Inhibitory effect of synthetic C-C biflavones on various phospholipase A2s activity

Article abstract of DOI:10.1016/j.bmc.2007.07.054

Several prototypes of C-C biflavones (a-f) were synthesized and evaluated their inhibitory activity against phospholipase A₂s (PLA₂s) activity. The synthetic C-C biflavones (a-f) showed rather different inhibitory activity against va

Full text of DOI:10.1016/j.bmc.2007.07.054

Bioorganic & Medicinal Chemistry 15 (2007) 7138–7143
Inhibitory effect of synthetic C–C biflavones on
various phospholipase A2s activity
Tae Chul Moon,a Zhejiu Quan,a Jeongsoo Kim,b Hyun Pyo Kim,b Ichiro Kudo,c
Makodo Murakami,c Haeil Parkb,* and Hyeun Wook Changa,*
aCollege of Pharmacy, Yeungnam University, Gyeongsan 712-749, Republic of Korea
bCollege of Pharmacy, Kangwon National University, Chunchon 200-701, Republic of Korea
cDepartment of Health Chemistry, School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai,
Shinagawa-ku, Tokyo 142-8555, Japan
Received 3 April 2007; revised 2 July 2007; accepted 6 July 2007
Available online 22 August 2007
Abstract—Several prototypes of C–C biflavones (a–f) were synthesized and evaluated their inhibitory activity against phospholipase
A2s (PLA2s) activity. The synthetic C–C biflavones (a–f) showed rather different inhibitory activity against various PLA2s. Most
synthetic C–C biflavonoids exhibited potent and broad inhibitory activity against various sPLA2s and cPLA2 tested regardless of
their structural array. In particular, of natural and synthetic biflavonoids tested, the synthetic C–C biflavonoid (d) only showed
inhibitory activity against sPLA2 X. None of the natural and synthetic biflavonoids tested showed inhibitory activity against sPLA2
IB. Further chemical modification of these basic structures will be carried out in order to investigate the synthetic C–C biflavones
which possess more selective inhibitory activity against isozymes of PLA2.
Ó 2007 Published by Elsevier Ltd.
1. Introduction
PLA2 is a growing family of distinct enzymes that shows
different substrate specificities, cofactor requirements,
subcellular localization, and cellular functions.8 sPLA2
has a low molecular weight (14–18 kDa) with a rigid ter-
tiary structure that is configured by 6–8 disulfide
bridges. Thus far, 10 genes encoding structurally related
and enzymatically active sPLA2s have been identified in
mammals (groups IB, IIA, IIC, IID, IIF, V, X, and
XII).9 sPLA2-IIA involvement in the inflammatory pro-
cess has also been investigated by administering particu-
lar inhibitors or antibodies that are fairly specific to
Biflavonoid, flavonoid dimer connected with either a
C–C or C–O–C bond, is one of the subclasses of flavo-
noid. Although many biflavonoids have been discovered
from various plants, only a few data on their biological
and pharmacological properties have been available so
far. Previously, certain biflavonoids were reported to in-
hibit phosphodiesterase,1 lens aldose reductase,2 release
of histamine from mast cells3 and have anticancer activ-
ity.4 In addition, some C–C biflavonoids were synthe-
sized and their antimicrobial activities were reported.5
During our study to investigate potential anti-inflamma-
tory plant drugs, several biflavones such as amentoflav-
one, ginkgetin and ochnaflavone (Fig. 1) were
demonstrated to be inhibitors of group II secretory
phospholipase A2(sPLA2IIA).6 Moreover, morelloflav-
one (Fig. 1), a flavone-flavanone dimer, was also
reported to be a sPLA2 inhibitor.7
sPLA2-IIA into inflamed sites. Group
V sPLA2
(sPLA2-V) is expressed mainly in rats, the human heart,
and various mouse tissues.10,11 This enzyme may
compensate for sPLA2-IIA under particular conditions
because it is induced in many tissues by pro-inflamma-
tory agents12,13 in a similar way to sPLA2-IIA. sPLA2-
IIA and -V, whose genes are clustered on the same
chromosome locus, share several enzymatic and func-
tional features.14sPLA2 inhibitors might show favorable
anti-inflammatory activity since sPLA2 is a key enzyme
that generates arachidonic acid, which is converted to
proinflammatory eicosanoids. Indeed, some of these
biflavonoids have been found to possess promising
anti-inflammatory activity in vivo.7,15 In a previous
Keywords: Synthetic C–C biflavones; Suzuki C–C cross-coupling
reaction; Phospholipase A2(PLA2) isozymes; Inflammation.
*
Corresponding authors. Tel.: +82 33 250 6920; fax: + 82 33 255 7865
(H.P.); e-mail addresses: haeilp@kangwon.ac.kr; hwchang@yu.ac.kr
0968-0896/$ - see front matter Ó 2007 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2007.07.054

T. C. Moon et al. / Bioorg. Med. Chem. 15 (2007) 7138–7143
7139
OH
OH
O
O
OH
HO
O
O
OH
OH
O
OH
HO
O
O
OH
O
O
HO
OH
HO
Amentoflavone
Ochnaflavone
OH
OMe
OH
OH
OH
MeO
O
O
O
O
OH
O
OH
O
OH
O
O
HO
OH
HO
Ginkgetin
Figure 1. Structures of naturally occurring biflavonoids.
Morelloflavone
report, various biflavonoids were synthesized and their
PLA inhibitory activity was examined.16 In this respect,
this study evaluated several synthetic biflavonoids for
their sPLA2-IIA enzyme inhibitory activity.
and rheumatoid arthritis.20–22 The correlation between
the sPLA2-IIA levels and the disease status has led to
the development of various pharmacological agents that
can inhibit the sPLA2-IIA activity, which would be ben-
eficial for mitigating various diseases. As a result of the
ongoing efforts aimed at developing new sPLA2 inhibi-
tors from natural products through an activity-guided
isolation procedure, new sPLA2-IIA biflavonoids were
isolated from natural products.6,7 Figure 1 shows the
chemical structures of naturally occurring biflavonoids.
In an attempt to develop more potent sPLA2 inhibitors
than natural biflavonoids, various C–C type biflavo-
noids were designed and synthesized (Scheme 1 and
Fig. 2). The synthetic biflavones showed somewhat dif-
ferent inhibitory activity against sPLA2-IIA.16 Among
them, a biflavone with a C–C 40-4000 (40,4000-BF, a) linkage
showed 7 times stronger inhibition than the natural bifl-
avonoid.16 However, the effect of the synthetic biflavo-
noids on other sPLA2 isozymes was not described.
Therefore, a comparative test was carried out to deter-
mine out how the synthetic biflavonoids affect various
mammalian sPLA2s isozymes. As shown in Table 1,
the synthetic C–C biflavonoids (a–f) were generally
more potent and showed broad inhibitory activity pro-
files against the PLA2s tested compared with those of
amentoflavone, a natural C–C biflavonoid. Natural bifl-
avonoids, amentoflavone and ochnaflavone, only
showed moderate inhibitory activity against sPLA2 V,
whereas synthetic C–C biflavonoids a–f showed inhibi-
tory activity against sPLA2 V and cPLA2. In particular,
among natural and synthetic biflavonoids tested, only
2. Chemistry
Six C–C biflavones (a–f) were prepared following the
synthetic pathway (Scheme 1) and evaluated for the
inhibitory activity against various secretoryvPLA2s.16
Treatment of bromoflavones (1–4) with commercially
available bis(pinacolato)diboron in the presence of cata-
lytic PdCl2(dppf) and K2CO3 in DMF at 90 °C provided
the corresponding pinacolato boronates (I–IV) as de-
scribed in the previous literature.16 Suzuki coupling
reactions of the pinacolato boronates I–IV (120 mol%)
with bromoflavones (1,2,3,4; 100 mol%) in standard
conditions [Pd(PPh3)4 (5 mol%), NaOH (400 mol%),
and DMF-water (9:1), 100 °C] gave C–C biflavones (a,
b, c, d, e, f) in 74%, 68%, 43%, 52%, 40%, and 45%
yields, respectively. Thus six C–C biflavones (a–f,
Fig. 2) were prepared and evaluated for their inhibitory
activity against PLA2s.
3. Results and discussion
Extracellular sPLA2-IIA activity has been reported in
the inflamed sites of several experimental animal mod-
els,17–19 as well as in human diseases such as pancreatitis
R
R
4
3
O
Pd(PPh )
3 4
Bromoflavones
(3, 6, 3', 4')
Biflavones
+
NaOH, heat
DMF-H O (9:1)
R
R
1
2
2
a: IV+4 (74%)
b: III+3 (68%)
c: II+4 (43%)
d: II+3 (52%)
e: II+2 (40%)
f: I+4 (45%)
O
1 R2=R3=R4=H, R1=Br
2 R1=R3=R4=H, R2=Br
3 R1=R2=R4=H, R3=Br
4 R1=R2=R3=H, R4=Br
I R2=R3=R4=H, R1=PB
II R1=R3=R4=H, R2=PB
III R1=R2=R4=H, R3=PB
IV R1=R2=R3=H, R4=PB
(PB=pinacolatoboronyl)
Scheme 1. Synthesis of Biflavones a–f by Suzuki C–C cross-coupling reaction.

7140
T. C. Moon et al. / Bioorg. Med. Chem. 15 (2007) 7138–7143
O
O
O
O
O
O
O
O
b (4',3'''-BF)
a (4',4'''-BF)
c (6,4'''-BF)
e (6,6''-BF)
O
O
O
O
O
O
O
O
d (6,3'''-BF)
O
O
O
O
O
O
O
O
f (3,4'''-BF)
Figure 2. Structures of synthesized C–C biflavones (a–f).
Table 1. IC50 values of synthetic biflavones on various PLA2s activity
Compounds
sPLA2 IB
sPLA2 IIAa
sPLA2
V
sPLA2
X
cPLA2
a (40,40-BF)
b (40,30-BF)
c (6,40-BF)
d (6,30-BF)
e (6,6-BF)
>100
>100
>100
>100
>100
>100
>100
>100
3.0 0.90
15.7 3.7
63.9 4.2
19.9 4.6
69.3 5.7
23.2 3.1
23.8 3.4
3.5 0.6
8.7 3.77
23.9 5.21
10.8 2.56
12.8 3.08
50.0 10.1
6.50 3.41
41.8 5.2
>100
>100
>100
6.0 1.92
38.7 6.3
11.9 3.07
16.8 3.74
48.1 10.6
8.42 5.08
>100
27.6 4.3
>100
>100
f (3,40-BF)
Amentoflavone
Ochanflavone
>100
>100
17.6 2.41
>100
a These results were already published.16 All data are arthmetic means SD (n = 3).
the C–C biflavonoid d showed inhibitory activity against
sPLA2 X. None of the natural and synthetic C–C bifl-
avonoids tested showed any inhibitory activity against
sPLA2 IB. The synthetic C–C biflavonoid a showed
equipotent inhibitory activity against sPLA2 IIA16 and
more potent inhibitory activity against sPLA2 V com-
pared with those of ochnaflavone, a natural C–O–C bifl-
avonoid that is known to be the most potent natural
biflavonoid against sPLA2 IIA and sPLA2 V. At this
moment, we do not have any reasonable ideas to explain
the selectivity differences between synthetic and natural
biflavones. We assume that phenol groups in natural bif-
lavone structures may play roles on selective interactions
with PLA2 isoforms.
avonoid d showed inhibitory activity against sPLA2 X.
Further chemical modification of these basic structures
will be carried out in order to investigate the synthetic
C–C biflavones which possess more selective inhibitory
activity against isozymes of sPLA2.
4. Experimental
4.1. 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 dehydrat-
ing agent under nitrogen gas. All solvents used for chro-
matography were purchased and directly applied
In summary, the synthetic C–C biflavonoids (a–f) exam-
ined in this study have potent and broad inhibitory
activity against various PLA2s tested regardless of their
structural array. We also found that only the C–C bifl-
1
without further purification. H NMR spectra were re-
corded on
a
(200 MHz) and a Bruker DPX 400 (400 MHz) spec-
Varian Gemini 2000 instrument

T. C. Moon et al. / Bioorg. Med. Chem. 15 (2007) 7138–7143
7141
trometers. Chemical shifts are reported in parts per mil-
4.3. General synthetic procedure for Suzuki C–C cross-
coupling reaction
lion (ppm) downfield relative to tetramethylsilane as an
internal standard. Peak splitting patterns are abbrevi-
ated 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) spectrometer, fully decoupled, and chemical
shifts are reported in parts per million (ppm) downfield
relative to tetramethylsilane as an internal standard.
Melting points were recorded on a Fisher-Johns micro-
scopic scale melting point apparatus. EI mass spectra
were recorded on an Autospec M363. Analytical thin-
layer chromatography (TLC) was performed using com-
mercial glass plate with silica gel 60F254 purchased from
Merck. Chromatographic purification was carried out
by flash chromatography using Kieselgel 60 (230–400
mesh, Merck).
The reaction mixture of tetrakis(triphenylphosphine)-
palladium (5 mol%), a flavone pinacolato boronate (I–
IV, 120 mol%), an aryl bromide (1–4, 100 mol%), and
NaOH (400 mol%) in a mixed solution of DMF and
water (9:1) was degassed by vacuum pump for 10 min
and saturated with nitrogen gas. The reaction was con-
ducted with stirring at 100 °C for 14 h. The mixture was
cooled to room temperature, filtered through a filtering
reagent, and the filtrate was extracted with ethyl acetate.
The organic layer was washed with brine and water,
dried over anhydrous MgSO4, and filtered with a glass
filter. The filtrate was concentrated in a reduced pres-
sure, and the crude residue was purified by column chro-
matography. The product was further purified by
recrystallization in hot methyl alcohol to afford the tar-
get compound (a–f).
4.2. General procedure for synthesis of flavone pinacolato
boronates (I-IV)
4.3.1. [40,4000]-Biflavone (40,4000-BF; a). The product was
obtained as a white solid in 74% yield; mp >300 °C;
1H NMR (400 MHz, CDCl3) d 8.25–8.28 (dd, 2H,
J = 1.5 Hz, 7.9 Hz, H5, H500), 8.06–8.08 (d, 4H,
J = 8.4 Hz, H30, H3000, H50, H5000), 7.82–7.84 (d, 4H,
J = 8.4 Hz, H20, H2000, H60, H6000), 7.72–7.76 (m, 2H,
H7, H700), 7.61–7.63 (d, 2H, J = 8.1 Hz, H8, H800),
7.44–7.47 (t, 2H, J = 7.4 Hz, H6, H600), 6.92 (s, 2H,
H3, H300); 13CNMR (100 MHz, CDCl3) d 178.84 (C-4,
C-400), 163.46 (C-2, C-200), 156.72 (C-9, C-900), 143.31
(C-40, C-4000), 134.46 (C-7, C-700), 131.79 (C-10, C-1000),
128.17 (C-30, C-3000, C-50, C-5000 ), 127.45 (C-20, C-2000,
C-60, C-6000), 126.20 (C-6, C-600), 125.88 (C-5, C-500),
124.25 (C-10, C-1000), 118.54 (C-8, C-800), 107.99 (C-3,
C-300); m/z 442.12 (M+, 100).
The reaction mixture of bis(pinacolato)diboron
(100 mol%), PdCl2(dppf) (5 mol%), and anhydrous
potassium carbonate (400 mol%) in freshly distilled
DMF was degassed by vacuum pump for 10 min and
saturated with nitrogen gas. The reaction was conducted
with stirring at 90 °C for 10 h. The mixture was cooled
to room temperature, filtered through a filtering reagent,
and the filtrate was extracted with ethyl acetate. The or-
ganic layer was washed with brine and water, dried over
anhydrous MgSO4, and filtered with a glass filter. The
filtrate was concentrated in a reduced pressure, and
the crude residue was purified by column chromatogra-
phy. The product was further purified by recrystalliza-
tion in hot methyl alcohol to afford the target
compound (I–IV).
4.3.2. [40,3000]Biflavone (40,3000-BF; b). The product was
obtained as a white solid in 68% yield; mp 267–269 °C;
1H NMR (400 MHz, CDCl3) d 8.25–8.27 (dd, 2H,
J = 1.5 Hz, 7.9 Hz, H5, H500), 8.19–8.20 (d, 1H,
J = 1.4 Hz, H20), 8.07–8.09 (d, 2H, J = 8.4 Hz, H3000,
H5000), 7.96–7.98 (d, 1H, J = 7.9 Hz, H60), 7.81–7.83 (d,
3H, J = 8.4 Hz, H2000, H6000, H40), 7.72–7.76 (ddd, 2H,
J = 1.7 Hz, 7.8 Hz, 1.3 Hz, H7, H700), 7.61–7.68 (m,
3H, H50, H8, H800), 7.44–7.47 (t, 2H, J = 7.5 Hz, H6,
H600), 6.92–6.93 (s, 2H, H3, H300); 13C NMR
(100 MHz, CDCl3) d 178.82 (C-4, C-400), 163.45 (C-2),
163.28 (C-200), 156.71 (C-9, C-900), 143.66 (C-4000),
141.24 (C-30), 134.35 (C-700), 134.31 (C-7), 133.11
(C-10), 131.70 (C-1000), 130.70 (C-20), 130.23 (C-40),
128.22 (C-3000, C-5000), 127.41 (C-2000, C-6000), 126.40 (C-60),
126.20 (C-6, C-600), 125.83 (C-500), 125.77 (C-50), 125.44
(C-5), 124.41 (C-10, C-1000), 118.55 (C-8), 118.54 (C-800),
108.45 (C-3), 108.13 (C-300); m/z 442 (M+, 100), 441 (23),
322 (14), 221 (14), 202 (21), 92 (14), 57 (22).
4.2.1. 3-Pinacolatoboronflavone (I). 78%; 1H NMR
(200 MHz, CDCl3) d 8.30 (d, 1H, J = 1.6 Hz, H5), 7.86
(d, 2H, J = 2.2 Hz, 4.4 Hz, H20, H60), 7.75 (t, 1H,
J = 1.8 Hz, 8.8 Hz, H7), 7.53–7.57 (m, 3H, H30, H40,
H50), 7.49 (t, 1H, J = 3.6 Hz, 4.4 Hz, 8.0 Hz, H6), 7.42
(d, 1H, J = 1.4 Hz, H8), 1.37 (s, 12H, 4 CH ).
3
*
4.2.2. 6-Pinacolatoboronflavone (II). 65%; 1H NMR
(200 MHz, CDCl3) d 8.35 (s, 1H, H5), 7.92 (d, 2H,
J = 3.2 Hz, 4.4 Hz, H20, H60), 7.79 (d, 1H, J = 2.6 Hz,
H7), 7.45–7.56 (m, 4H, H8, H30, H40, H50), 6.88 (s,
1H, H3), 1.38 (s, 12H, 4 CH ).
3
*
4.2.3. 30-Pinacolatoboronflavone (III). 86%; 1H NMR
(200 MHz, CDCl3) d 8.36 (d, 1H, J = 8.0 Hz, H5), 8.24
(d, 1H, J = 6.6 Hz, H40), 8.00 (t, 1H, J = 8.0 Hz, H7),
7.50–7.78 (m, 4H, H8, H30, H20, H50), 7.43 (t, 1H,
J = 6.0 Hz, H6), 6.88 (s, 1H, H3), 1.38 (s, 12H,
4 CH ).
3
4.3.3. [6,4000]Biflavone (6,4000-BF; c). The product was ob-
tained as a white solid in 43% yield; mp 291–293 °C; 1H
NMR (400 MHz, CDCl3) d 8.53 (s, 1H, H5), 8.24–8.26
(d, 1H, J = 7.8 Hz, H500), 8.04–8.06 (d, 2H, J = 7.8 Hz,
H3000, H5000), 8.00–8.02 (d, 1H, J = 8.8 Hz, H7), 7.96–
7.97 (d, 2H, J = 5.3 Hz, H20, H60), 7.84–7.86 (d, 2H,
J = 7.9 Hz, H2000, H6000), 7.70–7.75 (m, 2H, H8, H700),
*
4.2.4. 40-Pinacolatoboronflavone (IV). 84%; 1H NMR
(200 MHz, CDCl3) d 8.25 (d, 1H, J = 6.0 Hz, H5),
7.94–7.95 (m, 4H, H20, H60, H7, H8), 7.66 (d, 2H,
J = 5.0 Hz, 6.4 Hz, H30, H50), 7.44 (t, 1H, J = 6.4 Hz,
5.4 Hz, H6), 6.89 (s, 1H, H3), 1.38 (s, 12H, 4 CH ).
3
*

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T. C. Moon et al. / Bioorg. Med. Chem. 15 (2007) 7138–7143
7.61–7.63 (d, 1H, J = 8.4 Hz, H800), 7.55–7.57 (d, 3H,
J = 5.1 Hz, H30, H40, H50), 7.43–7.46 (t, 2H,
J = 7.5 Hz, H600), 6.89–6.90 (d, 2H, J = 4.5 Hz, H3,
H300); 13C NMR (100 MHz, CDCl3) d 178.74 (C-4,
C-400), 163.01 (C-2), 163.23 (C-200), 156.70 (C-900),
156.55 (C-9), 142.79 (C-4000), 137.26 (C-6), 134.26
(C-700), 132.86 (C-7), 132.21 (C-10), 132.02 (C-5),
131.55 (C-1000), 129.54 (C-40), 128.12 (C-3000, C-5000),
127.36 (C-2000, Cb-6000), 126.76 (C-30, C-50), 126.16
(C-600), 125.72 (C-500), 124.71 (C-10), 124.31 (C-10,
C-20, C-60), 119.41 (C-8), 118.56 (C-800), 108.12 (C-3 ,
C-300); m/z 442 (M+, 100), 441 (8), 340 (6), 322 (6), 220 (5).
126.53 (C-20, C-60), 126.10 (C-600), 125.79 (C-5), 125.63
(C-500), 124.41 (C-1000), 123.79 (C-10), 118.50 (C-800),
118.47 (C-8), 107.97 (C-3, C-300); m/z 442 (M+, 70), 441
(100), 322 (10), 321 (18), 202 (16), 121 (9).
4.4. Assay of phospholipase A2 inhibitory activity by
synthetic biflavonoids
The cDNA for rat sPLA2-IB, human sPLA2-IIA,
sPLA2-V, sPLA2-X, and cPLA2 was cloned into an
expression vector and transfected into human embry-
onic kidney 293 cells (HEK293 cells) using Lipofec-
AMINE PLUS (Gibco BRL, Gaithersburg, MD,
USA) as described previously.13,14
4.3.4. [6,3000]Biflavone (6,3000-BF; d). The product was
obtained as a yellowish solid in 52% yield; mp 250–
252 °C; 1H NMR (400 MHz, CDCl3) d 8.52 (s, 1H,
H5), 8.25–8.26 (d, 1H, J = 7.7 Hz, H500), 8.22 (s, 1H,
H2000), 7.93–8.01 (m, 4H, H7, H20, H60, H6000) 7.86–7.86
(d, 1H, J = 7.5 Hz, H4000), 7.71–7.75 (t, 2H, J = 6.8 Hz,
7.7 Hz, H700, H8), 7.62–7.66 (t, 2H, J = 8.0 Hz, 7.5 Hz,
H5000, H800), 7.55–7.57 (d, 3H, J = 5.8 Hz, H30, H40,
H50), 7.43–7.47 (t, 2H, J = 7.4 Hz, H600), 6.90–6.92 (s,
2H, H3, H300); 13C NMR (100 MHz, CDCl3) d 178.83
(C-400), 178.74 (C-4), 163.04 (C-2), 163.51 (C-200),
156.73 (C-900), 156.43 (C-9), 140.82 (C-3000), 137.73 (C-
6), 134.32 (C-700), 133.09 (C-1000), 133.05 (C-7), 132.22
(C-10), 132.04 (C-5), 130.82 (C-2000), 130.21 (C-4000),
129.55 (C-40), 126.79 (C-30, C-50), 126.17 (C-600,C-6000),
125.79 (C-5000), 125.45 (C-500), 124.66 (C-10), 124.42
(C-1000), 124.35 (C-20, C-60), 119.43 (C-8), 118.69
(C-800), 108.49 (C-300), 108.10 (C-3); m/z 442 (M+, 100),
340 (17), 220 (11), 192 (9), 163 (12).
The standard reaction mixture (200 ll) contained
100 mM Tris–HCl (pH 9.0), 6 mM CaCl2, 1% bovine
serum albumin, 2.5 lM of radiolabeled 1-acyl-2-[1-14C]
arachidonoyl-sn-glycerol phosphoethanolamine (48 mCi/
mmol, NEN, Boston, MA, USA), and synthetic biflavo-
noids. The reaction was started by the addition of an ali-
quot of the culture medium as an enzyme source and
carried out at 37 °C for 20 min, and [14C]arachidonic
acid released was extracted by the method described
previously.17
Under these conditions, the reaction mixture without
synthetic biflavonoids released 10% free fatty acid. Inhi-
bition was expressed as a percentage. Synthetic biflavo-
noids were dissolved in dimethylsulfoxide (DMSO) and
added to the enzyme assay tubes at 2% of the final vol-
ume. Control experiments showed that DMSO at con-
centrations up to 2% had no effect on enzymatic
activity. All determinations were duplicated and the
50% inhibitory concentration was obtained by linear
regression analysis at 1–100 lM of biflavones tested.
4.3.5. [6,600]Biflavone (6,600-BF; e). The product was
obtained as a white solid in 40% yield; mp > 300 °C;
1H NMR (400 MHz, CDCl3) d 8.53–8.53 (d, 2H,
J = 2.3 Hz, H5, H500), 8.07–8.10 (dd, 2H, J = 2.3 Hz,
8.8 Hz, H7, H700), 7.96–7.98 (m, 4H, H20, H2000, H60,
H6000), 7.70–7.72 (d, 2H, J = 8.8 Hz, H8, H800), 7.54–
7.57 (m, 6H, H30, H3000, H40, H4000, H50, H5000), 6.89 (s,
2H, H3, H300); 13C NMR (100 MHz, CDCl3) d 178.71
(C-4, C-4), 164.10 (C-2, C-200), 156.40 (C-9, C-900),
136.95 (C-6, C-600), 133.13 (C-7, C-700), 132.21 (C-10,
C-1000), 132.05 (C-5, C-5000), 129.54 (C-40, C-4000), 126.79
(C-30, C-3000, C-50, C-5000), 124.57 (C-10, C-1000), 124.14
(C-20, C-2000, C-60, C-6000), 119.44 (C-8, C-800) , 108.02
(C-3, C-300 ); m/z 442 (M+, 100), 340 (39), 210 (20), 126
(19), 102 (20).
Acknowledgments
This study was supported by Kangwon National
University and by the grant R01-2004-000-10134-0 from
the Basic Research Program of the Korea Science &
Engineering Foundation. The authors thank Phamacal
Research Institute and Central Laboratory of Kangwon
National University for the use of analytical instruments
and bioassay facilities.
4.3.6. [3,4000]Biflavone (3,4000-BF; f). The product was
obtained as a white solid in 45% yield; mp 231–233 °C;
1H NMR (400 MHz, CDCl3) d 8.292–8.316 (dd, 1H,
J = 1.5 Hz, 8.0 Hz, H500), 8.11–8.23 (dd, 1H,
J = 1.6 Hz, 8.0 Hz, H5), 7.86–7.89 (t, 2H, J = 1.9 Hz,
8.4 Hz, H20, H60), 7.66–7.75 (m, 2H, H7, H700), 7.53–
7.57 (dd, 2H, J = 8.2 Hz, H8, H800), 7.36–7.47 (m, 7H,
H30, H3000, H50, H5000, H2000, H6000, H40), 7.29–7.33 (m
2H, H6, H600), 6.82 (s, 1H, H300); 13C NMR (100 MHz,
CDCl3) d 178.85 (C-400), 178.36 (C-4), 163.52 (C-200),
162.48 (C-2), 156.65 (C-900), 156.45 (C-9), 137.19
(C-4000), 134.41 (C-700), 134.19 (C-7), 133.30 (C-10),
132.47 (C-3000, C-5000), 131.18 (C-1000), 130.92 (C-2000,
C-6000), 130.02 (C-40), 128.78 (C-30, C-50), 126.79 (C-6),
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