Design, synthesis and biological evaluation of biphenyl urea derivatives as novel VEGFR-2 inhibitors

Article abstract of DOI:10.1039/c3md00192j

VEGFR-2 plays a critical role in vasculogenesis and VEGFR-2 inhibitors have been widely used in the treatment of cancer. In our continued efforts to search for potent and novel VEGFR-2 inhibitors as antitumor agents, we have identified a potent lead compound (HMQ-16) bearing a biphenyl scaffold. Rearrangement and replacement of arylcarbamoyl in HMQ-16 with a urea moiety generated a series of novel VEGFR-2 inhibitors. In order to enhance the affinity with VEGFR-2, the 4′-acetyl group was converted to an oxime group. Fourteen biphenyl urea derivatives were designed and synthesized as potent VEGFR-2 inhibitors. Six of them (T2, T5, T7, T9, T11, T14) exhibited potent VEGFR-2 inhibitory activity comparable to that of sorafenib. Compound T7 was the most potent with an IC ₅₀ value of 1.08 nM. The enzymatic and cellular assays suggested that T7 has potential as a valuable lead compound for further optimization.

Full text of DOI:10.1039/c3md00192j

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Medicinal Chemistry Communications
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Design, synthesis and biological evaluation of biphenyl urea derivatives as novel VEGFR-2 inhibitors
DOI: 10.1039/C3MD00192J
Chen Wang, Jinyun Dong, Yanmin Zhang, Fang Wang, Hongping Gao, Pengfei Li, Sicen Wang, Jie Zhang
School of Medicine, Xi’an Jiaotong University, No. 76, Yanta West Road, Xi’an, Shaanxi Province, 710061, P.R. China.
 Corresponding author. Tel/Fax:+86-29-82655451; E-mail: zhj8623@mail.xjtu.edu.cn
Abstract: VEGFR-2 plays a critical role in vasculogenesis and VEGFR-2 inhibitors have been broadly used in the treatment of cancer. In our continued
efforts to search for potent and novel VEGFR-2 inhibitors as antitumor agents, we identified a potent lead compound (HMQ-16) bearing biphenyl
scaffold. Rearragement and replacement of arylcarbamoyl in HMQ-16 with urea moiety generated a series of novel VEGFR-2 inhibitors. In order to
enhance the affinity with VEGFR-2, 4'-acetyl group was converted to oxime group. Fourteen biphenyl urea derivatives were designed and synthesized
as potent VEGFR-2 inhibitors. Six of them (T2, T5, T7, T9, T11, T14) exhibited potent VEGFR-2 inhibitory activity comparable to that of Sorafinib.
Compound T7 was the most potent with an IC50 value of 1.08 nM. The enzymatic and cellular assays suggested that T7 has potential as a valuable lead
compound for further optimization.
Keywords: Taspine, VEGFR-2, Biphenyl, Urea, Antiangiogenesis.
Angiogenesis is essential for solid tumor growth and
dissemination. Blockade of angiogenesis is an attractive
approach for the treatment of cancer.1 Vascular endothelial
growth factor (VEGF) is one of the most important inducers
of angiogenesis and exerts its cellular effects by interacting
with VEGF receptors: VEGFR-1 and VEGFR-2 (KDR).
VEGFR-2 is the major positive signal transducer for
endothelial cell proliferation and differentiation.2 VEGFR-2
inhibition is considered to be important in regulating
angiogenesis which is vital for the survival and proliferation
of tumor cells. In recent years, VEGFR-2 inhibitors have
attracted great attention in cancer therapy.3
There has been a lot of research focusing on the development
of novel VEGFR-2 inhibitors. A number of VEGFR-2
inhibitors have been reported with potency to inhibit tumor
angiogenesis.4 Several small molecule VEGFR-2 inhibitors,
such as Sorafenib (BAY-43-9006, Bayer), Sunitinib (SU-
11248, Pfizer) and Vandetanib (ZD6474, AstraZeneca) have
been clinically approved for the treatment of several cancers.5
Vandetanib was approved by FDA in April 2011 for the
treatment of late-stage (metastatic) medullary thyroid cancer.
Moreover, numerous small molecules have progressed to the
clinical evaluation stage. Linifanib (ABT-869, Abbott) is a
potent inhibitor of members of the VEGFR-2 and PDGFR
families. Linifanib is currently in phase III trials. The
preclinical characteristics of Linifanib suggested that it may
offer distinct advantages in cancer therapy. Linifanib is a
novel, orally active multi-targeted receptor tyrosine kinase
(RTK) inhibitor that exhibits potent antitumor and
continues to be an intense area of investigation in anticancer
agents.6
NEt2
H
H
O
N
N
CF3
N
H
O
O
Cl
N
F
H
O
H
N
N
N
H
O
Sorafenib (BAY-93-4006)
Sunitinib (SU-11248)
F
Br
F
H
H
HN
NH2
N
N
N
HN
O
O
N
O
N
N
Vandetanib (ZD6474)
Linifanib (ABT-869)
Figure 1. Structures of VEGFR-2 inhibitors.
He et al found that taspine had good affinity characteristics in
human umbilical vein endothelial cell membrane
chromatography model. In a further study, they identified that
taspine could inhibit tumor angiogenesis through inhibition of
VEGFR-2.7 In our efforts to search for potent VEGFR-2
inhibitors with taspine as lead compound,8 we developed a
novel class of potent VEGFR-2 inhibitors bearing biphenyl
scaffold (Figure 2).9 We found that aniline which contains
one or more halogen substituents (fluoro, chloro, bome) are
useful for biological activity.The biological evaluation of
these biphenyls inspired us to search for potent inhibitors with
biphenyl scaffold targeting VEGFR-2.10 HMQ-16 from this
series was selected as novel lead compound for further
structural optimization. In this manuscript, we described
further optimization of biphenyl-based VEGFR-2 inhibitors in
order to enhance the structural complexity and diversity.
antiangiogenic activities against
experimental tumors and
patients. Development of novel VEGFR-2 inhibitors
a
broad spectrum of
malignancies in
Figure 2. Structural optimization of HMQ-16 and strucutres of novel biphenyls.

Medicinal Chemistry Communications
Page 2 of 4
According to the structural analysis, both Sorafenib and ABT-
The amide group of 3 was converted to amine through
869 contain aromatic urea fragment. The structures of
complexes between inhibitors and VEGFR-2 revealed that the
urea unit interacted with conserved Glu885 and Asp1046
through hydrogen bonds. The terminal phenyl moiety
occupies the hydrophobic pocket of receptor. Sufficient space
was available for the introduction of various substitutes to the
terminal aniline.11 Firstly, rearragement of arylcarbamoyl at 2-
position and amide at 2'-postion to the favorable position was
performed. In order to enhance the affinity with receptor, the
arylcarbamoyl was replaced with urea moiety and the acetyl
was optimized to oxime. Moreover, we introduced a water-
soluble tertiary amine side chain to terminal aniline. The
incorporation of tertiary amine could increase the solubility of
the compounds while retaining their biological activity. The
tertiary amine also mimiced the N,N-dimethylaminoethyl side
chain of taspine. Therefore, the preferable length of side chain
between oxygen and nitrogen was two or three atoms. We
further explored the influence of terminal side chain
variations. The optimization of the substituents at the urea
terminal phenyl ring afforded a series of biphenyl ureas as
novel VEGFR-2 inhibitors (Figure 2). We directed structural
diversity in various substituted aniline.
Hoffmann's degradation. Another intermediate boronate 7 was
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prepared by coupling of bis(pinacolato)diboron with bromide
6, derived from Fries rearrangement ofD3O-Ib: 1ro0.m10o3p9h/Ce3nMyDl 0a0c1e9t2aJte
and O-methylation. Without workup and further addition of
palladium catalyst, in-situ Suzuki coupling with 4 afforded the
key intermediate 8. Subsequently, compound 8 was treated
with triphosgene to produce isocyanate, followed by reacting
with various substituted anilines in dichloromethane to yield
ureas 9. The title compounds T1-T14 were prepared by
subsequent condensation of
9
with hydroxylamine
hydrochloride. All the title compounds were characterized by
NMR, mass spectroscopy and melting point analysis and their
purity were above 95% determined by LC-MS
(Supplementary Information).
All synthesized compounds were screened for kinase
inhibitory activity against VEGFR-2 and the results are
summarized in Table 1 and Table 2. Kinase enzymatic assays
were performed utilizing the homogeneous time-resolved
fluorescence (HTRF) protocol.13 Most of them exhibited
potent VEGFR-2 inhibitory activities. As shown in Tables,
the IC50 values of title compounds ranged from 1.08 nM to
203 nM, while the IC50 value of Sorafinib was 1.06 nM.
Compound T7 displayed the greatest activity with IC50 value
of 1.08 nM which was comparable to Sorafinib. Compounds
T2 and T14 also exhibited potent VEGFR-2 inhibitory
activities with IC50 values of 1.28 nM and 3.07 nM,
respectively. The unexpected loss of potency of T3 might be
due to the poor solubility. The inhibitory rates of T3 were
45.7% (0.032 nM), 64.0% (0.32 nM), 59.7% (3.2 nM), 45.7%
(32 nM), 53.0% (320 nM) and 28.1 (3200 nM). The
unexpected IC50 value of T3 was calculated according to
above inhibitory rates which were not concentration-
Based on our earlier work,12 a general synthesis of biphenyl
ureas followed the reaction pathway outlined in Scheme 1.
We used commercially available isovanillin as the starting
material.
dependent.
The
experimental
measurements
of
physicochemical properties of title compounds are being
performed to validate the hypothesis. Furthermore, changing
dimethylamine to morpholine (T7) restored potency 5-fold
greater than T1. T7 also displayed the highest inhibitory rate
against VEGFR-2 at 3.2 nM. The oxygen atom of morpholine
might interact with the active site of VEGFR-2 as a hydrogen-
bond donor. Moreover, morpholine could improve the
physicochemical properties such as solubility. Unfortunately,
the preliminary VEGFR-2 inhibitory activity of T6 was not
concentration-dependent. The inhibition ratios at low
concentration were higher. Therefore, the IC50 value of T6
could not be correctly calculated according to corresponding
inhibition ratios. Based on these results, we assumed that the
potency of these biphenyl ureas might be affected by the
length of side chain between benzene ring and nitrogen atom
as well as the terminal side chain variations.
Scheme 1. Preparation of title compounds.
Reagents and Conditions: (a) HCOOH, HCOONa, (NH3OH)2SO4,
98%; (b) (CH3)2SO4, K2CO3, Me2CO, 94%; (c) H2O2, NaOH, EtOH,
90%; (d) Br2, NaOH, 60%; (e) AlCl3, 80%; (f) (CH3)2SO4, K2CO3,
EtOH, 95%; (g) [BO2C2(CH3)4]2, dioxane, Pd(pddf)Cl2; (h)
Pd(pddf)Cl2, Na2CO3, H2O, dioxane, 65%; (i) triphosgene, Et3N,
DCM, 50-85%; (j) EtOH, NH4OH, H2O, 88%.
The key intermediate 4 was prepared in a four-step sequence
from 5-bromovanillin 1. Firstly, the aldehyde group of 1 was
converted to cyano via one-pot oxime formation and
dehydration, and subsequent O-methylation to yield 2.
Subsequent oxidative hydration of nitrile afforded amide 3.
Table 1. VEGFR-2 inhibitory activity of the biphenyl ureas (T1-T7)
Compound
R
VEGFR-2, IC50 (nM) (n=5) Inhibitory rate (%)b
N(CH3)2
5.77
1.28
203
7.01
4.08
NDa
1.08
1.06
20.7
52.4
59.7
52.2
55.9
51.0
63.3
68.5
T1
T2
T3
T4
T5
T6
T7
-
N(CH2CH3)2
CH2N(CH3)2
N(CH2)5
N(CH2)4
N(CH2)4O
CH2N(CH2)4O
-
Sorafinib
aND is not determined; b Inhibitory rate determined at 3.2 nM.

Page 3 of 4
Medicinal Chemistry Communications
Table 2. VEGFR-2 inhibitory activity of the biphenyl ureas (T8-T14)
Compound
R
VEGFR-2, IC50 (nM) (n=5) Inhibitory rate (%)b
NDa
3.90
9.87
3.77
8.90
NDa
3.07
1.06
34.9
View Article Online
N(CH3)2
T8
T9
N(CH2CH3)2
CH2N(CH3)2
N(CH2)5
DOI: 10.103594/C.73MD00192J
48.7
29.5
30.2
36.1
54.7
68.5
T10
T11
T12
T13
T14
-
N(CH2)4
N(CH2)4O
CH2N(CH2)4O
-
Sorafinib
aND is not determined; b Inhibitory rate determined at 3.2 nM.
Based on the results of the VEGFR-2 enzymatic assay, six
potent compounds were further evaluated in functional cell-
based assays using a variety of cancer cell lines.14 The results
were shown in Table 3. Most of the title compounds
displayed moderate antiproliferative activity against various
cancer cell lines. Specially, compound T7 showed the highest
activity against 7901 cell with IC50 value of 6.80 μM which
was much less than that of Sunitinib. Moreover, compound
T7 also exhibited the greatest activities against K562 and
SY5Y with IC50 values of 0.29μM and 4.91μM, respectively.
For MDA-MB-231 cell, compound T2 was the most potent.
However, the activity of these compounds against other cells
was less potent than that of Sunitinib. The antiproliferative
results indicated that these biphenyl ureas displayed higher
selectivity against K562 cells with IC50 values ranging from
0.29 μM to 15.8μM.
Table 3. Antiproliferative activities of title compounds on cancer cells
Cancer Cell Lines, IC50 (μM) (n=5)
Compound
7901a
102
34.2
58.1
ND*
6.80
ND
K562b
0.86
8.58
1.29
4.81
0.29
1.25
SY5Yc
5.73
6.20
18.4
ND
4.91
43.5
18.4
MDA-MB-231d
LOVOe
6.56
ND
SK-BR-3f
5.21
ND
A549g
3.36
ND
11.7
ND
11.7
11.2
23.7
7721h
9.86
ND
36.4
ND
9.88
ND
33.3
Helai
7.68
63.2
13.4
ND
92.3
ND
243
35.0
35.2
20.3
ND
Sunitinib
T1
48.4
49.2
ND
ND
38.9
ND
T2
T5
T7
T9
ND
ND
49.2
ND
ND
14.7
ND
ND
15.8
T14
a7901, human gastric cancer cell line; K562, human immortalised myelogenous leukemia line; SY5Y, human neuroblastoma cell line;
b
c
dMDA-MB-231, human breast carcinoma cell line; LOVO, human colon adenocarcinoma cell line; SK-BR-3, humanbreast adenocarcinoma
e
f
cell line; gA549, carcinomic human alveolar basal epithelial cells; h7721, human hepatocarcinoma cell line; iHela, Human epithelial
carcinoma cell line.
* ND is not determined.
Computational studies were performed to investigate the
potential binding mode of the title compounds with VEGFR-2.
The most active compound T7 was constructed and optimized
using Powell’s method with Tripos force field.15 The
convergence criterion was set at 0.05 kcal/(Å·mol), and
assigned with Gasteiger-Hückel charge.16 The docking study
was performed using Sybyl/Surflex-Dock module, the
residues in a radius of 6.5 Å around BAX 1500 of VEGFR-2
(PDB ID: 4ASD) were selected as the active site. The binding
mode of T7 into the active site of VEGFR-2 was shown in
Figure 3. There were eight H-bond interactions between urea
and oxime of T7 and amino acid residues of the enzyme. The
urea portion accessed the hydrophobic pocket adjacent to the
hinge region, with the urea carbonyl oxygen forming a
hydrogen bond with the NH of Asp1046 with length of 1.70
Å. The external NH of the urea unit formed two hydrogen
bonds to the carboxylate of Glu885 with distance of 2.27 Å
and 1.72 Å, respectively. The terminal oxime group acted as
essential hydrogen bond acceptor and donor for Asp814 and
Arg1027 in active site. The nitrogen of oxime acted as
hydrogen bond acceptor, forming two hydrogen bonds to
Arg1027 with distance of 2.21 Å and 2.83 Å, respectively.
The oxygen formed a hydrogen bond to Arg1027 with
distance of 2.56 Å while hydrogen formed two hydrogen
bonds to Asp814 carboxylate (2.17 Å and 2.54 Å). These
interactions between the urea moiety and VEGFR-2 were
consistent with the reports of other VEGFR-2 inhibitors of the
urea type. The capability to form hydrogen bonds of urea and
oxime contributed significantly to the affinity with the
receptor. Molecular insights based on molecular docking
indicated favorable binding interactions of these biphenyl
ureas with the active site of VEGFR-2.17 This binding
hypothesis could provide valuable information for structure-
based design of VEGFR-2 inhibitors.
Figure 3. Binding conformation of T7 with VEGFR-2 (PDB ID: 4ASD). Hydrogen bonds are shown with yellow dotted lines.

Medicinal Chemistry Communications
Page 4 of 4
In the current study, we described the design, synthesis and
preliminary evaluation of novel biphenyl ureas as potent
VEGFR-2 inhibitors. A molecular docking study was
performed to identify their binding mode with the receptor.
The results indicated that compound T7 was nicely bound to
VEGFR-2 with eight hydrogen bonds. The urea portion
interacted with the conserved pocket as previous report. It
also disclosed the importance of the urea and oxime for the
potency of these inhibitors.
View Article Online
DOI: 10.1039/C3MD00192J
In summary, we indentified an alternative substitution pattern
of biphenyl urea as novel VEGFR-2 inhibitors. Incorporation
of urea unit and oxime moiety to biphenyl afforded a series of
potent VEGFR-2 inhibitors. The molecular docking studies
demonstrated that oxime played important role in its rationale
interacting with VEGFR-2. Oxime group was identified to be
capable of acting as both hydrogen bond donor and acceptor
for the residues in active site. Compound T7 was identified as
a promising lead compound with novel scaffold. These
compounds have strong potential to be further developed as
novel VEGFR-2 inhibitors. The results suggest that they could
therefore be a promising starting point for further medicinal
chemistry efforts. Further modifications may be undertaken
on the biphenyl core and terminal benzene ring of the urea
unit. Our efforts in pursuing the optimization of the
physicochemical and biological properties of the biphenyl
ureas will be reported in due course.
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (Grant Number 30901839 and
81230079) and the Fundamental Research Funds for the
Central Universities.
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