Discovery and biological evaluation of proteolysis targeting chimeras (PROTACs) as an EGFR degraders based on osimertinib and lenalidomide

Article abstract of DOI:10.1016/j.bmcl.2020.127167

Epidermal growth factor receptor (EGFR) is one of the important and valuable drug targets. Overexpression of EGFR is associated with the development of many types of cancer. In this study, three PROTACs small molecules (16a–16c) were designed, synthesized and evaluated for their cytotoxicity against the growth in different NSCLC cell line and the degradation effect. The bioassay results indicated that 16c has a good inhibition in PC9 cells and H1975 cells, and the corresponding IC₅₀ value was 0.413 μM and 0.657 μM, respectively. Western blotting results demonstrated that compound 16c could serve as an effective EGFR^del19-targeting degrader in PC9 cells.

Full text of DOI:10.1016/j.bmcl.2020.127167

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Discovery and biological evaluation of proteolysis targeting chimeras (PRO‐
TACs) as an EGFR degraders based on osimertinib and lenalidomide
Kailun He, Zhuo Zhang, Wenbing Wang, Xiaoliang Zheng, Xiaoju Wang,
Xingxian Zhang
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DOI:
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https://doi.org/10.1016/j.bmcl.2020.127167
BMCL 127167
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Bioorganic & Medicinal Chemistry Letters
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Please cite this article as: He, K., Zhang, Z., Wang, W., Zheng, X., Wang, X., Zhang, X., Discovery and
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Discovery and biological evaluation of proteolysis targeting chimeras (PROTACs)
as an EGFR degraders based on osimertinib and lenalidomide
Kailun He a, Zhuo Zhang a, Wenbing Wang a, Xiaoliang Zheng b, Xiaoju Wangb, *, Xingxian Zhang a,*
(a College of Pharmaceutical Sciences, Zhejiang University of Technology, Hangzhou, Zhejiang 310014)
(b Center for Molecular Medicine, Zhejiang Academy of Medical Sciences, Hangzhou, Zhejiang 310032)
ARTICLE INFO
ABSTRACT
Keywords:
Proteolysis targeting chimera
CRBN
EGFR-TK degrader
Osimertinib
Lenalidomide
Epidermal growth factor receptor (EGFR) is one of the important and valuable drug targets.
Overexpression of EGFR is associated with the development of many types of cancer. In this
study, three PROTACs small molecules (16a-16c) were designed, synthesized and evaluated for
their cytotoxicity against the growth in different NSCLC cell line and the degradation effect.
The bioassay results indicated that 16c has a good inhibition in PC9 cells and H1975 cells, and
the corresponding IC50 value was 0.413 μM and 0.657 μM, respectively. Western blotting results
demonstrated that compound 16c could serve as an effective EGFRdel19-targeting degrader in
PC9 cells.
.
Epidermal growth factor receptor (EGFR), a receptor tyrosine kinase (RTK), belongs to the ErbB family, which plays a
fundamentally important role in cell proliferation, opposing apoptosis, survival and migration1. Overexpression of EGFR is associated
with the development of many types of cancer, such as pancreatic cancer, breast cancer, glioblastoma multiforme, head and neck cancer,
and non-small cell lung cancer (NSCLC)2. Overexpression and mutation of EGFR are closely related to the development of NSCLC,
and is also one of the important and valuable drug targets in NSCLC. The activating mutations in the EGFR tyrosine kinase domain
(L858R mutation and exon-19 deletion) have been identified as oncogenic drivers for NSCLC3, and the first-generation EGFR tyrosine
kinase inhibitors (EGFR-TKIs) gefitinib4 and erlotinib5 have approved for NSCLC patients with EGFR activation mutations. However,
most of NSCLC patients have developed acquired resistance after 9 to14 months with gefitinib or erlotinib clinical therapy treatment6, 7
,
a secondary threonine790 to methionine790 mutation (T790M), is also called “gatekeeper” T790M mutation8,9. Therefore, the
second-generation EGFR-TKIs afatinib10 and the third-generation EGFR-TKIs osimertinib (AZD9291)11 were developed to overcome
the resistance. In particular, osimertinib have achieved significant clinical response rate in NSCLC patients with EGFR T790M12, 13
.
However, there are several studies reported a tertiary Cys797 to Ser797 (C797S) point mutation with osimertinib clinical therapy up to
now14.
Proteolysis Targeting Chimeras (PROTACs) are heterobifunctional small molecules, first proposed by Crews groups15. It contains
one ligand for the protein of interest (POI), another for E3 ubiquitin ligase (cereblon (CRBN), von Hippel-Lindau (VHL) and mouse
double minute 2 (MDM2) et al) and connected by a linker16. Different with other small molecule inhibitors, PROTACs can target and
degrade the proteins rather than inhibits them17. At present, a series of small molecular compounds were synthesized and successfully
targeted and induced the degradation of kinases based on PROTACs technology, such as AR18, 19, BTK20, 21, ER22, 23 and BET24, 25, etc.
Specifically, the drug of ARV-110 targeted to the androgen receptors (AR) has been approved for clinical phase I and shows a good
tolerance26.
Epidermal growth factor-RTK (EGF-RTK), as a transmembrane protein, is hard to be degraded. Crews groups firstly demonstrated
that RTKs, such as HER2, c-Met and EGFR, were able to be degraded induced by PROTACs 27. In this study, lapatinib, gefitinib and
afatinib were used as a ligand for the POI and von Hippel-Lindau (VHL) was selected as an E3 ligase recruiting ubiquitin. Recently, the
Zhang group reported a series of PROTAC compounds to induce the degradation of EGFR on the basis of a novel fourth-generation
EGFR-TKI and VHL28. The Jin and Xiong groups described the discovery of a novel E3 ligase VHL-recruiting EGFR degrader (MS39)
and E3 ligase CRBN-recruiting EGFR degrader (MS154) base on gefitinib29. (Answer 4 to reviewer 1)
We employed the binding mode data that a published T790M structure (PDB code: 3IKA) with osimertinib (Fig. 1). (Answer 4 to
editor) The X-ray crystal structure of EGFRT790M bound to osimertinib showed that the acrylamide group of osimertinib forms a
covalent bond to Cys797 of EGFRT790M, the aminopyrimidine core forms two hydrogen bonds with Met793, the methylated indole is
adjacent to the gatekeeper Met790 and the dimethylamine moiety is exposed to the solvent channel 11, 30. (Answer 1 to reviewer 1) Thus,
the dimethylamine side chain in AZD9291 was considered as the binding site to lenalidomide via a linker for the design of potential

EGFR degraders. (Answer 2 to reviewer 1) To the best of our knowledge, studies concerning their discovery and biological evaluation
of EGFR degradation induced by PROTACs are relatively limited. Herein, we wish to develop a novel class of small-molecule EGFR
degraders employing lenalidomide PROTAC with EGFR inhibitor osimertinib as POI ligand through the different linkers. As well, their
corresponding biological activity was evaluated, such as anti-proliferative activity, degradation of EGFR and cell health and cycle.
Fig. 1. The binding interactions between osimertinib and EGFRT790M
The synthetic route of compounds 6a-6c was showed in Scheme 1. Compounds 1a-1c reacted with tosyl chloride (TsCl) provided
2a-2c, followed by the Williamson alkylation with tert-butyl bromoacetate to give 3a-3c in the presence of KOH and TBAB. The
treatment of 3a-3c with NaI in acetone produced the iodide compounds 4a-4c, which was subjected to remove the Boc- protecting
group by TFA. Then, the compounds 5a-5c reacted with oxaloyl chloride and in situ amidated with lenalidomide in one pot to give
compounds 6a-6c.
The synthetic route of intermediate 15 was described in Scheme 2. Nucleophilic substitution of compound 7 and 8 produced 9 under
acidic condition in an excellent yield. Subsequently, compound 9 reacted with tert-butyl methyl(2-(methylamino)ethyl) carbamate 10 to
give 11, followed by the reduction of nitro group with the treatment of iron powder and NH4Cl to afford compound 12. Furthermore,
the amidation of 12 with 3-chloropropionyl chloride and then elimination expelled HCl to produce acrylamide derivative 14 in the
presence of triethylamine under reflux. Finally, after removal of Boc- protecting group with treatment of TFA, alkylation of 15 with
iodide compounds 6a-6c resulted in the desired PROTACs 16a-16c, respectively (Scheme 2).
O
i
ii
O
O
O
HO
OH
TsO
OH
iv
TsO
O
n
n
n
O
1a-1c
, n=1-3
2a
c

3a 3c

v
O
OH
O
O
iii
I
O
I
O
n
n
O
O
4a-4c
5a-5c
O
I
O
O
n
NH
O
NH
N
O
O
6a-6c
, n=1-3
Scheme 1. General synthesis of compounds 6a-6c. Reagents and conditions: (i) TsCl, Et3N, CH2Cl2, 0 ℃ to r.t, 35%-48% yield; (ii) tert-butyl bromoacetate,
TBAB, KOH, toluene, r.t, 60%-73% yield; (iii) NaI, acetone, reflux, 92%-97% yield; (iv) TFA, CH2Cl2, r.t, 73%-80% yield; (v) a) (COCl)2, DMF(cat.), 0 ℃; b)
lenalidomide, DMF, r.t, 40%-50%. (Answer 3 to reviewer 1)

O
N
H
N
N
N
H2N
NO2
F
N
ii
O
NO2
O
NO2
10
MeO
F
N
N
N
N
8
N
O
i
N
N
N
N
N
Cl
H
H
OMe
11
OMe
9
7
Cl
N
N
NH2
O
iv
O
NH
O
v
iii
N
N
N
O
N
N
N
O
N
N
N
N
H
H
OMe
12
13
OMe
N
N
O
N
O
NH
O
NH
6a-6c
vi
N
N
N
O
N
NH
vii
N
N
N
N
H
H
OMe
14
OMe
15
O
O
N
N
NH
NH
O
O
O
NH
O
N
O
N
N
n
O
N
N
H
16a-16c,
n = 1-3
Scheme 2. Synthesis of PROTACs 16a~16c. Reagents and conditions: (i) p-TsOH, 1,4-dioxane, reflux, 95% yield; (ii) DIPEA, 90 ℃, 82% yield; (iii) Fe, NH4Cl,
EtOH, H2O, 57% yield; (iv) 3-chloropropionyl chloride, Et3N, DMF, 80% yield; (v) Et3N, MeCN, 80 ℃, 70% yield; (vi) TFA, DCM, 94% yield; (vii) 6a-6c,
DIPEA, DMF, 90 ℃, 53%-62% yield. (Answer 3 to reviewer 1)
Next, we evaluated the biological activity of these compounds in vitro. The antiproliferative activities in vitro of the PROTACs
16a-16c against human cancer cells PC9 cells (EGFRDel19), HCC827 cells (EGFRDel E746-A750, also known as EGFRDel19) and H1975 cells
(EGFRL858R/T790M) were evaluated by MTT assay. The results were summarized in Table 1. As shown in Table 1, the length of the linker
had pronounced effects on anti-proliferative activity. All the tested compounds exhibited good biological activities against human
cancer cells PC9 cells, HCC827 cells and H1975 cells. Especially, compound 16c has simultaneously better inhibitory effect on PC9
cells and H1975 cells and its corresponding IC50 value is 0.413 μM and 0.657 μM, respectively. Furthermore, compounds 16a-16c were
allowed to determine EGFR degradation activity against PC9 cells and H1975 cells.
The western blotting analysis revealed that all the tested compounds 16a-16c displayed moderate to potent degradation activity
against PC9 cells and H1975 cells at the concentration of 10 μM (Fig. 2). According to the anti-proliferative activity of the compounds
16a-16c, compound 16c was preferred to be selected to further determine the degradation of EGFR in PC9 cells. We set six
concentration gradients ranged from 0.1 μM to 10 μM to determine the degradation activity and calculated the DC50 values (Fig. 2B).
The results revealed that compound 16c can induce degradation of EGFR in a concentration dependent manner in PC9 cells. From the
concentration-dependent of compound 16c, we observed a distinct “hook effect’’ on EGFR degradation and reached the maximum
degradation rate (Dmax = 68%) with treatment for 6 h at the concentration of 0.3 μM. The corresponding DC50 value of compound 16c
is 0.161 μM. (Answer 1 to reviewer 2)
Moreover, the time-dependent degradation activities of compound 16c at the concentration of 0.3 μM were also investigated (Fig.
2C). The amount EGFR protein was decreased as the administration time was prolonged. Wherein compound 16c reached the
maximum degradation rate (Dmax = 56%) at 6 h. These data suggests that the degradation of transmembrane protein EGFR is a very
time-consuming process. Unexpectedly, compound 16c stimulated the PC9 cells to cause a stress effect and increased the expression of
EGFR for a certain period of time (Fig. 2C).
In order to further investigate the mechanism of EGFR protein degradation, the proteasome inhibitor MG-132 and lenalidomide
were pretreated with the cells before the treatment of the compound 16c, respectively. As shown in Fig. 2D, the protein degradation
property of compound 16c was disrupted and almost disappeared due to the presence of MG-132 and lenalidomide. The results
suggested that the EGFR protein was likely to be degraded through ubiquitination. In addition, the immunofluorescence microscopy
analysis illustrated that compound 16c could also effectively induce the degradation of EGFR protein (Fig. 3).
Table 1.
Antiproliferative activities of compounds against different cells (n = 3, X ± SD).
IC50(μM)
compound
PC9 Cells
HCC827 Cells
1.874 ± 0.098
1.611 ± 0.139
1.344 ± 0.112
H1975 Cells
1.388 ± 0.556
1.698 ± 0.614
0.657 ± 0.008
16a
16b
16c
1.425 ± 0.052
1.117 ± 0.105
0.413 ± 0.087

To explore the effects of cell health with degrader 16c, the PC9 cells cycle and apoptosis were analyzed by ImageXpress Micro
Confocal/Widefield after the treatment with 0.5 μM of compound 16c for 24 h. As shown in Fig. 4, the results demonstrated that the
degrader 16c could arrest the PC9 cells in G0/G1 phase and caused 38.6% of apoptosis in PC9 cell.
In conclusion, we have developed a new class of PROTACs EGFR degraders which were based on osimertinib and lenalidomide. All
of the compounds were evaluated for antiproliferative effects in vitro, and showed a significant inhibitory effect in PC9 cells, HCC827
cells and H1975 cells. The western blotting assays analyzed the effect of EGFR degradation and the results showed that compound 16c
could effectively degrade EGFR protein through ubiquitination and reached the maximum degradation rate (Dmax = 68%) in PC9 cells.
Meanwhile, compound 16c could significantly induce the apoptosis of PC9 cells and arrest the cells in G0/G1 phase. These findings
demonstrated that EGFRDel19 in PC9 cells can be effectively targeted for degradation by our designed PROTACs. On the basis of the
potent degradation activity on EGFRDel19 in PC9 cells demonstrated here, further evaluations of degradation activity on EGFRC797S are
ongoing in our lab and the data will be reported in due courses.
Fig. 2. The western blotting analysis of EGFR protein degradation. (A) PC9 Cells and H1975 cells were treated for 24 h with the concentration 10 μM. (B) The
concentration-dependent EGFR degradation by PROTAC 16c in PC9 cells. Cells were treated for 6h with the concentration from 0.1 μM to 10 μM. (C) The
time-dependent degradation by PROTAC 16c in PC9 cells. Cells were treated with 0.3 μM for indicated time points. (D) The mechanistic of EGFR degradation
with PROTAC 16c, PC9 Cells were pretreated with lenalidomide (len, 10 μM), and MG-132 (10 μM) for 2 h, followed by 6 h treatment with 0.3 μM of
PROTAC 16c.
Fig. 3. Representative immunofluorescence microscopy images of EGFR (green) internalization in response to compound 16c (0.3 μM) at 24 h and DAPI nuclear
stain in blue.

Fig. 4. The cell health and cycle of compound 16c in PC9 cells
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to
influence the work reported in this paper.
Acknowledgement
This work was supported by National Natural Science Foundation of China (21877099).
Appendix A. Supplementary data
Supplementary data to this article can be found online at (Answer 5 to editor)
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Declaration of interests
☒ 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 authors declare the following financial interests/personal relationships which may be considered as potential
competing interests:
Graphical Abstract
Discovery and biological evaluation of proteolysis targeting chimeras (PROTACs)
as an EGFR degraders based on osimertinib and lenalidomide
Kailun He a, Zhuo Zhang a, Wenbing Wang a, Xiaoliang Zheng b, Xiaoju Wang b,*, Xingxian Zhang a,*
(a College of Pharmaceutical Sciences, Zhejiang University of Technology, Hangzhou, Zhejiang 310032)
(b Center for Molecular Medicine, Zhejiang Academy of Medical Sciences, Hangzhou, Zhejiang 310032)
O
O
N
N
NH
NH
O
O
O
NH
O
N
O
N
N
n
=
O
N
N
H
16c,
n = 3
PC9: IC50
0.413 M
H1975: IC50 = 0.657 M

Highlights
 We designed and synthesized novel PROTACs by tethering the third generation EGFR-TKI
osimertinib with CRBN ligands.
 Compounds 16c (Dmax = 68%) potently degraded EGFRDel19 in PC9 cells.
 Compounds 16c could significantly induce the apoptosis of PC9 cells and arrest the cells in G0/G1
phase.