Ester and carbamate ester derivatives of Biochanin A: Synthesis and in vitro evaluation of estrogenic and antiproliferative activities

Article abstract of DOI:10.1016/j.bmc.2012.03.012

Biochanin A (BCA), a major isoflavone in red clover and many other legumes, has been reported to display estrogenic as well as cancer chemopreventive properties. Ingested BCA is known to display low bioavailability due to poor solubility, extensive metabolism and rapid clearance. Esters of bioactive isoflavones are known to increase metabolic stability and bioavailability following local rather than systemic administration. We synthesized BCA from phloroglucinol and p-methoxy-phenylacetic acid by a Friedel-Crafts reaction and cyclization. We also synthesized esters (1, 3) and carbamate esters (2, 4, 5) at position 7 of BCA using short aliphatic chains bearing a chlorine (1, 2) or a bromine atom (3, 4) or long aliphatic chains without such atoms (5). We tested the estrogenic and antiproliferative activities of 1-5 and BCA using human breast and endometrial adenocarcinoma cells. We found that 5 affects MCF-7 and Ishikawa cells in a manner providing for induction of gene expression to a level similar to 17β-estradiol and BCA but, unlike both of the latter, for suppression of cell proliferation as well. In addition, 5 appeared to display higher stability compared to 1-4 and BCA in both MCF-7 and Ishikawa cells. The inference is that 5 may represent a safer than BCA alternative to hormone replacement therapy.

Full text of DOI:10.1016/j.bmc.2012.03.012

Bioorganic & Medicinal Chemistry 20 (2012) 2962–2970
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Ester and carbamate ester derivatives of Biochanin A: Synthesis and
in vitro evaluation of estrogenic and antiproliferative activities
Nikolas Fokialakis a, , Xanthippi Alexi b, , Nektarios Aligiannis a, Despina Siriani b, Aggeliki K. Meligova b,
b,
⇑
Harris Pratsinis c, Sofia Mitakou a, , Michael N. Alexis
⇑
a Department of Pharmacognosy and Natural Products Chemistry, Faculty of Pharmacy, University of Athens, Panepistimiopolis, 157 71 Athens, Greece
b Molecular Endocrinology Programme, Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, 48 Vassileos Constantinou Ave.,
116 35 Athens, Greece
c Laboratory of Cell Proliferation & Ageing, Institute of Biology, NCSR ‘Demokritos’, GR-15310, Athens, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
Biochanin A (BCA), a major isoflavone in red clover and many other legumes, has been reported to display
estrogenic as well as cancer chemopreventive properties. Ingested BCA is known to display low bioavail-
ability due to poor solubility, extensive metabolism and rapid clearance. Esters of bioactive isoflavones
are known to increase metabolic stability and bioavailability following local rather than systemic admin-
istration. We synthesized BCA from phloroglucinol and p-methoxy-phenylacetic acid by a Friedel–Crafts
reaction and cyclization. We also synthesized esters (1, 3) and carbamate esters (2, 4, 5) at position 7 of
BCA using short aliphatic chains bearing a chlorine (1, 2) or a bromine atom (3, 4) or long aliphatic chains
without such atoms (5). We tested the estrogenic and antiproliferative activities of 1–5 and BCA using
human breast and endometrial adenocarcinoma cells. We found that 5 affects MCF-7 and Ishikawa cells
in a manner providing for induction of gene expression to a level similar to 17b-estradiol and BCA but,
unlike both of the latter, for suppression of cell proliferation as well. In addition, 5 appeared to display
higher stability compared to 1–4 and BCA in both MCF-7 and Ishikawa cells. The inference is that 5
may represent a safer than BCA alternative to hormone replacement therapy.
Received 12 December 2011
Revised 1 March 2012
Accepted 3 March 2012
Available online 10 March 2012
Keywords:
Biochanin A
Carbamate
Ester
Estrogenic activity
Chemoprevention
MCF-7
Ishikawa cells
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
oxyisoflavone), an ERb-selective isoflavone in red clover and many
other legumes, is commercially available as a nutraceutical and is
Hormone replacement therapy (HRT) of postmenopausal symp-
toms using combined estrogen–progestin or estrogen-only regi-
mens is associated with increased breast and endometrial cancer
risk.1,2 However, menopausal symptoms such as hot flashes, poor
wound healing and vaginal dyspareunia may be treated by local
rather than systemic administration of estrogen, thus limiting ad-
verse side effects of HRT. Estrogen action is primarily mediated by
known to display cancer chemopreventive properties.6 Although
BCA is thought to exert its beneficial effects predominantly
through ER
a and ERb, BCA agonism of estrogen-related receptors
(ERR) is also well documented.7
Phytoestrogen-containing dietary supplements are widely con-
sumed for putative health benefits. However, although preclinical
studies suggest that dietary intake of phytoestrogens can protect
from postmenopausal symptoms and endocrine-related degenera-
tive diseases, including endocrine-related cancer,8 clinical trials
suggest that their health benefits are inconsistent or marginal.9–11
Pharmacokinetic studies in rats suggest that plasma BCA concentra-
two estrogen receptor (ER) subtypes, ER
a
and ERb, with ERb func-
tioning as tumor suppressor by opposing the mitogenic action of
ERa
.3 Accordingly, in human breast, malignant progression is asso-
ciated with down-regulation of ERb expression.4 Phytoestrogens
are plant derived natural products that have functional similarities
with estrogen and are considered as a safe alternative to HRT.5 Iso-
flavones are a major group of phytoestrogens that have been the
focus of many studies. Biochanin A (BCA, 5,7-dihydroxy-40-meth-
tions 60.01 lM are attainable with a daily oral intake of 5 mg per kg
of body weight.12 Following ingestion, BCA is known to display low
bioavailability due to poor solubility, extensive metabolism and ra-
pid clearance.12 BCA is primarily metabolized to the isoflavone gen-
istein and to BCA and genistein conjugates.12,13 Esters and especially
carbamate esters of bioactive phenols are known to increase bio-
availability and/or metabolic stability.14,15 Interestingly, interven-
tion studies have shown that it is possible to introduce intact
genistein ester molecules in sufficiently high concentrations by sub-
cutaneous but not through oral administration.16 The present study
⇑
Corresponding authors. Tel.: +30 210 7274598; fax: +30 210 7274594 (S.M.);
tel.: +30 210 7273741; fax: +30 210 7273677 (M.N.A.)
E-mail addresses: mitakou@pharm.uoa.gr (S. Mitakou), mnalexis@eie.gr
(M.N. Alexis).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.03.012

N. Fokialakis et al. / Bioorg. Med. Chem. 20 (2012) 2962–2970
2963
Table 1
describes the preparation of esters and carbamate esters of BCA and
their biological evaluation using human breast and endometrial
adenocarcinoma cells.
Relative ERa
- and ERb-binding affinity of BCA and its derivativesa
Compound
RBAa
RBAb
Estradiol
100
100
PPTb
MPPc
DPNd
BCAe
1
2
3
4
5
50.2 2.4
20.2 4.3
0.32 0.04
0.24 0.11
0.03 0.01
0.05 0.01
0.03 0.01
0.05 0.02
0.06 0.01
1.4 0.3
0.3 0.1
2. Results
13.4 2.5
0.92 0.16
0.09 0.02
0.06 0.01
n.d.
2.1. Chemistry
In order to synthesize BCA, starting from phloroglucinol and the
addition of p-methoxy phenylacetic acid, a Friedel–Crafts reaction
catalyzed by boron trifluoride took place to afford the intermediate
n.d.
0.08 0.02
product
1-(2,4,6-trihydroxyphenyl)-2-(4-methoxyphenyl)etha-
a
The RBA values (mean SEM of at least three independent experiments) of
Biochanin A (BCA) and its derivatives for ER (RBA ) and ERb (RBAb) were calcu-
none. Then a cyclization to BCA was performed using CH3SO2Cl
in DMF and freshly distilled BF3ꢀEt2O as catalyst under argon
(Scheme 1).17 Then BCA was mixed with the corresponding acyl-
chlorides or isocyanates in order to prepare the esters (1, 3) and
carbamate esters (2, 4 and 5) at position 7 of BCA.
a
a
lated by [RBA = (IC50 estradiol/IC50 test compound) ꢁ 100], where IC50 values are
estradiol or test compound concentrations capable of inhibiting the binding of the
fluorescent estrogen ES2 (1 nM) to ER
ER
a
and ERb by 50%. IC50 values of estradiol for
and RBAb of estradiol
a
and ERb were 2.22 and 3.3 nM, respectively. The RBA
a
were set equal to 100.
b
PPT (propyl-pyrazol-triol), ER
MPP (methyl-piperidino-pyrazole), ERa-specific antagonist.
DPN (diaryl-propionitrile), potency-selective ERb agonist.
BCA, Biochanin A.
a
-specific agonist.
2.2. Assessment of estrogenic and anticancer activities of BCA
and its derivatives
c
d
e
Initially we used fluorescence polarization to assess the ability
of BCA and its ester (1, 3) and carbamate ester (2, 4, 5) derivatives
to compete with fluorescent estradiol for binding to purified re-
of 10 on average. The previously reported RBA
a
and RBAb values
-specific
combinant human ER
affinity (RBA) values of BCA for ER
0.24 0.11 and 0.92 0.16, respectively (Table 1). The RBA
a
and ERb. Relative (to estradiol) binding
and ERb were found equal to
and
of the ER -specific agonist PPT (propyl-pyrazol-triol), ER
a
a
a
antagonist MPP (methyl-piperidino-pyrazole) and the potency-
selective ERb agonist DPN (diaryl-propionitrile) are also shown
for comparison.18,19
a
RBAb values of 1–5 were found to be lower than BCA by factors
(a) Reagents for compound 1: Py, ClCH2COCl,
for compound 2: Py, ClCH2CH2NCO,
for compound 3: Py, BrCH2COCl,
for compound 4: Py, ΒrCH2CH2NCO
for compound 5: Py, CH3(CH2)10NCO,
HO
OH
HO
OH
HO
O
BF3Et2O
80 ºC
BF3Et2O
OH
MeSO 2Cl
DMF
+
HO
OH
O
OH
O
OCH
3
OCH
3
O
BCA
OCH
3
a
RO
O
O
1 R = ClCH2CO
2 R = ClCH2CH2NCO
3 R = BrCH2CO
4 R = BrCH2CH2NCO
5
=
CH3(CH2)10NCO
R
OH
OCH
3
Scheme 1. Synthesis of BCA derivatives. (a) Reagents for compound 1: Py, ClCH2COCl, for compound 2: Py, ClCH2CH2NCO, for compound 3: Py, BrCH2COCl, for compound 4:
Py, BrCH2CH2NCO for compound 5: Py, CH3(CH2)10NCO.

2964
N. Fokialakis et al. / Bioorg. Med. Chem. 20 (2012) 2962–2970
The ability of 1–5 and BCA to induce estrogen response element
efficacies of test compounds compared to estradiol were not due
to differences in viable cell numbers (data not shown).
(ERE)-dependent gene expression was assessed using MCF-7:D5L
cells, a previously reported clone of MCF-7 cells.20 As expected
from previous findings,20–22 treatment of estrogen-free MCF-
7:D5L cells with estradiol concentrations P0.1 nM resulted in full
(3- to 4-fold) induction of luciferase expression in a manner that
was fully inhibited by the ER destabilizer fulvestrant (Fig. 1A and
We also determined the potential of 1–5 and BCA to stimulate
the proliferation of MCF-7 cells. These cells are known to cease
proliferating following transfer to estrogen-free media supple-
mented with fetal bovine serum which has been treated with dex-
tran coated charcoal to remove endogenous estrogens.23 Estradiol
is known to stimulate the proliferation of MCF-7 cells primarily
B). Figure 1A shows, in addition, that the ER
PPT displayed an induction efficacy similar to estradiol; and that
the ER -specific antagonist MPP and an equimolar combination
a-specific agonist
through ERa.
24 Treatment with 1–4 or BCA stimulated cell prolifer-
a
ation to levels significantly lower (t-test) than 0.1 nM estradiol in
an ER-dependent manner, as assessed using fulvestrant, while 5
was totally ineffective in this respect (Fig. 2 and data not shown).
The rank order of proliferation stimulation efficacies (Table 2,
of MPP and the potency-selective ERb agonist DPN failed to induce
luciferase activity. Interestingly, treatment of MCF-7:D5L cells with
1–5 or BCA induced luciferase expression to a level considerably
higher than 0.1 nM estradiol in ER-dependent manner, as assessed
using fulvestrant (Fig. 1B and data not shown). The rank order of
columns 6 and 7) at 1
lM test compound was, E2 > BCA P 3 P
1 ꢂ 4 P 2 > 5 ꢂ vehicle (t-test), while at 10
l
M test compound
luciferase induction efficacies (Table 2, columns 3 and 4) at 1
test compound was, 1 > 2 P 3 P 4 P 5 P BCA > E2 > vehicle (t-
test), while at 10 >
lM
was, E2 > BCA ꢂ 2 P 1 ꢂ 3 P 4 > 5 ꢂ vehicle (t-test). We also
determined the effect of 1–5 and BCA on the proliferation of
MCF-7 cells in the presence of 0.1 nM estradiol (Fig. 3), a concen-
tration of the hormone similar to that usually found in the plasma
of postmenopausal women.25 We observed partial suppression of
l
M
test compound was, 1 ꢂ 3 > 2 P 4
5 P BCA > E2 > vehicle (t-test). Assessment of the number of viable
cells using MTT suggested that the higher luciferase induction
estrogen stimulation of cell proliferation with 10
10 M 1 or 3 and 1 M 4. Notably, however, 4 at 10
concentrations P1 M fully suppressed the proliferation of MCF-7
cells (Fig. 3). We also tested 1–5 and BCA against MDA-MB-231
cells which are known to lack ER expression and to proliferate
in an estrogen-independent manner. Carbamate ester 5 inhibited
by 21% the proliferation of MDA-MB-231 cells at 10 M but was
totally ineffective at 1 M.
Finally, we determined the ability of test compounds to induce
AlkP expression in Ishikawa cells. As expected from previous find-
ings,20,21,26 treatment of estrogen-free Ishikawa cells with estradiol
at concentrations P0.1 nM resulted in full (ꢃ4.4-fold) induction of
AlkP expression in a manner that was fully inhibited by the ER
destabilizer fulvestrant (Fig. 4A and B). Figure 4A shows, in addi-
tion, that PPT induced and MPP failed to induce AlkP activity in
Ishikawa cells, much like luciferase activity in MCF-7:D5L cells;
however, unlike MCF-7:D5L cells, Ishikawa cells responded to the
equimolar combination of MPP and DPN by inducing AlkP to a level
ꢃ70% of that of estradiol. Treatment of Ishikawa cells with increas-
ing concentrations of 1–5 or BCA induced AlkP expression to a level
similar (2, 4) or significantly lower (1, 3, 5, BCA) than 0.1 nM estra-
diol in an ER-dependent manner, as assessed using fulvestrant
(Fig. 4B and data not shown). The rank order of induction efficacies
l
M BCA, 1 and
l
l
l
lM and 5 at
400
A
a
*
*
l
300
200
100
0
l
vehicle
E2
E2+Fulv
PPT
MPP MPP+DPN
1400
1200
1000
800
600
400
200
0
B
1
2
3
4
(Table 2, columns
9
and 10) at
1
l
M
test compound was,
M test
5
BCA
0.1 nM E2
E2 > 3 P 1 ꢂ BCA ꢂ 4 ꢂ 2 > 5 > vehicle (t-test), while at 10
l
compound was, E2 ꢂ 2 ꢂ 4 > 3 P 5 P 1 P BCA > vehicle (t-test).
We also determined the effect of 1–5 and BCA on the proliferation
of Ishikawa cells. BCA stimulated the proliferation of Ishikawa cells
at 10
l
M, while 5 displayed a significant (t-test) inhibitory effect at
M (Fig. 5). In contrast, estra-
the concentration of 1 as well as 10
l
diol and fulvestrant failed to significantly affect the proliferation of
Ishikawa cells, in accordance with previous findings.26,27
3. Discussion
0.01
0.1
1
10
High affinity ligand binding to ERa and ERb largely depends on
two hydroxyl groups with an O–O distance similar to estradiol
Concentration (μM)
(10.8 Å) or the potency-selective ERb agonist genistein
Figure 1. Induction of luciferase expression in MCF-7:D5L cells: (A) by 0.1 nM
estradiol (E2) in the absence or presence of 100 nM fulvestrant, 100 nM PPT and
100 nM MPP in the absence or presence of 100 nM DPN. Data are mean SEM of
three independent experiments carried out in triplicate. ⁄ p 60.05 versus vehicle (t-
test). (B) By increasing concentrations of Biochanin A (BCA) and its ester (1, 3) and
carbamate ester (2, 4, 5) derivatives. Luciferase activity is expressed as % of that of
cells treated only with vehicle (compound diluent). Luciferase activity in the
presence of 0.1 nM estradiol (E2) is shown for comparison. Data are mean of three
independent experiments carried out in triplicate with an inter-assay variation
(mean SEM) similar to that shown for BCA.
(12.1 Å).20,28 The RBA
a and RBAb values of BCA are lower than
those previously reported for genistein by factors of approx. 4
and 48, respectively.21 The 40-OH and the 7-OH of genistein are
known to mimic the 3-OH and 17-OH of estradiol in forming H
bonds with the side chains of Glu353-Arg394 (Glu305-Arg346 in
ERb) and His524 (His475), respectively.20,28 H bond formation with
Glu353 and Arg394 is presumably not possible for BCA due to the

N. Fokialakis et al. / Bioorg. Med. Chem. 20 (2012) 2962–2970
2965
methylation of its 40-OH. The lower RBA values of 1–5 compared to
BCA could reflect inability of H bond formation between the 7-OH
group and His524 as well. Based on previous findings with several
other isoflavones,21 the above RBA values indicate that BCA con-
centrations P0.1 lM are deemed necessary for full induction of
gene expression in Ishikawa and MCF-7:D5L cells; and that the
necessary concentrations of 1–5 could be even higher than BCA.
Notably, however, compared to BCA 1–5 displayed considerably
higher luciferase induction potencies, that is, lower EC50 (Table 2,
column 2), than would have been predicted on the basis of their
RBAa values, suggesting that their estrogenic activity was probably
the result of ester hydrolysis.
We observed that luciferase expression of MCF-7:D5L cells was
fully induced by the ER
combination of the ER
selective ERb agonist DPN was totally ineffective in this respect, in
a-specific agonist PPT, while the equimolar
a-specific antagonist MPP and the potency-
accordance with reports that MCF-7 cells express ER
a but hardly
any ERb.19,29 We also observed that treatment of MCF-7:D5L cells
with 1–5 or BCA induced luciferase expression to a level consider-
ably higher than 0.1 nM estradiol in ER-dependent manner. It has
been reported that while estradiol is unable to bind to any one of
the three subtypes of ERR (ERRa, ERRb and ERRc), BCA and its
major metabolite genistein are ERR agonists;27 that BCA agonism
of ERR and ER occur under comparable conditions;27 and that
ER
estradiol can be enhanced by ERR possibly through increased
recruitment of co-activators to ERa 30
In the light of these reports
a-dependent induction of ERE-driven luciferase reporters by
.
the data of Figure 1B are taken to suggest that, in MCF-7:D5L cells,
BCA agonism of ERR enhanced isoflavone induction of luciferase
through ER
as in wild-type MCF-7 cells. Luciferase induction efficacies at
10 M test compound are thought to reflect levels of free BCA in-
side MCF-7:D5L cells under the conditions of the assay. Hence, it
appears that 2 and 4 at 10 M released lower amounts of BCA com-
a, the predominant form of ER in these cells as well
l
l
pared to 1 and 3, but higher amounts compared to 5; and that
metabolism and rapid efflux of BCA likely prevented the free iso-
flavone from mounting a luciferase response as high as 1–4. Conse-
quently, it appears that esters (1, 3) and carbamate esters (2, 4, 5)
of BCA display different stabilities in the specific cell context and
hence different abilities to penetrate and remain in MCF-7:D5L
cells, with the carbamate esters being more stable and therefore
more effective in controlling metabolism and rapid efflux of the
isoflavone. Presumably, BCA released in the cytoplasm following
hydrolysis of 1–5 will be equally accessible to ER and ERR, since
these nuclear receptors display intensive ligand-independent
nucleocytoplasmic shuttling.31,32
The data of Figures 1B and 2 show that 5 induced ERE-depen-
dent gene expression to a level similar to estradiol and BCA, but
failed to stimulate the proliferation of MCF-7 cells. We have previ-
ously reported that some deoxybenzoins can exhibit a similar de-
gree of dissociation between gene expression and cell
proliferation stimulation efficacies.20 In addition, the data of Fig-
ure 3 show that 5 fully inhibited estradiol-dependent proliferation
of MCF-7 cells. This is taken to indicate that 5 displays higher abil-
ity to penetrate and remain inside MCF-7 cells, crossing cell mem-
branes easily and partitioning more effectively in critical cellular
compartments compared to the remainder test compounds. The
inhibitory potency of 5 could reflect the potential of lipophilic
compounds, including lipophilic esters of isoflavones,16 to pene-
trate cell membranes and thus be more capable of reaching targets
critical for cell proliferation and/or survival, for example, inside
mitochondria. These organelles are known to accumulate lipophilic
drugs.33 Delivery of BCA to mitochondria may inhibit cell prolifer-
ation through ERb, as already shown for resveratrol.34 Alterna-
tively, 1–5 may also enhance delivery of genistein, the major
metabolite of BCA, to mitochondria. The intracellular level and

2966
N. Fokialakis et al. / Bioorg. Med. Chem. 20 (2012) 2962–2970
220
200
180
160
140
120
100
80
A
1
2
3
4
500
*
5
BCA
0.1 nM E2
400
300
200
100
*
*
60
0.01
0.1
1
10
0
Cοncentration (μΜ)
vehicle
E2
E2+Fulv
PPT
MPP MPP+DPN
Figure 2. Stimulation of proliferation of estrogen-free MCF-7 cells by increasing
concentrations of Biochanin A (BCA) and its ester (1, 3) and carbamate ester (2, 4, 5)
derivatives. Stimulation in the presence of 0.1 nM estradiol (E2) is shown for
comparison. Absorbance of MTT–formazan (reflects the number of viable cells) is
expressed as % of that of cells treated only with vehicle (compound diluent). Data
are mean of three independent experiments carried out in triplicate with an inter-
assay variation (mean SEM) similar to that shown for BCA.
B
500
400
300
200
100
0
1
2
3
4
5
BCA
0.1 nM E2
E2 + 1 μM compound
E2 + 10 μM compound
250
200
150
100
50
0.1 nM E2
*
*
*
*
#
*
#
*
#
*
#
*
#
*
#
0.01
0.1
1
10
#
#
#
Concentration (μΜ)
Figure 4. Induction of alkaline phosphatase expression in Ishikawa cells: (A) by
0.1 nM estradiol (E2) in the absence or presence of 100 nM fulvestrant, 100 nM PPT
and 100 nM MPP in the absence or presence of 100 nM DPN. Data are mean SEM
of three independent experiments carried out in triplicate. ⁄ p 60.05 versus vehicle
(t-test). (B) Increasing concentrations of Biochanin A (BCA) and its ester (1, 3) and
carbamate ester (2, 4, 5) derivatives. Alkaline phosphatase activity is expressed as %
of that of cells treated only with vehicle (compound diluent). Alkaline phosphatase
activity in the presence of 0.1 nM estradiol (E2) is shown for comparison. Data are
mean of three independent experiments carried out in triplicate with an inter-assay
variation (mean SEM) similar to that shown for BCA.
0
1
2
3
4
5
BCA
Compound
Figure 3. Effects of Biochanin A (BCA) and its ester (1, 3) and carbamate ester (2, 4,
5) derivatives on the proliferation of MCF-7 cells in the presence of 0.1 nM estradiol
(E2). Absorbance of MTT–formazan (reflects the number of viable cells) is expressed
as % of that of cells treated only with vehicle (compound diluent). Absorbance in the
presence of 0.1 nM (E2 is shown for comparison. Data are mean SEM of three
independent experiments carried out in triplicate. p 60.05 versus vehicle, # p
60.05 versus 0.1 nM E2; t-test.
⁄
respond to the combination of MPP and DPN cannot exclude the
presence of active ERb in the mitochondria of these cells. We also
tested 1–5 and BCA against MDA-MB-231 cells. These cells are of
activity of free BCA and its major metabolites, genistein and BCA
and genistein conjugates is dependent on cell-type specific metab-
olism. Since free hydroxyl groups are subject to conjugation, which
in turn provides for rapid efflux of the conjugates, ester and in par-
ticular carbamate esters of BCA are likely to limit the level of free
BCA that is available for conjugation and efflux. In addition, efflux
of free BCA is likely to lack behind that of genistein. Thus, it is pos-
sible that using 1–5 rather than free BCA or genistein could provide
for a more effective distribution of the latter between different cel-
lular compartments and hence increase its impact on cell prolifer-
ation and viability. Notably, the mitochondria of MCF-7 cells are
known to harbor functional ERb,35,36 the mitochondrial ERb is said
to be a vulnerability factor in these cells,35 and genistein is a po-
tency-selective ERb agonist that may challenge cell viability
through ERb. That luciferase activity of MCF-7:D5L cells failed to
the triple negative phenotype i.e. negative for ER
receptor and HER2 but positive for ERb.37 We found that prolifera-
tion of MDA-MB-231 cells was only marginally inhibited by 10
5. Whether the higher sensitivity of MCF-7 cells to 5 as compared
a, progesterone
lM
to MDA-MB-231 cells is associated with expression of ER
former but not the latter cells is presently unknown.
a in the
BCA and its derivatives were also tested to determine their
estrogenic and cancer chemopreventive activities using Ishikawa
cells. In these cells, isoflavones induce AlkP expression through
ER in a manner that is indicative of their intrinsic estrogenic activ-
ity.38 We observed that the AlkP expression of Ishikawa cells was
induced by the equimolar combination of MPP and DPN as well
as by PPT, in accordance with reports that uterine epithelial cells
and Ishikawa cells in particular express ERb as well as ERa 39,40
The AlkP induction efficacies of test compounds may be taken to
.

N. Fokialakis et al. / Bioorg. Med. Chem. 20 (2012) 2962–2970
2967
180
160
140
120
100
80
Importantly, lipophilic carbamate ester 5 appeared to display high-
er stability compared to 1–4 and BCA in both MCF-7:5L and Ishik-
awa cells (EC50 = 0.32 and 1.70 lM, respectively). It should be said
here that in order to experimentally support the above stability
claims it would be necessary to separate free from esterified BCA
by several extraction and chromatographic steps using radiola-
belled BCA as internal standard and to analyze free BCA using
dedicated methods and tools, similar to those developed for
genistein.16
E2 + 1 μM compound
E2 + 10 μM compound
0.1 nM E2
#
#
#
Taken together, Figures 1–5 suggest that 5 affects MCF-7 and
Ishikawa cells in a manner providing for induction of gene expres-
sion to a level similar to estradiol and BCA and, at the same time,
for suppression of cell proliferation. In contrast, estradiol and
BCA stimulated the proliferation of MCF-7 cells and BCA that of
Ishikawa cells. By and large, our findings indicate that 5 could be
as effective as estradiol and BCA HRT wise but more effective than
the free isoflavone chemoprevention wise. Since systemic adminis-
tration of isoflavone esters is ineffective, while the opposite is
likely the case with local administration,16 5 could be used to rem-
edy post-menopausal symptoms such as vaginal dyspareunia, skin
aging and poor wound healing. Skin is an established estrogen tar-
60
40
20
0
1
2
3
4
5
BCA
Compound
Figure 5. Effects of Biochanin A (BCA) and its ester (1, 3) and carbamate ester (2, 4,
5) derivatives on the proliferation of Ishikawa cells in the presence of 0.1 nM
estradiol (E2). Absorbance of MTT–formazan (reflects the number of viable cells) is
expressed as % of that of cells treated only with vehicle (compound diluent).
Absorbance in the presence of 0.1 nM E2 is shown for comparison. Data are
mean SEM of three independent experiments carried out in triplicate. # p ꢄ0.05
versus 0.1 nM E2; t-test.
get organ that expresses both ERa
and ERb.43,44 Notably, it was re-
cently shown that exogenous estrogen replacement promotes
cutaneous wound healing via ERb in a preclinical model of
menopause.43
indicate levels of free BCA inside Ishikawa cells under the condi-
tions of the assay, as already assumed for luciferase induction effi-
cacies in MCF-7:D5L cells. The AlkP induction efficacies of the
carbamate esters (2, 4) exceeded those of the respective esters
(1, 3). Assessment of the number of viable cells using MTT
(Fig. 5) suggested that the lower AlkP induction efficacies of 1, 3
and BCA were not due to inhibition of cell proliferation. We spec-
ulate that due to more extensive hydrolysis in Ishikawa cells com-
pared to MCF-7 cells, 1 and 3 allowed BCA metabolism and efflux
to an extent comparable to that observed with free BCA. The AlkP
induction efficacy of 5 was also lower than 2 and 4 and comparable
to 1, 3 and to free BCA. However, since the induction potency of 5
was much lower than that of 1 and 3, its low induction efficacy is
probably due to limited BCA release rather than the opposite. Nota-
bly, only 5 was able to inhibit the proliferation of Ishikawa cells
(Fig. 5). Again, the stability and lipophilicity of 5 could allow BCA
to penetrate and remain inside Ishikawa cells to comparatively
higher level, promoting isoflavone distribution to key cellular tar-
gets in Ishikawa and MCF-7 cells alike.
Our interpretation of the induction efficacy data of Figures 1B
and 4B implies that carbamate esters (2, 4, 5) are better shielded
from hydrolysis compared to carboxylic acid esters (1, 3). The
induction efficacy data of Figure 2 were not taken into account gi-
ven the complexity of proliferative response which among other
signaling entities and cascades is known to involve membrane
ER.41 Carbamate esters have been used in the design of prodrugs
in order to protect hydroxyl groups from first-pass and systemic
metabolism leading to drug clearance.14,15,42 The therapeutic po-
tential of a drug is reportedly increased by reducing the rate of
hydrolysis of the carbamate ester prodrug. In addition, the stability
of carbamate ester prodrugs is known to depend on the substitu-
tion of the carbamic acid shield.15 Accordingly, hydrolysis of chlo-
rinated ester 1 appeared to proceed faster than that of the
brominated ester 3 in MCF-7:D5L cells, as indicated by the higher
luciferase induction potency of the former compared to the latter
(EC50 = 0.14 and 18 nM, respectively). This appears to be also the
4. Conclusions
We have synthesized two ester (1, 3) and three carbamate ester
derivatives (2, 4, 5) of BCA as well as the free isoflavone and eval-
uated their estrogenic and cancer chemopreventive activity using
cell model systems of human breast and endometrial adenocarci-
noma. We found that carbamate ester 5 displayed estradiol-like
stimulation of gene expression as well as inhibition of proliferation
of breast and endometrial cancer cells, implying that it may repre-
sent a safe alternative to HRT, especially if administered locally
rather than systemically. Further investigation is needed to sub-
stantiate this hypothesis.
5. Experimental part
5.1. General chemistry methods
All chemicals were of the highest available commercial purity.
Starting materials were purchased from Aldrich (analytical reagent
grades) and used without further purification. All anhydrous reac-
tions were carried out under argon atmosphere. Solvents were
dried by distillation prior to use. Analytical thin-layer chromatog-
raphy (TLC) was conducted on Merck silica gel 60 F254 and spots
visualized using UV light and vanillin–sulfuric acid reagent. Liquid
chromatography was performed on columns containing Si gel 60
Merck (40–63 lm). Solvent mixtures employed in chromatography
are reported as volume to volume ratios. 1H NMR (400 MHz) and
13C NMR (100 MHz) data were recorded on a Bruker DRX400 spec-
trometer. COSY, HMQC, and HMBC NMR data were obtained using
standard Bruker microprograms. Mass spectrometry HRMS (ESI+)
were run on a Thermo Scientific LTQ Orbitrap Discovery mass spec-
trometer with the infusion method.
5.2. Synthesis of 5,7–dihydroxy-3-(4-methoxyphenyl)-4H-
chromen-4-one (Biochanin A, BCA)
case in Ishikawa cells (EC50 = 0.47 and 0.52
rinated carbamate ester 2 appeared less stable than brominated 4
in MCF-7:D5L cells (EC50 = 0.14 and 0.22 M, respectively) but
not in Ishikawa cells (EC50 = 0.66 and 0.62 M, respectively).
lM, respectively). Chlo-
Synthesis of BCA was carried out as described by Wahala and
Hase.17 Briefly phloroglucinol (0.050 mol) and p-methoxy phenyla-
cetic acid (0.050 mol) were dissolved into freshly distilled BF3ꢀEt2O
l
l

2968
N. Fokialakis et al. / Bioorg. Med. Chem. 20 (2012) 2962–2970
under argon. The mixture was stirred and heated at 80 °C on an
oil-bath. The corresponding intermediate product 1-(2,4,6-trihydr-
oxyphenyl)-2-(4-methoxyphenyl)ethanone was isolated by wash-
ing of the collected material thoroughly with aq NaHCO3 and
ethyl acetate. The residue was purified by column chromatography
on silica gel using a mixture of dichloromethane and methanol. In a
solution of the intermediate product (450 mg, 2 mmol) in DMF
(6 mL), freshly distilled BF3ꢀEt2O (6.3 mL) was added under argon.
The mixture was heated to 50 °C, and a solution of methanesulfo-
nyl chloride (1 mL) in dry DMF (1.5 mL) was added slowly. After
reaction at 80 °C, for 1 h, the mixture was cooled to room temper-
ature and poured into a large volume of ice-cold aq sodium acetate
(12 g/100 mL). Then the mixture was extracted with ethyl acetate,
and the organic layer was dried and concentrated. The residue was
purified by column chromatography on silica gel using a mixture of
dichloromethane and methanol.
5.3.4. 5-Hydroxy-3-(4-methoxyphenyl)-4-oxo-4H-chromen-7-yl
2-bromooacetate (3)
Yield 58%. 1H NMR (600 MHz, CDCl3, d ppm): 13.0 (1H, s, OH),
7.99 (1H, s, H-2), 7.42 (2H, d, J = 8 Hz, H-20, H-60), 6.98 (2H, d,
J = 8 Hz, H-30, H-50), 6.74 (1H, d, J = 1.5 Hz, H-8), 6.57 (1H, d,
J = 1.5 Hz, H-6), 4.05 (2H, s, CH2), 3.81 (3H, s, OCH3). 13C NMR
(100 MHz, CDCl3, d ppm): 181.2 (C-4), 169.7 (CH2COO), 164.9 (C-
7), 163.4 (C-5), 158.5 (C-8a), 158.0 (C-40), 154.0 (C-2), 130.0 (C-20,
C-60), 123.7 (C-3) 123.1 (C-10), 115.4 (C-30, C-50), 108.9 (C-4a),
105.7 (C-6) 100.5 (C-8), 55.8 (OCH3), 25.5 (CH2). HRMS (ESI+) m/z
404.9967 [M+H]+, calculated for 404.9974, C18H14BrO6
5.3.5. 5-Hydroxy-3-(4-methoxyphenyl)-4-oxo-4H-chromen-7-yl
2-bromooethylcarbamate (4)
Yield 63%. 1H NMR (600 MHz, CDCl3, d ppm): 13.0 (1H, s, OH),
7.92 (1H, s, H-2), 7.42 (2H, d, J = 8.0 Hz, H-20/H-60), 6.97 (2H, d,
J = 8.0 Hz, H-30/H-50), 6.80 (1H, d, J = 1.5 Hz, H-8), 6.60 (1H, d,
J = 1.5 Hz, H-6), 5.52 (1H, t, J = 5.5 Hz, CH2NH), 3.83 (3H, s, OCH3),
3.69 (2H, q, J = 5.5 Hz, CH2NH), 3.52 (2H, t, BrCH2). 13C NMR
(100 MHz, CDCl3, d ppm): 181.0 (C-4), 165.0 (C-7), 163.2 (C-5),
160.0 (C-40), 157.1 (C-8a), 156.4 (OCONH), 153.5 (C-2), 130.0 (C-
20, C-60), 124.1 (C-3) 123.9 (C-10), 114.5 (C-30, C-50), 109.2 (C-4a),
105.0 (C-6) 100.5 (C-8), 55.6 (OCH3), 43.2 (CH2NH), 32.3 (CH2Br).
HRMS (ESI+) m/z 433.0170 [M+H]+, calculated for 433.0161,
5.3. Synthesis of BCA derivatives
For the preparation of the esters and the carbamide esters of
BCA, in a solution of BCA (100 mg, 0.35 mmol) in 20 mL ether
which was cooled to 0 °C, 0.8 mL of pyridine and 0.50 mL of
the corresponding acylchlorides or isocyanates were added.
After the completion of the reaction in room temperature, the
organic solvents were evaporated and the residue was chromato-
graphed on a silica gel column chromatography using as mobile
phase a gradient CH2Cl2/MeOH mixture, to afford the relative
compound.
C19H16BrNO6.
5.3.6. 5-Hydroxy-3-(4-methoxyphenyl)-4-oxo-4H-chromen-7-yl
undecylcarbamate (5)
Yield 65%. 1H NMR (600 MHz, CDCl3, d ppm): 12.8 (1H, s, OH),
7.98 (1H, s, H-2), 7.48 (2H, d, J = 8 Hz, H-20, H-60), 7.01 (2H, d,
J = 8 Hz, H-30, H-50), 6.86 (1H, d, J = 1.5 Hz, H-8), 6.62 (1H, d,
J = 1.5 Hz, H-6), 5.10 (1H, t, J = 5.5 Hz, CH2NH), 3.83 (3H, s, OCH3).
3.82 (3H, s, OCH3), 3.32 (2H, q, J = 1.5 Hz, CH2NH), 1.58–1.25
(18H, m, CH2), 0.91 (3H, t, J = 1.5 Hz, CH3). 13C NMR (100 MHz,
CDCl3, d ppm): 181.2 (C-4), 162.2 (C-7), 159.8 (C-5), 156.9 (C-8a),
156.5 (C-40), 153.2 (C-2), 153.0 (NHCOO), 130.1 (C-20, C-60), 123.9
(C-3) 122.6 (C-10), 114.1 (C-30, C-50), 109.1 (C-4a), 104.9 (C-6),
100.4 (C-8), 55.3 (OCH3), 41.3 (CH2NH), 31.9 (CH2CH2NH), 29.7–
29.2 (5 CH2), 22.7 (CH3CH2), 26.7 (CH3CH2CH2), 14.1 (CH3CH2).
HRMS (ESI+) m/z 482.2550 [M+H]+, calculated for 482.2543,
C28H36NO6.
5.3.1. Biochanin A (BCA)
Yield 72%. 1H NMR (600 MHz, CDCl3, d ppm): 13.0 (1H, s, OH),
8.22 (1H, s, H-2), 7.54 (2H, d, J = 8.0 Hz, H-20/H-60), 7.01 (2H, d,
J = 8,0 Hz, H-30/H-50), 6.42 (1H, d, J = 1.5 Hz, H-8), 6.30 (1H, d,
J = 1.5 Hz, H-6), 3.89 (3H, s, OCH3). 13C NMR (100 MHz, CDCl3, d
ppm): 181.6 (C-4), 165.0 (C-7), 163.9 (C-5), 161.7 (C-40), 159.0
(C-8a), 154.5 (C-2), 131.1 (C-20, C-60), 124.2 (C-3) 123.8 (C-10),
114.5 (C-30, C-50), 106.2 (C-4a), 100.0 (C-6) 94.5 (C-8), 55.5
(OCH3). HRMS (ESI+) m/z 285.0756 [M+H]+, calculated for
285.0763, C16H13O5.
5.3.2. 5-Hydroxy-3-(4-methoxyphenyl)-4-oxo-4H-chromen-7-yl
2-chloroacetate (1)
Yield 68%. 1H NMR (600 MHz, CDCl3, d ppm): 12.9 (1H, s, OH),
7.99 (1H, s, H-2), 7.48 (2H, d, J = 8.0 Hz, H-20/H-60), 7.01 (2H, d,
J = 8,0 Hz, H-30/H-50), 6.82 (1H, d, J = 1.5 Hz, H-8), 6.65 (1H, d,
J = 1.5 Hz, H-6), 4.38 (2H, s, CH2), 3.87 (3H, s, OCH3). 13C NMR
(100 MHz, CDCl3, d ppm): 180.9 (C-4), 164.5 (C-7), 162.9 (C-5),
160.1 (C-40), 156.9 (C-8a), 153.4 (C-2), 130.1 (C-20, C-60), 124.2
(C-3) 122.3 (C-10), 114.2 (C-30, C-50), 109.8 (C-4a), 105.2 (C-6),
100.5 (C-8), 55.3 (OCH3), 40.7 (ClCH2CO). HRMS (ESI+) m/z
361.0482 [M+H]+, calculated for 361.0479, C18H14ClO6.
5.4. Biological evaluation
5.4.1. Cells, cell culture and fine chemicals
If not stated otherwise, fine chemicals and culture media
were from Sigma–Aldrich (Steinheim, Germany). Fulvestrant
(ICI182,780), DPN (diaryl-propionitrile), MPP (methylpiperidino-
pyrazole) and PPT (propyl-pyrazol-triol) were from Tocris Biosci-
ence (Bristol, UK). Fetal bovine serum (FBS) was from Invitrogen
Corporation (Carlsbad, USA). Ishikawa human endometrial adeno-
carcinoma cells, MCF-7 and MDA-MD-231 human breast adenocar-
cinoma cells and MCF-7:D5L cells, a clone of MCF-7 cells stably
transfected with the estrogen-responsive reporter plasmid pERE-
Gl-Luciferase, were maintained in culture as already described.20,45
DCC-FBS, that is, FBS that was treated with 10% dextran-coated
charcoal (DCC) to remove endogenous steroids was prepared as al-
ready described.23
5.3.3. 5-Hydroxy-3-(4-methoxyphenyl)-4-oxo-4H-chromen-7-yl
2-chloroethylcarbamate (2)
Yield 81%. 1H NMR (600 MHz, CDCl3, d ppm): 12.8 (1H, s, OH),
7.92 (1H, s, H-2), 7.43 (2H, d, J = 8.0 Hz, H-20/H-60), 6.98 (2H, d,
J = 8.0 Hz, H-30/H-50), 6.81 (1H, d, J = 1.5 Hz, H-8), 6.61 (1H, d,
J = 1.5 Hz, H-6), 5.51 (1H, t, J = 5.5 Hz, CH2NH), 3.83 (3H, s, OCH3),
3.67 (2H, q, J = 5.5 Hz, CH2NH), 3.51 (2H, t, ClCH2). 13C NMR
(100 MHz, CDCl3, d ppm): 181.2 (C-4), 165.4 (C-7), 164.0 (C-5),
160.0 (C-40), 158.0 (C-8a), 156.5 (OCONH), 153.8 (C-2), 131.0 (C-
20, C-60), 124.0 (C-3) 123.5 (C-10), 114.0 (C-30, C-50), 109.2 (C-4a),
105.3 (C-6) 100.0 (C-8), 55.0 (OCH3), 43.5 (CH2NH), 42.0 (CH2Cl).
HRMS (ESI+) m/z 389.0648 [M+H]+, calculated for 389.0666,
5.4.2. Test compound effects on luciferase expression of MCF-
7:D5L cells
The effects of BCA and its derivatives (hereafter collectively re-
ferred to as test compounds) on the regulation of estrogen re-
sponse element (ERE)-dependent luciferase gene expression of
MCF-7:D5L cells were assessed as already described.20 Briefly,
C19H16ClNO6.

N. Fokialakis et al. / Bioorg. Med. Chem. 20 (2012) 2962–2970
2969
the cells were plated in 96-well microculture plates at a density of
12,000 cells per well in MEM devoid of phenol-red and supple-
mented with 1 g/mL insulin and 5% DCC-FBS. Increasing concen-
trations (up to 10 M) of test compounds or vehicle (DMSO to a
final concentration of 0.1%) were administered to the cells 72 h
after plating and luciferase expression was assessed 16 h later
using a Safire II microplate reader (Tecan). Cells treated with 0.1
and 1 nM 17b-estradiol (estradiol) or only with vehicle served as
controls.
Supplementary data
l
Supplementary data (NMR spectra of all compounds) associated
with this article can be found, in the online version, at doi:10.1016/
j.bmc.2012.03.012.
l
References and notes
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5% DCC-FBS. Increasing concentrations (up to 10
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5.4.5. Relative (to estradiol) binding affinity of test compounds
for ERa and ERb
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polarization assay kits (P2698 and P2700, Invitrogen) and a Beacon
2000 Fluorescence Polarization Reader (Invitrogen) as previously
described.21,22
5.4.6. Statistics
The statistical significance of differences in cell proliferation
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Acknowledgments
This work was supported in part by the Greek Framework Pro-
gramme Competitiveness—Research network 05DSBEPRO-32 from
the General Secretariat for Research and Technology. In addition,
another part of the work was supported by SYNERGASIA 2009 PRO-
GRAMME. This Programme is co-funded by the European Regional
Development Fund and National Resources (Project code: 09SYN-
13-1076). Dr. BR Steele is gratefully acknowledged for reviewing
the manuscript.
31. Leclercq, G.; Lacroix, M.; Laïos, I.; Laurent, G. Curr. Cancer Drug Targets 2006, 6,
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