Synthesis and antioxidant activity of a procyanidin B3 analogue

Article abstract of DOI:10.1016/j.bmcl.2016.12.067

Proanthocyanidin, an oligomer of catechin, is a natural antioxidant and a potent inhibitor of lectin-like oxidized LDL receptor-1, which is involved in the pathogenesis of arteriosclerosis. We synthesized proanthocyanidin analogue 1, in which the geometry of one catechin molecule in procyanidin B3, a dimer of (+)-catechin, is constrained to be planar. The antioxidant activities of the compounds were evaluated in terms of their capacities to scavenge galvinoxyl radicals, and results demonstrate that while procyanidin was 3.8 times more potent than (+)-catechin, the radical scavenging activity of proanthocyanidin analogue 1 was further increased to 1.9 times that of procyanidin B3. This newly designed proanthocyanidin analogue 1 may be a promising lead compound for the treatment of arteriosclerosis and related cerebrovascular diseases.

Full text of DOI:10.1016/j.bmcl.2016.12.067

Accepted Manuscript
Synthesis and antioxidant activity of a procyanidin B3 analogue
Mirei Mizuno, Ikuo Nakanishi, Satoko Matsubayashi, Kohei Imai, Takuya Arai,
Ken-ichiro Matsumoto, Kiyoshi Fukuhara
PII:
S0960-894X(16)31340-3
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.12.067
BMCL 24556
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
1 October 2016
14 December 2016
23 December 2016
Please cite this article as: Mizuno, M., Nakanishi, I., Matsubayashi, S., Imai, K., Arai, T., Matsumoto, K-i., Fukuhara,
K., Synthesis and antioxidant activity of a procyanidin B3 analogue, Bioorganic & Medicinal Chemistry Letters
(2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.12.067
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Synthesis and antioxidant activity of a procyanidin B3 analogue
Mirei Mizunoa, Ikuo Nakanishib, Satoko Matsubayashia, Kohei Imaia, Takuya Araia,c, Ken-ichiro
Matsumotob, Kiyoshi Fukuharaa,*
aSchool of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo
142-8555, Japan
bQuantitative RedOx Sensing Team (QRST), Department of Basic Medical Sciences for
Radiation Damages, National Institute of Radiological Sciences (NIRS), Inage-ku, Chiba
263-8555, Japan
cGraduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori,
Mizuho-ku, Nagoya, Aichi 467-8603, Japan
*Corresponding author:
Kiyoshi Fukuhara
School of Pharmaceutical Sciences, Showa University,
1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan
Tel.: +81-3-3784-8186; Fax: +81-3-3784-8252
E-mail address: fukuhara@pharm.showa-u.ac.jp

Abstract
Proanthocyanidin, an oligomer of catechin, is a natural antioxidant and a potent inhibitor of
lectin-like oxidized LDL receptor-1, which is involved in the pathogenesis of arteriosclerosis.
We synthesized proanthocyanidin analogue 1, in which the geometry of one catechin molecule
in procyanidin B3, a dimer of (+)-catechin, is constrained to be planar. The antioxidant
activities of the compounds were evaluated in terms of their capacities to scavenge galvinoxyl
radicals, and results demonstrate that while procyanidin was 3.8 times more potent than
(+)-catechin, the radical scavenging activity of proanthocyanidin analogue 1 was further
increased to 1.9 times that of procyanidin B3. This newly designed proanthocyanidin analogue 1
may be a promising lead compound for the treatment of arteriosclerosis and related
cerebrovascular diseases.
Keywords
proanthocyanidin, catechin, arteriosclerosis LOX-1, radical scavenging activity

Arteriosclerosis, which is an important pathologic cause of cerebrovascular disease and
heart disease, is characterized by oxidized low-density lipoprotein (OxLDL)-induced lipid
accumulation within blood vessels, leading to arterial narrowing and/or obstruction. Suppression
of outbreak of the arteriosclerosis enables several diseases to inhibit the onset of them. However,
there are few reports of effective therapeutic agents for arteriosclerosis. Thus, development of
novel therapeutic agents is of utmost importance.
One of the markers of arteriosclerosis onset is endothelial dysfunction, which is caused by
OxLDL binding to lectin-like oxidized LDL receptor-1 (LOX-1).1 LOX-1 is expressed at
relatively low levels in healthy adults during the resting state. However, LOX-1 levels are
elevated under pathological conditions, such as hypertension2, hyperlipidaemia3, diabetes4 and
arteriosclerosis.5 LOX-1 is found not only on endothelial cells, but also on macrophages,6
platelets7 and other areas. Ligand binding to LOX-1 causes the activation of endothelial
dysfunction, cell proliferation, apoptosis and arteriosclerosis by intracellular signaling. One
such LOX-1 binding ligand is OxLDL, which is generated during the oxidation of LDL. OxLDL
binding to LOX-1 activates NADPH oxidase to cause rapid generation of reactive oxygen
species (ROS)8, resulting in further production of OxLDL. The LOX-1-mediated action of
OxLDL also induces a decrease in levels of NO and CYP450, which are typical changes seen in
endothelial dysfunction.9 Furthermore, OxLDL-mediated LOX-1 expression has been reported
to stimulate the NF-κB signaling pathway to promote arteriosclerosis.10 This pathway is known
to increase the expression of the pro-inflammatory gene. Thus, generation of OxLDL by ROS
and the successive binding to LOX-1 contribute to the activation of several pathways that are
key to the progression of arteriosclerosis.
Proanthocyanidins, which are oligomers of catechin, are very powerful antioxidants found in
grape seeds and nutshells.11 Recently, it was reported that proanthocyanidins significantly inhibit
OxLDL binding to LOX-1. The LOX-1 inhibitory activity of proanthocyanidin depends on the
degree of catechin polymerization, with a higher degree of catechin polymerization resulting in
stronger inhibition of OxLDL binding to LOX-1, as well as more potent free radical scavenging
activities. In this context, several beneficial functions have been attributed to highly polymerized
proanthocyanidin, such as prevention of arteriosclerosis12 and cataracts,13 and improvement for
antioxidative activity for LDL. However, in the development of arteriosclerosis
chemopreventive agents, such highly polymerized compounds are often inappropriate for
targeting cerebrovascular disease because they exhibit high molecular masses, which do not
allow them to pass through the blood–brain barrier.
Previously, we synthesized a catechin analogue, called “planar catechin,” in which the
geometry of (+)-catechin was constrained to be planar by a bridge between the 3-hydroxyl

group and C6' on the C-ring.14
Figure 1. Structures of (+)-catechin, procyanidin B3, and its analogue, proanthocyanidin
analogue 1
Planar catechin exhibited potent radical scavenging activities, which were 5-fold greater
than catechin.15 Several biological activities were also reported, including remarkable
α-glucosidase inhibition, and anti-virus and anti-tumor activities.14,16 Thus, when considering a
new type of LOX-1 inhibitor, we focused on the α-glucosidase inhibitory activity of planar
catechin because of the specific structure required to exhibit an affinity for the substrate-binding
site of α-glucosidase. That is, we hypothesized that if the catechin moiety in proanthocyanidin
was constrained to be planar, both efficient LOX-1 inhibitory activity as well as enhanced
antioxidative activity might result, even at low degrees of polymerization, leading to the
development of an arteriosclerosis chemopreventive agent capable of crossing the blood–brain
barrier. Therefore, we designed proanthocyanidin analogue 1 based on the structure of
procyanidin B3, a dimer of (+)-catechin, by constraining the geometry of one catechin molecule
to be planar. Compared with procyanidin B3, proanthocyanidin analogue 1 may prevent the
formation of OxLDL by oxidative stress and improved binding affinity toward the lectin-like
domain in LOX-1, thereby inhibiting the activation of LOX-1. Herein, we describe the highly
stereoselective synthesis of proanthocyanidin analogue 1, as well as its radical scavenging
activity.

Scheme 1. Reagents and conditions: (a) BnBr, K2CO3, DMF, 0° C → rt, 67 %; (b) Ac2O,
pyridine, DMAP, rt, 97 %; (c) ethylene glycol, DDQ, CH2Cl2, rt, 59 %; (d) acetone, TMSOTf,
THF, -20°C, quant.; (e) BnBr, K2CO3, DMF, 0° C → rt, 79%; (f) TMSOTf, CH2Cl2, -78° C,
94%; (g) K2CO3, toluene/MeOH, rt, 60 %; (h) H2, Pd(OH)2, THF/MeOH/H2O, rt, 60 %
Proanthocyanidin analogue 1 was synthesized according to a procedure for the synthesis of
procyanidin B3.17 That is, the synthesis is based on a regio- and enantioselective Friedel-Crafts
type alkylation process to connect the benzylic C-ring C4 position of an electrochemically
activated (+)-catechin derivative (4) with the A-ring C8 center of a nucleophilic planar catechin
derivative (6). As shown in Scheme 1, the four phenolic hydroxyl groups of (+)-catechin were
protected as benzyloxy moieties by reaction with benzyl bromide18 and the alcoholic hydroxyl
group was subsequently protected by acetic anhydride.19 The desired product (3) formed in
quantitative yield was then oxidized with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) in
the presence of ethylene glycol to yield a C4 activated electrophile (4) with high
stereoselectivity.17

The stereoselective synthesis is attributed to the ability of DDQ to form a charge transfer
complex20,21 with the phloroglucinol-type A-ring, in which the electron density is higher than that
of the pyrocatechol-type B-ring.22 In this context, unfavorable steric interactions between H-2(C)
and the bulky oxidant facilitates association of DDQ with the alpha-face of the A-ring, leading
to asymmetric oxygenation of the pro-R diastereotopic benzylic hydrogen. The incipient
carbocationic species is then synchronously attacked from the opposite side by ethylene glycol
as the nucleophilic trapping agent. Although introduction of different leaving groups, such as
methoxy and ethoxyethyl groups, was also attempted using methanol and ethoxyethanol,
respectively, the (+)-catechin derivative (4) containing a 2-hydroxyethoxy group was found to
be excellent in its availability and ability to accomplish the following condensation reaction
with the planar catechin derivative (6) in high yield and very high stereoselectivity. The
synthesis of the nucleophilic planar catechin derivative (6) was readily accomplished through
the Oxa-Pictet–Spengler reaction23 in which (+)-catechin and acetone were treated with
TMSOTf in THF14 and subsequent protection of the four hydroxyl groups by benzyl bromide.
Saito et al.19 reported that Lewis acid-catalyzed intermolecular condensation of the benzylated
catechin with 4-alkoxy catechin derivatives afforded the 4-8 linked dimer with high
stereochemical preference for the 3,4-trans form of procyanidin B3.24 Therefore, the
condensation of the electrophilic catechin derivative (4) and the nucleophilic planar catechin (6)
was performed under the same conditions using TMSOTf as a Lewis acid. The used
nucleophilic planar catechin (6) was in 4.5-fold excess to avoid the formation of oligomeric
catechins, resulting in the highly stereoselective formation of the 3,4-trans isomer (7) in high
yield. Although other Lewis acids, such as Yb(OTf)3 and TiCl4, are also known to yield
procyanidin B3 with high selectivity for the 3,4-trans isomer,25 conducting the reaction with
these Lewis acids resulted in the formation of the condensation products in lower yield, despite
the fact that the reactions proceeded in highly regio- and stereoselective manners. Finally, the
condensed product (7) was subjected to hydrolysis of the acetate with K2CO3 in
methanol-toluene solvents and subsequent deprotection of the benzyl groups by
Pd(OH)2-catalyzed hydrogenation furnished the desired proanthocyanidin analogue 1.26
The radical scavenging activities of proanthocyanidin analogue 1 and procyanidin B3 were
evaluated in a non-aqueous system using galvinoxyl radical (GO・) as an oxyl radical species.
Because of its odd electron, GO・ exhibits a strong absorption band at 428 nm, and a solution of
GO・ appears yellow in color. As the electron is paired, the absorption vanishes, and the
resulting decolorization is stoichiometric with respect to the number of electrons that are
scavenged. Taking advantage of the color change of GO・ in the presence of an antioxidant, the

rates of radical scavenging of proanthocyanidin analogue 1 and procyanidin B3 toward
galvinoxyl were measured. As shown in Figure 2, the concentration of proanthocyanidin
analogue 1 or procyanidin B3 was maintained at more than 10-fold excess relative to the GO・
concentration. The pseudo first-order rate constant (kobs) exhibited first-order dependence with
respect to the concentration of proanthocyanidin analogue 1 or procyanidin B3 as shown in
Figure 3. From the linear plot of kobs vs the concentration of procyanidin B3 or
proanthocyanidin analogue 1, the second-order rate constants (k) for radical scavenging toward
G・ were determined to be 1.00 × 102 mol-1 s-1 and 1.89 × 102 mol-1 s-1 for procyanidin B3 and
proanthocyanidin analogue 1, respectively. We previously reported the k value for (+)-catechin
determined under the same manner to be 2.6 × 10 mol-1 s-1,15 indicating that dimerization of the
catechin monomer led to a 3.8 fold further increase in radical scavenging activity. In general, the
structures of highly polymerized catechins are much more capable of exerting antioxidant
activities and inhibiting OxLDL binding to LOX-1. The synergistic radical scavenging activity
of procyanidin B3, a dimer of catechin, implies that efficient antioxidative effects on
arteriosclerosis might be expected even by catechins with a low degree of polymerization,
leading to the development of blood–brain barrier permeable procyanidin derivatives for the
treatment of cerebrovascular diseases. Indeed, proanthocyanidin analogue 1, in which the
geometry of one catechin molecule is constrained to be planar, increased the radical scavenging
activity compared to procyanidin B3 by 1.9 times, or a 7.2-fold increase over catechin. The
enhanced radical scavenging activity of procyanidin analogue 1 may be ascribed to the stability
of the radical cation of planar catechin in 1, which is produced in the electron transfer from 1 to
GO・. That is, the electron-donating i-propyl group at the planar catechin significantly stabilizes
1•+, resulting in the acceleration of the electron transfer step.27 Since the radical scavenging
activity of the monomeric planar catechin is 5-fold more potent than that of catechin, the
additive effect between catechin and planar catechin may be attained in radical scavenging
activity of compound 1 as opposed to the synergistic radical scavenging activities of two
catechins in procyanidin B3. The differences in radical scavenging mechanism between
procyanidin B3 and 1 are currently under investigation.
Finally, proanthocyanidin analogue 1, originally designed to increase the binding affinity of
procyanidin B3 with LOX-1, was found to be a potential antioxidant that is expected to reduce
the oxidative stress generated during the progression of arteriosclerosis. This is the first report of
the enhanced radical scavenging activities of procyanidin B3 and its synthesized derivative
proanthocyanidin analogue 1. Investigations are currently underway to better understand the
effect on OxLDL binding to LOX-1 to assess how these compounds may be better modified to
develop promising therapeutic agents for cerebrovascular diseases.

Figure 2. Spectral changes observed during the reaction between 1 (0.1 mM) and GO・(9.7 μM)
in deaerated MeCN at 298 K. Inset: the first-order plot based on the absorbance change at 428
nm due to GO・.
1
procyanidin B3
[procyanidin B3 or 1] / M

Figure 3. Plot of the pseudo-first-order rate constants (kobs) vs. [procyanidin B3 or 1] for the
reaction with GO・(9.7 μM) in deaerated MeCN at 298 K.

Acknowledgments
This work was partially supported by Grants-in-Aid for Scientific Research (No. 26460153)
form the Ministry of Education, Culture, Sports, Science and Technology, Japan.

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