Design, synthesis and biological evaluation of biphenylurea derivatives as VEGFR-2 kinase inhibitors (II)

Article abstract of DOI:10.1016/j.cclet.2015.10.004

Inhibition of VEGFR-2 signaling pathway is one of the most promising approaches for the treatment of cancer. In this paper, we reported the design, synthesis, and biological evaluation of a series of biphenylurea derivatives as VEGFR-2 inhibitors. Among t

Full text of DOI:10.1016/j.cclet.2015.10.004

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Chinese Chemical Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Original article
Design, synthesis and biological evaluation of biphenylurea
derivatives as VEGFR-2 kinase inhibitors (II)
Guo-Rui Gao a, Meng-Yuan Li b,d, Yong-Cong Lv c, Su-Fen Cao a,
Lin-Jiang Tong b, Li-Xin Wei d, Jian Ding b, Hua Xie b, Wen-Hu Duan a,c,
*
a School of Pharmacy, East China University of Science & Technology, Shanghai 200237, China
b Division of Anti-tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai
201203, China
c Department of Medicinal Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
d Pharmacology and Safety Evaluation Key Laboratory of Tibetan Medicine in Qinghai Province, Northwest Institute of Plateau Biology, Chinese Academy of
Sciences, Xining 810008, China
A R T I C L E I N F O
A B S T R A C T
Article history:
Inhibition of VEGFR-2 signaling pathway is one of the most promising approaches for the treatment of
cancer. In this paper, we reported the design, synthesis, and biological evaluation of a series of
biphenylurea derivatives as VEGFR-2 inhibitors. Among these compounds, 39 exhibited potent
inhibitory activity against VEGFR-2 both in vitro and in vivo. The antiangiogenesis activity of 39 was
further confirmed by both tube formation assay and chick chorioallantoic membrane assay.
ß 2015 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.
Published by Elsevier B.V. All rights reserved.
Received 23 June 2015
Received in revised form 12 July 2015
Accepted 17 September 2015
Available online xxx
Keywords:
Angiogenesis
Kinase
Inhibitor
VEGFR-2
1. Introduction
efforts have been made on the development of drugs targeting
VEGFR-2, leading to several launched anticancer drugs, such as
Angiogenesis, the formation of new blood vessels from pre-
existing vessels, is a normal and indispensable process in growth
and development as well as in wound healing and female
reproductive cycling [1,2]. Pathological angiogenesis has been
correlated with a variety of diseases, such as retinopathies,
diabetes, rheumatoid arthritis, psoriasis, and cancers [3,4]. Tumor
angiogenesis is vital for tumor growth as it is a fundamental step in
the growth and metastasis of cancer [5], making angiogenesis
inhibition a promising therapeutic strategy against cancer.
VEGFs and their receptors (especially VEGFR-2) are very
important in the direct regulation of angiogenesis, and the
overexpression of VEGFR-2 has been closely implicated in the
angiogenesis of solid tumors [6]. Neutralization of the circulating
VEGF by antibodies and inhibition of VEGF receptor tyrosine
kinases with small molecules have been found to effectively inhibit
angiogenesis, tumor progression and dissemination [7]. Enormous
sunitinib [8,9], sorafenib [10], vandetanib [11], pazopanib [12] and
axitinib [13]. Nevertheless, intolerable adverse effects that cause
dose reductions and treatment discontinuations may potentially
negate the life-prolonging effects of these drugs [14], indicating an
unmet need for safer VEGFR-2 inhibitors with high efficacy. As a
part of our continuing program on VEGFR-2 inhibitors, we reported
here the discovery and structural optimization of biphenylurea
derivatives as new VEGFR-2 inhibitors. Sorafenib and pazopanib
are now being used in clinic for the treatment of advanced renal
cell carcinoma (RCC). A close look of their binding modes may
provide some useful information; in general, both molecules bind
to the ATP binding site, penetrated into the extended hydrophobic
pocket and filled the RDP (regulatory domain pocket) region,
locking the protein in a ‘‘DFG-out’’ conformation (Fig. 1); residing
in the ATP binding site, Cys919 forms H-bonds with the pyridine
nitrogen and amide N–H of sorafenib and the anilinopyrimidine
scaffold of pazopanib. Glu885 is served as a H-bond donor to both
molecules; Asp1046, however, only interacted with sorafenib [15].
Direct H-bonds with Glu885 and Asp1046 were considered a
significant opportunity for better selectivity, and greater molecular
size in the RDP site would cause a reduction of efficacy [15]. We
*
Corresponding author at: School of Pharmacy, East China University of Science
& Technology, Shanghai 200237, China.
E-mail address: whduan@simm.ac.cn (W.-H. Duan).
http://dx.doi.org/10.1016/j.cclet.2015.10.004
1001-8417/ß 2015 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: G.-R. Gao, et al., Design, synthesis and biological evaluation of biphenylurea derivatives as VEGFR-2
kinase inhibitors (II), Chin. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.cclet.2015.10.004

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with an IC50 value of 69 nmol/L. Inspired by this promising result,
we then synthesized a series of biphenylurea analogs of 1 to
investigate the structure–activity relationships (SARs).
2. Experimental
The synthesis of analogs was illustrated in Scheme 1. 2,3 (or
4,6)-Dichloropyrimidine (2 and 3) were coupled with substituted
p-nitrophenol to give intermediates 4–7 which were subsequently
reduced to corresponding amine 8–11. Ureas 12–19 were prepared
through a well-established method [16]. All the derivatives (1, 20–
40) were prepared through an acid-catalyzed SNAr reaction of urea
intermediates with substituted anilines in the final step. All
derivatives listed in Tables 1–3 were evaluated for their enzymatic
activities against VEGFR-2 and cellular activities against adeno-
carcinomic human alveolar basal epithelial cells (A549) and
human microvascular endothelial cell (HMEC-1).
3. Results and discussion
Fig. 1. Binding models of sorafenib (PDB ID: 4ASD) and pazopanib with VEGFR-2.
Reproduced with the permission from Ref. [15].
We first explored the effect of variation in rings B and C of
compound 1 (Table 1). Incorporation of F atom on an aromatic ring
could increase lipophilicity, mask a potential metabolic site and
offer a polarized C–F bond that may participate in binding
interaction [17]. Regorafenib, for example, was invented as a
fluoro derivative of sorafenib with better activities. The derivatives
envisioned that an additional hydrogen bond to the Asp1046 by
grafting the biphenylurea moiety from sorafenib to the anilino-
pyrimidine structure of pazopanib would hopefully provide a new
scaffold with high selectivity. Tuning the size of the 3-chloro-4-
(trifluoromethyl)benzene ring may yield better efficacy (Fig. 2). To
our delight, compound 1 inhibited VEGFR-2 at the enzymatic level
with fluoro substitution on the
B ring (20 and 21) were
synthesized. Unfortunately, no improvement of activity was
observed. We also altered the substitution pattern of the
H
H
N
N
CF3
Cl
O
O
N
H
H
N
N
H
N
O
Cl
O
N
O
Soraf enib
CF3
N
N
SO2NH2
N
H
N
N
N
1
N
N
SO2NH2
H
Pazopanib
Fig. 2. Design of hybrid compound 1.
Scheme 1. Synthesis of compounds 1, and 20–40. Reagents and conditions: (a) substituted p-nitrophenol, aq. sodium hydroxide, acetone, 0–80 8C; (b) Fe, aq. ammonium
chloride, ethanol, 70 8C; (c) substituted anilines, triphosgene, triethyl amine, methylene chloride, r.t.; and (d) substituted anilines, 36% HCl, isopropanol, sealed tube, 100 8C.
Please cite this article in press as: G.-R. Gao, et al., Design, synthesis and biological evaluation of biphenylurea derivatives as VEGFR-2
kinase inhibitors (II), Chin. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.cclet.2015.10.004

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3
Table 1
SAR of ring B and ring C.
H
H
3
R
F3C
N
N
2
Ring B
O
Cl
O
N
.
Y
X
Ring C
N
SO2NH2
H
Compd.
R
X
Y
IC50 (mmol/L)
VEGFR-2
A549
HMEC-1
1
H
N
N
N
C
C
C
C
N
–
0.069 Æ 0.033
0.072 Æ 0.007
0.079 Æ 0.004
0.123 Æ 0.058
0.002 Æ 0.001
5.12 Æ 1.08
>100
5.92 Æ 1.35
>100
20
2-F
3-F
H
21
19.22 Æ 15.68
10.46 Æ 3.84
0.10 Æ 0.01
18.33 Æ 14.63
11.02 Æ 1.45
0.12 Æ 0.03
22
Su11248
–
–
pyrimidine ring to move the nitrogen closer to the amino acid
residues in the hinge region, hoping for an extra interaction.
Compound 22, the 4,6-disubstitued pyrimidine analog of 1, was
synthesized and examined. The result indicated that 22 displayed a
1.8-fold reduction in enzymatic activity (IC50: 123 nmol/L). These
facts implied that unmodified rings B and C in compound 1 were
favorable for maintaining activity.
Table 2
SAR of 2-substituents on the pyrimidine ring.
H
H
F3
C
N
N
O
Cl
O
N
.
N
R
Compd.
R
IC50 (mmol/L)
Next, we investigated the effect of substitution pattern of the
aniline at pyrimidine ring on the activity. At first, a set of five
sulfonamides including four meta-sulfonamides (23, 25–27) and
one para-sulfonamide (24) were explored. Although these
derivatives exhibited high inhibitory potency against VEGFR-2
with IC50 values ranging from 4 nmol/L to 69 nmol/L, they were
inactive on cancer cell proliferation assay. Then, the synthesis of a
series of derivatives with various substituted anilines on pyrimi-
dine ring led to 34 (3,4-dimethyl substitution) which exhibited
potent activities in both enzymatic and cellular assays (IC50 values
VEGFR-2
A549
HMEC-1
23
0.039 Æ 0.006
>100
>100
N
SO NH
H
H
N
24
0.069 Æ 0.033
>100
>100
SO NH
25
26
0.004 Æ 0.001
0.064 Æ 0.016
>100
>100
>100
>100
N
SO NH
H
of 7 nmol/L, 2.88
HMEC-1, respectively). Interestingly, unsubstituted aniline deriv-
ative 36 showed high potency against VEGFR-2 and A549 (IC50
5 nmol/L and 8.4 mol/L, respectively) but low potency against
HMEC-1 (IC50: 46.28 mol/L). Taken together, the aniline group at
mmol/L and 2.81 mmol/L for VEGFR-2, A549 and
O
N
H
SO NH
:
m
Cl
27
0.007 Æ 0.001
>100
>100
m
N
H
SO NH
the 2-position of pyrimidine was tolerable to modification. 3,4-
dimethoxy substituted analog was proven to be the best in this
series of compounds at both enzymatic and cellular levels, and
therefore further investigation was focused on the 3,4-dimethoxy
substituted analogs.
Finally, a set of analogs of 34 was prepared to explore the
influence of the substituent on ring A, and this exploration led to
analogs 37–40 as shown in Table 3. Among them, 39 exhibited high
enzymatic inhibitory activity with an IC50 value of 0.4 nmol/L,
which was one of the most potent VEGFR-2 inhibitors as far as we
knew. The privileged compound 39 was further examined in
HUVEC proliferation assay and it exhibited high inhibitory potency
with an IC50 value of 20.36 nmol/L.
28
29
0.007 Æ 0.008
0.014 Æ 0.001
>100
>100
>100
>100
O
O
S
N
H
N
H
O
N
H
30
31
0.006 Æ 0.010
0.004 Æ 0.002
7.92 Æ 2.34
>100
7.87 Æ 2.01
>100
O
O
S
N
H
O
O
S
N
H
H
32
33
2.269 Æ 1.852
0.048 Æ 0.024
>100
>100
>100
>100
N
Compound 39 was then profiled against a panel of tyrosine
kinases. The result demonstrated that 39 inhibited a distinctive,
narrow range of kinases at pharmacologically relevant concentra-
tions (as shown in Table 4). In addition to its high inhibitory
potency on VEGFR-2, 39 also exhibited great potency against
O
O
S
N
O
H
VEGFR-1, PDGFR-
a and PDGFR-b with IC50 values of 3.4, 4.4 and
34
35
0.007 Æ 0.002
0.025 Æ 0.004
2.88 Æ 0.89
>100
2.81 Æ 1.02
>100
O
O
3.6 nmol/L, respectively. Simultaneous inhibition of these distinct
proangiogenic receptors may enhance the antitumor efficacy of 39
and overcome resistance to VEGF- and VEGFR-2-pathway targeted
agents. Moreover, 39 showed high selectivity over RET (1574-fold,
IC50: 629.7 nmol/L) and excellent selectivity (>25,000-fold) over
ErbB2, ErbB4, EGFR, EGFR (T790M/L858R), ABL, EPH-A2, FGFR-1,
FGFR-2 and IGF-1R.
Subsequently, inhibition of VEGF stimulated of cell proliferation
of HUVEC by 39 (positive control: AZD2171 [18]) was tested. As
shown in Fig. 3, the VEGF-induced phosphorylation of VEGFR2
N
H
O
O
O
N
H
36
0.005 Æ 0.006
0.002 Æ 0.001
8.40 Æ 3.039
0.10 Æ 0.01
46.28 Æ 2.78
0.12 Æ 0.03
N
H
Su11248
–
Please cite this article in press as: G.-R. Gao, et al., Design, synthesis and biological evaluation of biphenylurea derivatives as VEGFR-2
kinase inhibitors (II), Chin. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.cclet.2015.10.004

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Table 3
SAR of ring A.
H
H
N
N
R
O
O
N
.
O
O
N
Ring A
N
H
Compd.
R
IC50
(
mmol/L)
VEGFR-2
A549
HMEC-1
37
3-CF3
3-CH3
4-F
0.014 Æ 0.001
0.020 Æ 0.004
0.0004 Æ 0.0002
0.0016 Æ 0.0001
0.002 Æ 0.001
5.83 Æ 0.45
4.06 Æ 1.48
2.65 Æ 1.30
1.37 Æ 0.45
0.10 Æ 0.01
5.07 Æ 0.78
4.24 Æ 1.65
2.93 Æ 1.45
1.38 Æ 1.16
0.12 Æ 0.03
38
39
40
2,4-difluoro
–
Su11248
Table 4
Kinase-selectivity profiling of compound 39.
Fig. 3. Inhibition of VEGFR-2 phosphorylation and related signaling pathways by
compound 39. HUVEC cells were starved for 12 h and treated with indicated
concentration of compounds for 2 h and then stimulated by 100 ng/mL VEGF for
15 min. Cells were harvested and subjected to Western blot analysis.
Enzyme
IC50 (nmol/L)
Enzyme
EGFR
IC50 (nmol/L)
VEGFR-1
VEGFR-2
VEGFR-3
RET
3.4
0.4
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
EGFR(T790M/L858R)
ABL
215.8
629.7
4.4
EPH-A2
antiangiogenic effect of 39 was performed, using chick chorioal-
lantoic membrane (CAM) assay, a method to evaluate in vivo
neovascularization. The results (Fig. 4B) demonstrated that
vascularization in chick embryos was significantly inhibited
when the embryos were treated with 39 (1 and 10 nmol per egg).
These results proved that 39 possessed potent antiangiogenic
activities both in vitro and in vivo.
PDGFR-
PDGFR-
ErbB2
a
b
FGFR-1
3.6
FGFR-2
>10,000
>10,000
IGF-1R
ErbB4
(p-VEGFR-2) was substantially decreased in the presence of 39 in a
dose-dependent manner. Meanwhile, Akt and Erk, the downstream
effector of VEGF/VEGFR, were also attenuated in a dose-dependent
manner.
4. Conclusion
In summary, we designed and synthesized
a series of
Eventually, the anti-angiogenic effect of 39 was also exam-
ined. In vitro tube formation assay of HUVECs showed that
HUVECs formed a complete network structure within 6 h. After
treatment of 39, the tube formation was suppressed in a dose-
dependent manner (Fig. 4A). Further examination of the
compounds as VEGFR2 inhibitors. A highly potent VEGFR-2
inhibitor 39 was identified with an IC50 value of 0.4 nmol/L.
Kinase profiling study indicated that 39 also inhibited three
distinct proangiogenic receptors, VEGFR-1, PDGFR-a and PDGFR-b
with high selectivity over other kinases. More importantly, 39
Fig. 4. Antiangiogenesis activities of compound 39. A. Compound 39 inhibited in vitro tube formation of HUVECs. HUVECs containing with or without indicated concentration
of compound were seeded in 96-well plates which were coated with Matrigel. After 6 h, the tube was examined with an inverted phase contrast microscope. B. Compound 39
showed anti-angiogenesis in CAM model. Glasscover-slip saturated with or without compound 39 was placed on the right side of per field. Arrows indicated the edge line of
the coverglass.
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5
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We thank the National Natural Science Foundation of China
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Technology Major Project ‘‘Key New Drug Creation and
Manufacturing Program’’ (Nos. 2012ZX09103101-024,
&
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the Shanghai Science and Technology Commission (No.
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Please cite this article in press as: G.-R. Gao, et al., Design, synthesis and biological evaluation of biphenylurea derivatives as VEGFR-2
kinase inhibitors (II), Chin. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.cclet.2015.10.004