Synthesis of biflavones having a 6-O-7'' linkage and effects on cyclooxygenase-2 and inducible nitric oxide synthase

Article abstract of DOI:10.1016/j.bmcl.2008.11.017

In order to establish anti-inflammatory potential of biflavonoids, 17 biflavone derivatives having a 6-O-7″ linkage were synthesized and their effects on cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) were evaluated. The basic molecul

Full text of DOI:10.1016/j.bmcl.2008.11.017

Bioorganic & Medicinal Chemistry Letters 19 (2009) 74–76
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of biflavones having a 6-O-7’’ linkage and effects on
cyclooxygenase-2 and inducible nitric oxide synthase
Haiyan Che a, Byung Kyu Park a, Hyun Lim a, Hyun Pyo Kim a, Hyeun Wook Chang b, Jin-Hyun Jeong c,
Haeil Park a,
*
a College of Pharmacy, Kangwon National University, Chunchon, 200-701, Republic of Korea
b College of Pharmacy, Yeungnam University, Gyongsan 712-749, Republic of Korea
c College of Pharmacy, Kyung Hee University, Seoul 130-701, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
In order to establish anti-inflammatory potential of biflavonoids, 17 biflavone derivatives having a 6-O-700
linkage were synthesized and their effects on cyclooxygenase-2 (COX-2) and inducible nitric oxide syn-
thase (iNOS) were evaluated. The basic molecule (6-O-700 biflavone) potently inhibited COX-2-mediated
Article history:
Received 6 May 2008
Revised 21 September 2008
Accepted 7 November 2008
Available online 12 November 2008
PGE2 production (IC50: < 2 lM), being less active on iNOS-mediated NO production (IC50: > 50 lM) from
lipopolysaccharide-treated RAW 264.7 cells, a mouse macrophage cell line. Generally, the hydroxyl/
methoxyl substitution(s) on the basic biflavone (6-O-700) reduced the inhibitory activity of PGE2 produc-
tion, while the effects on NO production were varied. It is suggested that the basic biflavone (6-O-700) may
have a potential for new anti-inflammatory agent.
Dedicated to Prof. Moon Woo Chun on the
occasion of his retirement.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
6-O-700 biflavonoid
Cyclooxygenase
Nitric oxide synthase
Anti-inflammation
Inducible isoforms of cyclooxygenase (COX-2) and nitric oxide
synthase (iNOS) produce large amount of prostaglandins (PG)
and nitric oxide (NO), respectively, which have an important role
to provoke and maintain inflammatory response.1 The compounds
having inhibitory capacity of these enzymes may show anti-
inflammatory effect.
yflavone analogs as key intermediates. 6-Halogenoflavone and
7-hydroxyflavone analogs were prepared from 5-bromo-2-
hydroxyacetophenone following the general procedures as
described in the earlier publications in our group.11 The mixture
of a 6-bromoflavone analog (100 mol%), a 7-hydroxyflavone analog
(100 mol%), CuI (20 mol%), N,N-dimethylglycine (8 mol%), cesium
carbonate (8 mol%) in dry dioxane was reacted at 100 °C for 24 h.
Purification of the crude product by column chromatography
(CHCl3:MeOH = 100 ꢀ 20:1) gave biflavones (671-1, 672-1, 673-1,
674-1, 675-1, 676-1, 677-1 and 678-1) as white solid in 10–16%
yields, respectively. Demethylation of the C–O–C biflavones with
BBr3 in CHCl3 (2.0–8.0 equiv) at 0 °C provided crude products. The
crude products were purified by recrystallization in MeOH gave
the desired biflavones (671-2, 672-2, 673-2, 674-2, 675-2, 676-2,
677-2 and 678-2) as pale yellow solid in 65–75% yields, respectively
(Scheme 1).
Biflavonoids belong to flavonoid family and they are dimers of
monomeric flavonoid. Many different chemical structures of bifl-
avonoids have been isolated from plants and some of them were
found to possess anti-inflammatory activity in vivo and in vitro.
Their anti-inflammatory action mechanisms include inhibition of
mast cell histamine release,2 lymphocyte proliferation3 and phos-
pholipase A2 (PLA2),4 and down-regulation of proinflammatory
molecule expression such as COX-2 and iNOS.5,6 Recently, several
C–C biflavones were chemically synthesized and some of them
showed promising anti-inflammatory activity.7,8 To extend these
findings and to find structurally optimized derivatives, 17 C–O–C
biflavone derivatives having 6-O-700 linkage (Fig. 1) were prepared
and their effects on cyclooxygenase-2 (COX-2) and inducible nitric
oxide synthase (iNOS) were evaluated.
These biflavonoids were evaluated for their anti-inflammatory
activity as followings. RAW 264.7 cells obtained from American
Type Culture Collection (ATCC) were cultured with DMEM supple-
mented with 10% FBS and 1% antibiotics (penicillin, 100 U/ml and
The 6-O-700 biflavones were prepared via intermolecular Ull-
mann ether syntheses9,10 of 6-bromoflavone analogs and 7-hydrox-
streptomycin, 100 lg/ml) under 5% CO2 at 37 °C and activated with
LPS based on the previously described procedures.12 Briefly, cells
were plated in 96-well plates (2 ꢁ 105 cells/well). After pre-incu-
bation for 2 h, test compounds and LPS (1 lg/ml) were added
* Corresponding author.
and incubated for 24 h, unless otherwise specified. Test com-
E-mail address: haeilp@kangwon.ac.kr (H. Park).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.11.017

H. Che et al. / Bioorg. Med. Chem. Lett. 19 (2009) 74–76
75
R2O
R2O
O
O
O
O
O
O
O
O
O
O
O
O
OR1
OR1
O
O
O
OR3
671-1: R1 = R2 = Me
672-1: R1 = R2 = R3 = Me
672-2: R1 = R2 = R3 = H
6-O-7"
671-2: R1 = R2 = H
R2O
R1O
R1O
O
O
O
O
O
O
O
O
OR1
O
O
O
O
O
O
O
R3O
OR4
OR2
675-1: R1 = R2 = Me
673-1: R1 = R2 = R3 = R4 = Me
673-2: R1 = R2 = R3 = R4 = H
674-1: R1 = Me
674-2: R1 = H
675-2: R1 = R2 = H
R1O
O
O
O
O
O
O
O
O
O
OR1
OR1
O
O
O
O
O
O
OR2
R2O
OR3
677-1: R1 = Me
676-1: R1 = R2 = R3 = Me
676-2: R1 = R2 = R3 = H
678-1: R1 = R2 = Me
678-2: R1 = R2 = H
677-2: R1 = H
Figure 1. Synthetic biflavones with 6-O-700 linkage.
O
O
(RO)m
(RO)m
O
CsCO , CuI
3
N,N-dimethylglycine
O
+
HO
O
dioxane, reflux
(OR)n
O
O
(OR)n
Br
O
(m,n=0, 1, 2)
O
Scheme 1. Typical intermolecular Ullmann ether syntheses for 6-O-700 biflavones.
Table 1
pounds dissolved in DMSO were diluted with serum-free DMEM
into appropriate concentrations. Final concentration of DMSO
was adjusted to 0.1% (v/v). Cell viability was assessed with MTT as-
say as described previously.13 For determination of NO concentra-
tion, 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 manufac-
turer’s recommendation. N-[2-Cyclohexyloxy-4-nitrophenyl]-
methane sulfonamide (NS-398) was obtained from Biomol
(Plymouth Meeting, PA). 2-Amino-5,6-dihydro-6-methyl-4H-1,3-
thiazine hydrochloride (AMT) was purchased from Tocris Cookson,
Ltd (UK). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) and lipopolysaccharide (LPS, Escherichia coli
0127:B8) were purchased from Sigma Chem. (St. Louis, MO). Lipo-
fecAMINE PLUS, DMEM and other cell culture reagents including
FBS were products of Gibco BRL (Grand Island, NY). Protein assay
kit was purchased from Bio-Rad Lab. (Hercules, CA).
Inhibition of PGE2 and NO production from LPS-treated RAW264.7 cells.
Compound14
IC50 (lM)
PGE2 production
NO production
6-O-700
671-1*
671-2
672-1
672-2
673-1
673-2*
674-1
674-2*
675-1
675-2
676-1
676-2*
677-1
677-2
678-1
678-2
<2
>50
—
—
>50
>50
>50
10–50
—
—
—
—
>50
50
2–10
>50
>50
>50
50
10–50
2–10
—
—
>50
>50
>50
>50
>50
—
—
—
—
10–50
>50
It is well established that RAW 264.7 cells, a mouse macrophage
cell line, induce COX-2 and iNOS which produce large amounts of
PGE2 and NO, respectively, when activated with LPS. As in Table 1,
IC50 values were calculated by linear regression analysis using the inhibition data of
three concentrations of test compounds (2, 10 and 50 M). PGE2 and NO concen-
trations of the control group without LPS were 1.0 0.3 nM and 1.2 0.1 M,
respectively, while the LPS-treated control group produced 105.4 3.2 nM of PGE2
and 32.5 1.8 M of NO (arithmetic mean SD, n = 3). As reference compounds,
NS-398 (selective COX-2 inhibitor) showed 99.3% inhibition of PGE2 production at
l
l
LPS treatment (1 lg/ml) to RAW cells for 24 h increased PGE2 and
NO production. Under this condition, synthetic 6-O-700 biflavones
showed more or less inhibitory activity on these two parameters.
For COX-2-mediated PGE2 production, the basic molecule (6-O-700
biflavone without any substitution) showed most potent inhibitory
activity. The hydroxyl/methoxyl substitutions on the basic biflavone
abolished/diminished inhibitory activity. Only 671-1 and 674-1
possessed slightly less inhibitory activity compared to the basic
molecule. For iNOS-mediated NO production, all compounds tested
did not show potent inhibition. Some of them possessed weak inhi-
l
0.1
1.0
l
l
M and AMT (iNOS inhibitor) showed 92.5% inhibition of NO production at
M. *MTT bioassay indicated that 671-1, 673-2, 674-2 and 676-2 reduced cell
viability of 2%, 50%, 3% and 9% at 50 lM, respectively.
bition of NO production. Among the derivatives, 678-2 possessed
slightly higher inhibitory activity than the basic molecule. It is sig-

76
H. Che et al. / Bioorg. Med. Chem. Lett. 19 (2009) 74–76
nificant to note that the potent PGE2 inhibitory biflavonoids are
methoxylated derivatives (671-1, 673-1 and 674-1) among the
substituted derivatives while meaningful NO inhibitory biflavonoid
is hydroxylated derivative (678-2).
References and notes
1. Gallin, J. I.; Snyderman, R. Overview. In Inflammation: Basic Principles and
Clinical Correlates; Gallin, J. I., Snyderman, R., Eds., third ed.; Lippincott Williams
& Wilkins: Philadelphia, 1999; pp 1–4.
In the present investigation, anti-inflammatory activity of the
biflavonoids having a 6-O-7000 linkage with/without substituents
at C-20, 2000, 40 and 4000 was examined in vitro. Although various sub-
stitutions were tried, their anti-inflammatory activities were not
satisfactory. Only the basic molecule has the potential for new
ant-inflammatory agent. The substitutions at different position
should be tried further.
2. Amella, M.; Bronner, F.; Hagg, M.; Anton, R.; Landry, Y. Planta Med. 1985,
16.
3. Lee, S. J.; Choi, S. H.; Son, K. H.; Chang, H. W.; Kang, S. S.; Kim, H. P. Life Sci. 1995,
57, 551.
4. Chang, H. W.; Baek, S. H.; Chung, K. W.; Son, K. H.; Kim, H. P.; Kang, S. S.
Biochem. Biophys. Res. Commun. 1994, 205, 843.
5. Kwak, W.-J.; Han, C. K.; Son, K. H.; Chang, H. W.; Kang, S. S.; Park, B. K.; Kim, H.
P. Planta Med. 2002, 68, 316.
6. Cheon, B. S.; Kim, Y. H.; Son, K. H.; Chang, H. W.; Kang, S. S.; Kim, H. P. Planta
Med. 2000, 66, 596.
7. Chen, J.; Chang, H. W.; Kim, H. P.; Park, H. Bioorg. Med. Chem. Lett. 2006, 16,
2373.
8. Park, H.; Kim, Y. H.; Chang, H. W.; Kim, H. P. J. Pharm. Pharmacol. 2006, 58, 1661.
9. Sawyer, J. S. Tetrahedron 2000, 56, 5045.
10. Frlan, R.; Kikelj, D. Synthesis 2006, 14, 2271.
11. Dao, T. T.; Chi, Y. S.; Kim, J.; Kim, H. P.; Kim, S.; Park, H. Bioorg. Med. Chem. Lett.
2004, 14, 1165.
12. Chi, Y. S.; Cheon, B. S.; Kim, H. P. Biochem. Pharmacol. 2000, 61, 1195.
13. Mossman, T. J. Immunol. Methods 1983, 65, 55.
14. Kim, H. P.; Park, H.; Chang, H. W. Korean Patent Application No. 10-2006-
005964, 2006.
Acknowledgments
The present investigation was supported by Grant R01-2004-
000-10134-0 from the Basic research program of the Korea Science
and Engineering Foundation and post-BK21 project. The authors
thank the Pharmacal Research Institute and the Central Laboratory
of Kangwon National University for the use of analytical
instruments.