Note: Descriptions are shown in the official language in which they were submitted.
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Synthetic metabolites of Fluoro substituted Omega-Carboxyaryl Diphenyl Urea
for the
Treatment and Prevention of Diseases and Conditions
Field of the Invention
This invention relates to novel compounds, pharmaceutical compositions
containing such
compounds and the use of those compounds or compositions for treating diseases
and
conditions mediated by abnormal VEGFR, PDGFR, raf, p38, and/or flt-3 kinase
signaling,
either alone or in combination with anti-cancer agents.
Background of the Invention
Activation of the ras signal transduction pathway indicates a cascade of
events that have a
profound impact on cellular proliferation, differentiation, and
transformation. Raf kinase, a
downstream effector of ras, is recognized as a key mediator of these signals
from cell surface
receptors to the cell nucleus (Lowy, D. R.; Willumsen, B. M. Ann. Rev.
Biochem. 1993, 62,
851; Bos, J. L. Cancer Res. 1989, 49, 4682). It has been shown that inhibiting
the effect of
active ras by inhibiting the raf kinase signaling pathway by administration of
deactivating
antibodies to raf kinase or by co-expression of dominant negative raf kinase
or dominant
negative MEK, the substrate of raf kinase, leads to the reversion of
transformed cells to the
normal growth phenotype (see: Daum et al. Trends Biochem. Sci. 1994, 19, 474-
80; Fridman
et al. J. Biol. Chem. 1994, 269, 30105-8. Kolch et al. (Nature 1991, 349, 426-
28) have
further indicated that inhibition of raf expression by antisense RNA blocks
cell proliferation
in membrane-associated oncogenes. Similarly, inhibition of raf kinase (by
antisense
oligodeoxynucleotides) has been correlated in vitro and in vivo with
inhibition of the growth
of a variety of human tumor types (Monia et al., Nat. Med. 1996, 2, 668-75).
To support progressive tumor growth beyond the size of 1-2 mm3, it is
recognized that tumor
cells require a functional stroma, a support structure consisting of
fibroblast, smooth muscle
cells, endothelial cells, extracellular matrix proteins, and soluble factors
(Folkman, J., Semin.
Oncol. 2002. 29(6 Suppl 16), 15-8). Tumors induce the formation of stromal
tissues through
the secretion of soluble growth factors such as PDGF and transforming growth
factor-beta
(TGF-beta), which in turn stimulate the secretion of complimentary factors by
host cells such
as fibroblast growth factor (FGF), epidermal growth factor (EGF), and vascular
endothelial
growth factor (VEGF). These stimulatory factors induce the formation of new
blood vessels,
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or angiogenesis, which brings oxygen and nutrients to the tumor and allows it
to grow and
provides a route for metastasis. It is believed some therapies directed at
inhibiting stroma
formation will inhibit the growth of epithelial tumors from a wide variety of
histological
types. (George, D. Semin. Oncol. 2001. 28(5 Suppl 17), 27-33; Shaheen, R.M.,
et al., Cancer
Res. 2001, 61(4), 1464-8; Shaheen, R.M., et al. Cancer Res. 1999, 59(21), 5412-
6). However,
because of the complex nature and the multiple growth factors involved in
angiogenesis
process and tumor progression, an agent targeting a single pathway may have
limited
efficacy. It is desirable to provide treatment against a number of key
signaling pathways
utilized by tumors to induce angiogenesis in the host stroma. These include
PDGF, a potent
stimulator of stroma formation (Ostman, A. and C.H. Heldin, Adv. Cancer Res.
2001, 80, 1-
38), FGF, a chemo-attractant and mitogen for fibroblasts and endothelial
cells, and VEGF, a
potent regulator of vascularization.
PDGF is a key regulator of stromal formation, which is secreted by many tumors
in a
paracrine fashion and is believed to promote the growth of fibroblasts, smooth
muscle and
endothelial cells, promoting stroma formation and angiogenesis. PDGF was
originally
identified as the v-sis oncogene product of the simian sarcoma virus (Heldin,
C.H., et al., J.
Cell. Sci. Suppl. 1985, 3, 65-76). The growth factor is made up of two peptide
chains,
referred to as A or B chains which share 60% homology in their primary amino
acid
sequence. The chains are disulfide cross linked to form the 30 kDa mature
protein composed
of either AA, BB or AB homo- or heterodimmers. PDGF is found at high levels in
platelets,
and is expressed by endothelial cells and vascular smooth muscle cells. In
addition, the
production of PDGF is up regulated under low oxygen conditions such as those
found in
poorly vascularized tumor tissue (Kourembanas, S., et al., Kidney Int. 1997,
51(2), 438-43).
PDGF binds with high affinity to the PDGF receptor, a 1106 amino acid 124 kDa
transmembrane tyrosine kinase receptor (Heldin, C.H., A. Ostman, and L.
Ronnstrand,
Biochim. Biophys. Acta 1998, 1378(1), 79-113). PDGFR is found as homo- or
heterodimer
chains which have 30% homology overall in their amino acid sequence and 64%
homology
between their kinase domains (Heldin, C.H., et al.. Embo J. 1988, 7(5), 1387-
93). PDGFR is
a member of a family of tyrosine kinase receptors with split kinase domains
that includes
VEGFR-2 (KDR), V~- i1 R flat-4 ii c-kit, and flt-3. The PDGF receptor is
expressed
primarily on fibroblasts, smooth muscle cells, and pericytes and to a lesser
extent on neurons,
kidney mesangial, Leydig, and Schwann cells of the central nervous system.
Upon binding to
the receptor, PDGF induces receptor dimerization and undergoes auto- and trans-
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phosphorylation of tyrosine residues which increase the receptors' kinase
activity and
promotes the recruitment of downstream effectors through the activation of SH2
protein
binding domains. A number of signaling molecules form complexes with activated
PDGFR
including PI-3-kinase, phospholipase C-gamma, src and GAP (GTPase activating
protein for
p21-ras) (Soskic, V., et al. Biochemistry 1999, 38(6), 1757-64). Through the
activation of PI-
3-kinase, PDGF activates the Rho signaling pathway inducing cell motility and
migration,
and through the activation of GAP, induces mitogenesis through the activation
of p21-ras and
the MAPK signaling pathway.
In adults, it is believed the major function of PDGF is to facilitate and
increase the rate of
wound healing and to maintain blood vessel homeostasis (Baker, E.A. and D.J.
Leaper,
Wound Repair Regen. 2000, 8(5), 392-8, and Yu, J., A. Moon, and H.R. Kim,
Biochem.
Biophys. Res. Commun. 2001, 282(3), 697-700). PDGF is found at high
concentrations in
platelets and is a potent chemoattractant for fibroblast, smooth muscle cells,
neutrophils and
macrophages. In addition to its role in wound healing PDGF is known to help
maintain
vascular homeostasis. During the development of new blood vessels, PDGF
recruits pericytes
and smooth muscle cells that are needed for the structural integrity of the
vessels. PDGF is
thought to play a similar role during tumor neovascularization. As part of its
role in
angiogenesis PDGF controls interstitial fluid pressure, regulating the
permeability of vessels
through its regulation of the interaction between connective tissue cells and
the extracellular
matrix. Inhibiting PDGFR activity can lower interstitial pressure and
facilitate the influx of
cytotoxics into tumors improving the anti-tumor efficacy of these agents
(Pietras, K., et al.
Cancer Res. 2002, 62(19), 5476-84; Pietras, K., et al. Cancer Res. 2001,
61(7), 2929-34).
PDGF can promote tumor growth through either the paracrine or autocrine
stimulation of
PDGFR receptors on stromal cells or tumor cells directly, or through the
amplification of the
receptor or activation of the receptor by recombination. Over expressed PDGF
can transform
human melanoma cells and keratinocytes (Forsberg, K., et al. Proc. Natl. Acad.
Sci. U S A.
1993, 90(2), 393-7; Skobe, M. and N.E. Fusenig, Proc. Natl. Acad. Sci. U S A.
1998, 95(3),
1050-5), two cell types that do not express PDGF receptors, presumably by the
direct effect
of PDGF on stroma formation and induction of angiogenesis. This paracrine
stimulation of
tumor stroma is also observed in carcinomas of the colon, lung, breast, and
prostate
(Bhardwaj, B., et al. Clin. Cancer Res. 1996, 2(4), 773-82; Nakanishi, K., et
al. Mod. Pathol.
1997, 10(4), 341-7; Sundberg, C., et al. Am. J. Pathol. 1997, 151(2), 479-92;
Lindmark, G., et
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al. Lab. Invest. 1993, 69(6), 682-9; Vignaud, J.M., et al, Cancer Res. 1994,
54(20), 5455-63)
where the tumors express PDGF, but not the receptor. The autocrine stimulation
of tumor cell
growth, where a large faction of tumors analyzed express both the ligand PDGF
and the
receptor, has been reported in glioblastomas (Fleming, T.P., et al. Cancer
Res. 1992, 52(16),
4550-3), soft tissue sarcomas (Wang, J., M.D. Coltrera, and A.M. Gown, Cancer
Res. 1994,
54(2), 560-4) and cancers of the ovary (Henriksen, R., et al. Cancer Res.
1993, 53(19), 4550-
4), prostate (Fudge, K., C.Y. Wang, and M.E. Stearns, Mod. Pathol. 1994, 7(5),
549-54),
pancreas (Funa, K., et al. Cancer Res. 1990, 50(3), 748-53) and lung
(Antoniades, H.N., et
al., Proc. Natl. Acad. Sci. U S A 1992, 89(9), 3942-6). Ligand independent
activation of the
receptor is found to a lesser extent but has been reported in chronic
myelomonocytic
leukemia (CMML) where the a chromosomal translocation event forms a fusion
protein
between the Ets-like transcription factor TEL and the PDGF receptor. In
addition, activating
mutations in PDGFR have been found in gastrointestinal stromal tumors in which
c-kit
activation is not involved (Heinrich, M.C., et al., Science 2003, 9, 9).
Another major regulator of angiogenesis and vasculogenesis in both embryonic
development
and some angiogenic-dependent diseases is vascular endothelial growth factor
(VEGF; also
called vascular permeability factor, VPF). VEGF represents a family of
isoforms of mitogens
existing in homodimeric forms due to alternative RNA splicing. The VEGF
isoforms are
highly specific for vascular endothelial cells (for reviews, see: Farrara et
al. Endocr. Rev.
1992, 13, 18; Neufield et al. FASEB J. 1999, 13, 9).
VEGF expression is induced by hypoxia (Shweiki et al. Nature 1992, 359, 843),
as well as by
a variety of cytokines and growth factors, such as interleukin-1, interleukin-
6, epidermal
growth factor and transforming growth factor. To date, VEGF and the VEGF
family
members have been reported to bind to one or more of three transmembrane
receptor tyrosine
kinases (Mustonen et al. J. Cell Biol. 1995, 129, 895), VEGF receptor-1 (also
known as flt-1
(fms-like tyrosine kinase-1)), VEGFR-2 (also known as kinase insert domain
containing
receptor (KDR); the murine analogue of VEGFR-2 is known as fetal liver kinase-
1 (flk-1)),
and VEGFR-3 (also known as flt-4). VEGFR-2 and flt-1 have been shown to have
different
signal transduction properties (Waltenberger et al. J. Biol. Chem. 1994, 269,
26988); Park et
al. Oncogene 1995, 10, 135). Thus, VEGFR-2 undergoes strong ligand-dependant
tyrosine
phosphorylation in intact cells, whereas flt-1 displays a weak response. Thus,
binding to
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VEGFR-2 is believed to be a critical requirement for induction of the full
spectrum of VEGF-
mediated biological responses.
In vivo, VEGF plays a central role in vasculogenesis, and induces angiogenesis
and
permeabilization of blood vessels. Deregulated VEGF expression contributes to
the
development of a number of diseases that are characterized by abnormal
angiogenesis and/or
hyperpermeability processes. It is believed that regulation of the VEGF-
mediated signal
transduction cascade by some agents can provide a useful control of abnormal
angiogenesis
and/or hyperpermeability processes. Tumorigenic cells within hypoxic regions
of tumors
respond by stimulation of VEGF production, which triggers activation of
quiescent
endothelial cells to stimulate new blood vessel formation. (Shweiki et al.
Proc. Nat'l. Acad.
Sci. 1995, 92, 768). In addition, VEGF production in tumor regions where there
is no
angiogenesis may proceed through the ras signal transduction pathway (Grugel
et al. J. Biol.
Chem. 1995, 270, 25915; Rak et al. Cancer Res. 1995, 55, 4575). In situ
hybridization
studies have demonstrated VEGF mRNA is strongly upregulated in a wide variety
of human
tumors, including lung (Mattern et al. Br. J. Cancer 1996, 73, 931), thyroid
(Viglietto et al.
Oncogene 1995, 11, 1569), breast (Brown et al. Human Pathol. 1995, 26, 86),
gastrointestinal
tract (Brown et al. Cancer Res. 1993, 53, 4727; Suzuki et al. Cancer Res.
1996, 56, 3004),
kidney and bladder (Brown et al. Am. J. Pathol. 1993, 1431, 1255), ovary
(Olson et al. Cancer
Res. 1994, 54, 1255), and cervical (Guidi et al. J. Nat'l Cancer Inst. 1995,
87, 12137)
carcinomas, as well as angiosarcoma (Hashimoto et al. Lab. Invest. 1995, 73,
859) and
several intracranial tumors (Plate et al. Nature 1992, 359, 845; Phillips et
al. Int. J. Oncol.
1993, 2, 913; Berkman et al. J. Clin. Invest. 1993, 91, 153). Neutralizing
monoclonal
antibodies to VEGFR-2 have been shown to be efficacious in blocking tumor
angiogenesis
(Kim et al. Nature 1993, 362, 841; Rockwell et al. Mol. Cell. Differ. 1995, 3,
315).
Overexpression of VEGF, for example under conditions of extreme hypoxia, can
lead to
intraocular angiogenesis, resulting in hyperproliferation of blood vessels,
leading eventually
to blindness. Such a cascade of events has been observed for a number of
retinopathies,
including diabetic retinopathy, ischemic retinal-vein occlusion, and
retinopathy of
prematurity (Aiello et al. New Engl. J. Med. 1994, 331, 1480; Peer et al. Lab.
Invest. 1995,
72, 638), and age-related macular degeneration (AMD; see, Lopez et al. Invest.
Opththalmol.
Vis. Sci. 1996, 37, 855).
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In rheumatoid arthritis (RA), the in-growth of vascular pannus may be mediated
by
production of angiogenic factors. Levels of immunoreactive VEGF are high in
the synovial
fluid of RA patients, while VEGF levels were low in the synovial fluid of
patients with other
forms of arthritis of with degenerative joint disease (Koch et al. J. Immunol.
1994, 152,
4149). The angiogenesis inhibitor AGM-170 has been shown to prevent
neovascularization
of the joint in the rat collagen arthritis model (Peacock et al. J. Exper.
Med. 1992, 175, 1135).
Increased VEGF expression has also been shown in psoriatic skin, as well as
bullous
disorders associated with subepidermal blister formation, such as bullous
pemphigoid,
erythema multiforme, and dermatitis herpetiformis (Brown et al. J. Invest.
Dermatol. 1995,
104, 744).
The vascular endothelial growth factors (VEGF, VEGF-C, VEGF-D) and their
receptors
(VEGFR-2, VEGFR-3) are not only key regulators of tumor angiogenesis, but also
lymphangiogenesis. VEGF, VEGF-C and VEGF-D are expressed in most tumors,
primarily
during periods of tumor growth and, often at substantially increased levels.
VEGF expression
is stimulated by hypoxia, cytokines, oncogenes such as ras, or by inactivation
of tumor
suppressor genes (McMahon, G. Oncologist 2000, 5(Suppl. 1), 3-10; McDonald,
N.Q.;
Hendrickson, W.A. Cell 1993, 73, 421-424).
The biological activities of the VEGFs are mediated through binding to their
receptors.
VEGFR-3 (also called flt-4) is predominantly expressed on lymphatic
endothelium in normal
adult tissues. VEGFR-3 function is needed for new lymphatic vessel formation,
but not for
maintenance of the pre-existing lymphatics. VEGFR-3 is also upregulated on
blood vessel
endothelium in tumors. Recently VEGF-C and VEGF-D, ligands for VEGFR-3, have
been
identified as regulators of lymphangiogenesis in mammals. Lymphangiogenesis
induced by
tumor-associated lymphangiogenic factors could promote the growth of new
vessels into the
tumor, providing tumor cells access to systemic circulation. Cells that invade
the lymphatics
could find their way into the bloodstream via the thoracic duct. Tumor
expression studies
have allowed a direct comparison of VEGF-C, VEGF-D and VEGFR-3 expression with
clinicopathological factors that relate directly to the ability of primary
tumors to spread (e.g.,
lymph node involvement, lymphatic invasion, secondary metastases, and disease-
free
survival). In many instances, these studies demonstrate a statistical
correlation between the
expression of lymphangiogenic factors and the ability of a primary solid tumor
to metastasize
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(Skobe, M. et al. Nature Med. 2001, 7(2), 192-198; Stacker, S.A. et al..
Nature Med. 2001,
7(2), 186-191; Makinen, T. et al. Nature Med. 2001, 7(2), 199-205; Mandriota,
S.J. et al.
EMBO J. 2001, 20(4), 672-82; Karpanen, T. et al. Cancer Res. 2001, 61(5), 1786-
90; Kubo,
H. et al. Blood 2000, 96(2), 546-53).
Hypoxia appears to be an important stimulus for VEGF production in malignant
cells.
Activation of p38 MAP kinase is required for VEGF induction by tumor cells in
response to
hypoxia (Blaschke, F. et al. Biochem. Biophys. Res. Commun. 2002, 296, 890-
896;
Shemirani, B. et al. Oral Oncology 2002, 38, 251-257). In addition to its
involvement in
angiogenesis through regulation of VEGF secretion, p38 MAP kinase promotes
malignant
cell invasion, and migration of different tumor types through regulation of
collagenase
activity and urokinase plasminogen activator expression (Laferriere, J. et al.
J. Biol. Chem.
2001, 276, 33762-33772; Westermarck, J. et al. Cancer Res. 2000, 60, 7156-
7162; Huang, S.
et al. J. Biol. Chem. 2000, 275, 12266-12272; Simon, C. et al. Exp. Cell Res.
2001, 271,
344-355).
Inhibition of the mitogen-activated protein kinase (MAPK) p38 has been shown
to inhibit
both cytokine production (e.g., TNF, IL-1, IL-6, IL-8) and proteolytic enzyme
production
(e.g., MMP-1, MMP-3) in vitro and/or in vivo. The mitogen activated protein
(MAP) kinase
p38 is involved in IL-1 and TNF signaling pathways (Lee, J. C.; Laydon, J. T.;
McDonnell, P.
C.; Gallagher, T. F.; Kumar, S.; Green, D.; McNulty, D.; Blumenthal, M. J.;
Heys, J. R.;
Landvatter, S. W.; Stricker, J. E.; McLaughlin, M. M.; Siemens, I. R.; Fisher,
S. M.; Livi, G.
P.; White, J. R.; Adams, J. L.; Yound, P. R. Nature 1994, 372, 739).
Clinical studies have linked tumor necrosis factor (TNF) production and/or
signaling to a
number of diseases including rheumatoid arthritis (Maini. J. Royal Coll.
Physicians London
1996, 30, 344). In addition, excessive levels of TNF have been implicated in a
wide variety
of inflammatory and/or immunomodulatory diseases, including acute rheumatic
fever (Yegin
et al. Lancet 1997, 349, 170), bone resorption (Pacifici et al. J. Clin.
Endocrinol. Metabol.
1997, 82, 29), postmenopausal osteoporosis (Pacifici et al. J. Bone Mineral
Res. 1996, 11,
1043), sepsis (Blackwell et al. Br. J. Anaesth. 1996, 77, 110), gram negative
sepsis (Debets et
al. Prog. Clin. Biol. Res. 1989, 308, 463), septic shock (Tracey et al. Nature
1987, 330, 662;
Girardin et al. New England J. Med. 1988, 319, 397), endotoxic shock (Beutler
et al. Science
1985, 229, 869; Ashkenasi et al. Proc. Nat'l. Acad. Sci. USA 1991, 88, 10535),
toxic shock
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syndrome, (Saha et al. J. Immunol. 1996, 157, 3869; Lina et al. FEMS Immunol.
Med.
Microbiol. 1996, 13, 81), systemic inflammatory response syndrome (Anon. Crit.
Care Med.
1992, 20, 864), inflammatory bowel diseases (Stokkers et al. J. Inflamm. 1995-
6, 47, 97)
including Crohn's disease (van Deventer et al. Aliment. Pharmacol. Therapeu.
1996, 10
(Suppl. 2), 107; van Dullemen et al. Gastroenterology 1995, 109, 129) and
ulcerative colitis
(Masuda et al. J. Clin. Lab. Immunol. 1995, 46, 111), Jarisch-Herxheimer
reactions (Fekade
et al. New England J. Med. 1996, 335, 311), asthma (Amrani et al. Rev. Malad.
Respir. 1996,
13, 539), adult respiratory distress syndrome (Roten et al. Am. Rev. Respir.
Dis. 1991, 143,
590; Suter et al. Am. Rev. Respir. Dis. 1992, 145, 1016), acute pulmonary
fibrotic diseases
(Pan et al. Pathol. Int. 1996, 46, 91), pulmonary sarcoidosis (Ishioka et al.
Sarcoidosis
Vasculitis Diffuse Lung Dis. 1996, 13, 139), allergic respiratory diseases
(Casale et al. Am. J.
Respir. Cell Mol. Biol. 1996, 15, 35), silicosis (Gossart et al. J. Immunol.
1996, 156, 1540;
Vanhee et al. Eur. Respir. J. 1995, 8, 834), coal worker's pneumoconiosis
(Borm et al. Am.
Rev. Respir. Dis. 1988, 138, 1589), alveolar injury (Horinouchi et al. Am. J.
Respir. Cell
Mol. Biol. 1996, 14, 1044), hepatic failure (Gantner et al. J. Pharmacol. Exp.
Therap. 1997,
280, 53), liver disease during acute inflammation (Kim et al. J. Biol. Chem.
1997, 272, 1402),
severe alcoholic hepatitis (Bird et al. Ann. Intern. Med. 1990, 112, 917),
malaria (Grau et al.
Immunol. Rev. 1989, 112, 49; Taverne et al. Parasitol. Today 1996, 12, 290)
including
Plasmodium falciparum malaria (Perlmann et al. Infect. Immunit. 1997, 65, 116)
and cerebral
malaria (Rudin et al. Am. J. Pathol. 1997, 150, 257), non-insulin-dependent
diabetes mellitus
(NIDDM; Stephens et al. J. Biol. Chem. 1997, 272, 971; Ofei et al. Diabetes
1996, 45, 881),
congestive heart failure (Doyama et al. Int. J. Cardiol. 1996, 54, 217;
McMurray et al. Br.
Heart J. 1991, 66, 356), damage following heart disease (Malkiel et al. Mol.
Med. Today
1996, 2, 336), atherosclerosis (Parums et al. J. Pathol. 1996, 179, A46),
Alzheimer's disease
(Fagarasan et al. Brain Res. 1996, 723, 231; Aisen et al. Gerontology 1997,
43, 143), acute
encephalitis (Ichiyama et al. J. Neurol. 1996, 243, 457), brain injury (Cannon
et al. Crit. Care
Med. 1992, 20, 1414; Hansbrough et al. Surg. Clin. N. Am. 1987, 67, 69; Marano
et al. Surg.
Gynecol. Obstetr. 1990, 170, 32), multiple sclerosis (M.S.; Coyle. Adv.
Neuroimmunol.
1996, 6, 143; Matusevicius et al. J. Neuroimmunol. 1996, 66, 115) including
demyelation and
oligiodendrocyte loss in multiple sclerosis (Brosnan et al. Brain Pathol.
1996, 6, 243),
advanced cancer (MucWierzgon et al. J. Biol. Regulators Homeostatic Agents
1996, 10, 25),
lymphoid malignancies (Levy et al. Crit. Rev. Immunol. 1996, 16, 31),
pancreatitis (Exley et
al. Gut 1992, 33, 1126) including systemic complications in acute pancreatitis
(McKay et al.
Br. J. Surg. 1996, 83, 919), impaired wound healing in infection inflammation
and cancer
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(Buck et al. Am. J. Pathol. 1996, 149, 195), myelodysplastic syndromes (Raza
et al. Int. J.
Hematol. 1996, 63, 265), systemic lupus erythematosus (Maury et al. Arthritis
Rheum. 1989,
32, 146), biliary cirrhosis (Miller et al. Am. J. Gasteroenterolog. 1992, 87,
465), bowel
necrosis (Sun et al. J. Clin. Invest. 1988, 81, 1328), psoriasis
(Christophers. Austr. J.
Dermatol. 1996, 37, S4), radiation injury (Redlich et al. J. Immunol. 1996,
157, 1705), and
toxicity following administration of monoclonal antibodies such as OKT3 (Brod
et al.
Neurology 1996, 46, 1633). TNF levels have also been related to host-versus-
graft reactions
(Piguet et al. Immunol. Ser. 1992, 56, 409) including ischemia reperfusion
injury (Colletti et
al. J. Clin. Invest. 1989, 85, 1333) and allograft rejections including those
of the kidney
(Maury et al. J. Exp. Med. 1987, 166, 1132), liver (Imagawa et al.
Transplantation 1990, 50,
219), heart (Bolling et al. Transplantation 1992, 53, 283), and skin (Stevens
et al. Transplant.
Proc. 1990, 22, 1924), lung allograft rejection (Grossman et al. Immunol.
Allergy Clin. N.
Am. 1989, 9, 153) including chronic lung allograft rejection (obliterative
bronchitis;
LoCicero et al. J. Thorac. Cardiovasc. Surg. 1990, 99, 1059), as well as
complications due to
total hip replacement (Cirino et al. Life Sci. 1996, 59, 86). TNF has also
been linked to
infectious diseases (review: Beutler et al. Crit. Care Med. 1993, 21, 5423;
Degre. Biotherapy
1996, 8, 219) including tuberculosis (Rook et al. Med. Malad. Infect. 1996,
26, 904),
Helicobacter pylori infection during peptic ulcer disease (Beales et al.
Gastroenterology
1997, 112, 136), Chaga's disease resulting from Trypanosoma cruzi infection
(Chandrasekar
et al. Biochem. Biophys. Res. Commun. 1996, 223, 365), effects of Shiga-like
toxin resulting
from E. coli infection (Harel et al. J. Clin. Invest. 1992, 56, 40), the
effects of enterotoxin A
resulting from Staphylococcus infection (Fischer et al. J. Immunol. 1990, 144,
4663),
meningococcal infection (Waage et al. Lancet 1987, 355; Ossege et al. J.
Neurolog. Sci.
1996, 144, 1), and infections from Borrelia burgdorferi (Brandt et al. Infect.
Immunol. 1990,
58, 983), Treponema pallidum (Chamberlin et al. Infect. Immunol. 1989, 57,
2872),
cytomegalovirus (CMV; Geist et al. Am. J. Respir. Cell Mol. Biol. 1997, 16,
31), influenza
virus (Beutler et al. Clin. Res. 1986, 34, 491a), Sendai virus (Goldfield et
al. Proc. Nat'l.
Acad. Sci. USA 1989, 87, 1490), Theiler's encephalomyelitis virus (Sierra et
al. Immunology
1993, 78, 399), and the human immunodeficiency virus (HIV; Poli. Proc. Nat'l.
Acad. Sci.
USA 1990, 87, 782; Vyakaram et al. AIDS 1990, 4, 21; Badley et al. J. Exp.
Med. 1997,
185, 55).
A number of diseases are thought to be mediated by excess or undesired matrix-
destroying
metalloprotease (MMP) activity or by an imbalance in the ratio of the MMPs to
the tissue
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inhibitors of metalloproteinases (TIMPs). These include osteoarthritis
(Woessner et al. J.
Biol. Chem. 1984, 259, 3633), rheumatoid arthritis (Mullins et al. Biochim.
Biophys. Acta
1983, 695, 117; Woolley et al. Arthritis Rheum. 1977, 20, 1231; Gravallese et
al. Arthritis
Rheum. 1991, 34, 1076), septic arthritis (Williams et al. Arthritis Rheum.
1990, 33, 533),
tumor metastasis (Reich et al. Cancer Res. 1988, 48, 3307; Matrisian et al.
Proc. Nat'l. Acad.
Sci., USA 1986, 83, 9413), periodontal diseases (Overall et al. J. Periodontal
Res. 1987, 22,
81), corneal ulceration (Burns et al. Invest. Opthalmol. Vis. Sci. 1989, 30,
1569), proteinuria
(Baricos et al. Biochem. J. 1988, 254, 609), coronary thrombosis from
atherosclerotic plaque
rupture (Henney et al. Proc. Nat'l. Acad. Sci., USA 1991, 88, 8154),
aneurysmal aortic
disease (Vine et al. Clin. Sci. 1991, 81, 233), birth control (Woessner et al.
Steroids 1989, 54,
491), dystrophobic epidermolysis bullosa (Kronberger et al. J. Invest.
Dermatol. 1982, 79,
208), degenerative cartilage loss following traumatic joint injury,
osteopenias mediated by
MMP activity, tempero mandibular joint disease, and demyelating diseases of
the nervous
system (Chantry et al. J. Neurochem. 1988, 50, 688).
Because inhibition of p38 leads to inhibition of TNF production and MMP
production, it is
believed inhibition of mitogen activated protein (MAP) kinase p38 enzyme can
provide an
approach to the treatment of the above listed diseases including osteoporosis
and
inflammatory disorders such as rheumatoid arthritis and COPD (Badger, A. M.;
Bradbeer, J.
N.; Votta, B.; Lee, J. C.; Adams, J. L.; Griswold, D. E. J. Pharm. Exper.
Ther. 1996, 279,
1453).
Hypoxia appears to be an important stimulus for VEGF production in malignant
cells.
Activation of p38 kinase is required for VEGF induction by tumor cells in
response to
hypoxia (Blaschke, F. et al. Biochem. Biophys. Res. Commun. 2002, 296, 890-
896;
Shemirani, B. et al. Oral Oncology 2002, 38, 251-257). In addition to its
involvement in
angiogenesis through regulation of VEGF secretion, p38 kinase promotes
malignant cell
invasion, and migration of different tumor types through regulation of
collagenase activity
and urokinase plasminogen activator expression (Laferriere, J. et al. J. Biol.
Chem. 2001,
276, 33762-33772; Westermarck, J. et al. Cancer Res. 2000, 60, 7156-7162;
Huang, S. et al.
J. Biol. Chem. 2000, 275, 12266-12272; Simon, C. et al. Exp. Cell Res. 2001,
271, 344-355).
Therefore, inhibition of p38 kinase is also expected to impact tumor growth by
interfering
with signaling cascades associated with both angiogenesis and malignant cell
invasion.
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Certain ureas have been described as having activity as serine-threonine
kinase and/or as
tyrosine kinase inhibitors. In particular, the utility of certain ureas as an
active ingredient in
pharmaceutical compositions for the treatment of cancer, angiogenesis
disorders,
inflammatory disorders, has been demonstrated.
For cancer and angiogenesis, see:
Smith et al., Bioorg. Med. Chem. Lett. 2001, 11, 2775-2778.
Lowinger et al., Clin. Cancer Res. 2000, 6(suppl.), 335.
Lyons et al., Endocr.-Relat. Cancer 2001, 8, 219-225.
Riedl et al., Book of Abstracts, 92nd AACR Meeting, New Orleans, LA, USA,
abstract 4956.
Khire et al., Book of Abstracts, 93rdAACR Meeting, San Francisco, CA, USA,
abstract 4211.
Lowinger et al., Curr. Pharm. Design 2002, 8, 99-110.
Carter et al., Book of Abstracts, 92ndAACR Meeting, New Orleans, LA, USA,
abstract 4954.
Vincent et al., Book of Abstracts, 38th ASCO Meeting, Orlando, FL, USA,
abstract 1900.
Hilger et al., Book of Abstracts, 38th ASCO Meeting, Orlando, FL, USA,
abstract 1916.
Moore et al., Book of Abstracts, 38th ASCO Meeting, Orlando, FL, USA, abstract
1816.
Strumberg et al., Book of Abstracts, 38th ASCO Meeting, Orlando, FL, USA,
abstract 121.
For p38 mediated diseases, including inflammatory disorders, see:
Redman et al., Bioorg. Med. Chem. Lett. 2001, 11, 9-12.
Dumas et al., Bioorg. Med. Chem. Lett. 2000, 10, 2047-2050.
Dumas et al., Bioorg. Med. Chem. Lett. 2000, 10, 2051-2054.
Ranges et al., Book of Abstracts, 220th ACS National Meeting, Washington, DC,
USA,
MEDI 149.
Dumas et al., Bioorg. Med. Chem. Lett. 2002, 12, 1559-1562.
Regan et al., J. Med. Chem. 2002, 45, 2994-3008.
Pargellis et al., Nature Struct. Biol. 2002, 9(4), 268-272.
Madwed J. B., Book of Abstracts, Protein Kinases: Novel Target Identification
and
Validation for Therapeutic Development, San Diego, CA, USA, March 2002.
Pargellis C. et al., Curr. Opin. Invest. Drugs 2003, 4, 566-571.
Branger J. et al., J. Immunol. 2002, 168, 4070-4077.
Branger J. et al., Blood 2003, 101, 4446-4448.
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Omega-Carboxyaryl diphenyl ureas are disclosed in WO00/42012, published: July
20, 2000,
WO00/41698, published: July 20, 2000, the following published U.S.
applications:
US2002-0165394-A1, published November 7, 2002,
US2001-003447-A1, published October 25, 2001,
US2001-0016659-A1, published August 23, 2001,
US2002-013774-A1, published September 26, 2002,
and copending U.S. applications:
09/758,547, filed January 12, 2001,
09/889,227, filed July 12, 2001,
09/993,647, filed November 27, 2001,
10/042,203, filed January 11, 2002 and
10/071,248, filed February 11, 2002,
Description of the Invention
It has been discovered that synthetic metabolites of omega-carboxyaryl
diphenyl urea of
Formula I below are potent inhibitors of raf kinase, VEGFR kinase, p38 kinase,
and PDGFR
kinase, which are all molecular targets of interest for the treatment and
prevention of
osteoporosis, inflammatory disorders, hyper-proliferatrive disorders, and
angiogenesis
disorders, including cancer.
The present invention provides, e.g.,
(i) novel synthetic metabolites of the compound of Formula (I), salts,
prodrugs, and
metabolites thereof,
(ii) pharmaceutical compositions containing such compounds, and
(iii) use of such synthetic metabolites or compositions for treating diseases
and conditions
mediated by raf, VEGFR, PDGFR, flt-3, and p38, either as a sole agent or in
combination
with cytotoxic therapies.
The compound of the Formula I below, salts, prodrugs and metabolites thereof
is collectively
referred to as the "compounds of the invention." Formula I is as follows:
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CF3 O
CI IOI / I O I NH ',,CH 3
I I
H H F
The synthetic metabolites of the compound of this invention include oxidized
derivatives of
Formula I wherein one or more of the urea nitrogens are substituted with a
hydroxy group.
The synthetic metabolites of the compound of this invention also include
analogs where the
methylamide group of the compound of Formula I is hydroxylated then de-
methylated by
metabolic degradation. The synthetic metabolites of the compound of this
invention further
include oxidized derivatives where the pyridine nitrogen atom is in the N-
oxide form (e.g.
carries a hydroxy substituent) leading to those structures referred to in the
art as 1-oxo-
pyridine and 1-hydroxy-pyridine.
Where the plural form of the word compounds, salts, and the like, is used
herein, this is taken
to mean also a single compound, salt, or the like.
In particular, the present invention relates to synthetic forms of M2, M3, M4
and M5
metabolites of the compounds of Formula I, whose structures appear below:
;ELY , 5-y 198) (CAY : 5-;'49S)
+ N . si.8 2
#'r1 :'t4 bo[ to rN= -5
MVV 484.i )
id W, rnoie=:_uiarb;eight
The relationship of the aforementioned synthetic metabolites with the parent
compound of
Formula I is illustrated in Fig. 1.
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Particularly preferred are the M2 and M5 metabolites of the compounds of
Formula I.
IL CH
Metabolite M-2
z 0"
Metabolite M-5
As an embodiment of the present invention, the biotransformation of the
compound of
Formula I was investigated in vitro with liver microsomes and hepatocytes, and
in vivo in the
plasma of several species. In man, the N-oxide (M-2) and the demethylated N-
oxide (M-5)
are of significance. Both metabolites in synthetic form show systemic exposure
(area under
the curve [AUC], mg*h/L) at steady state similar to regorafenib (the parent
compound) in
patients at a dose of 160 mg in a 3 weeks on/1 week off Phase I study.
The pharmacologic activities of the synthetic metabolites of the compounds of
formula I,
particularly, the M-2 and M-5 metabolites, are also of relevance. In vitro
biochemical and
cellular kinase phosphorylation assays reveal that the synthetic metabolites
of compounds of
formula I are broad-spectrum kinase inhibitors. In in vivo preclinical models
the metabolites
were effective against tumor growth and further inhibited tumor vasculature.
The synthetic
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metabolites also demonstrated acute effects on vascular endothelial growth
factor (VEGF)-
induced hypotension in pharmacodynamic rat models.
The term "synthetic" is art-recognized and refers to production by in vitro
chemical or
enzymatic synthesis.
As used herein, the term "isolated" means that the referenced material is
removed from its
native environment, e.g., a cell, or a tissue, or from the body. Thus, an
isolated metabolite can
be free of some or all cellular components, i.e., components of the cells in
which the native
material occurs naturally (e.g., cytoplasmic or membrane component, such as,
for example,
microsomes).
The term "purified" as used herein refers to material that has been isolated
under conditions
that reduce or eliminate the presence of unrelated materials, i.e.,
contaminants, including
precursor materials from which the material is obtained. For example, a
purified metabolite is
preferably substantially free of other metabolites or the precursor compound
from which it is
derived. As used herein, the term "substantially free" is used operationally,
in the context of
analytical testing of the material. Preferably, purified material is
substantially free of
contaminants is at least 50% pure; more preferably, at least 90% pure, and
more preferably
still at least 99% pure. Purity can be evaluated by chromatography, gel
electrophoresis,
HPLC, NMR or mass-spectroscopy analysis, composition analysis, biological
assays, and
other methods known in the art.
Salts
Pharmaceutically acceptable salts of the synthetic metabolites of Forumla I
are also within
the scope of this invention. The term "pharmaceutically acceptable salt"
refers to a relatively
non-toxic, inorganic or organic acid addition salt of a compound of the
present invention.
For example, see S. M. Berge, et al. "Pharmaceutical Salts," J. Pharm. Sci.
1977, 66, 1-19.
Representative salts of the compound of this invention include the
conventional non-toxic
salts, for example, from inorganic or organic acids by means well known in the
art. For
example, such acid addition salts include acetate, adipate, alginate,
ascorbate, aspartate,
benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate,
camphorsulfonate,
cinnamate, cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, fumarate,
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glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate,
hydrochloride,
hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, itaconate, lactate,
maleate,
mandelate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,
oxalate, pamoate,
pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate,
succinate, sulfonate,
tartrate, thiocyanate, tosylate, and undecanoate.
The salts or prodrugs of the compounds of Formula I may contain one or more
asymmetric
centers. Asymmetric carbon atoms may be present in the (R) or (S)
configuration or (R,S)
configuration. Substituents on a ring may also be present in either cis or
trans form. It is
intended that all such configurations (including enantiomers and
diastereomers), are included
within the scope of the present invention. Preferred isomers are those with
the configuration
which produces the more desirable biological activity. Separated, pure or
partially purified
isomers or racemic mixtures of the compounds of this invention are also
included within the
scope of the present invention. The purification of said isomers and the
separation of said
isomeric mixtures can be accomplished by standard techniques known in the art.
The particular process to be utilized in the preparation of the synthetic
metabolites used in
this embodiment of the invention is described in Example 4. Salt forms of the
synthetic
metabolites of Formula (I) are described in the Examples.
Methods of use
The present invention provides compounds which are capable of modulating one
or more
signal transduction pathways involving raf, VEGFR, PDGFR, p38, and/or flt-3
kinases. Raf
is an important signaling molecule involved in the regulation of a number of
key cellular
processes, including cell growth, cell survival and invasion. It is a member
of the
Ras/raf/MEK/ERK pathway. This pathway is present in most tumor cells. VEGFR,
PDGFR,
and flt-3 are transmembrane receptor molecules which, when stimulated by an
appropriate
ligand, trigger the Ras/raf/MEK/ERK cell signaling pathway, leading to a
cascade of cellular
events. Each of these receptor molecules have tyrosine kinase activity.
The VEGFR receptors are stimulated by vascular endothelial growth factors
(VEGF), and are
important control points in the regulation of endothelial cell development and
function. The
PDGF-beta receptor regulates cell proliferation and survival in a number of
cell types,
including mesenchymal cells. Flt-3 is a receptor for the FL ligand. It is
structurally similar
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to c-kit, and modulates the growth of pluripotent haemopoietic cells,
influencing the
development of T-cells, B-cells, and dendritic cells.
Any gene or isoform of raf, VEGFR, PDGFR, p38, and/or flt-3 can be modulated
in
accordance with present invention, including both wild-type and mutant forms.
Raf or raf-1
kinase is a family of serine/threonine kinases which comprise at least three
family members,
a-raf, b-raf, and c-raf or raf-1. See, e.g., Dhillon and Kolch, Arch. Biochem.
Biophys. 2002,
404, 3-9. C-raf and b-raf are preferred targets for compounds of the present
invention.
Activating b-raf mutations (e.g., V599E mutant) have been identified in
various cancers,
including melanoma, and the compounds described herein can be utilized to
inhibit their
activity.
By the term "modulate", it is meant that the functional activity of the
pathway (or a
component of it) is changed in comparison to its normal activity in the
absence of the
compound. This effect includes any quality or degree of modulation, including,
increasing,
agonizing, augmenting, enhancing, facilitating, stimulating, decreasing,
blocking, inhibiting,
reducing, diminishing, antagonizing, etc.
The compounds of the present invention can also modulate one or more of the
following
processes, including, but not limited to, e.g., cell growth (including, e.g.,
differentiation, cell
survival, and/or proliferation), tumor cell growth (including, e.g.,
differentiation, cell
survival, and/or proliferation), tumor regression, endothelial cell growth
(including, e.g.,
differentiation, cell survival, and/or proliferation), angiogenesis (blood
vessel growth),
lymphangiogenesis (lymphatic vessel growth), and/or hematopoiesis (e.g., T-
and B-cell
development, dendritic cell development, etc.).
While not wishing to be bound by any theory or mechanism of action, it has
been found that
compounds of the present invention possess the ability to modulate kinase
activity. The
methods of the present invention, however, are not limited to any particular
mechanism or
how the compounds achieve their therapeutic effect. By the term "kinase
activity", it is
meant a catalytic activity in which a gamma-phosphate from adenosine
triphosphate (ATP) is
transferred to an amino acid residue (e.g., serine, threonine, or tyrosine) in
a protein substrate.
A compound can modulate kinase activity, e.g., inhibiting it by directly
competing with ATP
for the ATP-binding pocket of the kinase, by producing a conformational change
in the
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enzyme's structure that affects its activity (e.g., by disrupting the
biologically-active three-
dimensional structure), etc.
Kinase activity can be determined routinely using conventional assay methods.
Kinase
assays typically comprise the kinase enzyme, substrates, buffers, and
components of a
detection system. A typical kinase assay involves the reaction of a protein
kinase with a
peptide substrate and an ATP, such as 32P-ATP, to produce a phosphorylated end-
product
(for instance, a phosphoprotein when a peptide substrate is used). The
resulting end-product
can be detected using any suitable method. When radioactive ATP is utilized, a
radioactively
labeled phosphoprotein can be separated from the unreacted gamma-32P-ATP using
an
affinity membrane or gel electrophoresis, and then visualized on the gel using
autoradiography or detected with a scintillation counter. Non-radioactive
methods can also be
used. Methods can utilize an antibody which recognizes the phosphorylated
substrate, e.g.,
an anti-phosphotyrosine antibody. For instance, kinase enzyme can incubated
with a
substrate in the presence of ATP and kinase buffer under conditions which are
effective for
the enzyme to phosphorylate the substrate. The reaction mixture can be
separated, e.g.,
electrophoretically, and then phosphorylation of the substrate can be
measured, e.g., by
Western blotting using an anti-phosphotyrosine antibody. The antibody can be
labeled with a
detectable label, e.g., an enzyme, such as HRP, avidin or biotin,
chemiluminescent reagents,
etc. Other methods can utilize ELISA formats, affinity membrane separation,
fluorescence
polarization assays, luminescent assays, etc.
An alternative to a radioactive format is time-resolved fluorescence resonance
energy transfer
(TR-FRET). This method follows the standard kinase reaction, where a
substrate, e.g.,
biotinylated poly(GluTyr), is phosphorylated by a protein kinase in the
presence of ATP. The
end-product can then detected with a europium chelate phosphospecific antibody
(anti-
phosphotyrosine or phosphoserine/threonine), and streptavidin-APC, which binds
the
biotinylated substrate. These two components are brought together spatially
upon binding,
and energy transfer from the phosphospecific antibody to the acceptor (SA-APC)
produces
fluorescent readout in the homogeneous format.
The compounds of the present invention can be used to treat and/or prevent any
disease or
condition mediated by one or more cellular signal transduction pathways
involving raf,
VEGFR, PDGFR, p38, and/or flt-3 kinases. The term "treating" is used
conventionally, e.g.,
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the management or care of a subject for the purpose of combating, alleviating,
reducing,
relieving, improving the condition of, etc., of a disease or disorder. The
compounds can also
be described as being used to prevent and/or treat diseases and/or condition
mediated by the
signaling molecules. The term "mediated" indicates, e.g., that the signaling
molecule is part
of the pathway which is aberrant or disturbed in the disease and/or condition.
Diseases and conditions that can be treated include any of those mentioned
above and below,
as well as:
Raf associated diseases include, e.g., cell-proliferation disorders, cancer,
tumors, etc.;
VEGFR-2 associated diseases include, e.g., cancer, tumor growth, inflammatory
disease,
rheumatoid arthritis, retinopathy, psoriasis, glomerulonephritis, asthma,
chronic bronchitis,
atherosclerosis, transplant rejection, conditions involving angiogenesis,
etc.;
VEGFR-3 associated diseases include, e.g., cancer, corneal disease, inflamed
cornea (e.g.,
Hamrah, Am. J. Path. 2003, 163, 57-68), corneal transplantation (Cursiefen et
al., Cornea
2003, 22, 273-81), lymphatic hyperplasia, conditions involving
lymphangiogenesis, etc.;
PDGFR-beta associated diseases include, e.g., diseases or conditions
characterized by cell
proliferation, cell matrix production, cell movement, and/or extracellular
matrix production.
Specific examples, include, e.g., tumors, malignancies, cancer, metastasis,
chronic myeloid
leukemia, inflammation, renal disease, diabetic nephropathy, mesangial
proliferative
glomerulonephritis, fibrotic conditions, atherosclerosis, restenosis,
hypertension-related
arteriosclerosis, venous bypass graft arteriosclerosis, scleroderma,
interstitial pulmonary
diseases, synovial disorders, arthritis, leukemias, lymphomas, etc
Flt-3 associated diseases include, e.g., immune-related disorders, blood cell
disorders,
conditions involving hematopoietic cell development (e.g., T-cells, B-cells,
dendritic cells,
cancer, anemia, HIV, acquired immune deficiency syndrome, etc.
p38 associated diseases include inflammatory disorders, immunomodulatory
disorders, and
other disorders that have been linked to abnormal cytokine production,
especially TNF-alpha,
or abnormal MMP activity. These disorders include, but are not limited to,
rheumatoid
arthritis, COPD, osteoporosis, Crohn's disease and psoriasis.
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In addition, compounds of the present invention can be used to treat
conditions and disorders
disclosed in U.S. Pat. No. 6,316,479, e.g., glomerular sclerosis, interstitial
nephritis,
interstitial pulmonary fibrosis, atherosclerosis, wound scarring and
scleroderma.
The compounds of this invention also have a broad therapeutic activity to
treat or prevent the
progression of a broad array of diseases, such as inflammatory conditions,
coronary
restenosis, tumor-associated angiogenesis, atherosclerosis, autoimmune
diseases,
inflammation, certain kidney diseases associated with proliferation of
glomerular or
mesangial cells, and ocular diseases associated with retinal vessel
proliferation. psoriasis,
hepatic cirrhosis, diabetes, atherosclerosis, restenosis, vascular graft
restenosis, in-stent
stenosis, angiogenesis, ocurlar diseases, pulmonary fibrosis, obliterative
bronchiolitis,
glomerular nephritis, rheumatoid arthritis.
The present invention also provides for treating, preventing, modulating,
etc., one or more of
the following conditions in humans and/or other mammals: retinopathy,
including diabetic
retinopathy, ischemic retinal-vein occlusion, retinopathy of prematurity and
age related
macular degeneration; rheumatoid arthritis, psoriasis, or bullous disorder
associated with
subepidermal blister formation, including bullous pemphigoid, erythema
multiforme, or
dermatitis herpetiformis, rheumatic fever, bone resorption, postmenopausal
osteoperosis,
sepsis, gram negative sepsis, septic shock, endotoxic shock, toxic shock
syndrome, systemic
inflammatory response syndrome, inflammatory bowel disease (Crohn's disease
and
ulcerative colitis), Jarisch-Herxheimer reaction, asthma, adult respiratory
distress syndrome,
acute pulmonary fibrotic disease, pulmonary sarcoidosis, allergic respiratory
disease,
silicosis, coal worker's pneumoconiosis, alveolar injury, hepatic failure,
liver disease during
acute inflammation, severe alcoholic hepatitis, malaria (Plasmodium falciparum
malaria and
cerebral malaria), non-insulin-dependent diabetes mellitus (NIDDM), congestive
heart
failure, damage following heart disease, atherosclerosis, Alzheimer's disease,
acute
encephalitis, brain injury, multiple sclerosis (demyelation and
oligiodendrocyte loss in
multiple sclerosis), advanced cancer, lymphoid malignancy, pancreatitis,
impaired wound
healing in infection, inflammation and cancer, myelodysplastic syndromes,
systemic lupus
erythematosus, biliary cirrhosis, bowel necrosis, radiation injury/ toxicity
following
administration of monoclonal antibodies, host-versus-graft reaction (ischemia
reperfusion
injury and allograft rejections of kidney, liver, heart, and skin), lung
allograft rejection
(obliterative bronchitis), or complications due to total hip replacement, ad
an infectious
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disease selected from tuberculosis, Helicobacter pylori infection during
peptic ulcer disease,
Chaga's disease resulting from Trypanosoma cruzi infection, effects of Shiga-
like toxin
resulting from E. coli infection, effects of enterotoxin A resulting from
Staphylococcus
infection, meningococcal infection, and infections from Borrelia burgdorferi,
Treponema
pallidum, cytomegalovirus, influenza virus, Theiler's encephalomyelitis virus,
and the human
immunodeficiency virus (HIV), papilloma, blastoglioma, Kaposi's sarcoma,
melanoma, lung
cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, astrocytoma,
head cancer,
neck cancer, bladder cancer, breast cancer, colorectal cancer, thyroid cancer,
pancreatic
cancer, gastric cancer, hepatocellular carcinoma, leukemia, lymphoma,
Hodgkin's disease,
Burkitt's disease, arthritis, rheumatoid arthritis, diabetic retinopathy,
angiogenesis, restenosis,
in-stent restenosis, vascular graft restenosis, pulmonary fibrosis, hepatic
cirrhosis,
atherosclerosis, glomerulonophritis, diabetic nephropathy, thrombic
micoangiopathy
syndromes, transplant rejection, psoriasis, diabetes, wound healing,
inflammation, and
neurodegenerative diseases. hyperimmune disorders, hemangioma, myocardial
angiogenesis,
coronary and cerebral collateral vascularization, ischemia, corneal disease,
rubeosis,
neovascular glaucoma, macular degeneration retinopathy of prematurity, wound
healing,
ulcer Helicobacter related diseases, fractures, endometriosis, a diabetic
condition, cat scratch
fever, thyroid hyperplasia, asthma or edema following burns, trauma, chronic
lung disease,
stroke, polyps, cysts, synovitis, chronic and allergic inflammation, ovarian
hyperstimulation
syndrome, pulmonary and cerebral edema, keloid, fibrosis, cirrhosis, carpal
tunnel syndrome,
adult respiratory distress syndrome, ascites, an ocular condition, a
cardiovascular condition,
Crow-Fukase (POEMS) disease, Crohn's disease, glomerulonophritis,
osteoarthritis, multiple
sclerosis, graft rejection, Lyme disease, sepsis, von Hippel Lindau disease,
pemphigoid,
Paget's disease, polycystic kidney disease, sarcoidosis, throiditis,
hyperviscosity syndrome,
Osler-Weber-Rendu disease, chronic occlusive pulmonary disease, radiation,
hypoxia,
preeclampsia, menometrorrhagia, endometriosis, infection by Herpes simplex,
ischemic
retinopathy, corneal angiogenesis, Herpes Zoster, human immunodeficiency
virus,
parapoxvirus, protozoa, toxoplasmosis, and tumor-associated effusions and
edema.
The compounds of this invention can possess more than one of the mentioned
activities, and
therefore can target a plurality of signal transduction pathways. Thus, these
compounds can
achieve therapeutic and prophylactic effects which normally are only obtained
when using a
combination of different compounds. For instance, the ability to inhibit both
new vessel
formation (e.g., associated with VEGFR-2 and VEGFR-3 function) (e.g., blood
and/or
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lymph) and cell-proliferation (e.g., associated with raf and PDGFR-beta
function) using a
single compound is especially beneficial in the treatment of cancer, and other
cell-
proliferation disorders that are facilitated by neo-vascularization. Thus, the
present invention
relates specifically to compounds which possess at least anti-cell
proliferation and anti-
angiogenic (i.e., inhibits angiogenesis) activity. Any disorder or condition
that would benefit
from inhibiting vessel growth and cell proliferation can be treated in
accordance with the
present invention. Using a single compound is also advantageous because its
range of
activities can be more precisely defined.
As indicated above, the present invention relates to methods of treating
and/or preventing
diseases and conditions; and/or modulating one or more of the pathways,
polypeptides, genes,
diseases, conditions, etc., associated with raf, VEGFR, PDGFR, p38, and/or flt-
3. These
methods generally involve administering effective amounts of compounds of the
present
invention, where an effective amount is the quantity of the compound which is
useful to
achieve the desired result. Compounds can be administered in any effective
form by any
effective route, as discussed in more detail below.
Methods include modulating tumor cell proliferation, including inhibiting cell
proliferation.
The latter indicates that the growth and/or differentiation of tumor cells is
reduced, decreased,
diminished, slowed, etc. The term "proliferation" includes any process which
relates to cell
growth and division, and includes differentiation and apoptosis. As discussed
above, raf
kinases play a key role in the activation of the cytoplasmic signaling cascade
involved in cell
proliferation, differentiation, and apoptosis. For example, studies have found
that inhibiting
c-raf by anti-sense oligonucleotides can block cell proliferation (see above).
Any amount of
inhibition is considered therapeutic.
Included in the methods of the present invention is a method for using
synthetic metabolites
of the compound described above (Compound of Formula I), including salts,
prodrugs, and
compositions thereof, to treat mammalian hyper-proliferative disorders
comprising
administering to a mammal, including a human in need thereof, an amount of a
synthetic
metabolite of a compound of this invention, pharmaceutically acceptable salt,
and
composition thereof, which is effective to treat the disorder. Hyper-
proliferative disorders
include but are not limited to solid tumors, such as cancers of the breast,
respiratory tract,
brain, reproductive organs, digestive tract, urinary tract, eye, liver, skin,
head and neck,
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thyroid, parathyroid and their distant metastases. Those disorders also
include lymphomas,
sarcomas, and leukemias.
Any tumor or cancer can be treated, including, but not limited to, cancers
having one or more
mutations in raf, ras, and/or flt-3, as well as any upstream or downstream
member of the
signaling pathways of which they are a part. As discussed earlier, a cancer
can be treated
with a compound of the present invention irrespective of the mechanism which
is responsible
for it. Cancers of any organ can be treated, including cancers of, but are not
limited to, e.g.,
colon, pancreas, breast, prostate, bone, liver, kidney, lung, testes, skin,
pancreas, stomach,
colorectal cancer, renal cell carcinoma, hepatocellular carcinoma, melanoma,
etc.
Examples of breast cancer include, but are not limited to, invasive ductal
carcinoma, invasive
lobular carcinoma, ductal carcinoma in situ, and lobular carcinoma in situ.
Examples of cancers of the respiratory tract include, but are not limited to,
small-cell and
non-small-cell lung carcinoma, as well as bronchial adenoma and
pleuropulmonary blastoma.
Examples of brain cancers include, but are not limited to, brain stem and
hypophtalmic
glioma, cerebellar and cerebral astrocytoma, medulloblastoma, ependymoma, as
well as
neuroectodermal and pineal tumor.
Tumors of the male reproductive organs include, but are not limited to,
prostate and testicular
cancer. Tumors of the female reproductive organs include, but are not limited
to, endometrial,
cervical, ovarian, vaginal, and vulvar cancer, as well as sarcoma of the
uterus.
Tumors of the digestive tract include, but are not limited to, anal, colon,
colorectal,
esophageal, gallbladder, gastric, pancreatic, rectal, small-intestine, and
salivary gland
cancers.
Tumors of the urinary tract include, but are not limited to, bladder, penile,
kidney, renal
pelvis, ureter, and urethral cancers.
Eye cancers include, but are not limited to, intraocular melanoma and
retinoblastoma.
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Examples of liver cancers include, but are not limited to, hepatocellular
carcinoma (liver cell
carcinomas with or without fibrolamellar variant), cholangiocarcinoma
(intrahepatic bile duct
carcinoma), and mixed hepatocellular cholangiocarcinoma.
Skin cancers include, but are not limited to, squamous cell carcinoma,
Kaposi's sarcoma,
malignant melanoma, Merkel cell skin cancer, and non-melanoma skin cancer.
Head-and-neck cancers include, but are not limited to, laryngeal,
hypopharyngeal,
nasopharyngeal, and/or oropharyngeal cancers, and lip and oral cavity cancer.
Lymphomas include, but are not limited to, AIDS-related lymphoma, non-
Hodgkin's
lymphoma, cutaneous T-cell lymphoma, Hodgkin's disease, and lymphoma of the
central
nervous system.
Sarcomas include, but are not limited to, sarcoma of the soft tissue,
osteosarcoma, malignant
fibrous histiocytoma, lymphosarcoma, and rhabdomyosarcoma.
Leukemias include, but are not limited to, acute myeloid leukemia, acute
lymphoblastic
leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and
hairy cell
leukemia.
In addition to inhibiting the proliferation of tumor cells, compounds of the
present invention
can also cause tumor regression, e.g., a decrease in the size of a tumor, or
in the extent of
cancer in the body.
The present invention also relates to methods of modulating angiogenesis
and/or
lymphangiogenesis in a system comprising cells, comprising administering to
the system an
effective amount of a compound described herein. A system comprising cells can
be an in
vivo system, such as a tumor in a patient, isolated organs, tissues, or cells,
in vitro assays
systems (CAM, BCE, etc), animal models (e.g., in vivo, subcutaneous, cancer
models), hosts
in need of treatment (e.g., hosts suffering from diseases having angiogenic
and/or
lymphangiogenic component, such as cancer), etc.
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Inappropriate and ectopic expression of angiogenesis can be deleterious to an
organism. A
number of pathological conditions are associated with the growth of extraneous
blood
vessels. These include, e.g., diabetic retinopathy, neovascular glaucoma,
psoriasis, retrolental
fibroplasias, angiofibroma, inflammation, etc. In addition, the increased
blood supply
associated with cancerous and neoplastic tissue, encourages growth, leading to
rapid tumor
enlargement and metastasis. Moreover, the growth of new blood and lymph
vessels in a
tumor provides an escape route for renegade cells, encouraging metastasis and
the
consequence spread of the cancer.
Useful systems for measuring angiogenesis and/or lymphangiogenesis, and
inhibition thereof,
include, e.g., neovascularization of tumor explants (e.g., U.S. Pat. Nos.
5,192,744;
6,024,688), chicken chorioallantoic membrane (CAM) assay (e.g., Taylor and
Folkman,
Nature 1982, 297, 307-312; Eliceiri et al., J. Cell Biol. 1998, 140, 1255-
1263), bovine
capillary endothelial (BCE) cell assay (e.g., U.S. Pat. No. 6,024,688;
Polverini, P. J. et al.,
Methods Enzymol. 1991, 198, 440-450), migration assays, and HUVEC (human
umbilical
cord vascular endothelial cell) growth inhibition assay (e.g., U.S. Pat. No.
6,060,449), and
use of the rabbit ear model (e.g., Szuba et al., FASEB J. 2002, 16(14), 1985-
7).
Modulation of angiogenesis can be determined by any other method. For example,
the
degree of tissue vascularity is typically determined by assessing the number
and density of
vessels present in a given sample. For example, microvessel density (MVD) can
be estimated
by counting the number of endothelial clusters in a high-power microscopic
field, or
detecting a marker specific for microvascular endothelium or other markers of
growing or
established blood vessels, such as CD31 (also known as platelet-endothelial
cell adhesion
molecule or PECAM). A CD31 antibody can be employed in conventional
immunohistological methods to immunostain tissue sections as described by,
e.g., U.S. Pat.
No. 6,017,949; Dellas et al., Gyn. Oncol. 1997, 67, 27-33; and others. Other
markers for
angiogenesis, include, e.g., Vezf1 (e.g., Xiang et al., Dev. Bio. 1999, 206,
123-141),
angiopoietin, Tie-1, and Tie-2 (e.g., Sato et al., Nature 1995, 376, 70-74).
Additionally, the present invention relates to methods of screening patients
to determine their
sensitivity to compounds of the present invention. For example, the invention
relates to
methods of determining whether a condition can be modulated by a compound
disclosed
herein, comprising measuring the expression or activity of raf, VEGFR-2, VEGFR-
3,
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PDGFR-beta, p38, and/or flt-3 in a sample comprising cells or a cell extract,
wherein said
sample has been obtained from a cell or subject having said condition. When
the results of
the determination indicate that one or more of the mentioned genes (and/or
polypeptides
which they encode) differ from the normal state, this identifies the condition
as being
treatable with a compound of the present invention, i.e., whereby said
disorder or condition
can be modulated by the compound when said expression or activity is increased
in said
condition as compared to a normal control. The method can further comprise a
step of
comparing the expression in a sample with a normal control, or expression in a
sample
obtained from normal or unaffected tissue. Comparing can be done manually,
against a
standard, in an electronic form (e.g., against a database), etc. The normal
control can be a
standard sample that is provided with the assay; it can be obtained from
adjacent, but
unaffected, tissue from the same patient; or, it can be pre-determined values,
etc. Gene
expression, protein expression (e.g., abundance in a cell), protein activity
(e.g., kinase
activity), etc., can be determined.
For instance, a biopsy from a cancer patient can be assayed for the presence,
quantity, and/or
activity of raf, VEGFR-2, VEGFR-3, PDGFR-beta, p38, and/or flt-3. Increased
expression or
activity of one or more of these can indicate that the cancer can be targeted
for treatment by a
compound of the present invention. For example, as described in the examples
below, raf
activity can be monitored by its ability to initiate the cascade leading to
ERK phosphorylation
(i.e., raf/MEK/ERK), resulting in phospho-ERK. Increased phospho-ERK levels in
a cancer
specimen shows that its raf activity is elevated, suggesting the use of
compounds of the
present invention to treat it.
Measuring expression includes determining or detecting the amount of the
polypeptide
present in a cell or shed by it, as well as measuring the underlying mRNA,
where the quantity
of mRNA present is considered to reflect the quantity of polypeptide
manufactured by the
cell. Furthermore, the genes for raf, VEGFR-2, VEGFR-3, PDGFR-beta, p38,
and/or Flt-3
can be analyzed to determine whether there is a gene defect responsible for
aberrant
expression or polypeptide activity.
Polypeptide detection can be carried out by any available method, e.g., by
Western blots,
ELISA, dot blot, immunoprecipitation, RIA, immunohistochemistry, etc. For
instance, a
tissue section can be prepared and labeled with a specific antibody (indirect
or direct and
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visualized with a microscope. Amount of a polypeptide can be quantitated
without
visualization, e.g., by preparing a lysate of a sample of interest, and then
determining by
ELISA or Western the amount of polypeptide per quantity of tissue. Antibodies
and other
specific binding agents can be used. There is no limitation on how detection
is performed.
Assays can be utilized which permit quantification and/or presence/absence
detection of a
target nucleic acid (e.g., genes, mRNA, etc., for raf, VEGFR, PDGFR, p38,
and/or flt-3) in a
sample. Assays can be performed at the single-cell level, or in a sample
comprising many
cells, where the assay is "averaging" expression over the entire collection of
cells and tissue
present in the sample. Any suitable assay format can be used, including, but
not limited to,
e.g., Southern blot analysis, Northern blot analysis, polymerase chain
reaction ("PCR") (e.g.,
Saiki et al., Science 1988, 241, 53; U.S. Pat. Nos. 4,683,195, 4,683,202, and
6,040,166; PCR
Protocols: A Guide to Methods and Applications, Innis et al., eds., Academic
Press, New
York, 1990), reverse transcriptase polymerase chain reaction ("RT-PCR"),
anchored PCR,
rapid amplification of cDNA ends ("RACE") (e.g., Schaefer in Gene Cloning and
Analysis:
Current Innovations, Pages 99-115, 1997), ligase chain reaction ("LCR") (EP
320 308), one-
sided PCR (Ohara et al., Proc. Natl. Acad. Sci. 1989, 86, 5673-5677), indexing
methods (e.g.,
U.S. Pat. No. 5,508,169), in situ hybridization, differential display (e.g.,
Liang et al., Nucl.
Acid. Res. 1993, 21, 3269 3275; U.S. Pat. Nos. 5,262,311, 5,599,672 and
5,965,409;
W097/18454; Prashar and Weissman, Proc. Natl. Acad. Sci., 93:659-663, and U.S.
Pat. Nos.
6,010,850 and 5,712,126; Welsh et al., Nucleic Acid Res., 20:4965-4970, 1992,
and U.S. Pat.
No. 5,487,985) and other RNA fingerprinting techniques, nucleic acid sequence
based
amplification ("NASBA") and other transcription based amplification systems
(e.g., U.S. Pat.
Nos. 5,409,818 and 5,554,527; WO 88/10315), polynucleotide arrays (e.g., U.S.
Pat. Nos.
5,143,854, 5,424,186; 5,700,637, 5,874,219, and 6,054,270; PCT WO 92/10092;
PCT WO
90/15070), Qbeta Replicase (PCT/US87/00880), Strand Displacement Amplification
("SDA"), Repair Chain Reaction ("RCR"), nuclease protection assays,
subtraction-based
methods, Rapid-Scan, etc. Additional useful methods include, but are not
limited to, e.g.,
template-based amplification methods, competitive PCR (e.g., U.S. Pat. No.
5,747,251),
redox-based assays (e.g., U.S. Pat. No. 5,871,918), Taqman-based assays (e.g.,
Holland et al.,
Proc. Natl. Acad, Sci. 1991, 88, 7276-7280; U.S. Pat. Nos. 5,210,015 and
5,994,063), real-
time fluorescence-based monitoring (e.g., U.S. Pat. 5,928,907), molecular
energy transfer
labels (e.g., U.S. Pat. Nos. 5,348,853, 5,532,129, 5,565,322, 6,030,787, and
6,117,635; Tyagi
and Kramer, Nature Biotech., 14:303-309, 1996). Any method suitable for single
cell
27 BAYER-0183-WO
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analysis of gene or protein expression can be used, including in situ
hybridization,
immunocytochemistry, MACS, FACS, flow cytometry, etc. For single cell assays,
expression products can be measured using antibodies, PCR, or other types of
nucleic acid
amplification (e.g., Brady et al., Methods Mol. & Cell. Biol. 1990, 2, 17-25;
Eberwine et al.,
Proc. Natl. Acad. Sci. 1992, 89, 3010-3014; U.S. Pat. No. 5,723,290). These
and other
methods can be carried out conventionally, e.g., as described in the mentioned
publications.
Activity of raf, VEGFR-2, VEGFR-3, PDGFR-beta, p38, and/or flt-3 can be
assessed
routinely, e.g., as described in the examples below, or using standard assays
for kinase
activity.
The present invention also provides methods of assessing the efficacy of a
compound of the
present invention in treating a disorder, comprising one or more of the
following steps in any
effective order, e.g., administering an amount of a compound, measuring the
expression or
activity of raf, VEGFR-2, VEGFR-3, PDGFR-beta, p38, and/or flt-3 (see above),
determining
the effect of said compound on said expression or activity. For instance,
biopsy samples can
be removed from patients who have been treated with a compound of the present
invention,
and then assayed for the presence and/or activity of the mentioned signaling
molecules.
Similarly, as discussed above, decreases in the levels of phospho-ERK in the
cancer tissue
(e.g., compared to normal tissue or before treatment) indicate that the
compound is exerting
in vivo efficacy and a therapeutic effect. The method can be used to determine
appropriate
dosages and dosing regimens, e.g., how much compound to administer and at what
frequency
to administer it. By monitoring its effect on the signaling molecules in the
tissue, the
clinician can determine the appropriate treatment protocol and whether it is
achieving the
desired effect, e.g., on modulating or inhibiting the signal transduction
pathway.
Compounds of the present invention also can be used as markers to determine
the presence
and quantity of raf, VEGFR-2, VEGFR-3, PDGFR-beta, p38, and/or flt-3, in a
sample
comprising a biological material. This comprises one or more of the following
steps in any
effective order: (i) contacting said sample comprising a biological material
with a compound
of the present invention, and (ii) determining whether said compound binds to
said material.
The compound can be labeled, or it can be used as a competitor to a labeled
compound, such
as labeled-ATP.
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The invention also provides methods for treating, preventing, modulating,
etc., diseases and
conditions in mammals comprising administering a compound of this invention
with another
modulator of the signal transduction pathway comprising, but not limited to
raf, VEGFR,
PDGFR, p38, and/or flt-3. These can be present in the same composition or in
separate
formulations or dosage units. Administration can be the same or different
routes, and can be
simultaneous or sequential.
The following publications relate to VEGFR-3 modulation and are incorporated
herein for
their description of disease states mediated by VEGFR-3 and assays to
determine such
activity.
W095/33772 Alitalo, et. al.
W095/33050 Charnock-Jones, et. al..
W096/39421 Hu, et. al.
W098/33917 Alitalo, et. al.
W002/057299 Alitalo, et. al.
W002/060950 Alitalo, et. al.
W002/081520 Boesen, et. al.
The following publications relate to VEGFR-2 modulation and are incorporated
herein for
their description of disease states mediated by VEGFR-2 and assays to
determine such
activity.
EP0882799 Hanai, et. al.
EP1167384 Ferraram, et, al.
EP1086705 Sato, et. al.
EP11300032 Tesar, et. al.
EP1166798 Haberey, et. al.
EP1166799 Haberey, et. al.
3o EP1170017 Maini, et. al.
EP1203827 Smith
W002/083850 Rosen, et. al.
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The following publications relate to flt-3 modulation and are incorporated
herein for their
description of disease states mediated by flt-3 and assays to determine such
activity.
2002/0034517 Brasel, et. al.
2002/0107365 Lyman, et. al.
2002/0111475 Graddis, et. al.
EP0627487 Beckermann, et. al.
W09846750 Bauer, et. al.
W09818923 McWherter, et. al.
lo W09428391 Beckermann, et al.
W09426891 Birnbaum, et. al.
The following patents and publication relate to PDGF/PDGFR modulation and are
incorporated herein for their description of the disease states mediated by
PDGFR-beta and
assays to determine such activity.
5,094,941 Hart, et. al.
5,371,205 Kelly, et. al.
5,418,135 Pang
5,444,151 Vassbotn, et. al.
5,468,468 LaRochelle, et. al.
5,567,584 Sledziewski, et. al.
5,618,678 Kelly, et. al.
5,620,687 Hart, et. al.
5,648,076 Ross, et. al.
5,668,264 Janjic, et. al.
5,686,572 Wolf, et. al.
5,817,310 Ramakrishnan, et. al.
5,833,986 LaRochelle, et. al.
5,863,739 LaRochelle, et. al.
5,872,218 Wolf, et. al.
5,882,644 Chang, et. al.
5,891,652 Wolf, et. al.
5,976,534 Hart, et. al.
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5,990,141 Hirth, et. al.
6,022,854 Shuman
6,043,211 Williams, et. al.
6,110,737 Escobedo, et. al.
6,207,816B 1 Gold, et. al.
6,228,600B 1 Matsui, et. al.
6,229,002B1 Janjic, et. al.
6,316,603131 McTigue, et. al.
6,372,438131 Williams, et. al.
6,403,769B 1 La Rochelle, et. al.
6,440,445B 1 Nowak, et. al.
6,475,782B 1 Escobedo, et. al.
W002/083849 Rosen, et. al.
W002/083704 Rosen, et. al.
W002/081520 Boesen, et. al.
W002/079498 Thomas, et. al.
W002/070008 Rockwell, et. al.
W009959636 Sato, et. al.
W009946364 Cao, et. al.
W009940118 Hanai, et. al.
W09931238 Yabana, et. al.
W09929861 Klagsbrun, et. al.
W09858053 Kendall, et. al.
W09851344 Maini, et. al.
W09833917 Alitalo, et. al.
W09831794 Matsumoto, et. al.
W09816551 Ferrara, et. al.
W09813071 Kendall, et al.
W09811223 Martiny-Baron, et. al.
W09744453 Chen, et. al.
W09723510 Plouet, et. al.
W09715662 Stinchcomb, et. al.
W09708313 Ferrara, et. al.
W09639515 Cao, et. al.
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W09623065 Smith, et. al.
W09606641 Fleurbaaij, et. al.
W09524473 Cao, et. al.
W09822316 Kyowa
W09521868 Rockwell, et. al.
W002/060489 Xia, et. al.
PDGFR-beta
1o EP0869177 Matsui, et. al.
W009010013 Matsui, et. al.
W09737029 Matsui, et. al.
PDGFR-alpha
EP1000617 Lammers, et. al.
EP0869177 Matsui, et. al.
EP0811685 Escobedo, et. al.
Pharmaceutical compositions based on the compounds of the present invention
This invention also relates to pharmaceutical compositions containing a
compound of the
present invention and pharmaceutically acceptable salts thereof. These
compositions can be
utilized to achieve the desired pharmacological effect by administration to a
patient in need
thereof. A patient, for the purpose of this invention, is a mammal, including
a human, in need
of treatment for the particular condition or disease. Therefore, the present
invention includes
pharmaceutical compositions which are comprised of a pharmaceutically
acceptable carrier
and a pharmaceutically effective amount of a compound, or salt thereof, of the
present
invention. The term "pharmaceutically acceptable carrier" is meant as any
carrier which is
relatively non-toxic and innocuous to a patient at concentrations consistent
with effective
activity of the active ingredient so that any side effects ascribable to the
carrier do not vitiate
the beneficial effects of the active ingredient. A pharmaceutically effective
amount of
compound is that amount which produces a result or exerts an influence on the
particular
condition being treated. The compound of the present invention can be
administered with
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pharmaceutically-acceptable carriers well known in the art using any effective
conventional
dosage unit forms, including immediate, slow and timed release preparations,
orally,
parenterally, topically, nasally, ophthalmically, optically, sublingually,
rectally, vaginally,
and the like.
For oral administration, the compound can be formulated into solid or liquid
preparations
such as capsules, pills, tablets, troches, lozenges, melts, powders,
solutions, suspensions, or
emulsions, and may be prepared according to methods known to the art for the
manufacture
of pharmaceutical compositions. The solid unit dosage forms can be a capsule
which can be
of the ordinary hard- or soft-shelled gelatin type containing, for example,
surfactants,
lubricants, and inert fillers such as lactose, sucrose, calcium phosphate, and
corn starch.
In another embodiment, the compounds of this invention may be tableted with
conventional
tablet bases such as lactose, sucrose and cornstarch in combination with
binders such as
acacia, corn starch or gelatin, disintegrating agents intended to assist the
break-up and
dissolution of the tablet following administration such as potato starch,
alginic acid, corn
starch, and guar gum, gum tragacanth, acacia, lubricants intended to improve
the flow of
tablet granulation and to prevent the adhesion of tablet material to the
surfaces of the tablet
dies and punches, for example talc, stearic acid, or magnesium, calcium or
zinc stearate,
dyes, coloring agents, and flavoring agents such as peppermint, oil of
wintergreen, or cherry
flavoring, intended to enhance the aesthetic qualities of the tablets and make
them more
acceptable to the patient. Suitable excipients for use in oral liquid dosage
forms include
dicalcium phosphate and diluents such as water and alcohols, for example,
ethanol, benzyl
alcohol, and polyethylene alcohols, either with or without the addition of a
pharmaceutically
acceptable surfactant, suspending agent or emulsifying agent. Various other
materials may be
present as coatings or to otherwise modify the physical form of the dosage
unit. For instance
tablets, pills or capsules may be coated with shellac, sugar or both.
Dispersible powders and granules are suitable for the preparation of an
aqueous suspension.
They provide the active ingredient in admixture with a dispersing or wetting
agent, a
suspending agent and one or more preservatives. Suitable dispersing or wetting
agents and
suspending agents are exemplified by those already mentioned above. Additional
excipients,
for example those sweetening, flavoring and coloring agents described above,
may also be
present.
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The pharmaceutical compositions of this invention may also be in the form of
oil-in-water
emulsions. The oily phase may be a vegetable oil such as liquid paraffin or a
mixture of
vegetable oils. Suitable emulsifying agents may be (1) naturally occurring
gums such as gum
acacia and gum tragacanth, (2) naturally occurring phosphatides such as soy
bean and
lecithin, (3) esters or partial esters derived form fatty acids and hexitol
anhydrides, for
example, sorbitan monooleate, (4) condensation products of said partial esters
with ethylene
oxide, for example, polyoxyethylene sorbitan monooleate. The emulsions may
also contain
sweetening and flavoring agents.
Oily suspensions may be formulated by suspending the active ingredient in a
vegetable oil
such as, for example, arachis oil, olive oil, sesame oil or coconut oil, or in
a mineral oil such
as liquid paraffin. The oily suspensions may contain a thickening agent such
as, for example,
beeswax, hard paraffin, or cetyl alcohol. The suspensions may also contain one
or more
preservatives, for example, ethyl or n-propyl p-hydroxybenzoate; one or more
coloring
agents; one or more flavoring agents; and one or more sweetening agents such
as sucrose or
saccharin.
Syrups and elixirs may be formulated with sweetening agents such as, for
example, glycerol,
propylene glycol, sorbitol or sucrose. Such formulations may also contain a
demulcent, and
preservative, such as methyl and propyl parabens and flavoring and coloring
agents.
The compounds of this invention may also be administered parenterally, that
is,
subcutaneously, intravenously, intraocularly, intrasynovially,
intramuscularly, or
interperitoneally, as injectable dosages of the compound in a physiologically
acceptable
diluent with a pharmaceutical carrier which can be a sterile liquid or mixture
of liquids such
as water, saline, aqueous dextrose and related sugar solutions, an alcohol
such as ethanol,
isopropanol, or hexadecyl alcohol, glycols such as propylene glycol or
polyethylene glycol,
glycerol ketals such as 2,2-dimethyl-1,1-dioxolane-4-methanol, ethers such as
poly(ethylene
glycol) 400, an oil, a fatty acid, a fatty acid ester or, a fatty acid
glyceride, or an acetylated
fatty acid glyceride, with or without the addition of a pharmaceutically
acceptable surfactant
such as a soap or a detergent, suspending agent such as pectin, carbomers,
methycellulose,
hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agent
and other
pharmaceutical adjuvants.
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Illustrative of oils which can be used in the parenteral formulations of this
invention are those
of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil,
soybean oil,
sesame oil, cottonseed oil, corn oil, olive oil, petrolatum and mineral oil.
Suitable fatty acids
include oleic acid, stearic acid, isostearic acid and myristic acid. Suitable
fatty acid esters are,
for example, ethyl oleate and isopropyl myristate. Suitable soaps include
fatty acid alkali
metal, ammonium, and triethanolamine salts and suitable detergents include
cationic
detergents, for example dimethyl dialkyl ammonium halides, alkyl pyridinium
halides, and
alkylamine acetates; anionic detergents, for example, alkyl, aryl, and olefin
sulfonates, alkyl,
olefin, ether, and monoglyceride sulfates, and sulfosuccinates; non-ionic
detergents, for
example, fatty amine oxides, fatty acid alkanolamides, and poly(oxyethylene-
oxypropylene)s
or ethylene oxide or propylene oxide copolymers; and amphoteric detergents,
for example,
alkyl-beta-aminopropionates, and 2-alkylimidazoline quarternary ammonium
salts, as well as
mixtures.
The parenteral compositions of this invention will typically contain from
about 0.5% to about
25% by weight of the active ingredient in solution. Preservatives and buffers
may also be
used advantageously. In order to minimize or eliminate irritation at the site
of injection, such
compositions may contain a non-ionic surfactant having a hydrophile-lipophile
balance
(HLB) of from about 12 to about 17. The quantity of surfactant in such
formulation ranges
from about 5% to about 15% by weight. The surfactant can be a single component
having the
above HLB or can be a mixture of two or more components having the desired
HLB.
Illustrative of surfactants used in parenteral formulations are the class of
polyethylene
sorbitan fatty acid esters, for example, sorbitan monooleate and the high
molecular weight
adducts of ethylene oxide with a hydrophobic base, formed by the condensation
of propylene
oxide with propylene glycol.
The pharmaceutical compositions may be in the form of sterile injectable
aqueous
suspensions. Such suspensions may be formulated according to known methods
using
suitable dispersing or wetting agents and suspending agents such as, for
example, sodium
carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, sodium
alginate,
gum tragacanth and gum acacia; dispersing or wetting agents which may be a
naturally
occurring phosphatide such as lecithin, a condensation product of an alkylene
oxide with a
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fatty acid, for example, polyoxyethylene stearate, a condensation product of
ethylene oxide
with a long chain aliphatic alcohol, for example, heptadeca-
ethyleneoxycetanol, a
condensation product of ethylene oxide with a partial ester derived form a
fatty acid and a
hexitol such as polyoxyethylene sorbitol monooleate, or a condensation product
of an
ethylene oxide with a partial ester derived from a fatty acid and a hexitol
anhydride, for
example polyoxyethylene sorbitan monooleate.
The sterile injectable preparation may also be a sterile injectable solution
or suspension in a
non-toxic parenterally acceptable diluent or solvent. Diluents and solvents
that may be
employed are, for example, water, Ringer's solution, isotonic sodium chloride
solutions and
isotonic glucose solutions. In addition, sterile fixed oils are conventionally
employed as
solvents or suspending media. For this purpose, any bland, fixed oil may be
employed
including synthetic mono- or diglycerides. In addition, fatty acids such as
oleic acid can be
used in the preparation of injectables.
A composition of the invention may also be administered in the form of
suppositories for
rectal administration of the drug. These compositions can be prepared by
mixing the drug
with a suitable non-irritation excipient which is solid at ordinary
temperatures but liquid at
the rectal temperature and will therefore melt in the rectum to release the
drug. Such material
is, for example, cocoa butter and polyethylene glycol.
Another formulation employed in the methods of the present invention employs
transdermal
delivery devices ("patches"). Such transdermal patches may be used to provide
continuous or
discontinuous infusion of the compounds of the present invention in controlled
amounts. The
construction and use of transdermal patches for the delivery of pharmaceutical
agents is well
known in the art (see, e.g., US Patent No. 5,023,252, issued June 11, 1991,
incorporated
herein by reference). Such patches may be constructed for continuous,
pulsatile, or on
demand delivery of pharmaceutical agents.
Controlled release formulations for parenteral administration include
liposomal, polymeric
microsphere and polymeric gel formulations which are known in the art.
It may be desirable or necessary to introduce the pharmaceutical composition
to the patient
via a mechanical delivery device. The construction and use of mechanical
delivery devices
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for the delivery of pharmaceutical agents is well known in the art. Direct
techniques for, for
example, administering a drug directly to the brain usually involve placement
of a drug
delivery catheter into the patient's ventricular system to bypass the blood-
brain barrier. One
such implantable delivery system, used for the transport of agents to specific
anatomical
regions of the body, is described in US Patent No. 5,011,472, issued April 30,
1991.
The compositions of the invention can also contain other conventional
pharmaceutically
acceptable compounding ingredients, generally referred to as carriers or
diluents, as
necessary or desired. Conventional procedures for preparing such compositions
in
appropriate dosage forms can be utilized. Such ingredients and procedures
include those
described in the following references, each of which is incorporated herein by
reference:
Powell, M.F. et al, "Compendium of Excipients for Parenteral Formulations" PDA
Journal
of Pharmaceutical Science & Technology 1998, 52(5), 238-311; Strickley, R.G
"Parenteral
Formulations of Small Molecule Therapeutics Marketed in the United States
(1999)-Part-1"
PDA Journal of Pharmaceutical Science & Technology 1999, 53(6), 324-349; and
Nema, S.
et al, "Excipients and Their Use in Injectable Products" PDA Journal of
Pharmaceutical
Science & Technology 1997, 51(4), 166-171.
Commonly used pharmaceutical ingredients which can be used as appropriate to
formulate
the composition for its intended route of administration include:
acidifying agents (examples include but are not limited to acetic acid, citric
acid, fumaric
acid, hydrochloric acid, nitric acid);
alkalinizing agents (examples include but are not limited to ammonia solution,
ammonium
carbonate, diethanolamine, monoethanolamine, potassium hydroxide, sodium
borate, sodium
carbonate, sodium hydroxide, triethanolamine, trolamine);
adsorbents (examples include but are not limited to powdered cellulose and
activated
charcoal);
aerosol propellants (examples include but are not limited to carbon dioxide,
CC12F2, F2C1C-
CC1F2 and CC1F3)
air displacement agents (examples include but are not limited to nitrogen and
argon);
antifungal preservatives (examples include but are not limited to benzoic
acid, butylparaben,
ethylparaben, methylparaben, propylparaben, sodium benzoate);
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antimicrobial preservatives (examples include but are not limited to
benzalkonium chloride,
benzethonium chloride, benzyl alcohol, cetylpyridinium chloride,
chlorobutanol, phenol,
phenylethyl alcohol, phenylmercuric nitrate and thimerosal);
antioxidants (examples include but are not limited to ascorbic acid, ascorbyl
palmitate,
butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorus acid,
monothioglycerol, propyl gallate, sodium ascorbate, sodium bisulfite, sodium
formaldehyde
sulfoxylate, sodium metabisulfite);
binding materials (examples include but are not limited to block polymers,
natural and
synthetic rubber, polyacrylates, polyurethanes, silicones, polysiloxanes and
styrene-butadiene
copolymers);
buffering agents (examples include but are not limited to potassium
metaphosphate,
dipotassium phosphate, sodium acetate, sodium citrate anhydrous and sodium
citrate
dihydrate)
carrying agents (examples include but are not limited to acacia syrup,
aromatic syrup,
aromatic elixir, cherry syrup, cocoa syrup, orange syrup, syrup, corn oil,
mineral oil, peanut
oil, sesame oil, bacteriostatic sodium chloride injection and bacteriostatic
water for injection)
chelating agents (examples include but are not limited to edetate disodium and
edetic acid)
colorants (examples include but are not limited to FD&C Red No. 3, FD&C Red
No. 20,
FD&C Yellow No. 6, FD&C Blue No. 2, D&C Green No. 5, D&C Orange No. 5, D&C Red
No. 8, caramel and ferric oxide red);
clarifying agents (examples include but are not limited to bentonite);
emulsifying agents (examples include but are not limited to acacia,
cetomacrogol, cetyl
alcohol, glyceryl monostearate, lecithin, sorbitan monooleate, polyoxyethylene
50
monostearate);
encapsulating agents (examples include but are not limited to gelatin and
cellulose acetate
phthalate)
flavorants (examples include but are not limited to anise oil, cinnamon oil,
cocoa, menthol,
orange oil, peppermint oil and vanillin);
humectants (examples include but are not limited to glycerol, propylene glycol
and sorbitol);
levigating agents (examples include but are not limited to mineral oil and
glycerin);
oils (examples include but are not limited to arachis oil, mineral oil, olive
oil, peanut oil,
sesame oil and vegetable oil);
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ointment bases (examples include but are not limited to lanolin, hydrophilic
ointment,
polyethylene glycol ointment, petrolatum, hydrophilic petrolatum, white
ointment, yellow
ointment, and rose water ointment);
penetration enhancers (transdermal delivery) (examples include but are not
limited to
monohydroxy or polyhydroxy alcohols, mono-or polyvalent alcohols, saturated or
unsaturated fatty alcohols, saturated or unsaturated fatty esters, saturated
or unsaturated
dicarboxylic acids, essential oils, phosphatidyl derivatives, cephalin,
terpenes, amides, ethers,
ketones and ureas)
plasticizers (examples include but are not limited to diethyl phthalate and
glycerol);
solvents (examples include but are not limited to ethanol, corn oil,
cottonseed oil, glycerol,
isopropanol, mineral oil, oleic acid, peanut oil, purified water, water for
injection, sterile
water for injection and sterile water for irrigation);
stiffening agents (examples include but are not limited to cetyl alcohol,
cetyl esters wax,
microcrystalline wax, paraffin, stearyl alcohol, white wax and yellow wax);
suppository bases (examples include but are not limited to cocoa butter and
polyethylene
glycols (mixtures));
surfactants (examples include but are not limited to benzalkonium chloride,
nonoxynol 10,
oxtoxynol 9, polysorbate 80, sodium lauryl sulfate and sorbitan mono-
palmitate);
suspending agents (examples include but are not limited to agar, bentonite,
carbomers,
carboxymethylcellulose sodium, hydroxyethyl cellulose, hydroxypropyl
cellulose,
hydroxypropyl methylcellulose, kaolin, methylcellulose, tragacanth and
veegum);
sweetening agents (examples include but are not limited to aspartame,
dextrose, glycerol,
mannitol, propylene glycol, saccharin sodium, sorbitol and sucrose);
tablet anti-adherents (examples include but are not limited to magnesium
stearate and talc);
tablet binders (examples include but are not limited to acacia, alginic acid,
carboxymethylcellulose sodium, compressible sugar, ethylcellulose, gelatin,
liquid glucose,
methylcellulose, and pregelatinized starch);
tablet and capsule diluents (examples include but are not limited to dibasic
calcium
phosphate, kaolin, lactose, mannitol, microcrystalline cellulose, powdered
cellulose,
precipitated calcium carbonate, sodium carbonate, sodium phosphate, sorbitol
and starch);
tablet coating agents (examples include but are not limited to liquid glucose,
hydroxyethyl
cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,
methylcellulose,
ethylcellulose, cellulose acetate phthalate and shellac);
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tablet direct compression excipients (examples include but are not limited to
dibasic calcium
phosphate);
tablet disintegrants (examples include but are not limited to alginic acid,
carboxymethylcellulose calcium, microcrystalline cellulose, polacrillin
potassium, sodium
alginate, sodium starch glycollate and starch);
tablet glidants (examples include but are not limited to colloidal silica,
corn starch and talc);
tablet lubricants (examples include but are not limited to calcium stearate,
magnesium
stearate, mineral oil, stearic acid and zinc stearate);
tablet/capsule opaquants (examples include but are not limited to titanium
dioxide);
tablet polishing agents (examples include but are not limited to carnauba wax
and white
wax);
thickening agents (examples include but are not limited to beeswax, cetyl
alcohol and
paraffin);
tonicity agents (examples include but are not limited to dextrose and sodium
chloride);
viscosity increasing agents (examples include but are not limited to alginic
acid, bentonite,
carbomers, carboxymethylcellulose sodium, methylcellulose, sodium alginate and
tragacanth); and
wetting agents (examples include but are not limited to heptadecaethylene
oxycetanol,
lecithin, sorbitol monooleate, polyoxyethylene sorbitol monooleate, and
polyoxyethylene
stearate).
Pharmaceutical compositions according to the present invention can be
illustrated as follows:
Sterile IV Solution: a 5 mg/mL solution of the desired compound of this
invention is made
using sterile, injectable water, and the pH is adjusted if necessary. The
solution is diluted for
administration to 1 - 2 mg/mL with sterile 5% dextrose and is administered as
an IV infusion
over 60 minutes.
Lyophilized powder for IV administration: A sterile preparation can be
prepared with (i) 100
- 1000 mg of the desired compound of this invention as a lypholized powder,
(ii) 32- 327
mg/mL sodium citrate, and (iii) 300 - 3000 mg Dextran 40. The formulation is
reconstituted
with sterile, injectable saline or dextrose 5% to a concentration of 10 to 20
mg/mL, which is
further diluted with saline or dextrose 5% to 0.2 - 0.4 mg/mL, and is
administered either IV
bolus or by IV infusion over 15 - 60 minutes.
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Intramuscular suspension: The following solution or suspension can be
prepared, for
intramuscular injection:
50 mg/mL of the desired, water-insoluble compound of this invention
5 mg/mL sodium carboxymethylcellulose
4 mg/mL Tween 80
9 mg/mL sodium chloride
9 mg/mL benzyl alcohol
Hard Shell Capsules: A large number of unit capsules are prepared by filling
standard two-
piece hard galantine capsules each with 100 mg of powdered active ingredient,
150 mg of
lactose, 50 mg of cellulose and 6 mg of magnesium stearate.
Soft Gelatin Capsules: A mixture of active ingredient in a digestible oil such
as soybean oil,
cottonseed oil or olive oil is prepared and injected by means of a positive
displacement pump
into molten gelatin to form soft gelatin capsules containing 100 mg of the
active ingredient.
The capsules are washed and dried. The active ingredient can be dissolved in a
mixture of
polyethylene glycol, glycerin and sorbitol to prepare a water miscible
medicine mix.
Tablets: A large number of tablets are prepared by conventional procedures so
that the
dosage unit was 100 mg of active ingredient, 0.2 mg of colloidal silicon
dioxide, 5 mg of
magnesium stearate, 275 mg of microcrystalline cellulose, 11 mg of starch, and
98.8 mg of
lactose. Appropriate aqueous and non-aqueous coatings may be applied to
increase
palatability, improve elegance and stability or delay absorption.
Immediate Release Tablets/Capsules: These are solid oral dosage forms made by
conventional and novel processes. These units are taken orally without water
for immediate
dissolution and delivery of the medication. The active ingredient is mixed in
a liquid
containing ingredient such as sugar, gelatin, pectin and sweeteners. These
liquids are
solidified into solid tablets or caplets by freeze drying and solid state
extraction techniques.
The drug compounds may be compressed with viscoelastic and thermoelastic
sugars and
polymers or effervescent components to produce porous matrices intended for
immediate
release, without the need of water.
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Dosage of the pharmaceutical compositions of the present invention
Based upon standard laboratory techniques known to evaluate compounds useful
for the
treatment of any of the aforementioned disorders, by standard toxicity tests
and by standard
pharmacological assays for the determination of treatment of the conditions
identified above
in mammals, and by comparison of these results with the results of known
medicaments that
are used to treat these conditions, the effective dosage of the compounds of
this invention can
readily be determined for treatment of each desired indication. The amount of
the active
ingredient to be administered in the treatment of one of these conditions can
vary widely
according to such considerations as the particular compound and dosage unit
employed, the
mode of administration, the period of treatment, the age and sex of the
patient treated, and the
nature and extent of the condition treated.
The total amount of the active ingredient to be administered can range from
about 0.001
mg/kg to about 200 mg/kg, and preferably from about 0.1 mg/kg to about 50
mg/kg body
weight per day. A unit dosage may preferably contain from about 5 mg to about
4000 mg of
active ingredient, and can be administered one or more times per day. The
daily dosage for
oral administration will preferably be from 0.1 to 50 mg/kg of total body
weight. The daily
dosage for administration by injection, including intravenous, intramuscular,
subcutaneous
and parenteral injections, and use of infusion techniques will preferably be
from 0.1 to 10
mg/kg of total body weight. The daily rectal dosage regimen will preferably be
from 0.1 to 50
mg/kg of total body weight. The daily vaginal dosage regimen will preferably
be from 0.1 to
50 mg/kg of total body weight. The daily topical dosage regimen will
preferably be from 0.1
to 10 mg/kg administered between one to four times daily. The transdermal
concentration
will preferably be that required to maintain a daily dose of from 0.1 to 10
mg/kg. The daily
inhalation dosage regimen will preferably be from 0.1 to 10 mg/kg of total
body weight.
Other dosages and amounts can be selected routinely.
The specific initial and continuing dosage regimen for each patient will vary
according to the
nature and severity of the condition as determined by the attending
diagnostician, the activity
of the specific compound employed, the age and general condition of the
patient, time of
administration, route of administration, rate of excretion of the drug, drug
combinations, and
the like. The desired mode of treatment and number of doses of a compound of
the present
invention or a pharmaceutically acceptable salt or ester or composition
thereof can be
ascertained by those skilled in the art using conventional treatment tests.
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Combination of the compounds and compositions of the present invention with
additional
active ingredients
Compounds of this invention can be administered as the sole pharmaceutical
agent or in
combination with one or more other pharmaceutical agents where the combination
causes no
unacceptable adverse effects. This may be of particular relevance for the
treatment of hyper-
proliferative diseases such as cancer. In this instance, the compound of this
invention can be
combined with known cytotoxic agents, signal transduction inhibitors, or with
other anti-
cancer agents, as well as with admixtures and combinations thereof.
In one embodiment, the compounds of the present invention can be combined with
cytotoxic
anti-cancer agents. Examples of such agents can be found in the 11th Edition
of the Merck
Index (1996). These agents include, by no way of limitation, asparaginase,
bleomycin,
carboplatin, carmustine, chlorambucil, cisplatin, colaspase, cyclophosphamide,
cytarabine,
dacarbazine, dactinomycin, daunorubicin, doxorubicin (adriamycine),
epirubicin, etoposide,
5-fluorouracil, hexamethylmelamine, hydroxyurea, ifosfamide, irinotecan,
leucovorin,
lomustine, mechlorethamine, 6-mercaptopurine, mesna, methotrexate, mitomycin
C,
mitoxantrone, prednisolone, prednisone, procarbazine, raloxifen, streptozocin,
tamoxifen,
thioguanine, topotecan, vinblastine, vincristine, and vindesine.
Other cytotoxic drugs suitable for use with the compounds of the invention
include, but are
not limited to, those compounds acknowledged to be used in the treatment of
neoplastic
diseases in Goodman and Gilman's The Pharmacological Basis of Therapeutics
(Ninth
Edition, 1996, McGraw-Hill). These agents include, by no way of limitation,
aminoglutethimide, L-asparaginase, azathioprine, 5-azacytidine cladribine,
busulfan,
diethylstilbestrol, 2', 2'-difluorodeoxycytidine, docetaxel,
erythrohydroxynonyladenine,
ethinyl estradiol, 5-fluorodeoxyuridine, 5-fluorodeoxyuridine monophosphate,
fludarabine
phosphate, fluoxymesterone, flutamide, hydroxyprogesterone caproate,
idarubicin, interferon,
medroxyprogesterone acetate, megestrol acetate, melphalan, mitotane,
paclitaxel, pentostatin,
N-phosphonoacetyl-L-aspartate (PALA), plicamycin, semustine, teniposide,
testosterone
propionate, thiotepa, trimethylmelamine, uridine, and vinorelbine.
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Other cytotoxic anti-cancer agents suitable for use in combination with the
compounds of the
invention also include newly discovered cytotoxic principles such as
oxaliplatin, gemcitabine,
capecitabine, epothilone and its natural or synthetic derivatives,
temozolomide (Quinn et al.,
J. Clin. Oncology 2003, 21(4), 646-651), tositumomab (Bexxar), trabedectin
(Vidal et al.,
Proceedings of the American Society for Clinical Oncology 2004, 23, abstract
3181), and the
inhibitors of the kinesin spindle protein Eg5 (Wood et al., Curr. Opin.
Pharmacol. 2001, 1,
370-377).
In another embodiment, the compounds of the present invention can be combined
with other
signal transduction inhibitors. Of particular interest are signal transduction
inhibitors which
target the EGFR family, such as EGFR, HER-2, and HER-4 (Raymond et al., Drugs
2000, 60
(Suppl.1), 15-23; Harari et al., Oncogene 2000, 19 (53), 6102-6114), and their
respective
ligands. Examples of such agents include, by no way of limitation, antibody
therapies such as
Herceptin (trastuzumab), Erbitux (cetuximab), and pertuzumab. Examples of such
therapies
also include, by no way of limitation, small-molecule kinase inhibitors such
as ZD-1839 /
Iressa (Baselga et al., Drugs 2000, 60 (Suppl. 1), 33-40), OSI-774 / Tarceva
(Pollack et al. J.
Pharm. Exp. Ther. 1999, 291(2), 739-748), CI-1033 (Bridges, Curr. Med. Chem.
1999, 6,
825-843), GW-2016 (Lackey et al., 92nd AACR Meeting, New Orleans, March 24-28,
2001,
abstract 4582), CP-724,714 (Jani et al., Proceedings of the American Society
for Clinical
Oncology 2004, 23, abstract 3122), HKI-272 (Rabindran et al., Cancer Res.
2004, 64, 3958-
3965), and EKB-569 (Greenberger et al., 11th NCI-EORTC-AACR Symposium on New
Drugs in Cancer Therapy, Amsterdam, November 7-10, 2000, abstract 388).
In another embodiment, the compounds of the present invention can be combined
with other
signal transduction inhibitors targeting receptor kinases of the split-kinase
domain families
(VEGFR, FGFR, PDGFR, flt-3, c-kit, c-fms, and the like), and their respective
ligands. These
agents include, by no way of limitation, antibodies such as Avastin
(bevacizumab). These
agents also include, by no way of limitation, small-molecule inhibitors such
as STI-571 /
Gleevec (Zvelebil, Curr. Opin. Oncol., Endocr. Metab. Invest. Drugs 2000,
2(1), 74-82),
PTK-787 (Wood et al., Cancer Res. 2000, 60(8), 2178-2189), SU-11248 (Demetri
et al.,
Proceedings of the American Society for Clinical Oncology 2004, 23, abstract
3001), ZD-
6474 (Hennequin et al., 92nd AACR Meeting, New Orleans, March 24-28, 2001,
abstract
3152), AG-13736 (Herbst et al., Clin. Cancer Res. 2003, 9, 16 (suppl 1),
abstract C253),
KRN-951 (Taguchi et al., 95th AACR Meeting, Orlando, FL, 2004, abstract 2575),
CP-
44 BAYER-0183-WO
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547,632 (Beebe et al., Cancer Res. 2003, 63, 7301-7309), CP-673,451 (Roberts
et al.,
Proceedings of the American Association of Cancer Research 2004, 45, abstract
3989),
CHIR-258 (Lee et al., Proceedings of the American Association of Cancer
Research 2004,
45, abstract 2130), MLN-518 (Shen et al., Blood 2003, 102, 11, abstract 476),
and AZD-2171
(Hennequin et al., Proceedings of the American Association of Cancer Research
2004, 45,
abstract 4539).
In another embodiment, the compounds of the present invention can be combined
with
inhibitors of the Raf/MEK/ERK transduction pathway (Avruch et al., Recent
Prog. Horm.
1o Res. 2001, 56, 127-155), or the PKB (akt) pathway (Lawlor et al., J. Cell
Sci. 2001, 114,
2903-2910). These include, by no way of limitation, PD-325901 (Sebolt-Leopold
et al.,
Proceedings of the American Association of Cancer Research 2004, 45, abstract
4003), and
ARRY-142886 (Wallace et al., Proceedings of the American Association of Cancer
Research
2004, 45, abstract 3891).
In another embodiment, the compounds of the present invention can be combined
with
inhibitors of histone deacetylase. Examples of such agents include, by no way
of limitation,
suberoylanilide hydroxamic acid (SAHA), LAQ-824 (Ottmann et al., Proceedings
of the
American Society for Clinical Oncology 2004, 23, abstract 3024), LBH-589 (Beck
et al.,
Proceedings of the American Society for Clinical Oncology 2004, 23, abstract
3025), MS-
275 (Ryan et al., Proceedings of the American Association of Cancer Research
2004, 45,
abstract 2452), and FR-901228 (Piekarz et al., Proceedings of the American
Society for
Clinical Oncology 2004, 23, abstract 3028).
In another embodiment, the compounds of the present invention can be combined
with other
anti-cancer agents such as proteasome inhibitors, and m-TOR inhibitors. These
include, by no
way of limitation, bortezomib (Mackay et al., Proceedings of the American
Society for
Clinical Oncology 2004, 23, Abstract 3109), and CCI-779 (Wu et al.,
Proceedings of the
American Association of Cancer Research 2004, 45, abstract 3849).
Generally, the use of cytotoxic and/or cytostatic anti-cancer agent in
combination with a
compound or composition of the present invention for the treatment of cancer
will serve to:
(1) yield better efficacy in reducing the growth of a tumor or even eliminate
the tumor as
compared to administration of either agent alone,
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(2) provide for the administration of lesser amounts of the administered
chemotherapeutic
agents,
(3) provide for a chemotherapeutic treatment that is well tolerated in the
patient with fewer
deleterious pharmacological complications than observed with single agent
chemotherapies
and certain other combined therapies,
(4) provide for treating a broader spectrum of different cancer types in
mammals, especially
humans,
(5) provide for a higher response rate among treated patients,
(6) provide for a longer survival time among treated patients compared to
standard
chemotherapy treatments,
(7) provide a longer time for tumor progression, and/or
(8) yield efficacy and tolerability results at least as good as those of the
agents used alone,
compared to known instances where other cancer agent combinations produce
antagonistic
effects.
Aspects of the instant invention include, but are not limited to:
In one embodiment, the present invention provides for the following aspects
Aspect 1. A synthetic metabolite of Formula (I) or a salt, or a prodrug or an
isolated
stereoisomer thereof
CF3 O
CI IOI / I O I NH,,CH3 I
: N )
NN J
I I
H H F
Aspect 2. A synthetic M2, M3, M4, or M5 metabolite of a compound of Formula I
or a
salt of said metabolite, wherein the structures of said M2, M3, M4, and M5
metabolites are:
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~\sA ~.,
t U M2
1011
r .- rte, raw. - .
MetaboLlte M
rr
ti
Meta bolas
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Metabolite M
Aspect 3. A pharmaceutically acceptable salt of synthetic metabolite according
to Aspect 2
which is
a) a basic salt of an organic acid or inorganic acid which is hydrochloric
acid,
hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid,
trifluoromethanesulfonic acid, benzenesulfonic acid, p-toluene sulfonic acid
(tosylate salt),
1-napthalene sulfonic acid, 2-napthalene sulfonic acid, acetic acid,
trifluoroacetic acid, malic
acid, tartaric acid, citric acid, lactic acid, oxalic acid, succinic acid,
fumaric acid, maleic acid,
benzoic acid, salicylic acid, phenylacetic acid, or mandelic acid; or
b) an acid salt of an organic or inorganic base containing an alkali metal
cation,
an alkaline earth metal cation, an ammonium cation, an aliphatic substituted
ammonium
cation or an aromatic substituted ammonium cation.
Aspect 4. The synthetic metabolite according to Aspect 2 which is a metabolite
of 4{4-
[3-(4-chloro-3-trifluoromethylphenyl)-ureido]-3-fluorophenoxy } -pyridine-2-
carboxylic acid
methylamide.
Aspect 5. A pharmaceutically acceptable salt of a synthetic metabolite
according to
Aspect 4 which is a basic salt of hydrochloric acid, hydrobromic acid,
sulfuric acid,
phosphoric acid, methanesulfonic acid, trifluoromethanesulfonic acid,
benzenesulfonic acid,
p-toluene sulfonic acid (tosylate salt), 1-napthalene sulfonic acid, 2-
napthalene sulfonic acid,
acetic acid, trifluoroacetic acid, malic acid, tartaric acid, citric acid,
lactic acid, oxalic acid,
succinic acid, fumaric acid, maleic acid, benzoic acid, salicylic acid,
phenylacetic acid, or
mandelic acid.
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Aspect 6. A compound which is which is a hydrochloride, benzenesulfonate, or
methanesulfonate salt of the synthetic metabolite according to Aspect 2.
Aspect 7. A pharmaceutical composition comprising a synthetic metabolite
according to
Aspect 2 and a physiologically acceptable carrier.
Aspect 8. A pharmaceutical composition comprising a synthetic metabolites
according
to Aspect 4 and a physiologically acceptable carrier.
Aspect 9. A pharmaceutical composition for the treatment of a disease in a
human or
other mammal regulated by a protein kinase, associated with an aberration in
the protein
kinase signal transduction pathway comprising a synthetic metabolite according
to Aspect 2
and a physiologically acceptable carrier.
Aspect 10. A pharmaceutical composition for the treatment of a hyper-
proliferative
disorder comprising a synthetic metabolite according to Aspect 4 and a
physiologically
acceptable carrier.
Aspect 11. A pharmaceutical composition for the treatment of a cancerous cell
growth
comprising a synthetic metabolite according to Aspect 2 and a physiologically
acceptable
carrier.
Aspect 12. A method for regulating tyrosine kinase signal transduction
comprising
administering to a human or other mammal a synthetic metabolite according to
Aspect 2.
Aspect 13. A method for treating or preventing a disease in a human or other
mammal
which is regulated by tyrosine kinase and associated with an aberration in the
tyrosine kinase
signal transduction pathway, said method comprising administering to a human
or other
mammal a synthetic metabolite according to Aspect 2.
Aspect 14. A method for treating or preventing a disease in a human and/or
other
mammal which is a VEGFR-2 mediated disorder, said method comprising
administering to a
human or other mammal a synthetic metabolite according to Aspect 2.
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Aspect 15. A method for treating or preventing a disease in a human and/or
other
mammal which is a PDGFR mediated disorder, said method comprising
administering to a
human or other mammal a synthetic metabolite according to Aspect 2.
Aspect 16. A method for treating or preventing a disease in a human or other
mammal
which is a raf-mediated disorder, said method comprising administering to a
human or other
mammal a synthetic metabolite according to Aspect 2.
Aspect 17. A method for treating or preventing a disease in a human or other
mammal
which is a p38-mediated disorder, said method comprising administering to a
human or other
mammal a synthetic metabolite according to Aspect 2.
Aspect 18. A method for treating or preventing a disease in a human or other
mammal
which is a VEGF-mediated disorder, said method comprising administering to a
human or
other mammal a synthetic metabolite according to Aspect 2.
Aspect 19. A method for treating or preventing a disease in a human or other
mammal
which is a hyper-proliferative, inflammatory and/or angiogenesis disorder
which comprises
administering to a human or other mammal a synthetic metabolite according to
Aspect 2.
Aspect 20. A method for treating or preventing a disease in a human or other
mammal
which is a hyper-proliferative disorder which comprises administering to a
human or other
mammal a synthetic metabolite according to Aspect 2.
Aspect 21. A method as in aspect 20, wherein the hyper-proliferative disorder
is cancer.
Aspect 22. A method as in aspect 21, wherein said method comprises
administering to a
human or other mammal a synthetic metabolite according to Aspect 2 in
combination with
one or several additional anti-cancer agents.
Aspect 23. A method for treating or preventing a disease in a human or other
mammal
characterized by abnormal angiogenesis or hyperpermiability processes
comprising
administering to a human or other mammal a synthetic metabolite according to
Aspect 2.
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Aspect 24. A method as in aspect 23, for treating or preventing a disease in a
human or
other mammal characterized by abnormal angiogenesis or hyperpermeability
processes,
comprising administering to a human or other mammal, a synthetic metabolite
according to
Aspect 2 simultaneously with another anti-angiogenesis agent, either in the
same formulation
or in separate formulations.
Aspect 25. A method for treating or preventing one or more of the following
conditions in
humans and/or other mammals:
tumor growth, retinopathy, ischemic retinal-vein occlusion, retinopathy of
prematurity, age related macular degeneration; rheumatoid arthritis,
psoriasis, a bullous
disorder associated with subepidermal blister formation, including bullous
pemphigoid,
erythema multiforme, or dermatitis herpetiformis, rheumatoid arthritis,
osteoarthritis, septic
arthritis, tumor metastasis, periodontal disease, cornal ulceration,
proteinuria and coronary
thrombosis from atherosclerotic plaque, aneurismal aortic, birth control,
dystrophobic
epidermolysis bullosa, degenerative cartilage loss following traumatic joint
injury,
osteopenias mediated by MMP activity, tempero mandibular joint disease or
demyelating
disease of the nervous system,
said method comprising administering to a human or other mammal, a synthetic
metabolite according to Aspect 2.
Aspect 26. A method for treating or preventing one or more of the following
conditions in
humans and/or other mammals: tumor growth, retinopathy, ischemic retinal-vein
occlusion,
retinopathy of prematurity, age related macular degeneration; rheumatoid
arthritis, psoriasis,
a bullous disorder associated with subepidermal blister formation, including
bullous
pemphigoid, erythema multiforme, or dermatitis herpetiformis;
in combination with another condition selected from the group consisting of:
rheumatic fever, bone resorption, postmenopausal osteoporosis, sepsis, gram
negative sepsis,
septic shock, endotoxic shock, toxic shock syndrome, systemic inflammatory
response
syndrome, inflammatory bowel disease (Krohn's disease and ulcerative colitis),
Jarisch-
Herxheimer reaction, asthma, adult respiratory distress syndrome, acute
pulmonary fibrotic
disease, pulmonary sarcoidosis, allergic respiratory disease, silicosis, coal
worker's
pneumoconiosis, alveolar injury, hepatic failure, liver disease during acute
inflammation,
severe alcoholic hepatitis, malaria (Plasmodium falciparum malaria and
cerebral malaria),
non-insulin-dependent diabetes mellitus (NIDDM), congestive heart failure,
damage
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following heart disease, atherosclerosis, Alzheimer's disease, acute
encephalitis, brain injury,
multiple sclerosis (demyelation and oligiodendrocyte loss in multiple
sclerosis), advanced
cancer, lymphoid malignancy, pancreatitis, impaired wound healing in
infection,
inflammation and cancer, myelodysplastic syndromes, systemic lupus
erythematosus, biliary
cirrhosis, bowel necrosis, radiation injury/ toxicity following administration
of monoclonal
antibodies, host-versus-graft reaction (ischemia reperfusion injury and
allograft rejections of
kidney, liver, heart, and skin), lung allograft rejection (obliterative
bronchitis) and
complications due to total hip replacement,
said method comprising administering to a human or other mammal a synthetic
metabolite according to Aspect 2.
Aspect 27. A method for treating or preventing one or more of the following
conditions in
humans and/or other mammals: tumor growth, retinopathy, diabetic retinopathy,
ischemic
retinal-vein occlusion, retinopathy of prematurity, age related macular
degeneration;
rheumatoid arthritis, psoriasis, bullous disorder associated with subepidermal
blister
formation, bullous pemphigoid, erythema multiforme, and dermatitis
herpetiformis,
in combination with an infectious disease selected from the group consisting
of :
tuberculosis, Helicobacter pylori infection during peptic ulcer disease,
Chaga's disease
resulting from Trypanosoma cruzi infection, effects of Shiga-like toxin
resulting from E. coli
infection, effects of enterotoxin A resulting from Staphylococcus infection,
meningococcal
infection, and infections from Borrelia burgdorferi, Treponema pallidum,
cytomegalovirus,
influenza virus, Theiler's encephalomyelitis virus, and the human
immunodeficiency virus
(HIV);
said method comprising administering to a human or other mammal a synthetic
metabolite
according to Aspect 2.
Aspect 28. A method as in aspect 22 wherein the additional anti-cancer agent
is selected
from the group consisting of asparaginase, bleomycin, carboplatin, carmustine,
chlorambucil,
cisplatin, colaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin,
daunorubicin, doxorubicin (adriamycine), epirubicin, etoposide, 5-
fluorouracil,
hexamethylmelamine, hydroxyurea, ifosfamide, irinotecan, leucovorin,
lomustine,
mechlorethamine, 6-mercaptopurine, mesna, methotrexate, mitomycin C,
mitoxantrone,
prednisolone, prednisone, procarbazine, raloxifen, streptozocin, tamoxifen,
thioguanine,
topotecan, vinblastine, vincristine, vindesine, aminoglutethimide, L-
asparaginase,
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azathioprine, 5-azacytidine cladribine, busulfan, diethylstilbestrol, 21,21-
difluorodeoxycytidine, docetaxel, erythrohydroxynonyl adenine, ethinyl
estradiol, 5-
fluorodeoxyuridine, 5-fluorodeoxyuridine monophosphate, fludarabine phosphate,
fluoxymesterone, flutamide, hydroxyprogesterone caproate, idarubicin,
interferon,
medroxyprogesterone acetate, megestrol acetate, melphalan, mitotane,
paclitaxel, pentostatin,
N-phosphonoacetyl-L-aspartate (PALA), plicamycin, semustine, teniposide,
testosterone
propionate, thiotepa, trimethylmelamine, uridine, and vinorelbine,
oxaliplatin, gemcitabine,
capecitabine, epothilone and its natural or synthetic derivatives,
tositumomab, trabedectin,
and temozolomide. trastuzumab, cetuximab, bevacizumab, pertuzumab, ZD-1839
(Iressa),
OSI-774 (Tarceva), CI-1033, GW-2016, CP-724,714, HKI-272, EKB-569, STI-571
(Gleevec), PTK-787, SU-11248, ZD-6474, AG-13736, KRN-951, CP-547,632, CP-
673,451,
CHIR-258, MLN-518, AZD-2171, PD-325901, ARRY-142886, suberoylanilide
hydroxamic
acid (SAHA), LAQ-824, LBH-589, MS-275, FR-901228, bortezomib, and CCI-779.
Aspect 29. A method as in aspect 22 wherein the additional anti-cancer agent
is a
cytotoxic agent selected from the group consisting of DNA topoisomerase I and
II inhibitors,
DNA intercalators, alkylating agents, anti-metabolites, cell-cycle blockers,
microtubule
disruptors, and Eg5 inhibitors.
Aspect 30. A method as in aspect 22 wherein the additional anti-cancer agent
is selected
from the group consisting of inhibitors of growth factor receptor signaling,
histone
deacetylase inhibitors, inhibitors of the PKB pathway, inhibitors of the
Raf/MEK/ERK
pathway, inhibitors of the mTOR pathway, and proteasome inhibitors.
Aspect 31. A method for treating or preventing one or more of the following
conditions in
humans and/or other mammals:
rheumatic fever, bone resorption, postmenopausal osteoporosis, sepsis, gram
negative
sepsis, septic shock, endotoxic shock, toxic shock syndrome, systemic
inflammatory response
syndrome, inflammatory bowel disease (Krohn's disease and ulcerative colitis),
Jarisch-
Herxheimer reaction, asthma, adult respiratory distress syndrome, acute
pulmonary fibrotic
disease, pulmonary sarcoidosis, allergic respiratory disease, silicosis, coal
worker's
pneumoconiosis, alveolar injury, hepatic failure, liver disease during acute
inflammation,
severe alcoholic hepatitis, malaria (Plasmodium falciparum malaria and
cerebral malaria),
non-insulin-dependent diabetes mellitus (NIDDM), congestive heart failure,
damage
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following heart disease, atherosclerosis, Alzheimer's disease, acute
encephalitis, brain injury,
multiple sclerosis (demyelation and oligiodendrocyte loss in multiple
sclerosis), advanced
cancer, lymphoid malignancy, pancreatitis, impaired wound healing in
infection,
inflammation and cancer, myelodysplastic syndromes, systemic lupus
erythematosus, biliary
cirrhosis, bowel necrosis, psoriasis, radiation injury/ toxicity following
administration of
monoclonal antibodies, host-versus-graft reaction (ischemia reperfusion injury
and allograft
rejections of kidney, liver, heart, and skin), lung allograft rejection
(obliterative bronchitis) or
complications due to total hip replacement,
said method comprising administering to a human or other mammal, a synthetic
metabolite according to Aspect 2.
Aspect 32. A method for treating or preventing one or more of the following
conditions in
humans and/or other mammals:
tuberculosis, Helicobacter pylori infection during peptic ulcer disease,
Chaga's
disease resulting from Trypanosoma cruzi infection, effects of Shiga-like
toxin resulting from
E. coli infection, effects of enterotoxin A resulting from Staphylococcus
infection,
meningococcal infection, and infections from Borrelia burgdorferi, Treponema
pallidum,
cytomegalovirus, influenza virus, Theiler's encephalomyelitis virus, and the
human
immunodeficiency virus (HIV) ,
said method comprising administering to a human or other mammal, a synthetic
metabolite according to Aspect 2.
Aspect 33. A method for treating or preventing osteoporosis, inflammation, and
angiogenesis
disorders, with the exclusion of cancer, in a human and/or other mammal by
administering an
effective amount of a synthetic metabolite according to Aspect 2 to said
mammal.
Aspect 34. A method for treating or preventing cancer in a human or other
mammal which
comprises administering to a human or other mammal a single active principle
combining
inhibition of tumor cell proliferation mediated by the raf / MEK / ERK
pathway, and
inhibition of angiogenesis mediated by PDGF and VEGF, wherein said active
principle
comprises a synthetic metabolite according to Aspect 2.
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Aspect 35. A method of aspect 34 where said inhibition of tumor cell
proliferation is caused
by inhibition of raf kinase, and said inhibition of angiogenesis is caused by
dual inhibition of
PDGFR-beta and VEGFR-2 kinases.
Aspect 36. A method for treating or preventing cancer in a human or other
mammal which
comprises administering to a human or other mammal a single active principle
combining
inhibition of tumor cell proliferation mediated by the raf pathway, and
inhibition of
angiogenesis mediated by PDGF or VEGF, wherein said active principle comprises
a
synthetic metabolite according to Aspect 2.
Aspect 37. A method of treating and/or preventing a disease and/or condition
in a subject in
need thereof, comprising administering an effective amount of a synthetic
metabolite
according to Aspect 2.
Aspect 38. A method of aspect 37, wherein said method comprises causing tumor
regression
in a subject or cells therefrom.
Aspect 39. A method of aspect 37, wherein said method comprises inhibiting
lymphangiogenesis.
Aspect 40. A method of aspect 37, wherein said method comprises inhibiting
angiogenesis.
Aspect 41. A method of aspect 37, wherein said method comprises inhibiting
lymphangiogenesis and angiogenesis.
Aspect 42. A method of aspect 37, wherein said method comprises stimulating
the
proliferation of hematopoietic progenitor cells.
Aspect 43. A method of aspect 37, wherein said method comprises treating a
disorder in a
mammalian subject mediated by raf, VEGFR-2, VEGFR-3, PDGFR-beta, p38 and/or
flt-3.
Aspect 44. A method of aspect 37, wherein said method comprises determining
whether a
condition can be modulated by said compound, comprising measuring the
expression or
activity of raf, VEGFR-2, VEGFR-3, PDGFR-beta, p38 and/or flt-3, in a sample
comprising
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cells or a cell extract, wherein said ample is obtained from a subject or cell
having said
condition, whereby said condition can be modulated by said compound when said
expression
or activity is different in said condition as compared to a normal control.
Aspect 45. A method of aspect 44, further comprising comparing the expression
in said
sample to said normal control.
Aspect 46. A method of aspect 37, wherein said method comprises assessing the
efficacy of
said compound disorder, comprising administering said compound, measuring the
expression
or activity of raf, VEGFR-2, VEGFR-3, PDGFR-beta, p38, and/or flt-3, and
determining the
effect of said compound on said expression or activity.
Aspect 47. A method of aspect 37, wherein said method comprises determining
the
presence of raf, VEGFR-2, VEGFR-3, PDGFR-beta, p38 and/or flt-3 in a sample of
a
biological material, contacting said sample with said compound, and
determining whether
said compound binds to said material.
Aspect 48. A method of aspect 37, wherein said method comprises treating a
tumor in a
subject in need thereof, comprising administering an effective amount of said
compound
wherein said amount is effective to inhibit tumor cell proliferation and
neovascularization.
Aspect 49. The synthetic metabolite according to aspect 2, which is an M-2 or
an M-5
metabolite having the following structural formula:
M bo h -2
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Metabolite Mm
or a salt, or a prodrug or an isolated stereoisomer thereof.
Aspect 50. A method for treating or preventing a tumor or vascular disease in
a subject,
comprising administering to said subject, a synthetic metabolite according to
aspect 49.
Claim 51. A method of making the synthetic metabolite according to aspect 2,
comprising contacting the compound of formula I or a salt thereof with a liver
microsome
preparation under conditions sufficient for the metabolism of said compound of
Formula I
and isolating the metabolites from said preparation.
BRIEF DESCRIPTION OF THE DRAWINGS
Various features and attendant advantages of the present invention will be
more fully
appreciated as the same becomes better understood when considered in
conjunction with the
accompanying drawings, in which like reference characters designate the same
or similar
parts throughout the several views, and wherein:
Fig. 1 shows metabolites of regorafenib from in vitro and in vivo studies.
Fig. 2 shows kinase selectivity profile of regorafenib and its N-oxide (M-2)
and desmethyl-N-
oxide (M-5) synthetic metabolites.
Fig. 3 shows that M-2 and M-5 synthetic metabolites significantly inhibit
tumor growth of
human xenografts in mice.
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Fig. 4 shows that regorafenib and its synthetic metabolites M-2 and M-5
inhibit the acute
hypotensive effect of VEGF in anesthetized rats.
Fig. 5 shows that the M-2 synthetic metabolite of regorafenib affects
vasculature
extravasation in a rat GS9L glioblastoma model after a single oral dose of 7.5
mg/kg. Assays
were performed using kinetic DCE-MRI analysis.
Fig. 6 shows the plasma concentrations of regorafenib, M-2 and M-5 synthetic
metabolite on
day 1 and day 21 following administration of 160 mg regorafenib co-precipitate
tablet to
patients with colo-rectal cancer (n = 9, preliminary data). Data are derived
from the same
study as shown in Table 4.
EXAMPLES
The present invention is further illustrated with the following non-limiting
examples.
Abbreviations used in this specification are as follows:
HPLC high pressure liquid chromatography
MS mass spectrometry
ES electrospray
DMSO dimethylsulfoxide
MP melting point
NMR nuclear resonance spectroscopy
TLC thin layer chromatography
rt room temperature
Preparation of 4-amino-3-fluorophenol
OH
F
NH2
To a dry flask purged with Argon was added 10% Pd/C (80 mg) followed by 3-
fluoro-4-
nitrophenol (1.2 g, 7.64 mmol) as a solution in ethyl acetate (40 mL). The
mixture was
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stirred under an H2 atmosphere for 4 h. The mixture was filtered through a pad
of Celite and
the solvent was evaporated under reduced pressure to afford the desired
product as a tan solid
(940 mg, 7.39 mmol; 97 % yield); 1H-NMR (DMSO-d6) 4.38 (s, 2H), 6.29-6.35 (m,
1H),
6.41 (dd, J=2.5, 12.7, 1H), 6.52-6.62 (m, 1H), 8.76 (s, 1H).
Preparation of 4-(4-amino-3-fluorophenoxy)pyridine-2-carboxylic acid
methylamide
O
N
H2N 01I N H
F
A solution of 4-amino-3-fluorophenol (500 mg, 3.9 mmol) in N,N-
dimethylacetamide (6 mL)
cooled to 0 C was treated with potassium tert-butoxide (441 mg, 3.9 mmol),
and the brown
solution was allowed to stir at 0 C for 25 min. To the mixture was added 4-
chloro-N-
methyl-2-pyridinecarboxamide (516 mg, 3.0 mmol) as a solution in
dimethylacetamide (4
mL). The reaction was heated at 100 C for 16 h. The mixture was cooled to
room
temperature, quenched with H2O (20 mL), and extracted with ehtylacetate (4 x
40 mL). The
combined organics were washed with H2O (2 x 30 mL), dried (MgSO4), and
evaporated to
afford a red-brown oil. 1H-NMR indicated the presence of residual
dimethylacetamide, thus
the oil was taken up in diethylether (50 mL) and was further washed with brine
(5 x 30 mL).
The organic layer was dried (MgSO4) and concentrated to give 950 mg of the
desired product
as a red-brown solid, which was used in the next step without purification.
A method of preparing 4-chloro-N-methyl-2-pyridinecarboxamide is described in
Bankston
et al., Org. Proc. Res. Dev. 2002, 6(6), 777-781.
Example 1: Preparation of 4{4-[3-(4-chloro-3-trifluoromethylphenyl)-ureidol-3-
fluorophenoxy } -pyridine-2-carboxylic acid methylamide
CF3 O
CI N O N, O Ni
I N H
H H F
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To a solution of 4-(4-amino-3-fluorophenoxy)pyridine-2-carboxylic acid
methylamide (177
mg, 0.68 mmol) in toluene (3 mL) was added 4-chloro-3-(trifluoromethyl)phenyl
isocyanate
(150 mg, 0.68 mmol). The mixture was stirred at rt for 72 h. The reaction was
concentrated
under reduced pressure and the residue was triturated with diethylether. The
resulting solid
was collected by filtration and dried in vacuo for 4 h to afford the title
compound (155 mg,
0.32 mmol; 47% yield); 1H-NMR (DMSO-d6) 2.78 (d, J=4.9, 3H), 7.03-7.08 (m,
1H), 7.16
(dd, J=2.6, 5.6, 1H), 7.32 (dd, J=2.7, 11.6, 1H), 7.39 (d, J=2.5, 1H), 7.60
(s, 2H), 8.07-8.18
(m, 2H), 8.50 (d, J=5.7, 1H), 8.72 (s, 1H), 8.74-8.80 (m, 1H), 9.50 (s, 1H);
MS (HPLC/ES)
483.06 m/z = (M + 1).
Example 2: Preparation of 4{4-[3-(4-chloro-3-trifluoromethylphenyl)-ureidol-3-
fluorophenoxy } -pyridine-2-carboxylic acid methylamide hydrochloride
The compound of example 1 as a free base (2.0 g) was dissolved in anhydrous
tetrahydrofuran (15 mL) and a 4M HCl/dioxane was added (excess). The solution
was then
concentrated in vacuo to afford 2.32 grams of off-white solids. The crude salt
was dissolved
in hot ethanol (125 mL), activated carbon was added and the mixture heated at
reflux for 15
minutes. The hot suspension was filtered through a pad of Celite 521 and
allowed to cool to
room temperature. The flask was placed in a freezer overnight. The crystalline
solids were
collected by suction filtration, washed with ethanol, then hexane and air-
dried. The mother
liquors were concentrated down and crystallization (in freezer) allowed taking
place
overnight. A second crop of solids was collected and combined with the first
crop. The
colorless salt was dried in a vacuum oven at 60 C over two days. Yield of
hydrochloride salt
obtained 1.72 g (79%).
Melting point: 215 C
Elemental analysis:
Calcd. Found
C 48.57 48.68
3o H 3.11 2.76
N 10.79 10.60
Cl 13.65 13.63
F 14.63 14.88
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Example 3: Preparation of 4{4-[3-(4-chloro-3-trifluoromethylphenyl)-ureido]-3-
fluorophenoxy}-pyridine-2-carboxylic acid methylamide mesylate
The compound of example 1 as a free base (2.25 g) was dissolved in ethanol
(100 mL) and a
stock solution of methanesulfonic acid (excess) was added. The solution was
then
concentrated in vacuo to afford a yellow oil. Ethanol was added and
concentration repeated,
affording 2.41 g of off-white solids. The crude salt was dissolved in hot
ethanol (-125 mL)
and then cooled slowly to crystallize. After reaching room temperature, the
flask was placed
in a freezer overnight. The colorless crystalline material was collected by
suction filtration;
the filter cake was washed with ethanol, then hexane and air-dried, to afford
2.05 g of
material, which was dried in a vacuum oven at 60 C overnight.
Melting point: 231 C
Elemental analysis:
Calcd. Found
C 45.64 45.34
H 3.31 3.08
N 9.68 9.44
Cl 6.12 6.08
F 13.13 13.42
S 5.54 5.59
Example 4: Preparation of 4{4-[3-(4-chloro-3-trifluoromethylphenyl)-ureido]-3-
fluorophenoxy}-pyridine-2-carboxylic acid methylamide phenylsulfonate
The compound of example 1 as a free base (2.25 g) was suspended in ethanol (50
mL) and
benzensulfonic acid (0.737 g) in ethanol (50 mL) was added. The mixture was
heated with
vigorous stirring. All solid material dissolved to give a reddish solution.
The solution was
allowed to cool to room temperature and the flask scratched. Crystal formation
was difficult
to achieve, some seeds were found, added to solution and placed in freezer
overnight.
Grayish-tan solids had formed in the flask; the material was broken up &
collected by suction
filtration. The solids were washed with ethanol, then hexane and air-dried.
Weighed
product: 2.05 g, 69% yield.
Melting point: 213 C
Elemental Analysis:
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Calcd. Found
C 50.59 50.24
H 3.30 3.50
N 8.74 8.54
F 11.86 11.79
Cl 5.53 5.63
S 5.00 5.16
Example 5: Preparation of synthetic metabolites from compounds of Formula I
As a representative example, the synthetic metabolites of the compounds of
Formula I can be
obtained by incubating the parent regorafenib compound with liver microsomes.
The metabolites of the present application can also be synthetically obtained.
The metabolites of the present application can be purified using techniques
that are known in
the art.
Example 6: Metabolite profiles in animals
N-oxide (M-2) and hydroxymethyl (M-3) metabolites were identified as
metabolites of
regorafenib in vitro upon incubation with liver enzymes of various mammalian
species (man,
dog, rat, mouse). Studies reveal that and its metabolites show high protein
binding in man and
animal species.
The metabolite profiles are presented in Fig. 1 and Table 1, below.
Table 1. Metabolite profiles in incubations of [14C] regorafenib (20 M) with
liver
microsomes of different species (protein concentration 0.5 mg/mL, 60 min).
...............................................................................
...............................................................................
............ .
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-
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...............................................................................
...............................................................................
............ .
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Example 7: Pharmacological profiling
After oral administration of 10 mg/kg regorafenib to mice for 5 days, the N-
oxide (M-2)
exposure accounted for -16%of the total AUC (R+M-2+M-5), whereas the
contribution of
M-5 was -2% relative to total AUC. Data are shown in Table 2, below. Following
oral
administration of 10 mg/kg M-2 to mice, regorafenib exposure reached -17% of
total
exposure, indicating reduction of the N-oxide to be a relevant metabolic
pathway in vivo,
whereas M-5 accounted for 5% of total exposure.
Table 2. Pharmacokinetic parameters of regorafenib and its metabolites M-2 and
M-5 at
steady state after their oral administration to female NMRI-Foxn-1 mice.
nn p
\ti `'
Example 8. Kinome-wide selectivity profile
A kinome-wide selectivity profiling of regorafenib and its metabolites M-2 and
M-5 was
performed by Ambit Biosciences (San Diego, CA, US) using an active-site
competitive
binding assay (Fabian et al. Nat Biotechnol 2005; 23: 329-336). A total of 402
kinases were
analyzed using a single dose of 1 M of compound. Binding inhibition
activities are
displayed as the percentage of the kinase that remained bound compared with
the DMSO-
treated control. The potency of the compound is reflected by the size of the
circle.
In the competitive binding assay, M-2 and M-5 showed kinase selectivity
profiles similar to
but distinct from regorafenib. Results are shown in Fig. 2.
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Example 9. Biochemical characterization of M-2 and M-5 metabolites
Characterization of both M-2 and M-5 demonstrated potent pharmacologic
activities. In
biochemical kinase assays, M-2 and M-5 showed an inhibition profile similar to
but distinct
from regorafenib. In cellular assays, M-2 and M-5 inhibited key targets such
as vascular
endothelial growth factor (VEGF) receptor 2, TIE-2, and mutant and wild-type c-
KIT and B-
RAF, with IC50 values very similar to regorafenibfor M-2 and somewhat higher
for M-5.
Data are presented in Table 3.
Table 3. Pharmacologic activity of regorafenib, M-2 and M-5in cellular kinase
phosphorylation assays.
R\:. ~~=`: : ;tics,?~\'w:\\: Mv. ~~\.v ~s=,. ~. :M ,.
KUM NH~-13M 13 (1) 30 , j", 20 lit
...
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . .
G AT viz.
Example 10. Tumor growth inhibitory effects of M-2 and M-5 metabolites
Human and rat tumor models were to determine the in vivo effects of M-2 and M-
5 synthetic
metabolites on tumor growth and on tumor vasculature.
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In these studies, mice bearing xenografts of the human breast cancer cell
lineMDA-MB-231
(K-RASG13D, B-RAFG464V) or the human colorectal cancer cell line HT-29 (B-
RAFV600E) were treated orally (qd x 27) with 10 mg/kg of regorafenib, M-2 or M-
5, starting
at day 11 after tumor inoculation. Tumor growth inhibition is given as
relative tumor area (p
< 0.05 vs vehicle in all instances; n = 8 mice/group). The tumor growth
inhibitory assays
were performed using DCE-MRI.
As shown in Fig. 3, both synthetic metabolites dosed orally exhibited potent
dose-dependent
tumor growth inhibition (TGI) in preclinical murine HT-29 colorectal and MDA-
MB-231
breast cancer xenografts, achieving significant TGI of 62/58% and 54/50%,
respectively,
compared with vehicle controls at 10 mg/kg (Fig. 3).
Example 11. Inhibition of VEGF-induced hypotension
The acute effect of the synthetic metabolites on vascular endothelial growth
factor (VEGF)-
induced hypotension was studied using a rat pharmacodynamic model.
In these studies, a bolus i.v. of VEGF (9 g/kg) was initially used to induce
a transient
hypotension in rats (see inset of Fig. 4). The antagonistic effects of
regorafenib and its
synthetic metabolites on VEGF-induced hypotension were then studied. To this
end,
following a 10 minute i.v. pretreatment with vehicle, regorafenib, M-2 or M-5
were
administered at the indicated doses (Fig. 4). Reduction of diastolic (blue
bars) and systolic
(orange bars) blood pressure (difference between pre-injection and minimum
value) were
calculated. Results are presented in Fig. 4.
Example 12. Effect on tumor vasculature
The pharmacodynamic effect of regorafenib and M-2 on the tumor vasculature in
vivo was
analyzed in GS9L rat glioblastoma tumor-bearing Fischerrats by DCE-MRI
analysis using
the macromolecular contrast agent Gadomer-17. In these studies, rats carrying
glioblastoma
tumors in their thighs were treatedat a single oral dose of 7.5 mg/kg of M-2
or regorafenib
and inhibition of the extravasation of Gadomer-17 was analyzed 2, 6 and 24
hours
aftertreatment.
Results are presented in Fig. 5. The left panel shows results for regorafenib
and the right
panel shows results for the M-2 synthetic metabolite. The left y-axis
indicates tumor AUC
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values, which were normalized to thigh muscle. The right y-axis shows serum
levels of
regorafenib, M-2, M-4 and M-5. The contribution of regorafenib, M-2, M-4 and M-
5 could
therefore be independently assessed.
Example 13. In vivo pharmacological studies
In colon cancer patients treated daily with 160 mg coprecipitate tablets for a
period of 21
days, M-2 and M-5showed systemic exposures very similar to regorafenib.
Results are
presented in Table 4 and Figure 6(B). As shown in Table 4, bio-transformation
of regorafenib
in patientswith colorectal cancer results in significant elevation of
demethylated and oxidized
M-2 and M-5 synthetic metabolite levels. The synthetic metabolites were
observed after
single or multiple qd dosing with 160 mg co-precipitate tablets.
Continuous qd dosing in patients with colorectal cancer for 19 days resulted
in a 25- and 2-
fold increase in the AUC of M-5 and M-2 synthetic metabolites, respectively.
Cmax values
were similarly increased 42-fold and 5-fold, respectively. Values are
reflected in comparison
to the first dose.
In order to further characterize the metabolites, plasma concentrations of
regorafenib, M-2
and M-5 were observed on a daily basis on day 1 and day 21 following
administration of 160
mgregorafenib co-precipitate tablet to patients with colorectal cancer (n = 9,
preliminary
data). Results are shown in Fig. 6. Multiple plasma peaks of regorafenib, M-2
and M-5 were
observed, most likelyas a result of enterohepatic cycling. Combined Cmax level
of the parent
compound and M2/M5 metabolites was 11 mg/L on day 21. The combined
pharmacologically relevant plasma concentration was evaluated to -2.5 mg/L,
remaining 3
days after the last dosing.
Table 4. Pharmacokinetic parameters of regorafenib, M-2 and M-5 on day 1 and
day 21,
following administration of 160 mg regorafenib co-precipitate tablet to
patients with colo-
rectal cancer (geometric mean (%CV), preliminary data)
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...............................................................................
...............................................................................
...................................................... .
...............................
N~,,:qorafi mb 242 611 017 as 17
, * e RA - \ '
(10 1) fit) I
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . .
...............................................................................
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.....................................................
Example 14. c-raf (raf-1) Biochemical Assay
The c-raf biochemical assay is performed with a c-raf enzyme that was
activated
(phosphorylated) by Lck kinase. Lck-activated c-raf (Lck/c-raf) is first
produced in Sf9 insect
cells by co-infecting cells with baculoviruses expressing, under the control
of the polyhedrin
promoter, GST-c-raf (from amino acid 302 to amino acid 648) and Lck (full-
length). Both
baculoviruses are used at the multiplicity of infection of 2.5 and the cells
are harvested 48 h
post infection.
MEK-1 protein is produced in Sf9 insect cells by infecting cells with the
baculovirus
expressing GST-MEK-1 (full-length) fusion protein at the multiplicity of
infection of 5 and
harvesting the cells 48 hours post infection. Similar purification procedure
is used for GST-
c-raf 302-648 and GST-MEK-1.
Transfected cells are suspended at 100 mg of wet cell biomass per mL in a
buffer containing
10 mM sodium phosphate, 140 mM sodium chloride pH 7.3, 0.5% Triton X-100 and
the
protease inhibitor cocktail. The cells are disrupted with Polytron homogenizer
and
centrifuged 30,000g for 30 minutes. The 30,000g supernatant is applied onto
GSH-
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Sepharose. The resin is washed with a buffer containing 50 mM Tris, pH 8.0,
150 mM NaCl
and 0.01% Triton X-100. The GST-tagged proteins are eluted with a solution
containing 100
mM Glutathione, 50 mM Tris, pH 8.0, 150 mM NaCl and 0.01% Triton X-100. The
purified
proteins are dialyzed into a buffer containing 20 mM Tris, pH 7.5, 150 mM NaCl
and 20%
Glycerol.
Test compounds are serially diluted in DMSO using three-fold dilutions to
stock
concentrations ranging typically from 50 M to 20 nM (final concentrations in
the assay
range from 1 M to 0.4 nM). The c-Raf biochemical assay is performed as a
radioactive
filtermat assay in 96-well Costar polypropylene plates (Costar 3365). The
plates are loaded
with 75 L solution containing 50 mM HEPES pH 7.5, 70 mM NaCl, 80 ng of Lck/c-
raf and
1 g MEK-1. Subsequently, 2 L of the serially diluted individual compounds
are added to
the reaction, prior to the addition of ATP. The reaction is initiated with 25
L ATP solution
containing 5 M ATP and 0.3 VCi [33P]-ATP. The plates are sealed and incubated
at 32 oC
for 1 h. The reaction is quenched with the addition of 50 L of 4 % Phosphoric
Acid and
harvested onto P30 filtermats (PerkinElmer) using a Wallac Tomtec Harvester.
Filtermats
are washed with 1 % Phosphoric Acid first and deinonized H2O second. The
filters are dried
in a microwave, soaked in scintillation fluid and read in a Wallac 1205
Betaplate Counter
(Wallac Inc., Atlanta, GA, U.S.A.). The results are expressed as percent
inhibition.
% Inhibition = [100-(Tib/Ti)] x 100 where
Tib = (counts per minute with inhibitor)-(background)
Ti = (counts per minute without inhibitor)-(background)
The compound of the present invention shows potent inhibition of raf kinase in
this assay.
Example 15: p38 kinase in vitro assay
Purified and His-tagged p38 (expressed in E. Coli) is activated in vitro by
MMK-6 to a high
specific activity. Using a microtiter format, all reactions are conducted in
100 L volumes
with reagents diluted to yield 0.05 g/well of activated p38 and 10 g/well of
myelin basic
protein in assay buffer (25 mM HEPES 7.4, 20 mM MgC12, 150 mM NaCI). Test
compounds (5 L of a 10% DMSO solution in water) are prepared and diluted into
the assay
to cover a final concentration range from 5 nM to 2.5 M. The kinase assay is
initiated by
addition of 25 L of an ATP cocktail to give a final concentration of 10 M
cold ATP and
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0.2 VCi [gamma-33P] ATP per well (200-400 dpm/pmol of ATP). The plate is
incubated at
32 oC for 35 min., and the reaction quenched with 7 L of a 1 N aq HCl
solution. The
samples are harvested onto a P30 Filtermat (Wallac, Inc.) using a TomTec 1295
Harvester
(Wallac, Inc.), and counted in a LKB 1205 Betaplate Liquid Scintillation
Counter (Wallac,
Inc.). Negative controls include substrate plus ATP alone. SW1353 cellular
assay: SW1353
cells (human chondro-sarcoma) are seeded (1000 cells/100 L DMEM 10% FCS/well)
into
96-well plates and incubated overnight. After medium replacement, cells are
exposed to test
compounds for 1 h at 37 oC, at which time human IL-1 (1 ng/mL, Endogen,
Woburn, WA)
and recombinant human TNFalpha (10 ng/mL) are added. Cultures are incubated
for 48 h at
37 oC, then supernatant IL-6 values are determined by ELISA. The compounds of
this
invention show significant inhibition of p38 kinase.
Example 16: Bio-Plex Phospho-ERK'h immunoassay.
A 96 well pERK immunoassay, using laser flow cytometry (Bio-Rad) platform has
been
established to measure inhibition of basal pERK in breast cancer cell line.
MDA-MB-231
cells are plated at 50,000 cells per well in 96 well microtitre plates in
complete growth media.
For effects of test compounds on basal pERK1/2 inhibition, the next day after
plating, MDA-
MB-231 cells are transferred to DMEM with 0.1% BSA and incubated with test
compounds
diluted 1:3 to a final concentration of 3 M to 12 nM in 0.1% DMSO. Cells are
incubated
with test compounds for 2 h, washed, and lysed in Bio-Plex whole cell lysis
buffer A.
Samples are diluted with buffer B 1:1 (v/v) and directly transferred to assay
plate or frozen at
-80 C degrees until processed. 50 L of diluted MDA-MB-231 cell lysates are
incubated
with about 2000 of 5 micron Bio-Plex beads conjugated with an anti-ERK1/2
antibody
overnight on a shaker at room temperature. The next day, biotinylated phospho-
ERK1/2
sandwich immunoassay is performed, beads are washed 3 times during each
incubation and
then 50 L of PE-strepavidin is used as a developing reagent. The relative
fluorescence units
of pERK1/2 are detected by counting 25 beads with Bio-Plex flow cell (probe)
at high
sensitivity. The IC50 is calculated by taking untreated cells as maximum and
no cells (beads
only) as background using in an Excel spreadsheet based program.
The compound of this invention shows significant inhibition in this assay.
Example 17: Flk-1 (murine VEGFR-2) Biochemical Assay
This assay is performed in 96-well opaque plates (Costar 3915) in the TR-FRET
format.
Reaction conditions are as follows: 10 M ATP, 25 nM poly GT-biotin, 2 nM Eu-
labelled
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phospho-Tyr Ab, 10 nM APC, 7 nM Flk-1 (kinase domain), 1% DMSO, 50 mM HEPES pH
7.5, 10 mM MgC12, 0.1 mM EDTA, 0.015% BRIJ, 0.1 mg/mL BSA, 0.1% mercapto-
ethanol). Reaction is initiated upon addition of enzyme. Final reaction volume
in each well
is 100 L. Plates are read at both 615 and 665 nM on a Perkin Elmer Victor V
Multilabel
counter at about 1.5- 2.0 hours after reaction initiation. Signal is
calculated as a ratio: (665
nm / 615 nm) * 10000 for each well.
The compounds of this invention show significant inhibition of VEGFR2 kinase.
Example 18: Murine PDGFR FRET biochemical assay
This assay is formatted in a 96-well black plate (Costar 3915). The following
reagents are
used: Europium-labeled anti-phosphotyrosine antibody pY20 (Perand streptavidin-
APC; poly
GT-biotin from, and mouse PDGFR. The reaction conditions are as follows: 1 nM
mouse
PDGFR is combined with 20 M ATP, 7 nM poly GT-biotin, 1 nM pY20 antibody, 5
nM
streptavidin-APC, and 1% DMSO in assay buffer (50 mM HEPES pH 7.5, 10 mM
MgC12,
0.1 mM EDTA, 0.015% BRIJ 35, 0.1 mg/mL BSA, 0.1% mercaptoethanol). Reaction is
initiated upon addition of enzyme. Final reaction volume in each well is 100
L. After 90
minutes, the reaction is stopped by addition of 10 tL/well of 5 M
staurosporine. Plates are
read at both 615 and 665 nm on a Perkin Elmer VictorV Multilabel counter at
about 1 hour
after the reaction is stopped. Signal is calculated as a ratio: (665 nm / 615
nm) * 10000 for
each well. The compounds of this invention show significant inhibition of
PDGFR kinase.
For IC50 generation for both PDGFR and Flk-1, compounds are added prior to the
enzyme
initiation. A 50-fold stock plate was made with compounds serially diluted 1:3
in a 50%
DMSO/50% dH2O solution. A 2 L addition of the stock to the assay gives final
compound
concentrations ranging from 10 M - 4.56 nM in 1% DMSO. The data are expressed
as
percent inhibition: % inhibition = 100-((Signal with inhibitor-
background)/(Signal without
inhibitor - background)) * 100
Example 19: MDA-MB231 proliferation assay
Human breast carcinoma cells (MDA MB-23 1, NCI) are cultured in standard
growth medium
(DMEM) supplemented with 10% heat-inactivated FBS at 37oC in 5% CO2 (vol/ vol)
in a
humidified incubator. Cells are plated at a density of 3000 cells per well in
90 L growth
medium in a 96 well culture dish. In order to determine TOh CTG values, 24
hours after
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plating, 100 L of CellTiter-Glo Luminescent Reagent (Promega) is added to
each well and
incubated at room temperature for 30 minutes. Luminescence is recorded on a
Wallac Victor
II instrument. The CellTiter-Glo reagent results in cell lysis and generation
of a luminescent
signal proportional to the amount of ATP present, which, in turn is directly
proportional to
the number of cells present.
Test compounds are dissolved in 100% DMSO to prepare 10 mM stocks. Stocks are
further
diluted 1:400 in growth medium to yield working stocks of 25 M test compound
in 0.25%
DMSO. Test compounds are serially diluted in growth medium containing 0.25%
DMSO to
maintain constant DMSO concentrations for all wells. 60 L of diluted test
compound are
added to each culture well to give a final volume of 180 L. The cells with
and without
individual test compounds are incubated for 72 hours at which time ATP
dependent
luminescence is measured, as described previously, to yield T72h values.
Optionally, the
IC50 values can be determined with a least squares analysis program using
compound
concentration versus percent inhibition.
% Inhibition = [1-(T72h test-TOh)/(T72h ctrl-TOh)] x 100, where
T72h test = ATP dependent luminescence at 72 hours in the presence of test
compound
T72h ctrl = ATP dependent luminescence at 72 hours in the absence of test
compound
TOh = ATP dependent luminescence at Time Zero
The compound of this invention shows significant inhibition of proliferation
using this assay.
Example 20: pPDGFR-beta sandwich ELISA in AoSMC cells
100K P3-P6 Aortic SMC are plated in each well of 12-well cluster in 1000 L
volume/ well
of SGM-2 using standard cell culture techniques. Next day, cells are rinsed
with 1000 L D-
PBS once, then serum starved in 500 L SBM (smooth muscle cell basal media)
with 0.1%
BSA overnight. Compounds are diluted at a dose range from (10 M to 1 nM in 10-
fold
dilution steps in DMSO. Final DMSO concentration 0.1%). Remove old media by
inversion
into the sink quickly then add 100 L of each dilution to corresponding well
of cells for 1 h at
37 OC. Cells are then stimulated with 10 ng/mL PDGF-BB ligand for 7 min at 37
OC. The
media is decanted and 150 L of isotonic lysis buffer with protease inhibitor
tablet
(Complete; EDTA-free) and 0.2 mM Na vanadate is added. Cells are lysed for 15
min at 4 OC
on shaker in cold room. Lysates are put in eppendorf tubes to which 15 L of
agarose-
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conjugated anti-PDGFR-beta antibody is added and incubated at 4 OC overnight.
Next day,
beads are rinsed in 50-volumes of PBS three times and boiled in lx LDS sample
buffer for 5
minutes. Samples are run on 3-8% gradient Tris-Acetate gels and transferred
onto
Nitrocellulose. Membranes are blocked in 1% BSA/TBS-T for 1 hr. before
incubation in
anti-phospho-PDGFR-b (Tyr-857) antibody in blocking buffer (1:1000 dilution)
for 1 h. After
three washes in TBS-T, membranes are incubated in Goat anti-rabbit HRP IgG
(1:25000
dilution) for 1 hr. Three more washes followed before addition of ECL
substrate. Membranes
are exposed to Hyperfilm-ECL. Subsequently, membranes are stripped and
reprobed with
anti-PDGFR-beta antibody for total PDGFR-beta.
Table A illustrates the results of a representative in vitro kinase
biochemical assay for p38
kinase, PDGFR kinase and VEGFR2 kinase. These three kinase targets are all
involved in
stroma activation and endothelial cell proliferation, leading to angiogenesis,
and providing
blood supply to the tumor tissue.
Table A
mPDGFR mVEGFR2 p38
IC50, nM IC50, nM IC50, nM
Example 1 83 5.5 24
Table B illustrates representative results of two cellular assays for raf
kinase activity, which
are (i) inhibition of pERK in MDA-MB231 cells, a mechanistic readout of raf
kinase activity,
and (ii) a proliferation assay of MDA-MB231 cells, a functional assay of raf
kinase activity.
In addition, Table B illustrates the results of PDGFR driven phosphorylation
of PDGFR-beta
in aortic smooth muscle cells, which is a mechanistic readout of PDGFR kinase
inhibition.
Table B
pERK in cells Proliferation pPDGFR
(MDA-MB- (MDA-MB-231) (AoSMC)
231) IC50, nM IC50, nM
IC50, nM
Example 1 22 600 43.6
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Overall, compounds of the present invention provide a unique combination of
inhibition of
angiogenesis and tumor cell proliferation. They also possess an improved
inhibition profile
against several key kinase targets such as raf, p38, PDGFR, and VEGFR-2, which
are all
molecular targets of interest for the treatment of osteoporosis, inflammatory
diseases, and
hyper-proliferative diseases, including cancer.
It is believed that one skilled in the art, using the preceding information
and information
available in the art, can utilize the present invention to its fullest extent.
It should be apparent
to one of ordinary skill in the art that changes and modifications can be made
to this
invention without departing from the spirit or scope of the invention as it is
set forth herein.
All publications, applications and patents cited above and below are
incorporated herein by
reference.
The topic headings set forth above and below are meant as guidance where
certain
information can be found in the application, but are not intended to be the
only source in the
application where information on such topic can be found.
Without further elaboration, it is believed that one skilled in the art can,
using the preceding
description, utilize the present invention to its fullest extent. The
preceding preferred specific
embodiments are, therefore, to be construed as merely illustrative, and not
limitative of the
remainder of the disclosure in any way whatsoever.
In the foregoing and in the examples, all temperatures are set forth
uncorrected in degrees
Celsius and, all parts and percentages are by weight, unless otherwise
indicated.
The entire disclosures of all applications, patents and publications, cited
herein and the
following publications are incorporated by reference herein.
1. Wilhelm S et al. "Regorafenib (BAY 73-4506): identification of clinically
relevant
metabolites and their preclinical pharmacology." American Society of Clinical
Oncology (ASCO) Annual Meeting, 2010. Abstract no. 1666 (enclosed).
2. Wilhelm S et al. Mol Cancer Ther 2009; 8 (suppl): abs B42.
3. Eisen T et al. Eur J Cancer Suppl 2009; 7: 424, abs 0-71053.
4. Strumberg D et al. J Clin Oncol (Meeting Abstracts) 2009; 27: 161s, abs
35604.
5. Fabian MA et al. Nat Biotechnol 2005; 23: 329-336
73 BAYER-0183-WO
CA 02796744 2012-10-17
WO 2011/130728 PCT/US2011/032835
The preceding examples can be repeated with similar success by substituting
the generically
or specifically described reactants and/or operating conditions of this
invention for those used
in the preceding examples.
From the foregoing description, one skilled in the art can easily ascertain
the essential
characteristics of this invention and, without departing from the spirit and
scope thereof, can
make various changes and modifications of the invention to adapt it to various
usages and
conditions.
74 BAYER-0183-WO
