Chemical degradation of androgen receptor (Ar) using bicalutamide analog–thalidomide protacs

Article abstract of DOI:10.3390/molecules26092525

A series of PROTACs (PROteolysis-TArgeting Chimeras) consisting of bicalutamide analogs and thalidomides were designed, synthesized, and biologically evaluated as novel androgen receptor (AR) degraders. In particular, we found that PROTAC compound 13b could successfully demonstrate a targeted degradation of AR in AR-positive cancer cells and might be a useful chemical probe for the investigation of AR-dependent cancer cells, as well as a potential therapeutic candidate for prostate cancers.

Full text of DOI:10.3390/molecules26092525

molecules
Article
Chemical Degradation of Androgen Receptor (AR) Using
Bicalutamide Analog–Thalidomide PROTACs
Ga Yeong Kim 1,†, Chae Won Song 2,†, Yo-Sep Yang 1, Na-Rae Lee 1, Hyung-Seok Yoo 1, Seung Hwan Son 1,
Soo Jin Lee 1, Jong Seon Park 1, Jong Kil Lee 1, Kyung-Soo Inn 1,2,* and Nam-Jung Kim 1,2,
*
1
College of Pharmacy, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, Korea;
gagoyo1234@khu.ac.kr (G.-Y.K.); didtmdrl316@khu.ac.kr (Y.-S.Y.); nrl9758@khu.ac.kr (N.-R.L.);
yoohs@khu.ac.kr (H.-S.Y.); thstmd09@khu.ac.kr (S.H.S.); eldtn05@naver.com (S.J.L.);
airfixdog@naver.com (J.S.P.); jklee3984@khu.ac.kr (J.K.L.)
Department of Life and Nanopharmaceutical Sciences, Graduate School, Kyung Hee University,
26 Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, Korea; chae1@khu.ac.kr
Correspondence: innks@khu.ac.kr (K.-S.I.); kimnj@khu.ac.kr (N.-J.K.)
2
*
†
These authors contributed equally to this work.
Abstract: A series of PROTACs (PROteolysis-TArgeting Chimeras) consisting of bicalutamide analogs
and thalidomides were designed, synthesized, and biologically evaluated as novel androgen receptor
(AR) degraders. In particular, we found that PROTAC compound 13b could successfully demonstrate
a targeted degradation of AR in AR-positive cancer cells and might be a useful chemical probe for
the investigation of AR-dependent cancer cells, as well as a potential therapeutic candidate for
prostate cancers.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Kim, G.Y.; Song, C.W.;
Yang, Y.-S.; Lee, N.-R.; Yoo, H.-S.; Son,
S.H.; Lee, S.J.; Park, J.S.; Lee, J.K.; Inn,
K.-S.; et al. Chemical Degradation of
Androgen Receptor (AR) Using
Bicalutamide Analog–Thalidomide
PROTACs. Molecules 2021, 26, 2525.
https://doi.org/10.3390/
Keywords: bicalutamide; protein degradation; PROTAC; androgen receptor (AR); prostate cancer
1. Introduction
Chemical degraders of specific protein targets, also known as PROTACs (PROteolysis-
TArgeting Chimeras), have recently been introduced and considered as part of a novel
approach for the treatment of various diseases, especially focusing on pathologies that
molecules26092525
cannot be alleviated via classical pharmacological therapeutics [1–3]. PROTACs are het-
Academic Editor: James
T. Hodgkinson
erodimeric small molecules consisting of a protein target binder, linker, and E3 ligase
recruiter. These types of molecules can bind both E3 ligase complexes and target proteins,
as well as providing proximity between them. This interaction forms a ternary complex
consisting of proteins and enables the target proteins to be ubiquitinated, leading to their
proteasomal degradation. In particular, PROTACs require only transient binding to the
surface of the target, leading to the catalytic induction of ubiquitination. This catalytic
ability of PROTACs enables the target protein to be degraded iteratively and thus address
the resistance issues of the existing pharmacological inhibitors, which are generally related
to increases or mutations of the target proteins, especially frequently problematic in cancer
diseases. Therefore, for the past decade, most of these PROTAC approaches have been
developed for targeting refractory cancers including castration-resistant prostate cancer
Received: 19 February 2021
Accepted: 23 April 2021
Published: 26 April 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
(CRPC) [4–6]. In the United States, prostate cancer is one of the most prevalent cancers for
men as well as the second leading cause of death by cancer. Once prostate cancer occurs in
patients, anti-androgens (also known as androgen receptor (AR) antagonists, which inhibit
the signaling mediated by AR that plays a key role in the development of prostate cancer,
thus acting as a type of chemical castration) are preferentially prescribed as a first-line
therapy for the treatment of these diseases because most prostate cancer cells grow in an
androgen-dependent manner [7–9] (Figure 1). Alternatively, physical castration to suppress
the production of natural androgens can also be carried out for the inhibition of cancer
progression. These different types of castration therapies are generally effective in early
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
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Attribution (CC BY) license (https://
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4.0/).
Molecules 2021, 26, 2525. https://doi.org/10.3390/molecules26092525
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Molecules 2021, 26, 2525
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prostate cancer patients. However, as the treatment time goes by, hormone-dependent
prostate cancer becomes progressively worse and is transformed into CRPC, for which the
prognosis is very poor. For the treatment of CRPC, next-generation AR antagonists such
as enzalutamide have been clinically used [7,8,10,11], but their efficacy is still limited by
resistance mechanisms, including increased AR expression and/or AR point mutations for
the more potent binding of androgens.
Figure 1. Structures of representative androgen receptor (AR) modulators.
To overcome these unmet medical needs, several PROTACs targeting AR for degra-
dation, not just inhibition, were recently disclosed [4,5,12,13]. Among the AR-targeted
PROTACs, ARV-110 was spotlighted and recently entered into clinical trial (Phase 1) in
2019. Given that chemical AR degradation by PROTACs can provide advantages over
simple inhibition by existing AR antagonists and that several PROTACs targeting AR have
recently been introduced and shown therapeutic potential, we considered it worthwhile to
develop novel AR-targeted PROTACs as a potential approach for the treatment of refractory
prostate cancers. In this study, we developed a series of novel PROTACs to degrade AR
in AR-positive cancer cells. In an effort to successfully develop AR degraders, we prefer-
entially designed and synthesized a series of novel AR antagonists, such as AR ligands
(Scheme 1). Upon using a novel AR antagonist as a warhead for AR, we prepared novel
PROTACs by conjugating the antagonist to thalidomide, a representative E3 ligase recruiter
known as the ligands of cereblon (CRBN) that forms an E3 ubiquitin ligase complex with
Cullin-4A (CUL4A) and other compartments, through biologically compatible linking and
biologically evaluated them as AR degraders in AR-positive cancer cells (Figure 2).
Scheme 1. Design strategy for novel AR-targeted PROTACs.

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Figure 2. Strategy for androgen receptor (AR) degradation using PROTACs consisting of a bicalu-
tamide analog and thalidomide.
2. Results and Discussion
2.1. Synthesis
To design and synthesize AR-targeting PROTACs, we initially tried to select a nons-
teroidal AR antagonist that was clinically more useful than a steroidal AR antagonist in
terms of oral availability and off-target side effects as a warhead for targeting AR [14].
Among the various nonsteroidal AR antagonists, bicalutamide, which has been widely
used for prostate cancer and acts as an AR antagonist with minor side effects, was selected
as an initial template for PROTAC synthesis in our study [10
the amide nitrogen and the hydroxyl group of bicalutamide have crucial roles in its anti-
androgen activity through hydrogen bonding to the androgen receptor [16 17]. In addition,
,15]. It was reported that
,
in 2000, the Van Dort group reported that DTIB where the trifluoromethyl group of RU
59063 was substituted with the hydrophobic iodine had a better binding affinity than RU
59063. Recently, the sulfoxide and fluorine of bicalutamide were reported not to be crucial
for the anti-AR activity of bicalutamide [18]. In addition, an attachable moiety of the AR
ligand was also necessary to enable it to be conjugated with thalidomide. Collectively,
given these previous reports we designed and synthesized a series of AR antagonists such
as 5a and 7a
for PROTAC synthesis (Schemes 2 and 3).
Benzonitrile was synthesized from commercially available 2-iodo-4-nitroaniline
Sandmeyer reaction [19]. The nitro reduction of gave 4-aminobenzonitirle , which was
followed by acylation with methacryloyl chloride to obtain phenylacrylamide 19 20].
Next, sequential epoxidation and ring opening reactions with hydroquinone were per-
formed to afford 5a 18 20]. Similarly, from commercially available methacrylamide epoxide
bearing trifluoromethyl , a series of thioethers and ethers (7a ) were prepared. Based on
the preliminary screening results of the AR antagonists that we designed and synthesized,
PROTACs 11a and 13a were synthesized according to the concise and unified synthetic
routes described in Scheme 3. PROTACs 8a were synthesized by the alkylation of phenol
5a with alkyl bromide containing several ethylene glycol units [21]. Phthalimide moieties
such as and 10 were prepared as shown in the supporting information to be conjugated
as ligands for CRBN. The desired PROTAC compounds such as 11a were finally ob-
tained by the carbamate coupling of 8a–d with CRBN ligand 9 under basic conditions. To
conjugate 8a with 4-hydroxythalidomide 10 to synthesize the desired PROTACs 13a
primary alcohol moieties in the intermediates 8a were converted into the corresponding
–c, which are expected to provide better AR antagonists as well as a template
2
1 by
2
3
4
[ ,
[
,
6
–c
–
d
–d
–
d
9
–
d
–d
–d,
–
d

Molecules 2021, 26, 2525
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methanesulfonyl groups [22]. The resultant O-mesylates 12a
–
d
were reacted with 10 under
the basic conditions to yield other PROTACs such as 13a–d.
Scheme 2. Synthesis of compounds 5a and 7a–c. Conditions and reagents: (a) NaNO2, NaCN, CuCN, H2SO4, H2O, r.t.,
50%; (b) SnCl2
•
HCl, EtOH, reflux, 84%; (c) methacryloyl chloride, CH2Cl2,◦r.t., 80%; (d) formic acid, hydrogen peroxide,
◦
◦
40 C; (e) hydroquinone, NaH, DMF, 0 to 90 C, 55%; (f) NaH, DMF, 0 to 90 C, 31–95%.
◦
Scheme 3. Synthesis of compounds 11a
–
d
and 13a
–d
. Conditions and reagents: (f) K2CO3, dry DMF, 90 C, 54–80%;
◦
(g) NEt3, CH2Cl2, r.t., 17–53%; (h) methanesulfonyl chloride, NEt3, CH2Cl2, r.t., 66–81%; (i) K2CO3, DMF, 90 C, 27–33%.
2.2. Biology
At first, an experiment was conducted to select the most effective AR antagonist among
the synthesized antagonists as an AR ligand for PROTAC synthesis. LNCaP cells, one of
the AR-dependent prostate cancer cell lines, were treated with 5a and 7a–c and incubated
for 24 h. Additionally, the mRNA levels of AR-dependent transcriptional genes such as
prostate-specific antigen (PSA) and transmembrane protease serine subtype 2 (TMPRSS2)
in these cells were investigated (Figure 3A,B). As a result, we found that compound 5a
was shown to reduce both the levels of PSA and TMPRSS2 in the most effective manner.
Thus, 5a was found to be more potent than compound 7a, which was reported to have a
slightly better AR antagonistic activity than bicalutamide [18]. This result suggested that
the substitution of the trifluoromethyl group and sulfide group with the iodine and ether,
respectively, could significantly improve the AR antagonistic activity. In addition, in the

Molecules 2021, 26, 2525
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case of incorporating the hydroxy group instead of fluorine in the terminal phenyl moiety
located at the solvent exposure site, we observed that the AR antagonistic activity was not
decreased, indicating that the linker moiety for conjugating the AR ligand with thalidomide
could be introduced as an ether linkage on the hydroxy group in that terminal site. With
these results in hand, we synthesized a series of AR PROTACs where AR antagonist 5a
was assembled using biocompatible ethylene glycol linkers. Additionally, we evaluated the
efficacy of AR degradation by these PROTAC compounds in AR-overexpressing HEK293T
cells. Our immunoblotting results indicated that all the compounds could achieve the
targeted degradation of AR at the protein level (Figure 3C,D). Among them, compounds
13b and 13c reduced the AR protein in highly efficient manners (Figure 3D). Based on these
results, we selected compounds 13b and 13c as candidate compounds for further study.
Figure 3. Effects of the novel AR antagonists and novel AR PROTACs. (
and TMPRSS2 in LNCaP cells treated with AR antagonists 5a and 7a (10
various AR PROTAC compounds (10 M) on the levels of ectopically expressed AR in HEK293T cells
as determined by immunoblotting. ( ) Quantitation of protein levels by the treatment of compounds
A
,
B) mRNA levels of PSA
–
c
µ
M) (n = 3). ( ) Effects of
C
µ
D
using densitometry. The experiments were repeated at least three times. The data represent mean
values ± SEM. *** p < 0.001, ** p < 0.01. Ctrl, control.
Next, we examined whether the mRNA expression levels of the AR and AR-target
genes in LNCaP cells could be affected by the selected PROTACs, such as 13b and 13c
As expected, the mRNA levels of AR were rarely altered by the PROTACs. However,
compound 13b suppressed the expression of the PSA gene, while 13c did not suppress
the mRNA level of PSA. In other words, the AR expression was not decreased but the
AR-dependent transcription was decreased by PROTAC 13b at the mRNA level (Figure 4A).
To confirm whether 13b is a targeted AR degrader, a series of immunoblotting analyses was
conducted. As shown in Figure 4B, the levels of ectopically expressed AR were decreased
.
by the treatment of compound 13b in a dose-dependent manner, indicating that the 13b
-
mediated decrease in AR is not due to transcriptional regulation via the AR signaling
pathway. In a similar manner, AR was shown to be significantly degraded in LNCaP cells
after the 13b treatment in dose-dependent and time-dependent manners (Figure 4C,D). The
DC50 (degradation concentration 50) value in LNCaP cells was 5.211 µM. The effect of 13b
on AR degradation was further confirmed using 22Rv1, a prostate cancer cell line derived
from castration-recurrent xenograft [23]. Interestingly, the AR variant 7 (AR-V) form was
much more susceptible to 13b-mediated degradation (Figure 4E). The AR-V of 22Rv1 is a
splicing variant of AR and constitutively active form which enables the prostate tumor to
be grown under an environment with insufficient androgen [24]. The result suggested the
possible use of the compound as a therapeutic agent to treat castration-resistant prostate
cancer through the expression of AR variants. To further analyze the anti-tumor effect

Molecules 2021, 26, 2525
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of 13b, the proapoptotic effect of the compound was investigated. Treatment with 13b
resulted in the increment of Bax and the decrement of c-Myc in LNCaP cells, whereas
those levels were not significantly affected in androgen receptor-negative DU-145 cells,
suggesting that 13b exerts a proapoptotic effect on AR-dependent cells (Figure 4F). Indeed,
robust apoptosis was observed in 13b-treated LNCaP cells but not in DU-145 cells, as
determined by TUNEL staining (Figure 4G). Collectively, our results clearly indicate that
PROTAC 13b enables AR to be targeted and degraded through the direct binding of 13b
with AR, and it does not modulate the mRNA expression level of AR.
Figure 4. Effects of the novel AR PROTACs. (
with AR PROTACs (10 M) (n = 3). ( ) Degradation of ectopically expressed AR in HEK293T cells
treated with different concentrations of 13b for 24 h and the quantitative analysis by densitometry.
) Dose-dependent degradation of endogenous AR protein in LNCaP cells treated with 13b for
24 h. DC50: 5.211 M. ( ) Time-kinetics of 13b-mediated degradation of AR in LNCaP cells treated
with 13b (10 M). All protein intensity analyses were performed in triplicate. ( ) Effect of 13b on
the protein levels of AR and AR variant (AR-V). 22Rv1 cells were treated with increasing amounts
of 13b for 24 h and the protein levels were analyzed by immunoblotting. ( ) AR transcriptional
A) mRNA levels of AR and PSA in LNCaP cells treated
µ
B
(C
µ
D
µ
E
F,G
activity-dependent proapoptotic effects of 13b on prostate cancer cells. LNCaP and DU-145 cells
were treated with 13b for 24 h and subjected to immunoblotting to determine the levels of Bax and
c-Myc (
F
). Analysis of apoptosis by TUNEL staining. LNCaP and DU-145 cells were treated with
13b (5
µ
M) for 24 h and subjected to TUNEL staining ( ). The data represent mean values SEM.
G
±
*** p < 0.001, ** p < 0.01, * p < 0.05.
2.3. Molecular Modeling
To investigate the binding mode of 5a, which was identified as a novel AR inhibitor in
our study, we conducted a molecular docking study of 5a with AR. Using the X-ray crystal
structure of (R)-bicalutamide, known as an active enantiomer within AR (PDB code: 1Z95),
we performed a molecular modeling of 5a and observed that the overall binding mode of
the compound might be quite similar to that of bicalutamide (Figure 5). The estimated free
binding energy of 5a
cating that 5a might have an energetically more favored conformation than 7a
In detail, a tertiary alcohol of 5a, known as a key pharmacophore for improving the AR an-
(
−
10.71 kcal/mol) was lower than that of 7a
(
−9.70 kcal/mol), indi-
(
Figure S1).

Molecules 2021, 26, 2525
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tagonistic activity of bicalutamide, forms hydrogen bonds with the amide bonds of Leu701,
Asn705, and Thr877, which looks similar to the binding mode of bicalutamide. When
fluorine on the terminal phenyl group was replaced with a hydroxyl group, the binding
modes of the compounds within AR were likely to be similar [16]. In other words, like
fluorine atoms, the phenol group is oriented toward a solvent exposure site, and it seems
that phenolic OH can be an optimal site to be conjugated to the linker and thalidomide.
Figure 5. Molecular docking model of 5a (orange) with the projection of bicalutamide (sky blue) in
androgen receptor (PDB code: 1Z95), which was visualized using Chimera 1.10 (UC13bSF Chimera).
With the preliminary modeling result of 5a in hand, to explain the capability of PRO-
TAC 13b to provide proximity and sequentially induce ubiquitination, we also performed
a molecular modeling of 13b and investigated the feasibility of being assembled into the
ternary complex in silico using both the crystal structure of AR bound to (R)-bicalutamide
and CRBN bound to pomalidomide (Figure 6). In the molecular modeling, AR PROTAC
13b could be located at the active site of AR and freely extrude the linked thalidomide to
the solvent exposure site (Figure 6A). Additionally, it was estimated that the thalidomide
region of 13b could be bound within CRBN (cereblon), appropriately providing proximity
between CRBN and AR without mutual steric interactions. For this reason, it was estimated
that 13b might induce the targeted ubiquitination of AR and its resultant degradation at
the protein level (Figure 6B).
Figure 6.
(
A
) Superposition of 13b (orange) with (R)-bicalutamide (purple) bound to androgen
) Cartoon representation
receptor (AR, PBD code: 1Z95). The interacting residues are shown in gray. (
B
of the ternary complex of 13b (orange) with AR (PDB Code: 1Z95) and CRBN (PDB Code: 4CI3),
which were visualized using Chimera 1.10 (UCSF Chimera).

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3. Materials and Methods
3.1. Chemistry
3.1.1. General Experimental Methods
Unless noted otherwise, all the starting materials and reagents were obtained from
commercial suppliers (Aldrich, Acros Organics, Alfa Aesar, and TCI) and were used
without further purification. All the solvents used for the routine isolation of products and
chromatography were of reagent grade. Reaction flasks were dried at 80 ◦C. Analytical
thin-layer chromatography (TLC) was performed using Merck silica gel glass plates with
an F-254 indicator, visualized by UV light (254, 365 nm) and in some cases stained with
ninhydrin or p-anisaldehyde, followed by heating. Flash column chromatography was
performed using silica gel 60 (230–400 mesh) with the indicated solvents. NMR spectra were
recorded and obtained using a Bruker 400 (400 MHz for 1H NMR) and JMTC 500/54/JJ
(126 MHz for 13C NMR and 500 MHz for 1H NMR) spectrometer, respectively. 1H and 13
C
were reported in parts per million (ppm) relative to tetramethylsilane, with the residual
solvent peak used as an internal reference. Signals are reported as m (multiplet), s (singlet),
d (doublet), t (triplet), q (quartet), br (broad signal), and td (triplet of doublets); the coupling
constants (J) are reported in hertz (Hz). High-resolution mass spectrometry (HRMS) data
were obtained with a JEOL JMS-700 instrument (EI-quadrupole). Molecular docking model
of 5a (pink) with projection of 7a (sky blue) in androgen receptor (PDB Code: 1Z95), which
was visualized using Chimera 1.10 (UC13bSF Chimera) and the synthesis of Pomalidomide
and Derivatives are available in Supplementary Materials.
3.1.2. Synthesis
2-Iodo-4-nitrobenzonitrile (2)
2-iodo-4-nitroaniline (5.0 g, 18.9 mmol), H2SO4 (6 mL), and AcOH (22.5 mL) were
added to a shading flask at 0 ◦C. Copper(I) cyanide (2.61 g, 37.8 mmol) in distilled water
(4.5 mL) was slowly dropped to the mixture and stirred for 30 min. Then, the mixture
was added to copper(I) nitrite (3.39 g, 37.8 mmol), sodium nitrite (1.86 g, 37.8 mmol), and
sodium bicarbonate (10.0 g) in distilled water (20 mL). After 1 h, water was added to the
flask and extracted with CH2Cl2. The organic layers were dried with MgSO4 and the
filtrate was concentrated under reduced pressure. The crude mixture was purified with
flash column chromatography to afford compound
2
as a yellow solid (Yield: 61%, 2.09 g).
8.76 (s, 1H), 8.32 (d, J = 8.5 Hz, 1H), 7.82 (d, J = 8.5 Hz,
150.09, 136.43, 133.91, 125.42, 124.02, 118.99, 101.89.
1H NMR (400 MHz, Chloroform-d),
δ
1H). 13C NMR (126 MHz, DMSO-d6),
δ
4-Amino-2-iodobenzonitrile (3)
Tin(II) chloride dihydrate (2.57 g, 11.4 mmol) and compound 2 (626 mg, 2.28 mmol)
were dissolved in ethanol (5 mL). The mixture was refluxed for 3 h. Then, the solvent was
removed under reduced pressure, followed by extraction with EtOAc and distilled water.
The organic layer was dried with Na2SO4. The filtrate was concentrated under reduced
pressure. The crude mixture was purified with flash column chromatography to afford
compound
δ
3 as a white solid (Yield: 81%, 445 mg). 1H NMR (400 MHz, Chloroform-d),
7.32 (s, 1H), 7.14 (d, J = 2.3 Hz, 2H), 6.63 (d, J = 2.3 Hz, 1H), 6.61 (d, J = 2.3 Hz, 1H). 13
C
NMR (126 MHz, DMSO-d6), δ 154.21, 135.71, 123.25, 121.77, 113.50, 103.67, 101.30.
N-(4-Cyano-3-iodophenyl)methacrylamide (4)
Methacryolyl chloride (209.06
µ
L, 1.8 mmol) was added to a flask and stirred under
◦
Ar at 0 C. Compound
3 (443 mg, 1.8 mmol) was dissolved in DMA dropwise. The mixture
was quenched with sodium bicarbonate, followed by extraction with EtOAc and distilled
water. The organic layer was dried with MgSO4.The filtrate was concentrated under
reduced pressure. The crude mixture was purified with flash column chromatography to
afford compound 3 as a white solid (Yield: 81%, 430 mg). 1H NMR (500 MHz, DMSO-d6),
δ
10.24 (s, 1H), 8.45 (d, J = 2.0 Hz, 1H), 7.91 (dd, J = 8.6, 2.0 Hz, 1H), 7.80 (d, J = 8.6 Hz, 1H),

Molecules 2021, 26, 2525
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5.88 (s, 1H), 5.65 (d, J = 1.6 Hz, 1H), 1.96 (s, 3H). 13C NMR (126 MHz, DMSO-d6),
δ
167.95,
144.31, 140.25, 135.57, 129.67, 122.08, 119.64, 113.33, 100.77, 19.03.
N-(4-Cyano-3-iodophenyl)-2-hydroxy-3-(4-hydroxyphenoxy)-2-methylpropanamide (5a
)
Formic acid (2.3 mL) and 35% hydrogen peroxide (1.4 mL) were dropped ◦in a flask
with compound
4 (100 mg, 0.32 mmol). The mixture was stirred for 4 h at 40 C. After
cooling, the reaction was quenched by 2N sodium hydroxide solution. The solution was
washed with CH2Cl2 and dried with MgSO4. The filtrate was concentrated under reduced
pressure. The crude mixture of N-(4-cyano-3-iodophenyl)-2-methyloxirane-2-carboxamide
was used for the next step without further purification. Sodium hydride (60% dispersion
in mineral oil, 19.2 mg, 0.480 mmol) was added to hydroquinone (70.5 mg, 0.64 mmol) in
anhydrous DMF. A solution of N-(4-cyano-3-iodophenyl)- 2-methyloxirane-2-carboxamide
in anhydrous DMF was slowly added to the reaction mixture and stirred for 1 h at 90 ◦C.
After cooling, the reaction mixture was quenched by 2N HCl solution. The solution was
washed with CH2Cl2 and the organic layer was dried with MgSO4. The filtrate was
concentrated under reduced pressure. The crude mixture was purified with flash column
chromatography to afford compound 5a as a white solid (Yield: 55%, 70.1 mg). 1H NMR
(500 MHz, DMSO-d6),
δ 10.26 (s, 1H), 8.96 (s, 1H), 8.63 (d, J = 2.1 Hz, 1H), 8.02 (dd,
J = 8.6, 2.0 Hz, 1H), 7.81 (d, J = 8.6 Hz, 1H), 6.80–6.71 (m, 2H), 6.71–6.63 (m, 2H), 6.19 (s, 1H),
4.15–4.12 (m, 1H), 3.87 (d, J = 9.5 Hz, 1H), 1.42 (s, 3H). 13C NMR (126 MHz, DMSO-d6),
δ
174.38, 151.42, 151.39, 143.28, 135.02, 129.12, 119.88, 119.20, 115.68, 115.67, 113.03, 100.31,
74.94, 74.33, 23.05. HR-MS (EI) calculated for (M+) 438.0766; found 438.0077.
N-(4-Cyano-3-(trifluoromethyl)phenyl)-2-hydroxy-3-((4-hydroxyphenyl)thio)-2-
methylpropanamide (7a)
Sodium hydride (60% dispersion in mineral oil, 60.1 mg, 0.56 mmol) was added to
4-fluorobenzenethiol (78.9 mg, 0.74 mmol) in anhydrous DMF. A solution of N-(4-cyano-3-
(trifluoromethyl)phenyl)-2-methyloxirane-2-carboxamide ( ◦
6) (101.2 mg, 0.37 mmol) was
slowly added to the reaction mixture and stirred for 6 h at 100 C. After cooling, the reaction
mixture was quenched by 2N HCl solution. The solution was washed with CH2Cl2 and the
organic layer was dried with MgSO4. The filtrate was concentrated under reduced pressure.
The crude mixture was purified with flash column chromatography to afford compound 7a
as a white solid (Yield: 95.7%, 141 mg). 1H NMR (500 MHz, DMSO-d6),
δ 10.51 (s, 1H), 8.45
(d, J = 2.0 Hz, 1H), 8.23 (dd, J = 8.6, 2.0 Hz, 1H), 8.09 (d, J = 8.6 Hz, 1H), 7.44–7.37 (m, 2H),
7.13–6.98 (m, 2H), 3.43 (d, J = 13.3 Hz, 1H), 3.36 (s, 1H), 3.26 (d, J = 13.3 Hz, 1H), 1.47 (s, 3H).
13C NMR (126 MHz, DMSO-d6),
δ 174.84, 161.78, 159.84, 143.14, 136.22, 131.91, 131.89,
131.67, 131.61, 122.73, 117.38, 117.34, 115.87, 115.69, 101.88, 75.22, 44.69, 25.83; HR-MS (EI)
calculated for (M+) 398.0712; found 398.0710.
N-(4-Cyano-3-(trifluoromethyl)phenyl)-3-(4-fluorophenoxy)-2-hydroxy-2-
methylpropanamide (7b)
Compound 7b was prepared using a similar method for the synthesis of 7a (16.7%).
1H NMR (500 MHz, DMSO-d6),
Hz, 1H), 7.78 (d, J = 8.5 Hz, 1H), 7.01–6.82 (m, 4H), 4.42 (d, J = 8.9 Hz, 1H), 3.95 (d, J = 8.9
Hz, 1H), 3.56 (s, 1H), 1.58 (s, 3H). 13C NMR (126 MHz, DMSO-d6),
174.65, 157.57, 155.69,
δ 9.18 (s, 1H), 8.11 (d, J = 2.1 Hz, 1H), 7.95 (dd, J = 8.5, 2.2
δ
154.88, 143.29, 136.38, 122.74, 117.42, 117.38, 116.03, 115.97, 115.88, 115.93, 115.74, 101.93,
74.91, 74.21, 23.03; HR-MS (EI) calculated for (M+) 382.0941; found 382.0939.
N-(4-Cyano-3-(trifluoromethyl)phenyl)-2-hydroxy-3-(4-hydroxyphenoxy)-2-
methylpropanamide (7c)
Compound 7c was prepared using a similar method for the synthesis of 7a (31.4%).
1H NMR (500 MHz, DMSO-d6),
δ 9.13 (s, 1H), 8.10 (d, J = 2.2 Hz, 1H), 7.96 (dd, J = 8.5,

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2.2 Hz, 1H), 7.81 (d, J = 8.5 Hz, 1H), 6.84–6.72 (m, 4H), 4.70 (s, 1H), 4.40 (d, J = 9.0 Hz, 1H),
3.92 (d, J = 9.1 Hz, 1H), 3.47 (s, 1H), 1.57 (s, 3H). 13C NMR (126 MHz, DMSO-d6),
δ 174.79,
151.44, 151.37, 143.33, 136.37, 122.69, 117.39, 117.35, 115.90, 115.68, 101.88, 74.99, 74.34, 23.06;
HR-MS (EI) calculated for (M+) 380.0984; found 380.0984.
N-(4-Cyano-3-(trifluoromethyl)phenyl)-3-((4-fluorophenyl)thio)-2-hydroxy-2-
methylpropanamide (8a)
A solution of acetone (2 mL) and DMF (0.5 mL) was added to N-(4-cyano-3-iodophenyl)-
2-hydroxy-3-(4-hydroxyphenoxy)-2-methylpropanamide (5a) (190 mg, 0.434 mmol), potas-
sium carbonate (120 mg, 0.868 mmol), and tetrabutylammonium iodide (13.7 mg, 0.043
mmol). The mixture was stirred for 30 min at ambient temperature. 2-Bromoethan-1-ol
(0.5 mL) was added to the mixture and refluxed overnight. After cooling, the reaction
mixture was quenched by 2N HCl solution. The solution was washed with CH2Cl2 and the
organic layer was dried with MgSO4. The filtrate was concentrated under reduced pressure.
The crude mixture was purified with flash column chromatography to afford compound
8a as a yellow oil. (113 mg, 54%). 1H NMR (400 MHz, Chloroform-d), δ 8.98 (s, 1H), 8.27
(s, 1H), 7.69 (d, J = 5.4 Hz, 1H), 7.55 (dd, J = 8.4, 3.4 Hz, 1H), 6.84–6.72 (m, 4H), 5.45 (s, 1H),
4.41–4.30 (m, 1H), 3.96–3.82 (m, 1H), 3.60 (s, 1H), 2.96 (d, J = 3.5 Hz, 2H), 2.89 (d, J = 3.5 Hz,
2H), 1.55 (s, 3H).
N-(4-Cyano-3-iodophenyl)-2-hydroxy-3-(4-(2-(2-hydroxyethoxy)ethoxy)phenoxy)-2-
methylpropanamide (8b)
Compound 8b was prepared using a similar method for the synthesis of 8a (56.4%).
1H NMR (400 MHz, DMSO-d6),
(d, J = 8.6 Hz, 1H), 6.85 (s, 4H), 6.20 (s, 1H), 4.64 (s, 1H), 4.15 (d, J = 9.5 Hz, 1H), 4.02
t, J = 4.4 Hz, 2H), 3.91 (d, J = 9.5 Hz, 1H), 3.74–3.67 (m, 2H), 3.52 (d, J = 3.8 Hz, 2H), 3.49
δ 10.25 (s, 1H), 8.62 (s, 1H), 8.01 (d, J = 8.6 Hz, 1H), 7.79
(
(d, J = 4.0 Hz, 2H), 1.42 (s, 3H).
N-(4-Cyano-3-iodophenyl)-2-hydroxy-3-(4-(2-(2-(2-
hydroxyethoxy)ethoxy)ethoxy)phenoxy)-2-methylpropanamide (8c)
Compound 8c was prepared using a similar method for the synthesis of 8a (54.8%).
1H NMR (400 MHz, DMSO-d6), δ 10.23 (s, 1H), 8.60 (s, 1H), 8.00 (m, 1H), 7.78 (dd, J = 8.6,
0.9 Hz, 1H), 6.83 (s, 4H), 6.18 (s, 1H), 4.59 (t, J = 5.7 Hz, 1H), 4.13 (d, J = 10.4 Hz, 1H),
4.02–3.96 (m, 2H), 3.89 (d, J = 10.7 Hz, 1H), 3.73–3.65 (m, 2H), 3.58–3.52 (m, 4H), 3.48
(t, J = 5.1 Hz, 2H), 3.42 (t, J = 5.5 Hz, 2H) 1.40 (s, 3H).
N-(4-Cyano-3-iodophenyl)-2-hydroxy-3-(4-((5-hydroxypentyl)oxy)phenoxy)-2-
methylpropanamide (8d)
Compound 8d was prepared using a similar method for the synthesis of 8a (80%).
1H NMR (400 MHz, Chloroform-d),
δ 8.98 (s, 1H), 8.27 (s, 1H), 7.72–7.67 (m, 1H), 7.54
(d, J = 8.5 Hz, 1H), 6.75 (s, 4H), 5.42 (s, 1H), 5.10 (s, 1H), 4.36 (d, J = 9.0 Hz, 1H), 3.89 (t,
J = 7.2 Hz, 2H), 3.69 (t, J = 6.4 Hz, 2H), 2.92 (d, J = 29.3 Hz, 1H), 1.81–1.75 (m, 2H), 1.68–1.59
(m, 4H), 1.54 (s, 3H).
2-(4-(3-((4-Cyano-3-iodophenyl)amino)-2-hydroxy-2-methyl-3-
oxopropoxy)phenoxy)ethyl
(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)carbamate (11a)
Compound 8a (25 mg, 0.048 mmol), compound 9 (19 mg, 0.438 mmol), and NEt3 (6 µL,
0.043 mmol) in DMF were added to a flask and stirred under Ar at room temperature.
After 1 h, the mixture was quenched with ammonium chloride, followed by extraction
with EtOAc and distilled water. The organic layer was dried with MgSO4 and the filtrate
was concentrated under reduced pressure. The crude mixture was purified with flash

Molecules 2021, 26, 2525
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column chromatography to afford compound 11a as a white solid (20 mg, 53%). 1H NMR
(500 MHz, Chloroform-d),
δ 8.95 (d, J = 2.7 Hz, 1H), 8.77 (s, 1H), 8.51 (d, J = 8.3 Hz, 1H),
8.31–8.21 (m, 2H), 7.74–7.64 (m, 2H), 7.53 (dd, J = 8.5, 4.3 Hz, 1H), 7.48 (ddd, J = 20.2, 7.3,
0.7 Hz, 1H), 6.89 –6.76 (m, 4H), 5.29 (s, 1H), 4.94 (ddd, J = 12.5, 6.0, 3.0 Hz, 1H), 4.63–4.42
(m, 2H), 4.24–4.15 (m, 3H), 3.87 (dd, J = 9.1, 7.9 Hz, 1H), 3.68 (s, 1H), 2.96–2.85 (m, 1H),
2.84–2.68 (m, 2H), 2.19–2.11 (m, 1H), 1.53 (s, 3H). 13C NMR (126 MHz, Chloroform-d),
δ
172.68, 170.74, 168.71, 168.17, 166.82, 153.54, 152.71, 152.33, 141.57, 137.82, 136.46, 134.82,
131.39, 129.48, 123.74, 119.43, 118.78, 117.84, 116.17, 116.00, 115.97, 115.91, 115.66, 114.89,
99.00, 75.69, 73.03, 66.75, 64.62, 49.22, 31.36, 22.98, 22.75; HR-MS (EI) calculated for (M+)
781.0881; found 781.0887.
2-(2-(4-(3-((4-Cyano-3-iodophenyl)amino)-2-hydroxy-2-methyl-3-
oxopropoxy)phenoxy)ethoxy)ethyl
(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)carbamate (11b)
Compound 11b was prepared using a similar method for the synthesis of 11a (43%).
1H NMR (500 MHz, Chloroform-d),
δ 8.98 (d, J = 7.6 Hz, 1H), 8.77 (d, J = 14.3 Hz, 1H), 8.51
(d, J = 8.6 Hz, 1H), 8.29 (d, J = 2.1 Hz, 1H), 8.23 (d, J = 14.8 Hz, 1H), 7.74–7.63 (m, 2H), 7.54
(d, J = 8.5 Hz, 1H), 7.45 (ddd, J = 7.3, 2.4, 0.7 Hz, 1H), 6.87 –6.71 (m, 4H), 5.29 (s, 1H), 4.93
(ddd, J = 12.7, 5.5, 3.5 Hz, 1H), 4.45–4.28 (m, 2H), 4.07 (ddd, J = 5.4, 3.9, 2.7 Hz, 2H), 3.84
(dtd, J = 6.0, 4.4, 1.7 Hz, 5H), 2.94–2.85 (m, 1H), 2.83–2.68 (m, 2H), 2.16 (s, 1H), 2.19–2.11
(m, 1H), 1.52 (d, J = 1.5 Hz, 3H). 13C NMR (126 MHz, Chloroform-d)
δ 172.76, 170.81,
168.66, 168.05, 166.83, 153.85, 152.88, 152.00, 141.61, 137.91, 136.37, 134.81, 131.33, 129.50,
123.85, 119.45, 118.80, 117.74, 116.10, 115.95, 115.82, 115.76, 115.65, 114.87, 98.99, 73.45, 73.22,
69.74, 69.30, 68.17, 65.01, 49.21, 31.38, 22.98, 22.70; HR-MS (EI) calculated for (M+) 825.1143;
found 825.1143.
2-(2-(2-(4-(3-((4-Cyano-3-iodophenyl)amino)-2-hydroxy-2-methyl-3-
oxopropoxy)phenoxy)ethoxy)ethoxy)ethyl
(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)carbamate (11c)
Compound 11c was prepared using a similar method for the synthesis of 11a (27%).
1H NMR (500 MHz, Chloroform-d), δ 8.97 (s, 1H), 8.84 (d, J = 5.6 Hz, 1H), 8.53 (d, J = 8.7 Hz,
1H), 8.28 (d, J = 1.8 Hz, 1H), 8.16–8.07 (m, 1H), 7.73–7.64 (m, 2H), 7.54 (dd, J = 8.5, 2.4 Hz,
1H), 7.47 (d, J = 7.4 Hz, 1H), 6.79 (q, J = 9.6 Hz, 4H), 5.29 (s, 1H), 4.85 (ddd, J = 12.5, 5.3,
2.4 Hz, 1H), 4.40–4.31 (m, 3H), 4.07 (dd, J = 5.8, 4.1 Hz, 2H), 3.91–3.82 (m, 3H), 3.78–3.68
(m, 5H), 3.69 (d, J = 3.5 Hz, 1H), 3.65 (d, J = 3.5 Hz, 1H), 2.91–2.84 (m, 1H), 2.82–2.64 (m, 2H),
2.14–2.07 (m, 1H), 1.60 (s, 3H). 13C NMR (126 MHz, Chloroform-d),
δ 172.99, 170.89, 168.73,
168.08, 166.79, 153.96, 152.97, 151.94, 141.61, 137.97, 136.45, 134.85, 131.37, 129.52, 123.83,
119.48, 118.82, 117.86, 115.91, 115.73, 99.02, 75.87, 73.26, 70.88, 70.80, 69.96, 69.15, 68.08,
65.05, 49.24, 31.04, 23.02, 22.65; HR-MS (EI) calculated for (M+) 869.1405; found 869.1403.
5-(4-(3-((4-Cyano-3-iodophenyl)amino)-2-hydroxy-2-methyl-3-
oxopropoxy)phenoxy)pentyl
(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)carbamate (11d)
Compound 11d was prepared using a similar method for the synthesis of 11a (17%).
1H NMR (500 MHz, Chloroform-d), δ 8.95 (d, J = 4.1 Hz, 1H), 8.80 (d, J = 2.7 Hz, 1H), 8.54
(d, J = 8.5 Hz, 1H), 8.27 (dd, J = 2.1, 1.1 Hz, 1H), 8.13 (d, J = 5.9 Hz, 1H), 7.74–7.66 (m, 2H),
7.54 (d, J = 8.5 Hz, 1H), 7.49 (d, J = 7.2 Hz, 1H), 6.80 (m, 4H), 4.93 (dd, J = 12.2, 5.4 Hz,
1H), 4.36 (dd, J = 9.0, 4.0 Hz, 1H), 4.23 (tt, J = 6.6, 3.4 Hz, 1H), 3.94–3.86 (m, 3H), 3.53 (d,
J = 6.6 Hz, 1H), 2.96–2.86 (m, 1H), 2.85–2.69 (m, 2H), 2.19–2.11 (m, 1H), 1.84–1.71 (m, 4H),
1.64–1.56 (m, 2H), 1.57 (m, 3H). 13C NMR (126 MHz, Chloroform-d),
δ 172.76, 170.73, 168.89,
167.91, 166.77, 154.20, 153.14, 151.76, 141.55, 138.15, 136.44, 134.82, 131.34, 129.49, 123.82,
119.43, 118.78, 117.76, 116.05, 116.02, 115.69, 115.57, 115.55, 114.83, 98.99, 75.80, 73.26, 68.17,
65.89, 49.25, 31.40, 28.87, 28.47, 22.99, 22.72, 22.47; HR-MS (EI) calculated for (M+) 823.1350;
found 823.1347.

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2-(4-(3-((4-Cyano-3-iodophenyl)amino)-2-hydroxy-2-methyl-3-
oxopropoxy)phenoxy)ethyl methanesulfonate (12a)
Mathanesulfonyl chloride (18.7 µL, 0.242 mmol) dropwise added to compound 8a
(77.8 mg, 0.161 mmol) and anhydrous CH2Cl2 (3 mL) in an ice bath. After 1 h, the solvent
was evaporated in vacuo and the crude mixture was purified with flash column chro-
1
matography to afford compound 12a as a white solid (59.6 mg, 66%). H NMR (400 MHz,
Chloroform-d), δ 8.95 (s, 1H), 8.29 (s, 1H), 7.70 (d, J = 8.0 Hz, 1H), 7.567 (t, J = 6.8 Hz, 1H),
6.85 (m, 4H), 4.55 (t, J = 4.2 Hz, 2H), 4.19 (t, J = 4.0 Hz, 2H), 3.45 (brs, 1H), 3.23–3.21 (m, 1H),
3.11–3.08 (m, 5H), 1.56 (s, 3H).
2-(2-(4-(3-((4-Cyano-3-iodophenyl)amino)-2-hydroxy-2-methyl-3-
oxopropoxy)phenoxy)ethoxy)ethyl methanesulfonate (12b)
Compound 12b was prepared using a similar method for the synthesis of 12a (81%).
1H NMR (400 MHz, DMSO-d6),
δ 10.28 (s, 1H), 8.39 (d, J = 1.9 Hz, 1H), 7.92 (dd, J = 8.6,
2.0 Hz, 1H), 7.82 (d, J = 8.6 Hz, 1H), 6.96–6.83 (m, 4H), 4.45–4.36 (m, 2H), 4.35–4.29 (m, 2H),
4.06–4.00 (m, 2H), 3.79–3.63 (m, 5H), 3.18 (s, 3H), 1.86 (s, 3H).
2-(2-(2-(4-(3-((4-Cyano-3-iodophenyl)amino)-2-hydroxy-2-methyl-3-
oxopropoxy)phenoxy)ethoxy)ethoxy)ethyl methanesulfonate (12c)
Compound 12c was prepared using a similar method for the synthesis of 12a (74%).
1H NMR (400 MHz, DMSO-d6), δ 10.31 (s, 1H), 8.41 (s, 1H), 7.93 (dd, J = 8.7, 2.0 Hz, 1H),
7.84 (d, J = 8.6 Hz, 1H), 6.96–6.86 (m, 4H), 4.45–4.38 (m, 2H), 4.35–4. 30 (m, 2H), 4.07–4.01
(m, 3H), 3.75–3.71 (m, 2H), 3.71–3.66 (m, 2H), 3.60 (s, 4H), 3.19 (s, 3H), 1.87 (s, 3H).
5-(4-(3-((4-Cyano-3-iodophenyl)amino)-2-hydroxy-2-methyl-3-
oxopropoxy)phenoxy)pentyl methanesulfonate (12d)
Compound 12d was prepared using a similar method for the synthesis of 12a (67%).
1H NMR (400 MHz, DMSO-d6),
δ 10.30 (s, 1H), 8.41 (d, J = 1.9 Hz, 1H), 7.93 (dd, J = 8.6,
1.9 Hz, 1H), 7.84 (d, J = 8.6 Hz, 1H), 6.94–6.84 (m, 4H), 4.45–4.37 (m, 2H), 4.23 (d, J = 12.8 Hz
,
2H), 4.05 (q, J = 7.1 Hz, 1H), 3.91 (t, J = 6.4 Hz, 2H), 1.87 (s, 3H), 1.75–1.71 (m, 2H), 1.55–1.45
(m, 2H).
N-(4-Cyano-3-iodophenyl)-3-(4-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-
yl)oxy)ethoxy)phenoxy)-2-hydroxy-2-methylpropanamide (13a)
Compound 10 (9.9 mg, 0.036 mmol), potassium carbonate (12.4 mg, 0.068 mmol), and
◦
anhydrous DMF (0.8 mL) were added to a flask at 90 C. After 30 min, compound 12a
(25.4 mg, 0.453 mmol) was added to the mixture and stirred for 3 h. The mixture was
diluted with H2O, followed by extraction with EtOAc and distilled water. The organic
layer was dried with MgSO4, and the filtrate was concentrated under reduced pressure.
The crude mixture was purified with flash column chromatography to afford compound
13a as a white solid (9.0 mg, 27%). 1H NMR (500 MHz, Chloroform-d),
δ 8.96 (d, J = 2.3 Hz,
1H), 8.27 (dd, J = 2.8, 2.1 Hz, 1H), 8.22–8.14 (m, 1H), 7.72–7.62 (m, 2H), 7.53 (dd, J = 8.5,
2.2 Hz, 1H), 7.48 (dt, J = 7.3, 0.7 Hz, 1H), 7.30 (ddd, J = 8.6, 3.9, 0.7 Hz, 1H), 6.92–6.85 (m,
2H), 6.88–6.76 (m, 2H), 5.29 (s, 1H), 4.97–4.89 (m, 1H), 4.52 (td, J = 4.5, 2.2 Hz, 2H), 4.41–4.31
(m, 2H), 3.94–3.84 (m, 1H), 3.54 (d, J = 9.3 Hz, 1H), 2.91–2.84 (m, 1H), 2.84–2.66 (m, 2H),
2.60 (s, 1H), 1.53 (d, J = 1.6 Hz, 3H). 13C NMR (126 MHz, Chloroform-d),
δ 172.79, 170.95,
168.19, 166.94, 165.60, 156.22, 153.56, 152.34, 136.58, 134.81, 133.85, 129.50, 119.66, 118.81,
117.54, 116.53, 116.17, 116.13, 116.09, 116.03, 98.98, 75.81, 73.19, 68.52, 67.25, 49.14, 31.40,
23.00, 22.64; HR-MS (EI) calculated for (M+) 738.0823; found 738.0830.

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N-(4-Cyano-3-iodophenyl)-3-(4-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-
yl)oxy)ethoxy)ethoxy)phenoxy)-2-hydroxy-2-methylpropanamide (13b)
Compound 13b was prepared using a similar method for the synthesis of 13a (33%).
1H NMR (500 MHz, Chloroform-d), δ 8.98 (d, J = 6.4 Hz, 1H), 8.28 (dt, J = 4.3, 2.2 Hz, 1H),
8.15 (s, 1H), 7.73–7.59 (m, 2H), 7.54 (dd, J = 8.5, 0.9 Hz, 1H), 7.48–7.39 (m, 1H), 7.25–7.22 (m,
1H), 6.88–6.74 (m, 4H), 4.91 (dd, J = 12.2, 5.5 Hz, 1H), 4.42–4.30 (m, 2H), 4.20–4.02 (m, 4H),
4.02–3.93 (m, 2H), 3.89 (s, 1H), 3.71–3.57 (m, 1H), 2.90–2.83 (m, 1H), 2.82–2.65 (m, 2H),
2.15–2.06 (m, 1H)1.53 (s, 3H). 13C NMR (126 MHz, Chloroform-d),
δ 172.82, 171.09, 168.12,
167.07, 165.54, 156.39, 153.85, 151.98, 136.55, 134.82, 133.74, 129.50, 119.37, 118.81, 117.28,
116.22, 115.98, 115.93, 98.98, 75.83, 73.36, 70.38, 69.56, 69.45, 68.30, 49.10, 29.75, 22.99, 22.61;
HR-MS (EI) calculated for (M+) 782.1085; found 782.1087.
N-(4-Cyano-3-iodophenyl)-3-(4-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-
4-yl)oxy)ethoxy)ethoxy)ethoxy)phenoxy)-2-hydroxy-2-methylpropanamide (13c)
Compound 13c was prepared using a similar method for the synthesis of 13a (29%).
1H NMR (500 MHz, Chloroform-d), δ 8.99 (d, J = 2.1 Hz, 1H), 8.28 (t, J = 2.1 Hz, 1H), 8.11 (d,
J = 15.2 Hz, 1H), 7.69 (ddd, J = 8.6, 2.1, 0.8 Hz, 1H), 7.63 (dd, J = 8.5, 7.3 Hz, 1H), 7.53 (dd,
J = 8.5, 1.2 Hz, 1H), 7.42 (dt, J = 7.4, 0.8 Hz, 1H), 7.26–7.21 (m, 2H), 6.84–6.74 (m, 4H), 4.86
(dd, J = 12.4, 5.3 Hz, 1H), 4.35 (t, J = 9.0 Hz, 1H), 4.32 (ddd, J = 6.8, 3.5, 1.8 Hz, 2H), 4.05
(dd, J = 5.7, 4.0 Hz, 2H), 3.94 (ddd, J = 6.1, 3.7, 1.8 Hz, 2H), 3.88 (dd, J = 9.1, 2.6 Hz, 1H),
3.85–3.74 (m, 4H), 3.73 (dd, J = 5.8, 3.2 Hz, 2H), 3.68 (s, 1H), 2.90–2.61 (m, 3H), 2.11–2.00
(m, 1H), 1.56 (s, 3H). 13C NMR (126 MHz, Chloroform-d),
δ 172.83, 171.10, 168.13, 167.06,
165.57, 156.46, 153.93, 151.97, 136.55, 134.81, 133.73, 129.50, 119.46, 119.41, 118.81, 117.25,
116.17, 115.90, 115.75, 98.97, 75.83, 73.25, 71.19, 70.85, 69.79, 69.36, 69.23, 68.10, 49.07, 29.74,
22.98, 22.58; HR-MS (EI) calculated for (M+) 826.1347; found 826.1346.
N-(4-Cyano-3-iodophenyl)-3-(4-((5-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-
yl)oxy)pentyl)oxy)phenoxy)-2-hydroxy-2-methylpropanamide (13d)
Compound 13d was prepared using a similar method for the synthesis of 13a (31%).
1H NMR (500 MHz, Chloroform-d), δ 8.23 (d, J = 2.1 Hz, 1H), 7.98 (s, 1H), 7.71–7.62 (m, 2H),
7.61–7.50 (m, 1H), 7.44 (dd, J = 7.3, 2.0 Hz, 2H), 7.19 (d, J = 8.6 Hz, 2H), 6.82–6.68 (m, 4H),
4.93 (dd, J = 12.0, 5.2 Hz, 1H), 4.22–4.14 (m, 2H), 4.11 (q, J = 7.1 Hz, 1H), 3.99 (d, J = 9.8 Hz,
1H), 3.91 (q, J = 6.2 Hz, 2H), 2.81–2.39 (m, 4H), 2.03 (d, J = 1.9 Hz, 2H), 1.97–1.89 (m, 2H),
1.86–1.80 (m, 2H), 1.69 (s, 3H). 13C NMR (126 MHz, Chloroform-d),
δ 172.15, 170.84, 168.01,
167.09, 165.41, 156.66, 153.89, 152.14, 136.59, 134.29, 133.84, 132.06, 121.12, 118.92, 118.31,
117.15, 115.93, 115.41, 101.70, 81.53, 74.04, 69.25, 68.24, 49.11, 29.75, 28.96, 28.68, 22.66, 22.56,
19.40; HR-MS (EI) calculated for (M+) 780.1292; found 780.1298.
3.2. Biology
3.2.1. Cell Culture and Treatment
Human embryonic kidney 293T (HEK 293T) cells and 22Rv1 cells were maintained
in Dulbecco’s modified Eagle medium (DMEM, Hyclone
with 10% fetal bovine serum (FBS, Hyclone , SH30084.03), 100 units/mL penicillin, and
100 g/mL streptomycin (Hyclone , SV30010). LNCaP cells and DU-145 cells were
maintained in Roswell Park Memorial Institute 1640 medium (RPMI1640, Hyclone
SH30027.01) supplemented with 10% fetal bovine serum (FBS, Hyclone , SH30084.03),
, SV30010). All the
™, SH30243.01) supplemented
™
µ
™
™,
™
100 units/mL penicillin, and 100 µg/mL streptomycin (Hyclone™
cells were cultured in a humidified atmosphere of 5% CO2 and 95% air at 37 ◦C. All the
◦
compounds were stored in DMSO at -20 C with a concentration of 10 mM. When treating
cells, a compound was diluted in DMEM and RPMI1640 according to the concentration to
use for treatment. After removing the cell medium, the compound was replaced with the
diluted medium and cultured according to the desired incubation time.

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3.2.2. Transfection
Calcium-phosphate transfection (calcium chloride, 2
methods were performed for the overexpression of AR in HEK293T cells. Plasmid DNA
in distilled water was mixed with 2M calcium chloride. The mixture was mixed using a
×
HEPES Buffered Saline (HBS))
vortex mixer and mixed with 2
×
HBS. The final mixture was incubated at RT for 10 min
and then carefully treated on the prepared HEK293T cells. HEK293T cells were incubated
for 12 h in an CO2 incubator to stabilize and overexpression was sufficiently achieved,
then used for experiments. AR plasmid (pIRESneoFLAGhAR Cat.89116) was purchased
from Addgene.
3.2.3. Immunoblotting
After the treatment was completed according to the experimental conditions, the cells
were lightly chilled with cold PBS, separated from the cell culture plate, and centrifuged
at 1200 rpm for 3 min. The pellet was sampled with 2
×
sample buffer (1M TrisHCl pH
6.8, 50% glycerol, 10% SDS, 2-mercaptoethanol, 1% bromophenol blue) and then boiled
◦
at 100 C for 10 min. After that, electrophoresis was performed with sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE) suitable for the kDa of the protein
to be detected, then transferred to a PVDF membrane activated with EtOH. Blocking was
performed for a specific binding reaction between◦the protein of the PVDF membrane
and the antibody. Blocking was performed at 4 C overnight in a 5% bovine serum
albumin (BSA, RMbio, Missoula, MT, USA) solution. The BSA solution was discarded,
and the membrane was washed with PBST (PBS, 0.5% Tween-20). The specific binding
◦
reaction between the membrane protein and the antibody was performed at 4 C overnight
and washed with PBST. Finally, horse radish peroxidase (HRP)-conjugated secondary
antibodies (Cell Signaling Technology, Danvers, MA, USA) were diluted in PBST and
reacted for two hours at room temperature (RT). Photographing was performed using an
HRP substrate (Luminata Forte (Millipore, Burlington, MA, USA)). For immunoblotting,
anti-Flag rabbit antibody (Cell Signaling Technology, 14793), anti-AR rabbit antibody (Cell
signaling Technology, 5153), anti-GST antibody (Abcam, Cambridge, UK), and anti-actin
mouse antibody (Santacruz, D0419, Dallas, TX, USA) were used. The ImageJ program was
used for intensity comparison and the analysis of all the proteins.
3.2.4. RT-PCR
The mRNA levels of AR, PSA, and TMPRSS2 were analyzed by qRT-PCR. Cellular
total RNA was isolated using an RNA isolation and preparation kit (GeneAll, Lisbon,
Portugal, Hybrid-R 100 prep 305–101) according to the instructions provided by the man-
ufacturers. The concentration and purity of the isolated total RNA were measured with
a spectrophotometer. A total of 1000 ng of total RNA was synthesized to cDNA. Synthe-
sis was performed as indicated in the protocol of the cDNA synthesis kit (Enzynomix,
TOPsript RT DryMIX (dT 18 plus) RT200). Synthesized cDNA was subjected to qRT-PCR
with SYBR Green Mix using the CFX connect real-time PCR system. The mRNA level was
analyzed using AR (androgen receptor) primer, PSA (prostate-specific antigen) primer, and
TMPRSS2 (transmembrane protease serine subtype2) primer. All values were normalized
to the β-actin mRNA level as a control. The primer sequence of Table 1. was used as the
primer used in all RT-PCR.
3.2.5. Transferase-Mediated Deoxyuridine Triphosphate (dUTP)-Digoxigenin Nick End
Labeling (TUNEL) Assay
The apoptotic effects of 13b in the DU-145 and LNCaP cell lines were determined using
the transferase-mediated deoxyuridine triphosphate (dUTP)-digoxigenin nick end labeling
assay. Briefly, DU-145 and LNCaP cells were seeded on poly-L-lysine pre-coated 24-well
cover glasses at 1
×
105 cells/well. Following treatment with vehicle or 13b, the cells
were fixed and subjected to TUNEL staining using a commercial kit (In Situ Direct DNA
Fragmentation (TUNEL) Assay Kit; abcam66108) in accordance with the manufacturer’s

Molecules 2021, 26, 2525
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instructions. Apoptotic cells were detected as localized bright red cells (positive cells) in
DIC images using a Leica DM IL microscope.
Table 1. Primer sequences for RT-PCR.
Gene
Forward Primer Sequence
Reverse Primer Sequence
AR
PSA
TMPRSS2
β-actin
50-CAGTGGATGGGCTGAAAAAT-30
50-ACGCTGGACAGGGGGCAAAAG-30
50-CAGGAGTGTACGGGAATGTGATGGT-30
50-AGTTGCGTTACACCCTTTCTTG-30
50-GGAGCTTGGTGAGCTGGTAG-30
50-GGGCAGGGCACATGGTTCACT-30
50-GATTAGCCGTCTGCCCTCATTTGT-30
50-GCTGTCACCTTCACCGTTCC-30
3.3. Molecular Modeling
Docking Studies
The in silico docking of 5a with the 3D coordinates of the crystal structure of the
androgen receptor ligand-binding domain W741L mutant complex with (R)-bicalutamide
(AR, PDB code: 1Z95) was accomplished using the AutoDock 4.2 program downloaded
from the Molecular Graphics Laboratory of the Scripps Research Institute. The AutoDock
program was chosen because it uses a genetic algorithm to generate the poses of the
ligand inside a known or predicted binding site, utilizing the Lamarckian version of the
genetic algorithm where the changes in conformations adopted by molecules after in situ
optimization are used as subsequent poses for the offspring. In the docking experiments
carried out, water was removed from the 3D X-ray coordinates while Gasteiger charges
were placed on the X-ray structures of the active site of AR along with 5a using tools from
the AutoDock suite. A grid box centered on the AR binding domain with definitions of
11.466, 18.150, and 32.019 points and a 0.37 Å spacing was chosen for the ligand docking
experiments. The docking parameters consisted of setting the population size to 150, the
number of generations to 27,000, and the number of evaluations to 25,000,000, while the
number of docking runs was set to 50 with a cutoff of 1 Å for the root mean square tolerance
for the grouping of each docking run. The docking model of androgen receptor ligand-
binding domain W741L mutant complex with compound 5a is depicted in Figure 5; the
rendering of the picture was generated using Chimera 1.10 (UCSF Chimera, San Francisco,
CA, USA).
4. Conclusions
In conclusion, we developed a series of novel PROTACs to induce the targeted degra-
dation of AR as the potential therapeutics for the treatment of CRPC. We designed, syn-
thesized, and biologically evaluated novel AR antagonists based on the structure of bi-
calutamide, which has been used clinically as an anti-androgen agent (Scheme 1). By
using a novel bicalutamide analog 5a identified as a potent AR antagonist in the cellular
experiments, we prepared novel PROTACs through the conjugation of 5a, an AR-targeting
warhead, with thalidomide through a biologically compatible ethylene glycol linkage.
Several novel PROTACs that we synthesized were investigated with regard to their abilities
to induce targeted AR degradation. In particular, PROTAC 13b was shown to significantly
induce targeted AR degradation in a dose- and time-dependent manner. Further biological
studies, such as antiproliferative activity experiments and in vivo xenograft experiments
using PROTAC compounds such as 13b, remain to be conducted.
Supplementary Materials: The following are available online, Figure S1. Molecular docking model
of 5a (pink) with projection of 7a (sky blue) in androgen receptor (PDB Code: 1Z95), which was
visualized using Chimera 1.10 (UC13bSF Chimera).; Scheme S1. The synthesis of Pomalidomide
and Derivatives. References from supporting material [25–28].
Author Contributions: Conceptualization, N.-J.K. and K.-S.I.; methodology, G.Y.K., C.W.S., Y.-S.Y.;
investigation, N.-R.L., S.J.L., and J.S.P.; writing—original draft preparation, N.-J.K. and K.-S.I.;
writing—review and editing, H.-S.Y., S.H.S., and N.-J.K.; supervision, N.-J.K., K.-S.I., and J.K.L.;

Molecules 2021, 26, 2525
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project administration, J.K.L.; funding acquisition, N.-J.K. All authors have read and agreed to the
published version of the manuscript.
Funding: This research was supported by the Basic Science Research Program through the National
Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2020R1F1A1064755).
This research was also supported by the Basic Research Laboratory Program (BRL) of the Korean
National Research Foundation funded by the Korean Ministry of Science, ICT and Future Planning
(NRF-2020R1A4A1016142).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is contained within the article.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Winter, G.E.; Buckley, D.L.; Paulk, J.; Roberts, J.M.; Souza, A.; Dhe-Paganon, S.; Bradner, J.E. DRUG DEVELOPMENT. Phthalimide
conjugation as a strategy for in vivo target protein degradation. Science 2015, 348, 1376–1381. [CrossRef] [PubMed]
Sakamoto, K.M.; Kim, K.B.; Kumagai, A.; Mercurio, F.; Crews, C.M.; Deshaies, R.J. Protacs: Chimeric molecules that target
proteins to the Skp1-Cullin-F box complex for ubiquitination and degradation. Proc. Natl. Acad. Sci. USA 2001, 98, 8554–8559.
[CrossRef]
2.
3.
4.
Burslem, G.M.; Crews, C.M. Small-Molecule Modulation of Protein Homeostasis. Chem. Rev. 2017, 117, 11269–11301. [CrossRef]
Salami, J.; Alabi, S.; Willard, R.R.; Vitale, N.J.; Wang, J.; Dong, H.; Jin, M.; McDonnell, D.P.; Crew, A.P.; Neklesa, T.K.; et al.
Androgen receptor degradation by the proteolysis-targeting chimera ARCC-4 outperforms enzalutamide in cellular models of
prostate cancer drug resistance. Commun. Biol. 2018, 1, 100. [CrossRef]
5.
Han, X.; Wang, C.; Qin, C.; Xiang, W.; Fernandez-Salas, E.; Yang, C.Y.; Wang, M.; Zhao, L.; Xu, T.; Chinnaswamy, K.; et al.
Discovery of ARD-69 as a Highly Potent Proteolysis Targeting Chimera (PROTAC) Degrader of Androgen Receptor (AR) for the
Treatment of Prostate Cancer. J. Med. Chem. 2019, 62, 941–964. [CrossRef] [PubMed]
6.
7.
Rodriguez-Gonzalez, A.; Cyrus, K.; Salcius, M.; Kim, K.; Crews, C.M.; Deshaies, R.J.; Sakamoto, K.M. Targeting steroid hormone
receptors for ubiquitination and degradation in breast and prostate cancer. Oncogene 2008, 27, 7201–7211. [CrossRef]
Helsen, C.; van den Broeck, T.; Voet, A.; Prekovic, S.; van Poppel, H.; Joniau, S.; Claessens, F. Androgen receptor antagonists for
prostate cancer therapy. Endocr. Relat. Cancer 2014, 21, T105–T118. [CrossRef] [PubMed]
8.
9.
Heinlein, C.A.; Chang, C. Androgen receptor in prostate cancer. Endocr. Rev. 2004, 25, 276–308. [CrossRef]
Taplin, M.E.; Bubley, G.J.; Shuster, T.D.; Frantz, M.E.; Spooner, A.E.; Ogata, G.K.; Keer, H.N.; Balk, S.P. Mutation of the
androgen-receptor gene in metastatic androgen-independent prostate cancer. N. Engl. J. Med. 1995, 332, 1393–1398. [CrossRef]
[PubMed]
10. Masiello, D.; Cheng, S.; Bubley, G.J.; Lu, M.L.; Balk, S.P. Bicalutamide functions as an androgen receptor antagonist by assembly
of a transcriptionally inactive receptor. J. Biol. Chem. 2002, 277, 26321–26326. [CrossRef]
11. Chandrasekar, T.; Yang, J.C.; Gao, A.C.; Evans, C.P. Mechanisms of resistance in castration-resistant prostate cancer (CRPC).
Transl. Androl. Urol. 2015, 4, 365–380.
12. Neklesa, T.; Snyder, L.B.; Willard, R.R.; Vitale, N.; Raina, K.; Pizzano, J.; Gordon, D.; Bookbinder, M.; Macaluso, J.; Dong, H.; et al.
Abstract 5236: ARV-110: An androgen receptor PROTAC degrader for prostate cancer. Cancer Res. 2018, 78 (Suppl. 13), 5236.
13. Haven, N. Meeting. A Proof-of-Concept with PROTACs in Prostate Cancer. Cancer Discov. 2020, 10, 1084.
14. Gooren, L.J.G.; Bunck, M.C.M. Androgen Replacement Therapy. Drugs 2004, 64, 1861–1891. [CrossRef]
15. Furr, B.J.A.; Tucker, H. The preclinical development of bicalutamide: Pharmacodynamics and mechanism of action. Urology 1996
,
47 (Suppl. 1), 13–25. [CrossRef]
16. Bohl, C.E.; Gao, W.; Miller, D.D.; Bell, C.E.; Dalton, J.T. Structural basis for antagonism and resistance of bicalutamide in prostate
cancer. Proc. Natl. Acad. Sci. USA 2005, 102, 6201–6206. [CrossRef]
17. van Dort, M.E.; Robins, D.M.; Wayburn, B. Design, Synthesis, and Pharmacological Characterization of 4-[4,4-Dimethyl-3-(4-
hydroxybutyl)-5-oxo-2-thioxo-1-imidazolidinyl]- 2-iodobenzonitrile as a High-Affinity Nonsteroidal Androgen Receptor Ligand.
J. Med. Chem. 2000, 43, 3344–3347. [CrossRef] [PubMed]
18. Bassetto, M.; Ferla, S.; Pertusati, F.; Kandil, S.; Westwell, A.D.; Brancale, A.; McGuigan, C. Design and synthesis of novel
bicalutamide and enzalutamide derivatives as antiproliferative agents for the treatment of prostate cancer. Eur. J. Med. Chem.
2016, 118, 230–243. [CrossRef] [PubMed]
19. Habibi, D.; Heydari, S.; Nasrollahzadeh, M. Synthesis of Aryl Nitriles using the Stable Aryl Diazonium Silica Sulfates. J. Chem.
Res. 2012, 36, 573–574. [CrossRef]
20. Parent, E.E.; Dence, C.S.; Jenks, C.; Sharp, T.L.; Welch, M.J.; Katzenellenbogen, J.A. Synthesis and Biological Evaluation of
[18F]Bicalutamide, 4-[76Br]Bromobicalutamide, and 4-[76Br]Bromo-thiobicalutamide as Non-Steroidal Androgens for Prostate
Cancer Imaging. J. Med. Chem. 2007, 50, 1028–1040. [CrossRef]

Molecules 2021, 26, 2525
17 of 17
21. Wood, P.M.; Woo, L.W.L.; Labrosse, J.-R.; Trusselle, M.N.; Abbate, S.; Longhi, G.; Castiglioni, E.; Lebon, F.; Purohit, A.; Reed,
M.J.; et al. Chiral Aromatase and Dual Aromatase Steroid Sulfatase Inhibitors from the Letrozole Template: Synthesis, Absolute
−
Configuration, and In Vitro Activity. J. Med. Chem. 2008, 51, 4226–4238. [CrossRef] [PubMed]
22. Romeiro, L.A.S.; Ferreira, M.d.; da Silva, L.L.; Castro, H.C.; Miranda, A.L.P.; Silva, C.L.M.; Noël, F.; Nascimento, J.B.; Araújo,
C.V.; Tibiriçá, E.; et al. Discovery of LASSBio-772, a 1,3-benzodioxole N-phenylpiperazine derivative with potent alpha 1A/D-
Adrenergic receptor blocking properties. Eur. J. Med. Chem. 2011, 46, 3000–3012. [CrossRef] [PubMed]
23. Sramkoski, R.M.; Pretlow, T.G.; Giaconia, J.M.; Pretlow, T.P.; Schwartz, S.; Sy, M.; Marengo, S.R.; Rhim, J.S.; Zhang, D.; Jacobberger,
J.W. A new human prostate carcinoma cell line, 22R v1. In Vitro Cell. Dev. Biol. Anim. 1999, 35, 403–409. [CrossRef] [PubMed]
24. Sharp, A.; Coleman, I.; Yuan, W.; Sprenger, C.; Dolling, D.; Rodrigues, D.N.; Russo, J.W.; Figueiredo, I.; Bertan, C.; Seed, G.
Androgen receptor splice variant-7 expression emerges with castration resistance in prostate cancer. J. Clin. Invest. 2019, 129,
192–208. [CrossRef]
25. Orrego, F.; Villanueva, S. The chemical nature of the main central excitatory transmitter: A critical appraisal based upon release
studies and synaptic vesicle localization. Neuroscience 1993, 56, 539–555.
26. Capitosti, S.M.; Hansen, T.P.; Brown, M.L. Facile synthesis of an azido-labeled thalidomide analogue. Org. Lett. 2003, 5, 2865–2867.
27. Ruchelman, A.L.; Man, H.; Zhang, W.; Chen, R.; Capone, L.; Kang, J.; Parton, A.; Corral, L.; Schafer, P.H.; Babusis, D. Isosteric
analogs of lenalidomide and pomalidomide: Synthesis and biological activity. Bioorg. Med. Chem. Lett. 2013, 23, 360–365.
28. Phillips, A.J.; Nasveschuk, C.G.; Henderson, J.A.; Liang, Y.; Fitzgerald, M.E.; He, M.; Michael, R.E. Heterocyclic degronimers for
target protein degradation. U.S. Patent No 10,646,575, 12 May 2020.