Synthesis and binding affinity to human α and β estrogen receptors of various 7-hydroxycoumarins substituted at 4- and 3,4- positions

Article abstract of DOI:10.1016/j.farmac.2004.08.004

The study of the relative binding affinity (RBA) to the human α and β estrogen receptors (ERs) of various 7-hydroxycoumarins substituted at 4- and 3,4- positions is weak and lacks in selectivity for both ERα and ERβ. The 4-(4-hydroxyphenyl)-7-hydroxycoumarin shows a weak RBA to ERβ and 3,4-diphenyl-7-hydroxycoumarin presents a stronger RBA to ERα than ERβ.

Full text of DOI:10.1016/j.farmac.2004.08.004

IL FARMACO 59 (2004) 981–986
http://france.elsevier.com/direct/FARMAC/
Short communication
Synthesis and binding affinity to human a and b estrogen receptors
of various 7-hydroxycoumarins substituted at 4- and 3,4- positions
Serge Kirkiacharian a,*, Anh Tuan Lormier a, Henri Chidiack a,
Françoise Bouchoux b, Evelyne Cérède b
a Faculté de Pharmacie de Paris-Sud, Laboratoire de Chimie Thérapeutique, 5, rue Jean-Baptiste-Clément; 92296 Chatenay Malabry cedex, France
b Laboratoires Aventis, 111, route de Noisy, F 93230 Romainville cedex, France
Received 6 June 2004; accepted 6 August 2004
Available online 21 September 2004
Abstract
The study of the relative binding affinity (RBA) to the human a and b estrogen receptors (ERs) of various 7-hydroxycoumarins substituted
at 4- and 3,4- positions is weak and lacks in selectivity for both ERa and ERb. The 4-(4-hydroxyphenyl)-7-hydroxycoumarin shows a weak
RBA to ERb and 3,4-diphenyl-7-hydroxycoumarin presents a stronger RBA to ERa than ERb.
© 2004 Elsevier SAS. All rights reserved.
Keywords: Substituted-7-hydroxycoumarins; Binding affinity; a and b estrogen receptors
1. Introduction
Among the pure antiestrogens, only Fulvestrant 3 is now
used in the treatment of advanced breast cancer [8,9] al-
though the pure antiestrogen RU 58,668 4 might present the
same therapeutic potential [10].
The bone formation and resorption is influenced by cyto-
kines, growth factors and hormones. Most of the health
benefits of estrogens in the treatment of postmenopausal
osteoporosis are associated with their ability to regulate bone
turnover and to decrease bone resorption. Estrogens regulate
bone remodeling by modulating the synthesis of cytokines
and growth factors, decreasing interleukine 6 (Il-6) mRNA
expression [11] and inhibiting Il-6 induction by tumor necro-
sis factor [12].
Since many years, important research studies are devoted
to natural phyto-estrogens and to their synthetic analogues.
These derivatives can bind to the ERs and lead to agonist or
antagonist activities through the activation or inactivation of
certain genes [13–15]. In the case of Ipriflavone 6a and
Genisteine 6b (Fig. 1) [16], their bone resorption inhibitory
potency is already used in some countries and involves the
indirect increasing of calcitonin secretion by enhancing the
effect of estrogen [17].
The selective estrogen receptor modulators (SERMs) re-
present a wide class of compounds ranging from the mixed
agonist/antagonist derivatives exemplified by Tamoxifen 1
and Raloxifene 2, till the pure antagonists represented by ICI
182,780 (Fulvestrant) 3 and RU 58,668 4 [1] (Fig. 1).
Tamoxifen 1, is the first SERM belonging to the tria-
rylethylene group, that has been widely used for the adjuvant
treatment of hormone-dependant breast cancer, due to its
antiestrogen action on mammary cells [2]. Furthermore, like
estradiol, Tamoxifen presents additional health benefits such
as the prevention of postmenopausal decrease of bone mine-
ral density leading to osteoporosis, fractures, vertebral crush
[3] and to coronary heart disease [4]. However, its prolifera-
tive properties on the endometrium [5] and the increase in the
incidence of endometrial carcinoma [6] limits its long-term
clinical use. Raloxifene 2 is another SERM presenting mixed
agonist/antagonist activities. It is now currently recommen-
ded for hormone replacement therapy to increase bone mine-
ral density and its serum cholesterol lowering properties
would be interesting for the prevention of postmenopausal
risk factors [7].
The availability of new SERMs presenting agonist and/or
antagonist activities would thus be of great therapeutic inte-
rest. Furthermore, the identification and cloning of the se-
* Corresponding author.
E-mail address: serge.kirkiacharian@cep.u-psud.fr (S. Kirkiacharian).
0014-827X/$ - see front matter © 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2004.08.004

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S. Kirkiacharian et al. / IL FARMACO 59 (2004) 981–986
Fig. 1. Reference structures.
cond estrogen receptor (ERb) in addition to the known (ERa)
[18] opens up the perspective to prepare new selective li-
gands to either ERa and/or ERb, with new potential biologic
and pharmacologic applications, in relationship with the tar-
geted tissue [19]. The research in this area has already led to
ligands exhibiting a greater selectivity to ERa [20,21] and/or
to ERb [22] as well as for synthetic derivatives than for
natural phytoestrogens [23].
the RBAs to the ERs of various substituted coumarins was
decided. Compounds 9a–e were selected in order to study the
influence of a 4-ethyl or a 4-hydroxy group and derivatives
11a–c were chosen to study the effect of the presence of a
4-aryl and that of a 3,4-diphenyl group substituted on
7-hydroxycoumarin.
Previous research dedicated to the 3-substituted coumarin
7 [24] and to coumestrol 8 [25] (Fig. 1) showed an estrogenic
activity related to their structural analogy with the natural
hormone estradiol 5. Furthermore, a study of the rela-
tionships between the structure of various substituted couma-
rins and their RBAs to the ERs, showed the importance of the
substituents at positions 3 and 7 [26,27] and in the case of
3-(4-hydroxyphenyl)-7-hydroxycoumarin, the presence of a
five times more potent RBA for the ERb than for the ERa
[28].
Taking into consideration these results, we decided to
investigate the relationships between the structure and the
RBAs to the human ERa and ERb of some 4- and 3,4-
disubstituted coumarins. Our objective was to look out for
compounds capable to present selective RBAs to either ERa
or Erb which could lead to the design of potential tissue-
selective SERMs. Therefore, the synthesis and the study of
2. Synthesis
The preparation of the 3-aryl-4-ethyl-7-hydroxycou-
marins 9a–d with various substitution patterns was perfor-
med using the modified Perkin–Oglialoro reaction [29] by
condensation of 2,4-dihydroxypropiophenone with conve-
niently substituted phenylacetic acids in presence of triethy-
lamine and acetic anhydride. The reaction leads to a mixture
of 7-acetoxy derivatives. After acid hydrolysis, the corres-
ponding 7-hydroxycoumarins 9a–d were obtained accompa-
nied always by 2,3-dimethylchromone 10 (Scheme 1).
These results can be explained by the participation of two
different reactions:
• the first one, is the Perkin–Oglialoro reaction leading to
the formation of the expected coumarins 9a–d [30,31],
• the second one, is the result of the acetylation of 2,4-
dihydroxypropiophenone and that of its enol group lea-

S. Kirkiacharian et al. / IL FARMACO 59 (2004) 981–986
983
6 gives a doublet of doublet due to the ortho and meta
couplings, respectively, with protons 5 and 8.
The infrared spectra (IR) were recorded on a Bruker “Vec-
tor 22” (m in cm–1). They present characteristic bands of the
hydroxyl and the conjugated carbonyl groups.
The 3-(4-hydroxyphenyl)-4,7-dihydroxycoumarin 9e
[34], 4-aryl-7-hydroxycoumarins 11a–b [35,36] and 3,4-
diphenyl-7-hydroxycoumarin 11c [37,38] are obtained ac-
cording to the previously described procedures.
3.1.1. Preparation of 3-aryl-4-ethyl-7-hydroxycoumarins
9a–d and 2,3-dimethyl-chromone 10
Scheme 1
.
3.1.1.1. 3-(2-Hydroxyphenyl)-4-ethyl-7-hydroxycoumarin
9d (C17 H14 O4). Typical procedure
In a round bottom dry flask, equipped with a magnetic
stirring bar and a reflux condenser topped by a calcium
chloride drying tube, a solution of 2,4-dihydroxypro-
piophenone (1.66 g, 10 mmol), 2-hydroxyphenylacetic acid
(3.04 g, 20 mmol), triethylamine (5.62 ml, 40 mmol) and
acetic anhydride (4.71 ml, 50 mmol) are refluxed at 160 °C
during 23 h. After cooling, the reaction mixture is poured in
cold water (100 ml) and the product recovered by filtration,
washed with water and dried. The hydrolysis is performed
during 5 h (monitored by TLC) with a mixture of 10%
sulfuric acid (20 ml) in a solution of ethanol (80 ml) and
water (100 ml). After cooling, the mixture is poured in cold
water (100 ml) and the solid is filtered off, washed till
neutrality with water and dried. Recrystallization in an ether–
chloroform mixture (7/3) gives 2,3-dimethylchromone 10.
Yield: 114 mg (6.0%). mp 270 °C, Litt. 270 °C [39];
271–272 °C [33].
Scheme 2
.
ding to a mixture of 1-(2,4-diacetoxyphenyl)propane-1-
one and 2-(a-acetoxy-propenyl)benzene-1,3-diol diace-
tate. These latter form by a Baker–Venkataraman rearran-
gement the intermediate 1,3-diketone which gives by
cyclization the 2,3-dimethylchromone 10 [32,33]
(Scheme 2). The mechanism of this reaction was confir-
med by the acetylation of 2,4-dihydroxypropiophenone
without the presence of the arylacetic acid. Only the mix-
ture of 1-(2,4-diacetoxyphenyl)propane-1-one (83%) and
2-(a-acetoxy-propenyl)benzene-1,3-diol diacetate (17%)
was obtained. On treatment of this mixture of acetates
with triethylamine followed by acidification led to the
expected pure 2,3-dimethylchromone 10 in 94% yield
(Scheme 2).
IR: 3185, 1631, 1568, 1405, 1245, 859, 690.
1H-NMR (DMSO-d6): 1.9 (s, 3H, CH3); 2.35 (s, 3H,
CH3); 6.75 (d, 1H, J = 2.2 Hz, arom.); 6.85 (d, 1H, J = 8.8 Hz,
arom.); 7.85 (d, 1H, J = 8.8 Hz, arom.); 10.6 (s, 1H, OH).
13C-NMR (DMSO-d6): 176.17, 162.41, 161.61, 157.39,
127.08, 115.35, 114.84, 102.05, 18.44, 9.99.
The structure of the prepared coumarins 9a–d and that of
the chromone 10 was established by elemental analysis, IR,
1H-NMR and 13C-NMR spectroscopy.
The recovered solution upon concentration gives a crys-
talline solid of the expected coumarin 9d. Yield 535 mg
(19%) mp 262–264 °C (ethanol–water: 8/2).
3. Experimental
IR: 3307, 1674, 1614, 1559, 1141, 1000, 757.
1H-NMR (DMSO-d6): 1.0 (t, 3H, CH3), 2.5 (q, 2H, CH2),
6.7–6.9 (m, 4H, arom.), 7.0 (d, 1H, arom.), 7.1 (t, 1H, arom.),
7.7 (d, 1H, arom.).
3.1. Chemistry
13C-NMR (DMSO-d6): 160.45, 155.45, 154.94, 154.42,
131.44, 131.12, 129.43, 127.04, 122.33, 119.67, 119.16,
115.99, 113.41, 111.25, 102.59, 22.86, 13.80.
The purity of all the compounds was routinely checked on
the “Riedel-de Haën 60 F254 special” silica gel plates
(0.2 mm) and spots were located by UV lamp and/or by
iodine vapors. Melting points were taken on a Kofler bench
and are not corrected. Analyses (C, H) are within 0.4% of
3.1.1.2. 3-(2-Methoxyphenyl)-4-ethyl-7-hydroxycoumarin
9b (C18 H16 O4). Performed according to the typical proce-
dure.
Yields: 123 mg (6.5%) of 2,3-dimethylchromone 10 and
590 mg (20%) of coumarin 9b mp: 199 °C (ethanol–water:
8/2).
1
the theoretical values. The H-NMR spectra were recorded
on a Brucker AC 300 in CDCl3 or DMSO-d6 using tetra-
methylsilane (TMS) as internal reference. Chemical shifts d
are in ppm and J in Hz. Splitting patterns are described as
follows: (s) singlet, (d) doublet, (dd) doublet of doublet, (t)
triplet, (q) quadruplet; (m) multiplet. The proton at position
IR: 3412, 1683, 1566, 1470, 1245, 855, 755.

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S. Kirkiacharian et al. / IL FARMACO 59 (2004) 981–986
1H-NMR (DMSO-d6): 1.2 (t, J = 7.4 Hz, 3H, CH3), 2.6 (q,
1H-NMR (CDCl3) of 1-(2,4-diacetoxyphenyl)propane-1-
one: 1.10 (t, 3H, CH3), 2.28 (s, 3H, CH3), 2.30 (s, 3H, CH3),
2.9 (q, 2H, CH2), 6.9 (d, 1H, arom.), 7.10 (dd, 1H, arom.), 7.8
(d, 1H, arom.).
2H, CH2), 3.7 (s, 3H, OCH3), 6.74 (d, J = 2.2 Hz, 1H arom.),
6.83 (dd, J = 8.8 Hz and 2.4 Hz, 1H arom.), 6.97–7.19 (m, 3H
arom.), 7.39 (td, J = 8.8 Hz and 1.8 Hz, 1H arom.), 7.65 (d,
J = 8.8 Hz, 1H arom.)-7.6 (m, 7H, arom.), 10.47 (s, 1H, OH).
13C-NMR (DMSO-d6): 161.02, 160.23, 157.33, 154.82,
154.32, 131.30, 129.86, 127.15, 123.98, 120.66, 119.46,
113.42, 111.64, 111.19, 102.61, 55.69, 22.84, 13.74.
1H-NMR (CDCl3) of 2-(a-acetoxy-propenyl)benzene-
1,3-diol diacetate: 1.7 (d, 3H, CH3), 2.0 (s, 3H, CH3), 2.10 (s,
3H, CH3),), 2.20 (s, 3H, CH3), 5.70 (m, 1H, ethylenic), 6.90
(d, 1H, arom.), 7.00 (dd, 1H, arom.), 7.4 (d, 1H arom.).
To the previously obtained mixture of acetates (910 mg),
triethylamine (1.01 ml, 7.2 mmol) is added and the mixture
refluxed overnight under anhydrous conditions. After coo-
ling, the reaction mixture is poured on a cold N hydrochloric
acid solution (200 ml) and the mixture stirred during 4 h. The
formed precipitate is recovered by filtration and washed with
an ethanol–water mixture (9–1) giving 640 mg (94%) of pure
7-hydroxy-2,3-dimethylchromone 10, (physico-chemical
data reported previously).
3.1.1.3. 3-(4-Dimethylaminophenyl)-4-ethyl-7-hydroxycou-
marin 9a (C19 H19 N O3). A mixture of 2,4-dihydroxypro-
piophenone (1.66 g, 10 mmol), 4-dimethylamino-phenyl-
acetic acid (3.58 g, 20 mmol), acetic anhydride (4.2 ml,
50 mmol) and triethylamine (4.72 ml, 40 mmol) is refluxed
during 23 h under anhydrous conditions according to the
typical procedure. After cooling, the mixture is poured on
cold water (100 ml) and the precipitate recovered by filtra-
tion. The black residue is hydrolysed overnight with a mix-
ture of 10% sulfuric acid (50 ml) in a solution of ethanol
(140 ml) and water (80 ml). The reaction mixture is poured in
cold water (100 ml) and stirred during 4 h. The precipitate of
2,3-dimethylchromone 10 is collected and washed with wa-
ter (yield 1.04 g, 54%). The solution after neutralization with
sodium bicarbonate (4% solution) gives a brown precipitate
of 3-(4-dimethylaminophenyl)-4-ethyl-7-hydroxycoumarin
9a, which is recovered by filtration and washed with water.
Yield 1.40 g (46%); mp: 233 °C (ethanol–water: 8/2).
IR: 3367, 1674, 1611, 1559, 1518, 1347, 1136, 1097, 819,
803, 788.
3.2. Biological assay
The determination of the RBAs to the human ERa and
ERb was performed according to previously reported proce-
dures [15,42,43] using Cos cells transiently transfected with
expression plasmids for human estrogen receptors
(pSG5ERa and pSG5ERb, obtained from Prof. P. Chambon,
IGBMC, Strasbourg, France). RBAs were determined by
incubating Cos cell cytosol for 24 h at 0 °C with either
[3H]-estradiol (NEN Life Science products) with or without
different concentrations of competitor steroids. Bound and
free ligands were separated by the dextran-coated charcoal
method [44]. The estradiol was taken as reference compound
with RBAs of 100% for both ERa and ERb.
1H-NMR (CDCl3): 1.1 (t, 3H, CH3); 2.8 (q, 2H, CH2); 3.0
(s, 6H, N(CH3) 2; 6.7 -7.8 (m, 7H, arom.)
13C-NMR (DMSO-d6): 161.21, 160.77, 154.88, 153.34,
150.01, 130.72 (2C), 127.18, 122.65, 122.52, 113.35, 112.17
(2C), 111.43, 106.56, 102.51, 39.89, 22.62, 14.51.
4. Results and discussion
3.1.1.4. 3-(4-Hydroxyphenyl)-4-ethyl-7-hydroxycoumarin
9c (C17 H14 O4). Performed according to the typical proce-
dure.
Yields: 2,3-dimethylchromone 10: 110 mg (5.8%), and
coumarin 9c 507 mg (18%); mp: 315–317 °C (ethanol–water
8/2); 316–320°C [40,41].
IR: 3356, 1687, 1609, 1554, 1511, 1220, 1137, 829, 789.
1H-NMR (DMSO-d6): 1.2 (t, 3H, CH3), 2.7 (q, 2H, CH2),
6.8–7.1 (m, 4H, arom.), 7.2 (d, 2H, arom.), 7.8 (d, 1H,
arom.), 9.7 (s br, 1H, OH), 10.7 (s br, 1H, OH).
The obtained results are reported in Table 1 for the
3-phenyl-4-ethyl-7-hydroxycoumarins 9a–d and for 3-(4-
hydroxyphenyl)-4,7-dihydroxycoumarin 9e and Table 2 for
the derivatives 11a–c.
The examination of the data reported in Table 1 indicates
that the RBAs of these derivatives to both ERa and ERb are
weak and lacking in selectivity. However, the 3-(4-hydro-
xyphenyl)-4-ethyl-7-hydroxycoumarin 9c shows stronger
RBAs to both ERa and ERb than the coumarins 9b, 9d and
9a substituted, respectively, by 3-(2-methoxyyphenyl), 3-(2-
hydroxyphenyl) and by 3-(4-dimethylaminophenyl) groups.
The same result is also observed with the coumarin 9e pre-
senting a 4-hydroxy substituent instead of the 4-ethyl group.
The RBAs obtained with the coumarins 11a–c presenting
a 4-phenyl or 3,4-diphenyl group are reported in Table 2.
The data show that 4-phenyl-7-hydroxycoumarin 11a is
devoid of RBA to any ER. However, the substitution of the
4-phenyl ring by a 4-hydroxy group as in the case of 4-(4-
hydroxyphenyl)-7-hydroxycoumarin 11b leads to the appea-
rance of a weak RBA to the ERb. This result is interesting as
3.1.2. Preparation of 2,3-dimethyl-7-hydroxychromone 10
(C11 H10 O3)
A mixture of 2,4-dihydroxypropiophenone (1.66 g,
10 mmol) acetic anhydride (38 ml, 400 mmol) and sodium
acetate (4.92 g, 60 mmol) is refluxed during 3 h. After
cooling, the solution is filtered and the solvent evaporated. A
visquous oil is obtained (1.10 g) constituted by a mixture of
two acetates: 1-(2,4-diacetoxyphenyl)propane-1-one (83%)
and 2-(a-acetoxy-propenyl)benzene-1,3-diol diacetate
(17%).

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985
Table 1
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4-ethyl-7-hydroxycoumarins
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R1
R2
Human estrogen receptors
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Compounds
R
a receptors b receptors
9a
9b
9c
9d
9e
C2H5 N(CH3)2
C2H5
C2H5 OH
H
0.03
0.2
<0.02
0.08
0.65
0.04
0.1
H
OCH3
H
0.7
C2H5
H
OH
0.02
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OH
OH
H
0.02
a Estradiol, 100 for both a and b estrogen receptors.
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Table 2
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Compound
Ar1
Ar2
Human estrogen receptors
RBA 24 h at 0 °C a
a receptors
b receptors
11a
11b
11c
C6H5
H
00
00
00
0.2
5.0
(p)HO-C6H4
H
C6H5
C6H5
56.0
a Estradiol, 100 for both a and b estrogen receptors.
ERb signalling might induce opposite effects to those of ERa
[45] and play significant roles in the central nervous, cardio-
vascular and immune systems as well as on urogenital tract,
bone, kidney and lung [46]. The substitution by a second
phenyl group at position 4 as in the case of 3,4-diphenyl-7-
hydroxycoumarin 11c leads to a significant improvement of
the RBAs to both ERs but with 10 times more selectivity to
ERa than ERb.
5. Conclusion
This study indicates that in comparison to estradiol, the
3-phenyl-4-ethyl-7-hydroxycoumarins and 3-(4-hydroxy-
phenyl)-4,7-dihydroxycoumarin present weak RBAs to both
a and b ERs lacking in selectivity. The substitution by a
second phenyl group at position 4 as for 3,4-diphenyl-7-
hydroxycoumarin increases the RBAs to both estrogen re-
ceptors but with more selectivity to ERa than ERb. More
work is now in progress in the study of the RBAs of couma-
rins presenting various substitution patterns.
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