Discovery of novel anti-angiogenesis agents. Part 11: Development of PROTACs based on active molecules with potency of promoting vascular normalization

Article abstract of DOI:10.1016/j.ejmech.2020.112654

Our recent investigation is focused on the discovery of anti-angiogenesis agents. Vascular normalization induced by anti-angiogenic agent appears to be a promising strategy. We have developed novel angiogenesis inhibitors with potency of promoting vascular normalization. Herein, we reported the design, synthesis and preliminary evaluation of proteolysis-targeting chimera (PROTACs) based on the previously developed anti-angiogenesis agents. Two PROTACs exhibited potent VEGFR-2 inhibition and anti-proliferative activity against HUVECs. Moreover, they were capable of reducing protein levels of VEGFR-2 in EA.hy926 cells without significant cytotoxicity against HEK293 cells. The novel PROTACs could be used to normalize the abnormal vessels, resulting in efficient delivery of drugs.

Full text of DOI:10.1016/j.ejmech.2020.112654

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Discovery of novel anti-angiogenesis agents. Part 11: Development of PROTACs
based on active molecules with potency of promoting vascular normalization
Yuanyuan Shan, Ru Si, Jin Wang, Qingqing Zhang, Jing Li, Yuexiang Ma, Jie Zhang
PII:
S0223-5234(20)30626-7
DOI:
https://doi.org/10.1016/j.ejmech.2020.112654
EJMECH 112654
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 12 June 2020
Revised Date: 5 July 2020
Accepted Date: 7 July 2020
Please cite this article as: Y. Shan, R. Si, J. Wang, Q. Zhang, J. Li, Y. Ma, J. Zhang, Discovery of novel
anti-angiogenesis agents. Part 11: Development of PROTACs based on active molecules with potency
of promoting vascular normalization, European Journal of Medicinal Chemistry, https://doi.org/10.1016/
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Discovery of novel anti-angiogenesis agents. Part 11: Development of PROTACs
based on active molecules with potency of promoting vascular normalization
Yuanyuan Shana,†, Ru Sib,†, Jin Wangb, Qingqing Zhangb, Jing Lib, Yuexiang Mab, Jie Zhangb,
aDepartment of Pharmacy, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an,
710061, China
bSchool of Pharmacy, Health Science Center, Xi'an Jiaotong University, Xi’an, 710061, China
As a continuation to our previous research, a series of novel PROTACs were developed based
on the potent anti-angiogenesis agents.
Corresponding author. E-mail: zhj8623@mail.xjtu.edu.cn (Jie Zhang).
† Both authors contributed equally to this work and should be regarded as joint first authors.

European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Discovery of novel anti-angiogenesis agents. Part 11: Development of PROTACs based on active
molecules with potency of promoting vascular normalization
Yuanyuan Shana,†, Ru Sib,†, Jin Wangb, Qingqing Zhangb, Jing Lib, Yuexiang Mab, Jie Zhangb,
aDepartment of Pharmacy, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, 710061, China
bSchool of Pharmacy, Health Science Center, Xi'an Jiaotong University, Xi’an, 710061, China
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Vascular normalization induced by anti-angiogenic agent appears to be a
promising strategy. Our recent investigation is focused on the discovery of anti-
angiogenesis agents with potency of promoting vascular normalization. Herein, we
reported the design, synthesis and preliminary evaluation of proteolysis-targeting
chimera (PROTACs) based on the previously developed anti-angiogenesis agents.
Two PROTACs exhibited potent VEGFR-2 inhibition and anti-proliferative
activity against HUVECs. Moreover, they were capable of reducing protein levels
of VEGFR-2 in EA.hy926 cells without significant cytotoxicity. These novel
PROTACs could be used to normalize the abnormal vessels, resulting in efficient
delivery of drugs.
Available online
Keywords:
PROTACs
Anti-angiogenesis agents
Active molecules
Vascular normalization
2009 Elsevier Ltd. All rights reserved
.
———
Corresponding author. E-mail: zhj8623@mail.xjtu.edu.cn (Jie Zhang).
† Both authors contributed equally to this work and should be regarded as joint first authors.
1

function by targeting protein destruction [9]. The targeted
degradation of proteins via the ubiquitin-proteasome
system (UPS) is an emerging modality in small molecule
drug discovery. Recent reports of proteolysis targeting
chimeras (PROTACs) have demonstrated that this
technology can effectively decrease the cellular levels of
target protein [10]. PROTACs are bifunctional molecules
composed of target protein ligand and E3 ubiquitin ligase
ligand via various linkers (Figure 2). These hetero-
bifunctional molecules could recruit an E3 ubiquitin ligase
to a given substrate protein resulting in ubiquitination and
1. Introduction
Pathological angiogenesis plays a critical role in numerous
diseases and has been recognized as
a defining
characteristic of cancers. Vasculature abnormal is
inadequate for delivery of drugs or oxygen and is
becoming an urgent problem in clinic [1]. Vascular
normalization induced by anti-angiogenesis agents appears
to be a promising strategy and has been proposed as an
emerging paradigm in the intervention for pathological
angiogenesis [2].
Our recent study is focused on the discovery of novel anti-
angiogenesis agents. We have identified biphenyl-aryl
ureas incorporated with salicyladoxime as angiogenesis
inhibitors. Subsequently, we developed diaryl ureas
incorporated with aromatic-heterocyclics as next
generation angiogenesis inhibitors via structure-guided
core-refining approach. Biological evaluations identified a
novel anti-angiogenesis agent, S7 (Figure 1) [3-7]. It
degradation [11].
PROTAC
Ubiquitin
2
E
2
E
Target Protein
E3 Ligase
Proteasome
displayed
selective
inhibitory
activities
against
angiogenesis-related receptor tyrosine kinase (RTK) and
proliferation of human umbilical vein endothelial cells
Fig. 2. The design strategy of novel PROTAC based on
active anti-angiogenesis agent.
(HUVECs).
Moreover,
S7
exhibited
vascular
Our findings suggested that anti-angiogenesis agents were
potential candidates for treatment of vasculature abnormal.
Emerging PROTACs technique converts target inhibitors
into advantageous target degraders. With the aims to
develop novel PROTACs for vasculature abnormal [12].
We designed and synthesized six PROTACs based on our
previously developed anti-angiogenesis agents as well as
the potent VEGFR-2 inhibitor, ABT-869. The
pharmacophores and scaffolds of two inhibitors are similar
with each other. The novel PROTACs are composed of
anti-angiogenesis agents and E3 ubiquitin ligases ligand
(VH 032) with different linkers. As the linker in a
PROTAC plays a key role for its degradation potency and
efficacy, we have performed extensive optimization of the
linker (Figure 3).
normalization potency in transgenic zebrafish model. In
summary, we developed a novel anti-angiogenesis agent
with potency of promoting vascular normalization
.
Fig. 1. Discovery of active anti-angiogenesis agent with
vascular normalization potential.
As well known, traditional occupancy-based RTK
inhibitors are designed to specifically activate or inactivate
protein functions. They have inherent limitations owing to
high concentration needed to ensure sufficient inhibition
and potential off-target side effects [8]. Moreover,
resistance to RTK inhibitors is responsible for the limited
benefit in clinic. As an alternative, induced protein
degradation might overcome these limitations and offers a
novel catalytic mechanism to irreversibly inhibit protein
2

the abnormal vessels, resulting in efficient delivery of
drugs and oxygen.
2. Results and discussion
2.1 Chemistry
The length and composition of linker is one of the critical
parameters affecting degrader potency [13]. Therefore, we
focused on extensive optimization of the linker. The
general synthetic procedures for the title PROTACs 1-6
were outlined in Scheme 1 and Scheme 2 [14]. VH032 is
commonly functionalized as von Hippel-Lindau (VHL)
ligand for PROTACs. According to the synthetic schemes,
E3 ubiquitin ligases ligand (VH032) was prepared as
previously described [15]. Target protein ligand S7 and
ABT-869 were prepared according to the synthetic scheme
previously reported [16]. Condensation of target protein
ligand with various di-acid in the presence of PyBop
afforded the key intermediates (6) or (7). Finally, the title
compounds (PROTAC-1~PROTAC-6) were yielded by
connecting the key intermediates with VHL ligand [17].
Fig. 3. The structures of novel PROTAC based on potent
anti-angiogenesis inhibitors.
Herein, we performed the design, synthesis, and biological
evaluation of novel PROTACs based on new angiogenesis
inhibitors. Among them, PROTAC-2 and PROTAC-5
displayed potent inhibition against VEGFR-2 and anti-
proliferative activities against human umbilical vein
endothelial cells (EA.hy926). They were capable of
specifically reducing protein levels of VEGFR-2.
Moreover, the new PROTACs exhibited no significant
cytotoxicity. These PROTACs could be used to normalize
N
OH
N
OH
OH
S
S
OH
b
c
N
d
e
HN
f
Boc
N
Boc
N
N
S
S
N
H
H2N
N
S
H
O
N
O
O
H
O
S
O
N
H
N
N
O
H
N
CN
1
H2N
N
5
3
4
2
k
VHL Ligand
a
O
O
HO
OH
n
Br
I
OH
H2N
N
S
H2N
CN
j
O
g
I
F
N
N
O
n
N
H2N
N
N
N
O
N
H
H
O
CN
i
H
NH
N
H
N
6
H
O
OH
h
O
B
+
O
O
S7
H2N
B
N
N
H
H
N
NH2
O
HN
O
H2N
S
N
N
O
O
N
O
H
n
N
N
N
O
H
H
PROTAC-1 n=3
PROTAC-2 n=8
PROTAC-3 n=12
Scheme 1. Synthetic route of the title PROTACs PROTAC-1~PROTAC-3.
Reagents and conditions: a. Pd(OAc)2, KOAc, DMAc, 150℃; b. NaBH4, CoCl2, MeOH, 0℃; c. Boc-Hyp-OH, DIPEA,
HATU, THF; d. TFA, CH2Cl2; e. Boc-L-tert-leucine, DIPEA, HATU, THF; f. TFA, CH2Cl2; g. EtOH, NaHCO3,
NH2NH2·H2O; h. BTC, Et3N, DCM; i. Pd(PPh3)4, Na2CO3, H2O, dioxane; j. PyBop, TEA, DMF; k. HATU, DIPEA, DMF.
3

OH
OH
OH
N
Boc
HN
N
OH
Boc
N
S
N
S
S
H
O
d
e
N
N
H
O
f
O
H
O
H
N
N
N
N
H2N
3
4
5
O
S
N
O
H
N
c
h
N
O
O
N
S
S
HO
O
OH
b
n
H
OH
H2N
O
N
N
N
g
O
n
NH
H
N
H2N
O
H2N
N
NH
CN
1
F
NH
F
2
7
OH
a
N
Br
H
HN
O
H2N
O
N
S
S
N
N
O
N
O
H
n
NH
N
N
O
CN
F
PROTAC-4 n=3
PROTAC-5 n=8
PROTAC-6 n=12
Scheme 2. Syntheitc route of the title PROTACs PROTAC-4~PROTAC-6.
Reagents and conditions: a. Pd(OAc)2, KOAc, DMAc, 150℃; b.NaBH4, CoCl2, MeOH, 0℃; c.Boc-Hyp-OH, DIPEA,
HATU, THF, rt; d.TFA, CH2Cl2, rt; e. Boc-L-tert-leucine, DIPEA, HATU, THF, rt; f. TFA, CH2Cl2, rt; g. PyBop, TEA,
DMF,
rt;
h.
HATU,
DIPE
A, DMF, rt.
action mechanism of these novel PROTAVs [18].
All the title compounds are characterized by HPLC, LC-
MS, 1H-NMR, 13C-NMR, and melting point analysis
(Supplementary Material).
2.2.1. Angiogenic receptor tyrosine kinases inhibition
assay
The inhibitory activities of PROTACs on VEGFR-2 were
evaluated using ADP-Glo Kinase Assay Kit (Promega,
Wisconsin, USA) [19]. The kinase inhibition evaluation
results were depicted in Table 1. Kinase assays indicated
that most of PROTACs exhibited considerable enzymetic
inhibitory potency. In special, PROTAC-2, PROTAC-5,
PROTAC-6 displayed potent inhibitory activity against
VEGFR-2. The results indicated that the new PROTACs
could still exhibit VEGFR-2 inhibition, especially for
PROTAC-6. However, three of them exhibited the largest
magnitude of reduction in enzymatic inhibition.
2.2 Biology assays
In order to demonstrate that these PROTACs retained the
biological activities of anti-angiogenesis agents, we firstly
tested the enzymatic inhibitory activity of PROTACs
against VEGFR-2. Subsequently, their inhibition against
viability of HUVECs (EA.hy926) was evaluated to prove
the anti-angiogenic potency. In parallel, ABT-869 was
used as positive control. Moreover, molecular docking,
western blot assay, cell cycle assay, and apoptosis
detection assay were also performed to investigate the
Table 1. Structures and kinase inhibitory activities of title PROTACs (n=3).
Compounds
n
IC50 (nM)
>1 ꢀM
Compounds
PROTAC-4
n
IC50 (nM)
PROTAC-1
PROTAC-2
PROTAC-3
S7
3
3
>1 ꢀM
8
208.151±49.62
>1 ꢀM
PROTAC-5
PROTAC-6
ABT-869
8
208.657±15.421
80.418±1.678
8.404±0.759
12
12
6.86±0.916
2.2.2. Effects of PROTACs on EA.hy926 and toxicity assay
With the aim to determining the anti-angiogenic effect of
4

these PROTACs, we evaluate the anti-proliferative activity
against HUVECs (EA.hy926, VEGFR-2 positive) [20].
Highly consistent with RTK inhibitory activity, three of
PROTACs displayed dose-dependent inhibition against
EA.hy926 viability with IC50 values ranging from 32 ꢀM
to 133 ꢀM (Table 2). Both PROTAC-2 and PROTAC-5
concentration-dependently inhibited proliferation of
HUVECs with similar potencies (IC50=32.793±4.971 ꢀM
and 38.650±3.876 ꢀM, respectively). On the other hand,
PROTAC-1 and PROTAC-3 were much less active at anti-
proliferative potency, which was consistent to their weaker
activities in the VEGFR-2 inhibition. The results
demonstrated that these PROTACs might display anti-
angiogenic potency through decreasing viability of
HUVECs (EA.hy926).
To identify the binding mode of PROTACs with E3
ubiquitin ligases and VEGFR-2, molecular docking studies
were performed using Sybyl-X (Version 2.0, Tripos Inc.
St. Louis, MO) [21]. PROTAC-2 and PROTAC-5 were
constructed and optimized using Powell’s method with a
Tripos force field. Molecular modeling was performed
using Surflex-dock module. E3 ubiquitin ligases (PDB ID:
1LM8) and VEGFR-2 (PDB ID: 4ASD) were identified as
the active site. The binding mode of PTOTAC-2 and
PROTAC-5 with E3 ubiquitin ligases and VEGFR-2 were
described respectively in Figure 5. As expected, VHL
ligand and small molecule inhibitors in PROTACs bound
tightly with E3 ubiquitin ligases and VEGFR-2,
respectively. Therefore, the length and flexibility of linker
are suitable for the binding of inhibitors with
corresponding protein.
Table 2. Anti-proliferative activity of title PROTACs
against EA.hy926 cells (n=3).
Compounds
PROTAC-1
PROTAC-2
PROTAC-3
S7
IC50 (ꢀM)
>100
Compounds
IC50 (ꢀM)
PROTAC-4 133.248±15.463
32.793±4.971
>100
PROTAC-5
PROTAC-6
ABT-869
38.650±3.876
ND*
51.554±1.511
2.480±0.480
*ND = Not Determined.
Given the potent activity of these PROTACs in decreasing
viability of HUVECs (EA.hy926) bearing VEGFR-2, we
also assessed the anti-proliferative activity against HEK
293 cells (VEGFR-2 negative) to ensure against general
toxicity. According to the results shown in Figure 4, these
PROTACs displayed negligible toxicity toward HEK 293
cells. The results suggested that all PROTACs exhibited no
significant cytotoxicity against VEGFR-2 negative cells,
which indicated that their potency was VEGFR-2
dependent.
Fig. 5. The binding mode of PROTACs with E3 ubiquitin
ligases (A and B) and VEGFR-2 (C and D).
2.2.4. Western blot assay
In order to demonstrate the protein degradation activity of
PROTACs, we investigated the abundance of VEGFR-2 in
EA.hy926 cells [22]. Based on the results of kinase assay
and anti-proliferation assay, potent PROTACs were
selected for protein degradation assay. We performed dose-
response and time-course investigation with PROTACs
and evaluated their degradation kinetics in EA.hy926 cells
in a time course. As shown in Figure 6, these PROTACs
could reduce protein levels of VEGFR-2. PROTAC-2 was
capable of reducing protein levels by 70% at 80 ꢀmol/L,
efficient degradation of VEGFR-2 was observed at 72 h
after treatment; PROTAC-5 was capable of reducing
Fig. 4. Cell proliferation assays and toxicity of PROTACs
against HEK 293 cells (n=3).
2.2.3. Molecular docking study
5

protein levels by 60% at 40 ꢀmol/L, the efficient
degradation of VEGFR-5 was observed as early as 24 h
after treatment and was nearly complete afer 48 h.
PROTAC-5 exhibited concentration-dependent VEGFR-2
degradation potency.
Fig. 6. Dose- and Time-dependent degradation of VEGFR-
2 for PROTAC-2 and PROTAC-5 (**P<0.01, ***P<0.001).
2.2.5. Cell cycle assay
To further explore the effects of PROTAC-5 on the cell
cycle of HUVECs, cell-cycle profiles of EA.hy926 were
assessed with flow cytometry analysis [23]. As shown in
Figure 7, the results indicated that PROTAC-5 could
obviously prolong the S phase at 20 ꢀM. PROTAC-5
treatment could induce a significant increase of cells in the
S-phase and corresponding decreased proportion of cells in
G1-phase. Therefore, the anti-proliferative activities of
PROTAC-5 might due to cell cycle arrest.
Fig. 7. Effects of PROTAC-5 on HUVECs (EA.hy926) cell cycle progress.
2.2.4. Apoptosis detection assay
percentage of late apoptotic cells was 6.94%, 8.32%,
To further investigate the effect of PROTAC-5 on the
apoptosis of HUVECs (EA.hy926), we performed flow
cytometry analysis and apoptosis detection assays [24]. A
substantial and dose-dependent proapoptotic activity in
HUVECs for 72 h was observed. PROTAC-5 exhibited
pro-apoptosis potency on HUVECs. The late apoptotic
HUVECs (5.34%) of negative control group were depicted
in Figure 8. After treatment with PROTAC-5 for 72 h, the
15.6%, 48.7%, and 64.2%, respectively. The results
suggested that PROTAC-5 could induce late apoptosis of
HUVECs in a dose dependent manner with concentration
of 10 ꢀM-40 ꢀM.
6

distilled prior to use. Melting points were measured on an
X-4 micromelting apparatus. 1H NMR and 13C NMR
spectra were measured on a Bruker Advance 400 at 400
and 100 HZ, respectively. Chemical shifts (δ) in DMSO-d6
were referenced to tetra-methyl silane (TMS 0.0 ppm). The
solvent peak and H2O peak were set to 2.50 ppm and 3.33
ppm. Mass spectra were partly measured using liquid
chromatography mass spectrometry (LC-MS) with
electrospray ionization (ESI), while others were measured
using gas chromatography mass spectrometry (GC-MS)
with electron impact ionization (EI). All reactions progress
was monitored by TLC (GF254), and visualized with UV
(254 nm) except Boc-protection of amino acids using
ninhydrin reaction. All compounds were purified by
chromatography with silica gel (300-400 mesh).
Fig. 8. Effects of PROTAC-5 on HUVECs (EA.hy926) cell
apoptosis by flow cytometry measurement.
3. Conclusion
PROTACs is a powerful approach for inducing protein
degradation and is an emerging modality in small molecule
drug discovery. In this investigation, we have applied the
PROTACs concept to explore the potential of anti-
angiogenesis agents in vascular normalization. Six novel
PROTACs were developed based on previously reported
anti-angiogenesis agents. Two of them, PROTAC-2 and
PROTAC-5, displayed potent inhibitory activity against
VEGFR-2 and considerable anti-proliferative activities
against human umbilical vein endothelial cells (EA.hy926).
In addition, they were capable of specifically reducing
protein levels of VEGFR-2. Meanwhile, all the PROTACs
exhibited no significant cytotoxicity against HEK293 cells.
PTOTAC-5 could induce the increase of cells in the S-
phase. It exhibited pro-apoptosis effects on VEGFR-2
positive HUVECs with negligible cytotoxicity against
VEGFR-2 negative HEK 293 cells. Taken together, these
results are in keeping with our previous findings and
provide strong evidences to support the potential
therapeutic usage of PROTAC-2 and PROTAC-5 in
vascular normalization.
4.1.1 General procedure for the synthesis of title
compounds PROTAC-1~PROTAC-6.
4.1.2 4-(4-methylthiazol-5-yl)benzonitrile (1)
To a solution of 4-bromobenzonitrile (3.0 g, 16.48 mmol, 1
eq) and Pd(OAc)2 (4 mg, 0.16mmol, 0.1mol%) in DMAc
(16mL) were added KOAc (3.24g, 33 mmol, 2 eq) and 4-
methylthiazole (3.26 g, 2.98 mL, 33 mmol, 2 eq). The
resulting mixture was heated to 150 ℃ and stirred 10
hours. The mixture was diluted with water and extracted
with DCM (3×). The combined organic phases were
washed with saturated sodium chloride solution and dried
overnight with NaSO4, and evaporated under reduced
pressure to give the corresponding cyano derivate as oil
substance, and the white product (1) was obtained by
column chromatography (2.84 g, 86.16%). LC-MS (EI,
m/z): 200.04.
4.1.3 (4-(4-methylthiazol-5-yl)phenyl)methanamine (2)
The compound (1) (0.54 g, 2.7mmol, 1eq) in methanol
(15mL) was cooled to 0 ℃. CoCl2 (0.52 g, 4.05 mmol, 1.5
eq) was added, followed by portionwise addition of NaBH4
(0.51g, 13.5 mmol, 5 eq). The resulting mixture was stirred
for 90 min, the reaction was quenched with water and
ammonium hydroxide, and the mixture was extracted with
DCM (6×). The combined organic phases were dried over
NaSO4 and evaporated under reduced pressure to give a
4. Experimental section
4.1. Chemistry: General procedure
All commercial chemicals and solvents were purchased
and used without further purification unless otherwise
indicated. All anhydrous solvents were dried according to
the standard methods and partly of them were freshly
7

dark-brown oil which was purified by column
chromatography to yield the product (2) as a yellow oil
(0.18 g, 32.63%). LC-MS (EI, m/z): 204.07.
extracted with DCM (3×). The combined organic phases
were washed with brine (2×), dried over NaSO4, and
evaporated under reduced pressure to give the
corresponding crude, which was purified by column
chromatography purification to yield the key intermediate
(5) (0.82 g, 75.45%). LC-MS (ESI) [M+H]+: 531.30.
4.1.7 (2S,4R)-1-((S)-2-Acetamido-3,3-dimethyl butanoyl)-
4-hydroxy-N-(4-(4-methylthiazol-5-yl) benzyl)pyrrolidine-
2-carboxamide (VHL ligand)
4.1.4 (2S,4R)-tert-Butyl-4-hydroxy-2-((4-(4-methylthiazol-
5-yl)benzyl)carbamoyl)pyrrolidine-1-carboxylate (3)
Boc-L-Hyp-OH (0.68 g, 2.94 mmol, 1 eq), PyBOP (1.68 g,
3.23 mmol, 1.1 eq) was added in DMF (10 mL), and the
solution was stirred at 0 ℃. TEA (0.814mL, 5.87 mmol, 2
eq) was added dropwise, and the mixture was stirred for 5
min at 0 ℃, then the compound (2) (0.6 g, 2.94 mmol, 1
eq) was added, and the mixture was stirred overnight at
room temperature. Water was added, and the mixture was
extracted with ethyl acetate (3×). The combined organic
phases were washed with brine (2×), dried over NaSO4,
and evaporated under reduced pressure to give the
corresponding crude, which was purified by column
chromatography purification to yield the key intermediate
(3) (1.22 g, 99.49%). LC-MS (EI, m/z): 417.17.
The compound (5) (0.82 g, 1.55 mmol)) was dissolved and
stirred in DCM (30 mL) at 0 ℃, and triethylamine (3 mL)
was added to the solution. After stirring the mixture for 2
hour at room temperature, the reaction was completed, and
the pH was adjusted to 7 with saturated NaHCO3 solution.
Then the mixture was extracted with DCM (3×), dried over
NaSO4, and the solvents were evaporated under reduced
pressure to give the corresponding crude which was
purified by column chromatography purification to yield
the VHL ligand (0.53 g, 79.73%). LC-MS (ESI) [M+ H]+:
4.1.5 (2S,4R)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)
pyrrolidine-2-carboxamide (4)
1
431.21. H NMR (400MHz, DMSO-d6) δ 9.00 (s, 1H),
8.74-8.77 (t, 1H), 7.40 (s, 4H), 5.31 (s, 1H), 4.52-4.57 (t,
1H), 4.39-4.47 (m, 2H), 4.21-4.27 (q, 1H), 4.00-4.06 (q,
1H), 3.90 (s, 1H), 2.45 (s, 1H), 2.09-2.14 (m, 1H), 1.16-
1.19 (t, 2H), 1.02 (s, 9H).
The compound (3) (0.86 g, 2.06 mmol)) was dissolved and
stirred in DCM (30 mL) at 0 ℃, and trifluoroacetic acid (3
mL) was added to the solution. After stirring the mixture
for 1 hour at room temperature, the reaction was
completed, and the pH was adjusted to 7 with saturated
NaHCO3 solution. Then the mixture was extracted with
DCM (3×), dried over NaSO4, and the solvents were
evaporated under reduced pressure to give the
corresponding crude compound (4) (0.65 g). LC-MS (EI,
m/z): .17.12.
4.1.8
5-(3-amino-4-(3-(3-(3-
-1H-indazol-1-yl)-5-
isopropylphenyl)ureido)phenyl)
oxopentanoic acid (6)
Compound (S7) and ABT-869 were prepared according to
the synthetic scheme previously reported. Subsequently,
heptanedioic acid (0.12 g, 0.778 mmol, 2eq), PyBOP (0.33
g, 0.648 mmol, 2 eq) was added in DCM (30 mL), and the
solution was stirred at 0 ℃ . TEA (4 eq) was added
4.1.6
tert-Butyl((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-
methylthiazole-5-yl)-benzyl)carbamoyl)pyrrolidin-1-yl)-
3,3-dimethyl-1-oxobutan-2-yl)-carbamate (5)
dropwise, and the mixture was stirred for 5 min at 0 ℃,
then S7 (0.1 g, 0.259 mmol) or ABT-869 (1 eq) was added,
and the mixture was stirred overnight at room temperature.
The volatile organic solvent is spun out under vacuum
conditions. Then water was added, and the mixture was
extracted with ethyl acetate (3×). The combined organic
phases were washed with brine (2×), dried over NaSO4,
and evaporated under reduced pressure to give the
Boc-L-tert-leucine (0.47 g, 2.04 mmol, 1 eq), HATU (0.85
g, 2.24 mmol 1.1 eq) was added in DCM (30 mL), and the
solution was stirred at 0 ℃. DIPEA (1.425mL, 8.16 mmol,
4 eq) was added dropwise, and the mixture was stirred for
10 min at 0 ℃, then the compound 10 (0.65 g, 2.04 mmol,
1 eq) was added, and the mixture was stirred overnight at
room temperature. Water was added, and the mixture was
8

corresponding crude, which was purified by column
chromatography purification to yield the key intermediate
(6) (0.11 g, 80.36%). LC-MS (ESI) [M+ H]+: 528.30.
4.1.10
(2S,4R)-1-((S)-2-(12-(3-amino-4-(3-(3-(3-
isopropylphenyl)ureido)phenyl)-1H-indazol-1-yl)-12-
oxododecanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-
(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide
(PROTAC-2)
4.1.9
(2S,4R)-1-((S)-2-(7-(3-amino-4-(3-(3-(3-
isopropylphenyl)ureido)phenyl)-1H-indazol-1-yl)-7-
oxoheptanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-
(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide
(PROTAC-1)
Yield: 48.9%, mp: 107-109 ℃, LC-MS (ESI) [M+H]+:
1
1010.45. H NMR (400MHz, DMSO-d6): δ 8.98 (s, 1H),
8.86 (s, 1H), 8.71 (s, 1H), 8.55-8.58 (t, 1H), 8.34-8.36 (d,
1H), 7.83-7.86 (d, 1H), 7.68 (s, 1H), 7.60-7.64 (t, 1H),
7.36-7.47 (m, 7H), 7.19-7.25 (m, 3H), 7.07- 7.09 (d, 1H),
6.86-6.88 (d, 1H), 5.28 (s, 2H), 5.13-5.13 (d, 1H), 4.54-
4.56 (d, 1H), 4.41-4.47 (q, 2H), 4.36 (s, 1H), 4.20-4.25 (q,
1H), 4.01-4.06 (q, 1H), 2.97-3.00 (t, 2H), 2.81-2.88 (m,
1H), 2.45 (s, 3H), 2.00-2.14 (m, 3H), 1.68-1.72 (m, 2H),
1.19-1.50 (m, 22H), 0.94 (s, 9H). 13C NMR (101 MHz,
DMSO) δ 172.58, 172.43, 172.27, 170.18, 153.13, 152.05,
151.91, 149.52, 148.18, 140.45, 140.31, 139.97, 139.95,
138.78, 136.80, 131.63, 130.21, 130.10, 129.80, 129.14,
129.10, 127.88, 125.25, 122.68, 120.51, 118.99, 118.56,
116.77, 116.55, 116.42, 114.72, 69.33, 59.15, 56.81, 56.74,
42.11, 38.41, 35.67, 35.33, 34.87, 33.96, 29.42, 29.38,
29.33, 29.22, 29.20, 29.12, 26.83, 25.91, 24.70, 24.34,
16.41.
The compound (6) (0.15 g, 0.284 mmol), HATU (0.16 g,
0.426 mmol) was added in DCM (30 mL), and the solution
was stirred at 0 ℃. DIPEA (193 ꢀL, 1.137 mmol) was
added dropwise, and the mixture was stirred for 10 min at
0 ℃, then VHL ligand (0.13 g, 0.284 mmol) was added,
and the mixture was stirred overnight at room temperature.
The volatile organic solvent is spun out under vacuum
conditions. Then water was added, and the mixture was
extracted with DCM (3×). The combined organic phases
were washed with brine (2×), dried over NaSO4, and
evaporated under reduced pressure to give the
corresponding crude, which was purified by column
chromatography purification to yield the final PROTAC-1
(0.12 g, 44.9%). mp: 115-117 ℃, LC-MS (ESI) [M+H]+:
1
940.55. H NMR (400MHz, DMSO-d6) δ 8.98 (s, 1H),
4.1.11
(2S,4R)-1-((S)-2-(16-(3-amino-4-(3-(3-(3-
8.86 (s, 1H), 8.71 (s, 1H), 8.55-8.58 (t, 1H), 8.34-8.36 (d,
1H), 7.87-7.89 (d, 1H), 7.68 (s, 1H), 7.60-7.64 (t, 1H),
7.35-7.47 (m, 6H), 7.17-7.26 (m, 3H), 7.07-7.09(d,1H),
6.85-6.88 (d, 2H), 5.29 (s, 2H), 5.13-5.14 (d, 1H), 4.53-
4.59 (t, 1H),4.41-4.45 (t, 2H), 4.35 (s, 1H), 4.19-4.25 (q,
1H), 4.01-4.06 (q, 1H), 2.96-3.00 (t, 2H), 2.79-2.88 (m,
1H), 2.45 (s, 3H), 2.12-2.19 (t, 3H), 1.36 (s, 8H), 1.18-1.20
(d, 6H), 0.93 (s, 9H). 13C NMR (101 MHz, DMSO) δ
172.54, 172.43, 172.22, 170.19, 153.13, 152.08, 151.86,
149.52, 148.19, 140.46, 140.32, 139.97, 139.49, 138.78,
136.81, 132.99, 131.64, 130.38, 130.21, 130.11, 129.80,
129.11, 128.82, 127.89, 126.53, 125.38, 125.25, 122.68,
120.51, 119.00, 118.57, 116.78, 116.58, 116.43, 114.73,
69.34, 59.16, 56.81, 42.12, 38.41, 35.66, 34.85, 34.78,
33.96, 30.89, 28.83, 26.85, 25.72, 24.43, 24.34, 16.41.
The title compounds PROTAC-2~PROTAC-6 were
prepared by using the general procedure described above.
isopropylphenyl)ureido)phenyl)-1H-indazol-1-yl)-16-
oxohexadecanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-
(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide
(PROTAC-3)
Yield: 54.1%, mp: 100-102 ℃, LC-MS (ESI) [M+H]+:
1
1066.60. H NMR (400MHz, DMSO-d6): δ 8.98 (s, 1H),
8.86 (s, 1H), 8.70 (s, 1H), 8.55-8.58 (t, 1H), 8.34-8.36 (d,
1H), 7.83-7.86 (d, 1H), 7.68 (s, 1H), 7.60-7.64 (t, 1H),
7.35-7.47 (m, 7H), 7.19-7.26 (m, 3H), 7.08-7.09 (d, 1H),
6.85-6.87 (d, 1H), 5.27 (s, 2H), 5.13-5.14 (d, 1H), 4.53-
4.56 (d, 1H), 4.41- 4.47 (q, 2H), 4.35 (s, 1H), 4.18-4.24 (m,
1H), 4.01-4.06 (q, 1H), 2.96-3.00 (t, 2H), 2.79-2.87 (m,
1H), 2.45 (s, 3H), 1.99-2.13 (m, 3H), 1.68-1.71 (m, 2H),
1.23-1.49 (m, 24H), 1.18-1.20 (d, 6H), 0.94 (s, 9H). 13C
NMR (101 MHz, DMSO) δ 172.59, 172.42, 172.28,
170.19, 153.12, 152.05, 151.91, 149.52, 148.18, 140.45,
9

140.32, 139.97, 139.95, 138.78, 136.80, 131.63, 130.20,
129.80, 129.14, 129.10, 127.89, 125.24, 122.68, 120.52,
119.00, 118.58, 116.78, 116.56, 116.43, 114.72, 69.33,
59.15, 56.80, 56.74, 42.13, 38.41, 35.68, 35.33, 34.85,
33.96, 29.48, 29.33, 29.21, 29.16, 29.12, 26.84, 25.90,
24.70, 24.33, 21.84, 16.40, 12.25.
3H), 2.00-2.12 (m, 3H), 1.91-1.92 (m, 1H), 1.66-1.71 (m,
2H), 1.43-1.55 (m, 2H), 1.17-1.36 (m, 14H), 0.93 (s, 9H).
13C NMR (151 MHz, DMSO) δ 172.57, 172.42, 172.24,
170.19, 152.62, 152.16, 151.91, 151.57, 149.99, 148.19,
140.34, 140.16, 139.98, 136.62, 134.02, 131.63, 131.57,
130.20, 130.11, 129.98, 129.11, 127.89, 127.47, 127.40,
125.43, 121.44, 118.53, 116.65, 115.09, 114.96, 114.38,
69.33, 59.16, 56.81, 56.74, 42.12, 38.42, 35.67, 35.33,
34.86, 29.42, 29.37, 29.33, 29.21, 29.20, 29.12, 26.84,
25.91, 24.72, 21.25, 16.41.
4.1.12 (2S,4R)-1-((S)-2-(7-(3-amino-4-(4-(3-(2-fluoro-5-
methylphenyl)ureido)phenyl)-1H-indazol-1-yl)-7-
oxoheptanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-
(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide
(PROTAC-4)
4.1.14 (2S,4R)-1-((S)-2-(16-(3-amino-4-(4-(3-(2-fluoro-5-
methylphenyl)ureido)phenyl)-1H-indazol-1-yl)-16-
oxohexadecanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-
(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide
(PROTAC-6)
Yield: 61.2%, mp: 124-126 ℃, LC-MS (ESI) [M+H]+:
1
930.45. H NMR (400MHz, DMSO-d6): δ 9.28 (s, 1H),
8.98 (s, 1H), 8.57 (s, 2H), 8.31- 8.33 (d, 1H), 8.00-8.02 (d,
1H), 7.87-7.89 (d, 1H), 7.58-7.64 (m, 3H), 7.37-7.43 (m,
6H), 7.10-7.19 (m, 2H), 6.81-6.84 (m, 1H), 5.21 (s, 2H),
5.14 (d, 1H), 4.53-4.56 (d, 1H), 4.41- 4.47 (t, 2H), 4.35 (s,
1H), 4.19-4.25 (q, 1H), 3.63-3.70 (t, 2H), 2.45 (s, 3H), 2.29
(s, 3H), 2.12-2.19 (m, 1H), 2.02-2.06 (m, 1H), 1.87-1.94
(m, 1H), 1.67-1.75 (m, 3H), 1.50-1.61 (m, 2H), 1.34-1.39
(m, 2H), 1.24 (m, 2H), 0.91 (s, 9H). 13C NMR (101 MHz,
DMSO) δ 172.57, 172.44, 172.20, 170.19, 152.62, 152.18,
151.90, 149.61, 148.18, 140.34, 140.15, 139.96, 136.61,
134.00, 131.64, 131.57, 130.19, 130.10, 129.97, 129.10,
127.89, 127.47, 127.36, 125.43, 123.33, 123.26, 121.46,
118.55, 116.66, 114.92, 114.38, 69.35, 59.17, 56.81, 42.13,
38.40, 35.66, 35.24, 34.76, 28.83, 26.84, 25.72, 24.44,
21.24, 16.40.
Yield: 55.6%, mp: 105-107 ℃, LC-MS (ESI) [M+H]+:
1
1056.60. H NMR (400MHz, DMSO-d6): δ 9.27 (s, 1H),
8.98 (s, 1H), 8.57 (s, 2H), 8.30-8.32 (d, 1H), 8.00-8.02 (d,
1H), 7.83-7.86 (d, 1H), 7.57-7.64 (m, 3H), 7.37-7.43 (m,
6H), 7.10-7.19 (m, 2H), 6.82 (m, 1H), 5.19 (s, 2H), 5.13-
5.14 (d, 1H), 4.53-4.56 (d, 1H), 4.41-4.47 (t, 2H), 4.35 (s,
1H), 4.19-4.24 (q, 1H), 3.63-3.70 (t, 2H), 2.45 (s, 3H), 2.29
(s, 3H), 1.99-2.12 (m, 2H), 1.87-1.94 (m, 1H), 1.67-1.71
(m, 2H), 1.23-1.49 (m, 25H), 0.94 (s, 9H). 13C NMR (101
MHz, DMSO) δ 172.60, 172.42, 172.24, 170.19, 152.61,
152.15, 151.97, 151.90, 148.18, 140.34, 140.15, 139.97,
136.61, 133.99, 131.63, 131.57, 130.19, 130.10, 129.97,
129.10, 127.89, 127.48, 127.37, 125.42, 123.24, 121.44,
118.53, 116.64, 115.11, 114.92, 114.36, 69.33, 59.16,
56.80, 56.74, 42.11, 38.41, 35.68, 35.33, 34.84, 29.48,
29.33, 29.21, 29.17, 29.12, 26.84, 25.90, 24.71, 21.24,
16.40.
4.1.13 (2S,4R)-1-((S)-2-(12-(3-amino-4-(4-(3-(2-fluoro-5-
methylphenyl)ureido)phenyl)-1H-indazol-1-yl)-12-
oxododecanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-
(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide
(PROTAC-5)
4.2. In vitro VEGFR-2 inhibition assays [24]
Yield: 58.1%, mp: 116-118 ℃, LC-MS (ESI) [M+H]+:
The in vitro enzymatic inhibition assays against VEGFR-2
was carried out by ADP-Glo™ kinase assay (Promega,
Madison, WI) according to the manufacturer’s instructions,
with ABT-869 as positive control. General procedures
were as follows: VEGFR-2 kinases (0.6 ng/ꢀL) was
incubated with substrates (0.2 ꢀg/ꢀL), tested title
compounds (1.2 ×10−4-12 ꢀM) and ATP (50 ꢀM) in a final
1
1000.55. H NMR (400MHz, DMSO-d6): δ 9.28 (s, 1H),
8.99 (s, 1H), 8.58 (s, 2H), 8.31-8.33 (d, 1H), 8.01-8.03 (d,
1H), 7.85-7.87 (d, 1H), 7.58-7.64 (m, 3H), 7.40-7.43 (m,
6H), 7.10-7.19 (m, 2H), 6.83 (m, 1H), 5.21 (s, 2H), 5.14 (s,
1H), 4.54-4.56 (d,1H), 4.43-4.45 (t, 2H), 4.36 (s, 1H),
4.21-4.25 (q, 1H), 3.63-3.70 (t, 2H), 2.45 (s, 3H), 2.29 (s,
10

buffer of Tris 40 mM, MgCl2 10 mM, BSA 0.1 mg/mL,
and DTT 1 mM in a 384-well plate with a total volume of
5 ꢀL. The assay plate was incubated at 30°C for 1 h. Once
the kinase reaction was complete, 5 ꢀL of ADP-Glo
reagent was added into each well to terminate the reaction
and deplete the remaining ATP. The mixture was
incubated at room temperature for another 40 min. At the
end, 10 ꢀL of kinase detection reagent was added into each
well and incubated for 30 min, to convert ADP to ATP and
allow the newly synthesized ATP to be measured using
luciferase/luciferin reaction. The luminescence generated
was read by a VICTORX multiple plate reader. The signal
was correlated with the amount of ATP present in the
reaction and was inversely correlated with the kinase
activity.
blot analyses, the following antibodies were used: GAPDH
(1:10000, Proteintech), VEGFR-2 (1:500, CST).
Quantification was performed using the software
distribution ImageJ. EA.hy926 cells were treated with
vehicle (DMSO) or tested title compounds at desired
concentration for 48 h. The cells were lysed in SDS sample
buffer, collected, and normalized using BCA protein assay
kit before being diluted in SDS loading buffer. Then the
samples containing equal amounts of protein were
separated by SDS-PAGE. After electrophoresis, proteins
were transferred to PVDF membranes and blocked with
5% nonfat milk in Tris-buffered saline with 0.1% Tween-
20. Membranes were incubated with antibodies Bcr-Abl
and p-Bcr-Abl at 4℃ overnight. The membranes were
washed with PBS three times, and incubated with the
appropriate anti-HRP secondary antibodies for 2 h at room
temperature. Finally, immunoreactive proteins were
visualized using the enhanced chemiluminescence system
from Pierce Chemical.
4.3. Anti-proliferative activity against human vascular
endotheial cell (EA.hy926) [25].
The viability of HUVEC (EA.hy926) was assessed using
standard MTT method with ABT-869 as positive control.
Briefly, EA.hy926 cells were harvested and plated in a 96-
well plate at the density of 1×105 cells for each well, and
cultured in DMEM medium with 10% FBS in humidified
5% CO2. For MTT assay, the operation detail was as
follows: The plate was incubated in 5% CO2 at 37℃ for
48 h. The test compounds at serial diluted concentrations
were added into the cells solution and incubated for 24 h.
Subsequently, MTT was added into each well at a final
concentration of 0.5 mg/mL and incubated for 2 h at 37℃.
Supernatant was discarded, and 150 ꢀL DMSO was added
to each well. After shaking 15 min, absorbance values at
450 nm were determined by a microplate reader (Bio-Rad,
Hercules, USA). The viability rate was calculated as
experimental OD value/control OD value. The IC50 values
were calculated according to inhibition ratios.
4.5. Molecular docking modeling [27]
In order to investigate the interaction between the title
compounds and VEGFR-2 kinases, molecular docking was
performed using Surflex-Dock Module of Sybyl-X
(version 2.0, Tripos Inc, St.Louis, MO). Crystal structures
of VEGFR-2 (PDB ID: 4ASD) and E3 ubiquitin ligases
(PDB ID: 1LM8) were imported, and protein structure
preparation was carried out as follows: removing waters
and other small molecules, repairing sidechain, and adding
hydrogen atoms, and minimized with Pullman charges
under Tripos Force Field. Then the corresponding ligand in
the prepared protein structure was extracted and used to
define the binding cavity for molecules. The residues in a
radius 5.0 Å around the ligand were selected as active site
and generated as docking promotal. The title molecules
were drawn with Sketch and minimized by Powell's
method for 1000 iterations under Tripos Force Field with
Gasteiger-Huckel charges. In the end, the optimized
molecules were docking into the active sites of protein
promotal using ligand-based mode with other docking
parameters as default.
4.4. Western blotting assay [26].
Protein content was determined with a bicinchoninic acid
(BCA) assay and equal protein amounts were separated by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
at a constant voltage. Protein was transferred onto an
Immobilon-P transfer membrane (Millipore). For western
11

4.6. Cell Cycle Assay.
L. Zhang, J. Zhang, Discovery of novel anti-
angiogenesis agents. Part 10: Multi-target inhibitors of
VEGFR-2, Tie-2 and EphB4 incorporated with 1,2,3-
triazol, Eur J Med Chem. 163 (2019) 1-9.
HUVEC cells (5×105 cells/mL) were seeded in six-well
plates and treated with compounds at different
concentrations for 24 h. The cells were then harvested by
trypsinization and washed twice with cold PBS. After
centrifugation and removal of the supernatants, cells were
resuspended in 400 ꢀL of 1×PBS buffer. After adding 10
ꢀL of PI the cells were incubated at room temperature for
15 min in the dark. The stained cells were analyzed by a
flow cytometer (BD Accuri C6).
4. Y. Shan, R. Si, J. Wang, Q. Zhang, H. Zhou, J. Song,
J. Zhang, Q. Chen, Discovery of novel anti-
angiogenesis agents. Part 9: Multiplex inhibitors
suppressing compensatory activations of RTKs, Eur J
Med Chem. 164 (2019) 440-447.
5. Y. Sun, Y. Shan, C. Li, R. Si, X. Pan, B. Wang, J.
Zhang, Discovery of novel anti-angiogenesis agents.
Part 8: Diaryl thiourea bearing 1H-indazole-3-amine
as multi-target RTKs inhibitors, Eur J Med Chem. 141
(2017) 373-385.
4.7. Apoptosis Detection Assay.
HUVEC cells (5×105 cells/mL) were seeded in six-well
plates and treated with compounds at different
concentrations for 48 h. The cells were then harvested by
trypsinization and washed twice with cold PBS. After
centrifugation and removal of the supernatants, cells were
resuspended in 400 ꢀL of 1×binding buffer which was then
added to 5 ꢀL of annexin V-FITC and incubated at room
temperature for 15 min. After adding 10ꢀL of PI the cells
were incubated at room temperature for another 15 min in
the dark. The stained cells were analyzed by a flow
cytometer (BD Accuri C6).
6. C. Li, Y. Shan, Y. Sun, R. Si, L. Liang, X. Pan, B.
Wang, J. Zhang, Discovery of novel anti-angiogenesis
agents. Part 7: Multitarget inhibitors of VEGFR-2,
TIE-2 and EphB4, Eur J Med Chem. 141 (2017) 506-
518.
7. L. Zhang, Y. Shan, C. Li, Y. Sun, P. Su, J. Wang, L. Li,
X. Pan, J. Zhang, Discovery of novel anti-
angiogenesis agents. Part 6: Multi-targeted RTK
inhibitors, Eur J Med Chem. 127 (2017) 275-285.
8. F.W. Peng, D.K. Liu, Q.W. Zhang, Y.G. Xu, L. Shi,
VEGFR-2 inhibitors and the therapeutic applications
thereof: a patent review (2012-2016), Expert Opin
Ther Pat. 27 (2017) 987-1004.
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (NSFC, No. 81573285), the Natural
Science Basic Research Plan in Shaanxi Province (No.
2020SF-240 and 2018JM7071), and the Fundamental
9. M. Pettersson, C.M. Crews, PROteolysis TArgeting
Chimeras (PROTACs)-Past, present and future. Drug
Discov Today Technol. 31 (2019) 15-27.
Research
Funds
for
the
Central Universities
(xzy032020003).
10. M.G. Jaeger, G.E. Winter, Expanding the Degradable
Proteome: Designing PROTACs by the Book, Cell
Chem Biol. 27 (2020) 14-16.
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13

Highlights
ꢀ Six PROTACs have been developed based on anti-angiogenesis agents.
ꢀ PROTACs exhibited potent VEGFR-2 inhibition and anti-proliferative activity.
ꢀ PROTACs exhibited no significant cytotoxicity against HEK293 cells.

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: