5-Substituted pyrido[2,3-d]pyrimidine, an inhibitor against three receptor tyrosine kinases

Article abstract of DOI:10.1016/j.bmcl.2008.12.023

NP506, the 3-{2,4-dimethyl-5-[2-oxo-5-(N′-phenylhydrazinocarbonyl)-1,2-dihydro-indol-3-ylidenemethyl]-1H-pyrrol-3-yl}-propionic acid, was designed as FGF receptor 1 inhibitor by computational study and found to be more active against endothelial proliferation of HUVEC after the rhFGF-2 stimulation than SU6668 with minimum effective dose of 10 μM. NP506 inhibited the tyrosine phosphorylation in FGF, VEGF, and PDGF receptors and the activation of extracellular signal-regulated kinase (ERK), c-Jun-N-terminal-kinase (JNK) and AKT after the rhFGF-2 stimulation. The introduction of the phenyl hydrazide motif to the position 5 of the pyrido[2,3-d]pyrimidine scaffold led to the inhibitory effect in two signaling pathways: inhibition of AKT activation in the phosphatidyl inositol 3′-kinase (PI13K)/AKT signaling pathway and the inhibition of ERK and JNK activation in MAPK pathway.

Full text of DOI:10.1016/j.bmcl.2008.12.023

Bioorganic & Medicinal Chemistry Letters 19 (2009) 745–750
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
5-Substituted pyrido[2,3-d]pyrimidine, an inhibitor against three receptor
tyrosine kinases
Naparat Kammasud a, Chantana Boonyarat b, Kingkan Sanphanya a, Maleeruk Utsintong a, Satoshi Tsunoda c,
Hiroaki Sakurai c, Ikuo Saiki c, Isabelle André d, David S. Grierson d, Opa Vajragupta a,
*
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Mahidol University, 447 Sri-Ayudhya Road, Bangkok 10400, Thailand
b Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand
c Division of Pathogenic Biochemistry, Department of Bioscience, Institute of Natural Medicine, University of Toyama, Japan
d UMR 176 CNRS, Institut Curie, Section Recherche, Centre Universitaire, Bat.110-112, 91405 Orsay cedex, France
a r t i c l e i n f o
a b s t r a c t
NP506, the 3-{2,4-dimethyl-5-[2-oxo-5-(N0-phenylhydrazinocarbonyl)-1,2-dihydro-indol-3-ylidenem-
ethyl]-1H-pyrrol-3-yl}-propionic acid, was designed as FGF receptor 1 inhibitor by computational study
and found to be more active against endothelial proliferation of HUVEC after the rhFGF-2 stimulation
Article history:
Received 15 August 2008
Revised 3 December 2008
Accepted 5 December 2008
Available online 10 December 2008
than SU6668 with minimum effective dose of 10 lM. NP506 inhibited the tyrosine phosphorylation in
FGF, VEGF, and PDGF receptors and the activation of extracellular signal-regulated kinase (ERK), c-Jun-
N-terminal-kinase (JNK) and AKT after the rhFGF-2 stimulation. The introduction of the phenyl hydrazide
motif to the position 5 of the pyrido[2,3-d]pyrimidine scaffold led to the inhibitory effect in two signaling
pathways: inhibition of AKT activation in the phosphatidyl inositol 30-kinase (PI13K)/AKT signaling path-
way and the inhibition of ERK and JNK activation in MAPK pathway.
Keywords:
FGFR-1
FGFR-1 inhibitor
SU6668
5-Substituted indolin-2-one
Virtual screening
Docking
Ó 2008 Elsevier Ltd. All rights reserved.
Binding mode
Anti-proliferation
Anti-angiogenesis
We have previously reported the 6-substituted indolin-2-one,
NP603,1 as a novel inhibitor of FGFR. The binding affinity and selec-
tivity for FGFR1 is attained through modification of the phenyl
group attached to the 6-position of the pyrido[2,3-d]pyrimidine
scaffold. In this report, we describe our efforts to explore the mod-
ification of the group attached to the 5-position of the pyrido[2,3-
d]pyrimidine scaffold.
To study the role of structure on the inhibitory activity through
FGFR1 binding which is essential for structure-based drug design,
virtual screening was carried out. FGFR-1 target was constructed
starting from X-ray structure of FGFR-1 in complex with SU4984
(PDB:1AGW2). The poorly ordered P-loop missing in 1AGW was re-
built to give 1AGWRB, which was used as our protein model. The
prepared FGFR1 template was validated by docking (FlexX, Tripos)
with the three active inhibitors: SU5402, SU4984, and PD1730741
for which the bound crystal structures are currently available.
RMSD values when redocking with the 3 inhibitors were com-
puted. In the docked poses with 1AGWRB, the three inhibitors
were found to adopt the same configuration as in the crystal struc-
tures with RMSD less than 2 Å (0.69, 0.73, and 1.61 Å). Thus,
1AGWRB was a good model of FGFR1 protein template for virtual
screening.
Virtual chemical libraries (A, B, C, and D) with variation of frag-
ments substituted on the specified positions (Fig. 1) of oxindole
were created. The diverse side chains corresponded to groups or
fragments from an in-house database and from the 2003 version
of MDL. Virtual combinatorial 2D-libraries were constructed by
CombiLibMaker (Tripos), and all 3D structures were generated by
Corina (Molecular Networks GmbH) and initially optimized with
O
O
OH
OH
O
C
H
N
N
H
N
H
5
N
H
O
O
O
N
H
N
H
6
NP603
NP506
O
* Corresponding author. Tel.: +66 2 6448677; fax: +66 2 6448695.
E-mail address: pyovj@mahidol.ac.th (O. Vajragupta).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.12.023

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N. Kammasud et al. / Bioorg. Med. Chem. Lett. 19 (2009) 745–750
22 structures or hits with full consensus of 3, for example, hydra-
Library A
R
106 fragments
Library B
R4
zine, hydrazide, phenyl urea, cinnamyl, cinnamoyl, and carbeth-
oxy. Phenyl urea motif was in the 25% top ranked fragment but
not as good as hydrazide motif whereas amide motif in position
ꢀ5 was unable to form H-bonding with Glu562 and Ala564 leading
to poor docking result. Ten fragments from the 22 hits were se-
lected to be the fragments substituted at position 4, 5, or 6 of oxin-
dole in Library C. In the 25% top ranked structures from virtual
screening of Library B (295 structures), there were 153 hits with
full consensus of 3. Ninety-six fragments from the 153 hits were
selected to be the fragments substituted at position 3 of oxindole
in Library C. Therefore, the selected fragments in Library C were
ten fragments from Library A and ninety-six from Library B. In Li-
brary C, there are 1539 molecules with 106 substituents which
lead to 7021 conformations in total. When focusing on the 25%
top ranked structures from virtual screening in Library C, there
were 911 hits with full consensus of 3. Compounds with hydrazide
159 fragments
R
R3'
R5
R6
32 fragments
4
N
H
5
R4'
O
HN
131
3
6
N
HN
131
fragments
R5'
HN
H
O
N
H
R = 3 x 131 fragments
fragments
Virtual Screening
Virtual Screening
96 fragments
10 fragments
2 fragments
R
5 fragments
R4
Library C
2 fragments
R
R3'
R5
4
7
15 fragments
3
5
fragments
R4'
O
N
H
6
HN
42
fragments
R6
HN
HN
1593 molecules
R5'
32
1 fragment
fragments
Virtual Screening
substituent at position 5 and the 2-carboxyethyl fragment in posi-
0
tion ꢀ3 of the vinyl pyrrole (R4 in Fig. 1) were the top of the list.
911 hits
From virtual screening results, the pharmacophore model of
oxindole analogs binding at FGFR1 binding site was generated
(Fig. 2). This model revealed the role of substituents at position
3, 4, 5, and 6 on the oxindole ring to binding in the ATP site.
Among the oxindoles bearing substitutes at these positions, the
modification at position 5 offers the best possibility to introduce
a fragment bearing functional groups that could allow additional
hydrogen bonding interactions with the Lys514 and/or a neigh-
boring H2O, and also reach the Asp641 for additional interac-
tions. Similar interactions could be achieved from substitution
at position 4; however position 5 remains the closest from
Lys514 and Asp641 side chains.
Structure-based design
Library D
O
OH
O
N
H
5
R
O
N
H
R
= Phenylhydrazine
294 molecules
Synthesis
Library D was constructed on the basis of the structural infor-
mation obtained. In Library D, position 5 of oxindole was substi-
Biological test
Figure 1. The procedure in library construction and virtual screening.
tuted with phenyl hydrazide fragments and position
3 was
substituted with vinyl pyrrole fragment. The selected fragments
in Library D corresponded to 294 fragments taken from commer-
cial phenyl hydrazine fragments from MDL. NP506 and compounds
1–5 proved to be the most pertinent virtual hits in Library D. The
binding energies of NP506 and compounds 1–5 with FGFR1 tem-
plate were shown in Table 1. Among five compounds of interest,
NP506 and compound 5 have the lowest docking energy.
The binding mode of NP506 with substitution of phenyl hydra-
zide motif at the 5-position of indolin-2-one was found to enhance
the binding capacity via the hydrophobic interaction at the recep-
tor pocket (Fig. 3).
SYBYL7.0. All protons were added. Charges were assigned using the
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 carbons identified, and lastly, the rigid root
and rotatable bonds were defined.
Library A was prepared to study the role of substitutions at po-
sition 4, 5, and 6 on the oxindole ring while Library B was created
in order to study the role of substituents at position 3 as well as to
search for potential fragments. The oxindole at position 4, 5, and 6
of compounds in Library A were substituted with 131 fragments in
each position. Library B, the oxindole, at position 3 were substi-
tuted with 106 fragments, 159 vinyl fragments, and 393 vinyl pyr-
role fragments. The virtual screening process involved docking
each molecule in the generated 3D-Libraries into the binding site
of the FGFR1 tyrosine kinase model using a docking program,
FlexX. All computational work was performed on a SGI Octane
workstation equipped with the IRIX 6.5 operating system. The con-
sensus-scoring module, consensus score (CScore) in SYBYL, version
7.0, made up of five scoring functions (G Score, D Score, PMF Score,
Chem Score, and F Score), was used to estimate and rank the bind-
ing affinities. In iterative way, the fragments of the 25% top ranked
structures from virtual screening were focused for further design
(Fig. 1).
The results from virtual screening using Library A and B led to
preparation of Library C. There are 393 molecules in Library A with
131 substituents which lead to 513 conformations in total. In Li-
brary B, there are 658 molecules with 396 substituents which lead
to 1187 conformations in total. In the 25% top ranked structures
from virtual screening of Library A (126 structures), there were
Figure 2. Model of the generated pharmacophore. HBA, hydrogen bond acceptor;
HBD, hydrogen bond donor.

N. Kammasud et al. / Bioorg. Med. Chem. Lett. 19 (2009) 745–750
747
Table 1
Virtual hits and effect on the proliferation of HUVEC after the rhFGF-2 stimulation
O
OH
N
R
H
O
N
H
Compound
SU6668
R
Docking energy (kcal/mol)
Min effective
10.0
H
ꢀ10.2
O
O
H
N
1
ꢀ11.7
50.0
N
H
Cl
H
N
NP506
ꢀ13.0
ꢀ12.7
ꢀ11.6
10.0
>50.0
50
N
H
O
H
N
3
4
N
H
NC
O
H
N
N
H
NO2
O
H
N
5
ꢀ13.1
50
N
H
F3C
Figure 3. The binding modes of the FGFR1 kinase and NP506 (green) showing
hydrogen bonds (green spheres) (a) and hydrophobic pocket (b).
The oxindone scaffold in NP506 structure makes three hydro-
gen bonds to the protein backbone of FGFR1: between N-1-oxin-
dole and the carbonyl oxygen of Glu562, between carbonyl
oxygen of the oxindole and the amide nitrogen of Ala564, and be-
tween N-1 of the pyrrole and the carbonyl oxygen of Ala564. These
same two residues Glu562 and Ala564, located in the hinge region
of FGFR1, are involved in hydrogen bonds to N-1 and N-6 in the
adenine ring of ATP. The phenyl hydrazide substituted at position
5 locates into the hydrophobic pocket and forms two additional
H-bonding interactions. The first is between carbonyl oxygen next
to hydrazine and the NH2 of Lys514, and the second involves the
hydrazide NH (CONHNHPh) and carbonyl oxygen of Asp641, van
der Waals contacts are made between the phenyl group and the
side chains of Lys514, Ile545, Ala640, and Phe642 (Fig. 3b). The
docking studies confirmed that the added substituent locates in
the pocket and contributes to the hydrophobic type and hydrogen
bonding interactions as expected.
CH(COOCH3)2
CH2COOH
c
F
a
b
O
N
NO2
NO2
NO2
H
[6]
[7]
[8]
d
O
H
N
C
HOOC
f
ClH2COC
N
O
O
H
e
O
N
N
N
H
H
H
[11]
[9]
[10]
Scheme 1. Synthesis of 5-substituted indolinone. Reagents and conditions: (a)
CH2(COOCH3)2, NaH, THF, 100 °C, 3 h; (b) 6 N HClaq, overnight; (c) Pd/C, H2, HOAc,
rt, 4 h; (d) AlCl3, CH2Cl2, 0 °C, ClCH2COCl, 15 min, 45 °C, 2 h; (e) 1—pyridine 70–
80 °C, 3 h; 2—2.5 N NaOH 70–80 °C, 3 h; 3—0.5 N HClaq, pH 2; (f) EDCI, HOBT, Et3N,
NH2NHPh, DMF, rt, overnight.
O
O
O
O
O
O
O
O
As NP506 demonstrated good binding with FGFR1 in silico, it
was synthesized along with related compounds (1, 3–5) and
SU6668 and evaluated in vitro for their kinase inhibition activity.
The synthesis of NP5064 was achieved by the synthetic Schemes
1 and 2.
O
O
O
c
a
b
N
H
N
H
N
N
O
HO
H
H
H
Bzl
O
d
OH
The 5-substituted indolin-2-one 11 was prepared in six steps
(260 mg, 38%), involving: reaction of dimethyl malonate (3 g,
23 mmol) and 1-fluoro-2-nitro-benzene (1.63 g, 11.6 mmol) to
give 2-(2-nitro-phenyl)-malonic acid 6 (1.71 g, 66%), hydrolytic
decarboxylation using 6 N aqueous hydrochloric acid to give (2-ni-
tro-phenyl)-acetic acid 7 (1.4 g, 82%), reduction of nitro group
using Pd-C as catalyst and cyclization to give indolin-2-one 8
(0.65 g, 70%), addition of chloroacetyl chloride (0.15 mol, 11.9 ml)
to give 9, followed by nucleophilic substitution of 9 using 2.5 N
NaOH as nucleophile to give 2-oxo-2,3-dihydro-1H-indole-5-car-
boxylic acid 10 (5.05 g, 38%), and finally, coupling 10 with phen-
ylhydrazine hydrochloride (368 mg, 2.56 mmol) to give 11
(260 mg, 38%) (Scheme 1). NP506 (84 mg, 77%) was obtained by
condensation of 5-substituted indolin-2-one 11 (67 mg,
O
OH
O
C
H
N
N
H
[11]
N
O
O
H
N
N
H
e
H
H
NP506
[12]
Scheme 2. Synthesis of NP506. Reagents and conditions: (a) Pd/C, H2, EtOH, rt, 2 h;
(b) TFA, reflux, 1 h; (c) TFA, trimethyl orthoformate, reflux, 1 h; (d) NaOHaq, reflux,
2 h; (e) 5-substituted indolin-2-one, piperidine 11, EtOH, reflux, 3 h, then 2 N
HClaq.
0.25 mmol) with pyrrole aldehyde 12 (49 mg, 0.25 mmol) which
was prepared from methyl-5-(benzyloxycarbonyl) 2,4-dimethyl-
3-pyrrole propionate as shown (Scheme 2). The structures of syn-

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N. Kammasud et al. / Bioorg. Med. Chem. Lett. 19 (2009) 745–750
thesized compound were characterized by their melting points,
FTIR, 1H NMR, 13C NMR, MS, and elemental analyses of C, H, and
N. Spectral data (FTIR, NMR, and MS) were compatible with the as-
signed structures in all cases.
The in vitro inhibition of human FGFR1 kinase activity (ex-
pressed in insect cells) by NP506 was quantified by measuring
the phosphorylation of the substrate biotinyl-bAbAbAAAEEYFFL-
FAKKK and the HTRF detection method.2 The IC50 against FGFR1
was found to be 0.1 lM, slightly better than NP603. FGF-2 is
known to be one of the potent mitogens of vascular endothelial
cells and to play an important role in the tumor-induced angiogen-
esis. Therefore, we determined the effect of NP506 and related
compounds on the proliferation of human umbilical vein endothe-
lial cells (HUVEC) after the recombinant human FGF-2 (rhFGF-2)
treatment. All the tested compounds inhibited the rhFGF-2-in-
duced cellular proliferation of HUVEC. NP506 has an inhibitory
equipotent to SU6668. The minimum effective concentration of
NP506 is 10
lM (Table 1). The minimum cytotoxic concentration
of NP506 in the absence of rhFGF-2 was found to be 50
lM, which
is five times greater than the minimum effective dose. Compounds
3–5 with the minimum effective anti-proliferation concentrations
of 50 lM were considered inactive as the concentrations were
Figure 4. The binding modes of NP506 (green) and NP603 (magenta) with FGFR1
kinase (a) and VEGFR2 in different views (b).
close to their cytotoxic concentrations.
Selectivity of NP506 against other kinases such as VEGF,5
PDGF,6 and EGFR7 were performed and the results are summarized
in Table 2. NP503 exhibited good potency against 3 RTKs, FGFR1,
VEGFR2, and PDGFRb with different profile from SU6668 and
NP603. The inhibition of PDGF signaling attributed to SU66686
oxindole scaffold while the inhibition of FGFR1 and VEGFR2 signal-
ing were from the hydrophobic interaction of the phenylhydrazide
motif at the receptor pocket in the same manner as 3,5-dimethoxy-
phenyl moiety in NP603 and PD173074 structures. Despite of two
H-bonding with the same amino acid residues (Asp641 and
Lys514), the phenylhydrazide motif in NP506 structure locates
deeper in the pocket and confers the pi–pi interaction with
Phe642 (Fig. 4a), leading to 5-fold increase in potency towards
FGFR1.
rhFGF-2 treatment were found to be comparable which is due to
the less cell accessibility of NP506, logP value of 2.292 versus
3.39 of NP603. Furthermore, NP506 showed moderate inhibition
against EGFR.
The effect on the intracellular signaling in HUVEC after the
rhFGF-2 treatment was further investigated by Western blot anal-
ysis. The serine/threonine kinase AKT or PKB, extracellular signal-
regulated kinase (ERK) and c-Jun-N-terminal-kinase (JNK) play an
important role in angiogenesis process for tumor growth and
metastasis. Intracellular signaling cascade is the result of activation
of AKT, ERK, and JNK which are the growth stimulating mediators.
Phosphorylation of these mediators is necessary for tumor growth
and metastasis. The growth factor induced activation of these
mediators after the phosphorylation in FGF receptors. AKT has
been implicated as anti-apoptoic mediator and its activation in-
volved in endothelial cell survival and cell migration toward vari-
ous matrix proteins.9,10 Furthermore, resistance to radiation has
been linked to the phosphatidyl inositol 30-kinase (PI13K)/AKT sig-
naling pathway.11 It is increasingly evident that AKT is central to
regulation of multiple pathways in endothelial cells and fibro-
blasts. While activation of ERK and JNK in mitogen-activated pro-
tein kinase (MAPK) pathway has been associated in growth factor
induced cell proliferation and cell migration.10,12,13 The result from
Western blot analysis demonstrated that the NP506 treatment
caused reduced phosphorylation of growth factor stimulating sig-
naling mediators: AKT, ERK, and JNK in HUVEC cells. The level of
When docking both compounds with VEGFR2 (PDB:2OH4),8 the
NP603 structure was found to align in opposite direction, the car-
boxylic acid in position ꢀ3 of the pyrrole locates in the receptor
pocket instead of the 3,5-dimethoxyphenyl group. It is because
VEGFR2 contains larger Cys917 and Val914 residues corresponding
to Ala640 and Ile545 residues of FGFR1,3 respectively, and conse-
quently make the gate of the pocket too shallow to accommodate
the bulky dimethoxyphenyl group (Fig. 4b). The increase in po-
tency of NP506 to nM level against PDGFRb was possibly due to
additional interaction provided by cysteine residues of PDGFRb
corresponding to Ala640 and Ala564 of FGFR1. The effect of
NP506 and NP603 in inhibiting the proliferation of HUVEC after
Table 2
Inhibition of tyrosine kinase activity and HUVEC proliferation after the rhFGF-2 stimulation
O
OH
N
H
O
R
N
H
Inhibition of tyrosine kinase activity (IC50, lM)
ID
R
FGFR1
VEGFR2
PDGFRb
EGFR
Inhibition of cell proliferation (IC50, lM)
SU6668
NP506
NP603
H
1.81
0.10
0.52
0.68
0.07
0.93
0.05
0.04
0.78
>100
3.48
>100
>50
27.4
18.2
5-(CO-NHNHphenyl)
6-(3,5-diOCH3phenyl)

N. Kammasud et al. / Bioorg. Med. Chem. Lett. 19 (2009) 745–750
749
phosphorylated AKT and JNK (pAKT and pJNK) were inhibited com-
pletely by NP506 after FGF-2 stimulation while NP603 and SU6668
reduced the phosphorylation in the lesser extent. The decrease in
phosphorylation of ERK by NP506 was more pronounced than
SU6668 but in the lesser degree than NP603 (Fig. 5). Compared
to SU6668, NP506 showed a stronger inhibitory effect of AKT,
ERK, and JNK activation. The changing in position of the substitu-
tion from position 6 to position 5 resulted in inhibition of multiple
signaling pathways implicated in cancer and cancer metastasis via
angiogenesis. In addition to inhibition of MAPK signaling pathway,
NP506 was able to inhibit (PI13K)/AKT signaling pathway.
These results indicate that NP506 has potent inhibitory activity
on the FGF-2-induced tyrosine phosphorylation of 3 TK receptors,
which resulted in the inhibition of ERK and JNK activation in MAPK
pathway and AKT activation in (PI13K)/AKT pathway, leading to
cellular proliferation in HUVEC.
To evaluate the effect of NP506 on angiogenesis, the HUVEC
tube formation assay was performed in which the lengths of the
capillary-like tubes formed were measured. NP506 suppressed HU-
VEC tubes formation in a dose dependent manner, in the presence
of NP506, HUVECs formed incomplete and narrow tube-like struc-
tures (Fig. 6). In contrast, following stimulation with FGF-2, forma-
tion of elongated and robust tube-like structures was observed
which were organized by a greater number of cells as compared
FGF-2
control
1 μM
25 μM
3 μM
10 μM
120
100
80
60
40
20
0
NP 506
∗
∗
∗∗
0
1
3
10
25
Concentration, μM
to the control. Treatment with 1, 3, 10, and 25 lM of NP506 re-
sulted in the inhibition of tube formation as compared with the
control group. NP506 significantly inhibited tube formation in a
concentration-dependent manner.
It was apparent that NP506, NP603, and SU6668 had compara-
ble effect on angiogenesis according to the HUVEC tube formations
assay. However, the profile of inhibition in signaling pathways are
different. When comparing to SU6668, the effect of NP506 on angi-
Figure 6. Effect of NP506 on the tube-like formations of HUVEC. HUVEC were
seeded onto 96-well plates, which had been coated with 40 l of Matrigel per well,
in the presence of NP506. After a 4-h incubation period at 37 °C, the culture was
fixed by glutaraldehyde, and the length of the tube-like formations was evaluated.
Original magnification: 5ꢁ. A representative experiment is shown, with means + SD
of four wells. *P < 0.05, **P < 0.01.
l
ogenesis appeared to be comparable despite the greater inhibition
against tyrosine kinase and HUVEC proliferation after the rhFGF-2
stimulation. Hence, the contribution from other factors, that is,
ADME might govern the same efficacy observed.
The introduction of the phenyl hydrazide motif at position 5 of
the pyrido[2,3-d]pyrimidine scaffold has led to the inhibition of
AKT activation in the phosphatidyl inositol 30-kinase (PI13K)/AKT
signaling pathway and the inhibition of ERK and JNK activation
in MAPK pathway. The (PI13K)/AKT and MAPK pathway are the
main signal transduction pathways implicated in cancer and can-
cer metastasis via angiogenesis.
Acknowledgments
This work was funded by the Royal Golden Jubilee Project,
Thailand Research Fund, The Commission of Higher Education,
Ministry of Education, Thailand and the 21st Century COE project
from the Ministry of Education, Culture, Sports, Science and
Technology, Japan.
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acid as a mustard yellow solid: mp 232–234 °C; FTIR (KBr) (cmꢀ1): 3308 (NꢀH
st.), 3101 (aromatic CꢀH st.), 2935 (aliphatic CꢀH st.), 1759, 1632 (C@O st.),
1547 (C@C st.), 1437 (bending C–H), 1146 (C–O st.); 1HNMR (300 MHz, DMSO-
d6) d 13.35 (s, 1H, NH), 10.08 (s, 1H, NH), 8.29 (s, 1H, H4), 7.78 (s, 1H, NH), 7.77
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 JNK, phospho-specific JNK, ERK1/2, phospho-specific ERK, AKT, and phopho-
specific AKT.

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N. Kammasud et al. / Bioorg. Med. Chem. Lett. 19 (2009) 745–750
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