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.
References and notes
1. Kammasud, N.; Boonyarat, C.; Tsunoda, S.; Sakurai, H.; Saiki, I.; Grierson, D. S.;
Vajragupta, O. Bioorg. Med. Chem. Lett. 2007, 17, 4812.
2. Mohammadi, M.; Froum, S.; Hamby, J. M.; Schroeder, M. C.; Panek, R. L.; Lu, G.
H.; Dliseenkova, A. V.; Green, D.; Schlessinger, J.; Hubbard, S. R. EMBO J. 1998,
17, 5896.
3. Mohammadi, M.; McMahon, G.; Sun, L.; Tang, C.; Hirth, P.; Yeh, B. K.; Hubbard,
S. R.; Schlessinger, J. Science 1997, 276, 955.
4. Melting point, IR, 1H, and 13C NMR, mass, and elemental analysis (C, H, and N)
determination. For NP506, 3-{2,4-dimethyl-5-[2-oxo-5-(N0-phenyl hydra-
zinocarbonyl)-1,2-dihydro-indol-3-ylidene-methyl]-1H-pyrrol-3-yl}-propionic
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.