Biocatalytic production of acyclic bis[bibenzyls] from dihydroresveratrol by crude Momordica charantia peroxidase

Article abstract of DOI:10.1002/cbdv.200800229

Biotransformation of dihydroresveratrol by crude Momordica charantia peroxidase provided six new acyclic bis[bibenzyls] 1-6. Their structures were established on the basis of NMR and MS analyses as C-C, C-O-C, and C-CH ₂-C dimers of dihydroresv

Full text of DOI:10.1002/cbdv.200800229

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
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Biocatalytic Production of Acyclic Bis[bibenzyls] from Dihydroresveratrol by
Crude Momordica charantia Peroxidase
by Chun-Feng Xiea), Hui-Qing Yuanb), Jian-Bo Qua), Jie Xinga), Bei-Bei Lꢀa), Xiao-Ning Wanga),
Mei Jia), and Hong-Xiang Lou*a)
a) School of Pharmaceutical Sciences, Shandong University, No. 44 West Wenhua Road, Jinan 250012,
P. R. China (phone: þ86-531-8382012; fax: þ86-531-8382019; e-mail: louhongxiang@sdu.edu.cn)
b) School of Medicine, Shandong University, 44 West Wenhua Road, Jinan 250012, P. R. China
Biotransformation of dihydroresveratrol by crude Momordica charantia peroxidase provided six new
acyclic bis[bibenzyls] 1–6. Their structures were established on the basis of NMR and MS analyses as
CꢀC, CꢀOꢀC, and CꢀCH2ꢀC dimers of dihydroresveratrol. Compounds 1–6 were tested for
antiproliferative activity against human prostate cancer PC3 cell line in vitro, and 2 and 6 were found to
be more potent than the parent compound.
Introduction. – Biotransformation is considered to be an economical ꢀgreenꢁ
technology used to convert and diversify large-scale, readily available materials into
various derivatives for biological assessments [1]. Momordica charantia peroxidase
(MCP) is an easily obtainable peroxidase from the fruits of M. charantia. MCP has
several advantages including economical source, enzyme pH stability, and thermo-
stability, compared to the widely used horseradish peroxidase [2]. MCP has been a
useful tool to facilitate oxidative coupling of substrates for new compounds with
possible pharmacological activities [3–5]. In the previous study [6], we have
successfully synthesized eight oligomers of resveratrol and ferulic acid including one
new heterocoupling oligomer by crude peroxidase extraction from M. charantia.
Acyclic bis[bibenzyls] are naturally occurring stilbenoids found in liverworts [7–9],
ferns [10][11], Moraceae [12][13], and Orichidaceae [14][15]. This family of natural
molecules has exhibited anticancer [16], antioxidant [17], antiplatelet [18], and
tyrosinase inhibitory [11] activities. However, limited availability and poor structural
diversity among natural sources has hindered detailed pharmacological studies. Acyclic
bis[bibenzyls] can be biosynthesized through free radical-mediated dimerization of
phenolic bibenzyls [19]. The liverwort peroxidase may even participate in the
biogenesis of naturally occurring acyclic bis[bibenzyls] in plants [20].
Dihydroresveratrol (DR) is a major metabolite of resveratrol in humans, and
preliminary experiments showed antiproliferative effects of DR on the human oral
epithelial SCC-9 cells [21]. Here, we report an easy method to prepare new acyclic
bis[bibenzyl] derivatives utilizing DR as starting material and crude MCP as a catalyst.
This coupling reaction provided six compounds, and their antiproliferative activities
against PC3 human cancer cell line were evaluated.
ꢂ 2009 Verlag Helvetica Chimica Acta AG, Zꢃrich

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
Results and Discussion. – 1. HPLC-DAD/MS Analysis of the Reaction Products
from Crude MCP/H2O2 Oxidation of DR. The substrate DR was prepared by catalytic
hydrogenation of resveratrol according to the procedure described in [22]. The HPLC
chromatogram of the reaction mixture of the MCP/H2O2 oxidation of DR revealed the
formation of seven major products (Fig. 1). The Q1 scan spectra of the LC eluted
compounds 1–7 in the negative-ion mode showed [MꢀH]ꢀ peaks at m/z 457, 457, 471,
457, 457, 457, and 457, respectively, which suggested the presence of DR dimerization
products. Among them, peak 3 was a special one with an additional 14 atom mass units.
The six major products 1–6 (Fig. 2), corresponding to peaks 1–6, were isolated by
semi-preparative HPLC, and their structures were established by MS and NMR
techniques.
Fig. 1. The HPLC-DAD chromatogram obtained from the reaction mixture formed by peroxidase/H2O2
oxidation of dihydroresveratrol
2. Structure Elucidation of Compounds 1–6. Compound 1 was obtained as a
yellowish amorphous powder. HR-ESI-MS Analysis of 1 gave a molecular formula of
1
C28H26O6 (m/z 459.1806, [MþH]þ ). The H-NMR spectrum of 1 was very similar to
that of DR except for the disappearance of the HꢀC(14) resonance (Table 1). Based
on the molecular formula and NMR spectra, compound 1 appeared to be a symmetric
dimer of DR. The HMBC analysis confirmed that compound 1 comprised two 14-
substituted1) DR moieties. Thus, compound 1 was established as 6,6’-bis[2-(4-
hydroxyphenyl)ethyl]-1,1’-biphenyl-2,2’,4,4’-tetraol or 14–14’-diDR. Compound 1 is
an acyclic bis[bibenzyl] of the plagilin type [13][23].
Compound 2 was isolated as a yellowish amorphous powder with the same
molecular formula as 1 determined by HR-ESI-MS and NMR analysis (Table 2), but
with an asymmetrical linkage of two molecules of DR. The HꢀC(5’)1) signal at d(H)
6.96 showed HMBC correlations with the 13C resonances at d(C) 154.3 (C(1’)), 129.3
(C(3’)), 37.8 (C(7’)), and 116.6 (C(14)), which confirmed the formation of the ꢀ14,6’-
biphenylꢁ bond. Consequently, the structure of 2 was concluded to be 5’-[2-(3,5-
1
)
Arbitrary C-atom numbering.

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
1195
Fig. 2. The structures of compounds 1–6
Table 1. 1H- and 13C-NMR Data of Compounds 1, 3, and 4. At 600 and 150 MHz, resp., in (D6)acetone; d
in ppm, J in Hz.
Positiona)
1
3
4
d(H)
d(C)
d(H)
d(C)
d(H)
d(C)
1,1’
2,2’
3,3’
4,4’
5,5’
6,6’
7,7’
8,8’
156.2
115.7
129.9
134.1
129.9
115.7
36.7
156.1
115.7
130.2
134.0
130.2
115.7
37.6
127.0
117.2
129.5
134.8
132.6
152.7
37.6
6.64 (d, J¼8.4)
6.68 (d, J¼8.4)
6.69 (d, J¼8.4)
6.94 (d, J¼8.4)
6.90 (d, J¼8.1)
7.10 (dd, J¼8.1, 2.1)
6.68 (d, J¼8.4)
6.64 (d, J¼8.4)
2.43–2.58 (m)
2.43–2.58 (m)
6.94 (d, J¼8.4)
6.69 (d, J¼8.4)
2.42–2.44 (m)
2.75–2.78 (m)
7.04 (d, J¼2.1)
2.75–2.78 (m)
2.84–2.87 (m)
37.6
37.7
39.1
9,9’
144.8
108.5
158.6
101.2
157.3
114.0
144.4
109.5
156.8
101.4
156.6
118.2
21.4
145.1
107.8
159.3
101.1
159.3
107.8
10,10’
11,11’
12,12’
13,13’
14,14’
14,14’-CH2
6.41 (d, J¼2.3)
6.36 (d, J¼2.3)
6.30 (d, J¼2.5)
6.26 (d, J¼2.5)
6.25 (d, J¼2.1)
6.22 (t, J¼2.1)
6.25 (d, J¼2.1)
4.02 (s)
a) Arbitrary C-atom numbering as indicated in formula 1.

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
dihydroxyphenyl)ethyl]-6-[2-(4-hydroxylphenyl)ethyl]-1,1’-biphenyl-2,2’,4-triol
14,6’-diDR. It is an isoplagilin-type bis[bibenzyl] [23].
or
HR-ESI-MS of compound 3 indicated a molecular formula of C29H28O6. The NMR
spectra of 3 were similar to those of 1 except for additional signals at d(H) 4.02 (s, 2 H)
and d(C) 21.4 (Table 1), indicating the presence of the bis[benzylic] CH2 group
[24][25]. The location of this CH2 bridge must be between C(14) and C(14’) based on
the long-range correlations of CH2 with C(14) (d(C) 118.2), C(9) (d(C) 144.4), and
C(13) (d(C) 156.6) in the HMBC spectrum. Therefore, the structure of 3 was finally
identified as 4,4’-methylenebis{5-[2-(4-hydroxyphenyl)ethyl]benzene-1,3-diol} or 14,14’-
methylene-diDR. Compound 3 is an exceptional example from the reaction possessing
a diphenylmethane moiety. This is the first report of an acyclic bis[bibenzyl] with a
CꢀCH2ꢀC linkage of the benzene nucleus in a bibenzyl monomer.
Table 2. 1H- and 13C-NMR Data of Compounds 2, 5, and 6. At 600 and 150 MHz, resp., in (D6)acetone; d
in ppm, J in Hz.
Positiona)
2
5
6
d(H)
d(C)
d(H)
d(C)
d(H)
d(C)
1
2
3
4
5
6
7
8
156.1
115.7
130.0
133.9
130.0
115.7
37.1
158.0
130.0
130.1
135.8
130.1
130.0
37.9
157.1
117.4
130.2
136.7
130.2
117.4
37.4
6.64 (d, J ¼ 8.4)
6.76 (d, J ¼ 8.4)
6.75 (d, J ¼ 8.6)
7.15 (d, J ¼ 8.6)
6.77 (d, J ¼ 8.5)
7.17 (d, J ¼ 8.6)
6.76 (d, J ¼ 8.4)
6.64 (d, J ¼ 8.4)
2.46–2.83 (m)
2.46–2.83 (m)
7.15 (d, J ¼ 8.6)
6.75 (d, J ¼ 8.6)
2.58–2.82 (m)
2.58–2.82 (m)
7.17 (d, J ¼ 8.6)
6.77 (d, J ¼ 8.5)
2.74–2.84 (m)
2.74–2.84 (m)
37.6
39.1
38.7
9
144.2
108.7
128.4
101.3
156.8
116.6
154.3
116.4
129.3
133.8
133.2
124.0
37.8
145.1
107.7
159.3
101.0
159.3
107.7
156.3
115.8
115.5
133.5
115.5
115.8
36.3
145.0
107.6
159.3
101.1
159.3
107.6
143.8
117.6
125.6
134.6
121.7
147.9
37.4
10
11
12
13
14
1’
2’
3’
4’
5’
6.36 (d, J ¼ 2.3)
6.33 (d, J ¼ 2.3)
6.25 (d, J ¼ 2.1)
6.18 (t, J ¼ 2.1)
6.25 (d, J ¼ 2.1)
6.24 (d, J ¼ 2.1)
6.18–6.20 (m)
6.24 (d, J ¼ 2.1)
6.88 (d, J ¼ 8.1)
6.68 (d, J ¼ 8.4)
6.89 (d, J ¼ 8.4)
6.90 (d, J ¼ 0.9)
6.90 (d, J ¼ 0.9)
7.11 (dd, J ¼ 8.1, 2.2)
6.96 (d, J ¼ 2.2)
6.89 (d, J ¼ 8.4)
6.68 (d, J ¼ 8.4)
2.58–2.82 (m)
2.58–2.82 (m)
6.74 (t, J ¼ 0.9)
6’
7’
8’
2.46–2.83 (m)
2.46–2.83 (m)
2.69–2.76 (m)
2.69–2.76 (m)
39.4
33.8
38.8
9’
145.3
107.7
159.3
101.1
159.3
107.7
137.3
108.4
155.9
102.5
151.7
133.9
144.8
107.8
159.2
101.1
159.2
107.8
10’
11’
12’
13’
14’
6.27 (d, J ¼ 2.1)
6.18 (t, J ¼ 2.1)
6.27 (d, J ¼ 2.1)
6.30 (d, J ¼ 2.8)
6.39 (d, J ¼ 2.8)
6.16 (d, J ¼ 2.1)
6.18–6.20 (m)
6.16 (d, J ¼ 2.1)
a) Arbitrary C-atom numbering as indicated in formula 1.

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
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Compound 4 had the same molecular formula and a symmetric structure as 1, based
1
on the HR-MS and NMR evidences. The H-NMR spectrum of 4 was very similar to
that of DR except for the disappearance of the HꢀC(6) resonance (Table 1). The
1H-NMR spectrum also indicated the presence of 1,2,4-substituted aromatic rings
(d(H) 7.10 (dd, J ¼ 2.1, 8.1), 7.04 (d, J ¼ 2.1), and 6.9 (d, J ¼ 8.1)) in the molecule,
suggesting a 6,6’-linkage of two substrate molecules. HMQC and HMBC spectra also
confirmed the formation of 5,5’-[2,2’-(6,6’-dihydroxy-1,1’-biphenyl-3,3’-diyl)bis(ethane-
2,1-diyl)]di[benzene-1,3-diol] (6,6’-diDR). Compound 4 belongs to the family of
isoperrottetin-type bis[bibenzyls] [26][27].
The molecular formula of compound 5, C28H26O6, was deduced by HR-ESI-MS
1
(m/z 459.1801, [MþH]þ ). In the H-NMR spectrum, the absence of one OH signal
indicated a possible biphenyl ether linkage. According to their coupling patterns, three
H-atoms (d(H) 6.25 (d, J ¼ 2.1, HꢀC(10), HꢀC(14)) and 6.18 (t, J ¼ 2.1, HꢀC(12)))
were on a 1,3,5-trisubstituted benzene ring (ring B), two H-atoms (d(H) 6.39 (d, J ¼
2.8, HꢀC(12’)) and 6.30 (d, J ¼ 2.8, HꢀC(10’))) belonged to a 1,2,3,5-tetrasubstituted
benzene ring (ring D), and eight H-atoms (d(H) 7.15 (d, J ¼ 8.6, HꢀC(3), HꢀC(5)),
6.89 (d, J ¼ 8.4, HꢀC(3’), HꢀC(5’)), 6.75 (d, J ¼ 8.6, HꢀC(2), HꢀC(6)), and 6.68 (d,
J ¼ 8.4, HꢀC(2’), HꢀC(6’))) were on two 1,4-disubstituted benzene rings (rings A and
C), respectively. Furthermore, the downfield-shifted resonance for C(14’) (d(C) 133.9)
confirmed the linkage between rings A and D as a C(1)ꢀOꢀC(14’) bridge. Thus,
compound 5 was characterized as 4-{4-[2-(3,5-dihydroxyphenyl)ethyl]phenoxy}-5-[2-
(4-hydroxyphenyl)ethyl]benzene-1,3-diol or C(1)ꢀOꢀC(14’)-diDR. This is the first
example of acyclic bis[bibenzyls] with a C(1)ꢀOꢀC(14’) bridge.
The molecular formula C28H26O6 of compound 6 was determined by HR-ESI-MS
(m/z 459.1802, [MþH]þ ). The similar disappearance of the one OH signal in the
1H-NMR spectrum as in that of compound 5 indicated the formation of a diarylether
bond. The characteristic resonance at d(H) 6.74 (HꢀC(5’)) suggested that C(1) (d(C)
157.1) and C(6’) (d(C) 147.9) were linked by an ether bridge [28]. Compound 6 was thus
assigned as 5-[2-(4-{5-[2-(3,5-dihydroxyphenyl)ethyl]-2-hydroxyphenoxy}phenyl)e-
thyl]benzene-1,3-diol or C(1)ꢀOꢀC(6’)-diDR, which is an acyclic bis[bibenzyl]
belonging to perrottetin derivatives [9].
Compounds 1–6 were all produced by the coupling of DR in a manner readily
rationalized by free-radical reactions. One-electron oxidation mediated by crude MCP
can lead to the formation of C(ortho or para)–C(ortho or para) bonds and ether
linkages [19]. Compound 3 was an exception due to the appearance of a bisbenzylic
CH2 bridge in this molecule. The structure was possibly achieved by a carbene-
mediated radical process, which needed to be further confirmed. The possible
biotransformation pathway for compounds 1, 2, and 4–6 catalyzed by crude MCP is
shown in the Scheme. Besides the obtained six derivatives, another peak 7 with the
molecular weight 458 was not purified successfully. However, its structure was
proposed as a dimer of DR based on a first-order MS spectrum, possibly resulting from
.
the radical reactions between D6 and other radical types of DR.
3. Antiproliferative Effect on PC3 Human Prostate Cancer Cells. Our ongoing
research on the anticancer program [29] led us to test the antiproliferative effect of DR
and the transformed products 1–6 on PC3 human prostate cancer cell line. MTT Test
results (Table 3) indicated that compounds 2 and 6 exhibited more potent inhibitory

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
Scheme. A Proposed Biotransformation Pathway of Dihydroresveratrol to Compounds 1, 2, and 4–6 by
crude Momordica charantia Peroxidase
effect compared to DR in vitro, while other compounds were essentially inactive with
IC50 values up to 50 mm. These data suggested that the introduction of C(14)ꢀC(6’) and
C(1)ꢀOꢀC(6’) linkages increased the antiproliferative effect. These results might
provide useful information for the synthesis of acyclic bis[bibenzyl] of pharmaceutical
interest.
Table 3. Antiproliferative Effect of DR and Compounds 1–6 on PC3 Cancer Cell Line
Substrate and products
IC50 [mm]a)
Dihydroresveratrol
35ꢁ5
>50
1
2
3
4
5
6
14ꢁ2
>50
>50
>50
17ꢁ2
a) The results are meansꢁstandard deviation of three independent replicates.

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
1199
This work was financially supported by the National Natural Science Foundation of China (Nos.
30672531 and 30730109) and Shandong Provincial Foundation for Science Research (No.
2006GG1102023 and No. 2005GG3202107).
Experimental Part
General. The fresh fruits of M. charantia, identified by Dr. Xue-Sen Wen of Shandong University,
were purchased from a local supermarket. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
hydrobromide (MTT) was obtained from Sigma Chemical Co. (St. Louis, MO), and the manufacturers
directions were followed to measure the cancer cell viability. Human prostate cancer PC3 cell line was
obtained from The American Type Culture Collection (Rockville, MD). Spots on TLC plates were
visualized by spraying with 10% H2SO4/EtOH, followed by heating. Silica gel (200–300 mesh) for
column chromatography (CC) and high-performance TLC plates precoated with silica gel GF254 were
purchased from Qingdao Haiyang Chemical Co., Ltd. IR Spectra: in KBr disks, Thermo-Nicolet 670
spectrophotometer; in cmꢀ1. 1H- and 13C-NMR spectra: Bruker Avance-600 spectrometer at 600 (1H) and
150 (13C) MHz, chemical shifts in ppm as d rel. to Me4Si (internal standard), J in Hz. HR-ESI-MS, ESI-
MS, and HR-EI-MS: Agilent LC/time-of-flight (TOF) MS instrument, API 4000, and Waters GCT mass
spectrometer, resp; in m/z (rel.).
Preparation of Dihydroresveratrol. The substrate DR was prepared by catalytic hydrogenation of
commercially available resveratrol (5.0 g; Shanxi Scidoor Hi-Tech Biology Co., Ltd., P. R. China) with
5% Pd/C catalyst in MeOH (120 ml) for 12 h at r.t. and atmospheric pressure. The catalyst was filtered off
and washed with MeOH. The solvent was then removed under reduced pressure, and the residue was
chromatographed over silica gel (hexane/AcOEt 7:3) to obtain DR (95% yield). The ESI-MS and
1H-NMR for DR were consistent with this structure and compatible with the published data [30].
Extraction of MCP from M. charantia. The extraction of MCP from M. charantia was carried out
according to the protocol described in [6]. The protein content was 0.86 mg/ml, and the specific activity
was 3790 U/mg.
HPLC-DAD/MS Analysis of the Reaction Products from Crude MCP/H2O2 Oxidation of DR. High-
performance liquid chromatography (HPLC; the LC/MS system was composed of an Agilent HPLC
system (1100 series) equipped with an online degasser and API 4000 triplestage quadrupole mass
spectrometer (Applied Biosystems). Analysis was performed using ESI at the negative-ion mode with the
following settings: collision energy, 30 eV; spray voltage, 4.5 kV; detector voltage, 30 V; and source
temp., 5508. All gases used were N2. Pressures for the curtain gas, nebulizer gas, and heater gas were 15,
50, and 50 psi, resp. A column C18 (Phenomenex; 5 mm, 150 mmꢂ4.60 mm) was used as the stationary
phase. The mobile phases consisted of MeCN and 0.1% HCOOH in H2O 40 :60 (v/v) at 280 nm. The flow
rate was set to 0.6 ml/min. The injection volume was 10 ml, and the elute was monitored at 280 nm. The
HPLC/UV chromatograms, total ion current, and the mass chromatograms were recorded.
Biotransformation of DR by Crude MCP. Crude MCP (9 ml) was dissolved in 90 ml of AcONa/
AcOH buffer (100 mmol/l, pH 5.0) in a 500-ml flask, followed by the addition of 260 ml of 1,4-dioxane.
DR (500 mg) dissolved in additional 10 ml of 1,4-dioxane was then dripped to the flask. The reaction was
initiated by adding 0.4 ml of H2O2 (3.0%, aq. sol.) in portions 10 times at 1 h intervals. The mixture was
maintained under electro-magnetic stirring for 10 h in total at 308, and products were extracted by
AcOEt. The org. layer was washed with a sat. NaCl soln., dried (Na2SO4), and filtered. The filtrate was
concentrated under vacuum. A preliminary separation of the residue (488 mg) was performed by CC
eluted with hexane/AcOEt 7:3 to obtain the fraction with no substrate (120 mg). This fraction was then
subjected to semi-prep. HPLC (YMC-Pack ODS-A; 250ꢂ20 mm, S-10 mm, 12 nm) eluted with MeOH/
H2O 35 :65 at a flow rate of 4.0 ml/min to afford 1 (12.9 mg, 2.58%, tR 7.0 min), 2 (36.6 mg, 7.32%, tR
21.5 min), 3 (10.2 mg, 2.04%, tR 26.2 min), 4 (35.1 mg, 7.02%, tR 28.6 min), 5 (6.6 mg, 1.32%, tR 65.7 min),
and 6 (12.6 mg, 2.52%, tR 84.5 min).
Dihydroresveratrol. White amorphous powder. 1H-NMR ((D6)acetone): 2.69–2.72 (m, CH2(7));
2.76–2.78 (m, CH2(8)); 6.20 (t, J¼2.2, HꢀC(12)); 6.22 (d, J¼2.2, HꢀC(10), HꢀC(14)); 6.74 (br. dt, J¼

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
2.8, 9.3, HꢀC(2), HꢀC(6)); 7.05 (br. d, J¼11.1, HꢀC(3), HꢀC(5)). ESI-MS 231.4 (100, [MþH]þ ).
HR-EI-MS: 230.0945 (Mþ, C14H14O3þ ; calc. 230.0943).
6,6’-Bis[2-(4-hydroxyphenyl)ethyl]-1,1’-biphenyl-2,2’,4,4’-tetraol (1). Yellowish amorphous powder.
IR (KBr): 3355, 1613, 1513, 1455, 1155, 832. 1H- and 13C-NMR ((D6)acetone): see Table 1. HR-ESI-MS:
459.1806 ([MþH]þ , C28H27Oþ6 ; calc. 459.1808).
5’-[2-(3,5-Dihydroxyphenyl)ethyl]-6-[2-(4-hydroxylphenyl)ethyl]-1,1’-biphenyl-2,2’,4-triol (2). Yel-
lowish amorphous powder. IR (KBr): 3356, 1598, 1513, 1454, 1151, 834. 1H- and 13C-NMR ((D6)acetone):
see Table 2. HR-ESI-MS: 459.1811 ([MþH]þ , C28H27O6þ ; calc. 459.1808).
4,4’-Methylenebis{5-[2-(4-hydroxyphenyl)ethyl]benzene-1,3-diol} (3). Yellowish amorphous powder.
IR: 3355, 1612, 1513, 1451, 1234, 1004, 831. 1H- and 13C-NMR ((D6)acetone): see Table 1. HR-ESI-MS:
473.1961 ([MþH]þ , C29H29Oþ6 ; calc. 473.1964).
5,5’-(2,2’-(6,6’-Dihydroxy-1,1’-biphenyl-3,3’-diyl)bis(ethane-2,1-diyl))di[benzene-1,3-diol] (4). Yel-
lowish amorphous powder. IR (KBr): 3330, 1601, 1495, 1150, 835, 691. 1H- and 13C-NMR ((D6)acetone):
see Table 1. HR-ESI-MS: 459.1799 ([MþH]þ , C28H27O6þ ; calc. 459.1808).
4-{4-[2-(3,5-Dihydroxyphenyl)ethyl]phenoxy}-5-[2-(4-hydroxyphenyl)ethyl]benzene-1,3-diol (5).
Yellowish amorphous powder. IR (KBr): 3358, 1609, 1510, 1217, 1146, 836. 1H- and 13C-NMR
((D6)acetone): see Table 2. HR-ESI-MS: 459.1801 ([MþH]þ , C28H27O6þ ; calc. 459.1808).
5-[2-(4-{5-[2-(3,5-Dihydroxyphenyl)ethyl]-2-hydroxyphenoxy}phenyl)ethyl]benzene-1,3-diol (6).
Yellowish amorphous powder. IR (KBr): 3357, 1599, 1506, 1154, 837, 691. 1H- and 13C-NMR
((D6)acetone): see Table 2. HR-ESI-MS: 459.1802 ([MþH]þ , C28H27O6þ ; calc. 459.1808).
Antiproliferative Effect on Human Prostate Cancer PC3 Cell Line. Human prostate cancer PC3 cell
line was seeded in 100-mm dishes in RPMI 1640 medium supplemented with 5% fetal bovine serum and
incubated at 378 with 5% CO2. Cells were maintained in serum-free RPMI 1640 medium for 24 h. Cells
were next treated with tested compounds with mibolerone (Mib) in RPMI 1640 medium containing 5%
charcoal-stripped serum. Mib and the compounds were dissolved in EtOH and DMSO, resp. The MTT
assay was used to evaluate the cell growth according to standard protocols [31][32]. The antiproliferative
activity was recorded as IC50 values.
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Received July 2, 2008