4′-Bromo-5,6,7-trimethoxyflavone represses lipopolysaccharide-induced iNOS and COX-2 expressions by suppressing the NF-κB signaling pathway in RAW 264.7 macrophages

Article abstract of DOI:10.1016/j.bmcl.2011.10.067

The regulations of the NO and PGE₂ productions are research topics of interest in the field of anti-inflammatory drug development. In the present study, 5,6,7-trimethoxy- and 5,6,7-trihydroxyflavones 3a-3g were synthesized from cinnamic acid derivatives. In particular, 4′-bromo-5,6,7- trimethoxyflavone (3b) most potently inhibited the productions of NO and PGE₂ in LPS-treated RAW 264.7 cells (IC₅₀ = 14.22 ± 1.25 and 10.98 ± 6.25 μM, respectively), and these inhibitory effects were more potent than those of oroxylin A or baicalein. Consistent with these findings, 3b concentration-dependently reduced the LPS-induced expressions of iNOS and COX-2 at the protein and mRNA levels. In addition, the release of TNF-α, IL-6, and IL-1β and the mRNA expressions of these cytokines were reduced by 3b in a concentration-dependent manner. Furthermore, 3b attenuated the LPS-induced transcriptional activities of NF-κB and this was accompanied by parallel reductions in the degradation and phosphorylation of IκB-α, and consequently by a decrease in the nuclear translocation of the p65 subunit of NF-κB. Taken together, these results suggest that suppressions of the expressions of iNOS, COX-2, TNF-α, IL-6, and IL-1β via NF-κB inactivation are responsible for the anti-inflammatory effects of 3b.

Full text of DOI:10.1016/j.bmcl.2011.10.067

Bioorganic & Medicinal Chemistry Letters 22 (2012) 700–705
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
40-Bromo-5,6,7-trimethoxyflavone represses lipopolysaccharide-induced
iNOS and COX-2 expressions by suppressing the NF-
in RAW 264.7 macrophages
jB signaling pathway
Dong Han Kim a, , Chang Hyeon Yun a, , Min Hwan Kim a, Ch. Naveen Kumar a, Bo Hee Yun a, Ji-Sun Shin a,b
,
Hyo Jin An a, Young Hun Lee a, Yong Don Yun a, Hong-Kun Rim a,b, Min-Sang Yoo a,b, Kyung-Tae Lee a,b,
,
⇑
Yong Sup Lee a,
⇑
a Department of Life and Nanopharmaceutical Science & Department of Pharmaceutical Science, College of Pharmacy, Kyung Hee University, Seoul 130 701, Republic of Korea
b Department of Biomedical Science, College of Medical Science, Kyung Hee University, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
The regulations of the NO and PGE2 productions are research topics of interest in the field of anti-inflam-
matory drug development. In the present study, 5,6,7-trimethoxy- and 5,6,7-trihydroxyflavones 3a–3g
were synthesized from cinnamic acid derivatives. In particular, 40-bromo-5,6,7-trimethoxyflavone (3b)
most potently inhibited the productions of NO and PGE2 in LPS-treated RAW 264.7 cells (IC50 = 14.22
Received 12 August 2011
Revised 10 October 2011
Accepted 12 October 2011
Available online 31 October 2011
1.25 and 10.98 6.25
lin A or baicalein. Consistent with these findings, 3b concentration-dependently reduced the LPS-induced
expressions of iNOS and COX-2 at the protein and mRNA levels. In addition, the release of TNF- , IL-6, and
IL-1b and the mRNA expressions of these cytokines were reduced by 3b in a concentration-dependent
manner. Furthermore, 3b attenuated the LPS-induced transcriptional activities of NF- B and this was
accompanied by parallel reductions in the degradation and phosphorylation of I B- , and consequently
by a decrease in the nuclear translocation of the p65 subunit of NF- B. Taken together, these results suggest
, IL-6, and IL-1b via NF- B inactivation are
lM, respectively), and these inhibitory effects were more potent than those of oroxy-
Keywords:
a
5,6,7-Trimethoxyflavone
Oroxylin A
iNOS
j
j
a
COX-2
j
NF-jB
Anti-inflammatory
that suppressions of the expressions of iNOS, COX-2, TNF-
a
j
responsible for the anti-inflammatory effects of 3b.
Ó 2011 Elsevier Ltd. All rights reserved.
Inflammation, characterized by redness, swelling, pain, and a
sensation of heat, is one of the most important components of the
host defense mechanisms against invading pathogens. However,
inflammation may also aid microbial pathogeneses because the
inflammatory responses elicited by invading microorganisms can
result in considerable host damage, make nutrients available, and
provide access to host tissues.1
In the inflammatory state, activated immune cells secrete
excessive amounts of NO, PGE2, and pro-inflammatory cytokines.
NO and PGs are pleiotypic mediators produced at inflammatory
sites by iNOS and COX-2, respectively. The overproduction of NO
is mainly caused by iNOS, which is up-regulated in macrophages
by cytokines and/or bacterial LPS.2,3 Furthermore, despite its ben-
eficial role in host defense, sustained NO production can be delete-
rious to the host, for example, NO has been implicated in the
pathogeneses of various inflammatory diseases, such as, arthritis,
asthma, multiple sclerosis, inflammatory bowel disease, and ath-
erosclerosis.1,2,4 On the other hand, elevated COX-2 expression
causes the excessive production of inflammatory PGs. In particular,
PGE2 is a major COX-2 product at inflammatory sites, where it con-
tributes to increases in local blood flow, edema formation, and pain
sensitization.5
The pathogenesis of inflammation is highly complex process in
cells of the immune system, which in turn is regulated by cyto-
kine networks and a plethora of pro-inflammatory genes. Further-
more, the release of pro-inflammatory cytokines by macrophages
importantly regulates inflammatory responses, and macrophages
are the major cellular source of TNF-a, IL-1, and IL-6, which in
turn participate in the mediation of acute phase responses to in-
jury.6 Accordingly, the regulations of NO, PGE2, and cytokines,
such as IL-1b, IL-6, and TNF-
a are considered important targets
by those trying to develop anti-inflammatory agents.7–9
Scutellaria baicalensis Georgi (Huang Qui) is an important
medicinal herb, which is often used to treat inflammatory dis-
eases, hepatitis, tumors, and diarrhea in China, Korea, Taiwan,
and Japan.10,11 Furthermore, this plant has been reported to con-
tain a large number of flavonoids. Oroxylin A (1, 5,7-dihydroxy-6-
methoxyflavone) is the major component of Scutellariae Radix,
and has been shown to possess diverse pharmacological activities,
which include anti-cancer, hepatoprotective, anti-allergic, and
⇑
Corresponding authors. Tel.: +82 2 961 0370; fax: +82 2 966 3885.
E-mail addresses: ktlee@khu.ac.kr (K.-T. Lee), kyslee@khu.ac.kr (Y.S. Lee).
These authors contributed equally to this paper.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.10.067

D. H. Kim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 700–705
701
R2
HO
O
O
HO
HO
O
O
R1O
R1O
O
O
MeO
OH
OH
OR1
1, oroxylin A
2, baicalein
3
Figure 1. Chemical structures of oroxylin A (1), baicalein (2) and 5,6,7-trimethoxyflavones (3).
R2
R2
1. SOCl2
benzene, reflux
MeO
MeO
O
MeO
MeO
OH
I2
COOH
DMSO
2. 3,4,5-trimethoxyphenol
R2
.
BF3 OEt2, toluene
MeO
MeO
O
O
4
, R2 = H, 98%
, R2 = H, 80%
5a
5b
3a
3b
2
2
, R = Br, 56%
, R = Br, 59%
5c, R2 = NO2, 61%
3c, R2 = NO2, 89%
3d, R2 = NH2, 73%
SnCl2, EtOH,
reflux, 73%
R2
HO
HO
O
47%HBr
AcOH, 130oC
OH
O
2
2
, R = H, 92%
, R2 = Br,73%
3e
3f, R2 = NO2, 43%
3g, R2 = NH2, 84%
Scheme 1. Synthetic route to 5,6,7-trimethoxy- and 5,6,7-trihydroxyflavones 3a–3g.
Table 1
The cytotoxicities and inhibitory effects of oroxylin A, baicalein, and compounds 3a–3g on the LPS-induced productions of NO and PGE2
R2
R1O
R1O
O
O
OR1
3a-g
Compounds
R1
R2
Cell viability, IC50
(lM)
% Inhibition at 25
NO production
12.10 0.93
l
Ma
PGE2 production
42.19 3.83
3a
3b
3c
3d
3e
3f
3g
CH3
CH3
CH3
CH3
H
H
Br
>100
69.88 5.32
>100
>100
57.11 + 1.91
>100
94.13 6.71
16.73 1.35
>100
89.63 7.32 (14.22 l.25)b
17.41 + 1.53
89.61 6.25 (10.98 6.25)b
84.41 5.32
NO2
NH2
Br
NO2
NH2
23.10 + 1.26
27.03 + 2.06
7.65 0.61
6.98 0.48
12.44 + 1.02
11.13 + 0.88
69.54 4.95
29.87 2.32
29.87 2.01
H
H
83.11 5.99
c
1 (Oroxylin A)
2 (Baicalein)
—
H
H
60.87 3.91
a
b
c
Values represent means SDs of three independent experiments.
Numbers in parenthesis are IC50 values in M.
No inhibition at less than the cytotoxic concentration.
l
anti-inflammatory effects.12–14 It has also been reported to inhibit
the LPS-induced expressions of iNOS and COX-2 by suppressing
NF-
anti-inflammatory agents by examining their inhibitory effects
j
B activation.15 However, during our efforts to identify new

702
D. H. Kim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 700–705
on the productions of NO and PGE2, we realized that structural
modification of oroxylin A was probably needed to increase its
anti-inflammatory effect. In fact, in a previous study involving
structural modifications of oroxylin A, it was observed that acyl-
ation of its 7-hydroxyl group significantly enhanced its antibacte-
rial activity.16 Methylations of the hydroxyl groups of oroxylin A
(1) or baicalein (2) to produce 5,6,7-trimethoxyflavone, were also
found to enhance the anticancer,17 anti-aggregatory,18 and anti-
inflammatory activities19 of the parent compound. However,
although the inhibitory effects of 5,6,7-trimethoxyflavones deriv-
atives were screened with respect to LPS-induced NO production,
and arachidonic acid and collagen-induced aggregation, the
underlying mechanism involved was not elucidated. Accordingly,
we synthesized various baicalein and oroxylin A derivatives with
the aim to producing adducts that more potently inhibit the LPS-
induced productions of inflammatory mediators in RAW 264.7
cells. In addition, we also attempted to identify the mechanisms
underlying their anti-inflammatory effects. Structural modifica-
tions were carried out by methylating the 5- and 7-hydroxyl
groups or by demethylating the 6-O-methyl group on the A-ring,
and by introducing substituents on the B-ring of oroxylin A
(Fig. 1).
Oroxylin A derivatives were synthesized as shown in Scheme 1
using
modification of previously reported procedures.18,20
Cinnamic acid derivatives were transformed to cinnamoyl
a
4
chlorides by reaction with thionyl chloride in benzene and coupled
with 3,4,5-trimethoxyphenol in the presence of BF3ꢀEt2O to afford
the corresponding chalcones 5a–c. Intramolecular oxidative cycli-
zation of these chalcones with iodine in DMSO then provided the
5,6,7-trimethoxyflavones 3a–c. The 40-nitro group in 3c was
reduced using tin(II) chloride to give the 40-amino-5,6,7-trimeth-
oxyflavone (3d). The methyls of the methoxy groups on the A-ring
of the trimethoxyflavones 3a–d were removed by treating them
with 47% HBr in glacial acetic acid to provide 5,6,7-trihydroxyflav-
ones, baicalein (2) and 3e–g.21
Initially, the synthesized flavones 3a–3g were screened for their
inhibitory effects on the LPS-induced productions of NO and PGE2 in
RAW 264.7 cells. The cytotoxic effects and inhibitory activities of the
oroxylin A derivatives 3a–3g on the productions of NO and PGE2 are
listed in Table 1; data on oroxylin A (1) and baicalein (2) were in-
cluded for comparison purposes. In most cases, the 5,6,7-trimeth-
oxyflavones (3a–3d) and 5,6,7-trihydroxyflavones (3e–3g) showed
much less cytotoxicity (IC50 > 50 lM) than the parent compound
oroxylin A (1, IC50 = 16.73
lM). In our assay systems, oroxylin A
A
B
30
10
#
#
RAW 264.7
25
RAW 264.7
7.5
20
*
**
15
10
5
5
**
**
**
2.5
0
***
25
***
0
Con
NIL
6.25
12.5
25
Con
NS398 6.25
12.5
25
25
3b (μM)
3b (μM)
LPS (1μg/ml)
LPS (1μg/ml)
C
D
#
30
25
20
15
10
5
4
3
2
1
0
#
Peritoneal macrophages
Peritoneal macrophages
**
**
**
**
**
**
***
***
0
Con
NIL
6.25
12.5
25
25
Con
NS398
6.25
12.5
25
25
3b (μM)
3b (μM)
LPS (10μg/ml)
LPS (10μg/ml)
Figure 2. Effects of 3b on LPS-induced NO and PGE2 production in RAW 264.7 macrophages. (A) Cells were pretreated with different concentrations (6.25, 12.5 or 25
3b for 1 h, then with LPS (1 g/ml), and incubated for 24 h. NO levels in culture media were determined using Griess assays. -N6-(1-Iminoethyl) lysine ( -NIL, 10 M) was
used as a positive NO production inhibitor. (B) Samples were treated as described in the legend of panel A. N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide (NS-
lM) of
l
L
L
l
398, 10 lM) was used as a positive PGE2 production inhibitor. Level of PGE2 in culture media was quantified using EIA kits. (C) Peritoneal macrophages isolated from C57BL/6
mice were treated as described in the legend of panel A. Values represent the means SDs of three independent experiments. #p < 0.05 as compared with the control cells, and
⁄p < 0.05, ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001 as compared with the LPS-only treated cells. ANOVA and Dunnett’s post-hoc test were used to determine the significances of differences.

D. H. Kim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 700–705
703
(1) exhibitedno noticeable effect on NO or PGE2 production under its
cytotoxic concentration and baicalein inhibited only the production
of PGE2 by 60.87% at 25 lM concentration. On the other hand,
compounds 3b–3d and 3g more potently inhibited PGE2 production
(by 69.54–89.61%) than baiclein at the same concentration. In
particular, 40-bromo-5,6,7-trimethoxyflavone (3b) potently inhib-
ited the productions of NO and PGE2, and these inhibitory effects
of 3b on LPS-induced NO and PGE2 production in peritoneal macro-
phages isolated from thioglycolate-injected C57BL/6 mice. 3b was
found to inhibit the LPS-induced productions of NO and PGE2 con-
centration-dependently in these cells (Fig. 2C and D). We then inves-
tigated whether the inhibitory effects of 3b on the production of NO
and PGE2 are related to changes in the expressional levels of iNOS
and COX-2. In unstimulated RAW 264.7 cells, iNOS and COX-2
protein levels were undetectable, but after treatment with LPS
(IC50 = 14.22 1.25, and 10.98 6.25 lM, respectively) were greater
than those of oroxylin A (1) and baicalein (2). As shown in Figure 2A
and B, 3b significantly and concentration-dependently inhibited
LPS-induced NO and PGE2 production in RAW 264.7 cells. The syn-
thesized compounds 3a–3g did not affect RAW 264.7 cell viability
in either the presence or absence of LPS at concentrations up to
(1 lg/ml), they were markedly upregulated. Furthermore, 3b signif-
icantly and concentration-dependently inhibited these expressions
(Fig. 3A). Because changes in iNOS and COX-2 levels could reflect
altered protein synthesis or degradation, qRT-PCR was performed
to determine whether 3b suppresses LPS-induced iNOS and COX-2
expression via a pre-translational mechanism. After treating RAW
50 lM for 24 h, indicating that modification or substitution of the
A- or B-rings of oroxylin A may reduce cytotoxicity and enhance
inhibition of NO and PGE2 production in LPS-induced RAW 264.7
cells. Accordingly, we sought to identify the mechanisms responsi-
ble for the anti-inhibitory effect of 3b in RAW 264.7 cells.
To determine whether 3b has inhibitory effects on the LPS-in-
duced productions of NO and PGE2 in vivo, we examined the effects
264.7 macrophages with 3b (6.25, 12.5, or 25 lM) and LPS (1 lg/ml)
for 4 h, iNOS and COX-2 mRNA levels were elevated about five and
seven-fold, respectively. Furthermore, 3b significantly suppressed
the gene expressions of iNOS and COX-2 (Fig. 3B). These results
indicate that the inhibition of iNOS and COX-2 expressions by 3b
occurs in parallel with inhibition of the productions of NO and
A
B
LPS (1μg/ml)
8
iNOS
#
3b (μM)
COX-2
6
4
2
0
6.25 12.5 25
25
#
*
iNOS
COX-2
β-actin
**
**
**
Con
6.25
12.5
25
25
3b (μM)
LPS (1μg/ml)
D
C
20
15
10
5
300
250
200
150
100
50
#
#
*
**
**
***
***
25
0
0
Con
6.25
12.5
3b (μM)
LPS (1μg/ml)
25
25
Con
6.25
12.5
25
3b (μM)
LPS (1μg/ml)
Figure 3. Effects of 3b on LPS-induced iNOS and COX-2 protein and mRNA expressions, and promoter activity in RAW 264.7 macrophages. (A) Cells were pretreated with
different concentrations (6.25, 12.5, or 25 M) of 3b for 1 h, then treated with LPS (1 g/ml), and incubated for 24 h. Control (Con) values were obtained in the absence of LPS
and 3b. Total cellular proteins (30 g) were resolved by SDS–PAGE, transferred to nitrocellulose, and detected using specific antibodies for iNOS and COX-2. The data shown
are representative of three independent experiments, and the immunoblot is representative of three separate experiments. (B) Total RNA was prepared for qRT-PCR analysis
l
l
l
of iNOS and COX-2 from cells stimulated with LPS (1 lg/ml) with/without 3b (6.25, 12.5, or 25 lM) for 4 h. Steady-state mRNA levels of iNOS, COX-2 and b-actin were
determined by qRT-PCR using the Takara thermal cycler diceÒ (Takara Bio Inc., Japan). The results were expressed as the ratio of optimal density to b-actin. Values represent
the means SDs of three independent experiments. #p < 0.05 as compared with the control cells, and ⁄p < 0.05, ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001 as compared with the LPS-only treated
cells. ANOVA and Dunnett’s post-hoc test were used to determine the significances of differences. (C) Cells were transiently co-transfected with piNOS-luc reporter or pCOX-
2-luc reporter with pRL-TK-luc either not pretreated (Con) or pretreated with different concentrations (6.25, 12.5, or 25 lM) of 3b for 1 h. Cells were then harvested and
luciferase activities were determined using a Promega luciferase assay system and a luminometer. Values represent the means SDs of three independent experiments.
#p < 0.05 as compared with the control cells, and ⁄p < 0.05, ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001 as compared with the LPS-only treated cells. ANOVA and Dunnett’s post-hoc test were used
to determine the significances of differences.

704
D. H. Kim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 700–705
A
B
30
25
20
15
10
5
8
#
#
*
6
4
*
*
**
2
***
0
0
Con
6.25
12.5
25
25
Con
6.25
12.5
25
25
3b (μM)
3b (μM)
LPS (1μg/ml)
LPS (1μg/ml)
#
C
D
250
200
150
100
50
25
20
15
10
5
TNF-α
IL-6
#
#
#
IL-1β
*
*
*
**
**
**
***
0
0
Con
6.25
12.5
3b (μM)
LPS (1μg/ml)
25
25
Con
6.25
12.5
25
25
3b (μM)
LPS (1μg/ml)
Figure 4. Effects of 3b on LPS-induced TNF-
a, IL-6, and IL-1b production and gene expressions. (A–C) Cells were pretreated with different concentrations (6.25, 12.5, or
25 M) of 3b for 1 h, then with LPS (1 lg/ml), and incubated for 24 h. The levels of TNF-a, IL-6 and IL-1b in macrophage culture medium were measured by EIA kits according
l
to the manufacturer’s instructions (R&D Systems). Values represent the means SDs of three independent experiments. #p < 0.05 as compared with the control cells, and
⁄p < 0.05, ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001 as compared with the LPS-only treated cells. ANOVA and Dunnett’s post-hoc test were used to determine the significances of differences. (D)
Total RNA was prepared for qRT-PCR analysis of iNOS and COX-2 from cells stimulated with LPS (1 lg/ml) with/without 3b (6.25, 12.5, or 25 lM) for 4 h. Steady-state mRNA
levels of TNF-a
, IL-6, IL-1b and b-actin were determined by qRT-PCR using the Takara thermal cycler diceÒ (Takara Bio Inc., Japan). The results were expressed as the ratio of
optimal density to b-actin. Values represent the means SDs of three independent experiments. #p < 0.05 as compared with the control cells, and ⁄p < 0.05, ⁄⁄p < 0.01,
⁄⁄⁄p < 0.001 as compared with the LPS-only treated cells. ANOVA and Dunnett’s post-hoc test were used to determine the significances of differences.
PGE2 (Fig. 2A and B). In addition, 3b dose-dependently inhibited
LPS-induced iNOS and COX-2 promoter luciferase activity (Fig. 3C
and D).
phosphorylation was significantly blocked by 3b. Accordingly, we
investigated whether 3b prevents the translocation of the subunit
of NF-jB (p65) to the nucleus, and found that pretreatment with
In the inflammatory state, macrophages release pro-inflamma-
3b dose-dependently attenuated LPS-induced p65 translocation
to the nucleus (Fig. 5A). These findings show that 3b prevents
tory cytokines like TNF-a
, IL-6, and IL-1b.22 We examined the pro-
ductions and expressions of these cytokines by ELISA and qRT-PCR,
respectively, and found that pre-treatment with 3b considerably
reduced LPS-induced TNF-a, IL-6, and IL-1b release (Fig. 4A–C)
NF-
tion of I
after only 3b treatment is remained for further studies.
j
B-activation by inhibition the phosphorylation and degrada-
j
B- . However, the enhancing expression of I B- band
a
j a
and their mRNA expressions (Fig. 4D). These findings suggest that
the decreased protein levels of these cytokines are responsible for
the reduced transcription level.
Summarizing, we synthesized 5,6,7-trimethoxy- and 5,6,7-tri-
hydroxyflavones 3a–3g from cinnamic acid derivatives to enhance
inhibitory effects on LPS-induced NO and PGE2 production versus
oroxylin A and baicalein in RAW 264.7 cells. Of the compounds
synthesized, 40-bromo-5,6,7-trimethoxyflavone (3b) was found to
significantly inhibit the productions of NO and PGE2 production
by LPS. Consistent with these results, 3b inhibited the LPS-stimu-
Because the activation of NF-
expressions of iNOS, COX-2, TNF-
, IL-6, and IL-1b,23 we examined
the effect of 3b using an LPS-stimulated NF- B-dependent reporter
gene assay. Analysis of reporter gene expression using pNF- B-luc
demonstrated that 3b concentration-dependently inhibited NF-
B-dependent luciferase activity (Fig. 5A). In general, NF- B is
inactive in the cytosol because it is bound by I B and becomes ac-
tive after I B is phosphorylated and degraded, which enables the
subsequent nuclear translocation of NF-
B.24,25 Thus, we explored
whether 3b could inhibit the LPS-stimulated phosphorylation and
degradation of I B- in RAW 264.7 cells by Western blotting.
Figure 5B shows that the LPS-induced I B- degradation and
jB is critical for LPS-induced
a
j
j
lated expressions of iNOS, COX-2, TNF-
induced attendant reductions in the productions of NO, PGE2,
TNF- , IL-6, and IL-1b in RAW 264.7 cells. Furthermore, our
molecular data suggest that 3b inhibits these anti-inflammatory
modulators by suppressing NF- B activation via the inhibition of
B- phosphorylation and degradation. Accordingly, our findings
a, IL-6, and IL-1b and
j
j
j
a
j
j
j
Ij a
j
a
provide a partial description of the mechanism underlying the
anti-inflammatory effects of 3b.
j
a

D. H. Kim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 700–705
705
LPS (1μg/ml)
A
B
3b (μM)
5
4
3
2
1
0
6.25 12.5 25
25
#
p-IκBα
IκBα
**
***
25
β-actin
p65 (N)
PARP (N)
Con
6.25
12.5
25
3b (μM)
LPS (1μg/ml)
Figure 5. Effects of 3b on the NF-
transiently co-transfected with pNF-
LPS (1
j
B promoter activity, phosphorylation and degradation of I
j
B
a
, and p65 nuclear translocation in RAW 264.7 macrophages. (A) Cells were
jB-luc reporter and pRL-TK-luc and either not pretreated or pretreated with different concentrations (6.25, 12.5, or 25
l
M) of 3b for 1 h.
l
g/ml) was then added, and cells were further incubated for 1 h. Cells were collected and luciferase activities were determined using a Promega luciferase assay system
and a luminometer. Values represent the means SDs of three independent experiments. #p < 0.05 as compared with the control cells, and ⁄p < 0.05, ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001 as
compared with the LPS-only treated cells. ANOVA and Dunnett’s post-hoc test were used to determine the significances of differences. (B) Cells were pretreated with different
concentrations (6.25, 12.5, or 25
blotting by using a specific p-I
l
M) of 3b for 1 h, then with LPS (1
lg/ml), and incubated for 15 min or 1 h. Cytosol and nuclear extracts were then prepared for the Western
j
Ba
, I and NF- B p65 specific antibodies. A representative immunoblot of three separate experiments is shown. PARP and b-actin were
jB
a
j
used as internal controls. The data shown are representative of three independent experiments, and the immunoblot is representative of three separate experiments.
13. Zhao, L.; Chen, Z.; Wang, J.; Yang, L.; Zhao, Q.; Qi, Q.; Mu, R.; You, Q. D.; Guo, Q.
Acknowledgments
L. Cancer Chemother. Pharmacol. 2010, 65, 481.
14. Kim, D. H.; Jeon, S. J.; Son, K. H.; Jung, J. W.; Lee, S.; Yoon, B. H.; Lee, J. J.;
This study was partly supported by the Korea Healthcare Tech-
nology R&D Project, Ministry for Health, Welfare & Family Affairs
(#A090487) and the Seoul Research and Business Development
Program (#10524), Republic of Korea.
Cho, Y. W.; Cheong, J. H.; Ko, K. H.; Ryu, J. H. Neurobiol. Learn. Mem. 2007,
87, 536.
15. Chen, Y.; Yang, L.; Lee, T. J. Biochem. Pharmacol. 2000, 59, 1445.
16. Suresh Babu, K.; Hari Babu, T.; Srinivas, P. V.; Sastry, B. S.; Hara Kishore, K.;
Murty, U. S.; Madhusudana Rao, J. Bioorg. Med. Chem. Lett. 2005, 15, 3953.
17. Lee, Y.; Yeo, H.; Liu, S. H.; Jiang, Z.; Savizky, R. M.; Austin, D. J.; Cheng, Y. C. J.
Med. Chem. 2004, 47, 5555.
18. Liao, H. L.; Hu, M. K. Chem. Pharm. Bull. 2004, 52, 1162.
19. Huang, W. H.; Lee, A. R.; Chien, P. Y.; Chou, T. C. J. Pharm. Pharmacol. 2005, 57,
219.
References and notes
1. Oberyszyn, T. M. Front Biosci. 2007, 12, 2993.
2. Nathan, C.; Xie, Q. W. Cell 1994, 78, 915.
20. Huang, W. H.; Chien, P. Y.; Yang, C. H.; Lee, A. R. Chem. Pharm. Bull. 2003, 51,
339.
3. Liu, R. H.; Hotchkiss, J. H. Mutat. Res. 1995, 339, 73.
4. Pan, M. H.; Lai, C. S.; Wang, Y. J.; Ho, C. T. Biochem. Pharmacol. 2006, 72, 1293.
5. Hinz, B.; Brune, K. J. Pharmacol. Exp. Ther. 2002, 300, 367.
6. Bendtzen, K. Immunol. Lett. 1988, 19, 183.
7. Mercurio, F.; Zhu, H.; Murray, B. W.; Shevchenko, A.; Bennett, B. L.; Li, J.; Young,
D. B.; Barbosa, M.; Mann, M.; Manning, A.; Rao, A. Science 1997, 278, 860.
8. Szabó, C. New Horiz. 1995, 3, 2.
9. MacMicking, J.; Xie, Q. W.; Nathan, C. Ann. Rev. Immunol. 1997, 15, 323.
10. Bochoráková, H.; Paulová, H.; Slanina, J.; Musil, P.; Táborská, E. Phytother. Res.
2003, 17, 640.
11. Aung, H. H.; Dey, L.; Mehendale, S.; Xie, J. T.; Wu, J. A.; Yuan, C. S. Cancer
Chemother. Pharmacol. 2003, 52, 453.
21. Spectral data of 3b: 1H NMR (400 MHz, DMSO-d6) d 8.07 (2H, d, J = 8.8 Hz), 7.83
(2H, d, J = 8.8 Hz), 7.29 (1H, s), 6.92 (1H, s), 4.01 (3H, s), 3.86 (3H, s), 3.83 (3H,
s); 13C NMR (100 MHz, DMSO-d6) d 175.5, 159.0, 157.6, 153.9, 151.5, 139.8,
132.0 (2C), 130.1, 127.9 (2C), 125.1, 112.1, 107.8, 97.3, 61.8, 60.9, 56.4; HR-
DART-MS m/z 391.0078 (calcd for C18H16BrO5 [M+H]+ , 391.0181).
22. Riedemann, N. C.; Guo, R. F.; Ward, P. A. Nat. Med. 2003, 9, 517.
23. Baeuerle, P. A.; Henkel, T. Ann. Rev. Immunol. 1994, 12, 141.
24. Hla, T.; Ristimäki, A.; Appleby, S.; Barriocanal, J. G. Ann. N. Y. Acad. Sci. 1993,
696, 197.
25. Mestre, J. R.; Chan, G.; Zhang, F.; Yang, E. K.; Sacks, P. G.; Boyle, J. O.; Shah, J. P.;
Edelstein, D.; Subbaramaiah, K.; Dannenberg, A. J. Ann. N. Y. Acad. Sci. 1999, 889,
62.
12. Shih, H. C.; Hsu, C. S.; Yang, L. L. J. Biomed. Sci. 2009, 16, 27.