Inhibitory effects of chalcone glycosides isolated from Brassica rapa L. 'hidabeni' and their synthetic derivatives on LPS-induced NO production in microglia

Article abstract of DOI:10.1016/j.bmc.2011.07.036

Activation of microglia induces the production of various inflammatory mediators including nitric oxide (NO), leading to neurodegeneration in many central nervous system diseases. In this study, we examined the effects of chalcone glycosides isolated from

Full text of DOI:10.1016/j.bmc.2011.07.036

Bioorganic & Medicinal Chemistry 19 (2011) 5559–5568
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Inhibitory effects of chalcone glycosides isolated from Brassica rapa
L. ‘hidabeni’ and their synthetic derivatives on LPS-induced NO production
in microglia
Hirokazu Hara a, , Yoko Nakamura a, Masayuki Ninomiya b, Ryosuke Mochizuki b, Tetsuro Kamiya a,
⇑
Elias Aizenman c, Mamoru Koketsu b, Tetsuo Adachi a
a Laboratory of Clinical Pharmaceutics, Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi, Gifu 501-1196, Japan
b Department of Materials Science and Technology, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
c Department of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Activation of microglia induces the production of various inflammatory mediators including nitric oxide
(NO), leading to neurodegeneration in many central nervous system diseases. In this study, we examined
the effects of chalcone glycosides isolated from Brassica rapa L. ‘hidabeni’ on lipopolysaccharide (LPS)-
Received 3 June 2011
Revised 19 July 2011
Accepted 20 July 2011
Available online 26 July 2011
induced NO production using rat immortalized microglia HAPI cells. 40-O-b- -Glucopyranosyl-30,
D
4-dimethoxychalcone (A2) inhibited LPS-induced inducible NO synthase (iNOS) expression and NO pro-
duction. However, A2 did not affect nuclear factor- B and mitogen-activated protein kinase pathways.
j
Keywords:
The signal transduction and activator of transcription 1 (STAT1), which is activated via production of
IFN-b by LPS, is an important transcription factor responsible for LPS-induced iNOS expression. A2 sup-
pressed LPS-induced phosphorylation and nuclear translocation of STAT1, although it had no effects on
LPS-induced IFN-b expression. These results indicate that the inhibitory effect of A2 is due to the preven-
tion of STAT signaling. Moreover, structure–activity relationship studies on newly synthesized ‘hidabeni’
Brassica rapa L. ‘hidabeni’
Chalcone glycoside
Microglia
Inducible nitric oxide synthase
Signal transduction and activator of
transcription 1
chalcone derivatives showed that 40-O-b-
functional groups in the B-ring, inhibits LPS-induced NO production more potently than A2.
Ó 2011 Elsevier Ltd. All rights reserved.
D
-glucopyranosyl-30-methoxychalcone (A11), which has no
1. Introduction
microglia. Increasing evidence suggests that the excess NO released
from activated microglia results in neuronal cell death.3,4
Microglia, resident immune cells in the central nervous system
(CNS), are involved in the progression of neurodegeneration in many
CNS disorders, such as ischemic stroke and Alzheimer’s disease.1,2 It
has been reported that cerebral ischemia stimulates activation of
microglia in the injured area and elicits a robust neuroinflammatory
response.1 Activated microglia produce inflammatory mediators
including nitric oxide (NO). NO, which is a multifunctional effector
molecule, is produced by inducible NO synthase (iNOS) in activated
Toll-like receptor 4 (TLR4) plays a key role in innate immunity.
TLR4-mediated signaling stimulates expression of various inflam-
matory response genes including iNOS and proinflammatory cyto-
kines, tumor necrosis factor-a (TNF-a
) and interleukin-1b (IL-1b).5
Recent studies suggest that TLR4 signaling exacerbates ischemic
outcomes.5,6 For example, it was reported that a loss of function
of TLR4 in TLR4 mutant mice protects against focal cerebral ische-
mia-induced neurodegeneration.7 Therefore, activation of TLR4
signaling caused by ischemic stroke is thought to be responsible
for neuronal cell death associated with brain ischemia. Lipopoly-
saccharide (LPS), one of the major constituents of the outer mem-
brane of Gram-negative bacteria, is a ligand of TLR4 and triggers
expression of iNOS and proinflammatory cytokines through activa-
Abbreviations: AlCl3, aluminium chloride; Ag2CO3, silver carbonate; BF3ꢀEt2O,
boron trifluoride-etherate; CNS, central nervous system; CHCl3, chloroform; CC,
column chromatography; ESI, electrospray ionization; EtOAc, ethyl acetate; ERK,
extracellular signal-regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehy-
drogenase; iNOS, inducible NO synthase; IFN-b, interferon-b; IL-1b, interleukin-1b;
LPS, lipopolysaccharide; JAK, Janus kinase; JNK, c-Jun N-terminal kinase; LC, liquid
tion of TLR4. Nuclear factor-jB (NF-jB) and signal transduction
and activator of transcription 1 (STAT1) have been shown to be
key transcription factors responsible for LPS-induced iNOS
expression.8
chromatography;
activated protein kinase; NO, nitric oxide; NF-
quadrupole time of flight; STAT1, signal transduction and activator of transcription
L-NAME, N(G)-nitro-
L-arginine methyl ester; MAPK, mitogen-
j
B, nuclear factor- B; QTOF,
j
NF-
inhibitor-
degradation of I-
j
B is sequestered in the cytoplasm by the inhibitory protein,
B (I- B), under normal conditions. LPS promotes the
B, and in turn stimulates the translocation of
1; TLR4, toll-like receptor 4; TNF-
a
, tumor necrosis factor-
a
.
j
j
⇑
Corresponding author. Tel.: +81 58 230 8100; fax: +81 58 230 8105.
E-mail address: harah@gifu-pu.ac.jp (H. Hara).
j
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.07.036

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H. Hara et al. / Bioorg. Med. Chem. 19 (2011) 5559–5568
NF-
j
B to the nucleus.9 The binding site of NF-
jB is reported to be
2.2. Effect of A2 on the NF-jB and MAPK pathways
located in the regulatory region of iNOS gene.10 On the other hand,
activation of STAT1 is mainly regulated via an autocrine loop
involving interferon-b (IFN-b) produced by LPS-activated macro-
phages.11,12 Secreted IFN-b stimulates phosphorylation of STAT1,
and in turn promotes formation of STAT1 dimerization and its
translocation to the nucleus. Since LPS-induced iNOS expression
has been shown to be suppressed in macrophages prepared from
STAT1ꢁ/ꢁ mice,13 SATA1 is known to be indispensable for this
phenomenon.
In the next step, we addressed the mechanism by which A2
inhibits iNOS expression induced by LPS. It has been reported that
LPS triggers iNOS expression via NF-jB and mitogen-activated pro-
tein kinase (MAPK) pathways.8,19 We investigated the effects of A2
on the activation of both pathways caused by LPS. To determine
whether A2 inhibits translocation of NF-
jB induced by LPS, we
performed immunoblot analysis using nuclear extracts. As shown
in Figure 4A, A2 had no effects on the translocation of NF-jB to
Chalcone, a subgroup of flavonoids, has been demonstrated to
possess anti-inflammatory,14 anti-allergic15 and anti-oxidant16
activities. Recently, we reported that chalcone glycosides isolated
from Brassica rapa L. ‘hidabeni’ have inhibitory effects on antigen-
induced degranulation in basophilic leukemia RBL-2H3 cells.17,18
In this study, we found that ‘hidabeni’ chalcones and their synthetic
derivatives suppress LPS-induced iNOS expression and NO produc-
tion in rat immortalized microglia, HAPI cells. Furthermore, we also
addressed the mechanism underlying the inhibitory effects of these
compounds on LPS-induced iNOS expression.
the nucleus.
To investigate the effect of A2 on the activation of MAPKs
caused by LPS, we performed immunoblot analysis for phosphory-
lation of ERK, JNK and p38 using cell lysates. As shown in Figure 4B,
although ERK was constitutively phosphorylated in HAPI cells,
phosphorylation of JNK and p38 was induced by the treatment
with LPS. However, A2 did not affect the phosphorylation state of
MAPKs. These results indicate that A2 suppresses LPS-induced
iNOS expression independently of the NF-jB and MAPK pathways.
2.3. A2 suppressed IFN-b signal triggered by LPS
2. Results
The production of IFN-b is up-regulated in response to LPS in
macrophages. Secreted IFN-b binds to type I IFN receptor (IFN-R),
and in turn stimulates phosphorylation of STAT1 via the recep-
tor-associated Janus kinase (JAK).20 The JAK-STAT pathway has
been shown to be responsible for LPS-induced iNOS expres-
sion.11,13 To investigate the effect of the compounds on LPS-in-
duced IFN-b mRNA expression in HAPI cells, we performed RT-
2.1. Effects of compounds on LPS-induced iNOS expression
To confirm whether LPS induces iNOS expression under our
conditions, HAPI cells were treated with 100 ng/mL LPS. As shown
in Figure 1A, the induction of iNOS mRNA reached a peak at 6 h.
LPS induced iNOS mRNA in a dose-dependent manner (Fig. 1B).
Next, to test the effects of six compounds isolated from Brassica
rapa L. ‘hidabeni’ (A1–A6, Fig. 2) on LPS-induced iNOS expression,
we performed RT-PCR. HAPI cells were pretreated with each com-
PCR. HAPI cells were pretreated with each compound (50 lM) for
30 min, followed by the treatment with LPS (100 ng/mL) for 6 h.
However, as shown in Figure 5A, these compounds hardly affected
LPS-induced IFN-b mRNA expression. Next, we examined the effect
of A2 on phosphorylation of SATA1 caused by LPS. As shown in Fig-
ure 5B, immunoblot analysis revealed that LPS induced phosphor-
ylation of STAT1 and A2 suppressed the phosphorylation.
Phosphorylation of STAT1 promotes translocation of STAT1 to the
nucleus. Therefore, we examined the effect of A2 on LPS-induced
nuclear translocation of STAT1. As expected, A2 suppressed trans-
location of STAT1 to the nucleus (Fig. 5C). Finally, to test the possi-
bility that down-regulation of IFN-R by A2 results in reduction of
LPS-induced STAT1 activation, we examined whether A2 sup-
presses expression of IFN-R. As shown in Figure 5D, A2 had no ef-
fects on expression of IFN-R.
pound (50 lM) for 30 min, followed by the treatment with LPS
(100 ng/mL). As shown in Figure 3A, A2 reduced the expression
of iNOS mRNA induced by LPS and other compounds had weak
inhibitory activities. In addition, we examined the effect of these
compounds on LPS-induced NO production. As shown in Figure
3B, treatment of HAPI cells with LPS promoted generation of NO.
A1 and A2 significantly prevented LPS-induced NO production.
These results indicated that A2 most potently suppressed LPS-in-
duced iNOS expression. Although we tested the effect of the com-
pounds on LPS-induced IL-1b expression, only A2 had a weak,
albeit significant, effect (Fig. 3A).
2.4. Synthesis of chalcone glycoside derivatives
As a continuation to the above-mentioned studies, we were
interested in the structure–activity relationships of chalcone glyco-
side derivatives with a focus on the B-ring structures against NO
production. Comparative analyses with a variety of chalcone glyco-
side derivatives having different functional groups are necessary to
examine the inhibitory effects of the most potent compounds.
Therefore, we synthesized a series of chalcone glycoside deriv-
atives through the Claisen–Schmidt reaction of acetophenone 1
and benzaldehydes 2a–2g using BF3ꢀEt2O,21 followed by Koenigs–
Knorr glycosylation (Scheme 1). Of the many methods available
for the synthesis of chalcones, the most widely used is the base-
catalyzed Claisen–Schmidt reaction. Although we first endeavored
to prepare the chalcone using the NaOH aq method, the yield was
quite low. Next, we tried to prepare the chalcone via acid-catalyzed
methodologies using AlCl3 or BF3ꢀEt2O. Both conditions gave the
chalcone, and we chose the BF3ꢀEt2O method, which produced
the product in higher yield than the AlCl3 method. Treatment of
acetophenone 1 with 2 equiv of benzaldehydes 2a–2g was carried
out in the presence of BF3ꢀEt2O (5 equiv) in 1,4-dioxane for 19 h.
Figure 1. LPS induces iNOS mRNA expression in HAPI cells. (A) Time-course study.
HAPI cells were treated with or without LPS (100 ng/mL) for 3, 6, 9, 12 and 24 h.
After the treatment, total RNA was extracted, and then RT-PCR was performed. A
representative result from three independent experiments is shown. (B) Dose-
dependent study. HAPI cells were treated with or without LPS (1, 10, 100 and
1000 ng/mL) for 6 h. After the treatment, total RNA was extracted, and then RT-PCR
was performed. A representative result from three independent experiments is
shown.

H. Hara et al. / Bioorg. Med. Chem. 19 (2011) 5559–5568
5561
3
OMe
OH
O
4
OH
OH
O
2
1
OMe
OMe
3'
HO
HO
HO
HO
O
O
4'
5'
5
β
2'
OH
OH
6
α
1'
6'
O
O
4'-O- -D-Glucopyranosyl
4'-O- -D-Glucopyranosyl
β
β
-4-hydroxy-3'-methoxychalcone (A1)
-3',4-dimethoxychalcone (A2)
HO
O
OH
O
OH
OH
OMe
HO
O
HO
O
HO
OH
O
β
4,4'-Di-O- -D-glucopyranosyl
-3'-methoxychalcone (A3)
HO
O
HO
OMe
O
O
HO
O
OH
OH
OH
O
O
OH
OH
OH
OH
HO
HO
O
HO
OMe
O
O
Picein (A4)
Glucoacetosyringone (A5)
Corchoionoside C (A6)
Figure 2. Chemical structure of isolated compounds from Brassica rapa L. ‘hidabeni’.
The reaction was carried out successfully at room temperature, and
the desired chalcones 3a–3g were obtained after the usual workup
of the reaction mixtures. For glycosylation, we first attempted to
employ the Schmidt glycosylation conditions. However, the prod-
3. Discussion
NO is thought to be an important mediator of progression of
neurodegeneration in many CNS diseases. Excessive NO release un-
der pathological conditions results in neuronal cell death. We have
demonstrated here that A2 from the aerial parts of Brassica rapa L.
‘hidabeni’ prevents LPS-induced iNOS expression and NO produc-
tion in HAPI cells. In addition, we addressed the mechanism by
which this compound suppresses LPS-induced iNOS expression,
and found that it inhibits the activation of STAT1 caused by LPS.
Binding LPS to TLR4 elicits a variety of biological functions
including the expression of pro-inflammatory gene products such
as cytokines and iNOS. The NF-
shown to be critically involved in LPS-induced iNOS expression.8,19
In fact, we also confirmed that the NF- B inhibitor, BAY11-7082,
and the JNK inhibitor, SP600125, suppressed LPS-induced iNOS
mRNA expression in HAPI cells (data not shown). Therefore, both
pathways are thought to be involved, at least in part, in the induc-
tion of iNOS mRNA by LPS in HAPI cells. However, A2 had no effects
uct was an a/b anomer mixture of the glucopyranose. Finally, chal-
cones were glycosylated using tetra-O-acetylglucopyranosyl
bromide with Ag2CO3 as a promoter in pyridine. The acetyl groups
of products 4a–4g were deprotected with sodium methoxide to
yield the corresponding chalcone glycoside derivatives A1, A2
and A7–A11.
2.5. Structure–activity relationships of chalcone glycoside
derivatives
jB and MAPK pathways have been
We examined the effects of newly synthesized compounds on
LPS-induced NO production and iNOS mRNA expression. As
shown in Figure 6A, A11 that has no functional groups in the
B-ring potently inhibited NO production to an extent similar to
j
that of the iNOS inhibitor, N(G)-nitro-L-arginine methyl ester (L-
NAME). As expected, it also suppressed iNOS mRNA induction
caused by LPS (Fig. 6B). As described above, A2 that has one
methoxy group at the 4-position of the B-ring had inhibitory ef-
fects, while A1 having one hydroxy at the same position and
other compounds having more than one hydroxy and/or methoxy
groups at the 3-, 4- and/or 5-positions of the B-ring exerted weak
or no effects.
on LPS-induced nuclear translocation of NF-jB and phosphoryla-
tion of JNK. Therefore, these pathways activated by LPS are unlikely
to contribute to the inhibitory effect of A2 on LPS-induced iNOS
mRNA expression. In addition, we also found that the inhibitory ef-
fect of A2 on IL-1b mRNA induction by LPS was weak compared
with the inhibitory effect on iNOS mRNA induction. Because NF-
j
B plays an important role in LPS-induced IL-1b expression,23 this
result concerning IL-1b supports the finding that A2 has only a
2.6. A2 and A11 suppress interferon-
expression
c (IFN-c)-induced iNOS
weak inhibitory activity toward the NF- B pathway. On the other
j
hand, it has been reported that the inhibitory effects of various
chalcone derivatives on LPS-induced iNOS mRNA expression are
It was reported that IFN-
pathway in microglia.22 Therefore, we determined whether A2 and
A11 suppress IFN- -induced iNOS expression. HAPI cells were
treated with IFN- in the presence or absence of these compounds,
and expression of iNOS mRNA was analyzed using RT-PCR. As
shown in Figure 7, A2 and A11 suppressed iNOS expression in-
c induces iNOS expression via the STAT
due to their function of preventing LPS-induced NF-jB and/or
MAPK activation.16,24–27 Therefore, these findings suggest that
the differences between these chalcone derivatives and A2 in
terms of the mechanisms of action might be due to differences in
chemical structures because the chalcones isolated from B. rapa
L. ‘hidabeni’ have a unique structure.17
c
c
duced by IFN-
A11, at least in part, inhibit activation of STAT signaling.
c. This result indicates the possibility that A2 and
NF-
jB signaling alone is not sufficient to accomplish maximal
induction of iNOS by LPS in macrophages. IFN-b that is secreted

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H. Hara et al. / Bioorg. Med. Chem. 19 (2011) 5559–5568
Figure 4. Effect of A2 on NF-
j
B and MAPK pathways. (A) Effect of A2 on nuclear
translocation of NF-jB. HAPI cells were pretreated with A2 (50 lM) or vehicle for
30 min, followed by treatment with or without LPS (100 ng/mL) for 3 h. Nuclear
extracts were prepared from the treated cells, and then subjected to immunoblot
analysis. A representative blot from three independent experiments is shown. (B)
Effect of A2 on phosphorylation of MAPKs. HAPI cells were pretreated with A2
(50 lM) or vehicle for 30 min, followed by treatment with or without LPS (100 ng/
mL) for 1 h. Whole cell lysates were prepared from the treated cells, and then
subjected to immunoblot analysis. A representative blot from three independent
experiments is shown.
pathways. Several binding sites for transcription factors including
NF-
region of IFN-b gene.28 We also confirmed that the pharmacological
inhibitor of NF- B or JNK, which is associated with AP-1 activation,
jB and activator protein-1 (AP-1) are located in the regulatory
j
Figure 3. Effects of compounds on LPS-induced iNOS and IL-1b mRNA expression
(A) and NO production (B). (A) HAPI cells were pretreated with each compound
reduced LPS-induced IFN-b mRNA expression in HAPI cells (data
(A1–A6, 50 lM) for 30 min, followed by treatment with or without LPS (100 ng/mL)
for 6 h. Each compound was present 30 min prior to and during LPS treatment. After
the treatment, total RNA was extracted, and then RT-PCR was performed. Values
not shown). As described above, however, A2 did not prevent
LPS-induced activation of both NF-jB and JNK pathways. There-
fore, our finding that A2 did not affect LPS-induced IFN-b mRNA
expression seems reasonable.
(mean SEM, n = 3) are expressed as
a percentage compared with the mRNA
expression level in cells treated with LPS alone. ꢂ and ꢂꢂ, significant differences (P
Binding of secreted IFN-b to IFN-R stimulates the activation of
STAT1. It was reported that LPS-induced iNOS expression is mark-
edly suppressed in macrophages prepared from STAT1ꢁ/ꢁ mice.13
Therefore, STAT signal plays a key role in iNOS induction caused
by LPS. We demonstrated here that A2 prevents LPS-induced acti-
vation of STAT1 including phosphorylation and translocation to the
nucleus. It has been reported that Tyr-70129 and Ser-72729,30
residues were identified as phosphorylation sites in STAT1.
Phosphorylation of Tyr-701 results in dimerization and nuclear
translocation of STAT1, whereas phosphorylation of Ser-727 is
required for the augmentation of transcriptional ability of STAT1.
In this study, we employed anti-phospho Tyr-701 antibody to
detect the phosphorylation state of STAT1. Because JAK is respon-
sible for the phosphorylation of Tyr-701, it is likely that A2 inhibits
JAK activation. Although ERK31 or p3832 is reported to be involved
<0.05 and P <0.01, respectively) compared with cells treated with LPS alone. (B)
HAPI cells were pretreated with each compound (A1–A6, 50 lM) for 30 min,
followed by treatment with or without LPS (100 ng/mL) for 24 h. Each compound
was present 30 min prior to and during LPS treatment. Nitrite level in culture
medium was measured by the Griess method. Values (mean SD, n = 3) are
expressed as the fold induction relative to untreated cells. ꢂ and ꢂꢂ, significant
differences (P <0.05 and P <0.01, respectively) compared with cells treated with LPS
alone.
in response to LPS has been shown to be an essential mediator for
iNOS induction.11,12 For example, it was reported that neutralizing
antibody against IFN-b attenuates induction of iNOS mRNA caused
by LPS in macrophages.11 However, A2 failed to suppress the
induction of IFN-b mRNA by LPS. Transcriptional regulation of
IFN-b gene has been shown to be mediated via various signaling

H. Hara et al. / Bioorg. Med. Chem. 19 (2011) 5559–5568
5563
either of these signaling pathways. Because A2 suppressed LPS-in-
duced STAT1 activation, we speculate that it may inhibit activation
of the JAK-STAT pathway. On the other hand, other tyrosine protein
kinases such as c-Src have been reported to be involved in phos-
phorylation of STAT1.33 Moreover, it was also demonstrated that
butein (20,3,4,40-tetrahydroxychalcone) induces expression of the
protein tyrosine phosphatase SHP-1 that is involved in the down-
regulation of STAT1 phosphorylation.34 Therefore, at present, we
cannot rule out the possibility that kinases other than JAK or pro-
tein tyrosine phosphatases contribute to the inhibitory effect of A2.
On the basis of the structure of A2, we synthesized five new chal-
cone glycoside derivatives. Among compounds tested, A11 that
had no hydroxy and methoxy groups in the B-ring most potently
inhibited LPS-induced NO production and iNOS mRNA expression.
The addition of two or more functional groups to the B-ring re-
duced the inhibitory activities of the chalcone derivatives. We also
found that A2 and A11 reduced IFN-
c-induced iNOS expression.
Because IFN- regulates induction of iNOS via the STAT signaling
c
pathway, this result strongly indicates that these compounds inhi-
bit activation of STAT signaling caused by LPS. However, further
studies are needed to identify target molecule(s) of these
compounds.
In conclusion, we demonstrated here that A2, a ‘hidabeni’ chal-
cone derivative, prevents LPS-induced NO production in immortal-
ized microglia HAPI cells. We also found that inhibitory effect of A2
on iNOS induction by LPS is probably due to abrogation of LPS-in-
duced STAT1 activation (Fig. 8). Moreover, structure–activity rela-
tionship studies led to the identification of A11, which potently
inhibited NO production and iNOS induction. Because microglia
play a key role in the progression of neurodegeneration in CNS dis-
orders, these compounds might be good candidates for the devel-
opment of novel neuroprotective drugs.
4. Experimental section
4.1. Biochemical methods
4.1.1. Materials
LPS was purchased from Sigma (St. Louis, MO). Anti-STAT1,
anti-phospho JNK (Thr183/Thr185), anti-JNK, anti-phospho ERK,
anti-ERK, anti-phospho p38 and anti-p38 antibodies were pur-
chased from Cell Signaling Technology (Denvers, MA). Anti-phos-
pho-STAT1 (Thr701) antibody was purchased from Signalway
Figure 5. A2 suppressed activation of IFN-b signal triggered by LPS. (A) Effect of
compounds on LPS-induced IFN-b mRNA expression. HAPI cells were treated with
or without LPS (100 ng/mL) for 6 h. Each compound (A1–A6, 50 lM) or vehicle was
present 30 min prior to and during LPS treatment. After the treatment, total RNA
was extracted, and then RT-PCR was performed. Values (mean SEM, n = 3) are
expressed as a percentage compared with the mRNA expression level in cells
treated with LPS alone. (B) Effect of A2 on LPS-induced phosphorylation of STAT1.
Antibody (Pearland, TX). Anti-NF-
from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-actin anti-
body was purchased from Millipore (Billerica, MA). -NAME was
jB p65 antibody was purchased
L
purchased from Wako Pure Chemical Industries (Osaka, Japan).
HAPI cells were pretreated with A2 (50 lM) or vehicle for 30 min, followed by
incubation with or without LPS (100 ng/mL) for 3 h. Whole cell lysates were
prepared from the treated cells, and then subjected to immunoblot analysis. A
representative blot from three independent experiments is shown. (C) Effect of A2
on LPS-induced nuclear translocation of STAT1. HAPI cells were pretreated with A2
4.1.2. Cell culture
Rat immortalized-microglia HAPI cells were generously do-
nated by Dr. J. Connor (Pennsylvania State University, Hershey,
PA). HAPI cells were cultured in DMEM containing 10% heat-inac-
tivated fetal calf serum (FCS), 4 mM glutamine, 100 units/mL pen-
icillin G and 0.1 mg/mL streptomycin in a humidified 5% CO2/95%
air incubator at 37 °C.
(50 lM) or vehicle for 30 min, followed by incubation with or without LPS (100 ng/
mL) for 3 h. Nuclear extracts were prepared from the treated cells, and then
subjected to immunoblot analysis. A representative blot from three independent
experiments is shown. (D) Effect of A2 on IFN-R mRNA expression. HAPI cells were
treated with or without LPS (100 ng/mL) for 6 h. A2 (50 lM) or vehicle was present
30 min prior to and during LPS treatment. After the treatment, total RNA was
extracted, and then RT-PCR was performed. Values (mean SEM, n = 3) are
expressed as fold change compared with untreated cells.
4.1.3. RT-PCR
HAPI cells were seeded in a 6-cm-diameter dish at a density of
ꢃ7.0 ꢄ 105 cells in DMEM containing 1% FCS. The next day, cells were
treated with LPS at various concentrations of LPS for the time indi-
cated in the figure legends. Total RNA was extracted from the treated
cells with TRIzol reagent (Invitrogen, Carlsbad, CA). First-strand cDNA
in phosphorylation at Ser-727, A2 had no inhibitory effects on acti-
vation of both kinases. Therefore, A2 is unlikely to influence the
phosphorylation of STAT1 at Ser-727 caused by LPS.
A number of chalcones have been reported to possess anti-
inflammatory activities. As described above, the properties of these
chalcones are attributable to the inhibitory activities toward the
was synthesized from 4
reaction mixture (1 L) were amplified with primers specific for rat
iNOS (forward primer, 50-TTGCTTCTGTGCTAATGCGG-30; reverse
lg of total RNA. Aliquots of transcription
l
NF-j
B and MAPK pathways.16,24–27 However, A2 hardly affected

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H. Hara et al. / Bioorg. Med. Chem. 19 (2011) 5559–5568
R1
OMe
R2
R3
R1
OMe
3a R1=R3=H, R2=OAc (40 %)
HO
BF3 Et2O
R2
R3
3b R1=R3=H, R2=OMe (58 %)
HO
+
R1=R2=OMe, R3=H (65 %)
3c
3d
3e
1,4-dioxane
R1=R2=R3=OMe (39 %)
OHC
AcO
O
R1=R3=OMe, R2=OAc (50 %)
O
2a-2g
3f R1=OAc, R2=OMe, R3=H (50 %)
3g R1=R2=R3=H (47 %)
1
3a-3g
OAc
O
AcO
AcO
R1
R1
AcO
Br
R2
R3
R2
R3
OAc
OH
O
OMe
OMe
Ag2CO3
O
NaOMe
MeOH
HO
HO
O
O
AcO
pyridine
OAc
OH
O
O
4a-4g
A1, A2, and A7-A11
4a R1=R3=H, R2=OAc (32 %)
A1 R1=R3=H, R2=OH (Quant.)
R1=R3=H, R2=OMe (90 %)
A2 R1=R3=H, R2=OMe (Quant.)
4b
4c
4d
4e
R1=R2=OMe, R3=H (85 %)
R1=R2=R3=OMe (76 %)
A7
A8
A9
R1=R2=OMe, R3=H (Quant.)
R1=R2=R3=OMe (83 %)
R1=R3=OMe, R2=OAc (56 %)
R1=R3=OMe, R2=OH (Quant.)
4f R1=OAc, R2=OMe, R3=H (53 %)
4g R1=R2=R3=H (47 %)
A10 R1=OH, R2=OMe, R3=H (84 %)
A11 R1=R2=R3=H (92 %)
Scheme 1. Synthesis of chalcone glycoside derivatives.
primer, 50-CAGAACTGAGGGTACATGCT-30), IL-1b (forward primer,
50-AGTGTCTGAAGCAGCTATGG-30; reverse primer, 50-TCATCATCC-
CACGA GTCACA-30), IFN-b (forward primer, 50-ATCGACTACAAG-
CAGCTCCA-30; reverse primer, 50-ACCTTTGTACCCTCCAGTAA-30),
IFN-R (forward primer, 50-AGAGGTGCTTCGTCATTGAG-30; reverse
primer, 50-CATCCACATGTACTCCAGAC-30), and glyceraldehyde-3-
phosphate dehydrogenase (GAPDH) (forward primer, 50-ACCACAGT
CCATGCCATCAC-30; reverse primer, 50-TCCACCACCCTGTTGCTGTA-
30). For amplification of iNOS, PCR was carried out using EX Taq (Taka-
ra Bio, Shiga, Japan) as follows: 2 min at 94 °C, one cycle; 40 s at 94 °C,
40 s at 58 °C, 1 min at 72 °C, 27 cycles. For amplification of IFN-b, PCR
was carriedout asfollows:2 min at 94 °C, one cycle;40 s at 94 °C, 40 s
at 54 °C, 1 min at 72 °C, 35 cycles. For amplification of IL-1b and GAP-
DH, PCR was carried out as follows: 2 min at 94 °C, one cycle; 40 s at
94 °C, 40 s at 58 °C, 1 min at 72 °C, 30 or 18 cycles, respectively. For
amplification of IFN-R, PCR was carried out using KOD FX (Toyobo,
Osaka, Japan) as follows: 2 min at 94 °C, one cycle; 10 s at 98 °C,
30 s at 55 °C, 40 s at 68 °C, 35 cycles. Aliquots of the PCR mixtures
wereseparatedon 2% agarosegelandstained with ethidiumbromide.
Densitometric analyses were performed using Multi Gauge (Fuji Film,
Tokyo, Japan). The iNOS, IL-1b, IFN-b, and IFN-R mRNA levels were
normalized relative to the GAPDH mRNA level in each sample.
NaCl, 10% glycerol, 5 lg/mL leupeptin, 0.5 mM PMSF and 2 mM
DTT), and stood for 30 min on ice. The nuclear extracts were centri-
fuged at 14,000 rpm for 10 min at 4 °C to remove cellular debris. The
protein content of the supernatants was determined using Bio-Rad
protein assay reagent.
4.1.5. Immunoblotting
Whole cell lysates (40 lg) or nuclear extracts (10 lg) were sep-
arated by SDS–PAGE on 10% or 12% (w/v) polyacrylamide gels.
After being transferred onto a PVDF membrane, the blotted mem-
brane was blocked using PBS containing 1% BSA. The membrane
was incubated with each primary antibody (1:1,000), biotin-conju-
gated second antibody (1:2000) and ABC reagents (Vector Labora-
tories, Inc., Burlingame, CA) (1:5000), and then visualized using the
Super-signal West Pico Chemiluminescent Substrate (Thermo Fish-
er Scientific Inc., Waltham, MA).
4.1.6. Measurement of nitrite production
HAPI cells were seeded in a 24-well plate at a density of
ꢃ1.0 ꢄ 105 cells in DMEM containing 1% FCS. The next day, the
cells were treated with 100 ng/mL LPS in the presence or absence
of compounds for 24 h. The nitrite concentrations in the medium
were measured using NO2/NO3 Assay Kit-C II (Dojin Chemicals,
Kumamoto, Japan) according to the manufacturer’s protocol.
4.1.4. Preparation of whole cell and nuclear extracts
HAPI cells were treated with 100 ng/mL LPS in the presence or ab-
sence of compounds for the time indicated in the figure legends. The
incubation time in each experiment was determined from results of
preliminaryexperiments. After thetreatment, the cells were washed
twice with ice-cold PBS. For preparation of whole cell extracts, the
4.1.7. Statistical analysis
Data were analyzed using ANOVA followed by post hoc Bonfer-
roni tests or Student’s t-test. A P value less than 0.05 was consid-
ered significant.
cells were collected using 150
7.4, containing 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 10 mM
NaF, 1 mM Na3VO4, 20 mM b-glycerophosphate, 5 g/mL leupeptin,
lL of buffer A (20 mM Tris–HCl, pH
4.2. General chemistry methods
l
1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 mM dithiothre-
itol (DTT)), and were lysed on ice for 30 min. The lysates were centri-
fuged at 14,000 rpm for 10 min at 4 °C to remove cellular debris. For
preparation of nuclear extracts, the cells were collected using buffer
B (20 mM HEPES-NaOH, pH 7.8, containing 15 mM KCl, 2 mM MgCl2,
All solvents and reagents were purchased from the suppliers
and used without further purification. IR spectra were recorded
on a JASCO FT/IR-460 Plus spectrophotometer. 1H (400 MHz) and
13C (100 MHz) NMR spectra were recorded with a JEOL ECX 400
spectrometer with tetramethylsilane as an internal standard. MS
spectra were obtained using a Waters Xero™ QTOFMS. Optical
rotations were measured using a HORIBA SEPA-300 polarimeter
at a specified temperature and concentration in the solvents indi-
cated. Silica gel column chromatography (CC) was performed on
5 lg/mL leupeptin, 0.5 mM PMSF and 2 mM DTT), and centrifuged.
The cells were lysed in buffer C (buffer B containing 0.2% Nonidet
P-40) for 5 min on ice, and centrifuged. Finally, the pellets were sus-
pended in buffer D (20 mM HEPES-NaOH, pH 7.8, containing 0.4 M

H. Hara et al. / Bioorg. Med. Chem. 19 (2011) 5559–5568
5565
Figure 7. Effect of A2 and A11 on IFN-
HAPI cells were pretreated with A2 (50
followed by treatment with or without IFN-
was present 30 min prior to and during IFN-
c
l
-induced iNOS expression in HAPI cells.
M), A11 (50 lM), or vehicle for 30 min,
c
(100 ng/mL) for 8 h. Each compound
c
treatment. After the treatment, total
RNA was extracted, and then RT-PCR was performed. Values (mean SEM, n = 3) are
expressed as a percentage compared with the mRNA expression level in cells
treated with IFN-
respectively) compared with cells treated with IFN-
c
alone. ꢂ and ꢂꢂ, significant differences (P <0.05 and P <0.01,
c
alone.
Figure 6. Effects of synthetic ‘hidabeni’ chalcone derivatives on LPS-induced NO
production (A) and iNOS mRNA expression (B). (A) HAPI cells were pretreated with
each compound (A1, A2, A7–A11, 50 lM; L-NAME 1 mM) or vehicle for 30 min,
followed by treatment with or without LPS (100 ng/mL) for 24 h. Each compound
was present 30 min prior to and during LPS treatment. Nitrite level in culture
medium was measured by the Griess method. Values (mean SD, n = 4) are
expressed as the fold induction relative to untreated cells. ꢂ and ꢂꢂ, significant
differences (P <0.05 and P <0.01, respectively) compared with cells treated with LPS
alone. L-N,
A7–A11, 50
L
-NAME. (B) HAPI cells were pretreated with each compound (A1, A2,
M) or vehicle for 30 min, followed by treatment with or without LPS
l
(100 ng/mL) for 6 h. Each compound was present 30 min prior to and during LPS
treatment. After the treatment, total RNA was extracted, and then RT-PCR was
performed. Values (mean SEM, n = 3) are expressed as a percentage compared
with the mRNA expression level in cells treated with LPS alone. ꢂ and ꢂꢂ, significant
differences (P <0.05 and P <0.01, respectively) compared with cells treated with LPS
alone.
Figure 8. Schematic diagram for inhibitory mechanism of A2 and A11.
silica gel N-60 (40–50 lm). Thin-layer chromatography (TLC) spots
on plates pre-coated with Silica Gel 60 F254 were detected with a
UV lamp (254 nm). Fractionations for all CCs were based on TLC
analyses.
room temperature. After stirring for 19 h, the resultant solution
was partitioned with EtOAc, washed with 10% HCl aq, distilled
water, brine, dried over anhydrous Na2SO4 and concentrated in va-
cuo. The residue was purified using silica gel CC eluted with n-hex-
ane/CHCl3 (2/1) to yield chalcones 3a–3g.
4.3. Isolated compounds from Brassica rapa L. ‘hidabeni’
The detailed isolation procedure was carried out as described
previously. The spectral data of isolated compounds A1–A6 have
been mentioned in the literature.17,18
4.4.1. 4-O-Acetyl-40-hydroxy-30-methoxychalcone (3a)
Yellow powder, 40% yield. IR (film): 3372, 1653, 1590 cmꢁ1 1H
.
NMR (400 MHz, (CD3)2CO): d 7.87 (1H, d, J = 15.6 Hz, H-b), 7.86
(2H, m, H-2 and H-6), 7.80 (1H, dd, J = 8.2, 2.3 Hz, H-60), 7.75 (1H,
4.4. Synthesis of chalcones 3a–3g
d, J = 15.6 Hz, H-a
), 7.70 (1H, s, H-20), 7.21 (2H, d, J = 9.2 Hz, H-3
and H-5), 6.97 (1H, d, J = 8.2 Hz, H-50), 3.94 (3H, s, 30-OMe), 2.28
(3H, s, 4-OAc). 13C NMR (100 MHz, (CD3)2CO): d 187.8, 169.5,
153.3, 152.4, 148.5, 142.6, 133.8, 131.4, 130.4, 124.4, 123.2,
122.9, 115.4, 112.1, 56.3, 20.9.
Boron trifluoride-etherate (6.0 mmol) was added to a stirred
solution of 40-hydroxy-30-methoxyacetophenone
1
(1.2 mmol)
and benzaldehydes 2a–2g (2.4 mmol) in 1,4-dioxane (10 mL) at

5566
H. Hara et al. / Bioorg. Med. Chem. 19 (2011) 5559–5568
4.4.2. 30,4-Dimethoxy-40-hydroxychalcone (3b)
resultant solution was partitioned with EtOAc, washed with 5%
Yellow powder, 58% yield. IR (film): 3403, 1645, 1593 cmꢁ1
.
1H
HCl aq, distilled water, brine, dried over anhydrous Na2SO4 and
concentrated in vacuo. The residue was purified using silica gel
CC eluted with n-hexane/CHCl3 (1/1) to yield acetylated chalcone
glycosides 4a–4g.
NMR (400 MHz, CDCl3): d 7.78 (1H, d, J = 15.8 Hz, H-b), 7.65–7.63
(2H, m, H-20 and H-60), 7.59 (2H, d, J = 8.7 Hz, H-2 and H-6), 7.43
(1H, d, J = 15.8 Hz, H-a
) 6.99 (1H, d, J = 8.2 Hz, H-50), 6.93 (2H, d,
J = 8.7 Hz, H-3 and H-5), 3.84 (3H, s, 30-OMe), 3.95 (3H, s, 4-
OMe). 13C NMR (100 MHz, CDCl3): d 188.6, 161.5, 150.2, 146.8,
143.8, 131.1, 130.0, 127.7, 123.5, 119.2, 114.3, 113.8, 110.5, 56.0,
55.3.
4.5.1. 40-(200,300,400,600-Tetra-O-acetyl-b-
D
-glucopyranosyl)-4-O-
acetyl-30-methoxychalcone (4a)
Yellow powder, 32% yield. IR (film): 1650, 1593 cmꢁ1
.
1H NMR
(400 MHz, (CD3)2CO): d 7.79–7.72 (2H, m, H-60 and H-b), 7.71–
7.68 (3H, m, H-2, H-6, and H-
), 7.65 (1H, s, H-20), 7.32 (1H, d,
4.4.3. 40-Hydroxy-3,30,4-trimethoxychalcone (3c)
a
Yellow powder, 65% yield. IR (film): 3404, 1645, 1587 cmꢁ1
.
1H
J = 8.7 Hz, H-50), 6.92 (2H, d, J = 8.7 Hz, H-3 and H-5), 5.45 (1H, d,
J = 7.8 Hz, H-100), 5.40 (1H, t, J = 9.6 Hz, H-400), 5.27–5.23 (1H, m,
H-300), 5.15 (1H, t, J = 9.6 Hz, H-200), 4.34–4.29 (1H, m, H-500), 4.22–
4.17 (2H, m, H-6a00 and H-6b00), 3.92 (3H, s, 30-OMe), 2.15 (3H, s,
4-OAc), 2.05–1.97 (12H, m, 200-OAc, 300-OAc, 400-OAc, and 600-OAc).
13C NMR (100 MHz, (CD3)2CO): d 188.4, 170.6, 170.3, 170.0,
169.6, 160.8, 151.2, 150.9, 144.8, 135.3, 131.5, 127.6, 122.9,
119.5, 118.1, 116.7, 113.2, 100.1, 73.2, 72.7, 71.9, 69.2, 62.7, 56.6,
20.6.
NMR (400 MHz, (CD3)2CO): d 8.48 (1H, bs), 7.81–7.66 (3H, m, H-20,
H-60, H-
, and H-b), 7.48 (1H, d, J = 1.8 Hz, H-2), 7.34 (1H, dd,
a
J = 8.6, 2.3 Hz, H-6), 7.02 (1H, d, J = 8.1 Hz, H-50), 6.94 (1H, d,
J = 8.1 Hz, H-5), 3.94 (3H, s, 30-OMe), 3.90 (3H, s, 3-OMe), 3.87
(3H, s, 4-OMe). 13C NMR (100 MHz, (CD3)2CO): d 187.1, 151.8,
151.4, 149.8, 147.7, 143.3, 128.3, 123.4, 123.2, 119.6, 114.5,
111.6, 111.2, 110.8, 55.5, 55.4.
4.4.4. 40-Hydroxy-3,30,4,5-tetramethoxychalcone (3d)
Yellow powder, 39% yield. IR (film): 3371, 1654, 1587 cmꢁ1
.
1H
4.5.2. 40-(200,300,400,600-Tetra-O-acetyl-b- -glucopyranosyl)-30,4-
D
NMR (400 MHz, (CD3)2CO): d 7.81 (1H, d, J = 15.6 Hz, H-b), 7.76
dimethoxychalcone (4b)
(1H, dd, J = 8.2, 3.2 Hz, H-60), 7.69 (1H, d, J = 15.6 Hz, H-
a), 7.67
Yellow powder, 90% yield. IR (film): 1753, 1655, 1595 cmꢁ1 1H
.
(1H, d, J = 3.2 Hz, H-20), 7.16 (2H, s, H-2 and H-6), 6.95 (1H, d,
J = 8.5 Hz, H-50), 3.94 (3H, s, 30-OMe), 3.91 (6H, s, 3-OMe and 5-
OMe), 3.78 (3H, s, 4-OMe). 13C NMR (100 MHz, (CD3)2CO): d
187.9, 154.6, 152.3, 148.6, 144.1, 141.3, 131.7, 131.6, 124.3,
122.0, 115.3, 112.0, 107.0, 60.6, 56.6, 56.3.
NMR (400 MHz, CD3OD): d 7.65 (1H, d, J = 16.0 Hz, H-b), 7.59 (2H,
d, J = 8.7 Hz, H-2 and H-6), 7.54–7.47 (3H, m, H-20, H-60, and H-
a
), 7.16 (1H, d, J = 8.7 Hz, H-50), 6.90 (2H, d, J = 8.7 Hz, H-3 and
H-5), 5.32 (1H, d, J = 9.6 Hz, H-100), 5.16 (1H, t, J = 11.0 Hz, H-300),
5.04 (1H, t, J = 9.6 Hz, H-200), 4.24 (1H, dd, J = 11.0, 5.5 Hz, H-6a00),
4.13–4.09 (1H, m, H-6b00), 3.98–3.93 (1H, m, H-500), 3.80 (3H, s,
30-OMe), 3.77 (3H, s, 4-OMe), 2.04–1.96 (12H, m, 200-OAc, 300-OAc,
400-OAc, and 600-OAc). 13C NMR (100 MHz, CD3OD): d 189.1, 171.0,
170.3, 170.0, 169.8, 162.0, 150.2, 150.1, 144.6, 133.9, 130.4,
127.5, 122.4, 118.7, 116.8, 114.2, 112.0, 99.2, 72.6, 71.8, 71.2,
68.4, 61.8, 55.4, 54.7, 19.4, 19.3.
4.4.5. 4-O-Acetyl-40-hydroxy-3,30,5-trimethoxychalcone (3e)
Yellow powder, 50% yield. IR (film): 3392, 1645, 1593 cmꢁ1 1H
.
NMR (400 MHz, (CD3)2CO): d 7.89 (1H, d, J = 15.8 Hz, H-b), 7.77
(1H, dd, J = 8.3, 1.8 Hz, H-60), 7.71 (1H, d, J = 15.8 Hz, H-
a), 7.68
(1H, d, J = 1.8 Hz, H-20), 7.22 (2H, s, H-2 and H-6), 6.95 (1H, d,
J = 8.2 Hz, H-50), 3.94 (3H, s, 30-OMe), 3.90 (6H, s, H-3 and H-5),
2.81 (1H, bs), 2.26 (3H, s, 4-OAc). 13C NMR (100 MHz, (CD3)2CO):
d 187.1, 167.6, 152.7, 151.6, 147.8, 142.8, 133.7, 130.6, 123.6,
122.2, 114.6, 111.2, 105.3, 55.8, 55.5, 19.4.
4.5.3. 40-(200,300,400,600-Tetra-O-acetyl-b-
D
-glucopyranosyl)-3,30,4-
trimethoxychalcone (4c)
Yellow powder, 85% yield. IR (film): 1755, 1643, 1592 cmꢁ1
.
1H
NMR (400 MHz, CD3OD): d 7.72–7.68 (2H, m, H-60 and H-b), 7.60
4.4.6. 3-O-Acetyl-30,4-dimethoxy-40-hydroxychalcone (3f)
(1H, d, J = 4.6 Hz, H-20), 7.57 (1H, d, J = 15.6 Hz, H-
a), 7.32 (1H, s,
Yellow powder, 50% yield. IR (film): 3392, 1650, 1592 cmꢁ1
.
1H
H-2), 7.25 (1H, dd, J = 8.2, 1.8 Hz, H-6), 7.19 (1H, d, J = 8.7 Hz, H-
50), 6.95 (1H, d, J = 8.7 Hz, H-5), 5.36 (1H, t, J = 9.6 Hz, H-300), 5.26
(1H, d, J = 8.2 Hz, H-100), 5.20 (1H, t, J = 9.6 Hz, H-200), 5.09 (1H, t,
J = 9.6 Hz, H-400), 4.29 (1H, dd, J = 12.8, 5.0 Hz, H-6a00), 4.15 (1H,
dd, J = 12.4, 4.9 Hz, H-6b00), 4.02–3.99 (1H, m, H-500), 3.88 (3H, s,
30-OMe), 3.85 (6H, s, 3-OMe and 4-OMe), 2.15–1.99 (12H, m, 200-
OAc, 300-OAc, 400-OAc, and 600-OAc). 13C NMR (100 MHz, CD3OD): d
189.2, 170.9, 170.2, 169.9, 151.8, 150.2, 149.4, 145.0, 134.0,
128.0, 123.5, 122.4, 119.0, 116.9, 112.1, 111.2, 110.7, 99.2, 72.6,
71.8, 71.2, 68.3, 61.7, 55.4, 55.2, 55.1, 19.4, 19.3.
NMR (400 MHz, (CD3)2CO): d 7.88 (1H, d, J = 15.6 Hz, H-b), 7.79
(1H, dd, J = 8.2, 1.8 Hz, H-60), 7.74 (1H, d, J = 15.6 Hz, H-
a), 7.69
(1H, d, J = 1.8 Hz, H-20), 7.59 (1H, d, J = 1.8 Hz, H-2), 7.41 (1H, dd,
J = 8.2, 1.8 Hz, H-6), 7.13 (1H, d, J = 8.2 Hz, H-50), 6.95 (1H, d,
J = 8.2 Hz, H-5), 3.95 (3H, s, 30-OMe), 3.92 (3H, s, 4-OMe), 2.27
(3H, s, 3-OAc). 13C NMR (100 MHz, (CD3)2CO): d 187.0, 168.0,
151.8, 151.6, 147.5, 142.3, 141.2, 134.3, 130.9, 123.6, 123.3,
122.2, 121.5, 114.6, 112.1, 111.2, 55.6, 55.5, 19.9.
4.4.7. 40-Hydroxy-30-methoxychalcone (3g)
Yellow powder, 47% yield. IR (film): 3367, 1651, 1584 cmꢁ1
.
1H
4.5.4. 40-(200,300,400,600-Tetra-O-acetyl-b-
D
-glucopyranosyl)-3,30,4,5-
NMR (400 MHz, (CD3)2CO): d 7.89 (1H, d, J = 14.0 Hz, H-b), 7.86–
tetramethoxychalcone (4d)
7.69 (3H, m, H-20, H-60, and H-
a), 7.46–7.43 (3H, m. H-3, H-4,
Yellow powder, 76% yield. IR (film): 1751, 1663, 1583 cmꢁ1 1H
.
and H-5), 7.25–7.24 (1H, m, H-50), 6.97 (2H, d, J = 8.2 Hz, H-2 and
H-6), 3.95 (3H, s, 30-OMe). 13C NMR (100 MHz, (CD3)2CO): d
188.1, 152.4, 148.5, 143.7, 136.1, 131.3, 130.9, 129.6, 129.2,
124.4, 122.7, 115.4, 112.0, 56.3.
NMR (400 MHz, (CD3)2CO): d 7.82–7.76 (2H, m, H-60 and H-b), 7.70
(1H, d, J = 14.2 Hz, H-
), 7.69 (1H, s, H-20), 7.32 (1H, d, J = 8.7 Hz, H-
a
50), 7.17 (2H, s, H-2 and H-6), 5.47 (1H, d, J = 8.2 Hz, H-100), 5.41 (1H,
t, J = 9.6 Hz, H-300), 5.26 (1H, t, J = 7.8 Hz, H-200), 5.16 (1H, t,
J = 9.6 Hz, H-400), 4.31 (1H, dd, J = 12.8, 5.5 Hz, H-6a00), 4.22–4.18
(1H, m, H-6b00), 3.92 (3H, s, 30-OMe), 3.91 (9H, s, 3-OMe, 4-OMe,
and 5-OMe), 2.04 (12H, m, 200-OAc, 300-OAc, 400-OAc, and 600-OAc).
13C NMR (100 MHz, (CD3)2CO): d 187.6, 169.9, 169.5, 169.2,
168.7, 153.8, 150.4, 150.3, 144.1, 140.6, 134.2, 130.7, 122.3,
121.0, 117.2, 112.4, 106.3, 99.2, 72.3, 71.9, 71.0, 68.4, 61.8, 59.9,
55.8, 55.7, 19.8, 19.8, 19.7.
4.5. Synthesis of acetylated chalcone glycosides 4a–4g
Silver carbonate (2.57 mmol) was added to a stirred solution
of tetra-O-acetylglucopyranosyl bromide (1.45 mmol) and chal-
cones 3a–3g (0.65 mmol) in pyridine (10 mL) at room tempera-
ture under shaded conditions. After stirring for 18 h, the

H. Hara et al. / Bioorg. Med. Chem. 19 (2011) 5559–5568
5567
4.5.5. 40-(200,300,400,600-Tetra-O-acetyl-b-
acetyl-3,30,5-trimethoxychalcone (4e)
Yellow powder, 56% yield. IR (film): 1756, 1646, 1594 cmꢁ1
NMR (400 MHz, (CD3)2CO): d 7.85 (1H, d, J = 16.0 Hz, H-b), 7.79
(1H, dd, J = 8.7, 1.8 Hz, H-60), 7.73 (1H, d, J = 16.0 Hz, H-
), 7.69
D
-glucopyranosyl)-4-O-
119.4, 114.9, 111.8, 111.5, 110.7, 100.6, 77.2, 73.7, 70.3, 61.7,
55.6, 55.4, 55.3. LCESI-QTOFMS: m/z 499.1560 [M+Na]+ (calcd for
.
1H
C24H28O10Na, 499.1580).
a
4.6.2. 40-O-b-
D
-Glucopyranosyl-3,30,4,5-tetramethoxychalcone
(1H, d, J = 1.8 Hz, H-20), 7.33 (1H, d, J = 8.7 Hz, H-50), 7.22 (2H, s,
H-2 and H-6), 5.47 (1H, d, J = 8.2 Hz, H-100), 5.39 (1H, t, J = 9.6 Hz,
H-300), 5.25 (1H, dd, J = 9.6, 8.0 Hz, H-200), 5.16 (1H, t, J = 9.6 Hz, H-
400), 4.31–4.98 (1H, m, H-6a00), 4.21–4.17 (2H, m, H-500 and H-6b00),
3.92 (3H, s, 30-OMe), 3.89 (6H, s, 3-OMe and 5-OMe), 2.20–1.99
(12H, m, 200-OAc, 300-OAc, 400-OAc, and 600-OAc). 13C NMR
(100 MHz, (CD3)2CO): d 187.7, 169.9, 169.5, 169.2, 168.8, 167.8,
152.7, 150.4, 143.6, 134.1, 133.5, 130.7, 122.4, 122.2, 117.2,
112.4, 105.4, 99.2, 72.3, 72.0, 71.0, 68.4, 61.8, 55.8, 19.83, 19.78,
19.73, 19.5.
(A8)
21
589
Yellow powder, 83% yield. ½
a
ꢅ
ꢁ10.0 (c 0.10, MeOH). IR (film):
3391, 1640, 1586 cmꢁ1. 1H NMR (400 MHz, CD3OD): d 7.80 (1H, dd,
J = 8.7, 1.8 Hz, H-60), 7.70–7.65 (1H, m, H-20, H-
, and H-b), 7.24
a
(1H, d, J = 8.7 Hz, H-50), 7.05 (2H, s, H-2 and H-6), 5.06 (1H, d,
J = 7.8 Hz, H-100), 3.92 (3H, s, 30-OMe), 3.90 (9H, s, 3-OMe, 4-OMe,
and 5-OMe), 3.83–3.78 (3H, m, H-200, H-6a00, and H-6b00), 3.15–
3.48 (2H, m, H-300 and H-500), 3.31 (1H, t, J = 1.4 Hz, H-400). 13C
NMR (100 MHz, CD3OD): d 189.3, 153.5, 151.1, 149.1, 144.6,
140.1, 132.4, 130.9, 123.2, 120.7, 114.7, 111.5, 105.9, 103.6,
100.5, 77.0, 76.5, 73.4, 69.9, 61.1, 59.9, 55.4, 54.7. LCESI-QTOFMS:
m/z 507.1850 [M+H]+ (calcd for C25H31O11, 507.1866).
4.5.6. 40-(200,300,400,600-Tetra-O-acetyl-b-
D-glucopyranosyl)-3-O-
acetyl-30,4-dimethoxychalcone (4f)
Yellow powder, 53% yield. IR (film): 1776, 1644, 1595 cmꢁ1 1H
.
4.6.3. 40-O-b- -Glucopyranosyl-4-hydroxy-3,30,5-
D
NMR (400 MHz, (CD3)2CO): d 8.05–7.09 (7H, m, H-2, H-20, H-50, H-
6, H-60, H- , and H-b), 5.44–5.15 (4H, m, H-100, H-300, H-200, and H-
trimethoxychalcone (A9)
a
21
589
400), 4.43–4.02 (3H, m, H-500, H-6a00, and H-6b00), 3.91 (6H, s, 30-
OMe and 4-OMe), 2.18–1.99 (12H, m, 200-OAc, 300-OAc, 400-OAc,
and 600-OAc). 13C NMR (100 MHz, (CD3)2CO): d 187.6, 169.9,
169.6, 169.3, 168.9, 168.1, 151.8, 150.4, 143.2, 141.9, 134.0,
133.9, 123.4, 122.4, 122.0, 121.6, 117.2, 112.4, 112.2, 99.2, 72.4,
72.0, 71.0, 68.4, 61.9, 55.8, 55.6, 19.8.
Yellow powder, Quantitative. ½
aꢅ
ꢁ10.0 (c 0.10, MeOH). IR
(film): 3372, 1649, 1512 cmꢁ1 1H NMR (400 MHz, CD3OD): d
.
7.77 (1H, dd, J = 8.7, 1.8 Hz, H-60), 7.69 (1H, d, J = 15.6 Hz, H-b),
7.64 (1H, d, J = 1.8 Hz, H-20), 7.60 (1H, d, J = 15.6 Hz, H-
), 7.22
a
(1H, d, J = 8.7 Hz, H-50), 7.03 (2H, s, H-2 and H-6), 5.06 (1H, d,
J = 7.4 Hz, H-100), 3.92 (3H, s, 30-OMe), 3.90 (6H, s, 3-OMe and 5-
OMe), 3.73–3.69 (2H, m, H-200 and H-6a00), 3.56–3.42 (3H, m, H-
300, H-500, and H-6b00), 3.30 (1H, t, J = 1.4 Hz, H-40). 13C NMR
(100 MHz, CD3OD): d 189.4, 150.9, 149.3, 148.1, 145.6, 138.9,
132.6, 125.9, 123.0, 118.5, 114.7, 111.5, 106.1, 100.5, 77.0, 76.5,
73.4, 69.9, 61.1, 55.5, 55.3. LCESI-QTOFMS: m/z 515.1514
[M+Na]+ (calcd for C24H28O11Na, 515.1529).
4.5.7. 40-(200,300,400,600-Tetra-O-acetyl-b- -glucopyranosyl)-30-
D
methoxychalcone (4g)
Yellow powder, 47% yield. IR (film): 1754, 1649, 1587 cmꢁ1 1H
.
NMR (400 MHz, (CD3)2CO): d 7.88 (1H, d, J = 16.0 Hz, H-b), 7.84–
7.76 (2H, m, H-60 and H- ), 7.73 (1H, d, J = 1.9 Hz, H-20), 7.47–
a
7.45 (4H, m, H-3, H-4, H-5, and H-50), 7.34 (2H, d, J = 8.7 Hz, H-2
and H-6), 5.48 (1H, d, J = 7.8 Hz, H-100), 5.41 (1H, t, J = 9.6 Hz, H-
300), 5.29–5.23 (1H, m, H-200), 5.16 (1H, t, J = 9.6 Hz, H-400), 4.33–
4.26 (1H, m, H-6a00), 4.23–4.18 (2H, m, H-500 and H-6b00), 3.93
(3H, s, 30-OMe), 2.04–1.98 (12H, m, 200-OAc, 300-OAc, 400-OAc, and
600-OAc). 13C NMR (100 MHz, (CD3)2CO): d 187.6, 169.9, 169.5,
169.3, 168.9, 150.4, 143.7,135.3, 134.0, 130.5, 129.4, 129.0, 128.7,
128.3, 122.4, 121.9, 117.2, 117.0, 112.4, 112.2, 99.2, 72.4, 72.0,
71.1, 68.4, 61.9, 55.8, 19.9, 19.81, 19.76.
4.6.4. 40-O-b- -Glucopyranosyl-30,4-dimethoxy-3-
D
hydroxychalcone (A10)
22
589
Yellow powder, 84% yield. ½
aꢅ
ꢁ15.0 (c 0.10, MeOH). IR (film):
3372, 1644, 1593, 1512 cmꢁ1 1H NMR (400 MHz, CD3OD): d 7.77
.
(1H, dd, J = 8.7, 1.8 Hz, H-600), 7.72 (1H, d, J = 16.0 Hz, H-b), 7.66
(1H, d, J = 1.8 Hz, H-20), 7.61 (1H, d, J = 16.0 Hz, H-
a
), 7.35 (1H, d,
J = 1.8 Hz, H-2), 7.26 (1H, d, J = 8.7 Hz, H-5), 7.22 (1H, dd, J = 8.7,
1,8 Hz, H-6), 6.84 (1H, d, J = 8.2 Hz, H-5), 5.06 (1H, d, J = 7.3 Hz,
H-100), 3.93 (3H, s, 30-OMe), 3.92–3.88 (1H, m, H-6a00), 3.71–3.68
(1H, m, H-6b00), 3.59–3.48 (3H, m, H-200, H-300, and H-500), 3.31
(1H, t, J = 1.8 Hz, H-400). 13C NMR (100 MHz, CD3OD): d 189.5,
150.9, 149.7, 149.4, 148.1, 145.4, 132.7, 127.0, 123.6, 122.9,
118.2, 115.2, 114.8, 111.5, 110.9, 100.5, 77.0, 76.5, 73.4, 69.9,
61.1, 55.3, 55.2. LCESI-QTOFMS: m/z 485.1408 [M+Na]+ (calcd for
4.6. Synthesis of chalcone glycosides A1, A2 and A7–A11
Acetylated chalcone glycosides 4a–4g were stirred in 10 mg/ml
sodium methoxide/methanol solution (10 ml) for 10 min. The reac-
tion mixture was neutralized using a Dowex Marathon C (H+ form),
filtrated and concentrated in vacuo. The residue was purified using
silica gel CC eluted with CHCl3/MeOH (10/1) to yield chalcone gly-
cosides A1, A217 and A7-A11.
C23H26O10Na,485.1424).
4.6.5. 40-O-b-
Yellow powder, 92% yield. ½
3373, 1666, 1599, 1511 cmꢁ1
7.74 (5H, m, H-2, H-6, H-60, H-
D
-Glucopyranosyl-30-methoxychalcone (A11)
22
aꢅ
ꢁ75.0 (c 0.10, MeOH). IR (film):
589
.
1H NMR (400 MHz, CD3OD): d 7.80–
, and H-b,), 7.68 (1H, d, J = 2.3 Hz,
4.6.1. 40-O-b-
D
-Glucopyranosyl-3,30,4-trimethoxychalcone (A7)
21
a
Yellow powder, Quantitative. ½
aꢅ
ꢁ25.0 (c 0.10, MeOH). IR
589
(film): 3404, 1647, 1587, 1512 cmꢁ1
(CD3)2CO): d 7.79–7.76 (2H, m, H-60 and H-b), 7.73 (1H, d,
J = 16.3 Hz, H-
), 7.67 (1H, d, J = 2.1 Hz, H-20), 7.49 (1H, d,
.
1H NMR (400 MHz,
H-20), 7.45–7.42 (3H, m, H-3, H-4, and H-5), 7.27 (1H, d,
J = 8.7 Hz, H-50), 5.07 (1H, d, J = 7.8 Hz, H-100), 3.94 (3H, s, 30-
OMe), 3.86 (1H, dd, J = 12.4, 2.5 Hz, H-6a00), 3.69 (1H, dd, J = 12.4,
6.9 Hz, H-6b00), 3.56 (1H, t, J = 7.8 Hz, H-200), 3.52–3.39 (2H, m H-
300 and H-500), 3.30 (1H, t, J = 8.9 Hz, H-400). 13C NMR (100 MHz,
CD3OD): d 189.3, 151.1, 149.5, 144.4, 135.0, 130.3, 128.7, 128.4,
128.0, 123.1, 121.3, 114.8, 111.4, 100.5, 77.0, 76.5, 73.4,69.9,
61.1, 55.3. LCESI-QTOFMS: m/z 439.1357 [M+Na]+ (calcd for
a
J = 2.1 Hz, H-50), 7.34 (1H, dd, J = 8.9, 2.1 Hz, H-60), 7.25 (1H, d,
J = 8.7 Hz, H-2), 7.02 (1H, d, J = 8.9 Hz, H-5), 5.13 (1H, d,
J = 7.3 Hz, H-100), 3.92 (3H, s, 30-OMe), 3.90 (1H, s, 3-OMe), 3.87
(1H, s, 4-OMe), 3.85–3.69 (3H, m, H-200, H-6a00, and H-6b00), 3.61–
3.49 (3H, m, H-300, H-400, and H-500). 13C NMR (100 MHz, (CD3)2CO):
d 187.4, 151.0, 149.7, 149.6, 143.8, 132.8, 128.2, 123.5, 122.6,
C22H24O8Na,439.1369).

5568
H. Hara et al. / Bioorg. Med. Chem. 19 (2011) 5559–5568
15. Yamamoto, T.; Yoshimura, M.; Yamaguchi, F.; Kouchi, T.; Tsuji, R.; Saito, M.;
Acknowledgment
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This work was supported by a Grant-in-Aid for Scientific Re-
search (C) from the Japan Society for the Promotion of Science
(20590543 to H.H.).
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