Y. Miyazaki et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1773–1778
1777
11. Analytical data of compound 8a: 1H NMR (400 MHz,
DMSO-d6) ppm 7.39–7.42 (m, 1H), 7.47 (d, J = 8.6 Hz,
2H), 7.49–7.54 (m, 1H), 7.63 (d, J = 8.8 Hz, 2H), 7.95
(s, 1H), 8.26 (s, 1H), 8.63 (d, J = 7.1 Hz, 1H), 8.99 (s,
1H), 9.39 (s, 1H). LC/MS: m/z 432 (M + 1)+, 430
(M À 1)À.
for angiogenesis. The profile data of compound 8a are
shown in Table 4.18
In conclusion, as a part of our research for anti-angio-
genic agents for cancer therapy, we synthesized 4-ami-
no-furo[2,3-d]pyrimidine derivatives, and evaluated
their activities as VEGFR2 and Tie-2 kinase inhibitors.
Compound 8a exhibited not only potency in both en-
zyme and cellular assays, but also exhibited anti-angio-
genic and anti-tumor activity in HT-29 tumor
xenograft models. These results indicate that com-
pounds of furopyrimidines bearing diarylurea moieties
potentially possess promising features as drug candi-
dates for anti-angiogenic cancer therapy.
12. 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. The kinases were purified as the intracellular
domain of human Tie-2 or VEGFR2 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 synthetic peptide as a substrate, biotin-
C6-LEARLVAYEGWVAGKKK-amide, and biotin-
aminohexyl-EEEEYFELVAKKKK-NH2, for Tie-2 and
VEGFR2, respectively. Phosphorylated substrate is
measured by streptavidin linked-APC (Molecular
Probes) and europium-labeled anti-phosphorylated tyro-
sine antibody (Perkin-Elmer). 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-VEGFR2 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.
Acknowledgments
We acknowledge Michael Hansbury and Roberta Bator-
sky for their assistance with the Tie-2 autophosphoryla-
tion results and Ron Wegrzyn, Hieu Do, Kevin
Kershner, and Jessica Ward for their contribution to
the HFF and HT-29 proliferation data. We acknowl-
edge CVU Preclinical Drug Discovery DMPK for mea-
surement of systemic exposure, Screening & Compound
Profiling for kinase panel assays. The authors thank Jo-
seph H. Chan, Karen E. Lackey, and Stephen V. Frye
for their guidance and support.
Supplementary data
Supplementary data associated with this article can be
13. Dai, Y.; Guo, Y.; Frey, R. R.; Ji, Z.; Curtin, M. L.;
Ahmed, A. A.; Albert, D. H.; Arnold, L.; Arries, S. S.;
Barlozzari, T.; Bauch, J. L.; Bouska, J. J.; Bousquet, P.
F.; Cunha, G. A.; Glaser, K. B.; Guo, J.; Li, J.;
Marcotte, P. A.; Marsh, K. C.; Moskey, M. D.; Pease,
L. J.; Stewart, K. D.; Stoll, V. S.; Tapang, P.; Wishart,
N.; Davidsen, S. K.; Michaelides, M. R. J. Med. Chem.
2005, 48, 6066.
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