Discovery of a PROTAC targeting ALK with in vivo activity

Article abstract of DOI:10.1016/j.ejmech.2020.113150

Anaplastic lymphoma kinase (ALK) was involved in the development of various cancer types. Although several ALK inhibitors have been advanced to clinical trials, the emergence of drug resistance has limited the clinical application of them. To overcome the drug resistance, proteolysis targeting chimeras (PROTACs) could be an alternative strategy. In this study, a series of ALK degraders were designed and synthesized. The degraders were developed through the conjugation of LDK378 and CRBN E3 ubiquitin ligase ligands. Among all the molecules, compound B3 showed potent selective inhibitory activity to ALK and can decrease the cellular levels of ALK fusion proteins in a concentration- and time-dependent manner in H3122 cell line. Meanwhile, B3 showed improved anticancer activity in vitro comparing with LDK378 and the antiproliferative activity to xenograft tumor model was acceptable. All the results demonstrated that ALK degrader B3 with in vitro and in vivo anti-cancer activities was valuable for further investigation.

Full text of DOI:10.1016/j.ejmech.2020.113150

European Journal of Medicinal Chemistry 212 (2021) 113150
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Discovery of a PROTAC targeting ALK with in vivo activity
,
b
,
,
,
Guoyi Yan a 1, Xinxin Zhong a 1, Lin Yue a 1, Chunlan Pu a, Huifang Shan a, Suke Lan a,
Meng Zhou c, Xueyan Hou d, Jie Yang a, Rui Li a
,
*
a State Key Laboratory of Biotherapy, Collaborative Innovation Center of Biotherapy and Cancer Center, West China Hospital of Sichuan University, Chengdu,
China
b Department of Hepatobiliary Pancreatic Surgery, Henan Provincial People’s Hospital, Henan University, Zhengzhou, China
c Engineering Research Center for the Development and Application of Ethnic Medicine and TCM, Ministry of Education & State Key Laboratory of Functions
and Applications of Medicinal Plants, Guizhou Medical University, Guiyang, China
d College of Pharmacy, Xinxiang Medical University, Xinxiang, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Anaplastic lymphoma kinase (ALK) was involved in the development of various cancer types. Although
several ALK inhibitors have been advanced to clinical trials, the emergence of drug resistance has limited
the clinical application of them. To overcome the drug resistance, proteolysis targeting chimeras (PRO-
TACs) could be an alternative strategy. In this study, a series of ALK degraders were designed and syn-
thesized. The degraders were developed through the conjugation of LDK378 and CRBN E3 ubiquitin
ligase ligands. Among all the molecules, compound B3 showed potent selective inhibitory activity to ALK
and can decrease the cellular levels of ALK fusion proteins in a concentration- and time-dependent
manner in H3122 cell line. Meanwhile, B3 showed improved anticancer activity in vitro comparing
with LDK378 and the antiproliferative activity to xenograft tumor model was acceptable. All the results
demonstrated that ALK degrader B3 with in vitro and in vivo anti-cancer activities was valuable for
further investigation.
Received 10 November 2020
Received in revised form
17 December 2020
Accepted 29 December 2020
Available online 2 January 2021
Keywords:
ALK
Degrader
PROTAC
Oral anticancer bioactivities
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
Crizotinib and ceritinib (LDK378) are potent ALK inhibitors in
clinical trials [8,9] (Fig. 1). NCCN recommended crizotinib as first-
Lung cancer has become the leading cause of cancer deaths in
the urban population in China, among them, non-small cell lung
cancer (NSCLC) accounts for 80% [1]. 75% of the patients were
diagnosed in the middle or advanced stage, and the 5-year survival
rate is low [2]. ALK was first discovered in a subtype of anaplastic
large cell lymphoma (ALCL) [3], after that, several other ALK fusion
partners have been described in multiple malignancies [4]. It has
been proved that ALK is a strong driver gene enhancing the
development of various cancers [5]. The incidence of ALK gene
rearrangement in NSCLC is 3e7%, most of which were fused with
EML4 [2]. It was reported that the inhibition of EML4-ALK can lead
to the reduction of cancer loading [6]. Although the incidence of
ALK rearrangement in NSCLC is significantly lower than that of
ALCL or inflammatory myofibroblastic tumor (IMT) [7], given to the
significant morbidity of lung cancer worldwide, NSCLC patients
constitute the largest group of ALK rearrangement.
line treatment for NSCLC patients with ALK rearrangements, and
ceritinib for the treatment of recurrence or intolerance after treated
with crizotinib. However, drug resistance of ceritinib has been
observed in clinical trials [10], suggesting that the subsequent
development of ALK inhibitors is needed to overcome drug resis-
tance in clinical trials.
PROTAC method is a chemical knockdown strategy in which a
heterobifunctional molecule recruits a specific protein target to an
E3 ubiquitin ligase, resulting in the target’s ubiquitination and
degradation [11]. PROTACs behave catalytically in their ability to
induce the ubiquitination of proteins, which is a key advantage over
small molecule inhibitors [12]. To date, PROTAC method has been
successfully applied in the degradation of variety of targets such as
BRD4 [13e16], RIPK2 [11], Akt [17], HDAC6 [18e20], BCR-ABL [21],
BCL-XL [22,23], Smad3 [24], STAT3 [25,26], CDK9 [27], Estrogen
Receptor a [28], TANK-Binding Kinase 1 [29], Sirt2 [30] and Tau [31].
Among the targets of PROTACs, kinases attracted much attention
for the predominant regulatory roles in nearly every aspect of cell
biology [32].
In recent years, degraders of ALK also have been reported
(Fig. 2): Gray et al. reported TL13-112 can induce the degradation of
* Corresponding author.
E-mail address: lirui@scu.edu.cn (R. Li).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.ejmech.2020.113150
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

G. Yan, X. Zhong, L. Yue et al.
European Journal of Medicinal Chemistry 212 (2021) 113150
Fig. 1. Chemical structures of Crizotinib and Ceritinib.
ALK at half-maximal degrading concentration (DC50) value of
10 nM [33]; Hwang’s group reported TD-004 can significantly
reduce the amount of ALK protein over 90% in SU-DHL-1 at 1 mM
of linkers were applied to discuss the structure activity relationship
(SAR). Based on the x-ray co-crystal structure of ALK kinase domain
and LDK378 (pdb: 4MKC) [39], nitrogen atom of piperidine is sol-
vent exposed and therefore can serve as proper attachment site for
a linker, which is consistent with the reported works.
[34]; Two potent ALK degraders (5 and 6) were reported by Jin’s
group [35]; Jiang’s group reported SIAIS117 with DC50 of 51 nM in
H2228 cell line [36]. It is noteworthy that ALK degraders in Fig. 2
did not share much similarity in linker part, the phenomenon
may suggest that the optional linker may be different varying on
different ALK inhibitors and E3 ligase ligands utilized.
As far as we learned, most of the reported PROTAC molecules
lack of oral bioactivity, which limited the clinical application
[37,38]. In this study, we describe our efforts in the discovery of
potent ALK degraders utilizing CRBN E3 ligase and resulted in a
promising compound with in vitro and in vivo anticancer activities.
2.2. Chemistry
The synthetic route of target compounds was summarized in
Scheme 1. The compounds in this work shared similar synthetic
methods: the preparation of target molecules was started from
LDK378, michael addition reaction of methyl acrylate and the
subsequently hydrolysis under basic conditions provided inter-
mediate K2. After that, the intermediate product was connected
with CRBN ligands through different linkers to generate the cor-
responding compounds.
2. Results and discussion
Reagents and conditions: (a): Chloroacetyl chloride, THF, reflux,
5h, yield 70%; (b) Acryloyl chloride, THF, reflux, 5h, yield 63%; (c)
K2CO3, tert-Butyl bromoacetate, DMF, rt, 15h; CF3COOH, 1h, yield
70%; (d) DIPEA, NPM, 90 ꢀC, 10 h, yield 52e68%; (e) K2CO3, THF,
66 ꢀC, 5h, yield 61e72%; (f) Triethylamine, THF, 90 ꢀC, 12 h, yield
56e73%; (g) HATU, DIPEA, DMF, rt, yield 35e60%; (h) Methyl
acrylate, Triethylamine, MeOH, rt, 8h; NaOH, MeOH/H2O,10h, yield
95%; (i) HATU, DIPEA, DMF, rt, 15h, yield 21e51%.
2.1. Molecular design
In this work, a series of PROTACs targeting ALK were designed and
synthesized. LDK378wasselected asthe partof ALK inhibitorbecause
of the success of previously reported works. Among the reported ALK
degraders, only Hwang’s group reported TD004 with in vivo anti-
cancer activities [34]. TD004 was designed by linking Von Hippel-
Lindau (VHL) E3 ligase ligand with LDK378. However, we suggested
it is possible to get orallyactive molecules by utilizing CRBN ligands to
recruit the E3 ligase and careful optimization of linker part.
2.3. In vitro bioassays
To get novel PROTACs with in vivo bioactivities, different linkers
and CRBN E3 ligase ligands were utilized. Different type and length
The binding affinities of the compounds to ALK were evaluated
through mobility shift assay with ATP at concentration of Km
Fig. 2. Chemical structures of the previously reported ALK degraders.
2

G. Yan, X. Zhong, L. Yue et al.
European Journal of Medicinal Chemistry 212 (2021) 113150
Scheme 1. Syntheses of ALK PROTACs.
(30.3
m
M). The IC50 values were summarized in Table 1. Most of the
addition, as can be demonstrated in kinase assay, none of the two
molecules (B2, B3) showed superior inhibitory activity against ALK
than LDK378, indicating that the potent anti-cancer activity may
due to the degradation-inducing activity. The phenomenon was
consistent with the reported ALK degraders.
The toxicity of inhibitors has always aroused great concern in
drug development. The biologically active molecules were tested
against normal human liver cell line LO2 by MTT for 72h to learn
the toxicities. As a result, all the compounds showed less inhibitory
activities to the cells comparing with LDK378 (Fig. 3), indicating
that the degraders of ALK do not increase the toxicity to normal
cells. The in vitro bioassays of the compounds showed that the cells
with ALK-fusion proteins were more sensitive to ALK inhibitors or
degraders than other cells [39,40].
degraders exhibited inhibitory activities at low IC50 values
demonstrating that the linkers applied in this study were feasible
to maintain the ALK inhibitory activity of the synthesized de-
graders. Additionally, the type of linker may have little affection on
the binding affinity of the molecules to ALK: as can be depicted in
Table1, in most cases, the molecules with chains of B can show
better inhibitory activities comparing with other ones, which may
result from the better flexibility and solubility provided by the
chains.
To evaluate the antiproliferative effect of degraders against
cancer cells, we performed MTT assay for 72h of synthesized
compounds on several tumor cell lines, which including H3122
(NSCLC cell line), H2228 (NSCLC cell line), H1299 (NSCLC cell line),
A549 (lung cancer cell line) and Hela (human cervical cancer cell).
The IC50 values of the degraders were summarized in Table 2 and
Supplementary Table 1. Two of the molecules (B2, B3) showed
better anti-proliferation effect than LDK378 in H3122 cells. In
2.4. B3 effectively induce ALK degradation
To evaluate the extent of ALK degradation induced by PROTACs,
western blot was conducted. H3122 was selected because the
expression of ALK fusion proteins and the cell line was highly
sensitive in cytotoxicity assay [39].
Table 1
The IC50 (nM) value of compounds to ALK.
NO.
IC50
NO.
IC50
Table 2
Antiproliferative activity of the compounds (IC50: mM).
A1
A2
A3
B1
B2
B3
B4
C1
C2
C3
D1
D2
D3
19.3 1.52
15.8 1.31
19.8 1.73
10.2 0.96
2.3 0.22
1.6 0.12
11.4 0.73
8.3 0.76
50.9 2.81
12.8 0.92
4.6 0.15
4.8 0.17
5.4 0.13
E1
E2
E3
F1
1.5 0.09
12.6 0.93
8.2 0.71
1.4 0.06
1.9 0.09
15.8 1.06
2.1 0.16
5.4 0.14
3.6 0.22
1.9 0.11
1.5 0.09
3.9 0.12
0.81 0.07
NO.
H3122
H2228
H1299
A549
HeLa
B2
B3
C1
C2
C3
D1
D2
F1
0.7 0.05
0.3 0.02
3.52 0.35 8.7 0.56
1.1 0.04 3.1 0.14
2.66 0.06 15.1 0.67 >20
1.83 0.18 5.74 0.26 7.23 0.38
2.34 0.11 4.51 0.23 10.86 0.92 5.81 0.32
4.86 0.24 5.2 0.26 12.28 0.74 10.9 0.59
3.5 0.21
0.9 0.03
7.59 0.34
2.84 0.23
>20
2.6 0.11
1.6 0.09
13.38 0.85 8.15 0.62
5.13 0.38
>20
2.23 0.09
1.18 0.06
F2
G1
G2
G3
H1
H2
H3
K2
>20
4.36 0.25
7.67 0.28
5.78 0.21
5.86 0.25
7.98 0.37
6.99 0.49
0.94 0.04
6.73 0.24
G1
1.3 0.11
>20
1.3 0.14
9.33 0.13
2.92 0.19
>20
1.32 0.06
LDK378
LDK378 1.1 0.21
3

G. Yan, X. Zhong, L. Yue et al.
European Journal of Medicinal Chemistry 212 (2021) 113150
Fig. 3. Antiproliferative activities of compounds to human normal cell line LO2.
To determine if B2 and B3 were ALK degraders, we treated
H3122 cells with increasing concentrations of B2/B3 for 8 h (h) and
the lysates were subjected to western blot analyses with antibodies
for EML4-ALK and GAPDH. LDK378 was utilized as control in this
assay. As depicted in Fig. 4, B2 and B3 can degrade ALK in a dose
dependent manner. At the concentration of 50 nM, B2 and B3
effectively degraded ALK in contrast with LDK378. Moreover, B3
demonstrated a more potent ability to degrade ALK comparing
with B2, at the concentration of 200 nM, B3 could degrade all the
ALK protein in H3122.
Next, we conducted time and concentration-course studies to
assess the degradation of ALK fusing proteins and the inhibition of
signaling pathways by compound B3 in H3122 cell line (Fig. 5).
After 16 h treatment of B3 at concentration of 100 nM, significant
amount (>80%) of EML4-ALK degradation was achieved, mean-
while, maximum degradation of EML4-ALK degradation was not
observed until treatment of B3 for 24 h. At the same time, p-ALK
was significantly inhibited after 24 h treatment, while the level of
p-STAT3 was slightly downregulated after treatment. In order to
identify the degradation activity of B3 in different concentrations,
ALK degradation assay was conducted with a fixed incubation time
of 24 h. The results revealed that B3 could induce the degradation
of ALK in a concentration dependent manner, at a concentration of
50 nM, maximum degradation of EML4-ALK degradation was
achieved. Meanwhile, the level of p-ALK and p-STAT3 was also
downregulated in a concentration dependent manner. In summary,
it can be concluded that B3 can decrease the cellular levels of ALK
fusion proteins in a concentration- and time-dependent manner.
The results indicated that B3 is a potent ALK degrader comparable
with the reported ones.
2.5. Selectivity of B3
Among all the molecules, B3showed significant increase ininvitro
bioactivity in contrary with LDK378 while keeping low toxicity to
normal cells, therefore this molecule was selected for further inves-
tigation. B3 could potently inhibit ALK with low IC50 value, mean-
while, high selectivity may be the important feature in the design of a
safe and effective drug. In this study, to learn the selectivity of B3,
several reported main targets of LDK378 (IGF1R, INSR, FLT3 and
FGFR2) were selected [41]. As a result, B3 showed high selectivity to
ALK (Fig. 6), maintaining moderate inhibition activity to IGF1R and
INSR. In order to learn ifB3 couldinducethe degradation ofIGF1R and
INSR inH3122, westernblot wasconducted withconcentrationsofB3
from 0 to 500 nM. The result showed that no significant degradation
was observed (Fig. S1), demonstrating high selectivity of B3 to ALK in
inhibition and degradation activities.
2.6. Bioactivity of B3 to ALK mutant cell lines
To assay the possible anti-drug resistant activity of B3, ALK
mutant cell lines BaF3-ALK-L1196M and BaF3-ALK-G1202R were
employed. As shown in Fig. 7, to BaF3-ALK-L1196M cells, B3 was
slightly more active than LDK378, and BaF3-ALK-G1202R was less
sensitive to B3 comparing with LDK378, which demonstrated that
B3 may be a potential treatment for ALK mutant cancer cells.
2.7. Pharmacokinetics evaluation of B3 in rats
We performed a pharmacokinetics (PK) analysis in rats before
assessing the antitumor activity of B3 in vivo, and the results were
listed in Fig. 8. In this experiment, a single dose of B3 at 1 mg/kg
Fig. 4. B2 and B3 could significantly reduce the ALK protein levels in a concentration-dependent manner.
4

G. Yan, X. Zhong, L. Yue et al.
European Journal of Medicinal Chemistry 212 (2021) 113150
Fig. 5. B3 could significantly reduce the ALK protein levels and inhibit the ALK down-stream signaling in a time and concentration-dependent manner.
25 mg/kg and 50 mg/kg, respectively. LDK378 as positive control in
this assay have a TGI of 49% at dose of 25 mg/kg. In addition, B3 did
not cause significant weight loss and toxicity during the treatment
period, suggesting a safe candidate for further investigation.
3. Conclusions
In this work, by linking LDK378 to CRBN ligands through
different linkers, a series of PROTACs targeting ALK were designed
and synthesized. Among all the molecules, B3 could effectively
induce the degradation of ALK in H3122 cell line, which is com-
parable with the reported ALK degraders. In addition, B3 displayed
a clear advantage in inhibiting the growth of cell line H3122
comparing with LDK378 and the in vivo anticancer activity is
acceptable. In summary, this is a novel reported ALK PROTAC
molecule that can inhibit the growth of NSCLC in vitro and in vivo.
Fig. 6. Selectivity of B3 against ALK, IGF1R, INSR, FLT3 and FGFR2.
4. Experimental section
was taken by intravenous injection followed by plasma level
quantitation. The data showed that B3 with a half-life of 4.09 h
(n ¼ 3) has a concentration of 10 ng/mL in plasma at 10 h. Based on
the acceptable half-life and concentration in plasma, we suggest B3
could be an effective candidate for in vivo cancer treatment.
4.1. Chemistry
Unless otherwise noted, all reagents and solvents obtained from
commercial sources were used without further purification. Flash
column chromatography was performed using silica gel from
Qingdao Haiyang. 1H NMR and 13C NMR spectra were recorded on a
Bruker AMX 400 spectrometer and were calibrated using TMS or
residual deuterated solvent as an internal reference (CDCl3: 1H,
2.8. In vivo antiproliferative activity of compound B3
d
¼ 7.26 ppm; 13C,
d
¼ 77.16 ppm, DMSO‑d6 2.50 ppm, CD3OD
The anti-tumor activity in vivo of B3 was conducted, BALB/c
nude mice bearing H3122 xenograft tumors were treated with B3
by intragastric administration for 15 days. As can be depicted in
Fig. 9, the growth of xenograft tumors can be inhibited by B3 in a
dose-dependent manner. Tumor growth inhibitions (TGIs) of 37%
and 48% were observed in the H3122 xenograft model at dose of
3.31 ppm). All of the target compounds were examined by HPLC,
and the purity of the biologically tested compounds was ꢁ95%.
4.1.1. Synthesis of K2
To a solution of LDK378 (330 mg, 0.6 mmol) in MeOH (50 mL),
methyl acrylate (78 mg, 0.9 mmol) and Triethylamine (240 mg,
2.4 mmol) were added. The resulting reaction mixture was stirred
at room temperature for 8 h and monitored by TLC. Upon
completion, the methanol was removed by vacuum distillation, the
reaction was then extracted with EtOAc, the organic layer was
washed twice with water, dried over anhydrous sodium sulfate,
filtered and concentrated under reduced pressure. The crude ma-
terial was purified by column chromatography, and the product
was obtained as white solid. The white solid (320 mg, 0.5 mmol)
was then suspended in 1:1 v/v MeOH/H2O (50 mL), to this solution
was added NaOH (200 mg, 5 mmol). The resulting reaction mixture
was stirred at room temperature for 8 h and monitored by TLC.
Upon completion, the methanol was removed by vacuum distilla-
tion, and the pH of water layer was adjusted to 2 with dilute hy-
drochloric acid. The reaction was then extracted with EtOAc, the
organic layer was washed twice with water, dried over anhydrous
Fig. 7. Bioactivity of B3 to BaF3 models of ALK mutations.
5

G. Yan, X. Zhong, L. Yue et al.
European Journal of Medicinal Chemistry 212 (2021) 113150
Fig. 8. PK study of B3 in rats (n ¼ 3 per group).
Fig. 9. Pharmacodynamic profile of B3 in vivo. (A) Growth inhibitory effect of B3 on established H3122 xenografts in female BALB/c nude mice (N ¼ 6 per group); (B) Body weight of
the mice during the dosage period.
sodium sulfate, filtered and concentrated under reduced pressure.
The crude material was purified by column chromatography, and
the product was obtained as white powder (yield 95%). HRMS
(DART-TOF) calculated for C31H41ClN5O5S [M þ H]þ m/z 630.2517,
found 630.2508.
(101 MHz, CDCl3)
d
¼ 171.83, 171.20, 169.59, 168.67, 167.55, 157.42,
155.37, 146.87, 144.80, 138.48, 136.23, 134.62, 132.48, 131.30, 128.11,
127.17, 124.92, 123.59, 123.16, 120.82, 116.72, 111.63, 111.06, 110.06,
105.96, 71.88, 60.40, 55.49, 53.86, 48.91, 42.16, 31.71, 26.93, 26.59,
22.23, 15.37. HRMS (DART-TOF) calculated for C48H59ClN9O8S [M þ
H]þ m/z 956.3896, found 956.3958.
4.1.2. Synthesis of 3-(4-(4-((5-chloro-4-((2-(isopropylsulfonyl)
phenyl)amino)pyrimidin-2-yl)amino)-5-isopropoxy-2-
methylphenyl)piperidin-1-yl)-N-(4-((2-(2,6-dioxopiperidin-3-yl)-
1,3-dioxoisoindolin-4-yl)amino)butyl)propenamide (A1)
4.1.3. Synthesis of 3-(4-(4-((5-chloro-4-((2-(isopropylsulfonyl)
phenyl)amino) pyrimidin-2-yl)amino)-5-isopropoxy-2-
methylphenyl)piperidin-1-yl)-N-(6-((2-(2,6-dioxopiperidin-3-yl)-
1,3-dioxoisoindolin-4-yl)amino)hexyl)propenamide (A2)
A1-M was prepared as reported [42]. Next, to a solution of A1-M
(90 mg, 0.2 mmol) in DMF (10 mL), K2 (63 mg, 0.1 mmol), HATU
(45.6 mg, 0.12 mmol) and DIPEA (38.7 mg, 0.3 mmol) were added.
The resulting reaction mixture was stirred at room temperature for
15 h and monitored by TLC. Upon completion, the reaction was then
quenched with water and extracted with EtOAc. The organic layer
was washed twice with water, dried over anhydrous sodium sul-
fate, filtered and concentrated under reduced pressure. The crude
material was purified by column chromatography to afford target
molecule as yellow powder (yield 40%). 1H NMR (400 MHz, CDCl3)
The title compound was obtained from tert-butyl (6-
aminohexyl)carbamate following similar synthesis procedure of
A1 (yellow powder, yield 35%). 1H NMR (400 MHz, CDCl3)
d
¼ 9.46
(s, 1H, CONHCO), 8.51 (d, J ¼ 8.3, 1H, AreH), 8.09 (s, 1H, Ar-NH), 7.89
(s, 1H, AreH), 7.85 (dd, J ¼ 8.0, 1.5, 1H, AreH), 7.79 (s, 1H, Ar-NH),
7.62 (s, 1H, AreH), 7.56e7.49 (m, 1H, AreH), 7.43e7.36 (m, 1H,
AreH), 7.16 (d, J ¼ 7.9, 1H, AreH), 7.00 (d, J ¼ 7.1, 1H, AreH), 6.78 (d,
J ¼ 8.5, 1H, AreH), 6.68 (s, 1H, AreH), 4.83 (dd, J ¼ 12.3, 5.3, 1H,
COCHN), 4.53e4.37 (m, 1H, OCH), 3.29e3.07 (m, 4H, NCH2),
2.86e2.58 (m, 8H, CH2), 2.19 (s, 3H, Ar-CH3), 2.13e2.01 (m, 4H,
CH2), 1.59 (dt, J ¼ 13.9, 6.8, 4H, CH2), 1.23 (m, 12H, CH3). 13C NMR
d
¼ 9.45 (s, 1H, CONHCO), 8.51 (d, J ¼ 8.3, 1H, AreH), 8.09 (s, 1H, Ar-
NH), 7.94 (s,1H, AreH), 7.90e7.78 (m,1H, AreH), 7.53 (m, 3H, AreH,
Ar-NH), 7.41 (dd, J ¼ 15.8, 8.3, 1H, AreH), 7.21e7.10 (m, 1H, AreH),
7.00 (d, J ¼ 7.1, 1H, AreH), 6.81 (d, J ¼ 8.5, 1H, AreH), 6.70 (s, 1H,
AreH), 4.94e4.71 (m, 1H, COCHN), 4.47 (dt, J ¼ 11.9, 5.9, 1H, OCH),
3.39e3.05 (m, 4H, N-CH2), 2.96e2.29 (m, 8H, CH2), 2.08 (s, 3H, Ar-
CH3), 1.84e1.53 (m, 8H, CH2), 1.37e1.22 (m, 12H, CH3). 13C NMR
(101 MHz, CDCl3)
d
¼ 171.98, 171.40, 169.60, 168.91, 167.67, 157.47,
155.38, 155.19, 146.92, 144.97, 138.48, 136.12, 134.62, 132.54, 131.27,
127.82, 126.98, 124.87, 123.61, 123.10, 121.15, 116.57, 111.41, 110.96,
109.98, 105.80, 71.59, 60.40, 55.48, 54.14, 53.81, 48.98, 42.50, 39.12,
37.58, 32.38, 31.93, 31.52, 29.69, 29.44, 29.06, 26.81, 26.67, 22.80,
6

G. Yan, X. Zhong, L. Yue et al.
European Journal of Medicinal Chemistry 212 (2021) 113150
22.69, 22.12, 18.90, 15.36, 14.20, 14.12. HRMS (DART-TOF) calculated
CH2), 3.69e3.58 (m, 8H, CH2), 3.55 (t, J ¼ 5.0, 2H, CH2), 3.44 (td,
J ¼ 10.5, 5.2, 5H, CH2, CH), 3.26 (dt, J ¼ 13.7, 6.8, 1H, CH), 2.91e2.69
(m, 6H, CH2), 2.18e2.08 (m, 5H, CH2, Ar-CH3), 1.85 (d, J ¼ 12.5, 4H,
CH2), 1.33 (dd, J ¼ 10.9, 6.4, 12H, CH3).
for C50H63ClN9O8S [M þ H]þ m/z 984.4209, found 984.4210.
4.1.4. Synthesis of 3-(4-(4-((5-chloro-4-((2-(isopropylsulfonyl)
phenyl)amino) pyrimidin-2-yl)amino)-5-isopropoxy-2-
methylphenyl)piperidin-1-yl)-N-(8-((2-(2,6-dioxopiperidin-3-yl)-
1,3-dioxoisoindolin-4-yl)amino)octyl)propanamide (A3)
13C NMR (101 MHz, CDCl3)
d
¼ 172.26, 172.05, 169.54, 169.45,
167.62, 157.45, 155.35, 155.29, 146.73, 144.80, 138.46, 136.17, 134.68,
132.52, 131.28, 127.96, 127.18, 124.85, 123.59, 123.19, 120.88, 116.82,
111.73, 110.95, 110.30, 71.74, 70.70, 70.03, 69.83, 69.12, 60.40, 55.50,
53.82, 48.91, 42.20, 39.44, 31.38, 22.95, 22.21, 21.05, 18.92, 15.37,
14.20. HRMS (DART-TOF) calculated for C50H63ClN9O10S [M þ H]þ
m/z 1016.4107, found 1016.4116.
The title compound was obtained from tert-butyl (8-
aminooctyl)carbamate following similar synthesis procedure of
A1 (yellow powder, yield 41%). 1H NMR (400 MHz, CDCl3)
d
¼ 9.53
(s, 1H, CONHCO), 8.58 (d, J ¼ 8.3, 1H, AreH), 8.20 (s, 1H, Ar-NH), 7.95
(s, 1H, AreH), 7.92 (dd, J ¼ 8.0, 1.4, 1H, AreH), 7.76 (s, 1H, AreH),
7.63e7.55 (m, 1H, AreH), 7.51e7.44 (m, 1H, AreH), 7.27e7.21 (m,
2H, AreH, Ar-NH), 7.07 (d, J ¼ 7.1, 1H, AreH), 6.86 (d, J ¼ 8.6, 1H,
AreH), 6.73 (s, 1H, AreH), 4.91 (dd, J ¼ 12.2, 5.3, 1H, COCHN),
4.58e4.45 (m, 1H, OCH), 3.36e3.22 (m, 6H, N-CH2), 2.95e2.64 (m,
8H, CH2), 2.09 (s, 3H, Ar-CH3), 1.90e1.56 (m, 8H, CH2), 1.40e1.29
4.1.7. Synthesis of 3-(4-(4-((5-chloro-4-((2-(isopropylsulfonyl)
phenyl)amino) pyrimidin-2-yl)amino)-5-isopropoxy-2-
methylphenyl)piperidin-1-yl)-N-(2-(2-(2-(2-((2-(2,6-
dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)
ethoxy)ethoxy) ethyl)propenamide (B3)
The title compound was obtained from tert-butyl (2-(2-(2-(2-
aminoethoxy)ethoxy)ethoxy)ethyl)carbamate following similar
synthesis procedure of A1 (yellow powder, yield 45%). 1H NMR
(m, 20H, CH2, CH3). 13C NMR (101 MHz, CDCl3)
d
¼ 172.17, 171.49,
169.57, 169.01, 167.72, 157.47, 155.40, 155.09, 146.96, 145.07, 138.47,
137.17, 136.05, 134.63, 132.57, 131.25, 127.68, 126.83, 124.87, 123.64,
123.09, 121.38, 116.53, 111.29, 110.79, 109.95, 105.74, 71.43, 60.39,
55.48, 54.19, 53.77, 48.96, 42.40, 39.18, 37.76, 32.62, 32.29, 31.55,
29.70, 29.46, 29.19, 29.05, 27.01, 26.75, 22.77, 22.07, 22.03, 21.04,
18.92, 15.37, 15.35, 14.20. HRMS (DART-TOF) calculated for
(400 MHz, CDCl3)
d
¼ 9.51 (s, 1H, CONHCO), 8.57 (d, J ¼ 8.4, 1H,
AreH), 8.16 (s, 1H, Ar-NH), 7.99 (s, 1H, AreH), 7.92 (d, J ¼ 7.9, 1H,
AreH), 7.83 (s, 1H, Ar-NH), 7.67e7.54 (m, 2H, AreH), 7.46 (t, J ¼ 7.7,
1H, AreH), 7.27e7.20 (m, 1H, AreH), 7.06 (d, J ¼ 7.0, 1H, AreH), 6.89
(d, J ¼ 8.6, 1H, AreH), 6.77 (s, 1H, AreH), 6.46 (d, J ¼ 5.1, 1H, Ar-NH),
4.93 (dd, J ¼ 11.7, 5.7, 1H, COCHN), 4.56 (dt, J ¼ 12.0, 6.0, 1H, OCH),
3.78e3.52 (m, 12H, OCH2), 3.45 (d, J ¼ 4.7, 4H, NCH2), 3.25 (dd,
J ¼ 13.4, 6.7, 3H, NCH2, Ar-CH), 2.95e2.65 (m, 6H, CH2), 2.59e2.33
(m, 4H, CH2), 2.17e2.11 (m, 4H, Ar-CH3, SO2CH), 1.80 (d, J ¼ 21.0,
4H, CH2), 1.41e1.28 (m, 12H, CH3). 13C NMR (101 MHz, CDCl3)
C
52H67ClN9O8S [M þ H]þ m/z 1012.4522, found 1012.4512.
4.1.5. Synthesis of 3-(4-(4-((5-chloro-4-((2-(isopropylsulfonyl)
phenyl)amino) pyrimidin-2-yl)amino)-5-isopropoxy-2-
methylphenyl)piperidin-1-yl)-N-(2-(2-((2-(2,6-dioxopiperidin-3-
yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethyl)propenamide (B1)
The title compound was obtained from tert-butyl (2-(2-
aminoethoxy)ethyl)carbamate following similar synthesis proced-
ure of A1 (yellow powder, yield 46%).1H NMR (400 MHz, CDCl3)
d
¼ 172.74, 172.07, 171.16, 169.44, 169.41, 167.65, 157.46, 155.34,
155.27, 146.75, 144.83, 138.46, 136.14, 134.69, 132.45, 131.26, 127.85,
127.12, 124.81, 123.59, 123.17, 120.93, 116.89, 111.67, 110.96, 110.26,
105.79, 71.69, 70.43, 70.27, 70.07, 69.96, 69.36, 60.39, 55.50, 54.08,
53.79, 48.90, 42.22, 39.14, 37.22, 31.81, 31.39, 22.75, 22.19, 21.04,
d
¼ 9.49 (s, 1H, CONHCO), 8.57 (d, J ¼ 8.3, 1H, AreH), 8.15 (s, 1H, Ar-
NH), 7.97 (s, 1H, AreH), 7.92 (dd, J ¼ 7.9, 1.3, 1H, AreH), 7.67e7.58
(m, 1H, AreH), 7.54 (s, 1H, AreH), 7.53e7.47 (m, 1H, AreH), 7.24 (d,
J ¼ 7.8, 1H, AreH), 7.13 (d, J ¼ 7.1, 1H, AreH), 6.86 (d, J ¼ 8.5, 1H,
AreH), 6.77 (s, 1H, AreH), 4.89 (dd, J ¼ 11.9, 5.6, 1H, COCHN),
4.61e4.46 (m, 1H, OCH), 3.80e3.21 (m, 12H, OCH2, NCH2),
2.90e2.45 (m, 9H, CH2, Ar-CH), 2.24e2.10 (m, 5H, Ar-CH3, CH2),
18.91,15.36,14.20. HRMS (DART-TOF) calculated for C52H67ClN9O11
S
[M þ H]þ m/z 1060.4369, found 1060.4349.
4.1.8. Synthesis of 3-(4-(4-((5-chloro-4-((2-(isopropylsulfonyl)
phenyl)amino)pyrimidin-2-yl)amino)-5-isopropoxy-2-
methylphenyl)piperidin-1-yl)-N-(14-((2-(2,6-dioxopiperidin-3-yl)-
1,3-dioxoisoindolin-4-yl)amino)-3,6,9,12-tetraoxatetradecyl)
propenamide (B4)
The title compound was obtained from tert-butyl (14-amino-
3,6,9,12-tetraoxatetradecyl)carbamate following similar synthesis
procedure of A1 (yellow powder, yield 47%). 1H NMR (400 MHz,
1.37e1.30 (m, 12H, CH3). 13C NMR (101 MHz, CDCl3)
d
¼ 172.53,
171.83, 171.14, 170.11, 167.47, 157.51, 155.33, 146.75, 144.73, 138.48,
136.32, 134.67, 132.51, 131.24, 127.70, 127.15, 124.88, 123.71, 123.13,
120.76, 116.79, 111.92, 111.32, 110.49, 105.69, 71.81, 70.16, 68.31,
60.39, 55.46, 54.54, 53.88, 53.22, 48.91, 41.67, 39.17, 37.72, 32.56,
31.74, 31.48, 29.69, 23.65, 22.27, 22.26, 21.04, 18.93, 15.37, 14.20.
HRMS (DART-TOF) calculated for C48H59ClN9O9S [M þ H]þ m/z
972.3845, found 972.3887.
MeOD)
d
¼ 8.35 (d, J ¼ 8.5, 1H, AreH), 8.05 (d, J ¼ 1.5, 1H, AreH),
7.82 (d, J ¼ 8.0, 1H, AreH), 7.66 (t, J ¼ 6.0, 1H, AreH), 7.60e7.54 (m,
1H, AreH), 7.43 (ddd, J ¼ 9.6, 8.5, 6.0, 1H, AreH), 7.26 (t, J ¼ 7.7, 1H,
AreH), 7.03e6.88 (m, 2H, AreH), 6.74 (d, J ¼ 3.8, 1H, AreH),
6.20e6.08 (m, 1H, Ar-NH), 5.00e4.90 (m, 1H, COCHN), 4.56e4.43
(m, 1H, OCH), 3.66e3.25 (m, 20H, OCH2, N-CH2), 2.92e2.33 (m, 8H,
CH2), 2.09e1.99 (s, 3H, Ar-CH3), 1.27e1.14 (m, 12H, CH3). HRMS
(DART-TOF) calculated for C54H71ClN9O12S [M þ H]þ m/z 1104.4631,
found 1104.4629.
4.1.6. Synthesis of 3-(4-(4-((5-chloro-4-((2-(isopropylsulfonyl)
phenyl)amino) pyrimidin-2-yl)amino)-5-isopropoxy-2-
methylphenyl)piperidin-1-yl)-N-(2-(2-(2-((2-(2,6-dioxopiperidin-
3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethyl)
propenamide (B2)
The title compound was obtained from tert-butyl (2-(2-(2-
aminoethoxy)ethoxy)ethyl) carbamate following similar synthesis
procedure of A1 (yellow powder, yield 41%). 1H NMR (400 MHz,
4.1.9. Synthesis of 3-(4-(4-((5-chloro-4-((2-(isopropylsulfonyl)
phenyl)amino) pyrimidin-2-yl)amino)-5-isopropoxy-2-
methylphenyl)piperidin-1-yl)-N-(4-((2-((2-(2,6-dioxopiperidin-3-
yl)-1,3-dioxoisoindolin-4-yl)amino)-2-oxoethyl)amino)butyl)
propenamide (C1)
CDCl3)
d
¼ 9.51 (s, 1H, CONHCO), 8.57 (d, J ¼ 8.3, 1H, AreH), 8.15 (s,
1H, Ar-NH), 8.00 (s, 1H, AreH), 7.93 (dd, J ¼ 8.0, 1.4, 1H, AreH), 7.62
(dd, J ¼ 12.4, 3.2, 2H, AreH, Ar-NH), 7.52e7.45 (m, 1H, AreH),
7.27e7.21 (m, 1H, AreH), 7.08 (d, J ¼ 7.0, 1H, AreH), 6.90 (d, J ¼ 8.5,
1H, AreH), 6.82 (s, 1H, AreH), 6.52 (t, J ¼ 5.4, 1H, AreH), 4.98e4.90
(m, 1H, COCHN), 4.60 (dt, J ¼ 12.0, 6.0, 1H, OCH), 3.72 (t, J ¼ 5.2, 2H,
To a solution of Pomalidomide (546 mg, 2 mmol) in THF
(100 mL), Chloroacetyl chloride (452 mg, 4 mmol) was added. The
7

G. Yan, X. Zhong, L. Yue et al.
European Journal of Medicinal Chemistry 212 (2021) 113150
resulting reaction mixture was stirred at 66 ꢀC for 5 h and moni-
tored by TLC. Upon completion, EtOAc (200 mL) was added and the
mixture was washed with water (3 x 100 mL). The organic layer was
dried over anhydrous sodium sulfate, filtered and concentrated
under reduced pressure. The crude material was purified by column
chromatography, and the product was obtained as white solid
(yield 70%). Then the product (70 mg, 0.2 mmol) was dissolved in
THF (10 mL), to this solution tert-butyl (4-aminobutyl)carbamate
(113 mg, 0.6 mmol) and K2CO3 (83 mg, 0.6 mmol) was added and
stirred at 66 ꢀC for 5 h and monitored by TLC. Upon completion, the
reaction was then quenched with 30 mL H2O and extracted with
EtOAc. The organic layer was washed twice with water, dried over
anhydrous sodium sulfate, filtered and concentrated under reduced
pressure. The crude material was purified by column chromatog-
raphy, and the product was obtained as white solid (yield 69%).
Then the product of this step was dissolved in 5 mL trifluoroacetic
acid and stirred at room temperature for 5 h. Upon completion, the
reaction was then quenched with DCM (10 mL) and the solution
was removed by vacuum distillation to get C1-M (brown oil). C1-M
was applied in synthesis without further purification. Next, to a
solution of C1-M (92 mg, 0.2 mmol) in DMF (10 mL), K2(63 mg,
0.1 mmol), HATU (45.6 mg, 0.12 mmol) and DIPEA (38.7 mg,
0.3 mmol) were added. The resulting reaction mixture was stirred
at room temperature for 15 h and monitored by TLC. Upon
completion, the reaction was then quenched with water and
extracted with EtOAc. The organic layer was washed twice with
water, dried over anhydrous sodium sulfate, filtered and concen-
trated under reduced pressure. The crude material was purified by
column chromatography to afford target molecule as yellow pow-
m/z 1041.4423, found 1041.4438.
4.1.11. Synthesis of 3-(4-(4-((5-chloro-4-((2-(isopropylsulfonyl)
phenyl)amino)pyrimidin-2-yl)amino)-5-isopropoxy-2-
methylphenyl)piperidin-1-yl)-N-(8-((2-((2-(2,6-dioxopiperidin-3-
yl)-1,3-dioxoisoindolin-4-yl)amino)-2-oxoethyl)amino)octyl)
propenamide (C3)
The title compound was obtained from tert-butyl (8-
aminooctyl)carbamate following similar synthesis procedure of
C1 (yellow powder, yield 48%). 1H NMR (400 MHz, CDCl3)
d
¼ 9.52
(s, 1H, CONHCO), 8.58 (d, J ¼ 8.3, 1H, AreH), 8.17 (d, J ¼ 6.8, 1H,
AreH), 8.17 (d, J ¼ 6.8, 1H, AreH), 8.01 (d, J ¼ 9.4, 1H, AreH), 7.93 (d,
J ¼ 7.9, 1H, AreH), 7.71 (dt, J ¼ 12.6, 6.4, 1H, AreH), 7.66e7.50 (m,
3H, AreH, Ar-NH), 7.24 (d, J ¼ 7.7, 1H, AreH), 4.96 (dd, J ¼ 12.0, 5.1,
1H, COCHN), 4.52 (dd, J ¼ 12.0, 5.9, 1H, OCH), 3.57e3.38 (m, 2H,
CH2), 3.33e3.13 (m, 5H, CH2, NH), 2.95e2.40 (m, 10H, CH2), 2.15 (s,
3H, Ar-CH3), 1.90e1.70 (m, 4H, CH2), 1.32 (ddd, J ¼ 26.8, 14.8, 6.7,
24H, CH2, CH3). HRMS (DART-TOF) calculated for C54H70ClN10O9S
[M þ H]þ m/z 1069.4736, found 1069.4791.
4.1.12. Synthesis of 3-(4-(4-((5-chloro-4-((2-(isopropylsulfonyl)
phenyl)amino)pyrimidin-2-yl)amino)-5-isopropoxy-2-
methylphenyl)piperidin-1-yl)-N-(2-(2-((2-((2-(2,6-dioxopiperidin-
3-yl)-1,3-dioxoisoindolin-4-yl)amino)-2-oxoethyl)amino)ethoxy)
ethyl) propenamide (D1)
The title compound was obtained from tert-butyl (2-(2-
aminoethoxy)ethyl)carbamate following similar synthesis proced-
ure of C1 (yellow powder, yield 47%). 1H NMR (400 MHz, CDCl3)
d
¼ 11.18 (s, 1H, CONH), 9.51 (s, 1H, CONHCO), 8.87 (d, J ¼ 8.5, 1H,
der (yield 46%). 1H NMR (400 MHz, CDCl3)
d
¼ 9.51 (s,1H, CONHCO),
AreH), 8.58 (d, J ¼ 8.3,1H, AreH), 8.14 (d, J ¼ 11.3,1H, AreH), 7.99 (s,
1H, Ar-NH), 7.92 (dd, J ¼ 7.9, 1.3, 1H, AreH), 7.75e7.66 (m, 1H,
AreH), 7.65e7.58 (m, 1H, AreH), 7.58e7.50 (m, 2H, AreH, Ar-NH),
7.25 (d, J ¼ 7.6, 1H, AreH), 6.77 (s, 1H, AreH), 4.96 (dd, J ¼ 12.1,
5.4, 1H, COCHN), 4.60e4.48 (m,1H, OCH), 3.78e3.53 (m, 4H, OCH2),
3.53e3.38 (m, 4H, NCH2), 3.34e3.07 (m, 2H, CH2), 2.96e2.61 (m,
8H, CH2), 2.47 (t, J ¼ 6.4, 2H, CH2), 2.15 (s, 3H, Ar-CH3), 1.88e1.61
(m, 4H, CH2), 1.38e1.30 (m, 12H, CH3). 13C NMR (101 MHz, CDCl3)
8.89 (d, J ¼ 8.4, 1H, AreH), 8.57 (d, J ¼ 8.3, 1H, AreH), 8.16 (s, 1H, Ar-
NH), 8.01 (d, J ¼ 4.5, 1H, AreH), 7.93 (d, J ¼ 7.8, 1H, AreH), 7.75e7.65
(m, 1H, AreH), 7.65e7.59 (m, 1H, AreH), 7.55 (d, J ¼ 6.7, 1H, AreH),
7.25 (d, J ¼ 9.3, 3H, AreH, Ar-NH), 6.80e6.72 (m, 1H, AreH),
5.06e4.86 (m, 1H, COCHN), 4.55 (dd, J ¼ 12.0, 5.9, 1H, OCH), 3.30
(ddd, J ¼ 20.4, 15.4, 7.6, 6H, NCH2), 3.00e2.61 (m, 7H, CH2, NH),
2.21e2.13 (s, 3H, Ar-CH3), 1.97e1.49 (m, 4H, CH2), 1.38e1.29 (m,
12H, CH3). HRMS (DART-TOF) calculated for C50H62ClN10O9S [M þ
H]þ m/z 1013.4110, found 1013.4082.
d
¼ 172.72, 171.80, 171.45, 168.55, 168.50, 166.89, 157.47, 155.34,
144.76, 138.49, 137.02, 136.23, 134.65, 131.47, 131.27, 127.85, 127.25,
125.14, 124.87, 123.62, 123.12, 120.87, 118.53, 116.03, 111.18, 105.83,
71.83, 70.23, 69.83, 60.40, 55.47, 54.12, 53.99, 53.83, 53.30, 49.57,
49.28, 39.02, 37.83, 32.45, 31.42, 29.69, 22.83, 22.69, 22.26, 21.04,
18.92, 15.37, 14.20, 14.12. HRMS (DART-TOF) calculated for
4.1.10. Synthesis of 3-(4-(4-((5-chloro-4-((2-(isopropylsulfonyl)
phenyl)amino) pyrimidin-2-yl)amino)-5-isopropoxy-2-
methylphenyl)piperidin-1-yl)-N-(6-((2-((2-(2,6-dioxopiperidin-3-
yl)-1,3-dioxoisoindolin-4-yl)amino)-2-oxoethyl)amino)hexyl)
propenamide (C2)
C
50H60ClN10O10S [M þ H]þ m/z 1029.4060, found 1029.4071.
The title compound was obtained from tert-butyl (6-
aminohexyl)carbamate following similar synthesis procedure of
4.1.13. Synthesis of 3-(4-(4-((5-chloro-4-((2-(isopropylsulfonyl)
phenyl)amino) pyrimidin-2-yl)amino)-5-isopropoxy-2-
methylphenyl)piperidin-1-yl)-N-(2-(2-(2-((2-((2-(2,6-
dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-2-oxoethyl)
amino) ethoxy)ethoxy)ethyl)propenamide (D2)
The title compound was obtained from tert-butyl (2-(2-(2-
aminoethoxy)ethoxy)ethyl)carbamate following similar synthesis
procedure of C1 (yellow powder, yield 51%). 1H NMR (400 MHz,
C1 (yellow powder, yield 45%). 1H NMR (400 MHz, CDCl3)
d
¼ 11.12
(s, 1H, CONHAr), 9.44 (s, 1H, CONHCO), 8.83 (d, J ¼ 8.4, 1H, AreH),
8.50 (d, J ¼ 8.2, 1H, AreH), 8.09 (s, 1H, Ar-NH), 7.92 (s, 1H, AreH),
7.86 (d, J ¼ 7.8, 1H, AreH), 7.63 (t, J ¼ 7.7, 1H, AreH), 7.58e7.44 (m,
3H, AreH, Ar-NH), 7.18 (d, J ¼ 11.8, 2H, AreH), 6.70 (s, 1H, AreH),
4.90 (dd, J ¼ 11.6, 5.1, 1H, COCHN), 4.47 (dt, J ¼ 11.7, 5.8, 1H, OCH),
3.35 (q, J ¼ 17.6, 2H, CH2), 3.27e3.06 (m, 5H, CH2, CH), 2.95e2.51
(m, 10H, CH2), 2.19 (s, 3H, Ar-CH3), 1.47 (ddd, J ¼ 22.2, 16.0, 7.8, 6H,
CH2), 1.26 (dd, J ¼ 13.5, 6.4, 12H, CH3). 13C NMR (101 MHz, CDCl3)
CDCl3)
d
¼ 11.21 (s,1H, CONH), 9.50 (s,1H, CONHCO), 8.88 (d, J ¼ 8.5,
1H, AreH), 8.57 (d, J ¼ 8.4,1H, AreH), 8.14 (s, 1H, Ar-NH), 7.97 (s,1H,
AreH), 7.92 (d, J ¼ 7.5, 2H, AreH), 7.75e7.66 (m, 1H, AreH),
7.66e7.57 (m, 2H, AreH, Ar-NH), 7.55 (d, J ¼ 7.2, 1H, AreH), 7.24 (d,
J ¼ 7.6, 1H, AreH), 6.78 (s, 1H, AreH), 4.96 (dd, J ¼ 12.1, 5.3, 1H,
COCHN), 4.54 (dt, J ¼ 12.1, 6.0, 1H, OCH), 3.77e3.38 (m, 14H, OCH2,
NCH2), 3.26 (dt, J ¼ 13.7, 6.8, 1H, CH), 3.13 (d, J ¼ 11.2, 2H, CH2),
2.97e2.61 (m, 4H, CH2), 2.45 (t, J ¼ 6.5, 2H, CH2), 2.15 (d, J ¼ 6.4, 7H,
CH2, Ar-CH3), 1.75 (d, J ¼ 11.4, 6H, CH2), 1.38e1.29 (m, 12H, CH3).
d
¼ 172.01, 171.73, 168.63, 168.33, 166.98, 157.45, 155.29, 144.78,
138.48, 137.08, 136.13, 134.63, 131.52, 131.28, 127.88, 127.11, 125.16,
124.94, 123.63, 123.13, 120.87, 118.43, 116.02, 111.01, 105.86, 100.00,
71.74, 60.40, 55.47, 54.10, 53.81, 53.53, 50.44, 49.27, 39.24, 37.59,
32.42, 31.57, 29.96, 29.54, 26.96, 26.77, 22.74, 22.22, 18.92, 15.37,
14.20. HRMS (DART-TOF) calculated for C52H66ClN10O9S [M þ H]þ
8

G. Yan, X. Zhong, L. Yue et al.
European Journal of Medicinal Chemistry 212 (2021) 113150
13C NMR (101 MHz, CDCl3)
d
¼ 172.51, 171.91, 171.40, 168.49, 168.45,
4.1.17. Synthesis of 3-(4-(4-((5-chloro-4-((2-(isopropylsulfonyl)
phenyl)amino) pyrimidin-2-yl)amino)-5-isopropoxy-2-
methylphenyl)piperidin-1-yl)-N-(2-(2-((3-((2-(2,6-dioxopiperidin-
3-yl)-1,3-dioxoisoindolin-4-yl)amino)-3-oxopropyl)amino)ethoxy)
ethyl)propenamide (F1)
The title compound was obtained from tert-butyl (2-(2-
aminoethoxy)ethyl)carbamate following similar synthesis proced-
ure of E1 (yellow powder, yield 39%). 1H NMR (400 MHz, CDCl3)
166.92, 157.47, 155.34, 155.26, 144.80, 138.48, 137.41, 137.08, 136.19,
134.66, 131.45, 131.26, 127.70, 127.09, 125.15, 124.86, 123.63, 123.11,
120.88, 118.45, 116.01, 111.12, 105.76, 71.67, 70.55, 70.23, 70.06,
69.97, 55.47, 54.20, 53.91, 53.18, 49.40, 49.30, 38.97, 37.95, 32.85,
32.52, 31.52, 29.69, 22.71, 22.22, 18.93, 15.36, 14.20. HRMS (DART-
TOF) calculated for C52H66ClN10O11S [M þ H]þ m/z 1073.4322, found
1073.4313.
d
¼ 9.52 (s, 1H, CONHCO), 8.79 (d, J ¼ 8.2, 1H, AreH), 8.58 (d, J ¼ 8.0,
1H, AreH), 8.16 (s, 1H, Ar-NH), 8.05e7.97 (m, 1H, AreH), 7.93 (dd,
J ¼ 8.0, 1.5, 1H, AreH), 7.70e7.64 (m, 1H, AreH), 7.64e7.58 (m, 1H,
AreH), 7.57 (s, 1H, Ar-NH), 7.53 (d, J ¼ 6.8, 1H, AreH), 7.26e7.23 (m,
1H, AreH), 6.77 (s, 1H, AreH), 4.94 (dd, J ¼ 12.4, 5.4, 1H, COCHN),
4.54 (dt, J ¼ 12.1, 6.1, 1H, OCH), 3.71e3.54 (m, 4H, OCH2), 3.49e3.43
(m, 2H, CH2), 3.29e3.18 (m, 2H, CH2), 3.04 (t, J ¼ 5.9, 2H, CH2), 2.78
(dddd, J ¼ 21.3, 18.4, 10.7, 5.0, 11H, CH2, CH), 2.53 (t, J ¼ 6.2, 2H,
CH2), 2.14 (s, 3H, Ar-CH3), 1.33 (dd, J ¼ 11.8, 6.4, 12H, CH3). 13C NMR
4.1.14. Synthesis of 14-(3-(4-(4-((5-chloro-4-((2-(isopropylsulfonyl)
phenyl) amino)pyrimidin-2-yl)amino)-5-isopropoxy-2-
methylphenyl)piperidin-1-yl)propanamido)-N-(2-(2,6-
dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)-6,9,12-trioxa-3-
azatetradecanamide (D3)
The title compound was obtained from tert-butyl (2-(2-(2-(2-
aminoethoxy)ethoxy)ethoxy)ethyl)carbamate following similar
synthesis procedure of C1 (yellow powder, yield 49%). 1H NMR
(101 MHz, CDCl3)
d
¼ 172.27, 171.65, 171.41, 168.53, 168.53, 168.26,
(400 MHz, CDCl3)
d
¼ 9.51 (s, 1H, CONHCO), 8.77 (d, J ¼ 7.7, 1H,
166.84, 157.45, 155.35,144.80, 138.49, 137.33,136.04, 134.64, 134.64,
131.48, 131.28, 127.94, 127.94, 127.12, 126.18, 124.89, 123.60, 123.13,
120.85, 118.47, 116.18, 111.07, 105.88, 71.79, 69.74, 55.48, 53.86,
49.25, 48.77, 44.77, 39.07, 37.43, 31.93, 31.45, 29.70, 29.36, 22.69,
22.24, 18.93, 15.37, 14.12. HRMS (DART-TOF) calculated for
AreH), 8.56 (d, J ¼ 8.2, 1H, AreH), 8.15 (s, 1H, Ar-NH), 7.99 (s, 1H,
AreH), 7.92 (d, J ¼ 8.1, 1H, AreH), 7.68 (d, J ¼ 7.3, 1H, AreH),
7.65e7.59 (m, 1H, AreH), 7.55 (d, J ¼ 11.0, 1H, AreH), 7.52 (s, 1H, Ar-
NH), 7.29e7.21 (m, 4H, AreH, Ar-NH), 6.74 (s, 1H, AreH), 4.63e4.51
(m, 1H, OCH), 3.64 (m, 12H, OCH2), 3.39 (m, 4H, NCH2), 2.84 (m, 6H,
CH2), 2.57 (m, 4H, CH2), 2.13 (m, 5H, CH2, Ar-CH3),1.32 (dd, J ¼ 11.0,
C
51H64ClN10O10S [M þ H]þ m/z 1043.4216, found 1043.4178.
6.4, 12H, CH3). HRMS (DART-TOF) calculated for C54H70ClN10O12
S
[M þ H]þ m/z 1117.4584, found 1117.4648.
4.1.18. Synthesis of 3-(4-(4-((5-chloro-4-((2-(isopropylsulfonyl)
phenyl)amino) pyrimidin-2-yl)amino)-5-isopropoxy-2-
methylphenyl)piperidin-1-yl)-N-(2-(2-(2-((3-((2-(2,6-
dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-3-oxopropyl)
amino) ethoxy)ethoxy)ethyl)propenamide (F2)
The title compound was obtained from tert-butyl (2-(2-(2-
aminoethoxy)ethoxy)ethyl)carbamate following similar synthesis
procedure of E1 (yellow powder, yield 31%). 1H NMR (400 MHz,
4.1.15. Synthesis of 3-(4-(4-((5-chloro-4-((2-(isopropylsulfonyl)
phenyl)amino) pyrimidin-2-yl)amino)-5-isopropoxy-2-
methylphenyl)piperidin-1-yl)-N-(4-((3-((2-(2,6-dioxopiperidin-3-
yl)-1,3-dioxoisoindolin-4-yl)amino)-3-oxopropyl)amino)butyl)
propenamide (E1)
The title compound was obtained from Acryloyl chloride and
tert-butyl (4-aminobutyl)carbamate following similar synthesis
procedure of C1 (yellow powder, yield 46%). 1H NMR (400 MHz,
CDCl3)
d
¼ 9.51 (s, 1H, CONHCO), 8.82 (d, J ¼ 8.0, 1H, AreH), 8.58 (d,
J ¼ 8.3,1H, AreH), 8.17e8.13 (m,1H, AreH), 8.00 (dd, J ¼ 8.6, 4.2,1H,
AreH), 7.93 (dd, J ¼ 8.0, 1.5, 1H, AreH), 7.68 (dd, J ¼ 8.5, 7.3, 1H,
AreH), 7.65e7.58 (m, 2H, AreH, Ar-NH), 7.53 (d, J ¼ 6.7, 1H, AreH),
7.26e7.22 (m, 1H, AreH), 6.78 (d, J ¼ 4.4, 1H, AreH), 5.01e4.88 (m,
1H, COCHN), 4.55 (dt, J ¼ 12.2, 6.0, 1H, OCH), 3.79e3.36 (m, 12H,
OCH2, NCH2), 3.04e2.98 (m, 1H, CH), 2.92e2.83 (m, 2H, CH2),
2.82e2.69 (m, 4H, CH2), 2.62e2.55 (m, 1H, CH), 2.46 (t, J ¼ 6.5, 2H,
CH), 2.25e2.11 (s, 3H, Ar-CH3), 1.76 (d, J ¼ 17.4, 4H, CH2), 1.39e1.31
CDCl3)
d
¼ 9.51 (s, 1H, CONHCO), 8.81e8.65 (m, 1H, AreH), 8.56 (t,
J ¼ 9.4, 1H, AreH), 8.20e8.10 (m, 1H, AreH), 8.08e7.98 (m, 1H,
AreH), 7.93 (d, J ¼ 7.5, 1H, AreH), 7.71 (d, J ¼ 5.2, 1H, AreH),
7.67e7.48 (m, 3H, AreH, Ar-NH), 7.24 (s,1H, AreH), 6.80 (d, J ¼ 10.0,
1H, AreH), 4.96 (s, 1H, COCHN), 4.57 (s, 1H, OCH), 3.77 (dd, J ¼ 22.6,
16.6, 2H, CH2), 3.60e3.12 (m, 6H, CH2), 2.81 (dd, J ¼ 91.0, 52.5, 8H,
CH2), 2.14 (s, 3H, Ar-CH3), 2.09e1.52 (m, 8H, CH2), 1.40e1.28 (m,
12H, CH3). HRMS (DART-TOF) calculated for C51H64ClN10O9S [M þ
H]þ m/z 1027.4267, found 1027.4261.
(m, 12H, CH3). 13C NMR (101 MHz, CDCl3)
d
¼ 172.05, 171.32, 168.47,
168.21, 166.85, 157.49, 155.35, 144.80, 138.51, 137.49, 137.31, 136.04,
134.64, 131.28, 127.73, 127.16, 127.07, 126.27, 124.88, 123.64, 123.10,
118.39, 116.15, 111.12, 71.68, 70.20, 70.13, 70.00, 55.47, 53.99, 48.74,
44.83, 38.95, 37.93, 37.30, 32.88, 32.47, 31.93, 29.70, 29.36, 22.69,
22.23, 18.94, 15.37. HRMS (DART-TOF) calculated for
4.1.16. Synthesis of 3-(4-(4-((5-chloro-4-((2-(isopropylsulfonyl)
phenyl)amino) pyrimidin-2-yl)amino)-5-isopropoxy-2-
methylphenyl)piperidin-1-yl)-N-(6-((3-((2-(2,6-dioxopiperidin-3-
yl)-1,3-dioxoisoindolin-4-yl)amino)-3-oxopropyl)amino)hexyl)
propenamide (E2)
C
53H68ClN10O11S [M þ H]þ m/z 1087.4478, found 1087.4503.
The title compound was obtained from tert-butyl (6-
aminohexyl)carbamate following similar synthesis procedure of
4.1.19. Synthesis of 3-(4-(4-((5-chloro-4-((2-(isopropylsulfonyl)
phenyl)amino) pyrimidin-2-yl)amino)-5-isopropoxy-2-
methylphenyl)piperidin-1-yl)-N-(4-(2-((2-(2,6-dioxopiperidin-3-
yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)butyl)propanamide
(G1)
E1 (yellow powder, yield 31%). 1H NMR (400 MHz, CDCl3)
d
¼ 9.52
(s, 1H, CONHCO), 8.57 (t, J ¼ 8.6, 1H, AreH), 8.16 (d, J ¼ 1.8, 1H,
AreH), 8.05 (d, J ¼ 13.6, 1H, AreH), 7.93 (dd, J ¼ 8.0, 1.5, 1H, AreH),
7.62 (t, J ¼ 7.9, 1H, AreH), 7.56 (d, J ¼ 4.2, 1H, AreH), 7.36e7.25 (m,
4H, AreH, Ar-NH), 6.74 (d, J ¼ 8.0, 1H, AreH), 4.60 (td, J ¼ 12.5, 6.3,
1H, OCH), 4.05e3.95 (m, 1H, CH), 3.77 (d, J ¼ 13.4, 1H, CH),
3.34e2.89 (m, 6H, CH2), 2.84e2.67 (m, 1H, CH), 2.20 (s, 3H, Ar-
CH3), 1.94e1.49 (m, 8H, CH2), 1.40e1.21 (m, 16H, CH2, CH3).
HRMS (DART-TOF) calculated for C53H68ClN10O9S [M þ H]þ m/z
1055.4580, found 1055.4595.
To
a
solution of 2-(2,6-dioxopiperidin-3-yl)-4-hydroxyi
soindoline-1,3-dione (411 mg, 0.15 mmol) in DMF (100 mL), tert-
Butyl bromoacetate (293 mg, 0.15 mmol) and K2CO3 (621 mg,
4.5 mmol) were added. The resulting reaction mixture was stirred at
room temperature for 15 h and monitored by TLC. Upon completion,
thereactionmixturewasthen quenched withH2O and extracted with
EtOAc, then the organic layer was washed with water (100 mL) for
9

G. Yan, X. Zhong, L. Yue et al.
European Journal of Medicinal Chemistry 212 (2021) 113150
three times. The organic layer was dried over anhydrous sodium
sulfate, filtered and concentrated under reduced pressure. The crude
material was purified by column chromatography, and the product
was obtained as white powder (yield 70%). The product (78 mg,
0.2 mmol) was dissolved in trifluoroacetic acid (5 mL) and stirred at
room temperature for 5 h. Upon completion, the reaction was then
quenched with 10 mL DCM and the solution was removed by vacuum
distillation to get white solid. The solid was dissolved in DMF (10 mL),
to this solution tert-butyl (4-aminobutyl)carbamate (54 mg,
0.3 mmol), HATU (91 mg, 0.24 mmol) and DIPEA (38.7 mg, 0.3 mmol)
were added and stirred at room temperature for 10 h and monitored
by TLC. Upon completion, the reactionwas then quenched with water
and extracted with EtOAc. The organic layer was washed twice with
water, driedoveranhydrous sodiumsulfate, filteredandconcentrated
under reduced pressure. The crude material was purified by column
chromatography, and the product was obtained as yellow powder
(yield 56%). Then the product of this step was dissolved in trifluoro-
acetic acid (5 mL) and stirred at room temperature for 5 h. Upon
completion, the reaction was then quenched with DCM (10 mL) and
the solution was removed by vacuum distillation to get G1-M (brown
oil). G1-M wasapplied insynthesis without further purification. Next,
to a solution of G1-M (100 mg, 0.2 mmol) in DMF (10 mL), K2 (63 mg,
0.1mmol),HATU(45.6mg,0.12mmol)andDIPEA(38.7mg,0.3mmol)
were added. The resulting reaction mixture was stirred at room
temperature for 15 h and monitored by TLC. Upon completion, the
reaction was then quenched with water and extracted with EtOAc.
The organiclayerwaswashed twicewith water, dried overanhydrous
sodium sulfate, filtered and concentrated under reduced pressure.
The crude material was purified by column chromatography to afford
target molecule as yellow powder (yield 39%). 1H NMR (400 MHz,
124.86, 123.61, 123.12, 120.95, 119.65, 118.21, 117.35, 110.97, 105.82,
71.73, 68.10, 60.38, 55.48, 54.20, 53.82, 49.36, 39.11, 38.97, 37.67,
32.40, 32.15, 31.52, 29.54, 29.05, 26.69, 26.39, 22.71, 22.23, 21.04,
18.91,15.36,14.20. HRMS (DART-TOF) calculated for C52H65ClN9O10
S
[M þ H]þ m/z 1042.4264, found 1042.4293.
4.1.21. Synthesis of 3-(4-(4-((5-chloro-4-((2-(isopropylsulfonyl)
phenyl)amino) pyrimidin-2-yl)amino)-5-isopropoxy-2-
methylphenyl)piperidin-1-yl)-N-(8-(2-((2-(2,6-dioxopiperidin-3-
yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)octyl)propenamide
(G3)
The title compound was obtained from tert-butyl (8-
aminooctyl)carbamate following similar synthesis procedure of
G1 (yellow powder, yield 21%). 1H NMR (400 MHz, CDCl3)
d
¼ 9.52
(s, 1H, CONHCO), 8.58 (d, J ¼ 8.2, 1H, AreH), 8.16 (s, 1H, Ar-NH), 7.99
(s, 1H, AreH), 7.93 (dd, J ¼ 8.0, 1.5, 1H, AreH), 7.73 (dd, J ¼ 10.1, 5.7,
1H, AreH), 7.62 (dd, J ¼ 10.4, 3.2, 2H, AreH, Ar-NH), 7.55 (t, J ¼ 6.0,
1H, AreH), 7.39 (t, J ¼ 5.6, 1H, AreH), 7.26e7.23 (m, 1H, AreH), 7.19
(d, J ¼ 8.4, 1H, AreH), 6.75 (s, 1H, AreH), 4.96 (dd, J ¼ 12.3, 5.3, 1H,
COCHN), 4.63 (t, J ¼ 7.8, 2H, CONH2), 4.59e4.45 (m, 1H, OCH), 3.29
(dddd, J ¼ 27.1, 23.9, 16.5, 8.6, 6H, NCH2), 2.98e2.67 (m, 6H, CH2),
2.49 (d, J ¼ 5.7, 2H, CH2), 2.23 (d, J ¼ 21.1, 2H, CH2), 2.15 (s, 3H, Ar-
CH3),1.89e1.69 (m, 4H, CH2),1.62e1.49 (m, 4H, CH2),1.44e1.29 (m,
18H, CH3, CH2). 13C NMR (101 MHz, CDCl3)
d
¼ 171.39, 168.35,
166.68, 166.61, 166.01, 157.46, 155.37, 155.27, 154.49, 144.86, 138.48,
137.03, 134.63, 133.61, 131.28, 124.91, 123.63, 123.13, 120.94, 119.49,
118.13,117.33,110.93,105.87, 71.71, 68.00, 55.48, 53.85, 49.39, 39.09,
31.58, 29.70, 29.35, 29.12, 29.01, 26.78, 26.62, 22.59, 22.21, 18.93,
15.37. HRMS (DART-TOF) calculated for C54H69ClN9O10S [M þ H]þ
m/z 1070.4577, found 1070.4604.
CDCl3)
d
¼ 9.51 (s,1H, CONHCO), 8.58 (d, J ¼ 8.0,1H, AreH), 8.16 (s,1H,
Ar-NH), 8.00 (s, 1H, AreH), 7.93 (dd, J ¼ 8.0, 1.5, 1H, AreH), 7.73 (dd,
J ¼ 8.3, 7.4, 1H, AreH), 7.66e7.59 (m, 2H, AreH, Ar-NH), 7.55 (s, 2H,
AreH), 7.27e7.23 (m,1H, AreH), 7.19 (d, J ¼ 8.3,1H, AreH), 6.77 (s,1H,
AreH), 5.05e4.93 (m, 1H, COCHN), 4.60 (dt, J ¼ 12.2, 10.2, 3H, OCH,
COCH2), 2.98e2.64 (m, 6H, CH2), 2.50 (d, J ¼ 6.2, 2H, CH2), 2.14 (s, 3H,
Ar-CH3),1.78 (d, J ¼ 18.6, 4H, CH2),1.70e1.53 (m, 4H, CH2),1.42e1.29
4.1.22. Synthesis of 3-(4-(4-((5-chloro-4-((2-(isopropylsulfonyl)
phenyl)amino)pyrimidin-2-yl)amino)-5-isopropoxy-2-
methylphenyl)piperidin-1-yl)-N-(2-(2-(2-((2-(2,6-dioxopiperidin-
3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)ethoxy)ethyl)
propenamide (H1)
The title compound was obtained from tert-butyl (2-(2-
aminoethoxy)ethyl)carbamate following similar synthesis proced-
ure of G1 (yellow powder, yield 23%). 1H NMR (400 MHz, CDCl3)
(m, 12H, CH3). 13C NMR (101 MHz, CDCl3)
d
¼ 171.49, 168.82, 166.81,
166.59, 166.45, 157.48, 155.34, 154.76, 144.74, 138.50, 137.12, 134.65,
133.51, 131.27, 127.85, 127.10, 124.89, 123.63, 123.12, 120.79, 120.33,
118.41, 117.63, 111.03, 105.83, 71.80, 68.66, 60.39, 55.48, 53.92, 53.78,
49.44, 38.84, 38.75, 32.14, 31.53, 26.83, 26.62, 22.79, 22.24, 21.04,
d
¼ 9.51 (s, 1H, CONHCO), 8.58 (d, J ¼ 8.1, 1H, AreH), 8.15 (d, J ¼ 4.4,
1H, AreH), 7.99 (s, 1H, AreH), 7.92 (dd, J ¼ 8.0, 1.5, 1H, AreH),
7.76e7.71 (m, 1H, AreH), 7.67 (d, J ¼ 4.9, 1H, AreH), 7.64e7.59 (m,
1H, AreH), 7.58e7.50 (m, 3H, AreH, Ar-NH, CONH), 7.25 (dd, J ¼ 9.5,
2.3,1H, AreH), 7.22e7.16 (m, 1H, AreH), 5.05e4.92 (m, 1H, COCHN),
4.63 (s, 2H, COCH2O), 4.54 (dt, J ¼ 12.1, 6.0, 1H, OCH), 3.68e3.42 (m,
9H, OCH2, NCH2), 3.33e3.13 (m, 4H, CH2), 2.91e2.62 (m, 8H, CH2),
2.14 (s, 3H, Ar-CH3), 1.37e1.29 (m, 16H, CH2, CH3). 13C NMR
18.92, 15.37, 14.20. HRMS (DART-TOF) calculated for C50H61ClN9O10
S
[M þ H]þ m/z 1014.3951, found 1014.3975.
4.1.20. Synthesis of 3-(4-(4-((5-chloro-4-((2-(isopropylsulfonyl)
phenyl)amino) pyrimidin-2-yl)amino)-5-isopropoxy-2-
methylphenyl)piperidin-1-yl)-N-(6-(2-((2-(2,6-dioxopiperidin-3-
yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)hexyl)propenamide
(G2)
(101 MHz, CDCl3)
d
¼ 171.67, 171.12, 168.96, 166.71, 166.67, 166.15,
157.48, 155.32, 154.28, 144.77, 138.49, 137.02, 134.65, 133.66, 131.25,
127.78, 127.08, 124.86, 123.63, 123.11, 120.82, 119.16, 117.97, 117.25,
111.07, 105.78, 71.75, 69.58, 69.37, 67.64, 60.38, 55.47, 54.09, 53.95,
53.84, 49.39, 39.37, 38.83, 37.73, 32.72, 32.05, 31.52, 29.68, 22.91,
22.24, 21.04, 18.92, 15.37, 14.20. HRMS (DART-TOF) calculated for
The title compound was obtained from tert-butyl (6-
aminohexyl)carbamate following similar synthesis procedure of
G1 (yellow powder, yield 28%). 1H NMR (400 MHz, CDCl3)
d
¼ 9.52
(s, 1H, CONHCO), 8.58 (d, J ¼ 8.3, 1H, AreH), 8.16 (s, 1H, Ar-NH), 7.99
(s, 1H, AreH), 7.92 (dd, J ¼ 8.0, 1.4, 1H, AreH), 7.77e7.69 (m, 1H,
AreH), 7.66e7.59 (m, 1H, AreH), 7.58 (s, 1H, Ar-NH), 7.56e7.52 (m,
1H, AreH), 7.49 (t, J ¼ 5.4, 1H, AreH), 7.25 (d, J ¼ 8.0, 1H, AreH), 7.19
(d, J ¼ 8.4,1H, AreH), 6.75 (s, 1H, AreH), 5.07e4.92 (m, 1H, COCHN),
4.67e4.59 (m, 2H, COCH2O), 4.60e4.47 (m, 1H, OCH), 3.51e3.11 (m,
7H, NCH2, CH), 2.97e2.67 (m, 6H, CH2), 2.48 (t, J ¼ 6.0, 2H, CH2),
2.28 (dd, J ¼ 22.1, 11.6, 2H, CH2), 2.18 (d, J ¼ 21.2, 4H, Ar-CH, NH),
1.90e1.67 (m, 4H, CH2), 1.55 (ddd, J ¼ 26.9, 13.5, 6.7, 4H, CH2), 1.34
C
50H61ClN9O11S [M þ H]þ m/z 1030.3900, found 1030.3875.
4.1.23. Synthesis of 3-(4-(4-((5-chloro-4-((2-(isopropylsulfonyl)
phenyl)amino)pyrimidin-2-yl)amino)-5-isopropoxy-2-
methylphenyl)piperidin-1-yl)-N-(2-(2-(2-(2-((2-(2,6-
dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)
ethoxy)ethoxy)ethyl) propenamide (H2)
The title compound was obtained from tert-butyl (2-(2-(2-
aminoethoxy)ethoxy)ethyl)carbamate following similar synthesis
procedure of G1 (yellow powder, yield 26%). 1H NMR (400 MHz,
(dd, J ¼ 15.3, 6.5, 12H, CH3). 13C NMR (101 MHz, CDCl3)
d
¼ 172.42,
171.67, 171.13, 168.61, 166.71, 166.67, 166.10, 157.47, 155.34, 155.29,
154.50, 144.80, 138.48, 137.01, 134.64, 133.59, 131.26, 127.84, 127.15,
CDCl3)
d
¼ 9.51 (s, 1H, CONHCO), 8.58 (d, J ¼ 8.0, 1H, AreH),
8.21e8.09 (m, 1H, AreH), 7.98 (s, 1H, AreH), 7.92 (dd, J ¼ 8.0, 1.5, 1H,
10

G. Yan, X. Zhong, L. Yue et al.
European Journal of Medicinal Chemistry 212 (2021) 113150
AreH), 7.73 (dd, J ¼ 8.3, 7.5, 1H, AreH), 7.65e7.59 (m, 2H, Ar-NH),
7.58 (s, 1H, AreH), 7.53 (d, J ¼ 7.2, 1H, AreH), 7.27 (d, J ¼ 9.8, 1H,
AreH), 7.19 (d, J ¼ 8.4, 1H, AreH), 6.79 (s, 1H, AreH), 5.04e4.93 (m,
1H, COCHN), 4.66 (d, J ¼ 14.3, 2H, COCH2O), 4.60e4.51 (m, 1H,
OCH), 3.73e3.52 (m, 10H, OCH2, NCH2), 3.45 (dd, J ¼ 10.7, 5.3, 2H,
NCH2), 3.27 (dq, J ¼ 13.7, 6.8, 1H, CH), 3.16 (d, J ¼ 10.6, 2H, CH2),
2.92e2.63 (m, 6H, CH2), 2.18e2.12 (m, 4H, Ar-CH3, CH), 1.38e1.28
4.4. Western blot assay
H3122 cells were treated with B3 at different concentrations at
37 ꢀC, then the cells were washed with PBS before being lysed with
RIPA buffer, protease inhibitors, phosphatase cocktails A and B, and
PMSF (1 mM). Protein in lysate was determined by the BCA Protein
Assay Kit (Beyotime#p0012s). The samples were subjected to
SDSꢂPAGE and then transferred onto PVDF membranes (Millpore).
The membranes were incubated overnight at 4 ꢀC with the primary
antibody in 5% BSA/TBST buffer with gentle shaking, then washed
with 1 ꢃ TBS/T 3 times, followed by incubation for 1 h with a 1/
5000 dilution of secondary HRP antibody in 5% nonfat milk/TBST.
Primary antibodies to anti ALK (no. 3633T), pALK (no. 3341s), IGF1R
(no. 9750T), INSR (no.23413T) and GAPDH (no. 2118s) were from
Cell Signaling Technology, primary antibodies to STAT3 (no. 10253-
2-ap) were from Proteintech Group and primary antibodies to p-
STAT3 (Af3293) were from Affinity. The target blots were detected
with chemiluminescence system.
(m, 16H, CH3). 13C NMR (101 MHz, CDCl3)
d
¼ 171.63, 171.12, 168.67,
166.89,166.66, 165.92, 157.49, 155.33,155.29, 154.43, 144.81, 138.49,
136.98, 134.65, 133.68, 131.25, 127.72, 127.03, 124.84, 123.62, 123.10,
120.86, 119.38, 118.03, 117.29, 111.12, 105.76, 71.67, 70.33, 69.92,
69.53, 67.95, 60.38, 55.47, 54.13, 53.94, 49.35, 39.05, 39.00, 37.84,
32.75, 32.34, 31.52, 22.71, 22.23, 21.04, 18.93, 15.36, 14.20. HRMS
(DART-TOF) calculated for C52H65ClN9O12S [M þ H]þ m/z 1074.4162,
found 1074.4153.
4.1.24. Synthesis of 3-(4-(4-((5-chloro-4-((2-(isopropylsulfonyl)
phenyl)amino)pyrimidin-2-yl)amino)-5-isopropoxy-2-
methylphenyl)piperidin-1-yl)-N-(1-((2-(2,6-dioxopiperidin-3-yl)-
1,3-dioxoisoindolin-4-yl)oxy)-2-oxo-6,9,12-trioxa-3-azatetradecan-
14-yl)propenamide (H3)
4.5. Pharmacokinetics determination
The title compound was obtained from tert-butyl (2-(2-(2-(2-
aminoethoxy)ethoxy)ethoxy)ethyl)carbamate following similar
synthesis procedure of G1 (yellow powder, yield 25%). 1H NMR
The pharmacokinetic properties were determined at XPiscoric
lnc., China. We carried out pharmacokinetics determinations using
adult SD rats (180e250 g, 6e7 weeks, N ¼ 3 per group). B3 was
formulated in DMA/Solutol HS 15/saline (5:10:85, v/v/v) and
administered intravenously at a dose of 1 mg/kg. Plasma samples
were collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, and 12 h after treatment.
Plasma was obtained from the blood samples by centrifugation
(8000 rpm for 6 min at 2e8 ꢀC) and stored at ꢂ80 ꢀC. All samples of
the compound were determined by LCꢂMS/MS (Shimadzu; API
4000).
(400 MHz, CDCl3)
d
¼ 9.44 (s, 1H, CONHCO), 8.51 (d, J ¼ 8.3, 1H,
AreH), 8.09 (s, 1H, Ar-NH), 7.91 (s, 1H, Ar-NH), 7.85 (dd, J ¼ 7.9, 1.5,
1H, AreH), 7.72 (d, J ¼ 4.9, 1H, AreH), 7.65 (dd, J ¼ 10.7, 5.0, 1H,
AreH), 7.59 (s, 1H, AreH), 7.57e7.53 (m, 1H, AreH), 7.52 (s, 1H,
AreH), 7.45 (dd, J ¼ 7.3, 2.0, 1H, Ar-NH), 7.20e7.15 (m, 1H, AreH),
7.15e7.08 (m, 1H, AreH), 6.76e6.66 (m, 1H, AreH), 4.90 (dd,
J ¼ 11.9, 5.4, 1H, COCHN), 4.57 (s, 2H, COCH2O), 4.47 (dt, J ¼ 12.1, 6.1,
1H, OCH), 3.78e3.44 (m, 16H, OCH2, NCH2), 3.09 (d, J ¼ 10.3, 2H,
CH2), 2.90e2.54 (m, 7H, CH2, CH), 1.39e1.25 (m, 16H, CH3). 13C
4.6. In vivo xenograft studies
NMR (101 MHz, CDCl3)
d
¼ 172.37, 171.76, 168.78, 166.92, 166.64,
165.91, 157.46, 155.31, 155.28, 154.43, 144.78, 138.45, 137.03, 134.69,
133.62, 131.25, 127.66, 127.04, 124.78, 123.61, 123.13, 120.86, 119.42,
117.99, 117.27, 111.02, 105.73, 71.60, 70.32, 70.20, 69.93, 69.43, 67.89,
60.40, 55.45, 54.29, 53.94, 49.28, 39.06, 38.95, 37.76, 32.62, 32.32,
31.45, 29.69, 22.73, 22.22, 21.07,18.96. HRMS (DART-TOF) calculated
for C54H69ClN9O13S [M þ H]þ m/z 1118.4424, found 1118.4403.
The female BALB/c nude mice were purchased from Beijing HFK
Bioscience Co. ltd., (Beijing, China). H3122 cells (5 ꢃ 106) suspended
in 0.1 mL of serum were transplanted to the mice at 6e7 weeks old
and weighed 18e22 g. The mice were divided randomly (6 mice for
each group) when the size of tumors reached 60e100 mm3. The
mice were dosed orally with B3 (25, 50 mg/kg/d, dissolved in 10%
NMP/90% PEG300), vehicle (10% NMP/90% PEG300), and LDK378
(positive control, 25 mg/kg/d, dissolved in 10% NMP/90% PEG300).
The body weight and tumor volume were measured every 3 days.
The tumor volume was determined with Vernier calipers and
calculated as follows: tumor volume ¼ a ꢃ b2/2 (a, long diameter; b,
short diameter). All animal experiments have been approved by
Institutional Animal Care and Treatment Committee of Sichuan
University in China.
4.2. ALK inhibition assays
The ALK activity assay was conducted by Sundia MediTech
Company, Ltd. (China). Briefly, the compound, ALK enzyme (Carna,
08e518), Kinase substrate22 and ATP (Km, Sigma) were diluted in
kinase buffer to the indicated concentrations. The assay plate was
covered and incubated at room temperature. The data were
collected on Caliper EZ Reader and the curves were fitted by
Graphpad Prism 5.0.
Declaration of competing interest
4.3. Cell viability assay
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
The human cancer cell lines were obtained from the American
Type Culture Collection (Manassas, VA, USA). Cells were cultured in
RPMI 1640 or DMEM supplemented with 10% (v/v) fetal bovine
serum (Gibco) and 1% (v/v) penicillinꢂ streptomycin (HyClone) at
37 ꢀC with 5% CO2. Cells were seeded in a 96-well plate for 24 h and
then an equal volume of medium containing various concentrations
of compounds were added to each well. After 72 h, MTT was added,
and the cells were incubated for an additional 1e4 h. The absorbance
values (OD) of the 96-well plate were measured at 450 nm using a
Spectra MAX M5 microplate spectrophotometer (Molecular De-
vices). The IC50 values were the means of at least three independent
experiments and calculated by GraphPad Prism5 software.
Acknowledgement
We greatly appreciate the financial support from National Nat-
ural Science Foundation of China (grants 81773195 and 81472780).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.113150.
11

G. Yan, X. Zhong, L. Yue et al.
European Journal of Medicinal Chemistry 212 (2021) 113150
PROTAC BCL-XL degraders as potent anticancer agents with low on-target
platelet toxicity, Eur. J. Med. Chem. 192 (2020 Apr 15) 112186. PubMed
PMID: 32145645. Pubmed Central PMCID: 7433031.
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