Discovery of 1,3-Diaryl-pyridones as Potent VEGFR-2 Inhibitors: Design, Synthesis, and Biological Evaluation

Article abstract of DOI:10.1111/cbdd.12703

In this study, we described the design, synthesis, and biological evaluation of 1,3-diaryl-pyridones as vascular endothelial growth factor receptor-2 (VEGFR-2) inhibitors. The 1,3-diaryl-pyridones were synthesized via Chan-Lam and Suzuki coupling reactions. Two representative compounds, 17 and 35h, displayed excellent enzymatic inhibitory activities, with IC₅₀ values of 3.5 and 3.0 nm, respectively. Furthermore, compounds 17 and 35h blocked the tube formation and suppressed the VEGF-induced phosphorylation of VEGFR-2 and downstream extracellular signal-regulated kinases (Erk) in human umbilical vein endothelial cells (HUVECs) at 10 nm concentration. The docking simulation showed that compound 17 bound well into the active site of VEGFR-2 via two hydrogen bonds and hydrophobic interactions.

Full text of DOI:10.1111/cbdd.12703

Chem Biol Drug Des 2016; 87: 694–703
Research Article
Discovery of 1,3-Diaryl-pyridones as Potent VEGFR-2
Inhibitors: Design, Synthesis, and Biological
Evaluation
Wei Yan1,†, Zhaoru Huang2,3,†, Zhengyu Wang4,
Angiogenesis, the process of forming new blood vessels
from existing ones, is a normal physiological process that
occurs during embryogenesis, wound healing, and the
menstrual cycle (1). Aberrant angiogenesis, however, plays
Sufen Cao1, Linjiang Tong2, Tao Zhang2, Chen
Wang5, Lin Zhou3, Jian Ding2, Cheng Luo5,*,
Jinpei Zhou4,*, Hua Xie2,* and Wenhu Duan1,6,
*
a critical role in tumor cell proliferation, invasion, and
metastasis (2–4). New blood capillaries are required by
solid tumors to supply nutrient, remove metabolic waste,
and facilitate metastasis formation (2,3,5). Therefore, the
inhibition of tumor angiogenesis has been proven to be a
reliable approach for the treatment of cancers (6,7).
1School of Pharmacy, East China University of Science &
Technology, Shanghai 200237, China
2Division of Anti-Tumor Pharmacology, State Key
Laboratory of Drug Research, Shanghai Institute of Materia
Medica, Shanghai 201203, China
3Department of Gastroenterology, The First Affiliated
Hospital of Zhengzhou University, Zhengzhou, Henan
Province 450014, China
Vascular endothelial growth factor (VEGF) and its receptor
(VEGFR) are settled to be key intermediates of angiogene-
sis. Overexpression of VEGF correlates with poor progno-
sis and clinical stage in patients with solid tumors (8–10).
Binding of VEGF to VEGFR-2 triggers a cascade of down-
stream signaling pathway that finally leads to angiogenesis,
tumor proliferation, and migration (11,12). Inhibition of
angiogenesis by the blockage of VEGF/VEGFR-2 signaling
pathway has been utilized as a valuable approach in the
treatment of cancers. In the past decade, several small
molecule VEGFR-2 inhibitors have been approved, for
example, sunitinib (13), sorafenib (14), and pazopanib (15),
or have entered late stage clinical trials, for example, tivo-
zanib (16) and linifanib (17).
4Department of Medicinal Chemistry, China
Pharmaceutical University, Nanjing 210009, China
5Drug Discovery and Design Center, State Key Laboratory
of Drug Research, Shanghai Institute of Materia Medica,
Shanghai 201203, China
6Department of Medicinal Chemistry, Shanghai Institute of
Materia Medica, Shanghai 201203, China
*Corresponding authors: Cheng Luo, cluo@simm.ac.cn;
Jinpei Zhou, jpzhou668@163.com; Hua Xie,
hxie@simm.ac.cn; Wenhu Duan, whduan@simm.ac.cn
†These authors contributed equally to this work.
In this study, we described the design, synthesis, and
biological evaluation of 1,3-diaryl-pyridones as vascu-
lar endothelial growth factor receptor-2 (VEGFR-2) inhi-
bitors. The 1,3-diaryl-pyridones were synthesized via
Chan-Lam and Suzuki coupling reactions. Two repre-
sentative compounds, 17 and 35h, displayed excellent
enzymatic inhibitory activities, with IC50 values of 3.5
and 3.0 nM, respectively. Furthermore, compounds 17
and 35h blocked the tube formation and suppressed
the VEGF-induced phosphorylation of VEGFR-2 and
downstream extracellular signal-regulated kinases (Erk)
in human umbilical vein endothelial cells (HUVECs) at
10 nM concentration. The docking simulation showed
that compound 17 bound well into the active site of
VEGFR-2 via two hydrogen bonds and hydrophobic
interactions.
Recent studies of VEGFR-2 inhibitors have been focused
on exploring different scaffolds that bind to the hinge
region, and several new scaffolds have been utilized to
generate potent VEGFR-2 inhibitors, for example, quinoxa-
lines (18), pyrazolo[3,4-d]pyrimidines (19,20), pyrazolylami-
nes (21), nicotinamides (22), and thiazolo[5,4-d]pyrimidines
(23). However, modifications on the linker between hinge
region and the hydrophobic pocket were rarely reported.
The urea moiety was mainly kept unchanged because
it could form three hydrogen bonds with Clu-885 and
Asp-1046 (24). Therefore, the urea moiety seemed to be a
‘restricted area’ for modification.
Researchers from Amgen Inc. (Thousand Oaks, CA, USA)
have developed two series of potent VEGFR-2 inhibitors
(25,26). In both series, one of the NHs in the urea moiety
is cyclized with the middle phenyl ring either to form naph-
thamides or to form 2,3-dihydro-4H-benzo[b][1,4]oxazine-
4-carboxamides while maintaining the potency (IC50 values
Key words: 1,3-Diaryl-pyridone, angiogenesis, anticancer
agent, Chan-Lam coupling, VEGFR-2 inhibitors
Received 15 July 2015, revised
4 November 2015 and
accepted for publication 3 December 2015
694
ª 2015 John Wiley & Sons A/S. doi: 10.1111/cbdd.12703

1,3-Diaryl-pyridones as Potent VEGFR-2 Inhibitors
of 0.5 nM for compounds 1 and 2) (Figure 1, cyclization
pattern 1). This phenomenon was also found to be consis-
tent with our previous study (27). In view of these facts,
we envisioned that cyclization of the urea moiety to form
pyridones (Figure 1, cyclization pattern 2) would also be
acceptable. Herein, we described the discovery of 1,3-dia-
ryl-pyridone derivatives as potent VEGFR-2 inhibitors.
Compounds 7b–g were prepared following the same pro-
cedure as described for the synthesis of compound 7a,
and the analytical data were summarized in supporting
information.
Synthesis of 1-benzyl-3-bromopyridin-2(1H)-one
(7h)
Sodium hydride (60% in mineral oil, 172 mg, 4.3 mmol)
was suspended in DMF (10 mL), and 3-bromopyridin-2
(1H)-one (5) (500 mg, 2.9 mmol) was added portionwise at
0 °C. Benzyl bromide (983 mg, 5.7 mmol) was added,
and the mixture was stirred at ambient temperature for
2 h. The mixture was quenched with water and extracted
with ethyl acetate. The organic layer was dried over
sodium sulfate, filtered, and concentrated under reduced
pressure. The resulting residue was purified by silica gel
chromatography (CH2Cl2/MeOH 99:1 v:v) to give 7h
(530 mg, 72%) as an amber oil. 1H NMR (300 MHz,
CDCl3) d 7.71 (dd, J = 7.2, 1.9 Hz, 1H), 7.35 (m, 5H),
7.29 (dd, J = 7.2, 1.9 Hz, 1H), 6.05 (t, J = 7.0 Hz, 1H),
5.18 (s, 2H).
Methods and Materials
Chemistry
All reagents and solvents were purchased from commer-
cial suppliers and used without further purification unless
otherwise noted. Flash chromatography was performed
using silica gel (300–400 mesh). 1H NMR spectral data
were recorded on Varian Mercury 300 or 400 MHz NMR
spectrometer, and 13C NMR spectral data were recorded
on Varian Mercury 101 or 126 MHz NMR spectrometer.
Chemical shifts (d) were reported in parts per million using
TMS as the internal standard. The high-resolution mass
spectra were recorded on a Finnigan/MAT95 spectrome-
ter.
Synthesis of 3-bromo-1-(4-methoxybenzyl)pyridin-
2(1H)-one (7i)
General procedure for the synthesis of
compounds 7a–g
To a mixture of 3-bromopyridin-2(1H)-one (5) (174 mg,
1 mmol), (4-fluorophenyl)boronic acid (210 mg, 1.5 mmol),
and pyridine (158 mg, 2 mmol) in CH2Cl2 (10 mL) was
Compound 7i was prepared from 4-methoxybenzyl bro-
mide and compound 5 following the same procedure as
described for the synthesis of compound 7h. White solid
(81%). 1H NMR (400 MHz, CDCl3) d 7.69 (d, J = 7.2 Hz,
1H), 7.29 (dd, J = 5.5, 3.2 Hz, 3H), 6.87 (d, J = 8.5 Hz,
2H), 6.04 (t, J = 7.0 Hz, 1H), 5.11 (s, 2H), 3.79 (s, 3H).
ꢀ
added Cu(OAc)2 (40 mg, 0.2 mmol) and 4 A molecular
sieves (50 mg). The resulting mixture was stirred in the
open air overnight. The reaction mixture was filtered, and
the filtrate was concentrated under reduced pressure. The
resulting residue was purified by silica gel chromatography
(CH2Cl2/MeOH 99:1 v:v) to give 7a (206 mg, 77%) as a
white solid (28). 1H NMR (300 MHz, CDCl3) d 7.81 (dd,
J = 7.2, 1.9 Hz, 1H), 7.40–7.30 (m, 3H), 7.17 (t,
J = 8.5 Hz, 2H), 6.16 (t, J = 7.0 Hz, 1H).
Synthesis of 3-bromo-1-ethylpyridin-2(1H)-one (7j)
A
mixture of 3-bromopyridin-2(1H)-one (5) (200 mg,
1.2 mmol), potassium carbonate (497 mg, 3.6 mmol), and
iodoethane (561 mg, 3.6 mmol) in DMF (10 mL) was
heated at 80 °C for 2 h. The reaction was concentrated
Figure 1: Different cyclization patterns of urea moiety.
Chem Biol Drug Des 2016; 87: 694–703
695

Yan et al.
under reduced pressure, and the resulting residue was
purified by silica gel chromatography (CH2Cl2/MeOH 99:1
v:v) to give 7j (115 mg, 52%) as an amber oil. 1H NMR
(400 MHz, CDCl3) d 7.72 (d, J = 7.2 Hz, 1H), 7.29 (d,
J = 6.7 Hz, 1H), 6.08 (t, J = 7.0 Hz, 1H), 4.04 (q,
J = 7.2 Hz, 2H), 1.37 (t, J = 7.2 Hz, 3H).
added one drop of concentrated HCl. The reaction mixture
was heated at 120 °C in a sealed tube for 2 h. The reac-
tion mixture was filtrated, and the filter cake was washed
with ethyl acetate to give 33c (130 mg, 81%) as a pale-
yellow solid. 1H NMR (400 MHz, DMSO) d 10.71 (s, 1H),
8.37 (d, J = 6.9 Hz, 1H), 8.14 (s, 1H), 7.75 (d, J = 8.6 Hz,
2H), 7.46 (d, J = 9.0 Hz, 3H), 6.78 (d, J = 6.9 Hz, 1H),
4.00 (s, 3H), 3.98 (s, 3H).
Synthesis of 3-bromo-1-methylpyridin-2(1H)-one
(7k)
Compound 7k was prepared from 5 and iodomethane fol-
lowing the same procedure as described for the synthesis
of compound 7j. Amber oil (60%). 1H NMR (300 MHz,
CDCl3) d 7.73 (d, J = 7.2 Hz, 1H), 7.29 (d, J = 6.7 Hz,
1H), 6.06 (t, J = 7.4 Hz, 1H), 3.61 (s, 3H).
General procedure for the synthesis of
compounds 11 and 34
A mixture of 10 (500 mg, 1.4 mmol), bis(pinacolato)di-
boron (529 mg, 2.1 mmol), and potassium acetate
(409 mg, 4.2 mmol) was bubbled with argon for 10 min.
Pd(dppf)2Cl2 (50 mg, 0.07 mmol) was added, and the
reaction mixture was heated at 90 °C for 2 h. The reaction
mixture was concentrated under reduced pressure, and
the resulting residue was purified by silica gel chromatog-
raphy (CH2Cl2/MeOH 99:1–98:2 v:v) to give 11 (560 mg,
99%) as a yellow solid. 1H NMR (300 MHz, CDCl3) d 8.48
(d, J = 5.3 Hz, 1H), 7.90 (d, J = 8.6 Hz, 2H), 7.52 (s, 1H),
7.42 (s, 1H), 7.17 (d, J = 8.6 Hz, 2H), 6.49 (d, J = 5.3 Hz,
1H), 4.04 (s, 3H), 4.02 (s, 3H), 1.36 (s, 12H).
General procedure for the synthesis of
compounds 10, 33a and 33d–i
A
mixture of 4-chloro-6,7-dimethoxyquinoline (8) (1 g,
4.5 mmol) and 4-bromophenol (2.3 g, 13.4 mmol) was
heated at 170 °C for an hour. The resulting mixture was
dissolved in ethyl acetate (50 mL) and washed with water
and brine successively. The organic layer was dried over
sodium sulfate, filtered, and concentrated under reduced
pressure. The resulting residue was purified by silica gel
chromatography (CH2Cl2/MeOH 98:2 v:v) to give 10
(1.44 g, 89%) as a yellow solid. 1H NMR (300 MHz,
CDCl3) d 8.49 (d, J = 5.2 Hz, 1H), 7.56 (d, J = 9.0 Hz,
2H), 7.49 (s, 1H), 7.41 (s, 1H), 7.06 (d, J = 8.9 Hz, 2H),
6.46 (d, J = 5.3 Hz, 1H), 4.04 (s, 3H), 4.02 (s, 3H).
Compounds of 34 were prepared following the same pro-
cedure as described for the synthesis of compound 11,
and the analytical data were summarized in supporting
information.
General procedure for the synthesis of
Compounds 33a and 33d–i were prepared following the
same procedure as described for the synthesis of com-
pound 10, and the analytical data were summarized in the
supporting information.
compounds 3, 12–21 and 35
A mixture of 11 (81 mg, 0.2 mmol), 7a (50 mg, 0.2 mmol),
and cesium carbonate (409 mg, 0.4 mmol) was bubbled
with argon for 10 min. Pd(PPh3)2Cl2 (9 mg, 0.01 mmol)
was added, and the reaction mixture was heated at 60 °C
for 2 h. The reaction mixture was concentrated under
reduced pressure, and the resulting residue was purified
by silica gel chromatography (CH2Cl2/MeOH 99:1–98:2 v:
v) to give 3 (52 mg, 56%) as a white solid, mp: 200–
Synthesis of 4-((4-bromophenyl)thio)-6,7-
dimethoxyquinoline (33b)
To a mixture of 8 (200 mg, 0.9 mmol) and 4-bromobenze-
nethiol (338 mg, 1.8 mmol) in DMF (10 mL) was added tri-
ethylamine (270 mg, 2.8 mmol). The reaction mixture was
stirred at ambient temperature for 3 h. The reaction mix-
ture was concentrated under reduced pressure, and the
resulting residue was purified by silica gel chromatography
(CH2Cl2/MeOH 99:1–97:3 v:v) to give 33b (200 mg, 60%)
as a white solid. 1H NMR (300 MHz, CDCl3) d 8.47 (d,
J = 4.9 Hz, 1H), 7.55 (d, J = 8.5 Hz, 2H), 7.41 (s, 1H),
7.37 (m, 3H), 6.81 (d, J = 4.8 Hz, 1H), 4.04 (s, 3H), 4.01
(s, 3H).
1
202 °C. H NMR (300 MHz, CDCl3) d 8.49 (d, J = 5.3 Hz,
1H), 7.84 (d, J = 8.7 Hz, 2H), 7.63 (dd, J = 7.0, 2.0 Hz,
1H), 7.55 (s, 1H), 7.46–7.36 (m, 4H), 7.24–7.16 (m, 4H),
6.56 (d, J = 5.3 Hz, 1H), 6.39 (t, J = 6.9 Hz, 1H), 4.05 (s,
6H). 13C NMR (126 MHz, CDCl3)
d
162.4 (d, JC–
= 287.0 Hz), 161.6, 160.5, 154.5, 153.0, 149.7, 149.1,
F
147.1, 138.1, 137.3, 137.2 (d, JC–F = 2.8 Hz), 133.8,
131.7, 130.7 (2C), 128.7 (d, JC–F = 8.7 Hz, 2C), 120.7
(2C), 116.4, 116.4 (d, JC–F = 23.2 Hz, 2C), 108.0, 106.4,
104.1, 99.6, 56.3 (2C). HRMS (EI+) m/z calcd for
C28H21FN2O4 468.1485; found 468.1475.
Synthesis of N-(4-bromophenyl)-6,7-
dimethoxyquinolin-4-amine (33c)
To a mixture of 8 (100 mg, 0.45 mmol) and 4-bromoani-
line (93 mg, 0.54 mmol) in isopropyl alcohol (5 mL) was
Compounds 12–21 and 35 were prepared following the
same procedure as described for the synthesis of com-
pound 3, and the analytical data were summarized in sup-
porting information.
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Chem Biol Drug Des 2016; 87: 694–703

1,3-Diaryl-pyridones as Potent VEGFR-2 Inhibitors
Synthesis of 3-(4-fluorophenyl)-1-(4-
methoxyphenyl)pyridin-2(1H)-one (22)
inhibit the activity of VEGFR-2 was tested by ELISA. The
substrate of poly (Glu, Tyr) 4:1 (20 lg/mL) (Sigma, St.
Louis, MO, USA) was precoated in 96-well ELISA plates,
and the kinase was incubated with the compounds in
reaction buffer (50 mM HEPES pH 7.4, 20 mM MgCl2,
0.1 mM MnCl2, 0.2 mM Na3VO4, 1 mM DTT) containing
10 lM ATP at 37 °C for an hour. The plates were washed
with PBS and incubated with antiphosphotyrosine (PY99)
antibody (Santa Cruz Biotechnology, Santa Cruz, CA,
USA) and horseradish peroxidase-conjugated antibody
(Calbiochem, SanDiego, CA, USA) successively. The
results were visualized using o-phenylenediamine (OPD)
and read on a multiwell spectrophotometer (VERSAmaxTM;
Molecular Devices, Sunnyvale, CA, USA).
Compound 22 was prepared from 6a and 7d following the
same procedure as described for the synthesis of com-
pound 3. White solid (77%). 1H NMR (300 MHz, CDCl3) d
7.72 (dd, J = 8.7, 5.5 Hz, 2H), 7.53 (dd, J = 7.0, 2.0 Hz,
1H), 7.38–7.30 (m, 3H), 7.07 (t, J = 8.7 Hz, 2H), 6.99 (d,
J = 8.9 Hz, 2H), 6.32 (t, J = 6.9 Hz, 1H), 3.85 (s, 3H).
Synthesis of 3-(4-fluorophenyl)-1-(4-
hydroxyphenyl)pyridin-2(1H)-one (23)
Boron tribromide (424 mg, 1.7 mmol) was added dropwise
to a solution of 22 (100 mg, 0.3 mmol) in CH2Cl2 (5 mL)
at 0 °C. The resulting reaction mixture was stirred at ambi-
ent temperature overnight. CH2Cl2 (20 mL) was added to
the mixture, and the resulting solution was washed with
saturated aq NaHCO3, water and brine successively. The
organic layer was dried over sodium sulfate, filtered, and
concentrated under reduced pressure. The resulting resi-
due was purified by silica gel chromatography (CH2Cl2/
MeOH 99:1–97:3 v:v) to give 23 (75 mg, 79%) as a white
Western blotting
The HUVECs were obtained from ScienCell Research Lab-
oratories (Santiago, CA, USA). To determine the inhibitory
activities of the compounds on the VEGF-induced VEGFR
phosphorylation, HUVECs were seeded in the 6-well
plates and were cultured overnight. Then, the cells were
starved for 24 h with endothelial cell basal medium which
contained no serum. The HUVECs were treated with the
compounds for 2 h and then stimulated with 100 ng/mL
recombinant human VEGF165 (R&D Systems, Abingdon,
UK) for 15 min. Subsequently, the HUVECs were lysed
with 1 9 SDS lysis buffer and were processed for Western
blot analysis. The intensities of each band on Western blot
results were quantified by IMAGE J software (National Insti-
tutes of Health, Bethesda, MD, USA).
1
solid. H NMR (300 MHz, DMSO) d 9.76 (s, 1H), 7.74 (dd,
J = 8.4, 5.8 Hz, 2H), 7.71–7.58 (m, 2H), 7.27–7.15 (m,
4H), 6.86 (d, J = 8.6 Hz, 2H), 6.38 (t, J = 6.9 Hz, 1H).
Synthesis of 1-(4-((6,7-dimethoxyquinolin-4-yl)oxy)
phenyl)-3-(4-fluorophenyl)pyridin-2(1H)-one (4)
A mixture of 8 (22 mg, 0.1 mmol), 23 (28 mg, 0.1 mmol),
and 4-dimethylaminopyridine (12 mg, 0.1 mmol) in toluene
(2 mL) and chloroform (1 mL) was heated to 180 °C for an
hour under microwave irradiation. The reaction mixture
was concentrated, and the residue was purified by silica
gel chromatography (CH2Cl2/MeOH 99:1–97:3 v:v) to give
4 (30 mg, 64%) as a white solid, mp: 228–230 °C. 1H
NMR (300 MHz, CDCl3) d 8.53 (d, J = 5.2 Hz, 1H), 7.76–
7.69 (m, 2H), 7.60–7.49 (m, 4H), 7.46–7.41 (m, 2H), 7.31
(d, J = 8.8 Hz, 2H), 7.09 (t, J = 8.8 Hz, 2H), 6.62 (d,
J = 5.2 Hz, 1H), 6.40 (t, J = 6.9 Hz, 1H), 4.05 (s, 6H). 13C
NMR (126 MHz, CDCl3) d 162.6 (d, JC–F = 248.2 Hz),
161.5, 160.1, 154.6, 153.1, 149.8, 148.7, 146.9, 138.1,
137.9, 136.9, 132.4 (d, JC–F = 3.2 Hz), 131.7, 130.5 (d,
JC–F = 8.1 Hz, 2C), 128.7 (2C), 121.5 (2C), 116.3, 115.0
(d, JC–F = 21.3 Hz, 2C), 107.7, 106.5, 104.3, 99.3, 56.2
(2C). HRMS (EI+) m/z calcd for C28H21FN2O4 468.1485;
found 468.1481.
Tube formation assay
The standard BD Matrigel matrix (BD Biosciences, Biller-
ica, MA, USA) was coated into 96-well plates and incu-
bated at 37 °C. HUVECs were seeded into Matrigel-
coated plates together with VEGF165 (100 ng/mL) and dif-
ferent concentration of compounds. After the HUVECs
were incubated at 37 °C for 8 h, photographs of the tube
network formation were taken on a OLYMPUS IX51 stere-
oscope (Tokyo, Japan). The numbers of the intact tubes
were counted manually of each full field.
Molecular modeling
Prior to the docking study, protein structure of VEGFR-2
(PDB ID: 3B8Q) was prepared using the Protein Prepara-
tion Wizard Workflow provided in MAESTRO 9.0 and the
default settings were useda. A grid box was built with the
Compounds 8 and 24 were synthesized according to liter-
ature procedures (29).
ꢀ3
size of 20 A , centered on the cocrystal ligand located in
the ATP-binding site of VEGFR-2. Both compound 17 and
Biological evaluation
the ligand from the cocrystal structure were prepared with
b
LIGPREP to generate low-energy 3D conformations and to
Enzyme-linked immunosorbent assay (ELISA)
determine probable ionization states at pH 7.0 ꢀ 2.0. A
redocking run was performed to evaluate the performance
of the docking protocol employing GLIDE 5.5c with extra
precision (XP) (30) mode. Upon validation (RMSD = 0.134),
The kinase domain of VEGFR-2 was expressed using the
Bac-to-BacTM baculovirus expression system and purif-
ied on Ni-NTA columns. The ability of the compounds to
Chem Biol Drug Des 2016; 87: 694–703
697

Yan et al.
compound 17 was docked into the ATP-binding site of
VEGFR-2 using the same method. Figure 4 was generated
by PYMOL (Version 1.3r1)d.
stituents on the terminal phenyl ring, displayed a slight
decrease in potency compared with compound 3, implying
that inclusion of substituents on the terminal phenyl ring
had favorable impacts on the activity.
Results and Discussion
Compound 12 served as a benchmark during the initial
optimization stage. Several compounds with different sub-
stitution patterns were designed and synthesized (Table 1).
The 3-fluorine analogue 13 was equipotent to compound
3. Slight increases in potency were observed in the meth-
oxyl analogues 14 and 15. Displacement of fluorine with
chlorine led to a twofold enhancement in potency (16 and
17). Benzyl derivative 18 was fivefold less active than 12,
while the IC50 value of 4-methoxyl benzyl derivative 19
was > 100 nM. These results indicated that the distance
between pyridone and the terminal phenyl ring was strictly
restricted. Displacement of the terminal phenyl (12) with
ethyl (20) or methyl (21) resulted in significant losses of
activity (IC50 > 100 nM), which indicated the small lipophilic
substituents were insufficient to occupy the hydrophobic
pocket. To further explore the structure activity relation-
ships (SARs), we selected 3-position chlorine as the
default substituent of the terminal phenyl ring.
Chemistry
As depicted in Schemes 1 and 2, intermediates 7a–k were
prepared from 5 via Chan-Lam coupling reaction (7a–g) or
nucleophilic substitution reactions (7h–k) in good to excel-
lent yields. Conversion from 4-chloro-6,7-dimethoxyquino-
line (8) to bromide 10 with 4-bromophenol and a following
boration reaction with bis(pinacolato)diboron gave boronic
ester 11, which was then coupled with 7a–k to generate
compounds 3 and 12–21. Coupling of 6a with 7d gave
ether 22, which was subsequently treated with boron tri-
bromide to furnish phenol 23. Compound 4 was obtained
by heating the mixture of 8 and 23 under microwave irradi-
ation at 180 °C in toluene and chloroform using
4-dimethylaminopyridine as the catalyst. Derivatives 35
were smoothly obtained following the same route as
described for compound 3 by starting from the appropriate
chlorides (8 or 24) and nucleophiles (9 or 25–32) as illus-
trated in Scheme 3. As observed in the Suzuki coupling
reaction step, the yields (60–72%) of the 30-substituted
derivatives (35d, 35f, and 35h) were comparable to the
unsubstituted derivative 17, while the 20-substituted deriva-
tives (35e, 35g, and 35i) gave much lower yields (11–25%)
due to the steric hindrance effect.
Quinazoline was also utilized as an important scaffold in
VEGFR-2 inhibitors (31,32). However, as detailed in
Table 2, the quinazoline derivative 35a displayed a twofold
decline in potency compared with quinoline 17. Optimiza-
tion of the linker atom between the 6,7-dimethoxyquinoline
core and the middle phenyl ring demonstrated that an
oxygen linker is preferred to the sulfur or nitrogen one.
Compared with the oxygen linker derivative 17, the sulfur
or amino linker derivatives (35b–c) exhibited twofold to six-
fold drops in potency.
Kinase inhibitory activity
Isomers 3 and 4 were prepared as proof of concept com-
pounds. As detailed in Table 1, compound 3 demon-
strated potent VEGFR-2 inhibitory activity possessing an
IC50 value of 6.9 nM. However, the inhibitory activity of
compound 4 was much weaker (IC50 > 100 nM). This
result indicated that the substitution pattern of the pyri-
done moiety was an important factor that influenced
VEGFR-2 inhibitory activity. Compound 12, with no sub-
The effects of substituents on the middle phenyl group
were also investigated. The electronic properties of the sub-
stituents had minimal influence on potency; chlorine (35d
and 35e) or methoxyl (35f and 35g) derivatives displayed
similar inhibitory effects against VEGFR-2 (Table 2). But
they were sixfold to ninefold less active compared with the
ꢀ
Scheme 1: Synthesis of compounds 7a–k. Reagents and conditions: (a) Cu(OAc)2, Py, 4 A molecular sieve, O2, CH2Cl2, r.t., 75%–88%;
(b) for 7h–i, NaH, DMF, 0 °C to r.t., 72–81%; for 7j–k, K2CO3, DMF, 80 °C, 52–60%.
698
Chem Biol Drug Des 2016; 87: 694–703

1,3-Diaryl-pyridones as Potent VEGFR-2 Inhibitors
A
B
Scheme 2: Synthesis of compounds 3, 4, and 12–21. Reagents and conditions: (a) neat, 160 °C, 89%; (b) B2pin2, Pd(dppf)2Cl2, KOAc,
dioxane, 90 °C, 95%; (c) Cs2CO3, Pd(PPh3)2Cl2, DMF, 60 °C, 45–80%; (d) BBr3, CH2Cl2, 0 °C to r.t., 79%; (e) 8, 4-DMAP, toluene,
CHCl3, 180 °C, microwave irradiation, 64%.
Scheme 3: Synthesis of compounds 35a–i. Reagents and conditions: (a) for 33a and 33d–i, neat, 170 °C, 80–89%; for 33b, Et3N, DMF,
r.t., 60%; for 33c, conc. HCl, i-PrOH, 120 °C sealed tube, 81%; (b) B2pin2, Pd(dppf)2Cl2, KOAc, dioxane, 90 °C, 80–99%; (c) 7g,
Cs2CO3, Pd(PPh3)2Cl2, DMF, 60 °C, 11–72%.
unsubstituted compound 17, which may suggest bulky
substituents were not well tolerated at this site: The chlorine
or methoxyl may be relatively too big to occupy the binding
pocket around the middle phenyl ring; the steric hindrance
effect between the chlorine or methoxyl and the pyridone
moiety may destabilize the active conformations. On the
contrary, the smaller fluorine derivatives (35h and 35i)
slightly increased or retained VEGFR-2 potency. In consid-
eration of their excellent VEGFR-2 inhibitory activities, we
selected compounds 17 and 35h for further evaluation.
Chem Biol Drug Des 2016; 87: 694–703
699

Yan et al.
Table 1: VEGFR-2 kinase inhibitory activity of compounds 3, 4,
and 12–21
stream Erk in HUVECs. As illustrated in Figure 2, com-
pounds 17 and 35h dose dependently suppressed the
VEGF-induced phosphorylations of VEGFR-2 and Erk in
HUVECs. The approved multikinase inhibitor sorafenib was
used as a positive control. It was found that both com-
pounds 17 and 35h were more potent than sorafenib at
the concentration of 100 nM and compound 35h signifi-
cantly inhibited VEGFR-2 phosphorylation at the concen-
tration of 10 nM. These results indicated that compounds
17 and 35h could block the VEGFR and Erk signaling
pathways.
N
R
O
O
O
N
O
Compound
R
VEGFR-2 IC50 (nM)a
Tube formation by endothelial cells plays an important role
in angiogenesis. Therefore, we investigated the in vitro
anti-angiogenic effect of compounds 17 and 35h in a tube
formation assay. HUVECs were planted on Matrigel and
treated with compounds 17 or 35h at different concentra-
tions in the presence of VEGF. As shown in Figure 3, both
compounds dose dependently inhibited the VEGF-induced
HUVEC tube formation and were more potent than sorafe-
nib. Meanwhile, the cytotoxicity of both compounds was
also tested, and they showed no obvious cytotoxicity on
HUVECs under the same experimental condition (data not
shown), which ruled out the possibility that cytotoxicity
was involved in the inhibition of tube formation. These find-
ings revealed that compounds 17 and 35h were potent
VEGFR-2 inhibitors at the cellular level.
3
4
4-F-Ph
6.9 ꢀ 1.6
> 100
N
O
O
F
12
13
Ph
3-F-Ph
8.0 ꢀ 1.5
6.1 ꢀ 0.7
5.3 ꢀ 1.1
4.8 ꢀ 0.0
4.0 ꢀ 0.7
3.5 ꢀ 0.1
41.9 ꢀ 0.4
> 100
> 100
> 100
13.0 ꢀ 1.1
14
15
4-OMe-Ph
3-OMe-Ph
4-Cl-Ph
3-Cl-Ph
Bn
4-OMe-Bn
Et
Me
16
17
18
19
20
21
Su11248
–
aIC50 values were represented as the mean ꢀ SD of two or more
independent experiments.
Molecular docking study
To gain insight into the ligand–protein interactions, we per-
formed a molecular docking study of compound 17 using
a reported crystal structure of VEGFR-2 kinase domain
(PDB ID: 3B8Q) (25). As depicted in Figure 4, compound
17 bound to the ATP-binding site of VEGFR-2. The 6,7-
dimethoxyquinoline core engaged the linker region of the
enzyme in a hydrogen bond between the nitrogen and
Table 2: VEGFR-2 kinase inhibitory activity of compounds 17
and 35
R'
N
O
2'
Y
N
3'
O
O
Cl
X
ꢀ
Cys-919 (3.0 A). The carbonyl group of the pyridone par-
ticipated in a hydrogen bond contact with the backbone
ꢀ
NH of Asp-1046 (2.7 A), enabling the terminal 3-chloro-
Compound
X
Y
R’
VEGFR-2 IC50 (nM)a
phenyl ring to reside in the extended hydrophobic pocket.
Additional van der Waals interaction was seen with the
central phenyl ring and the surrounding lipophilic residues.
This binding model disclosed the interaction patterns of
the 1,3-diaryl-pyridone derivatives with VEGFR-2 and was
consistent with our experimental data.
17
C
N
C
C
C
C
C
C
C
C
–
O
O
S
H
H
H
H
30-Cl
20-Cl
30-OMe
20-OMe
30-F
3.5 ꢀ 0.1
7.8 ꢀ 0.9
7.6 ꢀ 1.2
23.6 ꢀ 2.2
25.0 ꢀ 3.2
36.6 ꢀ 3.8
22.8 ꢀ 3.1
26.5 ꢀ 4.5
3.0 ꢀ 1.2
3.6 ꢀ 0.6
13.0 ꢀ 1.1
35a
35b
35c
35d
35e
35f
NH
O
O
O
O
O
O
–
35g
35h
35i
Conclusion
20-F
–
In conclusion, we have developed a series of 1,3-diaryl-
pyridone derivatives as novel VEGFR-2 inhibitors. Com-
pounds 17 and 35h exhibited excellent VEGFR-2 inhibitory
activity in enzymatic assays with IC50 values of 3.5 and
3.0 nM, respectively. In addition, these two compounds
inhibited VEGF-induced HUVEC tube formation and sup-
pressed activation of VEGFR-2 and downstream Erk sig-
naling. Docking simulation showed that compound 17 was
Su11248
aIC50 values were represented as the mean ꢀ SD of two or more
independent experiments.
Western blotting and tube formation study
We subsequently examined the ability of compounds 17
and 35h to inhibit the activation of VEGFR-2 and down-
700
Chem Biol Drug Des 2016; 87: 694–703

1,3-Diaryl-pyridones as Potent VEGFR-2 Inhibitors
A
B
C
Figure 2: (A) Compounds 17 and 35h inhibited the VEGF-induced activation of VEGFR-2 and Erk signaling in HUVECs. (B) The density
of p-VEGFR bands was measured, and the ratios to VEGFR were calculated. (C) The density of p-ERK bands was measured, and the
ratios to ERK were calculated. Sor: Sorafenib.
A
B
Figure 3: (A) Compounds 17 and 35h inhibited the tube formation in HUVECs. (B) Numbers of the blood vessels in HUVEC assay were
counted. Sor: Sorafenib. The asterisk (*) indicates a significant decrease of tube formation of cells treated with compounds compared with
the DMSO-treated control group.
promising scaffold for the development of novel VEGFR-2
inhibitors.
Acknowledgments
We thank the National Natural Science Foundation of China
(Nos. 81273365, 81173080 and 81321092), National
Science & Technology Major Project ‘Key New Drug Creation
and Manufacturing Program’ (Nos. 2012ZX09103101-024
and 2014ZX09304002-008-001), Chinese National Programs
for High Technology Research and Development (No.
2012AA020302), and the Shanghai Science and Technology
Commission (No. 1315431901300) for their financial support.
Conflict of Interests
The authors declared no conflict of interests.
Figure 4: Predicted binding mode for compound 17 within the
ATP-binding site of VEGFR-2.
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