Oroxylin A analogs exhibited strong inhibitory activities against iNOS-mediated nitric oxide (NO) production

Article abstract of DOI:10.1016/j.bmcl.2012.01.135

A number of oroxylin A analogs were prepared and evaluated for their inhibitory activities against iNOS-mediated nitric oxide (NO) production from LPS-stimulated BV2 cells. The analogs were synthesized from purchased 2′-hydroxy-4,5,6-trimethoxyacetophenone and aldehydes in 3 steps. Among the tested compounds, several analogs (3b, 3c, 3d, 3f) exhibited strong inhibitory activities. Especially, the analog with 4-nitrophenyl group (3b) showed stronger inhibitory activity (IC₅₀ = 4.73 μM) than that of wogonin (IC₅₀ = 7.80 μM).

Full text of DOI:10.1016/j.bmcl.2012.01.135

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2534–2535
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Bioorganic & Medicinal Chemistry Letters
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Oroxylin A analogs exhibited strong inhibitory activities against
iNOS-mediated nitric oxide (NO) production
Tuan-Ahn. N. Pham a, Haiyan Che a, Phuong-Thuy T. Phan a, Jae-Won Lee b, Sung-Soo Kim b, Haeil Park a,
⇑
a College of Pharmacy, Kangwon National University, Chunchon 200-701, Republic of Korea
b College of Medicine, Kangwon National University, Chunchon 200-701, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A number of oroxylin A analogs were prepared and evaluated for their inhibitory activities against iNOS-
mediated nitric oxide (NO) production from LPS-stimulated BV2 cells. The analogs were synthesized from
purchased 20-hydroxy-4,5,6-trimethoxyacetophenone and aldehydes in 3 steps. Among the tested com-
pounds, several analogs (3b, 3c, 3d, 3f) exhibited strong inhibitory activities. Especially, the analog with
Received 5 December 2011
Revised 29 January 2012
Accepted 31 January 2012
Available online 8 February 2012
4-nitrophenyl group (3b) showed stronger inhibitory activity (IC50 = 4.73
(IC50 = 7.80 M).
lM) than that of wogonin
l
Keywords:
Oroxylin A and its analogs
Wogonin
Ó 2012 Published by Elsevier Ltd.
Inhibition of NO production
Anti-inflammatory activity
Oroxylin A (5,7-dihydroxy-6-methoxyflavone) is a plant origi-
nated flavone from Scutellaria baicalensis, which has been tradition-
ally used as medicinal herb to improve memory and cognition.
Several major components of this herb were isolated and identified
as flavones such as wogonin, baicalin, baicalein, oroxylin A and so
forth.1,2 Behavior studies have demonstrated that water extract of
S. baicalensis exhibited potent anxiolysis in mice without sedative
and myorelaxant effects.3 Flavones from S. baicalensis may possess
diverse biological activities such as anxiolysis, anticonvulsion,
muscle-relaxation and sedative effects due to their binding affinity
with benzodiazepine or GABA receptors.4 Recently, oroxylin A was
reported to improve attention-deficit hyperactivity disorder
(ADHD) related behaviors in spontaneously hypertensive rats.5
Recently wogonin (5,7-dihydroxy-8-methoxyflavone), one of
major constituents of S. baicalensis, was reported to possess strong
anti-inflammatory activity.6 Wogonin and oroxylin A have similar
chemical structures except the position of methoxy group in their
structures as shown in Figure 1. The structural difference may re-
sult in different biological activities of wogonin and oroxylin A.
Our previous results showed that 6,8-disubstituted chrysin ana-
logs (5,7-dihydroxy-6,8-disubstituted flavones) retained strong
inhibitory activities against COX-2 catalyzed PGE2 and iNOS-med-
iated NO production.7
significantly reduced anti-inflammatory activity.8,9 These results
imply that the C8 methoxy group of wogonin plays very important
roles for strong anti-inflammatory activity. Oroxylin A also pos-
sesses a methoxy group at C6 of its structure. Since the C6 methoxy
group of oroxylin A was also expected to contribute for strong anti-
inflammatory activity, a number of oroxylin A analogs were prepared
and evaluated their inhibitory activities against iNOS-mediated NO
production, a major chemical mediator for inflammation.
Oroxylin A and its analogs were synthesized from commercial
starting materials in 3 steps. The reaction of 20-hydroxy-40,50,60-tri-
methoxyacetophenone, purchased aldehydes and KOH in metha-
nol yielded the corresponding chalcones in 65%–96% yields.
Reaction of chalcones (1aꢀ1i) in iodine/DMSO condition afforded
flavones (2aꢀ2i) in 81%–98% yields. Demethylation of flavones
(2aꢀ2i) in 48% HBr/AcOH produced oroxylin A (3a) and its analogs
(3bꢀ3i) in 36%–97% yields (Scheme 1).7,10
Inhibition of iNOS mediated NO production from LPS-stimu-
lated BV2 cells by oroxylin A analogs was determined according
to the published procedure.6 RAW 264.7 cells obtained from Amer-
ican Type Culture Collection were cultured in DMEM supple-
mented with 10% FBS and 1% CO2 at 37 °C and activated with
LPS. Briefly, cells were plated in 96-well plates (2 ꢁ 105 cells/well).
Oroxylin A analog and LPS (200 ng/mL) were added and incubated
for 24 h. Cell viability was assessed with MTT assay based on the
experimental procedures described previously. All experiments
were carried out at least twice and they gave similar results. The
inhibitory activities of analogs on iNOS mediated NO production
from LPS-stimulated BV-2 cells were estimated and the results
are shown in Table 1.
These results showed that di-substitutions at C6 and C8 of chry-
sin were tolerable for anti-inflammatory activity. Deletion of the
methoxy group at C8 of wogonin produced chrysin, which exhibited
⇑
Corresponding author.
E-mail address: haeilp@kangwon.ac.kr (H. Park).
0960-894X/$ - see front matter Ó 2012 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2012.01.135

Tuan-Ahn. N. Pham et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2534–2535
2535
OCH3
R
HO
O
O
HO
O
O
HO
O
HO
R
O
O
H3CO
OH
OH
OH
O
OH
wogonin
chrysin
oroxylin A
6,8-disubstituted chrysin
Figure 1. Structures of chrysin, oroxylin A, wogonin and 6,8-disubstituted chrysin analogs.
MeO
MeO
OH
I2, DMSO,
100ºC
KOH, MeOH,
rt or
MeO
MeO
OH
+
R
R
OHC
KOH, MeOH/
THF, 40ºC
OMe O
OMe O
1
R
R
R :
HO
O
MeO
MeO
O
HBr (48%)
AcOH
(a) 4-H (b) 4-NO2 (c) 4-F
(d) 3-F (e) 2-F (f) 3-Br
(g) 2,4-F2 (h) 3,4-Cl2
(i) 3-OMe, 4-OH
MeO
reflux
OH
O
OMe O
3
2
Scheme 1. Synthesis of oroxylin A and its analogs.
In conclusion, structural modification of oroxylin A provided
synthetic oroxylin A analogs possessing strong inhibitory activities
against iNOS-mediated NO production from LPS-stimulated BV2
cells. Strong inhibitory activity was observed from the analogs
with mono-substitution at 30 or 40 position of oroxylin A regardless
of atoms or functional groups. Further SAR study with a large num-
ber of analogs possessing diverse functional groups with different
electronic and physical parameters is under progress.
Table 1
Inhibition of NO production from LPS-stimulated BV2 cells by oroxylin A analogs at
10
Ma,b,c
l
No
% of Inhibition (IC50
,
lM)
No
% of Inhibition (IC50, lM)
3a
3b
3c
3d
3e
26.0
3f
45.2 (10.84)
10.6
13.1
8.2
40.0 (7.80)
45.7 (4.73)
40.8 (8.58)
39.9 (11.42)
14.2
3g
3h
3i
wgn
Acknowledgments
a
All compounds were treated at 10
NO production from the basal level.
lM. Treatment of LPS to BV2 cells increased
b
This work was supported by the grant (2009-0072124: Synthe-
sis of Biologically Active Flavonoids Analogs) from the Korea Sci-
ence & Engineering Foundation. The authors thank to Pharmacal
Research Institute and Central Laboratory of Kangwon National
University for the use of analytical instruments and bioassay
facilities.
% inhibition = 100 ꢁ [1 ꢂ (NO of LPS with the analogs treated group ꢂ NO of the
basal)/(NO of LPS stimulated group ꢂ NO of the basal)].
c
Wogonin (wgn) was used as the reference compound.
As demonstrated in Table 1, synthetic oroxylin A (3a) exhibited
less potent inhibitory activity against iNOS-mediated NO produc-
tion from LPS-stimulated BV2 cells compared to that of wogonin.
However, several analogs (3b, 3c, 3d, 3f) showed equivalent to
more potent inhibitory activities than that of wogonin. Mono-
substitutions at 40 (3b, 3c), or 30 (3d, 3f) position increased the
bioactivities, while mono-substitution at 20 position exhibited sig-
nificantly reduced bioactivity (3e) compared to those of analogs at
30 (3d) and 40 (3c) positions. Moreover, di-substitutions at 20/40 (3g)
and 30/40 (3h, 3i) positions resulted in significantly decreased bio-
activities regardless of atoms or functional group. Our present re-
References and notes
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sults imply that anti-inflammatory activity of oroxylin
A is
largely dependent on structural modification of B ring. Also the
methoxyl group of oroxylin A is essential for strong inhibitory
activity since deletion of the methoxyl group resulted in signifi-
cantly reduced bioactivity as shown in the previous results.8,9