Synthesis of phospholipase A2 inhibitory biflavonoids

Article abstract of DOI:10.1016/j.bmcl.2006.01.117

A series of C-C biflavones was designed to investigate the relationship between structural array of different flavone-flavone subunit linkage and the inhibitory activity against phospholipase A₂ (PLA₂). Among six classes of C-C bifla

Full text of DOI:10.1016/j.bmcl.2006.01.117

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2373–2375
Synthesis of phospholipase A2 inhibitory biflavonoids
Jianjun Chen,a Hyeun Wook Chang,b,* Hyun Pyo Kima and Haeil Parka,*
aCollege of Pharmacy, Kangwon National University, Chunchon 200-701, Republic of Korea
bCollege of Pharmacy, Yeungnam University, Gyeongsan 712-749, Republic of Korea
Received 14 September 2005; revised 25 January 2006; accepted 30 January 2006
Available online 28 February 2006
Abstract—A series of C–C biflavones was designed to investigate the relationship between structural array of different flavone–fla-
vone subunit linkage and the inhibitory activity against phospholipase A2 (PLA2). Among six classes of C–C biflavones designed,
four classes of C–C biflavones, which have flavone–flavone subunit linkages at A ring–A ring, A ring–B ring, B ring–B ring, and B
ring–C ring, were synthesized. The synthetic biflavones exhibited somewhat different inhibitory activities against sPLA2-IIA. Among
them, the biflavone a having a C–C 40–40 linkage showed comparable inhibitory activity with that of the natural biflavonoid, och-
naflavone, and 7-fold stronger activity than that of amentoflavone. Further chemical modification is being carried out in order to
obtain the chemically optimized biflavonoids.
Ó 2006 Elsevier Ltd. All rights reserved.
Biflavonoids are flavonoid dimers connected with a C–C
or C–O–C bond. Although a wealth of biflavonoids
have been discovered from various plant species, their
biological and pharmacological data are limited. Previ-
ously, certain biflavonoids were reported to inhibit
phosphodiesterase,1 lens aldose reductase,2 and mast cell
histamine release,3 and to show anticancer activity.4
Recently, some C–C biflavonoids were synthesized and
their antimicrobial activities were demonstrated.5 Dur-
ing our investigations to find potential anti-inflammato-
ry plant drugs, several biflavones such as amentoflavone
and ochnaflavone (Fig. 1) were for the first time demon-
strated as inhibitors of group II secretory phospholipase
A2 (sPLA2IIA).6 Later, morelloflavone, a flavone–flava-
none dimer, was also revealed as sPLA2 inhibitors7
(Fig. 1).
XII).9 Since sPLA2 is a pivotal enzyme to generate ara-
chidonic acid that is converted further to proinflamma-
tory eicosanoids, sPLA2 inhibitors may show favorable
anti-inflammatory activity. Actually, some of these bifl-
avonoids were found to possess promising anti-inflam-
matory activity in vivo.7,10 In this respect, several basic
biflavonoids have been synthesized and their inhibitory
activities on sPLA2-IIA were evaluated in this
investigation.
A series of C–C biflavones was designed (Fig. 2) to
investigate the relationship between structural array of
a different flavone–flavone subunit linkage and the
inhibitory activity against sPLA2-IIA. Among six classes
of C–C biflavones designed, four classes of C–C biflav-
ones, which have flavone–flavone subunit linkages at
A ring–A ring, A ring–B ring, B ring–B ring, and B
ring–C ring, were synthesized.
PLA2 is a growing family of distinct enzymes that exhib-
it different substrate specificities, cofactor requirement,
subcellular localization, and cellular functions.8 sPLA2
has low molecular weights (14–18 kDa) with a rigid ter-
tiary structure configured by 6–8 disulfide bridges. Thus
so far, 10 genes coding for structurally related and enzy-
matically active sPLA2s have been identified in mam-
mals (groups IB, IIA, IIC, IID, II, IIF, III, V, X, and
The total synthesis of C–C biflavones was approached
via construction of two flavone analogs, one substituted
with halogen atom (bromo) and the other substituted
with groups that could be coupled using transition
metal-catalyzed cross-coupling methodology.
Two typical methods, Suzuki coupling reaction11 and
Stille coupling reaction,12 were applied to connect two
flavone units via a biaryl linkage (Scheme 1). Halogenof-
lavones (Ar-X) were prepared from halogen-substituted
2-hydroxyacetophenones or flavones by reacting with
halogenating reagents following the general procedures
Keywords: Biflavonoids; C–C cross-coupling reaction; Phospholipase
A2 inhibition.
*
Corresponding authors. Tel.: +82 33 250 6920; fax: +82 33 255 7865
(H.P.); tel.: +82 53 810 2811; fax: +82 53 810 4654 (H.W.C.); e-mail
addresses: hwchang@yu.ac.kr; haeilp@kangwon.ac.kr
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.01.117

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J. Chen et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2373–2375
OH
OH
O
OH
O
O
OH
OH
O
OH
HO
O
O
HO
O
O
OH
O
OH
O
OH HO
O
O
OH
OH
O
HO
OH
HO
OH
Amentoflavone
Ochnaflavone
Morelloflavone
Figure 1. Structures of amentoflavone, ochnaflavone, and morelloflavone.
O
O
O
O
O
O
O
O
O
O
O
O
A-A
A-B
A-C
O
O
O
O
O
O
O
O
O
O
O
O
C-C
B-C
B-B
Figure 2. Biflavones with different flavone–flavone subunit linkages.
SnR3
FLA'
kidney 293 cells (HEK293 cells) using LipofectAMINE
PLUS (Gibco-BRL, Gaithersburg, MD, USA) as de-
scribed previously.19,20
Pd-caltalyst
or
FLA-X
+
FLA
FLA'
(a-f)
O
B
FLA'
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]-
arachidonyl-sn-glycerol phosphoethanolamine (48 mCi/
mmole, NEN, Boston, MA, USA), and synthetic
biflavonoids. The reaction was started by the addition
of an aliquot 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.21 Under these conditions,
the reaction mixture without synthetic biflavonoids re-
leased 10% free fatty acid. Inhibition was expressed as
a percentage. Synthetic biflavonoids were dissolved in
dimethylsulfoxide (DMSO) and added to the enzyme as-
say tubes at 2% of the final volume. Control experiments
showed that DMSO at concentrations 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
biflavones tested.
O
Scheme 1. Typical cross-coupling reactions for C–C biflavones.
as described in the earlier publications.13–18 Also tribu-
tyltinflavones and boronates of flavones were prepared
from the corresponding halogenoflavones following the
procedures and conditions as described in the previous
literatures.13,17
Treatment of bromoflavones (40-, 6-, and 30-) with com-
mercially available hexa(n-butyl)ditin in the presence of
catalytic Pd(PPh3)4 in refluxing toluene afforded the
tributyltinflavones. Stille coupling of tributyltinflavones
(1.3 equiv) with bromoflavones (3-, 6-, 30-, and 40-, 1.0
equiv) in the presence of 5 mol% Pd(PPh3)4 in refluxing
toluene gave C–C biflavones (a, b, d, and e) in 25–50%
yields. Treatment of 4-bromoflavone with bis(pinacola-
to)diboron in the presence of catalytic PdCl2(dppf)
and K2CO3 in DMF at 90 °C provided the correspond-
ing pinacolato boronate. Suzuki coupling reactions of
the pinacolato boronate (1.2 equiv) with bromoflavones
(30- and 3-, 1.0 equiv) in standard conditions [Pd(PPh3)4
(5 mol%), NaOH (4.0 equiv)3, DMF–water (9:1), 90 °C]
gave C–C biflavones (c and f) in 31% and 21% yields,
respectively. Thus six C–C biflavones (Fig. 3) were pre-
pared and evaluated for their inhibitory activity against
phospholipase A2.
As demonstrated in Table 1, the synthetic biflavonoids
exhibited somewhat different inhibitory activities against
sPLA2-IIA depending on their chemical structures.
Among them, the biflavone a having a C–C 40–40 linkage
showed a potent inhibition. The inhibitory potency of a
was comparable with that of the natural biflavonoid,
ochnaflavone, and 7-fold stronger than that of
amentoflavone. The biflavones, b, d, and f, possess
the similar inhibitory potency with amentoflavone.
However, the potency of inhibition of c and e was weaker.
The cDNA for human sPLA2-IIA was cloned into an
expression vector and transfected into human embryonic

J. Chen et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2373–2375
2375
O
a
c
b
O
O
O
O
O
O
O
O
O
O
O
e
d
f
O
O
O
O
O
O
O
O
O
O
O
O
Figure 3. Structures of synthesized C–C biflavones (a–f).
Table 1. Inhibition of sPLA2-IIA by synthetic biflavonoids a–e
3. Amella, M.; Bronner, C.; Briancon, F.; Hagg, M.; Anton,
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Compound
IC50 (lM)a
Amentoflavone
Ochnaflavone
23.8 3.4
3.5 0.6
3.0 0.9
a
b
c
d
e
f
15.5 3.7
63.9 4.2
19.9 4.6
69.3 5.7
23.2 3.1
a All data are arithmetic means SD (n = 3).
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Further chemical modification of these basic structures is
being carried out for increasing their pharmacological
activities.
Acknowledgments
This work was supported by Grant R01-2004-000-
10134-0 from the Basic Research Program of the Korea
Science and Engineering Foundation. The authors
thank the Pharmacal Research Institute and the Central
Laboratory of Kangwon National University for the use
of analytical instruments.
References and notes
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