VEGFR-2 Tyrosine Kinase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5 1365
reported.30 All assays were carried out in quadruplicate with
1-10 µM ATP.
(9) Strawn, L. M.; McMahon, G.; App, H.; Schreck, R.; Kuchler, W.
R.; Longhi, M. P.; Hui, T. H.; Tang, C.; Levitzki, A.; Gazit, A.;
Chen, I.; Keri, G.; Orfi, L.; Risau, W.; Flamme, I.; Ullrich, A.;
Hirth, K. P.; Shawver, L. K. Flk-1 as a Target for Tumor Growth
Inhibition. Cancer Res. 1996, 56, 3540-3545.
(10) Millauer, B.; Longhi, M. P.; Plate, K. H.; Shawver, L. K.; Risau,
W.; Ullrich, A.; Strawn, L. M. Dominant-negative inhibition of
Flk-1 suppresses the growth of many tumor types in vivo. Cancer
Res., 1996, 56, 1615-1620.
(11) Kim, K. J.; Li, B.; Winer, J.; et al. Inhibition of vascular endo-
thelial growth factor-induced angiogenesis suppresses tumor
growth in vivo. Nature 1993, 362, 841-844.
(12) Prewett, M.; Huber, J.; Li, Y.; et al. Antivascular endothelial
growth factor receptor (fetal liver kinase 1) monoclonal antibody
inhibits tumor angiogenesis and growth of several mouse and
human tumors. Cancer Res. 1999, 59, 5209-5218.
(13) Dumas, J. Protein kinase inhibitors: emerging pharmacophores
1997-2000. Exp. Opin. Ther. Pat. 2001, 11, 405-429.
(14) Connell, R. D.; Beebe, J. S. Patent focus on cancer chemothera-
peutics. II Angiogenesis agents: April 2000-September 2000.
Exp. Opin. Ther. Pat. 2001, 11, 77-114.
(15) Sun, L.; Tran, N.; Tang, F.; App, H.; Hirth, P.; McMahon, G.;
Tang, C. Synthesis and Biological Evaluations of 3-Substituted
Indolin-2-ones: A Novel Class of Tyrosine Kinase Inhibitors that
Exhibit Selectivity toward Particular Receptor Tyrosine Kinases.
J. Med. Chem. 1998, 41, 2588-2603.
(16) Fong, T. A. T.; Shawver, L. K.; Sun, L.; Tang, C.; App, H.; Powell,
T. J.; Kim, Y. H.; Schreck, R.; Wang, X.; Risau, W.; Ullrich, A.;
Hirth, K. P.; McMahon, G. SU5416 Is a Potent and Selective
Inhibitor of the Vascular Endothelial Growth Factor Receptor
(Flk-1/KDR) That Inhibits Tyrosine Kinase Catalysis, Tumor
Vascularization, and Growth of Multiple Tumor Types. Cancer
Res. 1999, 59, 99-106.
(17) Sun, L.; Tran, N.; Liang, C.; Tang, F.; Rice, A.; Schreck, R.;
Waltz, K.; Shawver, L. K.; McMahon, G.; Tang, C. Design,
synthesis and evaluation of substituted 3-[(3- or 4-carboxy-
ethylpyrrol-2-yl)methylidenyl]indolin-2-ones as inhibitors of
VEGF, FGF, and PDGF receptor tyrosine kinases. J. Med. Chem.
1999, 42, 5120-5130.
Inhibition of VEGF-Stimulated HUVEC Proliferation.
HUVECs were placed at a density of 4000 cells/200 µL/well
on collagene type I precoated 96-well plates in M199 medium
with 5% fetal bovine serum (FBS). After 24 h, the cells were
incubated for 1 h in the presence or absence of 6ab; then the
cells were stimulated by rhVEGF (20 ng/mL). The cultures
were incubated at 37 °C for 72 h, then pulsed with 1 µCi/well
[3H]thymidine and reincubated for 14 h. Cells were assayed
for the incorporation of tritium using a beta counter.
Antitumor Assays Using Human Tumor Xenografts in
Nude Mice. Human glioma (GLO7) was transplanted into
nude mice. When the tumor grew to a volume of about 100
mm3, the nude mice were divided into several groups of four
animals so as to equalize the average tumor volumes of each
group. The compounds were administered orally to mice in the
experimental groups once a day for 9 consecutive days at 20
mg/kg, and the vehicle was administered to control animals.
Tumor volumes were monitored twice weekly by measuring
the perpendicular diameters (taking the length to be the
longest diameter (L), the width to be the shortest diameter
(W) and the height (H) to be the longest perpendicular
diameter) and calculated using the formula (L × W × H)/2.
The tumor growth inhibition (TGI) was calculated as follows:
TGI(%) ) 1 - (RTV of the treated group at the day of
measurement)/(RTV of control-group at the day of measure-
ment) × 100. RTV ) (Tumor volume at the day of measure-
ment)/(Tumor volume at the initial day)
To determine the effects of 6ab on tumor growth, its effects
were tested against various tumors, human stomach carci-
noma (St-4), human lung carcinoma (LC-6), human colon
carcinoma (DLD-1) and human melanoma (A375), using hu-
man tumor xenografts in nude mice.
Antitumor Assay Using Human Xenograft in Nude
Rats. Human lung carcinoma (LC-6) was transplanted into
nude rats. When the tumor grew to a volume of about 700
mm3, the nude rats were divided into several groups of three
animals so as to equalize the average tumor volumes of each
group. Body weights were also measured twice weekly at the
same time of measurement of the tumor volume.
(18) Laird, A. D.; Vajkoczy, P.; Shawver, L. K.; Thurnher, A.; Liang,
C.; Mohammadi, M.; Schlessinger, J.; Ullrich, A.; Hubbard, S.
R.; Blake, R. A.; Fong, T. A. T.; Strawn, L. M.; Sun, L.; Tang,
C.; Hawtin, R.; Tang, F.; Shenoy, N.; Hirth, K. P.; McMahon,
G.; Cherrington, J. M. SU6668 Is a Potent Antiangiogenic and
Antitumour Agent That Induces Regression of Established
Tumors. Cancer Res. 2000, 60, 4152-4160.
(19) Hennequin, L. F.; Thomas, A. P.; Johnstone, C.; Stokes, E. S.
E.; Ple, P.; Lohmann, J.-J. M.; Ogilvie, D. J.; Dukes, M.; Wedge,
S. R.; Curwen, J. O.; Kendrew, J.; Lambert-van-der-Brempt, C.
Design and Structure-Activity Relationship of a New Class of
Potent VEGF Receptor Tyrosine Kinase Inhibitors. J. Med.
Chem. 1999, 42, 5369-5389.
Acknowledgment. We thank Prof. Masabumi
Shibuya (Division of Genetics, Institute of Medical
Science, University of Tokyo, Japan) for useful sugges-
tions. We also thank Yasunobu Murakami, Hiroaki
Ohno and Tetsuya Yoshino for their technical support.
(20) Wedge, S. R.; Ogilvie, D. J.; Dukes, M.; Kendrew, J.; Curwen,
J. O.; Hennequin, L. F.; Thomas, A. P.; Stokes, E. S. E.; Curry,
B.; Richmond, G. H.; Wadsworth, P. F. Cancer Res. 2000, 60,
970-975.
(21) Hennequin, L. F.; Stokes, E. S. E.; Thomas, A. P.; Johnstone,
C.; E.; Ple, P.; Ogilvie, D. J.; Dukes, M.; Wedge, S. R.; Kendrew,
J.; Curwen, J. O. Novel 4-Anilinoquinazolines with C-7 Basic
Side Chains: Design and Structure-activity Relationship of a
Series of Potent, Orally Active, VEGF Receptor TyrosineKinase
Inhibitors. J. Med. Chem. 2002, 45, 1300-1312.
(22) Wedge, S. R.; Ogilvie, D. J.; Dukes, M.; Kendrew, J.; Chester,
R.; Jackson, J. A.; Boffey, S. J.; Valentine, P. J.; Curwen, J. O.;
Musgrove, H. L.; Graham, G. A.; Hughes, G. D.; Thomas, A. P.;
Stokes, E. S. E.; Curry, B.; Richmond, G. H. P.; Wadsworth, P.
F.; Bigley, A. L.; Hennequin, L. F. Cancer Res. 2002, 62, 4645-
4655.
(23) Bold, G.; Altmann, K.-H.; Frei, J.; Lang, M.; Manley, P. W.;
Traxler, P.; Wietfeld, B.; Bruggen, J.; Buchdunger, E.; Cozens,
R.; Ferrari, S.; Furet, P.; Hofmann, F.; Martiny-Baron, G.;
Mestan, J.; Rosel, J.; Sills, M.; Stover, D.; Acemoglu, F.; Boss,
E.; Emmenegger, R.; Lasser, L.; Masso, E.; Roth, R.; Schlachter,
C.; Vetterli, W.; Wyss, D.; Wood, J. M. New Anilinophthalazines
as Potent and Orally well Absorbed Inhibitors of the VEGF
Receptor Tyrosine Kinases Useful as Antagonists of Tumor-
Driven Angiogenesis. J. Med. Chem. 2000, 43, 2310-2323.
(24) Wood, J. M.; Bold, G.; Buchdunger, E.; Cozens, R.; Ferrari, S.;
Frei, J.; Hofmann, F.; Mestan, J.; Mett, H.; O’Reilly, T.; Persohn,
E.; Rosel, 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,
2178-2189.
Supporting Information Available: Analysis data not
included in the Experimental Section and a table of elemental
analyses data. This material is available free of charge via the
Internet at http//pubs.acs.org.
References
(1) Folkman, J. Anti-Angiogenesis: New Concept for Therapy of
Solid Tumors. Ann. Surg. 1972, 175, 409-416.
(2) Liotta, L. A.; Kleinerman, J.; Saidel, G. M. Quantitative Rela-
tionships of Intravascular Tumor Cells, Tumor Vessels, and
Pulmonary Metastases following Tumor Implantation. Cancer
Res. 1974, 34, 997-1004.
(3) Eckhardt, S. G. Angiogenesis Inhibitors as Cancer Therapy.
Hosp. Pract. 1999, 63-84.
(4) Bergers, G.; Javaherian, K.; Lo, K.-M.; Folkman, J.; Hanahan,
D. Effects of Angiogenesis Inhibitors on Multistage Carcinogen-
esis in Mice. Science 1999, 284, 808-812.
(5) Fan, T.-P. D.; Jaggar, R.; Bicknell, R. Controlling the vascula-
ture: angiogenesis, anti-angiogenesis and vascular targeting of
gene therapy. Trends Pharmacol. Sci. 1995, 16, 57-66.
(6) Kim, K. J.; Li, B.; Winer, J.; Armanini, M.; Gillett, N.; Phillips,
H. S.; Ferrara, N. Inhibition of vascular endothelial growth
factor-induced angiogenesis suppresses tumour growth in vivo.
Nature 1993, 362, 841-844.
(7) Kolch, W.; Martiny-Baron, G.; Kieser, A.; Marme, D. Regulation
of the expression of VEGF/VPS and its receptors: role in tumor
angiogenesis. Breast Cancer Res. Treat. 1995, 36, 139-155.
(8) Leenders, W. P. J. Targetting VEGF in anti-angiogenic and anti-
tumour therapy: Where are we now? Int. J. Exp. Pathol. 1998,
79, 339-346.
(25) Fernand, N. H.; Hurwitz H. I.; Inhibition of vascular endothelial
growth factor in the treatment of colorectal cancer. Semin. Oncol.
2003, 3 Suppl 6, 39-50.