Designed, synthesized and biological evaluation of proteolysis targeting chimeras (PROTACs) as AR degraders for prostate cancer treatment

Article abstract of DOI:10.1016/j.bmc.2021.116331

As a continuation of our research on developing potent and potentially safe androgen receptor (AR) degrader, a series of novel proteolysis targeting chimeras (PROTACs) containing the phthalimide degrons with different linker were designed, synthesized and evaluated for their AR degradation activity against LNCaP (AR+) cell line. Most of the synthesized compounds displayed moderate to satisfactory AR binding affinity and might lead to antagonist activity against AR. Among them, compound A16 exhibited the best AR binding affinity (85%) and degradation activity against AR. Due to the strong fluorescence properties of pomalidomide derivatives, B10 was found to be effectively internalized and visualized in LNCaP (AR + ) cells than PC-3 (AR-) cells. Moreover, the molecular docking of A16 with AR and the active site of DDB1-CRBN E3 ubiquitin ligase complex provides guidance to design new PROTAC degrons targeting AR for prostate cancer therapy. These results represent a step toward the development of novel and improved AR PROTACs.

Full text of DOI:10.1016/j.bmc.2021.116331

Bioorg. Med. Chem. 45 (2021) 116331
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Designed, synthesized and biological evaluation of proteolysis targeting
chimeras (PROTACs) as AR degraders for prostate cancer treatment
,
,
,
b
,
,
b
,
,b,c
Jian-Jia Liangd 1, Hang Xiee 1, Rui-Hua Yanga c, Ni Wanga c, Zi-Jun Zhenga
,
Chen Zhoua c, Ya-Lei Wanga c, Zhi-Jia Wanga c, Hong-Min Liua
*, Li-Hong Shana
,
,
b
,
,
b,
,
b
,
,
b,
c,
,b,c,*
Yu Kea
,b,c,*
a School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, Henan 450001, PR China
b Collaborative Innovation Center of New Drug Research and Safety Evaluation, Henan Province, Zhengzhou 450001, PR China
c Key Laboratory of Advanced Drug Preparation Technologies, Ministry of Education of China, PR China
d School of Pharmacy, Wuhan University, Wuhan, Hubei 430072, PR China
e School of Pharmacy, Fudan University, Shanghai 201203, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
As a continuation of our research on developing potent and potentially safe androgen receptor (AR) degrader, a
series of novel proteolysis targeting chimeras (PROTACs) containing the phthalimide degrons with different
linker were designed, synthesized and evaluated for their AR degradation activity against LNCaP (AR+) cell line.
Most of the synthesized compounds displayed moderate to satisfactory AR binding affinity and might lead to
antagonist activity against AR. Among them, compound A16 exhibited the best AR binding affinity (85%) and
degradation activity against AR. Due to the strong fluorescence properties of pomalidomide derivatives, B10 was
found to be effectively internalized and visualized in LNCaP (AR + ) cells than PC-3 (AR-) cells. Moreover, the
molecular docking of A16 with AR and the active site of DDB1-CRBN E3 ubiquitin ligase complex provides
guidance to design new PROTAC degrons targeting AR for prostate cancer therapy. These results represent a step
toward the development of novel and improved AR PROTACs.
Androgen receptor
PROTAC
Phthalimide
Degrons
Prostate cancer
1. Introduction
promotes their androgen dependent proliferation8. Consequently,
downregulation of the AR level is regarded as a promising strategy to
Prostate cancer (PCa) is the second most frequently diagnosed can-
cer, after lung cancer, in men worldwide1. The activation of the
androgen receptor (AR) signaling is crucial for PCa development and
androgen-independent growth2,3. As such, androgen deprivation ther-
apy (ADT), via either surgical or chemical castration, has been the
mainstay of treatment for advanced PCa for decades4,5. At present,
nonsteroidal AR antagonists such as nilutamide, flutamide, and bicalu-
tamide are the most commonly prescribed ADT drugs whose mechanism
of action consists of competitively inhibiting androgen-AR binding.
However eventually metastatic castration-resistant prostate cancer
(CRPC) occurs approximately 18–24 months after the start of therapy,
and no cure is currently available6,7. Although the mechanisms
responsible for the progression of PCa to CRPC are not well-known, it
has become clear that AR is often overexpressed in CRPC cells and
treat CRPC9,10
.
Inducing protein degradation by small molecules has recently
become a hot spot for drug discovery. Unlike mRNA degradation with
siRNA, this technology is best exemplified by Proteolysis Targeting
Chimeras (PROTACs), which exploit the unique characteristics of the
ubiquitin proteasome system in eukaryotic cells11. PROTAC is a heter-
obifunctional molecule composed of a ligand for the protein of interest, a
recognition motif for an E3 ubiquitin ligase and two different ligands
connected by a linker12–16. To date, several PROTAC molecules targeting
AR have been reported (Fig 1). In 2008, Crews et al developed the first
entirely small-molecule AR PROTAC employed the known MdM2 ligand
nutlin-3a as an E3 ligase recruiting moiety17. This complex induced AR
degradation was observed at a concentration of 10
μM in cells. Another
small-molecule based PROTAC approach for targeted AR degradation,
* Corresponding authors at: Zhengzhou University, Zhengzhou 450001, PR China.
E-mail addresses: liuhm@zzu.edu.cn (H.-M. Liu), shlh@zzu.edu.cn (L.-H. Shan), ky@zzu.edu.cn (Y. Ke).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.bmc.2021.116331
Received 29 June 2021; Received in revised form 20 July 2021; Accepted 21 July 2021
Available online 26 July 2021
0968-0896/© 2021 Elsevier Ltd. All rights reserved.

J.-J. Liang et al.
Bioorganic & Medicinal Chemistry 45 (2021) 116331
Fig. 1. Structures of representative AR degrader and E3 ligase (red box).
termed cellular inhibitor of apoptosis protein 1 (cIAP1), was reported in
2017 by the Naito group18. However, its maximally effective only in the
micromolar range and numerous off-target effects were observed, which
limit the further development of compound 3. In 2019, ARD-69 has been
recently reported as a new class of small-molecule AR PROTAC
degrader, which was designed using several different AR antagonists
and a von Hippel-Lindau (VHL) ligand19. ARD-69 was shown to be more
potent and effective than AR antagonists at inhibiting proliferation and
inducing apoptosis of CRPC.
1,2,3-triazole linker to a CRBN ligand28. Furthermore, we also investi-
gated the effects of length and diversity of linker moieties and other
functional group for optimization of these degraders. In addition, we
found that pomalidomide can be used as a fluorescent agent to assess the
cellular uptake and accumulation of PROTACs.
2. Result and discussion
2.1. Chemistry
Very recently, cereblon/cullin 4A neddylation degradation systems
have been used successfully in the design of PROTAC degraders for
different proteins11,20-26. The strategy has also been applied to AR
PROTAC, such as ARV-110 (Fig 1, 4), which is discovered by Arvinas,
and now is being used in the treatment of PCa in the second phase of
clinical. Thus, the phthalimide family is often employed as a part of
PROTACs to hijack CRBN to target proteins27. In this study, we designed
and synthesized a series of heterobifunctional compounds based on the
high-affinity AR agonist RU59063 derivatives connected through a
The target compounds were obtained through Huisgen 1,3-dipolar
cycloaddition of Ru59063 azide derivatives with different phthalimide
terminal alkyne moiety. Along with the promising biological activity of
1,2,3-triazole derivatives make them attractive targets in synthetic
organic chemistry29,30. Compounds 2a-c and 3 have been reported in
our early literature31,32. With potassium carbonate as the base, com-
pound 4 and 7 were obtained with a mild yield by reacting the methyl
amino acid ester, which were further reacted with 3-fluoro-4-
Scheme 1. Synthesis of AR ligands intermediates a. a Reagents and conditions: (a) K2CO3, DMF, 45 ◦C, 5 h; (b) Dibromo alkane, NaH, dry DMF, 0 ◦C, 2 h; (c) NaN3,
acetone, 80 ◦C, 6 h; (d) 1-azido-4-(bromomethyl) benzene, NaH, dry DMF, 0 ◦C, 1 h. (e, i) Bromo-1-butanol, 1-azido-4-(bromomethyl) benzene, K2CO3, CH3CN, 40
◦C, 2–6 h; (f) 3-fluoro-4-methylphenylisothiocyanate, iPOH, triethylamine, rt, 3 h;(g) TsCl, DABCO, CH2Cl2, 0 ◦C, 1 h;(h) NaN3, acetone, 80 ◦C, 5 h.
2

J.-J. Liang et al.
Bioorganic & Medicinal Chemistry 45 (2021) 116331
Scheme 2. Synthesis of CRBN ligands intermediates a. a Reagents and conditions: (a) CH3COOH, CH3COONa, 110 ◦C. (b) NaHCO3, DMF, 80 ◦C, 5–8 h. (c) 1. CH3OH,
rf, 3 h. 2. NaHCO3, CH3I, DMF, 60 ◦C, 3 h, 90%. (d) Pd/C, H2, Actone, 95%. (e) CH3COOH, DCM, Sodium triacetoxyborohydride. (f) NaOH, THF, MeOH, r.f. (g) 2,3-
amino piperidine-2, 6-dione, pyridine, 110 ◦C, 15 h, 55%.
methylphenylisothiocyanate to afford 5 and 8 with good yield. More-
over, the compound 5 was treated with p-TsCl and NaN3 to yield 6
(Scheme 1A and B).
Overall, it was determined that all compounds possessed potent binding
affinity and might display antagonist activity against AR.
Entry
Cmpd.
affinity %a (10
μ
M)
Entry
Cmpd.
affinity %a (10
μM)
Pathalimide intermediates 12a-h were synthesized as shown in
scheme 2. The commercially available 4-hydroxyphthalic anhydride
mixed with 3-amino-piperidine-2,6-dione catalyzed by sodium acetate
in acetic acid at 110 ◦C to provide 9, and then reacted with propargyl-
tosylates containing varying aliphatic alcohol or ethylene glycol units
afforded the requisite library of cereblon ligase ligands (12a-g). Besides,
a pomalidomide derivative (12h) containing diethylene glycol unit was
prepared, 4-nitrophthalic anhydride was treated with the methyl
alcohol to generate 10. Then, the intermediate 11 was obtained by the
reduction of 10 in 10% Pd/C. Finally, the key intermediate 12h was
gained through the reaction of 11 with 2-(prop-2-ynyloxy) acetaldehyde
in the presence of sodium triacetoxyborohydride and following treat-
ment with 3-amino-piperidine-2,6-dione.
1
A1
74.12
51.40
29.32
61.42
68.61
56.35
72.92
50.47
52.10
47.26
73.41
57.25
45.78
36.30
74.53
16
17
18
19
20
21
22
23
24
25
26
27
28
29
A16
A17
A18
B1
85.33
76.46
72.19
53.22
42.17
48.64
56.38
39.21
22.97
53.41
41.84
57.37
61.47
89.15
2
A2
3
A3
4
A4
5
A5
B2
6
A6
B3
7
A7
B4
8
A8
B5
9
A9
B6
10
11
12
13
14
15
A10
A11
A12
A13
A14
A15
B7
B8
B9
B10
Enza.
With both azide and alkyne components in hand, we then mixed
them to typical conditions for a click reaction (Scheme 3). This involved
CuSO4/sodium ascorbate as catalyst, and THF/H2O to aid in solubilizing
the copper catalyst. The reactions were typically very clean and often
went to completion within 6 h, and all of the compounds gave moderate
to good yields.
2.2.2. Confocal microscopy
Due to the strong fluorescence properties of Pomalidomide de-
rivatives (A5-6, B9-10). We employed LNCaP (AR+) and PC-3 (ARꢀ ) for
cell imaging to demonstrate the potential of PROTACs as targeted
theranostic in cells and the fluorescence response by laser scanning
confocal microscopy. As shown in Figure S1, PC-3 (ARꢀ ) cells treated
with B10 showed weak fluorescence. Instead, LNCaP (AR+) cells
exhibited strong fluorescence intensity upon incubation with B10 for 24
h, confirming the remarkable cellular uptake of B10 by LNCaP cells
(Figure 3). On the other hand, the cells showed faint fluorescence under
the same treatment with pomalidomide. As the stronger fluorescence
often means higher cellular uptake, we could deem that the AR targeted
ligand could significantly increase the uptake of B10 in AR overex-
pressed LNCaP cells and the complex could apply for cell imaging.
2.2. Biological evaluation
2.2.1. AR binding affinity
In order to evaluate the AR binding affinity of these new PROTAC
derivatives, the fluorescence polarization (FP) based binding assay by
competition with the fluorescent tracer was carried out, and the results
were shown in Figure 2. The affinities are presented as the relative
binding affinity (RBA) values, with the binding affinity of testosterone
being set at 100%.
In general, most of the synthesized PROTACs showed good RBA af-
finity values and compound A16 (85%) displayed the highest AR affinity
among the whole series. More interestingly, the binding affinity values
were closely related to the length of the R2 groups, too long or too short
chains reduced the binding affinity of the compounds (A1-18). Com-
pounds B1-10 (red bars, Figure 2) with phenyl group showed modest
activity except for B6 (22%) that with a lowest AR affinity. It is worth
noting that a sulfur atom on the W substituent dramatically improved
compound activity (A15-18). Moreover, a decreased efficacy was
observed when the propylene glycol moiety of derivative A11 was
extended to tetraethylene glycol (A14). Furthermore, changing the R2
alkyl substituent (A1-4) to PEG unit (A7-A14) led to a weak decrease of
the affinity, which indicated that substituents on the thalidomide ligand
moiety might have little impact on determining its binding affinity.
2.2.3. AR degradation assay
The AR degradation assay of the synthesized PROTACs has been
further explored. Considering the AR RBA of this series of compounds,
we used 20 μM of some high RBA compounds to test the AR level. The
Western blot results of new PROTACs have been presented in Figure S2.
Among them, compound A16 showed equal or better degradation ac-
tivity compared to other compounds. However, all selected compounds
have only a modest effect in reducing the level of AR protein, with the
AR degradations ranging from 6% to 32%, respectively (Fig S2), which
indicated that these hybrids were essentially inactive against AR
degradation. Lastly, we chose the best compound A16 of the series at
three different concentrations (10, 20, and 30
μM) for 24 h, untreated
cells were applied as the control. Figure 4A and B revealed that
3

J.-J. Liang et al.
Bioorganic & Medicinal Chemistry 45 (2021) 116331
R2 or
R2 or
Yield
(%)
Yield
(%)
Comp.
m
X
W
Comp
m
X
W
A1
A2
A3
A4
A5
A6
A7
A8
3
4
4
4
3
4
3
3
O
O
O
O
N
N
O
O
(CH2)2
(CH2)0
(CH2)1
(CH2)2
(CH2)1
(CH2)2
(CH2)3O
n=2
O
O
O
O
O
O
O
O
76%
60%
73%
75%
65%
58%
64%
59%
A10
A11
A12
A13
A14
A15
A16
A17
4
5
5
4
4
3
3
3
O
O
O
O
O
O
O
O
n=2
(CH2)3O
n=2
O
O
O
O
O
S
47%
59%
52%
71%
57%
68%
43%
54%
n=3
n=4
(CH2)3O
n=2
S
n=3
S
A9
4
O
(CH2)3O
O
74%
A18
3
O
n=4
S
43%
R2
R2
Yield
(%)
Yield
(%)
Comp.
X
W
Comp.
X
W
B1
B2
B3
B4
B5
O
O
O
O
O
(CH2)3O
O
O
O
O
S
85%
82%
67%
75%
89%
B6
B7
O
O
O
N
N
2
3
4
2
2
S
S
S
O
S
84%
85%
80%
58%
55%
2
3
4
B8
B9
(CH2)3O
B10
Scheme 3. Synthesis of PROTAC derivatives A1-18, B1-10 a. a Reagents and conditions: (a) CuSO4, Na-ascorbate, THF/H2O, rt, 2–10 h.
compound A16 could dose-dependently down-regulate the AR, and it
estimated the binding effect of compound A16 to AR (PDB: 2axa). As
shown in Figure 5A, possessing a similar binding mode to other
nonsteroidal AR ligands33, compound A16 could also fit into the binding
site by the formation of the key hydrogen bond between the cyano group
and Gln711, Arg752, the hydrogen bond lengths were 1.82 Å and 1.72 Å,
respectively. Moreover, the thalidomide carbanyl group could form a
1.53 Å hydrogen bond with Met742. By analyzing 2D binding mode
(Figure 5B), we could see that the polarity of the hybrid core structure is
significantly increased. In addition, thalidomide ring of A16 displayed
showed good degradation activity at 30 μM. Taken together, these data
suggest that more studies are needed to determine if the cereblon/cullin
4A E3 ligase system can be used for the design of highly potent and
effective AR degraders.
2.2.4. Molecule docking
To better illustrate the mechanism of compound A16 degradation at
the molecular level, we performed computer modeling using Molecular
Operating Environment (MOE version 2014) software. Firstly, we
π-π stacking effect with Trp741.
4

J.-J. Liang et al.
Bioorganic & Medicinal Chemistry 45 (2021) 116331
Fig 2. a Relative binding affinity (RBA) values are determined by competitive fluorometric binding assays. We set the RBA value of testosterone as 100%.
bond length were 2.38 Å and 2.09 Å, respectively. Moreover, piperidone
ring of thalidomide displayed π-H stacking effect with Trp382. From
these interaction patterns found in the computer modeling, we can
provide the basis for the discovery of new and simpler PROTACs.
Fig 3. (A) Structure of compound B10. (B) Confocal microscopy images of
LNCaP cells treated with 10 μM compound B10 (d ꢀ f). Fluorescence from DAPI
staining nucleus appears as blue signals (Ex: 340 nm; Em: 450–470 nm) in the
left column while fluorescence from B10 appear as green signals (Ex: 355 nm;
Em: 490–520 nm) in the middle column. The right column was the merge of
both above. Scale bar: 100 μm.
Then, we verified the binding effect of compound A16 to DDB1-
CRBN E3 ubiquitin ligase (PDB: 4ci3). As shown in Figure 5C and D,
we noticed that AR ligand can’t fit into the binding site, and mostly
exposure to the protein (blue region). In contrast, thalidomide ligand
could form hydrogen bonds with Asn353 and Trp402, and the hydrogen
Fig 5. (A) Computer modeling of conjugates A16 in the active site of AR (PDB:
2axa). (B) 2D complex structures of the AR with A16. (C) Docking study of
compound A16 bound to DDB1-CRBN E3 ubiquitin ligase (PDB: 4ci3). (D) 2D
complex structures of the CRBN with A16, in 2D graph, bright blue region
indicates ligand exposure, and hydrogen bonds are shown as green arrow lines.
Fig 4. (A) Western blot assay of AR treated with A16 in different doses from 0 μM to 30 μM. (B) The graph exhibits AR values for A16. The whole proteins were
extracted, and the AR protein levels were analyzed by Western blotting. (*) P < 0.05 was considered statistically significant. (**) P < 0.01 was considered statistically
highly significant.
5

J.-J. Liang et al.
Bioorganic & Medicinal Chemistry 45 (2021) 116331
3. Conclusion
116.93, 116.67, 113.05, 64.60, 58.50, 52.77, 48.05, 36.61, 29.68,
24.53, 24.11, 23.91. ESI-HRMS: m/z cacld. For C32H27F3N8O7 [M+H]+:
693.2033, found 693.2021.
Herein, we have designed, synthesized, and evaluated a type of
PROTAC AR degraders using RU59063 derivatives and ligands for cer-
eblon/cullin 4A E3 ligases. We also developed a mild and efficient
CuAAC process that allows access to a wide library of new 1,2,3-triazole
derivatives with antagonist activity and degradation against the AR. FP
competitive experiments showed that most PROTACs displayed satis-
factory AR binding affinity and antagonist activity against AR, and
compound A16 (85% VS 89%, Enzalutamide) proved to be the most
promising of the series. Additionally, there was greater accumulation of
B10, in LNCaP (AR+) cells than in PC-3 (ARꢀ ) cells. Such differences in
accumulation contributed to the selective AR activity in vivo. Moreover,
the internalization of pomalidomide could be easily visualized by
confocal microscopy due to its fluorescence property, hinting at its po-
tential as a targeting theranostic agent. Western blotting assays analyzed
the effect of AR degradation and the results showed that PROTAC A16
have only a modest effect in reducing the level of AR protein in LNCaP
cells. Thus, whether the cereblon/cullin 4A E3 ligase system can be used
for the design of highly potent and effective AR degraders are needed
more studies to determine. Further design and mechanistic studies on
these PROTAC conjugates are currently in progress in our group.
4-(3-(4-(4-(((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-
yl)oxy)methyl)-1H-1,2,3-triazol-1-yl)butyl)-4,4-dimethyl-2,5-diox-
oimidazolidin-1-yl)-2-(trifluoromethyl)benzonitrile (A2). White
solid, 60% yield, m.p.:133–135 ◦C, 1H NMR (400 MHz, DMSO‑d6) δ
11.10 (s, 1H), 8.31 (d, J = 8.4 Hz, 1H), 8.17 (d, J = 1.7 Hz, 1H), 8.11 (s,
1H), 8.02 (dd, J = 8.4, 1.8 Hz, 1H), 7.81 (dd, J = 8.4, 7.4 Hz, 1H), 7.54
(d, J = 8.5 Hz, 1H), 7.45 (d, J = 7.2 Hz, 1H), 5.07 (dd, J = 12.8, 5.4 Hz,
1H), 4.44 (t, J = 7.1 Hz, 3H), 3.37 (dd, J = 13.6, 6.2 Hz, 2H), 3.16 (t, J =
6.4 Hz, 2H), 2.87 (ddd, J = 14.2, 5.4 Hz, 1H), 2.25 – 2.10 (m, 2H), 2.06 –
2.01 (m, 1H), 1.43 (s, 6H). 13C NMR (100 MHz, DMSO‑d6) δ 173.71,
172.68, 169.97, 169.74, 168.61, 155.81, 155.71, 151.61, 138.65,
136.92, 133.20, 132.75, 132.32, 131.76, 123.67, 118.92, 118.87,
116.95, 116.93, 116.67, 114.73, 113.05, 68.74, 64.60, 52.77, 48.05,
36.61, 29.68, 27.72, 24.53, 24.11, 23.91. ESI-HRMS: m/z cacld. For
C
33H29F3N8O7 [M+H]+: 707.2111, found 707.2120.
4-(3-(4-(4-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-
4-yl)oxy)ethyl)-1H-1,2,3-triazol-1-yl)butyl)-4,4-dimethyl-2,5-diox-
oimidazolidin-1-yl)-2-(trifluoromethyl)benzonitrile (A3). White
solid, 73% yield, m.p.:131-133 ◦C, 1H NMR (400 MHz, CDCl3) δ 8.54 (s,
1H), 8.12 (s, 1H), 7.99 (d, J = 8.2 Hz, 1H), 7.92 (d, J = 8.4 Hz, 1H), 7.82
(s, 1H), 7.69 (t, J = 7.8 Hz, 1H), 7.52 – 7.44 (m, 2H), 5.45 (s, 2H), 4.96
(dd, J = 11.2, 4.8 Hz, 1H), 4.45 (t, J = 6.7 Hz, 2H), 3.39 (t, J = 7.4 Hz,
2H), 2.94 – 2.82 (m, 1H), 2.77 (t, J = 11.8 Hz, 2H), 2.17 – 2.05 (m, 1H),
4. Experimental section
4.1. Materials and methods
2.07 – 1.98 (m, 2H), 1.76 – 1.64 (m, 2H), 1.47 (d, J = 12.6 Hz, 6H). 13
C
Melting points were determined on an X-5 micro-melting apparatus
and are uncorrected. Reagents and solvents were purchased from com-
mercial sources and used without further purification. Thin-layer chro-
matography (TLC) was carried out on glass plates coated with silica gel
(Qingdao Haiyang Chemical Co., G60F-254) and visualized by UV light
(254 nm). The products were purified by column chromatography over
silica gel (Qingdao Haiyang Chemical Co., 200–300 mesh). 1H NMR and
13C NMR spectra were recorded on a Bruker 400 MHz and 101 MHz
spectrometer, respectively. High resolution mass spectra (HRMS) of all
derivatives were recorded on a Waters Micromass Q-T of Micromass
spectrometer by electrospray ionization (ESI). The more details of syn-
thesis of intermediate compounds and spectroscopic characterizations
were supplied in the Supporting Information.
NMR (100 MHz, CDCl3) δ 173.71, 172.68, 169.97, 169.74, 168.61,
155.81, 155.78, 142.78, 138.65, 135.83, 134.08, 132.75, 132.32,
131.76, 123.67, 121.27, 120.04, 118.89, 118.26, 116.93, 116.67,
113.05, 64.60, 58.50, 52.77, 51.79, 40.72, 29.68, 27.72, 24.88, 24.53,
24.11. ESI-HRMS: m/z cacld. For C34H31F3N8O7 [M+H]+: 721.2368,
found 721.2359.
4-(3-(4-(4-(3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-
4-yl)oxy)propyl)-1H-1,2,3-triazol-1-yl)butyl)-4,4-dimethyl-2,5-
dioxoimidazolidin-1-yl)-2-(trifluoromethyl)benzonitrile
(A4).
White solid, 75% yield, m.p.:154-156 ◦C, 1H NMR (400 MHz, CDCl3) δ
8.68 (s, 1H), 8.13 (s, 1H), 8.00 (d, J = 8.3 Hz, 1H), 7.92 (d, J = 8.4 Hz,
1H), 7.67 (t, J = 7.9 Hz, 1H), 7.52 (s, 1H), 7.45 (d, J = 7.2 Hz, 1H), 7.22
(d, J = 8.5 Hz, 1H), 4.96 (dd, J = 11.9, 5.2 Hz, 1H), 4.38 (t, J = 6.8 Hz,
2H), 4.32 – 4.16 (m, 2H), 3.37 (t, J = 7.3 Hz, 2H), 3.00 (dd, J = 13.8, 7.0
Hz, 2H), 2.85 (dd, J = 22.0, 9.3 Hz, 2H), 2.76 (t, J = 7.4 Hz, 2H), 2.29
(dd, J = 12.8, 6.4 Hz, 2H), 2.15 (dd, J = 15.7, 7.9 Hz, 1H), 2.01 – 1.92
(m, 2H), 1.70 (dt, J = 14.8, 7.4 Hz, 2H), 1.49 (s, 6H). 13C NMR (100
MHz, CDCl3) δ 173.71, 172.68, 169.97, 169.74, 168.61, 155.81, 155.71,
150.80, 138.65, 136.92, 133.20, 132.75, 132.32, 131.76, 123.67,
120.49, 118.92, 118.87, 116.95, 116.93, 116.67, 113.05, 69.99, 64.60,
52.77, 51.79, 40.72, 29.68, 27.72, 25.79, 24.88, 24.53, 24.11, 23.95.
ESI-HRMS: m/z cacld. For C35H33F3N8O7 [M+H]+: 735.2424, found
735.2421.
4.2. Chemistry
4.2.1. General procedures for click reaction (A1-18 and B1-10)
Into a round bottom flask, alkyne derivatives (1.0 equiv) with
different side chain lengths (12a-h) and appropriate azides (2a-c, 3, 6
and 8, 1.2 equiv) were dissolved in THF (4 mL), then put 1 mL H2O in it,
finally, CuSO4 (1.0 equiv) and Na-ascorbate (3.0 equiv) as catalytic
agent to react 2–10 h. Then insoluble matter was filtered and condensed,
subsequently, extracted with EtOAc and washed with brine, dried over
anhydrous Na2SO4, filtered, evaporated and the crude product was pu-
rified by column chromatography to give products.
4-(3-(3-(4-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-
4-yl)amino)ethyl)-1H-1,2,3-triazol-1-yl)propyl)-4,4-dimethyl-2,5-
dioxoimidazolidin-1-yl)-2-(trifluoromethyl)benzonitrile (A5). Yel-
low solid, 65% yield, m.p.:158-159 ◦C, 1H NMR (400 MHz, DMSO‑d6) δ
8.31 (d, J = 8.4 Hz, 1H), 8.19 (d, J = 1.6 Hz, 1H), 8.04 (d, J = 8.4 Hz,
1H), 7.94 (d, J = 32.8 Hz, 1H), 7.47 (dd, J = 8.4, 7.1 Hz, 1H), 7.01 (dt, J
= 7.7, 2.7 Hz, 2H), 6.52 (s, 1H), 5.13 (dd, J = 13.0, 5.4, 2.7 Hz, 1H), 4.43
(dd, J = 11.6, 7.0 Hz, 2H), 4.00 – 3.82 (m, 1H), 3.37 (t, J = 7.2 Hz, 2H),
3.08 – 2.87 (m, 1H), 2.77 (dd, J = 21.4, 13.5 Hz, 2H), 2.60 (dd, J = 16.0,
8.5 Hz, 1H), 2.25 – 2.10 (m, 2H), 2.04 (dt, J = 16.5, 13.1 Hz, 1H), 1.78
(dd, J = 14.5, 7.4 Hz, 1H), 1.44 (d, J = 3.8 Hz, 6H), 1.37 – 1.13 (m, 2H).
13C NMR (100 MHz, DMSO‑d6) δ 174.60, 171.48, 169.54, 168.51,
167.32, 152.72, 146.72, 136.75, 136.01, 135.49, 131.92, 129.99,
124.05, 122.48, 121.74, 115.19, 111.02, 108.43, 61.69, 49.05, 47.03,
36.76, 31.16, 29.64, 27.22, 23.48, 22.34, 21.35. ESI-HRMS: m/z cacld.
For C33H30F3N9O6 [M+H]+: 706.2349, found 706.2322.
4.2.2. Characterization data for final compounds A1-18 and B1-10
4-(3-(3-(4-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-
4-yl)oxy)ethyl)-1H-1,2,3-triazol-1-yl)propyl)-4,4-dimethyl-2,5-
dioxoimidazolidin-1-yl)-2-(trifluoromethyl)benzonitrile
(A1).
White solid, 76% yield, mp 141-143 ◦C, 1H NMR (400 MHz, DMSO‑d6) δ
11.09 (s, 1H), 8.32 (t, J = 4.2 Hz, 2H), 8.19 (d, J = 1.7 Hz, 1H), 8.04 (dd,
J = 8.4, 1.8 Hz, 1H), 7.84 (dd, J = 8.5, 7.3 Hz, 1H), 7.78 – 7.69 (m, 1H),
7.50 (dd, J = 13.2, 7.5 Hz, 1H), 5.44 (s, 2H), 5.07 (dd, J = 12.8, 5.4 Hz,
1H), 4.51 (t, J = 7.2 Hz, 3H), 3.47 – 3.35 (m, 5H), 2.88 (ddd, J = 13.9,
5.5 Hz, 1H), 2.56 (s, 2H), 2.25 – 2.16 (m, 2H), 2.06 – 1.97 (m, 1H), 1.45
(s, 6H). 13C NMR (100 MHz, DMSO‑d6) δ 173.71, 172.68, 169.97,
169.74, 168.61, 155.81, 155.78, 142.78, 138.65, 135.83, 134.08,
132.75, 132.32, 131.76, 123.67, 121.27, 120.04, 118.89, 118.26,
6

J.-J. Liang et al.
Bioorganic & Medicinal Chemistry 45 (2021) 116331
4-(3-(3-(4-(3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-
22.65.ESI-HRMS: m/z cacld. For C36H35F3N8O8 [M+H]+: 765.2530,
found 765.2521.
4-yl)amino)propyl)-1H-1,2,3-triazol-1-yl)propyl)-4,4-dimethyl-
2,5-dioxoimidazolidin-1-yl)-2-(trifluoromethyl)benzonitrile (A6).
Yellow solid, 58% yield, m.p.:192.2–193.8 ◦C, 1H NMR (400 MHz,
CDCl3) δ 8.11 (d, J = 13.1 Hz, 1H), 7.99 (d, J = 8.4 Hz, 1H), 7.92 (d, J =
8.4 Hz, 1H), 7.44 (dd, J = 8.2, 7.3 Hz, 1H), 7.17 (dd, J = 14.7, 7.5 Hz,
1H), 6.88 (d, J = 8.3 Hz, 1H), 5.26 (s, 1H), 4.88 (dd, J = 11.8, 5.5 Hz,
1H), 4.46 (t, J = 12.1 Hz, 2H), 3.88 (t, J = 11.8 Hz, 2H), 3.40 (m, 2H),
2.94 (t, J = 12.1 Hz, 1H), 2.82 – 2.70 (m, 3H), 2.36 (m, 2H), 2.09 (d, J =
2.9 Hz, 1H), 1.99 (s, 2H), 1.69 (m, 2H), 1.47 (s, 6H). 13C NMR (100 MHz,
CDCl3) δ 174.39, 171.02, 168.90, 167.64, 153.14, 145.65, 136.31,
135.64, 135.32, 132.34, 127.94, 122.98, 121.48, 113.18, 110.84,
108.39, 77.24, 62.07, 49.72, 39.85, 37.69, 32.05, 29.76, 23.34, 22.11.
ESI-HRMS: m/z cacld. For C34H32F3N9O6 [M+H]+: 720.2506, found
720.2511.
4-(3-(4-(4-((2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindo-
lin-4-yl)oxy)ethoxy)ethoxy)methyl)-1H-1,2,3-triazol-1-yl)butyl)-
4,4-dimethyl-2,5-dioxoimidazolidin-1-yl)-2-(trifluoromethyl)ben-
zonitrile (A10).
White solid. 47% yield, m.p.:197.2–198.6 ◦C, 1H NMR (400 MHz,
CDCl3) δ 8.48 (s, 1H), 8.13 (d, J = 1.8 Hz, 1H), 7.99 (dd, J = 8.5, 1.9 Hz,
1H), 7.92 (d, J = 8.4 Hz, 1H), 7.67 (dd, J = 8.4, 7.4 Hz, 1H), 7.59 (s, 1H),
7.46 (d, J = 7.2 Hz, 1H), 7.29 – 7.23 (m, 2H), 4.95 (dd, J = 12.1, 5.3 Hz,
1H), 4.67 (s, 2H), 4.40 (t, J = 6.9 Hz, 2H), 4.35 (t, J = 4.6 Hz, 2H), 3.95 –
3.90 (m, 2H), 3.80 (dd, J = 5.7, 2.8 Hz, 2H), 3.74 – 3.69 (m, 2H), 3.48 (s,
1H), 3.40 – 3.33 (m, 2H), 2.85 (dd, J = 18.3, 16.0 Hz, 1H), 2.80 – 2.69
(m, 2H), 2.16 – 2.08 (m, 1H), 2.02 – 1.94 (m, 2H), 1.71 (t, J = 7.3 Hz,
2H), 1.50 (s, 6H). 13C NMR (100 MHz, DMSO‑d6) δ 174.62, 172.80,
169.90, 166.80, 155.80, 153.16, 145.15, 138.37, 137.05, 136.79,
136.02, 133.10, 130.15, 128.94, 124.22, 121.99, 119.87, 116.14,
115.23, 65.76, 63.21, 62.04, 48.69, 28.75, 22.60, 21.94. ESI-HRMS: m/z
cacld. For C37H37F3N8O9 [M+H]+: 795.2636, found 795.2616.
4-(3-(5-(4-((3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-
4-yl)oxy)propoxy)methyl)-1H-1,2,3-triazol-1-yl)pentyl)-4,4-
dimethyl-2,5-dioxoimidazolidin-1-yl)-2-(trifluoromethyl)benzoni-
trile (A11).
4-(3-(3-(4-((3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-
4-yl)oxy)propoxy)methyl)-1H-1,2,3-triazol-1-yl)propyl)-4,4-
dimethyl-2,5-dioxoimidazolidin-1-yl)-2-(trifluoromethyl)benzoni-
trile (A7).
White solid. 64% yield, m.p.:196.4–197.5 ◦C, 1H NMR (400 MHz,
DMSO‑d6) δ 11.11 (s, 1H), 8.31 (d, J = 8.4 Hz, 1H), 8.19 (d, J = 1.7 Hz,
1H), 8.13 (s, 1H), 8.04 (dd, J = 8.4, 1.8 Hz, 1H), 7.80 (dd, J = 8.4, 7.4
Hz, 1H), 7.50 (d, J = 8.5 Hz, 1H), 7.44 (d, J = 7.2 Hz, 1H), 5.09 (dd, J =
12.8, 5.4 Hz, 1H), 4.53 (s, 2H), 4.45 (t, J = 7.2 Hz, 2H), 4.26 (t, J = 6.2
Hz, 2H), 3.65 (t, J = 6.2 Hz, 2H), 3.39 (t, J = 7.3 Hz, 2H), 2.97 – 2.80 (m,
1H), 2.65 – 2.52 (m, 2H), 2.19 (dd, J = 13.7, 6.6 Hz, 2H), 2.08 – 2.03 (m,
1H), 2.02 – 2.00 (m, 1H), 1.46 (d, J = 5.3 Hz, 6H). 13C NMR (100 MHz,
CDCl3) δ 174.32, 171.13, 168.53, 167.01, 165.82, 156.54, 153.38,
136.64, 136.20, 135.39, 133.74, 133.44, 128.07, 123.08, 123.03,
119.11, 117.08, 115.92, 114.98, 108.44, 77.25, 66.45, 66.08, 64.37,
62.12, 49.13, 47.81, 37.62, 31.40, 29.87, 29.20, 23.37, 23.34, 22.64.
ESI-HRMS: m/z cacld. For C35H33F3N8O8 [M+H]+: 751.2373, found
751.2365.
White solid. 59% yield, m.p.:176.2–177.3 ◦C, 1H NMR (400 MHz,
CDCl3) δ 8.36 (s, 1H), 8.07 (s, 1H), 7.91 (t, J = 11.6 Hz, 1H), 7.85 (d, J =
8.3 Hz, 1H), 7.65 – 7.57 (m, 1H), 7.39 (t, J = 8.0 Hz, 2H), 7.17 (d, J =
8.4 Hz, 1H), 4.88 (dd, J = 12.1, 5.1 Hz, 2H), 4.33 – 4.18 (m, 6H), 3.84 –
3.59 (m, 4H), 3.28 – 3.18 (m, 2H), 2.89 – 2.77 (m, 2H), 2.68 (dd, J =
28.5, 13.0 Hz, 2H), 2.20 – 2.09 (m, 2H), 2.05 (dd, J = 17.0, 9.2 Hz, 2H),
1.89 (m, 2H), 1.65 (m, 3H), 1.44 (s, 6H), 1.32 (dd, J = 24.7, 11.8 Hz,
2H), 0.83 – 0.74 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 174.32, 171.13,
168.53, 167.01, 165.82, 156.54, 153.38, 136.64, 136.20, 135.39,
133.74, 133.44, 128.07, 123.08, 123.03, 119.11, 117.08, 115.92,
114.98, 108.44, 77.25, 66.45, 66.08, 64.37, 62.12, 49.13, 47.81, 37.62,
31.40, 29.87, 29.20, 23.37, 23.34, 22.64. ESI-HRMS: m/z cacld. For
4-(3-(3-(4-((2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindo-
lin-4-yl)oxy)ethoxy)ethoxy)methyl)-1H-1,2,3-triazol-1-yl)propyl)-
4,4-dimethyl-2,5-dioxoimidazolidin-1-yl)-2-(trifluoromethyl)ben-
zonitrile (A8).
C
37H37F3N8O8 [M+H]+: 779.2686, found 779.2682.
4-(3-(5-(4-((2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindo-
White solid. 59% yield, m.p.:206.4–207.9 ◦C, 1H NMR (400 MHz,
CDCl3) δ 8.49 (s, 1H), 8.12 (s, 1H), 7.99 (dd, J = 8.4, 1.8 Hz, 1H), 7.93
(d, J = 8.5 Hz, 1H), 7.71 – 7.64 (m, 2H), 7.46 (d, J = 7.3 Hz, 1H), 7.27 (d,
J = 1.7 Hz, 1H), 4.95 (dd, J = 12.2, 5.3 Hz, 1H), 4.69 (s, 3H), 4.44 (dd, J
= 13.4, 6.7 Hz, 2H), 4.38 – 4.32 (m, 2H), 3.97 – 3.90 (m, 3H), 3.80 (dd,
J = 5.5, 2.6 Hz, 3H), 3.76 – 3.68 (m, 4H), 3.47 – 3.39 (m, 2H), 2.94 –
2.81 (m, 2H), 2.79 – 2.69 (m, 2H), 2.35 (dd, J = 13.9, 7.2 Hz, 2H), 2.15 –
2.07 (m, 1H), 1.91 – 1.82 (m, 2H), 1.50 (s, 6H). 13C NMR (100 MHz,
DMSO‑d6) δ 174.62, 172.80, 169.90, 166.80, 155.80, 153.16, 145.15,
138.37, 137.05, 136.79, 136.02, 133.10, 130.15, 128.94, 124.22,
121.99, 119.87, 116.14, 115.23, 65.76, 63.21, 62.04, 48.69, 28.75,
22.60, 21.94. ESI-HRMS: m/z cacld. For C36H35F3N8O9 [M+H]+:
780.2479, found 780.2469.
lin-4-yl)oxy)ethoxy)ethoxy)methyl)-1H-1,2,3-triazol-1-yl)pentyl)-
4,4-dimethyl-2,5-dioxoimidazolidin-1-yl)-2-(trifluoromethyl)ben-
zonitrile (A12).
White solid. 52% yield, m.p.:196.3–197.8 ◦C, 1H NMR (400 MHz,
CDCl3) δ 8.47 (s, 1H), 8.14 (s, 1H), 8.00 (d, J = 8.4 Hz, 1H), 7.92 (d, J =
8.4 Hz, 1H), 7.27 (s, 4H), 4.95 (dd, J = 11.9, 5.1 Hz, 1H), 4.36 (s, 5H),
3.94 (s, 2H), 3.79 (d, J = 24.9 Hz, 4H), 3.38 – 3.28 (m, 2H), 2.93 – 2.81
(m, 1H), 2.74 (dd, J = 22.6, 10.9 Hz, 2H), 2.17 – 2.07 (m, 1H), 1.97 (s,
2H), 1.73 (s, 2H), 1.51 (s, 6H), 1.38 (s, 2H), 0.93 – 0.79 (m, 2H). 13C
NMR (100 MHz, CDCl3) δ 174.32, 171.13, 168.53, 167.01, 165.82,
156.54, 153.38, 136.64, 136.20, 135.39, 133.74, 133.44, 128.07,
123.08, 123.03, 119.11, 117.08, 115.92, 114.98, 108.44, 77.25, 66.45,
66.08, 64.37, 62.12, 49.13, 47.81, 37.62, 31.40, 29.87, 29.20, 23.37,
23.34, 22.64. ESI-HRMS: m/z cacld. For C38H39F3N8O9 [M+H]+:
809.2792, found 809.2791.
4-(3-(4-(4-((3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-
4-yl)oxy)propoxy)methyl)-1H-1,2,3-triazol-1-yl)butyl)-4,4-
dimethyl-2,5-dioxoimidazolidin-1-yl)-2-(trifluoromethyl)benzoni-
trile (A9).
4-(3-(3-(4-((2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoi-
soindolin-4-yl)oxy)ethoxy)ethoxy)ethoxy)methyl)-1H-1,2,3-tri-
azol-1-yl)propyl)-4,4-dimethyl-2,5-dioxoimidazolidin-1-yl)-2-(tri-
fluoromethyl)benzonitrile (A13).
White solid. 74% yield, m.p.:186.4–187.9 ◦C, 1H NMR (400 MHz,
CDCl3) δ 8.36 (s, 1H), 8.13 (d, J = 1.7 Hz, 1H), 7.99 (dd, J = 8.5, 1.9 Hz,
1H), 7.92 (d, J = 8.5 Hz, 1H), 7.68 (dd, J = 8.4, 7.4 Hz, 1H), 7.57 (s, 1H),
7.46 (d, J = 7.3 Hz, 1H), 7.23 (d, J = 8.5 Hz, 1H), 4.96 (dd, J = 12.2, 5.3
Hz, 1H), 4.65 (s, 2H), 4.39 (t, J = 6.9 Hz, 2H), 4.28 (t, J = 6.0 Hz, 2H),
3.78 (t, J = 5.9 Hz, 2H), 3.41 – 3.32 (m, 2H), 2.93 – 2.82 (m, 2H), 2.79
(d, J = 10.3 Hz, 2H), 2.34 – 2.21 (m, 1H), 2.19 – 2.09 (m, 3H), 2.02 –
1.94 (m, 2H), 1.71 (t, J = 7.6 Hz, 2H), 1.50 (s, 6H), 0.95 – 0.82 (m, 2H).
13C NMR (100 MHz, CDCl3) δ 175.32, 172.13, 166.53, 162.01, 158.82,
156.54, 151.37, 134.32, 133.27, 132.39, 131.74, 131.44, 127.07,
124.08, 125.03, 118.11, 116.08, 114.92, 112.98, 107.44, 77.25, 66.45,
67.08, 64.38, 62.12, 50.33, 47.81, 37.62, 32.45, 29.63, 29.21, 23.34,
White solid. 71% yield, m.p.:216.3–217.8 ◦C, 1H NMR (400 MHz,
DMSO‑d6) δ 11.11 (s, 1H), 8.32 (d, J = 8.3 Hz, 1H), 8.19 (d, J = 1.5 Hz,
1H), 8.12 (s, 1H), 8.04 (d, J = 8.3 Hz, 1H), 7.83 – 7.76 (m, 1H), 7.52 (d,
J = 8.5 Hz, 1H), 7.45 (d, J = 7.2 Hz, 1H), 5.08 (dd, J = 12.8, 5.3 Hz, 1H),
4.51 (s, 2H), 4.46 (t, J = 7.1 Hz, 2H), 4.36 – 4.31 (m, 2H), 3.84 – 3.77 (m,
2H), 3.67 – 3.61 (m, 2H), 3.52 (d, J = 8.3 Hz, 6H), 2.89 (dd, J = 22.6,
8.4 Hz, 1H), 2.59 (d, J = 18.1 Hz, 2H), 2.17 (dd, J = 12.3, 5.0 Hz, 2H),
2.06 – 1.98 (m, 1H), 1.46 (s, 6H). 13C NMR (100 MHz, DMSO‑d6) δ
174.62, 172.80, 169.90, 166.80, 155.80, 153.16, 145.15, 138.37,
137.05, 136.79, 136.02, 133.10, 130.15, 128.94, 124.22, 121.99,
7

J.-J. Liang et al.
Bioorganic & Medicinal Chemistry 45 (2021) 116331
119.87, 116.14, 115.23, 65.76, 63.21, 62.04, 48.69, 28.75, 22.60,
21.94. ESI-HRMS: m/z cacld. For C38H39F3N8O10 [M+H]+: 825.2741,
found 825.2743.
66.08, 64.37, 62.12, 49.13, 47.81, 37.62, 31.40, 29.87, 29.20, 23.37,
23.34, 22.64. ESI-HRMS: m/z cacld. For C38H39F3N8O9S [M+H]+:
841.2513, found 841.2516.
4-(3-(3-(4-(13-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindo-
lin-4-yl)oxy)-2,5,8,11-tetraoxatridecyl)-1H-1,2,3-triazol-1-yl)pro-
pyl)-4,4-dimethyl-2,5-dioxoimidazolidin-1-yl)-2-(trifluoromethyl)
benzonitrile (A14).
4-(3-(3-(4-(13-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindo-
lin-4-yl)oxy)-2,5,8,11-tetraoxatridecyl)-1H-1,2,3-triazol-1-yl)pro-
pyl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl)-2-(tri-
fluoromethyl)benzonitrile (A18).
White solid. 57% yield, m.p.:212.6–213.7 ◦C, 1H NMR (400 MHz,
CDCl3) δ 8.78 (s, 1H), 8.12 (d, J = 1.6 Hz, 1H), 7.99 (dd, J = 8.4, 1.9 Hz,
1H), 7.93 (d, J = 8.5 Hz, 1H), 7.72 (s, 1H), 7.67 (dd, J = 8.3, 7.5 Hz, 1H),
7.45 (d, J = 7.2 Hz, 1H), 7.26 (s, 1H), 4.95 (dd, J = 12.0, 5.4 Hz, 1H),
4.68 (s, 2H), 4.46 (t, J = 6.7 Hz, 2H), 4.33 (dd, J = 11.7, 6.8 Hz, 3H),
3.98 – 3.90 (m, 3H), 3.77 (dd, J = 5.6, 3.6 Hz, 3H), 3.67 (ddd, J = 8.6,
5.5, 2.0 Hz, 13H), 3.48 – 3.40 (m, 2H), 2.85 (dd, J = 10.2, 8.3 Hz, 1H),
2.80 – 2.70 (m, 2H), 2.39 – 2.31 (m, 2H), 2.15 – 2.07 (m, 1H), 1.50 (s,
6H). 13C NMR (100 MHz, DMSO‑d6) δ 174.62, 172.80, 169.90, 166.80,
155.80, 153.16, 145.15, 138.37, 137.05, 136.79, 136.02, 133.10,
130.15, 128.94, 124.22, 121.99, 119.87, 116.14, 115.23, 65.76, 63.21,
62.04, 48.69, 28.75, 22.60, 21.94. ESI-HRMS: m/z cacld. For
White solid. 43% yield, m.p.:207.3–208.5 ◦C, 1H NMR (400 MHz,
CDCl3) δ 8.70 (s, 1H), 7.95 (t, J = 7.4 Hz, 1H), 7.89 (s, 1H), 7.77 (dd, J =
8.3, 1.7 Hz, 1H), 7.71 – 7.63 (m, 2H), 7.45 (d, J = 7.3 Hz, 2H), 7.26 (s,
2H), 4.95 (dd, J = 11.7, 5.2 Hz, 1H), 4.70 (s, 2H), 4.48 (t, J = 6.5 Hz,
2H), 4.35 (dd, J = 10.3, 5.8 Hz, 3H), 3.94 (dd, J = 9.2, 4.5 Hz, 3H), 3.78
(dd, J = 8.7, 5.1 Hz, 5H), 3.68 – 3.62 (m, 11H), 2.91 – 2.80 (m, 2H), 2.79
– 2.72 (m, 3H), 2.54 – 2.45 (m, 2H), 2.15 – 2.05 (m, 3H), 1.54 (d, J = 6.5
Hz, 6H). 13C NMR (100 MHz, CDCl3) δ 178.74, 175.04, 171.26, 168.36,
167.01, 165.61, 156.41, 136.97, 136.53, 135.24, 133.73, 132.12,
127.01, 119.51, 117.26, 116.13, 114.82, 77.28, 72.49, 71.12, 70.57,
70.55, 70.51, 70.45, 69.80, 69.34, 69.29, 65.25, 64.59, 61.64, 49.12,
47.96, 41.49, 31.40, 29.68, 28.32, 23.47, 23.02, 22.61, 21.56. ESI-
HRMS: m/z cacld. For C40H43F3N8O10S [M+H]+: 885.2775, found
885.2779.
C
40H43F3N8O11 [M+H]+: 869.3003, found 869.3005.
4-(3-(3-(4-((3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-
4-yl)oxy)propoxy)methyl)-1H-1,2,3-triazol-1-yl)propyl)-4,4-
dimethyl-5-oxo-2-thioxoimidazolidin-1-yl)-2-(trifluoromethyl)
benzonitrile (A15).
4-(3-(4-(4-((3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-
4-yl)oxy)propoxy)methyl)-1H-1,2,3-triazol-1-yl)benzyl)-4,4-
dimethyl-2,5-dioxoimidazolidin-1-yl)-2-(trifluoromethyl)benzoni-
trile (B1).
White solid. 68% yield, m.p.:211.4–212.6 ◦C, 1H NMR (400 MHz,
CDCl3) δ 8.25 (s, 1H), 7.95 (t, J = 7.1 Hz, 2H), 7.88 (d, J = 1.9 Hz, 1H),
7.80 – 7.73 (m, 2H), 7.70 – 7.63 (m, 3H), 7.45 (d, J = 7.2 Hz, 2H), 7.23
(d, J = 8.4 Hz, 3H), 4.96 (dd, J = 12.2, 5.3 Hz, 2H), 4.66 (d, J = 10.6 Hz,
3H), 4.46 (t, J = 6.1 Hz, 3H), 4.29 (t, J = 6.0 Hz, 4H), 3.78 (dd, J = 13.3,
7.2 Hz, 6H), 2.87 (dd, J = 15.8, 13.2 Hz, 3H), 2.82 – 2.71 (m, 4H), 2.50
(dd, J = 14.7, 7.3 Hz, 2H), 2.34 – 2.23 (m, 2H), 2.15 (dd, J = 13.0, 7.8
Hz,2H), 1.53 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 174.32, 171.13,
168.53, 167.01, 165.82, 156.54, 153.38, 136.64, 136.20, 135.39,
133.74, 133.44, 128.07, 123.08, 123.03, 119.11, 117.08, 115.92,
114.98, 108.44, 77.25, 66.45, 66.08, 64.37, 62.12, 49.13, 47.81, 37.62,
31.40, 29.87, 29.20, 23.37, 23.34, 22.64. ESI-HRMS: m/z cacld. For
Brown solid. 85% yield, m.p.:215.2–216.1 ◦C, 1H NMR (400 MHz,
DMSO‑d6) δ 11.10 (s, 1H), 8.77 (s, 1H), 8.34 (d, J = 8.4 Hz, 1H), 8.26 (d,
J = 1.5 Hz, 1H), 8.11 (d, J = 8.4 Hz, 1H), 7.82 (d, J = 8.4 Hz, 1H), 7.79
(dd, J = 8.4, 7.4 Hz, 1H), 7.64 (d, J = 8.5 Hz, 2H), 7.50 (d, J = 8.5 Hz,
1H), 7.41 (d, J = 7.2 Hz, 1H), 5.05 (dd, J = 12.7, 5.4 Hz, 1H), 4.69 (s,
2H), 4.62 (s, 2H), 4.27 (t, J = 6.1 Hz, 2H), 3.71 (t, J = 6.2 Hz, 2H), 2.95 –
2.81 (m, 1H), 2.58 (d, J = 17.8 Hz, 1H), 2.28 – 2.15 (m, 1H), 2.03 (dd, J
= 11.5, 5.5 Hz, 2H), 1.44 (s, 6H). 13C NMR (100 MHz, DMSO‑d6) δ
174.62, 172.80, 169.90, 166.80, 155.80, 153.16, 145.15, 138.37,
137.05, 136.79, 136.02, 133.10, 130.15, 128.94, 124.22, 121.99,
119.87, 116.14, 115.23, 65.76, 63.21, 62.04, 48.69, 28.75, 22.60,
21.94. ESI-HRMS: m/z cacld. For C39H33F3N8O8 [M+H]+: 799.2373,
found 799.2329.
C
35H33F3N8O9S [M+H]+: 767.2145, found 767.2139.
4-(3-(3-(4-((2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindo-
lin-4-yl)oxy)ethoxy)ethoxy)methyl)-1H-1,2,3-triazol-1-yl)propyl)-
4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl)-2-(trifluoromethyl)
benzonitrile (A16).
4-(3-(4-(4-((2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindo-
lin-4-yl)oxy)ethoxy)ethoxy)methyl)-1H-1,2,3-triazol-1-yl)benzyl)-
4,4-dimethyl-2,5-dioxoimidazolidin-1-yl)-2-(trifluoromethyl)ben-
zonitrile (B2).
White solid. 43% yield, m.p.:217.6–218.9 ◦C, 1H NMR (400 MHz,
CDCl3) δ 8.49 (s, 1H), 7.96 (d, J = 8.3 Hz, 1H), 7.88 (s, 1H), 7.76 (d, J =
8.3 Hz, 1H), 7.67 (t, J = 7.8 Hz, 1H), 7.44 (t, J = 13.7 Hz, 1H), 7.26 (s,
2H), 4.96 (d, J = 6.9 Hz, 1H), 4.61 (d, J = 79.0 Hz, 4H), 4.36 (s, 2H),
3.94 (s, 2H), 3.88 – 3.65 (m, 7H), 2.88 (d, J = 16.7 Hz, 2H), 2.82 – 2.70
(m, 2H), 2.52 (s, 2H), 2.11 (d, J = 5.4 Hz, 1H), 1.53 (s, 6H). 13C NMR
(100 MHz, CDCl3) δ 178.82, 175.03, 171.18, 168.34, 166.96, 165.67,
136.98, 136.58, 135.23, 133.77, 132.12, 127.00, 119.51, 117.32,
116.22, 114.81, 77.26, 71.14, 69.93, 69.40, 65.27, 60.41, 49.16, 41.54,
31.42, 29.70, 28.30, 23.09, 22.64, 21.05, 14.21. ESI-HRMS: m/z cacld.
For C36H35F3N8O8S [M+H]+: 797.2651, found 797.2654.
Brown solid. 82% yield, m.p.:226.5–227.9 ◦C, 1H NMR (400 MHz,
DMSO‑d6) δ 11.10 (s, 1H), 8.76 (s, 1H), 8.34 (d, J = 8.4 Hz, 1H), 8.26 (s,
1H), 8.11 (d, J = 8.3 Hz, 1H), 7.86 (d, J = 8.4 Hz, 2H), 7.82 – 7.74 (m,
1H), 7.65 (d, J = 8.5 Hz, 2H), 7.52 (d, J = 8.5 Hz, 1H), 7.44 (t, J = 7.0
Hz, 1H), 5.07 (dd, J = 12.6, 5.4 Hz, 1H), 4.70 (s, 2H), 4.63 (s, 2H), 4.38 –
4.30 (m, 2H), 3.85 – 3.79 (m, 2H), 3.70 (dd, J = 5.9, 3.2 Hz, 2H), 3.65
(dd, J = 5.7, 3.2 Hz, 2H), 2.93 – 2.80 (m, 1H), 2.63 – 2.51 (m, 2H), 2.31 –
2.22 (m, 2H), 2.08 – 1.99 (m, 1H), 1.42 (d, J = 15.1 Hz, 6H). 13C NMR
(100 MHz, DMSO‑d6) δ 174.62, 172.80, 169.90, 166.80, 155.80, 153.16,
145.15, 138.37, 137.05, 136.79, 136.02, 133.10, 130.15, 128.94,
124.22, 121.99, 119.87, 116.14, 115.23, 65.76, 63.21, 62.04, 48.69,
28.75, 22.60, 21.94. ESI-HRMS: m/z cacld. For C40H35F3N8O9 [M+H]+:
829.2479, found 829.2470.
4-(3-(3-(4-((2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoi-
soindolin-4-yl)oxy)ethoxy)ethoxy)ethoxy)methyl)-1H-1,2,3-tri-
azol-1-yl)propyl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl)-
2-(trifluoromethyl)benzonitrile (A17).
4-(3-(4-(4-((2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoi-
soindolin-4-yl)oxy)ethoxy)ethoxy)ethoxy)methyl)-1H-1,2,3-tri-
azol-1-yl)benzyl)-4,4-dimethyl-2,5-dioxoimidazolidin-1-yl)-2-(tri-
fluoromethyl)benzonitrile (B3).
White solid. 54% yield, m.p.:221.3–222.6 ◦C, 1H NMR (400 MHz,
CDCl3) δ 8.67 (s, 1H), 7.95 (t, J = 6.9 Hz, 1H), 7.88 (d, J = 1.5 Hz, 1H),
7.80 – 7.73 (m, 1H), 7.67 (dd, J = 8.7, 7.1 Hz, 2H), 7.46 (t, J = 6.0 Hz,
1H), 7.25 (s, 1H), 4.96 (dd, J = 11.3, 4.9 Hz, 1H), 4.70 (s, 2H), 4.47 (t, J
= 6.6 Hz, 2H), 4.37 – 4.31 (m, 3H), 3.94 (dd, J = 10.8, 6.0 Hz, 3H), 3.81
– 3.75 (m, 5H), 3.74 – 3.64 (m, 8H), 2.90 – 2.81 (m, 2H), 2.80 – 2.70 (m,
White solid. 67% yield, m.p.:214.1–215.6 ◦C, 1H NMR (400 MHz,
CDCl3) δ 8.47 (s, 1H), 8.19 (d, J = 1.5 Hz, 1H), 8.10 – 8.02 (m, 2H), 7.94
(d, J = 8.4 Hz, 1H), 7.74 (d, J = 8.3 Hz, 2H), 7.69 – 7.62 (m, 1H), 7.53 (d,
J = 8.3 Hz, 2H), 7.44 (d, J = 7.2 Hz, 1H), 7.25 (d, J = 8.5 Hz, 1H), 4.93
(dd, J = 11.7, 5.4 Hz, 1H), 4.77 (s, 2H), 4.67 (s, 2H), 4.37 – 4.30 (m, 2H),
3.97 – 3.90 (m, 2H), 3.79 (dd, J = 9.1, 4.2 Hz, 2H), 3.76 (d, J = 14.2 Hz,
3H), 3.69 (dd, J = 10.0, 5.1 Hz, 5H), 2.85 (dd, J = 12.1, 8.4 Hz, 1H),
2H), 2.55 – 2.44 (m, 2H), 2.12 (dd, J = 7.4, 4.9 Hz, 2H), 1.53 (s, 6H). 13
C
NMR (100 MHz, CDCl3) δ 174.32, 171.13, 168.53, 167.01, 165.82,
156.54, 153.38, 136.64, 136.20, 135.39, 133.74, 133.44, 128.07,
123.08, 123.03, 119.11, 117.08, 115.92, 114.98, 108.44, 77.25, 66.45,
8

J.-J. Liang et al.
Bioorganic & Medicinal Chemistry 45 (2021) 116331
2.81 – 2.68 (m, 2H), 2.27 – 2.18 (m, 1H), 2.10 (dd, J = 12.8, 7.9 Hz, 1H),
1.46 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 174.32, 171.13, 168.53,
167.01, 165.82, 156.54, 153.38, 136.64, 136.20, 135.39, 133.74,
133.44, 128.07, 123.08, 123.03, 119.11, 117.08, 115.92, 114.98,
108.44, 77.25, 66.45, 66.08, 64.37, 62.12, 49.13, 47.81, 37.62, 31.40,
29.87, 29.20, 23.37, 23.34, 22.64. ESI-HRMS: m/z cacld. For
7.59 (t, J = 7.8 Hz, 2H), 7.43 (d, J = 7.2 Hz, 1H), 7.25 (d, J = 8.5 Hz,
1H), 5.17 (d, J = 6.1 Hz, 2H), 4.95 (dd, J = 11.6, 5.4 Hz, 1H), 4.78 (d, J
= 8.3 Hz, 2H), 4.37 – 4.26 (m, 2H), 3.98 – 3.88 (m, 2H), 3.78 (dd, J =
10.0, 5.1 Hz, 2H), 3.75 (d, J = 5.2 Hz, 2H), 3.73 – 3.68 (m, 5H), 2.90 –
2.79 (m, 2H), 2.79 – 2.70 (m, 2H), 2.10 (dd, J = 15.0, 10.4 Hz, 2H), 1.50
(s, 6H). 13C NMR (100 MHz, CDCl3) δ 174.32, 171.13, 168.53, 167.01,
165.82, 156.54, 153.38, 136.64, 136.20, 135.39, 133.74, 133.44,
128.07, 123.08, 123.03, 119.11, 117.08, 115.92, 114.98, 108.44, 77.25,
66.45, 66.08, 64.37, 62.12, 49.13, 47.81, 37.62, 31.40, 29.87, 29.20,
C
42H39F3N8O10 [M+H]+: 873.2741, found 873.2751.
4-(3-(4-(4-(13-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindo-
lin-4-yl)oxy)-2,5,8,11-tetraoxatridecyl)-1H-1,2,3-triazol-1-yl)
benzyl)-4,4-dimethyl-2,5-dioxoimidazolidin-1-yl)-2-(tri-
23.37, 23.34, 22.64. ESI-HRMS: m/z cacld. For
[M+H]+: 889.2513, found 889.2519.
C42H39F3N8O9S
fluoromethyl)benzonitrile (B4).
White solid. 75% yield, m.p.:236.3–237.5 ◦C. 1H NMR (400 MHz,
CDCl3) δ 8.43 (s, 1H), 8.19 (d, J = 1.5 Hz, 1H), 8.11 – 8.02 (m, 1H), 7.95
(d, J = 8.5 Hz, 1H), 7.74 (d, J = 8.5 Hz, 1H), 7.66 (dd, J = 8.3, 7.4 Hz,
1H), 7.54 (d, J = 8.5 Hz, 1H), 7.44 (d, J = 7.2 Hz, 1H), 7.25 (s, 1H), 4.93
(dd, J = 12.0, 5.3 Hz, 1H), 4.79 (d, J = 12.9 Hz, 1H), 4.68 (s, 1H), 4.35 –
4.28 (m, 1H), 3.96 – 3.88 (m, 1H), 3.79 – 3.72 (m, 2H), 3.71 – 3.64 (m,
3H), 2.90 – 2.79 (m, 1H), 2.73 (dd, J = 33.7, 18.4, 11.7 Hz, 1H), 2.34 (t,
J = 6.7 Hz, 1H), 2.15 – 2.05 (m, 1H), 1.87 (dt, J = 12.7, 6.2 Hz, 2H), 1.46
(s, 6H). 13C NMR (100 MHz, DMSO‑d6) δ 174.62, 172.80, 169.90,
166.80, 155.80, 153.16, 145.15, 138.37, 137.05, 136.79, 136.02,
133.10, 130.15, 128.94, 124.22, 121.99, 119.87, 116.14, 115.23, 65.76,
63.21, 62.04, 48.69, 28.75, 22.60, 21.94. ESI-HRMS: m/z cacld. For
4-(3-(4-(4-(13-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindo-
lin-4-yl)oxy)-2,5,8,11-tetraoxatridecyl)-1H-1,2,3-triazol-1-yl)
benzyl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl)-2-(tri-
fluoromethyl)benzonitrile (B8).
Brown solid. 80% yield, m.p.:223.3–224.5 ◦C, 1H NMR (400 MHz,
CDCl3) δ 8.36 (d, J = 17.7 Hz, 1H), 8.08 (s, 1H), 7.99 (d, J = 8.2 Hz, 2H),
7.95 (s, 1H), 7.84 (d, J = 8.2 Hz, 1H), 7.75 (d, J = 8.3 Hz, 3H), 7.65 (dd,
J = 14.5, 6.4 Hz, 2H), 7.59 (d, J = 8.2 Hz, 2H), 7.46 (t, J = 8.0 Hz, 1H),
7.26 (d, J = 9.6 Hz, 6H), 5.17 (s, 3H), 4.94 (dd, J = 11.8, 5.0 Hz, 2H),
4.77 (s, 2H), 4.33 (s, 2H), 3.96 – 3.89 (m, 2H), 3.76 (dd, J = 9.8, 5.0 Hz,
4H), 3.73 – 3.62 (m, 12H), 2.85 (dd, J = 25.6, 8.7 Hz, 1H), 2.76 (dd, J =
16.4, 7.2 Hz, 1H), 2.15 – 2.09 (m, 2H), 1.50 (s, 6H). 13C NMR (100 MHz,
CDCl3) δ 174.32, 171.13, 168.53, 167.01, 165.82, 156.54, 153.38,
136.64, 136.20, 135.39, 133.74, 133.44, 128.07, 123.08, 123.03,
119.11, 117.08, 115.92, 114.98, 108.44, 77.25, 66.45, 66.08, 64.37,
62.12, 49.13, 47.81, 37.62, 31.40, 29.87, 29.20, 23.37, 23.34, 22.64.
ESI-HRMS: m/z cacld. For C44H43F3N8O10S [M+H]+: 933.2775, found
933.2765.
C
44H43F3N8O11 [M+H]+: 917.3003, found 917.3014.
4-(3-(4-(4-((3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-
4-yl)oxy)propoxy)methyl)-1H-1,2,3-triazol-1-yl)benzyl)-4,4-
dimethyl-5-oxo-2-thioxoimidazolidin-1-yl)-2-(trifluoromethyl)
benzonitrile (B5).
White solid. 89% yield, m.p.:231.2–232.7 ◦C. 1H NMR (400 MHz,
CDCl3) δ 8.50 (s, 1H), 8.00 (s, 1H), 7.97 (d, J = 6.1 Hz, 2H), 7.85 (dd, J
= 8.2, 1.6 Hz, 1H), 7.69 (d, J = 8.3 Hz, 2H), 7.67 – 7.60 (m, 1H), 7.57 (d,
J = 8.3 Hz, 2H), 7.39 (d, J = 7.2 Hz, 1H), 7.22 (d, J = 8.4 Hz, 1H), 5.15
(s, 2H), 4.93 (dd, J = 11.9, 5.3 Hz, 1H), 4.72 (s, 2H), 4.28 (t, J = 5.4 Hz,
2H), 3.83 (t, J = 5.2 Hz, 2H), 2.89 – 2.82 (m, 1H), 2.75 (ddd, J = 19.5,
14.1, 4.1 Hz, 2H), 2.20 – 2.13 (m, 2H), 2.09 (dd, J = 11.2, 6.1 Hz, 1H),
1.51 (d, J = 1.5 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ 174.32, 171.13,
168.53, 167.01, 165.82, 156.54, 153.38, 136.64, 136.20, 135.39,
133.74, 133.44, 128.07, 123.08, 123.03, 119.11, 117.08, 115.92,
114.98, 108.44, 77.25, 66.45, 66.08, 64.37, 62.12, 49.13, 47.81, 37.62,
31.40, 29.87, 29.20, 23.37, 23.34, 22.64. ESI-HRMS: m/z cacld. For
4-(3-(4-(4-((2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindo-
lin-4-yl)amino)ethoxy)ethoxy)methyl)-1H-1,2,3-triazol-1-yl)
benzyl)-4,4-dimethyl-2,5-dioxoimidazolidin-1-yl)-2-(tri-
fluoromethyl)benzonitrile (B9).
Yellow solid. 58% yield, m.p.:225.8–226.9 ◦C, 1H NMR (400 MHz,
CDCl3) δ 8.48 (s, 1H), 8.18 (d, J = 1.8 Hz, 1H), 8.10 (s, 1H), 8.04 (dd, J
= 8.4, 1.9 Hz, 1H), 7.94 (d, J = 8.4 Hz, 1H), 7.73 (d, J = 8.5 Hz, 2H),
7.50 (d, J = 8.8 Hz, 3H), 7.48 – 7.42 (m, 1H), 7.06 (d, J = 6.9 Hz, 1H),
6.91 (d, J = 8.5 Hz, 1H), 6.53 (s, 1H), 4.86 – 4.80 (m, 1H), 4.79 (s, 3H),
4.67 (d, J = 5.4 Hz, 2H), 3.78 (dd, J = 5.7, 2.8 Hz, 3H), 3.73 (dt, J = 5.9,
4.1 Hz, 4H), 3.48 (d, J = 3.6 Hz, 2H), 2.88 – 2.73 (m, 2H), 2.71 – 2.58
C
39H33F3N8O7S [M+H]+: 815.2145, found 815.2139.
(m, 2H), 2.10 – 2.04 (m, 1H), 1.77 (s, 3H), 1.47 (d, J = 5.2 Hz, 6H). 13
C
4-(3-(4-(4-((2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindo-
NMR (100 MHz, DMSO‑d6) δ 174.62, 172.80, 169.90, 166.80, 155.80,
153.16, 145.15, 138.37, 137.05, 136.79, 136.02, 133.10, 130.15,
128.94, 124.22, 121.99, 119.87, 116.14, 115.23, 65.76, 63.21, 62.04,
48.69, 28.75, 22.60, 21.94. ESI-HRMS: m/z cacld. For C40H36F3N9O8
[M+H]+: 828.2639, found 828.2631.
lin-4-yl)oxy)ethoxy)ethoxy)methyl)-1H-1,2,3-triazol-1-yl)benzyl)-
4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl)-2-(trifluoromethyl)
benzonitrile (B6).
White solid. 84% yield, m.p.:219.2–220.6 ◦C. 1H NMR (400 MHz,
CDCl3) δ 8.49 (s, 1H), 8.06 (s, 1H), 7.98 (t, J = 7.4 Hz, 1H), 7.95 (s, 1H),
7.84 (dd, J = 8.2, 1.8 Hz, 1H), 7.72 (d, J = 8.4 Hz, 2H), 7.67 – 7.61 (m,
1H), 7.56 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 7.3 Hz, 1H), 7.25 (d, J = 8.5
Hz, 1H), 5.17 (d, J = 7.6 Hz, 2H), 4.93 (dd, J = 12.1, 5.3 Hz, 1H), 4.78
(d, J = 8.4 Hz, 2H), 4.39 – 4.30 (m, 2H), 3.99 – 3.91 (m, 2H), 3.83 (d, J
= 2.7 Hz, 2H), 3.77 (d, J = 2.7 Hz, 2H), 2.82 (dd, J = 22.9, 9.8 Hz, 1H),
4-(3-(4-(4-((2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindo-
lin-4-yl)amino)ethoxy)ethoxy)methyl)-1H-1,2,3-triazol-1-yl)
benzyl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl)-2-(tri-
fluoromethyl)benzonitrile (B10).
Yellow solid. 55% yield, m.p.:248.1–249.7 ◦C, 1H NMR (400 MHz,
DMSO‑d6) δ 11.08 (s, 1H), 8.76 (s, 1H), 8.39 (d, J = 8.2 Hz, 1H), 8.34 (d,
J = 1.4 Hz, 1H), 8.12 (dd, J = 8.2, 1.4 Hz, 1H), 7.86 (d, J = 8.6 Hz, 2H),
7.69 (d, J = 8.6 Hz, 2H), 7.56 (dd, J = 8.4, 7.3 Hz, 1H), 7.14 (d, J = 8.7
Hz, 1H), 7.01 (d, J = 7.0 Hz, 1H), 6.61 (t, J = 5.8 Hz, 1H), 5.18 (s, 2H),
5.04 (dd, J = 12.9, 5.4 Hz, 1H), 4.63 (s, 2H), 3.68 – 3.60 (m, 7H), 3.51 –
3.44 (m, 2H), 2.92 – 2.81 (m, 1H), 2.55 (dd, J = 18.2, 10.2 Hz, 2H), 2.07
– 1.97 (m, 1H), 1.50 (s, 6H). 13C NMR (100 MHz, DMSO‑d6) δ 174.62,
172.80, 169.90, 166.80, 155.80, 153.16, 145.15, 138.37, 137.05,
136.79, 136.02, 133.10, 130.15, 128.94, 124.22, 121.99, 119.87,
116.14, 115.23, 65.76, 63.21, 62.04, 48.69, 28.75, 22.60, 21.94. ESI-
HRMS: m/z cacld. For C40H36F3N9O7S [M+H]+: 844.2410, found
844.2419.
2.77 – 2.67 (m, 2H), 2.09 (dd, J = 12.7, 7.2 Hz, 1H), 1.50 (s, 6H). 13
C
NMR (100 MHz, CDCl3) δ 174.32, 171.13, 168.53, 167.01, 165.82,
156.54, 153.38, 136.64, 136.20, 135.39, 133.74, 133.44, 128.07,
123.08, 123.03, 119.11, 117.08, 115.92, 114.98, 108.44, 77.25, 66.45,
66.08, 64.37, 62.12, 49.13, 47.81, 37.62, 31.40, 29.87, 29.20, 23.37,
23.34, 22.64. ESI-HRMS: m/z cacld. For C40H35F3N8O8S [M+H]+:
845.2251, found 845.2246.
4-(3-(4-(4-((2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoi-
soindolin-4-yl)oxy)ethoxy)ethoxy)ethoxy)methyl)-1H-1,2,3-tri-
azol-1-yl)benzyl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl)-
2-(trifluoromethyl)benzonitrile (B7).
Brown solid. 85% yield, m.p.:198.5–199.8 ◦C, 1H NMR (400 MHz,
CDCl3) δ 8.53 (s, 1H), 8.06 (s, 1H), 7.99 (d, J = 8.3 Hz, 1H), 7.95 (s, 1H),
7.84 (d, J = 8.2 Hz, 1H), 7.73 (d, J = 8.4 Hz, 2H), 7.69 – 7.61 (m, 1H),
9

J.-J. Liang et al.
Bioorganic & Medicinal Chemistry 45 (2021) 116331
4.3. AR binding affinity
Appendix A. Supplementary data
Relative binding affinities were determined by a competitive fluo-
rometric binding assay. Briefly, the assay entails titration of the test
compound against a preformed complex of Fluormone AL Green and the
AR-LBD (GST). The assay mixture was allowed to equilibrate at room
temperature in 384-well black plates for 4 h in the dark, after which the
fluorescence polarization values were measured in a SpectraMax Para-
digm Multi-Mode Detection Platform (Molecular Devices) using an
excitation wavelength of 485 nm and an emission wavelength of 535
nm.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmc.2021.116331.
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Declaration of Competing Interest
26 Qin C, Hu Y, Zhou B, et al. Discovery of QCA570 as an Exceptionally Potent and
Efficacious Proteolysis Targeting Chimera (PROTAC) Degrader of the Bromodomain
and Extra-Terminal (BET) Proteins Capable of Inducing Complete and Durable
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The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
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Acknowledgement
This work was supported by the National Natural Sciences Founda-
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11