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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%.