Synthesis of aurones and their inhibitory effects on nitric oxide and PGE2 productions in LPS-induced RAW 264.7 cells

Article abstract of DOI:10.1016/j.bmcl.2011.05.117

Sulfuretin is one of major constituents of Rhus verniciflua that exerts anti-inflammatory activities. Some of aurones were synthesized as sulfuretin derivatives and evaluated for their abilities to inhibit NO and PGE₂ production in LPS-induced RAW 264.7 cells in order to reveal the relationship. Of the aurones synthesized in the present study, 2h and 2i, which possess C-6 hydroxyl group in A-ring and methoxy substituents in B-ring, more potently inhibited NO and PGE₂ production and were less cytotoxic than sulfuretin.

Full text of DOI:10.1016/j.bmcl.2011.05.117

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4520–4523
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of aurones and their inhibitory effects on nitric oxide and PGE2
productions in LPS-induced RAW 264.7 cells
⇑
Seo Young Shin, Min Cheol Shin, Ji-Sun Shin, Kyung-Tae Lee, Yong Sup Lee
Department of Pharmaceutical Science, College of Pharmacy, Kyung Hee University, Seoul 130-701, Republic of Korea
Department of Life and Nanopharmaceutical Sciences, 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:
Sulfuretin is one of major constituents of Rhus verniciflua that exerts anti-inflammatory activities. Some of
aurones were synthesized as sulfuretin derivatives and evaluated for their abilities to inhibit NO and PGE2
production in LPS-induced RAW 264.7 cells in order to reveal the relationship. Of the aurones synthesized
in the present study, 2h and 2i, which possess C-6 hydroxyl group in A-ring and methoxy substituents in
B-ring, more potently inhibited NO and PGE2 production and were less cytotoxic than sulfuretin.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 23 April 2011
Revised 28 May 2011
Accepted 31 May 2011
Available online 12 June 2011
Keywords:
Sulfuretin
Aurone
Nitric oxide
Prostaglandin E2
Anti-inflammatory
Inflammation is one of the most important aspects of host
defense mechanisms against invading pathogens.1 In the inflamma-
tory state, activated immune cells secrete large amounts of nitric
oxide (NO), prostaglandin E2 (PGE2), and cytokines. However, high
levels of NO production can induce various pathological conditions
such as, inflammation, systemic septic shock and carcinogenesis.2
Prostaglandins are also potent pro-inflammatory mediators,
and produced from arachidonic acid by cyclooxygenase (COX). Of
the prostanoids, production of PGE2 is related to two isozymes
which are present as COX-1 and COX-2.3 COX-1 is responsible for
PGE2 production under normal physiological conditions, whereas
COX-2 is responsible at sites of inflammation.1 Accordingly, these
relations regarding the productions of NO and PGE2 have attracted
the interest of researchers attempting to develop novel anti-
inflammatory drugs.
Sulfuretin (1) is an amorphous orange powder that is obtained
from the lacquer tree (Anacardiaceae, Rhus verniciflua)4,5 and the
bark of the silk tree (Leguminosae, Albizzia julibrissin).6 Structurally,
sulfuretin is an aurone flavonoid derivative with three hydroxyl
groups on C-6, C-30, and C-40 (6,30,40-trihydroxyaurone). Sulfuretin
has been known to possess diverse biological activities, such
as anti-nociceptive,5 anti-oxidant,6 anti-mutagenic,7 and anti-
diabetic activities.8,9 Accordingly, several derivatives of sulfuretin
have been synthesized and their anti-viral,10 anti-oxidant,11
anti-cancer,12 and tyrosinase inhibitory activities13 have been
evaluated. Recently, we found that sufuretinis an anti-inflammatory
constituent of R. verniciflua because it reduces the productions of NO
and PGE2 induced by LPS. Furthermore, we demonstrated that the
anti-inflammatory effect of sulfuretin in LPS-induced RAW 264.7
macrophages is associated with the suppression of NF-jB transcrip-
tional activity via the inhibitory regulation of IKKb phosphoryla-
tion.14 However, to our best knowledge, there is no report on the
evaluation of the anti-inflammatory activities of sulfuretin
derivatives, aurones.
In the present study, we prepared aurones 2a–2q with the aim
to study structure–activity relationships, and thereby, to provide
new leads possessing enhanced inflammatory activity and reduced
cytotoxicity than sulfuretin (Figure 1). More specifically, we syn-
thesized aurones possessing hydroxyl or methoxy substituent at
the 6-position of the A-ring and introduced hydroxyl or methoxy
substituents at various position on the B-ring. The anti-inflamma-
tory activities of aurones were evaluated by measuring the inhibi-
tions of the productions of NO and PGE2 in LPS- induced RAW
264.7 macrophages.
R5
R4
HO
OH
4'
3'
R3
B
R1
HO
O
O
C
6
R2
A
O
O
sufuretin, 1
target compounds, 2
⇑
Corresponding author. Tel.: +82 2 961 0370; fax: +82 2 966 3885.
E-mail address: kyslee@khu.ac.kr (Y.S. Lee).
Figure 1. Structures of sulfuretin and target compounds.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.05.117

S. Y. Shin et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4520–4523
4521
The synthesis of aurones 2a–2q was accomplished in a simple
procedure as shown in Scheme 1. 6-Hydroxy-3-coumaranone 3a,
which already possesses A- and C rings of aurones, was used as a
starting material. The C-6 hydroxy-substituted aurones 2a–2j were
prepared by cross-aldol condensation of coumaranone 3a with var-
ious substituted-benzaldehydes 4. The condensation reaction was
carried out using two methods involving reaction under acidic
(12N HCl in ethanol)15 or basic conditions (50% KOH in metha-
nol).13,16–18
mediators is a potential strategy for the treatment of many acute
and chronic inflammatory diseases. Recently, we found that sul-
furetin dose-dependently reduced the productions of NO and
PGE2. In the present study, we synthesized several aurone deriva-
tives to determine whether any had better anti-inflammatory
activity and less cytotoxicity than sulfuretin. The synthesized auro-
nes 2a–2q were assessed in terms of their abilities to inhibit the
productions of inflammatory mediators, NO and PGE2, in LPS-
induced RAW 264.7 cells (Table 1). The screen for activity was
For the synthesis of C-6 methoxy-substituted aurones 2k–2q,
starting 6-methoxy-3-coumaranone 3b was prepared by methyla-
tion of the hydroxyl group in 3a using methyl iodide in the pres-
ence of potassium carbonate in DMF.19 Finally, compound 3b was
condensed with benzaldehydes 4 in basic condition to provide
aurones 2k–2q.20 Compound 2d was also prepared under the basic
condition.
Inflammation is a protective immune response against tissue
damage induced by external irritation or other injury in the body.1
However, inflammatory response can also result in considerable
damage to the host, because microbial components like lipopoly-
saccharide (LPS) stimulates macrophages to produce pro-inflam-
matory cytokines, chemokines, iNOS, and COX-2. In particular,
the large amounts of NO and PGE2 secreted by activated immune
cells in the inflammatory state, can induce various pathological
conditions. Thus, blocking the effects of pro-inflammatory
performed in a dose–response to determine the IC50 value (lM)
as previously described.21–23 Sulfuretin (1) was used as a reference
for comparisons. The cytotoxic effects of the aurons were also eval-
uated using the MTT assay24 to test whether the inhibitory effects
on the productions of NO and PGE2 were due to cytotoxic effects.
Most compounds synthesized showed less cytotoxicities than
sulfuretin on RAW 264.7 cells with varying abilities to inhibit the
productions of NO and PGE2. Generally, C-6 hydroxy-substituted
aurones exhibited inhibitory effects on NO production that were
similar to that of the parent compound, sulfuretin (1). However,
compounds 2a, 2c, and 2g–2j more potently inhibited the produc-
tion of PGE2 with IC50 in the range of 1.60–2.90
(IC50 = 5.90 M). On the other hand, C-6 methoxy-substituted
aurones 2m and 2o were about three times more potent than sul-
furetin on the inhibition of NO production (IC50 = 9.30–10.53
lM than sulfuretin
l
l
M
vs 28.97 lM). Compound 2j and 2k also potently inhibited PGE2
R5
R4
R2
O
R2
R5
R1
R3
R4
O
A or B
R3
H
R1
+
O
O
O
1
3a
3b
, R = OH
MeI, K2CO3,
DMF, 58%
4
2a-2q
1
, R = OCH3
R5
R1
R2
R3
R4
Compd
conditions
Yields (%)
from 3a or 3b
2a
2b
2c
2d
2e
A
A
A
B
A
OH
OH
OH
OH
OH
OH
H
H
H
H
75
74
78
19
72
H
H
OH
H
H
OH
OH
OH
H
OH
H
H
H
OH
OH
2f
OCH3
H
A
A
OH
OH
H
H
H
H
H
78
92
2g
OCH3
2h
2i
A
A
A
B
OH
OH
OH
H
H
OCH3
OCH3
H
H
87
50
67
76
H
OCH3
OCH3
H
H
2j
H
OCH3
H
2k
2l
OH
H
OCH3
OCH3
OCH3
B
B
H
H
H
H
H
27
39
2m
2n
H
OH
B
B
B
B
OCH3
OCH3
OCH3
OCH3
OH
H
H
OH
OH
H
H
H
H
H
12
58
69
36
2o
2p
OH
H
OCH3
H
2q
OCH3
H
Scheme 1. Reagents and conditions: (A): 12N HCl, ethanol, 60–70 °C; (B) 50% KOH, methanol, 60 °C.

4522
S. Y. Shin et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4520–4523
12. Valclavikova, R.; Kondrova, E.; Ehrlichova, M.; Boumendjel, A.; Kovar, J.; Stopka,
Table 1
P.; Soucek, P.; Gut, I. Bioorg. Med. Chem. 2008, 16, 2034.
13. Okombi, S.; Rival, D.; Bonnet, S.; Mariotte, A.-M.; Perrier, E.; Boumendjel, A. J.
Med. Chem. 2006, 49, 329.
14. Shin, J.-S.; Park, Y. M.; Choi, J.-H.; Park, H.-J.; Shin, M. C.; Lee, Y. S.; Lee, K.-T. Int.
Immunopharmacol. 2010, 10, 943.
In vitro cytotoxicity, NO and PGE2 production inhibitory activities of aurones 2a–2q in
LPS-induced RAW 267.4 cells
a
Compds
IC50
(lM)
NO production
PGE2 production
Cytotoxicity
15. General procedure
A for the synthesis of aurones: To a solution of 3a
(2.0 mmol) and benzaldehyde derivative 4 (2.0 mmol) in ethanol was added
dropwise 12N HCl (3 mL) and heated at 60–70 °C until the reaction was
finished from TLC monitoring. The mixture was poured into H2O and the
resulting precipitate was filtered and dried in vacuo. The crude products
were purified by silica gel column chromatography (eluting solvent systems:
ethyl acetate/hexanes or methanol/methylene chloride) to give aurones.
General procedure B for the synthesis of aurones: To a solution of compound
3a or 3b (2.0 mmol) in methanol (20 mL) was added successively an
aqueous 50% potassium hydroxide (3 mL) and benzaldehyde derivatives 4
(3.0 mmol). The mixture was heated at 60 °C and then solvent was
evaporated. The residue was diluted in water and extracted with ethyl
acetate. The organic layer was dried over anhydrous MgSO4 and filtered. The
filtrate was concentrated and purified by silica gel column chromatography
to give aurones.
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2q
1
46.29 9.45
47.23 3.17
38.70 3.73
113.26 26.20
99.36 6.84
121.12 17.32
23.38 13.03
23.51 5.90
23.92 6.97
22.64 9.87
>100
105.97 16.35
10.53 6.60
20.18 3.05
9.30 3.87
130.48 23.39
28.58 9.85
28.97 10.81
1.80 0.12
9.30 0.65
1.06 0.13
18.62 1.62
37.62 2.48
18.10 1.11
3.79 0.22
2.00 0.11
2.90 0.19
1.67 0.09
2.22 0.14
13.15 1.10
35.82 2.23
8.80 0.77
4.90 0.38
59.50 3.99
2.50 0.15
5.90 0.44
131.40 2.49
168.29 8.58
99.79 1.93
186.27 1.73
>200
186.63 12.80
32.85 2.53
74.84 2.09
>200
55.11 4.58
48.83 8.35
211.31 13.67
115.47 14.63
85.83 0.51
77.43 11.58
>200
16. Boumendjel, A.; Beney, C.; Deka, N.; Mariotte, A.-M.; Lawson, M. A.; Trompier,
D.; Baubichon-Cortay, H.; Pietro, A. D. Chem. Pharm. Bull. 2002, 50, 854.
17. Lee, C.-Y.; Chew, E.-H.; Go, M.-L. Eur. J. Med. Chem. 2010, 45, 2957.
18. Wong, E. Phytochemistry 1967, 6, 1227.
111.64 12.94
80.68 3.03
19. Laczkowski, K. Z.; Pakulski, M. M.; Krzeminski, M. P.; Jaisankar, P.; Zaidlewicz,
M. Tetrahedron: Asymmetry 2008, 19, 788.
20. The 1H NMR (400 MHz) and 13C NMR (100 MHz) data of aurones: (Z)-6-Hydroxy-
2-(2-hydroxybenzylidene)benzofuran-3(2H)-one (2a): 1H NMR (DMSO-d6) d 11.2
(br s, 1H, OH), 10.3 (br s, 1H, OH), 8.11 (dd, 1H, J = 8.0, 4.0 Hz, H-60), 7.63 (d, 1H,
J = 8.0 Hz, H-4), 7.25–7.23 (m, 1H, H-50), 7.10 (s, 1H, H-10), 6.96–6.93 (m, 2H,
H-30, H-40), 6.80 (d, 1H, J = 1.6 Hz, H-7), 6.73 (dd, 1H, J = 8.0, 1. Hz, H-5); 13C
NMR (DMSO-d6) d 181.9, 186.2, 166.9, 157.6, 147.3, 131.8, 131.5, 126.3, 120.1,
a
Data are presented as the means SDs of three independent experiments.
production inhibition with IC50 value of 1.67 and 2.20 lM, respec-
tively, but their cytotoxicities were higher than that of sulfuretin.
Of the compounds, 2h, 2i and 2o exhibited well-balanced activity
profiles with respect to the inhibitions of the productions of NO
and PGE2 and cytotoxicity. In particular, compound 2i had no cyto-
119.3,
116.2,
113.4
(2C),
105.1,
99.1;
(Z)-6-hydroxy-2-(3-
hydroxybenzylidene)benzofuran-3(2H)-one (2b): 1H NMR (DMSO-d6) d 11.3 (s,
1H, OH), 9.71 (s, 1H, OH), 7.68 (d, 1H, J = 8.4 Hz, H-4), 7.43 (d, 1H, J = 1.6 Hz, H-
20), 7.40 (d, 1H, J = 8.0 Hz, H-60), 7.34 (t, 1H, J = 8.0 Hz, H-50), 6.90–6.87 (m, 1H,
H-40), 6.82 (d, 1H, J = 1.8 Hz, H-7), 6.77 (dd, 1H, J = 8.4, 1.8 Hz, H-5), 6.73 (s, 1H,
H-10); 13C NMR (DMSO-d6) d 181.9, 168.3, 167.1, 158.0, 147.7, 133.6, 130.4,
126.5, 122.8, 117.8, 117.5, 113.5, 113.2, 111.1, 99.0; (Z)-6-hydroxy-2-(4-
hydroxybenzylidene)benzofuran-3(2H)-one (2c): 1H NMR (DMSO-d6) d 11.1 (s,
1H, OH), 10.1 (s, 1H, OH), 7.83 (d, 2H, J = 8.6 Hz, H-20, H-60), 7.61 (d, 1H,
J = 8.4 Hz, H-4), 6.90 (d, 2H, J = 8.6 Hz, H-30, H-50), 6.78 (d, 1H, J = 1.6 Hz, H-7),
6.73 (s, 1H, H-10), 6.72 (dd, 1H, J = 8.4, 1.6 Hz, H-5); 13C NMR (DMSO-d6) d
181.7, 168.0, 166.6, 159.7, 146.2, 133.7 (2C), 126.2, 123.5, 116.5 (2C), 113.6,
113.3, 111.9, 99.0; (Z)-2-(2,4-dihydroxybenzylidene)-6-hydroxybenzofuran-
3(2H)-one (2d): 1H NMR (DMSO-d6) d 10.3 (br s, 1H, OH), 10.0 (br s, 1H, OH),
7.97 (d, 1H, J = 8.0 Hz, H-60), 7.59 (d, 1H, J = 8.4 Hz, H-4), 7.05 (s, 1H, H-10), 6.76
(d, 1H, J = 2.0 Hz, H-7), 6.70 (dd, 1H, J = 8.4, 2.0 Hz, H-5), 6.40–6.37 (m, 2H, H-30,
H-50); 13C NMR (DMSO-d6) d 181.0, 167.1, 165.8, 160.8, 159.0, 145.1, 132.4,
125.5, 113.3, 112.7, 110.5, 108.2, 105.8, 102.2, 98.4; (Z)-6-hydroxy-2-(3,4,5-
trihydroxybenzylidene)benzofuran-3(2H)-one (2e): 1H NMR (DMSO-d6) d 11.15
(br s, 1H, OH), 9.18 (m, 2H, OH), 7.61 (d, 1H, J = 8.4 Hz, H-4), 6.95 (s, 2H, H-20,
H-60), 6.73–6.69 (m, 2H, H-5, H-7), 6.54 (s, 1H, H-10); 13C NMR (DMSO-d6) d
181.6, 167.8, 166.5, 146.5 (2C), 146.2, 136.8, 126.2, 122.6, 113.7, 113.3, 112.9,
111.3 (2C), 98.7; (Z)-6-hydroxy-2-(2-methoxybenzylidene)benzofuran-3(2H)-
one (2f): 1H NMR (DMSO-d6) d 11.20 (s, 1H, OH), 8.17 (dd, 1H, J = 8.0, 1.6 Hz, H-
60), 7.64 (d, 1H, J = 8.4 Hz, H-4), 7.45 (td, 1H, J = 8.0, 1.6 Hz, H-50), 7.13–7.07 (m,
2H, H-30, H-40), 7.07 (s, 1H, H-10), 6.80 (d, 1H, J = 2.0 Hz, H-7), 6.73 (dd, 1H,
J = 8.4, 2.0 Hz, H-5), 3.90 (s, 3H, OCH3); 13C NMR (DMSO-d6) d 181.4, 167.8,
166.5, 158.0, 147.3, 131.5, 130.9, 125.9, 120.9, 120.3, 113.0, 112.8, 111.5, 103.6,
98.6, 55.8; (Z)-6-hydroxy-2-(3-methoxybenzylidene)benzofuran-3(2H)-one (2g):
1H NMR (DMSO-d6) d 7.63 (d, 1H, J = 8.4 Hz, H-4), 7.54 (d, 1H, J = 8.0 Hz, H-60),
7.48 (d, 1H, J = 2.4 Hz, H-20), 7.41 (t, 1H, J = 8.0 Hz, H-50), 7.02 (dd, 1H, J = 8.0,
2.4 Hz, H-40), 6.80 (d, 1H, J = 2.0 Hz, H-7), 6.74 (s, 1H, H-10), 6.73 (dd, 1H,
J = 8.4, 2.0 Hz, H-5), 3.80 (s, 3H, OCH3); 13C NMR (DMSO-d6) d 182.0, 168.5,
167.1, 159.9, 147.9, 133.7, 130.4, 126.5, 123.9, 116.8, 115.8, 113.6, 113.1, 110.8,
99.1, 55.6; (Z)-6-hydroxy-2-(4-methoxybenzylidene)benzofuran-3(2H)-one (2h):
1H NMR (DMSO-d6) d 11.22 (s, 1H, OH) 8.00 (d, 2H, J = 8.8 Hz, H-20, H-60), 7.70
(d, 1H, J = 8.4 Hz, H-4), 7.14 (d, 2H, J = 8.8 Hz, H-30, H-50), 6.87 (d, 1H, J = 2.0 Hz,
H-7), 6.84 (s, 1H, H-10), 6.80 (dd, 1H, J = 8.4, 2.0 Hz, H-5), 3.90 (s, 3H, OCH3);
13C NMR (DMSO-d6) d 181.7, 168.1, 166.7, 160.9, 146.7, 133.4 (2C), 126.3,
125.1, 115.1 (2C), 113.5, 113.4, 111.2, 99.0, 55.8; (Z)-2-(3,4-
dimethoxybenzylidene)-6-hydroxybenzofuran-3(2H)-one (2i): 1H NMR (DMSO-
d6) d 7.61 (d, 1H, J = 8.4 Hz, H-4), 7.57 (d, 1H, J = 8.0 Hz, H-60), 7.55 (s, 1H, H-20),
7.08 (d, 1H, J = 8.0 Hz, H-50), 6.79 (s, 1H, H-10), 6.76 (s, 1H, H-7) 6.72 (d, 1H,
J = 8.4 Hz, H-5), 3.83 (s, 6H, OCH3); 13C NMR (DMSO-d6) d 181.7, 168.1, 166.7,
150.8, 149.2, 146.7, 126.2, 125.5, 125.2, 114.6, 113.5, 113.0, 112.3, 111.6, 99.0,
56.0 (2C); (Z)-2-(3,5-dimethoxybenzylidene)-6-hydroxybenzofuran-3(2H)-one
(2j): 1H NMR (pyridine-d5) d 7.84 (d, 1H, J = 8.4 Hz, H-4), 7.29 (d, 2H, J =
2.2 Hz, H-20, H-60), 7.04 (s, 1H, H-10), 7.02 (d, 1H, J = 2.0 Hz, H-7), 6.94 (dd, 1H,
J = 8.4, 2.0 Hz, H-5), 6.71 (t, 1H, J = 2.2 Hz, H-40), 3.73 (s, 6H, OCH3); 13C NMR
(pyridine-d5) d 182.3, 168.9, 168.0, 161.2 (2C), 148.5, 134.6, 126.1, 113.6 (2C),
110.7, 109.4 (2C), 102.1, 99.4, 55.1 (2C); (Z)-2-(2-hydroxybenzylidene)-6-
methoxybenzofuran-3(2H)-one (2k): 1H NMR (DMSO-d6) d 10.4 (s, 1H, OH),
8.15 (dd, 1H, J = 8.0, 1.2 Hz, H-60), 7.70 (d, 1H, J = 8.4 Hz, H-4), 7.31 (td, 1H,
toxic effect on RAW 264.7 cells at concentrations of <200 lM.
In conclusion, we synthesized sufuretin derivatives in order to
explore the relationship between structures of aurones and
anti-inflammatory activities. Aurones were synthesized by con-
densation of 6-hydroxy- or 6-methoxy-coumaranone with various
benzaldehydes and evaluated for their inhibition of NO and PGE2
productions in LPS-induced RAW 267.4 cells. Compounds 2a, 2c,
and 2g–2j, which possess a C-6 hydroxy substituent, more potently
inhibited the production of PGE2 than sulfuretin. On the other
hand, C-6 methoxy-substituted aurones 2m and 2o were more po-
tent than sulfuretin on the inhibition of NO production. Among
synthesized, compounds 2h–2j and 2o exhibited increased inhibi-
tory activities than sulfuretin on the productions of both NO and
PGE2 in LPS-induced RAW 264.7 cells.
Acknowledgment
This work was supported by the Basic Science Research
Program through the National Research Foundation of Korea
(NRF) funded by MEST (2011–0002998).
Reference and notes
1. Oberyszyn, T. M. Front. Biosci. 2007, 12, 2993.
2. Yun, H. Y.; Dawson, V. L.; Dawson, T. M. Crit. Rev. Neurobiol. 1996, 10, 291.
3. Hinz, B.; Brune, K. J. Pharmacol. Exp. Ther. 2002, 300, 367.
4. Choi, J. W.; Yoon, B. J.; Han, Y. N.; Lee, S. K.; Lee, K.-T.; Park, H.-J. Nat. Prod. Sci.
2003, 9, 97.
5. Kim, I. T.; Park, Y. M.; Shin, K. M.; Ha, J. H.; Choi, J. W.; Jung, H. J.; Park, H.-J.; Lee,
K.-T. J. Ethnopharmacol. 2004, 94, 165.
6. Jung, M. J.; Chung, H. Y.; Kang, S. S.; Choi, J. H.; Bae, K. S.; Choi, J. S. Arch. Pharm.
Res. 2003, 6, 458.
7. Park, K. Y.; Jung, G. O.; Lee, K.-T.; Choi, J. W.; Choi, M. Y.; Kim, G. T.; Jung, H. J.;
Park, H.-J. J. Ethnopharmacol. 2004, 90, 73.
8. Lee, E. H.; Song, D. G.; Lee, J. Y.; Pan, C. H.; Um, B. H.; Jung, S. H. Biol. Pharm. Bull.
2008, 31, 1626.
9. Song, M.-Y.; Jeong, G.-S.; Kwon, K.-B.; Ka, S.-O.; Jang, H.-Y.; Park, J.-W.; Kim, Y.-
C.; Park, B.-H. Exp. Mol. Med. 2010, 42, 628.
10. Liu, A.-L.; Wang, H.-D.; Lee, S. M.; Wang, Y.-T.; Du, G.-H. Bioorg. Med. Chem.
2008, 16, 7141.
11. Detsi, A.; Majdalani, M.; Kontogiorgis, C. A.; Hadjipavlou-Litina, D.; Kefalas, P.
Bioorg. Med. Chem. 2009, 17, 8073.

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J = 8.0, 1.2 Hz, H-50), 7.17 (d, 1H, J = 2.0 Hz, H-7), 7.16 (s, 1H, H-10), 6.98–6.93
(m, 2H, H-30, H-40), 6.87 (dd, 1H, J = 8.4, 2.0 Hz, H-5), 3.93 (s, 3H, OCH3); 13C
NMR (DMSO-d6) d 181.6, 167.8, 167.2, 157.2, 146.8, 131.5, 131.0, 125.4, 119.6,
118.7, 115.8, 114.0, 112.6, 105.2, 97.1, 56.4; (Z)-2-(3-hydroxybenzylidene)-6-
methoxybenzofuran-3(2H)-one (2l): 1H NMR (DMSO-d6) d 9.73 (s, 1H, OH), 7.72
(d, 1H, J = 8.4 Hz, H-4), 7.41–7.40 (m, 2H, H-20, H-60) 7.32 (t, 1H, J = 8.0 Hz, H-
50), 7.15 (d, 1H, J = 2.0, H-7), 6.88–6.86 (m, 2H, H-5, H-40), 6.76 (s, 1H, H-10),
3.94 (s, 3H, OCH3); 13C NMR (DMSO-d6) d 182.2, 168.5, 167.9, 158.1, 147.6,
133.5, 130.4, 126.0, 122.9, 118.0, 117.7, 114.3, 113.2, 111.7, 97.6, 56.9; (Z)-2-
6.96 (m, 1H, J = 8.0, 2.0 Hz, H-40), 6.79–6.75 (m, 3H, H-5, H-7, H-10), 3.93 (s, 3H,
OCH3), 3.88 (s, 3H, OCH3); 13C NMR (CDCl3) d 183.0, 168.6, 167.5, 159.8, 148.0,
133.7, 129.8, 125.9, 124.1, 116.5, 115.4, 114.9, 112.2, 111.7, 96.7, 56.1, 55.4.
21. Cell culture and sample treatment. The RAW 264.7 macrophage cell line was
obtained from the Korea Cell Line Bank (Seoul). Cells were grown at 37 °C in
Dulbecco0s Modified Eagle Medium (DMEM) supplemented with 10% fetal
bovine serum (FBS), penicillin (100 units/ml), and streptomycin sulfate
(100 lg/ml) in a humidified 5% CO2 atmosphere. Cells were incubated with
various concentrations of tested samples and then stimulated with LPS 1
for the indicated time.
lg/ml
(4-hydroxybenzylidene)-6-methoxybenzofuran-3(2H)-one
(2m):
1H
NMR
(DMSO-d6) d 10.2 (br s, 1H, OH), 7.87 (d, 2H, J = 8.8 Hz, H-20,H-60), 7.69 (d,
1H, J = 8.4 Hz, H-4), 7.16 (d, 1H, J = 2.0 Hz, H-7), 6.91 (d, 2H, J = 8.8 Hz, H-30,H-
50), 6.87 (dd, 1H, J = 8.4, 2.0 Hz, H-5), 6.80 (s, 1H, H-10), 3.93 (s, 3H, OCH3); 13C
NMR (DMSO-d6) d 181.4, 167.5, 167.0, 159.4, 145.6, 133.4 (2C), 125.2, 122.9,
116.0 (2C), 114.2, 112.4, 112.0, 97.0, 56.3; (Z)-2-(2,4-dihydroxybenzylidene)-6-
methoxybenzofuran-3(2H)-one (2n): 1H NMR (DMSO-d6) d 10.4 (s, 1H, OH),
10.1 (s, 1H, OH), 8.02 (d, 1H, J = 8.4 Hz, H-60), 7.67 (d, 1H, J = 8.8 Hz, H-4), 7.14
(d, 1H, J = 2.4 Hz, H-7), 7.10 (s, 1H, H-10), 6.86 (dd, 1H, J = 8.8, 2.4 Hz, H-5),
6.42–6.38 (m, 2H, H-30, H-50), 3.90 (s, 3H, OCH3); 13C NMR (DMSO-d6) d 181.1,
167.2, 166.7, 161.0, 159.3, 145.0, 132.5, 125.0, 114.5, 112.3, 110.5, 108.3, 106.5,
102.3, 97.0, 56.3; (Z)-2-(3,4-dihydroxybenzylidene)-6-methoxybenzofuran-
22. Nitrite determination. RAW 264.7 cells were plated at 4 ꢀ 105 cells /well in 24
well-plates and then incubated with or without LPS (1 lg/mL) in the absence
or presence of various concentrations (3.15, 6.25, 12.5, 25, 50 and 100
tested samples for 24 h. Nitrite levels in culture media were determined using
the Griess reaction and presumed to reflect NO levels. Briefly, 100 L of cell
culture medium was mixed with 100 L of Griess reagent [equal volumes of 1%
lM) of
l
l
(w/v) sulfanilamide in 5% (v/v) phosphoric acid and 0.1% (w/v)
naphtylethylenediamine-HCl], and incubated at room temperature for
10 min. Absorbance was then measured at 540 nm using a microplate reader
(Perkin Elmer Cetus, Foster City, CA, USA). Fresh culture media were used as
blanks in all experiments. The amount of nitrite in the samples was measured
with the sodium nitrite serial dilution standard curve and nitrite production
was measured.
1
3(2H)-one (2o): H NMR (methanol-d4) d 7.68 (dd, 1H, J = 8.8, 4.4 Hz, H-60),
7.52 (s, 1H, H-20), 7.29 (dd, 1H, J = 8.4, 2.4 Hz, H-4), 6.95 (d, 1H, J = 2.4 Hz, H-7),
6.86–6.82 (m, 2H, H-5, H-10), 6.73 (d, 1H, J = 4.4 Hz, H-50), 3.96 (s, 3H, OCH3);
13C NMR (DMSO-d6) d 181.3, 167.4, 166.9 (2C), 148.2, 145.5, 125.2, 124.6,
123.3, 118.2, 116.0, 114.3, 112.5, 112.4, 96.9, 56.3; (Z)-6-methoxy-2-(2-
methoxybenzylidene)benzofuran-3(2H)-one (2p): 1H NMR (CDCl3) d 8.27 (dd,
1H, J = 8.0, 2.0 Hz, H-60), 7.72 (d, 1H, J = 8.4 Hz, H-4), 7.40 (s, 1H, H-10), 7.38 (td,
1H, J = 8.0, 2.0 Hz, H-50), 7.07 (t, 1H, J = 8.0 Hz, H-40), 6.94 (d, 1H, J = 8.0 Hz, H-
30), 6.77–6.73 (m, 2H, H-5, H-7), 3.92 (s, 3H, OCH3), 3.90 (s, 3H, OCH3); 13C NMR
(CDCl3) d 183.0, 168.4, 167.2, 158.7, 147.9, 131.8, 131.1, 125.8, 121.5, 120.8,
115.1, 112.0, 110.8, 106.1, 96.6, 56.0, 55.6; (Z)-6-methoxy-2-(3-
23. PGE2 assay. RAW 264.7 cells were pretreated with tested samples for 1 h and
then stimulated with LPS (1 lg/mL) for 24 h. Levels of PGE2 in the culture
media was quantified using EIA kits (R&D Systems, Minneapolis, MN. USA)
according to the manufacturer0s instructions.
24. MTT assay for cell viability. RAW 264.7 cells were plated at a density of 105 cells/
well in 96-well plates. To determine the appropriate concentration not toxic to
cells, cytotoxicity studies were performed 24 h after treating cells with various
concentrations of tested compounds. Cell viabilities were determined using
colorimetric MTT assays, as described previously.25
methoxybenzylidene)benzofuran-3(2H)-one (2q): 1H NMR (CDCl3)
1H, J = 8.0 Hz, H-4), 7.48–7.46 (m, 2H, H-20, H-60), 7.38 (t, 1H, J = 8.0 Hz, H-50),
d
7.72 (d,
25. Kim, J. Y.; Park, S. J.; Yun, K. J.; Cho, Y. W.; Park, H. J.; Lee, K.-T. Eur. J. Pharmacol.
2008, 584, 175.