Selective Human Estrogen Receptor Partial Agonists (ShERPAs) for Tamoxifen-Resistant Breast Cancer

Article

pubs.acs.org/jmc

Selective Human Estrogen Receptor Partial Agonists (ShERPAs) for

Tamoxifen-Resistant Breast Cancer

Rui Xiong,^†,∥Hitisha K. Patel,^†,∥Lauren M. Gutgesell,^†Jiong Zhao,^†Loruhama Delgado-Rivera,^†

Thao N. D. Pham,^‡Huiping Zhao,^‡Kathryn Carlson,^§Teresa Martin,^§John A. Katzenellenbogen,^§

Terry W. Moore,^†Debra A. Tonetti,^‡and Gregory R. J. Thatcher*^,†

^†Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 833 S. Wood Street, Chicago, Illinois

60612, United States

^‡Department of Biopharmaceutical Sciences, University of Illinois at Chicago, 833 S. Wood Street, Chicago, Illinois 60612, United

States

^§Department of Chemistry, University of Illinois, UrbanaChampaign, 600 South Mathews Avenue, Urbana, Illinois 61801, United

States

S

* Supporting Information

ABSTRACT: Almost 70% of breast cancers are estrogen receptor α (ERα) positive. Tamoxifen, a selective estrogen receptor

modulator (SERM), represents the standard of care for many patients; however, 30−50% develop resistance, underlining the

need for alternative therapeutics. Paradoxically, agonists at ERα such as estradiol (E₂) have demonstrated clinical eﬃcacy in

patients with heavily treated breast cancer, although side eﬀects in gynecological tissues are unacceptable. A drug that selectively

mimics the actions of E₂in breast cancer therapy but minimizes estrogenic eﬀects in other tissues is a novel, therapeutic

alternative. We hypothesized that a selective human estrogen receptor partial agonist (ShERPA) at ERα would provide such an

agent. Novel benzothiophene derivatives with nanomolar potency in breast cancer cell cultures were designed. Several showed

partial agonist activity, with potency of 0.8−76 nM, mimicking E₂in inhibiting growth of tamoxifen-resistant breast cancer cell

lines. Three ShERPAs were tested and validated in xenograft models of endocrine-independent and tamoxifen-resistant breast

cancer, and in contrast to E₂, ShERPAs did not cause signiﬁcant uterine growth.

advanced ER+ breast cancer but were discontinued due to

unacceptable, adverse eﬀects.^6,7

INTRODUCTION

■

The estrogen receptors, ERα and ERβ, are transcription factors

that regulate genes involved in cell proliferation, diﬀerentiation,

and migration. Dysregulation of ERα signaling is associated with

breast cancer development, which has made ERα an attractive

drug target.^1,2The archetype selective estrogen receptor

modulator (SERM) tamoxifen is the standard of care for many

patients with ER-positive (ER+) breast cancer and is intended to

antagonize the actions of estrogens at ERα in the breast, although

it poses an increased risk of endometrial cancer.³⁻⁵Aromatase

inhibitors that block estrogen biosynthesis are presently used as

an alternative to tamoxifen; however, both tamoxifen and

aromatase inhibitors cause compliance problems, due to side

eﬀects such as hot ﬂashes, arising from antiestrogenic actions in

nongynecological tissues. Paradoxically, before the advent of

tamoxifen as standard of care, estrogen and the full ERα agonist,

diethylstilbesterol (DES), were used for the treatment of

Most importantly, in about 30−50% of women undergoing

tamoxifen treatment, resistance occurs, leaving these women

with no alternative but cytotoxic chemotherapy.^4,8The cause of

tamoxifen resistance and endocrine independence of ER+ breast

cancer is not fully deﬁned and may include remodeling of several

cellular systems; for example, an overexpression of growth factor

receptors, increased activity of downstream kinase pathways,

phosphorylation of ER and its coregulators, and mutations in

ER.⁹⁻¹¹The clinical beneﬁts of estrogen or compounds with

estrogenic activity have been largely forgotten or overlooked in

the treatment of breast cancer,^12,13despite demonstrated eﬃcacy

in recent clinical trials in patients with heavily pretreated,

Received: August 14, 2015

A

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Figure 1. Structures of representative estrogen receptor ligands emphasizing the 2−4 carbon bond linking 2 phenol groups.

Figure 2. Design of partial agonists. (A) Crystal structure of BPAF (6) (PDB code 3UUA) bound to ER LBD shows two binding modes with hydrogen

bonding switch from His-524 to Thr-347, suggestive of alternate binding modes leading to agonist and antagonist conformations that contributes to

partial agonist activity. (B) Overlay of E₂(1) and tamoxifen (2) crystal structures (PDB codes 1GWR and 3ERT) shows the movement of helix 11 (His-

524 and Leu-525) that can lead to strain of helix 12, and a partial agonist conformation. (C) Truncation of the side chain of raloxifene and substitution of

phenolic OH leads to design 1. Docking of ligand 19 shows two binding poses with alternate hydrogen bonding. (D) Insertion of a linker and

diversifying substituents at C2 of the benzothiophene leads to design 2. Docking of ligand 30a shows the eﬀect of the elongation of the 2-substituent

compatible with a shift of helix 11.

metastatic ER+ breast cancer.¹⁴⁻¹⁶In the present study, we

sought to exploit the beneﬁcial aspects of estrogenic compounds

while minimizing the adverse eﬀects. Selective human estrogen

receptor partial agonists (ShERPAs) stand out as a novel

approach toward this goal. Ideally, these partial ERα agonists

would mimic estradiol’s eﬃcacy in treating tamoxifen-resistant

breast cancer while attenuating adverse eﬀects that result from

full estrogenicity.

B

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a

Scheme 1. Synthesis of 2-(4-Fluorophenyl)- and 3-(4-Fluorophenyl)benzothiophene Cores

a

Reagents and conditions: (a) KOH, EtOH/H₂O, 3-methoxythiophenol, rt, 2 h; (b) PPA, 120 °C, 3 h, 55% over two steps; (c) BF₃·OEt₂, DCM, rt,

overnight, 73% over two steps; (d) BBr₃, DCM, −78 °C to rt, overnight.

SERMs are able to act as tissue-selective ER ligands because of

the ability of liganded-ER to cause gene activation or silencing

when complexed with coactivators or corepressors.¹⁷⁻¹⁹

However, the extensive search for clinical SERMs has been

focused on compounds that display ERα antagonist activity in

breast tissues and estrogenic, agonist activity in bone tissues in

postmenopausal women.²⁰The SERM, raloxifene, in clinical use

for postmenopausal osteoporosis for over a decade, appears safe

and noncarcinogenic and is clinically indicated for chemo-

prevention of invasive breast cancer. Arzoxifene, an analogue of

raloxifene and a “third generation” SERM, demonstrated a

favorable proﬁle in preclinical studies,²¹and its active metabolite

is more potent than raloxifene.²²⁻²⁶The indole SERM,

bazedoxifene, was recently approved in Europe for postmeno-

pausal osteoporosis.²⁷⁻²⁹We have focused upon a deeper

understanding of ER ligands based upon the benzothiophene

scaﬀold, common to raloxifene and arzoxifene.³⁰⁻⁴¹Recently, we

demonstrated in two diﬀerent animal models of tamoxifen-

resistant breast cancer that a prototype benzothiophene ShERPA

caused regression of established xenografts in the absence of the

uterine weight gain shown by E₂.⁴²Herein we report the design,

synthesis, optimization, and characterization of potent ShERPAs

with potential for treatment of endocrine-independent and

tamoxifen-resistant breast cancer.

cellular targets of genistein.⁴⁷⁻⁴⁹ERα partial agonist activity has

also been reported for ligands in hepatocarcinoma cell lines

transfected with ER or endometrial cell lines; however, these

observations have not been followed up in mammary cells

containing native, functional ER or to conﬁrm pharmacological

partial agonism.⁵⁰⁻⁵³Relatively weak and partial agonist activity

was reported for bisphenol AF (BPAF)⁵⁴and WAY-169916

congeners,⁵⁵compatible with stabilization of suboptimal

conformations of ER, which might form weaker complexes

with coregulators. The crystal structure with WAY-169916 (PDB

code 2qzo)⁵⁶showed a conformation structurally diﬀerent from

the ERα/E₂complex due to a shift of helix 11 toward helix 12. A

similar shift at helix 11 by placing a 4-methoxyphenyl group at the

11β position of E₂was also proposed as a potential mechanism

for inducing ligand transition from an agonist to a partial

agonist.⁵³In the case of ERα/BPAF crystal structures, two

conformations were revealed in the same crystal structure, with

one monomer of the homodimer occupying an “antagonist”

conformation and the other an “agonist” conformation. This

emphasizes that ligand:ER complexes can adopt multiple stable

binding modes and that these might correspond to agonist,

antagonist, or partial agonist conformations (Figure 2A).⁵⁴

ER ligands commonly possess two phenol groups linked by

two to four chemical bonds, with phenolic-OH separation of 11−

12 Å. The central scaﬀold is usually planar and hydrophobic

(Figure 1).⁴³An overlay of the E₂and tamoxifen-bound ERα

structures demonstrates the large shift in the terminal residues of

helix 11, caused by the tamoxifen side chain, resulting in

displacement of helix 12 and antagonist activity (Figure 2B). A

closer look at the structure of ERα complexed with WAY-169916

shows that the smaller shift of helix 11, repositioning His-524 and

Leu-525, strains helix 12 without complete displacement. The

ERα:BPAF structures show a switch from His-524 to Thr-347 as

a key hydrogen bonding residue with the ligand, demonstrating a

dynamic hydrogen bonding network that can accommodate

diverse agonists or antagonists in various poses (Figure 2A). We

hypothesized that novel ligands could maintain high aﬃnity and

potency while acting as partial agonists by exerting small shifts in

helix 11 and exploiting the dynamic hydrogen bonding network

for stabilization.

STRUCTURE DESIGN

■

The plasticity of the ER ligand-binding domain (LBD) is

reﬂected by the structural diversity of ER ligand scaﬀolds (Figure

1) and lends itself to design of novel pharmacological

modulators.^43,44The volume of the ER ligand-binding pocket

is ∼450 Å³and can accommodate ligands as small as 250 Å³(e.g.,

estradiol, E₂; PDB code 1gwr) or as large as 380 Å³(e.g., 17R-

(2E-triﬂuoromethylphenylvinyl)-E₂; PDB code 2p15).⁴⁵The

ligand-binding domain of ER comprises 12 α helices and a β

sheet. These secondary structures create an interior hydrophobic

cavity forming a ligand binding site and a hydrophobic, solvent-

exposed surface to which transcriptional coregulators bind.⁴⁶In

the “mousetrap” model of ER, helix 12 is closed, allowing

coregulator binding (agonist mode), or open and hindering

coregulator binding (antagonist mode).⁴⁶This binary model

may have limited pursuit of partial agonists at ER; however, even

in this mousetrap view, a ligand that can stabilize both agonist

and antagonist conformations may act as a partial agonist.

Partial estrogenic activity (deﬁned by maximal activity

signiﬁcantly lower than E₂) has been reported for genistein in

cell cultures; however, this submaximal activity at micromolar

concentrations may be associated with the many other known

To develop partial agonists, we chose the benzothiophene

scaﬀold, common to the SERMs arzoxifene and raloxifene, the

latter having a superior safety proﬁle to tamoxifen. As depicted in

Figure 2C,D, the initial design step was to truncate the SERM

side chain to avert complete displacement of helix 12 and to

avoid a pure antagonist. A small probe library was prepared using

interchange and substitution of phenolic substituents to explore

the eﬀect on activity of truncation and replacing phenolic OH.

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a

Scheme 2. Synthesis of 2-(4-Fluorophenyl)benzothiophenes

a

Reagents and conditions: (a) N-bromoacetamide, DCM/EtOH, rt, 2 h, 78%; (b) H₂O₂, TFA, DCM, 0 °C, 2 h, 75%; (c) 4-methoxyphenol, NaH,

DMF, rt, 3 h, 93%; (d) LiAlH₄, THF, 0 °C, 2 h, 71%; (e) BBr₃, DCM, −78 °C to rt, overnight; (f) AlCl₃, R₁COCl, DCM, rt, 2−48 h.

We hypothesized that partial agonist activity could result from

alternate binding modes, as seen in BPAF complexes, which

might correspond to agonist and antagonist conformations

(design 1, Figure 2C), or alternatively, from a shift in helix 11,

causing strain in helix 12 and consequently suboptimal

coregulator binding (design 2, Figure 2D). We inserted an

ether or ketone linker at C2 intended to displace the 2-

benzothiophene substituent to interact directly with the terminal

residues of helix 11, causing strain in helix 12 (design 2).

ﬁnal product. Regioisomers were veriﬁed by 2D NMR, and one

of the key intermediates was recrystallized for X-ray crystallog-

raphy for structural conﬁrmation (Supporting Information

Figure 1).

BIOLOGICAL TESTING

■

Ligand aﬃnity and estrogenic potency were evaluated,

respectively, in a ﬂuorescence polarization (FP) binding assay

using full-length ERα and a cell-based assay using an estrogen

response element (ERE) luciferase reporter (Table 1). Ligand

aﬃnity of selected ShERPAs was also studied by radioligand

displacement (Table 2). Potency and eﬃcacy (E_max) for ERE-

luciferase activation were measured in endocrine-dependent

ER+ MCF-7:ws8 cells, the response being normalized to control

treated cells (0%) and E₂(1 nM) treated cells (100%).

CHEMISTRY

■

2-(4-Fluorophenyl)-6-methoxybenzo[b]thiophene (9) was pre-

pared by the cyclization rearrangement induced by polyphos-

phoric acid (PPA) as reported by Jones and colleagues.⁵⁷The

regioisomer 3-(4-ﬂuorophenyl)-6-methoxybenzo[b]thiophene

(10) was synthesized with BF₃·OEt₂in dichloromethane

(Scheme 1). Aroylation at C3 of benzothiophene was

accomplished by direct Friedel−Crafts acylation. Coupling

with the acid chloride bearing a strong electron withdrawing

group, e.g., 14b, gave multiple isomers and minor products, and a

C18 reverse-phase column was used to purify the ﬁnal products

(Scheme 2). A trans-halogenation minor product, 14c, was

isolated from the preparation of 14b with 3% isolated yield,

possibly from a S_NAr displacement of ﬂuoride by chloride.^58,59

C2 benzothiophene aroylation used a similar strategy with the

exception of products containing strong electron withdrawing

groups, which were obtained using n-butyllithium to generate an

organic lithium salt that reacts directly with the acid chloride

(Scheme 3). The synthesis of ether-linked derivatives was

accomplished by adapting a reported procedure (Scheme 4).³²

Bromination of the benzothiophene core with N-bromoaceta-

mide provided exclusively the monobrominated product in high

yield, which was activated for S_NAr reaction by oxidation to the

sulfoxide under H₂O₂. Introduction of the substituted phenol

was accomplished in the presence of NaH in DMF. Sulfoxide

reduction was performed under LiAlH₄in THF. Deprotection of

the methyl group was achieved by BBr₃or BF₃·SMe₂to give the

Pharmacological partial agonists are deﬁned by the submax-

imal eﬃcacy compared to the preferred, endogenous ligand and

by antagonism of the activity of the endogenous agonist.

Antagonism has rarely been tested in the literature for putative

partial agonists at ER. By deﬁnition, a ShERPA must antagonize

the actions of E₂in breast cancer cell cultures in which it exerts

partial agonist activity. The therapeutic rationale for exploring

ShERPAs is based upon the need for an ER ligand that mimics

the actions of estrogen in killing tamoxifen-resistant breast cancer

cells. Therefore, the ability of these mimics to inhibit growth of

endocrine-independent, tamoxifen-resistant MCF-7:5C cells, on

the ninth day after treatment, was assessed by MTS assay

(conﬁrmed by DNA assay). Results were reported as cell viability

relative to vehicle (100%) (Figure 3) or as inhibition of cell

growth relative to vehicle (0%) and E₂(100%) (Table 3).

The three ERα ligands for which data are depicted in Figure 3

represent examples of a full agonist (1), SERM antagonist (3),

and a ShERPA (30a). Panel A shows ERE-luciferase response in

MCF-7:ws8 cells. Panel B shows FP measured by displacement

of ﬂuorescent E₂from ERα, and panel C shows viability of treated

tamoxifen-resistant MCF-7:5C cells.

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a

Scheme 3. Synthesis of 3-(4-Fluorophenyl)benzothiophene Compounds with a Ketone Linker

a

Reagents and conditions: (a) AlCl₃, R₁COCl, DCM, rt, 2−48 h; (b) BBr₃, DCM, −78 °C to rt, 4−48 h for 21a−e and 26a,b; BF₃·SMe₂, DCM, 0

°C to rt, 24 h for 21g and 23; (c) N-bromoacetamide, DCM/EtOH, rt, 89%, 2 h; (d) n-BuLi, R₂COCl, THF, −78 °C to rt, 4 h; (e) Et₃N,

Pd(PPh₃)₂Cl₂, DMF, CuI, ethynyltrimethylsilane, 4 h; (f)TBAF, THF, rt, 1 h, 50% over two steps.

ERE-Luciferase Guided Structure Optimization. A small

probe library was prepared to explore side chain truncation and

substitution of phenolic OH in derivatives containing the 2-

phenylbenzothiophene core common to arzoxifene and

raloxifene. Truncation and substitution of a methyl group at

the 4′-benzoyl position switched an antagonist (raloxifene) to a

potent agonist (14a): EC₅₀= 2.6 nM, eﬃcacy (E_max) of ∼100%

(Table 1). Substitution of the phenolic OH by F in 14b reduced

potency and aﬃnity but retained full agonist activity. Similarly,

the chloro congener (14c) was a full agonist, and 19, containing

an ether linkage and 4′-OH replaced by F, was again a potent full

agonist in MCF-7 cells: EC₅₀= 6.2 nM. Side chain truncation was

conﬁrmed as a route to ERα agonists, and replacement of

phenolic OH groups was tolerated while retaining nanomolar

potency. In the FP assay using ligand displacement from full

length ERα, all truncations and substitutions of raloxifene

reduced relative binding aﬃnity compared to raloxifene itself

(RBA_ral). Even in this small conserved library of structures,

correlation of aﬃnity with potency in cell cultures was poor,

driving use of the luciferase reporter assay for optimization.

Having demonstrated the simple hypothesis that side chain

truncation would produce ER agonists, we proceeded to test the

proposal that insertion and elongation of the 2-benzothiophene

substituent would lead to partial agonist activity. The 3-

ﬂuorophenylbenzothiophene series (21) yielded agonists that

as in the probe library showed reduced RBA_ralbut nanomolar,

and in one case picomolar, potency for ERE activation. For

example, compound 21b with two phenolic substituents was a

potent full agonist (EC₅₀= 0.2 nM) but with RBA_ralonly 14%

that of raloxifene. One derivative in this series, 21c, proved to be a

potent partial agonist (EC₅₀= 30 nM). Moreover, we were able

to replace the 2-benzoyl group with sp³alkanoyl substituents

while maintaining potency (EC₅₀= 1−14 nM). Within the 2-

benzoyl and 2-alkanoyl benzothiophene series, 3 of 10

compounds (21c, 23, 26a) demonstrated potent partial agonist

activity but with relatively low RBA_ral: for example, the

triﬂuoromethyl derivative (26a), E_max= 80%, EC₅₀= 5 nM,

and the alkynyl derivative (23), E_max= 65%, EC₅₀= 5 nM. A

further series of 3-phenylbenzothiophenes with a 2-ether linkage

(30) showed a similar pattern of activity, with relatively low

RBA_ral, contrasting with nanomolar potency as agonists (0.8−76

nM). Directly comparing the ether- and ketone-linked series

(21b vs 30a and 21e vs 30c) revealed bis-phenolic derivatives to

have relatively high potency (<1 nM) and RBA_ral, whereas both

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a

Scheme 4. Synthesis of 3-(4-Fluorophenyl)benzothiophene Compounds with an Ether Linker

a

Reagents and conditions: (a) N-bromoacetamide, DCM/EtOH, rt, 89%, 2 h; (b) H₂O₂, TFA, DCM, 0 °C, 2 h, 84%; (c) R₃OH, NaH, DMF, rt, 6 h

for 28a; Cs₂CO₃, DMF, 80 °C, overnight for 28b−d; (d) LiAlH₄, THF, 0 °C, 2 h; (e) BBr₃, DCM, −78 °C to rt, 4−48 h.

bis-ﬂuorophenyl derivatives had lower RBA_raland 100-fold

reduced potency (Table 1). We used G15, the inhibitor of

GPER/GPR30, to test for any involvement of this extranuclear

receptor in ShERPA activity. Cotreatment of cells with G15 and

ShERPAs did not signiﬁcantly reduce observed luciferase

reporter activity (Supporting Information Figure 2).

The relatively low RBA_ralvalues obtained from the FP assay

accompanied by nanomolar potency in the reporter assay led to

the testing of the three ShERPAs, 23, 26a, and 30a, in the

classical radioligand displacement assay (Table 2). It was seen

that the potent ShERPA, 30a, exhibited 92% RBA relative to

estradiol (RBA = 100%) while ShERPAs 23 and 26a exhibited

8% and 1% RBA, respectively. While our compounds exhibit

strong aﬃnity in this assay, a lack of correlation between

biochemical data and cellular response remains, conﬁrming the

use of the reporter assay as the optimal route for structure

optimization.

Characterization of ShERPA Partial Agonism. All 2-

phenyl and 3-phenyl benzothiophene derivatives obtained

according to the design principles described in Figure 2 acted

as ERα agonists. As deﬁned by ERE-luciferase activation in ER+

breast cancer cell lines, ﬁve of these were ShERPA candidates:

21c, 23, 26a, 30a, and 30b. When compared to full agonists,

these partial agonists did not uniformly display weaker aﬃnity

(RBA_ral) or reduced potency for ERE activation; for example, 30a

is a partial agonist with picomolar potency and aﬃnity for

isolated ERα (RBA) not signiﬁcantly diﬀerent from E₂itself. In

addition to submaximal eﬃcacy, a partial agonist should by

deﬁnition antagonize the action of the endogenous agonist. The

partial agonists 30a, 23, and 30b, showing a range of potency

(EC₅₀= 0.8 nM, 5.3 nM, and 76.1 nM, respectively), were tested

as antagonists in MCF-7:ws8 cells. Pharmacological partial

agonist activity was validated by the antagonism of E₂induced

ERE activation (Figure 4).

ER in an agonist conformation. The recruitment, assembly, and

disassembly of protein complexes at DNA control transcriptional

events triggered by liganded-ER.⁶⁰⁻⁶²Recruitment of steroid

receptor coregulator-3 (SRC3 or AIB1, ampliﬁed in breast cancer

1) is a key component of ERα-mediated transcription. The

interaction of ERα-LBD with SRC3 coactivator is therefore a

minimal predictor of phenotype, as a necessary step in the

canonical nuclear ER-transactivation pathway.^63,64Time-re-

solved ﬂuorescence (or Forster) resonance energy transfer

̈

(TR-FRET) assay of the interaction of ERα-LBD with an LxxLL-

containing recognition sequence of SRC3, in the presence of E₂,

has been used to identify small molecules that directly inhibit

protein−protein binding.^65,66Ligand stabilization of the ERα/

SRC3 complex is an initial and essential step in transmitting a

functional, transcriptional signal and, unlike RBA measurements,

will deﬁne agonist and antagonist activity. A full agonist is

predicted to stabilize the ERα/SRC3 complex in a similar

conformation to the E₂-bound complex, yielding a similar TR-

FRET signal, whereas an antagonist in the presence of E₂will

disrupt the ERα/SRC3 complex, destroying the FRET signal.

In the TR-FRET assay, the increase of FRET signal on titration

of SRC3 into solutions of E₂, 23, or 30a was indicative of

formation and stabilization of the ERα/SRC3 complex (Figure

5A). In the presence of 23 and 30a, the maximal FRET signal was

less than 50% of that generated by the E₂-liganded ERα-LBD/

SRC3 complex, compatible with 23 and 30a acting as partial

agonists. In a second TR-FRET assay, conducted in the presence

of a submaximal concentration of E₂(10 nM), titration of E₂, 23,

or 30a led to diﬀerent outcomes. While E₂enhanced the FRET

signal by increasing complexation, both 23 and 30a reduced

FRET signal, compatible with weak antagonist activity.

Observations in TR-FRET, although replicating only a minimal

portion of the ERE-bound ERα complex, are compatible with the

designation of ShERPA activity.

The mechanics preceding ERE activation by liganded ER

include dimerization, nuclear translocation/accumulation, and

binding to DNA at the ERE of a multiprotein complex containing

Inhibition of Growth of Tamoxifen-Resistant Breast

Cancer Cells. To further test the candidate ShERPAs, it was

necessary to conﬁrm their ability to mimic the actions of E₂in

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Table 1. Transcriptional Activity and Relative Binding Aﬃnity of Benzothiophene Analogs

a

In MCF-7:ws8 cells, estrogenic activity was measured using a luciferase reporter assay and normalized to Ctrl (0%) and E₂1 nM (100%). Data

b

show the mean SEM determined from an average of at least three passages. NA = no activity. RBA_ralmeasuring reduction in FP caused by

displacement of ﬂuorescein-E₂from ER. Data are normalized to raloxifene binding at 100% SEM.

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was equivalent or superior to the maximum inhibition observed

after treatment with E₂. Compound 21b gave 100% inhibition at

1 nM, and both 21g and 30a gave 100% inhibition at 10 nM. Of

the candidate ShERPAs, only one did not show eﬃcacy

comparable to E₂in inhibition of MCF-7:5C cell growth. It is

important to emphasize that inhibition of growth of MCF-7:5C

cells is not caused by any general cytotoxicity of SEMs and

ShERPAs. This was clearly demonstrated in the parent estrogen-

responsive MCF-7:ws8 cells, in which all compounds (100 nM

and 1 μM) supported growth at a level higher than DMSO

control (data not shown).

Table 2. Relative Binding Aﬃnity of Selected ShERPAs in

Radioligand Displacement Assay

a

compd

RBA

100

E₂

26a

23

1.3 0.3

7.9 0.3

92.4 21

30a

a

Relative binding aﬃnity (RBA) values, determined by radiometric

assays, are expressed as {[(IC₅₀estradiol)/(IC₅₀compound)] ×100}

the range or standard deviation (RBA, estradiol = 100%).

Regression of Tamoxifen-Resistant Xenografts Un-

coupled from Uterine Growth. We have previously reported

that treatment with ShERPA, 30a, resulted in dramatic

regression in the two tamoxifen-resistant breast cancer xenograft

models, T47D:A18/PKCα and T47D:A18-TAM1, comparable

to that observed on treatment with E₂.⁴²The eﬃcacy of

ShERPAs 23 and 26a was evaluated in the T47D:A18/PKCα

model of tamoxifen resistance. The tumor xenografts were

allowed to establish for 12 weeks prior to treatment. Although

dose optimization was not carried out, at the single dose studied,

the tumor size was shown to reduce rapidly on ShERPA

treatment and within 2 weeks to shrink to half the size of control

tumors (Figure 6A). Adverse estrogenic eﬀects in gynecological

tissues provided the rationale for development of ShERPAs.

Estrogens are known uterotrophic agents, and even tamoxifen

shows tissue-selective agonist activity in the uterus. In the

T47D:A18/PKCα xenograft study, tumor regression induced by

inhibiting growth of tamoxifen-resistant MCF-7 cells. A number

of tamoxifen-resistant cell lines have been created by long-term

exposure to tamoxifen (T47D:A18-TAM1), long-term estrogen

deprivation (MCF-7:5C), or ectopic upregulation of PKCα

(T47D:A18/PKCα).^42,67,68These stable cell lines are charac-

terized by endocrine independence (growth independent of

estrogens) and resistance to any growth inhibitory eﬀects of

tamoxifen. Many of these lines are also characterized by a

paradoxical inhibition of cell proliferation on treatment by E₂at

nanomolar concentrations. In the case of MCF-7:5C cell

cultures, treatment with E₂causes a signiﬁcant reduction in cell

viability measured at 9 days. E₂reduced cell viability by

approximately 60% compared to vehicle control (Figure 3C).

Inhibition of cell growth relative to vehicle (0%) and to E₂

(100%) was measured for the candidate selective estrogen

mimics (SEMs) and ShERPAs (Table 3). For compounds 14a,b,

21b−g, and 30a at ≤100 nM, the percent of growth inhibition

Figure 3. Proﬁling ERα ligands: endogenous agonist E₂(1); SERM antagonist raloxifene (3); benzothiophene partial agonist (ShERPA) (30a). (A)

Activation of ERE (estrogen response element) in MCF-7:ws8 cells. (B) RBA_ralfrom FP assay of displacement of ﬂuorescent-E₂from ERα. (C) Cell

viability of tamoxifen-resistant MCF-7:5C breast cancer cells 9 days after drug treatment, normalized to vehicle (100%). Data show the mean and SEM.

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Table 3. Cell Viability of MCF-7:5C Cells Treated with E₂and Test Compounds

a

Cell death percentage was normalized to control (0%) and E₂(100%) assessed in a 9-day cell viability assay with drug treatments renewed every 3

days. Data represented as mean SEM are an average of at least three cell passages (triplicates in each passage).

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Figure 4. Partial agonist activity of three ShERPAs (30a, 23, and 30b) assessed by activation of ERE (estrogen response element) in MCF-7:ws8 cells

(normalized to 0.1 nM E₂= 100%) using a luciferase assay after treatment for 18 h. The antagonist action of these ShERPAs was assessed in the presence

of 0.1 nM E₂, and a dose dependent reduction in E₂luciferase activity is seen with increasing concentrations of ShERPAs (100−500 nM). Data show the

mean and SEM from at least three cell passages.

Figure 5. (A) Agonist mediated recruitment of ERα coactivator (SRC3). Estradiol (1) induces an increase in FRET readout with increasing

concentrations of SRC3. ShERPAs 23 and 30a also show an increase in FRET signal with increasing concentration of SRC3, but maximal activation is

less than 50% of E₂. (B) Increasing concentrations of ShERPAs 23 and 30a decrease the FRET signal induced by 10 nM E₂, attributed to the antagonist

action of these molecules. The FRET signal is ampliﬁed in the presence of increasing concentrations of added E₂(1) as is expected of an agonist. Data

show the mean and SEM.

Figure 6. (A) T47D:A18/PKCα tumors were established as described in the Experimental Section. Tumors were grown to an average size of 0.3 cm².

Mice were then randomized into three treatment groups: Ctrl (n = 4), 23 and 26a (n = 6). Administration of drug was by oral gavage daily (100 mg/kg).

Within 2 weeks of treatment, a regression of established tumors was observed, while control tumors continued to grow. (B) Measured uterine weight (g)

of Ctrl (n = 4) and 26a (n = 6) treatment groups showed no signiﬁcant diﬀerence, suggesting an improved side eﬀect proﬁle. Data show the mean and

SEM.

30a was not accompanied by a signiﬁcant increase in uterine

weight, in contrast to E₂.⁴²Uterine weight was measured in the

xenograft study of 26a, presented herein, again showing no

signiﬁcant increase in uterine weight compared to vehicle control

mice (Figure 6B). It is tempting to link the lack of uterotrophic

activity of ShERPAs in these studies with partial agonist,

submaximal eﬃcacy at ERα in uterine tissues. This hypothesis

cannot be conﬁrmed in vitro because there is no cell-based model

that reliably reproduces the uterotrophic actions of ER ligands.

However, these observations do predict a reduced side eﬀect

proﬁle for ShERPAs relative to full estrogens.

DISCUSSION

■

The “classic” genomic actions of ERα as a nuclear receptor and

transcription factor have dominated thinking on the role of

estrogen in cancer. ERα is thought to mediate diﬀerentiation,

apoptosis, and proliferation in normal mammary epithelial cells;

however, in malignant tissues, a shift to proliferation contributes

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to progression. Paradoxically, in a variety of mammary cell lines

that have developed tamoxifen resistance, the proliferative

phenotype of estrogen switches to an antiproliferative and

apoptotic phenotype, compatible with the clinical eﬃcacy of E₂in

advanced ER+ breast cancer.^14−16,42

EXPERIMENTAL SECTION

■

Cell Lines and Culture Conditions. MCF-7:ws8 cells were

obtained from American Type Culture Collection (Manassas, VA).

MCF-7:ws8 cells were cultured in phenol red containing RPMI 1640

medium supplemented with 10% fetal bovine serum, 1% Glutamax, 1%

nonessential amino acids, insulin (10 μg/mL), 1% antibiotic−

antimycotic, and 5% CO₂at 37 °C. Treatment medium was prepared

by supplementing phenol red free RPMI 1640 medium with charcoal-

dextran treated fetal bovine serum, while other supplements remained

the same. MCF-7:5C cells were a gift from Dr. Tonetti’s lab. These cells

served as a tamoxifen resistance model and were obtained by long-term

estrogen deprivation of MCF-7:ws8 cells. These cells were maintained

in phenol-red free RPMI 1640 medium supplemented with 10%

charcoal-dextran treated fetal bovine serum, 1% Glutamax, 1%

nonessential amino acids, insulin (10 μg/mL), 1% antibiotic−

antimycotic, and 5% CO₂at 37 °C.

Array and ChIP technologies have allowed mapping of ER to

regulatory regions of activated and repressed target genes,

showing that liganded-ER may cause gene activation or silencing,

dependent on ligand stabilization of multiprotein transcriptional

complexes, containing either coactivators or corepressors.¹⁷⁻¹⁹

ERα ligands may therefore act in a cell and tissue selective

manner as exempliﬁed by the SERMs raloxifene and tamoxifen:

both SERMs act as estrogen antagonists in breast tissue but as

estrogen agonists in the bone tissue of postmenopausal women.

In contrast to SERM antagonists, the goal of this study was to

design agonists at ERα, selectively to mimic the actions of

estrogen in ER+ tamoxifen-resistant breast cancer.^69,70This goal

was founded on the clinical eﬃcacy of E₂and the ER agonist,

DES, in advanced ER+ breast cancer.^6,7Cognizant of the

unacceptable side eﬀects of these estrogens, it was hypothesized

that ShERPAs, as partial agonists, would minimize excessive

estrogenic activity in other gynecological tissues, for example, the

uterus.

The design of ShERPAs, utilizing the benzothiophene core of

raloxifene, was guided by structural information from published

ERα-LBD crystal structures. In tamoxifen-resistant breast cancer

cells, a ShERPA must stabilize ERα as part of a multiprotein

complex at ERE, mimicking E₂; however, partial agonism is

desired to minimize full estrogenic side eﬀects. Hypothetically,

an ERα ligand that yields a less stable multiprotein complex than

the E₂-bound complex, or a ligand that stabilizes both activated

and silenced complexes would produce partial agonist activity.

Such ligands are predicted to have lower potency than E₂itself;

however, potency similar to raloxifene should be achievable. The

ShERPA ligands discovered in this study were proﬁled as

pharmacological partial agonists with potency for ERE activation

of 0.8−76 nM. Neither RBA_ralnor RBA for isolated ERα

correlated with potency in cell cultures. Various studies have

made similar observations on the discordance between

biochemical aﬃnity and potency in cell cultures,^66,71−73and

alternative methodologies have been proposed.⁷⁴Poor correla-

tion was observed between RBA and growth inhibition in MCF-7

cells for a series of raloxifene analogues with similar RBAs but up

to 100-fold diﬀerences in potency in cell cultures.⁷¹A lack of

correlation between binding aﬃnities (RBA) and both potency

for coactivator recruitment and cellular ERE-reporter activation

was also apparent from a study of ERα ligands commonly used as

chemical probes.⁶⁶This is compatible with the thermodynamics

of ligand binding to ERα in a multiprotein complex being

inﬂuenced by other binding partners in that complex.

ER Binding Studies. The ligand binding studies for test compounds

to full length ER were performed using a PolarScreen nuclear receptor

competitor assay (Invitrogen, Carlsbad, CA) according to the

manufacturer’s instructions. Brieﬂy, serial dilutions of test compounds

were performed using a Biomek 3000 Laboratory Automation

Workstation in a 384 deep well polypropylene block. This served as

the 100× serial dilution plate of all test compounds in DMSO where

dilutions of test compounds were 100× the ﬁnal concentration. The 2×

dilution plate was prepared by adding 2 μL of the stocks from the 100×

plate to 98 μL of assay buﬀer and mixed well. The ﬁnal assay plate was

prepared by adding 10 μL of 2× stocks to 10 μL of ER + ﬂuorescein-

labeled E₂(ﬁnal concentration of ER = 25 nM, ﬂuorescein-labeled E₂=

4.5 nM) and mixing well. The plate was then incubated for 2 h in the

dark, and ﬂuorescence polarization was measured on a SynergyH4

hybrid multimode microplate reader (Biotek). The ﬂuorescence

polarization was computed by measuring parallel and perpendicular

polarizations at an excitation wavelength of 485 nm and an emission

wavelength of 535 nm. IC₅₀values were computed for unlabeled E₂,

raloxifene, and test compounds, and the relative binding aﬃnities were

computed using the following formula: RBA_ral= [(IC₅₀of raloxifene)/

(IC₅₀of test compounds)] × 100. Relative binding aﬃnities of 23, 26a,

30a were also determined by a competitive radiometric binding assay

with 2 nM [³H]estradiol as tracer (PerkinElmer, Waltham, MA) and

full-length puriﬁed human ERα (Pan Vera/Invitrogen, Carlsbad, CA), as

described previously.^75,76The RBA values were determined using the

following equation: [(IC₅₀estradiol)/(IC₅₀compound)] × 100.

Induction of Estrogen Response Elements in MCF-7 Cells.

Activation of ERα signaling by test compounds was measured using

MCF-7:ws8 cells which were kept in stripped medium 3 days prior to

treatment. Cells were plated at a density of 6 × 10⁵cells/well in 12-well

plates and were transfected with 3 μg of the pERE-luciferase plasmid,

which contains three copies of the Xenopus laevisvitellogenin A2 ERE

upstream of ﬁreﬂy luciferase. To normalize for cell viability and

transfection eﬃciency, 1 μg of pRL-TK plasmid (Promega, Madison,

WI) containing a cDNA encoding Renilla luciferase was cotransfected

along with ERE plasmid. Transfection was performed for 6 h using the

Lipofectamine 2000 transfection reagent (Invitrogen) in Opti-MEM

medium according to the manufacturer’s instructions. Cells were treated

with test compounds after 6 h, and the luciferase activity was measured

in cell lysates after 18 h of treatment using the dual luciferase assay

system (Promega) with FLUOstar OPTIMA (BMG LABTECH,

Durham, NC). Antiestrogenic activity of ShERPAs was studied by

cotreating with 0.1 nM E₂to observe inhibition of E₂response. Data are

represented as an average of three passages reported as the relative

luciferase percentage with 1 nM E₂treatment set as 100% and control

treated cells as 0%. The initial luciferase activity values were calculated

by dividing the ﬁreﬂy luciferase (ERE) reading by the Renilla luciferase

(pRL-TK) reading.

ShERPAs were eﬀective in killing tamoxifen-resistant breast

cancer cells in culture. Three ShERPAs were further validated in

tamoxifen-resistant xenograft models; these mice show no

signiﬁcant increase in uterine weight, a key side eﬀect of estrogen

treatment.⁴²It would be unsafe without further research to assign

these observations entirely to the partial agonist activity of

ShERPAs; for example, the involvement of extranuclear ER and

ERβ needs to be considered. Nevertheless, we have demon-

strated that potent, partial agonists at ERα can be created and

that these ShERPAs have activity in vivo that is of potential

therapeutic beneﬁt in treatment of a subset of ER+ breast

cancers.

TR-FRET. FRET assay buﬀers (A and B) were prepared as previously

reported.⁶⁶In the SRC3 titration assay, the 3× concentrations of

ﬂuorescein-labeled SRC3 (5 μL) prepared in buﬀer A was added to a 96-

well black microplate containing premixed streptavidin−terbium (SA-

Tb) and biotinylated ERα LBD (5 μL). This was followed by the

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addition of 3× concentration of ligands 1, 23, and 30a (5 μL) prepared

in buﬀer B. The ﬁnal 15 μL assay volume contained 1 nM ERα LBD, 20

μM ligand, and 0.25 nM SA-Tb. Assay solutions were mixed and

incubated for 1 h at room temperature in the dark before being

measured for TR-FRET. Parallel incubations without the biotinylated

ERα LBD were performed to correct for diﬀusion-enhanced FRET and

to correct for nonspeciﬁc binding. The background was subtracted from

the total FRET values obtained from the test samples and plotted against

log SRC3 concentrations. In the ligand titration assay, serial dilutions of

1, 23, and 30a were added to premixed 0.25 nM streptavidin−terbium, 1

nM ERα LBD, 100 nM ﬂuorescein-labeled SRC3, and 10 nM E₂. The

plate was mixed and incubated in the dark for 1 h before measurement of

the FRET signal. The donor SA-Tb was excited at 340/80 nm, and

emissions from the donor and the acceptor ﬂuorescein were monitored

at 495/20 and 520/25 nm, respectively, with a 100 μs delay. TR-FRET

was measured on a Wallac Victor II plate reader (Molecular Devices,

Sunnyvale, CA).

Cell Viability Assays. MCF-7:5C tamoxifen resistant cells were

plated overnight at a density of 5000 cells/well. Cells were then treated

with either E₂or test compounds at 1, 10, and 100 nM. Treatments were

renewed every 3 days, and the cell viability was tested at day 9 using a

CellTiter 96 AQueous One solution cell proliferation assay (MTS). The

absorbance was measured at 490 nm using a SynergyH4 hybrid

multimode microplate reader (Biotek), and cell viability was normalized

to control (DMSO) treated cells at 100%. Data are an average of at least

triplicates from three diﬀerent passages. The cell toxicity in MCF-7:5C

was corroborated with a second cell viability DNA content assay. Brieﬂy,

cells were plated in 96-well plates at a density of 5000 cells/well and

treated with test compounds for 6 days and then frozen in dH₂O for 24

h. Cells were then lysed with TNE buﬀer (100 μL/well) containing 50

mL of TE buﬀer, 1 M NaCl, and 100 μL of Hoescht DNA stain. The

ﬂuorescence was then measured at an excitation wavelength of 355 nm

and emission wavelength of 460 nm using the SynergyH4 hybrid mode

microplate reader (Biotek). MCF-7:ws8 cells were stripped 3 days prior

to plating a density of 5000 cells/well in 96-well plates and allowed to

attach overnight. Cells were then treated with DMSO, E₂(1 nM), and

test compounds (100 nM) for 3 days. Cell viability was measured using

an MTT assay. Brieﬂy, MTT powder was dissolved in PBS (5 mg/mL

stock solution) and was added to cell medium to a ﬁnal concentration of

0.5 mg/mL. The medium was removed after incubation, and 100 μL of

DMSO was added to each well. After shaking, the absorbance of the

plate was measured at 570 nm using the SynergyH4 hybrid mode

microplate reader (Biotek), and cell viability was normalized to control

at 100%.

(LR) mass spectra were acquired on an Agilent 6300 ion-trap LC−MS

instrument. High resolution mass spectral data were collected in-house

using a Shimadzu IT-TOF. All compounds submitted for biological

testing were conﬁrmed to be ≥95% pure by analytical HPLC. Synthetic

procedures, spectral data, and HRMS for ﬁnal compounds and novel

intermediates are described below.

3-(4-Fluorophenyl)-6-methoxybenzo[b]thiophene (10). Me-

thoxybenzenethiol (65 g, 7.1 mmol) was added to a round-bottom ﬂask

charged with a freshly prepared solution containing 300 mL of ethanol,

100 mL of water, and 30 g of KOH (538 mmol). The solution was

cooled in an ice−water bath, and a solution of 2-bromo-1-(4-

ﬂuorophenyl)ethanone (100 g, 461 mmol) in 100 mL of ethyl acetate

was slowly added. The reaction mixture was monitored by TLC until the

ﬁnish. The solvents were evaporated under reduced pressure, and the

residue mixture was partitioned between water and ethyl acetate. The

combined organic phase was washed with brine and dried by Na₂SO₄to

give 120 g of 1-(4-ﬂuorophenyl)-2-(3-methoxyphenylsulfanyl)-

ethanone, which was used in the next step without further puriﬁcation.

BF₃·OEt₂was slowly added to a ﬂask charged with 1-(4-ﬂuorophenyl)-2-

(3-methoxyphenylsulfanyl)ethanone (120 g) under argon atmosphere

in an ice bath. The reaction mixture was stirred until starting material

was consumed as monitored by TLC. The reaction mixture was poured

into saturated NaHCO₃/ice−water, stirred 30 min, and extracted with

dichloromethane. The crude product was puriﬁed by silica gel

chromatography (5% dichloromethane in hexane). The combined

fractions from the column were concentrated and recrystallized to give

75 g of pale yellow solid with a 54% yield over the two steps. ¹H NMR

(400 MHz, CDCl₃) δ 7.71 (d, J = 8.9 Hz, 1H), 7.56−7.48 (m, 2H), 7.38

(d, J = 2.2 Hz, 1H), 7.21−7.12 (m, 3H), 7.02 (dd, J = 8.9, 2.2 Hz, 1H),

3.90 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 162.49 (d, J_C−F= 246.7

Hz), 157.73, 142.24, 136.79, 132.34, 132.14, 130.30 (d, J_C−F= 8.0 Hz),

123.47, 120.85, 115.78 (d, J_C−F= 21.4 Hz), 114.70, 105.42, 55.80.

2-(4-Fluorophenyl)benzo[b]thiophen-6-ol (11). 2-(4-Fluoro-

phenyl)-6-methoxybenzo[b]thiophene (9)³²(4.8 g, 18.6 mmol) was

dissolved in 100 mL of anhydrous dichloromethane and cooled to −78

°C under a dry ice−acetone bath. BBr₃(1.0 M in CH₂Cl₂, 55 mL, 55

mmol) was added dropwise to this solution. The reaction mixture was

stirred until starting material was consumed as monitored by TLC and

then quenched by saturated NaHCO₃/ice−water. The solution was

extracted with ethyl acetate and washed with brine. The organic extracts

were combined, dried over anhydrous Na₂SO₄, concentrated in vacuum,

and then puriﬁed by ﬂash chromatography (5−30% ethyl acetate in

hexane) to give 3.3 g of white solid (yield, 73%). ¹H NMR (400 MHz,

CD₃OD) δ 7.72−7.65 (m, 2H), 7.60 (d, J = 8.6 Hz, 1H), 7.47 (s, 1H),

7.20 (d, J = 2.0 Hz, 1H), 7.14 (t, J = 8.8 Hz, 2H), 6.86 (dd, J = 8.6, 2.2 Hz,

1H). ¹³C NMR (100 MHz, CD₃OD) δ 163.81 (d, J_C−F= 246.1 Hz),

156.52, 142.33, 140.46, 135.45, 132.45, 128.77 (d, J_C−F= 8.1 Hz),

125.39, 120.41, 116.74 (d, J_C−F= 22.1 Hz), 115.73, 108.05. ESI-HRMS

(m/z): [M − H]⁻calcd for C₁₄H₉FOS, 243.0280; observed, 243.0289.

3-(4-Fluorophenyl)benzo[b]thiophen-6-ol (12). This com-

pound was prepared using similar method as 11 and gave 780 mg of

white solid (yield, 82%). ¹H NMR (400 MHz, CD₃OD) δ 7.59 (d, J =

8.8 Hz, 1H), 7.47 (dd, J = 8.6, 5.5 Hz, 2H), 7.28 (d, J = 2.2 Hz, 1H),

7.15−7.10 (m, 3H), 6.90 (dd, J = 8.8, 2.2 Hz, 1H). ¹³C NMR (100 MHz,

CD₃OD) δ 163.58 (d, J_C−F= 245.1 Hz), 156.35, 143.61, 137.62, 133.77

(d, J_C−F= 3.3 Hz) 132.32, 131.23 (d, J_C−F= 8.0 Hz), 124.15, 121.15,

116.39 (d, J_C−F= 21.6 Hz), 115.54, 108.64. ESI-HRMS (m/z): [M −

H]⁻calcd for C₁₄H₈FOS, 243.0280; observed, 243.0276.

Animal Experiments. T47D:A18/PKCa tumors were established

as previously described.⁷⁷23 and 26a were administered per os at a dose

of 100 mg/kg daily for 2 weeks in a formulation of 0.1% Tween 80, 10%

PEG400, and 0.5% CMC solution. Drinking water was replaced with a

hydrogel suspension of drugs at a concentration of 0.25 mg/mL to

maintain continuous drug exposure. Tumor cross-sectional area was

determined weekly using vernier calipers and calculated using the

formula (length/2) × (width/2) × π. Mean tumor area was plotted

against time (in weeks) to monitor tumor growth. The mice were

sacriﬁced by CO₂inhalation and cervical dislocation, and tumors and

uteri were excised, cleaned of connective tissue, and immediately

weighed. The Animal Care and Use Committee of UIC approved all of

the procedures involving animals.

General. All chemicals and solvents were purchased from Sigma-

Aldrich, Fisher Scientiﬁc, or Matrix Scientiﬁc and were used without

further puriﬁcation. Synthetic intermediates were puriﬁed by Biotage

(2-(4-Fluorophenyl)-6-methoxybenzo[b]thiophen-3-yl)(4-

(triﬂuoromethyl)phenyl)methanone (13b). To an oven-dried ﬂask

charged with 2-(4-ﬂuorophenyl)-6-methoxybenzo[b]thiophene (258

mg, 1 mmol) and 4-(triﬂuoromethyl)benzoyl chloride (250 mg, 1.2

mmol) in 10 mL of dichloromethane, AlCl₃(400 mg, 3 mmol) was

added in three portions over 10 min. The mixture was stirred until all the

starting material was consumed and then poured into ice−water. The

layers were separated, and the aqueous layer was extracted three times

with dichloromethane. The organic layers were combined, washed by

brine, and dried over Na₂SO_4.The crude product was puriﬁed by silica

gel chromatography (1−10% ethyl acetate in hexane) to give 160 mg of

yellow solid (yield, 37%). ¹H NMR (400 MHz, CDCl₃) δ 7.81 (d, J = 8.1

1

ﬂash chromatography on 230−400 mesh silica gel. H and ¹³C NMR

spectra were recorded on Bruker DPX-400 or AVANCE-400

spectrometer at 400 and 100 MHz, respectively. NMR chemical shifts

were reported in δ (ppm) using residual solvent peaks as standard

(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); acetone-

d₆, 2.05 ppm (¹H), 29.84 ppm (¹³C)). Data are reported as follows:

chemical shift, multiplicity (s = singlet, d = doublet, dd = doublet of

doublet, t = triplet, q = quartet, br = broad, m = multiplet, abq = ab

quartet), number of protons, and coupling constants. Low-resolution

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Hz, 2H), 7.68 (d, J = 8.9 Hz, 1H), 7.51 (d, J = 8.1 Hz, 2H), 7.35−7.26

(m, 3H), 7.04 (dd, J = 9.0, 2.4 Hz, 1H), 6.90 (t, J = 8.6 Hz, 2H), 3.91 (s,

3H). ¹³C NMR (100 MHz, CDCl₃) δ 192.87, 163.07 (d, J_C−F= 250.5

Hz), 158.30, 144.42, 140.47, 134.40 (q, J_C−F= 32.6 Hz), 133.43, 131.25

(d, J_C−F= 8.4 Hz), 130.72, 130.18, 129.43 (d, J_C−F= 3.5 Hz), 125.46 (q,

J_C−F= 3.7 Hz), 124.55, 123.60 (q, J_C−F= 272.9 Hz), 116.02, 115.70 (d,

J_C−F= 20.7 Hz), 104.65, 55.82.

(2-(4-Fluorophenyl)-6-hydroxybenzo[b]thiophen-3-yl)(4-

(triﬂuoromethyl)phenyl)methanone (14b). This compound was

prepared by a procedure identical with the preparation of 11 except a

C18 reverse phase column was used to get the pure compound (2.5 g,

yield 61%). ¹H NMR (400 MHz, CD₃OD) δ 7.77 (d, J = 8.1 Hz, 2H),

7.65 (d, J = 8.8 Hz, 1H), 7.57−7.49 (m, 2H), 7.32−7.28 (m, 3H), 6.97−

6.86 (m, 3H). ¹³C NMR (100 MHz, CD₃OD) δ 194.20, 164.30 (d, J_C−F

= 248.8 Hz), 157.41, 145.51, 142.29, 141.84, 134.95 (q, J_C−F= 32.5 Hz),

133.65, 132.56 (d, J_C−F= 8.5 Hz), 131.85, 131.29, 130.97 (d, J_C−F= 3.4

Hz), 126.36 (q, J_C−F= 3.8 Hz), 125.45, 125.02(q, J_C−F= 272 Hz),

116.67, 116.54 (d, J_C−F= 22.3 Hz), 107.87. ESI-HRMS (m/z): [M −

H]⁻calcd for C₂₂H₁₁F₄O₂S, 415.0416; observed, 415.0409.

153.68, 152.10, 142.36, 138.54, 130.31, 130.24 (d, J_C−F= 8.0 Hz),

128.33, 125.19, 123.45, 117.35, 117.04, 116.52 (d, J_C−F= 21.9 Hz),

115.67, 108.73. ESI-HRMS (m/z): [M + H]⁺calcd for C₂₀H₁₃FO₃S,

353.0648; observed, 353.0632.

(3-(4-Fluorophenyl)-6-methoxybenzo[b]thiophen-2-yl)(4-

(methylsulfonyl)phenyl)methanone (20a). This compound was

prepared by a procedure identical to the preparation of 13b (180 mg,

yield, 21%). ¹H NMR (400 MHz, CDCl₃) δ 7.71 (d, J = 8.3 Hz, 2H),

7.61 (d, J = 8.3 Hz, 2H), 7.55 (d, J = 9.0 Hz, 1H), 7.37 (d, J = 2.1 Hz,

1H), 7.17−7.13 (m, 2H), 7.04 (dd, J = 9.0, 2.1 Hz, 1H), 6.88 (t, J = 8.5

Hz, 2H), 3.94 (s, 3H), 2.97 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ

189.78, 162.61 (d, J_C−F= 249.3 Hz), 160.49, 143.73, 142.96, 142.68,

142.37, 135.72, 133.52, 132.38 (d, J_C−F= 8.2 Hz), 130.45 (d, J_C−F= 3.5

Hz), 129.93, 126.95, 126.43, 116.70, 115.45 (d, J_C−F= 21.6 Hz), 104.40,

55.92, 44.54.

(3-(4-Fluorophenyl)-6-methoxybenzo[b]thiophen-2-yl)(4-

methoxyphenyl)methanone (20b). This compound was prepared

by a procedure identical to the preparation of 13b (488 mg, yield 52%).

¹H NMR (400 MHz, CDCl₃) δ 7.63−7.57 (m, 3H), 7.35 (d, J = 2.2 Hz,

(2-(4-Chlorophenyl)-6-hydroxybenzo[b]thiophen-3-yl)(4-

(triﬂuoromethyl)phenyl)methanone (14c). This compound was

isolated from the preparation of 14b as a minor product, (130 mg, yield

3%). ¹H NMR (400 MHz, CD₃OD) δ 7.79 (d, J = 8.2 Hz, 2H), 7.65 (d, J

= 8.8 Hz, 1H), 7.56 (d, J = 8.2 Hz, 2H), 7.31 (d, J = 2.2 Hz, 1H), 7.27 (d,

J = 8.5 Hz, 2H), 7.19 (d, J = 8.5 Hz, 2H), 6.95 (dd, J = 8.8, 2.2 Hz, 1H).

¹³C NMR (100 MHz, CD₃OD) δ 194.18, 157.53, 145.00, 142.23,

141.94, 135.98, 135.06 (q, J_C−F= 32.5 Hz).133.65, 133.35, 132.13,

131.91, 131.32, 129.79, 126.44 (q, J_C−F= 3.8 Hz), 125.50, 125.10 (q,

J_C−F= 272 Hz), 116.76, 107.88. ESI-HRMS (m/z): [M − H]⁻calcd for

C₂₂H₁₁ClF₃O₂S, 431.0120; observed, 431.0113.

1H), 7.29−7.21 (m, 2H), 7.02 (dd, J = 9.0, 2.3 Hz, 1H), 6.94 (t, J = 8.7

Hz, 2H), 6.68 (d, J = 8.8 Hz, 2H), 3.91 (s, 3H), 3.78 (s, 3H). ¹³C NMR

(100 MHz, CDCl₃) δ 189.74, 163.19, 162.48 (d, J_C−F= 248.6 Hz),

159.49, 142.55, 139.65, 135.81, 133.37, 132.17, 131.98 (d, J_C−F= 8.2

Hz), 130.86 (d, J_C−F= 3.4 Hz), 130.51, 125.65, 115.96, 115.42 (d, J_C−F

=

21.6 Hz), 113.31, 104.41, 55.81, 55.53.

(4-(Dimethylamino)phenyl)(3-(4-ﬂuorophenyl)-6-methoxy-

benzo[b]thiophen-2-yl)methanone (20c). This compound was

prepared by a procedure identical to the preparation of 13b (393 mg,

yield 48%). ¹H NMR (400 MHz, CDCl₃) δ 7.66−7.58 (m, 3H), 7.36−

7.30 (m, 3H), 7.09−6.92 (m, 3H), 6.44 (d, J = 9.0 Hz, 2H), 3.92 (s, 3H),

3.00 (s, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 188.84, 162.37 (d, J_C−F

=

2-(4-Fluorophenyl)-6-methoxy-3-(4-methoxyphenoxy)-

benzo[b]thiophene 1-Oxide (17). NaH (380 mg, 9.5 mmol, 60%

dispersion in mineral oil) was added in three portions to a ﬂask charged

with a solution of 4-methoxyphenol (0.85 g, 6.9 mmol) in 10 mL of

anhydrous DMF under argon atmosphere at room temperature. After

the mixture was stirred for 30 min, 3-bromo-2-(4-ﬂuorophenyl)-6-

methoxybenzo[b]thiophene 1-oxide³²(2.2 g, 6.3 mmol) was added in

small portions. After the mixture was stirred for 2 h, ethyl acetate and

water were added, and the organic layer was washed several times with

water and brine, then dried over Na₂SO₄. The residue was puriﬁed by

ﬂash chromatography (5−60% ethyl acetate in hexane) to yield 2.4 g

247.1 Hz), 159.00, 153.43, 142.02, 137.96, 136.20, 133.30, 132.52,

131.85 (d, J_C−F= 8.1 Hz), 131.16 (d, J_C−F= 3.3 Hz), 125.18, 125.05,

115.58, 115.41 (d, J_C−F= 21.5 Hz), 110.37, 104.49, 55.80, 40.09.

(3-(4-Fluorophenyl)-6-methoxybenzo[b]thiophen-2-yl)(p-

tolyl)methanone (20d). This compound was prepared by a procedure

identical to the preparation of 13b (390 mg, yield 49%). ¹H NMR (400

MHz, CDCl₃) δ 7.58 (d, J = 9.0 Hz, 1H), 7.50 (d, J = 8.1 Hz, 2H), 7.35

(d, J = 2.2 Hz, 1H), 7.25−7.19 (m, 2H), 7.06−6.96 (m, 3H), 6.93 (t, J =

8.7 Hz, 2H), 3.93 (s, 3H), 2.30 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ

190.85, 162.51 (d, J_C−F= 247.8 Hz), 159.62, 143.18, 142.74, 140.33,

135.82, 135.27, 133.46, 131.98 (d, J_C−F= 8.2 Hz), 130.77 (d, J_C−F= 3.4

Hz), 129.83, 128.64, 125.81, 116.02, 115.31 (d, J_C−F= 21.6 Hz), 104.36,

55.79, 21.66.

(4-Fluorophenyl)(3-(4-ﬂuorophenyl)-6-methoxybenzo[b]-

thiophen-2-yl)methanone (20e). This compound was prepared by a

procedure identical to the preparation of 13b (510 mg, yield 67%). ¹H

NMR (400 MHz, CDCl₃) δ 7.64−7.54 (m, 3H), 7.36 (d, J = 2.2 Hz,

1H), 7.25−7.18 (m, 2H), 7.03 (dd, J = 9.0, 2.3 Hz, 1H), 6.94 (t, J = 8.6

Hz, 2H), 6.85 (t, J = 8.6 Hz, 2H), 3.92 (s, 3H). ¹³C NMR (100 MHz,

CDCl₃) δ 189.72, 165.14 (d, J_C−F= 254.4 Hz), 162.61 (d, J_C−F= 248.7

Hz), 159.87, 142.96, 140.74, 135.54, 134.24 (d, J_C−F= 3.0 Hz), 133.43,

1

(yield, 93%) of the title compound. H NMR (400 MHz, CDCl₃) δ

7.78−7.72 (m, 2H), 7.50 (d, J = 2.3 Hz, 1H), 7.09−6.97 (m, 5H), 6.91

(dd, J = 8.5, 2.3 Hz, 1H), 6.83−6.76 (m, 2H), 3.88 (s, 3H), 3.76 (s, 3H).

¹³C NMR (100 MHz, CDCl₃) δ 162.68 (d, J_C−F= 249.6 Hz), 161.12,

156.29, 150.13, 149.01, 144.76, 130.26 (d, J_C−F= 8.2 Hz), 130.19,

126.40, 125.94, 124.11, 118.24, 118.18, 116.04 (d, J_C−F= 21.8 Hz),

115.03, 112.08, 56.10, 55.84.

2-(4-Fluorophenyl)-6-methoxy-3-(4-methoxyphenoxy)-

benzo[b]thiophene (18). LiAlH₄(372 mg, 9.8 mmol) was added in

small portions to a solution of 17b (2.6 g, 6.6 mmol) in 30 mL of

anhydrous THF under argon atmosphere at 0 °C. After the mixture was

stirred for 2 h, 0.4 mL of water was slowly added followed by 0.4 mL of

15% NaOH to quench the reaction. The precipitate was then ﬁltered

through Celite and washed with ethyl acetate. The ﬁltrate was washed

with water and brine three times. The organic layers were combined,

dried over anhydrous Na₂SO₄, and then puriﬁed by ﬂash chromatog-

raphy (1−30% ethyl acetate in hexane) to yield 1.8 g (yield, 71%)

product. ¹H NMR (400 MHz, CDCl₃) δ 7.76−7.68 (m, 2H), 7.28−7.24

(m, 2H), 7.04 (t, J = 8.7 Hz, 2H), 6.94−6.85 (m, 3H), 6.83−6.75 (m,

2H), 3.87 (s, 3H), 3.75 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 162.30

(d, J_C−F= 248.0 Hz), 158.27, 155.06, 151.73, 140.81, 137.24, 129.34 (d,

132.16 (d, J_C−F= 9.5 Hz), 132.07 (d, J_C−F= 8.5 Hz), 130.61 (d, J_C−F

3.4 Hz), 125.96, 116.24, 115.49 (d, J_C−F= 21.7 Hz), 115.11 (d, J_C−F

22.0 Hz), 104.42, 55.84.

=

Cyclopropyl(3-(4-ﬂuorophenyl)-6-methoxybenzo[b]-

thiophen-2-yl)methanone (20f). This compound was prepared by a

procedure identical to the preparation of 13b (450 mg, yield 69%). ¹H

NMR (400 MHz, CDCl₃) δ 7.48−7.41 (m, 2H), 7.37 (d, J = 9.0 Hz,

1H), 7.31 (d, J = 2.3 Hz, 1H), 7.20 (dd, J = 12.0, 5.4 Hz, 2H), 6.97 (dd, J

= 9.0, 2.3 Hz, 1H), 3.91 (s, 3H), 1.84−1.73 (m, 1H), 1.22−1.10 (m,

2H), 0.73 (dq, J = 7.1, 3.5 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃) δ

195.69, 163.02 (d, J_C−F= 248.0 Hz), 159.99, 142.64, 140.19, 138.65,

134.86, 131.96 (d, J_C−F= 8.1 Hz), 131.35, 126.28, 116.02, 115.84 (d,

J_C−F= 21.6 Hz), 104.34, 55.83, 20.65, 12.58.

Cyclohexyl(3-(4-ﬂuorophenyl)-6-methoxybenzo[b]-

thiophen-2-yl)methanone (20g). This compound was prepared by a

procedure identical to the preparation of 13b (520 mg, yield 91%). ¹H

NMR (400 MHz, CDCl₃) δ 7.37−7.31 (m, 2H), 7.28 (d, J = 2.2 Hz,

1H), 7.25 (d, J = 9.0 Hz, 1H), 7.21−7.17 (m, 2H), 6.93 (dd, J = 9.0, 2.3

J_C−F= 8.0 Hz), 128.38, 128.01, 125.52, 122.63, 116.52, 115.91 (d, J_C−F

=

21.6 Hz), 114.95, 114.68, 105.54, 55.80, 55.78.

2-(4-Fluorophenyl)-3-(4-hydroxyphenoxy)benzo[b]-

thiophen-6-ol (19). This compound was prepared by a procedure

1

identical to the preparation of 11 (1.3 g, white solid, yield 78%). H

NMR (400 MHz, CD₃OD) δ 7.77−7.66 (m, 2H), 7.22−7.15 (m, 2H),

7.07 (t, J = 8.7 Hz, 2H), 6.83−6.72 (m, 3H), 6.71−6.64 (m, 2H). ¹³C

NMR (100 MHz, CD₃OD) δ 163.43 (d, J_C−F= 246.6 Hz), 157.29,

M

DOI: 10.1021/acs.jmedchem.5b01276

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Hz, 1H), 3.88 (s, 3H), 2.47 (ddd, J = 11.7, 7.3, 2.8 Hz, 1H), 1.77−1.60

(m, 4H), 1.54 (d, J = 13.2 Hz, 1H), 1.44−1.27 (m, 2H), 1.17−1.02 (m,

1H), 0.93−0.75 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 199.25,

162.96 (d, J_C−F= 248.2 Hz), 159.99, 142.49, 139.95, 137.33, 135.15,

131.45, 131.43 (d, J_C−F= 8.1 Hz), 126.33, 115.72 (d, J_C−F= 21.6 Hz),

115.61, 104.25, 55.80, 48.44, 29.38, 25.81, 25.77.

(3-(4-Fluorophenyl)-6-methoxybenzo[b]thiophen-2-yl)(4-

iodophenyl)methanone (20h). This compound was prepared by a

procedure identical to the preparation of 13b (982 mg, yield 26%). ¹H

NMR (400 MHz, CDCl₃) δ 7.57−7.51 (m, 3H), 7.34 (d, J = 2.1 Hz,

1H), 7.26 (d, J = 8.3 Hz, 2H), 7.21−7.18 (m, 2H), 7.02 (dd, J = 8.7, 2.1

Hz, 1H), 6.94 (t, J = 8.5 Hz, 2H), 3.91 (s, 3H). ¹³C NMR (100 MHz,

CDCl₃) δ 190.27, 162.69 (d, J_C−F= 248.9 Hz), 159.94, 143.07, 141.17,

137.34, 137.18, 135.31, 133.43, 132.02 (d, J_C−F= 8.2 Hz), 130.84, 130.46

(d, J_C−F= 3.4 Hz), 126.06, 116.30, 115.50 (d, J_C−F= 21.7 Hz), 104.34,

99.73, 55.83.

Cyclopropyl(3-(4-ﬂuorophenyl)-6-hydroxybenzo[b]-

thiophen-2-yl)methanone (21f). NaO^tBu (280 mg, 3.6 mmol) was

added to an oven-dried round-bottom ﬂask charged with 2-

(dimethylamino)ethanethiol hydrochloride (305 mg, 1.8 mmol) in 3

mL of DMF in an ice bath. After 15 min, the mixture was allowed to

equilibrate to room temperature, and one portion of 20f (400 mg, 1.2

mmol) was added, and the mixture was heated to reﬂux. The reaction

was monitored by TLC and quenched by ice−water. Ethyl acetate and

water were added, and the organic layer was washed several times with

water and brine, then dried over Na₂SO₄. The residue was puriﬁed by

ﬂash chromatography (5−40% ethyl acetate in hexane) to yield 94 mg

(yield, 25%) of the title compound. ¹H NMR (400 MHz, acetone-d₆) δ

7.62−7.51 (m, 2H), 7.39 (d, J = 2.1 Hz, 1H), 7.38−7.31 (m, 3H), 7.00

(dd, J = 8.9, 2.2 Hz, 1H), 1.80 (tt, J = 7.8, 4.6 Hz, 1H), 1.05−0.97 (m,

2H), 0.75−0.69 (m, 2H). ¹³C NMR (100 MHz, acetone-d₆) δ 195.38,

163.89 (d, J_C−F= 246.2 Hz), 158.91, 143.34, 141.12, 138.83, 135.01,

133.14 (d, J_C−F= 8.3 Hz), 132.43 (d, J_C−F= 3.4 Hz), 127.34, 116.86,

116.57 (d, J_C−F= 21.7 Hz), 108.01, 20.97, 12.32. ESI-HRMS (m/z): [M

+ H]⁺calcd for C₁₈H₁₄FO₂S, 313.0699; observed, 313.0696.

(3-(4-Fluorophenyl)-6-hydroxybenzo[b]thiophen-2-yl)(4-

(methylsulfonyl)phenyl)methanone (21a). This compound was

prepared by a procedure identical to the preparation of 11 (120 mg, light

1

green solid, yield 85%). H NMR (400 MHz, DMSO-d₆) δ 10.32 (s,

Cyclohexyl(3-(4-ﬂuorophenyl)-6-hydroxybenzo[b]thiophen-

2-yl)methanone (21g). 20g (6.56 g, 18 mmol) was dissolved in 50 mL

of anhydrous dichloromethane and cooled to −78 °C in a dry ice−

acetone bath. BF₃·SMe₂(23.4 g, 180 mmol) was added dropwise to this

solution. The reaction mixture was stirred until starting material was

consumed as monitored by TLC and then quenched by saturated

NaHCO₃/ice−water. The reaction mixture was extracted with ethyl

acetate and washed with brine. The organic extracts were combined,

dried over anhydrous Na₂SO₄, concentrated in vacuo, and then puriﬁed

by ﬂash chromatography (5−60% ethyl acetate in hexane) to give 4.8 g

of white powder (yield, 76%). ¹H NMR (400 MHz, CD₃OD) δ 7.46−

7.38 (m, 2H), 7.31 (t, J = 8.8 Hz, 2H), 7.23 (d, J = 2.2 Hz, 1H), 7.19 (d, J

= 8.9 Hz, 1H), 6.88 (dd, J = 8.9, 2.2 Hz, 1H), 2.51 (tt, J = 11.5, 2.9 Hz,

1H), 1.68−1.64 (m, 4H), 1.57−1.54 (m, 1H), 1.37−1.23 (m, 2H),

1.19−1.05 (m, 1H), 0.92−0.77 (m, 2H). ¹³C NMR (100 MHz,

CD₃OD) δ 201.15, 164.43 (d, J_C−F= 246.9 Hz), 159.63, 144.07, 142.15,

137.60, 135.51, 133.00, 132.74 (d, J_C−F= 8.2 Hz), 127.58, 117.02, 116.64

(d, J_C−F= 21.9 Hz), 107.78, 49.50, 30.40, 26.79, 26.76. ESI-HRMS (m/

z): [M + H]⁺calcd for C₂₁H₂₀FO₂S, 355.1168; observed, 355.1150.

(4-Ethynylphenyl)(3-(4-ﬂuorophenyl)-6-methoxybenzo[b]-

thiophen-2-yl)methanone (22). To a mixture of 20h (244 mg, 0.5

mmol) and ethynyltrimethylsilane (108 mg, 0.55 mmol) in dry

trimethylamine (5 mL) were added Pd(PPh₃)₂Cl₂(5% mol) and CuI

(5% mol) under argon atmosphere. The mixture was heated at 70 °C.

After cooling the mixture to room temperature, ethyl acetate was added

and the suspension was ﬁltered through a pad of silica gel. The ﬁltrate

was collected, and the crude product was treated with 1 M TBAF (1.5

equiv) in THF. After 30 min, the reaction was quenched by pouring into

ice−water and extracted with ethyl acetate. The organic phase was

washed with brine and dried over Na₂SO₄. The mixture was puriﬁed by

ﬂash chromatography (1−30% ethyl acetate in hexane) to give the title

product (96 mg, yield 50%). ¹H NMR (400 MHz, CDCl₃) δ 7.57 (d, J =

9.0 Hz, 1H), 7.51 (d, J = 8.4 Hz, 2H), 7.35 (d, J = 2.2 Hz, 1H), 7.28 (d, J

= 8.3 Hz, 2H), 7.24−7.16 (m, 2H), 7.03 (dd, J = 9.0, 2.3 Hz, 1H), 6.93 (t,

J = 8.6 Hz, 2H), 3.92 (s, 3H), 3.18 (s, 1H). ¹³C NMR (100 MHz,

CDCl₃) δ 190.35, 162.67 (d, J_C−F= 248.8 Hz), 159.93, 143.10, 141.10,

137.90, 135.53, 133.46, 132.06 (d, J_C−F= 8.2 Hz), 131.61, 130.51 (d,

J_C−F= 3.5 Hz), 129.41, 126.06, 125.94, 116.29, 115.49 (d, J_C−F= 21.7

Hz), 104.37, 82.92, 80.08, 55.84.

1H), 7.67 (d, J = 8.4 Hz, 2H), 7.60 (d, J = 8.4 Hz, 2H), 7.45−7.43 m,

2H), 7.23 (dd, J = 8.5, 5.6 Hz, 2H), 7.02−6.98 (m, 3H), 3.14 (s, 3H). ¹³C

NMR (100 MHz, DMSO-d₆) δ 189.53, 161.80 (d, J_C−F= 246.0 Hz),

158.49, 142.71, 142.69, 142.43, 142.27, 134.29, 132.50 (d, J_C−F= 8.5

Hz), 132.15, 129.96, 129.35, 126.53, 126.26, 116.59, 114.94 (d, J_C−F

=

21.6 Hz), 107.22, 43.38. ESI-HRMS (m/z): [M − H]⁻calcd for

C₂₂H₁₄FO₄S₂, 425.0318; observed, 425.0326.

(3-(4-Fluorophenyl)-6-hydroxybenzo[b]thiophen-2-yl)(4-

hydroxyphenyl)methanone (21b). This compound was prepared by

a procedure identical to the preparation of 11 (284 mg, white solid, yield

53%). ¹H NMR (400 MHz, CD₃OD) δ 7.51−7.44 (m, 3H), 7.29 (d, J =

2.0 Hz, 1H), 7.25−7.22 (m, 2H), 7.02−6.88 (m, 3H), 6.57 (d, J = 8.7 Hz,

2H). ¹³C NMR (100 MHz, CD₃OD) δ 192.04, 163.79 (d, J_C−F= 243.1

Hz), 163.31, 158.86, 143.82, 141.34, 135.99, 133.64, 133.48, 133.28 (d,

J_C−F= 8.3 Hz), 132.34 (d, J_C−F= 3.4 Hz), 130.33, 126.72, 116.95, 116.13

(d, J_C−F= 21.9 Hz), 115.75, 107.96. ESI-HRMS (m/z): [M + H]⁺calcd

for C₂₁H₁₄FO₃S, 365.0648; observed, 365.0638.

(4-(Dimethylamino)phenyl)(3-(4-ﬂuorophenyl)-6-hydroxy-

benzo[b]thiophen-2-yl)methanone (21c). This compound was

prepared by a procedure identical to the preparation of 11 (205 mg,

white solid, yield 54%). ¹H NMR (400 MHz, CD₃OD) δ 7.54 (dd, J =

9.0, 2.9 Hz, 3H), 7.37−7.27 (m, 3H), 7.01 (t, J = 8.8 Hz, 2H), 6.96 (dd, J

= 8.8, 2.2 Hz, 1H), 6.51 (d, J = 9.1 Hz, 2H), 3.00 (s, 6H). ¹³C NMR (100

MHz, CD₃OD) δ 191.43, 163.72 (d, J_C−F= 246.2 Hz), 158.47, 155.26,

143.39, 139.88, 136.14, 133.63, 133.52, 133.15 (d, J_C−F= 8.3 Hz),

132.61, 126.30, 125.62, 116.76, 116.20 (d, J_C−F= 21.9 Hz), 111.47,

107.99, 40.04. ESI-HRMS (m/z): [M + H]⁺calcd for C₂₃H₁₉FNO₂S,

392.1121; observed, 392.1113.

(3-(4-Fluorophenyl)-6-hydroxybenzo[b]thiophen-2-yl)(p-

tolyl)methanone (21d). This compound was prepared by a procedure

identical to the preparation of 11 (190 mg, white solid, yield 52%). ¹H

NMR (400 MHz, DMSO-d₆) δ 10.18 (s, 1H), 7.52−7.38 (m, 4H),

7.32−7.27 (m, 2H), 7.11−7.02 (m, 4H), 6.99 (dd, J = 8.9, 2.1 Hz, 1H),

2.26 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 190.37, 162.18 (d, J_C−F

= 245.3 Hz), 158.18, 142.94, 142.32, 140.76, 135.37, 134.48, 132.57 (d,

J_C−F= 8.3 Hz), 132.27, 130.98 (d, J_C−F= 3.1 Hz), 129.70, 128.90, 126.24,

116.74, 115.51 (d, J_C−F= 21.6 Hz), 107.61, 21.50. ESI-HRMS (m/z):

[M + H]⁺calcd for C₂₂H₁₆FO₂S, 363.0855; observed, 363.0860.

(4-Fluorophenyl)(3-(4-ﬂuorophenyl)-6-hydroxybenzo[b]-

thiophen-2-yl)methanone (21e). This compound was prepared by a

procedure identical to the preparation of 11 (211 mg, green solid, yield

61%). ¹H NMR (400 MHz, acetone-d₆) δ 7.68−7.59 (m, 2H), 7.56 (d, J

= 8.9 Hz, 1H), 7.47 (d, J = 2.2 Hz, 1H), 7.39−7.29 (m, 2H), 7.09−7.02

(m, 3H), 7.01−6.94 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 189.93,

165.28 (d, J_C−F= 251.6 Hz), 163.04 (d, J_C−F= 247.5 Hz), 158.70,

143.53, 141.59, 135.85, 135.49 (d, J_C−F= 3.0 Hz), 133.55, 133.30 (d,

J_C−F= 8.3 Hz) 132.85 (d, J_C−F= 9.3 Hz), 131.69 (d, J_C−F= 3.3 Hz),

126.95, 116.95, 115.92 (d, J_C−F= 21.8 Hz), 115.60 (d, J_C−F= 22.2 Hz),

108.02. ESI-HRMS (m/z): [M − H]⁻calcd for C₂₁H₁₁F₂O₂S, 365.0448;

observed, 365.0444.

(4-Ethynylphenyl)(3-(4-ﬂuorophenyl)-6-hydroxybenzo[b]-

thiophen-2-yl)methanone (23). This compound was prepared by a

procedure identical to the preparation of 21g (704 mg, yellow solid,

yield 56%). ¹H NMR (400 MHz, acetone-d₆) δ 7.57−7.52 (m, 3H), 7.47

(d, J = 2.2 Hz, 1H), 7.37−7.28 (m, 4H), 7.08−7.00 (m, 3H), 3.81 (s,

1H). ¹³C NMR (100 MHz, acetone-d₆) δ 190.56, 163.39 (d, J_C−F= 246.4

Hz), 158.76, 143.71, 141.93, 139.09, 135.87, 133.64, 133.31 (d, J_C−F

=

8.3 Hz), 132.15, 131.67 (d, J_C−F= 3.4 Hz), 130.12, 127.06, 126.43,

116.99, 115.93 (d, J_C−F= 21.8 Hz), 108.03, 83.44, 81.75. ESI-HRMS

(m/z): [M + H]⁺calcd for C₂₃H₁₄FO₂S, 373.0699; observed, 373.0686.

2-Bromo-3-(4-ﬂuorophenyl)-6-methoxybenzo[b]thiophene

(24). N-Bromoacetamide (1.46 g, 10.5 mmol) was added in small

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portions to a solution of 10 (2.58 g, 10 mmol) in 300 mL of CH₂Cl₂and

20 mL of ethanol at room temperature. After the mixture was stirred for

1 h, the solvent was removed in vacuo. The residue was titrated with

pure ethanol and ﬁltered to give 3.0 g (yield, 89%) of white powder. ¹H

NMR (400 MHz, CDCl₃) δ 7.47−7.41 (m, 2H), 7.39 (d, J = 8.9 Hz,

1H), 7.24 (d, J = 2.3 Hz, 1H), 7.23−7.17 (m, 2H), 6.94 (dd, J = 8.9, 2.4

vigorously for 30 min and then concentrated in vacuo. The residue was

partitioned between CH₂Cl₂and saturated aqueous NaHCO₃solution.

The layers were separated, and the organic layer was washed with water,

saturated NaHCO₃, and water and then dried over anhydrous Na₂SO_4.

The residue was titrated with diethyl ether and ﬁltered to give 2.1 g

1

(yield, 84%) of pale yellow powder. H NMR (400 MHz, CDCl₃) δ

Hz, 1H), 3.87 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 162.63 (d, J_C−F

=

7.52−7.45 (m, 3H), 7.25−7.20 (m, 2H), 7.16 (d, J = 8.5 Hz, 1H), 6.97

247.5 Hz), 157.89, 157.88, 141.13, 135.81, 132.81, 131.79 (d, J_C−F= 8.1

Hz), 130.10 (d, J_C−F= 3.3 Hz), 123.51, 115.78 (d, J_C−F= 21.6 Hz),

114.72, 104.66, 55.80.

(dd, J = 8.5, 2.4 Hz, 1H), 3.89 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ

163.45 (d, J_C−F= 250.6 Hz), 160.84, 146.91, 144.07, 130.87 (d, J_C−F

=

8.7 Hz), 130.86, 127.36 (d, J_C−F= 3.5 Hz), 125.09, 124.75, 118.07,

116.35 (d, J_C−F= 21.9 Hz), 112.84, 56.13.

(3-(4-Fluorophenyl)-6-methoxybenzo[b]thiophen-2-yl)(4-

(triﬂuoromethyl)phenyl)methanone (25a). To an oven-dried ﬂask

charged with 24 (7.4 g, 21.8 mmol) in 40 mL of THF in a dry ice−

acetone bath, n-BuLi (1.6 M, 15 mL) was added dropwise under argon

atmosphere. After stirring for 30 min, a solution of 4-(triﬂuoromethyl)-

benzoyl chloride (5 g, 24 mmol) in 10 mL of THF was added dropwise.

The reaction was allowed to warm to room temperature and was

monitored by TLC. Upon the consumption of the starting material, the

reaction was poured into ice−water. The mixture was extracted with

ethyl acetate, washed with brine, and then dried over anhydrous Na₂SO_4.

The crude product was puriﬁed by ﬂash chromatography (1−30% ethyl

acetate in hexane) to give the title product (5.3 g, yield 57%). ¹H NMR

(400 MHz, CDCl₃) δ 7.59−7.55 (m, 3H), 7.42−7.36 (m, 3H), 7.18−

7.13 (m, 2H), 7.03 (dd, J = 9.0, 2.1 Hz, 1H), 6.88 (t, J = 8.5 Hz, 2H), 3.93

(s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 190.14, 162.71 (d, J_C−F= 249.5

3-(4-Fluorophenyl)-6-methoxy-2-(4-methoxyphenoxy)-

benzo[b]thiophene 1-Oxide (28a). This compound was prepared by

a procedure identical to the preparation of 17 (2.47g, yellow solid, yield

89%). ¹H NMR (400 MHz, CDCl₃) δ 7.51−7.48 (m, 2H), 7.41 (d, J =

2.3 Hz, 1H), 7.26 (d, J = 8.5, 1H), 7.17−7.05 (m, 4H), 6.99 (dd, J = 8.5,

2.3 Hz, 1H), 6.81 (d, J = 9.0 Hz, 2H), 3.88 (s, 3H), 3.77 (s, 3H). ¹³C

NMR (100 MHz, CDCl₃) δ 163.14 (d, J_C−F= 249.7 Hz), 160.47, 157.89,

156.42, 150.83, 142.13, 130.80 (d, J_C−F= 8.3 Hz), 129.34, 128.00, 126.13

(d, J_C−F= 3.4 Hz), 124.35, 118.76, 118.01, 116.14 (d, J_C−F= 21.7 Hz),

114.92, 112.92, 56.06, 55.80.

2-(4-Chlorophenoxy)-3-(4-ﬂuorophenyl)-6-methoxybenzo-

[b]thiophene 1-Oxide (28b). To an oven-dried ﬂask charged with 4-

chlorophenol in 10 mL of DMF, Cs₂CO₃(1.3 g, 4 mmol) was added in a

portion. After 15 min, 27 (702 mg, 2 mmol) was added slowly. The

reaction was heated to 80 °C until the consumption of starting material.

The mixture was quenched by ice−water and extracted with ethyl

acetate and washed with water and brine. The crude material was then

puriﬁed by ﬂash chromatography (5−40% ethyl acetate in hexane) to

give 403 mg (yield, 50%) of the title compound. ¹H NMR (400 MHz,

CDCl₃) δ 7.50−7.44 (m, 3H), 7.31−7.24 (m, 3H), 7.20−7.08 (m, 4H),

7.03 (dd, J = 8.5, 2.5 Hz, 1H), 3.91 (s, 3H). ¹³C NMR (100 MHz,

CDCl₃) δ 163.29 (d, J_C−F= 250.32 Hz), 160.82, 156.39, 155.68, 142.42,

130.72 (d, J_C−F= 8.36 Hz), 130.10, 129.90, 129.35, 128.75, 125.76 (d,

J_C−F= 4 Hz), 124.73, 118.48, 118.18, 116.28 (d, J_C−F= 21.8 Hz), 112.89,

56.10.

Hz), 160.27, 143.46, 142.08, 141.28, 135.58, 133.59, 133.20 (q, J_C−F

=

32.7 Hz), 132.13 (d, J_C−F= 8.2 Hz), 130.29 (d, J_C−F= 3.4 Hz), 129.51,

126.35, 124.84 (q, J_C−F= 3.7 Hz), 123.63 (q, J_C−F= 272.9 Hz), 116.50,

115.46 (d, J_C−F= 21.8 Hz), 104.37, 55.87.

4-(3-(4-Fluorophenyl)-6-methoxybenzo[b]thiophene-2-

carbonyl)benzonitrile (25b). This compound was prepared by a

procedure identical to the preparation of 25a (420 mg, yield 54%). ¹H

NMR (400 MHz, CDCl₃) δ 7.56 (t, J = 8.1 Hz, 3H), 7.44 (d, J = 8.4 Hz,

2H), 7.36 (d, J = 2.3 Hz, 1H), 7.20−7.13 (m, 2H), 7.04 (dd, J = 9.0, 2.3

Hz, 1H), 6.95−6.88 (m, 2H), 3.93 (s, 3H). ¹³C NMR (100 MHz,

CDCl₃) δ 189.50, 162.61 (d, J_C−F= 250.0 Hz), 160.22, 143.41, 141.99,

141.66, 135.04, 133.30, 132.03 (d, J_C−F= 8.2 Hz), 131.52, 130.09 (d,

J_C−F= 3.5 Hz), 129.51, 126.21, 117.87, 116.48, 115.46 (d, J_C−F= 21.7

Hz), 114.91, 104.23, 55.75.

(3-(4-Fluorophenyl)-6-hydroxybenzo[b]thiophen-2-yl)(4-

(triﬂuoromethyl)phenyl)methanone (26a). This compound was

prepared by a procedure identical to the preparation of 11 (3.8 g, light

green powder, yield 82%). ¹H NMR (400 MHz, acetone-d₆) δ 7.65 (d, J

= 8.0 Hz, 2H), 7.53 (t, J = 9.1 Hz, 3H), 7.48 (d, J = 2.2 Hz, 1H), 7.33−

7.22 (m, 2H), 7.06 (dd, J = 8.9, 2.2 Hz, 1H), 6.97 (t, J = 8.8 Hz, 2H). ¹³C

NMR (100 MHz, acetone-d₆)) δ 190.61, 163.52 (d, J_C−F= 246.9 Hz),

159.21, 144.17, 143.13, 142.97, 136.13, 133.92, 133.55 (d, J_C−F= 8.4

Hz), 132.93 (q, J_C−F= 32.2 Hz) 131.52 (d, J_C−F= 3.4 Hz), 130.47,

127.51, 125.65 (q, J_C−F= 3.9 Hz), 124.93 (q, J_C−F= 271.1 Hz), 117.22,

115.99 (d, J_C−F= 21.9 Hz), 108.13. ESI-HRMS (m/z): [M + H]⁺calcd

for C₂₂H₁₃F₄O₂S, 417.0572; observed, 417.0563.

2-(4-Fluorophenoxy)-3-(4-ﬂuorophenyl)-6-methoxybenzo-

[b]thiophene 1-Oxide (28c). This compound was prepared by a

procedure identical to the preparation of 28b (240 mg, yield 63%). ¹H

NMR (400 MHz, CDCl₃) δ 7.49−7.44 (m, 2H), 7.43 (d, J = 2.4 Hz,

1H), 7.27 (d, J = 8.6 Hz, 1H), 7.18−7.07 (m, 4H), 7.04−6.91 (m, 3H),

3.88 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 163.20 (d, J_C−F= 250.1

Hz), 160.67, 159.28 (d, J_C−F= 242.7 Hz), 157.04, 152.95 (d, J_C−F= 2.5

Hz), 142.22, 130.73 (d, J_C−F= 8.3 Hz), 129.24, 128.92, 125.84 (d, J_C−F

=

3.5 Hz), 124.59, 118.72 (d, J_C−F= 8.4 Hz), 118.10, 116.44 (d, J_C−F= 24.2

Hz), 116.21 (d, J_C−F= 22.3 Hz), 112.88, 56.07.

3-(4-Fluorophenyl)-6-methoxy-2-(4-methoxyphenoxy)-

benzo[b]thiophene (29a). This compound was prepared by a

procedure identical to the preparation of 18 (1.2 g, white solid, yield

67%). ¹H NMR (400 MHz, CDCl₃) δ 7.56−7.48 (m, 3H), 7.20 (d, J =

2.4 Hz, 1H), 7.15−7.10 (m, 2H), 7.05−7.01 (m, 2H), 6.97 (dd, J = 8.8,

2.4 Hz, 1H), 6.85−6.77 (m, 2H), 3.86 (s, 3H), 3.78 (s, 3H). ¹³C NMR

(100 MHz, CDCl₃) δ 162.19 (d, J_C−F= 246.5 Hz), 157.42, 156.22,

153.10, 152.53, 134.57, 131.21 (d, J_C−F= 8.0 Hz), 131.08, 129.05,

123.11, 120.80, 118.61, 115.71 (d, J_C−F= 21.4 Hz), 114.80, 114.22,

106.03, 55.86, 55.82.

2-(4-Chlorophenoxy)-3-(4-ﬂuorophenyl)-6-methoxybenzo-

[b]thiophene (29b). This compound was prepared by a procedure

identical to the preparation of 18 (206 mg, yield 53%). ¹H NMR (400

MHz, CDCl₃) δ 7.54 (d, J = 8.8 Hz, 1H), 7.49−7.43 (m, 2H), 7.26−7.21

(m, 3H), 7.11 (t, J = 8.8 Hz, 2H), 7.02−6.96 (m, 3H), 3.88 (s, 3H). ¹³C

NMR (100 MHz, CDCl₃) δ 162.32 (d, J_C−F= 247.2 Hz), 157.80, 157.28,

150.75, 135.01, 131.12 (d, J_C−F= 8.1 Hz), 130.78, 129.75, 128.77, 128.62

(d, J_C−F= 3.4 Hz), 123.50, 122.84, 118.08, 115.83 (d, J_C−F= 21.5 Hz),

114.52, 105.98, 55.87.

4-(3-(4-Fluorophenyl)-6-hydroxybenzo[b]thiophene-2-

carbonyl)benzonitrile (26b). This compound was prepared by a

1

procedure identical to the preparation of 11 (95 mg, yield 23%). H

NMR (400 MHz, CDCl₃) δ 7.60−7.54 (m, 3H), 7.45 (d, J = 8.5 Hz,

2H), 7.34 (d, J = 2.2 Hz, 1H), 7.21−7.13 (m, 2H), 6.98 (dd, J = 8.9, 2.3

Hz, 1H), 6.96−6.86 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 189.77,

162.81 (d, J_C−F= 250.1 Hz), 156.43, 143.41, 142.17, 141.78, 135.24,

133.72, 132.17 (d, J_C−F= 8.2 Hz), 131.72, 130.21, 129.68, 126.82,

117.99, 116.22, 115.65 (d, J_C−F= 21.7 Hz), 115.14, 107.73. ESI-HRMS

(m/z): [M − H]⁻calcd for C₂₂H₁₁FNO₂S, 372.0495; observed,

372.0487.

2-Bromo-3-(4-ﬂuorophenyl)-6-methoxybenzo[b]thiophene

1-Oxide (27). Triﬂuoroacetic acid (13 mL) was added dropwise to a

solution of 24 (2.4 g, 7 mmol) in 13 mL of anhydrous CH₂Cl₂in an ice

bath. The mixture was stirred for 5 min, H₂O₂(1.0 mL, 7 mmol, 30%

aqueous solution) was added dropwise, and the resulting mixture was

allowed to warm to room temperature and stirred for 2 h. Upon the

consumption of starting material, sodium bisulﬁte (0.3 g) was added to

the solution followed by 5 mL of water. The mixture was stirred

2-(4-Fluorophenoxy)-3-(4-ﬂuorophenyl)-6-methoxybenzo-

[b]thiophene (29c). This compound was prepared by a procedure

identical to the preparation of 18 (340 mg, yield 83%). ¹H NMR (400

MHz, CDCl₃) δ 7.53 (d, J = 8.8 Hz, 1H), 7.51−7.42 (m, 2H), 7.23 (d, J =

2.4 Hz, 1H), 7.12 (t, J = 8.7 Hz, 2H), 7.06−6.92 (m, 5H), 3.87 (s, 3H).

O

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Journal of Medicinal Chemistry

Article

¹³C NMR (100 MHz, CDCl₃) δ 162.10 (d, J_C−F= 247.0 Hz), 158.87 (d,

AUTHOR INFORMATION

■

J_C−F= 242.2 Hz), 157.51, 154.46 (d, J_C−F= 2.5 Hz), 151.58, 134.63,

130.99 (d, J_C−F= 8.0 Hz), 130.71, 128.58 (d, J_C−F= 3.3 Hz), 123.19,

121.90, 118.19 (d, J_C−F= 8.3 Hz), 116.12 (d, J_C−F= 23.6 Hz), 115.60 (d,

J_C−F= 21.5 Hz), 114.25, 105.83, 55.68.

Corresponding Author

*Phone: 312-355-5282. E-mail: thatcher@uic.edu.

Author Contributions

^∥R.X. and H.K.P. contributed equally.

2-(3,4-Diﬂuorophenoxy)-3-(4-ﬂuorophenyl)-6-methoxy-

benzo[b]thiophene (29d). This compound was prepared by a

The manuscript was written through contributions of all

authors. All authors have given approval to the ﬁnal version of the

manuscript.

1

procedure identical to the preparation of 18 (330 mg, yield 73%). H

NMR (400 MHz, CDCl₃) δ 7.54 (d, J = 8.8 Hz, 1H), 7.47−7.44 (m,

2H), 7.24 (d, J = 2.1 Hz, 1H), 7.15−7.03 (m, 3H), 7.00 (dd, J = 8.9, 2.1

Hz, 1H), 6.92−6.83 (m, 1H), 6.77 (d, J = 9.1 Hz, 1H), 3.88 (s, 3H).

3-(4-Fluorophenyl)-2-(4-hydroxyphenoxy)benzo[b]-

thiophen-6-ol (30a). This compound was prepared by a procedure

identical to the preparation of 11 (1.1 g, white powder, yield 75%).¹H

NMR (400 MHz, acetone-d₆) δ 7.62−7.57 (m, 2H), 7.46 (d, J = 8.7 Hz,

1H), 7.30−7.19 (m, 3H), 7.05−6.97 (m, 2H), 6.95 (dd, J = 8.7, 2.3 Hz,

1H), 6.86−6.78 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 162.57 (d,

J_C−F= 245.0 Hz). 155.67, 154.69, 153.54, 152.12, 135.01, 131.97 (d, J_C−F

= 8.1 Hz), 130.64, 130.04 (d, J_C−F= 3.2 Hz), 123.50, 120.60, 119.42,

116.76, 116.03 (d, J_C−F= 21.5 Hz), 115.35, 108.84. ESI-HRMS (m/z):

[M − H]⁻calcd for C₂₀H₁₂FO₃S, 351.0491; observed, 351.0490.

2-(4-Chlorophenoxy)-3-(4-ﬂuorophenyl)benzo[b]thiophen-

6-ol (30b). This compound was prepared by a procedure identical to

the preparation of 11 (140 mg, white powder, yield 73%). ¹H NMR (400

MHz, CDCl₃) 7.51 (d, J = 8.7 Hz, 1H), 7.49−7.42 (m, 2H), 7.26−7.21

(m, 2H), 7.20 (d, J = 2.3 Hz, 1H), 7.11 (t, J = 8.7 Hz, 2H), 7.03−6.97 (m,

2H), 6.91 (dd, J = 8.7, 2.4 Hz, 1H). ¹³C NMR (100 MHz, CDCl₃) δ

162.34 (d, J_C−F= 247.3 Hz), 157.20, 153.43, 150.89, 135.05, 131.13 (d,

J_C−F= 8.1 Hz), 129.76, 128.85, 128.55 (d, J_C−F= 3 Hz), 123.68, 122.71,

118.14, 115.84 (d, J_C−F= 21.5 Hz), 114.61, 108.53. ESI-HRMS (m/z):

[M + H]⁺calcd for C₂₀H₁₁ClFO₂S, 369.0152; observed, 369.0144.

2-(4-Fluorophenoxy)-3-(4-ﬂuorophenyl)benzo[b]thiophen-

6-ol (30c). This compound was prepared by a procedure identical to the

preparation of 11 (248 mg, white solid, yield 79%). ¹H NMR (400 MHz,

CDCl₃) δ 7.56−7.41 (m, 3H), 7.18 (d, J = 2.3 Hz, 1H), 7.12 (t, J = 8.7

Hz, 2H), 7.06−6.92 (m, 4H), 6.90 (dd, J = 8.7, 2.4 Hz, 1H), 4.83 (s, 1H).

¹³C NMR (100 MHz, CDCl₃) δ 162.12 (d, J_C−F= 247.1 Hz), 158.91 (d,

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

This work was suppored by NIH Grants R01 CA188017 and R01

CA102590, the University of Illinois Cancer Center, UIC Center

for Clinical and Translational Science Grant UL1RR029879. We

also thank Dr. Bernard D. Santarsiero from the Center for

Pharmaceutical Biotechnology and the Department of Medicinal

Chemistry and Pharmacognosy at UIC for his help with X-ray

crystallography experiments.

ABBREVIATIONS USED

■

ShERPA, selective human estrogen receptor partial agonist; ERα,

estrogen receptor α; ERβ, estrogen receptor β; E₂, estradiol;

SERM, selective estrogen receptor modulator; DES, diethyl-

stilbesterol; LBD, ligand-binding domain; BPAF, bisphenol AF;

PPA, polyphosphoric acid; FP, ﬂuorescence polarization; ERE,

estrogen response element; RBA_ral, relative binding aﬃnity to

raloxifene; RBA, relative binding aﬃnity; SRC3, steroid receptor

coregulator 3; AIB1, ampliﬁed in breast cancer 1; TR-FRET,

time-resolved ﬂuorescence (or Forster) resonance energy

̈

transfer; SEM, selective estrogen mimic; ChIP, chromatin

immunoprecipitation; SA-Tb, streptavidin−terbium; CMC,

carboxymethylcellulose; TLC, thin layer chromatography

J_C−F= 242.4 Hz), 154.39, 153.15, 151.73, 134.68, 131.00, 130.99 (d, J_C−F

= 8.1 Hz), 128.53 (d, J_C−F= 3.4 Hz), 123.36, 121.76, 118.27 (d, J_C−F

=

8.3 Hz), 116.14 (d, J_C−F= 23.6 Hz), 115.61 (d, J_C−F= 21.5 Hz), 114.37,

108.33. ESI-HRMS (m/z): [M − H]⁻calcd for C₂₀H₁₁F₂O₂S, 353.0448;

observed, 353.0451.

REFERENCES

■

(1) Deroo, B. J.; Korach, K. S. Estrogen receptors and human disease. J.

Clin. Invest. 2006, 116, 561−570.

(2) Sommer, S.; Fuqua, S. A. Estrogen receptor and breast cancer.

Semin. Cancer Biol. 2001, 11, 339−352.

(3) Jordan, V. C. Fourteenth Gaddum Memorial Lecture. A current

view of tamoxifen for the treatment and prevention of breast cancer. Br.

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(4) Jordan, V. C. Tamoxifen: toxicities and drug resistance during the

treatment and prevention of breast cancer. Annu. Rev. Pharmacol.

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competing causes of mortality in breast cancer survivors, with analysis of

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(6) Ingle, J.; Ahmann, D.; Green, S.; Edmonson, J.; Bisel, H.; Kvols, L.;

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2002, 4, 133−136.

2-(3,4-Diﬂuorophenoxy)-3-(4-ﬂuorophenyl)benzo[b]-

thiophen-6-ol (30d). This compound was prepared by a procedure

1

identical to the preparation of 11 (83 mg, yield 61%). H NMR (400

MHz, CDCl₃) δ 7.50 (d, J = 8.7 Hz, 1H), 7.45 (dd, J = 8.2, 5.9 Hz, 2H),

7.21 (d, J = 2.1 Hz, 1H), 7.17−7.00 (m, 3H), 6.97−6.83 (m, 2H), 6.79−

6.75(m, 1H), 4.85 (s, 1H). ¹³C NMR (100 MHz, CDCl₃) δ 162.4 (d,

J_C−F= 247.4 Hz), 154.46 (dd, J_C−F= 8.0, 2.4 Hz), 153.59, 150.60 (dd,

J_C−F= 249.9, 14.2 Hz), 150.59 (d, J_C−F= 249.7 Hz), 146.91 (dd, J_C−F

=

244.0, 13.0 Hz), 135.07, 131.13 (d, J_C−F= 8.1 Hz), 130.97, 128.41 (d,

J_C−F= 3.4 Hz), 123.81, 123.05, 117.61 (dd, J_C−F= 18.9, 1.1 Hz), 115.9

(d, J_C−F= 21.5 Hz), 114.73, 112.32 (dd, J_C−F= 6.0, 3.8 Hz), 108.54,

106.7 (d, J_C−F= 20.9 Hz). ESI-HRMS (m/z): [M − H]⁻calcd for

C₂₀H₁₀F₃O₂S, 371.0354; observed, 371.0346.

ASSOCIATED CONTENT

■

S

* Supporting Information

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.jmed-

chem.5b01276.

(8) Schafer, J. M.; Lee, E. S.; Dardes, R. C.; Bentrem, D.; O’Regan, R.

M.; De Los Reyes, A.; Jordan, V. C. Analysis of cross-resistance of the

selective estrogen receptor modulators arzoxifene (LY353381) and

LY117018 in tamoxifen-stimulated breast cancer xenografts. Clin.

Cancer Res. 2001, 7, 2505−2512.

X-ray crystallography veriﬁcation for 29a and eﬀect of

GPER antagonist, G15, on ShERPA luciferase activity

(PDF)

Crystallographic data (CIF)

(9) Roodi, N.; Bailey, L. R.; Kao, W. Y.; Verrier, C. S.; Yee, C. J.;

Dupont, W. D.; Parl, F. F. Estrogen receptor gene analysis in estrogen

Molecular formula strings (CSV)

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J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Selective Human Estrogen Receptor Partial Agonists (ShERPAs) for Tamoxifen-Resistant Breast Cancer

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