Discovery of potent small molecule PROTACs targeting mutant EGFR

Article abstract of DOI:10.1016/j.ejmech.2020.112781

Epidermal growth factor receptor (EGFR) is an important therapeutic target for the treatment of non-small cell lung cancer. A number of efficacious EGFR tyrosine kinase inhibitors have been developed. However, acquired drug resistance largely encumbered their clinical practicability. Therefore, there is an urgent need to develop new therapeutic regime. Herein, we designed and synthesized a set of EGFR-targeting small molecule PROTACs which showed promising efficacy. In particular, VHL-recruiting compound P3 showed potent anti-proliferative activity against HCC827 and H1975 cell lines with IC₅₀ values of 0.83 and 203.01 nM, respectively. Furthermore, both EGFR^del19 and EGFR^L858R/T790M could be significantly induced to be degraded under treatment of P3 with DC₅₀ values of 0.51 and 126.2 nM, respectively. Compound P3 was able to dramatically suppress EGFR pathway signal transduction. Moreover, compound P3 could significantly induce cell apoptosis, arrest cell cycle and suppress cell colony formation. In addition, we identified that ubiquitination was indispensable in the degradation process, and found that the degradation was related to autophagy. Our work would provide an alternative approach for development of potentially effective EGFR degraders and give a new clue for investigation of PROTAC-induced protein degradation.

Full text of DOI:10.1016/j.ejmech.2020.112781

European Journal of Medicinal Chemistry 208 (2020) 112781
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Discovery of potent small molecule PROTACs targeting mutant EGFR
Hong-Yi Zhao a, Xue-Yan Yang a, Hao Lei a, Xiao-Xiao Xi a, She-Min Lu b, Jun-Jie Zhang c,
,
, **
Minhang Xin a *, San-Qi Zhang a
a Department of Medicinal Chemistry, School of Pharmacy, Xi0an Jiaotong University, Xi0an, Shaanxi, 710061, PR China
b School of Basic Medical Sciences, Xi0an Jiaotong University Health Science Center, Xi0an, Shaanxi, 710061, PR China
c School of Science, Xi0an Jiaotong University, Xi0an, Shaanxi, 710049, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Epidermal growth factor receptor (EGFR) is an important therapeutic target for the treatment of non-
small cell lung cancer. A number of efficacious EGFR tyrosine kinase inhibitors have been developed.
However, acquired drug resistance largely encumbered their clinical practicability. Therefore, there is an
urgent need to develop new therapeutic regime. Herein, we designed and synthesized a set of EGFR-
targeting small molecule PROTACs which showed promising efficacy. In particular, VHL-recruiting
compound P3 showed potent anti-proliferative activity against HCC827 and H1975 cell lines with IC50
values of 0.83 and 203.01 nM, respectively. Furthermore, both EGFRdel19 and EGFRL858R/T790M could be
significantly induced to be degraded under treatment of P3 with DC50 values of 0.51 and 126.2 nM,
respectively. Compound P3 was able to dramatically suppress EGFR pathway signal transduction.
Moreover, compound P3 could significantly induce cell apoptosis, arrest cell cycle and suppress cell
colony formation. In addition, we identified that ubiquitination was indispensable in the degradation
process, and found that the degradation was related to autophagy. Our work would provide an alter-
native approach for development of potentially effective EGFR degraders and give a new clue for
investigation of PROTAC-induced protein degradation.
Received 25 July 2020
Received in revised form
20 August 2020
Accepted 21 August 2020
Available online 26 August 2020
Keywords:
EGFR
PROTAC
Degradation
Autophagy
© 2020 Elsevier Masson SAS. All rights reserved.
1. Introduction
TKI has been exploited for EGFR C797S mutation-driven resistance.
However, development of new generation of EGFR-TKI is con-
Non-small cell lung cancer (NSCLC), one of the most aggressive
cancers, is closely related to aberrant EGFR signaling, which made
small molecular EGFR inhibitors exceedingly attractive for
anticancer-drug development. To date, a panel of small molecular
inhibitors has been discovered, and some have achieved remark-
able antitumor efficacy. Gefitinib is a first-generation EGFR tyrosine
kinase inhibitor (EGFR-TKI) targeting activating mutant EGFR, and
affords dramatically clinical benefit [1]. Unfortunately, acquired
resistance develops after short-term treatment [2]. To overcome
the drug resistance caused by T790 M mutation of EGFR-TK, sec-
ond-generation and third-generation EGFR-TKIs such as afatinib
and osimertinib (AZD9291) have been developed [3]. However,
new acquired drug resistance has been identified such as EGFR
C797S mutation [4e7]. In this regard, the fourth-generation EGFR-
fronted with great challenge due to the continuously heteroge-
neous mutations. Therefore, it is required to explore new therapies
for complete treatment of NSCLC.
Recent years have witnessed tremendous advance of PROTAC
(PROteolysis TArgeting Chimera) induced protein degradation.
PROTAC, capable of inducing protein degradation, is a bifunctional
molecule consisting of two linker-combined warheads as recruiting
elements for E3 ligase and targeted protein. The distinct mecha-
nism, which is different from kinase inhibitor, confers PROTAC
potential of excelling at overcoming drug-resistance, targeting
undruggable protein, and working with low dose-dependent
toxicity [8e13]. Since Crews group described the notion of PRO-
TAC through an elegant work of artificial manipulation of UPS
(Ubiquitin-Proteasome System) for METAP2 degradation in 2001
[14], many different kinds of proteins have been targeted by small
molecules for destruction, and the number of different PROTACs
has boomed. Proteins like BET family [15e22], CDK [23e25], ALK
[26,27], etc. [10,28e36] have been validated to be arrested and
destroyed by small molecule PROTACs. Importantly, the androgen
* Corresponding author.
** Corresponding author.
E-mail addresses: xmhcpu@163.com (M. Xin), sqzhang@xjtu.edu.cn
(S.-Q. Zhang).
https://doi.org/10.1016/j.ejmech.2020.112781
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

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H.-Y. Zhao et al. / European Journal of Medicinal Chemistry 208 (2020) 112781
receptor targeting PROTAC ARV-110 has been shown with signifi-
cant efficacy in the clinical trials for treatment of patients suffering
from metastatic castrate-resistant prostate cancer following enza-
lutamide or abiraterone, which fueled the development of PROTACs
[37].
introduced lenalidomide (CRBN ligand, Fig. 2B) or VHL-L (VHL
ligand, Fig. 2B) to the solvent-exposed terminal piperazine ring of
compound F by different linkers to design a set of EGFR-targeting
PROTACs, and investigated their ability of inducing EGFR degrada-
tion, antitumor activity and mechanism of action (Fig. 2).
Recently, EGFR-targeting PROTACs were exploited for treatment
of NSCLC based on the consideration of their therapeutic potential
for solving drug resistance and their advantages over EGFR-TKI.
Crews group synthesized several PROTACs (Fig. 1A and B) target-
ing EGFR for destruction based on the first or second-generation
EGFR-TKI in 2018 [38]. Jin group also designed gefitinib-based
PROTAC (Fig. 1C) [39]. Very recently, Ding group reported their
work on EGFRL858R/T790M-targeting PROTAC (Fig. 1D) displaying
nanomolar DC50 against EGFRL858R/T790M and submicromolar anti-
proliferative activity against H1975 cell line [40]. Our laboratory
also reported EGFR-targeting PROTAC based on fourth-generation
EGFR-TKI (Fig. 1E) [41]. However, although pioneer EGFR-
targeting PROTACs were investigated, those PROTACs still have
some defects such as no effect on double mutant EGFR or poor anti-
proliferative activity.
In this study, we designed a set of EGFR-targeting small mole-
cule PROTACs by combining a reversible EGFR-TKI with purine
scaffold and CRBN or VHL ligand. These small molecule PROTACs
showed potent anti-proliferative activity against HCC827 and
H1975 cell lines and excellent activity of inducing mutant EGFR
degradation. More importantly, we also reported that EGFR
degradation induced by PROTAC was related to autophagy pathway
for the first time.
The synthesis of target compounds was depicted in Scheme 1, 2
and 3. Scheme 1 shows synthetic route of the key intermediate 6.
Initially, commercially available 2,4-dichloro-5-nitropyrimidine (1)
underwent nucleophilic substitution reaction with cyclopentyl-
amine to afford compound 2 which was subsequently reacted with
tert-butyl 4-(4-aminophenyl) piperinzine-1-carboxylate to afford
compound 3. Then, reduction of 3 by catalytic hydrogenation gave
compound 4.4 underwent cyclization reaction to generate 5 fol-
lowed by deprotection employing TFA to release the key interme-
diate 6.
The synthetic routes of target compounds P1eP4 were outlined
in Scheme 2. The reaction between 6 and ethyl 7-bromoheptanoate
produced 7 which was subsequently converted to 8 via hydrolysis.
Then, compounds P1 and P3 were obtained through acylation re-
action of 8 with lenalidomide and VHL-L, respectively. Lenalido-
mide or VHL-L was transformed to 9a or 9b via acylation with 8-
bromooctanoic acid. P2 or P4 was generated from 9a or 9b
through nucleophilic substitution with intermediate 6.
Compounds P5eP10 with different polyethylene glycol linkers
were synthesized according to Scheme 3. Intermediate 6 reacted
with polyethylene glycol di-p-toluenesulfonate to produce 11a-11c
which were converted to 12a-12c via nucleophilic substitution with
10. Subsequently, deprotection of 12a-12c followed by acylation
with lenalidomide generated target compounds P5eP7. Reaction of
polyethylene glycol di-p-toluenesulfonate with VHL-L gave inter-
mediate 13a-13c. Finally, P8eP10 were obtained from 13a-13c via
nucleophilic substitution with 6. Thus, we synthesized ten target
compounds with different linkers and E3-recruiting elements.
2. Results and discussions
2.1. Design and synthesis of EGFR-targeting PROTACs
It was reported that purine-containing derivatives showed
effective inhibitory activity against EGFR-TK [42e45]. In particular,
compound F (Fig. 2) was discovered as a highly potent EGFR-TKI
which was considered as an ideal EGFR-binding module for the
design of EGFR-targeting PROTAC [42]. Therefore, in this paper, we
2.2. Evaluation of the anti-proliferative activity in vitro
With target compounds in hand, we firstly evaluated their anti-
proliferative activity against HCC827 cell line (Table 1 and Fig. S1).
Fig. 1. Structures of EGFR-targeting PROTACs and their activity profiles.

H.-Y. Zhao et al. / European Journal of Medicinal Chemistry 208 (2020) 112781
3
Fig. 2. (A) Docking of compound F with EGFR (PDB: 3IKA) using Sybyl-X 2.0. (B) Design of EGFR-targeting small molecule PROTAC.
Scheme 1. Synthesis of key intermediate 6. Reagents and reaction conditions: (i) cyclopentylamine, TEA, DCM, ꢀ20 ꢁC ~ rt, 80%; (ii) tert-butyl 4-(4-aminophenyl) piperinzine-1-
carboxylate, Na2CO3, EtOH, rt, 88%; (iii) Pd/C, H2, MeOH, 50 ꢁC, 94%; (iv) phenyl isothiocyanate, EDCI, DIPEA, 1,2-dichloroethane, 60 ꢁC, 92%; (v) DCM, TFA, rt; (vi) saturated Na2CO3
(aq), 90% overall yield.
Compound F and AZD9291 were used as positive controls. As
illustrated in Table 1, compounds P1eP7 dominantly inhibited
growth of HCC827 cell line, which was comparable to AZD9291 and
parent compound F. Among them, compounds with alkyl linker and
VHL-L components (P3eP4) were a little more effective than the
others with IC50 values of 0.83 and 0.76 nM, respectively. Moreover,
the activity of compounds with PEG linker and lenalidomide E3-
recruiting element (P1eP2) was almost indistinguishable with
that of their counterparts (P5eP7). Converting carbonyl-containing
alkyl linker to polyethylene glycol chain resulted in the diminished
activity when using VHL-L as E3-recuiting element (P3eP4,
P8eP10). Subsequently, we tested inhibitory activity of the
compounds against H1975 cell line harboring drug-resistant mu-
tation (L858R/T790 M). Compound P3 displayed submicromolar
inhibitory activity against H1975 (IC50 ¼ 203 nM) which was about
twice as good as that of compound F (IC50 ¼ 430 nM) while the
others were less effective (Table 1 and Fig. S1). Next, A431 cell line
was selected to evaluate their cellular selectivity as it expressed
high level of wild-type EGFR. P1eP4 exhibited submicromolar
inhibitory activity against A431 indicating good selectivity against
HCC827 (Table 1 and Fig. S1). As compound F was reported to have
significant inhibitory activity against VEGFR2 [42], we tested our
compounds on HepG2 cell line. Our experiment demonstrated the
synthesized compounds were almost ineffective on HepG2 cell line

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H.-Y. Zhao et al. / European Journal of Medicinal Chemistry 208 (2020) 112781
Scheme 2. Synthesis of target compounds P1eP4. Reagents and reaction conditions: (i) Br(CH2)6COOEt, K2CO3, KI, DMF, 60 ꢁC, 50%; (ii) NaOH, EtOH, H2O, rt; (iii) 1 M HCl, 79%
overall yield; (iv) lenalidomide, HATU, DIPEA, DMF, rt, 51%; (v) VHL-L, HATU, DIPEA, DMF, rt, 51%; (vi) Br(CH2)7COOH, EDCI, DMF, 40 ꢁC, 35%; (vii) 6, DIPEA, KI, NMP, 100 ꢁC, 26%; (viii)
Br(CH2)7COOH, HATU, DIPEA, DCM, rt, 83%; (ix) 6, K2CO3, KI, CH3CN, reflux, 63%.
indicating their high selectivity against lung cancer cells. These
results indicated that the composition of linker had negligible
impact on anti-proliferative activity when using lenalidomide as
E3-recruiting element, and that the PROTACs decorated with VHL
ligand were more responsive to EGFRL858R/T790M than those with
CRBN ligand.
Induced EGFR degradation was time-dependent as shown in
Fig. 3B. The maximum effect of the compounds was achieved
almost after 48 h. Furthermore, P3, P4 and P6 all displayed excel-
lent activity of inducing EGFRdel19 degradation with nanomolar
DC50 values of 0.51, 3.54 and 1.91 nM, respectively, while their
parental compound F was ineffective at the concentration of
100 nM (Fig. 3C and D).
It remains a challenge to develop effective EGFRL858R/T790M-tar-
geting PROTAC. Then the induced EGFR degradation in H1975 cell
line was studied. As shown in Fig. 4, P3 and its analogue P4 were
responsive to double mutant EGFR in H1975 cell line (DC50 ¼ 126.2
and 151.2 nM, respectively. Dmax ¼ 90.3% and 80.3%, respectively.
Fig. 4A and C). Additionally, almost no degradation on wild-type
EGFR (EGFRWT) was examined in A431 cell line under treatment
of P3 or P6 indicating its excellent selectivity (Fig. 4B and Fig. S2).
Subsequently, we determined duration time of their effect. After
being washed out for 24 h, P3 and P4 still suppressed EGFR level in
HCC827 cell line, which was more long-lasting than the effect of
CRBN-recruiting P6 (Fig. 5A). Furthermore, EGFR level almost
2.3. PROTAC-induced EGFR degradation
Having determined anti-proliferative activity of the compounds
against tumor cells, we evaluated their capability of inducing EGFR
degradation (Fig. 3A). Obviously, compounds P1eP7 with different
linkers or E3-recruiting elements were able to mediate EGFR
degradation. Treatment at the concentration of 333 nM impaired
the activity because of hook effect. However, compounds P8eP10
were barely incompetent in inducing EGFR degradation, which is
consistent with their cellular activity. Based on both cellular and
degradative activity, we selected compounds P3, P4 and P6 for
further study.

H.-Y. Zhao et al. / European Journal of Medicinal Chemistry 208 (2020) 112781
5
Scheme 3. Synthesis of target compounds P5eP10. Reagents and reaction conditions: (i) methylamine, CH3CN, 0 ꢁC ~ rt, 46%; (ii) TsO(CH2CH2O)nTs, CH3CN, K2CO3, 60 ꢁC, 22e36%;
(iii) CH3CN, K2CO3, reflux, 36e68%; (iv) TFA, DCM; (v) lenalidomide, HATU, DIPEA, DMF, rt, 28%~31%; (vi) VHL-L, CH3CN, K2CO3, 60 ꢁC, 25e35%; (vii) 6, K2CO3, CH3CN, reflux, 28e57%.
Table 1
In vitro anti-proliferative activity of compounds P1eP10 against cancer cells (n ¼ 3,x SD).
compds
Linker
IC50 (nM)
A431 (EGFRWT
)
HCC827 (EGFRdel19
1.41 0.15
)
H1975 (EGFRL858R/T790M
9301 57
)
HepG2 (VEGFR2)
P1
P2
220 28
>5000
240 40
2.61 1.58
3776 347
>5000
P3
P4
245 30
330 35
0.83 0.30
0.76 0.33
203 21
>5000
>5000
970 162
P5
P6
P7
2667 315
1856 346
2723 368
1.39 0.23
2.00 0.72
2.83 0.38
1211 140
3176 223
3336 460
1100 283
>5000
>5000
>5000
P8
ND
ND
ND
53.49 0.49
54.38 3.00
62.00 1.86
>5000
>5000
>5000
P9
2100 57
2894 345
430 85
P10
F
e
e
163 25
742 53
0.82 0.69
0.97 0.10
>5000
ND
AZD9291
42
7
“ND”, not determined.

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H.-Y. Zhao et al. / European Journal of Medicinal Chemistry 208 (2020) 112781
recovered after 48 h whether treated with P3 or P6. In contrast,
double mutant EGFR (EGFRL858R/T790M) level was constantly sup-
pressed even after 48 h (Fig. 4B).
To explore the effect of the degrader on downstream signaling,
we detected the level of phosphorylated EGFR and Akt in HCC827
and H1975 cell lines. As seen in Fig. 6, the phosphorylation of EGFR
and its downstream effector Akt was dramatically reduced in
HCC827 and H1975 cell lines when treated with P3 at concentra-
tions as low as 3 and 100 nM, respectively, while Akt remained
intact (Fig. 6A and B). These results indicated that P3 was able to
inhibit signal transduction of EGFR pathway.
2.4. Mechanism study
UPS was reported to be involved in the PROTAC-induced protein
degradation in which the formation of ternary complex (protein-
PROTAC-E3) was indispensable for protein ubiquitination and
subsequent split by proteasome. Therefore, we firstly inverted the
hydroxyproline stereochemistry on the VHL-L to synthesize dia-
stereomer P11 (Fig. 7C) to abolish its ability to bind VHL. P11 dis-
played much weaker anti-proliferative and degradative activity
than P3 indicating that VHL binding was crucial for ternary com-
plex formation in the degradative process (Fig. 7AeC). Further-
more, as shown in Fig. 8, no degradation of EGFR was detected upon
treatment of ubiquitination inhibitor MLN4924 despite the pres-
ence of CRBN-recruiting P6 (Fig. 8A and S3A) or VHL-recruiting P3
(Fig. 8B and S3B) implying the necessity of EGFR ubiquitination for
destruction. As another evidence of ternary complex formation,
EGFR-TKI F (Fig. 8A and B, S3A-S3B) or E3 ligands lenalidomide
(Fig. 8A and S3A) was also capable of preventing EGFR degradation.
Unexpectedly, VHL-L was unable to antagonize the effect of com-
pound P3 possibly due to its weak VHL-binding affinity (Fig. 8B and
S3B). Subsequently, we synthesized acetylated VHL-L, namely,
VHL-Ac (Fig. 8D), and discovered that VHL-Ac attenuated the effect
of P3 suggesting its stronger VHL-binding affinity (Fig. 8C and S3C).
Proteasome was widely supposed to be the intracellular protein-
digesting machinery in the process of PROTAC-induced protein
degradation. Nonetheless, proteasome inhibitor MG132 had no
impact on induced EGFR destruction at concentration of 10 mM in
our research (Fig. 8A and B, S3A-S3B). This prompted us to turn our
attention to autophagic-lysosomal system, another protein-
hydrolyzing pathway.
We next explored the relationship between autophagy and
EGFR degradation. Chloroquine, a widely used autophagy inhibitor,
slightly impaired the effect of P6 or P3 in HCC827 cell line as well as
in H1975 cell line indicating the EGFR degradation was lysosome-
dependent (Figs. 8C and 9A, S3C-S3D). We supposed that the
weak inhibition of chloroquine on EGFR degradation was partly due
to its ability of inducing autophagy (Fig. S4). Subsequently, we
devoted to exploring whether enhancement of autophagy could
amplify the effect of P3. Serum deprivation was proved to induce
autophagy, so we used medium without fetal bovine serum (FBS) to
culture cells and discovered that EGFR degradation was facilitated
under the starvation condition (Fig. 9B, S5A-S5B). LC3-II/I ratio and
p62 level confirmed the enhancement of autophagy. Rapamycin, a
mammalian target of rapamycin (mTOR) inhibitor, was also capable
of inducing autophagy. As shown in Fig. 9C and S5C, rapamycin
accelerated EGFR degradation in H1975 cells line. Thus, it was
ensured that induced EGFR degradation initiated from protein
ubiquitination upon formation of ternary complex, and that
autophagic-lysosomal system was implicated in the process.
Fig. 3. PROTAC-induced EGFRdel19 degradation in HCC827 cell line detected with
Western blotting (A) The cell line was treated with compounds P1eP10 with indicated
concentrations for 24 h. (B) The cell line was treated with compounds P3, P4 or P6 at
33 nM, and protein was collected at each indicated time point. (C, D) The cell line was
treated with P3, P4 or P6 with indicated concentrations for 48 h (n ¼ 3).
2.5. Cell apoptosis assay
We next determined whether compound P3 could induce cell

H.-Y. Zhao et al. / European Journal of Medicinal Chemistry 208 (2020) 112781
7
Fig. 5. Investigation of EGFR level after the compounds were removed. (A) HCC827 cell
line was treated with indicated compounds (33 nM) for 24 h. (B) H1975 cell line was
treated with indicated compounds (333 nM) for 48 h. Then, the medium was removed
and fresh medium without compounds was added. Protein was collected after indi-
cated time and analyzed by Western blotting (n ¼ 2).
its different mode of action from traditional enzyme inhibitors.
Although several EGFR-targeting PROTACs were developed, they
suffered from no effect on double mutant EGFR or poor anti-
proliferative activity. Herein, we have discovered
a novel
degrader (compound P3) capable of effectively inducing mutant
EGFR degradation and dramatically suppressing the growth of
HCC827 and H1975 cell lines as well as EGFR pathway signal
transduction. Additionally, compound P3 could prompt cell
apoptosis, arrest cell cycle and suppress cell colony formation. We
further confirmed that induced EGFR degradation initiated from
protein ubiquitination upon formation of ternary complex, and
reported for the first time that PROTAC-induced EGFR degradation
was related to autophagy. In short, compound P3 was finally
discovered as a potent EGFR degrader and antitumor agent. Our
work would provide an alternative approach for development of
clinically effective EGFR degraders and give a new clue for inves-
tigation of PROTAC-induced protein degradation.
Fig. 4. PROTAC-induced EGFR degradation in H1975 (A, C) and A431 (B) cell lines
detected with Western blotting. Cell lines were treated with compound P3 or P4 under
indicated concentrations for 48 h (n ¼ 3).
apoptosis. Compound P3 induced 31.07% and 44.80% of HCC827 cell
line to undergo apoptosis at concentration of 10 and 100 nM,
respectively (Fig. 10 and S6). However, it was not competent in
inducing apoptosis of H1975 cell line.
4. Experimental section
4.1. Chemistry
2.6. Cell cycle assay
Unless specified otherwise, all the starting materials, reagents
and solvents are commercially available. All the reactions were
monitored by thin-layer chromatography on silica gel plates
(GF254) and visualized with UV light (254 nm and 365 nm). NMR
spectra were recorded on a 400 Bruker NMR spectrometer with
tetramethylsilane (TMS) as an internal reference. All chemical shifts
are reported in parts per million (ppm). The following abbrevia-
tions were used to describe peak splitting patterns when appro-
priate: s (singlet), d (doublet), t (triplet), m (multiplet), br (broad
signal), dd (doublet of doublets). Coupling constants (J) are
expressed in hertz unit (Hz). Mass spectrum (MS) was obtained by
ESI-MS (Skyray instrument, LC-MS 1000). High resolution mass
spectrum (HRMS) was obtained by electrospray ionization (positive
mode) on an Ultra performance liquid chromatography-
Quadrupole-time of flight Mass Spectrometer (WATERS I-Class
VION IMS Q-TOF).
Flow cytometry was employed to examine the impact of P3 on
cycle of tumor cells. As depicted in Fig. 11 and S7, the degrader P3
was able to arrest both HCC827 and H1975 cell lines at G1 phase.
2.7. Colony formation assay
As shown in Fig. 12 and S8, Colony formation assay confirmed
that P3 could significantly inhibit cell cloning of HCC827 and
H1975 cell lines at concentrations as low as 1 and 100 nM,
respectively. These results indicated that P3 could effectively
inhibit the growth of tumor cells.
3. Conclusions
Emerging PROTAC technology displays many advantages due to

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H.-Y. Zhao et al. / European Journal of Medicinal Chemistry 208 (2020) 112781
Fig. 6. P3 significantly reduced the level of phosphorylated EGFR (P-EGFR) and Akt (P-Akt). HCC827 (A) and H1975 (B) cell lines were treated with compound P3 with indicated
concentrations for 48 h. The protein was collected and analyzed by Western blotting (n ¼ 2).
Fig. 7. HCC827 (A) or H1975 (B) cell lines were treated with P3 or its diastereomer P11 with indicated concentrations for 24 h. EGFR level was analyzed by Western blotting. (C)
Chemical structure of P3 and P11. (n ¼ 3).
4.1.1. 2-Chloro-N-cyclopentyl-5-nitropyrimidin-4-amine (2)
(50 mL) was added dropwise. The resulted mixture was washed
with water (3 ꢂ 15 mL) and saturated brine (20 mL), dried with
anhydrous sodium sulfate and evaporated under vacuum to afford
compound 2 without further purification. Yellow solid, 80% yield,
To a round bottom flask were added DCM (50 mL) and 2,4-
dichloro-5-nitro-pyrimidine (5.00 g, 25.8 mmol). The solution
was stirred at ꢀ20 ꢁC for 10 min, and a solution of cyclopentylamine
(2.42 g, 28.4 mmol) and triethylamine (3.92 g, 38.7 mmol) in DCM
1H NMR (400 MHz, CDCl3),
d 9.03 (s, 1H), 8.39 (s, 1H), 4.60 (dd,

H.-Y. Zhao et al. / European Journal of Medicinal Chemistry 208 (2020) 112781
9
Fig. 8. Degradation mechanism investigation. HCC827 cell line was pretreated with indicated compounds or DMSO for 2 h. Then P3 or P6 was added. And protein was collected
after 24 h and analyzed by Western blotting (AeC). (D) Chemical structures of VHL-L and VHL-Ac. (n ¼ 3).
J ¼ 13.9, 6.9 Hz,1H), 2.32e2.11 (m, 2H),1.90e1.65 (m, 4H),1.64e1.48
(m, 2H). MS (ESI): m/z calcd for C9H12ClN4O2: 243.1 [MþH]þ; found:
455.5.
yield, 1H NMR (400 MHz, CDCl3),
d 9.01 (s, 1H), 8.53 (s, 1H), 7.69 (s,
1H), 7.55 (s, 2H), 6.95 (d, J ¼ 8.8 Hz, 2H), 4.55e4.45 (m, 1H),
3.68e3.55 (m, 4H), 3.22e3.06 (m, 4H), 2.17e2.04 (m, 2H), 1.85e1.76
(m, 2H), 1.73e1.59 (m, 4H), 1.49 (s, 9H). MS (ESI): m/z calcd for
C
24H34N7O4: 484.3 [MþH]þ; found: 484.4.
4.1.2. Tert-butyl 4-(4-((4-(cyclopentylamino)-5-nitropyrimidin-2-
yl)amino)phenyl) piperazine-1-carboxylate (3)
Tert-butyl 4-(4-aminophenyl)piperazine-1-carboxylate (3.82 g,
13.8 mmol) was dissolved in EtOH (25 mL). Then, Na2CO3 (2.19 g,
20.7 mmol) and 2 were added. The resulted mixture was stirred at
room temperature overnight. The precipitated solid was filtered
and washed with EtOH (3 ꢂ 3 mL). The filter cake was mixed with
water (20 mL) followed by filtration to produce 3. Yellow solid, 88%
4.1.3. Tert-butyl 4-(4-((5-amino-4-(cyclopentylamino)pyrimidin-2-
yl)amino)phenyl)pi-perazine-1-carboxylate (4)
Compound 3 (5.00 g, 10.34 mmol) and Pd/C (1.00 g, 5%) were
mixed with MeOH (70 mL). The suspension was stirred under H2
atmosphere at 55 ꢁC overnight. The mixture was filtered through
Celite, and the filtrate was evaporated under vacuum to produce

10
H.-Y. Zhao et al. / European Journal of Medicinal Chemistry 208 (2020) 112781
Fig. 9. Exploring relationship between EGFR degradation and autophagy. (A) H1975 cell line was pretreated with chloroquine for 2 h followed by 24 h treatment with P3. (B)
HCC827 or H1975 cell lines were pre-cultured using medium with or without FBS for 8 h followed by 16 h treatment with P3 under indicated concentrations. (C) H1975 cell line was
pretreated with rapamycin under indicated concentrations for 8 h followed by 16 h treatment with P3. Protein level was analyzed by Western blotting (n ¼ 3).
Fig. 10. Flow cytometry analysis of cell apoptosis induced by P3 and its parental compound F. Cell lines were treated with indicated concentration for 48 h (n ¼ 2).

H.-Y. Zhao et al. / European Journal of Medicinal Chemistry 208 (2020) 112781
11
Fig. 11. The effect of P3 and its parental compound F on cell cycle analyzed by flow cytometry. Cell lines were treated with indicated concentrations for 48 h (n ¼ 2).
Fig. 12. Colony formation assay. Cell were seeded on 6-well plate and treated with P3 under indicated concentrations for 15 days. And the cells were stained with crystal violet.
Colony number was counted (n ¼ 3, *P < 0.05, **P < 0.01, ***P < 0.001).
compound 4 without further purification. Gray solid, 94% yield, 1H
NMR (400 MHz, CDCl3)
J ¼ 8.9 Hz, 2H), 6.77 (s, 1H), 5.20 (d, J ¼ 6.8 Hz, 1H), 4.44e4.26 (m,
1H), 3.69e3.53 (m, 4H), 3.13e2.98 (m, 4H), 2.16e2.05 (m, 2H),
1.80e1.62 (m, 4H), 1.55e1.50 (m, 2H), 1.48 (s, 9H). MS (ESI): m/z
calcd for C24H36N7O2: 454.3 [MþH]þ; found: 454.5.
Subsequently, the pH of the residue was adjusted to 10 using
saturated aqueous Na2CO3. The precipitate was filtered to obtain
intermediate 6. Yellow solid, 90% yield, 1H NMR (400 MHz,
d
7.54 (s, 1H), 7.51 (d, J ¼ 8.9 Hz, 2H), 6.89 (d,
DMSO‑d6)
d
9.03 (d, J ¼ 4.7 Hz, 2H), 8.36 (s, 1H), 7.81 (d, J ¼ 8.0 Hz,
2H), 7.65 (d, J ¼ 8.9 Hz, 2H), 7.33 (t, J ¼ 7.9 Hz, 2H), 6.98 (t, J ¼ 7.3 Hz,
1H), 6.90 (d, J ¼ 9.0 Hz, 2H), 5.01e4.90 (m, 1H), 3.13 (s, 4H), 3.11 (s,
4H), 2.05 (s, 5H), 1.69 (d, J ¼ 5.1 Hz, 3H). MS (ESI): m/z calcd for
C
26H31N8: 455.3 [MþH]þ; found: 455.5.
4.1.4. Tert-butyl 4-(4-((9-cyclopentyl-8-(phenylamino)-9H-purin-
2-yl)amino)phenyl)pi perazine-1-carboxylate (5)
To a round bottom flask were added 4 (4.00 g, 8.82 mmol), EDCI
(2.03 g, 10.58 mmol), 1,2-dichloroethane (50 mL), phenyl isothio-
cyanate (1.43 g, 10.58 mmol) and DIPEA (1.37 g, 10.58 mmol). The
resulted mixture was stirred at 65 ꢁC for 5 h. Product 5 was isolated
using column chromatography (DCM:MeOH ¼ 50:1). Pale yellow
4.1.6. Ethyl 7-(4-(4-((9-cyclopentyl-8-(phenylamino)-9H-purin-2-
yl)amino)phenyl)pi-perazin-1-yl)heptanoate (7)
To a flask were added 6 (0.30 g, 0.66 mmol), ethyl 7-
bromoheptanoate (0.30 g, 1.32 mmol), K2CO3 (0.18 g, 1.32 mmol),
NaI (0.04 g, 0.26 mmol) and CH3CN. The resulted mixture was
heated at reflux for 3 h. The solvent was evaporated under reduced
pressure, and the residue was purified using column chromatog-
raphy (DCM: MeOH ¼ 30:1) to produce 7. Pale yellow solid, 50%
solid, 92% yield, 1H NMR (400 MHz, CDCl3),
d 8.41 (s, 1H), 7.54 (dd,
J ¼ 7.6, 5.3 Hz, 4H), 7.38 (t, J ¼ 8.0 Hz, 2H), 7.22 (s, 1H), 7.09 (t,
J ¼ 7.4 Hz, 1H), 6.94 (d, J ¼ 8.9 Hz, 2H), 6.33 (s, 1H), 4.75e4.59 (m,
1H), 3.69e3.53 (m, 4H), 3.20e3.01 (m, 4H), 2.52e2.37 (m, 2H),
2.19e2.01 (m, 4H), 1.83e1.72 (m, 2H), 1.49 (s, 9H). MS (ESI): m/z
calcd for C31H39N8O2: 555.3 [MþH]þ; found: 555.6.
yield, 1H NMR (400 MHz, CDCl3),
d
8.45 (s, 1H), 7.53 (t, J ¼ 8.2 Hz,
4H), 7.37 (t, J ¼ 7.9 Hz, 2H), 7.08 (t, J ¼ 7.4 Hz, 1H), 6.93 (d, J ¼ 9.0 Hz,
3H), 6.35 (s, 1H), 4.73e4.60 (m, 1H), 4.13 (q, J ¼ 7.1 Hz, 2H), 3.20 (s,
4H), 2.68 (s, 4H), 2.51e2.38 (m, 4H), 2.30 (t, J ¼ 7.5 Hz, 2H),
2.18e2.01 (m, 4H), 1.82e1.71 (m, 2H), 1.68e1.61 (m, 2H), 1.61e1.53
(m, 2H), 1.40e1.32 (m, 4H), 1.26 (t, J ¼ 7.1 Hz, 3H). MS (ESI): m/z
calcd for C35H47N8O2: 611.4 [MþH]þ; found: 611.5.
4.1.5. 9-Cyclopentyl-8-phenylamino-2-(4-(piperazin-1-yl)
phenylamino)-9H-purine (6)
To a solution of 5 (2.86 g, 5.16 mmol) in DCM (15 mL) were
added TFA (10 mL), and the solution was stirred at room temper-
ature overnight. The solvent was evaporated under vacuum.

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H.-Y. Zhao et al. / European Journal of Medicinal Chemistry 208 (2020) 112781
4.1.7. 7-(4-(4-((9-cyclopentyl-8-(phenylamino)-9H-purin-2-yl)
amino)phenyl)pipe-razin-1-yl)heptanoic acid (8)
4.1.11. 2-(2-(2-(4-(4-((9-cyclopentyl-8-(phenylamino)-9H-purin-2-
yl)amino)phen-yl)piperazin-1-yl)ethoxy)ethoxy)ethyl 4-
methylbenzenesulfonate (11a)
A flask was charged with 6 (0.40 g, 0.88 mmol), tri (ethyl-
eneglycol) di-p-toluenesulfonate (0.82 g, 1.76 mmol), K2CO3 (0.25 g,
1.76 mmol), KI (0.03 g, 0.18 mmol) and CH3CN (5 mL). The resulted
suspension was heated at 65 ꢁC overnight. The reaction mixture
To a flask were added 7 (0.20 g, 0.33 mmol), NaOH (0.26 g,
6.6 mmol), H2O (2 mL) and EtOH (4 mL). The resulted suspension
was stirred at room temperature for 6 h. The solvent was evapo-
rated under reduced pressure, and the pH of the residue was
adjusted to 5 using 1 M HCl. The precipitate was filtered to obtain
compound 8. Yellow solid, 79% yield, 1H NMR (400 MHz, DMSO‑d6)
was
(DCM:MeOH ¼ 50:1e30:1) to produce compound 11a. Yellow solid,
26% yield, 1H NMR (400 MHz, CDCl3)
8.45 (s, 1H), 7.80 (d,
isolated
using
column
chromatography
d
12.02 (s, 1H), 10.35 (s, 1H), 9.13 (s, 1H), 8.36 (s, 1H), 7.83 (d,
J ¼ 7.8 Hz, 2H), 7.66 (d, J ¼ 8.9 Hz, 2H), 7.33 (t, J ¼ 7.9 Hz, 2H), 6.99 (t,
J ¼ 7.4 Hz, 1H), 6.95 (d, J ¼ 8.8 Hz, 2H), 5.08e4.94 (m, 1H), 3.69 (d,
J ¼ 11.7 Hz, 2H), 3.55 (d, J ¼ 11.1 Hz, 2H), 3.15e2.99 (m, 6H), 2.23 (t,
J ¼ 7.3 Hz, 2H), 2.04 (s, 5H), 1.70 (s, 5H), 1.55e1.48 (m, 2H), 1.32 (s,
4H). MS (ESI): m/z calcd for C33H43N8O2: 583.4 [MþH]þ; found:
583.6.
d
J ¼ 8.2 Hz, 2H), 7.58e7.49 (m, 4H), 7.40e7.31 (m, 4H), 7.07 (t,
J ¼ 7.3 Hz, 1H), 6.99e6.89 (m, 3H), 6.38 (s, 1H), 4.74e4.60 (m, 1H),
4.20e4.12 (m, 2H), 3.72e3.68 (m, 2H), 3.66 (t, J ¼ 5.7 Hz, 2H),
3.62e3.55 (m, 4H), 3.21e3.13 (m, 4H), 2.77e2.65 (m, 6H), 2.44 (s,
3H), 2.16e2.00 (m, 5H), 1.80e1.69 (m, 3H). MS (ESI): m/z calcd for
C
39H49N8O5S: 741.4 [MþH]þ; found: 741.5.
4.1.8. 8-Bromo-N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-
yl)octanamide (9a)
4.1.12. 2-(2-(2-(2-(4-(4-((9-cyclopentyl-8-(phenylamino)-9H-
purin-2-yl)amino)ph-enyl)piperazin-1-yl)ethoxy)ethoxy)ethoxy)
ethyl 4-methylbenzenesulfonate (11b)
Lenalidomide (0.50 g, 1.93 mmol), 8-bromooctanoic acid (0.50 g,
2.31 mmol), EDCI (0.44 g, 2.31 mmol), DMAP (0.28, 2.31 mmol) and
DMF (10 mL) were mixed in a flask. The suspension was stirred in
an oil bath at 40 ꢁC for 4 h. The solvent was evaporated under
vacuum. The residue was isolated using column chromatography
(DCM:MeOH ¼ 50:1) to give compound 9a. White solid, 35% yield,
Compound 11b was prepared similarly as described for 11a.
Yellow oil, 22% yield, 1H NMR (400 MHz, CDCl3)
d 8.44 (s, 1H), 7.80
(d, J ¼ 8.3 Hz, 2H), 7.62e7.48 (m, 4H), 7.42e7.31 (m, 4H), 7.07 (t,
J ¼ 7.4 Hz, 1H), 6.99e6.88 (m, 3H), 6.45 (s, 1H), 4.72e4.61 (m, 1H),
4.16 (t, J ¼ 4.1 Hz, 2H), 3.71e3.65 (m, 6H), 3.63 (s, 4H), 3.60 (s, 2H),
3.23e3.10 (m, 4H), 2.78e2.61 (m, 6H), 2.44 (s, 3H), 2.17e1.99 (m,
5H), 1.88 (s, 3H). MS (ESI): m/z calcd for C41H53N8O6S: 785.4
[MþH]þ; found: 785.5.
1H NMR (400 MHz, DMSO‑d6)
d 11.03 (s, 1H), 9.77 (s, 1H), 7.81 (dd,
J ¼ 7.0, 1.7 Hz, 1H), 7.54e7.46 (m, 2H), 5.15 (dd, J ¼ 13.3, 5.0 Hz, 1H),
4.36 (q, J ¼ 17.5 Hz, 2H), 3.63 (t, J ¼ 6.6 Hz, 2H), 3.00e2.84 (m, 1H),
2.61 (d, J ¼ 16.8 Hz, 1H), 2.41e2.31 (m, 3H), 2.09e1.99 (m, 1H),
1.76e1.66 (m, 2H), 1.66e1.56 (m, 2H), 1.47e1.24 (m, 6H).
4.1.13. 2-(2-(2-(2-(2-(4-(4-((9-cyclopentyl-8-(phenylamino)-9H-
purin-2-yl)amino)-phenyl)piperazin-1-yl)ethoxy)ethoxy)ethoxy)
ethoxy)ethyl 4-methylbenzenesulfonate (11c)
4.1.9. (2S,4R)-1-((S)-2-(8-bromooctanamido)-3,3-
dimethylbutanoyl)-4-hydroxy- N-(4-(4-methylthiazol-5-yl)benzyl)
pyrrolidine-2-carboxamide (9b)
Compound 11c was prepared similarly as described for 11a.
Yellow oil, 36% yield, 1H NMR (400 MHz, DMSO‑d6)
d 9.10e8.94 (m,
To a solution of 8-bromooctanoic acid (0.12 g, 0.56 mmol) and
VHL-L (0.20 g, 0.46 mmol) in DCM (3 mL) were added HATU (0.21 g,
0.56 mmol) and DIPEA (0.12 g, 0.92 mmol). The mixture was stirred
at room temperature for 3.5 h. The reaction mixture was diluted
with DCM (80 mL) and washed with water (3 ꢂ 20 mL). The organic
phase was washed with saturated brine (20 mL), dried with
anhydrous sodium sulfate and isolated using column chromatog-
raphy to give compound 9b. White solid, 83% yield, 1H NMR
2H), 8.36 (s, 1H), 7.87e7.76 (m, 3H), 7.69 (d, J ¼ 8.9 Hz, 1H), 7.61 (d,
J ¼ 8.9 Hz, 1H), 7.48 (dd, J ¼ 8.0, 4.4 Hz, 2H), 7.33 (t, J ¼ 7.9 Hz, 2H),
7.11 (d, J ¼ 7.8 Hz, 1H), 6.98 (t, J ¼ 7.3 Hz, 2H), 6.86 (d, J ¼ 8.9 Hz, 1H),
5.03e4.84 (m, 1H), 4.59 (t, J ¼ 5.5 Hz, 2H), 3.58e3.55 (m, 4H),
3.52e3.50 (m,12H), 3.48 (s, 2H), 3.45 (s, 2H), 3.42 (d, J ¼ 5.0 Hz, 4H),
3.03 (s, 2H), 2.51 (s, 3H), 2.41 (s,1H), 2.28 (s, 1H), 2.04 (s, 4H),1.69 (s,
2H). MS (ESI): m/z calcd for C43H57N8O7S: 829.4 [MþH]þ; found:
829.6.
(400 MHz, DMSO‑d6)
d
9.00 (s, 1H), 8.58 (t, J ¼ 6.0 Hz, 1H), 7.86 (d,
J ¼ 9.2 Hz, 1H), 7.56e7.46 (m, 2H), 7.40 (q, J ¼ 8.3 Hz, 2H), 4.55 (d,
J ¼ 9.3 Hz, 2H), 4.48e4.40 (m, 3H), 4.35 (s, 1H), 4.22 (dd, J ¼ 15.9,
5.4 Hz, 1H), 3.69e3.63 (m, 1H), 3.52 (t, J ¼ 6.7 Hz, 2H), 2.52e2.49
(m, 4H), 2.45 (s, 3H), 1.94e1.84 (m, 2H), 1.82e1.73 (m, 2H), 1.36 (s,
6H), 0.94 (s, 9H). MS (ESI): m/z calcd for C30H43BrN4NaO4S: 657.2
[MþNa]þ; found: 657.4 (79Br), 659.4 (81Br).
4.1.14. Tert-butyl 2-((N-(2-(2-(2-(4-(4-((9-cyclopentyl-8-
(phenylamino)-9H-purin-2- yl)amino)phenyl)piperazin-1-yl)
ethoxy)ethoxy)ethyl)-N-methyl)amino)acetate (12a)
11a (0.15 g, 0.20 mmol), 10 (0.0637 g, 0.40 mmol), K2CO3
(0.0552 g, 0.40 mmol) and NaI (0.0120 g, 0.08 mmol) were sus-
pended in CH3CN (3 mL). The resulted mixture was heated at reflux
under argon atmosphere for 11 h. The solvent was evaporated
under vacuum and the residue was isolated using column chro-
matography (DCM:MeOH ¼ 30:1e15:1) to give product 12a. Yellow
4.1.10. Tert-butyl 2-methylaminoacetate (10)
To a solution of methylamine (0.80 g, 25.6 mmol) in CH3CN
(3 mL) were added NaI (0.15 g, 1.02 mmol), and the suspension was
stirred in an ice bath for 5 min. Subsequently, tert-butyl 2-
bromoacetate (0.50 g, 2.56 mmol) in CH3CN (7 mL) was added
dropwise. The resulted mixture was stirred at room temperature
overnight. The solvent was evaporated under vacuum, and the
water was added (10 mL). The water phase was extracted with ethyl
acetate (4 ꢂ 20 mL), and the combined organic phase was washed
with saturated brine and dried with anhydrous sodium sulfate.
Compound 10 was obtained by filtration followed by evaporating
the solvent without further purification. Yellow oil, 46% yield, 1H
solid, 64% yield, 1H NMR (400 MHz, DMSO‑d6)
d
8.99 (d, J ¼ 7.2 Hz,
2H), 8.35 (s, 1H), 7.81 (d, J ¼ 7.8 Hz, 2H), 7.61 (d, J ¼ 9.0 Hz, 2H), 7.33
(t, J ¼ 7.9 Hz, 2H), 6.98 (t, J ¼ 7.3 Hz, 1H), 6.86 (d, J ¼ 9.0 Hz, 2H),
5.02e4.89 (m, 1H), 3.56 (t, J ¼ 5.7 Hz, 2H), 3.53e3.46 (m, 6H), 3.19
(s, 2H), 3.05 (s, 4H), 2.65 (t, J ¼ 5.9 Hz, 4H), 2.61 (s, 4H), 2.31 (s, 3H),
2.04 (s, 5H), 1.81e1.61 (m, 3H), 1.41 (s, 9H). MS (ESI): m/z calcd for
C
39H56N9O4: 714.4 [MþH]þ; found: 714.6.
4.1.15. Tert-butyl 2-((N-(2-(2-(2-(2-(4-(4-((9-cyclopentyl-8-
(phenylamino)-9H-purin- 2-yl)amino)phenyl)piperazin-1-yl)
ethoxy)ethoxy)ethoxy)ethyl)-N-methyl)amino)ace-tate (12b)
Compound 12b was prepared similarly as described for 12a.
NMR (400 MHz, CDCl3)
d 3.26 (s, 2H), 2.43 (s, 3H), 1.76 (s, 1H), 1.47
(s, 9H). MS (ESI): m/z calcd for C7H16NO2: 146.1 [MþH]þ; found:
146.4.
Yellow oil, 68% yield, 1H NMR (400 MHz, DMSO‑d6)
d 9.01 (s, 2H),

H.-Y. Zhao et al. / European Journal of Medicinal Chemistry 208 (2020) 112781
13
8.35 (s, 1H), 7.81 (d, J ¼ 7.8 Hz, 2H), 7.62 (d, J ¼ 8.9 Hz, 2H), 7.33 (t,
J ¼ 7.9 Hz, 2H), 6.98 (t, J ¼ 7.3 Hz, 1H), 6.88 (d, J ¼ 8.9 Hz, 2H),
5.02e4.87 (m, 1H), 3.61 (s, 2H), 3.56e3.46 (m, 10H), 3.20 (s, 2H),
3.11 (s, 4H), 2.70e2.62 (m, 4H), 2.54 (s, 4H), 2.31 (s, 3H), 2.04 (s, 5H),
1.70 (s, 3H), 1.40 (s, 9H). MS (ESI): m/z calcd for C41H60N9O5: 758.5
[MþH]þ; found: 758.6.
brine (20 mL). The organic phase was dried with anhydrous sodium
sulfate and isolated using column chromatography
(DCM:MeOH ¼ 30:1e50:1) followed by wash with ethyl acetate to
give compound P2. Yellow solid, 26% yield, 1H NMR (400 MHz,
DMSO‑d6)
d 11.06 (s, 1H), 9.83 (s, 1H), 9.07 (s, 1H), 9.00 (s, 1H), 8.36
(s, 1H), 7.84 (d, J ¼ 7.7 Hz, 3H), 7.63 (d, J ¼ 8.9 Hz, 2H), 7.56e7.45 (m,
2H), 7.33 (t, J ¼ 7.9 Hz, 2H), 6.98 (t, J ¼ 7.3 Hz,1H), 6.88 (d, J ¼ 9.0 Hz,
2H), 5.16 (dd, J ¼ 13.3, 5.1 Hz, 1H), 5.05e4.92 (m, 1H), 4.38 (q,
J ¼ 17.5 Hz, 2H), 3.12 (s, 4H), 2.98e2.88 (m, 1H), 2.70 (s, 4H),
2.65e2.58 (m, 2H), 2.47 (s, 2H), 2.40e2.30 (m, 3H), 2.05 (s, 5H),
1.76e1.58 (m, 5H), 1.52 (s, 2H), 1.33 (s, 6H). 13C NMR (100 MHz,
4.1.16. Tert-butyl 2-((N-(2-(2-(2-(2-(2-(4-(4-((9-cyclopentyl-8-
(phenylamino)-9H-pu- rin-2-yl)amino)phenyl)piperazin-1-yl)
ethoxy)ethoxy)ethoxy)ethoxy)ethyl)-N-methyl)amino)acetate (12c)
Compound 12c was prepared similarly as described for 12a.
Yellow solid, 36% yield, 1H NMR (400 MHz, DMSO‑d6)
d
9.02 (d,
DMSO‑d6) d 173.3,171.9,171.6,168.3,154.8,152.9,149.6,145.5,143.3,
J ¼ 3.0 Hz, 2H), 8.35 (s, 1H), 7.81 (d, J ¼ 8.0 Hz, 2H), 7.64 (d,
J ¼ 8.9 Hz, 2H), 7.33 (t, J ¼ 7.9 Hz, 2H), 6.98 (t, J ¼ 7.3 Hz,1H), 6.90 (d,
J ¼ 8.9 Hz, 2H), 5.01e4.88 (m, 1H), 3.64e3.44 (m, 16H), 3.23 (s, 2H),
3.17 (s, 4H), 2.94 (s, 4H), 2.72e2.63 (m, 4H), 2.33 (s, 3H), 2.04 (s, 5H),
1.70 (s, 3H), 1.40 (s, 9H). MS (ESI): m/z calcd for C43H64N9O6: 802.5
[MþH]þ; found: 802.7.
140.9, 134.6, 134.3, 134.2, 133.1, 129.1 (2C), 127.4, 125.7, 122.0, 119.6
(2C), 119.4, 118.9 (2C), 116.6 (2C), 57.8, 55.4, 54.8, 52.8, 52.0 (2C),
49.0, 47.0 (2C), 36.3, 31.7, 29.5 (2C), 29.1 (2C), 27.1, 25.5, 24.9 (2C),
23.1, 21.5. HRMS (ESI): m/z calcd for C47H56N11O4: 838.45167
[MþH]þ; found: 838.45145.
4.1.20. (2S,4R)-1-((S)-2-(7-(4-(4-((9-cyclopentyl-8-(phenylamino)-
9H-purin-2-yl)ami-no)phenyl)piperazin-1-yl)heptanamido)-3,3-
dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)
pyrrolidine-2-carboxamide (P3)
4.1.17. (2S,4R)-1-((S)-2-acetamido-3,3-dimethylbutanoyl)-4-
hydroxy-N-(4-(4-methyl-thiazol-5-yl)benzyl)pyrrolidine-2-
carboxamide (VHL-Ac)
To a solution of VHL-L (0.12 g, 0.25 mmol) in DCM (2 mL) were
added acetic anhydride (0.04 g, 0.38 mmol). The resulted solution
was stirred at room temperature for 1 h. Compound VHL-Ac was
isolated using column chromatography (DCM:MeOH ¼ 50:1e30:1).
Compound P3 was prepared similarly as described for P1 using
VHL-L as E3-recruitment element. Yellow solid, 51% yield, 1H NMR
(400 MHz, DMSO‑d6)
d 9.14 (s, 1H), 9.05 (s, 1H), 8.99 (s, 1H), 8.61 (t,
J ¼ 5.7 Hz, 1H), 8.37 (s, 1H), 7.93e7.81 (m, 3H), 7.66 (d, J ¼ 8.7 Hz,
2H), 7.46e7.37 (m, 4H), 7.33 (t, J ¼ 7.7 Hz, 2H), 6.98 (t, J ¼ 7.3 Hz,1H),
6.92 (d, J ¼ 8.7 Hz, 2H), 5.19 (s, 1H), 5.08e4.95 (m, 1H), 4.57 (d,
J ¼ 9.3 Hz, 1H), 4.49e4.41 (m, 2H), 4.37 (s, 1H), 4.23 (dd, J ¼ 15.8,
5.1 Hz,1H), 3.67 (s, 2H), 3.27 (s, 4H), 3.08 (s, 4H), 2.85 (s, 2H), 2.45 (s,
5H), 2.34e2.24 (m, 1H), 2.22e2.12 (m, 1H), 2.05 (s, 5H), 1.96e1.87
(m, 1H), 1.75e1.60 (m, 4H), 1.56e1.46 (m, 2H), 1.38e1.20 (m, 4H),
White solid, 56% yield, 1H NMR (400 MHz, DMSO‑d6)
d 8.99 (s, 1H),
8.58 (t, J ¼ 6.0 Hz,1H), 7.96 (d, J ¼ 9.4 Hz,1H), 7.40 (q, J ¼ 8.4 Hz, 4H),
5.13 (d, J ¼ 3.5 Hz, 1H), 4.54 (d, J ¼ 9.5 Hz, 1H), 4.47e4.40 (m, 2H),
4.35 (s, 1H), 4.21 (dd, J ¼ 15.9, 5.4 Hz, 1H), 3.70e3.62 (m, 2H), 2.44
(s, 3H), 2.07e1.98 (m, 1H), 1.89 (s, 3H), 0.94 (s, 9H). MS (ESI): m/z
calcd for C24H22N4O4S: 473.2 [MþH]þ; found: 473.4.
0.96 (s, 9H). 13C NMR (100 MHz, DMSO‑d6)
d 172.5, 172.4, 170.2,
4.1.18. 7-(4-(4-((9-cyclopentyl-8-(phenylamino)-9H-purin-2-yl)
amino)phenyl)pipera-zin-1-yl)-N-(2-(2,6-dioxopiperidin-3-yl)-1-
oxoisoindolin-4-yl)heptanamide (P1)
154.8, 152.9, 151.9, 149.7, 148.2, 144.8, 143.3, 140.9, 140.0, 135.1,
131.6, 130.1, 129.1 (2C), 129.1 (2C), 127.9 (2C), 127.5, 122.0, 119.6 (2C),
119.0 (2C), 116.9 (2C), 69.4, 59.2, 56.8, 56.8 (2C), 54.8, 51.9 (2C), 47.8,
42.1, 38.5, 35.7 (2C), 35.2, 29.5 (2C), 28.7 (2C), 26.9 (3C), 26.6, 25.7,
24.9 (2C), 16.41. HRMS (ESI): m/z calcd for C55H71N12O4S: 995.54419
[MþH]þ; found: 995.54146.
A flask was charged with 8 (0.0701 g, 0.12 mmol), lenalidomide
(0.0467 g, 0.18 mmol), HATU (0.0684 g, 0.18 mmol), DIPEA
(0.0310 g, 0.24 mmol) and DMF (1 mL). The resulted mixture was
stirred at room temperature overnight. The solvent was evaporated
under vacuum, and the residue was isolated using column chro-
matography (DCM:MeOH ¼ 20:1e8:1) to give target compound P1
which was subsequently washed with ethyl acetate to obtain pure
product. Yellow solid, 51% yield, 1H NMR (400 MHz, DMSO‑d6)
4.1.21. (2S,4R)-1-((S)-2-(8-(4-(4-((9-cyclopentyl-8-(phenylamino)-
9H-purin-2-yl)ami-no)phenyl)piperazin-1-yl)octanamido)-3,3-
dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)
pyrrolidine-2-carboxamide (P4)
d
11.05 (s, 1H), 9.92 (s, 1H), 9.14 (s, 1H), 9.07 (s, 1H), 8.37 (s, 1H), 7.85
A flask was charged with 6 (0.09 g, 0.19 mmol), 9b (0.12 g,
0.19 mmol), K2CO3 (0.05 g, 0.38 mmol), KI (0.0063 g, 0.04 mmol)
and CH3CN (3 mL). The mixture was heated at 80 ꢁC in an oil bath
overnight. The solvent was evaporated under vacuum, and the
(d, J ¼ 8.0 Hz, 3H), 7.67 (d, J ¼ 8.7 Hz, 2H), 7.52 (t, J ¼ 7.5 Hz, 2H), 7.33
(t, J ¼ 7.8 Hz, 2H), 6.99 (t, J ¼ 7.3 Hz, 1H), 6.93 (d, J ¼ 8.8 Hz, 2H), 5.17
(dd, J ¼ 13.2, 4.9 Hz,1H), 5.09e4.96 (m, 1H), 4.40 (q, J ¼ 17.5 Hz, 2H),
3.19 (d, J ¼ 10.6 Hz, 4H), 3.11e2.84 (m, 5H), 2.69e2.56 (m, 2H), 2.46
(s, 2H), 2.43e2.33 (m, 3H), 2.05 (s, 5H), 1.78e1.60 (m, 7H), 1.36 (s,
residue
was
purified
using
column
chromatography
(DCM:MeOH ¼ 30:1e10:1) followed by wash with ethyl acetate to
4H). 13C NMR (100 MHz, DMSO‑d6)
d 173.4, 171.8, 171.6, 168.3, 154.7,
produce P4. Yellow solid, 63% yield, 1H NMR (400 MHz, DMSO‑d6)
152.9, 149.7, 144.5, 143.2, 140.9, 135.2, 134.3, 134.2, 133.1, 129.1 (2C),
127.5, 125.8, 122.0, 119.6 (2C), 119.5, 190.0 (2C), 117.0 (2C), 56.3, 54.9,
52.0, 51.6 (2C), 47.4 (2C), 47.1, 36.1, 31.7, 29.5 (2C), 28.7 (2C), 26.5,
25.3, 24.9 (2C), 24.0, 23.1. HRMS (ESI): m/z calcd for C46H54N11O4:
824.43602 [MþH]þ; found: 824.43462.
d
9.03 (d, J ¼ 7.6 Hz, 2H), 8.98 (s, 1H), 8.59 (t, J ¼ 5.9 Hz, 1H), 8.36 (s,
1H), 7.88 (d, J ¼ 9.4 Hz, 1H), 7.83 (d, J ¼ 7.9 Hz, 2H), 7.64 (d,
J ¼ 8.8 Hz, 2H), 7.45e7.37 (m, 4H), 7.33 (t, J ¼ 7.9 Hz, 2H), 6.99 (t,
J ¼ 7.3 Hz, 1H), 6.89 (d, J ¼ 8.8 Hz, 2H), 5.16 (s, 1H), 5.03e4.91 (m,
1H), 4.57 (d, J ¼ 9.4 Hz,1H), 4.49e4.41 (m, 2H), 4.37 (s,1H), 4.23 (dd,
J ¼ 15.8, 5.3 Hz, 1H), 3.72e3.63 (m, 2H), 3.37e3.30 (m, 2H), 3.12 (s,
4H), 2.71 (s, 4H), 2.45 (s, 5H), 2.33e2.24 (m, 1H), 2.18e2.11 (m, 1H),
2.05 (s, 5H), 1.95e1.88 (m, 1H), 1.70 (s, 2H), 1.56e1.44 (m, 4H), 1.28
4.1.19. 8-(4-(4-((9-cyclopentyl-8-(phenylamino)-9H-purin-2-yl)
amino)phenyl)pipera-zin-1-yl)-N-(2-(2,6-dioxopiperidin-3-yl)-1-
oxoisoindolin-4-yl)octanamide (P2)
(s, 6H), 0.95 (s, 9H). 13C NMR (100 MHz, DMSO‑d6)
d 172.6, 172.4,
To a flask were added 6 (0.10 g, 0.22 mmol), 9a (0.15 g,
0.33 mmol), DIPEA (0.0568 g, 0.44 mmol), KI (0.0073 g,
0.044 mmol) and NMP (2 mL). The suspension was heated at 100 ꢁC
in an oil bath overnight. The reaction mixture was diluted with
DCM (80 mL) and washed with water (5 ꢂ 20 mL) and saturated
170.2, 154.8, 152.9, 151.9, 149.6, 148.2, 145.4, 143.3, 140.9, 140.0,
134.6, 131.6, 130.1, 129.1 (2C), 129.1 (2C), 127.9 (2C), 127.4, 122.0,
119.6 (2C), 118.9 (2C), 116.6 (2C), 69.3, 59.2, 56.8, 56.8 (2C), 54.8,
52.8 (2C), 49.0, 42.1, 38.4, 35.7 (2C), 35.3, 29.5 (2C), 29.1 (2C), 27.1,
26.9, 26.9 (3C), 25.8, 24.9 (2C), 16.4. HRMS (ESI): m/z calcd for

14
H.-Y. Zhao et al. / European Journal of Medicinal Chemistry 208 (2020) 112781
C56H73N12O4S: 1009.55984 [MþH]þ; found: 1009.55876.
143.3, 140.9, 134.5, 134.5, 133.7, 133.1, 129.2, 129.1 (2C), 127.4, 125.9,
122.0, 119.8, 119.6 (2C), 118.9 (2C), 116.5 (2C), 70.2, 70.2 (4C), 70.1,
70.1, 68.8, 61.4, 57.3, 56.9 (2C), 54.8, 53.4, 52.0 (2C), 49.3, 46.7, 43.4,
31.7, 29.5 (2C), 24.9 (2C), 23.0. HRMS (ESI): m/z calcd for
4.1.22. 2-((2-(2-(2-(4-(4-((9-cyclopentyl-8-(phenylamino)-9H-
purin-2-yl)amino)phen-yl)piperazin-1-yl)ethoxy)ethoxy)ethyl)
(methyl)amino)-N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-
yl)acetamide (P5)
C
52H67N12O8: 987.52048 [MþH]þ; found: 987.51632.
To a solution of 12a (0.08 g, 0.11 mmol) in DCM (1 mL) were
added TFA (1 mL). The resulted solution was stirred at room tem-
perature for 8 h. The solvent was evaporated under high vacuum. To
the residue was added DMF (1 mL), DIPEA (0.28 g, 2.2 mmol), HATU
(0.08 g, 0.22 mmol) and lenalidomide (0.0570 g, 0.22 mmol). The
mixture was stirred at room temperature for 4 h. To the reaction
mixture was added DCM (100 mL), and the solution was washed
with water (3 ꢂ 20 mL) and saturated brine (20 mL) and dried with
anhydrous sodium sulfate. Compound P5 was obtained using col-
umn chromatography (DCM:MeOH ¼ 20:1e16:1) followed by wash
using PE:EA (5:1). Yellow solid, 31% yield, 1H NMR (400 MHz,
4.1.25. (2S,4R)-1-((S)-2-((2-(2-(2-(4-(4-((9-cyclopentyl-8-
(phenylamino)-9H-purin-2- yl)amino)phenyl)piperazin-1-yl)
ethoxy)ethoxy)ethyl)amino)-3,3-dimethylbutanoyl)-4-hydroxy-N-
(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (P8)
VHL-L (0.25 g, 0.58 mmol), (ethane-1,2-diylbis (oxy))bis
(ethane-2,1-diyl)
bis(4-methylbenzenesulfonate)
(0.53
g,
1.16 mmol), K2CO3 (0.16 g, 1.16 mmol), NaI (0.0348 g, 0.23 mmol)
and CH3CN (25 mL) were mixed in a flask. The resulted suspension
was heated at 80 ꢁC in an oil bath for 48 h. The solvent was evap-
orated under vacuum and the residue was isolated using column
chromatography (DCM:MeOH ¼ 40:1e20:1) to give compound 13a
as colorless oil. 33% yield, MS (ESI): m/z calcd for C35H49N4O8S2:
717.3 [MþH]þ; found: 717.5.
DMSO‑d6)
d
11.05 (s, 1H), 9.72 (s, 1H), 9.00 (d, J ¼ 10.5 Hz, 2H), 8.36
(s, 1H), 7.92e7.76 (m, 3H), 7.61 (d, J ¼ 8.6 Hz, 2H), 7.57e7.48 (m, 2H),
7.33 (t, J ¼ 7.6 Hz, 2H), 6.98 (t, J ¼ 7.2 Hz, 1H), 6.85 (d, J ¼ 8.7 Hz, 2H),
5.16 (dd, J ¼ 13.0, 4.7 Hz, 1H), 5.04e4.89 (m, 1H), 4.39 (q, J ¼ 17.3 Hz,
2H), 3.63e3.56 (m, 2H), 3.53 (s, 2H), 3.46 (d, J ¼ 5.6 Hz, 4H), 3.25 (s,
2H), 3.00 (s, 4H), 2.94e2.85 (m, 1H), 2.68 (s, 2H), 2.65e2.56 (m, 1H),
2.51 (s, 4H), 2.46e2.36 (m, 7H), 2.04 (s, 6H), 1.69 (s, 2H). 13C NMR
To a solution of 13a (0.15 g, 0.21 mmol) in CH3CN were added 6
(0.12 g, 0.27 mmol), K2CO3 (0.06 g, 0.42 mmol) and NaI (0.0126 g,
0.084 mmol). The resulted suspension was heated at 65 ꢁC in an oil
bath for 24 h. The solvent was evaporated under vacuum, and the
(100 MHz, DMSO‑d6)
d 173.3, 171.5, 169.7, 168.3, 154.9, 152.9, 149.6,
residue
was
purified
using
column
chromatography
145.9, 143.3, 140.9, 134.5, 134.3, 133.7, 133.2, 129.2, 129.1 (2C), 127.4,
125.8, 122.0, 119.8, 119.7 (2C), 118.9 (2C), 116.4 (2C), 70.1, 70.0, 68.8,
68.8, 61.5, 57.6, 56.9, 54.8 (2C), 53.6, 52.0 (2C), 49.7, 46.7, 43.5, 31.7,
29.5 (2C), 24.9 (2C), 23.0. HRMS (ESI): m/z calcd for C48H59N12O6:
899.46805 [MþH]þ; found: 899.46376.
(DCM:MeOH ¼ 20:1e10:1) followed by wash with PE:EA (10:1) to
give compound P8. Yellow solid, 57% yield, 1H NMR (400 MHz,
DMSO‑d6)
d
9.03 (s, 1H), 8.98 (d, J ¼ 8.8 Hz, 2H), 8.59 (d, J ¼ 5.4 Hz,
1H), 8.36 (s, 1H), 7.83 (d, J ¼ 7.9 Hz, 2H), 7.62 (d, J ¼ 8.4 Hz, 2H),
7.47e7.37 (m, 4H), 7.33 (t, J ¼ 7.7 Hz, 2H), 6.98 (t, J ¼ 7.2 Hz,1H), 6.87
(d, J ¼ 8.7 Hz, 2H), 5.09 (s, 1H), 5.02e4.90 (m, 1H), 4.61e4.51 (m,
1H), 4.45e4.33 (m, 2H), 4.33e4.22 (m, 1H), 3.70e3.60 (m, 2H),
3.58e3.47 (m, 8H), 3.24 (s, 1H), 3.17 (s, 1H), 3.06 (s, 4H), 2.63 (s, 6H),
2.47e2.42 (m, 5H), 2.04 (s, 6H), 1.96e1.88 (m, 1H), 1.68 (s, 3H), 0.94
4.1.23. 2-(2-(2-(2-(2-(4-(4-((9-cyclopentyl-8-(phenylamino)-9H-
purin-2-yl)amino)ph-enyl)piperazin-1-yl)ethoxy)ethoxy)ethoxy)
ethyl) (methyl)amino-N-(2-(2,6-dioxopiperidin-3-yl)-1-
oxoisoindolin-4-yl)acetamide (P6)
(s, 9H). 13C NMR (100 MHz, DMSO‑d6)
d 172.7, 172.5, 154.9, 152.9,
Compound P6 was prepared similarly as described for P5. Yel-
151.9, 149.6, 148.2, 145.8, 143.3, 140.9, 140.0, 134.4, 131.6, 130.1, 129.1
(2C), 129.1 (2C), 127.9 (2C), 127.4, 122.0, 119.6 (2C), 118.9 (2C), 116.5
(2C), 70.9, 70.1, 70.1 69.4, 68.5, 66.8, 59.0, 57.6, 56.7, 54.8, 53.6 (2C),
49.5 (2C), 48.2, 42.1, 38.2, 35.4, 29.5 (2C), 27.0 (3C), 24.9 (2C), 16.4.
HRMS (ESI): m/z calcd for C54H71N12O5S: 999.53911 [MþH]þ;
found: 999.53746.
low solid, 28% yield, 1H NMR (400 MHz, DMSO‑d6)
d 11.03 (s, 1H),
9.71 (s, 1H), 9.02 (s, 1H), 8.98 (s, 1H), 8.35 (s, 1H), 7.83 (t, J ¼ 8.1 Hz,
3H), 7.61 (d, J ¼ 8.9 Hz, 2H), 7.57e7.47 (m, 2H), 7.33 (t, J ¼ 7.9 Hz,
2H), 6.98 (t, J ¼ 7.3 Hz, 1H), 6.86 (d, J ¼ 9.0 Hz, 2H), 5.15 (dd, J ¼ 13.2,
5.0 Hz, 1H), 5.01e4.85 (m, 1H), 4.39 (q, J ¼ 17.3 Hz, 2H), 3.57 (t,
J ¼ 5.4 Hz, 2H), 3.53e3.48 (m, 6H), 3.44 (s, 4H), 3.24 (s, 2H), 3.03 (s,
4H), 2.97e2.87 (m, 1H), 2.68 (t, J ¼ 5.5 Hz, 2H), 2.63 (s, 1H), 2.56 (s,
4H), 2.49e2.40 (m, 4H), 2.38 (s, 3H), 2.02 (s, 6H), 1.75e1.63 (m, 2H).
13C NMR (100 MHz, DMSO‑d6)
d
173.3, 171.5, 169.6, 168.3, 154.9,
4.1.26. (2S,4R)-1-((S)-2-((2-(2-(2-(2-(4-(4-((9-cyclopentyl-8-
(phenylamino)-9H-purin -2-yl)amino)phenyl)piperazin-1-yl)
ethoxy)ethoxy)ethoxy)ethyl)amino)-3,3-dimethylbutanoyl)-4-
hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-
carboxamide (P9)
152.9,149.6,145.9,143.3,140.9,134.5,134.3,133.7,133.1,129.2,129.1
(2C), 127.4, 125.9, 122.0, 119.8, 119.7 (2C), 118.9 (2C), 116.4 (2C), 70.2
(2C), 70.1, 70.1, 68.8, 68.7, 61.5, 57.6, 56.9, 54.8, 53.7 (2C), 52.0, 49.7
(2C), 46.71, 43.4, 31.7, 29.5 (2C), 24.9 (2C), 23.0. HRMS (ESI): m/z
calcd for C50H63N12O7: 943.49427 [MþH]þ; found: 943.49047.
Compound P9 was prepared similarly as described for P8. Yel-
low solid, 28% yield, 1H NMR (400 MHz, DMSO‑d6)
d 9.02 (s, 1H),
4.1.24. 2-(2-(2-(2-(2-(2-(4-(4-((9-cyclopentyl-8-(phenylamino)-
9H-purin-2-yl)amino)- phenyl)piperazin-1-yl)ethoxy)ethoxy)
ethoxy)ethoxy)ethyl) (methyl)amino-N-(2-(2,6-dioxopiperidin-3-
yl)-1-oxoisoindolin-4-yl)acetamide (P7)
8.98 (d, J ¼ 5.0 Hz, 2H), 8.58 (t, J ¼ 5.7 Hz, 1H), 8.35 (s, 1H), 7.82 (d,
J ¼ 8.0 Hz, 2H), 7.62 (d, J ¼ 8.7 Hz, 2H), 7.46e7.37 (m, 4H), 7.33 (t,
J ¼ 7.9 Hz, 2H), 6.98 (t, J ¼ 7.3 Hz,1H), 6.86 (d, J ¼ 8.8 Hz, 2H), 5.07 (s,
1H), 5.01e4.90 (m, 1H), 4.62e4.50 (m, 1H), 4.45e4.33 (m, 2H),
4.33e4.20 (m, 1H), 3.72e3.59 (m, 2H), 3.58e3.42 (m, 14H), 3.23 (s,
1H), 3.12 (s,1H), 3.04 (s, 4H), 2.59 (s, 4H), 2.54 (s, 2H), 2.48e2.40 (m,
5H), 2.02 (s, 5H), 1.97e1.87 (m, 1H), 1.69 (s, 2H), 0.92 (s, 9H). 13C
Compound P7 was prepared similarly as described for P6. Yel-
low solid, 28% yield, 1H NMR (400 MHz, DMSO‑d6)
d 11.03 (s, 1H),
9.73 (s, 1H), 9.04 (s, 1H), 8.99 (s, 1H), 8.35 (s, 1H), 7.83 (dd, J ¼ 6.9,
4.6 Hz, 3H), 7.62 (d, J ¼ 8.9 Hz, 2H), 7.56e7.48 (m, 2H), 7.33 (t,
J ¼ 7.9 Hz, 2H), 6.98 (t, J ¼ 7.3 Hz, 1H), 6.87 (d, J ¼ 9.0 Hz, 2H), 5.15
(dd, J ¼ 13.3, 5.1 Hz, 1H), 5.02e4.91 (m, 1H), 4.38 (q, J ¼ 17.3 Hz, 2H),
3.57 (t, J ¼ 5.4 Hz, 4H), 3.52e3.47 (m, 8H), 3.44 (s, 4H), 3.26 (s, 2H),
3.08 (s, 4H), 2.97e2.86 (m, 1H), 2.74e2.58 (m, 9H), 2.48e2.42 (m,
2H), 2.39 (s, 3H), 2.02 (s, 6H), 1.75e1.63 (m, 2H). 13C NMR (100 MHz,
NMR (100 MHz, DMSO‑d6)
d 173.4, 172.6, 154.9, 152.9, 151.9, 149.6,
148.2, 145.9, 143.3, 140.9, 140.0, 134.3, 131.6, 130.1, 129.1 (2C), 129.1
(2C), 127.9 (2C), 127.4, 122.0, 119.6 (2C), 118.9 (2C), 116.4 (2C), 71.0,
70.2, 70.2, 70.2, 70.1, 69.4, 68.7, 66.8, 59.0, 57.6, 56.6, 54.8, 53.6 (2C),
49.6 (2C), 48.2, 42.1, 38.2, 35.4, 29.5 (2C), 27.0 (3C), 24.9 (2C), 16.4.
HRMS (ESI): m/z calcd for C56H75N12O6S: 1043.56532 [MþH]þ;
found: 1043.56471.
DMSO‑d6)
d 173.3, 171.5, 169.5, 168.3, 154.9, 152.9, 149.6, 145.7,

H.-Y. Zhao et al. / European Journal of Medicinal Chemistry 208 (2020) 112781
15
4.1.27. (2S,4R)-1-((S)-2-((2-(2-(2-(2-(2-(4-(4-((9-cyclopentyl-8-
4.2.3. Western blotting assay
(phenylamino)-9H- purin-2-yl)amino)phenyl)piperazin-1-yl)
ethoxy)ethoxy)ethoxy)ethoxy)ethyl)amino)-3,3-dimethylbutanoyl)-
4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-
carboxamide (P10)
Cells (0.5e1 ꢂ 106/well) were seeded in 6-well plates and
incubated for 24 h. Cells were treated with different concentrations
of compounds for indicated time and lysed by lysis buffer (Beyo-
time) with protease and phosphatase inhibitors. The suspension
was centrifuged at 12,000 rpm for 20 min, and the insoluble ma-
Compound P10 was prepared similarly as described for P8.
Yellow solid, 36% yield, 1H NMR (400 MHz, DMSO‑d6)
d
9.03 (s, 1H),
terial was removed. Protein (about 15 mg) was separated by 8% SDS-
8.98 (d, J ¼ 6.0 Hz, 2H), 8.59 (t, J ¼ 5.7 Hz, 1H), 8.36 (s, 1H), 7.83 (d,
J ¼ 8.0 Hz, 2H), 7.62 (d, J ¼ 8.9 Hz, 2H), 7.45e7.37 (m, 4H), 7.33 (t,
J ¼ 7.9 Hz, 2H), 6.98 (t, J ¼ 7.3 Hz,1H), 6.87 (d, J ¼ 8.9 Hz, 2H), 5.08 (s,
1H), 5.03e4.88 (m, 1H), 4.62e4.52 (m, 1H), 4.46e4.33 (m, 2H),
4.32e4.20 (m, 1H), 3.70e3.62 (m, 2H), 3.61e3.47 (m, 16H),
3.47e3.44 (m, 2H), 3.25e3.20 (m, 1H), 3.17 (s, 1H), 3.07 (s, 4H), 2.65
(s, 6H), 2.48e2.40 (m, 5H), 2.04 (s, 5H),1.96e1.87 (m,1H),1.78e1.63
PAGE and transferred to PVDF membranes (Millpore). Aafter incu-
bation with primary and secondary antibodies (Xi’an Zhuangzhi
Biotechnology Co., Ltd.), Membranes were imaged by a ChemiDoc
MP Imaging system (BIO-RAD) and organized with Image Lab
software.
The antibodies against EGFR (4267,1:1000), Akt (4691T,1:1000),
P-EGFR (3777, 1:1000) and P-Akt (4060T, 1:1000) were purchased
from Cell Signaling. PP62 (ab109012, 1:1000) and LC3B (ab192890,
(m, 2H), 0.93 (s, 9H). 13C NMR (100 MHz, DMSO‑d6)
d 172.7, 172.5,
154.9, 152.9, 151.9, 149.6, 148.2, 145.8, 143.3, 140.9, 140.0, 134.4,
131.6, 130.1, 129.1 (2C), 129.1 (2C), 127.9 (2C), 127.4, 122.0, 119.6 (2C),
118.9 (2C),116.5 (2C), 70.9, 70.3 (3C), 70.2, 70.1 (2C), 69.4, 68.4, 66.8,
59.0, 57.5, 56.7, 54.8, 53.5 (2C), 49.4 (2C), 48.1, 42.1, 38.2, 35.4, 29.5
(2C), 27.0 (3C), 24.9 (2C), 16.4. HRMS (ESI): m/z calcd for
1:2000) were purchased from abcam.
was purchased from proteintech.
b-actin (20536-1-AP, 1:1000)
4.2.4. Cell apoptosis assay
Cells (0.5e1 ꢂ 106/well) were seeded in 6-well plates and
incubated for 24 h. And compounds at indicated concentrations
were added. Having been incubated for further 48 h, cells were
collected and disposed with an Annexin V-FITC apoptosis detection
kit (Becton Dikinson) according to the instructions and apoptosis
rate was determined by the Flow Cytometry.
C
58H79N12O7S: 1087.59154 [MþH]þ; found: 1087.58916.
4.1.28. (2S,4S)-1-((S)-2-(7-(4-(4-((9-cyclopentyl-8-(phenylamino)-
9H-purin-2-yl)ami-no)phenyl)piperazin-1-yl)heptanamido)-3,3-
dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)
pyrrolidine-2-carboxamide (P11)
Compound P11 was prepared similarly as described for P1 using
4.2.5. Cell cycle assay
VHL-L as E3-recruitment element. Yellow solid, 30% yield, 1H NMR
Cells (0.5e1 ꢂ 106/well) were seeded into 6-well plates and
incubated for 24 h. And compounds at indicated concentrations
were added. Having been incubated for further 48 h, cells were
collected and washed twice with PBS. Cold ethanol (75%, 1.5 mL)
was added dropwise and the cells were fixed at 4 ꢁC for 20 h. The
samples were centrifuged at 5000 rpm, and the supernatant was
discarded completely. And the collected cells were treated with
(400 MHz, DMSO‑d6)
d
9.12 (s, 1H), 9.00 (d, J ¼ 11.7 Hz, 2H), 8.67 (s,
1H), 8.36 (s, 1H), 7.85 (s, 3H), 7.65 (s, 2H), 7.41 (s, 4H), 7.32 (s, 2H),
6.98 (s, 1H), 6.89 (s, 2H), 5.47 (s, 1H), 5.01 (s, 1H), 4.53e4.36 (m, 2H),
4.32e4.15 (m, 2H), 3.96 (s, 1H), 3.45 (s, 2H), 3.16 (s, 4H), 3.06 (s, 2H),
2.80 (s, 4H), 2.45 (s, 5H), 2.34 (s,1H), 2.24 (s,1H), 2.04 (s, 5H),1.91 (s,
1H), 1.69 (s, 2H), 1.52 (s, 4H), 1.27 (s, 4H), 0.97 (s, 9H). MS (ESI): m/z
calcd for C55H71N12O4S: 995.5 [MþH]þ; found: 995.5.
500 m
L PI/RNase (Becton Dikinson, #550825), incubated at 37 ꢁC for
15 min and analyzed on a FACS CaliburTM flow cytometer (Becton
4.2. Biology
Dickinson).
4.2.1. Cell lines and agents
4.2.6. Cell colony formation assay
HCC827, A549 and H1975 cells were provided by Stem Cell Bank,
Chinese Academy of Sciences. HCC827 cell line were culture in
RPMI-1640 supplemented with 10% heat inactivated fetal bovine
serum (FBS), 1% sodium pyruvate, 1% glutamine and 1% penicillin-
streptomycin. H1975 cell line were cultured in RPMI-1640 supple-
mented with 10% heat inactivated fetal bovine serum (FBS) and 1%
penicillin-streptomycin. A549 were cultured in DMEM supple-
mented with 10% heat inactivated fetal bovine serum (FBS) and 1%
penicillin-streptomycin. All cells were cultured in a humidified
atmosphere containing 5% CO2 at 37 ꢁC.
Cells (200/well) were seeded into 6-well plates and incubated
for 24 h. And compounds at indicated concentrations were added.
Having been incubated for 15 days, the cells were washed with PBS
three times and fixed with cold ethanol (75%, 1.5 mL/well). The
ethanol was removed followed by addition of crystal violet solution
(0.1% in water) and incubation for 1 h. Colony number was counted
and the photos were taken by a camera.
4.2.7. Statistical analysis
Data was analyzed using GraphPad Prism 5.0. DC50 (concen-
tration that caused deletion of 50% of EGFR) and IC50 (concentration
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)”.
MLN4924 (S7109) was purchased from Selleck. MG132
(M832899-5 mg) and Chloroquine (B20714-50 mg) were purchased
from Macklin. Rapamycin (C843545-20 mg) was obtained from
Shang Hai Yuan Ye.
4.2.2. Cell proliferation inhibition assay
The anti-proliferative activities of compounds against tumor
cells were determined by MTT assay. Cells (3000e8000/well) were
Declaration of competing interest
seeded in 96-well plates (200
24 h. And the cells were treated with a series of concentrations of
compounds and incubated for further 72 h 20 L of MTT solution
(5 mg/mL in PBS) was added to each well and incubated for 4 h. The
supernatant in each well was removed carefully, and 150 L of
m
L medium/well) and incubated for
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.
m
m
Acknowledgement
DMSO was added. The optical density of each well was determined
by Varioskan Flash Multimode Reader (Thermo scientific) at
490 nm or 570 nm wavelength.
The financial support from The National Natural Science Foun-
dation of China (Grant No. 81673354) is gratefully acknowledged.

16
H.-Y. Zhao et al. / European Journal of Medicinal Chemistry 208 (2020) 112781
H. Dong, K. Siu, J.D. Winkler, A.P. Crew, C.M. Crews, K.G. Coleman, PROTAC-
Appendix A. Supplementary data
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