Synthesis and inhibitory activity against COX-2 catalyzed prostaglandin production of chrysin derivatives

Article abstract of DOI:10.1016/j.bmcl.2003.12.087

A series of chrysin derivatives were prepared and evaluated for their inhibitory activities of cyclooxygenase-2 catalyzed prostaglandin production. Chrysin derivatives were prepared from 2-hydroxyacetophenone, 2,4-dihydroxyacetophenone and 2,6-dihydroxyacetophenone in 2 to 4 steps, respectively. Methxoylated chrysin derivatives were converted to the corresponding hydroxylated chrysin derivatives by the reaction with BBr ₃ in good yields. The inhibitory activity of the chrysin derivatives against prostaglandin production from lipopolysaccharide-treated RAW 264.7 cells was measured. We found that chrysin derivatives with 3′,4′- dichloro substituents (5e, 6e and 7e) exhibited good inhibitory activity of prostaglandin production.

Full text of DOI:10.1016/j.bmcl.2003.12.087

Bioorganic & Medicinal Chemistry Letters 14(2004) 1165–1167
Synthesis and inhibitory activity against COX-2 catalyzed
prostaglandin production of chrysin derivatives
Tran Thanh Dao,a Yeon Sook Chi,a Jeongsoo Kim,a Hyun Pyo Kim,a Sanghee Kimb and
Haeil Parka,*
aCollege of Pharmacy, Kangwon National University, Chunchon 200-701, South Korea
bCollege of Pharmacy, Seoul National University, Seoul 110-460, South Korea
Received 14September 2003; accepted 18 December 2003
Abstract—A series of chrysin derivatives were prepared and evaluated for their inhibitory activities of cyclooxygenase-2 catalyzed
prostaglandin production. Chrysin derivatives were prepared from 2-hydroxyacetophenone, 2,4-dihydroxyacetophenone and 2,6-
dihydroxyacetophenone in 2 to 4steps, respectively. Methxoylated chrysin derivatives were converted to the corresponding
hydroxylated chrysin derivatives by the reaction with BBr3 in good yields. The inhibitory activity of the chrysin derivatives against
prostaglandin production from lipopolysaccharide-treated RAW 264.7 cells was measured. We found that chrysin derivatives with
30,40-dichloro substituents (5e, 6e and 7e) exhibited good inhibitory activity of prostaglandin production.
# 2004Elsevier Ltd. All rights reserved.
Inflammatory process comprises of several aspects pro-
voked by different chemicals/biologicals including
proinflammatory enzymes/cytokines, small molecular
chemicals such as eicosanoids and tissue degradation
enzymes. Among these factors, cyclooxygenase (COX)
catalyzes the conversion of arachidonic acid to pros-
taglandins (PGs), a key proinflammatory eicosanoid.
COX exists in two isoforms. COX-1 is a constitutive
enzyme processing homeostasis function, while COX-2
is an inducible one and known as a major isoform found
in the inflammatory lesions.1 Recently developed COX-2
inhibitors show promising results in clinical use. There-
fore, a search for COX-2 inhibitors or modulators may
be important to develop new anti-inflammatory agents.
Flavonoids from plant origin possess anti-inflammatory
activity. In addition to the inhibitory activity of some
flavonoids against COX-1 and/or COX-2,2,3 recent
studies have shown that several flavone analogues such
as apigenin, wogonin and tectorigenin (Fig. 1) down-
regulate COX-2 expression,4ꢀ6 suggesting a potential for
new class of anti-inflammatory agents. Chrysin (Fig. 1),
a natural flavonoid widely distributed in plants, has
been reported to have various biological activities such
as anti-oxidant,7 anti-anxiolytic,8 and anti-cancer9 effects.
Furthermore chrysin has been reported to possess anti-
inflammatory effects.10 In order to improve the anti-
inflammatory activity of chrysin, 25 derivatives of chry-
sin with serial deletion and methylation of the phenol
Figure 1. Structures of some naturally occurring polyhydroxyflavonoids.
Keywords: Chrysin derivatives; Prostaglandin production; COX-2; Anti-inflammatory activity.
* Corresponding author. Tel.: +82-33-250-6920; fax: +82-33-255-7865; e-mail: haeilp@kangwon.ac.kr
0960-894X/$ - see front matter # 2004Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.12.087

1166
T. T. Dao et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1165–1167
groups on the A ring and/or substitution of the B ring
were prepared. Herein, we described the synthesis of
chrysin derivatives and their inhibitory activities against
COX-2 catalyzed PGE2 production from LPS-induced
RAW 264.7 cells.
96-well plates (2ꢂ105 cells/well). Each synthetic flavone
was dissolved in dimethyl sulfoxide (DMSO) and LPS
(1 mg/mL) were added and incubated for 24h. Cell via-
bility was assessed with MTT assay based on the
experimental procedures described previously.15 All tes-
ted compounds showed no or less than 10% reduction
of MTT assay, indicating that they were not sig-
nificantly cytotoxic to RAW 264.7 cells in the presence
or absence of LPS. Therefore, the inhibition of PGE2
production by flavone derivatives might be not asso-
ciated with their cytotoxicity. PGE2 concentration in the
medium was measured using EIA kit for PGE2 accord-
ing to the manufacturer’s recommendation. All experi-
ments were carried out at least twice and they gave
similar results. The inhibitory activities of synthetic fla-
vones on COX-2 catalyzed PGE2 production from LPS-
induced RAW 264.7 cells were estimated and the results
are shown in Table 1.
Commercially available 2,4- and 2,6-dihydroxy-
acetophenones were treated with anhydrous potassium
carbonate and dimethyl sulfate (1 equiv) in acetone to
give 4-methoxy and 6-methoxy-2-hydroxyacetophenone
in good yields, respectively.11 These compounds were
reacted with aryl aldehydes in methanolic KOH solu-
tion to afford the corresponding chalcones (2a, b, d, e,
and 3a–e). Treatment of the chalcones with catalytic
amount of iodine in dimethyl sulfoxide gave 5-methoxy-
flavones (6a–e) and 7-methoxyflavones (5a, b, d,
and e).12 Reaction of 2-hydroxyacetophenone with aryl
aldehydes followed by the flavone ring formation in
same conditions gave the flavone analogues (4a–e)
without any phenol group on A ring. Reaction of the
methoxyflavones with BBr3 in dichloromethane gave
5-hydroxyflavones (8a, b, d and e) and 7-hydroxy-
flavones (7a, b, d and e), respectively.13 Reaction of the
40,5-dimethoxyflavone (6c) with AlCl3 gave the
5-hydroxyflavones (8c).14 The synthetic procedure and
reaction conditions are shown in Scheme 1. For the
synthesis of 7-hydroxy-40-methoxyflavone (7c), we pro-
tected the 4-hydroxyl group of 2,4-dihydroxyaceto-
phenone with benzyl group in the standard conditions.11
Reaction of 4-benzyloxy-2-hydroxyacetophenone in
methanolic KOH yielded the chalcone (2c). The chal-
cone was converted to 7-benzyloxy-40-methoxyflavone
(5c) and the removal of the protecting group gave the
compound (7c).
Most chrysin derivatives showed better biological activ-
ities than chrysin against COX-2 catalyzed PGE2 pro-
duction from LPS-induced RAW 264.7 cells. Among
the chrysin derivatives tested, 7-methoxyflavone analo-
gues (5a–e) generally showed potent inhibitory activities
regardless of the substituent on B ring, whereas other
series of synthetic flavones mostly exhibited moderate to
little inhibitory activities as demonstrated in Table 1.
Also flavones with 30,40-dichloro substituents on B ring
(6e and 7e) exhibited strong inhibitory activities. Fla-
vones without any substituent on A ring showed mod-
erate to little inhibitory activities. 5-Hydroxyflavones
were inactive regardless of B ring substituent. Further
bioassays at lower concentrations were performed for
the active analogues (4a, 5d, e, 6e and 7e) which exhib-
ited more than 90% inhibition of PGE2 production at
10 mM concentration and the results were shown in
Table 2. The compounds (4a, 5d, e, 6e and 7e) were
proved to possess more potent inhibitory activities
(IC50=0.1–0.5 mM) than wogonin (IC50=1.08 mM), a
plant-originated flavone with anti-inflammatory activ-
ity. Thus, we found that the 30,40-dichloro-7-methoxy-
The bioassays were performed according to the pub-
lished procedure.6 RAW 264.7 cells obtained from
American Type Culture Collection were cultured with
DMEM supplemented with 10% FBS and 1% CO2 at
37 ꢁC and activated with LPS (Lipopolysaccharide,
Escherichia coli O127:B8). Briefly, cells were plated in
Scheme 1. Synthesis of flavone analogues: (a) dimethyl sulfate, K2CO3, acetone, reflux, >90%; (b) aryl aldehydes, KOH, MeOH, rt, 70–90%; (c) I2,
DMSO, heat, 60–70%; (d) BBr3, CH2Cl2, 0 ꢁC to rt, 80–85%; for 8c, AlCl3, CH2Cl2, rt, 83%.

T. T. Dao et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1165–1167
1167
Table 1. Inhibition of COX-2 catalyzed PGE2 production from LPS-
induced RAW 264.7 cells by chrysin derivativesa
In summary, we prepared chrysin derivatives modified
on A and B rings and evaluated their inhibitory activ-
ities on COX-2 catalyzed PGE2 production from LPS-
induced RAW 264.7 cells. We found that some chrysin
derivatives (5e, 6e and 7e) possessed strong inhibitory
activities. Our results are not enough to identify the
structural requirement of chrysin for better biological
activity at this moment, however, 30,40-dichloro sub-
stituents on the B ring of flavones enhance biological
activity based on this experiment. Further SARs study
on the A ring and the B ring of chrysin is currently
under investigation.
Compd
% Inhibition of PGE2
productionb
4a
4b
4c
4d
4e
5a
5b
5c
5d
5e
6a
6b
6c
6d
92.2d
69.0d
38.9d
45.6d
46.2d
36.2d
87.2d
84.2d
92.1d
96.7d
28.0d
29.9d
33.0d
4.9d
Acknowledgements
We thank Ministry of Health and Social Welfare of
Korea for financial support (Grant 01-PJ2-PG6-
01NA01-0002). We also thank KNU Institute of
Phamacal Research for the use of NMR and bioassay
facilities.
6e
7a
7b
7c
7d
7e
8a
8b
8c
8d
8e
NS-398c
Chrysin
Wogonin
98.8
70.8
19.9
9.8
8.9
97.4
45.0
72.4
84.5
50.4
0.0
98.3
11.0
98.8
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a All compounds were treated at 10 mM. Treatment of LPS to RAW
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b % inhibition=100ꢂ[1ꢀ(PGE2 of LPS with the flavones treated
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c NS-398, N-(2-cyclohexyloxy)-4-nitrophenylmethanesulfonamide, was
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Table 2. IC50 values of the chrysin derivatives on COX-2
Compd
4a 5d
5e
6e
7e
NS-398 Wogonin
0.05 1.08
IC50 (mM) 0.5 0.5 0.1–0.5 0.1–0.5 0.1–0.5
flavone (5e), 30,40-dichloro-5-methoxyflavone (6e) and
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tion from LPS-induced RAW 264.7 cells. These results
may imply that the 30,40-dichloro substituents contribute
to the bioactivity regardless of the substitution patterns
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