Novel inhibitor for fibroblast growth factor receptor tyrosine kinase

Article abstract of DOI:10.1016/j.bmcl.2007.06.058

NP603, the 6-dimethoxy phenyl indolin-2-one, was designed as FGF receptor 1 inhibitor by computational study. NP603 was synthesized and found to be more active against endothelial proliferation of HUVEC after the rhFGF-2 stimulation than SU6668 with minim

Full text of DOI:10.1016/j.bmcl.2007.06.058

Bioorganic & Medicinal Chemistry Letters 17 (2007) 4812–4818
Novel inhibitor for fibroblast growth factor receptor tyrosine kinase
Naparat Kammasud,a Chantana Boonyarat,b Satoshi Tsunoda,c Hiroaki Sakurai,c
Ikuo Saiki,c David S. Griersond and Opa Vajraguptaa,*
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Mahidol University,
447 Sri-Ayudhya Road, Bangkok 10400, Thailand
bFaculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand
cDivision of Pathogenic Biochemistry, Department of Bioscience, Institute of Natural Medicine, University of Toyama, Japan
dUMR 176 CNRS, Institut Curie, Section Recherche, Centre Universitaire, Bat.110-112, 91405 Orsay cedex, France
Received 16 March 2007; revised 8 June 2007; accepted 18 June 2007
Available online 26 June 2007
Abstract—NP603, the 6-dimethoxy phenyl indolin-2-one, was designed as FGF receptor 1 inhibitor by computational study.
NP603 was synthesized and found to be more active against endothelial proliferation of HUVEC after the rhFGF-2 stimulation
than SU6668 with minimum effective dose of 0.4 lM but with similar potency as SU16g. NP603 inhibited the tyrosine phosphor-
ylation in FGF receptor and the activation of extracellular signal-regulated kinase and c-Jun-N-terminal-kinase after the rhFGF-
2 stimulation. The increase in activity of NP603 supports the role of Lys514 movement in ligand–receptor binding in modeling
study as the movement accommodates the hydrophobic interaction at the receptor pocket leading to the enhancement of binding
capacity.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Receptor tyrosine kinases (RTKs) have been shown to
be important mediators of signal transduction in
cells.1–5 These transmembrane molecules characteristi-
cally consist of an extracellular ligand-binding domain
connected through a segment in the plasma membrane
to an intracellular tyrosine kinase domain. Binding of
the ligand to the receptor results in receptor dimeriza-
tion and stimulation of the receptor-associated tyro-
sine kinase activity, which leads to phosphorylation
of tyrosine residues on both the receptor and other
intracellular molecules. These changes in tyrosine
phosphorylation initiate a signaling cascade leading
to a variety of cellular responses, one of which is angi-
ogenesis.6,7 Growth factors, including the vascular
endothelial growth factors (VEGF), the fibroblast
growth factors (FGF), and the platelet-derived growth
factors (PDGF) and their associated receptor tyrosine
kinases, are major regulators of angiogenesis.
RTKs are important therapeutic targets for cancer
drug discovery and development. Amongst the many
inhibitors undergoing development, 3-substituted indo-
lin-2-ones were found to exhibit high potency and
selectivity against PDGF and VEGF (Flk-1) RTKs.
SU5402 and SU5416 have been shown to be specific
inhibitors of the kinase activity of the fibroblast
growth factor receptor (FGFR) and vascular endothe-
lial growth factor receptor (VEGFR), respectively,
whereas SU6668 showed inhibitory activity against
FGFR, VEGFR, and PDGFR.8,9 The pyrido[2,3-
d]pyrimidine PD173074, a potential drug candidate,
was similarly found to be highly selective for the
FGFR1. This molecule is a nanomolar inhibitor of
FGFR1 and is also a submicromolar inhibitor of
VEGFR2. Structure–activity relationship (SAR) data
for compounds in the pyrido[2,3-d]pyrimidine series
indicate that binding affinity and selectivity for
FGFR1 is attained through modification of the phenyl
group attached to the 6-position of the pyrido[2,3-
d]pyrimidine scaffold. In general, derivatization at the
3- and 5-positions of the phenyl ring increases selectiv-
ity for FGFR1, especially when the attached group is
larger than a methyl. The SAR data are in good
agreement with the surface observed in the structure
for PD173074 bound in the FGFR1 kinase domain
(PDB accession code 2FGI).10
Keywords: FGFR1; FGFR1 inhibitor; SU6668; Phenyl indolin-2-one;
Docking; Binding mode; Antiproliferation; Antiangiogenesis.
*
Corresponding author. Tel.: +66 2 6448677; fax: +66 2 6448695;
e-mail: pyovj@mahidol.ac.th
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.06.058

N. Kammasud et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4812–4818
4813
pounds with different kinase inhibition profiles for
VEGFR2 (Flk-1) and FGFR1. However, the structural
information from the protein binding study of
PD173074 indicated that the contribution of the
dimethoxy phenyl group to tight binding in 1FGFR is
important. Therefore, structural modification of the
indolin-2-ones to include this dimethoxy phenyl motif
at position 6 should, in principle, lead to a potent
1FGFR inhibitor. This reasoning suggested that the no-
vel C-6 substituted oxindole analog (NP603) may be an
interesting probe in modeling studies to ascertain the
role of Lys514 movement in ligand–receptor binding.
O
N
N
H
N
H
O
O
N
H
N
H
SU5416
SU4984
NEt
2
O
OH
HN
O
HN
N
N
NH
N
H
O
N
O
N
H
In this study, the proposed compound NP603 was flex-
ibly docked to the model of the constructed FGF1
receptor template. The goal was to study the effects of
the added substituent at 6-position on the structure
and the orientation of the ligand–receptor complex. In
addition to docking studies, NP603 was synthesized
and evaluated for its activity.
SU6668
PD173074
O
O
OH
N
H
O
O
N
H
The crystal structure of FGFR1 bound to the inhibitor
PD173074 (Protein Data Bank code: 2FGI10) was se-
lected for the construction of the docking template. In
order to prepare the target protein as a template, the li-
gand and crystallographic water were removed. Hydro-
gens and Kollman unified charges were added by using
AutoDockTools (ADT).11 Cubic affinity grid maps for
each atom type in the ligand set (plus an electrostatics
map) centered on the cavity with dimensions of
NP603
O
Through superposition of the SU5402-FGFR1 structure
(1FGI) with 2FGI it was determined that the conserved
Lys514 undergoes significant movement upon
PD173074 binding. The side chain of Lys514 adopts
an alternative conformation which allows the dimethoxy
phenyl substituent of PD173074 access to the back of
the ATP-binding cleft.
˚
˚
60 · 60 · 60 A and 0.375 A spacing between grid points
were computed using AutoGrid 3.0. The constructed
FGFR1 template was validated by redocking with the
native ligand, PD173074, AutoDock 3.0.5 was employed
to perform the docking calculation. All ligands were cre-
ated and initially optimized with SYBYL7.0. All pro-
tons were added, charges were assigned using the
On comparison of indolinones, such as SU4984,
SU5402, and SU6668 to PD173074, certain structural
similarity may be noticed, and in particular, a NH
(hydrogen donor) and an aromatic group. Figure 1 pro-
vides the schematic drawing of the binding mode be-
tween FGFR1 and the inhibitors based on these
different scaffolds.
Gasteiger–Huckel method, and ligand energies were
¨
minimized using the TRIPOS force field. During final
preparations Kollman charges were added, nonpolar
hydrogens and lone pairs were merged, aromatic car-
bons identified, and lastly, the rigid root and rotatable
bonds were defined. The 3D configuration of
PD173074, obtained from docking with the constructed
FGFR1 template, was compared with the crystallo-
graphic pose. PD173074 docked in the binding pocket
SAR studies on the indolin-2-one series suggested that
modifications at the C-6 position would lead to com-
O
HO
NEt2
HN
HN
O
N
H
N
N
NH
O
O
N
N
H
O
O
H
H
O
N
N
O
Ala 564
Ala 564
O
O
NH
O
NH
Glu 562
HN
HN
PD173074
Glu 562
SU5402
Figure 1. The binding modes of the FGFR1 kinase and the inhibitors: SU5402 and PD173047.

4814
N. Kammasud et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4812–4818
in the conformation found in the crystal structure. The
result showed the same configuration with RMSD of
˚
1.6 A when compared with the crystal structure of PD
type interaction as expected. The docking energy of
NP603 is ꢀ13.3 kcal/mol, the lowest amongst the inhib-
itors studied (Table 1).
173074 in complex with the tyrosine kinase domain of
FGFR1.
Figure 3 shows the superposition of the oriented confor-
mations of the inhibitors (SU6668 and NP603) docked
to the FGFR template, with the conformation of
PD173074 from X-ray crystal structure. It was observed
that the docked conformations of the inhibitors showed
very similar orientations of binding on FGFR1.
In order to study the binding mode, SU6668, NP603,
and related analogs (unsubstituted phenyl and methoxy
substituted phenyl at position 6, SU16f and SU16g)
were docked with the validated FGFR1 template. A
computational docking study using Autodock supports
that the binding cleft for the ATP domain of FGFR1
is the target area for the inhibitors. The binding mode
of NP603, which was designed by substitution of dime-
thoxy phenyl group at the 6-position of indolin-2-one to
enhance the binding capacity via the hydrophobic inter-
action at the receptor pocket, is shown in Figure 2. The
docking studies confirmed that the added substituent lo-
cates in the pocket and contributes to the hydrophobic
The docked orientation of NP603 showed that the
substituted moiety at position 6 located near to the ac-
tive binding site of the substrate. This molecule makes
three hydrogen bonds to the protein backbone of
O
OH
NP603
N
H
Lys514
O
O
N
H
O
H
O
N
O
Ala 564
O
NH
Hydrophobic pocket
HN
Figure 3. Superposition of crystallographic PD173074 binding posi-
tion (red stick model) and docked orientations of inhibitors: NP603
(green) and SU6668 (blue) bound to FGFR1 template.
Glu 562
Figure 2. The binding modes of the FGFR1 kinase and NP603.
Table 1. Effect on the proliferation of HUVEC after the rhFGF-2 stimulation
O
OH
N
H
O
N
H
R
R
H
Binding energy (kcal/mol)
Docking energy (kcal/mol)
Concentration (lM)
IC50 Min effective
SU6668
SU16f9
ꢀ8.49
ꢀ11.3
ꢀ10.2
ꢀ13.2
>50.0
10
>50.0
10
SU16g9
NP603
ꢀ11.0
ꢀ11.5
ꢀ13.0
ꢀ13.3
17.9
18.2
0.4
OMe
MeO
0.4
OMe

N. Kammasud et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4812–4818
4815
in this study was developed from the crystal structure
of PD173074 with the alternative conformation involv-
ing Lys514 movement to accommodate hydrophobic
interaction. For PD173074 and NP603, the docking re-
sults showed that the dimethoxy phenyl substitution at
position 6 fitted the hydrophobic pocket creating van
der Waals interactions between the 6-substituted aro-
matic ring and Val559 and Val561. According to the
docked conformation of NP603, a total of seven hydro-
gen bonding interactions were found.
As NP603 demonstrated good binding with FGFR1 in
silico, NP603 and the related compounds SU6668,
SU16f, and SU16g were synthesized and evaluated for
their activity in vitro. The synthesis of NP60312 was
accomplished by the synthetic routes A and B. The 6-
substituted indolin-2-one [4] was prepared in four steps
(1.1 g, 25%), involving: reaction of 3,5-dimethoxyphenyl
boronic acid (6.9 g, 56.5 mmol) and 5-bromo-2-fluoroni-
trobenzene (0.2 ml, 14.1 mmol) under Suzuki condi-
tions, to give 5-aryl-2-fluoronitrobenzene [1] (2.51 g,
75%), displacement of the o-fluoro substituent by di-
methyl malonate (1.38 g, 34.5 mmol) followed by hydro-
lytic decarboxylation using 6 N aqueous hydrochloric
acid and cyclization (Scheme 1). NP603 (80 mg, 20%)
was obtained by condensation of 6-substituted indolin-
2-one [4] (15 mg, 1.1 mmol) with pyrrole aldehyde [5]
(22 mg, 1.1 mol) (Scheme 2). The structure of synthe-
sized compound was characterized by their melting
Figure 4. Hydrogen bonds (green spheres) between NP603 (blue) and
FGFR1 template.
FGFR1: between N-1 of the oxindole and the carbonyl
oxygen of Glu562, between carbonyl oxygen of the oxin-
dole and the amide nitrogen of Ala564, and between N-1
of the pyrrole and the carbonyl oxygen of Ala564
(Fig. 4). This same two residues Glu562 and Ala564, lo-
cated in the hinge region of FGFR1, are involved in
hydrogen bonds to N-1 and N-6 in the adenine ring of
ATP. The hydrophobic residues, Val559 and Val561,
appear to be involved in the interaction with the 3,5-
dimethoxy group of NP603. In addition to three hydro-
gen bonds involving the oxindole scaffold, the oxygen
atoms of the two methoxy groups in NP603 contribute
two hydrogen bonds. One is made between the methoxy
group of NP603 and the amide nitrogen of Asp641, and
the other between the other methoxy group of NP603
and the amide nitrogen of Lys514. van der Waals con-
tacts are made between the dimethoxy phenyl group
and the side chains of Lys514, Glu531, Met535, Ile545,
Val559, Val561, Ala640, and Phe642 (Fig. 4).
1
points, FTIR, H NMR, 13C NMR, MS, and elemental
analyses of C, H, and N. Spectral data (FTIR, NMR,
and MS) were compatible with the assigned structures
in all cases.
The in vitro kinase assay of NP603 was performed. The
effect on the activity of the human FGFR1 kinase ex-
pressed in insect cells was quantified by measuring the
phosphorylation of the substrate biotinyl-bAbA-
bAAAEEEYFFLFAKKK and the HTRF detection
method.13 The IC50 against FGFR1 was found to be
0.4 lM, three times less than that of SU16g (IC50
1.2 lM). FGF-2 is known to be one of the potent mito-
gens of vascular endothelial cells and to play an impor-
tant role in the tumor-induced angiogenesis. Therefore,
According to the derived docking model of SU6668 (the
indolinone scaffold without substitution on position 6)
two hydrogen bond interactions were formed between
the N and O of the oxindole with Ala564. We found that
the docked conformation of SU6668 was flipped when
compared with the crystal structure of SU5402 in
1FGI (Fig. 1). This was because the FGFR1 template
O
B(OH)2
O
F
CH(COOCH3)2
NO2
O
F
O
NO2
b
a
Br
NO2
O
O
[1]
[2]
c
CH2COOH
NO2
O
O
O
N
H
d
O
[3]
O
[4]
Scheme 1. Synthesis of 6-substituted indolinone. Reagents and conditions: (a) (Ph3P)4Pd, NaHCO3, toluene–ethanol, reflux, 2 h; (b)
CH2(COOCH3)2, NaH, THF, 100 ꢁC, 2 h; (c) 6 N HClaq, overnight; (d) Pd/C, H2, HOAc, rt, 2 h.

4816
N. Kammasud et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4812–4818
O
O
O
O
O
O
b
O
O
a
N
H
N
H
N
H
O
HO
Bzl
O
c
OH
O
O
O
N
H
OH
O
O
O
[4]
e
O
N
H
d
N
N
H
[5]
H
H
H
NP603
O
Scheme 2. Synthesis of NP603. Reagents and conditions: (a) Pd/C, H2, ethanol, rt, 2 h; (b) TFA, reflux, 1 h; (c) TFA, trimethyl orthoformate, reflux,
1 h; (d) NaOHaq, reflux, 2 h; (e) 6-substituted indolin-2-one, piperidine, ethanol, reflux, 3 h, then 2 N HClaq
.
we determined the effect of NP603 and related com-
pounds on the proliferation of human umbilical vein
Cpd treated for 3 hrs.
a
b
endothelial cells (HUVEC) after the recombinant hu-
man FGF-2 (rhFGF-2) treatment. All the tested com-
pounds inhibited the rhFGF-2-induced cellular
proliferation of HUVEC. NP603 has the highest inhibi-
tory effect among the compounds. The SU16g9 and
NP603 bearing methoxyl group on phenyl ring were
highly active against endothelial proliferation, as com-
pared with SU6668 and the unsubstituted SU16f9. The
minimum effective concentrations of NP603 and
SU16g are 0.4 lM. The degree of inhibition correlates
with the binding energy obtained from the docking
study (Table 1). The minimum cytotoxic concentration
of NP603 in the absence of rhFGF-2 was found to be
50 lM, which is one hundred times greater than the min-
imum effective dose.
Cpd treated for 3 hrs
pERK
ERK1/2
pJNK
pERK
ERK1/2
JNK
rhFGF2
Cpds
(50 μM)
−
−
+
−
+
+
+
+
+
+
+
+
rhFGF2
Cpds
(10 μM)
−
−
+
−
+
+
+
+
Figure 5. Effect on the intracellular signaling in HUVEC after the
rhFGF-2 stimulation, Western blot of total proteins in HUVEC cell
lysates with antibodies against ERK1/2, JNK, and phospho-specific
ERK1/2 and JNK.
The effect on the intracellular signaling in HUVEC after
the rhFGF-2 treatment was further investigated by Wes-
tern blot analysis. The extracellular signal-regulated ki-
nase (ERK) and c-Jun-N-terminal-kinase (JNK) play
an important role in angiogenesis process for tumor
growth and metastasis. rhFGF-2 induced activation of
ERK after the phosphorylation in FGF receptors. The
ERK activation was inhibited completely by NP603
and to a lesser extent by SU16g and SU6668 (Fig. 5).
These results indicate that NP603 has potent inhibitory
activity on the FGF-2-induced tyrosine phosphorylation
of FGF receptor, which resulted in the inhibition of
ERK and JNK activation and cellular proliferation in
HUVEC.
the inhibition of tube formation as compared with the
control group. NP603 significantly inhibited tube forma-
tion in a concentration-dependent manner. However,
when compared with SU6668, NP 603 inhibited tube
formation better than SU6668 but not significantly
different.
The activity observed lends strong support to the role of
Lys514 movement in ligand–receptor binding in order to
accommodate the presence of the 3,5-dimethoxyphenyl
substituent at the C-6 position in NP603 to locate in
the receptor pocket. This leads to an enhancement of
the binding capacity. In this regard, the 3,5-dimethoxy-
phenyl moiety at the C-6 position of indolinone in-
creased the inhibitory potency against FGFR1 as
predicted by the modeling study. In order to support
this notion, the analogs of mimicking substitution at
the C-6 position of the indolin-2-one core (L1–L5) were
docked with FGFR1, they all gave better docking en-
ergy than SU6668 as expected (Table 2). The binding
orientations of L1–L8 docked with FGFR1 are as same
as NP603 (Fig. 7). The docking energies of L4 and L8
with 3,5-di-substitution of cyano and with hydroxy-
To evaluate the effect of NP603 on angiogenesis, the
HUVEC tube formation assay was performed in which
the lengths of the capillary-like tubes formed were mea-
sured. NP603 suppressed HUVEC tube formation in a
dose dependent manner, in the presence of NP603 HU-
VECs formed incomplete and narrow tube-like struc-
tures (Fig. 6). In contrast, following stimulation with
FGF-2, formation of elongated and robust tube-like
structures was observed which were organized by a
greater number of cells as compared to the control.
Treatment with 1, 3, 10, and 25 lM NP603 resulted in

N. Kammasud et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4812–4818
4817
120
NP603
100
*
1 μM
control
FGF-2
80
60
40
20
0
**
**
10 μM
3 μM
25 μM
0
1
3
10
25
Concentration, μM
Figure 6. Effect of NP603 on the tube-like formations of HUVEC. HUVEC were seeded onto 96-well plates, which had been coated with 40 ll of
Matrigel per well, in the presence of NP603. After a 5-h incubation period at 37 ꢁC, the culture was fixed by glutaraldehyde, and the length of the
tube-like formations was evaluated. Original magnification: 5·. A representative experiment is shown, with means SD of four wells. *P < 0.05,
**P < 0.01.
Table 2. Docking results of the analogs of mimicking substitution at
the C-6 position of the indolin-2-one core
R
Binding energy Docking energy
(kcal/mol)
(kcal/mol)
SU6668
NP603
H
ꢀ8.49
ꢀ10.2
H3CO
ꢀ11.5
ꢀ11.5
ꢀ13.3
ꢀ13.5
OCH3
L1
N
N
N
Figure 7. The superposition of docked conformations of L1 (red), L4
(green), L8 (turquoise), and N603 (blue) bound with FGFR1.
L2
L3
ꢀ11.4
ꢀ12.4
ꢀ13.3
ꢀ14.4
NC
trons across two cyano groups. For L8, in addition to
fitting in the pocket, the inductive effect of the hydroxy-
methyl groups substituted on benzene is the additional
factor resulting in the enhanced binding leading to the
lowest docking energy. Thus, L4 and L5 were chosen
for further synthesis and activity testing. The result in
Table 2 demonstrated the role of Lys514 movement in
ligand–receptor binding to accommodate the hydropho-
bic interaction at the receptor pocket leading to the
enhancement of binding capacity.
NC
L4
ꢀ12.8
ꢀ15.4
CN
HOH2C
L5
L6
ꢀ11.9
ꢀ12.2
ꢀ13.9
ꢀ14.2
HO
HO
Acknowledgments
L7
L8
ꢀ12.9
ꢀ13.0
ꢀ14.7
ꢀ15.6
This work was funded by the Royal Golden Jubilee Pro-
ject, Thailand Research Fund, and the 21st Century
COE project from the Ministry of Education, Culture,
Sports, Science and Technology, Japan.
OH
HOH2C
C H2OH
References and notes
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NP603, ꢀ15.4 kJ/mol for L4 and ꢀ15.6 kJ/mol for L8.
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4818
N. Kammasud et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4812–4818
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