Exploring the PROTAC degron candidates: OBHSA with different side chains as novel selective estrogen receptor degraders (SERDs)

Article abstract of DOI:10.1016/j.ejmech.2019.03.058

As the mutant estrogen receptor (ER) continues to be characterized, breast cancer is becoming increasingly difficult to cure when treated with hormone therapy. In this regard, a strategy to selectively and effectively degrade the ER might be an effective alternative to endocrine therapy for breast cancer. In a previous study, we identified a novel series of 7-oxabicyclo[2.2.1]heptene sulfonamide (OBHSA) compounds as full ER antagonists while lacking the prototypical ligand side chain that has been widely used to induce antagonism of ERα. Further crystal structure studies and phenotypic assays revealed that these compounds are selective estrogen receptor degraders (SERDs) with a new mechanism of action. However, from a drug discovery point of view, there still is room to improve the potency of these OBHSA compounds. In this study, we have developed new classes of SERDs that contain the OBHSA core structure and different side chains, e.g., basic side chains, long alkyl acid side chains, and glycerol ether side chains, to simply mimic the degrons of proteolysis targeting chimera (PROTAC) and then investigated the structure-activity relationships of these PROTAC-like hybrid compounds. These novel SERDs could effectively inhibit MCF-7 cell proliferation and demonstrated good ERα degradation efficacy. Among the SERDs, compounds 17d, 17e and 17g containing a basic side chain with a N-trifluoroethyl substituent and a para methoxyl group at the phenyl group of the sulfonamide turned out to be the best candidates for ER degraders. A further docking study of these compounds with ERα elucidates their structure-activity relationships, which provides guidance to design new PROTAC degrons targeting ER for breast cancer therapy. Lastly, easy modification of these PROTAC-like SERDs enables further fine-tuning of their pharmacokinetic properties, including oral availability.

Full text of DOI:10.1016/j.ejmech.2019.03.058

Accepted Manuscript
Exploring the PROTAC degron candidates: OBHSA with different side chains as
novel selective estrogen receptor degraders (SERDs)
Yuanyuan Li, Silong Zhang, Jing Zhang, Zhiye Hu, Yuan Xiao, Jian Huang, Chune
Dong, Shengtang Huang, Hai-Bing Zhou
PII:
S0223-5234(19)30286-7
DOI:
https://doi.org/10.1016/j.ejmech.2019.03.058
EJMECH 11228
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 January 2019
Revised Date: 23 March 2019
Accepted Date: 24 March 2019
Please cite this article as: Y. Li, S. Zhang, J. Zhang, Z. Hu, Y. Xiao, J. Huang, C. Dong, S. Huang, H.-B.
Zhou, Exploring the PROTAC degron candidates: OBHSA with different side chains as novel selective
estrogen receptor degraders (SERDs), European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.03.058.
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ACCEPTED MANUSCRIPT
Exploring the PROTAC Degron Candidates: OBHSA with
Different Side Chains as Novel Selective Estrogen Receptor
Degraders (SERDs)
Yuanyuan Li, Silong Zhang, Jing Zhang, Zhiye Hu, Yuan Xiao, Jian Huang, Chune Dong,*
Shengtang Huang,* and Hai-Bing Zhou*
A series of novel SERDs with excellent ER degradation efficacy have been
discovered. These findings simplified the structure of currently available degrons and
provide new possibility for discovering novel PROTACs.

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Exploring the PROTAC Degron Candidates: OBHSA with
Different Side Chains as Novel Selective Estrogen
Receptor Degraders (SERDs)
Yuanyuan Li,1† Silong Zhang,1† Jing Zhang,1 Zhiye Hu,1 Yuan Xiao,1 Jian Huang,3 Chune
Dong,1* Shengtang Huang,2* and Hai-Bing Zhou1*
1 State Key Laboratory of Virology, Hubei Province Engineering and Technology Research
Center for Fluorinated Pharmaceuticals, Key Laboratory of Combinatorial Biosynthesis and
Drug Discovery (Wuhan University), Ministry of Education, Wuhan University School of
Pharmaceutical Sciences, Wuhan 430071, China; E-mails: zhouhb@whue.du.cn;
cdong@whu.edu.cn
2 The Lab of Cardiovascular, Cerebrovascular and Metabolic Disorder, Hubei University of
Science and Technology, Xianning 437100, China; E-mail: huangst6511@hbust.edu.cn
3 College of Life Sciences, Wuhan University, Wuhan 430072, China
† The authors contributed equally to this work.

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Abstract
As the mutant estrogen receptor (ER) continues to be characterized, breast cancer is
becoming increasingly difficult to cure when treated with hormone therapy. In this regard, a
strategy to selectively and effectively degrade the ER might be an effective alternative to
endocrine therapy for breast cancer. In a previous study, we identified a novel series of
7-oxabicyclo[2.2.1]heptene sulfonamide (OBHSA) compounds as full ER antagonists while
lacking the prototypical ligand side chain that has been widely used to induce antagonism of
ERα. Further crystal structure studies and phenotypic assays revealed that these compounds
are selective estrogen receptor degraders (SERDs) with a new mechanism of action. However,
from a drug discovery point of view, there still is room to improve the potency of these
OBHSA compounds. In this study, we have developed new classes of SERDs that contain the
OBHSA core structure and different side chains, e.g., basic side chains, long alkyl acid side
chains, and glycerol ether side chains, to simply mimic the degrons of proteolysis targeting
chimera (PROTAC) and then investigated the structure-activity relationships of these
PROTAC-like hybrid compounds. These novel SERDs could effectively inhibit MCF-7 cell
proliferation and demonstrated good ERα degradation efficacy. Among the SERDs,
compounds 17d, 17e and 17g containing a basic side chain with a N-trifluoroethyl substituent
and a para methoxyl group at the phenyl group of the sulfonamide turned out to be the best
candidates for ER degraders. A further docking study of these compounds with ERα
elucidates their structure-activity relationships, which provides guidance to design new
PROTAC degrons targeting ER for breast cancer therapy. Lastly, easy modification of these
PROTAC-like SERDs enables further fine-tuning of their pharmacokinetic properties,
including oral availability.
Keywords: SERDs, side chains, degrons, ER degradation

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1. Introduction
It is well-known that the overexpression of estrogen receptor α (ERα) may lead to ER
positive breast cancer, which accounts for 70% of breast cancer [1, 2]. Contraposing this
target, clinical pharmacists generally use endocrine therapy, in which the drugs can be
classified into two types: aromatase inhibitors (AIs) [3-5] and selective estrogen receptor
modulators (SERMs) [2, 6-8]. However, the recurrence of breast cancer is difficult to treat,
and the long-term use of SERMs, such as tamoxifen, is prone to lead to the development
ovarian cancer and drug resistance [9-11], which is usually fatal. For patients who are on
long-term medication, the limited number of effective drugs encourages the development of
new compounds that reduce the likelihood of disease recurrence and drug resistance through
various means.
Inducing protein degradation by small molecules has recently become a hot spot for drug
discovery [12, 13]. To date, as the emerging and efficient treatments for breast cancer, there
have been two approaches targeting ERα protein degradation: selective estrogen receptor
downregulators (SERDs) and proteolysis targeting chimeras (PROTACs). SERDs are
recognized as pure ER antagonists that can cause spatial structural instability and activate the
ubiquitination pathway to degrade ERα protein [14-18]. In 2007, the FDA approved the first
SERD fulvestrant for the treatment of advanced breast cancer [19]. Nevertheless, this SERD’s
low oral bioavailability limited its application [20]. Therefore, medicinal chemists hope to
obtain SERDs with better oral bioavailability through structural modification and
optimization. To date, a variety of SERDs have been identified [21-26], most of which have
different side chains, e.g., long alkyl side chains, acrylic side chains, and basic side chains
(Figure 1). Fulvestrant (Figure 1, compound 1) is one typical drug that introduced a sulfinyl
pentafluoro alkyl chain onto a E2 skeleton; the hydrophobic chain was exposed to the surface
of the protein when binding to ER and destabilized the active ligand binding domain (LBD)
conformer, thereby leading to protein degradation. Therefore, Kurihara et al. reported that a
derivative of tamoxifen with a long alkyl side chain (Figure 1, compound 2) could effectively
reduce ER protein levels in breast cancer cells and have an antagonistic effect [26]. Later,

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these researchers expanded the range of currently available SERDs by attaching a decyl group
to the amine moiety of raloxifene and revealed that the ER degradation efficacy of this
compound is 1 µM [25]. GDC0810 (Figure 1, compound 3), a second-generation SERD,
contains an acrylic side chain, which directly interacted with the peptide backbone of ERα
and induced a conformational change that exposed a hydrophobic surface on the receptor [27].
GDC0810 has undergone a clinical phase II trial and was effective in endocrine therapy to
treat advanced metastatic ER+ breast cancer; unfortunately, its clinical trial was recently
discontinued. In 2018, Smith’s group reported that derivatives of EM-652 that contained a
basic side chain were highly potent and efficacious SERDs [18]. The best compound (Figure
1, compound 4) of this series demonstrated robust activity with a 91% ERα degradation
efficacy in a xenograft model of tamoxifen-resistant breast cancer. Moreover, the crystal
structure study revealed that the side chains of these compounds may play an essential role in
the degradation of ER, e.g., the acrylic acid side chain of 3-type compounds formed a
hydrogen bond with D351 in the ligand binding domain of ER, which blocked the proper
positioning of the critical helix 12. Thus, hydrophobic patches were exposed in solution, the
receptor was recognized by the ubiquitin-proteasome system and degradation proceeded [28].
For fulvestrant-type SERDs, when binding to ER, helix 12 appeared to be completely
distorted, and the hydrophobic regions were exposed to solution for ubiquitination targeting.
As a result, the side chain is a major driving factor of SERDs and plays a critical role in the
degradation of ER.

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OH
HO
O
(CH2)11CH3
N
H
H
O
S
F
F
H
H
HO
CF3
Fulvestrant
Tamoxifen derivative
1
2
Cl
F
HO
OH
O
N
N
OH
O
N
H
O
GDC0810
EM-652 derivative
3
4
Figure 1. Four representative SERDs with typical side chains.
Another method for compounds capable of inducing protein degradation is proteolysis
targeting chimeras (PROTACs). PROTAC is a bifunctional compound possesses a ligand for
a targeted protein and a recognition motif for E3 ubiquitin ligase recruitment that ultimately
promotes ubiquitination-dependent proteasomal degradation of the target protein. In 2010, an
ER-targeting PROTAC that consisted of an estradiol and a hypoxia-inducing factor 1α
(HIF-1α)-derived synthetic pentapeptide was reported [29], which showed good ER binding
affinity (10% binding affinity relative to E2) and effective ER degradation efficacy (60% ER
degradation). Kurihara et al. disclosed a complex comprised of 4-hydroxytamoxifen and
bestatin (an inhibitor of the cIAP1) via an alkyl linker [30]. This complex induced
cIAP1-mediated ubiquitination of ERα with proteasomal degradation at 10 µM. Recently, Li
et al. reported a novel ERα-targeting PROTAC that was composed of an N-terminal aspartic
acid cross-linked stabilized peptide ERα modulator (TD-PERM) and the Von Hippel-Lindau
(VHL) E3 ubiquitin ligase through a pentapeptide [31]. The TD-PROTAC approach utilized a
peptide stabilization strategy, which could provide peptide conjugates with satisfactory
stability and cellular uptake. However, the effective dose for degradation is 20 µM, which still
needs improvement. Generally, PROTACs are under rapid development as a novel and
promising technology for drug discovery [32-34]. Nonetheless, there is a very narrow

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selection of the structural species for PROTACs targeting ER degradation, and their low
potency limits their clinical trials.
Despite the different mechanisms for degrading ER by SERDs and PROTACs, these
compounds have a molecular skeleton that induces ER degradation and could be called a
degron. A degron is reported to be a part of the ligand-induced degradation (LID) domain in
known works, and it functions as a cryptic degradation sequence [35, 36]. Recently, Sharma's
group reported a series of new SERDs that extended the currently available library of
PROTAC-type scaffolds, which may be useful for the degradation of a variety of other
therapeutically important proteins [16]. The researchers defined the special motif that can
induce ER degradation as a degron in both SERDs and PROTAC molecules. As the skeletons
of the PROTAC degrons are limited and always complicated, there is an urge to explore novel
degrons with diverse and simple structures that allow the ER degrader to display drug-like
properties and more potent efficacy. In 2012, our group reported oxabicycloheptene
sulfonamide (OBHSA, Figure 2) compounds as full ER antagonists [37], and further study
on the crystal structures of complexes of these ligands with ERα revealed a new mechanism
of action of these SERD compounds [38]. The large R2 groups (N-trifluoroethyl group and
N-ethyl group) clashed strongly with Leu525, inducing a 2.5 Å shift in h11 and leading to the
complete disorder of the C terminus of h11. Thus, h12 Leu544 moved out of the hydrophobic
groove. In addition, the 4-methoxyphenyl substitution flipped the ethyl and aryl groups
around the sulfonamide linker and interacted with the loop between h7 and h8 by attacking
the backbone carbonyl of Glu419. Therefore, under the influence of the disordered h11 and
h12 exposed in the hydrophilic environment, the ER protein would become a target for
ubiquitination. Considering the important role played by the sulfonamide moiety in this new
mechanism, we turn our attention to the phenol group on the other side, hoping to develop
new degrons on the phenolic group that would contribute to the degradation activity. As part
of our long-term interests in the development of ER ligands [39-41], we utilized the OBHSA
as a core structure and explored alternatives of degrons to, for example, the basic side chains,
long alkyl acid side chains, and glycerol ether side chains, and then, we investigated their

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inhibition efficacy of MCF-7 cell proliferation and ERα degradation activity. In the process,
we uncovered some remarkable structure-activity relationships (SARs) in which compounds
17d, 17e and 17g containing basic side chains with N-trifluoroethyl and para methoxy
substituents displayed outstanding ERα degradation efficacy and anti-proliferation activity.
Further docking analysis illustrates that the basic side chain could distort helix 3 by forming a
hydrogen bond with Thr347 and a hydrophobic repulsion with the backbone of Met343. The
N-trifluoroethyl group and the para methoxyl group may also be beneficial to the ERα
degradation efficacy by influencing the loop of helix 7 and helix 8. The results indicate that
the basic side chain proved to be the best degron and provides new possibilities in the
development of more effective PROTACs.
Figure 2. Design of OBHSA derivatives with different degrons as SERDs.
2. Results and discussion
2.1 Chemistry
The synthesis of the final OBHSA derivatives 17a-t involved a Diels-Alder reaction between
a series of furan analogues and ethylene sulfonamide derivatives (Scheme 4), and the furan
intermediate 7 was synthesized according to our previous work [42]. To introduce different
degrons onto the OBHSA pharmacophore core, we synthesized the key intermediates 8a-j,
and the synthetic route is shown in Scheme 1. The furan analogues 8a-g were obtained by
Williamson etherification reactions with different halogenated compounds, and the hydrolysis
of the compounds 8e-g created the furan analogues 8h-j.

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Scheme 1. Synthesis of furan derivatives 8a-ja.
a
Reagents and conditions: (a) 2-chloro-N,N-dimethylethanamine hydrochloride,
1-(2-chloroethyl)pyrrolidine hydrochloride, 1-(2-chloroethyl)piperidine hydrochloride,
3-chloropropane-1,2-diol, ethyl 11-bromoundecanoate, ethyl 8-bromooctanoate, or ethyl
7-bromoheptanoate, KOH, CH3CN, 60 °C, 6 h; (b) LiOH, MeOH, rt, 4 h
.
In a previous study, it was indicated that the trifluoroethyl group and para methoxyl group
played an important role for the binding and degradation of the OBHSA compounds [38], and
as such, our synthesis mainly focused on the N-trifluoroethyl group and para substitution of
the phenyl of sulfonamide. Ethylene sulfonamide derivatives 14a-f were synthesized as
shown in Scheme 2. The substituted anilines 9a-d yielded analogs 10a-e through an
amidation reaction with acetic anhydride or trifluoroacetic anhydride in the presence of
DMAP. The compound 10a was treated with methyl iodide to yield N-methyl
phenylacetamide 11. Then, the intermediate 12 was obtained by the deacylation of 11 after
refluxing for 24 h in 10% HCl and glycol. Finally, the key intermediate 14a was gained
through the reaction of 12 with 2-chloroethanesulfonyl chloride in the presence of TEA. For
the synthesis of ethylene sulfonamides 14b-f, the route was slightly different. Compounds
10a-e were reduced by BH3 SMe2 in THF at 60 °C to give 13a-e. Then, following the reaction
with 2-chloroethanesulfonyl chloride, dienophiles 14b-f were obtained.
Scheme 2. Synthesis of dienophiles 14a-ga.

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a
Reagents and conditions: (a) acetic anhydride or trifluoroacetic anhydride, rt, 3 h; (b) MeI,
NaH, THF, 0 °C, 4 h; (c) 10% HCl, HO(CH2)2OH, reflux, 3 h; (d) 2-chloroethanesulfonyl
chloride, 20% NaOH, DCM, 0 °C, 24 h; (e) BH3 SMe2, THF, 60 °C, 24 h.
The ethylene sulfonamides with different side chains were obtained from the intermediate 14b.
After the demethylation of compound 14b with BBr3, product 15 interacted with
3-chloropropane-1,2-diol, alkyl ester bromides, or 1-(2-chloroethyl)piperidine hydrochloride
through Williamson etherification and yielded the dienophiles 16a-e. Compounds 16f-h with
long alkyl acid chains were obtained by the hydrolysis of esters 16c-e (Scheme 3).
Scheme 3. Synthesis of ethylene sulfonamides with different side chainsa.
a Reagents and conditions: (a) BBr3, CH2Cl2, -20 °C, 12 h; (b) 3-chloropropane-1,2-diol, ethyl
11-bromoundecanoate,
ethyl
8-bromooctanoate,
ethyl
7-bromoheptanoate,
or
1-(2-chloroethyl)piperidine hydrochloride, KOH, CH3CN, 60 °C, 6 h; (c) LiOH, MeOH, rt, 4
h.
The final step was the Diels-Alder reaction between different furan analogues and dienophiles
(Scheme 4). All of the compounds gave moderate to good yields. It is worth noting that a high

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stereoselectivity was obtained in the cycloaddition reaction, as we have observed previously
[43, 44]. In this study, the thermodynamically favorable exo products of 17a-t were more
easily generated through this Diels−Alder reaction, which was presumed to be due to the high
rate and easy reversibility, while the endo isomers were hardly found. In addition, among
compounds 17a-q, compounds 17b, 17d, 17f, 17g and 17j are mixtures of two regioisomers,
while the others are obtained as single isomers. The ratios of regioisomers were determined
1
from the corresponding H NMR, and the structures and stereoisomeric assignments of the
single isomers were analyzed by NOESY-NMR (see the Experimental Section and Supporting
Information for details).
Scheme 4. Synthesis of OBHSA derivatives 17a-t.
2.2 Biological testing
To explore the biological activities of these compounds, we utilized three assays to evaluate
the biological properties. Their relative binding affinities for ERα were tested by the

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competitive fluorometric receptor-binding assay. Their anti-proliferative activities on the
MCF-7
cell
line
were
evaluated
using
the
MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, and their ERα
degradation was assessed by Western blot assays.
2.2.1 Relative binding affinity
All of the binding affinities of the synthesized compounds for both ERα and ERβ were
determined by a competitive fluorometric receptor-binding assay using methods that have
been previously described [41], and the results are presented in Table 1 (absolute affinities
for estradiol: Kd 3.49 nM on ERα and 4.12 nM on ERβ). The affinities are presented as the
relative binding affinity (RBA) values, with the binding affinity of estradiol being set at
100%.
In general, most of the synthesized compounds were ERα selective and exhibited modest
affinities toward ERα, as well as low affinities toward ERβ. From entries 1-3 and 16, we can
find that the compounds with a glycerol ether side chain demonstrated modest binding
activities for ERα (RBA values were 0.26-1.94) and low affinities for ERβ (RBA values were
less than 0.6). Furthermore, compound 17c, which had no affinity toward ERβ, displayed the
highest ERα selectivity among the whole series (α/β ratio was greater than 162), while analog
17p showed slight ERβ selectivity (α/β ratio was 0.87).
Compounds with basic side chains (compounds 17d-i) exhibited relatively high binding
affinities for ERα and poor affinities for ERβ (Table 1, entries 4-9, RBA values were
0.95-3.25 for ERα and 0.12-0.81 for ERβ), except for compound 17e (RBA values were 2.12
for ERα and 1.81 for ERβ). The best compound 17f showed binding affinities of 3.25 for ERα
and 0.12 for ERβ, respectively, thus providing a high ERα selectivity of 27. Nevertheless, the
analogue 17h with a N-methyl group demonstrated a relatively lower binding affinity for ERα
(Table 1, entry 8, RBA value was 0.95) compared to other analogs with basic side chains.
Comparing the size of the N-substituents (analogues 17h, 17i and 17d-g), we also found that
the higher binding affinity comes as a consequence of increasing the size of the N-substitute

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from CH3 to CH2CF3, which may be caused by that the trifluoroethyl substituent can stretch
into the loop of helix 7 and helix 8 in the pocket of ERα and lead to steric strain (see
discussion in the molecular docking section for details).
Compounds 17j-o and 17r-t (Table 1, entries 10-15 and entries 18-20) that contain long alkyl
acidic chains displayed poor to modest binding activities toward ERα (RBA values were
0.06-3.21) and poor affinities toward ERβ (RBA values were less than 0.5). Comparison of
the ERα binding affinities of 17l, 17n, 17o and 17t (RBA values were 0.06-0.93) to those of
17j, 17k, 17m, 17r and 17s (RBA values were 1.20-3.21) indicates that the longer the chain
length, the lower the binding affinity.
Table 1. Relative binding affinities (RBAs) of the compounds 17a-t to ERα and ERβ.
RBAa (%)
ERα
RBAa (%)
ERβ
Entry Cmpd Side chain
R1
R2
n
α/β ratio
1
2
3
CH2CH3
CH2CF3
CH2CF3
H
OCH3
Cl
0.26 ± 0.03
1.94 ± 0.31
1.62 ± 0.10
0.28 ± 0.02
0.26 ± 0.05
< 0.01
0.94
7.52
17a
17b
17c
> 162
4
5
CH2CF3
CH2CF3
OCH3
OCH3
1.71 ± 0.41
0.40 ± 0.07
4.28
1.17
17d
17e
2.12 ± 0.68
3.25 ± 0.75
1.81 ± 0.08
0.12 ± 0.03
6
7
CH2CF3
CH2CF3
CH3
CH3
OCH3
H
27.08
7.95
1.21
3.10
5.39
3.00
7.10
13.68
> 7
17f
17g
17h
17i
2.94 ± 0.22 0.37 ± 0.001
8
0.95 ± 0.17
2.51 ± 0.17
2.48 ± 0.05
1.20 ± 0.63
0.79 ± 0.11
0.81 ± 0.01
0.46 ± 0.02
0.40 ± 0.04
9
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CF3
CH2CF3
CH2CF3
H
10
11
12
13
14
15
H
6
7
17j
H
17k
17l
H
10
6
0.93 ± 0.08 0.13 ± 0.002
OCH3
OCH3
OCH3
1.82 ± 0.04
0.13 ± 0.07
< 0.01
17m
17n
17o
7
0.07 ± 0.001
10 0.06 ± 0. 002 0.70 ± 0.20
0.09

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16
17
0.46 ± 0.07
0.53 ± 0.20
0.25 ± 0.07
0.87
2.28
17p
17q
0.58 ± 0.09
18
19
20
6
7
1.22 ± 0.03
3.21 ± 0.52
0.57 ± 0.01
0.41 ± 0.03
0.42 ± 0.03
< 0.01
2.98
7.64
> 57
17r
17s
17t
10
aRelative binding affinity (RBA) values are determined by competitive fluorometric binding assays and are
expressed as IC50estradiol/IC50compound ×100 ± the range (RBA, estradiol = 100%).
2.2.2 Cell viability assay.
To evaluate their inhibition of breast cancer cell proliferation, all of the compounds were
screened against the MCF-7 breast cancer cell line (Table 2). As a global observation, all
compounds exhibited moderate to good anti-proliferation activity except for compound 17r
(Table 2, entry 18, IC50 value was greater than 50 µM), and some of them performed better
than 4-hydroxytamoxifen.
Compounds with a glycerol ether side chain (Table 2, entries 1-3 and 16, compounds 17a-c
and 17p) displayed modest potencies, and the anti-proliferative activities of 17b and 17c (IC50
values were 17.0 and 16.8 µM, respectively) were superior to those of 17a and 17p (IC50
values were 33.6 and 32.3 µM, respectively), which may be explained by the favorable
interaction of the trifluoroethyl group with Met421 in the ER binding pocket, and this will be
discussed in the molecular docking section.
Among the OBHSA derivatives that contain different basic side chains (Table 2, entries 4-9
and 17, compounds 17d-i and 17q), compounds 17f and 17g demonstrated the best
anti-proliferative activities with IC50 values of 3.0 and 2.8 µM, respectively, which were also
the most potent analogs among all compounds. In terms of compounds 17b, 17d, 17e, 17g,
17m, 17n and 17o (Table 2, entries 2, 4-5, 7 and 13-15), one can see that the compounds 17d,
17e, and 17g with basic side chains (Table 2, entries 4-5, and 7) showed better
anti-proliferative activities (IC50 values were 8.4, 6.1, and 2.8 µM, respectively) than
compounds with long alkyl acidic side chains (17m, 17n, and 17o, IC50 values were 15.9,
40.1, and 14.3 µM, respectively) and the glycerol ether side chain (17b, IC50 value was 17.0
µM). When the trifluoroethyl group was introduced on the N-position of the sulfonamide,

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comparing compounds 17f-i (Table 2, entries 6-9), the better efficacies of 17f and 17g than
those of 17h and 17i were revealed in an anti-proliferation assay (Table 2, entries 6-7 vs 8-9).
In general, among all of the compounds investigated except 17n, the compounds with a
trifluoroethyl group demonstrated good to the best activity against MCF-7 cells.
Among the analogues that contain long alkyl acidic side chains (Table 2, entries 10-15 and
18-20), the best compounds were 17l, 17o and 17t with IC50 values of 10.3, 14.3 and 18.4 µM,
respectively, which may uncover that the undecanoic acid chain (n = 10) was better than the
octanoic acid chain (n = 7) and the heptanoic acid chain (n = 6).
Table 2. The anti-proliferative activity against MCF-7 cells.
Entry
Comp
17a
17b
Side chain
R1
CH2CH3
CH2CF3
CH2CF3
R2
H
OCH3
Cl
n
IC50 (µM)a
33.6 ± 1.83
17 ± 1.39
1
2
3
16.8 ± 2.27
17c
4
5
CH2CF3
CH2CF3
OCH3
OCH3
8.4 ± 0.55
6.1 ± 0.86
17d
17e
6
7
8
CH2CF3
CH2CF3
CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CF3
CH2CF3
CH2CF3
CH3
OCH3
H
3.0 ± 0.03
2.8 ± 0.25
17f
17g
17h
17i
17j
17k
17l
17m
17n
17o
15.2 ± 0.16
12.9 ± 0.74
34.6 ± 0.41
27.4 ± 0.54
10.3 ± 0.20
15.9 ± 0.41
40.3 ± 4.81
14.3 ± 0.76
9
H
H
H
H
10
11
12
13
14
15
6
7
10
6
7
10
OCH3
OCH3
OCH3
16
32.3 ± 3.16
17p

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17
5.4 ± 0.29
17q
18
19
20
21
6
7
10
>50b
24.9 ± 0.29
18.4 ± 0.44
10.1 ± 0.34
17r
17s
17t
4-OHT
a IC50 values are an average of at least three independent experiments ± standard deviation (mean ± SD). b
IC50 not determinable up to the highest concentrations tested.
2.2.3 ERα degradation assay
The ERα degradation assay of the synthesized compounds has been further investigated.
Considering the anti-proliferation ability of this series of compounds, we used 10 µM of each
compound to test the ERα level. The Western blot results of synthesized OBHSA analogs
with different degrons have been presented in Figure 3A-C. All of the bands were
quantitatively analyzed by Quantity One, and the results are given in Figure 3D. Comparing
compounds 17a-c and 17p that contained the glycerol ether side chain (Figure 3A),
compounds 17b-c with a N-trifluoroethyl group demonstrated modest degradation efficacies
(the ERα levels were 41% and 38%, respectively). For the analogs that contained basic side
chains (Figure 3B), compounds 17d, 17e, and 17g could efficiently degrade ERα with 99%,
99% and 81% degradation efficacies, respectively. However, when treated with compounds
17f, 17h, 17i and 17q, the ERα levels were 102%, 98%, 95% and 89%, which indicated that
these analogs were essentially inactive against ERα degradation. A further docking study
showed that the basic side chain could influence helix 3 with noncovalent repulsion to Thr347
and Met343. The N-trifluoroethyl group and the para methoxyl group may also support ERα
degradation by clashing with Met421 on the loop of helix 7 and helix 8. These combined
influences might contribute to the good degradation activities of compounds 17d, 17e and 17g.
From the results when treated with compounds containing long alkyl acidic side chains
(Figure 3C), the compounds 17m, 17n and 17t (side chain lengths were 6, 7 and 10,
respectively) showed modest activities (ER levels were 32%, 42% and 29%, respectively),
which indicated that the length of the long alkyl acidic side chain had no influence on the ERα
degradation efficacy.

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Lastly, in order to verify that the degradation activities of these novel PROTACs are likely
mediated mainly by the side chains, we chose the best compound 17e of the series for
comparison with the parent compound OBHSA-1 and conducted an ERα degradation assay
with different concentrations hoping to obtain an approximate ERα degradation potency of
this compound. We chose concentrations of 0.1 µM, 0.5 µM, 1 µM, 5 µM, and 10 µM and
compared them with the essential control OBHSA-1 (Figure 3E). The results revealed that
compound 17e demonstrated better results than OBHSA-1. Compound 17e can completely
degrade ERα at 1 µM, and it showed good degradation activity at 0.5 µM. Meanwhile,
OBHSA-1 showed complete degradation at 5 µM, which indicated that the basic side chain of
compound 17e played an important role in increasing the degradation potency on ERα.

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Figure 3. Western blot assay of ERα in MCF-7 cells treated by compounds with (A) a
glycerol ether side chain, (B) basic side chains and (C) long alkyl acidic side chains. (D) The
graph exhibits ERα values for 17a-t. Cells were incubated with DMSO or the compound (10
µM) for 20 h. The whole proteins were extracted, and the ERα protein levels were analyzed
by Western blotting. (E) Western blot assay of ERα treated with OBHSA-1 and 17e in
different doses from 0.1 µM to 10 µM.
2.4 Structure-activity relationships of the OBHSA derivatives
To more intuitively observe the effects of different degrons on the biological activities of the
compounds, we made a scatter plot based on the anti-proliferative activity and ERα
degradation efficacy (Figure 4A). For the ERα degradation assay, as a global observation,
compounds that contain basic side chains (17d, 17e, and 17g, red spots) demonstrated
excellent ERα degradation efficacies, which indicated that the basic side chains were the best
degrons for ER degradation. Most of the green spots and the blue spots were distributed in the
middle or up the figure, from which we can conclude that the long alkyl acidic side chain and
the glycerol ether side chain possess a weak influence on ER degradation. However,
compounds 17f, 17q, 17i and 17h, which also contain basic chains, didn’t demonstrate
potency to degrade ER. Our data suggests that the basic side chain degron might be a major
driving factor of ERα degradation activity; however, it appears to require a combination of
influences of other substituents in these types of compounds through a distinct mechanism of
action, which could be illustrated in the further docking analysis. Additionally, the length of

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the alkyl acidic side chain did not affect the ERα degradation efficacy, as indicated by
comparing compounds 17j-o (green spots).
In terms of the anti-proliferative activity, a similar trend as the efficacy of ERα degradation
was observed. All of the compounds that contain basic side chains (red spots) demonstrated
less than 20 µM IC50 values against MCF-7 cell lines, which indicated that the basic chain
could help to inhibit the growth of breast cancer cells. Meanwhile, compounds with the long
alkyl acidic side chain (green spots) and the glycerol ether side chain (blue spots) showed
modest activities, and the majority of them exhibited IC50 values greater than 20 µM.
Moreover, comparison of analogs 17l, 17o and 17t (green spots, chain length = 10) with 17j,
17k, 17n, 17r and 17s (green spots, chain length = 6 and 7) may uncover that the longer the
chain length, the more potent the anti-proliferative activity. Taking all of the results and data
into account, we summarized the structure-activity relationships of the OBHSA compounds
in Figure 4B.

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Figure 4. (A) Correlation between the ERα degradation and anti-proliferation activities of
different degrons. (B) Structure-activity relationships of the novel SERD compounds.
2.5 Molecular docking
As mentioned above, the obtained compound 17e turned out to be a single regioisomer, and
thus, we analyzed the docking mode of the 2D and 3D interactions between the two
enantiomers of compound 17e and ERα (Figure 5). Although we did not obtain another
regioisomer 17e', we still analyzed the docking mode of the two enantiomers of 17e' hoping
to provide a reference for explaining the biological activity of compound 17e. The results
demonstrated that 17e' showed less favored interactions with ERα compared to 17e, and the
details are given in the Supporting Information.
In the case of the (1R, 2S, 4R)-enantiomer of compound 17e, similar to other ER ligands, the
phenolic hydroxyl group mimics the A ring of E2 and forms a hydrogen bond with Glu353 on
helix 3. At the same time, the basic side chain, as a protein degrading degron, appears to
display important interactions with ERα. It forms a hydrogen bond between the nitrogen atom
of the basic side chain with Thr347 on helix 3; thus, the entire chain folds in a constrained
manner in the binding pocket. In addition, the distance between the ortho-methylene group on
pyrrolidine and the Met343 is 3.64 Å (Supporting Information), which also develops
hydrophobic repulsion with the backbone of Met343 on helix 3 (Figure 5A, 5B). These

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noncovalent interactions may distort the position of helix 3 so that it could not fold properly,
and the ubiquitination of ER will proceed. In contrast, the OBHS analogs with basic side
chains published in 2017[45] as ER antagonists formed a hydrogen bond between the basic
side chain with Asp351, which stuck out of the pocket toward helix 12. These different
interaction modes may illustrate a new mechanism of the basic side chain for degrading ER
protein. Moreover, with only a 4.03 Å distance, the N-trifluoroethyl substituent demonstrates
a hydrophobic interaction with the backbone of Met421 in the loop between helix 7 and helix
8, which is a little different from the crystal structures of OBHSA compounds that were
reported recently (the para substitution on the aryl group would clash with the backbone
carbonyl of Glu419 in the loop between helix 7 and helix 8) [38]. For the (1R, 2S,
4R)-enantiomer of compound 17e, the para methoxyl group on the phenyl group is very close
to Trp383 and Gly521, which may also clash with the position of helix 11 and thus further
enhance the degradation of ERα.
Compared to the (1R, 2S, 4R)-enantiomer, the docking mode of the (1S, 2R, 4S)-enantiomer
of compound 17e is less robust and unstable (Figure 5C, 5D). Although the position of the
two enantiomers is very similar, there are some differences, which influence their docking
mode to ER. The CH2 group on the basic side chain instead of the nitrogen atom of the (1R,
2S, 4R)-enantiomer interacts with Thr347 but loses the hydrophobic repulsion with the
backbone of Met343 on helix 3. Furthermore, the phenolic hydroxyl group of the (1S, 2R,
4S)-configuration failed to form a hydrogen bond with ERα. Although the oxygen atom on the
sulfonamide group displays an interaction with Gly521 on helix 11, the (1S, 2R,
4S)-enantiomer displays less interactions than the (1R, 2S, 4R)-enantiomer overall. As a result,
only one enantiomer, which possesses the 1R, 2S, 4R absolute configuration, could be docked
effectively into the ligand binding pocket, which predicts greater degradation activity for this
enantiomer.
From these interaction patterns found in the molecular docking, we can conclude that a new
mechanism of the basic side chains on the OBHSA scaffold to degrade ER proteins may be
discovered, thereby providing the basis for the discovery of new and simpler PROTACs.

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Figure 5. Docking study of two enantiomers of compound 17e bound to ERα (PDB code:
5kcc). (A) Cartoon schematic of the interactions between the residues of the ERα ligand
binding domain with the (1R, 2S, 4R)-enantiomer of compound 17e. It shows the side chain
acceptors of Glu353 and Thr347 in green arrows and the backbone acceptors of Met343 and
Met 421 in blue arrows. (B) The docking analysis of the (1R, 2S, 4R)-enantiomer of
compound 17e bound to ERα. The phenolic hydroxyl group forms a hydrogen bond (2.31 Å)
with Glu353 on helix 3. The basic side chain forms a hydrogen bond (2.66 Å) between the

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nitrogen atom with Thr347, and the ortho-methylene group on pyrrolidine develops
hydrophobic repulsion with the backbone of Met343 (3.64 Å) on helix 3. In addition, the
N-trifluoroethyl substituent demonstrates a hydrophobic interaction with the backbone of
Met421 (4.03 Å) in the loop between helix 7 and helix 8. (C) Cartoon schematic of the
interactions between the residues of the ERα ligand binding domain with the (1S, 2R,
4S)-enantiomer of compound 17e. It shows the side chain acceptor of Thr347 in a green arrow
and the backbone acceptor of Gly521 in a blue arrow. (D) The docking analysis of the (1S, 2R,
4S)-enantiomer of compound 17e bound to ERα. The CH2 group on the basic side chain
develops hydrophobic repulsion with the backbone of Thr347, and the oxygen atom on
sulfonamide group displays interaction with the Gly521 on helix 11.
2.6 Pharmacokinetic profile of 17e in rats
Since 17e showed good cell antiproliferative activity and complete degradation activity, both
the oral and intravenous pharmacokinetic profiles of compound 17e have been investigated,
and the results are shown in Table 3. Intravenous dosing with a 3 mg/kg solution of 17e
resulted in high clearance (46 mL/min/kg), and the half-lives of iv and po are 6.2 h and 3.7 h,
respectively. The exposure of the compound is good for intravenous injection (1.0 h·µg/mL),
while it is moderate by the oral route (0.33 h·µg/mL). The oral bioavailability of 17e is 9.2%,
which is modest compared to other reported SERDs. However, these moderate
pharmacokinetic parameters make it possible for further optimal modification of these
compounds to improve bioavailability.
Table 3. Pharmacokinetics of 17e in ratsa
Administration
Doseb
(mg/kg)
3
CL
(mL/min/kg)
46
T1/2
(h)
Cmax
(µg/mL)
1.0
AUC
Vss
F
route
iv
(h*µg/mL) (L/kg)
(%)
6.2
3.7
1.08
0.33
9.55
po
10
0.05
9.2
aSprague-Dawley rats were used (n = 3). Plasma samples were measured for drug exposure by
LC-MS/MS. bDosed intravenously at 3 mg/kg and orally at 10 mg/kg in 5% DMSO and 40%
PEG400 in saline.

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3. Conclusions
As drug resistance is continuously being identified in the treatment of breast cancer, new
strategies based on small molecule-induced protein degradation have developed rapidly.
However, most of the candidates involve very limited scaffolds and are still in clinical trials,
and none of them has been approved for marketing. Therefore, there is still an urgent need to
develop novel compounds with good ER degradation efficacy. In this work, we directed our
attention toward discovering new degrons that favor ER degradation and explored the
possibility of different side chains (basic side chain, long alkyl acid side chain and glycerol
ether side chain) to be the degron of the ER degrader. We designed and synthesized a series of
novel OBHSA derivatives that contained different side chains and investigated the
structure-activity relationships of these compounds. As a result, the basic side chain was
confirmed as the appropriate degron because compounds 17d, 17e and 17g with
N-trifluoroethyl and para methoxyl groups exhibited the best anti-proliferative activities and
good ERα degradation efficacies. The docking study demonstrates a new mechanism of the
basic side chain by forming a hydrogen bond with Thr347 and hydrophobic repulsion with the
backbone of Met343, which would distort helix 3. The N-trifluoroethyl group and the para
methoxyl group may also be beneficial to the ERα degradation efficacy by influencing the
loop of helix 7 and helix 8. Although these compounds showed micromolar activities and
modest oral bioavailability, the novel skeleton and easy modification of these PROTAC-like
SERDs allow further fine-tuning of their pharmacokinetic properties including oral
availability. In summary, these findings simplified the structure of currently available degrons
and provide new possibilities for discovering novel scaffolds in the development of novel
PROTACs.
4. Experimental section
4.1 General chemical methods.
Starting materials, reagents and solvents are purchased from commercial sources and used
directly unless otherwise noted. THF, DCM and acetonitrile are redistilled and dried to avoid

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water. Glassware was oven-dried, assembled while hot, and cooled under an inert atmosphere.
Reaction progress was monitored using analytical thin-layer chromatography (TLC).
Visualization was achieved by UV light (254 nm and 365 nm). Silica gel (230−400 mesh) was
1
used for column chromatography purifications. A Bruker Biospin AV400 (400 MHz, H
1
NMR; 100 MHz, 13C NMR) instrument was used to measure the H NMR and 13C NMR
spectra.
The synthesis of intermediate compounds 8a-j, 15, 16a-h is reported in the Supporting
Information.
4.2 Chemistry.
4.2.1 General procedure for the synthesis of final compounds17a-t
We used the distilled THF (2 mL) as cosolvent and added furans 8a-d or 8h-j (0.6 mmol) and
dienophiles 14a or 14c-f (0.6 mmol) to the round-bottom flask. The reaction mixture was
heated to 90 °C and stirred for 8 h under argon. The crude product was purified by silica gel
column chromatography (DCM-MeOH, 50:1-20:1).
4.2.2 Characterization data for final compounds 17a-t
6-(4-(2,3-Dihydroxypropoxy)phenyl)-N-ethyl-5-(4-hydroxyphenyl)-N-phenyl-7-oxabicyclo[2.2
.1]hept-5-ene-2-sulfonamide (17a): Yellow solid, 61% yield, mp 96-98 °C. 1H NMR (400
MHz, MeOD) δ 7.34 – 7.27 (m, 5H), 7.24 – 7.16 (m, 2H), 7.12 (dt, J = 14.9, 7.5 Hz, 2H),
6.88 (dd, J = 17.1, 8.7 Hz, 2H), 6.73 (dd, J = 18.5, 8.6 Hz, 2H), 5.44 (d, J = 10.7 Hz, 1H),
5.28 (t, J = 3.8 Hz, 1H), 4.08 – 4.02 (m, 1H), 4.02 – 3.95 (m, 2H), 3.84 – 3.75 (m, 2H), 3.67
(ddd, J = 12.3, 10.2, 5.0 Hz, 2H), 3.46 (td, J = 8.4, 4.5 Hz, 1H), 2.20 (dd, J = 7.6, 4.5 Hz, 1H),
1.99 (s, 1H), 1.04 (t, J = 7.0 Hz, 3H). 13C NMR (101 MHz, MeOD) δ 158.76, 157.50, 141.54,
140.51, 138.82, 137.70, 136.65, 129.06, 128.97, 128.94, 128.76, 128.35, 128.16, 127.65,
125.39, 123.08, 115.43, 114.42, 84.41, 82.72, 70.37, 68.97, 62.79, 60.20, 46.39, 19.57, 13.61.
HRMS (ESI) calcd for C29H31NO7S [M + H]+, 560.1713; found 560.1713.

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6-(4-(2,3-Dihydroxypropoxy)phenyl)-5-(4-hydroxyphenyl)-N-(4-methoxyphenyl)-N-(2,2,2-trifl
uoroethyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonamide (17b, Mixture of 4:1 Isomers):
Yellow solid, 74% yield, mp 99-100 °C. 1H NMR (400 MHz, MeOD) δ 7.32 – 7.18 (m, 4H),
7.18 – 7.08 (m, 2H), 7.00 – 6.90 (m, 2H), 6.87 (d, J = 8.8 Hz, 1H), 6.83 – 6.78 (m, 2H), 6.73
– 6.68 (m, 1H), 5.47 (d, J = 5.3 Hz, 1H), 5.31 (s, 1H), 4.42 (q, J = 8.5 Hz, 2H), 4.08 – 4.03 (m,
1H), 4.02 – 3.95 (m, 2H), 3.78 (s, 3H), 3.73 – 3.65 (m, 2H), 3.51 – 3.42 (m, 1H), 2.28 – 2.19
(m, 1H), 2.09 – 2.03 (m, 1H). 13C NMR (101 MHz, MeOD) δ 159.66, 159.01, 158.79, 157.64,
157.38, 141.75, 140.71, 137.61, 136.46, 131.36, 130.15, 129.24, 129.01, 128.18, 127.98,
1
125.32, 124.61, 124.16 (d, JCF = 279.6 Hz), 123.63, 122.98, 115.43, 115.14, 114.64, 114.37,
2
114.18, 84.40, 84.25, 82.70, 82.65, 70.36, 69.04, 68.95, 62.73, 61.14 (d, JCF = 30.9 Hz),
60.16, 54.61, 54.56, 53.42, 51.93, 22.35, 19.49. HRMS (ESI) calcd for C30H30F3NO8S [M +
Na]+, 644.1536; found 644.1531.
N-(4-Chlorophenyl)-6-(4-(2,3-dihydroxypropoxy)phenyl)-5-(4-hydroxyphenyl)-N-(2,2,2-trifluo
roethyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonamide(17c): Yellow solid, 56% yield, mp
95-97 °C. 1H NMR (400 MHz, MeOD) δ 7.42 – 7.26 (m, 4H), 7.21 (dd, J = 12.8, 6.1 Hz, 2H),
7.17 – 7.08 (m, 2H), 6.98 – 6.86 (m, 2H), 6.81 – 6.69 (m, 2H), 5.45 (d, J = 18.9 Hz, 1H), 5.32
(s, 1H), 4.50 (dd, J = 16.3, 8.1 Hz, 2H), 4.06 (dd, J = 12.5, 7.5 Hz, 1H), 4.03 – 3.94 (m, 2H),
3.69 (dd, J = 11.2, 4.9 Hz, 2H), 3.50 (d, J = 7.8 Hz, 1H), 2.22 – 2.13 (m, 1H), 2.03 (s, 1H).
13C NMR (101 MHz, MeOD) δ 159.02, 157.42, 140.79, 137.96, 136.37, 134.01, 130.20,
1
129.23, 129.11, 128.23, 124.06 (d, JCF = 280.3 Hz), 115.41, 115.14, 114.63, 114.37, 84.38,
2
82.73, 70.36, 69.02, 68.94, 62.19(d, JCF = 32.9 Hz), 62.02, 61.83, 60.16, 30.18, 19.48.
HRMS (ESI) calcd for C29H27ClF3NO7S [M + Na]+, 648.1041; found 648.1038.
6-(4-(2-(Dimethylamino)ethoxy)phenyl)-5-(4-hydroxyphenyl)-N-(4-methoxyphenyl)-N-(2,2,2-t
rifluoroethyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonamide (17d, Mixture of 5:1 Isomers):
Yellow solid, 75% yield, mp 99-101 °C. 1H NMR (400 MHz, MeOD) δ 7.29 – 7.20 (m, 4H),
7.13 (dd, J = 8.6, 7.2 Hz, 2H), 6.97 (d, J = 8.7 Hz, 1H), 6.92 (d, J = 8.8 Hz, 1H), 6.84 (d, J =
9.0 Hz, 1H), 6.82 – 6.75 (m, 2H), 6.71 (d, J = 8.6 Hz, 1H), 5.48 (s, 1H), 5.32 (dd, J = 7.0, 4.1
Hz, 1H), 4.42 (dd, J = 16.9, 8.5 Hz, 2H), 4.27 (dt, J = 12.8, 4.6 Hz, 2H), 3.78 (d, J = 2.9 Hz,

ACCEPTED MANUSCRIPT
3H), 3.47 (ddd, J = 17.7, 8.3, 4.4 Hz, 1H), 3.29 (t, J = 5.9 Hz, 2H), 2.75 (t, J = 4.8 Hz, 6H),
13
2.27 – 2.19 (m, 1H), 2.07 – 2.03 (m, 1H). C NMR (101 MHz, MeOD) δ 159.68, 158.01,
157.81, 157.76, 157.51, 142.16, 140.61, 137.98, 136.31, 131.42, 131.30, 130.16, 129.27,
1
128.99, 128.25, 128.08, 126.04, 124.15(d, JCF = 280.0 Hz), 123.48, 115.47, 115.18, 114.68,
114.44, 114.31, 114.18, 113.79, 84.41, 84.36, 82.76, 82.62, 63.14, 61.46, 61.21 (d, 2JCF = 30.7
Hz), 61.03, 60.15, 56.71, 54.57, 43.21, 30.10, 29.39, 21.25, 19.49.HRMS (ESI) calcd for
C31H33F3N2O6S [M + Na]+, 642.1937; found 642.1928.
5-(4-Hydroxyphenyl)-N-(4-methoxyphenyl)-6-(4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)-N-(2,2,2-t
rifluoroethyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonamide (17e): Yellow solid, 72% yield,
mp 115-117 °C. 1H NMR (400 MHz, MeOD) δ 7.45 – 7.34 (m, 1H), 7.35 – 7.17 (m, 4H), 7.18
– 7.08 (m, 2H), 7.00 – 6.91 (m, 2H), 6.83 (dd, J = 12.6, 3.6 Hz, 1H), 6.79 (dd, J = 8.7, 5.0 Hz,
1H), 6.71 (dd, J = 8.5, 3.7 Hz, 1H), 5.49 (d, J = 8.1 Hz, 1H), 5.30 (dd, J = 10.1, 4.0 Hz, 1H),
4.48 – 4.34 (m, 2H), 4.29 (dt, J = 10.0, 5.1 Hz, 2H), 3.78 (dd, J = 14.8, 2.8 Hz, 3H), 3.52 (dd,
J = 10.2, 5.7 Hz, 2H), 3.49 – 3.43 (m, 1H), 3.32 (dd, J = 4.6, 3.0 Hz, 4H), 2.29 – 2.16 (m, 1H),
13
2.10 – 2.04 (m, 4H), 2.04 – 1.99 (m, 1H). C NMR (101 MHz, MeOD) δ 159.68, 157.89,
1
157.07, 140.10, 138.01, 131.40, 130.18, 129.45, 129.03, 128.30, 124.24 (d, JCF = 282.1 Hz),
2
115.51, 115.23, 114.54, 114.24, 84.43, 82.78, 63.76, 61.32
(
d, JCF = 31.8 Hz), 61.06, 54.67,
54.30, 53.89, 30.15, 22.61. HRMS (ESI) calcd for C33H35F3N2O6S [M + Na]+, 667.2060;
found 667.2047.
5-(4-Hydroxyphenyl)-6-(4-(2-(piperidin-1-yl)ethoxy)phenyl)-N-(p-tolyl)-N-(2,2,2-trifluoroethy
l)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonamide (17f, Mixture of 4:1 Isomers): Yellow solid,
1
81% yield, mp 101-103 °C. H NMR (400 MHz, MeOD) δ 7.27 – 7.18 (m, 4H), 7.12 (d, J =
8.2 Hz, 2H), 6.97 – 6.91 (m, 1H), 6.88 (d, J = 8.7 Hz, 1H), 6.80 – 6.74 (m, 1H), 6.71 (d, J =
8.5 Hz, 1H), 5.47 (d, J = 3.7 Hz, 1H), 5.31 (t, J = 3.9 Hz, 1H), 4.45 (dd, J = 17.0, 8.5 Hz, 2H),
4.19 (dt, J = 16.8, 5.3 Hz, 2H), 3.47 (ddd, J = 12.9, 8.3, 4.3 Hz, 1H), 2.97 (dt, J = 16.2, 5.7 Hz,
2H), 2.76 (s, 4H), 2.35 (d, J = 18.9 Hz, 3H), 2.23 – 2.16 (m, 1H), 2.02 (dd, J = 13.7, 4.3 Hz,
13
1H), 1.71 (dd, J = 10.5, 5.3 Hz, 4H), 1.55 (s, 2H). C NMR (101 MHz, MeOD) δ 158.52,
158.37, 157.69, 157.52, 138.59, 137.76, 137.25, 136.53, 136.44, 136.38, 130.00, 129.81,

ACCEPTED MANUSCRIPT
1
129.70, 129.15, 129.04, 128.94, 128.51, 128.25, 128.08, 125.52, 124.24 (d, JCF = 281.1 Hz),
123.53, 123.05, 115.43, 115.19, 114.62, 114.40, 84.38, 84.35, 82.73, 82.63, 64.07, 63.96,
61.49, 61.45 (d, 2JCF = 31.2 Hz), 60.15, 56.93, 56.89, 54.18, 53.44, 30.17, 29.40, 24.32, 24.27,
22.86, 22.82, 19.75, 19.71. HRMS (ESI) calcd for C34H37F3N2O5S [M + H]+, 643.2448; found
643.2456.
5-(4-Hydroxyphenyl)-N-(4-methoxyphenyl)-6-(4-(2-(piperidin-1-yl)ethoxy)phenyl)-N-(2,2,2-tr
ifluoroethyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonamide (17g, Mixture of 3:1 Isomers):
Yellow solid, 75% yield, mp 106-108 °C. 1H NMR (400 MHz, MeOD) δ 7.27 – 7.19 (m, 4H),
7.13 (t, J = 8.6 Hz, 2H), 6.93 (d, J = 8.8 Hz, 1H), 6.90 – 6.82 (m, 2H), 6.79 (dd, J = 8.7, 4.7
Hz, 2H), 6.71 (d, J = 8.6 Hz, 1H), 5.48 (s, 1H), 5.31 (t, J = 3.7 Hz, 1H), 4.42 (dd, J = 17.0, 8.4
Hz, 2H), 4.17 (dd, J = 11.5, 5.5 Hz, 2H), 3.81 – 3.75 (m, 3H), 3.51 – 3.42 (m, 1H), 2.92 (dt, J
= 10.5, 5.3 Hz, 2H), 2.70 (s, 4H), 2.23 (ddd, J = 11.2, 7.6, 4.3 Hz, 1H), 2.01 (dd, J = 8.3, 4.1
Hz, 1H), 1.69 (dd, J = 10.3, 5.1 Hz, 5H), 1.53 (s, 2H). 13C NMR (101 MHz, MeOD) δ 159.65,
158.53, 158.31, 157.68, 157.42, 141.84, 140.65, 137.62, 136.37, 131.35, 131.27, 130.16,
1
129.50, 129.27, 129.03, 128.21, 128.03, 125.46, 124.15 (d, JCF = 279.9 Hz), 122.89, 115.47,
115.17, 114.61, 114.35, 114.18, 84.36, 84.21, 83.05, 82.63, 64.43, 64.35, 61.16 (d, 2JCF = 28.9
Hz), 60.20, 57.14, 54.58, 54.31, 54.17, 53.44, 52.29, 51.94, 31.36, 30.21, 28.73, 24.63, 24.39,
23.16. HRMS (ESI) calcd for C34H37F3N2O6S [M + Na]+, 681.2217; found 681.2217.
5-(4-Hydroxyphenyl)-N-methyl-N-phenyl-6-(4-(2-(piperidin-1-yl)ethoxy)phenyl)-7-oxabicyclo
[2.2.1]hept-5-ene-2-sulfonamide (17h): Yellow solid, 70% yield, mp 96-98 °C. 1H NMR (400
MHz, MeOD) δ 7.48 – 7.44 (m, 1H), 7.40 – 7.33 (m, 4H), 7.26 – 7.20 (m, 2H), 7.16 – 7.08 (m,
2H), 6.92 (dd, J = 13.2, 8.9 Hz, 2H), 6.78 – 6.69 (m, 2H), 5.43 (s, 1H), 5.33 – 5.27 (m, 1H),
4.24 (dt, J = 10.8, 5.3 Hz, 2H), 3.53 (td, J = 8.7, 4.4 Hz, 1H), 3.37 (t, J = 5.6 Hz, 3H), 3.16 –
3.06 (m, 2H), 2.89 (s, 4H), 2.21 (d, J = 7.4 Hz, 1H), 2.19 – 2.15 (m, 1H), 1.75 (dt, J = 10.7,
13
5.4 Hz, 4H), 1.59 (s, 4H), 0.92 (t, J = 6.8 Hz, 2H). C NMR (101 MHz, MeOD) δ 158.49,
157.44, 141.69, 137.72, 129.40, 128.84, 128.52 (dd, J = 42.1, 15.4 Hz), 126.97, 126.48,
124.90, 123.62, 115.36, 115.17, 114.54, 114.36, 84.31, 82.77, 64.64, 60.55, 60.15, 57.29,
54.39, 38.07, 24.77, 23.33, 19.49. HRMS (ESI) calcd for C32H36N2O5S [M + H]+, 561.2418;

ACCEPTED MANUSCRIPT
found 561.2418.
N-Ethyl-5-(4-hydroxyphenyl)-N-phenyl-6-(4-(2-(piperidin-1-yl)ethoxy)phenyl)-7-oxabicyclo[2
1
.2.1]hept-5-ene-2-sulfonamide (17i): Yellow solid, 72% yield, mp 97-100 °C. H NMR (400
MHz, MeOD) δ 7.32 (dd, J = 8.4, 6.3 Hz, 4H), 7.26 – 7.19 (m, 2H), 7.18 – 7.07 (m, 2H), 6.93
– 6.84 (m, 2H), 6.74 (dd, J = 19.6, 8.6 Hz, 2H), 5.46 (s, 1H), 5.30 (t, J = 3.1 Hz, 1H), 4.14 (dt,
J = 9.1, 5.5 Hz, 2H), 3.80 (dt, J = 17.5, 7.1 Hz, 2H), 3.52 – 3.43 (m, 1H), 2.82 (dt, J = 7.9, 5.2
Hz, 2H), 2.61 (s, 4H), 2.27 – 2.14 (m, 1H), 2.03 (ddd, J = 14.0, 8.5, 3.8 Hz, 1H), 1.66 (dd, J =
13
9.5, 5.0 Hz, 4H), 1.51 (s, 2H), 1.09 – 1.03 (m, 3H). C NMR (101 MHz, MeOD) δ 158.60,
157.42, 141.58, 138.84, 136.59, 129.05, 128.90, 128.77, 128.31, 128.16, 127.63, 125.44,
123.68, 115.18, 114.56, 84.40, 82.77, 64.85, 61.35, 60.17, 57.40, 54.50, 46.34, 24.92, 23.49,
19.50, 13.55. HRMS (ESI) calcd for C33H38N2O5S [M + Na]+, 597.2394; found 597.2398.
7-(4-(6-(N-Ethyl-N-phenylsulfamoyl)-3-(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-2-en-2-yl)
phenoxy)heptanoic acid (17j, Mixture of 3:1 Isomers): Yellow solid, 60% yield, mp
1
103-105 °C. H NMR (400 MHz, Acetone-d6) δ 7.47 – 7.41 (m, 1H), 7.40 – 7.33 (m, 4H),
7.27 (d, J = 8.8 Hz, 2H), 7.23 – 7.15 (m, 2H), 6.90 (dd, J = 14.5, 8.8 Hz, 2H), 6.85 – 6.79 (m,
2H), 5.49 (s, 1H), 5.36 – 5.31 (m, 1H), 4.04 – 3.98 (m, 2H), 3.92 – 3.80 (m, 2H), 3.53 (ddd, J
= 7.6, 4.3, 3.0 Hz, 1H), 2.33 (dd, J = 10.2, 4.5 Hz, 2H), 2.18 (tt, J = 11.3, 5.7 Hz, 1H), 2.09
(dd, J = 7.7, 4.5 Hz, 1H), 1.84 – 1.76 (m, 2H), 1.68 – 1.61 (m, 2H), 1.52 (dd, J = 17.5, 10.7
13
Hz, 2H), 1.48 – 1.41 (m, 2H), 1.08 – 0.98 (m, 3H). C NMR (101 MHz, MeOD) δ176.31,
159.13, 158.98, 157.53, 157.35, 141.33, 140.54, 138.84, 137.50, 131.66, 131.41, 129.06,
128.89, 128.72, 128.27, 128.09, 127.62, 125.00, 124.37, 123.76, 123.10, 115.35, 115.12,
114.74, 114.48, 114.25, 113.74, 84.37, 84.13, 83.18, 82.77, 67.53, 67.47, 61.31, 61.19, 60.65,
60.15, 59.66, 46.35, 33.49, 28.79, 28.58, 25.47, 24.63, 19.48, 13.54, 13.37. HRMS (ESI)
calcd for C33H37NO7S [M + Na]+, 614.2183; found 614.2184.
8-(4-(6-(N-Ethyl-N-phenylsulfamoyl)-3-(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-2-en-2-yl)
1
phenoxy)octanoic acid (17k): Yellow solid, 63% yield, mp 88-90 °C. H NMR (400 MHz,
Acetone-d6) δ 7.41 – 7.34 (m, 4H), 7.27 (d, J = 8.7 Hz, 2H), 7.19 (dd, J = 10.6, 9.4 Hz, 2H),
6.90 (dd, J = 14.5, 8.8 Hz, 2H), 6.81 (dt, J = 17.3, 7.3 Hz, 2H), 5.49 (s, 1H), 5.34 (d, J = 4.1