EP1066257A2 - Familles heterocycliques de composes destinees a la modulation de la tyrosine-kinase - Google Patents

Familles heterocycliques de composes destinees a la modulation de la tyrosine-kinase

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Publication number
EP1066257A2
EP1066257A2 EP99915018A EP99915018A EP1066257A2 EP 1066257 A2 EP1066257 A2 EP 1066257A2 EP 99915018 A EP99915018 A EP 99915018A EP 99915018 A EP99915018 A EP 99915018A EP 1066257 A2 EP1066257 A2 EP 1066257A2
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Prior art keywords
group
alkyl
ring
compound
formula
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German (de)
English (en)
Inventor
Annie Fong
Alison Hannah
David G. Harris
Peter Hirth
Li Sun
Peter Langecker
Congxin Liang
Gerald Mcmahon
Peng C. Tang
Axel Ullrich
Laura K. Shawver
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Max Planck Institut fuer Biochemie
Sugen LLC
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Max Planck Institut fuer Biochemie
Sugen LLC
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    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • 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
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    • 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
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • 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
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/30—Indoles; Hydrogenated indoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to carbon atoms of the hetero ring
    • C07D209/32—Oxygen atoms
    • C07D209/34—Oxygen atoms in position 2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/56—Ring systems containing three or more rings
    • C07D209/80—[b, c]- or [b, d]-condensed
    • C07D209/82—Carbazoles; Hydrogenated carbazoles
    • C07D209/86—Carbazoles; Hydrogenated carbazoles with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to carbon atoms of the ring system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms

Definitions

  • Cellular signal transduction is a fundamental mechanism whereby extracellular stimuli are relayed to the interior of cells and subsequently regulate diverse cellular processes.
  • One of the key biochemical mechanisms of signal transduction involves the reversible phosphorylation of proteins. Phosphorylation of polypeptides regulates the activity of mature proteins by altering their structure and function. Phosphate most often resides on the hydroxyl moiety (-OH) of serine, threonine, or tyrosine amino acids in proteins.
  • Enzymes that mediate phosphorylation of cellular effectors generally fall into two classes.
  • the first class consists of protein kinases which transfer a phosphate moiety from adenosine triphosphate to protein substrates.
  • the second class consists of protein phosphatases which hydrolyze phosphate moieties from phosphoryl protein substrates. The converse functions of protein kinases and protein phosphatases balance and regulate the flow of signals in signal transduction processes.
  • Protein kinases and protein phosphatases are generally divided into two groups - receptor and non-receptor type proteins. Most receptor-type protein tyrosine phosphatases contain two conserved catalytic domains, each of which encompasses a segment of 240 amino acid residues. Saito et al, 1991, Cell Growth andDiff. 2:59-65. Receptor protein tyrosine phosphatases can be subclassified further based upon the amino acid sequence diversity of their extracellular domains. Saito et al, supra; Kr eger et al, 1992, Proc. Natl. Acad. Sci. USA 89:1411-142 .
  • Protein kinases and protein phosphatases are also typically divided into three classes based upon the amino acids they act upon. Some catalyze the addition or hydrolysis of phosphate on serine or threonine only, some catalyze the addition or hydrolysis of phosphate on tyrosine only, and some catalyze the addition or hydrolysis of phosphate on serine, threonine, and tyrosine.
  • Tyrosine kinases can regulate the catalytic activity of other protein kinases involved in cell proliferation. Protein kinases with inappropriate activity are also involved in some types of cancer. Abnormally elevated levels of cell proliferation are associated with receptor and non-receptor protein kinases with unregulated activity.
  • protein kinases are thought to be involved in cellular differentiation processes.
  • Cell differentiation occurs in some cells upon nerve growth factor (NGF) or epidermal growth factor (EGF) stimulation.
  • NGF nerve growth factor
  • EGF epidermal growth factor
  • Cellular differentiation is characterized by rapid membrane ruffling, cell flattening, and increases in cell adhesion. Chao, 1992, Cell 68:995-991.
  • biomedical researchers and chemists have designed, synthesized, and tested molecules that inhibit the function of protein kinases.
  • Some small organic molecules form a class of compounds that modulate the function of protein kinases.
  • Examples of molecules that have been reported to inhibit the function of protein kinases are bis-monocyclic, bicyclic or heterocyclic aryl compounds (PCT WO 92/20642), vinylene-azaindole derivatives (PCT WO 94/14808), l-cyclo ⁇ ropyl-4-pyridyl-quinolones (U.S. Patent No. 5,330,992), styryl compounds (by Levitzki, et al., U.S. Patent No. 5,217,999, and entitled "Styryl Compounds which Inhibit EGF Receptor Protein Tyrosine Kinase), styryl-substituted pyridyl compounds (U.S. Patent No. 5,302,606), certain quinazoline derivatives (EP
  • the compounds that can traverse cell membranes and are resistant to acid hydrolysis are potentially advantageous therapeutics as they can become highly bioavailable after being administered orally to patients.
  • many of these protein kinase inhibitors only weakly inhibit the function of protein kinases.
  • the present invention is directed in part towards tricyclic-based indolinone compounds, pyrazolylamide-based compounds, imidazoyl 2-indolinone derivatives, phenyl 2-indolinone derivatives, and methods of modulating the function of protein kinases (PKs) with these compounds.
  • the methods incorporate cells that express a protein kinase.
  • the invention describes methods of preventing and treating protein kinase-related abnormal conditions in organisms with a compound identified by the methods described herein.
  • the invention pertains to pharmaceutical compositions comprising compounds identified by methods of the invention.
  • the present invention features indolinone compounds that potently inhibit protein kinases and related products and methods.
  • Inhibitors of protein kinases can be obtained by adding chemical substituents to an indolinone compound.
  • the compounds of the invention represent a new generation of therapeutics for diseases associated with one or more functional or non-functional protein kinases. Neuro-degenerative diseases and certain types of cancer fall into this class of diseases.
  • the compounds can be modified such that they are specific to their target or targets and will subsequently cause few side effects and thus represent a new generation of potential cancer therapeutics. These properties are significant improvements over the currently utilized cancer therapeutics that cause multiple side effects and deleteriously weaken patients.
  • the compounds of the invention will minimize and obliterate solid tumors by inhibiting the activity of the protein kinases, or will at least modulate or inhibit tumor growth and or metastases.
  • Protein kinases regulate proliferation of blood vessels during angiogenesis, among other functions. Increased rates of angiogenesis accompany cancer tumor growth in cells as cancer tumors must be nourished by oxygenated blood during growth. Therefore, inhibition of the protein kinase and the corresponding decreases in angiogenesis will starve tumors of nutrients and most likely obliterate them.
  • PKs e.g., the fibroblast growth factor receptor 1 (FGFR1)
  • FGFR1 fibroblast growth factor receptor 1
  • the compounds are believed to interact with the amino acids of the PKs' catalytic region.
  • PKs typically possess a bi-lobate structure, and 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 to the PKs through non-covalent interactions such as hydrogen bonding, Van der Waals interactions, and ionic bonding, in the same general region that ATP binds to the PKs.
  • the oxyindole component of the compounds of the present invention binds in the same general space occupied by the adenine ring of ATP.
  • Specificity of an indolinone PK inhibitor for a particular PK may be conferred by interactions between the constituents around the oxyindole core with amino acid domains specific to individual PKs.
  • different indolinone substitutents may contribute to preferential binding to particular PKs.
  • the ability to select those compounds active at different ATP binding sites makes them useful in targeting any protein with such a site, not only protein tyrosine kinases, but also serine/threonine kinases and protein phosphatases.
  • Unregulated blood vessel growth due to angiogenesis and vascularization, which is at least in part responsible for diseases and disorders such as rheumatoid arthritis, endometriosis, ocular diseases, cancer and metastases, psoriasis, arterial thickening and restenosis, and excessive scarring during wound healing, is dependent on phosphorylation of substrate molecules by activated tyrosine kinases.
  • the methods of the invention can minimize angiogenesis and vascularization of tissues by specifically inhibiting the activity of protein kinases, which regulate proliferation of blood vessels during angiogenesis.
  • This inhibition can result in a blockade to tyrosine signal transduction and an interruption of abnormal cell proliferation and thus disease progression.
  • it may be useful to increase the activity of protein kinases in order to increase the proliferation of blood vessels through angiogenesis.
  • the compounds of the invention may potently inhibit the action of phosphatases and may represent a new generation of therapeutics for diseases associated with defects in said phosphatases.
  • the compounds of the present invention may be used to modulate the functions of protein phosphatases and may be used for preventing and treating protein phosphatase related abnormal conditions in organisms.
  • Terms defined herein with respect to kinases have a similar meaning to one skilled in the art with respect to phosphatases.
  • the present invention also provides for methods of preventing and treating sexual dysfunction in mammals, especially humans.
  • the invention provides methods for administering a compound to a mammal in need thereof and monitoring the progress of the mammal's sexual activity, including, in males, erectile function, the ability to perform intercourse, maintaining an erection subsequent to penetration, and orgasm, and in females, increased vaginal lubrication and orgasm.
  • compound refers to the compound or a pharmaceutically acceptable salt, ester, amide, prodrug, isomer, or metabolite, thereof.
  • isomers Different compounds that have the same molecular formula are called "isomers.” Isomers have the same numbers of the same kinds of atoms, but the atoms are attached to one another in different ways. Thus, all stereoisomers of the indolinone compounds are within the scope of the invention.
  • pharmaceutically acceptable salt refers to a formulation of a compound that does not abrogate the biological activity and properties of the compound.
  • Pharmaceutical salts can be obtained by reacting a compound of the invention with inorganic or organic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, /j-toluenesulfonic acid, salicylic acid and the like.
  • prodrug refers to an agent that 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.
  • An example, without limitation, of a prodrug would be 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 which 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.
  • indolinone is used as that term is commonly understood in the art and includes a large subclass of substituted or unsubstituted compounds that are capable of being synthesized from an aldehyde moiety and a oxindole moiety.
  • oxindole refers to an oxindole compound optionally substituted with chemical substituents. Oxindole compounds are of the general structure:
  • pyrazolylamide is used as that term is commonly understood in the art and includes a large subclass of substituted or unsubstituted compounds that are formed by linking an aryl moiety with a pyrazol moiety through an amide bond, where such linking takes place at position 3 of the pyrazol ring.
  • pyrazol refers to a pyrazol compound substituted with chemical substituents. Pyrazol compounds are of the general structure:
  • substituted in reference to the invention, refers to a compound that is derivatized with any number of chemical substituents.
  • alkyl refers to saturated or unsaturated alkyl optionally substituted with substituents selected from the group consisting of halogen, trihalomethyl, carboxy late, amino, nitro, ester, and a five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moiety, where the ring moiety is optionally substituted with one, two, or three substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, amino, nitro, and ester moieties.
  • saturated alkyl refers to an alkyl moiety that does not contain any alkene or alkyne moieties.
  • the alkyl moiety may be branched or non-branched.
  • unsaturated alkyl refers to an alkyl moiety that contains at least one alkene or alkyne moiety.
  • the alkyl moiety may be branched or non-branched.
  • a "cycloalkyl” group refers to an all-carbon monocyclic or fused ring (i.e., rings which share an adjacent pair of carbon atoms) group wherein one or more of the rings does not have a completely conjugated pi-electron system.
  • examples, without limitation, of cycloalkyl groups are cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, cyclohexadiene, cycloheptane and, cycloheptatriene.
  • a cycloalkyl group may be substituted or unsubstituted.
  • the substituent group(s) is preferably one or more individually selected from alkyl, aryl, heteroaryl, heteroalycyclic, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, halo, carbonyl, thiocarbonyl, carboxy, O-carbamyl, N-carbamyl, C-amido, N-amido, nitro, amino and NR10R11 with R ⁇ 0 and Rn as previously defined herein.
  • alkenyl refers to an alkyl group, as defined herein, consisting of at least two carbon atoms and at least one carbon-carbon double bond.
  • alkynyl refers to an alkyl group, as defined herein, consisting of at least two carbon atoms and at least one carbon-carbon triple bond.
  • aromatic refers to an aromatic group which has at least one ring having a conjugated pi electron system and includes both carbocyclic aryl (e.g. phenyl) and heterocyclic aryl groups (e.g. pyridine).
  • carbocyclic refers to a compound which contains one or more covalently closed ring structures, and that the atoms forming the backbone of the ring are all carbon atoms. The term thus distinguishes carbocyclic from heterocyclic rings in which the ring backbone contains at least one atom which is different from carbon.
  • heterocyclic refers to an aromatic group which contains at least one heterocyclic ring.
  • aromatic or heteroaromatic rings defined herein may optionally be substituted with one, two, or three substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, amino, nitro, and ester moieties.
  • aliphatic ring refers to a compound which contains one or more covalently closed ring structures, and that at least one of the atoms forming the backbone is a saturated carbon atom (e.g. cyclohexane).
  • heteroaliphatic ring refers to a ring system in which at least one of the atoms forming the backbone is a heteroatom (e.g. tetrahydropyran).
  • the aliphatic or heteroaliphatic rings defined herein may optionally be substituted with one, two, or three substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, amino, nitro, ester, and an aromatic or heteroaromatic ring optionally substituted with one, two, or three substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, amino, nitro, and ester moieties.
  • a “fused heteroarylxycloalkyl/heteroalicyclic” group refers to a heteroaryl group in which two adjacent carbon atoms of the heteroaryl ring are connected to one another, in addition to the direct covalent bond of each of them to the heteroaryl ring, by a five-atom or a six-atom chain which may consist of all carbon atoms or carbon atoms combined with heteroatoms such as nitrogen, oxygen and or sulfur; this second connection results in the formation of a second ring.
  • fused heteroary cycloalkyl heteroalicyclic groups are:
  • the fused heteroaryhcycloalkyl/heteroalicyclic groups may be unsubstituted or substituted.
  • substitutent(s) on a heteroaryl moiety of a fused heteroarylxycloalkyl/heteroalicyclic group are selected from those set forth above in the definition of the heteroaryl group.
  • substituents on a cycloalkyl or a heteroalicyclic moiety of a fused heteroaryhcycloalkyl/hetero- alicyclic group are selected from those set forth in the definitions of cycloalkyl and heteroalicyclic.
  • amine refers to a chemical moiety of formula -(X ⁇ ) n ⁇ -NX 2 X 3 , where X ⁇ is selected from the group consisting of saturated or unsaturated alkyl, and five-membered or six-membered aromatic, heteroaromatic, or aliphatic ring moieties and where nl is 0, 1, or 2, and where X and X 3 are independently selected from the group consisting of hydrogen, saturated or unsaturated alkyl, five-membered or six-membered aromatic, heteroaromatic, or aliphatic ring moieties, and where X 2 and X together form a five- membered or six-membered heteroaromatic or heteroaliphatic ring, as those rings are defined herein.
  • halogen refers to an atom selected from the group consisting of fluorine, chlorine, bromine, and iodine.
  • trihalomethyl refers to the -CX 3 group, where X is a halogen.
  • ketone refers to a chemical moiety with formula -(X ) n4 -CO-X 5 , where
  • X and X 5 are independently selected from the group consisting of alkyl optionally substituted with halogen, trihalomethyl, carboxylate, amino, nitro, ester, and five- membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties, where the ring moieties are optionally substituted with one, two, or three substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, amino, nitro, and ester moieties and where n4 is 0, 1, or, 2.
  • carboxylic acid refers to a chemical moiety with formula -(X 6 ) n6 -
  • esters refers to a chemical moiety with formula -(X 7 ) n7 -COO-X 8 .
  • X 6 , X 7 , and X 8 and are independently selected from the group consisting of alkyl and five- membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties and where n6 and n7 are independently 0, 1, or, 2.
  • An "O-carboxy” group refers to a group, with X as defined herein.
  • alcohol refers to a chemical substituent of formula -(X 9 ) n9 -OH
  • alkoxyalkyl refers to a chemical substituent of formula -(X ⁇ o) n io-O-X ⁇ , where X 9 , X 10 , and Xn are independently selected from the group consisting of saturated or unsaturated alkyl, and five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties, where the ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, and ester and where n9 and nlO are independently 0, 1, or, 2. When n is 0, then the alkoxyalkyl moiety is called an "alkoxy moiety".
  • alkoxyalkoxy refers to a chemical substituent of formula -O-(X 2 o) n2 o-O-X 2 , where X 20 and X 2 are each independently selected from the group consisting of alkyl, five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, or ester, and where n20 is 0 or 1.
  • amide refers to a chemical substituent of formula -(X 12 ) n ⁇ 2 -NHCOX 13 , i.e., "N-amido", or of formula -(X ⁇ ) n ⁇ -CONXi 5 X ⁇ , i.e., "C-amido", where X J2 and X 1 are each independently selected from the group consisting of alkyl, and five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties, where the ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, and ester and where nl2 and nl4 are independently 0, 1, or, 2, and where X ⁇ 3 , X ⁇ , and X ⁇ 6 are each independently selected from the group consisting of hydrogen, alkyl, hydroxyl, and five- membered or six
  • cyano refers to a chemical moiety with formula -C ⁇ N.
  • cyanato refers to a chemical moiety with formula -CNO.
  • isocyanato refers to a chemical moiety with formula -NCO.
  • thiocyanato refers to a chemical moiety with formula -CNS.
  • isothiocyanato refers to a chemical moiety with formula -NCS.
  • sulfonamide refers to a chemical moiety with formula -(X ⁇ 7 ) n ⁇ 7 - SO 2 NX ⁇ 8 Xi 9 , where X ⁇ 8 , and X ⁇ 9 are independently selected from the group consisting of alkyl, five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, or ester, and where Xj 8 and X 19 taken together form a five-membered or six-membered aliphatic or heteroaliphatic ring optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, amino, nitro, and ester, and X] 7 is selected from the group consisting of alkyl, five-membered or six-membered
  • aldehyde refers to a chemical moiety with formula -(X 20 ) n2 o-CO-H where X 20 is selected from the group consisting of saturated or unsaturated alkyl, and five- membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties, where the ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, amino, nitro, and ester and where n20 is 0, 1, or, 2.
  • sulfone refers to a chemical moiety with formula -(X 2 ⁇ ) n2 ⁇ -SO 2 -X 22 , X 2 ⁇ and X 22 are independently selected from the group consisting of saturated or unsaturated alkyl, and five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties, where the ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, amino, nitro, and ester and where n21 is 0, 1, or, 2.
  • thiol refers to a chemical moiety with formula-(X 23 ) n23 -SH
  • thioether refers to a chemical moiety of the formula -(X 24 ) n24 -S-X 25 , where X 23 , X 2 , and X 25 are independently selected from the group consisting of saturated or unsaturated alkyl, and five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties, where the ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, amino, nitro, and ester and where n23 and n24 are independently 0, 1, or, 2.
  • acyl refers to chemical moieties of the general formula -C(O)R.
  • R is hydrogen the molecule containing the acyl group is an aldehyde.
  • R is an alkyl, an aliphatic ring, or an aromatic ring, then the molecule containing the acyl group is a ketone.
  • N-carbamyl refers to a chemical moiety with formula
  • heavy metal refers to elements in the transition metal series of the periodic table of the elements, and include elements number 21-30, 39-48, and 57-82.
  • the heavy metal may be substituted with a five-membered or six-membered aromatic or heteroaromatic ring moieties, where the ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, amino, nitro, and ester.
  • ferrocene refers to an organometallic sandwich complex containing an atom of iron sandwiched by two cyclopentadienyl groups.
  • the cyclopentadienyl rings can be optionally substituted with substituents described herein. Ferrocene is normally depicted using one of the two structures shown below, structure A and structure B.
  • X can be carbon, nitrogen, oxygen or sulfur.
  • a “combinatorial library” refers to all the compounds formed by the reaction of each compound in one dimension of a multi-dimensional array with a compound in each of the other dimensions of the multi-dimensional array.
  • the multidimensional array is two dimensional, one dimension being all the oxindoles of this invention and the other dimension being all the aldehydes of this invention.
  • Each oxindole may be reacted with each of the aldehydes to form a 2-indolinone. All 2-indolinone compounds formed in this manner are within the scope of this invention.
  • smaller combinatorial libraries formed by the reaction of some of the oxindoles of this invention with all the aldehydes of this invention or all of the oxindoles with some of the aldehydes or some of the oxindoles with some of the aldehydes.
  • the temperature range is preferably ⁇ 10 °C, more preferably ⁇ 5 °C, and most preferably ⁇ 2 °C.
  • the temperature range is preferably 50 ⁇ 10 °C, more preferably 50 ⁇ 5 °C, and most preferably 50 ⁇ 2 °C.
  • composition refers to a mixture of an indolinone compound of the invention with other chemical components, such as diluents, excipients, or carriers.
  • the pharmaceutical composition facilitates administration of the compound to an organism. Multiple techniques of administering a compound exist in the art including, but not limited to, oral, injection, aerosol, parenteral, and topical administration.
  • Pharmaceutical compositions can also be obtained by reacting compounds with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, -toluenesulfonic acid, salicylic acid and the like.
  • pharmaceutically acceptable refers to solutions or components of the formulation that do not prevent the therapeutic compound from exerting a therapeutic effect and do not cause unacceptable adverse side effects. Examples of pharmaceutically acceptable reagents are provided in The United
  • carrier defines a chemical compound that facilitates the incorporation of a compound into cells or tissues.
  • DMSO dimethyl sulfoxide
  • dimethyl sulfoxide (DMSO) is a commonly utilized carrier as it facilitates the uptake of many organic compounds into the cells or tissues of an organism.
  • diluent defines chemical compounds diluted in water that will dissolve the compound of interest as well as stabilize the biologically active form of the compound. Salts dissolved in buffered solutions are utilized as diluents in the art.
  • One commonly used buffered solution is phosphate buffered saline because it mimics the salt conditions of human blood. Since buffer salts can control the pH of a solution at low concentrations, a buffered diluent rarely modifies the biological activity of a compound.
  • the term "function" refers to the cellular role of a protein kinase.
  • the protein kinase family includes members that regulate many steps in signaling cascades, including cascades controlling cell growth, migration, differentiation, gene expression, muscle contraction, glucose metabolism, cellular protein synthesis, and regulation of the cell cycle.
  • catalytic activity in the context of the invention, defines the rate at which a protein kinase phosphorylates a substrate. Catalytic activity can be measured, for example, by determining the amount of a substrate converted to a product as a function of time. Phosphorylation of a substrate occurs at the active-site of a protein kinase.
  • the active-site is normally a cavity in which the substrate binds to the protein kinase and is phosphorylated.
  • substrate refers to a molecule phosphorylated by a protein kinase.
  • the substrate is preferably a peptide and more preferably a protein.
  • the term “activates” refers to increasing the cellular function of a protein kinase.
  • the protein kinase function is preferably the interaction with a natural binding partner and most preferably catalytic activity.
  • the term “inhibit” refers to decreasing the cellular function of a protein kinase.
  • the protein kinase function is preferably the interaction with a natural binding partner and most preferably catalytic activity.
  • modulates refers to altering the function of a protein kinase by increasing or decreasing the probability that a complex forms between a protein kinase and a natural binding partner. Such modulation may take place either in vivo or in vitro.
  • a modulator preferably increases the probability that such a complex forms between the protein kinase and the natural binding partner, more preferably increases or decreases the probability that a complex forms between the protein kinase and the natural binding partner depending on the concentration of the compound exposed to the protein kinase, and most preferably decreases the probability that a complex forms between the protein kinase and the natural binding partner.
  • a modulator preferably activates the catalytic activity of a protein kinase, more preferably activates or inhibits the catalytic activity of a protein kinase depending on the concentration of the compound exposed to the protein kinase, or most preferably inhibits the catalytic activity of a protein kinase.
  • complex refers to an assembly of at least two molecules bound to one another. Signal transduction complexes often contain at least two protein molecules bound to one another.
  • the term "natural binding partner” refers to polypeptides that bind to a protein kinase in cells. Natural binding partners can play a role in propagating a signal in a protein kinase signal transduction process. A change in the interaction between a protein kinase and a natural binding partner can manifest itself as an increased or decreased probability that the interaction forms, or an increased or decreased concentration of the protein kinase/natural binding partner complex.
  • a protein kinase natural binding partner can bind to a protein kinase' s intracellular region with high affinity. High affinity represents an equilibrium binding constant on the order of 10 "6 M or less.
  • a natural binding partner can also transiently interact with a protein kinase intracellular region and chemically modify it.
  • Protein kinase natural binding partners are chosen from a group that includes, but is not limited to, SRC homology 2 (SH2) or 3 (SH3) domains, other phosphoryl tyrosine binding (PTB) domains, guanine nucleotide exchange factors, protein phosphatases, and other protein kinases. Methods of determining changes in interactions between protein kinases and their natural binding partners are readily available in the art.
  • contacting refers to mixing a solution comprising a compound of the invention or other compounds such as VEGF, FGF, or PDGF, with a liquid medium bathing the cells of the methods.
  • the solution comprising the compound may also comprise another component, such as dimethylsulfoxide (DMSO), which facilitates the uptake of the indolinone compound or compounds into the cells of the methods.
  • DMSO dimethylsulfoxide
  • the solution comprising the indolinone compound may be added to the medium bathing the cells by utilizing a delivery apparatus, such as a pipet-based device or syringe- based device.
  • monitoring refers to observing the effect of adding the compound to the cells of the method.
  • the effect can be manifested in a change in cell phenotype, cell proliferation, protein kinase catalytic activity, or in the interaction between a protein kinase and a natural binding partner.
  • the term “monitoring” is further used herein in reference to an effect on rats in the adjuvant arthritis model to include general disease symptoms including ear nodulation, tail nodulation, nose swelling, paw swelling, and balanitis.
  • An arthritis index can be calculated from these measurements as defined in the
  • effect describes a change or an absence of a change in cell phenotype or cell proliferation.
  • Effect can also describe a change or an absence of a change in the catalytic activity of the protein kinase.
  • Effect can also describe a change or an absence of a change in an interaction between the protein kinase and a natural binding partner.
  • cell phenotype refers to the outward appearance of a cell or tissue or the function of the cell or tissue.
  • cell phenotype examples include cell size (reduction or enlargement), cell proliferation (increased or decreased numbers of cells), cell differentiation (a change or absence of a change in cell shape), cell survival, apoptosis (cell death), or the utilization of a metabolic nutrient (e.g., glucose uptake). Changes or the absence of changes in cell phenotype are readily measured by techniques known in the art.
  • antibody refers to an antibody (e.g., a monoclonal or polyclonal antibody), or antibody fragment, having specific binding affinity to protein kinase or its fragment.
  • specific binding affinity is meant that the antibody binds to target (protein kinase) polypeptides with greater affinity than it binds to other polypeptides under specified conditions.
  • Antibodies having specific binding affinity to a protein kinase may be used in methods for detecting the presence and/or amount of a protein kinase in a sample by contacting the sample with the antibody under conditions such that an immunocomplex forms and detecting the presence and/or amount of the antibody conjugated to the protein kinase.
  • Diagnostic kits for performing such methods may be constructed to include a first container containing the antibody and a second container having a conjugate of a binding partner of the antibody and a label, such as, for example, a radioisotope.
  • the diagnostic kit may also include notification of an FDA approved use and instructions therefor.
  • polyclonal refers to antibodies that are heterogenous populations of antibody molecules derived from the sera of animals immunized with an antigen or an antigenic functional derivative thereof.
  • various host animals may be immunized by injection with the antigen.
  • Various adjuvants may be used to increase the immunological response, depending on the host species.
  • Monoclonal antibodies are substantially homogenous populations of antibodies to a particular antigen. They may be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture. Monoclonal antibodies may be obtained by methods known to those skilled in the art. See, for example, Kohler, et al., Nature 256:495-497 (1975), and U.S. Patent No. 4,376,110.
  • antibody fragment refers to a portion of an antibody, often the hypervariable region and portions of the surrounding heavy and light chains, that displays specific binding affinity for a particular molecule.
  • a hypervariable region is a portion of an antibody that physically binds to the polypeptide target.
  • inhibition in conjunction with a signal transduction process, refers to a protein kinase that is over- or under-expressed in an organism, mutated such that its catalytic activity is lower or higher than wild-type protein kinase activity, mutated such that it can no longer interact with a natural binding partner, is no longer modified by another protein kinase or protein phosphatase, or no longer interacts with a natural binding partner.
  • promoting or disrupting the abnormal interaction refers to a method that can be accomplished by administering a compound of the invention to cells or tissues in an organism.
  • a compound can promote an interaction between a protein kinase and natural binding partners by forming favorable interactions with multiple atoms at the complex interface.
  • a compound can inhibit an interaction between a protein kinase and natural binding partners by compromising favorable interactions formed between atoms at the complex interface.
  • preventing refers to administering a composition to a patient before an abnormal condition manifests itself in that patient, and at least partially stopping or reducing the abnormal condition that would otherwise result.
  • treating refers to the method of the invention having a therapeutic effect and at least partially alleviating or abrogating an abnormal condition in the organism.
  • rheumatoid arthritis refers to a chronic systemic disease primarily of the joints, usually polyarticular, marked by inflammatory changes in the synovial membranes and articular structures and by muscle atrophy and rarefaction of the bones. Forms of rheumatoid arthritis include, but are not limited to, juvenile, chronic villous, cricoarytenoid, deformans, degenerative, mutilans, and proliferative.
  • endometriosis refers to a condition in which tissue containing typical endometrial granular and stromal elements occurs aberrantly in various locations in the pelvic cavity or some other area of the body (most commonly the peritoneal cavity).
  • ocular disease refers to diseases of, pertaining to, or affecting the eye, specifically those where new capillaries in the retina invade the vitreous, bleed, and can cause blindness. Examples include, but are not limited to, senile macular degeneration and diabetic retinopathy.
  • cancer and metastases refers to a new growth of tissue in which the multiplication of cells is uncontrolled and progressive and a growth of abnormal cells distant from the site primarily involved by the morbid process.
  • psoriasis refers to a common chronic, squamous dermatosis with polygenic inheritance and a fluctuating course. Methods of diagnosis are well-known to those in the art. It is a chronic skin disorder characterized by hyperproliferation of the epidermis, inflammation and angiogenesis.
  • arterial thickening refers to thickening of the arterial wall as part of the atherosclerotic process or the result of treatments for coronary occlusions, for example.
  • Atherosclerosis refers to an extremely common form of arteriosclerosis in which deposits of yellowish plaques are formed in arteries.
  • restenosis refers to recurrent stenosis, especially of a valve of the heart, after surgical correction of the primary condition.
  • Stenosis refers to narrowing or stricture of a duct or canal.
  • tissue ischemia refers to a deficiency of blood in tissue, usually due to a functional constriction or actual obstruction of a blood vessel.
  • excessive scarring during wound healing refers to the result of uncontrolled angiogenesis leading to neovascularization during wound healing.
  • An example is keloid formation. A keloid is a sharply elevated, irregularly-shaped, progressively enlarging scar.
  • the invention features a tricyclic-based indolinone compound having a structure set forth in formula I or II:
  • ring A, ring B, and ring R are independently selected from the group consisting of an aromatic ring, a heteroaromatic ring, an aliphatic ring, a heteroaliphatic ring, and a fused aromatic or aliphatic ring system, where the heteroaromatic ring and heteroaliphatic ring each independently contain 0, 1, 2, or 3 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; and
  • ring A, ring B, ring Q, and ring R are each independently and optionally substituted with one, two, or three substituents independently selected from the group consisting of (i) alkyl; (ii) an aromatic or heteroaromatic ring; (iii) an aliphatic or heteroaliphatic ring; (iv) an amine; (v) a nitro of formula -NO 2 ; (vi) a halogen or trihalomethyl; (vii) a ketone; (viii) a carboxylic acid or ester; (ix) an alcohol or an alkoxyalkyl moiety; (x) an amide; (xi) a sulfonamide; (xii) an aldehyde; (xiii) a sulfone; (xiv) a thiol or a thioether; and (xv) a heavy metal; and
  • X is selected from the group consisting of CH and oxygen.
  • ring A and ring B of the tricyclic-based indolinone compound of formula I are each independently selected from the group consisting of a 5- membered ring, a 6-membered ring, a 7-membered ring, and an 8-membered ring. More preferably, ring A and ring B are each a 6-membered ring.
  • ring R in formula I is selected from the group consisting of a 5-membered ring, a 6-membered ring, a 7-membered ring, an 8-membered ring, and a bicyclic or tricyclic fused ring system. More preferably, R is a 5-membered ring, even more preferably, R is a 6-membered ring. In a preferred embodiment, Ring R is a bicyclic fused ring system comprising preferably 8, more preferably 9, and even more preferably 10, and most preferably 13 atoms in the ring backbone. In other preferred embodiments, R is an optionally substituted ferrocene. Ring R is most preferrably derived from an aldehyde, ketone, or lactone selected from the group consisting of the compounds set forth in Table 8.
  • the invention features a library, e.g., a combinatorial library of at least 10 tricyclic-based indolinone compounds that can be formed by reacting an oxindole with an aldehyde, ketone, or lactone, where the oxindole has a structure set forth in formula III
  • rings A, B, Q, and R are as defined herein or any of the subgroups thereof set forth herein.
  • the oxindole of the combinatorial library preferably has a structure set forth in formula VII, VIII, or IX:
  • each ring in said formulae is optionally substituted with one or more substituents independently selected from the group consisting of (i) saturated or unsaturated alkyl; (ii) an aromatic or heteroaromatic ring; (iii) an aliphatic or heteroaliphatic ring; (iv) an amine of formula -(X ⁇ ) n] -NX 2 X 3 , where Xj, X 2 , and X 3 are independently selected from the group consisting of hydrogen, saturated or unsaturated alkyl, and five-membered or six- membered aromatic, heteroaromatic, or aliphatic ring moieties and where nl is 0, 1, or 2; (v) a nitro of formula -NO 2 ; (vi) a halogen or trihalomethyl; and (vii) an alcohol of formula or an alkoxyalkyl moiety of formula -(X ⁇ o) n io
  • the aldehyde, ketone, or lactone of the combinatorial library is selected from the group consisting of the compounds set forth in Table 8.
  • the invention features a method for synthesizing a tricyclic- based indolinone compound comprising the steps of:
  • the first reactant preferably is an oxindole having the structure set forth in formula III:
  • oxindole has the structure set forth in formula VII, VIII, or IX:
  • the second reactant is preferably an aldehyde, ketone, or lactone, where the aldehyde has a structure set forth in formula X, the ketone has a structure set forth in formula XI, and the lactone has a structure set forth in formula XII
  • the second reactant is an aldehyde, ketone, or lactone selected from the group consisting of the compounds set forth in Table 8.
  • the base is preferably a nitrogen base, and most preferably, the base is piperidine.
  • nitrogen bases are commonly used in the art and are selected from acyclic and cyclic amines. Examples of nitrogen bases include, but are not limited to, ammonia, methyl amine, trimethylamine, aniline, and piperidine. Those skilled in the art know which nitrogen base would match the requirements of the reaction conditions.
  • the solvent of the reaction is preferably an alcohol or an amide, and most preferably, the solvent is ethanol or dimethylformamide (DMF).
  • the synthetic method of the invention calls for the reaction to take place at elevated temperatures.
  • elevated temperatures refers to temperatures that are greater than room temperature. More preferably, the elevated temperature is about 50 °C, and most preferably the elevated temperature is about 90 °C.
  • This method may be accompanied by the step of screening a library for a compound of the desired activity and structure - thus, providing a method of synthesis of a compound by first screening for a compound having the desired properties and then chemically synthesizing that compound.
  • the invention provides for a pyrazolylamide-based compound having a structure set forth in formula X: where
  • Ri and R 2 are independently selected from the group consisting of (i) hydrogen; (ii) alkyl; (iii) an aromatic or heteroaromatic ring; (iv) an aliphatic or heteroaliphatic ring; (v) an amine; (vi) a nitro of formula -NO 2 ; (vii) a halogen; (viii) a ketone; (ix) a carboxylic acid or ester; (x) an alcohol or an alkoxyalkyl moiety; (xi) an amide; (xii) a sulfonamide; (xiii) an alkoxyalkoxy; and (xiv) a sulfone; and
  • R 4 and R 5 are each independently selected from the group consisting of (i) hydrogen; (ii) alkyl; (iii) an aromatic ring; (iv) a heteroaromatic ring; (v) an aliphatic or heteroaliphatic ring; (vi) an amine; (vii) a ketone; (viii) a carboxylic acid or ester; (ix) an alcohol or an alkoxyalkyl moiety; (x) an amide; (xi) a sulfonamide; (xii) a sulfone; and (xiii) an alkoxyalkoxy; and
  • R 3 is selected from the group consisting of (i) hydrogen; (ii)alkyl; (iii)an aromatic or heteroaromatic ring; (iv) an aliphatic or heteroaliphatic ring; (v) a halogen and trihalomethyl; (vi) an amine; (vii) an amide; (viii) an alcohol or an alkoxyalkyl moiety; (ix) a carboxylic acid or ester; (x)a cyano of formula -CN; and (xi) a sulfonamide;
  • K and L are each independently selected from the gruop consisting of (i) hydrogen; (ii) alkyl; and (iii) K and L taken together form a three-membered, four- membered, five-membered, or six-membered aliphatic ring.
  • the invention provides for a pyrazolylamide-based compound having a structure set forth in formula X:
  • Ri, R , and R 5 are independently selected from the group consisting of (i) hydrogen; (ii) alkyl; (iii) an aromatic or heteroaromatic ring; (iv) an aliphatic or heteroaliphatic ring; (v) an amine; (vi) a nitro of formula -NO 2 ; (vii) a halogen; (viii) a ketone; (ix) a carboxylic acid or ester; (x) an alcohol or an alkoxyalkyl moiety; (xi) an amide of formula; (xii) a sulfonamide; (xiii) an alkoxyalkoxy; and (xiv) a sulfone; and where at least one of R4 and R 5 is an aromatic or heteroaromatic ring, as described herein; and
  • R 2 is selected from the group consisting of (i) alkyl; (ii) an aromatic or heteroaromatic ring; (iii) an aliphatic or heteroaliphatic ring; (iv) an amine; (v) a ketone; (vi) a carboxylic acid or ester; (vii) an alcohol or an alkoxyalkyl moiety; (viii) an amide; (ix) a sulfonamide; and (x) a sulfone; and
  • R 3 is selected from the group consisting of (i) hydrogen; (ii) alkyl; (iii) an aromatic or heteroaromatic ring; (iv) an aliphatic or heteroaliphatic ring; and (v) a halogen and trihalomethyl; (vi) an amine; (vii) an amide; (viii) an alcohol or an alkoxyalkyl moiety; (ix) a carboxylic acid or ester; (x) a cyano of formula -CN; and (xi) a sulfonamide;
  • K and L are each independently selected from the gruop consisting of (i) hydrogen; (ii) alkyl; and (iii) K and L taken together form a three-membered, four- membered, five-membered, or six-membered aliphatic ring.
  • the invention provides for a pyrazolylamide based compound where Ri is selected from the group consisting of (i) hydrogen; (ii) alkyl; (iii) an aromatic or heteroaromatic ring; and (iv) an aliphatic or heteroaliphatic ring.
  • Ri is selected from the group consisting of hydrogen and optionally substituted saturated or unsaturated alkyl. Most preferably, Ri is selected from the group consisting of hydrogen, methyl, «-propyl, t-propyl, w-butyl, isobutyl, sec-butyl, and t-butyl. Thus, in certain preferred embodiments, Ri is methyl, whereas in certain other preferred embodiments Ri is w-propyl, and still in other preferred embodiments Ri is t-butyl.
  • the R 2 group of the pyrazolylamide-based compounds of the invention is preferably selected from the group consisting of hydrogen, alkyl, and halogen. More preferably, the R 2 group is selected from the group consisting of hydrogen and halogen; it is most preferably hydrogen, and even most preferably bromine.
  • R and R 5 are each independently selected preferably from the group consisting of hydrogen, alkyl, an aromatic or heteroaromatic ring, and an aliphatic or heteroaliphatic ring.
  • R 4 and R 5 are each independently selected from the group consisting of hydrogen and an aromatic or heteroaromatic ring optionally substituted with one, two, or three substituents independently selected from the group consisting of alkyl and alkoxy moieties. Even most preferably, R and R 5 are each independently selected from the group consisting of hydrogen, methyl, phenyl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, 3-methyl-pyridin-2-yl, 4-methyl-pyridin-2-yl, 5-trifluoromethyl-pyridin-2-yl, 5-trifluoromethyl-pyridin-3-yl, 4,6- dimethyl-pyridin-3-yl, 2,6-dimethoxy-pyridin-3-yl, 2,3,5,6-tetrafluoiO-pyridin-4-yl, quinolin-3-yl, 3-methyl-quinolin-4-yl, isoquinolin-1-yl, isoquinol
  • R and R 5 are each independently selected from the group consisting of 2-trifluoromethyl-phenyl, 3-trifluoromethyl-phenyl, and 4- trifluoromethyl-phenyl.
  • the R of the pyrazolylamide-based compound is selected from the group consisting of hydrogen, alkyl, halogen, and trihalomethyl.
  • R 3 is preferably selected from the group consisting of hydrogen, methyl, and halogen. More preferably, R is hydrogen, even more preferably R 3 is methyl, and most preferably R 3 is chlorine.
  • p and q are each independently 0, 1, 2, or 3. More preferably, p is 1 , and most preferably q is 1.
  • the pyrazolylamide-based compounds of the invention are selected from the group consisting of the pyrazolylamide-based compounds listed in Table 10, below, and numbered AP-001 through AP-033. TABLE 10
  • the invention features a method for synthesizing a pyrazolylamide- based compound comprising the steps of: (a) reacting a first reactant with a second reactant and a base in a solvent at a set temperature; and
  • the first reactant is preferably a substituted pyrazole, and more preferably is selected from the group consisting of l-benzyl-3-tert-butylpyrazole-5-carbonyl chloride, l-benzyl-3- tert-butylpyrazole-5-carboxylic acid, l-(4-methylphenyl)-3-methylpyrazole-5 -carboxylic acid, l-(4-methylbenzyl)-3-methylpyrazole-5 -carboxylic acid, and l-(4-chlorobenzyl)-3- methylpyrazole-5-carboxylic acid.
  • the second reactant is preferably an amine, and more preferably is selected from the group consisting of 2-trifluoromethyl-aniline, 3-trifluoromethyl-aniline, 4- trifluoromethyl-aniline, 2-aminopyridine, 3-aminopyridine, 4-aminopyridine, 2-amino-3- methyl-pyridine, 2-amino-4-methyl-pyridine, 2-amino-5-trifluoromethyl-pyridine, 3- amino-5-trifluoromethyl-pyridine, 3-amino-4,6-dimethyl-pyridine, 3-amino-2,6- dimethoxy-pyridine, 4-amino-2,3,5,6-tetrafluoro-pyridine, 3-amino-quinolin, 4-amino-3- methyl-quinolin, 1-amino-isoquinolin, 3-amino-isoquinolin, 5-amino-benzo[l,3]dioxol, 3- amino-4-methoxy-biphenyl, 3-
  • the base is preferably an amine, and more preferably it is diisopropylamine.
  • the solvent is preferably a polar solvent, and more preferably it is selected from the group consisting of methylene chloride and dimethylformamide.
  • the reaction takes place at a set temperature.
  • the set temperature preferably is room temperature, more preferably the temperature is higher than room temperature.
  • the temperature is higher than room temperature, it is preferably about 50 °C, more preferably about 55 °C, and most preferably about 90 °C.
  • the invention provides an indolinone compound having a structure set forth in formula XI:
  • Ri, R , and R 3 are independently selected from the group consisting of (i) hydrogen; (ii) alkyl; (iii) an aromatic or heteroaromatic ring; (iv) an aliphatic or heteroaliphatic; (v) an amine; (vi) a nitro of formula -NO 2 ; (vii) a halogen or trihalomethyl; (viii) a ketone; (ix) a carboxylic acid or an ester; (x) an alcohol or an alkoxyalkyl moiety; (xi) an amide; (xii) a sulfonamide; (xiii) an aldehyde; and (xiv) a sulfone; and (xv) a thiol or a thioether; (b) A, B, D, and E are selected from the group consisting of carbon and nitrogen; and
  • R4, R 5 , R , and R 7 are independently selected from the group consisting of (i) hydrogen; (ii) alkyl; (iii) an aromatic or heteroaromatic ring; (iv) an aliphatic or heteroaliphatic ring; (v) an amine; (vi) a nitro of formula -NO 2 ; (vii) a halogen or trihalomethyl; (viii) a ketone; (ix) a carboxylic acid or an ester; (x) an alcohol or an alkoxyalkyl moiety; (xi) an amide; (xii) a sulfonamide; (xiii) an aldehyde; (xiv) a sulfone; and (xv) a thiol or a thioether; and
  • X is selected from the group consisting of NX 26 , sulfur, SO, SO 2 , and oxygen, where X 26 is selected from the group consisting of (i) hydrogen; (ii) alkyl; (iii) an aryl optionally substituted with one, two, or three substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester moieties; (iv) a sulfone of formula -SO 2 -X 7 , where X 27 is selected from the group consisting of saturated or unsaturated alkyl and five-membered or six-membered aryl or heteroaryl moieties; and (v) an acyl of formula -C(O)X 28 , where X 28 is selected from the group consisting of hydrogen, saturated and unsaturated alkyl, aryl, and a five- membered or six-membered ring moiety;
  • ring Y is selected from the group consisting of five- membered, six-membered, and seven-membered aromatic, heteroaromatic, or non- aromatic rings, where the heteroaromatic ring contains a heteroatom selected from the group consisting of nitrogen, oxygen, and sulfur, and where the non- aromatic ring in combination with R optionally forms a carbonyl functionality;
  • G, J, and L are selected from the group consisting of carbon and nitrogen;
  • the invention relates to an indolinone compound of formula XI, where Ri and R 2 are selected from the group consisting of (i) hydrogen; (ii) saturated alkyl optionally substituted with halogen, trihalomethyl, carboxylate, nitro, ester, and an aliphatic or heteroaliphatic ring optionally substituted with halogen, trihalomethyl, carboxylate, nitro, and ester moieties; (iii) an aromatic ring optionally substituted with one, two, or three substituents independently selected from the group consisting of alkyl, alkoxy, halogen, and nitro moieties; (iv) an amine of formula -(X !
  • Xi is optionally substituted saturated alkyl and where nl is 0 or 1, and where X 2 , and X 3 are independently selected from the group consisting of hydrogen and optionally substituted saturated alkyl; (v) a nitro of formula -NO 2 ; (vi) a halogen or trihalomethyl; (vii) a ketone of formula -( ) n4 -CO-X 5 , where X 4 and X 5 are alkyl and where n4 is 0 or 1 ; (viii) a carboxylic acid of formula -(X 6 ) n6 -COOH or ester of formula -(X 7 ) n7 -COO-X 8 , where X 6 , X 7 , and X 8 are alkyl and where n6 and n7 are independently 0 or 1 ; (ix) an alcohol of formula -(X 9 ) n9 -OH or
  • R ⁇ and R 2 of the indolinone of the invention are selected from the group consisting of (i) hydrogen; (ii) methyl, ethyl, propyl, and butyl groups optionally substituted with halogen, trihalomethyl, cyano, and nitro moieties; (iii) phenyl optionally substituted with one, two, or three substituents independently selected from the group consisting of alkyl, alkoxy, halogen, and nitro moieties; (iv) an amine of formula
  • X 2 and X 3 are independently selected from the group consisting of hydrogen and optionally substituted saturated alkyl, and Xi is optionally substituted saturated alkyl, and where n is 0 or 1;
  • X a nitro of formula -NO 2 a halogen or trihalomethyl;
  • a ketone of formula -CO-Xi where X4 is selected from the group consisting of methyl, ethyl, propyl, and butyl;
  • X ⁇ 8 and X19 are independently selected from the group consisting of hydrogen, methyl, ethyl, phenyl optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, and trihalomethyl, and where X ⁇ 8 and X 19 taken together form a six-membered heteroaliphatic ring moiety.
  • E in formula XIII is nitrogen.
  • the indolinone compounds of the invention are those whose structure is set forth in formula XI, where Q is selected from the group consisting of type Q oxindoles.
  • type Q oxindoles is meant oxindole compounds which are selected from the list of oxindoles numbered O-l through O-60, and are depicted as follows. v o Br YVVo cl YYVo (O-l) ⁇ " (0-2) ⁇ -» (0-3) ⁇ B
  • the indolinone of the invention has a structure as set forth in formula XI, where R ⁇ and R 5 are independently selected from the group consisting of
  • (x) an alcohol of formula or an alkoxyalkyl moiety of formula -(X ⁇ o) n io-O-Xn, where X 9 , and X 10 are independently selected form the group consisting of methylene, ethylene, and propylene, and where X ⁇ is independently selected from the group consisting of methyl, ethyl, and propyl, and where n9 and nlO are independently 0 or 1 ;
  • a sulfone of formula -(X 2 ⁇ ) n2 ⁇ -SO -X 22 where X 2 is selected from the group consisting of hydroxide, saturated or unsaturated alkyl, and five-membered or six-membered aryl or heteroaryl moieties, and where X 2] is saturated alkyl, and where n21 is 0 or 1; and (x) a thioether of formula -(X 24 ) n24 -S-X 25 , where X 24 is independently selected from the group consisting of methylene, ethylene, and propylene, and where X 25 is independently selected from the group consisting of methyl, ethyl, propyl, and phenyl, and where n24 is 0 or 1.
  • R 4 and R are each independently selected from the group consisting of (i) hydrogen; (ii) methyl and ethyl; (iii) an amine of formula -(X ⁇ ) nl -NX 2 X 3 , where X 2 and X 3 are independently selected from the group consisting of hydrogen methyl and ethyl, and Xi is methylene or ethylene, and where nl is 0 or 1, or where X 2 and X 3 taken together form a substituted ring selected from the group consisting of
  • R and R 7 groups of the indolinone compounds of the invention are independently selected from the group consisting of (i) hydrogen; (ii) methyl, ethyl, propyl, and butyl groups optionally substituted with halogen, trihalomethyl, cyano, and nitro moieties; (iii) an amine of formula -(X ⁇ ) n ⁇ -NX 2 X 3 , where X 2 and X 3 are independently selected from the group consisting of hydrogen and substituted saturated alkyl, and X ⁇ is an optionally substituted saturated alkylene, and where nl is 0 or 1; (iv) a halogen or trihalomethyl; (v) an alcohol of formula -(Xg) n9 -OH or an alkoxyalkyl moiety of formula -(X ⁇ o) n i(rO-Xn, where X9, and X10 are independently selected form the group consisting of methylene, ethylene, and propylene
  • Re and R 7 are independently selected from the group consisting of (i) hydrogen; (ii) methyl and ethyl; (iii) an amine of formula -(X ⁇ ) nl -NX X 3 , where X 2 and X 3 are independently selected from the group consisting of hydrogen, methyl, and ethyl, and Xj is selected from the group consisting of methylene and ethylene, and where nl is 0 or 1; (iv) a halogen; (v) a hydroxy -OH or an alkoxy moiety of formula -O-Xn, where Xn is independently selected form the group consisting of methyl, ethyl, and propyl.
  • the Y ring of the indolinone compound of the invention is a six- membered aromatic or heteroaromatic ring. If Y is an aromatic ring, then the moiety set forth in formula XII would take the form set forth in formula XII-A
  • Y is a heteroaromatic ring
  • at least one of carbon atoms of the Y ring of the structure set forth in formula V is a heteroatom (e.g. nitrogen).
  • the Y ring of the indolinone compounds is a six-membered aliphatic or heteroaliphatic ring. Then, the Y ring in the structure set forth
  • formula XII takes the form of, for example, optionally substituted or
  • G, J, and L are independently nitrogen.
  • X may also preferably be oxygen, nitrogen, optionally substituted with an alkyl, or be selected from the group consisting of sulfur, SO, and SO 2 .
  • the precursor to the T moiety of the indolinone compound of the invention set forth in formula XI is selected from the group consisting of type T aldehydes.
  • type T aldehyde aldehyde compounds which are selected from the list of aldehydes numbered A-l through A-95, and are depicted as follows.
  • the invention relates to a combinatorial library of at least 10 indolinone compounds that can be formed by reacting oxindoles with aldehydes.
  • the oxindoles are those that have a structure set forth in formula XIII as defined herein or any of the subgroups thereof set forth herein.
  • the oxindoles are preferably selected from the group consisting of type Q oxindoles.
  • the aldehydes of the combinatorial library of the invention preferably have a structure set forth in formula XIV:
  • the aldehydes are selected from the group consisting of type T aldehydes.
  • the present invention also features novel oxindole compounds.
  • the invention features an oxindole compound of formula XV
  • R 8 is selected from the group consisting of (i) saturated alkyl, optionally substituted with a substituent selected from the group consisting of alkoxy, trihalomethyl, nitro, and cyano moieties, provided that the alkyl is not methyl; (ii) an amine; (iii) an iodine; (iv) a ketone of formula -(X 4 ) n -CO-X 5 , where Xj and X 5 are independently alkyl and where n4 is 0 or 1 ; (v) a carboxylic acid or ester; (vi) an amide; and (vii) a sulfonamide.
  • the oxindole compound of formula XV is preferably selected from the group consisting of
  • the invention features an oxindole compound of formula XVI
  • Rg is selected from the group consisting of (i) an amine; (ii) a nitro of formula - NO 2 ; (iii) a chlorine, bromine, or iodine; (iv) a ketone; (v) a carboxylic or ester; (vi) an amide; and (vii) a sulfonamide.
  • the oxindole compound of formula XVI is preferably selected from the group consisting of
  • the invention features an oxindole compound of formula XVII where Rio is selected from the group consisting of (i) an aromatic or heteroaromatic ring; (ii) an aliphatic or heteroaliphatic ring; (iii) an amine; (iv) a nitro of formula -NO 2 ; (v) a bromine; (vi) a ketone; (vii) a carboxylic acid or ester; (viii) a sulfonamide.
  • Rio is selected from the group consisting of (i) an aromatic or heteroaromatic ring; (ii) an aliphatic or heteroaliphatic ring; (iii) an amine; (iv) a nitro of formula -NO 2 ; (v) a bromine; (vi) a ketone; (vii) a carboxylic acid or ester; (viii) a sulfonamide.
  • the oxindole compound of formula XVII is preferably selected from the group consisting of
  • the invention features an oxindole compound, where the oxindole compound is selected from the group consisting of
  • the invention features a method for synthesizing an indolinone compound comprising the steps of:
  • the first reactant is preferably selected from the group consisting of type Q oxindoles and the second reactant is preferably selected from the group consisting of type T aldehydes.
  • the base is preferably a nitrogen base, and most preferably, the base is piperidine.
  • nitrogen bases are commonly used in the art and are selected from acyclic and cyclic amines. Examples of nitrogen bases include, but are not limited to, ammonia, methyl amine, trimethylamine, aniline, and piperidine. Those skilled in the art know which nitrogen base would match the requirements of the reaction conditions.
  • the solvent of the reaction is preferably an alcohol, and most preferably, the solvent is ethanol.
  • the synthetic method of the invention calls for the reaction to take place at elevated temperatures.
  • elevated temperatures refers to temperatures that are greater than room temperature. More preferably, the elevated temperature is about 90 °C.
  • the compounds of the present invention relate to imidazoyl 2- indolinone derivatives having the chemical structure of the formula XIX:
  • A, B, D and E are independently selected from the group consisting of carbon and nitrogen wherein it is understood that, when A, B, D or E is nitrogen, R ⁇ , R 7 , R 8 , or R 9 , respectively, does not exist and there is no bond.
  • G and J are selected from the group consisting of nitrogen and carbon such that, when G is nitrogen, J is carbon and when J is nitrogen, G is carbon. When either G or J is nitrogen then R 5 or R 5 -, respectively, does not exist.
  • R and the imidazolyl ring may exchange places on the double bond; i.e., compound of formula XIX may exist in the E or Z configuration about the double bond at the 3-position of the 2-indolinone.
  • Ri and R 3 are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, hydroxy, alkoxy, C-carboxy, O-carboxy, C-amido, C-thioamido, sulfonyl and trihalomethylsulfonyl.
  • R 2 is selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, heteroaryl and halo.
  • I t , R 5 , and R 5 > are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, halo, trihalomethyl, hydroxy, alkoxy, aryloxy, C-carboxy, O-carboxy, carbonyl, nitro, cyano, S-sulfonamido, amino and -NRioRn-
  • Rio and Rn are independently selected from the group consisting of alkyl, cycloalkyl, aryl, carbonyl, sulfonyl, tri-halomethanesulfonyl and, combined, a five- member or a six-member heteroalicyclic ring.
  • R 6 , R 7 , R 8 , and Rg are independently selected from the group consisting of hydrogen, alkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, S- sulfonamido, N-sulfonamido, N-trihalomethanesulfonamido, carbonyl, C-carboxy, O- carboxy, cyano, nitro, halo, cyanato, isocyanato, thiocyanato, isothiocyanato, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, amino and -NRio
  • R 6 and R 7 or R and R 8 or R 8 and Rg may form a five or six-membered aromatic, heteroaromatic, alicyclic or heteroalicyclic ring such as, by way of example and not limitation, a methylenedioxy or an ethylenedioxy ring.
  • Ri is hydrogen.
  • A, B, D, and E are carbon in preferred compounds of this invention.
  • R 2 is also hydrogen in preferred compounds of this invention.
  • R 3 is hydrogen in preferred compounds of this invention.
  • Ri, R 2 , and R 3 are hydrogen and A, B, D and E are carbon.
  • R 6 , R 7 , R 8 and R 9 are selected from the group consisting of hydrogen, unsubstituted lower alkyl, lower alkyl substituted with a group selected from the group consisting of halo, C-carboxy and -NRioRn; unsubstituted lower alkoxy, lower alkoxy substituted with a group selected from the group consisting of halo, C-carboxy and -NRioRn; trihalomethyl, unsubstituted alkenyl, unsubstituted alkynyl, unsubstituted aryl, aryl substituted with one or more groups independently selected from the group consisting of unsubstituted lower alkyl, lower alkyl substituted with one or more halo groups, halo, C-carboxy, unsubstituted alkoxy, amino, S-sulfonamido or -NRioRn; unsubstituted heteroalicycl
  • R , R 5 and Ry are preferrably independently selected from the group consisting of hydrogen, unsubstituted lower alkyl, trihalomethyl, lower alkyl substituted on the carbon furthest from the point of attachment to the ring with a C- carboxy group, halo, hydroxy, unsubstituted alkoxy, O-carboxy, C-carboxy, amino, C- amido, N-amido, S-sulfonamido, nitro, amino and -NRioRn-
  • the chemical formulae referred to herein may exhibit the phenomena of tautomerism and structural isomerism.
  • the compounds described herein may adopt and E or a Z configuration about the double bond connecting the 2-indolinone moiety to the imidazoyl moiety or they may be a mixture of E and Z.
  • the formulae shown herein are drawn with wavy lines on the imidazoyl side of the double bond to signify that the positions of R 2 and the imidazoyl group are interchangeable.
  • 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.
  • the invention provides for a method for synthesizing an imidazoyl-2-indolinone comprising the steps of:
  • the solid substrate comprises a resin. More preferably, the resin comprises a 2-chlorotrityl resin. In other preferred embodiments, the method of synthesis comprises washing the solid substrate-bound imidazoyl-2-indolinone with acetone, water, dichloromethane and methanol.
  • the compounds of the present invention relate to phenyl 2- indolinone derivatives having the chemical structure of formula XX:
  • A, B and D are independently selected from the group consisting of carbon and nitrogen wherein it is understood that, when A, B or D is nitrogen, R 3 , R 4 or R 5 , respectively, does not exist.
  • Ri is selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, hydroxy, alkoxy, C-carboxy, O-carboxy, C-amido, C-thioamido, sulfonyl and trihalomethylsulfonyl.
  • R 2 is selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, and heteroaryl.
  • R 3 , R 4 , R , R ⁇ , R 7 , R 8 , R9 and Rio are independently selected from the group consisting of hydrogen, alkyl, trihalomethyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, S-sulfonamido, N-sulfonamido, N-trihalomethanesulfonamido, carbonyl, C- carboxy, O-carboxy, cyano, azido, nitro, halo, cyanato, isocyanato, thiocyanato, isothiocyanato, O-carbamyl, N-carbamyl, 0-thiocarbamyl, N-thiocarbamyl, C-amido
  • R 3 and R or R ⁇ and R or R 7 and R 8 or R 8 and R 9 or R 9 and R ⁇ 0 may combine to form a methylenedioxy or an ethylenedioxy group.
  • Q is selected from the group consisisting of aryl, heteroaryl and fused heteroaryhcycloalkyl heteroalicyclic.
  • Rn and Rj 2 are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, carbonyl, acetyl, sulfonyl, trihalomethanesulfonyl and, combined, a five-member or a six-member heteroalicyclic ring.
  • a preferred embodiment of this invention is a compound in which Ri and R 2 are hydrogen.
  • a further preferred embodiment of this invention is a compound in which A, B and
  • Ri and R are hydrogen and A, B and D are carbon. It is also a preferred embodiment of this invention that R 3 , R4 and R 5 are hydrogen. It is likewise a preferred embodiment of this invention that R ⁇ , R 7 , R 8 , Rg and Rio are independently selected from the group consisting of hydrogen and unsubstituted lower alkoxy.
  • R ⁇ R 7 , R 8 , R 9 or Rio is unsubstituted lower alkoxy.
  • Another preferred embodiment of this invention is that, when Q is aryl, the aryl group is substituted with one or more groups independently selected from the groups consisting of hydrogen, unsubstituted lower alkyl, unsubstituted lower alkoxy and heteroalicylic, in particular, 4-formylpiperazin-l-yl. It is a preferred embodiment of this invention that when Q is heteroaryl, the heteroaryl group is selected from the group consisting of pyrrol-2-yl, imidazo-4-yl and thiophen-2-yl.
  • the heteroaryl moiety is preferably selected from pyrrolo, thiopheno, furano, thiazolo, oxazolo, pyridino and imidazolo.
  • Q is 4,5,6,7- tetrahydroindol-2-yl.
  • Q is preferably substituted with one or more groups independently selected from the group consisting of hydrogen, unsubstituted lower alkyl, unsubstituted lower alkoxy, carboxy, carboxy salt, carboxyalkyl and carboxyalkyl salt where, in the carboxyalkyl or carboxyalkyl salt, r is 1 or 2.
  • Another aspect of this invention is a combinatorial library of at least ten (10) compounds formed by reacting an oxindole having the general chemical structure of formula XXI:
  • Ri, R 3 , R 4 , R 5 , R ⁇ , R 7 , R 8 , R 9 and Rio are as previously defined above.
  • G, J, L, M and P are independently selected from the group consisting of carbon and nitrogen, n is 0 or 1 and it is understood that the 5- or 6-member ring formed when any of G, J, L, M and/or P is nitrogen is one known in the chemical arts.
  • R ⁇ , R ⁇ 5 , R ⁇ 6 , R ⁇ 7 and R] 8 are independently selected from the group consisting of hydrogen, alkyl, trihalomethyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, S-sulfonamido, N-sulfonamido, N-trihalomethanesulfonamido, carbonyl, C- carboxy, O-carboxy, cyano, azido, nitro, halo, cyanato, isocyanato, thiocyanato, isothiocyanato, O-carbamyl, N-carbamyl, O-thiocarbamyl, C-amido, N- amido, amino and
  • Ri 4 and R 15 or R 15 and R )6 or R 16 and R] or R ]7 and R ⁇ 8 may combine to form a 5- or 6-menber cycloalkyl or heteroalicyclic ring.
  • Method of Modulating Cell Proliferation Another aspect of the invention features a method of modulating cell proliferation, comprising administering to a patient in need of such treatment a pharmaceutically acceptable composition comprising a therapeutically effective amount of one or more indolinone compounds of formula XVIII:
  • Ri is H or alkyl; R 2 is O or S; R 3 is H;
  • R , R 5 , R ⁇ , and R are each independently selected from the group consisting of hydrogen alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(0)R, S0 2 NRR ⁇ S0 3 R, SR, N0 2 , NRR', OH, CN, C(0)R, OC(0)R, NHC(0)R, (CH 2 ) n C0 2 R, CONRR' , and (CH 2 ) n ONRR' ;
  • A is selected from the group consisting of a 4,5,6,7-tetrahydroindole and a five- membered heteroaryl ring, where the five-membered ring is selected from the group consisting of thiophene, pyrrole, pyrazole, imidazole, 1,2,3-triazole, 1,2,4-triazole, oxazole, isoxazole, thiazole, isothiazole, 2-sulfonylfuran, 4-alkylfuran, 1,2,3-oxadiazole, 1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,3,4-oxadiazole, 1,2,3,4,-oxatriazole, 1,2,3,5- oxatriazole, 1,2,3-thiadiazole, 1,2,4-thiadiazole, 1,2,5-thiadiazole, 1,3,4-thiadiazole, 1,2,3,4-thiatriazole, 1,2,3,5-thiatri
  • abnormal cell proliferation or "cell proliferative disorder” as used herein refers to a disorder where an excess cell proliferation of one or more subset of cells in a multicellular organism occurs resulting in harm (e.g., discomfort or decreased life expectancy) to the multicellular organism.
  • the excess cell proliferation can be determined by reference to the general population and or by reference to a particular patient (e.g. , at an earlier point in the patient's life).
  • Hyper-proliferative cell disorders can occur in different types of animals and in humans, and produce different physical manifestations depending upon the affected cells.
  • Hyper-proliferative cell disorders include cancer and metastases, autoimmune disorders, rheumatoid arthritis, endometriosis, ocular disease, arterial thickening and restenosis, inflammatory disorders such as psoriasis, and fibrotic disorders, such as aberrant wound healing.
  • a therapeutic effect refers to one or more of the following: (a) a reduction in hyperproliferation; (b) inhibition of hyperproliferation; (c) inhibition (i.e., slowing or stopping) of tumor metastasis; and (d) relieving to some extent one or more of the symptoms associated with the abnormal condition.
  • signal transduction refers to a fundamental mechanism whereby external stimuli that regulate diverse cellular processes are relayed to the interior of cells.
  • One of the key biochemical mechanisms of signal transduction involves the reversible phosphorylation of proteins by kinases, which enables regulation of the activity of mature proteins by altering their structure and function.
  • the protein kinases involved in signal transduction include tyrosine kinases which phosphorylate proteins on the alcohol moiety of tyrosine residues, and serine/threonine kinases which phosphorylate on serine/threonine residues.
  • signal transduction pathway refers to the molecules that propagate an extracellular signal through the cell membrane to become intracellular signals. These signals can then stimulate a cellular response.
  • the polypeptide molecules involved in signal transduction processes are typically receptor and non-receptor protein kinases, receptor and non-receptor protein phosphatases, adaptor molecules, nucleotide exchange factors, and transcription factors.
  • solvent refers to a chemical compound that facilitates the solubilization of compounds.
  • solvents include, but are not limited to, pharmaceutically acceptable alcohols, such as ethanol; polyoxyhydrocarbyl compounds, such as poly(ethylene glycol); pharmaceutically acceptable surfactants such as CREMOPHOR EL®; polyglycolized lipids, such as GELUCIRE® and LABRASOL®; and pharmaceutically acceptable oils, such as miglyol 812.
  • pharmaceutically acceptable alcohols such as ethanol
  • polyoxyhydrocarbyl compounds such as poly(ethylene glycol)
  • pharmaceutically acceptable surfactants such as CREMOPHOR EL®
  • polyglycolized lipids such as GELUCIRE® and LABRASOL®
  • oils such as miglyol 812.
  • pharmaceutically acceptable alcohol refers to alcohols which are liquids at about room temperature (approximately 20 °C). These include propylene glycol, ethanol, 2-(2-ethoxyethoxy)ethanol (TRANSCUTOL®, Gattefosse, Westwood, NJ 07675), and glycerol.
  • polyoxyhydrocarbyl compound refers to a water soluble carbohydrate such as glucose, sucrose, maltotriose, and the like; water soluble carbohydrate derivatives such as gluconic acid and mannitol, and oligosaccharides; and water soluble polymers such as polyvinylpyrrolidone, poly( vinyl alcohol), and in particular, polyethers such as other polyoxyalkylenes including poly( ethylene glycol) or other water soluble mixed oxyalkylene polymers and the polymeric form of ethylene glycol.
  • polyoxyhydrocarbyl compounds preferably contain more than one carbon, oxygen, and hydrogen atom, some molecules such as poly(ethylene imine) are also included.
  • a particularly preferred class of solubilizing polyoxyhydrocarbyl moieties comprises poly(ethylene glycol) (PEG) and PEG derivatives, such as PEG monomethyl ether.
  • PEG derivatives include PEG-silicon derived ethers.
  • PEG-silicon derived ethers are commercially available in a variety of molecular weights. Others may be conveniently prepared from commercially available materials, such as by coupling of amino-PEG moiety to a haloalkyl silyl or silane moiety.
  • Suitable PEGs may vary in molecular weight from about 200 g/mol to about 20,000 g/mol or more, more preferably 200 g/mol to 5,000 g/mol, even more preferably 250 g/mol to 1,000 g/mol, and most preferably 250 g/mol to 500 g/mol.
  • the choice of a particular molecular weight may depend on the particular indolinone compound chosen and its molecular weight and degree of hydrophobicity, as well as the particular application for which the formulation is to be used.
  • pharmaceutically acceptable surfactant refers to a compound that can solubilize or aid in solubilization of indolinone compounds into aqueous solutions.
  • the surfactant is a non-ionic surfactant.
  • examples of pharmaceutically acceptable surfactants include POLYSORBATE 80® and other polyoxyethylene sorbitan fatty acid esters, glyceryl monooleate, polyvinyl alcohol, ethylene oxide copolymers such as PLURONIC® (a polyether) and TETRONIC® (BASF), polyol moieties, and sorbitan esters.
  • ethoxylated castor oils such as CREMOPHOR® EL, are used for the formulation of indolinone compounds.
  • ethoxylated castor oil refers to castor oil that is modified with at least one oxygen containing moiety.
  • the term refers to castor oil comprising at least one ethoxyl moiety.
  • pharmaceutically acceptable surfactant includes pharmaceutically acceptable non-ionic surfactants (for example polyoxyethylenepolypropylene glycol, such as POLOXAMER® 68 (BASF Corp.) or a mono fatty acid ester of polyoxyethylene (20) sorbitan monooleate (TWEEN® 80), polyoxyethylene (20) sorbitan monostearate (TWEEN® 60), polyoxyethylene (20) sorbitan monopalmitate (TWEEN® 40), polyoxyethylene (20) sorbitan monolaurate (TWEEN® 20) and the like); polyoxyethylene castor oil derivatives (for example, polyoxyethyleneglycerol-triricinoleate or polyoxyl 35 castor oil (CREMOPHOR® EL, BASF Corp.), polyoxyethyleneglycerol oxystearate (CREMOPHOR® RH 40 (polyethyleneglycol 40 hydrogenated castor oil) or CREMOPHOR® RH 60
  • pharmaceutically acceptable non-ionic surfactants for example polyoxyethylenepol
  • polyglycolized lipids refers to mixtures of monoglycerides, diglycerides, or triglycerides and polyethyleneglycol monoesters and diesters formed by the partial alcoholysis of vegetable oil using PEG of 200 g/mol to 2,000 g/mol or by the esterification of fatty acids using PEG 200 g/mol to 2,000 g/mol and glycerols.
  • these include GELUCIRE® 35/10, GELUCIRE® 44/14,
  • GELUCIRE® 46/07 GELUCIRE® 46/07
  • GELUCIRE® 50/13 GELUCIRE® 53/10
  • LABRASOL® LABRASOL®
  • oils such as mineral oil or vegetable oil (including safflower oil, peanut oil, and olive oil), fractionated coconut oil, propylene glycol monolaurate, mixed triglycerides with caprylic acid and capric acid, and the like.
  • Preferred embodiments of the invention feature mineral oil, vegetable oil, fractionated coconut oil, mixed triglycerides with caprylic acid, and capric acid.
  • a highly preferred embodiment of the invention features Miglyol 812 (available from Huls America, USA).
  • the composition consists of compounds of formula XVIII, where A is selected from the group consisting of pyrrole, thiophene, and 4,5,6,7-tetrahydroindole, optionally substituted with one or more substituents selected from the group consisting of alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO 2 NRR', SO 3 R, SR, NO 2 , NRR', OH CN, C(O)R, OC(O)R, NHC(O)R, (CH 2 ) n CO 2 R, CONRR', and (CH 2 ) n ONRR; n is 0-3; R is selected from the group consisting of H, alkyl, and aryl; and
  • R' is selected from the group consisting of H, alkyl, and aryl, wherein the alkyl is optionally substituted with a six-membered heteroaliphatic ring, and where the six- membered ring is optionally substituted at one or more positions with substituents selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, NO 2 , and (CH 2 ) n CO 2 R.
  • A is pyrrole.
  • indolinone compounds of Formula XVIII are preferably selected from the group consisting of Compound AV-002, Compound AV-003, Compound AV-004 , Compound AV-005, Compound AV-006, Compound AV-007, and Compound AV-008.
  • Compound AV-002 is meant either the E or Z isomer of the indolinone compound, 5-amino-3-(3,5-diethyl-lH-pyrrol-2-ylmethylene)-l,3-dihydro-indol-2-one.
  • Compound AV-003 is meant either the E or Z isomer of the indolinone compound, 4-methyl-3-(3-methyl-thiophen-2-ylmethylene)-l,3-dihydro-indol-2-one.
  • Compound AV-004 is meant either the E or Z isomer of the indolinone compound, 5-chloro-3-(3,5-dimethyl-lH-pyrrol-2-ylmethylene)-l,3-dihydro-indol-2-one.
  • Compound AV-005" is meant either the E or Z isomer of the indolinone compound, 3-[4-methyl-5-(2-oxo-l,2-dihydro-indol-3-ylidenemethyl)-H-pyrrol-3-yl]- propionic acid.
  • Compound AV-006 is meant the indolinone compound, 3-[2,4-dimethyl-5- (2-oxo-l,2-dihydro-indol-3-ylidenemethyl)-lH-pyrrol-3-yl]-propionic acid.
  • Compound AV-007 is meant either the E or Z isomer of the indolinone compound, N-(2-Morpholin-4-yl-ethyl)-3-[2-(2-oxo- 1 ,2-dihydro-indol-3-ylidenemethyl)- 4,5,6,7-tetrahydro-lH-indol-3-yl]-propionamide.
  • Compound AV-008 is meant either the E or Z isomer of the indolinone compound, 3-[2-(2-Oxo-l,2-dihydro-indol-3-ylidenemethyl)-4,5,6,7-tetrahydro-lH-indol- 3 -yl] -propionic acid.
  • E or Z isomer refers to the positioning of the two atoms or groups for each doubly-bonded carbon atom compared with the positioning of the two atoms or groups for the other doubly-bonded carbon.
  • the priority of the two atoms or groups is identified for each doubly-bonded carbon atom, and then the positioning relative to each other is determined, i.e. whether the higher priority atoms or groups for each doubly-bonded carbon atom are on the same or opposite sides.
  • Z is meant on the same side, and by E, on the opposite side.
  • “Priority” refers to atomic number, with the atom of higher atomic weight getting the higher priority.
  • the atom of higher mass number has the higher priority. If the relative priority of the two groups attached to the doubly-bonded carbon cannot be decided, a similar comparison is made with the atoms attached to these groups, and so on.
  • the composition further comprises one or more pharmaceutically acceptable excipients in a formulation, where the formulation is selected from the group consisting of, but not limited to, a parenteral, a topical and an oral formulation.
  • the effective amount of the compound in the composition comprises 1 to 1000 mg/m 2 /day, preferably 10 to 500 mg/m /day, and most preferably 10 to 250 mg/m /day of one or more indolinone compounds.
  • the patient is a mammal; more preferably the mammal is a human.
  • the mammal can be a rat, in which case altered activity of VEGF, FGF, and/or PDGF can be induced.
  • mice preferably refers to such organisms as mice, rats, rabbits, guinea pigs, goats, sheep, horses, and cows, for example; more preferably to dogs, cats, monkeys, and apes; and most preferably to humans.
  • Another aspect of the invention features methods of modulating the activity of
  • VEGF, FGF, and/or PDGF on cells in vitro, or more preferably, in vivo, comprising administering to said cells a pharmaceutically acceptable composition comprising a therapeutic amount of one or more indolinone compounds of formula XVIII:
  • the composition further comprises one or more pharmaceutically acceptable excipients in the formulation.
  • the formulation is selected from the group consisting of a parenteral, an oral, and a topical formulation and the effective amount of the compound in the composition comprises 1 to 1000 mg/m /day, preferably 10 to 500 mg/m 2 /day, and most preferably 10 to 250 mg/m 2 /day of one or more indolinone compounds.
  • the patient is a mammal; more preferably the mammal is a human.
  • the mammal can be a rat, in which case altered activity of VEGF, FGF, and or PDGF can be induced.
  • the invention also features in another aspect, methods of modulating tyrosine kinase signal transduction, comprising administering to a patient in need of such treatment a pharmaceutically acceptable composition comprising a therapeutically effective amount of one or more indolinone of formula XVIII:
  • the composition further comprises one or more pharmaceutically acceptable excipients in the formulation.
  • the formulation is selected from the group consisting of a parenteral, an oral, and a topical formulation and the effective amount of the compound in the composition comprises 1 to 1000 mg/m 2 /day, preferably 10 to 500 mg/m 2 /day, and most preferably 10 to 250 mg/m 2 /day of one or more indolinone compounds selected from the group consisting of Compound AV-002, Compound AV- 003, and Compound AV-004.
  • the patient is a mammal; more preferably the mammal is a human.
  • the mammal can be a rat, in which case altered activity of VEGF, FGF, and/or PDGF can be induced.
  • Another aspect of the invention features methods of identifying one or more indolinone compounds that inhibit growth factor-stimulated or platelet-derived growth factor-stimulated cell proliferation comprising the following steps: (a) contacting cells with one or more indolinone compounds of formula XVIII; (b) contacting the cells with one or more growth factors selected from the group consisting of VEGF, PDGF, and FGF; and (c) monitoring an effect upon the cells.
  • the growth factor is VEGF and the cells are endothelial cells, or PDGF and the cells are smooth muscle cells, or FGF.
  • the effect is monitored colorimetrically, for example using a change in absorbance.
  • the indolinone compounds of the invention preferably modulate the activity of the protein kinase in vitro. These compounds preferably show positive results in one or more in vitro assays for an activity co ⁇ esponding to treatment of the disease or disorder in question (such as the assays described in the Examples below).
  • the invention also features methods of identifying one or more indolinone compounds that are active in an adjuvant arthritis model in rats comprising the following steps: (a) administration of one or more indolinone compounds of formula XVIII to the rats; and (b) monitoring an effect upon the rats.
  • the compounds are administered at a concentration of 1 to 1000 mg/m 2 /day, preferably 10 to 500 mg/m 2 /day, and most preferably 10 to 250 mg/m 2 /day of one or more indolinone compounds and the effect on the rats' disease is selected from the group consisting of ear nodulation, tail nodulation, nose swelling, paw swelling, and balanitis.
  • adjuvant arthritis model is used herein to refer to rats, preferably Wistar-Lewis or other rat strains commonly known to those skilled in the art, in which disease was induced by injecting 0.1 mL Freund's adjuvant into the base of the tail.
  • This adjuvant arthritis model is only one example of an animal model that can be used to test the compounds of the invention.
  • Oliver & Brahn (1996) J. Rheumatol. 23:56-60, hereby enclosed herein by reference in its entirety, including any drawings, figures, or tables.
  • An additional aspect of the invention features methods of modulating abnormal cell proliferation, the activity of VEGF, FGF, or PDGF on cells in vivo or in vitro, or tyrosine kinase signal transduction, comprising administering to a patient in need of such treatment a pharmaceutically acceptable composition comprising a therapeutically effective amount of one or more indolinone compounds of formula XVIII identified by their ability to inhibit VEGF-, FGF-, or PDGF-stimulated cell proliferation, or inhibit one or more of the effects of adjuvant arthritis in rats.
  • the compounds are administered in a composition that further comprises one or more pharmaceutically acceptable excipients in a formulation that can be, but is not limited to being, administered orally, parenterally, or topically.
  • the compounds may be administered at a concentration comprising 1 to 1000 mg/m 2 /day, preferably 10 to 500 mg/m 2 /day, and most preferably 10 to 250 mg/m 2 /day of one or more indolinone compounds, preferably to a mammal, and more preferably to a human.
  • Another aspect of the invention features methods of treating or preventing an abnormal condition by administering to a patient in need of such treatment a pharmaceutically acceptable composition comprising a therapeutically effective amount of one or more indolinone compounds of formula XVIII:
  • Additional methods of treating or preventing an abnormal condition include, administering to a patient in need of such treatment a pharmaceutically acceptable composition comprising a therapeutically effective amount of one or more compounds identified by previously described methods for identifying compounds that modulate VEGF-,FGF-, or PDGF-activity, or that are active in an adjuvant arthritis model in rats, where the abnormal condition is selected from the group consisting of rheumatoid arthritis, endometriosis, ocular diseases, cancer and metastases, psoriasis, arterial thickening and restenosis, tissue ischemia, and excessive scarring during wound healing.
  • the disease is preferably endometriosis or rheumatoid arthritis
  • the composition further comprises one or more pharmaceutically acceptable excipients in a formulation that is selected from the group consisting of an intra-muscular, a depot, a parenteral, an oral, and a topical formulation.
  • the compounds inhibit tyrosine kinase activity in vitro
  • the patient is a mammal, or preferably a human.
  • the mammal can be a rat, in which case the stated diseases or disorders can be induced.
  • the invention provides for a method of increasing the sexual function of a mammal by administering to the mammal a formulation containing an indolinone compound.
  • the invention also provides for a method of treating sexual dysfunction in a mammal in need thereof, comprising the steps of (a) administering to the mammal a formulation containing an indolinone compound; (b) monitoring the sexual activity of the mammal; and (c) adjusting the dose of the compound to match the specific needs of the mammal.
  • Another aspect of the invention provides for a method of preventing sexual dysfunctions in mammals, comprising the steps of (a) administering to a mammal a formulation containing an indolinone compound; (b) monitoring the sexual activity of the mammal; and (c) adjusting the dose of the compound to match the specific needs of the mammal.
  • “Mammal” generally refers to such organisms as mice, rats, rabbits, dogs, cats, pigs, cows, sheep, goats, more preferably monkeys and apes, and most preferably humans. Both male and female mammals are contemplated as subjects for treatment by the methods of the present invention.
  • “Sexual function” refers to performing any sexual acitivity, including sexual desire, intercourse, and orgasm.
  • sexual dysfunction refers to any malady that would result in loss of sexual activity or the inability of the subject to perform a sexual act.
  • the sexual dysfunctions contemplated by this invention include those dysfunctions that have an organic basis (e.g., impotence) rather than a psychosexual basis. A common sexual dysfunction is impotence.
  • Impotence is a sexual dysfunction that occurs in male mammals and consists of the inability of the male to achieve or maintain an erection, which is the elongation and hardening of the penis, sufficient to penetrate the vagina of a female or to perform a sexual act. Impotence does not necessarily involve lack of sexual desire. In general, impotent males have sexual desire but are not able to perform the sexual act. Therefore, impotence refers to two different conditions: one in which the male cannot obtain an erection, and the second in which, once an erection is obtained, the male cannot maintain it long enough for the completion of the sexual act. The treatment of both of these conditions is within the scope of the present invention.
  • sexual dysfunctions in mammals include loss of sexual desire and the inability to reach orgasm, and in female mammals, vaginal dryness.
  • orgasm is normally coincident with ejaculation, which is the ejection of semen through the penis.
  • administering relates broadly to the provision to an organism and more specifically to a method of introducing a compound into cells or tissues of an organism. Many techniques exist in the art to administer compounds to an organism, including (but not limited to) oral, parenteral, dermal, injection, and aerosol applications.
  • the indolinone compound used in the methods of the invention is 3- [(2,4-dimethylpyrrol-5-yl)methylene]-2-indolinone, which has the structure set forth in formula XXIII.
  • the methods of the invention also include a pharmaceutical composition comprising an indolinone compound and a pharmaceutically acceptable diluent or carrier.
  • a pharmaceutical composition comprising an indolinone compound and a pharmaceutically acceptable diluent or carrier.
  • the sexual activity of the mammal may be monitored and any changes or improvements may be noted.
  • monitoring refers to observing the effect of adding a compound of the invention to a mammal. The effect can be manifested in a change in the sexual activity of the mammal.
  • “Sexual activity” can be, for example, the mammal's sexual desire, its ability to perform a sexual act, or its ability to have an orgasm.
  • the invention provides a method of identifying an indolinone compound which increases sexual function in a mammal, comprising the steps of (a) administering to the mammal a formulation containing the indolinone compound; (b) monitoring the sexual activity of the mammal; and (c) identifying an indolinone compound which modulates the sexual activity of the mammal.
  • the invention also provides a method for synthesizing an indolinone compound that was identified by the methods of the invention to be a modulator of mammalian sexual activity.
  • modulates refers to altering the sexual activity of a mammal by, for example, increasing or decreasing the frequency of having sexual desires, obtaining an erection, or achieving orgasm.
  • a modulator preferably increases the sexual activity of the mammal.
  • the invention provides a method of increasing sexual function in a mammal in need of such increase comprising the steps of (a) identifying the mammal in need of the increase in sexual function; and (b) administering to the mammal a formulation containing an indolinone compound.
  • the identification step may be conducted by a health care professional as part of the mammal's medical diagnosis, or, if the mammal is a human, he or she may self-diagnose the need for the increase in his or her sexual function.
  • the invention features a pharmaceutical composition
  • a pharmaceutical composition comprising (i) a physiologically acceptable carrier, diluent, or excipient; and (ii) an indolinone compound as described herein, or a salt thereof.
  • the invention also features a method of modulating the function of a protein kinase with a compound of the invention, comprising the step of contacting cells expressing the protein kinase with the compound.
  • the compounds of the invention preferably modulate the activity of the protein kinase in vitro. These compounds preferably show positive results in one or more in vitro assays for an activity corresponding to treatment of the disease or disorder in question (such as the assays described in the Examples below).
  • the invention also features a method of identifying indolinone compounds that modulate the function of protein kinase, comprising the following steps: (a) contacting cells expressing the protein kinase with the compound; and (b) monitoring an effect upon the cells.
  • the effect upon the cells is preferably a change or an absence of a change in cell phenotype, more preferably it is a change or an absence of a change in cell proliferation, even more preferably it is a change or absence of a change in the catalytic activity of the protein kinase, and most preferably it is a change or absence of a change in the interaction between the protein kinase with a natural binding partner, as described herein.
  • the invention features a method for identifying the indolinone compounds of the invention, comprising the following steps: (a) lysing the cells to render a lysate comprising protein kinase; (b) adsorbing the protein kinase to an antibody; (c)incubating the adsorbed protein kinase with a substrate or substrates; and (d) adsorbing the substrate or substrates to a solid support or antibody; where the step of monitoring the effect on the cells comprises measuring the phosphate concentration of the substrate or substrates.
  • the invention features a method for treating a disease related to unregulated tyrosine kinase signal transduction, where the method includes the step of administering to a subject in need thereof a therapeutically effective amount of an indolinone compound as described herein.
  • the invention also features a method of regulating tyrosine kinase signal transduction comprising administering to a subject a therapeutically effective amount of a compound as described herein.
  • the invention features a method of preventing or treating an abnormal condition in an organism, where the abnormal condition is associated with an aberration in a signal transduction pathway characterized by an interaction between a protein kinase and a natural binding partner, where the method comprises the following steps: (a) administering a compound as described herein; and (b) promoting or disrupting the abnormal interaction.
  • the organism is preferably a mammal and the abnormal condition is preferably cancer.
  • Table 1 shows a summary of IC 50 determinations of the inhibition of VEGF- stimulation by the indolinone compounds, Compound AV-002, Compound AV-003, and Compound AV-004, of human umbilical vein endothelial cells (HUVEC), in the absence and presence of exogenously applied VEGF. The effect of doxorubicin is included for comparison.
  • Table 2 shows a summary of IC 5 o determinations of the activity of the indolinone compounds, Compound AV-002, Compound AV-003, and Compound AV-004, for embryonic thoracic aorta smooth muscle cells (A10 cells), in the absence of exogenously applied PDGF.
  • Table 3 shows a summary of IC 5 0 determinations of the activity of the indolinone compounds, Compound AV-002, Compound AV-003, and Compound AV-004, with pre- incubation, for A10 cells, in the absence of exogenously applied PDGF. The effect of doxorubicin is included for comparison.
  • Table 4 shows a summary of IC 5 0 determinations of the inhibition of PDGF- stimulation by the indolinone compounds, Compound AV-002, Compound AV-003, and Compound AV-004, of A10 cells without pre-incubation.
  • Table 5 shows a summary of IC 50 determinations of the inhibition of PDGF- stimulation by the indolinone compounds, Compound AV-002, Compound AV-003, and Compound AV-004, of A10 cells with pre-incubation. The effect of doxorubicin is included for comparison.
  • Table 6 shows a summary of the effects of test compounds (Compound AV-002, Compound AV-003, and Compound AV-004) in the adjuvant arthritis rat model compared to arthritis control (vehicle only).
  • Table 7 shows a summary of the effects of test compounds (Compound AV-002, Compound AV-003, and Compound AV-004) in the adjuvant arthritis rat model compared to arthritis control (vehicle only) as an arthritis index.
  • Figure 1 shows the effect of Compound AV-002 on paw volume of adjuvant arthritis rats.
  • Figure 2 shows the effect of Compound AV-003 on paw volume of adjuvant arthritis rats.
  • Figure 3 shows the effect of Compound AV-004 on paw volume of adjuvant arthritis rats.
  • Figure 4 shows the effect of test compounds on body weight of adjuvant arthritis rats.
  • the present invention relates to compounds capable of regulating and/or modulating protein kinase signal transduction and more particularly receptor and non- receptor protein kinase signal transduction.
  • 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 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 to the extracellular microenvironment). See, Schlessinger and Ullrich, 1992, Neuron 9:303-391.
  • tyrosine phosphorylation sites in growth factor receptors function as high-affinity binding sites for SH2 (src homology) domains of signaling molecules. Fantl et ⁇ /., 1992, Cell 69: 13-423; Songyang et al, 1994, Mol. Cell. Biol. 14:2111-2185); Songyang et al, 1993, Cell 72:161-118; and Koch et al, 1991, Science 252:668-618.
  • substrate proteins that associate with receptor tyrosine kinases have been identified. They may be divided into two principal groups: (1) substrates which have a catalytic domain; and (2) substrates which lack such domain but serve as adapters and associate with catalytically active molecules.
  • Tyrosine kinase signal transduction results in, among other responses, cell proliferation, differentiation and metabolism.
  • Abnormal cell proliferation may result in a wide array of disorders and diseases, including the development of neoplasia such as carcinoma, sarcoma, leukemia, glioblastoma, hemangioma, psoriasis, arteriosclerosis, arthritis and diabetic retinopathy (or other disorders related to uncontrolled angiogenesis and/or vasculogenesis).
  • This invention is therefore directed to compounds which regulate, modulate and/or inhibit tyrosine kinase signal transduction by affecting the enzymatic activity of the RTKs and/or the non-receptor tyrosine kinases and interfering with the signal transduced such proteins.
  • the present invention is directed to compounds which regulate, modulate and/or inhibit the RTK and/or non-receptor tyrosine kinase mediated signal transduction pathways as a therapeutic approach to cure many kinds of solid tumors, including but not limited to carcinoma, sarcoma, leukemia, erythroblastoma, glioblastoma, meningioma, astrocytoma, melanoma and myoblastoma.
  • 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.
  • the compounds described herein are useful for treating disorders related to unregulated tyrosine kinase signal transduction, including cell proliferative disorders, fibrotic disorders and metabolic disorders.
  • Cell proliferative disorders which can be treated or further studied by the present invention include cancers, blood vessel proliferative disorders and mesangial cell proliferative disorders.
  • Blood vessel proliferative disorders refer to angiogenic and vasculogenic disorders generally resulting in abnormal proliferation of blood vessels.
  • 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.
  • disorders related to the shrinkage, contraction or closing of blood vessels, such as restenosis are also implicated.
  • Fibrotic disorders refer to the abnormal formation of extracellular matrix.
  • fibrotic disorders include hepatic cirrhosis and mesangial cell proliferative disorders.
  • Hepatic cirrohis is characterized by the increase in extracellular matrix constituents resulting in the formation of a hepatic scar.
  • Hepatic cirrhosis can cause diseases such as cirrhosis of the liver.
  • An increased extracellular matrix resulting in a hepatic scar can also be caused by viral infection such as hepatitis.
  • Lipocytes appear to play a major role ion hepatic cirrhosis.
  • Other fibrotic disorders implicated include atherosclerosis (see, below).
  • 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, malignant nephrosclerosis, thrombotic microangiopathy syndromes, transplant rejection, and glomerulopathies.
  • the PDGF-R has been implicated in the maintenance of mesangial cell proliferation. Floege et al, 1993, Kidney International 43:41 S-54S.
  • PKs have been associated with such cell proliferative disorders. For example, some members of the RTK family have been associated with the development of cancer. Some of these receptors, like the EGFR (Tuzi et al, 1991, Br. J. Cancer 63:221-233; Torp et al, 1992, APMIS 100:113-119) HER2/neu (Slamon et al, 1989, Science 244:101- 712) and the PDGF-R (Kumabe et al, 1992, Oncogene 7:627-633) are overexpressed in many tumors and or persistently activated by autocrine loops.
  • EGFR Tuzi et al, 1991, Br. J. Cancer 63:221-233; Torp et al, 1992, APMIS 100:113-119
  • HER2/neu Semon et al, 1989, Science 244:101- 712
  • the PDGF-R Kerabe et al, 1992, Oncogene 7:627-63
  • the EGFR receptor 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.
  • the PDGF-R has been associated with glioblastoma, lung, ovarian, melanoma and prostate.
  • the RTK c-met has been generally associated with hepatocarcinogenesis and thus hepatocellular carcinoma. Additionally, c-met has been linked to malignant tumor formation.
  • the RTK c-met has been associated with, among other cancers, colorectal, thyroid, pancreatic and gastric carcinoma, leukemia and lymphoma. Additionally, over-expression of the c- met gene has been detected in patients with Hodgkins disease, Burkitts disease, and the lymphoma cell line.
  • 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. 54:1418-1423) and small lung tumor cells (Macauley et ⁇ /., 1990, Cancer Res. 50:251 -2511).
  • IGF-I integrally involved in the normal growth and differentiation of the nervous system, appears to be an autocrine stimulator of human gliomas. Sandberg-Nordqvist et al, 1993, Cancer Res. 53:2415-2418.
  • IGF-IR insulin growth factor-IR
  • fibroblasts epithelial cells, smooth muscle cells, T-lymphocytes, myeloid cells, chondrocytes, osteoblasts, the stem cells of the bone marrow
  • IGF-I Eukaryotic Gene Expression 7:301-326.
  • Baserga even suggests that IGF-I-R plays a central role in the mechanisms of transformation and, as such, could be a preferred target for therapeutic interventions for a broad spectrum of human malignancies. Baserga, 1995, Cancer Res.
  • RTKs have been associated with metabolic diseases like psoriasis, diabetes mellitus, wound healing, inflammation, and neurodegenerative diseases.
  • These diseases include, but are not limited to hypertension, depression, generalized anxiety disorder, phobias, post-traumatic stress syndrome, avoidant personality disorder, sexual dysfunction, eating disorders, obesity, chemical dependencies, cluster headache, migraine, pain, Alzheimer's disease, obsessive-compulsive disorder, panic disorder, memory disorders, Parkinson's disease, endocrine disorders, vasospasm, cerebellar ataxia, and gastrointestinal tract disorders.
  • the EGF-R is indicated in corneal and dermal wound healing. Defects in the Insulin-R and the 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 7:334-339.
  • tyrosine kinases Not only receptor type tyrosine kinases, but also many cellular tyrosine kinases (CTKs) including src, abl, fps, yes, fyn, lyn, lck, blk, hck, fgr, yrk (reviewed by Bolen et al , 1992, FASEB J. (5:3403-3409) are involved in the proliferative and metabolic signal transduction pathway and thus in indications of the present invention. For example, mutated src (v-src) has been demonstrated as an oncoprotein (pp60 v"src ) in chicken.
  • CTKs tyrosine kinases
  • pp60 c"src transmits oncogenic signals of many receptors.
  • overexpression of EGF-R or HER2/neu in tumors leads to the constitutive activation of pp60 c ⁇ src , which is characteristic for the malignant cell but absent from the normal cell.
  • mice deficient for 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.
  • Zap 70 is implicated in T-cell signaling.
  • CTK modulating compounds to augment or even synergize with RTK aimed blockers is an aspect of the present invention.
  • RTKs and non-receptor type kinases have been connected to hyperimmune disorders.
  • Alterations in the function of a protein kinase that normally regulates cell proliferation can lead to enhanced or decreased cell proliferative conditions evident in certain diseases.
  • Aberrant cell proliferative conditions include angiogenic and vasculogenic disorders including rheumatoid arthritis, endometriosis, ocular diseases, cancer and metastases, psoriasis, arterial thickening and restenosis, tissue ischemia, and excessive scarring during wound healing.
  • Neovascularization is an important step in the transition from hyperplasia to neoplasia and it must occur for tumors to grow beyond 1 to 2 mm 3 (Folkman, J. Natl. Cancer Inst. 1990, 82, 4-6; Folkman, et al, Nature 1989, 339, 58-61).
  • a correlation between microvessel density and severity of disease has been observed in a number of different tumor types including malignant glioma (Plate & Risau, GLIA 1995, 75, 339-347), and breast (Horak, et al,
  • VEGF tumor endothelial growth factor
  • Flk-1/KDR Flk-1/KDR
  • VEGF is secreted by a number of human tumor cell lines in culture, including glioma (Tsai, et al, J. Neurosurg 1995, 82, 864-867), melanoma (Claffey, et al, Cancer Res. 1996, 56, 172-181.) Kaposi sarcoma, and epidermoid carcinoma cells (Myoken, et al, Proc. Natl. Acad. Sci. USA 1991, 55, 5819-5823).
  • VEGF transcripts or protein has been identified by in situ hybridization or immunohistochemistry in primary gliomas (Plate, et al, Lab Invest. 1992, 67, 529-534; Plate, et al, Int. J. Cancer 1994, 59, 520-529), hemangioblastomas (Hatva, et al, Amer. J. Pathol 1996, 148, 763-775) and breast (Toi, et al, Jpn. J. Cancer Res. 1994, 55, 1045- 1049; Anan, et al, Surgery 1996, 779, 333-339; Yoshiji, et al, Cancer Res.
  • Rat C6 glioma and human U87MG glioblastoma cells secrete VEGF and grow subcutaneously in athymic mice (Saleh, et al, Cancer Res. 1996, 56, 393-401; Cheng, et al, Proc. Natl Acad. Sci. USA 1996, 93, 8502-8507).
  • the introduction of antisense constructs to VEGF mRNA into these cell lines reduces their in vivo growth, as well as the degree of neovascularization.
  • Monoclonal antibodies against VEGF inhibit the subcutaneous growth of human rhabdomyosarcoma, glioblastoma, leiomyosarcoma (Kim, et al, Nature 1993, 362, 841-844) and fibrosarcoma (Asano, et al, Cancer Res. 1995, 55, 5296-5301) in athymic mice. Metastasis of fibrosarcoma (Asano, et al, Cancer Res. 1995, 55, 5296-5301) and colon cancer tumors (Warren, et al, J. Clin. Invest. 1995, 95, 1789-1797) was also blocked by anti- VEGF antibodies.
  • VEGF vascular endothelial growth factor
  • Flk-1/KDR and Fit- 1 VEGF receptors
  • their mRNA has been detected in tumors such as gliomas (Plate, et al, Lab Invest. 1992, 67, 529-534), Plate, et al, Int. J. Cancer 1994, 59, 520-529), hemangioblastomas (Hatva, et al, Am. J.
  • Flk-1 The capacity of Flk-1 to act as a modulator of tumor growth has also been studied in animal tumor models.
  • Athymic mice were co-implanted with tumor cells and virus- producing cells that produced viral DNA encoding a truncated flk-1 gene (Millauer, et al, Nature 1994, 367, 516-519; Millauer, Cancer Res. 1996, 56, 1615-1620).
  • the co-implantation allowed the introduction of mutant receptor into endothelial cells where it acted in a dominant-negative fashion to block activation of Flk-1 and effect the growth of the tumor.
  • the subcutaneous growth of a variety of human, rat and mouse tumor cells was shown to be inhibited.
  • the microvessel density was shown to be reduced in the small tumors that did form, confirming the connection between Flk-1, angiogenesis, and tumor growth.
  • Flk-1/KDR is an excellent target for the development of novel anticancer agents.
  • Specific inhibitors of Flk-1 /KDR would be expected to have fewer side effects than cytotoxic chemotherapy drugs, since angiogenesis is thought to rarely occur in healthy adults (with the exception of angiogenesis that occurs following wound injury or during cyclical changes in the endometrium and ovary).
  • Monoclonal antibodies specific for VEGF Kim, et al, Nature 1993, 362, 841-844
  • Flk-1 Rockwell, et al, Proc. Am. Assc. Cancer Res. 1997, 38, 266) have been shown to inhibit tumor growth in animals by disrupting binding of VEGF to the receptor.
  • FGFl/aFGF and FGF2/bFGF Acidic and basic fibroblast growth factors
  • FGF2/bFGF Acidic and basic fibroblast growth factors
  • a human renal cell carcinoma cell line has been shown to secrete FGF2/bFGF (Singh, et al. , Cell Growth Diffl 1996, 7, 397-404) and two human prostate tumor cell lines were found to make and respond to FGF2/bFGF (Nakamoto, et al. , Cancer Res. 1992, 52, 511 -577).
  • FGF2/bFGF Neutralizing antibodies specific for FGF2/bFGF have been used to investigate the role of FGF2/bFGF in cancer. FGF-induced mitogenesis of SCI 15 mouse mammary carcinoma cells in response to bFGF was shown to be inhibited by anti-FGF antibodies (Lu, et al, Cancer Res. 1989, 49, 4963-4967). Similarly, an anti-FGF2/bFGF monoclonal antibody blocked the growth of U-87MG and T98G human glioblastoma cells in culture and as xenografts in nude mice (Takahashi, et al, FEBSLett. 1991, 255, 65-71).
  • PDGF receptors have also been identified in various tumors and tumor cell lines and contribute to the transformation of cells. As with the FGF receptors, they may contribute to the growth of tumors by promoting angiogenesis and tumor cell proliferation and survival.
  • PDGF is the most potent mitogen for cells of mesenchymal origin. Kidney glomerular mesangial cells were the targets of disrupted PDGF-B or
  • PDGF- ⁇ receptor (Leveen, P., et al, Genes Dev. 1994, 5, 1875-1887; Soriano, P., Genes Dev. 1994, 5, 1888-1896) genes in mice and were found to lead to the development of lethal hemorrhage and edema in late embryogenesis.
  • Mesangial cells are related to microvascular pericytes, another target of the disrupted PDGF-B gene.
  • Pericytes encircle the microvessels in many different tissues. They are contractile cells and therefore may contribute to the mechanical stability of the capillary wall. Pericytes express PDGF receptors and respond to PDGF in vitro. Pericytes may also regulate endothelial cell function. Thus, PDGF and its receptors may function to support vascular integrity.
  • Rheumatoid arthritis is an inflammatory joint disease that is characterized by cellular infiltration of synovial fluid by neutrophils, and of the synovial membrane by T lymphocytes and macrophages, hyperproliferation of cells of the synovial membrane which results in formation of a pannus, and destruction of cartilage and bone (Feldman, et al, Ann. Rev. Immunol. 1996, 74, 397-440; Paleolog, Br. J. Rheumatol. 1996, 35, 917-920). Angiogenesis is thought to have an important role in the pathogenesis of RA (Colville-Nash & Scott, Annals.
  • RA synovial fluid or tissue include VEGF (Fava, et al, J. Exp. Med. 1994, 750, 341-346; Koch, et al. J. Immunol.
  • VEGF expression is significantly higher in synovial fluid and tissue from RA patients than from patients with other types of arthritis (Fava, et al, J. Exp. Med. 1994, 750, 341-346; Koch, et al. J. Immunol. 1994, 752, 4149-4156).
  • the source of this VEGF appears to be elevated expression in synovial lining cells, subsynovial macrophages, fibroblasts surrounding microvessels, and vascular smooth muscle cells (Fava, et al, J. Exp. Med. 1994, 750, 341-346; Koch, et al. J. Immunol. 1994, 752, 4149-4156;
  • Synovial lining cells, macrophages, endothelial cells, and vascular smooth muscle cells of rheumatoid joints (Hosaka, et al, Pathobiol 1995, 63, 249-56), as well as mast cells in rheumatoid synovium (Qu, et al, Am. J. Pathol 1995, 747, 564-573), have been reported to express FGF2/bFGF, but it does not appear to be elevated in RA synovial fluid (Hosaka, 1995, supra).
  • FGFl/aFGF is abundantly expressed in synovial tissues from RA patients (Byrd, et al, Arthritis & Rheumatism 1996, 39, 914-922), and expression of FGF-R1, the receptor for FGF1 and FGF2 (Ornitz, et al, J. Biol Chem. 1996, 277, 15292-15297), is enhanced on CD4 + T cells (Byrd, 1996, supra).
  • Psoriasis is a chronic skin disorder that is characterized by hyperproliferation of the epidermis, inflammation, and angiogenesis.
  • Angiogenesis appears to be crucial in the pathogenesis of psoriasis, and microvascular changes are one of the earliest detectable events in developing psoriatic lesions (for a review see Creamer & Barker, Clin. Exp. Dermatol. 1995, 20, 6-9).
  • Several reports have implicated the epidermis as the origin of angiogenic factors (Nishioka & Ryan, J. Invest. Dermatol. 1972, 55, 33-45; Wolf & Harrison, J. Invest. Dermatol 1973, 59, 40-43; Barnhill, et al, Br. J.
  • VEGF has been the best characterized as a direct inducer of angiogenesis.
  • VEGF is overexpressed in keratinocytes of psoriatic skin, but only minimally expressed in normal epidermis (Detmar, et al, J. Exp. Med. 1994, 750, 1141-1146).
  • VEGF is also overexpressed in other skin diseases such as bullous pemphigoid, dermatitis herpetiformis, and erythema multiforme (Brown, et al, Invest.
  • Mast cells have also been reported to be a major source of FGF2/bFGF in chronic inflammatory diseases (Qu, et al, Am. J. Pathol 1995, 747, 564-573), which may contribute to the link between inflammation and angiogenesis.
  • angiogenic factors from the ischemic retina has been hypothesized to be the central stimulus for retinal neovascularization.
  • Glaucoma vitreous hemorrhage and retinal detachment, secondary to intraocular neovascularization, accounts for the resultant vision loss in several ocular disorders such as retinopathy of prematurity, age-related macular degeneration, and diabetic retinopathy.
  • the release of angiogenic factors by the ischemic retina to induce new blood vessel growth and increase the oxygen supply to the area turns out to be harmful as the new vessels do not grow with normal architecture.
  • angiogenic factors such as IGF-1, FGF and VEGF have elevated expression in vitreous and neo vascular membranes from patients with retinal disorders (Grant, et al. , Diabetes 1986, 35, 416-20; Sivalingam, et al, Arch Ophthalmol. 1990, 705, 869-72; Aiello, et al, New Engl J. Med. 1994, 331, 1480-1487; Malecaze, et al, Arch. Ophthalmol. 1994, 772, 1476-82; Adamis, et al, Amer. J. Ophthalmology 1994, 775, 445-50). Therefore, these growth factors are candidates for the angiogenic factors which modulate or initiate intraocular neovascularization in retinal disorders.
  • VEGF is constitutively expressed in the vascularized tissues of the normal eye (Adamis, et al, Arch. Ophthalmol 1996, 774, 66-71). Intraocular VEGF gene expression is increased in disease states like diabetic retinopathy (Adamis, et al, Amer. J. Ophthalmology 1994, 775, 445-450; Malecaze, et al, Arch. Ophthalmology 1994, 772, 1476-1482; Aiello, 1994, supra; Pe'er, et al, Lab Invest. 1995, 72, 638-645).
  • VEGF vascular endothelial growth factor
  • VEGF into normal eyes caused retinal edema, microaneurysms, hemorrhage and intraretinal neovascularization (Pierce, et al, Arch. Ophthalmol. 1995, 774, 964-970; Miller, et al, Am. J. Pathol. 1994, 745, 574-584; Tolentino, 1996, supra).
  • Decreased levels of VEGF paralleled the regression of proliferative retinopathy.
  • Stimuli associated with oxygen deprivation such as hypoxia (Pierce, 1995, supra; Miller, 1994, supra; Shweiki, et al, Nature 1992, 359, 843-845; Plate, et al, Lab Invest.
  • VEGF signaling including antisense VEGF reagents (Smith, IBC Conference on Angiogenesis Inhibitors and Other Novel Therapeutic Strategies for Ocular Diseases of Neovascularization 1996) and soluble VEGF receptor chimeric proteins (Aiello, et al, Proc. Natl Acad. Sci.
  • FGF2/bFGF appears to play a role in diabetic retinopathy (Sivalingam, et al, Arch Ophthalmol 1990, 705, 869-872; Hanneken, et al, Arch. Ophthalmol 1991, 709, 1005-1011).
  • FGF2/bFGF induces endothelial cell proliferation (Gospodarowitz, Prog. Clin. Biol. Res. 1976, 9, 1-19; D'Armore & Klagsbrun, J. Cell
  • FGF2/bFGF induces corneal neovascularization (Gospodarowitz, et al, Exp. Eye Res. 1979, 25, 501-14; Risau, Proc. Natl. Acad Sci. USA 1986, 83, 3855-3859) as well as retinal fibrovascular proliferation with an enhanced fibrotic component compared to IGF-1 when injected into the vitreous cavity (Grant, et al, Reg. Peptides 1993, 48, 267-278).
  • the development of retinal capillary basement membrane thickening and subsequent retinal traction and detachment is similar to those occurring in humans and animals with diabetes.
  • Arterial Thickening and Restenosis Arterial injury as part of the atherosclerotic process or as a consequence of balloon-mediated injury to treat coronary occlusions is known to be accompanied by thickening of the arterial wall. This response is also observed in cases of organ rejection where chronic vascular injury is associated with thickening of the arteries at the site of the organ transplant.
  • RTKs have been implicated as important players within the disease process associated with the injury response.
  • both PDGF and FGFs are associated with the hyperproliferation of the arterial smooth muscle cell. When these cell layers become ischemic, they require new blood vessel formation in order to support their hyperproliferation.
  • mitogen-activated protein kinase has been shown to be associated with PDGF-dependent cell movement (Graf, et al, Hypertension 1997, 29, 334-339).
  • a synthetic compound that blocks binding of PDGF to its receptor has been shown to inhibit chemotaxis of smooth muscle cells and inhibit neo-intimal formation in restenotic lesions following balloon-injury of carotid arteries in rats (Mullins, et al, Arterioscler Thromb 1994, 74, 1047-1055).
  • PDGF mRNA levels have been shown to be increased in human cardiac allografts (Zhao, et al, J. Clin. Invest. 1994, 94, 992-1003).
  • PDGF and FGF have been shown to be expressed in the injury response following cardiac (Zhao, 1994, supra; Zhao, et al, Circulation 1994, 90, 677-685) and renal (Alpers, et al, Am. J. Pathol. 1996, 745, 439-451; Abboud, Annu. Rev. Physiol 1995, 57, 297-309) transplantation.
  • Atherosclerosis is a disease associated with the formation of arterial lesions or atheromas consisting of endothelium-covered fibro-fatty plaques. Beneath the endothelial layer exists smooth muscle cells and extracellular matrix components containing variable amounts of serum proteins. This overlies an area characterized by collections of lipid- laden macrophages. Significant numbers of lymphocytes, particularly T cells, are also present and may contribute to lymphocyte-mediated angiogenesis (Kaminski, M. & Auerbach, R., Proc. Soc. Exp. Biol. Med. 1988, 755. 440-443). Both macrophages and lymphocytes traverse the endothelium in order to enter the atheroma lesion.
  • the lesion is characterized by a flux of blood cells including platelets that enter or exit the endothelium. Similar to the restenotic lesion, the build up of smooth muscle cells is supported by angiogenesis and is likely to be stimulated by factors released from the smooth muscle cells when they become hypoxic. In contrast to atheromas, thrombi of arteries and veins are characterized by structures containing a fibrin mesh in which blood cells are entrapped. The presence of platelets and other cells in the lesion have led to a number of studies that implicate growth factors and their cognate receptor tyrosine kinases as players in the disease (Chabrier, Int. Angiol. 1996, 75, 100-103). The most prominent player in this regard is PDGF.
  • PDGF has been detected in atherosclerotic lesions of rat (Waltenbeger, et al, Arterioscler Thromb. Vase. Biol 1996, 16, 1516-1523), rabbit (Agapitos, et al, Int. Angiol. 1996, 75, 249-251), and human (Billett, et al, Arterioscler Thromb. Vase. Biol. 1996, 16, 399-406; Ito, et al, Neurol Res. 1995, 77, 345-348) origin.
  • the release of PDGF from platelets and other lymphocytes is thought to elicit a pleiotrophy of paracrine effects on blood cells and smooth muscle cells in the vicinity of the lesion.
  • the chemoattractant, cell survival, migratory and mitogenic properties of PDGF receptor function in cells may contribute directly or indirectly to different activities on different cell types.
  • PDGF RNA expression has been shown to be associated with the presence of circulating mononuclear cells in hypercholesterolemic patients (Billett, 1996, supra). The presence of mononuclear and other blood cells in the atherosclerotic lesion has been shown to also elaborate the expression of VEGF and FGF growth factors. In smooth muscle cells, PDGF has been shown to exert a number of effects that may directly or indirectly effect the angiogenic process (Newby, A.C. & George, S.J., Curr. Opin. Cardiol. 1996, 77, 574-582).
  • PDGF signaling may upregulate the expression of VEGF in smooth muscle cells (Stavri, et al, FEBS Lett. 1995, 358, 311-315).
  • PDGF is a player in myointimal proliferation associated with formation of atherosclerotic plaques.
  • FGF has been considered to be an important player in hyperprohferative and angiogenic aspects of this lesion.
  • FGFl/aFGF and FGF2/bFGF or receptor expression has been shown to be increased in rat aortic smooth muscle cells (van Neck, et al, Biochim. Biophys. Acta. 1995, 1261, 210-214), human cardiac allografts (Zhao, 1994, supra; Zhao, 1994, supra), CD4+ T cells (Zhao, et al, J Immunol. 1995, 755, 3904-3911), and atherosclerotic human arteries (Hughes, Cardiovasc. Res. 1996, 32, 557-569).
  • RTK-mediated signaling events may be important in the development and maintenance of the lesion. Interruption of these signaling systems inhibit the formation or disrupt the remodeling or integrity of the lesion. In the fibrotic thrombus, targeting of these receptors may be advantageous to reduce the number of connective tissues cells involved in fibrin deposition and development of the clot.
  • VEGF vascular endothelial growth factor
  • FGF FGF
  • PDGF receptor systems where these receptors effect the growth and survival of endothelial cells, pericytes, and arterial smooth muscle cells.
  • tissue ischemia the induction of new blood vessel growth may be advantageous for the treatment of specific human diseases where oxygen and nutrient limitation is linked to the disease pathology.
  • VEGF gene therapy trials have been initiated to measure improved vascular function (Isner, Lancet 1996, 345, 370-374).
  • Endometriosis also requires angiogenesis and neovascularization to permit the establishment and growth of endometriotic cells outside the uterus.
  • Endometriosis is the most prevalent cause of female infertility, and affects approximately 6 million women in the U.S. The disease is most commonly characterized by the growth of endometrium in the peritoneal cavity where it proliferates, invades, secretes, desquamates, and accumulates with each ovarian cycle. Abdominal pain, fibrosis, rare ascites formation, and adhesions of intra-abdominal structures, result.
  • Endometriosis has been linked to increased levels of angiogenic factors and specifically to elevated VEGF levels (L. Brown et al, Lab. Invest, 1997, 76, 245-255; J. Shifren et ⁇ /., J. Clin. Endocrinol Metab., 1996, 57, 3112-3118; j. McLaren et al, Hum. Reprod., 1996, 77, 220-223).
  • expression of Fit is constant but that of kinase insert domain containing receptor (FLK-1 /KDR) is increased in the luteal phase, at which time the cells migrate in response to VEGF.
  • the compounds of the invention can be used for the treatment of several kinds of diseases which include, but not limited to, cancers, such as, squamous cell carcinoma, astrocytoma, glioblastoma, lung cancer, bladder cancer, head and neck cancer, melanoma, ovarian cancer, prostate cancer, breast cancer, gastrointestinal cancer, colorectal cancer, small-cell lung cancer and glioma, and immunological disorders, hyperproliferation disorders, cardiovascular disorders, inflammatory disorders, restenosis, fibrosis, psoriasis, osteoarthritis, rheumatoid arthritis, atherosclerosis, diabetes, and angiogenesis.
  • cancers such as, squamous cell carcinoma, astrocytoma, glioblastoma, lung cancer, bladder cancer, head and neck cancer, melanoma, ovarian cancer, prostate cancer, breast cancer, gastrointestinal cancer, colorectal cancer, small-cell lung cancer and glioma
  • immunological disorders hyperproliferation disorders
  • Suitable routes of administration may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intranasal, or intraocular injections.
  • parenteral delivery including intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intranasal, or intraocular injections.
  • parenteral delivery including intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intranasal, or intraocular injections.
  • parenteral delivery including intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intranasal, or intraocular injections.
  • parenteral delivery including intra
  • the liposomes will be targeted to and taken up selectively by the tumor.
  • compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • compositions for use in accordance with the present invention thus 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 agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • physiologically compatible buffers such as Hanks's 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 readily 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, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
  • Pharmaceutical preparations for oral use can be obtained solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable 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, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • 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.
  • 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 filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • 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. All formulations for oral administration should be in dosages suitable for such administration.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of e.g. 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 be formulated for parenteral administration by injection, 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 formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which 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, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • 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.
  • the cosolvent system may be 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.
  • the proportions of a 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 usually 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 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.
  • PK modulating compounds of the invention may be provided as salts with pharmaceutically compatible counterions.
  • Pharmaceutically compatible salts may be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms.
  • compositions suitable for use in the present invention include compositions where the active ingredients are contained in an amount effective to achieve its intended purpose. More specifically, 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 dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC 5 o 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 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., for determining the LD 5 o (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD 50 and ED 5 o.
  • Compounds which exhibit high therapeutic indices are preferred.
  • the data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range 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 moiety which are sufficient to maintain the kinase modulating effects, or minimal effective concentration (MEC).
  • MEC minimal effective concentration
  • 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 the kinase using the assays described herein. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, 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 which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%. In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.
  • composition administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.
  • compositions may, if desired, be presented in a pack or dispenser device 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 with a notice associated with the container in 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 polynucleotide for human or veterinary administration.
  • Such notice for example, may be the labeling approved by the U.S. Food and Drug Administration for prescription drugs, or the approved product insert.
  • 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 also relates to a method of increasing sexual function in mammals by administering to the mammal a compound of the invention.
  • the present invention involves monitoring the effect of the compound on the mammal.
  • Methods of synthesis of the compounds of the invention, methods of administration of those compounds to mammals, methods of preparing pharmaceutical compositions containing those compounds, and some biological activities of those compounds are described in detail in the International Application No. PCT/US98/04134, by Shenoy et al., entitled “FORMULATIONS FOR HYDROPHOBIC PHARMACEUTICAL AGENTS",(Lyon & Lyon Docket No. 231/299), and U.S. Provisional Application Serial No.
  • the cells used in the methods are commercially available.
  • the nucleic acid vectors harbored by the cells are also commercially available and the sequences of genes for the various protein kinases are readily accessible in sequence data banks.
  • a person of ordinary skill in the art can readily recreate the cell lines in a timely manner by combining the commercially available cells, the commercially available nucleic acid vectors, and the protein kinase genes using techniques readily available to persons of ordinary skill in the art.
  • EXAMPLE 1 PROCEDURES FOR SYNTHESIZING THE TRICYCLIC-BASED INDOLINONE COMPOUNDS OF THE INVENTION
  • the compounds are prepared by condensation of any one of the aldehyde/ketone/lactone compounds of Table 8 and any one of the tricyclic based indolin- 2-ones, depicted in formulae VII, VIII, and IX, as described herein. If aldehyde is one of the starting materials for preparing the compounds, Method 1 should be used. If ketone or lactone is one of the starting materials, Method 2 should be used for preparing the final product. Method 1:
  • a reaction mixture of the proper tricyclic indolin-2-ones (1.0 equiv.), the appropriate aldehyde (1.2 equiv.), and piperidine (0.1 equiv.) in ethanol (1-2 mL/1.0 mmol oxindole) is stirred at 90 °C for 3-5 h. After cooling, the precipitate is filtered, washed with cold ethanol, and dried to yield the target compound.
  • Aldehydes which are not commercially available could be prepared by conventional Vilsmeier formylation (See, for example, Org. Synth. Coll., Vol. IV, 1963, 831).
  • EXAMPLE 2 PROCEDURES FOR SYNTHESIZING THE PYRAZOLYLAMIDE- BASED COMPOUNDS OF THE INVENTION
  • reaction mixture was stirred at room temperature for 30 minutes, added with 130 mg of 4- triflurormethylaniline followed by 0.6 mL of diisopropylethylamme.
  • the mixture was heated at 50 °C for 6 hrs, diluted with 50 mL of water and extracted with ethyl acetate.
  • 5-Chloro-2-oxindole is commercially available from Aldrich Chemicals.
  • 2-Oxindole is commercially available from Aldrich Chemicals.
  • the solid was suspended in 200 mL of denatured ethanol and slurry-washed by stirring and heating to reflux for 10 minutes. The mixture was cooled in an ice bath to 10 °C. The solid product was collected by vacuum filtration, washed with 25 mL of ethanol and dried under vacuum at 44 °C to give 12.7 g (51% yield) of 7-bromo-5-chloro-2-oxindole.
  • 5-Fluoroisatin (8.2 g) was dissolved in 50 mL of hydrazine hydrate and was heated to refulux for 1 hr. The reaction mixtures were then poured in ice water. The precipitate was then filtered, washed with water and dried under vacuum oven to give the title compound.
  • 2-Oxindole (6.5 g) was dissolved in 25 mL of concentrated sulfuric acid and the mixture maintained at -10 - 15 °C while 2.1 mL of fuming nitric acid was added dropwise.
  • 5-Iodo-2-oxindole (O-l It 2-Oxindole (82.9 g) was suspended in 630 mL of acetic acid with mechanical stirring and the mixture cooled to 10 °C in an ice water bath. Solid N-iodosuccinimide (175 g) was added in portions over 10 minutes. After the addition was complete the mixture was stirred for 1 hour at 10 °C. The suspended solid which was always present became very thick at this time. The solid was collected by vacuum filtration, washed with 100 mL of 50% acetic acid in water and then with 200 mL of water and sucked dry for 20 minutes in the funnel. The product was dried under vacuum to give 93.5 g (36%) of 5- iodo-2-oxindole.
  • 6-Chlorooxindole is commercially available from Finorga.
  • the diester was dissolved in a mixture of 6.4 g of lithium chloride and 2.7 mL of water in 100 mL of dimethylsulfoxide and heated to 100 °C for 3 hours. The reaction was cooled and poured into a mixture of ethyl acetate and brine. The organic phase was washed with brine, dried with sodium sulfate, concentrated and chromatographed on silica gel in 10% ethyl acetate in hexane. The fractions containing product were evaporated to give 25.7 g of methyl 2-nitro-4-trifluoromethylphenylacetate.
  • Methyl 2-nitro-4-trifluoromethylphenylacetate (26 mg) was hydrogenated over 10% palladium on carbon and then heated at 100 °C for 3 hours. The catalyst was removed by filtration and the solvent evaporated to give the title compound.
  • 5-Chloroacetyl-2-oxindole (prepared by the same procedure used for the preparation of O-53, except starting from chloroacetyl chloride) (4.18 g) in 30 mL of trifluoroacetic acid in an ice bath was treated with 4.65 g of triethylsilane and stirred at room temperature for 3 hours. The mixture was poured into 150 mL of water and the precipitate collected by vacuum filtration, washed with 50 mL of water and dried to give 2.53 g (65% yield) of 5-(2-chloroethyl)-2-oxindole as a reddish-brown solid.
  • 5-Iodo-2-oxindole (17 g) was was heated to refulux with 2 g of palladium diacetate, 18.2 g of triethylamine, 150 mL of methanol, 15 mL of dimethylsulfoxide and 2.6 g of DPPP in an atmosphere saturated with carbon monoxide. After 24 hours, the reaction was filtered to remove the catalyst and the filtrate concentrated. The concentrate was chromatographed on silica gel in 30% ethyl acetate in hexane. The fractions containing product were concentrated and allowed to stand. The precipitated product was collected by vacuum filtration to give 0.8 g (7%) of the title compound as an off-white solid.
  • 2-Oxindole (6.7 g) was added to a stirred suspension of 23 g of aluminum chloride in 30 mL of dichloroethane in an ice bath. Chloroacetyl chloride (11.3 g) was slowly added and hydrogen chloride gas was evolved. After ten minutes of stirring, the reaction was warmed to 40 - 50 °C for 1.5 hours. Thin layer chromatography (ethyl acetate, silica gel) showed no remaining starting material. The mixture was cooled to room temperature and poured into ice water. The precipitate was collected by vacuum filtration, washed with water and dried under vacuum to give 10.3 g (98%) of 5-chloroacetyl-2-oxindole as an off-white solid.
  • a suspension of 9.3 g of 5-chloroacetyl-2-oxindole was stirred in 90 mL pyridine at 80 - 90 °C for 3 hours then cooled to room temperature.
  • the precipitate was collected by vacuum filtration and washed with 20 mL of ethanol.
  • the solid was dissolved in 90 mL of 2.5 N sodium hydroxide and stirred at 70 - 80 °C for 3 hours.
  • the mixture was cooled to room temperature and acidified to pH 2 with 0.5 N hydrochloric acid.
  • the precipitate was collected by vacuum filtration and washed thoroughly with water to give crude 5-carboxy-2-oxindole as a dark brown solid.
  • 3,3-Dibromo-7-azaoxindole (2.9 g) was dissolved in a mixture of 20 mL of acetic acid and 30 mL of acetonitrile. To the solution was added 6.5 g of zinc dust. The mixture was stirred for 2 hrs at room temperature. The solid was filtered from the mixture and the solvent evaporated. The residue was treated with ethyl acetate. The ethyl acetate solution containing insoluble solid was passed through a short column of silica gel. The collected ethyl acetate solution was evaporated and the residue dried under vacuum to give 1.8 g (yield 91%) of 7-azaoxindole acetic acid salt.
  • 6-Methoxyoxindole is commercially available from Finorga.
  • Iron powder (2.6 g) was added all at once to 4.5 g of 3-nitrobiphenyl-4-acetic acid in 40 mL of acetic acid. The mixture was heated to refulux for 2 hours, concentrated to dryness and taken up in ethyl acetate. The solids were removed by filtration and the filtrate washed twice with 1 N hydrochloric acid and brine and dried over anhydrous sodium sulfate. The filtrate was concentrated to give 3.4 g (93% yield) of 6-phenyl-2- oxindole as a light brown solid.
  • 6-(2-MethoxyphenvD-2-oxindole (O-47 Tetrakis(triphenylphosphine)palladium (1 g) was added to a mixture of 5 g of 2- methoxyphenylboronic acid, 6.6 g of 5-bromo-2-fluoronitrobenzene and 30 mL of 2 M sodium carbonate solution in 50 mL of toluene and 50 mL of ethanol. The mixture was heated to refulux for 2 hours, concentrated, and the residue extracted twice with ethyl acetate. The ethyl acetate layer was washed with water, brine, dried, and concentrated to give a dark green oil which solidified on standing, crude 4-fluoro-2'-methoxy-3- nitrobiphenyl.
  • Dimethyl malonate 14 mL was added dropwise to 2.9 g of sodium hydride suspended in 50 mL of dimethylsulfoxide. The mixture was heated at 100 °C for 15 minutes and cooled to room temperature. Crude 4-fluoro-2'-methoxy-3-nitrobiphenyl in 60 mL of dimethylsulfoxide was added and the mixture was heated at 100 °C for 2 hours. The reaction mixture was cooled and quenched with 300 mL of saturated sodium chloride solution and extracted twice with ethyl acetate.
  • Iron powder (5 g) was added in one portion to 9.8 g of 2'-methoxy-3- nitrobiphenyl-4-acetic acid in 50 mL of glacial acetic acid was heated to 100 °C for 3 hours.
  • the reaction mixture was concentrated to dryness, sonicated in ethyl acetate and filtered to remove the insolubles.
  • the filtrate was washed twice with 1 N hydrochloric acid, water, brine, dried over anhydrous sodium sulfate and concentrated.
  • Tetrakis(triphenylphosphine)palladium (0.8 g) was added to a mixture of 5 g of 3- methoxyphenylboronic acid, 5 g of 5-bromo-2-fluoronitrobenzene and 11 mL of 2 M sodium carbonate solution in 100 mL of toluene. The mixture was heated to refulux for 2 hours, diluted with water and extracted with ethyl acetate. The ethyl acetate was washed with saturated sodium bicarbonate, brine, dried, and concentrated to give an oily solid.
  • reaction mixture was cooled and quenched with 300 mL of saturated ammonium chloride solution and extracted twice with ethyl acetate. The extracts were combined, washed with brine, dried over anhydrous sodium sulfate and concentrated to give crude dimethyl 3'-methoxy-3-nitrobiphenyl-4-malonate as a pale yellow solid.
  • 3'-Methoxy-3-nitrobiphenyl-4-acetic acid (5.2 g) was dissolved in methanol and hydrogenated over 0.8 g of 10% palladium on carbon for 3 hours at room temperature. The catalyst was removed by filtration, washed with methanol and the filtrates combined and concentrated to give a brown solid. The solid was chromatographed on silica gel in ethyl acetate:hexane:acetic acid 33:66:1 to give 3.0 g (75% yield based on 4-fluoro-3'- methoxy-3-nitrobiphenyl) of 6-(3-methoxypheny)-2-oxindole as a pink solid.
  • Tetrakis(triphenylphosphine)palladium (1 g) was added to a mixture of 5 g of 4- methoxyphenylboronic acid, 6.6 g of 5-bromo-2-fluoronitrobenzene and 30 mL of 2 M sodium carbonate solution in 50 mL of toluene and 50 mL of ethanol. The mixture was heated to refulux for 2 hours, concentrated, and the residue extracted twice with ethyl acetate. The ethyl acetate layer was washed with water, brine, dried, and concentrated to give a brown oily solid.
  • reaction mixture was cooled and quenched with 300 mL of saturated sodium chloride solution and extracted three times with ethyl acetate. The extracts were combined, washed with saturated ammonium chloride, water and brine, dried over anhydrous sodium sulfate and concentrated to give crude dimethyl 4'-methoxy-3- nitrobiphenyl-4-malonate as a yellow oil.
  • Iron powder (3.6 g) was added in one portion to 7.2 g of 4'-methoxy-3- nitrobiphenyl-4-acetic acid in 50 mL of glacial acetic acid and heated at 100 °C overnight.
  • the reaction mixture was concentrated to dryness, sonicated in ethyl acetate and filtered to remove the insolubles.
  • the filtrate was washed twice with 1 N hydrochloric acid, brine, dried over anhydrous sodium sulfate and concentrated to give 2.7 g (54% yield based on - I l l -
  • 6-(3-Ethoxyphenvn-2-oxindole (O-50 Tetrakis(triphenylphosphine)palladium (0.8 g) was added to a mixture of 4.2 g of
  • Dimethyl malonate (11.4 mL) was added dropwise to 4.0 g of sodium hydride suspended in 20 mL of dimethylsulfoxide. The mixture was heated to 100 °C for 10 minutes and cooled to room temperature. Crude 4-fluoro-3'-ethoxy-3-nitrobiphenyl (5.3 g) in 25 mL of dimethylsulfoxide was added and the mixture was heated at 100 °C for 2 hours. The reaction mixture was cooled and quenched with 300 mL of saturated amonium chloride solution and extracted three times with ethyl acetate.
  • 6-Aminooxindole was synthesized using the procedure set forth in Helv. Chem. Acta 31:1381, 1948.
  • 6-(Morpholin-4-ylV2-oxindole (O-58) 6-Amino-2-oxindole (2.2 g), 4.0 g of 2,2'-dibromoethyl ether and 7.9 g of sodium carbonate were refluxed in 20 mL of ethanol overnight, concentrated and diluted with 50 mL of water. The mixture was extracted three times with 50 mL of ethyl acetate each time and the organic extracts combined, washed with 20 mL of brine, dried over anhydrous sodium sulfate and concentrated to dryness.
  • 6-Acetylamino-2-oxindole was synthesized using the procedure set forth in Helv. Chem. Acta 20:373, 1937.
  • 3-Aminophenylboronic acid (3.9 g), 5 g of 5-bromo-2-fluronitrobenzene, 0.8 g of tetrakis(triphenylphosphine)-palladium and 23 mL of 2 M sodium bicarbonate solution in 50 mL of toluene under nitrogen was refluxed for 2.5 hours. The reaction was poured into 200 mL of ice water and the mixture extracted three times with 50 mL of ethyl acetate each time.
  • Trifluoroacetic anhydride (5.4 mL) was slowly added to a stirred solution of 9.7 g, of 2-fluoro-5-(3-aminophenyl)-nitrobenzene and 5.3 mL of triethylamine in 50 mL of dichloromethane at 0 °C and the mixture was stirred for an additional 20 minutes. The mixture was concentrated and the residue chromatographed on a column of silica gel eluting with 10% ethyl acetate in hexanes to give 8.6 g (65% yield) of 2-fluoro-5-(3- trifluoroacetyamidophenyl)-nitrobenzene as a pale orange oil which solidified on standing.
  • Dimethyl malonate (9.6 mL) was added dropwise to a stirred suspension of 3.2 g of 60% sodium hydride in mineral oil in 40 mL of anhydrous dimethylsulfoxide under nitrogen. The mixture was stirred for 10 minutes and 2-fluoro-5-(3- trifluoroacetamidophenyl)nitrobenzene in 20 mL of dimethylsulfoxide was added. The resulting dark red mixture was heated to 100 °C for 2 hours. The reaction was quenced by pouring into 100 mL of saturated ammonium chloride solution and extracted twice with 50 mL of ethyl acetate each time.
  • Substituted 9H-carbazole-3-carbaldehydes, dibenzothiphen-2-carboxaldehydes, and dibenzofuran-2-carboxaldehydes are prepared by conventional Vilsmeier formylation starting from commercially available substituted 9 ⁇ -carbazole, dibenzothiphen, dibenzofuran, 6,7,8,9-tetrahydro-dibenzofuran, 9H ⁇ ca rboline, 9H-2,4,9-triaza-fluorene, 9H-Pyrido[2,3-b]indole-6-carbaldehyde, 9-Thia-l,5,7-triaza-fluorene-3-carbaldehyde, and Furo[3,2-b;4,5-b ']dipyridine.
  • EXAMPLE 4 PROCEDURES FOR SYNTHESIZING THE IMIDAZOYL
  • the appropriately substituted 2-indolinone (1 equiv.), the appropriately substituted carbonylimidazole (1.2 equiv.) and piperidine (0.1 equiv.) are mixed with ethanol (1-2 mL/mmol 2-indolinone) and the mixture is then heated at 90 °C for 3 to 5 hours. After cooling, the precipitate is filtered, washed with cold ethanol and dried to yield the target compound.
  • the 2-indolinone (6.5 g) was dissolved in 25 mL of concentrated sulfuric acid and the mixture maintained at -10-15 °C while 2.1 mL of fuming nitric acid was added dropwise. After the addition of the nitric acid the reaction mixture was stirred at 0 °C for 0.5 hr and poured into ice-water. The precipitate was collected by filtration, washed with water and crystallized from 50% of acetic acid. The final crystal was then filtered, washed with water and dried under vacuum to give 6.3 g (70%) of 5-nitro-2-indolinone.
  • Step 2 Synthesis of 5-Chloroethyl-2 -indolinone (indolinone-041) 5-Chloroacetyl-2-indolinone (7.0 g) was added to 25 mL of trifluoroacetic acid and the mixture cooled in an ice bath with stirring. Triethylsilane (12.3 mL) was added drop- wise over 2 minutes. The reaction was then stirred at room temperature for 4 hours and poured into ice water. Hexane was added, the mixture stirred vigorously, and the solid collected by vacuum filtration and washed with hexane to give 5.9 g (91%) yield) of the product as a white solid.
  • Step 3 Synthesis of 5-Cyanoethyl-2-indolinone
  • Step 4 Synthesis of 5-Carboxyethyl-2-indolinone 5-Cyanoethyl-2-indolinone (4.02 g) in 10 mL of water containing 25 mL of concentrated hydrochloric acid was refluxed for 4 hours. The mixture was cooled, water added and the resulting solid collected by vacuum filtration, washed with water and dried to give 1.9 g (44%) yield) of the title compound as a yellow solid.
  • Step 2 Synthesis of 5-Aminosulfonyl-2-indolinone.
  • 5-Cholorosulfonyl-2 -indolinone (2.1 g) was added to 10 mL of ammonium hydroxide in 10 mL of ethanol and stirred at room temperature overnight. The mixture was concentrated and the solid collected by vacuum filtration to give 0.4 g (20% yield) of the title compound as an off-white solid.
  • the compounds of this invention may be synthesized using the procedure described below. Other approaches to the synthesis of the compounds and/or precursors to the compounds of this invention may become apparent to those skilled in the art based on the disclosures herein. Such alternate procedures are within the scope and spirit of this invention.
  • the exemplary syntheses which follow are not to be construed as limiting the scope of this invention in any manner.
  • Tetrakis(triphenylphosphine)palladium (0.8 g) was added to a mixture of 5 g of 3- methoxyphenylboronic acid, 5 g of 5-bromo-2-fluoronitrobenzene and 11 mL of 2 M sodium carbonate in 100 mL of toluene. The mixture was refluxed for 2 hours, diluted with water and extracted with ethyl acetate. The ethyl acetate layer was separated, washed with saturated sodium bicarbonate then with brine, dried and concentrated to give an oily solid.
  • 3'-Methoxy-3-nitrobiphenyl-4-acetic acid (5.2 g) was dissolved in methanol and hydrogenated at room temperature for 3 hours using 0.8 g of 10 % palladium on carbon as the hydrogenation catalyst. The catalyst was removed by filtration, washed with methanol and the filtrates combined and concentrated to give a brown solid. The solid was chromatographed on silica gel using ethyl acetate:hexane:acetic acid 33:66:1 as the eluent to give 3.0 g (75 % yield based on 4-fluoro-3'-methoxy-3-nitrobiphenyl) of 6-(3- methoxyphenyl)-2-oxindole as a pink solid.
  • Acetic acid 34 mL
  • 18 g 2-acetylcyclohexanone 18 g diethyl aminomalonate hydrochloride and 11.6 g sodium acetate were charged to a 250 mL three neck flask equipped with a reflux condenser and magnetic stirring.
  • the flask was placed in an oil bath and the bath temperature was raised to 130 °C over 20 minutes.
  • At 80 °C carbon dioxide began to evolve. Fifteen minutes after reaching 130 °C, gas evolution ceased.
  • the mixture was cooled in an ice bath and 68 mL water was slowly added with stirring.
  • 2-Carboxy-3-methyl-4, 5, 6, 7-tetrahydroindole (14.5 g) was placed in a 50 mL round bottom flask equipped with magnetic stirring. The flask was placed in an oil bath and heated to a bath temperature of 140-150 °C for 20 minutes. Water was seen to condense at the neck of the flask. Thin layer chromatography (dichloromethane, silica gel, Rf 0.9) showed complete conversion to product. The dark liquid was cooled, taken up in 30 mL of hexane, dried over sodium sulfate and evaporated to give 7.5 g of 3-methyl- 4,5, 6, 7-tetrahydroindole as a mixture of crystals and dark oil. To a sti ⁇ ed suspension of 5.0 g of Vilsmeier reagent in 40 mL dichloromethane at
  • Tetrakis(triphenylphosphine)palladium (0.8 g) was added to a mixture of 4.2 g 3- ethoxyphenylboronic acid, 5.0 g 5-bromo-2-fluoronitrobenzene and 22 mL 2 M sodium carbonate in 50 mL toluene and 50 mL ethanol. The mixture was refluxed for 2 hours, concentrated, water was added and the mixture was extracted twice with ethyl acetate. The ethyl acetate layer was washed with water, then with brine and then dried and concentrated.
  • the crude dimethyl 3'-ethoxy-3-nitrobiphenyl-4-malonate was added to 60 mL 6 N hydrochloric acid, the mixture was heated to 100 °C and stirred at that temperature for 4 days. The reaction mixture was then cooled to room temperature. The precipitate which formed was collected by filtration, washed with water and hexane, and dried to give 4.7 g (77 % yield based on 5-bromo-2-fluoronitrobenzene) of crude 3'-ethoxy-3-nitrobiphenyl- 4-acetic acid as a light tan solid.
  • Iron powder (2.4 g) was added in one portion to 4.6 g 3'-ethoxy-3-nitrobiphenyl-4- acetic acid in 40 mL glacial acetic acid and the mixture was refluxed for 2 hours. The reaction mixture was then concentrated to dryness, treated repeatedly with ethyl acetate and filtered to remove insoluble materials. The filtrate was washed twice with 1 N hydrochloric acid then with brine. It was then dried over anhydrous sodium sulfate and concentrated to give 3.5 g (91 % yield) of 6-(3-ethoxyphenyl)-2-oxindole as a light brown solid.
  • Tetrakis(triphenylphosphine)palladium (1 g) was added to a mixture of 5 g 4- methoxyphenylboronic acid, 6.6 g 5-bromo-2-fluoronitrobenzene and 30 mL of 2 M sodium carbonate in 50 mL toluene and 50 mL ethanol. The mixture was refluxed for 2 hours, concentrated, and the residue extracted twice with ethyl acetate. The ethyl acetate layer was washed with water and brine, dried, and concentrated to give a brown oily solid.
  • 6-(3-methoxyphenyl)-2-oxindole and 1 drop piperidine in 2 mL ethanol were heated to 90 °C and held at that temperature overnight.
  • the reaction mixture was cooled and concentrated.
  • the residue was suspended in 6 N hydrochloric acid and the precipitate which formed was filtered, washed with water until the pH of the wash was approximately 6 and then dried in a vacuum oven overnight to give 156 mg of the title compound (82%) as a brown solid.
  • Tetrakis(triphenylphosphine)palladium (1 g) was added to a mixture of 5 g 2- methoxyphenylboronic acid, 6.6 g 5-bromo-2-fluoronitrobenzene and 30 mL 2 M sodium carbonate in 50 mL toluene and 50 mL ethanol. The mixture was refluxed for 2 hours, concentrated, and the residue extracted twice with ethyl acetate. The ethyl acetate extracts were combined, washed with water and brine, dried, and concentrated to give crude 4- fluoro-2'-methoxy-3-nitrobiphenyl as a dark green oil which solidified on standing.
  • Iron powder (5 g) was added in one portion to 9.8 g 2'methoxy-3-nitrobiphenyl-4- acetic acid in 50 mL glacial acetic acid and the mixture was heated to 100 °C and stirred at that temperature for 3 hours. The reaction mixture was then concentrated to dryness, sonicated in ethyl acetate and filtered to remove insoluble materials. The filtrate was washed twice with 1 N hydrochloric acid, then with water, then with brine. The filtrate was then dried over anhydrous sodium sulfate and concentrated.
  • in vitro 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 cellular/catalytic assays described herein are performed in an ELISA format.
  • the general procedure is as follows: a compound is introduced to cells expressing the test kinase, either naturally or recombinantly, for some period of time after which, if the test kinase is a ligand known to activate the receptor's activity is added. 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 phoshorylation on the substrate is detected with an antibody specifically recognizing phoshotyrosine compared with control cells that were not contacted with a test compound.
  • the cellular/biologic assays described herein measure the amount of DNA made in response to activation of a test kinase, which is a general measure of a proliferative response.
  • the general procedure for this assay is as follows: a compound is introduced to cells expressing the test kinase, either naturally or recombinantly, for some period of time after which, if the test kinase is a receptor, a ligand known to activate the receptor's activity is added. After incubation at least overnight, a DNA labeling reagent such as Bromodeoxy-uridine (BrdU) or 3H-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.
  • a DNA labeling reagent such as Bromodeoxy-uridine (BrdU) or 3H-thymidine is added.
  • EGF Receptor kinase activity in cells genetically engineered to express human EGF-R is measured as described below:
  • EGF 201 TOYOBO, Co., Ltd.
  • Japan. b. 05-101 (UBI) (a monoclonal antibody recognizing an EGFR extracellular domain).
  • Anti-phosphotyosine antibody (anti-Ptyr) (polyclonal).
  • Detection antibody Goat anti-rabbit IgG horse radish peroxidase conjugate,
  • Citric Acid 100 mM NA 2 HPO 4 250 mM HCl, cone. 4.0 pH ABTS * 0.5 mg/mL Keep solution in dark at 4 °C until used. h. Stock reagents of:
  • Pre-coat ELISA Plate 1 Coat ELISA plates (Corning, 96 well, Cat. #25805-96) with 05-101 antibody at 0.5 ⁇ g per well in PBS, 150 ⁇ L final volume/well, and store overnight at 4 °C. Coated plates are good for up to 10 days when stored at 4 °C.
  • NIH 3T3/C7 cell line (Honegger, et al., Cell 51:199-209, 1987) can be used for this assay.
  • EGF ligand dilute stock EGF in DMEM so that upon transfer of 10 ⁇ L dilute EGF (1 :12 dilution), 25 nM final concentration is attained. *
  • HNTG* comprises: HNTG stock (2.0 mL), milli-Q H 2 0 (7.3 mL), EDTA, 100 mM, pH 7.0 (0.5 mL), Na 3 V0 4 0.5 M (0.1 mL) and Na 4 (P 2 0 7 ), 0.2 M (0.1 mL).
  • the maximal phosphotyrosine signal is determined by subtracting the value of the negative controls from the positive controls. The percent inhibition of phosphotyrosine content for extract-containing wells is then calculated, after subtraction of the negative controls. The IC 5 o values measured in the EGF receptor phosphorylation assay for the compound of formula XXIV was found to be > 100 ⁇ M.
  • the following protocol is used to measure phosphotyrosine level on IGF-1 receptor, which indicates IGF-1 receptor tyrosine kinase activity.
  • the following materials and reagents are used: a.
  • the cell line used in this assay is 3 T3 -LI /IGF-IR, a cell line genetically engineered to overexpresses IGF-1 receptor.
  • b. NIH3T3/IGF-1R is grown in an incubator with 5% C0 2 at 37 °C. The growth media is DMEM + 10% FBS (heat inactivated)+ 2 mM L-glutamine.
  • D-PBS D-PBS:
  • Blocking Buffer TBST plus 5% Milk (Carnation Instant Non-Fat Dry Milk).
  • TBST buffer 5% Milk (Carnation Instant Non-Fat Dry Milk).
  • ABTS solution should be kept in dark and 4 °C. The solution should be discarded when it turns green. o. Hydrogen Peroxide: 30% solution is kept in the dark at 4 °C.
  • All the following steps are conducted at room temperature unless it is specifically indicated. All ELISA plate washings are performed by rinsing the plate with tap water three times, followed by one TBST rinse. Pat plate dry with paper towels.
  • the cells grown in tissue culture dish (Corning 25020- 100) to 80-90% confluence, are harvested with Trypsin-EDTA (0.25%, 0.5 mL/D-100, GIBCO).
  • EXAMPLE 8 ASSAY MEASURING THE PDGF RECEPTOR KINASE ACTIVITY
  • All cell culture media, glutamine, and fetal bovine serum were purchased from Gibco Life Technologies (Grand Island, NY) unless otherwise specified. All cells were grown in a humid atmosphere of 90-95% air and 5-10% C0 2 at 37 °C. All cell lines were routinely subcultured twice a week and were negative for mycoplasma as determined by the Mycotect method (Gibco).
  • cells (U1242, obtained from Joseph schlessinger, NYU) were grown to 80-90% confluency in growth medium (MEM with 10% FBS, NEAA, 1 mM NaPyr and 2 mM GLN) and seeded in 96-well tissue culture plates in 0.5% serum at 25,000 to 30,000 cells per well. After overnight incubation in 0.5% serum-containing medium cells were changed to serum-free medium and treated with test compound for 2 hr in a 5% C0 2 , 37 °C incubator.
  • Cells were then stimulated with ligand for 5-10 minute followed by lysis with HNTG (20 mM Hepes, 150 mMNaCl, 10% glycerol, 5 mM EDTA, 5 mM Na 3 V0 4 , 0.2% Triton X-100, and 2 mM NaPyr).
  • HNTG 20 mM Hepes, 150 mMNaCl, 10% glycerol, 5 mM EDTA, 5 mM Na 3 V0 4 , 0.2% Triton X-100, and 2 mM NaPyr.
  • Cell lysates (0.5 mg/well in PBS) were transferred to ELISA plates previously coated with receptor-specific antibody and which had been blocked with 5% milk in TBST (50 mM Tris-HCl ph 7.2, 150 mM NaCl and 0.1% Triton X-100) at room temperature for 30 min. Lysates were incubated with shaking for 1 hour at room temperature.
  • the plates were washed with TBST four times and then incubated with polyclonal anti-phosphotyrosine antibody at room temperature for 30 minutes. Excess anti-phosphotyrosine antibody was removed by rinsing the plate with TBST four times. Goat anti-rabbit IgG antibody was added to the ELISA plate for 30 min at room temperature followed by rinsing with TBST four more times.
  • ABTS 100 mM citric acid, 250 mM Na 2 HP0 4 and 0.5 mg/mL 2,2'-azino-bis(3-ethybenzthiazoline-6- sulfonic acid) plus H 2 0 2 (1.2 mL 30% H 2 0 2 to 10 mL ABTS) was added to the ELISA plates to start color development. Absorbance at 410 nm with a reference wavelength of 630 nm was recorded about 15 to 30 min after ABTS addition.
  • the IC 5 o value measured in the PDGF receptor phosphorylation assay for the compound of formula XXIV (Example 6) was found to be 49.29 ⁇ M.
  • HER2 kinase activity in whole EGFR-NIH3T3 cells was measured as described below.
  • b. 05-101 (UBI) a monoclonal antibody recognizing an EGFR extracellular domain).
  • Anti-phoshotyrosine antibody anti-Ptyr
  • Detection antibody Goat anti-rabbit IgG horse radish peroxidase conjugate, TAGO, Inc., Burlingame, CA.
  • TEST buffer
  • Citric Acid 100 mM Na 2 HP0 4 250 mM HCl, cone. 0. 5 pM ABTS* 0.5mg/mL
  • Coated plates are good for up to 10 days when stored at 4 °C.
  • An NIH3T3 cell line overexpressing a chimeric receptor containing the EGFR extracellular domain and intracellular HER2 kinase domain can be used for this assay.
  • DMEM seeding medium
  • seeding medium 0.5% bovine serum
  • seed cells in DMEM medium (0.5% bovine serum) at a density of 10,000 cells per well, 100 ⁇ L per well, in a 96 well microtiter plate.
  • EGF ligand dilute stock EGF in DMEM so that upon transfer of 10 41 dilute EC37 :12 dilution), 100 nM final concentration is attained. 3. Prepare fresh HNTG sufficient for 100 ⁇ L per well; and place on ice.
  • HNTG stock 2.0 mL milli-Q H 2 O 7.3 mL

Abstract

L'invention concerne certains composés à base d'indolinone et de pyrazolylamide, leur méthode de synthèse, et des banques combinatoires constituées de ces composés. L'invention concerne également des méthodes utilisant ces composés qui permettent de moduler la fonction de protéines kinases, ainsi que des méthodes de traitement de maladies consistant à moduler la fonction des protéines kinases et les voies de transduction du signal associées.
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WO1999048868A3 (fr) 2000-02-24
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CA2325935A1 (fr) 1999-09-30
AU3363599A (en) 1999-10-18

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