A chrysin analog exhibited strong inhibitory activities against both PGE2 and NO production

Article abstract of DOI:10.1016/j.ejmech.2011.04.044

A number of 8-substituted chrysin analogs have been prepared and evaluated for their inhibitory activities of cyclooxygenase-2 catalyzed prostaglandin E₂ and iNOS-mediated NO production. Analogs were prepared via Suzuki-Miyaura C-C cross coupli

Full text of DOI:10.1016/j.ejmech.2011.04.044

European Journal of Medicinal Chemistry 46 (2011) 4657e4660
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
A chrysin analog exhibited strong inhibitory activities against both PGE2 and NO
production
*
Haiyan Che, Hyun Lim, Hyun Pyo Kim, Haeil Park
College of Pharmacy, Kangwon National University, 192-1 Hyoja-2-dong, Chuncheon 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 8-substituted chrysin analogs have been prepared and evaluated for their inhibitory
activities of cyclooxygenase-2 catalyzed prostaglandin E2 and iNOS-mediated NO production. Analogs
were prepared via SuzukieMiyaura CeC cross coupling reaction of 8-iodo-5,7-dimethoxyflavone and
alkyl/areneboronic acids as a key reaction. Among the analogs tested, 5,7-dihydroxy-8-(pyridine-4-yl)
flavone (3d) showed strong biological activities against COX-2 catalyzed PGE2 and iNOS-mediated NO
production from LPS-induced RAW 264.7 cells compared to those of chrysin.
Received 28 November 2010
Received in revised form
13 April 2011
Accepted 13 April 2011
Available online 21 April 2011
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
8-Substituted chrysin analogs
Wogonin
Inflammation
PGE2
NO
SuzukieMiyaura reaction
1. Introduction
bioactivity regardless the number and position of substituents.
Therefore, series of flavone analogs with various substituents at 8-
Chrysin (5,7-dihydroxyflavone) is a naturally occurring flavo-
noid that possesses a very broad spectrum of biological activities
[1e4]. Action mechanism of chrysin has been known as an agonist
position were prepared and evaluated their biological effects on
inflammation mediators [7e9]. As a continuing study in this point
of the view, 8-substituted chrysin analogs were synthesized as
congeners of chrysin and evaluated for their inhibitory activities
against COX-2 catalyzed PGE2 and iNOS-mediated NO production
from LPS-induced RAW 264.7 cells Fig. 1.
of PPAR-g which results in down regulation of the key pro-
inflammatory enzymes, inducible nitric oxide synthase (iNOS)
and cyclooxygenase-2 (COX-2) [5]. From comparison between the
chemical structures and anti-inflammatory activities of several
natural flavonoids such as baicalein, oroxylin A and wogonin, the
extra 6- or 8-substituent group at the A-ring of the flavones
structures seems to play very important roles to possess strong
anti-inflammatory activity. Therefore, structural elaboration of A-
ring of chrysin seems to be tolerable to the bioactivity. As an
attempt to discover novel synthetic flavones with potent anti-
inflammatory activity, recently we have synthesized 6,8-disubsti-
tuted chrysin derivatives which showed very strong inhibitory
activities of prostaglandin E2 (PGE2) production from LPS-induced
RAW 264.7 cells [6]. These results implied that introduction of
a substituent at 6- and/or 8-position is tolerable to bioactivity.
The results also implied that the electronic and steric charac-
teristics of the substituent seemed to play more important roles to
2. Chemistry
8-Substituted chrysin analogs (3ae3e) were prepared by the
synthetic pathway as shown in Scheme 1. 8-Iodo-5,7-dimethoxy-
flavone 1 was prepared following the procedure as described in the
precedent literature by us [8]. Introduction of heteroaryl and
methyl groups at 8-position was successively conducted via Suzu-
kieMiyaura carbonecarbon cross coupling reaction of 8-iodo-5,7-
dimethoxyflavone and alkyl/areneboronic acids [10]. Reaction
mixture of iodoflavone 1, boronic acids (3-furan, 3-thiophene,
3-pyridine, 4-pyridine) (2 equiv), K2CO3 (aqueous) and Pd(PPh3)4
catalyst dissolved in anhydrous DMF was stirred at room temper-
ature for 1 h under N2 gas. After 1 h, another portion of alkyl/are-
neboronic acid (1 equiv) was added to the reaction mixture and the
reaction mixture was refluxed for overnight. Purification of the
crude products by flash column chromatography provided CeC
coupled compounds 2ae2e (50e79%). Demethylation of 2ae2e
* Corresponding author. Tel.: þ82 33 250 6920; fax: þ82 33 255 7865.
E-mail address: haeilp@kangwon.ac.kr (H. Park).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.04.044

4658
H. Che et al. / European Journal of Medicinal Chemistry 46 (2011) 4657e4660
OCH3
R
HO
O
HO
O
O
HO
O
O
HO
HO
O
O
HO
O
O
H3CO
OH
O
OH
OH
OH
OH
wogonin
chrysin
Baicalein
oroxylin A
Fig. 1. Structures of chrysin, baicalein, oroxylin A, wogonin and 8-substituted chrysin analogs.
with BBr3 in CHCl3 gave 8-substituted chrysin analogs (3ae3e) in
44e81%, respectively.
From MTTassay, all the tested compounds did not show any notable
cytotoxicity. Our previous [8,9,12] and present study indicates that
the functional group at the 8-position of flavones seems to play
very important roles for the bioactivity. Further pharmacological
study of 5,7-dihydroxy-8-(pyridine-4-yl)flavone (3d) is currently
under investigation.
3. Pharmacology
3.1. RAW 264.7 cell culture and measurement of NO and PGE2
concentrations
5. Experimental
RAW 264.7 cells obtained from American Type Culture Collec-
tion (ATCC, Rockville, MD) were cultured in DMEM supplemented
with 10% FBS and 1% antibiotics under 5% CO2 at 37 ꢀC based on the
previously described procedures [3]. Briefly, cells were plated in 96-
well plates (2 ꢁ 105 cells/well). After pre-incubation for 2 h, the test
5.1. Materials and methods
All chemicals were obtained from commercial suppliers, and
used without further purification. All solvents used for reaction
were freshly distilled from proper dehydrating agent under
nitrogen gas. All solvents used for chromatography were purchased
and directly applied without further purification. 1H NMR spectra
were recorded on a Bruker Avance 300 (300 MHz), Bruker DPX 400
(400 MHz) and Bruker Avance 600 (150 MHz) spectrometers.
Chemical shifts are reported in parts per million (ppm) downfield
relative to tetramethylsilane as an internal standard. Peak splitting
patterns are abbreviated as m (multiplet), s (singlet), bs (broad
singlet), d (doublet), bd (broad doublet), t (triplet), dd (doublet of
doublets) and ddd (doublet of double doublet). 13C NMR spectra
were recorded on a Bruker DPX 400 (100 MHz) and Bruker Avance
600 (150 MHz) spectrometer, fully decoupled and chemical shifts
are reported in parts per million (ppm) downfield relative to tet-
ramethylsilane as an internal standard. Melting points were
recorded on a Fisher-Johns microscopic scale melting point appa-
ratus. EI mass spectra were recorded on an Autospec M363.
Analytical thin-layer chromatography (TLC) was performed using
commercial glass plate with silica gel 60F254 purchased from Merck.
Chromatographic purification was carried out by flash chroma-
tography using Kieselgel 60 (230e400 mesh, Merck).
compounds and LPS (1 mg/ml) were added and incubated for 24 h.
From the media, NO and PGE2 concentrations were measured. For
determination of NO concentration, the stable conversion product
of NO, nitrite (NO2ꢂ), was measured using Griess reagent and
optical density was checked at 550 nm. PGE2 concentration in the
medium was measured using ELISA kit for PGE2 (Cayman Chem.
Co.) according to the manufacturer’s recommendation. Cell viability
was assessed with MTT assay as described previously [11]. All tested
compounds showed no or less than 10% reduction of MTT assay at
the tested concentrations, indicating that they were not signifi-
cantly cytotoxic to RAW 264.7 cells in the presence or absence of
LPS. Therefore, the inhibition of PGE2 production by 8-substituted
chrysin analogs might be not associated with their cytotoxicity. The
inhibitory activities of synthetic flavones on COX-2 catalyzed PGE2
and NO production from LPS-induced RAW 264.7 cells were esti-
mated and the results are shown in Table 1.
4. Results and discussion
As shown in Table 1, only 5,7-dihydroxy-8-(pyridine-4-yl)
flavone (3d) showed strong biological activities against both COX-2
catalyzed PGE2 production and iNOS-mediated NO production from
LPS-induced RAW 264.7 cells, comparable to those of wogonin, the
most potent natural flavones. Other tested chrysin analogs (3a, 3b,
3c and 3e) exhibited low to moderate inhibitory activities against
both PGE2 and NO production. The present results of the analog 3d
are somewhat different from our previous results that synthetic
chrysin analogs generally exhibited strong inhibitory activities only
against COX-2 catalyzed PGE2 production and generally little to low
inhibitory activities against iNOS-mediated NO production [7].
5.2. General synthetic conditions for CeC cross coupling reaction
(SuzukieMiyaura reaction)
To a solution of 8-iodo-5,7-dimethoxyflavone (1, 5 mmol) and
alkyl/areneboronic acid derivatives (6 mmol) in 20 mL of dry N,
N-dimethylformamide (DMF) was added Pd (PPh3)4 (0.2 mmol).
The resulting mixture was degassed and stirred at ambient
Table 1
Inhibitory activities against COX-2 catalyzed PGE2 and iNOS-mediated NO produc-
tion from LPS-induced RAW 264.7 cells.
Flavonesa
% Inhibition
R
I
MeO
O
MeO
HO
O
R-B(OH)2, Pd(PPh3)4
BBr3, CHCl3
reflux
PGE2 productionb
NO production
K CO (aq), DMF
2
3
3a
3b
3c
3d
3e
Chrysin
26.7
31.4
48.1
92.4
44.3
74.0
8.3
7.6
16.7
29.1
3.7
OMeO
2
OMeO
1
R
O
O
S
N
c
a
b
8.9
N
CH
3
a
All compounds were treated at 10
m
M. Treatment of LPS to raw cells increased
O
OH
e
d
PGE2 production (10 mM) from the basal level of 0.5 mM.
3
b
% Inhibition ¼ 100 ꢁ [1 ꢂ (PGE2 of LPS with the flavones treated group ꢂ PGE2 of
Scheme 1. Synthetic pathway for 8-substituted chrysin analogs.
the basal)/(PGE2 of LPS treated group ꢂ PGE2 of the basal)].

H. Che et al. / European Journal of Medicinal Chemistry 46 (2011) 4657e4660
4659
temperature for 20 min before adding saturated aqueous K2CO3
solution (10 mL). The mixture was degassed again and then stirred
under nitrogen gas for 1 h. Another portion of alkyl/areneboronic
acid (10 mmol) was added and the reaction mixture was heated at
80 ꢀC for overnight. After cooling to room temperature, the mixture
was diluted with dichloromethane (40 mL) and water (20 mL), the
organic phase was separated, washed with water and dried over
magnesium sulfate, filtered and evaporated in reduced pressure.
The residue was purified by flash column to obtain CeC cross
coupled products (2ae2e).
35), 320 (100), 319 (30), 293 (74), 292 (52), 291 (70), 279 (39); HR-
MS (C19H12O5) Calcd: 320.0685. Found: 320.0684.
5.3.2. 5,7-Dihydroxy-8-(thiophen-3-yl)flavone (3b)
Yellow solid, 48%; mp: 215e216 ꢀC, 1H NMR (400 MHz,
CDCl3 þ MeOD):
d
7.72e7.74 (m, 2H, J ¼ 8.3 Hz, 1.6 Hz, H20, H60),
7.43e7.53 (m, 5H, H30, H40, H50, H2-thiophen, H4-thiophen),
7.35e7.36 (d, 1H, J ¼ 5.0 Hz, H5-thiophen), 6.72 (s, 1H, H3), 6.44 (s,
1H, H6); 13C NMR (100 MHz, CDCl3 þ MeOD):
d 183.31 (C-4), 164.42
(C-7),161.86 (C-2),160.76 (C-5),155.11 (C-9),132.22 (C-1-thiophen),
131.36 (C-5-thiophen), 131.12 (C-10), 130.25 (C-40), 129.41 (C-30,
C-50), 126.65 (C-20, C-60), 125.56 (C-4-thiophen), 124.99 (C-2-thio-
phen), 105.27 (C-3, C-8), 104.50 (C-10), 99.55 (C-6); m/z 338 (Mþ,
44), 337 (69), 336 (100), 308 (27), 234 (51), 201 (54), 57 (66); HR-
MS (C19H12O4S) Calcd.: 336.0458. Found: 336.0277.
5.2.1. 8-(Furan-3-yl)- 5,7-dimethoxyflavone (2a)
White solid, 76%; 1H NMR (300 MHz, CDCl3):
d 7.76e7.79 (m, 3H,
H4-furan, H20, H60), 7.58e7.60 (t, 1H, J ¼ 1.7 Hz, H5-furan),
7.42e7.50 (m, 3H, H30, H40, H50), 6.77e6.78 (d, 1H, J ¼ 1.3 Hz, H2-
furan), 6.71 (s, 1H, H3), 6.52 (s, 1H, H6), 4.06 (s, 3H, OMe), 3.98 (s,
3H, OMe).
5.3.3. 5,7-Dihydroxy-8-(pyridin-3-yl)flavone (3c)
Yellow solid, 80%; mp>300 ꢀC, 1H NMR (600 MHz, DMSO-d6):
5.2.2. 5,7-Dimethoxy-8-(thiophen-3-yl)flavone (2b)
d 13.17 (s, 1H, OH), 11.53 (s, 1H, OH), 9.21 (s, 1H, H2-pyridine),
White solid, 50%; 1H NMR (300 MHz, CDCl3):
d
7.63e7.66 (m, 2H,
8.95e8.96 (d, 1H, J ¼ 5.5 Hz, H4-pyridine), 8.78e8.79 (d, 1H,
J ¼ 8.0 Hz, H6-pyridine), 8.18e8.20 (dd, J ¼ 7.9 Hz, 5.7 Hz, H5-
pyridine), 7.74e7.75 (d, 2H, J ¼ 7.5 Hz, H20, H60), 7.56e7.58 (t, 1H,
J ¼ 7.5 Hz, H40), 7.48e7.50 (t, 2H, J ¼ 7.6 Hz, H30, H50), 7.07 (s, 1H,
H20, H60), 7.37e7.47 (m, 5H, H30, H40, H50, H2-thiophen, H4-thio-
phen), 7.27e7.29 (dd, 1H, J ¼ 3.7 Hz, 2.6 Hz, H5-thiophen), 6.72 (s,
1H, H3), 6.52 (s, 1H, H6), 4.07 (s, 3H, OMe), 3.94 (s, 3H, OMe).
H3), 6.52 (s, 1H, H6); 13C NMR (150 MHz, DMSO-d6):
d 181.97 (C-4),
5.2.3. 5,7-Dimethoxy-8-(pyridin-3-yl)flavone (2c)
163.40 (C-7), 161.81 (C-2), 161.40 (C-5), 154.34 (C-9), 147.20 (C-2-
pyridine), 144.00 (C-4-pyridine), 141.19 (C-6-pyridine), 131.98 (C-1-
pyridine), 131.11 (C-10), 130.48 (C-5-pyridine), 129.06 (C-30, C-50),
126.27 (C-20, C-40, C-60), 105.41 (C-3), 103.96 (C-10), 101.59 (C-8),
98.63 (C-6); m/z 332 (Mþ, 84), 331 (100), 330 (72), 303 (68), 229
(73), 228 (95), 77 (78), 70 (89); HR-MS (C20H13NO4) Calcd.:
331.0845. Found: 331.0767.
White solid, 54%; 1H NMR (300 MHz, CDCl3):
d 8.71 (d, 1H,
J ¼ 1.5 Hz, H2-pyridine), 8.66e8.68 (dd, 1H, J ¼ 4.9 Hz, 1.7 Hz, H4-
pyridine), 7.77e7.81 (ddd, 1H, J ¼ 7.8 Hz, 2.0 Hz, H6-pyridine),
7.49e7.52 (m, 2H, H20, H60), 7.33e7.46 (m, 4H, H30, H40, H50, H5-
pyridine), 6.69 (s, 1H, H3), 6.55 (s, 1H, H6), 4.08 (s, 3H, OMe), 3.92
(s, 3H, OMe).
5.2.4. 5,7-Dimethoxy-8-(pyridin-4-yl)flavone (2d)
5.3.4. 5,7-Dihydroxy-8-(pyridin-4-yl)flavone (3d)
White solid, 79%; 1H NMR (300 MHz, CDCl3):
d
8.73e8.75 (d, 2H,
Yellow solid, 53%; mp>300 ꢀC, 1H NMR (400 MHz, DMSO-d6):
J ¼ 5.6 Hz, H3-pyridine, H5-pyridine), 749e7.53 (m, 2H, H20, H60),
7.34e7.46 (m, 5H, H30, H40, H50, H2-pyridine, H6-pyridine), 6.70 (s,
1H, H3), 6.53 (s, 1H, H6), 4.08 (s, 3H, OMe), 3.92 (s, 3H, OMe).
d 13.12 (s, 1H, 5eOH), 11.24 (s, 1H, 7eOH), 8.70e8.73 (m, 2H, H3-
pyridine, H5-pyridine), 7.76e7.78 (m, 2H, H2-pyridine, H6-pyri-
dine), 7.47e7.59 (m, 5H, Ph), 7.05 (s, 1H, H3), 6.45 (s, 1H, H6); 13C
NMR (100 MHz, DMSO-d6):
d 182.60 (C-4), 163.71 (C-7), 161.79 (C-
5.2.5. 5,7-Dimethoxy-8-methylflavones (2e)
2), 161.49 (C-5), 154.50 (C-9), 149.53 (C-3-pyridine, C-5-pyridine),
140.64 (C-1-pyridine), 132.43 (C-10), 131.02 (C-40), 129.48 (C-30,
C-50), 126.77 (C-20, C-60), 126.60 (C-2-pyridine, C-6-pyridine),
105.84 (C-8), 105.49 (C-3), 104.39 (C-10), 99.16 (C-6); m/z 332 (Mþ,
84), 331 (100), 330 (73), 303 (67), 268 (81), 229 (72), 228 (95); HR-
MS (C20H13NO4) Calcd.: 331.0845. Found: 331.0733
White solid, 50%; 1H NMR (300 MHz, CDCl3):
d 7.89e7.92 (m, 2H,
H20, H60), 7.50e7.52 (m, 3H, H30, H40, H50), 6.68 (s, 1H, H3), 6.42 (s,
1H, H6), 4.00 (s, 3H, OMe), 3.96 (s, 3H, OMe), 2.34 (s, 3H, Me).
5.3. Demethylation conditions
5.3.5. 5,7-Dihydroxy-8-methylflavones (3e)
To a solution of compound 2 in dry chloroform was added cold
diluted solution of BBr3 (5e10 equiv) in dry chloroform at 0 ꢀC. The
reaction mixture was stirred at 0 ꢀC for 30 min and refluxed for
overnight. The reaction mixture was cooled to room temperature
and excess BBr3 was destroyed by adding MeOH. The solid precipi-
tated out was filtered and the solid residue was washed with satu-
rated aqueous Na2CO3 solution and water, filtered and washed with
MeOH and CHCl3 to get 8-substituted chrysin analogs (3ae3e).
Yellow solid, 81%; mp:241 ꢀC, 1H NMR (600 MHz,
CDCl3 þ MeOD):
H30, H40, H50), 6.68 (s, 1H, H3), 6.34 (s, 1H, H6), 2.20 (s, 3H, Me). 13C
186.94 (C-4), 167.90 (C-7), 166.17
d
7.94e7.95 (m, 2H, H20, H60), 7.54e7.58 (m, 3H,
NMR (150 MHz, CDCl3 þ MeOD):
d
(C-2), 162.77 (C-5), 159.35 (C-9), 135.73 (C-10), 135.45 (C-40), 133.01
(C-30, C-50), 130.15 (C-20, C-60), 108.74 (C-3), 108.53 (C-10), 107.07 (C-
6), 102.56 (C-8), 11.44 (Me); m/z 269 (Mþ, 19), 268 (100), 267 (46),
239 (8), 165 (9), 138 (18), 120 (8); HR-MS (C16H12O4) Calcd:
268.0736. Found: 268.0730.
5.3.1. 8-(Furan-3-yl)-5,7-dihythoxyflavone (3a)
Yellow solid, 44%; mp: 140e141 ꢀC, 1H NMR (400 MHz, DMSO-
Acknowledgments
d6):
d 13.06 (s, 1H, 5eOH), 7.98 (s, 1H, H4-furan), 7.94e7.96 (m, 2H,
H20, H60), 7.80e7.81 (t, 1H, J ¼ 1.6 Hz, H5-furan), 7.53e7.59 (m, 3H,
This research was supported by Basic Science Research Program
through the National Research Foundation of Korea (NRF) funded
by the Ministry of Education, Science, and Technology (Grant 2010-
C1007296: Development of Anti-inflammatory Synthetic Biflavo-
noids). The authors thank to Pharmacal Research Institute and
Central Laboratory of Kangwon National University for the use of
analytical instruments and bioassay facilities.
H30, H40, H50), 7.01 (s, 1H, H3), 6.87e6.88 (d, 1H, J ¼ 1.3 Hz, H2-
furan), 6.42 (s, 1H, H6); 13C NMR (100 MHz, DMSO-d6):
d 183.11
(C-4), 164.49 (C-7), 162.67 (C-2), 160.84 (C-5), 155.07 (C-9), 143.07
(C-4-furan, C-2-furan), 132.84 (C-10), 131.81 (C-40), 130.00 (C-30,
C-50), 127.34 (C-20, C-60), 115.72 (C-1-furan), 113.27 (C-5-furan),
106.08 (C-3), 104.97 (C-10), 100.39 (C-8), 99.70 (C-6); m/z 321 (Mþ,

4660
H. Che et al. / European Journal of Medicinal Chemistry 46 (2011) 4657e4660
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