Quinoxalinone (Part II). Discovery of (Z)-3-(2-(pyridin-4-yl)vinyl)quinoxalinone derivates as potent VEGFR-2 kinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2016.03.008

Inhibition of VEGFR-2 kinase has been highlighted as one of the well-defined strategies to suppress tumor growth via blockade of angiogenesis. Guided by the principles of bioisosteric replacement and pharmacophoric fragment migration, a series of novel quinoxalinone derivates were designed, synthesized and evaluated for their VEGFR-2 inhibitory potencies. Among them, compounds 7c, 8b, 8c, 8e and 10b displayed antiangiogenic abilities via the in vitro tube formation assay (cellular level) and ex vivo rat aortic ring assay (tissue level) at a low concentration (0.1 μM). By means of in vivo zebrafish embryo model, two (Z)-3-(2-(pyridin-4-yl)vinyl)quinoxalinone derivates 8c and 8e showed significant antiangiogenesis effects, suggesting they have potentials to be developed into antiangiogenesis agents via further structural optimization. Moreover, these two compounds also demonstrated potent inhibition toward VEGFR-2 and B-raf kinases in a low concentration (1 μM). A possible interpretation of our evaluation result has been presented by a molecular docking study by docking representative compound 8c with VEGFR-2.

Full text of DOI:10.1016/j.bmc.2016.03.008

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Quinoxalinone (Part II). Discovery of (Z)-3-(2-(pyridin-4-yl)vinyl)
quinoxalinone derivates as potent VEGFR-2 kinase inhibitors q
Leilei Shi a, Jianfeng Zhou b, Jifeng Wu c, Junya Cao d, Yuemao Shen a, Hua Zhou e, Xun Li a,
⇑
a School of Pharmaceutical Sciences, Key Laboratory of Chemistry and Chemical Biology (Ministry of Education), Shandong University, Ji’nan, Shandong 250012, PR China
b School of Medicine and Pharmacy, Ocean University of China, Qingdao 266100, PR China
c Institute of Criminal Science and Technology, Ji’nan Public Security Bureau, Ji’nan 250100, PR China
d School of Medicine, Shandong University, Ji’nan 250012, PR China
e Department of Chemistry, Texas A & M University, College Station, TX 77842, USA
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 kinase has been highlighted as one of the well-defined strategies to suppress tumor
growth via blockade of angiogenesis. Guided by the principles of bioisosteric replacement and pharma-
cophoric fragment migration, a series of novel quinoxalinone derivates were designed, synthesized and
evaluated for their VEGFR-2 inhibitory potencies. Among them, compounds 7c, 8b, 8c, 8e and 10b dis-
played antiangiogenic abilities via the in vitro tube formation assay (cellular level) and ex vivo rat aortic
Received 12 January 2016
Revised 1 March 2016
Accepted 3 March 2016
Available online xxxx
ring assay (tissue level) at a low concentration (0.1 lM). By means of in vivo zebrafish embryo model, two
Keywords:
(Z)-3-(2-(pyridin-4-yl)vinyl)quinoxalinone derivates 8c and 8e showed significant antiangiogenesis
effects, suggesting they have potentials to be developed into antiangiogenesis agents via further struc-
tural optimization. Moreover, these two compounds also demonstrated potent inhibition toward
VEGFR-2 inhibitor
Quinoxalinone derivates
Antiangiogenesis
Pyridyl motif
VEGFR-2 and B-raf kinases in a low concentration (1 lM). A possible interpretation of our evaluation
result has been presented by a molecular docking study by docking representative compound 8c with
Activity evaluation
VEGFR-2.
Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction
therapy in the treatment of carcinoma, especially for metastatic
or advanced solid tumors.2
Abnormal angiogenesis plays a critical role in carcinogenesis
and metastasis of tumors, and is also the necessary path through
which tumor transition from benign to malignant. Among the
identified angiogenic factors that are involved in tumor angiogen-
esis, vascular endothelial growth factor receptor-2 (VEGFR-2), also
known as kinase insert domain receptor (KDR) for human and Flk-
1 for murine, is believed to be the primary and the most pivotal
regulator.1 VEGFR-2 can drive angiogenesis through binding with
its natural ligand VEGF, a known promoter of angiogenesis and
selective mitogen mediating the mitogenic signaling of endothelial
cells, resulting in the stimulation of downstream signaling cas-
cades and some endothelial responses, such as vessel permeability
increase and endothelial cell proliferation enhancing. Conse-
quently, the blockage of the VEGF/VEGFR-2 mediated signaling
transduction represents a promising strategy for antiangiogenic
In the past decades, efforts on aiming at the interference of
VEGF/VEGFR-2 have exploited numerous tumor angiogenesis inhi-
bitors (TAIs) with high selectivity and inhibitory efficacies, such as
the anti-VEGF monoclonal antibody Bevacizumab, and small-mole-
cule inhibitors such as ATP-competitive antiangiogenic agents, as
shown in Figure 1,3 confirming the attractiveness of VEGFR-2
targeting inhibitors for cancer therapy.
From the binding modes of these small molecular inhibitors
with VEGFR-2, several structural features are required for achiev-
ing a potent efficacy:4 (i) a basic skeletal structure that contains at
least one H-bond acceptor (N atom is preferred, followed by the
O atom) so as to interact with the NH of Cys919, the key amino acid
residue in the catalytic ATP-binding domain. Monocyclic ring com-
pounds (e.g., pyridine and pyrimidine) and several 5–6- or 6–6-
bicyclic heterocycles, including but not limited to quinazoline,
quinoline, benzimidazole, and indolin-2-one motifs, are common
chemotypes utilized as basic backbones; (ii) a functional group
acting as pharmacophore (e.g., amide or urea) that possesses both
H-bond acceptor and donor in order to bind with two crucial
residues (Glu885 and Asp1046) in the DFG (Asp-Phe-Gly) motif,
q
Part I has been reported in BMC (Ref. 6).
⇑
Corresponding author.
E-mail address: tjulx2004@sdu.edu.cn (X. Li).
http://dx.doi.org/10.1016/j.bmc.2016.03.008
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Shi, L.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.03.008

2
L. Shi et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
SO2NH2
NH
H
N
H
N
H
N
H
N
CF3
Cl
CF3
O
O
N
O
NH
O
N
O
F
Cl
O
N
N
H
N
N
H
N
N
H
N
N
O
S
O
H
N
N
NH2
H
N
N
H3C
O
N
N
O
O
N
Motesanib
Pazopanib
Sorafenib
Regorafenib
Apatinib
H
N
F
Br
H
N
F
Br
N
F
Br
HN
N
O
HN
HN
N
O
N
O
O
O
O
F
O
O
N
O
N
F
O
O
N
O
O
N
N
O
OH
N
N
N
O
N
Cediranib
Vandetanib
Brivanib
Cl
Erlotinib
Erlotinib
H
N
H
N
H
N
H
H
N
Cl
Cl
N
O
O
O
N
O
O
O
N
Cl
O
O
N
F
O
NH
O
N
O
O
NC
O
O
H2N
N
N
O
N
O
O
Cabozantinib
Tivozanib
Lenvatinib
Bosutinib
N
N
N
O
O
N
O
N
N
H
N
N
H
NH
N
H
N
F
N
H
H
F
O
N
NH
O
O
O
N
H
O
N
S
N
H
N
H
O
N
H
N
H
N
H2N
N
H
H
F
NH
O
O
Linifanib
Sunitinib
Toceranib
Axitinib
Semaxanib
Nintedanib
Figure 1. Representative VEGFR-2 inhibitors that have been approved or in various clinical trials.
an essential tripeptide sequence in the active kinase domain; (iii) a
hydrophobic substituent that can embed into the deep cavity of
hydrophobic pocket and further stabilizes the interaction between
the inhibitor and target enzyme via hydrophobic van der Waals
contact. All this binding information has provided guidance for
developing novel VEGFR-2 tyrosine kinase inhibitors.
TAIs do, but rather occupies the hydrophobic areas of the enzyme
using the central phthalazine scaffold as well as the terminal Cl-
containing aryl moiety (Fig. 2). Moreover, a water-bridged indirect
H-bond can be observed between the NH and Glu915/Cys917.14
On the basis of the structure–activity relationship (SAR) analy-
sis of Axitinib and Vatalanib, it is reasonable to come to a conclu-
sion that pyridyl, aryl and amide moieties are favorable
contributors for selective VEGFR-2 inhibition. These pharma-
cophoric elements were therefore appropriately assembled in the
quinoxalinone core so as to achieve potent VEGFR-2 inhibition
and also the molecular diversity for further SAR investigation: (i)
incorporating a pyridyl motif (Part A) into 3-position of quinoxali-
none core via an aldol condensation to build a conjugated system
as Axitinib does; (ii) installing an amide (Part B) and substituted
aryl moiety (Part C) to the 1-N of quinoxalinone via nucleophilic
substitution to maintain a similar molecular orientation as Vata-
lanib does; (iii) introducing an adjacent phenyl ring that links with
the 1-N of quinoxalinone and amide (Part D) to simulate the struc-
tural pattern of the VEGFR-2 inhibitor Sorafenib and afford possible
As a continuous work for quinoxalinone-derived medicinal
chemistry in our lab,5–7 the 6–6-bicyclic quinoxalinone (or quinox-
alin-2-one) core, which contains both electron donor and acceptor,
acts as a bioisostere with the validated scaffolding structures of
several VEGFR-2 inhibitors, such as quinazoline (e.g., Vandetanib),
quinoline (e.g., Cabozantinib), phthalazine (e.g., Vatalanib), and
indoline-2,3-dione (e.g., Sunitinib), etc. It is therefore developed
into new VEGFR-2 inhibitors as an attractive building block.
Axitinib (AG013736) is an approved VEGFR-2 inhibitor for renal
cell carcinoma (RCC) with promising long-term safety profile8 and
inhibitory potency for BCR-ABL1-driven drug-resistant leukemia.9
It has been proved that the conjugated pyridyl portion contributes
to the solubility, metabolic stability, and pharmacokinetics.10 The
same pyridyl group can also be seen in Vatalanib (PTK787), another
selective VEGFR-2 inhibitor which can sensitize multidrug resis-
tant cancer cells to chemotherapy under hypoxic conditions and
has entered phase III clinical trials as a second-line therapy in sev-
eral types of metastatic or advanced cancers.11–13 The pyridyl part
contributes largely to the kinase selectivity in addition to solubil-
ity. This is because the pyridyl N can form pivotal H-bonding inter-
action with Lys1060, a residue of the kinase activation loop which
plays an important role in selective recognition of the identified
tyrosine kinase subtypes. In addition to this, Vatalanib doesn’t
form any other direct H-bonds with the ATP hinge region as most
p–p stacking interaction with Phe1047 in the DFG area, as Sorafe-
nib does;15 (iv) hydrophobic substituents (R) were fixed at various
positions of terminal aryl portion so as to penetrate into the
extended hydrophobic pocket of the kinase domain of VEGFR-2.
To test our designing hypothesis, these quinoxalinone derivates
were consequently subjected to the in vitro and in vivo antiangio-
genesis evaluation. It resulted that compounds 8b, 8c, 8e, 10b were
promising inhibitors against VEGFR-2 and might be viewed as
potential leads for further structural modification. Preliminary
SAR investigation and molecular modeling study of these deriva-
tives were also discussed.
Please cite this article in press as: Shi, L.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.03.008

L. Shi et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
Glu915
Cl
H
O
Potency
Glu917
H
H
N
Cys917
Glu885
H
H
N
Asp1046
Cys919
N
N
N
N
Hydrophobic
interaction
O
S
Pharmacokinetics,
metabolic stability,
and solubility
N
Selectivity,
solubility
N
Pharmacophore
Lys1060
Axitinib
Vatalanib
Part C
Part B
O
O
R
R
N
H
Part D
N
O
Part C
1
H
N
Part B
2
1N
O
2
3
N
3
4
N
4
Part A
N
Part A
N
Target compounds
Figure 2. The design concepts of targeted compounds.
condensation of the commercially available o-phenylenediamine 1
with ethyl pyruvate led to the formation of quinoxalinone core 2.
Owing to the keto–enol tautomerization of amide bond in quinoxali-
none scaffold, subsequent nucleophilic substitution by using methyl
2-bromoacetate in the presence of K2CO3 provided a mixture of N-
substituted ester compound 3 with relatively high yield (>50%)
2. Results and discussion
2.1. Synthesis
To achieve the target quinoxalinone derivates 7–10, the general
synthetic steps outlined in Scheme 1 were adopted. Briefly, the
O
O
O
O
H
R1
R1
NH2
NH2
R1
R1
N
O
R1
R1
N
N
N
N
O
O
a
b
N
1
1
1
2a:
2b:
R =H;
1
3a:
3b:
R =H;
4a:
R =H
1
1
R =OMe
R =OMe
O
R2
O
R2
O
N
O
H
R1
R1
N
N
N
OH
O
H
O
R1
R1
N
N
R1
R1
N
N
e
d
N
1
1
2
1
2
5a:
5b:
R =H;
7a:
7b:
7c
R =H; R =H
8a:
8b:
8c
R =H; R =H
1
1
2
1
2
R =OMe
R =H; R =4'-F
R =H; R =4'-F
1
2
1
2
R =H; R =4'-Cl
R =H; R =4'-Cl
7d: R1=H; R2=2'-Me
7e: R1=H; R2=3',4'-Cl
8d: R1=H; R2=2'-Me
8e: R1=H; R2=3',4'-Cl
c
7f: R1=H; R2=3',4'-OMe
7g: R1=OMe; R2=H
8f: R1=H; R2=3',4'-OMe
8g: R1=OMe; R2=H
O
R3
R3
N
O
O
O
N
H
N
O
N
N
O
OH
N
H
N
e
d
N
N
3
3
10a:
10b:
R =Me
6a
9a:
9b:
R =Me
3
3
R =Cl
R =Cl
Scheme 1. General procedures for the synthesis of quinoxalinone derivates. Reagents and conditions: (a) Ethyl pyruvate, anhydrous EtOH, rt; (b) methyl bromoacetate,
K2CO3, acetone, reflux; (c) LiOH, Dioxane/H2O (3:1, v:v); (d) substituted aniline, isobutyl chloroformate, 4-methylmorpholine, anhydrous THF, 0 °C–rt; (e) 4-
pyridinecarboxaldehyde, concentrated sulfuric acid, acetic acid, 50 °C.
Please cite this article in press as: Shi, L.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.03.008

4
L. Shi et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
O
O
O
O
H
N
O
O
a
OH
b
N
N
O
Br
Br
N
2a
11
12
R
O
O
H
N
N3
OH
O
N
O
N
N
O
N
O
c
d
e
N
N
15a:
15b:
R=Me;
R=Cl
14
13
Scheme 2. Reagents and conditions: (a) acetyl chloride, anhydrous methanol, reflux; (b) K2CO3, KI, acetone, reflux; (c) KOH, anhydrous alcohol, H2O, rt; (d) DPPA, dioxane,
TEA, reflux; (e) substituted anilines, TEA, rt.
and the O-substituted ester compounds 4 with relatively low yield
(<30%). After purification by column chromatography method, the
separated ester compounds were respectively submitted to the fol-
lowing hydrolization under basic condition to give the correspond-
ing carboxylic acids 5 and 6. This was followed by coupling with
various anilines in the existence of isobutyl chloroformate and N-
methylmorpholine (NMM) in anhydrous THF to afford the quinox-
alinone amides 7 and 9. In this reaction, apart from THF, other apro-
tic polar solvents such as dichloromethane (CH2Cl2) and N,N-
dimethylformamide (DMF) are possible alternatives. Treatment of
quinoxalinone amides 7 or 9 with 4-pyridinecarboxaldehyde using
a small amount of concentrated sulfuric acid as catalyst via an aldol
condensation furnished the final products 8 and 10. In these two
final products, the coupling constants for the vinyl protons
(J = 5.1 Hz) clearly indicate that double bonds is cis-configuration.
The synthesis of compounds 15a and 15b is illustrated in
Scheme 2. Nucleophilic substitution of quinoxalinone core 2a with
methyl 4-(bromomethyl)benzoate (11), which was obtained via
esterification of the commercially available 4-(bromomethyl)ben-
zoic acid, gave the ester compound 12. The subsequent ester
hydrolization led to the carboxylic acid 13, which was subjected
to the treatment of diphenylphosphoryl azide (DPPA) and was con-
verted to the acyl azide 14. The intermediate was treated in situ
with anilines via nucleophilic substitution to yield the amide com-
pounds 15.
the cytotoxic evaluation against a panel of human tumor cell lines
by MTT assay, which was performed in triplicates. As summarized
in Table 1, all the tested compounds displayed no or weak cyto-
toxic effects against six kinds of tumor cells, MCF-7 (breast carci-
noma), 3AO (ovarian carcinoma), PC-3 (prostatic carcinoma),
K562 (human chronic myeloid leukemia), HepG-2 (hepatic carci-
noma) and Hela (cervix adenocarcinoma) under the testing condi-
tions, as compared with the approved VEGFR-2 inhibitor Pazopanib
(GW786034). In this regard, the target compounds are proved to be
potentially cytostatic rather than cytotoxic agents.
2.2.2. In vitro antiangiogenic ability in a tube formation assay
Given tube formation of vascular endothelial cells is an impor-
tant process during tumor angiogenesis, and human umbilical vein
endothelial cells (HUVECs) can form capillary-like structures in the
presence of Matrigel under the stimulation of VEGF to simulate the
tumor angiogenetic process, the transmutation of HUVECs in
Matrigel is thereby utilized as a validated in vitro model to evalu-
ate the antiangiogenic ability. Compounds 7b, 7c, 8a–8g, 9, 10 and
15 were subsequently submitted to the tube formation inhibitory
assay, using Pazopanib as a positive control. As shown in Figure 3,
five compounds 7c, 8b, 8c, 8e and 10b were effectively inhibited
VEGF-induced tube formation of HUVECs in a dose-dependent
manner.
Actually, our original intention is to introduce the diaryl urea
functionality, a well-defined pharmacophoric moiety in VEGFR-2
inhibitors by forming essential H-bonding interactions with DFG
motif of VEGFR-2,16,17 to 1-N position of quinoxalinone core to fur-
ther explore the SAR. However, in our synthetic condition as shown
in Scheme 2, when the carboxylic acid 13 worked with DPPA, the
azido-substituted intermediate 14 rather than our assumed iso-
cyanate was obtained. The possible reason might be that the reac-
tion temperature is not high enough using dioxane as solvent.
Based on this, the diaryl urea derivatives are underway in our lab
and the results will be reported later.
2.2.3. Ex vivo antiangiogenic ability in rat aortic ring
Rat aortic ring microvessel growth is another practical and cost
effective assay of angiogenesis, which offers an intuitive observa-
tion of the ex vivo antiangiogenic effect and bridges the gap
between in vitro and in vivo models. By using intact vascular
explants that were cultured in collagen gel, the rat aortic ring
model can reproduce the microvascular networks more accurately
in which angiogenesis can take place rather than those with iso-
lated endothelial cells. The assay results demonstrated that five
compounds, 7c, 8b, 8c, 8e and 10b, with anti-angiogenetic activi-
ties at the cellular level, also had capacities to inhibit microvessel
sprouting in the mimetic angiogenic process at the tissue level
under the experimental conditions. As evinced in Figure 4, the
antiangiogenic effects of five test compounds were even better
than that exerted by the reference Pazopanib at the same
concentrations.
2.2. Biological evaluation
2.2.1. Antiproliferation assay
Considering VEGFR-2-targeted inhibitors are inherently cyto-
static agents that can only delay tumor growth via inhibition of
newly formed blood vessels but not completely eliminate tumor
cells as traditional cytotoxic agents (chemotherapeutics) do, target
compounds 7b, 7c, 8a–8g, 9, 10, and 15 were initially submitted to
2.2.4. In vivo antiangiogenic ability in zebrafish model
The ideal animal model to evaluate the antiangiogenesis effect
should provide easily visible blood vessels growing in their natural
Please cite this article in press as: Shi, L.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.03.008

L. Shi et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
Table 1
The anti-proliferative results of target compounds
Compds/cell lines
MCF-7a (IC50
/
l
M)
3AOa (IC50
>100
/
l
M)
PC-3a (IC50
/
l
M)
K562a (IC50
/
l
M)
Helaa (IC50
/l
M)
BEAS-2Ba (IC50
/lM)
7b
7c
8a
8b
8c
8d
8e
8f
8g
9a
9b
10a
10b
15a
15b
Pazopanib
>100
>100
>100
>100
>100
>100
59.93 1.75
>100
>100
>100
>100
>100
>100
68.05 2.11
>100
>100
>100
>100
>100
55.45 2.31
>100
>100
>100
>100
>100
>100
>100
>100
74.56 0.21
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
40.23 1.92**
>100
55.52 3.11***
>100
49.04 1.18***
>100
>100
>100
50.44 2.45***
>100
>100
69.65 0.32***
>100
47.57 2.47***
>100
>100
>100
55.23 0.14***
>100
>100
>100
>100
55.65 3.09*
>100
>100
>100
>100
>100
>100
>100
>100
>100
50.23 0.98
60.22 1.44***
>100
49.86 3.45***
>100
>100
47.57 4.47***
76.78 0.019
39.56 0.23
>100
>100
89.32 0.23
>100
>100
a
*
Results are presented as the mean SD for at least three experiments for each group.
P <0.05.
P <0.01.
P <0.001.
**
***
environment, and rapid responses of live organism to drugs, allow-
ing rapid screening of the test compounds.18 In this context, the
in vivo vascular studies of the compounds 7c, 8b, 8c, 8e and 10b
were further submitted to the anti-angiogenetic evaluation on a
well-accepted vertebrate zebrafish (Danio rerio) embryos model,
using vatalanib as the positive control.
To attain an optimal visualization, the Tg(flk1:GFP) transgenic
zebrafish embryos expressing enhanced green fluorescent protein
(GFP) in their endothelial cells under the control of flk1/VEGFR-2
promoter was used in the assay, with a clear view of vasculature
under the fluorescence microscopy.19 The main vascular structures
of zebrafish embryos include the dorsal longitudinal anastomotic
vessel (DLAV), posterior cardinal vein (PCV), dorsal aorta (DA)
and intersegmental vessel (ISV), as manifested in Figure 5. The
anti-angiogenic effect was assessed according to the inhibitory
effects of different doses of test compounds on ISV formation in
the zebrafish embryos. In this study, after post-fertilization of zeb-
rafish embryos at 24 h, and treated embryos with test compounds
(7c, 8b, 8c, 8e and 10b) for 48 h, 8c and 8e showed significantly
dose-dependent inhibition of ISV formation at the concentrations
by the 7c, and 8b gave the least potency, which were consistent
with that of their in vitro and in vivo activities. Moreover, all the
test compounds exhibited weaker activities than the reference
standards (staurosporine and sorafenib), however, they displayed
selective inhibition against VEGFR-2 as compared with other
angiogenesis-related kinases. It is worth mentioning that com-
pounds 8c, 8e and 10b, in addition to exerting VEGFR-2 inhibition,
also displayed partly inhibitory abilities towards B-RAF, another
critical kinase in mediating tumor proliferation and angiogenesis,
and also has close correlation with tumor multidrug resistance
(MDR).21 As a result, these analogues might be expected to develop
into multi-targeted anti-tumor agents after further chemical
optimization.
2.3. Structure–activity relationships (SARs)
Collectively, the in vitro and in vivo results supported an impor-
tant role of the introduced pyridyl, amide and aryl motifs, which
have been validated as pharmacophoric structures and favorable
contributors for selective VEGFR-2 inhibition. Typically, when
amide and aryl substituents were present at 1-N position of the
quinoxalinone skeleton, compounds 8a–g, to a greater extent, gen-
erated most potent VEGFR-2 inhibition, in comparison with com-
pound 7 (without pyridyl motif), or compounds 9 and 10, in
which the amide aryl substituents were present at 2-O position
of the quinoxalinone skeleton. However, in the cases of compounds
15a and 15b, the introduction additional phenyl ring between
quinoxalinone core and amide functionality did not exert an
expected improved potency, but a total loss of activity. This might
be derived from the less effectiveness of biphenyl amide structure,
as compared with the diaryl urea functionality which has been rec-
ognized as an critical pharmacophore in designing potential
VEGFR-2 inhibitors.22
Comparing with the impacts of substituents on quinoxalinone
core, we can conclude that introducing substituent (e.g., methoxyl)
to the quinoxalinone core is unfavorable for the bioactivity. Next,
we examined the impact of the hydrophobic substituent variations
on the terminal phenyl ring on their bioactivities. Considering
methyl and halogen groups in meta- and/or para-position are the
most frequent substitution pattern in type II VEGFR-2 inhibitors,23
five groups (H, F, Cl, CH3, OCH3) are investigated in our target com-
pounds 7, 8 and 10. As a consequence, compounds 7c, 8b, 8c, 8e
of 10, 50 and 100 lM, 10b gave a moderate inhibitory effect, while
8b presented a weak inhibition in comparison with the vehicle-
treated control (0.2% DMSO). As for the positive control (vatalanib),
the embryos displayed complete ISV regression at the concentra-
tion of 10
lM. As far as compound 7c was concerned, ISV malfor-
mation was observed at 100
lM
concentration. These data
clearly demonstrated the anti-angiogenic property of target com-
pounds, largely owing to the blood vessel inhibition rather than
the toxicity of the existing vasculature.
2.2.5. Enzymatic inhibition assay
Compounds that revealing obvious antiangiogenic activities (7c,
8b, 8c, 8e and 10b) were further evaluated for their inhibitory effi-
cacies on several kinases that involved in angiogenesis. In vitro
enzymatic inhibition percentage (%) of compounds at 1 lM against
recombinant human kinase insert domain receptor (KDR/VEGFR-
2), serine/threonine protein kinase B-RAF, tyrosine kinases FGFR1
and EGFR were performed.20 The levels of phosphorylation of the
kinase-specific ligand peptides at 1 lM of compounds were mea-
sured, and the kinase inhibition profile was shown in Table 2.
Among them, compounds 8e, 8c, 10b displayed the most potent
inhibition toward VEGFR-2 in a low concentration (1 lM), followed
Please cite this article in press as: Shi, L.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.03.008

6
L. Shi et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
1 µM
10 µM
0.1 µM
Negative
control
Pazopanib
7c
8b
8c
8e
10b
Figure 3. Anti-angiogenesis assay results on HUVEC.
and 10b presented similar anti-angiogenetic bioactivity, implying
the halogen-substitution is the most preferred substitutions
against VEGFR-2. And else, different halogens in aromatic ring have
similar levels of impacts on their bioactivities. We envisioned that
the small halogens might provide an effective binding interaction
with amino acid residues in target protein, leading to a favorable
occupation into the hydrophobic pocket. Additionally, activity of
substitution in para-position is better than meta-position.
(ii) the introduced carbonyl O of amide functionality acts as a
hydrogen bond acceptor to interact with Lys866 with distance of
1.63 Å; (iii) the para-Cl on the terminal phenyl ring orients this
moiety to penetrate deeply into hydrophobic pocket of enzyme
by forming two hydrogen bonds with Asp1026 and Asn1031, with
distances of 2.10 Å and 2.22 Å, respectively, and (iv) the formation
of non-covalent
p–p stacking interaction between the aromatic
quinoxalinone core and Phe1045 further stabilizes the binding
interactions. Based on these docking information, the (Z)-3-(2-
(pyridin-4-yl)vinyl)quinoxalinone structure can be regarded as a
favorable motif for new inhibitor design owing to its contribution
to effective hinge-binding interaction.
2.4. Molecular docking study
To gain insight into the binding pattern of these compounds,
one of the active VEGFR-2 inhibitors, 8c, was chosen to dock into
the active site of VEGFR-2 tyrosine kinase (PDB ID: 3EWH) using
Autodock program. The resulting optimal binding mode of 8c, as
exhibited in Figure 6, unraveled several favorable interactions: (i)
the N atom of pyridyl motif, acting as a hydrogen bond acceptor,
interacts with the key Cys917 residue with distance of 1.87 Å;
3. Conclusion
In summary, we report the design, synthesis, and biological
evaluation of novel (Z)-3-(2-(pyridin-4-yl)vinyl)quinoxalinone
derivatives as a new class of VEGFR-2 kinase inhibitors. SAR
Please cite this article in press as: Shi, L.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.03.008

L. Shi et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7
Negative (0.1µM)
Pazopanib (0.1µM)
7c (0.1µM)
10 b (0.1µM)
8e (0.1µM)
8c (0.1µM)
8b (0.1µM)
Figure 4. Anti-angiogenesis assay results on arterial ring of rat.
investigations manifested that this novel skeletal structure was
tolerable to the introduction of favorable contributors for selective
VEGFR-2 inhibition, such as pyridyl and amide moieties, leading to
moderate to improved potencies. These derivatives did not exert
cytotoxic effect. However, compounds 7c, 8b, 8c, 8e and 10b
clearly exhibited anti-angiogenesis effects in in vitro anti-angio-
genetic assessment via both HUVEC tube formation assay and
ex vivo rat aortic ring assay. Among these four compounds, 8b,
8c, 8e and 10b were withstood the in vivo test in zebrafish
embryos model. Further kinase inhibition screening proved that
although the inhibitory potencies of all of tested inhibitors did
not surpass the reference standard, they exerted considerable
selectivity profiling against other angiogenesis-related kinases,
such as EGFR and FGFR1. Three test compounds, 8c, 8e and 10b,
presented clear inhibition percentage against VEGFR-2 in a low
(TLC) on 0.25-mm silica gel plates (grade 60, GF254) and visualized
with UV light (254 nm), or iodine vapor. Flash chromatography
was performed on silica gel (Qingdao Haiyang Inc., 300–400 mesh).
Melting points were determined using X-6 digital display binocular
microscope (uncorrected). If not specified, 1H NMR and 13C NMR
spectra were recorded on a Bruker Advance DRX-600 using CDCl3
or DMSO-d6 as solvents. If it has been indicated as 400 MHz or
300 MHz, AVANCEII-400 or AVANCEII-300 spectrometer were
employed, and TMS as an internal standard. Chemical shift values
(d) are reported in parts per million (ppm) and coupling constants
(J) are given in hertz (Hz), and splitting patterns are designated as
follows: s, single; d, doublet; t, triplet; q, quartet; m, multiplet.
Elemental analyses were carried out on a Perkin–Elmer C, H, N
elemental analyzer. Electrospray ionization mass spectrometry
(ESI-MS) data were collected on a Micromass Qtof-Micro LC–MS
instrument.
concentration (1 lM) and moderate inhibition toward B-Raf.
Molecular modeling studies suggested that the inhibitor adopt sev-
eral similar H-binding interactions as the designing prototype (axi-
4.1.2. General synthetic procedure for the preparation of 3-
methylquinoxalin-2(1H)-one (2)
tinib and vatalanib) do, the newly presented
p–p stacking
interaction also contributes to the binding conformation.24,25 Over-
all, we envisioned that 8c, 8e and 10b might be promising candi-
dates to be considered for further chemical optimization.
o-Phenylenediamine (0.02 mol, 2.16 g) was suspended in anhy-
drous ethanol (20 mL). The mixture was then cooled in an ice bath.
A solution of ethyl pyruvate (0.022 mol, 25.52 g) in anhydrous
ethanol (5 mL) was added dropwise over a period of 20 min under
stirring. The resulting solution was allowed to react at room tem-
perature for 6 h until the TLC showed the reaction has completed.
The resulting precipitate was filtered and washed with anhydrous
ethanol (2 Â 5 mL), and dried in vacuum to give the product 2a
(3.04 g, 95%) as a white solid which was pure enough for further
reaction. Mp: 241.0–242.7 °C; ESI-MS m/z: 161.6 [M+H]+. Anal.
Calcd for C9H8N2O: C, 67.49; H, 5.03; N, 17.49; Found: C, 67.50;
H, 5.05; N, 17.48.
4. Experimental section
4.1. Chemistry
4.1.1. General
All the starting materials including reagents and solvents,
unless otherwise specified, were analytically pure and obtained
from commercial suppliers. They were used without further purifi-
cation and, when necessary, were purified and dried by standard
protocols. All reactions except those in aqueous media were car-
ried out by standard techniques for the exclusion of moisture. All
reaction progress was monitored by thin-layer chromatography
For 6,7-dimethoxy-3-methylquinoxalin-2(1H)-one (2b): light
brown powder, yield: 43%, ESI-MS m/z: 221.8 [M+H]+. Mp:
261.0–262.0 °C. Anal. Calcd for C11H12N2O3: C, 59.99; H, 5.49; N,
12.72; Found: C, 60.01; H, 5.47; N, 12.74.
Please cite this article in press as: Shi, L.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.03.008

8
L. Shi et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
DLAV
ISV
PCV
DA
Vatalanib (10µmol/L)
Vehicle-treated control (0.2% DMSO)
ISV is totally
inhibited
ISV is significantly
inhibited
ISV is further inhibited
0.2% DMSO
8e (10µM)
8e (50µM)
8e (100µM)
ISV is inhibited
ISV is totally
inhibited
ISV is further inhibited
0.2% DMSO
0.2% DMSO
8c (10µM)
8c (50µM)
8c (100µM)
ISV is inhibited
ISV is inhibited
ISV is inhibited
10b (10µM)
10b (50µM)
10b (100µM)
ISV is weakly
inhibited
ISV is inhibited
ISV is inhibited
8b (10µM)
7c (50µM)
8b (50µM)
8b (100µM)
0.2% DMSO
0.2% DMSO
ISV malformation
is observed
7c (100µM), magnified view
7c (100µM), lateral view
Figure 5. Anti-angiogenic activities of target compounds using zebrafish model.
Table 2
Kinase inhibitory activities of (Z)-3-(2-(pyridin-4-yl)vinyl)quinoxalinone derivates
a
a
a
a
Comps (1
lM)
VEGFR-2 (% inhibition)
EGFR (% inhibition)
B-RAF (% inhibition)
FGFR1 (% inhibition)
7c
8b
8c
8e
10b
19
6
1
0
2
7
0
70
—
8
4
25
20
17
—
0
2
5
8
0
—
—
44
56
37
82
90
Staurosporine
Sorafenib
58
a
Results are given as average inhibitory values.
Please cite this article in press as: Shi, L.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.03.008

L. Shi et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
9
resulting solution was allowed to react at room temperature for
3 h. The organic solvent was removed in vacuo and the resulting
water phase was washed with EtOAc (2 Â 10 mL), the combined
water was acidified by 1 N HCl until white solid was precipitated.
Filtered and recrystallized with 70% methanol to give the pure pro-
duct 5a (4.0 g, 92%) as a off-white solid. Mp: 225–226 °C, ESI-MS
m/z: 217.5 [MÀH]À. Anal. Calcd for C11H10N2O3: C, 60.55; H,
4.62; N, 12.84; Found: C, 60.57; H, 4.63; N, 12.86.
For
2-(6,7-dimethoxy-3-methyl-2-oxoquinoxalin-1(2H)-yl)
acetic acid 5b: 87%, white solid, mp 233–234 °C, ESI-MS m/z:
277.6 [MÀH]À. Anal. Calcd for C13H14N2O5: C, 56.11; H, 5.07; N,
10.07; Found: C, 56.12; H, 5.06; N, 10.08.
4.1.6. 2-((3-Methylquinoxalin-2-yl)oxy)acetic acid (6a)
This compound was prepared from 4a using the same experi-
mental procedure as for 5. It afforded 1.89 g (87%) of 6a as light
yellow solid, ESI-MS m/z: 217.3 [MÀH]À. Anal. Calcd for
C11H10N2O3: C, 60.55; H, 4.62; N, 12.84; Found: C, 60.56; H, 4.64;
N, 12.86.
4.1.7. General procedure for 2-(3-methyl-2-oxoquinoxalin-1
(2H)-yl)-N-substituted phenylacetamide (7a–g)
A round-bottom flask charged with compound 5 (2 mmol) and
15 mL of anhydrous THF was successively added isobutyl chloro-
formate (2.4 mmol, 1.2 equiv), 4-methylmorpholine (2.4 mmol,
1.2 equiv) and stirred in an ice-salt baths for 30 min. Then the
ice-salt bath was removed and appropriate aniline (2.4 mmol,
1.2 equiv) was added, the mixture was then allowed to react at
room temperature under nitrogen atmosphere for about 4 h until
the TLC showed the reaction has been completed. The reaction
mixture was concentrated in vacuo and the resulting residue was
extracted with ethyl acetate (3 Â 50 mL), washed with water and
dried over anhydrous MgSO4. After filtration and concentration in
vacuo, the obtained residue was purified by flash chromatography
(PE/EtOAc = 4:1, v:v) to afford the desired products.
Figure 6. (a) Molecular docking result of compound 8c with VEGFR-2 (PDB entry:
3EWH). (b) The observed interactions between 8c and target enzyme shown by
LIGPLOT.
4.1.3. General procedure for methyl 2-(3-methyl-2-oxoquinoxalin-
1(2H)-yl)acetate (3)
Compound 2a (10 mmol, 1.6 g), K2CO3 (12 mmol, 1.66 g) and KI
(1 mmol, 0.17 g) were suspended in anhydrous acetone (15 mL) at
0 °C. Then a solution of methyl bromoacetate (12 mmol, 1.84 g) in
anhydrous acetone (10 mL) was added dropwise into the above
mixture. The resulting mixture was then stirred on oil bath at
62 °C for 8 h under nitrogen atmosphere. After completion of reac-
tion, the solvent was removed under vacuum and the residue was
partitioned between EtOAc (30 mL) and distilled water (10 mL).
The organic layer was separated and washed with brine
(2 Â 15 mL), dried over anhydrous MgSO4, filtered, and concen-
trated in vacuo to afford the crude product, which was further
purified by silica gel flash chromatography (PE:EA = 8:1, v:v) to
furnish 1.28 g of 3a as a white solid, yield 52%. Mp: 120.0–
121.1 °C, ESI-MS m/z: 247.1 [M+H]+; 1H NMR: (CDCl3, ppm) d:
7.05–7.86 (m, 4H, ArH), 5.05 (s, 2H, NCH2C@O), 3.18 (s, 3H,
OCH3), 2.62 (s, 3H, N@CCH3). Anal. Calcd for C12H12N2O3: C,
62.06; H, 5.21; N, 12.06; Found: C, 62.07; H, 5.22; N, 12.04.
For methyl 2-(6,7-dimethoxy-3-methyl-2-oxoquinoxalin-1
(2H)-yl)acetate (3b): 47%, light yellow solid, mp 144–145 °C, ESI-
MS m/z: 291.6 [MÀH]À. Anal. Calcd for C14H16N2O5: C, 57.53; H,
5.52; N, 9.58; Found: C, 57.52; H, 5.55; N, 9.57.
4.1.7.1.
etamide (7a).
2-(3-Methyl-2-oxoquinoxalin-1(2H)-yl)-N-phenylac-
1.22 g as white solid, yield: 81%; mp = 294–
295 °C; 1H NMR (CDCl3): d = 10.47 (s, 1H, ArNHC@O), 7.08–7.10
(m, 2H, ArH), 7.19–7.68 (m, 6H, ArH), 7.87–7.89 (d, J = 12.0 Hz,
1H, ArH), 5.02 (s, 2H, NCH2C@O), 2.67 (s, 3H, N@CCH3). ESI-MS
m/z: 294.4 [M+H]+. Anal. Calcd for C17H15N3O2: C, 69.61; H, 5.15;
N, 14.33; Found: C, 69.62; H, 5.14; N, 14.34.
4.1.7.2.
(2H)-yl)acetamide (7b).
N-(4-Fluorophenyl)-2-(3-methyl-2-oxoquinoxalin-1
0.29 g as white solid, yield: 48%,
mp = 280–281 °C; 1H NMR (DMSO-d6): d = 10.50 (s, 1H, ArNHC@O),
7.80–7.813 (d, J = 7.8 Hz, 1H, ArH), 7.54–7.61 (m, 4H, ArH), 7.36–
7.39 (m, 1H, ArH), 7.13–7.19 (m, 2H), 5.11 (s, 2H, NCH2C@O),
2.47 (s, 3H, N@CCH3). ESI-MS m/z: 312.4 [M+H]+; 334.4 [M+Na]+.
Anal. Calcd for C17H14FN3O2: C, 65.59; H, 4.53; N, 13.50; Found:
C, 65.60; H, 4.54; N, 13.48.
4.1.7.3.
(2H)-yl)acetamide (7c).
N-(4-Chlorophenyl)-2-(3-methyl-2-oxoquinoxalin-1
0.34 g as white solid, yield: 52%;
mp = 267–268.5 °C; 1H NMR (DMSO-d6): d = 10.56 (s, 1H,
ArNHC@O), 7.81–7.8235 (d, J = 8.1 Hz, 1H, ArH), 7.61–7.62 (m,
2H, ArH), 7.48–7.59 (m, 2H, ArH), 7.34–7.40 (m, 3H, ArH), 5.12 (s,
2H, NCH2C@O), 2.41 (s, 3H, N@CCH3). ESI-MS m/z: 328.4 [M+H]+;
350.4 [M+Na]+; 366.7 [M+K]+. Anal. Calcd for C17H14ClN3O2: C,
62.30; H, 4.31; N, 12.82; Found: C, 62.31; H, 4.32; N, 12.80.
4.1.4. Methyl 2-((3-methylquinoxalin-2-yl)oxy)acetate (4a)
This compound was prepared from 2a using the same experi-
mental procedure as for 3. Owing to the keto–enol tautomeriza-
tion, the O-substituted ester compounds 4a was isolated with
relatively low yield (28 %) as a light yellow solid. ESI-MS m/z:
233.6 [M+H]+. Anal. Calcd for C12H12N2O3: C, 62.06; H, 5.21; N,
12.06; Found: C, 62.07; H, 5.22; N, 12.07.
4.1.7.4.
etamide (7d).
2-(3-Methyl-2-oxoquinoxalin-1(2H)-yl)-N-(o-tolyl)ac-
4.1.5. 2-(3-Methyl-2-oxoquinoxalin-1(2H)-yl)acetic acid (5)
Compound 3a (20 mmol, 4.64 g) and KOH (2.24 g, 40 mmol)
were suspended in 40 mL of dioxane and water (3:1, v:v), the
2.22 g as white solid, yield: 77%; mp = 254–
255 °C; 1H NMR (CDCl3): d = 10.52 (s, 1H, ArNHC@O), 7.91–7.92
(d, J = 6.0 Hz, 1H, ArH), 7.22–7.39 (m, 3H, ArH), 7.02–7.18 (m,
Please cite this article in press as: Shi, L.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.03.008

10
L. Shi et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
4H), 5.04 (s, 2H, NCH2C@O), 2.46 (s, 3H, N@CCH3), 1.78 (s, 3H,
ArCH3). ESI-MS m/z: 308.5 [M+H]+. Anal. Calcd for C18H17N3O2: C,
70.34; H, 5.58; N, 13.67; Found: C, 70.33; H, 5.59; N, 13.66.
0.18 g, yield 51%. Mp = 183–184 °C, 1H NMR (300 MHz, DMSO-
d6): d = 10.54 (s, 1H, ArNHC@O), 8.52–8.542 (d, J = 6.6 Hz, 2H,
ArH), 7.81–7.843 (m, J = 9.9 Hz, 1H, ArH), 7.53–7.63 (m, 4H, ArH),
7.36–7.42 (m, 3H, ArH), 7.14–7.19 (m, 2H, ArH), 5.65–5.666 (d,
J = 4.8 Hz, 1H, CH@CHAr), 5.31–5.326 (d, J = 4.8 Hz, 1H,
N@CACH@CH), 5.14 (s, 2H, NCH2C@O) ppm; 13C NMR (75 MHz,
DMSO-d6): d = 165.2, 159.8, 157.6, 154.7, 154.6, 149.9, 135.5,
133.4, 132.4, 130.5, 129.6, 124.0, 121.5, 121.4, 116.0, 115.8,
115.2, 45.7 ppm. ESI-MS m/z: 418.4 [M+NH4]+. Anal. Calcd for C23
H17FN4O2: C, 68.99; H, 4.28; N, 13.99; Found: C, 69.00; H, 4.27;
N, 14.01.
4.1.7.5. N-(3,4-Dichlorophenyl)-2-(3-methyl-2-oxoquinoxalin-1
(2H)-yl)acetamide (7e).
0.46 g as white solid, yield: 65%,
mp = 274–274.5 °C, 1H NMR (DMSO-d6): d = 10.74 (s, 1H,
ArNHC@O), 7.93–7.933 (d, J = 1.8 Hz, 1H, ArH), 7.79 (dd,
J1 = 8.4 Hz, J2 = 1.8 Hz, 1H, ArH), 7.59 (d, J = 8.4 Hz, 1H, ArH), 7.58
(dd, J1 = 8.4 Hz, J2 = 1.8 Hz, 1H, ArH), 7.50 (m, 2H, ArH), 7.38 (d,
J = 8.4 Hz, 1H, ArH), 5.12 (s, 2H, NCH2C@O), 2.47 (s, 3H, N@CCH3).
ESI-MS m/z: 362.3 [M+H]+. Anal. Calcd for C17H13Cl2N3O2: C,
56.37; H, 3.62; N, 11.60; Found: C, 56.38; H, 3.64; N, 11.59.
4.1.8.3.
(Z)-N-(4-Chlorophenyl)-2-(2-oxo-3-(2-(pyridin-4-yl)
White powder
vinyl)quinoxalin-1(2H)-yl)acetamide (8c).
4.1.7.6.
N-(3,4-Dimethoxyphenyl)-2-(3-methyl-2-oxoquinox-
0.39 g as white solid, yield:
0.18 g (68%), mp = 205–206 °C, 1H NMR (300 MHz, DMSO-d6):
d = 10.64 (s, 1H, ArNHC@O), 8.52–8.5387 (d, J = 5.6 Hz, 2H, ArH),
7.78–7.81 (d, J = 9.0 Hz, 1H, ArH), 7.54–7.63 (m, 4H, ArH), 7.37–
7.42 (m, 5H, ArH), 5.64–5.653 (d, J = 5.1 Hz, 1H, CH@CHAr), 5.30–
5.313 (d, J = 5.1 Hz, 1H, N@CACH = CH), 5.15 (s, 2H, NCH2C@O)
ppm; 13C NMR (75 MHz, DMSO-d6): d = 165.8, 154.7, 151.4,
150.8, 143.4, 139.2, 135.4, 133.1, 131.6, 131.3, 130.1, 126.9,
125.6, 124.6, 122.2, 120.9, 119.7, 115.5, 46.3 ppm, ESI-MS m/z:
436.7 [M+NH4]+. Anal. Calcd for C23H17ClN4O2: C, 66.27; H, 4.11;
N, 13.44; Found: C, 66.28; H, 4.13; N, 13.42.
alin-1(2H)-yl)acetamide (7f).
56%, mp = 230–231 °C, 1H NMR (CDCl3): d = 10.49 (s, 1H,
ArNHC@O), 7.87–7.88 (d, J = 6.0 Hz, 1H, ArH), 7.21 (s, 1H, ArH),
7.07–7.17 (m, 3H, ArH), 7.04–7.043 (d, 1H, J = 1.8 Hz, ArH), 6.82–
6.833 (d, 1H, J = 7.8 Hz, ArH), 4.99 (s, 2H, NCH2C@O), 3.67 (s, 6H,
2 Â OCH3), 2.44 (s, 3H, N@CCH3), ESI-MS m/z: 354.8 [M+H]+. Anal.
Calcd for C19H19N3O4: C, 64.58; H, 5.42; N, 11.89; Found: C, 64.59;
H, 5.43; N, 11.88.
4.1.7.7. 2-(6,7-Dimethoxy-3-methyl-2-oxoquinoxalin-1(2H)-yl)-
N-phenylacetamide (7g).
0.49 g as light yellow floccule,
4.1.8.4. (Z)-2-(2-Oxo-3-(2-(pyridin-4-yl)vinyl)quinoxalin-1(2H)-
yield: 70%, mp = 257–258 °C, 1H NMR (DMSO-d6): d = 10.42 (s,
1H, ArNHC@O), 7.57–7.583 (t, J = 7.8 Hz, 2H, ArH), 7.33–7.41 (m,
2H, ArH), 7.22–7.23 (s, 1H, ArH), 7.08–7.12 (dd, J1 = J2 = 12.0 Hz,
1H, ArH), 6.99 (s, 1H, ArH), 5.15 (s, 2H, NCH2C@O), 3.77 (s, 6H,
2 Â OCH3), 2.47 (s, 3H, N@CCH3). ESI-MS m/z: 354.5 [M+H]+;
376.8 [M+Na]+. Anal. Calcd for C19H19N3O4: C, 64.58; H, 5.42; N,
11.89; Found: C, 64.58; H, 5.44; N, 11.87.
yl)-N-(o-tolyl)acetamide (8d).
White solid 1.48 g (77%),
mp = 192–193 °C, 1H NMR (400 MHz, DMSO-d6): d = 9.85 (s, 1H,
ArNHC@O), 8.51–8.5247 (d, J = 5.88 Hz, 2H, ArH), 7.81–7.83 (d,
J = 8.0 Hz, 1H, ArH), 7.59–7.63 (m, 2H, ArH), 7.36–7.51 (m, 4H,
ArH), 7.07–7.23 (m, 3H, ArH), 5.63–5.643 (d, J = 5.1 Hz, 1H,
CH = CHAr), 5.31–5.323 (d, J = 5.1 Hz, 1H, N@CACHN@CH), 5.20
(s, 2H, NCH2C@O), 2.24 (s, 3H, ArCH3) ppm; 13C NMR (100 MHz,
DMSO-d6): d = 165.4, 157.8, 154.8, 154.6, 149.9, 136.2, 133.3,
132.5, 132.3, 130.9, 130.4, 129.6, 126.5, 126.0, 125.3, 124.0,
121.5, 115.1, 69.4, 45.5, 18.3 ppm. ESI-MS m/z: 414.6 [M+NH4]+.
Anal. Calcd for C24H20N4O2: C, 72.71; H, 5.08; N, 14.13; Found: C,
72.72; H, 5.07; N, 14.14.
4.1.8. General procedure for substituted (Z)-2-(2-oxo-3-(2-
(pyridin-4-yl)vinyl)quinoxalin-1(2H)-yl)-N-substituted
phenylacetamide (8a–g)
To a solution of compound 7a–g (0.6 mmol) in acetic acid
(15 mL) was added 4-pyridinecarboxaldehyde (0.72 mmol,
1.2 equiv) dropwise, and catalytic amount of concentrated sulfuric
acid. The resulting solution was heated to 50 °C and reacted for 2 h
until the completion of reaction via TLC. The reaction mixture was
concentrated in vacuo and then water (15 mL) and ethyl acetate
(30 mL) was added, the organic layers were separated and the
water phase was basified by K2CO3 to pH to 7. The resulting water
phase was extracted with ethyl acetate (50 mL), the combined
organic phase was washed with water and dried over anhydrous
MgSO4. The obtained residue was purified by flash chromatogra-
phy (PE/EtOAc = 3:1–1:3, v:v) to afford the desired products.
4.1.8.5. (Z)-N-(3,4-Dichlorophenyl)-2-(2-oxo-3-(2-(pyridin-4-yl)
vinyl)quinoxalin-1(2H)-yl)acetamide (8e).
Yellow powder
0.48 g (56%); mp = 282–283 °C; 1H NMR (400 MHz, DMSO-d6):
d = 10.61 (s, 1H, ArNHC@O), 8.50–8.522 (d, J = 8.8 Hz, 2H, ArH),
7.82–7.8395 (d, J = 7.8 Hz, 1H, ArH), 7.51–7.61 (m, 4H, ArH),
7.36–7.41 (m, 5H, ArH), 5.61–5.6227 (d, J = 5.08 Hz, 1H,
CHN@CHAr), 5.31–5.3227 (J = 5.08 Hz, 1H, N@CACH = CH), 5.14
(s, 2H, NCH2C@O) ppm; 13C NMR (100 MHz, DMSO-d6): d = 165.4,
157.6, 154.7, 154.6, 149.9, 138.1, 133.4, 132.4, 130.5, 129.6,
129.3, 127.6, 124.0, 121.5, 121.2.7, 115.2, 45.7 ppm. ESI-MS m/z:
451.5 [M+H]+. Anal. Calcd for C23H16Cl2N4O2: C, 61.21; H, 3.57; N,
12.41; Found: C, 61.22; H, 3.58; N, 12.43.
4.1.8.1. (Z)-2-(2-Oxo-3-(2-(pyridin-4-yl)vinyl)quinoxalin-1(2H)-
yl)-N-phenylacetamide (8a).
0.89 g as white powder, yield:
71%; mp = 182–184 °C; 1H NMR (400 MHz, DMSO-d6): d = 10.48
(s, 1H, ArNHC@O), 8.51–8.53 (m, 2H, ArH), 7.82–7.84 (d,
J = 8.0 Hz, 1H, ArH), 7.57–7.60 (m, 3H, ArH), 7.52–7.5396 (d,
J = 7.84 Hz, 1H, ArH), 7.40–7.4022 (d, J = 0.88 Hz, 2H, ArH), 7.31–
7.36 (m, 3H, ArH), 7.07 (m, 1H, ArH), 5.65–5.6628 (d, J = 5.12 Hz,
1H, CH@CHAr), 5.31–5.323 (d, J = 5.1 Hz, 1H, N@CACH@CH), 5.16
(s, 2H, NCH2C@O) ppm; 13C NMR (100 MHz, DMSO-d6): d = 165.2,
157.7, 154.7, 154.6, 149.9, 139.1, 133.4, 132.4, 130.5, 129.6,
129.3, 124.1, 124.0, 121.5, 119.6, 115.2, 69.4, 45.7 ppm. ESI-MS
m/z: 400.4 [M+NH4]+. Anal. Calcd for C23H18N4O2: C, 72.24; H,
4.74; N, 14.65; Found: C, 72.22; H, 4.75; N, 14.66.
4.1.8.6. (Z)-N-(3,4-Dimethoxyphenyl)-2-(2-oxo-3-(2-(pyridin-4-
yl)vinyl)quinoxalin-1(2H)-yl)acetamide (8f).
Light yellow
powder 0.62 g (70%); mp = 277–278 °C; 1H NMR (400 MHz,
DMSO-d6): d = 10.89 (s, 1H, ArNHC@O), 8.63–8.64 (d, J = 4.0 Hz,
2H, ArH), 8.03–8.07 (d, J = 16.0 Hz, 1H, ArH), 7.85–7.92 (m, 2H,
ArH), 7.71–7.73 (m, 2H, ArH), 7.56–7.65 (m, 2H, ArH), 7.41–7.45
(m, 1H, ArH), 7.37–7.3755 (d, J = 2.2 Hz, 1H, ArH), 7.06–7.09 (m,
1H, ArH), 6.89–6.912 (d, J = 8.76 Hz, 1H, ArH), 5.61–5.623 (d,
J = 5.08 Hz, 1H, CHN@CHAr), 5.31–5.323 (J = 5.08 Hz, 1H,
N@CACH = CH), 5.22 (s, 2H, NCH2C@O), 3.71 (s, 3H, OCH3), 3.69
(s, 3H, OCH3) ppm; 13C NMR (100 MHz, DMSO-d6): d = 164.7,
154.7, 151.4, 150.8, 149.0, 145.4, 143.4, 135.2, 133.5, 133.0,
132.9, 131.4, 130.1, 127.1, 124.4, 122.2, 115.4, 112.5, 111.5,
104.7, 56.2, 55.8, 45.8 ppm. ESI-MS m/z: 443.6 [M+H]+. Anal. Calcd
4.1.8.2.
(Z)-N-(4-Fluorophenyl)-2-(2-oxo-3-(2-(pyridin-4-yl)
White powder
vinyl)quinoxalin-1(2H)-yl)acetamide (8b).
Please cite this article in press as: Shi, L.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.03.008

L. Shi et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
11
for C25H22N4O4: C, 67.86; H, 5.01; N, 12.66; Found: C, 67.88; H,
5.02; N, 12.67.
J = 15 Hz, 1H, NCH2C@O), 2.25 (s, 3H, ArCH3) ppm; 13C NMR
(75 MHz, DMSO-d6): d = 166.1, 155.5, 154.5, 149.9, 148.5, 139.2,
138.8, 136.4, 133.0, 130.1, 129.7, 128.6, 127.5, 127.0, 121.5,
120.0, 70.0, 65.0, 20.9 ppm. ESI-MS m/z: 414.6 [M+NH4]+. Anal.
Calcd for C24H20N4O2: C, 72.71; H, 5.08; N, 14.13; Found: C,
72.73; H, 5.09; N, 14.12.
4.1.8.7.
(Z)-2-(6,7-Dimethoxy-2-oxo-3-(2-(pyridin-4-yl)vinyl)
Light red
quinoxalin-1(2H)-yl)-N-phenylacetamide (8g).
powder 0.23 g (76%); mp = 215–216 °C, 1H NMR (300 MHz,
DMSO-d6): d = 10.47 (s, 1H, ArNHC@O), 8.73–8.751 (d, J = 6.3 Hz,
2H, ArH), 7.96–8.01 (m, 4H, ArH), 7.60–7.626 (d, J = 7.8 Hz, 2H,
ArH), 7.30–7.36 (m, 3H, ArH), 7.09–7.102 (d, J = 3.6 Hz, 2H, ArH),
5.60–5.617 (d, J = 5.08 Hz, 1H, CHN@CHAr), 5.31–5.327
(J = 5.08 Hz, 1H, N@CACHN@CH), 5.21 (s, 2H, NCH2C@O), 3.90 (s,
3H, OCH3), 3.89 (s, 3H, OCH3) ppm; 13C NMR (75 MHz, DMSO-
d6): d = 165.2, 157.7, 145.6, 151.1, 149.9, 139.1, 133.4, 132.4,
130.5, 129.6, 128.0, 124.1, 123.0, 121.6, 119.6, 109.7, 69.4, 56.2,
55.1 ppm. ESI-MS m/z: 443.8 [M+H]+. Anal. Calcd for C25H22N4O4:
C, 67.86; H, 5.01; N, 12.66; Found: C, 67.85; H, 5.03; N, 12.68.
4.1.10.2.
(Z)-N-(4-Chlorophenyl)-2-((3-(2-(pyridin-4-yl)vinyl)
White crystal 0.13 g
quinoxalin-2-yl)oxy)acetamide (10b).
(67%); mp = 218–219 °C; 1H NMR (300 MHz, DMSO-d6): d = 10.36
(s, 1H, ArNHC@O), 8.50–8.52 (m, 3H, ArH), 7.75 (m, 5H, ArH),
7.44 (m, 4H, ArH), 5.75–5.768 (d, J = 5.4 Hz, 1H, CHN@CHAr),
5.38–5.398 (d, J = 5.4 Hz, 1H, N@CACHN@CH), 5.16–5.209 (d,
J = 14.7 Hz, 1H, NCH2C@O), 2.26 (s, 3H, ArCH3) ppm; 13C NMR
(75 MHz, DMSO-d6): d = 166.1, 154.9, 154.0, 149.4, 147.9, 138.7,
138.3, 137.4, 129.6, 128.7, 128.1, 127.2, 127.0, 126.5, 121.0,
120.9, 69.4, 64.5 ppm; ESI-MS m/z: 434.7 [M+H]+. Anal. Calcd for
4.1.9. General procedure for N-(4-substitutedphenyl)-2-((3-
methylquinoxalin-2-yl)oxy)acetamide (9a–b)
C23H17ClN4O2: C, 66.27; H, 4.11; N, 13.44; Found: C, 66.28; H,
4.09; N, 13.45.
In a round-bottom flask, compound 6a (2 mmol) was dissolved
in 20 mL of anhydrous dichloromethane at 0 °C. Then isobutyl
chloroformate (2.4 mmol, 1.2 equiv) and 4-methylmorpholine
(2.4 mmol, 1.2 equiv) were successively added and the resulting
solution was stirred in an ice-salt baths for 30 min. The ice-salt
bath was then removed and appropriate aniline (2.4 mmol,
1.2 equiv) was added, the mixture was then allowed to react at
room temperature under nitrogen atmosphere for about 3–4 h
until the completion of reaction. The reaction mixture was diluted
with distilled water and extracted with CH2Cl2 (3 Â 20 mL), the
combined organic phase was successively washed with 5% KHSO4,
saturated Na2CO3, water, and brine, then dried over anhydrous
MgSO4. After filtration and concentration in vacuo, the obtained
residue was purified by flash chromatography (PE/EtOAc = 4:1, v:
v) to afford the desired products.
4.1.11. Methyl 4-(bromomethyl)benzoate (11)
In a round-bottom flask, 4-(bromomethyl)benzoic acid (3.4 g,
20 mmol) was dissolved in 50 mL of anhydrous methanol at 0 °C.
After being stirred for 10 min, 40 mL of acetyl chloride was added
dropwise to the above mixture and continued to react for 15 min.
Then the ice-bath was removed and the reaction mixture was
heated to 70 °C and reacted for additional 6 h. The mixture was
concentrated in vacuo and afforded the desired product as oil
which was used directly without any further purification.
4.1.12. Methyl 4-((3-methyl-2-oxoquinoxalin-1(2H)-yl)methyl)
benzoate (12)
A round-bottom flask charged with 3-methylquinoxalin-2(1H)-
one (compound 2a, 1.6 g, 10 mmol), K2CO3 (1.7 g, 12 mmol), and KI
(170 mg, 1 mmol), 30 mL of acetone was then added. The resulting
suspension was added compound 11 (1.84 g, 12 mmol) dropwise
and then flushed with nitrogen gas. After heating and reacting at
62 °C for 8 h under nitrogen atmosphere, the solvent was removed
under reduced pressure. The residue was extracted with ethyl acet-
ate (3 Â 20 mL), washed with water and brine, and dried over anhy-
drous MgSO4. Filtration and concentration in vacuo afforded the
crude product which was purified by chromatography column (PE:
EtOAc = 8:1, v:v) to afford 2.4 g (71%) of pure product as white crys-
tal. ESI-MS m/z: 309.5 [M+H]+, 331.4 [M+Na]+. Anal. Calcd for
4.1.9.1. 2-((3-Methylquinoxalin-2-yl)oxy)-N-(p-tolyl)acetamide
(9a).
Off-white powder 0.39 g, yield: 64%; 1H NMR
(300 MHz, DMSO-d6): d = 10.32 (s, 1H, ArNHC@O), 7.94–7.96 (m,
2H, ArH), 7.74–7.76 (m, 2H, ArH), 7.48–7.54 (m, 2H, ArH), 7.28–
7.31 (m, 2H, ArH), 5.15 (s, 2H, NCH2C@O), 2.67 (s, 3H, ArCH3),
2.47 (s, 3H, N@CCH3) ppm; ESI-MS m/z: 308.5 [M+H]+. Anal. Calcd
for C18H17N3O2: C, 70.34; H, 5.58; N, 13.67; Found: C, 70.33; H,
5.56; N, 13.68.
4.1.9.2. N-(4-Chlorophenyl)-2-((3-methylquinoxalin-2-yl)oxy)
acetamide (9b).
C18H16N2O3: C, 70.12; H, 5.23; N, 9.09; Found: C, 70.13; H, 5.22; N,
9.08.
White powder 0.33 g, yield: 51%; 1H NMR
(300 MHz, DMSO-d6): d = 10.32 (s, 1H, ArNHC@O), 7.93–7.95 (m,
2H, ArH), 7.72–7.75 (m, 2H, ArH), 7.38–7.40 (m, 2H, ArH), 7.30–
7.33 (m, 2H, ArH), 5.15 (s, 2H, NCH2C@O), 2.45 (s, 3H, N@CCH3)
ppm; ESI-MS m/z: 328.8 [M+H]+. Anal. Calcd for C17H14ClN3O2: C,
62.30; H, 4.31; N, 12.82; Found: C, 62.31; H, 4.33; N, 12.81.
4.1.13. 4-((3-Methyl-2-oxoquinoxalin-1(2H)-yl)methyl)benzoic
acid (13)
Compound 12 (1.54 g, 5 mmol) and KOH (0.34 g, 6 mmol) was
suspended in 50% ethanol solution (40 mL), the resulting solution
was reacted at room temperature for 3 h. The organic solvent
was removed in vacuo and the resulting water phase was washed
with EtOAc (10 mL), the combined water was acidified by 1 N HCl
until a large amount of white solid was precipitated. Filtered and
recrystallized with 70% methanol to give the pure product 1.38 g
(94%) as white solid. Mp: 211–212 °C, ESI-MS m/z: 293.6 [MÀH]À.
Anal. Calcd for C17H14N2O3: C, 69.38; H, 4.79; N, 9.52; Found: C,
69.37; H, 4.77; N, 9.54.
4.1.10. General procedure for (Z)-N-(4-substitutedphenyl)-2-((3-
(2-(pyridin-4-yl)vinyl)quinoxalin-2-yl)oxy)acetamide (10a–b)
The synthetic procedure of compounds 10a–b was similar with
that of compounds (8a–g).
4.1.10.1. (Z)-2-((3-(2-(Pyridin-4-yl)vinyl)quinoxalin-2-yl)oxy)-
N-(p-tolyl)acetamide (10a).
Light yellow crystal 0.22 g
(52%); mp = 179–180 °C; 1H NMR (300 MHz, DMSO-d6): d = 10.11
(s, 1H, ArNHC@O), 8.49–8.51 (m, 2H, ArH), 7.80–7.93 (dd,
J1 = 7.8 Hz, J2 = 0.9 Hz, 1H, ArH), 7.75–7.78 (m, 1H, ArH), 7.69–
7.71 (m, 1H, ArH), 7.60–7.62 (m, 1H, ArH), 7.48–7.508 (d,
J = 8.4 Hz, 2H, ArH), 7.43 (d, J = 6.0 Hz, 2H, ArH), 7.12–7.148 (d,
J = 8.4 Hz, 2H, ArH), 5.74–5.758 (d, J = 5.4 Hz, 1H, CHN@CHAr),
5.25–5.27 (d, J = 5.4 Hz, 1H, N@CACHN@CH), 5.19–5.24 (d,
4.1.14. General synthetic protocol for 4-substituted-N-(4-((3-
methyl-2-oxoquinoxalin-1(2H)-yl)methyl)phenyl)benzamide
(15)
To a solution of compound 13 (0.3 g, 1.1 mmol) in 20 mL of
dioxane was added successively DPPA (330 mg, 1.2 mmol) and
triethylamine (120 mg, 1.2 mmol). The resulting solution was
Please cite this article in press as: Shi, L.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.03.008

12
L. Shi et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
heated to 110 °C and refluxed at the same temperature for 6 h.
Then the reaction was cooled to room temperature and added
appropriate substituted phenylamines (1.2 mmol, 1.1 equiv). The
mixture was then stirred at rt for additional 12 h. After comple-
tion of the reaction, it was quenched with distilled water
(20 mL). The obtained suspension was diluted with EtOAc
(30 mL) and washed with water and brine, and dried over anhy-
drous Na2SO4. The residue was purified by flash chromatography
(PE:EtOAc = 5:1, v:v) to afford the desired amide products. One
thing to note here is that if the solvent dioxane was changed into
other solvents with higher boiling point (e.g., toluene), the urea
products rather than amide compounds will be generated, which
is on-going in our lab.
4.2.2. Tubular structure formation assay in HUVEC
Before the HUVEC tubular structure formation assay, Matrigel
(BD Bioscience, Bedford, MA) was polymerized in 24-well plates
at 37 °C for 1 h. Then HUVEC was trypsinized and seeded onto
Matrigel at a concentration of 105 cells per well and then incubated
with various concentration of Pazopanib or test compounds (0.1, 1,
and 10 lg/mL) for 8 h at 37 °C in a CO2 incubator until the tube
network formed. Cellular morphology was then taken under a
microscope (OLYMPUS IX51) in five random fields per well at a
four magnification. Experiment was repeated 3 times.
4.2.3. Ex vivo antiangiogenic assay in rat aortic ring
The aortas were harvested from 6-week-old Sprague–Dawley
rats. Each aorta was cut into 1-mm slices and embedded in 70
Matrigel in 96-well plates. The aortic rings were then fed with
100 L of RPMI-1640 culture medium without FBS (with 0.1%
DMSO in final concentration) with or without different concentra-
tions of Pazopanib or test compounds (0.1 g/mL), and pho-
tographed on the 6th day. The quantity of microvessels is valued
by relative area covered with microvessels using Image-Pro
Express.
lL
4.1.14.1. 4-Methyl-N-(4-((3-methyl-2-oxoquinoxalin-1(2H)-yl)
methyl)phenyl)benzamide (15a).
White solid (0.31 g, 71%);
l
mp = 242–243 °C; 1H NMR (400 MHz, DMSO-d6): d = 10.11 (s, 1H,
ArNHC@O), 7.85–7.87 (m, 2H, ArH), 7.78–7.80 (d, J = 8.0 Hz, 1H,
ArH), 7.61–7.631 (d, J = 8.4 Hz, 2H, ArH), 7.32–7.48 (m, 5H, ArH),
7.12–7.14 (m, 2H, ArH), 5.58 (s, 2H, NCH2C@O), 2.53 (s, 3H, Ar
CH3), 2.26 (s, 3H, N@CCH3) ppm; 13C NMR (100 MHz, DMSO-d6):
d = 165.5, 158.5, 155.1, 140.0, 137.1, 134.7, 133.1, 132.8, 130.0,
129.4, 128.5, 127.2, 124.0, 120.8, 115.4, 45.2, 21.8, 20.9 ppm; ESI-
MS m/z: 384.7 [M+H]+. Anal. Calcd for C24H21N3O2: C, 75.18; H,
5.52; N, 10.96; Found: C, 75.17; H, 5.54; N, 10.94.
l
4.2.4. In vivo antiangiogenic assay in zebrafish model
Two zebrafish types were used as the in vivo model, they are
the transgenic zebrafish Tg(flk1:GFP) which express strong green
fluorescent protein (GFP) in their endothelial cell and also the
wild-type zebrafish. Both of them were kindly donated by Prof
Jianfeng Zhou from Ocean University of China. Both stocks were
maintained as described in the zebrafish handbook and all animal
experiments were performed in accordance with the ethical guide-
lines of the Shandong University. In brief, stocks were maintained
in a controlled environment at a temperature of 28 °C on 1
14 h:10 h light:dark cycle. The fish were fed twice daily with arte-
mia nauplii in the morning and afternoon, and occasionally with
general tropical fish food. The embryos were collected in the morn-
ing and cultured at 28 °C in embryo medium. One day after
fertilization, the embryos were dechorionated with tweezers in a
12-well plate coated with 1% agarose and distributed into per well
with 3 mL of assay solution. The embryos receiving a solution of
DMSO (0.2%) was utilized as the vehicle-treated control and the
concentrations of test compounds were prepared as 10, 50, and
4.1.14.2. 4-Chloro-N-(4-((3-methyl-2-oxoquinoxalin-1(2H)-yl)
methyl)phenyl)benzamide (15b).
White solid (0.28 g, 63%);
mp = 254–255 °C; 1H NMR (400 MHz, DMSO-d6): d = 10.32 (s, 1H,
ArNHC@O), 7.77–7.80 (m, 2H, ArH), 7.76–7.78 (m, 3H, ArH),
7.46–7.50 (m, 1H, ArH), 7.31–7.42 (m, 6H, ArH), 5.56 (s, 2H, NCH2
C@O), 2.53 (s, 3H, N@CCH3) ppm; 13C NMR (100 MHz, DMSO-d6):
d = 165.8, 158.5, 155.1, 140.2, 138.6, 134.4, 132.8, 132.7, 130.0,
129.4, 129.0, 128.6, 127.7, 127.3, 124.0, 122.2, 115.4, 45.2,
21.8 ppm; ESI-MS m/z: 404.8 [M+H]+. Anal. Calcd for C23H18ClN3O2:
C, 68.40; H, 4.49; Cl, 8.78; N, 10.40; Found: C, 68.41; H, 4.50; Cl,
8.77, N, 10.41.
4.2. Biological protocols
4.2.1. In vitro antiproliferative assay
The in vitro cytotoxicity of test compounds was determined
by evaluating the growth inhibition rates on certain concentra-
tions by MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-
tetrazolium bromide) method. In brief, tumor cells were plated
in a 96-well plate (8000 cells per well) in RPMI-1640 culture
medium with 10% (v/v) heat-inactivated fetal bovine serum
(FBS), and allowed to adhere and spread for 10 h. Then the cul-
100 lM. All the experiments were repeated at least three times,
with 10 embryos per well. Before the assay, the embryos that have
precultured with embryo rearing solution (ERS) were treated with
0.1% methylenedi, and for the transgenic group, 0.1% N-phenylth-
iourea (PTU) was supplemented. After treatment with the com-
pound for 24 h, the embryos were observed for morphological
changes using an Olympus Spinning Disk Confocal Microscope Sys-
tem (Nikon SMZ1500) at 50- and 100-fold magnification,
respectively.
ture medium was removed and 100 lg/mL of positive control
drug, test compounds in RPMI-1640 culture medium with 1%
FBS (with 0.1% DMSO as fluxing agent, also in the control the
zero-setting wells) were added, and then the cells were cultured
4.2.5. Kinase inhibition assay
for 24 h at 37 °C in a CO2 incubator. MTT solution (10
l
L of
The ability of compounds to inhibit the phosphorylation of a
peptide substrate by the recombinant human enzymes (Sigma)
was evaluated in a microtiter plate format using luciferase analy-
5 mg/mL) was added per well and the cells were cultured for
additional 4 h. The blue formazan crystals formed from MTT
was pelleted to the bottom of well by centrifugation, then add-
sis. Briefly, 15
tion buffer (40 mM Tris, pH 7.4; 10 mM MgCl2; 0.1 mg/mL BSA;
1 mM DTT; 10 M ATP) and kinase substrate was incubated at
30 °C for 5 min in 96-well microtiter plates. After addition of speci-
fic kinase, the samples (final volume of 50 L) were incubated for
lL of reaction mixture containing the kinase reac-
ing 100 lL of DMSO per well and mixing for another 15 min. The
optical density (OD) values were measured at 590 nm. The
growth inhibition rate was calculated as [(ODc À ODt)/
(ODc À ODz)] Â 100. ODc represents the OD values of the control
group, ODt represents the OD values of the treating groups, and
ODz represents the OD values of the zero-setting groups. Two
separate experiments with triplicate data were performed to
achieve the cytotoxic results, and the IC50 values were calculated
according to the inhibition ratios.
l
l
further 50 min in the absence (control) or in the presence of differ-
ent concentrations of test compounds and during incubation the
fluorescence intensity (kex = 355 nm, kem = 460 nm) was recorded
on a MD-SpectraMax M5 microplate reader. % activity = {(Lu no
kinase À Lu compound)/(Lu no kinase À Lu kinase)} Â 100%.
Please cite this article in press as: Shi, L.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.03.008

L. Shi et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
13
4.3. Statistical analysis
References and notes
1. Lv, Y. C.; Li, M. Y.; Liu, T.; Tong, L. J.; Peng, T.; Wei, L. X.; Ding, J.; Xie, H.; Duan,
W. H. ACS Med. Chem. Lett. 2014, 5, 592.
Experimental values were expressed as mean SD. Statistical
analysis was performed with two-tailed Student’s t-test or
Mann–Whitney test using Prism 6.0 (GraphPad Software, Inc.,
San Diego, CA). A P-value <0.05 was considered statistically
significant.
2. Abdel-Rahman, O. Crit. Rev. Oncol. Hemat. 2015, 93, 18.
3. Musumeci, F.; Radi, M.; Brullo, C.; Schenone, S. J. Med. Chem. 2012, 55, 10797.
4. Eskander, R. N.; Tewari, K. S. Gynecol. Oncol. 2014, 132, 496.
5. Shi, L. L.; Zhou, H.; Wu, J. F.; Li, X. Mini Rev. Org. Chem. 2015, 12, 96.
6. Li, Y. G.; Zhang, J.; Xu, W. F.; Zhu, H. W.; Li, X. Bioorg. Med. Chem. 2010, 18, 1516.
7. Yuan, H. Y.; Li, X.; Qu, X. J.; Sun, L. R.; Xu, W. F.; Tang, W. Med. Chem. Res. 2009,
18, 671.
8. Zakharia, Y.; Zakharia, K.; Rixe, O. Expert Opin. Drug Disc. 2015, 10, 925.
9. Pemovska, T.; Johnson, E.; Kontro, M.; Repasky, G. A.; Chen, J.; Wells, P.; Cronin,
C. N.; McTigue, M.; Kallioniemi, O.; Porkka, K.; Murray, B. W.; Wennerberg, K.
Nature 2015, 519, 102.
4.4. Molecular modeling study
AutoDock 4.2 program was used to simulate a binding model by
docking into the active site of VEGFR-2 (PDB code: 3EWH), and the
docking study was performed roughly as follows. (1) Protein tem-
plate preparation: X-ray crystal structure of VEGFR-2 was down-
loaded from RCSB. Two missing loops, Asp857-Thr861 and
Arg1051-Asp1056, were reconstructed by SuperLooper web server.
The energy of reconstructed template was minimized by MOE
using AMBER94 force field. Nonpolar hydrogens of new template
3EWHOK were merged before adding Gasteiger Huckel charges
by AutoDockTools (ADT) 1.5.2. After that, types of atoms were
assigned and grid representation of 3EWHOK was prepared by
AutoGrid4. (2) Ligand preparation: compound 8c was optimized
with Chem3D Ultra 12.0. Ligands AAX, GIG, K11, LIF, 877 and 900
were extracted from crystal poses. These ligands were prepared
with ADT1.5.2 as follow: Gasteiger charges were assigned, nonpo-
lar hydrogens were merged, aromatic carbons were identified, and
the rigid root and rotatable bonds were defined. (3) Docking proce-
dures: The docking simulations were performed using the Lamarck-
ian genetic algorithm (LGA) with 100 solutions for each ligand
obtained, in which the default values of the other parameters were
used. The binding model with the lowest binding free energy for
each inhibitor is selected as the best model.
10. Escudier, B.; Gore, M. Drugs R. D. 2011, 11, 113.
11. Gaumann, A. K.; Drexler, H. C.; Lang, S. A.; Stoeltzing, O.; Diermeier-Daucher,
S.; Buchdunger, E.; Wood, J.; Bold, G.; Breier, G. Int. J. Oncol. 2014, 45, 2267.
12. Dragovich, T.; Laheru, D.; Dayyani, F.; Bolejack, V.; Smith, L.; Seng, J.; Burris, H.;
Rosen, P.; Hidalgo, M.; Ritch, P.; Baker, A. F.; Raghunand, N.; Crowley, J.; Von
Hoff, D. D. Cancer Chemother. Pharmacol. 2014, 74, 379.
13. To, K. K.; Poon, D. C.; Wei, Y.; Wang, F.; Lin, G.; Fu, L. W. Biochem. Pharmacol.
2015, 97, 27.
14. Bold, G.; Altmann, K. H.; Frei, J.; Lang, M.; Manley, P. W.; Traxler, P.; Wietfeld,
B.; Brüggen, J.; Buchdunger, E.; Cozens, R.; Ferrari, S.; Furet, P.; Hofmann, F.;
Martiny-Baron, G.; Mestan, J.; Rösel, J.; Sills, M.; Stover, D.; Acemoglu, F.; Boss,
E.; Emmenegger, R.; Lässer, L.; Masso, E.; Roth, R.; Schlachter, C.; Vetterli, W. J.
Med. Chem. 2000, 43, 2310.
15. Potashman, M. H.; Bready, J.; Coxon, A., Jr.; DeMelfi, T. M.; DiPietro, L.; Doerr,
N.; Elbaum, D.; Estrada, J.; Gallant, P.; Germain, J.; Gu, Y.; Harmange, J. C.;
Kaufman, S. A.; Kendall, R.; Kim, J. L.; Kumar, G. N.; Long, A. M.; Neervannan, S.;
Patel, V. F.; Polverino, A.; Rose, P.; Plas, Sv.; Whittington, D.; Zanon, R.; Zhao, H.
J. Med. Chem. 2007, 50, 4351.
16. Su, P.; Wang, J.; Shi, Y.; Pan, X.; Shao, R.; Zhang, J. Bioorg. Med. Chem. 2015, 23,
3228.
17. Wang, C.; Gao, H.; Dong, J.; Zhang, Y.; Su, P.; Shi, Y.; Zhang, J. Bioorg. Med. Chem.
2014, 22, 277.
18. Quaife, N. M.; Watson, O.; Chico, T. J. Curr. Opin. Pharmacol. 2012, 12, 608.
19. Cho, Y. S.; Jung, H. G.; Seok, S. H.; Payumo, A. Y.; Chen, J. K.; Kwon, H. J. Biochem.
Biophys. Res. Commun. 2013, 433, 396.
20. Wilhelm, S. M.; Carter, C.; Tang, L.; Wilkie, D.; McNabola, A.; Rong, H.; Chen, C.;
Zhang, X.; Vincent, P.; McHugh, M.; Cao, Y.; Shujath, J.; Gawlak, S.; Eveleigh, D.;
Rowley, B.; Liu, L.; Adnane, L.; Lynch, M.; Auclair, D.; Taylor, I.; Gedrich, R.;
Voznesensky, A.; Riedl, B.; Post, L. E.; Bollag, G.; Trail, P. A. Cancer Res. 2004, 64,
7099.
Acknowledgments
21. Yang, W.; Chen, Y.; Zhou, X.; Gu, Y.; Qian, W.; Zhang, F.; Han, W.; Lu, T.; Tang,
W. Eur. J. Med. Chem. 2015, 89, 581.
22. Gao, H.; Su, P.; Shi, Y.; Shen, X.; Zhang, Y.; Dong, J.; Zhang, J. Eur. J. Med. Chem.
2015, 90, 232.
23. Vijayan, R. S.; He, P.; Modi, V.; Duong-Ly, K. C.; Ma, H.; Peterson, J. R., Jr.;
Dunbrack, R. L.; Levy, R. M. J. Med. Chem. 2015, 58, 466.
24. Shi, L.; Wu, T. T.; Wang, Z.; Xue, J. Y.; Xu, Y. G. Eur. J. Med. Chem. 2014, 84, 698.
25. Shi, L.; Gao, H.; Dong, J.; Zhang, Y.; Su, P.; Shi, Y.; Zhang, J. Bioorg. Med. Chem.
2014, 22, 277.
This work was partially supported by the Natural Science Foun-
dation of Shandong Province (No. ZR2013HM101), Program for
Changjiang Scholars and Innovative Research Team in University
(PCSIRT, No. IRT13028) and China–Australia Centre for Health
Sciences Research Program (No. 2015GJ07). Ms. Ying Feng has con-
tributed a lot for the in vivo zebrafish assay, and Dr. Qiang Wang’s
helpful discussion with regard to the docking study is also
appreciated.
Please cite this article in press as: Shi, L.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.03.008