Discovery of novel anti-angiogenesis agents. Part 7: Multitarget inhibitors of VEGFR-2, TIE-2 and EphB4

Article abstract of DOI:10.1016/j.ejmech.2017.10.030

Herein, we embarked on a structural optimization campaign aiming at the discovery of second generation anti-angiogenesis agents with our previously reported BPS-7 as lead compound. A library of 27 compounds has been afforded based on the highly conserved ATP-binding pocket of VEGFR-2, Tie-2, and EphB4. Several title compounds exhibited simultaneous inhibitory effects against three angiogenic RTKs. These compounds with a ‘triplet’ inhibition profile have been identified as novel anti-angiogenic and anticancer agents. The representative VDAU11 displayed prominent anti-angiogenic and anticancer potency and could be considered as a candidate for further optimization. These results indicate that N-(pyridin-2-yl)acrylamide could serve as a novel hinge-binding group of triple inhibitors.

Full text of DOI:10.1016/j.ejmech.2017.10.030

Accepted Manuscript
Discovery of novel anti-angiogenesis agents. Part 7: Multitarget inhibitors of
VEGFR-2, TIE-2 and EphB4
Chuansheng Li, Yuanyuan Shan, Ying Sun, Ru Si, Liyuan Liang, Xiaoyan Pan,
Binghe Wang, Jie Zhang
PII:
S0223-5234(17)30822-X
10.1016/j.ejmech.2017.10.030
EJMECH 9822
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 June 2017
Revised Date: 3 August 2017
Accepted Date: 10 October 2017
Please cite this article as: 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,
European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.10.030.
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ACCEPTED MANUSCRIPT
Discovery of novel anti-angiogenesis agents. Part 7: Multitarget inhibitors of
VEGFR-2, TIE-2 and EphB4
Chuansheng Lia,†, Yuanyuan Shanb,†, Ying Suna, Ru Sia, Liyuan Lianga, Xiaoyan Pana, Binghe
Wangc, Jie Zhanga,
aSchool of Pharmacy, Health Science Center, Xi'an Jiaotong University, No. 76, Yanta West Road,
Xi’an, 710061, China
bDepartment of Pharmacy, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an,
710061, China
cDepartment of Chemistry and Center for Diagnostics and Therapeutics, Georgia State University,
Atlanta, Georgia 30303, United States
O
H
N
O
Hydrophobic pocket
Gatekeeper
DFG-Motif (Asp)
COO-
O
Ar
Ar
N
N
HN
HN
H
H
O
HN
O
N
H
O
O
O
O
N
R
N
H
O
N
H2N
N
N
O
N
O
H
N
N
H
N
H
H
O
H2N
N
N
O
H
O
O
X
O
O
O
Conserved Glu
R
HN
H2N
N
N
Selective site
H
N
Hinge region
Structural optimization of lead compound BPS-7
As a continuation to our previous research, several triple RTK inhibitors of VEGFR-2,
Tie-2 and EphB4 were designed and synthesized as novel anti-angiogenesis agents.
Corresponding author. Tel/Fax: +86-29-82655451; E-mail: zhj8623@xjtu.edu.cn (Jie Zhang).
† Both authors contributed equally to this work and should be regarded as joint first authors.

ACCEPTED MANUSCRIPT
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Discovery of novel anti-angiogenesis agents. Part 7: Multitarget inhibitors of VEGFR-2, TIE-2 and
EphB4
Chuansheng Lia,†, Yuanyuan Shanb,†, Ying Suna, Ru Sia, Liyuan Lianga, Xiaoyan Pana, Binghe Wangc, Jie Zhanga,
aSchool of Pharmacy, Health Science Center, Xi'an Jiaotong University, No. 76, Yanta West Road, Xi’an, 710061, China
bDepartment of Pharmacy, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, 710061, China
cDepartment of Chemistry and Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, Georgia
30303, United States
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Herein, we embarked on a structural optimization campaign aiming at the
discovery of second generation anti-angiogenesis agents with our previously
reported BPS-7 as lead compound.
A library of 27 compounds has been
afforded based on the highly conserved ATP-binding pocket of VEGFR-2,
Tie-2, and EphB4. Several title compounds exhibited simultaneous inhibitory
effects against three angiogenic RTKs. These compounds with a ‘triplet’
inhibition profile have been identified as novel anti-angiogenic and anticancer
agents. The representative VDAU11 displayed prominent anti-angiogenic and
anticancer potency and could be considered as a candidate for further
optimization. These results indicate that N-(pyridin-2-yl)acrylamide could
Available online
Keywords:
Anti-angiogenesis Agents
Triple Inhibitors
Angiogenic RTKs
Hinge-binding group
N-(pyridin-2-yl)acrylamide
serve as a novel hinge-binding group of triple inhibitors.
2009 Elsevier Ltd. All rights reserved
.
———
Corresponding author. Tel/Fax: +86-29-82655451; E-mail: zhj8623@xjtu.edu.cn (Jie Zhang).
† Both authors contributed equally to this work and should be regarded as joint first authors.
1

(Stivarga, Bayer), Linifanib (ABT-869, Abbott) [9],
ACCEPTED MANUSCRIPT
1. Introduction
Angiogenesis plays a significant role in the pathogenesis of
a variety of disorders including cancer, proliferative
retinopathies, rheumatoid arthritis or psoriasis [1]. In
addition, angiogenesis is widely identified as a crucial
factor in metastasis, which is a major contributor to cancer-
related death. Therefore, inhibition of angiogenesis has
been considered as an attractive strategy for the treatment
of cancers. Many anti-angiogenesis agents have been under
clinical trial, or approved for clinical use. On the opposite,
many limitations have been disclosed including resistance,
enhancing hypoxia, and reducing delivery of drugs.
Intracellular signals for angiogenesis including cell growth,
proliferation, migration, and survival are frequently
triggered by receptor tyrosine kinases (RTKs). Cancer cells
secrete various RTKs involved in the process of
angiogenesis [2]. Interaction of vascular endothelial
growth factors (VEGFs) and its receptor (VEGF Receptor-
2, VEGFR-2) promotes epithelial cell (EC) survival,
proliferation, and migration [3]. Angiopoietin (Ang) and its
receptor (tyrosine kinase with Ig and epidermal growth
factor homology domain-2, Tie-2) are essential for vessel
maturation, stabilization, and remodeling of vasculature
[4]. Activation of erythropoietin-producing hepatoma
receptor B4 (EphB4) by its membrane-associated ligand,
ephrinB2, is critical for vessel maturation, remodeling, and
stabilization [5].
Vandetanib
(Caprelsa,
AstraZeneca)
[10],
and
Cabozantinib (Cabometyx, Exelixis) [11] (Figure 1).
Encouraged by previous results, we described here the
discovery of a series of multiplex inhibitors of VEGFR-
2/EphB4/TIE-2 as novel anti-angiogenesis agents.
<Insert Figure 1 here>
To the best of our knowledge, all the three RTKs contain a
highly conserved catalytic site binding with ATP. The
catalytic domains and ATP-binding site in the active
conformation of these RTKs are notably similar.
Moreover, it is reasonable to assume that all the ATP-
competitive RTKIs with anti-angiogenesis potency bind to
the ATP binding site of RTKs. Overlay of the crystal
structures of the three RTKs is depicted in Figure 2.
Structural alignment indicated that there was no significant
difference. The RMSD values for them are 0.849 Å, 0.725
Å, and 0.706 Å, respectively. As the structures display a
similar two-lobed architecture and highly similar to each
other, we believe that the similarity of kinase domains
makes the multiplex inhibitors a feasible drug discovery
strategy.
<Insert Figure 2 here>
In our previous research, we have focused on the discovery
of novel VEGFR-2 inhibitors as anti-angiogenesis agents.
Numbers of biphenyl-aryl urea incorporated with
salicylaldoxime including BPS-7 have been developed as
potent and selective VEGFR-2 inhibitors (Figure 3) [12-
17]. Based on conserved active sites of three angiogenic
RTKs (VEGFR-2, Tie-2, and EphB4) and similar
interaction conformation of inhibitors with them, we
propose that the rational design of triple inhibitors of
The possibility of inhibiting angiogenic RTK has led to
development of specific RTK inhibitors as anti-
angiogenesis and anticancer agents. However, acquired
resistance caused by compensatory activation of multiple
RTKs limit the benefit of these drugs [6]. It has been
revealed that simultaneous inhibition and combinatorial
targeting of multiple pro-angiogenic RTKs have been
recognized as valuable strategies to maximize the
therapeutic potential [7]. There are several small molecular
multi-target RTK inhibitors in clinical trials as anti-
angiogenesis agents. Five drugs with different inhibition
profile against angiogenic RTKs have been approved
including Sorafenib (Nexavar, Bayer) [8], Regorafenib
VEGFR-2/Tie-2/EphB4 is
a promising strategy for
discovery of anti-angiogenesis agents. Extensive structural
optimization was conducted to expand structural diversity
and improve activity using following strategies (Figure 3):
(i) We explored various aromatic-heterocyclics as hinge
binding group (HBG) via core-refining approach. Pyridin-
2-amine, N-(pyridin-2-yl)acrylamide, quinoxalin-2-amine,
and quinazolin-4(3H)-one were introduced as novel HBG.
2

aminophenylboronic
These heterocyclics bearing various hydroAgeCn bCoEndPdToEnoDrs MANUSCRIPT
acid
3.
Coupling
of
1-
or acceptors could retain the essential hydrogen bonds with
hinge region of RTKs. We supposed that these groups
might simultaneously form hydrogen bonds with hinge
region of three RTKs and therefore provide an opportunity
to improve affinity with receptors; (ii) The two methoxyl
groups on biphenyl were removed to reduce the steric
hindrance of ligands binding with receptors. In addition,
we investigated the effect of position of urea on their
biological activity; (iii) Inspired by the classic bioisosteric
paradigm, urea moiety was replaced with cyclopropane-
1,1-dicarboxamide which bears more hydrogen bond
donors and acceptors; (iv) Aniline bearing halogen
substituents is beneficial for antitumor potency and
halogen could enhance the persistence and lipid solubility
[18]. Therefore, various anilines bearing halogen were
incorporated to develop novel anti-angiogenesis agents. In
addition, other anilines containing morpholine side chain,
tert-butyl, alkynyl, or benzo[d][1,3]dioxole were also
incorporated at terminal as fragments interacting with
active site.
cyclopropanedicarboxylate 5 with substituted aniline 6 in
the presence of thionyl chloride gave intermediates 7.
Finally, treatment of intermediates 7 with 4 in the presence
of triethylamine and 1-[Bis(dimethylamino)methylene]-
1H-1,2,3-triazolo
[4,5-b]pyridinium
3-oxide
hexafluorophosphate (HATU) afforded cyclopropyl-
malonic amide derivatives CPMA1-11. The synthetic
route for title compounds VDAU1-11 is described in
Scheme 2. Firstly, the key intermediates 9 were prepared
from commercial available 4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)aniline 8. Various substituted anilines
were treated with BTC in anhydrous CH2Cl2 to yield
isocyanate, followed by reacting with 8 to generate diaryl
ureas 9. Pd(PPh3)4catalyzed Suzuki reaction of diaryl ureas
9 with 2-amino-5-bromopyridine 10 in presence of cesium
carbonate in acetonitrile/H2O afforded the key
intermediates 11. Finally, the title compounds VDAU1-11
were prepared through acylation with acryloyl chloride.
The synthetic route of quinazolinone diaryl urea
derivatives QZAU1-5 is shown in Scheme 3. 2-Amino-5-
bromobenzoic acid 12 was converted to intermediate 13 by
cyclization with formamide under atmospheric microwave
heating at 150°C. The coupling substrates 15 were
obtained by condensation of amino-boronate 14 with
various isocyanates, which were prepared from reaction of
anilines with BTC. Subsequently, the title commands
QZAU1-5 were prepared from 13 and 15 by Suzuki
coupling reaction. All the title compounds characterized by
1H-NMR, 13C-NMR, high resolution mass spectrum
(HRMS), and melting point analysis and their purity were
above 95% determined by LC-MS (Supplementary
Material).
<Insert Figure 3 here>
Herein, we performed the design, synthesis, and biological
evaluation of three classes of triple inhibitors of VEGFR-2,
Tie-2, and EphB4. Several biphenyl-aryl ureas
incorporated with quinazolin-4(3H)-one displayed
promising anti-angiogenic and anti-cancer potency. The
representative compound VDAU11 could be considered as
a
promising lead compound for further structural
optimization.
2. Results and discussion
2.1 Chemistry
The synthetic routes of title compounds were illustrated in
Scheme 1-3. The synthetic route for cyclopropyl-malonic
amide derivatives CPMA1-11 was outlined in Scheme 1.
Commercially available 5-bromo-2-fluorobenzaldehyde 1
was converted to 6-bromoaminoquinazoline 2 by treatment
with guanidine carbonate in N,N-dimethylacetamide. The
critical intermediate 2 was subsequently converted to 4
through Pd-catalyzed Suzuki coupling with 4-
<Insert Scheme 1-3 here>
2.2 RTK inhibitory activity
All the title compounds were evaluated for their inhibitory
potency against VEGFR-2, TIE-2 and EphB4 with
sorafenib as a positive control (Table 1-3). Tyrosine kinase
inhibition were tested by luminescent ADP-GloTM assay.
We began our exploration with optimization of urea core
3

and hinge binding group of BPS-7. Firstly, we investigated
RTK inhibition. In addition, two title compounds (QZAU4,
QZAU5) displayed simultaneously inhibitory potency
against VEGFR-2, Tie-2, and EphB4. In particular,
QZAU4 bearing 3,4-difluoroaniline was the most potent
multi-target RTKs inhibitors with IC50 values of 28.95 nM
(VEGFR-2), 16.55 nM (Tie-2), and 38.52 nM (EphB4),
respectively. This study identified that quinazolin-4(3H)-
one was a promising hinge binding group for triple
inhibitors of VEGFR-2, Tie-2, and EphB4 as anti-
angiogenesis and anticancer agents.
ACCEPTED MANUSCRIPT
the replacement of urea unit with cyclopropane-1,1-
dicarboxamide. Meanwhile, quinazolin-2-amine was
incorporated as a novel hinge binding group. The results in
Table 1 showed that the majority of these compounds
exhibited significantly decreased RTK inhibitory activities
compared with lead compound except for CPMA1,
CPMA5 and CPMA7. These results indicated that the urea
moiety was critical and the replacement of urea resulted in
a significant loss in RTK inhibition. In addition,
quinazolin-2-amine was not a favorable hinge binding
group for multi-target RTK inhibitors. Compound CPMA7
bearing 3,4-difluoroaniline exhibited the highest RTKs
inhibitory activities with IC50 values of 21.37 nM
(VEGFR-2), 14.49 nM (Tie-2), and 37.78 nM (EphB4),
respectively. The results suggested that 3,4-difluoro
substituents led to improvement in simultaneous inhibition
of VEGFR-2, Tie-2, and EphB4.
<Insert Table 3 here>
2.3. RTK selectivity assay
In order to determine the RTK selectivity profile of the
most potent compounds, VDAU11, a broad panel kinase
screening was further conducted by evaluating the
inhibition against other ten RTKs including VEGFR-1,
VEGFR-3, EGFR, FGFR-1, FGFR-4, PDGFR-β, IGF1-R,
B-Raf, c-Kit, and c-Met (Figure 4 and Table 4). Besides
VEGFR-2, Tie-2, and EphB4, VDAU11 displayed
moderate inhibitory activity against FGFR-1 and PDGFR-
β. Interestingly, they also exhibited potent inhibition
against VEGFR-1, VEGFR-3, and B-Raf with IC50 values
less than 50 nM. The results confirmed that these triple
inhibitors exhibited good selectivity on VEGFR-2/TIE-
2/EphB4 relative to other RTKs including EGFR, FGFR-1,
FGFR-4, IGF1-R, c-Kit, and c-Met.
<Insert Table 1 here>
Inspired by the above investigation, N-(pyridin-2-
yl)acrylamide was introduced onto diaryl urea scaffold as
novel hinge binding group (Table 2). The activity data
indicated that two title compounds (VDAU2, VDAU11,)
exhibited simultaneous and potent inhibition against
VEGFR-2, Tie-2, and EphB4. In particular, VDAU11
bearing 3-chlorine substituent at terminal aniline was the
most potent multi-target RTK inhibitor with IC50 values of
2.35 nM (VEGFR-2), 5.63 nM (Tie-2), and 3.87 nM
(EphB4), respectively. The results suggested that N-
(pyridin-2-yl)acrylamide could serve as a novel and potent
hinge binding group for triple inhibitors of VEGFR-2, Tie-
2, and EphB4.
<Insert Figure 4 here>
<Insert Table 4 here>
2.4. Cell growth inhibition
In order to determine the potential anti-angiogenic effect of
these triple inhibitors, we evaluate the inhibition of title
compounds on HUVECs (EA.hy926) viability using cell
counting kit-8 (CCK-8) method [19]. As shown in Table 5,
majority of them displayed moderate to high
antiproliferative activities with IC50 values ranging from
3.53 µM to 197.44 µM. Three compounds (CPMA5,
CPMA7, and VDAU11) exhibited potent inhibition against
the growth of human vascular endothelial cell with IC50
values less than 10 µM. Particularly, compound VDAU11
with the most potent RTK inhibitory activities exhibited
<Insert Table 2 here>
Encouraged by the promising multiple RTK inhibition of
diaryl ureas, we chose to continue our structural
optimization keeping urea unit constant. Quinazolin-4(3H)-
one was incorporated onto diaryl urea core as a novel hinge
binding group of multi-target inhibitors (Table 3). The
results indicated that meta-substitution of quinazolin-
4(3H)-one on diaryl ureas were favorable for angiogenic
4

the highest cell growth inhibition with IC50 value of 3.53
ID: 4ASD), Tie-2 (PDB ID: 2P4I) and EphB4 (PDB ID:
2X9F) were selected as the active site. The binding mode
of CDAU-2 with the ATP-pocket of VEGFR-2 (A), TIE-2
(B), EphB4 (C) were depicted in Figure 6-8. Favorable
binding interactions of VDAU11 with the active site of
VEGFR-2 included three hydrogen bonds (Figure 6): N-
(pyridin-2-yl)acrylamide in VDAU11 forms one hydrogen
bond with Cys 919 in the hinge region of VEGFR-2 with
distance of 2.37 Å. In addition, the NH of urea formed one
hydrogen bond with conserved Glu 885 for the bond length
of 1.95 Å, while CO formed a hydrogen bond with Asp
1046 of DFG-motif with distance of 1.76 Å. As for TIE-2,
the binding model was described in Figure 7. NH of
acrylamide also formed a hydrogen bond with Ala 905 in
hinge region of TIE-2 with the bond length of 1.92 Å.
While the CO of acrylamide forms another hydrogen bond
with Tyr 904 in hinge region with distances of 2.34 Å. As
shown in Figure 8, N-(pyridin-2-yl)acrylamide in
VDAU11 forms one hydrogen bond with Thr 693 in the
hinge region of EphB4 with distance of 2.62 Å. N-(pyridin-
2-yl)acrylamide could form one or two hydrogen bonds
with hinge region of three angiogenic RTKs. These
molecular docking results indicated that the N-(pyridin-2-
yl)acrylamide is beneficial for affinity of inhibitors with
VEGFR-2, TIE-2, and EphB4. It could be considered as
novel hinge-binding group for further discovery of triple
RTK inhibitors.
ACCEPTED MANUSCRIPT
µM. This compound represents the promising candidate
with a "triplet" inhibition profile as well as anti-
angiogenesis potency. It might not only inhibit the process
of angiogenesis, but also prevent the occurrence of
resistance.
<Insert Table 5 here>
2.5. Anti-proliferative activity against cancer cells
RTKs and angiogenesis played critical role in the
proliferation of various cancers cells and RTK inhibition
generally translated well into anti-proliferation of cancer
cells. In this regard, in order to investigate the potential
anticancer potency of these multi-target RTK inhibitors,
the most potent VDAU11 was selected to examine its anti-
proliferative activity against fifteen cancer cells including
human hepatic cancer cell lines (97h, BEL-7402, Hep3B,),
human breast cancer cell lines (MCF-7, ZR-75-30), human
pancreatic cancerous cell lines (PANC-1), human lung
cancer cell (A549), human colon carcinoma cell line
(LOVO), and human cervical cancer cell line (HeLa).
Highly consistent with the RTK inhibition, we are pleased
to find that VDAU11 significantly inhibited growth of all
nine cancer cell lines with IC50 values ranging from 2.70
µM to 22.93 µM (Figure 5). It displayed comparable anti-
proliferative potency with sorafenib. The highest activity
for VDAU11 were found in human breast cancer cell lines
(MCF-7) and human lung cancer cell (A549) with IC50
values of 2.70 µM. In summary, VDAU11 displayed
significant anti-proliferative activity against a broad
spectrum of cancer cell lines.
<Insert Figure 6-8 here>
Molecular docking results provided a better understanding
of VDAU11’s interaction with three angiogenic RTKs. Our
docking results might explain why VDAU11 displayed
potent inhibitory activities against all three proteins. In
addition, N-(pyridin-2-yl)acrylamide was beneficial for
simultaneous inhibition of RTKs.
<Insert Figure 5 here>
2.6 Molecular docking study
For further structural optimization and investigation of the
potential binding mode, molecular modeling studies were
performed using Sybyl-X (Version 2.0, Tripos Inc. St.
Louis, MO). The most potent compound, VDAU11, was
constructed and optimized using Powell’s method with a
Tripos force field. The molecular modeling was performed
using Sybyl-X/Surflex-dock module, and the residues in a
5.0 Å radius around the natural ligand of VEGFR-2 (PDB
3. Conclusion
In summary, we described the triple inhibitors of VEGFR-
2/TIE-2/EphB4 as potential anti-angiogenesis and
anticancer agents. N-(pyridin-2-yl)acrylamide was firstly
introduced to diaryl urea core as hinge-binding group of
triple inhibitors. The biological results indicated that
5

VDAU11 displayed stimultaneous inhibition of VEGFR-2,
After cooling to room temperature, 120 mL water was
added and the reaction mixture was allowed to stir for
another 2 h at room temperature. The product was
collected by filtration and dried under vacuum to afford 6-
bromo-2-quinazolinamine (2) (5.0 g, 60%).
ACCEPTED MANUSCRIPT
TIE-2 and EphB4 and is marginally better than the positive
control (Sorafenib) in two of three inhibitors. Meanwhile,
it displayed the most potent antiproliferative activity
against human vascular endothelial cell (EA. hy926). In
addition, VDAU11 exhibited comparable anticancer
activity compared with sorafenib. Several title compounds
(CPMA1, CPMA5, CPMA6, CPMA7, VDAU2, VDAU11,
QZAU4, and QZAU5) exhibited simultaneous inhibition
against VEGFR-2, TIE-2 and EphB4, three angiogenic
RTK. They might be considered as novel lead compound
for further discovery of triple RTK inhibitors as anti-
angiogenesis and anticancer agents. They might not only
inhibit the process of angiogenesis, but also prevent drug
resistance. Moreover, biological evaluation and molecular
docking indicated that N-(pyridin-2-yl)acrylamide was
beneficial for potency of these triple inhibitors. It could be
considered as a novel hinge-binding group for further
discovery of novel anti-angiogenesis agents.
4.2.2. 7-(4-Aminophenyl)- 2-quinazolinamine (4).
Pd(PPh3)4 (1.03 g, 0.89 mmol) was added to a degassed
solution of 4-aminophenyl boronic acid (1.54 g, 8.9 mmol),
K2CO3 (2.8 g, 26.7 mmol), 6-bromo-2-quinazolinamine (2)
(2.0 g, 8.9 mmol) in 120 mL 1,4-dioxane and 40 mL water.
The reaction mixture was heated at 100°C in an oil bath
and stirred under nitrogen for 24 h. The mixture was
dissolved in H2O and then extracted with ethyl acetate (30
mL×3). The combined organic layer was washed with
brine, dried over Na2SO4, filtered, and concentrated in
vacuo to give the crude product, which was isolated by
silica gel flash chromatography (PE/AcOEt =3:1)to yield
the key intermediate 4 (0.8 g, yield 28%).
4.2.3.
1-((3,5-dimethylphenyl)carbamoyl)cyclopropane
carboxylic acid (7).
4. Experimental section
4.1. Chemistry: General procedure
1,1-Cyclopropanedicarboxylic acid (5) (12.0 g, 15.4 mmol)
was dissolved in anhydrous THF (50 mL). Then
triethanolamine (2.0 mL,13.9mmol) was added to the
mixture and the mixture was stirred on the ice-bath for 30
min. SOCl2 (1.2 mL, 16.66 mmol) was then added. Stirring
was continued for 2h, a solution of 3,5-dimethylaniline 6
(3.6 g, 15.4 mmol) in anhydrous THF (10 mL) was added
and continued stirring for 2h, after that, the ice bath was
removed, and the mixture was stirred at room temperature
overnight. After the completion of the reaction, the mixture
was filtered and concentrated in vacuo. The residues was
Reagents and solvents are purified according to the
standard procedure. The reactions except those in aqueous
are performed by standard techniques for the exclusion of
moisture. Reactions are monitored by thin layer
chromatography (TLC) on 0.25-mm silica gel plates
(60GF-254) and visualized with UV light. Melting points
are determined on electrothermal melting point apparatus
1
and are uncorrected. H NMR and 13C NMR spectra are
measured at 400 MHz on a Bruker Advance AC 400
instrument with TMS as an internal standard. High
resolution mass spectra (HRMS) are obtained on a
Shimadzu LCMS-IF-TOF instrument.
purified
by
silica
gel
flash
chromatography
(PE/AcOEt=5:1) to yield 7 as white solid (0.8 g, 30.7%).
4.2.4. N-(4-(2-aminoquinazolin-7-yl)phenyl)-N-(3-bromo-
5-(trifluoromethyl)phenyl)cyclopropane-1,1-
4.2. General procedure for the synthesis of cyclopropyl-
malonic amide derivatives CPMA1-11.
4.2.1. 6-Bromoquinazolin-2-amine (2).
dicarboxamide (CPMA1).
2-Fluoro-5-bromobenzaldehyde (10 g, 49.2 mmol) and
bisguanidinium carbonate (13.3 g, 74 mmol) were
dissolved in N,N-dimethylacetamide (50 mL). The
resulting mixture was heated to reflux and stirred for 5 h.
To a mixture of 7 (0.24 g, 0.70 mmol) and 4 (0.38 g, 0.70
mmol) dissolved in 5 mL of THF in ice-bath, HATU (0.48
mg, 1.26 mmol) was added. After stirring for 30 min,
triethanolamine (0.10 mL, 0.56 mmol) was dropped. The
6

DMSO) δ 11.31 (s, 1H), 9.79 (s, 1H), 9.17 (s, 1H), 8.12
reaction was warmed to the room temperAatuCreCaEndPTstiErrDed MANUSCRIPT
overnight. After the completion of the reaction, the mixture
was filtered and the filtrate distilled by rotary evaporation
to remove THF. Water (50 mL) was added and the product
was extracted with AcOEt (30 mL×3), washed with water,
and dried over Na2SO4. After filtration and concentration
in vacuo, the residue was purified by silica gel flash
chromatography (PE/AcOEt = 3:1) to afford CPMA1 as
white solid. HRMS m/z calcd for C26H19BrF3N5O2
([M+H]+) 570.0674, found570.0654.m.p.=234-236°C,1H
NMR (400 MHz, DMSO) δ 11.49 (s, 1H), 9.74 (s, 1H),
9.16 (s, 1H), 8.44 (s, 1H), 8.12 (d, J = 2.0 Hz, 1H), 8.05
(m,1H), 7.76 (d, J = 8.8 Hz, 2H), 7.72 (d, J = 8.8 Hz, 2H),
7.50 (d, J = 8.8 Hz, 1H), 7.43 (s, 2H), 6.90 (s, 2H), 1.76
(m, 2H), 1.63 (m, 2H).13C NMR (101 MHz, DMSO) δ
170.78, 168.99, 163.07, 161.35, 151.65, 138.40, 137.77,
135.57, 133.37, 133.17, 132.98, 127.94, 126.92, 126.04,
125.57, 125.20, 123.75, 122.29, 121.78, 120.46, 120.15,
29.84, 18.16.
(m, 2H), 8.05 (m, 2.1 Hz, 1H), 7.74 (d, J = 5.4 Hz, 4H),
7.70 (s, 1H), 7.63 (m, 1H), 7.50 (d, J = 8.8 Hz, 1H), 6.91
(s, 2H), 1.76-1.72 (m, 2H), 1.62 (m, 2H).13C NMR (101
MHz, DMSO) δ 170.70, 168.86, 163.07, 161.35, 151.65,
139.75, 137.81, 135.53, 133.39, 133.17, 131.50, 131.15,
126.92, 125.57, 125.44, 125.19, 124.89, 122.20, 121.80,
120.15, 116.06, 29.88, 17.98.
4.2.7. N-(4-(2-aminoquinazolin-7-yl)phenyl)-N-(5-bromo-
2-(trifluoromethoxy)phenyl)cyclopropane-1,1-
dicarboxamide (CPMA4).
HRMS m/z calcd for C26H19BrF3N5O3 ([M+H]+) 586.0623,
1
found 586.0674. m.p.=239-240°C, H NMR (400 MHz,
DMSO) δ 11.48 (s, 1H), 9.74 (s, 1H), 9.16 (s, 1H), 8.44 (d,
J = 0.8 Hz, 1H), 8.12 (d, J = 2.0 Hz, 1H), 8.05 (m, 1H),
7.76 (d, J = 8.9 Hz, 2H), 7.72 (d, J = 8.9 Hz, 2H), 7.50 (d,
J = 8.8 Hz, 1H), 7.44 (s, 2H), 6.90 (s, 2H), 1.76 (m, 2H),
1.63 (m, 2H).13C NMR (101 MHz, DMSO) δ 170.78,
168.99, 163.07, 161.35, 151.65, 138.41, 137.77, 135.57,
133.37, 133.17, 132.98, 127.95, 126.92, 126.04, 125.57,
125.20, 123.75, 122.29, 121.78, 120.46, 120.15, 29.85,
18.16.
Compounds CPMA2-11 were prepared from the key
intermediate 4 and 7 with a similar procedure as described
for compound CPMA1.
4.2.5. N-(4-(2-aminoquinazolin-7-yl)phenyl)-N-(3-bromo-
5-(trifluoromethoxy)phenyl)cyclopropane-1,1-
4.2.8. N-(4-(2-aminoquinazolin-7-yl)phenyl)-N-(2-bromo-
5-(trifluoromethoxy)phenyl)cyclopropane-1,1-
dicarboxamide (CPMA2).
dicarboxamide (CPMA5).
HRMS m/z calcd for C26H19BrF3N5O3 ([M+H]+) 586.0623,
HRMS m/z calcd for C26H19BrF3N5O3 ([M+H]+) 586.0623,
1
1
found 586.0676. m.p.=224-225°C, H NMR (400 MHz,
found 586.0673. m.p.=191-192°C, H NMR (400 MHz,
DMSO) δ 11.31 (s, 1H), 9.80 (s, 1H), 9.16 (s, 1H), 8.13
(m, 2H), 8.05 (m, 1H), 7.76 (m, 2H), 7.74-7.69 (m, 3H),
7.63 (m, 1H), 7.49 (d, J = 8.8 Hz, 1H), 6.92 (s, 2H), 1.74
(m, 2H), 1.66-1.57 (m, 2H).13C NMR (101 MHz, DMSO)
δ 170.68, 168.86, 163.09, 161.34, 151.64, 139.76, 137.82,
135.51, 133.37, 133.18, 131.53, 131.15, 126.92, 125.56,
125.47, 125.19, 124.93, 122.18, 120.14, 116.09, 29.90,
17.96.
DMSO) δ 9.17 (s, 1H), 8.19 (d, J = 2.0 Hz, 1H), 8.10 (d, J
= 1.8 Hz, 1H), 8.04 (m, 1H), 7.81 (d, J = 8.8 Hz, 1H),
7.78-7.69 (m, 4H), 7.50 (d, J = 8.8 Hz, 1H), 7.12 (d, J =
8.0 Hz, 1H), 6.90 (s, 2H), 1.72 (d, J = 10.8 Hz, 2H), 1.66
(s, 2H).13C NMR (101 MHz, DMSO) δ 170.49, 169.30,
163.07, 161.34, 151.65, 148.01, 137.84, 135.55, 134.31,
133.45, 133.19, 126.97, 125.59, 125.18, 122.40, 121.69,
120.15, 119.14, 116.31, 113.51, 60.21, 30.05, 18.16.
4.2.6. N-(4-(2-aminoquinazolin-7-yl)phenyl)-N-(2-bromo-
5-(trifluoromethoxy)phenyl)cyclopropane-1,1-
4.2.9.
N-(4-(2-aminoquinazolin-7-yl)phenyl)-N-(2,4-
dichlorophenyl)cyclopropane-1,1-dicarboxamide
dicarboxamide (CPMA3).
(CPMA6).
HRMS m/z calcd for C26H19BrF3N5O3 ([M+H]+) 586.0623,
HRMS m/z calcd for C25H19Cl2N5O2 ([M+H]+) 492.0916,
1
1
found 586.0675. m.p.=212-214°C, H NMR (400 MHz,
found 492.0967. m.p.=261-263°C, H NMR (400 MHz,
7

DMSO) δ 10.68 (s, 1H), 10.16 (s, 1H), 9.17 (s, 1H), 8.39
DMSO) δ 10.99 (s, 1H), 9.93 (s, 1H), 9.17A(sC, 1CHE),P8.T09E(Dd, MANUSCRIPT
J = 8.7 Hz, 2H), 8.04 (d, J = 8.8 Hz, 1H), 7.74 (m, 4H),
7.70-7.68 (m, 1H), 7.50 (d, J = 8.7 Hz, 1H), 7.44 (m, 1H),
6.90 (s, 2H), 1.71 (s, 2H), 1.64 (s, 2H).13C NMR (101
MHz, DMSO) δ 170.35, 168.99, 163.07, 161.34, 151.65,
137.86, 135.53, 134.60, 133.44, 133.19, 129.23, 129.20,
128.18, 126.99, 126.27, 125.68, 125.59, 125.56, 125.17,
122.23, 120.15, 30.07, 17.86.
(s, 2H), 8.09 (d, J = 1.9 Hz, 1H), 8.03 (m, 1H), 7.78 (d, J =
8.6 Hz, 3H), 7.72 (d, J = 8.8 Hz, 2H), 7.50 (d, J = 8.8 Hz,
1H), 6.90 (s, 2H), 1.52 (s, 4H).13C NMR (101 MHz,
DMSO) δ 169.52, 167.79, 163.04, 161.32, 151.59, 141.47,
138.80, 134.86, 133.53, 133.14, 131.13, 130.81, 126.91,
125.56, 125.11, 124.99, 122.40, 121.20, 120.42, 120.16,
116.59, 32.82, 15.89.
4.2.10.
N-(4-(2-aminoquinazolin-7-yl)phenyl)-N-(3,4-
4.2.13. N-(4-(2-aminoquinazolin-7-yl)phenyl)-N-(3-fluoro-
4-(3-morpholinopropoxy)phenyl)cyclopropane-1,1-
dicarboxamide (CPMA10).
difluorophenyl)cyclopropane-1,1-dicarboxamide
(CPMA7).
HRMS m/z calcd for C25H19F2N5O2 ([M+H]+) 460.1507,
HRMS m/z calcd for C32H33FN6O4 ([M+H]+) 585.2547,
1
1
found 460.1558. m.p.=263-266°C, H NMR (400 MHz,
found 585.2560. m.p.=213-214°C, H NMR (400 MHz,
DMSO) δ 10.24 (s, 1H), 10.11 (s, 1H), 9.16 (s, 1H), 8.09
(d, J = 2.0 Hz, 1H), 8.03 (m, 1H), 7.86-7.80 (m, 1H), 7.76
(d, J = 8.9 Hz, 2H), 7.74-7.70 (m, 2H), 7.49 (d, J = 8.8 Hz,
1H), 7.39 (m, 2H), 6.89 (s, 2H), 1.50 (s, 2H), 1.48 (s,
2H).13C NMR (101 MHz, DMSO) δ 168.86, 168.34,
163.06, 161.30, 151.56, 138.71, 136.48, 136.39, 134.84,
133.53, 133.16, 126.90, 125.55, 125.01, 121.28, 120.16,
117.68, 117.51, 117.19, 110.04, 109.82, 32.32, 15.90.
DMSO) δ 10.17 (s, 1H), 10.02 (s, 1H), 9.16 (s, 1H), 8.09
(d, J = 2.0 Hz, 1H), 8.03 (m, 1H), 7.80 (s, 1H), 7.76 (d, J =
8.8 Hz, 2H), 7.72 (d, J = 8.9 Hz, 2H), 7.63 (m, 1H), 7.49
(d, J = 8.8 Hz, 1H), 7.31 (d, J = 8.9 Hz, 1H), 7.14 (m, 1H),
6.89 (s, 2H), 3.63-3.51 (m, 6H), 2.51 (m, 4H), 2.48 (s, 4H),
1.49 (s, 4H).13C NMR (101 MHz, DMSO) δ 168.66,
168.55, 163.05, 161.31, 151.58, 142.72, 138.69, 134.83,
133.53, 133.16, 133.00, 132.90, 126.91, 125.56, 125.00,
121.25, 120.16, 116.94, 115.69, 109.64, 109.41, 67.44,
66.63, 57.38, 54.06, 32.00, 15.96.
4.2.11.
N-(4-(2-aminoquinazolin-7-yl)phenyl)-N-(3,4-
dichlorophenyl)cyclopropane-1,1-dicarboxamide
(CPMA8).
4.2.14. N-(4-(2-aminoquinazolin-7-yl)phenyl)-N-(benzo[d]
[1,3]dioxol-5-yl)cyclopropane-1,1-dicarboxamide
(CPMA11).
HRMS m/z calcd for C25H19Cl2N5O2 ([M+H]+) 492.0916,
1
found 492.0924. m.p.=253-254°C, H NMR (400 MHz,
DMSO) δ 10.32 (s, 1H), 10.11 (s, 1H), 9.17 (s, 1H), 8.09
(d, J = 2.0 Hz, 1H), 8.06 (d, J = 2.1 Hz, 1H), 8.03 (m, 1H),
7.77 (d, J = 8.8 Hz, 2H), 7.71 (d, J = 8.8 Hz, 2H), 7.60 (m,
1H), 7.56 (d, J = 8.8 Hz, 1H), 7.49 (d, J = 8.8 Hz, 1H),
6.90 (s, 2H), 1.52 (d, J = 10.2 Hz, 2H), 1.48 (d, J = 10.1
Hz, 2H).13C NMR (101 MHz, DMSO) δ 169.01, 168.23,
163.05, 161.32, 151.58, 139.57, 138.73, 134.86, 133.54,
133.15, 131.19, 130.84, 126.90, 125.56, 125.40, 125.00,
122.08, 121.29, 120.75, 120.17, 32.49, 15.97.
HRMS m/z calcd for C26H21N5O4 ([M+H]+) 468.1627,
1
found 468.1637. m.p.=283-285°C, H NMR (400 MHz,
DMSO) δ 10.21 (s, 1H), 9.91 (s, 1H), 9.17 (s, 1H), 8.09 (d,
J = 1.8 Hz, 1H), 8.03 (m, 1H), 7.76 (d, J = 8.8 Hz, 2H),
7.72 (d, J = 8.8 Hz, 2H), 7.49 (d, J = 8.8 Hz, 1H), 7.32 (d,
J = 1.5 Hz, 1H), 7.02 (m, 1H), 6.90 (s, 2H), 6.86 (d, J = 8.4
Hz, 1H), 6.00 (s, 2H), 1.49 (s, 4H).13C NMR (101 MHz,
DMSO) δ 168.71, 168.64, 163.05, 161.31, 151.58, 147.36,
143.76, 138.65, 134.84, 133.55, 133.47, 133.16, 126.92,
125.56, 125.01, 121.25, 120.17, 114.15, 108.24, 103.44,
101.44, 67.48, 31.78, 16.03.
4.2.12.
N-(4-(2-aminoquinazolin-7-yl)phenyl)-N-(3,5-
bis(trifluoromethyl)phenyl)cyclopropane-1,1-
dicarboxamide (CPMA9).
HRMS m/z calcd for C27H19F6N5O2 ([M+H]+) 560.1443,
4.2.15. General procedure for the synthesis of N-(pyridin-
2-yl)acrylamidediaryl urea derivatives VDAU1-11.
4.2.16. 1-(4-(tert-butyl)phenyl)-3-(4-(4,4,5,5-tetramethyl-
1
found 560.1492. m.p.=218-219°C, H NMR (400 MHz,
8

1,3,2-dioxaborolan-2-yl)phenyl)urea (9).
1.33mmol) was dropped. The reaction was warmed to the
room temperature and stirred overnight. After the
completion of the reaction, the mixture was filtered and the
filtrate distilled by rotary evaporation to remove THF.
After filtration and concentration in vacuo, the residues
was purified by silica gel flash chromatography
(PE/AcOEt = 1:1) yielding VDAU-1 as white solid (yield
21.7%), HRMS m/z calcd for C25H26N4O2 ([M+H]+)
415.0156, found 415.0151. m.p.=180-182°C, 1H NMR
(400 MHz, DMSO) δ 10.82 (s, 1H), 8.78 (s, 1H), 8.66 (d, J
= 2.4 Hz, 1H), 8.63 (s, 1H), 8.27 (d, J = 8.7 Hz, 1H), 8.10
(m, J = 8.7, 2.5 Hz, 1H), 7.66 (d, J = 8.7 Hz, 2H), 7.57 (d, J
= 8.7 Hz, 2H), 7.39 (d, J = 8.7 Hz, 2H), 7.31 (d, J = 8.8 Hz,
2H), 6.64 (m, J = 17.0, 10.2 Hz, 1H), 6.33 (m, J = 17.0, 1.9
Hz, 1H), 5.83 – 5.77 (m, 1H), 1.27 (s, 10H). 13C NMR
(101 MHz, DMSO) δ 164.08, 152.97, 151.20, 145.80,
144.69, 140.09, 137.43, 135.98, 132.01, 131.60, 130.39,
128.20, 127.21, 125.87, 118.97, 118.60, 114.07, 34.37,
31.73.
ACCEPTED MANUSCRIPT
Triphosgene (1.77 g, 6.03 mmol) was dissolved in
anhydrous CH2Cl2 (30 mL) and the mixture was stirred on
the ice-bath for 5 min. Tert-butyl-aniline (2.0 g, 13.4mmol)
in anhydrous CH2Cl2 was added dropwise to the mixture
and stirring was continued for 15 min. Then
triethanolamine (2.2 mL, 16.1 mmol) diluted with CH2Cl2
(20 mL) was added onto the mixture. Stirring was
continued for 15 min, a solution of triethanolamine (2.2
mL, 16.1 mmol) and 4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)aniline 8 (2.35 g, 10.72mmol) in
anhydrous CH2Cl2 (30 mL) was added and continued
stirring for 20 min. Subsequently, the ice bath was
removed, and the mixture was reacted at room temperature
overnight. After completion of the action, the reaction was
quenched with dilute NaHCO3. The organic layer was
washed with water and brine, and dried over Na2SO4. After
filtration and concentration in vacuo, the residue was
purified by silica gel flash chromatography (PE/AcOEt =
3:1) yielding 10 as white solid (3.4 g, 64.4%).
The title compounds VDAU2-11 were prepared from the
key intermediate 11 and acryloyl chloride with a similar
procedure as described for compounds VDAU1.
4.2.17.
1-(4-(6-aminopyridin-3-yl)phenyl)-3-(4-(tert-
butyl)phenyl)urea (11).
A flask charged with Pd(PPh3)4 (0.30 g, 0.26mmol),
cesium carbonate (1.71 g, 5.26mmol), and intermediate 9
(1.00 g, 2.63mmol) and 2-amino-5-bromopyridine 10 (0.45
g, 2.63mmol) were flushed with nitrogen and suspended in
1,4-dioxane (90 mL) and water (30 mL). The mixture was
then refluxed overnight under nitrogen. The hot suspension
was filtered and the filtrate distilled by rotary evaporation
to remove 1,4-dioxane. Water (50 mL) was added and the
product was extracted with AcOEt (30 mL×3), washed
with water, and dried over Na2SO4. After filtration and
concentration in vacuo, the residue was purified by silica
gel flash chromatography (PE/AcOEt = 3:1) affording 11
(0.56 g, 45.5%) as yellow solid.
4.2.19.
N-(5-(4-(3-(3-isopropylphenyl)ureido)phenyl)
pyridin-2-yl)acrylamide (VDAU2).
White solid (yield 47.6%), HRMS m/z calcd for
C24H24N4O2 ([M+H]+) 400.1899, found 400.9911.
1
m.p.=180-181°C, H NMR (400 MHz, DMSO) δ 10.82 (s,
1H), 8.79 (s, 1H), 8.67 (d, J = 1.9 Hz, 2H), 8.28 (d, J = 8.6
Hz, 1H), 8.10 (m, J = 8.6, 2.3 Hz, 1H), 7.67 (d, J = 8.6 Hz,
2H), 7.58 (d, J = 8.6 Hz, 2H), 7.37 (s, 1H), 7.28 (d, J = 8.0
Hz, 1H), 7.20 (t, J = 7.8 Hz, 1H), 6.87 (d, J = 7.4 Hz, 1H),
6.65 (m, J = 17.0, 10.2 Hz, 1H), 6.38 – 6.29 (m, 1H), 5.84
– 5.76 (m, 1H), 2.93 – 2.78 (m, 1H), 1.19 (t, J = 11.5 Hz,
6H). 13C NMR (101 MHz, DMSO) δ 164.09, 152.94,
151.22, 149.51, 145.81, 140.03, 135.99, 132.02, 131.60,
130.47, 129.16, 128.18, 127.21, 120.47, 119.04, 116.70,
116.36, 114.08, 33.97, 24.35.
4.2.18.
N-(5-(4-(3-(4-(tert-butyl)phenyl)ureido)phenyl)
pyridin-2-yl)acrylamide(VDAU1).
To a mixture of 11 (0.20 g, 0.55mmol) and triethanolamine
(0.31 mL, 2.20 mmol) dissolved in 20 mL of THF in ice-
bath. After stirring for 30 min, acryloyl chloride (0.11 mL,
4.2.20.
N-(5-(4-(3-(4-chloro-3-(trifluoromethyl)phenyl)
ureido)phenyl)pyridin-2-yl)acrylamide(VDAU3).
White solid (yield70.6%), HRMS m/z calcd for
9

C22H16ClF3N4O2 ([M]+) 460.0914, found 460.9700.
131.55, 130.78, 128.18, 127.23, 119.25, 114.46, 114.08,
108.55, 105.51, 105.25.
ACCEPTED MANUSCRIPT
1
m.p.=193-194°C, H NMR (400 MHz, DMSO) δ 10.82 (s,
1H), 9.22 (s, 1H), 9.01 (s, 1H), 8.66 (d, J = 2.1 Hz, 1H),
8.28 (d, J = 8.7 Hz, 1H), 8.13 (d, J = 2.3 Hz, 1H), 8.10 (m,
J = 8.7, 2.5 Hz, 1H), 7.68 (d, J = 8.7 Hz, 2H), 7.66 (d, J =
2.3 Hz, 1H), 7.63 (s, 1H), 7.59 (d, J = 8.7 Hz, 2H), 6.65
(m, J = 17.0, 10.2 Hz, 1H), 6.34 (m, J = 17.0, 1.8 Hz, 1H),
5.80 (m, J = 10.2, 1.8 Hz, 1H). 13C NMR (101 MHz,
DMSO) δ 164.09, 152.81, 151.29, 145.86, 139.78, 139.47,
136.05, 132.47, 132.00, 131.50, 131.01, 128.19, 127.23,
124.65, 123.57, 122.81, 119.48, 117.30, 114.07.
4.2.23. N-(5-(4-(3-(4-fluorophenyl)ureido)phenyl)pyridin-
2-yl)acrylamide (VDAU6).
White solid (yield 15.6%), HRMS m/z calcd for
C21H17FN4O2 ([M]+) 376.1336, found 376.9213. m.p.=256-
1
258°C, H NMR (400 MHz, DMSO) δ 10.81 (s, 1H), 8.83
(s, 1H), 8.77 (s, 1H), 8.66 (d, J = 2.0 Hz, 1H), 8.27 (d, J =
8.7 Hz, 1H), 8.09 (m, J = 8.7, 2.4 Hz, 1H), 7.67 (d, J = 8.7
Hz, 2H), 7.57 (d, J = 8.7 Hz, 2H), 7.50 (d, J = 5.0 Hz, 1H),
7.48 (d, J = 4.9 Hz, 1H), 7.14 (t, J = 8.9 Hz, 2H), 6.65 (m, J
= 17.0, 10.2 Hz, 1H), 6.34 (m, J = 17.0, 1.8 Hz, 1H), 5.80
(m, J = 10.2, 1.8 Hz, 1H). 13C NMR (101 MHz, DMSO) δ
164.09, 159.04, 153.03, 151.22, 145.81, 139.97, 136.42,
135.99, 132.02, 131.59, 130.54, 128.18, 127.21, 120.55,
120.47, 119.11, 115.87, 115.65, 114.07.
4.2.21.
N-(5-(4-(3-(3-chloro-4-
pyridin-2-yl)acrylamide
methylphenyl)ureido)phenyl)
(VDAU4).
White solid (yield 51.4%), HRMS m/z calcd for
C22H19ClN4O2 ([M]+) 406.1197, found 406.9234.
1
m.p.=214-215°C, H NMR (400 MHz, DMSO) δ 10.82 (s,
4.2.24.
N-(5-(4-(3-(3-(trifluoromethyl)phenyl)ureido)
1H), 8.89 (s, 1H), 8.84 (s, 1H), 8.66 (d, J = 2.2 Hz, 1H),
8.27 (d, J = 8.7 Hz, 1H), 8.09 (m, J = 8.7, 2.5 Hz, 1H), 7.72
(d, J = 1.9 Hz, 1H), 7.67 (d, J = 8.7 Hz, 2H), 7.58 (d, J =
8.7 Hz, 2H), 7.25 (d, J = 8.4 Hz, 1H), 7.21 (m, J = 8.3, 2.0
Hz, 1H), 6.65 (m, J = 17.0, 10.2 Hz, 1H), 6.34 (m, J = 17.0,
1.8 Hz, 1H), 5.80 (m, J = 10.2, 1.8 Hz, 1H), 2.27 (s, 3H).
13C NMR (101 MHz, DMSO) δ 164.09, 152.84, 151.24,
145.83, 139.80, 139.30, 136.00, 133.59, 132.01, 131.64,
131.55, 130.67, 128.76, 128.19, 127.21, 119.19, 118.64,
117.48, 114.07, 19.29.
phenyl)pyridin-2-yl)acrylamide (VDAU7).
White solid (yield 53.1%), HRMS m/z calcd for
C22H17F3N4O2 ([M]+) 426.1304, found 426.9504.
1
m.p.=193-195°C, H NMR (400 MHz, DMSO) δ 10.82 (s,
1H), 9.12 (s, 1H), 8.97 (s, 1H), 8.67 (d, J = 2.1 Hz, 1H),
8.28 (d, J = 8.7 Hz, 1H), 8.10 (m, J = 8.7, 2.4 Hz, 1H), 8.04
(s, 1H), 7.68 (d, J = 8.7 Hz, 2H), 7.60 (d, J = 7.4 Hz, 2H),
7.59 (s, 1H), 7.53 (t, J = 7.9 Hz, 1H), 7.33 (d, J = 7.6 Hz,
1H), 6.65 (m, J = 17.0, 10.2 Hz, 1H), 6.34 (m, J = 17.0, 1.8
Hz, 1H), 5.80 (m, J = 10.2, 1.8 Hz, 1H). 13C NMR (101
MHz, DMSO) δ 164.10, 152.92, 151.27, 145.85, 141.01,
139.63, 136.04, 132.01, 131.53, 130.88, 130.39, 130.16,
129.85, 128.19, 127.23, 122.37, 119.36, 118.58, 114.66,
114.07.
4.2.22. N-(5-(4-(3-(3-fluorophenyl)ureido)phenyl)pyridin-
2-yl)acrylamide (VDAU5).
White solid (yield 34.4%), HRMS m/z calcd for
C21H17FN4O2 ([M]+) 376.1336, found 376.9202. m.p.=199-
1
201°C, H NMR (400 MHz, DMSO) δ 10.82 (s, 1H), 8.99
4.2.25. N-(5-(4-(3-(2-chlorophenyl)ureido)phenyl)pyridin-
2-yl)acrylamide (VDAU8).
(s, 1H), 8.93 (s, 1H), 8.66 (d, J = 2.1 Hz, 1H), 8.28 (d, J =
8.7 Hz, 1H), 8.10 (m, J = 8.7, 2.4 Hz, 1H), 7.68 (d, J = 8.7
Hz, 2H), 7.58 (d, J = 8.7 Hz, 2H), 7.55 – 7.49 (m, 1H),
7.32 (m, J = 15.2, 8.1 Hz, 1H), 7.15 (d, J = 8.1 Hz, 1H),
6.80 (m, J = 8.4, 2.1 Hz, 1H), 6.65 (m, J = 17.0, 10.2 Hz,
1H), 6.34 (m, J = 17.0, 1.8 Hz, 1H), 5.80 (m, J = 10.2, 1.8
Hz, 1H). 13C NMR (101 MHz, DMSO) δ 164.10, 161.68,
152.80, 151.26, 145.84, 141.96, 139.71, 136.02, 132.02,
White solid (yield 14.3%), HRMS m/z calcd for
C21H17ClN4O2 ([M]+) 392.1040, found 392.9006.
1
m.p.=190-192°C, H NMR (400 MHz, DMSO) δ 10.82 (s,
1H), 9.57 (s, 1H), 8.67 (d, J = 2.1 Hz, 1H), 8.36 (s, 1H),
8.28 (d, J = 8.7 Hz, 1H), 8.19 (d, J = 8.3 Hz, 1H), 8.11 (m,
J = 8.7, 2.4 Hz, 1H), 7.69 (d, J = 8.6 Hz, 2H), 7.59 (d, J =
8.6 Hz, 2H), 7.50 – 7.44 (m, 1H), 7.32 (t, J = 7.8 Hz, 1H),
10

(s, 1H), 7.68 (d, J = 8.7 Hz, 2H), 7.58 (d, J = 8.7 Hz, 2H),
7.09 – 7.01 (m, 1H), 6.65 (m, J = 17.0, 10A.2CHCz,E1HP)T, E6.D34 MANUSCRIPT
(m, J = 17.0, 1.6 Hz, 1H), 5.80 (m, J = 10.2, 1.6 Hz, 1H).
13C NMR (101 MHz, DMSO) δ 164.10, 152.54, 151.27,
145.85, 139.69, 136.36, 136.03, 132.01, 131.52, 130.85,
129.71, 128.20, 128.07, 127.32, 123.86, 122.47, 121.83,
119.08, 114.08.
7.31 (d, J = 4.1 Hz, 1H), 7.29 (d, J = 5.6 Hz, 1H), 7.03 (m,
J = 6.8, 2.2 Hz, 1H), 6.65 (m, J = 17.0, 10.2 Hz, 1H), 6.34
(m, J = 17.0, 1.8 Hz, 1H), 5.80 (m, J = 10.2, 1.8 Hz, 1H).
13C NMR (101 MHz, DMSO) δ 164.10, 152.80, 151.26,
145.84, 141.69, 139.70, 136.02, 133.68, 132.02, 131.54,
130.87, 130.80, 128.18, 127.23, 121.98, 119.27, 118.08,
117.17, 114.08.
4.2.26.
N-(5-(4-(3-(4-(trifluoromethoxy)phenyl)ureido)
phenyl)pyridin-2-yl)acrylamide (VDAU9).
White solid (yield26.5%), HRMS m/z calcd for
C22H17F3N4O3 ([M]+) 442.1253, found 442.9704.
4.2.29. General procedure for the synthesis of
quinazolinone diaryl urea derivatives QZAU1-5.
1
m.p.=220-222°C, H NMR (400 MHz, DMSO) δ 10.81 (s,
4.2.30. 7-Bromoquinazolin-4(3H)-one (13).
1H), 8.94 (s, 1H), 8.89 (s, 1H), 8.66 (d, J = 2.1 Hz, 1H),
8.27 (d, J = 8.7 Hz, 1H), 8.10 (m, J = 8.7, 2.4 Hz, 1H), 7.68
(d, J = 8.7 Hz, 2H), 7.58 (d, J = 8.8 Hz, 4H), 7.30 (d, J =
8.6 Hz, 2H), 6.65 (m, J = 17.0, 10.2 Hz, 1H), 6.34 (m, J =
17.0, 1.8 Hz, 1H), 5.80 (m, J = 10.2, 1.8 Hz, 1H). 13C
NMR (101 MHz, DMSO) δ 164.09, 152.88, 151.25,
145.83, 143.10, 139.79, 139.42, 136.01, 132.02, 131.56,
130.72, 128.18, 127.23, 122.20, 119.93, 119.40, 119.21,
114.07.
Around bottom two flask charged with 2-amino-5-
bromobenzoicacid 12 (5.00 g, 23.14 mmol) was flushed
with nitrogen and suspended in HCONH2 (80 mL, 2.01
mmol), the mixture was stirred at 150°C for 1.5 h by
atmospheric microwave heating. The product was
extracted with AcOEt (50 mL ×3), washed with water, and
dried over Na2SO4. After filtration and concentration in
vacuo, the residue was purified by silica gel flash
chromatography (PE/AcOEt=5:1) affording 13 (1.46 g,
41.24%) as white solid. mp: 259~261°C.
4.2.27.
N-(5-(4-(3-(2,6-dimethylphenyl)ureido)phenyl)
pyridin-2-yl)acrylamide (VDAU10).
4.2.31. (3-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)
phenyl)boronic acid (15).
White solid (yield 35.4%), HRMS m/z calcd for
C23H22N4O2 ([M]+) 386.1743, found 386.9670. m.p.=290-
Triphosgene (0.94 g, 3.2 2mmol) was dissolved in
anhydrous CH2Cl2 (20 mL) and the mixture was stirred on
the ice-bath for 5 min. A solution of 4-chloro-3-
(trifluoromethyl)aniline (1.40 g, 7.16 mmol) in anhydrous
CH2Cl2 was added dropwise to the above mixture and
stirring was continued for 15 min. Then triethanolamine
(1.20 mL,17.18 mmol) diluted with CH2Cl2 (10 mL) was
then added onto the mixture. Stirring was continued for 15
min, a solution of triethanolamine (1.20 mL, 17.18 mmol)
and 3-aminophenylboronic acid 14 (0.89 g,5.73 mmol) in
anhydrous CH2Cl2 (10 mL) was added and continued
stirring for 20min. Subsequently, the ice bath was
removed, and the mixture was reacted at room temperature
overnight. After completion of the action, the reaction was
quenched with dilute NaHCO3.The organic layer was
washed with water and brine, and dried overNa2SO4. After
filtration and concentration in vacuo, the residue was
1
292°C, H NMR (400 MHz, DMSO) δ 10.81 (s, 1H), 8.97
(s, 1H), 8.65 (d, J = 2.1 Hz, 1H), 8.27 (d, J = 8.7 Hz, 1H),
8.08 (m, J = 8.7, 2.4 Hz, 1H), 7.85 (s, 1H), 7.65 (d, J = 8.7
Hz, 2H), 7.58 (d, J = 8.8 Hz, 2H), 7.08 – 7.04 (m, 3H),
6.65 (m, J = 17.0, 10.2 Hz, 1H), 6.38 – 6.30 (m, 1H), 5.80
(m, J = 10.2, 1.8 Hz, 1H), 2.23 (s, 6H). 13C NMR (101
MHz, DMSO) δ 164.08, 153.57, 151.15, 145.74, 140.67,
136.04, 135.91, 135.77, 132.03, 131.68, 130.02, 128.20,
127.12, 126.44, 118.79, 114.07, 18.75.
4.2.28. N-(5-(4-(3-(3-chlorophenyl)ureido)phenyl)pyridin-
2-yl)acrylamide (VDAU11).
White solid (yield 40.9%), HRMS m/z calcd for
C21H17ClN4O2 ([M]+) 392.1040, found 392.9001.
1
m.p.=197-199°C, H NMR (400 MHz, DMSO) δ 10.82 (s,
1H), 8.97 (s, 1H), 8.94 (s, 1H), 8.66 (d, J = 2.3 Hz, 1H),
8.28 (d, J = 8.7 Hz, 1H), 8.10 (m, J = 8.7, 2.4 Hz, 1H), 7.74
11

purified
by
silica
gel
flash
chromatography
7.45 (s, 1H). 13C NMR (101 MHz, DMSO) δ 161.01,
ACCEPTED MANUSCRIPT
(PE/AcOEt=5:1) yielding 15 as white solid (0.84 g,
32.68%).
152.99, 149.75, 146.39, 146.30, 142.27, 140.30, 139.84,
131.33, 131.00, 130.11, 127.15, 125.80, 125.17, 125.14,
122.43, 122.09, 121.87, 119.52, 118.55, 118.52, 117.96,
114.89.
4.2.32. 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(3-(4-oxo-
3,4-dihydroquinazolin-7-yl)phenyl)urea (QZAU1).
A flask charged with Pd(PPh3)4 (0.39 g, 0.34 mmol),
potassium carbonate (1.43 g, 10.35 mmol), and the key
intermediate 15 (1.40 g, 3.45 mmol) and 13 (0.78 g, 3.45
mmol) were flushed with nitrogen and suspended in 1,4-
dioxane (90 mL) and water (30 mL). The mixture was then
refluxed overnight under nitrogen. The hot suspension was
filtered and the filtrate distilled by rotary evaporation to
remove 1,4-dioxane. Water (50 mL) was added and the
product was extracted with AcOEt (30 mL×3), washed
with water, and dried over Na2SO4. After filtration and
concentration in vacuo, the residue was purified by silica
gel flash chromatography (PE/AcOEt=2:1) affording
QZAU1 (0.21g) as white solid (yield 30.8%), HRMS m/z
calcd for C22H14ClF3N4O2([M+H]+) 459.0757, found
4.2.34. 1-(3-(4-oxo-3,4-dihydroquinazolin-7-yl)phenyl)-3-
(3-(trifluoromethyl)phenyl)urea (QZAU3).
White solid (yield 14.4%), HRMS m/z calcd for
C22H15F3N4O2 ([M+H]+) 425.1147, found 425.1176. m.p.
>300°C, 1H NMR (400 MHz, DMSO) δ 12.30 (s, 1H), 9.28
(s, 1H), 9.11 (s, 1H), 8.22 (d, J = 8.3 Hz, 1H), 8.15 (s, 1H),
8.05 (s, 1H), 7.98 (s, 1H), 7.88 (s, 1H), 7.81 (m, J = 8.3,
1.4 Hz, 1H), 7.62 (d, J = 8.3 Hz, 1H), 7.51 (m, J = 9.9, 7.6
Hz, 2H), 7.45 (d, J = 4.3 Hz, 1H), 7.44 (d, J = 2.1 Hz, 1H),
7.33 (d, J = 7.6 Hz, 1H). 13C NMR (101 MHz, DMSO) δ
161.04, 153.12, 149.75, 146.39, 141.03, 140.66, 139.84,
130.40, 130.15, 129.84, 127.17, 126.04, 125.82, 125.16,
123.34, 122.37, 122.07, 121.51, 119.11, 118.62, 117.52,
114.64, 40.59, 40.38, 40.18, 39.97, 39.76, 39.55, 39.34.
1
459.0786. m.p. >300°C, H NMR (400 MHz, DMSO) δ
4.2.35.
1-(3,4-difluorophenyl)-3-(3-(4-oxo-3,4-
12.30 (s, 1H), 9.26 (s, 1H), 9.02 (s, 1H), 8.21 (d, J = 8.3
Hz, 1H), 8.15 (d, J = 3.3 Hz, 1H), 8.13 (d, J = 2.4 Hz, 1H),
7.97 (s, 1H), 7.88 (d, J = 1.3 Hz, 1H), 7.80 (m, J = 8.3, 1.7
Hz, 1H), 7.68 (m, J = 8.8, 2.4 Hz, 1H), 7.61 (d, J = 8.8 Hz,
1H), 7.53 – 7.48 (m, 1H), 7.45 (d, J = 2.3 Hz, 1H), 7.44 (d,
J = 1.9 Hz, 1H). 13C NMR (101 MHz, DMSO) δ 161.01,
152.97, 149.75, 146.40, 146.34, 140.48, 139.83, 139.76,
132.44, 130.12, 127.16, 125.79, 125.16, 124.64, 123.64,
122.86, 122.08, 121.66, 119.29, 117.73, 117.35, 117.29.
The title compounds QZAU2 -5 were prepared from the
key intermediate 13 and 15 with a similar procedure as
described for compound QZAU1.
dihydroquinazolin-7-yl)phenyl)urea (QZAU4).
White solid (yield 20.2%), HRMS m/z calcd for
C21H14F2N4O2 ([M+H]+) 393.1085, found 393.1125. m.p.
>300°C, 1H NMR (400 MHz, DMSO) δ 12.30 (s, 1H), 9.00
(s, 1H), 8.93 (s, 1H), 8.15 (d, J = 2.9 Hz, 1H), 7.96 (s, 1H),
7.87 (s, 0H), 7.80 (m, J = 8.3, 1.6 Hz, 1H), 7.70 (m, J =
13.4, 7.5, 2.6 Hz, 1H), 7.52 – 7.46 (m, 1H), 7.44 (d, J = 4.3
Hz, 1H), 7.43 (s, 1H), 7.35 (m, J = 19.6, 9.2 Hz, 1H), 7.21
– 7.13 (m, 1H). 13C NMR (101 MHz, DMSO) δ 161.01,
153.03, 149.75, 146.41, 140.67, 139.81, 137.30, 137.18,
130.13, 127.15, 125.80, 125.14, 122.08, 121.45, 119.09,
117.92, 117.75, 117.52, 114.98, 107.88, 107.67.
4.2.33. 1-(3,5-bis(trifluoromethyl)phenyl)-3-(3-(4-oxo-3,4-
dihydroquinazolin-7-yl)phenyl)urea (QZAU2).
4.2.36.
1-(3-chlorophenyl)-3-(3-(4-oxo-3,4-
dihydroquinazolin-7-yl)phenyl)urea (QZAU5).
White solid (yield 25.2%), HRMS m/z calcd for
C23H14F6N4O2 ([M+H]+) 493.1021, found 493.1053. m.p.
>300°C, 1H NMR (400 MHz, DMSO) δ 12.30 (s, 1H), 9.49
(s, 1H), 9.16 (s, 1H), 8.21 (d, J = 8.3 Hz, 1H), 8.16 (s, 2H),
8.15 (s, 1H), 7.98 (s, 1H), 7.88 (s, 1H), 7.80 (d, J = 8.3 Hz,
1H), 7.64 (s, 1H), 7.53 (d, J = 4.1 Hz, 1H), 7.46 (s, 1H),
White solid (yield 25.4%), HRMS m/z calcd for
C21H15ClN4O2 ([M+H]+) 391.0884, found 391.0928. m.p.
>300°C, 1H NMR (400 MHz, DMSO) δ 12.30 (s, 1H), 9.00
(s, 1H), 8.95 (s, 1H), 8.22 (d, J = 8.3 Hz, 1H), 8.15 (s, 1H),
7.98 (s, 1H), 7.88 (d, J = 1.1 Hz, 1H), 7.80 (m, J = 8.3, 1.6
Hz, 1H), 7.75 (s, 1H), 7.52 – 7.46 (m, 1H), 7.44 (d, J = 5.0
12

the density of 1×105 cells for each well and cultured in
Hz, 1H), 7.43 (s, 1H), 7.31 (s, 1H), 7.30A(sC, C1HE)P, 7T.0E6D– MANUSCRIPT
7.01 (m, 1H). 13C NMR (101 MHz, DMSO) δ 161.03,
152.96, 149.76, 146.39, 141.67, 140.68, 139.82, 133.68,
130.85, 130.13, 127.16, 125.80, 125.15, 122.08, 122.01,
121.46, 119.07, 118.14, 117.50, 117.23.
DMEM containing 10% FBS in humidified 5% CO2
atmosphere. After incubation at 37 ºC for 48 h, the cells
were treated with tested compounds at various
concentrations for 24 h. Subsequently, premixed CCK-8
and medium (10 µL) were added into the 96-well plates to
monitor cell viability and were incubated at 37 ºC for 2 h.
The number of viable cells was assessed by measurement
of absorbance at 450 nm by a microplate reader. The
viability rate was calculated as experimental OD
value/control OD value.
4.3. Angiogenic RTK inhibition evaluation [20]
The inhibitory activity against VEGFR-2, TIE-2, and
EphB4 of all the title compounds were evaluated using the
ADP-Glo™ kinase assay kit (Promega, Madison) with
sorafenib as positive control. The kinase assay was carried
in duplicate in a reaction mixture of final volume of 10 µL.
General procedures are as the following: for VEGFR-2
assays, the kinase (0.6 ng/mL) were incubated with
substrates (0.2 mg/mL), title compounds (1.2×10-4~12 µM)
and ATP (50 µM) in a final buffer of Tris 40 mM, MgCl2
10 mM, BSA 0.1 mg/mL, DTT 1 mM in 384-well plate
with the total volume of 5 µL. The assay plate was
incubated at 30°C for 60 min and cooled at room
temperature for 5 min, 5 µL of ADP-Glo reagent was
added to stop the reaction and consume the remaining ADP
within 40 min. At the end, 10 µL of kinase detection
reagent was added and incubated for 30 min to produce a
luminescence signal. As for TIE-2 and EphB4 assays, the
kinase (2.4 ng/mL) were incubated with substrates (0.2
mg/mL), title compounds (1.2 ×10-4~12 µM) and ATP (50
µM) in a final buffer of Tris 40 mM, MgCl2 10 mM, BSA
0.1 mg/mL, DTT 1 mM in 384-well plate with the total
volume of 5 µL. The assay plate was incubated at 30°C for
4 h and cooled at room temperature for 5 min, 5 µL of
ADP-Glo reagent was added to stop the reaction and
consume the remaining ADP within 1 h. At the end, 10 µL
of kinase detection reagent was added and incubated for 30
min to produce a luminescence signal. The luminescence
was read by VICTOR-X multi- label plate reader.
4.3. Cell growth inhibitory activity in cancer cell lines [22]
The antiproliferative activity of title compounds were
evaluated against nine cancer cell lines. All the title
compounds were evaluated using MTT assay to assess cell
proliferation. Exponentially growing cells were harvested
and plated in 96-well plates at a concentration of 1×104
cells/well, and then incubated for 24 h at 37 °C. The cells
in wells were treated with title compounds respectively at
various concentrations for 48 h. Then, 22 mL fresh MTT (5
mg/mL) was added to each well and incubated for 4 h at 37
°C. Supernatant was discarded, and 150 mL DMSO was
added to each well. Absorbance values were determined by
a microplate reader (Bio-Rad Instruments) at 490 nm. The
IC50 values were calculated according to inhibition ratios.
4.4. Molecular docking modeling [23]
In order to understand the binding mode of inhibitors with
VEGFR-2/TIE-2/EphB4, molecule docking was performed
using Sybyl-X/Surflex-dock module based on the crystal
structures of VEGFR-2 (PDB ID: 4ASD), TIE-2 (PDB ID:
2P4I) and EphB4 (PDB ID: 2X9F). Hydrogen was added
and minimized using the Tripos force field and Pullman
charges. The most potent compound (VDAU11) was
depicted with the Sybyl-X/Sketch module (Tripos Inc.) and
optimized applying Powell's method with the Tripos force
field with convergence criterion set at 0.05 kcal/(Åmol),
and assigned with the Gasteiger-Hückel charge. The
docking studied was carried out using Surflex-dock
module. The residues in a radius 5.0 Å around the natural
ligand of VEGFR-2/TIE-2/ EphB4 in the crystal complex
4.3. Human vascular endothelial cell (EA.hy926) viability
assay [21].
The viability of HUVEC (EA.hy926) was assessed using
the cell counting kit-8 (CCK-8, Sigma, USA)assay
according to the manufacturer's instruction. In brief, EA-
hy926 cells were harvested and plated in a 96-wellplate at
13

were selected as the active site. Other docking parameters
784-793.
ACCEPTED MANUSCRIPT
11. Y.L. Chiu, D.M. Carlson, R.S. Pradhan, J.L. Ricker,
were kept at default.
Exposure-response (safety) analysis to identify
linifanib dose for a Phase III study in patients with
hepatocellular carcinoma, Clin Ther. 35 (2013) 1770-
1777.
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (NSFC, Grant No. 81573285) and the
Fundamental Research Funds for the Central Universities
(2015qngz13).
12. J. Zhang, Y. Zhang, S. Zhang, S. Wang, L. He,
Discovery of novel taspine derivatives as
antiangiogenic agents, Bioorg Med Chem Lett. 20
(2010) 718-721.
13. J. Zhang, Y. Zhang, Y. Shan, N. Li, W. Ma, L. He,
Synthesis and preliminary biological evaluation of
novel taspine derivatives as anticancer agents, Eur J
Med Chem. 45 (2010) 2798-2805.
14. C. Wang, H. Gao, J. Dong, Y. Zhang, P. Su, Y. Shi, J.
Zhang, Biphenyl derivatives incorporating urea unit
as novel VEGFR-2 inhibitors: design, synthesis and
biological evaluation, Bioorg Med Chem. 22 (2014)
277-284.
15. H. Gao, P. Su, Y. Shi, X. Shen, Y. Zhang, J. Dong, J.
Zhang, Discovery of novel VEGFR-2 inhibitors. Part
II: biphenyl urea incorporated with salicylaldoxime,
Eur J Med Chem. 90 (2015) 232-240.
16. W. Lu, P. Li, Y. Shan, P. Su, J. Wang, Y. Shi, J. Zhang,
Discovery of biphenyl-based VEGFR-2 inhibitors.
Part 3: design, synthesis and 3D-QSAR studies,
Bioorg Med Chem. 23 (2015) 1044-1054.
17. P. Su, J. Wang, Y. Shi, X. Pan, R. Shao, J. Zhang,
Discovery of biphenyl-aryl ureas as novel VEGFR-2
inhibitors. Part 4: exploration of diverse hinge-
binding fragments, Bioorg Med Chem. 23 (2015)
3228-3236.
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14

ACCEPTED MANUSCRIPT
Table 1. Structures and RTKs inhibitory activities of title compounds (CPMA1-11)
(IC50, nM)
H
H
R1
R2
N
N
O
O
N
NH2
N
Compound
CPMA1
CPMA2
CPMA3
CPMA4
CPMA5
CPMA6
CPMA7
CPMA8
CPMA9
CPMA10
R1
3-CF3
2-OCF3
2- Br
2-OCF3
2-Br
R2
5-Br
4- Br
VEGFR-2
40.37
>500
Tie-2
65.26
>500
>500
>500
14.65
54.90
14.49
>500
21.20
>500
EphB4
78.92
>500
>500
>500
26.43
160.10
37.78
>500
>500
>500
4-OCF3
5-Br
>500
>500
5-OCF3
4-Cl
125.32
108.08
21.37
>500
2-Cl
3-F
4-F
3-Cl
4-Cl
3-CF3
3-F
5-CF3
>500
>500
4-
CPMA11
Sorafinib
>500
0.85
432.35
6.77
>500
4.46

ACCEPTED MANUSCRIPT
Table 2. Structures and RTKs inhibitory activities of title compounds (VDAU1-11)
(IC50, nM)
R1
R2
Compound
VDAU1
VDAU2
VDAU3
VDAU4
VDAU5
VDAU6
VDAU7
VDAU8
VDAU9
VDAU10
VDAU11
Soranifib
VEGFR-2
>500
2.10
Tie-2
21.27
9.19
EphB4
24.92
23.55
>500
29.28
>500
NDa
H
H
4-C(CH3)3
3-CH(CH3)2
3-CF3
4-Cl
3-Cl
H
>500
>500
>500
7.97
9.77
4-CH3
3-F
14.20
90.86
105.07
3.59
H
4-F
H
3-CF3
325.82
>500
>500
>500
2.35
>500
ND
H
2-Cl
7.97
H
4-OCF3
6-CH3
3-Cl
59.93
>500
5.63
ND
2-CH3
H
133.73
3.87
0.85
6.77
4.46
aND = Not Determined.

ACCEPTED MANUSCRIPT
Table 3. Structures and RTKs inhibitory activities of title compounds (QZAU1-5)
(IC50, nM)
R1
R2
Compound
QZAU1
QZAU2
QZAU3
QZAU4
QZAU5
Soranifib
VEGFR-2
>500
Tie-2
>500
245.28
12.81
16.55
24.75
6.77
EphB4
491.93
>500
>500
38.52
47.96
4.46
3-CF3
5-CF3
3-CF3
4-F
4-Cl
3-CF3
>500
H
3-F
H
>500
28.95
34.68
0.85
3-Cl

ACCEPTED MANUSCRIPT
Table 4. RTKs selectivity profile of VDAU11 (IC50, nM)
VEGFR EGFR
-3
IGF1-R
40
VEGFR
-1
PDGFR
Compound
VDAU11
FGFR-1 FGFR-4
200 1076
B-Raf c-Kit c-Met
32 68.9 57.8
-β
45
8.7
25
180

ACCEPTED MANUSCRIPT
Table 5. Inhibition of compounds on human vascular endothelial cell viability (IC50,
µM)
Compound
CPMA1
CPMA2
CPMA3
CPMA4
CPMA5
CPMA6
CPMA7
CPMA8
CPMA9
EA.hy 926
15.68
28.99
76.72
42.44
5.76
Compound
CPMA10
CPMA11
VDAU1
VDAU2
VDAU3
VDAU4
VDAU5
VDAU6
VDAU7
EA. hy926
32.21
Compound
VDAU8
VDAU9
VDAU11
QZAU1
QZAU2
QZAU3
QZAU4
QZAU5
Sorafenib
EA. hy926
72.31
29.20
83.59
23.33
3.53
127.23
30.27
78.78
141.30
197.44
53.10
11.22
4.42
113.86
67.42
96.55
112.13
57.53
22.99
125.96
24.36

ACCEPTED MANUSCRIPT
Figure 1. Multi-target RTK inhibitors currently approved or in clinical trials.

ACCEPTED MANUSCRIPT
Figure 2. Protein structure alignment of three angiogenic RTKs to each other. VEGFR-2 (Red),
EphB4 (Cyan), and TIE-2 (Green).

ACCEPTED MANUSCRIPT
O
H
N
O
Hydrophobic pocket
Gatekeeper
DFG-Motif (Asp)
COO-
O
Ar
Ar
N
N
HN
HN
H
H
O
HN
O
N
H
O
O
O
O
N
R
N
H
O
N
H2N
N
N
O
N
O
H
N
N
H
N
H
H
O
H2N
N
N
O
H
O
O
X
O
O
O
Conserved Glu
R
HN
H2N
N
N
Selective site
H
N
Hinge region
Structural optimization of lead compound BPS-7
Figure 3. Design strategy and structures of novel anti-angiogenesis agents derived from BPS-7.

ACCEPTED MANUSCRIPT
Figure 4. The selectivity of inhibitory activity of VDAU11 against various RTKs.

ACCEPTED MANUSCRIPT
Figure 5. Inhibition of VDAU11 (Red) on the growth of nine cancer cell lines
compared with sorafenib (Blue).

ACCEPTED MANUSCRIPT
Figure 6. Docked molecule (VDAU11) and residues within 5Å in the crystal structure
of VEGFR-2 (PDB ID: 4ASD).

ACCEPTED MANUSCRIPT
Figure 7. Docked molecule (VDAU11) and residues within 5Å in the crystal structure
of Tie-2 (PDB ID: 2P4I).

ACCEPTED MANUSCRIPT
Figure 8. Docked molecule (VDAU11) and residues within 5Å in the crystal structure
of EphB4 (PDB ID: 2X9F).

ACCEPTED MANUSCRIPT
Scheme 1. Synthetic scheme of cyclopropyl-malonic amide derivatives (CPMA1-11)
Reagents and conditions: (a) guanidine carbonate, DMA, 140°C; (b) Pd(PPh3)4, Na2CO3, H2O,
dioxane; (c) SOCl2, Et3N, DCM; (d) HATU, Et3N, DCM, 0°C to rt.