Discovery of novel anti-angiogenesis agents. Part 6: Multi-targeted RTK inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.12.059

Angiogenesis is modulated by a multitude of pro-angiogenic factors including VEGFR-2, Tie-2, and EphB4. Moreover, their crosstalk also had been well elaborated. We have identified several diarylurea-based VEGFR-2 inhibitors as potential anti-angiogenesis agents. As a continuation to our previous research, two series of diaryl malonamide and diaryl thiourea derivatives have been developed as multiplex VEGFR-2/Tie-2/EphB4 inhibitors. Interestingly, the biological evaluation indicated that several compounds bearing trifluoromethyl or trifluoromethoxyl exhibited promising multiplex inhibition against angiogenesis-related VEGFR-2, Tie-2, and EphB4. The representative compound (18a) displayed both potent multi-targeted RTK inhibition and considerable antiproliferative activities against human umbilical vein endothelial cells (EA.hy926). These results will contribute to the discovery of novel muti-targeted anti-angiogenesis agents.

Full text of DOI:10.1016/j.ejmech.2016.12.059

European Journal of Medicinal Chemistry 127 (2017) 275e285
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Discovery of novel anti-angiogenesis agents. Part 6: Multi-targeted
RTK inhibitors
,
,
Lin Zhang a 1, Yuanyuan Shan b 1, Chuansheng Li a, Ying Sun a, Ping Su a, Jinfeng Wang a,
Lisha Li a, Xiaoyan Pan a, Jie Zhang a
,
*
a School of Pharmacy, Health Science Center, Xi'an Jiaotong University, No. 76, Yanta West Road, Xi'an, Shaanxi Province, 710061, PR China
b Department of Pharmacy, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi Province, 710061, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Angiogenesis is modulated by a multitude of pro-angiogenic factors including VEGFR-2, Tie-2, and
EphB4. Moreover, their crosstalk also had been well elaborated. We have identified several diarylurea-
based VEGFR-2 inhibitors as potential anti-angiogenesis agents. As a continuation to our previous
research, two series of diaryl malonamide and diaryl thiourea derivatives have been developed as
multiplex VEGFR-2/Tie-2/EphB4 inhibitors. Interestingly, the biological evaluation indicated that several
compounds bearing trifluoromethyl or trifluoromethoxyl exhibited promising multiplex inhibition
against angiogenesis-related VEGFR-2, Tie-2, and EphB4. The representative compound (18a) displayed
both potent multi-targeted RTK inhibition and considerable antiproliferative activities against human
umbilical vein endothelial cells (EA.hy926). These results will contribute to the discovery of novel muti-
targeted anti-angiogenesis agents.
Received 26 October 2016
Received in revised form
29 December 2016
Accepted 30 December 2016
Available online 2 January 2017
Keywords:
Anti-angiogenesis agents
Multi-targeted
RTK inhibitors
© 2017 Elsevier Masson SAS. All rights reserved.
Diaryl malonamide
Diaryl thiourea
1. Introduction
studies have indicated that these three RTKs play an essential role
in both vasculogenesis and angiogenesis [3]. VEGF is one of the
Angiogenesis plays an essential role in a number of physiological
and pathological processes, including embryonic development,
wound healing, tumor growth, and certain ocular diseases. Under
normal physiological conditions, angiogenesis is strictly modulated
by a multitude of pro-angiogenic and anti-angiogenic factors [1]. In
tumor growth, to acquire and sustain microvascular network,
cancer cells secrete many growth factors which could induce
angiogenesis and promote tumor progression. Inhibition of angio-
genesis is a promising and clinically validated approach for treat-
ment of cancer. Angiogenesis is primarily mediated by growth
factors that cause signal transduction, for the most part, via re-
ceptor tyrosine kinases (RTKs). Several angiogenic ligands and their
corresponding RTKs play critical roles in angiogenesis. Moreover,
the crosstalk between various RTKs had also been well elaborated
[2]. Three subfamilies out of at least 14 RTK subfamilies, VEGF/
VEGFR-2, Ang/Tie-2, and Ephrin B2/EphB4 are highly expressed in
the endothelial cells (ECs) from embryonic to adulthood. Extensive
most potent angiogenic growth factors, and its binding to VEGFR-2
induces signal transduction that promotes ECs survival, prolifera-
tion, and migration. Angiogenic growth factors (Ang) and their
tyrosine kinase receptor (Tie-2) have been implicated in vessel
stabilization, maturation, and remodeling of vasculature [4]. Recent
reports indicated that Ephrin receptor tyrosine kinase (EphB4) and
its transmembrane-type Ephrin B2 ligand are crucial for vascular
development [5]. Therefore, these three RTKs, VEGFR-2, Tie-2, and
EphB4, are considered to be promising targets for design and dis-
covery of novel anti-angiogenesis agents.
In our previous research, we have focused on the discovery of
novel VEGFR-2 inhibitors as anti-angiogenesis agents. Numbers of
biphenyl-aryl ureas incorporated with salicylaldoxime have been
developed as potent and selective VEGFR-2 inhibitor through
structural optimization of Taspine (Fig. 1) [6e9]. Several nanomolar
VEGFR-2 inhibitors with potential anti-angiogenesis activities have
been identified [10e12]. These biphenyl-aryl ureas exhibited
significantly VEGFR-2 inhibitory activity as well as anticancer po-
tency. Among them, BPS-7 was found to possess a reasonable anti-
angiogenic potency. Further evaluation indicated that BPS-7 could
inhibit phosphorylation of VEGFR-2 and the formation of blood
vessels. Moreover, it also significantly inhibited the proliferation
* Corresponding author.
E-mail address: zhj8623@xjtu.edu.cn (J. Zhang).
Both authors contributed equally to this work.
1
http://dx.doi.org/10.1016/j.ejmech.2016.12.059
0223-5234/© 2017 Elsevier Masson SAS. All rights reserved.

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L. Zhang et al. / European Journal of Medicinal Chemistry 127 (2017) 275e285
Fig. 1. Previous research on the discovery of biphenyl-aryl urea as novel VEGFR-2 inhibitors.
and migration of human umbilical vein endothelial cells [13].
Therefore, diaryl urea (BPS-7) could be considered as a promising
lead compound for further optimized as novel anti-angiogenesis
agents.
Angiogenesis is a complex process which are highly related with
VEGFR-2, Tie-2, and EphB4. These three RTKs could stimulate
endothelial proliferation in angiogenesis. Each of these RTKs has
been considered as valid target of anti-angiogenesis agents [14].
This realization and the drug resistance of single target drugs have
led researchers to speculate compounds which simultaneously
have been yielded by using Pd-catalyzed Suzuki coupling reaction
as key step. The preparation of title compounds (10a~10n) started
with the synthesis of aminoindazole (2) from commercially avail-
able 2-fluoro-6-iodo-benzonitrile (1) by using five equiv of hydra-
zine monohydrate in refluxing ethanol under the presence of
NaHCO3 [15]. Subsequently, aminoindazole (2) and 4-
aminophenylboronic acid (3) or 3-aminophenylboronic acid (4)
were coupled by classical Pd-catalyzed Suzuki coupling reaction
affording intermediates (5) and (6). Condensation of various aniline
(8a~8j) with 1,1-cyclopropanedicarboxylic acid afforded (9a~9j).
Finally, treatment of (5) or (6) with various intermediates (9a~9j) in
the presence of triethylamine and 1-[Bis(dimethylamino)methy-
inhibit multiple RTKs (multi-targeted inhibitors) might be
a
promising strategy. Encouraged by previous results, our further
efforts focused on the discovery of novel anti-angiogenesis agents
with multiplex inhibition profile. First of all, the urea moiety in lead
compound was replaced with malonamide bearing more
hydrogen-bond donors and acceptors. The two methoxyl groups on
biphenyl have been removed in order to reduce the steric hindrance
of inhibitors when binding with receptors. Meanwhile, re-
placements of salicylaldoxime by 1H-indazol-3-amine and quina-
zolin-2-amine were carried out to enhance the affinity of inhibitors
with hinge region of RTKs. These heterocyclic amines bearing more
hydrogen bond acceptors could retain the essential hydrogen
bonds with hinge region. Moreover, various diverse substituents
such as halogen, trifluoromethyl, trifluoromethoxyl and methyl-
enedioxy were incorporated to terminal aniline. The design strat-
egy and structures of the title compounds were represented in
Fig. 2.
In our continuous efforts on the discovery of novel anti-
angiogenesis agents, we designed and synthesized fourteen diaryl
malonamides and ten diaryl thioureas as multi-targeted VEGFR-2/
Tie-2/EphB4 inhibitors via structural optimization of previously
reported VEGFR-2 inhibitors. Biological evaluations including
enzymatic inhibition and antiproliferative potency were performed
to yield novel anti-angiogenesis agents with multiplex inhibition
profile. Molecular modeling studies were also carried out to ratio-
nalize the efficiency of the potent inhibitors. These derivatives
exhibited potent anti-angiogenesis activity and could be consid-
ered as promising lead compounds for further development of
novel anti-angiogenesis agents.
lene]-1H-1,2,3-triazolo
[4,5-b]pyridinium
3-oxide
hexa-
fluorophosphate (HATU) afforded the title compounds (10a~10n)
[16] (see Scheme 1).
Diaryl thiourea (18a~18j) were readily prepared using the
following synthetic route (Scheme 2). Commercially available
aldehyde (11) was converted to 6-bromoaminoquinazoline (12) by
treatment with guanidine carbonate heating in N,N-dimethylace-
tamide (DMA). Intermediate (12) was subsequently converted to
(14) through Suzuki coupling with 4-aminophenylboronic acid (13)
[17]. Various anilines were treated with CS2 to yield (16a~16j), and
then reacted with triphosgene (BTC) to produce various iso-
thiocyanates (17a~17j) [18]. Finally, these isothiocyanates were
treated with intermediate (14) in DCM to afford the second series of
title compounds (18a~18j).
All of the title compounds were characterized by 1H-NMR, 13C-
NMR, mass spectroscopy and melting point. 1H-NMR, 13C-NMR,
mass spectroscopy, and melting point of all the title compounds are
described in Supplementary Material.
2.2. RTK inhibitory activity
Enzymatic inhibition evaluation of title compounds against
VEGFR-2, Tie-2, and EphB4 were performed using ADP-Glo Kinase
Assay Kit (Promega, Wisconsin, USA). The biological evaluation
results were depicted in Table 1 and Table 2 with Sorafenib as
positive control. The majority of the title compounds exhibited
moderate to good inhibitory potency. As shown in Table 1,
replacement of urea with malonamide yielded several potent
multi-targeted compounds with multiplex inhibition profile. Four
compounds (10a, 10b, 10g, and 10i) of them exhibited promising
activity with nanomolar IC50 values less than 50 nM. Compound
(10g) displayed comparable RTK inhibitory potency to Sorafenib
with IC50 values less than 25 nM. It also showed the most potent
2. Results and discussion
2.1. Synthesis of title compounds
Two series of diaryl malonamide and diaryl thiourea derivatives

L. Zhang et al. / European Journal of Medicinal Chemistry 127 (2017) 275e285
277
Fig. 2. Design strategies and structures of multi-targeted anti-angiogenesis agents.
Scheme 1. Synthetic route of the title compounds (10a~10j).
Reagents and conditions: (a) EtOH, NaHCO3, NH2NH2, H2O; (b) Pd(PPh3)4, Na2CO3, H2O, dioxane; (c) SOCl2, Et3N, DCM; (d) HATU, Et3N, DCM, 0 ꢀC to r.t.
inhibition against EphB4 with IC50 value of 0.13 nM. Compound
(10i) displayed the highest VEGFR-2 inhibitory activity with IC50
value of 1.35 nM while compound (10k) was the most potent Tie-2
inhibitor with IC50 value of 1.95 nM.
As observed in Table 2, the majority of diaryl thioureas displayed
potent and selective inhibition against Tie-2. Interestingly, four
compounds (18a, 18b, 18c, and 18d) exhibited promising multiplex
inhibitory activity against VEGFR-2, Tie-2, and EphB4. These com-
pounds bearing with trifluoromethoxy displayed more potent RTK
inhibitory activity which suggested that trifluoromethoxy might be
beneficial for RTK inhibition. The most potent compound (18a)
inhibited VEGFR-2, Tie-2, and EphB4 with IC50 values of 1.05 nM,
2.47 nM, and 0.27 nM, respectively. These results suggested that
aminoquinazoline could be considered as a novel and useful
pharmacophore in the design of multi-targeted RTK inhibitors. The
biological results indicated that these diaryl thioueas could be
considered as the first class of multiplex inhibitors of VEGFR-2/TIE-
2/EphB4 with a “triplet” inhibition profile.
2.3. Cell growth inhibition
On the basis of RTK inhibition assay, several potent compounds

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L. Zhang et al. / European Journal of Medicinal Chemistry 127 (2017) 275e285
Scheme 2. Synthetic route of the title compounds (18a~18j).
Reagents and conditions: (a) guanidine carbonate, DMA, 140 ꢀC; (b) Pd(PPh3)4, Na2CO3, H2O, dioxane; (c) Dabco, CS2, toluene; (d) BTC, DCM; (e) DCM, r.t.
Table 2
Table 1
Structures and RTK inhibitory activities of title compounds (18a-18j) (IC50, nM).
Structures and RTK inhibitory activities of title compounds (10a-10n) (IC50, nM).
Compound
R1
R2
VEGFR-2
TIE-2
EphB4
Compound
R1
R2
VEGFR-2
TIE-2
EphB4
18a
18b
18c
18d
18e
18f
18g
18h
18i
3-CF3
2-OCF3
2-Br
2-OCF3
2-Br
2-Cl
3-F
3-Cl
3-CF3
5-Br
4-Br
4-OCF3
5-Br
5-OCF3
4-Cl
4-F
4-Cl
5-CF3
1.05
1.42
0.31
11.41
33.02
>1000
>1000
>1000
>1000
>1000
2.47
15.68
9.68
0.27
ND
10a
10b
10c
10d
10e
10f
10g
10h
10i
3-CF3
2-OCF3
2- Br
2-OCF3
2-Br
2-Cl
3-F
3-Cl
3-CF3
5-Br
4-Br
4-OCF3
5-Br
5-OCF3
4-Cl
4-F
4-Cl
5-CF3
34.67
5.58
>1000
35.75
ND
7.80
1.40
700.53
1.35
>1000
13.22
134.12
>1000
6.05
904.52
>1000
23.78
3.49
9.22
4.53
3.60
ND
787.89
383.59
0.13
170.11
38.14
1.26
48.53
92.59
>1000
>1000
>1000
179.60
ND
0.60
87.38
164.35
52.04
1.14
0.16
0.13
49.97
6.72
18j
ND
10j
Sorafinib
0.45
0.83
0.20
10k
10l
10m
10n
Sorafinib
3-CF3
2-OCF3
2-Cl
5-Br
5-Br
4-Cl
4-Cl
>1000
32.37
>1000
ND
1.95
6.34
>1000
>1000
0.83
613.00
>1000
>1000
>1000
0.20
ND ¼ Not Determined.
3-Cl
0.45
positive control.
As depicted in Table 3, it was found that five of selected com-
pounds (10d, 10j, 18a, 18c and 18i) displayed potent anti-
proliferative activities with IC50 values ranging from 2.68 to
ND ¼ Not Determined.
were screened for their antiproliferative potency against human
umbilical vein endothelial cells (EA.hy926) in vitro. Cell prolifera-
tion was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) method with Sorafenib as
7.56
Sorafenib. Particularly, 18i showed the highest antiproliferative
activity with IC50 values of 2.68 M. The antiproliferative activity
was mostly in accordance with their enzymatic inhibition against
mM. Their potency were comparable with the reference drug
m

L. Zhang et al. / European Journal of Medicinal Chemistry 127 (2017) 275e285
279
Table 3
distance was 2.02 Å; 2) the second forming between nitrogen atom
of aminoquinazoline and NH2 of Ala 905 with the distance of 2.19 Å;
3) the third was observed between trifluoromethyl of (18a) and
NH2 of Arg 987 with the distance of 1.90 Å, which suggested that
trifluoromethyl might be an important element. Therefore, these
results indicated that aminoquinazoline could be considered as a
favorable hinge-binding fragment of Tie-2. Moreover, tri-
fluoromethyl was identified as a critical structural requirement. The
binding mode of (18a) with EphB4 was similar with that of native
ligand (Fig. 5). Nitrogen of aminoquinazoline forms a hydrogen
bond with Thr 693 with distance of 2.13 Å. In summary, molecular
docking results indicated that compound (18a) bound in a similar
fashion with native ligands of three RTK complexes. The molecular
modeling results indicated that compound (18a) occupied the same
region in the binding sites with native ligand, which clarified the
rationality of the design strategy of multi-targeted RTK inhibitors as
anti-angiogenesis agents.
Antiproliferative activities of title compounds against human umbilical vein endo-
thelial cells (IC50
, mM).
Compound
Ea.HY926
Compound
Ea.HY926
Sorafinib
10a
10b
10d
10g
24.36
17.40
30.22
4.88
42.11
13.86
230.53
10j
18a
18b
18c
18d
18i
18j
4.37
7.56
25.86
6.58
336.28
2.68
ND
10h
10i
VEGFR-2, Tie-2, or EphB4. In summary, compound (18a) was a
promising potent anti-angiogenesis agent and worth of further
investigation.
2.4. Molecular docking study
In order to investigate the interactions between inhibitors with
corresponding RTKs, docking studies were performed using
Surflex-Dock Module of Sybyl-X (Version 2.0, Tripos Inc. St. Louis,
MO). The most potent compound (18a) was drawn with Sketch
module and minimized under Tripos Force field. The ligand of
protein-ligand complex was used to define the binding cavity and
generate the protomol [19].
The most reasonable binding configuration of (18a) with three
RTKs were carried out and depicted in Fig. 3, Fig. 4, and Fig. 5. It was
nicely bound to VEGFR-2 and presented the similar binding
conformation with Sorafenib which was displayed as purple
(Fig. 3). The two nitrogen atoms of thiourea unit formed two
hydrogen bonds with Asp 1046 with distance of 2.38 Å and 1.96 Å,
respectively. Favorable binding interactions of (18a) with the active
site of Tie-2 including three hydrogen bonds (Fig. 4): 1) the first
forming between NH2 of aminoquinazoline and C¼O of Ala 905, the
3. Conclusion
Multiplex inhibition of angiogenesis-related RTK pathways has
provided a promising strategy in the discovery of novel anti-
angiogenesis agents [20e23]. Herein, we have identified several
multi-targeted inhibitors of VEGFR-2/EphB4/TIE-2 with a “triplet”
inhibition profile. They might be considered as potent anti-
angiogenesis agents which might be useful to prevent the resis-
tance of single-target drugs.
The biological evaluation indicated that several title compounds
bearing trifluoromethyl or trifluoromethoxyl exhibited promising
inhibitory activity against three angiogenesis-related RTKs and
EA.hy926 proliferation. In particular, compound (18a) exhibited the
most potent inhibition against VEGFR-2, Tie-2, and EphB4 with IC50
values of 1.05 nM, 2.47 nM, and 0.27 nM, respectively. In addition to
their enzymatic activities, five compounds of them displayed
Fig. 3. Superimposition of the docking poses for VEGFR-2 with compound (18a) and Sorafenib (purple). Hydrogen bonds were depicted in dashed yellow lines. (For interpretation of
the references to colour in this figure legend, the reader is referred to the web version of this article.)

280
L. Zhang et al. / European Journal of Medicinal Chemistry 127 (2017) 275e285
Fig. 4. Superimposition of the docking poses for Tie-2 with compound (18a) and 2-(Pyridin-2-yl)-1,3,5-triazine (purple). Hydrogen bonds were depicted in dashed yellow lines. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 5. Superimposition of the docking poses for EphB4 with compound (18a) and bis-anilinopyrimidine (purple). Hydrogen bonds were depicted in dashed yellow lines. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
potent antiproliferative activity at micromolar concentrations
against human umbilical vein endothelial cells (EA.hy926). Mo-
lecular docking results suggested that these novel multi-targeted
inhibitors shared the similar binding conformation with native li-
gands of complexes. In conclusion, we disclosed our progress on
identifying novel multi-targeted inhibitors. They displayed effec-
tive antiproliferative and antiangiogenic activities by targeting
multiplex RTKs. Our results identified the rationality of design
strategies of multi-targeted VEGFR-2, Tie-2, and EphB4 inhibitors as
novel anti-angiogenesis agents. Among them, compound (18a)

L. Zhang et al. / European Journal of Medicinal Chemistry 127 (2017) 275e285
281
could be considered as a promising starting point for further
4.1.4. N-[4-(3-amino-1H-indazol-4-yl)phenyl]-N-[3-bromo-5-
optimization. Our findings may contribute to the discovery of novel
anti-angiogenesis agents for the intervention of pathological
angiogenesis-related diseases. Further biological evaluation and
structural optimization of these promising anti-angiogenesis
agents are currently under investigation and will be reported in
due course.
(trifluoromethyl)phenyl]cyclopropane-1,1-dicarboxamide (10a)
4-(4-aminophenyl)-1H-indazol-3-amine (5) (0.38 g, 0.7 mmol),
intermediate (9a) was dissolved in anhydrous CH2Cl2 on the ice
bath and HATU (0.48 g, 1.26 mmol) was added. The mixture was
stirred on the ice-bath for 30 min, then 0.1 mL anhydrous trie-
thylamine in anhydrous CH2Cl2 was added dropwise to the above
mixture. Subsequently, the ice bath was removed, and the mixture
was reacted at room temperature for 8 h. The product was
extracted with CH2Cl2 (30 mL ꢁ 3), washed with water and brine
(100 mL ꢁ 3), and dried over Na2SO4. After filtration and concen-
tration in vacuo, the residues was purified by silica gel flash chro-
matogrphy gave (10a) (0.12 g, 20%). m.p. ¼ 186e188 ꢀC, TLC
(CHCl2:MeOH, 80:20 v/v): Rf ¼ 0.35, MS (ESI) [MþH]þ: m/
4. Experimental section
4.1. Chemistry:General procedure
The reactions except those in aqueous media are carried out by
standard techniques for the exclusion of moisture. Reaction prog-
ress was monitored by thin layer chromatography (TLC) on 0.25-
mm silica gel plates (60 GF-254) and visualized with UV light.
Column chromatography was performed with silica gel. Melting
points (mp.) are determined on electrothermal melting point
apparatus and are uncorrected. 13C-NMR and 1H-NMR spectra are
measured at 400 MHz on a Bruker Advance AC 400 instrument with
TMS as an internal standard. Mass spectra are obtained on a Shi-
madzu HPLC-MS-QP2010 instrument.
z ¼ 558.35. 1H NMR (400 MHz, CDCl3)
8.32 (d, J ¼ 8.3 Hz, 2H), 7.55
d
(m, 1H, Ar-H), 7.33e7.14 (m, 4H, Ar-H), 7.11 (d, J ¼ 8.5 Hz, 1H, Ar-H),
6.85 (m, 2H, Ar-H), 4.54 (d, J ¼ 4.9 Hz, 2H), 1.73e1.58 (m, 4H). 13
C
NMR (101 MHz, CDCl3)
d 172.93, 171.05, 157.21, 151.17, 144.64,
142.25, 141.19, 135.75, 133.97, 133.75, 131.54, 131.25, 130.21, 129.98,
125.81, 124.20, 120.96, 119.16, 118.03, 36.99, 19.21.
Title compounds 10b~10j were prepared by using the general
procedure described above.
4.1.5. N-[4-(3-amino-1H-indazol-4-yl)phenyl]-N-[4-bromo-2-
(trifluoromethoxy)phenyl]cyclopropane-1,1-dicarboxamide (10b)
m.p. ¼ 96e98 ꢀC, TLC (CHCl2:MeOH, 80:20 v/v): Rf ¼ 0.30, MS
4.1.1. 4-Iodine -1H-indazol-3-ylamine (2)
2-Fluoro-6-iodobenzonitrile was dissolved in ethanol (70 mL),
then hydrazine hydrate (12.5 mL), NaHCO3 (5.2 g) was added. The
resulting mixture was heated to reflux and stirred for 8 h. After
cooling to room temperature, 50 mL water was added and the re-
action mixture was allowed to stir for another 2 h at room tem-
perature. The product was collected by filtration and dried under
vacuum to afford 4-iodine -1H-indazol-3-ylamine (2) as slight
yellow solid (8 g, 77%).
(ESI) [MþH]þ: m/z ¼ 574.30. 1H NMR (400 MHz, CDCl3)
d 8.58 (s,
1H), 8.38 (d, J ¼ 8.3 Hz, 1H), 8.27 (d, J ¼ 8.9 Hz, 1H), 7.57 (m,1H, Ar-
H), 7.39 (m,1H, Ar-H), 7.24 (d, J ¼ 8.1 Hz, 2H, Ar-H), 7.19 (d, J ¼ 7.3 Hz,
1H, Ar-H), 6.81 (d, J ¼ 8.1 Hz, 2H, Ar-H), 4.32 (s, 2H), 1.72 (s, 4H). 13
C
NMR (101 MHz, CDCl3)
d 167.94, 167.19, 152.81, 146.76, 140.95,
138.28, 136.87, 130.55, 130.27, 130.24, 130.01, 127.61, 126.09, 123.52,
123.24, 116.87, 115.54, 115.06, 114.56, 33.33, 16.04.
4.1.6. N-[4-(3-amino-1H-indazol-4-yl)phenyl]-N-[2-bromo-4-
(trifluoromethoxy)phenyl]cyclopropane-1,1-dicarboxamide(10c)
m.p. ¼ 162e164 ꢀC, TLC (CHCl2:MeOH, 80:20 v/v): Rf ¼ 0.28, MS
4.1.2. 4-(4-minophenyl)-1H-indazol-3-amine (5)
Pd(PPh3)4 (3.3 g, 2.89 mmol) was added to a degassed solution
of 4-aminophenylboronic acid (5 g, 28.9 mmol), K2CO3 (9.2 g,
86.7 mmol), 4-iodine -1H-indazol-3-ylamine (2) (7.5 g, 28.9 mmol)
in 150 mL 1,4- dioxane and 50 mL water.The reaction mixture was
heated at 90 ꢀ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 for overnight, filtered, and concentrated in
vacuo to give the crude product, which was isolated by silica gel
flash chromatography (PE/AcOEt ¼ 3:1)to obtain the title com-
pound 3.2 g in 45% yield.
(ESI) [MþH]þ: m/z ¼ 574.35. 1H NMR (400 MHz, CDCl3)
d 8.40 (d,
J ¼ 8.2 Hz,1H), 8.29 (d, J ¼ 9.1 Hz,1H), 8.24 (s, 1H), 7.60e7.54 (m,1H,
Ar-H), 7.35 (d, J ¼ 2.1 Hz, 1H, Ar-H), 7.25 (d, J ¼ 8.3 Hz, 2H, Ar-H),
7.21e7.19 (m, 2H, Ar-H), 6.81 (d, J ¼ 8.3 Hz, 2H), 4.34 (s, 2H),
1.75e1.71 (m, 2H), 1.71e1.67 (m, 2H). 13C NMR (101 MHz, CDCl3)
d
168.15, 167.04, 152.77, 146.66, 144.66, 140.95, 136.87, 134.98,
130.21, 130.06, 127.65, 126.06, 124.98, 122.81, 121.01, 117.03, 115.15,
114.45, 113.23, 33.42, 15.77.
4.1.7. N-[4-(3-amino-1H-indazol-4-yl)phenyl]-N-[5-bromo-2-
(trifluoromethoxy)phenyl]cyclopropane-1,1-dicarboxamide(10d)
m.p. ¼ 188e190 ꢀC, TLC (CHCl2:MeOH, 80:20 v/v): Rf ¼ 0.32, MS
4.1.3. 1-({[3-Bromo-5-(trifluoromethyl)phenyl]amino} carbonyl)
cyclopropanecarboxylic acid (9a)
(ESI) [MþH]þ: m/z ¼ 574.35. 1H NMR (400 MHz, CDCl3)
d 8.65 (s,
1,1-Cyclopropanedicarboxylic acid (2 g, 15.4 mmol) was dis-
solved in anhydrous CH2Cl2, anhydrous triethylamine (2.2 mL) was
added under nitrogen. The mixture was stirred on the ice-bath for
30 min. Thionyl chloride (1.2 mL) in anhydrous CH2Cl2 was added
dropwise to the above mixture and stirring for 2 h. 5-bromo-3-
(trifluoromethyl)-benzenamine (3.6 g, 15.4 mmol)dissolved in
anhydrous CH2Cl2 was added into the mixture. Stirring was
continued for 2 h and the solution was adjusted to pH 10 with
NaOH (2 mol/L), after filtration and concentration in vacuo, an
appropriate amount of water was added to the residues.The prod-
uct was extracted with ethyl acetate (50 mL), The organic layer was
separated and the aqueous layer was adjusted to pH 2 with HCl
(2 mol/L). The product was extracted with ethyl acetate (50 mL ꢁ 3)
again. The combined organic layer was concentrated in vacuo, total
yielding 1-[[(3,5-dimethylphenyl)amino]carbonyl] (9) (0.8 g, 37%).
1H), 8.60 (d, J ¼ 2.2 Hz, 1H), 8.37 (d, J ¼ 8.3 Hz, 1H), 7.58e7.52 (m,
1H, Ar-H), 7.23 (d, J ¼ 8.3 Hz, 2H, Ar-H), 7.19 (d, J ¼ 3.4 Hz, 1H, Ar-H),
7.04 (m, 1H, Ar-H), 6.79 (d, J ¼ 8.3 Hz, 2H, Ar-H), 4.35 (s, 2H), 1.73 (s,
4H). 13C NMR (101 MHz, CDCl3)
d 171.21, 168.02, 167.15, 152.85,
146.84, 140.94, 136.90, 132.22, 130.21, 130.00, 127.54, 126.73, 126.08,
124.87, 121.62, 120.72, 116.88, 115.04, 114.53, 33.27, 16.18.
4.1.8. N-[4-(3-amino-1H-indazol-4-yl)phenyl]-N-[2-bromo-5-
(trifluoromethoxy)phenyl]cyclopropane-1,1-dicarboxamide(10e)
m.p. ¼ 156e157 ꢀC, TLC (CHCl2:MeOH, 80:20 v/v): Rf ¼ 0.40, MS
(ESI) [MþH]þ: m/z ¼ 574.35. 1H NMR (400 MHz, CDCl3)
d 8.41 (d,
J ¼ 8.1 Hz, 1H), 8.34 (s, 1H), 8.31 (d, J ¼ 2.4 Hz,1H), 7.60e7.54 (m, 1H,
Ar-H), 7.45 (d, J ¼ 8.8 Hz,1H, Ar-H), 7.24 (d, J ¼ 8.4 Hz, 2H, Ar-H), 7.20
(d, J ¼ 7.1 Hz, 1H, Ar-H), 6.80 (d, J ¼ 8.4 Hz, 2H, Ar-H), 4.34 (s, 2H),
1.75 (m, 2H), 1.70 (m, 2H). 13C NMR (101 MHz, CDCl3)
d 168.20,

282
L. Zhang et al. / European Journal of Medicinal Chemistry 127 (2017) 275e285
166.94, 152.84, 148.73, 148.71, 146.82, 140.96, 137.18, 136.94, 132.74,
130.22, 130.04, 127.51, 126.08, 117.05, 115.08, 114.81, 114.43, 110.44,
33.48, 15.93.
heated at 90 ꢀ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 for overnight, filtered, and concentrated in
vacuo to give the crude product, which was isolated by silica gel
flash chromatography (PE/AcOEt ¼ 3:1) to obtain the title com-
pound 2.9 g with yield of 40%.
4.1.9. N-[4-(3-amino-1H-indazol-4-yl)phenyl]-N-(2,4-
dichlorophenyl)cyclopropane-1,1-dicarboxamide(10f)
m.p. ¼ 153e155 ꢀC, TLC (CHCl2:MeOH, 80:20 v/v): Rf ¼ 0.35, MS
(ESI) [MþH]þ: m/z ¼ 480.35. 1H NMR (400 MHz, CDCl3)
d 8.40 (s,
1H), 8.38 (s, 1H), 8.22 (d, J ¼ 8.9 Hz, 1H), 7.57 (m, 1H, Ar-H), 7.30 (d,
J ¼ 2.4 Hz, 1H, Ar-H), 7.27e7.24 (m, 2H, Ar-H), 7.21 (d, J ¼ 2.4 Hz, 1H,
Ar-H), 6.84e6.80 (m, 2H, Ar-H), 4.32 (s, 2H), 1.73 (m, 2H), 1.68 (m,
4.1.15. 1-[[(3,5-dimethylphenyl)amino]carbonyl] (9)
1,1-cyclopropanedicarboxylic acid (2 g, 15.4 mmol) was dis-
solved in anhydrous CH2Cl2 (15 mL), anhydrous triethylamine
(2.2 mL) was added under nitrogen atmosphere, the mixture was
stirred on the ice-bath for 30 min. Thionyl chloride (1.2 mL) in
anhydrous CH2Cl2 (10 mL) was added dropwise to the above
2H). 13C NMR (101 MHz, CDCl3)
d 167.96, 167.12, 152.71, 146.61,
140.95, 136.88, 133.70, 130.22, 130.07, 128.91, 128.63, 127.72, 127.68,
126.05, 123.52, 122.87, 116.91, 115.16, 114.50, 33.43, 15.75.
mixture and stirring continued for
2
h. 3-bromo-5-(tri-
4.1.10. N-[4-(3-amino-1H-indazol-4-yl)phenyl]-N-(3,4-
difluorophenyl)cyclopropane-1,1-dicarboxamide (10g)
m.p. ¼ 105e107 ꢀC, TLC (CHCl2:MeOH, 80:20 v/v): Rf ¼ 0.30, MS
fluoromethyl)-benzenamine (3.5 g, 13.9 mmol) dissolved in anhy-
drous CH2Cl2 was then added into the mixture. Stirring was
continued for 2 h and the solution was adjusted to pH 10 with
NaOH (2 mol/L), after filtration and concentration in vacuo, an
appropriate amount of water was added to the residues. The
product was extracted with ethyl acetate (50 mL). The organic layer
was separated and the aqueous layer was adjusted to pH 2 with HCl
(2 mol/L) The product was extracted with ethyl acetate (50 mL ꢁ 3)
The combined organic layer was concentrated in vacuo, total
yielding (9) (0.8 g, 35%).
(ESI) [MþH]þ: m/z ¼ 447.45. 1H NMR (400 MHz, DMSO)
d 9.86 (s,
1H), 8.25 (d, J ¼ 8.2 Hz, 1H), 7.69 (d, J ¼ 3.8 Hz, 1H, Ar-H), 7.56 (m,
1H, Ar-H), 7.29 (d, J ¼ 9.0 Hz, 1H, Ar-H), 7.26 (s, 1H, Ar-H), 7.11 (m,
3H, Ar-H), 6.70 (d, J ¼ 8.4 Hz, 2H), 5.13 (s, 2H), 1.51 (m, 2H), 1.45 (m,
2H). 13C NMR (101 MHz, DMSO)
d 168.12, 167.27, 152.31, 149.11,
140.73, 137.65, 136.87, 136.78, 130.00, 125.57, 125.04, 117.46, 117.29,
117.06, 116.90, 114.43, 113.98, 110.02, 109.81, 33.07, 14.60.
4.1.11. N-[4-(3-amino-1H-indazol-4-yl)phenyl]-N-(3,4-
dichlorophenyl)cyclopropane-1,1-dicarboxamide (10h)
m.p. ¼ 103e104 ꢀC, TLC (CHCl2:MeOH, 80:20 v/v): Rf ¼ 0.25, MS
4.1.16. N-[3-(3-amino-1H-indazol-4-yl)phenyl]-N-[3-bromo-5-
(trifluoromethyl)phenyl]cyclopropane-1,1-dicarboxamide (10k)
Compound (6) (0.38 g, 0.7 mmol), 1-[[(3,5-dimethyl phenyl)
amino]carbonyl] (0.34 g, 0.92 mmol) was dissolved in anhydrous
CH2Cl2 (10 mL), and HATU (0.63 g,1.66 mmol) was added, the
mixture was stirred on the ice-bath for 30 min, then 0.13 mL
anhydrous triethylamine in anhydrous CH2Cl2 was slowly added
dropwise to the above mixture, after that, the ice bath was
removed, and the mixture was reacted at room temperature for 8 h.
The product was extracted with CH2Cl2 (30 mL ꢁ 3), washed with
water and brine (100 mL ꢁ 3), and dried over Na2SO4. After filtra-
tion and concentration in vacuo, the residues was purified by silica
gel flash chromatogrphy gave (10k) (0.12 g, 25%). m.p. ¼171e173 ꢀC,
TLC (CHCl2:MeOH, 80:20 v/v): Rf ¼ 0.23, MS (ESI) [MþH]þ: m/
(ESI) [MþH]þ: m/z ¼ 480.35. 1H NMR (400 MHz, DMSO)
d 9.95 (s,
1H), 8.26 (d, J ¼ 8.1 Hz, 1H), 7.91 (s, 1H, Ar-H), 7.59e7.54 (m, 1H, Ar-
H), 7.49 (s, 2H, Ar-H), 7.12 (d, J ¼ 8.2 Hz, 3H, Ar-H), 6.73 (d, J ¼ 8.4 Hz,
2H), 5.12 (s, 2H), 1.53 (m, 2H), 1.46 (m, 2H). 13C NMR (101 MHz,
CDCl3) d 168.29,167.14,152.33,148.68,140.72,139.98,137.60,131.10,
130.71, 130.03, 125.60, 125.36, 124.95, 121.92, 120.69, 116.90, 114.68,
114.02, 33.16, 14.73.
4.1.12. N-[4-(3-amino-1H-indazol-4-yl)phenyl]-N-[3,5-
bis(trifluoromethyl)phenyl]cyclopropane-1,1-dicarboxamide (10i)
m.p. ¼ 135e137 ꢀC, TLC (CHCl2:MeOH, 80:20 v/v): Rf ¼ 0.30, MS
(ESI) [MþH]þ: m/z ¼ 547.45. 1H NMR (400 MHz, CDCl3)
d
9.27 (s,
z ¼ 558.35. 1H NMR (400 MHz, CDCl3)
d 8.64e8.59 (m, 2H), 8.42 (d,
1H), 8.18 (d, J ¼ 8.3 Hz, 1H), 8.06 (s, 2H), 7.56 (s, 1H, Ar-H), 7.44e7.38
(m, 1H, Ar-H), 7.23 (d, J ¼ 8.4 Hz, 2H, Ar-H), 7.15e7.11 (m, 1H, Ar-H),
6.81 (d, J ¼ 8.4 Hz, 2H), 4.33 (s, 2H), 1.74 (d, J ¼ 3.1 Hz, 2H), 1.71 (d,
J ¼ 8.3 Hz,1H), 7.61e7.54 (m,1H, Ar-H), 7.31e7.25 (m,1H, Ar-H), 7.22
(d, J ¼ 7.3 Hz, 1H, Ar-H), 7.04 (m, 1H, Ar-H), 6.83e6.77 (m, 2H), 6.74
(s, 1H), 4.38 (s, 2H), 1.73 (s, 4H). 13C NMR (101 MHz, CDCl3)
d 167.97,
J ¼ 3.1 Hz, 2H). 13C NMR (101 MHz, CDCl3)
d
168.41, 167.58, 165.70,
167.21, 152.63, 146.63, 140.85, 139.00, 136.96, 136.77, 132.21, 130.18,
129.75, 126.74, 125.75, 124.83, 121.65, 120.77, 118.97, 116.65, 115.41,
115.10, 115.06, 33.28, 16.19.
Title compounds 10l~10n were prepared by using the general
procedure described above.
152.67, 146.68, 140.91, 140.12, 136.96, 132.25, 131.92, 130.00, 127.53,
126.06, 124.53, 121.82, 119.82, 117.02, 116.85, 115.17, 114.36, 33.06,
15.97.
4.1.13. N-[4-(3-amino-1H-indazol-4-yl)phenyl]-N-1,3-benzodioxol-
5-ylcyclopropane-1,1-dicarboxamide (10j)
4.1.17. N-[3-(3-amino-1H-indazol-4-yl)phenyl]-N-[5-bromo-2-
(trifluoromethoxy)phenyl]cyclopropane-1,1-dicarboxamide (10l)
m.p. ¼ 173e174 ꢀC, TLC (CHCl2:MeOH, 80:20 v/v): Rf ¼ 0.27, MS
m.p. ¼ 116e118 ꢀC, TLC (CHCl2:MeOH, 80:20 v/v): Rf ¼ 0.38, MS
(ESI) [MþH]þ: m/z ¼ 455.45. 1H NMR (400 MHz, CDCl3)
d 8.28 (d,
J ¼ 7.4 Hz, 1H), 8.08 (s, 1H), 7.47 (m, 1H, Ar-H), 7.24 (d, J ¼ 8.2 Hz, 2H,
Ar-H), 7.15 (d, J ¼ 7.1 Hz, 2H, Ar-H), 6.79 (d, J ¼ 8.2 Hz, 2H, Ar-H), 6.70
(d, J ¼ 11.8 Hz, 1H), 5.90 (s, 2H), 4.39 (s, 2H), 1.67 (s, 2H), 1.60 (s, 2H).
(ESI) [MþH]þ: m/z ¼ 574.35. 1H NMR (400 MHz, CDCl3)
d 8.62 (s,
1H), 8.61 (d, J ¼ 2.3 Hz, 1H), 8.41 (d, J ¼ 8.1 Hz, 1H), 7.60e7.55 (m,
1H, Ar-H), 7.31e7.26 (m, 1H, Ar-H), 7.22 (d, J ¼ 6.9 Hz,1H, Ar-H), 7.04
(m, 1H, Ar-H), 6.79 (m, 2H), 6.74 (d, J ¼ 1.7 Hz, 1H), 4.38 (s, 2H), 1.73
13C NMR (101 MHz, CDCl3)
d 167.85, 152.53, 147.65, 146.82, 144.23,
140.87, 136.90, 132.44, 130.06, 127.56, 125.91, 120.63, 116.87, 115.06,
114.41, 113.70, 107.91, 103.43, 101.17, 38.63, 33.08, 15.18.
(s, 4H). 13C NMR (101 MHz, CDCl3)
d 167.97, 167.21, 152.64, 146.72,
140.84, 139.00, 136.97, 136.79, 132.21, 130.17, 129.75, 126.74, 125.75,
124.83, 121.65, 121.44, 120.76, 118.90, 116.65, 115.36, 115.05, 33.28,
16.19.
4.1.14. 4-(4-aminophenyl)-1H-indazol-3-amine (6)
Pd(PPh3)4 (3.4 g, 3.65 mmol) was added to a degassed solution
of 3-aminobenzeneboronic acid (4 g, 36.5 mmol), K2CO3 (9.3 g,
87.6 mmol), 4-iodine -1H-indazol-3-ylamine (2) (7.6 g, 36.5 mmol)
in 150 mL 1,4-dioxane and 50 mL water. The reaction mixture was
4.1.18. N-[3-(3-amino-1H-indazol-4-yl)phenyl]-N-(2,4-
dichlorophenyl)cyclopropane-1,1-dicarboxamide (10m)
m.p. ¼ 109e111 ꢀC, TLC (CHCl2:MeOH, 80:20 v/v): Rf ¼ 0.25, MS

L. Zhang et al. / European Journal of Medicinal Chemistry 127 (2017) 275e285
283
(ESI) [MþH]þ: m/z ¼ 480.35. 1H NMR (400 MHz, CDCl3)
d
8.43 (d,
4.1.24. N-[4-(2-aminoquinazolin-7-yl)phenyl]-N'-[3-bromo-5-
(trifluoromethyl)phenyl]thiourea (18a)
7-(4-aminophenyl)-2-quinazolinamine (9) (0.2 g, 0.85 mmol)
was dissolved in anhydrous CH2Cl2 (10 mL). 1-bromo-3-
J ¼ 8.3 Hz, 1H), 8.38 (s, 1H), 8.22 (d, J ¼ 8.8 Hz, 1H), 7.59 (m, 1H, Ar-
H), 7.30e7.28 (m, 3H, Ar-H), 7.25e7.20 (m, 2H, Ar-H), 6.82 (d,
J ¼ 7.6 Hz, 1H, Ar-H), 6.77 (d, J ¼ 9.7 Hz, 1H), 4.37 (s, 2H), 1.72 (s, 2H),
1.69 (s, 2H). 13C NMR (101 MHz, CDCl3)
d
167.94, 167.16, 152.49,
isothiocyanato-5-(trifluoromethyl)benzene
(17)
(0.23
g,
146.58,140.84,138.98,136.78, 133.68, 130.16,129.76,128.93,128.63,
127.74, 125.72, 122.83, 119.06, 116.69, 115.47, 115.12, 115.02, 38.63,
33.43.
0.77 mmol) in anhydrous CH2Cl2 (10 mL) was added dropwise to
the above mixture, and stirring on the ice-bath continued for 2 h.
After stirring at room temperature for 18 h. The organic layer was
washed with water and brine (100 mL ꢁ 3), and dried over Na2SO4.
After filtration and concentration in vacuo, the residue was purified
by silica gel flash chromatography, yielding (18a) (0.12 g, 30%).
m.p. ¼ 263e265 ꢀC, TLC (CHCl2:MeOH, 90:10 v/v): Rf ¼ 0.32, MS
4.1.19. N-[3-(3-amino-1H-indazol-4-yl)phenyl]-N-[3,5-
bis(trifluoromethyl)phenyl]cyclopropane-1,1-dicarboxamide (10n)
m.p. ¼ 112e114 ꢀC, TLC (CHCl2:MeOH, 80:20 v/v): Rf ¼ 0.24, MS
(ESI) [MþH]þ: m/z ¼ 518.35. 1H NMR (400 MHz, DMSO)
d 10.39 (s,
(ESI) [MþH]þ: m/z ¼ 547.45. 1H NMR (400 MHz, CDCl3)
d 9.18 (s,
1H), 10.23 (s, 1H), 9.18 (s, 1H), 8.13 (d, J ¼ 6.8 Hz, 2H), 8.05 (d,
J ¼ 8.7 Hz, 1H), 8.00e7.89 (m, 2H), 7.76 (d, J ¼ 7.4 Hz, 2H, Ar-H), 7.69
(s, 1H, Ar-H), 7.59 (d, J ¼ 7.4 Hz, 1H, Ar-H), 7.51 (d, J ¼ 8.7 Hz, 1H, Ar-
1H), 8.13 (d, J ¼ 8.3 Hz, 1H), 8.06 (s, 2H), 7.57 (s, 1H, Ar-H), 7.35 (m,
1H, Ar-H), 7.28 (m, 1H, Ar-H), 7.13 (d, J ¼ 7.3 Hz, 1H, Ar-H), 6.80 (d,
J ¼ 7.7 Hz, 2H), 6.72 (s, 1H), 4.40 (s, 2H), 1.76 (d, J ¼ 9.7 Hz, 2H), 1.71
H), 6.99 (s, 2H). 13C NMR (101 MHz, DMSO)
d 179.99, 163.18, 161.33,
(d, J ¼ 9.7 Hz, 2H). 13C NMR (101 MHz, CDCl3)
d 168.28, 167.76,
142.61, 138.53, 136.33, 133.39, 133.27, 129.86, 127.50, 127.17, 125.57,
125.38, 124.70, 124.32, 121.99, 120.12, 119.25, 114.78.
Title compounds 18b~18j were prepared by using the general
procedure described above.
152.62,146.55,140.73,139.99, 138.78, 136.85, 132.33,132.00,130.01,
129.84, 125.85, 124.50, 121.78, 119.74, 119.01, 116.67, 115.39, 115.26,
114.79, 33.09, 16.03.
4.1.20. 6-Bromo-2-quinazolinamine (12)
4.1.25. N-[4-(2-aminoquinazolin-7-yl)phenyl]-N'-[4-bromo-2-
(trifluoromethoxy)phenyl]thiourea (18b)
Compound 2-Fluoro-5-bromobenzaldehyde (10.0 g, 49.2 mmol)
bisguanidiniu 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. After cooling to room temperature,
120 mL of 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 yield 6-Bromo-2-
quinazolinamine (12) (5 g, 60%).
m.p. ¼ 257e259 ꢀC, TLC (CHCl2:MeOH, 90:10 v/v): Rf ¼ 0.30, MS
(ESI) [MþH]þ: m/z ¼ 534.35. 1H NMR (400 MHz, DMSO)
d 10.24 (s,
1H), 9.62 (s,1H), 9.18 (s,1H), 8.12 (s,1H), 8.06 (d, J ¼ 8.8 Hz,1H), 7.75
(m, 3H, Ar-H), 7.70 (s, 1H), 7.63 (d, J ¼ 8.5 Hz, 3H, Ar-H), 7.51 (d,
J ¼ 8.8 Hz, 1H, Ar-H), 6.98 (s, 2H). 13C NMR (101 MHz, DMSO)
d
180.67,163.23,161.28,143.47,138.82,136.14,133.41,133.28,132.18,
131.25, 130.89, 127.00, 125.51, 125.36, 124.77, 124.54, 120.11, 119.12,
118.52.
4.1.21. 7-(4-Aminophenyl)- 2-quinazolinamine (14)
Pd(PPh3)4 (1.03 g, 0.89 mmol) was added to a degassed solution
of 4-aminophenylboronic acid (1.54 g, 8.9 mmol), K2CO3 (2.8 g,
26.7 mmol), 6-bromo-2-quinazolinamine (12) (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 for overnight, filtered, and concentrated in
vacuo to give the crude product, which was isolated by silica gel
flash chromatography (PE/AcOEt ¼ 3:1)to obtain the title com-
pound 0.8 g with yield of 28%.
4.1.26. N-[4-(2-aminoquinazolin-7-yl)phenyl]-N'-[2-bromo-4-
(trifluoromethoxy)phenyl]thiourea (18c)
m.p. ¼ 251e253 ꢀC, TLC (CHCl2:MeOH, 90:10 v/v): Rf ¼ 0.29, MS
(ESI) [MþH]þ: m/z ¼ 534.35. 1H NMR (400 MHz, DMSO)
d 10.27 (s,
1H), 9.59 (s,1H), 9.18 (s,1H), 8.12 (s,1H), 8.06 (d, J ¼ 8.8 Hz,1H), 7.80
(s, 1H, Ar-H), 7.76 (d, J ¼ 8.5 Hz, 2H, Ar-H), 7.69 (d, J ¼ 5.8 Hz, 2H, Ar-
H), 7.66 (s, 1H, Ar-H), 7.51 (d, J ¼ 8.8 Hz, 1H, Ar-H), 7.44 (d, J ¼ 8.3 Hz,
1H, Ar-H), 6.98 (s, 2H). 13C NMR (101 MHz, DMSO)
d 180.68, 163.25,
161.26,138.83, 138.07, 136.05, 133.44, 133.29, 131.89, 127.51, 127.02,
125.77, 125.48, 125.34, 124.57, 122.53, 121.06, 120.11, 114.82.
4.1.27. N-[4-(2-aminoquinazolin-7-yl)phenyl]-N'-[5-bromo-2-
(trifluoromethoxy)phenyl]thiourea (18d)
4.1.22. (3-Bromo-5-(trifluoromethyl)phenyl) carbamodithioic acid
(16)
m.p. ¼ 253e255 ꢀC, TLC (CHCl2:MeOH, 90:10 v/v): Rf ¼ 0.32, MS
(ESI) [MþH]þ: m/z ¼ 534.35. 1H NMR (400 MHz, DMSO)
d 10.32 (s,
3-bromo-5-(trifluoromethyl)aniline (11) (2.5 g, 10.4 mmol),
DABC (1.4 g, 12.5 mmol) was dissolved in toluene (40 mL). Then
1.9 mL carbon disulphide was added dropwise to the above
mixture, after that, the mixture was reacted at room temperature
for 8 h. The product was collected by filtration and dried under
vacuum to give (3-bromo-5-(trifluoromethyl)phenyl)carbamodi-
thioic acid (16) as slight yellow solid (1.2 g, 40%).
1H), 9.67 (s, 1H), 9.18 (s, 1H), 8.13 (d, J ¼ 1.8 Hz, 1H), 8.08e8.04 (m,
2H), 7.76 (d, J ¼ 8.6 Hz, 2H, Ar-H), 7.64 (d, J ¼ 8.5 Hz, 2H, Ar-H), 7.55
(m,1H, Ar-H), 7.51 (d, J ¼ 8.8 Hz,1H, Ar-H), 7.42 (d, J ¼ 8.8 Hz,1H, Ar-
H), 6.98 (s, 2H). 13C NMR (101 MHz, DMSO)
d 180.55, 163.32, 161.21,
142.15, 138.73, 136.21, 134.03, 133.44, 133.32, 131.70, 130.00, 128.18,
127.03, 125.40, 124.58, 123.48, 120.09, 119.34,116.58.
4.1.23. 1-Bromo-3-isothiocyanato-5-(trifluoromethyl) benzene (17)
(3-bromo-5-(trifluoromethyl)phenyl)carbamodithioic acid (16)
(0.8 g, 2.5 mmol) was dissolved in anhydrous CH2Cl2 (20 mL). Then
triphosgene (0.88 g, 3 mmol) in anhydrous CH2Cl2 was added
dropwise to the above mixture on the ice-bath. Then stirring
continued for 2 h at room temperature. After completion of the
reaction, the organic layer was washed with water and brine
(100 mL ꢁ 3), and dried over Na2SO4. After filtration and concen-
tration in vacuo, the residue was purified by silica gel flash chro-
matography, yielding (17) (0.5 g, 30%).
4.1.28. N-[4-(2-aminoquinazolin-7-yl)phenyl]-N'-[2-bromo-5-
(trifluoromethoxy)phenyl]thiourea (18e)
m.p. ¼ 256e258 ꢀC, TLC (CHCl2:MeOH, 90:10 v/v): Rf ¼ 0.35, MS
(ESI) [MþH]þ: m/z ¼ 534.35. 1H NMR (400 MHz, DMSO)
d 10.41 (s,
1H), 9.63 (s, 1H), 9.20 (s, 1H), 8.14 (s, 1H), 8.07 (d, J ¼ 8.8 Hz, 1H),
7.82 (d, J ¼ 8.8 Hz,1H, Ar-H), 7.77 (d, J ¼ 8.4 Hz, 2H, Ar-H), 7.73 (s,1H,
Ar-H), 7.69 (d, J ¼ 8.4 Hz, 2H, Ar-H), 7.52 (d, J ¼ 8.7 Hz, 1H, Ar-H),
7.25 (d, J ¼ 7.0 Hz, 1H, Ar-H), 7.09 (s, 2H). 13C NMR (101 MHz, DMSO)
d
180.32, 163.46, 161.08, 147.46, 139.94, 138.68, 136.13, 134.30,
133.53, 133.39, 127.07, 125.41, 125.26, 124.61, 122.74, 120.60, 120.07,

284
L. Zhang et al. / European Journal of Medicinal Chemistry 127 (2017) 275e285
119.73,116.58.
procedures are as the following: Kinases (1.5 ng/mL) were incubated
with substrates (1.0
mg/m
L), compounds (1.2 ꢁ 10ꢂ4 - 12
mM) and
4.1.29. N-[4-(2-aminoquinazolin-7-yl)phenyl]-N'-(2,4-
dichlorophenyl)thiourea(18f)
ATP (250 mM) 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
L. After the assay plate was incubated at 30 ꢀC for 60 min and
cooled for 5 min at room temperature, 5 L of ADP-Glo™ reagent
m.p. ¼ 243e245 ꢀC, TLC (CHCl2:MeOH, 90:10 v/v): Rf ¼ 0.40, MS
5 m
(ESI) [MþH]þ: m/z ¼ 440.35. 1H NMR (400 MHz, DMSO)
d
10.25 (s,
m
1H), 9.62 (s, 1H), 9.19 (d, J ¼ 3.9 Hz, 1H), 8.13 (d, J ¼ 1.9 Hz, 1H), 8.06
(m, 1H), 7.80e7.73 (m, 2H, Ar-H), 7.71 (d, J ¼ 2.4 Hz, 1H, Ar-H), 7.67
(s, 1H, Ar-H), 7.63 (d, J ¼ 9.1 Hz, 2H, Ar-H), 7.52 (d, J ¼ 8.8 Hz, 1H, Ar-
H), 7.45 (m, 1H, Ar-H), 7.07 (s, 2H). 13C NMR (101 MHz, DMSO)
was added into each well to stop the kinase reaction and deplete
the unconsumed ATP. After an additional incubation for 40 min at
room temperature, 10 mL of kinase detection reagent was added
into the well to convert ADP to ATP. The mixture was incubated for
30 min to produce a luminescence signal. The luminescence was
measured using a VICTOR-X multi-label plate reader. The signal was
correlated with the amount of ATP present in the reaction and was
inversely correlated with the kinase activity.
d
180.65,163.40,161.11,138.89,136.19,135.99,133.54,133.36,131.74,
131.48, 131.38, 129.39, 127.84, 127.01, 125.37, 125.31, 124.54,
124.27,120.08.
4.1.30. N-[4-(2-aminoquinazolin-7-yl)phenyl]-N'-(3,4-
difluorophenyl)thiourea(18g)
4.3. Antiproliferative activity against human umbilical vein
endothelial cells [25]
m.p. ¼ 255e257 ꢀC, TLC (CHCl2:MeOH, 90:10 v/v): Rf ¼ 0.28, MS
(ESI) [MþH]þ: m/z ¼ 407.45. 1H NMR (400 MHz, DMSO)
d 10.47 (s,
1H), 10.44 (s, 1H), 9.53 (s, 1H), 8.39 (s, 1H), 8.30 (d, J ¼ 7.3 Hz, 1H),
7.77 (d, J ¼ 8.6 Hz, 3H, Ar-H), 7.73 (s, 1H, Ar-H), 7.70 (d, J ¼ 8.6 Hz,
2H, Ar-H), 7.43 (d, J ¼ 10.3 Hz, 2H, Ar-H), 7.28 (d, J ¼ 8.8 Hz, 1H, Ar-
The antiproliferative activity of title compounds was evaluated
against EA.hy926 cell line by the standard MTT assay in vitro. Hu-
man umbilical vein endothelial cells (EA.hy926) were purchased
from the Cell Bank of Type Culture Collection of Chinese Academy
of Science (Shanghai, China). EA.hy926 cells were cultured in
DMEM supplemented with 10% (v/v) heat-inactivted fetal calf
serum, penicillin (100 U/mL) and streptomycin (100 U/mL). Cul-
tures were maintained at 37 ꢀC in a humidified atmosphere con-
H). 13C NMR (101 MHz, DMSO)
d 179.95, 168.70, 161.46, 139.77,
137.79, 135.82, 134.56, 132.20, 131.02, 127.19, 126.47, 124.26, 120.44,
119.22, 117.94, 117.59, 117.40, 113.23, 113.04.
4.1.31. N-[4-(2-aminoquinazolin-7-yl)phenyl]-N'-(3,4-
dichlorophenyl)thiourea (18h)
taining 5% CO2. Briefly, 2 ꢁ 104 cells in 100
mL DMEM supplemented
m.p. ¼ 152e154 ꢀC, TLC (CHCl2:MeOH, 90:10 v/v): Rf ¼ 0.27, MS
(ESI) [MþH]þ: m/z ¼ 440.35. 1H NMR (400 MHz, DMSO)
d 10.62 (s,
with 2% (v/v) heat-inactivated FBS, penicillin (100 U/ml) and
streptomycin (100 U/ml) were seeded into 96-well plates. After
incubation at 37 ꢀC for 24 h, the test compounds at indicated final
concentrations were added to the culture medium and the cells
cultures were continued for 48 h. The same volume of double-
1H), 9.44 (d, J ¼ 6.4 Hz,1H), 8.30 (s, 1H), 8.23 (d, J ¼ 8.5 Hz, 2H), 8.03
(s, 1H), 7.75 (s, 1H, Ar-H), 7.73e7.68 (m, 2H, Ar-H), 7.64 (d, J ¼ 8.7 Hz,
2H, Ar-H), 7.59 (s, 1H, Ar-H), 7.55 (s, 1H, Ar-H). 13C NMR (101 MHz,
DMSO)
d 179.69, 168.84, 140.27,135.22, 135.20, 130.90, 130.68,
distilled water was used as the negative control. Then, 22
MTT (5 mg/mL) was added to each well and incubated for 4 h at
37 ꢀC. The formazan crystals were dissolved in 100
L DMSO each
well. Supernatant was discarded, and 150 L DMSO was added to
mL fresh
127.91, 127.54, 127.16, 126.22, 125.71, 124.61, 124.26, 124.21, 123.48,
119.44, 118.91, 114.80.
m
m
4.1.32. N-[4-(2-aminoquinazolin-7-yl)phenyl]-N'-[3,5-
bis(trifluoromethyl)phenyl]thioura(18i)
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.
m.p. ¼ 252e254 ꢀC, TLC (CHCl2:MeOH, 90:10 v/v): Rf ¼ 0.35, MS
(ESI) [MþH]þ: m/z ¼ 507.45. 1H NMR (400 MHz, DMSO)
d 10.46 (s,
1H), 10.32 (s, 1H), 9.18 (s, 1H), 8.29 (s, 2H), 8.12 (d, J ¼ 1.7 Hz, 1H),
8.05 (m, 1H), 7.83 (s, 1H, Ar-H), 7.77 (d, J ¼ 8.5 Hz, 2H, Ar-H), 7.58 (d,
4.4. Molecular docking study [26]
J ¼ 8.5 Hz, 2H, Ar-H), 7.51 (d, J ¼ 8.8 Hz, 1H, Ar-H), 6.97 (s, 2H). 13
C
NMR (101 MHz, DMSO)
d 180.13, 163.17, 161.35, 142.34, 138.37,
Surflex-Dock module of Sybyl-X (Version 2.0, Tripos Inc. St.
Louis, MO) was used in molecular docking study. The crystal
structures of VEGFR-2 (PDB ID: 4ASD), Tie-2 (PDB ID: 2P4I) [27],
and EphB4 (PDB ID: 2X9F) [28] were extracted from the Protein
Data Bank. Prior to docking, the ligand was extracted from complex
structure and was regarded as the reference molecule. The other
ligands and water molecules were removed. All the hydrogen
atoms and AMBER7 FF99 charges were added. Compound (12o)
was depicted and optimized using Powell's method with the Tripos
force field with convergence criterion set at 0.05 kcal/(Åmol). Then
Gasteiger-Hückel charges were assigned to the small molecule. The
residues in a radius 5.0 Å around BAX (the ligand of VEGFR-2 in the
crystal complex) were considered as the active site. The optimized
conformation of (12o) was then docked into the active site of
VEGFR-2. Other docking parameters were kept at default.
136.49, 133.34, 133.26, 130.62, 130.29, 127.24, 125.61, 125.41, 124.82,
124.00, 122.37, 120.12, 119.62, 118.29.
4.1.33. N-[4-(2-aminoquinazolin-7-yl)phenyl]-N0-1,3-benzodioxol-
5-ylthiourea(18j)
m.p. ¼ 230e231 ꢀC, TLC (CHCl2:MeOH, 90:10 v/v): Rf ¼ 0.33, MS
(ESI) [MþH]þ: m/z ¼ 415.45. 1H NMR (400 MHz, DMSO)
d 9.82 (s,
1H), 9.77 (s, 1H), 9.18 (s, 1H), 8.10 (d, J ¼ 1.9 Hz, 1H), 8.04 (m, 1H),
7.72 (d, J ¼ 8.6 Hz, 2H, Ar-H), 7.60 (d, J ¼ 8.5 Hz, 2H, Ar-H), 7.50 (d,
J ¼ 8.8 Hz, 1H, Ar-H), 7.12 (s, 1H, Ar-H), 6.97 (s, 2H), 6.90 (d,
J ¼ 8.3 Hz, 1H), 6.83 (d, J ¼ 1.6 Hz, 1H), 6.04 (s, 2H).13C NMR
(101 MHz, DMSO)
d 180.18, 163.15, 161.29, 147.37, 133.74, 133.56,
133.54,133.27, 126.92, 126.83, 125.54, 125.22, 124.63,124.46,120.13,
118.10, 108.28, 106.95, 101.70, 99.98.
4.2. RTK inhibitory activity assay [24]
Acknowledgments
The in vitro RTK inhibition assays 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 performed
This work was supported by the National Natural Science
Foundation of China (NSFC, Grant No. 81573285) and the Funda-
mental Research Funds for the Central Universities (2015qngz13).
in duplicate in a reaction mixture of final volume of 10 mL. General

L. Zhang et al. / European Journal of Medicinal Chemistry 127 (2017) 275e285
285
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