Potent estrogen receptor ligands based on bisphenols with a globular hydrophobic core

Article abstract of DOI:10.1021/jm050195r

Candidate estrogen receptor (ER) ligands with two phenolic residues on a three-dimensional hydrophobic core structure (carborane, bicyclo[2.2.2]octene, or adamantane) were synthesized and biologically evaluated. The biological properties of the ligands we

Full text of DOI:10.1021/jm050195r

J. Med. Chem. 2005, 48, 3941-3944
3941
Chart 1. Structures of ER Ligands. in Icosahedral Cage
Structures throughout This Paper, Closed Circles
Represent Carbon Atoms and Other Vertices Represent
BH Units
Potent Estrogen Receptor Ligands Based
on Bisphenols with a Globular
Hydrophobic Core
Yasuyuki Endo,*,† Tomohiro Yoshimi,†
Kiminori Ohta,† Tomoharu Suzuki,‡ and
Shigeru Ohta‡
Faculty of Pharmaceutical Sciences, Tohoku Pharmaceutical
University, 4-4-1, Komatsushima, Aoba-ku,
Sendai 981-8558, Japan, and Graduate School of Medical
Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku,
Hiroshima 734-8551, Japan
Received March 3, 2005
Abstract: Candidate estrogen receptor (ER) ligands with two
phenolic residues on a three-dimensional hydrophobic core
structure (carborane, bicyclo[2.2.2]octene, or adamantane)
were synthesized and biologically evaluated. The biological
properties of the ligands were markedly dependent on the
nature of the hydrophobic core structure. Bis(4-hydroxy-
phenyl)-o-carborane (6) was a partial agonist/antagonist for
ER. 1,2-Bis(4-hydroxyphenyl)bicyclo[2.2.2]octene (10) exhibited
potent agonist activity for ER, even though the two phenolic
groups are located similarly to those of 6.
(carborane)5 as a hydrophobic component of biologically
active molecules. The carboranes are icosahedral carbon-
containing boron clusters with characteristic properties,
such as spherical geometry and hydrophobicity. We have
demonstrated that the hydrophobicity of the carboranes
is comparable with that of hydrocarbons,6 and their
spherical hydrophobic surface effectively interacts with
the hydrophobic surface of the ligand-binding domain
of nuclear receptors.7-10 Recently, we have reported a
potent estrogen agonist bearing a carborane, 1-hydroxy-
methyl-12-(4-hydroxyphenyl)-1,12-dicarba-closo-dodeca-
borane, BE120 (2),9 which has an activity greater than
that of E2 in transcriptional assay using an ERR and
in ERR binding assay.10 The compound also showed
potent in vivo effects on the recovery of uterine weight
and bone loss in OVX mice.10 These results suggested
that the hydrophobic interaction along the spherical
carborane cage produces a stronger interaction than
that in the case of E2. We have utilized the strong
binding of the spherical carborane cage to the cavity of
ERRLBD to develop a potent ER antagonist, BE361
(3),11 which has two hydroxyphenyl groups and a basic
side chain, like that of tamoxifen (4) and 4-hydroxy-
tamoxifen (5). During our studies of ER antagonists, we
also found that the simple bis(hydroxyphenyl)-o-car-
borane, BE360 (6) exhibited antiestrogenic activity,
although the antagonist activity was somewhat weaker
than that of 3 bearing the basic side chain. Further-
more, in an in vivo evaluation, compound 6 exhibited
estrogenic action in bone, preventing bone loss without
inducing estrogenic action in the uterus,12 suggesting
its possible application as a new type of SERM to treat
osteoporosis.
The estrogen receptor (ER) is a member of the
superfamily of ligand-dependent transcriptional factors.
Endogenous estrogen, 17â-estradiol (1, E2), plays an
important role in the female and male reproductive
systems and also in bone maintenance, in the central
nervous system, and in the cardiovascular system. The
first step in the appearance of estrogenic activity is the
binding of agonist ligands to ERR1 and â2, resulting in
a conformational change. The resulting ligand-bound
ER then dimerizes, forms complexes with various
cofactors, and binds to specific promoter elements of
DNA to initiate gene transcription. Antagonist ligands
form ER-ligand complexes with different conforma-
tions, thereby inhibiting the interactions with cell- and
promoter-specific factors. Differences of distribution and
function of these factors among tissues seems to be
connected with the tissue selectivity of certain ER
ligands, which are called selective estrogen receptor
modulators (SERM).3 Therefore, even minor differences
in the conformation of ER-ligand complexes depending
on the structure of the ligand may be important in
determining whether the ligand exhibits agonist or
antagonist effects in a certain tissue.4
Binding of ligands to the ER ligand binding domain
(LBD) primarily requires a phenolic ring, with an
appropriate hydrophobic group adjacent to the phenolic
ring. The hydrophobic group should closely match the
hydrophobic surface of the ER, serving to increase the
binding affinity. The hydrophobic structure also plays
a role as a scaffold, fixing the spatial positions of
hydrogen-bonding functional groups. In our studies to
develop new hydrophobic core structures for drug
design, we have focused on dicarba-closo-dodecaborane
Therefore, control of the geometry of the two hydroxy-
phenyl groups and modification of the three-dimensional
hydrophobic core structure should provide a basis for
developing various ER ligands with agonist or antago-
nist activities. These considerations led us to design,
synthesize, and biologically evaluate compounds having
various three-dimensional hydrophobic cores bearing
hydroxyphenyl groups (7-12), as shown in Figure 1. We
presumed that the first phenolic hydroxyl group acts
as an anchor at the hydrogen-bonding site of ER, while
the three-dimensional hydrophobic core fills the hydro-
phobic cavity of ER. The position and angle of the second
* To whom correspondence should be addressed. Phone, +81-22-
234-4181; fax, +81-22-275-2013; e-mail, yendo@tohoku-pharm.ac.jp.
† Tohoku Pharmaceutical University.
‡ Hiroshima University.
10.1021/jm050195r CCC: $30.25 © 2005 American Chemical Society
Published on Web 05/12/2005

3942 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12
Letters
Scheme 2a
Figure 1. Designed bisphenolic ER ligands (6-12) with
carborane and globular hydrocarbon cores.
Scheme 1a
a Reagents: (a) DIBAL/THF; (b) PBr3, pyridine/Et2O; (c)
[CH(CO2Et)2]2, NaH/THF; (d) LiAlH4/THF; (e) MsCl/pyridine;(f)
LiBr/2-ethoxyethanol; (g) NaBH4/HMPA; (h) BBr3/CH2Cl2; (i)
4-methoxyphenylboronic acid, Pd(PPh3)4, Na2CO3/toluene-EtOH-
H2O.
Scheme 3a
a Reagents: (a) (1) n-BuLi/benzene-Et2O, (2) 4-TBSO-benzyl
bromide or 4-TBSO-phenylethyl bromide; (b) HCl/MeOH.
hydroxyphenyl group are presumed to determine the
nature of the estrogenic action. Therefore, we designed
BE380 (7) and BE381 (8), which have one or two
methylene groups inserted between the second hydroxy-
phenyl group and the carborane cage of 6. We also
designed BE1054 (9) and BE1060 (10) bearing a three-
dimensional hydrocarbon unit, 4,4,5,5-tetramethyl-
cyclohexene or bicyclo[2.2.2]octene, in place of the
carborane cage of 6, while retaining similar geometry
of the two hydroxyphenyl groups.13 BE1080 (11) and
BE1081 (12) with an adamantane unit are analogues
of 7 and 8, respectively.
The designed molecules 7 and 8 were synthesized
from 1-(4-MEMO-phenyl)-o-carborane (14) as shown in
Scheme 1. Compound 14, which was prepared by trans-
formation of 1-ethynyl-4-MEMO-benzene with deca-
borane (14)5 in 37% yield, was treated with n-BuLi and
then reacted with an electrophile,14 4-TBSO-benzyl
bromide or 4-TBSO-phenethyl bromide, to afford 15 or
16, respectively, in 23-36% yield. The O-protected
groups of 15 and 16 were deprotected under an acidic
condition to give 7 and 8, respectively.
The synthesis of the designed compounds 9 and 10 is
summarized in Scheme 2. Reduction of 3,4-bis(4-meth-
oxyphenyl)-5H-furan-2-one (17)15 with DIBAL in THF
afforded the diol 18 in 67% yield. Replacement of the
hydroxyl groups of the diol 18 with bromines, followed
by reaction with tetraethyl ethane-1,1,2,2-tetracarbox-
ylate in the presence of NaH in THF, gave the cyclo-
hexene tetracarboxylate 19 in 39% yield.
The tetracarboxylate 19 was converted into the tetra-
bromide 2016 in 30% yield by reduction with LiAlH4,
methanesulfonylation employing mesyl chloride, and
then substitution of the mesyl groups with bromo
groups. Reduction of the bromide19 with NaBH4 in
HMPA followed by demethylation afforded 9 in 69%
a Reagents: (a) 4-methoxyphenylethyltriphenylphosphonium
bromide, n-BuLi/Et2O; (b) BBr3/CH2Cl2; (c) methyltriphenylphos-
phonium bromide, NaHMDS/THF; (d) 4-iodoanisole, Pd(OAc)2,Ph3P,
Ag2CO3/DMF; (e) C2H5SNa/DMF.
yield. Compound 10 was prepared by Miyaura-Suzuki
coupling of 1,2-dibromobicyclo[2.2.2]octene (21)17 with
4-methoxyphenylboronic acid in the presence of
Pd(PPh3)4, followed by demethylation using BBr3 (47%,
two steps). In the demethylation, formation of 6,7-bis-
(4-hydroxyphenyl)bicyclo[3.2.1]oct-6-ene (23) as a re-
arranged byproduct was observed. The synthesis of the
designed compounds 11 and 12 was started from
1-(4-methoylphenyl)adamantan-2-one (24)18 and is sum-
marized in Scheme 3. The Wittig reaction of the ada-
mantanone 24 with 4-methoxyphenethyltriphenyl-
phosphorus ylide afforded an (E) and (Z) mixture of 1-(4-
methoxyphenyl)-2-[2-(4-methoxyphenyl)ethylidene]ada-
mantane (25) (21%). After isolation of the (Z)-isomer of
25 by recrystallization, treatment with BBr3 resulted
in isomerization of the double bond to give the (E)-
isomer of 12 in a quantitative yield. In the synthesis of
12, the ketone of 24 was transformed into a methylene
group by Wittig reaction with methyltriphenylphos-
phorus ylide, followed by Heck reaction with 4-iodo-

Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12 3943
Table 1. Relative Binding Affinity (RBA) of Test Compounds
versus Specific [3H]Estradiol (4 nM) Binding with Human
Recombinant ERR.
compound
RBAa
compound
RBAa
6
48 ( 5
45 ( 8
9
10
11
12
20 ( 6
7
47 ( 5
8
15 ( 3
9.3 ( 0.8
1.8 ( 0.3
tamoxifen
2.1 ( 0.2
a The relative binding affinity of estradiol is taken as 100.
Values represent the average range or SD of two or three
independent experiments.
Figure 3. Inhibition of transcriptional activation of estradiol
by the test compounds. MCF-7 cells were transfected with ERE
(SV-40)-LUC and phRL/CMV and incubated with estradiol
(10-10 M) and test compounds (10-8-10-5 M). Results are
shown as means ( SD for triplicate transfection. The values
given are expressed as a percent of the response with 10-10
estradiol.
M
binding of ligands is primarily the result of interaction
of the receptor with a phenolic residue and an appropri-
ate hydrophobic group adjacent to the phenolic ring. The
compounds based on carborane (6, 7, and 8) showed
potent binding affinity. This is reasonable, because
4-hydroxyphenyl-o-carborane (13) itself binds strongly
to ERR with a Ki of 1.1 nM.20 The high affinity suggests
that the hydrophobic van der Waals contacts along the
spherical carborane cage with the hydrophobic cavity
of ER produce a strong interaction. In view of the
expected fixation by the 4-hydroxyphenyl-o-carborane
moiety in the cavity of ER, the notable differences of
the transcriptional characteristics of 6, 7, and 8 can be
well interpreted in terms of differences of the direction
of the second hydroxyphenyl moiety and the distance
between it and the carborane cage. The conformation
of the ER-ligand complex determines the agonist-
antagonist nature of a particular ligand in the interac-
tion with cell- and promoter-specific factors. The partial
agonist-antagonist nature of 6 is supposed that 6-ER
complex is suitable for the appearance of the activity.
The insertion of one methylene group alters the direc-
tion of the second hydroxyphenyl group with respect to
the 4-hydroxyphenyl-o-carborane moiety, so that the
conformation of 7-ER complex corresponds to the
agonist conformation. The insertion of two methylene
groups again alters the direction of the second hydroxy-
phenyl group and its distance from the 4-hydroxy-
phenyl-o-carborane moiety, producing an antagonist
conformation of the 8-ER complex.
Another remarkable change of transcriptional nature
was observed upon altering the 3D hydrophobic core
from carborane to bicyclo[2.2.2]octene. The compound
10 bearing bicyclo[2.2.2]octene, exhibited potent agonist
activity, although 10 and 6 have a similar geometry of
the two hydroxyphenyl groups, and show similar bind-
ing affinity to ERR. The change of the transcriptional
nature seems to be caused by the difference of shape
between the carborane with its smooth spherical surface
and the bicyclooctene with an uneven surface. This
difference of the molecular surface apparently leads to
the formation of distinct ER-ligand complexes with
opposite transcriptional effects. On the other hand, the
compound 9 bearing 4,4,5,5-tetramethylcyclohexene,
which has a markedly nonspherical shape, was a partial
Figure 2. Transcriptional activation by the test compounds.
MCF-7 cells were transfected with ERE (SV-40)-LUC and
phRL/CMV and incubated with test compounds (10-9-10-6 M).
The values given are averages for duplicate transfections and
are expressed as a percent of the response with 10-10
estradiol.
M
anisole to give (E)-2-(4-methoxybenzylidene)-1-(4-meth-
oxyphenyl)adamantane (27) in 70% yield. Demethyl-
ation of 27 employing BBr3 failed, because of formation
of the benzyl cation and rearrangement of the adaman-
tane skeleton. Therefore, the demethylation was per-
formed with C2H5SNa in DMF to afford 11 in 68% yield.
A competitive binding assay using [6,7-3H]17â-estra-
diol (Kd ) 0.4 nM) and human recombinant ERR
(PanVera) was employed for initial screening of the
synthesized compounds.10 The binding affinity data are
summarized in Table 1. Insertion of a methylene group
into 6 afforded 7, which retained potent ER affinity. The
potencies of 6 and 7 were equivalent and were some-
what weaker than that of E2. The affinity of 8, into
which two methylene units had been inserted, was 10
times weaker than that of E2. Alteration of the hydro-
phobic core from carborane to bicyclo[2.2.2]octene (10)
or 4,4,5,5-tetramethyl-cyclohexene (9) resulted in equiva-
lent affinity to that of 6. On the other hand, the affinities
of 11 and 12, bearing adamantane, were decreased 10-
fold compared with those of 7 and 8 bearing a carborane
cage.
To evaluate the activity of the synthesized compounds
as agonists and antagonists, transcriptional assay was
done with ERE/Luci (firefly luciferase) and phRL/CMV
(Renilla luciferase) plasmid-cotransfected MCF-7 cells.19
The results of the transcriptional activation and inhibi-
tion are summarized in Figure 2 (agonist) and Figure 3
(antagonist). A remarkable difference of activity among
the three analogues bearing a carborane cage (6, 7, and
8) was observed. The compound 6 exhibited partial
agonist/antagonist activity, while 7, with an additional
methylene group, showed weak agonist activity. Com-
pound 8, with two additional methylene groups, was an
antagonist with a higher potency than that of 6. ER

3944 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12
Letters
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The compound 11 bearing adamantane in place of the
carborane cage of 7 exhibited agonist activity 10 times
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In summary, we have synthesized and biologically
evaluated novel ER ligands bearing two phenolic resi-
dues on a three-dimensional hydrophobic core structure
(carborane, bicyclo[2.2.2]octene, or adamantane). Among
the carborane-containing compounds (6, 7, and 8), a
dramatic change of agonist/antagonist balance was
observed depending on the direction of the second
hydroxyphenyl moiety and the distance between it and
the carborane cage. In general, replacement of the
carborane cage with 3D hydrocarbon cores increases the
agonist nature of the ligand. The compound 10 exhibited
potent agonist activity for ER, even though the two
phenolic groups appear to be similarly directed to those
of the partial agonist/antagonist 6. The results obtained
with these three-dimensional hydrophobic core struc-
tures raise the possibility that structure-function stud-
ies could lead to the development of more selective
estrogen agonists and antagonists, which could be useful
as therapeutic agents for a wide variety of conditions.
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Acknowledgment. This work was supported by a
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from the Ministry of Education, Culture, Sports, Science
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Supporting Information Available: Details of synthesis,
spectral data for compounds 7-12, stereo representations for
6-8, 9-12, and experimental procedures of biological evalu-
ations. This material is available free of charge via the Internet
at http://pubs.acs.org.
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