Genistein derivatives as selective estrogen receptor modulators: Sonochemical synthesis and in vivo anti-osteoporotic action

Article abstract of DOI:10.1016/j.bmc.2005.04.082

Genistein derivatives were synthesized from genistein through a facile sonochemical approach in high yields. The bioassay was performed on ovariectomized (OVX) rats in terms of bone mineral density (BMD) and the weight of bone ash (WBA) to lead to the discovery of eight novel genistein-based selective estrogen receptor modulators. Attention to the structure-activity relationship disclosed that the newly introduced 2-hydroxyethylthio scaffolds were essential for the anti-osteoporotic activity. Moreover, the anti-osteoporotic action of genistein, deprivable by methylation, could be restored and enhanced by subsequent sulfonation. The most promising compound was 4′,5,7-tri[3-(2-hydroxyethylthio)propoxy]isoflavone, displaying 24% (or 8%) increment in BMD and 31% (or 11%) increase in WBA of the femora relative to those discerned with the OVX (or genistein) group. Acute toxicity test showed that none of the active compounds was acutely toxic.

Full text of DOI:10.1016/j.bmc.2005.04.082

Bioorganic & Medicinal Chemistry 13 (2005) 4880–4890
Genistein derivatives as selective estrogen receptor modulators:
Sonochemical synthesis and in vivo anti-osteoporotic action
Shi F. Wang,a,b Qing Jiang,a Yong H. Ye,a Yang Lia and Ren X. Tana,*
aInstitute of Functional Biomolecules, State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University,
Nanjing 210093, PR China
bSchool of Chemistry and Chemical Engineering, Anhui University of Technology, Maanshan 243002, PR China
Received 10 March 2005; revised 30 April 2005; accepted 30 April 2005
Available online 9 June 2005
Abstract—Genistein derivatives were synthesized from genistein through a facile sonochemical approach in high yields. The bioas-
say was performed on ovariectomized (OVX) rats in terms of bone mineral density (BMD) and the weight of bone ash (WBA) to
lead to the discovery of eight novel genistein-based selective estrogen receptor modulators. Attention to the structure–activity rela-
tionship disclosed that the newly introduced 2-hydroxyethylthio scaffolds were essential for the anti-osteoporotic activity. Moreover,
the anti-osteoporotic action of genistein, deprivable by methylation, could be restored and enhanced by subsequent sulfonation. The
most promising compound was 40,5,7-tri[3-(2-hydroxyethylthio)propoxy]isoflavone, displaying 24% (or 8%) increment in BMD and
31% (or 11%) increase in WBA of the femora relative to those discerned with the OVX (or genistein) group. Acute toxicity test
showed that none of the active compounds was acutely toxic.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
of the repeated HRT have already emerged including the
increased risk in the secondary incidence of endometrial
cancer, breast tumor, and venous thrombosis.16–19
Accordingly, the current major emphasis in this regard
has been re-put on searching for novel lead compounds,
which can selectively regulate bone metabolism with
negligible estrogenic action on uterus even after long-term
administrations. Raloxifene can be reckoned as a success
under this notion. However, a recently found neuronal
toxicity of raloxifene at higher concentrations could be
its drawback20 necessitating continuous research and
clinic attention to phytoestrogens such as isoflavones
and their derivatives for more reliable selective estrogen
receptor modulators (SERMs).
Osteoporosis, presently standing as a major public health
problem,1,2 is characterized by low bone mass and micro-
architectural deterioration of bone tissue resulting in the
bone fragility and a consequent increment in fracture
risks.3,4 It is anticipated that the cost for the hip fracture
therapy in the United States will approach 250 billion
US dollars by 2050.5 Furthermore, the incidence of
fractures continues to increase remarkably as the aged
population is growing worldwide.6,7 Concerning the
management of osteoporosis, the hormone replacement
therapy (HRT) by prescribing estrogen hormone(s) has
been widely accepted for relieving climacteric symptoms
of postmenopausal women since the regulative role of
some estrogen hormones in maintaining bone mass in
women has been unambiguously ascertained.8–11 More-
over, this HRT practice is believed to improve womenÕs
quality of life owing to its prevention of spinal deformity
or hip fracture.12–15 In spite of the confirmed curative
evidence of the therapeutical approach, severe side effects
Genistein (40,5,7-trihydroxyisoflavone, 1), a major iso-
flavone phytochemical in some plants belonging to the
Leguminosae family, is known as a phytoestrogen that
is capable of binding to the estrogen receptor.21 Much
attention has been focused on the role of genistein in
preventing bone loss resulted at least in part from the
estrogen deficiency.22–24 Furthermore, the finding that
genistein acts as an SERM on osteoblastic cells25 and
in ovariectomized (OVX) mice26 highlights the possibil-
ity that this isoflavone phytochemical hardly affect
the uterus in some dose ranges at which it does exert the
estrogen-like regulative action on bone and bone marrow
Keywords: Genistein derivatives; SERMs; Synthesis; Osteoporosis;
Ovariectomized rat.
*
Corresponding author. Tel.: +86 25 8359 2945; fax: +86 25 8330
2728; e-mail: rxtan@nju.edu.cn
0968-0896/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.04.082

S. F. Wang et al. / Bioorg. Med. Chem. 13 (2005) 4880–4890
4881
metabolisms. However, few reports have been dedicated
to the improvement of the SERM activity and the struc-
ture–activity understanding of genistein derivatives. We
therefore started a project aiming at the recognition of
novel genistein derivatives possessing more intensive
efficacy in preventing bone loss. And during the study,
special attention was paid as well to the development of
a facile sonochemical approach for the selective synthesis
of genistein derivatives without any protecting group
strategy. We hereby wish to present the results regarding
the synthesis and anti-osteoporotic evaluation of novel
genistein derivatives as SERMs in vivo.
tude was set constantly at 40 kHz, at which the reaction
underwent better in the study. The ideal condition for
the present preparation of new genistein derivatives
was detailed in Table 1 and Scheme 1. In Scheme 1,
the five novel intermediates 2–6, bearing the active bro-
mine at the scaffold termini of the molecules, can readily
react subsequently with nucleophiles to give multifari-
ously designed novel analogs carrying would-be phar-
macophores with nitrogen, sulfur and oxygen atoms.
Thus, mercaptoethanol was allocated to react at 48 °C
separately with genistein derivatives 2–6 in ethanol in
the presence of differentiated amounts of triethylamine
to give the corresponding sulfur-containing genistein
derivatives 7–11 (Scheme 1, step b). Furthermore, differ-
ently controlled methylations of genistein with MeI in
the presence of K2CO3 were performed to afford com-
pounds 12–14 (Scheme 2, step c) which were trans-
formed subsequently in high yield at 0 °C under
ultrasonic irradiation into the corresponding target sul-
fonated products 15–17 (Scheme 2, step d) using concen-
trated sulfuric acid (98%). Technically, the separation of
the obtained genistein sulfonic acids (15–17) was accom-
plished by taking the advantage of the difference in their
solubilities in H2SO4 solutions. Specifically, the majority
(>85%) of sulfonic acids 15–17 was able to precipitate
from 60% H2SO4, and the pure products were afforded
after recrystallizations of precipitated powders in water.
These three sulfonic acids were transformed quantita-
tively by titration with NaOH into their sodium sulfo-
nates for the bioassay.
2. Results and discussion
2.1. Chemistry
Previous attempts at the regioselective preparation of
genistein derivatives were limited to the phase transfer
catalyzed synthesis and sequential silylations/acyla-
tions.27 Although regioselective acylation can be carried
out by taking the advantage of the subtle difference in
acidity of the phenolic hydroxyls or by using tert-butyl-
dimethylsilyl as protecting group,28,29 the poor solubility
of the intermediates (genistein salts) in organic solvents
and/or the removal of the protecting groups often com-
plicate or shallow the selective reactivity in most cases.
Moreover, its acylated derivatives cannot be accepted
as intermediates for further synthesis since the acylation
usually blocks the reactivity of hydroxyls in genistein
(1). We have very recently reported that ultrasonic irra-
diation could accelerate hydrolyzation of genistein esters
and improve the stability of pyrone ring of genistein es-
ters.30 In order to investigate the application of ultra-
sound in the selective structure modification of natural
polyphenols, ultrasound was applied hereby for the
semi-synthesis of other genistein analogs. Alkyl dibro-
mides such as 1,2-dibromoethane or 1,3-dibromopro-
pane were used to introduce scaffolds through
nucleophilic substitution reaction into the parent com-
pound with the rest bromine ÔactiveÕ enough for further
derivations. As illustrated in Table 1, the sonochemical
synthetic approach could, under the same or similar
reaction conditions, reduce remarkably the reaction
time with discernible improvements in the regioselectiv-
ity. It is noteworthy that the application of ultrasound in
the modification of natural products is of particular
interest since this physical technique is more convenient,
economic and manageable, and gives good to excellent
yields and selectivities, as exemplified by the present
preparation of genistein derivatives (Schemes 1 and 2)
and other reports.31,32
All of the semi-synthetic products gave satisfactory ana-
lytical and spectroscopic data, which were in the full
accordance with their formulated structures.
2.2. Biological evaluation
All semi-synthetic genistein derivatives except for bro-
mides 2–6 (assumed to be highly toxic owing to the pres-
ence of Ôactive bromineÕ) were assessed in vivo for the
inhibitory effect on bone resorption on the OVX rat
model by inspecting bone mineral density (BMD), the
weight of bone ash (WBA), the uterine weight (as a sys-
temic estrogen parameter), body weight and the bio-
chemical parameters in serum.
Success of ovariectomy was confirmed by means of va-
gina smear (Fig. 1) and by observation of marked atro-
phy of uterine horns. The vagina smears were evaluated
by morphological observation of cell with phase-con-
trast microscope. Sham-operated rats were standing in
oestrus. Many dead keratinocytes were observed in the
smear (Fig. 1A). Only epithelia were observed in the va-
gina smears of OVX rats which demonstrated that the
operation of OVX rats was successful (Fig. 1B).
Starting from genistein (1), novel compounds 2–11 were
synthesized as depicted in Scheme 1. Thus, derivatives
2–6 were prepared through Ôstep aÕ under ultrasound
irradiation by treating 1 with excessive amounts of 1,2-
dibromoethane or 1,3-dibromopropane. Accordingly,
the 7-mono-, 40,7-di- and 40,5,7-tri-substituted analogs
of genistein were obtained in high yields by optimizing
the molar rate of 1 to K2CO3 as well as duration and
temperature of the ultrasonic irradiation whose magni-
From the radiograms of the femora (Fig. 2) and the re-
sults of BMD, WBA (Fig. 3A and B), it could been seen
that the 2-hydroxyethylthio side chains in the sulfur-
containing derivatives 7–11 are important for the inhibi-
tion of bone loss since they were all stronger than that of
the parent compound (1). However, the dependence of
the activity on the 2-hydroxyethylthio chain numbers

4882
S. F. Wang et al. / Bioorg. Med. Chem. 13 (2005) 4880–4890
Table 1. The synthetic conditions and yields for compounds 2–17 with and without (data in bracket) ultrasonic irradiation
Compounds
Structures
Time (h)
—
Temperature (°C)
Yield (%)
—
1
—
2
3
4
5
6
1.5 (8)
3 (16)
1.5 (8)
3 (16)
4 (48)
40 (40)
60 (60)
40 (40)
60 (60)
80 (80)
86 (85)
82 (70)
85 (88)
80 (75)
90 (86)
7
2 (12)
2 (12)
2 (12)
2 (12)
2 (12)
48 (48)
48 (48)
48 (48)
48 (48)
48 (48)
85 (80)
80 (77)
85 (80)
80 (70)
92 (75)
8
9
10
11
12
13
14
15
1 (8)
50 (50)
50 (50)
50 (50)
0 (5)
78 (68)
80 (75)
85 (80)
80 (76)
1.5 (12)
3 (24)
0.5 (8)

S. F. Wang et al. / Bioorg. Med. Chem. 13 (2005) 4880–4890
4883
Table 1 (continued)
Compounds
Structures
Time (h)
0.5 (8)
Temperature (°C)
Yield (%)
16
17
0 (5)
0 (5)
85 (70)
85 (75)
0.5 (8)
Scheme 1. The synthetic route for compounds 2–11. Reagents: (a) BrCH2CH2Br or BrCH2CH2CH2Br, K2CO3, DMF; (b) HSCH2CH2OH,
triethylamine, ethanol.
Scheme 2. The synthetic route for compounds 12–17. Reagents: (c) MeI, K2CO3, acetone; (d) 98% H2SO4.

4884
S. F. Wang et al. / Bioorg. Med. Chem. 13 (2005) 4880–4890
Figure 1. Vagina smears of rats: (A) Vagina smear of sham-operated rat. (B) Vagina smear of OVX rat.
was not very striking although the fully substituted gen-
istein analog 11 was substantially more active than its
congeners 7–10. Furthermore, the anti-osteoporotic ac-
tion of the methylated derivatives 12–14 was trivial.
Any of them was not as effective as the parent com-
pound (1). This observation implied that the methyla-
tion at any of the three hydroxyl groups in the starting
material would attenuate the bone loss inhibitory activ-
ity. As an extreme, permethylation of genistein could de-
prive the isoflavone of the anti-osteoporotic activity, but
the lost activity of methylated derivatives could be
restored and substantially enhanced by sulfonation as
demonstrated by the anti-osteoporotic data of 15–17
(bioassayed as the corresponding sodium salts). It is
noteworthy that the direct sulfonation of genistein gave
complex mixture (data not shown) suggesting that the
Figure 2. Radiograms of the femora. Sham and OVX expressed the
femora taken from sham-operated and OVX rats, and E2, G and 11
stood for those from OVX rats treated after surgery with 17b-estradiol
at 0.5 lmol/kg per day, with genistein at 25 lmol/kg per day, and with
compound 11 at 25 lmol/kg per day, respectively. The marked bone
loss, observed in the distal metaphysis of the femoral cancellous bone
in OVX rat, was prevented by treatments with 17b-estradiol, genistein
and compound 11.
Figure 3. Effect of genistein, its analogs 7–17 and 17b-estradiol on BMD (A), WBA (B), uterine weight (C) and body weight (D). BMD, WBA,
uterine weight and body weight values were expressed as mean SEM based on six independent experiments. The p values were obtained using
StudentÕs t-test. (*) p < 0.01, compared with the value of OVX group; and (^) p < 0.01, compared with the value of genistein group.

S. F. Wang et al. / Bioorg. Med. Chem. 13 (2005) 4880–4890
4885
derivation strategy was of very limited practical value.
The aforementioned observations highlighted that the
sulfur element could be most possibly helpful for inten-
sifying the anti-osteoporotic activity of genistein. The
magnitude of 11 in inhibiting bone resorption has a
24% increment in BMD and a 31% increase in WBA
of the femora when compared with those of the OVX
control (p < 0.01). Relative to those of genistein (1),
compound 11 increased BMD and WBA of femora by
8% and 11% (p < 0.01), respectively.
As expected, the weights of uterus in OVX rats were sig-
nificantly decreased when compared with that in sham-
operated rats (Fig. 3C), but on the other hand, OVX
animals gained more body weight (Fig. 3D) during the
study period than that of sham rats (p < 0.01). The
gained body weight due to ovarian hormone deficiency
was prevented by estrogen, genistein, or its derivativesÕ
administration. The uterine weight of OVX rats was in-
creased by approximately fourfolds after peroral admin-
istration of 17b-estradiol at a dose of 0.5 lmol/kg per
day. However, the OVX rats treated with genistein or
its derivatives at 25 lmol/kg per day did not show any
increment in uterine weights. This observation indicates
that a big merit of genistein and its derivatives lies in
exerting, irrespective of the uterus, the estrogen-like reg-
ulation on bone and bone marrow metabolisms.
With regard to the bone metabolic markers, the activity of
serum alkaline phosphatase (ALP) associated with bone
formation was significantly increased after ovariectomy.
Although the serum calcium phosphorus level did not dif-
fer significantly among the sham, OVX control group,
and the other treated groups (Fig. 4A and B), treatment
with 17b-estradiol or the derivatives 9, 11, 15, 16 for
two months significantly decreased the level of ALP
(Fig. 4C). Serum concentration of ALP, an index of bone
formation, was significantly greater in the OVX group
than in the sham-operated group.33,34] Both 17b-estradiol
and genistein derivatives decreased serum ALP in the
OVX rats in our study. The results from bone metabolic
makers, BMD, WBA and X-ray observation indicated
that bone turnover was increased after ovariectomy and
the incidence of the bone turnover could be decreased
by peroral administration of genistein or its derivatives.
To get the knowledge about the acute toxicity of the ac-
tive materials, compounds 1, 7–9, 11 and 16 were orally
administrated to mice (n = 10) at 27 mmol/kg (a 500-
fold effective dosage). The tested animals appeared nor-
mal immediately after the administration and did not
exhibit any indication of acute toxicity for 14 days after-
ward (Table 2). The body weights in treated mice were
similar to that of the control mice dosed with an equal
volume of vehicle. The result from this acute toxicity
determination demonstrated that the genistein and its
derivatives (7–9, 11 and 16) are not acutely toxic.
Figure 4. Serum biochemical analysis on calcium (A), phosphorus (B)
and alkaline phosphatase (ALP) (C). Values were expressed as
mean SEM based on six independent experiments. The p values
were obtained using StudentÕs t-test. (*) p < 0.01, compared with the
value of OVX group.
approach with the desired regioselectivity allowed by the
subtle difference in acidity and regioaccessibility of
hydroxyls co-anchored on the isoflavone skeleton. An-
other strategic key point lies in the selective introduction
of Ôactive bromineÕ-bearing scaffolds into the designed
key bromides of genistein, from which further deriva-
tions can be realized with the target compounds afforded
in high yields. These methodological merits may shed a
new light on the potent application in the selective struc-
3. Conclusion
A set of novel anti-osteoporotic genistein derivatives
have been synthesized through an efficient sonochemical

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S. F. Wang et al. / Bioorg. Med. Chem. 13 (2005) 4880–4890
Table 2. The acute toxic determination of compounds 1, 7–9, 11 and 16
Tested compounds
Number of mice
Limited (mmol/kg)
Limited (mg/kg)
Body weighta on day 1
Body weighta on day 14
Genistein (1)
7
10
10
10
10
10
10
10
27
27
27
27
27
27
—
7290
10,098
12,906
10,476
16,848
10,800
—
20.0 1.0
19.8 0.8
20.5 0.6
20.1 0.4
19.5 0.5
20.3 0.9
20.4 0.7
29.1 0.3
28.9 0.7
28.5 0.7
28.7 0.4
29.0 1.0
28.6 0.5
28.3 0.9
8
9
11
16
Vehicle
a Body weights were mean SEM (n = 10).
ture modification of natural polyphenols such as resve-
ratrol and fisetin, both being of current special con-
cerns.35 The anti-osteoporotic activity of the new
derivatives was evaluated in vivo to find eight new
SERMs (7–11 and 15–17), each being more active than
genistein. The attention to the structure–activity
relationship of the series of semi-synthetic isoflavone
derivatives demonstrated that the newly introduced
2-hydroxyethylthio motif was a key pharmacophore
for the inhibition of bone loss.
agent Company (Shanghai, China). Genistein (1,
>96%) provided by Shanxi Huike Botanical Develop-
ment Co. Ltd was further refined prior to use by its
recrystallization from ethanol as pale yellow needles,
mp 298–299 °C; IR mmax cmꢀ1: 3411 (OH), 1650
(C@O); 1H NMR d: 6.21 (d, J = 1.9 Hz, 1H, H-8),
6.37 (d, J = 1.9 Hz, 1H, H-6), 6.80 (d, J = 8.5 Hz, 2H,
H-30 and H-50), 7.35 (d, J = 8.5 Hz, 2H, H-20 and H-
60), 8.32 (s, 1H, H-2), 9.64 (s, 1H, 40-OH), 10.94 (s,
1H, 7-OH), 12.96 (s, 1H, 5-OH); HRMS (ESI) Calcd
C15H10O5 [MꢀH]ꢀ 269.0447. Found 269.0449. Anal.
Calcd for C15H10O5: C, 66.67; H, 3.73. Found: C,
66.68; H, 3.87.
4. Experimental
4.1. General
The purity of each active compound mentioned in the
study was checked to be >99% through elemental anal-
yses plus HRMS and H NMR spectra.
1
Reaction and the resulted products were monitored by
thin-layer chromatography (TLC) on Merck pre-coated
silica gel F254 plates with separated compounds visual-
ized at 254 nm under a UV lamp and/or by spraying
with a diluted ferric chloride solution. Melting points
(uncorrected) were determined on a XT4 MP appara-
tus (Taike Corp., Beijing, China). The IR spectra were
recorded on a Perkin-Elmer FT-IR spectrometer (KBr
pellets). ESI mass spectra were obtained on a Mariner
4.2.1. 40,5-Dihydroxy-7-(2-bromoethoxy)isoflavone (2).
Genistein (0.27 g, 1 mmol), dibromoethane (4.7 g,
25 mmol) and potassium carbonate (0.07 g, 0.5 mmol)
in 60 ml ofdry DMF were sonicated. After the completion
of reaction, the resultant mixture was cooled to room tem-
perature and filtered. The filtrate was distilled to form
yellow solid. Recrystallization of the solid from 15 ml
acetone gave compound 2 as yellow needle, mp 175–
178 °C; IR mmax cmꢀ1: 3421 (OH), 2922 (CH2), 1665
1
System 5304 high-resolution mass instrument, and H
NMR spectra were recorded in DMSO-d6 on a Bruker
DPX500 or DPX300 spectrometer with TMS and sol-
vent signals allotted as internal standards. Elemental
analyses were performed on a CHN–O-Rapid instru-
ment and were within 0.4% of the theoretical values.
Sonication was performed in a Kunshan KQ 500E
ultrasonic cleaner (Jiangsu, China) with irradiation
delivered at 40 kHz and 500 W. The reaction flask
was positioned in the maximum-energy area in the
cleaner with cycled water running to control the tem-
perature of the water bath. BMD measurements were
performed for the whole femur on a Lunar DPX-IQ
dual-energy X-ray densitometer. Serum alkaline phos-
phatase (ALP), serum total calcium (Ca) and phospho-
rus (P) were measured with spectrophotometer using
kits (kits were purchased from Nanjing Jiancheng Bio-
engineering Institute, China).
1
(C@O); H NMR d: 3.81 (t, J = 5.5 Hz, 2H, –CH2Br),
4.43 (t, J = 5.5 Hz, 2H, –OCH2–), 6.41 (d, J = 2.0 Hz,
1H, H-8), 6.67 (d, J = 2.0 Hz, 1H, H-6), 6.82 (dd,
J = 8.5 Hz, 1.0 Hz, 2H, H-30 and H-50), 7.38 (d,
J = 8.5 Hz, 2H, H-20 and H-60), 8.39 (d, J = 1.0 Hz,
1H, H-2), 9.64 (s, 1H, 40-OH), 12.95 (s, 1H, 5-OH);
HRMS (ESI) Calcd C17H13BrO5 [MꢀH]ꢀ 374.9864.
Found 374.9886. Anal. Calcd for C17H13BrO5: C,
54.13; H, 3.47. Found: C, 54.06; H, 3.51.
4.2.2. 40,7-Di(2-bromoethoxy)-5-hydroxyisoflavone (3).
Genistein (0.27 g, 1 mmol), dibromoethane (9.4 g,
50 mmol) and potassium carbonate (0.14 g, 1 mmol) in
60 ml of dry DMF were sonicated. After the completion
of reaction, the afforded mixture was cooled to room
temperature and filtered. The filtrate is distilled to yield
yellow solid. Recrystallization of the solid from 15 ml
acetone gave compound 3 as pale yellow needle, mp
4.2. Chemistry
140–142 °C; IR m
cmꢀ1: 3447 (OH), 2929 (CH2),
1663 (C@O); H NMR d: 3.80 (overlapped multiplets,
4H, 2(–CH2Br)), 4.34 (t, J = 5.5 Hz, 2H, –OCH2–),
4.44 (t, J = 5.4 Hz, 2H, –OCH2–), 6.44 (t, J = 1.5 Hz,
1,2-Dibromoethane, 1,3-dibromopropane, triethyl-
amine, mercaptoethanol, acetone, ethanol, DMF, MeI,
H2SO4 (98%), NaOH and K2CO3, all being of A. R.
grade, were purchased from Shanghai Chemical Re-
max
1

S. F. Wang et al. / Bioorg. Med. Chem. 13 (2005) 4880–4890
4887
1H, H-8), 6.70 (t, J = 1.5 Hz, 1H, H-6), 7.03 (d,
J = 8.5 Hz, 2H, H-30 and H-50), 7.50 (d, J = 8.5 Hz,
2H, H-20 and H-60), 8.42 (s, 1H, H-2), 12.93 (s, 1H, 5-
OH); HRMS (ESI) Calcd C19H16Br2O5 [M+H]+
482.9437. Found 482.9415. Anal. Calcd for
C19H16Br2O5: C, 47.14; H, 3.33. Found: C, 47.19; H,
3.45.
Found 630.9722. Anal. Calcd for C24H25Br3O5: C,
45.53; H, 3.98. Found: C, 45.48; H, 3.83.
4.2.6. 40,5-Dihydroxy-7-[2-(2-hydroxyethylthio)ethoxy]iso-
flavone (7). Compound 2 (0.37 g, 1 mmol), mercap-
toethanol (0.15 g, 2 mmol) and triethylamine (0.11 g,
1 mmol) in 50 ml of dry ethanol were sonicated. After
the completion of reaction, the resulted mixture was
distilled to give pale yellow residue which was washed
with water. Recrystallization of the washed solid from
20 ml acetone afforded compound 7 as white needle, mp
4.2.3. 40,5-Dihydroxy-7-(3-bromopropoxy)isoflavone (4).
Genistein (0.27 g, 1 mmol), dibromopropane (5.1 g,
25 mmol) and potassium carbonate (0.07 g, 0.5 mmol)
in 60 ml of dry DMF were sonicated. After the completion
of reaction, the obtained mixture was cooled to room tem-
perature and filtered. The filtrate is distilled to give yellow
solid. Recrystallization of the solid from 15 ml acetone
gave compound 4 as yellow needle, mp 124–126 °C; IR
132–135 °C; IR mmax cmꢀ1
:
3420 (OH), 2926
1
(CH2), 1664 (C@O), 1047 (C–OH); H NMR d: 2.66 (t,
J = 6.5 Hz, 2H, –SCH2CH2O–), 2.91 (t, J = 6.5 Hz, 2H,
HOCH2CH2S–), 3.55 (t, J = 6.5 Hz, 2H, –CH2OH),
4.24 (t, J = 6.5 Hz, 2H, –OCH2–), 6.40 (d, J = 1.5 Hz,
1H, H-8), 6.66 (d, J = 1.5 Hz, 1H, H-6), 6.82 (d,
J = 8.5 Hz, 2H, H-30 and H-50), 7.38 (d, J = 8.5 Hz, 2H,
H-20 and H-60), 8.40 (s, 1H, H-2), 9.62 (s, 1H, 40-OH),
12.95 (s, 1H, 5-OH); HRMS (ESI) Calcd C19H18O6S
[MꢀH]ꢀ 373.0740. Found 373.0742. Anal. Calcd for
C19H18O6S: C, 60.95; H, 4.85. Found: C, 60.99; H, 4.89.
1
mmax cmꢀ1: 3423 (OH), 2934 (CH2), 1663 (C@O); H
NMR d: 2.24 (m, 2H, –CH2CH2CH2–), 3.65 (t,
J = 5.5 Hz, 2H, –CH2Br), 4.18 (t, J = 5.5 Hz, 2H,
–OCH2–), 6.42 (s, 1H, H-8), 6.67 (s, 1H, H-6), 6.82 (d,
J = 8.5 Hz, 2H, H-30 and H-50), 7.38 (d, J = 8.5 Hz, 2H,
H-20 and H-60), 8.40 (s, 1H, H-2), 9.63 (s, 1H, 40-OH),
12.95 (s, 1H, 5-OH); HRMS (ESI) Calcd C18H15BrO5
[MꢀH]ꢀ 389.0020. Found 389.0008. Anal. Calcd for
C18H15BrO5: C, 55.26; H, 3.86. Found: C, 55.14; H, 3.80.
4.2.7. 40,7-Di[2-(2-hydroxyethylthio)ethoxy]-5-hydroxy-
isoflavone (8). Compound 3 (0.48 g, 1 mmol), mercap-
toethanol (0.30 g, 4 mmol) and triethylamine (0.22 g,
2 mmol) in 50 ml of dry ethanol were sonicated. After
the completion of reaction, the afforded mixture was
distilled to give pale yellow residue which was washed
with water. Recrystallization of the washed solid from
15 ml acetone gave compound 8 as white needle, mp
66–68 °C; IR mmax cmꢀ1: 3431 (OH), 2924, 2854
4.2.4. 40,7-Di(3-bromopropoxy)-5-hydroxyisoflavone (5).
Genistein (0.27 g, 1 mmol), dibromopropane (10.2 g,
50 mmol) and potassium carbonate (0.14 g, 1 mmol) in
60 ml of dry DMF were sonicated. After the completion
of reaction, the yielded mixture was cooled to room tem-
perature and filtered. The filtrate is distilled to give yellow
solid. Recrystallization of the solid from 10 ml acetone
gave compound 5 as pale yellow needle, mp 128–130 °C;
IR mmax cmꢀ1: 3447 (OH), 2927, 2875 (CH2), 1661
1
(CH2), 1663 (C@O), 1048 (C–OH); H NMR d: 2.66
(t, J = 6.5 Hz, 4H, 2(–SCH2CH2O–)), 2.90 (q, 4H,
2(HOCH2CH2S–)),
3.55
(t,
J = 6.5 Hz,
4H,
2(–CH2OH)), 4.15 (t, J = 6.5 Hz, 2H, –OCH2–), 4.25
(t, J = 6.5 Hz, 2H, –OCH2–), 6.42 (d, J = 2.0 Hz, 1H,
H-8), 6.69 (d, J = 2.0 Hz, 1H, H-6), 7.00 (d,
J = 8.5 Hz, 2H, H-30 and H-50), 7.50 (d, J = 8.5 Hz,
2H, H-20 and H-60), 8.46 (s, 1H, H-2), 12.92 (s, 1H,
5-OH); HRMS (ESI) Calcd C23H26O7S2 [MꢀH]ꢀ
477.1033. Found 477.1002. Anal. Calcd for
C23H26O7S2: C, 57.72; H, 5.48. Found: C, 57.51; H,
5.52.
1
(C@O); H NMR d: 2.24 (m, 4H, 2(–CH2CH2CH2–)),
3.65 (overlapped multiplets, 4H, 2 (–CH2Br)), 4.08 (over-
lapped multiplets, 4H, 2(–OCH2–)), 6.41 (br s, 1H, H-8),
6.66 (br s, 1H, H-6), 7.01 (d, J = 8.5 Hz, 2H, H-30 and
H-50), 7.50 (d, J = 8.5 Hz, 2H, H-20 and H-60), 8.43 (s,
1H, H-2), 12.91 (s, 1H, 5-OH); HRMS (ESI) Calcd
C21H20Br2O5 [M+H]+ 510.9749. Found 510.9790. Anal.
Calcd for C21H20Br2O5: C, 49.24; H, 3.94. Found: C,
49.34; H, 3.88.
4.2.8. 40,5-Dihydroxy-7-[3-(2-hydroxyethylthio)propoxy]iso-
flavone (9). Compound 4 (0.39 g, 1 mmol), mercap-
toethanol (0.15 g, 2 mmol) and triethylamine (0.11 g,
1 mmol) in 30 ml of dry ethanol were sonicated. After
the completion of reaction, the resultant mixture was
distilled to give yellow residue which was washed with
water. Recrystallization of the washed solid from 20 ml
acetone to yield compound 9 as white needle, mp 152–
154 °C; IR mmax cmꢀ1: 3434 (OH), 2942, 2920, 2876
4.2.5. 40,5,7-Tri(3-bromopropoxy)isoflavone (6). Genistein
(0.27 g, 1 mmol), dibromopropane (15.3 g, 75 mmol) and
potassium carbonate (0.49 g, 3.5 mmol) in 60 ml of dry
DMF were sonicated. After the completion of reaction,
the reaction mixture was cooled to room temperature
and filtered. The filtrate is distilled and yellow solid was
formed. Recrystallization from 10 ml acetone gave
pure compound 6 in colorless needle, mp 110–112 °C;
IR mmax cmꢀ1: 3447 (OH), 2930, 2874 (CH2), 1644
(C@O);1H NMR d: 2.22 (overlapped multiplets, 6H,
3(–CH2CH2CH2–)), 3.66 (t, J = 6.4 Hz, 4H, 2 (–CH2Br)),
3.82 (t, J = 6.4 Hz, 2H, –CH2Br), 4.08 (overlapped multi-
plets, 4H, 2(–OCH2–)), 4.23 (t, J = 5.9 Hz, 2H, –OCH2–),
6.53 (d, J = 2.1 Hz, 1H, H-8), 6.69 (d, J = 2.1 Hz, 1H,
H-6), 6.97 (d, J = 8.5 Hz, 2H, H-30 and H-50), 7.41
(d, J = 8.5 Hz, 2H, H-20 and H-60), 8.20 (s, 1H, H-2);
HRMS (ESI) Calcd C24H25Br3O5 [M+H]+ 630.9322.
1
(CH2), 1664 (C@O), 1042 (C–OH); H NMR d: 1.95
(overlapped multiplets, 2H, –SCH2CH2CH2O–), 2.56
(t, J = 6.8 Hz, 2H, –SCH2CH2CH2O–), 2.64 (t,
J = 7.1 Hz, 2H, HOCH2CH2S–), 3.50 (t, J = 7.1 Hz,
2H, HOCH2–), 4.14 (t, J = 6.2 Hz, 2H, –CH2O–),
6.39 (d, J = 2.0 Hz, 1H, H-8), 6.65 (d, J = 2.0 Hz, 1H,
H-6), 6.80 (d, J = 8.5 Hz, 2H, H-30 and H-50), 7.37
(d, J = 8.5 Hz, 2H, H-20 and H-60), 8.40 (s, 1H, H-2),
9.62 (s, 1H, 40-OH), 12.95 (s, 1H, 5-OH); HRMS

4888
S. F. Wang et al. / Bioorg. Med. Chem. 13 (2005) 4880–4890
(ESI) Calcd C20H20O6S [MꢀH]ꢀ 387.0896. Found
387.0872. Anal. Calcd for C20H20O6S: C, 61.84; H,
5.19. Found: C, 61.86; H, 5.20.
283.0599. Anal. Calcd for C16H12O5: C, 67.60; H,
4.26. Found: C, 67.64; H, 4.37.
4.2.12. 40,7-Dimethoxy-5-hydroxyisoflavone (13). Geni-
stein (0.27 g, 1 mmol), iodomethane (0.26 ml, 2 mmol)
and potassium carbonate (0.14 g, 1 mmol) in 50 ml of
dry acetone were sonicated. After the completion of
reaction, the given mixture was cooled to room temper-
ature followed by filtration. The filtrate was distilled to
give a yellow solid. Recrystallization of the solid from
20 ml hot acetone afforded compound 13 as pale yellow
needle, mp 138–139 °C; IR mmax cmꢀ1: 3434 (OH), 2958,
4.2.9. 40,7-Di[3-(2-hydroxyethylthio)propoxy]-5-hydroxy-
isoflavone (10). Compound 5 (0.51 g, 1 mmol), mercap-
toethanol (0.30 g, 4 mmol) and triethylamine (0.22 g,
2 mmol) in 30 ml of dry ethanol were sonicated. After
the completion of reaction, the mixture was distilled to
give pale yellow residue which was washed with water.
Recrystallization of the washed solid from 15 ml acetone
afforded compound 10 as colorless needle, mp 102–
105 °C; IR mmax cmꢀ1: 3421 (OH), 2923, 2877 (CH2),
1671 (C@O), 1049 (C–OH); 1H NMR d: 1.93 (over-
lapped multiplets, 4H, 2(–SCH2CH2CH2O–)), 2.56 (t,
J = 6.8 Hz, 4H, 2(–SCH2CH2CH2O–)), 2.64 (t,
J = 6.8 Hz, 4H, 2(HOCH2CH2S–)), 3.51 (t, J = 6.8 Hz,
4H, 2(HOCH2–)), 4.14 (t, J = 6.0 Hz, 4H, 2(–OCH2–)),
6.39 (d, J = 2.0 Hz, 1H, H-8), 6.64 (d, J = 2.0 Hz, 1H,
H-6), 6.80 (d, J = 8.5 Hz, 2H, H-30 and H-50), 7.36 (d,
J = 8.5 Hz, 2H, H-20 and H-60), 8.39 (s, 1H, H-2),
12.92 (s, 1H, 5-OH); HRMS (ESI) Calcd C25H30O7S2
[M+Na]+ 529.1293. Found 529.1360. Anal. Calcd for
C25H30O7S2: C, 59.27; H, 5.97. Found: C, 59.19; H,
5.93.
1
2972 (CH3), 1650 (C@O); H NMR d: 3.84 and 3.89 (s,
3H each, 40, 7-OCH3), 6.40 (d, J = 2.0 Hz, 1H, H-8),
6.42 (d, J = 2.0 Hz, 1H, H-6), 6.99 (d, J = 9.0 Hz, 2H,
H-30 and H-50), 7.42 (d, J = 9.0 Hz, 2H, H-20 and H-
60), 7.88 (s, 1H, H-2), 12.89 (s, 1H, 5-OH); HRMS
(ESI) Calcd C17H14O5 [M+H]+ 299.0915. Found
299.0899. Anal. Calcd for C17H14O5: C, 68.45; H, 4.73.
Found: C, 68.45; H, 4.82.
4.2.13. 40,5,7-Trimethoxyisoflavone (14). Genistein
(0.27 g, 1 mmol), iodomethane (0.62 ml, 3.5 mmol) and
potassium carbonate (0.41 g, 3 mmol) in 50 ml of dry
acetone were sonicated. After the completion of reac-
tion, the yielded mixture was cooled to room tempera-
ture and filtered. The filtrate was distilled to afford a
yellow solid. Recrystallization of the solid from 20 ml
hot acetone gave compound 14 as colorless needle, mp
161–163 °C; IR mmax cmꢀ1: 2968, 2938 (CH3), 1633
4.2.10. 40,5,7-Tri[3-(2-hydroxyethylthio)propoxy]isoflav-
one (11). Compound 6 (0.63 g, 1 mmol), mercaptoethanol
(0.30 g, 4 mmol) and triethylamine (0.33 g, 3 mmol) in
30 ml of dry ethanol were sonicated. After the completion
of reaction, the reaction mixture was distilled. The pale
residue was washed by water and recrystallized from
10 ml acetone to give pure compound 10 in colorless nee-
dle, mp 78–80°C; IR mmax cmꢀ1: 3430 (OH), 2922, 2872
1
(C@O); H NMR d: 3.77, 3.82 and 3.87 (s, 3H each,
40, 5,7-OCH3), 6.50 (d, J = 2.3 Hz, 1H, H-8), 6.66 (d,
J = 2.3 Hz, 1H, H-6), 6.94 (d, J = 8.8 Hz, 2H, H-30
and H-50), 7.40 (d, J = 8.8 Hz, 2H, H-20 and H-60),
8.20 (s, 1H, H-2); HRMS (ESI) Calcd C18H16O5
[M+H]+ 313.1071. Found 313.1051. Anal. Calcd for
C18H16O5: C, 69.22; H, 5.16. Found: C, 69.25; H, 5.39.
1
(CH2), 1639 (C@O), 1051 (C–OH); H NMR d: 1.96
(overlapped multiplets, 6H, 3(–SCH2CH2CH2O–)), 2.56
(m, 6H, 3(–SCH2CH2CH2O–)), 2.66 and 2.67 (t, J =
7.0 Hz, 2H each, 2(HOCH2CH2S–)), 2.78 (t, J = 7.0 Hz,
2H, HOCH2CH2S–), 3.53 (t, J = 7.1 Hz, 4H, 2(HOCH2–)),
3.51 (t, J = 7.1 Hz, 2H, HOCH2–), 4.19, 4.12 and 4.07
(t, J = 6.1 Hz, 2H each, 3(–OCH2–)), 6.50 (d, J =
2.1 Hz, 1H, H-8), 6.64 (d, J = 2.1 Hz, 1H, H-6), 6.95 (d,
J = 8.8 Hz, 2H, H-30 and H-50), 7.40 (d, J = 8.8 Hz, 2H,
H-20 and H-60), 8.16 (s, 1H, H-2); HRMS (ESI) Calcd
C30H40O8S3 [M+H]+ 625.1959. Found 625.1962. Anal.
Calcd for C30H40O8S3: C, 57.67; H, 6.45. Found: C,
57.68; H, 6.39.
4.2.14. 40,5-Dihydroxy-7-methoxyisoflavone-30-sulfonic
acid (15). Concentrated sulfuric acid (0.5 ml, 98%) and
compound 12 (0.28 g, 1 mmol) in an erlenmeyer flask
were sonicated in an ice bath. After the completion of
reaction, the mixture was diluted with 0.5 ml of distilled
water to give white solid as precipitate. Recrystallization
of the solid from 5 ml water afforded compound 15 as
white needle, mp 365 °C (dec); IR mmax cmꢀ1: 3419
1
(OH), 2956 (CH3), 1655 (C@O), 1179 (S@O); H NMR
d: 3.86 (s, 3H, 7-OCH3), 6.41 (d, J = 2.1 Hz, 1H, H-8),
6.66 (s, 1H, H-6), 6.84 (d, J = 8.5 Hz, 1H, H-50), 7.39
(dd, J = 8.5, 1.2 Hz, 1H, H-60), 7.70 (d, J = 1.2 Hz, 1H,
H-20), 8.43 (s, 1H, H-2); HRMS (ESI) Calcd
C16H12O8S [MꢀH]ꢀ 363.0170. Found 363.0130. Anal.
Calcd for C16H12O8S: C, 52.75; H, 3.32. Found: C
52.61; H, 3.17. Compound 15 was neutralized with
NaOH to obtain its corresponding sodium sulfonate
for bioassay.
4.2.11. 40,5-Dihydroxy-7-methoxyisoflavone (12). Geni-
stein (0.27 g, 1 mmol), iodomethane (0.13 ml, 1 mmol)
and potassium carbonate (0.07 g, 0.5 mmol) in 50 ml
of dry acetone were sonicated. After the completion
of reaction, the afforded mixture was cooled to room
temperature followed by filtration. The filtrate was dis-
tilled to form a yellow solid. Recrystallization of the
solid from 20 ml hot ethanol yielded compound 12 as
pale yellow needle, mp 240–242 °C; IR mmax cmꢀ1
:
3412 (OH), 2964 (CH3), 1654 (C@O); 1H NMR d:
3.86 (s, 3H, 7-OCH3), 6.41 (br s, 1H, H-8), 6.65 (br
s, 1H, H-6), 6.82 (d, J = 8.0 Hz, 2H, H-30 and H-50),
7.38 (d, J = 8.0 Hz, 2H, H-20 and H-60), 8.40 (s, 1H,
H-2), 9.61 (s, 1H, 40-OH), 12.95 (s, 1H, 5-OH); HRMS
(ESI) Calcd C16H12O5 [MꢀH]ꢀ 283.0603. Found
4.2.15. 40,7-Dimethoxy-5-hydroxyisoflavone-30-sulfonic
acid (16). Concentrated sulfuric acid (0.5 ml, 98%) and
compound 13 (0.30 g, 1 mmol) in an erlenmeyer flask
were sonicated in an ice bath. After the completion of
reaction, the mixture was diluted with 0.5 ml of distilled
water to form white solid. Recrystallization of the solid

S. F. Wang et al. / Bioorg. Med. Chem. 13 (2005) 4880–4890
4889
from 5 ml water yielded compound 16 as white needle, mp
360 °C (dec); IR mmax cmꢀ1: 3421 (OH), 2957 (CH3), 1654
calcium diet to induce rapid osteoporosis,4,36 whereas
sham-operated animals were given conventional food.
1
(C@O), 1199 (S@O); H NMR d: 3.78 and 3.86 (s, 3H
each, 40, 7-OCH3), 6.41 (br s, 1H, H-8), 6.67 (br s, 1H,
H-6), 7.01 (d, J = 8.6 Hz, 1H, H-50), 7.45 (dd, J = 8.6,
2.3 Hz, 1H, H-60), 7.89 (d, J = 2.3 Hz, 1H, H-20), 8.42
(s, 1H, H-2), 12.92 (s, 1H, 5-OH); HRMS (ESI) Calcd
C17H14O8S [MꢀH]ꢀ 377.0326. Found 377.0346. Anal.
Calcd for C17H14O8S: C, 53.97; H, 3.73. Found: C,
53.88; H, 3.59. Compound 16 was neutralized with NaOH
to obtain its corresponding sodium sulfonate for
bioassay.
Two months later, OVX (n = 6) and sham-operated
(n = 6) rats were sacrificed by bleeding followed by a
midline abdominal laparotomy to excise total uterus.
Each uterus was put on individual labeled bibulous pa-
per to suck off the liquor on the surface before weighed.
Blood was obtained by cardiac puncture and serum was
kept frozen at ꢀ20 °C for biochemical analysis. After
this, the femora were taken out immediately with the
accompanying soft tissue cleaned up and stored in a
freezer at ꢀ80 °C until examination. The right femora
were used for the measurement of the bone mineral den-
sity (BMD), X-ray analysis and then burnt to ash for the
determination of the weight of bone ash (WBA) for eval-
uating inorganic matter content of bone.
4.2.16. 40,5,7-Trimethoxyisoflavone-30-sulfonic acid (17).
Concentrated sulfuric acid (0.5 ml, 98%) and compound
14 (0.31 g, 1 mmol) in an erlenmeyer flask were soni-
cated in an ice bath. After the completion of reaction,
the mixture was diluted with 0.5 ml of distilled water
to give white solid. Recrystallization of the solid from
5 ml water yielded compound 17 as white needle, mp
360 °C (dec); IR mmax cmꢀ1: 3431 (OH), 2955 (CH3),
4.4. Acute toxicity assessment
Every mouse (19–22 g) was given orally a single dose
(25 ml/kg) of each of 1, 7, 8, 9, 11, and 16 at 27 mmol/
kg which was 500-fold effective dosage, and the control
animal an equal volume of vehicle (DMSO/water (1:9)
mixture). Mice were observed for adverse effects imme-
diately, 2, 4, 8, and 24 h after dosing, followed by daily
observation till the 14th day.
1
1653 (C@O), 1180 (S@O); H NMR d: 3.66, 3.72 and
3.81 (s, 3H each, 40, 5,7-OCH3), 6.26 (br s, 1H, H-8),
6.36 (br s, 1H, H-6), 7.00 (d, J = 9.0 Hz, 1H, H-50),
7.32 (dd, J = 9.0 Hz, 2.0 Hz, 1H, H-60), 7.72 (d,
J = 2.0 Hz, 1H, H-20), 7.94 (s, 1H, H-2); HRMS (ESI)
Calcd C18H16O8S [MꢀH]– 391.0482. Found 391.0462.
Anal. Calcd for C18H16O8S: C, 55.10; H, 4.11. Found:
C, 54.98; H, 4.23. Compound 17 was neutralized with
NaOH to obtain its corresponding sodium sulfonate
for bioassay.
Acknowledgments
The financial support from the Ministry of Education
(Key Grant No. 104195) is acknowledged. The authors
are grateful to Ms. Xin Chen and Mr. Wei Guo Li at
Gulou hospital (Nanjing, China) for their assistance in
determination of BMD and X-ray analysis.
4.3. Bioassay on the OVX rat model
Ninety-six female Sprague–Dawley rats were obtained
from Nanjing University of Traditional Chinese Medi-
cine, aged 2 months, with an average weight of
202 2.5 g (mean SEM). Bilateral ovariectomies were
performed from a dorsal approach with a small midline
dorsal skin incision. The sham-operated rats were sub-
ject to sham surgery exposing, but the ovaries were
not removed. Success of ovariectomy was confirmed
by means of vagina smear and by observation of marked
atrophy of uterine horns. All rats were housed in a metal
cage on a 12 h light/dark cycle at 22 °C and 60% humid-
ity with free access to tap water. The rats were weighed
every week during the experiments.
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