Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
  • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
  • C07D403/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/06—Antipsoriatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
  • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
  • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
  • C07D471/04—Ortho-condensed systems

Definitions

• the present invention relates to certain 3- (4- amidopyrrol-2-ylmethylidene) -2-indolinone derivatives which modulate the activity of protein kinases ("PKs") .
• PKs protein kinases
• the compounds of this invention are therefore useful in treating disorders related to abnormal PK activity.
• Pharmaceutical compositions comprising these compounds, methods of treating diseases utilizing pharmaceutical compositions comprising these compounds and methods of preparing them are also disclosed.
• PKs are enzymes that catalyze the phosphorylation of hydroxy groups on tyrosine, serine and threonine residues of proteins.
• the consequences of this seemingly simple activity are staggering; cell growth, differentiation and proliferation, i.e., virtually all aspects of cell life in one way or another depend on PK activity.
• abnormal PK activity has been related to a host of disorders, ranging from relatively non life threatening diseases such as psoriasis to extremely virulent diseases such as glioblastoma (brain cancer) .
• the PKs can be conveniently broken down into two classes, the protein tyrosine kinases (PTKs) and the serine-threonine kinases ( STKs ) .
• PTKs protein tyrosine kinases
• STKs serine-threonine kinases
• growth factor receptors are cell-surface proteins. When bound by a growth factor ligand, growth factor receptors are converted to an active form which interacts with proteins on the inner surface of a cell membrane. This leads to phosphorylation on tyrosine residues of the receptor and other proteins and to the formation inside the cell of complexes with a variety of cytoplasm signaling molecules that, in turn, effect numerous cellular responses such as cell division (proliferation) , cell differentiation, cell growth, expression of metabolic effects to the extracellular microenvironment, etc.
• cytoplasm signaling molecules that, in turn, effect numerous cellular responses such as cell division (proliferation) , cell differentiation, cell growth, expression of metabolic effects to the extracellular microenvironment, etc.
• RTKs receptor tyrosine kinases
• HER receptor tyrosine kinases
• RTK subfamily consists of insulin receptor (IR) , insulin-like growth factor I receptor (IGF-1R) and insulin receptor related receptor (IRR) .
• IR and IGF-lR interact with insulin, IGF-I and IGF-II to form a heterotetramer of two entirely extracellular glycosylated ⁇ subunits and two ⁇ subunits which cross the cell membrane and which contain the tyrosine kinase domain.
• a third RTK subfamily is referred to as the platelet derived growth factor receptor (“PDGFR”) group, which includes PDGFR ⁇ , PDGFR ⁇ , CSFIR, c-kit and c-fms. These receptors consist of glycosylated extracellular domains composed of variable numbers of immunoglobin-like loops and an intracellular domain wherein the tyrosine kinase domain is interrupted by unrelated amino acid sequences.
• flk fetus liver kinase
• KDR/FLK-1, VEGF-R2 kinase insert domain-receptor fetal liver kinase-1
• flk-lR flk-lR
• flk-4 fms-like tyrosine kinase 1
• a further member of the tyrosine kinase growth factor receptor family is the fibroblast growth factor ("FGF") receptor subgroup.
• FGF fibroblast growth factor
• This group consists of four receptors, FGFR1-4, and seven ligands, FGF1-7. While not yet well defined, it appears that the receptors consist of a glycosylated extracellular domain containing a variable number of immunoglobin-like loops and an intracellular domain in which the tyrosine kinase sequence is interrupted by regions of unrelated amino acid sequences.
• VEGF vascular endothelial growth factor receptor subgroup.
• VEGF is a dimeric glycoprotein similar to PDGF but has different biological functions and target cell specificity in vivo. In particular, VEGF is presently thought to play an essential role is vasculogenesis and angiogenesis.
• CTK non-receptor tyrosine kinases
• cellular tyrosine kinases This latter designation, abbreviated “CTK, " will be used herein.
• CTKs do not contain extracellular and transmembrane domains.
• Sc, Frk, Btk, Csk, Abl, Zap70, Fes, Fps, Fak, Jak and Ack have been identified.
• the Src subfamily appear so far to be the largest group of CTKs and includes Src, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk.
• Src Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk.
• STKs serine/threonine kinases
• CTKs receptor kinases
• STKs are the most common of the cytosolic kinases; i.e., kinases that perform their function in that part of the cytoplasm other than the cytoplasmic organelles and cytoskelton.
• the cytosol is the region within the cell where much of the cell's intermediary metabolic and biosynthetic activity occurs; e.g., it is in the cytosol that proteins are synthesized on ribosomes.
• RTKs, CTKs and STKs have all been implicated in a host of pathogenic conditions including, significantly, cancer.
• Other pathogenic conditions which have been associated with PTKs include, without limitation, psoriasis, hepatic cirrhosis, diabetes, angiogenesis, restenosis, ocular diseases, rheumatoid arthritis and other inflammatory disorders, immunological disorders such as autoimmune disease, cardiovascular disease such as atherosclerosis and a variety of renal disorders.
• PK regulated functions known to be PK regulated. That is, it has been suggested that malignant cell growth results from a breakdown in the mechanisms that control cell division and/or differentiation. It has been shown that the protein products of a number of proto-oncogenes are involved in the signal transduction pathways that regulate cell growth and differentiation. These protein products of proto-oncogenes include the extracellular growth factors, transmembrane growth factor PTK receptors (RTKs), cytoplasmic PTKs (CTKs) and cytosolic STKs, discussed above.
• RTKs transmembrane growth factor PTK receptors
• CTKs cytoplasmic PTKs
• STKs cytosolic STKs
• RNA ligands (Jelinek, et al., Biochemistry, 33:10450-56); Takano, et al., Mol. Bio. Cell 4:358A (1993); Kinsella, et al . , Exp. Cell Res. 199:56-62 (1992); Wright, et al . , J. Cellular Phys . , 152:448- 57) and tyrosine kinase inhibitors (WO 94/03427; WO 92/21660; WO 91/15495; WO 94/14808; U.S. Pat. No. 5,330,992; Mariani, et al., Proc. Am. Assoc. Cancer Res., 35:2268 (1994)).
• the present invention is directed to certain 3- (4- amidopyrrol-2-ylmethylidene) -2-indolinone derivatives which exhibit PK modulating ability and are therefore useful in treating disorders related to abnormal PK activity.
• One embodiment of this invention is a compound of Formula (I) :
• R 1 is selected from the group consisting of hydrogen, halo, alkyl, haloalkoxy, cycloalkyl, heteroalicyclic, hydroxy, alkoxy, -C(0)R 8 , -NR 9 R 10 and -C (0) NR 1 R 13 ;
• R 2 is selected from the group consisting of hydrogen, halo, alkyl, trihalomethyl, hydroxy, alkoxy, cyano, -NR 9 R 10 ,
• R 3 , R 4 and R 5 are independently hydrogen or alkyl
• Z is aryl, heteroaryl, heterocycle, or -NR 15 R 16 wherein R 15 and R 16 are independently hydrogen or alkyl; or R 15 and R 16 together with the nitrogen atom to which they are attached from a heterocycloamino group;
• R 6 is selected from the group consisting of hydrogen or alkyl
• R 7 is selected from the group consisting of hydrogen, alkyl, aryl, heteroaryl, and -C(0)R 17 ;
• R 8 is selected from the group consisting of hydroxy, alkoxy, and aryloxy
• R 9 and R 10 are independently selected from the group consisting of hydrogen, alkyl, cyanoalkyl, cycloalkyl, aryl and heteroaryl; or
• R 12 and R 13 are independently selected from the group consisting of hydrogen, alkyl, hydroxyalkyl, and aryl; or R 12 and R 13 together with the nitrogen atom to which they are attached form a heterocycloamino;
• R 17 is selected from the group consisting of hydroxy, alkyl, cycloalkyl, aryl and heteroaryl; or a pharmaceutically acceptable salt thereof.
• R 1 is selected from the group consisting of hydrogen, halo, alkyl, cycloalkyl, heteroalicyclic, hydroxy, alkoxy, -C(0)R 8 , -NR 9 R 10 and -C (0) NR 12 R 13 ;
• R 2 is selected from the group consisting of hydrogen, halo, alkyl, trihalomethyl, hydroxy, alkoxy, cyano, -NR 9 R 10 , -NR 9 C(0)R 10 , -C(0)R 8 , -S(0) 2 NR 9 R 10 and -S0 2 R 14 (wherein R 14 is alkyl, aryl, aralkyl, heteroaryl and heteroaralkyl) ;
• R 3 , R 4 and R 5 are independently hydrogen or alkyl;
• Z is aryl, heteroaryl, heterocycle, or -NR 15 R 16 wherein R 15 and R 16 are independently hydrogen or alkyl; or R 15 and R 16 together with the nitrogen atom to which they are attached form a heterocycloamino group;
• R 6 is selected from the group consisting of hydrogen or alkyl
• R 7 is selected from the group consisting of hydrogen, alkyl, aryl, heteroaryl, and -C(0)R 17 ;
• R 8 is selected from the group consisting of hydroxy, alkoxy, and aryloxy;
• R 9 and R 10 are independently selected from the group consisting of hydrogen, alkyl, cyanoalkyl, cycloalkyl, aryl and heteroaryl; or
• R 9 and R 10 combine to form a heterocyclo group
• R 12 and R 13 are independently selected from the group consisting of hydrogen, alkyl and aryl, or R 12 and R 13 together with the nitrogen atom to which they are attached form a heterocycle;
• R 17 is selected from the group consisting of hydroxy, alkyl, cycloalkyl, aryl and heteroaryl; or a pharmaceutically acceptable salt thereof.
• Another embodiment is compound of Formula ( la)
• R 1 , R , R,and R are hydrogen
• R 2 is fluoro and is located at the 5-position of the indolinone ring
• Z is morpholin-4-yl; R 6 and R 7 are methyl.
• the stereochemistry at the *C is (S) .
• Another embodiment is compound of Formula (II)
• R is hydrogen or alkyl
• R 1 is selected from the group consisting of hydrogen, halo, alkyl, haloalkoxy, cycloalkyl, heteroalicyclic, hydroxy, alkoxy, -C(0)R 8 , -NR 9 R 10 and -C (0) NR 12 R 13 ;
• R 2 is selected from the group consisting of hydrogen, halo, alkyl, trihalomethyl, hydroxy, alkoxy, cyano, -NR 9 R 10 , -NR 9 C(0)R 10 , -C(0)R 8 , -S(0) 2 NR 9 R 10 and -S0 2 R 14 (wherein R 14 is alkyl, aryl, aralkyl, heteroaryl and heteroaralkyl) ;
• R 3 , R 4 and R 5 are independently hydrogen or alkyl;
• Z is aryl, heteroaryl, heterocycle, or -NR 15 R 16 wherein R 15 and R 16 are independently hydrogen or alkyl; or R 15 and R 16 together with the nitrogen atom to which they are attached from a heterocycloamino group;
• R 6 is selected from the group consisting of hydrogen or alkyl
• R 7 is selected from the group consisting of hydrogen, alkyl, aryl, heteroaryl, and -C(0)R 17 ;
• R 8 is selected from the group consisting of hydroxy, alkoxy, and aryloxy;
• R 9 and R 10 are independently selected from the group consisting of hydrogen, alkyl, cyanoalkyl, cycloalkyl, aryl and heteroaryl; or
• R 9 and R 10 combine to form a heterocycloamino group
• R 12 and R 13 are independently selected from the group consisting of hydrogen, alkyl, hydroxyalkyl, and aryl; or R 12 and R 13 together with the nitrogen atom to which they are attached form a heterocycloamino;
• R 17 is selected from the group consisting of hydroxy, alkyl, cycloalkyl, aryl and heteroaryl; or a pharmaceutically acceptable salt thereof.
• Another embodiment is a pharmaceutical composition, comprising a compound or salt of Formulas I, la, or II and a pharmaceutically acceptable carrier or excipient.
• Another embodiment is a method for the modulation of the catalytic activity of a protein kinase, comprising contacting the protein kinase with a compound or salt of Formulas I, la, or II.
• the protein kinase for this method can be a receptor tyrosine kinase, a non-receptor tyrosine kinase and a serine- threonine kinase.
• Another embodiment is a method for treating or preventing a protein kinase related disorder in an organism, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a compound or salt • of Formulas I, la, or II and a ' pharmaceutically acceptable carrier or excipient to the organism.
• the protein kinase for this method can be a receptor tyrosine kinase, a non-receptor tyrosine kinase and a serine-threonine kinase.
• the protein kinase related disorder can be an EGFR related disorder, a PDGFR related disorder, an IGFR related disorder and a flk related disorder.
• the protein kinase disorder can also be squamous cell carcinoma, astrocytoma, Kaposi's sarcoma, glioblastoma, lung cancer, bladder cancer, head and neck cancer, melanoma, ovarian cancer, prostate cancer, breast cancer, small-cell lung cancer, glioma, colorectal cancer, genitourinary cancer and gastrointestinal cancer.
• the protein kinase disorder can also be diabetes, an autoimmune disorder, a hyperproliferation disorder, restenosis, fibrosis, psoriasis, von Heppel-Lindau disease, osteoarthritis, rheumatoid arthritis, angiogenesis, an inflammatory disorder, an immunological disorder and a cardiovasc ⁇ lar disorder. These methods can be used to treat humans .
• this invention is directed to methods of preparing compounds of Formula (I) .
• this invention is also directed to identifying a chemical compound that modulates the catalytic activity of a protein kinase by contacting cells expressing the protein kinase with a compound or a salt of the present invention and then monitoring the cells for an effect.
• Alkyl refers to a saturated aliphatic hydrocarbon radical including straight chain and branched chain groups of 1 to 20 carbon atoms (whenever a numerical range; e.g. "1-20", is stated herein, it means that the group, in this case the alkyl group, may contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc. up to and including 20 carbon atoms) . More preferably, it is a medium size alkyl having 1 to 10 carbon atoms e.g., methyl, ethyl, propyl, 2-propyl, n-butyl, .iso- butyl, tert-butyl, pentyl, and the like.
• Alkyl having 1 to 4 carbon atoms e.g., methyl, ethyl, propyl, 2-propyl, n-butyl, iso-butyl, or tert-butyl, and the like.
• Alkyl may be substituted or unsubstituted, and when substituted the substituent group (s) is preferably halo, hydroxy, lower alkoxy, aryl, aryloxy, heteroaryl, heteroalicyclic, C(0)R 8 , NR 9 R 10 , and C(0)NR 9 R 10 .
• Cycloalkyl refers to a 3 to 8 member all-carbon monocyclic ring, an all-carbon 5-member/6-member or 6- member/6-member fused bicyclic ring or a multicyclic fused ring (a "fused" ring system means that each ring in the system shares an adjacent pair of carbon atoms with each other ring in the system) group wherein one or more of the rings may contain one or more double bonds but none of the rings has a completely conjugated pi-electron system.
• cycloalkyl groups examples, without limitation, are cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, cyclohexadiene, adamantane, cycloheptane, cycloheptatriene, and the like.
• a cycloalkyl group may be substituted or unsubstituted.
• the substituent group (s) is preferably one or more, more preferably one or two substituents, independently selected from the group consisting of lower alkyl, trihaloalkyl, halo, hydroxy, lower alkoxy, aryl optionally substituted with one or more, preferably one or two groups independently of each other halo, hydroxy, lower alkyl or lower alkoxy groups, aryloxy optionally substituted with one or more, preferably one or two groups independently of each other halo, hydroxy, lower alkyl or lower alkoxy groups, 6-member heteroaryl having from 1 to 3 nitrogen atoms in the ring, the carbons in the ring being optionally substituted with one or more, preferably one or two groups independently of each other halo, hydroxy, lower alkyl or lower alkoxy groups, 5-member heteroaryl having from 1 to 3 heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, the carbon and nitrogen atoms of the group being optionally substituted with one or more, preferably one or
• Alkenyl refers to an alkyl group, as defined herein, consisting of at least two carbon atoms and at least one carbon-carbon double bond. Representative examples include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, 1-, 2-, or 3-butenyl, and the like.
• Alkynyl refers to an alkyl group, as defined herein, consisting of at least two carbon atoms and at least one carbon-carbon triple bond. Representative examples include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-, 2-, or 3-butynyl, and the like.
• Aryl refers to an all-carbon monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups of 1 to 12 carbon atoms having a completely conjugated pi-electron system. Examples, without limitation, of aryl groups are phenyl, naphthalenyl and anthracenyl . The aryl group may be substituted or unsubstituted.
• the substituted group (s) is preferably one or more, more preferably one, two or three, even more preferably one or two, independently selected from the group consisting of lower alkyl, trihaloalkyl, halo, hydroxy, lower alkoxy, mercapto, (lower alkyl)thio, cyano, acyl, thioacyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, nitro, N-sulfonamido, S-sulfonamido, R 9 S(0)-, R 9 S(0) 2 -, - C(0)0R 9 , R 9 C(0)0-, and -NR 9 R 10 , with R 9 and R 10 as defined above.
• the aryl group is optionally substituted with one or two substituents independently selected from halo, lower alkyl, trihaloalkyl, hydroxy, mercapto, cyano, N-amido, mono or dialkylamino, carboxy, or N-sulfonamido.
• substituents independently selected from halo, lower alkyl, trihaloalkyl, hydroxy, mercapto, cyano, N-amido, mono or dialkylamino, carboxy, or N-sulfonamido.
• Heteroaryl refers ' to a monocyclic or fused ring (i.e., rings which share an adjacent pair of atoms) group of 5 to 12 ring atoms containing one, two, three or four ring heteroatoms selected from N, 0, or S, the remaining ring atoms being C, and, in addition, having a completely conjugated pi-electron system.
• unsubstituted heteroaryl groups are pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline, purine, tetrazole, triazine, and carbazole.
• the heteroaryl group may be substituted or unsubstituted.
• the substituted group (s) is preferably one or more, more preferably one, two, or three, even more preferably one or two, independently selected from the group consisting of lower alkyl, trihaloalkyl, halo, hydroxy, lower alkoxy, mercapto, (lower alkyl) thio, cyano, acyl, thioacyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, nitro, N-sulfonamido, S-sulfonamido, R 9 S (0) -, R 9 0) 2 -, -C(0)OR 9 , R 9 C(0)0-, and -NR 9 R 10 , with R 9 and R 10 as defined above.
• the heteroaryl group is optionally substituted with one or two substituents independently selected from halo, lower alkyl, trihaloalkyl, hydroxy, mercapto, cyano, N-amido, mono or dialkylamino, carboxy, or N-sulfonamido.
• substituents independently selected from halo, lower alkyl, trihaloalkyl, hydroxy, mercapto, cyano, N-amido, mono or dialkylamino, carboxy, or N-sulfonamido.
• Heteroalicyclic refers to a monocyclic or fused ring group having in the ring(s) of 5 to 9 ring atoms in which one or two ring atoms are heteroatoms selected from N, 0, or S(0) n
• n is an integer from 0 to 2
• the rings may also have one or more double bonds. However, the rings do not have a completely conjugated pi- electron system. Examples, without limitation, of unsubstituted heteroalicyclic groups are pyrrolidino, piperidino, piperazino, morpholino, thiomorpholino, homopiperazino, and the like.
• the heteroalicyclic ring may be substituted or unsubstituted.
• the substituted group (s) is preferably one or more, more preferably one, two or three, even more preferably one or two, independently selected from ' the group consisting of lower alkyl, trihaloalkyl, halo, hydroxy, lower alkoxy, mercapto, (lower alkyl) thio, cyano, acyl, thioacyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, nitro, N-sulfonamido, S-sulfonamido, R 9 S(0)-, R 9 S(0) 2 -, -
• heteroalicyclic group is optionally substituted with one or two substituents independently selected from halo, lower alkyl, trihaloalkyl, hydroxy ' , mercapto, cyano, N-amido, mono or dialkylamino, carboxy, or N- sulfonamido.
• Heterocycle means a saturated cyclic radical of 3 to 8 ring atoms in which one or two ring atoms are heteroatoms selected from N, 0, or S(0) n (where n is an integer from 0 to
• the heterocyclyl ring may be optionally substituted independently with one, two, or three substituents selected from lower alkyl optionally substituted one or two substituents independently selected from carboxy or ester group, haloalkyl, cyanoalkyl, halo, nitro, cyano, hydroxy, alkoxy, amino, monoalkylamino, dialkylamino, aralkyl, heteroaralkyl, and -COR (where R is alkyl) .
• heterocyclyl includes, but is not limited to, tetrahydropyranyl, 2, 2-dimethyl-l, 3- dioxolane, piperidino, N-methylpiperidin-3-yl, piperazino, N- methylpyrrolidin-3-yl, pyrrolidino, morpholino, thiomorpholino, thiomorpholino-1-oxide, thiomorpholino-1, 1- dioxide, 4-ethyloxycarbonylpiperazino, 3-oxopiperazino, 2- imidazolidone, 2-pyrrolidinone, 2-oxohomopiperazino, tetrahydropyrimidin-2-one, and the derivatives thereof.
• the heterocycle group is optionally substituted with one or two substituents independently selected from halo, lower alkyl, lower alkyl substituted with carboxy, ester hydroxy, or mono or dialkylamino.
• Heterocycloamino means a saturated cyclic radical of 3 to 8 ring atoms in which at least one of the ring atoms is nitrogen and optionally where one or two additionally ring atoms are heteroatoms selected from N, 0, or S(0) n (where n is an integer from 0 to 2) , the remaining ring atoms being C, where one or two C atoms may optionally be replaced by a carbonyl group.
• the heterocycloamino ring may be optionally substituted independently with one, two, or three substituents selected from lower alkyl optionally substituted one or two substituents independently selected from carboxy or ester group, haloalkyl, cyanoalkyl, halo, nitro, cyano, hydroxy, alkoxy, amino, monoalkylamino, dialkylamino, aralkyl, heteroaralkyl, and -COR (where R is alkyl.
• heterocycloamino includes, but is not limited to, piperidinl-yl, piperazin-1-yl, pyrrolidin-1-yl, morpholin-4- yl, thiomorpholin-4-yl, thiomorpholino-1-oxide, thiomorpholino-1, 1-dioxide, 4-ethyloxycarbonylpiperazin-l-yl, 3-oxopiperazin-l-yl, 2-imidazolidon-l-yl, 2-pyrrolidinon-l-yl, 2-oxohomopiperazino, tetrahydropyrimidin-2-one, and the derivatives thereof.
• the heterocycle group is optionally substituted with one or two substituents independently selected from halo, lower alkyl, lower alkyl substituted with carboxy or ester, hydroxy, or mono or dialkylamino.
• the heterocycloamino group is a subset of the heterocycle group defined above.
• Hydrophilicity refers to an -OH group.
• Alkoxy refers to both an -0- (alkyl) and an -0- (unsubstituted cycloalkyl) group. Representative examples include, but are not limited to, e.g., methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like.
• Haloalkoxy refers to both an -0- (haloalkyl) group. Representative examples include, but are not limited to, e.g., trifluoromethoxy, tribromomethoxy, and the like.
• Aryloxy refers to both an -O-aryl and an -O-heteroaryl group, as defined herein. Representative examples include, but are not limited to, phenoxy, pyridinyloxy, furanyloxy, thienyloxy, pyrimidinyloxy, pyrazinyloxy, and the like, and derivatives thereof.
• Alkylthio refers to both an -S- (alkyl) and an -S- (unsubstituted cycloalkyl) group. Representative examples include, but are not limited to, e.g., methylthio, ethylthio, propylthio, butylthio, cyclopropylthio, cyclobutylthio, cyclopentylthio, cyclohexylthio, and the like.
• Arylthio refers to both an -S-aryl and an -S-heteroaryl group, as defined herein. Representative examples include, but are not limited to, phenylthio, pyridinylthio, furanylthio, thienylthio, pyri idinylthio, and the like and derivatives thereof.
• Acyl refers to a -C(0)-R" group, where R" is selected from the group consisting of hydrogen, lower alkyl, trihalomethyl, unsubstituted cycloalkyl, aryl optionally substituted with one or more, preferably one, two, or three substituents selected from the group consisting of lower alkyl, trihalomethyl, lower alkoxy, halo and -NR 9 R 10 groups, heteroaryl (bonded through a ring carbon) optionally substituted with one or more, preferably one, two, or three substitutents selected from the group consisting of lower alkyl, trihaloalkyl, lower alkoxy, halo and -NR 9 R 10 groups and heteroalicyclic (bonded through a ring carbon) optionally substituted with one or more, preferably one, two, or three substituents selected from the group consisting of lower alkyl, trihaloalkyl, lower alkoxy, halo and -NR 9 R 10 groups.
• Aldehyde refers to an acyl group in which R" is hydrogen.
• Thioacyl refers to a -C(S)-R” group, with R" as defined herein.
• Ester refers to a -C(0)0-R" group with R" as defined herein except that R" cannot be hydrogen.
• Alcohol refers to a -C(0)CH 3 group.
• Halo group refers to fluorine, chlorine, bromine or iodine, preferably fluorine or chlorine.
• Trihalomethyl refers to a -CX 3 group wherein X is a halo as defined above.
• Cyano refers to a -C ⁇ N group.
• S-sulfonamido refers to a -S (0) 2 NR 9 R 10 group, with R 9 and R 10 as defined herein.
• N-sulfonamido refers to a -NR 9 S (0) 2 R 10 group, with R 9 and R 10 as defined herein.
• O-carbamyl refers to a -OC (0) NR 12 R 13 group with R 12 and R 13 as defined herein.
• N-carbamyl refers to an R 9 OC(0)NR 10 - group, with R 9 and R 10 as defined herein.
• O-thiocarbamyl refers to a -OC (S) NR 12 R 13 group with R 12 and R 13 as defined herein.
• N-thiocarbamyl refers to a R 9 OC(S)NR 10 - group, with R 9 and R 10 as defined herein.
• Amino refers to an -NR 9 R 10 group, wherein R 9 and R 10 " are both hydrogen.
• C-amido refers to a -C(0)NR 9 R 10 group with R 9 and R 10 as defined herein.
• N-amido refers to a R 9 C(0)NR 10 - group, with R 9 and R 10 as defined herein.
• Haloalkyl means an alkyl, preferably lower alkyl as defined above that is substituted with one or more same or different halo atoms, e.g., -CH 2 C1, -CF 3 , -CH 2 CF 3 , -CH 2 CC1 3 , and the like.
• Hydroxyalkyl means an alkyl, preferably lower alkyl as defined above that is substituted with one, two, or three hydroxy groups, e.g., hyroxymethyl, 1 or 2-hydroxyethyl, 1,2-, 1,3-, or 2, 3-dihydroxypropyl, and the like.
• Alkyl means alkyl, preferably lower alkyl as defined above which is substituted with an aryl group as defined above, e.g., -CH 2 phenyl, - (CH 2 ) 2 phenyl, - (CH 2 ) 3 phenyl,
• Heteroaralkyl means alkyl, preferably lower alkyl as defined above which is substituted with a heteroaryl group, e.g., -CH 2 pyridinyl, - (CH ) 2 pyrimidinyl, - (CH ) 3 imidazolyl, and the like, and derivatives thereof.
• “Monoalkylamino” means a radical -NHR where R is an alkyl or unsubstituted cycloalkyl group as defined above, e.g., methylamino, (1-methylethyl) amino, cyclohexylammo, and the like.
• “Dialkylamino” means a radical -NRR where each R is independently an alkyl or unsubstituted cycloalkyl group as defined above, e.g., dimethylamino, diethylamino, (1-methylethyl) -ethyla ino, cyclohexylmethylamino, cyclopentylmethylamino, and the like.
• heterocycle group optionally substituted with an alkyl group means that the alkyl may but need not be present, and the description includes situations where the heterocycle group is substituted with an alkyl group and situations where the heterocyclo group is not substituted with the alkyl group.
• isomers Compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or the arrangement of their atoms in space are termed “isomers”. Isomers that differ in the arrangement of their atoms in space are termed “stereoisomers”. Stereoisomers that are not mirror images of one another are termed “diastereomers” and those that are non-superimposable mirror images of each other are termed “enantiomers”. When a compound has an asymmetric center, for example, it is bonded to four different groups, a pair of enantiomers is possible.
• An enantiomer can be characterized by the absolute configuration of its asymmetric center and is described by the R- and S-sequencing rules of Cahn and Prelog, or by the manner in which the molecule rotates the plane of polarized light and designated as dextrorotatory or levorotatory (i.e., as (+) or (-) -isomers respectively) .
• a chiral compound can exist as either individual enantiomer or as a mixture thereof. A mixture containing equal proportions of the enantiomers is called a "racemic mixture".
• the compounds of this invention may possess one or more asymmetric centers; such compounds can therefore be produced as individual (R)- or (S)- stereoisomers or as mixtures thereof.
• the carbon atom carrying the hydroxy group in -CONHCHR 3 -CR 4 (OH) CR 5 Z in a compound of formula (I) is an asymmetric center and therefore the compound of Formula (I) can exist as an (R) - or (S) -stereoisomer .
• the description or naming of a particular compound in the specification and claims is intended to include both individual enantiomers and mixtures, racemic or otherwise, thereof.
• the methods for the determination of stereochemistry and the separation of stereoisomers are well-known in the art (see discussion in Chapter 4 of "Advanced Organic Chemistry", 4th edition J. March, John Wiley and Sons, New York, 1992) .
• the compounds of Formula (I) may exhibit the phenomena of tautomerism and structural isomerism.
• the compounds described herein may adopt an E or a Z configuration about the double bond connecting the 2-indolinone moiety to the pyrrole moiety or they may be a mixture of E and Z.
• This invention encompasses any tautomeric or structural isomeric form and mixtures thereof which possess the ability to modulate RTK, CTK and/or STK activity and is not limited to any one tautomeric or structural isomeric form.
• a “pharmaceutical composition” refers to a mixture of one or more of the compounds described herein, or physiologically/pharmaceutically acceptable salts or prodrugs thereof, with other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients.
• the purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
• the compound of Formula (I) may also act as a prodrug.
• a “prodrug” refers to an agent which is converted into the parent drug in vivo. Prodrugs are often useful because, in some situations, they may be easier to administer than the parent drug. They may, for instance, be bioavailable by oral administration whereas the parent drug is not.
• the prodrug may also have improved solubility in pharmaceutical compositions over the parent drug.
• prodrug a compound of the present invention which is administered as an ester (the "prodrug") to facilitate transmittal across a cell membrane where water solubility is detrimental to mobility but then is metabolically hydrolyzed to the carboxylic acid, the active entity, once inside the cell where water solubility is beneficial.
• a further example of a prodrug might be a short polypeptide, for example, without limitation, a 2 - 10 amino acid polypeptide, bonded through a terminal amino group to a carboxy group of a compound of this invention wherein the polypeptide is hydrolyzed or metabolized in vivo to release the active molecule.
• the prodrugs of a compound of Formula (I) are within the scope of this invention.
• a compound of Formula (I) would be metabolized by enzymes in the body of the organism such as a human being to generate a metabolite that can modulate the activity of the protein kinases. Such metabolites are within the scope of the present invention.
• a physiologically/pharmaceutically acceptable carrier refers to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the- administered compound.
• An “pharmaceutically acceptable excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
• salts As used herein, the term “pharmaceutically acceptable salt” refers to those salts which retain the biological effectiveness and properties of the parent compound. Such salts include:
• PK refers to receptor protein tyrosine kinase (RTKs) , non- receptor or “cellular” tyrosine kinase (CTKs) and serine- threonine kinases (STKs) .
• RTKs receptor protein tyrosine kinase
• CTKs non- receptor or “cellular” tyrosine kinase
• STKs serine- threonine kinases
• Method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by, practitioners of the chemical, pharmaceutical, biological, biochemical and medical arts.
• Modulation refers to the alteration of the catalytic activity of RTKs, CTKs and STKs.
• modulating refers to the activation of the catalytic activity of RTKs, CTKs and STKs, preferably the activation or inhibition of the catalytic activity of RTKs, CTKs and STKs, depending on the concentration of the compound or salt to which the RTK, CTK or STK is exposed or, more preferably, the inhibition of the catalytic activity of RTKs, CTKs and STKs.
• Catalytic activity refers to the rate of phosphorylation of tyrosine under the influence, direct or indirect, of RTKs and/or CTKs or the phosphorylation of serine and threonine under the influence, direct or indirect, of STKs.
• Contacting refers to bringing a compound of this invention and a target PK together in such a manner that the compound can affect the catalytic activity of the PK, either directly, i.e., by interacting with the kinase itself, or indirectly, i.e., by interacting with another molecule on which the catalytic activity of the kinase is dependent.
• Such "contacting” can be accomplished “in vi tro, " i.e., in a test tube, a petri dish or the like. In a test tube, contacting may involve only a compound and a PK of interest or it may involve whole cells. Cells may also be maintained or grown in cell culture dishes and contacted with a compound in that environment.
• the ability of a particular compound to affect a PK related disorder can be determined before use of the compounds in vivo with more complex living organisms is attempted.
• IC 50 of the compound i.e., the IC 50 of the compound, defined below.
• cells outside the organism multiple methods exist, and are well-known to those skilled in the art, to get the PKs in contact with the compounds including, but not limited to, direct cell microinjection and numerous transmembrane carrier techniques .
• " In vitro" refers to procedures performed in an artificial environment such as, e.g., without limitation, in a test tube or culture medium.
• In vivo refers to procedures performed within a living • organism such as, without limitation, a mouse, rat or rabbit.
• PK related disorder "PK driven disorder”
• abnormal PK activity all refer to a condition characterized by inappropriate, i.e., under or, more commonly, over, PK catalytic activity, where the particular PK can be an RTK, a CTK or an STK.
• Inappropriate catalytic activity can arise as the result of either: (1) PK expression in cells which normally do not express PKs, (2) increased PK expression leading to unwanted cell proliferation, differentiation and/or growth, or, (3) decreased PK expression leading to unwanted reductions in cell proliferation, differentiation and/or growth.
• Over-activity of a PK refers to either amplification of the gene encoding a particular PK or production of a level of PK activity which can correlate with a cell proliferation, differentiation and/or growth disorder (that is, as the level of the PK increases, the severity of one or more of the symptoms of the cellular disorder increases) . Under-activity is, of course, the converse, wherein the severity of one or more symptoms of a cellular disorder increase as the level of the PK activity decreases.
• Treatment refer to a method of alleviating or abrogating a PK mediated cellular disorder and/or its attendant symptoms.
• Monitoring means observing or detecting the effect of contacting a compound with a cell expressing a particular PK.
• the observed or detected effect can be a change in cell phenotype, in the catalytic activity of a PK or a change in the interaction of a PK with a natural binding partner. Techniques for observing or detecting such effects are ' well- known in the art.
• the above-referenced effect is selected from a change or an absence of change in a cell phenotype, a change or absence of change in the catalytic activity of said protein kinase or a change or absence of change in the interaction of said protein kinase with a natural binding partner in a final aspect of this invention.
• Cell phenotype refers to the outward appearance of a cell or tissue or the biological function of the cell or tissue. Examples, without limitation, of a cell phenotype are cell size, cell growth, cell proliferation, cell differentiation, cell survival, apoptosis, and nutrient uptake and use. Such phenotypic characteristics are measurable by techniques well-known in the art.
• Natural binding partner refers to a polypeptide that binds to a particular PK in a cell. Natural binding partners can play a role in propagating a signal in a PK-mediated signal transduction process. A change in the interaction of the natural binding partner with the PK can manifest itself as an increased or decreased concentration of the PK/natural binding partner complex and, as a result, in an observable change in the ability of the PK to mediate signal transduction.
• a preferred group of compounds of Formula (I) is that wherein: R 6 is selected from the group consisting of hydrogen and alkyl, preferably hydrogen, methyl, ethyl, isopropyl, tert-butyl, isobutyl, or n-butyl, more preferably hydrogen or methyl; and R 7 is selected from the group consisting of hydrogen, alkyl, aryl, heteroaryl, and -C(0)R 17 wherein R 17 is hydroxy, alkyl, cycloalkyl, aryl, or heteroaryl, and more preferably R 7 is hydrogen, methyl, ethyl, isopropyl, n-, iso or tert-butyl, phenyl, benzoyl, acetyl or carboxy, even more preferably methyl, hydrogen or phenyl.
• R 6 is selected from the group consisting of hydrogen and alkyl, preferably hydrogen, methyl, ethyl, isopropyl, tert-butyl, isobutyl, or n-butyl, more preferably hydrogen or methyl, most preferably methyl;
• R 7 is selected from the group consisting of hydrogen, alkyl, aryl, heteroaryl, and -C(0)R 17 wherein R 17 is hydroxy, alkyl or aryl, and R 7 is more preferably hydrogen, methyl, ethyl, isopropyl, n-, iso or tert-butyl, phenyl, benzoyl, acetyl or carboxy, even more preferably methyl, hydrogen or phenyl; and R 3 , R 4 , and R 5 are hydrogen; and Z is aryl.
• R 6 is selected from the group consisting of hydrogen and alkyl, preferably hydrogen, methyl, ethyl, isopropyl, tert-butyl, isobutyl, or n-butyl, more preferably hydrogen or methyl, most preferably methyl;
• R 7 is selected from the group consisting of hydrogen, alkyl, aryl, heteroaryl, and -C(0)R 17 wherein R 17 is hydroxy, alkyl or aryl, and R 7 is more preferably hydrogen, methyl, ethyl, isopropyl, n-, iso or tert-butyl, phenyl, benzoyl, acetyl or carboxy, even more preferably methyl, hydrogen or phenyl, most preferably methyl; and R 3 , R 4 , and R 5 are hydrogen; and
• Z is heteroaryl, preferably triazinyl, tetrazolyl, imidazolyl, pyridinyl, pyrimidinyl or pyrazinyl.
• R 6 is selected from the group consisting of hydrogen and alkyl, preferably hydrogen, methyl, ethyl, isopropyl, tert-butyl, isobutyl, or n-butyl, more ' preferably hydrogen or methyl, most preferably methyl;
• R 7 is selected from the group consisting of hydrogen, alkyl, aryl, heteroaryl, and -C(0)R 17 wherein R 17 is hydroxy, alkyl or aryl, and R 7 is more preferably hydrogen, methyl, ethyl, isopropyl, n-, iso or tert-butyl, phenyl, benzoyl, acetyl or carboxy, even more preferably methyl, hydrogen or phenyl; and R 3 , R 4 , and R 5 are hydrogen; and Z is heterocycle.
• R 6 is selected from the group consisting of hydrogen and alkyl, preferably hydrogen, methyl, ethyl, isopropyl, tert-butyl, isobutyl, or n-butyl, more preferably hydrogen or methyl, most preferably methyl;
• R 7 is selected from the group consisting of hydrogen, alkyl, aryl, heteroaryl, and -C(0)R 17 wherein R 17 is hydroxy, alkyl or aryl, and R 7 is more preferably hydrogen, methyl, ethyl, isopropyl, n-, iso or tert-butyl, phenyl, benzoyl, acetyl or carboxy, even more preferably methyl, hydrogen or phenyl, most preferably methyl; and
• R 3 , R 4 , and R 5 are hydrogen; and Z is -NR 15 R 16 wherein R 15 and R 16 combine to form heterocyclamino, preferably piperidin-1-yl, N-methylpiperidin- 1-yl, piperazin-1-yl, N-methylpyrrolidin-1-yl, pyrrolidin-1- yl, morpholin-4-yl, thiomorpholin-4-yl, thiomorpholino-1- oxide, thiomorpholino-1, 1-dioxide, 4- ethyloxycarbonylmethylpiperazin-1-yl, 3-oxopiperazin-l-yl, imidazolidin-l-yl-2-one, pyrrolidin-l-yl-2-one, 2- oxohomopiperazin-1-yl, or tetrahydropyrimidin-l-yl-2-one, more preferably morpholin-4-yl.
• R 6 is selected from the group consisting of hydrogen and 'alkyl, preferably hydrogen, methyl, ethyl, isopropyl, tert-butyl ' , isobutyl, or n-butyl, more preferably hydrogen or methyl;
• R 7 is selected from the group consisting of hydrogen, alkyl, aryl, heteroaryl, and -C(0)R 17 wherein R 17 is hydroxy, alkyl or aryl, and R 7 is more preferably hydrogen, methyl, ethyl, isopropyl, n-, iso or tert-butyl, phenyl, benzoyl, acetyl or carboxy, even more preferably methyl, hydrogen or phenyl; and R 3 , R 4 , and R 5 are hydrogen; and
• Z is -NR 15 R 16 wherein R 15 and R 16 are alkyl, preferably diethylamino, dimethylamino, or ethylamino.
• R 1 is hydrogen, alkyl, -C (0)NR 12 R 13 , unsubstituted cycloalkyl, preferably hydrogen, 3,4- dimethoxyphenylaminocarbonyl, 4-methoxy-3-chlorophenyl- aminocarbonyl, even more preferably hydrogen or methyl, most preferably hydrogen; and
• R 2 is hydrogen, cyano, halo, lower alkoxy, or -S(0) 2 NR 9 R 10 wherein R 9 is hydrogen and R 10 is hydrogen, aryl or alkyl and is at the 5-position of the oxindole ring, preferably R 2 is hydrogen, chloro, bromo, fluoro, methoxy, ethoxy, phenyl, dimethylaminosulfonyl, 3-chlorophenyl-aminosulfonyl, carboxy, methoxy, aminosulfonyl, methylaminosulfonyl, phenylaminosulfonyl, pyridin-3-yl-aminosulfonyl, dimethylaminosulfonyl, isopropylamino-sulfonyl, more preferably hydrogen, fluoro, or bromo. Most preferably R 2 is fluoro and is located at the 5-position of the indolinone ring.
• the stereochemistry at the carbon atom carrying the hydroxy group in the -CONHCH (R 3 ) *CR 4 (OH) CR 5 Z chain and indicated by a * is either RS, R, or S, more preferably S.
• the PKs whose catalytic activity is modulated by the compounds of this invention include protein tyrosine kinases of which there are two types, receptor tyrosine kinases (RTKs) and cellular tyrosine kinases (CTKs), and serine-threonine kinases (STKs) .
• RTK mediated signal transduction is initiated by extracellular interaction with a specific growth factor (ligand) , followed by receptor dimerization, transient stimulation of the intrinsic protein tyrosine kinase activity and phosphorylation.
• Binding sites are thereby created for intracellular signal transduction molecules and lead to the formation of complexes with a spectrum of cytoplasmic signaling molecules that facilitate the appropriate cellular response (e.g., cell division, metabolic effects on the extracellular microenvironment, etc.). See, Schlessinger and Ullrich, 1992, Neuron 9:303-391.
• each RTK is determined not only by its pattern of expression and ligand availability but also by the array of downstream signal transduction pathways that are activated by a particular receptor.
• phosphorylation provides an important regulatory step which determines the selectivity of signaling pathways recruited by specific growth factor receptors, as well as differentiation factor receptors.
• STKs being primarily cytosolic, affect the internal biochemistry of the cell, often as a down-line response to a PTK event. STKs have been implicated in the signaling process which initiates DNA synthesis and subsequent mitosis leading to cell proliferation.
• PK signal transduction results in, among other responses, cell proliferation, differentiation, growth and metabolism.
• Abnormal cell proliferation may result in a wide array of disorders and diseases, including the development of neoplasia such as carcinoma, sarcoma, glioblastoma and hemangioma, disorders such as leukemia, psoriasis, arteriosclerosis, arthritis and diabetic retinopathy and other disorders related to uncontrolled angiogenesis and/or vasculogenesis .
• neoplasia such as carcinoma, sarcoma, glioblastoma and hemangioma
• disorders such as leukemia, psoriasis, arteriosclerosis, arthritis and diabetic retinopathy and other disorders related to uncontrolled angiogenesis and/or vasculogenesis .
• a precise understanding of the mechanism by which the compounds of this invention inhibit PKs is not required in order to practice the present invention. However, while not hereby being bound to any particular mechanism or theory, it is believed
• PKs typically possess a bi- lobate structure wherein ATP appears to bind in the cleft between the two lobes in a region where the amino acids are conserved among PKs.
• Inhibitors of PKs are believed to bind by non-covalent interactions such as hydrogen bonding, van der Waals forces and ionic interactions in the same general region where the aforesaid ATP binds to the PKs. More specifically, it is thought that the 2-indolinone component of the compounds of this invention binds in the general space normally occupied by the adenine ring of ATP.
• Specificity of a particular ' molecule for a particular -PK may then arise as the result of additional interactions between the various substituents on the 2-indolinone core and the amino acid domains specific to particular PKs.
• different indolinone substituents may contribute to preferential binding to particular PKs.
• the ability to select compounds active at different ATP (or other nucleotide) binding sites makes the compounds of this invention useful for targeting any protein with such a site.
• the compounds disclosed herein thus have utility in in vi tro assays for such proteins as well as exhibiting in vivo therapeutic effects through interaction with such proteins.
• the compounds of the present invention provide a therapeutic approach to the treatment of many kinds of solid tumors, including but not limited to carcinomas, sarcomas including Kaposi's sarcoma, erythroblastoma, glioblastoma, meningioma, astrocytoma, melanoma and myoblastoma.
• Treatment or prevention of non-solid tumor cancers such as leukemia are also contemplated by this invention.
• Indications may include, but are not limited to brain cancers, bladder cancers, ovarian cancers, gastric cancers, pancreas cancers, colon cancers, blood cancers, lung cancers and bone cancers.
• cell proliferative disorders which may be prevented, treated or further studied by the present invention include cancer, blood vessel proliferative disorders and mesangial cell proliferative disorders.
• Blood vessel proliferative disorders refer to disorders related to abnormal vasculogenesis (blood vessel formation) and angiogenesis (spreading of blood vessels) .
• vasculogenesis and angiogenesis play important roles in a variety of normal physiological processes such as embryonic development, corpus luteum formation, wound healing and organ regeneration, they also play a pivotal role in cancer development where they result in the formation of new capillaries needed to keep a tumor alive.
• Other examples of blood vessel proliferation disorders include arthritis, where new capillary blood vessels invade the joint and destroy cartilage, and ocular diseases, like diabetic retinopathy, where new capillaries in the retina invade the vitreous, bleed and cause blindness.
• VEGF vascular endothelial growth factor
• VEGF is not only responsible for endothelial cell proliferation, but also is the prime regulator of normal and pathological angiogenesis. See generally, Klagsburn & Soker, 1993, Current Biology, 3(10)699-702; Houck, et al., 1992, J_ ⁇ Biol. Chem., 267:26031-26037.
• vasculogenesis and angiogenesis play important roles in a variety of physiological processes such as embryonic development, wound healing, organ regeneration and female reproductive processes such as follicle development in the corpus luteum during ovulation and placental growth after pregnancy.
• Folkman & Shing 1992, J. Biological Chem. , 267 (16) : 10931-34.
• Uncontrolled vasculogenesis and/or angiogenesis has been associated with diseases such as diabetes as well as with malignant solid tumors that rely on vascularization for growth. Klagsburn & Soker, 1993, Current Biology, 3 (10) : 699-702.; Folkham, 1991, J. Natl. Cancer Ins .
• VEGF endothelial growth factor
• VEGF endothelial growth factor
• vasculogenesis indicates an important role for the KDR/FLK-1 receptor in these processes.
• Diseases such as diabetes mellitus (Folkman, 198, in XI th Congress of Thrombosis and Haemostasis (Verstraeta, et al., eds.), pp. 583-596, Leuven University Press, Leuven) and arthritis, as well as malignant tumor growth may result from uncontrolled angiogenesis. See e.g., Folkman, 1971, N.
• VEGF vascular endothelial growth factor
• the receptors to which VEGF specifically binds are an important and powerful therapeutic target for the regulation and modulation of vasculogenesis and/or angiogenesis and a variety of severe diseases which involve abnormal cellular growth caused by such processes. Plowman, et al . , 1994, DN&P, 7 (6) :334-339. More particularly, the KDR/FLK-1 receptor's highly specific role in neovascularization make it a choice target for therapeutic approaches to the treatment of cancer and other diseases which involve the uncontrolled formation of blood vessels.
• the present invention provides compounds capable of regulating and/or modulating tyrosine kinase signal transduction including KDR/FLK-1 receptor signal transduction in order to inhibit or promote angiogenesis and/or vasculogenesis, that is, compounds that inhibit, prevent, or interfere with the signal transduced by KDR/FLK-1 when activated by ligands such as VEGF.
• KDR/FLK-1 receptor signal transduction in order to inhibit or promote angiogenesis and/or vasculogenesis
• the compounds of the present invention act on a receptor or other component along the tyrosine kinase signal transduction pathway, they may also act directly on the tumor cells that result from uncontrolled angiogenesis.
• the nomenclature of the human and murine counterparts of "the generic "flk-I" receptor differ, they are, in many respects, interchangeable.
• the murine receptor, Flk-1, and its human counterpart, KDR share a sequence homology of 93.4% within the intracellular domain.
• murine FLK-I binds human VEGF with the same affinity as mouse VEGF, and accordingly, is activated by the ligand derived from either species. Millauer et al . , 1993, Cell, 72:835-846; Quinn et al., 1993, Proc. Natl. Acad. Sci. USA, 90:7533-7537.
• FLK-1 also associates with and subsequently tyrosine phosphorylates human RTK substrates (e.g., PLC- ⁇ or p85) when co-expressed in 293 cells (human embryonal kidney fibroblasts) .
• Models which rely upon the FLK-1 receptor therefore are directly applicable to understanding the KDR receptor.
• use of the murine FLK-1 receptor in methods which identify compounds that regulate the murine signal transduction pathway are directly applicable to the identification of compounds which may be used to regulate the human signal transduction pathway, that is, which regulate activity related to the KDR receptor.
• chemical compounds identified as inhibitors of KDR/FLK-1 in vitro can be confirmed in suitable in vivo models. Both in vivo mouse and rat animal models have been demonstrated to be of excellent value for the examination of the clinical potential of agents acting on the KDR/FLK-1 induced signal transduction pathway.
• the present invention provides compounds that regulate, modulate and/or inhibit vasculogenesis and/or angiogenesis by affecting the enzymatic activity of the KDR/FLK-1 receptor and interfering with the signal transduced by KDR/FLK-1.
• the present invention provides a therapeutic approach to the treatment of many kinds of solid tumors including, but not limited to, glioblastoma, melanoma and Kaposi's sarcoma, and ovarian, lung, mammary, prostate, pancreatic, colon and epidermoid carcinoma.
• data suggests the administration of compounds which inhibit the KDR/Flk-1 mediated signal transduction pathway may also be used in the treatment of hemangioma, restenois and diabetic retinopathy.
• this invention relates to the inhibition of vasculogenesis and angiogenesis by other receptor-mediated pathways, including the pathway comprising the flt-1 receptor.
• Receptor tyrosine kinase mediated signal transduction is initiated by extracellular interaction with a specific growth factor (ligand) , followed by receptor dimerization, transient stimulation of the intrinsic protein tyrosine kinase activity and autophosphorylation. Binding sites are thereby created for intracellular signal transduction molecules which leads to the formation of complexes with a spectrum of cytoplasmic signalling molecules that facilitate the appropriate cellular response, e.g., cell division and metabolic effects to the extracellular microenvironment . See, Schlessinger and Ullrich, 1992, Neuron, 9:1-20.
• the close homology of the intracellular regions of KDR/FLK-1 with that of the PDGF- ⁇ receptor (50.3% homology) and/or the related flt-1 receptor indicates the induction of overlapping signal transduction pathways.
• the PDGF- ⁇ receptor members of the src family (Twamley et al., 1993, Proc. Natl. Acad. Sci. USA, 90:7696-7700), phosphatidylinositol-3 ' -kinase (Hu et al., 1992, Mol. Cell. Biol. , 12:981-990), phospholipase c ⁇ (Kashishian & Cooper, 1993, Mol. Cell.
• a still further aspect of this invention relates to the use of the organic compounds described herein to modulate angiogenesis and vasculogenesis as such processes are controlled by these pathways.
• disorders related to the shrinkage, contraction or closing of blood vessels, such as restenosis are also implicated and may be treated or prevented by the methods of this invention.
• Fibrotic disorders refer to the abnormal formation of extracellular matrices. Examples of fibrotic disorders include hepatic cirrhosis and mesangial cell proliferative disorders. Hepatic cirrhosis is characterized by the increase in extracellular matrix constituents resulting in the formation of a hepatic scar.
• An increased extracellular matrix resulting in a hepatic scar can also be caused by a viral infection such as hepatitis.
• Lipocytes appear to play a major role in hepatic cirrhosis.
• Other fibrotic disorders implicated include atherosclerosis.
• Mesangial cell proliferative disorders refer to disorders brought about by abnormal proliferation of mesangial cells.
• Mesangial proliferative disorders include various human renal diseases such as glomerulonephritis, diabetic nephropathy and malignant nephrosclerosis as well as such disorders as thrombotic microangiopathy syndromes, transplant rejection, and glomerulopathies.
• the RTK PDGFR has been implicated in the maintenance of mesangial cell proliferation. Floege et al., 1993, Kidney International 43:47S-54S.
• PKs have been associated with cell proliferative disorders.
• PKs such as, for example, members of the RTK family have been associated with the development of cancer.
• EGFR has been associated with squamous cell carcinoma, astrocytoma, glioblastoma, head and neck cancer, lung cancer and bladder cancer.
• HER2 has been associated with breast, ovarian, gastric, lung, pancreas and bladder cancer.
• PDGFR has been associated with glioblastoma and melanoma as well as lung, ovarian and prostate cancer.
• the RTK c-met has also been associated with malignant tumor formation.
• c-met has been associated with, among other cancers, colorectal, thyroid, pancreatic, gastric and hepatocellular carcinomas and lymphomas. Additionally c-met has been linked to leukemia. Over-expression of the c-met gene has al'so been detected in patients with Hodgkins disease and Burkitts disease.
• IGF-IR in addition to being implicated in nutritional support and in type-II diabetes, has also been associated with several types of cancers.
• IGF-I has been implicated as an autocrine growth stimulator for several tumor types, e.g. human breast cancer carcinoma cells (Arteaga et al., 1989, J. Clin. Invest. 84:1418-1423) and.small lung tumor cells (Macauley et al., 1990, Cancer Res. , 50:2511-2517).
• IGF-I while integrally involved in the normal growth and differentiation of the nervous system, also appears to be an autocrine stimulator of human gliomas. Sandberg-Nordqvist et al., 1993, Cancer Res. 53:2475-2478.
• IGF-IR insulin growth factor-IR
• fibroblasts epithelial cells, smooth muscle cells, T-lymphocytes, myeloid cells, chondrocytes and osteoblasts (the stem cells of the bone marrow)
• IGF-I insulin growth factor-I
• Baserga and Coppola suggest that IGF-IR plays a central role in the mechanism of transformation and, as such, could be a preferred target for therapeutic interventions for a broad spectrum of human malignancies. Baserga, 1995, Cancer Res .
• STKs have been implicated in many types of cancer including, notably, breast cancer (Cance, et al., Int. ' J. Cancer, 54:571-77 (1993)).
• RTKs have been associated with diseases such as psoriasis, diabetes mellitus, endometriosis, angiogenesis, atheromatous plaque development, Alzheimer's disease, restenosis, von Hippel-Lindau disease, epidermal hyperproliferation, neurodegenerative diseases, age- related macular degeneration and hemangiomas.
• diseases such as psoriasis, diabetes mellitus, endometriosis, angiogenesis, atheromatous plaque development, Alzheimer's disease, restenosis, von Hippel-Lindau disease, epidermal hyperproliferation, neurodegenerative diseases, age- related macular degeneration and hemangiomas.
• EGFR has been indicated in corneal and dermal wound healing. Defects in Insulin-R and IGF-IR are indicated in type-II diabetes mellitus.
• a more complete correlation between specific RTKs and their therapeutic indications is set forth in Plowman et al . , 1994, DN&P 7:3
• CTKs including, but not limited to, src, abl, fps, yes, fyn, lyn, lck, blk, hck, fgr and yrk (reviewed by Bolen et al., 1992, FASEB J. , 6:3403-3409) are involved in the proliferative and metabolic signal transduction pathway and thus could be expected, and have been shown, to be involved in many PTK-mediated disorders to which the present invention is directed.
• mutated src v-src
• pp60 v_src oncoprotein
• pp60 c ⁇ src transmits oncogenic signals of many receptors.
• Over-expression of EGFR or HER2/neu in tumors leads to the constitutive activation of pp60 c_SEC , which is characteristic of malignant cells but absent in normal cells.
• mice deficient in the expression of c-src exhibit an osteopetrotic phenotype, indicating a key participation of c-src in osteoclast function and a possible involvement in related disorders.
• Zap70 has been implicated in T-cell signaling which may relate to autoimmune disorders.
• STKs have been associated with inflammation, autoimmune disease, immunoresponses, and hyperproliferation disorders such as restenosis, fibrosis, psoriasis, osteoarthritis and rheumatoid arthritis.
• PKs have also been implicated in embryo implantation.
• the compounds of this invention may provide an effective method of preventing such embryo implantation and thereby be useful as birth control agents.
• Additional disorders which may be treated or prevented using the compounds of this invention are immunological disorders such as autoimmune disease, AIDS and cardiovasular disorders such as atherosclerosis.
• a compound of the present invention or a pharmaceutically acceptable salt thereof can be administered as such to a human patient or can be administered in pharmaceutical compositions in which the foregoing materials are mixed with suitable carriers or excipient (s) .
• suitable carriers or excipient s
• Techniques for formulation and administration of drugs may be found in "Remington's Pharmacological Sciences,” Mack Publishing Co., Easton, PA., latest edition.
• administer refers to the delivery of a compound of Formula (I) or a pharmaceutically acceptable salt thereof or of a pharmaceutical composition containing a compound of Formula (I) or a pharmaceutically acceptable salt thereof of this invention to an organism for the purpose of prevention or treatment of a PK-related disorder.
• Suitable routes of administration may include, without limitation, oral, rectal, transmucosal or intestinal administration or intramuscular, subcutaneous, intramedullary, intrathecal, direct intraventricular, intravenous, intravitreal, intraperitoneal, intranasal, or intraocular injections.
• the preferred routes of administration are oral and parenteral.
• compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee- making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
• Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
• the compounds of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer.
• physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer.
• penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
• the compounds can be formulated by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
• Such carriers enable the compounds of the invention to be formulated as tablets, pills, lozenges, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient.
• Pharmaceutical preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding other suitable auxiliaries if desired, to obtain tablets or dragee cores.
• Useful excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol, cellulose preparations such as, for example, maize starch, wheat starch, rice starch and potato starch and other materials such as gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl- cellulose, sodium carboxymethylcellulose, and/or polyvinyl- pyrrolidone (PVP) .
• disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid. A salt such as sodium alginate may also be used.
• Dragee cores are provided with suitable coatings.
• suitable coatings For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
• Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses .
• Pharmaceutical compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
• the push-fit capsules can contain the active ingredients in admixture with a filler such as lactose, a binder such as starch, and/or a lubricant such as talc or' magnesium stearate and, optionally, stabilizers.
• a filler such as lactose
• a binder such as starch
• a lubricant such as talc or' magnesium stearate
• the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
• Stabilizers may be added in these formulations, also.
• compositions which may also be used include hard gelatin capsules.
• the active compound capsule oral drug product formulation may be as 50 and 200 mg dose strengths. The two dose strengths are made from the same granules by filling into different size hard gelatin capsules, size 3 for the 50 mg capsule and size 0 for the 200 mg capsule.
• the composition of the formulation may be, for example, as indicated in Table 2.
• the capsules may be packaged into brown glass or plastic bottles to protect the active compound from light.
• the containers containing the active compound capsule formulation must be stored at controlled room temperature ( 15-30 °C) .
• the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray using a pressurized pack or a nebulizer and a suitable propellant, e.g., without limitation, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetra- fluoroethane or carbon dioxide.
• a suitable propellant e.g., without limitation, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetra- fluoroethane or carbon dioxide.
• the dosage unit may be controlled by providing a valve to deliver a metered amount.
• Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
• the compounds may also be formulated for parenteral administration, e.g., by bolus injection or continuous infusion.
• Formulations for injection may be presented -in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
• the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulating materials such as suspending, stabilizing and/or dispersing agents.
• compositions for parenteral administration include aqueous solutions of a water soluble form, such as, without limitation, a salt, of the active compound.
• suspensions of the active compounds may be prepared in a lipophilic vehicle.
• Suitable lipophilic vehicles include fatty oils such as sesame oil, synthetic fatty acid esters such as ethyl oleate and triglycerides, or materials such as liposomes.
• Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
• the suspension may also contain suitable stabilizers and/or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
• the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.
• a suitable vehicle e.g., sterile, pyrogen-free water
• the compounds may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
• the compounds may also be formulated as depot preparations.
• Such long acting formulations may be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection.
• a compound of this invention may be formulated for this route of administration with suitable polymeric or hydrophobic materials (for instance, in an emulsion with a pharamcologically acceptable oil) , with ion exchange resins, or as a sparingly soluble derivative such as, without limitation, a sparingly soluble salt.
• a non-limiting example of a pharmaceutical carrier for the hydrophobic compounds of the invention is a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer and an aqueous phase such as the VPD co-solvent system.
• VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.
• the VPD co-solvent system (VPD:D5W) consists of VPD diluted 1:1 with a 5% dextrose in water solution. This co- solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration.
• co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics.
• identity of the co-solvent components may be varied: for example, other low- toxicity nonpolar surfactants may be used instead of Polysorbate 80, the fraction size of polyethylene glycol may be varied, other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone, and ' other sugars or polysaccharides may substitute for dextrose.
• hydrophobic pharmaceutical compounds may be employed.
• Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs.
• certain organic solvents such as dimethylsulfoxide also may be employed, although often at the cost of greater toxicity.
• the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent.
• sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.
• compositions herein also may comprise suitable solid or gel phase carriers or excipients.
• suitable solid or gel phase carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
• Many of the PK modulating compounds of the invention may be provided as physiologically acceptable salts wherein the claimed compound may form the negatively or the positively charged species.
• salts in which the compound forms the positively charged moiety include, without limitation, quaternary ammonium (defined elsewhere herein) , salts such as the hydrochloride, sulfate, carbonate, lactate, tartrate, malate, maleate, succinate wherein the nitrogen atom of the quaternary ammonium group is a nitrogen of the selected compound of this invention which has reacted with the appropriate acid.
• Salts in which a compound of this invention forms the negatively charged species include, without limitation, the sodium, potassium, calcium and magnesium salts formed by the reaction of a carboxylic acid group in the compound with an appropriate base (e.g. sodium hydroxide (NaOH), potassium hydroxide (KOH) , Calcium hydroxide (Ca(OH) 2 ), etc. ) .
• compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an amount sufficient to achieve the intended purpose, e.g., the modulation of PK activity or the treatment or prevention of a PK-related disorder.
• a therapeutically effective amount means an amount of compound effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
• the therapeutically effective amount or dose can be estimated initially from cell culture assays. Then, the dosage can be formulated for use in animal models so as to achieve a circulating concentration range that includes the IC 50 as determined in cell culture (i.e., the concentration of the test compound which achieves a half-maximal inhibition of the PK activity) . Such information can then be used to more accurately determine useful doses in humans.
• Toxicity and therapeutic efficacy of the compounds described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the IC 50 and the LD 50 (both of which are discussed elsewhere herein) for a subject compound.
• the data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
• the dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l).
• Dosage amount and interval may be adjusted individually to provide plasma levels of the active species which are sufficient to maintain the kinase modulating effects. These plasma levels are referred to as minimal effective concentrations (MECs) .
• MECs minimal effective concentrations
• the MEC will vary for each compound but can be estimated from in vitro data, e.g., the concentration necessary to achieve 50-90% inhibition of a kinase may be ascertained using the assays described herein. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. HPLC assays or bioassays can be used to determine plasma concentrations.
• Dosage intervals can also be determined using MEC value.
• Compounds should be administered using a regimen that maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%.
• the therapeutically effective amounts of compounds of Formulas I, la, or II may range from approximately 25 mg/m 2 to 1500 mg/m 2 per day; preferably about 3 mg/m 2 /day. Even more preferably 50mg/qm qd till 400 mg/qd.
• the effective local concentration of the drug may not be related to plasma concentration and other procedures known in the art may be employed to determine the correct dosage amount and interval.
• compositions may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient.
• the pack may for example comprise metal or plastic foil, such as a blister pack.
• the pack or dispenser device may be accompanied by instructions for administration.
• the pack or dispenser may also be accompanied by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or of human or veterinary administration.
• compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition. Suitable conditions indicated on the label may include treatment of a tumor, inhibition of angiogenesis, treatment of fibrosis, diabetes, and the like.
• the present invention can be administered with a CMC suspension vehicle.
• An exemplary CMC suspension is listed below in Table 3.
• a protocol for a 1.0 Lit of CMC suspension vehicle is as follows. Calculate the appropriate amount of excipients required to make the vehicle formulation using the table showing the composition of vehicle formulation and the batch size. Weigh a suitable empty container, such as a clean wide mouthed glass bottle, or a polyethylene bottle. Add about 600 mL of water to the container. Weigh carboxymethylcellulose sodium (5 gms) and transfer to the container. Stir using a magnetic stir bar or a laboratory stirrer with propeller until homogenous (about 2-3 hours) . Weigh NaCl and add to the container. Continue mixing until dissolved (about 10 mins) . Add polysorbate-80. Mix until the solution is' homogenous (about 20 mins) . Add benzyl alcohol.
• the suspension formulation can be manufactured as follows. Grind the API using a mortar and pestle to obtain a homogenous looking powder with small particulate size (no chunks or large particulates - ideally should pass through a US Standard Sieve >80 i.e. ⁇ 180 ⁇ m size). Weigh the calculated amount of API into the container. Add about 90% of the total required amount of (CMC suspension vehicle) into the container. Suspend compounds in the vehicle using a laboratory stirrer with propeller or equivalent. The diameter of the propeller blades should match the diameter of the bottom of the container to ensure efficient mixing. Stir at 50 rpm for 30 mins or until the drug is well suspended.
• a compound, salt or prodrug of this invention might be combined with alkylating agents such as fluorouracil (5-FU) alone or in further combination with leukovorin; or other alkylating agents such as, without limitation, other pyrimidine analogs such as UFT, capecitabine, gemcitabine and cytarabine, the alkyl sulfonates, e.g., busulfan (used in the treatment of chronic granulocytic leukemia) , improsulfan and piposulfan; aziridines, e.g., benzodepa, carboquone, meturedepa and uredepa; ethyleneimines and methylmelamines, e.g., altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine; and the nitrogen mustards, e.g., chlorambucil (used in the treatment of chronic lymphocytic leukemia, primary macroglobulor
• a compound, salt or prodrug of this invention can also be used in combination with other antimetabolite chemotherapeutic agents such as, without limitation, folic acid analogs, e.g. methotrexate (used in the treatment of acute lymphocytic leukemia, choriocarcinoma, mycosis fungiodes breast cancer, head and neck cancer and osteogenic sarcoma) and pteropterin; and the purine analogs such as mercaptopurine and thioguanine which find use in the treatment of acute granulocytic, acute lymphocytic and chronic granulocytic leukemias.
• folic acid analogs e.g. methotrexate (used in the treatment of acute lymphocytic leukemia, choriocarcinoma, mycosis fungiodes breast cancer, head and neck cancer and osteogenic sarcoma) and pteropterin
• purine analogs such as mercaptopurine and thioguan
• a compound, salt or prodrug of this invention can also be used in combination with natural product based chemotherapeutic agents such as, without limitation, the vinca alkaloids, e.g., vinblastin (used in the treatment of breast and testicular cancer) , vincristine and vindesine; the epipodophylotoxins, e.g., etoposide and teniposide, both of which are useful in the treatment of testicular cancer and Kaposi's sarcoma; the antibiotic chemotherapeutic agents, e.g., daunorubicin, doxorubicin, epirubicin, mitomycin (used to treat stomach, cervix, colon, breast, bladder and pancreatic cancer) , dactinomycin, temozolomide, plicamycin, bleomycin (used in the treatment of skin, esophagus and genitourinary tract cancer) ; and the enzymatic chemotherapeutic agents such as
• a compound, salt or prodrug of this invention could also be used in combination with the platinum coordination complexes (cisplatin, etc.); substituted ureas such as hydroxyurea; methylhydrazine derivatives, e.g., procarbazine; adrenocortical suppressants, e.g., mitotane, aminoglutethimide; and hormone and hormone antagonists such as the adrenocorticosteriods (e.g., prednisone) , progestins (e.g., hydroxyprogesterone caproate) ; estrogens (e.g., diethylstilbesterol) ; antiestrogens such as tamoxifen; androgens, e.g., testosterone propionate; and aromatase inhibitors such as anastrozole.
• substituted ureas such as hydroxyurea
• methylhydrazine derivatives e.g., procarbazin
• a compound of this invention will be effective in combination with mitoxantrone, paclitaxel, cyclooxygenase-2 inhibitors known in the art, in particular Celebrex®, Paracoxib®, Vioxx®, Abbott's Cox-189 disclosed in PCT Publication No. WO 99/11605, topoisomerase inhibitors such as Camptosar®, Her-2 receptor antagonist such as Herceptin®, endostatin, Gleevac®, ImClone VEGF receptor antagonist IMC C225® for the treatment of solid tumor cancers or leukemias such as, without limitation, acute myelogenous (non-lymphocytic) leukemia.
• the base may be an organic or an inorganic base. If an organic base is used, preferably it is a nitrogen base. Examples of organic nitrogen bases include, but are not limited to, diisopropylamine, trimethylamine, triethylamine, aniline, pyridine, 1, 8-diazabicyclo [5.4.1] undec-7-ene,- pyrrolidine and piperidine.
• inorganic bases are, without limitation, ammonia, alkali metal or alkaline earth hydroxides, phosphates, carbonates, bicarbonates, bisulfates and amides.
• the alkali metals include, lithium, sodium and potassium while the alkaline earths include calcium, magnesium and barium.
• the base is an alkali metal or an alkaline earth inorganic base, preferably, a alkali metal or an alkaline earth hydroxide.
• the solvent in which the reaction is carried out may be a protic or an aprotic solvent, preferably it is a protic solvent.
• a * "protic solvent” is a solvent which has hydrogen atom(s) covalently bonded to oxygen or nitrogen atoms which renders the hydrogen atoms appreciably acidic and thus capable of being “shared” with a solute through hydrogen bonding.
• Examples of protic solvents include, without limitation, water and alcohols.
• aprotic solvent may be polar or non-polar but, in either case, does not contain acidic hydrogens and therefore is not capable of hydrogen bonding with solutes.
• non-polar aprotic solvents are pentane, hexane, benzene, toluene, methylene chloride and carbon tetrachloride.
• polar aprotic solvents are chloroform, tetrahydrofuran, dimethylsulfoxide and dimethylformamide.
• the solvent is a protic solvent, preferably water or an alcohol such as ethanol.
• the reaction is carried out at temperatures greater than room temperature.
• the temperature is generally from about 30° C to about 150° C, preferably about 80°C to about 100° C, most preferable about 60° C to about 85° C, which is about the boiling point of ethanol.
• about is meant that the temperature range is preferably within 10 degrees Celcius of the indicated temperature, more preferably within 5 degrees Celcius of the indicated temperature and, most preferably, within 2 degrees Celcius of the indicated temperature.
• 75° C is meant 75° C ⁇ 10° C, preferably 75° C ⁇ 5° C and most preferably, 75° C ⁇ 2° C.
• Amines of formula ZCH (R 5 ) -CR 4 (OH) - CHR 3 NH 2 are commercially available or they can be prepared by method well known in the art. Some such procedures are described herein below. It will be appreciated by those skilled in the art that other synthetic pathways for forming the compounds of the invention are available and that the following is offered by way of example and not limitation.
• 5-Formyl-2 4-dimethyl-lH-pyrrole-3-carboxylic acid ethyl ester (2 g, 10 mmol) was added to a solution of potassium hydroxide (3 g, 53 mmol) dissolved in methanol (3 mL) and water (10 mL) . The mixture was refluxed for 3 hours, cooled to room temperature and acidified with 6 N hydrochloric acid to pH 3. The solid was collected by filtration, washed with water and dried in a vacuum oven overnight to give 5-formyl-2, 4- dimethyl-lH-pyrrole-3-carboxylic acid (1.6 g , 93%).
• Step 3 5- (5-Fluoro-2-oxo-l, 2-dihydro-indol-3-ylidenemethyl) - 2, 4-dimethyl-li ⁇ -pyrrole-3-carboxylic acid (120 mg, 0.4 mmol) was condensed with l-amino-3-morpholin-4-yl-propan-2-ol (74 mg, 0.48 mmol) to precipitate 5- [5-fluoro-2-oxo-l, 2-dihydro- indol- (3Z) -ylidenemethyl] -2, 4-dimethyl-lH-pyrrole-3- carboxylic acid (2-hydroxy-3-morpholin-4-yl-propyl) -amide (65 mg, 36%) .
• the reaction was assayed by GC (dilute 5 drops of reaction mixture into 1 ml of ethanol and inject onto a 15m DB-5 capillary GC column with the following run parameters, Injector 250°C, detector 250°C, initial oven temperature 28 °C warming to 250 °C at 10 °C per minute.) The reaction was complete with less than 3% morpholine remaining. The reaction was concentrated on the rotoevaporated at 50 °C with full house vacuum until no more distillate could be condensed. The resulting oil was stored at room temperature for 24-48 hours or until a significant mass of crystals was observed (seeded will speed up the process) . The slurry was diluted with 250ml of acetone and filtered.
• the reaction was assayed by GC (dilute 5 drops of reaction mixture into 1 ml of ethanol and inject onto a 15m DB-5 capillary GC column with the following run parameters, Injector 250°C, detector 250°C, initial oven temperature 28 °C warming to 250 °C at 10 °C per minute) .
• the reaction was complete with less than 3% morpholine remaining.
• the solution was cooled to 10°C and a 20 wt% solution of potassium t-butoxide in THF (576g) was added dropwise keeping the temperature less than 15°C.
• the resulting white slurry was stirred at 10-15°C for 2 hours and checked by GC using the above conditions. None of the chlorohydrin could be observed.
• the mixture was concentrated on the rotoevaporated using 50°C bath and full house vacuum.
• the resulting mixture was diluted with water (500ml) and methylene chloride.
• the phases were separated and the aqueous phase washed with methylene chloride (500ml) .
• the combined organic layers were dried over sodium sulfate and concentrated to a clear, colorless oil. This provided 145g, 97% yield of the epoxide.
• the reaction was assayed by GC (dilute 5 drops of reaction mixture intol ml of ethanol and inject onto a 15m DB-5 capillary GC column with the following run parameters, Injector 250°C, detector 250°C, initial oven temperature 28°C warming to 250°C at 10°C per minute.) The reaction was complete with less than 3% morpholine remaining. The solution was cooled to 10°C and a 25 wt . %solution of sodium methoxide in methanol (233g, 1.08 mole, 247 ml) was added dropwise keeping the temperature less than 15°C. The resulting white slurry was stirred at 10-15°C for 2 hours and checked by GC using the above conditions. None of the chlorohydrin could be observed.
• the mixture was concentrated on the rotoevaporator using 50°C bath and full house vacuum.
• the resulting mixture was diluted with water (500ml) and methylene chloride.
• the phases were separated and the aqueous phase washed with methylene chloride (500ml) .
• the combined organic layers were dried over sodium sulfate and concentrated to a clear, colorless oil. This provided 145g, 97% yield of l,2-epoxy-3-morpholin-4-ylpropane.
• the R and S stereoisomers can be prepared as follows
• Imidazole amide (7.0 g, 32.3 mmol), amine (15.5 g ' , 96.9 mmol), 5-chlorooxindole (5.48 g, 32.6 mmol), triethylamine (14 ml), and THF (88 ml) were mixed and heated to 60°C. A red solution formed. After stirring for 16 h at 60 °C, the yellow slurry was cooled to rt and filtered. The cake was washed with 2 x 50 ml THF and dried overnight at 50 °C under house vacuum. 4.36 g were obtained with 29% chemical yield.
• Imidazole amide (6.8 g, 31.3 mmol), amine (10.0 g, 62.5 mmol), 5-chlorooxindole (5.3 g, 31.6 mmol), and THF (100 ml) were mixed and heated to 60 °C. A red solution formed. After stirring for 68 h at 60°C, triethylamine (14ml) was added and stirred for 5 h at 60°C. Reaction was not complete. Add 4.6 g of amine side chain, and stirred for 20 h at 60 °C. The yellow slurry was cooled to rt and filtered. The cake was washed with 2 x 50 ml THF and dried overnight at 50 °C under house vacuum. 5.48 g were obtained with 38% chemical yield.
• the first fraction provided pure 3- (2-H-tetrazol-2-yl) -2-hydroxy-l-chloropropane, 6.215g (colorless oil, 38%Y)
• the second fraction yielded 9.208g of pure 3- (1-H-tetrazol-l-yl) ] -2-hydroxyl-l- chloropropane (sticky gum; 57%Y) .
• the obtained solution was evaporated on highvac and purified on a column of silica in a mixture of chloroform-methanol (a gradient 5 to 20% of methanol) to obtain 5.572g of the chloro-compound as a white amorphous solid (90% Y) .
• the chloride was transformed into amine as follows.
• the obtained hydroxy-chloro intermediate was dissolved in methanolic ammonia (saturated with ammonia gas), potassium carbonate was added and the mixture was stirred in closed flask for 2 days.
• the reaction mixture was filtered, filtrates evaporated.
• the residue was, purified on a silica column in a mixture chloroform-methanol-conc. aqueous ammonia 80:15:1.5.
• the Robbins Block is removed from the oven.
• the top seal of the block is removed and 800 uL of DMSO is added to the mixture.
• the block is resealed and again placed in the 60 °C oven where it rotates continuously for 1 hour.
• the Robbins Block is removed from the oven and allowed to cool.
• the bottom seal of the Robbins Block is carefully removed and the entire block is fitted into the filtration device, which enables the newly synthesized compounds to be filtered away from the resin.
• the first fractions provided 3- [1-H- (benztriazolyl) -oxy] -2-hydroxy-l-chloropropane 10.570 g (pale yellow honey; 51.5%Y), followed by fraction of 3- [1-H- (benztriazolyl-3-N-oxido) ] -2-hydroxy-l-chloropropane 9.990g (white crystalline solid, 48.5% Y)
• the following assays may be used to determine the level of activity and effect of the different compounds of the present invention on one or more of the PKs. Similar assays can be designed along the same lines for any PK using techniques well known in the art.
• the cells are lysed and the lysate is transferred to the wells of an ELISA plate previously coated with a specific antibody recognizing the substrate of the enzymatic phosphorylation reaction.
• Non-substrate components of the cell lysate are washed away and the amount of phosphorylation on the substrate is detected with an antibody specifically recognizing phosphotyrosine compared with control cells that were not contacted with a test compound.
• a DNA labeling reagent such as 5-bromodeoxyuridine (BrdU) or H 3 -thymidine is added.
• the amount of labeled DNA is detected with either an anti- BrdU antibody or by measuring radioactivity and is compared to control cells not contacted with a test compound.
• This assay analyzes the tyrosine kinase activity of GST- Flkl on poly (glu, tyr) peptides.
• Materials and Reagents 1. Corning 96-well ELISA plates (Corning Catalog No. 5805-96) .
• PBS Buffer for 1 L, mix 0.2 g KH 2 P0 4 , 1.15 g Na 2 HP0 4 , 0.2 g KC1 and 8 g NaCl in approx. 900ml dH 2 0. When all reagents have dissolved, adjust the pH to 7.2 with HCl. Bring total volume to 1 L with dH 2 0.
• PBST Buffer to 1 L of PBS Buffer, add 1.0 ml Tween- 20.
• TBB - Blocking Buffer for 1 L, mix 1.21 g TRIS,
• KDB Kinase Dilution Buffer
• EDTA mix 14.12 g ethylenediaminetetraacetic acid (EDTA) to approx. 70 ml dH 2 0. Add 10 N NaOH until EDTA dissolves. Adjust pH to 8.0. Adjust total volume to 100 ml with dH 2 0.
• This assay is used to measure the in vitro kinase activity of HA epitope-tagged full length pyk2 (FL.pyk2-HA) in an ELISA assay.
• PBS Dulbecco's Phosphate-Buffered Saline (Gibco Catalog # 450-1300EB)
• TBST Buffer for 1 L, mix 8.766 g NaCl, 6.057 g TRIS and 1 ml of 0.1% Triton X-100 in approx. 900 ml dH 2 0. Adjust pH to 7.2, bring volume to 1 L.
• Blocking Buffer for 1 L, mix 100 g 10% BSA, 12.1 g 100 mM TRIS, 58.44 g IM NaCl and 10 mL of 1% TWEEN-
• Antibody dilution buffer for 100 mL, 1 mL 5% BSA/PBS and 1 mL 10% Tween-20 in 88 mL TBS.
• This assay is used to measure the in vitro kinase activity of FGFl-R in an ELISA assay.
• PBS Gibco Catalog # 450-1300EB
• 50 mM Hepes Buffer Solution 4. 50 mM Hepes Buffer Solution.
• Blocking Buffer 5% BSA/PBS).
• step 10 Wash as in step 10. 14. Add 100 ⁇ l per well of Biosource Goat anti-rabbit IgG peroxidase conjugate (1:6000 dilution in ADB). Incubate, with shaking for 1 hr. at room temperature.
• This assay is used to the in vitro kinase activity of FGF1-R in an ELISA assay.
• Materials and Reagents 1. Corning 96-well Elisa plates.
• Blocking Buffer for 100 ml, mix 5.0 g Carnation Instant Non-fat Milk® with 100 ml of PBS. 6. A431 cell lysate (SUGEN, Inc.).
• TBS + 10% DMSO for IL, mix 1.514 g TRIS, 2.192 g NaCl and 25 ml DMSO; bring to 1 liter total volume with dH 2 0.
• ATP Adiosine-5 ' -triphosphate, from Equine muscle, Sigma Cat. No. A-5394
• ATP/MnCl 2 phosphorylation mix to make 10 ml, mix 300 ⁇ l of 1 mM ATP, 500 ⁇ l MnCl 2 and 9.2 ml dH 2 0. Prepare just prior to use, keep on ice.
• Blocking Buffer (same as for EGFR bioassay) .
• PDGFR- ⁇ expressing NIH 3T3 cell lysate (SUGEN, Inc. ) .
• Kinase buffer phosphorylation mix for 10 ml, mix 250 ⁇ l IM TRIS, 200 ⁇ l 5M NaCl, 100 ⁇ l IM MnCl 2 and 50 ⁇ l 100 mM Triton X-100 in enough dH 2 0 to make 10 ml .
• This assay is used to measure HER-2 kinase activity in whole cells in an ELISA format.
• SUMO 1 monoclonal anti-EGFR antibody (SUGEN, Inc. ) .
• Blocking Buffer 5% Carnation Instant Milk® in PBS.
• EGF Ligand EGF-201, Shinko American, Japan. Suspend powder in 100 uL of lOmM HCl. Add lOOuL lOmM
• IX HNTG* mix 2 ml HNTG, 100 ⁇ L 0. IM Na 3 V0 4 , 250 ⁇ L 0.2M Na 4 P 2 0 7 and 100 ⁇ L EDTA.
• Test compounds are dissolved in 4% DMSO. Samples are then further diluted directly on plates with starve-DMEM. Typically, this dilution will be 1:10 or greater. All wells are then transferred to the cell plate at a further 1:10 dilution (lO ⁇ l sample and media into 90 ⁇ l of starve media. The final DMSO concentration should be 1% or lower. A standard serial dilution may also be used. 9. . Incubate ' under 5% C0 2 at 37°C for 2 hours. 10. Prepare EGF ligand by diluting stock EGF (16.5 uM) in warm DMEM to 150 nM.
• EGF ligand to cells, 50 ul per well, for a final concentration of 50 nM. Positive control wells receive the same amount of EGF. Negative controls do not receive EGF. Incubate at 37° C for 10 min. 13. Remove test compound, EGF, and DMEM. Wash cells once with PBS. 14. Transfer HNTG* to cells, 100 ul per well. Place on ice for 5 minutes. Meanwhile, remove blocking buffer from ELISA plate and wash. 15. Scrape cells from plate with a micropipettor and homogenize cell material by repeatedly aspirating and dispensing the HNTG* lysis buffer. Transfer lysate to a coated, blocked, washed ELISA plate.
• This assay is used to measure the in vitro serine/threonine kinase activity of human cdk2/cyclin A in a Scintillation Proximity Assay (SPA) . Materials and Reagents.
• Peptide/ATP Mixture for 10 ml, mix 9.979 ml dH 2 0, 0.00125 ml "cold” ATP, 0.010 ml Debtide and 0.010 ml ⁇ 33 P ATP. The ultimate concentration per well will be 0.5 ⁇ M "cold” ATP, 0.1 ⁇ g Debtide and 0.2 ⁇ Ci ⁇ 33 P ATP.
• Stop solution For 10 ml, mix 9.25 ml PBS, 0.005 ml 100 mM ATP, 0.1 ml 0.5 M EDTA, 0.1 ml 10% Triton X- 100 and 1.25 ml of 20 mg/ml SPA beads. Procedure:
• This assay is used to measure phosphotyrosine levels on a poly (glutamic acid: tyrosine (4:1)) substrate as a means for identifying agonists/antagonists of met transphosphorylation of the substrate.
• Blocking Buffer Dissolve 25 g Bovine Serum Albumin, Sigma Cat. No A-7888, in 500 ml PBS, filter through a 4 ⁇ m filter.
• Antibody Dilution Buffer for 100 mL, mix 10 mL 5% BSA/PBS, 0.5 mL 5% Carnation Instant Milk® in PBS and 0.1 mL 0.1 M sodium orthovanadate in 88.4 mL
• ABTS Solution for 1 L, mix 19.21 g citric acid, 35.49 g Na 2 HP0 and 500 mg ABTS with sufficient dH 2 0 to make 1 L.
• ABTS/H 2 0 2 mix 15 mL ABST solution with 2 ⁇ L H 2 0 2 five minutes before use.
• This assay is used to measure the phosphotyrosine level in poly (glutamic acid: tyrosine) (4:1) for the identification of agonists/antagonists of gst-IGF-1 transphosphorylation- of a substrate.
• TBB Blocking Buffer for 1 L, mix 100 g BSA, 12.1 gTRIS (pH 7.5), 58.44 g sodium chloride and 10 mL l%TWEEN-20. 6. Purified GST fusion protein containing the IGF-1 kinase domain (Sugen, Inc.) 7. TBST Buffer: for 1 L, mix 6.057 g Tris, 8.766 g sodium chloride and 0.5 ml TWEEN-20 with enough dH 2 0 to make 1 liter.
• Antibody Dilution Buffer for 100 mL, mix 10. mL 5% BSA in PBS, 0.5 mL 5% Carnation Instant Non-fat
• the following assays use cells engineered to express a selected receptor and then evaluate the effect of a compound of interest on the activity of ligand-induced DNA synthesis by determining BrdU incorporation into the DNA.
• BrdU Labeling Reagent 10 mM, in PBS (pH 7.4) (Boehringer Mannheim, Germany) .
• FixDenat fixation solution (ready to use) (Boehringer Mannheim, Germany) .
• Anti-BrdU-POD mouse monoclonal antibody conjugated with peroxidase (Boehringer Mannheim, Germany) .
• TMB Substrate Solution tetramethylbenzidine (TMB, Boehringer Mannheim, Germany) .
• PBS Washing Solution IX PBS, pH 7.4.
• Cells are seeded at 8000 cells/well in 10% CS, 2 mM Gin in DMEM, in a 96 well plate. Cells are incubated o overnight at 37 C in 5% C02 •
• test compounds are prepared in serum free DMEM with ligand in a 96 well plate, and serially diluted for 7 test concentrations .
• the medium is removed by decanting and tapping the inverted plate on a paper towel.
• FixDenat solution is added (50 ⁇ l/well) and the plates are incubated at room temperature for 45 minutes on a plate shaker.
• FixDenat solution is thoroughly removed by decanting and tapping the inverted plate on a paper towel. Milk is added (5% dehydrated milk in PBS, 200 ⁇ l/well) as a blocking ' solution and the plate is incubated for 30 minutes at room temperature on a plate shaker.
• the blocking solution is removed by decanting and the wells are washed once with PBS.
• Anti-BrdU-POD solution (1:200 dilution in PBS, 1% BSA) is added (50 ⁇ l/well) and the plate is incubated for 90 minutes at room temperature on a plate shaker.
• the antibody conjugate is thoroughly removed by decanting and rinsing the wells 5 times with PBS, and the plate is dried by inverting and tapping on a paper towel.
• TMB substrate solution is added (100 ⁇ l/well) and incubated for 20 minutes at room temperature on a plate shaker until color development is sufficient for photometric detection. 10.
• the absorbance of the samples are measured at 410 nm (in "dual wavelength” mode with a filter reading at 490 nm, as a reference wavelength) on a Dynatech ELISA plate reader.
• EGF-Induced Her-2-driven BrdU Incorporation Assay Materials and Reagents: i. Mouse EGF, 201 (Toyobo Co., Ltd., Japan).
• Cells are seeded at 9000 cells/well in RPMI 10% FBS in a 96 well plate. Cells are incubated overnight at 37 °C in 5% C02. 2. After 24 hours, the cells are washed with PBS, and then are serum starved in 100 ⁇ l serum-free medium (RPMI with 0.1% BSA) for 24 hours.
• test compounds are prepared at 5 times their final concentration in serum-free RPMI with ligand in a 96 well plate, and serially diluted to give 7 test concentrations. Typically, the highest final concentration of test compound is 100 ⁇ M, and 1:3 dilutions are used (i.e. final test compound concentration range is 0.137-100 ⁇ M) .
• This assay is used to measure a compound's activity against PDGF-R, FGF-R, VEGF, aFGF or Flk-1/KDR, all of which are naturally expressed by HUV-EC cells. DAY 0
• HUV-EC-C cells human umbilical vein endothelial cells, (American Type Culture Collection, catalogue no. 1730 CRL) . Wash with Dulbecco's phosphate- buffered saline (D-PBS, obtained from Gibco BRL, catalogue no. 14190-029) 2 times at about 1 ml/10 cm 2 of tissue culture flask. Trypsinize with 0.05% trypsin-EDTA in non-enzymatic cell dissociation solution (Sigma Chemical Company, catalogue no. C-1544) .. The 0.05% trypsin is made by diluting 0.25% trypsin/1 mM EDTA (Gibco, catalogue no.
• test compound titrations make up two-fold test compound titrations in separate 96-well plates, generally 50 ⁇ M on down to 0 ⁇ M. Use the same assay medium as mentioned in day 0, step 2 above. Titrations are made by adding 90 ⁇ l/well of test compound at 200 ⁇ M (4X the final well concentration) to the top well of a particular plate column. Since the stock test compound is usually 20 mM in DMSO, the 200 ⁇ M drug concentration contains 2% DMSO.
• a diluent made up to 2% DMSO in assay medium (F12K + 0.5% fetal bovine serum) is used as diluent for the test compound titrations in order to dilute the test compound but keep the DMSO concentration constant. Add this diluent to the remaining wells in the column at 60 ⁇ l/well. Take 60 ⁇ l from the 120 ⁇ l of 200 ⁇ M test compound dilution in the top well of the column and mix with the 60 ⁇ l in the second well of the column. Take 60 ⁇ l from this well and mix with the 60 ⁇ l in the third well of the column, and so on until two-fold titrations are completed.
• VEGF vascular endothelial cell growth factor
• aFGF acidic fibroblast growth factor
• the growth factor concentrations are 4X the desired final concentration.
• the 4X concentrations of test compound and growth factors become IX once everything has been added to the wells. DAY 2
• Bioassays which have been or can be used to evaluate compounds are described in detail below. Compounds 1-9 were tested and found active in flkGST, FGFRl and PDGF assays.
• human tumors to grow as xenografts in athymic mice (e.g., Balb/c, nu/nu) provides a useful in vivo model for studying the biological response to therapies for human tumors. Since the first successful xenotransplantation of human tumors into athymic mice, (Rygaard and Povlsen, 1969, Acta Pathol. Microbial . Scand. 77:758-760), many different human tumor cell lines (e.g., mammary, lung, genitourinary, gastro-intestinal, head and neck, glioblastoma, bone, and malignant melanomas) have been transplanted and successfully grown in nude mice.
• human tumor cell lines e.g., mammary, lung, genitourinary, gastro-intestinal, head and neck, glioblastoma, bone, and malignant melanomas
• the following assays may be used to determine the level of activity, specificity and effect of the different compounds of the present invention.
• Three general types of assays are useful for evaluating compounds: cellular/catalytic, cellular/biological and in vivo.
• the object of the cellular/catalytic assays is to determine the effect of a compound on the ability of a TK to phosphorylate
• the object of the cellular/biological assays is to determine the effect of a compound on the biological response stimulated by a TK in a cell.
• the object of the in vivo assays is to determine the effect of a compound in an animal model of a particular disorder such as cancer.
• Suitable cell lines for subcutaneous xenograft experiments include C6 cells (glioma, ATCC # CCL 107) , A375 cells (melanoma, ATCC # CRL 1619) , A431 cells (epidermoid carcinoma, ATCC # CRL 1555), Calu 6 cells (lung, ATCC # HTB
• mice Female athymic mice (BALB/c, nu/nu) are obtained from BALB/c, nu/nu.
• Simonsen Laboratories (Gilroy, CA) . All animals are maintained under clean-room conditions in Micro-isolator cages with Alpha-dri bedding. They receive sterile rodent chow and water ad libitum. Cell lines are grown in appropriate medium (for example, MEM, DMEM, Ham's F10, or Ham's F12 plus 5% - 10% fetal bovine serum (FBS) and 2 mM glutamine (GLN) ) . All cell culture media, glutamine, and fetal bovine serum are purchased from Gibco Life Technologies (Grand Island, NY) unless otherwise specified. All cells are grown in a humid atmosphere of 90- 95% air and 5-10% C0 at 37 °C. All cell lines are routinely subcultured twice a week and are negative for mycoplasma as determined by the Mycotect method (Gibco) .
• Cells are harvested at or near confluency with 0.05% Trypsin-EDTA and pelleted at 450 x g for 10 min. Pellets are resuspended in sterile PBS or media (without FBS) to a particular concentration and the cells are implanted into the hindflank of the mice (8 - 10 mice per group, 2 - 10 x 10 6 cells/animal) . Tumor growth is measured over 3 to 6 weeks using venier calipers. Tumor volumes are calculated as a product of length x width x height unless otherwise indicated. P values are calculated using the Students t-test. Test compounds in 50 - 100 ⁇ L excipient (DMSO, or VPD:D5W) can be delivered by IP injection at different concentrations generally starting at day one after implantation. TUMOR INVASION MODEL
• 8 week old nude mice (female) (Simonsen Inc.) are used as experimental animals. Implantation of tumor cells can be performed in a laminar flow hood. For anesthesia, Xylazine/Ketamine Cocktail (100 mg/kg ketamine and 5 mg/kg Xylazine) are administered intraperitoneally. A midline incision is done to expose the abdominal cavity (approximately 1.5 cm in length) to inject 10 7 tumor cells in a volume of 100 ⁇ l medium. The cells are injected either into the duodenal lobe of the pancreas or under the serosa of the colon. The peritoneum and muscles are closed with a 6-0 silk continuous suture and the skin is closed by using wound clips. Animals are observed daily.
• mice are sacrificed, and the local tumor metastases to various organs (lung, liver, brain, stomach, spleen, heart, muscle) are excised and analyzed (measurement of tumor size, grade of invasion, immunochemistry, in situ hybridization determination, etc.).
• This assay is used to detect the level of c-kit tyrosine phosphorylation.
• M07E human acute myeloid leukemia
• M07E human acute myeloid leukemia cells are serum starved overnight in 0.1% serum. Cells are pre-treated with the compound (concurrent with serum starvation) , prior to ligand stimulation. Cells are stimulated with 250 ng/ml rh- SCF for 15 minutes. Following stimulation, cells were lysed and immunoprecipitated with an anti-c-kit antibody. Phosphotyrosine and protein levels were determined by Western blotting. MTT PROLIFERATION ASSAY
• M07E cells are serum starved and pre-treated with compound as described for the phosphorylation experiments.
• Cells areplated @ 4X10 5 cells/well in a 96 well dish, in 100 ⁇ l RPMI + 10% serum.
• rh-SCF 100 ng/mL is added and the plate is incubated for 48 hours.
• 10 ⁇ l of 5 mg/ml MTT [3- (4, 5-dimethythiazol-2-yl) -2, 5-diphenyl tetrazolium bromide) is added and allowed to incubate for 4 hours.
• Acid isopropanol 100 ⁇ l of 0.04N HCl in isopropanol
• the optical density was measured at a wavelength of 550 nm.
• M07E cells are incubated +/- SCF and +/- compound in 10% FBS with rh-GM-CSF(10ng/mL) and rh-IL-3 (lOng/mL) . Samples are assayed at 24 and 48 hours. To measure activated caspase-3, samples are washed with PBS and permeabiliz.ed with ice-cold 70% ethanol. The cells are then stained with PE-conjugated polyclonal rabbit anti-active caspase-3 and analyzed by FACS. To measure cleaved PARP, samples are lysed and analyzed by western blotting with an anti-PARP antibody. Additional assays
• Additional assays which may be used to evaluate the compounds of this invention include, without limitation, a bio-flk-1 assay, an EGF receptor-HER2 chimeric receptor assay in whole cells, a bio-src assay, a bio-lck assay and an assay measuring the phosphorylation function of raf .
• the protocols for each of these assays may be found in U. S. Application Ser. No. 09/099,842, which is incorporated by reference, including any drawings, herein.
• Measurement of Cell Toxicity Therapeutic compounds should be more potent in inhibiting receptor tyrosine kinase activity than in exerting a cytotoxic effect.
• a measure of the effectiveness and cell toxicity of a compound can be obtained by determining the therapeutic index, i.e., IC 5 0/LD50.
• IC 50 the dose required to achieve 50% inhibition
• LD 50 the dosage which results in 50% toxicity
• Compounds with a large therapeutic index are preferred.
• the therapeutic index should be greater than 2, preferably at least 10, more preferably at least 50.

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