New Class of Selective Estrogen Receptor Degraders (SERDs): Expanding the Toolbox of PROTAC Degrons

Article abstract of DOI:10.1021/acsmedchemlett.8b00106

An effective endocrine therapy for breast cancer is to selectively and effectively degrade the estrogen receptor (ER). Up until now, there have been largely only two molecular scaffolds capable of doing this. In this study, we have developed new classes of scaffolds that possess selective estrogen receptor degrader (SERD) and ER antagonistic properties. These novel SERDs potently inhibit MCF-7 breast cancer cell proliferation and the expression of ER target genes, and their efficacy is comparable to Fulvestrant. Unlike Fulvestrant, the modular protein-targeted chimera (PROTAC)-type design of these novel SERDs should allow easy diversification into a library of analogs to further fine-tune their pharmacokinetic properties including oral availability. This work also expands the pool of currently available PROTAC-type scaffolds that could be beneficial for targeted degradation of various other therapeutically important proteins.

Full text of DOI:10.1021/acsmedchemlett.8b00106

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Letter
A New Class of Selective Estrogen Receptor Degraders
(SERDs): Expanding the Toolbox of PROTAC Degrons
Lucia Wang, Valeria Sanabria Guillen, Naina Sharma, Kevin Flessa, Jian
Min, Kathryn E. Carlson, Weiyi Toy, Sara Braqi, Benita S. Katzenellenbogen,
John A Katzenellenbogen, Sarat Chandarlapaty, and Abhishek Sharma
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00106 • Publication Date (Web): 05 Jul 2018
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A New Class of Selective Estrogen Receptor Degraders (SERDs):
Expanding the Toolbox of PROTAC Degrons
Lucia Wang,§,† Valeria S. Guillen,§,‡ Naina Sharma,‡ Kevin Flessa,† Jian Min,‡ Kathryn E. Carlson,‡
Weiyi Toy,∥ Sara Braqi,† Benita S. Katzenellenbogen,‡ John A. Katzenellenbogen,‡ Sarat Chandarꢀ
lapaty∥ and Abhishek Sharma*,†
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†Department of Chemistry and Chemical Biology, Stevens Institute of Technology, Hoboken, New Jersey 07601 United
States
‡Department of Chemistry, Department of Molecular and Integrative Physiology, University of Illinois at Urbanaꢀ
Champaign, Urbana, Illinois 61801 United States
∥Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, United
States
ABSTRACT: An effective endocrine therapy for breast cancer is to selectively and effectively degrade the estrogen receptor (ER).
Up until now, there have been largely only two molecular scaffolds capable of doing this. In this study, we have developed new
classes of scaffolds that possess selective estrogen receptor degrader (SERD) and ER antagonistic properties. These novel SERDs
potently inhibit MCFꢀ7 breast cancer cell proliferation and the expression of ER target genes, and their efficacy is comparable to
Fulvestrant. Unlike Fulvestrant, the modular proteinꢀtargeted chimera (PROTAC)ꢀtype design of these novel SERDs should allow
easy diversification into a library of analogs to further fineꢀtune their pharmacokinetic properties including oral availability. This
work also expands the pool of currently available PROTACꢀtype scaffolds which could be beneficial for targeted degradation of
various other therapeutically important proteins.
KEYWORDS: Targeted protein degradation, estrogen receptor, antiproliferation, breast cancer, antagonist
OH
Estrogen receptorꢀalpha (ERα) is the target of endocrine theraꢀ
- The only SERD that is clinically approved
- Administered by a painful injection
- Difficult to make analogs libraries
Fulvestrant
(1991)
pies for treatment of the more than 70% of breast cancers that
are ERꢀpositive.1 Among these therapies, smallꢀmoleculeꢀ
induced, targeted degradation of ERs is the last line of
treatment, especially in metastatic breast cancer patients who
have become resistant to therapies that inhibit the function of
ER.2 This targeted destruction of ER is induced by molecules
(Selective Estrogen Receptor Degraders or SERDs) that
possess distinct structural elements for binding to the ligandꢀ
binding pocket (LBP) of ER and recruitment of the cellular
protein degradation machinery. Despite the immense
therapeutic importance of SERDs, the repertoire of molecular
scaffolds known to induce ER degradation (degrons) has been
rather limited, and their degron properties were discovered
serendipitously. Currently, there is only one clinically
approved SERD, Fulvestrant3 (Figure 1a), and some related
analogs,4 which possess a core for binding in the LBP and a
long alkyl side chain (degron) that induces ER degradation.3
The clinical utility of Fulvestrant is hampered due to its poor
oral bioavailability, so that it has to be administered as a large
painful intramuscular injection.5 Moreover, the bulky and
steroidal structure of Fulvestrant also limits further chemical
diversification to improve its bioactivity. A second structurally
distinct small molecule scaffold/degron known to confer
SERD properties is an acrylic acid based side chain, which
was first developed in 1994 (GW5638, Figure 1b),6 as well as
more recent versions having diversified ligand core elements
but the same side chain; this class of SERDs is still awaiting
full clinical evaluation.7ꢀ10
H
(a)
F
H
O
H
F
F
HO
S
6
F
F
O
OH
(b)
GW 5638 - Second known SERD scaffold
(1994) - Two analogs in clinical trials
OH
- A novel class of SERDs
This work
- Potent anti-proliferative activity
- Modular design and ease of synthesis
allows rapid chemical diversification
to fine-tune chemical properties
- Expands the tool-box of
(2018)
(c)
O
H
PROTAC degrons
N
O
n
R
n = 0-1, R = Various degrons
Figure 1. (aꢀb): Previously known SERD scaffolds and (c) the
newly developed class of SERDs. The number in parenthesis
represents the year of discovery of each SERD. The big time
gap between the last discovered SERD (b) and the latest (c)
illustrates the challenges in discovering new SERD scaffolds.
In view of the extremely limited pool of currently available
SERDs,11 there is an urgent need for development of
structurally novel classes of small molecules that are not only
capable of inducing ER degradation in breast cancer and
blocking cancer cell proliferation, but also ideally would allow
easy chemical diversification to fineꢀtune their physicochemiꢀ
cal and biological properties.
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Recently, there has been an increased interest in developing
Page 2 of 7
be projected outside the surface of ER through an exit channel
in the antagonist conformation of the ligandꢀbinding domain.
Thus, we used this second phenol to attach an alkyl linker
possessing an amine terminus for coupling with various
degron candidates.
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targeted protein degradation strategies for the treatment of
various types of cancers and other diseases.12ꢀ16 These proteolꢀ
ysis targeting chimeras (PROTACs) possess a targeting ligand
attached to a recognition motif (“degron”) that binds to E3
ubiquitin ligases or other proteins that ultimately promote
ubiquitinationꢀdependent proteasomal degradation of the
target protein. In this context, several interesting and useful
“degron motifs” have been developed for the targeted
destruction of clinically relevant proteins.12,13,17 The previous
PROTACs for ER comprised peptidic17 or bestatin esterꢀbased
degrons,18,19 which limited their drugꢀlike properties or
imparted offꢀtarget effects and low potency, besides requiring
specific E3 ligases for ubiquitin mediated degradation.17, 20 In
order to realize the full potential of PROTACs as a therapeutic
option for breast cancer and other diseases, it would be
beneficial to develop novel and simpler degrons that allow the
characterization and optimization of functional contributions
made by distinct regions of a degron to ultimately aid its cliniꢀ
cal application.
We initially selected Leu, Phe and Trp from the pool of amiꢀ
no acids that are known to participate in the Nꢀend rule
pathway.21 This selection was based on the assumption that
mild to strong hydrophobicity might also contribute to their
degron action on ER, as is the case with other SERDs. In view
of the easy availability of Bocꢀamino acids and the
hydrophobic character of Boc group,25 these Nꢀprotected (L)
amino acids were chosen as degronꢀtype elements along with
their unprotected versions for comparison. These amino acids
were converted into their NHS esters and then coupled to the
amine side chain of ER ligands to give compounds 2ꢀ4
(Scheme 1).
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Binding assays showed that installation of these 3 degron
side chains on this ligand core did not preclude them from
binding directly to ER (Table 1), although their relative bindꢀ
ing affinities (RBAs) decreased somewhat from that of the
parent bisphenolic core.24 Assessing cellular levels of ERα by
inꢀcell western (ICW) assays (Figure 2A, Table 1) showed that
only the BocꢀTrp analog (4) had high, subnanomolar potency,
Nature devised the ubiquitinꢀdependent degradation pathway
for removing unwanted or damaged cellular proteins. An
important component of this protein quality control machinery
is the Nꢀend rule pathway, wherein the Nꢀterminus of target
proteins is conjugated with a destabilizing amino acid (degron)
that is recognized by the ubiquitin proteasome system.21 In the
Table 1. Structureꢀactivity data of novel SERDs: Summary
of ERα binding affinity, potency and efficacy in antiprolifꢀ
eration and ERα downregulation assaysa
course of
our efforts to
develop novel ER
antagonists/SERDs,22,23 we became interested in exploring the
selective degradation of ERs using ligands that carry a destabiꢀ
lizing Nꢀend rule amino acid or other degrons. Herein, we
report on a novel class of PROTACs (Figure 1c) that (i)
effectively induce ER degradation and show potent antiprolifꢀ
erative activity; (ii) can be easily diversified to fineꢀtune their
biological/ADMET properties, and (iii) significantly expand
the repertoire of currently known PROTAC scaffolds.
Our molecular design for SERDs involved a bisphenolicꢀ
adamantyl system (Scheme 1) as the “core” to anchor our ligꢀ
NH2
n
OH
O
i) Cs2CO3
Antiproꢀ
liferation
IC50 (nM)
ERα
Downꢀ
regulation
IC50 (nM)
DMF, 12 h
OMs
+
BocHN
n = 1-3
n
RBA
(estradiol
= 100)
ii) TFA, CH2Cl2,
12 h
Structure
(R)
Entry
[% of
vehicle]b
[% of
HO
HO
O
vehicle]b
H
Et3N, DMF,
12 h
N
n
R
O
O
R
O-NHS
<0.1
<0.1
1
Fulvestrant
61 ± 1
H
[0]
[9]
N
R
O
n
Glu
Degron
O
Lipophilic amino acids as Degrons
O
36
636
2
14 ± 1
Arg
NHBoc
NHBoc
[20]
[56]
HO
Scheme 1. Divergent route for synthesis of novel SERDs.
Inset: Our model for design of ER targeting PROTACs.
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3
4
14 ± 4
20 ± 6
[71]
[25]
and inside the LBP of ER. We had earlier developed these
cyclic cores as very high affinity ligands (ca 2ꢀ3 times higher
affinity affinity than estradiol) for ER.24 Our molecular modelꢀ
ing studies suggested that one of the phenols of these ligandsꢀ
forms the canonical Hꢀbonds with Glu353 and Arg394 in ERα,
while a side chain attached on the other phenol is expected to
16
0.5
[45]
[16]
NHBoc
HN
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Trp analogs (3 and 4) being the most potent. Thus, the Boc
Tricyclic and Bicyclic Degrons
1
2
3
4
5
6
7
8
group as well as the amino acid side chain make distinct conꢀ
tributions to the ERα degradation and antiproliferative
activities.
17
NDc
5
6
21 ± 6
26 ± 6
[16]
[76]
(C2 linker, n=1)
(C3 linker, n=2)
1, Fulvestrant
Boc Amino Acids
Tricyclic & Bicyclic
Monocylic
A
B
C
125
100
75
2
3
4
9
3
[13]
[42]
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[51]
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6.0 ± 0.1
125
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[57]
[42]
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[42]
[71]
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[42]
[53]
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Monocyclic Degrons
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NDc
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[69]
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0.4
12
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28 ± 6
30 ± 5
[13]
[35]
ꢀ11
ꢀ10
ꢀ9
ꢀ8
ꢀ7
ꢀ6
ꢀ5
Log Concentration (M)
5
NDc
[15]
[76]
Figure 2. DoseꢀResponse of ERα Level in MCFꢀ7 Cells.
MCFꢀ7 cells in stripped medium were treated with indicated
concentrations of Fulvestrant (1) or compounds 2ꢀ15 for 24
hours, and ERα levels were determined by in cell Western
analysis. Values represent average ± SEM of 4 measurements.
100% represents ERα levels in vehicle treated samples. (For
representative Western Blots, see SI, Figure S1)
4
NDc
30 ± 0.1
10 ± 0.4
[20]
[67]
0.5
2
[13]
[31]
Encouraged by the initial success of our new PROTAC
model for ER, we sought to systematically investigate whether
the ER degrading/antiproliferative capability depends on
increased structural complexity/hydrophobicity of degron
motifs in PROTACs. Thus, we prepared a series of bridged
bicyclic and tricyclic analogs (5ꢀ10) and a variety of monocyꢀ
clic analogs (11ꢀ15); with the adamantyl26 candidate degron,
the length of the linker chain was also varied (5ꢀ7). The RBA
values of most of these compounds were comparable to those
of the previous series (1ꢀ4), with one (11) exceeding that of
fulvestrant (1).
an = 3 unless otherwise noted. bEfficacy values are expressed as %
of ERα level (Fig 2) or relative proliferation rate (Fig 3) at the
highest compound dose relative to vehicle control. Proliferation
with Fulvestrant is considered zero. cIC50 values could not be deꢀ
termined accurately due to limited ERα degradation.
although it was less efficacious than fulvestrant; removal of
the Boc group in 4 completely abolished its SERD activity
(not shown). All three of the Bocꢀamino acid PROTACs were
efficacious, low nanomolar inhibitors of MCFꢀ7 cell
proliferation (Table 1, Figure 3A), with the BocꢀPhe and Bocꢀ
All members of the biꢀ and tricyclic group (5ꢀ10) were low
nanomolar antiproliferative agents (Table 1, Figure 3B), with
overall the three compounds having an adamantane degron (5ꢀ
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ACS Medicinal Chemistry Letters
7) giving more complete suppression than those with other
degron candidates (8ꢀ10). The length of the linker in comꢀ
pounds 5ꢀ7 had only a modest effect on antiproliferative poꢀ
tency and efficacy. Although the potencies of these comꢀ
pounds in degrading ERα were good, the extent of degradation
varied (Table 1, Figure 2B), with compounds 6 and 8 being
more complete than the others, and 5 being the least potent
and efficacious.
Page 4 of 7
effect” is an important benefit of these novel PROTACs as
compared to many of the current PROTACs which show reꢀ
duced potency at higher concentrations.15 Overall, the comꢀ
pounds possessing degrons from Nꢀend rule pathway (2ꢀ4)
were less effective ERα degraders and antiproliferative agents
as compared to those possessing bicyclic and monocyclic
degrons (5ꢀ15). Further, the comparatively better potency of
Fulvestrant could be due to its higher affinity (RBA=61) toꢀ
wards the estrogen receptor compared to all of the new ligands
but compound 11.
1
2
3
4
5
6
7
8
140
120
100
80
1, Fulvestrant
Boc Amino Acids
Tricyclic and Bicyclic
Monocyclic
9
A
2
3
4
We selected for further characterization one compound of
interest from each category, based on the structural novelty of
the degron and a combination of their potency and efficacy in
antiproliferative and ERα degradation assays: these were the
BocꢀTrp (4), the C3ꢀlinked adamantane (6), and particularly
the trifluoromethyl cyclohexane (15). We assayed their activiꢀ
ty as antagonists on three estrogenꢀregulated genes, progesterꢀ
one receptor (PgR), pS2, and GREB1 (Figure 4). All of the
compounds reduced the low control vehicle level of agonist
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PgR
10
ligand +E2
10
60
40
20
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0
140
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C
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120
100
80
60
40
20
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0
ligand alone
pS2
4
ligand + E2
3
2
1
0
ꢀ11
ꢀ10
ꢀ9
ꢀ8
ꢀ7
ꢀ6
ꢀ5
Log Concentration (M)
Figure 3. DoseꢀResponse of MCFꢀ7 Cell Proliferation.
MCFꢀ7 cells in complete medium were treated with indicated
concentrations of Fulvestrant (1) or compounds 2ꢀ15 for 6
days, and cell densities were determined by MTT assays. Valꢀ
ues represent average ± SEM of 4 measurements. 100% repreꢀ
sents ERα levels in vehicle treated samples.
GREB1
ligand alone
ligand + E2
4
3
2
1
0
As a group, the monocyclic compounds (11ꢀ15) proved to be
more potent and complete as antiproliferative agents than the
bridged polycyclic group (Table 1, Figure 3C), with comꢀ
pounds 12 and 15 having subnanomolar IC50 values. This
group was also more uniformly and fully efficacious, and all
were as good as the best of the Bocꢀamino acids and bridged
biꢀ and tricyclic series. Again, as ERα degraders (Table 1,
Figure 2C), there were wider variations, with 12 and 15 again
having the most complete reduction of ERα levels. The turnoꢀ
ver of ER was inhibited by proteasome inhibitor (MG132) (see
Fig. S1). Because the extent of degradation by compounds 11,
13, and 14 (as well as 5 from the bridged cyclic group) was
modest, accurate IC50 values could not be determined for these
compounds.
Figure 4. Assessment of selected compounds as antagonists of
the expression of ERꢀRegulated Genes. MCFꢀ7 cells were
treated with 3 ꢁM of the indicated compounds or Fulvestrant,
either alone or in the presence of 1 nM estradiol (E2) for 24
hours. Gene expression level was determined by qRTꢀPCR.
activity (gray bars), and they all functioned as effective antagꢀ
onists of ERα transcriptional regulation of these genes in the
presence of E2 (stippled dark bars).
It is notable that the above dose response studies also sugꢀ
gest that the inhibitory activity of these PROTACs is not reꢀ
duced at higher concentrations. The absence of this “hook
We have previously shown that the bisphenolicꢀadamantyl
cores are high affinity ligands and partial agonists on ERα.24
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Therefore, the ERα degradation, antiproliferative and
has been a major bottleneck in clinical utility of SERDs Beꢀ
yond SERDs, this study also significantly expands the
currently available toolbox for PROTACs, and these novel
degrons could be useful for the development of new
PROTACs for targeted degradation of various other therapeuꢀ
tically relevant proteins.
1
2
3
4
5
6
7
8
antagonist activities of our novel PROTACs are likely mediatꢀ
ed mainly by the side chains (presumed to be degrons). Our
data suggests that hydrophobicity of the degron, alone, is not a
major driver of SERD or antiproliferative action of this new
class of SERDs. In fact, the degrons in the most potent comꢀ
pounds (4, 11, 12 and 15) had lower cLogP values (SI, Table
S1) as compared to other degrons. The mechanism of action of
our monoꢀBocꢀprotected amino acid degrons (2ꢀ4) also apꢀ
pears to be distinct from a recently described Boc3Arg degron
that requires all the three Boc groups to be present on an Argiꢀ
nine motif to retain its activity.27 Crews et al. have done pioꢀ
neering investigations of the adamantyl motif as a degron for
androgen receptor and some other proteins.13,26 These studies
suggest involvement of Heat Shock Proteins (Hsp 70 and 90)
in the degradation pathway. Because Hsp 90 is also known to
regulate folding/unfolding of ER,28 this pathway might be
playing a role in observed SERD behavior of some of our
compounds (5ꢀ8). In addition, other factors such as enhanced
cell permeability and/or inducement of distinct destabilizing
conformations of ER could also contribute to the increased
potency of our compounds. In fact, the observation that some
of our most potent antiproliferative agents (e.g., 12 and 15,
possessing cyclohexyl degrons) were not complete ERα deꢀ
graders (Table 1) suggests that these compounds could also be
operating by inducing an antagonist conformation of ERα, a
scenario previously shown in the case of Fulvestrant.29 Altꢀ
hough the precise mechanism of action of Fulvestrant is still
unclear, previous studies suggest that the long alkyl chain of
Fulvestrant interacts with a hydrophobic groove on ER that
otherwise recruits ER coactivator proteins.3 Subsequently, two
distinct pathways operate: exposure of hydrophobic residues
on ER and recruitment of corepressors to the hydrophobic
groove.3,30
ASSOCIATED CONTENTS
Supporting Information
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The Supporting Information is available free of charge on the
ACS Publications website.
Detailed experimental procedures for synthesis of comꢀ
pounds, characterization data for all compounds, cLogP data of
degrons, biological assay protocols, and molecular modelling
data/method (PDF).
AUTHOR INFORMATION
Corresponding Author
* Eꢀmail: abhishek.sharma@stevens.edu
ORCID
Abhishek Sharma: 0000ꢀ0001ꢀ6070ꢀ4403
Author Contributions
§These authors contributed equally. The manuscript was written
through contributions of all authors. All authors have given
approval to the final version of the manuscript.
ACKNOWLEDGMENTS
We thank Dr. Athula Attygalle for HRMS analysis and Ye Zheng
for assistance in some experiments. Financial support from Steꢀ
vens Institute of Technology (AS), the NIH (R01DK015556 to
JAK; predoctoral fellowship T32GM070421 to VSG), NCI Canꢀ
cer Center Support Grant (P30CA08748) to MSKCC and the
Breast Cancer Research Foundation (BCRF 17ꢀ082 to JAK and
BSK; BCRF 17ꢀ083 to BSK; BCRF 17ꢀ185 to SC) is gratefully
acknowledged.
Docking studies of our novel SERDs into ERα (SI, Figure
Sꢀ2 and Sꢀ3) reveal that unlike Fulvestrant, the degrons of our
compounds are unable to fully occupy the coactivator groove
due to the shorter linker lengths. After passing through an exit
channel, the Bocꢀamino acid degrons project in a direction
opposite to that of tricyclic and monocyclic degrons (Figure Sꢀ
3C). Interestingly, the tricyclic/monocyclic degrons are likely
projected towards the loop connecting the helixꢀ11 and 12
(Figure Sꢀ2, Sꢀ3A and B). Collectively, the above results indiꢀ
cate likely involvement of novel and decoupled mechanisms
for ER degradation and antagonism for our SERDs. Studies to
elucidate more precisely the mechanism of action of these
structurally distinct degrons are currently underway.
ABBREVIATIONS
E2, estradiol; ER, estrogen receptor; GREB1, growth regulation
by estrogen in breast cancer 1; Hsp, heat shock protein; ICW, inꢀ
cell western assay; LBP, ligand binding pocket; NHS, Nꢀ
hydroxysuccinimide; PgR, progesterone receptor; PROTAC, proꢀ
teolysis targeting chimera; RBA, relative binding affinity; SERD,
selective estrogen receptor degrader.
REFERENCES
(1) Ariazi, E. A; Ariazi, J. L.; Cordera, F.; Jordan, V.C. Estrogen
receptors as therapeutic targets in breast cancer. Curr. Top. Med.
Chem. 2006, 6, 181ꢀ202.
In conclusion, this work represents a new addition to the
very limited pool of distinct molecular scaffolds (Fulvestrant
and GW type compounds) that are known to induce selective
degradation of ERα and also antagonize ERα. Our molecular
design strategy involved a PROTACꢀbased model, wherein, a
synthetically tractable ligand core was attached to various
novel degronꢀtype side chains in a highly modular fashion.
Through these studies, we found members of three distinct
degron classes (lipophilic amino acids, bridged cyclic systems,
and simpler monocyclic systems) that had low nanomolar poꢀ
tencies as ERα degraders and antiproliferative agents that also
inhibited ER target gene expression. The modular design, ease
of synthesis and ready availability of ligand cores and degrons
of this class of SERDs lay the foundation for rapid chemical
diversification to optimize the ADMET properties, an area that
(2) Toy, W.; Shen, Y.; Won, H.; Green, B.; Sakr, R. A.; Will, M.;
Li, Z.; Gala, K.; Fanning, S.; King, T. A.; Hudis, C.; Chen, D.; Taran,
T.; Hortobagyi, G.; Greene, G.; Berger, M.; Baselga, J.; Chandarꢀ
lapaty, S. ESR1 ligandꢀbinding domain mutations in hormoneꢀ
resistant breast cancer. Nat. Genet. 2013, 45, 1439ꢀ1445.
(3) Pike, A. C.; Brzozowski, A. M.; Walton, J.; Hubbard, R. E.;
Thorsell, A. G.; Li, Y. L.; Gustafsson, J. A.; Carlquist, M. Structural
insights into the mode of action of a pure antiestrogen. Structure
2001, 9, 145ꢀ153.
(4) Misawa, T.; Fujisato, T.; Kanda, Y.; Ohoka, N.; Shoda, T.; Yoꢀ
rioka, M.; Makishima, M.; Sekino, Y.; Naito, M.; Demizu, Y.; Kuꢀ
rihara, M. Design and synthesis of novel selective estrogen receptor
degradation inducers based on the diphenylheptane skeleton. Med-
ChemComm. 2017, 8, 239ꢀ246.
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 6 of 7
(5) Robertson, J. F. R. Fulvestrant (Faslodex®) – how to make a
drug better. Oncologist 2007, 12, 774ꢀ784.
of estrogen receptor degradation inducer based on a protein knockꢀ
down strategy. Bioorg. Med. Chem. Lett. 2012, 22, 1793ꢀ1796.
(20) Jiang, Y.; Deng, Q.; Zhao, H.; Xie, M.; Chen, L.; Yin, F.; Qin,
X.; Zheng, W1.; Zhao, Y.; Li, Z. Development of stabilized peptideꢀ
based PROTACs against Estrogen Receptor α. ACS Chem. Biol. 2018
13, 628ꢀ635.
(21) Dougan, D. A.; Micevski, D.; Truscott, K. N. The Nꢀend rule
pathway: from recognition by Nꢀrecognins, to destruction by
AAA+proteases. Biochim. Biophys. Acta, Molec. Cell Res. 2012,
1823, 83ꢀ91.
(22) Min, J.; Guillen, V. S.; Sharma, A.; Zhao, Y.; Ziegler, Y.;
Gong, P.; Mayne, C. G.; Srinivasan, S.; Kim, S. H.; Carlson, K. E.
Nettles, K. W.; Katzenellenbogen, B. S.; Katzenellenbogen, J. A.
Adamantyl antiestrogens with novel side chains reveal a spectrum of
activities in suppressing estrogen receptor mediated activities in
breast cancer cells. J. Med. Chem. 2017, 60, 6321ꢀ6336.
(23) Zhao, Y.; Laws, M. J.; Guillen, V. S.; Ziegler, Y.; Min, J.;
Sharma, A.; Kim, S. H.; Chu, D.; Park, B. H.; Oesterreich, S. Mao,
C5.; Shapiro, D. J.; Nettles, K. W.; Katzenellenbogen, J. A.; Katzenꢀ
ellenbogen, B. S. Structurally novel antiestrogens elicit differential
responses from constitutively active mutant estrogen receptors in
breast cancer cells and tumors. Cancer Res. 2017, 77, 5602ꢀ5613.
(24) Muthyala, R. S.; Sheng, S.; Carlson, K. E.; Katzenellenbogen,
B. S.; Katzenellenbogen, J. A. Bridged bicyclic cores containing a 1,
1ꢀdiarylethylene motif are highꢀaffinity subtypeꢀselective ligands for
the estrogen receptor. J. Med. Chem. 2003, 46, 1589ꢀ1602.
(25) Long, M. J. C.; Gollapalli, D. R.; Hedstrom, L. Inhibitor meꢀ
diated protein degradation. Chem. Biol. 2012, 19, 629ꢀ637.
(26) Gustafson, J. L.; Neklesa, T. K.; Cox, C. S.; Roth, A. G.;
Buckley, D. L.; Tae, H. S.; Sundberg, T. B.; Stagg, D. B.; Hines, J.;
McDonnell, D. P.; Norris, J. D.; Crews, C. M. Smallꢀmoleculeꢀ
mediated degradation of the Androgen Receptor through hydrophobic
tagging. Angew. Chem. Int. Ed. 2015, 54, 9659ꢀ9662.
1
2
3
4
5
6
7
8
(6) Willson, T. M.; Henke, B. R.; Momtahen, T. M.; Charifson, P.
S.; Batchelor, K. W.; Lubahn, D. B.; Moore, L. B.; Oliver, B. B.;
Sauls, H. R.; Triantafillou, J. A.; Wolfe, S. G.; Baer, P. G. 3ꢀ[4ꢀ(1,2ꢀ
Diphenylbutꢀ1ꢀenyl)phenyl]acrylic Acid: A NonꢀSteroidal Estrogen
with Functional Selectivity for Bone over Uterus in Rats. J. Med.
Chem. 1994, 37, 1550ꢀ1552.
(7) Lai, A.; Kahraman, M.; Govek, S.; Nagasawa, J.; Bonnefous,
C.; Julien, J.; Douglas, K.; Sensintaffar, J.; Lu, N.; Lee, K. J.; Apariꢀ
cio, A.; Kaufman, J.; Qian, J.; Shao, G.; Prudente, R.; Moon, M. J.;
Joseph, J. D.; Darimont, B.; Brigham, D.; Grillot, K.; Heyman, R.;
Rix, P. J.; Hager, J. H.; Smith, N. D. Identification of GDCꢀ0810
(ARNꢀ810), an orally bioavailable Selective Estrogen Receptor Deꢀ
grader (SERD) that demonstrates robust activity in tamoxifenꢀ
resistant breast cancer xenografts. J. Med. Chem. 2015, 58, 4888ꢀ
4904.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(8) De Savi, C.; R. H.; Bradbury, A.; Rabow, A.; Norman, R. C.;
de Almeida, C.; Andrews, D. M.; Ballard, P.; Buttar, D.; Callis, R. J.;
Currie, G. S. Curwen, J. O.; Davies, C. D.; Donald, C. S.; Feron, L. J.;
Gingell, H.; Glossop, S. C.; Hayter, B. R.; Hussain, S.; Karoutchi, G.;
Lamont, S. G.; MacFaul, P.; Moss, T. A.; Pearson, S. E.; Tonge, M.;
Walker, G. E.; Weir, H. M.; Wilson, Z. Optimization of a novel bindꢀ
ing
motif
to
(E)ꢀ3ꢀ(3,5ꢀdifluoroꢀ4ꢀ((1R,3R)ꢀ2ꢀ(2ꢀfluoroꢀ2ꢀ
methylpropyl)ꢀ3ꢀmethylꢀ2,3,4,9ꢀtetrahydroꢀ1Hꢀpyrido[3,4ꢀb]indolꢀ1ꢀ
yl)phenyl)acrylic acid (AZD9496), a potent and orally bioavailable
Selective Estrogen Receptor Downregulator and antagonist. J. Med.
Chem. 2015, 58, 8128ꢀ8140.
(9) Govek, S. P.; Nagasawa, J. Y.; Douglas, K. L.; Lai, A. G.;
Kahraman, M.; Bonnefous, C.; Aparicio, A. M.; Darimont, B. D.;
Grillot, K. L.; Joseph, J. D.; Kaufman, J. A.; Lee, K. J.; Lu, N.; Moon
M. J.; Prudente, R. Y.; Sensintaffar, J.; Rix, P. J.; Hager, J. H.; Smith,
N. D. Optimization of an indazole series of selective estrogen receptor
degraders: Tumor regression in a tamoxifenꢀresistant breast cancer
xenograft. Bioorg. Med. Chem. Lett. 2015, 15, 5163ꢀ5167.
(10) 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.; Rowlinsonm 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 ERꢀPositive and ESR1ꢀmutant breast tumors in preclinical
models. Cancer Res. 2016, 76, 3307ꢀ3318.
(27) Shi, Y.; Long, M. J. C.; Rosenberg, M. M.; Li, S.; Kobjack,
A.; Lessans, P.; Coffey, R. T.; Hedstrom, L. Boc3Argꢀlinked ligands
induce degradation by localizing target proteins to the 20S proꢀ
teasome. ACS Chem. Biol. 2016, 11, 3328ꢀ3337.
(28) Aumais, J. P.; Lee, H. S.; Lin, R.; White, J. H. Selective interꢀ
action of hsp90 with an estrogen receptor ligandꢀbinding domain
containing a point mutation. J. Biol. Chem. 1997, 272, 12229ꢀ12235.
(29) Wardell, S. E.; Marks, J. R.; McDonnell, D. P. The turnover of
estrogen receptor α by the selective estrogen receptor degrader
(SERD) fulvestrant is a saturable process that is not required for anꢀ
tagonist efficacy. Biochem. Pharmacol. 2011, 82, 122ꢀ130.
(30) Weatherman, R. V.; Clegg, N. J.; Scanlan, T. S. Differential
SERM activation of the estrogen receptors (ERalpha and ERbeta) at
APꢀ1 sites. Chem. Biol. 2001, 8, 427ꢀ 436.
(11) For an interesting scaffold displaying mixed action (agoꢀ
nist/SERD), see: Wardell, S. E.; Nelson, E. R.; Chao, C. A.; Alley, H.
M.; McDonnell, D. P. Evaluation of the pharmacological activities of
RAD1901, a selective estrogen receptor degrader. Endocr. Relat.
Cancer 2015, 22, 713ꢀ724.
(12) Toure, M.; Crews, C. M. Smallꢀmolecule PROTACS: new
approaches to protein degradation Angew. Chem. Int. Ed. 2016, 55,
1966ꢀ1973.
(13) Salami, J.; Crews, C. M. Waste disposalꢀan attractive strategy
for cancer therapy. Science 2017, 355, 1163.
(14) Winter, G. E.; Buckley, D. L.; Paulk, J.; Roberts, J. M.; Souza,
A.; Paganon, S. D.; Bradner, J. E. Phthalimide conjugation as a stratꢀ
egy for in vivo target protein degradation. Science 2015, 348, 1376ꢀ
1381.
(15) Burslem, G. M.; Crews, C. M. Smallꢀmolecule modulation of
protein homeostasis. Chem. Rev. 2017, 117, 11269ꢀ11301.
(16) Lai, A. C.; Crews, C. M. Induced protein degradation: an
emerging drug discovery paradigm. Nat. Rev. Drug. Discov. 2017, 16,
101ꢀ11.
(17) Ottis, P.; Crews, C. M. ProteolysisꢀTargeting Chimeras: inꢀ
duced protein degradation as a therapeutic Strategy. ACS Chem. Biol.
2017, 12, 892ꢀ898.
(18) Itoh, Y.; Kitaguchi, R.; Ishikawa, M.;Naito, M.; Hashimoto,
Y. Design, synthesis and biological evaluation of nuclear receptorꢀ
degradation inducers. Bioorg. Med. Chem. 2011, 19, 6768ꢀ6778.
(19) Demizu, Y.; Okuhira, K.; Motoi, H.; Ohno, A.; Shoda, T.; Fuꢀ
kuhara, K.; Okuda, H.; Naito, M.; Kurihara, M. Design and synthesis
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