Symmetric 4,4′-(piperidin-4-ylidenemethylene)bisphenol derivatives as novel tunable estrogen receptor (ER) modulators

Article abstract of DOI:10.1016/j.bmc.2016.01.035

We designed and synthesized 4,4′-(piperidin-4-ylidenemethylene)bisphenol derivatives as novel tunable estrogen receptor (ER) modulators. The introduction of hydrophobic substituents on the nitrogen atom of the piperidine ring enhanced ERα binding affinity

Full text of DOI:10.1016/j.bmc.2016.01.035

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Symmetric 4,40-(piperidin-4-ylidenemethylene)bisphenol derivatives
as novel tunable estrogen receptor (ER) modulators
⇑
Manabu Sato, Kiminori Ohta , Asako Kaise, Sayaka Aoto, 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
We designed and synthesized 4,40-(piperidin-4-ylidenemethylene)bisphenol derivatives as novel tunable
estrogen receptor (ER) modulators. The introduction of hydrophobic substituents on the nitrogen atom of
Article history:
Received 16 December 2015
Revised 14 January 2016
Accepted 18 January 2016
Available online xxxx
the piperidine ring enhanced ER
adjacent to the piperidine ring nitrogen atom remarkably enhanced ER
2,2,6,6-tetramethylpiperidine derivative 3b showed high ER binding affinity, high MCF-7 cell prolifera-
tion inducing activity, and high metabolic stability in rat liver S9 fractions.
Ó 2016 Elsevier Ltd. All rights reserved.
a
binding affinity. In addition, the introduction of four methyl groups
a
binding affinity. N-Acetyl-
a
Keywords:
Estrogen receptor modulator
Piperidine
2,2,6,6-Tetramethylpiperidine
McMurry coupling
Hydrophobicity
1. Introduction
N
Tamoxifen (Fig. 1) has been used worldwide for more than
40 years for the treatment of breast cancer.1 It exhibits competitive
antagonism against endogenous estrogen 17b-estradiol (E2, Fig. 1)
on the estrogen receptor (ER). Tamoxifen produces a powerful
effect against ER-positive but not ER-negative breast cancer.1 In
addition, since E2 is an endogenous estrogen that plays important
roles in the female and male reproductive systems as well as in
bone maintenance, the central nervous system, and the cardiovas-
cular system, tamoxifen also exerts some biological actions in
those tissues.2 Interestingly, tamoxifen acts as either an agonist
or an antagonist depending on tissue type; it exhibits anti-
estrogenic action in breast cancer and hot flashes, and estrogenic
action in bone and cholesterol metabolism, and is a selective estro-
gen receptor modulator (SERM).3
O
OH
HO
O
R
17β-Estradiol (E2)
R = H Tamoxifen
R = OH 4-Hydroxytamoxifen
N
N
OAc
O
Following the success of tamoxifen, several triphenylethylene
derivatives, such as toremifene4 and clomifene5, were developed
as novel SERMs (Fig. 1). An alkylamino chain is attached to the ter-
minal of benzene ring where it plays a strategic role in the expres-
sion of antagonistic activity.6 The three SERMs show quite varied
biological activities because of the different hydrophobic side
chains attached to the ethylene moiety.7 The hydrophobic side
chain plays an important role in controlling SERM activity. The
triphenylethylene structure has geometric isomers E and Z, which
Cl
Clomifene
Cl
Toremifene
AcO
Cyclofenil
Figure 1. Chemical structures of 17b-estradiol (E2) and clinically used SERMs.
are due to the key alkylamino chain and the asymmetric
hydrophobic part. As the isomers easily isomerize between the E
and Z forms, problems arise in the synthesis, purification, and
preservation of the SERMs.8 Indeed, 4-hydroxytamoxifen, an active
⇑
Corresponding author.
E-mail address: k-ohta@tohoku-pharm.ac.jp (K. Ohta).
http://dx.doi.org/10.1016/j.bmc.2016.01.035
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
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M. Sato et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
OH
OH
tion of the cyclohexane ring of 1a into the 3,3,5,5-tetramethylcy-
clohexane ring enhanced ER binding affinity because of the
favorable hydrophobic interaction with the ER ligand binding
a
a
domain (LBD). 3,3,5,5-Tetramethylcyclohexane derivative 1b acts
as an ER partial agonist, whereas compound 1a is an ER agonist.
The cyclohexyl moiety in 1a and 1b plays an important role in the
hydrophobic interactions with the hydrophobic amino acid residues
HO
HO
1a
1b
of ER
a LBD, but is not easy tunable. A multi-tunable symmetric
RBA for ERα = 6.71
RBA for ERα = 28.4
structure would be a very attractive tool for ER ligand studies
because it would enable detailed SAR studies and not require isomer
separation, and its physicochemical properties would be control-
lable for optimum ADME. Therefore, we focused on the discovery
of readily available ER modulators with multi-tunable symmetric
structures and designed novel ER ligand candidates 2a–2d, 3a,
and 3b containing a piperidine ring (Fig. 3). In this paper, we
describe the synthesis of those compounds, their biological activi-
ER agonist
ER partial agonist
Figure 2. Chemical structures of cyclofenil derivatives 1a and 1b.
OH
OH
ties, such as ER
a binding affinity and ER-dependent proliferation
of MCF-7 cell line, and a metabolic study in rat liver S9 fractions.
N
N
HO
R
HO
R
2
3
R = H, CH3, COCH3, CO2Et
2. Results and discussion
2.1. Chemistry
Figure 3. Novel ER modulator candidates 2 and 3 having a tunable piperidine ring.
A structure common to compounds 2a–2d was synthesized from
piperidinone derivatives and 4,40-dimethoxybenzophenone by
means of the McMurry coupling.16 Scheme 1 summarizes the syn-
thesis of N-substituted piperidine derivatives 2a–2d. Commercially
available N-ethoxycarbonyl piperidin-4-one 4 was reacted with
4,40-dimethoxybenzophenone to afford 5 in 90% yield, which was
then demethylated with BBr3 to afford 2d in 78% yield. McMurry
coupling product 5 was hydrolyzed with KOH and then subjected
to spontaneous decarboxylation to afford key intermediate 6 in
90% yield. Compound 6 was transformed into 2a with BBr3 in 54%
yield. N-Methylated derivative 2b was obtained by reductive
amination using paraformaldehyde and NaBH4 in trifluoroethanol,
metabolite of tamoxifen, easily isomerizes from the active Z form
to the inactive E form in solution and in vitro studies (Fig. 1).9 In
this regard, novel SERMs having other skeletons, such as benzoth-
iophene,10 dihydronaphthalene,11 benzopyrane,12 and steroid,13
have been developed and used as antagonistic SERMs.
On the other hand, cyclofenil shows weak estrogenic activity
because of the absence of the alkylamino chain, and thus it has
been developed as an agonistic SERM (Fig. 1).14 Cyclofenil has a
C-2 symmetric structure and no E and Z isomer. Recently, we have
reported an SAR study of the cyclohexane ring of cyclofenil
reductant 1a, in which the diphenylmethane skeleton and the
cyclohexane ring are linked by a single bond (Fig. 2).15 Transforma-
OH
OMe
CO2Et
a
b
N
O
4
N
N
HO
CO2Et
MeO
CO2Et
5
2d
c
OMe
N
MeO
H
6
d, b
b
e, b
OH
OH
OH
N
N
HO
COCH3
HO
H
2c
2a
N
HO
Me
2b
Scheme 1. Synthesis of the designated derivatives 2a–2d. Reagents and conditions: (a) TiCl4, Zn, 4,40-dimethoxybenzophenone, THF, 90%; (b) BBr3, CH2Cl2, 23–90%; (c) KOH,
EtOH, 90%; (d) Ac2O, 93%; (e) (CHO)n, NaBH4, CF3CH2OH, 87%.
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M. Sato et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
OH
1.1
3b
1.0
0.9
0.8
H
COCH3
a
b
b, c
N
N
O
O
N
HO
COCH3
8
7
0.7
0.6
0.5
0.4
0.3
3b
OMe
OH
-13 -12 -11 -10
-9
-8
-7
-6
-5
c
Log concentration (M)
N
H
N
Figure 5. Dose–response curve of 3b in the MCF-7 cell proliferation assay. MCF-7
cells were incubated with 3b (1 ꢀ 10ꢁ6 to 1 ꢀ 10ꢁ12 M) for 5 days, and the results
are shown by relative cell number with the value for E2 taken as 1. Cell proliferation
assay was performed in triplicate (n = 3). Values are means SD of separate
experiments.
MeO
HO
H
9
3a
Scheme 2. Synthesis of 2,2,6,6-tetramethylpiperidine derivatives 3a and 3b.
Reagents and conditions: (a) Ac2O, 88%; (b) TiCl4, Zn, 4,40-dimethoxybenzophenone,
THF, 48–75%; (c) BBr3, CH2Cl2, 75–82%.
Testosterone
2c
3b
120
100
80
1.2
E2
2a
1.0
2b
2c
2d
3a
3b
0.8
0.6
0.4
0.2
60
40
20
0
10
20
30
40
50
Time (min)
0.1
1
10
100
1000
0
Figure 6. Percentages of 2c and 3b remaining in rat liver S9 fractions. The
percentages were estimated from peak area ratios of corticosterone as an internal
standard to the tested compounds.
Relative concentration
Figure 4. Binding affinities of test compounds 2a–2d, 3a, and 3b. Competitive
binding assay of compounds 2a–2d, 3a, and 3b with [3H]E2 (4 nM) for ER
. Binding
a
assays of the test compounds (0.4–4 lM) were conducted in the presence of
form in this assay buffer. Therefore, the extremely low binding
affinity of 2a and 2b might be caused by the polarity around the
protonated nitrogen atom on the piperidine ring rather than the
basicity of the nitrogen atom. N-Acetylated derivative 2c also
showed low binding affinity because of the high polarity (CLogP:
1.72).18 Compound 2d having an ethyl carbamate group dose-
[6,7-3H]17b-estradiol (4 nM). The assays were performed in duplicate (n = 2).
followed by deprotection with BBr3 in 20% yield over two steps.
N-Acetylated derivative 2c was synthesized by acetylation and
subsequent demethylation in 84% yield over two steps.
The McMurry coupling of 4,40-dimethoxybenzophenone with
N-acetyl tetramethylpiperidinone 8, which was obtained through
the acetylation of commercially available 2,2,6,6-tetramethyl-
piperidin-4-one 7 followed by deprotection with BBr3, afforded
3b in 38% yield over three steps (Scheme 2). To prepare 3a, we tried
to remove the N-acetyl group from 3b. However, deacetylation did
not proceed under both acidic and basic conditions due to the
steric hindrance of the four methyl groups adjacent to the nitrogen
atom of the piperidine ring. Therefore, direct McMurry coupling of
2,2,6,6-tetramethylpiperidine with 4,40-dimethoxyphenylben-
zophenone was carried out to afford corresponding coupling
product 9 in 75% yield. Compound 9 was transformed into 3a by
demethylation with BBr3 in 82% yield.
dependently bound to ERa and showed higher ERa binding affinity
than 2a–2c owing to the increase in hydrophobicity caused by the
ethyl group. Indeed, compound 2d had the highest CLogP value at
3.90 among 2a–2d.18 In addition, ethyl group of 2d would be
accommodated in the hydrophobic pocket of ERa. The steric
hinderance of the four methyl groups in 3a and 3b obscured the
physicochemical properties of the nitrogen atom, such as basicity
and hydrophilicity. Thus, 3a and 3b would show higher binding
affinity than corresponding normal piperidine derivatives 2a and
2c, if the sterically bulky tetramethyl moiety were tolerated in
the hydrophobic space of ER
of 3a was low, it seemed that the four methyl groups improved
incompatibility between the piperidine ring and ER LBD. Interest-
a LBD. Although the binding affinity
a
ingly, compound 3b showed more than 100 times higher binding
affinity than corresponding piperidine derivative 2c. The four
methyl groups exerted a strong positive effect on the binding of
2.2. Biological evaluations
the piperidine derivatives to ER
Next, cell functional assays of 3b were performed using the
MCF-7 cell line that showed ER
-dependent growth.17 Compound
3b demonstrated high cell proliferation inducing activity in a dose
dependent manner (Fig. 5), but no inhibition of cell proliferation
activity inducing by 0.1 nM of E2 (data not shown). The EC50 value
a LBD.
The ER
evaluated in a competitive binding assay using tritium-labeled E2
and recombinant hER
(Fig. 4).17 Unsubstituted piperidine deriva-
tive 2a showed no binding affinity for ER LBD, whereas N-methyl
and N-acetyl derivatives 2b and 2c bound very weakly to ER LBD.
Aliphatic amines 2a and 2b would be present in the protonated
a-binding affinity of the synthesized compounds was
a
a
a
a
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M. Sato et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
of 3b estimated from the sigmoidal dose response curve was
0.22 nM. The maximal efficacy for MCF-7 cell proliferation induc-
ing activity of 3b was similar to that of E2. Interestingly, 3b acted
as ER full agonist, unlike the lead compound 1b. We suggested that
ER full agonist activity of 3b would be caused by the nitrogen atom
or the spatial configuration of 2,2,6,6-tetramethylpiperidine ring.
To examine the effect of the four methyl groups on metabolic
stability, we measured the elimination rates of piperidine deriva-
tives 2c and 3b in rat liver S9 fractions (Fig. 6).19 The percentage
(%) of unmetabolized test compounds was estimated from the peak
areas of 2c and 3b in HPLC analyses. Approximately 80% of testos-
terone used as a positive control was metabolized at 50 min under
the assay conditions. Compounds 2c and 3b showed low metabolic
rates; approximately 10% and 20% were metabolized at 50 min,
respectively. Compound 3b was more rapidly metabolized than
2c and the tetramethyl moiety had no effect on the metabolic sta-
bility of these compounds. It seems that the piperidine rings of 2c
and 3b are quite stable under the assay conditions.
(2.37 mL, 21.6 mmol) under an argon (Ar) atmosphere. A mixture
was refluxed for 2.5 h and cooled to room temperature. To the
mixture was added a solution of 4,40-dimethoxybenzophenone
(1.42 g, 5.86 mmol) and ethyl 4-oxopiperidine-1-carboxylate
(1.01 g, 5.90 mmol) in 20 mL of dry THF, and it was refluxed for
12 h. The reaction mixture was poured into saturated NaHCO3
aqueous solution. Ether was added to the aqueous solution with
vigorous stirring, and insoluble materials were filtered off through
Celite. The filtrate was extracted with ether, dried over MgSO4, and
concentrated. The residue was purified by column chromatography
on silica gel with 40:1 n-hexane/AcOEt to afford the title com-
pound 5 (2.23 g, 99%) as a yellow liquid; 1H NMR (395 MHz, CDCl3)
d (ppm) 7.01 (d, 2H, J = 7.9 Hz), 6.82 (d, 2H, J = 7.9 Hz), 4.14 (q, 2H,
J = 7.9 Hz), 3.79 (s, 6H), 3.49 (t, 4H, J = 4.0 Hz), 2.35 (t, 4H,
J = 4.0 Hz), 1.26 (t, 3H, J = 7.9 Hz); 13C NMR (100 MHz, CDCl3) d
(ppm) 158.1, 155.5, 136.5, 134.8, 132.9, 130.7, 113.3, 61.2, 55.0,
44.8, 31.5, 14.6; MS (EI) m/z 381 (M+, 100%).
4.2.2. Methyl 4-[(4-methoxyphenyl)(piperidin-4-ylidene)
methyl]phenoxide (6)
3. Conclusion
A mixture of 5 (7.54 g, 19.8 mmol) and potassium hydroxide
(92.9 g, 1.66 mol) in 300 mL of ethanol was refluxed for 6 h under
an Ar atmosphere. The solvent was evaporated, water (50 mL) was
added to the residue, and then the aqueous mixture was extracted
with AcOEt. The organic phase was dried over Na2SO4 and
concentrated to afford the title compound 6 (5.52 g, 90%) as a
yellow solid. 1H NMR (395 MHz, CDCl3) d (ppm) 7.02 (d, 4H,
J = 7.9 Hz), 6.82 (d, 4H, J = 7.9 Hz), 3.79 (s, 6H), 2.90 (t, 4H,
J = 4.0 Hz), 2.33 (t, 4H, J = 4.0 Hz); 13C NMR (100 MHz, CDCl3) d
(ppm) 157.9, 136.3, 135.2, 130.9, 129.2, 113.3, 55.1, 48.5, 33.5;
MS (EI) m/z 309 (M+), 267 (100%).
In conclusion, we designed and synthesized novel tunable ER
modulators having the 4,40-(piperidin-4-ylidenemethylene)
bisphenol structure. In contrast to the unsubstituted piperidine
ring that was unfavorable for ER
a LBD, the introduction of
hydrophobic substituents on the nitrogen atom or the four methyl
groups adjacent to the nitrogen atom improved the binding affinity
of the piperidine ring for ER
a LBD. Compound 3b showed high
binding affinity for ER , high MCF-7 cell proliferation inducing
a
activity, and good metabolic stability in rat liver S9 fractions. We
are of the opinion that 2,2,6,6-tetramethylpiperidine ring is a
promising core skeleton for ER modulator studies. Further investi-
gations focusing on the chemical modification of the substituents
on the nitrogen atom of the 2,2,6,6-tetramethylpiperidine ring,
the introduction of alkylmino chains on the phenol group for the
development of ER antagonists and SERMs, and the biological eval-
uation of ER subtype and tissue selectivity, are in progress.
4.2.3. 4-[(4-Hydroxyphenyl)(piperidin-4-ylidene)methyl]phenol
(2a)
To a solution of 6 (2.95 g, 9.54 mmol) in 20 mL of CH2Cl2 was
added 1 M of BBr3 solution (17.5 mL, 17.5 mmol) at 0 °C under Ar
atmosphere. The mixture was stirred for 12 h at 0 °C, the solvent
was removed, water was added to the residue, and the mixture
was extracted with AcOEt. The organic phase was dried over
Na2SO4 and concentrated to afford the title compound 2a (1.44 g,
54%) as a yellow solid; colorless cubes (MeOH); mp 205–207 °C;
1H NMR (395 MHz, CD3OD) d (ppm) 6.88 (d, 4H, J = 7.9 Hz), 6.69
(d, 4H, J = 7.9 Hz), 2.83 (t, 4H, J = 4.0 Hz), 2.32 (t, 4H, J = 4.0 Hz);
13C NMR (100 MHz, CD3OD) d (ppm) 157.2, 137.6, 135.3, 134.3,
131.9, 115.8, 33.6, 32.2; MS (EI) m/z 281 (M+, 100%); Anal. Calcd
for C18H19NO2: C, 76.84; H, 6.81; N, 4.98. Found: C, 76.84; H,
6.81; N, 4.91.
4. Experimental section
4.1. General
Melting points were determined with a Yanaco micro melting
point apparatus and were uncorrected. 1H NMR and 13C NMR spec-
tra were recorded with JEOL JNM-LA-400 spectrometers. Chemical
shifts for 1H NMR spectra were referenced to tetramethylsilane
(0.0 ppm) as an internal standard. Chemical shifts for 13C NMR
spectra were referenced to residual 13C present in deuterated sol-
vents. The splitting patterns are designed as follows: s (singlet), d
(doublet), t (triplet), and q (quartet). Mass spectra were recorded
on a JEOL JMS-DX-303 spectrometer. Elemental analyses were per-
formed with a Perkin Elmer 2400 CHN spectrometer. Column chro-
matography was carried out using Fuji Silysia silica gel BW-80S
and TLC was performed on Merck silica gel F254. Reagents were
purchased from Wako Pure Chemical Industries, Ltd, Sigma-Aldrich
Co., and Tokyo Chemical Industry, Ltd (TCI). All solvents were of
reagent quality, purchased commercially, and were used without
further purification.
4.2.4. 4-[(4-Hydroxyphenyl)(N-methylpiperidin-4-ylidene)
methyl]phenol (2b)
A solution of paraformaldehyde (191 mg, 6.4 mmol) in 30 mL of
trifluoroethanol was stirred at 35–40 °C. To the stirred solution
was added 6 (1.18 g, 3.88 mmol), and NaBH4 (185 mg, 4.89 mmol)
was added 5 min later. After 30 min, the mixture was filtered
through Celite and the filtrate was concentrated. The residue was
purified by column chromatography on silica gel with AcOEt to
afford N-methylated compound (1.09 g, 87%) as a yellow solid.
1H NMR (395 MHz, DMSO) d (ppm) 6.96 (d, J = 7.9 Hz, 4H), 6.85
(d, 4H, J = 7.9 Hz), 3.72 (s, 6H), 2.33 (t, 4H, J = 4.0 Hz), 2.25 (t, 4H,
J = 4.0 Hz), 2.15 (s, 3H); 13C NMR (100 MHz, DMSO) d (ppm)
159.8, 137.7, 136.2, 133.3, 131.8, 114.4, 58.0, 55.7, 45.9, 32.1; MS
(EI) m/z 323 (M+, 100%). The N-methylated compound was
transformed into 2b by the same method that described the
synthesis of 2a; 23% yield; colorless needles (MeOH–CHCl3)
mp 255–257 °C; 1H NMR (395 MHz, CD3OD) d (ppm) 6.99 (d, 4H,
J = 8.0 Hz), 6.70 (d, 4H, J = 8.0 Hz), 2.52 (t, 4H, J = 8.0 Hz), 2.41
4.2. Synthesis
4.2.1. Methyl 4-[(4-methoxyphenyl)(N-ethyloxy-carbonylpiperi-
din-4-ylidene)methyl]phenoxide (5)
To a stirred suspension of Zn powder (2.83 g, 43.2 mmol) in
30 mL of dry THF was added slowly titanium(IV) tetrachloride
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M. Sato et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
(t, 4H, J = 4.0 Hz), 2.32 (s, 3H); 13C NMR (100 MHz, CD3OD) d (ppm)
157.1, 138.0, 135.3, 132.9, 131.9, 115.7, 58.1, 46.1, 32.2; MS (EI)
m/z 295 (M+, 100%).; Anal. Calcd for C19H21NO2: C, 76.45; H,
7.17; N, 4.74; Found: C, 76.45; H, 7.15; N, 4.69.
110–112 °C; 1H NMR (395 MHz, CD3OD) d (ppm) 6.96 (d, 4H,
J = 8.0 Hz), 6.70 (d, 4H, J = 8.0 Hz), 2.18 (s, 4H), 1.18 (s, 12H); 13C
NMR (100 MHz, CD3OD) d (ppm) 182.2, 176.4, 157.2, 140.4,
134.7, 131.9, 115.7, 58.4, 46.1, 30.3, 28.4; MS (EI) m/z 379 (M+),
224 (100%). Anal. Calcd for C22H27NO2 0.1 H2O: C, 77.88; H, 8.08;
N, 4.13. Found: C, 77.92; H, 8.19; N, 4.10.
4.2.5. 4-[(4-Hydroxyphenyl)(N-acetylpiperidin-4-ylidene)
methyl]phenol (2c)
A mixture of 6 (218 mg, 0.71 mmol) and 3 mL of acetic anhy-
dride was heated at 100 °C for 6 h. After cooling to room tempera-
ture, 10% NaOH aqueous solution was added and stirred. The
mixture was extracted with CH2Cl2, washed with brine, dried over
Na2SO4, and then concentrated. The residue was purified by
column chromatography on silica gel with 2:1 n-hexane/AcOEt to
afford N-acetylated compound (230 mg, 93%) as a yellow solid.
1H NMR (395 MHz, CD3OD) d (ppm) 7.02 (d, J = 7.9 Hz, 4H),
6.85 (d, 4H, J = 7.9 Hz), 3.77 (s, 6H), 3.60 (t, 2H, J = 4.0 Hz), 3.55
(t, 2H, J = 4.0 Hz), 2.43 (t, 2H, J = 4.0 Hz), 2.35 (t, 2H, J = 4.0 Hz),
2.11 (s, 3H); 13C NMR (100 MHz, CDCl3) d (ppm) 168.7, 158.0,
136.7, 134.5, 132.0, 130.5, 113.2, 54.9, 47.3, 42.7, 31.8; MS (EI)
m/z 351 (M+, 100%). The N-acetylated compound was transformed
into 2c by the same method that described the synthesis of 2a; 22%
yield; colorless leaflets (MeOH–CHCl3) mp 255–257 °C; 1H NMR
(395 MHz, CD3OD) d (ppm) 6.99 (d, 4H, J = 8.0 Hz), 6.70 (d, 4H,
J = 8.0 Hz), 2.52 (t, 4H, J = 8.0 Hz), 2.41 (t, 4H, J = 4.0 Hz), 2.32 (s,
3H); 13C NMR (100 MHz, CD3OD) d (ppm) 157.1, 138.0, 135.3,
132.9, 131.9, 115.7, 58.1, 46.1, 32.2; MS (EI) m/z 295 (M+, 100%).
Anal. Calcd for C20H21NO3: C, 74.28; H, 6.55; N, 4.33; Found: C,
74.28; H, 6.58; N, 4.25.
4.2.10. 4-[(4-Hydroxyphenyl)(2,2,6,6-tetramethyl-N-acetylpipe-
ridin-4-ylidene)methyl]phenol (3b)
The product obtained from McMurry coupling reaction of com-
pound 9 with 4,40-dimethoxybenzophenone was transformed into
3b by the same method that described the synthesis of 2a; 43%
yield over two steps; colorless cubes (Et2O) mp 138–140 °C; 1H
NMR (395 MHz, CD3OD) d (ppm) 6.93 (d, 4H, J = 7.9 Hz), 6.72
(d, 4H, J = 7.9 Hz), 2.64 (s, 4H), 2.17 (s, 3H), 1.45 (s, 12H); 13C
NMR (100 MHz, CD3OD) d (ppm) 182.2, 176.4, 157.2, 140.4,
134.7, 131.9, 115.7, 58.4, 46.1, 30.3, 28.4; MS (EI) m/z 379 (M+),
224 (100%). Anal. Calcd for C24H29NO3: C, 75.96; H, 7.70; N, 3.69.
Found: C, 75.91; H, 7.83; N, 3.73.
4.3. ERa binding assay
The ligand binding affinity of ER
a
was determined by the previ-
was diluted with an assay
ously reported method.17 Briefly, ER
a
buffer, which contains 20 mM Tris–HCl, 0.3 M NaCl, 10 mM 2-mer-
captoethanol, 0.2 mM phenylmethylsulfonyl fluoride, 1 mM EDTA,
and incubated with 4 nM [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 assay buffer. The membrane was washed
twice with buffer, and then with 25% EtOH in distilled water.
Radioactivity that remained on the membrane was measured in
Atomlight using a liquid scintillation counter.
4.2.6. 4-[(4-Hydroxyphenyl)(N-ethyloxycarbonyl-piperidin-4-
ylidene)methyl]phenol (2d)
Compound 2d was prepared by the same method that described
the synthesis of 2a; 90% yield; a yellow prisms (CH2Cl2) mp 105 °C;
1H NMR (395 MHz, CDCl3) d (ppm) 6.96 (d, 4H, J = 7.9 Hz), 6.75 (d,
4H, J = 7.9 Hz), 4.14 (q, 2H, J = 4.0 Hz), 3.48 (t, 4H, J = 4.0 Hz), 2.34
(t, 4H, J = 4.0 Hz), 1.26 (t, 3H, J = 7.9 Hz); 13C NMR (100 MHz,
CD3OD) d (ppm) 157.3, 157.2, 139.0, 135.2, 133.0, 131.9, 115.8,
62.7, 46.4, 32.6, 15.0; MS (EI) m/z 353 (M+, 100%). Anal. Calcd for
4.4. MCF-7 cell proliferation assay
The human breast adenocarcinoma cell line, MCF-7 was
routinely cultivated in DMEM supplemented with 10% FBS,
100 IU/mL penicillin and 100 mg/mL streptomycin at 37 °C in a
5% CO2 humidified incubator. Before an assay, MCF-7 cells were
switched to DMEM (low glucose phenol red-free supplemented
with 5% FBS, 100 UI/mL penicillin and 100 mg/mL streptomycin).
Cells were trypsinized from the maintenance dish with phenol
red-free trypsin–EDTA and seeded in a 96-well plate at a density
C21H23NO4: C, 71.37; H, 6.56; N, 3.96; Found: C, 71.11; H, 6.59;
N, 3.93.
4.2.7. N-Acetyl-2,2,6,6-tetramethyl-4-piperidone (8)
A
mixture of 2,2,6,6-tetramethyl-4-piperidone (4.91 g,
31.6 mmol) and 20 mL of acetic anhydride was heated at 100 °C
for 7 h. Solvent was removed, 10% NaOH aqueous solution was
added, and the mixture was stirred. The mixture was extracted
with CH2Cl2, washed with brine, dried over Na2SO4, and then con-
centrated. The residue was purified by column chromatography on
silica gel with 3:1 n-hexane/AcOEt to afford the title compound 8
(5.49 g, 88%) as a colorless liquid. 1H NMR (395 MHz, CDCl3) d
(ppm) 2.59 (s, 4H), 2.23 (s, 3H), 1.54 (s, 12H); MS (EI) m/z 197
(M+), 140 (100%).
of 2000 cells per final volume of 100
5% FBS, 100 UI/mL penicillin and 100 mg/mL streptomycin. After
24 h, the medium was exchanged into 90 L of fresh DMEM and
10 L of the drug solution, supplemented with serial dilutions of
lL DMEM supplemented with
l
l
3b or DMSO as dilute control in the presence or absence of 1 nM
E2, was added to triplicate microcultures. Cells were incubated
for 5 days, and medium with 3b or DMSO as dilute control in the
presence or absence of 1 nM E2 was exchanged once after 3 days.
At the end of the incubation time, proliferation was evaluated by
using the WST-8. 10 nM of WST-8 was added to microcultures
and cells were incubated for 4 h. The absorbance at 450 nm was
measured. This parameter relates to the number of living cells in
the culture.
4.2.8. Methyl 4-[(4-methoxyphenyl)(2,2,6,6-tetramethylpiperi-
din-4-ylidene)methyl]phenoxide (9)
Compound 9 was prepared by the same method that described
the synthesis of 5; 75% yield; 1H NMR (395 MHz, CD3OD) d (ppm)
7.07 (d, 4H, J = 8.0 Hz), 6.82 (d, 4H, J = 8.0 Hz), 3.78 (s, 6H), 2.14
(s, 4H), 1.18 (s, 12H); 13C NMR (CD3OD) d (ppm) 157.8, 137.3,
135.4, 132.4, 130.2, 113.4, 55.1, 52.8, 43.7, 31.2; MS (EI) m/z 365
(M+), 98 (100%).
4.5. Metabolism study of compounds 2c and 3b in rat liver S9
fractions
For the evaluation of metabolic stability, 40 lL of compound
4.2.9. 4-[(4-Hydroxyphenyl)(2,2,6,6-tetramethylpiperidin-4-
ylidene)methyl]phenol (3a)
Compound 3a was prepared by the same method that described
the synthesis of 2a; 82% yield; colorless cubes (MeOH–CHCl3) mp
was incubated with 1 mg/mL pooled rat liver S9 fractions in
0.1 M potassium phosphate buffer (pH 7.4) containing 3.3 mM
MgCl2, 1.3 mM NADP+, 3.3 mM glucose-6-phosphate, and
0.4 U/mL glucose-6-phosphatedehydrogenase at 37 °C for 0, 5, 10,
Please cite this article in press as: Sato, M.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.01.035

6
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This research was supported by a Grant-in-Aid of Strategic
Research Program for Private Universities (2010–2014), a Grant-
in-Aid for Young Scientists (B) (No. 21790116), and a Grant-in-
Aid for Scientific Research (C) (No. 26460151) from the Ministry
of Education, Culture, Sports, Science, and Technology, Japan.
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Please cite this article in press as: Sato, M.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.01.035