Effective degradation of EGFRL858R+T790M mutant proteins by CRBN-based PROTACs through both proteosome and autophagy/lysosome degradation systems

Article abstract of DOI:10.1016/j.ejmech.2021.113328

Targeted therapy of treating patients with specific tyrosine kinase inhibitors (TKIs) is currently the standard care for epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer. However, the inevitably developed drug resistance in patien

Full text of DOI:10.1016/j.ejmech.2021.113328

European Journal of Medicinal Chemistry 218 (2021) 113328
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Effective degradation of EGFRL858RþT790M mutant proteins by CRBN-
based PROTACs through both proteosome and autophagy/lysosome
degradation systems
,
b
,
f
,
,
c
,
f
,
,
*
,
,
, d, 2
Xiaojuan Qu a
1, Haixia Liu a
1, Xiaoling Song a 1, Ning Sun a 2, Hui Zhong a
,
Xing Qiu a e, Xiaobao Yang a ***, Biao Jiang a
,
,
, e, **
a Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, 393 Middle Huaxia Road, Shanghai, 201210, China
b School of Life Science, ShanghaiTech University, China
c School of Physical Science and Technology, ShanghaiTech University, China
d School of Pharmaceutical Engineering and Life Science, Changzhou University, Jiangsu, 213164, China
e Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai, 200032, China
f University of Chinese Academy of Sciences, Beijing, 100049, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Targeted therapy of treating patients with specific tyrosine kinase inhibitors (TKIs) is currently the
standard care for epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer. However,
the inevitably developed drug resistance in patients to EGFR TKIs is the biggest obstacle for cancer
targeted therapy. About 60% of drug resistance to the 1st generation of EGFR TKIs was resulted from an
acquired T790M mutation in the kinase domain of EGFR protein. Proteolysis targeting chimera (PROTAC)
is a lately-developed technology to target point of interest proteins for degradation. Because EGFR-
mutant lung cancers are highly dependent on EGFR proteins, designing specific PROTAC molecules to
degrade EGFR proteins from cancer cells provides a very promising strategy to treat such patients and
eradicate drug resistance. Currently, there is no cereblon (CRBN)-based PROTAC reported able to degrade
T790M-containing EGFR resistant proteins. In this study, we synthesized two novel CRBN-based EGFR
PROTACs, SIAIS125 and SIAIS126, based on EGFR inhibitor canertinib and cereblon ligand pomalidomide.
These two degraders displayed potent and selective antitumor activities in EGFR TKI resistant lung
cancer cells. Firstly, they could selectively degrade EGFRL858RþT790M resistant proteins in H1975 cells at
the concentration of 30e50 nM, and EGFREx19del proteins in PC9 cells. But they did not degrade
EGFREx19delþT790M mutant proteins in PC9Brca1 cells or wild type EGFR in A549 lung cancer cells. They
could also selectively inhibit the growth of EGFR mutant lung cancer cells but not that of normal cells or
A549 cells. Secondly, the degradation of EGFRL858RþT790M proteins was long lasting up to 72 h. Thirdly,
these degraders displayed better inhibition of EGFR phosphorylation in H1975 cells and PC9Brca1 cells
comparing to canertinib. Finally, these degraders could also induce significant apoptosis and cell cycles
arrest in H1975 cells. Pre-incubation with canertinib, pomalidomide or ubiquitination inhibitor MLN4924
totally blocked EGFR degradation by PROTACs. Mechanistic studies showed that PROTAC could induce
autophagy in lung cancer cells. PROTAC-induced EGFR degradation acted through both ubiquitin/pro-
teosome system and ubiquitin/autophagy/lysosome system. Elevating autophagy activities enhanced
EGFR degradation and cell apoptosis induced by PROTACs. Our research not only offered a novel PROTAC
tool to target EGFR TKI drug resistance in lung cancer, but also firstly demonstrated that the involvement
of autophagy/lysosome system in PROTAC- mediated target protein degradation.
Received 23 December 2020
Received in revised form
21 February 2021
Accepted 21 February 2021
Available online 7 March 2021
Keywords:
EGFR
PROTAC
Resistance
Non-small cell lung cancer
T790M
Autophagy/ lysosome
Degradation
© 2021 Elsevier Masson SAS. All rights reserved.
* Corresponding author. Room A402, Y building 199 Huanke Road, Pudong District, Shanghai institute for advanced immunochemical studies, ShanghaiTech University,
Shanghai, 201210, China.
** Corresponding author. Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, 393 Middle Huaxia Road, Shanghai, 201210, China.
*** Corresponding author.
E-mail addresses: songxl@shanghaitech.edu.cn (X. Song), yangxb@shanghaitech.edu.cn (X. Yang), jiangbiao@shanghaitech.edu.cn (B. Jiang).
These authors contributed equally.
These authors contributed equally.
1
2
https://doi.org/10.1016/j.ejmech.2021.113328
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

X. Qu, H. Liu, X. Song et al.
European Journal of Medicinal Chemistry 218 (2021) 113328
1. Introduction
system and ubiquitin/autophagy/lysosome system were involved in
the degradation of target proteins by PROTACs.
Lung cancer is the leading cause of cancer-related death in the
worldwide [1], of which about 90% is non-small cell lung cancer
(NSCLC) [2,3]. About 15%e45% of NSCLC patients carrying EGFR
activating mutations, and currently, targeted therapy is the stan-
dard care for such patients [4e7]. Treating patients with EGFR
specific tyrosine kinase inhibitors greatly improved the life quali-
ties of NSCLC patients comparing to traditional chemotherapy
[8e11]. However, drug resistance to these kinase inhibitors even-
tually occurs a period after initial treatment even when the latest
FDA approved EGFR TKI drug osimertinib is used [12e14]. The drug
resistanceproblem is currently a big obstacle for targeted therapy in
clinic. Effective therapeutic strategies that can overcome drug
resistance are in great need to treat such patients. Because a big
proportion of drug resistance is caused by alterations in EGFR
proteins [15], eradicating EGFR proteins from cancer cells provides
a promising way to treat such patients and overcome drug
resistance.
Proteolysis targeting chimera (PROTAC) is a brand new tech-
nology that can degrade oncogenic proteins and has great potential
to overcome drug resistance. Each product of PROTAC is a bi-
functional molecule that can bind both the protein of interest
(POI) and an E3 ubiquitin ligase simultaneously [16]. After entering
cells, it can recruit the E3 ubiquitin ligase to the proximity of the
target protein, leading to the ubiquitination of its target proteins
followed by protein degradation by proteasome [17e19]. Because
many onco-proteins act as the critical drivers to promote cancer
formation, eliminating these onco-proteins by PROTAC molecules
will lead to the death of these cancer cells and theoretically provide
an ideal way to treat such diseases. Many PROTACs have been
developed to target onco-proteins now, and the most successful
PROTAC compound is ARV110. Latest data from ASCO2020 had
shown that ARV110 was very effective to treat cancer patients in
clinic and it could induce tumor regression in drug resistant cancer
patients [20].
EGFR mutant lung cancer is found in about 15% of Caucasion
NSCLC patients and about 40e50% of Asian NSCLC patients [5e7].
This type of cancer is driven by EGFR onco-genic activating muta-
tions, and the majority of these mutations are either EGFR exon 21
point mutations (L858R) or exon 19 deletion mutations (Exdel19).
The majority of drug resistance to 1st generation EGFR TKIs is
caused by an acquired T790M mutation in the EGFR kinase domain
(about 60%) [15]. Effectively targeting EGFR proteins with acquired
T790M mutations is critical to evade such drug resistance. Several
EGFR-targeted PROTAC molecules have been developed to degrade
EGFR oncogenic proteins in lung cancer in the past couple of years
[17,21e24]. In these studies, some EGFR PROTACs could degrade
EGFRL858RþT790M mutant proteins. These PROTACs utilized either
VHL ligands or MDM2 ligands to recruit E3 ubiquitin ligase. So far,
there is no CRBN-based EGFR PROTAC reported able to degrade
EGFR proteins with acquired T790M mutations. Although three
different groups had tried to generate EGFR degraders using CRBN
ligands, all these CRBN-based PROTACs fail to degrade
EGFRL858RþT790M mutant proteins [21e23]. Herein, we successfully
synthesized two novel CRBN-recruiting EGFR PROTACs that could
effectively degrade EGFRL858RþT790M mutant proteins at the con-
centration of 30e50 nM. These degraders could also effectively
degrade EGFREx19del mutants in PC9 cells, but not EGFR wild type
protein in A549 lung cancer cells. The degradation mechanisms of
these PROTACs were also investigated in this study. Our study
provided the first evidence that both the ubiquitin/proteosome
2. Results
2.1. Synthesis of CRBN-based EGFR-targeting PROTACs
To generate EGFR PROTACs, pomalidomide was used in our
study as the ligand of E3 ubiquitin ligase. It is the second generation
of immuno-modulatory drug (IMiDs) and was approved by FDA to
treat multiple myeloma in 2013. It is orallyavailable and can induce
significant degradation of essential ikaros transcription factors
through interacting with cereblon E3 ubiquitin ligase [25].
Comparing to two other cereblon ligands, thalidomide and its de-
rivative lenalidomide, pomalidomide is the most potent immuno-
modulatory drug [26]. Previously, it has been successfully used to
design PROTACs (such as BRD4 targeting PROTAC ARV-825), leading
to fast and potent degradation of target proteins [27]. In our pre-
screening, CRBN-based PROTACs generated from canertinib could
effectively inhibit the growth of cancer cells and induce the
degradation of EGFRL858RþT790M mutant proteins. Canertinib
(Fig. 1A) is an effective and irreversible EGFR inhibitor that was
previously developed for cancer treatment by University of Auck-
land and Pfizer [28]. It is water soluble, orally-available, and highly
potent in repressing the growth of tumor in xenograft mouse
models [28]. Carbon linker is a very commonly used linker for
PROTAC technology. So it was used in our PROTAC design to
generate a series of canertinib-CRBN based PROTACs with different
lengths of linkers (Scheme 1).
The scheme of synthetic routes for generating these EGFR de-
graders was displayed in Scheme 1 and Experimental section 4.1.
All the starting materials were commercially available. Firstly, tert-
butyl piperazine-1-carboxylate and 3-bromopropan-1-ol form
compound 3 by nucleophilic substitution. Then, compound 3 was
reacted with N-(3-chloro-4-fluorophenyl)-7-fluoro-6- nitro-
quinazolin-4-amine which was purchased from the company to get
the key intermediate 5. The compound 5 was following hydroge-
nation and condensation with Acryloyl chloride and removed Boc
protection under TFA condition to afford Canertinib derivative 7
(SIAIS092). Meanwhile, 2-(2,6-dioxopiperidin-3-yl)- 4- fluo-
roisoindoline-1,3-dione was reacted with various amines(9a~9f) by
nucleophilic substitution following hydrolyzation and removed
tert-butyl protection to form intermediate 10a~10f. Finally, the
compounds 10a~10fcondensed with compound 7 (SIAIS092) to
afford the desired compounds SIAIS121~SIAIS126.
The degradation effects of our synthesized PROTACs on
EGFRL858RþT790M proteins were examined in H1975 cells (Fig. 1F),
and the effects on cell proliferation were evaluated in a bunch of
EGFR mutant or wild type cell lines (Table 1). Two PROTACs
SIAIS125 and SIAIS126 were identified to have the best degradation
capabilities for EGFRL858RþT790M mutants (Fig. 1F) and best anti-
proliferation of EGFR mutant cancer cells (Table 1) in our pre-
screening. Further increasing the length of protac linker did not
achieve better effects than SIAIS126. So, these two PROTACs were
mainly investigated in the following studies. The structures of
canertinib, pomalidomide and PROTACs, SIAIS125 and SIAIS126
were displayed in Fig. 1.
2.2. SIAIS125 & SIAIS126 induced effective degradation of mutant
EGFR proteins in lung cancer cells
The degradation capabilities of SIAIS125 and SIAIS126 were
2

X. Qu, H. Liu, X. Song et al.
European Journal of Medicinal Chemistry 218 (2021) 113328
Fig. 1. Structures of two best CRBN-based EGFR-targeting PROTACs and related compounds. A. canertinib (EGFR ligand); B. SIAIS092 (derivative of canertinib to make PROTACs); C:
pomalidomide (cereblon ligand to recruit E3 ubiquitin ligase); D&E: structures of CRBN-based EGFR PROTACs: SIAIS125 and SIAIS126. F: the degradation of EGFR by PROTACs in
H1975 cells after 16-h drug treatment.
Scheme 1. Synthetic Routes for EGFR Degraders SIAIS121~SIAIS126 a
.
aReagents and conditions: (a) K2CO3, NaI, Acetone, 80 ꢀC, 12 h; (b) NaH, DMF, 100 ꢀC, 2 h; (c) H2, Pd/C, CH3OH,
40 ꢀC, 12 h; (d) THF, 0 ꢀC, 10 min; (e) TFA, DCM, rt, 12 h; (f) DIPEA, DMF, microwave, 110 ꢀC, 2 h; (g) HATU, DIPEA, DMF, rt, overnight.
evaluated in a bunch of EGFR mutant or wild type lung cancer cell
lines. Results showed that turning canertinib into PROTAC de-
graders endowed it with great capabilities to degrade mutant EGFR
protein (Fig. 2). Firstly, SIAIS125 and SIAIS126 could degrade
oncogenic EGFR proteins. In PC9 cells, SIAIS125 and SIAIS126
induced significant degradation of EGFREx19del proteins after
treating cells for 48 h, and the degradation occurred at various
concentrations above 10 nM (Fig. 2A).
Secondly, these two degraders could very effectively degrade
EGFRL858RþT790M mutant proteins in H1975 lung cancer cells. After
treating H1975 cells for 16 h, SIAIS125 and SIAIS126 degraded
EGFRL858RþT790M mutant proteins at the minimum concentration of
about 50 nM and 30 nM respectively (Fig. 2B). The degradation
capability of SIAIS126 was stronger than that of SIAIS125. Treating
cells with EGFR warhead inhibitor canertinib did not reduce EGFR
protein level, but instead, it slightly increased EGFR total protein by
3

X. Qu, H. Liu, X. Song et al.
European Journal of Medicinal Chemistry 218 (2021) 113328
Table 1
The inhibition and degradation effects of SIAIS125 and SIAIS126 on different lung cancer cell lines.
Cell lines
Cell type
SIAIS092
SIAIS121
SIAIS122
SIAIS123
SIAIS124
SIAIS125
SIAIS126
Canertinib
IC50s
PC9
PB1
H1975
HCC827
A549
Ba/F3-LT
293T
NSCLC
NSCLC
NSCLC
NSCLC
NSCLC
1.115
33.38
162.1
ND
ND
ND
4.999
189.7
234.4
ND
ND
ND
0.9765
162.8
110.4
ND
ND
ND
1.382
145.5
209.3
ND
ND
ND
0.4517
122.1
35.4
ND
ND
ND
>10000
>10000
2.603
45.04
22.1
2.123
>10000
71.27
>10000
>10000
3.942
89.49
26.52
1.154
>10000
51.28
>10000
>10000
0.8585
70.19
11.34
0.3776
7162
9.2
Normal
Normal
21838
2745
>10000
>10000
>10000
>10000
14743
>10000
~6000
5612
BJ
DC50s
PC9
PB1
H1975
A549
Ba/F3-LT
NSCLC
NSCLC
NSCLC
NSCLC
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
100a
Not degrade
30e50
Not degrade
~100
30e100
Not degrade
<30
Not degrade
~100
Not degrade
Not degrade
Not degrade
Not degrade
ND
IC50: half-maximal inhibitory concentration (nM). ND: not tested. Experiments were repeated for more than 3 times. Notdegrade: tested by western blot but had no obvious
degradation observed. DC50s: half-maximal degradation concentrations, estimated values (nM).
a
Dmax: 40%.
Fig. 2. The degradation effects of SIAIS125 and SIAIS126 on mutant EGFR proteins in non-small cell lung cancers. Western blots were used to evaluate the degradation of EGFR
proteins after incubated cells with PROTACs at indicated concentrations for specific periods. A: Dose-dependent degradation of EGFREx19del in PC9 cells after 48-h treatment; B&D:
Dose-dependent degradation of EGFRL858RþT790M in H1975 cells after 16-h and 48-h treatment; C: Time-dependent degradation of EGFR by PROTACs. E&F: the effects on EGFR
Ex19delþT790M
proteins after treating PC9Brca1 cells with PROTACs for 16 or 48 h.
about 15e31% (Fig. 2B). The maximum extents of degradation for
these two degraders reachedabout 95e100%. Thirdly, the degra-
dation of EGFR proteins occurred in a dose- and time-dependent
manner (Fig. 2AeD). With the increase in PROTAC concentrations,
EGFR degradation effects were elevated. After treating H1975 cells
with PROTACs for 48 h, almost all EGFR proteins were degraded at
the concentrations above 30 nM (Fig. 2D). At the same time, the
degradation degrees were enhanced with the increase of incuba-
tion time, and the maximum degradation reached about 98%e100%
after 48 h degraders’ treatment (Fig. 2C).
Finally, the degradation effects on EGFR proteins were mutant-
specific. SIAIS125 and SIAIS126 did not degrade EGFREx19delþT790M
proteins after 16 h treatment in PC9Brca1 cells (Fig. 2E). Even after
treating cells with SIAIS125 or SIAIS126 for 48 h, EGFREx19delþT790M
mutant proteins were barely degraded (Fig. 2F). Additionally,
SIAIS125 and SIAIS126 barely affected the total level of wild type
EGFR protein in A549 human cancer cells (Suppl. Figure 1A). In
contrast, in Ba/F3 cells that were stably expressed human
EGFRL858RþT790M mutant proteins, the levels of EGFR protein were
also significantly reduced by SIAIS125 and SIAIS126 at 100 nM
concentration (Supplemental Figure 1B). A slightly weaker degra-
dation of EGFR protein in Ba/F3 cells than H1975 cells might be
caused by species difference when a mouse cell line instead of a
human cell line was used.
In summary, SIAIS125 and SIAIS126 could selectively induce the
degradation of EGFREx19del activating mutants and EGFRL858RþT790M
resistant proteins. These data also suggested that turning inhibitor
canertinib into a PROTAC could improve its target specificity on
EGFR mutant proteins and potentially reduce its toxic side effects.
2.3. Evaluation of the anti-proliferative activity in vitro
The effects of PROTACs on the proliferation of EGFR-mutant lung
cancer cells were also examined (Table 1). Results showed that
SIAIS125 and SIAIS126 not only inhibited the proliferation of non-
small cell lung cancer cells expressing EGFREx19del mutant
4

X. Qu, H. Liu, X. Song et al.
European Journal of Medicinal Chemistry 218 (2021) 113328
proteins such as PC9 and HCC827, but also inhibited the growth of
H1975 and PC9Brca1 cells that express EGFRL858RþT790M or
EGFRex19delþT790M mutants respectively (Table 1). SIAIS125 and
SIAIS126 displayed a slightly weaker inhibition of the proliferation
comparing to parent inhibitor canertinib, but were more potent
than canertinib derivative SIAIS092. For example, in H1975 cells,
the IC50s for SIAIS125 and SIAIS126 were 19.41 and 30.76 nM
respectively, which were close to that of canertinib (11.34 nM)but
better than that of canertinib derivative SIAIS092 (162.1 nM).
SIAIS125 and SIAIS126 also inhibited the proliferation of
EGFRL858RþT790M expressing Ba/F3 cells. However, they did not
affect the proliferation of A549 cells which express wild type EGFR.
We also examined the effects of SIAIS125 and SIAIS126 on the
proliferation of two normal cell lines: human embryo kidney cell
line 293T cells and human normal foreskin fibroblast BJ cells
(Table 1). Both degraders did not inhibit the proliferation of these
two cell lines. These data indicated that these two degraders had
potent and selective anti-tumor activities in EGFR mutant lung
cancer cells with very low side effects on EGFR wild type cells.
SIAIS125 or SIAIS126 after 24-h treatment (Fig. 4B). The degrada-
tion effects of SIAIS125 and SIAIS126 on EGFR proteins were long
lasting. After changing with fresh complete culture medium, EGFR
protein levels did not recover even at the time of 72 h after drug
removal (Fig. 4B).
2.6. degraders induced cell cycle arrest in cancer cells
The effects of SIAIS125 and SIAIS126 on cell doubling were
tested in H1975 cells by labeling cells with a fluorescent dye CFSE
(Fig. 5A). After cell duplication, the dye will split into two daughter
cells and the intensities of fluorescent dye will reduce by half. Re-
sults showed that in H1975 cells that were treated with 100 nM or
300 nM EGFR degraders, the intensities of fluorescence were about
similar to that in the DMSO treatment on day 1; but, on day 4 the
fluorescence levels were significantly higher than that of DMSO
control. This indicated that after PROTAC treatment, cell doubling
time was greatly increased comparing to DMSO control, and
consequently the intensities of cell fluorescence decreased
comparing to those treated with DMSO (Fig. 5A). SIAIS126 exhibi-
ted stronger repression effects on cell doubling than SIAIS125 did.
The proliferation of cells was slower in SIAIS126 treatment than
that in SIAIS125 treatment when both were used to treat cells at
2.4. Better inhibition of EGFR activation by degraders in EGFR
mutant lung cancer cells
EGFR promotes cell proliferation through phosphorylation itself
followed by the activation of downstream signaling pathways. Our
data showed that both canertinib and EGFR degraders could inhibit
the phosphorylation of EGFR in H1975 cells (Fig. 3A). Interestingly,
SIAIS125 and SIAIS126 exhibited stronger inhibition of EGFR
phosphorylation than canertinib did (Fig. 3A). Similar effects were
observed in PC9Brca1 cells that express EGFREx19delþT790M mutants
(Fig. 3B). Thus, turning an inhibitor canertinib into a PROTAC
degrader endowed the inhibitor with stronger inhibition
capabilities.
2.5. The degradation effects on EGFR proteins were long lasting
To study how long the effects on EGFR protein degradation
could last, a drug wash-out experiment was performed according
to the experimental scheme shown in Fig. 4A. H1975 cells were
firstly treated with 300 nM either SIAIS125 or SIAIS126 for 24 h.
Then, the condition medium was discarded and cells were washed
with PBS for 3 times. Fresh complete 1640 culture medium with
10% fetal calf serum was added to cells for maintenance. Finally,
cells were harvested at indicated time points and EGFR total protein
levels were evaluated by Western blot (Fig. 4A). Results showed
that EGFR proteins were degraded completely by 300 nM of
Fig. 4. Wash out experiments to examine the lasting effects on EGFR degradation. A:
the research scheme of drug wash-out experiment: after a brief treatment with
PROTACs for 24 h, condition medium was removed and replaced with fresh culture
medium. Cells were collected at indicated time points and subjected to Western blot
analysis B: Western blots to evaluate EGFR protein levels after drug treatment. A
sustained degradation effect on EGFR proteins was observed and the level was not
recovered even after removing drug for 72 h.
Fig. 3. Degrader SIAIS125 and SIAIS126 displayed better inhibition of EGFR phosphorylation than canertinib. Western blots were used to detect the phosphorylation of EGFR protein
after 16-h PROTAC treatment. A: H1975 cells; B: PC9Brca1 cells.
5

X. Qu, H. Liu, X. Song et al.
European Journal of Medicinal Chemistry 218 (2021) 113328
Fig. 5. The impacts of SIAIS125 and SIAIS126 on the cell cycle, apoptosis and migration of lung cancer cells. A: CFSE staining to examine the proliferation of H1975 cells after treated
with SIAIS125 and SIAIS126. B: The effects of PROTACs on cell cycle; C: the effects of SIAIS125 and SIAIS126 on cell apoptosis were evaluated by flow cytometry with Annexin V-FITC
assay. D: the effects of EGFR PROTACs on cell migration during wound healing; E: the effects of EGFR PROTACs on cell invasion via Boyden chamber invasion assay.
300 nM.
In the meanwhile, PROTAC induced cell cycle arrest in
With the increase in the degrader concentrations, less live cells
were presented and more apoptosis events were observed. Stron-
ger apoptosis effects were observed in cells treated with higher
concentration of SIAIS125 or SIAIS126 comparing to those treated
with canertinib (Fig. 5C).
H1975 cells (Fig. 5B). Results of flow cytometry experiment showed
that PROTAC treatment increased the proportion of cells in G0/G1
phase, and dramatically reduced the proportion of cells in S phase
(Fig. 5B). In summary, degrader treatment induced cell cycle arrest
and inhibited cell proliferation.
2.8. degraders affected cell migration
2.7. degraders promoted cell apoptosis in cancer cells
The effects of SIAIS125 and SIAIS126 on cell migration were
evaluated via wound healing assay (Fig. 5D). Briefly, H1975 cells
were seeded in a 24-well-plate. Cells were scratched at confluence
with the tip of a 0.2 mL pipette 24 h later, and then were washed for
3 times with PBS. Followed by incubating cells with PROTACs at the
concentration of 300 nM or 500 nM, six photos were taken from
each well at the indicated time points as shown in supplementary
To see whether SIAIS125 and SIAIS126 induced cell apoptosis,
H1975 cells were firstly incubated with PROTACs for 72 h, and then
were collected and analyzed by flow cytometry with Annexin V-
FITC apoptosis detection kit. Results showed that treating cells with
SIAIS125 or SIAIS126 induced the apoptosis of H1975 cells (Fig. 5C).
6

X. Qu, H. Liu, X. Song et al.
European Journal of Medicinal Chemistry 218 (2021) 113328
figure 2. ImageJ software was used to quantify the areas that were
not covered by cells. Results revealed that SIAIS126 affected cell
migration at the concentration of both 300 nM and 500 nM, while
SIAIS125 could only affect cell migration at 500 nM (Fig. 5D).
proteosome-mediated protein degradation, and then evaluated the
impacts on EGFR degradation (Fig. 6A and B). Pretreating cells with
100 nM MG132 only led to a partial recovery of EGFR protein level
in degrader treatments, which indicated that there might be other
mechanisms involved in PROTAC-induced EGFR degradation
(Fig. 6B). To further test this hypothesis, a second-generation irre-
versible proteosome inhibitor, carfilzomib, was used to examine
the impacts on EGFR degradation (Fig. 6C). Treating cells with 5 nM
of carfilzomib alone increased endogenous EGFR protein levels,
which indicated that proteosome-mediated pathway was involved
in endogenous EGFR protein degradation. When treated together
with EGFR PROTACs, carfilzomib also could not totally block the
degradation of EGFR by SIAIS125 or SIAIS126 (Fig. 6C). In summary,
above data indicated that other protein degradation pathways
might be also present and were equally important for EGFR
degradation by PROTACs.
Fourthly, an autophagy/lysosome-mediated protein degradation
system was required for EGFR protein degradation by PROTACs. It
has been demonstrated that inhibiting proteosome function could
induce a switch in the balance of target protein degradation from
an ubiquitination/proteosome -dependent pathway to an
ubiquitination/lysosomal-dependent protein degradation pathway
[29]. To examine whether lysosome pathway was involved in the
ubiquitin/proteosome independent degradation of EGFR,
H1975 cells were incubated with different concentrations of bafi-
lomycin A1, a lysosome inhibitor, for 16 h before treatment with
PROTACs (Fig. 7A). Western blot analysis showed that endogenous
EGFR protein levels were increased.
2.9. Degrader SIAIS126 blocked cancer cell invasion
The effects of degraders on cell invasion were evaluated via
transwell assay (Fig. 5E). H1975 cells that stably expressed GFP
proteins were seeded inside of the matrigel-coated transwell
chamber, and EGFR degraders were then added to cells at indicated
concentrations. Five photos were randomly taken from the lower
side of the chamber at 18-h and 42-h time points after initial drug
treatment. Three biological replicates were set up for each treat-
ment. Cell numbers were counted and were used to compare the
difference among different treatments (Fig. 5E). Results showed
that 18-h after drug treatment, both canertinib and SIAIS125 did
not show much effects on cell invasion comparing to DMSO control.
SIAIS126 significantly reduced the number of invaded cells at the
concentration of 500 nM (P < 0.05). There was about 38% of
reduction in the number of invaded cells after 18 h treatment, and
40% after 42-h drug treatment in SIAIS126 treatment (Fig. 5E).
Because both SIAIS125 and SIAIS126 could inhibit cell proliferation
and induced cell apoptosis at about similar extents, but only
SIAIS126 at high concentration affected cell migration and invasion,
we speculated that SIAIS126 might have the ability to block cell
invasion.
2.10. PROTACs degrade EGFR proteins through both ubiquitin/
proteosome system and autophagy/lysosome system
Dramatically after bafilomycin A1 treatment (Fig. 7B). This result
was consistent with literature that lysosome-mediated degradation
played an important role in endogenous EGFR protein degradation
[30,31]. Interestingly, when cells were pretreated with bafilomycin
The mechanisms of EGFR degradation by PROTACs were studied
in H1975 cells. Results revealed that the degradation acted through
not only the ubiquitin/proteosome system, but also the ubiquitin/
autophagy/lysome degradation system.
Firstly, the degradation of EGFR proteins relied on the recruit-
ment of E3 ubiquitin ligase to EGFR protein. In this experiment, two
A1 for 2 h and then treated with 0.3 mM SIAIS125 or SIAIS126 for
16 h, the degradation effects on EGFR protein were partially
reversed with increase in the concentration of lysosome inhibitor
(Fig. 7B). These data indicated that lysosome-mediated pathway
also took part in the PROTAC-induced EGFR degradation.
components of EGFR PROTAC molecules, canertinib (1
CRBN ligand pomalidomide (10 M), were added to cells before
treatment to compete with PROTACs (0.3 M) to bind to EGFR and
m
M) and
Previous studies by Zhao et al. showed that autophagy might be
involved in the degradation of EGFR by PROTAC molecules P3 [24].
So, we examined the activation of autophagy by EGFR degraders
(Fig. 7C). Both serum free treatment and rapamycin were used as a
positive control for the activation of autophagy, and the relative
protein levels of autophagy markers LC3B II to LC3B I were used to
evaluate the occurrence of autophagy. Results showed that both
serum starvation and rapamycin treatment greatly increased the
ratio of LC3B II to LC3B I protein level, which indicated that auto-
phagy occurred in these cases (Fig. 7C). Interestingly, treating cells
with 30, 100 or 300 nM of EGFR degrader SIAIS126 also increased
the ratio of LC3B II/LC3B I (Fig. 7C). These results indicated that
EGFR degraders induced autophagy in lung cancer cells. Combi-
nation treatment of cells with rapamycin and degrader SIAIS126
further increased the degradation of EGFR protein comparing to
SIAIS126 alone (Fig. 7C). Additionally, under serum starvation
condition that promoted autophagy, the number of live cells was
dramatically reduced when treated with SIAIS125 or SIAIS126 for
either 12 h or 24 h (Fig. 7D&E). While PROTACs barely induced
apoptosis after 24-h drug treatment in the complete medium with
10% FBS, under the condition of serum starvation, SIAIS125 and
SIAIS126 induced significant apoptosis in H1975 lung cancer cells
(Fig. 7F&G). These data suggested that autophagy could enhance
the apoptosis of lung cancer cells induced by PROTACs.
m
m
E3 ubiquitin ligase respectively (Fig. 6A and B). The aim was to
validate that the requirement of both EGFR ligand binding and E3
ubiquitin ligase binding for EGFR degradation. Results showed that
both canertinib and pomalidomide were able to out-compete EGFR
PROTAC molecules to bind to either EGFR protein or E3 ubiquitin
ligase (Fig. 6B). In the presence of these two ligands, the degrada-
tion effects on EGFR protein by degraders were eliminated. This
suggested that both EGFR ligand and E3 ligase ligand were required
for EGFR degradation by degraders SIAIS125 and SIAIS126.
Secondly, PROTAC-induced EGFR degradation relied on the
ubiquitination of EGFR protein (Fig. 6C). An inhibitor pevonedistat
(MLN4924) was added to cells to block the process of ubiquitina-
tion prior PROTAC treatment, and then the effects on EGFR degra-
dation were evaluated by Western blot (Fig. 6C). Results showed
that treating cells with degrader SIAIS125 or SIAIS126 reduced
EGFR protein levels. However, the effects were totally blocked by
the inhibitor MLN4924 (1 mM), which in turn led to a recovery at
EGFR total protein levels. This indicated that the process of ubiq-
uitination was an essential procedure for EGFR degradation by
PROTACs.
Thirdly, ubiquitin/proteosome system (UPS) was partially
responsible for EGFR degradation by PROTACs. Previous studies
showed that ubiquitin/proteosome/system was the major mecha-
nism responsible for PROTAC-induced EGFR degradation. So, we
Because Bafilomycin A1 inhibited the function of both auto-
phagy and lysosome by preventing their fusion, it was possible that
EGFR degradation by PROTACs might act through the autophagy/
lysosome pathway. To examine whether both proteosome system
used
a reversible proteosome inhibitor MG132 to stop the
7

X. Qu, H. Liu, X. Song et al.
European Journal of Medicinal Chemistry 218 (2021) 113328
Fig. 6. The degradation of EGFR relied on protein ubiquitination and proteosome system. A: the scheme of the experiments. Cells were firstly treated with indicated compounds for
2 h, and then treated with EGFR PROTACs for 16 h. Western blot were then used to examine the effect of compounds on EGFR degradation by PROTACs; B: The effects of canertinib
and pomalidomide on EGFR degradation; C: the effects of proteosome inhibition and ubiquitination inhibition on EGFR degradation; D: The effects of proteosome inhibition on
EGFR degradation.
and autophagy/lysosome pathway were both required in EGFR
degradation by PROTACs, we inhibited the function of both together
to see the effects on PROTAC-mediated EGFR degradation (Fig. 7H).
We found out that the inhibition of both pathways led to a signif-
icant recovery of EGFR protein amount (Fig. 7H).
of EGFR TKIs [15]. EGFR T790M-targeting PROTACs provide poten-
tial tools to overcome the drug resistance to EGFR tyrosine kinase
inhibitors. Right now, EGFRL858RþT790M mutant protein was the only
form of resistant proteins that were able to be degraded by PRO-
TACs. EGFREx19delþT790M mutants are as important as
EGFRL858RþT790M mutants in drug resistance. In addition, there was
no PROTAC developed against EGFR with acquired C797S mutation
yet. In the long run, developing PROTACs that are able to degrade
EGFR with these different resistant mutations is still in great need
to overcome drug resistance.
To study the relationships among EGFR ubiquitination, the
proteosome system and the autophagy/lysosome system, the in-
hibitor of ubiquitin ligase MLN4924 was treated together with the
inhibitors of proteosome or autophagy/lysosome, and then to
evaluate the effects on EGFR degradation by PROTACs (Fig. 8A).
Results showed that treating H1975 cells with SIAIS126 induced
EGFR degradation, treating cells with MLN4924 alone slightly
increased EGFR protein level (Fig. 8B). Inhibiting the function of
ubiquitin ligase alone or in combination with lysosome inhibition
totally blocked EGFR degradation by PROTACs (Fig. 8B). Because
Bafilomycin only slightly reversed the degradation effect of
SIAIS126 as shown in Fig. 7B - D, it suggested that ubiquitination
was an upstream event for lysosome degradation of EGFR. Similarly,
ubiquitination was also an upstream event for proteosome-
mediated degradation of EGFR protein by PROTAC (Fig. 8B).
Taken together, our data suggested that PROTAC-mediated EGFR
degradation acted through both ubiquitin/proteosome system and
ubiquitin/autophagy lysosome degradation system.
Both cereblon ligands and VHL ligands are E3 ubiquitin ligases
that respectively mediate the degradation of IKZF proteins and
HIF1a proteins in vivo [32,33]. They had also been successfully used
in PROTAC design to induce the degradation of many proteins.
Previously developed T790M-targeted EGFR PROTACs used either
VHL ligands or MDM2 ligands to recruit E3 ubiquitin ligase
[17,22,24]. None of reported CRBN-based EGFR PROTAC was able to
degrade EGFR with T790M mutation [22e24]. In these reports,
most of CRBN-based PROTACs used lenalidomide as the ligand to
recruit E3 ubiquitin ligase. In our experiments, pomalidomide was
used to develop EGFR PROTACs. However, because different war-
heads were used in these EGFR PROTACs, we cannot conclude that
pomalidomide is better than the other ligands in EGFR PROTAC
design. For example, Jin’s group found that a thalidomide-based
compound MS154 exhibited better EGFR degradation activities
comparing to pomalidomide-based compound 9 when the same
warhead was used [21]. Rational design of PROTACs with different
warheads and different ligands of E3 ubiquitin ligase is crucial for
fast developing a successful PROTAC compound. Computer-aided
3. Conclusions and discussion
Drug resistance is a big clinical problem in EGFR-targeted
therapy, and acquired T790M secondary mutation in EGFR is
responsible for about 60% of drug-resistance to the first generation
8

X. Qu, H. Liu, X. Song et al.
European Journal of Medicinal Chemistry 218 (2021) 113328
9

X. Qu, H. Liu, X. Song et al.
European Journal of Medicinal Chemistry 218 (2021) 113328
PROTAC design might be helpful to speed up the process of
designing of a good PROTAC.
Our data suggested that turning inhibitor canertinib into a
PROTAC degrader not only could endow it with extra degradation
capability, but also could improve its target specificity. For example,
SIAIS126 could inhibit the phoshorylation of both EGFRL858RþT790M
and EGFREx19delþT790M mutant proteins, however, it only could
degrade EGFR L858RþT790M proteins very well. The underline reason
is not clear yet. Knowing the mechanisms may help later to design a
better EGFR PROTAC to target both T790M containing resistant
proteins and the C797S-containing triple mutant proteins.
Most targets of developed PROTACs are intracellular proteins
such as BCR-ABL [34], EML4-ALK [19], and bromodomain and
extraterminal (BET) proteins [35]. The degradation mechanisms of
such PROTACs acted mainly through the proteosome system. Some
technologies such as LYTAC (lysosome-targeting chimeras) were
developed to specifically target membrane proteins for degrada-
tion, and reported successful targets included EGFR and PDL1 [36].
Although EGFR is mainly expressed on cell membrane, previous
studies showed it could also be effectively degraded by PROTACs
through the ubiquitin/proteosome system. In this study, we found
that EGFR PROTAC activated autophagy in cancer cells. We firstly
demonstrated that PROTACs could induce EGFR degradation
through the ubiquitin/autophagy/lysosome degradation system in
addition to the well known ubiquitin/proteosome system. Our
study will be very helpful for later PROTAC design and future
PROTAC applications.
Fig. 8. Degradation of EGFR by degrader required the ubiquitin-lysosome and
ubiquitin-proteosome system. A: the experimental scheme of drug treatment; B:
Western blot for EGFR protein level after compounds treatment. ImageJ was used to
quantify the intensity of each band and the relative ratios of EGFR protein to tubulin
protein levels were calculated and labeled below the bands.
3.81e3.74 (m, 2H), 3.53 (t, J ¼ 5.3 Hz, 4H), 2.79 (t, J ¼ 6.2 Hz, 2H),
2.67 (s, 4H), 1.87e1.76 (m, 2H), 1.44 (s, 9H).
4.1.2. Tert-butyl 4-(3-((4-((3-chloro-4-fluorophenyl)amino)-6-
nitroquinazolin-7-yl) oxy) propyl) piperazine-1-carboxylate (5)
To a solution of tert-butyl 4-(3-hydroxypropyl)piperazine-1-
carboxylate (1.7 g, 6.9 mmol) 3 in N,N-dimethylformamide (DMF,
10 mL) was added NaH (441.6 mg, 18.4 mmol) at 0 ꢀC under ni-
trogen atmosphere. After stirring for 30 min, N-(3-chloro-4-
4. Experimental section
4.1. Chemistry general procedure
fluorophenyl)-7-fluoro-6-nitroquinazolin-4-amine(1.54
g,
All chemicals were obtained from commercial suppliers (Ada-
mas and Alfa), and used without further purification, unless
otherwise indicated. N,N-diisopropylethylamine (DIPEA),dichloro-
4.6 mmol) was added to the mixture at 0 ꢀC. After striring at 70 ꢀC
for 2 h, the reaction mixture was poured into water and extracted
with EtOAc. The combined organic layers were washed with brine,
dried over Na2SO4, and concentrated. The resulting residue was
purified by silica gel column chromatography (5%e10% MeOH in
DCM) to provide the desired product (1.2 g, 48%) as yellow solid.
MS(ESI) m/z: 561.2020 [MþH]þ.1H NMR (500 MHz, Methanol-d4)
methane
(DCM),
Tetrahydrofuran
(THF)
and
N,N-
dimethylformamide (DMF) were dried by a 4 Å MS. Flash chro-
matography 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 compounds were characterized by 1H
NMR, HRMS. 1H NMR spectra were recorded on Bruker AVANCE III
500 MHZ (operating at 500 MHz for 1H NMR), chemical shifts were
reported in ppm relative to the residual DMSO‑d6 (2.50 ppm 1H),
and coupling constants (J) are given in Hz. Multiplicities of signals
are described as follows: s — singlet, br. s — broad singlet, d —
doublet, t — triplet, m — multiple. High Resolution Mass spectra
were recorded on AB Triple 4600 spectrometer with acetonitrile
and water as solvent.
d
8.98 (s, 1H), 8.59 (s, 1H), 8.05 (dd, J ¼ 6.7, 2.6 Hz, 1H), 7.74e7.68
(m, 1H), 7.35 (d, J ¼ 3.0 Hz, 1H), 7.27 (t, J ¼ 8.9 Hz, 1H), 4.41e4.32 (m,
2H), 3.45 (s, 4H), 2.63 (t, J ¼ 7.3 Hz, 2H), 2.47 (t, J ¼ 5.1 Hz, 4H),
2.12e2.07z (m, 2H), 1.46 (s, 9H).
4.1.3. Tert-butyl-(3-((6-amino-4-((3-chloro-4-fluorophenyl)amino)
quinazolin-7-yl) oxy)propyl)piperazine-1-carboxylate (6)
To a solution of tert-butyl 4-(3-((4-((3-chloro-4-fluorophenyl)
amino)
-6-
nitroquinazolin-7-yl)oxy)propyl)piperazine-1-
4.1.1. Tert-butyl 4-(3-hydroxypropyl)piperazine-1-carboxylate (3)
carboxylate (1.2 g, 2.1 mmol) 5 in CH3OH (30 mL) was added Pd/C
500 mg, at room temperature under H2 atmosphere. After stirring
at 40 ꢀC for 12 h, The reaction mixture was filtered through a
Buchner funnel to remove Pd/C. Then the filtrate was evaporated
under the reduced pressure and passed through a short silica gel
column chromatography (5%e10% MeOH in DCM) to provide the
desired product (802 mg, 72%) as yellow solid. m.p.
133.1e134.7 ꢀC.MS(ESI) m/z: 531.2278 [MþH]þ. 1H NMR (500 MHz,
To
a solution of tert-butyl piperazine-1-carboxylate (2 g,
11 mmol) in acetone (30 mL) were added 3-bromopropan-1-ol
(1.83 g, 13.2 mmol) and K2CO3 (3.8 g, 27.5 mmol) and NaI
(165 mg, 1.1 mmol) at room temperature. After stirring at 80 ꢀC for
12 h, the resulting mixture was filtered through a Buchner funnel.
The filtrate was evaporated under the reduced pressure and passed
through a short silica gel column (0%e5% CH3OH in DCM) to give
the desired product (1.7 g, 65%) as transparent liquid. MS(ESI) m/z:
Chloroform-d)
d
8.56 (s, 1H), 7.92 (dd, J ¼ 6.5, 2.7 Hz, 1H), 7.53e7.47
245.1865 [MþH]þ.1H NMR (500 MHz, Chloroform-d)
d 4.45 (s, 1H),
(m, 1H), 7.21e7.11 (m, 2H), 7.01 (s, 1H), 6.91 (s, 1H), 4.28 (s, 2H), 4.22
Fig. 7. Autophagy-lysosome system was involved in mutant EGFR degradation by PROTACs. A: the scheme of the experiments; B: the effects of autophagy/lysosome inhibition on
EGFR degradation; C: the involvement of autophagy in EGFR degradation by PROTACs. Serum starvation and rapamycin as the positive control of autophagy induction; D & E:
Relative cell number after PROTAC treatment for 12 h (D) or 24 h (E) under serum starvation condition; F: The percentage of apoptotic cells after PROTAC treatment for 12 or 24 h in
complete culture medium; G: The percentage of apoptotic cells after PROTAC treatment for 12 or 24 h under serum starvation condition; H: The effects of proteosome and lysosome
inhibition on EGFR degradation.
10

X. Qu, H. Liu, X. Song et al.
European Journal of Medicinal Chemistry 218 (2021) 113328
(t, J ¼ 6.3 Hz, 2H), 3.45 (t, J ¼ 5.0 Hz, 4H), 2.57 (t, J ¼ 7.2 Hz, 2H), 2.43
(s, 4H), 2.09 (t, J ¼ 6.9 Hz, 2H), 1.45 (s, 9H).
4.1.7. 4- ((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)
amino)butanoic acid(10c)
The title compound (yellow solid, 0.8 g, 61% yield over two
steps) was synthesized according to procedures for the preparation
of 10a. MS(ESI) m/z:360.1187 [MþH]þ. 1H NMR (500 MHz, DMSO)
4.1.4. N-(4-((3-chloro-4-fluorophenyl)amino)-7-(3-(piperazin-1-
yl)propoxy)quinazolin-6-yl)acrylamide (7)/(SIAIS092)
d
12.13 (s, 1H), 11.09 (s, 1H), 7.58 (dd, J ¼ 8.4, 7.2 Hz, 1H), 7.13 (d,
To
fluorophenyl)
carboxylate (802 mg, 1.5 mmol) 6 in THF (20 mL) was added
acryloyl chloride (140
l) at 0 ꢀC. The reaction mixture was then
a
solution of tert-butyl 4-(3-((6-amino-4-((3-chloro-4-
J ¼ 8.6 Hz, 1H), 7.02 (d, J ¼ 7.0 Hz, 1H), 6.65 (t, J ¼ 5.9 Hz, 1H), 5.05
(dd, J ¼ 12.7, 5.4 Hz, 1H), 3.34e3.30 (m, 2H), 2.94e2.84 (m, 1H),
2.60e2.52 (m, 2H), 2.31 (t, J ¼ 7.2 Hz, 2H), 2.07e2.00 (m, 1H),
1.82e1.75 (m, 2H).
amino)quinazolin-7-yl)oxy)propyl)piperazine-1-
m
warmed up to room temperature. After stirring for 10 min, 2 mL
H2O was added to the mixture to quench the reaction. The reaction
mixture was poured into water and extracted with DCM. The
combined organic layers were washed with brine, dried over
Na2SO4, and concentrated. The resulting residue was passed
through a short silica gel column (7% CH3OH in DCM) to give the
compound (465 mg, crude yield 54%) as a yellow solid. To a mixture
of crude compound (465 mg) and TFA solution (2 mL TFA in 10 mL
DCM) was stirred for 12 h at room temperature. When the reaction
was complete, the crude mixture was evaporated under the
reduced pressure, the residue was purified by C18 column chro-
matography (10e90% acetonitrile/0.05% HCl in H2O) to obtain
target compounds (411 mg, 97%) as yellow solid.
m.p.127.1e128.8 ꢀC.MS(ESI) m/z: 485.1861 [MþH]þ.1H NMR
4.1.8. 5- ((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)
amino)pentanoic acid(10d)
The title compound (yellow solid, 0.9 g, 50% yield over two
steps) was synthesized according to procedures for the preparation
of 10a. MS(ESI) m/z:374.1349 [MþH]þ.1H NMR (500 MHz, DMSO)
d
12.05 (s, 1H), 11.11 (s, 1H), 7.57 (dd, J ¼ 8.3, 7.4 Hz, 1H), 7.09 (d,
J ¼ 8.6 Hz, 1H), 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, 2H), 2.27e2.25 (m, 2H), 2.06e1.99 (m, 1H), 1.62e1.53
(m, 4H).
4.1.9. 6- ((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)
amino)hexanoic acid (10e)
(500 MHz, DMSO‑d6) d 9.73 (s, 1H), 8.99 (s, 1H), 8.71 (s, 2H), 8.08 (d,
The title compound (yellow solid, 1.26 g, 61% yield over two
steps) was synthesized according to procedures for the preparation
of 10a. MS(ESI) m/z: 388.1504 [MþH]þ. 1H NMR (500 MHz, DMSO)
J ¼ 7.2 Hz, 1H), 7.75 (s, 1H), 7.49 (t, J ¼ 9.0 Hz, 1H), 7.34 (s, 1H), 6.74
(dd, J ¼ 17.0, 10.3 Hz, 1H), 6.36 (dd, J ¼ 17.0, 1.9 Hz, 1H), 5.86 (dd,
J ¼ 10.2, 1.9 Hz, 1H), 4.32 (t, J ¼ 6.1 Hz, 2H), 3.26e3.1 (m, 10H), 2.09
(d, J ¼ 12.3 Hz, 2H),1.24e1.19 (m, 1H).
d
12.01 (s, 1H),11.09 (s, 1H), 7.57 (t, J ¼ 7.6 Hz,1H), 7.09 (d, J ¼ 8.5 Hz,
1H), 7.02 (d, J ¼ 6.8 Hz, 1H), 6.53 (s, 1H), 5.04 (dd, J ¼ 12.3, 5.0 Hz,
1H), 3.29 (s, 2H), 2.92e2.82 (m, 1H), 2.60e2.50 (m, 2H), 2.21 (t,
J ¼ 7.0 Hz, 2H), 2.06e1.99 (m, 1H), 1.58e1.52 (m, 4H), 1.40e1.30 (m,
2H).
4.1.5. (2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)glycine
(10a)
To
a
stirred solution of 2-(2,6-dioxopiperidin-3-yl)-4-
4.1.10. 7-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)
amino)heptanoic acid (10f)
fluoroisoindoline-1,3-dione (1.5 g, 5.4 mmol) in DMF (8 mL) were
added DIPEA (2.7 g, 21.6 mmol) and tert-butyl glycinate (848 mg,
6.48 mmol) 9a. Then the resulting mixture was stirred at 110 ꢀC for
2 h in the microwave under an atmosphere of nitrogen. When the
reaction was complete, the crude mixture was cooled down to
room temperature, diluted with 70% brine aqueous (50 mL),
extracted with EtOAc, the combined organic layer was washed with
water and brine, then dried over Na2SO4 and concentrated in vac-
uum to afford crude oil. After solvent evaporation, the residue was
purified by column chromatography on silica gel (25% EtOAc in PE)
to afford desired product as yellow solid. To a mixture of above
compound and TFA solution (4 mL TFA in 10 mL DCM) was stirred
for 12 h at room temperature. When the reaction was complete, the
mixture purified by preparative HPLC (10e90% acetonitrile/0.05%
HCl in H2O) to obtain target compound (1.2 g, 48%) as yellow solid.
The title compound (yellow solid, 1.3 g, 64% yield over two
steps) was synthesized according to procedures for the preparation
of 10a. MS(ESI) m/z: 402.1659 [MþH]þ.1H NMR (500 MHz, DMSO)
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), 2.92e2.84
(m, 1H), 2.65e2.51 (m, 2H), 2.19 (t, J ¼ 7.3 Hz, 2H), 2.05e2.00 (m,
1H), 1.59e1.55 (m, 2H), 1.53e1.46 (m, 2H), 1.37e1.28 (m, 4H).
4.1.11. N-(4-((3-chloro-4-fluorophenyl)amino)-7-(3-(4-((2-(2,6-
dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)glycyl)piperazin-1-yl)
propoxy)quinazolin-6-yl)acrylamide(SIAIS121)
To a solution of N-(4-((3-chloro-4-fluorophenyl)amino)-7-(3-
MS(ESI) m/z: 332.0872 [MþH]þ. 1H NMR (500 MHz, DMSO)
d
12.91
(piperazin-1-yl)propoxy)quinazolin-6-yl)acrylamide
(10
mg,
(s, 1H), 11.10 (s, 1H), 7.58 (dd, J ¼ 8.4, 7.3 Hz, 1H), 7.08 (d, J ¼ 7.0 Hz,
1H), 6.99 (d, J ¼ 8.6 Hz,1H), 6.85 (t, J ¼ 5.8 Hz,1H), 5.07 (dd, J ¼ 12.8,
5.4 Hz,1H), 4.11 (d, J ¼ 5.9 Hz, 2H), 2.93e2.85 (m,1H), 2.63e2.51 (m,
2H), 2.07e2.01 (m, 1H).
0.02 mmol) 7 in DMF (2 mL) were added (2-(2,6-dioxopiperidin-3-
yl) ꢁ1,3-dioxoisoindolin-4-yl)glycine (7.7 mg, 0.02 mmol) 10a,
HATU (9.12 mg, 0.024 mmol) and DIPEA(7.7 mg, 0.06 mmol).After
being stirred overnight at room temperature, the resulting mixture
was purified by preparative HPLC (10e90% acetonitrile/0.05% HCl in
H2O) to obtain target compound (7.9 mg 46%) as yellow solid. m.p.
281.8e282.7 ꢀC.MS(ESI) m/z: 798.2566 [MþH]þ.1H NMR (500 MHz,
4.1.6. 3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)
amino)propanoic acid(10b)
DMSO‑d6)
d 11.11 (s, 1H), 9.89 (s, 1H), 9.12 (s, 1H), 8.82 (s, 1H), 8.02
The title compound (yellow solid, 0.93 g, 39% yield over two
steps) was synthesized according to procedures for the preparation
of 10a. MS(ESI) m/z: 346.1030 [MþH]þ.1H NMR (500 MHz, DMSO)
(d, J ¼ 6.9 Hz, 1H), 7.70 (s, 1H), 7.63 (dd, J ¼ 8.5, 7.1 Hz, 1H), 7.53 (t,
J ¼ 9.0 Hz, 1H), 7.41e7.32 (m, 2H), 7.11 (dd, J ¼ 9.4, 7.8 Hz, 2H), 7.02
(d, J ¼ 5.0 Hz, 1H), 6.91 (t, J ¼ 8.5 Hz, 1H), 6.37 (dd, J ¼ 17.0, 1.9 Hz,
1H), 5.87 (dd, J ¼ 10.1, 1.9 Hz, 1H), 5.08 (dd, J ¼ 12.7, 5.4 Hz, 1H), 4.49
(d, J ¼ 14.2 Hz, 1H), 4.39 (t, J ¼ 5.8 Hz, 2H), 4.32 (s, 1H), 4.27e4.09
(m, 2H), 3.61 (s, 4H), 3.10e2.99 (m, 2H), 2.97e2.79 (m, 2H),
2.67e2.54 (m, 2H), 2.41e2.31 (m, 3H), 2.08e1.99 (m, 1H).
d
11.09 (s, 1H), 7.59 (dd, J ¼ 8.2, 7.5 Hz, 1H), 7.15 (d, J ¼ 8.6 Hz, 1H),
7.04 (d, J ¼ 7.0 Hz, 1H), 6.67 (t, J ¼ 6.0 Hz, 1H), 5.05 (dd, J ¼ 12.8,
5.4 Hz, 1H), 3.53 (dd, J ¼ 12.6, 6.3 Hz, 2H), 2.91e2.84 (m, 1H),
2.62e2.52 (m, 4H), 2.08e1.98 (m, 1H).
11

X. Qu, H. Liu, X. Song et al.
European Journal of Medicinal Chemistry 218 (2021) 113328
4.1.12. N-(4-((3-chloro-4-fluorophenyl)amino)-7-(3-(4-(3-((2-(2,6-
dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)propanoyl)
piperazin-1-yl)propoxy)quinazolin-6-yl)acrylamide(SIAIS122)
The title compound (yellow solid, 6.6 mg, 42% yield) was syn-
thesized according to procedures for the preparationþ 1of
SIAIS121.m.p.263.1e264.8 ꢀC. MS(ESI) m/z: 812.2734 [MþH] . H
134.84, 134.79, 132.64, 131.93, 129.14, 128.55, 126.97, 125.61 (d,
J ¼ 8.1 Hz), 119.74,119.59, 117.69, 117.46, 117.29, 110.92, 109.43,
107.70, 67.16, 53.23, 51.56, 51.13, 49.00, 42.21, 40.48, 38.33, 32.28,
31.43, 29.01, 26.49, 24.74, 23.29, 22.63.
4.1.16. N-(4-((3-chloro-4-fluorophenyl)amino)-7-(3-(4-(7-((2-(2,6-
dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)heptanoyl)
piperazin-1-yl)propoxy)quinazolin-6-yl)acrylamide(SIAIS126)
The title compound (yellow solid, 7.6 mg, 42% yield) was syn-
thesized according to procedures for the preparation of SIAIS121.
m.p.246.3e247.7 ꢀC.MS(ESI) m/z: 868.3341 [MþH]þ.1H NMR
NMR (500 MHz, DMSO‑d6)
d
11.09 (s, 1H), 9.97 (d, J ¼ 9.1 Hz, 1H),
9.19 (d, J ¼ 4.1 Hz,1H), 8.91 (d, J ¼ 3.1 Hz,1H), 7.98 (dt, J ¼ 6.9, 2.1 Hz,
1H), 7.67 (ddd, J ¼ 9.1, 4.3, 2.6 Hz, 1H), 7.63e7.49 (m, 2H), 7.44 (d,
J ¼ 9.5 Hz, 1H), 7.16 (d, J ¼ 8.7 Hz, 1H), 7.04 (d, J ¼ 7.0 Hz, 1H), 6.95
(dd, J ¼ 17.1, 9.8 Hz, 1H), 6.77 (s, 1H), 6.36 (dd, J ¼ 17.1, 1.8 Hz, 1H),
5.87 (dd, J ¼ 10.1, 1.9 Hz, 1H), 5.05 (dd, J ¼ 12.8, 5.4 Hz, 1H), 4.49 (d,
J ¼ 14.1 Hz, 1H), 4.38 (t, J ¼ 5.7 Hz, 2H), 4.07 (d, J ¼ 14.2 Hz, 1H), 3.56
(s, 4H), 3.18e3.08 (m, 3H), 2.97 (d, J ¼ 10.6 Hz, 1H), 2.92e2.84 (m,
1H), 2.73 (q, J ¼ 6.1 Hz, 2H), 2.64e2.59 (m, 1H), 2.58e2.54 (m, 1H),
2.54e2.52 (m, 2H), 2.39e2.30 (m, 2H), 2.15e1.92 (m, 1H).
(500 MHz, DMSO‑d6)
d 11.09 (s, 1H), 10.85 (s, 1H), 9.84 (s, 1H), 9.06
(s, 1H), 8.74 (s, 1H), 8.05 (d, J ¼ 6.9 Hz, 1H), 7.73 (d, J ¼ 8.9 Hz, 1H),
7.58 (dd, J ¼ 8.6, 7.0 Hz, 1H), 7.50 (t, J ¼ 9.1 Hz, 1H), 7.37 (s, 1H), 7.10
(d, J ¼ 8.7 Hz, 1H), 7.02 (d, J ¼ 7.0 Hz, 1H), 6.89 (dd, J ¼ 17.0, 10.3 Hz,
1H), 6.53 (t, J ¼ 6.0 Hz, 1H), 6.35 (dd, J ¼ 17.0, 1.9 Hz, 1H), 5.85 (dd,
J ¼ 10.1, 1.9 Hz, 1H), 5.05 (dd, J ¼ 12.8, 5.4 Hz, 1H), 4.46 (d,
J ¼ 14.1 Hz, 1H), 4.36 (t, J ¼ 5.8 Hz, 2H), 4.07 (d, J ¼ 14.1 Hz, 1H),
3.62e3.51 (m, 4H), 3.11e3.00 (m, 3H), 2.99e2.85 (m, 3H),
2.65e2.55 (m, 2H), 2.41e2.26 (m, 5H), 2.08e1.99 (m, 1H), 1.62e1.55
(m, 2H), 1.55e1.48 (m, 2H), 1.41e1.30 (m, 4H).13C NMR (126 MHz,
4.1.13. N-(4-((3-chloro-4-fluorophenyl)amino)-7-(3-(4-(4-((2-(2,6-
dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)butanoyl)
piperazin-1-yl)propoxy)quinazolin-6-yl)acrylamide(SIAIS123)
The title compound (yellow solid, 6.7 mg, 39% yield) was syn-
thesized according to procedures for the preparationþ 1of
SIAIS121.m.p.207.4e208.9 ꢀC. MS(ESI) m/z: 826.2875 [MþH] . H
DMSO‑d6)
d 173.34, 171.50, 170.60, 169.44, 167.79, 164.31, 159.15,
156.51,150.85, 146.91, 136.81, 132.64, 131.94, 129.18, 128.54, 127.02,
125.65 (d, J ¼ 6.8 Hz), 119.74,119.59, 117.68,117.46,117.29, 110.91,
109.44, 107.68, 67.17, 53.21, 51.53, 51.11, 49.00, 42.27,40.49,38.34,
32.32, 31.43, 29.05, 28.92, 26.62, 24.94, 23.29, 22.62.
NMR (500 MHz, DMSO‑d6)
d 11.10 (s, 1H), 9.86 (s, 1H), 9.11 (s, 1H),
8.82 (s, 1H), 8.03 (s, 1H), 7.71 (s, 1H), 7.60 (dd, J ¼ 8.6, 7.1 Hz, 1H),
7.55e7.48 (m, 1H), 7.36 (s, 1H), 7.18 (d, J ¼ 8.6 Hz, 1H), 7.03 (d,
J ¼ 7.0 Hz,1H), 6.88 (s,1H), 6.68 (s,1H), 6.36 (dd, J ¼ 17.0,1.9 Hz,1H),
5.87 (dd, J ¼ 10.1, 1.9 Hz, 1H), 5.05 (dd, J ¼ 12.8, 5.5 Hz, 1H), 4.49 (d,
J ¼ 14.1 Hz,1H), 4.36 (d, J ¼ 6.6 Hz, 2H), 4.08 (d, J ¼ 14.3 Hz,1H), 3.53
(d, J ¼ 26.3 Hz, 6H), 3.08 (s, 2H), 3.00e2.81 (m, 2H), 2.65e2.56 (m,
2H), 2.54 (s, 2H), 2.38e2.30 (m, 3H), 2.06e1.98 (m, 1H), 1.88e1.78
(m, J ¼ 7.2 Hz, 2H).
4.2. Biology
4.2.1. Cell lines and agents
Non-small cell lung cancer cell line HCC827(EGFR exon 19
deletion), H1975 (EGFR L858R þ T790M) and A549 (EGFR WT) were
purchased from ATCC. EGFR deletion mutant lung cancer cell line
PC9 (EGFR exon 19 deletion), PC9Brc1 (EGFR exon 19
deletion þ T790M) were received as previously [37]. All lung cancer
cells were maintained in RPMI 1640 medium supplemented with
10% fetal calf serum and 1% penicillin/streptomycin. Cells were
cultured in a humidified atmosphere containing 5% CO2 at 37 ꢀC. All
cells were tested asmycoplasma negative regularly with correct cell
identification (STR analysis).
4.1.14. N-(4-((3-chloro-4-fluorophenyl)amino)-7-(3-(4-(5-((2-(2,6-
dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)pentanoyl)
piperazin-1-yl)propoxy)quinazolin-6-yl)acrylamide(SIAIS124)
The title compound (yellow solid, 7.1 mg, 42% yield) was syn-
thesized according to procedures for the preparationþ 1of
SIAIS121.m.p.259.4e260.9 ꢀC. MS(ESI) m/z: 840.3038 [MþH] . H
NMR (500 MHz, DMSO‑d6)
d 11.09 (s, 1H), 9.94 (s, 1H), 9.16 (s, 1H),
8.87 (s, 1H), 8.02e7.97 (m, 1H), 7.68 (dt, J ¼ 6.9, 3.6 Hz, 1H),
7.63e7.49 (m, 2H), 7.45e7.40 (m, 1H), 7.11 (d, J ¼ 8.7 Hz,1H), 7.03 (d,
J ¼ 7.0 Hz, 1H), 6.95 (t, J ¼ 8.9 Hz, 1H), 6.57 (s, 1H), 6.36 (dd, J ¼ 17.0,
1.9 Hz,1H), 5.89e5.83 (m,1H), 5.05 (dd, J ¼ 12.8, 5.4 Hz,1H), 4.47 (d,
J ¼ 14.1 Hz, 1H), 4.38 (t, J ¼ 5.7 Hz, 2H), 4.09 (d, J ¼ 14.3 Hz, 1H), 3.55
(s, 4H), 3.19e3.02 (m, 3H), 2.98e2.83 (m, 2H), 2.65e2.55 (m, 2H),
2.54e2.52 (m, 2H), 2.44 (d, J ¼ 6.9 Hz, 2H), 2.38e2.32 (m, 2H),
2.09e2.01 (m, 1H), 1.61e1.54 (m, 4H).
The antibodies against EGFR (#4267S, 1:1000), P-EGFR (#3777S,
1:1000) alpha-tubulin (#9099S, 1:1000), beta-actin(#1260S,
1:1000) and GAPDH (#2118S, 1:1000) were purchased from Cell
Signaling Technology. LC3 antibodies were purchased from Sigma
Aldrich (#L7543, 1:1000).
MLN4924 (#S7109) was purchased from Selleck. MG132
(#M832899-5 mg), Cell Counting Kit-8 (CCK-8)(#C0005) and Car-
filzomib (#T1795, 5 mg) were purchased from Targetmol. Bafilo-
mycin A1(#88899-55-2, 1 mg) was obtained from Bide Pharmatech
Ltd. CellTrace™ CFSE Cell Proliferation Kit for flow cytometry was
purchased from ThermoFisher Scientific (#C34554).
4.1.15. N-(4-((3-chloro-4-fluorophenyl)amino)-7-(3-(4-(6-((2-(2,6-
dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)hexanoyl)
piperazin-1-yl)propoxy)quinazolin-6-yl)acrylamide(SIAIS125)
The title compound (yellow solid, 7.9 mg, 46% yield) was syn-
thesized according to procedures for the preparation of
SIAIS121.m.p.>300 ꢀC(decomposed).MS(ESI) m/z: 854.3187
4.2.2. Generation of stable cell line
Lentiviral vector containing human EGFR cDNA was mutated at
L858R/T790M
the sites of L858 and T790 to generate EGFR
double
[MþH]þ. 1H NMR (500 MHz, Methanol-d4)
d
9.05 (s, 1H), 8.65 (s,
mutant plasmid with KOD plus mutagenesis kit (Toyobo).
EGFRL858R/T790M plasmid was firstly packed in 293Tcells to generate
lentivirus, and then transduced Ba/F3 cells to establish stable Ba/F3
LT (EGFRL858R/T790M) cell line. Ba/F3 LT cells were cultured in DMEM
supplemented with 10% heat inactivated fetal bovine serum (FBS),
and 1% penicillin/streptomycin.
1H), 7.84 (dd, J ¼ 6.6, 2.6 Hz,1H), 7.62e7.54 (m,1H), 7.45 (dd, J ¼ 8.6,
7.1 Hz, 1H), 7.32e7.23 (m, 2H), 6.94 (dd, J ¼ 12.1, 7.8 Hz, 2H), 6.72
(dd, J ¼ 16.9, 10.3 Hz, 1H), 6.41 (dd, J ¼ 16.9, 1.5 Hz, 1H), 5.80 (dd,
J ¼ 10.3, 1.6 Hz, 1H), 4.95 (dd, J ¼ 12.4, 5.5 Hz, 1H), 4.38 (t, J ¼ 5.8 Hz,
2H), 4.11 (s,1H),3.56 (s, 3H),3.37 (t, J ¼ 8.0 Hz, 2H), 3.26 (t, J ¼ 6.8 Hz,
2H), 3.21e2.89 (m, 4H), 2.81e2.71 (m, 1H), 2.67e2.54 (m, 2H),
2.43e2.35 (m, 4H), 2.04e1.97 (m, 1H), 1.65e1.55 (m, 4H), 1.45e1.38
A GFP containing lenti-vector was firstly packed in 293T cells to
make lentivirus. Then, the lentiviruses were added to culture me-
(m, 2H).13C NMR (126 MHz, DMSO‑d6)
169.44, 167.79, 164.31, 159.12, 156.48,150.92, 149.75, 146.91, 136.83,
d
173.35, 171.45, 170.60,
dium with polybrene (8
mg/mL) to infect H1975 cells. Cells that
stably expresedGFP proteinswere sorted by flow cytometry.
12

X. Qu, H. Liu, X. Song et al.
European Journal of Medicinal Chemistry 218 (2021) 113328
4.2.3. Cell proliferation inhibition assay
scratch areas in each photo were analyzed with Image J software.
The anti-proliferative activities of compounds in different cell
lines were determined by CCK-8 reagent. Different cells were firstly
seeded in 96-well plates at the density of 3000 cells/well supple-
4.2.9. Transwell assay
Matrigel (Biocoat 354234) was firstly diluted 10 times with
mented with 100
were treated with different concentrations of compounds and
incubated for further 72 h. Then, cells were incubated with 10 L of
CCK-8 solution for 1e4 h, and the optical density of each well was
determined by EnVision multi-mode plate reader at 450 nm
wavelength.
mL culture medium per well. One day later, cells
serum free culture medium to make coating buffer. 100
buffer was then added into transwell insert (Corning FluoroBlok™
mL coating
m
24 well plate permeable Support with 8.0 mm Colored PET mem-
brane, #351152) and incubated at 37 ꢀC for 1 h. H1975 cells were
digested with tripsin and washed with PBS. After re-suspended in
150
m
L serum-free medium with different compounds, cells were
L RPMI1640 full growth
then seeded in Transwell inserts. 200
m
4.2.4. Western blot
medium were added into the lower chambers. The 24-well-plates
were incubated in a humidified atmosphere containing 5% CO2 at
37 ꢀC. InvasedH1975-GFP cells were visualized with Fluorescence
inverted microscope. Five areas were randomly chosen to take
photos from each Transwell. Image J software was used to quantify
the number of cells for each treatment. Data from 3 different re-
peats were presented.
Cells (1e3X105/well) were seeded in 12-well plates. One day
later, different concentrations of compounds were added to cells for
indicated time, and then cells lysates were collected with RIPA lysis
buffer containing protease inhibitors. Equal amount of protein from
each treatment (15
transferred to nitrocellulose membrane (AmershamTM ProtranTM
0.4 m NC, A21886265). Then, membranes were sequentially
mg) was separated by 8% SDS-PAGE gel and then
m
incubated with primary and secondary antibodies (Cell Signaling).
Chemistar ECL western blotting substrates were used to visualize
protein levels with an Amersham Imager 600 imaging system.
4.2.10. Statistical analysis
Data was analyzed using GraphPad Prism 8.0. DC50 (concen-
tration that caused deletion of 50% of EGFR) and IC50 (concentra-
tion that resulted in 50% of cell growth inhibition) were calculated
through the nonlinear regression-“log (inhibitor) vs response”
analytical protocol. Statistical test was performed through “t tests
(and nonparametric tests, *P < 0.05, **P < 0.01, ***P < 0.001)”.
4.2.5. CFSE staining
3X 10^6 H1975 cells were re-suspended in
warmed(37 ꢀC) phosphate-buffered saline (PBS), then mixed with
1 mL CFSE(10 M/mL: 2 L 5 mM CFSE stock solution diluted with
1 mL pre-
m
m
1 mL PBS) and incubated for 10 minutesat room temperature
(protected from light). Added 8 mL completemedium to the cells
and incubated for 5 minuteson ice. Pelleted the cells by centrifu-
gation and re-suspended them in fresh, ice-cold complete medium,
repeated the procedure for 3 times. Planted the cells in a 12-well-
plate with 1X10^5 cells/well. 24 h after cell seeding, treated above
cells with degraders at indicated concentrations and detected the
CFSE Fluorescence with the CytoFLEX at indicated time (H1975 cells
without CFSE staining were used as the negative control). Data
were analyzed with FlowJo software (BD).
Author contribution
XS and XY designed the work. XY was in charge of protac design
and synthesis, and XS was responsible for the biological charac-
terization of protac molecules. XQ, XS and HZ contributed to bio-
logical data collection. HL, NS and XQ synthesized the compound.
XS, XY, XQ and HL analyzed the data. XS, XQ and HL wrote the
manuscript. XS, XY and BJ supervised the whole work.
Declaration of competing interest
4.2.6. Cell apoptosis 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.
H1975 cells (8 ꢂ 104/well) were seeded in 12-well plates, and
one day later different compounds were added at indicated con-
centrations. After 72 h-incubation, cells were collected and evalu-
ated with an Annexin V-FITC apoptosis detection kit (Vazyme)
according to the instructions. The extent of apoptosis was deter-
mined by Flow Cytometry.
Acknowledgement
This research was supported by National Natural Science
Foundation of China (Grant No. 82072592 to X.S.), Science and
Technology Commission of Shanghai Municipality (Grant No.
20S11903100 to X.S. and Grant No.19ZR1433600 to X.Y.) and
ShanghaiTech University (to B.J.).
4.2.7. Cell cycle assay
H1975 cells (1 ꢂ 105/well) were seeded into 6-well plates. One
day later, different compounds were added to cells at indicated
concentrations. 48 h later, cells were collected and washed twice
with pre-cooled PBS. Cells were then re-suspended in 250
m
L pre-
Appendix A. Supplementary data
cooled PBS with dropwise addition of 50
mL cold ethanol. Cells
were then fixed at ꢁ20 ꢀC for over 24 h. Samples were then pelleted
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113328.
at 5000 rpm, and the supernatant was discarded completely. After
treated with 400 mL PI/RNase (4A Bioteck Co., Ltd, FXP0211-200),
cells were kept in dark place and incubated at 37 ꢀC for 30 min.
The proportion of cells at different phase were analyzed on a flow
cytometer (CytoFLEX, Beckman Coulter).
References
[1] L.A. Torre, F. Bray, R.L. Siegel, J. Ferlay, J. Lortet-Tieulent, A. Jemal, Global
cancer statistics, 2012, CA A Cancer J. Clin. 65 (2) (2015) 87e108.
[2] A. Jemal, F. Bray, M.M. Center, J. Ferlay, E. Ward, D. Forman, Global cancer
statistics, CA A Cancer J. Clin. 61 (2) (2011) 69e90.
[3] A. Kuykendall, A. Chiappori, Advanced EGFR mutation-positive non-small-cell
lung cancer: case report, literature review, and treatment recommendations,
Cancer Control 21 (1) (2014) 67e73.
[4] H. Otani, S. Toyooka, J. Soh, H. Yamamoto, H. Suehisa, N. Kobayashi, H. Gobara,
H. Mimura, K. Kiura, Y. Sano, S. Kanazawa, H. Date, Detection of EGFR gene
mutations using the wash fluid of CT-guided biopsy needle in NSCLC patients,
4.2.8. Cell migration assay
H1975 cells (3 ꢂ 105/well) were firstly seeded into 24-well
plates, and then treated with different compounds at indicated
concentrations. After 12 h and 24 h compound-administration,
photos were randomly taken from different areas for evaluation
and three biological repeats were set for each treatment. The
13

X. Qu, H. Liu, X. Song et al.
European Journal of Medicinal Chemistry 218 (2021) 113328
J. Thorac. Oncol. 3 (5) (2008) 472e476.
[21] M. Cheng, X. Yu, K. Lu, L. Xie, L. Wang, F. Meng, X. Han, X. Chen, J. Liu, Y. Xiong,
J. Jin, Discovery of potent and selective epidermal growth factor receptor
(EGFR) bifunctional small-molecule degraders, J. Med. Chem. 63 (3) (2020)
1216e1232.
[22] H. Zhang, H.Y. Zhao, X.X. Xi, Y.J. Liu, M. Xin, S. Mao, J.J. Zhang, A.X. Lu,
S.Q. Zhang, Discovery of potent epidermal growth factor receptor (EGFR)
degraders by proteolysis targeting chimera (PROTAC), Eur. J. Med. Chem. 189
(2020) 112061.
[23] X. Zhang, F. Xu, L. Tong, T. Zhang, H. Xie, X. Lu, X. Ren, K. Ding, Design and
synthesis of selective degraders of EGFR(L858R/T790M) mutant, Eur. J. Med.
Chem. 192 (2020) 112199.
[24] H.Y. Zhao, X.Y. Yang, H. Lei, X.X. Xi, S.M. Lu, J.J. Zhang, M. Xin, S.Q. Zhang,
Discovery of potent small molecule PROTACs targeting mutant EGFR, Eur. J.
Med. Chem. 208 (2020) 112781.
[25] A.K. Gandhi, J. Kang, C.G. Havens, T. Conklin, Y. Ning, L. Wu, T. Ito, H. Ando,
M.F. Waldman, A. Thakurta, A. Klippel, H. Handa, T.O. Daniel, P.H. Schafer,
R. Chopra, Immunomodulatory agents lenalidomide and pomalidomide co-
stimulate T cells by inducing degradation of T cell repressors Ikaros and
Aiolos via modulation of the E3 ubiquitin ligase complex CRL4(CRBN.), Br. J.
Haematol. 164 (6) (2014) 811e821.
[26] P.H. Schafer, A.K. Gandhi, M.A. Loveland, R.S. Chen, H.W. Man,
P.P. Schnetkamp, G. Wolbring, S. Govinda, L.G. Corral, F. Payvandi,
G.W. Muller, D.I. Stirling, Enhancement of cytokine production and AP-1
transcriptional activity in T cells by thalidomide-related immunomodulatory
drugs, J. Pharmacol. Exp. Therapeut. 305 (3) (2003) 1222e1232.
[27] J. Lu, Y. Qian, M. Altieri, H. Dong, J. Wang, K. Raina, J. Hines, J.D. Winkler,
A.P. Crew, K. Coleman, C.M. Crews, Hijacking the E3 ubiquitin ligase cereblon
to efficiently target BRD4, Chem Biol 22 (6) (2015) 755e763.
[28] J.B. Smaill, G.W. Rewcastle, J.A. Loo, K.D. Greis, O.H. Chan, E.L. Reyner, E. Lipka,
H.D. Showalter, P.W. Vincent, W.L. Elliott, W.A. Denny, Tyrosine kinase in-
hibitors. 17. Irreversible inhibitors of the epidermal growth factor receptor: 4-
(phenylamino)quinazoline- and 4-(phenylamino)pyrido[3,2-d]pyrimidine -6-
acrylamides bearing additional solubilizing functions, J. Med. Chem. 43 (7)
(2000) 1380e1397.
[29] H.J. Kim, S.Y. Kim, D.H. Kim, J.S. Park, S.H. Jeong, Y.W. Choi, C.H. Kim, Crosstalk
between HSPA5 arginylation and sequential ubiquitination leads to AKT
degradation through autophagy flux, Autophagy (2020) 1e19.
[5] S.Y. Ha, S.J. Choi, J.H. Cho, H.J. Choi, J. Lee, K. Jung, D. Irwin, X. Liu, M.E. Lira,
M. Mao, H.K. Kim, Y.S. Choi, Y.M. Shim, W.Y. Park, Y.L. Choi, J. Kim, Lung cancer
in never-smoker Asian females is driven by oncogenic mutations, most often
involving EGFR, Oncotarget 6 (7) (2015) 5465e5474.
[6] S.V. Sharma, D.W. Bell, J. Settleman, D.A. Haber, Epidermal growth factor re-
ceptor mutations in lung cancer, Nat. Rev. Canc. 7 (3) (2007) 169e181.
[7] A.M. Chapman, K.Y. Sun, P. Ruestow, D.M. Cowan, A.K. Madl, Lung cancer
mutation profile of EGFR, ALK, and KRAS: meta-analysis and comparison of
never and ever smokers, Lung Canc. 102 (2016) 122e134.
[8] C. Zhou, Y.-L. Wu, G. Chen, J. Feng, X.-Q. Liu, C. Wang, S. Zhang, J. Wang,
S. Zhou, S. Ren, S. Lu, L. Zhang, C. Hu, C. Hu, Y. Luo, L. Chen, M. Ye, J. Huang,
X. Zhi, Y. Zhang, Q. Xiu, J. Ma, L. Zhang, C. You, Erlotinib versus chemotherapy
as first-line treatment for patients with advanced EGFR mutation-positive
non-small-cell lung cancer (OPTIMAL, CTONG-0802): a multicentre, open-
label, randomised, phase 3 study, Lancet Oncol. 12 (8) (2011) 735e742.
[9] T.J. Lynch, D.W. Bell, R. Sordella, S. Gurubhagavatula, R.A. Okimoto,
B.W. Brannigan, P.L. Harris, S.M. Haserlat, J.G. Supko, F.G. Haluska, D.N. Louis,
D.C. Christiani, J. Settleman, D.A. Haber, Activating mutations in the epidermal
growth factor receptor underlying responsiveness of non-small-cell lung
cancer to gefitinib, N. Engl. J. Med. 350 (21) (2004) 2129e2139.
[10] J.G. Paez, P.A. Janne, J.C. Lee, S. Tracy, H. Greulich, S. Gabriel, P. Herman,
F.J. Kaye, N. Lindeman, T.J. Boggon, K. Naoki, H. Sasaki, Y. Fujii, M.J. Eck,
W.R. Sellers, B.E. Johnson, M. Meyerson, EGFR mutations in lung cancer:
correlation with clinical response to gefitinib therapy, Science 304 (5676)
(2004) 1497e1500.
[11] W. Pao, V. Miller, M. Zakowski, J. Doherty, K. Politi, I. Sarkaria, B. Singh,
R. Heelan, V. Rusch, L. Fulton, E. Mardis, D. Kupfer, R. Wilson, M. Kris,
H. Varmus, EGF receptor gene mutations are common in lung cancers from
"never smokers" and are associated with sensitivity of tumors to gefitinib and
erlotinib, Proc. Natl. Acad. Sci. U. S. A. 101 (36) (2004) 13306e13311.
[12] K.S. Thress, C.P. Paweletz, E. Felip, B.C. Cho, D. Stetson, B. Dougherty, Z. Lai,
A. Markovets, A. Vivancos, Y. Kuang, D. Ercan, S.E. Matthews, M. Cantarini,
J.C. Barrett, P.A. Janne, G.R. Oxnard, Acquired EGFR C797S mutation mediates
resistance to AZD9291 in non-small cell lung cancer harboring EGFR T790M,
Nat. Med. 21 (6) (2015) 560e562.
[13] G.R. Oxnard, Y. Hu, K.F. Mileham, H. Husain, D.B. Costa, P. Tracy, N. Feeney,
L.M. Sholl, S.E. Dahlberg, A.J. Redig, D.J. Kwiatkowski, M.S. Rabin, C.P. Paweletz,
K.S. Thress, P.A. Janne, Assessment of resistance mechanisms and clinical
implications in patients with EGFR T790M-positive lung cancer and acquired
resistance to osimertinib, JAMA Oncol 4 (11) (2018) 1527e1534.
[30] L. Duan, Y. Miura, M. Dimri, B. Majumder, I.L. Dodge, A.L. Reddi, A. Ghosh,
N. Fernandes, P. Zhou, K. Mullane-Robinson, N. Rao, S. Donoghue, R.A. Rogers,
D. Bowtell, M. Naramura, H. Gu, V. Band, H. Band, Cbl-mediated ubiq-
uitinylation is required for lysosomal sorting of epidermal growth factor re-
ceptor but is dispensable for endocytosis, J. Biol. Chem. 278 (31) (2003)
28950e28960.
[31] G. Levkowitz, H. Waterman, E. Zamir, Z. Kam, S. Oved, W.Y. Langdon,
L. Beguinot, B. Geiger, Y. Yarden, c-Cbl/Sli-1 regulates endocytic sorting and
ubiquitination of the epidermal growth factor receptor, Genes Dev. 12 (23)
(1998) 3663e3674.
[14] C. Mehlman, J. Cadranel, G. Rousseau-Bussac, R. Lacave, A. Pujals, N. Girard,
C. Callens, V. Gounant, N. Theou-Anton, S. Friard, J. Tredaniel, H. Blons,
C. Dujon, B. Duchemann, P.O. Schischmanoff, T. Chinet, E. Giroux Leprieur,
Resistance mechanisms to osimertinib in EGFR-mutated advanced non-small-
cell lung cancer: a multicentric retrospective French study, Lung Canc. 137
(2019) 149e156.
[15] H.A. Yu, M.E. Arcila, N. Rekhtman, C.S. Sima, M.F. Zakowski, W. Pao, M.G. Kris,
V.A. Miller, M. Ladanyi, G.J. Riely, Analysis of tumor specimens at the time of
acquired resistance to EGFR-TKI therapy in 155 patients with EGFR-mutant
lung cancers, Clin. Canc. Res. 19 (8) (2013) 2240e2247.
[16] 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
(15) (2001) 8554e8559.
[17] 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 (1) (2018) 67e77 e3.
[18] T. Ishida, A. Ciulli, E3 ligase ligands for PROTACs: how they were found and
how to discover new ones, SLAS Discov (2020), https://doi.org/10.1177/
2472555220965528. Epub ahead of print. PMID: 33143537.
[19] N. Sun, C. Ren, Y. Kong, H. Zhong, J. Chen, Y. Li, J. Zhang, Y. Zhou, X. Qiu, H. Lin,
X. Song, X. Yang, B. Jiang, Development of a Brigatinib degrader (SIAIS117) as a
potential treatment for ALK positive cancer resistance, Eur. J. Med. Chem. 193
(2020) 112190.
[20] D. Petrylak, A. Tewari, First-in-human phase I study of ARV-110, an androgen
receptor (AR) PROTAC degrader in patients (pts) with metastatic castrate-
resistant prostate cancer (mCRPC) following enzalutamide (ENZ) and/or
abiraterone (ABI), J. Clin. Oncol. 2020 (2020 May) 165s.
[32] Y.X. Zhu, E. Braggio, C.X. Shi, K.M. Kortuem, L.A. Bruins, J.E. Schmidt,
X.B. Chang, P. Langlais, M. Luo, P. Jedlowski, B. LaPlant, K. Laumann, R. Fonseca,
P.L. Bergsagel, J. Mikhael, M. Lacy, M.D. Champion, A.K. Stewart, Identification
of cereblon-binding proteins and relationship with response and survival
after IMiDs in multiple myeloma, Blood 124 (4) (2014) 536e545.
[33] P. Jaakkola, D.R. Mole, Y.M. Tian, M.I. Wilson, J. Gielbert, S.J. Gaskell, A. von
Kriegsheim, H.F. Hebestreit, M. Mukherji, C.J. Schofield, P.H. Maxwell,
C.W. Pugh, P.J. Ratcliffe, Targeting of HIF-alpha to the von Hippel-Lindau
ubiquitylation complex by O2-regulated prolyl hydroxylation, Science 292
(5516) (2001) 468e472.
[34] 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 (2) (2016) 807e810.
[35] K.M. DeMars, C. Yang, C.I. Castro-Rivera, E. Candelario-Jalil, Selective degra-
dation of BET proteins with dBET1, a proteolysis-targeting chimera, potently
reduces pro-inflammatory responses in lipopolysaccharide-activated micro-
glia, Biochem. Biophys. Res. Commun. 497 (1) (2018) 410e415.
[36] S.M. Banik, K. Pedram, S. Wisnovsky, G. Ahn, N.M. Riley, C.R. Bertozzi, Lyso-
some-targeting chimaeras for degradation of extracellular proteins, Nature
584 (7820) (2020) 291e297.
[37] X. Song, P.D. Fan, A. Bantikassegn, U. Guha, D.W. Threadgill, H. Varmus,
K. Politi, ERBB3-independent activation of the PI3K pathway in EGFR-mutant
lung adenocarcinomas, Cancer Res 75 (6) (2015) 1035e1045.
14