Design and synthesis of novel selective estrogen receptor degradation inducers based on the diphenylheptane skeleton

Article abstract of DOI:10.1039/c6md00553e

Estrogen receptors (ERs) are a family of nuclear receptors (NRs) that regulate physiological effects such as reproduction and bone homeostasis. It has been reported that approximately 70% of human breast cancers are hormone-dependent and ERα-positive. Rec

Full text of DOI:10.1039/c6md00553e

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RESEARCH ARTICLE
Design and synthesis of novel selective estrogen
receptor degradation inducers based on the
diphenylheptane skeleton†‡
a
Cite this: DOI: 10.1039/c6md00553e
Takashi Misawa, Takuma Fujisato,a Yasunari Kanda,a Nobumichi Ohoka,a
*
Takuji Shoda,a Momoko Yorioka,a Makoto Makishima,b Yuko Sekino,a
a
a
a
*
Mikihiko Naito, Yosuke Demizu and Masaaki Kurihara
Estrogen receptors (ERs) are a family of nuclear receptors (NRs) that regulate physiological effects such as
reproduction and bone homeostasis. It has been reported that approximately 70% of human breast cancers
are hormone-dependent and ERα-positive. Recently, novel anti-breast cancer drugs based on different
mechanisms of action have received significant attention. In this article, we have designed and synthesized
a selective ER degradation inducer based on the diphenylheptane skeleton. Western blotting analysis re-
vealed that PBP-NC10 degraded ERα through the ubiquitin–proteasome system. We also performed com-
putational docking analysis to predict the binding mode of PBP-NC10 to ERα.
Received 5th October 2016,
Accepted 17th November 2016
DOI: 10.1039/c6md00553e
www.rsc.org/medchemcomm
tion using small molecules have been reported such as
PROTAC,9 SNIPER,10 and hydrophobic tag systems.11
Introduction
Estrogen receptor (ER), a member of the nuclear receptor
(NR) family of transcription factors, relates to several biologi-
cal functions such as reproduction and bone physiology.1 The
binding of 17β-estradiol (E2) and its metabolites, estrone and
estriol, to the ER ligand binding domain (LBD) induces the
folding of Helix 12 (H12) and the subsequent interaction with
co-activators along the hydrophobic groove, resulting in ER-
mediated target gene transcriptions.2 The ER is divided into
two subtypes, ERα and ERβ, which are encoded by distinct
genes but are highly conserved in their LBD.3 It is well known
that approximately 70% of human breast cancers are
hormone-dependent and ERα-positive.4 Therefore, several
ERα-mediated transcriptional inhibitors such as tamoxifen
(ERα antagonist)5 and raloxifene6 are widely used for ERα-
positive breast cancer therapy (Fig. 1a).7 Tamoxifen is metabo-
lized to 4-hydroxytamoxifen (4-OHT) with a much higher bind-
ing affinity for ERα, and 4-OHT exerts its anti-estrogenic ac-
tions.8 However, it has been problematic that long-term
administration of tamoxifen might cause drug resistance in
breast cancer cells and also increases the risk factors for uter-
ine cancer because of its agonistic effects on the uterus.
Therefore, novel anti-breast cancer drugs based on different
mechanisms of action have received significant attention. Re-
cently, several methods to induce targeted protein degrada-
Among these, selective ER downregulators (SERDs) have
been discussed as anti-breast cancer drugs. Fulvestrant
(ICI182,780) and ICI164,384 (Fig. 1b), which are E2 deriva-
tives bearing a long alkyl chain on the 7α-position of E2, are
representative SERDs and are capable of inducing ER degra-
dation through the ubiquitin–proteasome system (UPS).12
Based on the X-ray structure analysis of ICI164,384 bound to
the ER-LBD, the ER-degradation mechanism of ICI164,384 is
speculated to involve binding of the long alkyl chain of
ICI164,384 to the hydrophobic groove of ER, sterically
preventing H12 folding in ER and the co-activator interaction
a National Institute of Health Sciences Setagaya, Tokyo, 158-8501, Japan.
E-mail: misawa@nihs.go.jp, masaaki@nihs.go.jp
b Nihon University Itabashi, Tokyo, 173-8610, Japan
† The authors declare no competing interests.
Fig. 1 (a) Chemical structures of 17β-estradiol, anti-estrogenic com-
‡ Electronic supplementary information (ESI) available. See DOI: 10.1039/c6md00553e
pounds and (b) selective estrogen receptor downregulators.
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with ER, resulting in destabilization of ER and subsequent
degradation.13
Previously, we reported that 4-OHT derivatives bearing
long alkyl chains (Fig. 1b) induced ER degradation similar to
fulvestrant.14 However, these synthesized compounds were
obtained and assayed as a mixture of geometric isomer E and
Z forms. It has been reported that the triphenylethylene skel-
eton of 4-OHT is easily isomerized from the active Z form to
an inactive E form in solution.15 Therefore, these findings
raise the possibilities that its isomerization decreases the ER-
antagonistic and ER-downregulating activities. On the other
hand, Hashimoto and Ishikawa et al. recently reported that
the diphenylalkane skeleton acted as a steroid skeleton
mimic and could be a key to the development of several NR
ligands.16 Among these, the ER antagonist PBP based on the
diphenylheptane skeleton has potent anti-estrogenic activity
with an IC50 value of 4.5 nM from a reporter gene assay.17
Moreover, the diphenylalkane skeleton has an advantage of
good chemical stability and for short-step synthesis over the
triphenylethylene skeleton of 4-OHT. Herein, we described
the development of PBP derivatives bearing several long alkyl
chains on the hydroxy group (Fig. 2), and evaluated their bio-
logical properties such as ER degradation, binding affinity,
transcriptional activity and anti-proliferation activity.
Scheme 1 Synthesis of compounds PBP-NH2, PBP-tam, PBP-C2 to
PBP-C12, and PBP-NC6 to PBP-NC16. Reagents and conditions: (a)
MsOH, rt, 3 days, 59%; (b) R-I (R = ethyl, n-hexyl, n-octyl, n-decyl,
n-dodecyl), K2CO3, MeCN, 60–100 °C, 16–47%; (c) 1,2-dichloroethane,
K2CO3, MeCN, 48 h, 19%; (d) R-NH2 (R = n-hexyl, n-octyl, n-decyl,
n-dodecyl, n-hexadecyl), MeCN, microwave, 120 °C, 4 h, 20–42%; (e)
benzyl (2-bromoethyl)carbamate or 2-chloro-N,N-dimethylethylamine
Cs2CO3, NaI, DMF, 100 °C, 21 h, 6%; (f) PdIJOH)2, H2, MeOH, rt, 26 h,
25%.
First, we investigated the effects of the synthesized com-
pounds on ERα-downregulating activity by western blotting
analysis using MCF-7 breast cancer cells. The potency of the
ERα-downregulating activity is described as the ratio of ERα
to standard proteins. As shown in Fig. 3, ERα downregulation
was observed by treatment with PBP-NC8, PBP-NC10, and
PBP-NC12 with amino alkyl chains (ERα downregulation ratio
at 10 μM; PBP-NC8: 69%, PBP-NC10: 32%, and PBP-NC12:
68%). In particular, the ERα expression level was dose-
dependently reduced in the presence of PBP-NC10 (ERα
downregulation ratio at 1 μM: PBP-NC10: 58%). On the other
hand, PBP-C2–C12, with simple alkyl chains, PBP-NC6 and
PBP-NC16, with short or long alkyl chains, and PBP-tam, with
a dimethylamino group, did not show ERα-downregulating
activity (Fig. 3a and b). These results indicated that the nitro-
gen atom and the optimal length of the alkyl chain of PBP-
NC derivatives play a pivotal role in the ERα-downregulating
activity.
Results and discussion
The synthetic routes to the designed compounds PBP-
C2–C12, PBP-NC6–NC16, PBP-NH2, and PBP-tam are shown
in Scheme 1. 4-Heptanone and o-cresol were combined under
acidic conditions to give the diphenylheptane skeleton
(PBP, 3). Alkylation of the 4-hydroxy group of PBP with the
corresponding 1-bromoalkanes afforded alkylated products
PBP-C2–C12. Compounds PBP-NC6–NC16 were synthesized
by mono-alkylation of PBP with 1,2-dichloroethane and subse-
quent alkylation of alkylamines with compound 4. After alkyl-
ation of PBP with benzyl (2-bromoethyl)carbamate, the Cbz
group was removed using PdIJOH)2 under H2 atmosphere to
give PBP-NH2. PBP-tam was obtained by the treatment of PBP
with 2-chloro-N,N-dimethylethylamine.
Next, we investigated the selectivity of PBP-NC10 over
other NRs such as the androgen receptor (AR), vitamin D re-
ceptor (VDR), and aryl hydrocarbon receptor (AhR) because
the diphenylalkane skeleton could act as several NR li-
gands.16 Moreover, we also performed the western blotting
analysis in the presence of proteasome inhibitor MG132 to
investigate whether ERα downregulation by PBP-NC10 was
mediated by UPS as hypothesized. As shown in Fig. 4, ERα
was downregulated by treatment with PBP-NC10 in a dose-
dependent manner, whereas the other NR expression levels
were unaffected. Furthermore, reductions in the expression
levels of ERα induced by PBP-NC10 were suppressed by
MG132, indicating that PBP-NC10 induced proteasomal
degradation.
Fig. 2 Chemical structures of the diphenylheptane skeleton (PBP) and
designed compounds PBP-NH2, PBP-tam, PBP-C2 to PBP-C12, and
PBP-NC6 to PBP-NC16 in this study.
Next, we investigated the effects of the synthesized com-
pounds on the binding affinity and transcriptional activity
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Fig. 3 (a and b) Western blotting analysis of estrogen receptor (ER) α levels in MCF-7 cells after 6 h of incubation with the synthesized com-
pounds. (c) Quantification of the levels of ERα is provided in a histogram. Data are expressed as means S.E. of three independent experiments
(*P < 0.05 versus control).
against ERα. To evaluate the ability of the compounds to
bind to ERα, a fluorescence polarization (FP)-based competi-
tive binding assay was conducted using a PolarScreen nuclear
receptor competitor assay kit (Life Technologies).14 As
reported previously, 4-OHT and PBP strongly bound to ERα
with IC50 values of 5.6 nM and 7.3 nM, respectively. Table 1
shows that PBP-C2 to PBP-C8 exhibited moderate binding af-
finity to ERα (IC50 = 40–400 nM), whereas PBP-C10 and PBP-
C12 did not bind to ERα. These data indicated that the intro-
duction of longer alkyl chains rather than an n-octyl group
lowered the binding affinity. On the other hand, PBP-NC8 to
PBP-NC12 showed pronounced ERα binding affinity (IC50
values of 2.4 nM for PBP-NC8, 4.9 nM for PBP-NC10, and 4.9
nM for PBP-NC12), which was similar to that of 4-OHT (IC50
Table 1 ERα binding affinity of PBP derivatives
Entry
Binding affinity IC50 (nM)
4-OHT
PBP
PBP-C2
5.6
7.3
40
PBP-C4
87
PBP-C6
PBP-C8
168
400
NAa
NAa
313
78
PBP-C10
PBP-C12
PBP-tam
PBP-NH2
PBP-NC6
PBP-NC8
PBP-NC10
PBP-NC12
PBP-NC16
78
2.4
4.9
4.9
20
Fig. 4 The effects of PBP-NC10 on nuclear receptor levels in MCF-7
cells with or without proteasome inhibitor MG132. ER, estrogen recep-
tor; AR, androgen receptor; VDR, vitamin D receptor; AhR, aryl hydro-
carbon receptor.
a
No activity at 1 μM.
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value of 5.6 nM) in the FP assay. Moreover, PBP-tam
exhibited lower binding affinity to ERα compared with PBP-
NH2 and PBP-NC derivatives (PBP-tam: IC50 = 313 nM, PBP-
NH2: IC50 = 78 nM). These results revealed that the hydrogen
atom on the nitrogen atom of PBP-NC derivatives was impor-
tant for binding to ERα.
Furthermore, the ERα transcriptional activity was evalu-
ated using a reporter gene assay with CMX-GAL4N-hERα as
the recombinant receptor gene, TK-MH100x4-LUC as a re-
porter gene, and the CMX-β-galactosidase gene for normaliza-
tion, as reported previously.18 Human Embryonic Kidney 293
(HEK293) cells were incubated with the synthesized com-
pounds in the absence or presence of E2 (0.3 nM). After incu-
bation, the cells were assayed for reporter gene and
β-galactosidase activities. As shown in Fig. 5, it was demon-
strated that PBP-NC10 exhibited potent ERα antagonistic ac-
tivity, whereas PBP-C12, which has the same length of alkyl
chain as PBP-NC10, showed slight ERα antagonistic activity
(ERα antagonistic activity: PBP-C12, 61% inhibition at 10 μM;
PBP-NC10, IC50 = 45 nM).
Fig. 6 Computational model of the estrogen receptor (ER) α ligand
binding domain and PBP-NC10, ICI164,384. The hydrophobic region is
colored orange, PBP-NC10 is colored green, and ICI164,384 is colored
blue.
alkyl chain was the best around a decyl group on the amino
group of PBP-NC10 (Fig. 6).
Finally, we performed a computational docking analysis to
predict the binding mode of PBP-NC10 to ERα as follows; the
X-ray co-crystal structure of ERα bound to 4-OHT was
obtained from the Protein Data Bank (PDB: 3ERT) and used
in docking studies.19 Docking models of PBP-NC10 bound to
ERα were constructed via a conformational search analysis
using Molecular Operating Environment (MOE). MMFF94S
was used as a force field. In the modeled structures, the
amino group of PBP-NC10 interacted with the side-chain car-
boxyl group of Glu351 and the alkyl chain of PBP-NC10 was
included in the hydrophobic groove composed of hydropho-
bic amino acids such as Leu354, Met357, ILe358, Leu379,
and Trp383 of ERα. Therefore, these results indicated that
the amino group of PBP-NC10 played an important role in
leading the alkyl chain to the hydrophobic groove. Further-
more, the results of the docking analysis suggested that the
length of the hydrophobic groove interacting with the long
Conclusions
We have developed a novel selective estrogen degradation in-
ducer PBP-NC10, which could be useful as a lead structure in
the development of compounds with more potent and selec-
tive ER degradation-inducing activities. We also revealed
that the amino group and optimal length of the long alkyl
chain of PBP-NC10 are essential for exerting its ERα-
downregulating activity. Furthermore, a docking study was
performed and predicted that the interaction between the hy-
drogen atom on the amino group of PBP-NC10 and the car-
boxylic acid of Glu351 of ERα leads the long alkyl chain to
bind to the hydrophobic groove of ERα.
Experimental section
Chemistry
4-Hydroxytamoxifen was purchased from Fluka. All other re-
agents and solvents were purchased from Sigma-Aldrich,
Wako Pure Chemical and Tokyo Chemical Industry, and were
used without purification. Analytical TLC was carried out
using Merck silica gel 60 F254 pre-coated plates and visual-
ized using a 254 nm UV lamp, phosphomolybdic acid,
p-anisaldehyde or ninhydrin staining. Column chromatogra-
phy was performed with silica gel (spherical, neutral) pur-
chased from Kanto Chemical. The microwave reactions were
1
carried out using a Biotage Initiator. H and 13C NMR spectra
were obtained on a Varian AS 400 Mercury spectrometer (400
MHz for 1H and 100 MHz for 13C). FT-IR spectra were
recorded on a JASCO FT/IR-4100 spectrometer at 1 cm−1 reso-
lution, with an average of 128 scans used for the solution
(CDCl3) method and a 0.1 mm path length for NaCl cells.
Chemical shifts are expressed as ppm downfield from a sol-
vent residual peak or internal standard tetramethylsilane
Fig. 5 ERα antagonistic activity of PBP, PBP-C12 and PBP-NC10 in
HEK293 cells. Data are expressed as means
independent experiments.
S.E. of three
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(TMS). High-resolution mass spectra were obtained on a
Shimadzu IT-TOF MS equipped with an electrospray ioniza-
tion source.
1H), 6.62 (d, J = 8.4 Hz, 1H), 4.81 (br s, 1H), 3.91 (t, J = 6.2
Hz, 2H), 2.19 (s, 3H), 2.16 (s, 3H), 1.91–1.95 (m, 4H), 1.75–
1.79 (m, 2H), 1.45–1.46 (m, 2H), 1.30–1.31 (m, 8H), 0.82–0.97
(m, 13H); 13C NMR (100 MHz, CDCl3): δ 155.1, 151.6, 142.2,
141.2, 130.7, 130.5, 126.9, 126.2, 125.8, 122.8, 114.3, 110.1,
68.2, 48.3, 40.7, 32.2, 29.9, 29.8, 29.7, 26.6, 23.1, 17.7, 16.9,
16.5, 15.3, 14.6, 14.5; [HR-ESIIJ+)]: m/z calcd for C29H45O2 [M +
H]+: 425.3414, found: 425.3392.
4-[4-(4-n-Decyloxy-3-methylphenyl)heptan-4-yl]-2-methyl-
phenol (PBP-C10). Compound PBP-C10 was prepared from 3
(95.3 mg, 0.31 mmol) using the procedure described for PBP-
C2. Yield 39%; colorless oil; IR (CDCl3, cm−1): 3602, 2957,
2929, 2871, 1607, 1503, 1468; 1H NMR (400 MHz, CDCl3): δ
6.90–6.93 (m, 3H), 6.85 (d, J = 8.4 Hz, 1H), 6.66 (d, J = 8.4 Hz,
1H), 6.61 (d, J = 8.4 Hz, 1H), 4.69 (br s, 1H), 3.91 (t, J = 6.8
Hz, 2H), 2.18 (s, 3H), 2.16 (s, 3H), 1.92–1.96 (m, 4H), 1.73–
1.80 (m, 2H), 1.42–1.48 (m, 2H), 1.23–1.34 (m, 12H), 0.81–
0.99 (m, 13H); 13C NMR (100 MHz, CDCl3): δ 154.8, 151.2,
141.9, 140.9, 130.4, 130.2, 126.5, 125.8, 125.5, 122.5, 114.0,
109.8, 67.8, 47.9, 40.4, 32.0, 29.7, 29.6, 29.5, 29.4, 26.2, 22.7,
17.3, 16.6, 16.1, 14.9, 14.2; [HR-ESIIJ+)]: m/z calcd for C31H49O2
[M + H]+: 453.3727, found: 453.3767.
4-[4-(4-Ethoxy-3-methylphenyl)heptan-4-yl]-2-methylphenol
(PBP-C2). A solution of 321 (95.5 mg, 0.31 mmol) and K2CO3
(47.4 mg, 0.34 mmol) in MeCN (2 mL) was added ethyl iodide
(47 mg, 0.34 mmol) and stirred at 60 °C for 6 h. The reaction
mixture was acidified with 3% HCl and extracted with Et2O.
The organic layer was washed with brine and dried over
Na2SO4. After filtration, concentration in vacuo, and purifica-
tion by silica gel flash column chromatography (AcOEt/hex-
ane = 1/4), PBP-C2 (35.5 mg, 34%) was obtained as colorless
oil; IR (CDCl3, cm−1): 3602, 2958, 2932, 2872, 1607, 1503,
1477; 1H NMR (400 MHz, CDCl3): δ 6.90–6.93 (m, 3H), 6.85
(d, J = 8.4 Hz, 1H), 6.67 (d, J = 8.4 Hz, 1H), 6.61 (d, J = 8.4 Hz,
1H), 4.72 (br s, 1H), 3.98 (q, J = 14.0 Hz, 2H), 2.18 (s, 3H),
2.16 (s, 3H), 1.92–1.96 (m, 4H), 1.39 (t, J = 7.2 Hz, 3H), 0.93–
0.98 (m, 4H), 0.81–0.85 (m, 6H); 13C NMR (100 MHz, CDCl3):
δ 154.9, 151.5, 142.1, 141.3, 130.6, 130.5, 126.8, 126.1, 125.7,
122.7, 114.3, 110.2, 63.7, 48.2, 40.6, 17.6, 16.9, 16.4, 15.3,
15.2; [HR-ESIIJ+)]: m/z calcd for C23H33O2 [M + H]+: 341.2475,
found: 341.2445.
4-[4-(4-n-Butoxy-3-methylphenyl)heptan-4-yl]-2-methyl-
phenol (PBP-C4). Compound PBP-C4 was prepared from 3
(62.0 mg, 0.20 mmol) using the procedure described for PBP-
C2. Yield 16%; colorless oil; IR (CDCl3, cm−1): 3603, 2959,
2933, 2872, 1605, 1503, 1468; 1H NMR (400 MHz, CDCl3): δ
6.89–6.93 (m, 3H), 6.86 (d, J = 8.0 Hz, 1H), 6.67 (d, J = 8.4 Hz,
1H), 6.64 (d, J = 8.4 Hz, 1H), 4.47 (br s, 1H), 3.93 (t, J = 6.2
Hz, 2H), 2.19 (s, 3H), 2.16 (s, 3H), 1.91–1.95 (m, 4H), 1.73–
1.79 (m, 2H), 1.48–1.53 (m, 2H), 0.91–0.99 (m, 7H), 0.82–0.85
(m, 6H); 13C NMR (100 MHz, CDCl3): δ 154.8, 151.1, 141.9,
140.8, 130.3, 130.1, 126.6, 125.8, 125.4, 122.3, 114.0, 109.7,
67.4, 47.9, 40.3, 31.6, 19.4, 17.3, 16.5, 16.1, 14.9, 14.0; [HR-
ESIIJ+)]: m/z calcd for C25H37O2 [M + H]+: 369.2788, found:
369.2826.
4-[4-(4-n-Dodecyloxy-3-methylphenyl)heptan-4-yl]-2-methyl-
phenol (PBP-C12). Compound PBP-C12 was prepared from 3
(92.8 mg, 0.30 mmol) using the procedure described for PBP-
C2. Yield 33%; colorless oil; IR (CDCl3, cm−1): 3607, 2927,
2855, 1604, 1505, 1457; 1H NMR (400 MHz, CDCl3): δ 6.89–
6.93 (m, 3H), 6.86 (d, J = 8.4 Hz, 1H), 6.66 (d, J = 8.4 Hz, 1H),
6.64 (d, J = 8.4 Hz, 1H), 4.47 (br s, 1H), 3.91 (d, J = 6.4 Hz,
2H), 2.19 (s, 3H), 2.16 (s, 3H), 1.91–1.95 (m, 4H), 1.75–1.79
(m, 2H), 1.44–1.46 (m, 2H), 1.24–1.34 (m, 16H), 0.82–0.98 (m,
13H); 13C NMR (100 MHz, CDCl3): δ 154.8, 151.1, 141.9,
140.8, 130.3, 130.1, 126.6, 125.8, 125.4, 122.3, 114.0, 109.7,
67.7, 47.9, 40.3, 31.9, 29.7, 29.6, 29.5, 29.4, 26.2, 22.7, 17.3,
16.5, 16.1, 14.9, 14.1; [HR-ESIIJ+)]: m/z calcd for C33H53O2 [M +
H]+: 481.4040, found: 481.4002.
4-[4-(4-n-Hexyloxy-3-methylphenyl)heptan-4-yl]-2-methyl-
phenol (PBP-C6). Compound PBP-C6 was prepared from 3
(62.0 mg, 0.20 mmol) using the procedure described for PBP-
C2. Yield 47%; colorless oil; IR (CDCl3, cm−1): 3602, 2958,
2932, 2871, 1607, 1503, 1468; 1H NMR (400 MHz, CDCl3): δ
6.89–6.93 (m, 3H), 6.86 (d, J = 8.4 Hz, 1H), 6.66 (d, J = 8.4 Hz,
1H), 6.62 (d, J = 8.4 Hz, 1H), 4.59 (br s, 1H), 3.92 (t, J = 6.6
Hz, 2H), 2.19 (s, 3H), 2.16 (s, 3H), 1.92–1.96 (m, 4H), 1.73–
1.80 (m, 2H), 1.43–1.49 (m, 2H), 1.31–1.36 (m, 4H), 0.88–0.98
(m, 7H), 0.84 (t, J = 7.0 Hz, 6H); 13C NMR (100 MHz, CDCl3):
δ 154.8, 151.2, 141.9, 140.9, 130.4, 130.2, 126.5, 125.8, 125.5,
122.4, 114.0, 109.8, 67.8, 47.9, 40.4, 31.7, 29.5, 25.9, 22.7,
17.3, 16.6, 16.1, 14.9, 14.1; [HR-ESIIJ+)]: m/z calcd for C27H41O2
[M + H]+: 397.3101, found: 397.3078.
4-[4-(4-n-Octyloxy-3-methylphenyl)heptan-4-yl]-2-methyl-
phenol (PBP-C8). Compound PBP-C8 was prepared from 3
(59.1 mg, 0.19 mmol) using the procedure described for PBP-
C2. Yield 13%; colorless oil; IR (CDCl3, cm−1): 3602, 2958,
2931, 2871, 1607, 1503, 1468; 1H NMR (400 MHz, CDCl3): δ
6.91–6.93 (m, 3H), 6.85 (d, J = 8.4 Hz, 1H), 6.66 (d, J = 8.4 Hz,
4-{4-[4-(2-Chloroethoxy)-3-methylphenyl]heptan-4-yl}-2-
methylphenol (4). To a solution of 3 (525.8 mg, 1.7 mmol) in
MeCN (10 mL) was added 1,2-dichloroethane (333 mg, 3.4
mmol) and K2CO3 (705 mg, 5.1 mmol) at room temperature.
The mixture was stirred for 48 h at room temperature, brine
was added and the whole mixture was extracted with ethyl ac-
etate. The organic layer was dried over Na2SO4, filtered, and
then concentrated in vacuo. The residue was purified by col-
umn chromatography (ethyl acetate/n-hexane = 1/4) to give 4
(117 mg, 19%) as colorless oil; IR (CDCl3, cm−1): 3602, 2958,
2932, 2071, 1607, 1503, 1457; 1H NMR (400 MHz, CDCl3): δ
6.89–6.95 (m, 3H), 6.83 (d, J = 8.4 Hz, 1H), 6.65 (t, J = 7.8 Hz,
2H), 5.58 (br s, 1H), 4.18 (t, J = 5.8 Hz, 2H), 3.79 (t, J = 5.8 Hz,
2H), 2.18 (s, 6H), 1.92–1.96 (m, 4H), 0.82–0.99 (m, 10H); 13C
NMR (100 MHz, CDCl3): δ 153.8, 151.6, 142.2, 141.4, 130.5,
130.3, 126.4, 125.9, 122.8, 114.0, 110.3, 68.1, 48.0, 42.3, 40.3,
31.6, 22.7, 17.3, 16.5, 16.2, 14.9; [HR-ESIIJ+)]: m/z calcd for
C23H32ClO2 [M + H]+: 375.2085, found: 375.2051.
4-(4-{4-[2-(n-Hexylamino)ethoxy]-3-methylphenyl}heptan-4-
yl)-2-methylphenol (PBP-NC6). A solution of 4 (49.0 mg, 0.13
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mmol) in MeCN (0.5 mL) was added n-hexylamine (66.0 mg,
0.908 mmol) and stirred at 120 °C for 4 h assisted microwave
irradiation. The reaction mixture was concentrated in vacuo
and purified by silica gel flash column chromatography
(dichloromethane) to give PBP-NC6 (24.2 mg, 0.055 mmol,
42%) as colorless oil. IR (CDCl3, cm−1): 3602, 2958, 2931,
2871, 1606, 1503, 1466; 1H NMR (400 MHz, CDCl3): δ 6.88–
6.93 (m, 3H), 6.71 (d, J = 8.0 Hz, 1H), 6.63 (d, J = 8.4 Hz, 1H),
6.55 (d, J = 8.4 Hz, 1H), 4.05 (t, J = 4.4 Hz, 2H), 3.03 (t, J = 4.2
Hz, 2H), 2.73 (t, J = 7.0 Hz, 2H), 2.17 (s, 3H), 2.13 (s, 3H),
1.91–1.94 (m, 4H), 1.54–1.57 (m, 2H), 1.28–1.32 (m, 6H),
0.82–0.95 (m, 13H); 13C NMR (100 MHz, CDCl3): δ 154.7,
152.6, 142.2, 141.3, 130.9, 130.7, 127.0, 126.4, 125.8, 123.5,
114.6, 110.4, 66.9, 50.0, 49.1, 48.4, 40.9, 32.2, 29.9, 27.4, 23.1,
17.8, 17.1, 16.9, 15.4, 14.6; [HR-ESIIJ+)]: m/z calcd for
C29H46NO2 [M + H]+: 440.3523, found: 440.3482.
4-(4-{4-[2-(n-Octylamino)ethoxy]-3-methylphenyl}heptan-4-
yl)-2-methylphenol (PBP-NC8). Compound PBP-NC8 was pre-
pared from 4 (57.0 mg, 0.15 mmol) using the procedure de-
scribed for PBP-NC6. Yield 34%; colorless oil; IR (CDCl3,
cm−1): 3606, 2958, 2930, 2871, 1605, 1504, 1457; 1H NMR
(400 MHz, CDCl3): δ 6.89–6.93 (m, 3H), 6.78 (d, J = 8.4 Hz,
1H), 6.65 (d, J = 8.4 Hz, 1H), 6.59 (d, J = 8.4 Hz, 1H), 4.08 (t, J
= 4.8 Hz, 2H), 3.06 (t, J = 4.8 Hz, 2H), 2.76 (t, J = 7.6 Hz, 2H),
2.18 (s, 3H), 2.15 (s, 3H), 1.91–1.95 (m, 4H), 1.56–1.60 (m,
2H), 1.26–1.29 (m, 10H), 0.82–0.98 (m, 13H); 13C NMR (100
MHz, CDCl3): δ 154.7, 152.1, 142.2, 141.8, 130.8, 130.7, 127.0,
126.4, 125.8, 123.2, 114.6, 110.5, 66.8, 50.0, 49.0, 48.4, 40.8,
32.3, 29.9, 29.8, 27.7, 23.2, 17.8, 17.1, 16.7, 15.4, 14.6; [HR-
ESIIJ+)]: m/z calcd for C31H50NO2 [M + H]+: 468.3836, found:
468.3815.
4-(4-{4-[2-(n-Decylamino)ethoxy]-3-methylphenyl}heptan-4-
yl)-2-methylphenol (PBP-NC10). Compound PBP-NC10 was
prepared from 4 (33 mg, 0.09 mmol) using the procedure de-
scribed for PBP-NC6. Yield 32%; colorless oil; IR (CDCl3,
cm−1): 3603, 2957, 2929, 2856, 1606, 1503, 1466; 1H NMR
(400 MHz, CDCl3): δ 6.88–6.93 (m, 3H), 6.74 (d, J = 8.4 Hz,
1H), 6.64 (d, J = 8.4 Hz, 1H), 6.57 (d, J = 8.4 Hz, 1H), 4.06 (br,
2H), 3.04 (br, 2H), 2.73 (t, J = 7.6 Hz, 2H), 2.18 (s, 3H), 2.14
(s, 3H), 1.91–1.94 (m, 4H), 1.54–1.56 (m, 2H), 1.25–1.29 (m,
14H), 0.82–0.95 (m, 13H); 13C NMR (100 MHz, CDCl3): δ
154.7, 152.4, 142.1, 141.4, 130.8, 130.6, 126.9, 126.4, 125.8,
123.4, 114.6, 110.4, 66.9, 50.0, 49.1, 48.4, 40.8, 40.2, 32.4,
30.1, 30.0, 29.8, 27.7, 27.4, 23.2, 17.8, 17.1, 16.8, 15.4, 14.6;
[HR-ESIIJ+)]: m/z calcd for C33H54NO2 [M + H]+: 496.4149,
found: 496.4101.
4-(4-{4-[2-(n-Dodecylamino)ethoxy]-3-methylphenyl}heptan-
4-yl)-2-methylphenol (PBP-NC12). Compound PBP-NC12 was
prepared from 4 (68.7 mg, 0.183 mmol) using the procedure
described for PBP-NC6. Yield 26%; colorless oil; IR (CDCl3,
cm−1): 3603, 2957, 2920, 2855, 1605, 1503, 1465; 1H NMR
(400 MHz, CDCl3): δ 6.88–6.93 (m, 3H), 6.77 (d, J = 8.4 Hz,
1H), 6.65 (d, J = 8.4 Hz, 1H), 6.59 (d, J = 8.4 Hz, 1H), 4.07 (t, J
= 4.8 Hz, 2H), 3.05 (t, J = 5.2 Hz, 2H), 2.75 (t, J = 7.6 Hz, 2H),
2.18 (s, 3H), 2.14 (s, 3H), 1.91–1.95 (m, 4H), 1.53–1.58 (m,
2H), 1.25–1.30 (m, 18H), 0.82–0.98 (m, 13H); 13C NMR (100
MHz, CDCl3): δ 154.2, 151.6, 141.6, 141.3, 130.3, 130.2, 126.5,
125.9, 125.3, 122.7, 114.1, 109.9, 66.4, 49.5, 48.6, 47.9, 40.3,
39.7, 31.9, 29.7, 29.6, 29.5, 29.4, 29.3, 27.2, 26.9, 22.7, 17.3,
16.6, 16.2, 14.9, 14.1; [HR-ESIIJ+)]: m/z calcd for C35H58NO2 [M
+ H]+: 524.4462, found: 524.4445.
4-(4-{4-[2-(n-Hexadecylamino)ethoxy]-3-methylphenyl}-
heptan-4-yl)-2-methylphenol (PBP-NC16). Compound PBP-
NC16 was prepared from 4 (28.1 mg, 0.08 mmol) using the
procedure described for PBP-NC6. Yield 34%; colorless oil; IR
(CDCl3, cm−1): 3603, 2957, 2927, 2855, 1606, 1503, 1466; 1H
NMR (400 MHz, CDCl3): δ 6.88–6.93 (m, 3H), 6.75 (d, J = 8.4
Hz, 1H), 6.63 (d, J = 8.8 Hz, 1H), 6.58 (d, J = 8.4 Hz, 1H), 4.05
(t, J = 4.8 Hz, 2H), 3.04 (t, J = 5.0 Hz, 2H), 2.73 (t, J = 7.4 Hz,
2H), 2.18 (s, 3H), 2.13 (s, 3H), 1.91–1.95 (m, 4H), 1.25–1.28
(m, 28H), 0.82–0.98 (m, 13H); 13C NMR (100 MHz, CDCl3): δ
154.2, 151.7, 141.6, 141.1, 130.3, 130.2, 126.5, 125.9, 125.3,
122.7, 114.1, 109.9, 66.4, 49.5, 48.6, 47.9, 40.3, 31.9, 29.7,
29.6, 29.5, 29.4, 27.2, 22.7, 17.3, 16.6, 16.3, 14.9, 14.2; [HR-
ESIIJ+)]: m/z calcd for C39H66NO2 [M + H]+: 580.5088, found:
580.5077.
4-{4-[4-(2-Aminoethoxy)-3-methylphenyl]heptan-4-yl}-2-
methylphenol (PBP-NH2). To a solution of 3 (614.3 mg, 1.97
mmol) and Cs2CO3 (321.3 mg, 0.99 mmol) in MeCN (5 mL)
was added benzyl (2-bromoethyl)carbamate (264.7 mg, 1.03
mmol), and the mixture was stirred at 40 °C for 16 h. The re-
action mixture was neutralized with 3% aqueous HCl and
extracted with ethyl acetate. The organic layer was washed
with brine, dried over Na2SO4, filtered, and then concentrated
in vacuo. The residue was dissolved in MeOH (6 mL) and
PdIJOH)2 was added (ca. 20 mg). The solution was stirred at
room temperature for 16 h under H2 atmosphere. The reac-
tion mixture was filtered through celite and concentrated in
vacuo. The residue was purified with silica gel chromatogra-
phy (AcOEt/n-hexane = 1/4) to afford PBP-NH2 (175 mg, 25%)
as colorless oil; IR (CDCl3, cm−1): 3602, 2958, 2932, 2871,
1
1606, 1503, 1465; H NMR (400 MHz, CDCl3): δ 6.91–6.94 (m,
3H), 6.80 (d, J = 8.4 Hz, 1H), 6.66 (d, J = 8.4 Hz, 1H), 6.61 (d,
J = 8.4 Hz, 1H), 3.98 (t, J = 5.0 Hz, 2H), 3.11 (t, J = 5.0 Hz,
2H), 2.19 (s, 3H), 2.16 (s, 3H), 1.91–1.95 (m, 4H), 0.91–0.99
(m, 4H), 0.84 (t, J = 7.0 Hz, 6H); 13C NMR (100 MHz, CDCl3):
δ 154.3, 151.7, 141.6, 141.2, 130.3, 130.2, 126.5, 125.9, 125.3,
122.7, 114.0, 109.8, 69.4, 47.9, 41.6, 40.3, 17.3, 16.6, 16.3,
14.9; [HR-ESIIJ+)]: m/z calcd for C23H34NO2 [M + H]+: 356.2584,
found: 356.2547.
4-(4-{4-[2-(N,N-Dimethylamino)ethoxy]-3-methylphenyl}-
heptan-4-yl)-2-methylphenol (PBP-tam). To a solution of 3
(265.4 mg, 0.85 mmol), K2CO3 (351.6 mg, 2.5 mmol) and NaI
(127.2 mg, 0.85 mmol) in DMF (5 mL) was added
2-chlorodimethylethylamine (122.2 mg, 0.85 mmol) and the
mixture was stirred at 100 °C for 16 h. The reaction mixture
was concentrated in vacuo, and the residue was purified with
silica gel chromatography (AcOEt/MeOH = 9/1) to afford PBP-
tam (18.8 mg, 0.05 mmol, 6%) as colorless oil; IR (CDCl3,
cm−1): 3607, 3020, 2959, 1382, 1212; 1H NMR (400 MHz,
CDCl3): δ 6.80–6.92 (m, 3H), 6.81 (d, J = 8.4 Hz, 1H), 6.65 (d, J
= 8.4 Hz, 1H), 6.59 (d, J = 8.4 Hz, 1H), 4.05 (t, J = 6.0 Hz, 2H),
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2.78 (t, J = 6.0 Hz, 2H), 2.37 (s, 6H), 2.18 (s, 3H), 2.13 (s, 3H),
1.91–1.95 (m, 4H), 0.93–0.98 (m, 4H), 0.83 (t, J = 6.8 Hz, 6H);
13C NMR (100 MHz, CDCl3): δ 154.8, 151.1, 141.9, 140.8,
130.3, 130.1, 126.6, 125.8, 125.4, 122.3, 114.0, 109.7, 67.4,
47.9, 40.3, 31.6, 19.4, 17.3, 16.5, 16.1, 14.9, 14.0; [HR-ESIIJ+)]:
m/z calcd for C25H38NO2 [M + H]+: 384.2897, found: 384.2898.
(DMEM) without phenol red containing 10% fetal bovine se-
rum (FBS) and antibiotic–antimycotic (Nacalai) at 37 °C in a
humidified atmosphere of 5% CO2 in air. Transfections were
performed by the calcium phosphate coprecipitation method.
Test compounds were added at 8 h after transfection. Cells
were harvested 16–20 h after the treatment, and luciferase
and β-galactosidase activities were assayed using
a
luminometer and a microplate reader. DNA cotransfection ex-
periments were performed with 50 ng of reporter plasmid,
15–20 ng of pCMX-β-galactosidase, 10–15 ng of each receptor
expression plasmid and pGEM carrier DNA to make a total of
150 ng of DNA per well in a 96-well plate. Luciferase data
were normalized to an internal β-galactosidase control, and
the reported values are means of triplicate assays. Antagonist
activity was measured in the presence of 0.3 nM 17β-
estradiol.
Biology
Cell culture. Human breast carcinoma MCF-7 cells were
maintained in Dulbecco's modified Eagle's medium (DMEM)
containing 10% fetal bovine serum (FBS) and 100 μg mL−1
kanamycin with 10 μg ml−1 insulin (Sigma, St. Louis, MO,
USA). The cells were treated with various concentrations of
the compounds for 6 h. Whole-cell lysates were analyzed
using western blotting with the indicated antibodies.
Western blotting. Cells were lysed with SDS lysis buffer
(0.1 M Tris-HCl at pH 8.0, 10% glycerol, 1% SDS) and boiled
for 10 min. The protein concentration was measured by the
BCA method (Pierce, Rockford, IL, USA) and the lysates
containing an equal amount of protein were separated by
SDS-PAGE, transferred to PVDF membranes (Millipore, Darm-
stadt, Germany) for western blotting using the appropriate
antibodies. The immunoreactive proteins were visualized
using the Immobilon Western chemiluminescent HRP sub-
strate (Millipore) or Clarity Western ECL substrate (Bio-Rad,
Hercules, CA, USA), and light emission was quantified with a
LAS-3000 lumino-image analyzer (Fuji, Tokyo, Japan). The
antibodies used in this study were anti-ERα rabbit monoclo-
nal antibody (mAb) (Cell Signaling Technology, Danvers, MA,
USA; 8644), anti-β-actin mouse mAb (Sigma; A5316), anti-AR
rabbit mAb antibody (Cell Signaling Technology; 5153), anti-
AhR rabbit mAb antibody (Cell Signaling Technology; 13790),
anti-VDR rabbit mAb antibody (Cell Signaling Technology;
12550), and anti-Lamin B goat pAb (Santa Cruz, Dallas, TX,
USA; sc-6216).
Fluorescence polarization assay. Test compounds were
dissolved in DMSO to prepare a stock solution (10 mM). Fluo-
rescence polarization-based competition binding assays were
conducted to determine the relative binding affinity of com-
pounds for ERα using commercially available kits (P2698,
Life Technologies) according to the manufacturer's instruc-
tions. Fluorescence polarization signals (mP values) were
then measured using an EnVision multiple plate reader
(Perkin Elmer) with a 470 nm excitation filter and a 535 nm
emission filter. The fraction of compound bound to ERα was
correlated with the mP value and plotted against values of
competitor concentrations.
Acknowledgements
This work was supported in part by a grant for the Research
on Development of New Drugs from Japan Agency for Medical
Research and Development (AMED; M. N.) and a Grant-in-Aid
for Young Scientists (B) from the Japan Society for the Promo-
tion of Science (T. M.).
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