Discovery of novel VEGFR-2 inhibitors. Part II: Biphenyl urea incorporated with salicylaldoxime

Article abstract of DOI:10.1016/j.ejmech.2014.11.032

A series of novel VEGFR-2 inhibitors containing oxime as hinge binding fragment were described. A strategy of pseudo six-membered ring formed through intramolecular hydrogen bond was employed to mimic the planar quinazoline. The oxime group was firstly introduced to interact with hinge region of VEGFR-2. Most of compounds tested showed moderate to high VEGFR-2 inhibitory activity. In particular, 12l, 12p and 12y exhibited significant enzymatic inhibitory activity as well as potent antiproliferative activity against cancer cells. Molecular docking suggested that the salicylaldoxime formed two hydrogen bonds with hinge region. These biphenylureas could serve as promising lead compounds for developing novel anticancer agents.

Full text of DOI:10.1016/j.ejmech.2014.11.032

European Journal of Medicinal Chemistry 90 (2015) 232e240
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Discovery of novel VEGFR-2 inhibitors. Part II: Biphenyl urea
incorporated with salicylaldoxime
Hongping Gao, Ping Su, Yaling Shi, Xiuxiu Shen, Yanmin Zhang, Jinyun Dong, Jie Zhang*
School of Pharmacy, Health Science Center, Xi'an Jiaotong University, No. 76, Yanta West Road, 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:
A series of novel VEGFR-2 inhibitors containing oxime as hinge binding fragment were described. A
strategy of pseudo six-membered ring formed through intramolecular hydrogen bond was employed to
mimic the planar quinazoline. The oxime group was firstly introduced to interact with hinge region of
VEGFR-2. Most of compounds tested showed moderate to high VEGFR-2 inhibitory activity. In particular,
12l, 12p and 12y exhibited significant enzymatic inhibitory activity as well as potent antiproliferative
activity against cancer cells. Molecular docking suggested that the salicylaldoxime formed two hydrogen
bonds with hinge region. These biphenylureas could serve as promising lead compounds for developing
novel anticancer agents.
Received 14 May 2014
Received in revised form
11 November 2014
Accepted 16 November 2014
Available online 18 November 2014
Keywords:
VEGFR-2
Biphenyl urea
© 2014 Elsevier Masson SAS. All rights reserved.
Intramolecular hydrogen bond
Salicylaldoxime
1. Introduction
VEGFR-2 inhibitors (Fig. 2) [11]. Molecular docking indicated that
the urea moiety formed four hydrogen bonds with DFG residue of
Tumor angiogenesis plays a central role in cancer cell survival,
tumor growth, and the development of distant metastasis [1].
Numerous regulators of angiogenesis have been identified and
characterized. Among them, vascular endothelial growth factor
(VEGF) and receptor (VEGFR-2) are identified as positive regulators
of pathological angiogenesis associated with tumors [2]. VEGFR-2
inhibition has been considered as effective strategy for the pre-
vention of angiogenesis [3]. Antiangiogenic therapy based on
VEGFR-2 inhibition is powerful clinical treatment of cancers and
VEGFR-2 is becoming attractive target for anticancer drug discovery
[4]. At present, many VEGFR-2 targeted agents have been approved
for cancer therapy [5]. There are various structure elements of
VEGFR-2 inhibitors (Fig. 1) such as urea (Sorafenib) [6], ABT-869
VEGFR-2. The results confirmed as known that urea moiety played
an essential role in enzymatic inhibitory activity. Moreover, these
biphenylureas have strong potential to be further optimized.
In continuing our search for novel biphenyl ureas with potent
VEGFR-2 inhibitory and anticancer activity, we extended the
structural diversity of biphenyl ureas. In order to enhance the af-
finity for VEGFR-2, the methoxy group was converted to hydroxyl
group while the acetyl group was optimized to oxime (Fig. 2). These
salicylaldoximes form a pseudo six-membered ring via a hydrogen
bond. They could mimic quinazoline of known ATP-competitive
inhibitors [12]. The intramolecular hydrogen bond stabilizes planar
conformation and makes the molecule considerable rigid. The
conformational similarity of salicylaldoxime to quinazoline led to
the hypothesis that both of them could act with hinge region of
VEGFR-2.
Based on our previous promising results, we described the
design, synthesis and biological evaluation of a series of biphenyl
ureas as novel VEGFR-2 inhibitors. They displayed potent anti-
cancer effect and could be considered as promising lead com-
pounds for further optimization. A molecular dock model and
preliminary SAR based on enzymatic inhibitory activity will also be
discussed.
[7];
3-substituted
indolinones
(Sunitinib)
[8];
4-
anilinoquinazolines (ZD4190) [9]. These outcomes encouraged us
to develop novel VEGFR-2 inhibitors as promising anticancer agents
[10].
To our knowledge, biphenyl urea and its derivatives are impor-
tant fine chemicals. They have found extensive useful applications
as tranquilizers and antibiotic drugs. Recently, we have described
the synthesis and biological evaluation of biphenyl urea-based
* Corresponding author.
E-mail address: zhj8623@mail.xjtu.edu.cn (J. Zhang).
http://dx.doi.org/10.1016/j.ejmech.2014.11.032
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

H. Gao et al. / European Journal of Medicinal Chemistry 90 (2015) 232e240
233
Fig. 1. VEGFR-2 inhibitors currently approved or in clinical trials.
Fig. 2. Structures of title compounds with intramolecular hydrogen bonds and quinazoline.
2. Results and discussion
2.2. Biological evaluation
2.1. Chemistry
The entire library of title compounds was evaluated against
VEGFR-2 with Sorafenib as positive control (Table 1). As shown in
Table 1, the majority of them displayed moderate to high VEGFR-2
inhibitory activity. Among them, 12l, 12p, 12y potently inhibited
VEGFR-2 with nanomolar potency (IC50 ¼ 8.7 nM, 5.3 nM, 4.2 nM).
Biphenyl ureas incorporated with 2-fluorine substituent on
terminal aniline displayed more potent activity than the others.
They showed potent VEGFR-2 inhibitory activity with IC50 values
ranging from 5.3 nM to 549.0 nM. 12l and 12p exhibited high po-
tency with IC50 values of 8.7 nM and 5.3 nM, respectively. Com-
pounds with hydrogen and methyl substituent on terminal
benzene displayed moderate or poor activity. 12f was the most
potent in these two series with IC50 value of 13.0 nM. Biphenyl
ureas with 2-chlorine substituent also exhibited potent activity
with IC50 values ranging from 55.9 nM to 949.0 nM.
In order to identify the importance of the o-hydroxyl group,
three biphenylureas (12w′e12y′) without hydroxy group were
synthesized and evaluated (Table 2). As shown in Table 2, the
incorporation of the hydroxy into the ortho position of oxime
resulted in an increase of potency. The salicylaldoximes (12we12y)
exhibited potent activities which were 10- to 60-fold higher than
their precursor (12w′e12y′), especially for 12y which is the most
potent VEGFR-2 inhibitor with IC50 value of 4.2 nM. These results
A convenient method for the synthesis of biphenyl ureas has
been described before [13]. Based on our previous research, the
general synthesis of title compounds was depicted in Scheme 1.
Commercial available vanillin was converted into 2-bromovanillin
by reaction with bromine [14], then aldehyde group was con-
verted to cyano group via a one-pot oxime formation, dehydration,
and subsequent O-methylation to afford (2). Compound (3) was
obtained by methylation and hydrolysis of (2) with sodium formate
and hydroxylamine sulfate in formic acid [15]. Then amide group
was converted to amine through Hoffmann's degradation. Com-
pound (6) was obtained by acylation of 3-bromophenol with acetic
anhydride and Fries rearrangement [16]. Hydroxyl group of (6) was
protected by benzyl to afford (7). Another intermediate (8) was
prepared by coupling of bis(pinacolato)diboron with bromide (7)
without further purification [17]. Critical intermediate (9) was
prepared from (8) and (4) by classical Suzuki coupling reaction [18].
Subsequently, intermediate (9) was treated with triphosgene (BTC)
to produce various isocyanate, followed by reacting with
substituted anilines yielding (10) [19]. After deprotection of benzyl
group, the title compounds (12ae12y) were obtained by subse-
quent condensation of (11) with hydroxylamine hydrochloride.

234
H. Gao et al. / European Journal of Medicinal Chemistry 90 (2015) 232e240
Scheme 1. Synthetic route of biphenyl ureas. Reagents and conditions: a: Br2, AcOH, AcONa, Fe, rt; b: HCOOH, HCOONa, (NH3OH)2SO4, 90 ꢀC; c: (CH3)2SO4, K2CO3, Me2CO, 50 ꢀC; d:
H2O2, NaOH, EtOH, 60 ꢀC; e: Br2, NaOH; f: Py, Ac2O, rt; g: AlCl3,160 ꢀC; h: BnCl, K2CO3, DMF, 100 ꢀC; i: B2O4(CH)4(CH3)8, dioxane, Pd(pddf)Cl2, KOAc, 100 ꢀC; j: Na2CO3, H2O,
dioxane,100 ꢀC; k: BTC, substituted anilines, Et3N, DCM, rt; l: Pd/C, MeOH, rt; m: EtOH, NH4OH, H2O.
indicated that the intramolecular hydrogen bond might be essen-
tial for the VEGFR-2 inhibitory activity.
3. Conclusion
Eight selected biphenyl ureas were evaluated for their anti-
proliferative activity against nine cancer cell lines with Sunitinib as
a standard. Their activity was measured by 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. The anti-
cancer potency (IC50) of them was shown in Table 3. The IC50 values
were the average of at least three independent experiments. Ma-
jority of title compounds could inhibit the growth of cancer cell
lines in a dose-dependent manner. It was observed from Table 3
that 12p displayed the most potent antiproliferative activity
against five cancer cell lines comparable to that of Sunitinib. In
particular, it showed higher potency against MDA-MB-435S with
In conclusion, twenty-eight biphenyl ureas were designed and
synthesized using a convergent synthetic route. These compounds
allowed us to introduce efficiently structural diversity at biphenyl
scaffold, especially at the moiety binding with hinge region. The
majority of them exhibited moderate to high inhibitory activity
against VEGFR-2. Three of them (12l, 12p and 12y) displayed sig-
nificant enzymatic inhibitory activities, with IC50 values of 8.7 nM,
5.3 nM and 4.2 nM. Moreover, 12p showed great antiproliferative
potency against A549, MCF-7, MDA-MB-435S, MDA-MB-231 and
HT29 cell lines. Docking study indicated three hydrogen bonds
between urea unit and salicylaldoxime with VEGFR-2. It could be
verified that the pseudo six-membered ring formed by intra-
molecular hydrogen bond played important role in biological ac-
tivity. These results suggested that not only urea moiety but also
salicylaldoxime were benefit for their anticancer potency. These
biphenyl ureas may possibly be used as lead compounds for
developing novel VEGFR-2 inhibitors. Further structural optimiza-
tion of these promising anticancer agents will be reported in due
course.
IC50 value of 0.35 mM.
2.3. Molecular docking study
A deeper structural interpretation of above biological results is
required for further optimization. In order to obtain more under-
standing of the interaction with receptor, a molecular modeling
study was performed using Sybyl-X 2.0. The most potent inhibitor
(12p) was docked into ATP binding site of VEGFR-2 (PDB ID: 3C7Q).
We chose 12p because, according to the biological results presented
in the previous section, it is one of the most potent compounds.
Molecular insights based on docking indicated favorable binding
interactions of 12p with the active site of VEGFR-2 (Fig. 3). We
found that hydroxy group of oxime formed two hydrogen bonds to
Cys917 with distance of 2.05 Å and 1.97 Å, respectively. Carbonyl
group of urea was identified to be involved in hydrogen bond with
the backbone NH of Asp 1044 with distance of 2.21 Å. The salicy-
laldoxime moiety bound to the hinge region (Cys917) of VEGFR-2,
while the urea was sandwiched between the activation loop of
Asp1044 and Glu883.
4. Experimental section
4.1. Chemistry
4.1.1. General procedure
Solvents and reagents are purified according to the standard
procedure. Reactions are monitored by thin layer chromatography
on 0.25-mm silica gel plates (60GF-254) and visualized with UV
light. The reactions except those in aqueous media are carried out
by standard techniques for the exclusion of moisture. Melting
points are determined on electrothermal melting point apparatus

H. Gao et al. / European Journal of Medicinal Chemistry 90 (2015) 232e240
235
Table 1
Structure and VEGFR-2 inhibitory activity of biphenyl ureas.
Compound
12a
R1
H
R2
4-
4-
IC50, nM
NDa
Compound
12n
R1
R2
IC50, nM
2-F
247.0
4
12b
12c
12d
H
H
H
225.0
197.6
240.0
12o
12p
12q
2-F
2-F
2-Cl
365.9
5.3
4-
4-
4-
4-
141.2
4-
4-
12e
12f
2-CH3
2-CH3
ND
12r
12s
2-Cl
2-Cl
55.9
4-
13.0
435.1
4-
5-
5-
12g
12h
12i
2-CH3
2-CH3
2-CH3
ND
12t
12u
12v
2-Cl
2-Cl
2-Cl
949.0
300.8
615.1
4-
4-
4-
63.0
270.0
5-
5-
5-
12j
2-CH3
2-F
ND
12w
12x
H
H
18.4
78.7
4-
4-
12k
43.2
4-
4-
12l
2-F
2-F
8.7
12y
H
4.2
2.2
4-
12m
549.0
Sorafenib
4-
a
ND ¼ not determined.
and are uncorrected. 1H NMR spectra are measured at 400 MHz on a
Bruker Advance AC 400 instrument. Mass spectra are obtained on a
Shimadzu HPLC-MS-QP2010 instrument.
the mixture was heated at 90 ꢀC for 5 h. The reaction was cooled to
room temperature and poured to a solution of sodium chloride
(120 g) in water (600 mL). The resultant solid was collected by
filtration, washed with water, and dried to give (2) (18.92 g, 84%) as
off-white solid. Mp: 159e160 ꢀC, MS (EI) [M]þ: m/z ¼ 230, 1H NMR
4.1.2. 3-bromo-4-hydroxy-5-methoxybenzonitrile (2)
(400 MHz, CDCl3)
d ppm: 7.38 (s, 1H), 6.62 (s, 1H), 4.00 (s, 3H).
To a mixture of (1) (30.0 g, 0.20 mol), NaOAc (33.0 g, 0.40 mol)
and iron power (1.05 g, 0.02 mol) was added glacial acetic acid
(180 mL). The mixture was stirred at room temperature for 30 min.
Br2 (10.5 mL, 0.021 mol) in glacial acetic acid (40 mL) was added
dropwise at 23e25 ꢀC. The mixture was stirred at the same tem-
perature overnight. Ice-water (300 mL) was added and stirred for
another one hour. The solid obtained was dried and recrystallized
from EtOH to give gray solid. A mixture of the obtained solid (23.0 g,
100 mmol), sodium formate (12.6 g, 200 mmol), and formic acid
(150 mL) was heated to 90 ꢀC. Then hydroxylamine sulfate (9.80 g,
60 mmol) was added in five equal portions at 30 min intervals, and
4.1.3. 3-bromo-4,5-dimethoxybenzamide (3)
A mixture of (2) (16.0 g, 70 mmol), potassium carbonate (32.1 g,
0.23 mol) and acetone (150 mL) was stirred at 50 ꢀC for 30 min.
Dimethyl sulfate (13.9 mL, 0.15 mol) was added dropwise at 50 ꢀC.
After completion of reaction, the solid was filtered off and the
solvent was evaporated. The solid obtained was dried to give off-
white solid. To a solution of the obtained solid (13.3 g, 55 mmol)
in ethanol (200 mL) was added NaOH (2.42 g, 60.5 mmol) and 30%
H2O2 (22 mL, 0.93 mol). The mixture was stirred at 60 ꢀC for 1 h and

236
H. Gao et al. / European Journal of Medicinal Chemistry 90 (2015) 232e240
Table 2
filtration, washed with water, and dried to give (7) (5.18 g, 85%) as
an off-white solid.
Structure and VEGFR-2 inhibitory activity of biphenyl ureas without intramolecular
hydrogen bonds.
4.1.7. 1-[50-amino-3-(benzyloxy)-20,30-dimethoxybiphenyl-4-yl]
ethanone (9)
A flask charged with Pd(pddf)Cl2 (0.48 g, 0.65 mmol), KOAc
(2.57 g, 26 mmol), and the bis(pinacolato)diboron (1.82 g,
7.2 mmol) and compound (7) (2 g, 6.5 mmol) was flushed with
nitrogen.1,4-dioxane (30 mL) was then added. After being stirred at
100 ꢀC for 5 h under nitrogen atmosphere, the mixture was cooled
to afford the black oil mixture without further purified. Compound
(4) (1.21 g, 5.2 mmol), sodium carbonate (1.39 g, 13.1 mmol), H2O
(7 mL) and 1,4-dioxane (5 mL) were then added to the mixture. It
was then refluxed overnight under nitrogen. The product was
extracted with AcOEt (50 mL ꢂ 3), washed with water, and dried
over Na2SO4. After filtration and concentration in vacuo, the resi-
dues was purified by silica gel flash chromatography (PE/
AcOEt ¼ 3:1) gave (9) (1.55 g, 63%) as slight yellow solid.
Compound
12w
R
R0
IC50, nM Compound
R
R0
IC50, nM
235.3
H
18.4
78.7
4.2
12w0
12x0
12y0
Me
12x
12y
H
H
Me
Me
1580.0
256.3
4.1.8. N-[40-acetyl-30-(benzyloxy)-5,6-dimethoxybiphenyl-3-yl]-N0-
{4-[2-(diethylamino)ethoxy]phenyl}urea (10)
acidified with concentrated HCl. Ethanol was evaporated and res-
idues was poured into water and filtered. The white solid obtain
was dried to give (3) (12.40 g, 86.8%).
Triphosgene (0.47 g, 1.6 mmol) was dissolved in anhydrous
CH2Cl2 (25 mL) and the mixture was stirred on the ice-bath for
15 min. A solution of the (9) (1.50 g, 4 mmol) in anhydrous CH2Cl2
(10 mL) was added dropwise to the above mixture and stirring
continued for 15 min. Et3N (0.48 mL, 3.44 mmol) diluted with
CH2Cl2 (5 mL) was then added into the mixture. Stirring was
continued for 15 min and a solution of Et3N (0.48 mL, 3.44 mmol),
4.1.4. 3-bromo-4,5-dimethoxyphenylamine (4)
Br2 (1.35 mL) was added dropwise into a solution of NaOH (4.0 g,
100 mmol) in distilled water (81 mL) at ꢁ5 ꢀC and was stirred for
another 10 min. Compound (3) (5.40 g, 21 mmol) was added into
the above solution in batches and stirring continued for 20 min. The
mixture was warmed to r.t. and kept for 30 min. Then the sus-
pension was heated at 40 ꢀC for 1 h. The mixture was cool to r.t. and
was poured into water and extracted with AcOEt (50 mL ꢂ 3). The
organic layer was collected and washed with water, brine, and
dried over Na2SO4. Filtration and concentration in vacuo afford
crude product. Further purification by silica gel flash chromatog-
raphy (PE/AcOEt ¼ 2:1) gave (4) (2.62 g, 54%) as yellow solid.
4-(2-diethylamino-ethoxy)-phenylamine (0.83 g,
4 mmol) in
anhydrous CH2Cl2 (25 mL) was added. After completion of the ac-
tion, 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 puri-
fied by silica gel flash chromatography (CH2Cl2/MeOH ¼ 30:1) gave
as yellow solid (10) (0.87 g, 35%).
4.1.9. N-(40-acetyl-30-hydroxy-5,6-dimethoxybiphenyl-3-yl)-N0-{4-
[2-(diethylamino) ethoxy]phenyl}urea (11)
4.1.5. 1-(4-Bromo-2-hydroxyphenyl)ethanone (6)
To a solution of (10) (0.61 g, 1 mmol) in methanol (20 ml) was
added Pd/C (0.03 g, 0.3 mmol), stirred for 24 h in room temperature
under the atmosphere of hydrogen. The mixture was filtered to
remove Pd/C, after filtration and concentration in vacuo to give (11)
(0.44 g, 85%) as slight yellow solid.
3-Bromophenol (5) (10.0 g, 58 mmol) was dissolved in pyridine
(30 mL) under ice-bath and was stirred for 30 min. Acetic anhydride
(8.3 mL, 88 mmol) was added dropwise into above mixture at the
same temperature. The ice-bath was removed after dropping and
the mixture was stirred for 2 h. Concentrated HCl and ice-water
(300 mL) was added to neutralize PH to 7. The mixture was
extracted with AcOEt (100 mL ꢂ 3). The organic layer was collected
and washed with brine and dried over Na2SO4. Filtration and con-
centration in vacuo afforded yellow oil. In a 250 mL round bottom
flask, the obtained yellow oil (6.45 g, 30 mmol) and anhydrous AlCl3
(12.02 g, 90 mmol) was mixed at 160 ꢀC for three hours. Reaction
mixture was poured into ice water and concentrated hydrochloric
acid solution was added to break complex formed during reaction.
The mixture was taken in AcOEt (50 mL ꢂ 3). The organic layer was
combined and 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 30:1)
give (6) (4.74 g, 73.5%) as white solid.
4.1.10. N-{4-[2-(diethylamino)ethoxy]phenyl}-N0-{30-hydroxy-40-
[(1E)-N-hydroxyethanimidoyl]-5,6-dimethoxybiphenyl-3-yl}urea
(12a)
Compound (11) (0.40 g, 0.7 mmol) was dissolved in ethanol
20 mL and the solution was heated to 50 ꢀC. After being stirred for
15 min, hydroxylamine hydrochloride was added to the above
mixture and stirring continued for 5 h. The mixture was poured
into cool water and neutralized with NaHCO3. The product was
extracted with CH2Cl2 (20 mL ꢂ 3), washed with water, and dried
over Na2SO4. Filtration and concentration in vacuo afford 12a
(0.36 g, 87%) as slight yellow solid. mp: 117e119 ꢀC. EI-MS (m/z):
536.3 (Mþ). 1H NMR (400 MHz, (CD3)2SO)
d 11.64 (s, 1H), 11.61 (s,
1H), 8.62 (s, 1H), 8.48 (s, 1H), 7.54 (d, J ¼ 4 Hz, 1H), 7.35 (s, 1H), 7.33
(s, 1H), 7.25 (s, 1H), 7.01 (d, J ¼ 4 Hz, 1H), 6.98 (s, 1H), 6.95 (s, 1H),
6.87 (s, 1H), 6.85 (s, 1H), 3.97 (t, J ¼ 6 Hz, 2H), 3.83 (s, 3H), 3.50 (s,
3H), 2.76 (t, J ¼ 6 Hz, 2H), 2.56 (m, J ¼ 8 Hz, 4H), 2.30 (s, 3H), 0.98 (t,
J ¼ 6 Hz, 6H).
4.1.6. 1-[2-(benzyloxy)-4-bromophenyl]ethanone (7)
To a mixture of (6) (4.3 g, 20 mmol), potassium carbonate
(8.28 g, 60 mmol) and DMF (60 mL) was added Benzyl chloride
(2.78 mL, 22 mmol), stirred for 1 h at 100 ꢀC under the atmosphere
of nitrogen. Ice-water (240 mL) was added to the mixture and
stirred for another 30 min. The resultant solid was collected by
Title compounds 12be12y0 were prepared by using the general
procedure described above.

H. Gao et al. / European Journal of Medicinal Chemistry 90 (2015) 232e240
237
Table 3
Antiproliferative activities of title compounds on cancer cells.
Compound
Cancer cell lines, IC50 (mM)
A549
MCF-7
ECV304
MDA-MB-435S
H1299
MDA-MB-231
A375
HT29
SMMC-7721
Sunitinib
12b
12c
12f
12k
12l
12p
12r
3.99
4.29
4.67
5.30
2.55
3.96
2.03
3.91
ND
9.81
1.90
4.64
4.98
3.46
ND
4.78
1.65
16.06
12.09
6.68
ND
0.35
12.86
67.58
1.76
7.57
3.84
3.16
3.63
2.63
19.09
4.04
6.82
4.55
4.98
3.88
10.01
5.13
5.32
3.73
5.48
4.13
2.12
5.85
ND
2.63
8.16
6.57
6.54
6.83
3.40
2.04
4.33
14.97
3.21
9.28
ND
27.65
31.00
28.40
6.55
15.18
2.87
15.89
NDa
12.91
13.01
9.68
9.52
10.71
4.75
ND
4.99
3.96
2.44
2.30
2.37
ND
6.25
3.64
ND
12y
a
ND ¼ Not determined.
Fig. 3. The structure of VEGFR-2 ATP site in complex with compound (12p).
4.1.10.1. N-{30-hydroxy-40-[(1E)-N-hydroxyethanimidoyl]-5,6-
dimethoxybiphenyl-3-yl}-N0-[4-(2-piperidin-1-ylethoxy)phenyl]urea
(12b). mp.: 131e133 ꢀC. EI-MS (m/z): 548.3 (Mþ). 1H NMR
yl}urea (12e). mp.: 148e150 ꢀC. EI-MS (m/z): 536.3 (Mþ). 1H NMR
(400 MHz, (CD3)2SO) 11.69 (s, 1H), 9.78 (s, 1H), 8.50 (s, 1H), 7.53 (d,
J ¼ 4 Hz, 1H), 7.49 (d, J ¼ 4 Hz, 1H), 7.34 (s, 1H), 7.00 (d, J ¼ 4 Hz, 1H),
6.97 (s, 2H), 6.76 (s, 1H), 6.70 (m, J ¼ 6 Hz, 1H), 3.94 (t, J ¼ 6 Hz, 2H),
3.82 (s, 3H), 3.49 (s, 3H), 2.34 (t, J ¼ 8 Hz, 2H), 2.29 (s, 3H), 2.22 (s,
3H), 2.14 (s, 6H), 1.83(t, J ¼ m, 10 Hz,2H).
d
(400 MHz, (CD3)2SO)
d 11.64 (s, 1H), 11.62 (s, 1H), 8.79 (s, 1H), 8.65
(s, 1H), 7.54 (d, J ¼ 4 Hz, 1H), 7.36 (s, 1H), 7.34 (s, 1H), 7.27 (s, 1H),
7.01 (d, J ¼ 4 Hz, 1H), 6.98 (s, 1H), 6.95 (s, 1H), 6.89 (s, 1H), 6.87 (s,
1H), 4.06 (s, 2H), 3.82 (s, 3H), 3.50 (s, 3H), 2.74 (t, J ¼ 22 Hz, 2H), 2.51
(s, 4H), 2.29 (s, 3H), 1.54 (s, 4H), 1.40 (s, 2H).
4.1.10.5. N-{5-[2-(diethylamino)ethoxy]-2-methylphenyl}-N0-{30-hy-
droxy-40-[(1E)-N-hydroxyethanimidoyl]-5,6-dimethoxybiphenyl-3-
yl}urea (12f). mp.: 160e161 ꢀC. EI-MS (m/z): 550.2 (Mþ). 1H NMR
4.1.10.2. N-{30-hydroxy-40-[(1E)-N-hydroxyethanimidoyl]-5,6-
dimethoxybiphenyl-3-yl}-N0-[4-(2-pyrrolidin-1-ylethoxy)phenyl]urea
(12c). mp.: 121e122 ꢀC. EI-MS (m/z): 534.2 (Mþ). 1H NMR
(400 MHz, (CD3)2SO) d 9.46 (s, 1H), 8.09 (s, 1H), 7.57 (s, 1H), 7.53 (d,
(400 MHz, (CD3)2SO) d 11.65 (s, 1H), 8.69 (s, 1H), 8.55 (s, 1H), 7.54 (d,
J ¼ 4 Hz, 1H), 7.25 (s, 1H), 7.03 (d, J ¼ 6 Hz, 2H), 7.00 (s, 1H), 6.98 (s,
1H), 6.50 (m, J ¼ 6 Hz, 1H), 3.94 (t, J ¼ 6 Hz, 2H), 3.84 (s, 3H), 3.50 (s,
3H), 2.75 (t, J ¼ 6 Hz, 2H), 2.54 (m, J ¼ 12 Hz, 4H), 2.29 (s, 3H), 2.17 (s,
J ¼ 4 Hz, 1H), 7.47 (d, J ¼ 4 Hz, 1H), 7.36 (s, 1H), 7.34 (s, 1H), 7.25 (d,
J ¼ 4 Hz, 1H), 7.01 (d, J ¼ 4 Hz, 1H), 6.97 (d, J ¼ 4 Hz, 1H), 6.87 (s, 1H),
6.85 (s, 1H), 4.01 (t, J ¼ 6 Hz,2H), 3.48 (d, J ¼ 6 Hz, 3H), 2.76 (t,
J ¼ 6 Hz, 2H), 2.51 (s, 4H), 2.29 (s, 3H), 1.68 (s, 4H).
3H), 0.97 (t, J ¼ 8 Hz, 6H). 13C NMR (101 MHz, (CD3)2SO)
d 157.62,
157.45, 157.24, 153.29, 153.12, 141.16, 140.31, 138.74, 136.58, 134.54,
131.01, 128.13, 120.06, 119.11, 118.77, 117.25, 111.35, 108.56, 107.41,
103.43, 66.74, 60.75, 56.12, 51.83, 47.48, 17.54, 12.37, 11.53.
4.1.10.3. N-{30-hydroxy-40-[(1E)-N-hydroxyethanimidoyl]-5,6-
dimethoxybiphenyl-3-yl}-N0-[4-(3-morpholin-4-ylpropþoxy)phenyl]
urea (12d). mp.: 160e162 ꢀC. EI-MS (m/z): 564.3 (M ). 1H NMR
(400 MHz, (CD3)2SO) d 11.64 (s,1H),11.62 (s,1H), 9.34 (s,1H), 9.18 (s,
4.1.10.6. N-{5-[3-(dimethylamino)propoxy]-2-methylphenyl}-N0-{30-
hydroxy-40-[(1E)-N-hydroxyethanimidoyl]-5,6-dimethoxyþbiphenyl-3-
yl}urea (12g). mp.: 181e182 ꢀC. EI-MS (m/z): 536.3 (M ). 1H NMR
1H), 7.54 (d, J ¼ 4 Hz, 1H),7.38 (s, 1H), 7.36 (s, 1H), 7.31 (s, 1H), 7.00
(d, J ¼ 4 Hz, 1H), 6.97 (s, 1H), 6.92 (s, 1H), 6.89 (s, 1H), 6.86 (s, 1H),
4.01 (t, J ¼ 6 Hz, 2H), 3.86 (s, 4H), 3.82 (s, 3H), 3.49 (s, 3H), 3.16 (s,
6H), 2.29 (s, 3H), 2.14 (m, J ¼ 12 Hz, 2H).
(400 MHz, (CD3)2SO) d 9.61 (s, 1H), 8.20 (s, 1H), 7.56 (s, 1H), 7.52 (d,
J ¼ 4 Hz, 1H), 7.25 (s, 1H), 7.04 (s, 1H), 7.02 (s, 1H), 6.99 (s, 1H), 6.97
(s, 1H), 6.50 (m, J ¼ 6 Hz, 1H), 3.92 (t, J ¼ 6 Hz, 2H), 3.83 (s, 3H), 3.50
(s, 3H), 2.34 (t, J ¼ 8 Hz, 2H), 2.18 (s, 3H), 2.13 (s, 6H), 1.81 (m,
J ¼ 12 Hz, 2H).
4.1.10.4. N-{4-[3-(dimethylamino)propoxy]-2-methylphenyl}-N0-{30-
hydroxy-40-[(1E)-N-hydroxyethanimidoyl]-5,6-dimethoxybiphenyl-3-

238
H. Gao et al. / European Journal of Medicinal Chemistry 90 (2015) 232e240
4.1.10.7. N-{30-hydroxy-40-[(1E)-N-hydroxyethanimidoyl]-5,6-
dimethoxybiphenyl-3-yl}-N0-[2-methyl-5-(2-piperidin-1-yletþhoxy)
phenyl]urea (12h). mp.: 162e164 ꢀC. EI-MS (m/z): 564.1 (M ). 1H
yl}urea (12n). mp.: 174e176 ꢀC. EI-MS (m/z): 552.2 (Mþ). 1H NMR
(400 MHz, (CD3)2SO)
2H),7.41 (s, 1H), 7.11 (d, J ¼ 4 Hz, 1H), 7.07 (d, J ¼ 4 Hz, 1H), 6.99 (d,
J ¼ 4 Hz,1H), 6.96 (d, J ¼ 4 Hz, 2H), 4.07 (t, J ¼ 6 Hz, 2H), 3.81 (s, 3H),
3.49 (s, 3H), 2.77 (t, J ¼ 6 Hz, 2H), 2.51 (s, 4H), 2.28 (s, 3H), 1.68 (s,
4H).
d
11.80 (s, 1H), 10.48 (s, 1H), 7.53 (t, J ¼ 8 Hz,
NMR (400 MHz, (CD3)2SO)
d 11.65 (s, 1H), 11.62 (s, 1H), 9.19 (s, 1H),
7.89 (s, 1H), 7.59 (s, 1H), 7.55 (d, J ¼ 4 Hz, 1H), 7.21 (s, 1H), 7.03 (s,
1H), 7.01 (s, 1H), 6.99 (s, 1H), 6.51 (m, J ¼ 6 Hz, 1H), 3.99 (t, J ¼ 6 Hz,
2H), 3.84 (s, 3H), 3.50 (s, 3H), 2.63 (t, J ¼ 6 Hz, 2H), 2.42 (s, 4H), 2.30
(s, 3H), 2.17 (s, 3H), 1.49 (s, 4H), 1.37 (d, J ¼ 4 Hz, 2H). 13C NMR
4.1.10.14. N-[2-fluoro-4-(2-morpholin-4-ylethoxy)phenyl]-N0-{30-hy-
droxy-40-[(1E)-N-hydroxyethanimidoyl]-5,6-dimethoxybiphenyl-3-
yl}urea (12o). mp.: 195e197 ꢀC. EI-MS (m/z): 568.4 (Mþ). 1H NMR
(101 MHz, (CD3)2SO)
d 157.68, 157.42, 157.21, 153.31, 153.06, 141.20,
140.32, 138.70, 136.52, 134.54, 131.01, 128.15, 120.09, 119.05, 118.74,
117.25, 111.36, 108.48, 107.43, 103.43, 65.97, 60.75, 57.88, 56.12,
54.90, 26.04, 24.39, 17.53, 11.53.
(400 MHz, (CD3)2SO)
d
9.24 (s, 2H), 7.50 (d, J ¼ 2 Hz,1H), 7.48 (s,1H),
7.28 (d, J ¼ 2 Hz, 1H), 7.07 (t, J ¼ 10 Hz, 2H), 6.95 (d, J ¼ 4 Hz, 3H),
4.10 (t, J ¼ 6 Hz, 2H), 3.82 (s, 3H), 3.58 (t, J ¼ 6 Hz, 2H), 3.49 (s, 3H),
2.68 (t, J ¼ 4 Hz, 2H), 2.47 (t, J ¼ 4 Hz, 2H), 2.29 (s, 3H), 2.30 (s, 3H).
4.1.10.8. N-{30-hydroxy-40-[(1E)-N-hydroxyethanimidoyl]-5,6-
dimethoxybiphenyl-3-yl}-N0-[2-methyl-5-(2-pyrrolidin-1-yletþhoxy)
phenyl]urea (12i). mp.: 144e146 ꢀC. EI-MS (m/z): 548.3 (M ). 1H
13C NMR (101 MHz, (CD3)2SO)
d 157.58, 157.29, 153.26; 153.21,
141.39, 141.28, 141.12, 140.28, 136.58, 134.49, 128.11, 120.02, 118.80,
117.25, 116.35, 114.46, 111.49, 107.33, 107.11, 103.57, 67.60, 66.65,
60.74, 57.48, 56.11, 54.08, 11.55.
NMR (400 MHz, (CD3)2SO)
d
11.63 (d, J ¼ 6 Hz, 1H), 9.17 (s, 1H), 7.89
(d, J ¼ 4 Hz, 1H), 7.61 (d, J ¼ 4 Hz, 1H), 7.47 (d, J ¼ 4 Hz, 1H), 7.43 (d,
J ¼ 4 Hz, 1H), 7.25 (s, 1H), 7.06 (d, J ¼ 2 Hz, 1H), 6.99 (d, J ¼ 2 Hz, 1H),
6.52 (d, J ¼ 4 Hz, 1H), 5.20 (s, 1H), 4.00 (t, J ¼ 6 Hz, 2H), 3.84 (s, 3H),
3.50 (d, J ¼ 6 Hz, 3H), 2.78 (d, J ¼ 2 Hz, 2H), 2.51 (s, 4H), 2.17 (s, 3H),
2.10 (d, J ¼ 6 Hz, 3H), 1.67 (d, J ¼ 2 Hz, 4H).
4.1.10.15. N-[2-fluoro-4-(3-morpholin-4-ylpropoxy)phenyl]-N0-{30-
hydroxy-40-[(1E)-N-hydroxyethanimidoyl]-5,6-dimethoxyþbiphenyl-3-
yl}urea (12p). mp.: 207e208 ꢀC. EI-MS (m/z): 582.3 (M ). 1H NMR
(400 MHz, (CD3)2SO)
d
11.52 (s, 2H), 9.41 (s, 2H), 7.54 (d, J ¼ 2 Hz,
4.1.10.9. N-{30-hydroxy-40-[(1E)-N-hydroxyethanimidoyl]-5,6-
dimethoxybiphenyl-3-yl}-N0-[2-methyl-5-(3-morpholin-4-ylpropoxy)
phenyl]urea (12j). mp.: 202e203 ꢀC. EI-MS (m/z): 578.4 (Mþ). 1H
1H), 7.49 (d, J ¼ 8 Hz, 1H), 7.31 (s, 1H), 7.07 (s, 1H), 7.06 (s, 1H), 7.01
(m, J ¼ 6 Hz, 1H), 6.97 (s, 1H), 6.95 (s, 1H), 4.02 (t, J ¼ 6 Hz, 2H), 3.82
(s, 3H), 3.57 (t, J ¼ 4 Hz, 2H), 3.50 (s, 3H), 2.41 (t, J ¼ 6 Hz, 2H), 2.36
(s, 4H), 2.29 (s, 3H), 1.86 (m, J ¼ 12 Hz,2H). 13C NMR (101 MHz,
NMR (400 MHz, (CD3)2SO)
d
11.63 (d, J ¼ 6 Hz, 1H), 11.07 (s, 1H), 9.14
(s, 1H), 7.87 (d, J ¼ 4 Hz, 1H), 7.59 (d, J ¼ 4 Hz, 1H), 7.46 (d, J ¼ 4 Hz,
1H), 7.42 (d, J ¼ 4 Hz,1H), 7.24 (d, J ¼ 2 Hz,1H), 7.04 (d, J ¼ 4 Hz, 1H),
6.98 (s, 1H), 6.50 (d, J ¼ 4 Hz, 1H), 5.19 (s, 1H), 3.93 (t, J ¼ 6 Hz, 2H),
3.86 (s, 3H), 3.57 (s, 4H), 3.49 (d, J ¼ 6 Hz, 3H), 2.40 (t, J ¼ 6 Hz, 2H),
2.30 (s, 4H), 2.16 (s, 3H), 2.08 (s,3H), 1.83 (m, J ¼ 14 Hz, 2H).
(CD3)2SO) d 157.57, 157.31, 153.31, 153.19, 141.48, 141.37, 141.09,
140.29, 136.67, 134.47, 128.09, 120.00, 118.79, 117.26, 116.25, 114.52,
111.52, 107.37, 107.14, 103.58, 67.98, 66.67, 60.73, 56.09, 55.21, 53.83,
26.41, 11.54.
4.1.10.16. N-{2-chloro-4-[2-(diethylamino)ethoxy]phenyl}-N0-{30-hy-
droxy-40-[(1E)-N-hydroxyethanimidoyl]-5,6-dimethoxybiphenyl-3-
yl}urea (12q). mp.: 136e138 ꢀC. EI-MS (m/z): 570.2 (Mþ). 1H NMR
4.1.10.10. N-{4-[2-(diethylamino)ethoxy]-2-fluorophenyl}-N0-{30-hy-
droxy-40-[(1E)-N-hydroxyethanimidoyl]-5,6-dimethoxybiphenyl-3-
yl}urea (12k). mp.: 163e165 ꢀC. EI-MS (m/z): 554.2 (Mþ). 1H NMR
(400 MHz, (CD3)2SO)
d
11.64 (s, 1H), 11.61 (s, 1H), 7.54 (d, J ¼ 4 Hz,
(400 MHz, (CD3)2SO)
d
11.64 (s, 2H), 8.90 (s, 2H), 7.54 (d, J ¼ 4 Hz,
1H), 7.35 (d, J ¼ 4 Hz, 1H), 7.26 (s, 1H), 7.09 (d, J ¼ 4 Hz, 1H), 7.01 (d,
J ¼ 4 Hz, 1H), 6.98 (s, 1H), 6.95 (s, 1H), 6.87 (d, J ¼ 4 Hz, 1H), 4.03 (d,
J ¼ 12 Hz, 2H), 3.83 (s, 3H), 3.50 (s, 3H), 2.61 (s, 2H), 2.51 (s, 4H),
2.29 (s, 3H), 1.00 (t, J ¼ 6 Hz,2H).
1H),7.47 (d, J ¼ 6 Hz, 1H), 7.27 (s, 1H), 7.08 (d, J ¼ 4 Hz, 2H), 7.01 (d,
J ¼ 4 Hz, 1H), 6.98 (s, 1H), 6.97 (s, 1H), 4.03 (t, J ¼ 6 Hz, 2H), 3.83 (s,
3H), 3.50 (s, 3H), 2.76 (t, J ¼ 6 Hz, 2H), 2.54 (m, J ¼ 12 Hz, 4H), 2.30
(s, 3H), 0.97 (t, J ¼ 8 Hz,6H).
4.1.10.17. N-{2-chloro-4-[3-(dimethylamino)propoxy]phenyl}-N0-{30-
hydroxy-40-[(1E)-N-hydroxyethanimidoyl]-5,6-dimethoxyþbiphenyl-3-
yl}urea (12r). mp.: 176e178 ꢀC. EI-MS (m/z): 556.2 (M ). 1H NMR
4.1.10.11. N-{4-[3-(dimethylamino)propoxy]-2-fluorophenyl}-N0-{30-
hydroxy-40-[(1E)-N-hydroxyethanimidoyl]-5,6-dimethoxyþbiphenyl-3-
yl}urea (12l). mp.: 204e205 ꢀC. EI-MS (m/z): 550.1 (M ). 1H NMR
(400 MHz, (CD3)2SO) d 11.66 (s, 2H), 8.73 (s,1H), 8.59 (s, 1H), 7.54 (d,
(400 MHz, (CD3)2SO)
d
11.70 (s, 2H), 8.91 (s, 2H), 7.53 (d, J ¼ 4 Hz,
J ¼ 4 Hz, 1H), 7.35 (d, J ¼ 6 Hz, 1H), 7.27 (s, 1H), 7.01 (d, J ¼ 4 Hz, 1H),
6.98 (s, 1H), 6.96 (s, 1H), 6.86 (s, 1H), 6.83 (s, 1H), 3.94 (d, J ¼ 6 Hz,
2H), 3.82 (s, 3H), 3.49 (s, 3H), 2.34 (d, J ¼ 8 Hz, 2H), 2.29 (s, 3H), 2.14
(s, 6H), 1.83 (t, J ¼ 10 Hz,2H).
1H),7.47 (d, J ¼ 6 Hz, 1H), 7.27 (s, 1H), 7.07 (s, 1H), 7.06 (s, 1H), 7.00
(d, J ¼ 4 Hz, 1H), 6.97 (s, 1H), 6.95 (s, 1H), 4.01 (t, J ¼ 6 Hz, 2H), 3.82
(s, 3H), 3.50 (s, 3H), 2.34 (t, J ¼ 8 Hz, 2H), 2.29 (s, 3H), 2.13 (s, 6H),
1.83 (t, J ¼ 12 Hz, 2H). 13C NMR (101 MHz, (CD3)2SO)
d 157.60,157.25,
153.21, 141.63, 141.52, 141.17, 140.28, 136.51, 134.48, 134.11, 128.12,
120.05, 118.78, 117.25, 116.24, 114.59, 111.57, 107.47, 107.24, 103.66,
67.98, 60.74, 56.11, 56.01, 45.66, 27.43, 11.54.
4.1.10.18. N-[2-chloro-4-(2-piperidin-1-ylethoxy)phenyl]-N0-{30-hy-
droxy-40-[(1E)-N-hydroxyethanimidoyl]-5,6-dimethoxybiphenyl-3-
yl}urea (12s). mp.: 147e149 ꢀC. EI-MS (m/z): 582.2 (Mþ). 1H NMR
(400 MHz, (CD3)2SO) d 11.66 (s, 1H), 11.65s, 1H), 9.53 (s, 1H), 9.36 (s,
4.1.10.12. N-[2-fluoro-4-(2-piperidin-1-ylethoxy)phenyl]-N0-{30-hy-
droxy-40-[(1E)-N-hydroxyethanimidoyl]-5,6-dimethoxybiphenyl-3-
yl}urea (12m). mp.: 120e122 ꢀC. EI-MS (m/z): 566.1 (Mþ). 1H NMR
1H), 7.54 (d, J ¼ 4 Hz, 1H), 7.37 (d, J ¼ 4 Hz, 1H), 7.32 (s, 1H), 7.00 (d,
J ¼ 4 Hz, 1H), 6.96 (s, 1H), 6.90 (s, 2H), 6.87 (s, 1H), 4.17 (s, 2H), 3.82
(s, 3H), 3.49 (s, 3H), 3.38 (s, 2H), 2.51 (s, 4H), 2.29 (s, 3H), 1.62 (s,
4H), 1.43 (s, 2H).
(400 MHz, (CD3)2SO)
d
11.64 (s, 2H), 8.94 (s, 2H), 7.54 (d, J ¼ 4 Hz,
1H),7.47 (d, J ¼ 6 Hz, 1H), 7.27 (s, 1H), 7.11 (d, J ¼ 4 Hz, 2H), 7.08 (s,
1H),7.01 (d, J ¼ 4 Hz, 1H), 6.98 (s, 1H), 6.97 (s, 1H), 4.08 (t, J ¼ 6 Hz,
2H), 3.83 (s, 3H), 3.50 (s, 3H), 2.64 (t, J ¼ 6 Hz, 2H), 2.42 (s, 4H), 2.30
(s, 3H), 1.49 (m, J ¼ 12 Hz,4H), 1.37 (d, J ¼ 2 Hz, 2H).
4.1.10.19. N-[2-chloro-4-(2-pyrrolidin-1-ylethoxy)phenyl]-N0-{30-hy-
droxy-40-[(1E)-N-hydroxyethanimidoyl]-5,6-dimethoxybiphenyl-3-
yl}urea (12t). mp.: 108e110 ꢀC. EI-MS (m/z): 568.2 (Mþ). 1H NMR
(400 MHz, (CD3)2SO)
d
11.64 (d, J ¼ 4 Hz,1H), 8.85s, 2H), 7.46 (s, 1H),
4.1.10.13. N-[2-fluoro-4-(2-pyrrolidin-1-ylethoxy)phenyl]-N0-{30-hy-
droxy-40-[(1E)-N-hydroxyethanimidoyl]-5,6-dimethoxybiphenyl-3-
7.42 (d, J ¼ 4 Hz, 1H), 7.35 (d, J ¼ 4 Hz, 1H), 7.30 (s, 1H), 7.23 (s, 1H),
7.09 (d, J ¼ 4 Hz, 1H), 7.03 (d, J ¼ 6 Hz, 1H), 6.94 (s, 1H), 5.20 (s, 1H),

H. Gao et al. / European Journal of Medicinal Chemistry 90 (2015) 232e240
239
4.09 (t, J ¼ 6 Hz, 2H), 3.83 (s, 3H), 3.49 (d, J ¼ 6 Hz, 3H), 2.80 (t,
J ¼ 4 Hz, 2H), 2.55 (s, 4H), 2.10 (d, J ¼ 4 Hz, 3H), 1.68 (s, 4H).
(s, 6H), 3.55 (s, 3H), 3.34 (s, 2H), 2.29 (s, 4H), 2.10 (s, 3H), 1.47 (d,
J ¼ 4 Hz,4H), 1.37 (s, 2H).
4.1.10.26. N-{40-[(1E)-N-hydroxyethanimidoyl]-30,5,6-
trimethoxybiphenyl-3-yl}-N0-[4-(morpholin-4-ylmethyl)phenyl]urea
(12x0). mp.: 169e171 ꢀC. EI-MS (m/z): 534.3 (Mþ). 1H NMR
4.1.10.20. N-[2-chloro-4-(2-morpholin-4-ylethoxy)phenyl]-N0-{30-
hydroxy-40-[(1E)-N-hydroxyethanimidoyl]-5,6-dimethoxyþbiphenyl-3-
yl}urea (12u). mp.: 122e124 ꢀC. EI-MS (m/z): 584.1 (M ). 1H NMR
(400 MHz, (CD3)2SO) d 11.07 (s, 1H), 8.68 (s, 2H), 7.41 (s, 1H), 7.39 (s,
(400 MHz, (CD3)2SO)
d
11.65 (s, 1H), 11.61 (s, 1H), 7.54 (d, J ¼ 4 Hz,
1H), 7.33 (s,1H), 7.27 (d, J ¼ 2 Hz,1H), 7.21 (s,1H), 7.19 (s,1H), 7.13 (s,
1H), 7.03 (d, J ¼ 4 Hz, 1H), 6.93 (s, 1H), 3.84 (s, 6H), 3.55 (s, 7H), 3.38
(s, 2H), 2.33 (s, 4H), 2.09 (s, 3H).
1H), 7.34 (d, J ¼ 6 Hz, 1H), 7.26 (s, 1H), 7.10 (d, J ¼ 4 Hz, 1H), 7.01 (d,
J ¼ 6 Hz,1H), 6.98 (s, 1H), 6.96 (d, J ¼ 2 Hz, 1H), 6.87 (d, J ¼ 4 Hz,1H),
4.12 (t, J ¼ 4 Hz, 1H), 4.03 (t, J ¼ 6 Hz, 1H), 3.83 (s, 3H), 3.58 (d,
J ¼ 2 Hz, 4H), 3.50 (s, 3H), 2.69 (m, J ¼ 14 Hz, 2H), 2.51 (s, 4H), 2.30
4.1.10.27. N-{40-[(1E)-N-hydroxyethanimidoyl]-30, 5, 6-
trimethoxybiphenyl-3-yl}-N0-{4-[(4-methylpiperazin-1-yl)mþethyl]
phenyl}urea (12y0). mp.: 109e111 ꢀC. EI-MS (m/z): 547.3 (M ). 1H
(s, 3H). 13C NMR (101 MHz, (CD3)2SO)
d 157.69, 157.19, 153.24,
153.14, 149.37, 141.27, 141.10, 140.29, 136.36, 134.46, 134.09, 128.16,
121.75, 120.57, 120.11, 118.75, 117.24, 115.11, 111.68, 103.81, 67.72,
66.67, 60.75, 57.33, 56.14, 54.17, 11.55.
NMR (400 MHz, (CD3)2SO)
d 11.08 (s, 1H), 8.74 (s, 1H), 8.72 (s, 1H),
7.41 (s,1H), 7.39 (s,1H), 7.33 (s,1H), 7.27 (d, J ¼ 4 Hz,1H), 7.19 (s,1H),
7.17 (s, 1H), 7.13 (s, 1H), 7.04 (d, J ¼ 4 Hz, 1H), 6.93 (s, 1H), 3.84 (s,
6H), 3.55 (s, 3H), 3.37 (s, 2H), 2.31 (s, 8H), 2.14 (s, 3H), 2.10 (s, 3H).
4.1.10.21. N-[2-chloro-4-(3-morpholin-4-ylpropoxy)phenyl]-N0-{30-
hydroxy-40-[(1E)-N-hydroxyethanimidoyl]-5,6-dimethoxyþbiphenyl-3-
yl}urea (12v). mp.: 211e213 ꢀC. EI-MS (m/z): 598.2 (M ). 1H NMR
4.2. Kinase assay [20]
(400 MHz, (CD3)2SO) d 11.65 (s,1H),11.63 (s,1H), 9.29 (s,1H), 9.14 (s,
1H), 7.54 (d, J ¼ 4 Hz,1H), 7.38 (d, J ¼ 6 Hz, 1H), 7.30 (d, J ¼ 2 Hz,1H),
7.00 (d, J ¼ 4 Hz, 1H), 6.97 (s, 1H), 6.93 (d, J ¼ 2 Hz, 1H), 6.89 (s, 1H),
6.87 (s, 1H), 4.01 (t, J ¼ 6 Hz, 2H), 3.95 (d, J ¼ 6 Hz, 4H), 3.82 (s, 3H),
3.49 (s, 3H), 3.45 (d, J ¼ 6 Hz, 2H), 3.24 (s, 2H), 3.07 (s, 2H), 2.29 (s,
3H), 2.18 (m, J ¼ 8 Hz, 2H).
The ability of compounds to inhibit the phosphorylation of a
peptide substrate by VEGFR-2 was evaluated in a microtiter plate
format using homogenous time-resolved fluorescence (HTRF).
Firstly, 2
m
L kinase (Km ¼ 0.003767 ng/
mL) and 2 mL substrate
(Km ¼ 121.4 nM) were separately added to a 384-well plate, and
variable concentrations of compounds (diluted in buffer) were then
4.1.10.22. N-{30-hydroxy-40-[(1E)-N-hydroxyethanimidoyl]-5,6-
dimethoxybiphenyl-3-yl}-N0-[4-(piperidin-1-ylmethyl)phenyl]urea
(12w). mp.: 153e155 ꢀC. EI-MS (m/z): 518.3 (Mþ). 1H NMR
added to the assay plate. ATP (2
m
L, Km ¼ 1.332
mM) was added and
the reaction was allowed to proceed at 37 ꢀC for 30 min. The TK-
Antibody labeled with Eu3þ-cryptate and streptavidin- XL665
were then added with EDTA to detect the phosphorylated product
at room temperature for 1 h. Then the fluorescence was measured
at 615 nm (cryptate) and 665 nm (XL665) using the PerkineElmer
victor 2030 multilabel plate reader. Finally, the results were
calculated as follows: ratio ¼ (OD665 nm/OD615 nm) ꢂ 104.
(400 MHz, (CD3)2SO) d 11.67 (s, 2H), 9.10 (s, 1H), 9.03 (s, 1H), 7.54 (d,
J ¼ 4 Hz, 1H), 7.41 (s, 1H), 7.38 (s, 1H), 7.30 (s, 1H), 7.17 (s, 1H), 7.15 (s,
1H), 7.00 (d, J ¼ 4 Hz, 1H), 6.97 (s, 1H), 6.95 (s, 2H), 3.82 (s, 3H), 3.50
(s, 3H), 3.33 (s, 2H), 2.29 (s, 7H), 1.46 (d, J ¼ 2 Hz, 4H), 1.38 (s, 2H).
13C NMR (101 MHz, (CD3)2SO)
d 157.62, 157.22, 153.28, 153.23,
141.04, 140.33, 138.99, 136.74, 134.48, 132.20, 129.68, 128.13, 120.08,
118.76, 118.33, 117.24, 111.31, 103.39, 62.97, 60.75, 56.10, 54.27,
26.03, 24.55, 11.55.
4.3. Cell growth inhibitory activity in cancer cell lines [21]
Growth inhibitory activities were evaluated on the following cell
lines: A549, MCF-7, ECV304, MDA-MB-435S, H1299, MDA-MB-231,
A375, HT29, and SMMC-7721. Eight potent VEGFR-2 inhibitors were
evaluated using MTT assay to assess cell proliferation. Exponen-
tially growing cells were harvested and plated in 96-well plates at a
concentration of 1 ꢂ 104 cells/well, and then incubated for 24 h at
37 ꢀC. The cells in wells were treated with title compounds
respectively at various concentrations for 48 h. Then, 20 mL MTT
(5 mg/mL) was added to each well and incubated for 4 h at 37 ꢀC.
Supernatant was discarded, and 150 mL DMSO was added to each
well. Absorbance values were determined by a microplate reader
(Bio-Rad Instruments) at 490 nm. The IC50 values were calculated
according to inhibition ratios.
4.1.10.23. N-{30-hydroxy-40-[(1E)-N-hydroxyethanimidoyl]-5,6-
dimethoxybiphenyl-3-yl}-N0-[4-(morpholin-4-ylmethyl)phenyl]urea
(12x). mp.: 153e155 ꢀC. EI-MS (m/z): 520.1 (Mþ). 1H NMR
(400 MHz, (CD3)2SO)
d 11.65 (s, 1H), 11.61 (s, 1H), 8.68 (s, 1H), 8.66
(s, 1H), 7.55 (d, J ¼ 4 Hz, 1H), 7.42 (s, 1H), 7.40 (s, 1H), 7.26 (s, 1H),
7.21 (s, 1H), 7.19 (s, 1H), 7.02 (d, J ¼ 4 Hz, 1H), 6.98 (s, 1H), 6.97 (d,
J ¼ 2 Hz, 2H), 3.83 (s, 3H), 3.56 (s, 4H), 3.50 (s, 3H), 3.38 (s, 2H), 2.33
(s, 4H), 2.30 (s, 3H).
4.1.10.24. N-{30-hydroxy-40-[(1E)-N-hydroxyethanimidoyl]-5,6-
dimethoxybiphenyl-3-yl}-N0-{4-[(4-methylpiperazin-1-yl)mþethyl]
phenyl}urea (12y). mp.: 189e190 ꢀC. EI-MS (m/z): 533.2 (M ). 1H
NMR (400 MHz, (CD3)2SO) d 11.69 (s, 2H), 10.00 (s, 1H), 9.93 (s, 1H),
7.54 (d, J ¼ 4 Hz, 1H), 7.44 (s, 1H), 7.42 (s, 1H), 7.38 (s, 1H), 7.16 (s,
1H), 7.14 (s, 1H), 7.00 (d, J ¼ 4 Hz, 1H), 6.96 (s, 2H), 3.82 (s, 3H), 3.49
(s, 3H), 3.36 (s, 2H), 2.51 (s, 4H), 2.29 (s, 7H), 2.14 (s, 3H). 13C NMR
(101 MHz, (CD3)2SO) d 157.52, 157.18, 153.60, 153.14, 140.78, 140.40,
139.57, 137.21, 134.43, 131.44, 129.67, 128.10, 120.09, 118.75, 118.20,
117.22, 111.18, 103.25, 62.24, 60.75, 56.09, 55.19, 52.95, 46.22, 11.56.
4.4. Molecular docking modeling [22]
In order to understand the binding mode of inhibitors with
VEGFR-2, molecule docking was performed using Sybyl/Surflex-
dock based on the crystal structures of VEGFR-2 (PDB ID: 3C7Q).
Hydrogen was added and minimized using the Tripos force field
and Pullman charges. Compound (12p) was depicted with the
Sybyl/Sketch module (Tripos Inc.) and optimized applying Powell's
method with the Tripos force field with convergence criterion set at
0.05 kcal/(Åmol), and assigned with the GasteigereHückel method.
The residues in a radius 5.0 Å around BIBF1120 (the ligand of
VEGFR-2 in the crystal complex) were selected as the active site.
Other docking parameters were kept at default.
4.1.10.25. N-{40-[(1E)-N-hydroxyethanimidoyl]-30, 5, 6-
trimethoxybiphenyl-3-yl}-N0-[4-(piperidin-1-ylmethylþ)phenyl]urea
(12w0). mp.: 118e120 ꢀC. EI-MS (m/z): 532.3 (M ). 1H NMR
(400 MHz, (CD3)2SO) d 11.07 (s, 1H), 8.73 (s, 1H), 8.69 (s, 1H), 7.40 (s,
1H), 7.38 (s, 1H), 7.33 (d, J ¼ 2 Hz, 1H), 7.27 (d, J ¼ 4 Hz, 1H), 7.18 (s,
1H), 7.16 (s, 1H), 7.13 (s, 1H), 7.03 (d, J ¼ 4 Hz, 1H), 6.93 (s, 1H), 3.84

240
H. Gao et al. / European Journal of Medicinal Chemistry 90 (2015) 232e240
factor (VEGF)-165 and semaphorin 3A-mediated cellular invasion and tumor
Acknowledgment
growth by the VEGF signaling inhibitor ZD4190 in human colon cancer cells
and xenografts, Mol. Cancer Ther. 5 (2006) 2070e2077.
This work was supported by the National Natural Science
Foundation of China (Grant no. 81302641 and 81302737) and the
Fundamental Research Funds for the Central Universities
(xjj2014140).
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.11.032.
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