystals, mp 100–101 °C. IR
(C-3), 127.1 (C-60), 133.9 (C-4), 138.2 (C-2), 138.6 (C-b), 145.7 (C-
5), 147.2 (C-50), 157.8 (C-20), 192.6 (C@O). EIMS (70 eV) m/z (%
rel. int.): 260 (46) [M]+.
(KBr) 3446, 1637, 1570 cmꢁ1 1H NMR (CDCl3): d 1.35 (6H, d,
;
4.2.2. General procedure for obtaining chalcones 2–8, 10–12
Appropriate 1, alkyl iodide and potassium carbonate were re-
acted in acetone at room temperature. The solvent was evaporated
under reduced pressure. The residue was dissolved in 10% HCl and
extracted with EtOAc. The organic layer was separated, washed
with H2O, dried and evaporated in vacuo. The residue was
J = 6.0 Hz, 2 ꢂ CH3), 2.55 (3H, s, CH3), 4.47 (1H, m,OCH), 6.78 (1H,
dd, J = 3.6, 1.2 Hz, H-3), 6.94 (1H, d, J = 8.8 Hz, H-30), 7.12 (1H, dd,
J = 8.8, 2.8 Hz, H-40), 7.21 (1H, d, J = 3.6 Hz, H-4), 7.31 (1H, d,
J = 15.2 Hz, H-a
), 7.35 (1H, d, J = 2.8 Hz, H-60), 7.96 (1H, d,
J = 15.2 Hz, H-b), 12.47 (1H, s, OH). 13C NMR (CDCl3): d 16.0
(CH3), 22.1 (2 ꢂ CH3), 71.7 (OCH), 116.8 (C-60), 117.4 (C-30), 119.1

J.-H. Cheng et al. / Bioorg. Med. Chem. 16 (2008) 7270–7276
7275
(C-
a
), 119.9 (C-10), 125.7 (C-3), 127.1 (C-40), 133.8 (C-4), 138.2 (C-
(1H, d, J = 2.8 Hz, H-60), 7.95 (1H, d, J = 15.2 Hz, H-b), 12.49 (1H, s,
13
2), 138.4 (C-b), 145.6 (C-5), 149.6 (C-20), 157.9 (C-50), 192.8 (C@O).
EIMS (70 eV) m/z (% rel. int.): 302 (45) [M]+.
OH). C NMR (CDCl3): d 15.9 (CH3), 56.0 (OCH3), 112.7 (C-60),
117.3 (C-30), 119.2 (C- ), 119.6 (C-10), 123.5 (C-3), 127.1 (C-40),
a
133.7 (C-4), 138.1 (C-2), 138.4 (C-b), 145.6 (C-5), 151.6 (C-20),
157.8 (C-50), 192.7 (C@O). EIMS (70 eV) m/z (% rel. int.): 274 (18)
[M]+.
4.2.7. 50-Butoxy-20-hydroxy-2-(5-methylthienyl)chalcone (6)
Compound 1 (2.95 g, 10.2 mmol), normal butyl iodide (1.88 g,
10.2 mmol) and potassium carbonate (1.41 g, 10.2 mmol) were
treated as in general method for the synthesis of chalcone to afford
6 (2.42 g, 7.7 mmol, 75%) as red crystals, mp 86–87 °C. IR (KBr)
4.2.11. 20-Ethoxy-50-methoxy-2-(5-methylthienyl)chalcone (10)
Compound
9
(2.88 g, 10.5 mmol), ethyl iodide (1.64 g,
3446, 1628, 1559 cmꢁ1
;
1H NMR (CDCl3): d 1.00 (3H, t, J = 7.2 Hz,
10.5 mmol) and potassium carbonate (1.45 g, 10.5 mmol) were
treated as in general method for the synthesis of chalcone to afford
10 (2.25 g, 7.5 mmol, 71%) as yellow crystal, mp 70–71 °C. IR (KBr)
1648, 1577 cmꢁ1;1H NMR (CDCl3): d 1.44 (3H, t, J = 6.8 Hz, CH3),
2.50 (3H, s, CH3), 3.80 (3H, s, OCH3), 4.09 (2H, q, J = 6.8 Hz,
OCH2), 6.72 (1H, dd, J = 3.6, 1.2 Hz, H-3), 6.90 (1H, d, J = 8.8 Hz,
H-30), 7.00 (1H, dd, J = 8.8, 3.2 Hz, H-40), 7.10 (1H, d, J = 3.6 Hz, H-
CH3), 1.52 (2H, m, CH2), 1.77 (2H, m, CH2), 2.54 (3H, s, CH3), 3.97
(2H, t, J = 6.4 Hz, OCH2), 6.78 (1H, dd, J = 3.6, 1.2 Hz, H-3), 6.94
(1H, d, J = 9.2 Hz, H-30), 7.12 (1H, dd, J = 9.2, 3.2 Hz, H-40), 7.21
(1H, d, J = 3.6 Hz, H-4), 7.28 (1H, d, J = 15.2 Hz, H-a), 7.31 (1H, d,
J = 3.2 Hz, H-60), 7.97 (1H, d, J = 15.2 Hz, H-b), 12.47 (1H, s, OH).
13C NMR (CDCl3): d 13.9 (CH3), 16.0 (CH3), 19.2 (CH2), 31.4 (CH2),
68.8 (OCH2), 113.8 (C-60), 117.4 (C-30), 119.1 (C-
a
), 119.7 (C-10),
4), 7.24 (1H, d, J = 3.2 Hz, H-60), 7.26 (1H, d, J = 15.6 Hz, H-
a), 7.72
124.0 (C-3), 127.1 (C-40), 133.8 (C-4), 138.2 (C-2), 138.4 (C-b),
145.6 (C-5), 151.1 (C-20), 157.7 (C-50), 192.8 (C@O). EIMS (70 eV)
m/z (% rel. int.): 316 (42) [M]+.
(1H, d, J = 15.6 Hz, H-b). 13C NMR (CDCl3): d 15.0 (CH3), 15.8
(CH3), 55.8 (OCH3), 65.1 (OCH2), 114.2 (C-60), 114.6 (C-30), 119.5
(C-a
), 124.8 (C-3), 126.7 (C-40), 129.6 (C-10), 132.4 (C-4), 135.6
(C-b), 138.9 (C-2), 143.9 (C-5), 152.2 (C-20), 153.5 (C-50), 191.3
(C@O). EIMS (70 eV) m/z (% rel. int.): 302 (4) [M]+.
4.2.8. 20-Hydroxy-50-(2-methylpropoxy)-2-(5-methylthienyl)-
chalcone (7)
Compound
1
(2.95 g, 10.2 mmol), 2-methylpropyl iodide
4.2.12. 50-Methoxy-20-propoxy-2-(5-methylthienyl)chalcone (11)
Compound 9 (2.88 g, 10.5 mmol), normal propyl iodide (1.78 g,
10.5 mmol) and potassium carbonate (1.45 g, 10.5 mmol) were
treated as in general method for the synthesis of chalcone to afford
(1.88 g, 10.2 mmol) and potassium carbonate (1.41 g, 10.2 mmol)
were treated as in general method for the synthesis of chalcone
to afford 7 (2.07 g, 6.5 mmol, 64%) as red crystals, mp 99–100 °C.
IR (KBr) 3446, 1635, 1569 cmꢁ1
;
1H NMR (CDCl3): d 1.06 (6H, d,
11 (2.16 g, 6.8 mmol, 65%) as orange oil. IR (KBr) 1650, 1573 cmꢁ1
;
J = 6.8 Hz, 2 ꢂ CH3), 2.10 (1H, m, CH), 2.55 (3H, s, CH3), 3.74 (2H,
d, J = 6.8 Hz, OCH2), 6.78 (1H, dd, J = 3.6, 1.6 Hz, H-3), 6.95 (1H, d,
J = 8.8 Hz, H-30), 7.12 (1H, dd, J = 8.8, 2.8 Hz, H-40), 7.21 (1H, d,
1H NMR (CDCl3): d 1.03 (3H, t, J = 7.2 Hz, CH3), 1.83 (2H, m, CH2),
2.50 (3H, s, CH3), 3.80 (3 H, s, OCH3), 3.96 (2H, t, J = 6.4 Hz,
OCH2), 6.72 (1H, dd, J = 3.6, 1.2 Hz, H-3), 6.90 (1H, d, J = 8.8 Hz,
H-30), 7.00 (1H, dd, J = 8.8, 2.8 Hz, H-40), 7.10 (1H, d, J = 3.6 Hz, H-
J = 3.6 Hz, H-4), 7.25 (1H, d, J = 15.6 Hz, H-a), 7.35 (1H, d,
J = 2.8 Hz, H-60), 7.97 (1H, d, J = 15.6 Hz, H-b), 12.46 (1H, s, OH).
13C NMR (CDCl3): d 16.0 (CH3), 19.3 (2 ꢂ CH3), 28.4 (CH), 75.6
4), 7.23 (1H, d, J = 2.8 Hz, H-60), 7.24 (1H, d, J = 15.6 Hz, H-
a), 7.72
(1H, d, J = 15.6 Hz, H-b). 13C NMR (CDCl3): d 10.8 (CH3), 15.9
(OCH2), 113.8 (C-60), 117.5 (C-30), 119.1 (C-
a
), 119.7 (C-10), 124.2
(CH3), 22.7 (CH2), 55.8 (OCH3), 71.0 (OCH2), 114.2 (C-60), 114.3
(C-3), 127.1 (C-40), 133.7 (C-4), 138.2 (C-2), 138.4 (C-b), 145.6 (C-
5), 151.3 (C-20), 157.7 (C-50), 192.8 (C@O). EIMS (70 eV) m/z (%
rel. int.): 316 (30) [M]+.
(C-30), 119.5 (C- ), 124.7 (C-3), 126.7 (C-40), 129.6 (C-10), 132.4
a
(C-4), 135.7 (C-b), 138.8 (C-2), 144.0 (C-5), 152.3 (C-20), 153.4 (C-
50), 191.5 (C@O). EIMS (70 eV) m/z (% rel. int.): 316 (13) [M]+.
4.2.9. 20-Hydroxy-50-pentanoxy-2-(5-methylthienyl)chalcone (8)
4.2.13. 20-Butoxy-50-methoxy-2-(5-methylthienyl)chalcone (12)
Compound 9 (2.88 g, 10.5 mmol), normal butyl iodide (1.93 g,
10.5 mmol) and potassium carbonate (1.45 g, 10.5 mmol) were
treated as in general method for the synthesis of chalcone to afford
Compound
1 (2.95 g, 10.2 mmol), normal pentanyl iodide
(2.02 g, 10.2 mmol) and potassium carbonate (1.41 g, 10.2 mmol)
were treated as in general method for the synthesis of chalcone
to afford 8 (2.29 g, 6.9 mmol, 68%) as red crystals, mp 82–83 °C.
12 (1.84 g, 5.6 mmol, 53%) as orange oil. IR (KBr) 1644, 1531 cmꢁ1
;
IR (KBr) 3446, 1627, 1550 cmꢁ1
;
1H NMR (CDCl3): d 0.95 (3H, t,
1H NMR (CDCl3): d 0.92 (3H, t, J = 7.2 Hz, CH3), 1.49 (2H, m, CH2),
1.79 (2H, m, CH2), 2.50 (3H, s, CH3), 3.80 (3H, s, OCH3), 4.00 (2H,
t, J = 6.4 Hz, OCH2), 6.72 (1H, dd, J = 3.2, 1.2 Hz, H-3), 6.90 (1H, d,
J = 8.8 Hz, H-30), 7.00 (1H, dd, J = 8.8, 3.2 Hz, H-40), 7.10 (1H, d,
J = 3.2 Hz, H-4), 7.23 (1H, d, J = 3.2 Hz, H-60), 7.24 (1H, d,
J = 7.2 Hz, CH3), 1.42 (4H, m, CH2), 1.48 (2H, m, CH2), 1.80 (2H, m,
CH2), 2.55 (3H, s, CH3), 3.97 (2H, t, J = 6.4 Hz, OCH2), 6.78 (1H, dd,
J = 3.2, 1.2 Hz, H-3), 6.95 (1H, d, J = 8.8 Hz, H-30), 7.12 (1H, dd,
J = 8.8, 2.8 Hz, H-40), 7.21 (1H, d, J = 3.2 Hz, H-4), 7.24 (1H, d,
J = 15.2 Hz, H-
a
), 7.31 (1H, d, J = 2.8 Hz, H-60), 7.97 (1H, d,
J = 15.2 Hz, H-
d 13.8 (CH3), 15.8 (CH3), 19.4 (CH2), 31.4 (CH2), 55.8 (OCH3), 69.1
a
), 7.72 (1H, d, J = 15.2 Hz, H-b). 13C NMR (CDCl3):
J = 15.2 Hz, H-b), 12.46 (1H, s, OH). 13C NMR (CDCl3): d 14.0
(CH3), 16.0 (CH3), 22.5 (CH2), 28.2 (CH2), 29.1 (CH2), 69.1 (OCH2),
(OCH2), 114.2 (C-60), 114.3 (C-30), 119.5 (C-
a), 124.8 (C-3), 126.6
113.9 (C-60), 117.5 (C-30), 119.1 (C-
a
), 119.7 (C-10), 124.1 (C-3),
(C-40), 129.6 (C-10), 132.4 (C-4), 135.6 (C-b), 138.8 (C-2), 144.0
(C-5), 152.4 (C-20), 153.5 (C-50), 191.4 (C@O). EIMS (70 eV) m/z (%
rel. int.): 330 (40) [M]+.
127.1 (C-40), 133.7 (C-4), 138.2 (C-2), 138.4 (C-b), 145.5 (C-5),
151.2 (C-20), 157.7 (C-50), 192.8 (C@O). EIMS (70 eV) m/z (% rel.
int.): 330 (63) [M]+.
4.2.14. 20,30-Dihydroxy-40-methoxy-2-(5-methylthienyl)-
chalcone (13)
4.2.10. 20-Hydroxy-50-methoxy-2-(5-methylthienyl)chalcone (9)
20-Hydroxy-50-methoxyacetophenone (4.2 g, 25 mmol), 3,4-
20,30-Dihydroxy-40-methoxyacetophenone (4.6 g, 25 mmol),
dihydro-
a
-pyran (13 mL) and pyridinium p-toluenesulfonate
3,4-dihydro-a-pyran (13 mL) and pyridinium p-toluenesulfonate
(0.15 g, 0.6 mmol) were treated as in the method described in
(0.15 g, 0.6 mmol) were reacted in CH2Cl2 (100 mL) to give 20,30-
di(tetra-hydropyran-2-yloxy)-40- methoxyacetophenone (13a).
Compound 13a (8.8 g, 25 mmol), 5-methylthiophene-2-aldehyde
(3.2 g, 25 mmol) and barium hydroxide octahydrate (4.29 g,
25 mmol) were treated as in the method described in preparation
of 1 to afford 13 (3.48 g, 12.0 mmol, 48%) as red crystals, mp 131–
preparation of 1 to afford 9 (3.77 g, 13.8 mmol, 55%) as red crystals,
mp 78–79 °C. IR (KBr) 3446, 1632, 1552 cmꢁ1 1H NMR (CDCl3): d
;
2.53 (3H, s, CH3), 3.83 (3H, s,OCH3), 6.76 (1H, dd, J = 3.6, 0.8 Hz,
H-3), 6.95 (1H, d, J = 8.8 Hz, H-30), 7.12 (1H, dd, J = 8.8, 2.8 Hz, H-
40), 7.19 (1H, d, J = 3.6 Hz, H-4), 7.25 (1H, d, J = 15.2 Hz, H-
a), 7.29

7276
J.-H. Cheng et al. / Bioorg. Med. Chem. 16 (2008) 7270–7276
132 °C. IR (KBr) 3446, 1636, 1558 cmꢁ1
;
1H NMR (CDCl3): d 2.53
4.6. Assay of XO activity
(3H, s, CH3), 3.96 (3H, s, OCH3), 5.59 (1H, s, OH), 6.53 (1H, d,
J = 9.2 Hz, H-50), 6.76 (1H, dd, J = 3.6, 1.6 Hz, H-3), 7.18 (1H, d,
The XO activity with xanthine as the substrate was measured at
J = 3.6 Hz, H-4), 7.19 (1H, d, J = 15.6 Hz, H-
a
), 7.43 (1H, d,
25 °C, according to the protocol of Kong and others23 with modifi-
J = 9.2 Hz, H-60), 7.93 (1H, d, J = 15.6 Hz, H-b), 13.30 (1H, s, OH).
13C NMR (CDCl3): d 15.9 (CH3), 56.2 (OCH3), 102.7 (C-50), 114.9
cation. The assay mixture consisting of 50
lL of test solution, 60
lL
of 70 mM phosphate buffer (pH 7.5), and 30
lL of enzyme solution
(C-10), 117.4 (C-
a
), 121.5 (C-30 and 60), 127.0 (C-3), 133.5 (C-4),
(0.1 unite/mL in 70 mM phosphate buffer (pH 7.5)) was prepared
immediately before use. After preincubation at 25 °C for 15 min,
137.6 (C-b), 138.2 (C-2), 145.3 (C-5), 151.4 (C-40), 151.9 (C-20),
192.0 (C@O).21 EIMS (70 eV) m/z (% rel. int.): 290 (9) [M]+.
the reaction was initiated by addition of 60
lL of substrate solution
(150 M xanthine in the same buffer). The reaction was monitored
for 5 min at 295 nm. The XO activity was expressed as micromoles
of uric acid per minute.
l
4.2.15. 20,30,40-Trihydroxy-2-(5-methylthienyl)chalcone (14)
20,30,40-Trihydroxyacetophenone (4.2 g, 25 mmol), 3,4-dihydro-
a
-pyran (13 mL) and pyridinium p-toluenesulfonate (0.15 g,
0.6 mmol) were reacted in CH2Cl2 (100 mL) to give 20,30,40-tri(te-
4.7. Statistical analysis
tra-hydropyran-2-yloxy)acetophenone (14a). Compound 14a
(10.5 g,
25 mmol),
5-methylthiophene-2-aldehyde
(3.2 g,
Data were expressed as the mean SD. Statistical analysis were
performed using the Bonferroni t-test method after ANOVA for
multigroup comparison and the student’s t-test method for two
group comparison. P < 0.05 was considered to be statistically
significant.
25 mmol) and barium hydroxide octahydrate (4.29 g, 25 mmol)
were treated as in the method described in preparation of 1 to af-
ford 14 (3.18 g, 11.5 mmol, 46%) as orange crystals, mp 182–
183 °C. IR (KBr) 3446, 1624, 1566 cmꢁ1 1H NMR (acetone-d6): d
;
2.54 (3H, s, CH3), 6.51 (1H, d, J = 8.8 Hz, H-50), 6.88 (1H, dd,
J = 3.6, 1.2 Hz, H-3), 7.41 (1H, d, J = 3.6 Hz, H-4), 7.46 (1H, d,
Acknowledgements
J = 15.2 Hz, H-a
), 7.60 (1H, d, J = 8.8 Hz, H-60), 7.83 (1H, s, OH),
7.94 (1H, d, J = 15.2 Hz, H-b), 8.70 (1H, s, OH), 13.56 (1H, s, OH).
This work was supported by a grant from the National Science
council of Republic of China (NSC 95–2320–B–037–037) and par-
tially supported by a found of KMU-EM-97-2.1.b.
13C NMR (acetone-d6): d 14.6 (CH3), 107.3 (C-50), 113.4 (C-10),
117.6 (C-a
), 121.9 (C-60), 122.9 (C-30), 127.0 (C-3), 133.3 (C-4),
136.6 (C-b), 138.1 (C-2), 145.0 (C-5), 151.7 (C-40), 153.1 (C-20),
191.6 (C@O).21 EIMS (70 eV) m/z (% rel. int.): 276 (14) [M]+.
References and notes
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Compounds were added to cells (1 ꢂ 107). At various time inter-
vals, the reactions were terminated by washing with PBS. The cells
were fixed with 4% paraformadehyde/PBS (pH 7.4) at room tem-
perature for 30 min. After certrifugation 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. Propidium 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–Dickinson, Mountain View, CA). A minimum of 5000 cell
counts were collected for the analysis by LYSIS II Software.7
4.5. Mast cell degranulation, neutrophil degranulation,
superoxide anion generation, macrophage cultures and drugs
treatment and NO determination
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The inhibitory assays for chemical mediator induced by various
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performed by the methods as described in the literature.22