Synthesis of genistein derivatives and determination of their protective effects against vascular endothelial cell damages caused by hydrogen peroxide

Article abstract of DOI:10.1016/j.bmcl.2007.11.097

A series of genistein derivatives, prepared by alkylation and difluoromethylation, were tested for their inhibitory effects on the hydrogen peroxide induced impairment in human umbilical vein endothelial (HUVE-12) cells in vitro. The HUVE-12 cells were pr

Full text of DOI:10.1016/j.bmcl.2007.11.097

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Bioorganic & Medicinal Chemistry Letters 18 (2008) 513–517
Synthesis of genistein derivatives and determination
of their protective effects against vascular endothelial cell
damages caused by hydrogen peroxide
Xiao-Hua Fu,a Li Wang,b Hong Zhao,a Hong-Lin Xianga and Jian-Guo Caoa,b,
*
aCollege of Medicine, Hunan Normal University, Changsha, Hunan 410006, PR China
bCollege of Medicine, University of South China, 28 Changsheng Rd, Hengyang, Hunan 421001, PR China
Received 9 August 2007; revised 13 November 2007; accepted 26 November 2007
Abstract—A series of genistein derivatives, prepared by alkylation and difluoromethylation, were tested for their inhibitory effects
on the hydrogen peroxide induced impairment in human umbilical vein endothelial (HUVE-12) cells in vitro. The HUVE-12 cells
were pretreated with either the vehicle solvent (DMSO), genistein, or different amounts of the genistein derivatives for 30 min before
exposed to 1 mM hydrogen peroxide for 24 h. Cell apoptosis was determined by flow cytometry with propidium iodide (PI) staining.
Cellular injury was estimated by measuring the lactate dehydrogenase (LDH) release. Data suggested that the genistein derivatives
possessed a protective effect on HUVE-12 cells from hydrogen peroxide induced apoptosis and reduced LDH release. Among these
derivatives, 7-difluoromethyl-5,40-dimethoxygenistein exhibited the strongest activity against hydrogen peroxide induced apoptosis
of HUVE-12 cells.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Soybeans and soy products have been an important part
of the oriental diet for centuries. In recent years, the
therapeutic effects of certain compounds in the soy
products have attracted increased attention among the
science community. These compounds have been indi-
cated to reduce the health risk of certain diseases, such
as the cardiovascular ailments and various types of
cancers.
Endothelial cells are crucial in maintaining the physio-
logical functions of the cardiovascular system and they
are also involved in the development of a variety of hu-
man diseases.1 The two leading cardiovascular diseases,
hypertension and atherosclerosis, are complex processes
that occur, at least in part, in response to the necrosis or
apoptosis resulted vascular injury.2,3 Increasing evi-
dences suggested that oxidative-stress and free radicals
produced during apoptosis were the major causes of
endothelial damage. Therefore, pharmacological inter-
ventions targeting the endothelial remodeling have be-
come a hot spot in the biomedical research.
Among these beneficiary compounds, phytochemicals
were considered to play the major role. Phytochemicals
are biologically active compounds in plants. Soybeans
are a rich source of many phytochemicals including iso-
flavones, saponins, phytic acid, and phytosterols. Iso-
flavones are a class of phenolic compounds, which
include daidzein, glycitein, and genistein. These com-
pounds exist in soybeans either as glucosides or in free
form (aglucons). Genistein is the major isoflavone in
soy.
Using natural products or extracts from plants for the
treatment of oxidative stress-induced cell injury has
been demonstrated by more and more researchers,
for example, isoflavone significantly decreased the
post menopause-related cardiovascular disease.4 Geni-
stein, an isoflavone compound, has been reported to
inhibit the apoptosis in cultured endothelial cells
and in animal models.5,6 However, the low absor-
bance of flavonoids in intestines resulted in the low
biological activities of this compound.7,8 Therefore,
designing new ready-to-absorb compounds using gen-
istein as the lead compound is essential for the geni-
stein bioactivity.
Keywords: Genistein derivatives; Protective effects; Vascular endothe-
lial cell; 7-Difluoromethylyl-5,40-dimethoxygenistein.
*
Corresponding author. Tel.: +86 734 8281510; fax: +86 734
8281305; e-mail: caojianguo2005@yahoo.com.cn
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.11.097

Table 1. Characterization of the synthesized compounds
Compound
MS (EI, 70 eV)
IRtmax (cmꢀ1, KBr)
1H NMR (300 MHz, CDCl3)
19F NMR (282 MHz)
2
m/z: 332
1448, 1519, 1662 (C@O), 3319, 3528 (OH)
6.599 (1H, d, J = 2.4 Hz), 6.820 (1H, t,
J = 12.0 Hz), 6.867 (1H, d, J = 2.4 Hz), 7.371 (2H,
d, J = 12.0 Hz), 7.427 (1H, t, J = 72.9 Hz), 8.244
(1H, s), 9.550 (1H, s), 12.994 (1H, s)
ꢀ84.372 (d, J = 72.9 Hz)
3
m/z: 370
m/z: 348
m/z: 376
m/z: 375
1430, 1448, 1498, 1653 (C@O), 3074 (OH)
1430, 1469, 1512, 1580, 1612, 1637 (C@O)
1476, 1581, 1613 (C@O), 1645, 2874, 2984
6.651 (1H, t, J = 73.8 Hz), 6.590 (1H, d,
J = 2.4 Hz), 6.638 (1H, t, J = 72.3 Hz), 6.684 (1H,
d, J = 2.4 Hz), 7.229 (2H, d, J = 9.0 Hz), 7.551
(2H, d, J = 9.0 Hz), 7.975 (1H, s)
ꢀ80.953 (d, J = 73.8 Hz), ꢀ82.414 (d,
J = 72.3 Hz)
4a
4b
4c
3.834 (3H, s),3.970 (3H, s), 6.540 (1H, d,
J = 2.1 Hz), 6.642 (1H, t, J = 72.3 Hz), 6.724 (1H,
d, J = 2.1 Hz), 6.940 (1H, d, J = 8.7 Hz), 7.472
(1H, d, J = 8.7 Hz), 7.811 (1H, s)
ꢀ82.150 (d, J = 72.3 Hz)
ꢀ82.085 (d, J = 72.6 Hz)
ꢀ82.142 (d, J = 72.6 Hz).
1.386–1.572 (6H, m), 4.023–4.181 (4H, m), 6.525
(1H, d, J = 2.4 Hz), 6.628 (1H, t, J = 72.6 Hz),
6.699 (1H, d, J = 2.4 Hz), 6.931 (2H, d,
J = 8.4 Hz), 7.436 (2H, d, J = 8.4 Hz)
1469, 1513, 1581, 1614, 1651 (C@O),
2880, 2968
0.972–1.120 (4H, m), 1.157 (3H, t, J = 7.5 Hz),
1.223 (3H, t, J = 7.5 Hz), 3.987–4.164 (4H, m),
6.643 (1H, d, J = 2.4 Hz), 6.761 (1H, t,
J = 72.6 Hz), 6.807 (1H, d, J = 2.4 Hz), 7.053 (2H,
d, J = 9.0 Hz), 7.547 (2H, d, J = 9.0 Hz)
4.636 (4H, s), 7.314–7.342 (18H, m)
4d
4e
m/z: 500
m/z: 431, 517 (M+)
1454, 1513, 1579, 1614, 1647 (C@O), 2926
1433, 1469, 1581, 1614, 1652 (C@O),
2859, 2956
ꢀ82.161 (d, J = 72.2 Hz)
ꢀ82.897 (d, J = 72.9 Hz)
0.906–2.016 (26H, m), 3.950 (2H, t, J = 6.6 Hz),
4.047 (2H, t, J = 6.6 Hz), 6.512 (1H, d,
J = 2.1 Hz), 6.648 (1H, d, J = 2.1 Hz), 6.764 (1H,
t, J = 72.9 Hz), 9.893 (2H, d, J = 9.0 Hz), 7.412
(2H, d, J = 9.0 Hz), 7.759 (1H, s)
4f
m/z: 544
1435, 1467, 1512, 1569, 1611, 1651
(C@O), 2858, 2929
0.860–1.218 (30H, m), 3.985–4.046 (4H, m), 6.378
(1H, d, J = 2.1 Hz), 6.423 (1H, d, J = 2.1 Hz),
6.531 (1H, t, J = 74.1 Hz), 7.155 (2H, d,
J = 8.7 Hz), 7.543 (2H, d, J = 8.7 Hz), 7.748 (1H,
s)
ꢀ80.555 (d, J = 74.1 Hz)
ꢀ82.052 (d, J = 72.3 Hz)
ꢀ82.348 (d, J = 72.9 Hz)
4g
4h
m/z: 473, 600 (M+)
1658 (C@O)
0.848–1.783 (38H, m), 3.966 (2H, t, J = 6.6 Hz),
4.040 (2H, t, J = 6.6 Hz), 6.513 (1H, d,
J = 2.1 Hz), 6.627 (1H, t, J = 72.3 Hz), 6.674 (1H,
d, J = 2.1 Hz), 6.925 (2H, d, J = 8.4 Hz), 7.423
(2H, d, J = 8.4 Hz), 7.753 (1H,s)
m/z: 432
1472, 1513, 1579, 1628, 1650 (C@O),
2876, 2963
0.809–1.057 (12H, m), 2.176–2.198 (2H, m), 3.759
(2H, t, J = 6.6 Hz), 3.783 (2H, t, J = 6.6 Hz), 6.568
(1H, d, J = 2.4 Hz), 6.627 (1H, t, J = 72.9 Hz),
6.656 (1H, d, J = 2.4 Hz), 6.984 (2H, d,
J = 9.0 Hz), 7.446 (2H, d, J = 9.0 Hz), 7.941 (1H,
s)

X.-H. Fu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 513–517
515
Figure 1. Synthetic scheme of the genistein derivatives.
It is well known that the introduction of the CHF2
group into organic molecules can often dramatically
change their physical and chemical properties.9 In addi-
tion, the extension or the branching of hydrocarbon
chain may affect their bioactivity.10 Thus we designed,
synthesized, and tested genistein derivatives that had
7-OH group substituted by –OCHF2 and 5-OH, 4-OH
groups substituted by alkoxylates. The activities of those
derivatives were assayed with the established model of
human umbilical vein endothelial (HUVE-12) cells
cultured in vitro and apoptosis induced by oxidative
stress, we discovered an active new chemical (7-dif-
luoromethylyl-5,40-dimethoxygenistein) that showed
high protective activity against oxidative stress induced
apoptosis of the vascular endothelial cells.
concentrations of 7-difluoromethylyl-5,40-di substituted
alkoxyl genistein (0.1, 1.0, and 10.0 lM) before being
challenged with 1 mM H2O2 for 24 h. HUVE-12 cells
were also treated with various concentrations of dFM-
GEN, genistein (0.1, 0.3, 1.0, 3.0, and 10.0 lM) before
being challenged with 1 mM H2O2 for 24 h. Cells were
then harvested with 0.25% trypsin and washed with
PBS. For the flow cytometry, cells at a density of
1 · 106/ml were fixed on ice with 70% ice-cold EtOH
in PBS for 20 min, washed with PBS, incubated in pro-
pidium iodide (PI) solution (69 mM PI, 388 mM sodium
citrate, 100 lg/ml RNase A) for 15 min at 37 ꢁC, and
immediately analyzed with the FAC Scan flow cytome-
ter (Becton–Dickinson, San Jose, USA).11 Lactate dehy-
drogenase (LDH) activity was determined using a
colorimetric assay kit obtained from Sigma (St. Louis,
MO). Before the cells were harvested, 100 ll of medium
from each well was removed and used for LDH activity
assay.
The readily available genistein (1) was used as the start-
ing material for the preparation of the 7-difluoromethy-
lated and 7,40-gem-difluoromethylated genistein
derivatives. Compound 1 (genistein) was mixed with
HCF2Cl/NaOH and incubated at 40 ꢁC for 24 h. Ethyl
acetate was then used for the extraction of the organic
products and the extracted products were separated with
column chromatography. A mixture of ethyl acetate and
petroleum ether in different ratios was used for elution.
The two compounds prepared were named 7-difluoro-
methyl-5,40-dihydroxygenistein (2) and 5-hydroxyl-7,40-
gem-difluoromethylgenistein (3). Compound 2 was then
mixed with K2CO3 and alkylating agents dissolved in
acetone, and the mixture was allowed to react for 8–
24 h at 40 ꢁC. Eight new compounds (Compounds 4a–
4h, Fig. 1) were prepared. All the new compounds were
characterized by detailed spectroscopic analysis of mass
The new compounds synthesized during the reaction
were characterized in detail by spectroscopic analysis
(Table 1). Compounds 4a–4h were identified as 7-
difluoromethyl-5,40-dimethoxygenistein (4a), 7-difluoro-
methyl-5,40-diethyloxygenistin (4b), 7-difluoromethyl- 5,
40-dipropeinoxygenistein (4c), 7-difluoromethyl-5,40-dib-
enzyloxygenistein (4d), 7-difluoromethyl -5,40-diheptane-
oxygenistein (4e), 7-difluoromethyl -5,40-dioctyloxygenistein
(4f), 7-difluoromethyl-5,40-dinonyloxygenistein (4g), and
7-difluoromethyl-5,40-diisohexenylglutaconyloxygenistein
(4h).
The flow cytometric analysis using propidium iodide
(PI) staining indicated that the incubation of H2O2 for
24 h promoted the apoptosis of HUVE-12 cells. When
cells were pretreated with 7-difluoromethylyl-5,40-di-
substituted alkoxyl isoflavones in different concentra-
tion, however, the rate of apoptosis decreased
(Fig. 2A). The LDH activity (Fig. 2B) was induced by
1 mM H2O2 significantly. However, genistein and its
derivatives reversed this effect. The derivative 7-dif-
luoromethylyl-5,40-dimethoxygenistein seemed to have
a better protective effect against the endothelium injury
from oxidative stress than the lead compound, genistein.
The derivative 7-difluoromethylyl-5,40-dimethoxygeni-
stein was the most potent among all the derivatives.
1
spectrometry (MS), infrared radiation (IR), and H and
19F nuclear magnetic resonance (NMR).
Human umbilical vein endothelial cell line (HUVE-12)
was obtained from the China Center for Type Culture
Collection (Wuhan, China). Cells were cultured in
DMEM (Clonetics, Walkersville, USA) supplemented
with 10% newborn calf serum, 100 U/mL penicillin,
and 100 lg/mL streptomycin (Life Technology, New
York, USA) at 37 ꢁC in a 5% CO2 incubator. The cul-
tured human umbilical vein endothelial cells (HUVE-
12) were incubated for 30 min in vitro with either the
vehicle solvent (0.02% DMSO), genistein or different

516
X.-H. Fu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 513–517
20
18
16
14
12
20
18
16
14
12
10
8
A
Compound 4a + H
GEN + H 1mM
2
O
2 1mM
2O2
10
**
*
*
8
*
*
6
**
**
6
4
2
0
**
4
**
**
**
2
**
0
*
Figure 3. 7-Difluoromethylyl-5,40-dimethoxygenistein suppressed
H2O2-induced apoptosis of vascular endothelial cells in a concentra-
tion dependent manner. Data shown here means from three indepen-
dent experiments. *P < 0.05 versus genistein + 1 mM H2O2, **P < 0.01
versus genistein + 1 mM H2O2.
160
140
120
100
80
B
LDH activity increase in the medium is a general marker
for cell damage.16 Results in our study suggested that
the derivative of 7-difluoromethylyl-5,40-dimethoxygeni-
stein reduced the release of LDH H2O2-induced HUVE-
12 cells. We also showed that this derivative suppressed
H2O2-induced apoptosis of vascular endothelial cells in
a concentration dependent manner (Fig. 3). All these
data demonstrated that 7-difluoromethylyl-5,40-dimeth-
oxygenistein could provide protection against vascular
endothelium injury caused by oxidative stress.
*
*
*
*
*
60
**
**
40
**
20
0
Acknowledgment
We thank the support by the grant from the Key Pro-
gram of the Department of Education of Hunan Prov-
ince, China (No. 05A035).
Figure 2. Protective effects of different genistein derivatives against
vascular endothelial cell damages caused by hydrogen peroxide. (A)
FCM has shown all genistein derivatives can decrease apoptosis rate of
H2O2-induced HUVE-12 cells. Among them, 7-difluoromethylyl-5,40-
References and notes
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*
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**
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