Design and synthesis of novel breast cancer therapeutic drug candidates based upon the hydrophobic feedback approach of antiestrogens

Article abstract of DOI:10.3390/molecules24213966

Based upon hydrophobic feedback approaches, we designed and synthesized novel sulfur-containing ERα modulators (4 and 5) as breast cancer therapeutic drug candidates. The tetrahydrothiepine derivative 5a showed the highest binding affinity toward ERα beca

Full text of DOI:10.3390/molecules24213966

molecules
Article
Design and Synthesis of Novel Breast Cancer
Therapeutic Drug Candidates Based upon the
Hydrophobic Feedback Approach of Antiestrogens
Kiminori Ohta 1,*, Asako Kaise 2 , Fumi Taguchi 2, Sayaka Aoto 2, Takumi Ogawa 2
and Yasuyuki Endo 2,
*
1
School of Pharmacy, Showa University, 1-5-8, Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan
Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, 4-4-1 Komatsushima,
Aoba-ku, Sendai 981-8558, Japan; kaise@tohoku-mpu.ac.jp (A.K.); f0u1m2i9@docomo.ne.jp (F.T.);
syk-at1213@docomo.ne.jp (S.A.); ogawa-takumi@pmda.go.jp (T.O.)
2
*
Correspondence: k-ohta@pharm.showa-u.ac.jp (K.O.); yendo@tohoku-mpu.ac.jp (Y.E.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 29 September 2019; Accepted: 30 October 2019; Published: 1 November 2019
Abstract: Based upon hydrophobic feedback approaches, we designed and synthesized novel
sulfur-containing ER
The tetrahydrothiepine derivative 5a showed the highest binding affinity toward ER
high hydrophobicity, and it acted as an agonist toward MCF-7 cell proliferation. The corresponding
alkylamino derivative 5d maintained high binding affinity to ER and potently inhibited MCF-7 cell
proliferation (IC50: 0.09 M). Docking simulation studies of compound 5d with the ER BD revealed
that the large hydrophobic moiety of compound 5d fit well into the hydrophobic pocket of the ER
LBD and that the sulfur atom of compound 5d formed a sulfur– interaction with the amino acid
residue His524 of the ER LBD. These interactions play important roles for the binding affinity of
α
modulators (
4
and
5) as breast cancer therapeutic drug candidates.
α
because of its
α
µ
α
α
π
α
compound 5d to the ERα LBD.
Keywords: sulfur; anticancer; estrogen
1. Introduction
17β-Estradiol (E2) is an endogenous female hormone and plays an important role in the female
reproductive system. Estrogens including E2 influence the growth, differentiation, and functioning of
many target tissues mediated by the binding to estrogen receptor α (ERα) [1–3]. Although activation
of ER
induce and cause progression of estrogen-dependent breast cancers [
been developed to prevent and control hormone-responsive breast cancer [
been used worldwide for more than 40 years for the treatment of estrogen-dependent breast cancer
(Figure 1) [11 13]. Interestingly, tamoxifen acts as either an agonist or an antagonist depending on tissue
type; tamoxifen exhibits antiestrogenic action in breast cancer and hot flashes, and estrogenic action
in bone and cholesterol metabolism, and is a selective estrogen receptor modulator (SERM) [11 13].
α
is essential for the maintenance of homeostasis of female functions, they can sometimes
]. Several antiestrogens have
10]. Tamoxifen has
4–6
7–
–
–
The dimethylaminoethyl chain of tamoxifen is attached to a benzene ring, where it plays a strategic
role in the expression of antiestrogenic activity. The triphenylethylene moiety of tamoxifen plays
an important role in controlling ERα binding affinity. The triphenylethylene moiety is a promising
structure for the development of an ER ligand. The triphenylethylene structure exhibits geometric
isomers E and Z that are caused by the key alkylamino chain, and the asymmetric hydrophobic part
and the isomers easily isomerize between the E and Z forms. Therefore, the isomerization often
become a big problem in the synthesis, purification, and preservation of an either isomer. Indeed,
Molecules 2019, 24, 3966; doi:10.3390/molecules24213966
www.mdpi.com/journal/molecules

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4-hydroxytamoxifen, an active metabolite of tamoxifen, easily isomerizes from the active Z form to the
inactive E form in solution and in in vitro studies [14 17]. In this regard, novel ER antagonists comprised
–
of other skeleton structures, such as benzothiophene [18], dihydronaphthalene [19], benzopyrane [20],
and steroid [21] structures, have been developed as ER antagonists.
Figure 1. Structures of estrogen receptor (ER) modulators and newly designed sulfur-containing ER
antagonist candidates (compounds 4a–4d and 5a–5d).
On the other hand, we found that a simple 1,2-bis(4-hydroxyphenyl)-o-carborane, BE360 (
exhibited high binding affinity toward ER and estrogenic action in bone without showing estrogenic
action in the uterus of ovariectomized (OVX) and orchidectomized (ORX) mice (Figure 1) [22].
That is, compound acted as an agonist in bone and an antagonist in female reproductive tissues,
1)
α
1
despite lack of an alkylamino chain. Based upon the hydrophobic feedback approaches, we focused on
three-dimensional hydrocarbon units as new hydrophobic pharmacophores to design and synthesize
BE1060 (
compound
compound
2
) and BE1054 (
acted as a partial agonist, similar to compound
showed potent estrogenic activity. Unlike the triphenylethylene structure, a vicinal
3
) (Figure 1) [23]. Although the tetramethylcyclohexene-based bisphenol
3
2
1, the bicyclo[2,2,2]octene-based bisphenol
bisphenol structure does not have a geometric isomer, even with the key alkylamino chain. We were
interested in the effects of ring size and sulfur atom in the hydrophobic structure on ER activity.
Furthermore, to understand the effects of sulfur, sulfone, and sulfoxide on the biological activity,
we designed dihydrothiophene and tetrahydrothiepine derivatives (4a–4d and 5a–5d) as novel ER
antagonist candidates. Herein, we describe the facile synthesis and biological activities of the designated
compounds 4a–4d and 5a–5d (Figure 1).
2. Results and Discussion
2.1. Chemistry
Synthesis of the dihydrothiophene derivatives (4a
available 2-bromo-40-methoxyacetophenone (
) was reacted with sodium sulfide (Na2S) to form the
sulfide compound in 95% yield [24]. Intramolecular McMurry coupling of resulted in formation
–4d) is summarized in Scheme 1. Commercially
6
7
7

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of the dihydrothiophene derivative
8
in 60% yield, which was demethylated with BBr3 to form the
target compound 4a in 97% yield [25]. Although the dihydrothiophene derivative
equimolar amount of m-chloroperbenzoic acid (m-CPBA) to form the sulfoxide compound
yield, followed by demethylation with BBr3 to form the decomposition product of compound
not result in the formation of compound 4b. Thus, we carried out oxidation of compound 4a with
m-CPBA to form the desired compound 4b in 73% yield. When compound was treated with two
8
was treated with 1
in 70%
, it did
9
9
8
equimolar amounts of m-CPBA, the corresponding sulfone compound 10 was obtained and was then
demethylated with BBr3 to form compound 4c in quantitative yield over two steps. Mitsunobu reaction
of compound 4a with 3-dimethylamino-1-propanol resulted in formation of compound 4d with a
dimethylaminopropyl chain in 37% yield [26].
Scheme 1. Synthesis of dihydrothiophene derivatives (compounds 4a
(a) Na2S 9H2O, acetone, H2O, (b) Zn, TiCl4, THF, (c) BBr3, CH2Cl2, (d) m-CPBA, CH2Cl2, (e)
3-dimethylamino-1-propanol, Ph3P, DEAD, THF.
–4d); Reagents and conditions:
·
Scheme 2 summarizes the synthesis of tetrahydrothiepine derivatives (5a
12) for the seven-membered ring derivative was synthesized by Friedel-Craft acylation of anisole
with acyl chloride 11, followed by sulfide formation with Na2S to form compound 13 in 84% yield
over two steps [24]. The tetrahydrothiepine derivatives (5a 5d) were synthesized in the same manner
as those of compounds 4a 4d. Briefly, intramolecular McMurry coupling of compound 13, followed
–5d). A starting material
(
5
–
–
by demethylation, resulted in formation of the sulfide compound 5a in a 66% yield [25]. Compound
5a was transformed into the sulfoxide compound 5b by oxidation with one equimolar amount of
m-CPBA in a 30% yield. Moreover, compound 5a was reacted with 3-dimethylamino-1-propanol under
Mitsunobu reaction conditions to form compound 5d in a 28% yield [26]. Treatment of compound 14
with two equimolar amounts of m-CPBA followed by demethylation resulted in the formation of the
sulfone compound 5c in a 49% yield over two steps.

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Scheme 2. Synthesis of tetrahydrothiepine derivatives (compounds 5a–5d); Reagents and conditions:
(a) anisol, AlCl3, 1,2-dichloroetane, (b) Na2S 9H2O, acetone, H2O, (c) Zn, TiCl4, THF, (d) BBr3, CH2Cl2,
·
(e) m-CPBA, CH2Cl2, (f) 3-dimethylamino-1-propanol, Ph3P, DEAD, THF.
2.2. Biological Evaluation
The binding affinities of the synthesized bisphenols (compounds 4a
–
4c and 5a
-estradiol and human
29]. Figure 2A,B shows dose-response curves for competitive binding of the
synthesized compounds 4a 4c and 5a 5c to ER , respectively. Compound 4a, containing a sulfide
group, moderately bound to ER and the binding affinity was about 100 times lower than that of E2.
The sulfoxide (4b) and sulfone (4c) compounds did not bind to the ER ligand-binding domain (LBD),
even at concentrations 1000 times higher than that of E2. The binding affinity of the tetrahydrothiepine
derivative toward ER showed a similar tendency to that of compound , and the sulfide derivative
5a bound to ER more potently than compound 4a. The tamoxifen-inspired derivatives 4d and 5d
both containing a dimethylaminopropyl group, showed similar binding affinities to compounds 4a
and 5a, respectively. With the binding affinity of E2 to ER taken as 100, the relative binding affinity
–5c) toward ERα
were evaluated by means of a competitive binding assay using [6,7-3H] 17
β
recombinant ERα [27–
–
–
α
α
α
5
α
4
α
,
α
(RBA) values of compounds 4d and 5d were 4.0 and 37.1, respectively (Table 1). It is suggested that the
binding affinities of compounds 4 and 5 depend on the hydrophobicity of the ring structures and the
polarity of the sulfur atoms, which can be understood from the CLogP values of compounds 4a
and 5a–5d in Table 1.
–4d
Figure 2. Dose-response curves obtained from a competitive binding assay of compounds 4a
and 5a 5c ) toward ER . All binding assays were performed in duplicate (n = 2). IC50 value of E2 for
ERa is 4 nM.
–4c (A)
–
(B
α

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Table 1. Biological profiles of compounds 4a–4d and 5a–5d.
RBA (%)
EC50
Emax (%)
IC50
CLogP d
Compound
Ring
X
R
a
c
(µM) b
(µM) b
4a
4b
4c
4d
5a
5b
5c
5d
a
5
S
SO
SO2
S
H
H
H
1.2
0.003
0.04
4.0
4.9
0.001
0.01
0.39
NT e
0.30
–
0.048
NT f
4.7
119
NT e
130
–
–
NT e
–
3.1
0.8
0.7
4.2
3.9
1.6
1.5
4.9
(CH2)3N(CH3)2
0.41
–
7
S
H
H
H
99
NT f
89
NT f
–
SO
SO2
S
(CH2)3N(CH3)2 37.1
–
–
0.090
Relative binding affinity (RBA) values of each compound were estimated from the sigmoidal dose-response curves
b
in Figure 2A,B. The binding affinity of E2 was taken as 100. EC50 and IC50 values of the test compounds were
estimated from the sigmoidal dose-response curves using GraphPad Prism 7 software. Emax indicates the maximal
efficacy of each compound relative to that of E2, which was taken as 100. CLogP values of each compound were
estimated using ChemBioDraw Ultra 12.0. “NT” indicates not tested.
c
d
e
Next, estrogenic and antiestrogenic activities of compounds 4a
5b (poor binding affinity toward ERa), were evaluated by means of a cell proliferation assay using the
human breast cancer cell line MCF-7, which shows ER-dependent growth [27 29]. Table 1 summarizes
–4d and 5a–5d, except for 4b and
–
the EC50, IC50, and Emax values estimated from the MCF-7 cell proliferation assays. Emax represents the
maximal efficacy of each compound toward cell proliferation and is an index of ER partial agonistic
activity. Emax was estimated relative to that of E2, which was taken as 100. Although the binding affinity
of compound 4a was ~10 times higher than that of compound 4c, both compounds showed estrogenic
activity with similar EC50 values. Based on the Emax values of compounds 4a and 4c, we suggest
that the complex formed between compound 4c and the ER
than that formed between compound 4a and the ER LBD. Compound 5a, which exhibited the most
potent binding affinity to ER LBD, also showed the most potent MCF-7 cell proliferation activity,
with an EC50 value of 0.048 µM. As expected from the weak binding affinity to ERα LBD, compound
α LBD is more active for transcription
α
α
5c showed weak estrogenic activity. On the other hand, the tamoxifen-inspired derivatives 4d and 5d
containing alkylamino chains, potently inhibited cell proliferation of MCF-7 cells induced by 0.1 nM of
E2, with IC50 values of 0.41 and 0.09 µM, respectively.
,
2.3. Docking Study
To understand the docking modes of the sulfur-containing ligands 4d and 5d with the ER
docking simulation studies of both compounds toward the human ER LBD (PDB: 1ERR) were carried
out using an automatic docking program (Discovery Studio 2018/CDOCKER) [30]. The docking
mode of compound 4d was superimposed onto that of compound 5d at the ER LBD (Figure 3A,B).
The docking mode of compound 4d toward the ER LBD was similar to that of compound 5d
α LBD,
α
α
α
.
Each phenolic hydroxy group of compounds 4d and 5d formed hydrogen bonds with the amino acid
residues Glu353 and Arg394 (Figure 3C), and each dimethylaminopropyl chain of compounds 4d and
5d was found to be located within the same space of the ER
moiety of compound 5d was spread widely across the hydrophobic pocket of the ER
α
LBD (Figure 3A). The hydrophobic
LBD and the
α
sulfur atom of the tetrahydrothiepine ring formed a sulfur–π interaction with the amino acid residue
His524 (Figure 3B,C). Docking scores (CDOCKER interaction energy) of compounds 4d and 5d toward
the ERα LBD were 51.1 and 54.0, respectively, and the order of docking score was correlated with
the results of the binding assay. Therefore, we attributed the potent binding affinity of compound 5d
toward the ERα LBD to both hydrophobic interactions and sulfur–π interactions.

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Figure 3. Docking modes of compounds 4d and 5d at the ER
) Superimposed models of compounds 4d and 5d; ( ) Interaction modes of compounds 4d and 5d
) A sulfur– interaction of 5d with the amino acid
α ligand-binding domain (LBD).
(
A
B
with the amino acid residues of the ER
α
LBD; (
C
π
residue His524 of the ERα LBD.
3. Materials and Methods
3.1. General Consideration
Melting points were determined with an MP-J3 micro melting point apparatus (Yanaco, Kyoto,
Japan) and were not corrected. 1H NMR and 13C NMR spectra were recorded with JNM-LA-400
spectrometer (JEOL, Tokyo, Japan). 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 solvents. The splitting patterns are designated as
follows: s (singlet), d (doublet), t (triplet), q (quartet) and m (multiplet). Mass spectra were recorded
on a JEOL JMS-DX-303 spectrometer (JEOL, Tokyo, Japan). Elemental analyses were performed with
a Perkin Elmer 2400 CHN spectrometer (PerkinElmer, Winter Street Waltham, MA, USA). Column
chromatography was carried out using Merck silica gel 60 (0.063–0.200
Merck silica gel F254.
µm) and TLC was performed on
3.2. Synthesis
1-(4-Methoxyphenyl)-2-[2-(4-methoxyphenyl)-2-oxo-ethylsulfanyl] ethanone (7). To a stirred solution of
6
(2.80 g, 12.2 mmol) in acetone (40 mL) was added a solution of Na2S 9H2O (1.50 g, 6.25 mmol) in
H2O (12 mL) at 0 ◦C. After completion of the addition, the mixture was warmed to room temperature
and stirred for 2.5 h. The solvents were removed, and the residue was recrystallized from MeOH to

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give
7
(1.91 g, 5.78 mmol, 95 %) as a colorless solid; 1H NMR (400 MHz, CDCl3)
δ
(ppm) 3.87 (6H, s),
3.93 (4H, s), 6.93 (4H, d, J = 8.8 Hz), 7.95 (4H, d, J = 8.8 Hz); 13C NMR (100 MHz, CDCl3)
δ (ppm) 37.4,
55.5, 113.9, 128.4, 131.0, 163.8, 193.0; MS (EI) m/z = 330 (M+), 135 (100%).
3,4-Bis-(4-methoxyphenyl)-2,5-dihydrothiophene (
in dry THF (60 mL) was added TiCl4 (1.3 mL, 12.0 mmol) at
for 2.5 h. A solution of
8
). To a suspension of Zn powder (1.57 g, 24.0 mmol)
30 ◦C, and the mixture was refluxed
(1.0 g, 3.0 mmol) in dry THF (45mL) was slowly added to the reaction
−
7
mixture at 0◦C. The reaction mixture was refluxed for 2 h and then quenched with 10% K2CO3 aqueous
solution. The insoluble materials were filtered through Celite, and the filtrate was extracted with
AcOEt. The organic layer was washed with brine, dried over Na2SO4, and concentrated. Purification
by silica gel column chromatography (eluent: AcOEt/n-hexane = 1/30) gave
1.84 mmol, 61 %); 1H NMR (400 MHz, CDCl3)
(ppm) 3.77 (6H, s), 4.22 (4H, s), 6.75 (4H, d, J = 8.8 Hz),
7.05 (4H, d, J = 8.8 Hz); 13C NMR (100 MHz, CDCl3)
(ppm) 43.8, 55.1, 113.6, 129.4, 129.7, 134.3, 158.6;
MS (EI) m/z = 298 (M+, 100%); Anal. Calcd for C18H18O2S: C, 72.45; H, 6.08, Found: C, 72.13; H, 6.20.
3,4-Bis-(4-hydroxyphenyl)-2,5-dihydrothiophene (4a). To a solution of (407 mg, 1.36 mmol) in dry
CH2Cl2 (9 mL) was added 1M of a solution of BBr3 in CH2Cl2 (3.4 mL, 3.4 mmol) at
80 ◦C, and the
8 as a colorless solid (0.55 g,
δ
δ
8
−
mixture was stirred at room temperature for 24 h. The mixture was poured onto ice and extracted with
AcOEt. The organic layer was washed with brine, dried over MgSO4, and concentrated. Purification by
silica gel column chromatography (eluent: AcOEt/n-hexane = 1/3) gave 4a as a colorless solid (358 mg,
1.33 mmol, 97 %); colorless needles (MeOH/CH2Cl2); mp 168.5–170.0 ◦C; 1H NMR (400 MHz, CD3OD)
δ
(ppm) 4.15 (4H, s), 6.62 (4H, d, J = 8.8 Hz), 6.95 (4H, d, J = 8.8 Hz); 13C NMR (100 MHz, CD3OD)
δ(ppm)
44.3, 116.0, 129.8, 130.8, 135.5, 157.7; MS (EI) m/z = 270 (M+, 100%); HRMS Calcd for C16H14O2S: 270.0715,
Found: 270.0707; Anal. Calcd for C16H14O2S 0.4 H2O: C, 69.23; H, 5.38, Found: C, 69.04; H, 5.66.
3,4-Bis-(4-methoxyphenyl)-2,5-dihydrothiophene-1-oxide (9). To a solution of 8 (406 mg, 1.36 mmol) in
dry CH2Cl2 (15 mL) was portionwise added m-CPBA (65%, 349 mg, 1.41 mmol) at 0 ◦C. The reaction
mixture was stirred at room temperature for 25 h and then quenched with saturated Na2SO3 aqueous
solution. The mixture was extracted with AcOEt, washed with saturated NaHCO3 aqueous solution
and brine, dried over MgSO4, and concentrated. Purification by silica gel column chromatography
(eluent: AcOEt/n-hexane = 1/1) gave
(CHCl3/n-hexane); mp 181.0–182.0 ◦C; 1H NMR (400 MHz, CDCl3)
J = 16.6 Hz), 4.34 (2H, d, J = 16.6 Hz), 6.77 (4H, d, J = 8.8 Hz), 7.10 (4H, d, J = 8.8 Hz); 13C NMR (100
MHz, CDCl3)
(ppm) 55.2, 64.3, 113.9, 128.1, 129.9, 130.1, 159.1; MS (EI) m/z = 296 (M+, 100%); HRMS
9
as a colorless solid (302 mg, 0.96 mmol, 70%); colorless needles
δ
(ppm) 3.78 (6H, s), 4.00 (2H, d,
δ
Calcd for C18H18O3S: 314.0977, Found: 314.0974; Anal. Calcd for C18H18O3S: C, 68.76; H, 5.77, Found:
C, 68.42; H, 5.88.
3,4-Bis-(4-hydroxyphenyl)-2,5-dihydrothiophene-1-oxide (4b). To a solution of 4a (402 mg, 1.49 mmol)
in dry CH2Cl2 (60 mL) was portion-wise added m-CPBA (65%, 368 mg, 1.49 mmol) at 0 ◦C. The reaction
mixture was stirred at room temperature for 20 h and then quenched with saturated Na2SO3 aqueous
solution. The mixture was extracted with AcOEt, washed with saturated NaHCO3 aqueous solution
and brine, dried over MgSO4, and concentrated. Purification by silica gel column chromatography
(eluent: AcOEt/n-hexane = 1/1) afforded 4b◦as a colorless solid (311 mg, 1.09 mmol, 73 %); colorless
1
needles (MeOH/CH2Cl2); mp 219.0–220.0 C; H NMR (400 MHz, CD3OD)
δ
(ppm) 3.94 (2H, d,
J = 17.1 Hz), 4.49 (2H, d, J = 17.1 Hz), 6.67 (4H, d, J = 8.8 Hz), 7.05 (4H, d, J = 8.8 Hz); 13C NMR
(100 MHz, CD3OD)
δ
(ppm) 64.9, 116.3, 128.4, 131.0, 131.1, 158.3; MS (EI) m/z = 286 (M+), 268 (100%);
HRMS Calcd for C16H14O3S: 286.0664, Found: 286.0665; Anal. Calcd for C16H14O3S 0.1 H2O: C, 66.69;
H, 4.97, Found: C, 66.47; H, 4.72.
3,4-Bis-(4-methoxyphenyl)-2,5-dihydrothiophene-1,1-dioxide (10). To a solution of 8 (601 mg, 2.02 mmol)
in dry CH2Cl2 (30 mL) was portionwise added m-CPBA (65%, 1.11 g, 4.51 mmol) at 0 ◦C. The reaction
mixture was stirred at room temperature for 3.5 h and then quenched with saturated Na2SO3 aqueous
solution. The mixture was extracted with AcOEt, washed with saturated NaHCO3 aqueous solution
and brine, dried over MgSO4, and concentrated. The residue was purified by recrystallization with
CHCl3/n-hexane to give 10 (377 mg, 1.14 mmol, 57 %) as a colorless needles; mp 141.0–141.5 ◦C; 1H

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NMR (400 MHz, CDCl3)
δ
(ppm) 3.78 (6H, s), 4.26 (4H, s), 6.77 (4H, d, J = 9.3 Hz), 7.03 (4H, d, J = 9.3 Hz);
13C NMR (100 MHz, CDCl3) (ppm) 55.2, 61.0, 114.0, 127.2, 129.2, 129.7, 159.4; MS (EI) m/z = 330 (M+),
δ
266 (100%); Anal. Calcd for C18H18O4S: C, 65.43; H, 5.49, Found: C, 65.15; H, 5.55.
3,4-Bis-(4-hydroxyphenyl)-2,5-dihydrothiophene-1,1-dioxide (4c). To a solution of 10 (203 mg, 0.61
mmol) in dry CH2Cl2 (10 mL) was added 1 M of a solution of BBr3 in CH2Cl2 (1.4 mL, 1.4 mmol) at
−
80 ◦C. The reaction mixture was stirred at room temperature for 24 h, poured onto ice, and then
extracted with AcOEt. The organic layer was washed with brine, dried over MgSO4, and concentrated.
Purification by silica gel column chromatography (eluent: AcOEt/n-hexane = 1/2) afforded 4c as a
colorless solid (185 mg, 0.61 mmol, quant); colorless needles (AcOEt/n-hexane); mp 180.5–181.0◦C; 1H
NMR (400 MHz, CD3OD)
NMR (100 MHz, CD3OD)
δ
(ppm) 4.27 (4H, s), 6.64 (4H, d, J = 8.8 Hz), 6.99 (4H, d, J = 8.8 Hz); 13
(ppm) 61.7, 116.2, 127.9, 130.5, 131.0, 158.6; MS (EI) m/z = 302 (M+), 238
C
δ
(100%); HRMS Calcd for C16H14O4S: 302.0613, Found: 302.0618; Anal. Calcd for C16H14O4S: C, 63.56;
H, 4.67, Found: C, 63.29; H, 4.75.
4-{4-[4-(3-dimethylaminopropoxy)phenyl]-2,5-dihydrothiophen-3-yl}phenol (4d). To a solution of
4a (100 mg, 0.37 mmol) in dry THF (5 mL), 3-dimethylamino-1-propanol (48
triphenylphosphine (97 mg, 0.37 mmol) was added a solution of diethyl azodicarboxylate in toluene
(40%, 161 L, 0.37 mmol) at room temperature, and the mixture was stirred for 22 h. The mixture was
µL, 0.41 mmol),
µ
concentrated and purified by silica gel column chromatography (eluent: MeOH/CHCl3 = 1/5) gave 4d
as a colorless solid (49 mg, 37%); colorless needles (AcOEt); mp 177.0–178.5◦C; 1H NMR (400 MHz,
DMSO-d6)
δ (ppm) 1.79 (2H, quint, J = 6.5 Hz), 2.11 (6H, s), 2.30 (2H, t, J = 7.2 Hz), 3.92 (2H, t, J = 6.0
Hz), 4.14 (4H, s), 6.60 (2H, d, J = 8.2 Hz), 6.76 (2H, d, J = 8.7 Hz), 6.93 (2H, d, J = 7.7 Hz), 7.03 (2H, d, J =
8.2 Hz), 9.45 (1H, s); 13C NMR (100 MHz, DMSO-d6)
δ (ppm) 26.9, 43.0, 45.2, 55.6, 65.6, 114.1, 115.1,
126.8, 128.2, 128.9, 129.5, 133.1, 134.1, 157.2, 157.6; MS (EI) m/z 355 (M+), 58 (100%); HRMS Calcd for
C21H25NO2S: 355.1606, Found: 355.1607.
3-Chloro-1-(4-methoxyphenyl)-propan-1-one (12). To a soltion of anisol (8.5 mL, 78.2 mmol) and AlCl3
(11 g, 82.5 mmol) in dry 1,2-dichloroethane (30 mL) was added 3-chloropropionyl chloride 11 (5.5 mL,
57.61 mmol) at 0 ◦C, and the mixture was stirred at room temperature for 2 h. The mixture was poured
onto ice and extracted with AcOEt. The organic layer was washed with brine, dried over MgSO4, and
concentrated. The residue was purified by recrystallization from CH2Cl2/n-hexane to give 12 (9.3 g,
46.7 mmol, 81%) as a colorless solid; 1H NMR (400 MHz, CDCl3)
δ (ppm) 3.40 (2H, t, J = 6.8 Hz), 3.87
(3H, s), 3.91 (2H, t, J = 6.8 Hz), 6.95 (2H, d, J = 8.8 Hz), 7.94 (2H, d, J = 8.8 Hz); 13C NMR (100 MHz,
CDCl3) δ (ppm) 38.9, 40.9, 55.5, 113.9, 129.5, 130.3, 163.8, 195.2; MS (EI) m/z = 198 (M+), 135 (100%).
7-Methoxy-1-[3-(4-methoxy-phenyl)-3-oxo-propylsulfanyl]-4-methyl-hepta-4,6-dien-3-one (13). To a
stirred solution of 12 (7.01 g, 35.4 mmol) in acetone (100 mL) was added a solution of Na2S 9H2O (4.12 g,
17.2 mmol) in H2O (12 mL) at 0 ◦C. After completion of the addition, the mixture was warmed to room
temperature and stirred for 21 h. The solvents were removed, and the residue was recrystallized from
MeOH to give 13 (6.79 g, 17.4 mmol, 98 %) as a colorless solid; 1H NMR (400 MHz, CDCl3)
δ (ppm) 3.0
(4H, t, J = 7.3 Hz), 3.2 (4H, t, J = 7.3 Hz), 3.9 (6H, s), 6.9 (4H, d, J = 8.8 Hz), 7.9 (4H, d, J = 8.8 Hz); 13
C
NMR (100 MHz, CDCl3)
(M+), 135 (100%).
δ (ppm) 26.7, 38.5, 55.5, 113.8, 129.7, 130.3, 163.6, 196.8. MS (EI) m/z = 358
4,5-Bis (4-methoxyphenyl)-2,3,6,7-tetrahydrothiepine (14). To a suspension of Zn powder (4.39 g,
◦
67.1 mmol) in THF (90 mL) was added TiCl4 (3.8 mL, 34.6 mmol) at
−
30 C, and the mixture was
refluxed for 2.5 h. A solution of 13 (3.01 g, 8.4 mmol) in THF (120 mL) was slowly added to the
reaction mixture at 0 ◦C. The reaction mixture was refluxed for 18 h and then quenched with 10%
K2CO3 aqueous solution. The insoluble materials were filtered through Celite, and the filtrate was
extracted with AcOEt. The organic layer was washed with brine, dried over MgSO4, and concentrated.
The residue was purified by column chromatography on silica gel with n-hexane to give 14 (2.72 g,
8.34 mmol, 99%) as a colorless solid; 1H NMR (400 MHz, CDCl3)
δ (ppm) 2.8 (4H, t, J = 5.3 Hz), 3.13
(4H, t, J = 5.3 Hz), 3.72 (6H, s), 6.65 (4H, d, J = 8.8 Hz), 6.88 (4H, d, J = 8.8 Hz); 13C NMR (100 MHz,
CDCl3) δ (ppm) 27.3, 40.0, 55.1, 113.2, 130.0, 136.8, 139.2, 157.5; MS (EI) m/z = 326 (M+, 100%).

Molecules 2019, 24, 3966
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3,4-Bis (4-hydroxyphenyl)-2,3,6,7-tetrahydrothiepine (5a). To a solution of 14 (1.01 g, 3.08 mmol) in
dry CH2Cl2 (15 mL) was added 1M of a solution of BBr3 in CH2Cl2 (7.6 mL, 7.6 mmol) at
80 ◦C,
−
and the mixture was stirred at room temperature for 16 h. The mixture was poured onto ice and
extracted with AcOEt. The organic layer was washed with brine, dried over MgSO4, and concentrated.
The residue was purified by column chromatography on silica gel with 1:2 AcOEt:n-hexane to give 5a
(413 mg, 2.02 mmol, 66 %) as a colorless solid; colorless needles (MeOH/CH2Cl2); mp 165.5–166.0 ◦C;
1H NMR (400 MHz, CD3OD)
Hz), 6.78 (4H, d, J = 8.8 Hz); 13C NMR (100 MHz, CD3OD)
δ (ppm) 2.73 (4H, t, J = 5.4 Hz), 3.09 (4H, t, J = 5.4 Hz), 6.51 (4H, d, J = 8.8
δ
(ppm) 28.1, 41.0, 115.5, 131.2, 137.4, 140.5,
156.3; MS (EI) m/z = 298 (M+, 100%); HRMS Calcd for C18H18O2S: 298.1028, Found: 298.1034; Anal.
Calcd for C18H18O2S: C, 72.45; H, 6.08, Found: C, 72.27; H, 6.15.
4,5-Bis (4-hydroxyphenyl)-2,3,6,7-tetrahydrothiepine-1-oxide (5b). To a solution of 5a (402 mg, 1.35
◦
mmol) in CH2Cl2 (60 mL) was portionwise added m-CPBA (65%, 399 mg, 1.62 mmol) at 0 C.
The reaction mixture was stirred at room temperature for 9 h and then quenched with saturated
Na2SO3 aqueous solution. The mixture was extracted with AcOEt, washed with saturated NaHCO3
aqueous solution and brine, dried over MgSO4, and concentrated. The residue was purified by column
chromatography on silica gel with 3:1 AcOEt:n-hexane to give 5b (127 mg, 0.40 mmol, 30 %) as a
colorless solid; colorless needles (MeOH/CH2Cl2); mp 190–191.5 ◦C; 1H NMR (400 MHz, CD3OD)
(ppm) 2.51 (2H, dd, J = 15.8 Hz), 3.00 (2H, dd, J = 14.2 Hz), 3.10 (2H, t, J = 14.2 Hz), 3.65 (2H, dd, J =
δ
15.6 Hz), 6.54 (4H, d, J = 8.3 Hz), 6.83 (4H, d, J = 8.3 Hz); 13C NMR (100 MHz, CD3OD)
δ (ppm) 26.0,
46.8, 115.9, 131.4, 136.2, 140.4, 157.1; MS (EI) m/z = 314 (M+), 251 (100%); HRMS Calcd for C18H18O3S:
314.0977, Found: 314.0976.
4,5-Bis (4-methoxyphenyl)-2,3,6,7-tetrahydrothiepine-1,1-dioxide (15). To a solution of 14 (1.00 mg,
3.07 mmol) in dry CH2Cl2 (20 mL) was portion-wise added m-CPBA (65%, 1.75 g, 7.11 mmol) at 0
◦C. The reaction mixture was stirred at room temperature for 22 h and then quenched with saturated
Na2SO3 aqueous solution. The mixture was extracted with AcOEt, washed with saturated NaHCO3
aqueous solution and brine, dried over MgSO4, and concentrated. The residue was purified by column
chromatography on silica gel with 1:3 AcOEt:n-hexane to give 15 (1.05 g, 2.93 mmol, 96 %) as colorless
solid; 1H NMR (400 MHz, CDCl3) δ (ppm) 3.02 (4H, t, J = 5.1 Hz), 3.17 (4H, t, J = 5.1 Hz), 3.74 (6H, s),
6.68 (4H, d, J = 8.8 Hz), 6.88 (4H, d, J = 8.8 Hz); 13C NMR (100 MHz, CDCl3)
113.7, 130.1, 135.1, 138.6, 158.3. MS (EI) m/z = 358 (M+), 292 (100%).
δ (ppm) 30.4, 54.0, 55.3,
4,5-Bis (4-hydroxyphenyl)-2,3,6,7-tetrahydrothiepine-1,1-dioxide (5c). To a solution of 15 (403 mg,
1.12 mmol) in dry CH2Cl2 (7 mL) was added 1M of a solution of BBr3 in CH2Cl2 (2.8 mL, 2.8 mmol)
at
80 ◦C. The reaction mixture was stirred at room temperature for 31 h, poured onto ice, and then
−
extracted with AcOEt. The organic layer was washed with brine, dried over MgSO4, and concentrated.
The residue was purified by column chromatography on silica gel with 1 : 2 AcOEt : n-hexane to give
5c (195 mg, 0.20 mmol, 53%) as a colorless solid; colorless needles (AcOEt/n-hexane); mp 128.5–130.0
◦C; 1H NMR (400 MHz, CD3OD)
δ (ppm) 2.97 (4H, t, J = 5.3 Hz), 3.21 (4H, t, J = 5.3 Hz), 6.54 (4H,
d, J = 8.8 Hz), 6.85 (4H, d, J = 8.8 Hz); 13C NMR (100 MHz, CD3OD)
δ (ppm) 31.2, 54.5, 115.7, 131.4,
135.8, 139.8, 157.0; MS (EI) m/z = 330 (M+), 264 (100%); HRMS Calcd for C18H18O4S: 330.0926, Found:
330.0920; Anal. Calcd for C18H18O4S: C, 65.43; H, 5.49, Found: C, 65.40; H, 5.59.
4-{5-[4-(3-Dimethylaminopropoxy)phenyl]-2,3,6,7-tetrahydrothiepin-4-yl}phenol (5d). To a solution
of 5a (200 mg, 0.67 mmol) in dry THF (10 mL), 3-dimethylamino-1-propanol (87
triphenylphosphine (176 mg, 0.67 mmol) was added a solution of diethyl azodicarboxylate in toluene
(40%, 292 L, 0.67 mmol) at room temperature, and the mixture was stirred for 22 h. After the solvent
µL, 0.74 mmol),
µ
was removed, the residue was purified by column chromatography on silica gel with 1:5 MeOH:CHCl3
to give 5d (71 mg, 28%) as a colorless solid; Colorless needles (CH2Cl2/MeOH); mp 176.0–177.0 ◦C; 1H
NMR (400 MHz, CDCl3) δ (ppm) 1.88 (2H, quint, J = 6.9 Hz), 2.25 (6H, s), 2.43 (2H, t, J = 7.7 Hz), 2.78
(4H, t, J = 4.8 Hz), 3.12 (4H, t, J = 5.1 Hz), 3.91 (2H, t, J = 6.3 Hz), 6.53 (2H, d, J = 8.7 Hz), 6.61 (2H, d, J =
8.7 Hz), 6.79 (2H, d, J = 8.7 Hz), 6.83 (2H, d, J = 8.7 Hz); 13C NMR (100 MHz, CDCl3)
δ (ppm) 27.1, 27.3,

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39.9, 45.2, 56.3, 66.0, 113.8, 115.0, 130.1, 130.2, 136.3, 137.0, 139.1, 139.5, 154.4, 156.7; MS (EI) m/z 383
(M+), 58 (100%); HRMS Calcd for C23H29NO2S: 383.1919, Found: 383.1931.
3.3. Competitive Binding Assay Using Human ER
The ligand binding activity of human estrogen receptor
filter binding assay method. ER was 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 floride) and
incubated with 4 nM [6, 7-3H]-17
-estradiol in the presence or absence of an unlabeled competitor at 4
α (ER α) was determined by a nitrocellulose
α
β
◦C for 18 h. The incubation mixture was absorbed by suction onto a nitrocellulose membrane that
had been soaked in binding assay buffer. The membrane was washed two times with buffer (20 mM
Tris-HCl pH 8.0, 0.15 M NaCl) and with 25% ethanol in distilled water. Radioactivity that remained in
the membrane was measured in Atomlight by using a liquid scintillation counter.
3.4. MCF-7 Cell Proliferation Assay
The human breast adenocarcinoma cell line MCF-7 was routinely cultivated in DMEM
◦
supplemented with 10% FBS and 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, and 100 IU/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 of 2000 cells per final volume of 100
stripped FBS and 100 IU/mL penicillin and 100 mg/mL streptomycin. After 24 h, the medium was
removed and 90 L of fresh medium and 10 L of drug solution, supplemented with serial dilutions of
µL DMEM supplemented with 5%
µ
µ
test compounds or DMSO as dilute control in the presence or absence of 0.1 nM E2, were added to
triplicate microcultures. Cells were incubated for four days. At the end of the incubation time, number
of cells was counted by using the WST-8, which was added to microcultures 10 µL each, and they were
incubated for 2–4 h. The absorbance at 450 nm was measured. This parameter relates to and number
of living cells in the culture.
3.5. Docking Simulation Study
Three-dimensional (3D) structures of protein-ligand complexes were predicted using the Discovery
Studio 2018/CDOCKER software (BIOVIA) with default settings. The 3D structures of ER used in this
study were retrieved from the RCSB Protein Data Bank (PDB ID: 1ERR). Missing hydrogen atoms in
the crystal structure were computationally added and the center of the active site was defined as the
center of raloxifen in 1ERR. The conformations of 4d and 5d were optimized using the CHARMm
force field, and the docking simulations of 4d and 5d with 1ERR were performed using the CDOCKER
protocol. The docking poses for 4d and 5d with the highest CDOCKER ENERGY were selected for the
discussions of 10 docking modes, respectively.
4. Conclusions
Novel sulfur-containing ER
therapeutic drug candidates were designed and synthesized based upon the hydrophobic feedback
approach for the simple bisphenols developed in our previous studies. Tetrahydrothiepine
derivatives (compounds 5a 5d) showed higher binding affinity toward ER than the corresponding
dihydrothiophene derivatives (compounds 4a 4d) because of the hydrophobicity of the
sulfur-containing ring structure. Although the bisphenol derivatives 4a 4c 5a, and 5c showed
α modulators (compounds 4 and 5) as potential breast cancer
1–3
–
α
–
,
,
estrogenic activity toward the MCF-7 breast cancer cell line, the corresponding alkylamino derivatives
(compounds 4d and 5d) acted as ER antagonists. In particular, compound 5d showed the most potent
antiestrogenic activity among the tested compounds with an IC50 value of 0.09 µM. A sulfur-containing
structure might be a promising scaffold for antiestrogen discovery, owing to ease of synthesis, binding
modes toward the ERα LBD, and availability of various substituted derivatives.

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Author Contributions: Conceptualization, K.O. and Y.E.; Data curation, A.K., F.T., S.A. and T.O.; Formal analysis,
K.O., A.K. and T.O.; Funding acquisition, K.O. and Y.E.; Investigation, K.O., A.K., F.T. and S.A.; Methodology, K.O.
and A.K.; Project administration, K.O.; Resources, K.O.; Software, A.K., F.T., S.A. and T.O.; Supervision, K.O. and
Y.E.; Validation, A.K.; Visualization, K.O. and A.K.; Writing—original draft, K.O.; Writing—review & editing, K.O.
Funding: This research was supported by a grant-in-aid from the Strategic Research Program for Private
Universities (2015–2019) and a grant-in-aid for Scientific Research (C) (26460151 and 15K08029) from the Japanese
Ministry of Education, Culture, Sports, Science, and Technology (MEXT).
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of all the compounds are available from the authors.
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2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).