Synthesis and anti-inflammatory effect of chalcones

Article abstract of DOI:10.1211/0022357001773814

The process of degranulation of mast cells and neutrophils contributes to inflammatory disorders. Activation of microglial cells and macrophages is believed to be involved in inflammatory, infectious and degenerative diseases of the CNS. Combining the pot

Full text of DOI:10.1211/0022357001773814

J. Pharm. Pharmacol. 2000, 52: 163±171
Received September 3, 1999
Accepted October 18, 1999
# 2000 J. Pharm. Pharmacol.
Synthesis and Anti-in¯ammatory Effect of Chalcones
HSIN-KAW HSIEH*{, LO-TI TSAO{, JIH-PYANG WANG{
AND CHUN-NAN LIN*
*School of Pharmacy, Kaohsiung Medical College, Kaohsiung, Taiwan 807, {China Junior College of
Medical Technology, Tainan Hsien, Taiwan 717 and {Department of Medical Research, Taichung Veterans
General Hospital, Taichung, Taiwan 407, Taiwan
Abstract
The process of degranulation of mast cells and neutrophils contributes to in¯ammatory
disorders. Activation of microglial cells and macrophages is believed to be involved in
in¯ammatory, infectious and degenerative diseases of the CNS. Combining the potent
inhibition of chemical mediators released by the degranulation of mast cells or neutrophils
and from the activated microglial cells or macrophages, would lead to a promising anti-
in¯ammatory agent for the treatment of peripheral and central in¯ammation. A series of
chalcone derivatives have been reported to have potent anti-in¯ammatory activity. In an
effort to continually develop potent anti-in¯ammatory agents, novel series of chalcones, 20-
hydroxy- and 20,50-dihydroxychalcones were synthesized and their inhibitory effects on the
activation of mast cells, neutrophils, microglial cells and macrophages were evaluated
in-vitro. The chalcones were prepared by Claisen-Schmidt condensation of appropriate
acetophenones with an appropriate aromatic aldehyde. The alkoxychalcones were prepared
with appropriate hydroxychalcones and alkyl iodide and the dihydroxychalcones were
prepared by hydrogenation of an appropriate chalcone with Pd=C.
Almost all of the hydroxychalcones exhibited potent inhibitory effects on the release of
b-glucuronidase and lysozyme from rat neutrophils stimulated with formyl-Met-Leu-
Phe=cytochalasin B (fMLP=CB). Of the hydroxychalcones, compound 1 was the most
potent inhibitor of the release of b-glucuronidase (IC50ˆ1Á6Æ 0Á2 mM) and lysozyme
(IC50ˆ1Á4Æ 0Á2 mM) from rat neutrophils stimulated with fMLP=CB. Almost all of the
20,50-dialkoxychalcones exhibited potent inhibitory effects on nitric oxide (NO) formation
from murine microglial cell lines N9 stimulated with lipopolysaccharide (LPS). Of these,
compound 11 showed the greatest effect (IC50ˆ0Á7Æ 0Á06 mM).
The present results demonstrated that most of the chalcone derivatives have an anti-
in¯ammatory effect. The inhibitory effects of dialkoxychalcones, 10±12 on in¯ammation
are probably not due to the inhibition of mast cells and neutrophil degranulation, but are
mediated through the suppression of NO formation from N9 cells.
The processes of degranulation of mast cells and
neutrophils contributes to in¯ammatory disorders
(Middleton & Kandaswami 1992). Combining
potent inhibition of chemical mediators released
from mast cell and neutrophil degranulation, would
suggest a promising anti-in¯ammatory agent. Our
previous reports indicated that some chalcones
exert potent anti-in¯ammatory effects, at least
partly, through the suppression of chemical med-
iators released from mast cell and neutrophil
degranulation (Lin et al 1997; Hsieh et al 1998).
These ®ndings suggested that some chalcones
might be promising anti-in¯ammatory agents. In
our current study, we further synthesize and
describe the anti-in¯ammatory effects of chalcones
using in-vitro in¯ammatory models.
Macrophages are important in nonspeci®c host
resistance to microbial pathogens and serve as
central regulators of the speci®c immune response
(Solbach et al 1991). Upon activation, nitric oxide
(NO), together with other chemical mediators, is
released in response to bacterial endotoxin (lipo-
polysaccharide, LPS) (Ding et al 1988). NO plays a
central role in macrophage-induced cytotoxicity,
Correspondence: C.-N. Lin, School of Pharmacy, Kaohsiung
Medical College, Kaohsiung, Taiwan 807, Taiwan.
E-Mail: lincna@cc.kmc.edu.tw

164
HSIN-KAW HSIEH ET AL
however; excess NO may contribute to the patho-
physiology of septic shock (Thiermermann & Vane
1990). Microglial cells, the brain's resident mac-
rophages, have detrimental as well as bene®cial
effects on the surrounding cells and are believed to
be involved in most in¯ammatory, infectious, and
degenerative diseases of the CNS (Mallat & Cha-
mak 1994; Gehrmann et al 1995). The release of
NO by activated microglial cells in response to LPS
is likely to have a crucial role in mediating the
interaction between microglia and other cells pre-
sent in the nervous system, as it is known to reg-
ulate in¯ammation, immune functions, blood vessel
dilation, neurotransmission and neural-cell survival
(Gebicke-Haerter et al 1989; Nathan 1992; Min-
ghetti et al 1997). Hence, inhibition of NO released
from microglial cells or macrophages is a rational
therapeutic approach to treat a variety of in¯am-
matory and neuronal conditions observed in ageing
and Alzheimer's disease (Meda et al 1995).
Hypaque separation and hypotonic lysis of the
residual erythrocytes, neutrophils were washed and
suspended in Hank's balanced salt solution (HBSS)
1
to 1 6 107 cells mL
(Boyum 1968). The cell
suspension was pre-incubated at 37ꢀC with 0Á5%
DMSO or drugs for 3 min, and then challenged with
formyl-Met-Leu-Phe=cytochalasin B (fMLP=CB;
1
1 mM=5 mg mL ). After 45 min, the lysozyme
(Micrococcus lysodeikticus as substrate, 450 nm)
and b-glucuronidase in the supernatant were
determined (Smith & Iden 1979). The total content
was measured after treatment of the cell suspension
with Triton X-100 and the percentage released was
calculated. The ®nal concentration of drugs in
DMSO was ®xed at 0Á5%.
Superoxide anion formation
Superoxide anion formation was measured in terms
of superoxide dismutase-inhibitable cytochrome c
reduction (Market et al 1984). Neutrophil suspen-
sion was pre-incubated with 0Á5% DMSO or drugs
for 3 min, and then superoxide dismutase or HBSS
was added into the test and blank wells, respec-
tively. After addition of cytochrome c, the reaction
was initiated by challenge with fMLP=CB
In this study, the anti-in¯ammatory activities of
a series of synthetic compounds were assessed
in-vitro by determining their inhibitory effects on
macrophages and microglial cells, as well as on
mast cells and neutrophils, and their structure±
activity relationships are discussed.
1
(0Á3 mM=5 mg mL ) or phorbol 12-myristate B
acetate (PMA) (3 nM). The reaction was terminated
after 30 min by centrifugation and the absorbance
changes of supernatant were monitored at 550 nm
in a microplate reader. The ®nal concentration of
drugs in DMSO was ®xed at 0Á5%.
Materials and Methods
Mast cell degranulation
Heparinized Tyrode's solution was injected into the
peritoneal cavity of exsanguinated rats (Sprague-
Dawley, 250±300 g). After abdominal massage,
cells in the peritoneal ¯uid were harvested and then
separated in 38% bovine serum albumin (BSA) in
glucose-free Tyrode's solution. Cell pellets were
washed and suspended in Tyrode's solution with
Macrophage cultures and drugs treatment
Raw 264Á7 mouse macrophage-like cell line
(American Type Culture Collection) was plated in
96-well tissue-culture plates in Dulbecco's Modi-
®ed Eagle Medium supplemented with 5% foetal
1
calf serum (FCS), 100 int. units mL of penicillin
1
0Á1% BSA to 1 6 106 cell mL . The cell suspen-
and streptomycin at 2 6 105 cells per 200 mL per
well. Cells were allowed to adhere overnight.
sion was then pre-incubated at 37ꢀC with 0Á5%
dimethylsulphoxide (DMSO) or drugs for 3 min.
Fifteen minutes after the addition of compound
Pretreatment of cells with test drugs at 37ꢀC for
1
1 h before stimulation with 1 mg mL
of LPS
1
48=80 (10 mg mL ), b-glucuronidase (phenol-
(Escherichia coli, serotype 0111 : B4) for 24 h, and
then the medium was collected and stored at
70ꢀC until used. The ®nal concentration of drugs
in DMSO was ®xed at 0Á5%.
phthalein-b-glucuronide as substrate, 550 nm) and
histamines
(o-phthadialdehyde
condensation,
350=450 nm) in the supernatant were determined
(Wang et al 1994). The total content was measured
after treatment of the cell suspension with Triton
X-100 and the percentage released was calculated.
The ®nal concentration of drugs in DMSO was
®xed at 0Á5%.
Microglial cell cultures and drugs treatment
Murine microglial cell lines N9 (Corradin et al
1993) (kindly provided by Dr P. Ricciardi-
Castagnoli, CNR Cellular and Molecular Pharma-
cology Center, Italy) was plated in 96-well
tissue-culture plates in Iscove's Modi®ed Dulbec-
co's medium containing 2% heat-inactivated FCS
Neutrophil degranulation
Blood was withdrawn from rat and mixed with
EDTA. After dextran sedimentation, Ficoll-
1
and antibiotics at 8 6 104 cells per 200 mL per

SYNTHESIS AND ANTI-INFLAMMATORY EFFECT OF CHALCONES
165
well. Pretreatment of cells with test drugs at 37ꢀC
for 1 h before stimulation with LPS (10 mg mL )
Analysis of the regression line was used to calcu-
late IC50 values.
1
for 24 h, and then the medium was collected and
stored at 70ꢀC until used. The ®nal concentration
of drugs in DMSO was ®xed at 0Á5%.
Chemistry
Melting points (uncorrected) were determined with
a Yanaco Micro-Melting Point apparatus. IR
spectra were determined with a Hitachi model 260-
30 IR spectrophotometer.1H and13C NMR spectra
(d, ppm; J, Hz) (Tables 2 and 3) were determined
with a Varian Gemini 200 MHz FT-NMR spectro-
meter. Mass spectra were determined with a Jeol
JMS-D-100 mass spectrometer. Elemental analyses
were within Æ 0Á4% of the theoretical values,
unless otherwise noted. Chromatography was per-
formed using a ¯ash-column technique on silica gel
60 supplied by E. Merck.
We prepared a number of new chalcones (1±9;
Table 1) using Claisen-Schmidt condensation
of appropriate acetophenones or hydroxy-
acetophenones, protected as tetrahydropyranyl
ether, with an appropriate aromatic aldehyde or
hydroxyaromatic aldehyde, protected as tetra-
hydropyranyl ether (Hsieh et al 1998). A mixture of
20,50-dihydroxy-4-chlorochalcone (16), appropriate
alkyl iodide and K2CO3 in N,N-dimethylformamide
NO determination
The production of NO was determined in cell
medium by measuring the content of nitrite (Min-
ghetti et al 1997) based on the Griess reaction.
Brie¯y, 40 mL of 5 mM sulphanilamide, 10 mL of
2 M HCl and 20 mL of 40 mM naphthylethylene-
diamine were added to 150 mL culture medium.
After 10 min of incubation at room temperature,
absorbance was measured at 550 nm in a microplate
reader. A standard nitrite curve was generated in
the same fashion using NaNO2. The ®nal con-
centration of drugs in DMSO was ®xed at 0Á5%.
Statistical analysis
Data are presented as the meansÆ s.e.m. Statistical
analyses were performed using the Least Sig-
ni®cant Difference Test method after analysis of
variance. P< 0Á05 was considered to be signi®cant.
Figure 1. Synthesis of 20,50-dihydroxy-4-chlorodihdrochalcone (14) and general synthesis of 20,50-dialkoxychalcones 10±12. (a)
Alkyl halide, K2CO3, DMF, room temperature; (b) EtOAc, H2, 5% Pd=C, room temperature.

166
HSIN-KAW HSIEH ET AL
Table 1. Chalcones 1±12 and dihydrochalcones 13±14.
Compound
R20
R50
R2
R3
R4
mp (ꢀC)
% Yield
Formula
Anal.
1
2
3
4
5
6
7
8
OH
OH
H
H
OH
H
H
H
OH
OH
OCH3
H
H
H
H
H
H
Cl
160±162
160±161
183±185
158±160
131±132
87±88
93±94
116±117
90±91
115±116
70±71
72±73
115±116
131±132
46a
38b
38c
48d
42b
46b
52b
34b
42b
62b
60b
52b
82e
88e
C15H12O3
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C15H11O2Cl
C13H10O3S
C17H16O5
C18H18O5
C17H16O4
C17H16O3
C16H13O3Cl
C17H15O3Cl
C19H19O3Cl
C21H23O3Cl
C23H27O3Cl
C15H14O3
OH
OH
OCH3
OCH3
CH3
Cl
Cl
Cl
Cl
Cl
OCH3
OH
OH
OH
OH
OCH3
OC2H5
OC3H7
OC4H9
OH
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OC2H5
OC3H7
OC4H9
OH
H
H
H
H
H
H
H
H
H
H
H
9
10
11
12
13
14
H
H
H
H
H
Cl
OH
OH
C15H13O3Cl
Solvents used for recrystallization: aCyclohexane±ethyl acetate. bMethanol. cBenzene. dCyclohexane±acetone. eBenzene±
petroleum ether.
(DMF) was stirred at room temperature to give
compounds 10±12 (Figure 1; Table 1). A solution
of dihydroxychalcone or 16 in EtOAc was hydro-
genated with Pd=C at room temperature to give 13
and 14 (Figure 1), respectively (Table 1).
chalcone (3b). Crude 3b and p-toluenesulphonic
acid (0Á2 g, 1Á05 mmol) were dissolved in MeOH
(100 mL). The reaction mixture was stirred for 4 h
at room temperature, and then evaporated in-vacuo.
Water (100 mL) was added to the mixture, neutra-
lized with 5% NaHCO3 (50 mL) and extracted with
EtOAc. The organic layer was separated, washed
with water, dried and evaporated in-vacuo. The
residue was eluted through a silica-gel column
with cyclohexane±EtOAc (4 : 1) to give 3 (2Á3 g,
General procedure for obtaining chalcones 1±9
(Hsieh et al 1998)
20-Thienyl-3,4-dihydroxychalcone (3). 3,4-Dihy-
droxybenzaldehyde (3Á45 g, 25 mmol) and pyridi-
nium p-toluenesulphonate (0Á15 g, 0Á6 mmol) were
stirred for 0Á5 h in methylene chloride (100 mL) and
then 3,4-dihydro-a-pyran in methylene chloride
(13 mL in 20 mL) was added dropwise. The reac-
tion mixture was stirred at room temperature for
4 h, washed twice with water, dried and evaporated
in-vacuo. The residue yielded crude 3,4-bis(tetra-
hydropyran-2-yloxy)benzaldehyde (3a). Crude 3a,
2-thiophenecarboxacetophenone (3Á15 g, 25 mmol)
and barium hydroxide octahydrate (8Á15 g,
25 mmol) were dissolved in MeOH (100 mL). The
reaction mixture was stirred for 12 h at 40ꢀC 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-vacuo. The residue yielded
crude 20-thienyl-3,4-bis(tetrahydropyran-2-yloxy)-
1
9Á5 mmol, 38%): IR (KBr) 3200, 1635 cm ; MS
‡
m=z 246 (M , 111).
20,2-Dihydroxychalcone (1). 2-Hydroxyacetophe-
none (3Á4 g, 25 mmol), 2-hydroxybenzaldehyde
(3Á05 g, 25 mmol) and barium hydroxide octahydrate
(8Á15 g, 25 mmol) were treated as in the synthesis of
3b to give 1 (2Á8 g, 11Á5 mmol, 46%): IR (KBr) 3256,
1
‡
1636 cm ; MS m=z 240 (M , 221).
20-Hydroxy-4-chlorochalcone (2). 2-Acetophenone
(3Á4 g, 25 mmol), 4-chlorobenzaldehyde (3Á5 g,
25 mmol) and barium hydroxide octahydrate
(8Á15 g, 25 mmol) were treated as in the synthesis
of 3b to give 2 (2Á7 g, 9Á5 mmol, 38%): IR (KBr)
1
‡
1620 cm ; MS m=z 258 (M , 147).
20,50-Dimethoxy-3,4-dihydroxychalcone (4). 3,4-
Dihydroxybenzaldehyde (3Á45 g, 25 mmol) and
pyridinium p-toluenesulphonate (0Á15 g, 0Á6 mmol)

SYNTHESIS AND ANTI-INFLAMMATORY EFFECT OF CHALCONES
167
were treated as in the synthesis of 3a to give crude
3,4-bis(tetrahydro-pyran-2-yloxy)benzaldehyde
(4a). Crude 4a, 2,5-dimethoxyacetophenone (4Á5 g,
25 mmol) and barium hydroxide octahydrate
(8Á15 g, 25 mmol) were treated as in the synthesis
of 3b to give 4 (3Á6 g, 12 mmol, 48%): IR (KBr)
with water and washed three times with water. The
organic phase was dried over sodium sulphate,
®ltered and concentrated in-vacuo to give the
product. Puri®cation via silica-gel column chroma-
tography provided 5Á15 g (15Á5 mmol, 62%) of 10
as a yellow microcrystalline solid: IR (KBr) 2980,
1
‡
1
‡
3400, 1610 cm ; MS m=z 300 (M , 77).
1640 cm ; MS m=z 330 (M , 164).
20-Hydroxy-50,3,4-trimethoxychalcone (5). 2-Hy-
droxy-5-methoxyacetophenone (4Á15 g, 25 mmol),
3,4-dimethoxybenzaldehyde (4Á2 g, 25 mmol) and
barium hydroxide octahydrate (8Á15 g, 25 mmol)
were treated as in the synthesis of 3b to give 5
(3Á3 g, 10Á5 mmol, 42%): IR (KBr) 2850,
20,50-Dipropoxy-4-chlorochalcone (11). 20,50-Dihy-
droxychalcone (6 g, 25 mmol), n-propyl iodide
(8Á8 g, 52 mmol) and potassium carbonate (15 g,
25 mmol) in DMF (50 mL) were treated as in the
synthesis of 10 to give 11 (5Á37 g, 15 mmol, 60%):
1
‡
IR (KBr) 3050, 1620 cm ; MS m=z 358 (M ,
1
‡
1640 cm ; MS m=z 314 (M , 164).
136).
20-Hydroxy-50,4-dimethoxychalcone (6). 20-Hy-
droxy-50-methoxyacetophenone (4Á15 g, 25 mmol),
4-methoxybenzaldehyde (3Á4 g, 25 mmol) and bar-
ium hydroxide octahydrate (8Á15 g, 25 mmol) were
treated as in the synthesis of 3b to give 6 (3Á2 g,
20,50-Dibutoxy-4-chlorochalcone (12). 20,50-Dihy-
droxychalcone (6 g, 25 mmol), n-butyl iodide
(9Á5 g, 52 mmol) and potassium carbonate (8Á15 g,
25 mmol) in DMF (50 mL) were treated as in the
synthesis of 10 to give 12 (5 g, 13 mmol, 52%): IR
1
1
‡
11Á5 mmol, 46%): IR (KBr) 3000, 1640 cm ; MS
(KBr) 2980, 1620 cm ; MS m=z 386 (M , 136).
‡
m=z 284 (M , 134).
General procedure for obtaining dihydro-
chalcones 13±14
20-Hydroxy-50-methoxy-4-methylchalcone (7). 2-
Hydroxy-5-methoxyacetophenone
(4Á15 g,
25
20,50-Dihydroxydihydrochalcone (13). A solution of
20,50-dihydroxychalcone (1Á7 g, 7 mmol) in ethyl
acetate (50 mL) was hydrogenated in an autoclave
mmol), 4-methylbenzaldehyde (3 g, 25 mmol) and
barium hydroxide octahydrate (8Á15 g, 25 mmol)
were treated as in the synthesis of 3b to give 7
1
3
(3Á5 g, 13 mmol, 52%): IR (KBr) 3100, 1640 cm ;
for 3 h at an initial pressure of 60 kg cm in the
‡
MS m=z 268 (M , 150).
presence of 5% Pd=C (300 mg) at room tempera-
ture. The catalyst was removed by ®ltration and
the ®ltrate was evaporated in-vacuo and puri®ed
by chromatography over a silica-gel column
using a 5 : 1 mixture of cyclohexane and ethyl
acetate as eluant to afford the crude product. The
crude product was recrystallized from benzene±
petroleum ether to give 13 (1Á4 g, 5Á74 mmol, 82%):
20-Hydroxy-50-methoxy-4-chlorochalcone (8). 20-
Hydroxy-50-methoxyacetophenone (4Á15 g, 25
mmol), 4-chlorobenzaldehyde (3Á5 g, 25 mmol)
and barium hydroxide octahydrate (8Á15 g,
25 mmol) were treated as in the synthesis of 3b to
give 8 (2Á4 g, 8Á5 mmol, 34%): IR (KBr) 3100,
1
‡
1
‡
1610 cm ; MS m=z 288 (M , 150).
IR (KBr) 3400, 1630 cm ; MS m=z 242 (M ,
137).
20,50-Dimethoxy-4-chlorochalcone (9). 2,5-Dimeth-
oxyacetophenone (3Á75 g, 25 mmol), 4-chlorobenz-
aldehyde (3Á5 g, 25 mmol) and barium hydroxide
octahydrate (8Á15 g, 25 mmol) were treated as in the
synthesis of 3b to give 9 (3Á1 g, 10Á5 mmol, 42%):
20,50-Dihydroxy-4-chlorodihydrochalcone (14). 20,50-
Dihydroxy-4-chlorochalcone (2 g, 7 mmol) and 5%
Pd=C (300 mg) in ethyl acetate were treated as in
the synthesis of 13 to give 14 (1Á7 g, 6Á2 mmol,
1
‡
1
IR (KBr) 3100, 1645 cm ; MS m=z 302 (M ,
88%): IR (KBr) 3440, 1660 cm ; MS m=z 276
‡
165).
(M , 137).
General procedure for obtaining alkoxychalcones
10±12
Results and Discussion
20,50-Diethoxy-4-chlorochalcone (10). A mixture
of 20,50-dihydroxychalcone (6 g, 25 mmol), ethyl
iodide (8Á1 g, 52 mmol) and potassium carbonate
(15 g, 25 mmol) in DMF (50 mL) was stirred at
room temperature for 18 h. The mixture was diluted
The anti-in¯ammatory activities of 1±14 (Table 1)
were studied in-vitro for their inhibitory effects on
chemical mediators released from mast cells, neu-
trophils, macrophages and microglial cells. Com-
pounds 1, 5±7 and 9 caused concentration-

168
HSIN-KAW HSIEH ET AL
Table 2. 1H NMR data for various chalcone and dihydrochalcone derivatives.
Compound
H-60
H-30
H-50
H-40
H-2
H-6
H-3
H-5
1a
8Á20(dd)
7Á91(dd)
7Á10(d)
7Á10(d)
7Á39(d)
7Á36(d)
7Á36(d)
7Á34(d)
7Á19(d)
7Á22(d)
7Á22(d)
7Á22(d)
7Á17(d)
7Á14(d)
6Á90±7Á04(m) 7Á27±7Á36(m)
7Á39±7Á62(m)
7Á87(dd)
6Á90±7Á04(m)
6Á91±7Á06(m)
6Á82(d)
2b
3c
8Á02(dd) 7Á84(dd)
6Á99(d)
7Á22(dd)
6Á96(dd)
7Á14(dd)
7Á13(dd)
7Á14(dd)
7Á16(dd)
7Á03(dd)
7Á02(dd)
7Á01(dd)
7Á02(dd)
7Á01(dd)
6Á97(dd)
7Á19(d) 7Á10(dd)
7Á06(d) 6Á98(dd)
7Á16(d) 7Á27(dd)
4b
6Á79(d)
5b
6Á97(d)
6Á91(d)
6b
6Á97(d)
6Á96(dd)
7b
6Á97(d)
7Á24(m)
8b
6Á98(d)
7Á40±7Á45(m)
9b
6Á93(d)
7Á26±7Á38(m)
10b
11b
12b
13c
14c
6Á91(d)
7Á37±7Á56(m)
6Á90(d)
7Á50±7Á52(m)
7Á35±7Á37(m)
7Á35±7Á37(m)
7Á20±7Á30(m)
6Á90(d)
7Á51±7Á74(m)
6Á87(d)
6Á87(d)
7Á16±7Á29(m)
Compound
H-4
H-a
H-b
OCH3
CH3
CH2
OCH2
1a
7Á51±7Á60(m)
8Á07(d)
8Á34(d)
7Á62(d)
7Á68(d)
7Á44(d)
7Á88(d)
7Á90(d)
7Á90(d)
7Á86(d)
7Á58(d)
7Á60(d)
7Á59(d)
7Á59(d)
3Á25(t)
3Á23(t)
2b
7Á87(d)
3c
7Á41(d)
7Á16(d)
7Á44(d)
7Á47(d)
7Á54(m)
7Á55(d)
7Á39(d)
7Á30(d)
7Á50(d)
7Á50(d)
3Á04(t)
3Á04(t)
4b
3Á73;3Á79
3Á84;3Á94;3Á97
3Á85;3Á87
3Á84(s)
5b
6b
7Á62(dd)
7b
2Á41(s)
7Á54(m)
8b
3Á85(s)
7Á57±7Á62(m)
7Á49±7Á54(m)
3Á98±4Á13(m)
3Á91(m)
9b
3Á81;3Á86
10b
11b
12b
13c
14c
1Á36±1Á43(m)
1Á0(m)
1Á8(m)
0Á92(m)
1Á45;1Á70(m)
3Á97(m)
b
c
aMeasured in (CD3)2CO. Measured in CDCl3. Measured in CD3OD.
dependent inhibition of the mast cell degranulation
stimulated with compound 48=80 (10 mg mL )
dent inhibitory effects on the activation of neu-
trophils (Table 5). Compound 9 showed signi®cant
inhibitory effects on the activation of neutrophils
while 8 did not. This clearly indicates that a
20-hydroxy- or 20,50-dihydroxychalcone with an
appropriate substitution in its B ring shows potent
inhibitory effects on the activation of neutrophils.
O-methylation of 16 slightly decreased the inhibi-
tion of fMLP=CB-induced neutrophil degranula-
tion, whereas with other types of O-alkylation of 16
it was abolished (Table 5), indicating that the
increase in lipophicity of 16 did not enhance the
inhibitory activity. Reduction of 15 and 16 to 13
and 14, respectively, was also shown not to
enhance the inhibitory effects on the fMLP=CB-
induced response (Hsieh et al 1998). Hence the
enone moiety of chalcones appears to be required
for inhibition of neutrophil degranulation. Tri-
¯uoperazine was used in this study as a postive
control and produced a dose-dependent inhibition
of neutrophil degranulation caused by fMLP=CB.
1
(41Á5Æ 1Á2 and 63Á7Æ 2Á7% release of b-glu-
curonidase and histamine, respectively, as con-
trol values) (Table 4), indicating that substituting
the 20,50-diphenolic or 20,50-dihydroxyphenyl or the
B ring of 20,50-dihydroxychalcone did not enhance
the inhibitory effects on mast cells degranulation
(Hsieh et al 1998). The ®nding that reduction of 15
to the 20,50-dihydroxydihydrochalcone (13) abol-
ished the inhibitory activity (Table 4) indicates that
the enone moiety of chalcones appears to be
required for the inhibition of mast cell degranula-
tion. Mepacrine was used in this study as a positive
control and produced a dose-dependent inhibition
of mast cell degranulation caused by compound
48=80.
1
fMLP=CB (1 mM=5 mg mL ) induced the release
of b-glucuronidase and lysozyme (21Á1Æ 1Á5 and
40Á6Æ 2Á0%, respectively) from rat neutrophils.
Compounds 1, 15 and 16 had potent and con-
centration-dependent inhibitory effects on the
activation of neutrophils while 2 did not have any
signi®cant inhibitory effects (Table 5). Compounds
4±7 also had signi®cant and concentration-depen-
1
fMLP=CB (0Á3 mM=5 mg mL ) or PMA (3 nM)
stimulated superoxide anion formation (1Á23Æ 0Á12
and 2Á58Æ 0Á13 nmol per 30 min per 106 cells,
respectively) from rat neutrophils. As shown in

SYNTHESIS AND ANTI-INFLAMMATORY EFFECT OF CHALCONES
Table 3. 13C NMR data for various chalcones and dihydrochalcones.
169
Compound
1a
2b
3c
4c
5b
6b
7b
8b
9b
10b
11b
12b
13b
14b
C-10
C-20
C-30
C-40
C-50
C-60
C-1
C-2
C-3
C-4
C-5
C-6
C-a
C-b
OCH3
121Á4 119Á9
123Á7 119Á8 119Á7 119Á7 119Á6 129Á5 128Á0 129Á6 128Á1 118Á9 118Á9
165Á1 163Á6 146Á9 154Á9 157Á8 157Á8 157Á9 158Á0 153Á6 153Á0 153Á2 152Á3 156Á2 156Á8
117Á8 118Á7 133Á6 116Á6 117Á8 117Á5 119Á0 119Á4 114Á4 115Á1 115Á2 115Á2 119Á2 119Á5
137Á8 136Á5 129Á6 124Á7 123Á5 123Á5 123Á7 124Á0 127Á3 127Á6 127Á6 127Á7 124Á7 124Á8
119Á6 118Á9 135Á4 153Á6 151Á6 151Á6 151Á7 151Á8 152Á6 152Á2 152Á3 153Á2 147Á5 147Á3
129Á8 129Á6
114Á6 113Á5 112Á9 112Á9 113Á0 113Á4 114Á7 114Á4 114Á3 114Á6 114Á5
123Á3 133Á1 128Á1 130Á9 127Á6 127Á3 131Á8 133Á1 133Á7 133Á8 133Á8 133Á8 140Á6 139Á1
158Á9 129Á8 115Á8 115Á4 110Á4 130Á5 128Á7 129Á8 129Á4 129Á1 129Á1 129Á1 128Á6 128Á7
117Á8 129Á3 146Á4 146Á8 149Á3 114Á5 129Á8 129Á4 129Á1 129Á3 129Á4 129Á4 128Á4 129Á8
131Á8 136Á9 150Á1 150Á0 151Á9 162Á0 141Á6 136Á9 136Á0 135Á9 135Á9 135Á9 126Á3 132Á1
121Á6 129Á3 116Á6 115Á5 111Á2 114Á5 129Á8 129Á4 129Á1 129Á3 129Á4 129Á4 128Á4 129Á8
133Á9 129Á8 123Á7 128Á2 123Á1 130Á5 128Á7 129Á8 129Á4 129Á1 129Á1 129Á1 128Á6 128Á7
120Á4 120Á6 119Á2 119Á4 119Á2 119Á2 119Á3 120Á6 119Á4 120Á4 120Á3 120Á4
142Á5 143Á9 146Á4 146Á5 145Á8 145Á4 145Á7 144Á1 141Á5 140Á9 140Á8 140Á8
40Á1
29Á9
39Á8
29Á2
56Á2;
56Á8
56Á2
55Á4;
56Á1
56Á1
56Á2
55Á8;
56Á5
OCH2
64Á1;
65Á2
70Á2;
71Á1
22Á6;
22Á7
68Á4;
69Á2
19Á2;
19Á4
31Á4;
31Á5
CH2
CH2CH2CH3
CˆO
196Á0 193Á5 184Á3 195Á1 193Á2 193Á0 193Á4 193Á1 192Á0 191Á8 191Á8 191Á9 204Á8 204Á8
CH3
21Á6
14Á8;
15Á0
10Á5;
10Á6
13Á7;
13Á8
b
c
aMeasured in (CD3)2CO. Measured in CDCl3. Measured in CD3OD.
Table 6, compounds 4±9 had potent inhibitory
effects on fMLP=CB-induced superoxide anion
formation from rat neutrophils, while 3, 4 and 8
also showed potent inhibitory effects on the PMA-
induced response. Compounds 10±12 (with two
alkyl chains of C2-C4 at the 20 and 50 positions of
16) and 9±12 (with two alkyl chains of C1-C4 at the
20 and 50 positions of 16) did not inhibit the
superoxide anion formation caused by fMLP=CB or
PMA, respectively. These results indicate that the
introduction of a lipophilic alkyl group at the 20 and
50 positions of 16 might attenuate its inhibitory
effects on fMLP=CB- and PMA-induced responses.
Reduction of 15 and 16 to 13 and 14, respectively,
did not inhibit fMLP=CB- or PMA-induced super-
oxide anion formation from rat neutrophils (Hsieh
et al 1998), suggesting that the enone moiety of
chalcones is also required for the inhibition of
superoxide anion formation from rat neutrophils.
Chalcones (except for 2, 4, 8, and 10±14) showed
different potencies for the inhibition of fMLP=CB-
and PMA-induced responses. It has been reported
that fMLP and PMA induce superoxide anion
formation by activating the same oxidase in
neutrophils but that they utilize different trans-
duction mechanisms and are regulated differently
(Segal & Abo 1993). Our results indicated that the
mechanisms by which compounds 1 and 3 inhibit
superoxide anion formation were different from
those of 5±7 and 9. These inhibitory compounds
exert better stimulant selectivity than tri-
¯uoperazine. Tri¯uoperazine was used in this study
as a positive control and produced a dose-depen-
Table 4. The inhibitory effects of chalcones on the release of
b-glucuronidase and histamine from rat peritoneal mast cells
stimulated with compound 48=80.
Compound
IC50 (mM)a
b-Glucuronidase
Histamine
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
45Á8Æ 7Á0
>30 (10Á2Æ 5Á0)
>10 (13Á9Æ 3Á7)
>30 (23Á0Æ 2Á0)
65Á3Æ 6Á4
54Á1Æ 9Á3
>30 ( 0Á1Æ 3Á2)
>10 (35Á5Æ 2Á8)
>30 (30Á6Æ 0Á2)
80Á8Æ 17Á2
81Á0Æ 6Á7
>100 (38Á2Æ 7Á9)
>100 (47Á6Æ 6Á5)
>30 (5Á1Æ 2Á2)
>100 (39Á2Æ 5Á9)
>100 (15Á2Æ 3Á5)
>100 (4Á5Æ 3Á0)
>100 (3Á6Æ 3Á7)
>100 (14Á3Æ 0Á5)
>30 (9Á0Æ 4Á2)
30Á1Æ 2Á96b
80Á1Æ 11Á9
>30 (16Á7Æ 5Á7)
88Á8Æ 8Á5
>100 (16Á2Æ 2Á5)
>100 (8Á6Æ 8Á2)
>100 (14Á9Æ 7Á9)
>100 (28Á1Æ 1Á5)
>30 (22Á0Æ 6Á4)
20Á8Æ 0Á84b
>100 (28Á7Æ 3Á7)c
21Á2Æ 2Á2
>100 (41Á7Æ 3Á0)c
18Á4Æ 2Á5
Mepacrine
a
Results are presented as averageÆ s.e.m. (nˆ3±5). When
50% inhibition could not be reached at the highest concentra-
b
tion, the % of inhibition is given in parentheses. Data from
c
Lin et al (1997). Data from Hsieh et al (1998).

170
HSIN-KAW HSIEH ET AL
Table 5. The inhibitory effects of chalcones on the release of
b-glucuronidase and lysozyme from rat neutrophils stimulated
with fMLP=CB.
Table 7. The inhibitory effects of chalcones on the accumu-
lation of NO2 from Raw 264Á7 cells stimulated with LPS and
N9 cells stimulated with LPS.
Compound
IC50 (mM)a
b-Glucuronidase
Compound
IC50 (mM)a
Lysozyme
Raw 264Á7 cells
N9 Cells
±
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
1Á6Æ 0Á2
>30 (33Á4Æ 4Á0)
>10 (45Á0Æ 11Á8)
16Á9Æ 2Á8
1Á4Æ 0Á2
1
2
3
4
5
6
7
8
±
>30 (19Á4Æ 2Á4)
>10 (30Á6Æ 12Á2)
13Á1Æ 2Á0
>30 (25Á9Æ 1Á7)
>30 ( 34Á3Æ 2Á3)
±
±
±
±
±
±
±
±
±
±
5Á6Æ 1Á4
7Á3Æ 1Á4
11Á9Æ 1Á4
16Á9Æ 2Á4
17Á4Æ 1Á8
7Á9Æ 2Á7
>30 (29Á1Æ 5Á1)
>30 (16Á0Æ 1Á4)
>30 (28Á2Æ 5Á7)
>10 (8Á0Æ 0Á8)
>3 (14Á9Æ 0Á4)
>3 (17Á5Æ 0Á7)
>10 (29Á7Æ 1Á9)
11Á8Æ 0Á9
>1 (12Á4Æ 1Á1)
1Á1Æ 0Á1
>30 ( 6Á5Æ 0Á6)
5Á8Æ 0Á2
1Á0Æ 0Á1
0Á7Æ 0Á06
1Á9Æ 0Á1
>3 (48Á8Æ 3Á3)
>3 (5Á9Æ 1Á5)
0Á3Æ 0Á01
11Á9Æ 1Á4
14Á7Æ 2Á8
9
10
11
12
13
14
L-NAME
>100 (34Á7Æ 3Á9)
>100 (30Á1Æ 4Á4)
>100 (25Á8Æ 4Á3)
47Á7Æ 7Á7
>100 (21Á9Æ 9Á1)
>100 (16Á3Æ 3Á4)
>100 (17Á0Æ 2Á9)
53Á4Æ 7Á7
16Á3Æ 0Á9
>30 (37Á2Æ 3Á2)
3Á6Æ 0Á3b
2Á3Æ 0Á2b
0Á6Æ 0Á1c
2Á6Æ 0Á2c
Tri¯uoperazine
15Á2Æ 2Á6
13Á8Æ 2Á2
a
Results are presented as averageÆ s.e.m. (nˆ3±5). When
50% inhibition could not be reached at the highest concentra-
tion, the % of inhibition is given in parentheses. ±, Not
determined.
a
Results are presented as averageÆ s.e.m. (nˆ3±5). When
50% inhibition could not be reached at the highest concentra-
b
tion, the % of inhibition is given in parentheses. Data from
c
Lin et al (1997). Data from Hsieh et al (1998).
ni®cant and concentration-dependent inhibitory
effects on NO accumulation from Raw 264Á7
mouse macrophage-like cells (70Á5Æ 1Á6 mM nitrite
accumulation as control value). LPS also activated
murine microglial cell lines N9 to produce NO
(Meda et al 1995). Compounds 9±12 showed
potent and concentration-dependent inhibition of
LPS-induced NO formation from N9 cells
(14Á7Æ 0Á6 mM nitrite accumulation as control
value), indicating that compounds with two alkyl
chains of C2-C4 at the 20 and 50 position of 16
exhibited stronger inhibitory effects on NO for-
mation from N9 cells. This shows that increasing
the lipophicity of a compound enhances its inhibi-
tory effects on NO formation from N9 cells.
Compound 13 (reduction from 15) exerted sig-
ni®cant inhibitory effects on NO formation from
Raw 264Á7 cells stimulated with LPS, while the
effects of 14 (reduction from 16) were insigni®cant.
NG-Nitro-L-arginine methylester (L-NAME) is a
competitive inhibitor of NO synthase (Crossin
1991), and hence inhibited the NO formation from
neutrophils.
dent inhibition of superoxide anion formation from
rat neutrophils with fMLP=CB and PMA, respec-
tively.
Following the activation of macrophages, NO
was generated in response to LPS (Ding et al 1988).
As shown in Table 7, compound 13 showed sig-
Table 6. The inhibitory effects of chalcones on superoxide
anion formation from rat neutrophils stimulated with
fMLP=CB or PMA.
Compound
IC50 (mM)a
fMLP=CB
PMA
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
>100 (43Á0Æ 10Á5)
>30 (28Á7Æ 7Á9)
>100 (15Á4Æ 8Á6)
6Á2Æ 3Á3
46Á6Æ 7Á4
>30 (11Á8Æ 9Á4)
3Á9Æ 0Á4
4Á9Æ 0Á8
2Á8Æ 0Á2
>100 (33Á4Æ 5Á5)
>100 ( 34Á3Æ 9Á9)
>100 ( 78Á9Æ 21Á7)
4Á2Æ 0Á6
35Á5Æ 10Á1
27Á7Æ 4Á9
4Á2Æ 0Á5
6Á3Æ 0Á5
>100 (39Á9Æ 7Á6)
>100 (2Á1Æ 7Á2)
>100 (15Á4Æ 11Á9)
>100 (16Á1Æ 6Á4)
>10 (34Á8Æ 7Á2)
>3 (45Á3Æ 7Á7)
±
>100 (29Á8Æ 4Á5)
>100 (7Á3Æ 8Á2)
>100 (27Á6Æ 9Á0)
>10 (10Á7Æ 8Á9)
>3 ( 26Á7Æ 6Á4)
2Á6Æ 0Á1b
Our previous report indicates that the anti-
in¯ammatory effect of 16 is not due to the release
of steroid hormones from the adrenal gland but to
the suppression of chemical mediators released
from mast cells and neutrophils (Hsieh et al 1998).
The present study has also demonstrated that most
of the chalcone derivatives studied have inhibitory
effects on in¯ammatory cells in-vitro. The NO
formation from N9 cells was inhibited by com-
>100 (19Á5Æ 1Á7)c
8Á0Æ 0Á2
>100 (40Á3Æ 14Á4)c
5Á0Æ 0Á3
Tri¯uoperazine
a
Results are presented as averageÆ s.e.m. (nˆ3±5). When
50% inhibition could not be reached at the highest concentra-
b
tion, the % of inhibition is given in parentheses. Data from
c
Lin et al (1997). Data from Hsieh et al (1998).

SYNTHESIS AND ANTI-INFLAMMATORY EFFECT OF CHALCONES
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