Promising core structure for nuclear receptor ligands: Design and synthesis of novel estrogen receptor ligands based on diphenylamine skeleton

Article abstract of DOI:10.1016/j.bmcl.2008.08.004

Novel diphenylamine-type estrogen receptor ligands were designed and synthesized, and their biological activities were evaluated by means of binding assays for estrogen receptor-α and -β and cell proliferation assay using MCF-7 cells. Compounds 4f, 11b, 12c, and 8 showed moderate estrogenic activities. We propose that the diphenylamine skeleton may be a privileged structure for various nuclear receptor ligands, including RAR, RXR, and AR ligands.

Full text of DOI:10.1016/j.bmcl.2008.08.004

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5050–5053
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Promising core structure for nuclear receptor ligands: Design and synthesis
of novel estrogen receptor ligands based on diphenylamine skeleton
*
Kiminori Ohta, Yuki Chiba, Takumi Ogawa, 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:
Novel diphenylamine-type estrogen receptor ligands were designed and synthesized, and their biological
Received 5 July 2008
Revised 29 July 2008
Accepted 1 August 2008
Available online 6 August 2008
activities were evaluated by means of binding assays for estrogen receptor-a and -b and cell proliferation
assay using MCF-7 cells. Compounds 4f, 11b, 12c, and 8 showed moderate estrogenic activities. We pro-
pose that the diphenylamine skeleton may be a privileged structure for various nuclear receptor ligands,
including RAR, RXR, and AR ligands.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Estrogen receptor ligand
Agonist
Diphenylamine
Bisphenol
Nuclear receptor ligand
Efficient synthetic methods for diphenylamines were developed
by Buckwald et al.1 and Hartwig,2 and the diphenylamine skeleton
has proved useful in medicinal chemistry.3 The nitrogen atom has
weak basicity (pKb = 13.2) and the two benzene rings are sterically
bulky, so diphenylamines do not readily form hydrogen bonds with
amino acid residues of receptors. Among related structures, diphen-
ylether and diphenylsulfide skeletons have less potential because it
is not possible to introduce a substituent on the heteroatoms. In the
case of diphenylmethanes, it is difficult to introduce substituents on
the linker carbon atom, and the C-substituted products can be enan-
tiomeric if the aromatic rings are different. Thus, the diphenylamine
skeleton has the advantages that the linker nitrogen atom can be
readily modified with various substituents and the products are
not enantiomeric, unless an asymmetric substituent is introduced,
so that synthesis is more straightforward. Thus, diphenylamine
structure is expected to be a useful skeleton for new drugs.
important roles in the regulation of the female and male reproduc-
tive systems, as well as in bone metabolism, the cardiovascular
systems, and the central nervous system. The first step in the
appearance of estrogenic activity is the binding of agonist ligands
to ERa
7 and ERb,8 resulting in a conformational change. The result-
ing ligand-bound ER then dimerizes, forms complexes with various
cofactors, and binds to specific promoter elements of DNA to initi-
ate gene transcription.9
A synthetic estrogen, diethylstilbestrol 1, acts like a native li-
gand, estradiol. Hydroxytamoxifen 2 has potent antagonistic activ-
OH
OH
H
HO
Diethylstilbestrol 1
H
H
We have already reported retinoic acid receptor (RAR)4 and ret-
inoid X receptor (RXR) modulators5 based on the diphenylamine
skeleton. In addition, an androgen receptor (AR) modulator based
on the diphenylamine skeleton has been reported.6 Since nuclear
transfer is an important process for nuclear receptor ligands, the
weak basicity of the nitrogen atom of diphenylamine (in other
words, the low polarity of its structure) should be favorable for
developing novel nuclear receptor ligands.
HO
17β-estradiol
OCH2CH2N(CH3)2
OH
The estrogen receptor (ER) is a member of the superfamily of
nuclear receptors. Endogenous estrogen, 17b-estradiol, plays
HO
Hydroxytamoxifen 2
HO
CF3
3
* Corresponding author. Fax: +81 22 275 2013.
E-mail address: yendo@tohoku-pharm.ac.jp (Y. Endo).
Figure 1. Structures of the native ligand and synthetic ligands for ER.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.08.004

K. Ohta et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5050–5053
5051
ity for ER and acts as a selective estrogen receptor modulator
OH
OMe
OMe
(SERM).10 Currently, various third- or fourth-generation SERMs
are under development.10 Recently, compound 3, which is a potent
antiestrogen without the alkylamino residue found in typical SER-
Ms, was reported11(see Fig. 1).
O
O
b
a
5
N
N
HO
OH
MeO
7
8
The development of ER ligands remains an important issue in the
field of medicinal chemistry because novel functions of estrogen are
still being found.12 Therefore, we initially designed and synthesized
N-alkylated diphenylamine derivatives 4 as ER ligand candidates
based upon their attractive chemical properties and easy availability
(Fig. 2). Here, we describe the synthesis and the structure–activity
relationships of these diphenylamine derivatives.
The synthesis of N-alkylated compounds 4a–4j is summarized
in Scheme 1. Using the usual conditions of Buckwald–Hartwig ami-
nation, a key intermediate, bis(4-methoxyphenyl)diphenylamine
5, was synthesized from 4-bromoanisole and p-anisidine, which
are commercially available. N-alkylated compounds 6 were ob-
tained by the reaction of 5 with the corresponding alkyl halides
in the presence of NaH as a base. Compounds 6 were reacted with
BBr3 to afford N-alkylated derivatives 4a–4j.
OH
c
N
HO
OH
4k
NH
2
d
b
N
N
HO
OH
MeO
OMe
4l
9
Scheme 2 summarizes the synthesis of compounds 4k–4m.
Compound 4k was synthesized by N-acylation of 5, demethylation,
and reduction of the amide group with LiAlH4, because N-(4-
methoxybenzyl)diphenyl-amine was decomposed in the demeth-
ylation with BBr3 as shown in Scheme 1. Compound 4l was synthe-
sized from cyclohexylamine by double Buckwald–Hartwig
amination using tBu3P as a ligand, followed by demethylation with
BBr3, since the sterically bulky N-cyclohexyl group could not be
introduced into the intermediate 5 shown in Scheme 1. An N-phe-
nyl derivative 4m was synthesized by Buckwald–Hartwig amina-
tion of the intermediate 5 with bromobenzene, followed by
demethylation with BBr3.
b
e
N
N
5
MeO
OMe
HO
OH
10
4m
Scheme 2. Synthetic scheme of 4k–4m. Reagents and conditions: (a) 4-methoxy-
benzoyl chloride, toluene, reflux, 87%; (b) BBr3, CH2Cl2, 53—97%; (c) LiAlH4, THF,
58%; (d) p-anisidine, Pd2(dba)3, tBu3P, NaOtBu, toluene, reflux, 91%; (e) bromoben-
zene, Pd2(dba)3, rac-BINAP, NaOtBu, toluene, reflux, 71%.
All of the synthesized compounds were evaluated with a com-
petitive binding assay using [2,4,6,7-3H]17b-estradiol (4 nM) and
R
N
human recombinant ER
sults of binding assay are summarized as relative binding affinity
(RBA) for ER and ERb, and selectivity ratio in Table 1. Compounds
a
and ERb as an initial screening.13 The re-
HO
OH
a
4a, 4b, and 4c did not bind to either of the ERs. When the hydro-
phobicity and bulkiness of the substituents on the nitrogen atom
4j
H
4a
4b
4c
4f
n
-C6H13
were increased, binding affinities for ER
pound 4h and 4k showed moderate binding affinity to ER
ERb. Compound 4j bound to ERb about 30 times more strongly than
to ER
a
and ERb increased. Com-
CH3
OH
4g
4k
a
and
a
.
4h
4l
4d
4e
The biological activities were evaluated by means of cell prolif-
Br
eration assay using human breast cancer MCF-7 cells.14 Although
4i
4m
Table 1
Figure 2. Designed compounds as ER ligand candidates based on diphenylamine
skeleton.
Binding affinity and receptor selectivity of compounds 4 for the human recombinant
ERa or ERb
Compound
RBAa for ER
a
RBAa for ERb
Selectivity ERba
4a
4b
4c
4d
4e
4f
4g
4h
4i
<0.001
<0.001
<0.001
0.054
0.129
0.153
0.890
3.643
0.551
0.655
4.260
1.130
1.295
<0.001
<0.001
<0.001
<0.001
2.339
0.102
6.696
6.836
0.565
18.134
4.050
4.299
1.912
—
—
—
—
18.132
0.667
7.523
1.876
1.025
27.685
0.951
3.804
1.476
H
Br
N
a
b
MeO
MeO
OMe
5
R
N
R
N
4j
4k
4l
c
MeO
OMe
HO
OH
6
4a-4j
4m
a
All binding assays were performed in triplicate (n = 3). IC50 value of estradiol for
and ERb is each 4 nM. Relative binding affinity (RBA) is IC50 (estradiol)/IC50
Scheme 1. Synthetic scheme of N-alkylated diphenylamine derivatives 4a–4j.
Reagents and conditions: (a) p-anisidine, Pd2(dba)3, rac-BINAP, NaOtBu, toluene,
reflux, 67%; (b) NaH, RX, DMF, 22—97%; (c) BBr3, CH2Cl2, 74%-quant.
ER
a
(compound) Â 100.

5052
K. Ohta et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5050–5053
Table 3
compounds 4a–4c were inactive, compounds 4d–4m allowed
MCF-7 cells to grow in dose-dependent manner. The EC50 values
of the tested compounds are summarized in Table 2. None of the
synthesized compounds exhibited antiestrogenic activity (data
not shown). Compounds 4f and 4j showed the best estrogenic
Binding affinity and receptor selectivity of compounds 8, 11a, 11b, and 12a—12c for
the human recombinant ER or ERb
a
Compound
RBAafor ER
a
RBAa for ERb
Selectivity ERba
11a
11b
8
12a
12b
12c
<0.001
0.193
0.336
<0.001
0.040
0.352
<0.001
1.258
0.063
<0.001
<0.001
0.004
—
activity (EC50 = 1.1 Â 10À8
M
and 6.7 Â 10À8 M, respectively),
6.518
0.188
—
—
0.011
although they were less potent than the native ligand, estradiol.
The estrogenic activity of compounds 4f and 4g showed higher effi-
cacy than that of estradiol.
Next, N-acyl 815 and 11 and N-sulfonyl 12 derivatives were de-
signed and synthesized to evaluate the effect of the linking group
on the activity. Based on the results of binding assay and cell pro-
liferation assay, compound 4j and 4k with the less flexible N-ben-
zyl substituents were selected. The methyl group as shown in the
structure of 4a was chosen as a negative control.
The structures and synthetic scheme of compounds 8, 11a, 11b,
and 12a–12c are summarized in Scheme 3. The structure and syn-
thetic scheme of compound 8 are shown in Scheme 2. As described
in the synthesis of compound 8, the reaction of 5 with the corre-
sponding acyl chlorides in toluene afforded the N-acylated com-
pounds, and demethylation with BBr3 gave compounds 11a and
11b. N-Sulfonylated compounds 12 were obtained by the reaction
of 5 with the corresponding sulfonyl chlorides in the presence of
NaH in THF, followed by demethylation with BBr3.
a
All binding assays were performed in triplicate (n = 3). IC50 value of estradiol for
and ERb is each 4 nM. Relative binding affinity (RBA) is IC50 (estradiol/IC50
ERa
(compound) Â 100.
Table 3 summarizes the binding affinity of N-acyl and N-sulfo-
nyl compounds. As expected, methyl derivatives 11a and 12a did
not bind to either ER
a or ERb. N-Acyl compounds showed higher
binding affinity to both ERs than did the corresponding N-sulfonyl
compounds. It seems that the binding affinity of these compounds
was decreased compared to those of the parent compounds owing
to the high hydrophilicity of the N-acyl and N-sulfonyl groups.16
The cell proliferation activity of compounds 8, 11a, 11b, and
12a–12c was evaluated using MCF-7 cells as described above.
Dose–response curves and EC50 values of the test compounds are
summarized in Figure 3 and Table 4, respectively. As expected,
the methyl derivatives 11a and 12a showed weak estrogenic activ-
ity (EC50 = 4.3 Â 10À5 M and 6.9 Â 10À6 M, respectively), being
more potent than the parent compound 4a. Both derivatives, N-
acyl and N-sulfonyl, showed the tendency that the compounds
with a 4-hydroxyphenyl substituent, 8 and 12c, exhibited more po-
tent estrogenic activity than the phenyl derivatives 11b and 12b,
although the parent 4-hydroxyphenyl compound 4k showed
weaker estrogenic activity than the phenyl compound 4j. Com-
Table 2
EC50 values of N-alkylated diphenylamine derivatives 4a—4m in cell proliferation
assay using MCF-7 cellsa
b
b
Compound
EC50 (M)
Compound
EC50 (M)
4a
4b
4c
4d
4e
4f
Inactive
Inactive
Inactive
Compound
5.7 Â 10À7 (31)
1.1 Â 10À8 (130)
2.1 Â 10À7 (109)
4h
4i
4j
4k
4l
4m
Estradiol
5.0 Â 10À7 (71)
1.1 Â 10À5 (77)
6.7 Â 10À8 (85)
3.7 Â 10À7 (42)
5.9 Â 10À7 (64)
1.2 Â 10À7 (87)
3.7 Â 10À12 (100)
11a
11b
8
350
300
250
200
150
100
50
4g
a
MCF-7 cells were incubated with the test compounds for 5 days. Cell prolifer-
ation assay was performed in triplicate (n = 3).
b
12a
12b
EC50 values of the test compounds were estimated from the sigmoidal dose–
response curves using GraphPad Prism 4 software. The values in parentheses
indicate the efficacy for cell proliferation with the value for estradiol taken as 100.
12c
O
R
O
R
N
N
c
0
MeO
OMe
HO
OH
13
-12 -11 -10 -9
-8
-7
-6
-5
-4
-3
R = CH (11a)
3
a
b
Log concentration (M)
Ph (11b)
4-OH-Ph (8)
Figure 3. MCF-7 cell growth in the presence of the synthesized compounds. MCF-7
cells were incubated with the test compounds (1 Â 10À4 to 1 Â 10À11 M) for 4 days,
and the results are shown as relative cell number, with the value for estradiol taken
as 100. Cell proliferation assay was performed in triplicate (n = 3). Values are
means SD for separate experiments.
5
R
O S O
N
R
O S O
N
c
Table 4
MeO
OMe
HO
OH
12
EC50 value of N-acylated and N-sulfonylated diphenylamine derivatives 8, 11a, 11b,
14
and 12a—12c in cell proliferation assay using MCF-7 cellsa
R = CH (12a)
3
Compound
EC50 (M)a
Compound
EC50 (M)a
Ph (12b)
4-OH-Ph (12c)
11a
11b
8
4.3 Â 10À5 (114)
1.4 Â 10À8 (114)
5.2 Â 10À9 (240)
12a
12b
12c
6.9 Â 10À6 (213)
6.3 Â 10À8 (98)
1.2 Â 10À8 (99)
Scheme 3. Structures and synthetic scheme of N-acylated 11 and N-sulfonylated
diphenylamine derivatives 8, 12a, and 12b. Reagents and conditions: (a) RCOCl,
toluene, reflux, 55—87%; (b) NaH, RSO2Cl, THF, reflux, 43—69%; (c) BBr3, CH2Cl2,
25—97%.
a
All experiments were performed according to the same method described in
Table 2.

K. Ohta et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5050–5053
5053
Kagechika, H. J. Med. Chem. 2002, 45, 3327; (d) Ohta, K.; Iijima, T.; Kawachi, E.;
Kagechika, H.; Endo, Y. Bioorg. Med. Chem. Lett. 2004, 14, 5913.
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Nature 1986, 320, 134.
pound 8 showed the most potent estrogen activity among the test
compounds, and the efficacy was 2.4 times higher than that of
estradiol.
These results suggest that the diphenylamine skeleton is a
suitable structure for the expression of estrogenic activity. The
greatest advantage of the diphenylamine skeleton as a platform
for drug design is the potential for control of the activity by easy
modification of N-substituents and aromatic substituents. The
diphenylamine skeleton appears to be a promising core structure
for ligands of various nuclear receptors, including ER, AR, RAR,
and RXR.
In conclusion, novel ER ligands were designed and synthesized
based on the diphenylamine skeleton. Compounds 4f, 8, 11b, and
12c showed moderate estrogenic activities. It is noteworthy that
the diphenylamine skeleton appears to be a suitable core structure
for ER ligands, as well as for RAR, RXR, and AR ligands, and we sug-
gest that this skeleton may be a privileged structure for nuclear
receptor ligands. Further, structural modification of 4, such as
introduction of a basic side chain as in 2, or a heteroatom contain-
ing-substituents on the nitrogen atom, may afford potent ER antag-
onists, SERMs or ERb-selective modulators. It may prove possible
to develop a wide range of nuclear receptor ligands based upon
the diphenylamine skeleton.
8. Kuiper, G. G. J. M.; Enmark, E.; Pelto-Huikko, M.; Nilsson, S.; Gustafsson, J. A.
Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 5925.
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Ohman, L.; Greene, G. L.; Gustafsson, J. A.; Carlquist, M. Nature 1997, 389, 753.
10. Fernand, L.; Claude, L.; Alain, B.; Jacques, S. In Selective Estrogen Receptor
Modulators; Andrea, M., Michael, V., Eds.; Humana Press Inc.: Totowa, NJ, 2002;
p 167.
11. Lubczyk, V.; Bachmann, H.; Gust, R. J. Med. Chem. 2002, 45, 5358.
12. (a) Van der Windt, D. J.; Kok, N. F. M.; Ijzermans, J. Science 2007, 318, 1239; (b)
Zhao, C.; Dahlman-Wright, K.; Gustafsson, J. A. Nucl. Rec. Sig. 2008, 6, 1; (c)
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13. Endo, Y.; Iijima, T.; Yamakoshi, Y.; Fukasawa, H.; Miyaura, C.; Inada, M.; Kubo,
A.; Itai, A. Chem. Biol. 2001, 8, 341: The ligand-binding activity of ER
was determined by the nitrocellulose filter binding assay method. ER
(0.5
a
a
and ERb
and ERb
l
g/tube) were diluted with a binding assay buffer (20 mM Tris–HCl, pH
8.0, 0.3 M NaCl, 1 mM EDTA, pH 8.0, 10 mM 2-mercaptoethanol, 0.2 mM
phenylmethylsulfonyl fluoride) and incubated with 4 nM [2,4,6,7-3H]17b-
estradiol in the presence or absence of an unlabeled competitor at 4 °C for 16 h.
The incubation mixture was absorbed by suction onto
a nitrocellulose
membrane that had been soaked in binding assay buffer. The membrane was
washed twice with buffer (20 mM Tris–HCl, pH 8.0, 0.3 M NaCl), and then with
25% ethanol in distilled water. Radioactivity that remained on the membrane
was measured in Atomlight using a liquid scintillation counter.
14. Human breast adenocarcinoma cell line MCF-7 was routinely cultivated in
DMEM (High Glucose) supplemented with 10% FBS, 100 IU/mL penicillin, and
100 lg/mL streptomycin at 37 °C in a 5% CO2 humidified incubator. On the day
before an assay, MCF-7 cells were switched to DMEM (Low Glucose, phenol red-
free) supplemented with 5% dextran-coated charcoal-stripped FBS (sFBS),
100 IU/mL penicillin, and 100 mg/mL streptomycin. Cells were trypsinized
from the maintenance dish with phenol red-free 0.25% trypsin-EDTA and seeded
Acknowledgments
This research was supported by a Grant-in-Aid for High Tech-
nology Research Program, a Grant-in-Aid for Scientific Research
(B) (No. 20390035), and Grant-in-Aid for Young Scientists (B)
(No. 18790089) from the Ministry of Education, Culture, Sport, Sci-
ence and Technology, Japan.
in a 96-well plate at a density of 2 Â 103 cells per final volume of 100
lL DMEM
Low Glucose phenol red-free supplemented with 5% sFBS, 100 IU/mL penicillin,
and 100
fresh DMEM Low Glucose phenol red-free supplemented with 5% sFBS, 100 IU/
mL penicillin, and 100 g/mL streptomycin, and 10 L of the drug solution,
lg/mL streptomycin. After 24 h, the medium was replaced with 90 lL of
l
l
which was supplemented with serial dilutions of compounds or DMSO as the
diluent control, was prepared and added in the presence or absence of
1 Â 10À10 M estradiol to triplicate microcultures. Cells were incubated for 6
days, and medium with compounds was changed once after 3 days. At the end of
the incubation time, proliferation was assessed using WST-8 according to the
manufacturers’ instructions; 10 lL of WST-8 was added to microcultures and
cells were incubated for 2–4 h. The absorbance at 450 nm was measured. This
parameter relates to the number of living cells in the culture.
References and notes
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15. Compound 8 has been the subject of a PCT application during our studies of
these compounds. However, their drug design was focused on benzanilide
structure, not on the diphenylamine skeleton.Dalton, J. T.; Barrett, C.; He, Y.;
Hong, S.-S.; Miller, D. D.; Mohler, M. L.; Narayanan, R.; Wu, Z. PCT Int. Appl.
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16. The ClogP values of N-acyl and N-sulfonyl compounds and the parent
compounds were calculated with ChemDraw Ultra 6.0. ClogP: 4j (4.51), 4k
(3.84), 11b (3.84), 8 (3.71), 12b (3.37), and 12c (3.23).