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2. (a) Daly, C.; Holash, J. Handbook of Cell Signaling, Vol. 2,
domain of human Tie-2, VEGFR2, or EphB4 fused by
GST and/or 6·His tags. In the case of VEGFR2, the
enzyme contains both GST and 6·His tags. The catalytic
activity of each kinase was detected by using a biotinylated
p 849; (b) Bilodeau, Mark T.; Fraley, Mark E.; Hartman,
George D. Expert Opin. Investig. Drugs 2002, 11, 737; (c)
Boyer, S. J. Curr. Top. Med. Chem. 2002, 2, 973.
3. (a) Cheng, N.; Brantley, D. M.; Chen, J. Cytokine Growth
Factor Rev. 2002, 13, 75; (b) Steinle, J. J.; Meininger, C. J.;
Forough, R.; Wu, G.; Wu, M. H.; Granger, H. J. J. Biol.
Chem. 2002, 277, 43830; There is recent report indicating
that ephrin-B2 mediated-activation of EphB4 inhibits
MDA-MB-435 cancer cell proliferation. (c) Noren, N.
K.; Lu, M.; Freeman, A. L.; Koolpe, M.; Pasquale, E. B.
Proc. Natl Acad. Sci. U.S.A. 2004, 101, 5583.
synthetic peptide as
RLVAYEGWVAGKKK-amide,
a
substrate, biotin-C6-LEA-
biotin-aminohexyl-
EEEEYFELVAKKKK-NH2, and biotin-aminohexyl-
MAHFENYEFFHAKKK-NH2 for Tie-2, VEGFR2,
and EphB4, respectively. Phosphorylated substrate is
measured by streptavidin-linked-APC (Molecular Probes)
and europium-labeled anti-phosphorylated tyrosine anti-
body (Perkin-Elmer) or by streptavidin-coated SPA beads
(Amersham-Pharmacia). Assay conditions are as follows.
Tie-2: GST-Tie-2 was preactivated with 800 lM ATP,
1 mM DTT, 0.1 mg/mL BSA, 10 mM MgCl2, 0.01%
Tween 20 in 100 mM HEPES, pH 7.5, for 1–2 h. The
preactivated enzyme was then incubated for 1–3 h with
1 lM peptide, 20 lM ATP, 5 mM MgCl2, 1 mM DTT,
0.1 mg/mL BSA, 0.01% Tween 20, and test compound in
100 mM HEPES, pH 7.4. VEGFR2: GST-6·His-VEG-
FR2 was preactivated with 100 lM ATP, 0.3 mM DTT,
0.1 mg/mL BSA, and 10 mM MgCl2 in 100 mM HEPES,
pH 7.5, for 20 min. The preactivated enzyme was then
incubated for 90 min with 360 nM peptide, 50 lM ATP,
10 mM MgCl2, 0.3 mM DTT, 0.1 mg/ml BSA, and test
compound in 100 mM HEPES, pH 7.5. EphB4: GST-
EphB4 was preactivated with 50 lM ATP and 10 mM
MgCl2 and was then incubated for 3 h with 8 lM peptide,
1 lM ATP, 5 lCi/mL 33P-ATP, 10 mM MgCl2, 1 mM
DTT, 5 mM KCl, 1 mM CHAPS, 0.1 mg/mL BSA, and
test compound in 100 mM HEPES.
4. Bevacizumab(Avastinꢂ): (a) Hurwitz, H. I.; Fehrenbacher,
L.; Hainsworth, J. D.; Heim, W.; Berlin, J.; Holmgren, E.;
Hambleton, J.; Novotny, W. F.; Kabbinavar, F. J. Clin.
Oncol. 2005, 23, 3502; BAY 43-9006: (b) 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; PTK787/
ZK222584: (c) Wood, J. M.; Bold, G.; Buchdunger, E.;
Cozens, R.; Ferrari, S.; Frei, J.; Hofmann, F.; Mestan, J.;
Mett, H.; O’Reilly, T.; Persohn, E.; Ro¨sel, J.; Schnell, C.;
Stover, D.; Theuer, A.; Towbin, H.; Wenger, F.; Woods-
Cook, K.; Menrad, A.; Siemeister, G.; Schirner, M.;
Thierauch, K.-H.; Schneider, M. R.; Drevs, J.; Martiny-
´
Baron, G.; Totzke, F.; Marme, D. Cancer Res. 2000, 60,
2178; See recent review: (d) Zakarija, A.; Soff, G. Curr.
Opin. Oncol. 2005, 17, 578.
5. Traxler, P.; Furet, P. Pharmacol. Ther. 1999, 82, 195.
6. Miyazaki, Y.; Matsunaga, S.; Tang, J.; Maeda, Y.; Nakano,
M.; Philippe, R. J.; Shibahara, M.; Liu, W.; Sato, H.; Wang,
L.; Nolte, R. T. Bioorg. Med. Chem. Lett. 2005, 15, 2203.
7. Miyazaki, Y. PCT Application, WO04100947, 2004.
Analytical data of compound 9f: 1H NMR (400 MHz,
DMSO-d6) ppm 8.12(t, J = 1.8 Hz, 1H), 8.0 (s, 1H), 7.91
(dt, J = 1.5, 7.6 Hz, 1H), 7.85 (dt, J = 1.4, 7.1 Hz, 1H),
7.75 (t, J = 7.8 Hz, 1H), 7.67 (s, 1H), 7.57 (t, J = 9.0 Hz,
1H), 7.51 (ddd, J = 2.2, 4.9, 8.5 Hz, 1H), 7.45 (s, 2H), 5.66
(s, 2H). MS: m/z 434 (M+1)+, 432 (MÀ1)À.
11. EphB4 autophosphorylation at the cellular level was
determined by ELISA using recombinant NIH3T3 cells
(cFms-EphB4) with the stable expression of the chimeric
receptors of cFms-derived extracellular domain and
EphB4-derived intracellular domain. The test compound
was incubated with cFms-EphB4 for 1 h after serum
starvation. The cells were stimulated with a specific ligand,
M-CSF (macrophage colony stimulating factor), to induce
the activation of the cFms-EphB4 receptors. The phos-
phorylation level of the receptors was measured by the
incubation of the ELISA plate capturing the protein via
anti-cFms antibody with anti-phosphotyrosine antibody
and visualized by incubating with chemiluminescent
substrates.
8. New, J. S.; Christopher, W. L.; Yevich, J. P.; Butler, R.;
Schlemmer, R. F.; VanderMaelen, C. P.; Cipollina, J. A.
J. Med. Chem. 1989, 32, 1147.
9. Frye, S. V. Chem. Biol. 1999, 6, R3.
12. Compounds 9f and 15c showed relatively weak inhibitory
activity (IC50 > 10 lM) against following targets. Akt3,
CDK2, ErbB2, GSK3b, IKK2, MAPKAPK2, and PLK1.
IC50 of 9f against VEGFR2 and Tie-2 is 1.9 lM and over
20 lM, respectively.
13. Compound 15c showed comparable potency against
VEGFR2 and Tie-2 against 4-amino-3-(4-((2-fluoro-5-
(trifluoromethyl)phenyl)amino-carbonylamino)phenyl)-2-
(4-methoxyphenyl)furo[2,3-d]pyrimidine, which was pub-
lished in our previous report (see Ref. 6). It should be
noted that assay condition was slightly modified from the
previous one.
10. For Tie-2 and VEGFR2, the enzyme assay was performed
by the HTRF (Homogeneous time-resolved fluorescence)
method using baculovirus-expressed recombinant protein.
HTRF is based on the proximity of a donor label
(europium chelate) and acceptor label (allophycocyanin,
APC) which have been brought together by a specific
binding reaction. When the two entities come into close
proximity and upon excitation, energy transfer occurs and
APC emits a specific long-lived fluorescence at 665 nm.
For EphB4, the enzyme assay used the scintillation
proximity method where the localization of a radiolabeled
phosphate near scintillant-containing beads generates a
signal. The kinases were purified as the intracellular
14. Bromination of compound 13 occurred at 2-position as
well as 7-position, resulting in low yield.