Bispecific Estrogen Receptor α Degraders Incorporating Novel Binders Identified Using DNA-Encoded Chemical Library Screening

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Bispeciﬁc Estrogen Receptor α Degraders Incorporating Novel

Binders Identiﬁed Using DNA-Encoded Chemical Library Screening

Jeremy S. Disch,* Jennifer M. Du ﬀ y, Esther C. Y. Lee, Diana Gikunju, Betty Chan, Benjamin Levin,

Michael I. Monteiro, Sarah A. Talcott, Anthony C. Lau, Fei Zhou, Anton Kozhushnyan,

Neil E. Westlund, Patrick B. Mullins, Yan Yu, Moritz von Rechenberg, Junyi Zhang, Yelena A. Arnautova,

Yanbin Liu, Ying Zhang, Andrew J. McRiner, Anthony D. Keefe, Anna Kohlmann, Matthew A. Clark,

John W. Cuozzo, Christelle Huguet, and Shilpi Arora *

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ABSTRACT: Bispeciﬁc degraders (PROTACs) of ERα are

expected to be advantageous over current inhibitors of ERα

signaling (aromatase inhibitors/SERMs/SERDs) used to treat ER+

breast cancer. Information from DNA-encoded chemical library

(DECL) screening provides a method to identify novel PROTAC

binding features as the linker positioning, and binding elements are

determined directly from the screen. After screening ∼120 billion

DNA-encoded molecules with ERα WT and 3 gain-of-function

(GOF) mutants, with and without estradiol to identify features that

enrich ERα competitively, the oﬀ-DNA synthesized small molecule

exemplar 7 exhibited nanomolar ERα binding, antagonism, and

degradation. Click chemistry synthesis on an alkyne E3 ligase

engagers panel and an azide variant of 7 rapidly generated bispeciﬁc nanomolar degraders of ERα, with PROTACs 18 and 21

inhibiting ER+ MCF7 tumor growth in a mouse xenograft model of breast cancer. This study validates this approach toward

identifying novel bispeciﬁc degrader leads from DECL screening with minimal optimization.

from Eli Lilly, Novartis, G1 Therapeutics, and Pﬁzer.⁴⁻⁷For

currently approved therapeutics, despite initial eﬃcacy, the

development of acquired resistance limits their utility. Gain-of-

function (GOF) mutations in ERα are enriched in one-third of

INTRODUCTION

■

Breast cancer is the most common cancer in women, with an

annual worldwide incidence of ∼2 million and accounting for

15% of all cancer-related deaths in women.¹Estrogen receptor

endocrine therapy-resistant recurrences, leading to ligand-

(ER) is a ligand-dependent nuclear hormone receptor, which,

upon activation, acts as a transcription factor in normal breast

cells. ERα, one of the two main subtypes of estrogen receptor,

is over-expressed in ∼70% of all breast cancers. ERα signaling

is responsible for cancer cell proliferation, survival, and

independent activation of the ERα pathway,^8,9thereby

conferring partial resistance to existing classes of endocrine

therapies. These acquired mutations are associated with

aggressive disease biology and reduced overall survival.¹⁰The

most common among these GOF mutations result in altering

metastasis in breast cancer.²Approved endocrine therapies

the amino acids Y537 and D538, generating a constitutively

include aromatase inhibitors (AIs) such as letrozole, selective

active ER.¹¹⁻¹³These GOF mutants are resistant to estrogen

ER modulators (SERMs) such as tamoxifen, and selective ER

deprivation and are less responsive to tamoxifen or Fulvestrant.

degrader (SERDs) such as Fulvestrant. These therapies have

Thus, there is a need to develop a next generation of ERα-

allowed for eﬀective management of ER+ breast cancer.³

targeted therapeutics that can address aberrant activities of

Fulvestrant’s approval as a ﬁrst line therapy for metastatic ER+

both wild-type and GOF mutants of ERα.

breast cancer suggests that degradation of estrogen receptor is

a valid and useful strategy for treating breast cancer.⁴Poor

Received: January 22, 2021

Published: April 12, 2021

pharmacokinetic properties of Fulvestrant have inspired the

development of second generation SERDs, both steroidal and

non-steroidal, that are currently in clinical trials or under

development. These include compounds in Phase 2/3 clinical

trials, Elacestrant (Radius), RG6171 (Roche), SAR439839

(Sanoﬁ), and AZD9833 (AstraZeneca), and others in Phase 1

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Figure 1. Schematic for DECL screening and PROTAC array platforms. (a) DNA-encoded chemical library (combinatorial synthesized small

molecules tethered to DNA tags) is incubated with ERα, aﬃnity captured, washed to remove non-binding library, and then enriched library

members are eluted. After repeating an additional round, the DNA tags are sequenced to identify the small molecule binders. (b) Azide variants of

the binders are prepared and reacted in a parallel click chemistry library of E3 binder alkynes to obtain an array of PROTACs for ERα.

Recently, bispeciﬁc degraders (PROTACs, proteolysis

targeting chimeras) have emerged as a novel therapeutic

strategy in drug discovery and development and the ﬁrst

PROTAC compounds have recently entered into clinical

trials.^14,15These bispeciﬁc degraders utilize two active domains

attached with a linker, allowing for the protein of interest

(POI) to be held in close proximity to a targeted E3 ligase,

thus targeting the former for degradation through the ubiquitin

proteasome system.^16,17Most ERα-targeting bifunctional

degraders are based on well-known binders to ERα, such as

raloxifene, tamoxifene, lasofoxifene, or estradiol, among

others.^4,18−23Though an immensely promising technology,

the ﬁeld acknowledges issues with optimization of these

molecules, such as linker presentation, which may be a limiting

factor in optimal design. However, one of the biggest

challenges is identifying novel POI and ubiquitin ligase

binders.²⁴The necessity for a tether may be best approached

using screening methodologies that utilize tethered molecules.

Thus, an approach like DNA-encoded chemical libraries

(DECL) screening may be the best option for identifying

such novel ligands.

Herein, we report the discovery of a series of novel ERα

bispeciﬁc degraders (PROTACs) that target both WT and

mutant ERα, by utilizing DECL screening to discover novel

ERα binders and antagonists, and quickly using this

information to develop novel POI engagers for generating

bifunctional degraders. DECL platforms have emerged as an

attractive approach to aﬃnity screen billions of compounds in

a single selection experiment.²⁵This platform uses split and

pool combinatorial chemistry to synthesize very large,

thousands to billions, member combinatorial libraries, in

which a unique DNA sequence is covalently tethered to each

small molecule. The DNA sequence is used to record the

library, building block, and reaction information during the

course of library synthesis. DECL technology has generated

several clinical candidates^2627−28and, most recently, has been

extended to screens in live cells²⁹and used to train machine

learning models.³⁰

In a typical DECL screening campaign (Figure 1a), DNA-

encoded libraries are incubated with a protein, aﬃnity

captured, and washed to enrich the library pool. The ﬁnal

selection output is ampliﬁed, sequenced, and translated to

identify the structures of the tethered small molecules. DECL

selections are frequently multiplexed, allowing for comparative

evaluation of enrichment to deﬁne a mechanism of action. For

instance, by screening homologs or including a known

competitor, substrate, or cofactor during the selection process,

one can focus on a particular binding proﬁle, mutant, or

species speciﬁcity.^25,29Once the structures are translated from

the DNA tag sequences, structures are ﬁltered by removing

promiscuous and matrix binders. After evaluating the enrich-

ment proﬁle across the multiplexed selection conditions, hits

are synthesized oﬀ-DNA to conﬁrm binding in assays.

A key advantage of the DECL platform is that a substantially

large collection of novel tethered molecules is being presented

to the target in their linked presentation, a key requirement for

the development of PROTACs. After identiﬁcation of a valid

binder to a protein of interest, the DECL information can be

directly leveraged to produce PROTACs very eﬃciently by

preparing an azide linked variant with the DNA tag replaced by

an azide and then cross-reacted with a series of E3 binder

alkynes utilizing click chemistry³¹(Figure 1b). Ultimately, this

would lead to sets of PROTACs, with novel POI binders

identiﬁed by large combinatorial space to progress into

biochemistry, cellular biology, ADME, and eﬃcacy studies.

RESULTS

■

Aﬃnity-Mediated Selection of DNA-Encoded Chem-

ical Libraries. To identify novel ERα binders, we conducted a

DNA chemical library aﬃnity screen utilizing X-Chem’s ∼120-

billion-member library collection. A collection of 44 distinct

DNA-encoded chemical libraries were combined and incu-

bated with no target, the ligand binding domains (LBD) of

ERα WT 7.7 μM to 24 nM, ERα WT at 7.7 μM with estradiol

at 15 μM, and each of three mutant forms of ERα, D538G,

S463P, and Y537S at 7.7 μM. Supplementary Figure S1 shows

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Figure 2. DECL selection output and oﬀ-DNA activity conﬁrmation of two related members. (a, b) Schematic of two structurally related 2-cycle

library members and their enrichment (ENRv1) suggests they bind ERα WT and mutants Y537S, S463P, and D538G. These library members do

not bind the matrix (NTC; no target control) and are competed oﬀ by the presence of Estradiol (E2), suggesting that these library members bind

in the ligand binding site. (c) Analogous “oﬀ-DNA” synthesized exemplars, with the linker and DNA truncated to a single methyl, bind in

ﬂuorescence polarization binding displacement assay. Assay results are an average of duplicate runs.

a denaturing PAGE of all the ERα LBD variants used in the

aﬃnity-mediated selection with the DNA-encoded chemical

libraries. Proteins and associated library members were

captured using an IMAC matrix followed by stringent washing

and elution at 85 °C. The output of the ﬁrst round of selection

was used as the input for the second round of selection with

fresh protein and competitor reagents, and the output of the

second round of selection was PCR-ampliﬁed and submitted

for Illumina sequencing. A total of 528 million single-end reads

were generated across all 44 libraries, and all selection

conditions averaged 1.2 million reads per library per selection

condition.

Selection Output Data Analysis and Hit ID. Individual

sequence reads were translated back into the corresponding

building block, and library schemes and statistical prevalence

data were calculated for all building block combinations in

each library across all selection conditions. A large number of

strongly enriched building block combinations were observed

across all libraries. During the process of deciding which

compounds to synthesize oﬀ-DNA for hit-conﬁrmation, a

range of parameters was considered including the extent of

enrichment, the proﬁle of enrichment across the diﬀerent

protein variants, whether competition was observed with

estradiol, how the enrichment extent varied across the dilution

series, and the extent to which “SAR” was observed for co-

enriching building blocks that manifest themselves in

structurally related compounds. Enrichment proﬁles were

calculated for each building block combination and the extent

to which individual building block combinations co-enriched.

Data are presented in Figure 2a,b for two individual building

block pairs (disynthons) that were resynthesized oﬀ-DNA,

determined to be active, and then used as the founder

members or hits to establish the series. The relative

enrichment of the two representative building block pairs

can be seen across the selection conditions with each showing

signiﬁcant enrichment at 7.7 and 1.6 μM ERα, competition

with estradiol, and also signiﬁcant enrichment against the

Y537S and S463P mutants with lesser enrichment against the

D538G mutant. ENRv1 represents a statistical metric of

enrichment signiﬁcance for each disynthon (negative log10 of

the asymptotic signiﬁcance value).³²

were evaluated. Two of the most potent oﬀ-DNA representa-

tive compounds (1 and 2) identiﬁed in the screen are shown in

Figure 2c. The potency of these ERα binders was assessed in

an in vitro biochemical FP (ﬂuorescence polarization) assay

that was established for ERα WT and three of the most

commonly occurring mutants, S463P, Y537S, and D538G.

Compounds identiﬁed to be active in the biochemical assay

were then tested in a dose titration in the ER+ cell line, MCF7,

in a proliferation assay in both antagonist and agonist mode.³³

The preferred phenotype in the cell-based assay is an anti-

proliferative eﬀect on ER-dependent cells in antagonist mode

(+E2), with no increase in growth of cells seen in hormone-

deprived media (agonist mode).³³In addition to the

proliferation assay, PROTACs were also tested in an ERα

degradation assay in MCF7 cells (in-cell western assay) to

conﬁrm the degradation of ERα in cells. ERα levels were

assessed in dose titration upon 24 or 48 h exposure to the

respective compounds.³³

The chlorophenol compounds (1 and 2) were potent

binders of WT and mutant (S463P, Y537S, and D538G) ERα

LBDs with 2 having 2- to 5-fold higher potency than 1 (Figure

2c), and both had modest cellular agonist activity (0.46−0.55

μM) in MCF7 cells (Table 1). With the guidance of the DECL

screening selection data and molecular docking, we designed,

prepared, and evaluated the in vitro small molecule SAR with

an emphasis on potency and antagonism. The DNA-encoded

library information indicated that the likely solvent accessible

exit vector was at the tertiary amine that provides the

connection between the encoded compounds and the linker

to DNA. However, docking also suggested other poses with the

ﬂuorophenyl ring projecting toward solvent, with the phenol

also buried deep in the pocket. We sought an approach to

build SAR by maintaining potency while modulating the

cellular activity from agonism to antagonism by appending a

polar amine group oﬀ these vectors to interact with Asp351,

which is known to introduce antagonism (see Table 1).

Initially replacing the methyl with phenyl at R₁gave compound

3, which maintained binding activity (15 nM); however, this

change resulted in producing a more potent cellular agonist (3,

GI₅₀< 5 nM). Progressively longer alkyl-piperidine chains 4−7

correlated with improvements in potency, with 7 having the

desired characteristics of notably potent antagonism, no

observed cellular agonism, and interestingly, degradation of

ERα as a small molecule in an in-cell western assay (DC₅₀= 15

Hit Conﬁrmation and SAR Follow-Up. To conﬁrm the

selection output, direct representative oﬀ-DNA compounds

were synthesized, and their biochemical and cellular activities

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a

capitalizes on Huisgen 1,3-dipolar cycloaddition reaction.

Commonly referred to as “click chemistry,” the cycloaddition

forms a triazole ring by reacting an azide and an alkyne moiety

under relatively mild conditions (Table 2). A toolbox of E3

ligase binders with varying linker lengths to alkynes³¹enabled

the parallel synthesis of PROTACs with the azide of a POI

binder in a facile manner. Our toolbox included alkynes linked

to cereblon, VHL, and IAP E3 ligase binders with one, two, or

three PEG units between the E3 binder and the azide moiety

(Table 2).

Table 1. Oﬀ-DNA Small Molecule SAR

From the literature, it is known that the indole portion of

bazedoxifene binds to the ligand-binding domain of the

estrogen receptor and thus the azepane points toward the

solvent.³⁴Therefore, an azide version of bazedoxifene was

prepared to react with the corresponding E3 binder alkynes to

produce PROTACs based on thalidomide (10); VHL amide

(11−13), VHL-phenoxy (14), IAP phenoxy (15), and IAP

amide (16). These compounds were tested in a binding assay

as well as the previously described cellular degradation and

viability assays. All seven compounds were potent in the

binding assay (Table 2, IC₅₀range 1.6−52 nM). However,

only the VHL amide (11−13), VHL phenoxy (14), and IAP

phenoxy (15)-based PROTACs had sub-micromolar cellular

degradation. These same compounds were also conﬁrmed to

be antagonists in the cellular viability assay with GI₅₀values

ranging from 34 to 335 nM (Table 2), demonstrating that

VHL-based PROTACs with bazedoxifene generate potent

degraders (11−14).

Based on the success of utilizing a VHL engager in the proof

of concept bazedoxifene series, we decided to focus on the

VHL ligands for designing PROTACs with our structurally

novel series. Click chemistry on an azide intermediate of 7,

where an azide replaced the piperidine in compound 7, was

used to prepare an array with three linker length alkynes of

PROTACs based on VHL amide (17−19) and VHL-phenoxy

(20−22). The binding aﬃnity of the VHL amide PROTACs

17−19 seemed less potent with increasing linker length (2- to

5-fold). However, the cellular degradation potency values

(DC₅₀) for all three PROTACs were single-digit nM. In the

VHL phenoxy series, the binding aﬃnities of 21 and 22 were

similar (IC₅₀= 32 and 44 nM), whereas the shortest linker 20

was less potent (IC₅₀= 198 nM). Unpredictably, the cellular

ERα degradation of 20 and 21 was the same (38 and 37 nM),

and the compound with the longest linker 22 was more potent

and with similar potency to the VHL amides at 5.3 nM (Table

2). These compounds were also tested in the corresponding

binding assay with mutants D538G, S463P, and Y537S and

showed pan inhibition with binding within 20-fold of WT. All

compounds were shown to be antagonists in the cell viability

assay (Table 2).

a

The structure−activity relationships for small molecules without

DNA barcodes in ERα binding FP assay, MCF7 agonism and

antagonism viability, and ERα degradation (DC₅₀). ND = not

determined. Results in the table represent an average of duplicate

runs.

Understanding the MOA and Kinetics of ERα

Inhibition and Degradation in MCF7 Cells. Two of the

most potent PROTACs from the two VHL variants (18 and

21) were utilized to understand the mechanism of action

(MOA) of ERα inhibition and degradation (structures shown

in Figure 3a). Biochemically, both compounds were very

potent in the WT and mutant ERα FP binding assays (Figure

3b and Table 2). In-cell western-based degradation assay in

MCF7 cells showed dose-dependent reduction in the levels of

ERα at 24 h, with both compounds tested, 18 (DC₅₀: 4.5 nM)

and 21 (DC₅₀: 20 nM; Figure 3c and Supplementary Figure

S2). These graphs represent normalized ERα levels. We did

not assess D_maxas a parameter in this study (due to the use of a

nM; D_max≈ 70%) in starved CSS media conditions. Curiously,

the other attachment points appeared to generate a

signiﬁcantly weaker binder (8) or potent agonist behavior

(9), indicating that there was sensitivity to the linker location.

We decided to further progress compound 7 forward as a

moiety in a bispeciﬁc PROTAC format.

Development of Potent ERα Binders into PROTACs.

To determine if our internal PROTAC strategy was feasible,

we started with a known ERα binder, bazedoxifene,^34,35to

rapidly identify which E3 ligands could be utilized to degrade

ERα protein. To concurrently explore the eﬀect of diﬀerent E3

targeting ligands and linker lengths for attaching the ERα

binding moiety, we utilized an exploration strategy that

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Table 2. PROTACs Utilizing a Bazedoxifene or Compound 7 Moeity as the POI Binder

PROTAC made from:

ERα binder azide E3 binder alkyne

ERα FP IC₅₀(nM)

MCF7 GI₅₀(μM)

MCF7 DC₅₀(μM)

ID

n

WT

1.6

11

22

5.3

7.3

52

17

53

97

198

32

44

90

S463P

Y537S

D538G

Ag.

Ant.

degradation

a

10

11

12

13

14

15

16

17

18

19

20

21

22

23

Baz azide

7 azide

Thalidomide

VHL amide

1

2

3

1

2

3

1

2

3

2

2.1

3.2

4.7

287

27

9.3

29

>10

3.25

2.5

a

21

8.1

0.247

0.335

0.211

0.034

0.051

0.020

0.009

0.027

a

18

38

a

VHL amide

6.0

15

b

VHL phenoxy

IAP phenoxy

IAP amide

14

52

b

24

61

40

0.174

c

ND

170

203

238

1129

143

134

122

ND

332

419

534

3720

527

302

341

ND

269

517

332

2205

329

261

199

>10

b

VHL amide

0.165

0.030

0.261

0.423

0.165

0.131

1.75

0.0042

b

7 azide

VHL amide

0.006

0.008

0.038

0.037

b

7 azide

VHL amide

b

7 azide

VHL phenoxy

VHL amide epimer

b

0.0053

b

de

,

1.78 , >10

a

b

c

d

e

f

Original assay. 48 h. 24 h. 6 h. 16 h. Individual compounds from the PROTAC array design were prepared utilizing click chemistry to obtain

PROTACs 10−23 with variable ERα and E3 binding elements and variable linker lengths (n). The compounds were assessed in FP binding assays

for WT and mutant ERα, MCF7 viability assay in agonist or antagonist mode, and in MCF7 in cell western ERα degradation. ND = not

determined. Results in the table represent an average of duplicate runs.

PROTAC control rather than an absolute control in the assay),

but upon comparing the two compounds, we saw at least 20%

more degradation of ERα with 21 than 18, demonstrating a

more robust degradation for 21. Next, the cellular viability

assay in MCF7 cells demonstrated negligible agonist eﬀects,

while both compounds demonstrated robust antagonistic

activity (Figure 3d). As ERα is a transcription factor, we

next tested its immediate downstream eﬀect on the gene

expression of its target progesterone receptor (PGR). The

results demonstrated a dose-dependent reduction in the levels

of PGR upon treatment with 18 (EC₅₀27 nM) and 21 (EC₅₀

90 nM) at 24 h (Figure 3e). The graphs represent normalized

PGR levels.

were treated with 1 μM Fulvestrant or 18 over a time course of

48 h. Results demonstrated degradation of ERα with

Fulvestrant 4 h after treatment, but complete degradation

with Fulvestrant under these conditions was not observed

(Figure 4a). In contrast, strong, near-complete degradation was

seen as early as 2 h with PROTAC-based degrader 18 (Figure

4a) (data for agonist mode not shown but was similar to

antagonist mode). Next, to understand the durability of ERα

degradation, we performed a wash-out experiment. Upon

compound wash-out, we observed a 10-fold loss in degradation

eﬃcacy with 18, but 21 demonstrated a 25-fold reduction

(Figure 4b). In order to conﬁrm that the observed degradation

is VHL-mediated, we performed a competition experiment

utilizing the E3 binder, VHL-298 (Figure 4e).³⁶Cells co-

treated with VHL-298 (5 μM) alongside ERα PROTACs (18

or 21) observed a DC₅₀shift of ∼8-fold, supporting the

hypothesis that VHL-engagement is required for proteasome-

mediated degradation of ERα (Figure 4c).

In order to assess the kinetics of PROTAC-mediated ERα

degradation, we performed time-course experiments followed

by western blotting to determine optimal ERα degradation

with PROTACs in both hormone-deprived media (agonist

mode) and full-serum media (antagonist mode). MCF7 cells

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Figure 3. Biochemical, cellular, and mouse PK properties of ERα PROTACs, 18 and 21. (a) Structures of 18 and 21. (b) Biochemical inhibition

using puriﬁed recombinant ERα WT and mutants D538G, Y537Y, and S463P. (c) Degradation of ERα in MCF7 cells treated for 24 h followed by

in-cell western analysis of ERα protein normalized to tubulin. The graphs represent normalized ERα levels. (d) MCF7 viability assay with cells

treated for 7 days followed by an end-point viability by CellTiter-Glo. (e) Downstream PGR expression analysis in MCF7 cells treated for 24 h

followed by RNA extraction, ﬁrst strand synthesis, and qPCR analysis for progesterone receptor RNA estimation, normalized to TBP (Tata-binding

protein); graphs represent normalized PGR levels. (f) Mean plasma concentration (μM) for 10 mpk (mg/kg) subcutaneous dosing in CD1 mouse

(n = 3). Curves shown are from a single run with technical replicates.

To additionally conﬁrm the PROTAC mechanism is

working via VHL engagement, we prepared the VHL prolinol

epimer 23 of compound 18 as a negative control. Although the

ERα binding potency for the negative control VHL amide

epimer 23 was 90 nM, the in-cell western degradation result at

48 h showed markedly less potent degradation (>770-fold)

than the corresponding active degrader 18 (Figure 4d). To

further explore the ameliorated activity of compound 23, we

also evaluated the degradation at shorter timepoints (6 and 16

h) to reduce any potential assay interference/toxicity. At these

shorter timepoints, compound 23 exhibited no protein

degradation. Therefore, we concluded that, at longer time-

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Figure 4. ERα PROTACs inhibit and degrade ERα in MCF7 cells. (a) Time course degradation of ERα treated with Fulvestrant (1 μM) or 18 (1

μM). Cells were lysed and assayed by western blot, and tubulin was used as a normalizer control. (b) In-cell western evaluating the persistence of

degradation eﬀect after a 2 h treatment, compound washout and cell collection after another 22 h, compared to full 24 h compound treatment.

Continued ERα reduction was observed after the washout. ERα normalized to tubulin control. (c) Proteasomal degradation of ERα is VHL-

mediated by observing that co-treating with VHL298 (5 μM, literature K_d= 90 nM) resulted in a higher DC₅₀compared to compound alone, 48 h

total treatment time followed by collection. (d) Degradation of ERα is signiﬁcantly impaired in the negative control prolinol epimer 23 compared

to the analogous active prolinol epimer 18, showing that degradation is proceeding through a VHL-based mechanism. (e) Structure of VHL298. All

traces and DC₅₀values are from a single experiment with technical replicates.

points (48 h), the slight reduction in protein levels was likely

due to compound toxicity, and the degradation of ERα of this

novel series proceeds through VHL-mediated degradation.

PROTACs Degrade ERα in Multiple ER-Positive Cell

Lines and Reduce Cell Viability. To demonstrate

degradation of ERα across multiple cell lines, we tested ERα

PROTACs, 18 and 21, in an additional ER-positive cell line,

T47D. MCF7 and T47D cell lines were treated with increasing

doses of 18 and 21 for 24 h. Cell lysates were prepared, and

western blotting was performed, with Actin used as a loading

control. Similar to observations with the MCF7 cell line, robust

ERα degradation was observed in T47D cells and the DC₅₀

values for the two cell lines were very similar (Figure 5a and

Supplementary Figure S3). Given the remarkable potency of

the compounds on all the GOF mutants, we would expect

those ERα mutant proteins to be degraded in the cells, but due

to unavailability of any commercially available cellular models

for these mutants, we did not test this.

Next, to test on-target vs oﬀ-target eﬀects, we tested various

breast cancer cell lines; ER+ (MCF7, T47D, and ZR-75-1),

ER-negative (MDA-MB-231), and an immortalized normal

breast cell line (MCF10A) in cellular viability assays. Various

breast cell lines were treated with increasing doses of ERα

PROTACs, 18 and 21, in full serum conditions for 7 days.

Relative cell numbers were assessed using CellTiter-Glo.

As expected, the results showed viability defects in all ER+

cell lines with GI₅₀values ranging between 13 and 170 nM for

18 and 34 and 154 nM for 21, while no eﬀect on viability was

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Figure 5. ERα PROTACs demonstrate on-target eﬀects in ER+ cell lines and not in ER− cell lines. (a) MCF7 and T47D cell lines were treated

with increasing doses of 18 and 21 for 24 h showing ERα degradation. Cell lysates were prepared and western blotted for ERα with Actin as a

loading control. (B) ER-positive (MCF7, T47D, and ZR-75-1) and ER-negative breast cancer cell lines (MBA-MB231) and immortalized breast

cell line (MCF10A) were treated with increasing doses of ERα PROTACs, 18 and 21, in full serum conditions for 7 days. Relative cell numbers

were assessed using CellTiter-Glo.

a

Table 3. In Vitro Clearance and In Vivo PK of Select Compounds in Male CD1 Mice

microsomal stability

i.v. PK CD1 mouse

s.c. PK CD1 mouse

AUC_inf

HLM CL_int(μL/min

MLM CL_int(μL/min

Cl_obs(mL/min

Vd_ss,obs

(L/kg)

ID

/mg)

/kg)

T_1/2(h)

C

_max(μM)

(μM·hr)

T_1/2(h)

%F

83

>100

20

17

18

20

21

31

59

42

47

199

249

162

139

20

23

1.2

0.9

1.24

4.82

0.12

0.18

2.7

8.7

2.2

4.7

1.50

2.13

2.08

3.55

9.8

14.6

44.5

53.9

21

17

21

17

25

a

Human (HLM) and mouse (MLM) liver microsome stability. All compounds were formulated in 40% HP-β-CD in water. Cl_obs, Vd_ssT_1/2

determined by 0.3 mg/kg CD1 mouse IV dose cassette dosing, C_max, AUC_inf, and T_1/2were determined by 10 mpk subcutaneous in CD1 mouse, %

F is reported for AUC_inf(sc/iv).

seen in ER-negative breast cancer cell line MBA-MB 231 and

the immortalized breast cell line MCF10A (Figure 5B). These

results conﬁrm the on-target eﬀects of this new series of ERα

PROTACs.

higher exposures for VHL-phenoxy PROTACs (20, 21: AUC_inf

= 44.5 and 53.9 μM·hr) over VHL amide PROTACs (17, 18:

AUC_inf= 9.8 and 14.6 μM·hr), as seen in the exposure curve

depicted in Figure 3f for 18 and 21. Minimal exposure was

observed when 18 and 21 were dosed orally (data not shown),

which was anticipated given their low solubility and

permeability, factors known to impede oral exposure.³⁸Despite

the low permeability, solubility, and high plasma protein

binding of 18 and 21, these two PROTACs present with

potent degradation activity (though 21 leads to at least 20%

greater D_maxin cells), signiﬁcant plasma exposures, yet

diﬀerentiated PK proﬁles when dosed subcutaneously that

warranted evaluation of ERα degradation in the in vivo setting.

In Vivo Eﬃcacy Studies. Based on reasonable subcuta-

neous single-dose PK data (10 mpk in CD1 mice) for 18 and

21 (Figure 3f) showing exposure over 24 h, we progressed

these two lead PROTACs, 18 and 21, into a mouse eﬃcacy

study. The MCF7 xenograft model was chosen, and we tested

the two compounds alongside Fulvestrant (reference com-

pound) in a 28 day eﬃcacy study (8 mice/arm). Fulvestrant

was highly eﬀective in suppressing MCF7 tumor growth and

on day 28, and TGI (tumor growth inhibition) of 91.6% was

seen, p < 0.001. Subcutaneous administration of 10 mg/kg qd

In Vitro ADME and PK. Overall, the amide and phenoxy

VHL-based compounds 17−22 were potent degraders of ERα

in ER+ cell lines, and we sought to evaluate the series further in

vivo. For both classes, we decided to progress the two shortest

of the three linker lengths 17, 18, 20, and 21 in in vitro ADME

assays and in mouse intra-venous and subcutaneous PK studies

(Table 3). In general, the optimization of ADME/PK of

PROTACs can be challenging.³⁷These compounds all have

low solubility (<0.2 μM), low permeability (MDCK P_appA-B <

0.1 cm/s × 10⁻⁶), and high PPB (>99.6%) (data not shown).

These compounds do have moderate in vitro clearance in

human and mouse microsomes (HLM CL_int= 31−59 mL/

min/kg; MLM CL_int139−249 mL/min/kg). In vivo, the IV

clearance of the VHL phenoxy compounds 20 and 21 (CL_obs

=

1.2 and 0.9 mL/min/kg) was much lower than the VHL amide

17 and 18 (20 and 23 mL/min/kg), and the volume of

distribution is much lower for the VHL phenoxy linked

PROTACs (20, 21: Vd_ss= 0.12 and 0.18 L/kg) compared the

VHL amide (17, 18: Vd_ss=1.24 and 4.82 L/kg), resulting in

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Figure 6. In vivo eﬃcacy and PK/PD study demonstrating eﬃcacy in MCF7 xenograft model. (a) Antitumor activity of 18 and 21 compared to

Fulvestrant in WT Estrogen-dependent MCF7 xenograft model. 18 and 21 were administered subcutaneously once daily at 10 mg/kg. Fulvestrant

was dosed SC QW at 3 mg/mouse. Data represent the mean tumor volume SEM (n = 8). *p < 0.05 versus vehicle control (Student’s t-test). (b)

All compounds were well tolerated with no changes in body weight. (c, d) Terminal Plasma PK and tumor PK of 18 and 21 at 3 h post-last dose

(Day 20; 18) and Day 27; 21). (e) PD analysis from tumor bearing mice treated with compounds mentioned above for 3 days. Western blot

analysis was performed to demonstrate reduction in ERα levels upon treatment with 18, 21, and Fulvestrant.

of 21 also demonstrated signiﬁcant tumor growth inhibition

(TGI) = 84.3%, p < 0.001. 18 at 10 mg/kg showed marginal

eﬀect on tumor volume (TGI = 30.8% p > 0.05), and as such,

dosing was discontinued at day 20 (Figure 6a). No changes in

average body weight in mice were recorded, and all

compounds appeared to be well tolerated (Figure 6b). We

collected plasma and tumor samples for end-point PK from the

18 and 21 arms, and the exposure in plasma and tumor

correlated well with the eﬃcacy seen in vivo, where 21 showed

much better exposure as compared to 18 (Figure 6c,d), though

high variability was observed in the levels of 21 measure in

tumor samples at the end of the study. We also collected

tumors from a separate set of mice treated with all three

compounds for 3 days. Western blot results demonstrate a

reduction in ERα levels in all treatment arms, though some

mouse-to-mouse variability in the levels is observed (Figure

6e). These results clearly demonstrated that 21 is an excellent

tool compound for inhibition and degradation of ERα both in

vitro and in vivo.

suitable PROTACs is ﬁnding an appropriate high aﬃnity novel

binder with a vector for attaching a linker to an E3 engager.

DECL screening has a distinct advantage as an appropriate

screening platform for designing PROTACs. Besides being an

aﬃnity (not activity)-based screening approach, the location of

the linker attachment point is known a priori; thus, in the

absence of structural information, the DNA-encoded chemistry

platform aﬀords a data-driven path forward to immediately

begin linker optimization and small molecule bait presentation

for a given POI. In conjunction with our DNA-encoded

chemistry platform, we also established a toolkit to readily

produce PROTACs based on well-known E3 ligase binders to

enable the facile generation of arrays of PROTACs, where in

one synthetic step, the POI binder, E3 binder, linker type and

length can be explored.

Using the information generated by DNA-encoded chem-

istry screening, we were able to generate a small set of

compounds with nM binding aﬃnity. Attachment of a basic

amine at the linker region converted these compounds from

potent agonists into potent antagonists and further validated

that we were targeting ERα directly.

Similar to bazedoxifene-based PROTACs, we also observed

robust degradation with our novel ERα ligand with two

peptidic ligands for VHL. We also demonstrated that this

degradation was VHL-mediated by using a VHL ligand as a

competitor and by also synthesizing the negative control

PROTAC incorporating an inactive stereoisomer in the VHL-

binding portion (see compound 18 vs 23). Though the VHL

amide and VHL phenoxy were nearly equivalent in their in

vitro assay proﬁles, they behaved quite diﬀerently in PK

studies, with markedly diﬀerent AUC, CL_obs, and Vd_ss. We

decided to progress a compound from each VHL variant (VHL

DISCUSSION AND CONCLUSIONS

■

Since its introduction in 2001, the proteolysis targeting

chimera (PROTAC) concept has been applied to many

targets, including ERα.^18,19,39,40Currently, there is one ERα

PROTAC in the clinic⁴¹(ARV-471, structure was recently

disclosed during review²³), which is based on the cereblon

binder lenalidomide and the SERM lasofoxifene. Although

there are few other PROTACs in the clinic, the drug discovery

path for PROTAC-based degraders is still being established,

and there are many reviews trying to bring understanding to

the community.⁴²⁻⁴⁵One of the challenges of discovering

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compounds were added to the cells in a 10-point dose series in the

same media. For antagonist mode, all wells (except the minimum

signal control wells) were treated with 0.1 nM estradiol for 30 min

followed by treatment with compounds. Plates were incubated a

further 7 days at 37 °C, 5% CO₂, and then the cellular ATP content

was measured using CellTiter-Glo 2.0 (Promega, Madison, WI) on a

Tecan M1000 (Mannedorf, Switzerland). Once we began testing the

PROTACs, the proliferation assay was run in complete media

followed by compound treatment and a 7 day incubation period

(results shown in Figure 3d).

Degradation Assay-ICW (Hormone-Deprived Media). MCF7

cells were seeded in hormone-deprived media and incubated for 72 h.

Test compounds were added to the cells at 10-point dose series in

hormone-deprived media. DMSO was dispensed into the maximum-

signal control wells, and a compound control (13) was dispensed into

the minimum-signal control wells to give a ﬁnal concentration of 1

μM and were incubated a further 48 h. Cells were ﬁxed with 4%

formaldehyde, permeabilized with 0.1% Triton-X 100, and blocked

with 3% ﬁsh gelatin. Primary antibodies (ERα and α-tubulin) were

incubated overnight and secondary antibodies for 1 h before imaging

on the Azure Biosystem Sapphire Biomolecular Imager (Dublin, CA).

Degradation Assay (Western Blotting). For western blots, 5 ×

10⁵MCF7 cells in 2 mL of media per well were seeded into six-well

plates 48 h before treatment. Cells were treated for the indicated

times and doses, and western blot images were obtained through

detection of rabbit anti-ERα (D6R2W, 1:1000, Cell Signaling

Technologies, Danvers, MA) and mouse anti-β-actin (1:10000,

Sigma) antibodies with goat anti-rabbit IRDye 800CW secondary

antibody (1:10000, LI-COR) and donkey anti-mouse IRDye 680RD

secondary antibody (1:10000, LI-COR) using an Azure Biosystems

Sapphire Biomolecular Imager (Dublin, CA).

amide and VHL-phenoxy) into the eﬃcacy study and, aligned

with the PK data, we observed, signiﬁcant shrinkage in tumor

volume with the phenoxy-linked PROTAC 21.

For ERα, both monovalent (Fulvestrant and other second-

generation SERDs) as well as PROTAC-based degraders seem

to be viable approaches to ERα degradation. Both approaches

have their advantages, wherein monovalent degraders are

usually smaller and Lipinski rule compliant. However the rules

of converting a target binding ligand into a monovalent

degrader are not very well understood and the levels of

degradation could vary widely. For PROTACs, their modular

design allows for relatively simple construction, but the

resulting molecules are usually large and need optimization

of physiochemical properties. PROTACs may oﬀer advantages

of catalytic activity⁴⁶and an acceptance of relatively weak POI

binders.^42,47

Overall, we have demonstrated that by utilizing our DECL

platform, we were able to quickly identify very potent novel

ERα binders that were eﬃciently incorporated into VHL-

engaging PROTACs which demonstrated nanomolar ERα

DC₅₀values with activity in ER-positive cells while

demonstrating no eﬀects in ER-negative cells. We also tested

these compounds in normal immortalized breast cells and

observed no oﬀ-target eﬀects. The compounds also demon-

strated suitable properties for in vivo applications and were

eﬃcacious in an ERα-dependent xenograft model. These

compounds are valuable tools to further the development of

novel ERα-based PROTAC-based therapeutics for patients

with breast cancer.

Gene Expression Analysis. MCF7 cells were seeded at 1.5 × 10⁴

cells per well in 96-well plates in a volume of 90 μL complete media.

Cell plates were incubated for 72 h. Test compounds were added in a

6-point dose series (10 μL). DMSO was dispensed into control wells,

and Fulvestrant was dispensed as a reference (100 nM). All

compounds were dosed for a ﬁnal concentration of 0.5% DMSO

(v/v). Cells with compound were further incubated for 24 h in

humidiﬁed chambers at 37 °C, 5% CO₂. RNA was extracted, cDNA

prepared, and qPCRs run using speciﬁc TaqMan probes (see

Supplemental Methods). Data were analyzed using the QuantStudio

Design & Analysis Software v1.4.3 using “Comparative C_T(ΔΔC_T)”

to analyze and export data to make dose−response curves in

GraphPad Prism (San Diego, CA).⁵⁰

ADME/PK. Determination of microsomal stability was performed

at Pharmaron (Beijing, China). Test compounds (at 2 μM ﬁnal

concentration) were subjected to pooled human or male mouse liver

microsomes, and % of compound remaining at 0, 15, 30, 45, and 60

min was measured and used to calculate an in vitro half-life (in

minutes). PK studies were also performed at Pharmaron (Beijing,

China) and were carried out according to the guidelines approved by

the Institutional Animal Care and Use Committee (IACUC) of

Pharmaron. Cassette iv (intravenous) PK was performed in male CD1

mice (3 mice per group) with up to ﬁve compounds each dosed at 0.3

mg/kg (dose volume of 2 mL/kg). The concentration of compounds

was measured in plasma at nine timepoints (2, 5, 15, and 30 min and

1, 2, 4, 6, and 8 h post-dose), with 0.03 mL of blood collected via the

dorsal metatarsal vein at each timepoint. Single dose sc (subcuta-

neous) PK was performed in male CD1 mice with each compound

dosed at 10 mg/kg (dose volume of 10 mL/kg). The concentration of

compounds was measured in plasma at nine timepoints (15 and 30

min and 1, 2, 4, 6, 8, 12, and 24 h post-dose), with 0.03 mL of blood

collected via the dorsal metatarsal vein at each timepoint.

EXPERIMENTAL SECTION

■

Construct Design and Cloning. The nucleotide sequences of

the ligand-binding domains (LBD), containing amino acids 307−554,

of ERα WT and the clinically relevant mutations D538G, S463P, and

Y537S were synthesized with an N-terminal Avi-Tev-His tandem

aﬃnity tag and cloned into a pD454-SR E. coli expression vector by

ATUM (Newark, CA, USA).

Protein Expression and Puriﬁcation. Proteins were expressed

in T7 Express cells for 12 h at 20 °C after induction with IPTG and

then puriﬁed using standard Ni-NTA aﬃnity chromatography

followed by size exclusion chromatography.

Aﬃnity-Mediated Selection of DNA-Encoded Chemical

Libraries. Selections were performed as previously described.^48,49

Forty-four diﬀerent DNA-encoded chemical libraries were combined

and incubated with ERα WT, D538G, S463P, or Y537S. Separate

incubations were set up with no target, ERα WT over a ﬁve-fold

dilution series down from 7.7 μM to 24 nM, ERα WT at 7.7 μM with

estradiol at 15 μM, and each of the D538G, S463P, and Y537S at 7.7

μM. After incubation, the mixtures were captured on His-Select

IMAC matrix. After capture, the matrices were washed and then heat-

eluted at 85 °C. The second round of selection was performed using

the round-one eluate as an input and fresh proteins and estradiol

repeating the conditions of the ﬁrst round. The encoding DNA in the

output of the second round of selection was ampliﬁed and then

sequenced.

FP Binding Assay. Inhibition values for ERα compounds were

determined using a ﬂuorescent polarization (FP) assay. Compounds

were pre-incubated with protein for 15 min, prior to the addition of

the ﬂuorescent ligand. The plates were further incubated at room

temperature for 1 h and read on Spectramax Paradigm (λex 485 nm,

λem 535 nm).

In Vivo Experiments. All the procedures were performed

according to the guidelines approved by the Institutional Animal

Care and Use Committee (IACUC) of WuXi AppTec, Shanghai,

China following the guidance of the Association for Assessment and

Accreditation of Laboratory Animal Care (AAALAC). Each mouse

was inoculated subcutaneously at the right ﬂank with exponentially

growing MCF-7 tumor cells (10 × 10⁶) in 0.2 mL of PBS mixed with

Cell Lines. Breast lines were obtained from the American Type

Culture Collection: MCF7, MDA-MB231, T47D, ZR-75-1, and

MCF10A and maintained in media speciﬁed by the ATCC.

Proliferation Assay. Cancer cells were seeded in hormone-

deprived media and incubated for 72 h. In agonist mode, test

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Matrigel (50:50) for tumor development. 17β-Estradiol (0.18 mg)

pellets (Innovative Research of America) were implanted 2 days

before cell inoculation. Sixty-four animals were randomized when the

average tumor volume reached 165 mm³for the eﬃcacy study. Each

group consisted of eight randomly assigned tumor-bearing mice. Mice

were dosed with vehicle, 18, 21, or Fulvestrant until the vehicle

volume reached 2000 mm³as per IACUC guidelines. Tumor size was

measured two times a week in two dimensions using a caliper, and the

tumor volume (V) was expressed in mm³using the formula: V = 0.5a

× b², where a and b were the long and short diameters of the tumor,

respectively. The mice were weighed every time prior to dosing.

Tumor growth inhibition was calculated using the formula TGI (%) =

(Vc − Vt)/(Vc −Vo) × 100, where Vc and Vt are the median of

control and treated groups at the end of the study, respectively, and

Vo at the start. Tumors were collected at the end of the study for PD

analysis.

mg, 2 mmol) in methanol (5 mL) was stirred at room temperature

overnight. The reaction mixture was concentrated under vacuum, and

the residue was puriﬁed by column chromatography (20% EtOAc in

petroleum ether with 1% TEA) to obtain N-((1-(2-ﬂuorophenyl)-

cyclopentyl)methyl)aniline (210 mg, 80%) as a colorless oil. MS:

(ESI) [M + H]⁺270.

A solution of N-((1-(2-ﬂuorophenyl)cyclopentyl)methyl)aniline

(210 mg, 0.8 mmol), 2-chloro-3-hydroxybenzaldehyde (125 mg, 0.8

mmol), NaBH₃CN (100 mg, 1.6 mmol), and acetic acid (100 mg, 1.6

mmol) in methanol (5 mL) was stirred at room temperature

overnight. The reaction mixture was concentrated under vacuum and

the crude product was puriﬁed by prep-HPLC to obtain the title

compound (30 mg, 9%) as a white solid. HPLC purity, 94.7%. MS:

(ESI) [M + H]⁺410.33; ¹H NMR (400 MHz, CD₃OD) δ 7.30−7.08

(m, 2H), 7.01 (ddd, J = 12.3, 6.7, 2.0 Hz, 4H), 6.87 (t, J = 7.9 Hz,

1H), 6.73 (dd, J = 8.0, 1.3 Hz, 1H), 6.60−6.49 (m, 3H), 6.42−6.34

(m, 1H), 4.19 (s, 2H), 3.74 (s, 2H), 2.25−2.12 (m, 2H), 2.08−1.96

(m, 2H), 1.93−1.79 (m, 2H), 1.77−1.63 (m, 2H).

2-Chloro-3-((((1-(2-ﬂuorophenyl)cyclopentyl)methyl)(2-(pi-

peridin-1-yl)ethyl)amino)methyl)phenol (4). To a solution of (1-

(2-ﬂuorophenyl)cyclopentyl)methanamine (300 mg, 1.55 mmol), 1-

(2-chloroethyl)piperidine (228 mg,1.55 mmol) in acetonitrile (10

mL) was added potassium carbonate (212 mg,1.55 mmol). Then, the

mixture was stirred at 80 °C for 2 h. The solution was concentrated

under vacuum, and the residue was puriﬁed by silica gel column

chromatography (5% MeOH in CH₂Cl₂) to obtain N-((1-(2-

ﬂuorophenyl)cyclopentyl)methyl)-2-(piperidin-1-yl)ethan-1-amine

(400 mg, 85%) as a white solid. MS: (ESI) [M + H]⁺305.

Synthetic Methods. All solvents and chemicals were used as

purchased without further puriﬁcation. All the reported yields are

1

isolated yields. H NMR spectra were recorded on a Bruker Advance

spectrometer at 400 MHz for ¹H NMR. The chemical shift is

expressed in ppm relative to tetramethylsilane (TMS) as an internal

standard, and CDCl₃, DMSO-d₆, and CD₃OD were used as solvents.

Multiplicity of peaks is expressed as s (singlet), d (doublet), t

(triplet), q (quartet), dd (doublet of doublets), td (triplet of

doublets), qd (quartet of doublets), dt (doublet of triplets), and m

(multiplet). Compound identity and purity was assessed by LCMS

using a Thermo UltiMate 3000 instrument, Phenomenex XB-C18 2.6

μM 2.1 × 30 mm column, mobile phase A: 99.9 acetonitrile/0.1

formic acid; mobile phase B: 99.9 water/0.1 formic acid. Elution was

accomplished via a 7.0 min gradient beginning at 90:10 A/B and

ending with 100% B with compounds identiﬁed by UV, λ = 254 nM,

and ESI positive ion mass spectrometry. High-resolution mass

spectrometry was acquired on an Agilent 6500 Q-TOF. All purities

were >95%, unless speciﬁed (see supplemental for tabulation). NMR

and MS data are consistent with in silico predicted values. See

To a solution of N-((1-(2-ﬂuorophenyl)cyclopentyl)methyl)-2-

(piperidin-1-yl)ethan-1-amine (400 mg,1.31 mmol), 2-chloro-3-

hydroxybenzaldehyde (204 mg,1.31 mmol) in MeOH (10 mL) was

added acetic acid (1 mL). The mixture was stirred at room

temperature for 5 h, and then NaBH₃CN (83 mg, 1.31 mmol) was

added. After stirring overnight, the reaction mixture was concentrated

under vacuum, and the residue was puriﬁed by prep-HPLC to obtain

the title compound (30 mg, 5%) as a white solid. MS: (ESI) [M +

1

Supporting Information for LC-MS traces (1−23) and H and ¹³C

1

H]⁺445.33; H NMR (400 MHz, CD₃OD) δ = 7.40 (s, 1H), 7.27

NMR spectra for 18 and 21.

(m, 1H), 7.07(m, 3H), 6.86 (m, 2H), 3.63 (s, 2H), 2.85(m, 8H), 2.52

(m, 1H), 2.01 (s, 2H), 1.87 (m, 2H), 1.45−1.57 (m, 9H)).

2-Chloro-3-(((1-cyclopentyl-2,2,2-triﬂuoroethyl)(methyl)-

amino)methyl)phenol (1). To a solution of 1-cyclopentyl-2,2,2-

triﬂuoroethan-1-amine hydrochloride (406 mg, 2 mmol) and 2-

chloro-3-hydroxybenzaldehyde (312 mg, 2 mmol) in MeOH (15 mL)

was added AcOH (3 drops) and NaBH₃CN (126 mg, 2 mmol). The

reaction mixture was stirred at room temperature overnight, and then

formaldehyde (2 mL, dissolved in water) was added to the solution,

and the reaction mixture was stirred for an additional 2 h. The solvent

was removed under reduced pressure to aﬀord the residue, which was

puriﬁed by prep-HPLC to obtain the title compound (164 mg, 25%)

2-Chloro-3-((((1-(2-ﬂuorophenyl)cyclopentyl)methyl)(3-(pi-

peridin-1-yl)propyl)amino)methyl)phenol (5). To a solution of

(1-(2-ﬂuorophenyl)cyclopentyl)methanamine (300 mg, 1.55 mmol),

1-(3-chloropropyl)piperidine (250 mg, 1.55 mmol) in CH₃CN (10

mL) was added K₂CO₃(212 mg, 1.55 mmol). The mixture was

stirred at 80 °C for 2 h and then concentrated under vacuum. The

residue was puriﬁed by column chromatography (5% MeOH in

CH₂Cl₂) to obtain N-((1-(2-ﬂuorophenyl)cyclopentyl)methyl)-3-

(piperidin-1-yl)propan-1-amine (400 mg, 81%) as a white solid.

MS: (ESI) [M + H]⁺319.

1

as a white solid. MS: (ESI) [M + H]⁺322.24; H NMR (400 MHz,

DMSO-d₆) δ 7.12 (t, J = 7.6 Hz, 1H), 6.89 (t, J = 8.8 Hz, 2H), 3.89

(s, 2H), 3.33−3.10 (m, 1H), 2.29 (s, 3H), 2.23−2.17 (m, 1H), 1.86−

1.24 (m, 8H).

To a solution of N-((1-(2-ﬂuorophenyl)cyclopentyl)methyl)-3-

(piperidin-1-yl)propan-1-amine (400 mg, 1.25 mmol), 2-chloro-3-

hydroxybenzaldehyde (295 mg,1.25 mmol) in methanol (10 mL) was

added AcOH (1 mL). The mixture was stirred at 25 °C for 5 h, and

then NaBH₃CN (79 mg, 1.25 mmol) was added to the solution. The

solution was concentrated under vacuum and the residue was puriﬁed

by prep-HPLC to obtain the title compound (30 mg, 5%) as a white

solid. MS: (ESI) [M + H]⁺459.24; ¹H NMR (400 MHz, CD₃OD) δ

= 10.07 (s, 1H), 7.29 (m, 2H), 7.06 (m, 3H), 76.89(d, 1H), 6.82 (d,

1H), 3.40 (s, 2H), 3.22(d, 2H), 2.68 (m, 4H), 2.58 (s, 2H), 2.07 (m,

2H), 1.95 (s, 2H)), 1.71 (m, 6H), 1.32−1.50 (m, 6H).

2-Chloro-3-((((1-(2-ﬂuorophenyl)cyclopentyl)methyl)(2-(2-

(piperidin-1-yl)ethoxy)ethyl)amino)methyl)phenol (6). To a

solution of 2-(2-(piperidin-1-yl)ethoxy)ethan-1-amine (172 mg, 1

mmol) and 1-(2-ﬂuorophenyl)cyclopentane-1-carbaldehyde (192 mg,

1 mmol) in MeOH (20 mL) was added acetic acid (0.2 mL). The

reaction mixture was stirred for 3 h, and then NaBH₃CN (124 mg, 2

mmol) was added, and the reaction mixture was stirred overnight at

room temperature. The reaction mixture was concentration under

vacuum, and the crude product was puriﬁed by column chromatog-

raphy (5% MeOH in CH₂Cl₂) to obtain N-((1-(2-ﬂuorophenyl)-

2-Chloro-3-((((1-(2-ﬂuorophenyl)cyclopentyl)methyl)-

(methyl)amino)methyl)phenol (2). To a solution of (1-(2-

ﬂuorophenyl)cyclopentyl)methanamine (386 mg, 2 mmol) and 2-

chloro-3-hydroxybenzaldehyde (312 mg, 2 mmol) in MeOH (15 mL)

was added AcOH (3 drops) followed by NaBH₃CN (126 mg, 2

mmol). The mixture was stirred at room temperature overnight, and

then formaldehyde (2 mL, dissolved in water) was added to the

solution, and the reaction mixture was stirred for an additional 2 h.

The solvent was removed under reduced pressure to obtain a residue,

which was puriﬁed by prep-HPLC to obtain the title compound (329

1

mg, 47%) as a white solid. MS: (ESI) [M + H]⁺348.34; H NMR

(400 MHz, DMSO-d₆) δ 7.46−7.36 (m, 2H), 7.23−7.17 (m, 3H),

7.09 (d, J = 7.6 Hz, 1H), 7.01 (d, J = 7.2 Hz, 1H), 4.30 (d, J = 44.1

Hz, 2H), 3.54 (d, J = 22.8 Hz, 2H), 2.49 (s, 3H), 2.01−1.91 (m, 4H),

1.57−1.34 (m, 4H).

2-Chloro-3-((((1-(2-ﬂuorophenyl)cyclopentyl)methyl)-

(phenyl)amino)methyl)phenol (3). A solution of 1-(2-

ﬂuorophenyl)cyclopentane-1-carbaldehyde (200 mg, 1 mmol), aniline

(93 mg, 1 mmol), NaBH₃CN (126 mg, 2 mmol), and acetic acid (120

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cyclopentyl)methyl)-2-(2-(piperidin-1-yl)ethoxy)ethan-1-amine (174

mg) as a yellow oil. MS: (ESI) [M + H]⁺349.

3.52−3.45 (m, 2H), 3.13−2.96 (m, 2H), 2.34−1.90 (m, 7H), 1.90−

1.73 (m, 5H), 1.73−1.47 (m, 2H).

2-Chloro-3-((methyl((1-(2-(2-(piperidin-1-yl)ethoxy)-

phenyl)cyclopentyl)methyl)amino)methyl)phenol (8). To a

solution of (1-(2-methoxyphenyl)cyclopentyl)methanamine (800

mg, 3.9 mmol) and NaHCO₃(982 mg, 11.7 mmol) in THF (10

mL) and water (10 mL) was added (Boc)₂O (1.7 g, 7.8 mmol). The

reaction mixture was stirred overnight at room temperature and then

concentrated in vacuo. The residue was extracted with dichloro-

methane (10 mL × 2), and the combined organic layers were dried

over Na₂SO₄, concentrated in vacuo, and puriﬁed by column

chromatography to obtain tert-butyl ((1-(2-methoxyphenyl)-

cyclopentyl)methyl)carbamate (925 mg, 78%) as an oil. MS: (ESI)

[M + Na]⁺328.1.

To a solution of N-((1-(2-ﬂuorophenyl)cyclopentyl)methyl)-2-(2-

(piperidin-1-yl)ethoxy)ethan-1-amine (174 mg, 0.5 mmol) and 2-

chloro-3-hydroxybenzaldehyde (78 mg, 0.5 mmol) in MeOH (20

mL) was added titanium(IV) isopropoxide (0.2 mL). The reaction

mixture was stirred for 3 h, then NaBH₃CN (92 mg, 1.5 mmol) was

added, and the mixture was stirred overnight at room temperature.

The reaction mixture was concentration under vacuum, and the crude

product was puriﬁed by column chromatography (5% MeOH in

CH₂Cl₂), and then by prep-HPLC to obtain the title compound (70

1

mg, 29%) as a white solid. MS: (ESI) [M + H]⁺489.34; H NMR

(400 MHz, CD₃OD) δ 7.31 (td, J = 8.0, 1.7 Hz, 1H), 7.21 (ddd, J =

7.2, 4.9, 1.7 Hz, 1H), 7.10−6.96 (m, 3H), 6.92 (d, J = 6.2 Hz, 1H),

6.81 (dd, J = 8.0, 1.5 Hz, 1H), 3.59−3.53 (m, 4H), 3.48−3.34 (m,

4H), 3.23−3.19 (m, 2H), 3.14−2.87 (m, 2H), 2.85 (s, 2H), 2.41 (q, J

= 6.4 Hz, 2H), 2.06 (d, J = 6.2 Hz, 2H), 1.95−1.67 (m, 7H), 1.62−

1.42 (m, 5H).

2-Chloro-3-((((1-(2-ﬂuorophenyl)cyclopentyl)methyl)(4-(2-

(piperidin-1-yl)ethoxy)phenyl)amino)methyl)phenol (7). To a

solution of 1-(2-ﬂuorophenyl)cyclopentane-1-carboxylic acid (1 g, 5

mmol) in methanol (10 mL) was added SOCl₂(1.2 g, 10 mmol)

dropwise, and the solution was stirred overnight and then

concentrated under vacuum. The residue was diluted with water

(20 mL) and extracted with ethyl acetate (10 mL × 3). The combined

organic layers were washed with NaHCO₃(15 mL × 2) and brine (15

mL) and then dried over with anhydrous Na₂SO₄. After ﬁltration, the

solution was concentrated under vacuum to give methyl 1-(2-

ﬂuorophenyl)cyclopentane-1-carboxylate (1 g, crude) as a colorless

oil, which was used directly in the next step. MS: (ESI) [M + H]⁺

223.

A solution of methyl 1-(2-ﬂuorophenyl)cyclopentane-1-carboxylate

(5 mmol) and LiAlH₄(tetrahydrofuran solution, 2 M, 5 mL) in

tetrahydrofuran (10 mL) was stirred for 2 h at room temperature. The

reaction was quenched by Na₂SO₄·10H₂O. After ﬁltration, the ﬁltrate

was concentrated under vacuum, and the crude product was puriﬁed

by column chromatography (1:15 methanol/dichloromethane) to

obtain (1-(2-ﬂuorophenyl)cyclopentyl)methanol (970 mg, 72%) as a

colorless oil. MS: (ESI) [M + H]⁺195.

A solution (1-(2-ﬂuorophenyl)cyclopentyl)methanol (970 mg, 5

mmol), 2-iodoxybenzoic acid (1680 mg, 6 mmol), and DMSO (1

drop) in ethyl acetate (10 mL) was stirred at 80 °C overnight. The

reaction mixture was ﬁltered, and the ﬁltrate was concentrated, diluted

with water (20 mL), and extracted with ethyl acetate (10 mL × 3).

The combined organic layers were washed with brine (15 mL × 3)

and dried over with anhydrous Na₂SO₄. After ﬁltration, the solution

was concentrated under vacuum to obtain 1-(2-ﬂuorophenyl)-

cyclopentane-1-carbaldehyde (1 g, crude) as a colorless oil. MS:

(ESI) [M + H]⁺193.

To a solution of tert-butyl ((1-(2-methoxyphenyl)cyclopentyl)-

methyl)carbamate (200 mg, 0.67 mmol) in THF (10 mL) at 0 °C was

added NaH (47 mg, 1.97 mmol). The mixture was stirred at 0 °C for

30 min, and then MeI (186 mg, 1.310 mmol) was added. The mixture

was stirred at room temperature overnight and then quenched with

ice water (20 mL) and extracted with ethyl acetate (15 mL × 3). The

combined organic layers were dried over Na₂SO₄, ﬁltered, and

concentrated, and the residue was puriﬁed by column chromatog-

raphy to obtain tert-butyl ((1-(2-methoxyphenyl)cyclopentyl)-

methyl)(methyl)carbamate (175 mg, 84%) as an oil. MS: (ESI) [M

+ Na]⁺342.1.

To a solution of tert-butyl ((1-(2-methoxyphenyl)cyclopentyl)-

methyl)(methyl)carbamate (175 mg, 0.56 mmol) in dichloromethane

(8 mL) was added BBr₃(278 mg, 1.11 mmol) at 0 °C. Then, the

mixture was stirred at room temperature for 3 h. The solution was

quenched with 1:9 ice water/methanol (10 mL) and extracted with

dichloromethane (15 mL × 3). The combined organic layers were

dried over Na₂SO₄, ﬁltered, and concentrated, and the residue was

puriﬁed by column chromatography to obtain 2-(1-((methylamino)-

methyl)cyclopentyl)phenol (52 mg, 46%) as an oil. MS: (ESI) [M +

H]⁺206.1.

To a solution of 2-(1-((methylamino)methyl)cyclopentyl)phenol

(52 mg, 0.253 mmol) and NaHCO₃(64 mg, 0.759 mmol) in THF (5

mL) and water (5 mL) was added (Boc)₂O (110 mg, 0.506 mmol).

The reaction mixture was stirred overnight at room temperature and

concentrated in vacuo, and the residue was acidiﬁed by 1 N HCl to

pH = 3. The solution was extracted with ethyl acetate (10 mL × 2),

and the combined organic layers were dried over Na₂SO₄, ﬁltered,

concentrated in vacuo, and puriﬁed by column chromatography to

obtain tert-butyl ((1-(2-hydroxyphenyl)cyclopentyl)methyl)(methyl)-

carbamate (50 mg, 77%) as an oil. MS: (ESI) [M + Na]⁺328.1.

To a solution of tert-butyl ((1-(2-hydroxyphenyl)cyclopentyl)-

methyl)(methyl)carbamate (47 mg, 0.154 mmol) and K₂CO₃(64 mg,

0.462 mmol) in acetonitrile (5 mL) was added 1-(2-chloroethyl)-

piperidine (34 mg, 0.231 mmol). The mixture was heated to 80 °C for

5 h and concentrated in vacuo, and the residue was puriﬁed by

column chromatography to obtain tert-butyl methyl((1-(2-(2-

(piperidin-1-yl)ethoxy)phenyl)cyclopentyl)methyl)carbamate (50

mg, 77%) as an oil. MS: (ESI) [M + H]⁺417.3.

A solution of 1-(2-ﬂuorophenyl)cyclopentane-1-carbaldehyde (200

mg, 1 mmol), 4-(2-(piperidin-1-yl)ethoxy)aniline (220 mg, 1 mmol),

NaBH₃CN (126 mg, 2 mmol), and acetic acid (120 mg, 2 mmol) in

methanol (5 mL) was stirred overnight at room temperature. The

reaction mixture was concentrated under vacuum, and the crude

product was puriﬁed by column chromatography (33% EtOAc in

petroleum ether with 1% TEA) to obtain N-((1-(2-ﬂuorophenyl)-

cyclopentyl)methyl)-4-(2-(piperidin-1-yl)ethoxy)aniline (300 mg,

75%) as a colorless oil. MS: (ESI) [M + H]⁺397.

A solution of tert-butyl methyl((1-(2-(2-(piperidin-1-yl)ethoxy)-

phenyl)cyclopentyl)methyl)carbamate (47 mg, 0.113 mmol) in HCl/

dioxane (5 mL) was stirred at for 2 h at room temperature. The

solution was concentrated in vacuo to obtain N-methyl-1-(1-(2-(2-

(piperidin-1-yl)ethoxy)phenyl)cyclopentyl)methanamine (50 mg,

crude) as an oil. LC-MS (M + H)⁺= 317.2.

A solution N-((1-(2-ﬂuorophenyl)cyclopentyl)methyl)-4-(2-(pi-

peridin-1-yl)ethoxy)aniline (300 mg, 0.8 mmol), 2-chloro-3-hydrox-

ybenzaldehyde (125 mg, 0.8 mmol), NaBH₃CN (100 mg, 1.6 mmol),

and acetic acid (100 mg, 1.6 mmol) in MeOH (5 mL) was stirred at

room temperature overnight. The reaction mixture was concentrated

under vacuum, and the crude product was puriﬁed by prep-HPLC to

obtain the title compound (40 mg, 10%) as a white solid. HPLC

A solution of N-methyl-1-(1-(2-(2-(piperidin-1-yl)ethoxy)phenyl)-

cyclopentyl)methanamine (47 mg, 0.149 mmol) and 2-chloro-3-

hydroxybenzaldehyde (35 mg, 0.224 mmol) in Ti(OPr)₄(2 mL) was

stirred for 4 h at room temperature. NaBH₃CN (28 mg, 0.447 mmol)

was added, and after stirring overnight at room temperature, the

reaction was quenched with methanol (20 mL) and water (1 mL).

The solid was removed by ﬁltration, and the solution was

concentrated in vacuo. The residue was puriﬁed by prep-HPLC to

obtain the title compound (2.6 mg, 4%) as an oﬀ-white solid. MS:

1

purity, 94.9%. MS: (ESI) [M + H]⁺537.47; H NMR (400 MHz,

CD₃OD) δ 7.26−7.16 (m, 2H), 7.04−6.94 (m, 2H), 6.90 (t, J = 7.9

Hz, 1H), 6.72 (dd, J = 26.9, 20.0 Hz, 5H), 6.49 (d, J = 7.5 Hz, 1H),

4.23 (d, J = 15.5 Hz, 4H), 3.77 (s, 2H), 3.59 (d, J = 12.1 Hz, 2H),

1

(ESI) [M + H]⁺457.27; H NMR (400 MHz, DMSO) δ 9.97 (s,

1H), 8.14 (s, 1H), 7.21−7.16 (m, 1H), 7.08−7.04 (m, 1H), 6.97 (d,

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1H), 6.88−6.76 (m, 3H), 4.19 (t, 2H), 3.38−3.32 (m, 4H), 3.18−

3.13 (m, 2H), 2.89−2.80 (m, 3H), 2.75 (s, 2H), 2.12−2.08 (m, 2H),

1.79−1.68 (m, 9H), 1.51−1.48 (m, 5H).

anhydrous Na₂SO₄, ﬁltrated, and concentrated under vacuum, and the

residue was puriﬁed by column chromatography followed by prep-

HPLC to obtain the title compound (35 mg, 27%) as a white solid.

MS: (ESI) [M + H]⁺475.27; ¹H NMR (400 MHz, MeOD) δ 7.33−

7.29 (m, 1H), 7.13−7.10 (m, 1H), 6.93−6.91 (m, 2H), 6.78−6.74

(m, 2H), 4.35 (s, 2H), 3.80 (s, 2.67H), 3.54−3.50 (m, 2.36H), 3.15

(s, 3H), 2.32 (s, 3H), 2.04−1.90 (m, 8H), 1.71−1.63 (m, 6H).

3-(((4-(2-Azidoethoxy)phenyl)((1-(2-ﬂuorophenyl)-

cyclopentyl)methyl)amino)methyl)-2-chlorophenol (Inter-

mediate 7-Azide). To a solution of 2-(4-aminophenoxy)ethan-1-ol

(306 mg, 2 mmol) and (Boc)₂O (436 mg, 2 mmol) in THF (20 mL)

was added K₂CO₃(828 mg, 6 mmol). The reaction mixture was

stirred overnight and then concentrated under vacuum. The residue

was diluted with water (30 mL) and extracted with ethyl acetate (30

mL × 3). The combined organic layers were washed with brine (25

mL × 3) and then dried over with anhydrous Na₂SO₄. After ﬁltration,

the solution was concentration under vacuum, and the crude product

was puriﬁed by column chromatography (5% MeOH in DCM) to

obtain tert-butyl (4-(2-hydroxyethoxy)phenyl)carbamate (540 mg,

crude) as a yellow oil. MS: (ESI) [M + H]⁺254.

To a solution of tert-butyl (4-(2-hydroxyethoxy)phenyl)carbamate

(540 mg, crude) in DCM (20 mL) was added MsCl (0.5 mL) and

DIEA (1 mL). The reaction mixture was stirred for 1 h, and then the

reaction mixture was washed with brine (25 mL × 3) and dried over

with anhydrous Na₂SO₄. After ﬁltration, the solution was concen-

tration under vacuum to a obtain 2-(4-((tert-butoxycarbonyl)amino)-

phenoxy)ethyl methanesulfonate (470 mg) as a yellow oil. MS: (ESI)

[M + H]⁺332.

To a solution of 2-(4-((tert-butoxycarbonyl)amino)phenoxy)ethyl

methanesulfonate (470 mg, crude) in DMF (10 mL) was added NaN₃

(650 mg, 10 mmol). The reaction mixture was stirred at 60 °C

overnight. The reaction mixture was diluted with water (50 mL) and

extracted with ethyl acetate (30 mL × 3). The combined organic layer

was washed with brine (30 mL × 3) and then dried over with

anhydrous Na₂SO₄. After ﬁltration, the solution was concentration

under vacuum to give a crude tert-butyl (4-(2-azidoethoxy)phenyl)-

carbamate (530 mg, crude) as a yellow oil. MS: (ESI) [M + H]⁺279.

To a solution of tert-butyl (4-(2-azidoethoxy)phenyl)carbamate

(200 mg, crude) in DMF (10 mL) was added TFA (1 mL). The

reaction mixture was stirred at room temperature for 2 h and then

concentrated under vacuum to obtain crude 4-(2-azidoethoxy)aniline

(350 mg, crude) as a yellow oil. MS: (ESI) [M + H]⁺179.

To a solution of 4-(2-azidoethoxy)aniline (300 mg, crude) and 1-

(2-ﬂuorophenyl)cyclopentane-1-carbaldehyde (192 mg, 1 mmol) in

MeOH (20 mL) was added acetic acid (0.2 mL). The reaction

mixture was stirred at room temperature for 3 h. Then, NaBH₃CN

(124 mg, 2 mmol) was added and the reaction mixture was stirred

overnight. The reaction mixture was concentration under vacuum,

and the crude product was puriﬁed by column chromatography (5%

MeOH in DCM) to obtain 4-(2-azidoethoxy)-N-((1-(2-

ﬂuorophenyl)cyclopentyl)methyl)aniline (390 mg) as a yellow oil.

MS: (ESI) [M + H]⁺355.

2-Chloro-3-((((1-(2-ﬂuoro-4-(2-(piperidin-1-yl)ethoxy)-

phenyl)cyclopentyl)methyl)(methyl)amino)methyl)phenol (9).

A solution of 2-(2-ﬂuoro-4-methoxyphenyl)acetonitrile (1650 mg, 10

mmol), 1,4-dibromobutane (3210 mg, 15 mmol), and NaH (360 mg,

15 mmol) in dried DMF (10 mL) was stirred at room temperature for

6 h. After the reaction was completed, water (20 mL) was added 0 °C.

The solution was concentrated, water was added, and the solution was

extracted with CH₂Cl₂(30 mL × 3). The combined organic layers

were dried over with anhydrous Na₂SO₄, ﬁltrated, and concentrated

under reduced pressure, and the residue was puriﬁed by column

chromatography to obtain 1-(2-ﬂuoro-4-methoxyphenyl)-

cyclopentane-1-carbonitrile (768 mg, 35%) as a white solid. MS:

(ESI) [M + H]⁺220.1.

A solution of 1-(2-ﬂuoro-4-methoxyphenyl)cyclopentane-1-carbon-

itrile (760 mg, 3.47 mmol) and LiAlH₄(132 mg, 3.47 mmol) in dried

THF (10 mL) was stirred at 0 °C for 3 h. The reaction was completed

by LC-MS, and Na₂SO₄·10 H₂O (1117 mg, 3.47 mmol) was added to

the solution at 0 °C. The solution was ﬁltrated and concentrated to

obtain (1-(2-ﬂuoro-4-methoxyphenyl)cyclopentyl)methanamine (610

mg, 79%) as a white solid. MS: (ESI) [M + H]⁺224.2.

To a solution of (1-(2-ﬂuoro-4-methoxyphenyl)cyclopentyl)-

methanamine (600 mg, 2.69 mmol) in dry CH₂Cl₂(10 mL) at 0

°C was added BBr₃(2 mL) dropwise, and then the mixture was

stirred for 2 h until the product was detected by LC-MS. After the

reaction was completed, water (10 mL) was added to the solution at 0

°C, and the solution extracted with CH₂Cl₂(30 mL × 3), and the

combined organic layer was dried over with anhydrous Na₂SO₄,

ﬁltrated, and then concentrated under vacuum to obtain 4-(1-

(aminomethyl)cyclopentyl)-3-ﬂuorophenol (368 mg, 65%) as a white

oil. MS: (ESI) [M + H]⁺210.1.

A solution of 4-(1-(aminomethyl)cyclopentyl)-3-ﬂuorophenol (360

mg, 1.72 mmol), Boc₂O (376 mg, 1.72 mmol), NaHCO₃(168 mg, 2

mmol) in THF (10 mL), and water (2 mL) was stirred at room

temperature for 3 h. The solution was concentrated, diluted with

water, and extracted with CH₂Cl₂(20 mL × 3), and the combined

organic layers were dried over with anhydrous Na₂SO₄, ﬁltrated, and

concentrated under vacuum, and the crude product was puriﬁed using

column chromatography to obtain tert-butyl ((1-(2-ﬂuoro-4-

hydroxyphenyl)cyclopentyl)methyl)carbamate (185 mg, 35%) as a

white solid. MS: (ESI) [M + H-tBu]⁺254.2.

A solution tert-butyl ((1-(2-ﬂuoro-4-hydroxyphenyl)cyclopentyl)-

methyl)carbamate (170 mg, 0.55 mmol), 1-(2-chloroethyl)piperidine

(88 mg, 0.6 mmol), and Cs₂CO₃(196 mg, 0.6 mmol) in DMF (10

mL) was stirred at 100 °C for 3 h. The reaction mixture was cooled,

concentrated, diluted with water, and extracted with CH₂Cl₂(20 mL

× 3). The combined organic layers were dried over with anhydrous

Na₂SO₄and ﬁltrated, and the residue was puriﬁed by column

chromatography to obtain tert-butyl ((1-(2-ﬂuoro-4-(2-(piperidin-1-

yl)ethoxy)phenyl)cyclopentyl)methyl)carbamate (118 mg, 51%) as a

white solid. MS: (ESI) [M + H]⁺421.3.

A solution of tert-butyl ((1-(2-ﬂuoro-4-(2-(piperidin-1-yl)ethoxy)-

phenyl)cyclopentyl)methyl)carbamate (118 mg, 0.28 mmol) in 1,4-

dioxane HCl (4 M, 5 mL) was stirred at room temperature. After 2 h,

the solution was concentrated to obtain (1-(2-ﬂuoro-4-(2-(piperidin-

1-yl)ethoxy)phenyl)cyclopentyl)methanamine (85 mg, 95%) as a

white solid. MS: (ESI) [M + H]⁺321.2.

A solution of 2-chloro-3-hydroxybenzaldehyde (47 mg, 0.3 mmol),

(1-(2-ﬂuoro-4-(2-(piperidin-1-yl)ethoxy)phenyl)cyclopentyl)-

methanamine (85 mg, 0.27 mmol) in AcOH (0.2 mL), and MeOH

(10 mL) was stirred for 2 h at room temperature. Then, NaBH₃CN

(19 mg, 0.3 mmol) was added, and the reaction mixture was stirred

overnight. Paraformaldehyde (12 mg, 0.4 mmol) was added, and the

mixture was stirred for 2 h. Then, another aliquot of NaBH₃CN (19

mg, 0.3 mmol) was added and stirring was continued at room

temperature for 5 h. After the reaction was completed, the solution

was concentrated diluted with water and extracted with CH₂Cl₂(20

mL × 3). The combined organic layers were dried over with

To a solution of 4-(2-azidoethoxy)-N-((1-(2-ﬂuorophenyl)-

cyclopentyl)methyl)aniline (350 mg, crude) and 1-(2-ﬂuorophenyl)-

cyclopentane-1-carbaldehyde (78 mg, 0.5 mmol) in MeOH (20 mL)

was added AcOH (0.2 mL), and the reaction mixture was stirred

overnight. Then, NaBH₃CN (124 mg, 2 mmol) was added, and the

reaction mixture was stirred overnight at room temperature. The

reaction mixture was concentrated and then puriﬁed by prep-HPLC

to obtain the title intermediate (110 mg) as a colorless oil. MS: (ESI)

[M + H]⁺489. ¹H NMR (400 MHz, CD₃OD) δ 7.30−7.16 (m, 2H),

7.04−6.97 (m, 2H), 6.90 (t, J = 7.9 Hz, 1H), 6.73 (dd, J = 8.0, 1.2 Hz,

1H), 6.70−6.64 (m, 2H), 6.54 (d, J = 9.2 Hz, 2H), 6.47 (d, J = 7.6

Hz, 1H), 4.15 (s, 2H), 4.05−4.00 (m, 2H), 3.69 (s, 2H), 3.52−3.48

(m, 2H), 2.23−2.13 (m, 2H), 2.05−1.95 (m, 2H), 1.88−1.76 (m,

2H), 1.75−1.63 (m, 2H).

General Procedure for Huisgen 1,3-Diploar Cycloaddition.

Equal volumes of 200 mM THPTA aqueous solution and 100 mM

CuSO₄·5H₂O aqueous solution were combined, vortexed and let

stand for 5 min. Separately, 1 mL of azide solution (25 mM in

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DMSO) and 1 mL of alkyne solution (25 mM) were added to a

reaction vial, vortexed, and then 75 μL of the premixed THPTA/

CuSO₄·5H₂O solution was added, followed by 225 μL of sodium

ascorbate solution (100 mM in water). The reaction mixture was

vortexed and then the reaction was monitored by LCMS. If necessary

the reaction was heated to 50 °C overnight. After reaction

completion, the cooled reaction mixture was loaded directly and

puriﬁed by a mass triggered Waters LCMS reversed phase puriﬁcation

system using water/acetonitrile/0.1% formic acid as an eluent. The

fractions were dried by lyophilization to aﬀord the title compound as

a solid.

2-(2,6-Dioxopiperidin-3-yl)-4-(2-((1-(2-(4-((5-hydroxy-2-(4-hy-

droxyphenyl)-3-methyl-1H-indol-1-yl)methyl)phenoxy)ethyl)-1H-

1,2,3-triazol-4-yl)methoxy)ethoxy)isoindoline-1,3-dione (10). Fol-

lowing General Procedure using 1-(4-(2-azidoethoxy)benzyl)-2-(4-

hydroxyphenyl)-3-methyl-1H-indol-5-ol (10.4 mg, 0.025 mmol) and

2-(2,6-dioxopiperidin-3-yl)-4-(2-(prop-2-yn-1-yloxy)ethoxy)-

isoindoline-1,3-dione (8.9 mg, 0.025 mmol) to obtain the title

compound (14.5 mg, 75% yield). MS: (ESI) [M + H]⁺771.42.

(2S,4R)-4-Hydroxy-1-((S)-2-(2-((1-(2-(4-((5-hydroxy-2-(4-hydroxy-

phenyl)-3-methyl-1H-indol-1-yl)methyl)phenoxy)ethyl)-1H-1,2,3-

triazol-4-yl)methoxy)acetamido)-3,3-dimethylbutanoyl)-N-((S)-1-

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

(11). Following General Procedure using 1-(4-(2-azidoethoxy)-

benzyl)-2-(4-hydroxyphenyl)-3-methyl-1H-indol-5-ol (10.4 mg,

0.025 mmol) and (2S,4R)-1-((S)-2-(1-ﬂuorocyclopropane-1-carbox-

amido)-3,3-dimethylbutanoyl)- 4-hydroxy-N-(4-(4-methylthiazol-5-

yl)-2-(2-(prop-2-yn-1-yloxy)ethoxy)benzyl)pyrrolidine-2-carboxa-

mide (13.5 mg, 0.025 mmol) to obtain the title compound (21.2 mg,

89% yield). MS: (ESI) [M + H]⁺955.43.

(2S,4R)-4-Hydroxy-1-((S)-2-(2-(2-((1-(2-(4-((5-hydroxy-2-(4-hy-

droxyphenyl)-3-methyl-1H-indol-1-yl)methyl)phenoxy)ethyl)-1H-

1,2,3-triazol-4-yl)methoxy)ethoxy)acetamido)-3,3-dimethylbuta-

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

carboxamide (12). Following General Procedure using 1-(4-(2-

azidoethoxy)benzyl)-2-(4-hydroxyphenyl)-3-methyl-1H-indol-5-ol

solution (10.4 mg, 0.025 mmol) and (2S,4R)-1-((S)-3,3-dimethyl-2-

(2-(2-(prop-2-yn-1-yloxy)ethoxy)acetamido)butanoyl)-4-hydroxy-N-

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

mide solution (14.6 mg, 0.025 mmol) to aﬀord the title compound

(22.2 mg, 89% yield). MS: (ESI) [M + H]⁺999.38.

(2S,4R)-1-((S)-12-(tert-Butyl)-1-(1-(2-(4-((5-hydroxy-2-(4-hydrox-

yphenyl)-3-methyl-1H-indol-1-yl)methyl)phenoxy)ethyl)-1H-1,2,3-

triazol-4-yl)-10-oxo-2,5,8-trioxa-11-azatridecan-13-oyl)-4-hydroxy-

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

boxamide (13). Following General Procedure using 1-(4-(2-

azidoethoxy)benzyl)-2-(4-hydroxyphenyl)-3-methyl-1H-indol-5-ol

(10.4 mg, 0.025 mmol) and (2S,4R)-1-((S)-2-(tert-butyl)-4-oxo-

6,9,12-trioxa-3-azapentadec-14-ynoyl)-4-hydroxy-N-((S)-1-(4-(4-

methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (15.7 mg,

0.025 mmol) to aﬀord the title compound (22.3 mg, 86%). MS: (ESI)

[M + H]⁺1043.43.

obtain the title compound as the formic salt. MS: (ESI) [M + H]⁺

995.47.

(S)-N-((S)-2-((S)-2-(4-(4-Fluorobenzoyl)thiazol-2-yl)pyrrolidin-1-

yl)-1-(1-(2-(2-((1-(2-(4-((5-hydroxy-2-(4-hydroxyphenyl)-3-methyl-

1H-indol-1-yl)methyl)phenoxy)ethyl)-1H-1,2,3-triazol-4-yl)-

methoxy)ethoxy)acetyl)piperidin-4-yl)-2-oxoethyl)-2-

(methylamino)propanamide (16). To a solution of 1-(4-(2-

azidoethoxy)benzyl)-2-(4-hydroxyphenyl)-3-methyl-1H-indol-5-ol

(22 mg, 0.053 mmol) and (S)-N-((S)-2-((S)-2-(4-(4-ﬂuorobenzoyl)-

thiazol-2-yl)pyrrolidin-1-yl)-2-oxo-1-(1-(2-(2-(prop-2-yn-1-yloxy)-

ethoxy)acetyl)piperidin-4-yl)ethyl)-2-(methylamino)propanamide

(21 mg, 0.033 mmol) in DMSO (2 mL) was added 0.08 mL of

premixed aqueous solution of CuSO₄·5H₂O (100 mM, 0.04 mL) and

THPTA (200 mM, 0.04 mL), and the mixture was stirred at room

temperature for 2 min followed by the addition of sodium ascorbate

(100 mM, 0.225 mL). The combined mixture was then shaken at 50

°C for 12 h. The cooled reaction mixture was loaded directly onto the

Waters LCMS reversed phase puriﬁcation system using water/

acetonitrile/0.1% FA as an eluent. The fractions were dried by

lyophilization to aﬀord the title compound as the formate salt (14.7

mg, 42% yield). MS: (ESI) [M + H]⁺1056.37.

(2S,4R)-1-((S)-2-(2-((1-(2-(4-((2-Chloro-3-hydroxybenzyl)((1-(2-

ﬂuorophenyl)cyclopentyl)methyl)amino)phenoxy)ethyl)-1H-1,2,3-

triazol-4-yl)methoxy)acetamido)-3,3-dimethylbutanoyl)-4-hy-

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

carboxamide (17). Following General Procedure using 3-(((4-(2-

azidoethoxy)phenyl)((1-(2-ﬂuorophenyl)cyclopentyl)methyl)-

amino)methyl)-2-chlorophenol (12.4 mg, 0.025 mmol) and (2S,4R)-

1-((S)-3,3-dimethyl-2-(2-(prop-2-yn-1-yloxy)acetamido)butanoyl)-4-

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

2-carboxamide (13.5 mg, 0.025 mmol) to aﬀord the title compound as

the formic acid salt (9.6 mg, 35% yield). MS: (ESI) [M + H]⁺

1035.35.

(2S,4R)-1-((S)-2-(2-(2-((1-(2-(4-((2-Chloro-3-hydroxybenzyl)((1-

(2-ﬂuorophenyl)cyclopentyl)methyl)amino)phenoxy)ethyl)-1H-

1,2,3-triazol-4-yl)methoxy)ethoxy)acetamido)-3,3-dimethylbuta-

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

pyrrolidine-2-carboxamide (18). To a solution of 3-(((4-(2-

azidoethoxy)phenyl)((1-(2-ﬂuorophenyl)cyclopentyl)methyl)-

amino)methyl)-2-chlorophenol(372 mg, 0.75 mmol) and (2S,4R)-1-

((S)-3,3-dimethyl-2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)acetamido)-

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

ethyl)pyrrolidine-2-carboxamide (422 mg, 0.723 mmol) in DMSO

(15 mL) was added 0.8 mL of premixed aqueous solution of CuSO₄(

100 mM, 0.4 mL) and THPTA (200 mM, 0.4 mL), and the mixture

was stirred at room temperature for 2 min followed by the addition of

sodium ascorbate (100 mM, 2.25 mL). The combined mixture was

then stirred at 25 °C for 12 h. The mixture was diluted with water (30

mL) and extracted with EtOAc (30 mL × 2), and the organic phase

was washed with brine (20 mL), dried over anhydrous Na₂SO₄,

concentrated to an oil, and puriﬁed over silica gel column

chromatography eluted with DCM/MeOH (gradient) to obtain the

title compound as a white solid (777 mg, 96% yield). MS: (ESI) [M +

H]⁺1079.37; HRMS (ES⁺) for C₅₇H₆₈ClFN₈O₈S (M + H)⁺: calcd

1079.4632; found 1079.4646; ¹H NMR (400 MHz, DMSO-d₆) δ

ppm 0.92 (s, 9H), 1.35 (d, J = 7.03 Hz, 3H), 1.57 (br s, 2H), 1.65−

1.81 (m, 3H), 1.84−1.96 (m, 2H), 1.97−2.10(m, 3H), 2.45 (s, 3H),

3.55−3.68 (m, 8H), 3.95 (s, 2H), 4.05 (s, 2H), 4.17−4.31 (m, 3H),

4.45 (t, J = 8.28 Hz, 1H), 4.52−4.62 (m, 3H), 4.66 (br t, J = 4.89 Hz,

2H), 4.90 (br t, J = 7.15 Hz, 1H), 5.13 (d, J = 3.51 Hz, 1H), 6.30 (d, J

= 7.53 Hz, 1H), 6.42−6.51 (m, 2H), 6.60−6.65 (m, 2H), 6.75 (d, J =

7.28 Hz, 1H), 6.90 (t, J = 7.78 Hz, 1H), 7.03−7.12 (m, 2H), 7.18−

7.29 (m, 2H), 7.33−7.46 (m, 5H), 8.12−8.17 (m, 1H), 8.44 (d, J =

7.53 Hz, 1H), 8.98 (s, 1 H), 10.03 (s, 1H).

(2S,4R)-1-((S)-2-(1-Fluorocyclopropane-1-carboxamido)-3,3-di-

methylbutanoyl)-4-hydroxy-N-(2-(2-((1-(2-(4-((5-hydroxy-2-(4-hy-

droxyphenyl)-3-methyl-1H-indol-1-yl)methyl)phenoxy)ethyl)-1H-

1,2,3-triazol-4-yl)methoxy)ethoxy)-4-(4-methylthiazol-5-yl)benzyl)-

pyrrolidine-2-carboxamide (14). Following General Procedure using

1-(4-(2-azidoethoxy)benzyl)-2-(4-hydroxyphenyl)-3-methyl-1H-

indol-5-ol and (2S,4R)-1-((S)-2-(1-ﬂuorocyclopropane-1-carboxami-

do)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)-2-

(2-(prop-2-yn-1-yloxy)ethoxy)benzyl)pyrrolidine-2-carboxamide to

obtain the title compound (55 mg, 71%). MS: (ESI) [M + H]⁺

1029.26.

(S)-N-((S)-1-Cyclohexyl-2-((S)-2-(4-(3-(2-((1-(2-(4-((5-hydroxy-2-

(4-hydroxyphenyl)-3-methyl-1H-indol-1-yl)methyl)phenoxy)ethyl)-

1H-1,2,3-triazol-4-yl)methoxy)ethoxy)benzoyl)thiazol-2-yl)-

pyrrolidin-1-yl)-2-oxoethyl)-2-(methylamino)propanamide (15).

Following General Procedure using 1-(4-(2-azidoethoxy)benzyl)-2-

(4-hydroxyphenyl)-3-methyl-1H-indol-5-ol and (S)-N-((S)-1-cyclo-

hexyl-2-oxo-2-((S)-2-(4-(3-(2-(prop-2-yn-1-yloxy)ethoxy)benzoyl)-

thiazol-2-yl)pyrrolidin-1-yl)ethyl)-2-(methylamino)propanamide to

(2S,4R)-1-((S)-12-(tert-Butyl)-1-(1-(2-(4-((2-chloro-3-

hydroxybenzyl)((1-(2-ﬂuorophenyl)cyclopentyl)methyl)amino)-

phenoxy)ethyl)-1H-1,2,3-triazol-4-yl)-10-oxo-2,5,8-trioxa-11-aza-

tridecan-13-oyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)-

phenyl)ethyl)pyrrolidine-2-carboxamide (19). Following General

Procedure using 3-(((4-(2-azidoethoxy)phenyl)((1-(2-ﬂuorophenyl)-

cyclopentyl)methyl)amino)methyl)-2-chlorophenol (12.4 mg, 0.025

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mmol) and (2S,4R)-1-((S)-2-(tert-butyl)-4-oxo-6,9,12-trioxa-3-aza-

pentadec-14-ynoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)-

phenyl)ethyl)pyrrolidine-2-carboxamide (15.7 mg, 0.025 mmol) to

obtain the title compound as a formate salt (1.5 mg, 5%). MS: (ESI)

[M + H]⁺1123.25.

(2S,4R)-N-(2-(2-((1-(2-(4-((2-Chloro-3-hydroxybenzyl)((1-(2-

ﬂuorophenyl)cyclopentyl)methyl)amino)phenoxy)ethyl)-1H-1,2,3-

triazol-4-yl)methoxy)ethoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-

((S)-2-(1-ﬂuorocyclopropane-1-carboxamido)-3,3-dimethylbutano-

yl)-4-hydroxypyrrolidine-2-carboxamide (20). Following General

Procedure using 3-(((4-(2-azidoethoxy)phenyl)((1-(2-ﬂuorophenyl)-

cyclopentyl)methyl)amino)methyl)-2-chlorophenol (12.4 mg, 0.025

mmol) and (2S,4R)-1-((S)-2-(1-ﬂuorocyclopropane-1-carboxamido)-

3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)-2-(2-

(prop-2-yn-1-yloxy)ethoxy)benzyl)pyrrolidine-2-carboxamide (15.4

mg, 0.025 mmol) to obtain the title compound as the formic acid

salt (9.4 mg, 29%). MS: (ESI) [M + H]⁺1109.38.

mide to aﬀord the title compound as the formic acid salt. MS: (ESI)

[M + H]⁺1079.40.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00127.

■

sı

Denaturing gel of proteins, LC-MS for compounds 1−

23 (parent ion, retention time, purity, and chromato-

1

grams), H and ¹³C NMR spectra for compounds 18

and 21, supplemental ﬁgures demonstrating puriﬁcation

of ERα proteins and degradation of ERα in ER+ breast

cancer cells, and supplemental methods giving detailed

protocols for protein production, biochemical and

cellular assays, and DECL screening (PDF)

(2S,4R)-N-(2-(2-(2-((1-(2-(4-((2-Chloro-3-hydroxybenzyl)((1-(2-

ﬂuorophenyl)cyclopentyl)methyl)amino)phenoxy)ethyl)-1H-1,2,3-

triazol-4-yl)methoxy)ethoxy)ethoxy)-4-(4-methylthiazol-5-yl)-

benzyl)-1-((S)-2-(1-ﬂuorocyclopropane-1-carboxamido)-3,3-dime-

thylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (21). To a

solution of 3-(((4-(2-azidoethoxy)phenyl)((1-(2-ﬂuorophenyl)-

cyclopentyl)methyl)amino)methyl)-2-chlorophenol (421 mg, 0.85

mmol) and (2S,4R)-1-((S)-2-(1-ﬂuorocyclopropane-1-carboxami-

do)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)-2-

(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)benzyl)pyrrolidine-2-car-

boxamide (519 mg, 0.788 mmol) in DMSO (15 mL) was added 0.8

mL of premixed aqueous solution of CuSO₄(100 mM, 0.4 mL) and

THPTA (200 mM, 0.4 mL), and the mixture was stirred at room

temperature for 2 min followed by the addition of sodium ascorbate

(100 mM, 2.25 mL). The combined mixture was then stirred at 25 °C

for 12 h. The mixture was diluted with water (30 mL), extracted with

EtOAc (30 mL x 2), the organic phase was washed with brine (20

mL), dried over anhydrous Na₂SO₄, concentrated to an oil, and

puriﬁed over silica gel column chromatography eluted with DCM/

MeOH (gradient) to obtain the title compound as a white solid (864

mg, 95%). MS: (ESI) [M + H]⁺1153.41; HRMS (ES⁺) for

C₆₀H₇₁ClF₂N₈O₉S (M + H)⁺: calcd 1153.4800; found 1153.4794; ¹H

NMR (400 MHz, DMSO-d₆) δ ppm 0.93 (s, 9H), 1.10−1.28 (m,

2H), 1.29−1.43 (m, 2H), 1.57 (m, 2H), 1.73 (m, 2H), 1.84−1.92 (m,

3H), 1.99−2.12 (m, 3H), 2.43 (s, 3H), 3.56−3.67 (m, 8H), 3.71−

3.83 (m, 2H), 4.04 (s, 2H), 4.12−4.23 (m, 5H), 4.25−4.38 (m, 2H),

4.46−4.55 (m, 3H), 4.56−4.70 (m, 3H), 5.17 (d, J = 3.51 Hz, 1H),

6.30 (d, J = 7.53 Hz, 1H), 6.45 (m, J = 9.03 Hz, 2H), 6.61 (m, J =

9.03 Hz, 2H), 6.75 (d, J = 7.03 Hz, 1H), 6.89 (t, J = 7.41 Hz, 1H),

6.96 (d, J = 7.26 Hz, 1H), 7.01−7.11 (m, 3H), 7.18−7.32 (m, 3H),

7.41 (d, J = 8.03 Hz, 1H), 8.09 (s, 1H), 8.49 (t, J = 5.90 Hz, 1H), 8.97

(s, 1H), 10.03 (s, 1H).

(2S,4R)-N-(2-(2-(2-(2-((1-(2-(4-((2-Chloro-3-hydroxybenzyl)((1-(2-

ﬂuorophenyl)cyclopentyl)methyl)amino)phenoxy)ethyl)-1H-1,2,3-

triazol-4-yl)methoxy)ethoxy)ethoxy)ethoxy)-4-(4-methylthiazol-5-

yl)benzyl)-1-((S)-2-(1-ﬂuorocyclopropane-1-carboxamido)-3,3-di-

methylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (22). Fol-

lowing General Procedure using 3-(((4-(2-azidoethoxy)phenyl)((1-

(2-ﬂuorophenyl)cyclopentyl)methyl)amino)methyl)-2-chlorophenol

(12.4 mg, 0.025 mmol) and (2S,4R)-1-((S)-2-(1-ﬂuorocyclopropane-

1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methyl-

thiazol-5-yl)-2-(2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethoxy)-

benzyl)pyrrolidine-2-carboxamide (17.6 mg, 0.025 mmol) to obtain

the title compound as a formate salt (5.5 mg, 18% yield). MS: (ESI)

[M + H]⁺1197.38.

(2S,4S)-1-((S)-2-(2-(2-((1-(2-(4-((2-Chloro-3-hydroxybenzyl)((1-(2-

ﬂuorophenyl)cyclopentyl)methyl)amino)phenoxy)ethyl)-1H-1,2,3-

triazol-4-yl)methoxy)ethoxy)acetamido)-3,3-dimethylbutanoyl)-4-

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

2-carboxamide (23). Following General Procedure using 3-(((4-(2-

azidoethoxy)phenyl)((1-(2-ﬂuorophenyl)cyclopentyl)methyl)-

amino)methyl)-2-chlorophenol and (2S,4S)-1-((S)-3,3-dimethyl-2-

(2-(2-(prop-2-yn-1-yloxy)ethoxy)acetamido)butanoyl)-4-hydroxy-N-

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

Molecular formula strings and biological data (CSV)

AUTHOR INFORMATION

Corresponding Authors

■

Jeremy S. Disch − X-Chem Inc., Waltham, Massachusetts

02453, United States; orcid.org/0000-0002-0357-1672;

Email: jdisch92@gmail.com

Shilpi Arora − X-Chem Inc., Waltham, Massachusetts 02453,

United States; Email: ssoniarora@gmail.com

Authors

Jennifer M. Duﬀy − X-Chem Inc., Waltham, Massachusetts

02453, United States

Esther C. Y. Lee − X-Chem Inc., Waltham, Massachusetts

02453, United States

Diana Gikunju − X-Chem Inc., Waltham, Massachusetts

02453, United States

Betty Chan − X-Chem Inc., Waltham, Massachusetts 02453,

United States

Benjamin Levin − X-Chem Inc., Waltham, Massachusetts

02453, United States

Michael I. Monteiro − X-Chem Inc., Waltham, Massachusetts

02453, United States

Sarah A. Talcott − X-Chem Inc., Waltham, Massachusetts

02453, United States

Anthony C. Lau − X-Chem Inc., Waltham, Massachusetts

02453, United States

Fei Zhou − X-Chem Inc., Waltham, Massachusetts 02453,

United States

Anton Kozhushnyan − X-Chem Inc., Waltham, Massachusetts

02453, United States

Neil E. Westlund − X-Chem Inc., Waltham, Massachusetts

02453, United States

Patrick B. Mullins − X-Chem Inc., Waltham, Massachusetts

02453, United States

Yan Yu − X-Chem Inc., Waltham, Massachusetts 02453,

United States

Moritz von Rechenberg − X-Chem Inc., Waltham,

Massachusetts 02453, United States

Junyi Zhang − X-Chem Inc., Waltham, Massachusetts 02453,

United States

Yelena A. Arnautova − X-Chem Inc., Waltham, Massachusetts

02453, United States

Yanbin Liu − X-Chem Inc., Waltham, Massachusetts 02453,

United States

Ying Zhang − X-Chem Inc., Waltham, Massachusetts 02453,

United States

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incidence and mortality worldwide for 36 cancers in 185 countries.

CA Cancer J. Clin. 2018, 68, 394−424.

Andrew J. McRiner − X-Chem Inc., Waltham, Massachusetts

02453, United States

Anthony D. Keefe − X-Chem Inc., Waltham, Massachusetts

02453, United States

Anna Kohlmann − X-Chem Inc., Waltham, Massachusetts

02453, United States

Matthew A. Clark − X-Chem Inc., Waltham, Massachusetts

02453, United States

John W. Cuozzo − X-Chem Inc., Waltham, Massachusetts

02453, United States; orcid.org/0000-0002-6229-0395

Christelle Huguet − X-Chem Inc., Waltham, Massachusetts

02453, United States

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Author Contributions

J.S.D., E.C.Y.L., J.Z., A.J.M., Y.Z., and M.A.C. contributed to

the design of compounds, the establishment of the E3 alkyne

toolkit, and evaluation of results; J.M.D. optimized and ran the

cell-based viability and degradation assays and other cell-based

assays; A.C.L. ran some of the degradation assays; S.A.T.

optimized and executed the gene expression analysis. D.G. and

B.C. planned and performed the biochemical assays; A.D.K.

designed and M.I.M. performed the selections; B.L. and M.v.R.

oversaw protein production and performed protein puriﬁca-

tions; J.W.C. and C.H. helped plan the biology and in vivo

experiments; A.K. and Y.A.A. performed the molecular

docking; compounds were synthesized by F.Z., A.K., N.E.W.,

P.B.M., and Y.Y.; Y.L. planned and oversaw the synthesis of the

small molecules; A.J.M. and E.C.Y.L. oversaw all outsourced

ADME and PK work; J.S.D., E.C.Y.L., A.J.M., and S.A. planned

the mouse xenograft eﬃcacy study; S.A. planned and conceived

all the biology experiments, oversaw the performance of the

outsourced mouse xenograft eﬃcacy study, and was the project

lead. All authors helped in writing and reviewing of the

manuscript.

Funding

The work included in the manuscript was performed as part of

internal drug discovery eﬀorts at X-Chem Inc. No grant

support was used.

Notes

The authors declare the following competing ﬁnancial

interest(s): All authors are current or former employees of

X-Chem Inc., which conducted the work.

ACKNOWLEDGMENTS

■

Authors would like to thank Marie-Aude Guié and Scott

Lindberg for providing scientiﬁc database management and

Surayya Sana, Masarah Taktak, and Autumn Jetson for

compound management. Some of the small molecule synthesis

was performed at BioDuro (China).

ABBREVIATIONS

(17) Li, X.; Song, Y. Proteolysis-targeting chimera (PROTAC) for

targeted protein degradation and cancer therapy. J. Hematol. Oncol.

2020, 13, 50.

■

ERα, estrogen receptor α; PROTAC, proteolysis targeting

chimera; VHL, von Hippel−Lindau E3 ubiquitin ligase; IAP,

inhibitor of apoptosis protein; DECL, DNA-encoded chemical

library; POI, protein of interest; TGI, tumor growth inhibition;

ENRv1, enrichment metric

(18) Dai, Y.; Yue, N.; Gong, J.; Liu, C.; Li, Q.; Zhou, J.; Huang, W.;

Qian, H. Development of cell-permeable peptide-based PROTACs

targeting estrogen receptor α . Eur. J. Med. Chem. 2020, 187, 111967.

(19) Flanagan, J. J.; Neklesa, T. K. Targeting Nuclear Receptors with

PROTAC degraders. Mol. Cell. Endocrinol. 2019, 493, 110452.

(20) Hu, J.; Hu, B.; Wang, M.; Xu, F.; Miao, B.; Yang, C. Y.; Wang,

M.; Liu, Z.; Hayes, D. F.; Chinnaswamy, K.; Delproposto, J.; Stuckey,

J.; Wang, S. Discovery of ERD-308 as a Highly Potent Proteolysis

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Article Doi

Bispecific Estrogen Receptor α Degraders Incorporating Novel Binders Identified Using DNA-Encoded Chemical Library Screening

DOI: 10.1021/acs.jmedchem.1c00127

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