Design and characterization of cereblon-mediated androgen receptor proteolysis-targeting chimeras

Article abstract of DOI:10.1016/j.ejmech.2020.112769

Proteolysis-targeting chimera (PROTAC)-mediated protein degradation is a rapidly emerging therapeutic intervention that induces the degradation of targeted proteins. Herein, we report the design and biological evaluation of a series of androgen receptor (AR) PROTAC degraders for the treatment of metastatic castration-resistant prostate cancer. Predominantly, instead of thalidomide, we utilized the TD-106 scaffold, a novel cereblon (CRBN) binder that was identified in our previous study. Our results suggest that the linker position in the TD-106 CRBN binder is critical for the efficiency of AR degradation. The compounds attached to the 6-position of TD-106 promoted better degradation of AR than those at the 5- and 7-positions. Among the synthesized AR PROTACs, the representative degrader 33c (TD-802) effectively induced AR protein degradation, with a degradation concentration 50% of 12.5 nM and a maximum degradation of 93% in LNCaP prostate cancer cells. Additionally, most AR PROTAC degraders, including TD-802, displayed good liver microsomal stability and in vivo pharmacokinetic properties. Finally, we showed that TD-802 effectively inhibited tumor growth in an in vivo xenograft study.

Full text of DOI:10.1016/j.ejmech.2020.112769

European Journal of Medicinal Chemistry 208 (2020) 112769
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design and characterization of cereblon-mediated androgen receptor
proteolysis-targeting chimeras
,
b
,
, f,
Akshay D. Takwale a 1, Seung-Hyun Jo e 1, Yeong Uk Jeon a, Hyung Soo Kim c,
Choong Hoon Shin d, Heung Kyoung Lee a, Sunjoo Ahn a, Chong Ock Lee a, Jae Du Ha a
,
,
**
Jeong-Hoon Kim e ***, Jong Yeon Hwang a
,
f
,
, b, *
a Therapeutics and Biotechnology Division, Korea Research Institute of Chemical Technology, Daejeon, 305-606, Republic of Korea
b Department of Medicinal Chemistry and Pharmacology, Korea University of Science and Technology, Daejeon, 34113, Republic of Korea
c Department of Chemistry, Korea Univeristy, Seoul, 02841, Republic of Korea
d Department of Chemistry, Sogang Univeristy, Seoul, 04107, Republic of Korea
e Disease Target Structure Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, 34141, Republic of Korea
f Department of Functional Genomics, Korea University of Science and Technology, Daejeon, 34113, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Proteolysis-targeting chimera (PROTAC)-mediated protein degradation is a rapidly emerging therapeutic
intervention that induces the degradation of targeted proteins. Herein, we report the design and bio-
logical evaluation of a series of androgen receptor (AR) PROTAC degraders for the treatment of metastatic
castration-resistant prostate cancer. Predominantly, instead of thalidomide, we utilized the TD-106
scaffold, a novel cereblon (CRBN) binder that was identified in our previous study. Our results suggest
that the linker position in the TD-106 CRBN binder is critical for the efficiency of AR degradation. The
compounds attached to the 6-position of TD-106 promoted better degradation of AR than those at the 5-
and 7-positions. Among the synthesized AR PROTACs, the representative degrader 33c (TD-802) effec-
tively induced AR protein degradation, with a degradation concentration 50% of 12.5 nM and a maximum
degradation of 93% in LNCaP prostate cancer cells. Additionally, most AR PROTAC degraders, including
TD-802, displayed good liver microsomal stability and in vivo pharmacokinetic properties. Finally, we
showed that TD-802 effectively inhibited tumor growth in an in vivo xenograft study.
Received 14 May 2020
Received in revised form
13 August 2020
Accepted 14 August 2020
Available online 26 August 2020
Keywords:
Androgen receptor
Proteolysis targeting chimera
Prostate cancer
Cereblon
TD-106
© 2020 Elsevier Ltd. All rights reserved.
1. Introduction
therefore, AR is a major target for prostate cancer therapies. Several
techniques, such as androgen deprivation therapy by castration and
The androgen receptor (AR), a member of the nuclear hormone
receptor superfamily, plays a vital role in the maintenance of male
secondary sexual characteristics and development of the prostate
gland. Upon binding to its natural ligand, dihydrotestosterone, AR
regulates target gene transcription [1e4]. Besides its role in
downstream signaling, AR is required for prostate cancer growth;
radiotherapy, are used as a standard of treatment [5,6]. Anti-
androgen small molecules (Fig. 1), including enzalutamide (1) and
apalutamide, which inhibit ligand binding to AR, are currently used
to treat AR-dependent prostate cancer [7e9]. Although anti-
androgen drugs are available, side effects as well as resistance
progressing to metastatic castration-resistant prostate cancer
(mCRPC) have been observed with these treatments, necessitating
novel anti-androgen modalities [10]. Targeted protein degradation
is a rapidly emerging therapeutic intervention that induces protein
degradation. Proteolysis-targeting chimera (PROTAC) is a hetero-
bifunctional molecule composed of a binder for target protein
binding, a binder for E3 ubiquitin ligase binding, and a linker for
connecting two binders [11]. PROTAC promotes the formation of a
ternary complex with the targeted protein and E3 ligase, leading to
ubiquitination of the targeted protein and subsequent proteasomal
* Corresponding author. Therapeutics and Biotechnology Division, Korea
Research Institute of Chemical Technology, Daejeon, 305-606, Republic of Korea.
** Corresponding author.
*** Corresponding author. Disease Target Structure Research Center, Korea
Research Institute of Bioscience and Biotechnology, Daejeon, 4141, Republic of
Korea.
E-mail addresses: jdha@krict.re.kr (J. Du Ha), jhoonkim@kribb.re.kr (J.-H. Kim),
jyhwang@krict.re.kr (J.Y. Hwang).
1
These authors contributed equally to this study.
https://doi.org/10.1016/j.ejmech.2020.112769
0223-5234/© 2020 Elsevier Ltd. All rights reserved.

A.D. Takwale, S.-H. Jo, Y.U. Jeon et al.
European Journal of Medicinal Chemistry 208 (2020) 112769
Fig. 1. Chemical structure of the reported androgen receptor (AR) antagonists.
degradation process. Currently, several E3 ubiquitin ligases,
including b-TRCP, MDM2, cIAP, VHL, and cereblon (CRBN), have
been used in PROTAC [12,13]. Among these, cIAP, VHL, and CRBN
have been successfully applied in the degradation of various targets
efficacy data of AR PROTAC have not been reported. The potency of
PROTAC is generally influenced by the use of the E3 ligase, linker,
and warhead independently. Therefore, we hypothesized that AR
PROTACs utilizing TD-106 as a CRBN binder can provide effective AR
degradation. Herein, we report the synthesis of AR degraders with
TD-106 and results from biological evaluations performed in vitro
and in vivo.
such as FKBP12, ERa, AR, BET, BCR-ABL, and RIPK2 [14e20].
Several groups have reported AR PROTAC degraders (Fig. 2).
ARV-110, developed by Arvinas, is the first clinical trial of a PROTAC
degrader for the treatment of patients with mCRPC [21]; however,
its structure remains unknown. Naito et al. have reported AR
degrader 4 by applying their targeted protein degradation platform,
named specific and nongenetic inhibitor of apoptosis protein (IAP)-
dependent protein erasers, for E3 ligase and pyrazole-based AR
antagonist 2 [22]. Furthermore, Crews et al. have reported AR
degrader 5 (ARCC-4), which utilizes a VHL ligand for the E3 ligase
and enzalutamide as the AR antagonist [23]. Recently, Wang et al.
have reported two degraders, 6 (ARD-69) and 7 (ARD-266), using
VHL ligands and tetramethylcyclobutane-based AR antagonist 3.
They systematically explored the effect of linkers and inhibitors,
and E3 ligases, achieving highly potent AR degraders with degra-
dation concentration 50% (DC50) values in the low nanomolar
ranges. Additionally, a single dose of ARD-69 effectively decreases
the number of AR in tumor tissues of xenograft mice [24].
Previously, we reported the novel CRBN binder 8 (TD-106) that
binds to CRBN and induces efficient degradation of well-known
neosubstrates, IKZF1/3. Moreover, we successfully validated its
applicability for PROTAC degraders, inducing BET protein degra-
dation [25]. As part of our efforts to develop targeted protein
degradation programs, we were interested in generating an effec-
tive AR PROTAC degrader mediated by TD-106 CRBN binder.
Although highly potent AR PROTAC degraders have been reported,
as mentioned above, no potent AR PROTAC degrader utilizing
thalidomide analogs to recruit CRBN E3 ligase has been reported.
Wang et al. reported only two compounds with very weak or no
degradation in LNCaP cells [24]. Moreover, in vivo antitumor
2. Results and discussion
2.1. Design and synthesis of AR-PROTAC
To generate a successful PROTAC degrader, the attachment of the
linker to the warhead, as well as the type of linker, is critical.
Although the X-ray co-crystal structure of the AR ligand-binding
domain with antagonist 3 has not been reported, we utilized the
molecular docking model of antagonist 3 published by Guo et al.
[26] as well as the AR co-crystal structure with agonists [27]. We
replaced the pyrazole-hydroxyethyl moiety of 3 with phenyl-
piperazine as an exit vector, as reported previously, as it was
exposed to solvent and was well-tolerated for structural modifi-
cations, making it suitable for conjugation to a CRBN ligand via
linkers (Fig. 3) [26]. For CRBN binding, we also explored the posi-
tion of the linkage at TD-106, particularly at the 5-, 6-, and 7-
positions.
Tetramethylcyclobutane-based AR antagonist 15 was prepared
from ethyl 4-fluorobenzoate 10 using a modified method, as shown
in Scheme 1. Briefly, acid 12 was prepared from starting material 10
by a substitution reaction with tert-butyl piperazine-1-carboxylate,
followed by hydrolysis. Amide coupling with intermediate 13,
prepared as previously described [26], provided 14, which was
transformed to the desired AR binder 15 by treatment with
hydrogen chloride.
CRBN binders 18, 20, and 26 were synthesized as shown in
Fig. 2. Chemical structure of the reported androgen receptor (AR) degraders.
2

A.D. Takwale, S.-H. Jo, Y.U. Jeon et al.
European Journal of Medicinal Chemistry 208 (2020) 112769
Fig. 3. Strategy for the tethering androgen receptor (AR) antagonist (3) and cereblon (CRBN) binder (8) in this study.
Scheme 1. Reagents and conditions; (a) DIPEA, DMSO, tert-butyl piperazine-1-carboxylate, 130 ꢀC; (b) LiOH$H2O, THF, H2O, MeOH, 40 ꢀC; (c) EDCI, HOBt, DIPEA, 13, DMF, room
temperature (rt); (d) 4.0 M HCl in dioxane, DCM, rt.
Scheme 2. Reagents and conditions: (a) EDCI, HOBt, DIPEA, 3-aminopiperidine-2,6-dione hydrochloride, DMF, room temperature (rt); (b) NaNO2, AcOH, rt; (c) DIPEA, DMF, 17, 90 ꢀC;
(d) TFA:DCM (1:1), rt. (e) Pd/C, H2, MeOH, rt; (f) K2CO3, ter-butyl-2-bromoacetate, DMF, rt.
3

A.D. Takwale, S.-H. Jo, Y.U. Jeon et al.
European Journal of Medicinal Chemistry 208 (2020) 112769
Scheme 2. Fluoro-substituted 2-aminobenzoic acid 16 was treated
with 3-aminopiperidine-2,6-dione hydrochloride in the presence
of EDCI, HOBt, and DIPEA at room temperature to provide the
corresponding amide 17. Cyclization of 17 by treatment with NaNO2
in AcOH furnished the desired CRBN binders 18. In the case of the
CRBN binder 20 bearing carboxylic acid linkage, 18b was treated
with the corresponding amines, that is, glycine tert-butyl ester,
azetidine carboxylic tert-butyl ester, and piperidine carboxylic tert-
butyl ester in the presence of DIPEA in DMF to furnish intermediate
19. The tert-butyl ester of 19 was converted to carboxylic acid 20 by
treatment with 50% trifluoroacetic acid (TFA) in DCM. For the gly-
colic acid linkage 26, intermediate 24 was prepared from hydrox-
ynitrobenzoic acid 21 using a similar protocol with 18. Then, 24 was
treated with tert-butyl-2-bromoacetate in DMF at room tempera-
ture to provide 25, which was converted to carboxylic acid 26 by
TFA treatment.
N-Boc-piperidine carboxaldehyde, N-Boc-2-oxo-7-azaspiro [3.5]
nonane, or tert-butyl 4-(2-oxoethyl)piperidine-1-carboxylate pro-
vided the corresponding 27a, 27b, and 27c, respectively. Alkylation
of 15 with tert-butyl 4-(2-iodoethoxy)piperidine-1-carboxylate in
the presence of K2CO3 at room temperature provided the corre-
sponding 27d. Following treatment with HCl, compounds of 27
were converted to 28. Finally, conjugation with CRBN binders 18,
20, and 26 provided the corresponding degraders 29e35, respec-
tively, via either nucleophilic substitution reaction or treatment
with EDCI, HOBt, and DIPEA in DMF. Compound 39 was synthesized
as shown in Scheme 4. CRBN binder 18b was treated with MeI in the
presence of K2CO3 in DMF at room temperature to provide 36.
Substitution reaction with tert-butyl piperidine-4-carboxylate hy-
drochloride in the presence of DIPEA in DMF furnished interme-
diate 37, which was converted to carboxylic acid 38 by treatment
with 50% TFA in DCM. Conjugation with 28b and 38 by treatment
with EDCI, HOBt, and DIPEA in DMF provided 39.
The synthesis of the AR PROTAC degraders is depicted in Scheme
3. We selected piperidine-based linkers of different lengths because
the cyclic linker decreases the flexibility of large chimeric mole-
cules, providing better physio-chemical properties, including the
number of single rotational bonds. Reductive alkylation of 15 with
2.2. Evaluation of AR PROTACs
All compounds synthesized in this study were evaluated by
Scheme 3. Reagents and conditions: (a) For 27a-27c, aldehyde/ketone, NaBH3CN, cat. AcOH, DCM, room temperature (rt). For 27d, halide, K2CO3, DMF, overnight (b) 4.0 M HCl in
dioxane, DCM, rt; (c) 18a, 18b, or 18c, DIPEA, DMF, 90 ꢀC; (d) 28a, 28b, 28c, or 28d, EDCI, HOBt, DIPEA, DMF, rt.
4

A.D. Takwale, S.-H. Jo, Y.U. Jeon et al.
European Journal of Medicinal Chemistry 208 (2020) 112769
Scheme 4. Reagents and conditions: (a) MeI, K2CO3, DMF, room temperature (rt); (b) tert-butyl piperidine-4-carboxylate hydrochloride, DIPEA, DMF, 90 ꢀC; (c) TFA:DCM (1:1), room
temperature; (d) 28b, EDCI, HOBt, DIPEA, DMF, room temperature.
western blotting for AR degradation at three concentrations (0.1, 1,
and 10 M). To examine the effects of the degraders, the anti-
case of the 7-position with the methylpiperazine linker, 31a
demonstrated poor AR degradation by 28% at 0.1 M. It was
m
m
proliferative activity of degraders was tested in AR-dependent
prostate cancer cell lines, LNCaP and VCaP. First, we investigated
the linkage position in TD-106 (Table 1). Three compounds of 29
(for the 5-position) and four compounds of 30 (for the 6-position)
were evaluated for AR degradation. Compound 29a, attached to
methylpiperidine at the 5-position, showed moderate AR degra-
observed that the linker conjugated to the 6-position of TD-106
(30) effectively reduced the AR protein level compared with
linkers conjugated at the 5- and 7-positions (29 and 31a). Next, we
evaluated the anti-proliferative activity of degraders in both LNCaP
and VCaP prostate cancer cell lines. Compound 30 showed a better
anti-proliferative effect in both cell lines than compound 29.
Overall, linker conjugation to the 6-position of TD-106 was superior
to conjugation to the 5- and 7-positions for both AR degradation
and anti-proliferative effects. In this study, VCaP cells were gener-
ally more sensitive than LNCaP cells to AR degraders.
dation by 77% at 1
robutylpiperidine and ethylpiperazine in the 5-position,
demonstrated weak AR degradation by 26% and 31% at 1 M,
mM, whereas 29b and 29c, attached with spi-
m
respectively. For the 6-position attachment, the methylpiperazine
linker 30a and ethyloxypiperidine linker 30d effectively reduced
Reportedly, the linker type and length of PRTOAC are critical for
effective and targeted protein degradation [28,29]. Therefore, we
further explored the linkers with different types of linkages with
the CRBN ligand. Here, we selected four linkages, glycine (X1),
azetidine carboxylic acid (X2), piperidine carboxylic acid (X3), and
the AR level by 79% and 81% at 0.1
30b and 30c, attached to the spirobutylpiperidine and ethyl-
piperidine linker, reduced the AR protein by 15% and 51% at 0.1 M,
mM, respectively. Compounds
m
respectively; thus, these were less potent than 30a and 30d. In the
Table 1
Investigation of linker attachment to TD-106 CRBN binder.
No.
29a
CRBN binder
AR degaradation in LNCaP(%)
Cyctotoxicity(CC50
, mM)
0.1
m
M
1
mM
10
m
M
LNCaP
VCaP
N.D.
15
77
78
0.613
29b
29c
0
0
26
31
71
90
13.4
N.D.
1.58
>10.0
30a
79
91
95
81
0.259
0.081
30b
30c
15
51
95
94
0.214
0.449
0.113
0.120
N.D.
87
30d
31a
86
28
89
60
0.523
N.D.
0.022
N.D.
83
5

A.D. Takwale, S.-H. Jo, Y.U. Jeon et al.
European Journal of Medicinal Chemistry 208 (2020) 112769
Table 3
glycolic acid (X4). We postulated that these linkages could affect
the conformation of PROTAC or pharmacological properties,
thereby providing superior molecules for in vivo studies. Using a
combinatorial matrix approach, a total of 16 degraders were syn-
thesized and evaluated for their potency in western blotting and
anti-proliferative assays, as described in Table 2. In western blotting
analyses, most compounds in this series effectively induced AR
Stability of AR PROTACs in mouse liver microsomes.
Compound
% of remaining after 30 mina
30a
30c
30d
32b
32c
33a
33c
33d
71.7 1.37
86.7 1.28
86.8 4.10
92.8 0.41
74.3 3.50
85.5 0.41
96.0 6.86
62.8 3.77
0.560 0.01
92.9 2.80
93.7 3.42
2.98 0.08
31.2 0.99
92.6 2.20
78.4 2.70
0.03 0.01
degradation by >60% at 0.1 mM, except for 33b, 34a, and 34d. The
anti-proliferative activity of AR PROTACs was evaluated in both
LNCaP and VCaP cells. Degraders 32b, 32c, and 33c demonstrated
good anti-proliferative activities against both cell lines.
34a
34b
Next, we investigated the mechanism of AR degradation with
the representative degrader 33c (TD-802). A dose-dependent
experiment was conducted to determine the DC50 and the
maximum degradation (Dmax) values, documented as 12.5 nM and
93%, respectively (Fig. 4A and B). Once androgen binds to the
ligand-binding domain (LBD) of AR, the AR-androgen dimer regu-
lates the transcription of its target genes. Some splice variants,
including AR-V7, lacking the LBD are major androgen-independent
drivers of AR-regulated gene expression in prostate cancer [30]. To
determine if AR degraders can degrade endogenously expressed
splice variant AR, TD-802 was treated in 22RV1 cells expressing
both full-length AR and the AR-V7 splice variant. Western blotting
analysis showed that TD-802 induced full-length AR protein
34c
34d
35a
35b
35c
Buspirone (positive control)
a
Mean SD, n ¼ 3.
induced AR degradation (Fig. 4D). Time-course experiments indi-
cated that TD-802 decreased AR levels by 80% after 4 h of incuba-
mM (Fig. 4E). However, 39 (TD-802-N-Me), which
supposedly does not bind to CRBN, did not induce AR degradation
(Fig. 4F) and exhibited a more 10-fold higher inhibition of prolif-
tion at 1
degradation, with a Dmax of 85% at 1
degradation with a Dmax of 34% at 10
m
M, but weak AR-V7 protein
M (Fig. 4C). To ascertain
m
eration than TD-802 in LNCaP (CC50 ¼ 1.6
mM) and VCaP
M) cell lines. These data indicate that the binding of
whether AR degradation by TD-802 is dependent on the ubiquitin-
proteasome system and Cullin-Ring ubiquitin ligase, we examined
the effects of bortezomib, the proteasome inhibitor, and MLN4924,
the neddylation inhibitor, along with TD-802. Pretreatment with
either bortezomib or MLN4924 completely inhibited TD-802-
(CC50 ¼ 0.499
m
TD-802 to CRBN is required for AR degradation and cell growth
inhibitory activity.
Table 2
Study of linkage effect for androgen receptor (AR) degradation and anti-proliferative activity.
No.
Linker
L1
X
AR degradation in LNCaP (%)
Anti-proliferative activity
(CC50 M)
,
m
0.1
m
M
1
m
M
10
m
M
LNCaP
VCaP
32a
32b
32c
32d
X1
X2
X3
X4
70
96
90
89
89
96
89
40
35
83
94
51
0.117
0.089
0.108
0.804
3.82
0.038
0.045
N$C.
33a
33b
33c
33d
L2
L3
L4
X1
X2
X3
X4
61
0
85
60
77
79
93
77
39
0
95
79
0.208
0.312
0.098
0.196
0.054
0.135
0.044
0.076
34a
34b
34c
34d
X1
X2
X3
X4
7
74
77
0
87
84
93
50
83
96
99
82
1.09
0.538
0.316
3.18
0.107
0.037
0.052
0.046
35a
35b
35c
35d
X1
X2
X3
X4
77
82
72
86
95
86
92
98
88
90
98
94
0.852
0.762
0.847
1.30
0.057
0.057
0.080
0.141
N$C., not calculated.
6

A.D. Takwale, S.-H. Jo, Y.U. Jeon et al.
European Journal of Medicinal Chemistry 208 (2020) 112769
Table 4
2.3. Pharmacokinetic studies of AR PROTACs
Pharmacokinetic parameters in male mice.
Based on the explicit effect of degraders in cells, we examined
whether degraders possessed appropriate liver microsomal stabil-
ity. Fifteen AR PROTACs were tested against mouse liver micro-
somes (Table 3). Most degraders were well-tolerated in the mouse
liver microsomes, whereas three degraders, 34a, 34d, and 35a,
were quickly metabolized with less than 30% remaining after a 30-
min incubation. A structure-microsomal stability relationship was
not observed with the linker in this series.
To ensure that the microsomal studies accurately predict in vivo
clearance, we assessed the in vivo pharmacokinetic (PK) properties
of a single dose. Four representative compounds (30d, 32b, 33c, and
35b) were selected and administered intravenously (IV), intraper-
itoneally (IP), and orally in this study (Fig. 5 and Table 4). The IV
Compound
30d
Injection
T
max (h) Cmax (mg/mL) T1/2 (h) AUCt (mg$h/L)
IV (5 mpk)
IP (5 mpk)
PO (30 mpk)
IV (5 mpk)
IP (5 mpk)
PO (30 mpk)
IV (5 mpk)
IP (5 mpk)
PO (30 mpk)
IV (5 mpk)
IP (5 mpk)
PO (30 mpk)
N/A
2.0
N/A
1.78
0.28
N/A
5.54
0.05
N/A
2.78
0.11
N/A
2.01
0.02
4.68
4.51
5.58
4.48
4.82
26.7
8.28
4.41
N/C
14.3
10.7
2.84
24.1
24.2
0.44
20.7
1.72
0.53
4.49
7.49
0.15
2.0
N/A
2.0
32b
33c
35b
0.67
N/A
0.03
4.0
N/A
1.67
0.7
4.35
4.24
19.7
N/A, not applicable; N/C, not calculated; IV, intravenous; IP, intraperitoneal; PO,
peroral.
Fig. 4. Characterization of androgen receptor (AR) degradation by 33c (TD-802) (A) A dose-dependent degradation of AR by TD-802. (B) Quantification of AR protein remaining and
DC50 graph from western blotting. (C) Degradation of full-length AR (AR-FL) and splicing variant (AR-V7) in 22RV1 cells. (D) UPS- and CRL-dependent AR degradation by TD-802. (E)
Time-dependent AR degradation by TD-802. (F) Degradation of AR by 39 (TD-802-N-Me). DC50, degradation concentration 50%; UPS, ubiquitin-proteasome system; CRL, Cullin-Ring
ubiquitin ligase.
Fig. 5. Plasma concentration-time profiles in male mice (n ¼ 3).
7

A.D. Takwale, S.-H. Jo, Y.U. Jeon et al.
European Journal of Medicinal Chemistry 208 (2020) 112769
Fig. 6. Effect of TD-802 on tumor growth (A) and body weight (B) of VCaP prostate cancer in SCID mice. *P ¼ 0.0440 vs. control group on the final day using Student’s t-test.
half-life of all compounds was longer than 4 h; in particular, 33c
showed a considerably longer half-life (t1/2 ¼ 8.28 h), which re-
evaluated in mouse liver microsomes and most degraders dis-
played good microsomal stability, with >70% remaining after
30 min incubation. Additionally, in vivo PK studies showed that AR
PROTACs displayed not only a long half-life of >4 h but also high
plasma exposure following both IV and IP injections. Finally, TD-
802 induced AR degradation demonstrated good antitumor effi-
cacy in an in vivo xenograft mouse model, suggesting that CRBN-
based AR degraders might be used for developing novel AR in-
terventions. Further optimization efforts are currently under
investigation for an orally bioavailable AR PROTAC degrader.
flected a high plasma exposure (AUCt ¼ 20.7
mg h/L). In the case of
IP administration, all compounds demonstrated similar half-lives of
4e5 h. Interestingly, 32b exhibited the highest Cmax value of
5.54
(AUCt ¼ 24.2
1.78e2.78 g h/L. Notably, 33c was quickly absorbed within 10 min
m
g/mL, which also reflected
a high plasma exposure
mg h/L); others demonstrated modest values of
m
after IP injection. Unfortunately, all degraders displayed extremely
poor absorption after oral injection.
4. Experimental section
2.4. In vivo studies of 33c (TD-802)
4.1. Chemistry
To validate the antitumor activity of TD-802 in vivo, SCID mice
(C$B-17/IcrCrj-scid, male) were injected with AR-positive VCaP
prostate cancer cells and grown until the tumor size could be
measured (~130 mm3). Then, vehicle (control, 20% PEG400 þ 3%
Tween 80 in PBS) or TD-802 were administered intraperitoneally
once a day for 10 days. Tumor volume and body weight were
measured every 1e3 days after compound administration. The tu-
mor growth rate was lower in mice administered TD-802 than in
those administered vehicle during drug treatment and the tumor
exhibited slow re-growth after stopping TD-802 treatment
(Fig. 6A). No additional loss in body weight or off-target effects was
observed (Fig. 6B). Collectively, these data demonstrated that the
AR degrader, TD-802, effectively inhibited the growth of AR-
positive tumors.
All reagents were obtained from commercial vendors and used
without further purification. Thin layer chromatography was
monitored by using pre-coated silica gel plates (Merck, art. 5715).
Purification of all compounds was achieved by normal phase
chromatographic technique (Silica gel 230e400 mesh).1H NMR and
13C NMR were denoted in parts per million (ppm) and were
recorded in Bruker Avance 300 and 500 using CDCl3, D2O, CD3OD,
and DMSO‑d6. LC/MS analysis was executed with electron spay
ionization technique with Agilent Technology 6130 quadrupole
instrument.
4.1.1. Synthesis of tert-butyl 4-(4-(ethoxycarbonyl)phenyl)
piperazine-1-carboxylate (11)
To a solution of ethyl 4-fluorobenzoate (5.0 g, 29.7 mmol) and
tert-butyl piperazine-1-carboxylate (6.7 g, 35.64 mmol) in DMSO
(40 mL) was added DIPEA (15.5 mL, 89.1 mmol). The mixture was
stirred at 130 ꢀC for 24 h. The reaction mixture was cooled to room
temperature and diluted with water and extracted with ethyl ac-
etate. The organic layer was washed with brine, dried over Na2SO4,
and concentrated under reduced pressure. The residue was purified
by column chromatography to afford 11 (5.5 g, 55%) as a white solid.
3. Conclusion
The AR protein plays a key role in prostate cancer. Thus, the
inhibition of AR signaling by blocking androgen synthesis (e.g.,
abiraterone) or ligand binding to the AR (e.g., enzalutamide and
apalutamide) is a common strategy for treating prostate cancer.
Most patients initially respond to these drugs but partially exhibit
drug resistance by genetic alterations such as gene amplification or
mutations. Given these mechanisms of resistance, degradation of
the AR protein by PROTAC can be considered an alternative method
for mCRPC treatment.
1H NMR (300 MHz, CDCl3)
d 7.97e7.88 (m, 2H), 6.89e6.82 (m, 2H),
4.33 (q, J ¼ 7.1 Hz, 2H), 3.58 (m, 4H), 3.30 (m, 4H), 1.49 (s, 9H), 1.37
(t, J ¼ 7.1 Hz, 3H).
In this study, we designed and evaluated a series of CRBN-
mediated AR PROTACs, in which a tetramethylcyclobutane-based
AR antagonist was linked with TD-106 CRBN binder. Most AR
PROTACs synthesized in this study reduced the AR protein levels in
LNCaP cells. In particular, we examined the effect of the linker
position in the CRBN binder, TD-106, and observed that the 6-
position in TD-106 was better than the 5-position for AR degrada-
tion. The elongation of the linker by different linkages in TD-106
improved AR degradation as well as proliferative inhibition of
LNCaP and VCaP cells. The metabolic stability of AR PROTACs was
4.1.2. Synthesis of 4-(4-(2-(1-(tert-butoxycarbonyl)piperidin-4-yl)
ethyl)piperazin-1-yl)benzoic acid (12)
To a solution of 11 (5.5 g, 16.44 mmol) in MeOH (10 mL), H2O
(10 mL) and THF (40 mL) was added LiOH,H2O (1 g, 24.66 mmol).
The mixture was stirred at 40 ꢀC for 12 h. The reaction mixture was
cooled to room temperature and concentrated. The crude residue
was diluted with water and extracted with ethyl acetate. The
aqueous layer was acidified with 1 N HCl solution, and the pre-
cipitate was washed with water and dried to afford 12 (1.6 g, 85%)
8

A.D. Takwale, S.-H. Jo, Y.U. Jeon et al.
European Journal of Medicinal Chemistry 208 (2020) 112769
as a white solid. 1H NMR (500 MHz, CDCl3)
d
8.20e7.68 (m, 2H),
(m, 1H), 2.09 (m, 1H), 2.00e1.89 (m, 1H).
7.02e6.73 (m, 2H), 3.59 (t, J ¼ 5.2 Hz, 4H), 3.34 (t, J ¼ 5.2 Hz, 4H),
1.49 (s, 9H).
4.1.8. Synthesis of 2-amino-N-(2,6-dioxopiperidin-3-yl)-4-
fluorobenzamide (17c)
4.1.3. Synthesis of 4-((1R, 3R)-3-amino-2,2,4,4-
tetramethylcyclobutoxy)-2-chlorobenzonitrile hydrogen chloride
(13)
Similar procedure of 17a was performed to give 17c (240 mg,
71%) as white solid. 1H NMR (500 MHz, DMSO‑d6)
d 10.85 (s, 1H),
8.50 (d, J ¼ 8.4 Hz, 1H), 7.64e7.54 (m, 1H), 6.77 (s, 2H), 6.48 (d,
J ¼ 11.1 Hz, 1H), 6.36 (t, J ¼ 8.3 Hz, 1H), 4.72 (m, 1H), 2.84e2.73 (m,
1H), 2.56 (m, 1H), 2.18e2.06 (m, 1H), 1.96 (m, 1H).
Compound 13 was prepared by a similar procedure reported in
the literature [26].1H NMR (400 MHz, D2O)
d
7.74 (d, J ¼ 8.8 Hz, 1H),
7.19 (d, J ¼ 2.4 Hz, 1H), 7.00 (dd, J ¼ 8.8, 2.5 Hz, 1H), 4.42 (s, 1H), 3.36
(s, 1H), 1.37 (s, 6H), 1.19 (s, 6H).
4.1.9. Synthesis of 3-(5-fluoro-4-oxobenzo[d][1,2,3] triazin-3(4H)-
yl)piperidine-2,6-dione (18a)
4.1.4. Synthesis of tert-butyl 4-(4-(((1R,3R)-3-(3-chloro-4-
cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl) carbamoyl)phenyl)
piperazine-1-carboxylate (14)
To a solution of 17a (5 g, 56.55 mmol) in AcOH (120 mL) was
added sodium nitrite (2.2 g, 32.04 mmol). The mixture was stirred
at room temperature for 2 h. The mixture was diluted with water
and the white precipitate was collected, washed with water, dried
To a solution of 12 (1.5 g, 4.75 mmol), 13 (2.9 g, 9.51 mmol),
EDCI,HCl (1.0 g, 5.22 mmol), and HOBt (0.705 g, 5.22 mmol) in DMF
(20 mL) was added DIPEA (3.3 mL, 19.0 mmol). The mixture was
stirred at room temperature for 12 h. The reaction mixture was
diluted with water and extracted with ethyl acetate. The organic
layer was washed with brine, dried over Na2SO4, and concentrated
under reduced pressure. The residue was purified by column
chromatography to afford 14 (2.2 g, 81%) as a white solid. 1H NMR
to afford 18a (4 g, 75%). 1H NMR (300 MHz, DMSO‑d6)
d 11.21 (s,1H),
8.24e8.03 (m, 2H), 7.86e7.72 (m, 1H), 5.97 (dd, J ¼ 12.1, 5.4 Hz, 1H),
2.96 (m, 1H), 2.76e2.58 (m, 2H), 2.35e2.16 (m, 1H).
4.1.10. Synthesis of 3-(6-fluoro-4-oxobenzo[d][1,2,3] triazin-3(4H)-
yl)piperidine-2,6-dione (18b)
Similar procedure of 18a was performed to give 18b (4.8 g, 88%)
(300 MHz, CDCl3)
d
7.78e7.64 (m, 2H), 7.57 (d, J ¼ 8.7 Hz, 1H), 6.97
as a yellow solid. 1H NMR (400 MHz, DMSO‑d6)
d 11.21 (s, 1H),
(d, J ¼ 2.4 Hz, 1H), 6.94e6.89 (m, 2H), 6.81 (dd, J ¼ 8.7, 2.5 Hz, 1H),
6.14 (d, J ¼ 8.1 Hz,1H), 4.15 (d, J ¼ 9.0 Hz,1H), 4.05 (s,1H), 3.65e3.56
(m, 4H), 3.27 (m, 4H), 1.49 (s, 9H), 1.27 (s, 6H), 1.22 (s, 6H).
8.46e8.34 (m, 1H), 8.04 (m, 2H), 6.00 (dd, J ¼ 12.4, 5.4 Hz, 1H),
3.05e2.90 (m, 1H), 2.81e2.62 (m, 2H), 2.39e2.21 (m, 1H).
4.1.11. Synthesis of 3-(7-fluoro-4-oxobenzo[d][1,2,3]triazin-3(4H)-
yl)piperidine-2,6-dione (18c)
4.1.5. Synthesis of N-((1R,3R)-3-(3-chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)-4-(piperazin-1-yl) benzamide (15)
To a solution of 14 (1.5 g, 2.64 mmol) in DCM (10 mL) was added
4.0 N HCl in dioxane (1.0 mL). The mixture was stirred at room
temperature for 3 h and concentrated under reduced pressure. The
crude residue was diluted with 5% K2CO3 and extracted with ethyl
acetate. The organic layer was washed with brine, dried over
Na2SO4, and concentrated under reduced pressured. The residue
was purified by column chromatography to afford 15 (1.0 g, 83%) as
Similar procedure of 18a was performed to give 18c (200 mg,
80%) as white solid. 1H NMR (300 MHz, DMSO‑d6)
d 11.22 (s, 1H),
8.37 (dd, J ¼ 8.8, 5.6 Hz, 1H), 8.17 (dd, J ¼ 8.9, 2.5 Hz, 1H), 7.87 (m,
1H), 6.01 (dd, J ¼ 12.2, 5.4 Hz, 1H), 3.08e2.89 (m, 1H), 2.81e2.57 (m,
2H), 2.30 (m, 1H).
4.1.12. Synthesis of tert-butyl (3-(2,6-dioxopiperidin-3-yl)-4-oxo-
3,4-dihydrobenzo[d][1,2,3] triazin-6-yl)glycinate (19a)
a white solid. 1H NMR (300 MHz, CDCl3)
d 7.70 (m, 2H), 7.57 (d,
To a solution of 18b (1 g, 3.6 mmol) and glycine tert-butyl ester
hydrochloride (902 mg, 5.4 mmol) in DMSO (20 mL) was added
DIPEA (2.5 mL, 14.4 mmol). The reaction mixture was stirred at
90 ꢀC for 2 h. The mixture was diluted with water and the white
precipitate was collected, washed with water, dried to afford 19a.
J ¼ 8.7 Hz, 1H), 6.97 (d, J ¼ 2.4 Hz, 1H), 6.94e6.88 (m, 2H), 6.81 (dd,
J ¼ 8.7, 2.4 Hz, 1H), 6.11 (d, J ¼ 8.2 Hz, 1H), 4.15 (d, J ¼ 8.1 Hz, 1H),
4.04 (s, 1H), 3.40e3.19 (m, 4H), 3.13e2.97 (m, 4H), 1.26 (m, 7H), 1.22
(s, 6H).
1H NMR (300 MHz, CDCl3)
d 8.12 (s, 1H), 7.95 (m, 1H), 7.17 (m, 2H),
4.1.6. Synthesis of 2-amino-N-(2,6-dioxopiperidin-3-yl)-6-
fluorobenzamide (17a)
5.79 (dd, J ¼ 11.5, 5.1 Hz, 1H), 5.24 (m, 1H), 3.94 (d, J ¼ 4.8 Hz, 2H),
3.08e2.76 (m, 3H), 2.44e2.32 (m, 1H), 1.52 (s, 9H).
To a solution of 2-amino-6-floro-benzoic acid (5 g, 32.23 mmol),
3-amino pyridine-2,6-dione hydrochloride (5.26 g, 35.45 mmol),
EDCI,HCl (6.8 g, 35.45 mmol), and HOBt (4.8 g, 35.45 mmol) in DMF
(150 mL) was added DIPEA (17 mL, 96.69 mmol). The mixture was
to stirred at room temperature for 12 h. The reaction mixture was
diluted with water and extracted with ethyl acetate. The organic
layer was washed with brine, dried over Na2SO4 and concentrated
under reduced pressure. The crude compound 17a was obtained as
a yellow solid (8.0 g, 94%) and used in the next step without further
4.1.13. Synthesis of tert-butyl 1-(3-(2,6-dioxopiperidin-3-yl)-4-oxo-
3,4-dihydrobenzo[d][1,2,3] triazin-6-yl)azetidine-3-carboxylate
(19b)
Similar procedure of 19a was performed to give 19b (800 mg,
53%) as a white solid. 1H NMR (500 MHz, CDCl3)
d 8.07 (s, 1H), 7.98
(dd, J ¼ 8.9, 1.4 Hz, 1H), 7.06 (m, 1H), 6.93 (m, 1H), 5.77 (dd, J ¼ 11.4,
5.4 Hz, 1H), 4.26 (t, J ¼ 8.4 Hz, 2H), 4.21 (t, J ¼ 7.0 Hz, 2H), 3.59e3.51
(m, 1H), 3.01e2.81 (m, 3H), 2.38 (m, 1H), 1.49 (s, 9H).
purification. 1H NMR (300 MHz, DMSO‑d6)
d 10.90 (s, 1H), 8.53 (dd,
J ¼ 8.2, 2.8 Hz, 1H), 7.10 (m, 1H), 6.52 (d, J ¼ 8.2 Hz, 1H), 6.33 (dd,
J ¼ 10.6, 8.1 Hz, 1H), 6.00 (s, 2H), 4.75 (m, 1H), 2.88e2.69 (m, 1H),
2.57 (m, 1H), 2.20e1.94 (m, 2H).
4.1.14. Synthesis of tert-butyl 2-(1-(3-(2,6-dioxopiperidin-3-yl)-4-
oxo-3,4-dihydrobenzo[d][1,2,3] triazin-6-yl)piperidin-4-yl)acetate
(19c)
4.1.7. Synthesis of N-(2,6-dioxopiperidin-3-yl)-5-fluoro-2-
nitrobenzamide (17b)
Similar procedure of 19a was performed to give 19c (850 mg,
53%) as a yellow solid. 1H NMR (300 MHz, DMSO‑d6)
d 11.16 (s, 1H),
Similar procedure of 17a was performed to give 17b (5 g, 58%) as
8.01 (d, J ¼ 9.2 Hz, 1H), 7.72 (dd, J ¼ 9.3, 2.9 Hz, 1H), 7.38 (d,
J ¼ 2.8 Hz, 1H), 5.89 (dd, J ¼ 12.3, 5.4 Hz, 1H), 4.09e3.94 (m, 3H),
3.12 (t, J ¼ 12.2 Hz, 2H), 3.02e2.88 (m, 1H), 2.76e2.60 (m, 2H), 2.23
(m, 1H), 1.91 (d, J ¼ 13.2 Hz, 2H), 1.61 (m, 2H), 1.41 (s, 9H).
a grey solid. 1H NMR (500 MHz, DMSO‑d6)
d 10.86 (s, 1H), 8.55 (d,
J ¼ 8.3 Hz, 1H), 7.37 (dd, J ¼ 10.5, 3.0 Hz, 1H), 7.08 (m, 1H), 6.73 (dd,
J ¼ 9.1, 4.9 Hz,1H), 6.33 (s, 2H), 4.73 (m,1H), 2.84e2.71 (m,1H), 2.55
9

A.D. Takwale, S.-H. Jo, Y.U. Jeon et al.
European Journal of Medicinal Chemistry 208 (2020) 112769
4.1.15. Synthesis of (3-(2,6-dioxopiperidin-3-yl)-4-oxo-3,4-
dihydrobenzo[d][1,2,3] triazin-6-yl)glycine (20a)
To a solution of 19a (1.0 g, 2.5 mmol) in DCM (15 mL) was added
TFA (15 mL). The reaction mixture was stirred at room temperature
for 1 h and concentrated. The crude residue was purified by silica
gel chromatography to afford 20a (600 mg, 70%) as a white solid. 1H
washed with brine, dried over Na2SO4, and concentrated under
reduced pressure. The residue was purified by column chroma-
tography to afford 24 (1 g, 40%) as an ivory solid. 1H NMR (500 MHz,
DMSO‑d6)
d
11.18 (s, 1H), 8.13 (d, J ¼ 8.8 Hz, 1H), 7.51 (dd, J ¼ 8.9,
2.7 Hz, 1H), 7.47 (d, J ¼ 2.6 Hz, 1H), 5.92 (dd, J ¼ 12.5, 5.4 Hz, 1H),
3.05e2.88 (m, 1H), 2.73e2.59 (m, 2H), 2.25 (m, 1H).
NMR (300 MHz, DMSO‑d6)
d 12.84 (s, 1H), 11.14 (s, 1H), 7.94 (d,
J ¼ 8.9 Hz, 1H), 7.50 (t, J ¼ 6.0 Hz, 1H), 7.38 (dd, J ¼ 8.9, 2.6 Hz, 1H),
7.11e6.97 (m, 1H), 5.86 (dd, J ¼ 12.2, 5.4 Hz, 1H), 4.06 (d, J ¼ 5.9 Hz,
2H), 2.96 (m, 1H), 2.66 (m, 2H), 2.23 (m, 1H).
4.1.21. Synthesis of tert-butyl 2-((3-(2,6-dioxopiperidin-3-yl)-4-
oxo-3,4-dihydrobenzo[d][1,2,3] triazin-6-yl)oxy)acetate (25)
To a solution of 24 (1 g, 3.64 mmol), mL) and potassium car-
bonate (0.497 g, 3.6 mmol) in DMF (10 mL) was added tert-butyl
bromoacetate (0.3 mL, 2.1 mmol). The reaction mixture was stirred
at room temperature for 3 h. The reaction mixture was quenched
with water and then extracted with ethyl acetate. The organic layer
was washed with brine, dried over Na2SO4, and concentrated under
reduced pressure. The residue was purified by column chroma-
tography to afford 25 (1 g, 72%) as a white solid. 1H NMR (500 MHz,
4.1.16. Synthesis of 1-(3-(2,6-dioxopiperidin-3-yl)-4-oxo-3,4-
dihydrobenzo[d][1,2,3] triazin-6-yl)azetidine-3-carboxylic acid
(20b)
Similar procedure of 20a was performed to give 20b (512 mg,
76%) as a white solid. 1H NMR (300 MHz, DMSO‑d6)
d 12.79 (s, 1H),
11.14 (s,1H), 8.01 (d, J ¼ 8.9 Hz,1H), 7.15 (dd, J ¼ 8.9, 2.6 Hz,1H), 6.91
(d, J ¼ 2.6 Hz, 1H), 5.88 (dd, J ¼ 12.2, 5.4 Hz, 1H), 4.28 (t, J ¼ 8.6 Hz,
2H), 4.15 (m, 2H), 3.72e3.55 (m, 1H), 2.96 (m, 1H), 2.76e2.59 (m,
2H), 2.29e2.16 (m, 1H).
DMSO‑d6)
d
11.22 (s, 1H), 8.23 (d, J ¼ 9.0 Hz, 1H), 7.72 (dd, J ¼ 9.0,
2.9 Hz, 1H), 7.53 (d, J ¼ 2.9 Hz, 1H), 6.03e5.91 (m, 1H), 5.00 (s, 2H),
3.02e2.91 (m, 1H), 2.75e2.63 (m, 2H), 2.31e2.23 (m, 1H), 1.44 (s,
9H).
4.1.17. Synthesis of 1-(3-(2,6-dioxopiperidin-3-yl)-4-oxo-3,4-
dihydrobenzo[d][1,2,3] triazin-6-yl)piperidine-4-carboxylic acid
(20c)
4.1.22. Synthesis of 2-((3-(2,6-dioxopiperidin-3-yl)-4-oxo-3,4-
dihydrobenzo[d][1,2,3] triazin-6-yl)oxy)acetic acid (26)
Similar procedure of 20a was performed to give 20c (500 mg,
To a solution of 25 (1.0 g, 2.5 mmol) in DCM (15 mL) was added
TFA (15 mL). The reaction mixture was stirred at room temperature
for 1 h. The reaction mixture was concentrated under vacuum to
afford crude solid. The crude solid was purified by silica gel chro-
matography to afford 26 (625 mg, 79%) as a white solid. 1H NMR
67%) as a white solid. 1H NMR (300 MHz, DMSO)
d 12.31 (s, 1H),
11.15 (s,1H), 8.01 (d, J ¼ 9.2 Hz,1H), 7.72 (dd, J ¼ 9.3, 2.9 Hz,1H), 7.38
(d, J ¼ 2.8 Hz, 1H), 5.89 (dd, J ¼ 12.2, 5.4 Hz, 1H), 4.01 (d, J ¼ 13.5 Hz,
2H), 3.20e3.06 (m, 2H), 3.01e2.88 (m, 1H), 2.76e2.56 (m, 3H), 2.24
(m, 1H), 1.98e1.86 (m, 2H), 1.62 (m, 2H).
(400 MHz, DMSO‑d6)
d
11.18 (s, 1H), 8.22 (d, J ¼ 9.0 Hz, 1H), 7.72 (dd,
J ¼ 9.0, 2.9 Hz, 1H), 7.55 (d, J ¼ 2.9 Hz, 1H), 6.01e5.90 (m, 1H), 5.01
4.1.18. Synthesis of N-(2,6-dioxopiperidin-3-yl)-5-hydroxy-2-
nitrobenzamide (22)
(s, 2H), 3.04e2.90 (m, 1H), 2.78e2.62 (m, 2H), 2.32e2.22 (m, 1H).
To a solution of 21 (5 g, 27.3 mmol), 3-aminopiperidine-2,6-
dione hydrochloride (3.5 g, 27.3 mmol), EDCI,HCl (5.8 g,
30.03 mmol), and HOBt (4 g, 30.03 mmol) in DMF (20 mL) was
added DIPEA (19 mL, 109.2 mmol). The reaction mixture was stirred
at room temperature for 12 h. The reaction mixture was diluted
with water and extracted with ethyl acetate. The organic layer was
washed with brine, dried over Na2SO4, and concentrated under
reduced pressure. The residue was purified by column chroma-
tography to afford 22 (7.5 g, 93%) as a yellow solid. 1H NMR
4.1.23. Synthesis of tert-butyl 4-((4-(4-(((1r,3r)-3-(3-chloro-4-
cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl) carbamoyl) phenyl)
piperazin-1-yl)methyl)piperidine-1-carboxylate (27a)
To
a mixture of 15 (700 mg, 1.49 mmol), tert-butyl 4-
formylpiperidine-1-carboxylate (1.3 g, 5.99 mmol), and AcOH
(1.0 mL) in MeOH (15 mL) was added NaBH3CN (561 mg,
8.94 mmol). The mixture was stirred at room temperature for 12 h.
The reaction mixture was concentrated under reduced pressure.
The residue was quenched with saturated NaHCO3 solution and
extracted with dichloromethane. The organic layer was washed
with brine, dried over Na2SO4, and concentrated under reduced
pressure. The residue was purified by column chromatography to
afford 27a (700 mg, 70%) as a white solid. 1H NMR (400 MHz, CDCl3)
(500 MHz, CD3OD
(dd, J ¼ 12.6, 5.2 Hz, 1H), 2.79e2.69 (m, 1H), 2.67e2.57 (m, 1H)
2.29e2.16 (m, 1H), 2.01 (m, 1H).
d
7.98 (d, J ¼ 8.7 Hz, 1H), 6.89e6.82 (m, 2H), 4.72
4.1.19. Synthesis of 2-amino-N-(2,6-dioxopiperidin-3-yl)-5-
hydroxybenzamide (23)
d
7.73e7.65 (m, 2H), 7.56 (d, J ¼ 8.7 Hz, 1H), 6.97 (d, J ¼ 2.4 Hz, 1H),
6.94e6.89 (m, 2H), 6.81 (dd, J ¼ 8.7, 2.4 Hz, 1H), 6.11 (d, J ¼ 8.2 Hz,
1H), 4.14 (m, 2H), 4.04 (m, 2H), 3.29 (t, J ¼ 5.0 Hz, 4H), 2.71 (t,
J ¼ 12.6 Hz, 2H), 2.56 (t, J ¼ 5.1 Hz, 4H), 2.23 (d, J ¼ 7.0 Hz, 2H), 1.75
(d, J ¼ 13.5 Hz, 2H),1.67 (m,1H),1.46 (s, 9H),1.26 (s, 6H),1.22 (s, 6H),
1.16e1.05 (m, 2H); LC/MS (M þ H)þ (m/z) 664.1.
To a solution of 22 (7.5 g, 25.5 mmol) in MeOH (4 mL) was added
10% Pd/C (0.725 g). The reaction mixture was stirred at room
temperature for 12 h under hydrogen gas environment. The solu-
tion was filtered through a pad of Celite and removed under vac-
uum to give crude oil. The crude residue was purified by silica gel
column chromatography to afford 23 (2.5 g, 38%) as a blue solid. 1H
NMR (500 MHz, DMSO‑d6)
d
10.84 (s, 1H), 8.62 (s, 1H), 8.42 (d,
4.1.24. Synthesis of tert-butyl 2-(4-(4-(((1R,3R)-3-(3-chloro-4-
cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl) carbamoyl) phenyl)
piperazin-1-yl)-7-azaspiro[3.5]nonane-7-carboxylate (27b)
Similar procedure of 27a was performed to give 27b (450 mg,
J ¼ 8.4 Hz, 1H), 6.93 (d, J ¼ 2.8 Hz, 1H), 6.71 (dd, J ¼ 8.7, 2.7 Hz, 1H),
6.57 (d, J ¼ 8.7 Hz, 1H), 5.72 (s, 2H), 4.71 (m, 1H), 2.78, (m, 1H), 2.54
(m, 1H), 2.09 (m, 1H), 1.97e1.83 (m, 1H).
93%) as a white solid. 1H NMR (500 MHz, CDCl3)
d
7.69 (d, J ¼ 8.4 Hz,
4.1.20. Synthesis of 3-(6-hydroxy-4-oxobenzo[d][1,2,3] triazin-
3(4H)-yl)piperidine-2,6-dione (24)
To a solution of 23 (2.5 g, 9.1 mmol) in AcOH (120 mL) was added
sodium nitrite (1.3 g, 18.2 mmol). The reaction mixture was stirred
at room temperature for 2 h. The reaction mixture was diluted with
water and extracted with ethyl acetate. The organic layer was
2H), 7.57 (d, J ¼ 8.7 Hz,1H), 6.96 (t, J ¼ 1.8 Hz,1H), 6.91 (d, J ¼ 8.5 Hz,
2H), 6.83e6.75 (m, 1H), 6.10 (d, J ¼ 8.1 Hz, 1H), 4.15 (d, J ¼ 8.0 Hz,
1H), 4.04 (s, 1H), 3.37 (m, 2H), 3.30 (m, 6H), 2.74 (p, J ¼ 7.9 Hz, 1H),
2.47 (t, J ¼ 5.0 Hz, 4H), 2.05 (t, J ¼ 9.5 Hz, 2H),1.67 (t, J ¼ 9.6 Hz, 2H),
1.58 (m, 2H), 1.51 (t, J ¼ 5.7 Hz, 2H), 1.45 (s, 9H), 1.26 (s, 6H), 1.22 (s,
6H).; LC/MS (M þ H)þ (m/z) 690.2.
10

A.D. Takwale, S.-H. Jo, Y.U. Jeon et al.
European Journal of Medicinal Chemistry 208 (2020) 112769
4.1.25. Synthesis of tert-butyl 4-(2-(4-(4-(((1R,3R)-3-(3-chloro-4-
cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl) carbamoyl) phenyl)
piperazin-1-yl)ethyl)piperidine-1-carboxylate (27c)
1.58e1.48 (m, 2H), 1.31 (m, 8H), 1.25 (s, 6H).
4.1.30. Synthesis of N-((1R,3R)-3-(3-chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)-4-(4-(2-(piperidin-4-yloxy) ethyl)
piperazin-1-yl) benzamide hydrochloride (28d)
Similar procedure of 27a was performed to give 27c (400 mg,
92%) as a white solid. 1H NMR (500 MHz, CDCl3)
d
7.69 (d, J ¼ 8.6 Hz,
2H), 7.56 (d, J ¼ 8.7 Hz, 1H), 6.96 (d, J ¼ 2.4 Hz, 1H), 6.91 (d,
J ¼ 8.9 Hz, 2H), 6.81 (dd, J ¼ 8.7, 2.4 Hz, 1H), 6.13 (d, J ¼ 8.1 Hz, 1H),
4.15 (d, J ¼ 8.1 Hz, 1H), 4.05 (s, 1H), 3.37 (t, J ¼ 4.9 Hz, 4H), 2.71 (m,
6H), 2.53 (t, J ¼ 7.8 Hz, 3H), 1.67 (d, J ¼ 12.6 Hz, 2H), 1.57e1.49 (m,
2H), 1.46 (s, 9H), 1.26 (m, 8H), 1.22 (s, 6H), 1.13 (m, 2H); LC/MS
(M þ H)þ (m/z) 678.47.
Similar procedure of 28a was performed to give 28d (680 mg,
91%) as a white solid. 1H NMR (300 MHz, DMSO‑d6)
d 11.43 (s, 1H),
9.09 (s, 2H), 7.91 (d, J ¼ 8.7 Hz, 1H), 7.82 (d, J ¼ 8.5 Hz, 2H), 7.64 (d,
J ¼ 9.2 Hz, 1H), 7.21 (d, J ¼ 2.4 Hz, 1H), 7.05 (d, J ¼ 8.7 Hz, 1H), 7.01
(dd, J ¼ 8.8, 2.4 Hz, 1H), 4.35 (s, 1H), 4.07 (d, J ¼ 9.2 Hz, 1H),
4.00e3.82 (m, 4H), 3.75e3.53 (m, 4H), 3.38 (m, 4H), 3.24 (m, 3H),
3.03e2.87 (m, 2H), 2.00 (m, 2H), 1.77 (m, 2H), 1.23 (s, 6H), 1.13 (s,
6H).
4.1.26. Synthesis of tert-butyl 4-(2-(4-(4-(((1R,3R)-3-(3-chloro-4-
cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl) carbamoyl) phenyl)
piperazin-1-yl)ethoxy)piperidine-1-carboxylate (27d)
4.1.31. Synthesis of N-((1R,3R)-3-(3-chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)-4-(4-((1-(3-(2,6-dioxopiperidin-3-
yl)-4-oxo-3,4-dihydrobenzo[d][1,2,3]triazin-5-yl)piperidin-4-yl)
methyl)piperazin-1-yl)benzamide (29a)
To a solution of 13 (700 mg, 1.49 mmol) and potassium car-
bonate (412 mg, 2.98 mmol) in DMF (20 mL) was added - tert-butyl
4-(2-iodoethoxy) piperidine-1-carboxylate (793 mg, 2.2 mmol).
The mixture was stirred at room temperature overnight. The re-
action was quenched with water and extracted with ethyl acetate.
The organic layer was washed with brine, dried over Na2SO4 and
concentrated under reduced pressure. The residue was purified by
column chromatography to afford 27d (820 mg, 70%) as a white
To a solution of 28a (80 mg, 0.133 mmol) and 18a (44 mg,
0.159 mmol) in DMF (10 mL) was added DIPEA (0.092 mL,
0.532 mmol). The reaction mixture was stirred at 90 ꢀC for 12 h. The
reaction mixture was diluted with water and extracted with ethyl
acetate. The organic layer was washed with brine, dried over
Na2SO4 and concentrated under reduced pressure. The residue was
purified by column chromatography to afford 29a (68 mg, 62%) as a
solid. 1H NMR (300 MHz, CDCl3)
d
7.71 (d, J ¼ 8.8 Hz, 2H), 7.58 (d,
J ¼ 8.7 Hz, 1H), 6.98 (d, J ¼ 2.4 Hz, 1H), 6.93 (d, J ¼ 8.8 Hz, 2H), 6.83
(dd, J ¼ 8.7, 2.4 Hz, 1H), 6.13 (d, J ¼ 8.1 Hz, 1H), 4.17 (d, J ¼ 8.5 Hz,
1H), 4.06 (s, 1H), 3.88e3.72 (m, 2H), 3.67 (t, J ¼ 5.8 Hz, 2H), 3.48 (m,
1H), 3.34 (t, J ¼ 5.1 Hz, 4H), 3.11 (m, 2H), 2.70 (m, 6H), 1.93e1.78 (m,
2H), 1.56 (m, 2H), 1.48 (s, 9H), 1.28 (s, 6H), 1.24 (s, 6H).
yellow solid. 1H NMR (500 MHz, CDCl3)
d 8.27 (s, 1H), 7.80 (t,
J ¼ 8.0 Hz,1H), 7.70 (d, J ¼ 8.4 Hz, 2H), 7.67 (d, J ¼ 7.9 Hz,1H), 7.57 (d,
J ¼ 8.7 Hz, 1H), 7.25 (m, 1H), 6.97 (d, J ¼ 2.4 Hz, 1H), 6.92 (d,
J ¼ 8.5 Hz, 2H), 6.81 (dd, J ¼ 8.8, 2.4 Hz, 1H), 6.13 (d, J ¼ 8.1 Hz, 1H),
5.84e5.77 (m, 1H), 4.15 (d, J ¼ 8.1 Hz, 1H), 4.05 (s, 1H), 3.50 (t,
J ¼ 11.3 Hz, 2H), 3.32 (t, J ¼ 5.0 Hz, 4H), 2.99e2.78 (m, 5H), 2.61 (m,
4H), 2.34 (m, 3H), 1.91 (d, J ¼ 12.7 Hz, 2H), 1.72 (m, 2H), 1.61 (m, 1H),
4.1.27. Synthesis of N-((1R,3R)-3-(3-chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)-4-(4-(piperidin-4-ylmthyl)
piperazin-1-yl) benzamide hydrochloride (28a)
1.27 (s, 6H), 1.22 (s, 6H); 13C NMR (101 MHz, CDCl3)
d 170.92, 168.11,
A mixture of 27a (700 mg, 1.053 mmol) and 4.0 N HCl in dioxane
(2 mL) in DCM (10 mL) was stirred at room temperature for 2 h. The
mixture was concentrated under reduced pressure to afford 28a
167.14, 162.66, 154.07, 153.57, 152.76, 146.83, 138.27, 135.51, 135.08,
128.27, 120.75, 116.77, 116.38, 114.25, 114.19, 111.26, 105.01, 84.91,
77.25, 64.53, 58.44, 58.17, 53.61, 53.38, 47.86, 40.32, 33.05, 31.09,
30.96, 23.61, 23.53, 23.43; LC/MS (M þ H)þ(m/z) 820.1, (M ꢁ H)- (m/
z) 818.0.
(600 mg, 94%) as a white solid. 1H NMR (500 MHz, CD3OD)
d 7.81 (d,
J ¼ 8.3 Hz, 2H), 7.72 (d, J ¼ 8.7 Hz, 1H), 7.16e7.05 (m, 3H), 6.98 (d,
J ¼ 8.8 Hz,1H), 4.29 (s, 1H), 4.14 (s,1H), 4.02 (d, J ¼ 13.7 Hz, 2H), 3.76
(m, 2H), 3.65 (m, 3H), 3.46 (d, J ¼ 13.1 Hz, 2H), 3.37 (t, J ¼ 12.7 Hz,
2H), 3.23 (d, J ¼ 6.8 Hz, 2H), 3.09 (t, J ¼ 12.9 Hz, 2H), 2.15 (d,
J ¼ 14.5 Hz, 2H),1.59 (q, J ¼ 12.8 Hz, 2H),1.28 (s, 6H),1.22 (s, 6H); LC/
MS (M þ H)þ (m/z) 564.1.
4.1.32. Synthesis of N-((1R,3R)-3-(3-chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)-4-(4-(7-(3-(2,6-dioxopiperidin-3-
yl)-4-oxo-3,4-dihydrobenzo[d][1,2,3]triazin-5-yl)-7-azaspiro[3.5]
nonan-2-yl)piperazin-1-yl)benzamide (29b)
Similar procedure of 29a was performed to give 29b (48 mg,
4.1.28. Synthesis of 4-(4-(7-azaspiro [3.5] nonan-2-yl) piperazin-1-
yl)-N-((1R3R)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-
44%) as a yellow solid. 1H NMR (500 MHz, CDCl3)
d 8.51 (s, 1H), 7.79
(t, J ¼ 8.0 Hz, 1H), 7.70 (d, J ¼ 8.5 Hz, 2H), 7.66 (d, J ¼ 7.9 Hz,1H), 7.56
(d, J ¼ 8.7 Hz, 1H), 7.25 (d, J ¼ 8.2 Hz, 1H), 6.97 (d, J ¼ 2.3 Hz, 1H),
6.92 (d, J ¼ 8.6 Hz, 2H), 6.81 (dd, J ¼ 9.0, 2.4 Hz, 1H), 6.13 (d,
J ¼ 8.1 Hz, 1H), 5.81 (dd, J ¼ 11.8, 5.3 Hz, 1H), 4.15 (d, J ¼ 8.0 Hz, 1H),
4.05 (s, 1H), 3.34 (t, J ¼ 5.0 Hz, 4H), 3.08 (d, J ¼ 34.9 Hz, 4H),
2.99e2.77 (m, 4H), 2.52 (m, 4H), 2.35 (m, 1H), 2.11 (dd, J ¼ 11.3,
7.5 Hz, 2H), 1.89 (t, J ¼ 5.3 Hz, 2H), 1.83 (m, 2H), 1.77 (m, 2H), 1.26 (s,
tetramethylcyclobutyl) benzamide hydrochloride (28b)
Similar procedure of 28a was performed to give 28b (350 mg,
90%) as a white solid. 1H NMR (300 MHz, CD3OD)
d 7.83 (d,
J ¼ 8.8 Hz, 2H), 7.74 (d, J ¼ 8.8 Hz,1H), 7.15 (d, J ¼ 2.4 Hz,1H), 7.12 (d,
J ¼ 8.8 Hz, 2H), 7.01 (dd, J ¼ 8.8, 2.4 Hz, 1H), 4.32 (s, 1H), 4.16 (s, 1H),
4.07 (d, J ¼ 13.7 Hz, 2H), 3.86 (t, J ¼ 8.3 Hz, 1H), 3.78e3.58 (m, 3H),
3.22 (t, J ¼ 5.7 Hz, 2H), 3.18e3.04 (m, 4H), 2.50e2.30 (m, 4H), 1.96
(q, J ¼ 6.5 Hz, 4H), 1.87e1.74 (m, 1H), 1.30 (s, 6H), 1.24 (s, 6H); LC/MS
(M þ H)þ (m/z) 590.1.
6H), 1.22 (s, 6H); 13C NMR (101 MHz, CDCl3)
d 171.01, 168.22, 167.12,
162.66, 154.04, 152.67, 146.82, 138.26, 135.47, 135.07, 128.28, 120.76,
116.77, 116.36, 114.36, 114.19, 111.22, 105.01, 84.92, 77.26, 58.46,
55.32, 50.47, 50.02, 49.52, 47.50, 40.32, 39.55, 36.74, 31.10, 31.01,
23.61, 23.52; LC/MS (M þ H)þ (m/z) 846.1, (M ꢁ H)- (m/z) 844.0.
4.1.29. Synthesis of 4-(4-(7-azaspiro [3.5] nonan-2-yl) piperazin-1-
yl)-N-((1R,3R)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-
tetramethylcyclobutyl) benzamide hydrochloride (28c)
4.1.33. Synthesis of N-((1R,3R)-3-(3-chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)-4-(4-(2-(1-(3-(2,6-dioxopiperidin-3-
yl)-4-oxo-3,4-dihydrobenzo[d][1,2,3]triazin-5-yl)piperidin-4-yl)
ethyl)piperazin-1-yl)benzamide (29c)
Similar procedure of 28a was performed to give 28c (330 mg,
90%) as a white solid. 1H NMR (500 MHz, CD3OD)
d 7.83 (d,
J ¼ 8.3 Hz, 2H), 7.74 (d, J ¼ 8.7 Hz, 1H), 7.15 (m, 1H), 7.12 (d,
J ¼ 8.4 Hz, 2H), 7.01 (m, 1H), 4.32 (s, 1H), 4.17 (s, 1H), 4.07 (m, 2H),
3.79e3.66 (m, 3H), 3.44 (d, J ¼ 12.9 Hz, 2H), 3.32e3.26 (m, 4H), 3.04
(t, J ¼ 12.6 Hz, 2H), 2.04 (d, J ¼ 14.0 Hz, 2H), 1.91e1.83 (m, 2H),
Similar procedure of 29a was performed to give 29c (55 mg,
51%) as a pink solid. 1H NMR (500 MHz, CDCl3)
d
8.20 (s, 1H), 7.80 (t,
J ¼ 8.0 Hz,1H), 7.71 (d, J ¼ 8.5 Hz, 2H), 7.67 (d, J ¼ 7.9 Hz,1H), 7.57 (d,
11

A.D. Takwale, S.-H. Jo, Y.U. Jeon et al.
European Journal of Medicinal Chemistry 208 (2020) 112769
J ¼ 8.7 Hz, 1H), 7.25 (d, J ¼ 8.4 Hz, 1H), 6.97 (d, J ¼ 2.4 Hz, 1H), 6.92
(d, J ¼ 8.5 Hz, 2H), 6.81 (m, 1H), 6.13 (d, J ¼ 8.1 Hz, 1H), 5.81 (dd,
J ¼ 11.7, 5.4 Hz,1H), 4.15 (d, J ¼ 8.0 Hz,1H), 4.05 (s, 1H), 3.48 (m, 5H),
3.01e2.63 (m, 10H), 2.36 (m, 1H), 1.83 (m, 2H), 1.65 (m, 5H), 1.27 (m,
116.77, 116.36, 114.31, 105.24, 105.02, 84.92, 77.26, 58.46, 58.12,
55.92, 52.97, 47.90, 40.32, 34.22, 33.25, 31.66, 31.07, 23.61, 23.52,
23.27; LC/MS (M þ H)þ (m/z) 834.20, (M ꢁ H)- (m/z) 832.31.
8H), 1.22 (s, 6H); 13C NMR (101 MHz, CDCl3)
d
171.09, 168.29, 167.05,
4.1.37. Synthesis of N-((1R,3R)-3-(3-chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)-4-(4-(2-((1-(3-(2,6-dioxopiperidin-
3-yl)-4-oxo-3,4-dihydrobenzo[d][1,2,3]triazin-6-yl)piperidin-4-yl)
oxy)ethyl)piperazin-1-yl)benzamide (30d)
162.66, 154.08, 152.69, 146.80, 138.26, 135.53, 135.08, 128.36,
120.88, 120.80, 116.78, 116.37, 114.70, 114.19, 111.27, 105.01, 84.89,
77.27, 58.50, 58.05, 56.00, 53.55, 53.37, 52.54, 47.16, 40.33, 33.84,
32.24, 31.11, 23.62, 23.53, 23.45; LC/MS (M þ H)þ (m/z) 834.14,
(M ꢁ H)- (m/z) 832.31.
Similar procedure of 29a was performed to give 30d (42 mg,
62%) as an ivory solid. 1H NMR (500 MHz, CDCl3)
d 8.33 (s, 1H), 7.99
(d, J ¼ 9.2 Hz, 1H), 7.69 (d, J ¼ 8.4 Hz, 2H), 7.56 (d, J ¼ 8.7 Hz, 1H),
7.55e7.51 (m, 1H), 7.44 (dd, J ¼ 9.2, 2.8 Hz, 1H), 6.99e6.95 (m, 1H),
6.91 (d, J ¼ 8.5 Hz, 2H), 6.84e6.77 (m, 1H), 6.12 (d, J ¼ 8.1 Hz, 1H),
5.81e5.73 (m,1H), 4.15 (d, J ¼ 8.1 Hz,1H), 4.04 (s,1H), 3.83e3.74 (m,
2H), 3.70 (t, J ¼ 5.8 Hz, 2H), 3.66e3.59 (m, 1H), 3.38e3.34 (m, 1H),
3.34e3.26 (m, 5H), 3.00e2.78 (m, 3H), 2.75e2.64 (m, 6H),
2.42e2.33 (m, 1H), 2.04e1.94 (m, 2H), 1.80e1.71 (m, 2H), 1.26 (s,
4.1.34. Synthesis of N-((1R,3R)-3-(3-chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)-4-(4-((1-(3-(2,6-dioxopiperidin-3-
yl)-4-oxo-3,4-dihydrobenzo[d][1,2,3]triazin-6-yl)piperidin-4-yl)
methyl)piperazin-1-yl)benzamide (30a)
Similar procedure of 29a was performed to give 30a (45 mg,
41%) as a yellow solid. 1H NMR (500 MHz, CDCl3)
d 8.36 (s, 1H), 7.98
(d, J ¼ 9.1 Hz,1H), 7.70 (d, J ¼ 8.4 Hz, 2H), 7.57 (d, J ¼ 8.7 Hz,1H), 7.52
(d, J ¼ 2.8 Hz, 1H), 7.45 (dd, J ¼ 9.2, 2.9 Hz, 1H), 6.99e6.94 (m, 1H),
6.92 (d, J ¼ 8.5 Hz, 2H), 6.81 (dd, J ¼ 8.2, 2.3 Hz, 1H), 6.14 (d,
J ¼ 8.1 Hz, 1H), 5.77 (dd, J ¼ 11.6, 5.4 Hz, 1H), 4.15 (d, J ¼ 8.1 Hz, 1H),
4.07 (s, 1H), 4.05 (m, 2H), 3.32 (t, J ¼ 5.0 Hz, 4H), 3.02 (t, J ¼ 12.5 Hz,
2H), 2.98e2.81 (m, 3H), 2.59 (t, J ¼ 5.0 Hz, 4H), 2.40e2.34 (m, 1H),
2.28 (d, J ¼ 7.1 Hz, 2H), 1.99e1.92 (m, 2H), 1.86 (m, 1H), 1.33 (m, 2H),
6H), 1.22 (s, 6H); 13C NMR (126 MHz, CDCl3)
d 171.20, 168.23, 167.10,
162.61, 156.02, 153.40, 152.95, 138.21, 136.39, 135.06, 130.34, 128.25,
123.89, 121.67, 121.44, 116.72, 116.40, 114.25, 114.14, 105.31, 104.90,
84.78, 74.14, 66.05, 58.37, 57.99, 53.31, 47.69, 44.94, 40.26, 31.07,
30.27, 23.58, 23.49, 23.23; LC/MS (M þ H)þ (m/z) 850.14, (M ꢁ H)-
(m/z) 848.45.
1.27 (s, 6H), 1.22 (s, 6H); 13C NMR (101 MHz, CDCl3)
d
171.03, 168.09,
4.1.38. Synthesis of N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)-4-(4-((1-(3-(2,6-dioxopiperidin-3-
yl)-4-oxo-3,4-dihydrobenzo[d][1,2,3]triazin-7-yl)piperidin-4-yl)
methyl)piperazin-1-yl)benzamide (31a)
167.13, 162.66, 156.09, 153.51, 153.29, 138.27, 136.37, 135.08, 130.34,
128.28, 123.93, 121.73, 121.51, 116.77, 116.37, 114.28, 114.19, 105.33,
105.01, 84.90, 64.23, 58.46, 58.12, 53.37, 47.79, 40.32, 33.30, 31.05,
30.13, 23.61, 23.52, 23.27; LC/MS (M þ H)þ (m/z) 820.1, (M ꢁ H)- (m/
z) 818.1.
Similar procedure of 29a was performed to give 31a (4 mg, 20%)
as a yellow solid. 1H NMR (500 MHz, CDCl3)
d
8.13 (d, J ¼ 9.1 Hz, 2H),
7.73e7.67 (m, 2H), 7.57 (d, J ¼ 8.7 Hz, 1H), 7.38 (d, J ¼ 2.6 Hz, 1H),
7.32 (dd, J ¼ 9.1, 2.6 Hz, 1H), 6.97 (d, J ¼ 2.4 Hz, 1H), 6.95e6.90 (m,
2H), 6.81 (dd, J ¼ 8.7, 2.4 Hz, 1H), 6.13 (d, J ¼ 8.1 Hz, 1H), 5.79 (dd,
J ¼ 11.7, 5.3 Hz, 1H), 4.15 (d, J ¼ 8.1 Hz, 1H), 4.04 (m, 3H), 3.32 (m,
4H), 3.09e2.84 (m, 5H), 2.60 (m, 4H), 2.42e2.35 (m, 1H), 2.30 (d,
J ¼ 7.1 Hz, 2H),1.96 (d, J ¼ 13.3 Hz, 2H),1.34 (m, 2H),1.27 (s, 6H),1.25
(m, 1H), 1.22 (s, 6H); LC/MS (M þ H)þ (m/z) 820.2, (M ꢁ H)- (m/z)
818.1.
4.1.35. Synthesis of N-((1R,3R)-3-(3-chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)-4-(4-(7-(3-(2,6-dioxopiperidin-3-
yl)-4-oxo-3,4-dihydrobenzo[d][1,2,3]triazin-6-yl)-7-azaspiro[3.5]
nonan-2-yl)piperazin-1-yl)benzamide (30b)
Similar procedure of 29a was performed to give 30b (45 mg,
41%) as a yellow solid. 1H NMR (500 MHz, CDCl3)
d 8.63 (s, 1H), 7.98
(d, J ¼ 9.1 Hz,1H), 7.70 (d, J ¼ 8.4 Hz, 2H), 7.56 (d, J ¼ 8.7 Hz,1H), 7.51
(d, J ¼ 2.8 Hz, 1H), 7.43 (dd, J ¼ 9.2, 2.8 Hz, 1H), 6.97 (m, 1H), 6.92 (d,
J ¼ 8.5 Hz, 2H), 6.81 (dd, J ¼ 8.8, 2.4 Hz, 1H), 6.14 (d, J ¼ 8.1 Hz, 1H),
5.76 (dd, J ¼ 11.7, 5.4 Hz, 1H), 4.15 (d, J ¼ 8.1 Hz, 1H), 4.05 (s, 1H),
3.53e3.46 (m, 4H), 3.34 (t, J ¼ 4.9 Hz, 4H), 2.98e2.79 (m, 4H), 2.52
(m, 4H), 2.37 (m, 1H), 2.11 (dd, J ¼ 11.4, 7.5 Hz, 2H), 1.76 (m, 4H), 1.72
(t, J ¼ 5.6 Hz, 2H), 1.26 (s, 6H), 1.22 (s, 6H); 13C NMR (101 MHz,
4.1.39. Synthesis of N-((1R,3R)-3-(3-chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)-4-(4-((1-((3-(2,6-dioxopiperidin-3-
yl)-4-oxo-3,4-dihydrobenzo[d][1,2,3]triazin-6-yl)glycyl)piperidin-
4-yl)methyl)piperazin-1-yl)benzamide (32a)
To a solution of 28a (50 mg, 0.0832 mmol), 20a (33 mg,
0.0998 mmol), EDCI,HCl (18 mg, 0.0915 mmol), and HOBt (12 mg,
0.0915 mmol) in DMF (5 mL) was added DIPEA (0.072 mL,
0.416 mmol). The reaction mixture was stirred at room temperature
for 12 h. The reaction mixture was diluted with water and extracted
with ethyl acetate. The organic layer was washed with brine, dried
over Na2SO4, and concentrated under reduced pressure. The res-
idue was purified by column chromatography to afford 32a (50 mg,
CDCl3) d 171.13, 168.19, 167.11, 162.66, 156.06, 153.19, 138.26, 136.34,
135.08, 130.34, 128.29, 121.63, 121.52, 116.77, 116.36, 114.38, 114.19,
105.27, 105.01, 84.91, 77.26, 58.47, 58.16, 55.08, 49.49, 47.49, 44.93,
44.77, 40.32, 38.62, 36.46, 35.96, 31.49, 31.07, 23.61, 23.52, 23.26;
LC/MS (M þ H)þ (m/z) 846.1, (M ꢁ H)- (m/z) 844.0.
4.1.36. Synthesis of N-((1R,3R)-3-(3-chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)-4-(4-(2-(1-(3-(2,6-dioxopiperidin-3-
yl)-4-oxo-3,4-dihydrobenzo[d][1,2,3]triazin-6-yl)piperidin-4-yl)
ethyl)piperazin-1-yl)benzamide (30c)
68%) as a white solid. 1H NMR (500 MHz, CDCl3)
d 9.03 (s, 1H), 7.90
(d, J ¼ 8.9 Hz, 1H), 7.70 (d, J ¼ 8.5 Hz, 2H), 7.56 (d, J ¼ 8.7 Hz, 1H),
7.25 (dd, J ¼ 9.0, 2.6 Hz, 1H), 7.13 (d, J ¼ 2.7 Hz, 1H), 6.97 (d,
J ¼ 2.4 Hz, 1H), 6.91 (d, J ¼ 8.6 Hz, 2H), 6.81 (dd, J ¼ 8.7, 2.4 Hz, 1H),
6.18 (d, J ¼ 8.2 Hz, 1H), 6.12 (t, J ¼ 3.8 Hz, 1H), 5.81 (m, 1H), 4.63 (d,
J ¼ 13.1 Hz, 1H), 4.15 (d, J ¼ 8.1 Hz, 1H), 4.05 (s, 1H), 4.00 (m, 2H),
3.83 (d, J ¼ 13.2 Hz, 1H), 3.31 (t, J ¼ 4.8 Hz, 4H), 3.06 (t, J ¼ 12.8 Hz,
1H), 2.96e2.81 (m, 3H), 2.71 (t, J ¼ 12.6 Hz, 1H), 2.58 (t, J ¼ 5.1 Hz,
4H), 2.39e2.33 (m, 1H), 2.26 (d, J ¼ 7.0 Hz, 2H), 1.94 (m, 3H), 1.27 (s,
6H), 1.22 (s, 6H), 1.20e1.10 (m, 2H); 13C NMR (101 MHz, CDCl3)
Similar procedure of 29a was performed to give 30c (48 mg,
44%) as a white solid. 1H NMR (500 MHz, CDCl3)
d 8.10 (s, 1H), 7.98
(d, J ¼ 9.1 Hz,1H), 7.70 (d, J ¼ 8.4 Hz, 2H), 7.57 (d, J ¼ 8.7 Hz,1H), 7.51
(d, J ¼ 2.8 Hz, 1H), 7.43 (dd, J ¼ 9.1, 2.9 Hz, 1H), 6.96 (m, 1H), 6.92 (d,
J ¼ 8.5 Hz, 2H), 6.81 (dd, J ¼ 8.6, 2.1 Hz, 1H), 6.11 (d, J ¼ 8.1 Hz, 1H),
5.76 (dd, J ¼ 11.5, 5.4 Hz, 1H), 4.15 (d, J ¼ 8.1 Hz, 1H), 4.09e3.98 (m,
3H), 3.32 (t, J ¼ 4.8 Hz, 4H), 2.98 (m, 4H), 2.84 (m, 1H), 2.61 (m, 4H),
2.47 (t, J ¼ 7.6 Hz, 2H), 2.38 (m, 1H), 1.87 (d, J ¼ 13.1 Hz, 2H), 1.66 (m,
1H), 1.53 (m, 2H), 1.40e1.33 (m, 2H), 1.26 (s, 6H), 1.22 (s, 6H); 13C
d
171.53, 168.52, 167.18, 165.71, 162.66, 156.13, 153.47, 150.51, 138.23,
136.87, 135.09, 130.26, 128.30, 123.94, 122.81, 121.91, 116.78, 116.38,
114.26,114.19,104.97,101.30, 84.89, 77.30, 64.04, 58.47, 58.16, 58.06,
53.34, 47.77, 44.51, 44.38, 42.60, 40.33, 33.56, 31.04, 30.30, 23.62,
NMR (101 MHz, CDCl3)
d 171.09, 168.15, 167.11, 162.66, 156.09,
153.45, 153.22, 138.26, 136.33,135.07, 130.34, 128.28, 124.00, 121.64,
12

A.D. Takwale, S.-H. Jo, Y.U. Jeon et al.
European Journal of Medicinal Chemistry 208 (2020) 112769
23.51, 23.29; LC/MS (M þ H)þ (m/z) 877.1, (M ꢁ H)- (m/z) 875.0.
7.92 (d, J ¼ 8.8 Hz, 2H), 7.76 (d, J ¼ 8.6 Hz, 2H), 7.55e7.48 (m, 2H),
7.25 (t, J ¼ 5.2 Hz, 1H), 7.22 (d, J ¼ 2.4 Hz, 1H), 7.14 (m, 1H), 7.01 (dd,
J ¼ 8.8, 2.4 Hz, 1H), 6.98 (d, J ¼ 8.6 Hz, 2H), 5.87 (m, 1H), 4.33 (s, 1H),
4.18e4.14 (m, 2H), 4.06 (d, J ¼ 9.1 Hz,1H), 3.48 (m, 2H), 3.40 (m, 2H),
3.26 (m, 4H), 3.02e2.92 (m, 1H), 2.80e2.63 (m, 3H), 2.40 (m, 4H),
2.22 (m, 1H), 2.03 (m, 2H), 1.64 (m, 3H), 1.55 (m, 2H), 1.45 (m, 1H),
4.1.40. Synthesis of N-((1R,3R)-3-(3-chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)-4-(4-((1-(1-(3-(2,6-dioxopiperidin-
3-yl)-4-oxo-3,4-dihydrobenzo[d][1,2,3]triazin-6-yl)azetidine-3-
carbonyl)piperidin-4-yl)methyl)piperazin-1-yl)benzamide (32b)
Similar procedure of 32a was performed to give 32b (50 mg,
1.23 (s, 6H), 1.13 (s, 6H); 13C NMR (101 MHz, DMSO‑d6)
d 173.22,
66%) as a white solid. 1H NMR (400 MHz, CDCl3)
d
8.10 (s, 1H), 7.97
170.32, 167.24, 166.71, 163.11, 155.54, 153.42, 152.51, 137.33, 136.55,
136.03, 129.97, 129.34, 124.25, 121.81, 117.29, 116.76, 115.14, 114.01,
104.07, 79.65, 58.51, 55.38, 54.88, 54.78, 49.45, 47.54, 44.59, 41.80,
41.66, 36.52, 31.70, 31.24, 24.44, 23.60, 23.19; LC/MS (M þ H)þ (m/z)
903.1, (M ꢁ H)- (m/z) 901.0.
(d, J ¼ 8.8 Hz, 1H), 7.70 (d, J ¼ 8.6 Hz, 2H), 7.57 (d, J ¼ 8.7 Hz, 1H),
7.05 (d, J ¼ 2.6 Hz, 1H), 6.99e6.86 (m, 4H), 6.81 (dd, J ¼ 8.7, 2.4 Hz,
1H), 6.11 (d, J ¼ 8.1 Hz, 1H), 5.80e5.71 (m, 1H), 4.63 (d, J ¼ 13.5 Hz,
1H), 4.38e4.24 (m, 4H), 4.15 (d, J ¼ 8.0 Hz, 1H), 4.04 (s, 1H), 3.79 (p,
J ¼ 7.5 Hz, 1H), 3.59 (d, J ¼ 13.6 Hz, 1H), 3.31 (t, J ¼ 5.0 Hz, 4H),
3.12e3.02 (m, 1H), 3.00e2.80 (m, 3H), 2.71e2.53 (m, 5H), 2.37 (m,
1H), 2.27 (d, J ¼ 7.0 Hz, 2H), 1.93 (m, 1H), 1.86 (m, 2H), 1.26 (s, 6H),
4.1.44. Synthesis of N-((1Rr,3R)-3-(3-chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)-4-(4-(7-(1-(3-(2,6-dioxopiperidin-3-
yl)-4-oxo-3,4-dihydrobenzo[d][1,2,3]triazin-6-yl)azetidine-3-
carbonyl)-7-azaspiro[3.5]nonan-2-yl)piperazin-1-yl)benzamide
(33b)
1.22 (s, 6H), 1.13 (m, 2H); 13C NMR (126 MHz, CDCl3)
d 171.12,
168.88, 168.12, 167.08, 162.59, 155.85, 153.44, 152.80, 138.21, 136.42,
135.05, 130.42, 128.24, 123.85, 121.35, 118.29, 116.71, 116.39, 114.18,
104.90, 101.98, 84.77, 64.06, 58.37, 57.89, 53.72, 53.30, 47.77, 45.48,
42.31, 40.27, 33.59, 32.14, 31.39, 31.02, 30.32, 23.59, 23.50, 23.26.;
LC/MS (M þ H)þ (m/z) 903.1, (M ꢁ H)- (m/z) 901.0.
Similar procedure of 32a was performed to give 33b (45 mg,
61%) as a white solid. 1H NMR (500 MHz, DMSO‑d6)
d 11.20 (s, 1H),
8.07 (dd, J ¼ 8.9, 1.4 Hz, 1H), 7.97 (d, J ¼ 8.7 Hz, 1H), 7.81 (d,
J ¼ 8.5 Hz, 2H), 7.58 (d, J ¼ 9.2 Hz, 1H), 7.27 (d, J ¼ 2.4 Hz, 1H), 7.21
(dt, J ¼ 8.9, 2.4 Hz, 1H), 7.07 (dd, J ¼ 8.8, 2.4 Hz, 1H), 7.04 (d,
J ¼ 8.7 Hz, 2H), 6.96 (t, J ¼ 2.3 Hz, 1H), 5.95 (dd, J ¼ 12.1, 5.6 Hz, 1H),
4.38 (s, 1H), 4.35 (q, J ¼ 8.1 Hz, 2H), 4.24 (m, 2H), 4.12 (d, J ¼ 9.1 Hz,
1H), 3.98 (m, 1H), 3.54 (m, 1H), 3.48 (m, 1H), 3.29 (m, 6H),
3.06e2.96 (m, 1H), 2.83e2.78 (m, 1H), 2.76e2.68 (m, 2H), 2.45 (m,
4H), 2.29 (m, 1H), 2.06 (m, 2H), 1.67 (m, 3H), 1.58 (m, 2H), 1.50 (m,
4.1.41. Synthesis of N-((1R,3R)-3-(3-chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)-4-(4-((1-(1-(3-(2,6-dioxopiperidin-
3-yl)-4-oxo-3,4-dihydrobenzo[d][1,2,3]triazin-6-yl)piperidine-4-
carbonyl)piperidin-4-yl)methyl)piperazin-1-yl)benzamide (32c)
Similar procedure of 32a was performed to give 32c (48 mg,
62%) as a pink solid. 1H NMR (400 MHz, CDCl3)
d 8.05e7.95 (m, 2H),
7.69 (d, J ¼ 8.8 Hz, 2H), 7.57 (d, J ¼ 8.7 Hz, 1H), 7.52 (d, J ¼ 2.9 Hz,
1H), 7.45 (dd, J ¼ 9.2, 2.9 Hz, 1H), 6.96 (d, J ¼ 2.4 Hz, 1H), 6.94e6.88
(m, 2H), 6.81 (dd, J ¼ 8.8, 2.4 Hz, 1H), 6.11 (d, J ¼ 8.1 Hz, 1H), 5.77
(dd, J ¼ 11.5, 5.3 Hz, 1H), 4.64 (d, J ¼ 13.2 Hz, 1H), 4.15 (d, J ¼ 8.1 Hz,
1H), 4.10e4.03 (m, 3H), 3.95 (m, 1H), 3.30 (t, J ¼ 5.1 Hz, 4H), 3.10 (t,
J ¼ 12.4 Hz, 3H), 2.96 (m, 2H), 2.88e2.75 (m, 2H) 2.58 (m, 5H),
2.41e2.34 (m, 1H), 2.27 (d, J ¼ 6.9 Hz, 2H), 1.88 (m, 7H), 1.26 (s, 6H),
1H),1.28 (s, 6H),1.19 (s, 6H); 13C NMR (101 MHz, DMSO‑d6)
d 173.20,
170.30, 169.35, 167.23, 163.11, 155.32, 153.41, 153.32, 137.33, 136.54,
136.02, 130.43, 129.34, 124.25, 121.46, 119.21, 117.29, 116.75, 115.13,
114.01, 104.07, 101.20, 84.40, 79.65, 58.51, 54.85, 49.45, 47.54, 40.78,
36.51, 31.78, 31.74, 31.24, 24.44, 23.60, 23.22; LC/MS (M þ H)þ (m/z)
929.0, (M ꢁ H)- (m/z) 927.0.
1.22 (s, 6H), 1.13 (m, 2H); 13C NMR (101 MHz, CDCl3)
d
172.35,
4.1.45. Synthesis of N-((1R,3R)-3-(3-chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)-4-(4-(7-(1-(3-(2,6-dioxopiperidin-3-
yl)-4-oxo-3,4-dihydrobenzo[d][1,2,3]triazin-6-yl)piperidine-4-
carbonyl)-7-azaspiro[3.5]nonan-2-yl)piperazin-1-yl)benzamide
(33c)
171.34, 168.30, 167.18, 162.67, 156.02, 153.47, 153.16, 138.21, 136.52,
135.08, 130.27, 128.30, 123.92, 121.81, 121.42, 116.78, 116.38, 114.25,
114.19, 105.44, 104.94, 84.89, 77.31, 64.16, 58.47, 58.02, 53.33, 47.79,
47.09, 45.64, 42.11, 40.33, 37.85, 31.76, 31.06, 30.51, 28.06, 27.80,
23.62, 23.50, 23.29; LC/MS (M þ H)þ (m/z) 931.1, (M ꢁ H)- (m/z)
929.1.
Similar procedure of 32a was performed to give 33c (170 mg,
56%) as a white solid. 1H NMR (400 MHz, CDCl3)
d 8.20 (s, 1H), 7.99
(d, J ¼ 9.1 Hz, 1H), 7.70 (d, J ¼ 8.7 Hz, 2H), 7.56 (d, J ¼ 8.7 Hz, 1H),
7.52 (d, J ¼ 2.8 Hz, 1H), 7.45 (dd, J ¼ 9.3, 2.8 Hz, 1H), 6.96 (d,
J ¼ 2.4 Hz, 1H), 6.94e6.87 (m, 2H), 6.81 (dd, J ¼ 8.8, 2.4 Hz, 1H), 6.11
(d, J ¼ 8.1 Hz, 1H), 5.77 (dd, J ¼ 11.5, 5.4 Hz, 1H), 4.15 (d, J ¼ 8.1 Hz,
1H), 4.06 (m, 3H), 3.64e3.39 (m, 4H), 3.32 (t, J ¼ 5.0 Hz, 4H), 3.09 (t,
J ¼ 12.7 Hz, 2H), 3.00e2.72 (m, 5H), 2.49 (t, J ¼ 4.9 Hz, 4H),
2.42e2.32 (m, 1H), 2.09 (m, 2H), 1.94 (m, 2H), 1.85 (m, 2H), 1.74 (m,
2H), 1.64 (m, 2H), 1.56 (m, 2H), 1.26 (s, 6H), 1.22 (s, 6H); 13C NMR
4.1.42. Synthesis of N-((1R,3R)-3-(3-chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)-4-(4-((1-(2-((3-(2,6-dioxopiperidin-
3-yl)-4-oxo-3,4-dihydrobenzo[d][1,2,3]triazin-6-yl)oxy)acetyl)
piperidin-4-yl)methyl)piperazin-1-yl)benzamide (32d)
Similar procedure of 32a was performed to give 32d (15 mg,
26%) as a yellow solid. 1H NMR (500 MHz, CDCl3)
d 8.15 (d,
J ¼ 8.1 Hz, 2H), 7.69 (d, J ¼ 8.4 Hz, 2H), 7.64 (d, J ¼ 7.3 Hz, 2H), 7.56
(d, J ¼ 8.7 Hz, 1H), 6.96 (m, 1H), 6.91 (d, J ¼ 8.5 Hz, 2H), 6.81 (d,
J ¼ 8.8 Hz, 1H), 6.11 (d, J ¼ 8.1 Hz, 1H), 5.84e5.72 (m, 1H), 4.90 (m,
2H), 4.58 (d, J ¼ 13.2 Hz, 1H), 4.15 (d, J ¼ 8.1 Hz, 1H), 4.04 (s, 1H),
3.91e3.78 (m, 1H), 3.48 (q, J ¼ 6.9 Hz, 1H), 3.31 (m, 4H), 3.13 (t,
J ¼ 12.9 Hz, 1H), 3.04e2.90 (m, 2H), 2.89e2.82 (m, 1H), 2.68 (t,
J ¼ 13.0 Hz, 1H), 2.58 (m, 4H), 2.39 (m, 1H), 2.27 (m, 2H), 1.95 (d,
J ¼ 13.2 Hz, 1H), 1.86 (m, 1H), 1.26 (s, 6H), 1.22 (s, 6H), 1.20 (m, 2H);
LC/MS (M þ H)þ (m/z) 878.2, (M ꢁ H)- (m/z) 876.1.
(101 MHz, DMSO‑d6)
d 173.21, 172.41, 170.29, 167.23, 163.11, 155.45,
153.42, 153.21, 137.33, 136.55, 136.02, 130.34, 129.33, 124.24, 122.55,
121.53, 117.29, 116.76, 115.14, 114.01, 104.33, 104.07, 84.40, 79.65,
58.51, 49.46, 47.54, 46.94, 40.78, 37.25, 36.60, 31.82, 31.23, 28.01,
24.44, 23.60, 23.20; LC/MS (M þ H)þ (m/z) 957.1, (M ꢁ H)- (m/z)
955.0.
4.1.46. Synthesis of N-((1R,3R)-3-(3-chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)-4-(4-(7-(2-((3-(2,6-dioxopiperidin-
3-yl)-4-oxo-3,4-dihydrobenzo[d][1,2,3]triazin-6-yl)oxy)acetyl)-7-
azaspiro[3.5]nonan-2-yl)piperazin-1-yl)benzamide (33d)
4.1.43. Synthesis of N-((1R,3R)-3-(3-chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)-4-(4-(7-((3-(2,6-dioxopiperidin-3-
yl)-4-oxo-3,4-dihydrobenzo[d][1,2,3]triazin-6-yl)glycyl)-7-azaspiro
[3.5]nonan-2-yl)piperazin-1-yl)benzamide (33a)
Similar procedure of 32a was performed to give 33d (13 mg,
22%) as a white solid. 1H NMR (300 MHz, CDCl3)
d 8.34 (s, 1H), 8.14
Similar procedure of 32a was performed to give 33a (42 mg,
(d, J ¼ 9.8 Hz, 1H), 7.66 (m, 4H), 7.57 (d, J ¼ 8.7 Hz, 1H), 6.96 (d,
55%) as a white solid. 1H NMR (500 MHz, DMSO‑d6)
d 11.15 (s, 1H),
J ¼ 2.4 Hz, 1H), 6.92 (d, J ¼ 8.5 Hz, 2H), 6.81 (dd, J ¼ 8.7, 2.4 Hz, 1H),
13

A.D. Takwale, S.-H. Jo, Y.U. Jeon et al.
European Journal of Medicinal Chemistry 208 (2020) 112769
6.12 (d, J ¼ 8.1 Hz, 1H), 5.79 (m, 1H), 4.89 (d, J ¼ 2.7 Hz, 2H), 4.15 (d,
J ¼ 8.1 Hz, 1H), 4.04 (s, 1H), 3.56 (m, 1H), 3.44 (m, 1H), 3.33 (m, 5H),
3.14 (m, 1H), 3.01e2.88 (m, 2H), 2.83e2.72 (m, 1H), 2.49 (m, 4H),
2.43e2.34 (m, 1H), 2.16e1.98 (m, 4H), 1.74 (m, 5H), 1.26 (s, 6H), 1.22
(s, 6H); LC/MS (M þ H)þ (m/z) 904.1, (M ꢁ H)- (m/z) 902.0.
135.08, 130.29, 128.29, 121.81, 121.43, 116.78, 116.37, 114.29, 114.19,
105.46, 104.98, 84.90, 77.28, 58.46, 58.00, 55.90, 52.95, 47.76, 47.11,
45.76, 42.23, 40.32, 37.81, 34.60, 33.32, 33.22, 32.01, 31.08, 28.04,
27.82, 23.61, 23.51, 23.30; LC/MS (M þ H)þ (m/z) 945.1, (M ꢁ H)- (m/
z) 943.0.
4.1.47. Synthesis of N-((1R,3R)-3-(3-chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)-4-(4-(2-(1-((3-(2,6-dioxopiperidin-
3-yl)-4-oxo-3,4-dihydrobenzo[d][1,2,3]triazin-6-yl)glycyl)
piperidin-4-yl)ethyl)piperazin-1-yl)benzamide (34a)
4.1.50. Synthesis of N-((1R,3R)-3-(3-chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)-4-(4-(2-(1-(2-((3-(2,6-
dioxopiperidin-3-yl)-4-oxo-3,4-dihydrobenzo[d][1,2,3]triazin-6-yl)
oxy)acetyl)piperidin-4-yl)ethyl)piperazin-1-yl)benzamide (34d)
Similar procedure of 32a was performed to give 34d (18 mg,
Similar procedure of 32a was performed to give 34a (45 mg,
62%) as a pink solid. 1H NMR (500 MHz, CDCl3)
d 8.31 (m, 1H), 7.95
42%) as a pink solid. 1H NMR (500 MHz, CDCl3)
d
8.14 (d, J ¼ 8.8 Hz,
(d, J ¼ 8.9 Hz, 1H), 7.70 (d, J ¼ 8.4 Hz, 2H), 7.57 (d, J ¼ 8.7 Hz, 1H),
7.26e7.23 (m, 1H), 7.13 (m, 1H), 6.97 (d, J ¼ 2.4 Hz, 1H), 6.92 (d,
J ¼ 8.5 Hz, 2H), 6.81 (dd, J ¼ 8.8, 2.4 Hz, 1H), 6.12 (d, J ¼ 8.1 Hz, 1H),
6.03 (m, 1H), 5.77 (m, 1H), 4.64 (d, J ¼ 13.2 Hz, 1H), 4.15 (d,
J ¼ 8.1 Hz, 1H), 4.05 (s, 1H), 3.99 (m, 2H), 3.79 (d, J ¼ 13.7 Hz, 1H),
3.36 (m, 4H), 3.10 (t, J ¼ 12.9 Hz, 1H), 3.01e2.80 (m, 4H), 2.70 (m,
4H), 2.39 (m, 3H), 1.85 (dd, J ¼ 30.7, 13.2 Hz, 2H), 1.64 (m, 3H), 1.26
1H), 7.70 (d, J ¼ 8.4 Hz, 2H), 7.65e7.60 (m, 2H), 7.56 (d, J ¼ 8.7 Hz,
1H), 6.97 (d, J ¼ 2.4 Hz, 1H), 6.92 (dd, J ¼ 8.5, 5.4 Hz, 2H), 6.81 (dd,
J ¼ 8.7, 2.4 Hz, 1H), 6.15 (d, J ¼ 8.4 Hz, 1H), 5.76 (m, 1H), 5.00e4.84
(m, 2H), 4.56 (d, J ¼ 13.3 Hz, 1H), 4.15 (d, J ¼ 8.1 Hz, 1H), 4.05 (s, 1H),
3.83 (m, 1H), 3.46 (m, 4H), 3.19e2.86 (m, 5H), 2.84e2.46 (m, 6H),
2.42e2.33 (m, 1H), 1.81 (m, 3H), 1.26 (m, 9H), 1.22 (m, 7H), 1.19e1.11
(m, 1H); LC/MS (M þ H)þ (m/z) 892.0, (M ꢁ H)- (m/z) 890.0.
(m, 7H), 1.22 (m, 7H); 3C NMR (101 MHz, CDCl3)
d 171.48, 168.56,
167.10, 165.65, 162.65, 156.11, 150.56, 138.25, 136.87, 135.08, 130.29,
128.35, 122.81, 121.89, 116.77, 116.36, 114.57, 114.19, 105.00, 101.32,
84.88, 77.26, 58.50, 55.66, 52.64, 47.29, 44.47, 42.64, 40.33, 34.21,
32.40, 31.73, 31.10, 23.62, 23.52, 23.32; LC/MS (M þ H)þ (m/z) 891.1,
(M ꢁ H)- (m/z) 889.0.
4.1.51. Synthesis of N-((1R,3R)-3-(3-chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)-4-(4-(2-((1-((3-(2,6-dioxopiperidin-
3-yl)-4-oxo-3,4-dihydrobenzo[d][1,2,3]triazin-6-yl)glycyl)
piperidin-4-yl)oxy)ethyl)piperazin-1-yl)benzamide (35a)
Similar procedure of 32a was performed to give 35a (26 mg,
48%) as an ivory solid. 1H NMR (500 MHz, CDCl3)
d 8.56 (d,
4.1.48. Synthesis of N-((1Rr,3R)-3-(3-chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)-4-(4-(2-(1-(1-(3-(2,6-
dioxopiperidin-3-yl)-4-oxo-3,4-dihydrobenzo[d][1,2,3]triazin-6-yl)
azetidine-3-carbonyl)piperidin-4-yl)ethyl)piperazin-1-yl)
benzamide (34b)
J ¼ 14.4 Hz, 1H), 7.94 (d, J ¼ 8.9 Hz, 1H), 7.69 (d, J ¼ 8.7 Hz, 2H), 7.56
(d, J ¼ 8.7 Hz, 1H), 7.25 (dd, J ¼ 8.9, 2.7 Hz, 1H), 7.13 (d, J ¼ 2.6 Hz,
1H), 6.96 (d, J ¼ 2.4 Hz, 1H), 6.91 (d, J ¼ 8.8 Hz, 2H), 6.81 (dd, J ¼ 8.7,
2.4 Hz, 1H), 6.12 (d, J ¼ 8.1 Hz, 1H), 6.03 (t, J ¼ 3.9 Hz, 1H), 5.79 (dd,
J ¼ 11.5, 5.3 Hz, 1H), 4.15 (d, J ¼ 8.1 Hz, 1H), 4.04 (s, 1H), 4.00 (d,
J ¼ 3.7 Hz, 2H), 3.90 (m, 1H), 3.66 (m, 4H), 3.59e3.51 (m, 1H), 3.33
(m, 5H), 3.02e2.79 (m, 3H), 2.70 (m, 6H), 2.38 (m, 1H), 1.97e1.82
(m, 2H), 1.78e1.63 (m, 2H), 1.26 (s, 6H), 1.22 (s, 6H); 13C NMR
Similar procedure of 32a was performed to give 34b (35 mg,
47%) as a white solid. 1H NMR (500 MHz, CDCl3)
d 8.17 (m, 1H), 7.97
(d, J ¼ 8.9 Hz, 1H), 7.70 (d, J ¼ 8.5 Hz, 2H), 7.57 (d, J ¼ 8.8 Hz, 1H),
7.04 (dd, J ¼ 5.4, 2.6 Hz, 1H), 7.00e6.88 (m, 4H), 6.81 (dd, J ¼ 8.8,
2.4 Hz, 1H), 6.11 (d, J ¼ 8.1 Hz, 1H), 5.82e5.70 (m, 1H), 4.61 (d,
J ¼ 13.2 Hz, 1H), 4.29 (m, 4H), 4.15 (d, J ¼ 8.1 Hz, 1H), 4.04 (s, 1H),
3.78 (m, 1H), 3.57 (d, J ¼ 13.3 Hz, 1H), 3.33 (m, 4H), 3.07 (t,
J ¼ 12.8 Hz, 1H), 2.99e2.89 (m, 2H), 2.89e2.76 (m, 1H), 2.66 (d,
J ¼ 12.4 Hz, 1H), 2.61 (m, 4H), 2.44 (m, 2H), 2.41e2.34 (m, 1H), 1.82
(dd, J ¼ 22.0, 13.5 Hz, 2H),1.60 (m, 1H), 1.52 (m, 2H), 1.26 (s, 6H), 1.22
(101 MHz, CDCl3)
d 171.40, 168.44, 167.13, 165.73, 162.66, 156.13,
153.42,150.45,138.25,136.93,135.07,130.30,128.28,124.02,122.85,
121.91, 116.77, 116.36, 114.29, 114.19, 105.00, 101.27, 84.91, 77.27,
73.77, 66.19, 58.47, 57.98, 53.33, 47.75, 44.33, 41.44, 40.32, 39.48,
31.19, 31.12, 30.42, 23.61, 23.52, 23.31; LC/MS (M þ H)þ (m/z)
907.38, (M ꢁ H)- (m/z) 905.56.
(s, 6H), 1.17 (m, 2H); 13C NMR (101 MHz, CDCl3)
d 171.14, 168.86,
168.16, 167.11, 162.66, 155.88, 153.44, 152.86, 138.26, 136.48, 135.07,
130.46, 128.28, 124.03, 121.41, 118.30, 116.77, 116.36, 114.31, 114.19,
105.02, 102.01, 84.92, 58.47, 57.91, 55.83, 53.77, 52.96, 47.78, 45.61,
42.47, 40.32, 34.47, 33.18, 32.95, 32.15, 31.88, 31.05, 23.61, 23.51,
23.31; LC/MS (M þ H)þ (m/z) 917.24, (M ꢁ H)- (m/z) 915.48.
4.1.52. Synthesis of N-((1R,3R)-3-(3-chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)-4-(4-(2-((1-(1-(3-(2,6-
dioxopiperidin-3-yl)-4-oxo-3,4-dihydrobenzo[d][1,2,3]triazin-6-yl)
azetidine-3-carbonyl)piperidin-4-yl)oxy)ethyl)piperazin-1-yl)
benzamide (35b)
4.1.49. Synthesis of N-((1Rr,3R)-3-(3-chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)-4-(4-(2-(1-(1-(3-(2,6-
dioxopiperidin-3-yl)-4-oxo-3,4-dihydrobenzo[d][1,2,3]triazin-6-yl)
piperidine-4-carbonyl)piperidin-4-yl)ethyl)piperazin-1-yl)
benzamide (34c)
Similar procedure of 32a was performed to give 35b (13 mg,
43%) as an ivory solid. 1H NMR (500 MHz, CDCl3)
d 8.24 (d,
J ¼ 5.2 Hz, 1H), 7.97 (d, J ¼ 8.8 Hz, 1H), 7.73e7.66 (m, 2H), 7.57 (d,
J ¼ 8.7 Hz, 1H), 7.06e7.02 (m, 1H), 6.96 (d, J ¼ 2.4 Hz, 1H), 6.96e6.93
(m, 1H), 6.93e6.89 (m, 2H), 6.81 (dd, J ¼ 8.7, 2.4 Hz, 1H), 6.12 (d,
J ¼ 8.1 Hz, 1H), 5.80e5.72 (m, 1H), 4.32 (t, J ¼ 7.1 Hz, 2H), 4.30e4.24
(m, 2H), 4.15 (d, J ¼ 8.1 Hz, 1H), 4.04 (s, 1H), 3.94e3.84 (m, 1H),
3.84e3.74 (m,1H), 3.73e3.63 (m, 2H), 3.64e3.57 (m,1H), 3.57e3.49
(m, 1H), 3.50e3.42 (m, 1H), 3.38e3.27 (m, 4H), 3.23e3.14 (m, 1H),
3.00e2.78 (m, 3H), 2.76e2.62 (m, 6H), 2.42e2.33 (m, 1H),1.92e1.80
(m, 2H), 1.71e1.59 (m, 2H), 1.26 (s, 6H), 1.22 (s, 6H); 13C NMR
Similar procedure of 32a was performed to give 34c (35 mg,
47%) as a white solid. 1H NMR (500 MHz, CDCl3)
d 8.05 (s, 1H), 8.00
(d, J ¼ 9.1 Hz,1H), 7.70 (d, J ¼ 8.5 Hz, 2H), 7.57 (d, J ¼ 8.7 Hz,1H), 7.52
(d, J ¼ 2.8 Hz, 1H), 7.45 (dd, J ¼ 9.2, 2.9 Hz, 1H), 6.98e6.95 (m, 1H),
6.92 (d, J ¼ 8.5 Hz, 2H), 6.81 (dd, J ¼ 8.7, 2.4 Hz, 1H), 6.11 (d,
J ¼ 8.1 Hz,1H), 5.77 (dd, J ¼ 11.4, 5.4 Hz,1H), 4.62 (d, J ¼ 13.3 Hz,1H),
4.15 (d, J ¼ 8.1 Hz, 1H), 4.06 (m, 3H), 3.93 (d, J ¼ 13.4 Hz, 1H), 3.32
(m, 4H), 3.09 (m, 3H), 3.01e2.89 (m, 2H), 2.88e2.76 (m, 2H), 2.60
(m, 5H), 2.45 (t, J ¼ 7.4 Hz, 2H), 2.39 (m, 1H), 1.96 (m, 2H), 1.86 (m,
3H), 1.78 (d, J ¼ 13.2 Hz, 1H), 1.62 (m, 1H), 1.52 (m, 2H), 1.26 (s, 6H),
(101 MHz, CDCl3)
d 171.39, 169.01, 168.39, 167.14, 162.67, 155.85,
153.41, 152.81, 138.22, 136.45, 135.08, 130.40, 128.30, 123.99, 121.38,
118.28, 116.79, 116.38, 114.27, 114.18, 104.95, 102.01, 84.90, 77.30,
73.96, 66.13, 58.47, 57.95, 57.85, 53.74, 53.30, 47.71, 42.44, 40.32,
39.25, 32.06, 31.57, 31.10, 30.42, 23.62, 23.51, 23.32; LC/MS (M þ H)þ
(m/z) 933.37, (M ꢁ H)- (m/z) 931.48.
1.22 (s, 6H), 1.20e1.08 (m, 2H); 13C NMR (101 MHz, CDCl3)
d
172.25,
171.26, 168.25, 167.13, 162.66, 156.02, 153.44, 153.17, 138.23, 136.55,
14

A.D. Takwale, S.-H. Jo, Y.U. Jeon et al.
European Journal of Medicinal Chemistry 208 (2020) 112769
4.1.53. Synthesis of N-((1R,3R)-3-(3-chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)-4-(4-(2-((1-(1-(3-(2,6-
dioxopiperidin-3-yl)-4-oxo-3,4-dihydrobenzo[d][1,2,3]triazin-6-yl)
piperidine-4-carbonyl)piperidin-4-yl)oxy)ethyl)piperazin-1-yl)
benzamide (35c)
2H), 1.82 (m, 2H), 1.46 (s, 9H).
4.1.57. Synthesis of 1-(3-(1-methyl-2,6-dioxopiperidin-3-yl)-4-oxo-
3,4-dihydrobenzo[d][1,2,3]triazin-6-yl)piperidine-4-carboxylic acid
(38)
Similar procedure of 32a was performed to give 35c (13 mg,
Similar procedure of 20a was performed to give 38 as crude
white solid. The crude product was used in the next step without
purification.
43%) as an ivory solid. 1H NMR (500 MHz, CDCl3)
d 8.36 (s, 1H), 7.99
(d, J ¼ 9.1 Hz, 1H), 7.73e7.66 (m, 2H), 7.57 (d, J ¼ 8.7 Hz, 1H), 7.52 (d,
J ¼ 2.8 Hz, 1H), 7.45 (dd, J ¼ 9.1, 2.8 Hz, 1H), 6.97 (d, J ¼ 2.4 Hz, 1H),
6.94e6.88 (m, 2H), 6.81 (dd, J ¼ 8.7, 2.4 Hz, 1H), 6.13 (d, J ¼ 8.1 Hz,
1H), 5.81e5.73 (m, 1H), 4.15 (d, J ¼ 8.1 Hz, 1H), 4.10e4.02 (m, 3H),
3.98e3.89 (m, 1H), 3.81e3.72 (m, 1H), 3.72e3.63 (m, 2H),
3.63e3.56 (m, 1H), 3.41e3.28 (m, 6H), 3.15e3.05 (m, 2H),
3.00e2.88 (m, 2H), 2.88e2.77 (m, 2H), 2.76e2.64 (m, 6H),
2.42e2.33 (m, 1H), 2.01e1.80 (m, 6H), 1.70e1.55 (m, 2H), 1.26 (s,
4.1.58. Synthesis of N-((1r,3r)-3-(3-chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)-4-(4-(7-(1-(3-(1-methyl-2,6-
dioxopiperidin-3-yl)-4-oxo-3,4-dihydrobenzo[d][1,2,3]triazin-6-yl)
piperidine-4-carbonyl)-7-azaspiro[3.5]nonan-2-yl)piperazin-1-yl)
benzamide (39)
To a solution of 28b (15 mg, 0.0239 mmol), 38 (14 mg,
0.0359 mmol), EDCI,HCl (5 mg, 0.0262 mmol), and HOBt (4 mg,
0.0262 mmol) in DMF (2 mL) was added DIPEA (0.021 mL,
0.119 mmol). The reaction mixture was stirred at room temperature
for 12 h. The reaction mixture was diluted with water and extracted
with ethyl acetate. The organic layer was washed with brine, dried
over Na2SO4 and concentrated under reduced pressure. The residue
was purified by column chromatography to afford 39 (13 mg, 56%)
6H), 1.22 (s, 6H); 13C NMR (101 MHz, CDCl3)
d 172.35, 171.07, 168.10,
167.10, 162.66, 156.01, 153.44, 153.16, 138.26, 136.58, 135.07, 130.33,
128.28, 124.01, 121.83, 121.45, 116.77, 114.29, 114.19, 105.50, 105.02,
84.91, 77.25, 74.35, 66.16, 58.46, 58.02, 53.36, 47.79, 47.10, 42.70,
40.32, 39.13, 37.76, 32.05, 31.07, 30.62, 27.92, 23.61, 23.52, 23.30; LC/
MS (M þ H)þ (m/z) 961.45, (M ꢁ H)- (m/z) 959.56.
4.1.54. Synthesis of N-((1R,3R)-3-(3-chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)-4-(4-(2-((1-(2-((3-(2,6-
dioxopiperidin-3-yl)-4-oxo-3,4-dihydrobenzo[d][1,2,3]triazin-6-yl)
oxy)acetyl)piperidin-4-yl)oxy)ethyl)piperazin-1-yl)benzamide
(35d)
as a white solid. 1H NMR (500 MHz, CDCl3)
d 7.99 (m, 1H), 7.73e7.66
(m, 2H), 7.57 (d, J ¼ 8.7 Hz, 1H), 7.52 (d, J ¼ 2.7 Hz, 1H), 7.45 (d,
J ¼ 9.2 Hz, 1H), 6.97 (d, J ¼ 2.4 Hz, 1H), 6.95e6.88 (m, 2H), 6.81 (dd,
J ¼ 8.8, 2.5 Hz, 1H), 6.12 (d, J ¼ 8.1 Hz, 1H), 5.78 (dd, J ¼ 11.7, 5.4 Hz,
1H), 4.15 (d, J ¼ 8.0 Hz, 1H), 4.07 (m, 1H), 4.06e4.02 (m, 2H), 3.59
(m, 1H), 3.53e3.44 (m, 2H), 3.40 (m, 1H), 3.33 (m, 4H), 3.24 (s, 3H),
3.12e3.01 (m, 3H), 2.93e2.82 (m, 2H), 2.84e2.75 (m, 2H), 2.51 (m,
4H), 2.38e2.30 (m, 1H), 2.10 (m, 2H), 1.98e1.82 (m, 6H), 1.76 (m,
2H), 1.67 (m, 1H), 1.57 (m, 1H), 1.26 (s, 6H), 1.22 (s, 6H).
Similar procedure of 32a was performed to give 35d (7 mg, 21%)
as a white solid. 1H NMR (500 MHz, CDCl3)
d 8.28 (m, 1H), 8.14 (d,
J ¼ 8.6 Hz, 1H), 7.70 (d, J ¼ 8.4 Hz, 2H), 7.63 (m, 2H), 7.57 (d,
J ¼ 8.7 Hz, 1H), 6.97 (d, J ¼ 2.3 Hz, 1H), 6.92 (d, J ¼ 8.5 Hz, 2H), 6.81
(dd, J ¼ 8.7, 2.3 Hz, 1H), 6.13 (d, J ¼ 8.1 Hz, 1H), 5.78 (dd, J ¼ 11.6,
5.4 Hz, 1H), 4.91 (s, 2H), 4.15 (d, J ¼ 8.0 Hz, 1H), 4.05 (s, 1H),
3.93e3.60 (m, 5H), 3.51e3.27 (m, 5H), 3.04e2.68 (m, 8H), 2.38 (m,
1H), 1.90 (m, 2H), 1.68 (m, 4H), 1.26 (s, 6H), 1.22 (s, 6H); LC/MS
(M þ H)þ (m/z) 908.33, (M ꢁ H)- (m/z) 906.50.
4.2. Biology
4.2.1. Cell culture
VCaP cells were purchased from the American Type Culture
Collection (ATCC). LNCaP and 22RV1 cells were purchased from the
Korea Research Institute of Bioscience & Biotechnology Bio-
Evaluation Center (KRIBB). LNCaP and VCaP cells were grown in
IMDM (Invitrogen), and 22RV1 cells were grown in RPMI 1640
(Invitrogen). All of the cells were supplemented with 10% fetal
bovine serum (Gibco, #16000e044) and Antibiotic-Antimycotic
(Gibco, #15240062) at 37 ꢀC in a humidified 5% CO2 incubator.
DMSO was used as a vehicle.
4.1.55. Synthesis of 3-(6-fluoro-4-oxobenzo[d][1,2,3]triazin-3(4H)-
yl)-1-methylpiperidine-2,6-dione (36)
To a solution of 18b (400 mg, 1.44 mmol) and K2CO3 (398 mg,
2.88 mmol) in DMF (5 mL) was added iodomethane (0.135 mL,
2.17 mmol). The reaction mixture was stirred at room temperature
for 12 h. The reaction mixture was diluted with water and extracted
with ethyl acetate. The organic layer was washed with brine, dried
over Na2SO4 and concentrated under reduced pressure. The residue
was purified by column chromatography to afford 36 (264 mg, 63%)
4.2.2. Western blot analysis
as a white solid. 1H NMR (500 MHz, DMSO‑d6)
d
8.40 (m, 1H),
LNCaP and 22RV1 cells were lysed in RIPA buffer (50 mM Tris-
HCl, pH7.5, 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, 0.1% SDS,
0.5% sodium deoxycholate and protease inhibitor cocktail) and
protein concentration was quantified by Bradford assay. Total pro-
8.08e8.00 (m, 2H), 6.07 (dd, J ¼ 12.8, 5.3 Hz, 1H), 3.05 (m, 4H), 2.85
(m, 1H), 2.71 (m, 1H), 2.33e2.25 (m, 1H).
4.1.56. Synthesis of tert-butyl 1-(3-(1-methyl-2,6-dioxopiperidin-3-
yl)-4-oxo-3,4-dihydrobenzo[d][1,2,3]triazin-6-yl)piperidine-4-
carboxylate (37)
tein lysates (30 mg) were separated with SDS-PAGE, and transferred
to NC membrane (GE healthcare). The blots were probed with the
indicated primary antibodies followed by HRP-conjugated anti-
rabbit (Cell signaling, #7074S) or anti-mouse IgG antibody (sigma,
#A9044). Detection was performed with chemiluminescent HRP
substrate (Thermo, #34580). Antibodies for western blots were:
anti-AR (Santacruz, #sc-7305) and anti-tubulin (Sigma, #T5168).
To a solution of 36 (100 mg, 0.344 mmol) and tert-butyl piper-
idine-4-carboxylate hydrochloride (114 mg, 0.516 mmol) in DMSO
(5 mL) was added DIPEA (0.18 mL, 1.032 mmol). The reaction
mixture was stirred at 90 ꢀC for 12 h. The reaction mixture was
cooled to room temperature, diluted with water, and extracted with
ethyl acetate. The organic layer was washed with brine, dried over
Na2SO4, and concentrated under reduced pressure. The residue was
purified by column chromatography to afford 37 (130 mg, 82%) as a
4.2.3. Cell viability assay
LNCaP and VCaP cells were seeded into each well of a 96 well
plate with 1.5 ꢂ 103 (LNCaP) and 4 ꢂ 103 (VCaP) cells. Next day,
chemicals were treated in each well to a final concentration of
white solid. 1H NMR (500 MHz, CDCl3)
(d, J ¼ 2.9 Hz,1H), 7.44 (dd, J ¼ 9.2, 2.9 Hz,1H), 5.79 (m,1H), 3.95 (dt,
J ¼ 13.9, 4.3 Hz, 2H), 3.25 (s, 3H), 3.12 (m, 2H), 3.07e3.01 (m, 1H),
2.93e2.82 (m, 2H), 2.54e2.45 (m, 1H), 2.37e2.30 (m, 1H), 2.02 (m,
d
7.99 (d, J ¼ 9.1 Hz, 1H), 7.53
0.3 nM, 1 nM, 3 nM, 10 nM, 30 nM, 100 nM, 300 nM, 1
10 M. After 6 days of treatment, cell viability was measured by a
CellTiter-Glo (Promega, #G7570) following the manufacturer’s
mM, 3 mM,
m
15

A.D. Takwale, S.-H. Jo, Y.U. Jeon et al.
European Journal of Medicinal Chemistry 208 (2020) 112769
instructions. The viability was quantified compared to DMSO alone.
The IC50s were calculated using GraphPad Prism version 6 for
Windows. The curves were fitted using a nonlinear regression
model with a log (inhibitor) versus response formula.
biology experiments. SA, HJK, HKL, COL designed and conducted
some experiments. JHK, and JYH wrote the manuscript. JDH, JHK,
and JYH contributed to experimental design and supervision.
Declaration of competing interest
4.2.4. Liver microsomal metabolic stability
NADPH-dependent metabolism of a test compound in mouse
liver microsomes was evaluated. The reaction mixture consisted of
liver microsomes (0.5 mg protein/mL) in 100 mM PBS (pH 7.4) and
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.
the final concentration of a test compound was 1 mM. The metabolic
reaction was initiated by the addition of NADPH-regenerating so-
lution, containing 1.3 mM NADPþ, 3.3 mM glucose-6-phosphate,
0.4 U/mL glucose-6-phosphate dehydrogenase, and 3.3 mM MgCl2.
After the 30 min sample incubation at 37 ꢀC, the reaction was
stopped by adding ice-cold acetonitrile and the samples were then
centrifuged at 13,000 g for 10 min. The supernatant was stored
at ꢁ20 ꢀC until the analysis.
Acknowledgements
This work was supported by the grant (CAP-15-11-KRICT) from
National Research Council of Science and Technology, the grant
(NRF-2019M3E5D4069882) from the National Research Founda-
tion, Ministry of Science and ICT and future planning, and the grant
from the KRIBB Initiative program.
4.2.5. Pharmacokinetic studies
Appendix A. Supplementary data
In male ICR mice (7 weeks), 5 mg/kg, 5 mg/kg and 30 mg/kg of
compound were administered via the intravenous (IV), intra-
peritonea (IP), and oral (PO) route, respectively. The composition of
dosing vehicles was 5% DMSO, 52% PEG400, and 3% Tween 80 in
water for IV and IP, and 3% Tween8 and 20% PEG400 in water for PO.
Blood samples were collected at different time points after drug
administration from retro-orbital venous plexus. After obtaining
plasma samples by centrifugation of blood samples, protein pre-
cipitation was performed by adding acetonitrile at a 1:10 sample
dilution. After vortex mixing and centrifugation, the supernatant
was collected and analyzed by LC-MS/MS. Mean plasma
concentration-time data were analyzed using noncompartmental
methods (Pheonix WinNonlin software, Pharsight Corporation,
Mountain View, CA). Standard pharmacokinetic parameters were
calculated and included maximal plasma concentration (Cmax),
time to reach Cmax (Tmax), area under the plasma concentration-
time curve from time 0 to time of last measurable concentration
(AUClast), area under the plasma concentration-time curve from
time 0 to infinity (AUCinf), half-life (T1/2), clearance rate (CL), and
volume of distribution (Vdss). The bioavailability (F) was calculated
as (AUCnon-intravenous ꢂ dosei.v.)/(AUCi.v. ꢂ dosenon-intravenous).
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.112769.
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ꢀ
^ ꢀ
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17