Biomimetic synthesis of active isorhapontigenin dimers

Article abstract of DOI:10.1080/10286020.2014.913030

Synthetic isorhapontigenin was treated with several kinds of inorganic reagents and peroxidase so as to prepare active stilbene dimers. Among them, silver acetate in methanol gave two new isorhapontigenin dimers 4 and 5, together with four known natural stilbene dimers 2, 3, 6, and 7. Their structures and relative configurations were determined on the basis of spectral analysis, and their possible formation mechanisms were discussed, respectively. Compounds 2, 6, and 7 were artificially synthesized for the first time. All the products were evaluated for anti-inflammatory activities.

Full text of DOI:10.1080/10286020.2014.913030

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Biomimetic synthesis of active
isorhapontigenin dimers
Xian-Fen Wanga, Yuan Zhangab, Ming-Bao Lina, Qi Houa, Chun-Suo
Yaoa & Jian-Gong Shia
a State Key Laboratory of Bioactive Substance and Function of
Natural Medicines, Institute of Materia Medica, Chinese Academy
of Medical Sciences & Peking Union Medical College, Beijing
100050, China
b College of Chemistry and Material Science, Hebei Normal
University, Shijiazhuang 050024, China
Published online: 02 May 2014.
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To cite this article: Xian-Fen Wang, Yuan Zhang, Ming-Bao Lin, Qi Hou, Chun-Suo Yao & Jian-
Gong Shi (2014) Biomimetic synthesis of active isorhapontigenin dimers, Journal of Asian Natural
Products Research, 16:5, 511-521, DOI: 10.1080/10286020.2014.913030
To link to this article: http://dx.doi.org/10.1080/10286020.2014.913030
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Journal of Asian Natural Products Research, 2014
Vol. 16, No. 5, 511–521, http://dx.doi.org/10.1080/10286020.2014.913030
Biomimetic synthesis of active isorhapontigenin dimers
Xian-Fen Wanga, Yuan Zhanga,b, Ming-Bao Lina, Qi Houa, Chun-Suo Yaoa* and
Jian-Gong Shia*
aState Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of
Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing
100050, China; bCollege of Chemistry and Material Science, Hebei Normal University,
Shijiazhuang 050024, China
(Received 22 January 2014; final version received 3 April 2014)
Synthetic isorhapontigenin was treated with several kinds of inorganic reagents and
peroxidase so as to prepare active stilbene dimers. Among them, silver acetate in
methanol gave two new isorhapontigenin dimers 4 and 5, together with four known
natural stilbene dimers 2, 3, 6, and 7. Their structures and relative configurations were
determined on the basis of spectral analysis, and their possible formation mechanisms
were discussed, respectively. Compounds 2, 6, and 7 were artificially synthesized for
the first time. All the products were evaluated for anti-inflammatory activities.
Keywords: isorhapontigenin; biomimetic synthesis; stilbene dimer; silver acetate
1. Introduction
through one-electron oxidative coupling
reaction of the stilbene monomers in plants
[5]. Therefore, oxidative polymerization
with inorganic metals and enzymes as
oxidants was believed to be a concise way
to obtain active stilbene oligomers with
various structural skeletons. Shegansu B
(2), a natural isorhapontigenin dimer with
benzofuran framework, was isolated from
traditional Chinese medicine Brlamctmcla
chinensis and Gnetaceae, which showed
multi-faced activities, such as anti-virus,
anti-oxidant, and anti-inflammatory[6–11].
Nevertheless, to the best of our knowledge,
until now this compound was obtained only
through oxidative dimerization of isorha-
pontigenin using various oxidants and
enzymes. In our previous research, the
oxidative coupling reaction of isorhaponti-
genin using FeCl3·6H2O as an oxidant has
been investigated, and benzofuran deriva-
tives were found to be the major products
[12,13]. Recently, according to the litera-
ture, oxidative coupling reaction of
Oligostilbene is a type of natural product
possessing complex structures, which is
considered to be the oligomers of different
stilbene monomers. Isorhapontigenin (1)
and its oligomers are a class of plant
polyphenols that have attracted intense
interest over the past 30 years on account
of their intricate structures and diverse
biological activities [1,2]. However, as
many of these compounds are exclusively
obtained by extraction from natural
sources, the studies on their biological
properties are limited by their very scarce
availabilities [3]. Currently, the synthesis
of stilbene dimers and their analogs has
been a popular research topic. However,
only a few syntheses have so far been
reported because of the complex molecular
architectures of dimeric stilbenes. Thus,
their total synthesis remains a great
challenge for chemists [4].
In view of the biogenetic relationship,
oligostilbenes were supposed to be formed
*Corresponding authors. Email: yaocs@imm.ac.cn; shijg@imm.ac.cn
q 2014 Taylor & Francis

512
X.-F. Wang et al.
resveratrol using silver acetate as an oxidant
led to an exclusive dimer with high yield
[14]. Thereafter, silver acetate was sup-
posed to be an effective oxidant in oxidative
polymerization of the stilbene. In order to
obtain more isorhapontigenin oligomers
(especially dimers) with various structures
for research on the bioactivity properties,
studies on the oxidative coupling reaction of
isorhapontigenin using silver acetate as
oxidizing reagent resulted in the isolation
and identification of two new isorhaponti-
genin dimers 4 and 5, and three naturally
occurring isorhapontigenin dimers 3, 6, and
7, except for shegansu B (2, Figure 1).
Among the products, three natural dimers 3,
6, and 7 were synthesized for the first time.
On the basis of spectral analysis, their
structures were determined and their
possible formation mechanisms were also
discussed.
reported in the literature [15], the starting
material 3,5-dihydroxybenzoic acid (8)
was successively esterified, and benzy-
lated, followed by a reduction in the
methoxyl carbonyl group with lithium
aluminum hydride to afford 3,5-bis(benzy-
loxy)benzyl alcohol (11). Compound 11
was then converted to the benzyl iodide
(12), which was transformed into phospho-
nate (13). At last, isorhapontigenin was
yielded through the reaction of 13 and 4-
(benzyloxy)-3-methoxybenzaldehyde (15),
which was prepared from 3-methoxy-4-
hydroxybenzaldehyde (14), followed by
deprotection.
2.2 Transformation of isorhapontigenin
with oxidizing reagents, and horseradish
peroxidase and hydrogen peroxide
To our knowledge, until now the treatment
of isorhapontigenin with various oxidants
and peroxidase will produce the major
product of shegansu B [12,13,15,16].
Therefore, the content of shegansu B in
the reaction mixture was regarded as a
reference of biomimetic synthesis. Isorha-
pontigenin was treated with three inorganic
oxidizing reagents [Ag2O, AgOAc, and
2. Results and discussion
2.1 Synthesis of isorhapontigenin
In this study, synthetic isorhapontigenin
under the procedure shown in Scheme 1
was used for the material of biomimetic
synthesis. According to the method
H3CO
3a
MeO
OH
4a
MeO
4a
OMe
3b
MeO
HO
3a
A1
O
1a
7a
7a
1a
O
H3CO
HO
b4
1b
7b
HO
8a
HO
13b
9a
OH
13a
1b
7b
B1
O
OH
12b
HO
HO
9a
A2
4b
9b
B2
3b
9b
12a
11a
HO
OH
OCH3
OH
11b
HO
OH
OH
OH
3
4
2
H3CO
H
HO
4a
3a
H3CO
OH
H
H3CO
HO
OCH3
7a
1a
9a
OH
H3CO
A1
OH
HO
H3CO
12b
OH
7a
7b
9b
12a
8a
OH
1b
HO
12b
O
A2
OH
7b
B1
OH
4b
OH
1b
3b
H3CO
B2
3b
4b
OH
9b
OCH3
H3CO
OH
OH
HO
7
5
6
Figure 1. Structures of compounds 2–7.

Journal of Asian Natural Products Research
513
OBn
OH
OBn
O
e
a,b,c,d
COOH
I
P(OEt)2
BnO
HO
BnO
12
8
13
OBn
g,h
1
OH
HCO3
HCO3
f
CHO
CHO
14
15
Scheme 1. Synthesis of compound 1. Reaction conditions (a) CH3OH, H2SO4, reflux;
(b) PhCH2Br, K2CO3, acetone; (c) LiAlH4, Et2O, 08C; (d) PPh3, I2, Imidazole, Et2O, CH3CN,
08C; (e) P(OEt)3, tetra-n- butylammonium iodide, 1208C; (f) PhCH2Br, K2CO3, CH3COCH3, reflux;
(g) NaH, THF, r.t.; (h) AlCl3, PhNMe2, 08C.
K3Fe(CN)6], and horseradish peroxidase
and hydrogen peroxide in certain solvents
as shown in Table 1. The reaction mixtures
were then analyzed by HPLC equipped
with ODS column using a mixture of
acetonitrile/water 32:68 (v/v) as a mobile
phase at the flow rate of 1 ml/min. As a
result of analysis, horseradish peroxidase
and hydrogen peroxide in acetone/H2O
produced shegansu B in the 87.8% yield,
and nearly no minor products were
observed. Silver oxide and potassium
hexacyanoferrate (III) in acetone or
acetone/H2O at r.t. generated shegansu B
in 79.9% and 65.2% yield, respectively,
and several minor products were observed
in the HPLC chromatograms. However, the
reaction of isorhapontigenin and silver
acetate in methanol at 2108C produced
five side products except for the major one
shegansu B (Figure 2), and the higher
contents of the side products in the
mixtures were observed obviously. In
addition, the oxidative coupling reaction
of resveratrol with silver acetate as an
oxidant was reported to generate an
exclusive product with high yield [14],
but for isorhapontigenin, there is no such
investigation reported. In view of the
above, silver acetate was used to the
biomimetic synthesis of isorhapontigenin
oligostilbenes.
2.3 Biomimetic synthesis of
isorhapontigenin dimers with AgOAc as
an oxidant
Isorhapontigenin was treated with two
equivalent silver acetate in dry methanol
at 2108C to produce a mixture, which was
further isolated through silica gel column
chromatography (CC), preparative HPLC,
Table 1. Treatment of isorhapontigenin (1) with oxidizing reagents.
Reagents
Solvent
Temperature (8C)
Time
2 (%)a
AgOAc
Ag2O
HRP/H2O2
K3Fe(CN)6
MeOH
Acetone
Acetone/H2O
Acetone/H2O
210
25
25
25
80 min
24 h
1 h
60.6
79.9
87.8
65.2
4 days 17 h
a The percentage in this table means the “degree of transformation” (%DT) (see Section 3.3).

514
X.-F. Wang et al.
Figure 2. Analysis of isorhapontigenin oxidation products by AgOAc (32% CH3CN/H2O,
l ¼ 230 nm).
and semi-preparative HPLC to obtain six
isorhapontigenin dimers 2–6. Among
them, shegansu B (2) was the most
abundant product, and cis shegansu B (3),
one of minor products, was its isomers with
a cis olefinic bond. Products 4 and 5, two
diastereoisomers, were determined to be
new minor products with chain structure,
and products 6 and 7 were determined to be
minor products with indane framework.
Compounds 3 [6], 6 [8], and 7 [17],
possessing the same structure with the
corresponding natural products, were arti-
ficially synthesized for the first time.
Nevertheless, the optical inactivation indi-
cated that they were obtained as racemates,
not as optically active natural products.
genin dimer. Its UV spectrum indicated
the absorption bands at lmax (log 1) 288
(sh), 324 (3.99)nm, displaying the presence
of a long conjugated system. The IR
spectrum revealed the presence of hydroxyl
(nmax 3334 cm21), olefinic bond (nmax
1655cm21), and aromatic functionalities
(nmax 1603, 1512, 1454cm21). The NMR
spectrum of 4 (Table 2) showed two AB2
systems at dH 6.04 (1H, t, J ¼ 2.4 Hz, H-
12a), 6.10 (2H, d, J ¼ 2.4 Hz, H-10a, 14a),
and dH 6.15 (1H, t, J ¼ 1.8 Hz, H-12b),
6.43 (2H, d, J ¼ 1.8 Hz, H-10b, 14b) for
rings A2 and B2; two ABX systems at dH
6.63–6.64 (2H, m, H-2a, 6a), 6.67 (1H, d,
J ¼ 7.8 Hz, H-5a), and dH 7.10 (1H, d,
J ¼ 1.8 Hz, H-2b), 6.74 (1H, d, J ¼ 8.4 Hz,
H-5b), 6.86 (1H, dd, J ¼ 8.4, 1.8 Hz, H-6b)
for rings A1 and B1; two coupled olefinic
bond proton signals at dH 6.81 (1H, d,
J ¼ 16.2 Hz, H-7b) and 6.91 (1H, d,
J ¼ 16.2Hz, H-8b), two coupled aliphatic
protons at dH 4.49 (1H, d, J ¼ 7.8 Hz, H-
7a) and 5.10 (1H, d, J ¼ 7.8 Hz, H-8a), and
three methoxyl protons at dH 3.32 (3H, s,
OCH3-7a), 3.72 (3H, s, OCH3-3a), and 3.93
2.4 Structural identification of the
products
Compound 4 was obtained as an amorphous
powder. Its negative ion HR-ESI-MS data
at m/z 545.1819 [M 2 H]2 showed the
molecular formula of C31H30O9, which
suggested that 4 should be an isorhaponti-

Journal of Asian Natural Products Research
Table 2. 1H and 13C NMR spectroscopic data of 4 and 5.
515
4
5
Position
dC
dH
dC
dH
1a
2a
3a
4a
5a
6a
7a
8a
130.1
112.8
148.6
147.3
115.6
121.9
86.4
130.4
113.3
148.6
147.3
115.2
122.8
88.1
6.63–6.64 (m)
6.98 (br s)
6.67 (d, 7.8)
6.63–6.64 (m)
4.49 (d, 7.8)
5.10 (d, 7.8)
6.70 (d, 8.5)
6.68 (br d, 8.5)
4.41 (d, 4.5)
5.19 (d, 4.5)
85.7
84.7
9a
141.6
107.4
159.2
102.9
132.3
111.4
151.3
148.9
116.7
120.8
129.3
128.1
141.1
105.9
159.7
102.9
56.3
141.9
107.4
159.1
102.9
132.3
111.6
151.4
148.9
116.6
120.8
129.3
128.1
141.1
105.9
159.7
102.8
56.3
10 (14)a
11 (13)a
12a
1b
2b
3b
4b
5b
6b
7b
8b
9b
10 (14)b
11 (13)b
12b
3a-OCH3
3b-OCH3
7a-OCH3
6.10 (d, 2.4)
6.04 (t, 2.4)
7.10 (d, 1.8)
6.20 (d, 2.0)
6.13 (t, 2.0)
7.07 (br s)
6.74 (d, 8.4)
6.66 (d, 8.5)
6.86 (dd, 8.4, 1.8)
6.81 (d, 16.2)
6.91 (d, 16.2)
6.84 (br d, 8.5)
6.89 (d, 16.5)
6.79 (dd, 16.5)
6.43 (d, 1.8)
6.42 (d, 2.0)
6.15 (t, 1.8)
3.72 (s)
3.93 (s)
6.15 (t, 2.0)
3.82 (s)
3.84 (s)
56.8
57.4
56.9
57.2
3.32 (s)
3.23 (s)
Notes: Data (d) were measured in CD3OD for 1H NMR at 600 MHz, and for 13C NMR at 150 MHz. The
assignments were based on DEPT, 1H–1HCOSY, gHSQC, and HMBC experiments.
(3H, s, OCH3-3b). The above proton
signals, combined with 30 carbon signals
in the 13C NMR spectrum (Table 2),
indicated that 4 was a chain isorhaponti-
genin dimer connected with C-8a and 4b-
OH. Comparison of NMR spectral data of 4
with those reported in the literature [3]
exhibited that they had similar skeleton.
The HMBC correlations (Figure 3) of 4
between H-7a and C-2a, C-9a, OCH3-7a
substantiated that the methoxyl should be
located at C-7a; the HMBC correlations
between H-8a and C-1a, C-4b, C-10(14)a
confirmed that OH-4b linked to C-8a. Thus,
the structure of 4 was determined as shown
in Figure 1.
bonding of the benzylic hydroxyl and
aryloxyl groups, the large and small J values
for H-7 and H-8 of 8-O-40 neolignan
diastereoisomers correspond to the threo
formandtheerythroform, respectively[18].
Although the lack of hydroxyl group at C-7
made it impossible to become intramole-
cular hydrogen bonding in 4, compared with
8-O-40 neolignan reported in the literature,
conformational analysis of 4 revealed that
they had the similar conformers due to the
large substitution group of the aryl at C-8 in
place of the hydroxyl ethyl. Therefore, the
observation of the large coupling constant
(J7a,8a ¼ 7.8 Hz) in 4 suggested that C-7a
and C-8a should be in the threo form.
In terms of the possible staggered
conformers with intramolecular hydrogen
Compound 5 was obtained as an
amorphous powder. Its negative ion HR-

516
X.-F. Wang et al.
H3CO
H3CO
OH
OH
OH
H3CO
H3CO
HO
OH
HO
HO
O
O
OCH3
OCH3
HO
HO
HO
4
5
Figure 3. Significant HMBC correlations of 4 and 5.
ESI-MS data at m/z 545.1819 [M 2 H]2
showed the molecular formula of C31H30O9,
The other products 2, 3, 6, and 7 were
identified as shegansu B [6], cis shegansu
B [8], lehmbachol A, and lehmbachol B
[17], respectively, on the basis of spectro-
scopic data analysis (including NOESY
spectra for 3, 6, and 7) and comparison
with data reported in the literature.
which suggested that 5 should be an
1
isorhapontigenin dimer. The H and 13C
NMR spectra (Table 2) of 5 were nearly
identical to those of 4, while the main
difference was a small coupling constant
(J7a,8a ¼ 4.5 Hz) in 5 (Table 1) compared
with the large one (J7a,8a ¼ 7.8 Hz) in 4.
Extensive 2D NMR analyses, including
1H–1H COSY, HSQC, HMBC (Figure 3),
and NOE, suggested that 5 had the same
gross structure as that of 4, and they were
determined as a pair of diastereoisomers.
According to the same method as 4, the
relative configuration of 5 was determined
as the erythro form on the basis of the
observation of the small coupling constant
(J7a,8a ¼ 4.5 Hz), contrasting with the
threo form of 4. Thus, the structure of 5
was determined as a threo diastereoisomer
of 4, as depicted in Figure 1.
2.5 Discussion of the probable coupling
reaction mechanisms
On the basis of the results obtained, a
mechanism for the formation of the
different dimeric derivatives can be
proposed. The dimeric structures indicated
that the oxidation reactions took place at
the 4-OH position of the stilbene moieties
[3,19]. In turn, the formed phenoxy
radicals could delocalize themselves as
reported in Figure 4. The diversity of
coupling modes leads to the structural
complexity of the oligomers.
O
O
HO
O
OMe
OMe
OMe
OMe
Ag+
OH
HO
OH
OH
OH
HO
HO
HO
[A]
Figure 4. Important radicals of isorhapontigenin.
[B]
[C]

Journal of Asian Natural Products Research
517
O
OMe
MeO
OMe
OMe
O
O
HO
O
O
MeO
HO
MeO
HO
H
OH
OH
2
OH
OH
OH
HO
OH
OH
OMe
OH
isomerization
HO
[C]
[B]
HO
O
MeO
HO
HO
OH
OH
3
MeO
O
O
OH
OH
OMe
MeO
OMe
MeO
O
OMe
HO
H
HO
HO
HO
O
O
O
Me
HO
OMe
HO
OH
HO
HO
HO
OH
4 and 5
[C]
[A]
O
HO
O
MeO
OH
HO
H3CO
HO
HOMe
H3CO
HO
OCH3
OH
OH
OH
OH
OH
OH
HO
OH
MeO
O
H3CO
H3CO
O
[C]
OH
6 and 7
[C]
Figure 5. Proposed coupling mechanism for compounds 2–7.
Accordingly, the coupling of radical
[B] and radical [C], followed by tautomeric
rearrangement and intramolecular nucleo-
philic attack to the intermediate quinone,
gave the dihydrofuran dimer 2, which
isomerized to form its isomer 3. The
formation of the chain dimers 4 and 5 can
be easily explained by the coupling of
radical [A] with radical [C], followed by an
addition of a methanol molecule to the
intermediate quinones. Finally, the coup-
ling of two radicals [C], followed by
intramolecular cyclization, and an addition
of a methanol molecule to the intermediate
quinones, generated 6 and its isomer 7
(Figure 5). In general, free radical reaction
is nonstereoselective. In the addition
reaction of methanol, the chances of
addition to Re face or Si face of the
intermediates are equal. Therefore, all of
the products were found to be racemates.
2.6 Anti-inflammatory activities of the
products
All the products were assessed for anti-
inflammatory activities in the in vitro
bioassay. Among them, compounds 2 and
6 showed weak activity against nitrogen
oxide (NO) production in macrophages
induced by lipopolysaccharide (LPS) at
the concentration of 1025 M, with inhi-
bition ratios of 24.1% and 36.0%, respect-
ively, but the other four compounds were
inactive. The positive control dexametha-

518
X.-F. Wang et al.
sone (DEX) gave a 82.2% inhibition at the
same concentration.
3.3 Evaluation of the reactivity
Degree of transformation (DT) means the
amount of isorhapontigenin (1), which was
consumed to form stilbene dimers, and the
values (%DT) are quantified on the basis
of the area of the peak from the analysis
results of the HPLC [20].
3. Experimental
3.1 General experimental procedures
Optical rotations were measured on a
P2000 polarimeter (JASCO, Tokyo,
Japan). UV spectra were obtained on a
JASCOP650 spectrometer (JASCO). IR
spectra were recorded on a Nicolet 5700
FT-IR microscope instrument (FT-IR
microscope transmission; Thermo Electron
Corporation, Madison, WI, USA). 1D and
2D NMR spectra were acquired at 500 or
3.4 Treatment of isorhapontigenin with
oxidizing reagents
3.4.1 Treatment with silver acetate
To a solution of isorhapontigenin (1,
100 mg, 0.3876 mmol) in dry methanol
(30 ml), AgOAc (64.7 mg, 0.388 mmol)
was added at 2108C. After stirring for
80 min at 2108C, the reaction mixture was
diluted with water and extracted with ethyl
acetate. The organic layer was then
washed with brine, water, and dried over
anhydrous sodium sulfate and evaporated.
The residue was analyzed by HPLC with
ODS column using a mixed solvent of
acetonitrile/water (32:68) as an eluent at
the flow rate of 1 ml/min, and the amount
of a product was quantified on the basis of
the area of the peak, shegansu B (2, 60.6%
DT). Then, the crude product was
subjected to preparative HPLC equipped
with ODS column using a mixed solvent of
methanol/water (47:53) as an eluent to
afford fraction 1 (11.7 mg), fraction 2
(7.8 mg, recovered isorhapontigenin,
7.80%), fraction 3 (26.1 mg), and fraction
4 (53.2 mg). Fraction 1 was submitted to
preparative HPLC (acetonitrile/water
25:75) to give compounds 6 (3.1 mg,
2.94%) and 7 (2.8 mg, 2.63%). Fraction 3
was submitted to preparative HPLC
(acetonitrile/water 25:75) to afford com-
pounds 4 (5.0 mg, 4.74%) and 5 (6.1 mg,
5.77%). Fraction 4 was submitted to
preparative HPLC (methanol/water
47:53) to yield compounds 2 (47.6 mg,
47.43%) and 3 (4.5 mg, 4.51%).
1
600 MHz for H and 125 or 150 MHz for
13C, respectively, on Varian INOVA
500 MHz, or Bruker AVANCE III HD
600 MHz (Bruker Corporation, Karlsruhe,
Germany), in chloroform, acetone-d6, or
methanol-d4, with solvent peaks as refer-
ences. ESI-MS and HR-ESI-MS data were
measured
using
an
AccuToFCS
JMST100CS spectrometer (Agilent Tech-
nologies, Ltd, Santa Clara, CA, USA). CC
was carried out with silica gel (200–300
mesh, Qingdao Marine Chemical, Inc.,
Qingdao, China) and Pharmadex LH-20
(Amersham Biosciences, Inc., Shanghai,
China). Preparative TLC separation was
carried out with high-performance silica
gel TLC plates (HSGF254, glass precoated;
Yantai Jiangyou Silica Gel Development
Co., Ltd, Yantai, China). HPLC separation
was carried out on an instrument consisting
of a Waters 515 pump and a Waters 2487
dual l absorbance detector (Waters Cor-
poration, Milford, MA, USA) with a YMC
(250mm £ 10 mm i.d.) semi-preparative
column packed with C18 (5 mM). TLC
was carried out with glass precoated silica
gel GF254 plates (Qingdao Marine Chemi-
cal, Inc.). Spots were visualized under UV
light or by spraying with 7% H2SO4 in 95%
EtOH followed by heating.
3.2 Synthesis of isorhapontigenin
Isorhapontigeninwassynthesizedaccording
to the method reported in the literature [15].
3.4.1.1 Shegansu B (2). Gray amorphous
1
powder; H NMR (500 MHz, CD3COCD3)

Journal of Asian Natural Products Research
519
d: 7.16 (1H, br s), 7.04 (1H, br s), 7.03 (1H, d,
J ¼ 16.0 Hz), 6.92 (1H, d, J ¼ 16.0 Hz), 6.84
(1H, br d, J ¼ 9.5 Hz), 6.82 (1H, br s), 6.81
(1H, d, J ¼ 8.0Hz), 6.52(2H, d,J ¼ 1.5 Hz),
6.27 (1H, t, J ¼ 1.5 Hz), 6.25 (1H, t, J ¼ 1.5
Hz), 6.19 (2H, d, J ¼ 1.5 Hz), 5.44 (1H, d,
J ¼ 8.5 Hz), 4.51 (1H, d, J ¼ 8.5 Hz), 3.96
(3H, s), 3.92 (3H, s); ESI-MS m/z: 515
[M þ H]þ, 537 [M þ Na]þ, 553 [M þ K]þ.
2937, 1654, 1604, 1512, 1454, 1349, 1270,
1154, 1033, 1008, 847, 710, 689 cm21; for
1H NMR (600 MHz, CD3OD) and 13C NMR
(150 MHz, CD3OD) spectral data, see
Table 2; HR-ESI-MS m/z: 545.1819
[M 2 H]2 (calcd for C31H29O9, 545.1817).
3.4.1.5 Lehmbachol A (6). Light brown
20
amorphous powder; ½aꢀD 0 (c ¼ 1.10,
1
MeOH); H NMR (500 MHz, CD3OD) d:
3.4.1.2 cis-Shegansu B (3). Yellow amor-
1
phous powder; H NMR (500 MHz, CD3-
6.63 (1H, d, J ¼ 8.5 Hz), 6.61 (1H, d,
J ¼ 1.0Hz), 6.59 (1H, d, J ¼ 8.0 Hz), 6.46
(1H, br s), 6.33 (2H, br d, J ¼ 7.5 Hz), 6.26
(1H, br s), 6.20 (1H, d, J ¼ 1.0Hz), 5.94(1H,
t, J ¼ 1.5 Hz), 5.67 (2H, d, J ¼ 1.5 Hz), 4.11
(1H, d, J ¼ 2.5 Hz), 3.87 (1H, d, J ¼ 9.0 Hz),
3.67 (3H, s), 3.56 (3H, s), 3.03 (3H, s), 2.65
(1H, t, J ¼ 2.0 Hz); 13C NMR (125 MHz,
CD3OD) d: 159.5 (3 £ C), 155.7, 151.5,
150.1, 149.0 (2 £ C), 147.4, 145.9, 139.1,
132.8, 123.1, 122.2, 121.2, 116.1, 115.9,
112.8, 112.7, 106.6 (2 £ C), 103.0, 101.5,
89.8, 61.1, 60.2, 57.1, 56.8, 56.6, 56.5; ESI-
MS m/z: 569.2 [M þ Na]þ, 545.4
[M 2 H]2.
COCD3) d: 7.01 (1H, br s), 6.94 (1H, br s),
6.81 (2H, m), 6.54 (2H, br s), 6.44 (1H, d,
J ¼ 12.0 Hz), 6.35 (1H, d, J ¼ 12.0 Hz), 6.34
(2H, br s), 6.23 (2H, br s), 6.14 (2H, d,
J ¼ 1.5 Hz), 5.36 (1H, d, J ¼ 8.5 Hz), 4.46
(1H, d, J ¼ 8.0 Hz), 3.82 (3H, s), 3.66 (3H,
s); 13C NMR (125 MHz, CD3COCD3) d:
159.6 (2 £ C), 159.2 (2 £ C), 148.5, 148.4,
147.7 (2 £ C), 144.5, 140.5, 132.4, 132.0,
131.6, 130.9, 129.2, 120.4, 119.5, 115.6,
113.4, 110.9, 108.1 (2 £ C), 107.5 (2 £ C),
102.5, 102.4, 94.5, 58.1, 56.3, 55.9; ESI-MS
m/z: 515.4 [M þ H]þ, 553.2 [M þ K]þ.
3.4.1.3 threo-5-[1-[4-[(1E)-2-(3,5-Dihy-
droxyphenyl)ethenyl]-3-methoxyphenoxy]
-2-methoxy-2-(4-hydroxy-3-methoxyphe-
nyl)ethyl]-1,3-benzenediol (4). Gray amor-
3.4.1.6 Lehmbachol B (7). Light brown
20
20
amorphous powder; ½aꢀD 0 (c ¼ 1.07,
phous powder; ½aꢀD 0 (c ¼ 1.05, MeOH);
1
MeOH); H NMR (500 MHz, CD3OD) d:
UV (MeOH) lmax (log 1): 288 (sh), 324
(3.99) nm. IR (film) nmax: 3334, 2972, 2934,
1655, 1603, 1512, 1454, 1349, 1266, 1223,
1154, 1035, 1011, 978, 961, 876, 846, 696
cm21; for 1H NMR (500 MHz, CD3OD) and
13C NMR (125 MHz, CD3OD) spectral data,
see Table 2; HR-ESI-MS m/z: 545.1819
[M 2 H]2 (calcd for C31H29O9, 545.1817).
6.75 (1H, br s), 6.64 (1H, d, J ¼ 8.0 Hz),
6.55 (1H, d, J ¼ 8.0 Hz), 6.54 (2H, br d,
J ¼ 9.0 Hz), 6.32 (1H, br s), 6.28 (2H, br d,
J ¼ 8.0 Hz), 6.08 (1H, br s), 6.05 (1H, br
s), 6.04 (1H, m), 6.03 (2H, br s), 4.08 (1H,
d, J ¼ 5.0 Hz), 3.94 (1H, d, J ¼ 7.5 Hz),
3.64 (3H, s), 3.60 (3H, s), 3.48(1H, t,
J ¼ 6.0 Hz), 3.11 (1H, t, J ¼ 5.0 Hz), 3.02
(3H, s); 13C NMR (125 MHz, CD3OD) d:
159.7 (2 £ C), 159.1, 155.7, 150.5, 149.1,
149.0 (2 £ C), 147.6, 147.3, 145.8, 139.2,
132.5, 124.2, 123.4, 121.5, 115.9, 115.7,
113.0, 112.2, 107.2, 106.4, 102.9, 101.7,
88.3, 60.9, 60.3, 57.4, 57.1, 56.6, 56.5;
ESI MS m/z: 569.3 [M þ Na]þ, 545.4
[M 2 H]2.
3.4.1.4 erythro-5-[1-[4-[(1E)-2-(3,5-
Dihydroxyphenyl)ethenyl]-3-methoxyphe-
noxy]-2-methoxy-2-(4–hydroxy-3-methoxy-
phenyl)ethyl]-1,3-benzenediol (5). Gray
20
amorphous powder; ½aꢀD 0 (c ¼ 0.918,
MeOH); UV (MeOH) lmax (log 1): 288
(sh), 324 (4.07) nm; IR (film) nmax: 3359,

520
X.-F. Wang et al.
3.4.2 Treatment with silver (II) oxide
3.4.5 Inhibition assay of NO production
in macrophage
To a solution of isorhapontigenin (1,
10 mg, 0.0388 mmol) in dry acetone
(3.3 ml), Ag2O (10 mg, 0.0432 mmol)
was added at r.t. After stirring for 24 h,
the reaction mixture was filtered and
evaporated under reduced pressure. The
residue was then analyzed by HPLC with
ODS column using a mixture of aceto-
nitrile/water 32:68 as a mobile phase at
the flow rate of 1 ml/min, and the
amount of product was quantified on
the basis of the area peak, Shegansu B
(2, 56.7%DT).
Mouse peritoneal macrophages (PEMTM
were treated with carrier control
(DMSO only), LPS, or test compounds
(LPS þ compounds of 1 £ 1026 M, final
concentration), or DEX (1 £ 1026 M, final
concentration) [21]. After cells (5 £ 105
cells) were incubated at 378C, 5% CO2 for
24 h, NO was monitored by measuring
nitrite levels in culture media using the
Griess reagent. Absorbance was measured
at 540 nm after incubating culture media
with the Griess reagent for 10 min. Treated
cells were incubated with 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide (2 mg/ml) for 4 h. Media
were then removed, and absorbance was
assayed at 540 nm. Analysis of variance
was used for all statistical analyses with
independent experiments and data are
represented as means ^ standard errors of
the means. Individual values were com-
pared by Student’s t-test, and a P-value of
,0.05 is considered as significant.
)
3.4.3 Treatment with horseradish
peroxidase
To a solution of isorhapontigenin (1,
10 mg, 0.0388 mmol) and HRP (0.1 mg)
in the mixture of acetone (0.6 ml) and water
(0.2 ml), 3% H2O2 (0.25 ml) was added at r.
t. The reaction mixture was stirred for 1 h,
diluted with water, and extracted with ethyl
acetate. Then, the organic layer was
washed with brine, water, and evaporated.
The residue was analyzed with the same
method mentioned above.
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3.4.4 Treatment with potassium
hexacyanoferrate (III)
To a solution of isorhapontigenin (1,
10 mg, 0.0388 mmol) in acetone (2 ml), a
solution of K3Fe(CN)6 (15.0 mg,
0.0456 mmol) and NaOAc (14 mg,
0.1707 mmol) in water (2.5 ml) was
added dropwise at 08C under N2 atmos-
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08C for 1 h, then raised to r.t. After
stirring for 4 days 17 h, the mixture was
diluted with water and extracted with
ethyl acetate. The organic layer was
then washed with brine, water, and
evaporated in vacuo. Then, the residue
was analyzed with the same method
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