Design and synthesis of novel benzothiophene analogs as selective estrogen receptor covalent antagonists against breast cancer

Article abstract of DOI:10.1016/j.ejmech.2021.113543

Endocrine therapy (ET) has benefited patients with estrogen receptor alpha (ERα) positive breast cancer for decades. Selective estrogen receptor modulator (SERM) such as Tamoxifen represents the clinical standard of care (SoC). Despite the therapeutic importance of current SoC agents, 30–50% of prolonged treatment patients inevitably generated resistant tumor cells, usually eventually suffered tumor relapse and developed into metastatic breast cancer (MBC), which was the leading cause of female cancer-related mortality. Among these, most resistant tumors remained dependent on ERα signaling, which reignited the need for the next generation of ERα related agents. We hypothesized that selective estrogen receptor covalent antagonists targeting ERα would provide a therapeutic alternative. In the current work, series of novel benzothiophene hybrids bearing electrophile moieties were synthesized and biologically evaluated. The representative analogue 15c exhibited potent anti-proliferative effect in MCF-7 cell lines in vitro, and further mechanism studies confirmed the necessity of covalent bonding. More importantly, 15c could attenuate the expression of TFF-1, GREB-1 and downregulate the levels of cellular ERα protein.

Full text of DOI:10.1016/j.ejmech.2021.113543

European Journal of Medicinal Chemistry 221 (2021) 113543
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Design and synthesis of novel benzothiophene analogs as selective
estrogen receptor covalent antagonists against breast cancer
,
,
,
,
Chengfeng Bai a b, Shengnan Ren a b, Shuangjie Wu a b, Meiqi Zhu a b, Guoshun Luo a,
,
*
Hua Xiang a
a State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing, 210009,
China
b Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing, 210009, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Endocrine therapy (ET) has benefited patients with estrogen receptor alpha (ERa) positive breast cancer
Received 9 February 2021
Received in revised form
4 May 2021
Accepted 10 May 2021
Available online 14 May 2021
for decades. Selective estrogen receptor modulator (SERM) such as Tamoxifen represents the clinical
standard of care (SoC). Despite the therapeutic importance of current SoC agents, 30e50% of prolonged
treatment patients inevitably generated resistant tumor cells, usually eventually suffered tumor relapse
and developed into metastatic breast cancer (MBC), which was the leading cause of female cancer-
related mortality. Among these, most resistant tumors remained dependent on ER
reignited the need for the next generation of ER related agents. We hypothesized that selective estrogen
receptor covalent antagonists targeting ER would provide a therapeutic alternative. In the current work,
a signaling, which
a
Keywords:
Breast cancer
Estrogen receptor alpha (ER
Endocrine-resistance
Covalent antagonist
Benzothiophene
a
series of novel benzothiophene hybrids bearing electrophile moieties were synthesized and biologically
evaluated. The representative analogue 15c exhibited potent anti-proliferative effect in MCF-7 cell lines
in vitro, and further mechanism studies confirmed the necessity of covalent bonding. More importantly,
a)
15c could attenuate the expression of TFF-1, GREB-1 and downregulate the levels of cellular ERa protein.
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
intramuscular injection [4]. Recently, considerable non-steroidal
SERDs with oral bioavailability are in clinical trials, addressing
both local breast cancer and peripheral metastases, such as LSZ102
(Novartis) [5], RAD1901 (Radius) [6], AZD9496 (AstraZeneca) [7],
GDC-0810 (Genentech) [8], etc. (Fig. 1). Notwithstanding these
successes in the clinic, intrinsic and acquired drug resistance re-
mains the persistent challenge. Generally, patients who generated
resistant tumors would inevitably progress to an antiestrogen-
resistant state [9], making TAM ineffective. Patients those
burdened with advanced endocrine metastatic diseases are
currently incurable and only have a less than 5-year survival [10].
Mechanisms of resistance have been included but not limited to
Breast cancer is the most common type of invasion cancers
among women all over the world. Nearly 70% of these patients
express ERa (encoded by ESR1) which is the critical driver in
oncogenic proliferation and metastasis [1,2]. Over the past 40 years,
the treatment has long been relied on endocrine therapies, tar-
geting ER
as tamoxifen (TAM) [3], or preventing the synthesis of estrogens
under aromatase catalysis. Despite TAM's clinical success, its ER
a either by blocking estrogen binding to the receptor, such
a
agonist activity observed in endometrium and associated with an
increased risk of developing endometrial cancer remain great
concerns. The efforts on creating novel ER
a
antagonists without
the following aspects: 1) aberrant expression of ER
a coactivators/
this risk finally led to the discovery of selective estrogen receptor
degrader (SERD), such as Fulvestrant (Fig. 1). However, considering
its poor physicochemical properties and oral bioavailability, Ful-
vestrant can only be clinically administered via monthly
corepressors [11]; 2) “cross-talk” between ER and other kinases
a
[12]; 3) hotspot mutations in ESR1 which occupied about 30% of
endocrine-resistance [13e15]. Of note is the resistance after TAM
treatment, it is associated with hotspot mutations activating of
Y537 S/C/N and D538G. Mutations that occurred in ligand binding
domain (LBD) could prompt activation of ER
a in a ligand-
independent manner, stabilize an active conformation of Helix 12
(H12), and favor the recruitment of coactivator even in the absence
* Corresponding author.
E-mail address: xianghua@cpu.edu.cn (H. Xiang).
https://doi.org/10.1016/j.ejmech.2021.113543
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

C. Bai, S. Ren, S. Wu et al.
European Journal of Medicinal Chemistry 221 (2021) 113543
2

C. Bai, S. Ren, S. Wu et al.
European Journal of Medicinal Chemistry 221 (2021) 113543
of estradiol (E2). Consequently, these mutations could result in
tumor resistance and reduced potency of antagonists [16]. For the
resistance settings, up to 90% of endocrine therapy-resistance tu-
linkers. On the other hand, influenced in part by the crystal struc-
ture of H3B-6545 bound to the ER LBD, various “Michael acceptors”
(amine electrophile moieties) that similar to H3B-6545 were
introduced and expected to interact with cysteine at 530 position of
Helix 11. To explore an optimal binding model and a meaningful
structure-activity relationship (SAR), both ethyl and azetidine
linkers were respectively applied in compounds 15(aef) and
21(aee), as depicted in Fig. 2. Moreover, through replacement of
the amide warheads, different acrylamide analogs (23a-c and 25a-
c) were also investigated. For the biological assessment, in order to
verify the pharmacophore role of “Michael acceptor,” compound
16, the saturated analogue of 15c, was designed as the negative
control and well biologically evaluated. Structure details of all the
newly prepared compounds were depicted in Table 1.
mors remain dependent on ER
tasis. Hence, there still exists an urgent need to develop novel
binding modes of ER antagonism and overcome resistance of
a signaling for survival and metas-
a
current SoC endocrine therapies.
Targeted covalent inhibitors (TCIs) have emerged as efficacious
pharmacological modalities and been demonstrated multiple ad-
vantages, including improved potency, selectivity, and efficacy over
conventional reversible inhibitors. As documented, the pocket
volume of ER ligand-binding is ~450 Å3 and suitable for ligands
250e380 Å3. Additionally, the plasticity of ER-LBD is reflected by
the diversity of ER ligand scaffolds and suits itself to discover novel
pharmacological modulators [17]. Consistent with previous reports,
four cysteines (amino acids 381, 417, 447, and 530) in LBD were
2.2. Chemistry
demonstrated as covalent targets of ER
a [18]. Further analysis of the
X-ray crystal structure of ER confirmed that both C381 and C530
a
Synthesis of synthons 2, 4, and 6(aef) followed the procedures
depicted in Scheme 1. For the construction of compound 2, 4-
benzyloxyphenol was reacted with tert-butyl (2-bromoethyl)
carbamate in the presence of CS2CO3, followed by deprotection of
benzyl group under Pd/C and H2, intermediate 4 was obtained with
the similar method.
Bromination of (E)-but-2-enoic acid (compound 5) occurred in
the presence of NBS and benzoyl peroxide. Subsequently, in-
termediates 6(aef) were obtained by coupling different types of
amines to carboxyl of compound 5 (Scheme 1).
could be engaged by an electrophile in the ligand-binding pocket,
while only C530 is directly related to ligand-dependent activation
and suitable for the discovery of effective antagonists [19,20].
Mechanistically, the covalent interaction targeting a nonconserved
cysteine at 530 position of Helix 11 might overcome the stabilizing
effects of mutations [21]. Consistent with the hypothesis, Puyang
et al. (2018) identified a representative selective estrogen receptor
covalent antagonist (SERCA) named H3B-6545 (Fig. 1). The novel
antagonist emerged with the ability to inactivate both ER
ER
MUT (e.g., Y537S, D538G) in vitro and in vivo, through enforcing a
unique antagonist conformation in ER . Crystal structure of H3B-
a
WT and
a
Reagents and conditions. (a) tert-butyl (2-bromoethyl)carba-
mate, Cs2CO3, DMF, 50 ꢀC. (b) H2, Pd/C, MeOH, rt. (c) 1-Boc-3-iodo-
azetidine, Cs2CO3, DMF, 130 ꢀC. (d) H2, Pd/C, MeOH, rt. (e). NBS,
benzoyl peroxide, CCl4. (f) Oxalyl chloride, Na2CO3, amine, CH2Cl2.
Reagents and conditions. (a) KOH, EtOH. (b) PPA, 110e120 ꢀC.
(c) N-bromoacetamide, CH2Cl2. (d) H2O2, CF3COOH, CH2Cl2. (e)
Compound 2, Cs2CO3, DMF, 50 ꢀC. (f) LiAlH4, THF, 0 ꢀC (g) HCl/
MeOH, rt. (h) 6(aef), DIPEA, DMF. (i) BBr3, CH2Cl2, 0 ꢀC. (j) H2, Pd/C,
MeOH.
a
6545 bound to the ER LBD showed that the continuous electron
density between C530 and the Michael acceptor in the cocrystal
structure was observed, which directly confirmed the covalent
engagement. Currently, H3B-6545 has been evaluated in an
ongoing phase 1/2 study in women with locally advanced or met-
astatic ERþ, HER2-breast cancer (NCT03250676). The compelling
activity of H3B-6545 supports the notion of a covalent antagonist,
and it also shares some pharmacologic features with SERMs
[22e24].
Over the past decades, our group engaged in discovering novel
small molecule agents targeting ER-related diseases. For the pre-
sent study, we identified a series of novel SERCAs that bearing novel
skeletons and more satisfactory biological activity.
Reagents and conditions. (a) Compound 4, Cs2CO3, DMF, 50 ꢀC.
(b) LiAlH4, THF, 0 ꢀC (c) HCl/MeOH, rt. (d) 6(aee), DIPEA, DMF. (e)
BBr3, CH2Cl2, 0 ꢀC.
Reagents and conditions. (a) various acid chlorides, CH2Cl2,
DIPEA. (b) BBr3, CH2Cl2, 0 ꢀC.
Scheme 2 outlined the synthesis of target compounds 15(aef)
and the saturated analogue 16, which was utilized as the negative
control in the bioassay assays. As documented, through nucleo-
philic substitution and cyclization reaction, compound 8 was ob-
tained in good yield [30]. As initially expected, bromination of 8
should be accomplished with N-bromosuccinimide, followed by
introducing fragment 2 or 4 to the core. Unfortunately, both com-
pounds 2 and 4 failed to couple with the brominated intermediate 9
through nucleophilic aromatic substitution reaction. In order to
activate the reaction site, the thiophene fragment of compound 9
was oxidized to the corresponding sulfoxide 10. Desired nucleo-
philic substitution was accomplished without formation of obvious
by-products. Having served its functionality, the sulfoxide group of
11 was removed via reduction in the presence of LiAlH4. Subse-
quently, deprotection of Boc protecting group with HCl/MeOH so-
lution afforded compound 13, which was then undergoing SNAr
reaction with various synthons 6(a-f) to obtain compounds
14(aef). Finally, desired compounds 15(aef) were obtained
through demethylation in the presence of BBr3/CH2Cl2 solution.
Additionally, the negative control 16 was obtained through the
hydrogenation of 15c. Synthesis details for 21(aee) were outlined
in Scheme 3, which were quite similar to the procedure of 15(aef).
Meanwhile, amide analogs 23(aec) and 25(aec) were prepared
from compounds 13 and 19 through coupling with the
2. Results and discussion
2.1. Structure design
To date, Raloxifene (Fig. 1) has been applied in the therapy of
breast cancer and postmenopausal osteoporosis for decades. Its
benzothiophene scaffold was deemed safe and valid in the design
of novel ER-targeted ligands [25]. With the purpose of improving
potency and pharmacokinetic (PK) properties, various chemical
modifications have been accomplished and yielded different ER
ligands with satisfactory activity. For instance, arzoxifene [26,27],
G1T48 (G1) [28] and LSZ-102 (Novartis) [5] have been demon-
strated as potent antineoplastic agents across breast cancer model
(Fig. 1).
Based on structural knowledge gained from Raloxifene, its
benzothiophene scaffold was chosen as the core structure, as
depicted in Fig. 2. Additionally, fluorine substituted at the 40-posi-
tion of the skeleton was expected to attenuate the toxicity of ligand
while maintaining intrinsic ER
activity [29]. To further improve the anti-ER
a
binding affinity and modulating
potency of designed
a
antagonists, our chemistry efforts on structural optimization
focused on modifying the electrophile warheads and length of
3

C. Bai, S. Ren, S. Wu et al.
European Journal of Medicinal Chemistry 221 (2021) 113543
Fig. 2. Design of novel benzothiophene-based SERCAs, compound 15c was constituted with the core structure that similar to Raloxifene and side chains common to H3B-6545.
Table 1
Antiproliferation activity of analogs in breast cancers.
R1
R2
R3
R4
Antiproliferation (IC50
, mM)
Comp.
MCF-7
Ishikawa
MDA-MB-231
15a
15b
15c
H
-CH3
-CH3
-CH3
e
-
-
e
-
-
16.1
13.83
7.34
15.53
11.91
6.56
>50
>50
>50
15d
15e
15f
-
-
-
-
-
-
7.61
8.24
16.38
11.25
18.55
8.43
>50
>50
>50
21a
21b
21c
H
-CH3
-CH3
-CH3
-
-
-
-
-
-
>25
22.33
>25
>25
11.21
5.45
>50
>50
>50
21d
21e
-
-
-
-
8.45
8.90
9.15
9.84
>50
>50
23a
23b
23c
25a
25b
25c
16
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
H
H
-CH3
H
H
-CH3
-
-
-
H
-CH3
H
H
-CH3
H
-
-
-
22.96
>25
21.65
23.53
>25
27.96
>25
19.94
-
>25
>25
12.44
12.26
14.20
9.40
>25
12.39
16.78
>50
>50
>50
>50
>50
>50
>50
>50
>50
Raloxifene
Tamoxifen
a. IC50: Concentration that inhibits 50% of cell growth.
corresponding amine and demethylation reactions, details outlined
in Scheme 4.
4

C. Bai, S. Ren, S. Wu et al.
European Journal of Medicinal Chemistry 221 (2021) 113543
2.3. Antiproliferative activity in vitro
The in vitro antiproliferative efficacy of all the newly prepared
compounds was evaluated by Cell Counting Kit-8 (CCK-8) assay in
MCF-7, Ishikawa, and MDA-MB-231 (ERa-) cells. Results expressed
as IC50 were summarized in Table 1, and both Raloxifene and
Tamoxifen were used as the baseline control.
The result showed that most of the synthesized compounds
exerted moderate to potent antiproliferative activity. Of the ethane
series, compound 15c showed significantly more potent activity
than Raloxifene against both MCF-7 and Ishikawa cells
(IC50 ¼ 7.34
mM and 6.56 mM, respectively). On the other hand,
compounds 15d and 15e bearing different terminal amides
exhibited very similar potency and efficacy to 15c. To further
explore the necessity of electrophile moiety in 15c, compound 16
designed from 15c was synthesized and evaluated in vitro. The
saturated analogue 16 notably abolished the anti-proliferative ac-
tivity previously observed in 15c, revealing the indispensable
pharmacophore role of electrophile moiety in 15c.
Additionally, compounds 21(aee), through substituting the
ethane linker of 15(aef) for azetidines, were also investigated. As
shown in Table 1, 21d and 21e showed almost 2e3 folds more
potent activity than Raloxifene against both MCF-7 and Ishikawa
cells, while marginally inferior to 15d and 15e. As also observed for
21(aec), all these alterations had a slightly negative effect on anti-
proliferation potency relative to their corresponding ethane de-
rivatives, suggesting that ethane linker was conducive to antitumor
activity. On account of the non-significant activity difference be-
tween 15e and 15f, only 21e that corresponding to analogue 15e
and with morpholine “tail”, was synthesized and biologically
evaluated. A closer analysis of the binding model between 15c and
21c through molecular docking indicated that ethane linker facili-
tated the electrophile moiety closer to target residue Cys 530
(Fig. 6c).
Besides, reversing the carboxyl from terminal acrylamides to
directly coupling to the linker led to entirely different “warheads”,
compounds 23(aec) and 25(aec). However, a notable activity
reduction was observed. Having evaluated the antiproliferative
activity on ER
negative cells (MDA-MB-231) was accomplished. As expected, all
the synthesized compounds resulted with IC50 > 50 M.
a positive cells, a further investigation focused on ERa
m
These above-mentioned results collectively revealed that 15c
was a potent inhibitor for ER þ tumor cells. More importantly, its
antiproliferative activity was closely related to its electrophile
moiety.
2.4. ERa binding affinities of the synthetic compounds
To further assess whether the attractive activity of analogs was
correlated with the interaction between ER and the ligands, we
evaluated the ER binding affinities of these agents via a fluores-
a
a
cence polarization protocol. (ER Alpha Competitor Assay Kit,
Green).
Several compounds with more potent antiproliferation than
Raloxifene were chosen and tested at the concentration of 1
results illustrated in Table 2 (see Table 2).
mM,
As shown in Table 2, all the tested compounds showed higher
binding affinity for ER than the positive control Raloxifene, among
a
which compound 15c exhibited the most potent binding inhibition
effect. Additionally, the ethane series 15d, 15e, and 15f were more
potent than azetidine derivatives 21d and 12e, which was consis-
tent with the in vitro anti-proliferative activity. Based on the data
collected in antiproliferative activity and ER
prioritized for further study.
a binding assay,15c was
5

C. Bai, S. Ren, S. Wu et al.
European Journal of Medicinal Chemistry 221 (2021) 113543
6

C. Bai, S. Ren, S. Wu et al.
European Journal of Medicinal Chemistry 221 (2021) 113543
2.5. Effects of compound 15c on ER
a level of MCF-7 cell
To profile the impact of covalent engagement on cellular ER
a
protein, both western blotting and immunofluorescence micro-
scopy were utilized. Immunofluorescence microscopy analysis was
performed at concentration 5
tential effects of compound 15c on downregulating the ER
(Fig. 3A and B). Further investigation at lower concentrations was
accomplished by western blotting assay in the ER
WT-expressing
MCF-7 parental cell line. In contrast to Fulvestrant, following 24 h
treatment, compound 15c showed slightly decreased effects on ER
level at 0.5 M (Fig. 3c).
mM, and the result revealed the po-
a
level
a
a
m
2.6. Effects of compound 15c on ERa-dependent transcription
To further profile the impact of covalent interaction, quantita-
tive real-time polymerase chain reaction (RT-PCR) was adopted to
interrogate gene expression of two independent ER
GREB1 and TFF1. Estradiol (E2) was used to stimulate ER
a
target genes,
signaling
a
in MCF-7 cells before co-incubated with compounds. As expected,
compound 15c could dramatically decrease GREB1 and TFF1 gene
expression in a dose-dependent manner. Simultaneously, com-
pound 16 showed distinctly reduced potency relative to 15c. These
data collectively revealed that the cellular potency observed for 15c
was driven by its covalent binding (Fig. 4).
2.7. Potential SERMs-like activity of 15c on Ishikawa cells
It is pretty clear that 4-hydroxytamoxifen (4-OHT) could induce
ERa activity and stimulate signaling in Ishikawa endometrial car-
cinoma lines, result noted by the increased expression of PGR and
proliferation of tumor cells [31].
After three days' exposure to 15c in Ishikawa cells, both 15c and
16 significantly inhibited the expression of PGR in E2-stimulated
models (Fig. 5B). Additionally, following 3 days' incubation with
15c at tested concentrations varying from 156 nM to 5000 nM,
proliferation data showed no inducing impact on Ishikawa prolif-
eration, which was in sharp contrast to 4-OHT (Fig. 5A). These data
collectively indicated that 15c didn't share the potential liability of
SERMs in endometrial carcinoma cells.
2.8. Compound 15c was dependent on covalent engagement for
enhanced ERa antagonism
To investigate the binding model of 15c, molecular docking was
utilized to reveal the details of the interaction between ER and 15c
a
(PDB code: 6chw). As shown in Fig. 6A, the hydrogen-bonding
network occurred between hydroxyl moiety of A-ring and resi-
dues Arg394, Glu353, as well as an irreversible covalent engage-
ment of electrophile targeting Cys530 was observed (Fig. 6A).
Comparisons of docking details between saturated analogue 16 and
15c confirmed similar binding modes for core structure, while
linker without electrophile appeared higher degree of flexibility, as
shown in Fig. 6B.
Furthermore, to elucidate the binding site of 15c, a model re-
action between 15c and cysteine was carried out under basic con-
ditions, simulating the interaction between C530 residue, the
product was analyzed using 1H NMR. The disappearance of
hydrogen in double bond of Michael acceptor demonstrated that
cysteine reacted with the
b carbon atom of a, b-unsaturated
carbonyl in 15c (Fig. S1).
7

C. Bai, S. Ren, S. Wu et al.
European Journal of Medicinal Chemistry 221 (2021) 113543
Scheme 4. Synthesis of compounds 23 (aec) and 25 (aec).
2.9. Compound 15c induced cell cycle G0/G1 phase arrest and
apoptosis in MCF-7 cells
Table 2
ERa binding affinity of compounds 15c, 15d, 15e, 15f, 21d, and 21e.
Comp.
Inh% (1
m
M)a
Comp.
Inh% (1 m
M)a
Having demonstrated that the covalent engagement drove the
antiproliferative effects of 15c. Next, we further investigated the
impact of 15c on the cell progression of MCF-7. As illustrated in
Fig. 7B, the cell cycle was blocked at G0/G1 phase in a dose-
dependent manner in MCF-7 cells, and percentage of cells in G0/
G1 phase varied from 22.44 to 42.95%. To profile whether growth
inhibition of breast cancer cells was related to induction of
apoptosis, 15c-induced MCF-7 cells were stained with Annexin V-
FITC and propidium iodide (PI). The result indicated that 15c could
15c
15d
15e
15f
98.48
97.87
90.57
85.81
21d
21e
Raloxifene
88.80
86.81
74.81
a
Percent inhibition of each compound was calculated from the polarization
values.
Fig. 3. Effects of 15c on ERa level. (A) Immunofluorescence microscopy. MCF-7 cells were treated for 24 h with 15c at 5 mM. (B) fluorescence intensity of ERa. (C) Western blot
analysis. MCF-7 cells were incubated with 15c and Fulvestrant respectively at 125, 250 and 500 nM for 24 h.
8

C. Bai, S. Ren, S. Wu et al.
European Journal of Medicinal Chemistry 221 (2021) 113543
Fig. 4. Dose-response suppression of E2-stimulated model. (A) Expression of TFF-1 gene when treated with 15c and 16 respectively at 0.25, 0.5, 1, and 10
1 genes when treated with 15c and 16 at the indicated concentration. MCF-7 cells were co-incubated with agents for 24 h before determined.
mM. (B) Expression of GREB-
Fig. 5. (A). Proliferation data for Ishikawa cells following two days of treatment respectively with compound 4-OHT or 15c at 156, 312, 625, 1250, 2500, and 5000 nM. Data
presented as mean fold change relative to DMSO treatments. (B). Dose-response suppression of E2-stimulated expression of PGR genes by compound 15c and 16 at 0.25, 0.5, 1, and
10 mM.
induce a dose-dependent increment in both early- and late-stage
apoptosis of MCF-7 cells (2, 11.7, and 25.7%, respectively). (Fig. 7A).
significant concerns. Most recently, evidence demonstrated that
SERCAs, through targeting C530 residue of ER , might inactivate
ER -positive tumors and provide an alternative approach for pa-
tients bearing current endocrine resistance.
Our present study identified a series of novel covalent antago-
nists with preferable binding model to ER . Most of them acted as
suitable ER ligands and displayed excellent antagonistic activity,
among which compound 15c showed significant potential in ER
antagonistic effect. Moreover, 15c showed downregulated impacts
on the level of cellular ER
Mechanically, the loss of potency in compound 16 that relative
to 15c, assisted to reveal the indispensable role of Michael accep-
tors. Meanwhile, the covalent engagement between C530 residue
and 15c was also clearly observed in docking models. Besides, the
reaction model between 15c and cysteine under basic condition
confirmed the binding site of 15c, simulating the interaction be-
tween ER residue.
a
a
2.10. Early ADME properties assessment of 15c in vitro
a
We finally set out to assess in vitro ADME properties of 15c,
evaluating its pharmacological performance as a candidate drug. As
shown in Table 3, the evaluation of 15c thorough standard micro-
somal stability and plasma stability assay suggested that the half-
life of metabolism was moderate, and it was especially quite sta-
ble in rat plasma. Comprehensively considering the two tests,
compound 15c possessed well in vitro pharmacodynamic
parameters.
a
a
a
.
3. Conclusion
For the long term of breast cancer endocrine therapy, SERMs and
SERDs are standard-of-care representatives. However, both the
high recurrence rate and undeniable side effects remained
Our current study extends the understanding and discovery of
ERa-targeted covalent antagonists. Further studies focusing on the
9

C. Bai, S. Ren, S. Wu et al.
European Journal of Medicinal Chemistry 221 (2021) 113543
Fig. 6. (A). Compound 15c was docked to ER LBD (PDB ID: 6chw), demonstrating covalent engagement with C530 of ERa. (B). Comparison of docking details between 15c and 16.
The red structure stands for compound 16, and the gray structure stands for 15c. (C). Docking details between 15c and 21c. Distance between Cys530 residue and Michael acceptor
was measured, resulting with 4.3 Å and 4.7 Å individually. The yellow structure stands for compound 21c, and the gray structure stands for compound 15c.
improvement of in vivo activity and direct covalent binding evi-
dence have been accomplished, and additional findings will be
reported in due course.
with water and resulting mixture was extracted with CH2Cl2
(2 ꢁ 20 mL). The combined organic layer was dried over anhydrous
Na2SO4 and concentrated under vacuum to deliver the title com-
pound as white solid (620 mg, 72% yield).
Tert-butyl (2-(4-hydroxyphenoxy)ethyl)carbamate (2)
4. Experimental section
To a solution of compound 1 (1 g, 2.9 mmol) dissolved in 10 mL
MeOH, a catalytic amount of Pd/C was added. The solution was
stirred at rt under H2 protected for 2h. After completion, the re-
action mixture was filtered, filtrate was concentrated to dryness
and purified by column chromatography (SiO2, CH2Cl2: MeOH ¼ 15
: 1e10 : 1) to obtain compound 2 (515 mg, 70% yield) as off-white
4.1. General
All chemicals and solvents were purchased from Energy-
chemical, Sigma-Aldrich, and Alladin-chemistry, and used
without further purification. 1H NMR and 13C NMR spectra were
recorded with a Bruker Avance 300 MHz/400 MHz spectrometer at
300 K, using TMS as an internal standard. NMR chemical shifts are
solid. 1H NMR (300 MHz, CDCl3)
d 6.95e6.80 (m, 4H), 4.03 (t,
J ¼ 5.1 Hz, 2H), 3.60e3.57 (m, 3H), 1.58 (s, 9H).
Tert-butyl
3-(4-(benzyloxy)phenoxy)azetidine-1-carboxylate
described in
d (ppm) using residual solvent peaks as standard
(3)
(CDCl3, 7.26 ppm (1H), 77.16 ppm (13C); CD3OD, 3.31 ppm (1H),
49.00 ppm (13C); DMSO‑d6, 2.50 ppm (1H), 39.52 ppm (13C). MS
spectra was recorded on a Shimadzu GC-MS 2050 (ESI) or an Agi-
lent 1946A-MSD (ESI) Mass Spectrum. The TianGuang RY-1 appa-
ratus was used to test melting points (m.p.).
4-(benzyloxy)phenol (500 mg, 2.5 mmol) and tert-butyl 3-
iodoazetidine-1-carboxylate (849 mg, 3 mmol) were dissolved in
10 mL DMF, CS2CO3 (1.63 g, 5 mmol) was added. The solution was
stirred at 130 ꢀC for 3 h. After completion, the reaction was
quenched with water and resulting mixture was extracted with
CH2Cl2 (2 ꢁ 20 mL). The combined organic layer was dried and
concentrated under vacuum to deliver the title compound as white
solid which was used in the next step without further purification.
Tert-butyl (2-(4-(benzyloxy)phenoxy)ethyl)carbamate (1)
4-(benzyloxy)phenol (500 mg, 2.5 mmol) and tert-butyl (2-
bromoethyl) carbamate (669 mg, 3 mmol) were dissolved in
10 mL DMF, CS2CO3 (1.63 g, 5 mmol) was added. The solution was
stirred at 50 ꢀC for 3 h. After completion, the reaction was quenched
1H NMR (300 MHz, CDCl3)
d 7.46e7.29 (m, 5H), 6.94e6.86 (m, 2H),
10

Fig. 7. Compound 15c induced cell cycle G0/G1 phase arrest and apoptosis in MCF-7 cells. (A) After treated with 15c for 24 h, MCF-7 cells were harvested and stained with PI, analyzed by flow cytometry. (B) After treated with 15c at 1, 5,
and 10 M for 48 h, flow cytometry analysis was performed in MCF-7 cells.
m

C. Bai, S. Ren, S. Wu et al.
European Journal of Medicinal Chemistry 221 (2021) 113543
Table 3
Rat plasma and liver microsome were used in the two assays, Ketanserin and Benfluorex were used as the control. -: Stands for not performed.
Compd.
LogD
T
1/2 (min, plasma)
T1/2 (min, Liver microsomes)
CLint (mL/min/Kg)
Ketanserin
Benfluorex
15c
e
e
18.83
e
131.91
e
e
28.31
1017.95
1.67
38.15
65.11
6.71e6.63 (m, 2H), 5.01 (s, 2H), 4.81e4.79 (m, 1H), 4.26e4.24 (m,
2H), 3.99e3.96 (m, 2H).
partitioned between water and ethyl acetate. The aqueous layer
was isolated and extracted several times with ethyl acetate, com-
bined extracts were washed with consecutive portions of 10% HCl
aqueous solution, water and saturated NaHCO3 aqueous solution,
dried over anhydrous Na2SO4. The final crude product was used in
the next step without further purification. 1H NMR (400 MHz,
Tert-butyl 3-(4-hydroxyphenoxy)azetidine-1-carboxylate (4)
To a solution of compound 3 (1 g, 2.8 mmol) dissolved in 10 mL
MeOH, catalytic amount of Pd/C was added. The solution was stir-
red at rt under H2 protected for 2h. The reaction mixture was
filtered, and filtrate was concentrated to dryness and purified by
column chromatography (CH2Cl2: MeOH ¼ 15 : 1e10 : 1) to obtain
compound 4 (800 mg, 80% yield). 1H NMR (300 MHz, CDCl3)
CDCl3)
d 7.99e7.94 (m, 2H), 7.21e7.10 (m, 3H), 6.98e6.90 (m, 2H),
6.76e6.75 (m, 1H), 4.24 (s, 2H), 3.77 (s, 3H).
2-(4-fluorophenyl)-6-methoxybenzo[b]thiophene (8)
A 50 mL beaker fitted with a mechanical stirrer was placed in an
oil bath with temperature of 85 ꢀC, polyphosphoric acid (50 g) was
d
6.81e6.73 (m, 2H), 6.62e6.54 (m, 2H), 4.77e4.73 (m, 1H),
4.30e4.16 (m, 2H), 3.97e3.94 (m, 2H).
(E)-4-bromo-N-methylbut-2-enamide (6a)
placed
into
the
beaker.
1-(4-fluorophenyl)
2-(3-
To
a
solution of (E)-4-bromobut-2-enoic acid (500 mg,
methoxyphenylsulfanyl) ethanone (5 g, 18 mmol) was added
portion wise at a rate such that the temperature never exceeded
100 ꢀC. After the addition was completed, the reaction mixture was
stirred at ~115 ꢀC for 1 h, and followed by slowly poured into the
rapidly stirring ice water. The aqueous solution was extracted
several times with ethyl acetate, and the combined organic layer
was successively washed with water, saturated NaHCO3 aqueous
solution, brine, and dried over anhydrous Na2SO4. The organic layer
was concentrated in vacuo, and the residue was purified by flash
chromatography (CH2Cl2: MeOH ¼ 30 : 1e25 : 1) to give the desired
compound as white solid (3.2 g, 68% yield). 1H NMR (300 MHz,
3.0 mmol) dissolved in 10 mL CH2Cl2 under N2 protected at 0 ꢀC,
0.1 mL DMF and oxalyl chloride (571 mg, 4.5 mmol) were added.
The solution was stirred at 0 ꢀC for 2 h, followed by addition of
Na2CO3 (954 mg,
9 mmol) and methylamine hydrochloride
(303 mg, 4.5 mmol). Resulting solution was stirred at 0 ꢀC until
completion. The mixture was quenched with 10 mL H2O and then
extracted with CH2Cl2. Organic layer was washed with 2 ꢁ 20 mL
brine, then dried and concentrated, purified by column chroma-
tography (SiO2, CH2Cl2: MeOH ¼ 15 : 1e10 : 1) to obtain the title
compound as yellow solid (358 mg, 67% yield). 1H NMR (300 MHz,
CDCl3)
d
6.94e6.84 (m, 1H), 6.05e5.99 (m, 1H), 4.02 (d, J ¼ 7.2 Hz,
DMSO‑d6)
2H), 7.01e6.97 (m, 1H).
d
7.79e7.70 (m, 4H), 7.56 (d, J ¼ 2.3 Hz,1H), 7.34e7.27 (m,
2H), 2.88 (d, J ¼ 4.9 Hz, 3H).
(E)-4-bromo-N, N-dimethylbut-2-enamide (6b)
Compounds 6b was synthesized with the procedure similar to
3-bromo-2-(4-fluorophenyl)-6-methoxybenzo[b]thiophene (9)
To a solution of 2-(4-fluorophenyl)-6-methoxybenzo [b]thio-
phene (2.58 g, 10 mmol) dissolved in 30 mL CH2Cl2, N-Bromoace-
tamide (1.46 g, 10.5 mmol) was added dropwise. The mixture was
stirred at room temperature for 1 h, the solvent was removed in
vacuo. Subsequently, the residue was titrated with ethanol and
filtered to give desired compound as a white solid (3.13 g, 92%
6a (53% yield).1H NMR (400 MHz, CDCl3)
d 6.88e6.83 (m, 1H),
6.57e6.53 (m, 1H), 4.19e4.02 (m, 2H), 3.08 (s, 3H), 3.00 (s, 3H).
(E)-1-(azetidin-1-yl)-4-bromobut-2-en-1-one (6c)
Compounds 6c was synthesized with the procedure similar to
6a (57% yield). 1H NMR (300 MHz, CDCl3)
d 7.0e6.82 (m, 1H),
6.22e5.83 (m, 1H), 4.33e4.05 (m, 6H), 2.34e2.29 (m, 3H).
(E)-4-bromo-1-(pyrrolidin-1-yl)but-2-en-1-one (6d)
yield). 1H NMR (300 MHz, DMSO‑d6)
(m, 2H), 7.18e7.14 (m, 1H), 3.85 (s, 3H).
3-bromo-2-(4-fluorophenyl)-6-methoxybenzo[b]thiophene 1-
oxide (10)
d 7.79e7.64 (m, 4H), 7.42e7.35
Compounds 6d was synthesized with the procedure similar to
6a (73% yield).1H NMR (400 MHz, CDCl3)
d 7.01e6.93 (m, 1H),
6.43e6.39 (m, 1H), 4.21e4.19 (m, 2H), 3.58e3.53 (m, 4H), 2.02e1.96
(m, 2H), 1.92e1.88 (m, 2H).
Trifluoacetic acid (10 mL) was added dropwise to a solution of 3-
bromo-2-(4-fluorophenyl)-6-methoxybenzo [b]thiophene (2.4 g,
7 mmol) in 10 mL of anhydrous CH2Cl2. After the mixture was
stirred for 5 min, H2O2 (1.0 mL, 7 mmol, 30% aqueous solution) was
added dropwise, and the resulting mixture was stirred for 2 h at
room temperature. Sodium bisulfite (0.3 g) was added into the
solution followed by 5 mL of water. The mixture was stirred
vigorously for 15 min and then concentrated in vacuo. The residue
was partitioned between CH2Cl2 and saturated NaHCO3 aqueous
solution. Combined organic layer was then dried over anhydrous
Na2SO4 and concentrated in vacuo to give 2.1 g (84% yield) of the
(E)-4-bromo-1-morpholinobut-2-en-1-one (6e)
Compounds 6e was synthesized with the procedure similar to
6a (59% yield). 1H NMR (400 MHz, DMSO‑d6)
d 6.83e6.66 (m, 2H),
4.43e4.22 (m, 2H), 3.60e3.54 (m, 6H), 3.52e3.48 (m, 2H).
(E)-4-bromo-1-(piperidin-1-yl)but-2-en-1-one (6f)
Compounds 6f was synthesized with the procedure similar to 6a
(41% yield). 1H NMR (300 MHz, CDCl3)
d 6.83e6.79 (m, 1H),
6.58e6.40 (m, 1H), 4.21e4.01 (m, 2H), 3.52e3.48 (m, 4H), 1.59e1.47
(m, 6H).
1-(4-fluorophenyl)-2-((3-methoxyphenyl)thio)ethan-1-one (7)
3-Methoxybenzenethiol (1 g, 7.1 mmol) was added in one
portion to a freshly prepared solution of 7.5 mL of ethanol, 3 mL of
water, and 470 mg of KOH (8.4 mmol). The solution was cooled to
5e10 ꢀC. followed by addition of 2-bromo-1-(4-fluorophenyl)
ethanone (1.54 g, 7.1 mmol) solution in 2.5 mL of EtOAc at a rate
such that the temperature did not exceed 25 ꢀC. The final mixture
was allowed to stir overnight at room temperature. After comple-
tion, removed solvent under reduced pressure, and the residue was
desired compound. 1H NMR (300 MHz, DMSO‑d6)
d 7.83e7.79 (m,
3H), 7.63e7.60 (m, 1H), 7.46e7.39 (m, 2H), 7.33e7.31 (m, 1H), 3.90
(s, 3H).
Tert-butyl (2-(4-((2-(4-fluorophenyl)-6-methoxy-1-oxidobenzo
[b]thiophen-3-yl)oxy)phenoxy)ethyl)carbamate (11)
To a solution of 3-bromo-2-(4-fluorophenyl)-6-methoxybenzo
[b]thiophene 1-oxide (2 g, 5.7 mmol) dissolved in 15 mL DMF,
C
S2CO3 (3.7 g, 11.4 mmol) and tert-butyl (2-(4-hydroxyphenoxy)
ethyl)carbamate (1.44 g, 5.7 mmol) were added. The solution was
12

C. Bai, S. Ren, S. Wu et al.
European Journal of Medicinal Chemistry 221 (2021) 113543
stirred at 50 ꢀC for 3 h and quenched with NH4Cl aqueous solution,
extracted with CH2Cl2 and collected organic layer was washed with
10% LiCl aqueous solution. The combined organic layer was dried
over anhydrous Na2SO4 and concentrated to dryness to obtain the
crude product which was purified by column chromatography
(SiO2, n-hexane: ethyl acetate ¼ 10:1e5:1) to give title compound
as white solid (2.49 g, 82% yield). 1H NMR (300 MHz, CDCl3)
(49% yield). 1H NMR (300 MHz, CDCl3)
d 7.75e7.71 (m, 2H), 7.29 (d,
J ¼ 2.1 Hz, 2H), 7.08e7.05 (m, 2H), 6.97e6.80 (m, 6H), 6.14e6.12 (m,
1H), 4.24 (d, J ¼ 8.4 Hz, 2H), 4.11e4.09 (m, 4H), 3.91 (s, 3H),
3.61e3.57 (m, 2H), 3.11 (t, J ¼ 4.9 Hz, 2H), 2.34e2.28 (m, 2H).
(E)-4-((2-(4-((2-(4-fluorophenyl)-6-methoxybenzo[b]thio-
phen-3-yl)oxy)phenoxy)ethyl)amino)-1-(pyrrolidin-1-yl)but-2-
en-1-one (14d)
d
7.71e7.66 (m, 2H), 7.45e7.44 (m, 1H), 7.02e6.83 (m, 6H),
Compound 14d was synthesized using the similar method to
6.78e6.67 (m, 2H), 3.92e3.88 (m, 2H), 3.47e3.41 (m, 2H), 1.39 (s,
9H).
Tert-butyl (2-(4-((2-(4-fluorophenyl)-6-methoxybenzo[b]thio-
phen-3-yl)oxy)phenoxy)ethyl)carbamate (12)
14a (41% yield). 1H NMR (300 MHz, CDCl3)
d 7.75e7.66 (m, 2H),
7.26e7.21 (m, 2H), 7.08e6.99 (m, 2H), 6.98e6.91 (m, 1H), 6.90e6.84
(m, 3H), 6.82e6.75 (m, 2H), 6.31e6.28 (m, 1H), 4.01 (t, J ¼ 5.0 Hz,
2H), 3.86 (s, 3H), 3.54e3.46 (m, 6H), 3.00 (t, J ¼ 5.0 Hz, 2H),
2.01e1.85 (m, 4H).
(E)-4-((2-(4-((2-(4-fluorophenyl)-6-methoxybenzo[b]thio-
phen-3-yl)oxy)phenoxy)ethyl)amino)-1-morpholinobut-2-en-1-
one (14e)
To
a
solution of tert-butyl (2-(4-((2-(4-fluorophenyl)-6-
[b]thiophen-3-yl)oxy)phenoxy)ethyl)
methoxy-1-oxidobenzo
carbamate (2 g, 3.81 mmol) dissolved in 10 mL THF, LiAlH4
(289.6 mg, 7.62 mmol) was added at 0 ꢀC under N2 protected. After
30 min, the reaction was quenched with NH4Cl aqueous solution
and then extracted with CH2Cl2, the combined organic layer was
collected and concentrated to dryness, purified by column chro-
matography (SiO2, n-hexane: ethyl acetate ¼ 10 : 1e5 : 1) to obtain
the desired compound (1.7 g, 90% yield). 1H NMR (300 MHz, CDCl3)
Compound 14e was synthesized using the similar method to 14a
(39% yield). 1H NMR (300 MHz, CDCl3)
d 7.79e7.70 (m, 2H), 7.30 (d,
J ¼ 2.0 Hz, 2H), 7.12e7.04 (m, 2H), 7.03e6.95 (m, 1H), 6.95e6.90 (m,
3H), 6.86e6.79 (m, 2H), 6.49e6.43 (m, 1H), 4.06e4.01 (m, 2H),
3.75e3.69 (m, 6H), 3.62e3.59 (m, 2H), 3.55e3.49 (m, 2H),
3.09e3.02 (m, 2H).
d
7.74e7.66 (m, 2H), 7.26e7.23 (m, 2H), 7.09e6.97 (m, 2H),
6.88e6.86 (m, 3H), 6.80e6.75 (m, 2H), 3.94 (t, J ¼ 5.1 Hz, 2H), 3.86
(E)-4-((2-(4-((2-(4-fluorophenyl)-6-methoxybenzo[b]thio-
phen-3-yl)oxy)phenoxy)ethyl)amino)-1-(piperidin-1-yl)but-2-en-
1-one (14f)
(s, 3H), 3.52e3.47 (m, 2H), 1.44 (s, 9H).
2-(4-((2-(4-fluorophenyl)-6-methoxybenzo[b]thiophen-3-yl)
oxy)phenoxy)ethan-1-amine (13)
Compound 14f was synthesized using the similar method to 14a
Tert-butyl (2-(4-((2-(4-fluorophenyl)-6-methoxybenzo [b]thio-
phen-3-yl)oxy)phenoxy)ethyl)carbamate (2 g, 3.9 mmol) was dis-
solved in 10 mL hydrochloric acid solution (1 M in MeOH). The
mixture was stirred at rt for 1 h and then concentrated in vacuo to
remove most of solvent, the mixture was poured into 10 mL water
and adjust pH to 7e8, then extracted with CH2Cl2 three times
(30 mL each). The collected organic layer was concentrated to
dryness and used in the next step without further purification. 1H
(51% yield). 1H NMR (300 MHz, CDCl3)
d 7.77e7.63 (m, 2H),
7.30e7.19 (m, 2H), 7.05e7.02 (m, 2H), 6.93e6.74 (m, 6H), 6.56e6.43
(m, 1H), 4.11e3.94 (m, 2H), 3.87 (s, 3H), 3.59e3.52 (m, 2H),
1.64e1.51 (m, 8H), 0.87e8.81 (m, 4H).
(E)-4-((2-(4-((2-(4-fluorophenyl)-6-hydroxybenzo[b]thiophen-
3-yl)oxy)phenoxy)ethyl)amino)-N-methylbut-2-enamide (15a)
To
a
solution of ((E)-4-((2-(4-((2-(4-fluorophenyl)-6-
[b]thiophen-3-yl)oxy)phenoxy)ethyl)amino)-N-
methoxybenzo
NMR (300 MHz, CDCl3)
d
7.74e7.66 (m, 2H), 7.25 (d, J ¼ 3.0 Hz, 2H),
methylbut-2-enamide (300 mg, 0.59 mmol) dissolved in 10 mL
anhydrous CH2Cl2 at 0 ꢀC under N2 protected, BBr3 (2.36 mL, 1 M in
CH2Cl2) was added. The solution was stirred at 0 ꢀC for 1 h and then
quenched with MeOH. The mixture was concentrated to dryness
and purified by column chromatography (SiO2, CH2Cl2: MeOH ¼ 30
: 1e15 : 1) to obtain the desired compound as white solid in 81%
7.09e7.00 (m, 2H), 6.93e6.83 (m, 3H), 6.82e6.74 (m, 2H), 3.91 (t,
J ¼ 5.1 Hz, 2H), 3.04 (t, J ¼ 5.1 Hz, 2H).
(E)-4-((2-(4-((2-(4-fluorophenyl)-6-methoxybenzo[b]thio-
phen-3-yl)oxy)phenoxy)ethyl)amino)-N-methylbut-2-enamide
(14a)
2-(4-((2-(4-fluorophenyl)-6-methoxybenzo [b]thiophen-3-yl)
oxy)phenoxy)ethan-1-amine (500 mg, 1.22 mmol) and DIPEA
(1.57 g, 12.2 mmol) were added into 5 mL DMF, (E)-4-bromo-N-
methylbut-2-enamide (192 mg, 1.09 mmol) was added. The solu-
tion was stirred at rt for18 h and then poured into CH2Cl2. The
organic layer was washed with 10% LiCl aqueous solution, followed
by concentrated to dryness which was purified by column chro-
matography (SiO2, CH2Cl2: MeOH ¼ 20 : 1e10 : 1) to obtain the
desired compound (380 mg, 62% yield) as white solid. 1H NMR
yield, mp:164e166 ꢀC. 1H NMR (300 MHz, MeOD)
d 7.74e7.69 (m,
2H), 7.24e7.14 (m, 2H), 7.11e7.03 (m, 2H), 6.85e6.73 (m, 6H),
6.11e6.05 (m,1H), 4.00 (t, J ¼ 5.2 Hz, 2H), 3.45e3.41 (m, 2H), 2.95 (t,
J ¼ 5.2 Hz, 2H), 2.79 (s, 3H). 13C NMR (101 MHz, MeOD)
d 168.63,
164.64, 162.19, 157.39, 155.49, 153.11, 142.08, 141.29, 138.55, 130.27,
128.16, 125.98, 125.37, 123.35, 117.33, 116.66, 116.46, 115.76, 108.80,
68.28, 50.65, 26.32. MS ESI m/z 493.3 [M þ H]þ.
(E)-4-((2-(4-((2-(4-fluorophenyl)-6-methoxybenzo[b]thiophen-3-
yl)oxy)phenoxy)ethyl)amino)-N,N-dimethylbut-2-enamide (15b)
Compound 15b was prepared from (E)-4-((2-(4-((2-(4-
(300 MHz, MeOD)
d 8.70e8.78 (m, 2H), 8.04e8.79 (m, 2H),
7.93e7.84 (m, 4H), 7.79 (d, J ¼ 9.1 Hz, 2H), 6.99 (d, J ¼ 15.4 Hz, 1H),
5.02 (t, J ¼ 5.0 Hz, 2H), 4.48e4.43 (m, 2H), 4.01 (t, J ¼ 4.9 Hz, 2H),
3.87 (d, J ¼ 4.8 Hz, 3H).
fluorophenyl)-6-methoxybenzo
[b]thiophen-3-yl)oxy)phenoxy)
ethyl)amino)-N,N-dimethylbut-2-enamide similarly as the pro-
cedure for 15a. Desired compound 15b was obtained as white solid
in 47% yield, mp: 172e174 ꢀC. 1H NMR (300 MHz, MeOD)
(E)-4-((2-(4-((2-(4-fluorophenyl)-6-methoxybenzo[b]thio-
phen-3-yl)oxy)phenoxy)ethyl)amino)-N,N-dimethylbut-2-
enamide (14b)
d
7.72e7.63 (m, 1H), 7.20e7.12 (m, 2H), 7.07e6.98 (m, 2H),
6.82e6.72 (m, 6H), 6.57e6.54 (m, 1H), 3.98e3.94-3.89 (m, 2H),
Compound 14b was synthesized using the similar method to 14a
3.49e3.43 (m, 2H), 3.06 (s, 3H), 2.95e2.89 (m, 5H). 13C NMR
(53% yield). 1H NMR (300 MHz, CDCl3)
d
7.75e7.66 (m, 2H),
(101 MHz, MeOD) d 168.52, 164.60, 162.15, 157.41, 155.50, 153.04,
7.28e7.25 (m, 2H), 7.06e7.04 (m, 2H), 6.94e6.83 (m, 4H), 6.81e6.73
(m, 2H), 6.49e6.44 (m, 1H), 4.03e4.00 (m, 2H), 3.86 (s, 3H),
3.51e3.49 (m, 2H), 3.06 (s, 3H), 3.02e2.99 (m, 5H).
(E)-1-(azetidin-1-yl)-4-((2-(4-((2-(4-fluorophenyl)-6-
methoxybenzo[b]thiophen-3-yl)oxy)phenoxy)ethyl)amino)but-2-
en-1-one (14c)
143.72, 142.07, 138.54, 130.26, 130.18, 128.13, 125.35, 123.37, 122.60,
117.33, 116.68, 116.64, 116.46, 115.79, 108.83, 68.40, 50.92, 37.77,
35.99. MS ESI m/z 507.3 [M þ H]þ.
(E)-1-(azetidin-1-yl)-4-((2-(4-((2-(4-fluorophenyl)-6-
hydroxybenzo[b]thiophen-3-yl)oxy)phenoxy)ethyl)amino)but-2-
en-1-one (15c)
Compound 14c was synthesized using the similar method to 14a
Compound 15c was prepared from (E)-1-(azetidin-1-yl)-4-((2-
13

C. Bai, S. Ren, S. Wu et al.
European Journal of Medicinal Chemistry 221 (2021) 113543
(4-((2-(4-fluorophenyl)-6-methoxybenzo
phenoxy)ethyl)amino)but-2-en-1-one similarly as the procedure
[b]thiophen-3-yl)oxy)
MeOH ¼ 20 : 1e25 : 1) to obtain the desired compound (90 mg, 92%
yield) as a white solid, mp: 171e174 ꢀC. 1H NMR (300 MHz, MeOD)
for 15a. Desired compound 15c was obtained as white solid in 81%
d 7.75e7.66 (m, 2H), 7.18e7.13 (m, 2H), 7.10e7.07 (m, 2H),
yield, mp: 182e184 ꢀC. 1H NMR (300 MHz, MeOD)
d
7.74e7.68 (m,
6.85e6.82 (m, 4H), 6.78e7.74 (m, 1H), 4.24e4.15 (m, 2H), 4.04e3.91
(m, 4H), 2.92 (t, J ¼ 5.2 Hz, 2H), 2.65 (t, J ¼ 7.3 Hz, 2H), 2.26e2.21 (m,
2H), 2.17e2.13 (m, 2H), 1.79e1.75 (m, 2H). 13C NMR (101 MHz,
2H), 7.20e7.11 (m, 2H), 7.11e7.01 (m, 2H), 6.88e6.74 (m, 6H),
6.18e6.11 (m, 1H), 4.29e4.24 (m, 2H), 4.10e3.93 (m, 4H), 3.47e3.45
(m, 2H), 2.95 (t, J ¼ 5.2 Hz, 2H), 2.35e2.24 (m, 2H). 13C NMR
MeOD)
d 175.02, 164.63, 162.18, 158.53, 155.61, 153.13, 142.19,
(75 MHz, MeOD)
d
167.35, 165.03, 161.75, 157.42, 155.50, 153.06,
138.64, 130.24, 130.16, 127.70, 123.26, 117.34, 116.67, 116.45, 116.24,
109.02, 68.40, 51.55, 49.81, 30.77, 29.55, 25.51, 15.80. MS ESI m/z
543.3 [M þ Na]þ.
143.13, 142.09, 138.55, 130.28, 130.17, 128.14, 125.37, 123.38, 120.56,
117.36, 116.73, 116.66, 116.44, 115.80, 108.85, 68.38, 51.51, 50.75,
16.05. MS ESI m/z 519.2 [M þ H]þ.
Tert-butyl 3-(4-((2-(4-fluorophenyl)-6-methoxy-1-oxidobenzo
[b]thiophen-3-yl)oxy)phenoxy)azetidine-1-carboxylate (17)
To a solution of 3-bromo-2-(4-fluorophenyl)-6-methoxybenzo
[b]thiophene 1-oxide 10 (2 g, 5.7 mmol) dissolved in 15 mL DMF,
(E)-4-((2-(4-((2-(4-fluorophenyl)-6-hydroxybenzo[b]thiophen-
3-yl)oxy)phenoxy)ethyl)amino)-1-(pyrrolidin-1-yl)but-2-en-1-
one (15d)
Compound 15d was prepared from (E)-4-((2-(4-((2-(4-
CS2CO3 (3.7 g, 11.4 mmol) and tert-butyl 3-(4-hydroxyphenoxy)
fluorophenyl)-6-hydroxybenzo
[b]thiophen-3-yl)oxy)phenoxy)
azetidine-1-carboxylate 4 (1.51 g, 5.7 mmol) were added. The so-
lution was stirred at 50 ꢀC for 3 h and quenched with NH4Cl
aqueous solution. The mixture was extracted with CH2Cl2, and
collected organic layer was washed with 10% LiCl aqueous solution,
dried and concentrated to dryness to obtain the crude product
which was purified by column chromatography (SiO2, n-hexane:
ethyl acetate ¼ 5 : 1e3 : 1) to give title compound as white solid
ethyl)amino)-1-morpholinobut-2-en-1-one similarly as the pro-
cedure for 15a. Desired compound 15d was obtained as white solid
in 66% yield, mp: 168e170 ꢀC. 1H NMR (300 MHz, MeOD)
d
7.72e7.67 (m, 2H), 7.21e7.12 (m, 2H), 7.09e7.02 (m, 2H),
6.90e6.73 (m, 6H), 6.55e6.45 (m, 1H), 4.08 (t, J ¼ 5.1 Hz, 2H),
3.65e3.63 (m, 2H), 3.55 (t, J ¼ 6.6 Hz, 2H), 3.49e3.43 (m, 2H), 3.14
(t, J ¼ 5.1 Hz, 2 H), 2.01e1.82 (m, 4H). 13C NMR (101 MHz, MeOD)
(2.1 g, 68% yield). 1H NMR (300 MHz, CDCl3)
d 7.75e7.65 (m, 2H),
d
164.44,163.27,160.81,156.07,153.77,151.94,140.66,138.22,137.19,
7.12e6.84 (m, 6H), 6.62 (d, J ¼ 9 Hz, 2H), 4.82e4.75 (m, 1H),
128.91, 128.83, 126.74, 124.84, 124.04, 121.95, 116.03, 115.40, 115.34,
115.12, 114.44,107.47, 65.61, 46.57, 48.69, 45.79, 25.52, 23.80. MS ESI
m/z 533.3 [M þ H]þ.
4.30e4.21 (m, 2H), 3.98e3.94 (m, 5H), 1.44 (s, 9H).
Tert-butyl 3-(4-((2-(4-fluorophenyl)-6-methoxybenzo[b]thio-
phen-3-yl)oxy)phenoxy)azetidine-1-carboxylate (18)
(E)-4-((2-(4-((2-(4-fluorophenyl)-6-hydroxybenzo[b]thiophen-3-
yl)oxy)phenoxy)ethyl)amino)-1-morpholinobut-2-en-1-one (15e)
Compound 15e was prepared from (E)-4-((2-(4-((2-(4-
To
a
solution of tert-butyl 3-(4-((2-(4-fluorophenyl)-6-
methoxy-1-oxidobenzo [b]thiophen-3-yl)oxy)phenoxy)azetidine-
1-carboxylate (2 g, 3.72 mmol) dissolved in 10 mL THF, LiAlH4
(289.6 mg, 7.62 mmol) was added at 0 ꢀC under N2 protected. After
30 min, the reaction was quenched with NH4Cl aqueous solution
and then extracted with CH2Cl2. The combined organic layer was
collected and concentrated to dryness, purified by column chro-
matography (SiO2, n-hexane: ethyl acetate ¼ 5 : 1e3 : 1) to obtain
the desired compound (1.1 g, 57% yield). 1H NMR (300 MHz, CDCl3)
fluorophenyl)-6-hydroxybenzo
[b]thiophen-3-yl)oxy)phenoxy)
ethyl)amino)-1-(piperidin-1-yl)but-2-en-1-one (546 mg, 1 mmol)
similarly as the procedure for 15a. Desired compound 15e was
obtained as white solid in 83% yield, mp: 182e184 ꢀC. 1H NMR
(300 MHz, MeOD) d 7.74e7.64 (m, 2H), 7.19e7.14 (m, 2H), 7.09e6.99
(m, 2H), 6.86e6.72 (m, 6H), 6.59e6.52 (m, 1H), 3.99e3.97 (m, 2H),
3.65e3.6 (m, 8H), 3.48e3.44 (m, 2H),2.96e2.91 (m, 2H). 13C NMR
d
7.74e7.65 (m, 2H), 7.26e7.21 (m, 2H), 7.04 (t, J ¼ 8.7 Hz, 2H),
(101 MHz, MeOD)
d
175.54, 167.12, 164.68, 157.43, 155.57, 153.11,
6.88e6.82 (m, 3H), 6.67e6.56 (m, 2H), 4.77e4.74 (m, 1H),
4.26e4.23 (m, 2H), 3.99e3.95 (m, 2H), 3.86 (s, 3H), 1.44 (s, 9H).
3-(4-((2-(4-fluorophenyl)-6-methoxybenzo[b]thiophen-3-yl)
oxy)phenoxy)azetidine (19)
143.49, 142.12, 138.57, 130.30, 130.22, 128.17, 125.37, 123.35, 122.89,
117.35, 116.70, 116.48, 115.77, 108.78, 68.45, 50.98, 47.40, 27.77,
25.47. MS ESI m/z 549.3 [M þ H]þ.
(E)-4-((2-(4-((2-(4-fluorophenyl)-6-hydroxybenzo[b]thiophen-
3-yl)oxy)phenoxy)ethyl)amino)-1-(piperidin-1-yl)but-2-en-1-one
(15f)
Tert-butyl 3-(4-((2-(4-fluorophenyl)-6-methoxybenzo [b]thio-
phen-3-yl)oxy)phenoxy)azetidine-1-carboxylate (2 g, 3.84 mmol)
was dissolved in 10 mL hydrochloric acid solution (1 M in MeOH).
The mixture was stirred at rt for 1 h and then concentrated in vacuo
to remove most of solvent, the mixture was poured into 10 mL
water and adjust pH to 7e8, then extracted with CH2Cl2 three times
(30 mL each). The collected organic layer was concentrated to
dryness and used in the next step without further purification.
(E)-4-(3-(4-((2-(4-fluorophenyl)-6-methoxybenzo[b]thiophen-
3-yl)oxy)phenoxy)azetidin-1-yl)-N-methylbut-2-enamide (20a)
3-(4-((2-(4-fluorophenyl)-6-methoxybenzo [b]thiophen-3-yl)
oxy)phenoxy)azetidine (513 mg, 1.22 mmol) and DIPEA (1.57g,
12.2 mmol) were added into 5 mL DMF, (E)-4-bromo-N-methylbut-
2-enamide (192 mg, 1.09 mmol) was added. The solution was stir-
red at rt for 18 h and then partitioned between water and ethyl
acetate. The organic layer was washed with 10% LiCl aqueous so-
lution, followed by concentrated to dryness to obtain the crude
product which was purified by column chromatography (SiO2,
CH2Cl2: MeOH ¼ 20 : 1e10 : 1). Desired compound was obtained as
Compound 15f was prepared from (E)-4-((2-(4-((2-(4-
fluorophenyl)-6-hydroxybenzo
ethyl)amino)-1-(piperidin-1-yl)but-2-en-1-one (546 mg, 1 mmol)
similarly as the procedure for 15a. Desired compound 15f was
obtained as white solid in 63% yield, mp: 169e173 ꢀC. 1H NMR
[b]thiophen-3-yl)oxy)phenoxy)
(300 MHz, MeOD)
d 7.76e7.67 (m, 2H), 7.21e7.02 (m, 4H),
6.87e6.68 (m, 6H), 6.61e6.55 (m, 1H), 4.00 (t, J ¼ 5.2 Hz, 2H),
3.58e3.51 (m, 4H), 3.47e3.44 (m, 2H), 2.94 (t, J ¼ 5.2 Hz, 2H),
1.68e1.62 (m, 2H), 1.58e1.50 (m, 4H). 13C NMR (101 MHz, MeOD)
d
167.32, 164.64, 157.41, 155.53, 153.08, 144.51, 142.08, 138.55,
130.28, 130.22, 128.14, 125.37, 123.35, 122.01, 117.35, 116.70, 116.48,
115.77, 108.78, 68.45, 67.69, 50.98, 47.40, 43.62 MS ESI m/z 547.2
[M þ H]þ.
1-(azetidin-1-yl)-4-((2-(4-((2-(4-fluorophenyl)-6-
hydroxybenzo[b]thiophen-3-yl)oxy)phenoxy)ethyl)amino)butan-
1-one(16)
To a solution of 15c (100 mg, 0.19 mmol) dissolved in 5 mL
MeOH, catalytic amount of 10% Pd/C was added. The solution was
stirred at rt under hydrogen protected. After completion, resulting
solution was filtered to remove Pd/C, and filtrate was concentrated
to dryness and purified by chromatography (SiO2, CH2Cl2:
white solid in 53% yield. 1H NMR (300 MHz, CDCl3)
d 7.69e7.62 (m,
2H), 7.24e7.18 (m, 2H), 7.05e6.95 (m, 2H), 6.86e6.79 (m, 3H),
6.65e6.57 (m, 2H), 6.07e5.85 (m, 2H), 4.66e4.61 (m, 1H), 3.83 (s,
3H), 3.80e3.73 (m, 2H), 3.23e3.19 (m, 2H), 3.13e3.04 (m, 2H),
2.82e2.81 (m, 3H).
14

C. Bai, S. Ren, S. Wu et al.
European Journal of Medicinal Chemistry 221 (2021) 113543
(E)-4-(3-(4-((2-(4-fluorophenyl)-6-methoxybenzo[b]thiophen-
3-yl)oxy)phenoxy)azetidin-1-yl)-N,N-dimethylbut-2-enamide
(20b)
(m, 2H), 3.08 (s, 3H), 2.96 (s, 3H). 13C NMR (101 MHz, MeOD)
d
168.36, 157.44, 153.63, 153.40, 140.99, 138.57, 130.30, 130.22,
128.12, 125.47, 123.30, 123.81, 117.50, 116.85, 116.72, 116.50, 115.79,
108.79, 68.24, 62.12, 60.75, 37.82, 35.99, 30.76. MS ESI m/z 519.3
[M þ H]þ.
Compound 20b was prepared using the method similar to the
preparation of 20a. Desired compound 20b was obtained as white
solid in 52% yield. 1H NMR (300 MHz, CDCl3)
d
7.71e7.63 (m, 2H),
(E)-1-(azetidin-1-yl)-4-(3-(4-((2-(4-fluorophenyl)-6-
hydroxybenzo[b]thiophen-3-yl)oxy)phenoxy)azetidin-1-yl)but-2-
en-1-one (21c)
7.23e7.21 (m, 2H), 7.05e6.97 (m, 2H), 6.89e6.81 (m, 3H), 6.74e6.58
(m, 1H), 6.67e6.58 (m, 2H), 6.37e6.24 (m, 1H), 4.70 (m, 1H), 3.84 (s,
3H), 3.83e3.77 (m, 2H), 3.28e3.23 (m, 2H), 3.15e3.08 (m, 2H), 3.05
(s, 3H), 2.98 (s, 3H).
Compound 21c was prepared from (E)-1-(azetidin-1-yl)-4-(3-
(4-((2-(4-fluorophenyl)-6-methoxybenzo
[b]thiophen-3-yl)oxy)
(E)-1-(azetidin-1-yl)-4-(3-(4-((2-(4-fluorophenyl)-6-
methoxybenzo[b]thiophen-3-yl)oxy)phenoxy)azetidin-1-yl)but-2-
en-1-one (20c)
phenoxy)azetidin-1-yl)but-2-en-1-one (544 mg, 1 mmol) similarly
as the procedure for 21a. Desired compound 21c was obtained as
white solid in 79% yield, mp: 174e176 ꢀC. 1H NMR (300 MHz,
Compound 20c was synthesized using the method similar to
MeOD) d 7.75e7.64 (m, 2H), 7.24e7.11 (m, 2H), 7.11e7.01 (m, 2H),
20a (51% yield). 1H NMR (300 MHz, CDCl3)
d
7.73e7.63 (m, 2H),
6.88e6.77 (m, 3H), 6.75e6.56 (m, 3H), 6.08e6.06 (m, 1H),
4.87e4.75 (m, 1H), 4.31e4.28 (m, 1H), 4.07e4.04 (m, 1H), 3.87e3.82
(m, 2H), 3.48e3.44 (m, 1H), 3.41e3.33 (m, 4H), 3.29e3.28 (m, 1H),
2.38e1.99 (m, 2H). MS ESI m/z 531.2 [M þ H]þ.
7.24e7.21 (m, 2H), 7.06e6.97 (m, 2H), 6.90e6.72 (m, 4H), 6.66e6.57
(m, 2H), 5.96e5.91 (m, 1H), 4.69e4.61 (m, 1H), 4.20e4.18 (m, 2H),
4.07e4.02 (m, 2H), 3.85 (s, 3H), 3.80e3.71 (m, 2H), 3.28e3.26 (m,
2H), 3.15e3.05 (m, 2H), 2.27e2.25 (m, 2H).
(E)-4-(3-(4-((2-(4-fluorophenyl)-6-methoxybenzo[b]thiophen-
3-yl)oxy)phenoxy)azetidin-1-yl)-1-(pyrrolidin-1-yl)but-2-en-1-
one (20d)
(E)-4-(3-(4-((2-(4-fluorophenyl)-6-hydroxybenzo[b]thiophen-
3-yl)oxy)phenoxy)azetidin-1-yl)-1-(pyrrolidin-1-yl)but-2-en-1-
one (21d)
Compound 21d was prepared from (E)-4-(3-(4-((2-(4-
Compound 20d was synthesized using the method similar to
fluorophenyl)-6-methoxybenzo
azetidin-1-yl)-1-(pyrrolidin-1-yl)but-2-en-1-one
[b]thiophen-3-yl)oxy)phenoxy)
(558 mg,
20a (44% yield). 1H NMR (300 MHz, CDCl3)
d 7.72e7.62 (m, 2H),
7.23e7.21 (m, 2H), 7.02e7.69 (m, 2H), 6.89e6.73 (m, 4H), 6.66e6.58
(m, 2H), 6.23e6.21 (m, 1H), 4.70e4.64 (m, 1H), 3.85 (s, 5H),
3.50e3.47 (m, 4H), 3.37e3.25 (m, 2H), 3.12e3.09 (m, 2H), 1.93e1.89
(m, 2H), 1.84e1.79 (m, 2H).
(E)-4-(3-(4-((2-(4-fluorophenyl)-6-methoxybenzo[b]thiophen-
3-yl)oxy)phenoxy)azetidin-1-yl)-1-morpholinobut-2-en-1-one
(20e)
1 mmol) similarly as the procedure for 21a. Desired compound 21d
was obtained as white solid in 69% yield, mp: 168e170 ꢀC. 1H NMR
(300 MHz, MeOD) d 7.72e7.63 (m, 2H), 7.20e7.10 (m, 2H), 7.06e7.00
(m, 2H), 6.85e6.72 (m, 3H), 6.69e6.60 (m, 3H), 6.36e6.30 (m, 1H),
4.69e4.67 (m, 1H), 3.77e3.72 (m, 2H), 3.53 (t, J ¼ 6.7 Hz, 2H), 3.44
(t, J ¼ 6.8 Hz, 2H), 3.31e3.29 (m, 2H), 3.20e3.13 (m, 2H), 1.99e1.83
(m, 4H). 13C NMR (101 MHz, DMSO‑d6)
d 163.16, 160.26, 156.28,
Compound 20e was synthesized from 3-(4-((2-(4-
152.09, 151.20, 140.37, 139.74, 136.58, 128.93, 125.89, 123.52, 123.24,
122.06, 116.34, 116.24, 116.02, 115.71, 115.22, 108.09, 66.63, 60.79,
59.55, 46.01, 45.48, 25.66, 23.86. MS ESI m/z 545.3 [MþH]þ.
(E)-4-(3-(4-((2-(4-fluorophenyl)-6-hydroxybenzo[b]thiophen-
3-yl)oxy)phenoxy)azetidin-1-yl)-1-morpholinobut-2-en-1-
one(21e)
fluorophenyl)-6-methoxybenzo
azetidine using the method similar to 20a (47% yield). 1H NMR
(300 MHz, CDCl3) 7.79e7.68 (m, 2H), 7.29e7.25 (m, 2H), 7.12e7.03
[b]thiophen-3-yl)oxy)phenoxy)
d
(m, 2H), 6.95e6.78 (m, 4H), 6.74e6.64 (m, 2H), 6.40e6.37 (m, 1H),
4.75e4.71 (m, 1H), 3.91e3.85 (m, 3H), 3.86e3.83 (m, 2H), 3.72 (s,
6H), 3.60 (s, 2H), 3.35e3.29 (m, 2H), 3.18e3.16 (m, 2H).
Compound 21e was prepared from (E)-4-(3-(4-((2-(4-
(E)-4-(3-(4-((2-(4-fluorophenyl)-6-hydroxybenzo[b]thiophen-
3-yl)oxy)phenoxy)azetidin-1-yl)-N-methylbut-2-enamide (21a)
fluorophenyl)-6-methoxybenzo
[b]thiophen-3-yl)oxy)phenoxy)
azetidin-1-yl)-1-morpholinobut-2-en-1-one (574 mg, 1 mmol)
similarly as the procedure for 21a. Desired compound 21e was
obtained as white solid in 69% yield, mp: 145e150 ꢀC. 1H NMR
To
a
solution
of
(E)-4-(3-(4-((2-(4-fluorophenyl)-6-
methoxybenzo [b]thiophen-3-yl)oxy)phenoxy)azetidin-1-yl)-N-
methylbut-2-enamide (518 mg, 1 mmol) dissolved in 10 mL anhy-
drous CH2Cl2 at 0 ꢀC under N2 protected, BBr3 (4 mL, 1 M in CH2Cl2)
was added. The solution was stirred at 0 ꢀC for 1h and then
quenched with MeOH. The mixture was concentrated to dryness
and purified by column chromatography (SiO2, CH2Cl2: MeOH ¼ 30
: 1e15 : 1) to obtain the desired compound as white solid in 83%
(300 MHz, MeOD) d 7.71e7.67 (m, 2H), 7.19e7.13 (m, 2H),7.08e7.02
(m, 2H), 6.83e6.75 (m, 3H), 6.70e6.62 (m, 3H), 6.53e6.47 (m, 1H),
4.73e4.70 (m, 1H), 3.79e3.74 (m, 2H), 3.65e3.61 (m, 4H),
3.61e3.58 (m, 4H), 3.35e3.30 (m, 2H), 3.23e3.14 (m, 2H). 13C NMR
(101 MHz, MeOD)
d 167.10, 164.63, 157.41, 153.59, 153.31, 141.95,
138.54, 130.27, 130.19, 128.09, 125.46, 123.33, 123.03, 117.49, 116.80,
116.71, 116.50, 115.82, 108.84, 68.22, 62.10, 60.84, 47.43, 43.62. MS
ESI m/z 561.3 [M þ H]þ.
yield, mp: >200 ꢀC. 1H NMR (300 MHz, MeOD)
d 7.71e7.67 (m, 2H),
7.20e7.03 (m, 4H), 6.86e6.69 (m, 5H), 6.67e6.58 (m, 1H), 6.16e6.11
(m, 1H), 4.82e4.81 (m, 1H), 4.05e4.00 (m, 2H), 3.56e3.47 (m, 4H),
N-(2-(4-((2-(4-fluorophenyl)-6-methoxybenzo[b]thiophen-3-
yl)oxy)phenoxy)ethyl)but-3-enamide (22a)
2.78 (s, 3H). 13C NMR (101 MHz, MeOD)
d 167.92, 164.53, 162.08,
157.27, 153.46, 153.10, 141.79, 138.45, 136.53, 130.20, 128.43, 127.99,
125.44, 123.20, 117.48, 116.95, 116.65, 116.43, 115.76, 108.80, 67.82,
61.84, 59.05, 26.42. MS ESI m/z 505.2 [M þ H]þ.
To a solution of 13 (409 mg, 1 mmol) dissolved in 10 mL CH2Cl2,
DIPEA (154 mg, 1.2 mmol) and acryloyl chloride (108 mg, 1.2 mmol)
were added at 0 ꢀC under N2 protected. The solution was stirred at
(E)-4-(3-(4-((2-(4-fluorophenyl)-6-hydroxybenzo[b]thiophen-3-
yl)oxy)phenoxy)azetidin-1-yl)-N,N-dimethylbut-2-enamide (21b)
Compound 21b was prepared from (E)-4-(3-(4-((2-(4-
0
ꢀC for 2 h then quenched with 10 mL water. The mixture was
extracted CH2Cl2 (10 mL x 2), combined organic layer was dried
over anhydrous Na2SO4 and concentrated to dryness, used in the
next step without further purification (83% yield). 1H NMR
fluorophenyl)-6-methoxybenzo
azetidin-1-yl)-N,N-dimethylbut-2-enamide (532 mg,
[b]thiophen-3-yl)oxy)phenoxy)
mmol)
1
(300 MHz, CDCl3)
d
7.74e7.65 (m, 2H), 7.25 (d, J ¼ 2.6 Hz, 2H),
similarly as the procedure for 21a. Desired compound 21a was
7.11e6.99 (m, 2H), 6.88e6.82 (m, 3H), 6.82e6.74 (m, 2H), 6.37e6.30
(m, 1H), 6.17e6.06 (m, 1H), 5.68e5.64 (m, 1H), 4.14e4.97 (m, 2H),
3.87 (s, 3H), 3.74e3.72 (m, 2H).
obtained as white solid in 77% yield, mp: 171e173 ꢀC. 1H NMR
(300 MHz, MeOD) d 7.70e7.62 (m, 2H), 7.20e7.11 (m, 2H), 7.06e7.00
(m, 2H), 6.82e6.74 (m, 3H), 6.69e6.56 (m, 3H), 6.52e6.47 (m, 1H),
4.71e4.69 (m, 1H), 3.81e3.71 (m, 2H), 3.30e3.29 (m, 2H), 3.21e3.16
N-(2-(4-((2-(4-fluorophenyl)-6-methoxybenzo[b]thiophen-3-
yl)oxy)phenoxy)ethyl)methacrylamide (22b)
15

C. Bai, S. Ren, S. Wu et al.
European Journal of Medicinal Chemistry 221 (2021) 113543
Compound 22b was prepared using the method similar to the
1-(3-(4-((2-(4-fluorophenyl)-6-methoxybenzo[b]thiophen-3-
yl)oxy)phenoxy)azetidin-1-yl)-2-methylprop-2-en-1-one (24b)
Compound 24b was obtained with the similar method to the
synthesis of 22a.1H NMR (300 MHz, MeOD)
d 7.74e7.65 (m, 2H),
7.22e7.00 (m, 4H), 6.88e6.71 (m, 5H), 5.67e5.63 (m,1H), 5.35e5.31
(m, 1H), 3.99e3.91 (m, 2H), 3.57e3.52 (m, 2H), 1.91 (d, J ¼ 1.3 Hz,
3H).
(E)-N-(2-(4-((2-(4-fluorophenyl)-6-methoxybenzo[b]thiophen-
3-yl)oxy)phenoxy)ethyl)but-2-enamide (22c)
synthesis of 24a (41% yield). 1H NMR (300 MHz, CDCl3)
d 7.77e7.70
(m, 2H), 7.30e7.27 (m, 2H), 7.13e7.04 (m, 2H), 6.93e6.89 (m, 3H),
6.71e6.63 (m, 2H), 5.41e5.39 (m, 2H), 4.89e4.85 (m, 1H),
4.50e4.45 (m, 2H), 4.33e4.08 (m, 2H), 3.91 (s, 3H),1.97 (t, J ¼ 1.3 Hz,
3H).
Compound 22c was prepared using the method similar to the
synthesis of 22a.1H NMR (300 MHz, MeOD)
d
7.73e7.61 (m, 2H),
(E)-1-(3-(4-((2-(4-fluorophenyl)-6-methoxybenzo[b]thiophen-
3-yl)oxy)phenoxy)azetidin-1-yl)but-2-en-1-one (24c)
Compound 24c was obtained with the similar method to the
7.24e7.10 (m, 2H), 7.02e7.68 (m, 2H), 6.85e6.71 (m, 6H), 5.94e5.89
(m, 1H), 3.93e3.89 (m, 2H), 3.54e3.49 (m, 2H), 1.82e1.79 (m, 3H).
N-(2-(4-((2-(4-fluorophenyl)-6-hydroxybenzo[b]thiophen-3-
yl)oxy)phenoxy)ethyl)acrylamide (23a)
To a solution of 22a (463 mg, 1 mmol) dissolved in 10 mL
anhydrous CH2Cl2 at 0 ꢀC under N2 protected, BBr3 (4 mL, 1 M in
CH2Cl2) was added. The solution was stirred at 0 ꢀC for 1h and then
quenched with MeOH. The resulting mixture was concentrated to
dryness and purified by column chromatography (SiO2, CH2Cl2:
MeOH ¼ 30:1e15 : 1) to obtain the desired compound as white
solid (320 mg, 69% yield), mp: 171e173 ꢀC. 1H NMR (300 MHz,
synthesis of 24a (54% yield). 1H NMR (300 MHz, CDCl3)
d 7.77e7.70
(m, 2H), 7.29 (d, J ¼ 2.9 Hz, 2H), 7.12e7.05 (m, 2H), 6.98e6.87 (m,
4H), 6.71e6.64 (m, 2H), 5.90e5.85 (m, 1H), 4.90e4.85 (m, 1H),
4.62e4.37 (m, 2H), 4.29e4.05 (m, 2H), 3.91 (s, 3H), 1.90e1.87 (m,
3H).
1-(3-(4-((2-(4-fluorophenyl)-6-hydroxybenzo[b]thiophen-3-yl)
oxy)phenoxy)azetidin-1-yl)prop-2-en-1-one (25a)
To a solution of 1-(3-(4-((2-(4-fluorophenyl)-6-methoxybenzo
[b]thiophen-3-yl)oxy)phenoxy)azetidin-1-yl)prop-2-en-1-one
(500 mg, 1.07 mmol) dissolved in 10 mL anhydrous CH2Cl2 at 0 ꢀC
under N2 protected, BBr3 (4.28 mL, 1 M in CH2Cl2) was added. The
solution was stirred at 0 ꢀC for 1h and then quenched with MeOH.
The mixture was concentrated to dryness and purified by column
chromatography (SiO2, CH2Cl2: MeOH ¼ 30 : 1e15 : 1) to obtain the
desired compound as white solid in 78% yield, mp: 168e172 ꢀC. 1H
MeOD)
d 7.71e7.64 (m, 2H), 7.18e6.98 (m, 4H), 6.79e6.75 (m, 5H),
6.28e6.17 (m, 2H), 5.65e5.61 (m,1H), 3.95 (t, J ¼ 5.4 Hz, 2H), 3.57 (t,
J ¼ 5.4 Hz, 2H). 13C NMR (101 MHz, MeOD)
d 168.31, 164.57, 162.12,
157.28, 155.38, 153.05, 142.03, 138.52, 131.78, 130.23, 128.15, 126.92,
125.37, 123.35, 117.30, 116.63, 116.41, 115.71, 108.79, 67.85, 40.16. MS
ESI m/z 472.1 [M þ Na]þ.
N-(2-(4-((2-(4-fluorophenyl)-6-hydroxybenzo[b]thiophen-3-
yl)oxy)phenoxy)ethyl)methacrylamide (23b)
NMR (300 MHz, MeOD) d 7.78e7.66 (m, 2H), 7.23e7.02 (m, 4H),
6.91e6.69 (m, 5H), 6.40e6.19 (m, 2H), 5.73e5.69 (m, 1H), 4.97e4.92
(m, 1H), 4.69e4.63 (m, 1H), 4.40 (d, J ¼ 1.6 Hz, 1H), 4.27e4.18 (m,
Compound 23b was prepared from 1-(3-(4-((2-(4-
fluorophenyl)-6-methoxybenzo
[b]thiophen-3-yl)oxy)phenoxy)
1H), 4.04e3.93 (m, 1H). 13C NMR (101 MHz, DMSO‑d6)
d 164.70,
azetidin-1-yl)-2-methylprop-2-en-1-one (489 mg, 1 mmol) simi-
larly as the procedure for 23a. Desired compound 23b was obtained
as white solid in 64% yield, mp: 174e176 ꢀC. 1H NMR (300 MHz,
162.71, 160.26, 156.27, 151.65, 140.29, 136.59, 128.94, 126.92, 126.78,
125.84, 123.57, 122.05, 116.45, 116.23, 116.01, 115.89, 115.23, 108.10,
65.79, 56.90, 54.61. MS ESI m/z 486.2 [M þ Na]þ.
MeOD)
d
7.73e7.65 (m, 2H), 7.21e7.02 (m, 4H), 6.83e6.76 (m, 5H),
1-(3-(4-((2-(4-fluorophenyl)-6-hydroxybenzo[b]thiophen-3-yl)
oxy)phenoxy)azetidin-1-yl)-2 methylprop-2-en-1-one(25b)
Compound 25b was prepared from 1-(3-(4-((2-(4-
5.68e5.67 (m, 1H), 5.36e5.33 (m, 1H), 3.99 (t, J ¼ 5.7 Hz, 2H), 3.57
(t, J ¼ 5.7 Hz, 2H), 1.91 (s, 3H). 13C NMR (101 MHz, MeOD)
d 170.11,
163.28, 160.83, 156.00, 154.19, 151.17, 140.71, 139.77, 137.17, 128.90,
126.79, 124.00, 121.98, 119.26, 115.93, 115.34, 115.08, 114.36, 107.38,
66.42, 38.98, 17.37. MS ESI m/z 486.2 [M þ Na]þ.
fluorophenyl)-6-methoxybenzo
[b]thiophen-3-yl)oxy)phenoxy)
azetidin-1-yl)-2-methylprop-2-en-1-one (489 mg, 1 mmol) simi-
larly as the procedure for 25a. Desired compound 25b was obtained
as white solid in 72% yield, mp: >200 ꢀC. 1H NMR (300 MHz, MeOD)
(E)-N-(2-(4-((2-(4-fluorophenyl)-6-hydroxybenzo[b]thiophen-3-
yl)oxy)phenoxy)ethyl)but-2-enamide(23c)
d
7.72e7.71 (m, 2H), 7.21e7.04 (m, 4H), 6.89e6.70 (m, 5H),
Compound 23c was prepared from (E)-1-(3-(4-((2-(4-
5.48e5.35 (m, 2H), 4.96e4.95 (m, 1H), 4.63e4.61 (m, 1H),
4.39e4.37 (m, 1H), 4,25e4.23 (m, 1H), 3.96e3.94 (m, 1H), 1.90 (s,
fluorophenyl)-6-methoxybenzo
[b]thiophen-3-yl)oxy)phenoxy)
azetidin-1-yl)but-2-en-1-one (489 mg, 1 mmol) similarly as the
procedure for 23a. Desired compound 23c was obtained as white
solid in 61% yield, mp: 157e159 ꢀC. 1H NMR (300 MHz, MeOD)
3H). 13C NMR (101 MHz, DMSO‑d6)
d 170.00, 156.29, 151.69, 151.50,
140.30, 137.78, 136.59, 128.95, 128.87, 125.84, 123.56, 122.05, 119.89,
116.46, 116.25, 116.03, 115.85, 115.25, 108.10, 66.16, 59.44, 19.15. MS
ESI m/z 498.2 [M þ Na]þ.
d
7.73e7.62 (m, 2H), 7.23e7.09 (m, 2H), 7.05e7.00 (m, 2H),
6.84e6.72 (m, 6H), 5.97e5.90 (m, 1H), 3.93 (t, J ¼ 5.4 Hz, 2H), 3.54
(E)-1-(3-(4-((2-(4-fluorophenyl)-6-hydroxybenzo[b]thiophen-3-
yl)oxy)phenoxy)azetidin-1-yl)but-2-en-1-one(25c)
(t, J ¼ 5.4 Hz, 2H), 1.83e1.80 (m, 3H). 13C NMR (101 MHz, MeOD)
d
168.76,164.49,162.03,157.21,155.31,152.97,141.98,141.96,138.48,
Compound 25c was prepared from (E)-1-(3-(4-((2-(4-
130.18, 128.13, 125.80, 125.36, 123.35, 117.27, 116.59, 116.57, 115.69,
fluorophenyl)-6-methoxybenzo
[b]thiophen-3-yl)oxy)phenoxy)
108.81, 67.90, 40.02, 17.84. MS ESI m/z 486.2 [M þ Na]þ.
azetidin-1-yl)but-2-en-1-one similarly as the procedure for 25a.
Desired compound 25c was obtained as white solid in 78% yield,
1-(3-(4-((2-(4-fluorophenyl)-6-methoxybenzo[b]thiophen-3-
yl)oxy)phenoxy)azetidin-1-yl)prop-2-en-1-one (24a)
mp: 174e178 ꢀC. 1H NMR (300 MHz, MeOD)
d 7.74e7.67 (m, 2H),
To a solution of 19 (421 mg, 1 mmol) dissolved in 10 mL CH2Cl2,
DIPEA (154 mg, 1.2 mmol) and acryloyl chloride (108 mg, 1.2 mmol)
were added at 0 ꢀC under N2 protected. The solution was stirred at
0 ꢀC for 2h then quenched with 10 mL water. Resulting mixture was
extracted with CH2Cl2 (10 mL X 2), combined organic layer was
dried over anhydrous Na2SO4 and concentrated to dryness, used in
the next step without further purification. 1H NMR (300 MHz,
7.21e7.02 (m, 4H), 6.90e6.70 (m, 6H), 6.07e5.97 (m,1H), 5.00e4.92
(m, 1H), 4.65e4.56 (m, 1H), 4.40e4.37 (m, 1H), 4.20e4.18 (m, 1H),
3.97e3.92 (m, 1H), 1.89e1.86 (m, 3H). 13C NMR (101 MHz, MeOD)
d
166.82,162.93, 156.32, 152.27, 151.84, 141.61, 140.55, 137.21, 128.91,
128.83, 126.59, 121.87, 119.64, 116.21, 115.48, 115.34, 115.12, 114.53,
107.46, 65.64, 58.67, 54.75, 29.37, 16.69. MS ESI m/z 498.2 [M þ
Na]þ.
CDCl3)
d
7.78e7.70 (m, 2H), 7.29 (d, J ¼ 2.6 Hz, 2H), 7.15e7.03 (m,
2H), 6.92e6.89 (m, 3H), 6.86e6.77 (m, 2H), 6.35e6.31 (m, 1H),
6.14e6.12 (m, 1H), 5.70e5.64 (m, 1H), 4.14e3.97 (m, 2H), 3.91 (s,
3H), 3.76e3.74 (m, 2H).
16

C. Bai, S. Ren, S. Wu et al.
European Journal of Medicinal Chemistry 221 (2021) 113543
4.2. Biological evaluation
pre-cooled acetone. Further treatment was performed according to
the standard immunofluorescence procedures. The nucleus stained
by DAPI is blue under ultraviolet excitation, and the positive
expression is red light labeled with fluorescein.
4.2.1. Cell culture
MCF-7 and Ishikawa cells were cultured in Dulbecco's Modified
Eagle Medium (DMEM, KeyGEN Biotech), containing 10% fetal
bovine serum (FBS, GIBCO), 100U/ml Penicillin and 100 mg/mL
Streptomycin, at 37 ꢀC under 5% CO2 condition in humidified at-
mosphere. MDA-MB-231 cells were maintained in Leibovitz's L-15
media supplemented with 10% FBS.
4.2.7. ER
a
binding affinity assay
, fluorescent estradiol ligand and tested
The recombinant ER
a
compounds (10 mM, dissolved in DMSO) were diluted to the set
concentrations according to the protocol of ER Alpha Competitor
Assay Kit, Green (A15882) using ES2 Screening Buffer. The con-
4.2.2. Cytotoxicity assay
centrations of these three samples were 150 nM, 9.0 nM and 2
respectively. The diluted recombinant ER (5 l), tested compounds
(10 l) and fluorescent estradiol (5 l) were added to 394-well plate
(Corning, NO.4514) orderly and the mixture system was reacted for
2 h at room temperature in darkness. The estradiol (1 M) was set
mM,
Cytotoxicity assay of analogs on tumor cells was determined by
2-(2-Methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-
disulfophenyl)-2H-tetrazoliumsodiumsalt (CCK-8) assay. MCF-7
a
m
m
m
(4000 cells per well) were seeded into 96-well plates in 100
mL of
m
10% FBS containing DMEM media and incubated for 24 h. DMSO
stocks synthesized analogs were diluted in culture media (2% FBS
containing DMEM) to obtain a series of concentrations (100, 50, 25,
as a positive control group and the same volume buffer was set as
negative control group. After 2 h, the polarization values were read
by microplate reader (SpectraMax Paradigm) and were utilized to
calculated inhibition ratio of the tested compounds.
12.5, 6.25 and 3.125 mM). The final concentration of DMSO in the
treatment medium was 0.1% for all wells. After 48 h of treatment,
CCK-8 solution was added to each well (0.5 mg/mL) and incubated
at 37 ꢀC for 2 h. The optical density was determined at the wave-
length of 450 nm. For each time point, we have performed triplicate
data, and mean cell viability was calculated. IC50 values were
calculated using GraphPad Prism 8 Software.
4.2.8. Cellular apoptosis study
Apoptosis of MCF-7 cells was detected using a flow cytometric
assay. Briefly, cells were seeded in 6-well plates and incubated
overnight. The following day, cells were treated with different
concentrations of 15c for 48h. The cells and supernatants were
harvested and washed twice with cold PBS and then resuspended
4.2.3. Real-time polymerase chain reaction (RT-PCR)
in 100
added into each tube and the cells were then gently vortexed and
incubated for 15 min at 25 ꢀC in the dark. 400
buffer was then added to each tube. The stained cells were analyzed
using a flow cytometer.
m
L1 ꢁ binding buffer. 5
mL FITC Annexin V and 5 mL PI were
Real-time polymerase chain reaction (RT-PCR) RNA samples
were reverse transcribed to cDNA and the PCR reactions were
performed using TaKaRa SYBR Green Master Mix carried out in
StepOnePlusTM Real-Time PCR instrument. The program for
amplification was 1 cycle of 95 ꢀC for 10 min followed by 40 cycles
of 95 ꢀC for 5 s, 60 ꢀC for 60 s, and 95 ꢀC for 15 s. The PCR results
were normalized to GAPDH expression and were quantified by the
DDCT method.
mL of 1 ꢁ binding
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
4.2.4. Molecular docking
The crystal structure of ERaY537S in complex with antagonist
H3B-5942 (PDB ID: 6CHW) was downloaded from Protein Data
Bank (http://www.pdb.org). The molecular modeling was accom-
plished with Schrodinger 10.2/Glide docking protocol. Compounds
were drawn with ChemBioDraw 14.0 and visually presented bind-
ing model is typical sample of the highest scored conformations.
Acknowledgments
This work was supported by the Project Program of Jiangsu Key
Laboratory of Drug Design and Optimization at China Pharmaceu-
tical University, and the Program of “Double-top-class” University
Project (CPU2018PZQ02; CPU2018GY07).
4.2.5. Western blotting assay
MCF-7 cells were cultured in growth medium (DMEM/F12
phenol red-free media (Gibco) supplemented with 10% serum 72 h
prior to plating cells. MCF-7 cells treated with indicated com-
pounds were lysed in RIPA Lysis Buffer (Solarbio). After determi-
nation of protein concentration by BCA assay (Beyotime), equal
amounts of total protein were electrophoresed through 10% SDS-
polyacrylamide gels. The separated protein bands were trans-
ferred onto PVDF membranes and blotted against different anti-
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113543.
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