Synthesis and biological evaluation of novel m-carborane-containing estrogen receptor partial agonists as SERM candidates

Article abstract of DOI:10.1016/j.bmcl.2015.05.083

Abstract We designed and synthesized novel m-carborane-containing selective estrogen receptor modulator (SERM) candidates using previously reported m-carborane-containing ER partial agonist 1 as the lead compound. Biological activities were evaluated by m

Full text of DOI:10.1016/j.bmcl.2015.05.083

Bioorganic & Medicinal Chemistry Letters 25 (2015) 3213–3216
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of novel m-carborane-containing
estrogen receptor partial agonists as SERM candidates
⇑
Kiminori Ohta, Takumi Ogawa, Asako Kaise, Yasuyuki Endo
Faculty of Pharmaceutical Sciences, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We designed and synthesized novel m-carborane-containing selective estrogen receptor modulator
(SERM) candidates using previously reported m-carborane-containing ER partial agonist 1 as the lead
Received 12 May 2015
Revised 25 May 2015
Accepted 27 May 2015
Available online 3 June 2015
compound. Biological activities were evaluated by means of ER
cell proliferation assay. Re-positioning the N,N-dimethylaminoethyloxy group at the para position of 1
to the meta position enhanced the ER -binding affinity, and 4c showed the highest relative binding
a competitive binding assay and MCF-7
a
affinity (RBA: 83 vs 17b-estradiol = 100) among the tested compounds. Compound 4b showed the most
potent ER-agonist activity (EC50: 1.4 nM) and the lowest maximal efficacy (Emax: 50%) in MCF-7 cell pro-
Keywords:
Carborane
Estrogen receptor
Partial agonist
SERM
liferation assay. Inhibition of 0.1 nM 17b-estradiol-induced MCF-7 cell proliferation by 4b (IC50: 0.4
was at least 10 times more potent than that of the lead compound 1.
lM)
Ó 2015 Elsevier Ltd. All rights reserved.
Estrogens are involved in regulation of the female and male
reproductive systems, bone metabolism, and the cardiovascular
system, as well as the central nervous system, and these activities
are expressed through binding to and activation of nuclear estro-
including ER partial agonists, seem to be controlled by the overall
shape of the ER homodimer formed after ER-ligand binding.
Therefore, binding of different ligands to ER can facilitate or
impede the interaction of ER homodimer with various co-regula-
tors.9 The hydrophobic core structure of SERMs plays an important
role in determining their elaborate biological and pharmacokinetic
profiles. Therefore, development of ER modulators with novel
hydrophobic core structures is expected to afford unique SERMs
with distinctive biological properties.
We have developed several ER modulators having a carborane
cage as a novel hydrophobic pharmacophore.10 They exhibit
unique estrogen-related biological activities, different from those
of the endogenous estrogen 17b-estradiol (E2), or the above-
mentioned SERMs. BE360, an o-carborane-containing ER
modulator without an alkylamino side chain, showed partial
agonist activity in MCF-7 cell proliferation assay, and it increased
bone density with no effect on the uterus in ovariectomized mice
(Fig. 2).11 That is, BE360 is a carborane-containing SERM. We have
also reported that m-carborane derivative 1 bearing an alkylamino
group acted as a potent ER partial agonist in ER transactivation
assays (Fig. 2).12 However, the maximal efficacy of 1 is not low,
and so there is a risk that 1 might induce breast cancer. SAR studies
of the alkylamino chain of m-carborane derivative 2 revealed that
compounds with an alkyl, carbamate, or thiocarbamate group
instead of the alkylamino group acted as ER full agonists.13
Thus, the alkylamino group of 1 is essential for expression of ER
partial agonist activity, as has been observed with other SERM
candidates (Fig. 2).14 Recently, we have reported that the
gen receptor (ER).1 ER has two subtypes (
a and b), which show dif-
ferent patterns of tissue expression and mediate two different
signaling pathways: transcriptional regulation and non-genomic
membrane-associated transduction.1 Many of the physiological
effects of ER are subtype-specific. Non-steroidal and non-hormonal
ER antagonists, such as tamoxifen2 and raloxifene,3 are widely used
for treatment of breast cancer (Fig. 1). Tamoxifen and its active
metabolite, 4-hydroxytamoxifen, show ER agonism in endome-
trium and bone, whereas raloxifene acts as an antagonist in
endothelium and as an agonist in bone.4 Therefore, raloxifene has
no risk for cancer of the female reproductive system, and is used
as a protective agent against osteoporosis in post-menopausal
women.5 Compounds having tissue-specific ER agonist or antago-
nist activity are called selective estrogen receptor modulators
(SERMs).6 They exhibit considerable functional diversity: for exam-
ple, bazedoxifene contains an alkylamino side chain, which is crit-
ical for agonist and antagonist activities of SERMs, but its biological
activities are different from those of tamoxifen and raloxifene.7 In
addition, the binding mode of bazedoxifene to ERa is different
from that of 4-hydroxytamoxifen, as evaluated by docking simula-
tion study.8 The relative agonist/antagonist activities of SERMs,
⇑
Corresponding author. Tel.: +81 22 727 0142; fax: +81 22 275 2013.
E-mail address: yendo@tohoku-pharm.ac.jp (Y. Endo).
http://dx.doi.org/10.1016/j.bmcl.2015.05.083
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

3214
K. Ohta et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3213–3216
H3C
O
N
CH3
n
N
O
N
O
n = 0 ~ 2
N
O
HO
4
O
N
ER partial agonist candidates
O
OH
H
HO
R
S
HO
Figure 3. Structures of novel ER partial agonist candidates 4.
R = H Tamoxifen
R = OH 4-Hydroxytamoxifen
Raloxifene
Bazedoxifene
in 94% yield; its biological activity was evaluated and compared
with that of bisphenol 12, an intermediate of 1 (Scheme 1).
Compound 4c was obtained by stepwise synthesis involving two
SN2 reactions of dibromobutane. One bromine atom was changed
to a phenolic hydroxyl group (7) in 62% yield, and the other was
reacted with dimethylamine to afford 11 in 78% yield.
Demethylation of 11 with BBr3 afforded 4c in 34% yield.
Figure 1. Structures of clinically used SERMs.
9,10-dimethyl-m-carborane cage is effective for obtaining ER par-
tial agonist activity with very low maximal efficacy, as well as
for increasing ER-binding affinity (compound 3, Fig. 2).15 This
may be due to a geometry change of the alkylamino group remo-
tely induced by steric repulsion between the bulky hydrophobic
structure and amino acid residues surrounding the ER ligand-bind-
ing domain (LBD). Although the 9,10-dimethyl-m-carborane cage
of 3 seems to be more promising as a hydrophobic structure for
ER partial agonist discovery than m-carborane, preparation of
1,7-diaryl-9,10-dimethyl-m-carborane derivatives is synthetically
difficult in that coupling reaction of 9,10-dimethyl-m-carborane
with aryl iodides affords the products in very low yield.
ER-binding affinity was evaluated by means of competitive
binding assay using human recombinant ER
a
and [6,7-3H]
17b-estradiol. Relative binding affinity (RBA) values of the test
compounds are summarized in Table 1.17 Compounds 1 and 12
showed low and high RBA values of 1.5 and 110, respectively,
which are close to the previously reported values of 1.1 and 106,
respectively.12 Although the binding affinity of the parent bisphe-
nol 9 is similar to that of 12, compound 4a with an N,N-dimethy-
laminoethoxy side chain showed 5 times more potent ERa-
Therefore, we selected the m-carborane cage as a hydrophobic
structure for the preparation of ER partial agonists and designed
derivatives 4 in which the alkylamino side chain of 1 is transferred
to a neighboring carbon (Fig. 3).
Scheme 1 summarizes the synthesis of m-carborane-containing
ER partial agonist candidates 4. m-Carborane 5 was treated with n-
BuLi, and then transformed into C-copper-p-carborane, which was
reacted with 4-iodoanisole in the presence of pyridine as a ligand
binding than the corresponding p-substituted derivative 1. Our
previous results showed that extension of the alkylamino chain
of 1 has little influence on RBA values, but the same modification
of the alkylamino chain of 4 led to a remarkable enhancement of
RBA value, and the RBA of 4c was 83.18 These results suggest that
an alkylamino side chain at the meta position fits well into the cav-
ity of the ER
a
LBD, and the terminal tertiary amino group of 4c
LBD.
forms hydrogen bonds with amino acid residues of the ER
a
of copper to afford 4-methoxyphenyl-m-carborane
6 in 71%
Next, the functional activities of the test compounds were eval-
uated by means of cell proliferation assays using MCF-7 cell lines
that show ER-dependent growth.17 Table 2 summarizes EC50 and
IC50 values as parameters of the agonist and antagonist activities
of the test compounds, respectively. Agonist activity is also shown
as relative maximal efficacy (Emax), based on estradiol as 100%.
Bisphenol 9 showed similar EC50 and Emax values to 12. Both
bisphenols showed no ER-antagonist activity and acted as ER full
agonists, not as side-chainless partial agonists like BE360. The lead
compound 1 showed moderate agonist activity and its Emax value
was 78%, which means it has lower maximal efficacy than E2.
Compound 1 antagonized MCF-7 cell proliferation induced by
yield.16 Next, coupling reaction of 6 with TBS-protected iodophenol
under the same conditions, followed by deprotection of the TBS
group, afforded key intermediate diaryl-m-carborane 7 in 71%
yield. Dimethylaminoethyl and dimethylaminopropyl groups were
introduced into 7 by using the corresponding alkyl halides in 33%
and 24% yields, respectively. Demethylation of 8 with BBr3 afforded
the desired compounds 4a and 4b in 45% and 74% yields, respec-
tively. Compound 7 was reacted with BBr3 to afford bisphenol 9
HO
OH
0.1 nM of E2 with an IC50 value of 4.4 lM. Compound 4a, which
has the same side chain as the lead compound 1, but at the meta
position, showed potent ER agonist activity (EC50 = 4.7 nM) and a
= C
= BH
unmarked
vertices
HO
HO
low Emax value of 63%. The IC50 value of 4a was 6.5 lM, which is
BE360
17β-estradiol (E2)
similar to that of 1. Compound 4b with a dimethylaminopropyl
group showed the lowest EC50 value and the lowest Emax value
among the tested compounds. In addition, compound 4b showed
10 times more potent ER antagonist activity than the lead com-
pound 1. Although dimethylaminobutyl derivative 4c showed the
ER partial agonist (in vitro)
SERM (in vivo)
CH3
N
CH3
N
R
H3C
O
O
H3C
O
greatest ERa-binding affinity, its biological activities parameters,
EC50, Emax, and IC50, are similar to those of compound 4a. These
results confirm that the dimethylaminopropyl group is the most
suitable ER partial agonist activity-inducing substituent in the
series of m-carborane-containing m-substituted derivatives 4.
The ER-antagonist activity of 4b was more potent than that of 3,
which contains the 9,12-dimethyl-m-carborane cage (IC50 of
CH3
CH3
HO
HO
HO
1
2
3
ER partial agonist
ER agonist
Potent ER partial agonist
3 = 0.88 lM). The low IC50 value of 4b suggested that the side chain
serves to inhibit binding of co-activators by moving helix-12 of ER
Figure 2. Structures of 17b-estradiol (E2) and m-carborane-containing ER
to an unfavorable position. However, compound 4b has a higher
modulators.

K. Ohta et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3213–3216
3215
HO
O
O
N
n
N
n
H
H
a
b, c
HO
d
e
H
6
7
8
4a
n = 1
H3CO
H3CO
H3CO
5
HO
n = 2 4b
e
f
OH
O
O
O
Br
N
N
g
e
12
9
HO
HO
4c
10
11
H3CO
H3CO
HO
Scheme 1. Synthesis of novel ER modulators 4. Reagents and conditions: (a) n-BuLi, DME, then CuCl, pyridine, 4-iodoanisole, 71%; (b) n-BuLi, DME, then CuCl, pyridine, 3-tert-
butyldimethylsiloxyiodobenzen; (c) TBAF, THF, 71% over 2 steps; (d) N,N-dimethylaminoalkyl chloride, K2CO3, acetone, 24–38%; (e) BBr3, CH2Cl2, 34–94%; (f) 1,4-
dibromobutane, K2CO3, acetone, 62%; (g) dimethylamine, THF, 78%.
Table 1
Novel ER modulator 3 showed better partial agonist activity
than 1, even though it contains the same alkylamino chain and
Relative binding affinity (RBA) of test compounds versus specific [3H]estradiol (4 nM)
binding with human recombinant ER
a
has the same substitution position.15 Further syntheses of a series
of diaryl-m-carborane derivatives, docking simulation studies, and
biological investigations with other ER-expressing tissues, deter-
mination of ER expression levels, and examination of the influence
of 9,12-dimethyl-m-carborane structure on ER partial agonist
activity, as well as in vivo experiments, are in progress.
Compound
Position
Substituent
RBAa
1
p-
–O–(CH2)2–N(CH3)2
–O–(CH2)2–N(CH3)2
–O–(CH2)3–N(CH3)2
–O–(CH2)4–N(CH3)2
–OH
1.5
7.4
8.4
83
131
110
4a
4b
4c
9
m-
m-
m-
m-
p-
In conclusion, novel m-carborane-containing ER partial agonists
were synthesized and their biological activities were evaluated by
means of competitive ER binding assay and MCF-7 cell prolifera-
tion assay. All tested compounds 4 showed higher RBA values than
the corresponding para-substituted derivatives, suggesting that an
alkylamino side chain at the meta position fits well into the cavity
12
–OH
a
All binding assay were treated with the test compounds (0.4 nM to 4
lM) in the
presence of [6,7-3H]17b-estradiol (4 nM). The relative binding affinity is calculated
from IC50 values of E2 and test compounds taking that of E2 as 100%. Values rep-
resent the average of duplicate experiments.
of the ERa LBD. Compound 4b showed the most potent ER-agonist
Emax value than compound 3 (Emax of 3 = 23%), and might show
moderate estrogenic activity in breast tissue. A terminal cyclic
alkylamino group, such as piperidine or azepane (used in ralox-
ifene and bazedoxifene, respectively), can often enhance ER-antag-
onist activity, and thus introduction of these substituents might
afford better partial agonists or SERM candidates. Carborane cages
are promising hydrophobic core structure for the development of
ER modulators,10–13,15,17 and carborane-containing ER modulators
may show unique biological properties.
activity and the lowest Emax value among the tested compounds.
Moreover, the ER-antagonist activity of 4b was 10 times more
potent than that of the lead compound 1. As observed in the case
of bazedoxifene, the unique hydrophobic carborane cage structure
provide an entry into carborane-containing ER modulators with
various characteristics of ER up- or down-regulation, as well as dis-
tinctive pharmacokinetic properties. These findings should be
helpful for molecular design of further carborane-containing ER
modulators, including agonists, partial agonists, antagonists, and
SERMs as biological tools or candidate therapeutic agents for ER-
related diseases.
Table 2
Biological activities of the test compounds on MCF-7 cell proliferation
Acknowledgments
a
b
c
Compound Position Substituent
EC50
Emax
IC50
M)
(nM)
(%)
(
l
This research was supported by a Grant-in-Aid for the Strategic
Research Program for Private University (2010-2014) and a Grant-
in-Aid for Scientific Research (C) (No. 26460151) from the Ministry
of Education, Culture, Sports, Science, and Technology, Japan.
1
p-
–O–(CH2)2–
N(CH3)2
–O–(CH2)2–
N(CH3)2
–O–(CH2)3–
N(CH3)2
–O–(CH2)4–
N(CH3)2
–OH
8.2
78
63
50
62
4.4
6.5
0.4
4.3
4a
4b
4c
m-
m-
m-
4.7
1.4
2.6
References and notes
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derivatives
are
as
follows:
N,N-dimethylaminoethyl = 1.1,
N,N-
dimethylaminopropyl = 1.5, and N,N-dimethylaminobutyl = 1.7.