Discovery of ERD-308 as a Highly Potent Proteolysis Targeting Chimera (PROTAC) Degrader of Estrogen Receptor (ER)

Article

pubs.acs.org/jmc

Cite This: J. Med. Chem. XXXX, XXX, XXX −XXX

Discovery of ERD-308 as a Highly Potent Proteolysis Targeting

Chimera (PROTAC) Degrader of Estrogen Receptor (ER)

Jiantao Hu,^†,‡,#Biao Hu,^†,‡,#Mingliang Wang,^†,‡,#Fuming Xu,^†,‡,#Bukeyan Miao,^†,‡,#

Chao-Yie Yang,^†,‡Mi Wang,^†,‡Zhaomin Liu,^†,‡Daniel F. Hayes,^†,‡Krishnapriya Chinnaswamy,^⊥

James Delproposto,^⊥Jeanne Stuckey,^⊥and Shaomeng Wang*^{,†,‡,§,∥}

‡

§

^†The Rogel Cancer Center, Departments of Internal Medicine and Pharmacology, University of Michigan Medical School,

^∥Department of Medicinal Chemistry, College of Pharmacy, ^⊥Life Sciences Institute, University of Michigan, Ann Arbor, Michigan

48109, United States

S

* Supporting Information

ABSTRACT: The estrogen receptor (ER) is a validated target for the treatment of

estrogen receptor-positive (ER+) breast cancer. Here, we describe the design,

synthesis, and extensive structure−activity relationship (SAR) studies of small-

molecule ERα degraders based on the proteolysis targeting chimeras (PROTAC)

concept. Our eﬀorts have resulted in the discovery of highly potent and eﬀective

PROTAC ER degraders, as exempliﬁed by ERD-308 (32). ERD-308 achieves DC₅₀

(concentration causing 50% of protein degradation) values of 0.17 and 0.43 nM in

MCF-7 and T47D ER+ breast cancer cell lines, respectively, and induces >95% of

ER degradation at concentrations as low as 5 nM in both cell lines. Signiﬁcantly,

ERD-308 induces more complete ER degradation than fulvestrant, the only

approved selective ER degrader (SERD), and is more eﬀective in inhibition of cell

proliferation than fulvestrant in MCF-7 cells. Further optimization of ERD-308

may lead to a new therapy for advanced ER+ breast cancer.

inhibitors and SERMs, recent studies have demonstrated that

in most of the cases of drug-resistance, continued dependence

on ERα signaling for tumor growth and disease progression is

retained and the ER protein remains a principal driver in ER+

metastatic breast cancer.^8,9

INTRODUCTION

■

Breast cancer (BC) is one of the most common malignancies

in women worldwide. On the basis of the status of the tumor

receptor, breast cancer can be further subdivided into estrogen

receptor-positive (ER+), human epidermal growth factor

receptor 2 (HER2)-positive (HER2+), and triple-negative

subtypes.¹ER+ breast cancer occurs in approximately 80% of

newly diagnosed breast cancer cases.²As members of the

nuclear receptor family, estrogen receptors ERα and ERβ are

transcription factors regulating gene expression and mediating

the biological eﬀects of the estrogen. Both ERα and ERβ are

widely expressed in diﬀerent tissues, and ERα is considered to

be the major mediator which transduces the estrogen signaling

in the female reproductive tract and mammary glands.³ERα

has therefore been pursued as a promising therapeutic target in

multiple pathological settings, particularly in cancer and

osteoporosis, and this is highlighted by the clinical success of

tamoxifen for the treatment of ER+ BC and raloxifene for the

prevention and treatment of osteoporosis in postmenopausal

women.^4,5

Although inhibition of estrogen synthesis by aromatase

inhibitors and inhibition of ER pathway signaling by selective

estrogen receptor modulators (SERM) (Figure 1) have

demonstrated considerable clinical beneﬁts in the treatment

of ER+ BC, the development of intrinsic and acquired

resistance to those drug classes presents an impediment for

patients with advanced and metastatic breast cancer.^6,7While

there are clearly multiple resistance mechanisms to aromatase

Selective estrogen receptor degraders (SERD) are small

molecules that target ERα for proteasome-dependent degra-

dation. Currently, fulvestrant (5, Figure 1) is the only SERD

that has been approved for the treatment of postmenopausal

women with advanced ER+ breast cancer with standard

endocrine therapies.^10,11The clinical success enjoyed by

fulvestrant suggests that degradation of the ER protein is

beneﬁcial to patients with ER+ breast cancer, particularly those

whose disease continues to progress after standard endocrine

therapies. Because fulvestrant has poor solubility and is not

orally bioavailable, it is administered clinically by a monthly

intramuscular injection.^12,13To address the shortcomings of

fulvestrant, orally bioavailable SERD molecules have been

developed and a number of them, shown in Figure 1, are

currently being evaluated in clinical trials as new therapies for

the treatment of ER+, metastatic BC.¹⁴⁻¹⁹

The proposed mechanism of action for traditional SERDs

such as fulvestrant and those shown in Figure 1 is induction of

misfolding of the ER protein, which ultimately leads to

proteasome-dependent ERα protein degradation.²⁰The SERD

Received: October 9, 2018

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Figure 1. Representative SERMs and SERDs.

Figure 2. (A) A general scheme for the design of ER degraders based upon the PROTAC concept. (B) Structures of CRBN ligand and VHL ligand.

(C) Crystal structure of ERα ligand-binding domain in a complex with raloxifene (1) (PDB 1ERR). (D) Predicted binding models of N,N-

diethylamino analogue of raloxifene in complex with ERα LBD. Hydrogen bonds are depicted by yellow dashed lines.

molecules are typically potent and eﬀective in inducing

degradation of ER protein in ER+ breast cancer cells, but

they are only able to achieve partial degradation of the ER

protein.^21,22Consequently, we propose that novel therapeutic

agents, which can achieve more complete degradation of ER,

could be more eﬃcacious than the traditional SERD molecules

for the treatment of ER+ metastatic breast cancer.

The proteolysis targeting chimera (PROTAC) concept was

ﬁrst introduced in 2001,²³with the objective of induction of

selective target protein degradation by hijacking the cellular E3

ubiquitination ligase systems.²⁴⁻²⁸PROTACs are hetero-

bifunctional small molecules containing a ligand, which binds

to the target protein of interest, and another ligand for an E3

ligase system. These two ligands are tethered together by a

chemical linker (Figure 2A). The PROTAC strategy has

recently gained momentum due in part to the availability of

potent and druglike small-molecule ligands for a number of E3

ligase systems, and it has been employed for the design of

small-molecule degraders for a number of proteins.²⁹⁻⁴³

Recently, Naito et al. reported several PROTAC-like ERα

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Figure 3. Degradation of ER protein by PROTACs. (A) Structures of synthetic putative ER degraders. (B) Western blotting analysis of ER protein

in MCF-7 cells treated with putative PROTAC ER degraders and control compounds. Cells were treated with diﬀerent compounds for 4 h, and

whole cell lysates were then analyzed by Western blotting to examine the level of ER protein. The membranes were stripped and reblotted for

GAPDH as the loading control. The numbers below the panel represent the ERα/GADPH ratio normalized with the DMSO control at 100.

degraders, which were named speciﬁc and nongenetic IAP-

dependent protein erasers (SNIPERs).^44,45They designed

ERα SNIPER molecules using an ERα antagonist and a ligand

for inhibitors of apoptosis protein (IAPs), which are E3 ligases.

However, while SNIPER ER degraders eﬀectively induce

partial degradation of the ER protein, they also induce

autoubiquitylation and proteasomal degradation of the E3

ligase, the cIAP1 protein, thus limiting their degradation and

therapeutic eﬃcacy.

In the present study, we report our design, synthesis, and

evaluation of PROTAC degraders of ERα protein by

employing the cereblon/cullin 4A and VHL/cullin 2

neddylation degradation systems and several diﬀerent classes

of selective ER modulators/antagonists.⁴⁶Our study has

resulted in the discovery of highly potent PROTAC ERα

degraders (hereafter called ER degraders) with DC₅₀< 1 nM

and D_max> 95% in MCF-7 and T47D ER+ breast cancer cells.

This study lays a foundation for the development of a

completely new class of therapeutics for the treatment of ER+

metastatic breast cancer.

RESULTS AND DISCUSSION

■

Raloxifene (1, Figure 1), a highly potent SERM, has been

approved for the treatment of osteoporosis and breast cancer

in women and was employed in our design of PROTAC ER

degraders. Both cereblon/cullin 4A and VHL/cullin 2 have

been successfully used for the design of PROTAC

degraders.^47,48Therefore, we employed phthalimide analogues

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Figure 4. Optimization of linker length with linear aliphatic linkers. (A) Structures of ER degraders. (B) Western blotting analysis of ER protein in

the MCF-7 cells treated with indicated compounds at 1, 10, and 100 nM. MCF-7 cells were treated with diﬀerent compounds for 4 h and whole cell

lysates were analyzed by Western blotting to examine the level of ER protein. The membranes were stripped and reblotted for GAPDH as the

loading control. The numbers below the panel represent the ERα/GADPH ratio normalized with the DMSO control at 100.

to recruit cereblon and compound 11 to recruit VHL for the

design of putative PROTAC ERα degraders (Figure 2B).

The cocrystal structure of human ER ligand-binding domain

in a complex with raloxifene shows that the piperidine ring in

raloxifene is buried in the hydrophobic pocket of ERα (Figure

2C).⁴⁹For the synthesis of PROTAC ERα degraders, we

replaced the piperidine group with an N,N-diethylamino

group, similar to the N,N-dimethylamino group in tamoxifen

(2a, Figure 1). Our modeling shows that one of the ethyl

groups is exposed to solvent and is therefore suitable as the

linking site in the design of PROTAC ER degraders (Figure

2D).

compounds (12−15) were evaluated for their ability to induce

ER degradation in the MCF-7 ER+ breast cancer cell line, with

fulvestrant used as the control.

Western blotting data (Figure 3B) showed that, consistent

with its mode of action as a potent SERD molecule, fulvestrant

very eﬀectively induces ER protein degradation in MCF-7 cells.

Interestingly, while compounds 14 and 15, designed using a

VHL ligand, are very eﬀective in inducing ER degradation with

4 h treatment at concentrations as low as 1−10 nM in MCF-7

cells, compounds 12 and 13, designed using a cereblon ligand,

are completely ineﬀective in inducing ER degradation at

concentrations of 1−1000 nM. Although further investigation

is clearly needed before it can be concluded that the cereblon/

cullin 4A system is less eﬀective or ineﬀective for the design of

ER degraders, our data for compounds 14 and 15 clearly

demonstrate that the VHL/cullin 2 system can be successfully

We ﬁrst synthesized compounds 12 and 13 using a cereblon

ligand and two diﬀerent linkers and also prepared compounds

14 and 15 using the VHL ligand (11) and two diﬀerent linkers

as putative PROTAC ER degraders (Figure 3A). These four

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Figure 5. Optimization of N-substituent groups (A) Structures of ER degraders. (B) Western blotting analysis of ER protein in the MCF-7 cells

treated with indicated compounds at 1, 10, and 100 nM. MCF-7 cells were treated with diﬀerent compounds for 4 h, and whole cell lysates were

analyzed by Western blotting to examine the level of ER protein. The membranes were stripped and reblotted for GAPDH as the loading control.

The numbers below the panel represent the ERα/GADPH ratio normalized with the DMSO control at 100.

employed for the design of potent and highly eﬀective

PROTAC ER degraders. We have therefore focused on our

further design and optimization of PROTAC ER degraders

using VHL ligands.

Our previous studies on bromodomain and extra-terminal

(BET) degraders demonstrated that the length and chemical

composition of the linker in a PROTAC degrader molecule

were critical in achieving eﬀective protein degradation.^48,50,51

We therefore synthesized a series of analogues of compound

15 with the linker length varying from 3 to 9 atoms (Figure

4A). These compounds were evaluated for their ability to

induce ER degradation in MCF-7 cells at concentrations of 1,

10, and 100 nM, with compound 15, fulvestrant (5), another

SERD molecule RAD1901 (9), and raloxifene (1) included as

controls. Our Western blotting data (Figure 4B) showed that

compounds 16 and 17 with a (CH₂)₃or (CH₂)₄linker are

ineﬀective in inducing ER degradation at 1−100 nM

concentrations. However, compounds 15, 18, 19, 20, and

21, with linkers containing 6−9 carbon atoms, are all highly

eﬀective in inducing ER degradation at concentrations as low

as 1 nM. These compounds display similar potencies, with 15

being slightly more potent than the other compounds.

Fulvestrant eﬀectively induces ER degradation at concen-

trations of 1, 10, and 100 nM, but RAD1901 fails to induce ER

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Figure 6. Optimization of the linker composition. (A) Structures of the ER degraders. (B) Western blotting analysis of ER protein in the MCF-7

cells treated with indicated compounds at 1, 10, and 100 nM. MCF-7 cells were treated with diﬀerent compounds for 4 h, and whole cell lysates

were analyzed by Western blotting to examine the level of ER protein.The membranes were stripped and reblotted for GAPDH as the loading

control. The numbers below the panel represent the ERα/GADPH ratio normalized with the DMSO control at 100.

degradation at 1−100 nM. Consistent with its mode of action

as a SERM, raloxifene fails to induce any ER degradation.

Upon the basis of our modeling (Figure 2D), an N-ethyl

group in the ER antagonist portion of compound 15 binds to a

hydrophobic pocket in the ER protein and presumably

contributes to its binding to ER. We therefore synthesized

and tested a series of analogues of 15 with diﬀerent N-

substituent alkyl groups (Figure 5A). As shown in Figure 5B,

compound 22 with a N-methyl group is ineﬀective in inducing

ER degradation at 1 and 10 nM and induces only moderate ER

degradation at 100 nM. In contrast, compounds 23−29, with

an isopropyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl,

cyclohexyl, or cyclobutylmethyl group, respectively, all induce

essentially complete ER degradation in MCF-7 cells at

concentrations of 10 and 100 nM. Upon the basis of the

Western blotting data from these compounds at a 10 nM

concentration, compound 26 with a cyclobutyl group is the

most potent ER degrader among this series of compounds,

followed by compound 25 with a cyclopropyl group, and

compounds 15 and 24 with an ethyl or a tert-butyl group.

However, in view of the fact that compound 15 possesses the

lowest molecular weight (MW: 1002.3) and achieves an

excellent degradation potency, we employed it as the template

molecule for further optimization.

Our previous studies showed that in addition to the linker

length, the linker composition plays a key role for protein

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Figure 7. SAR study for ER ligands. (A) Structures of ER degraders. (B) Western blotting analysis of ER protein in the MCF-7 cells treated with

indicated compounds at 1, 10, and 100 nM. MCF-7 cells were treated with diﬀerent compounds for 4 h, and whole cell lysates were analyzed by

Western blotting to examine the level of ER protein. The membranes were stripped and reblotted for GAPDH as the loading control. The numbers

below the panel represent the ERα/GADPH ratio normalized with the DMSO control at 100.

degradation potencies in our BET PROTAC molecules.⁴⁸In

view of the fact that the linker in compound 15 is quite

hydrophobic and that this compound displays poor aqueous

solubility, we synthesized a series of new analogues with linkers

to improve solubility of the resulting compounds (Figure 6A).

These new analogues were evaluated together with 14 and 15

for their ability to induce ER degradation in MCF-7 cells

(Figure 6B).

Compounds 14 and 30−32 with a polyethylene glycol unit

(PEG) embedded in the linker are all eﬀective in inducing ER

degradation at 10 and 100 nM in MCF-7 cells. Upon the basis

of the Western blotting data from these compounds tested at 1

nM, compound 32 is the most potent compound, followed by

compound 31.

Compounds 33 and 34 containing a positively charged

piperidinyl or piperazinyl group are much less potent than

compound 15 and fail in fact to induce any ER degradation at

1 and 10 nM in MCF-7 cells. While 33 is still ineﬀective in

inducing ER degradation at 100 nM, compound 34 at 100 nM

clearly induces ER degradation.

Compound 15 has a very ﬂexible linker. We sought to

determine if introduction of a conformationally rigid linker

using a charge-neutral, aromatic ring can result in potent

PROTAC ER degraders. This led to the design and synthesis

of compounds 35−37. Western blotting analysis showed that

while compounds 35−37 eﬀectively induce ER degradation,

these three compounds are much less potent than compounds

15 and 32.

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Figure 8. SAR study for VHL ligands. (A) Structures of ER degraders and binding aﬃnities of relative VHL ligands against VHL protein by

ﬂuorescence polarization (FP) assay. (B) Western blotting analysis of ER protein in the MCF-7 cells treated with indicated compounds at 1, 10,

and 100 nM. MCF-7 cells were treated with diﬀerent compounds for 4 h, and whole cell lysates were analyzed by Western blotting to examine the

level of ER protein. The membranes were stripped and reblotted for GAPDH as the loading control. The numbers below the panel represent the

ERα/GADPH ratio normalized with the DMSO control at 100.

Compound 15 contains the core structure of the ER

antagonist raloxifene (1), and we next designed and

synthesized a series of ER degraders using the core structures

of several diﬀerent classes of selective ER modulators,

including tamoxifen (2a), 4-hydroxytamoxifen (2b), bazedox-

ifene (3), and lasofoxifene (4). These eﬀorts resulted in

compounds 38−41 (Figure 7A), which are all very eﬀective in

inducing ER degradation with compound 39, with the

tamoxifen core structure being the least potent. Our data

showed that compound 15 is still more potent than

compounds 38−41 in induction of ER degradation (Figure

7B).

We next investigated the eﬀects of the VHL ligand portion

in our PROTAC ER degraders on ER degradation, focusing

particularly on the benzylamine moiety because this portion is

known to be critical in achieving high binding aﬃnities to VHL

(Figure 8A).⁵²⁻⁵⁵First, we synthesized a series of VHL ligands.

We established a ﬂuorescence polarization (FP) assay for VHL

(Figure S1 in Supporting Information) and tested the binding

aﬃnities of VHL ligands 11 and 43a−48a, with a previously

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Figure 9. Further characterization of compound 32 (ERD-308). (A) ERα degradation dose-response for compound 32 with a treatment time of 4

h in MCF-7 cells. ER protein was examined by Western blotting and ER protein level was quantiﬁed by densitometry and normalized to the

corresponding density of GAPDH protein. (B) ERα degradation dose-response for compound 32 with a treatment time of 4 h in T47D cells. ER

protein was examined by Western blotting and ER protein level was quantiﬁed by densitometry and normalized to the corresponding density of

GAPDH protein. (C) Time course of ERα degradation by compound 32 (30 nM) and fulvestrant (30 nM) in the MCF-7 cells. (D) Time course of

ERα degradation by compound 32 (30 nM) and fulvestrant (30 nM) in the T47D cells.

reported VHL ligand (VH032)⁵⁴included as a control (Figure

8A, Figure S2 in Supporting Information). Among these VHL

ligands, compound 11 is twice as potent as VH032 with an

IC₅₀value of 196 nM in our FP assay. However, substitution of

the 4-methylthiazole group with other smaller aliphatic groups

decreases the binding aﬃnity signiﬁcantly. Among them, an

ethynyl group substituent displayed acceptable binding aﬃnity

with an IC₅₀value of 879 nM.

which lacks the chiral methyl group in 15, is much less potent

than 15 in inducing ER degradation. Replacement of the

methylthiazole group in 15 with other hydrophobic groups,

such as chloro (43), cyano (44), cyclopropyl (46), isopropyl

(47), and tert-butyl (48), led to signiﬁcantly reduced ER

degradation. An exception is compound 45 with an ethynyl

group, which displays good or moderate degradation activity at

10 and 100 nM. Collectively, our data show that, consistent

with the mechanism of action of PROTAC molecules, the

binding aﬃnity of degraders to VHL protein plays an

important role in induction of ER degradation.

Next, we employed those VHL ligands in the design of ER

degraders to investigate the inﬂuence of VHL binding aﬃnity

on ER degradation. As shown in Figure 8B, compound 42,

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Through extensive optimization of the three structural

moieties in our PROTAC ER degraders, we have obtained a

number of highly potent ER PROTAC molecules in MCF-7

cells, as exempliﬁed by 32 (ERD-308). We next performed

further investigations to characterize the action of compound

32.

We ﬁrst examined the ER degradation by compound 32 in a

wide range of concentrations to determine its DC₅₀

(concentration to achieve 50% of protein degradation) in

MCF-7 cells (Figure 9A). Quantiﬁcation of our Western

blotting data showed that compound 32 achieves a DC₅₀value

of 0.17 nM in the MCF-7 cells with a 4 h treatment.

Compound 32 achieves a maximum ER degradation of >95%

based upon our quantiﬁcation at concentrations as low as 5

nM. It is noteworthy that compound 32 induces less ER

degradation at 1 μM than at lower concentrations, a known

“hook” eﬀect that has been observed previously in PROTAC

degraders.^56,57

We also evaluated compound 32 for its ability to induce ER

degradation in the T47D ER+ breast cancer cell line. As shown

in Figure 9B, compound 32 achieves a DC₅₀value of 0.43 nM

and a maximum degradation of >95% at 5 nM. Compound 32

at 1 μM also demonstrates a hook eﬀect in the T47D cells.

Next, we examined the kinetics of ER degradation induced

by compound 32 in MCF-7 cells. As shown in Figure 9C, at a

concentration of 30 nM, compound 32 reduces >80% of the

ER protein level with a 1 h treatment and achieves essentially

complete ER degradation at the 3 h time-point, indicating fast

kinetics. In comparison, fulvestrant, a conventional SERD, has

only a modest eﬀect on reduction of the level of ER at 1 h and

achieves a maximum of approximately 90% of ER degradation

after a 24 h treatment. The kinetic data obtained for 32 and

fulvestrant in the T47D cells were similar to those observed in

MCF-7 cells (Figure 9D). Taken together, our data

demonstrate that compound 32 displays faster degradation

kinetics and more complete degradation than fulvestrant.

Upon the basis of the PROTAC design, degradation of ER

protein induced by the PROTAC degrader 32 should depend

upon its binding to both the ER protein and the VHL protein.

Furthermore, the induced ER degradation by a PROTAC ER

degrader such as 32 should be proteasome-dependent. We

therefore investigated the mechanism of action of ER

degradation induced by 32 (Figure 10). Consistent with the

PROTAC design, ER degradation induced by compound 32 at

a 30 nM concentration is signiﬁcantly reduced by addition of 1

μM of raloxifene or 1 μM of the proteasome inhibitor

carﬁlzomib, but raloxifene or carﬁlzomib alone have no eﬀect

on the ER protein levels. Interestingly, 1 μM of the VHL ligand

(11) blocks the degradation by 30 nM of compound 32 only

slightly (Figure 10A). To further conﬁrm that the degradation

is VHL-dependent, we performed a dose−response experiment

with VHL ligand 11. As shown in Figure 10B, the degradation

by compound 32 was completely blocked with 5 or 10 μM of

11. Taken together, these mechanistic data provide clear

evidence that compound 32 acts as a bona ﬁde PROTAC ER

degrader.

Figure 10. Degradation is dependent on VHL, ER, and proteasome.

(A) MCF-7 cells were pretreated with VHL ligand 11 (1 μM), or ER

ligand raloxifene (1) (1 μm), or the proteasome inhibitor carﬁlzomib

(1 μM) for 2 h, followed by treatment with DMSO or compound 32

(30 nM) for 4 h. Then whole-cell lysates were analyzed by Western

blotting. (B) MCF-7 cells were pretreated with VHL ligand 11 (+, 0.5

μM; ++, 1 μM; +++, 5 μM; ++++, 10 μM) for 2 h, followed by

treatment with DMSO or compound 32 (30 nM) for 4 h. Then

whole-cell lysates were analyzed by Western blotting.

maximum inhibition (I_max) of 57.5% in MCF-7 cells.

Fulvestrant is also very eﬀective and potent in inhibition of

cell proliferation, but it achieves an I_maxvalue of 43.8%.

Raloxifene, on the other hand, achieves an I_maxvalue of only

34.0%. Interestingly, RAD1901, a previously reported SERD

molecule,¹⁸achieves an I_maxvalue of only 25.7%. Hence the

more complete ER degradation achieved by compound 32

translates into a greater maximum cell growth inhibition than

that of fulvestrant or RAD1901, two conventional SERD

molecules. Furthermore, compound 32 achieves a much

greater maximum cell growth inhibition than raloxifene, a

SERM molecule. As expected, compound 32 does not exhibit

the cell proliferation inhibition eﬀects in triple-negative breast

cancer cell MDA-MB-231 (Figure S3 in Supporting

Information) and primary human mammary epithelial cells

(Figure S4 in Supporting Information).

To visually evaluate the cellular eﬀect, we conducted a

crystal violet staining experiment for compound 32 at 10, 100,

and 300 nM with raloxifene and fulvestrant as controls (Figure

11B). Consistent with the WST-8 cell proliferation assay,

treatment of MCF-7 cells with compound 32 reduced cell

proliferation more signiﬁcantly than raloxifene or fulvestrant at

all three of the concentrations tested (Figure 11B).

We next performed quantitative reverse transcription-

polymerase chain reaction (qRT-PCR) analysis to evaluate

the ability of compound 32 to suppress the mRNA levels of

pGR and GREB1, two ER-regulated genes in MCF-7 cells. As

shown in Figures 11C,D, the expression of both genes is

strongly suppressed by compound 32. Compound 32 is only

slightly more eﬀective than fulvestrant in suppressing the

expression of pGR and GREB1 at both 10 and 100 nM, but it

is signiﬁcantly more eﬀective than raloxifene.

Because compound 32 is highly potent and eﬀective in

induction of ER degradation, we used the WST-8 cell

proliferation assay to evaluate its ability to inhibit cell

proliferation in MCF-7 cells, with raloxifene and fulvestrant

included as controls (Figure 11A). Our data showed that

compound 32 is highly potent and eﬀective in inhibition of cell

proliferation and achieves an IC₅₀value of 0.77 nM and a

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Figure 11. Cellular eﬀects of compound 32 (ERD-308) in MCF-7 cells. (A) Cell viability assay. Cells were treated with indicated doses for 4 days.

Cell viability was determined by colorimetric WST-8 assay. GI₅₀values were calculated using the GraphPad Prism 7 software. (B) Crystal violet

staining. Cells were treated with indicated doses for 5 days, and then ﬁxed and stained with crystal violet. (C,D) qRT-PCR analysis for pGR gene

and GREB1 mRNA expression in MCF-7 cells. Cells were treated with DMSO, raloxifene (1), fulvestrant (5), or compound 32 at indicated doses

for 8 h. Multiple student’s t tests were conducted using the Holm−Sidak method. * Signiﬁcant diﬀerence between the compound treated group and

the vehicle treated control group (DMSO). ^&Signiﬁcant diﬀerence between the compound treated group and the ER inhibitor (raloxifene) treated

#

group. Signiﬁcant diﬀerence between fulvestrant and PROTAC degrader treated groups.

with excess ethylamine to aﬀord the secondary amine (52).

Cleavage of both aryl methoxy ethers in 52 with boron

tribromide furnished the dihydroxy intermediate 53. Following

the literature reported procedure,⁴⁷the synthesis of compound

58 commenced with the tert-butyloxycarbonyl protection of

commercial (S)-1-(4-bromophenyl)ethan-1-amine (54). Sub-

sequent Suzuki coupling with 4-methylthiazole aﬀorded

compound 55, and this was followed by deprotection under

acidic conditions and amide coupling with commercial

(2S,4R)-1-(tert-butoxycarbonyl)-4-hydroxypyrrolidine-2-car-

CHEMISTRY

■

The syntheses of the presented ﬁnal compounds were outlined

in Schemes 3−5. First, key common intermediates 53 and 58

were synthesized as shown in Schemes 1 and 2, respectively.

The commercial 4-acetoxybenzoic acid (49) was converted to

the acyl chloride, which, after Friedel−Crafts acylation of

commercial 6-methoxy-2-(4-methoxyphenyl)benzo-[b]-

thiophene, furnished compound 50. After deacetylation of

compound 50 under basic and aqueous conditions, compound

51 was converted to the alkyl bromide, which was substituted

K

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a

Scheme 1. Synthesis of the Key Intermediate 53

a

Reagents and conditions: (a) oxalyl chloride, DMF, DCM, 0 °C to RT, 1 h; (b) 6-methoxy-2-(4-methoxyphenyl)benzo[b]thiophene, AlCl₃,

DCM, 0 °C to RT, 1 h; (c) NaOAc, EtOH/H₂O, 80 °C, 12 h; (d) 1,2-dibromoethane, Cs₂CO₃, MeCN, reﬂux, 12 h; (e) EtNH₂, DIPEA, DMF, 80

°C, 12 h; (f) BBr₃, DCM, 0 °C to RT, 1 h.

a

Scheme 2. Synthesis of the Key Intermediate 58

a

Reagents and conditions: (a) Boc₂O, NaHCO₃, EtOAc/H₂O, 2 h; (b) 4-methylthiazole, Pd(OAc)₂, KOAc, DMA, 90 °C, 12 h; (c) 4 N HCl in

dioxane/MeOH, RT, 12 h; (d) (2S,4R)-1-(tert-butoxycarbonyl)-4-hydroxypyrrolidine-2-carboxylic acid, HATU, DIPEA, DMF, 0 °C to RT, 12 h;

(e) 4 N HCl in dioxane/MeOH, RT, 12 h; (f) (S)-1-(tert-butoxycarbonyl)piperidine-2-carboxylic acid, HATU, DIPEA, DMF, 0 °C to RT, 12 h;

(g) 4 N HCl in dioxane/MeOH, RT, 12 h.

a

Scheme 3. Synthesis of Compounds 12 and 13

a

Reagents and conditions: (a) MsCl, TEA, DCM, 0 °C to RT, 1 h; (b) 53, DIPEA, DMF, 80 °C, 12 h; (c) 3-(4-iodo-1-oxoisoindolin-2-

yl)piperidine-2,6-dione, Pd(PPh₃)Cl₂, CuI, DMF/TEA, 80 °C, 1 h.

boxylic acid. Under the same conditions, compound 56 was

deprotected, followed by amide coupling with commercial (S)-

1-(tert-butoxycarbonyl)piperidine-2-carboxylic acid to aﬀord

compound 57, which after acidic deprotection aﬀorded

compound 58.

As shown in Scheme 3, synthesis of compound 12

commenced with the conversion of commercial 2-(2-(2-

(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethan-1-ol (59a) to com-

pound 60a using methanesulfonyl chloride with trimethyl-

amine as base. Nucleophilic substitution of 60a with

compound 53 under mild basic conditions aﬀorded the N-

substituted compound (61a). Sonogashira coupling of

compound 61a with the previously published compound 3-

(4-iodo-1-oxoisoindolin-2-yl)piperidine-2,6-dione⁵⁰aﬀorded

the compound 12 in high yield. Compound 13 was synthesized

using the procedure described for the synthesis of compound

12 with oct-7-yn-1-ol 59b as the starting material.

As shown in Scheme 4, diversity of the linkers commenced

with the preparation of compound 63 or 65, which are

commercially available or can be prepared from 62 or 64,

respectively. The substitution reaction of compound 63 or 65

with compound 53 furnished compound 66, which underwent

acidic deprotection to aﬀord the acid (67). Amide coupling

between compound 67 and 58 aﬀorded the ﬁnal compounds

14−21 and 30−37 in high yields.

As shown in Scheme 5, the common intermediate 51 was

used for the SAR studies of the N-substituent groups.

Compound 51 was ﬁrst converted to the alkyl bromide,

which underwent nucleophilic attack with excess primary

amines furnishing compound 68. Substitution reaction of

compound 68 with tert-butyl 8-bromooctanoate (65)

furnished the linker attached intermediate, which underwent

boron tribromide-mediated demethylation and deprotection to

aﬀord the acid (69). Amide coupling between compounds 69

L

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Article

a

Scheme 4. Synthesis of Compounds 14−21, 30−37

a

Reagents and conditions: (a) TsCl or MsCl, TEA, DCM, 0 °C to RT, 1 h; (b) TFAA, tert-BuOH, DCM, 0 °C to RT, 12 h; (c) 53, DIPEA, DMF,

80 °C, 12 h; (d) TFA/DCM, 0 °C to RT, 6 h; (e) 58, HATU, DIPEA, DMF, RT, 12 h.

a

Scheme 5. Synthesis of Compounds 22−29

a

Reagents and conditions: (a) 1,2-dibromoethane, Cs₂CO₃, MeCN, reﬂux, 12 h; (b) RNH₂, K₂CO₃, MeCN, 80 °C; (c) 65, K₂CO₃, DMF, 80 °C,

12 h; (d) BBr₃, DCM, 0 °C to RT, 1 h; (e) 58, HATU, DIPEA, DMF, RT, 12 h.

and 58 aﬀorded the ﬁnal compounds 22−29 in high yields.

Compounds 38-48 were synthesized using the general

procedure used to prepare compound 15.

EXPERIMENTAL SECTION

■

General Chemical Methods. Unless otherwise noted, all

purchased reagents were used as received without further puriﬁcation.

¹H NMR and ¹³C NMR spectra were recorded on a Bruker Advance

1

400 MHz spectrometer. H NMR spectra are reported in parts per

CONCLUSIONS

million (ppm) downﬁeld from tetramethylsilane (TMS). All ¹³C

■

1

We have designed, synthesized, and evaluated a series of

PROTAC ER degraders. Through extensive optimization of

three structural components in our designed ER degraders, we

have successfully obtained a number of highly potent ER

degraders, exempliﬁed by 32 (ERD-308). With 4 h treatment,

compound 32 achieves DC₅₀values of 0.17 and 0.43 nM in

MCF-7 and T47D cells, respectively, and induces over 95% of

ER degradation at concentrations as low as 5 nM. In direct

comparison, compound 32 can achieve more complete

degradation than the only approved SERD molecule

fulvestrant. Consistently, compound 32 achieves more

complete cell growth inhibition than fulvestrant in MCF-7

cells. Our data suggest that further optimization of PROTAC

ER degraders may lead to a class of completely new and

eﬀective therapeutic agents for the treatment of advanced and

metastatic ER+ breast cancer.

NMR spectra are reported in ppm and obtained with H decoupling.

In the spectral data reported, the format (δ) chemical shift

(multiplicity, J values in Hz, integration) was used with the following

abbreviations: s = singlet, d = doublet, t = triplet, q = quartet, m =

multiplet. MS analyses were carried out with a H2Os UPLC−mass

spectrometer. The ﬁnal compounds were all puriﬁed by C18 reverse

phase preparative HPLC column with solvent A (0.1% TFA in H₂O)

and solvent B (0.1% TFA in MeCN) as eluents. The purities of all the

ﬁnal compounds were determined to be over 95% by UPLC-MS.

(2S,4R)-1-((S)-2-Acetamido-3,3-dimethylbutanoyl)-4-hydroxy-N-

((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxa-

mide (11). HATU (21 mg, 0.055 mmol, 1.1 equiv) was added to a

mixture of compound 58 (23 mg, 0.05 mmol, 1.0 equiv), acetic acid

(4 μL, 0.06 mmol, 1.2 equiv), and DIPEA (26 μL, 0.15 mmol, 3.0

equiv) in DMF (2 mL) at 0 °C under N₂. The mixture was stirred at

ambient temperature for 1 h, then the crude mixture was puriﬁed by

reversed-phase preparative HPLC to aﬀord the title compound as a

1

white solid (19 mg, 80% yield). H NMR (CD₃OD, 400 MHz) δ

(ppm) 9.02 (s, 1H), 7.47−7.42 (m, 4H), 5.04−4.98 (m, 1H), 4.62−

M

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4.55 (m, 2H), 4.43−4.41 (m, 1H), 3.88 (d, J = 1.08 Hz, 1H), 3.74

(dd, J = 10.8 Hz, J = 4.0 Hz, 1H), 2.50 (s, 3H), 2.22−2.16 (m, 1H),

2.00 (s, 3H), 1.98−1.91 (m, 1H), 1.51 (d, J = 6.8 Hz, 3H), 1.05 (s,

9H). ¹³C NMR (CD₃OD, 100 MHz) δ (ppm) 173.26, 173.11, 172.28,

153.34, 148.20, 146.01, 133.91, 131.04, 130.51, 127.69, 127.52, 70.97,

60.55, 59.22, 57.97, 50.14, 38.77, 36.41, 26.99, 22.38, 22.29, 15.41.

UPLC-MS (ESI⁺) calcd for C₂₅H₃₅N₄O₄S [M + 1]⁺: 487.24, found

487.43. Purity 98.5% (HPLC).

(m, 2H), 1.33 (t, J = 7.2 Hz, 3H). ¹³C NMR (CD₃OD, 100 MHz) δ

(ppm) 195.28, 174.58, 172.22, 170.98, 163.03, 159.27, 156.87,

145.24, 144.38, 141.48, 135.77, 134.20, 133.51, 132.92, 132.75,

131.47, 130.99, 129.62, 126.02, 124.72, 123.74, 120.90, 116.46,

116.12, 115.38, 107.92, 97.08, 77.41, 63.59, 54.42, 53.66, 52.53, 50.31,

32.33, 29.39, 27.05, 24.68, 24.08, 19.94, 9.10. UPLC-MS (ESI⁺) calcd

for C₄₆H₄₆N₃O₇S [M + 1]⁺: 784.31, found 784.27. Purity 98.9%

(HPLC).

(2S,4R)-1-((S)-17-(tert-Butyl)-3-ethyl-1-(4-(6-hydroxy-2-(4-

hydroxyphenyl)benzo[b]thiophene-3-carbonyl)phenoxy)-15-oxo-

6,9,12-trioxa-3,16-diazaoctadecan-18-oyl)-4-hydroxy-N-((S)-1-(4-

(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

(14). This compound was prepared using a procedure similar to that

used for compound 32. ¹H NMR (CD₃OD, 400 MHz) δ (ppm) 8.87

(s, 1H), 7.72 (d, J = 8.8 Hz, 2H), 7.44−7.39 (m, 5H), 7.26 (d, J = 2.0

Hz, 1H), 7.19 (d, J = 8.4 Hz, 2H), 6.89−6.85 (m, 3H), 6.63 (d, J =

8.4 Hz, 2H), 5.02−4.96 (m, 1H), 4.64−4.54 (m, 2H), 4.43−4.41 (m,

1H), 4.20 (t, J = 5.2 Hz, 2H), 3.85 (d, J = 11.2 Hz, 1H), 3.75−3.64

(m, 5H), 3.60−3.55 (m, 8H), 3.20−2.93 (m, 6H), 2.54−2.46 (m,

5H), 2.20−2.17 (m, 1H), 1.98−1.92 (m, 1H), 1.49 (d, J = 7.2 Hz,

3H), 1.67 (t, J = 7.2 Hz, 3H), 1.02 (s, 9H). UPLC-MS (ESI⁺) calcd

for C₅₇H₇₀N₅O₁₁S₂[M + 1]⁺: 1064.45, found 1064.74. Purity 96.4%

(HPLC).

3-(4-(3-Ethyl-1-(4-(6-hydroxy-2-(4-hydroxyphenyl)benzo[b]-

thiophene-3-carbonyl)phenoxy)-6,9,12-trioxa-3-azapentadec-14-

yn-15-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (12). Methane-

sulfonyl chloride (0.35 mL, 4.5 mmol, 1.5 equiv) and triethylamine

(0.84 mL, 6.0 mmol, 2.0 equiv) were added sequentially to a solution

of commercial compound 2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)-

ethoxy)ethan-1-ol (59a) (565 mg, 3.0 mmol, 1.0 equiv) in DCM

(10 mL) at 0 °C. The mixture was warmed to rt and stirred for 1 h.

After concentration, the residue was puriﬁed by silica gel ﬂash column

chromatography with hexane:EtOAc (2:1−1:2) to aﬀord the title

1

compound (60a) as a colorless oil (710 mg, 89% yield). H NMR

(CDCl₃, 400 MHz) δ (ppm) 4.21−4.18 (m, 2H), 4.01 (d, J = 2.4 Hz,

2H), 3.61−3.58 (m, 2H), 3.51−3.46 (m, 8H), 2.92 (s, 3H), 2.41 (t, J

= 2.4 Hz, 1H). ¹³C NMR (CDCl₃, 100 MHz) δ (ppm) 79.71, 74.91,

70.33, 70.25, 70.09, 69.51, 68.90, 68.77, 58.09, 37.43.

(2S,4R)-1-((S)-2-(8-(Ethyl(2-(4-(6-hydroxy-2-(4-hydroxyphenyl)-

benzo[b]thiophene-3-carbonyl)phenoxy)ethyl)amino)-

octanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-

methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (15,

ERD-148). Triﬂuoroacetic anhydride (3.80 mL, 27.34 mmol, 2.0

equiv) was added at 0 °C to a solution of commercial 8-

bromooctanoic acid 64 (3.05 g, 13.67 mmol, 1.0 equiv) in 50 mL

of DCM. The solution was stirred at rt for 2 h. Then tert-butanol

(3.92 mL, 41.01 mmol, 3.0 equiv) was added. The solution was stirred

at rt for 12 h. Saturated aqueous NaHCO₃was added. The organic

layer was separated and dried over anhydrous Na₂SO₄. After ﬁltration

and concentration, the residue was puriﬁed by silica gel ﬂash column

chromatography with hexane:EtOAc (20:1−5:1) to aﬀord tert-butyl

DIPEA (0.09 mL, 0.5 mmol, 5.0 equiv) was added to a solution of

compound 53 (44 mg, 0.1 mmol, 1.0 equiv) and 60a (40 mg, 0.15

mmol, 1.5 equiv) in DMF (3.0 mL). The solution was stirred at 100

°C for 12 h. After cooling to rt, the residue was puriﬁed by reversed-

phase preparative HPLC to aﬀord the title compound (61a) as a

white solid (30 mg, 50% yield). UPLC-MS (ESI⁺) calcd for

C₃₄H₃₈NO₇S [M + 1]⁺: 604.24, found 604.30.

3-(4-Iodo-1-oxoisoindolin-2-yl)piperidine-2,6-dione (19 mg, 0.05

mmol, 1.0 equiv) was added to a solution of compound 61a (30 mg,

0.05 mmol, 1.0 equiv) in DMF (2.0 mL). The solution was purged

and reﬁlled with nitrogen three times with sonication then

Pd(PPh₃)₂Cl₂(3.5 mg, 0.005 mmol, 0.1 equiv), CuI (2.0 mg, 0.01

mmol, 0.2 equiv), and trimethylamine (2.0 mL) were added

sequentially. The solution was purged and reﬁlled with N₂again.

The solution was stirred at 80 °C for 1 h and was then cooled to rt.

EtOAc and H₂O were added, and the aqueous layer was extracted

with EtOAc twice. The combined organic layer was dried over

anhydrous Na₂SO₄. After ﬁltration and concentration, the residue was

puriﬁed by reversed-phase preparative HPLC to aﬀord the title

compound (12) as a yellow solid (18 mg, 43% yield). ¹H NMR

(CD₃OD, 400 MHz) δ (ppm) 7.76−7.71 (m, 3H), 7.58 (d, J = 7.6

Hz, 1H), 7.46−7.40 (m, 2H), 7.25 (d, J = 2.0 Hz, 1H), 7.16 (d, J =

8.8 Hz, 2H), 6.89 (d, J = 8.8 Hz, 2H), 6.85 (dd, J = 8.8 Hz, J = 2.0 Hz,

1H), 6.60 (d, J = 8.8 Hz, 2H), 5.12 (dd, J = 13.2 Hz, J = 5.2 Hz, 1H),

4.42−4.34 (m, 6H), 3.81−3.78 (m, 2H), 3.70−3.59 (m, 10H), 3.43

(t, J = 4.8 Hz, 2H), 3.35 (q, J = 7.6 Hz, 2H), 2.90−2.81 (m, 1H),

2.74−2.68 (m, 1H), 2.37−2.30 (m, 1H), 2.11−2.05 (m, 1H), 1.32 (t,

J = 7.6 Hz, 3H). ¹³C NMR (CD₃OD, 100 MHz) δ (ppm) 195.30,

174.53, 172.14, 170.68, 163.12, 159.25, 156.85, 145.48, 144.12,

141.47, 135.99, 134.20, 133.51, 133.10, 132.62, 131.44, 130.98,

129.77, 126.01, 124.75, 124.71, 119.34, 116.50, 116.12, 115.49,

107.94, 91.94, 82.61, 71.34, 71.24, 70.43, 65.49, 63.61, 59.69, 54.01,

53.58, 53.13, 51.37, 32.30, 24.02, 9.07. UPLC-MS (ESI⁺) calcd for

C₄₇H₄₈N₃O₁₀S [M + 1]⁺: 846.31, found 846.52. Purity 99.1%

(HPLC).

3-(4-(8-(Ethyl(2-(4-(6-hydroxy-2-(4-hydroxyphenyl)benzo[b]-

thiophene-3-carbonyl)-phenoxy)ethyl)amino)oct-1-yn-1-yl)-1-ox-

oisoindolin-2-yl)piperidine-2,6-dione (13). This compound was

prepared using a procedure similar to that used for compound 12.

¹H NMR (CD₃OD, 400 MHz) δ (ppm) 7.73−7.71 (m, 3H), 7.56 (d,

J = 7.2 Hz, 1H), 7.47−7.42 (m, 2H), 7.26 (d, J = 2.0 Hz, 1H), 7.16

(d, J = 8.4 Hz, 2H), 6.91−6.86 (m, 3H), 6.60 (d, J = 8.8 Hz, 2H),

5.16 (dd, J = 13.6 Hz, J = 5.2 Hz, 1H), 4.50 (d, J = 17.2 Hz, 1H), 4.44

(d, J = 17.2 Hz, 1H), 4.35 (t, J = 4.4 Hz, 2H), 3.58 (t, J = 4.4 Hz, 2H),

3.32−3.30 (m, 2H), 3.21 (q, J = 7.6 Hz, 2H), 2.93−2.84 (m, 1H),

2.77−2.73 (m, 1H), 2.50−2.44 (m, 3H), 2.17−2.12 (m, 1H), 1.79−

1.72 (m, 2H), 1.68−1.61 (m, 2H), 1.58−1.51 (m, 2H), 1.47−1.42

1

8-bromooctanoate (65) as a colorless oil (2.48 g, 65% yield). H

NMR (CDCl₃, 400 MHz) δ (ppm) 3.34 (t, J = 6.8 Hz, 2H), 2.14 (t, J

= 7.6 Hz, 2H), 1.83−1.75 (m, 2H), 1.54−1.49 (m, 2H), 1.40−1.36

(m, 11H), 1.29−1.25 (m, 4H). ¹³C NMR (CDCl₃, 100 MHz) δ

(ppm) 173.08, 79.88, 35.47, 33.81, 32.73, 28.86, 28.44, 28.12, 27.99,

24.95;

Compound 15 (ERD-148) was prepared using a procedure similar

to that used for compound 32 with intermediate 65 instead of

compound 63 as the starting material. ¹H NMR (CD₃OD, 400 MHz)

δ (ppm) 9.09 (s, 1H), 7.75 (d, J = 8.8 Hz, 2H), 7.47−7.41 (m, 5H),

7.27 (d, J = 2.4 Hz, 1H), 7.19−7.15 (m, 2H), 6.92 (d, J = 8.8 Hz,

2H), 6.87 (dd, J = 8.8 Hz, J = 2.4 Hz, 1H), 6.63−6.59 (m, 2H), 5.02−

4.90 (m, 1H), 4.64−4.54 (m, 2H), 4.43−4.41 (m, 1H), 4.35 (t, J =

4.4 Hz, 2H), 3.88 (d, J = 11.2 Hz, 1H), 3.74 (dd, J = 11.2 Hz, J = 4.0

Hz, 1H), 3.60 (t, J = 4.8 Hz, 2H), 3.31−3.17 (m, 4H), 2.50 (s, 3H),

2.32−2.17 (m, 3H), 1.98−1.91 (m, 1H), 1.75−1.65 (m, 2H), 1.65−

1.55 (m, 2H), 1.50 (d, J = 6.8 Hz, 3H), 1.43−1.29 (m, 9H), 1.03 (s,

9H). ¹³C NMR (CD₃OD, 100 MHz) δ (ppm) 193.96, 174.53, 171.84,

170.93, 161.69, 157.88, 155.47, 152.13, 144.63, 142.99, 140.09,

132.82, 132.14, 131.39, 130.07, 129.62, 129.12, 126.33, 124.64,

123.31, 115.08, 114.74, 114.01, 106.51, 69.57, 62.17, 59.21, 57.61,

56.61, 53.15, 51.19, 48.85, 48.76, 37.41, 35.10, 28.54, 28.41, 25.93,

25.65, 25.33, 23.31, 20.96, 13.89, 7.67. UPLC-MS (ESI⁺) calcd for

C₅₆H₆₈N₅O₈S₂[M + 1]⁺: 1002.45, found 1002.51. Purity 97.5%

(HPLC).

(2S,4R)-1-((S)-2-(4-(Ethyl(2-(4-(6-hydroxy-2-(4-hydroxyphenyl)-

benzo[b]thiophene-3-carbonyl)phenoxy)ethyl)amino)-

butanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-

methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (16).

This compound was prepared using a procedure similar to that

used for compound 15. ¹H NMR (CD₃OD, 400 MHz) δ (ppm) 8.87

(s, 1H), 7.73 (d, J = 8.8 Hz, 2H), 7.45−7.39 (m, 5H), 7.26 (d, J = 2.0

Hz, 1H), 7.18 (d, J = 8.8 Hz, 1H), 6.90 (d, J = 8.8 Hz, 2H), 6.86 (dd,

J = 8.8 Hz, J = 2.4 Hz, 1H), 6.62 (d, J = 8.8 Hz, 2H), 5.00−4.95 (m,

1H), 4.56−4.50 (m, 2H), 4.38−4.36 (m, 1H), 4.26 (t, J = 4.8 Hz,

N

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2H), 3.83 (d, J = 11.2 Hz, 1H), 3.66 (dd, J = 10.8 Hz, J = 4.0 Hz,

1H), 3.06−2.99 (m, 4H), 2.47 (s, 3H), 2.43 (t, J = 6.4 Hz, 2H),

2.20−2.15 (m, 1H), 1.97−1.89 (m, 3H), 1.49 (d, J = 6.8 Hz, 3H),

1.23 (t, J = 7.2 Hz, 3H), 1.00 (s, 9H). UPLC-MS (ESI⁺) calcd for

C₅₂H₆₀N₅O₈S₂[M + 1]⁺: 946.39, found 946.41. Purity 97.4%

(HPLC);

decanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-

methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (21).

This compound was prepared using a procedure similar to that

used for compound 15. ¹H NMR (CD₃OD, 400 MHz) δ (ppm) 8.86

(s, 1H), 7.72 (d, J = 8.8 Hz, 2H), 7.43−7.36 (m, 5H), 7.26 (d, J = 2.0

Hz, 1H), 7.17 (d, J = 8.8 Hz, 2H), 6.89−6.84 (m, 3H), 6.61 (d, J =

8.8 Hz, 2H), 5.02−4.97 (m, 1H), 4.64−4.55 (m, 2H), 4.43−4.41 (m,

1H), 4.27 (t, J = 4.8 Hz, 2H), 3.88 (d, J = 11.2 Hz, 1H), 3.74 (dd, J =

11.2 Hz, J = 4.0 Hz, 1H), 3.39 (t, J = 4.4 Hz, 2H), 3.15−3.09 (m,

2H), 3.03−2.98 (m, 2H), 2.46 (s, 3H), 2.33−2.17 (m, 3H), 1.98−

1.92 (m, 1H), 1.69−1.50 (m, 4H), 1.49 (d, J = 7.2 Hz, 3H), 1.33−

1.23 (m, 13H), 1.03 (s, 9H). UPLC-MS (ESI⁺) calcd for

C₅₈H₇₂N₅O₈S₂[M + 1]⁺: 1030.48, found 1030.46. Purity 96.4%

(HPLC).

(2S,4R)-4-Hydroxy-1-((S)-2-(8-((2-(4-(6-hydroxy-2-(4-

hydroxyphenyl)benzo[b]thiophene-3-carbonyl)phenoxy)ethyl)-

(methyl)amino)octanamido)-3,3-dimethylbutanoyl)-N-((S)-1-(4-(4-

methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (22).

This compound was prepared using a procedure similar to that

used for compound 15. ¹H NMR (CD₃OD, 400 MHz) δ (ppm) 8.95

(s, 1H), 7.74 (d, J = 9.2 Hz, 2H), 7.45−7.42 (m, 5H), 7.27 (d, J = 2.0

Hz, 1H), 7.17 (d, J = 8.4 Hz, 2H), 6.92 (d, J = 8.8 Hz, 2H), 6.87 (dd,

J = 8.8 Hz, J = 2.4 Hz, 1H), 6.61 (d, J = 8.8 Hz, 2H), 5.02−4.97 (m,

1H), 4.64−4.54 (m, 2H), 4.42−4.37 (m, 3H), 3.87 (d, J = 11.2 Hz,

1H), 3.74 (dd, J = 11.2 Hz, J = 4.4 Hz, 1H), 3.66−3.48 (m, 2H),

3.23−3.13 (m, 2H), 2.93 (s, 3H), 2.48 (s, 3H), 2.34−2.17 (m, 3H),

1.98−1.91 (m, 1H), 1.80−1.70 (m, 2H), 1.63−1.55 (m, 2H), 1.50 (d,

J = 7.2 Hz, 3H), 1.45−1.35 (m, 6H), 1.03 (s, 9H). UPLC-MS (ESI⁺)

calcd for C₅₅H₆₆N₅O₈S₂[M+1]⁺: 988.44, found 988.54. Purity 95.0%

(HPLC).

(2S,4R)-4-Hydroxy-1-((S)-2-(8-((2-(4-(6-hydroxy-2-(4-

hydroxyphenyl)benzo[b]thiophene-3-carbonyl)phenoxy)ethyl)-

(isopropyl)amino)octanamido)-3,3-dimethylbutanoyl)-N-((S)-1-(4-

(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

(23). This compound was prepared using a procedure similar to that

used for compound 15. ¹H NMR (CD₃OD, 400 MHz) δ (ppm) 8.86

(s, 1H), 7.72 (d, J = 8.8 Hz, 2H), 7.43−7.38 (m, 5H), 7.26 (d, J = 2.0

Hz, 1H), 7.17 (d, J = 8.8 Hz, 2H), 6.88−6.84 (m, 3H), 6.62 (d, J =

8.8 Hz, 2H), 5.02−4.97 (m, 1H), 4.63−4.55 (m, 2H), 4.43−4.41 (m,

1H), 4.16 (t, J = 5.2 Hz, 2H), 3.87 (d, J = 11.2 Hz, 1H), 3.74 (dd, J =

10.8 Hz, J = 4.0 Hz, 1H), 3.37−3.35 (m, 1H), 2.86−2.82 (m, 2H),

2.46 (s, 3H), 2.31−2.16 (m, 3H), 1.98−1.92 (m, 1H), 1.59−1.56 (m,

4H), 1.49 (d, J = 6.8 Hz, 3H), 1.33−1.27 (m, 10H), 1.18 (d, J = 6.8

Hz, 6H), 1.03 (s, 9H). UPLC-MS (ESI⁺) calcd for C₅₇H₇₀N₅O₈S₂[M

+ 1]⁺: 1016.47, found 1016.55. Purity 96.0% (HPLC).

(2S,4R)-1-((S)-2-(8-(tert-Butyl(2-(4-(6-hydroxy-2-(4-

hydroxyphenyl)benzo[b]thiophene-3-carbonyl)phenoxy)ethyl)-

amino)octanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-

(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

(24). This compound was prepared using a procedure similar to that

used for compound 15. ¹H NMR (CD₃OD, 400 MHz) δ (ppm) 8.97

(s, 1H), 7.76 (d, J = 8.8 Hz, 2H), 7.47−7.40 (m, 5H), 7.26 (d, J = 2.4

Hz, 1H), 7.18 (d, J = 8.8 Hz, 2H), 6.92−6.86 (m, 3H), 6.62−6.60 (m,

2H), 5.03−4.97 (m, 1H), 4.62 (s, 1H), 4.59−4.55 (m, 1H), 4.45−

4.41 (m, 1H), 4.32 (t, J = 4.4 Hz, 2H), 3.91−3.83 (m, 2H), 3.74 (dd,

J = 11.2 Hz, J = 4.0 Hz, 1H), 3.49−3.38 (m, 2H), 3.14−3.10 (m, 1H),

2.48 (s, 3H), 2.33−2.17 (m, 3H), 1.99−1.92 (m, 1H), 1.83−1.73 (m,

1H), 1.70−1.55 (m, 4H), 1.51−1.47 (m, 3H), 1.47−1.45 (m, 9H),

1.39−1.29 (m, 6H), 1.03 (s, 9H). ¹³C NMR (CD₃OD, 100 MHz) δ

(ppm) 195.34, 175.92, 173.23, 172.33, 163.09, 159.30, 156.89,

153.17, 148.54, 145.80, 144.45, 141.51, 134.22, 133.59, 132.77,

131.50, 131.25, 130.98, 130.51, 127.67, 127.45, 126.04, 124.72,

116.47, 116.15, 115.30, 107.91, 70.97, 67.43, 65.32, 60.63, 59.02,

58.00, 53.38, 51.70, 50.16, 49.71, 38.82, 36.50, 29.99, 29.86, 27.63,

27.53, 27.06, 26.76, 24.94, 22.37, 15.57. UPLC-MS (ESI⁺) calcd for

C₅₈H₇₂N₅O₈S₂[M + 1]⁺: 1030.48, found 1030.52.

(2S,4R)-1-((S)-2-(5-(Ethyl(2-(4-(6-hydroxy-2-(4-hydroxyphenyl)-

benzo[b]thiophene-3 carbonyl)phenoxy)ethyl)amino)-

pentanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-

methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (17).

This compound was prepared using a procedure similar to that

used for compound 15. ¹H NMR (CD₃OD, 400 MHz) δ (ppm) 8.87

(s, 1H), 7.76−7.73 (m, 1H), 7.45−7.38 (m, 5H), 7.27 (d, J = 2.0 Hz,

1H), 7.20−7.14 (m, 2H), 6.94−6.92 (m, 2H), 6.89−6.85 (m, 1H),

6.62−6.59 (m, 2H), 5.01−4.97 (m, 1H), 4.59−4.52 (m, 2H), 4.43−

4.41 (m, 1H), 4.36 (t, J = 4.8 Hz, 2H), 3.86 (d, J = 11.2 Hz, 1H), 3.72

(dd, J = 10.8 Hz, J = 4.0 Hz, 1H), 3.59 (t, J = 4.8 Hz, 2H), 3.31−3.21

(m, 4H), 2.47 (s, 3H), 2.37 (t, J = 6.8 Hz, 2H), 2.21−2.16 (m, 1H),

1.98−1.91 (m, 1H), 1.79−1.68 (m, 4H), 1.48 (d, J = 7.2 Hz, 3H),

1.34 (t, J = 7.2 Hz, 3H), 1.04 (s, 9H). UPLC-MS (ESI⁺) calcd for

C₅₃H₆₂N₅O₈S₂[M + 1]⁺: 960.40, found 960.84. Purity 96.9%

(HPLC).

(2S,4R)-1-((S)-2-(6-(Ethyl(2-(4-(6-hydroxy-2-(4-hydroxyphenyl)-

benzo[b]thiophene-3-carbonyl)phenoxy)ethyl)amino)-

hexanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-

methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (18).

This compound was prepared using a procedure similar to that

used for compound 15. ¹H NMR (CD₃OD, 400 MHz) δ (ppm) 8.87

(s, 1H), 7.73 (d, J = 9.2 Hz, 2H), 7.44−7.38 (m, 5H), 7.26 (d, J = 2.4

Hz, 1H), 7.16 (d, J = 8.8 Hz, 2H), 6.90 (d, J = 9.2 Hz, 2H), 6.87 (dd,

J = 8.8 Hz, J = 2.0 Hz, 1H), 6.61 (d, J = 8.8 Hz, 2H), 5.03−4.96 (m,

1H), 4.61−4.54 (m, 2H), 4.41−4.39 (m, 1H), 4.35 (t, J = 4.8 Hz,

2H), 3.87−3.84 (m, 1H), 3.73−3.71 (m, 1H), 3.58 (t, J = 4.4 Hz,

2H), 3.31−3.17 (m, 4H),2.47 (s, 3H), 2.37−2.26 (m, 2H), 2.21−2.16

(m, 1H), 1.97−1.91 (m, 1H), 1.77−1.62 (m, 4H), 1.49 (d, J = 7.2 Hz,

3H), 1.45−1.30 (m, 5H), 1.02 (s, 9H). UPLC-MS (ESI⁺) calcd for

C₅₄H₆₄N₅O₈S₂[M + 1]⁺: 974.42, found 974.63. Purity 99.6%

(HPLC).

(2S,4R)-1-((S)-2-(7-(Ethyl(2-(4-(6-hydroxy-2-(4-hydroxyphenyl)-

benzo[b]thiophene-3-carbonyl)phenoxy)ethyl)amino)-

heptanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-

methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (19).

This compound was prepared using a procedure similar to that

used for compound 15. ¹H NMR (CD₃OD, 400 MHz) δ (ppm) 8.86

(s, 1H), 7.71 (d, J = 8.8 Hz, 2H), 7.44−7.38 (m, 5H), 7.26 (d, J = 2.0

Hz, 1H), 7.17 (d, J = 8.8 Hz, 2H), 6.89−6.80 (m, 3H), 6.62 (d, J =

8.8 Hz, 2H), 5.02−4.97 (m, 1H), 4.63−4.55 (m, 2H), 4.43−4.41 (m,

1H), 4.24 (t, J = 4.8 Hz, 2H), 3.87 (d, J = 10.8 Hz, 1H), 3.74 (dd, J =

10.8 Hz, J = 4.0 Hz, 1H), 3.32−3.30 (m, 2H), 3.04−2.90 (m, 4H),

2.47 (s, 3H), 2.34−2.16 (m, 3H), 1.98−1.91 (m, 1H), 1.63−1.60 (m,

4H), 1.49 (d, J = 7.2 Hz, 3H), 1.37−1.35 (m, 4H), 1.25−1.18 (m,

3H), 1.01 (s, 9H). UPLC-MS (ESI⁺) calcd for C₅₅H₆₆N₅O₈S₂[M +

1]⁺: 988.44, found 988.60. Purity 96.2% (HPLC).

(2S,4R)-1-((S)-2-(9-(Ethyl(2-(4-(6-hydroxy-2-(4-hydroxyphenyl)-

benzo[b]thiophene-3-carbonyl)phenoxy)ethyl)amino)-

nonanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-

methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (20).

This compound was prepared using a procedure similar to that for

compound 15. ¹H NMR (CD₃OD, 400 MHz) δ (ppm) 8.87 (s, 1H),

7.75−7.70 (m, 2H), 7.44−7.39 (m, 5H), 7.26 (d, J = 2.4 Hz, 1H),

7.18−7.15 (m, 2H), 6.90 (d, J = 8.8 Hz, 2H), 6.87 (dd, J = 8.8 Hz, J =

2.4 Hz, 1H), 6.61 (d, J = 8.8 Hz, 2H), 5.04−4.88 (m, 1H), 4.64−4.55

(m, 2H), 4.45−4.40 (m, 1H), 4.35 (t, J = 4.8 Hz, 2H), 3.89−3.86 (m,

1H), 3.74 (dd, J = 10.8 Hz, J = 4.0 Hz, 1H), 3.58 (t, J = 4.8 Hz, 2H),

3.31−3.28 (m, 2H), 3.21−3.15 (m, 2H), 2.47 (s, 3H), 2.31−2.17 (m,

3H), 1.98−1.92 (m, 1H), 1.75−1.55 (m, 4H), 1.50 (d, J = 7.2 Hz,

3H), 1.36−1.31 (m, 11H), 1.03 (s, 9H). UPLC-MS (ESI⁺) calcd for

C₅₇H₇₀N₅O₈S₂[M + 1]⁺: 1016.47, found 1016.53. Purity 95.7%

(HPLC).

(2S,4R)-1-((S)-2-(8-(Cyclopropyl(2-(4-(6-hydroxy-2-(4-

hydroxyphenyl)benzo[b]thiophene-3-carbonyl)phenoxy)ethyl)-

amino)octanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-

(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

(25). This compound was prepared using a procedure similar to that

(2S,4R)-1-((S)-2-(10-(Ethyl(2-(4-(6-hydroxy-2-(4-hydroxyphenyl)-

benzo[b]thiophene-3-carbonyl)phenoxy)ethyl)amino)-

O

DOI: 10.1021/acs.jmedchem.8b01572

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

used for compound 15. ¹H NMR (CD₃OD, 400 MHz) δ (ppm) 9.04

(s, 1H), 7.73 (d, J = 9.2 Hz, 2H), 7.46−7.41 (m, 5H), 7.26 (d, J = 2.0

Hz, 1H), 7.16 (d, J = 8.8 Hz, 2H), 6.91−6.86 (m, 3H), 6.61 (d, J =

8.8 Hz, 2H), 5.03−4.97 (m, 1H), 4.64−4.61 (m, 1H), 4.57 (t, J = 8.4

Hz, 1H), 4.42−4.29 (m, 3H), 3.87 (d, J = 11.2 Hz, 1H), 3.76−3.65

(m, 3H), 3.32−3.30 (m, 1H), 2.89−2.84 (m, 1H), 2.49 (s, 3H),

2.33−2.17 (m, 3H), 1.99−1.92 (m, 1H), 1.85−1.75 (m, 2H), 1.63−

1.55 (m, 2H), 1.50 (d, J = 7.2 Hz, 3H), 1.45−1.35 (m, 6H), 1.03 (s,

9H), 1.00−0.90 (m, 4H). ¹³C NMR (CD₃OD, 100 MHz) δ (ppm)

195.30, 175.92, 173.23, 172.33, 162.98, 159.26, 156.84, 153.43,

145.96, 144.57, 141.46, 134.21, 133.53, 132.77, 131.50, 131.02,

130.49, 127.70, 126.01, 124.75, 116.45, 116.14, 115.38, 107.92, 70.95,

63.22, 60.61, 59.01, 57.98, 57.85, 55.33, 50.14, 38.78, 38.54, 36.47,

29.94, 29.81, 27.42, 27.04, 26.72, 24.72, 22.34, 15.31. UPLC-MS

(ESI⁺) calcd for C₅₇H₆₈N₅O₈S₂[M + 1]⁺: 1014.45, found 1014.61.

Purity 96.1% (HPLC).

(2S,4R)-1-((S)-2-(8-(Cyclobutyl(2-(4-(6-hydroxy-2-(4-

hydroxyphenyl)benzo[b]thiophene-3-carbonyl)phenoxy)ethyl)-

amino)octanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-

(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

(26). This compound was prepared using a procedure similar to that

used for compound 15. ¹H NMR (CD₃OD, 400 MHz) δ (ppm) 9.04

(s, 1H), 7.74 (d, J = 8.8 Hz, 2H), 7.44−7.42 (m, 5H), 7.26 (d, J = 2.4

Hz, 1H), 7.17 (d, J = 8.8 Hz, 2H), 6.91−6.85 (m, 3H), 6.62 (d, J =

8.8 Hz, 2H), 5.03−4.97 (m, 1H), 4.64−4.55 (m, 2H), 4.45−4.41 (m,

1H), 4.32−4.29 (m, 2H), 3.91−3.86 (m, 2H), 3.74 (dd, J = 11.2 Hz, J

= 4.0 Hz, 1H), 3.49 (t, J = 4.8 Hz, 2H), 3.09 (t, J = 8.8 Hz, 2H), 2.48

(s, 3H), 2.35−2.15 (m, 7H), 1.99−1.92 (m, 1H), 1.87−1.55 (m, 6H),

1.50 (d, J = 7.2 Hz, 3H), 1.40−1.30 (m, 6H), 1.03 (s, 9H). ¹³C NMR

(CD₃OD, 100 MHz) δ (ppm) 195.32, 175.91, 173.22, 172.33, 163.01,

159.27, 156.85, 153.42, 145.95, 144.39, 141.46, 134.22, 133.54,

132.73, 131.45, 131.01, 130.49, 127.70, 125.99, 124.73, 116.49,

116.15, 115.37, 107.94, 70.95, 63.46, 60.60, 59.61, 59.01, 57.98, 52.11,

50.49, 50.14, 38.78, 36.49, 36.47, 29.94, 29.79, 27.40, 27.05, 26.71,

24.30, 22.35, 15.33, 14.18. UPLC-MS (ESI⁺) calcd for C₅₈H₇₀N₅O₈S₂

[M + 1]⁺: 1028.47, found 1029.18. Purity 97.7% (HPLC).

(2S,4R)-1-((S)-2-(8-(Cyclopentyl(2-(4-(6-hydroxy-2-(4-

hydroxyphenyl)benzo[b]thiophene-3-carbonyl)phenoxy)ethyl)-

amino)octanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-

(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

(27). This compound was prepared using a procedure similar to that

used for compound 15. ¹H NMR (CD₃OD, 400 MHz) δ (ppm) 8.93

(s, 1H), 7.75 (d, J = 8.8 Hz, 2H), 7.45−7.41 (m, 5H), 7.26 (d, J = 2.4

Hz, 1H), 7.18 (d, J = 8.8 Hz, 2H), 6.92−6.85 (m, 3H), 6.61 (d, J =

8.8 Hz, 2H), 5.02−4.98 (m, 1H), 4.64−4.55 (m, 2H), 4.43−4.41 (m,

1H), 4.37−4.33 (m, 2H), 3.89−3.73 (m, 3H), 3.65−3.55 (m, 2H),

3.22 (t, J = 8.4 Hz, 2H), 2.48 (s, 3H), 2.30−2.15 (m, 5H), 2.03−1.94

(m, 1H), 1.84−1.57 (m, 10H), 1.50 (d, J = 7.2 Hz, 3H), 1.45−1.30

(m, 6H), 1.03 (s, 9H). ¹³C NMR (CD₃OD, 100 MHz) δ (ppm)

195.34, 175.89, 173.21, 172.34, 163.06, 159.29, 156.87, 153.06,

148.67, 145.73, 144.37, 141.49, 134.22, 133.55, 132.78, 131.46,

131.32, 131.01, 130.49, 127.65, 127.42, 126.03, 124.71, 116.48,

116.14, 115.36, 107.92, 70.96, 67.31, 63.94, 60.62, 59.00, 57.99, 54.01,

52.09, 38.80, 36.50, 29.97, 29.84, 29.20, 29.14, 27.39, 27.05, 26.72,

24.85, 24.81, 24.66, 22.34, 15.61. UPLC-MS (ESI⁺) calcd for

C₅₉H₇₂N₅O₈S₂[M + 1]⁺: 1042.48, found 1042.39. Purity >99.5%

(HPLC).

144.35, 141.49, 134.23, 133.56, 133.34, 131.53, 131.47, 131.03,

130.50, 127.62, 127.41, 126.04, 116.47, 116.13, 115.36, 107.91, 70.96,

60.62, 59.01, 58.00, 53.41, 51.06, 50.15, 49.28, 38.81, 37.63, 36.50,

30.00, 29.89, 27.58, 27.05, 26.20, 26.19, 26.18, 26.17, 26.15, 26.14,

22.36, 15.79. UPLC-MS (ESI⁺) calcd for C₆₀H₇₄N₅O₈S₂[M + 1]⁺:

1056.50, found 1056.54.

(2S,4R)-1-((S)-2-(8-((Cyclobutylmethyl)(2-(4-(6-hydroxy-2-(4-

hydroxyphenyl)benzo-[b]thiophene-3-carbonyl)phenoxy)ethyl)-

amino)octanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-

(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

(29). This compound was prepared using a procedure similar to that

used for compound 15. ¹H NMR (CD₃OD, 400 MHz) δ (ppm) 8.98

(s, 1H), 7.73 (d, J = 8.8 Hz, 2H), 7.44−7.36 (m, 5H), 7.26 (d, J = 2.0

Hz, 1H), 7.17 (d, J = 8.4 Hz, 2H), 6.90−6.85 (m, 3H), 6.61(d, J = 8.4

Hz, 2H), 5.02−4.97 (m, 1H), 4.64−4.55 (m, 2H), 4.45−4.41 (m,

1H), 4.33 (t, J = 4.4 Hz, 2H), 3.87 (d, J = 11.2 Hz, 1H), 3.74 (dd, J =

11.2 Hz, J = 4.0 Hz, 1H), 3.54−3.52 (m, 2H), 3.28−3.25 (m, 2H),

3.16−3.11 (m, 2H), 2.80−2.73 (m, 1H), 2.48 (s, 3H), 2.32−2.14 (m,

5H), 2.04−1.84 (m, 5H), 1.75−1.65 (m, 2H), 1.65−1.55 (m, 2H),

1.49 (d, J = 7.2 Hz, 3H), 1.42−1.30 (m, 6H), 1.03 (s, 9H). ¹³C NMR

(CD₃OD, 100 MHz) δ (ppm) 195.33, 175.89, 173.20, 172.32, 163.05,

159.27, 156.84, 153.25, 148.24, 145.84, 144.30, 141.45, 134.22,

133.52, 132.72, 131.43, 131.07, 131.01, 130.48, 127.67, 125.97,

124.72, 116.50, 116.14, 115.39, 107.95, 70.94, 63.50, 60.60, 60.11,

59.00, 57.98, 55.21, 51.06, 50.13, 38.78, 36.48, 31.78, 29.94, 29.81,

28.20, 28.09, 27.35, 27.05, 26.71, 24.52, 22.34, 19.42, 15.46. UPLC-

MS (ESI⁺) calcd for C₅₉H₇₂N₅O₈S₂[M + 1]⁺: 1042.48, found

1042.82. Purity >99.5% (HPLC).

(2S,4R)-1-((S)-14-(tert-Butyl)-3-ethyl-1-(4-(6-hydroxy-2-(4-

hydroxyphenyl)benzo[b]-thiophene-3-carbonyl)phenoxy)-12-oxo-

6,9-dioxa-3,13-diazapentadecan-15-oyl)-4-hydroxy-N-((S)-1-(4-(4-

methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (30).

This compound was prepared using a procedure similar to that

used for compound 32. ¹H NMR (CD₃OD, 400 MHz) δ (ppm) 8.86

(s, 1H), 7.74 (d, J = 8.8 Hz, 2H), 7.44−7.37 (m, 5H), 7.27 (d, J = 2.4

Hz, 1H), 7.18 (d, J = 8.8 Hz, 2H), 6.93 (d, J = 8.8 Hz, 2H), 6.87 (dd,

J = 8.8 Hz, J = 2.4 Hz, 1H), 6.62 (d, J = 8.4 Hz, 2H), 5.02−4.95 (m,

1H), 4.57−4.53 (m, 2H), 4.40−4.37 (m, 3H), 3.85−3.80 (m, 3H),

3.73−3.58 (m, 9H), 3.45 (t, J = 8.8 Hz, 2H), 3.37 (q, J = 7.2 Hz, 2H),

2.55−2.44 (m, 5H), 2.22−2.17 (m, 1H), 1.97−1.91 (m, 1H), 1.48 (d,

J = 7.2 Hz, 3H), 1.35 (t, J = 7.2 Hz, 3H), 1.01 (s, 9H). UPLC-MS

(ESI⁺) calcd for C₅₅H₆₆N₅O₁₀S₂[M + 1]⁺: 1020.43, found 1020.77.

Purity 97.2% (HPLC).

(2S,4R)-1-((S)-2-(tert-Butyl)-12-ethyl-14-(4-(6-hydroxy-2-(4-

hydroxyphenyl)benzo[b]-thiophene-3-carbonyl)phenoxy)-4-oxo-

6,9-dioxa-3,12-diazatetradecanoyl)-4-hydroxy-N-((S)-1-(4-(4-meth-

ylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (31). This

compound was prepared using a procedure similar to that used for

compound 32. ¹H NMR (CD₃OD, 400 MHz) δ (ppm) 9.11 (s, 1H),

7.73 (d, J = 8.4 Hz, 2H), 7.44−7.37 (m, 5H), 7.26 (d, J = 2.0 Hz,

1H), 7.17 (d, J = 8.8 Hz, 2H), 6.92−6.84 (m, 3H), 6.61 (d, J = 8.4

Hz, 2H), 4.97−4.91 (m, 1H), 4.71−4.68 (m, 1H), 4.57−4.54 (m,

1H), 4.41−4.38 (m, 3H), 4.02−3.40 (m, 16H), 2.48 (s, 3H), 2.36−

2.20 (m, 1H), 1.95−1.89 (m, 1H), 1.45 (d, J = 7.2 Hz, 2H), 1.37 (t, J

= 7.6 Hz, 3H), 1.01 (s, 9H). UPLC-MS (ESI⁺) calcd for

C₅₄H₆₄N₅O₁₀S₂[M + 1]⁺: 1006.41, found 1006.66. Purity 95.1%

(HPLC).

(2S,4R)-1-((S)-2-(2-((5-(Ethyl(2-(4-(6-hydroxy-2-(4-

hydroxyphenyl)benzo[b]thiophene-3-carbonyl)phenoxy)ethyl)-

amino)pentyl)oxy)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-

N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-car-

boxamide (32, ERD-308). Sodium hydroxide (4.0 g, 100.0 mmol,

10.0 equiv) and tetrabutyl ammonium chloride (2.78 g, 10.0 mmol,

1.0 equiv) were added sequentially to a solution of 5-(benzyloxy)-

pentan-1-ol (1.94 g, 10.0 mmol, 1.0 equiv) and tert-butyl 2-

bromoacetate (3.90 g, 20.0 mmol, 2.0 equiv) in H₂O (20 mL) and

DCM (20 mL). The solution was stirred vigorously at rt overnight

when TLC showed that the reaction was complete. The mixture was

partitioned between DCM (100 mL) and H₂O (100 mL), and the

organic layer was collected, washed with brine (100 mL), dried over

anhydrous sodium sulfate, and concentrated under reduced pressure

(2S,4R)-1-((S)-2-(8-(Cyclohexyl(2-(4-(6-hydroxy-2-(4-

hydroxyphenyl)benzo[b]thiophene-3-carbonyl)phenoxy)ethyl)-

amino)octanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-

(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

(28). This compound was prepared using a procedure similar to that

used for compound 15. ¹H NMR (CD₃OD, 400 MHz) δ (ppm) 8.87

(s, 1H), 7.75 (d, J = 8.8 Hz, 2H), 7.45−7.39 (m, 5H), 7.26 (d, J = 2.0

Hz, 1H), 7.18 (d, J = 8.4 Hz, 2H), 6.91−6.84 (m, 3H), 6.62−6.60 (m,

2H), 5.02−4.97 (m, 1H), 4.64−4.54 (m, 2H), 4.45−4.41 (m, 1H),

4.32 (t, J = 4.4 Hz, 2H), 3.89−3.85 (m, 1H), 3.74 (dd, J = 11.2 Hz, J

= 4.0 Hz, 1H), 3.59−3.45 (m, 2H), 3.18−3.08 (m, 2H), 2.47 (s, 3H),

2.34−2.16 (m, 3H), 2.03−1.90 (m, 5H), 1.74−1.31 (m, 20H), 1.03

(s, 9H). ¹³C NMR (CD₃OD, 100 MHz) δ (ppm) 195.37, 175.94,

173.22, 172.33, 163.29, 159.29, 156.87, 152.87, 149.07, 145.61,

P

DOI: 10.1021/acs.jmedchem.8b01572

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

to give a residue that was puriﬁed by silica gel ﬂash column

chromatography with hexane:EtOAc (10:1−5:1) to aﬀord tert-butyl

2-((5-(benzyloxy)pentyl)oxy)acetate as a colorless oil (987 mg, 32%

yield).

= 3.6 Hz, 1H), 3.56 (t, J = 6.8 Hz, 2H), 3.45 (t, J = 4.8 Hz, 2H),

3.11−3.07 (m, 2H), 2.47 (s, 3H), 2.22−2.19 (m, 1H), 1.98−1.92 (m,

1H), 1.76−1.66 (m, 4H), 1.57−1.46 (m, 5H), 1.02 (s, 9H). ¹³C NMR

(CD₃OD, 100 MHz) δ (ppm) 195.36, 173.13, 173.05, 171.91, 171.80,

163.11, 159.29, 156.87, 152.90, 149.01, 145.60, 144.31, 141.47,

134.22, 133.55, 132.72, 131.45, 131.02, 130.49, 127.61, 127.38,

126.00, 124.72, 116.50, 116.13, 115.45, 107.93, 72.40, 70.90, 70.73,

69.08, 63.61, 60.68, 58.14, 54.33, 52.59, 50.26, 38.90, 37.80, 37.13,

29.90, 26.93, 24.56, 24.40, 22.43, 15.79, 9.18. UPLC-MS (ESI⁺) calcd

for C₅₅H₆₆N₅O₉S₂[M + 1]⁺: 1004.43, found 1004.11. Purity 97.4%

(HPLC).

(2S,4R)-1-((S)-2-(4-(4-(2-(Ethyl(2-(4-(6-hydroxy-2-(4-

hydroxyphenyl)benzo[b]thiophene-3-carbonyl)phenoxy)ethyl)-

amino)ethyl)piperidin-1-yl)butanamido)-3,3-dimethylbutanoyl)-4-

hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-

2-carboxamide (33). This compound was prepared using a procedure

similar to that used for compound 32. ¹H NMR (CD₃OD, 400 MHz)

δ (ppm) 8.95 (s, 1H), 7.74 (d, J = 8.8 Hz, 2H), 7.45−7.40 (m, 5H),

7.27 (d, J = 2.4 Hz, 1H), 7.17 (d, J = 8.4 Hz, 2H), 6.93 (d, J = 9.2 Hz,

2H), 6.87 (dd, J = 8.8 Hz, J = 2.4 Hz, 1H), 6.60 (d, J = 8.4 Hz, 2H),

5.03−4.90 (m, 1H), 4.59−4.53 (m, 2H), 4.43−4.41 (m, 1H), 4.37 (t,

J = 4.8 Hz, 2H), 3.90 (d, J = 10.8 Hz, 1H), 3.74 (dd, J = 10.8 Hz, J =

4.0 Hz, 1H), 3.61−3.56 (m, 4H), 3.35−3.27 (m, 4H), 3.10 (t, J = 6.8

Hz, 2H), 2.94−2.86 (m, 2H), 2.51 (t, J = 6.4 Hz, 2H), 2.48 (s, 3H),

2.22−2.17 (m, 1H), 2.01−1.95 (m, 5H), 1.78−1.68 (m, 3H), 1.59−

1.57 (m, 2H), 1.50 (d, J = 7.2 Hz, 3H), 1.34 (t, J = 7.6 Hz, 3H), 1.06

(s, 9H). UPLC-MS (ESI⁺) calcd for C₅₉H₇₃N₆O₈S₂[M + 1]⁺:

1057.49, found 1057.90. Purity 99.1% (HPLC).

(2S,4R)-1-((S)-2-(4-(4-(2-(Ethyl(2-(4-(6-hydroxy-2-(4-

hydroxyphenyl)benzo[b]thiophene-3-carbonyl)phenoxy)ethyl)-

amino)ethyl)piperazin-1-yl)butanamido)-3,3-dimethylbutanoyl)-4-

hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-

2-carboxamide (34). This compound was prepared using a procedure

similar to that used for compound 32. ¹H NMR (CD₃OD, 400 MHz)

δ (ppm) 8.94 (s, 1H), 7.75 (d, J = 8.8 Hz, 2H), 7.45−7.40 (m, 5H),

7.27 (d, J = 2.4 Hz, 1H), 7.18 (d, J = 8.4 Hz, 2H), 6.93 (d, J = 9.2 Hz,

2H), 6.87 (dd, J = 8.8 Hz, J = 2.4 Hz, 1H), 6.61 (d, J = 8.4 Hz, 2H),

5.03−4.99 (m, 1H), 4.58−4.54 (m, 2H), 4.44−4.39 (m, 3H), 3.90 (d,

J = 10.8 Hz, 1H), 3.76−3.70 (m, 3H), 3.51−3.48 (m, 2H), 3.42−3.37

(m, 2H), 3.14−3.12 (m, 2H), 2.85 (t, J = 6.4 Hz, 2H), 2.53 (t, J = 6.4

Hz, 2H), 2.48 (s, 3H), 2.23−2.18 (m, 1H), 2.01−1.92 (m, 3H), 1.50

(d, J = 6.8 Hz, 3H), 1.36 (t, J = 7.2 Hz, 3H), 1.06 (s, 9H). UPLC-MS

(ESI⁺) calcd for C₅₈H₇₂N₇O₈S₂[M + 1]⁺: 1058.49, found 1058.72.

Purity 99.3% (HPLC).

A mixture of tert-butyl 2-((5-(benzyloxy)pentyl)oxy)acetate (770

mg, 2.5 mmol, 1.0 equiv) and 10 wt % palladium on carbon (100 mg)

in MeOH (20 mL) was stirred at rt overnight under a hydrogen

atmosphere. TLC showed that the reaction was complete and the

solution was ﬁltered through Celite and washed with MeOH. The

combined ﬁltrate was concentrated under reduced pressure. The

residue was puriﬁed by silica gel ﬂash column chromatography with

hexane:EtOAc (2:1−1:1) to aﬀord tert-butyl 2-((5-hydroxypentyl)-

1

oxy)acetate (62) as a colorless oil (671 mg, 95% yield). H NMR

(CDCl₃, 400 MHz) δ (ppm) 3.58 (s, 2H), 3.21 (t, J = 6.8 Hz, 2H),

3.15 (t, J = 6.8 Hz, 2H), 1.31−1.14 (m, 4H), 1.12−1.05 (m, 12H).

¹³C NMR (CDCl₃, 100 MHz) δ (ppm) 169.51, 80.94, 71.18, 68.20,

61.54, 31.96, 28.97, 27.67, 21.95.

4-Toluenesulfonyl chloride (879 mg, 4.6 mmol, 1.5 equiv) and

Et₃N (0.86 mL, 6.14 mmol, 2.0 equiv) were added sequentially to a

solution of tert-butyl 2-((5-hydroxypentyl)oxy)acetate (62) (671 mg,

3.07 mmol, 1.0 equiv) in DCM (10 mL) at 0 °C. The mixture was

warmed to rt and stirred for 1 h. After concentration, the residue was

puriﬁed by silica gel ﬂash column chromatography with hexane:E-

tOAc (5:1−2:1) to aﬀord the intermediate tert-butyl 2-((5-(tosyloxy)-

1

pentyl)oxy)acetate (63) as a colorless oil (1.02 g, 89% yield). H

NMR (CDCl₃, 400 MHz) δ (ppm) 7.75 (d, J = 8.0 Hz, 2H), 7.32 (d,

J = 8.0 Hz, 2H), 3.99 (t, J = 6.4 Hz, 2H), 3.88 (s, 2H), 3.43 (t, J = 6.4

Hz, 2H), 2.42 (s, 3H), 1.68−1.61 (m, 2H), 1.57−1.50 (m, 2H), 1.44

(s, 9H), 1.42−1.36 (m, 2H). ¹³C NMR (CDCl₃, 100 MHz) δ (ppm)

169.76, 144.75, 133.16, 129.89, 127.92, 81.54, 71.19, 70.55, 68.76,

28.99, 28.65, 28.15, 22.07, 21.67. UPLC-MS (ESI⁺) calcd for

C₁₈H₂₈NaO₆S [M + 23]⁺: 395.15, found 395.36.

DIPEA (0.18 mL, 1.0 mmol, 5.0 equiv) was added to a solution of

intermediate 53 (87 mg, 0.2 mmol, 1.0 equiv) and tert-butyl 2-((5-

(tosyloxy)pentyl)oxy)acetate 63 (223 mg, 0.6 mmol, 3.0 equiv) in

DMF (3.0 mL). The solution was stirred at 80 °C for 12 h. After

cooling to temperature, the solution was diluted with EtOAc and

H₂O. The organic layer was separated and dried over anhydrous

Na₂SO₄. After ﬁltration and concentration, the residue was puriﬁed by

silica gel ﬂash column chromatography with DCM:MeOH (10:1) to

aﬀord the intermediate (66) as colorless oil (114 mg, 90% yield). ¹H

NMR (CDCl₃, 400 MHz) δ (ppm) 7.65 (d, J = 8.8 Hz, 2H), 7.42 (d,

J = 7.2 Hz, 1H), 7.19 (s, 1H), 7.08 (d, J = 8.0 Hz, 2H), 6.81 (d, J =

9.2 Hz, 1H), 6.59−6.54 (m, 4H), 3.99−3.95 (m, 2H), 3.92 (s, 2H),

3.48−3.40 (m, 4H), 2.86−2.82 (m, 2H), 2.64 (q, J = 6.8 Hz, 2H),

2.55−2.51 (m, 2H), 1.59−1.52 (m, 2H), 1.44 (s, 9H), 1.31−1.25 (m,

2H), 1.01 (t, J = 6.8 Hz, 3H). ¹³C NMR (CDCl₃, 100 MHz) δ (ppm)

194.16, 170.28, 162.73, 157.39, 154.83, 143.60, 140.09, 133.37,

132.59, 130.47, 129.90, 125.01, 124.06, 116.02, 115.57, 114.20,

107.67, 82.05, 71.67, 68.75, 53.54, 53.32, 51.72, 47.86, 29.78, 29.38,

28.18, 24.01, 10.64. UPLC-MS (ESI⁺) calcd for C₃₆H₄₄NO₇S [M +

23]⁺: 634.28, found 634.18.

Triﬂuoroacetic acid (5.0 mL) was added to a solution of

intermediate 66 (114 mg, 0.18 mmol) in DCM (10 mL) at 0 °C.

The solution was stirred at rt for 6 h. After concentration, the residue

was puriﬁed by reversed-phase preparative HPLC to aﬀord the title

compound (67) as a slightly yellow solid (81 mg, 78% yield). UPLC-

MS (ESI⁺) calcd for C₃₂H₃₆NO₇S [M + 23]⁺: 578.22, found 578.06.

HATU (53 mg, 0.14 mmol, 1.0 equiv) was added to a solution of

intermediate 67 (81 mg, 0.14 mmol, 1.0 equiv), intermediate 58 (67

mg, 0.15 mmol, 1.1 equiv), and DIPEA (0.12 mL, 0.70 mmol, 5.0

equiv) in DMF (2 mL). The mixture was stirred at ambient

temperature for 1 h then was puriﬁed by reversed-phase preparative

HPLC to aﬀord the title compound 32 (ERD-308) as a yellow solid

(56 mg, 40% yield). ¹H NMR (CD₃OD, 400 MHz) δ (ppm) 8.86 (s,

1H), 7.74 (d, J = 9.2 Hz, 2H), 7.43−7.35 (m, 5H), 7.26 (d, J = 2.0

Hz, 1H), 7.18 (d, J = 8.4 Hz, 2H), 6.91−6.85 (m, 3H), 6.62 (d, J =

8.4 Hz, 2H), 4.98−4.95 (m, 1H), 4.89 (s, 2H), 4.69−4.64 (m, 1H),

4.59−4.53 (m, 1H), 4.45−4.41 (m, 1H), 4.31 (t, J = 4.8 Hz, 2H),

4.02−3.92 (m, 2H), 3.84 (d, J = 11.2 Hz, 1H), 3.74 (dd, J = 10.8 Hz, J

(2S,4R)-1-((S)-2-(3-(4-(5-(Ethyl(2-(4-(6-hydroxy-2-(4-

hydroxyphenyl)benzo[b]thiophene-3-carbonyl)phenoxy)ethyl)-

amino)pent-1-yn-1-yl)-1H-pyrazol-1-yl)propanamido)-3,3-dime-

thylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)-

ethyl)pyrrolidine-2-carboxamide (35). This compound was prepared

1

using a procedure similar to that used for compound 32. H NMR

(CD₃OD, 400 MHz) δ (ppm) 9.13 (s, 1H), 7.69 (d, J = 8.8 Hz, 2H),

7.62 (s, 1H), 7.46−7.41 (m, 6H), 7.27 (d, J = 2.0 Hz, 1H), 7.17 (d, J

= 8.4 Hz, 2H), 6.90−6.86 (m, 3H), 6.61 (d, J = 8.8 Hz, 2H), 5.01−

4.96 (m, 1H), 4.59−4.55 (m, 2H), 4.42−4.26 (m, 5H), 3.86 (d, J =

10.8 Hz, 1H), 3.72 (dd, J = 10.8 Hz, J = 4.0 Hz, 1H), 3.62−3.57 (m,

2H), 3.41−3.30 (m, 4H), 2.92−2.85 (m, 1H), 2.75−2.70 (m, 1H),

2.54 (t, J = 6.8 Hz, 2H), 2.50 (s, 3H), 2.22−2.17 (m, 1H), 2.01−1.91

(m, 3H), 1.50 (d, J = 6.8 Hz, 3H), 1.36 (t, J = 7.2 Hz, 3H), 0.95 (s,

9H). ¹³C NMR (CD₃OD, 100 MHz) δ (ppm) 195.31, 173.25, 172.24,

172.10, 163.01, 159.26, 156.86, 153.68, 146.13, 144.43, 142.84,

141.47, 134.21, 133.48, 132.76, 131.49, 131.00, 130.51, 127.74,

126.02, 124.77, 116.48, 116.15, 115.42, 107.92, 104.32, 88.60, 74.07,

70.96, 63.58, 60.60, 59.18, 57.88, 53.41, 52.83, 50.53, 50.45, 38.77,

36.74, 36.42, 26.98, 23.92, 22.36, 17.27, 15.15, 9.16. UPLC-MS

(ESI⁺) calcd for C₅₉H₆₆N₇O₈S₂[M + 1]⁺: 1064.44, found 1064.89.

Purity 95.1% (HPLC).

(2S,4R)-1-((S)-2-(2-(4-(4-(Ethyl(2-(4-(6-hydroxy-2-(4-

hydroxyphenyl)benzo[b]thiophene-3-carbonyl)phenoxy)ethyl)-

amino)but-1-yn-1-yl)-1H-pyrazol-1-yl)acetamido)-3,3-dimethyl-

butanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)-

ethyl)pyrrolidine-2-carboxamide (36). This compound was prepared

Q

DOI: 10.1021/acs.jmedchem.8b01572

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

1

using a procedure similar to that used for compound 32. H NMR

(CD₃OD, 400 MHz) δ (ppm) 8.87 (s, 1H), 7.74−7.70 (m, 3H), 7.52

(s, 1H), 7.44−7.41 (m, 5H), 7.26 (d, J = 2.0 Hz, 1H), 7.18 (d, J = 8.4

Hz, 2H), 6.88−6.85 (m, 3H), 6.63 (d, J = 8.8 Hz, 2H), 5.01−4.98 (m,

1H), 4.62−4.52 (m, 2H), 4.42−4.39 (m, 3H), 3.82 (d, J = 11.2 Hz,

1H), 3.76−3.64 (m, 3H), 3.50−3.39 (m, 6H), 2.96 (t, J = 7.2 Hz,

2H), 2.47 (s, 3H), 2.26−2.15 (m, 1H), 1.96−1.90 (m, 1H), 1.49 (d, J

= 7.2 Hz, 3H), 1.38 (t, J = 7.2 Hz, 3H), 1.02 (s, 9H). UPLC-MS

(ESI⁺) calcd for C₅₇H₆₂N₇O₈S₂[M + 1]⁺: 1036.41, found 1035.92.

Purity 98.8% (HPLC).

methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (40).

This compound was prepared using a procedure similar to that

used for compound 15. ¹H NMR (CD₃OD, 400 MHz) δ (ppm) 9.08

(s, 1H), 7.45−7.40 (m, 4H), 7.12 (d, J = 8.4 Hz, 2H), 6.99 (d, J = 8.8

Hz, 1H), 6.91 (d, J = 2.4 Hz, 1H), 6.84−6.75 (m, 6H), 6.63 (dd, J =

8.8 Hz, J = 2.4 Hz, 1H), 5.11 (s, 2H), 5.02−4.97 (m, 1H), 4.64−4.62

(m, 1H), 4.56 (t, J = 8.4 Hz, 1H), 4.43−4.41 (m, 1H), 4.24 (t, J = 4.8

Hz, 1H), 3.87 (d, J = 11.2 Hz, 1H), 3.73 (dd, J = 11.2 Hz, J = 4.0 Hz,

1H), 3.54 (t, J = 4.8 Hz, 1H), 2.49 (s, 3H), 2.31−2.15 (m, 6H),

1.98−1.92 (m, 1H), 1.73−1.65 (m, 2H), 1.59−1.55 (m, 2H), 1.50 (d,

J = 7.2 Hz, 3H), 1.40−1.29 (m, 9H), 1.03 (s, 9H). ¹³C NMR

(CD₃OD, 100 MHz) δ (ppm) 175.91, 173.21, 172.33, 158.52, 157.92,

153.66, 151.65, 146.06, 139.86, 133.87, 133.08, 132.79, 131.04,

130.50, 128.68, 127.72, 124.47, 116.23, 115.54, 112.17, 111.59,

108.66, 103.92, 70.94, 63.27, 60.60, 59.00, 57.98, 54.43, 52.72, 50.15,

47.58, 38.78, 36.47, 29.90, 29.80, 29.76, 27.04, 24.69, 22.35, 15.21,

9.62, 9.05. UPLC-MS (ESI⁺) calcd for C₅₇H₇₃N₆O₇S [M + 1]⁺:

985.53, found 985.82. Purity >99.5% (HPLC).

(2S,4R)-1-((S)-2-(8-(Ethyl(2-(4-((1R,2S)-6-hydroxy-2-phenyl-

1,2,3,4-tetrahydronaphthalen-1-yl)phenoxy)ethyl)amino)-

octanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-

methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (41).

This compound was prepared using a procedure similar to that

used for compound 15. ¹H NMR (CD₃OD, 400 MHz) δ (ppm) 8.94

(s, 1H), 7.46−7.35 (m, 4H), 7.13−7.09 (m, 3H), 6.83−6.80 (m, 2H),

6.68−6.61 (m, 4H), 6.52 (dd, J = 8.4 Hz, J = 4.0 Hz 1H), 6.38 (d, J =

8.4 Hz, 2H), 5.03−4.98 (m, 1H), 4.64−4.54 (m, 2H), 4.43−4.41 (m,

1H), 4.25−4.20 (m, 3H), 3.89−3.86 (m, 1H), 3.75 (dd, J = 11.2 Hz, J

= 4.0 Hz, 1H), 3.56−3.53 (m, 2H), 3.37−3.35 (m, 1H), 3.23−3.16

(m, 2H), 3.06−2.99 (m, 2H), 2.49 (s, 3H), 2.34−2.14 (m, 4H),

1.99−1.92 (m, 1H), 1.79−1.50 (m, 8H), 1.38−1.29 (m, 9H), 1.03 (s,

9H). ¹³C NMR (CD₃OD, 100 MHz) δ (ppm) 175.89, 173.23, 172.31,

157.05, 156.64, 153.10, 148.68, 145.79, 138.82, 137.76, 132.43,

132.06, 131.33, 130.51, 129.21, 128.72, 127.66, 127.66, 127.44,

126.97, 126.63, 115.50, 114.68, 113.98, 70.96, 63.16, 61.04, 60.61,

58.98, 58.01, 54.48, 52.81, 52.24, 51.57, 50.15, 46.73, 38.82, 36.52,

36.49, 30.97, 29.97, 28.83, 27.38, 27.05, 26.74, 25.59, 24.73, 24.44,

23.24, 22.37, 15.62, 9.06. UPLC-MS (ESI⁺) calcd for C₅₇H₇₄N₅O₆S

[M + 1]⁺: 956.54, found 956.48.

(2S,4R)-1-((S)-2-(2-(4-(4-(Ethyl(2-(4-(6-hydroxy-2-(4-

hydroxyphenyl)benzo[b]thiophene-3-carbonyl)phenoxy)ethyl)-

amino)butyl)-1H-pyrazol-1-yl)acetamido)-3,3-dimethylbutanoyl)-

4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)-

pyrrolidine-2-carboxamide (37). This compound was prepared using

a procedure similar to that used for compound 32. ¹H NMR

(CD₃OD, 400 MHz) δ (ppm) 9.23 (s, 1H), 7.70 (d, J = 8.8 Hz, 2H),

7.49 (s, 1H), 7.44−7.37 (m, 6H), 7.25 (d, J = 2.4 Hz, 1H), 7.14 (d, J

= 8.8 Hz, 2H), 6.87−6.83 (m, 3H), 6.59 (d, J = 8.4 Hz, 2H), 4.99−

4.83 (m, 3H), 4.61−4.52 (m, 2H), 4.40−4.37 (m, 1H), 4.30 (t, J =

4.4 Hz, 2H), 3.81 (d, J = 11.2 Hz, 1H), 3.69 (dd, J = 11.2 Hz, J = 4.0

Hz, 1H), 3.54 (d, J = 4.0 Hz, 2H), 3.28−3.16 (m, 4H), 2.54−2.46 (m,

5H), 2.22−2.17 (m, 1H), 1.96−1.89 (m, 1H), 1.73−1.60 (m, 4H),

1.47 (d, J = 7.2 Hz, 3H), 1.30 (t, J = 7.2 Hz, 3H), 1.00 (s, 9H). ¹³C

NMR (CD₃OD, 100 MHz) δ (ppm) 195.28, 173.08, 171.67, 169.31,

163.05, 159.21, 156.78, 153.34, 147.93, 145.88, 144.26, 141.40,

140.79, 134.19, 133.92, 133.49, 132.59, 131.61, 131.41, 130.96,

130.89, 130.45, 127.65, 125.90, 124.74, 122.64, 116.52, 116.16,

115.42, 108.00, 70.90, 63.51, 60.56, 59.13, 57.96, 54.70, 54.16, 52.44,

50.14, 38.80, 36.70, 28.55, 26.93, 26.82, 24.24, 24.05, 22.38, 15.39,

9.07. UPLC-MS (ESI⁺) calcd for C₅₇H₆₆N₇O₈S₂[M + 1]⁺: 1040.44,

found 1040.17. Purity 98.7% (HPLC).

(2S,4R)-1-((S)-2-(8-(Ethyl(2-(4-((Z)-1-(4-hydroxyphenyl)-2-phe-

nylbut-1-en-1-yl)-phenoxy)ethyl)amino)octanamido)-3,3-dime-

thylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)-

ethyl)pyrrolidine-2-carboxamide (38). This compound was prepared

1

using a procedure similar to that used for compound 15. H NMR

(CD₃OD, 400 MHz) δ (ppm) 8.98 (s, 1H), 7.47−7.36 (m, 4H),

7.13−7.01 (m, 7H), 6.84−6.75 (m, 3H), 6.66−6.60 (m, 2H), 5.03−

4.98 (m, 1H), 4.64−4.62 (m, 1H), 4.56 (t, J = 8.4 Hz, 1H), 4.45−

4.41 (m, 1H), 4.22−4.18 (m, 2H), 3.89−3.86 (m, 1H), 3.74 (dd, J =

11.2 Hz, J = 4.0 Hz, 1H), 3.55−3.52 (m, 2H), 3.21−3.15 (m, 2H),

2.52−2.45 (m, 5H), 2.32−2.17 (m, 3H), 1.99−1.92 (m, 1H), 1.78−

1.56 (m, 5H), 1.52−1.49 (m, 3H), 1.45−1.30 (m, 9H), 1.04 (s, 9H),

0.91 (t, J = 7.2 Hz, 3H). ¹³C NMR (CD₃OD, 100 MHz) δ (ppm)

175.90, 173.22, 172.31, 157.41, 156.91, 153.20, 145.85, 144.03,

142.58, 139.49, 138.76, 136.07, 133.23, 131.55, 130.91, 130.51,

128.88, 127.67, 127.04, 115.92, 114.46, 70.96, 63.11, 60.60, 58.99,

58.00, 54.48, 52.76, 50.15, 38.82, 37.64, 36.51, 33.74, 29.95, 29.93,

29.86, 29.81, 27.36, 27.05, 26.73, 24.71, 22.37, 15.54, 13.86, 9.04.

UPLC-MS (ESI⁺) calcd for C₅₇H₇₄N₅O₆S [M + 1]⁺: 956.54, found

956.51.

(2S,4R)-1-((S)-2-(8-((2-(4-(1,2-Diphenylbut-1-en-1-yl)phenoxy)-

ethyl)(ethyl)amino)-octanamido)-3,3-dimethylbutanoyl)-4-hy-

droxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-

carboxamide (39). This compound was prepared using a procedure

similar to that used for compound 15. ¹H NMR (CD₃OD, 400 MHz)

δ (ppm) 9.08 (s, 1H), 7.47−7.42 (m, 4H), 7.36−7.32 (m, 1H), 7.28−

7.22 (m, 0.5H), 7.21−7.07 (m, 7H), 7.03−6.95 (m, 2.5H), 6.86−6.82

(m, 2H), 6.66−6.64 (m, 1H), 5.03−4.98 (m, 1H), 4.64−4.62 (m,

1H), 4.57 (t, J = 8.4 Hz, 1H), 4.43−4.41 (m, 1H), 4.38 (t, J = 4.8 Hz,

1H), 4.21 (t, J = 4.8 Hz, 1H), 3.88 (d, J = 10.8 Hz, 1H), 3.74 (dd, J =

10.8 Hz, J = 4.0 Hz, 1H), 3.64 (t, J = 4.8 Hz, 1H), 3.53 (t, J = 4.8 Hz,

1H), 3.38−3.34 (m, 1H), 3.31−3.17 (m, 3H), 2.49−2.42 (m, 5H),

2.30−2.19 (m, 3H), 1.99−1.95 (m, 1H), 1.70−1.56 (m, 4H), 1.50 (d,

J = 7.2 Hz, 3H), 1.40−1.30 (m, 9H), 1.04 (s, 9H), 0.94−0.88 (m,

3H). UPLC-MS (ESI⁺) calcd for C₅₇H₇₄N₅O₅S [M + 1]⁺: 940.54,

found 940.82. Purity 97.0% (HPLC).

(2S,4R)-1-((S)-2-(8-(Ethyl(2-(4-(6-hydroxy-2-(4-hydroxyphenyl)-

benzo[b]thiophene-3-carbonyl)phenoxy)ethyl)amino)-

octanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methyl-

thiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (42). This com-

pound was prepared using a procedure similar to that used for

compound 15. ¹H NMR (CD₃OD, 400 MHz) δ (ppm) 8.86 (s, 1H),

7.74 (d, J = 9.2 Hz, 2H), 7.46−7.39 (m, 5H), 7.27 (d, J = 2.4 Hz,

1H), 7.18 (d, J = 8.8 Hz, 2H), 6.91 (d, J = 8.8 Hz, 2H), 6.87 (dd, J =

8.8 Hz, J = 2.0 Hz, 1H), 6.61 (d, J = 8.8 Hz, 2H), 4.64 (d, J = 8.8 Hz,

1H), 4.58−4.49 (m, 3H), 4.38−4.33 (m, 3H), 3.90 (d, J = 11.2 Hz,

1H), 3.80 (dd, J = 10.8 Hz, J = 4.0 Hz, 1H), 3.59 (t, J = 4.8 Hz, 2H),

3.21−3.17 (m, 2H), 2.46 (s, 3H), 2.32−2.19 (m, 3H), 2.11−2.03 (m,

1H), 1.73−1.71 (m, 2H), 1.62−1.59 (m, 2H), 1.38−1.29 (m, 9H),

1.02 (s, 9H). UPLC-MS (ESI⁺) calcd for C₅₅H₆₆N₅O₈S₂[M + 1]⁺:

988.44, found 988.98. Purity 97.8% (HPLC).

(2S,4R)-N-((S)-1-(4-Chlorophenyl)ethyl)-1-((S)-2-(8-(ethyl(2-(4-(6-

hydroxy-2-(4-hydroxyphenyl)benzo[b]thiophene-3-carbonyl)-

phenoxy)ethyl)amino)octanamido)-3,3-dimethylbutanoyl)-4-hy-

droxypyrrolidine-2-carboxamide (43). This compound was prepared

1

using a procedure similar to that used for compound 15. H NMR

(CD₃OD, 400 MHz) δ (ppm) 7.75 (d, J = 8.8 Hz, 2H), 7.44 (d, J =

8.8 Hz, 1H), 7.32−7.27 (m, 5H), 7.21−7.15 (m, 2H), 6.95−6.86 (m,

3H), 6.65−6.60 (m, 2H), 4.95−4.87 (m, 1H), 4.63−4.61 (m, 1H),

4.53 (t, J = 8.4 Hz, 2H), 3.87−3.84 (m, 1H), 3.73 (dd, J = 11.2 Hz, J

= 4.0 Hz, 1H), 3.64−3.54 (m, 3H), 3.26−3.16 (m, 3H), 3.07−2.96

(m, 1H), 2.33−2.13 (m, 3H), 1.95−1.88 (m, 1H), 1.78−1.58 (m,

5H), 1.51 (d, J = 7.2 Hz, 2H), 1.41−1.29 (m, 11H), 1.02 (s, 9H). ¹³C

NMR (CD₃OD, 100 MHz) δ (ppm) 195.37, 175.91, 173.18, 172.29,

163.09, 159.28, 156.88, 144.38, 144.13, 141.49, 134.22, 133.71,

133.53, 132.77, 131.65, 131.47, 131.02, 129.56, 128.70, 128.70,

128.47, 126.04, 124.71, 116.59, 124.71, 116.59, 116.47, 116.13,

115.41, 107.91, 70.94, 63.57, 60.57, 58.99, 57.98, 54.55, 52.59, 50.24,

(2S,4R)-1-((S)-2-(8-(Ethyl(2-(4-((5-hydroxy-2-(4-hydroxyphenyl)-

3-methyl-1H-indol-1-yl)methyl)phenoxy)ethyl)amino)-

octanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-

R

DOI: 10.1021/acs.jmedchem.8b01572

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

49.82, 38.78, 36.49, 34.62, 29.95, 29.81, 27.33, 27.03, 26.73, 25.75,

24.71, 22.28, 9.06. UPLC-MS (ESI⁺) calcd for C₅₂H₆₄ClN₄O₈S [M +

1]⁺: 939.41, found 939.45.

(m, 1H), 4.62 (d, J = 8.8 Hz, 1H), 4.53 (d, J = 8.4 Hz, 1H), 4.43−

4.36 (m, 1H), 4.35−4.29 (m, 2H), 3.87−3.84 (m, 1H), 3.75−3.71

(m, 1H), 3.60−3.53 (m, 2H), 3.24−3.15 (m, 2H), 2.89−2.81 (m,

1H), 2.34−2.21 (m, 2H), 2.18−2.12 (m, 1H), 1.98−1.91 (m, 1H),

1.73−1.70 (m, 2H), 1.63−1.58 (m, 2H), 1.52−1.29 (m, 12H), 1.22

(d, J = 7.2 Hz, 6H), 1.02 (s, 9H). ¹³C NMR (CD₃OD, 100 MHz) δ

(ppm) 195.35, 175.89, 173.00, 172.30, 163.08, 159.29, 156.88,

148.92, 144.37, 142.57, 141.49, 134.22, 133.53, 132.79, 131.47,

131.01, 127.50, 126.76, 126.03, 124.71, 116.13, 115.40, 107.91, 70.94,

63.55, 60.63, 58.98, 57.98, 54.53, 52.57, 50.20, 50.10, 38.76, 36.51,

35.06, 29.94, 29.81, 27.34, 27.04, 26.72, 24.70, 24.45, 22.49, 9.06.

UPLC-MS (ESI⁺) calcd for C₅₅H₇₁N₄O₈S [M + 1]⁺: 947.50, found

947.53.

(2S,4R)-N-((S)-1-(4-Chlorophenyl)ethyl)-1-((S)-2-(8-(ethyl(2-(4-(6-

hydroxy-2-(4-hydroxyphenyl)benzo[b]thiophene-3-carbonyl)-

phenoxy)ethyl)amino)octanamido)-3,3-dimethylbutanoyl)-4-hy-

droxypyrrolidine-2-carboxamide (44). This compound was prepared

1

using a procedure similar to that used for compound 15. H NMR

(CD₃OD, 400 MHz) δ (ppm) 7.75 (d, J = 8.8 Hz, 2H), 7.68 (d, J =

8.8 Hz, 2H), 7.49−7.40 (m, 3H), 7.27 (d, J = 2.4 Hz, 1H), 7.18 (d, J

= 8.8 Hz, 2H), 6.94−6.86 (m, 3H), 6.64−6.60 (m, 2H), 5.01−4.96

(m, 1H), 4.63−4.61 (m, 1H), 4.47−4.38 (m, 1H), 4.35 (t, J = 4.8 Hz,

2H), 3.88−3.85 (m, 1H), 3.72 (dd, J = 11.2 Hz, J = 4.0 Hz, 1H),

3.60−3.58 (m, 2H), 3.23−3.17 (m, 2H), 2.32−2.16 (m, 3H), 1.94−

1.87 (m, 1H), 1.73−1.60 (m, 5H), 1.54−1.46 (m, 3H), 1.38−1.29

(m, 10H), 1.02 (s, 9H). ¹³C NMR (CD₃OD, 100 MHz) δ (ppm)

195.36, 175.91, 173.38, 172.30, 163.09, 159.28, 156.88, 151.22,

144.39, 141.49, 134.22, 131.47, 131.02, 128.06, 127.85, 126.04,

124.71, 119.67, 116.48, 116.13, 115.40, 111.78, 107.91, 70.95, 63.56,

60.51, 58.99, 57.99, 54.54, 52.58, 50.33, 50.22, 38.81, 36.49, 29.96,

29.82, 27.34, 27.02, 26.73, 24.72, 22.09, 9.06. UPLC-MS (ESI⁺) calcd

for C₅₃H₆₄N₅O₈S [M + 1]⁺: 930.45, found 930.48.

(2S,4R)-1-((S)-2-(8-(Ethyl(2-(4-(6-hydroxy-2-(4-hydroxyphenyl)-

benzo[b]thiophene-3-carbonyl)phenoxy)ethyl)amino)-

octanamido)-3,3-dimethylbutanoyl)-N-((S)-1-(4-ethynylphenyl)-

ethyl)-4-hydroxypyrrolidine-2-carboxamide (45). This compound

was prepared using a procedure similar to that used for compound 15.

¹H NMR (CD₃OD, 400 MHz) δ (ppm) 7.75 (d, J = 8.8 Hz, 2H),

7.46−7.38 (m, 3H), 7.29−7.22 (m, 3H), 7.19−7.15 (m, 2H), 6.93−

6.86 (m, 3H), 6.61(d, J = 8.8 Hz, 2H), 4.97−4.92 (m, 1H), 4.63−4.61

(m, 1H), 4.54 (t, J = 8.4 Hz, 2H), 4.42−4.39 (m, 1H), 4.36 (t, J = 4.8

Hz, 2H), 3.88−3.85 (m, 1H), 3.75−3.71 (m, 2H), 3.63−3.58 (m,

3H), 3.44 (s, 1H), 3.26−3.17 (m, 3H), 2.33−2.14 (m, 4H), 1.95−

1.89 (m, 1H), 1.80−1.65 (m, 3H), 1.67−1.54 (m, 3H), 1.46−1.30

(m, 10H), 1.02 (s, 9H). ¹³C NMR (CD₃OD, 100 MHz) δ (ppm)

195.38, 175.91, 173.20, 172.29, 163.10, 161.00, 159.28, 156.88,

155.70, 146.12, 144.39, 141.49, 134.22, 133.53, 133.18, 132.77,

131.47, 131.02, 130.05, 129.93, 127.14, 126.04, 124.71, 122.43,

116.47, 116.12, 115.41, 107.90, 84.19, 78.50, 70.95, 64.27, 60.58,

59.62, 58.99, 57.98, 55.93, 55.83, 55.65, 54.56, 53.65, 52.59, 50.17,

36.49, 29.94, 29.81, 27.03, 26.73, 24.71, 22.23, 18.70, 17.26, 13.17,

9.07. UPLC-MS (ESI⁺) calcd for C₅₃H₆₄N₅O₈S [M + 1]⁺: 929.45,

found 929.49.

(2S,4R)-N-((S)-1-(4-(tert-Butyl)phenyl)ethyl)-1-((S)-2-(8-(ethyl(2-

(4-(6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thiophene-3-carbonyl)-

phenoxy)ethyl)amino)octanamido)-3,3-dimethylbutanoyl)-4-hy-

droxypyrrolidine-2-carboxamide (48). This compound was prepared

1

using a procedure similar to that used for compound 15. H NMR

(CD₃OD, 400 MHz) δ (ppm) 7.75 (d, J = 8.8 Hz, 2H), 7.45 (d, J =

8.8 Hz, 1H), 7.34 (d, J = 8.4 Hz, 2H), 7.27 (d, J = 2.0 Hz, 1H), 7.22−

7.16 (m, 4H), 6.93−6.86 (m, 3H), 6.63−6.59 (m, 2H), 4.95−4.91

(m, 1H), 4.65−4.60 (m, 1H), 4.53 (t, J = 8.4 Hz, 1H), 4.42−4.40 (m,

1H), 4.35 (t, J = 4.4 Hz, 2H), 3.87−3.84 (m, 1H), 3.75−3.71 (m,

1H), 3.60−3.56 (m, 2H), 3.25−3.16 (m, 2H), 2.34−2.14 (m, 3H),

1.98−1.91 (m, 1H), 1.74−1.71 (m, 2H), 1.64−1.55 (m, 2H), 1.45−

1.32 (m, 1H), 1.29 (s, 9H), 1.02 (s, 9H). ¹³C NMR (CD₃OD, 100

MHz) δ (ppm) 193.36, 173.88, 171.01, 170.31, 161.08, 157.29,

154.88, 149.06, 142.37, 140.13, 139.49, 132.22, 131.53, 130.80,

129.47, 129.02, 124.71, 124.48, 124.39, 124.04, 122.70, 114.47,

114.13, 113.40, 105.91, 68.95, 61.56, 58.64, 56.98, 55.98, 52.54, 50.58,

48.21, 48.02, 36.77, 34.51, 33.26, 29.78, 27.94, 27.81, 25.34, 25.04,

24.72, 22.70, 20.45, 7.06. UPLC-MS (ESI⁺) calcd for C₅₆H₇₃N₄O₈S

[M + 1]⁺: 961.51, found 961.55.

4-(6-Methoxy-2-(4-methoxyphenyl)benzo[b]thiophene-3-

carbonyl)phenyl acetate (50). Oxalyl chloride (9.70 mL, 120 mmol,

3.0 equiv) was added dropwise under N₂to a solution of 4-

acetoxybenzoic acid (49) (7.206 g, 40 mmol, 1.0 equiv) in anhydrous

DCM (80 mL) at 0 °C. Then several drops of DMF were added. The

solution was warmed to rt and stirred for 1 h. The solution was

concentrated and dried to obtain the acyl chloride as a white solid.

The intermediate was dissolved in anhydrous DCM (150 mL), then

6-methoxy-2-(4-methoxyphenyl)-benzo[b]thiophene (8.65 g, 32

mmol, 0.8 equiv) was added followed by addition of AlCl₃(8.00 g,

60 mmol, 1.5 equiv) in three portions over a period of 5 min with

vigorous stirring at 0 °C under N₂. The mixture was warmed to rt and

stirred for 1 h. The reaction was quenched by slow addition of ice−

H₂O followed by 1 N HCl (aq). The layers were separated, and the

aqueous layer was extracted twice with DCM. The combined organic

layer was dried by anhydrous Na₂SO₄. After ﬁltration and

concentration, the residue was puriﬁed on a silica gel ﬂash column

with hexane:DCM (100:1−1:100) to aﬀord the intermediate (50) as

(2S,4R)-N-((S)-1-(4-Cyclopropylphenyl)ethyl)-1-((S)-2-(8-(ethyl(2-

(4-(6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thiophene-3-carbonyl)-

phenoxy)ethyl)amino)octanamido)-3,3-dimethylbutanoyl)-4-hy-

droxypyrrolidine-2-carboxamide (46). This compound was prepared

1

using a procedure similar to that used for compound 15. H NMR

(CD₃OD, 400 MHz) δ (ppm) 7.76 (d, J = 8.8 Hz, 2H), 7.45 (d, J =

8.8 Hz, 1H), 7.27 (d, J = 2.0 Hz, 2H), 7.20−7.10 (m, 4H), 7.02−6.98

(m, 2H), 6.94−6.86 (m, 3H), 6.63−6.59 (m, 2H), 4.95−4.91 (m,

1H), 4.63−4.61 (m, 1H), 4.54 (t, J = 8.4 Hz, 1H), 4.43−4.39 (m,

1H), 4.36−4.34 (m, 2H), 3.87−3.85 (m, 1H), 3.75−3.71 (m, 1H),

3.63−3.58 (m, 2H), 3.23−3.17 (m, 2H), 2.31−2.23 (m, 2H), 2.18−

2.11 (m, 1H), 1.96−1.83 (m, 2H), 1.72−1.59 (m, 4H), 1.51−1.28

(m, 12H), 1.02 (s, 9H), 1.00−0.90 (m, 2H), 0.64−0.60 (m, 2H). ¹³C

NMR (CD₃OD, 100 MHz) δ (ppm) 195.36, 175.88, 172.98, 172.30,

163.07, 159.29, 156.90, 144.39, 144.17, 142.11, 141.50, 134.22,

133.54, 131.48, 131.02, 126.95, 126.72, 126.06, 124.70, 116.47,

116.13, 115.40, 107.90, 70.94, 63.55, 60.64, 58.98, 57.98, 55.90, 54.57,

53.58, 52.62, 51.85, 51.29, 38.76, 36.51, 29.93, 29.83, 27.36, 27.04,

26.72, 24.72, 15.77, 9.51, 9.06. UPLC-MS (ESI⁺) calcd for

C₅₅H₆₉N₄O₈S [M + 1]⁺: 945.48, found 945.51.

1

a yellow solid (5.517 g, 40% yield). H NMR (CDCl₃, 400 MHz) δ

(ppm) 7.81 (d, J = 8.8 Hz, 2H), 7.61 (d, J = 8.8 Hz, 1H), 7.32−7.29

(m, 3H), 7.02−6.99 (m, 3H), 6.74 (d, J = 8.8 Hz, 2H), 3.86 (s, 3H),

3.73 (s, 3H), 2.25 (s, 3H). ¹³C NMR (CDCl₃, 100 MHz) δ (ppm)

193.15, 168.63, 159.99, 157.78, 154.38, 144.16, 140.10, 135.03,

133.76, 131.52, 130.48, 130.02, 125.76, 124.16, 121.54, 114.99,

114.13, 104.54, 55.65, 55.28, 21.16. UPLC-MS (ESI⁺) calcd for

C₂₅H₂₁O₅S [M + 1]⁺: 433.11, found 433.37.

(4-Hydroxyphenyl)(6-methoxy-2-(4-methoxyphenyl)benzo[b]-

thiophen-3-yl)methanone (51). Compound 50 (5.517 g, 12.76

mmol, 1.0 equiv) was dissolved in EtOH (70 mL) and H₂O (30 mL).

Then sodium acetate (5.23 g, 63.8 mmol, 5.0 equiv) was added. The

solution was heated to 90−100 °C and stirred for 12 h. The solution

was cooled to rt and concentrated. The residue was diluted in EtOAc

and H₂O. The organic layer was separated, and the aqueous layer was

extracted twice with EtOAc. The combined organic layer was dried by

anhydrous Na₂SO₄. After ﬁltration and concentration, the residue was

puriﬁed by silica gel ﬂash column chromatography with hexane:E-

tOAc (5:1−2:1) to aﬀord intermediate 51 as yellow oil (4.7 g, 95%

(2S,4R)-1-((S)-2-(8-(Ethyl(2-(4-(6-hydroxy-2-(4-hydroxyphenyl)-

benzo[b]thiophene-3-carbonyl)phenoxy)ethyl)amino)-

octanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-

isopropylphenyl)ethyl)pyrrolidine-2-carboxamide (47). This com-

pound was prepared using a procedure similar to that used for

1

compound 15. H NMR (CD₃OD, 400 MHz) δ (ppm) 7.75 (d, J =

8.8 Hz, 2H), 7.44 (d, J = 8.8 Hz, 2H), 7.27 (d, J = 2.0 Hz, 1H), 7.22−

7.15 (m, 6H), 6.93−6.86 (m, 3H), 6.63−6.59 (m, 2H), 4.96−4.91

S

DOI: 10.1021/acs.jmedchem.8b01572

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

1

yield). H NMR (CD₃OD, 400 MHz) δ (ppm) 7.64 (d, J = 9.2 Hz,

with H₂O (200 mL) and extracted with EtOAc (150 mL × 2). The

combined organic layer was washed with brine (200 mL) and dried

over Na₂SO₄. The organic solution was ﬁltered and concentrated. The

residue was puriﬁed by silica gel ﬂash column chromatography with

hexane:EtOAc (100:1−1:100), then DCM:MeOH (10:1) to aﬀord

2H), 7.43 (d, J = 8.8 Hz, 1H), 7.30 (d, J = 2.4 Hz, 1H), 7.24 (d, J =

8.8 Hz, 2H), 6.89 (dd, J = 8.8 Hz, J = 2.4 Hz, 1H), 6.69−6.64 (m,

4H), 3.73 (s, 3H), 3.59 (s, 3H). ¹³C NMR (CD₃OD, 100 MHz) δ

(ppm) 193.95, 162.85, 159.92, 157.81, 142.33, 140.08, 133.78,

132.55, 130.34, 129.91, 129.06, 125.78, 123.43, 115.04, 114.63,

113.79, 104.34, 54.78, 54.39. UPLC-MS (ESI⁺) calcd for C₂₃H₁₉O₄S

[M + 1]⁺: 391.10, found 391.42.

1

the intermediate (56) as white solid (10.98 g, 80% yield). H NMR

(CD₃OD, 400 MHz) δ (ppm) 8.84 (s, 1H), 7.43−7.37 (m, 4H),

5.11−5.07 (m, 1H), 4.44−4.37 (m, 2H), 3.60−3.46 (m, 2H), 2.44 (s,

3H), 2.27−2.22 (m, 1H), 1.98−1.91 (m, 1H), 1.50 (d, J = 7.2 Hz,

3H), 1.46 (s, 9H). UPLC-MS (ESI⁺) calcd for C₂₂H₃₀N₃O₄S [M +

1]⁺: 432.20, found 432.20.

(4-(2-(Ethylamino)ethoxy)phenyl)(6-methoxy-2-(4-

methoxyphenyl)benzo[b]thiophen-3-yl)methanone (52). 1,2-Di-

bromoethane (2.0 mL, 24.0 mmol, 2.0 equiv) and Cs₂CO₃(5.86 g,

18.0 mmol, 1.5 equiv) were added sequentially to a solution of

compound 51 (4.7 g, 12.0 mmol, 1.0 equiv) in 200 mL of MeCN.

The solution was heated to reﬂux for 12 h. The solution was ﬁltered

and washed with MeCN. The concentrated residue was used in next

step without further column puriﬁcation. Ethylamine (2.0 M in THF)

(60 mL, 120 mmol, 10.0 equiv) was added to a solution of the residue

in DMF. The solution was heated to 80 °C and stirred for 12 h. After

cooling to rt, the reaction mixture was diluted in EtOAc and saturated

brine. The aqueous layer was extracted with EtOAc twice. The

combined organic layer was dried and concentrated. The residue was

puriﬁed by silica gel ﬂash column chromatography with DCM:MeOH

(10:1) to aﬀord the compound 52 as a yellow solid (4.43 g, 80%

tert-Butyl ((S)-1-((2S,4R)-4-Hydroxy-2-(((S)-1-(4-(4-methylthia-

zol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-

oxobutan-2-yl)carbamate (57). This solid material (56), obtained as

described above was dissolved in 4 N HCl in dioxane (25 mL, 100

mmol) and 25 mL of MeOH, and the mixture was stirred at ambient

temperature for 12 h. The mixture was then concentrated, and the

residue was dried under vacuum to aﬀord intermediate, which was

used in next step without further puriﬁcation. UPLC-MS (ESI⁺) calcd

for C₁₇H₂₂N₃O₂S [M + 1]⁺: 332.14, found 332.11. HATU (1.37 g, 3.6

mmol, 1.2 equiv) was added to a solution of this intermediate (994

mg, 3.0 mmol, 1.0 equiv), (S)-2-((tert-butoxycarbonyl)amino)-3,3-

dimethylbutanoic acid (694 mg, 3.0 mmol, 1.0 equiv), and DIPEA

(1.57 mL, 9.0 mmol, 3.0 equiv) in DMF (10 mL) at 0 °C under N₂.

The mixture was stirred at ambient temperature for 12 h when TLC

showed that the reaction was complete. The reaction mixture was

quenched with H₂O (100 mL) and extracted with EtOAc (75 mL ×

2). The combined organic layer was washed with brine (100 mL) and

dried over Na₂SO₄. The organic solution was ﬁltered and

concentrated. The residue was puriﬁed by silica gel ﬂash column

chromatography with hexane:EtOAc then DCM:MeOH to aﬀord the

1

yield). H NMR (CD₃OD, 400 MHz) δ (ppm) 7.63 (d, J = 8.8 Hz,

2H), 7.42 (d, J = 8.8 Hz, 1H), 7.32 (d, J = 2.4 Hz, 1H), 7.20 (d, J =

8.8 Hz, 2H), 6.88 (dd, J = 8.8 Hz, J = 2.4 Hz, 1H), 6.71 (d, J = 8.8 Hz,

2H), 6.64 (d, J = 8.8 Hz, 2H), 3.93 (t, J = 4.2 Hz, 2H), 3.75 (s, 3H),

3.59 (s, 3H), 2.83 (t, J = 5.2 Hz, 2H), 2.59 (q, J = 7.2 Hz, 2H), 1.06

(t, J = 7.2 Hz, 3H). ¹³C NMR (CD₃OD, 100 MHz) δ (ppm) 194.88,

164.48, 161.27, 159.20, 143.95, 141.41, 135.02, 133.39, 131.61,

131.48, 131.26, 127.01, 124.76, 115.97, 115.30, 115.10, 105.67, 68.13,

56.10, 55.70, 49.65, 44.52, 14.68. UPLC-MS (ESI⁺) calcd for

C₂₇H₂₈NO₄S [M + 1]⁺: 462.17, found 462.27.

1

desired compound (57) as white solid (1.31 g, 80% yield). H NMR

(CDCl₃, 400 MHz) δ (ppm) 8.65 (s, 1H), 7.70 (d, J = 8.0 Hz, 1H),

7.35−7.31 (m, 4H), 5.29 (d, J = 9.2 Hz, 1H), 5.06−5.01 (m, 1H),

4.67 (t, J = 8.0 Hz, 1H), 4.46−4.44 (m, 1H), 4.22−4.19 (m, 1H),

3.91 (d, J = 17.2 Hz, 1H), 3.61−3.58 (m, 1H), 2.46 (s, 3H), 2.37−

2.30 (m, 1H), 2.04−1.99 (m, 1H), 1.44 (d, J = 7.2 Hz, 3H), 1.35 (s,

9H), 0.96 (s, 9H). ¹³C NMR (CDCl₃, 100 MHz) δ (ppm) 172.22,

170.13, 156.15, 150.56, 148.21, 143.43, 131.74, 130.59, 129.49,

126.46, 80.18, 69.91, 58.86, 56.58, 48.74, 38.60, 36.02, 35.48, 28.34,

26.39, 22.17, 15.95. UPLC-MS (ESI⁺) calcd for C₂₈H₄₁N₄O₅S [M +

1]⁺: 545.28, found 545.35.

(2S,4R)-1-((S)-2-Amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-

1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

(58). The solid material (57) obtained above was dissolved in 4 N

HCl in dioxane (4 mL, 16 mmol) and MeOH (4.0 mL), and the

mixture was stirred at ambient temperature for 12 h. The mixture was

then concentrated and the residue was dried under vacuum to aﬀord

crude compound, which was puriﬁed by reversed-phase preparative

HPLC to aﬀord the pure ﬁnal compound (58) as an oﬀ-white solid.

UPLC-MS (ESI⁺) calcd for C₂₃H₃₃N₄O₃S [M + 1]⁺: 445.23, found

445.44.

Cell Culture. Human breast cancer cell lines MCF-7 (ATCC

HTB-22) and T47D (ATCC HTB-133) were purchased from the

American Type Culture Collection (ATCC), Manassas, VA. They

were maintained and cultured in Dulbecco’s Modiﬁed Eagle’s medium

(DMEM) containing 10% fetal bovine serum, 1 unit/mL of penicillin,

and 1 μg/mL of streptomycin. Cells with 3−8 passages after

purchasing were used in experiments as indicated.

Western Blot Analysis. Western blot analysis was performed

essentially as described previously (Hu et al, 2015; PMID 26358219).

Cells treated with indicated compounds were lysed in Radio-

immunoprecipitation Assay Protein Lysis and Extraction Buﬀer (25

mmol/L Tris·HCl, pH 7.6, 150 mmol/L NaCl, 1% Nonidet P-40, 1%

sodium deoxycholate, and 0.1% sodium dodecyl sulfate) containing

proteinase inhibitor cocktail (Roche Diagnostics, Mannheim,

Germany). After determination of protein concentration by BCA

assay (Fisher Scientiﬁc, Pittsburgh, PA), equal amounts of total

protein were electrophoresed through 10% SDS-polyacrylamide gels.

The separated protein bands were transferred onto PVDF membranes

(4-(2-(Ethylamino)ethoxy)phenyl)(6-hydroxy-2-(4-

hydroxyphenyl)benzo[b]thiophen-3-yl)methanone (53). BBr₃sol-

ution (8.0 mL, 1.0 M in DCM) (8.0 mmol, 4.0 equiv) was slowly

added under N₂to a solution of 52 (923 mg, 2.0 mmol, 1.0 equiv) in

30 mL of anhydrous DCM at 0 °C. The dark-red solution was stirred

at rt for 2 h, then MeOH (1.0 mL) was added dropwise to quench the

reaction. The solution was concentrated, and the residue was

dissolved in EtOAc (50 mL) then aqueous saturated NaHCO₃(50

mL) and EtOH (5 mL) were added. The organic layer was separated

and dried over anhydrous Na₂SO₄. After ﬁltration, the solution was

concentrated and the residue was puriﬁed by silica gel ﬂash column

chromatography with DCM:MeOH (10:1−5:1) to aﬀord the key

1

intermediate (53) as a yellow solid (520 mg, 60% yield). H NMR

(CD₃OD, 400 MHz) δ (ppm) 7.72 (d, J = 8.8 Hz, 2H), 7.43 (d, J =

8.8 Hz, 1H), 7.27 (d, J = 2.0 Hz, 1H), 7.17 (d, J = 8.8 Hz, 2H), 6.91

(d, J = 8.8 Hz, 2H), 6.87 (dd, J = 8.8 Hz, J = 2.4 Hz, 1H), 6.61 (d, J =

8.8 Hz, 2H), 4.27 (t, J = 4.8 Hz, 2H), 3.42 (t, J = 4.8 Hz, 2H), 3.14

(q, J = 7.2 Hz, 2H), 1.33 (t, J = 7.2 Hz, 3H). ¹³C NMR (CD₃OD, 100

MHz) δ (ppm) 195.41, 163.41, 159.22, 156.80, 144.30, 141.45,

134.21, 133.45, 132.55, 131.42, 131.00, 125.99, 124.68, 116.43,

116.08, 115.39, 107.90, 64.68, 47.41, 44.36, 11.40. UPLC-MS (ESI⁺)

calcd for C₂₅H₂₄NO₄S [M + 1]⁺: 434.14, found 434.11.

tert-Butyl (2S,4R)-4-Hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)-

phenyl)ethyl)carbamoyl)-pyrrolidine-1-carboxylate (56). Com-

pound 55, synthesized using the reported procedure,⁴⁷was dissolved

in 4 N HCl in dioxane (25 mL, 100 mmol) and MeOH (25 mL), and

the mixture was stirred at ambient temperature for 12 h. The mixture

was concentrated, and the residue was dried under vacuum to aﬀord

the intermediate, which was used in next step without further

puriﬁcation.

HATU (14.51 g, 38.2 mmol, 1.2 equiv) was added to a solution of

the intermediate (55) obtained above (6.95 g, 31.8 mmol, 1.0 equiv),

(2S,4R)-1-(tert-Butoxycarbonyl)-4-hydroxypyrrolidine-2-carboxylic

acid (7.36 g, 31.8 mmol, 1.0 equiv), and DIPEA (11.08 mL, 63.6

mmol, 2.0 equiv) in DMF (36 mL) at 0 °C under nitrogen. The

mixture was stirred at ambient temperature for 12 h. TLC showed

that the reaction was complete. The reaction mixture was quenched

T

DOI: 10.1021/acs.jmedchem.8b01572

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(GE Healthcare Life Sciences, Marlborough, MA) and blotted against

diﬀerent antibodies as indicated. The human estrogen receptor α

antibody (AB16460) were purchased from Abcam, Inc., Cambridge,

MA. The membranes were reblotted with horseradish peroxidase-

conjugated anti-glyceraldehyde-3-phosphate dehydrogenase antibody

(G9295) from Sigma-Aldrich Corporation, St. Louis, MO. The blots

were scanned, and the band intensities were quantiﬁed using

GelQuant.NET software provided by biochemlabsolutions.com. The

relative mean intensity of target proteins was expressed after

normalization to the intensity of glyceraldehyde-3-phosphate

dehydrogenase bands from individual repeats.

Cell Growth Assay. MCF-7 cancer cells or human mammary

epithelial cells (HMEC) were seeded at 1500/well in 96-well plates

overnight. One day after the seeding, they were treated with indicated

doses of compounds, respectively. The growth of the cells was

evaluated by colorimetric wst-8 assay 4 days after the compound

treatment by following the instructions of the manufacturer, Cayman

Chemical, Ann Arbor, MI.

Molecular Modeling. The binding pose of the N,N-diethylamino

analogue of raloxifene with ER was modeled with the structure (PDB

1ERR)⁴⁹cocrystallized with raloxifene using MOE program. Residues

were rebuilt if atoms were missing based on the amber10 library in

MOE, and protons were added using the “protonate 3D” module

considered by setting pH at 7, temperature at 300 K, and salt

concentration at 0.1 mol/L. Docking simulations were then

performed using raloxifene to deﬁne the binding site with crystallized

H₂O molecules preserved. The ligand was placed by “Triangle

matcher” and evaluated by London dG scoring. DGVI/WSA dG

scoring was then applied to rank the poses, and the top ranked pose

was selected. Figures appeared in this paper were prepared using the

PyMOL program (www.pymol.org).

Cloning and Puriﬁcation of VHL-ElonginB/C Complex. The

DNA sequence of VHL (coding for residues 54−213) was

constructed by PCR and inserted into a His-TEV expression vector⁵⁸

using ligation-independent cloning. The DNA sequences of Elongin B

(encoding residues 1−118) and Elongin C (encoding residues 1−96)

were constructed by PCR and inserted into pCDFDuet 1 using

Gibson assembly.⁵⁹BL21(DE3) cells were transformed simulta-

neously with both plasmids and grown in Terriﬁc broth at 37 °C until

an OD600 of 1.2. The cells were induced overnight with 0.4 mM

IPTG at 24 °C. Pelleted cells were freeze−thawed then resuspended

in 20 mM Tris HCl pH 7.0, 200 mM NaCl, and 0.1% β-

mercaptoethanol (bME) containing protease inhibitors. The cell

suspension was lysed by sonication and debris removed via

centrifugation. The supernatant was incubated at 4 °C for 1 h with

Ni-NTA (Qiagen) prewashed in 20 mM Tris-HCl pH 7.0, 200 mM

NaCl, and 10 mM imidazole. The protein complex was eluted in 20

mM Tris-HCl pH 7.0, 200 mM NaCl, and 300 mM imidazole,

dialyzed into 20 mM Tris-HCl pH 7.0, 150 mM NaCl, and 0.01%

bME, and incubated with TEV protease overnight at 4 °C. The

protein sample was reapplied to the Ni-NTA column to remove the

His-tag. The ﬂow through containing the VHL complex was diluted to

75 mM NaCl and applied to a HiTrap Q column (GE Healthcare).

The sample was eluted with a salt gradient (0.075−1 M NaCl),

concentrated and further puriﬁed on a Superdex S75 column (GE

Healthcare) pre-equilibrated with 20 mM Bis-Tris 7.0, 150 mM NaCl,

and 1 mM DTT. Samples were aliquoted and stored at −80 °C.

Determination of Binding Aﬃnities of Synthesized VHL

Ligands to VHL-ElonginB/C Complex (VHL). We have developed

a competitive, ﬂuorescence polarization (FP) assay to determine the

binding aﬃnities of our synthesized VHL ligands to VHL-ElonginB/C

complex (VHL) protein. For this purpose, we synthesized a

ﬂuorencently tagged VHL ligand (VHL-F1) (Figure S1(A) in

Supporting Information) and determined its K_dvalue to be 2.3

0.3 nM (Figure S1(B) in Supporting Information).

Final concentrations of VHL protein and VHL-F1 ﬂuorescent probe

were both 5 nM. Negative controls containing protein/probe complex

only (equivalent to 0% inhibition) and positive controls containing

only the probe (equivalent to 100% inhibition) were included in each

assay plate. FP values in millipolarization units (mP) were measured

using the PHERAstar FSX HTS plate reader (BMG Labtech, 13000

Weston Parkway, Suite 109, Cary, NC 27513, USA) in 96-well, black,

ﬂat bottom microplates (Greiner Bio-One North America, Inc. 4238

Capital Drive Monroe, NC, 28110, USA) at an excitation wavelength

of 485 nm and an emission wavelength of 530 nm. IC₅₀values were

determined by nonlinear regression ﬁtting of the competition curves.

K_ivalues of competitive inhibitors were obtained directly by nonlinear

regression ﬁtting, based upon the K_Dvalues of the probe and

concentrations of the protein and probe in the competitive assays. All

the FP competitive experiments were performed in duplicate in three

independent experiments.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.jmed-

chem.8b01572.

■

S

Structure characterization for compound 11, VH032,

1

43a−48a; H NMR, ¹³C NMR, and UPLC-MS analysis

of compounds 15 (ERD-148) and 32 (ERD-308 (PDF)

Molecular string ﬁles for all the ﬁnal target compounds

(CSV)

AUTHOR INFORMATION

Corresponding Author

*Phone: (734)615-0362. Fax: (734)647-9647. E-mail:

shaomeng@umich.edu.

■

ORCID

Biao Hu: 0000-0002-4691-6490

Shaomeng Wang: 0000-0002-8782-6950

Author Contributions

^#Jiantao Hu, Biao Hu, Mingliang Wang, Fuming Xu, and

Bukeyan Miao contributed equally.

Notes

The authors declare the following competing ﬁnancial

interest(s): The University of Michigan has ﬁled a patent

application on these ER degraders, which has been licensed by

Oncopia Therapeutics LLC. S. Wang, J. Hu, B. Hu, M. Wang,

F. Xu, and B. Miao are co-inventors on this patent application.

The University of Michigan has received a research grant from

Oncopia and owns equity in Oncipia. S. Wang is a co-founder

of Oncopia, owns equity in Oncopia and is a paid consultant

for Oncopia.

ACKNOWLEDGMENTS

■

This study is supported in part by funding from Oncopia

Therapeutics, LLC, the University of Michigan Comprehensive

Cancer Center Strategic Fund for Breast Cancer (to S.W.), the

University of Michigan Comprehensive Cancer Center Core

Grant from the National Cancer Institute, NIH (grant

P30CA046592).

ABBREVIATIONS USED

■

BC, breast cancer; ER, estrogen receptor; HER2, human

epidermal growth factor 2; SERM, selective estrogen receptor

modulator; SERD, selective estrogen receptor degrader; VHL,

von Hippel-Lindau; CRBN, cereblon; FP, ﬂuorescence polar-

ization; GAPDH, glyceraldehyde 3-phosphate dehydrogenase;

IC₅₀and K_ivalues of VHL ligands to VHL protein were then

determined in competitive binding experiments. Mixtures of 5 μL of

each compound in DMSO and 95 μL of preincubated VHL protein/

VHL-F1 tracer complex solution were added into assay plates which

were incubated at room temperature for 60 min with gentle shaking.

U

DOI: 10.1021/acs.jmedchem.8b01572

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(17) Joseph, J. D.; Darimont, B.; Zhou, W.; Arrazate, A.; Young, A.;

Ingalla, E.; Walter, K.; Blake, R. A.; Nonomiya, J.; Guan, Z.; Kategaya,

L.; Govek, S. P.; Lai, A. G.; Kahraman, M.; Brigham, D.; Sensintaffar,

J.; Lu, N.; Shao, G.; Qian, J.; Grillot, K.; Moon, M.; Prudente, R.;

Bischoff, E.; Lee, K. J.; Bonnefous, C.; Douglas, K. L.; Julien, J. D.;

Nagasawa, J. Y.; Aparicio, A.; Kaufman, J.; Haley, B.; Giltnane, J. M.;

Wertz, I. E.; Lackner, M. R.; Nannini, M. A.; Sampath, D.; Schwarz,

L.; Manning, H. C.; Tantawy, M. N.; Arteaga, C. L.; Heyman, R. A.;

Rix, P. J.; Friedman, L.; Smith, N. D.; Metcalfe, C.; Hager, J. H. The

selective estrogen receptor downregulator GDC-0810 is efficacious in

diverse models of ER+ breast cancer. eLife 2016, 5, No. e15828.

(18) Bihani, T.; Patel, H. K.; Arlt, H.; Tao, N.; Jiang, H.; Brown, J.

L.; Purandare, D. M.; Hattersley, G.; Garner, F. Elacestrant

(RAD1901), a selective estrogen receptor degrader (SERD), has

antitumor activity in multiple ER+ breast cancer patient-derived

xenograft models. Clin. Cancer Res. 2017, 23, 4793−4804.

(19) Tria, G. S.; Abrams, T.; Baird, J.; Burks, H. E.; Firestone, B.;

Gaither, L. A.; Hamann, L. G.; He, G.; Kirby, C. A.; Kim, S.;

Lombardo, F.; Macchi, K. J.; McDonnell, D. P.; Mishina, Y.; Norris, J.

D.; Nunez, J.; Springer, C.; Sun, Y.; Thomsen, N. M.; Wang, C.;

Wang, J.; Yu, B.; Tiong-Yip, C. L.; Peukert, S. Discovery of LSZ102, a

potent, orally bioavailable selective estrogen receptor degrader

(SERD) for the treatment of estrogen receptor positive breast cancer.

J. Med. Chem. 2018, 61, 2837−2864.

PBS, phosphate buﬀered saline; SNIPER, speciﬁc and

nongenetic IAP-dependent protein erasers; PROTAC, pro-

teolysis targeting chimera; DMSO, dimethyl sulfoxide; HATU,

hexaﬂuorophosphate azabenzotriazole tetramethyl uronium;

DIPEA, N,N-diisopropylethylamine; DMF, N,N-dimethylfor-

mamide; DMA, N,N-dimethylacetamide; qPCR, quantitative

polymerase chain reaction; pGR, progesterone receptor;

GREB1, growth regulation by estrogen in breast cancer 1;

HMEC, human mammary epithelial cells.

REFERENCES

■

(1) Tong, C. W. S.; Wu, M.; Cho, W. C. S.; To, K. K. W. Recent

advances in the treatment of breast cancer. Front. Oncol. 2018, 8, 227.

(2) Anderson, W. F.; Katki, H. A.; Rosenberg, P. S. Incidence of

breast cancer in the United States: current and future trends. J. Natl.

Cancer Inst. 2011, 103, 1397−1402.

(3) Nilsson, S.; Koehler, K. F.; Gustafsson, J. A. Development of

subtype-selective oestrogen receptor-based therapeutics. Nat. Rev.

Drug Discovery 2011, 10, 778−792.

(4) Jordan, V. C. Tamoxifen: a most unlikely pioneering medicine.

Nat. Rev. Drug Discovery 2003, 2, 205−213.

(5) Das, S.; Crockett, J. C. Osteoporosis - a current view of

pharmacological prevention and treatment. Drug Des., Dev. Ther.

2013, 7, 435−448.

(20) Carlson, R. W. The history and mechanism of action of

fulvestrant. Clin. Breast Cancer 2005, 6, S5−S8.

(6) De Marchi, T.; Foekens, J. A.; Umar, A.; Martens, J. W.

Endocrine therapy resistance in estrogen receptor (ER)-positive

breast cancer. Drug Discovery Today 2016, 21, 1181−1188.

(7) AlFakeeh, A.; Brezden-Masley, C. Overcoming endocrine

resistance in hormone receptor-positive breast cancer. Curr. Oncol.

2018, 25, 18−27.

(8) Martin, L. A.; Ribas, R.; Simigdala, N.; Schuster, E.; Pancholi, S.;

Tenev, T.; Gellert, P.; Buluwela, L.; Harrod, A.; Thornhill, A.;

Nikitorowicz-Buniak, J.; Bhamra, A.; Turgeon, M. O.; Poulogiannis,

G.; Gao, Q.; Martins, V.; Hills, M.; Garcia-Murillas, I.; Fribbens, C.;

Patani, N.; Li, Z.; Sikora, M. J.; Turner, N.; Zwart, W.; Oesterreich, S.;

Carroll, J.; Ali, S.; Dowsett, M. Discovery of naturally occurring ESR1

mutations in breast cancer cell lines modelling endocrine resistance.

Nat. Commun. 2017, 8, 1865.

(21) Marsaud, V.; Gougelet, A.; Maillard, S.; Renoir, J. M. Various

phosphorylation pathways, depending on agonist and antagonist

binding to endogenous estrogen receptor α (ERα), differentially affect

ERα extractability, proteasome-mediated stability, and transcriptional

activity in human breast cancer cells. Mol. Endocrinol. 2003, 17,

2013−2027.

(22) Wittmann, B. M.; Sherk, A.; McDonnell, D. P. Definition of

functionally important mechanistic differences among selective

estrogen receptor down-regulators. Cancer Res. 2007, 67, 9549−9560.

(23) Sakamoto, K. M.; Kim, K. B.; Kumagai, A.; Mercurio, F.;

Crews, C. M.; Deshaies, R. J. Protacs: chimeric molecules that target

proteins to the Skp1-Cullin-F box complex for ubiquitination and

degradation. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 8554−8559.

(24) Toure, M.; Crews, C. M. Small-molecule PROTACS: new

approaches to protein degradation. Angew. Chem., Int. Ed. 2016, 55,

1966−1973.

(9) Nardone, A.; De Angelis, C.; Trivedi, M. V.; Osborne, C. K.;

Schiff, R. The changing role of ER in endocrine resistance. Breast

2015, 24, S60−S66.

(25) Lai, A. C.; Crews, C. M. Induced protein degradation: an

emerging drug discovery paradigm. Nat. Rev. Drug Discovery 2017, 16,

101−114.

(10) Robertson, J. F.; Harrison, M. Fulvestrant: pharmacokinetics

and pharmacology. Br. J. Cancer 2004, 90, S7−S10.

(11) Osborne, C. K.; Wakeling, A.; Nicholson, R. I. Fulvestrant: an

oestrogen receptor antagonist with a novel mechanism of action. Br. J.

Cancer 2004, 90, S2−S6.

(26) Burslem, G. M.; Crews, C. M. Small-molecule modulation of

protein homeostasis. Chem. Rev. 2017, 117, 11269−11301.

(27) Cromm, P. M.; Crews, C. M. Targeted protein degradation:

from chemical biology to drug discovery. Cell Chem. Biol. 2017, 24,

1181−1190.

(12) Howell, A.; Sapunar, F. Fulvestrant revisited: efficacy and safety

of the 500-mg dose. Clin. Breast Cancer 2011, 11, 204−210.

(13) Robertson, J. F.; Lindemann, J.; Garnett, S.; Anderson, E.;

Nicholson, R. I.; Kuter, I.; Gee, J. M. A good drug made better: the

fulvestrant dose-response story. Clin. Breast Cancer 2014, 14, 381−

389.

(28) Ottis, P.; Crews, C. M. Proteolysis-targeting chimeras: induced

protein degradation as a therapeutic strategy. ACS Chem. Biol. 2017,

12, 892−898.

(29) Rodriguez-Gonzalez, A.; Cyrus, K.; Salcius, M.; Kim, K.; Crews,

C. M.; Deshaies, R. J.; Sakamoto, K. M. Targeting steroid hormone

receptors for ubiquitination and degradation in breast and prostate

cancer. Oncogene 2008, 27, 7201−7211.

(30) Jiang, Y.; Deng, Q.; Zhao, H.; Xie, M.; Chen, L.; Yin, F.; Qin,

X.; Zheng, W.; Zhao, Y.; Li, Z. Development of stabilized peptide-

based PROTACs against estrogen receptor α. ACS Chem. Biol. 2018,

13, 628−635.

(31) Winter, G. E. B. D. L.; Buckley, D. L.; Paulk, J.; Roberts, J. M.;

Souza, A.; Dhe-Paganon, S.; Bradner, J. E. Phthalimide conjugation as

a strategy for in vivo target protein degradation. Science 2015, 348,

1376−1381.

(32) Lu, J.; Qian, Y.; Altieri, M.; Dong, H.; Wang, J.; Raina, K.;

Hines, J.; Winkler, J. D.; Crew, A. P.; Coleman, K.; Crews, C. M.

Hijacking the E3 ubiquitin ligase cereblon to efficiently target BRD4.

Chem. Biol. 2015, 22, 755−763.

(14) McDonnell, D. P.; Wardell, S. E.; Norris, J. D. Oral selective

estrogen receptor downregulators (SERDs), a breakthrough endo-

crine therapy for breast cancer. J. Med. Chem. 2015, 58, 4883−4887.

(15) Abdel-Magid, A. F. Selective estrogen receptor degraders

(SERDs): a promising treatment to overcome resistance to endocrine

therapy in ERα-positive breast cancer. ACS Med. Chem. Lett. 2017, 8,

1129−1131.

(16) Weir, H. M.; Bradbury, R. H.; Lawson, M.; Rabow, A. A.;

Buttar, D.; Callis, R. J.; Curwen, J. O.; de Almeida, C.; Ballard, P.;

Hulse, M.; Donald, C. S.; Feron, L. J.; Karoutchi, G.; MacFaul, P.;

Moss, T.; Norman, R. A.; Pearson, S. E.; Tonge, M.; Davies, G.;

Walker, G. E.; Wilson, Z.; Rowlinson, R.; Powell, S.; Sadler, C.;

Richmond, G.; Ladd, B.; Pazolli, E.; Mazzola, A. M.; D’Cruz, C.; De

Savi, C. AZD9496: an oral estrogen receptor inhibitor that blocks the

growth of ER-positive and ESR1-mutant breast tumors in preclinical

models. Cancer Res. 2016, 76, 3307−3318.

V

DOI: 10.1021/acs.jmedchem.8b01572

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(33) Bondeson, D. P.; Mares, A.; Smith, I. E.; Ko, E.; Campos, S.;

Miah, A. H.; Mulholland, K. E.; Routly, N.; Buckley, D. L.; Gustafson,

J. L.; Zinn, N.; Grandi, P.; Shimamura, S.; Bergamini, G.; Faelth-

Savitski, M.; Bantscheff, M.; Cox, C.; Gordon, D. A.; Willard, R. R.;

Flanagan, J. J.; Casillas, L. N.; Votta, B. J.; den Besten, W.; Famm, K.;

Kruidenier, L.; Carter, P. S.; Harling, J. D.; Churcher, I.; Crews, C. M.

Catalytic in vivo protein knockdown by small-molecule PROTACs.

Nat. Chem. Biol. 2015, 11, 611−617.

(34) Lai, A. C.; Toure, M.; Hellerschmied, D.; Salami, J.; Jaime-

Figueroa, S.; Ko, E.; Hines, J.; Crews, C. M. Modular PROTAC

design for the degradation of oncogenic BCR-ABL. Angew. Chem., Int.

Ed. 2016, 55, 807−810.

(35) Robb, C. M.; Contreras, J. I.; Kour, S.; Taylor, M. A.; Abid, M.;

Sonawane, Y. A.; Zahid, M.; Murry, D. J.; Natarajan, A.; Rana, S.

Chemically induced degradation of CDK9 by a proteolysis targeting

chimera (PROTAC). Chem. Commun. 2017, 53, 7577−7580.

(36) Zhang, C.; Han, X. R.; Yang, X.; Jiang, B.; Liu, J.; Xiong, Y.; Jin,

J. Proteolysis targeting chimeras (PROTACs) of anaplastic lymphoma

kinase (ALK). Eur. J. Med. Chem. 2018, 151, 304−314.

(37) Lu, M.; Liu, T.; Jiao, Q.; Ji, J.; Tao, M.; Liu, Y.; You, Q.; Jiang,

Z. Discovery of a Keap1-dependent peptide PROTAC to knockdown

Tau by ubiquitination-proteasome degradation pathway. Eur. J. Med.

Chem. 2018, 146, 251−259.

(38) Burslem, G. M.; Smith, B. E.; Lai, A. C.; Jaime-Figueroa, S.;

McQuaid, D. C.; Bondeson, D. P.; Toure, M.; Dong, H.; Qian, Y.;

Wang, J.; Crew, A. P.; Hines, J.; Crews, C. M. The advantages of

targeted protein degradation over inhibition: an RTK case study. Cell

Chem. Biol. 2018, 25, 67−77.

(39) Schiedel, M.; Herp, D.; Hammelmann, S.; Swyter, S.; Lehotzky,

A.; Robaa, D.; Olah, J.; Ovadi, J.; Sippl, W.; Jung, M. Chemically

induced degradation of sirtuin 2 (sirt2) by a proteolysis targeting

chimera (PROTAC) based on sirtuin rearranging ligands (SirReals).

J. Med. Chem. 2018, 61, 482−491.

(47) Raina, K.; Lu, J.; Qian, Y.; Altieri, M.; Gordon, D.; Rossi, A. M.

K.; Wang, J.; Chen, X.; Dong, H.; Siu, K.; Winkler, J. D.; Crew, A. P.;

Crews, C. M.; Coleman, K. G. PROTAC-induced BET protein

degradation as a therapy for castration-resistant prostate cancer. Proc.

Natl. Acad. Sci. U. S. A. 2016, 113, 7124−7129.

(48) Zhou, B.; Hu, J.; Xu, F.; Chen, Z.; Bai, L.; Fernandez-Salas, E.;

Lin, M.; Liu, L.; Yang, C. Y.; Zhao, Y.; McEachern, D.; Przybranowski,

S.; Wen, B.; Sun, D.; Wang, S. Discovery of a small-molecule degrader

of bromodomain and extra-terminal (BET) proteins with picomolar

cellular potencies and capable of achieving tumor regression. J. Med.

Chem. 2018, 61, 462−481.

(49) Brzozowski, A. M.; Pike, A. C. W.; Dauter, Z.; Hubbard, R. E.;

Bonn, T.; Engstrom, O.; Ohman, L.; Greene, G. L.; Gustafsson, J.-Å.;

Carlquist, M. Molecular basis of agonism and antagonism in the

oestrogen receptor. Nature 1997, 389, 753−758.

(50) Qin, C.; Hu, Y.; Zhou, B.; Fernandez-Salas, E.; Yang, C. Y.; Liu,

L.; McEachern, D.; Przybranowski, S.; Wang, M.; Stuckey, J.;

Meagher, J.; Bai, L.; Chen, Z.; Lin, M.; Yang, J.; Ziazadeh, D. N.;

Xu, F.; Hu, J.; Xiang, W.; Huang, L.; Li, S.; Wen, B.; Sun, D.; Wang, S.

Discovery of QCA570 as an exceptionally potent and efficacious

proteolysis targeting chimera (PROTAC) degrader of the bromodo-

main and extra-terminal (BET) proteins capable of inducing complete

and durable tumor regression. J. Med. Chem. 2018, 61, 6685−6704.

(51) Bai, L.; Zhou, B.; Yang, C. Y.; Ji, J.; McEachern, D.;

Przybranowski, S.; Jiang, H.; Hu, J.; Xu, F.; Zhao, Y.; Liu, L.;

Fernandez-Salas, E.; Xu, J.; Dou, Y.; Wen, B.; Sun, D.; Meagher, J.;

Stuckey, J.; Hayes, D. F.; Li, S.; Ellis, M. J.; Wang, S. Targeted

degradation of BET proteins in triple-negative breast cancer. Cancer

Res. 2017, 77, 2476−2487.

(52) Buckley, D. L.; Van Molle, I.; Gareiss, P. C.; Tae, H. S.; Michel,

J.; Noblin, D. J.; Jorgensen, W. L.; Ciulli, A.; Crews, C. M. Targeting

the von Hippel-Lindau E3 ubiquitin ligase using small molecules to

disrupt the VHL/HIF-1α interaction. J. Am. Chem. Soc. 2012, 134,

4465−4468.

(40) Shibata, N.; Nagai, K.; Morita, Y.; Ujikawa, O.; Ohoka, N.;

Hattori, T.; Koyama, R.; Sano, O.; Imaeda, Y.; Nara, H.; Cho, N.;

Naito, M. Development of protein degradation inducers of androgen

receptor by conjugation of androgen receptor ligands and inhibitor of

apoptosis protein ligands. J. Med. Chem. 2018, 61, 543−575.

(41) Crew, A. P.; Raina, K.; Dong, H.; Qian, Y.; Wang, J.; Vigil, D.;

Serebrenik, Y. V.; Hamman, B. D.; Morgan, A.; Ferraro, C.; Siu, K.;

Neklesa, T. K.; Winkler, J. D.; Coleman, K. G.; Crews, C. M.

Identification and characterization of von Hippel-Lindau-recruiting

proteolysis targeting chimeras (PROTACs) of TANK-binding kinase

1. J. Med. Chem. 2018, 61, 583−598.

(42) Sun, Y.; Zhao, X.; Ding, N.; Gao, H.; Wu, Y.; Yang, Y.; Zhao,

M.; Hwang, J.; Song, Y.; Liu, W.; Rao, Y. PROTAC-induced BTK

degradation as a novel therapy for mutated BTK C481S induced

ibrutinib-resistant B-cell malignancies. Cell Res. 2018, 28, 779−781.

(43) Yang, K.; Song, Y.; Xie, H.; Wu, H.; Wu, Y. T.; Leisten, E. D.;

Tang, W. Development of the first small molecule histone deacetylase

6 (HDAC6) degraders. Bioorg. Med. Chem. Lett. 2018, 28, 2493−

2497.

(44) Ohoka, N.; Okuhira, K.; Ito, M.; Nagai, K.; Shibata, N.; Hattori,

T.; Ujikawa, O.; Shimokawa, K.; Sano, O.; Koyama, R.; Fujita, H.;

Teratani, M.; Matsumoto, H.; Imaeda, Y.; Nara, H.; Cho, N.; Naito,

M. In vivo knockdown of pathogenic proteins via specific and

nongenetic inhibitor of apoptosis protein (IAP)-dependent protein

erasers (SNIPERs). J. Biol. Chem. 2017, 292, 4556−4570.

(45) Ohoka, N.; Morita, Y.; Nagai, K.; Shimokawa, K.; Ujikawa, O.;

Fujimori, I.; Ito, M.; Hayase, Y.; Okuhira, K.; Shibata, N.; Hattori, T.;

Sameshima, T.; Sano, O.; Koyama, R.; Imaeda, Y.; Nara, H.; Cho, N.;

Naito, M. Derivatization of inhibitor of apoptosis protein (IAP)

ligands yields improved inducers of estrogen receptor α degradation.

J. Biol. Chem. 2018, 293, 6776−6790.

(53) Buckley, D. L.; Gustafson, J. L.; Van Molle, I.; Roth, A. G.; Tae,

H. S.; Gareiss, P. C.; Jorgensen, W. L.; Ciulli, A.; Crews, C. M. Small-

molecule inhibitors of the interaction between the E3 ligase VHL and

HIF-1α. Angew. Chem., Int. Ed. 2012, 51, 11463−11467.

(54) Galdeano, C.; Gadd, M. S.; Soares, P.; Scaffidi, S.; Van Molle,

I.; Birced, I.; Hewitt, S.; Dias, D. M.; Ciulli, A. Structure-guided

design and optimization of small molecules targeting the protein-

protein interaction between the von Hippel-Lindau (VHL) E3

ubiquitin ligase and the hypoxia inducible factor (HIF) α subunit with

in vitro nanomolar affinities. J. Med. Chem. 2014, 57, 8657−8663.

(55) Soares, P.; Gadd, M. S.; Frost, J.; Galdeano, C.; Ellis, L.;

Epemolu, O.; Rocha, S.; Read, K. D.; Ciulli, A. Group-based

optimization of potent and cell-active inhibitors of the von Hippel-

Lindau (VHL) E3 ubiquitin ligase: structure-activity relationships

leading to the chemical probe (2S,4R)-1-((S)-2-(1-cyanocyclopropa-

necarboxamido)-3,3-dimethylbutanoyl)-4-hydroxy -N-(4-(4-methyl-

thiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (VH298). J. Med.

Chem. 2018, 61, 599−618.

(56) Gadd, M. S.; Testa, A.; Lucas, X.; Chan, K. H.; Chen, W.;

Lamont, D. J.; Zengerle, M.; Ciulli, A. Structural basis of PROTAC

cooperative recognition for selective protein degradation. Nat. Chem.

Biol. 2017, 13, 514−521.

(57) Long, M. J.; Poganik, J. R.; Aye, Y. On-demand targeting:

investigating biology with proximity-directed chemistry. J. Am. Chem.

Soc. 2016, 138, 3610−3622.

(58) Stols, L.; Gu, M.; Dieckman, L.; Raffen, R.; Collart, F. R.;

Donnelly, M. I. A new vector for high-throughput, ligation-

independent cloning encoding a tobacco etch virus protease cleavage

site. Protein Expression Purif. 2002, 25, 8−15.

(59) Benoit, R. M.; Ostermeier, C.; Geiser, M.; Li, J. S.; Widmer, H.;

Auer, M. Seamless insert-plasmid assembly at high efficiency and low

cost. PLoS One 2016, 11, No. e0153158.

(46) Ottis, P.; Toure, M.; Cromm, P. M.; Ko, E.; Gustafson, J. L.;

Crews, C. M. Assessing different E3 ligases for small molecule induced

protein ubiquitination and degradation. ACS Chem. Biol. 2017, 12,

2570−2578.

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J. Med. Chem. XXXX, XXX, XXX−XXX

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Discovery of ERD-308 as a Highly Potent Proteolysis Targeting Chimera (PROTAC) Degrader of Estrogen Receptor (ER)

DOI: 10.1021/acs.jmedchem.8b01572

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