Discovery of novel BCR-ABL PROTACs based on the cereblon E3 ligase design, synthesis, and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2021.113645

Protein degradation is a promising strategy for drug development. Proteolysis-targeting chimeras (PROTACs) hijacking the E3 ligase cereblon (CRBN) exhibit enormous potential and universal degradation performance due to the small molecular weight of CRBN ligands. In this study, the CRBN-recruiting PROTACs were explored on the degradation of oncogenic fusion protein BCR-ABL, which drives the pathogenesis of chronic myeloid leukemia (CML). A series of novel PROTACs were synthesized by conjugating BCR-ABL inhibitor dasatinib to the CRBN ligand including pomalidomide and lenalidomide, and the extensive structure-activity relationship (SAR) studies were performed focusing on optimization of linker parameters. Therein, we uncovered that pomalidomide-based degrader 17 (SIAIS056), possessing sulfur-substituted carbon chain linker, exhibits the most potent degradative activity in vitro and favorable pharmacokinetics in vivo. Besides, degrader 17 also degrades a variety of clinically relevant resistance-conferring mutations of BCR-ABL. Furthermore, degrader 17 induces significant tumor regression against K562 xenograft tumors. Our study indicates that 17 as an efficacious BCR-ABL degrader warrants intensive investigation for the future treatment of BCR-ABL⁺ leukemia.

Full text of DOI:10.1016/j.ejmech.2021.113645

European Journal of Medicinal Chemistry 223 (2021) 113645
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Discovery of novel BCR-ABL PROTACs based on the cereblon E3 ligase
design, synthesis, and biological evaluation
Haixia Liu c
,
a
,
e
,
1, Xinyu Ding
a
,
b
,
e
,
1
, Linyi Liu , Qianglong Mi a , Quanju Zhao ,
d
,
1
,
b
a
f
a,
b
a
a
g
a
YuBao Shao , Chaowei Ren , Jinju Chen , Ying Kong , Xing Qiu , Nicola Elvassore ,
Xiaobao Yang , Qianqian Yin
Shanghai Institute for Advanced Immunochemical Studies, China
School of Life Science and Technology, China
School of Physical Science and Technology, ShanghaiTech University, Shanghai, 201210, China
a
,
*
a
,
***
, Biao Jiang a
, g, **
a
b
c
d
Hunan Provincial Key Laboratory of Tumor Microenvironment Responsive Drug Research, School of Pharmaceutical Science, University of South China,
Hengyang City, 421001, China
e
University of Chinese Academy of Sciences, Beijing, 100049, China
Department of Histology and Embryology, Anhui Medical University, Hefei, 230032, China
CAS Key Laboratory of Synthetic Chemistry of Natural Substances, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling
f
g
Road, Shanghai, 200032, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 April 2021
Received in revised form
Protein degradation is a promising strategy for drug development. Proteolysis-targeting chimeras
(
PROTACs) hijacking the E3 ligase cereblon (CRBN) exhibit enormous potential and universal degradation
performance due to the small molecular weight of CRBN ligands. In this study, the CRBN-recruiting
PROTACs were explored on the degradation of oncogenic fusion protein BCR-ABL, which drives the
pathogenesis of chronic myeloid leukemia (CML). A series of novel PROTACs were synthesized by
conjugating BCR-ABL inhibitor dasatinib to the CRBN ligand including pomalidomide and lenalidomide,
and the extensive structure-activity relationship (SAR) studies were performed focusing on optimization
of linker parameters. Therein, we uncovered that pomalidomide-based degrader 17 (SIAIS056), pos-
sessing sulfur-substituted carbon chain linker, exhibits the most potent degradative activity in vitro and
favorable pharmacokinetics in vivo. Besides, degrader 17 also degrades a variety of clinically relevant
resistance-conferring mutations of BCR-ABL. Furthermore, degrader 17 induces significant tumor
regression against K562 xenograft tumors. Our study indicates that 17 as an efficacious BCR-ABL degrader
9
June 2021
Accepted 9 June 2021
Available online 25 June 2021
Keywords:
PROTAC
CRBN
Leukemia
BCR-ABL
Degradation
þ
warrants intensive investigation for the future treatment of BCR-ABL leukemia.
©
2021 Elsevier Masson SAS. All rights reserved.
1
. Introduction
a ligand targeting a protein of interest (POI), a ligand recruiting an
E3 ligase and a connecting linker [3,4]. PROTACs are distinguished
from classical inhibitors by inducing degradation of target proteins
and not merely inhibition. A line of evidence uncover that PROTACs
possess advantages over therapeutic inhibitors, including working
at very low doses [5,6], overcoming potential resistance to current
inhibitors [7,8], exerting improved selectivity [9,10] and having the
potential to degrade undruggable targets [11,12]. PROTACs have
exhibited remarkable biological activities in many target proteins
Proteolysis-targeting chimeras (PROTACs) have emerged as a
potential strategy for drug discovery and design [1,2]. PROTACs, also
known as degraders, are heterobifunctional molecules consisting of
*
Corresponding author. Shanghai Institute for Advanced Immunochemical
Studies, ShanghaiTech University, Shanghai, 201210, China.
* Corresponding author. Shanghai Institute for Advanced Immunochemical
Studies, ShanghaiTech University, Shanghai, 201210, China.
** Corresponding author. Shanghai Institute for Advanced Immunochemical
*
[
13,14] and ARV-110, a potential oral PROTAC targeting the
androgen receptor, has shown benefit in patients with metastatic
castration-resistant prostate cancer in early phase clinical trial [15].
Thalidomide, lenalidomide and pomalidomide are FDA
approved drugs for the treatment of multiple myeloma termed the
*
Studies, ShanghaiTech University, Shanghai, 201210, China.
E-mail addresses: xiaobaoyoung@126.com (X. Yang), yinqq@shanghaitech.edu.
cn (Q. Yin), jiangbiao@shanghaitech.edu.cn (B. Jiang).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.ejmech.2021.113645
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

H. Liu, X. Ding, L. Liu et al.
European Journal of Medicinal Chemistry 223 (2021) 113645
immunomodulatory drugs (IMiDs) in clinic, target is the cereblon
substituted carbon chain linker, achieves the most potent BCR-
ABL-degrading activity in vitro. Additionally, degrader 17 also in-
duces the degradation of several clinically relevant resistance-
conferring mutations of BCR-ABL. Moreover, degrader 17 exerts
favorable pharmacokinetics and induces substantial tumor
regression against K562 xenograft tumors in vivo. Collectively, our
study provided a deeper understanding for the development of
BCR-ABL PROTACs and novel potential therapeutic agents for CML
treatment.
(
CRBN) E3 ubiquitin ligase [16,17]. IMiDs have frequently been
utilized as CRBN ligands for the design of PROTACs, possibly due to
the relative safety and small molecular weight [18]. Furthermore,
evidences showed that CRBN-recruiting PROTACs showed broader
spectrum of protein degradation compared with VHL-based PRO-
TACs [10,19].
The chimeric oncogenic fusion protein BCR-ABL is the key
driving factor and therapeutic target of chronic myeloid leukemia
þ
(
CML) [20,21]. The progression of BCR-ABL CML can be effectively
controlled with the clinical application of various BCR-ABL in-
hibitors such as imatinib, dasatinib and ponatinib [22]. However,
several drawbacks including persistent leukemic stem cells reten-
tion and BCR-ABL kinase domain mutations as well as BCR-ABL
kinase-independent signaling activation scream for new therapies
2
. Results and discussion
2.1. Chemistry
The general reactions utilized for synthesis of the novel targeted
[
23e26]. Thus, exhaustive abrogation of BCR-ABL functions by
derivatives are outlined in Schemes 1-6. All the starting materials
were commercially available. First, the precursor of ABL inhibitor
dasatinib S2 was obtained from S1 by substitution reaction. As
shown in Scheme 1, the furnished 4-F-thalidomide S3 subsequently
reacted with various amines by nucleophilic substitution following
cleavage to form intermediates S5 or S7. Then derivatives 4e11, 13
were obtained through condensation of S2 with corresponding S5
or S7. Then thiolation and etherification of S3 produced the inter-
mediate S9, which was converted to S10 by removal of tert-butyl
group. After condensation of S2, S10 was transformed into deriva-
tive 12 (Scheme 2). Meanwhile, S9 was oxidized to afford S11, which
was followed by removal of tert-butyl group to get intermediate
S12. After condensation with S2, S12 was converted into derivative
PROTACs might have potential therapeutic benefits for CML treat-
ment. In recent years, PROTACs targeting BCR-ABL have been un-
covered to exhibit significant therapeutic potency [19,27e35].
Several PROTACs including 1 [19], 2 [30] and 3 [33] (Fig. 1), which
recruit the CRBN E3 ligase, were identified to significantly degrade
BCR-ABL at low nanomolar concentration. However, the structure-
activity relationship (SAR), pharmacokinetic properties as well as
the in vivo activity of CRBN- recruiting PROTACs targeting BCR-ABL
have not been well investigated. To design novel BCR-ABL PROTACs
hijacking CRBN and perform the SAR study, dasatinib was selected
in our study as the BCR-ABL ligand for novel PROTACs design due to
that dasatinib binds BCR-ABL both in its inactive and active
conformation and exerted much greater potency compared with
imatinib which binds BCR-ABL only in its inactive conformation,
and shows efficacy against the majority of imatinib-resistant mu-
tations (except the "gatekeeper" T315I) [36,37]. Herein, we
described the design, synthesis, and evaluation of novel PROTACs
targeting BCR-ABL by conjugating dasatinib to the CRBN ligands
including pomalidomide and lenalidomide. A line of evidence in
the field highlight the fact that linker composition and length play
crucial roles in modulation of binding kinetics and the subsequent
potency and selectivity [38e40]. Thus, a big challenge in PROTAC
design is the selection of the optimal linker to connect these two
binding ligands. In our previous work, we performed the SAR study
of VHL-recruiting PROTACs targeting BCR-ABL based on dasatinib
and identified SIAIS178, possessing a carbon chain linker, was the
most potent one among the VHL-based PROTACs by inducing more
robust ternary complex formation [28]. On this basis, the extensive
optimization of linker parameters such as length, hydrophilicity
and rigidity was investigated in our study. We uncovered that
pomalidomide-based degrader 17 (SIAIS056), possessing sulfur-
16 (Scheme 2).
As shown in Scheme 3, following condensation and ether-
ification, S13 was translated into S15 which was hydrolyzed
following condensation to get derivative 14. Further, after
condensation and palladium-catalyzed cross coupling reaction as
well as hydrogenation and hydrolysis, the intermediate S20 was
obtained from S17 through reacting with corresponding reagents.
Afterwards, derivative 15 was obtained through the condensation
of S20 with S2 (Scheme 3). The intermediate S23 derived from S21
was etherified with thiolated pomalidomide derivatives S8 or S8-1
as well as lenalidomide derivative S24 to afford corresponding 17,
24 or 19, respectively (Scheme 4). Following thiolation reaction and
the subsequent sulfide formation and the cleavage of tert-butyl
esters, lenalidomide was transformed into S27, which condensed
with S2 to obtain derivative 18 as depicted in Scheme 4.
Finally, S2 reacted with tert-butyl (4-bromobutyl) carbamate to
produce S28. After deprotection, the product reacted with S3 to
afford 20. Hereafter, derivative 21 was obtained from pomalido-
mide S29 following acylation reaction and electrophilic
Fig. 1. Reported effective BCR-ABL PROATCs based on CRBN.
2

H. Liu, X. Ding, L. Liu et al.
European Journal of Medicinal Chemistry 223 (2021) 113645
ꢀ
ꢀ
ꢀ
ꢀ
Scheme 1. Reagents and conditions: a) piperazine, DIPEA, n-BuOH, 110 C, 16 h; b) DIPEA, NMP, 110 C, MW, 2 h; c) 88% HCOOH, 25 C, 12 h; d) S2, HOAt, EDCI, NMM, DMF, 25 C,
2 h.
1
Scheme 2. Reagents and conditions: a) S8: Na
DMF, 25 C, 0.5 h; c) 88% HCOOH, 25 C, 12 h; d) S2, HOAt, EDCI, NMM, DMF, 25 C, 12 h; e) m-CPBA, DCM, 40 C, 4 h.
2
S.9H
S.9H
ꢀ ꢀ
2 2 3 3 2 2 2 2 3
O, DMF, 25 C, 0.5 h; S8-1: i, K CO , NaI, CH I, rt, 0.5 h; ii, Na O, DMF, N ,25 C, 3 h; b) tert-butyl 2-bromoacetate, K CO ,
ꢀ
ꢀ
ꢀ
ꢀ
substitution (Scheme 5). The syntheses of derivatives 22, 23 were
performed as depicted in Scheme 6. On one hand, lenalidomide S31
afforded the intermediate S32 following substitution and the
cleavage of tert-butyl esters. After that, S32 condensed with S2 to
yield compound 22. On the other hand, derivative 23 was yielded
through two condensation reactions from starting material S31. All
the synthesized compounds have been confirmed by NMR and
mass spectrometry.
3

H. Liu, X. Ding, L. Liu et al.
European Journal of Medicinal Chemistry 223 (2021) 113645
ꢀ
Scheme 3. Reagents and conditions: a) 3-aminopiperidine-2,6-dione, TEA, Toluene, reflux,12 h; b) tert-butyl 2-bromoacetate, NaHCO
3
, KI, DMF, 60 C, 12 h; c) TFA, DCM, rt, 2 h; d)
ꢀ
ꢀ
S2, HOAt, EDCI, NMM, DMF, 25 C, 12 h; e) 3-aminopiperidine-2,6-dione, NaOAc, AcOH, reflux,12 h; f) tert-butyl acrylate, Pd
2
(dba)
3
, Cy
2
NMe, HP(t-Bu)BF
4
, Dioxane, 55 C, 12 h; g) i:
10% Pd/C, Dioxane, rt, 12 h; ii: TFA, DCM, rt, 2 h.
ꢀ
ꢀ
.
O, CuSO.
Scheme 4. Reagents and conditions: a) sulfurous dichloride, Reflux, 12 h; b) DIPEA, DMF, 110 C, 12 h; c) NaI, K
2
CO
3
, DMF, 60 C, 8 h; d) BnCl, Na
2
S
2
O
3
5H
2
4
5H
2
O, Biby, t-
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
BuONO MeOH, H
2 h.
2
O, 80 C, 8 h; e) AlCl
3
, Toluene, 35 C,12 h; f) i: tert-butyl 2-bromoacetate, K
2
CO
3
, DMF, 25 C, 2 h; ii: 88% HCOOH, 25 C, 12 h; g) S2, HOAt, EDCI, NMM, DMF, 25 C,
1
ꢀ
ꢀ
Scheme 5. Reagents and conditions: a) tert-butyl (4-bromobutyl)carbamate, K
2
CO
3
, NaI, DMF, 110 C, 12 h; b) i: HCOOH, rt, 12 h; ii: S3, DIPEA, NMP, 110 C, 4 h; c) 5-bromopentanoyl
ꢀ
chloride, THF, reflux, 3 h; d) S2, NaI, K
2 3
CO , DMF, 60 C, 4 h.
4

H. Liu, X. Ding, L. Liu et al.
European Journal of Medicinal Chemistry 223 (2021) 113645
ꢀ
ꢀ
Scheme 6. Reagents and conditions: a) tert-butyl 5-bromopentanoate, DIPEA, NMP, 100 C, 12 h; b) TFA, DCM, rt, 1 h; c) S2, HOAt, EDCI, NMM, DMF, 25 C, 12 h; d) glutaric acid,
HOAt, EDCI, NMM, DCM/DMF, 0 Ce25 C, 12 h.
ꢀ
ꢀ
2
2
.2. Biological evaluation
inhibited K562 cell proliferation with the growth inhibitory po-
tency (IC50) value at low nanomolar concentration except for CRBN
ligands pomalidomide (poma) and lenalidomide (lena). Moreover,
compound 10 exhibited similar anti-proliferative activities to
dasatinib (Das). Furthermore, all these PROTACs induced BCR-ABL
degradation with the degradation potency (DC50) value at low
nanomolar concentrations. However, the ligands of BCR-ABL (Das)
and E3 ligase (poma and lena) didn't decrease the BCR-ABL protein
.2.1. Anti-proliferative activities and BCR-ABL-degrading activity
All synthesized compounds were evaluated for their anti-
proliferative activity on BCR-ABL driven CML cell line K562 cells
and degradative activity against BCR-ABL. For comparison, CRBN
ligands (pomalidomide and lenalidomide) and BCR-ABL inhibitor
dasatinib as well as reported CRBN-based PROTAC DAS-6-2-2-6-
CRBN (1) were also included as reference compounds. As shown
in Table 1, these initially synthesized compounds effectively
level even at 10
mM concentration. Among them, compounds 4, 9
and 11 were about 3 times more active than compound 1 in BCR-
Table 1
The anti-proliferative activities against K562 and BCR-ABL-degrading activity of derivatives 4e11 with PEG- or Carbon-based linkers.
Compound
Linker
X
K562
BCR-ABL
IC50 (nM)
DC50 (nM)
Pomalidomide
Lenalidomide
Dasatinib
e
e
e
e
>10
＞10
0.9
m
mM
M
>10
>10
>10
30.5
m
m
m
M
M
M
e
e
1
(DAS-6-2-2-6-CRBN)
CO
4.1
4
CO
CO
18.42
8.05
8.7
5
17.5
6
7
CO
CO
14.75
40.4
18.4
26.1
8
9
CO
CO
31.9
5.5
10.8
7.8
1
1
0
1
CO
CO
0.55
1.47
11
9
5

H. Liu, X. Ding, L. Liu et al.
European Journal of Medicinal Chemistry 223 (2021) 113645
ABL degradation. Interestingly, some compounds (4e9), possessing
better degradation potency, showed attenuated anti-proliferative
activities compared to 1, that is, the DC50 and IC50 are not paral-
lel. We speculated that the unparalleled DC50 and IC50 are possibly
due to that PROTAC-induced growth inhibition is a consequence of
the dual action of both inhibition and degradation of BCR-ABL
proteins, and there seems to be no good correlation between the
two capacities of PROTACs, just as the results reported by Crews
Table 3
The anti-proliferative activities against K562 and BCR-ABL-degrading activity of
derivatives 17e23 with or without acyl substitution.
Compound
Linker
X
K562
BCR-ABL
DC50 (nM)
0.18
IC50 (nM)
0.49
17(SIAIS056)
CO
CH
18
2
2
5.7
8.9
[
19], in which PROTACs exerting significant ABL inhibition (non-
phosphorylated or phosphorylated) didn't substantially degrade
BCR-ABL. Collectively, the results clarified that carbon-based linkers
can cause similar degradation efficacy to PEG-based linkers.
Meanwhile, these PROTACs composed of linkers of different lengths
all induced significant degradation of BCR-ABL, indicating that the
length of linkers seems not to be the key factor in BCR-ABL
degradation for CRBN-based PROTACs.
Hereafter, several derivatives which contain linkers of three
atoms length with varying heteroatoms (S, N, O) and carbon atom
connected to C-4 position of pomalidomide were synthesized and
investigated. As depicted in Table 2, all compounds exhibited sig-
nificant anti-proliferative activities and BCR-ABL-degrading activ-
ities in K562 cells. In detail, the sulfur-substituted compound 12
exhibited the best degradative activity with DC50 value about
1
2
9
0
CH
CO
0.51
0.28
2.7
2.7
21
CO
CH
4.6
4.2
10.1
11.1
2
2
3
2
2
2
CH
1.03
125
5
(
1
.6 nM. When sulfur atom is substituted by NH (compound 13) or O
compound 14), there was a slight decrease in the DC50 (about
0 nM). Substitution of S atom with C atom, the BCR-ABL-degrading
activity of the generated 15 is 17 times less potent than that of 12.
Inversion S atom in 12 with sulphone, the generated compound 16
was about 50 times less potent than 12 in BCR-ABL degradation.
Moreover, the anti-proliferative activity of 16 also significantly
decreased accompanied with reduction of degradation potency of
BCR-ABL. All these results suggested that the groups substituted at
C-4 of pomalidomide influence the BCR-ABL-degrading activity and
derivatives with substitution of S atom are more potent in BCR-ABL
degradation than those with substitution of other atoms.
Meanwhile, a series of acyl-substituted derivatives were syn-
thesized to explore the influence of the acyl group on biological
activities. As shown in Table 3, we found that the acyl substitution
at piperazine of dasatinib was adverse to the BCR-ABL-degrading
activity (12 vs 17, 18 vs 19, respectively). Compounds 17 and 19,
in which dasatinib was conjugated by alkylation substitution
exhibited more potent BCR-ABL-degrading activity with DC50 value
of 0.18 nM and 2.7 nM, respectively. Notably, 17 based on pomali-
domide possesses more potent degradative activity than 19 based
on lenalidomide. Furthermore, with insertion of carbonyl group at
amine group of pomalidomide or lenalidomide, the BCR-ABL-
degrading activity also decreased (20 vs 21, 22 vs 23, respec-
tively). In brief, PROTACs containing the alkylated linkers are more
potent in BCR-ABL degradation than those having linkers with the
acyl substitution group, while the acyl substitution on the end of
linkers was harmful to the BCR-ABL-degrading activity.
Herein, we summarized the SAR study according to the results
above (Fig. 2). First, the length of linkers based on either PEG-
2 2
containing (-O-CH -CH -O-) linkers or carbon-containing linkers
seem not to be an important factor in BCR-ABL degradation and this
will help us find potent PROTACs with smaller molecular weight.
Secondly, alkylated conjunction of dasatinib and CRBN ligands
Table 2
(pomalidomide and lenalidomide) exerts the better activity than
The anti-proliferative activities against K562 and BCR-ABL-degrading activity of
derivatives 12e16 having linkers with different atoms connected to C-4 of
pomalidomide.
acetylated conjunction between these two ligands in BCR-ABL
Compound
Linker
X
K562
BCR-ABL
DC50 (nM)
5.6
IC50 (nM)
3.2
12
13
14
15
16
CO
CO
CO
CO
CO
1.22
7
10
11.1
95.4
245.1
2.84
206
Fig. 2. A summary of SAR on BCR-ABL degradation.
6

H. Liu, X. Ding, L. Liu et al.
European Journal of Medicinal Chemistry 223 (2021) 113645
degradation. Moreover, the substitutions of heteroatoms and car-
bon atom at C-4 position of pomalidomide or lenalidomide provoke
different degradative ability and PROTACs based on sulfur-
substituted pomalidomide showed superior proliferative inhibi-
tory and BCR-ABL-degrading activities.
results showed that dramatic reduction of BCR-ABL was observed
after 4 h treatment of 17 at the 30 nM concentration (Fig. 4C).
Consistently, 17 also time-dependently inhibited the BCR-ABL
signaling, accompanied with decreased phosphorylation of BCR-
ABL and its downstream molecules STAT5 and CRKL. Additionally,
the time-course effects of 17 on turnover of BCR-ABL protein was
also assessed by adding a protein synthesis inhibitor cycloheximide
(CHX). The accelerated turnover rate of BCR-ABL and c-ABL proteins
were observed following treatment of 17 as compared to that of
vehicle treatment, further supporting the BCR-ABL-degrading effi-
cacy of 17 in K562 cells (Supporting Fig. S3A and Fig. S3B). More-
over, the pretreatment of pomalidomide or dasatinib, and
proteasome inhibitor MG132 as well as NEDD8-activating enzyme
(NAE) inhibitor MLN4924 significantly blocked the degrader 17-
induced degradation of BCR-ABL and c-ABL in a competition assay
(Fig. 4D), suggesting that 17-induced target protein degradation is
dependent on the CRBN ubiquitin ligase and consistent with the
nature of PROTAC principle.
As we mentioned in the introduction, this work builds on our
own previous work [28], in which the SAR study of VHL-recruiting
PROTACs targeting BCR-ABL was conducted and the most potent
degrader SIAIS178 was identified to induce effective degradation of
BCR-ABL protein. The comparison between 17 and SIAIS178, the
two most active BCR-ABL PROTACs of our CRBN- and VHL-
recruiting BCR-ABL PROTAC series respectively, is worthy of
further discussion. First, both of 17 and SIAIS178 possessed carbon
2.2.2. The linkers’ influence on PK properties of PROTACs
On the basis of chemical structure and biological evaluation
above, PROTACs including 5, 17 and 20 were further performed to
evaluate their pharmacokinetics (PK) properties. These compounds
were administered intravenously to the femoral vein of female
Wistar rats at 2 mg/kg. The results were shown in Table 4. Com-
pound 17 was shown to have superior pharmacokinetic profiles
relative to others in half-life (T1/2), and both of 17 and 20 displayed
lower clearance rate than 5. On the contrary, compound 5 exhibited
poor pharmacokinetic profiles. Unfortunately, the oral bioavail-
ability of all these compounds is unsatisfactory (data not shown).
Conclusively, the PK result indicated that the linkers of different
lengths and compositions could influence the PK properties of
PROTACs in vivo. Compound 17, containing the sulfur-substituted
linkers, resulted in a dramatically improved pharmacokinetic pro-
files in vivo, with a prolonged T1/2, increased plasma concentration,
and increased area under the plasma concentration-time curve
(
AUC), and will be utilized for further in vivo studies. The superior
pharmacokinetic profiles of compound 17 may be due to that its
linker parameters such as length, hydrophilicity and rigidity
possibly improved stability or cell membrane permeability in vivo.
Further ADME studies may be needed to be explored in the future.
2
alkyl chain (-(CH ) n-) linkers conjugating dasatinib to the corre-
sponding E3 ligands, which suggested that the alkylated chain
linker could be considered for the future design of novel PROTACs
to achieve effective BCR-ABL degradation. Secondly, the degrader
17 showed more potent in vitro anti-proliferative and BCR-ABL-
degrading activity than SIAIS178, further indicating that CRBN-
recruiting PROTACs exhibit more effective and broader spectrum
of protein degradation compared with VHL-based PROTACs, which
was in line with previous literatures [10,19]. Additionally, the
degrader 17, which presented smaller molecular weight and
shorter chain length than SIAIS178, to some extent, may have more
potential for the CML therapy and be worthy of further study.
2.2.3. Degrader 17 induced BCR-ABL degradation in dose and time
dependent manner
To confirm the mechanistic involvement of CRBN E3 ubiquitin
ligase for BCR-ABL degrading activity of compound 17 (SIAIS056),
we prepared inactive compound 24 (Fig. 3) which contained a
methyl group at N of glutarimide as a negative control. The inactive
compound 24 showed reduced anti-proliferative activity against
K562 cells and 32D cells exogenously expressing wide-type BCR-
ABL compared with 17 (Supporting Fig. S1). Moreover, the degrader
17 dramatically decreased the protein level of BCR-ABL and c-ABL at
dose dependent manner (Fig. 4A), while dasatinib and 24 failed to
induce BCR-ABL and c-ABL degradation (Fig. 4A and B). Meanwhile,
the BCR-ABL signaling activity, indicated as the phosphorylation of
BCR-ABL and its downstream proteins including STAT5 (signal
transducer and activator of transcription 5) and CRKL (Crk like
proto-oncogene), were significantly inhibited by both 17 and 24
2.2.4. Degrader 17 induced the degradation of BCR-ABL mutations
Degrader 17 was further investigated to evaluate the potential
ability to overcome resistance to currently clinical tyrosine kinase
inhibitors treatment primarily associated with the BCR-ABL muta-
tions. Herein, murine myeloid cell line 32D was transduced with
retroviral vector carrying wide-type BCR-ABL as well as various
clinically relevant BCR-ABL mutant isoforms and the degradation
was further evaluated by western blotting. Among them, G250E,
E255K, F317L and T315I were imatinib-resistant mutants. E255K,
V299L, F317V, F317L, T315A and T315I were associated with
(
Fig. 4A and B). Of note, degrader 17 also induced the degradation of
Src, but not PDGFR (Supporting Fig. S2), which are the potential
b
targets of dasatinib [36], indicating that the selectivity of PROTACs
was improved over parent inhibitor. Furthermore, the time-course
Table 4
The PK assay of compound 5, 17 and 20 in rat (iv).
a
b
c
AUC(0-t)d
e
Vzf
Clg
Compound
T
1/2
T
max
C
max
MRT(0-t)
h
V
ss
h
h
ng/mL
h*ng/mL
mL/kg
mL/kg
mL/h/kg
5
0.62
4.23
1.93
0.08
0.08
0.08
1142.78
1497.45
1449.44
496.43
926.29
1331.45
0.45
2.00
1.55
1929.82
5225.18
2783.19
3591.00
13046.83
4020.74
4026.80
2140.26
1448.68
1
2
7(SIAIS056)
0
Abbreviations: AUC, area under the plasma concentration-time curve; MRT, mean residence time.
a
T
T
1/2, elimination half-life.
max, time taken to reach peak plasma concentration.
b
c
d
e
f
Cmax, maximum (peak) plasma drug concentration.
AUC(0-t), AUC from time zero to time t.
V
V
ss, the apparent volume of distribution during steady state.
, apparent volume of distribution during the terminal phase.
z
g
Cl, apparent total body clearance of the drug from plasma.
7

H. Liu, X. Ding, L. Liu et al.
European Journal of Medicinal Chemistry 223 (2021) 113645
Fig. 3. The structure of compound 17 and its negative control 24.
Fig. 4. The BCR-ABL degradation induced by the degrader 17. (A, B) K562 cells were treated for 16 h with 17 or 24 at different concentrations, and dasatinib was included as control
compound. The protein levels were analyzed by western blotting. (C) K562 cells were treated with 17 for time as indicated. The BCR-ABL and c-ABL protein levels were determined
by Western blot. (D) K562 cells were pretreated with DMSO, pomalidomide (4
mM), dasatinib (100 nM), proteasome inhibitors MG132 (2 mM) or NEDD8-activating enzyme (NAE)
inhibitor MLN4924 (3 M) for 2 h and then followed by 17 treatment at 30 nM for 8 h.
m
dasatinib resistance [41e43]. As depicted in Fig. 5, besides of
degradation on wide-type BCR-ABL, degrader 17 also induced
degradation of BCR-ABL mutations, including G250E, E255V, V299L,
F317L, F317V and T315A at low nanomolar concentration. However,
the T315I mutant resulting from gatekeeper T315 residue replace-
ment with isoleucine, which sterically blocked the critical direct
binding with imatinib and dasatinib examined by the crystal
structure study [44], were as expectedly refractory to 17-induced
degradation.
78.9%, 93.8%, and 98.8% for the 1, 3, and 10 mg/kg dosages of
degrader 17, respectively. Of note, 17 at 3 mg/kg by intraperitoneal
administration exhibited similar anti-leukemic activity to that of
dasatinib at 5 mg/kg by oral administration (TGI: 93.8% versus
95.1%), although the comparison may be not so rational according
to the differences of administration methods and physicochemical
properties between the two compounds. Notably, the mice were
well tolerated even given compound 17 at the dose of 100 mg/kg
(data not shown) and the animal weight was preserved during the
treatment procedure above (Fig. 6B). The in vivo anti-CML efficacy
of 17 was further confirmed by bioluminescence imaging after 10
days of treatment (Fig. 6C and D). All the results indicated that 17
exerted potent activity and safety in vivo. Moreover, the BCR-ABL
protein degradation was also observed following the daily contin-
uous 4 days’ treatment, paralleled by suppression of BCR-ABL
phosphorylation (Supporting Fig. S4). Taken together, the results
clarified that PROTAC 17 induced effective degradation of BCR-ABL
protein and substantial anti-leukemic activity in vivo.
2.2.5. Anti-leukemic efficacy of 17 in vivo
Next, we then evaluated the tolerability and anti-CML efficacy of
17 in a murine xenograft model of K562 cells exogenously carrying
a luciferase reporter gene (termed K562-Luc) with dasatinib as
control compound. After 10 days of treatment, intraperitoneal
administration of 17 alleviated tumor progression in a dose-
dependent manner as determined by serial volumetric measure-
ment (Fig. 6A). A decrease in tumor burden was observed in
degrader 17-treated mice at 1 mg/kg and the complete tumor
regression was induced following injections of 17 at 10 mg/kg,
which was maintained even after drug withdrawal. The tumor
growth inhibition (TGI) is calculated at day 10 and shows values of
3. Conclusion
PROTACs represent
a
novel strategy for therapeutic
8

H. Liu, X. Ding, L. Liu et al.
European Journal of Medicinal Chemistry 223 (2021) 113645
Fig. 5. The degradative activity of 17 against BCR-ABL mutations. The western blotting analysis of wide-type and mutant isoforms of BCR-ABL exogenously expressed in 32D cells
following treatment of 17 at different concentrations.
Fig. 6. In vivo anti-leukemic efficacy of BCR-ABL degrader 17 in the K562-Luc xenograft models. (A) The tumor volume was measured following intraperitoneal administration of 17
(
IP) at different dose-schedules. Administration of dasatinib (PO) was served as controls. The dashed-line indicates the treatment period. (B) The body weight alteration was
measured following treatment as indicated. (C) Tumor burden was monitored by measuring the luciferase activity through bioluminescence imaging. (D) Quantitative results were
analyzed for each group (n ꢁ 6) and expressed as total photon values per second (Total Flux [p/s]). The symbols * indicate P < 0.05 compared with the vehicle-treated group.
intervention. The lingering problem of BCR-ABL-targeted therapy
such as persistent leukemic stem cells retention and resistance-
conferring mutations urge the exploration of potential BCR-ABL
degraders. In this study, we attempt to design novel BCR-ABL de-
graders by conjugating BCR-ABL inhibitor dasatinib and CRBN li-
gands and perform SAR studies to identify the potent and selective
PROTACs targeting BCR-ABL. We focused on the extensive optimi-
zation of linkers’ parameters and our results showed that
pomalidomide-based degrader 17 (SIAIS056), exerted the most
potent BCR-ABL-degrading activity in vitro and favorable pharma-
cokinetics in vivo. Besides, degrader 17 also degrades several clin-
ically relevant resistance-conferring mutations of BCR-ABL except
for T315I. Moreover, degrader 17 induces BCR-ABL degradation and
significant tumor regression against K562 xenograft tumors in vivo.
Our study indicates that 17 as an efficacious BCR-ABL degrader
warrants extensive further investigation for the treatment of BCR-
þ
ABL leukemia.
9

H. Liu, X. Ding, L. Liu et al.
European Journal of Medicinal Chemistry 223 (2021) 113645
4
. Experimental protocols
4.1.3. N-(2-chloro-6-methylphenyl)-2-((6-(4-(3-(2-((2-(2,6-
dioxopiperidin-3-yl)-1,3-dioxo- isoindolin-4-yl)amino)ethoxy)
propanoyl)piperazin-1-yl)-2-methylpyrimidin-4-yl)amino)thiazole-
4.1. Chemistry
5-carboxamide (4)
All chemicals were obtained from commercial suppliers (Ada-
Compound S2 (1.2 mmol, 1 equiv) and compound S5-
mas and Alfa), and used without further purification, unless
otherwise indicated. Flash chromatography was carried out on
silica gel (200e300 mesh). Analytical TLC was performed on
Haiyang ready-to-use plates with silica gel 60 (F254). All new
1 (1.2 mmol) were added into a dry round-bottom flask. Then HOAt
(1.8 mmol, 1.5 equiv), EDCI (1.8 mmol, 1.5 equiv), NMM (2.4 mmol, 2
ꢀ
equiv) and DMF (3 ml) were mixed and stirred at 25 C for 12 h. The
reaction was quenched with water (1.0 ml) followed by purification
1
13
1
compounds were characterized by H NMR and C NMR, HRMS. H
via preparative HPLC (100 g, C18 column) to afford the desired
1
NMR spectra were recorded on Bruker AVANCE III 500 MHZ
product 4. Yellow solid, 30.2 mg, (yield 82%). H NMR (500 MHz,
1
(
operating at 500 MHz for H NMR), chemical shifts were reported
CD
3
OD)
d
8.21 (s,1H), 7.55e7.47 (m,1H), 7.37 (dd, J ¼ 7.5, 1.5 Hz,1H),
in ppm relative to the residual DMSO‑d
6
(2.50 ppm 1H), and
7.30e7.21 (m, 2H), 7.04 (d, J ¼ 8.5 Hz, 1H), 6.95 (d, J ¼ 7.1 Hz, 1H),
6.13 (s, 1H), 5.02 (dd, J ¼ 12.8, 5.4 Hz, 1H), 3.84 (t, J ¼ 5.8 Hz, 2H),
3.79e3.62 (m, 10H), 3.52e3.45 (m, 2H), 2.87e2.79 (m, 1H),
2.77e2.61 (m, 4H), 2.54 (s, 3H), 2.33 (s, 3H), 2.13e2.05 (m, 1H).
NMR (126 MHz, DMSO‑d
167.26, 165.16, 162.53, 162.14, 159.91, 156.93, 146.39, 140.83, 138.83,
136.25, 133.52, 132.44, 132.08, 129.04, 128.19, 127.02, 125.78, 117.42,
110.68, 109.23, 82.75, 68.70, 66.70, 48.57, 44.30, 43.52, 43.17, 41.69,
40.50, 32.82, 30.98, 25.53, 22.16, 18.31. MS (ESI, m/z): 815 [MþH] .
coupling constants (J) are given in Hz. Multiplicities of signals are
described as follows: s ¼ singlet, d ¼ doublet, t ¼ triplet,
q ¼ quartet, m ¼ multiple. High Resolution Mass spectra were
recorded on AB Triple 4600 spectrometer with acetonitrile and
water as solvent. The final compounds were all purified by C18
reverse phase preparative HPLC column with solvent A (0.05% HCl
13
C
6
)
d
172.79, 170.08, 168.97 (d, J ¼ 4.0 Hz),
in H
compounds was confirmed to be >95% purity by HPLC
SHIMADZU).
2
O) and solvent B (MeCN) as eluents. The purity of all the final
þ
(
4.1.4. 3-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-
4.1.1. N-(2-chloro-6-methylphenyl)-2-((2-methyl-6-(piperazin-1-
yl)amino)ethoxy)ethoxy) propanoic acid (S5-2)
yl)pyrimidin-4-yl)amino)thiazole- 5-carboxamide (S2)
The synthesis of intermediate S5-2 was similar to S5-1. Yellow
1
In a round-bottom flask, to a stirred solution of compound S1
solid, 0.95 g, (yield 51%). H NMR (500 MHz, DMSO‑d
6
)
d
11.09 (s,
(
1.0 g, 2.54 mmol) in n-butyl alcohol (20 ml) DIPEA (4.9 g, 38 mmol)
1H), 7.58 (dd, J ¼ 8.0, 7.5 Hz, 1H), 7.14 (d, J ¼ 8.6 Hz, 1H), 7.04 (d, J ¼
7.0 Hz, 1H), 6.60 (t, J ¼ 5.7 Hz, 1H), 5.05 (dd, J ¼ 12.8, 5.4 Hz, 1H),
3.62e3.58 (m, 4H), 3.56e3.54 (m, 2H), 3.52e3.49 (m, 2H), 3.46 (dd,
J ¼ 11.1, 5.5 Hz, 2H), 2.92e2.84 (m, 1H), 2.66e2.51 (m, 2H), 2.42 (t,
and piperazine (0.55 g, 6.35 mmol) were added. Then the resulting
mixture was stirred for 16 h at 110 C under the nitrogen atmo-
sphere. Once the starting material disappeared, monitored by
LCMS, the mixture was filtered through Celite and washed with
MeOH. The collected solid was then added to a round-bottom flask
which was equipped with water (10 ml) and MeOH (30 ml). Then
the mixture was stirred for another 1 h at 60 C. The solid was
collected by filtration and washed with MeOH to furnish compound
S2 as a white solid (0.9 g, yield 80%). H NMR (500 MHz, DMSO‑d
ꢀ
þ
J ¼ 6.4 Hz, 2H), 2.06e1.98 (m, 1H). MS (ESI, m/z): 434 [MþH] .
4.1.5. N-(2-chloro-6-methylphenyl)-2-((6-(4-(3-(2-(2-((2-(2,6-
dioxopiperidin-3-yl)-1,3-dioxo- isoindolin-4-yl)amino)ethoxy)
ethoxy)propanoyl)piperazin-1-yl)-2-methylpyrimidin-4-yl)amino)
thiazole-5-carboxamide (5)
ꢀ
1
6
)
d
9.88 (s,1H), 8.23 (s,1H), 7.43e7.38 (m,1H), 7.31e7.24 (m, 2H), 6.04
The derivative 5 was obtained from the condensation of S5-2
(
3
4
s, 1H), 3.45 (d, J ¼ 4.6 Hz, 4H), 2.79e2.71 (m, 4H), 2.44e2.37 (m,
and S2 according the similar process of 4. Yellow solid, 13.7 mg,
þ
þ
1
H), 2.25 (s, 3H). HRMS (ESI) C20
44.1368; found, 444.1361.
H23ClN
7
OS [M þ H] , calcd
(yield 35%). H NMR (500 MHz, CD
3
OD)
d
8.18 (s, 1H), 7.49e7.43 (m,
1H), 7.38e7.34 (m, 1H), 7.29e7.21 (m, 2H), 6.97 (d, J ¼ 8.6 Hz, 1H),
6
.92 (d, J ¼ 7.1 Hz, 1H), 6.09 (s, 1H), 5.05 (dd, J ¼ 12.7, 5.5 Hz, 1H),
4
.1.2. 3-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)
3.79 (t, J ¼ 5.8 Hz, 4H), 3.70 (dd, J ¼ 11.6, 6.3 Hz, 6H), 3.64 (t, J ¼
3.8 Hz, 6H), 3.42 (t, J ¼ 5.2 Hz, 2H), 2.89e2.79 (m, 1H), 2.78e2.65
amino)ethoxy)propanoic acid (S5-1)
13
2
-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione
(m, 4H), 2.50 (s, 3H), 2.33 (s, 3H), 2.15e2.09 (m, 1H). C NMR
(
5
mmol, 1equiv), tert-butyl 3-(2-aminoethoxy)propanoate
6
(126 MHz, DMSO‑d ) d 172.81, 170.10, 169.13, 168.96, 167.28, 165.07,
(
6 mmol, 1.2 equiv) and DIPEA (25 mmol, 5 equiv) were put into the
162.52, 162.03, 159.89, 156.90, 146.39, 140.81, 138.83, 136.21, 133.50,
132.44, 132.08, 129.05, 128.20, 127.03, 125.76, 117.43, 110.67, 109.25,
82.79, 69.74, 68.88, 66.86, 48.57, 44.31, 43.58, 43.25, 41.71, 40.47,
32.88, 30.99, 25.46, 22.15, 18.31. MS (ESI, m/z): 859 [MþH] .
microwave reaction tube (30 ml), then added 8 ml NMP. And the
tube was ventilated with argon gas. The reaction mixture was
allowed to stir at 110 C microwave for 2 h. After that, the reaction
ꢀ
þ
solution was cooled to room temperature and poured into salt so-
lution. The reaction mixture was further extracted with ethyl ace-
tate (3 ꢂ 50 ml). The organic extracts were combined, washed with
4.1.6. 3-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-
4-yl)amino)ethoxy)ethoxy) ethoxy)propanoic acid (S5-3)
salt solution (50 ml), dried over Na
2
SO
4
, filtered, and evaporated
The synthesis of intermediate S5-3 was similar to S5-1. Yellow
1
under vacuum and was subjected to column purification (PE/
EA ¼ 1:1) to furnish the intermediate.
solid, 0.95 g, (yield 61%). H NMR (500 MHz, DMSO‑d
6
)
d
11.09 (s,
1H), 7.58 (dd, J ¼ 8.0, 7.0 Hz, 1H), 7.15 (d, J ¼ 8.6 Hz, 1H), 7.04 (d, J ¼
7.0 Hz, 1H), 6.61 (t, J ¼ 5.8 Hz, 1H), 5.05 (dd, J ¼ 12.8, 5.4 Hz, 1H),
3.63e3.48 (m, 14H), 2.92e2.83 (m, 1H), 2.64e2.52 (m, 2H), 2.18 (t,
The obtained intermediate was then added to a 50 ml singlet
bottle with 88% 20 ml of formic acid and stirred at room temper-
ature for 12 h. The reaction solvent is evaporated and water is
þ
J ¼ 8.1 Hz, 2H), 2.07e1.99 (m, 1H). MS (ESI, m/z): 478 [MþH] .
added to freeze-dry the final target compound S5-1. Yellow solid,
1
(
1
.0 g, (yield 48%). H NMR (500 MHz, DMSO‑d
6
)
d
12.17 (s, 1H), 11.09
4.1.7. N-(2-chloro-6-methylphenyl)-2-((6-(4-(3-(2-(2-(2-((2-(2,6-
dioxopiperidin-3-yl)-1,3-dioxo isoindolin4-yl)amino)ethoxy)
ethoxy)ethoxy)propanoyl)piperazin-1-yl)-2-methylpyrimidin-4-yl)
amino)thiazole-5-carboxamide (6)
The derivative 6 was obtained from the condensation of S5-3
and S2 according the similar process of 4. Yellow solid, 15.7 mg,
s, 1H), 7.57 (dd, J ¼ 8.5, 7.5 Hz, 1H), 7.13 (d, J ¼ 8.6 Hz, 1H), 7.04 (d,
J ¼ 7.0 Hz, 1H), 6.59 (t, J ¼ 5.7 Hz, 1H), 5.05 (dd, J ¼ 12.8, 5.4 Hz, 1H),
3
2
2
.65 (t, J ¼ 6.3 Hz, 2H), 3.59 (t, J ¼ 5.5 Hz, 2H), 3.46 (q, J ¼ 5.5 Hz,
H), 2.91e2.83 (m, 1H), 2.61e2.52 (m, 2H), 2.46 (t, J ¼ 6.3 Hz, 2H),
þ
.05e2.00 (m, 1H). MS (ESI, m/z): 390 [MþH] .
10

H. Liu, X. Ding, L. Liu et al.
European Journal of Medicinal Chemistry 223 (2021) 113645
1
(
yield 38%). H NMR (500 MHz, CD
3
OD)
d
8.17 (s, 1H), 7.54e7.46 (m,
4.1.12. 4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)
1
H), 7.37 (d, J ¼ 6.0 Hz, 1H), 7.29e7.22 (m, 2H), 6.99 (dd, J ¼ 14.0,
amino)butanoic acid (S7-1)
7
.8 Hz, 2H), 6.17 (s, 1H), 5.05 (dd, J ¼ 12.8, 5.5 Hz, 1H), 3.84e3.72 (m,
The synthesis of intermediate S7-1 was similar to S5-1. Yellow
1
10H), 3.68 (t, J ¼ 5.2 Hz, 2H), 3.65e3.62 (m, 6H), 3.61e3.59 (m, 2H),
solid, 0.8 g, (yield 61%). H NMR (500 MHz, DMSO‑d
6
)
d
12.14 (s, 1H),
3
.43 (t, J ¼ 5.2 Hz, 2H), 2.92e2.81 (m, 1H), 2.77e2.66 (m, 4H), 2.54
11.09 (s,1H), 7.58 (dd, J ¼ 8.4, 7.3 Hz,1H), 7.13 (d, J ¼ 8.6 Hz,1H), 7.02
þ
(
s, 3H), 2.33 (s, 3H), 2.15e2.09 (m, 1H). MS (ESI, m/z): 903 [MþH] .
(d, J ¼ 7.0 Hz, 1H), 6.65 (t, J ¼ 6.0 Hz, 1H), 5.05 (dd, J ¼ 12.8, 5.4 Hz,
1
2
H), 3.32 (dd, J ¼ 13.7, 6.7 Hz, 2H), 2.94e2.82 (m, 1H), 2.66e2.51 (m,
H), 2.30 (t, J ¼ 7.2 Hz, 2H), 2.05e2.00 (m, 1H), 1.82e1.75 (m, 2H).
4.1.8. 1-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)
þ
MS (ESI, m/z): 360 [MþH] .
amino)-3,6,9,12-tetraoxa pentadecan-15-oic acid (S5-4)
The synthesis of intermediate S5-4 was similar to S5-1. Yellow
4
.1.13. N-(2-chloro-6-methylphenyl)-2-((6-(4-(4-((2-(2,6-
1
solid, 0.87 g, (yield 53%). H NMR (500 MHz, DMSO‑d
6
)
d
11.09 (s,
dioxopiperidin-3-yl)-1,3-dioxoisoindolin -4-yl)amino)butanoyl)
piperazin-1-yl)-2-methylpyrimidin-4-yl)amino)thiazole-5-
carboxamide (9)
1
7
3
H), 7.58 (dd, J ¼ 8.5, 7.5 Hz, 1H), 7.15 (d, J ¼ 8.6 Hz, 1H), 7.04 (d, J ¼
.0 Hz, 1H), 6.60 (t, J ¼ 5.7 Hz, 1H), 5.05 (dd, J ¼ 12.8, 5.4 Hz, 1H),
.63e3.48 (m, 18H), 2.92e2.84 (m, 1H), 2.63e2.52 (m, 2H), 2.41 (t,
The derivative 9 was obtained from the condensation of S7-1
þ
J ¼ 6.4 Hz, 2H), 2.07e1.98 (m, 1H). MS (ESI, m/z): 522 [MþH] .
and S2 according the similar process of 4. Yellow solid, 31.2 mg,
1
(yield 88%). H NMR (500 MHz, DMSO‑d
6
)
d
11.53 (s, 1H), 11.09 (s,
1
H), 9.89 (s, 1H), 8.22 (s, 1H), 7.59 (dd, J ¼ 8.5, 7.1 Hz,1H), 7.40 (d, J ¼
4
.1.9. N-(2-chloro-6-methylphenyl)-2-((6-(4-(1-((2-(2,6-
7
.7 Hz, 1H), 7.31e7.23 (m, 2H), 7.19 (d, J ¼ 8.7 Hz, 1H), 7.02 (d, J ¼
dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-3,6,9,12-
tetraoxapentadecan-15-oyl)piperazin-1-yl)-2-methylpyrimidin-4-
yl)amino)thiazole-5-carboxamide (7)
7.0 Hz, 1H), 6.68 (s, 1H), 6.07 (s, 1H), 5.05 (dd, J ¼ 12.7, 5.4 Hz, 1H),
3
2
2
.51e3.48 (m, 8H), 3.34 (t, J ¼ 7.1 Hz, 2H), 2.92e2.84 (m, 1H),
.64e2.52 (m, 2H), 2.45 (t, J ¼ 7.1 Hz, 2H), 2.43 (s, 3H), 2.24 (s, 3H),
The derivative 7 was obtained from the condensation of S5-4
13
.04e2.00 (m, 1H), 1.86e1.79 (m, 2H). C NMR (126 MHz,
and S2 according the similar process of 4. Yellow solid, 18.6 mg,
DMSO‑d
62.51,162.03, 159.88, 156.92, 146.41, 140.79, 138.82, 136.25, 133.49,
32.43, 132.25, 129.05, 128.21, 127.02, 125.83, 117.30, 110.41, 109.11,
6
) d 172.83, 170.50, 170.12, 168.84, 167.33, 165.08,
1
(
yield 43%). H NMR (500 MHz, CD
3
OD)
d
8.17 (s, 1H), 7.50 (t, J ¼
1
1
7
2
3
2
2
1
1
1
.8 Hz, 1H), 7.36 (d, J ¼ 7.2 Hz, 1H), 7.31e7.21 (m, 2H), 7.06e6.95 (m,
H), 6.19 (s, 1H), 5.05 (dd, J ¼ 12.8, 5.5 Hz, 1H), 3.85e3.71 (m, 10H),
.70 (t, J ¼ 5.2 Hz, 2H), 3.66e3.57 (m, 12H), 3.44 (t, J ¼ 5.0 Hz, 2H),
.90e2.82 (m, 1H), 2.76e2.65 (m, 4H), 2.55 (s, 3H), 2.33 (s, 3H),
8
2
2.83, 48.54, 44.08, 43.53, 43.28, 41.60, 40.55, 30.99, 29.45, 25.45,
4.16, 22.17, 18.30. MS (ESI, m/z): 785 [MþH] .
þ
.16e2.07 (m, 1H). 13C NMR (126 MHz, DMSO‑d
d
172.82, 170.09,
69.13, 168.95, 167.30, 165.08, 162.54, 162.04, 159.89, 156.92, 146.41,
40.77,138.83, 136.22, 133.51, 132.44, 132.09, 129.05, 128.21, 127.03,
25.80,117.44, 110.68, 109.25, 82.82, 69.89e69.67 (m), 68.89, 66.78,
6
)
4.1.14. 5-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)
amino)pentanoic acid (S7-2)
The synthesis of intermediate S7-2 was similar to S5-1. Yellow
1
solid, 0.9 g, (yield 50%). H NMR (500 MHz, DMSO‑d
6
)
d
12.05 (s,
4
8.57, 44.32, 43.61, 43.28, 41.70, 40.47, 32.87, 30.99, 25.45, 22.16,
1
H), 11.11 (s, 1H), 7.57 (dd, J ¼ 8.3, 7.4 Hz, 1H), 7.09 (d, J ¼ 8.6 Hz,1H),
þ
18.31. MS (ESI, m/z): 947 [MþH] .
7.02 (d, J ¼ 7.0 Hz, 1H), 6.56 (t, J ¼ 5.9 Hz, 1H), 5.05 (dd, J ¼ 12.7,
5
.4 Hz, 1H), 3.32e3.28 (m, 2H), 2.94e2.82 (m, 1H), 2.62e2.51 (m,
2
H), 2.27e2.25 (m, 2H), 2.06e1.99 (m, 1H), 1.62e1.53 (m, 4H). MS
4
.1.10. 1-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)
þ
(ESI, m/z): 374 [MþH] .
amino)-3,6,9,12,15-pentaoxa octadecan-18-oic acid (S5-5)
The synthesis of intermediate S5-5 was similar to S5-1. Yellow
1
4.1.15. N-(2-chloro-6-methylphenyl)-2-((6-(4-(5-((2-(2,6-
dioxopiperidin-3-yl)-1,3-dioxoisoindolin -4-yl)amino)pentanoyl)
piperazin-1-yl)-2-methylpyrimidin-4-yl)amino)thiazole-5-
carboxamide (10)
solid, 0.8 g, (yield 51%). H NMR (500 MHz, DMSO‑d
6
)
d
11.09 (s, 1H),
7
6
8
1
.58 (t, J ¼ 8.0 Hz, 1H), 7.14 (d, J ¼ 8.6 Hz, 1H), 7.04 (d, J ¼ 7.0 Hz, 1H),
.60 (t, J ¼ 5.7 Hz, 1H), 5.05 (dd, J ¼ 12.8, 5.4 Hz, 1H), 3.63e3.54 (m,
H), 3.54e3.48 (m, 12H), 3.30 (dd, J ¼ 7.0 Hz, 4H), 2.92e2.84 (m,
The derivative 10 was obtained from the condensation of S7-2
H), 2.63e2.52 (m, 2H), 2.06e1.99 (m, 1H). MS (ESI, m/z): 947
þ
and S2 according the similar process of 4.Yellow solid, 10 mg, (yield
[MþH] .
1
28%). H NMR (500 MHz, CD
3
OD)
d
8.21 (s, 1H), 7.56 (dd, J ¼ 8.5,
7
.2 Hz, 1H), 7.37 (d, J ¼ 7.1 Hz, 1H), 7.26 (t, J ¼ 7.7 Hz, 2H), 7.08 (d, J ¼
4
.1.11. N-(2-chloro-6-methylphenyl)-2-((6-(4-(1-((2-(2,6-
8.6 Hz, 1H), 7.04 (d, J ¼ 7.1 Hz, 1H), 6.30 (s, 1H), 5.04 (dd, J ¼ 12.6,
5.5 Hz,1H), 3.83e3.69 (m, 8H), 3.40 (t, J ¼ 6.1 Hz, 2H), 2.89e2.80 (m,
1H), 2.76e2.67 (m, 2H), 2.61 (s, 3H), 2.53 (d, J ¼ 6.4 Hz, 2H), 2.32 (s,
dioxopiperidin-3-yl)-1,3-dioxoisoindolin -4-yl)amino)-3,6,9,12,15-
pentaoxaoctadecan-18-oyl)piperazin-1-yl)-2-methylpyrimidin-4-
yl)amino)thiazole-5-carboxamide (8)
13
3H), 2.13e2.07 (m, 1H), 1.80e1.72 (m, 4H). C NMR (126 MHz,
The derivative 8 was obtained from the condensation of S5-5
6
DMSO‑d ) d 172.81, 170.76, 170.11, 168.94, 167.30,163.92, 162.48,
and S2 according the similar process of 4. Yellow solid, 35 mg, (yield
159.73, 158.81, 156.80, 146.41, 139.80 (d, J ¼ 250.7 Hz), 136.27,
133.46, 132.32 (d, J ¼ 26.9 Hz), 129.04, 128.22, 127.03, 117.27, 110.39,
109.04, 83.04, 53.37, 48.54, 43.82 (d, J ¼ 63.3 Hz), 41.60, 31.89,
30.98, 28.30, 22.16, 21.96, 18.31, 18.00, 16.71. MS (ESI, m/z): 799
1
7
8%). H NMR (500 MHz, CD
3
OD)
d
8.18 (s, 1H), 7.54e7.48 (m, 1H),
7
.36 (d, J ¼ 7.4 Hz, 1H), 7.28e7.22 (m, 2H), 7.03 (dd, J ¼ 16.0, 7.8 Hz,
2
3
3
2
H), 6.23 (s, 1H), 5.04 (dd, J ¼ 12.8, 5.4 Hz, 1H), 3.86e3.73 (m, 10H),
.71 (t, J ¼ 5.1 Hz, 2H), 3.78e3.68 (m, 10H), 3.65e3.60 (m, 7H),
.59e3.57 (m, 9H), 3.46 (t, J ¼ 5.1 Hz, 2H), 2.89e2.81 (m, 1H),
þ
[MþH] .
1
3
.76e2.65 (m, 4H), 2.57 (s, 3H), 2.32 (s, 3H), 2.15e2.08 (m, 1H).
C
4.1.16. 7-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)
NMR (126 MHz, DMSO‑d
65.58,162.98,162.54, 160.35, 157.39, 146.88,141.25, 139.29, 136.69,
33.96, 132.90, 132.55, 129.51, 128.67, 127.49, 126.26,117.91, 111.14,
09.70, 83.27, 70.48e69.97 (m), 69.35, 67.25, 49.03, 44.77, 44.05,
6
)
d
173.27, 170.54, 169.58, 169.40, 167.76,
amino)heptanoic acid (S7-3)
1
1
1
The synthesis of intermediate S7-3 was similar to S5-1. Yellow
1
solid,1.3 g, (yield 64%). H NMR (500 MHz, DMSO‑d
6
)
d
12.04 (s,1H),
11.09 (s, 1H), 7.58 (dd, J ¼ 8.3, 7.3 Hz, 1H), 7.09 (d, J ¼ 8.6 Hz, 1H),
7.02 (d, J ¼ 7.0 Hz, 1H), 6.53 (t, J ¼ 5.9 Hz, 1H), 5.05 (dd, J ¼ 12.7,
5.4 Hz, 1H), 3.28 (dd, J ¼ 13.4, 6.7 Hz, 2H), 2.94e2.82 (m, 1H),
4
3.73, 42.16, 40.93, 33.33, 31.45, 25.94, 22.61, 18.76. MS (ESI, m/z):
þ
9
47 [MþH] .
11

H. Liu, X. Ding, L. Liu et al.
European Journal of Medicinal Chemistry 223 (2021) 113645
2
.65e2.51 (m, 2H), 2.19 (t, J ¼ 7.3 Hz, 2H), 2.05e2.00 (m, 1H),
4.1.21. N-(2-chloro-6-methylphenyl)-2-((6-(4-(2-((2-(2,6-
dioxopiperidin-3-yl)-1,3-dioxoisoindolin -4-yl)thio)acetyl)
piperazin-1-yl)-2-methylpyrimidin-4-yl)amino)thiazole-5-
carboxamide (12)
1.60e1.53 (m, 2H), 1.53e1.46 (m, 2H),1.37e1.28 (m, 4H). MS (ESI, m/
þ
z): 799 [MþH] .
Compounds S2 (27.2 mg, 0.06 mmol), S10 (20 mg, 0.06 mmol),
HOAt (16.3 mg, 0.12 mmol), EDCI (23 mg, 0.12 mmol), anhydrous
DMF (2 mL), and NMM (30 mg, 0.30 mmol) were added to the re-
action bottle and stirred overnight at room temperature. The re-
action was quenched with water (1.0 ml) followed by purification
via preparative HPLC (100 g, C18 column) to afford the desired
4
.1.17. N-(2-chloro-6-methylphenyl)-2-((6-(4-(7-((2-(2,6-
dioxopiperidin-3-yl)-1,3-dioxoisoindolin -4-yl)amino)heptanoyl)
piperazin-1-yl)-2-methylpyrimidin-4-yl)amino)thiazole-5-
carboxamide (11)
The derivative 11 was obtained from the condensation of S7-3
and S2 according the similar process of 4. Yellow solid, 10 mg, (yield
2
1
1
product 12. Yellow solid, 26.6 mg, (yield 56%). H NMR (500 MHz,
8%). H NMR (500 MHz, CD
3
OD)
d
7.54 (dd, J ¼ 8.5, 7.1 Hz, 1H), 7.36
DMSO‑d
.85e7.75 (m, 2H), 7.64 (d, J ¼ 6.4 Hz, 1H), 7.40 (d, J ¼ 7.2 Hz, 1H),
.34e7.21 (m, 2H), 6.09 (s, 1H), 5.13 (dd, J ¼ 12.9, 5.4 Hz, 1H), 4.34 (s,
H), 3.75e3.59 (m, 8H), 2.94e2.84 (m, 1H), 2.64e2.53 (m, 2H), 2.44
6
) d 11.56 (s, 1H), 11.15 (s, 1H), 9.91 (s, 1H), 8.23 (s, 1H),
(
6
d, J ¼ 6.1 Hz, 1H), 7.28e7.22 (m, 3H), 7.03 (dd, J ¼ 10.7, 7.8 Hz, 2H),
7
7
.15 (s,1H), 5.04 (dd, J ¼ 12.6, 5.5 Hz,1H), 3.89e3.62 (m, 8H), 3.34 (t,
J ¼ 7.5 Hz, 2H), 2.87e2.80 (m, 1H), 2.77e2.68 (m, 2H), 2.56 (s, 3H),
2
2
4
.45 (t, J ¼ 7.5 Hz, 2H), 2.32 (s, 3H), 2.13e2.08 (m, 1H), 1.72e1.64 (m,
1
3
13
(s, 3H), 2.24 (s, 3H), 2.10e2.02 (m, 1H). C NMR (126 MHz,
DMSO‑d
172.79, 169.89, 166.69 (d, J ¼ 6.8 Hz), 165.77, 165.24,
62.51, 162.24, 159.91, 156.99, 140.86, 138.82, 138.34, 134.81, 133.51,
32.36 (d, J ¼ 20.2 Hz), 131.29, 129.04, 128.19, 127.02, 125.79, 125.34,
6
H), 1.53e1.39 (m, 4H). C NMR (126 MHz,DMSO‑d ) d 172.80,
6
) d
1
1
1
70.87, 170.09, 168.94, 167.43, 167.29, 161.24, 159.55, 158.50, 156.90,
46.42, 140.81, 138.77, 136.28, 133.30, 132.38, 132.19, 129.06, 128.29,
27.03, 117.18, 110.37, 109.00, 103.44, 83.00, 48.52, 44.35, 43.64,
1
1
1
19.07, 82.86, 48.92, 44.68, 43.28 (d, J ¼ 49.7 Hz), 41.17, 33.39, 30.95,
4
2
3.11, 41.79, 40.42, 32.20, 30.96, 28.53 (d, J ¼ 11.3 Hz), 26.16, 25.16,
þ
þ
þ
25.59, 21.96, 18.31. HRMS (ESI) m/z: calcd C
H
35 33
ClN O S [MþH] ,
4.64, 22.14, 18.28 (d, J ¼ 2.4 Hz). MS (ESI, m/z): 827 [MþH] .
9 6 2
7
74.1678; found, 774.1688.
4
.1.18. 2-(2,6-dioxopiperidin-3-yl)-4-mercaptoisoindoline-1,3-
4
.1.22. (2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)
dione (S8)
Compound S3 (20 g, 72.4 mmol) was dissolved in DMF (150 ml)
at room temperature, then Na S 9H O (28 g, 108.6 mmol) was
2 2
added and the mixture were stirred for 3 h. After that the reaction
solution were poured into ice water (400 ml), and adjust the pH to
2
cake was washed with water for 3 times, then beaten with 100 mL
anhydrous acetone, and filtered. The filter cake was washed with
acetone for 3 times to afford hoary solid S8, 14 g, 67%. H NMR
glycine (S7-4)
The synthesis of intermediate S7-4 was similar to S5-1. Yellow
.
1
solid, 1.2 g, (yield 48%). H NMR (500 MHz, DMSO‑d
6
)
d
11.10 (s, 1H),
7
8
.59 (dd, J ¼ 15.9, 8.5 Hz, 1H), 7.07 (d, J ¼ 7.0 Hz, 1H), 6.99 (d, J ¼
.6 Hz, 1H), 6.86 (t, J ¼ 5.7 Hz, 1H), 5.06 (dt, J ¼ 15.1, 7.6 Hz, 1H), 4.08
e3 at 6 N HCl solution. The white solid was precipitated. Filter
(
(
d, J ¼ 5.7 Hz, 2H), 2.92e2.84 (m, 1H), 2.63e2.52 (m, 2H), 2.07e2.02
þ
m, 1H). MS (ESI, m/z): 322 [MþH] .
1
4.1.23. N-(2-chloro-6-methylphenyl)-2-((6-(4-((2-(2,6-
(
500 MHz, DMSO‑d
6
)
d
11.16 (s, 1H), 7.79 (d, J ¼ 7.8 Hz, 1H), 7.69 (t,
dioxopiperidin-3-yl)-1,3-dioxoisoindolin -4-yl)glycyl)piperazin-1-
yl)-2-methylpyrimidin-4-yl)amino)thiazole-5-carboxamide (13)
The derivative 13 was obtained from the condensation of S7-
J ¼ 7.6 Hz, 1H), 7.64 (d, J ¼ 7.1 Hz, 1H), 6.30 (s, 1H), 5.14 (dd, J ¼ 12.9,
5
1
.4 Hz, 1H), 2.93e2.84 (m, 1H), 2.62e2.52 (m, 2H), 2.09e2.02 (m,
þ
H). MS (ESI, m/z): 291 [MþH] .
4
and S2 according the similar process of 4. Yellow solid, 19.1 mg,
1
3
(yield 56%). H NMR (500 MHz, CD OD) d 8.11 (s, 1H), 7.51e7.46 (m,
4.1.19. tert-butyl 2-((2-(2,6-dioxopiperidin-3-yl)-1,3-
1
6
4
2
H), 7.27 (d, J ¼ 7.9 Hz,1H), 7.19e7.12 (m, 2H), 7.01 (d, J ¼ 7.0 Hz,1H),
.94 (d, J ¼ 8.5 Hz, 1H), 6.19 (s, 1H), 4.98 (dd, J ¼ 12.6, 5.3 Hz, 1H),
.19 (s, 2H), 3.83e3.63 (m, 8H), 2.82e2.73 (m, 1H), 2.69e2.61 (m,
dioxoisoindolin-4-yl)thio)acetate (S9)
The compound S8 (1.0 g, 3.5 mmol) was added to a 100 ml
bottle, followed by anhydrous DMF (10 ml) and anhydrous K
0.97 g, 7.0 mmol). Then tert-butyl bromoacetate (0.82 g, 4.2 mmol)
was added slowly and stirred at room temperature for 0.5 h. Water
50 ml) was poured into the reaction mixture which was extracted
with EA (2 ꢂ 50 ml), and washed with water (3 ꢂ 20 mL), saturated
salt solution (50 ml), then dried over Na SO , filtered, and evapo-
rated under vacuum and was subjected to column purification
2
CO
3
13
H), 2.50 (s, 3H), 2.22 (s, 3H), 2.06e1.99 (m, 1H). C NMR (126 MHz,
(
DMSO‑d
62.14, 159.90, 156.99, 145.48, 138.83, 136.21, 133.50, 132.44, 132.04,
29.05, 128.20, 127.03,125.82, 118.21, 110.88, 109.60, 82.91, 48.60,
6
) d 172.83, 170.09, 168.83, 167.36, 166.73,165.18, 162.52,
1
1
4
(
3.73, 43.19 (d, J ¼ 28.1 Hz), 41.06, 31.00, 25.53, 22.16, 18.30. MS
þ
2
4
(ESI, m/z): 757 [MþH] .
(
(
DCM/EA ¼ 20:1) to furnish the intermediate S9. Light yellow, 1.1 g,
4
(
.1.24. 2-(2,6-dioxopiperidin-3-yl)-4-hydroxyisoindoline-1,3-dione
S14)
-hydroxyisobenzofuran
.05 mmol),3-aminopiperidine-2, 6-dione (502 mg, 3.05 mmol)
1
6
yield 79%). H NMR (500 MHz, DMSO‑d ) d 11.13 (s, 1H), 7.80 (dd,
J ¼ 8.1, 7.3 Hz, 1H), 7.67 (dd, J ¼ 7.6, 4.8 Hz, 2H), 5.13 (dd, J ¼ 12.9,
4
ꢃ1,3-dione
S13
(500
mg,
5
2
.4 Hz, 1H), 4.07 (s, 2H), 2.94e2.83 (m, 1H), 2.65e2.51 (m, 2H),
3
þ
.09e2.04 (m, 1H), 1.39 (s, 9H). MS (ESI, m/z): 349 [MþH] .
and triethylamine (340 mg, 3.36 mmol) were added to a 100 ml
egg-shaped bottle, followed by anhydrous toluene (20 mL). The
reaction solution was then slowly raised to 110 C and stirred for
ꢀ
4
.1.20. 2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)
thio)acetic acid (S10)
12 h. After that, the reaction solution was reduced to room tem-
perature, and a large number of white and brown solids were
precipitated. Filtrated, the filter cake was added to the egg-shaped
bottle containing 30% water (10 mL) and methanol (2 mL), and
washed with ethyl acetate/petroleum ether for stirring 0.25 h. After
filtrated, the filter cake was washed with petroleum ether to drying
The compound S9 (1.0 g, 2.5 mmol) was added to a 25 ml bottle,
followed by 88% formic acid (10 ml) and stirred at room tempera-
ture for 12 h. After evaporated the solvent, water was added and the
residue was freeze-drying to afford the crude product (S10), pale
yellow solid, 0.69 g, (yield 80%). H NMR (500 MHz, DMSO‑d
1
6
)
1
d
13.06 (s, 1H), 11.15 (s, 1H), 7.80 (dd, J ¼ 8.1, 7.3 Hz, 1H), 7.66 (t, J ¼
compound S14, white solid, 770 mg, (yield 92%). H NMR (500 MHz,
7
1
.9 Hz, 2H), 5.13 (dd, J ¼ 12.9, 5.4 Hz,1H), 4.09 (s, 2H), 2.92e2.85 (m,
DMSO‑d
6
)
d
11.17 (s, 1H), 11.08 (s, 1H), 7.65 (dd, J ¼ 8.3, 7.3 Hz, 1H),
H), 2.66e2.51 (m, 2H), 2.08e2.03 (m, 1H). MS (ESI, m/z): 349
7.32 (d, J ¼ 7.1 Hz, 1H), 7.25 (d, J ¼ 8.3 Hz, 1H), 5.07 (dd, J ¼ 12.8,
þ
[MþH] .
5.4 Hz, 1H), 2.92e2.85 (m, 1H), 2.62e2.51 (m, 2H), 2.07e1.96 (m,
12

H. Liu, X. Ding, L. Liu et al.
European Journal of Medicinal Chemistry 223 (2021) 113645
1
H). MS (ESI, m/z): 275 [MþH]þ.
0.38 mmol), n-methyl dicyclohexylamine (250 mg, 1.28 mmol),
Pd (dba) (163 mg, 0.64 mmol) and anhydrous dioxane (6 ml) were
2 3
4
.1.25. 2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)
added to a 50 ml egg-shaped bottle and stirred at room tempera-
ture for 30 min. The compound S18 (300 mg, 0.89 mmol) and tert-
butyl acrylate (228 mg, 1.98 mmol) were then added. After that, the
reaction liquid was slowly raised to 55 C under nitrogen protection
and stirred for 12 h. Then the reaction solvent was steamed by
decompression, the silica gel sample was mixed, and the compound
oxy)acetic acid (S16)
The compound S14 (412 mg, 1.50 mmol), tert-butyl bromide
ꢀ
(
2
350 mg, 1.80 mmol), anhydrous sodium bicarbonate (190 mg,
.25 mmol), potassium iodide (25 mg, 0.15 mmol) and anhydrous
DMF (10 mL) were added to a 50 ml egg-shaped bottle and slowly
raised to 60 C and stirred for 12 h. After that, water was added to
ꢀ
S19 was purified by column chromatography (eluent: 40% EA/PE).
1
the reaction bottle. The reaction mixture was extracted with ethyl
acetate, and washed with saturated salt solution, dried with
anhydrous sodium sulfate. After evaporated the solvent, the com-
pound was purified by column chromatography (eluent: 40% EA/
PE) to get S15 with a pale yellow solid, 520 mg, (yield 89%).
The obtained intermediate S15 (500 mg, 1.29 mmol), TFA (2 mL)
and anhydrous dichloromethane (10 mL) were added to a 50 mL
egg-shaped bottle and stirred for 2 h at room temperature. After
that, the reaction solvent was removed by decompression, and the
crude product was prepared by C18 reversed phase column to
Yellow solid, 270 mg, (yield 79%). H NMR (500 MHz, CDCl
3
)
d
8.11
(s, 1H), 8.02 (s, 1H), 7.89 (d, J ¼ 7.7 Hz, 1H), 7.84 (dd, J ¼ 7.8, 1.2 Hz,
1H), 7.65 (d, J ¼ 16.0 Hz, 1H), 6.54 (d, J ¼ 16.0 Hz, 1H), 5.00 (dd, J ¼
12.5, 5.3 Hz, 1H), 2.94e2.72 (m, 3H), 2.20e2.13 (m, 1H), 1.54 (s, 9H).
þ
þ
HRMS (ESI) m/z: calcd C20
385.1389.
21
H N
2
O
6
[MþH] , 385.1394; found,
4.1.29. 3-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)
propanoic acid (S20)
The compound S19 (250 mg, 0.65 mmol) was added to a 100 ml
egg-shaped bottle, followed by dioxide-hexacyclic (20 ml) and 10%
1
afford compound (S16) with a white solid, 400 mg, (yield 92%). H
NMR (500 MHz, DMSO‑d
6
)
d
13.25 (s, 1H), 11.11 (s, 1H), 7.79 (dd, J ¼
wet Pd/C, respectively. The reaction system was replaced with H
2
8
.5, 7.3 Hz, 1H), 7.47 (d, J ¼ 7.2 Hz, 1H), 7.39 (d, J ¼ 8.6 Hz, 1H), 5.10
(25 psi) for three times, and the reaction liquid was stirred over-
night at room temperature. After that, the filtrate was extracted and
washed with the dioxy-hexane ring. The filtrate was concentrated
under decompression to produce a yellowish oily substance. The
crude product was not further purified and was directly used in the
next step of reaction. The obtained crude, TFA (1 ml) and anhydrous
dichloromethane (5 ml) were added to a 50 ml egg-shaped bottle,
and then stirred at room temperature for 2 h. After the reaction was
complete, the reaction solvent was removed by decompression,
and the crude product was prepared in a reversed phase column of
(
(
[
dd, J ¼ 12.8, 5.4 Hz, 1H), 4.98 (s, 2H), 2.93e2.85 (m, 1H), 2.63e2.51
þ
m, 2H), 2.08e2.00 (m, 1H). HRMS (ESI) m/z: calcd C15
H
13
N
2
O
7
þ
MþH] , 333.0717; found, 333.0719.
4.1.26. N-(2-chloro-6-methylphenyl)-2-((6-(4-(2-((2-(2,6-
dioxopiperidin-3-yl)-1,3-dioxoisoindolin -4-yl)oxy)acetyl)
piperazin-1-yl)-2-methylpyrimidin-4-yl)amino)thiazole-5-
carboxamide (14)
The derivative 14 was obtained from the condensation of S16
and S2 according the similar process of 4. Yellow solid, 18.5 mg,
C18 to afford compound S20 after freeze-drying. White solid,
1
1
(
8
(
2
yield 52%). H NMR (500 MHz, CD
3
OD)
d
8.21 (s, 1H), 7.77 (dd, J ¼
180 mg, (two-step, yield 84%). H NMR (500 MHz, DMSO‑d
6
)
d
12.22
.5, 7.3 Hz, 1H), 7.51 (d, J ¼ 7.1 Hz, 1H), 7.42 (d, J ¼ 8.5 Hz, 1H), 7.36
dd, J ¼ 7.2, 1.9 Hz, 1H), 7.28e7.22 (m, 2H), 6.30 (s, 1H), 5.17 (d, J ¼
.3 Hz, 2H), 5.14e5.08 (m, 1H), 3.80 (d, J ¼ 29.3 Hz, 8H), 2.90e2.80
m, 1H), 2.78e2.68 (m, 2H), 2.61 (s, 3H), 2.31 (s, 3H), 2.16e2.10 (m,
(s, 1H), 11.12 (s, 1H), 7.86e7.79 (m, 2H), 7.77e7.70 (m, 1H), 5.13 (dd,
J ¼ 12.8, 5.4 Hz, 1H), 3.01 (t, J ¼ 7.4 Hz, 2H), 2.93e2.85 (m, 1H), 2.64
(t, J ¼ 7.4 Hz, 2H), 2.62e2.50 (m, 2H), 2.08e2.02 (m,1H). HRMS (ESI)
þ
þ
(
m/z: calcd C16
H
15
N
2
O
6
[MþH] , 331.0925; found, 331.0919.
13
1
6
H). C NMR (126 MHz,DMSO‑d ) d 172.80, 169.95, 166.81, 165.37,
1
1
65.28, 160.73, 159.61, 156.67, 155.59, 138.79, 136.57, 133.41, 133.10,
32.42, 129.05, 128.24, 127.03, 120.26, 116.16, 115.51, 109.53, 83.40,
4.1.30. N-(2-chloro-6-methylphenyl)-2-((6-(4-(3-(2-(2,6-
dioxopiperidin-3-yl)-1,3-dioxoisoindolin -4-yl)propanoyl)piperazin-
1-yl)-2-methylpyrimidin-4-yl)amino)thiazole-5-carboxamide (15)
The derivative 15 was obtained from the condensation
76.61, 66.10, 48.77, 43.94, 43.75, 43.18, 40.71, 30.96, 22.00, 18.32.
þ
þ
HRMS (ESI) m/z: calcd C35
H33ClN
9
O
7
S
[MþH] , 758.1907; found,
7
58.1913.
of S20 and S2 according the similar process of 4. White solid,
1
2
4.2 mg, (yield 47%). H NMR (500 MHz, DMSO‑d
6
)
d
11.12 (s, 1H),
4
.1.27. 4-bromo-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione
9.94 (s, 1H), 8.25 (s, 1H), 7.88e7.82 (m, 2H), 7.78 (dd, J ¼ 7.8, 1.1 Hz,
1H), 7.40 (d, J ¼ 7.7 Hz, 1H), 7.31e7.23 (m, 2H), 6.12 (s, 1H), 5.14 (dd,
J ¼ 12.8, 5.4 Hz, 1H), 3.59 (s, 6H), 3.49 (dd, J ¼ 7.8, 3.0 Hz, 1H), 3.41
(dd, J ¼ 7.8, 3.0 Hz, 1H), 3.04 (t, J ¼ 7.5 Hz, 2H), 2.93e2.85 (m, 1H),
2.80 (t, J ¼ 7.5 Hz, 2H), 2.64e2.52 (m, 2H), 2.45 (s, 3H), 2.24 (s, 3H),
(
S18)
-bromoisobenzo -furan-1, 3-dione S17 (500 mg, 2.20 mmol), 3-
4
aminoperidol 2, 6-dione (400 mg, 2.42 mmol) and anhydrous so-
dium acetate (220 mg, 2.64 mmol) were added to a 100 ml egg-like
bottle, followed by glacial acetic acid (10 ml). The reaction solution
13
6
2.06e2.02 (m, 1H). C NMR (126 MHz, DMSO‑d ) d 172.79, 172.69,
ꢀ
was then slowly raised to 140 C and stirred for 12 h. After the
169.88 (d, J ¼ 8.7 Hz), 167.33, 167.14, 159.61, 156.58, 149.78, 149.11,
138.80, 135.04, 133.41, 132.43, 131.57 (d, J ¼ 7.2 Hz), 129.05, 128.25,
127.04, 123.61, 123.36, 83.36, 72.28, 48.97, 44.02 (d, J ¼ 39.0 Hz),
40.45, 34.62, 33.37, 30.96, 30.64, 22.04,18.32. HRMS (ESI) m/z: calcd
reaction was finished, the reaction liquid was reduced to room
temperature and a large amount of white solid was precipitated.
After filtrated the solvent, the solid was washed with the mixture of
water (10 mL) and methanol (2 mL) fully stirred for 0.5 h. then
filtration, the filter cake was washed with water to afford drying
þ
þ
C
36
H35ClN
9
O
6
S
[MþH] , 756.2114; found, 756.2114.
1
compound S18, white solid, 700 mg, (yield 94%). H NMR (500 MHz,
DMSO‑d
4.1.31. tert-butyl 2-((2-(2,6-dioxopiperidin-3-yl)-1,3-
dioxoisoindolin-4-yl)sulfonyl)acetate (S11)
6
)
d
11.14 (s, 1H), 8.14 (d, J ¼ 1.4 Hz, 1H), 8.09 (dd, J ¼ 7.9,
1
2
.7 Hz, 1H), 7.86 (d, J ¼ 7.9 Hz, 1H), 5.16 (dd, J ¼ 12.9, 5.4 Hz, 1H),
The compound S9 (0.45 g, 1.1 mmol) was added to a 100 ml egg-
shaped bottle, followed by an anhydrous DCM (20 ml), and then
.93e2.85 (m, 1H), 2.65e2.50 (m, 2H), 2.08e2.04 (m, 1H). HRMS
þ
þ
ꢀ
(
ESI) m/z: calcd C13
H10BrN
2
O
4
[MþH] , 336.9818; found, 336.9810.
slowly added to m-CPBA (0.76 g, 4.4 mmol) after stirring at 40 C for
4
h. After the reaction was completed, the reaction mixture cooled
4.1.28. tert-butyl (E)-3-(2-(2,6-dioxopiperidin-3-yl)-1,3-
to room temperature, with 10% NaHCO solution to adjust the
3
dioxoisoindolin-4-yl)acrylate (S19)
pH ¼ 8e9. After that, the mixture was extracted with DCM
(2 ꢂ 30 ml), organic phase was washed with water (2 ꢂ 20 ml) and
Tritert-butylphosphine tetrafluoroboric acid (110 mg,
13

H. Liu, X. Ding, L. Liu et al.
European Journal of Medicinal Chemistry 223 (2021) 113645
saturated salt water (50 ml), then dried with anhydrous Na
2
SO
4
.
4.1.36. N-(2-chloro-6-methylphenyl)-2-((6-(4-(2-((2-(2,6-
After removed the solvent, crude products was purified by column
dioxopiperidin-3-yl)-1,3-dioxoisoindolin -4-yl)thio)ethyl)piperazin-
1-yl)-2-methylpyrimidin-4-yl)amino)thiazole-5-carboxamide (17)
The compounds S23 (20 mg, 0.04 mmol), S8 (13 mg,
chromatography (eluent (v/v): PE/EA ¼ 1:1) to afford compound
1
S11, light yellow solid, 0.3 g, (yield 59%). H NMR (500 MHz, CDCl
3
)
d
8.42 (dd, J ¼ 7.9, 0.9 Hz, 1H), 8.19 (dd, J ¼ 7.5, 0.9 Hz, 1H), 8.12 (s,
2 3
0.044 mmol), anhydrous K CO (27.6 mg, 0.2 mmol) and NaI
1
H), 8.00 (t, J ¼ 7.7 Hz, 1H), 5.03 (dd, J ¼ 12.6, 5.4 Hz, 1H), 4.72e4.64
(30 mg, 0.2 mmol) were added to a 10 ml egg-shaped bottle, fol-
ꢀ
(
m, 2H), 2.96e2.72 (m, 3H), 2.26e2.17 (m, 1H), 1.30 (s, 9H). MS (ESI,
lowed by adding anhydrous DMF (3 mL), slowly rising to 60 C and
þ
m/z): 437 [MþH] .
stirring for 8 h. After the reaction was completed, the reaction
mixture was filtered, and the filtrate was prepared and separated by
HPLC to obtain the target compound 17. White solid, 20.2 mg, (yield
4
.1.32. 2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)
1
6
8%). H NMR (500 MHz, DMSO‑d
6
)
d
11.50 (s, 1H), 11.13 (s, 1H), 9.93
sulfonyl)acetic acid (S12)
(
s, 1H), 8.26 (s, 1H), 7.97 (d, J ¼ 8.3 Hz, 1H), 7.84 (t, J ¼ 7.7 Hz, 1H),
The synthesis of intermediate S12 was similar to S10. Yellow
1
7.71 (d, J ¼ 7.3 Hz, 1H), 7.40 (d, J ¼ 7.6 Hz, 1H), 7.32e7.23 (m, 2H),
solid, 0.25 g, (yield 96%). H NMR (500 MHz, DMSO‑d
6
)
d
11.20 (s,
6
3
.18 (s, 1H), 5.13 (dd, J ¼ 12.9, 5.4 Hz, 1H), 4.39e4.37 (m, 2H),
.73e3.66 (m, 4H), 3.44e3.35 (m, 4H), 3.18e3.07 (m, 2H),
1
5
2
H), 8.35e8.25 (m, 2H), 8.14 (t, J ¼ 7.7 Hz, 1H), 5.23 (dd, J ¼ 12.9,
.4 Hz, 1H), 4.96e4.73 (m, 2H), 2.93e2.86 (m, 1H), 2.66e2.51 (m,
þ
2.93e2.85 (m, 1H), 2.64e2.52 (m, 2H), 2.46 (s, 3H), 2.24 (s, 3H),
H), 2.13e2.08 (m, 1H). MS (ESI, m/z): 381 [MþH] .
1
3
2
1
1
1
1
C
.08e2.01 (m, 1H). C NMR (126 MHz, DMSO‑d
67.17, 165.68, 163.05, 162.77, 160.42, 157.42, 141.32, 139.41, 139.30,
35.43,134.00,132.94,130.89,129.50,128.64,127.48,126.19,125.90,
19.34, 83.14, 56.42, 52.45, 49.38, 43.99, 31.44, 27.74, 26.06, 22.45,
6
) d 173.27, 170.38,
4.1.33. N-(2-chloro-6-methylphenyl)-2-((6-(4-(2-((2-(2,6-
dioxopiperidin-3-yl)-1,3-dioxoisoindolin -4-yl)sulfonyl)acetyl)
piperazin-1-yl)-2-methylpyrimidin-4-yl)amino)thiazole-5-
carboxamide (16)
þ
8.79.MS (ESI, m/z): 760 [MþH] . HRMS (ESI) m/z: calcd
þ
þ
35
H35ClN
9
O
5
S
2
[MþH] , 760.1886; found, 760.1881.
The derivative 16 was obtained from the condensation
of S12 and S2 according the similar process of 12. Yellow solid,
4
.1.37. 3-(4-(benzylthio)-1-oxoisoindolin-2-yl)piperidine-2,6-dione
1
2
0
5.2 mg, (yield 69%). H NMR (500 MHz, CD
.9 Hz, 1H), 8.25 (dd, J ¼ 7.5, 0.9 Hz, 1H), 8.20 (s, 1H), 8.09 (t, J ¼
3
OD)
d
8.38 (dd, J ¼ 7.9,
(S26)
.
Na
2
S
5H
2
O
3
5H
O (77.4 mg, 0.31 mmol) and 2,2' -bipyridine (0.72 g,
.6 mmol) were added to a 500 ml egg-shaped bottle containing
2
O (53.7 g, 216.3 mmol), BnCl (27.4 g, 216.3 mmol),
7
.7 Hz, 1H), 7.37 (dd, J ¼ 7.4, 1.8 Hz, 1H), 7.31e7.19 (m, 2H), 6.24 (s,
CuSO.
4
4 2
1
3
2
H), 5.25 (dd, J ¼ 12.6, 5.5 Hz, 1H), 5.06 (dd, J ¼ 22.0, 14.0 Hz, 2H),
.95e3.86 (m, 4H), 3.80e3.66 (m, 4H), 2.94e2.85 (m, 1H),
methanol (120 ml) and water (120 ml), and then slowly heated to
8
room temperature, lenalidomide S25 (8.0 g, 30.9 mmol) was added,
and tert-butynitrite (4.78 g, 46.4 mmol) was slowly added. The
solution was then raised again to 80 C and stirred for 8 h. After the
1
3
.82e2.70 (m, 2H), 2.58 (s, 3H), 2.32 (s, 3H), 2.25e2.16 (m, 1H).
172.73, 169.50, 165.71, 164.61, 162.04,
60.07, 159.88, 158.20 (d, J ¼ 36.5 Hz), 156.97, 140.76, 138.82, 137.03,
35.73, 134.26, 133.50, 133.15, 132.43, 129.05, 128.25, 128.10, 127.02,
25.90, 82.95, 57.36, 49.39, 45.22, 43.59, 42.96, 41.20, 30.89, 25.48,
C
ꢀ
0 C and stirred for 2 h. The reaction solution was then brought to
6
NMR (126 MHz, DMSO‑d ) d
1
1
1
ꢀ
reaction was completed, the reaction liquid was reduced to room
temperature, water (200 ml) was added, extracted with
EA(2 ꢂ 200 mL), washed (2 ꢂ 50 mL) with saturated salt (50 ml),
Then the solvent was evaporated, and the crude was purified by
column chromatography (eluent (v/v): PE/EA ¼ 1:2) to obtain the
þ
21.84, 18.30. MS (ESI, m/z): 806 [MþH] .
4.1.34. 1-(2-chloroethyl)piperazine (S22)
2
SOCl (10 mL) was added to a 100 ml single-mouth bottle, fol-
lowed by a slow addition of the compound 1-(2-hydroxyethyl)
piperazine S21 (800 mg, 6.14 mmol), followed by reflux agitation
for 12 h. The reaction liquid was cooled to room temperature. After
removed the solvent, a large number of grey-yellow solids were
precipitated, then water was added, and the crude product was
freeze-dried. The crude product S22 was not purified and was
1
target compound S26. White solid, 6.8 g, (yield 60%). H NMR
(500 MHz, CDCl
3
)
d
8.07 (s, 1H), 7.75 (t, J ¼ 7.3 Hz, 1H), 7.55 (dd, J ¼
7
1
1
.4, 6.8 Hz, 1H), 7.49e7.41 (m, 1H), 7.27e7.17 (m, 5H), 5.20e5.17 (m,
H), 4.22 (d, J ¼ 16.5 Hz, 1H), 4.15e4.04 (m, 2H), 3.92 (d, J ¼ 16.5 Hz,
H), 2.95e2.74 (m, 2H), 2.32e2.22 (m, 1H), 2.17e2.11 (m, 1H). MS
þ
(ESI, m/z): 367 [MþH] .
1
directly used in the next step of the reaction, 810 mg, (yield 89%). H
NMR (500 MHz, DMSO‑d
6
) d 9.65 (s, 1H), 4.39e3.14 (m, 12H).
4
(
.1.38. 3-(4-mercapto-1-oxoisoindolin-2-yl)piperidine-2,6-dione
S24)
3
Anhydrous AlCl (2.61 g, 19.6 mmol) and anhydrous toluene
4.1.35. N-(2-chloro-6-methylphenyl)-2-((6-(4-(2-chloroethyl)
piperazin-1-yl)-2-methylpyrimidin-4 -yl)amino)thiazole-5-
carboxamide (S23)
(70 ml) were added to a 250 ml egg-shaped bottle, and slowly
added S26 (1.8 g, 4.9 mmol) after agitation. After the reaction was
completed, 20% citric acid solution was added slowly under agita-
tion, and a large amount of solid was precipitated. After filtration,
the precipitate was washed with water and EA respectively, and
The compound S2 (100 mg, 0.25 mmol), S22 (145 mg,
1.00 mmol) and N,N-diisopropyl ethylamine (130 mg, 1.00 mmol)
were added to a 50 ml egg-shaped bottle, followed by an anhydrant
ꢀ
DMF (3 ml), which was then slowly heated to 110 C under nitrogen
was dried to obtain the target compound S24, white solid, 1.15 g,
1
protection and stirred overnight. Then reaction liquid was cooled to
room temperature, then pour into 50 ml of water, and extracted
with EA (2 ꢂ 50 ml), organic phase was washed with water
(yield 85%). H NMR (500 MHz, DMSO‑d
6
)
d
11.01 (s, 1H), 7.82e7.39
(m, 3H), 5.73 (s, 1H), 5.21e5.04 (m, 1H), 4.40e4.20 (m, 2H),
2.99e2.85 (m, 1H), 2.67e2.56 (m, 1H), 2.47e2.30 (m, 1H), 2.05e1.95
þ
(
3 ꢂ 20 ml) and saturated salt water (20 ml), dried with anhydrous
(m, 1H). MS (ESI, m/z): 276 [MþH] .
2 4
Na SO then removed solvent, the crude products was purified by
column chromatography (eluent (v/v), DCM/MeOH ¼ 20:1) to
4.1.39. 2-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)thio)
acetic acid (S27)
afford product S23. White solid, 50 mg, (yield 39%). 1H NMR
(
500 MHz, DMSO‑d
6
)
d
11.47 (s, 1H), 9.87 (s, 1H), 8.22 (s, 1H), 7.40 (d,
The synthesis of intermediate S27 was similar to S10. white
1
J ¼ 6.6 Hz, 1H), 7.30e7.23 (m, 2H), 6.06 (s, 1H), 3.72 (t, J ¼ 6.4 Hz,
solid, 1.2 g, (65% yield over two steps) H NMR (500 MHz, DMSO‑d
6
)
2
2
H), 3.57e3.47 (m, 4H), 2.69 (t, J ¼ 6.3 Hz, 2H), 2.51e2.55 (m, 4H),
d
12.88 (s, 1H), 11.00 (s, 1H), 7.68e7.45 (m, 3H), 5.15e5.13 (m, 1H),
4.32 (dd, J ¼ 56.2, 17.3 Hz, 2H), 3.94 (s, 2H), 2.95e2.91 (m, 1H),
þ
.41 (s, 3H), 2.24 (s, 3H). MS (ESI, m/z): 505 [MþH] .
14

H. Liu, X. Ding, L. Liu et al.
European Journal of Medicinal Chemistry 223 (2021) 113645
2
(
1
.63e2.59 (m, 1H), 2.49e2.39 (m, 1H), 2.08e1.92 (m, 1H). 13C NMR
126 MHz, DMSO‑d 172.89, 170.95, 170.29, 167.70, 141.11, 132.06,
31.15, 130.92, 129.20, 120.81, 51.66, 46.66, 34.45, 31.22, 22.36. MS
egg-shaped bottle, followed by anhydrite DCM (1 ml) and TFA
(3 ml), and stirred at room temperature for 2 h. Then evaporated
the solvent and added the mixture of DCM/MeOH (10:1, ml), and
6
) d
þ
(
ESI, m/z): 334 [MþH] .
3
adjusted the pH to 8e9 with saturated NaHCO solution and
extracted with 10% of the DCM/methanol solution (2 ꢂ 11 ml).
4.1.40. N-(2-chloro-6-methylphenyl)-2-((6-(4-(2-((2-(2,6-
Organic phase was washed with saturated salt water (10 ml), dried
dioxopiperidin-3-yl)-1-oxoisoindolin-4 -yl)thio)acetyl)piperazin-1-
yl)-2-methylpyrimidin-4-yl)amino)thiazole-5-carboxamide (18)
The derivative 18 was obtained from the condensation of S27
2 4
with anhydrous Na SO . After removed the solvent, the obtained
white solid (90 mg) was used for the next step without further
purification.
and S2 according the similar process of 12. White solid, 10 mg,
The obtained crude compound (15 mg, 0.03 mmol), S3 (8 mg,
0.03 mmol) and DIPEA (37 mg, 0.3 mmol) were added to a 25 ml
egg-like bottle, followed by NMP (5 ml), which was slowly raised to
110 C and stirred for 12 h. After that, the reaction solution was
reduced to room temperature, poured into 50% salt water (30 ml),
1
(
yield 39%). H NMR (500 MHz, DMSO‑d
6
)
d
11.55 (s, 1H), 10.99 (s,
1
1
6
4
H), 9.90 (s, 1H), 8.23 (s, 1H), 7.76e7.71 (m, 1H), 7.60 (d, J ¼ 6.8 Hz,
ꢀ
H), 7.53 (t, J ¼ 7.6 Hz,1H), 7.40 (d, J ¼ 6.4 Hz,1H), 7.32e7.18 (m, 2H),
.09 (s, 1H), 5.13 (dd, J ¼ 13.3, 5.1 Hz, 1H), 4.42 (d, J ¼ 17.4 Hz, 1H),
.28 (d, J ¼ 17.4 Hz, 1H), 4.20 (s, 2H), 3.66e3.52 (m, 10H), 2.95e2.85
extracted with EA (3 ꢂ 30 ml), and washed with water (2 ꢂ 20 ml),
(
2
1
1
m, 1H), 2.64e2.57 (m, 1H), 2.45 (d, J ¼ 13.0 Hz, 5H), 2.24 (s, 3H),
2 4
saturated salt (20 ml), dried with anhydrous Na SO . After removed
.04e1.94 (m, 1H). 13C NMR (126 MHz, DMSO‑d
67.77, 166.23, 164.28, 162.54, 161.59, 159.83, 156.88, 141.49,140.88,
38.82, 133.48, 132.43, 131.95 (d, J ¼ 8.1 Hz), 131.22, 129.10 (d,
)
d
172.89, 170.98,
the solvent, the crude product was prepared and separated by HPLC
6
to obtained the final compound 20, yellow solid, 6.3 mg, (yield
1
28%). H NMR (500 MHz, CD
3
OD)
d
8.26 (s, 1H), 8.01 (s, 3H), 7.58
J ¼ 10.8 Hz), 128.23, 127.03,125.98, 120.86, 83.05, 51.67, 46.75, 44.67,
(dd, J ¼ 8.5, 7.1 Hz, 1H), 7.37 (dd, J ¼ 7.1, 2.2 Hz, 1H), 7.28e7.24 (m,
2H), 7.12 (d, J ¼ 8.6 Hz, 1H), 7.07 (d, J ¼ 7.1 Hz, 1H), 6.53 (s, 1H), 5.06
(dd, J ¼ 12.4, 5.5 Hz,1H), 4.82e4.58 (m, 2H), 3.75 (d, J ¼ 12.1 Hz, 2H),
4
3.72, 43.30, 41.03, 35.25, 31.23, 25.19,22.38, 18.31. MS (ESI, m/z):
þ
760 [MþH] .
3
.67e3.60 (m, 2H), 3.46 (t, J ¼ 6.6 Hz, 2H), 3.30e3.19 (m, 4H),
4.1.41. N-(2-chloro-6-methylphenyl)-2-((6-(4-(2-((2-(2,6-
2.86e2.80 (m, 1H), 2.76e2.69 (m, 2H), 2.66 (s, 3H), 2.32 (s, 3H),
2.14e2.08 (m,1H),1.98e2.00 (m, 2H),1.81e1.74 (m, 2H). MS (ESI, m/
z): 771 [MþH] .
dioxopiperidin-3-yl)-1-oxoisoindolin-4 -yl)thio)ethyl)piperazin-1-
yl)-2-methylpyrimidin-4-yl)amino)thiazole-5-carboxamide (19)
The derivative 19 was obtained from the condensation of S23
þ
and S24 according the similar process of 17. White solid, 6.0 mg,
4.1.44. 5-bromo-N-(2-(2,6-dioxopiperidin-3-yl)-1,3-
dioxoisoindolin-4-yl)pentanamide (S30)
1
(
2
2
1
yield 27%). H NMR (500 MHz, CD
3
OD)
d
8.22 (s, 1H), 7.79 (ddd, J ¼
4.9,11.2, 3.9 Hz, 2H), 7.60 (dd, J ¼ 17.9,10.2 Hz,1H), 7.36 (dd, J ¼ 7.2,
.0 Hz, 1H), 7.32e7.15 (m, 2H), 6.42 (d, J ¼ 29.7 Hz, 1H), 5.18 (dd, J ¼
The compound pomalidomide S29 (500 mg, 1.83 mmol) and
anhydrous THF (15 ml) were added to a 100 ml egg-shaped bottle,
stirred at room temperature and slowly added with 5-
bromopentanoyl chloride (1.83 g, 9.15 mmol), then slowly
warmed to reflux and stir for 3 h. After the reaction was completed,
the reaction liquid was cooled to room temperature, and 3 ml of
methanol was added to quench and stirred for 30min. Then the
reaction solvent was removed to obtain the solid crude product.
The solid was then beaten with 10 mL (EA/PE ¼ 10:1), filtered and
3.3, 5.2 Hz, 1H), 4.58e4.46 (m, 2H), 4.18e3.31 (m, 12H), 2.95e2.88
(
(
m, 1H), 2.79 (ddd, J ¼ 17.5, 4.5, 2.3 Hz, 1H), 2.64e2.46 (m, 4H), 2.31
13
s, 3H), 2.19 (ddd, J ¼ 10.4, 5.2, 2.6 Hz, 1H). C NMR (126 MHz,
172.90, 170.97, 167.65, 165.12, 161.53, 159.78, 157.18,
41.55, 138.82, 133.48, 132.42 (d, J ¼ 6.1 Hz), 130.88, 129.65, 129.49,
29.06, 128.24, 127.04,126.07, 121.16, 83.70, 66.46, 61.23, 54.12,
1.70, 50.00, 46.75, 40.94, 31.22, 24.89, 22.40, 18.33. MS (ESI, m/z):
6
DMSO‑d ) d
1
1
5
þ
7
46 [MþH] .
washed to produce a pale yellow solid S30. Pale yellow solid,
1
7
50 mg, (yield 94%). H NMR (500 MHz, DMSO‑d
6
)
d
11.14 (s, 1H),
4
.1.42. tert-butyl (4-(4-(6-((5-((2-chloro-6-methylphenyl)
9.73 (s,1H), 8.45 (d, J ¼ 8.3 Hz,1H), 7.88e7.76 (m, 1H), 7.68e7.57 (m,
1H), 5.14 (dd, J ¼ 12.9, 5.4 Hz, 1H), 3.58 (t, J ¼ 6.6 Hz, 2H), 2.93e2.86
(m, 1H), 2.65e2.57 (m, 1H), 2.56e2.51 (m, 2H), 2.08e2.04 (m, 1H),
1.91e1.85 (m, 2H), 1.80e1.69 (m, 2H). MS (ESI, m/z): 436 [MþH] .
carbamoyl)thiazol-2-yl)amino)-2-methyl pyrimidin-4-yl)piperazin-
1-yl)butyl)carbamate (S28)
þ
The compound S2 (200 mg, 0.45 mmol) was added to a 25 ml
egg-shaped bottle, followed by anhydrous DMF (5 ml), anhydrous
CO (125 mg, 0.90 mmol) and NaI (135 mg, 0.90 mmol), stirred at
K
2
3
4.1.45. N-(2-chloro-6-methylphenyl)-2-((6-(4-(5-((2-(2,6-
dioxopiperidin-3-yl)-1,3-dioxoisoindolin -4-yl)amino)-5-oxopentyl)
piperazin-1-yl)-2-methylpyrimidin-4-yl)amino)thiazole-5-
carboxamide (21)
The compound S2 (20 mg, 0.045 mmol) was added to a 25 ml
egg-shaped bottle, followed by anhydric DMF (2 ml), DIPEA (30 mg,
0.225 mmol) and NaI (13.5 mg, 0.09 mmol). Then S30 (39 mg,
0.09 mmol) was added and stirred at room temperature. After the
reaction was completed by LC-MS detection, the reaction liquid was
filtered, and the filtrate was prepared and separated by HPLC to
room temperature and then added to tert-butyl (4-bromobutyl)
carbamate (227 mg, 0.90 mmol), slowly heated to 110 C and stirred
ꢀ
overnight. After the reaction was completed, 30 ml of water was
poured into the reaction mixture, extracted with EA (2 ꢂ 30 ml),
organic phase was washed with water (3 ꢂ 10 ml) and saturated
2 4
salt (10 ml), dried with anhydrous Na SO , then the solvent was
evaporated, the crude was purified by column chromatography
eluent (v/v): DCM/MeOH ¼ 10:1) to afford compound S28, white
(
1
solid, 110 mg, (yield 52%). H NMR (500 MHz, DMSO‑d
6
)
d
11.46 (s,
H), 9.87 (s, 1H), 8.22 (s, 1H), 7.40 (d, J ¼ 6.5 Hz, 1H), 7.31e7.23 (m,
H), 6.85e6.80 (m, 1H), 6.05 (s, 1H), 3.54e3.47 (m, 4H), 2.98e2.84
1
2
obtained 21 after freeze-drying. White solid, 10.8 mg, (yield 29%).
1
H NMR (500 MHz, CD
3
OD)
d
8.59 (d, J ¼ 8.4 Hz, 1H), 8.21 (s, 1H),
(
m, 4H), 2.40 (s, 3H), 2.41e2.38 (m, 2H), 2.30e2.26 (m, 2H), 2.24 (s,
7.83e7.78 (m, 1H), 7.62 (d, J ¼ 7.1 Hz, 1H), 7.37 (d, J ¼ 5.6 Hz, 1H),
þ
3
H), 1.44e1.38 (m, 4H), 1.37 (s, 9H). MS (ESI, m/z): 615 [MþH] .
7.28e7.23 (m, 2H), 6.33 (s, 1H), 5.15 (dd, J ¼ 12.7, 5.5 Hz, 1H),
3
.75e3.72 (m, 2H), 3.50e3.40 (m, 2H), 3.35e3.27 (m, 4H),
4
.1.43. N-(2-chloro-6-methylphenyl)-2-((6-(4-(4-((2-(2,6-
3.23e3.17 (m, 2H), 2.89e2.84 (m,1H), 2.79e2.69 (m, 2H), 2.64 (t, J ¼
dioxopiperidin-3-yl)-1,3-dioxoisoindolin -4-yl)amino)butyl)
piperazin-1-yl)-2-methylpyrimidin-4-yl)amino)thiazole-5-
carboxamide (20)
6.9 Hz, 2H), 2.58 (s, 3H), 2.32 (s, 3H), 2.19e2.15 (m, 1H), 1.97e1.90
13
6
(m, 2H), 1.88e1.82 (m, 2H). C NMR (126 MHz, DMSO‑d ) d 172.79,
171.53, 169.81, 167.60, 166.67,165.13,162.36, 161.65, 159.81, 157.17,
138.81, 136.39, 136.11, 133.49, 132.44, 131.52, 129.05, 128.21, 127.03,
The compound S28 (110 mg, 0.18 mmol) was added to a 25 ml
15

H. Liu, X. Ding, L. Liu et al.
European Journal of Medicinal Chemistry 223 (2021) 113645
1
4
7
26.64, 126.07,123.75, 118.51, 117.35, 83.58, 55.12, 50.13, 48.93,
1.6 Hz, 1H), 7.54e7.46 (m, 2H), 7.40 (d, J ¼ 6.4 Hz, 1H), 7.34e7.21 (m,
2H), 6.13 (s, 1H), 5.14 (dd, J ¼ 13.3, 5.1 Hz, 1H), 4.38 (dd, J ¼ 22.0,
17.5 Hz, 2H), 3.62e3.51 (m, 8H), 2.97e2.86 (m, 1H), 2.63e2.58 (m,
0.79, 35.63, 30.95, 25.33, 22.54, 22.01, 21.83, 18.32. MS (ESI, m/z):
þ
99 [MþH] .
1
H), 2.45 (s, 3H), 2.47e2.40 (m, 4H), 2.39e2.30 (m, 1H), 2.24 (s, 3H),
13
4.1.46. 5-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)
6
2.06e1.99 (m, 1H), 1.91e1.82 (m, 2H). C NMR (126 MHz, DMSO‑d )
amino)pentanoic acid (S33)
d
172.90, 171.13 (d, J ¼ 9.2 Hz), 170.66, 167.88,163.68, 162.43, 160.28,
The compound lenalidomide S31 (259.3 mg, 1 mmol) was added
to a 25 ml egg-like bottle, followed by anhydrous NMP (5 ml), N,N-
diisopropyl ethylamine (387.7 mg, 3 mmol) and tert-butyl bromo-
pentate (284.6 mg, 1.2 mmol). The solution was then added, slowly
159.57, 156.55, 138.80, 133.79 (d, J ¼ 9.1 Hz), 133.39, 132.68, 132.43,
129.08, 128.62, 128.29, 127.06, 125.33, 119.00, 83.46, 51.57, 46.61,
44.15 (d, J ¼ 44.9 Hz), 40.36, 35.05, 31.68, 31.23, 24.29, 22.67, 20.63,
þ
18.33. MS (ESI, m/z): 799 [MþH] .
ꢀ
raised to 100 C and stirred overnight. After that, the reaction was
purified by C18 reverse phase column to afford target compound
S32 with a pale yellow solid, 260 mg, (yield 63%).
4.1.50. N-(2-chloro-6-methylphenyl)-2-((2-methyl-6-(4-(2-((2-(1-
methyl-2,6-dioxopiperidin-3-yl) ꢃ1,3-dioxoisoindolin-4-yl)thio)
ethyl)piperazin-1-yl)pyrimidin-4-yl)amino)thiazole-5-carboxamide
(24)
The obtained intermediate S32 (260 mg, 0.62 mmol), TFA (2 mL)
and anhydrous dichloromethane (10 mL) were added to a 50 mL
egg-shaped bottle and stirred for 2 h at room temperature. After
that, the reaction solvent was removed by decompression, and the
crude product was prepared by C18 reversed phase column to
To
a
solution of 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoi
CO
ndoline-1,3-dione (1 g, 3.6 mmol) S3 in DMF were added K
2
3
(1 g, 7.2 mmol) and MeI (760 mg, 5.4 mmol). After stirring at room
temperature for 30 min, the reaction mixture was poured into
water and extracted with EtOAc. The combined organic layers were
1
afford compound (S33) with a white solid, 210 mg, (yield 93%). H
NMR (500 MHz, DMSO‑d
6
)
d
11.00 (s, 1H), 7.28 (t, J ¼ 7.7 Hz, 1H),
6
5
.92 (t, J ¼ 10.9 Hz, 1H), 6.76 (d, J ¼ 8.0 Hz, 1H), 5.11 (dd, J ¼ 13.3,
washed with brine, dried over Na
resulting residue was purified by preparative HPLC (10e90%
acetonitrile/0.05% HCl in H O) to obtain intermediate (490 mg.47%)
2 4
SO , and concentrated. The
.1 Hz, 1H), 5.07e4.83 (m, 3H), 4.23 (d, J ¼ 17.2 Hz, 1H), 4.13 (d,
J ¼ 17.1 Hz, 1H), 3.13 (d, J ¼ 6.4 Hz, 2H), 2.97e2.87 (m, 1H), 2.61 (d,
2
J ¼ 16.7 Hz, 1H), 2.38e2.21 (m, 3H), 2.06e1.98 (m, 1H), 1.67e1.55
as a white solid. MS (ESI) m/z: 291. Then the intermediate (100 mg,
þ
(
m, 4H). MS (ESI, m/z): 360 [MþH] .
0.34 mmol) was dissolved in DMF (150 ml) at room temperature,
.
the Na
2
S 9H
2
O (125 mg, 0.52 mmol) was added and the mixture
atmosphere, The reaction was purified
by C18 column chromatography (10e90% acetonitrile/0.05% HCl in
O) to obtain S8-1 faint yellow, 70 mg (yield 68%). MS (ESI) m/z:
305.
The derivative 24 was obtained from the condensation of S23
and S8-1 according the similar process of 17. White solid, 6.2 mg,
4.1.47. N-(2-chloro-6-methylphenyl)-2-((6-(4-(5-((2-(2,6-
were stirred for 3 h under N
2
dioxopiperidin-3-yl)-1-oxoisoindolin-4 -yl)amino)pentanoyl)
piperazin-1-yl)-2-methylpyrimidin-4-yl)amino)thiazole-5-
carboxamide (22)
H
2
The derivative 22 was obtained from the condensation of S33
and S2 according the similar process of 4. White solid, 10.9 mg,
1
1
(
yield 41%). H NMR (500 MHz, DMSO‑d
6
)
d
11.63 (s, 1H), 11.00 (s,
6
(yield 40%). H NMR (500 MHz, DMSO‑d ) d 11.50 (s, 1H), 9.93 (s,
1
1
(
H), 9.93 (s, 1H), 8.25 (s, 1H), 7.40 (d, J ¼ 7.9 Hz, 1H), 7.28 (td, J ¼
5.2, 7.4 Hz, 3H), 6.96 (d, J ¼ 7.4 Hz, 1H), 6.80 (d, J ¼ 8.1 Hz, 1H), 6.12
s,1H), 5.11 (dd, J ¼ 13.2, 5.3 Hz,1H), 4.25 (d, J ¼ 16.7 Hz,1H), 4.15 (d,
1H), 8.26 (s, 1H), 7.97 (d, J ¼ 8.2 Hz, 1H), 7.84 (t, J ¼ 7.7 Hz, 1H), 7.71
(d, J ¼ 7.2 Hz, 1H), 7.40 (d, J ¼ 7.1 Hz, 1H), 7.33e7.23 (m, 2H), 6.18 (s,
1H), 5.20 (dd, J ¼ 13.1, 5.3 Hz, 1H), 4.38 (s, 2H), 3.71e3.63 (m, 4H),
3.42e3.39 (m, 4H), 3.18e3.08 (m, 2H), 3.02 (s, 3H), 2.99e2.90 (m,
1H), 2.80e2.74 (m, 1H), 2.59e2.53 (m, 1H), 2.46 (s, 3H), 2.24 (s, 3H),
J ¼ 17.3 Hz, 1H), 3.59 (s, 8H), 3.16 (s, 2H), 2.92 (t, J ¼ 12.8 Hz, 1H),
2
1
.61 (d, J ¼ 16.9 Hz, 1H), 2.45 (s, 3H), 2.41 (s, 2H), 2.30 (d, J ¼ 8.6 Hz,
13
H), 2.24 (s, 3H), 2.04 (s, 1H), 1.63 (s, 4H). MS (ESI, m/z): 785
2.12e2.04 (m, 1H). C NMR (126 MHz, DMSO‑d
6
) d 172.22, 170.10,
þ
[MþH] .
167.10 (d, J ¼ 9.2 Hz), 165.68, 163.05, 162.78, 160.42, 157.43, 141.32,
1
39.39 (d, J ¼ 21.2 Hz), 135.44, 134.01, 132.94 (d, J ¼ 4.8 Hz), 130.91,
4.1.48. 5-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)
129.49, 128.63, 127.48, 126.20, 125.87, 119.34, 83.14, 56.41, 52.45,
amino)-5-oxopentanoic acid (S34)
49.95, 43.99, 31.58, 27.77, 27.10, 26.06, 21.66, 18.79. HRMS (ESI) m/z:
þ
þ
Compound S31 (4 mmol, 1 equiv) and compound glutaric acid
calcd. C36
H
37ClN
9
O
S
5 2
[MþH] ,775.3195; found, 775.3191.
(
(
4 mmol) were added into a dry round-bottom flask. Then HOAt
6 mmol, 1.5 equiv), EDCI (6 mmol, 1.5 equiv), NMM (8 mmol, 2
4.2. Biological screening
ꢀ
equiv) and DMF (10 ml) were mixed and stirred at 25 C for 12 h.
The reaction was quenched with water (1.0 ml) followed by puri-
fication via preparative HPLC (100 g, C18 column) to afford the
4.2.1. Cell lines and cell culture
The erythroleukemia cell line K562, derived from a patient in
blast crisis CML, and the murine hematopoietic 32D cell was pur-
chased from American Type Culture Collection. All these cells were
1
desired product S34. White solid, 0.52 g, (yield 35%). H NMR
(
6
500 MHz, DMSO‑d ) d12.09 (s, 1H), 11.01 (s, 1H), 9.80 (s, 1H), 7.81
(
dd, J ¼ 7.2, 1.8 Hz, 1H), 7.52e7.45 (m, 2H), 5.14 (dd, J ¼ 13.3, 5.1 Hz,
cultured according to the provider's instructions and maintained at
ꢀ
1
1
4
2
H), 4.40 (d, J ¼ 17.5 Hz, 1H), 4.33 (d, J ¼ 17.5 Hz, 1H), 2.97e2.87 (m,
H), 2.64e2.56 (m, 1H), 2.41 (t, J ¼ 7.4 Hz, 2H), 2.35 (dd, J ¼ 13.1,
.5 Hz, 1H), 2.29 (t, J ¼ 7.3 Hz, 2H), 2.07e1.99 (m, 1H), 1.86e1.78 (m,
37 C in a humidified atmosphere containing 5% CO
2
in air.
4.2.2. Cell growth inhibition
þ
H). MS (ESI, m/z): 374 [MþH] .
For cell growth experiments, 3000 - 20,000 cells/well in 200 mL
were seeded into a 96-well tissue culture plate. Then compounds
were diluted in the corresponding medium and then 3-fold serially
4
.1.49. N-(2-chloro-6-methylphenyl)-2-((6-(4-(5-((2-(2,6-
ꢀ
dioxopiperidin-3-yl)-1-oxoisoindolin-4 -yl)amino)-5-oxopentanoyl)
piperazin-1-yl)-2-methylpyrimidin-4-yl)amino)thiazole-5-
carboxamide (23)
diluted into each well. Cells were incubated for 2 days at 37 C in an
2
atmosphere of 5% CO . Cell growth was evaluated utilizing CCK-8
assay (CK04, Dojindo Molecular Technologies, MD), incubated for
2e4 h in the cell culture incubator, and read at 450 nm in a
microporous plate detection system (PerkinElmer Envision, Cali-
fornia). The readings were normalized to the DMSO-treated cells
and fitted using a nonlinear regression analysis with the GraphPad
The derivative 23 was obtained from the condensation
of S34 and S2 according the similar process of 4. White solid,
1
1
1
3.1 mg, (yield 49%). H NMR (500 MHz, DMSO‑d
6
)
d
11.65 (s, 1H),
1.01 (s, 1H), 9.94 (s, 1H), 9.85 (s, 1H), 8.26 (s, 1H), 7.83 (dd, J ¼ 7.1,
16

H. Liu, X. Ding, L. Liu et al.
European Journal of Medicinal Chemistry 223 (2021) 113645
Prism 6 software to obtain the IC50 value for each compound.
For the establishment of cell lines that exogenously express BCR-
ABL proteins, the full-length BCR-ABL cDNA was cloned into
plasmid pMIGR1 (Addgen, #27490) and the BCR-ABL mutant iso-
forms harboring the point mutation G250E, E255K, E255V, F317L,
F317V, T315A, and T315I were obtained according to instructions of
Stratagene's QuikChange® Lightning Site-Directed Mutagenesis Kit.
Declaration of competing interest
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.
Acknowledgement
3
2D cells were transduced with retrovirus to express wide-type
and mutant isoforms of BCR-ABL and stable transfectants were
selected for GFP positive cells by FACS sorting.
We are grateful to the Discovery Technology Platform and
Analytical platform of Shanghai Institute for Advanced Immuno-
chemical Studies, ShanghaiTech University for technical assistance
with LC-MS, MS2, compound screen and flow cytometry experi-
ments. We appreciate the staff members of the National Facility for
Protein Science in Shanghai (NFPS), Zhangjiang Lab, China, for
providing technical support and assistance in animal handling, data
collection and analysis.
This work was supported, in part, by grants from the National
Natural Science Foundation of China (NSFC; Grant number
81702600), grants sponsored by Shanghai Sailing Program (Grant
number 17YF1412200), and grants sponsored by Shanghai Natural
Science Foundation (Grant number 19ZR1433600).
4
.2.3. Western blotting
5
3
ꢂ 10 cells/mL were plated in 24-well plates and treated with
compounds at the indicated concentrations and times. Cells were
collected, washed with cold 1 ꢂ PBS, and lysed in 1 ꢂ SDS buffer
containing protease inhibitor cocktails (no. 539134, Merck). Protein
in cell lysate was quantified by detergent compatible Bradford
assay kit (no. 23246, Thermo). Primary antibodies used in this study
include c-ABL antibody (no. 2862S, Cell Signaling Technology),
Phospho-c-ABL (Tyr245) antibody (no. 2861S, Cell Signaling Tech-
nology), Stat5 antibody (no. 9363S, Cell Signaling Technology),
Phospho-Stat5 (Tyr694) antibody (no. 4322S, Cell Signaling Tech-
nology), Src (no, 2108S, Cell Signaling Technology), Lck (no. 2752S,
Appendix A. Supplementary data
Cell Signaling Technology), PDGFR
b (no, 3162S, Cell Signaling
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113645.
Technology). The Millipore immobilon Western chem-
iluminescence substrate was used for signal development. Blots
were imaged in an Amersham Imager 600 (GE Healthcare).
References
[1] A.C. Lai, C.M. Crews, Induced protein degradation: an emerging drug discovery
paradigm, Nat. Rev. Drug Discov. 16 (2017) 101e114.
4
.2.4. Pharmacokinetic studies
The pharmacokinetic properties of compounds in rats were
[
2] M. Schapira, M.F. Calabrese, A.N. Bullock, C.M. Crews, Targeted protein
degradation: expanding the toolbox, Nat. Rev. Drug Discov. 18 (2019)
949e963.
determined as follows. The compound was administered intrave-
nously to the femoral vein of female Wistar rats as a solution in
NMP/PEG200 (1:9, v/v) at 2 mg/kg. Serial plasma samples were
collected by sublingual vein bleeding for 24 h (0.083, 0.25, 0.5, 1, 2,
[
3] K.M. Sakamoto, K.B. Kim, A. Kumagai, F. Mercurio, C.M. Crews, R.J. Deshaies,
Protacs: chimeric molecules that target proteins to the Skp1-Cullin-F box
complex for ubiquitination and degradation, Proc. Natl. Acad. Sci. U. S. A. 98
(2001) 8554e8559.
[
4] K.M. Sakamoto, K.B. Kim, R. Verma, A. Ransick, B. Stein, C.M. Crews,
R.J. Deshaies, Development of Protacs to target cancer-promoting proteins for
ubiquitination and degradation, Mol. Cell. Proteomics 2 (2003) 1350e1358.
4
, 6, 8, 12, 24 h) from one group of rats (n ¼ 3). Samples were
extracted and the concentration of compound was determined by
LCꢃMS/MS. LOQ was set to 2.5 ng/mL.
[5] G.M. Burslem, B.E. Smith, A.C. Lai, S. Jaime-Figueroa, D.C. McQuaid,
D.P. Bondeson, M. Toure, H. Dong, Y. Qian, J. Wang, A.P. Crew, J. Hines,
C.M. Crews, The advantages of targeted protein degradation over inhibition:
an RTK case study, Cell Chem. Biol. 25 (2018) 67e77, e63.
[6] Y. Zou, D. Ma, Y. Wang, The PROTAC technology in drug development, Cell
Biochem. Funct. 37 (2019) 21e30.
4.2.5. Establishment and analysis of K562 xenograft models
The K562 cell line was stably infected with the lentiviral vector
[
7] J. Peh, M.W. Boudreau, H.M. Smith, P.J. Hergenrother, Overcoming resistance
to targeted anticancer therapies through small-molecule-mediated MEK
degradation, Cell Chem. Biol. 25 (2018) 996e1005 e4.
pLVX-IRES-ZsGreen1 carrying the firefly luciferase cDNA and
termed K562-Luc cells. Each mouse was inoculated with approxi-
mately 200
5
mice. Treatments were initiated when the mean tumor volumes
were approximately 200 mm . The compound 17 was formulated in
DMSO/PEG 400/40% hydroxypropyl-
6
m
L of K562-Luc cell suspension (2 ꢂ 10 cells) contained
[8] Y. Sun, N. Ding, Y. Song, Z. Yang, W. Liu, J. Zhu, Y. Rao, Degradation of Bruton's
tyrosine kinase mutants by PROTACs for potential treatment of ibrutinib-
resistant non-Hodgkin lymphomas, Leukemia 33 (2019) 2105e2110.
0% Matrigel subcutaneously on the right subventral of NOD/SCID
[
9] S. Rana, M. Bendjennat, S. Kour, H.M. King, S. Kizhake, M. Zahid, A. Natarajan,
Selective degradation of CDK6 by a palbociclib based PROTAC, Bioorg. Med.
Chem. Lett 29 (2019) 1375e1379.
3
b-cyclodextrin (HP-b-CD)
[
[
10] D.P. Bondeson, B.E. Smith, G.M. Burslem, A.D. Buhimschi, J. Hines, S. Jaime-
Figueroa, J. Wang, B.D. Hamman, A. Ishchenko, C.M. Crews, Lessons in PROTAC
design from selective degradation with a promiscuous warhead, Cell Chem.
Biol. 25 (2018) 78e87 e75.
11] R.B. Kargbo, PROTAC-mediated degradation of KRAS protein for anticancer
therapeutics, ACS Med. Chem. Lett. 11 (2020) 5e6.
[12] L. Bai, H. Zhou, R. Xu, Y. Zhao, K. Chinnaswamy, D. McEachern, J. Chen,
C.Y. Yang, Z. Liu, M. Wang, L. Liu, H. Jiang, B. Wen, P. Kumar, J.L. Meagher,
D. Sun, J.A. Stuckey, S. Wang, A potent and selective small-molecule degrader
of STAT3 achieves complete tumor regression in vivo, Canc. Cell 36 (2019)
498e511, e417.
(
1:3:6, vol/vol) and applied daily by the intraperitoneal adminis-
tration to the tumor-bearing mice for 10 consecutive days at doses
of 1, 3, and 10 mg/kg/qd. Animals were weighed 2e3 times per
week during the treatment period. Tumor size was measured uti-
lizing electronic calipers 2e3 times per week during the treatment
2
period. Tumor volume was calculated as V ¼ L ꢂ W /2, where L is
the length and W is the width of the tumor. After 10 days, tumor
growth measurements were done by noninvasive imaging using a
Xenogen IVIS imaging system. Measurements were performed
[13] L.H. Jones, Small-Molecule kinase downregulators, Cell Chem. Biol. 25 (2018)
30e35.
10 min after intravenous injection of 150 mg/kg (15 mg/mL in PBS)
[
14] Y. Wang, X. Jiang, F. Feng, W. Liu, H. Sun, Degradation of proteins by PROTACs
D-luciferin. Mice were euthanized when tumors grew to larger
than 2000 mm . All animal experiments were conducted according
to the guidelines of the humane use and care of laboratory animals
and were approved by the ShanghaiTech University Animal Study
Committee (Approval number: 20201026001).
and other strategies, Acta Pharm. Sin. B 10 (2020) 207e238.
3
[15] T. Neklesa, L.B. Snyder, R.R. Willard, N. Vitale, J. Pizzano, D.A. Gordon, et al.,
ARV-110: an oral androgen receptor PROTAC degrader for prostate cancer,
J. Clin. Oncol. 37 (2019).
[
16] S. Yamshon, J. Ruan, IMiDs new and old, Curr. Hematol. Malig. Rep. 14 (2019)
414e425.
17

H. Liu, X. Ding, L. Liu et al.
European Journal of Medicinal Chemistry 223 (2021) 113645
degradation, ACS Med. Chem. Lett. 8 (2017) 1042e1047.
[32] N. Shibata, N. Miyamoto, K. Nagai, K. Shimokawa, T. Sameshima, N. Ohoka,
T. Hattori, Y. Imaeda, H. Nara, N. Cho, M. Naito, Development of protein
degradation inducers of oncogenic BCR-ABL protein by conjugation of ABL
kinase inhibitors and IAP ligands, Canc. Sci. 108 (2017) 1657e1666.
[33] Y. Yang, H. Gao, X. Sun, Y. Sun, Y. Qiu, Q. Weng, Y. Rao, Global PROTAC toolbox
for degrading BCR-ABL overcomes drug-resistant mutants and adverse effects,
J. Med. Chem. 63 (2020) 8567e8583.
[
17] T. Ito, H. Ando, T. Suzuki, T. Ogura, K. Hotta, Y. Imamura, Y. Yamaguchi,
H. Handa, Identification of a primary target of thalidomide teratogenicity,
Science 327 (2010) 1345e1350.
[18] T. Ishida, A. Ciulli, E3 ligase ligands for PROTACs: how they were found and
how to discover new ones, SLAS Discov. 26 (2020) 484e502.
[
19] A.C. Lai, M. Toure, D. Hellerschmied, J. Salami, S. Jaime-Figueroa, E. Ko, J. Hines,
C.M. Crews, Modular PROTAC design for the degradation of oncogenic BCR-
ABL, Angew Chem. Int. Ed. Engl. 55 (2016) 807e810.
[
20] J.D. Rowley, Letter, A new consistent chromosomal abnormality in chronic
myelogenous leukaemia identified by quinacrine fluorescence and Giemsa
staining, Nature 243 (1973) 290e293.
[34] B. Tong, J.N. Spradlin, L.F.T. Novaes, E. Zhang, X. Hu, M. Moeller, S.M. Brittain,
L.M. McGregor, J.M. McKenna, J.A. Tallarico, M. Schirle, T.J. Maimone,
D.K. Nomura, A nimbolide-based kinase degrader preferentially degrades
oncogenic BCR-ABL, ACS Chem. Biol. 15 (2020) 1788e1794.
[35] L. Jiang, Y. Wang, Q. Li, Z. Tu, S. Zhu, S. Tu, Z. Zhang, K. Ding, X. Lu, Design,
synthesis, and biological evaluation of Bcr-Abl PROTACs to overcome T315I
mutation, Acta Pharm. Sin. B 11 (2021) 1315e1328.
[
[
21] R. Ren, Mechanisms of BCR-ABL in the pathogenesis of chronic myelogenous
leukaemia, Nat. Rev. Canc. 5 (2005) 172e183.
22] F. Rossari, F. Minutolo, E. Orciuolo, Past, present, and future of Bcr-Abl in-
hibitors: from chemical development to clinical efficacy, J. Hematol. Oncol. 11
(
2018), 84.
[36] P.P. Piccaluga, S. Paolini, G. Martinelli, Tyrosine kinase inhibitors for the
treatment of Philadelphia chromosome-positive adult acute lymphoblastic
leukemia, Cancer 110 (2007) 1178e1186.
[37] M. Steinberg, Dasatinib: a tyrosine kinase inhibitor for the treatment of
chronic myelogenous leukemia and philadelphia chromosome-positive acute
lymphoblastic leukemia, Clin. Therapeut. 29 (2007) 2289e2308.
[38] A. Zagidullin, V. Milyukov, A. Rizvanov, E. Bulatov, Novel approaches for the
rational design of PROTAC linkers, Explor. Target Antitumor. Ther. 1 (2020)
381e390.
[
23] C.V. Ichim, Kinase-independent mechanisms of resistance of leukemia stem
cells to tyrosine kinase inhibitors, Stem Cells Transl. Med. 3 (2014) 405e415.
24] A. Hamilton, G.V. Helgason, M. Schemionek, B. Zhang, S. Myssina, E.K. Allan,
F.E. Nicolini, C. Muller-Tidow, R. Bhatia, V.G. Brunton, S. Koschmieder,
T.L. Holyoake, Chronic myeloid leukemia stem cells are not dependent on Bcr-
Abl kinase activity for their survival, Blood 119 (2012) 1501e1510.
25] F. Lee, A. Fandi, M. Voi, Overcoming kinase resistance in chronic myeloid
leukemia, Int. J. Biochem. Cell Biol. 40 (2008) 334e343.
[
[
[
26] Y. Ru, Q. Wang, X. Liu, M. Zhang, D. Zhong, M. Ye, Y. Li, H. Han, L. Yao, X. Li, The
chimeric ubiquitin ligase SH2-U-box inhibits the growth of imatinib-sensitive
and resistant CML by targeting the native and T315I-mutant BCR-ABL, Sci.
Rep. 6 (2016), 28352.
[39] C. Donoghue, M. Cubillos-Rojas, N. Gutierrez-Prat, C. Sanchez-Zarzalejo,
X. Verdaguer, A. Riera, A.R. Nebreda, Optimal linker length for small molecule
PROTACs that selectively target p38alpha and p38beta for degradation, Eur. J.
Med. Chem. 201 (2020), 112451.
[
[
[
[
[
27] G.M. Burslem, A.R. Schultz, D.P. Bondeson, C.A. Eide, S.L. Savage Stevens,
B.J. Druker, C.M. Crews, Targeting BCR-ABL1 in chronic myeloid leukemia by
PROTAC-mediated targeted protein degradation, Canc. Res. 79 (2019)
[40] K. Cyrus, M. Wehenkel, E.Y. Choi, H.J. Han, H. Lee, H. Swanson, K.B. Kim,
Impact of linker length on the activity of PROTACs, Mol. Biosyst. 7 (2011)
359e364.
[41] N.P. Shah, C. Tran, F.Y. Lee, P. Chen, D. Norris, C.L. Sawyers, Overriding imatinib
resistance with a novel ABL kinase inhibitor, Science 305 (2004) 399e401.
[42] J.S. Tokarski, J.A. Newitt, C.Y. Chang, J.D. Cheng, M. Wittekind, S.E. Kiefer,
K. Kish, F.Y. Lee, R. Borzillerri, L.J. Lombardo, D. Xie, Y. Zhang, H.E. Klei, The
structure of Dasatinib (BMS-354825) bound to activated ABL kinase domain
elucidates its inhibitory activity against imatinib-resistant ABL mutants, Canc.
Res. 66 (2006) 5790e5797.
[43] J. Cortes, E. Jabbour, H. Kantarjian, C.C. Yin, J. Shan, S. O'Brien, G. Garcia-
Manero, F. Giles, M. Breeden, N. Reeves, W.G. Wierda, D. Jones, Dynamics of
BCR-ABL kinase domain mutations in chronic myeloid leukemia after
sequential treatment with multiple tyrosine kinase inhibitors, Blood 110
(2007) 4005e4011.
[44] N.P. Shah, B.J. Skaggs, S. Branford, T.P. Hughes, J.M. Nicoll, R.L. Paquette,
C.L. Sawyers, Sequential ABL kinase inhibitor therapy selects for compound
drug-resistant BCR-ABL mutations with altered oncogenic potency, J. Clin.
Invest. 117 (2007) 2562e2569.
4744e4753.
28] Q. Zhao, C. Ren, L. Liu, J. Chen, Y. Shao, N. Sun, R. Sun, Y. Kong, X. Ding,
X. Zhang, Y. Xu, B. Yang, Q. Yin, X. Yang, B. Jiang, Discovery of SIAIS178 as an
effective BCR-ABL degrader by recruiting von hippel-lindau (VHL) E3 ubiq-
uitin ligase, J. Med. Chem. 62 (2019) 9281e9298.
29] Y. Demizu, N. Shibata, T. Hattori, N. Ohoka, H. Motoi, T. Misawa, T. Shoda,
M. Naito, M. Kurihara, Development of BCR-ABL degradation inducers via the
conjugation of an imatinib derivative and a cIAP1 ligand, Bioorg. Med. Chem.
Lett 26 (2016) 4865e4869.
30] N. Shibata, K. Shimokawa, K. Nagai, N. Ohoka, T. Hattori, N. Miyamoto,
O. Ujikawa, T. Sameshima, H. Nara, N. Cho, M. Naito, Pharmacological differ-
ence between degrader and inhibitor against oncogenic BCR-ABL kinase, Sci.
Rep. 8 (2018), 13549.
31] K. Shimokawa, N. Shibata, T. Sameshima, N. Miyamoto, O. Ujikawa, H. Nara,
N. Ohoka, T. Hattori, N. Cho, M. Naito, Targeting the allosteric site of onco-
protein BCR-ABL as an alternative strategy for effective target protein
18