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

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 Li^a,†, Yuanyuan Shan^b,†, Ying Sun^a, Ru Si^a, Liyuan Liang^a, Xiaoyan Pan^a, Binghe

Wang^c, Jie Zhang^a,

^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

N

HN

H

O

HN

O

N

H

O

N

R

N

H

O

N

H₂N

N

O

N

O

H

N

H

N

H

O

H₂N

N

O

H

O

X

O

Conserved Glu

R

HN

H₂N

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 Li^a,†, Yuanyuan Shan^b,†, Ying Sun^a, Ru Si^a, Liyuan Liang^a, Xiaoyan Pan^a, Binghe Wang^c, Jie Zhang^a,

^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.

.

———

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.

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.

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 hydro}A^geC^{n b}C^oE^ndP^dT^oE^noD^rsMANUSCRIPT

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 CH₂Cl₂to 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).

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-

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-Glo^TMassay.

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 IC₅₀values 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 IC₅₀values 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.

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 IC₅₀values

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.

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 IC₅₀values 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.

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 IC₅₀values 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

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 IC₅₀value 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.

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 IC₅₀values 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.

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.

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 temper}A^atuC^reC^aE^ndPT^stiE^rrD^edMANUSCRIPT

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,

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

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), 9.17}A^(sC^{, 1}C^HE^),P^8.T⁰⁹E⁽D^d,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

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 CH₂Cl₂was 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, 10}A^.2C^HC^z,E^1HP⁾T^,E^6.D³⁴MANUSCRIPT

(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

CH₂Cl₂was 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 1×10⁵cells for each well and cultured in

^{Hz, 1H), 7.43 (s, 1H), 7.31 (s, 1H), 7.30}A^(sC^,C^1HE⁾P^{, 7}T^.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, MgCl₂10 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

IC₅₀values 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

R₁

R₂

N

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

14.65

54.90

14.49

>500

21.20

>500

EphB4

78.92

>500

26.43

160.10

37.78

>500

4-OCF₃

5-Br

>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

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

ND^a

H

4-C(CH₃)₃

3-CH(CH₃)₂

3-CF₃

4-Cl

3-Cl

H

>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

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

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

N

HN

H

O

HN

O

N

H

O

N

R

N

H

O

N

H₂N

N

O

N

O

H

N

H

N

H

O

H₂N

N

O

H

O

X

O

Conserved Glu

R

HN

H₂N

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.

Article Doi

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

DOI: 10.1016/j.ejmech.2017.10.030

Source and publish data:

Authors:

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

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

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