Discovery of novel VEGFR-2 inhibitors. Part 5: Exploration of diverse hinge-binding fragments via core-refining approach

Article abstract of DOI:10.1016/j.ejmech.2015.08.045

Pathological angiogenesis plays a critical role in numerous diseases including malignancy. VEGFR-2 is the central regulators in angiogenesis and has become a promising target for anticancer drug design. We have identified a novel biphenyl-aryl urea incorporated with salicyladoxime (BPS-7) as potent VEGFR-2 inhibitor. As a continuation to our previous research, various aromatic-heterocyclic were introduced as hinge-binding fragment via a core-refining approach. Interestingly, many compounds exhibited comparable VEGFR-2 inhibition to Sorafenib. In particular, 12e and 12o displayed excellent VEGFR-2 inhibitory activity with IC₅₀ values of 0.50 nM and 0.79 nM, respectively. Several title compounds showed considerable antiproliferative activity against A549 and SMMC-7721 cells. In addition, molecular docking was performed to rationalize the efficiency of the better compounds. These results will be instructive for further inhibitor design and optimization.

Full text of DOI:10.1016/j.ejmech.2015.08.045

European Journal of Medicinal Chemistry 103 (2015) 80e90
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Discovery of novel VEGFR-2 inhibitors. Part 5: Exploration of diverse
hinge-binding fragments via core-refining approach
,
,
Yuanyuan Shan b 1, Hongping Gao a 1, Xiaowei Shao a, Jinfeng Wang 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:
Pathological angiogenesis plays a critical role in numerous diseases including malignancy. VEGFR-2 is the
central regulators in angiogenesis and has become a promising target for anticancer drug design. We
have identified a novel biphenyl-aryl urea incorporated with salicyladoxime (BPS-7) as potent VEGFR-2
inhibitor. As a continuation to our previous research, various aromatic-heterocyclic were introduced as
hinge-binding fragment via a core-refining approach. Interestingly, many compounds exhibited com-
parable VEGFR-2 inhibition to Sorafenib. In particular, 12e and 12o displayed excellent VEGFR-2 inhib-
itory activity with IC50 values of 0.50 nM and 0.79 nM, respectively. Several title compounds showed
considerable antiproliferative activity against A549 and SMMC-7721 cells. In addition, molecular docking
was performed to rationalize the efficiency of the better compounds. These results will be instructive for
further inhibitor design and optimization.
Received 8 April 2015
Received in revised form
19 August 2015
Accepted 23 August 2015
Available online 29 August 2015
Keywords:
VEGFR-2 inhibitors
Hinge-binding fragment
Urea
© 2015 Elsevier Masson SAS. All rights reserved.
Core-refining
Anti-angiogenesis
1. Introduction
inhibitors are also being evaluated in clinical trials for the treat-
ment of cancers.
As angiogenesis is essential for tumor growth and metastasis,
anti-angiogenesis is an integral modality of modern anti-cancer for
various malignancies [1]. Sprouting angiogenesis is regulated by
vascular endothelial growth factor (VEGF) and its receptors, espe-
cially VEGFR-2 [2]. VEGFR-2 has been considered to be the key and
attractive target for anti-angiogenesis drugs design [3]. In the last
decade, the importance of anti-angiogenesis agents in cancer
therapy has become increasingly clear. Anti-angiogenesis agents
including VEGFR-2 inhibitors have been widely used in clinical
treatment of cancers. So far, many structurally diverse VEGFR-2
inhibitors have emerged as promising anti-angiogenesis agents.
The clinical application of VEGFR-2 inhibitors has been validated as
a therapeutic strategy by positive results with Sorafenib (BAY-43-
9006, Bayer), Sunitinib (SU-11248, Pfizer), Vandetanib (ZD6474,
AstraZeneca), and Linifanib (ABT-869, Abbott) (Fig. 1) [4]. These
drugs act by competing with ATP for its binding site of VEGFR-2,
thereby preventing the phosphorylation. Many other VEGFR-2
Among these approved VEGFR-2 inhibitors, diaryl-ureas such as
Sorafenib and Linifanib have attracted considerable attention. In
our previous research, we have identified numbers of biphenyl-aryl
ureas incorporated with salicylaldoxime as potent and selective
VEGFR-2 inhibitor through structural optimization of Taspine
(Fig. 2) [5e7]. These biphenyl-aryl ureas displayed significantly
VEGFR-2 inhibitory activity as well as anticancer potency [8e10].
Among them, BPS-7 was the most potent VEGFR-2 inhibitor with
excellent selectivity. The biological evaluations indicated that BPS-
7 could inhibit phosphorylation of VEGFR-2 and the formation of
blood vessels. Moreover, it also significantly inhibited the prolif-
eration and migration of human umbilical vein endothelial cells
[11]. Therefore, BPS-7 can be considered as a promising lead
compound for further optimized as novel anti-angiogenesis agents.
Encouraged by previous results, further efforts have been
focused on expanding the structural diversity of these anti-
angiogenesis agents. In continuation of our pursuit of biphenyl-
aryl ureas as VEGFR-2 inhibitors, we explored various aromatic-
heterocyclics as hinge-binding fragments via core-refining
approach [12]. Our strategy was the replacement of salicylaldox-
ime by other aromatic-heterocyclics such as pyridine, pyrimidine,
pyridine, pyrimidine, indole, pyrazole and aminoindazole (Fig. 3).
* Corresponding author.
E-mail address: zhj8623@mail.xjtu.edu.cn (J. Zhang).
Both authors contributed equally to this work.
1
http://dx.doi.org/10.1016/j.ejmech.2015.08.045
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

Y. Shan et al. / European Journal of Medicinal Chemistry 103 (2015) 80e90
81
diaryl ureas incorporated with aromatic-heterocyclics as next-
generation VEGFR-2 inhibitors via structure-guided core-refining
approach. Biological evaluations including enzymatic inhibition
and antiproliferative potency were carried out. Molecular modeling
study of these novel VEGFR-2 inhibitors was also performed to
rationalize the efficiency of the better compounds. These de-
rivatives displayed potent VEGFR-2 inhibitory activity and could be
considered as promising lead compounds for further optimization.
2. Results and discussion
2.1. Synthesis of title compounds
The title compounds were prepared according to the synthetic
routes outlined in Scheme 1 and Scheme 2. For the synthesis of title
compounds (6a~6s), the key intermediates (4a~4d) were prepared
in a three-step sequence from commercially available 3-amino-p-
anisic acid (1). 3-Amino-p-anisic acid (1) was converted into cor-
responding ethanolamine esters (3a~3d) via Boc-protected product
(2) in one step. Dicyclohexylcarbodiimide (DCC) was used as
coupling agent in the presence of a catalytic amount of 4-(dime-
thylamino)pyridine (DMAP). Subsequent deprotection of the Boc
group of (3a~3d) with trifluoroacetic acid (TFA) provided the key
intermediates (4a~4d) [13]. Then various substituted m-bromoa-
niline were treated with triphosgene (BTC) to produce corre-
sponding isocyanate, followed by reacting with the key
intermediates (4a~4d) yielding compounds (5a~5g). Finally, the
title compounds (6a~6s) were prepared from (5a~5g) and
aromatic-heterocyclic boronic acids by classical Pd-catalyzed
Suzuki coupling reaction.
Fig. 1. Approved VEGFR-2 inhibitors with structural diversity.
These heterocyclics containing hydrogen bond acceptor nitrogen
could retain the essential hydrogen bonds with hinge region of
VEGFR-2. In addition, the design strategies of “hybridization” and
“mix-n-match” were also adopted for the other moieties with BPS-
7 as lead compound. Previous SAR exploration demonstrated that
urea moiety connecting biphenyl and aniline played a critical role in
VEGFR-2 inhibition. It could form three crucial hydrogen bonds
with DFG motif of VEGFR-2. Therefore, we maintained the urea
moiety in view of its paramount importance. In order to increase
the structural diversity and VEGFR-2 inhibitory potency, we
replaced the two methoxyls on biphenyl with trifluoromethyl or
trifluoromethoxyl, respectively. Moreover, various diverse sub-
stituents such as tertiary amines, halogen, ethanolamine ester, alkyl
or methylenedioxy were incorporated to terminal aniline. The
design strategy and structures of the title compounds were repre-
sented in Fig. 3.
The preparation of title compounds (12a~12s) commenced with
the preparation of aminoindazole (8) from commercially available
2-fluoro-6-iodo-benzonitrile (7) by using five equiv of hydrazine
monohydrate in refluxing ethanol in the presence of NaHCO3 [14].
In our continuous efforts to identify more potent anti-
angiogenesis agents, we designed and synthesized thirty eight
Fig. 2. Discovery of novel lead compound (BPS-7) through structural optimization of taspine.
Fig. 3. Expanding the structural diversity of VEGFR-2 inhibitors via core-refine approach.

82
Y. Shan et al. / European Journal of Medicinal Chemistry 103 (2015) 80e90
Scheme 1. Synthetic route of the title compounds (6ae6s). Reagents and conditions: (a) (Boc)2O, NaOH (1 mol/L), THF; (b) DCC, DMAP, THF; (c) TFA, DCM; (d) BTC, Et3N, DCM; (e)
Pd(PPh3)4, Na2CO3, H2O, dioxane.
Various anilines were treated with triphosgene (BTC) to produce
corresponding isocyanate. The coupling substrates (11a~11p) and
(11q~11s) were obtained by condensation of various isocyanates
and 3-aminophenylboronic acid pinacol ester (10) or 4-
aminophenylboronic acid pinacol ester (10′), respectively. Finally,
aminoindazole (8) and boronates (11a~11s) were coupled by clas-
sical Pd-catalyzed Suzuki coupling reaction to afford the title
compounds (12a~12s). All of the title compounds were character-
ized by 1H NMR, 13C NMR, mass spectroscopy and melting point. All
of the final compounds were purified to >95% purity which was
determined by High Performance Liquid Chromatography (HPLC).
HPLC analysis was performed on a Shimadzu LC-2010 HPLC system
with the use of C18 column (150 ꢀ 2.0 mm, 5 um). 1H NMR, 13C
NMR, mass spectroscopy, melting point and HPLC analysis chro-
matograms of all the title compounds are described in
Supplementary material.
2.2. Biological evaluation
The VEGFR-2 inhibitory activities of the newly synthesized title
compounds were depicted in Tables 1e3 with Sorafenib as positive
control. As shown in Table 1, replacement of salicyladoxime by
aromatic-heterocyclics markedly reduced the VEGFR-2 inhibitory
potency. Nine compounds of them displayed poor activity with IC50
values higher than 500 mM. Only two compounds (6a, 6c) exhibited
comparable VEGFR-2 inhibition to Sorafenib with IC50 values less
than 100 nM. Among them, compound 6a was the most potent one
with IC50 value of 28.0 nM. The activities of the title compounds
with aminoindazole as hinge-binding fragment were shown in
Tables 2 and 3. In general, their VEGFR-2 inhibitory activities were
much better than the first series. These results indicated that
aminoindazole on biphenyl core could enhance the activity with
comparison to aromatic-heterocyclics. Most of them exhibited
moderate to high VEGFR-2 inhibitory activities with IC50 values
ranging from 0.50 nM to 331.0 nM. Among them, 12e and 12o
potently inhibited VEGFR-2 with IC50 values of 0.50 nM and
0.79 nM, respectively. Only three compounds of them exhibited
poor activity with IC50 values higher than 500 nM. These com-
pounds could be considered as promising lead compounds for
further optimization.
As observed in Table 2, terminal aniline with substituents at
ortho position (12j, 12k, 12l) might significantly reduce VEGFR-2
inhibitory activities with IC50 values higher than 200 nM. Howev-
er, substituents at meta position or para position somewhat
enhanced VEGFR-2 inhibitory potency such as 12e, 12c, 12f, 12g,
and 12o. While the other compounds exhibited poor VEGFR-2
inhibitory activities compared to Sorafenib. The incorporation of
Scheme 2. Synthetic route of the title compounds (12a~12s). Reagents and conditions:
(a) EtOH, NaHCO3, NH2NH2$H2O; (b) MgSO4, THF; (c) BTC, Et3N, DCM; (d) Pd(PPh3)4,
Na2CO3, H2O, dioxane.

Y. Shan et al. / European Journal of Medicinal Chemistry 103 (2015) 80e90
83
Table 1
Structures and VEGFR-2 inhibitory activities of title compounds (6ae6s).
R1
R2
R3
IC50
,
m
M
R1
R2
R3
IC50, mM
6a
6b
6c
6d
6e
3-CF3
0.028
6k
6l
3-CF3
>500
>500
>500
>500
>500
3-CF3
>500
2-OCF3
2-OCF3
2-OCF3
2-OCF3
2-OCF3
2-OCF3
2-OCF3
0.062
6m
6n
6o
53.0
5.4
6f
2-OCF3
2-OCF3
2-OCF3
2-OCF3
3-CF3
51.9
6p
3-CF3
>500
>500
6g
6h
6i
0.16
6q
3-CF3
37.7
6r
2-OCF3
2-OCF3
4.7
>500
6s
14.9
6j
8.4
Sorafinib
0.48 nM
aromatic heterocyclics on the biphenyl somewhat reduced VEGFR-
2 inhibitory activity of these ureas. The biological results indicated
that these aromatic-heterocyclics were not favorable hinge-binding
group except for aminoindazole when considering VEGFR-2
inhibitory potency.
The antiproliferative activities of title compounds against hu-
man lung cancer cells A549 and human hepatoma cells SMMC-
7721 were further evaluated. The biological evaluation was per-
formed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) method with Sorafenib as positive control. As
shown in Table 4 and Table 5, it was found that majority of them
displayed potent antiproliferative activities with IC50 values
displayed in Fig. 4. It was nicely bound to VEGFR-2 and presented
the similar binding conformation with Sorafenib which was dis-
played as green. Several favorable interactions between (12o) and
VEGFR-2 were clearly observed. The hydrophobic 4-chloro-3-(tri-
fluoromethyl)-benzene moiety was buried in the end of VEGFR-2.
The binding conformation was stabilized mainly by the formation
of a network of four hydrogen bonds. The two nitrogen atoms of
urea unit made two hydrogen bonds with Asp 1046 with distance of
2.38 Å and 1.74 Å, respectively. Another two nitrogen atoms of
aminoindazole formed two hydrogen bonds with Glu 917 and Cys
919 with distance of 2.27 Å and 1.89 Å, respectively. Molecular in-
sights based on docking suggested favorable binding interactions of
(12o) with the active site of VEGFR-2. Therefore, aminoindazole
could be considered as a favorable hinge-binding fragment.
ranging from 0.28 to 15.81
mM. Many of them displayed potent
antiproliferative activity at submicromolar concentrations against
both cancer cell lines, such as 6a, 6d, 6e, 6g, 6h, 6j, 6k, 6l, 6m, 12g,
12n, 12q, 12r and 12s. Particularly, 6g was the most potent one with
3. Conclusion
IC50 values of 0.99 mM and 0.57 mM, respectively.
With the goal of expanding structural diversity of VEGFR-2 in-
hibitors, we described herein the exploration of various hinge-
binding fragments via core-refining approach. The pyridine, py-
rimidine, indole, pyrazole and aminoindazole were introduced as
novel hinge binding moieties. Several title compounds displayed
comparable VEGFR-2 inhibitory activity to that of Sorafenib. In
particular, compounds 12e and 12o potently inhibited VEGFR-2 at
subnanomolar potency with IC50 values of 0.50 nM and 0.79 nM,
2.3. Molecular docking study
To gain insight into the binding mode for these novel VEGFR-2
inhibitors, compound (12o) was docked into the active site of
VEGFR-2 (PDB ID: 4ASD). The docking study was carried out using
Surflex-Dock Module of Sybyl-X (Version 2.0, Tripos Inc. St. Louis,
MO). The most reasonable binding pose of (12o) was obtained and

84
Y. Shan et al. / European Journal of Medicinal Chemistry 103 (2015) 80e90
Table 2
Table 4
Structures and VEGFR-2 inhibitory activities of title compounds (12ae12p).
Antiproliferative activities of title compounds (6ae6s) (IC50, mM).
A549
SMCC-7721
A549
SMCC-7721
6a
6b
6c
6d
6e
6f
6g
6h
6i
2.49
1.66
1.01
0.95
0.91
14.69
0.99
1.73
>500
1.91
0.86
1.39
1.38
1.76
2.01
6.15
0.57
0.64
3.43
0.73
6k
6l
6m
6n
6o
6p
6q
6r
6s
1.57
2.06
3.97
1.52
>500
>500
1.24
1.28
>500
1.45
0.41
0.36
0.92
1.23
>500
11.09
1.77
10.85
2.55
0.65
R
IC50, nM
53.6
R
IC50, nM
21.0
12a
12b
12i
12j
6j
Sorafinib
59.2
>500
evaluation and structural optimization of these promising anti-
cancer agents are currently under investigation and will be re-
ported in due course. These results might contribute to the
discovery of novel anti-angiogenesis agents for the intervention of
pathological angiogenesis-related diseases.
12c
12d
2.15
12k
12l
>500
116.0
205.0
4. Experimental section
12e
0.50
12m
117.0
4.1. Chemistry
4.1.1. General procedure
12f
2.36
2.28
12n
12o
41.8
0.79
Commercial reagents and solvents were used without additional
purification. The reactions except those in aqueous media are car-
ried out by standard techniques for the exclusion of moisture. Re-
action progress 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 60
12g
12h
331.0
0.48
12p
>500
(particle size 70e200 mm). 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 Shimadzu HPLC-MS-QP2010 instrument.
Sorafinib
respectively. In addition to their enzymatic activities, many of them
exhibited potent antiproliferative activity at submicromolar con-
centrations against A549 and SMMC-7721 cancer cells. Molecular
docking results suggested that these VEGFR-2 inhibitors shared the
similar binding conformation with Sorafenib. Those results
confirmed that aminoindazole and urea moieties were benefit for
improving the biological activity. In conclusion, we disclosed our
progress on identifying novel VEGFR-2 inhibitors based on the lead
compound BPS-7. Several compounds had strong potential to be
further developed as novel VEGFR-2 inhibitors. Further biological
4.1.2. 3-[(Tert-butoxycarbonyl)amino]-4-methoxybenzoic acid (2)
3-Amino-p-anisic acid (1) (20.0 g,119 mmol) was suspended in a
solution of NaOH (1 mol/L, 180 mL) and THF (150 mL). The mixture
was cooled in an ice bath. A solution of di-tert-butyl dicarbonate
(Boc)2O (104.4 g, 479 mmol) in THF (70 mL) was then added
dropwise over a period of 30 min under stirring. The ice-bath was
removed after dropping and the mixture was stirred for another
12 h at room temperature. Thereafter, the THF was removed in
vacuo, and the remaining aqueous solution was diluted with water.
The resulting mixture was extracted three times with AcOEt
(70 mL ꢀ 3). The aqueous layer was acidified with concentrated HCl
to neutralize PH to 4e5 and extracted with AcOEt (80 mL ꢀ 3). The
combined organic layers were washed with water, and dried over
Na2SO4. Then filtration and concentration in vacuo gave (2) (20.78 g,
Table 3
Structures and VEGFR-2 inhibitory activities of title compounds (12qe12s).
Table 5
Antiproliferative activities of title compounds (12ae12s) (IC50, mM).
A549
SMCC-7721
A549
SMCC-7721
12a
12b
12c
12d
12e
12f
12g
12h
12i
1.17
1.36
>500
15.81
1.72
1.66
1.42
1.95
2.49
2.36
1.24
>500
>500
1.83
4.45
0.28
1.41
2.86
>500
12k
12l
12m
12n
12o
12p
12q
12r
12s
3.04
>500
1.51
1.75
3.41
>500
5.23
1.16
>500
1.45
5.88
1.90
3.54
0.51
1.15
0.55
0.53
0.43
0.60
0.65
R
IC50, nM
1.38
R
IC50, nM
12q
12s
12r
>500
3.47
Sorafinib
0.48
12j
229.19
Sorafinib

Y. Shan et al. / European Journal of Medicinal Chemistry 103 (2015) 80e90
85
Fig. 4. Superimposition of the docking poses for VEGFR-2 with compounds (12o) and Sorafenib (green). Hydrogen bonds were depicted in dashed yellow lines. (For interpretation
of the references to color in this figure legend, the reader is referred to the web version of this article.)
65%) as off-white solid, which was used for the next step without
further purification.
chromatography (PE/AcOEt ¼ 5:1) gave as white solid (5a) (1.36 g,
43%).
4.1.3. 2-(Dimethylamino)ethyl-3-[(tert-butoxycarbonyl) amino]-4-
methoxybenzoate (3a) [15]
4.1.6. 2-(Dimethylamino)ethyl 4-methoxy-3-(3-(3-(pyridin-3-yl)-
5-(trifluoromethyl)phenyl)ureido)benzoate (6a)
Compound (2) (8.0 g, 30 mmol) and DMAP (0.2 g, 1.6 mmol)
were dissolved in a solution of THF (50 mL). Then DCC (11.63 g,
56 mmol) in THF (20 ml) was added dropwise into above mixture
under ice-bath. The ice-bath was removed after dropping, 2-
(dimethylamino)ethanol (13.3 g, 149 mmol) in THF (30 mL) was
added dropwise into the above mixture. The mixture was stirred on
oil bath at 30 ꢁC for seventeen hours. After completion of reaction,
the reaction mixture was filtered off and the solvent was evapo-
rated. The mixture was taken in AcOEt (50 mL ꢀ 3). The organic
layer was combined and washed with water and brine, and dried
over Na2SO4. Filtration and concentration in vacuo gave (3a) (6.10 g,
60%) as a yellow viscous gel.
A flask charged with Pd(PPh3)4 (0.20 g, 0.17 mmol), K2CO3
(0.40 g, 2.9 mmol), and pyridin-3-ylboronic acid (0.30 g, 2.4 mmol)
and intermediate (5a) (0.60 g, 1.2 mmol) was flushed with nitrogen
and suspended in 1,4-dioxane (20 mL) and water (5 mL). The
mixture was then refluxed overnight under nitrogen. The hot sus-
pension was filtered and the filtrate distilled by rotary evaporation
to remove 1,4-dioxane. Water (150 mL) was added and 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 (CH2Cl2/
MeOH ¼ 20:1) gave 6a (0.33 g, 58%) as white solid. mp: 115e117 ꢁC,
MS (ESI) [MeH]þ: m/z ¼ 501.30. 1H NMR (400 MHz, DMSO-d6)
d
9.88 (s, 1H), 8.94 (d, J ¼ 2.3 Hz, 1H), 8.80 (d, J ¼ 2.1 Hz, 1H), 8.66
(dd, J ¼ 4.8, 1.5 Hz, 1H), 8.52 (s, 1H), 8.17e8.14 (m, 1H), 8.03 (s, 1H),
7.86 (s, 1H), 7.66 (dd, J ¼ 8.5, 2.1 Hz, 2H), 7.57e7.54 (m, 1H), 7.18 (d,
J ¼ 8.7 Hz,1H), 4.35 (t, J ¼ 5.8 Hz, 2H), 3.99 (s, 3H), 2.63 (t, J ¼ 5.8 Hz,
4.1.4. 2-(Dimethylamino)ethyl 3-amino-4-methoxybenzoate (4a)
[16]
The solution of compound (3a) (6.0 g, 17.7 mmol) in anhydrous
CH2Cl2 (100 mL) pre-cooled in ice-water bath was treated with TFA
(24 mL). The reaction mixture was stirred at room temperature for
2 h after the reaction was warmed up to room temperature from
2H), 2.24 (s, 6H). 13C NMR (101 MHz, DMSO-d6)
d166.03, 152.77,
151.98, 151.90, 149.74, 148.23, 141.71, 139.51, 134.97, 134.79, 131.21,
130.89, 128.67, 125.84, 124.75, 124.44, 123.13, 122.46, 122.29, 120.19,
119.59, 117.13, 113.99, 110.83, 62.71, 57.78, 56.67, 52.30, 45.81.
The title compounds 6b~6s were also prepared by using the
general procedure described above.
0
ꢁC. Dilute NaHCO3 was added to the mixture to neutralize pH to
alkalinity. The water layer was extracted with AcOEt (50 mL ꢀ 3).
The organic layer was combined and washed with water and brine,
and dried over Na2SO4. After filtration and concentration in vacuo to
give (4a) (3.10 g, 74%) as yellow oil.
4.1.7. 2-(Dimethylamino)ethyl 4-methoxy-3-(3-(3-(pyrimidin-5-
yl)-5-(trifluoromethyl)phenyl)ureido)benzoate (6b)
Yield 56% as white solid. mp: 155e156 ꢁC, MS (ESI) [MþH]þ: m/
4.1.5. 2-(Dimethylamino)ethyl-3-[({[3-bromo-5-(trifluoromethyl)
phenyl]amino}carbonyl)amino]-4-methoxybenzoate (5a)
z ¼ 504.10. 1H NMR (400 MHz, DMSO)
d 9.90 (s, 1H), 9.27 (s, 1H),
9.19 (s, 2H), 8.80 (d, J ¼ 2.1 Hz, 1H), 8.56 (s, 1H), 8.12 (s, 1H), 7.85 (s,
1H), 7.78 (s, 1H), 7.66 (dd, J ¼ 8.6, 2.1 Hz, 1H), 7.18 (d, J ¼ 8.7 Hz, 1H),
4.34 (t, J ¼ 5.8 Hz, 2H), 3.99 (s, 3H),2.61 (t, J ¼ 5.8 Hz, 2H), 2.22 (s,
Triphosgene (0.74 g, 2.5 mmol) was dissolved in anhydrous
CH2Cl2 (20 mL) and the mixture was stirred on the ice-bath for
15 min. A solution of the [3-bromo-5-(trifluoromethyl)phenyl]
amine (1.66 g, 6.9 mmol) in anhydrous CH2Cl2 (10 mL) was added
dropwise to the above mixture and stirring continued for 30 min.
Then triethanolamine (0.75 mL, 5.5 mmol) diluted with CH2Cl2
(10 mL) was then added into the mixture. Stirring was continued
for 20 min, a solution of triethanolamine (0.75 mL, 5.5 mmol) and
intermediate (4a) (1.50 g, 6.3 mmol) in anhydrous CH2Cl2 (10 mL)
was added. 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 con-
centration in vacuo, the residues was purified by silica gel flash
6H). 13C NMR (101 MHz, DMSO-d6)
d 166.01, 158.32, 155.49, 152.70,
151.94, 141.75, 136.27, 132.60, 131.35, 131.03, 128.60, 124.75, 122.46,
120.23, 119.57, 117.36, 114.69, 110.79, 62.77, 57.82, 56.66, 49.04,
45.86.
4.1.8. 2-(Dimethylamino)ethyl 4-methoxy-3-(3-(5-(pyridin-3-yl)-
2-(trifluoromethoxy)phenyl)ureido)benzoate (6c)
Yield 54% as white solid. mp: 103e105 ꢁC, MS (ESI) [MeH]þ: m/
z ¼ 517.20. 1H NMR (400 MHz, DMSO)
9.33 (d, J ¼ 4.1 Hz, 1H), 9.14
d
(d, J ¼ 5.4 Hz, 1H), 8.88 (s, 1H), 8.80 (d, J ¼ 2.1 Hz,1H), 8.62 (d,

86
Y. Shan et al. / European Journal of Medicinal Chemistry 103 (2015) 80e90
J ¼ 2.1 Hz, 1H), 8.60 (s, 1H), 8.07 (dd, J ¼ 8.2, 1.8 Hz, 1H), 7.64e7.68
(m, 1H), 7.54 (d, J ¼ 2.1 Hz, 1H), 7.52 (s, 1H), 7.47 (s, 1H), 7.18 (d,
J ¼ 8.7 Hz,1H), 4.33 (t, J ¼ 5.7 Hz, 2H), 3.99 (s, 3H), 2.61 (t, J ¼ 5.7 Hz,
z ¼ 575.30. 1H NMR (400 MHz, DMSO)
9.29 (d, J ¼ 3.3 Hz, 1H), 9.12
d
(d, J ¼ 4.1 Hz, 1H), 8.82 (dd, J ¼ 3.8, 2.1 Hz, 1H), 8.54 (dd, J ¼ 6.5,
2.2 Hz, 1H), 8.46 (d, J ¼ 1.9 Hz,1H), 8.00e7.96 (m, 1H), 7.67e7.63 (m,
1H), 7.47 (d, J ¼ 8.4 Hz, 1H), 7.37 (dd, J ¼ 8.5, 2.1 Hz, 1H), 7.17 (d,
J ¼ 8.6 Hz, 1H), 6.96e6.93 (m, 1H), 4.27 (t, J ¼ 5.9 Hz, 2H), 3.99 (s,
3H), 3.91 (s, 3H), 2.74 (t, J ¼ 5.8 Hz, 2H), 2.57e2.53 (m, 4H), 0.97 (t,
J ¼ 7.1 Hz, 6H).
3H), 2.22 (s, 6H). 13C NMR (101 MHz, DMSO-d6)
d 166.01, 158.32,
155.49, 152.70, 151.94, 141.75, 136.27, 132.60, 131.35, 131.03, 128.60,
124.75, 122.46, 120.23, 119.57, 117.36, 114.69, 110.79, 62.77, 57.82,
56.66, 49.04, 45.86.
4.1.9. 2-(Dimethylamino)ethyl 3-(3-(5-(1H-indol-5-yl)-2-
(trifluoromethoxy)phenyl)ureido)-4-methoxybenzoate (6d)
Yield 57% as white solid. mp: 110e112 ꢁC, MS (ESI) [MeH]þ: m/
4.1.14. 2-(Diethylamino)ethyl 4-methoxy-3-(3-(5-(pyrimidin-5-yl)-
2-(trifluoromethoxy)phenyl)ureido)benzoate (6i)
Yield 54% as white solid. mp: 198e200 ꢁC, MS (ESI) [MeH]þ: m/
z ¼ 555.25. 1H NMR (400 MHz, DMSO)
d
11.22 (s, 1H), 9.24 (s, 1H),
z ¼ 546.25. 1H NMR (400 MHz, DMSO)
d 9.35 (s, 1H), 9.25 (s, 1H),
9.11 (s, 1H), 8.82 (d, 2.1 Hz, 1H), 8.60 (d, J ¼ 2.1 Hz, 1H), 7.82 (s, 1H),
7.66 (d, J ¼ 2.1 Hz,1H), 7.64 (t, J ¼ 8.6 Hz,1H),7.51 (d, J ¼ 15.1 Hz,1H),
7.42 (s, 1H), 7.39 (d, J ¼ 15.1 Hz, 2H), 7.17 (d, J ¼ 15.1 Hz, 1H),6.53 (s,
1H), 4.34 (t, J ¼ 5.7 Hz, 2H), 4.00 (s, 3H), 2.62 (t, J ¼ 5.6 Hz, 2H), 2.24
9.13 (d, J ¼ 8.7 Hz, 1H), 9.11 (s, 2H), 8.82 (s, 1H), 8.64 (s, 1H),
7.64e7.66 (m, 1H), 7.56e7.59 (m, 1H),7.51e7.54 (m, 1H),7.18 (d,
J ¼ 8.7 Hz,1H), 4.27 (t, J ¼ 5.9 Hz, 2H), 4.00 (s, 3H), 2.74 (t, J ¼ 5.9 Hz,
2H), 2.55e2.51 (m, 4H), 0.97 (t, J ¼ 7.1 Hz, 6H). 13C NMR (101 MHz,
(s, 6H). 13C NMR (101 MHz, DMSO-d6)
d
166.09, 152.89, 152.26,
DMSO-d6) d 166.09, 158.07, 155.33, 155.28, 152.73, 152.19, 138.30,
141.97, 136.61, 136.17, 132.84, 130.80, 128.80, 128.72, 126.73, 124.75,
122.41, 122.12, 121.23, 120.78, 120.11, 118.73, 112.39, 110.90, 102.09,
74.00, 62.77, 57.77, 56.62, 45.83, 25.40.
133.93, 133.46, 133.12, 128.61, 124.79, 122.64, 122.48, 122.23, 121.97,
121.78, 120.37, 120.15, 119.41, 110.87, 63.32, 56.61, 52.26, 51.11, 47.53,
12.38.
4.1.10. 2-(Dimethylamino)ethyl 4-methoxy-3-(3-(5-(6-
methoxypyridin-3-yl)-2-(trifluoromethoxy)phenyl)ureido)benzoate
(6e)
4.1.15. 2-(Diisopropylamino)ethyl 4-methoxy-3-(3-(3-(pyridin-3-
yl)-5-(trifluoromethyl)phenyl)ureido)benzoate (6j)
Yield 51% as white solid. mp: 105e106 ꢁC, MS (ESI) [MeH]þ: m/
Yield 52% as white solid. mp: 104e106 ꢁC, MS (ESI) [MeH]þ: m/
z ¼ 557.30. 1H NMR (400 MHz, DMSO)
d 9.87 (s, 1H), 8.94 (d,
z ¼ 547.25. 1H NMR (400 MHz, DMSO)
d
9.28 (s, 1H), 9.11 (s, 1H),
J ¼ 1.9 Hz, 1H), 8.82 (d, J ¼ 2.1 Hz, 1H), 8.66 (dd, J ¼ 4.8, 1.5 Hz, 1H),
8.51 (s,1H), 8.16e8.13 (m,1H),8.04 (s,1H), 7.86 (s,1H), 7.71e7.63 (m,
2H), 7.57e7.53 (m, 1H),7.17 (d, J ¼ 8.7 Hz, 1H), 4.17 (t, J ¼ 6.5 Hz, 2H),
3.99 (s, 3H), 3.04e3.01 (m, 2H), 2.73 (t, J ¼ 6.5 Hz, 2H), 1.00 (s, 6H),
8.80 (d, J ¼ 2.1 Hz, 1H), 8.52 (s, 1H), 8.47 (s, 1H), 7.99 (dd, J ¼ 8.6,
2.6 Hz, 1H), 7.65 (dd, J ¼ 8.5, 2.1 Hz, 1H), 7.48 (d, J ¼ 7.3 Hz, 1H), 7.38
(dd, J ¼ 8.5, 2.2 Hz, 1H), 7.17 (d, J ¼ 8.7 Hz, 1H), 6.95 (d, J ¼ 8.6 Hz,
1H), 4.33 (t, J ¼ 5.7 Hz, 2H), 3.99 (s, 3H), 3.91 (s, 3H), 2.60 (t,
0.98 (s, 6H). 13C NMR (101 MHz, DMSO-d6)
d 166.13, 152.69, 151.83,
J ¼ 5.7 Hz, 2H), 2.22 (s, 6H). 13C NMR (101 MHz, DMSO-d6)
d
166.06,
149.73, 148.21, 141.71, 139.49, 134.94, 134.78, 131.22, 130.91, 128.61,
125.84, 124.67, 124.41, 123.13, 122.64, 120.12, 119.55, 117.13, 113.97,
110.74, 65.45, 56.63, 48.92, 43.60, 21.16.
163.79, 155.61, 152.80, 152.26, 145.33, 138.18, 137.18, 133.12, 129.02,
128.68, 124.83, 122.40, 121.18, 120.16, 119.94, 111.20, 110.97, 62.81,
57.82, 56.69, 53.81, 49.06, 45.90.
4.1.16. 2-(Diisopropylamino)ethyl 3-(3-(3-(1H-indol-5-yl)-5-
(trifluoromethyl)phenyl)ureido)-4-methoxybenzoate (6k)
Yield 52% as white solid. mp: 121e123 ꢁC, MS (ESI) [MeH]þ: m/
4.1.11. 2-(Dimethylamino)ethyl 4-methoxy-3-(3-(5-(pyrimidin-5-
yl)-2-(trifluoromethoxy)phenyl)ureido)benzoate (6f)
Yield 55% as white solid. mp: 221e222 ꢁC, MS (ESI) [MeH]þ: m/
z ¼ 595.35. 1H NMR (400 MHz, DMSO)
d 11.25 (s, 1H), 9.82 (s, 1H),
z ¼ 518.20. 1H NMR (400 MHz, DMSO)
d
9.35 (s, 1H), 9.24 (s, 1H),
8.83 (d, J ¼ 2.1 Hz, 1H), 8.45 (s, 1H), 7.91 (s, 1H), 7.89 (s, 1H), 7.86 (s,
1H), 7.66 (dd, J ¼ 8.5, 2.1 Hz, 1H), 7.56 (s, 1H), 7.53 (d, J ¼ 8.5 Hz, 1H),
7.45e7.42 (m, 2H), 7.17 (d, J ¼ 8.6 Hz, 1H), 6.54 (s, 1H), 4.17 (t,
J ¼ 6.5 Hz, 2H), 3.99 (s, 3H), 3.06e3.03 (m, 2H), 2.74 (t, J ¼ 6.5 Hz,
2H), 0.99 (d, J ¼ 6.5 Hz, 12H). 13C NMR (101 MHz, DMSO-d6)
9.13 (s, 1H), 9.12 (s, 2H), 8.80 (d, J ¼ 2.1 Hz, 1H), 8.62 (d, J ¼ 2.1 Hz,
1H), 7.67e7.64 (m, 1H), 7.59e7.56 (m, 1H), 7.54e7.51 (m, 1H),7.18 (d,
J ¼ 8.7 Hz,1H), 4.33 (t, J ¼ 5.7 Hz, 2H), 4.00 (s, 3H), 2.60 (t, J ¼ 5.7 Hz,
2H), 2.22 (s, 6H). 13C NMR (101 MHz, DMSO-d6)
d 166.02, 158.09,
155.33, 152.75, 152.26, 138.40, 133.97, 133.40, 133.13, 128.61, 124.88,
122.69, 122.41, 121.92, 120.56, 120.19, 119.41, 110.92, 62.76, 57.79,
56.65, 45.84.
d 166.20, 152.83, 152.16, 141.98, 136.54, 136.17, 132.88, 130.80,
128.76, 128.71, 126.71, 124.71, 122.58, 122.10, 121.17, 120.79, 120.16,
119.99, 118.73, 112.36, 110.83, 102.07, 65.42, 56.59, 48.90, 43.59,
21.20.
4.1.12. 2-(Diethylamino)ethyl 3-(3-(5-(1H-indol-5-yl)-2-
(trifluoromethoxy)phenyl)ureido)-4-methoxybenzoate (6g)
Yield 56% as slight yellow solid. mp: 114e115 ꢁC, MS (ESI)
4.1.17. 2-(Diisopropylamino)ethyl 3-(3-(5-(1H-indol-5-yl)-2-
(trifluoromethoxy)phenyl)ureido)-4-methoxybenzoate (6l)
Yield 53% as slight yellow solid. mp: 135e137 ꢁC, MS (ESI)
[MeH]þ: m/z ¼ 583.30. 1H NMR (400 MHz, DMSO)
d 11.21 (s, 1H),
9.23 (s, 1H), 9.11 (s, 1H), 8.84 (d, J ¼ 2.1 Hz, 1H), 8.61 (d, J ¼ 2.0 Hz,
1H), 7.82 (s, 1H), 7.65 (dd, J ¼ 8.5, 2.1 Hz, 1H), 7.50 (d, J ¼ 8.4 Hz, 1H),
7.42 (s, 1H), 7.40 (t, J ¼ 5.8 Hz, 2H), 7.38 (d, J ¼ 2.1 Hz, 1H),7.18 (d,
J ¼ 8.7 Hz, 1H), 6.53 (s, 1H), 4.28 (t, J ¼ 5.8 Hz, 2H), 4.00 (s, 3H), 2.75
[MeH]þ: m/z ¼ 611.30. 1H NMR (400 MHz, DMSO)
d 11.21 (s, 1H),
9.23 (s, 1H), 9.11 (s, 1H), 8.83 (d, J ¼ 2.0 Hz, 1H), 8.61 (d, J ¼ 1.9 Hz,
1H), 7.82 (s, 1H), 7.66 (dd, J ¼ 8.6, 2.0 Hz, 1H), 7.50 (d, J ¼ 8.0 Hz, 1H),
7.41 (d, J ¼ 2.0 Hz, 1H), 7.40 (d, J ¼ 2.0 Hz, 2H), 7.38 (s, 1H), 7.17 (d,
J ¼ 8.6 Hz, 1H), 6.53 (s, 1H), 4.16 (t, J ¼ 6.4 Hz, 2H), 3.99 (s, 3H),
3.03e3.00 (m, 2H), 2.73 (t, J ¼ 6.4 Hz, 2H), 0.99 (s, 6H), 0.97 (s, 6H).
(t, J ¼ 5.9 Hz, 2H), 2.58e2.53 (m, 4H), 0.98 (t, J ¼ 7.1 Hz, 6H). 13
C
NMR (101 MHz, DMSO-d6)
d 166.19, 152.85, 152.20, 141.97, 136.55,
136.17, 132.89, 130.80, 128.80, 128.72, 126.72, 124.68, 122.52, 122.10,
121.16, 120.78, 120.13, 119.99, 119.54, 118.73, 112.36, 110.88, 102.07,
63.48, 56.60, 51.13, 47.56, 12.52.
13C NMR (101 MHz, DMSO-d6)
d 166.18, 152.75, 151.81, 144.26,
141.42, 136.38, 130.84, 130.54, 130.25, 128.77, 128.73, 126.87, 126.10,
124.61, 123.39, 122.66, 120.67, 119.92, 119.50, 118.96, 116.79, 112.52,
112.20, 110.77, 102.19, 65.47, 56.65, 48.92, 43.62, 21.19.
4.1.13. 2-(Diethylamino)ethyl 4-methoxy-3-(3-(5-(6-
methoxypyridin-3-yl)-2-(trifluoromethoxy)phenyl)ureido)benzoate
(6h)
4.1.18. 2-(Diisopropylamino)ethyl 4-methoxy-3-(3-(5-(pyrimidin-
5-yl)-2-(trifluoromethoxy)phenyl)ureido)benzoate (6m)
Yield 57% as white solid. mp: 90e92 ꢁC, MS (ESI) [MeH]þ: m/
Yield 52% as white solid. mp: 185e187 ꢁC, MS (ESI) [MeH]þ: m/

Y. Shan et al. / European Journal of Medicinal Chemistry 103 (2015) 80e90
87
z ¼ 574.30. 1H NMR (400 MHz, DMSO)
d
9.35 (d, J ¼ 3.9 Hz, 1H), 9.24
NMR (101 MHz, DMSO-d6)
d
166.02, 158.06, 155.29, 152.75, 152.23,
(s, 1H), 9.13 (d, J ¼ 2.1 Hz, 1H), 9.11 (s, 2H), 8.81 (d, J ¼ 2.0 Hz, 1H),
8.64 (d, J ¼ 2.0 Hz, 1H), 7.66 (dd, J ¼ 8.6, 2.1 Hz, 1H), 7.56 (s, 1H), 7.53
(d, J ¼ 2.0 Hz, 1H),7.17 (dd, J ¼ 8.7, 2.1 Hz, 1H), 4.15 (t, J ¼ 6.4 Hz, 2H),
3.99 (s, 3H), 3.02e2.99 (m, 2H), 2.71 (t, J ¼ 6.4 Hz, 2H), 0.98 (s, 6H),
138.36, 133.96, 133.40, 133.12, 128.63, 124.84, 122.63, 122.42, 121.97,
121.88, 120.46, 120.17, 119.41, 110.92, 66.66, 62.32, 57.02, 56.63,
53.90.
0.96 (s, 6H). 13C NMR (101 MHz, DMSO-d6)
152.72, 152.19, 138.36, 134.00, 133.44, 133.14, 128.56, 124.86, 122.73,
122.55, 121.93, 120.50, 120.16, 119.41, 110.93, 65.40, 56.66, 48.93,
43.63, 21.18.
d
166.13, 158.11, 155.35,
4.1.24. 2-Morpholinoethyl 3-(3-(5-(1H-pyrazol-4-yl)-2-
(trifluoromethoxy)phenyl)ureido)-4-methoxybenzoate (6s)
Yield 50% as white solid. mp: 148e150 ꢁC, MS (ESI) [MeH]þ: m/
z ¼ 548.25. 1H NMR (400 MHz, DMSO)
d 13.01 (s, 1H), 9.16 (s, 1H),
9.07 (s, 1H), 8.82 (d, J ¼ 2.1 Hz, 1H), 8.40 (d, J ¼ 1.7 Hz, 1H), 7.65 (dd,
J ¼ 8.6, 2.1 Hz, 1H), 7.35 (d, J ¼ 2.1 Hz, 1H), 7.17 (d, J ¼ 8.7 Hz, 1H),
4.37 (t, J ¼ 5.7 Hz, 2H), 3.99 (s, 3H), 3.58 (t, J ¼ 8.7 Hz, 4H), 2.68 (t,
4.1.19. 2-(Diisopropylamino)ethyl 3-(3-(5-(1H-pyrazol-4-yl)-2-
(trifluoromethoxy)phenyl)ureido)-4-methoxybenzoate (6n)
Yield 51% as white solid. mp: 126e128 ꢁC, MS (ESI) [MeH]þ: m/
J ¼ 5.7 Hz, 2H), 2.50 (s, 4H). 13C NMR (101 MHz, DMSO-d6)
d 166.08,
z ¼ 562.30. 1H NMR (400 MHz, DMSO)
d
13.02 (s, 1H),9.16 (s, 1H),
152.81, 152.23, 136.34, 133.14, 132.92, 128.80, 124.74, 122.40, 122.35,
120.79, 120.18, 120.10, 118.77, 110.95, 66.68, 62.43, 57.02, 56.65,
53.94, 25.40.
9.06 (s, 1H), 8.81 (s, 1H), 8.41 (d, J ¼ 1.6 Hz, 1H), 7.68e7.65 (m, 1H),
7.34 (d, J ¼ 2.1 Hz, 2H), 7.17 (d, J ¼ 8.7 Hz, 1H), 4.17 (t, J ¼ 5.7 Hz,
2H),3.99 (s, 3H), 3.01 (t, J ¼ 5.7 Hz, 2H), 2.70 (t, J ¼ 6.5 Hz, 2H), 0.99
(s, 6H), 0.98 (s, 6H). 13C NMR (101 MHz, DMSO-d6)
d
166.17, 152.77,
4.1.25. 4-Iodo-1H-indazol-3-amine (8)
152.19, 136.32, 133.13, 132.94, 128.75, 124.74, 122.50, 122.36, 120.79,
120.16, 118.76, 110.89, 56.64, 49.08, 21.12.
2-Fluoro-6-iodobenzonitrile (7) (10.0 g, 40 mmol) and NaHCO3
(5.2 g, 62 mmol) were suspended in ethanol (60 mL). Aqueous
hydrazine monohydrate (12.5 mL) was added onto the above so-
lution. The resulting mixture was heated to reflux and stirred for
8 h. After cooling to room temperature, water (60 mL) was added to
the reaction mixture and stirred for another 2 h. The reaction
mixture was filtered off and washed with water to give (8) (8.6 g,
83%) as slight yellow solid. mp.: 150e152 ꢁC.
4.1.20. 2-(Diisopropylamino)ethyl 4-methoxy-3-(3-(5-(1-methyl-
1H-pyrazol-4-yl)-2-(trifluoromethoxy)phenyl)ureido) benzoate
(6o)
Yield 53% as white solid. mp: 100e102 ꢁC, MS (ESI) [MeH]þ: m/
z ¼ 576.30. 1H NMR (400 MHz, DMSO)
d 9.18 (s, 1H), 9.13 (s, 1H),
8.82 (d, J ¼ 2.1 Hz, 1H), 8.42 (d, J ¼ 2.1 Hz, 1H), 8.15 (s, 1H), 7.82 (s,
1H), 7.67 (s, 1H), 7.65 (s, 1H), 7.35 (s, 1H), 7.30 (d, J ¼ 2.0 Hz, 1H), 7.16
(d, J ¼ 8.6 Hz, 1H), 4.17 (t, J ¼ 5.7 Hz, 2H), 3.99 (s, 3H), 3.88 (s, 3H),
3.04e2.99 (m, 2H), 2.73 (t, J ¼ 5.7 Hz, 2H), 0.98 (s, 6H), 0.96 (s, 6H).
4.1.26. [3-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]
amine (10)
A round bottom flask charged with (3-aminophenyl)boronic
acid (9) (20.0 g, 148 mmol), Pinacol (19.2 g, 163 mmol) and MgSO4
(46.6 g, 388 mmol), THF (250 mL) was then added, the mixture was
stirred at room temperature for 5 h. After completion of reaction,
the reaction mixture was filtered off and the organic layer was
washed with brine twice, and dried over Na2SO4. Filtration and
concentration in vacuo afforded (10) (21.5 g, 76%) as yellow solid.
4.1.21. 2-Morpholinoethyl 4-methoxy-3-(3-(3-(pyridin-3-yl)-5-
(trifluoromethyl)phenyl)ureido)benzoate (6p)
Yield 52% as white solid. mp: 212e213 ꢁC, MS (ESI) [MeH]þ: m/
z ¼ 543.15. 1H NMR (400 MHz, DMSO)
d 9.88 (s, 1H), 8.94 (s, 1H),
8.82 (d, J ¼ 2.1 Hz, 1H), 8.66 (d, J ¼ 2.1 Hz, 1H), 8.52 (s, 1H), 8.15 (d,
J ¼ 8.7 Hz, 1H), 8.04 (s, 1H), 7.85 (s, 1H), 7.67e7.64 (m, 2H),
7.57e7.54 (m, 1H),7.18 (d, J ¼ 8.7 Hz,1H), 4.38 (t, J ¼ 5.7 Hz, 2H), 3.99
(s, 3H), 3.58 (t, J ¼ 8.7 Hz, 4H), 2.69 (t, J ¼ 5.7 Hz, 2H), 2.51 (t,
4.1.27. N-(2-fluorophenyl)-N0-[3-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)phenyl]urea (11a)
J ¼ 8.7 Hz, 4H). 13C NMR (101 MHz, DMSO-d6)
d
166.02, 152.73,
Triphosgene (0.80 g, 2.7 mmol) was dissolved in anhydrous
CH2Cl2 (20 mL) and the mixture was stirred on the ice-bath for
15 min. A solution of 2-fluoroaniline (0.76 g, 6.8 mmol) in anhy-
drous CH2Cl2 was added dropwise to the above mixture and stirring
was continued for 30 min. Then triethanolamine (0.83 mL,
8.2 mmol) diluted with CH2Cl2 (10 mL) was then added onto the
mixture. Stirring was continued for 20 min, a solution of trietha-
nolamine (0.83 mL, 8.2 mmol) and intermediate (10) (1.50 g,
6.8 mmol) in anhydrous CH2Cl2 (10 mL) was added. After comple-
tion 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 residues
was purified by silica gel flash chromatography (PE/AcOEt ¼ 3:1)
gave as white solid (11a) (1.06 g, 43.3%).
151.90, 149.74, 148.22, 141.68, 139.50, 134.96, 134.77, 131.23, 130.91,
128.67, 125.83, 124.70, 124.44, 123.12, 122.48, 120.15, 119.49, 117.14,
113.98, 110.84, 66.68, 62.44, 57.04, 56.67, 53.95.
4.1.22. 2-Morpholinoethyl 3-(3-(3-(1H-indol-5-yl)-5-
(trifluoromethyl)phenyl)ureido)-4-methoxybenzoate (6q)
Yield 54% as white solid. mp: 229e231 ꢁC, MS (ESI) [MeH]þ: m/
z ¼ 581.25. 1H NMR (400 MHz, DMSO)
d 11.25 (s, 1H), 9.82 (s, 1H),
8.84 (d, J ¼ 1.9 Hz, 1H), 8.46 (s, 1H), 7.89 (s, 2H), 7.87 (s, 1H), 7.65 (d,
J ¼ 8.7 Hz, 1H), 7.56 (s, 1H), 7.52 (s, 1H), 7.43 (s, 2H), 7.17 (d,
J ¼ 8.7 Hz, 1H), 6.54 (s, 1H), 4.38 (s, 2H), 3.99 (s, 3H), 3.59 (d,
J ¼ 8.7 Hz, 4H), 2.69 (s, 2H), 2.51 (s, 4H). 13C NMR (101 MHz, DMSO-
d6)
d 166.06, 152.79, 151.86, 144.28, 141.41, 136.40, 130.86, 130.55,
130.26, 128.80, 126.86, 126.11, 124.62, 123.40, 122.52, 120.68, 119.93,
119.45, 118.98, 116.83, 112.54, 112.17, 110.83, 102.22, 66.71, 62.46,
57.05, 56.66, 53.97.
4.1.28. N-[3-(3-amino-1H-indazol-4-yl)phenyl]-N0-(2-
fluorophenyl)urea (12a)
A flask charged with Pd(PPh3)4 (0.30 g, 0.267 mmol), sodium
carbonate (0.7 g, 6.6 mmol), and intermediates (8) (0.70 g,
2.7 mmol) and (11a) (1.0 g, 2.8 mmol) (0.60 g, 1.2 mmol) were
flushed with nitrogen and suspended in 1,4-dioxane (25 mL) and
water (5 mL). The mixture was then refluxed overnight under ni-
trogen. The hot suspension was filtered and the filtrate distilled by
rotary evaporation to remove 1,4-dioxane. Water (150 mL) was
added and the product was extracted with AcOEt (50 mL ꢀ 3),
washed with water, and dried over Na2SO4. After filtration and
4.1.23. 2-Morpholinoethyl 4-methoxy-3-(3-(5-(pyrimidin-5-yl)-2-
(trifluoromethoxy)phenyl)ureido)benzoate (6r)
Yield 51% as slight yellow solid. mp: 159e160 ꢁC, MS (ESI)
[MeH]þ: m/z ¼ 560.20. 1H NMR (400 MHz, DMSO)
d 9.35 (s, 1H),
9.24 (s, 1H), 9.14 (s, 1H), 9.12 (s, 2H), 8.81 (d, J ¼ 2.1 Hz, 1H), 8.62 (d,
J ¼ 2.1 Hz, 1H), 7.67e7.64 (m, 1H), 7.57 (s, 1H), 7.54e7.51 (m, 1H),
7.18 (d, J ¼ 8.7 Hz, 1H), 4.36 (t, J ¼ 5.7 Hz, 2H), 4.00 (s, 3H), 3.55 (t,
J ¼ 8.7 Hz, 4H), 2.67 (t, J ¼ 5.7 Hz, 2H), 2.48 (t, J ¼ 8.7 Hz, 4H). 13
C

88
Y. Shan et al. / European Journal of Medicinal Chemistry 103 (2015) 80e90
concentration in vacuo, the residue was purified by silica gel flash
chromatography (PE/AcOEt ¼ 1:1) affording 12a (0.56 g, 56%) as
slight yellow solid. mp: 107e109 ꢁC, MS (ESI) [MeH]þ: m/
119.20, 118.93, 117.93, 110.94, 109.46, 20.80.
4.1.34. N-[3-(3-amino-1H-indazol-4-yl)phenyl]-N0-(3-
isopropylphenyl)urea (12g)
z ¼ 360.10. 1H NMR (400 MHz, DMSO)
d 11.77 (s, 1H), 9.25 (s, 1H),
8.61 (s, 1H), 8.15 (t, J ¼ 8.2 Hz, 1H), 7.64 (s, 1H), 7.44 (d, J ¼ 4.3 Hz,
1H), 7.30 (d, J ¼ 4.3 Hz, 2H), 7.23 (d, J ¼ 8.0 Hz, 1H),7.16e7.10 (m,
Yield 54% as white solid. mp: 197e198 ꢁC, MS (ESI) [MeH]þ: m/
z ¼ 384.20. 1H NMR (400 MHz, DMSO)
d 11.77 (s, 1H), 8.81 (s, 1H),
2H), 7.02 (t, J ¼ 8.2 Hz, 1H),6.83 (t, J ¼ 3.9 Hz, 1H), 4.44 (s, 2H). 13
C
8.68 (s, 1H), 7.63 (d, J ¼ 1.8 Hz, 1H), 7.46e7.40 (m, 2H), 7.35 (d,
J ¼ 1.7 Hz, 1H), 7.30e7.28 (m, 2H), 7.27e7.25 (m, 1H), 7.19 (t,
J ¼ 7.8 Hz, 1H), 7.09e7.06 (m, 1H), 6.86 (d, J ¼ 7.5 Hz, 1H), 6.84e6.81
NMR (101 MHz, DMSO-d6)
d 164.10, 161.70, 153.07, 148.60, 142.59,
142.04, 141.93, 140.45, 139.82, 136.09, 130.81, 130.71, 129.51, 126.72,
123.04, 119.56, 119.44, 118.18, 114.52, 114.50, 110.93, 109.49, 108.81,
108.60, 105.62, 105.36.
(m, 1H), 4.45 (s, 2H), 2.88e2.81 (m, 1H), 1.19 (d, J ¼ 6.9 Hz, 6H). 13
C
NMR (101 MHz, DMSO-d6)
d 153.22, 149.55, 148.60, 142.59, 140.43,
The title compounds 12b~12s were also prepared by using the
general procedure described above.
140.17, 140.02, 136.17, 129.47, 129.15, 126.72, 122.73, 120.50, 119.53,
119.19, 117.97, 116.81, 116.45, 110.95, 109.46, 33.98, 24.33.
4.1.29. N-[3-(3-amino-1H-indazol-4-yl)phenyl]-N0-(3-
fluorophenyl)urea (12b)
4.1.35. N-[3-(3-amino-1H-indazol-4-yl)phenyl]-N0-[3-
(trifluoromethyl)phenyl]urea (12h)
Yield 52% as white solid. mp: 192e194 ꢁC, MS (ESI) [MeH]þ: m/
Yield 53% as white solid. mp: 187e188 ꢁC, MS (ESI) [MeH]þ: m/
z ¼ 360.10. 1H NMR (400 MHz, DMSO)
d
11.77 (s, 1H), 8.98 (s, 1H),
z ¼ 410.15. 1H NMR (400 MHz, DMSO)
d 11.78 (s, 1H), 9.12 (s, 1H),
8.93 (s, 1H), 7.63 (d, J ¼ 1.8 Hz, 1H), 7.53e7.49 (m, 1H), 7.48e7.41 (m,
2H), 7.34e7.28 (m, 3H), 7.15e7.09 (m, 2H),6.85e6.77 (m, 2H), 4.46
8.99 (s, 1H), 8.02 (s, 1H), 7.64 (s, 1H), 7.60 (d, J ¼ 12 Hz, 1H), 7.53 (d,
J ¼ 8 Hz, 1H), 7.48 (d, J ¼ 8 Hz, 1H), 7.44 (t, J ¼ 7.8 Hz, 1H), 7.31 (t,
J ¼ 7.8 Hz, 1H), 7.11 (d, J ¼ 8 Hz, 1H), 6.85e6.80 (m, 1H), 4.44 (s, 2H).
(s, 2H). 13C NMR (101 MHz, DMSO-d6)
d 153.75, 152.84, 151.35,
148.55, 142.58, 140.48, 139.83, 136.06, 129.56, 127.96, 127.86, 126.71,
124.99, 124.96, 123.10, 123.01, 121.18, 119.53, 119.12, 117.86, 115.54,
115.35, 110.89, 109.49, 74.02.
13C NMR (101 MHz, DMSO-d6)
d 160.18, 153.14, 148.60, 142.58,
141.29, 140.42, 140.04, 136.13, 130.03, 129.49, 126.73, 122.83, 119.55,
119.27, 118.04, 111.13, 110.93, 109.47, 107.83, 104.59, 55.40.
4.1.30. N-[3-(3-amino-1H-indazol-4-yl)phenyl]-N0-(4-
fluorophenyl)urea (12c)
4.1.36. N-[3-(3-amino-1H-indazol-4-yl)phenyl]-N0-(3-
methoxyphenyl)urea (12i)
Yield 51% as slight yellow solid. mp: 204e206 ꢁC, MS (ESI)
Yield 52% as white solid. mp: 166e167 ꢁC, MS (ESI) [MeH]þ: m/
[MeH]þ: m/z ¼ 360.10.1H NMR (400 MHz, DMSO)
d
11.77 (s, 1H),
z ¼ 372.10. 1H NMR (400 MHz, DMSO)
d 11.77 (s, 1H), 8.84 (s, 1H),
8.85 (s, 1H), 8.77 (s, 1H), 7.62 (s, 1H), 7.48 (dd, J ¼ 5.7, 3.5 Hz, 1H),
7.45 (dd, J ¼ 6.8, 2.6 Hz, 1H), 7.41 (d, J ¼ 8.1 Hz, 1H), 7.30 (d,
J ¼ 3.9 Hz, 2H), 7.12 (s, 1H), 7.10 (d, J ¼ 3.6 Hz, 1H), 6.84e6.80 (m,
8.75 (s, 1H), 7.63 (s, 1H), 7.46e7.40 (m, 2H), 7.30 (s, 1H), 7.29 (s,
1H),7.20e7.16 (m, 2H), 7.10e7.07 (m, 1H), 6.96e6.93 (m, 1H), 6.82 (t,
J ¼ 3.9 Hz, 1H), 6.56 (dd, J ¼ 8.1, 2.1 Hz, 1H), 4.45 (s, 2H), 3.73 (s, 3H).
1H), 4.48 (s, 2H). 13C NMR (101 MHz, DMSO-d6)
d
159.08, 156.71,
13C NMR (101 MHz, DMSO-d6)
d 153.16, 148.57, 142.56, 140.98,
153.30, 148.57, 142.58, 140.41, 140.07, 136.40, 136.38, 136.13, 132.54,
132.01, 131.91, 129.48, 129.29, 129.17, 126.71, 122.79, 120.65, 120.58,
119.52, 119.30, 118.04, 115.85, 115.63, 110.91, 109.46.
140.44,139.79,136.06,130.35,130.19,129.88,129.49,126.72,126.03,
123.32, 123.10, 122.41, 119.55, 119.44, 118.61, 118.24, 114.73, 114.69,
110.91, 109.48.
4.1.31. N-[3-(3-amino-1H-indazol-4-yl)phenyl]-N0-(2-
chlorophenyl)urea (12d)
4.1.37. N-[3-(3-amino-1H-indazol-4-yl)phenyl]-N0-(2,6-
dimethylphenyl)urea (12j)
Yield 54% as slight yellow solid. mp: 110e112 ꢁC, MS (ESI)
Yield 50% as white solid. mp: 239e241 ꢁC, MS (ESI) [MeH]þ: m/
[MeH]þ: m/z ¼ 376.15. 1H NMR (400 MHz, DMSO)
d
11.78 (s, 1H),
z ¼ 370.10. 1H NMR (400 MHz, DMSO)
d 11.74 (s, 1H), 8.94 (s, 1H),
9.61 (s, 1H), 8.37 (s, 1H), 8.17 (dd, J ¼ 8.3, 1.3 Hz, 1H), 7.65 (s, 1H),
7.48e7.45 (m, 3H), 7.32e7.28 (m, 3H), 7.12 (t, J ¼ 8.3 Hz,
1H),7.06e7.02 (m, 1H), 6.84 (t, J ¼ 3.9 Hz, 1H), 4.45 (s, 2H).
7.79 (s, 1H), 7.61 (d, J ¼ 3.5 Hz, 1H), 7.46 (d, J ¼ 8.5 Hz, 1H), 7.40 (t,
J ¼ 13.5 Hz, 1H),7.30e7.28 (m, 1H), 7.27 (s, 1H), 7.07 (s, 3H), 7.04
(d,J ¼ 8.5 Hz, 1H),6.80 (dd, J ¼ 4.3, 3.5 Hz, 1H), 4.46 (s, 2H), 2.22 (s,
6H). 13C NMR (101 MHz, DMSO-d6)
d13C NMR (101 MHz, DMSO)
4.1.32. N-[3-(3-amino-1H-indazol-4-yl)phenyl]-N0-(3-
chlorophenyl)urea (12e)
d 153.84, 148.58, 142.58, 140.69, 140.36, 136.24, 136.09, 135.69,
129.41, 128.20, 126.69, 126.51, 122.34, 119.49, 119.09, 117.73, 110.94,
109.41, 18.73.
Yield 51% as white solid. mp: 190e191 ꢁC, MS (ESI) [MeH]þ: m/
z ¼ 376.15. 1H NMR (400 MHz, DMSO)
d 11.78 (s, 1H), 8.96 (s, 1H),
8.94 (s, 1H), 7.73 (d, J ¼ 1.8 Hz, 1H), 7.63 (d, J ¼ 1.7 Hz, 1H), 7.48e7.41
(m, 2H), 7.33e7.26 (m, 4H), 7.12e7.09 (m, 1H), 7.04e7.01 (m, 1H),
6.85e6.80 (m, 1H), 4.46 (s, 2H). 13C NMR (101 MHz, DMSO-d6)
4.1.38. N-[3-(3-amino-1H-indazol-4-yl)phenyl]-N0-(3,4-
difluorophenyl)urea (12k)
Yield 53% as white solid. mp: 194e195 ꢁC, MS (ESI) [MeH]þ: m/
d
153.06, 148.59, 142.59, 141.66, 140.45, 139.82, 136.08, 133.72,
z ¼ 378.10. 1H NMR (400 MHz, DMSO)
d 11.78 (s, 1H), 8.97 (s, 1H),
130.83, 129.51, 126.72, 123.05, 122.03, 119.56, 119.44, 118.18, 117.22,
110.93, 109.49.
8.94 (s, 1H), 7.72e7.65 (m, 1H), 7.62 (s, 1H), 7.45 (d, J ¼ 2.1 Hz, 1H),
7.45 (t, J ¼ 5.7 Hz, 1H), 7.34 (d, J ¼ 12 Hz, 1H), 7.31e7.27 (m, 2H),
7.16e7.09 (m, 2H), 6.85e6.80 (m, 1H), 4.46 (s, 2H). 13C NMR
4.1.33. N-[3-(3-amino-1H-indazol-4-yl)phenyl]-N0-(4-
methylphenyl)urea (12f)
(101 MHz, DMSO-d6) d153.17, 148.62, 142.63, 140.44, 139.81, 137.27,
137.25, 137.18, 137.15, 136.10, 129.49, 126.72, 123.05, 119.59, 119.54,
118.25, 117.83, 117.65, 114.97, 114.91, 110.94, 109.50, 107.93, 107.71,
60.22, 21.11, 14.44.
Yield 52% as white solid. mp: 195e196 ꢁC, MS (ESI) [MeH]þ: m/
z ¼ 356.15. 1H NMR (400 MHz, DMSO)
d 11.76 (s, 1H), 8.80 (s, 1H),
8.62 (s, 1H), 7.62 (s, 1H), 7.45e7.39 (m, 2H), 7.35 (D, J ¼ 8.7 Hz, 2H),
7.30 (s, 1H), 7.29 (s, 1H), 7.10 (s, 1H), 7.08e7.06 (m, 2H), 6.82 (t,
J ¼ 3.9 Hz, 1H), 4.45 (s, 2H), 2.24 (s, 3H). 13C NMR (101 MHz, DMSO-
4.1.39. N-[3-(3-amino-1H-indazol-4-yl)phenyl]-N0-(2,4-
dichlorophenyl)urea (12l)
d6)
d13C NMR (101 MHz, DMSO)
d
153.26, 148.60, 142.60, 140.42,
Yield 55% as white solid. mp: 183e184 ꢁC, MS (ESI) [MeH]þ: m/
140.21, 137.48, 136.18, 131.28, 129.66, 129.48, 126.72, 122.67, 119.53,
z ¼ 410.00. 1H NMR (400 MHz, DMSO)
d 11.78 (s, 1H), 9.63 (s, 1H),

Y. Shan et al. / European Journal of Medicinal Chemistry 103 (2015) 80e90
89
8.45 (s, 1H), 8.21 (d, J ¼ 9.0 Hz, 1H), 7.64 (s, 2H),7.48e7.45 (m, 2H),
7.40e7.37 (m, 1H),7.29 (t, J ¼ 4.3 Hz, 2H), 7.14e7.11 (m, 1H), 6.83 (t,
2H), 7.43 (d, J ¼ 8.5 Hz, 2H), 7.30 (d, J ¼ 5.2 Hz, 2H), 7.27 (s, 1H), 6.88
(d, J ¼ 5.0 Hz, 1H), 6.81e6.79 (m, 1H), 4.35 (s, 2H), 2.31 (s, 3H). 13
C
J ¼ 3.9 Hz, 1H), 4.44 (s, 2H). 13C NMR (101 MHz, DMSO-d6)
d
152.63,
NMR (101 MHz, DMSO-d6) d 153.44, 152.78, 149.82, 148.61, 146.91,
142.58, 139.10, 135.86, 133.74, 129.84, 126.72, 119.58, 119.22, 119.13,
112.48, 110.99, 109.10, 21.33.
148.53,142.57, 140.49,139.66, 136.01, 135.62, 129.60, 129.02, 128.09,
126.69, 126.67, 123.20, 123.17, 122.65, 119.54, 119.28, 117.97, 110.87,
109.50.
4.1.46. N-[4-(3-amino-1H-indazol-4-yl)phenyl]-N0-{4-[2-
(dimethylamino)ethoxy]-3-fluorophenyl}urea (12s)
4.1.40. N-[3-(3-amino-1H-indazol-4-yl)phenyl]-N0-[3,5-
bis(trifluoromethyl)phenyl]urea (12m)
Yield 54% as slight yellow solid. mp: 136e138 ꢁC, MS (ESI)
Yield 51% as white solid. mp: 203e204 ꢁC, MS (ESI) [MeH]þ: m/
[MþH]þ: m/z ¼ 449.15. 1H NMR (400 MHz, DMSO)
d 11.73 (s, 1H),
z ¼ 478.10. 1H NMR (400 MHz, DMSO)
d
11.78 (s, 1H), 9.47 (s, 1H),
8.93 (s, 1H), 8.85 (s, 1H), 7.59 (d, J ¼ 8.4 Hz, 2H), 7.54e7.49 (m, 1H),
7.40 (d, J ¼ 8.4 Hz, 2H), 7.28e7.24 (m, 2H), 7.15e7.08 (m, 2H), 6.79
(dd, J ¼ 5.8, 1.9 Hz, 1H), 4.34 (s, 1H), 4.09 (t, J ¼ 5.8 Hz, 2H), 2.65 (t,
9.19 (s, 1H), 8.15 (s, 2H), 7.65 (t, J ¼ 4.3 Hz, 2H), 7.53e7.51 (m, 1H),
7.45 (t, J ¼ 4.3 Hz, 1H), 7.31 (t, J ¼ 4.3 Hz, 2H), 7.13 (d, J ¼ 7.5 Hz, 1H),
6.85e6.82 (m, 1H), 4.43 (s, 2H). 13C NMR (101 MHz, DMSO-d6)
J ¼ 5.7 Hz, 2H), 2.24 (s, 6H). 13C NMR (101 MHz, DMSO-d6)
d 153.08,
d
153.05, 148.55, 142.54, 142.29, 140.45, 139.49, 135.99, 131.69,
148.59, 142.55, 139.74, 135.95, 134.10, 133.09, 129.73, 126.70, 119.51,
118.50, 116.34, 114.60, 111.00, 109.01, 107.51, 107.28, 67.94, 58.07,
45.95.
131.36, 131.04, 130.71, 129.45, 126.67, 125.13, 123.43, 122.42, 119.75,
119.53, 118.56, 114.83, 110.92, 109.48.
4.1.41. N-[3-(3-amino-1H-indazol-4-yl)phenyl]-N0-(3-chloro-4-
methylphenyl)urea (12n)
4.2. Kinase assay [17]
Yield 56% as white solid. mp: 214e215 ꢁC, MS (ESI) [MeH]þ: m/
The in vitro VEGFR-2 inhibition assays of all the title compounds
were detected using the ADP-Glo™ kinase assay kit (Promega,
Madison) with Sorafenib as positive control. The kinase assay was
performed in duplicate in a reaction mixture of final volume of
z ¼ 390.10. 1H NMR (400 MHz, DMSO)
d 11.77 (s, 1H), 8.89 (s, 1H),
8.84 (s, 1H), 7.70 (d, J ¼ 2.0 Hz,1H), 7.62 (d, J ¼ 1.8 Hz, 1H), 7.47e7.40
(m, 2H), 7.30 (d, J ¼ 2.0 Hz, 2H), 7.24 (d, J ¼ 8.0 Hz,1H), 7.21e7.19 (m,
1H), 7.10e7.08 (m, 1H), 6.82 (t, J ¼ 3.9 Hz, 1H), 4.45 (s, 2H), 2.26 (s,
10
were incubated with substrates (1.0
e 12 M) and ATP (250 mM) in a final buffer of Tris 40 mM, MgCl2
mL. General procedures are as the following: Kinases (1.5 ng/mL)
3H). 13C NMR (101 MHz, DMSO-d6)
d
153.10, 148.59, 142.58, 140.44,
mg/m
L), compounds (1.2 ꢀ 10ꢂ4
139.94, 139.28, 136.11, 133.62, 131.61, 129.47, 128.82, 126.69, 122.92,
119.53, 119.35, 118.73, 118.09, 117.53, 110.94, 109.46, 19.28.
m
10 mM, BSA 0.1 mg/mL, DTT 1 mM in 384-well plate with the total
volume of 5
L. The assay plate was incubated at 30 ꢁC for 1 h. After
the plate was cooled for 5 min at room temperature, 5 L of ADP-
Glo reagent was added into each well to stop the reaction and
consume the remaining ADP within 40 min. At the end, 10 L of
m
4.1.42. N-[3-(3-amino-1H-indazol-4-yl)phenyl]-N0-[4-chloro-3-
(trifluoromethyl)phenyl]urea (12o)
m
Yield 54% as white solid. mp: 190e191 ꢁC, MS (ESI) [MeH]þ: m/
m
z ¼ 444.10. 1H NMR (400 MHz, DMSO)
d
11.78 (s, 1H), 9.23 (s, 1H),
kinase detection reagent was added into the well and incubated for
30 min to produce a luminescence signal. The luminescence was
read by VICTOR-X multi-label plate reader. The signal was corre-
lated with the amount of ATP present in the reaction and was
inversely correlated with the kinase activity.
9.04 (s, 1H), 8.11 (d, J ¼ 2.4 Hz, 1H), 7.68e7.65 (m, 1H), 7.62 (d,
J ¼ 12 Hz, 2H),7.49 (d, J ¼ 8 Hz,1H), 7.44 (t, J ¼ 4.9 Hz,1H), 7.32e7.27
(m, 2H), 7.12 (d, J ¼ 8 Hz, 1H), 6.85e6.80 (m, 1H), 4.44 (s, 2H). 13
C
NMR (101 MHz, DMSO-d6)
d 170.74, 153.06, 148.60, 142.62, 140.46,
139.75, 139.67, 136.06, 132.36, 129.45, 127.42, 127.11, 126.81, 126.69,
124.65, 123.54, 123.22, 122.93, 121.94, 119.61, 119.57, 118.37, 117.36,
117.31, 110.95, 109.49, 60.19, 21.11, 14.44.
4.3. Cell growth inhibitory activity in cancer cell lines [18]
The cell growth inhibitory activity of title compounds was
evaluated with A549 and SMMC-7721 cell lines by the standard
MTT assay in vitro. 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 test compounds at indicated
final concentrations were added to the culture medium and the
cells cultures were continued for 48 h. Then, 22 mL fresh MTT
(5 mg/mL) was added to each well and incubated for 4 h at 37 ꢁC.
4.1.43. N-[3-(3-amino-1H-indazol-4-yl)phenyl]-N0-1,3-thiazol-2-
ylurea (12p)
Yield 55% as white solid. mp: 233e234 ꢁC, MS (ESI) [MeH]þ: m/
z ¼ 349.05. 1H NMR (400 MHz, DMSO)
d 11.78 (s, 1H), 10.58 (s, 1H),
9.13 (s, 1H), 7.64 (s, 1H), 7.51e7.44 (m, 2H), 7.38 (d, J ¼ 3.4 Hz, 1H),
7.30 (d, J ¼ 3.7 Hz, 2H), 7.14 (d, J ¼ 9.0 Hz, 2H), 6.84e6.82 (m, 1H),
4.41 (s, 2H).
The formazan crystals were dissolved in 100 mL DMSO each well.
4.1.44. N-[4-(3-amino-1H-indazol-4-yl)phenyl]-N0-1,3-
benzodioxol-5-ylurea (12q)
Supernatant was discarded, and 150 mL DMSO was added to each
well. Absorbance values were determined by a microplate reader
(Bio-Rad Instruments) at 490 nm. The IC50 values were calculated
according to inhibition ratios.
Yield 52% as slight yellow solid. mp: 150e152 ꢁC, MS (ESI)
[MeH]þ: m/z ¼ 386.10. 1H NMR (400 MHz, DMSO)
d 11.73 (s, 1H),
8.77 (s, 1H), 8.63 (s, 1H), 7.58 (d, J ¼ 8.6 Hz, 2H), 7.39 (d, J ¼ 8.5 Hz,
2H), 7.27 (d, J ¼ 8.6 Hz, 2H), 7.24 (d, J ¼ 2.1 Hz, 1H), 6.58 (d,
J ¼ 8.6 Hz,1H), 6.80e6.78 (m, 2H), 5.99 (s, 2H), 4.35 (s, 2H). 13C NMR
4.4. Molecular docking study [19]
(101 MHz, DMSO-d6)
d13C NMR (101 MHz, DMSO)
d
153.16, 148.65,
Surflex-Dock module of Sybyl-X (Version 2.0, Tripos Inc. St.
Louis, MO) was used in molecular docking study. The crystal
structures of the kinase domain of VEGFR-2 in complex with Sor-
afenib (PDB ID: 4ASD) was 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
147.73, 142.60, 139.85, 135.99, 134.52, 133.02, 129.93, 129.74, 126.76,
119.58, 118.51, 114.21, 111.57, 111.01, 109.02, 108.61, 101.49, 101.31.
4.1.45. N-[4-(3-amino-1H-indazol-4-yl)phenyl]-N0-(4-
methylpyridin-2-yl)urea (12r)
Yield 51% as slight yellow solid. mp: 228e229 ꢁC, MS (ESI)
[MeH]þ: m/z ¼ 357.10. 1H NMR (400 MHz, DMSO)
d 11.74 (s, 1H),
10.89 (s, 1H), 9.50 (s, 1H), 8.17 (d, J ¼ 5.2 Hz, 1H), 7.68 (d, J ¼ 8.5 Hz,

90
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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
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Acknowledgment
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[(1E)-N-hydroxyethanimidoyl]-30,5,6-trimethoxybiphenyl-3-yl}-N0-[4-(3-
morpholin-4-ylpropoxy)phenyl]urea) screened by tissue angiogenesis model
and its activity evaluation on anti-angiogenesis, Phytomedicine 21 (2014)
1675e1683.
This work was supported by the National Natural Science
Foundation of China (NSFC, Grant No. 81573285 and 81302641), the
Natural Science Basic Research Plan in Shaanxi Province of China
(Program No. 2015JQ8312), and the Fundamental Research Funds
for the Central Universities (2015qngz13).
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Appendix A. Supplementary data
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dx.doi.org/10.1016/j.ejmech.2015.08.045.
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