US20010007033A1 - Novel 3-(substituted)-2-indolinones compounds and use thereof as inhibitors of protein kinase activity - Google Patents

Novel 3-(substituted)-2-indolinones compounds and use thereof as inhibitors of protein kinase activity Download PDF

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US20010007033A1
US20010007033A1 US09/516,948 US51694800A US2001007033A1 US 20010007033 A1 US20010007033 A1 US 20010007033A1 US 51694800 A US51694800 A US 51694800A US 2001007033 A1 US2001007033 A1 US 2001007033A1
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Prior art keywords
indolinone
group
dibromo
methyl
methylidenyl
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US09/516,948
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Peng Tang
Li Sun
Gerald McMahon
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Sugen LLC
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Sugen LLC
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Assigned to SUGEN, INC. reassignment SUGEN, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: MCMAHON, GERALD, SUN, LI, TANG, PENG CHO
Publication of US20010007033A1 publication Critical patent/US20010007033A1/en
Priority to US09/916,331 priority patent/US6506763B2/en
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    • 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

Definitions

  • the present invention relates generally to organic chemistry, biochemistry, pharmacology and medicine. More particularly, it relates to novel 3-(substituted)-2-indolinone compounds, and their physiologically acceptable salts and prodrugs, which modulate the activity of protein kinases (“PKs”) and, therefore, are expected to exhibit a salutary effect against a range of disorders related to abnormal PK activity.
  • PKs protein kinases
  • PKs are enzymes that catalyze the phosphorylation of hydroxy groups on tyrosine, serine and threonine residues of proteins.
  • the consequences of this seemingly simple activity are staggering; cell growth, differentiation and proliferation; i.e., virtually all aspects of cell life, in one way or another depend on PK activity.
  • abnormal PK activity has been related to a host of disorders, ranging from relatively non life threatening diseases such as psoriasis to extremely virulent diseases such as glioblastoma (brain cancer).
  • the PKs can conveniently be broken down into two classes, the protein tyrosine kinases (PTKs) and the serine-threonine kinases (STKs).
  • PTKs protein tyrosine kinases
  • STKs serine-threonine kinases
  • PK activity is involvement with growth factor receptors.
  • Growth factor receptors are cell-surface proteins. When bound by a growth factor ligand, a growth factor receptor is converted to an active form which interacts with proteins on the inner surface of a cell membrane. This leads to phosphorylation on tyrosine residues of the receptor as well as other proteins and to the formation inside the cell of complexes with a variety of cytoplasmic signaling molecules. These complexes, in turn, affect numerous cellular responses such as cell division (proliferation), cell differentiation, cell growth, expression of metabolic effects on the extracellular microenvironment, etc.
  • Schlessinger and Ullrich Neuron, 9:303-391 (1992) which is incorporated by reference, including any drawings, as if fully set forth herein.
  • RTKs receptor tyrosine kinases
  • RTK subfamily consists of insulin receptor (IR), insulin-like growth factor I receptor (IGF-1R) and insulin receptor related receptor (IRR).
  • IR and IGF-1R interact with insulin, IGF-I and IGF-II to form a heterotetramer composed of two entirely extracellular glycosylated ⁇ subunits and two ⁇ subunits which cross the cell membrane and which contain the tyrosine kinase domain.
  • a third RTK subfamily is referred to as the platelet derived growth factor receptor (“PDGFR”) group, which includes PDGFR ⁇ , PDGFR ⁇ , CSFIR, c-kit and c-fms. These receptors consist of glycosylated extracellular domains composed of variable numbers of immunoglobin-like loops and an intracellular domain wherein the tyrosine kinase domain is interrupted by unrelated amino acid sequences.
  • PDGFR platelet derived growth factor receptor
  • flk fetus liver kinase
  • KDR/FLK-1 kinase insert domain-receptor fetal liver kinase-1
  • flk-1R fetal liver kinase-1
  • flt-4 fms-like tyrosine kinase 1
  • FGF fibroblast growth factor
  • This group consists of four receptors, FGFR1-4, and seven ligands, FGF1-7. While not yet well characterized, it appears that the receptors consist of a glycosylated extracellular domain containing a variable number of immunoglobin-like loops and an intracellular domain in which the PTK sequence is interrupted by regions of unrelated amino acid sequences.
  • CTK non-receptor tyrosine kinases
  • cellular tyrosine kinases cellular tyrosine kinases
  • the Src subfamily appear so far to be the largest group of CTKs and includes Src, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk.
  • Src Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk.
  • STKs serine-threonine kinases
  • CTKs serine-threonine kinases
  • STKs are the most common of the cytosolic kinases; i.e., kinases which perform their function in that part of the cytoplasm other than the cytoplasmic organelles and cytoskelton.
  • the cytosol is the region within the cell where much of the cell's intermediary metabolic and biosynthetic activity occurs; e.g., it is in the cytosol that proteins are synthesized on ribosomes.
  • RTKs, CTKs and STKs have all been implicated in a host of pathogenic conditions including, significantly, cancer.
  • Other pathogenic conditions which have been associated with PTKs include, without limitation, psoriasis, hepatic cirrhosis, diabetes, atherosclerosis, angiogenesis, restenosis, ocular diseases, rheumatoid arthritis and other inflammatory disorders, autoimmune disease and a variety of renal disorders.
  • PK regulated functions known to be PK regulated. That is, it has been suggested that malignant cell growth results from a breakdown in the mechanisms that control cell division and/or differentiation. It has been shown that the protein products of a number of proto-oncogenes are involved in the signal transduction pathways that regulate cell growth and differentiation. These protein products of proto-oncogenes include the extracellular growth factors, transmembrane growth factor PTK receptors (RTKs), cytoplasmic PTKs (CTKs) and cytosolic STKs, discussed above.
  • RTKs transmembrane growth factor PTK receptors
  • CTKs cytoplasmic PTKs
  • STKs cytosolic STKs
  • RNA ligands (Jelinek, et al., Biochemistry, 33:10450-56); Takano, et al., Mol. Bio. Cell 4:358A (1993); Kinsella, et al., Exp. Cell Res. 199:56-62 (1992); Wright, et al., J. Cellular Phys., 152:448-57) and tyrosine kinase inhibitors (WO 94/03427; WO 92/21660; WO 91/15495; WO 94/14808; U.S. Pat. No. 5,330,992; Mariani, et al., Proc. Am. Assoc. Cancer Res., 35:2268 (1994)).
  • PKs are enzymes that catalyze the phosphorylation of hydroxy groups on tyrosine, serine and threonine residues of proteins.
  • the consequences of this seemingly simple activity are staggering; cell growth, differentiation and proliferation; i.e., virtually all aspects of cell life, in one way or another depend on PK activity.
  • abnormal PK activity has been related to a host of disorders, ranging from relatively non life threatening diseases such as psoriasis to extremely virulent diseases such as glioblastoma (brain cancer).
  • the PKs can conveniently be broken down into two classes, the protein tyrosine kinases (PTKs) and the serine-threonine kinases (STKs).
  • PTKs protein tyrosine kinases
  • STKs serine-threonine kinases
  • PK activity is involvement with growth factor receptors.
  • Growth factor receptors are cell-surface proteins. When bound by a growth factor ligand, a growth factor receptor is converted to an active form which interacts with proteins on the inner surface of a cell membrane. This leads to phosphorylation on tyrosine residues of the receptor as well as other proteins and to the formation inside the cell of complexes with a variety of cytoplasmic signaling molecules. These complexes, in turn, affect numerous cellular responses such as cell division (proliferation), cell differentiation, cell growth, expression of metabolic effects on the extracellular microenvironment, etc.
  • Schlessinger and Ullrich Neuron, 9:303-391 (1992) which is incorporated by reference, including any drawings, as if fully set forth herein.
  • RTKs receptor tyrosine kinases
  • HER receptor tyrosine kinases
  • RTK subfamily consists of insulin receptor (IR), insulin-like growth factor I receptor (IGF-1R) and insulin receptor related receptor (IRR).
  • IR and IGF-1R interact with insulin, IGF-I and IGF-II to form a heterotetramer composed of two entirely extracellular glycosylated ⁇ subunits and two ⁇ subunits which cross the cell membrane and which contain the tyrosine kinase domain.
  • a third RTK subfamily is referred to as the platelet derived growth factor receptor (“PDGFR”) group, which includes PDGFR ⁇ , PDGFR ⁇ , CSFIR, c-kit and c-fms. These receptors consist of glycosylated extracellular domains composed of variable numbers of immunoglobin-like loops and an intracellular domain wherein the tyrosine kinase domain is interrupted by unrelated amino acid sequences.
  • PDGFR platelet derived growth factor receptor
  • flk fetus liver kinase
  • KDR/FLK-1 kinase insert domain-receptor fetal liver kinase-1
  • flk-1R fetal liver kinase-1
  • flt-4 fms-like tyrosine kinase 1
  • FGF fibroblast growth factor
  • This group consists of four receptors, FGFR1-4, and seven ligands, FGF1-7. While not yet well characterized, it appears that the receptors consist of a glycosylated extracellular domain containing a variable number of immunoglobin-like loops and an intracellular domain in which the PTK sequence is interrupted by regions of unrelated amino acid sequences.
  • CTK non-receptor tyrosine kinases
  • cellular tyrosine kinases cellular tyrosine kinases
  • the Src subfamily appear so far to be the largest group of CTKs and includes Src, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk.
  • Src Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk.
  • STKs serine-threonine kinases
  • CTKs cytoplasmic kinases
  • STKs are the most common of the cytosolic kinases; i.e., kinases which perform their function in that part of the cytoplasm other than the cytoplasmic organelles and cytoskelton.
  • the cytosol is the region within the cell where much of the cell's intermediary metabolic and biosynthetic activity occurs; e.g., it is in the cytosol that proteins are synthesized on ribosomes.
  • RTKs, CTKs and STKs have all been implicated in a host of pathogenic conditions including, significantly, cancer.
  • Other pathogenic conditions which have been associated with PTKs include, without limitation, psoriasis, hepatic cirrhosis, diabetes, atherosclerosis, angiogenesis, restenosis, ocular diseases, rheumatoid arthritis and other inflammatory disorders, autoimmune disease and a variety of renal disorders.
  • PK regulated functions known to be PK regulated. That is, it has been suggested that malignant cell growth results from a breakdown in the mechanisms that control cell division and/or differentiation. It has been shown that the protein products of a number of proto-oncogenes are involved in the signal transduction pathways that regulate cell growth and differentiation. These protein products of proto-oncogenes include the extracellular growth factors, transmembrane growth factor PTK receptors (RTKs), cytoplasmic PTKs (CTKs) and cytosolic STKs, discussed above.
  • RTKs transmembrane growth factor PTK receptors
  • CTKs cytoplasmic PTKs
  • STKs cytosolic STKs
  • RNA ligands (Jelinek, et al., Biochemistry, 33:10450-56); Takano, et al., Mol. Bio. Cell 4:358A (1993); Kinsella, et al., Exp. Cell Res. 199:56-62 (1992); Wright, et al., J. Cellular Phys., 152:448-57) and tyrosine kinase inhibitors (WO 94/03427; WO 92/21660; WO 91/15495; WO 94/14808; U.S. Pat. No. 5,330,992; Mariani, et al., Proc. Am. Assoc. Cancer Res., 35:2268 (1994)).
  • the present invention relates generally to novel 3-(substituted)-2-indolinones which modulate the activity of both receptor (RTK) and non-receptor (CTK and STK) protein kinases (PKs).
  • PKs protein kinases
  • the present invention relates to the preparation and use of pharmacological compositions of the disclosed compounds and their physiologically acceptable salts and prodrugs in the treatment or prevention of PK driven disorders such as, by way of example and not limitation, cancer, diabetes, hepatic cirrhosis, atherosclerosis, angiogenesis and renal disease.
  • a “3-(substituted)-2-indolinone” refers to a chemical compound having the general structure shown in Formula 1, infra.
  • a “pharmacological composition” refers to a mixture of one or more of the compounds described herein, or physiologically acceptable salts thereof, with other chemical components, such as physiologically acceptable carriers and/or excipients.
  • the purpose of a pharmacological composition is to facilitate administration of a compound to an organism.
  • a “physiologically acceptable carrier” refers to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
  • excipient refers to an inert substance added to a pharmacological composition to further facilitate administration of a compound.
  • excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
  • a “prodrug” refers to an agent which is converted into the parent drug in vivo. Prodrugs are often useful because, in some situations, they may be easier to administer than the parent drug. They may, for instance, be bioavailable by oral administration whereas the parent drug is not. The prodrug may also have improved solubility in pharmacological 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 then is metabolically hydrolyzed to the carboxylic acid, the active entity, once inside the cell where water solubility is beneficial.
  • Table 1 is a list of aldehydes used in the synthesis of exemplary compounds of this invention.
  • Table 2 is a list of the oxindoles used in the synthesis of exemplary compounds of this invention.
  • Table 3 is a list of the 3-(substituted)-2-indolinone products obtained by the reaction of the aldehydes of Table 1 with the oxindoles of Table 2.
  • Table 4 shows the results of biochemical testing of the compounds of Table 3. The procedures for carrying out each of the indicated assays is set forth in detail below.
  • the present invention relates to 3-(substituted)-2-indolinones having the chemical structure shown in Formula 1:
  • 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 multi-dimensional 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.
  • 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 combinatorial library is formed by the reaction of the aldehydes shown in Table 1 with the oxindoles shown in Table 2.
  • the concept and practice of combinatorial library formation is well known to those of ordinary skill in the art.
  • the result of the combinatorial library reaction of the aldehydes of Table 1 with the oxindoles of Table 2 are the 3-(substituted)-2-indolinones shown in Table 3, which compounds are the subject of this invention.
  • R 1 , R 2 , R 3 and R 4 are independently selected from the group consisting of hydrogen, alkyl, halo and S-sulfonamido.
  • TABLE 1 STRUCTURE NAME a-tolualdehyde 3,4-difluorobenzaldehyde 3,5-difluorobenzaldehyde ⁇ , ⁇ , ⁇ -trifluoro-m-tolualdehyde 3,5-bis(trifluoromethyl)-benzaldehyde 2-cyanobenzaldehyde 2,5-difluorobenzaldehyde 1-naphthaldehyde 2,4-dichlorobenzaldehyde 4-biphenylcarboxaldehyde 4-chlorobenzaldehyde ⁇ , ⁇ , ⁇ -trifluoro-o-benzaldehyde m-tolualdehyde p-tolualdehyde 3-chlorobenzaldehyde 3-fluor
  • A is a ring selected from the group consisting of benzene, tetrahydrobenzene, naphthalene, tetrahydro-naphthalene, furan, tetrahydrofuran, thiophene, pyrrole, pyrazole, pyrrolidine, imidazole, pyridine, quinoline, indole, tetrahydroindole, isatin. chromone, fluorene, carbazole, benzo[b]furan, thieno[b]thiophene and uracil.
  • the “A” ring is substituted with one or more groups 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 —
  • a combinatorial library may be used to screen compounds of this invention for a desired activity.
  • verified activity is meant the ability to modulate the catalytic activity of a selected protein kinase.
  • screening is meant to contact an entire combinatorial library of compounds or any portion thereof with one or more target protein kinases and observe the effect of the compounds on the catalytic activity of the protein kinase.
  • a further aspect of this invention is a combinatorial library of at least 10, preferably at least 100, more preferably at least 1000 wherein the oxindole is selected from the group consisting of 5,7-dibromooxindole, 5-iodooxindole, 5-bromo-4-methyloxindole, 5-methylaminosulfonyloxindole, 5-(4-trifluoromethylanilinosulfonyl)oxindole, 5-(morpholin-4-yl)-sulfonyloxindole, 5-(2-chloroethyl)oxindindole and 5,7-dibromo-4-methyloxindole and the aldehyde is selected from the group consisting of unsubstituted and substituted benzaldehyde, 1-naphthaldehyde, 2-naphthaldehyde, 2-formyl-5,6,7,8-tetrahydronaphthalene, 3-furaldeh
  • a 3-(substituted)-2-indolinone may be made by testing the combinatorial library disclosed herein in the biological assays provided below, selecting one or more of the 3-(substituted)-2-indolinones which exhibit activity against a PK in the biological assays and then synthesizing those compounds.
  • test compound from the combinatorial library has a modulating effect on the specific PK which is the focus of the particular assay, when compared to an control not containing the test compound.
  • Yet another aspect of this invention is a compound which modulates protein kinase activity, in particular RTK, CTK or STK kinase catalytic activity.
  • alkyl refers to a saturated aliphatic hydrocarbon including straight chain and branched chain groups.
  • the alkyl group has 1 to 20 carbon atoms (whenever it appears herein, a numerical range such as “1 to 20” refers to each integer in the given range; e.g., “1 to 20 carbon atoms” means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc. up to and including 20 carbon atoms). More preferably, it is a medium size alkyl having 1 to 10 carbon atoms. Most preferably, it is a lower alkyl having 1 to 4 carbon atoms.
  • the alkyl group may be substituted or unsubstituted.
  • the substituent group(s) is preferably one or more individually selected from cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, halo, carbonyl, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, C-carboxy, C-carboxy salt, O-carboxy, carboxyalkyl, carboxyalkyl salt, cyanato, isocyanato, thiocyanato, isothiocyanato, nitro, silyl, amino and —NR 5 R 6 , R 5 and R 6 being as defined above.
  • 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 of 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, adamantane, 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, C-carboxy, C-carboxy salt, O-carboxy, carboxyalkyl, carboxyalkyl salt, O-carbamyl, N-carbamyl, C-amido, N-amido, nitro, amino and —NR 5 R 6 , with R 5 and R 6 being as defined above.
  • 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 group refers to an alkyl group, as defined herein, consisting of at least two carbon atoms and at least one carbon-carbon triple bond.
  • aryl group refers to an all-carbon monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups having a completely conjugated pi-electron system. Examples, without limitation, of aryl groups are phenyl, naphthalenyl and anthracenyl. The aryl group may be substituted or unsubstituted.
  • the substituted group(s) is preferably one or more selected from halo, trihalomethyl, alkyl, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, carbonyl, thiocarbonyl, C-carboxy, C-carboxy salt, O-carboxy, carboxyalkyl, carboxyalkyl salt, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, sulfinyl, sulfonyl, S-sulfonamido, N-sulfonamido, trihalomethanesulfonamido, amino and —NR 5 R 6 , R 5 and R 6 being as defined above.
  • heteroaryl group refers to a monocyclic or fused ring (i.e., rings which share an adjacent pair of atoms) group having in the ring(s) one or more atoms selected from the group consisting of nitrogen, oxygen and sulfur and, in addition, having a completely conjugated pi-electron system.
  • heteroaryl groups are pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline, purine and carbazole.
  • the heteroaryl group may be substituted or unsubstituted.
  • the substituted group(s) is preferably one or more selected from alkyl, cycloalkyl, halo, trihalomethyl, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, carbonyl, thiocarbonyl, sulfonamido, C-carboxy, C-carboxy salt, O-carboxy, carboxyalkyl, carboxyalkyl salt, sulfinyl, sulfonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, amino and —NR 5 R 6 , R 5 and R 6 being defined above.
  • a “heteroalicyclic” group refers to a monocyclic or fused ring group having in the ring(s) one or more atoms selected from the group consisting of nitrogen, oxygen and sulfur.
  • the rings may also have one or more double bonds. However, the rings do not have a completely conjugated pi-electron system.
  • the heteroalicyclic ring may be substituted or unsubstituted.
  • the substituted group(s) is preferably one or more selected from alkyl, cycloaklyl, halo, trihalomethyl, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, carbonyl, thiocarbonyl, C-carboxy, C-carboxy salt, O-carboxy, carboxyalkyl, carboxyalkyl salt, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, sulfinyl, sulfonyl, C-amido, N-amido, amino and —NR 5 R 6 , with R 5 and R 6 being as defined above.
  • a “hydroxy” group refers to an —OH group.
  • alkoxy refers to both an —O-alkyl and an —O-cycloalkyl group, as defined herein.
  • aryloxy refers to both an —O-aryl and an —O-heteroaryl group, as defined herein.
  • a “thiohydroxy” group refers to an —SH group.
  • a “thioalkoxy” group refers to both an S-alkyl and an —S-cycloalkyl group, as defined herein.
  • a “thioaryloxy” group refers to both an —S-aryl and an —S-heteroaryl group, as defined herein.
  • a “carbonyl” group refers to a —C( ⁇ O)—R′′ group, where R′′ is selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) and heteroalicyclic (bonded through a ring carbon), as defined herein.
  • aldehyde refers to a carbonyl group where R′′ is hydrogen.
  • a “thiocarbonyl” group refers to a —C( ⁇ S)—R′′ group, with R′′ as defined herein.
  • An “O-carboxy” group refers to a R′′C( ⁇ O)O— group, with R′′ as defined herein.
  • a “C-carboxy” group refers to a —C( ⁇ O)OR′′ groups with R′′ as defined herein.
  • a “C-carboxy salt” refers to a —C( ⁇ O)O ⁇ M + group wherein M + is selected from the group consisting of lithium, sodium, magnesium, calcium, potassium, barium, iron, zinc and quaternary ammonium.
  • acetyl refers to a —C( ⁇ O)CH 3 group.
  • a “trifluoroacetyl” group refers to a —C( ⁇ O)CF 3 group.
  • An “carboxyalkyl” group refers to —(CH 2 ) r C( ⁇ O)OR′′ wherein r is 1-6 and R′′ is as defined above.
  • An “carboxyalkyl salt” refers to a —(CH 2 ) r C( ⁇ O)O ⁇ M + wherein M + is selected from the group consisting of lithium, sodium, potassium, calcium, magnesium, barium, iron, zinc and quaternary ammonium.
  • a “carboxylic acid” group refers to a C-carboxy group in which R′′ is hydrogen.
  • an “ester” is a C-carboxy group, as defined herein, wherein R′′ is any of the listed groups other than hydrogen.
  • a “halo” group refers to fluorine, chlorine, bromine or iodine.
  • a “trihalomethyl” group refers to a —CX 3 group wherein X is a halo group as defined herein.
  • a “trihalomethanesulfonyl” group refers to a X 3 CS( ⁇ O) 2 — groups with X as defined above.
  • An “azido” group refers to a —N ⁇ N group.
  • a “cyano” group refers to a —C ⁇ N group.
  • a “cyanato” group refers to a —CNO group.
  • An “isocyanato” group refers to a —NCO group.
  • a “thiocyanato” group refers to a —CNS group.
  • An “isothiocyanato” group refers to a —NCS group.
  • a “sulfinyl” group refers to a —S( ⁇ O)—R′′ group, with R′′ as defined herein.
  • a “sulfonyl” group refers to a —S( ⁇ O) 2 R′′ group, with R′′ as defined herein.
  • a “sulfonamido” group refers to a —S( ⁇ O) 2 —NR 5 R 6 , with R 5 and R 6 as defined herein.
  • a “trihalomethanesulfonamido” group refers to a X 3 CS( ⁇ O) 2 NR 5 — group with X and R 5 as defined herein.
  • An “O-carbamyl” group refers to a —OC( ⁇ O)—NR 5 R 6 group with R 5 and R 6 as defined herein.
  • N-carbamyl refers to an R 5 OC( ⁇ O)NR 6 — group, with R 5 and R 6 as defined herein.
  • An “O-thiocarbamyl” group refers to a —OC( ⁇ S)—NR 5 R 6 group with R 5 and R 6 as defined herein.
  • N-thiocarbamyl refers to a R 5 OC( ⁇ S)NR 6 — group, with R 5 and R 6 as defined herein.
  • amino refers to an —NH 2 group.
  • a “C-amido” group refers to a —C( ⁇ O)—NR 5 R 6 group with R 5 and R 6 as defined herein.
  • N-amido refers to a R 5 C( ⁇ O)NR 6 — group, with R 5 and R 6 as defined herein.
  • a “nitro” group refers to a —NO 2 group.
  • a “quaternary ammonium” group refers to a — + NR 5 R 6 R 7 group wherein R 5 , R 6 and R 7 are independently selected from the group consisting of hydrogen and unsubstituted lower alkyl.
  • a “methylenedioxy” group refers to a —OCH 2 O— group wherein the oxygen atoms are bonded to adjacent ring carbon atoms.
  • An “ethylenedioxy” group refers to a —OCH 2 CH 2 O— group wherein the oxygen atoms are bonded to adjacent ring carbon atoms.
  • the substituents on the “A” ring are independently selected from the group consisting of hydrogen; unsubstituted lower alkyl; lower alkyl substituted with unsubstituted aryl, halo, hydroxy, unsubstituted lower alkyl C-carboxy and —NR 5 R 6 ; unsubstituted lower alkoxy; lower alkoxy substituted with a group selected from the group consisting of C-carboxy, —NR 5 R 6 , and halo; halo; nitro; cyano; hydroxy; acetyl; trihaloacetyl; trihalomethyl; unsubstituted aryl; aryl substituted with one or more groups selected from the group consisting of halo, hydroxy, unsubstituted lower alkyl, unsubstituted lower alkoxy, hydroxy and nitro; unsubstituted lower aryloxy; aryl
  • R 5 and R 6 are independently selected from the group consisting of hydrogen and unsubstituted lower alkyl.
  • aldehyde to be reacted with an oxindole of this invention is selected from the group consisting of:
  • the chemical formulae referred to herein may exhibit the phenomena of tautomerism and structural isomerism.
  • the compounds described herein may adopt a cis or trans configuration about the double bond connecting the 2-indolinone moiety to the “A” moiety or the compounds may be a mixture of cis and trans.
  • This invention encompasses any tautomeric or structural isomeric form and mixtures thereof which possess the ability to modulate RTK and/or CTK activity and is not limited to any one tautomeric or structural isomeric form.
  • the term “cis” refers to the structural configuration wherein the “A” group is on the same side of the double bond connecting it to the 2-indolinone ring as the 2-oxygen group of the 2-indolinone.
  • trans refers to the structural configuration wherein the “A” group is on the opposite side of the double bond connecting it to the 2-indolinone ring from the 2-oxygen group of the 2-indolinone.
  • this invention relates to a method for the modulation of the catalytic activity of PKs by contacting a PK with a compound of this invention or a physiologically acceptable salt or prodrug thereof.
  • a further embodiment of this invention is a method for identifying a compound which modulates the activity of a PK which method consists of contacting a cell which expresses the PK of interest with a compound and monitoring the effect of the compound on the cell.
  • PK RTKs, CTKs and STKs; i.e., the modulation of RTK, CTK and STK catalyzed signaling processes are contemplated by this invention.
  • method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by, practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • modulation refers to the alteration of the catalytic activity of RTKs, CTKs and STKs.
  • modulating refers to the activation of the catalytic activity of RTKs, CTKs and STKs, preferably the activation or inhibition of the catalytic activity of RTKs, CTKs and STKs, depending on the concentration of the compound or salt to which the RTK, CTK or STK is exposed or, more preferably, the inhibition of the catalytic activity of RTKs, CTKs and STKs.
  • catalytic activity refers to the rate of phosphorylation of tyrosine under the influence, direct or indirect, of RTKs and/or CTKs or the phosphorylation of serine and threonine under the influence, direct or indirect, of STKs.
  • contacting refers to bringing a compound of this invention and a target PK together in such a manner that the compound can affect the catalytic activity of the PK, either directly; i.e., by interacting with the kinase itself, or indirectly; i.e., by interacting with another molecule on which the catalytic activity of the kinase is dependent.
  • Such “contacting” can be accomplished in a test tube, a petri dish or the like. In a test tube, contacting may involve only a compound and a PK of interest or it may involve whole cells. Cells may also be maintained or grown in cell culture dishes and contacted with a compound in that environment.
  • the ability of a particular compound to affect a PK related disorder i.e., the IC 50 of the compound, defined below, can be determined before use of the compounds in vivo with more complex living organisms is attempted.
  • IC 50 of the compound defined below
  • cells outside the organism multiple methods exist, and are well-known to those skilled in the art, to get the PKs in contact with the compounds including, but not limited to, direct cell microinjection and numerous transmembrane carrier techniques.
  • monitoring is meant observing or detecting the effect of contacting a compound with a cell expressing a particular PK.
  • the observed or detected effect can be a change in cell phenotype, in the catalytic activity of a PK or a change in the interaction of a PK with a natural binding partner.
  • Techniques for observing or detecting such effects are well-known in the art.
  • Cell phenotype refers to the outward appearance of a cell or tissue or the biological function of the cell or tissue. Examples, without limitation, of a cell phenotype are cell size, cell growth, cell proliferation, cell differentiation, cell survival, apoptosis, and nutrient uptake and use. Such phenotypic characteristics are measurable by techniques well-known in the art.
  • a “natural binding partner” refers to a polypeptide that binds to a particular PK in a cell. Natural binding partners can play a role in propagating a signal in a PK-mediated signal transduction process. A change in the interaction of the natural binding partner with the PK can manifest itself as an increased or decreased concentration of the PK/natural binding partner complex and, as a result, in an observable change in the ability of the PK to mediate signal transduction.
  • RTK mediated signal transduction is initiated by extracellular interaction with a specific growth factor (ligand), followed by receptor dimerization, transient stimulation of the intrinsic protein tyrosine kinase activity and phosphorylation. Binding sites are thereby created for intracellular signal transduction molecules and lead to the formation of complexes with a spectrum of cytoplasmic signaling molecules that facilitate the appropriate cellular response (e.g., cell division, metabolic effects on the extracellular microenvironment, etc.). See, Schlessinger and Ullrich, 1992 , Neuron 9:303-391.
  • each RTK is determined not only by its pattern of expression and ligand availability but also by the array of downstream signal transduction pathways that are activated by a particular receptor.
  • phosphorylation provides an important regulatory step which determines the selectivity of signaling pathways recruited by specific growth factor receptors, as well as differentiation factor receptors.
  • STKs being primarily cytosolic, affect the internal biochemistry of the cell, often as a down-line response to a PTK event. STKs have been implicated in the signaling process which initiates DNA synthesis and subsequent mitosis leading to cell proliferation.
  • PK signal transduction results in, among other responses, cell proliferation, differentiation, growth and metabolism.
  • Abnormal cell proliferation may result in a wide array of disorders and diseases, including the development of neoplasia such as carcinoma, sarcoma, glioblastoma and hemangioma, disorders such as leukemia, psoriasis, arteriosclerosis, arthritis and diabetic retinopathy and other disorders related to uncontrolled angiogenesis and/or vasculogenesis.
  • PKs typically possess a bi-lobate structure wherein ATP appears to bind in the cleft between the two lobes in a region where the amino acids are conserved among PKs.
  • Inhibitors of PKs are believed to bind by non-covalent interactions such as hydrogen bonding, van der Waals forces and ionic interactions in the same general region where the aforesaid ATP binds to the PKs.
  • the 2-indolinone component of the compounds of this invention binds in the general space normally occupied by the adenine ring of ATP. Specificity of a particular molecule for a particular PK may then arise as the result of additional interactions between the various substituents on the 2-indolinone core and the amino acid domains specific to particular PKs. Thus, different indolinone substituents may contribute to preferential binding to particular PKs.
  • the ability to select compounds active at different ATP (or other nucleotide) binding sites makes the compounds of this invention useful for targeting any protein with such a site; i.e., not only PKs but protein phosphatases as well.
  • the compounds disclosed herein may thus have utility as in vitro assays for such proteins as well as exhibiting in vivo therapeutic effects through interaction with such proteins.
  • the protein kinase is a protein tyrosine kinase, more particularly, a receptor protein tyrosine kinase.
  • receptor protein tyrosine kinases whose catalytic activity can be modulated with a compound of this invention, or salt thereof, are, without limitation, EGF, HER2, HER3, HER4, IR, IGF-1R, IRR, PDGFR ⁇ , PDGFR ⁇ , CSFIR, C-Kit, C-fms, Flk-1R, Flk4, KDR/Flk-1, Flt-1, FGFR-1R, FGFR-2R, FGFR-3R and FGFR-4R.
  • the protein tyrosine kinase whose catalytic activity is modulated by contact with a compound of this invention, or a salt or a prodrug thereof, can also be a non-receptor or cellular protein tyrosine kinase (CTK).
  • CTKs such as, without limitation, Src, Frk, Btk, Csk, Abl, ZAP70, Fes, Fps, Fak, Jak, Ack, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk, may be modulated by contact with a compound or salt of this invention.
  • Still another group of PKs which may have their catalytic activity modulated by contact with a compound of this invention are the serine-threonine protein kinases such as, without limitation, CDK2 and Raf.
  • this invention relates to a method for treating or preventing a PK related disorder by administering a therapeutically effective amount of a compound of this invention, or a salt or a prodrug thereof, to an organism.
  • a pharmacological composition containing a compound of this invention or a salt or prodrug thereof is administered to an organism for the purpose of preventing or treating a PK related disorder.
  • PK related disorder As used herein, “PK related disorder,” “PK driven disorder,” and “abnormal PK activity” all refer to a condition characterized by inappropriate; i.e., under or, more commonly, over, PK catalytic activity, where the particular PK can be an RTK, a CTK or an STK. Inappropriate catalytic activity can arise as the result of either: (1) PK expression in cells which normally do not express PKs; (2) increased PK expression leading to unwanted cell proliferation, differentiation and/or growth; or, (3) decreased PK expression leading to unwanted reductions in cell proliferation, differentiation and/or growth.
  • Over-activity of a PK refers to either amplification of the gene encoding a particular PK or production of a level of PK activity which can correlate with a cell proliferation, differentiation and/or growth disorder (that is, as the level of the PK increases, the severity of one or more of the symptoms of the cellular disorder increases). Under-activity is, of course, the converse, wherein the severity of one or more symptoms of a cellular disorder increase as the level of the PK activity decreases.
  • the terms “prevent”, “preventing” and “prevention” refer to a method for barring an organism from acquiring a PK related disorder in the first place.
  • the terms “treat”, “treating” and “treatment” refer to a method of alleviating or abrogating a PK mediated cellular disorder and/or its attendant symptoms. With regard particularly to cancer, these terms simply mean that the life expectancy of an individual affected with a cancer will be increased or that one or more of the symptoms of the disease will be reduced.
  • organism refers to any living entity comprised of at least one cell.
  • a living organism can be as simple as, for example, a single eukariotic cell or as complex as a mammal, including a human being.
  • a therapeutically effective amount refers to that amount of the compound being administered which will relieve to some extent one or more of the symptoms of the disorder being treated.
  • a therapeutically effective amount refers to that amount which has the effect of (1) reducing the size of the tumor; (2) inhibiting (that is, slowing to some extent, preferably stopping) tumor metastasis; (3) inhibiting to some extent (that is, slowing to some extent, preferably stopping) tumor growth; and/or, (4) relieving to some extent (or, preferably, eliminating) one or more symptoms associated with the cancer.
  • This invention is therefore directed to compounds which modulate PK signal transduction by affecting the enzymatic activity of RTKs, CTKs and/or STKs, thereby interfering with the signals transduced by such proteins. More particularly, the present invention is directed to compounds which modulate RTK, CTK and/or STK mediated signal transduction pathways as a therapeutic approach to cure many kinds of solid tumors, including but not limited to carcinomas, sarcomas including Kaposi's sarcoma, erythroblastoma, glioblastoma, meningioma, astrocytoma, melanoma and myoblastoma.
  • Treatment or prevention of non-solid tumor cancers such as leukemia are also contemplated by this invention.
  • Indications may include, but are not limited to brain cancers, bladder cancers, ovarian cancers, gastric cancers, pancreas cancers, colon cancers, blood cancers, lung cancers and bone cancers.
  • disorders related to inappropriate PK activity are cell proliferative disorders, fibrotic disorders and metabolic disorders.
  • Cell proliferative disorders which may be prevented, treated or further studied by the present invention include cancer, blood vessel proliferative disorders and mesangial cell proliferative disorders.
  • Blood vessel proliferative disorders refer to disorders related to abnormal vasculogenesis (blood vessel formation) and angiogenesis (spreading of blood vessels). While vasculogenesis and angiogenesis play important roles in a variety of normal physiological processes such as embryonic development, corpus luteum formation, wound healing and organ regeneration, they also play a pivotal role in cancer development where they result in the formation of new capillaries needed to keep a tumor alive. Other examples of blood vessel proliferation disorders include arthritis, where new capillary blood vessels invade the joint and destroy cartilage, and ocular diseases, like diabetic retinopathy, where new capillaries in the retina invade the vitreous, bleed and cause blindness.
  • disorders related to the shrinkage, contraction or closing of blood vessels are also implicated and may be treated or prevented by the methods of this invention.
  • Fibrotic disorders refer to the abnormal formation of extracellular matrices.
  • fibrotic disorders include hepatic cirrhosis and mesangial cell proliferative disorders.
  • Hepatic cirrhosis is characterized by the increase in extracellular matrix constituents resulting in the formation of a hepatic scar.
  • An increased extracellular matrix resulting in a hepatic scar can also be caused by a viral infection such as hepatitis.
  • Lipocytes appear to play a major role in hepatic cirrhosis.
  • Other fibrotic disorders implicated include atherosclerosis.
  • Mesangial cell proliferative disorders refer to disorders brought about by abnormal proliferation of mesangial cells.
  • Mesangial proliferative disorders include various human renal diseases such as glomerulonephritis, diabetic nephropathy and malignant nephrosclerosis as well as such disorders as thrombotic microangiopathy syndromes, transplant rejection, and glomerulopathies.
  • the RTK PDGFR has been implicated in the maintenance of mesangial cell proliferation. Floege et al., 1993 , Kidney International 43:47S-54S.
  • PKs have been associated with cell proliferative disorders.
  • PKs such as, for example, members of the RTK family have been associated with the development of cancer.
  • EGFR has been associated with squamous cell carcinoma, astrocytoma, glioblastoma, head and neck cancer, lung cancer and bladder cancer.
  • HER2 has been associated with breast, ovarian, gastric, lung, pancreas and bladder cancer.
  • PDGFR has been associated with glioblastoma and melanoma as well as lung, ovarian and prostate cancer.
  • the RTK c-met has also been associated with malignant tumor formation.
  • c-met has been associated with, among other cancers, colorectal, thyroid, pancreatic, gastric and hepatocellular carcinomas and lymphomas. Additionally c-met has been linked to leukemia. Over-expression of the c-met gene has also been detected in patients with Hodgkins disease and Burkitts disease.
  • Flk has likewise been associated with a broad spectrum of tumors including, without limitation, mammary, ovarian and lung tumors as well as gliomas such as glioblastoma.
  • IGF-IR in addition to being implicated in nutritional support and in type-II diabetes, has also been associated with several types of cancers.
  • IGF-I has been implicated as an autocrine growth stimulator for several tumor types, e.g. human breast cancer carcinoma cells (Arteaga et al., 1989 , J. Clin. Invest. 84:1418-1423) and small lung tumor cells (Macauley et al., 1990 , Cancer Res., 50:2511-2517).
  • IGF-I while integrally involved in the normal growth and differentiation of the nervous system, also appears to be an autocrine stimulator of human gliomas.
  • IGF-IR insulin growth factor-IR
  • Baserga suggests that IGF-IR plays a central role in the mechanism of transformation and, as such, could be a preferred target for therapeutic interventions for a broad spectrum of human malignancies. Baserga, 1995 , Cancer Res., 55:249-252; Baserga, 1994 , Cell 79:927-930; Coppola et al., 1994 , Mol. Cell. Biol., 14:4588-4595.
  • STKs have been implicated in many types of cancer including, notably, breast cancer (Cance, et al., Int. J. Cancer, 54:571-77 (1993)).
  • RTKs have been associated with diseases such as psoriasis, diabetes mellitus, endometriosis, angiogenesis, atheromatous plaque development, Alzheimer's disease, epidermal hyperproliferation, neurodegenerative diseases, age-related macular degeneration and hemangiomas.
  • diseases such as psoriasis, diabetes mellitus, endometriosis, angiogenesis, atheromatous plaque development, Alzheimer's disease, epidermal hyperproliferation, neurodegenerative diseases, age-related macular degeneration and hemangiomas.
  • EGFR has been indicated in corneal and dermal wound healing. Defects in Insulin-R and IGF-1R 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.
  • CTKs including, but not limited to, src, abl, fps, yes, fyn, lyn, lck, blk, hck, fgr and yrk (reviewed by Bolen et al., 1992 , FASEB J., 6:3403-3409) are involved in the proliferative and metabolic signal transduction pathway and thus could be expected, and have been shown, to be involved in many PTK-mediated disorders to which the present invention is directed.
  • mutated src v-src
  • pp60 v-src oncoprotein
  • pp60 c-src transmits oncogenic signals of many receptors.
  • Over-expression of EGFR or HER2/neu in tumors leads to the constitutive activation of pp60 c ⁇ src , which is characteristic of malignant cells but absent in normal cells.
  • mice deficient in the expression of c-src exhibit an osteopetrotic phenotype, indicating a key participation of c-src in osteoclast function and a possible involvement in related disorders.
  • Zap70 has been implicated in T-cell signaling which may relate to autoimmune disorders.
  • STKs have been associated with inflamation, autoimmune disease, immunoresponses, and hyperproliferation disorders such as restenosis, fibrosis, psoriasis, osteoarthritis and rheumatoid arthritis.
  • PKs have also been implicated in embryo implantation.
  • the compounds of this invention may provide an effective method of preventing such embryo implantation and thereby be useful as birth control agents.
  • a compound of the present invention, a prodrug thereof or a physiologically acceptable salt of either the compound or its prodrug can be administered as such to a human patient or it can be administered in pharmacological compositions in which the foregoing materials are mixed with suitable carriers or excipient(s).
  • suitable carriers or excipient(s) suitable carriers or excipient(s).
  • administer refers to the delivery of a compound, salt or prodrug of the present invention or of a pharmacological composition containing a compound, salt or prodrug of this invention to an organism for the purpose of prevention or treatment of a PK-related disorder.
  • Suitable routes of administration may include, without limitation, oral, rectal, transmucosal or intestinal administration or intramuscular, subcutaneous, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
  • the preferred routes of administration are oral and parenteral.
  • the liposomes will be targeted to and taken up selectively by the tumor.
  • compositions of the present invention may be manufactured by processes well known in the art; e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the compounds of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer.
  • physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the compounds can be formulated by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the compounds of the invention to be formulated as tablets, pills, lozenges, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient.
  • Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding other suitable auxiliaries if desired, to obtain tablets or dragee cores.
  • Useful excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch and potato starch and other materials such as gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid. A salt such as sodium alginate may also be used.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings 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.
  • Pharmacological compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with a filler such as lactose, a binder such as starch, and/or a lubricant such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Stabilizers may be added in these formulations, also.
  • the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray using a pressurized pack or a nebulizer and a suitable propellant, e.g., without limitation, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane or carbon dioxide.
  • a suitable propellant e.g., without limitation, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane or carbon dioxide.
  • the dosage unit may be controlled by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • the compounds may also be formulated for parenteral administration, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulating materials such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of a water soluble form, such as, without limitation, a salt, of the active compound. Additionally, suspensions of the active compounds may be prepared in a lipophilic vehicle. Suitable lipophilic vehicles include fatty oils such as sesame oil, synthetic fatty acid esters such as ethyl oleate and triglycerides, or materials such as liposomes.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • the suspension may also contain suitable stabilizers and/or agents 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, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
  • the compounds may also be formulated as depot preparations. Such long acting formulations may be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection.
  • a compound of this invention may be formulated for this route of administration with suitable polymeric or hydrophobic materials (for instance, in an emulsion with a pharamcologically acceptable oil), with ion exchange resins, or as a sparingly soluble derivative such as, without limitation, a sparingly soluble salt.
  • the pharmacological compositions herein also may comprise suitable solid or gel phase carriers or excipients.
  • suitable solid or gel phase carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
  • PK modulating compounds of the invention may be provided as physiologically acceptable salts wherein the claimed compound may form the negatively or the positively charged species.
  • salts in which the compound forms the positively charged moiety include, without limitation, quaternary ammonium (defined elsewhere herein), salts such as the hydrochloride, sulfate, carbonate, lactate, tartrate, maleate, succinate wherein the nitrogen atom of the quaternary ammonium group is a nitrogen of the selected compound of this invention which has reacted with the appropriate acid.
  • Salts in which a compound of this invention forms the negatively charged species include, without limitation, the sodium, potassium, calcium and magnesium salts formed by the reaction of a carboxylic acid group in the compound with an appropriate base (e.g. sodium hydroxide (NaOH), potassium hydroxide (KOH), Calcium hydroxide (Ca(OH) 2 ), etc.).
  • compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an amount sufficient to achieve the intended purpose; i.e., the modulation of PK activity or the treatment or prevention of a PK-related disorder.
  • a therapeutically effective amount means an amount of compound effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated.
  • the therapeutically effective amount or dose can be estimated initially from cell culture assays. Then, the dosage can be formulated for use in animal models so as to achieve a circulating concentration range that includes the IC 50 as determined in cell culture (i.e., the concentration of the test compound which achieves a half-maximal inhibition of the PK activity). Such information can then be used to more accurately determine useful doses in humans.
  • Toxicity and therapeutic efficacy of the compounds described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the IC 50 and the LD 50 (both of which are discussed elsewhere herein) for a subject compound.
  • the data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch. 1 p.1).
  • Dosage amount and interval may be adjusted individually to provide plasma levels of the active specie which are sufficient to maintain the kinase modulating effects. These plasma levels are referred to as minimal effective concentrations (MECs).
  • MEC minimal effective concentrations
  • the MEC will vary for each compound but can be estimated from in vitro data; e.g., the concentration necessary to achieve 50-90% inhibition of a kinase may be ascertained using the assays described herein. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. HPLC assays or bioassays can be used to determine plasma concentrations.
  • Dosage intervals can also be determined using MEC value.
  • Compounds should be administered using a regimen which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90% .
  • the effective local concentration of the drug may not be related to plasma concentration and other procedures known in the art may be employed to determine the correct dosage amount and interval.
  • compositions administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
  • compositions may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may for example comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the pack or dispenser may also be accompanied by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or of human or veterinary administration.
  • Such notice for example, may be of the labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an 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.
  • this invention relates to novel 3-(substituted)-indolin-2-ones demonstrating the ability to modulate RTK, CTK and STK activity.
  • the following assays are employed to select those compounds demonstrating the optimal degree of the desired activity.
  • the phrase “optimal degree of the desired activity” refers to the lowest IC 50 , defined elsewhere herein, against a PTK related to a particular disorder so as to provide an organism, preferably a human, with a therapeutically effective amount of a compound of this invention at the lowest possible dosage.
  • Table 4 shows the results of biological assays of exemplary, but by no means limiting, compounds of this invention using the following assay procedures.
  • 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 a selected period of time after which, if the test kinase is a receptor, a ligand known to activate the receptor 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 phosphorylation on the substrate is detected with an antibody specifically recognizing phosphotyrosine 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 a selected period of time after which, if the test kinase is a receptor, a ligand known to activate the receptor is added. After incubation at least overnight, a DNA labeling reagent such as Bromodeoxyuridine (BrdU) or 3H-thymidine is added.
  • a DNA labeling reagent such as Bromodeoxyuridine (BrdU) or 3H-thymidine is added.
  • Enzyme linked immunosorbent assays may be used to detect and measure the presence of PK activity.
  • the ELISA may be conducted according to known protocols which are described in, for example, Voller, et al., 1980, “Enzyme-Linked Immunosorbent Assay,” In: Manual of Clinical Immunology, 2d ed., edited by Rose and Friedman, pp 359-371 Am. Soc. Of Microbiology, Washington, D.C.
  • the disclosed protocol may be adapted for determining activity with respect to a specific PK. That is, the preferred protocols for conducting the ELISA experiments for specific PKs is provided below. However, adaptation of these protocols for determining a compound's activity for other members of the RTK family, as well as for CTKs and STKs, is well within the scope of knowledge of those skilled in the art.
  • An ELISA assay is conducted to measure the kinase activity of the FLK-1 receptor and more specifically, the inhibition or activation of TK activity on the FLK-1 receptor. Specifically, the following assay can be conducted to measure kinase activity of the FLK-1 receptor in cells genetically engineered to express Flk-1.
  • Ethanolamine stock (10% ethanolamine (pH 7.0), stored at 4° C.);
  • HNTG buffer (20 mM HEPES buffer (pH 7.5), 150 mM NaCl, 0.2% Triton X-100, and 10% glycerol);
  • VEGF vascular endothelial growth factor
  • PeproTech, Inc. catalog no. 100-20
  • ABTS 2,2-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid
  • HNTG formulation includes sodium ortho vanadate, sodium pyrophosphate and EDTA.
  • HER2 kinase activity in whole EGFR-NIH3T3 cells are measured as described below:
  • EGF stock concentration: 16.5 ILM; EGF 201, TOYOBO, Co., Ltd. Japan.
  • UBI 05-101 (UBI) (a monoclonal antibody recognizing an EGFR extracellular domain).
  • Anti-Ptyr Anti-phosphotyrosine antibody (anti-Ptyr) (polyclonal) (see, Fendley, et al., supra).
  • Detection antibody Goat anti-rabbit lgG horseradish peroxidase conjugate, TAGO, Inc., Burlingame, CA. e.
  • TBST buffer Tris-HCl, pH 7.2 50 mM NaCl 150 mM Triton X-100 0.1 f.
  • HNTG 5X stock HEPES 0.1 M NaCl 0.75 M Glycerol 50% Triton X-100 1.0% g.
  • ABTS stock Citric Acid: 100 mM Na 2 HPO 4 250 mM HCl, conc. 0.5 mM ABTS* 0.5 mg/ml h.
  • An NIH3T3 cell line overexpressing a chimeric receptor containing the EGFR extracellular domain and intracellular HER2 kinase domain can be used for this assay.
  • [0270] Resuspend cells in seeding medium (DMEM, 0.5% bovine serum), and count the cells using trypan blue. Viability above 90% is acceptable. 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. Incubate seeded cells in 5% CO 2 at 37° C. for about 40 hours.
  • seeding medium DMEM, 0.5% bovine serum
  • EGF ligand dilute stock EGF in DMEM so that upon transfer of 10 ⁇ l dilute EGF (1:12 dilution), 100 nM final concentration is attained.
  • HNTG* (100 ⁇ l): HNTG stock 2.0 ml milli-Q H 2 O 7.3 ml EDTA, 100 mM, pH 7.0 0.5 ml Na 3 VO 4 (0.5 M) 0.1 ml Na 4 (P 2 O 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.
  • All cell culture media, glutamine, and fetal bovine serum can be purchased from Gibco Life Technologies (Grand Island, N.Y.) unless otherwise specified. All cells are grown in a humid atmosphere of 90-95% air and 5-10% CO 2 at 37° C. All cell lines are routinely subcultured twice a week and are negative for mycoplasma as determined by the Mycotect method (Gibco).
  • cells (U1242, obtained from Joseph Schlessinger, NYU) are 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 are changed to serum-free medium and treated with test compound for 2 hr in a 5% CO 2 , 37° C. incubator.
  • Cells are then stimulated with ligand for 5-10 minute followed by lysis with HNTG (20 mM Hepes, 150 mM NaCl, 10% glycerol, 5 mM EDTA, 5 mM Na 3 VO 4 , 0.2% Triton X-100, and 2 mM NaPyr).
  • HNTG 20 mM Hepes, 150 mM NaCl, 10% glycerol, 5 mM EDTA, 5 mM Na 3 VO 4 , 0.2% Triton X-100, and 2 mM NaPyr.
  • Cell lysates (0.5 mg/well in PBS) are 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 are incubated with shaking for 1 hour at room temperature.
  • the plates are washed with TBST four times and then incubated with polyclonal anti-phosphotyrosine antibody at room temperature for 30 minutes. Excess anti-phosphotyrosine antibody is removed by rinsing the plate with TBST four times. Goat anti-rabbit IgG antibody is 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 HPO 4 and 0.5 mg/mL 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) plus H 2 O 2 (1.2 mL 30% H 2 O 2 to 10 ml ABTS) is added to the ELISA plates to start color development. Absorbance at 410 nm with a reference wavelength of 630 nm is recorded about 15 to 30 min after ABTS addition.
  • the following protocol may be used to measure phosphotyrosine level on IGF-1 receptor, which indicates IGF-1 receptor tyrosine kinase activity.
  • the cell line used in this assay is 3T3/IGF-1R, a cell line genetically engineered to overexpresses IGF-1 receptor.
  • NIH3T3/IGF-1R is grown in an incubator with 5% CO 2 at 37° C.
  • the growth media is DMEM+10% FBS (heat inactivated)+2 mM L-glutamine.
  • D-PBS KH 2 PO 4 0.20 g/l KH 2 PO 4 2.16 g/l KCl 0.20 g/l NaCl 8.00 g/l (pH 7.2)
  • Blocking Buffer TBST plus 5% Milk (Carnation Instant Non-Fat Dry Milk).
  • TBST buffer Tris-HCl 50 mM NaCl 150 mM (pH 7.2/HCl 10 N) Triton X-100 0.1%
  • HNTG buffer HEPES 20 mM NaCl 150 mM (pH 7.2/HCl 1 N) Glycerol 10% Triton X-100 0.2%
  • ABTS (2,2′-azinobis(3-ethylbenzthiazolinesulfonic acid)) solution: Citric acid 100 mM Na 2 HPO 4 250 mM (pH 4.0/1 N HCl) ABTS 0.5 mg/ml
  • ABTS solution should be kept in dark and 4° C. The solution should be discarded when it turns green.
  • EGF Receptor kinase activity in cells genetically engineered to express human EGF-R can be measured as described below:
  • 05-101 (UBI) (a monoclonal antibody recognizing an EGFR extracellular domain).
  • Anti-phosphotyosine antibody (anti-Ptyr) (polyclonal).
  • Detection antibody Goat anti-rabbit lgG horse radish peroxidase conjugate, TAGO, Inc., Burlingame, Calif.
  • TBST buffer Tris-HCl, pH 7 50 mM NaCl 150 mM Triton X-100 0.1
  • HNTG 5 ⁇ stock HEPES 0.1 M NaCl 0.75 M Glycerol 50 Triton X-100 1.0%
  • ABTS stock Citric Acid 100 mM Na 3 VO 4 250 mM HCl, conc. 4.0 pH ABTS* 0.5 mg/ml
  • NIH 3T3/C7 cell line (Honegger, et al., Cell 51:199-209, 1987) can be use 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 O (7.3 ml), EDTA, 100 mM, pH 7.0 (0.5 ml), Na 3 VO 4 0.5 M (0.1 ml) and Na 4 (P 2 O 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.
  • This assay determines Met tyrosine kinase activity by analyzing Met protein tyrosine kinase levels on the Met receptor.
  • HNTG 5 ⁇ stock solution: Dissolve 23.83 g HEPES and 43.83 g NaCl in about 350 ml dH 2 O. Adjust pH to 7.2 with HCl or NaOH, add 500 ml glycerol and 10 ml Triton X-100, mix, add dH 2 O to 1 L total volume. To make 1 L of 1 ⁇ working solution add 200 ml 5 ⁇ stock solution to 800 ml dH 2 O, check and adjust pH as necessary, store at 4° C.
  • Blocking Buffer in 500 ml dH 2 O place 100 g BSA, 12.1 g Tris-pH7.5, 58.44 g NaCl and 10 ml Tween-20, dilute to 1 L total volume.
  • q. TBST Buffer to approx. 900 ml dH 2 O in a 1 L graduated cylinder add 6.057 g TRIS and 8.766 g NaCl, when dissolved, adjust pH to 7.2 with HCl, add 1.0 ml Triton X-100 and bring to 1 L total volume with dH 2 O.
  • EMR Transiently Transfected EGFR/Met chimeric cells
  • This procedure can be performed the night before or immediately prior to the start of receptor capture.
  • This assay is used to determine src protein kinase activity measuring phosphorylation of a biotinylated peptide as the readout.
  • Yeast cells expressing src are pelleted, washed once with water, re-pelleted and stored at ⁇ 80° C. until use.
  • N-terminus biotinylated EEEYEEYEEEYEEEYEEEY is prepared by standard procedures well known to those skilled in the art.
  • Vecastain ELITE ABC reagent Vector, Burlingame, Calif.
  • h. Anti-src (327) mab Schizosaccharomyces Pombe is used to express recombinant Src (Superti-Furga, et al., EMBO J., 12:2625-2634; Superti-Furga, et al., Nature Biochem., 14:600-605).
  • S. Pombe strain SP200 h-s leul.32 ura4 ade210
  • transformations are pRSP expression plasmids are done by the lithium acetate method (Superti-Furga, supra).
  • Cells are grown in the presence of 1 ⁇ M thiamine to repress expression from the nmtl promoter or in the absence of thiamine to induce expression.
  • PBS Dulbecco's Phosphate-Buffered Saline
  • GIBCO PBS GIBCO Cat. # 450-1300EB.
  • Blocking Buffer 5% Non-fat milk (Carnation) in PBS.
  • Carbonate Buffer Na 2 CO 4 from Fischer, Cat. # S495, make up 100 mM stock solution.
  • e. Lysis Buffer 5.0 HEPES (from 1M stock solution.); 2.74 ml NaCl (from 5M stock solution); 10 ml glycerol; 1.0 ml TX-100; 0.4 ml EDTA (from a 100 mM stock solution); 1.0 ml PMSF (from a 100 mM stock solution); 0.1 ml Na 3 VO 4 (from a 0.1 M stock solution); bring to 100 ml total volume with MilliQ H 2 O.
  • TRIS-HCl Fischer Cat. # BP 152-5, to 600 ml MilliQ H 2 O add 121.14 g material, adjust pH to 7.5 with HCl, bring to 1 L total volume with MilliQ H 2 O.
  • Na 3 VO 4 Fischer Cat. # S454-50; to 80 ml MilliQ H 2 O, add 1.8 g material; adjust pH to 10.0 with HCl or NaOH; boil in a microwave; cool; check pH, repeat pH adjustment until pH remains stable after heating/cooling cycle; bring to 100 ml total volume with MilliQ H 2 O; make 1 ml aliquots and store at ⁇ 80° C.
  • HEPES Fischer Cat. # BP 310-500; to 200 ml MilliQ H 2 O, add 59.6 g material, adjust pH to 7.5, bring to 250 ml total volume with MilliQ H 2 O, sterile filter (1M stock solution).
  • TBST Buffer To 900 ml dH 2 O add 6.057 g TRIS and 8.766 g NaCl; adjust pH to 7.2 with HCl, add 1.0 ml Triton-X100; bring to 1 L total volume with dH 2 O.
  • TBS TriS Buffered Saline
  • Biotin labeled EEEYEEYEEEYEEEYEEEYEEEY Make peptide stock solution (1 mM, 2.98 mg/ml) in water fresh just before use.
  • r. Vectastain ELITE ABC reagent To prepare 14 ml of working reagent, add 1 drop of reagent A to 15 ml TBST and invert tube several times to mix. Then add 1 drop of reagent B. Put tube on orbital shaker at room temperature and mix for 30 minutes.
  • 4G10 plate coat 0.5 ⁇ g/well 4G10 in 100 ⁇ l PBS overnight at 4° C. and block with 150 ⁇ l of 5% milk in PBS for 30 minutes at room temperature.
  • This assay is used to determine lck protein kinase activities measuring phosphorylation of GST- ⁇ as the readout.
  • Yeast transformed with lck Schizosaccharomyces Pombe is used to express recombinant Lck (Superti-Furga, et al., EMBO J, 12:2625-2634; Superti-Furga, et al., Nature Biotech., 14:600-605).
  • S. Pombe strain SP200 h-s leul.32 ura4 ade210
  • transformations with pRSP expression plasmids are done by the lithium acetate method (Superti-Furga, supra) Cells are grown in the presence of 1 ⁇ M thiamine to induce expression.
  • Yeast cells expressing lck are pelleted, washed once in water, re-pelleted and stored frozen at ⁇ 80° C. until use.
  • GST- ⁇ DNA encoding for GST- ⁇ fusion protein for expression in bacteria obtained from Arthur Weiss of the Howard Hughes Medical Institute at the University of California, San Francisco. Transformed bacteria are grown overnight while shaking at 25° C. GST- ⁇ is purified by glutathione affinity chromatography, Pharmacia, Alameda, Calif.
  • 96-Well ELISA plate Corning 96 Well Easy Wash, Modified Flat Bottom Plate, Corning Cat. #25805-96.
  • PBS Dulbecco's Phosphate-Buffered Saline 1 ⁇ solution: GIBCO PBS, GIBCO Cat. # 450-1300EB.
  • Blocking Buffer 100 g. BSA, 12.1 g. TRIS (pH7.5), 58.44 g NaCl, 10 ml Tween-20, bring up to 1 L total volume with MilliQ H 2 O.
  • Carbonate Buffer Na 2 CO 4 from Fischer, Cat. # S495; make up 100 mM solution with MilliQ H 2 O.
  • e. Lysis Buffer 5.0 HEPES (from 1M stock solution.); 2.74 ml NaCl (from 5M stock solution); 10 ml glycerol; 1.0 ml TX-100; 0.4 ml EDTA (from a 100 mM stock solution); 1.0 ml PMSF (from a 100 mM stock solution); 0.1 ml Na 3 VO 4 (from a 0.1 M stock solution); bring to 100 ml total volume with MilliQ H 2 O.
  • ATP Sigma Cat. # A-7699, make up 10 mM stock solution (5.51 mg/ml).
  • TRIS-HCl Fischer Cat. # BP 152-5, to 600 ml MilliQ H 2 O add 121.14 g material, adjust pH to 7.5 with HCl, bring to 1 L total volume with MilliQ H 2 O.
  • HEPES Fischer Cat. # BP 310-500; to 200 ml MilliQ H 2 O, add 59.6 g material, adjust pH to 7.5, bring to 250 ml total volume with MilliQ H 2 O, sterile filter (1M stock solution).
  • m. TBST Buffer To 900 ml dH 2 O add 6.057 g TRIS and 8.766 g NaCl; adjust pH to 7.2 with HCl, add 1.0 ml Triton-X100; bring to 1 L total volume with dH 2 O.
  • n. MnCl2 Fischer Cat. # M87-100, make up 1M stock solution with MilliQ H 2 O.
  • TBS TriS Buffered Saline
  • q. Kinase Reaction Mixture Amount per assay plate (100 wells): 1.0 ml Kinase Buffer, 200 ⁇ g GST- ⁇ , bring to final volume of 8.0 ml with MilliQ H 2 O.
  • UB40 plate 1.0 ⁇ g/well UB40 in 100 ⁇ l of PBS overnight at 4° C. and block with 150 ⁇ l of Blocking Buffer for at least 1 hour.
  • the following assay reports the amount of RAF-catalyzed phosphorylation of its target protein MEK as well as MEK's target MAPK.
  • the RAF gene sequence is described in Bonner et al., 1985 , Molec. Cell. Biol., 5:1400-1407, and is readily accessible in multiple gene sequence data banks. Construction of the nucleic acid vector and cell lines utilized for this portion of the invention are fully described in Morrison et al., 1988 , Proc. Natl. Acad. Sci. USA, 85:8855-8859.
  • RIPA buffer 20 mM Tris/HCl pH 7.4, 137 mM NaCl, 10% glycerol, 1 mM PMSF, 5 mg/L Aprotenin, 0.5% Triton X-100;
  • T-MEK Thioredoxin-MEK fusion protein
  • His-MAPK (ERK 2); His-tagged MAPK is expressed in XL1 Blue cells transformed with pUC18 vector encoding His-MAPK. His-MAPK is purified by Ni-affinity chromatography. Cat# 27-4949-01, Pharmacia, Alameda, Calif., as described herein.
  • RAF-1 protein kinase specific antibody URP2653 from UBI.
  • Coating buffer PBS; phosphate buffered saline, GIBCO-BRL, Gaithersburg, Md.
  • Wash buffer 50 mM Tris/HCL pH 7.2, 150 mM NaCl, 0.1% Triton X-100).
  • Block buffer TBST, 0.1% ethanolamine pH 7.4
  • ATP mix 100 mM MgCl 2 , 300 mM ATP, 10 mCi ⁇ 33 P ATP (Dupont-NEN)/mL.
  • Filter wash solution 1% phosphoric acid, Fisher, Pittsburgh, Pa.
  • ELISA plate coating ELISA wells are coated with 100 ml of Sheep anti mouse affinity purified antiserum (1 mg/100 mL coating buffer) over night at 4° C. ELISA plates can be used for two weeks when stored at 4° C.
  • This assay analyzes the protein kinase activity of CDK2 in exogenous substrate.
  • C. ATP solution (60 ⁇ M ATP, 300 ⁇ g/ml BSA, 3 mM DTT): 120 ⁇ l 10 mM ATP, 600 ⁇ l 10 mg/ml BSA to 20 ml, stored in 1 ml aliquots at ⁇ 80° C.
  • CDK2 solution cdk2/cyclin A in 10 mM HEPES pH 7.2, 25 mM NaCl, 0.5 mM DTT, 10% glycerol, stored in 9 ⁇ l aliquots at ⁇ 80° C.
  • PDGF human PDGF B/B; 1276-956, Boehringer Mannheim, Germany.
  • FixDenat fixation solution (ready to use), Cat. No. 1 647 229, Boehringer Mannheim, Germany.
  • Anti-BrdU-POD mouse monoclonal antibody conjugated with peroxidase, Cat. No. 1 647 229, Boehringer Mannheim, Germany.
  • TMB Substrate Solution tetramethylbenzidine (TMB), ready to use, Cat. No. 1 647 229, Boehringer Mannheim, Germany.
  • 3T3 cell line genetically engineered to express human PDGF-R.
  • ligand (PDGF, 3.8 nM, prepared in DMEM with 0.1% BSA) and test compounds are added to the cells simultaneously.
  • the negative control wells receive serum free DMEM with 0.1% BSA only; the positive control cells receive the ligand (PDGF) but no test compound.
  • Test compounds are prepared in serum free DMEM with ligand in a 96 well plate, and serially diluted for 7 test concentrations.
  • the blocking solution is removed by decanting and the wells are washed once with PBS.
  • Anti-BrdU-POD solution (1:100 dilution in PBS, 1% BSA) is added (100 ⁇ l/well) and the plate is incubated for 90 minutes at room temperature on a plate shaker.
  • TMB substrate solution is added (100 ⁇ l/well) and incubated for 20 minutes at room temperature on a plate shaker until color development is sufficient for photometric detection.
  • EGF mouse EGF, 201; Toyobo, Co., Ltd. Japan.
  • FixDenat fixation solution (ready to use), Cat. No. 1 647 229, Boehringer Mannheim, Germany.
  • Anti-BrdU-POD mouse monoclonal antibody conjugated with peroxidase, Cat. No. 1 647 229, Boehringer Mannheim, Germany.
  • TMB Substrate Solution tetramethylbenzidine (TMB), ready to use, Cat. No. 1 647 229, Boehringer Mannheim, Germany.
  • 3T3 cell line genetically engineered to express human EGF-R.
  • ligand EGF, 2 nM, prepared in DMEM with 0.1% BSA
  • test compounds are added to the cells simultaneously.
  • the negative control wells receive serum free DMEM with 0.1% BSA only; the positive control cells receive the ligand (EGF) but no test compound.
  • Test compounds are prepared in serum free DMEM with ligand in a 96 well plate, and serially diluted for 7 test concentrations.
  • TMB substrate solution is added (100 ⁇ l/well) and incubated for 20 minutes at room temperature on a plate shaker until color development is sufficient for photometric detection.
  • EGF mouse EGF, 201; Toyobo, Co., Ltd. Japan
  • FixDenat fixation solution (ready to use), Cat. No. 1 647 229, Boehringer Mannheim, Germany.
  • Anti-BrdU-POD mouse monoclonal antibody conjugated with peroxidase, Cat. No. 1 647 229, Boehringer Mannheim, Germany.
  • TMB Substrate Solution tetramethylbenzidine (TMB), ready to use, Cat. No. 1 647 229, Boehringer Mannheim, Germany.
  • 3T3 cell line engineered to express a chimeric receptor having the extra-cellular domain of EGF-R and the intra-cellular domain of Her2.
  • ligand (EGF 2 nM, prepared in DMEM with 0.1% BSA) and test compounds are added to the cells simultaneously.
  • the negative control wells receive serum free DMEM with 0.1% BSA only; the positive control cells receive the ligand (EGF) but no test compound.
  • Test compounds are prepared in serum free DMEM with ligand in a 96 well plate, and serially diluted for 7 test concentrations.
  • the blocking solution is removed by decanting and the wells are washed once with PBS.
  • Anti-BrdU-POD solution (1:100 dilution in PBS, 1% BSA) is added (100 ⁇ l/well) and the plate is incubated for 90 minutes at room temperature on a plate shaker.
  • TMB substrate solution is added (100 ⁇ l/well) and incubated for 20 minutes at room temperature on a plate shaker until color development is sufficient for photometric detection.
  • IGF1 Ligand human, recombinant; G511, Promega Corp, USA.
  • FixDenat fixation solution (ready to use), Cat. No. 1 647 229, Boehringer Mannheim, Germany.
  • Anti-BrdU-POD mouse monoclonal antibody conjugated with peroxidase, Cat. No. 1 647 229, Boehringer Mannheim, Germany.
  • TMB Substrate Solution tetramethylbenzidine (TMB), ready to use, Cat. No. 1 647 229, Boehringer Mannheim, Germany.
  • 3T3 cell line genetically engineered to express human IGF-1 receptor.
  • test compounds are added to the cells simultaneously.
  • the negative control wells receive serum free DMEM with 0.1% BSA only; the positive control cells receive the ligand (IGF1) but no test compound.
  • Test compounds are prepared in serum free DMEM with ligand in a 96 well plate, and serially diluted for 7 test concentrations.
  • TMB substrate solution is added (100 ⁇ l/well) and incubated for 20 minutes at room temperature on a plate shaker until color development is sufficient for photometric detection.
  • This assay measures FGF-induced DNA synthesis in 3Tc7/EGFr cells that express endogenous FGF receptors.
  • FGF human FGF2/bFGF (Gibco BRL, No. 13256-029).
  • Anti-BrdU-POD mouse monoclonal antibody conjugated with peroxidase, Boehringer Mannheim Cat. No. 1 647 229).
  • TMB tetramethylbenzidine, Boehringer Mannheim Cat. No. 1 647 229).
  • 3T3 engineered cell line 3T3c7/EGFr.
  • This assay measures the in vitro kinase activity of EGFR using ELISA.
  • Blocking Buffer Working Amount per Reagent M.W. Concentration 100 ml Carnation Instant 5% 5.0 g Non-Fat Milk PBS NA NA 100 ml
  • TBS Buffer Working Amount Reagent M.W. Concentration per L Tris 121.14 50 mM 6.057 g NaCl 58.44 150 mM 8.766 g
  • Adenosine-5′-triphosphate (ATP, from Equine muscle, Sigma Cat. No. A-5394).
  • This reagent should be prepared immediately before use and kept on ice
  • This assay measures the in vitro kinase activity of PDGFR using ELISA.
  • Adenosine-5′-triphosphate (ATP, from Equine muscle, Sigma Cat. No. A-5394).
  • Ethylenediaminetetraacetic acid (EDTA).
  • This assay measures in vitro kinase activity of the Myc-GyrB-FGFR fusion protein using ELISA.
  • This assay evaluates flk-1 autophosphorylation activity in vitro using ELISA.
  • Flk-1/NIH cells NIH fibroblast line over-expressing human flk-1 clone 3 (SUGEN, Inc., obtained from MPI, Martinsried, Germany).
  • Starvation medium DMEM plus 0.5% heat-inactivated FBS, 2 mM Glutamine (Gibco-BRL).
  • the following protocol may also be used to measure a compound's activity against PDGF-R, FGF-R, VEGF, aFGF or Flk-1/KDR, all of which are naturally expressed by HUV-EC cells.
  • HUV-EC-C cells human umbilical vein endothelial cells, (American Type Culture Collection; catalogue no. 1730 CRL). Wash with Dulbecco's phosphate-buffered saline (D-PBS; obtained from Gibco BRL; catalogue no. 14190-029) 2 times at about 1 ml/10 cm 2 of tissue culture flask. Trypsinize with 0.05% trypsin-EDTA in non-enzymatic cell dissociation solution (Sigma Chemical Company; catalogue no. C-1544). The 0.05% trypsin is made by diluting 0.25% trypsin/1 mnM EDTA (Gibco; catalogue no.
  • [0883] make up two-fold test compound titrations in separate 96-well plates, generally 50 ⁇ M on down to 0 ⁇ M. Use the same assay medium as mentioned in day 0, step 2 above. Titrations are made by adding 90 ⁇ l/well of test compound at 200 ⁇ M (4 ⁇ the final well concentration) to the top well of a particular plate column. Since the stock test compound is usually 20 mM in DMSO, the 200 ⁇ M drug concentration contains 2% DMSO.
  • a diluent made up to 2% DMSO in assay medium (F12K+0.5% fetal bovine serum) is used as diluent for the test compound titrations in order to dilute the test compound but keep the DMSO concentration constant. Add this diluent to the remaining wells in the column at 60 ⁇ l/well. Take 60 ⁇ l from the 120 ⁇ l of 200 ⁇ M test compound dilution in the top well of the column and mix with the 60 ⁇ l in the second well of the column. Take 60 ⁇ l from this well and mix with the 60 ⁇ l in the third well of the column, and so on until two-fold titrations are completed.
  • VEGF vascular endothelial cell growth factor
  • aFGF acidic fibroblast growth factor
  • [0886] In triplicate, add 50 ⁇ l/well of 80 ⁇ g/ml VEGF, 20 ng/ml ECGF, or media control to each test compound condition. As with the test compounds, the growth factor concentrations are 4 ⁇ the desired final concentration. Use the assay media from day 0 step 2 to make the concentrations of growth factors. Incubate approximately 24 hours at 37° C., 5% CO 2 . Each well will have 50 ⁇ l test compound dilution, 50 ⁇ l growth factor or media, and 100 ⁇ l cells, which calculates to 200 ⁇ l/well total. Thus the 4 ⁇ concentrations of test compound and growth factors become 1 ⁇ once everything has been added to the wells.
  • human tumors to grow as xenografts in athymic mice (e.g., Balb/c, nu/nu) provides a useful in vivo model for studying the biological response to therapies for human tumors. Since the first successful xenotransplantation of human tumors into athymic mice, (Rygaard and Povlsen, 1969 , Acta Pathol. Microbial. Scand. 77:758-760), many different human tumor cell lines (e.g., mammary, lung, genitourinary, gastro-intestinal, head and neck, glioblastoma, bone, and malignant melanomas) have been transplanted and successfully grown in nude mice.
  • human tumor cell lines e.g., mammary, lung, genitourinary, gastro-intestinal, head and neck, glioblastoma, bone, and malignant melanomas
  • the following assays may be used to determine the level of activity, specificity and effect of the different compounds of the present invention.
  • Three general types of assays are useful for evaluating compounds: cellular/catalytic, cellular/biological and in vivo.
  • the object of the cellular/catalytic assays is to determine the effect of a compound on the ability of a TK to phosphorylate tyrosines on a known substrate in a cell.
  • the object of the cellular/biological assays is to determine the effect of a compound on the biological response stimulated by a TK in a cell.
  • the object of the in vivo assays is to determine the effect of a compound in an animal model of a particular disorder such as cancer.
  • Suitable cell lines for subcutaneous xenograft experiments include C6 cells (glioma, ATCC # CCL 107), A375 cells (melanoma, ATCC # CRL 1619), A431 cells (epidermoid carcinoma, ATCC # CRL 1555), Calu 6 cells (lung, ATCC # HTB 56), PC3 cells (prostate, ATCC # CRL 1435), SKOV3TP5 cells and NIH 3T3 fibroblasts genetically engineered to overexpress EGFR, PDGFR, IGF-1R or any other test kinase.
  • the following protocol can be used to perform xenograft experiments:
  • mice Female athymic mice (BALB/c, nu/nu) are obtained from Simonsen Laboratories (Gilroy, Calif.). All animals are maintained under clean-room conditions in Micro-isolator cages with Alpha-dri bedding. They receive sterile rodent chow and water ad libitum.
  • Cell lines are grown in appropriate medium (for example, MEM, DMEM, Ham's F10, or Ham's F12 plus 5%-10% fetal bovine serum (FBS) and 2 mM glutamine (GLN)). All cell culture media, glutamine, and fetal bovine serum are purchased from Gibco Life Technologies (Grand Island, NY) unless otherwise specified. All cells are grown in a humid atmosphere of 90-95% air and 5-10% CO 2 at 37° C. All cell lines are routinely subcultured twice a week and are negative for mycoplasma as determined by the Mycotect method (Gibco).
  • appropriate medium for example, MEM, DMEM, Ham's F10, or Ham's F12 plus 5%-10% fetal bovine serum (FBS) and 2 mM glutamine (GLN)
  • All cell culture media, glutamine, and fetal bovine serum are purchased from Gibco Life Technologies (Grand Island, NY) unless otherwise specified. All cells are grown in a humid atmosphere of 90-95% air
  • 8 week old nude mice (female) (Simonsen Inc.) are used as experimental animals. Implantation of tumor cells can be performed in a laminar flow hood. For anesthesia, Xylazine/Ketamine Cocktail (100 mg/kg ketamine and 5 mg/kg Xylazine) are administered intraperitoneally. A midline incision is done to expose the abdominal cavity (approximately 1.5 cm in length) to inject 10 7 tumor cells in a volume of 100 ⁇ l medium. The cells are injected either into the duodenal lobe of the pancreas or under the serosa of the colon. The peritoneum and muscles are closed with a 6-0 silk continuous suture and the skin is closed by using wound clips. Animals are observed daily.
  • mice After 2-6 weeks, depending on gross observations of the animals, the mice are sacrificed, and the local tumor metastases to various organs (lung, liver, brain, stomach, spleen, heart, muscle) are excised and analyzed (measurement of tumor size, grade of invasion, immunochemistry, in situ hybridization determination, etc.).
  • Therapeutic compounds should be more potent in inhibiting receptor tyrosine kinase activity than in exerting a cytotoxic effect.
  • a measure of the effectiveness and cell toxicity of a compound can be obtained by determining the therapeutic index; i.e., IC 50 /LD 50 .
  • IC 50 the dose required to achieve 50% inhibition, can be measured using standard techniques such as those described herein.
  • LD 50 the dosage which results in 50% toxicity, can also be measured by standard techniques as well (Mossman, 1983 , J. Immunol. Methods, 65:55-63), by measuring the amount of LDH released (Korzeniewski and Callewaert, 1983 , J. Immunol.
  • the therapeutic index should be greater than 2, preferably at least 10, more preferably at least 50.
  • the compounds, methods and pharmacological compositions of the present invention modulate RTK, CTK and STK activity and therefore can be expected to be effective as therapeutic agents against a RTK-, CTK- and STK-related disorders.

Abstract

The present invention relates to novel 3-(substituted)-2-indolinones compounds and physiologically acceptable salts and prodrugs thereof which modulate the activity of protein kinases and therefore are expected to e useful in the prevention and treatment of protein kinase related disorders such as cancer.

Description

  • The present invention relates generally to organic chemistry, biochemistry, pharmacology and medicine. More particularly, it relates to novel 3-(substituted)-2-indolinone compounds, and their physiologically acceptable salts and prodrugs, which modulate the activity of protein kinases (“PKs”) and, therefore, are expected to exhibit a salutary effect against a range of disorders related to abnormal PK activity. [0001]
    • BACKGROUND OF THE INVENTION
  • The following is offered as background information only and is not admitted to be prior art to the present invention. [0002]
  • PKs are enzymes that catalyze the phosphorylation of hydroxy groups on tyrosine, serine and threonine residues of proteins. The consequences of this seemingly simple activity are staggering; cell growth, differentiation and proliferation; i.e., virtually all aspects of cell life, in one way or another depend on PK activity. Furthermore, abnormal PK activity has been related to a host of disorders, ranging from relatively non life threatening diseases such as psoriasis to extremely virulent diseases such as glioblastoma (brain cancer). [0003]
  • The PKs can conveniently be broken down into two classes, the protein tyrosine kinases (PTKs) and the serine-threonine kinases (STKs). [0004]
  • One of the prime aspects of PK activity is involvement with growth factor receptors. Growth factor receptors are cell-surface proteins. When bound by a growth factor ligand, a growth factor receptor is converted to an active form which interacts with proteins on the inner surface of a cell membrane. This leads to phosphorylation on tyrosine residues of the receptor as well as other proteins and to the formation inside the cell of complexes with a variety of cytoplasmic signaling molecules. These complexes, in turn, affect numerous cellular responses such as cell division (proliferation), cell differentiation, cell growth, expression of metabolic effects on the extracellular microenvironment, etc. For a more complete discussion, see Schlessinger and Ullrich, [0005] Neuron, 9:303-391 (1992) which is incorporated by reference, including any drawings, as if fully set forth herein.
  • Growth factor receptors with PK activity are known as receptor tyrosine kinases (“RTKs”). They comprise a large family of transmembrane receptors with diverse biological activity. At present, at least nineteen (19) distinct subfamilies of RTKs have been identified. An example of these is the subfamily designated the “HER” RTKs, which includes EGFR (epithelial growth factor receptor), HER2, HER3 and HER4. These RTKs consist of an extracellular glycosylated ligand binding domain, a transmembrane domain and an intracellular cytoplasmic catalytic domain that can phosphorylate tyrosine residues on proteins. [0006]
  • Another RTK subfamily consists of insulin receptor (IR), insulin-like growth factor I receptor (IGF-1R) and insulin receptor related receptor (IRR). IR and IGF-1R interact with insulin, IGF-I and IGF-II to form a heterotetramer composed of two entirely extracellular glycosylated α subunits and two β subunits which cross the cell membrane and which contain the tyrosine kinase domain. [0007]
  • A third RTK subfamily is referred to as the platelet derived growth factor receptor (“PDGFR”) group, which includes PDGFRα, PDGFRβ, CSFIR, c-kit and c-fms. These receptors consist of glycosylated extracellular domains composed of variable numbers of immunoglobin-like loops and an intracellular domain wherein the tyrosine kinase domain is interrupted by unrelated amino acid sequences. [0008]
  • Another group which, because of its similarity to the PDGFR subfamily, is sometimes subsumed in the later group, is the fetus liver kinase (“flk”) receptor subfamily. This group is composed of kinase insert domain-receptor fetal liver kinase-1 (KDR/FLK-1), flk-1R, flk-4 and fms-like tyrosine kinase 1 (flt-1). [0009]
  • One further member of the tyrosine kinase growth factor receptor family is the fibroblast growth factor (“FGF”)receptor group. This group consists of four receptors, FGFR1-4, and seven ligands, FGF1-7. While not yet well characterized, it appears that the receptors consist of a glycosylated extracellular domain containing a variable number of immunoglobin-like loops and an intracellular domain in which the PTK sequence is interrupted by regions of unrelated amino acid sequences. [0010]
  • A more complete listing of the known RTK subfamilies is described in Plowman et al., [0011] DN&P, 7(6):334-339 (1994) which is incorporated by reference, including any drawings, as if fully set forth herein.
  • In addition to the RTKs, there also exists a family of entirely intracellular PTKs called “non-receptor tyrosine kinases” or “cellular tyrosine kinases.” This latter designation, abbreviated “CTK”, will be used herein. CTKs do not contain extracellular and transmembrane domains. At present, over 24 CTKs in 11 subfamilies (Src, Frk, Btk, Csk, Abl, Zap70, Fes, Fps, Fak, Jak and Ack) have been identified. The Src subfamily appear so far to be the largest group of CTKs and includes Src, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk. For a more detailed discussion of CTKs, see Bolen, [0012] Oncogene, 8:2025-2031 (1993), which is incorporated by reference, including any drawings, as if fully set forth herein.
  • The serine-threonine kinases or STKs, like the CTKs, are predominantly intracellular although there are a few STK receptor kinases. STKs are the most common of the cytosolic kinases; i.e., kinases which perform their function in that part of the cytoplasm other than the cytoplasmic organelles and cytoskelton. The cytosol is the region within the cell where much of the cell's intermediary metabolic and biosynthetic activity occurs; e.g., it is in the cytosol that proteins are synthesized on ribosomes. [0013]
  • RTKs, CTKs and STKs have all been implicated in a host of pathogenic conditions including, significantly, cancer. Other pathogenic conditions which have been associated with PTKs include, without limitation, psoriasis, hepatic cirrhosis, diabetes, atherosclerosis, angiogenesis, restenosis, ocular diseases, rheumatoid arthritis and other inflammatory disorders, autoimmune disease and a variety of renal disorders. [0014]
  • With regard to cancer, two of the major hypotheses advanced to explain the excessive cellular proliferation that drives tumor development relate to functions known to be PK regulated. That is, it has been suggested that malignant cell growth results from a breakdown in the mechanisms that control cell division and/or differentiation. It has been shown that the protein products of a number of proto-oncogenes are involved in the signal transduction pathways that regulate cell growth and differentiation. These protein products of proto-oncogenes include the extracellular growth factors, transmembrane growth factor PTK receptors (RTKs), cytoplasmic PTKs (CTKs) and cytosolic STKs, discussed above. [0015]
  • In view of the apparent link between PK-related cellular activities and a number of human disorders, it is no surprise that a great deal of effort is being spent to identify ways to modulate PK activity. Some of these efforts have been directed at biomimetic approaches using large molecules patterned on those involved in the actual cellular processes (e.g., mutant ligands (U.S. app. Ser. No. 4,966,849); soluble receptors and antibodies (App. No. WO 94/10202, Kendall and Thomas, [0016] Proc. Nat'l Acad. Sci., 90:10705-09 (1994), Kim, et al., Nature, 362:841-844 (1993)); RNA ligands (Jelinek, et al., Biochemistry, 33:10450-56); Takano, et al., Mol. Bio. Cell 4:358A (1993); Kinsella, et al., Exp. Cell Res. 199:56-62 (1992); Wright, et al., J. Cellular Phys., 152:448-57) and tyrosine kinase inhibitors (WO 94/03427; WO 92/21660; WO 91/15495; WO 94/14808; U.S. Pat. No. 5,330,992; Mariani, et al., Proc. Am. Assoc. Cancer Res., 35:2268 (1994)).
  • More recently, attempts have been made to identify small molecules which act as PK inhibitors. For example, bis-monocylic, bicyclic and heterocyclic aryl compounds (PCT WO 92/20642), vinylene-azaindole derivatives (PCT WO 94/14808) and 1-cyclopropyl-4-pyridylquinolones (U.S. Pat. No. 5,330,992) have been described as PTK inhibitors. Styryl compounds (U.S. Pat. No. 5,217,999), styryl-substituted pyridyl compounds (U.S. Pat. No. 5,302,606), quinazoline derivatives (EP App. No. 0 566 266 A1), selenaindoles and selenides (PCT WO 94/03427), tricyclic polyhydroxylic compounds (PCT WO 92/21660) and benzylphosphonic acid compounds (PCT WO 91/15495) have all been described as PTK inhibitors useful in the treatment of cancer. [0017]
  • The following is offered as background information only and is not admitted to be prior art to the present invention. [0018]
  • PKs are enzymes that catalyze the phosphorylation of hydroxy groups on tyrosine, serine and threonine residues of proteins. The consequences of this seemingly simple activity are staggering; cell growth, differentiation and proliferation; i.e., virtually all aspects of cell life, in one way or another depend on PK activity. Furthermore, abnormal PK activity has been related to a host of disorders, ranging from relatively non life threatening diseases such as psoriasis to extremely virulent diseases such as glioblastoma (brain cancer). [0019]
  • The PKs can conveniently be broken down into two classes, the protein tyrosine kinases (PTKs) and the serine-threonine kinases (STKs). [0020]
  • One of the prime aspects of PK activity is involvement with growth factor receptors. Growth factor receptors are cell-surface proteins. When bound by a growth factor ligand, a growth factor receptor is converted to an active form which interacts with proteins on the inner surface of a cell membrane. This leads to phosphorylation on tyrosine residues of the receptor as well as other proteins and to the formation inside the cell of complexes with a variety of cytoplasmic signaling molecules. These complexes, in turn, affect numerous cellular responses such as cell division (proliferation), cell differentiation, cell growth, expression of metabolic effects on the extracellular microenvironment, etc. For a more complete discussion, see Schlessinger and Ullrich, [0021] Neuron, 9:303-391 (1992) which is incorporated by reference, including any drawings, as if fully set forth herein.
  • Growth factor receptors with PK activity are known as receptor tyrosine kinases (“RTKs”) . They comprise a large family of transmembrane receptors with diverse biological activity. At present, at least nineteen (19) distinct subfamilies of RTKs have been identified. An example of these is the subfamily designated the “HER” RTKs, which includes EGFR (epithelial growth factor receptor), HER2, HER3 and HER4. These RTKs consist of an extracellular glycosylated ligand binding domain, a transmembrane domain and an intracellular cytoplasmic catalytic domain that can phosphorylate tyrosine residues on proteins. [0022]
  • Another RTK subfamily consists of insulin receptor (IR), insulin-like growth factor I receptor (IGF-1R) and insulin receptor related receptor (IRR). IR and IGF-1R interact with insulin, IGF-I and IGF-II to form a heterotetramer composed of two entirely extracellular glycosylated α subunits and two β subunits which cross the cell membrane and which contain the tyrosine kinase domain. [0023]
  • A third RTK subfamily is referred to as the platelet derived growth factor receptor (“PDGFR”) group, which includes PDGFRα, PDGFRβ, CSFIR, c-kit and c-fms. These receptors consist of glycosylated extracellular domains composed of variable numbers of immunoglobin-like loops and an intracellular domain wherein the tyrosine kinase domain is interrupted by unrelated amino acid sequences. [0024]
  • Another group which, because of its similarity to the PDGFR subfamily, is sometimes subsumed in the later group, is the fetus liver kinase (“flk”) receptor subfamily. This group is composed of kinase insert domain-receptor fetal liver kinase-1 (KDR/FLK-1), flk-1R, flk-4 and fms-like tyrosine kinase 1 (flt-1). [0025]
  • One further member of the tyrosine kinase growth factor receptor family is the fibroblast growth factor (“FGF”)receptor group. This group consists of four receptors, FGFR1-4, and seven ligands, FGF1-7. While not yet well characterized, it appears that the receptors consist of a glycosylated extracellular domain containing a variable number of immunoglobin-like loops and an intracellular domain in which the PTK sequence is interrupted by regions of unrelated amino acid sequences. [0026]
  • A more complete listing of the known RTK subfamilies is described in Plowman et al., [0027] DN&P, 7(6):334-339 (1994) which is incorporated by reference, including any drawings, as if fully set forth herein.
  • In addition to the RTKs, there also exists a family of entirely intracellular PTKs called “non-receptor tyrosine kinases” or “cellular tyrosine kinases.” This latter designation, abbreviated “CTK”, will be used herein. CTKs do not contain extracellular and transmembrane domains. At present, over 24 CTKs in 11 subfamilies (Src, Frk, Btk, Csk, Abl, Zap70, Fes, Fps, Fak, Jak and Ack) have been identified. The Src subfamily appear so far to be the largest group of CTKs and includes Src, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk. For a more detailed discussion of CTKs, see Bolen, [0028] Oncogene, 8:2025-2031 (1993), which is incorporated by reference, including any drawings, as if fully set forth herein.
  • The serine-threonine kinases or STKs, like the CTKs, are predominantly intracellular although there are a few STK receptor kinases. STKs are the most common of the cytosolic kinases; i.e., kinases which perform their function in that part of the cytoplasm other than the cytoplasmic organelles and cytoskelton. The cytosol is the region within the cell where much of the cell's intermediary metabolic and biosynthetic activity occurs; e.g., it is in the cytosol that proteins are synthesized on ribosomes. [0029]
  • RTKs, CTKs and STKs have all been implicated in a host of pathogenic conditions including, significantly, cancer. Other pathogenic conditions which have been associated with PTKs include, without limitation, psoriasis, hepatic cirrhosis, diabetes, atherosclerosis, angiogenesis, restenosis, ocular diseases, rheumatoid arthritis and other inflammatory disorders, autoimmune disease and a variety of renal disorders. [0030]
  • With regard to cancer, two of the major hypotheses advanced to explain the excessive cellular proliferation that drives tumor development relate to functions known to be PK regulated. That is, it has been suggested that malignant cell growth results from a breakdown in the mechanisms that control cell division and/or differentiation. It has been shown that the protein products of a number of proto-oncogenes are involved in the signal transduction pathways that regulate cell growth and differentiation. These protein products of proto-oncogenes include the extracellular growth factors, transmembrane growth factor PTK receptors (RTKs), cytoplasmic PTKs (CTKs) and cytosolic STKs, discussed above. [0031]
  • In view of the apparent link between PK-related cellular activities and a number of human disorders, it is no surprise that a great deal of effort is being spent to identify ways to modulate PK activity. Some of these efforts have been directed at biomimetic approaches using large molecules patterned on those involved in the actual cellular processes (e.g., mutant ligands (U.S. app. Ser. No. 4,966,849); soluble receptors and antibodies (App. No. WO 94/10202, Kendall and Thomas, [0032] Proc. Nat'l Acad. Sci., 90:10705-09 (1994), Kim, et al., Nature, 362:841-844 (1993)); RNA ligands (Jelinek, et al., Biochemistry, 33:10450-56); Takano, et al., Mol. Bio. Cell 4:358A (1993); Kinsella, et al., Exp. Cell Res. 199:56-62 (1992); Wright, et al., J. Cellular Phys., 152:448-57) and tyrosine kinase inhibitors (WO 94/03427; WO 92/21660; WO 91/15495; WO 94/14808; U.S. Pat. No. 5,330,992; Mariani, et al., Proc. Am. Assoc. Cancer Res., 35:2268 (1994)).
  • More recently, attempts have been made to identify small molecules which act as PK inhibitors. For example, bis-monocylic, bicyclic and heterocyclic aryl compounds (PCT WO 92/20642), vinylene-azaindole derivatives (PCT WO 94/14808) and 1-cyclopropyl-4-pyridylquinolones (U.S. Pat. No. 5,330,992) have been described as PTK inhibitors. Styryl compounds (U.S. Pat. No. 5,217,999), styryl-substituted pyridyl compounds (U.S. Pat. No. 5,302,606), quinazoline derivatives (EP App. No. 0 566 266 A1), selenaindoles and selenides (PCT WO 94/03427), tricyclic polyhydroxylic compounds (PCT WO 92/21660) and benzylphosphonic acid compounds (PCT WO 91/15495) have all been described as PTK inhibitors useful in the treatment of cancer. [0033]
    • SUMMARY OF THE INVENTION
  • Our own efforts to identify small organic molecules which modulate PTK activity and which, therefore, should be useful in the treatment and prevention of disorders driven by abnormal PK activity, have led us to the discovery of novel 3-(substituted)-2-indolinone compounds which exhibit PK modulating ability and which are the subject of this invention. [0034]
  • The present invention relates generally to novel 3-(substituted)-2-indolinones which modulate the activity of both receptor (RTK) and non-receptor (CTK and STK) protein kinases (PKs). In addition, the present invention relates to the preparation and use of pharmacological compositions of the disclosed compounds and their physiologically acceptable salts and prodrugs in the treatment or prevention of PK driven disorders such as, by way of example and not limitation, cancer, diabetes, hepatic cirrhosis, atherosclerosis, angiogenesis and renal disease. [0035]
  • The terms “2-indolinone,” “indolin-2-one,” “2-oxindole” and “oxindole” are used interchangably herein; each refers to a chemical compound having the general structure: [0036]
    Figure US20010007033A1-20010705-C00001
  • A “3-(substituted)-2-indolinone” refers to a chemical compound having the general structure shown in Formula 1, infra. [0037]
  • A “pharmacological composition” refers to a mixture of one or more of the compounds described herein, or physiologically acceptable salts thereof, with other chemical components, such as physiologically acceptable carriers and/or excipients. The purpose of a pharmacological composition is to facilitate administration of a compound to an organism. [0038]
  • As used herein, a “physiologically acceptable carrier” refers to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. [0039]
  • An “excipient” refers to an inert substance added to a pharmacological composition to further facilitate administration of a compound. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. [0040]
  • A “prodrug” refers to an agent which is converted into the parent drug in vivo. Prodrugs are often useful because, in some situations, they may be easier to administer than the parent drug. They may, for instance, be bioavailable by oral administration whereas the parent drug is not. The prodrug may also have improved solubility in pharmacological 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 then is metabolically hydrolyzed to the carboxylic acid, the active entity, once inside the cell where water solubility is beneficial. [0041]
    • 1. BRIEF DESCRIPTION OF THE TABLES
  • Table 1 is a list of aldehydes used in the synthesis of exemplary compounds of this invention. [0042]
  • Table 2 is a list of the oxindoles used in the synthesis of exemplary compounds of this invention. [0043]
  • Table 3 is a list of the 3-(substituted)-2-indolinone products obtained by the reaction of the aldehydes of Table 1 with the oxindoles of Table 2. [0044]
  • Table 4 shows the results of biochemical testing of the compounds of Table 3. The procedures for carrying out each of the indicated assays is set forth in detail below. [0045]
    • 2. THE COMPOUNDS General Structural Features
  • In one aspect, the present invention relates to 3-(substituted)-2-indolinones having the chemical structure shown in Formula 1: [0046]
    Figure US20010007033A1-20010705-C00002
  • The “A” moiety is derived from the reaction of each of the group of aldehydes shown in Table 1 with each of 2-indolinones shown in Table 2 according to the following synthetic scheme: [0047]
    Figure US20010007033A1-20010705-C00003
  • The aldehydes and the 2-indolinones were reacted in a “combinatorial library” format. 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. As used herein, the multi-dimensional 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. Also within the scope of this invention are 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. In this invention, the combinatorial library is formed by the reaction of the aldehydes shown in Table 1 with the oxindoles shown in Table 2. The concept and practice of combinatorial library formation is well known to those of ordinary skill in the art. The result of the combinatorial library reaction of the aldehydes of Table 1 with the oxindoles of Table 2 are the 3-(substituted)-2-indolinones shown in Table 3, which compounds are the subject of this invention. [0048]
  • In Formula 1, R[0049] 1, R2, R3 and R4 are independently selected from the group consisting of hydrogen, alkyl, halo and S-sulfonamido.
    TABLE 1
    STRUCTURE NAME
    Figure US20010007033A1-20010705-C00004
         		a-tolualdehyde
    Figure US20010007033A1-20010705-C00005
         		3,4-difluorobenzaldehyde
    Figure US20010007033A1-20010705-C00006
         		3,5-difluorobenzaldehyde
    Figure US20010007033A1-20010705-C00007
         		α, α, α-trifluoro-m-tolualdehyde
    Figure US20010007033A1-20010705-C00008
         		3,5-bis(trifluoromethyl)-benzaldehyde
    Figure US20010007033A1-20010705-C00009
         		2-cyanobenzaldehyde
    Figure US20010007033A1-20010705-C00010
         		2,5-difluorobenzaldehyde
    Figure US20010007033A1-20010705-C00011
         		1-naphthaldehyde
    Figure US20010007033A1-20010705-C00012
         		2,4-dichlorobenzaldehyde
    Figure US20010007033A1-20010705-C00013
         		4-biphenylcarboxaldehyde
    Figure US20010007033A1-20010705-C00014
         		4-chlorobenzaldehyde
    Figure US20010007033A1-20010705-C00015
         		α,α,α-trifluoro-o-benzaldehyde
    Figure US20010007033A1-20010705-C00016
         		m-tolualdehyde
    Figure US20010007033A1-20010705-C00017
         		p-tolualdehyde
    Figure US20010007033A1-20010705-C00018
         		3-chlorobenzaldehyde
    Figure US20010007033A1-20010705-C00019
         		3-fluorobenzaldehyde
    Figure US20010007033A1-20010705-C00020
         		2-fluorobenzaldehdye
    Figure US20010007033A1-20010705-C00021
         		4-ethylbenzaldehyde
    Figure US20010007033A1-20010705-C00022
         		m-anisaldehyde
    Figure US20010007033A1-20010705-C00023
         		2-chlorobenzaldehyde
    Figure US20010007033A1-20010705-C00024
         		4-ethoxybenzaldehyde
    Figure US20010007033A1-20010705-C00025
         		2,4-dimethylbenzaldehyde
    Figure US20010007033A1-20010705-C00026
         		2,5-difluorobenzaldehyde
    Figure US20010007033A1-20010705-C00027
         		2,3-difluorobenzaldehyde
    Figure US20010007033A1-20010705-C00028
         		3-fluoro-p-anisaldehyde
    Figure US20010007033A1-20010705-C00029
         		2,5-dimethylbenzaldehyde
    Figure US20010007033A1-20010705-C00030
         		2-naphthaldehyde
    Figure US20010007033A1-20010705-C00031
         		4-phenoxybenzaldehyde
    Figure US20010007033A1-20010705-C00032
         		3-(4-t-butylphenoxy)benzaldehyde
    Figure US20010007033A1-20010705-C00033
         		3-(3-trifluoromethylphenoxy)benzaldehyde
    Figure US20010007033A1-20010705-C00034
         		3-(4-methylphenoxy)benzaldehyde
    Figure US20010007033A1-20010705-C00035
         		3-(3,4-dichlorophenoxy)benzaldehyde
    Figure US20010007033A1-20010705-C00036
         		3-hydroxy-4-methoxybenzaldehyde
    Figure US20010007033A1-20010705-C00037
         		5-hydroxy-2-nitrobenzaldehyde
    Figure US20010007033A1-20010705-C00038
         		2,3-dihydroxybenzaldehyde
    Figure US20010007033A1-20010705-C00039
         		3,5-dihydroxybenzaldehyde
    Figure US20010007033A1-20010705-C00040
         		3,4-dichlorobenzaldehyde
    Figure US20010007033A1-20010705-C00041
         		o-anisaldehyde
    Figure US20010007033A1-20010705-C00042
         		4-isopropylbenzaldehyde
    Figure US20010007033A1-20010705-C00043
         		4-propoxybenzaldehyde
    Figure US20010007033A1-20010705-C00044
         		3-methyl-p-anisaldehyde
    Figure US20010007033A1-20010705-C00045
         		3-benzyloxybenzaldehyde
    Figure US20010007033A1-20010705-C00046
         		4-benzyloxybenzaldehyde
    Figure US20010007033A1-20010705-C00047
         		1,4-benzodioxan-5-carboxaldehyde
    Figure US20010007033A1-20010705-C00048
         		2-chloro-4-hydroxybenaldehyde
    Figure US20010007033A1-20010705-C00049
         		3,4,5-trihydroxybenzaldehyde
    Figure US20010007033A1-20010705-C00050
         		3-(4-methoxyphenoxy)benzaldehyde
    Figure US20010007033A1-20010705-C00051
         		4-(3-dimethylaminopropoxy)benzaldehyde
    Figure US20010007033A1-20010705-C00052
         		3-furaldehyde
    Figure US20010007033A1-20010705-C00053
         		5-hydroxymethylfurfural
    Figure US20010007033A1-20010705-C00054
         		3-quinolinecarboxaldehyde
    Figure US20010007033A1-20010705-C00055
         		4-quinolinecarboxaldehyde
    Figure US20010007033A1-20010705-C00056
         		2-methoxy-1-pyrrolidinecarboxaldehyde
    Figure US20010007033A1-20010705-C00057
         		3-thiophenecarboxaldehyde
    Figure US20010007033A1-20010705-C00058
         		2-quinolinecarboxaldehyde
    Figure US20010007033A1-20010705-C00059
         		1-methylindole-3-carboxaldehyde
    Figure US20010007033A1-20010705-C00060
         		2-methylindole-3-carboxaldehyde
    Figure US20010007033A1-20010705-C00061
         		6-methyl-2-pyridine carboxaldehyde
    Figure US20010007033A1-20010705-C00062
         		4-bromo-2-thiophenecarboxaldehdye
    Figure US20010007033A1-20010705-C00063
         		4-butoxybenzaldehyde
    Figure US20010007033A1-20010705-C00064
         		2-ethoxybenzaldehyde
    Figure US20010007033A1-20010705-C00065
         		2-thiophenecarboxaldehyde
    Figure US20010007033A1-20010705-C00066
         		1-methylpyrrole-2-carboxaldehyde
    Figure US20010007033A1-20010705-C00067
         		4-fluorobenzaldehyde
    Figure US20010007033A1-20010705-C00068
         		Indole-3-carboxaldehyde
    Figure US20010007033A1-20010705-C00069
         		5-methylthiophene-2-carboxaldehyde
    Figure US20010007033A1-20010705-C00070
         		4-bromobenzaldehyde
    Figure US20010007033A1-20010705-C00071
         		pyrrole-2-carboxaldehyde
    Figure US20010007033A1-20010705-C00072
         		2-Hydroxy-6-methoxybenzaldehyde
    Figure US20010007033A1-20010705-C00073
         		3,4-Dibromo-5-methyl-2-pyrrolecarboxaldehyde
    Figure US20010007033A1-20010705-C00074
         		Ethyl-2,4-Dimethyl-5-formyl-3-pyrrolecarboxylate
    Figure US20010007033A1-20010705-C00075
         		3-Bromo-2-hydroxy-5-methoxybenzaldehyde
    Figure US20010007033A1-20010705-C00076
         		1-Hydroxy-2-naphthaldehyde
    Figure US20010007033A1-20010705-C00077
         		Ethyl-2(ethoxycarbonyl)-4-(ethoxycarbonylmethyl)-5-formyl-3-pyrrolepropionate
    Figure US20010007033A1-20010705-C00078
         		Ethyl-5-formyl-2-methyl-3-furancarboxylate
    Figure US20010007033A1-20010705-C00079
         		4-Formyl-3-methoycarbonymethyl-5-me-1H-pyrrole-2-carboxylic acid methyl ester
    Figure US20010007033A1-20010705-C00080
         		2,4-Dihydroxy-3-methylbenzaldehyde
    Figure US20010007033A1-20010705-C00081
         		2-furaldehyde
    Figure US20010007033A1-20010705-C00082
         		5-Nitro-2-furaldehyde
    Figure US20010007033A1-20010705-C00083
         		4-Ethoxy-3-methoxybenzaldehyde
    Figure US20010007033A1-20010705-C00084
         		3,4-Dihydroxybenzaldehyde
    Figure US20010007033A1-20010705-C00085
         		2,4-Dimethoxybenzaldehyde
    Figure US20010007033A1-20010705-C00086
         		3,5-Dimethyl-4-ethyl-2-pyrrolcarboxaldehyde
    Figure US20010007033A1-20010705-C00087
         		2,4,6-trimethoxybenzaldehyde
    Figure US20010007033A1-20010705-C00088
         		4-Hydroxybenzaldehyde
    Figure US20010007033A1-20010705-C00089
         		4-(Dimethylamino)-benzaldehyde
    Figure US20010007033A1-20010705-C00090
         		2-chloro-4-fluorobenzaldehyde
    Figure US20010007033A1-20010705-C00091
         		3-Nitrobenzaldehyde
    Figure US20010007033A1-20010705-C00092
         		4-Fluoro-2-(trifluoromethyl)benzaldehyde
    Figure US20010007033A1-20010705-C00093
         		2,4,6-Trifluorobenzaldehyde
    Figure US20010007033A1-20010705-C00094
         		4-Hydroxy-2-methoxybenzaldehyde
    Figure US20010007033A1-20010705-C00095
         		3,4-Dimethoxybenzaldehyde
    Figure US20010007033A1-20010705-C00096
         		Salicylaldehyde
    Figure US20010007033A1-20010705-C00097
         		Benzaldehyde
    Figure US20010007033A1-20010705-C00098
         		5-(Methylthio)thiophene-2-carboxaldehyde
    Figure US20010007033A1-20010705-C00099
         		2,4-Dihydroxy-6-methylbenzaldehyde
    Figure US20010007033A1-20010705-C00100
         		3-Ethoxy-4-hydroxybenzaldehyde
    Figure US20010007033A1-20010705-C00101
         		2-Hydroxy-5-methoxybenzaldehyde
    Figure US20010007033A1-20010705-C00102
         		2-Imidazolecarboxaldehyde
    Figure US20010007033A1-20010705-C00103
         		1-Methyl-2-formylbenzimidazole
    Figure US20010007033A1-20010705-C00104
         		4-Chloro-1-methylpyrazole-3-carboxaldehyde
    Figure US20010007033A1-20010705-C00105
         		2,3-dimethyl-5-formylthiophene
    Figure US20010007033A1-20010705-C00106
         		2-Formyl-4,5,6,7-tetrahydroindole
    Figure US20010007033A1-20010705-C00107
         		3-Chloromethyl-5-nitrosalicylaldehyde
    Figure US20010007033A1-20010705-C00108
         		5-Chlorothiophene-2-carboxaldehyde
    Figure US20010007033A1-20010705-C00109
         		3,5-dimethyl-5-formylpyrrole
    Figure US20010007033A1-20010705-C00110
         		3-t-Butyl-4-hydroxybenzaldehyde
    Figure US20010007033A1-20010705-C00111
         		3-t-Butyl-5-bromo-4-hydroxybenzaldehyde
    Figure US20010007033A1-20010705-C00112
         		3,5-Di-tert-butyl-4-hydroxybenzaldehyde hemihydrate
    Figure US20010007033A1-20010705-C00113
         		3-t-Butyl-4-hydroxy-5-nitrobenzaldehyde
    Figure US20010007033A1-20010705-C00114
         		2,4,6-Trihydroxybenzaldehyde
    Figure US20010007033A1-20010705-C00115
         		2-formyl-5-nitrothiophene
    Figure US20010007033A1-20010705-C00116
         		4-Carboxybenzaldehyde
    Figure US20010007033A1-20010705-C00117
         		2,4-difluorobenzaldehyde
    Figure US20010007033A1-20010705-C00118
         		3,5-Dimethyl-4-hydroxybenzaldehyde
    Figure US20010007033A1-20010705-C00119
         		3-Chloro-4-hydroxy-5-t-butylbenzaldehyde
    Figure US20010007033A1-20010705-C00120
         		2-Nitrothiophene-4-carboxaldehyde
    Figure US20010007033A1-20010705-C00121
         		4-(Dibutylamino)benzaldehyde
    Figure US20010007033A1-20010705-C00122
         		4-(Trifluoromethyl)benzaldehyde
    Figure US20010007033A1-20010705-C00123
         		4,6-Dimethoxy-salicylaldehyde
    Figure US20010007033A1-20010705-C00124
         		2,3,4-Trihydroxybenzaldehyde
    Figure US20010007033A1-20010705-C00125
         		2-Hydroxy-3-methoxybenzaldehyde
    Figure US20010007033A1-20010705-C00126
         		5-Bromo-3,4-dihydroxybenzaldehyde
    Figure US20010007033A1-20010705-C00127
         		3,4-Diacetoxybenzaldehyde
    Figure US20010007033A1-20010705-C00128
         		4-Hydroxy-3-methylbenzaldehyde
    Figure US20010007033A1-20010705-C00129
         		2-Bromobenzaldehyde
    Figure US20010007033A1-20010705-C00130
         		2,4-Dihydroxybenzaldehyde
    Figure US20010007033A1-20010705-C00131
         		2-Hydroxy-4-methoxybenzaldehyde
    Figure US20010007033A1-20010705-C00132
         		3-Bromobenzaldehyde
    Figure US20010007033A1-20010705-C00133
         		3,5-Di-tert-butyl-2-hydroxybenzaldehyde
    Figure US20010007033A1-20010705-C00134
         		4-Dimethylamino-1-naphthaldehyde
    Figure US20010007033A1-20010705-C00135
         		4-Hydroxy-3-nitrobenzaldehyde
    Figure US20010007033A1-20010705-C00136
         		3-Hydroxy-4-nitrobenzaldehyde
    2,3,6,7-Tetrahydro-8-hydroxy-1H,5H-benzo[ij]quinolizine.9
    Figure US20010007033A1-20010705-C00137
         		3,5-Diisopropyl-4-hydroxybenzaldehyde
    Figure US20010007033A1-20010705-C00138
         		Benzo[b]furan-2-carboxaldehyde
    Figure US20010007033A1-20010705-C00139
         		1-(4-chlorophenyl)pyrrole-2-carboxaldehyde
    Figure US20010007033A1-20010705-C00140
         		5-Ethyl-2-furaldehyde
    Figure US20010007033A1-20010705-C00141
         		3,4-Dimethylthieno[b]thiophene-2-carboxaldehyde
    Figure US20010007033A1-20010705-C00142
         		3-Bromothiophene-2-carboxaldehyde
    Figure US20010007033A1-20010705-C00143
         		6-Bromo-2-hydroxy-3-methoxybenzaldehyde
    Figure US20010007033A1-20010705-C00144
         		5-Methyrfurfural
    Figure US20010007033A1-20010705-C00145
         		3-Methyl-1H-Pyrazole-5-carboxaldehyde
    Figure US20010007033A1-20010705-C00146
         		6-Methoxy-4-methylsalicylaldehyde
    Figure US20010007033A1-20010705-C00147
         		4-(4-Formylpiperazine-1-yl)benzaldehyde
    Figure US20010007033A1-20010705-C00148
         		4-(4-Formylmorphorlino-1-yl)benzaldehyde
    Figure US20010007033A1-20010705-C00149
         		5-Chloro-3-methoxycarbonyl-4-methoxycarbonylmethyl-pyrrole-2-carboxaldehyde
    Figure US20010007033A1-20010705-C00150
         		1-(4-chlorobenzyl)-4-bromo-pyrazole-5-carboxaldehyde
    Figure US20010007033A1-20010705-C00151
         		Imidazole-4-carboxaldehyde
    Figure US20010007033A1-20010705-C00152
         		5-Ethoxycarbonyl-4-methyl-3-methylcarbonyl-pyrrole-2-carboxaldehyde
    Figure US20010007033A1-20010705-C00153
         		5-t-Butyl-4-hydroxy-3-iodobenzaldehyde
    Figure US20010007033A1-20010705-C00154
         		5-Bromofuran-2-carboxaldehyde
    Figure US20010007033A1-20010705-C00155
         		1,4-Dimethyl-3-formylcarbazole
    Figure US20010007033A1-20010705-C00156
         		1,4-Dihydroxy-2-formyl-5,6,7,8-tetrahydronaphthatene
    Figure US20010007033A1-20010705-C00157
         		5-fluoroisatin
    Figure US20010007033A1-20010705-C00158
         		isatin
    Figure US20010007033A1-20010705-C00159
         		5-ethyl-2-formylthiophene
    Figure US20010007033A1-20010705-C00160
         		4-methoxybenzaldehyde
    Figure US20010007033A1-20010705-C00161
         		4-diethylaminobenzaldehyde
    Figure US20010007033A1-20010705-C00162
         		3,5-diethylpyrrole-2-carboxaldehyde
    Figure US20010007033A1-20010705-C00163
         		3-Bromo-5-chloro-2-hydroxybenzaldehyde
    Figure US20010007033A1-20010705-C00164
         		2-(4-chlorophenylthio)benzaldehyde
    Figure US20010007033A1-20010705-C00165
         		6-Chloropiperonal
    Figure US20010007033A1-20010705-C00166
         		Chromone-3-carboxaldehyde
    Figure US20010007033A1-20010705-C00167
         		3-Cyanobenzaldehyde
    Figure US20010007033A1-20010705-C00168
         		4-Cyanobenzaldehyde
    Figure US20010007033A1-20010705-C00169
         		2,5-dihydroxybenzaldehyde
    Figure US20010007033A1-20010705-C00170
         		2,3-Dimethoxybenzaldehyde
    Figure US20010007033A1-20010705-C00171
         		2,5-Dimethoxybenzaldehyde
    Figure US20010007033A1-20010705-C00172
         		2,6-Dimethoxybenzaldehyde,
    Figure US20010007033A1-20010705-C00173
         		3,5-Dimethoxybenzaldehyde
    Figure US20010007033A1-20010705-C00174
         		4-Dimethylamino-2-methoxybenzaldehyde
    Figure US20010007033A1-20010705-C00175
         		Fluorene-2-carboxaldehyde
    Figure US20010007033A1-20010705-C00176
         		2-Fluoro-3-(trifluoromethyl)benzaldehyde
    Figure US20010007033A1-20010705-C00177
         		2-Fluoro-5-(trifluoromethyl)benzaldehyde
    Figure US20010007033A1-20010705-C00178
         		2-Fluoro-6-(trifluoromethyl)benzaldehyde
    Figure US20010007033A1-20010705-C00179
         		2-Formylphenoxyacetic acid
    Figure US20010007033A1-20010705-C00180
         		3-Methoxy-5,-methylenedioxybenzaldehyde
    Figure US20010007033A1-20010705-C00181
         		2-Methoxy-1-naphthaldehyde
    Figure US20010007033A1-20010705-C00182
         		4-Methoxy-1-naphthaldehyde
    Figure US20010007033A1-20010705-C00183
         		4-(Methylthio)benzaldehyde
    Figure US20010007033A1-20010705-C00184
         		3-Methylthiophene-2-carboxaldehyde
    Figure US20010007033A1-20010705-C00185
         		3-Phenoxybenzaldehyde
    Figure US20010007033A1-20010705-C00186
         		Pyridine-2-carboxaldehyde
    Figure US20010007033A1-20010705-C00187
         		Pyridine-3-carboxaldehyde
    Figure US20010007033A1-20010705-C00188
         		Pyridine-4-carboxaldehyde
    Figure US20010007033A1-20010705-C00189
         		4-Pyrrolidinobenzaldehyde, 98+%
    Figure US20010007033A1-20010705-C00190
         		1,2,3,6-Tetrahydrobenzaldehyde
    Figure US20010007033A1-20010705-C00191
         		2,3,4-Trimethoxybenzaldehyde
    Figure US20010007033A1-20010705-C00192
         		2,4,5-Trimethoxybenzaldehyde
    Figure US20010007033A1-20010705-C00193
         		3,4,5-Trimethoxybenzaldehyde
    Figure US20010007033A1-20010705-C00194
         		1-Acetyl-3-indolecarboxaldehyde
    Figure US20010007033A1-20010705-C00195
         		6-Chloro-3-formylchromone
    Figure US20010007033A1-20010705-C00196
         		5-(2-Chlorophenyl)furfural
    Figure US20010007033A1-20010705-C00197
         		2-Chloro-3-quinolinecarboxaldehyde
    Figure US20010007033A1-20010705-C00198
         		6,8-Dibromo-3-formylchromone
    Figure US20010007033A1-20010705-C00199
         		2,5-Dimethoxy-3-tetrahydrofuracarboxaldehyde
    Figure US20010007033A1-20010705-C00200
         		4,5-Dimethyl-2-furaldehyde
    Figure US20010007033A1-20010705-C00201
         		9-Ethyl-3-carbazolecarboxaldehyde
    Figure US20010007033A1-20010705-C00202
         		3-Formyl-6,7-dimethylchromone
    Figure US20010007033A1-20010705-C00203
         		3-formyl-6-isopropylchromone
    Figure US20010007033A1-20010705-C00204
         		3-formyl-6-methylchromone
    Figure US20010007033A1-20010705-C00205
         		3-formyl-6-nitrochromone
    Figure US20010007033A1-20010705-C00206
         		5-Formyluracil
    Figure US20010007033A1-20010705-C00207
         		5-Methoxyindole-3-carboxaldehyde
    Figure US20010007033A1-20010705-C00208
         		1-Methylisatin
    Figure US20010007033A1-20010705-C00209
         		5-(2-Nitrophenyl)furfural
    Figure US20010007033A1-20010705-C00210
         		(S)-(−)-Perillaldehyde
    Figure US20010007033A1-20010705-C00211
         		2-(Trifluoroacetyl)thiophene
    Figure US20010007033A1-20010705-C00212
         		3,5-diisopropyl-4-methoxybenzaldehyde
    Figure US20010007033A1-20010705-C00213
         		4-benzyloxy-3,5-diisopropylbenzaldehyde
    Figure US20010007033A1-20010705-C00214
         		3-t-butyl-4-methoxybenzaldehyde
    Figure US20010007033A1-20010705-C00215
         		4-benzyloxy-3-t-butylbenzaldehyde
    Figure US20010007033A1-20010705-C00216
         		3-bromo-5-t-butyl-4-methoxybenzaldehyde
    Figure US20010007033A1-20010705-C00217
         		4-benzyloxy-3-bromo-5-t-butylbenzaldehyde
    Figure US20010007033A1-20010705-C00218
         		3-t-butyl-5-chloro-4-methoxybenzaldehyde
    Figure US20010007033A1-20010705-C00219
         		4-benzyloxy-3-t-butyl-5-chlorobenzaldehyde
    Figure US20010007033A1-20010705-C00220
         		3-t-butyl-5-iodo-4-methoxybenzaldehyde
    Figure US20010007033A1-20010705-C00221
         		4-benzyloxy-3-t-butyl-5-iodobenzaldehyde
    Figure US20010007033A1-20010705-C00222
         		3-t-butyl-4-methoxy-5-nitrobenzaldehyde
    Figure US20010007033A1-20010705-C00223
         		3,5-di-t-butyl-4-methoxybenzaldehyde
    Figure US20010007033A1-20010705-C00224
         		4-benzyloxy-3,5-di-t-butylbenzaldehyde
    Figure US20010007033A1-20010705-C00225
         		3,5-dimethyl-4-methoxybenzaldehyde
    Figure US20010007033A1-20010705-C00226
         		4-benzyloxy-3,5-dimethylbenzaldehyde
    Figure US20010007033A1-20010705-C00227
         		5-bromo-2-hydroxy-3-methoxy-benzaldehyde
    Figure US20010007033A1-20010705-C00228
         		5-bromosalicyaldehyde 201
    Figure US20010007033A1-20010705-C00229
         		2-hydroxy-5-nitrobenzaldehyde
    Figure US20010007033A1-20010705-C00230
         		4-hydroxy-2-nitro-3-methoxybenzaldehyde
    Figure US20010007033A1-20010705-C00231
         		3-ethoxysalicyaldehyde
    Figure US20010007033A1-20010705-C00232
         		3,5-dichlorosalicylaldehyde
    Figure US20010007033A1-20010705-C00233
         		5-chlorosalicyaldehyde
    Figure US20010007033A1-20010705-C00234
         		4-(diethylamino)salicyaldehyde
    Figure US20010007033A1-20010705-C00235
         		3,5-dibromosalicyaldehyde
    Figure US20010007033A1-20010705-C00236
         		3-fluorosalicyaldehyde
    Figure US20010007033A1-20010705-C00237
         		3-bromo-4-hydroxybenzaldehyde
    Figure US20010007033A1-20010705-C00238
         		5-chlorosalicyaldehyde
    Figure US20010007033A1-20010705-C00239
         		2-nitrobenzaldehyde
    Figure US20010007033A1-20010705-C00240
         		4-nitrobenzaldehyde
    Figure US20010007033A1-20010705-C00241
         		4-tert-butylbenzaldehyde
    Figure US20010007033A1-20010705-C00242
         		5-bromo-2-thiophenecarboxaldehyde
  • [0050]
    TABLE 2
    STRUCTURE NAME
    Figure US20010007033A1-20010705-C00243
         		5,7-dibromo- oxindole
    Figure US20010007033A1-20010705-C00244
         		5-iodo- oxindole
    Figure US20010007033A1-20010705-C00245
         		5-bromo- 4-methyl- oxindole
    Figure US20010007033A1-20010705-C00246
         		5-methyl- aminosulf- onyloxindole
    Figure US20010007033A1-20010705-C00247
         		5-(4-tri- fluoro- methyl- anilino- sulfonyl)- oxindole
    Figure US20010007033A1-20010705-C00248
         		5-(morpho- lin-4-yl-sulf- onyl)ox- indole
    Figure US20010007033A1-20010705-C00249
         		5-(2-chloro- ethyl)ox- indole
    Figure US20010007033A1-20010705-C00250
         		5,7-Di- bromo-4- methylox- indole
  • [0051]
    TABLE 3
    COMPOUND
    NUMBER NAME
    10717/H02 3-(2-ethoxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10717/H03 3-[(thien-2-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10717/H04 3-[(1-nethylpyrrol-2-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10717/H05 3-(4-fluorobenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10717/H06 3-[(indol-3-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10717/H07 3-[(2-methylthien-5-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10717/H08 3-(4-bromobenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10717/H09 3-[(pyrrol-2-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10717/H10 3-(2-hydroxy-6-methoxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10717/H11 3-[(3,4-dibromo-2-methylpyrrol-5-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10718/H02 3-[(2,4-dimethyl-3-ethoxycarbonylpyrrol-5-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10718/H03 3-(3-bromo-2-hydroxy-5-methoxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10718/H04 3-[1-hydroxynapth-2-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10718/H05 3-[[2-ethoxycarbonyl-3-(2-ethoxycarbonyl)ethyl-4-(ethoxycarbonylmethyl)pyrrol-5-yl]methylidenyl]-
    5,7-dibromo-4-methyl-2-indolinone
    10718/H06 3-[(2-methyl-3-ethoxycarbonylfuran-5-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10718/H07 3-[(2,3-dimethoxycarbonyl-5-methylpyrrol-4-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10718/H08 3-(4-chloro-3-nitrobenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10718/H09 3-(2,4-dihydroxy-3-methylbenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10718/H10 3-[(furan-2-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10718/H11 3-[(2-nitrofuran-5-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10719/H02 3-(4-ethoxy-3-methoxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10719/H03 3-(3,4-dihydroxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10719/H04 3-(2,4-dimethoxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10719/H05 3-[(2,4-dimethyl-3-ethylpyrrol-5-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10719/H06 3-(2,4,6-trimethoxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10719/H07 3-(4-hydroxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10719/H08 3-(4-dimethylaminobenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10719/H09 3-(2-chloro-4-fluorobenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10719/H10 3-(3-nitrobenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10719/H11 3-[4-fluoro-2-(trifluoromethyl)benzylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10720/H02 3-(2,4,6-trifluorobenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10720/H03 3-(4-hydroxy-2-methoxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10720/H04 3-(3,4-dimethoxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10720/H05 3-(2-hydroxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10720/H06 3-benzylidenyl-5,7-dibromo-4-methyl-2-indolinone
    10720/H07 3-[(2-methylmercaptothien-5-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10720/H08 3-(2,4-dihydroxy-6-methylbenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10720/H09 3-(3-ethoxy-4-hydroxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10720/H10 3-(2-hydroxy-5-methoxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10720/H11 3-[(imidazol-2-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10721/H02 3-[(1-methylbenzimidazol-2-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10721/H03 3-[(4-chloro-1-methylpyrazol-3-yl)methylidenyl[-5,7-dibromo-4-methyl-2-indolinone
    10721/H04 3-[(2,3-dimethylthien-5-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10721/H05 3-[(4,5,6,7-tetrahydroindol-2-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10721/H06 3-(3-chloromethyl-2-hydroxy-5-nitrobenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10721/H07 3-[(2-chlorothien-5-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10721/H08 3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10721/H09 3-(3-t-butyl-4-hydroxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10721/H10 3-(3-bromo-5-t-butyl-4-hydroxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10721/H11 3-(3,5-di-t-butyl-4-hydroxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10722/H02 3-(3-t-butyl-4-hydroxy-5-nitrobenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10722/H03 3-(2,4,6-trihydroxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10722/H04 3-[(2-nitrothien-5-yl)methyidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10722/H05 3-(4-carboxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10722/H06 3-(2,4-difluorobenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10722/H07 3-(3,5-dimethyl-4-hydroxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10722/H08 3-(3-t-butyl-5-chloro-4-hydroxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10722/H09 3-[(2-nitrothien-4-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10722/H10 3-(4-di-n-butylaminobenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10722/H11 3-[4-(trifluoromethyl)benzylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10723/H02 3-(2,3,4-trihydroxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10723/H03 3-(2-hydroxy-3-methoxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10723/H04 3-(3-bromo-4,5-dihydroxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10723/H05 3-(3,4-diacetoxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10723/H06 3-(4-hydroxy-3-methylbenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10723/H07 3-(2-bromobenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10723/H08 3-(2,4-dihydroxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10723/H09 3-(2-hydroxy-4-methoxybenylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10723/H10 3-(3-bromobenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10723/H11 3-(3,5-di-t-butyl-2-hydroxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10724/H02 3-[(1-dimethylaminonapth-4-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10724/H03 3-(4-hydroxy-3-nitrobenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10724/H04 3-(3-hydroxy-4-nitrobenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10724/H05 3-[(8-hydroxy-2,3,6,7-tetrahydro-1H,5H-benzo[ij]quinolizin-9-yl(methylidenyl]-5,7-dibromo-4-methyl
    2-indolinone
    10724/H06 3-(3,5-diisopropyl-4-hydroxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10724/H07 3-[(benzo[b]furan-2-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10724/H08 3-(2-hydroxy-4,6-dimethoxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10724/H09 3-[1-(4-chlorophenyl)pyrrol-2-yl]methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10724/H10 3-[(2-ethylfuran-5-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10724/H11 3-[(3,4-dimethylthienol[2,3-b]thien-2-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10725/H02 3-[(3-bromothien-2-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10725/H03 3-(2-bromo-6-hydroxy-5-methoxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10725/H04 3-[(2-methylfuran-5-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10725/H05 3-[(3-methylpyrazol-5-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10725/H06 3-(2-hydroxy-6-methoxy-4-methylbenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10725/H07 3-[4-(4-formylpiperazin-1-yl)benzylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10725/H08 3-[4-(morpholin-1-yl)benzylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10725/H09 3-[[2-chloro-4-methoxycarbonyl-3-(methoxycarbonylmethyl)pyrrol-5-yl]methylidenyl]-5,7-dibromo-4-
    methyl-2-indolinone
    10725/H10 3-[[4-bromo-2-(4-chlorophenyl)pyrazol-3-yl]methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10725/H11 3-[(imidazol-4-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10726/H02 3-(2-nitrobenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10726/H03 3-[(2-ethoxycarbonyl-4-methoxycarbonyl-3-methylpyrrol-5-yl)methylidenyl]-5,7-dibromo-4-methyl-2-
    indolinone
    10726/H04 3-(3-t-butyl-4-hydroxy-5-methylbenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10726/H05 3-[(2-bromofuran-5-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10726/H06 3-[(1,3-dimethylpyrrol-4-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10726/H07 3-[(5,8-dihydroxy-1,2,3,4-tetrahydronapth-6-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10726/H08 3-(5-fluoro-2-oxindol-3-idenyl)-5,7-dibromo-4-methyl-2-indolinone
    10726/H09 3-(2-oxindol-3-idenyl)-5,7-dibromo-4-methyl-2-indolinone
    10726/H10 3-[(2-ethylthien-5-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10726/H11 3-(4-methoxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10727/H02 3-(4-diethylaminobenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10727/H03 3-[(2,4-diethylpyrrol-5-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10727/H04 3-(3-bromo-5-chloro-2-hydroxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10727/H05 3-[2-(4-chlorophenylmercapto)benzylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10727/H06 3-[(5-chlorobenzodioxolan-6-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10727/H07 3-[(1,4-benzopyranon-3-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10727/H08 3-(3-cyanobenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10727/H09 3-(4-cyanobenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10727/H10 3-(2,5-dihydroxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10727/H11 3-(2,3-dimethoxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10728/H02 3-(2,5-dimethoxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10728/H03 3-(2,6-dimethoxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10728/H04 3-(3,5-dimethoxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10728/H05 3-(4-dimethylamino-2-methoxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10728/H06 3-[(fluoren-2-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10728/H07 3-[2-fluoro-3-(trifluoromethyl)benzylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10728/H08 3-[2-fluoro-5-(trifluoromethyl)benzylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10728/H09 3-[2-fluoro-6-(trifluoromethyl)benzylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10728/H10 3-(2-carboxymethoxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10728/H11 3-[(4-methoxybenzodioxolan-6-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10729/H02 3-[(2-methoxynapth-1-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10729/H03 3-[(1-methoxynapth-4-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10729/H04 3-(4-methylmercaptobenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10729/H05 3-[(3-methylthien-2-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10729/H06 3-(3-phenoxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10729/H07 3-[(pyrid-2-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10729/H08 3-[(pyrid-3-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10729/H09 3-[(pyrid-4-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10729/H10 3-[4-(pyrrolidin-1-yl)benzylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10729/H11 3-[(cyclohexen-3-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10730/H02 3-(2,3,4-trimethoxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10730/H03 3-(2,4,5-trimethoxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10730/H04 3-(3,4,5-trimethoxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10730/H05 3-[(1-acetylindol-3-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10730/H06 3-[(6-chloro-1,4-benzofuranon-3-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10730/H07 3-[2-[(2-chlorophenyl)furan-5-yl]methyidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10730/H08 3-[(2-chloroquinolin-3-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10730/H09 3-[(6,8-dibromo-1,4-benzofuranon-3-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10730/H10 3-[(2,5-dimethoxytetrahydrofuran-3-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10730/H11 3-[(2,3-dimethylfuran-5-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10731/H02 3-[(9-ethylcarbazol-3-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10731/H03 3-[(6,7-dimethyl-1,4-benzopyron-3-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10731/H04 3-[[4-(propen-2-yl)cyclohexen-1-yl]methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10731/H05 3-[(6-isopropyl-1,4-benzopyron-3-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10731/H06 3-[(6-methyl-1,4-benzopyron-3-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10731/H07 3-[(6-nitro-1,4-benzopyron-3-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10731/H08 3-[(pyrimid-2,4-dion-5-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10731/H09 3-[(5-methoxyindol-3-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10731/H10 3-(1-methyl-2-oxindol-3-idenyl)-5,7-dibromo-4-methyl-2-indolinone
    10731/H11 3-[2-[2-(nitrophenyl)furan-5-yl]methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10732/H02 3-[2-(thien-2-yl)-2-(trifluoromethyl)ethylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10732/H03 3-(3,5-diisopropyl-4-methoxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10732/H04 3-(3,5-diisopropyl-4-phenoxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10732/H05 3-(3-t-butyl-4-methoxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10732/H06 3-(4-benzyloxy-3-t-butylbenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10732/H07 3-(3-bromo-5-t-butyl-4-methoxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10732/H08 3-(4-benzyloxy-3-bromo-5-t-butylbenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10732/H09 3-(3-t-butyl-5-chloro-4-methoxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10732/H10 3-(4-benzyloxy-5-t-butyl-3-chlorobenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10732/H11 3-(3-t-butyl-5-iodo-4-methoxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10733/H02 3-(4-benzyloxy-3-t-butyl-5-iodobenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10733/H03 3-(3-t-butyl-4-methoxy-5-nitrobenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10733/H04 3-(3,5-di-t-butyl-4-methoxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10733/H05 3-(4-benzyloxy-3,5-di-t-butylbenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10733/H06 3-(3,5-dimethyl-4-methoxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10733/H07 3-(4-benzyloxy-3,5-dimethylbenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10733/H08 3-(5-bromo-2-hydroxy-3-methoxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10733/H09 3-(5-bromo-2-hydroxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10733/H10 3-(2-hydroxy-5-nitrobenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10733/H11 3-(4-hydroxy-3-methoxy-2-nitrobenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10734/A02 3-(3-ethoxy-2-hydroxybenzylidenyl)-5,7-dibromo-2-indolinone
    10734/A03 3-(3,5-dichloro-2-hydroxybenzylidenyl)-5,7-dibromo-2-indolinone
    10734/A04 3-(5-chloro-2-hydroxybenzylidenyl)-5,7-dibromo-2-indolinone
    10734/A05 3-(4-diethylamino-2-hydroxybenzylidenyl)-5,7-dibromo-2-indolinone
    10734/A06 3-(4-nitrobenzylidenyl)-5,7-dibromo-2-indolinone
    10734/A07 3-(3-5,dibromo-2-hydroxybenzylidenyl)-5,7-dibromo-2-indolinone
    10734/A08 3-(3-fluoro-2-hydroxybenzylidenyl)-5,7-dibromo-2-indolinone
    10734/A09 3-(3-bromo-4-hydroxybenzylidenyl)-5,7-dibromo-2-indolinone
    10734/A10 3-(4-t-butylbenzylidenyl)-5,7-dibromo-2-indolinone
    10734/A11 3-[(2-bromothien-5-yl)methylidenyl]-5,7-dibromo-2-indolinone
    10734/B02 3-(3-ethoxy-2-hydroxybenzylidenyl)-5-iodo-2-indolinone
    10734/B03 3-(3,5-dichloro-2-hydroxybenzylidenyl)-5-iodo-2-indolinone
    10734/B04 3-(5-chloro-2-hydroxybenzylidenyl)-5-iodo-2-indolinone
    10734/B05 3-(4-diethylamino-2-hydroxybenzylidenyl)-5-iodo-2-indolinone
    10734/B06 3-(4-nitrobenzylidenyl)-5-iodo-2-indolinone
    10734/B07 3-(3,5-dibromo-2-hydroxybenzylidenyl)-5-iodo-2-indolinone
    10734/B08 3-(3-fluoro-2-hydroxybenzylidenyl)-5-iodo-2-indolinone
    10734/B09 3-(3-bromo-4-hydroxybenzylidenyl)-5-iodo-2-indolinone
    10734/B10 3-(4-t-butylbenzlidenyl)-5-iodo-2-indolinone
    10734/B11 3-[(2-bromothien-5-yl)methylidenyl]-5-iodo-2-indolinone
    10734/C02 3-(3-ethoxy-2-hydroxybenzylidenyl)-5-bromo-4-methyl-2-indolinone
    10734/C03 3-(3,5-dichloro-2-hydroxybenzylidenyl)-5-bromo-4-methyl-2-indolinone
    10734/C04 3-(5-chloro-2-hydroxybenzylidenyl)-5-bromo-4-methyl-2-indolinone
    10734/C05 3-(4-diethylamino-2-hydroxybenzylidenyl)-5-bromo-4-methyl-2-indolinone
    10734/C06 3-(4-nitrobenzylidenyl)-5-bromo-4-methyl-2-indolinone
    10734/C07 3-(3,5-dibromo-2-hydroxybenzylidenyl)-5-bromo-4-methyl-2-indolinone
    10734/C08 3-(3-fluoro-2-hydroxybenzylidenyl)-5-bromo-4-methyl-2-indolinone
    10734/C09 3-(3-bromo-4-hydroxybenzylidenyl)-5-bromo-4-methyl-2-indolinone
    10734/C10 3-(4-t-butylbenzylidenyl)-5-bromo-4-methyl-2-indolinone
    10734/C11 3-[(2-bromothien-5-yl)methylidenyl]-5-bromo-4-methyl-2-indolinone
    10734/D02 3-(3-ethoxy-2-hydroxybenzylidenyl)-5-methylaminosulfonyl-2-indolinone
    10734/D03 3-(3,5-dichloro-2-hydroxybenzylidenyl)-5-methylaminosulfonyl-2-indolinone
    10734/D04 3-(5-chloro-2-hydroxybenzylidenyl)-5-methylaminosulfonyl-2-indolinone
    10734/D05 3-(4-diethylamino-2-hydroxybenzylidenyl)-5-methylaminosulfonyl-2-indolinone
    10734/D06 3-(4-nitrobenzylidenyl)-5-methylaminosulfonyl-2-indolinone
    10734/D07 3-(3,5-dibromo-2-hydroxybenzylidenyl)-5-methylaminsulfonyl-2-indolinone
    10734/D08 3-(3-fluoro-2-hydroxybenzylidenyl)-5-methylaminosulfonyl-2-indolinone
    10734/D09 3-(3-bromo-4-hydroxybenzylidenyl)-5-methylaminosulfonyl-2-indolinone
    10734/D10 3-(4-t-butylbenzylidenyl)-5-methylaminosulfonyl-2-indolinone
    10734/D11 3-[(2-bromothien-5-yl)methylidenyl]-5-methylaminosulfonyl-2-indolinone
    10734/E02 3-(3-ethoxy-2-hydroxybenzylidenyl)-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10734/E03 3-(3,5-dichloro-2-hydroxybenzylidenyl)-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10734/E04 3-(5-chloro-2-hydroxybenzylidenyl)-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10734/E05 3-(4-diethylamino-2-hydroxybenzylidenyl)-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10734/E06 3-(4-nitrobenzylidenyl)-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10734/E07 3-(3,5-dibromo-2-hydroxybenzylidenyl)-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10734/E08 3-(3-fluoro-2-hydroxybenzylidenyl)-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10734/E09 3-(3-bromo-4-hydroxybenzylidenyl)-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10734/E10 3-(4-t-butylbenzylidenyl)-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10734/E11 3-[(2-bromothien-5-yl)methylidenyl]-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10734/F02 3-(3-ethoxy-2-hydroxybenzylidenyl)-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10734/F03 3-(3,5-dichloro-2-hydroxybenzylidenyl)-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10734/F04 3-(5-chloro-2-hydroxybenzylidenyl)-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10734/F05 3-(4-diethylamino-2-hydroxybenzylidenyl)-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10734/F06 3-(4-nitrobenzylidenyl)-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10734/F07 3-(3,5-dibromo-2-hydroxybenzylidenyl)-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10734/F08 3-(3-fluoro-2-hydroxybenzylidenyl)-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10734/F09 3-(3-bromo-4-hydroxybenzylidenyl)-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10734/F10 3-(4-t-butylbenzylidenyl)-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10734/F11 3-[(2-bromothien-5-yl)methylidenyl]-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10734/G02 3-(3-ethoxy-2-hydroxybenzylidenyl)-5-(2-chloroethyl)-2-indolinone
    10734/G03 3-(3,5-dichloro-2-hydroxybenzylidenyl)-5-(2-chloroethyl)-2-indolinone
    10734/G04 3-(5-chloro-2-hydroxybenzylidenyl)-5-(2-chloroethyl)-2-indolinone
    10734/G05 3-(4-diethylamino-2-hydroxybenzylidenyl)-5-(2-chloroethyl)-2-indolinone
    10734/G06 3-(4-nitrobenzylidenyl)-5-(2-chloroethyl)-2-indolinone
    10734/G07 3-(3,5-dibromo-2-hydroxybenzylidenyl)-5-(2-chloroethyl)-2-indolinone
    10734/G08 3-(3-fluoro-2-hydroxybenzylidenyl)-5-(2-chloroethyl)-2-indolinone
    10734/G09 3-(3-bromo-4-hydroxybenzylidenyl)-5-(2-chloroethyl)-2-indolinone
    10734/G10 3-(4-t-butylbenzylidenyl)-5-(2-chloroethyl)-2-indolinone
    10734/G11 3-[(2-bromothien-5-yl)methylidenyl]-5-(2-chloroethyl)-2-indolinone
    10734/H02 3-(3-ethoxy-2-hydroxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10734/H03 3-(3,5-dichloro-2-hydroxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10734/H04 3-(5-chloro-2-hydroxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10734/H05 3-(4-diethylamino-2-hydroxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10734/H06 3-(4-nitrobenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10734/H07 3-(3,5-dibromo-2-hydroxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10734/H08 3-(3-fluoro-2-hydroxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10734/H09 3-(3-bromo-4-hydroxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10734/H10 3-(4-t-butylbenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10734/H11 3-[(2-bromothien-5-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10735/A02 3-(2-methylbenzylidenyl)-5,7-dibromo-2-indolinone
    10735/A03 3-(3,4-difluorobenzylidenyl)-5,7-dibromo-2-indolinone
    10735/A04 3-(3,5-difluorobenzylidenyl)-5,7-dibromo-2-indolinone
    10735/A05 3-[(3-(trifluoromethyl)benzylidenyl]-5,7-dibromo-2-indolinone
    10735/A06 3-[(3,5-di-(trifluoromethyl)benzylidenyl]-5,7-dibromo-2-indolinone
    10735/A07 3-(2-cyanobenzylidenyl)-5,7-dibromo-2-indolinone
    10735/A08 3-(2,6-difluorobenzylidenyl)-5,7-dibromo-2-indolinone
    10735/A09 3-[(napth-1-yl)methylidenyl]-5,7-dibromo-2-indolinone
    10735/A10 3-(2,4-dichlorobenzylidenyl)-5,7-dibromo-2-indolinone
    10735/A11 3-[(biphenyl-4-yl)methylidenyl]-5,7-dibromo-2-indolinone
    10735/B02 3-(2-methylbenzylidenyl)-5-iodo-2-indolinone
    10735/B03 3-(3,4-difluorobenzylidenyl)-5-iodo-2-indolinone
    10735/B04 3-(3,5-difluorobenzylidenyl)-5-iodo-2-indolinone
    10735/B05 3-[(3-trifluoromethyl)benzylidenyl]-5-iodo-2-indolinone
    10735/B06 3-[(3,5-di(trifluoromethyl)benzylidenyl]-5-iodo-2-indolinone
    10735/B07 3-(2-cyanobenzylidenyl)-5-iodo-2-indolinone
    10735/B08 3-(2,6-difluorobenzylidenyl)-5-ioso-2-indolinone
    10735/B09 3-[(napth-1-yl)methylidenyl]-5-iodo-2-indolinone
    10735/B10 3-(2,4-dichlorobenzylidenyl)-5-iodo-2-indolinone
    10735/B11 3-[(biphenyl-4-yl)methylidenyl]-5-iodo-2-indolinone
    10735/C02 3-(2-methylbenzylidenyl)-5-bromo-4-methyl-2-indolinone
    10735/C03 3-(3,4-difluorobenzylidenyl)-5-bromo-4-methyl-2-indolinone
    10735/C04 3-(3,5-difluorobenzylidenyl)-5-bromo-4-methyl-2-indolinone
    10735/C05 3-[(3-trifluoromethyl)benzylidenyl]-5-bromo-4-methyl-2-indolinone
    10735/C06 3-[(3,5-di-trifluoromethyl)benzylidenyl]-5-bromo-4-methyl-2-indolinone
    10735/C07 3-(2-cyanobenzylidenyl)-5-bromo-4-methyl-2-indolinone
    10735/C08 3-(2,6-difluorobenzylidenyl)-5-bromo-4-methyl-2-indolinone
    10735/C09 3-[(napth-1-yl)methylidenyl]-5-bromo-4-methyl-2-indolinone
    10735/C10 3-(2,4-dichlorobenzylidenyl)-5-bromo-4-methyl-2-indolinone
    10735/C11 3-[(biphenyl-4-yl)methylidenyl]-5-bromo-4-methyl-2-indolinone
    10735/D02 3-(2-methylbenzylidenyl)-5-methylaminosulfonyl-2-indolinone
    10735/D03 3-(3,4-difluorobenzylidenyl)-5-methylaminosulfonyl-2-indolinone
    10735/D04 3-(3,5-difluorobenzylidenyl)-5-methylaminosulfonyl-2-indolinone
    10735/D05 3-[(3-trifluoromethyl)benzylidenyl]-5-methylaminosulfonyl-2-indolinone
    10735/D06 3-[(3,5-di-(trifluoromethyl)benzylidenyl]-5-methylaminosulfonyl-2-indolinone
    10735/D07 3-(2-cyanobenzylidenyl)-5-methylaminosulfonyl-2-indolinone
    10735/D08 3-(2,6-difluorobenzylidenyl)-5-methylaminosulfonyl-2-indolinone
    10735/D09 3-[(napth-1-yl)methylidenyl]-5-methylaminosulfonyl-2-indolinone
    10735/D10 3-(2,4-dichlorobenzylidenyl)-5-methylaminosulfonyl-2-indolinone
    10735/D11 3-[(biphenyl-4-yl)methylidenyl]-5-methylaminosulfonyl-2-indolinone
    10735/E02 3-(2-methylbenzylidenyl)-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10735/E03 3-(3,4-difluorobenzylidenyl)-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10735/E04 3-(3,5-difluorobenzylidenyl)-5-[4-(trifluoromethyl)phenylaminosulfonyl -2-indolinone
    10735/E05 3-[(3-(trifluoromethyl)benzylidenyl]-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10735/E06 3-[(3,5-di-(trifluoromethyl)benzylidenyl]-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10735/E07 3-(2-cyanobenzylidenyl)-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10735/E08 3-(2,6-difluorobenzylidenyl)-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10735/E09 3-[(napth-1-yl)methylidenyl]-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10735/E10 3-(2,4-dichlorobenzylidenyl-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10735/E11 3-[(biphenyl-4-yl)methylidenyl]-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10735/F02 3-(2-methylbenzylidenyl)-5-(morpholin-1-yl)sulfonyl]-2-indolinone
    10735/F03 3-(3,4-difluorobenzylidenyl)-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10735/F04 3-(3,5-difluorobenzylidenyl)-5-(morpholin-1-yl)sulfonyl]-2-indolinone
    10735/F05 3-[(3-trifluoromethyl)benzylidenyl]-5-(morpholin-1-yl)sulfonyl]-2-indolinone
    10735/F06 3-[(3,5-di-(trifluoromethyl)benzylidenyl]-5-(morpholin-1-yl)sulfonyl]-2-indolinone
    10735/F07 3-(2-cyanobenzylidenyl)-5-(morpholin-1-yl)sulfonyl]-2-indolinone
    10735/F08 3-(2,6-difluorobenzylidenyl)-5-(morpholin-1-yl)sulfonyl]-2-indolinone
    10735/F09 3-[(napth-1-yl)methylidenyl]-5-(morpholin-1-yl)sulfonyl]-2-indolinone
    10735/F10 3-(2,4-dichlorobenzylidenyl)-5-(morpholin-1-yl)sulfonyl]-2-indolinone
    10735/F11 3-[(biphenyl-4-yl)methylidenyl]-5-(morpholin-1-yl)sulfonyl]-2-indolinone
    10735/G02 3-(2-methylbenzylidenyl)-5-(2-chloroethyl)-2-indolinone
    10735/G03 3-(3,4-difluorobenzylidenyl)-5-(2-chloroethyl)-2-indolinone
    10735/G04 3-(3,5-difluorobenzylidenyl)-5-(2-chloroethyl)-2-indolinone
    10735/G05 3-[(3-trifluoromethyl)benzylidenyl]-5-(2-chloroethyl)-2-indolinone
    10735/G06 3-[(3,5-di(trifluoromethyl)benzylidenyl]-5-(2-chloroethyl)-2-indolinone
    10735/G07 3-(2-cyanobenzylidenyl)-5-(2-chloroethyl)-2-indolinone
    10735/G08 3-(2,6-difluorobenzylidenyl)-5-(2-chloroethyl)-2-indolinone
    10735/G09 3-[(napth-1-yl)methylidenyl]-5-(2-chloroethyl)-2-indolinone
    10735/G10 3-(2,4-dichlorobenzylidenyl)-5-(2-chloroethyl)-2-indolinone
    10735/G11 3-[(biphenyl-4-yl)methylidenyl]-5-(2-chloroethyl)-2-indolinone
    10735/H02 3-(2-methylbenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10735/H03 3-(3,4-difluorobenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10735/H04 3-(3,5-difluorobenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10735/H05 3-[(3-(trifluoromethyl)benzylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10735/H06 3-[(3,5-di-(trifluoromethyl)benzylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10735/H07 3-(2-cyanobenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10735/H08 3-(2,6-difluorobenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10735/H09 3-[(napth-1-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10735/H10 3-(2,4-dichlorobenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10735/H11 3-[(biphenyl-4-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10736/A02 3-(4-chlorobenzylidenyl)-5,7-dibromo-2-indolinone
    10736/A03 3-[2-(trifluoromethyl)benzylidenyl]-5,7-dibromo-2-indolinone
    10736/A04 3-(3-methylbenzylidenyl)-5,7-dibromo-2-indolinone
    10736/A05 3-(4-methylbenzylidenyl)-5,7-dibromo-2-indolinone
    10736/A06 3-(3-chlorobenzylidenyl)-5,7-dibromo-2-indolinone
    10736/A07 3-(3-fluorobenzylidenyl)-5,7-dibromo-2-indolinone
    10736/A08 3-(2-fluorobenzylidenyl)-5,7-dibromo-2-indolinone
    10736/A09 3-(4-ethylbenzylidenyl)-5,7-dibromo-2-indolinone
    10736/A10 3-(3-methoxybenzylidenyl)-5,7-dibromo-2-indolinone
    10736/A11 3-(2-chlorobenzylidenyl)-5,7-dibromo-2-indolinone
    10736/B02 3-(4-chlorobenzylidenyl)-5-iodo-2-indolinone
    10736/B03 3-[2-(trifluoromethyl)benzylidenyl]-5-iodo-2-indolinone
    10736/B04 3-(3-methylbenzylidenyl)-5-iodo-2-indolinone
    10736/B05 3-(4-methylbenzylidenyl)-5-iodo-2-indolinone
    10736/B06 3-(3-chlorobenzylidenyl)-5-iodo-2-indolinone
    10736/B07 3-(3-fluorobenzylidenyl)-5-iodo-2-indolinone
    10736/B08 3-(2-fluorobenzylidenyl)-5-iodo-2-indolinone
    10736/B09 3-(4-ethylbenzylidenyl)-5-iodo-2-indolinone
    10736/B10 3-(3-methoxybenzylidenyl)-5-iodo-2-indolinone
    10736/B11 3-(2-chlorobenzylidenyl)-5-iodo-2-indolinone
    10736/C02 3-(4-chlorobenzylidenyl)-5-bromo-4-methyl-2-indolinone
    10736/C03 3-[2-(trifluoromethyl)benzylidenyl]-5-bromo-4-methyl-2-indolinone
    10736/C04 3-(3-methylbenzylidenyl)-5-bromo-4-methyl-2-indolinone
    10736/C05 3-(4-methylbenzylidenyl)-5-bromo-4-methyl-2-indolinone
    10736/C06 3-(3-chlorobenzylidenyl)-5-bromo-4-methyl-2-indolinone
    10736/C07 3-(3-fluorobenylidenyl)-5-bromo-4-methyl-2-indolinone
    10736/C08 3-(2-fluorobenzylidenyl)-5-bromo-4-methyl-2-indolinone
    10736/C09 3-(4-ethylbenzylidenyl)-5-bromo-4-methyl-2-indolinone
    10736/C10 3-(3-methoxybenzylidenyl)-5-bromo-4-methyl-2-indolinone
    10736/C11 3-(2-chlorobenzylidenyl)-5-bromo-4-methyl-2-indolinone
    10736/D02 3-(4-chlorobenzylidenyl)-5-methylaminosulfonyl-2-indolinone
    10736/D03 3-[2-(trfluoromethyl)benzylidenyl]-5-methylaminosulfonyl-2-indolinone
    10736/D04 3-(3-methylbenzylidenyl)-5-methylaminosulfonyl-2-indolinone
    10736/D05 3-(4-methylbenzylidenyl)-5-methylaminosulfonyl-2-indolinone
    10736/D06 3-(3-chlorobenzylidenyl)-5-methylaminosulfonyl-2-indolinone
    10736/D07 3-(3-fluorobenzylidenyl)-5-methylaminosulfonyl-2-indolinone
    10736/D08 3-(2-fluorobenzylidenyl)-5-methylaminosulfonyl-2-indolinone
    10736/D09 3-(4-ethylbenzylidenyl)-5-methylaminosulfonyl-2-indolinone
    10736/D10 3-(3-methoxybenzylidenyl)-5-methylaminosulfonyl-2-indolinone
    10736/D11 3-(2-chlorobenzylidenyl)-5-methylaminosulfonyl-2-indolinone
    10736/E02 3-(4-chlorobenzylidenyl)-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10736/E03 3-[2-(trifluoromethyl)benzylidenyl]-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10736/E04 3-(3-methylbenzylidenyl)-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10736/E05 3-(4-methylbenzylidenyl)-5-[4-(trifluoromethyl)phenylaminosulfonyl)-2-indolinone
    10736/E06 3-(3-chlorobenzylidenyl)-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10736/E07 3-(3-fluorobenzylidenyl)-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10736/E08 3-(2-fluorobenzylidenyl)-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10736/E09 3-(4-ethylbenzylidenyl)-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10736/E10 3-(3-methoxybenzylidenyl)-5-[4-(trifluoromethyl)phenylaminsulfonyl]-2-indolinone
    10736/E11 3-(2-chlorobenzylidenyl)-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10736/F02 3-(4-chlorobenzylidenyl)-5-(morpholin-1-yl)sulfonyl-2-indolinyl
    10736/F03 3-[2-(trifluoromethyl)benzylidenyl]-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10736/F04 3-(3-methylbenzylidenyl)-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10736/F05 3-(4-methylbenzylidenyl)-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10736/F06 3-(3-chlorobenzylidenyl)-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10736/F07 3-(3-fluorobenzylidenyl)-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10736/F08 3-(2-fluorobenzylidenyl)-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10736/F09 3-(4-ethylbenzylidenyl)-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10736/F10 3-(3-methoxybenzylidenyl)-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10736/F11 3-(2-chlorobenzylidenyl)-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10736/G02 3-(4-chlorobenzylidenyl)-5-(2-chloroethyl)-2-indolinone
    10736/G03 3-[2-(trifluoromethyl)benzylidenyl]-5-(2-chloroethyl)-2-indolinone
    10736/G04 3-(3-methylbenzylidenyl)-5-(2-chloroethyl)-2-indolinone
    10736/G05 3-(4-methylbenzylidenyl)-5-(2-chloroethyl)-2-indolinone
    10736/G06 3-(3-chlorobenzylidenyl)-5-(2-chloroethyl)-2-indolinone
    10736/G07 3-(3-fluorobenzylidenyl)-5-(2-chloroethyl)-2-indolinone
    10736/G08 3-(2-fluorobenzylidenyl)-5-(2-chloroethyl)-2-indolinone
    10736/G09 3-(4-ethylbenzylidenyl)-5-(2-chloroethyl)-2-indolinone
    10736/G10 3-(3-methoxybenzylidenyl)-5-(2-chloroethyl)-2-indolinone
    10736/G11 3-(2-chlorobenzylidenyl)-5-(2-chloroethyl)-2-indolinone
    10736/H02 3-(4-chlorobenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10736/H03 3-[2-(trifluoromethyl)benzylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10736/H04 3-(3-methylbenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10736/H05 3-(4-methylbenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10736/H06 3-(3-chlorobenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10736/H07 3-(3-fluorobenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10736/H08 3-(2-fluorobenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10736/H09 3-(4-ethylbenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10736/H10 3-(3-methoxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10736/H11 3-(2-chlorobenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10737/A02 3-(4-ethoxybenzylidenyl)-5,7-dibromo-2-indolinone
    10737/A03 3-(2,4-dimethylbenzylidenyl)-5,7-dibromo-2-indolinone
    10737/A04 3-(2,5-difluorobenzylidenyl)-5,7-dibromo-2-indolinone
    10737/A05 3-(2,3-difluorobenzylidenyl)-5,7-dibromo-2-indolinone
    10737/A06 3-(3-fluoro-4-methoxybenzylidenyl)-5,7-dibromo-2-indolinone
    10737/A07 3-(2,5-dimethylbenzylidenyl)-5,7-dimbromo-2-indolinone
    10737/A08 3-[(napth-2-yl)methylidenyl]-5,7-dibromo-2-indolinone
    10737/A09 3-(4-phenoxybenzylidenyl)-5,7-dibromo-2-indolinone
    10737/A10 3-[3-(4-t-butylphenoxy)benzylidenyl]-5,7-dibromo-2-indolinone
    10737/A11 3-[3-[3-(trifluoromethyl)phenoxy]benzylidenyl]-5,7-dibromo-2-indolinone
    10737/B02 3-(4-ethoxybenzylidenyl)-5-iodo-2-indolinone
    10737/B03 3-(2,4-dimethylbenzylidenyl)-5-iodo-2-indolinone
    10737/B04 3-(2,5-difluorobenzylidenyl)-5-iodo-2-indolinone
    10737/B05 3-(2,3-difluorobenzylidenyl)-5-iodo-2-indolinone
    10737/B06 3-(3-fluoro-4-methoxybenzylidenyl)-5-iodo-2-indolinone
    10737/B07 3-(2,5-dimethylbenzylidenyl)-5-iodo-2-indolinone
    10737/B08 3-[(napth-2-yl)methylidenyl]-5-iodo-2-indolinone
    10737/B09 3-(4-phenoxybenzylidenyl)-5-iodo-2-indolinone
    10737/B10 3-[3-(4-t-butylphenoxy)benzylidenyl]-5-iodo-2-indolinone
    10737/B11 3-[3-[3-(trifluoromethyl)phenoxy]benzylidenyl]-5-iodo-2-indolinone
    10737/C02 3-(4-ethoxybenzylidenyl)-5-bromo-4-methyl-2-indolinone
    10737/C03 3-(2,4-dimethylbenzylidenyl)-5-bromo-4-methyl-2-indolinone
    10737/C04 3-(2,5-difluorobenzylidenyl)-5-bromo-4-methyl-2-indolinone
    10737/C05 3-(2,3-difluorobenzylidenyl)-5-bromo-4-methyl-2-indolinone
    10737/C06 3-(3-fluoro-4-methoxybenzylidenyl)-5-bromo-4-methyl-2-indolinone
    10737/C07 3-(2,5-dimethylbenzylidenyl)-5-bromo-4-methyl-2-indolinone
    10737/C08 3-[(napth-2-yl)methylidenyl]-5-bromo-4-methyl-2-indolinone
    10737/C09 3-(4-phenoxybenzylidenyl)-5-bromo-4-methyl-2-indolinone
    10737/C10 3-[3-(4-t-butylphenoxy)benzylidenyl]-5-bromo-4-methyl-2-indolinone
    10737/C11 3-[3-[3-(trifluoromethyl)phenoxy]benzylidenyl]-5-bromo-4-methyl-2-indolinonr
    10737/D02 3-(4-ethoxybenzylidenyl)-5-methylaminosulfonyl-2-indolinone
    10737/D03 3-(2,4-dimethylbenzylidenyl)-5-methylaminosulfonyl-2-indolinone
    10737/D04 3-(2,5-difluorobenzylidenyl)-5-methylaminosulfonyl-2-indolinone
    10737/D05 3-(2,3-difluorobenzylidenyl)-5-methylaminosulfonyl-2-indolinone
    10737/D06 3-(3-fluoro-4-methoxybenzylidenyl)-5-methylaminosulfonyl-2-indolinone
    10737/D07 3-(2,5-dimethylbenzylidenyl)-5-methylaminosulfonyl-2-indolinone
    10737/D08 3-[(napth-2-yl)methylidenyl]-5-methylaminosulfonyl-2-indolinone
    10737/D09 3-(4-phenoxybenzylidenyl)-5-methylaminosulfonyl-2-indolinone
    10737/D10 3-[3-(4-t-butylphenoxy)benzylidenyl]-5-methylaminosulfonyl-2-indolinone
    10737/D11 3-[3-[3-(trifluoromethyl)phenoxy]benzylidenyl]-5-methylaminosulfonyl-2-indolinone
    10737/E02 3-(4-ethoxybenzyidenyl)-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10737/E03 3-(2,4-dimethylbenzylidenyl)-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10737/E04 3-(2,5-difluorobenzylidenyl)-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10737/E05 3-(2,3-difluorobenzylidenyl)-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10737/E06 3-(3-fluoro-4-methoxybenzylidenyl)-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10737/E07 3-(2,5-dimethylbenzylidenyl)-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10737/E08 3-[(napth-2-yl)methylidenyl]-5-[4(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10737/E09 3-(4-phenoxybenzylidenyl)-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10737/E10 3-[3-(4-t-butylphenoxy)benzylidenyl]-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-
    indolinone
    10737/E11 3-[3-[3-(trifluoromethyl)phenoxy]benzylidenyl]-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-
    indolinone
    10737/F02 3-(4-ethoxybenzylidenyl)-5-(moprpholin-1-yl)sulfonyl-2-indolinone
    10737/F03 3-(2,4-dimethylbenzylidenyl)-5-(moprpholin-1-yl)sulfonyl-2-indolinone
    10737/F04 3-(2,5-difluorobenzylidenyl)-5-(moprpholin-1-yl)sulfonyl-2-indolinone
    10737/F05 3-(2,3-difluorobenzylidenyl)-5-(moprpholin-1-yl)sulfonyl-2-indolinone
    10737/F06 3-(3-fluoro-4-methoxybenzylidenyl)-5-(moprpholin-1-yl)sulfonyl-2-indolinone
    10737/F07 3-(2,5-dimethylbenzylidenyl)-5-(moprpholin-1-yl)sulfonyl-2-indolinone
    10737/F08 3-[(napth-2-yl)methylidenyl]-5-moprpholin-1-yl)sulfonyl-2-indolinone
    10737/F09 3-(4-phenoxybenzylidenyl)-5-(moprpholin-1-yl)sulfonyl-2-indolinone
    10737/F10 3-[3-(4-t-butylphenoxy)benzylidenyl]-5-(moprpholin-1-yl)sulfonyl-2-indolinone
    10737/F11 3-[3-[3-(trifluoromethyl)phenoxy]benzylidenyl]-5-(moprpholin-1-yl)sulfonyl-2-indolinone
    10737/G02 3-(4-ethoxybenzylidenyl)-5-(2-chloroethyl)-2-indolinone
    10737/G03 3-(2,4-dimethylbenzylidenyl)-5-(2-chloroethyl)-2-indolinone
    10737/G04 3-(2,5-dufluorobenzylidenyl)-5-(2-chloroethyl)-2-indolinone
    10737/G05 3-(2,3-difluorobenzylidenyl)-5-(2-chloroethyl)-2-indolinone
    10737/G06 3-(3-fluoro-4-methoxybenzylidenyl)-5-(2-chloroethyl)-2-indolinone
    10737/G07 3-(2,5-dimethylbenzylidenyl)-5-(2-chloroethyl)-2-indolinone
    10737/G08 3-[(napth-2-yl)methylidenyl]-5-(2-chloroethyl)-2-indolinone
    10737/G09 3-(4-phenoxybenzylidenyl)-5-(2-chloroethyl)-2-indolinone
    10737/G10 3-[3-(4-t-butylphenoxy)benzylidenyl]-5-(2-chloroethyl)-2-indolinone
    10737/G11 3-[3-[3-(trifluoromethyl)phenoxy]benzylidenyl]-5-(2-chloroethyl)-2-indolinone
    10737/H02 3-(4-ethoxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10737/H03 3-(2,4-dimethylbenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10737/H04 3-(2,5-difluorobenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10737/H05 3-(2,3-difluorobenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10737/H06 3-(3-fluoro-4-methoxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10737/H07 3-(2,5-dimethylbenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10737/H08 3-[(napth-2-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10737/H09 3-(4-phenoxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10737/H10 3-[3-(4-t-butylphenoxy)benzylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10737/H11 3-[3-[3-(trifluoromethyl)phenoxy]benzylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10738/A02 3-[3-(4-methylphenoxy)benzylidenyl]-5,7-dibromo-2-indolinone
    10738/A03 3[3-(3,4-dichlorophenoxy)benzylidenyl]-5,7-dibromo-2-indolinone
    10738/A04 3-(3-hydroxy-4-methoxybenzylidenyl)-5,7-dibromo-2-indolinone
    10738/A05 3-(5-hydroxy-2-nitrobenzylidenyl)-5,7-dibromo-2-indolinone
    10738/A06 3-(2,3-dihydrobenzylidenyl)-5,7-dibromo-2-indolinone
    10738/A07 3-(3,5-dihydroxybenzylidenyl)-5,7-dibromo-2-indolinone
    10738/A08 3-(3,4-dichlorobenzylidenyl)-5,7-dibromo-2-indolinone
    10738/A09 3-(2-methoxybenzylidenyl)-5,7-dibromo-2-indolinone
    10738/A10 3-(4-isopropylbenzylidenyl)-5,7-dibromo-2-indolinone
    10738/A11 3-(4-n-propoxybenzylidenyl)-5,7-dibromo-2-indolinone
    10738/B02 3-[3-(4-methylphenoxy)benzylidenyl]-5-iodo-2-indolinone
    10738/B03 3-[3-(3,4-dichlorophenoxy)benzylidenyl]-5-iodo-2-indolinone
    10738/B04 3-(3-hydroxy-4-methoxybenzylidenyl)-5-iodo-2-indolinone
    10738/B05 3-(5-hydroxy-2-nitrobenzylidenyl)-5-iodo-2-indolinone
    10738/B06 3-(2,3-dihydroxybenzylidenyl)-5-iodo-2-indolinone
    10738/B07 3-(3,5-dihydroxybenzylidenyl)-5-iodo-2-indolinone
    10738/B08 3-(3,4-dichlorobenzylidenyl)-5-iodo-2-indolinone
    10738/B09 3-(2-methoxybenzylidenyl)-5-iodo-2-indolinone
    10738/B10 3-(4-isopropylbenzylidenyl)-5-iodo-2-indolinone
    10738/B11 3-(4-n-propoxybenzylidenyl)-5-iodo-2-indolinone
    10738/C02 3-[3-(4-methylphenoxy)benzylidenyl]-5-bromo-4-methyl-2-indolinone
    10738/C03 3-[3-(3,4-dichlorophenoxy)benzylidenyl]-5-bromo-4-methyl-2-indolinone
    10738/C04 3-(3-hydroxy-4-methoxybenzylidenyl)-5-bromo-4-methyl-2-indolinone
    10738/C05 3-(5-hydroxy-2-nitrobenzylidenyl)-5-bromo-4-methyl-2-indolinone
    10738/C06 3-(2,3-dihydroxybenzylidenyl)-5-bromo-4-methyl-2-indolinone
    10738/C07 3-(3,5-dihydroxybenzylidenyl)-5-bromo-4-methyl-2-indolinone
    10738/C08 3-(3,4-dichlorobenzylidenyl)-5-bromo-4-methyl-2-indolinone
    10738/C09 3-(2-methoxybenzylidenyl)-5-bromo-4-methyl-2-indolinone
    10738/C10 3-(4-isopropylbenzylidenyl)-5-bromo-4-methyl-2-indolinone
    10738/C11 3-(4-n-propoxybenzylidenyl)-5-bromo-4-methyl-2-indolinone
    10738/D02 3-[3-(4-methylphenoxy)benzylidenyl]-5-methylaminosulfonyl-2-indolinone
    10738/D03 3-[3-(3,4-dichlorophenoxy)benzylidenyl]-5-methylaminsulfonyl-2-indolinone
    10738/D04 3-(3-hydroxy-4-methoxybenzylidenyl)-5-methylaminosulfonyl-2-indolinone
    10738/D05 3-(5-hydroxy-2-nitrobenzylidenyl)-5-methylaminosulfonyl-2-indolinone
    10738/D06 3-(2,3-dihydroxybenzylidenyl)-5-methylaminosulfonyl-2-indolinone
    10738/D07 3-(3,5-dihydroxybenzylidenyl)-5-methylaminosulfonyl-2-indolinone
    10738/D08 3-(3,4-dichlorobenzylidenyl)-5-methylaminosulfonyl-2-indolinone
    10738/D09 3-(2-methoxybenzylidenyl)-5-methylaminosulfonyl-2-indolinone
    10738/D10 3-(4-isopropylbenzylidenyl)-5-methylaminosulfonyl-2-indolinone
    10738/D11 3-(4-n-propoxybenzylidenyl)-5-methylaminosulfonyl-2-indolinone
    10738/E02 3-[3-(4-methylphenoxy)benzylidenyl]-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10738/E03 3-[3-(3,4-dichlorophenoxy)benzylidenyl]-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10738/E04 3-(3-hydroxy-4-methoxybenzylidenyl)-5-[4-(trifluoromethyl)phenylaminsulfonyl]-2-indolinone
    10738/E05 3-(5-hydroxy-2-nitrobenzylidenyl)-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10738/E06 3-(2,3-dihydroxybenzylidenyl)-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10738/E07 3-(3,5-dihydroxybenzylidenyl)-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10738/E08 3-(3,4-dichlorobenzylidenyl)-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10738/E09 3-(2-methoxybenzylidenyl)-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10738/E10 3-(4-isopropylbenzylidenyl)-5-[4(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10738/E11 3-(4-n-propoxybenzylidenyl)-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10738/F02 3-[3-(4-methylphenoxy)benzylidenyl]-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10738/F03 3-[3-(3,4-dichlorophenoxy)benzylidenyl]-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10738/F04 3-(3-hydroxy-4-methoxybenzylidenyl)-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10738/F05 3-(5-hydroxy-2-nitrobenzylidenyl)-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10738/F06 3-(2,3-dihydroxybenzylidenyl)-5-(morpholin-1-yl)sulfonyl-1-indolinone
    10738/F07 3-(3,5-dihydroxybenzylidenyl)-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10738/F08 3-(3,4-dichlorobenzylidenyl)-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10738/F09 3-(2-methoxybenzylidenyl)-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10738/F10 3-(4-isopropylbenzylidenyl)-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10738/F11 3-(4-n-propoxybenzylidenyl)-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10738/G02 3-[3-(4-methylphenoxy)benzylidenyl]-5-(2-chloroethyl)-2-indolinone
    10738/G03 3-[3-(3,4-dichlorophenoxy)benzylidenyl]-5-(2-chloroethyl)-2-indolinone
    10738/G04 3-(3-hydroxy-4-methoxybenzylidenyl)-5-(2-chloroethyl)-2-indolinone
    10738/G05 3-(5-hydroxy-2-nitrobenzylidenyl)-5-(2-chloroethyl)-2-indolinone
    10738/G06 3-(2,3-dihydroxybenzylidenyl)-5-(2-chloroethyl)-2-indolinone
    10738/G07 3-(3,5-dihydroxybenzylidenyl)-5-(2-chloroethyl)-2-indolinone
    10738/G08 3-(3,4-dichlorobenzylidenyl)-5-(2-chloroethyl)-2-indolinone
    10738/G09 3-(2-methoxybenzylidenyl)-5-(2-chloroethyl)-2-indolinone
    10738/G10 3-(4-isopropylbenzylidenyl)-5-(2-chloroethyl)-2-indolinone
    10738/G11 3-(4-n-propoxybenzylidenyl)-5-(2-chloroethyl)-3-indolinone
    10738/H02 3-[3-(4-methylphenoxy)benzylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10738/H03 3-[3-(3,4-dichlorophenoxy)benzylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10738/H04 3-(3-hydroxy-4-methoxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10738/H05 3-(5-hydroxy-2-nitrobenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10738/H06 3-(2,3-dihydroxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10738/H07 3-(3,5-dihydroxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10738/H08 3-(3,4-dichlorobenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10738/H09 3-(2-methoxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10738/H10 3-(4-isopropylbenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10738/H11 3-(4-n-propoxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10739/A02 3-(4-methoxy-3-methylbenzylidenyl)-5,7-dibromo-2-indolinone
    10739/A03 3-(3-benzyloxybenzylidenyl)-5,7-dibromo-2-indolinone
    10739/A04 3-(4-benzyloxybenzylidenyl)-5,7-dibromo-2-indolinone
    10739/A05 3-[(1,4-benzodioxan-3-yl)methylidenyl]-5,7-dibromo-2-indolinone
    10739/A06 3-(2-chloro-4-hydroxybenzylidenyl)-5,7-dibromo-2-indolinone
    10739/A07 3-(3,4,5-trihydroxybenzylidenyl)-5,7-dibromo-2-indolinone
    10739/A08 3-[3-(4-methoxyphenoxy)benzylidenyl]-5,7-dibromo-2-indolinone
    10739/A09 3-[4-(3-dimethylamino-n-propoxy)benzylidenyl]-5,7-dibromo-2-indolinone
    10739/A10 3-[(furan-3-yl)methylidenyl]-5,7-dibromo-2-indolinone
    10738/A11 3-[(2-hydroxymethylfuran-5-yl)methylidenyl]-5,7-dibromo-2-indolinone
    10739/B02 3-(4-methoxy-3-methylbenzylidenyl)-5-iodo-2-indolinone
    10739/B03 3-(3-benzyloxybenzylidenyl)-5-iodo-2-indolinone
    10739/B04 3-(4-benzyloxybenzylidenyl)-5-iodo-2-indolinone
    10739/B05 3-[(1,4-benzodioxan-3-yl)methylidenyl]-5-iodo-2-indolinone
    10739/B06 3-(2-chloro-4-hydroxybenzylidenyl)-5-iodo-2-indolinone
    10739/B07 3-(3,4,5-trihydroxybenzylidenyl)-5-iodo-2-indolinone
    10739/B08 3-[3-(4-methoxyphenoxy)benzylidenyl]-5-iodo-2-indolinone
    10739/B09 3-[4-(3-dimethylamino-n-propoxy)benzylidenyl]-5-iodo-2-indolinone
    10739/B10 3-[(furan-3-yl)methylidenyl]-5-iodo-2-indolinone
    10739/B11 3-[(2-hydroxymethylfuran-5-yl)methylidenyl]-5-iodo-2-indolinone
    10739/C02 3-(4-methoxy-3-methylbenzylidenyl)-5-bromo-4-methyl-2-indolinone
    10739/C03 3-(3-benzyloxybenzylidenyl)-5-bromo-4-methyl-2-indolinone
    10739/C04 3-(4-benzyloxybenzylidenyl)-5-bromo-4-methyl-2-indolinone
    10739/C05 3-[(1,4-benzodioxan-3-yl)methylidenyl]-5-bromo-4-methyl-2-indolinone
    10739/C06 3-(2-chloro-4-hydroxybenzylidenyl)-5-bromo-4-methyl-2-indolinone
    10739/C07 3-(3,4,5-trihydroxybenzylidenyl)-5-bromo-4-methyl-2-indolinone
    10739/C08 3-[3-(4-methoxyphenoxy)benzylidenyl]-5-bromo-4-methyl-2-indolinone
    10739/C09 3-[4-(3-dimethylamino-n-propoxy)benzylidenyl]-5-bromo-4-methyl-2-indolinone
    10739/C10 3-[(furan-3-yl)methylidenyl]-5-bromo-4-methyl-2-indolinone
    10739/C11 3-[(2-hydroxymethylfuran-5-yl)methylidenyl]-5-bromo-4-methyl-2-indolinone
    10739/D02 3-(4-methoxy-3-methylbenzylidenyl)-5-methylaminosulfonyl-2-indolinone
    10739/D03 3-(3-benzyloxybenzylidenyl)-5-methylaminosulfonyl-2-indolinone
    10739/D04 3-(4-benzyloxybenzylidenyl)-5-methylaminosulfonyl-2-indolinone
    10739/D05 2-[(1,4-benzodioxan-3-yl)methylidenyl]-5-methylaminosulfonyl-2-indolinone
    10739/D06 3-(2-chloro-4-hydroxybenzylidenyl)-5-methylaminosulfonyl-2-indolinone
    10739/D07 3-(3,4,5-trihydroxybenzylidenyl)-5-methylaminosulfonyl-2-indolinone
    10739/D08 3-[3-(4-methoxyphenoxy)benzylidenyl]-5-methylaminosulfonyl-2-indolinone
    10739/D09 3-[4-(3-dimethylamino-n-propoxy)benzylidenyl]-5-methylaminosulfonyl-2-indolinone
    10739/D10 3-[(furan-3-yl)methylidenyl]-5-methylaminosulfonyl-2-indolinone
    10739/D11 3-[(2-hydroxymethylfuran-5-yl)methylidenyl]-5-methylaminosulfonyl-2-indolinone
    10739/E02 3-(4-methoxy-3-methylbenzylidenyl)-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10739/E03 3-(3-benzyloxybenzylidenyl)-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10739/E04 3-(4-benzyloxybenzylidenyl)-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10739/E05 3-[(1,4-benzodioxan-3-yl)methylidenyl]-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10739/E06 3-(2-chloro-4-hydroxybenzylidenyl)-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10739/E07 3-(3,4,5-trihydroxybenzylidenyl)-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10739/E08 3-[3-(4-methoxyphenoxy)benzylidenyl]-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10739/E09 3-[4-(3-dimethylamino-n-propoxy)benzylidenyl]-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-
    indolinone
    10739/E10 3-[(furan-3-yl)methylidenyl]-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10739/E11 3-[(2-hydroxymethylfuran-5-yl)methylidenyl]-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10739/F02 3-(4-methoxy-3-methylbenzylidenyl)-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10739/F03 3-(3-benzyloxybenzylidenyl)-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10739/F04 3-(4-benzyloxybenzylidenyl)-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10739/F05 3-[(1,4-benzodioxan-3-yl)methylidenyl]-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10739/F06 3-(2-chloro-4-hydroxybenzylidenyl)-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10739/F07 3-(3,4,5-trihydroxybenzylidenyl)-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10739/F08 3-[3-(4-methoxyphenoxy)benzylidenyl]-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10739/F09 3-[4-(3-dimethylamino-n-propoxy)benzylidenyl]-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10739/F10 3-[(furan-3-yl)methylidenyl]-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10739/F11 3-[(2-hydroxymethylfuran-5-yl)methylidenyl]-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10739/G02 3-(4-methoxy-3-methylbenzylidenyl)-5-(2-chloroethyl)-2-indolinone
    10739/G03 3-(3-benzyloxybenzylidenyl)-5-(2-chloroethyl)-2-indolinone
    10739/G04 3-(4-benzyloxybenzylidenyl)-5-(2-chloroethyl)-2-indolinone
    10739/G05 3-[(1,4-benzodioxan-3-yl)methylidenyl]-5-(2-chloroethyl)-2-indolinone
    10739/G06 3-(2-chloro-4-hydroxybenzylidenyl)-5-2-chloroethyl)-2-indolinone
    10739/G07 3-(3,4,5-trihydroxybenzylidenyl)-5-(2-chloroethyl)-2-indolinone
    10739/G08 3-[3-(4-methoxyphenoxy)benzylidenyl]-5-(2-chloroethyl)-2-indolinone
    10739/G09 3-[4-(3-dimethylamino-n-propoxy)benzylidenyl]-5-(2-chloroethyl)-2-indolinone
    10739/G10 3-[(furan-3-yl)methylidenyl]-5-(2-chloroethyl)-2-indolinone
    10739/G11 3-[(2-hydroxymethylfuran-5-yl)methylidenyl]-5-(2-chloroethyl)-2-indolinone
    10739/H02 3-(4-methoxy-3-methylbenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10739/H03 3-(3-benzyloxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10739/H04 3-(4-benzyloxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10739/H05 3-[(1,4-benzodioxan-3-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10739/H06 3-(2-chloro-4-hydroxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10739/H07 3-(3,4,5-trihydroxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
    10739/H08 3-[3-(4-methoxyphenoxy)benzylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10739/H09 3-[4-(3-dimethylamino-n-propoxy)benzylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10739/H10 3-[(furan-3-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10739/H11 3-[(2-hydroxymethylfuran-5-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10740/A02 3-[(quinolin-3-yl)methylidenyl]-5,7-dibromo-2-indolinone
    10740/A03 3-[(quinolin-4-yl)methylidenyl]-5,7-dibromo-2-indolinone
    10740/A04 3-[(2-methoxypyrrolidin-1-yl)methylidenyl]-5,7-dibromo-2-indolinone
    10740/A05 3-[(thien-3-yl)methylidenyl]-5,7-dibromo-2-indolinone
    10740/A06 3-[(quinolin-2-yl)methylidenyl]-5,7-dibromo-2-indolinone
    10740/A07 3-[(1-methylindol-3-yl)methylidenyl]-5,7-dibromo-2-indolinone
    10740/A08 3-[(2-methylindol-3-yl)methylidenyl]-5,7-dibromo-2-indolinone
    10740/A09 3-[(1-methylpyrid-6-yl)methylidenyl]-5,7-dibromo-2-indolinone
    10740/A10 3-[(4-bromothien-2-yl)methylidenyl]-5,7-dibromo-2-indolinone
    10740/A11 3-(4-n-butoxybenzylidenyl]-5,7-dibromo-2-indolinone
    10740/B02 3-[(quinolin-3-yl)methylidenyl]-5-iodo-2-indolinone
    10740/B03 3-[(quinolin-4-yl)methylidenyl]-5-iodo-2-indolinone
    10740/B04 3-[(2-methoxypyrrolidin-1-yl)methylidenyl]-5-iodo-2-indolinone
    10740/B05 3-[(thien-3-yl)methylidenyl]-5-iodo-2-indolinone
    10740/B06 3-[(quinolin-2-yl)methylidenyl]-5-iodo-2-indolinone
    10740/B07 3-[(1-methylindol-3-yl)methylidenyl]-5-iodo-2-indolinone
    10740/B08 3-[(2-methylindol-3-yl)methylidenyl]-5-iodo-2-indolinone
    10740/B09 3-[(1-methylpyrid-6-yl)methylidenyl]-5-iodo-2-indolinone
    10740/B10 3-[(4-bromothien-2-yl)methylidenyl]-5-iodo-2-indolinone
    10740/B11 3-(4-n-butoxybenzylidenyl)-5-iodo-2-indolinone
    10740/C02 3-[(quinolin-3-yl)methylidenyl]-5-bromo-4-methyl-2-indolinone
    10740/C03 3-[(quinolin-4-yl)methylidenyl]-5-bromo-4-methyl-2-indolinone
    10740/C04 3-[(2-methoxypyrrolidin-1-yl)methylidenyl]-5-bromo-4-methyl-2-indolinone
    10740/C05 3-[(thien-3-yl)methylidenyl]-5-bromo-4-methyl-2-indolinone
    10740/C06 3-[(quinolin-2-yl)methylidenyl]-5-bromo-4-methyl-2-indolinone
    10740/C07 3-[(1-methylindol-3-yl)methylidenyl]-5-bromo-4-methyl-2-indolinone
    10740/C08 3-[(2-methylindol-3-yl)methylidenyl]-5-bromo-4-methyl-2-indolinone
    10740/C09 3-[(1-methylpyrid-6-yl)methylidenyl]-5-bromo-4-methyl-2-indolinone
    10740/C10 3-[(4-bromothien-2-yl)methylidenyl]-5-bromo-4-methyl-2-indolinone
    10740/C11 3-(4-n-butoxybenzylidenyl)-5-bromo-4-methyl-2-indolinone
    10740/D02 3-[(quinolin-3-yl)methylidenyl]-5-methylaminosulfonyl-2-indoline
    10740/D03 3-[(quinolin-4-yl)methylidenyl]-5-methylaminosulfonyl-2-indoline
    10740/D04 3-[(2-methoxypyrrolidin-1-yl)methylidenyl]-5-methylaminosulfonyl-2-indolinone
    10740/D05 3-[(thien-3-yl)methylidenyl]-5-methylaminosulfonyl-2-indolinone
    10740/D06 3-[(quinolin-2-yl)methylidenyl]-5-methylaminosulfonyl-2-indoline
    10740/D07 3-[(1-methylindol-3-yl)methylidenyl]-5-methylaminosulfonyl-2-indolinone
    10740/D08 3-[(2-methylindol-3-yl)methylidenyl]-5-methylaminosulfonyl-2-indolinone
    10740/D09 3-[(1-methylindol-6-yl)methylidenyl]-5-methylaminosulfonyl-2-indolinone
    10740/D10 3-[(4-bromothien-2-yl)methylidenyl]-5-methylaminosulfonyl-2-indolinone
    10740/D11 3-[(4-n-butoxybenylidenyl)-5-methylaminosulfonyl-2-indolinone
    10740/E02 3-[(quinolin-3-yl)methylidenyl]-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10740/E03 3-[(quinolin-4-yl)methylidenyl]-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10740/E04 3-[(2-methoxypyrrolidin-1-yl)methylidenyl]-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10740/E05 3-[(thien-3-yl)methylidenyl]-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10740/E06 3-[(quinolin-2-yl)methylidenyl]-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10740/E07 3-[(1-methylindol-3-yl)methylidenyl]-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10740/E08 3-[(2-methylindol-3-yl)methylidenyl]-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10740/E09 3-[(1-methylpyrid-6-yl)methylidenyl]-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10740/E10 3-[(4-bromothien-2-yl)methylidenyl]-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10740/E11 3-(4-n-butoxybenzylidenyl)-5-[4-(trifluoromethyl)phenylaminosulfonyl]-2-indolinone
    10740/F02 3-[(quinolin-3-yl)methylidenyl]-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10740/F03 3-[(quinolin-4-yl)methylidenyl]-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10740/F04 3-[(2-methoxypyrrolidin-1-yl)methylidenyl]-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10740/F05 3-[(thien-3-yl)methylidenyl]-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10740/F06 3-[(quinolin-2-yl)methylidenyl]-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10740/F07 3-[(1-methylindol-3-yl)methylidenyl]-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10740/F08 3-[(2-methylindol-3-yl)methylidenyl]-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10740/F09 3-[(1-methylpyrid-6-yl)methylidenyl]-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10740/F10 3-[(4-bromothien-2-yl)methylidenyl]-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10740/F11 3-(4-n-butoxybenzylidenyl)-5-(morpholin-1-yl)sulfonyl-2-indolinone
    10740/G02 3-[(quinolin-3-yl)methylidenyl]-5-(2-chloroethyl)-2-indolinone
    10740/G03 3-[(quinolin-4-yl)methylidenyl]-5-(2-chloroethyl)-2-indolinone
    10740/G04 3-[(2-methoxypyrrolidin-1-yl)methylidenyl]-5-(2-chloroethyl)-2-indolinone
    10740/G05 3-[(thien-3-yl)methylidenyl]-5-(2-chloroethyl)-2-indolinone
    10740/G06 3-[(quinolin-2-yl)methylidenyl]-5-(2-chloroethyl)-2-indolinone
    10740/G07 3-[(1-methylindol-3-yl)methylidenyl]-5-(2-chloroethyl)-2-indolinone
    10740/G08 3-[(2-methylindol-3-yl)methylidenyl]-5-(2-chloroethyl)-2-indolinone
    10740/G09 3-[(1-methylpyrid-6-yl)methylidenyl]-5-(2-chloroethyl)-2-indolinone
    10740/G10 3-[(4-bromothien-2-yl)methylidenyl]-5-(2-chloroethyl)-2-indolinone
    10740/G11 3-(4-n-butoxybenzylidenyl)-5-(2-chloroethyl)-2-indolinone
    10740/H02 3-[(quinolin-3-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10740/H03 3-[(quinolin-4-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10740/H04 3-[(2-methoxypyrrolidin-1-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10740/H05 3-[(thien-3-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10740/H06 3-[(quinolin-2-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10740/H07 3-[(1-methylindol-3-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10740/H08 3-[(2-methylindol-3-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10740/H09 3-[(1-methylpyrid-6-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10740/H10 3-[(4-bromothien-2-yl)methylidenyl]-5,7-dibromo-4-methyl-2-indolinone
    10740/H11 3-(4-n-butoxybenzylidenyl)-5,7-dibromo-4-methyl-2-indolinone
  • “A” is a ring selected from the group consisting of benzene, tetrahydrobenzene, naphthalene, tetrahydro-naphthalene, furan, tetrahydrofuran, thiophene, pyrrole, pyrazole, pyrrolidine, imidazole, pyridine, quinoline, indole, tetrahydroindole, isatin. chromone, fluorene, carbazole, benzo[b]furan, thieno[b]thiophene and uracil. [0052]
  • The “A” ring is substituted with one or more groups 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, N-thiocarbamyl, C-amido, N-amido, amino and —NR[0053] 5R6.
  • It is an aspect of this invention that a combinatorial library may be used to screen compounds of this invention for a desired activity. [0054]
  • By “desired activity” is meant the ability to modulate the catalytic activity of a selected protein kinase. [0055]
  • By “screening” is meant to contact an entire combinatorial library of compounds or any portion thereof with one or more target protein kinases and observe the effect of the compounds on the catalytic activity of the protein kinase. [0056]
  • Thus, a further aspect of this invention is a combinatorial library of at least 10, preferably at least 100, more preferably at least 1000 wherein the oxindole is selected from the group consisting of 5,7-dibromooxindole, 5-iodooxindole, 5-bromo-4-methyloxindole, 5-methylaminosulfonyloxindole, 5-(4-trifluoromethylanilinosulfonyl)oxindole, 5-(morpholin-4-yl)-sulfonyloxindole, 5-(2-chloroethyl)oxindindole and 5,7-dibromo-4-methyloxindole and the aldehyde is selected from the group consisting of unsubstituted and substituted benzaldehyde, 1-naphthaldehyde, 2-naphthaldehyde, 2-formyl-5,6,7,8-tetrahydronaphthalene, 3-furaldehyde, furfural, 2-quinolinecarboxaldehyde, 3-quinolinecarboxaldehyde, 4-quinonlinecarboxaldehyde, 1-pyrrolidinecarboxaldehyde, 3-thiophenecarboxaldehyde, indole-3-carboxaldehyde, 2-pyridinecarboxaldehyde, 2-thiophenecarboxaldehyde, 3-thiophenecarboxaldehyde, pyrrole-2-carboxaldehyde, pyrrole-3-carboxaldehyde, 2-imidazolecarboxaldehyde, 4-imidazolecarboxaldehyde, pyrazole-3-carboxaldehyde, 2-formylbenzimidazole, 2-formyl-4,5,6,7-tetrahydroindole, benzo[b]furan-2-carboxaldehyde, thieno[b]thiophene-2-carboxaldehyde, isatin, chromone-3-carboxaldehyde, fluorene-2-carboxaldehyde, pyridine-2-carboxaldehyde, pyridine-3-carboxaldehyde, 1,2,3,6-tetrahydro benzaldehyde, 3-tetrahydrofurancarboxaldehyde, carbazole-3-carboxaldehyde, 5-formyluracil, 2-(trifluoroacetyl)thiophene, wherein, when substituted, the substituent group(s) is one or more selected from the group consisting of unsubstituted lower alkyl; lower alkyl substituted with unsubstituted aryl, halo, hydroxy, unsubstituted lower alkyl C-carboxy and —NR[0057] 5R6; unsubstituted lower alkoxy; lower alkoxy substituted with a group selected from the group consisting of C-carboxy, —NR5R6, and halo; halo; nitro; cyano; hydroxy; acetyl; trihaloacetyl; trihalomethyl; unsubstituted aryl; aryl substituted with one or more groups selected from the group consisting of halo, hydroxy, unsubstituted lower alkyl, unsubstituted lower alkoxy, hydroxy and nitro; unsubstituted lower aryloxy; aryloxy substituted with one or more groups selected from the group consisting of unsubstituted lower alkyl, trihalomethyl, halo, unsubstituted lower alkoxy, and unsubstituted aryl; unsubstituted lower alkyl C-carboxy, unsubstituted lower alkyl thioalkoxy, carboxylic acid, unsubstituted lower alkyl O-carboxy, 4-formylpiperazinyl, 4-formylmorpholinyl and N-pyrrolidinyl.
  • It is further aspect of this invention that a 3-(substituted)-2-indolinone may be made by testing the combinatorial library disclosed herein in the biological assays provided below, selecting one or more of the 3-(substituted)-2-indolinones which exhibit activity against a PK in the biological assays and then synthesizing those compounds. [0058]
  • By “exhibiting activity” is meant that the test compound from the combinatorial library has a modulating effect on the specific PK which is the focus of the particular assay, when compared to an control not containing the test compound. [0059]
  • Yet another aspect of this invention is a compound which modulates protein kinase activity, in particular RTK, CTK or STK kinase catalytic activity. [0060]
  • As used herein, the term “alkyl” refers to a saturated aliphatic hydrocarbon including straight chain and branched chain groups. Preferably, the alkyl group has 1 to 20 carbon atoms (whenever it appears herein, a numerical range such as “1 to 20” refers to each integer in the given range; e.g., “1 to 20 carbon atoms” means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc. up to and including 20 carbon atoms). More preferably, it is a medium size alkyl having 1 to 10 carbon atoms. Most preferably, it is a lower alkyl having 1 to 4 carbon atoms. The alkyl group may be substituted or unsubstituted. When substituted, the substituent group(s) is preferably one or more individually selected from cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, halo, carbonyl, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, C-carboxy, C-carboxy salt, O-carboxy, carboxyalkyl, carboxyalkyl salt, cyanato, isocyanato, thiocyanato, isothiocyanato, nitro, silyl, amino and —NR[0061] 5R6, R5 and R6 being as defined above.
  • 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 of 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, adamantane, cyclohexadiene, cycloheptane and, cycloheptatriene. A cycloalkyl group may be substituted or unsubstituted. When substituted, 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, C-carboxy, C-carboxy salt, O-carboxy, carboxyalkyl, carboxyalkyl salt, O-carbamyl, N-carbamyl, C-amido, N-amido, nitro, amino and —NR[0062] 5R6, with R5 and R6 being as defined above.
  • An “alkenyl” group refers to an alkyl group, as defined herein, consisting of at least two carbon atoms and at least one carbon-carbon double bond. [0063]
  • An “alkynyl” group refers to an alkyl group, as defined herein, consisting of at least two carbon atoms and at least one carbon-carbon triple bond. [0064]
  • An “aryl” group refers to an all-carbon monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups having a completely conjugated pi-electron system. Examples, without limitation, of aryl groups are phenyl, naphthalenyl and anthracenyl. The aryl group may be substituted or unsubstituted. When substituted, the substituted group(s) is preferably one or more selected from halo, trihalomethyl, alkyl, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, carbonyl, thiocarbonyl, C-carboxy, C-carboxy salt, O-carboxy, carboxyalkyl, carboxyalkyl salt, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, sulfinyl, sulfonyl, S-sulfonamido, N-sulfonamido, trihalomethanesulfonamido, amino and —NR[0065] 5R6, R5 and R6 being as defined above.
  • As used herein, a “heteroaryl” group refers to a monocyclic or fused ring (i.e., rings which share an adjacent pair of atoms) group having in the ring(s) one or more atoms selected from the group consisting of nitrogen, oxygen and sulfur and, in addition, having a completely conjugated pi-electron system. Examples, without limitation, of heteroaryl groups are pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline, purine and carbazole. The heteroaryl group may be substituted or unsubstituted. When substituted, the substituted group(s) is preferably one or more selected from alkyl, cycloalkyl, halo, trihalomethyl, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, carbonyl, thiocarbonyl, sulfonamido, C-carboxy, C-carboxy salt, O-carboxy, carboxyalkyl, carboxyalkyl salt, sulfinyl, sulfonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, amino and —NR[0066] 5R6, R5 and R6 being defined above.
  • A “heteroalicyclic” group refers to a monocyclic or fused ring group having in the ring(s) one or more atoms selected from the group consisting of nitrogen, oxygen and sulfur. The rings may also have one or more double bonds. However, the rings do not have a completely conjugated pi-electron system. The heteroalicyclic ring may be substituted or unsubstituted. When substituted, the substituted group(s) is preferably one or more selected from alkyl, cycloaklyl, halo, trihalomethyl, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, carbonyl, thiocarbonyl, C-carboxy, C-carboxy salt, O-carboxy, carboxyalkyl, carboxyalkyl salt, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, sulfinyl, sulfonyl, C-amido, N-amido, amino and —NR[0067] 5R6, with R5 and R6 being as defined above.
  • A “hydroxy” group refers to an —OH group. [0068]
  • An “alkoxy” group refers to both an —O-alkyl and an —O-cycloalkyl group, as defined herein. [0069]
  • An “aryloxy” group refers to both an —O-aryl and an —O-heteroaryl group, as defined herein. [0070]
  • A “thiohydroxy” group refers to an —SH group. [0071]
  • A “thioalkoxy” group refers to both an S-alkyl and an —S-cycloalkyl group, as defined herein. [0072]
  • A “thioaryloxy” group refers to both an —S-aryl and an —S-heteroaryl group, as defined herein. [0073]
  • A “carbonyl” group refers to a —C(═O)—R″ group, where R″ is selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) and heteroalicyclic (bonded through a ring carbon), as defined herein. [0074]
  • An “aldehyde” group refers to a carbonyl group where R″ is hydrogen. [0075]
  • A “thiocarbonyl” group refers to a —C(═S)—R″ group, with R″ as defined herein. [0076]
  • An “O-carboxy” group refers to a R″C(═O)O— group, with R″ as defined herein. [0077]
  • A “C-carboxy” group refers to a —C(═O)OR″ groups with R″ as defined herein. [0078]
  • A “C-carboxy salt” refers to a —C(═O)O[0079] −M+group wherein M+is selected from the group consisting of lithium, sodium, magnesium, calcium, potassium, barium, iron, zinc and quaternary ammonium.
  • An “acetyl” group refers to a —C(═O)CH[0080] 3 group.
  • A “trifluoroacetyl” group refers to a —C(═O)CF[0081] 3 group.
  • An “carboxyalkyl” group refers to —(CH[0082] 2)rC(═O)OR″ wherein r is 1-6 and R″ is as defined above.
  • An “carboxyalkyl salt” refers to a —(CH[0083] 2)rC(═O)O−M+wherein M+is selected from the group consisting of lithium, sodium, potassium, calcium, magnesium, barium, iron, zinc and quaternary ammonium.
  • A “carboxylic acid” group refers to a C-carboxy group in which R″ is hydrogen. [0084]
  • As used herein, an “ester” is a C-carboxy group, as defined herein, wherein R″ is any of the listed groups other than hydrogen. [0085]
  • A “halo” group refers to fluorine, chlorine, bromine or iodine. [0086]
  • A “trihalomethyl” group refers to a —CX[0087] 3 group wherein X is a halo group as defined herein.
  • A “trihalomethanesulfonyl” group refers to a X[0088] 3CS(═O)2— groups with X as defined above.
  • An “azido” group refers to a —N≡N group. [0089]
  • A “cyano” group refers to a —C≡N group. [0090]
  • A “cyanato” group refers to a —CNO group. [0091]
  • An “isocyanato” group refers to a —NCO group. [0092]
  • A “thiocyanato” group refers to a —CNS group. [0093]
  • An “isothiocyanato” group refers to a —NCS group. [0094]
  • A “sulfinyl” group refers to a —S(═O)—R″ group, with R″ as defined herein. [0095]
  • A “sulfonyl” group refers to a —S(═O)[0096] 2R″ group, with R″ as defined herein.
  • A “sulfonamido” group refers to a —S(═O)[0097] 2—NR5R6, with R5 and R6 as defined herein.
  • A “trihalomethanesulfonamido” group refers to a X[0098] 3CS(═O)2NR5— group with X and R5 as defined herein.
  • An “O-carbamyl” group refers to a —OC(═O)—NR[0099] 5R6 group with R5 and R6 as defined herein.
  • An “N-carbamyl” group refers to an R[0100] 5OC(═O)NR6— group, with R5 and R6 as defined herein.
  • An “O-thiocarbamyl” group refers to a —OC(═S)—NR[0101] 5R6 group with R5 and R6 as defined herein.
  • An “N-thiocarbamyl” group refers to a R[0102] 5OC(═S)NR6— group, with R5 and R6 as defined herein.
  • An “amino” group refers to an —NH[0103] 2 group.
  • A “C-amido” group refers to a —C(═O)—NR[0104] 5R6 group with R5 and R6 as defined herein.
  • An “N-amido” group refers to a R[0105] 5C(═O)NR6— group, with R5 and R6 as defined herein.
  • A “nitro” group refers to a —NO[0106] 2 group.
  • A “quaternary ammonium” group refers to a —[0107] +NR5R6R7 group wherein R5, R6 and R7 are independently selected from the group consisting of hydrogen and unsubstituted lower alkyl.
  • A “methylenedioxy” group refers to a —OCH[0108] 2O— group wherein the oxygen atoms are bonded to adjacent ring carbon atoms.
  • An “ethylenedioxy” group refers to a —OCH[0109] 2CH2O— group wherein the oxygen atoms are bonded to adjacent ring carbon atoms.
  • By “combined” or “combined to form a 5- or 6-member ring” when referring to two adjacent “R” groups attached to a ring system, is meant that, the two R groups are linked together by one or two additional atoms; i.e., the structure becomes {—R—X—X—R—} such that a 5- or 6-member ring is formed. X can be carbon, nitrogen, oxygen or sulfur. [0110]
    • Preferred Structural Features
  • Presently preferred structural features of the compounds of this invention are that the substituents on the “A” ring are independently selected from the group consisting of hydrogen; unsubstituted lower alkyl; lower alkyl substituted with unsubstituted aryl, halo, hydroxy, unsubstituted lower alkyl C-carboxy and —NR[0111] 5R6; unsubstituted lower alkoxy; lower alkoxy substituted with a group selected from the group consisting of C-carboxy, —NR5R6, and halo; halo; nitro; cyano; hydroxy; acetyl; trihaloacetyl; trihalomethyl; unsubstituted aryl; aryl substituted with one or more groups selected from the group consisting of halo, hydroxy, unsubstituted lower alkyl, unsubstituted lower alkoxy, hydroxy and nitro; unsubstituted lower aryloxy; aryloxy substituted with one or more groups selected from the group consisting of unsubstituted lower alkyl, trihalomethyl, halo, unsubstituted lower alkoxy, and unsubstituted aryl; unsubstituted lower alkyl C-carboxy, unsubstituted lower alkyl thioalkoxy, carboxylic acid, unsubstituted lower alkyl O-carboxy, 4-formylpiperazinyl, 4-formylmorpholinyl and N-pyrrolidinyl.
  • In a presently preferred embodiment of this invention, R[0112] 5 and R6 are independently selected from the group consisting of hydrogen and unsubstituted lower alkyl.
  • Further presently preferred structural features of this invention are those in which the oxindole to be reacted with an aldehyde to form a 2-indolinone of this invention is selected from the group consisting of: [0113]
  • 5-iodoindolin-2-one [0114]
  • 4-methyl-5,7-dibromoindolin-2-one [0115]
  • 5-(4-trifluoromethylanilino)-indolin-2-one [0116]
  • Likewise, it is a further presently preferred feature of this invention that the aldehyde to be reacted with an oxindole of this invention is selected from the group consisting of: [0117]
  • 3,4,5-trihydroxybenzaldehyde [0118]
  • 2-hydroxy-3,5-dibromobenzaldehyde [0119]
  • 2,3-dihydroxybenzaldehyde [0120]
  • 2-hydroxy-3,5-dichlorobenzaldehyde [0121]
  • 3-bromo-4-hydroxybenzaldehyde [0122]
  • The chemical formulae referred to herein may exhibit the phenomena of tautomerism and structural isomerism. For example, the compounds described herein may adopt a cis or trans configuration about the double bond connecting the 2-indolinone moiety to the “A” moiety or the compounds may be a mixture of cis and trans. This invention encompasses any tautomeric or structural isomeric form and mixtures thereof which possess the ability to modulate RTK and/or CTK activity and is not limited to any one tautomeric or structural isomeric form. [0123]
  • As used herein, the term “cis” refers to the structural configuration wherein the “A” group is on the same side of the double bond connecting it to the 2-indolinone ring as the 2-oxygen group of the 2-indolinone. [0124]
  • As used herein, the term “trans” refers to the structural configuration wherein the “A” group is on the opposite side of the double bond connecting it to the 2-indolinone ring from the 2-oxygen group of the 2-indolinone. [0125]
    • 3. THE BIOCHEMISTRY
  • In yet another embodiment, this invention relates to a method for the modulation of the catalytic activity of PKs by contacting a PK with a compound of this invention or a physiologically acceptable salt or prodrug thereof. [0126]
  • A further embodiment of this invention is a method for identifying a compound which modulates the activity of a PK which method consists of contacting a cell which expresses the PK of interest with a compound and monitoring the effect of the compound on the cell. [0127]
  • By “PK” is meant RTKs, CTKs and STKs; i.e., the modulation of RTK, CTK and STK catalyzed signaling processes are contemplated by this invention. [0128]
  • The term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by, practitioners of the chemical, pharmacological, biological, biochemical and medical arts. [0129]
  • As used herein, the term “modulation” or “modulating” refers to the alteration of the catalytic activity of RTKs, CTKs and STKs. In particular, modulating refers to the activation of the catalytic activity of RTKs, CTKs and STKs, preferably the activation or inhibition of the catalytic activity of RTKs, CTKs and STKs, depending on the concentration of the compound or salt to which the RTK, CTK or STK is exposed or, more preferably, the inhibition of the catalytic activity of RTKs, CTKs and STKs. [0130]
  • The term “catalytic activity” as used herein refers to the rate of phosphorylation of tyrosine under the influence, direct or indirect, of RTKs and/or CTKs or the phosphorylation of serine and threonine under the influence, direct or indirect, of STKs. [0131]
  • The term “contacting” as used herein refers to bringing a compound of this invention and a target PK together in such a manner that the compound can affect the catalytic activity of the PK, either directly; i.e., by interacting with the kinase itself, or indirectly; i.e., by interacting with another molecule on which the catalytic activity of the kinase is dependent. Such “contacting” can be accomplished in a test tube, a petri dish or the like. In a test tube, contacting may involve only a compound and a PK of interest or it may involve whole cells. Cells may also be maintained or grown in cell culture dishes and contacted with a compound in that environment. In this context, the ability of a particular compound to affect a PK related disorder; i.e., the IC[0132] 50 of the compound, defined below, can be determined before use of the compounds in vivo with more complex living organisms is attempted. For cells outside the organism, multiple methods exist, and are well-known to those skilled in the art, to get the PKs in contact with the compounds including, but not limited to, direct cell microinjection and numerous transmembrane carrier techniques.
  • By “monitoring” is meant observing or detecting the effect of contacting a compound with a cell expressing a particular PK. The observed or detected effect can be a change in cell phenotype, in the catalytic activity of a PK or a change in the interaction of a PK with a natural binding partner. Techniques for observing or detecting such effects are well-known in the art. [0133]
  • “Cell phenotype” refers to the outward appearance of a cell or tissue or the biological function of the cell or tissue. Examples, without limitation, of a cell phenotype are cell size, cell growth, cell proliferation, cell differentiation, cell survival, apoptosis, and nutrient uptake and use. Such phenotypic characteristics are measurable by techniques well-known in the art. [0134]
  • A “natural binding partner” refers to a polypeptide that binds to a particular PK in a cell. Natural binding partners can play a role in propagating a signal in a PK-mediated signal transduction process. A change in the interaction of the natural binding partner with the PK can manifest itself as an increased or decreased concentration of the PK/natural binding partner complex and, as a result, in an observable change in the ability of the PK to mediate signal transduction. [0135]
  • RTK mediated signal transduction is initiated by extracellular interaction with a specific growth factor (ligand), followed by receptor dimerization, transient stimulation of the intrinsic protein tyrosine kinase activity and phosphorylation. Binding sites are thereby created for intracellular signal transduction molecules and lead to the formation of complexes with a spectrum of cytoplasmic signaling molecules that facilitate the appropriate cellular response (e.g., cell division, metabolic effects on the extracellular microenvironment, etc.). See, Schlessinger and Ullrich, 1992[0136] , Neuron 9:303-391.
  • It has been shown that tyrosine phosphorylation sites on growth factor receptors function as high-affinity binding sites for SH2 (src homology) domains of signaling molecules. Fantl et al., 1992[0137] , Cell 69:413-423; Songyang et al., 1994, Mol. Cell. Biol. 14:2777-2785); Songyang et al., 1993, Cell 72:767-778; and Koch et al., 1991, Science 252:668-678. Several intracellular substrate proteins that associate with RTKs 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 which serve as adapters and associate with catalytically active molecules. Songyang et al., 1993, Cell 72:767-778. The specificity of the interactions between receptors and SH2 domains of their substrates is determined by the amino acid residues immediately surrounding the phosphorylated tyrosine residue. Differences in the binding affinities between SH2 domains and the amino acid sequences surrounding the phosphotyrosine residues on particular receptors are consistent with the observed differences in their substrate phosphorylation profiles. Songyang et al., 1993, Cell 72:767-778. These observations suggest that the function of each RTK is determined not only by its pattern of expression and ligand availability but also by the array of downstream signal transduction pathways that are activated by a particular receptor. Thus, phosphorylation provides an important regulatory step which determines the selectivity of signaling pathways recruited by specific growth factor receptors, as well as differentiation factor receptors.
  • STKs, being primarily cytosolic, affect the internal biochemistry of the cell, often as a down-line response to a PTK event. STKs have been implicated in the signaling process which initiates DNA synthesis and subsequent mitosis leading to cell proliferation. [0138]
  • Thus, PK signal transduction results in, among other responses, cell proliferation, differentiation, growth and metabolism. Abnormal cell proliferation may result in a wide array of disorders and diseases, including the development of neoplasia such as carcinoma, sarcoma, glioblastoma and hemangioma, disorders such as leukemia, psoriasis, arteriosclerosis, arthritis and diabetic retinopathy and other disorders related to uncontrolled angiogenesis and/or vasculogenesis. [0139]
  • A precise understanding of the mechanism by which the compounds of this invention inhibit PKs is not required in order to practice the present invention. However, while not hereby being bound to any particular mechanism or theory, it is believed that the compounds interact with the amino acids in the catalytic region of PKs. PKs typically possess a bi-lobate structure wherein ATP appears to bind in the cleft between the two lobes in a region where the amino acids are conserved among PKs. Inhibitors of PKs are believed to bind by non-covalent interactions such as hydrogen bonding, van der Waals forces and ionic interactions in the same general region where the aforesaid ATP binds to the PKs. More specifically, it is thought that the 2-indolinone component of the compounds of this invention binds in the general space normally occupied by the adenine ring of ATP. Specificity of a particular molecule for a particular PK may then arise as the result of additional interactions between the various substituents on the 2-indolinone core and the amino acid domains specific to particular PKs. Thus, different indolinone substituents may contribute to preferential binding to particular PKs. The ability to select compounds active at different ATP (or other nucleotide) binding sites makes the compounds of this invention useful for targeting any protein with such a site; i.e., not only PKs but protein phosphatases as well. The compounds disclosed herein may thus have utility as in vitro assays for such proteins as well as exhibiting in vivo therapeutic effects through interaction with such proteins. [0140]
  • In another aspect, the protein kinase, the catalytic activity of which is modulated by contact with a compound of this invention, is a protein tyrosine kinase, more particularly, a receptor protein tyrosine kinase. Among the receptor protein tyrosine kinases whose catalytic activity can be modulated with a compound of this invention, or salt thereof, are, without limitation, EGF, HER2, HER3, HER4, IR, IGF-1R, IRR, PDGFRα, PDGFRβ, CSFIR, C-Kit, C-fms, Flk-1R, Flk4, KDR/Flk-1, Flt-1, FGFR-1R, FGFR-2R, FGFR-3R and FGFR-4R. [0141]
  • The protein tyrosine kinase whose catalytic activity is modulated by contact with a compound of this invention, or a salt or a prodrug thereof, can also be a non-receptor or cellular protein tyrosine kinase (CTK). Thus, the catalytic activity of CTKs such as, without limitation, Src, Frk, Btk, Csk, Abl, ZAP70, Fes, Fps, Fak, Jak, Ack, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk, may be modulated by contact with a compound or salt of this invention. [0142]
  • Still another group of PKs which may have their catalytic activity modulated by contact with a compound of this invention are the serine-threonine protein kinases such as, without limitation, CDK2 and Raf. [0143]
  • In another aspect, this invention relates to a method for treating or preventing a PK related disorder by administering a therapeutically effective amount of a compound of this invention, or a salt or a prodrug thereof, to an organism. [0144]
  • It is also an aspect of this invention that a pharmacological composition containing a compound of this invention or a salt or prodrug thereof is administered to an organism for the purpose of preventing or treating a PK related disorder. [0145]
  • As used herein, “PK related disorder,” “PK driven disorder,” and “abnormal PK activity” all refer to a condition characterized by inappropriate; i.e., under or, more commonly, over, PK catalytic activity, where the particular PK can be an RTK, a CTK or an STK. Inappropriate catalytic activity can arise as the result of either: (1) PK expression in cells which normally do not express PKs; (2) increased PK expression leading to unwanted cell proliferation, differentiation and/or growth; or, (3) decreased PK expression leading to unwanted reductions in cell proliferation, differentiation and/or growth. Over-activity of a PK refers to either amplification of the gene encoding a particular PK or production of a level of PK activity which can correlate with a cell proliferation, differentiation and/or growth disorder (that is, as the level of the PK increases, the severity of one or more of the symptoms of the cellular disorder increases). Under-activity is, of course, the converse, wherein the severity of one or more symptoms of a cellular disorder increase as the level of the PK activity decreases. [0146]
  • As used herein, the terms “prevent”, “preventing” and “prevention” refer to a method for barring an organism from acquiring a PK related disorder in the first place. [0147]
  • As used herein, the terms “treat”, “treating” and “treatment” refer to a method of alleviating or abrogating a PK mediated cellular disorder and/or its attendant symptoms. With regard particularly to cancer, these terms simply mean that the life expectancy of an individual affected with a cancer will be increased or that one or more of the symptoms of the disease will be reduced. [0148]
  • The term “organism” refers to any living entity comprised of at least one cell. A living organism can be as simple as, for example, a single eukariotic cell or as complex as a mammal, including a human being. [0149]
  • The term “therapeutically effective amount” as used herein refers to that amount of the compound being administered which will relieve to some extent one or more of the symptoms of the disorder being treated. In reference to the treatment of cancer, a therapeutically effective amount refers to that amount which has the effect of (1) reducing the size of the tumor; (2) inhibiting (that is, slowing to some extent, preferably stopping) tumor metastasis; (3) inhibiting to some extent (that is, slowing to some extent, preferably stopping) tumor growth; and/or, (4) relieving to some extent (or, preferably, eliminating) one or more symptoms associated with the cancer. [0150]
  • This invention is therefore directed to compounds which modulate PK signal transduction by affecting the enzymatic activity of RTKs, CTKs and/or STKs, thereby interfering with the signals transduced by such proteins. More particularly, the present invention is directed to compounds which modulate RTK, CTK and/or STK mediated signal transduction pathways as a therapeutic approach to cure many kinds of solid tumors, including but not limited to carcinomas, sarcomas including Kaposi's sarcoma, erythroblastoma, glioblastoma, meningioma, astrocytoma, melanoma and myoblastoma. Treatment or prevention of non-solid tumor cancers such as leukemia are also contemplated by this invention. Indications may include, but are not limited to brain cancers, bladder cancers, ovarian cancers, gastric cancers, pancreas cancers, colon cancers, blood cancers, lung cancers and bone cancers. [0151]
  • Further examples, without limitation, of the types of disorders related to inappropriate PK activity that the compounds described herein may be useful in preventing, treating and studying, are cell proliferative disorders, fibrotic disorders and metabolic disorders. [0152]
  • Cell proliferative disorders, which may be prevented, treated or further studied by the present invention include cancer, blood vessel proliferative disorders and mesangial cell proliferative disorders. [0153]
  • Blood vessel proliferative disorders refer to disorders related to abnormal vasculogenesis (blood vessel formation) and angiogenesis (spreading of blood vessels). While vasculogenesis and angiogenesis play important roles in a variety of normal physiological processes such as embryonic development, corpus luteum formation, wound healing and organ regeneration, they also play a pivotal role in cancer development where they result in the formation of new capillaries needed to keep a tumor alive. Other examples of blood vessel proliferation disorders include arthritis, where new capillary blood vessels invade the joint and destroy cartilage, and ocular diseases, like diabetic retinopathy, where new capillaries in the retina invade the vitreous, bleed and cause blindness. [0154]
  • Conversely, disorders related to the shrinkage, contraction or closing of blood vessels, such as restenosis, are also implicated and may be treated or prevented by the methods of this invention. [0155]
  • Fibrotic disorders refer to the abnormal formation of extracellular matrices. Examples of fibrotic disorders include hepatic cirrhosis and mesangial cell proliferative disorders. Hepatic cirrhosis is characterized by the increase in extracellular matrix constituents resulting in the formation of a hepatic scar. An increased extracellular matrix resulting in a hepatic scar can also be caused by a viral infection such as hepatitis. Lipocytes appear to play a major role in hepatic cirrhosis. Other fibrotic disorders implicated include atherosclerosis. [0156]
  • Mesangial cell proliferative disorders refer to disorders brought about by abnormal proliferation of mesangial cells. Mesangial proliferative disorders include various human renal diseases such as glomerulonephritis, diabetic nephropathy and malignant nephrosclerosis as well as such disorders as thrombotic microangiopathy syndromes, transplant rejection, and glomerulopathies. The RTK PDGFR has been implicated in the maintenance of mesangial cell proliferation. Floege et al., 1993[0157] , Kidney International 43:47S-54S.
  • Many cancers are cell proliferative disorders and, as noted previously, PKs have been associated with cell proliferative disorders. Thus, it is not surprising that PKs such as, for example, members of the RTK family have been associated with the development of cancer. Some of these receptors, like EGFR (Tuzi et al., 1991[0158] , Br. J. Cancer 63:227-233; Torp et al., 1992, APMIS 100:713-719) HER2/neu (Slamon et al., 1989, Science 244:707-712) and PDGF-R (Kumabe et al., 1992, Oncogene, 7:627-633) are over-expressed in many tumors and/or persistently activated by autocrine loops. In fact, in the most common and severe cancers these receptor over-expressions (Akbasak and Suner-Akbasak et al., 1992, J. Neurol. Sci., 111:119-133; Dickson et al., 1992, Cancer Treatment Res. 61:249-273; Korc et al., 1992, J. Clin. Invest. 90:1352-1360) and autocrine loops (Lee and Donoghue, 1992, J. Cell. Biol., 118:1057-1070; Korc et al., supra; Akbasak and Suner-Akbasak et al., supra) have been demonstrated. For example, EGFR has been associated with squamous cell carcinoma, astrocytoma, glioblastoma, head and neck cancer, lung cancer and bladder cancer. HER2 has been associated with breast, ovarian, gastric, lung, pancreas and bladder cancer. PDGFR has been associated with glioblastoma and melanoma as well as lung, ovarian and prostate cancer. The RTK c-met has also been associated with malignant tumor formation. For example, c-met has been associated with, among other cancers, colorectal, thyroid, pancreatic, gastric and hepatocellular carcinomas and lymphomas. Additionally c-met has been linked to leukemia. Over-expression of the c-met gene has also been detected in patients with Hodgkins disease and Burkitts disease.
  • Flk has likewise been associated with a broad spectrum of tumors including, without limitation, mammary, ovarian and lung tumors as well as gliomas such as glioblastoma. [0159]
  • IGF-IR, in addition to being implicated in nutritional support and in type-II diabetes, has also been associated with several types of cancers. For example, 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[0160] , J. Clin. Invest. 84:1418-1423) and small lung tumor cells (Macauley et al., 1990, Cancer Res., 50:2511-2517). In addition, IGF-I, while integrally involved in the normal growth and differentiation of the nervous system, also appears to be an autocrine stimulator of human gliomas. Sandberg-Nordqvist et al., 1993, Cancer Res. 53:2475-2478. The importance of IGF-IR and its ligands in cell proliferation is further supported by the fact that many cell types in culture (fibroblasts, epithelial cells, smooth muscle cells, T-lymphocytes, myeloid cells, chondrocytes and osteoblasts (the stem cells of the bone marrow)) are stimulated to grow by IGF-I. Goldring and Goldring, 1991, Eukaryotic Gene Expression, 1:301-326. In a series of recent publications, Baserga suggests that IGF-IR plays a central role in the mechanism of transformation and, as such, could be a preferred target for therapeutic interventions for a broad spectrum of human malignancies. Baserga, 1995, Cancer Res., 55:249-252; Baserga, 1994, Cell 79:927-930; Coppola et al., 1994, Mol. Cell. Biol., 14:4588-4595.
  • STKs have been implicated in many types of cancer including, notably, breast cancer (Cance, et al., [0161] Int. J. Cancer, 54:571-77 (1993)).
  • The association between abnormal PK activity and disease is not restricted to cancer. For example, RTKs have been associated with diseases such as psoriasis, diabetes mellitus, endometriosis, angiogenesis, atheromatous plaque development, Alzheimer's disease, epidermal hyperproliferation, neurodegenerative diseases, age-related macular degeneration and hemangiomas. For example, EGFR has been indicated in corneal and dermal wound healing. Defects in Insulin-R and IGF-1R 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[0162] , DN&P 7:334-339.
  • As noted previously, not only RTKs but CTKs including, but not limited to, src, abl, fps, yes, fyn, lyn, lck, blk, hck, fgr and yrk (reviewed by Bolen et al., 1992[0163] , FASEB J., 6:3403-3409) are involved in the proliferative and metabolic signal transduction pathway and thus could be expected, and have been shown, to be involved in many PTK-mediated disorders to which the present invention is directed. For example, mutated src (v-src) has been demonstrated to be an oncoprotein (pp60v-src) in chicken. Moreover, its cellular homolog, the proto-oncogene pp60c-src transmits oncogenic signals of many receptors. Over-expression of EGFR or HER2/neu in tumors leads to the constitutive activation of pp60c□src, which is characteristic of malignant cells but absent in normal cells. On the other hand, mice deficient in the expression of c-src exhibit an osteopetrotic phenotype, indicating a key participation of c-src in osteoclast function and a possible involvement in related disorders.
  • Similarly, Zap70 has been implicated in T-cell signaling which may relate to autoimmune disorders. [0164]
  • STKs have been associated with inflamation, autoimmune disease, immunoresponses, and hyperproliferation disorders such as restenosis, fibrosis, psoriasis, osteoarthritis and rheumatoid arthritis. [0165]
  • PKs have also been implicated in embryo implantation. Thus, the compounds of this invention may provide an effective method of preventing such embryo implantation and thereby be useful as birth control agents. [0166]
  • Finally, both RTKs and CTKs are currently suspected as being involved in hyperimmune disorders. [0167]
    • 4. PHARMACOLOGICAL COMPOSITIONS AND THERAPEUTIC APPLICATIONS
  • A compound of the present invention, a prodrug thereof or a physiologically acceptable salt of either the compound or its prodrug, can be administered as such to a human patient or it can be administered in pharmacological compositions in which the foregoing materials are mixed with suitable carriers or excipient(s). Techniques for formulation and administration of drugs may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition. [0168]
    • Routes of Administration
  • As used herein, “administer” or “administration” refers to the delivery of a compound, salt or prodrug of the present invention or of a pharmacological composition containing a compound, salt or prodrug of this invention to an organism for the purpose of prevention or treatment of a PK-related disorder. [0169]
  • Suitable routes of administration may include, without limitation, oral, rectal, transmucosal or intestinal administration or intramuscular, subcutaneous, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. The preferred routes of administration are oral and parenteral. [0170]
  • Alternatively, one may administer the compound in a local rather than systemic manner, for example, via injection of the compound directly into a solid tumor, often in a depot or sustained release formulation. [0171]
  • Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with tumor-specific antibody. The liposomes will be targeted to and taken up selectively by the tumor. [0172]
    • Composition/Formulation
  • Pharmacological compositions of the present invention may be manufactured by processes well known in the art; e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. [0173]
  • Pharmacological compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. [0174]
  • For injection, the compounds of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. [0175]
  • For oral administration, the compounds can be formulated by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, lozenges, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding other suitable auxiliaries if desired, to obtain tablets or dragee cores. Useful excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch and potato starch and other materials such as gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid. A salt such as sodium alginate may also be used. [0176]
  • Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses. [0177]
  • Pharmacological compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with a filler such as lactose, a binder such as starch, and/or a lubricant such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Stabilizers may be added in these formulations, also. [0178]
  • For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray using a pressurized pack or a nebulizer and a suitable propellant, e.g., without limitation, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be controlled by providing a valve to deliver a metered amount. Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. [0179]
  • The compounds may also be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulating materials such as suspending, stabilizing and/or dispersing agents. [0180]
  • Pharmacological compositions for parenteral administration include aqueous solutions of a water soluble form, such as, without limitation, a salt, of the active compound. Additionally, suspensions of the active compounds may be prepared in a lipophilic vehicle. Suitable lipophilic vehicles include fatty oils such as sesame oil, synthetic fatty acid esters such as ethyl oleate and triglycerides, or materials such as liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers and/or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. [0181]
  • Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use. [0182]
  • The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides. [0183]
  • In addition to the formulations described previously, the compounds may also be formulated as depot preparations. Such long acting formulations may be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection. A compound of this invention may be formulated for this route of administration with suitable polymeric or hydrophobic materials (for instance, in an emulsion with a pharamcologically acceptable oil), with ion exchange resins, or as a sparingly soluble derivative such as, without limitation, a sparingly soluble salt. [0184]
  • The pharmacological compositions herein also may comprise suitable solid or gel phase carriers or excipients. Examples of such 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. [0185]
  • Many of the PK modulating compounds of the invention may be provided as physiologically acceptable salts wherein the claimed compound may form the negatively or the positively charged species. Examples of salts in which the compound forms the positively charged moiety include, without limitation, quaternary ammonium (defined elsewhere herein), salts such as the hydrochloride, sulfate, carbonate, lactate, tartrate, maleate, succinate wherein the nitrogen atom of the quaternary ammonium group is a nitrogen of the selected compound of this invention which has reacted with the appropriate acid. Salts in which a compound of this invention forms the negatively charged species include, without limitation, the sodium, potassium, calcium and magnesium salts formed by the reaction of a carboxylic acid group in the compound with an appropriate base (e.g. sodium hydroxide (NaOH), potassium hydroxide (KOH), Calcium hydroxide (Ca(OH)[0186] 2), etc.).
    • Dosage
  • Pharmacological compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an amount sufficient to achieve the intended purpose; i.e., the modulation of PK activity or the treatment or prevention of a PK-related disorder. [0187]
  • 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. [0188]
  • 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. [0189]
  • For any compound used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from cell culture assays. Then, the dosage can be formulated for use in animal models so as to achieve a circulating concentration range that includes the IC[0190] 50 as determined in cell culture (i.e., the concentration of the test compound which achieves a half-maximal inhibition of the PK activity). Such information can then be used to more accurately determine useful doses in humans.
  • Toxicity and therapeutic efficacy of the compounds described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the IC[0191] 50 and the LD50 (both of which are discussed elsewhere herein) for a subject compound. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch. 1 p.1).
  • Dosage amount and interval may be adjusted individually to provide plasma levels of the active specie which are sufficient to maintain the kinase modulating effects. These plasma levels are referred to as minimal effective concentrations (MECs). The MEC will vary for each compound but can be estimated from in vitro data; e.g., the concentration necessary to achieve 50-90% inhibition of a kinase may be ascertained using the assays described herein. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. HPLC assays or bioassays can be used to determine plasma concentrations. [0192]
  • 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% . [0193]
  • In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration and other procedures known in the art may be employed to determine the correct dosage amount and interval. [0194]
  • The amount of a composition administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc. [0195]
    • Packaging
  • The compositions may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accompanied by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or of human or veterinary administration. Such notice, for example, may be of the labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an 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. [0196]
    • 5. SYNTHESIS
  • The compounds of this invention, as well as the precursor oxindoles and aldehydes, may be readily synthesized using techniques well known in the chemical arts. It will be appreciated by those skilled in the art that other synthetic pathways for forming the compounds of the invention are available and that the following is offered by way of example and not limitation. [0197]
    • General Synthetic Procedure
  • The following general methodology may be employed to prepare the compounds of this invention: [0198]
  • The appropriately substituted oxindole (1 equiv.), the appropriately substituted aldehyde (1.2 equiv.) and piperidine (0.1 equiv.) are mixed with ethanol (1-2 ml/mmol of 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. [0199]
  • Each of the compounds of Table 3 may be synthesized by the above procedure. [0200]
    • 6. BIOLOGICAL EVALUATION
  • It will be appreciated that, in any given series of compounds, a spectrum of biological activity will be afforded. In its preferred embodiments, this invention relates to novel 3-(substituted)-indolin-2-ones demonstrating the ability to modulate RTK, CTK and STK activity. The following assays are employed to select those compounds demonstrating the optimal degree of the desired activity. [0201]
  • As used herein, the phrase “optimal degree of the desired activity” refers to the lowest IC[0202] 50, defined elsewhere herein, against a PTK related to a particular disorder so as to provide an organism, preferably a human, with a therapeutically effective amount of a compound of this invention at the lowest possible dosage.
  • Table 4 shows the results of biological assays of exemplary, but by no means limiting, compounds of this invention using the following assay procedures. [0203]
    • Assay Procedures
  • The following 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. [0204]
  • 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 a selected period of time after which, if the test kinase is a receptor, a ligand known to activate the receptor 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 phosphorylation on the substrate is detected with an antibody specifically recognizing phosphotyrosine compared with control cells that were not contacted with a test compound. [0205]
  • 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 a selected period of time after which, if the test kinase is a receptor, a ligand known to activate the receptor is added. After incubation at least overnight, a DNA labeling reagent such as Bromodeoxyuridine (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. [0206]
    TABLE 4
    Biochem
    Flk Kinase EGFR PDGF Kinase Met Kinase
    Compound % Inhibition % Inhibition % Inhibition % Inhibition
    Number (P.110) (P.167) (P.119) (P.130)
    10717/H02 4.0 3.5 44.5
    10717/H03 8.6 25.7 18.3
    10717/H04 0.4 6.8 14.0
    10717/H05 5.0 −2.0 16.3
    10717/H06 12.5 −16.8 30.5
    10717/H07 1.1 91.2 9.1
    10717/H08 −8.4 33.4 23.0
    10717/H09 −17.5 5.4 8.8
    10717/H10 −5.0 0.5 52.3
    10717/H11 −0.4 55.6 58.2
    10718/H02 −1.4 −22.5 31.5
    10718/H03 −5.0 33.2 90.8
    10718/H04 −5.7 13.7 84.3
    10718/H05 1.0 0.2 13.5
    10718/H06 18.2 −5.1 32.2
    10718/H07 −3.0 −12.0 52.7
    10718/H08 −0.5 −2.5 14.4
    10718/H09 −5.0 34.2 73.6
    10718/H10 1.4 −5.7 19.9
    10718/H11 13.0 4.4 44.9
    10719/H02 22.0 43.9 36.9
    10719/H03 −40.4 25.1 67.0
    10719/H04 3.4 −0.5 0.7
    10719/H05 18.5 7.9 20.9
    10719/H06 17.8 −28.0 1.3
    10719/H07 2.4 −2.8 22.9
    10719/H08 11.6 2.9 20.0
    10719/H09 2.5 −33.5 5.8
    10719/H10 −0.7 14.6 −5.7
    10719/H11 12.3 −12.0 50.5
    10720/H02 9.7 17.0 64.1
    10720/H03 1.8 −13.9 40.0
    10720/H04 −5.8 −49.6 21.3
    10720/H05 −12.3 −29.0 51.0
    10720/H06 22.3 −52.4 24.4
    10720/H07 −2.9 −49.8 10.8
    10720/H08 −23.7 −55.8 40.0
    10720/H09 −4.5 −66.8 49.0
    10720/H10 −22.8 −62.8 48.2
    10720/H11 −8.8 −19.3 28.0
    10721/H02 5.5 20.1 39.7
    10721/H03 7.8 29.2 21.3
    10721/H04 0.7 −8.5 72.9
    10721/H05 −8.4 −12.4 33.6
    10721/H06 −7.9 43.3 90.3
    10721/H07 −7.0 −20.5 10.4
    10721/H08 −6.8 −6.8 42.5
    10721/H09 7.0 −10.4 36.7
    10721/H10 10.2 −3.9 46.9
    10721/H11 −6.0 −21.3 46.0
    10722/H02 4.2 24.8 19.2 37.8
    10722/H03 2.1 27.8 26.6 100.5
    10722/H04 3.8 8.6 −26.1 62.2
    10722/H05 4.3 1.1 0.9 83.2
    10722/H06 18.9 −2.0 18.6 70.3
    10722/H07 2.3 −24.8 −16.9 51.0
    10722/H08 −20.3 −5.7 −15.2 72.6
    10722/H09 3.3 −7.4 17.5 67.6
    10722/H10 −14.1 11.2 7.2 30.3
    10722/H11 −3.3 −16.3 15.7 66.1
    10723/H02 9.0 72.8 20.7 100.8
    10723/H03 6.1 6.3 −30.6 69.2
    10723/H04 −20.2 7.1 53.3 95.6
    10723/H05 −2.5 −17.3 −12.6 64.2
    10723/H06 10.6 −11.8 −17.4 44.9
    10723/H07 5.0 −11.3 −20.9 44.9
    10723/H08 −4.2 69.9 4.1 86.2
    10723/H09 −0.4 −2.6 15.9 62.1
    10723/H10 −13.4 −38.1 −22.3 46.4
    10723/H11 −5.1 −20.7 29.7 76.2
    10724/H02 11.5 17.6 2.2 51.9
    10724/H03 1.8 0.3 −7.6 26.1
    10724/H04 −3.0 14.0 −13.1 69.3
    10724/H05 −0.4 −1.3 −19.4 68.6
    10724/H06 −5.1 −8.2 −20.8 32.8
    10724/H07 1.1 −19.3 −39.6 8.6
    10724/H08 13.1 −19.7 −9.0 65.8
    10724/H09 1.3 −33.5 −13.1 4.2
    10724/H10 5.8 −25.5 −2.7 72.2
    10724/H11 8.6 −12.8 −31.2 9.8
    10725/H02 −7.6 −23.5 −19.9 15.8
    10725/H03 −5.8 −16.5 −12.8 58.3
    10725/H04 −5.1 17.2 −4.1 47.1
    10725/H05 21.6 −30.2 14.6 20.8
    10725/H06 16.1 0.3 6.9 33.0
    10725/H07 8.6 −13.3 2.6 14.2
    10725/H08 17.2 −3.7 13.6 5.7
    10725/H09 −6.1 −28.4 9.3 41.6
    10725/H10 7.8 9.1 12.2 21.8
    10725/H11 −40.8 0.5 16.5 17.8
    10726/H02 −8.5 −11.2 −8.3 66.6
    10726/H03 0.4 14.0 −17.8 44.8
    10726/H04 2.5 75.5 13.2 77.6
    10726/H05 −6.1 2.2 −38.0 80.2
    10726/H06 17.5 −9.2 −20.9 35.1
    10726/H07 −9.5 31.2 −30.4 80.9
    10726/H08 −15.0 −16.6 15.1 71.2
    10726/H09 1.5 −0.1 15.1 68.1
    10726/H10 11.0 12.0 6.9 45.7
    10726/H11 12.9 1.5 −0.1 40.8
    10727/H02 −1.2 −37.1 −33.6 55.5
    10727/H03 4.3 4.5 −20.5 49.2
    10727/H04 1.8 19.2 13.7 81.7
    10727/H05 5.5 0.4 −28.5 20.6
    10727/H06 −4.4 −35.8 −40.8 5.2
    10727/H07 −9.7 −17.3 −37.2 26.8
    10727/H08 2.9 −8.0 −27.0 12.3
    10727/H09 −1.4 −6.1 −11.7 31.1
    10727/H10 −8.3 83.6 10.1 89.7
    10727/H11 3.7 −3.5 2.8 17.9
    10728/H02 −13.3 −36.6 −9.3 25.8
    10728/H03 −2.1 3.5 −10.6 15.6
    10728/H04 −4.3 −0.6 −5.7 32.9
    10728/H05 1.1 −13.5 −10.6 41.2
    10728/H06 7.9 −22.8 −25.7 28.5
    10728/H07 1.5 −25.8 −1.7 45.8
    10728/H08 −4.4 −3.4 −5.3 33.9
    10728/H09 0.7 −6.1 −6.2 30.5
    10728/H10 −3.8 −22.4 4.5 65.2
    10728/H11 3.3 −25.6 −16.8 8.0
    10729/H02 14.3 −10.7 2.3 25.5
    10729/H03 18.3 30.3 −0.7 2.8
    10729/H04 1.1 −4.4 −5.6 25.1
    10729/H05 22.4 −4.4 −27.6 27.9
    10729/H06 11.0 −10.3 −12.1 28.4
    10729/H07 9.9 30.0 −43.1 81.0
    10729/H08 8.2 8.7 −1.5 44.0
    10729/H09 −2.7 8.0 0.1 45.1
    10729/H10 5.3 2.1 −1.5 12.8
    10729/H11 8.9 0.8 5.0 36.1
    10730/H02 21.2 12.6 1.0 32.0
    10730/H03 11.5 −3.4 −11.7 28.3
    10730/H04 12.1 −29.5 −1.2 37.1
    10730/H05 11.7 20.1 22.1 30.7
    10730/H06 15.7 −13.5 −9.9 13.4
    10730/H07 19.0 −3.2 0.6 38.7
    10730/H08 9.2 −15.8 6.3 57.8
    10730/H09 16.6 11.1 7.2 41.1
    10730/H10 18.3 1.7 23.0 24.9
    10730/H11 13.9 26.4 9.4 32.6
    10731/H02 19.7 −9.5 −18.7 16.4
    10731/H03 20.0 −1.5 1.8 23.3
    10731/H04 16.4 −1.7 −15.8 41.0
    10731/H05 23.6 −3.6 −1.6 32.2
    10731/H06 28.7 −7.8 −5.4 17.2
    10731/H07 74.5 −1.7 36.5 1.8
    10731/H08 31.4 −0.5 −10.1 10.9
    10731/H09 15.8 −2.7 −5.4 12.8
    10731/H10 25.9 3.5 40.3 28.4
    10731/H11 27.8 −3.1 8.9 6.9
    10732/H02 9.1 13.0 6.8 56.8
    10732/H03 8.4 1.4 −1.8 24.1
    10732/H04 4.9 −0.7 14.4 57.6
    10732/H05 12.8 −2.9 −25.6 13.3
    10732/H06 22.6 −6.9 −18.0 31.1
    10732/H07 9.7 −15.3 −22.0 26.7
    10732/H08 12.6 −14.0 7.8 38.7
    10732/H09 5.4 −7.4 −14.4 49.8
    10732/H10 3.3 −21.3 −21.0 28.0
    10732/H11 6.4 −15.1 −16.0 46.7
    10733/H02 −2.0 3.2 −5.6 38.9
    10733/H03 31.2 −16.5 16.8 0.6
    10733/H04 −8.5 −24.0 −14.5 11.9
    10733/H05 17.7 15.9 −15.3 5.6
    10733/H06 30.0 −10.5 −12.9 18.4
    10733/H07 7.6 −6.2 −19.0 −1.3
    10733/H08 4.8 0.3 −15.3 41.6
    10733/H09 44.8 −3.1 5.0 51.9
    10733/H10 12.6 −3.1 10.7 26.5
    10733/H11 29.1 7.0 −4.8 76.7
    10734/A02 4.1 0.0 −3.8 51.1
    10734/A03 10.1 18.5 22.3 87.8
    10734/A04 0.6 11.4 11.7 68.0
    10734/A05 9.1 22.6 2.9 69.9
    10734/A06 −6.4 −6.4 −9.9 46.9
    10734/A07 −9.4 15.2 21.9 89.3
    10734/A08 0.1 6.8 9.1 71.5
    10734/A09 −7.9 27.3 −9.5 88.4
    10734/A10 4.4 27.8 26.3 55.4
    10734/A11 6.3 12.5 5.5 51.9
    10734/B02 8.5 −8.9 −2.9 61.9
    10734/B03 21.7 45.1 13.0 90.1
    10734/B04 2.8 38.7 14.4 88.4
    10734/B05 −2.3 17.8 56.3 67.8
    10734/B06 −0.9 54.0 0.2 91.2
    10734/B07 9.1 65.5 18.8 93.2
    10734/B08 −0.5 19.3 −2.4 56.1
    10734/B09 −32.3 34.9 12.6 94.3
    10734/B10 15.7 60.7 7.7 82.6
    10734/B11 12.6 50.9 −5.5 83.6
    10734/C02 −3.9 −6.7 36.0 78.6
    10734/C03 −5.6 48.6 24.5 88.2
    10734/C04 −37.6 38.1 24.5 90.7
    10734/C05 −10.3 9.3 3.8 52.7
    10734/C06 −1.7 18.4 −0.2 48.3
    10734/C07 −2.7 41.0 32.0 96.8
    10734/C08 −34.2 35.1 5.5 93.2
    10734/C09 −13.3 36.2 11.3 88.6
    10734/C10 10.1 24.4 17.4 11.6
    10734/C11 6.3 18.0 9.1 23.9
    10734/D02 −11.5 5.2 6.4 46.0
    10734/D03 17.4 19.6 24.1 78.6
    10734/D04 6.1 −10.7 13.5 46.9
    10734/D05 2.4 −9.6 8.2 55.0
    10734/D06 0.0 1.3 −9.1 30.6
    10734/D07 14.7 51.1 28.1 92.0
    10734/D08 2.5 −13.7 2.0 49.8
    10734/D09 −11.6 21.0 42.2 88.0
    10734/D10 11.1 −4.4 9.9 20.6
    10734/D11 9.8 14.6 17.0 33.9
    10734/E02 7.9 −10.8 50.1 66.9
    10734/E03 26.9 55.0 27.2 99.3
    10734/E04 10.7 −7.8 26.7 88.9
    10734/E05 10.6 7.9 17.9 63.6
    10734/E06 28.4 6.3 13.5 50.6
    10734/E07 27.9 82.4 8.2 100.8
    10734/E08 26.6 24.2 13.0 89.1
    10734/E09 8.0 73.0 1.1 96.2
    10734/E10 18.5 −5.0 29.8 27.9
    10734/E11 19.2 −8.0 16.1 67.1
    10734/F02 6.2 0.7 10.4 55.0
    10734/F03 4.2 24.8 −24.1 86.8
    10734/F04 −2.6 3.9 25.0 76.5
    10734/F05 9.8 −15.6 −1.1 73.8
    10734/F06 11.0 −17.1 −21.4 64.2
    10734/F07 6.6 33.7 −5.1 95.7
    10734/F08 4.8 −23.6 −6.0 66.3
    10734/F09 −3.7 3.1 −17.4 93.9
    10734/F10 14.8 −8.3 25.8 44.8
    10734/F11 13.3 −15.6 16.1 47.3
    10734/G02 1.5 3.4 14.8 63.6
    10734/G03 18.7 82.8 −3.8 93.4
    10734/G04 −1.1 62.9 16.1 72.1
    10734/G05 7.4 −0.3 2.9 57.7
    10734/G06 16.5 12.5 −18.8 43.1
    10734/G07 21.9 51.1 −25.0 96.6
    10734/G08 11.6 33.7 0.2 82.2
    10734/G09 8.5 21.4 −7.7 84.7
    10734/G10 10.8 3.8 9.9 19.3
    10734/G11 8.7 4.1 −25.4 20.2
    10734/H02 0.5 −11.9 −10.8 48.3
    10734/H03 4.6 6.6 8.6 90.3
    10734/H04 −1.3 5.9 6.0 64.2
    10734/H05 1.6 4.7 −14.4 51.1
    10734/H06 2.6 −32.2/ −26.3 32.7
    10734/H07 1.4 6.6 −6.4 84.9
    10734/H08 −2.2 2.9 2.9 64.8
    10734/H09 −2.2 −4.2 −19.2 78.4
    10734/H10 3.9 −12.6 −17.9 23.5
    10734/H11 9.9 −8.5 −17.9 28.7
    10735/A02 −13.4 −14.3 14.3 26.9
    10735/A03 −1.0 28.8 21.9 53.0
    10735/A04 −20.8 5.0 18.8 48.0
    10735/A05 −12.2 41.5 0.9 46.1
    10735/A06 −8.5 24.9 4.7 58.9
    10735/A07 −1.3 18.7 0.5 61.9
    10735/A08 −19.6 10.9 5.3 50.8
    10735/A09 −6.3 13.0 1.9 19.2
    10735/A10 2.2 10.2 −0.2 41.4
    10735/A11 2.2 3.3 5.7 24.5
    10735/B02 1.1 39.1 22.6 72.4
    10735/B03 −14.3 42.9 16.4 73.3
    10735/B04 6.1 49.2 2.9 79.0
    10735/B05 7.4 61.6 17.4 83.7
    10735/B06 17.5 49.4 14.0 82.7
    10735/B07 −5.9 47.7 0.5 56.0
    10735/B08 1.6 24.5 9.8 53.4
    10735/B09 15.9 39.8 −0.5 46.8
    10735/B10 9.2 30.1 3.6 73.3
    10735/B11 15.7 52.4 −8.8 69.0
    10735/C02 0.1 1.6 4.7 27.7
    10735/C03 0.9 8.0 4.3 66.2
    10735/C04 17.2 5.8 12.9 75.8
    10735/C05 11.2 14.5 20.5 59.4
    10735/C06 18.2 8.5 25.0 47.0
    10735/C07 9.6 −4.9 11.9 49.1
    10735/C08 10.1 −11.3 −2.6 49.8
    10735/C09 24.5 −22.1 −2.6 24.1
    10735/C10 21.0 −14.4 4.0 37.8
    10735/C11 18.6 −6.4 −2.9 14.1
    10735/D02 0.9 20.3 11.6 32.6
    10735/D03 4.5 7.5 12.9 41.2
    10735/D04 9.1 3.8 8.5 25.6
    10735/D05 29.5 23.0 20.5 33.7
    10735/D06 16.2 8.0 14.7 36.9
    10735/D07 21.6 −17.6 3.6 33.7
    10735/D08 21.6 −1.5 −5.7 25.6
    10735/D09 22.6 −0.3 6.7 23.0
    10735/D10 15.1 46.5 3.3 35.0
    10735/D11 25.1 −16.5 0.5 18.5
    10735/E02 −2.0 16.7 14.7 13.2
    10735/E03 5.1 18.9 16.0 71.8
    10735/E04 12.2 14.3 91.6 85.2
    10735/E05 14.7 30.6 47.8 74.8
    10735/E06 23.8 8.7 32.3 33.3
    10735/E07 16.1 27.4 57.1 64.5
    10735/E08 14.4 10.2 25.4 35.7
    10735/E09 29.6 −3.9 37.1 11.3
    10735/E10 10.2 19.1 27.1 41.8
    10735/E11 21.9 10.1 11.9 7.2
    10735/F02 2.1 19.8 6.0 31.2
    10735/F03 11.0 19.1 7.8 36.1
    10735/F04 16.4 10.6 40.5 41.6
    10735/F05 30.2 5.5 37.1 26.4
    10735/F06 16.7 13.0 20.9 39.5
    10735/F07 34.0 28.9 19.2 39.7
    10735/F08 10.7 9.4 13.3 18.5
    10735/F09 40.2 0.0 19.8 7.0
    10735/F10 40.0 15.7 13.3 13.0
    10735/F11 34.6 14.8 4.0 16.6
    10735/G02 5.1 23.7 7.1 26.0
    10735/G03 6.8 30.3 6.4 24.3
    10735/G04 15.6 22.8 11.6 28.8
    10735/G05 8.8 16.7 13.6 22.2
    10735/G06 18.5 2.1 12.9 36.1
    10735/G07 17.6 4.6 12.6 37.1
    10735/G08 10.9 1.6 9.8 29.9
    10735/G09 15.9 19.8 2.2 16.8
    10735/G10 6.8 4.5 −8.8 37.8
    10735/G11 10.1 2.9 −5.3 23.0
    10735/H02 14.5 3.6 −4.7 29.2
    10735/H03 7.4 −1.0 1.2 40.8
    10735/H04 12.8 −9.3 1.2 42.9
    10735/H05 29.5 −0.3 9.1 49.8
    10735/H06 25.9 −14.2 6.4 57.2
    10735/H07 21.4 3.8 −0.9 48.5
    10735/H08 29.1 6.8 −2.6 59.2
    10735/H09 20.6 −10.7 6.4 22.4
    10735/H10 24.0 −16.3 −0.2 29.9
    10735/H11 28.3 −4.2 1.9 22.8
    10736/A02 −6.0 7.7 24.2 72.5
    10736/A03 5.7 24.2 31.6 42.6
    10736/A04 −13.0 14.1 13.2 75.2
    10736/A05 −19.4 80.1 8.4 73.6
    10736/A06 −18.2 34.4 32.1 69.7
    10736/A07 −12.6 30.2 8.4 55.2
    10736/A08 −13.4 27.6 11.0 58.7
    10736/A09 −8.7 20.0 −6.5 69.7
    10736/A10 −11.0 −0.1 5.8 65.0
    10736/A11 −12.4 −29.3 −5.6 46.1
    10736/B02 1.0 −38.8 −10.0 73.6
    10736/B03 11.3 −51.7 63.2 65.5
    10736/B04 3.0 −9.0 11.9 65.5
    10736/B05 −0.5 14.9 −3.0 63.9
    10736/B06 3.8 12.6 20.7 60.6
    10736/B07 4.0 8.4 20.7 87.2
    10736/B08 5.0 8.4 −0.4 59.3
    10736/B09 8.0 −3.5 7.1 55.2
    10736/B10 2.5 15.6 1.8 52.5
    10736/B11 7.3 −3.7 −15.3 47.0
    10736/C02 −0.9 −47.1 2.7 50.1
    10736/C03 6.5 0.3 25.5 9.9
    10736/C04 2.8 90.2 8.4 49.0
    10736/C05 4.6 16.8 16.7 44.6
    10736/C06 6.2 4.8 26.4 45.2
    10736/C07 4.4 −8.1 14.1 55.6
    10736/C08 3.1 −0.5 1.4 49.2
    10736/C09 11.2 4.6 −9.6 27.9
    10736/C10 1.1 −0.3 1.4 18.7
    10736/C11 7.2 −4.5 0.5 11.0
    10736/D02 5.8 1.8 −10.5 44.4
    10736/D03 5.4 −3.7 14.5 14.7
    10736/D04 4.3 24.9 15.0 8.1
    10736/D05 15.2 −7.9 3.6 3.9
    10736/D06 12.2 29.7 19.8 14.7
    10736/D07 7.0 47.1 4.4 15.4
    10736/D08 11.0 90.9 1.8 13.0
    10736/D09 8.5 66.8 −6.5 14.1
    10736/D10 −11.6 10.5 −2.1 16.9
    10736/D11 12.5 51.7 −4.3 22.6
    10736/E02 8.2 2.0 21.5 81.5
    10736/E03 17.7 90.2 96.1 33.0
    10736/E04 22.6 −17.2 24.6 61.8
    10736/E05 30.2 17.0 18.5 57.6
    10736/E06 20.1 47.3 10.6 66.6
    10736/E07 15.3 −26.7 18.5 45.9
    10736/E08 19.3 −47.3 7.9 52.3
    10736/E09 14.1 −40.1 −0.4 53.8
    10736/E10 27.2 −24.0 26.8 37.8
    10736/E11 20.5 −74.1 0.9 64.4
    10736/F02 23.9 −4.5 −14.4 69.4
    10736/F03 16.6 94.0 −3.0 23.9
    10736/F04 16.5 −21.7 3.6 45.2
    10736/F05 15.1 −6.0 −4.8 19.8
    10736/F06 14.5 25.1 12.3 49.0
    10736/F07 16.6 −12.1 −4.3 10.7
    10736/F08 7.4 −2.8 −20.1 −0.2
    10736/F09 24.0 6.3 2.7 40.2
    10736/F10 7.7 −0.7 11.0 65.3
    10736/F11 3.7 −23.4 4.4 25.2
    10736/G02 −7.2 −4.5 −5.2 48.3
    10736/G03 4.5 23.0 1.4 0.6
    10736/G04 13.0 −35.0 −8.3 −6.6
    10736/G05 2.3 −12.1 −9.2 0.0
    10736/G06 5.9 18.9 5.3 1.3
    10736/G07 9.4 −74.8 −14.8 −0.2
    10736/G08 −0.1 −9.2 −9.2 6.6
    10736/G09 −2.1 12.4 −3.5 16.2
    10736/G10 6.0 −4.5 −7.4 5.7
    10736/G11 0.0 −6.4 −1.7 10.3
    10736/H02 6.1 −32.0 −6.1 62.8
    10736/H03 15.1 −1.4 2.2 27.7
    10736/H04 14.0 −4.3 −10.9 27.2
    10736/H05 16.3 −28.9 −10.0 21.1
    10736/H06 15.5 12.6 9.3 27.4
    10736/H07 17.8 52.2 −21.9 31.0
    10736/H08 15.8 −20.2 −17.9 33.4
    10736/H09 13.2 35.7 −14.8 32.1
    10736/H10 2.1 45.6 −21.0 32.1
    10736/H11 17.6 11.1 −12.7 41.5
    10736/A02 −5.5 37.3 6.6 53.2
    10737/A03 −1.2 12.8 38.8 58.9
    10737/A04 −0.4 29.4 23.7 63.5
    10737/A05 −2.0 26.4 21.7 58.9
    10737/A06 −20.3 37.3 15.7 57.1
    10737/A07 3.4 25.3 7.0 30.1
    10737/A08 4.9 35.8 1.0 28.3
    10737/A09 −10.1 36.0 18.9 47.9
    10737/A10 6.0 10.9 −3.8 15.5
    10737/A11 6.2 10.8 28.1 25.3
    10737/B02 −3.0 −34.0 −28.9 85.0
    10737/B03 7.0 −34.9 29.7 86.3
    10737/B04 −8.2 7.1 12.5 88.9
    10737/B05 −4.6 17.4 19.7 89.5
    10737/B06 3.6 22.5 20.1 84.7
    10737/B07 10.5 18.1 18.9 82.5
    10737/B08 12.2 15.4 −0.6 84.1
    10737/B09 17.5 26.4 −14.1 77.0
    10737/B10 17.1 −7.3 −10.2 84.7
    10737/B11 12.8 −9.9 −10.2 78.8
    10737/C02 −2.9 −22.0 −2.2 30.6
    10737/C03 10.1 −0.7 7.0 52.5
    10737/C04 −20.1 14.4 25.7 82.0
    10737/C05 −3.3 13.3 12.5 81.8
    10737/C06 8.7 29.0 7.8 38.6
    10737/C07 1.6 27.9 8.6 57.5
    10737/C08 8.9 16.3 14.5 54.1
    10737/C09 8.4 13.0 −1.8 27.6
    10737/C10 16.0 −15.6 6.6 60.0
    10737/C11 13.3 −5.6 32.5 34.0
    10737/D02 10.6 —14.7 −4.2 27.8
    10737/D03 12.1 −21.5 −5.8 38.6
    10737/D04 12.0 27.7 9.4 33.5
    10737/D05 8.9 17.9 2.2 41.8
    10737/D06 13.0 33.8 −10.2 37.4
    10737/D07 12.9 16.1 −5.0 37.0
    10737/D08 15.3 5.6 3.0 42.0
    10737/D09 13.9 13.7 1.0 36.0
    10737/D10 23.2 −15.0 7.0 24.8
    10737/D11 17.2 −2.3 5.4 43.4
    10737/E02 14.3 6.7 12.9 53.0
    10737/E03 17.9 −4.5 39.2 13.4
    10737/E04 16.1 15.4 29.7 79.3
    10737/E05 14.9 −8.6 33.3 80.9
    10737/E06 11.1 −14.1 26.5 57.8
    10737/E07 20.1 −12.3 28.1 43.1
    10737/E08 21.3 −7.8 35.6 61.9
    10737/E09 11.9 −0.7 25.7 21.2
    10737/E10 18.9 −4.2 30.9 49.3
    10737/E11 18.3 −33.4 38.0 24.8
    10737/F02 15.1 21.4 9.0 67.4
    10737/F03 16.3 −3.1 7.8 28.0
    10737/F04 12.2 −23.3 3.8 62.8
    10737/F05 18.4 −24.0 1.0 67.1
    10737/F06 12.3 −13.7 11.3 62.8
    10737/F07 19.1 −25.5 12.9 33.3
    10737/F08 20.7 28.3 9.8 76.1
    10737/F09 15.4 23.5 9.8 76.5
    10737/F10 26.1 14.6 11.0 78.6
    10737/F11 31.6 −0.7 16.5 75.6
    10737/G02 8.7 12.4 −5.8 32.2
    10737/G03 13.6 −0.5 21.7 37.4
    10737/G04 12.1 −19.3 12.1 49.1
    10737/G05 16.4 −41.4 16.1 53.2
    10737/G06 12.8 −43.4 −10.2 50.7
    10737/G07 20.3 −25.0 24.1 41.8
    10737/G08 16.4 30.8 13.7 31.2
    10737/G09 15.2 23.6 8.2 37.9
    10737/G10 13.3 17.2 −0.2 22.8
    10737/G11 11.7 2.8 5.0 35.1
    10737/H02 3.1 −17.2 −18.1 42.9
    10737/H03 3.2 −22.6 −21.3 46.1
    10737/H04 11.6 −17.6 −21.3 63.0
    10737/H05 9.9 −17.4 −6.2 76.5
    10737/H06 5.8 −31.0 −1.8 28.7
    10737/H07 15.5 17.2 −17.7 51.8
    10737/H08 10.0 −36.6 −26.9 28.0
    10737/H09 5.5 −15.9 −11.7 38.1
    10737/H10 10.5 0.3 −12.9 51.6
    10737/H11 14.5 4.5 −16.5 31.7
    10738/A02 −14.2 16.7 10.0
    10738/A03 12.8 40.5 20.7
    10738/A04 −16.2 −17.5 55.9
    10738/A05 −24.5 33.6 33.1
    10738/A06 77.3 54.9 95.8
    10738/A07 −22.1 4.3 72.7
    10738/A08 −9.3 6.0 12.2
    10738/A09 −9.8 17.0 20.2
    10738/A10 −8.5 −0.9 35.7
    10738/A11 −7.5 4.5 41.7
    10738/B02 −8.5 −24.4 78.7
    10738/B03 −1.2 19.2 74.0
    10738/B04 55.0 51.2 70.7
    10738/B05 −13.1 57.6 67.6
    10738/B06 −44.1 22.9 83.6
    10738/B07 −22.7 28.7 66.7
    10738/B08 −0.7 33.9 70.9
    10738/B09 −6.2 20.9 79.4
    10738/B10 14.3 37.3 70.7
    10738/B11 8.8 18.0 75.4
    10738/C02 −0.1 −30.5 4.3
    10738/C03 28.0 8.2 22.7
    10738/C04 84.2 43.7 53.7
    10738/C05 −4.4 18.4 34.2
    10738/C06 −68.1 28.7 92.2
    10738/C07 −7.4 −19.0 51.2
    10738/C08 −7.4 −11.9 39.5
    10738/C09 4.5 31.9 8.9
    10738/C10 −2.5 5.2 3.2
    10738/C11 8.4 −1.1 12.0
    10738/D02 2.8 21.4 15.6
    10738/D03 13.6 47.6 16.0
    10738/D04 −13.1 7.4 32.2
    10738/D05 −12.5 4.3 42.4
    10738/D06 −26.7 32.4 96.0
    10738/D07 10.3 −8.7 68.3
    10738/D08 11.9 0.6 37.7
    10738/D09 9.4 12.1 22.7
    10738/D10 15.4 −12.4 25.3
    10738/D11 10.4 −13.1 4.3
    10738/E02 3.6 56.1 3.8
    10738/E03 7.8 82.6 21.1
    10738/E04 −11.5 13.1 64.1
    10738/E05 −4.8 52.0 61.0
    10738/E06 10.1 15.8 92.4
    10738/E07 43.4 57.1 89.3
    10738/E08 12.8 43.2 52.6
    10738/E09 21.2 35.3 18.0
    10738/E10 5.1 34.1 5.8
    10738/E11 10.2 15.8 11.8
    10738/F02 15.0 13.1 66.1
    10738/F03 9.0 45.6 20.0
    10738/F04 −32.3 −6.5 61.4
    10738/F05 3.3 51.5 33.5
    10738/F06 −12.6 −19.5 86.7
    10738/F07 6.4 9.1 52.3
    10738/F08 16.8 15.5 60.5
    10738/F09 1.7 0.1 28.9
    10738/F10 9.5 −4.8 47.9
    10738/F11 15.8 −14.8 52.6
    10738/G02 −10.5 19.9 16.9
    10738/G03 −4.1 31.9 32.8
    10738/G04 −57.2 12.1 42.6
    10738/G05 4.0 27.3 27.3
    10738/G06 −52.4 −8.2 86.5
    10738/G07 −19.3 −1.9 40.4
    10738/G08 −15.2 23.6 36.8
    10738/G09 4.1 16.7 16.9
    10738/G10 −17.0 26.5 12.5
    10738/G11 −8.1 19.4 22.2
    10738/H02 −14.6 −52.0 35.3
    10738/H03 −6.2 16.7 29.5
    10738/H04 −17.9 −7.0 72.9
    10738/H05 −17.9 5.5 73.6
    10738/H06 −44.8 −53.8 93.8
    10738/H07 −17.4 −13.4 77.2
    10738/H08 −12.7 −12.1 20.7
    10738/H09 −6.5 1.6 52.3
    10738/H10 1.7 11.4 27.7
    10738/H11 −4.1 −13.4 24.9
    10739/A02 1.2 0.0 21.7 41.8
    10739/A03 2.9 5.1 15.4 37.4
    10739/A04 −8.2 −6.3 23.2 45.2
    10739/A05 −11.0 10.7 15.9 36.5
    10739/A06 7.4 −5.5 17.8 70.1
    10739/A07 4.1 65.6 34.8 99.9
    10739/A08 −25.5 6.2 3.8 39.9
    10739/A09 −9.1 26.9 −0.6 60.4
    10739/A10 −2.5 20.7 −1.1 81.2
    10739/A11 3.9 −8.7 1.8 82.8
    10739/B02 2.1 18.1 5.2 68.1
    10739/B03 3.5 9.6 1.8 62.4
    10739/B04 13.2 17.5 19.8 65.1
    10739/B05 4.1 32.0 4.7 74.0
    10739/B06 9.3 9.2 6.7 68.1
    10739/B07 1.1 59.4 19.8 98.7
    10739/B08 6.5 19.8 −0.1 65.8
    10739/B09 −15.4 11.9 74.1 63.5
    10739/B10 −10.1 −2.1 −16.1 70.6
    10739/B11 12.5 −4.2 −9.8 84.9
    10739/C02 −10.8 −1.7 10.6 34.7
    10739/C03 16.4 18.3 10.1 10.9
    10739/C04 21.8 1.5 19.3 −3.6
    10739/C05 5.6 −1.7 31.4 34.7
    10739/C06 17.7 −17.0 33.8 56.1
    10739/C07 −14.5 34.7 44.5 93.0
    10739/C08 4.8 −1.7 11.0 9.1
    10739/C09 10.0 −2.7 105.6 76.3
    10739/C10 −7.1 −7.0 −18.6 66.3
    10739/C11 70.6 −17.0 9.1 61.0
    10739/D02 25.6 2.6 17.8 29.0
    10739/D03 −18.3 24.7 14.4 15.9
    10739/D04 11.8 −6.4 7.2 11.1
    10739/D05 12.2 −10.6 −6.9 30.0
    10739/D06 9.0 −19.8 −43.8 59.2
    10739/D07 −5.1 21.1 18.8 86.0
    10739/D08 14.3 −16.8 5.7 5.2
    10739/D09 −32.8 −15.1 4.7 66.7
    10739/D10 14.3 −7.6 13.0 20.4
    10739/D11 13.8 15.2 3.8 65.8
    10739/E02 26.7 −8.3 18.3 30.0
    10739/E03 −119.0 4.9 2.8 −3.4
    10739/E04 17.7 −35.5 5.2 −15.2
    10739/E05 28.0 −11.3 10.6 8.9
    10739/E06 34.3 33.5 72.7 66.3
    10739/E07 10.0 79.6 48.4 94.0
    10739/E08 17.2 −17.4 21.7 16.1
    10739/E09 24.5 15.2 18.3 33.8
    10739/E10 11.2 17.5 25.6 71.9
    10739/E11 15.3 −23.4 15.9 76.9
    10739/F02 25.5 −3.6 6.2 50.8
    10739/F03 24.5 −10.8 −7.4 38.8
    10739/F04 26.3 −7.0 −4.0 30.4
    10739/F05 8.7 −16.4 −4.5 −5.4
    10739/F06 13.7 −15.3 9.1 56.3
    10739/F07 0.6 62.4 23.7 100.5
    10739/F08 7.0 −19.1 9.1 13.4
    10739/F09 15.6 −19.6 3.3 −7.0
    10739/F10 12.3 −16.8 4.2 40.4
    10739/F11 11.9 −38.5 0.8 40.9
    10739/G02 5.9 −5.1 −12.2 12.9
    10739/G03 1.9 2.4 −6.9 9.3
    10739/G04 4.5 −12.3 −3.5 4.1
    10739/G05 10.7 −23.0 −5.9 27.7
    10739/G06 14.0 −31.2 −6.4 27.7
    10739/G07 −4.9 10.0 10.1 92.6
    10739/G08 15.0 −18.5 1.3 20.9
    10739/G09 3.1 −39.8 13.9 46.1
    10739/G10 −2.0 −38.1 −0.6 36.5
    10739/G11 9.8 −23.2 7.6 56.7
    10739/H02 2.2 −12.3 −18.6 26.6
    10739/H03 1.6 10.0 −19.5 10.4
    10739/H04 9.9 −21.9 −14.2 −2.7
    10739/H05 5.3 −8.3 −5.0 20.0
    10739/H06 35.6 −22.1 −17.1 42.2
    10739/H07 −1.4 10.9 14.9 93.7
    10739/H08 4.0 −28.5 −3.5 22.9
    10739/H09 10.0 −44.2 −6.4 45.6
    10739/H10 15.0 −26.6 −13.2 62.4
    10739/H11 −7.6 49.9 4.2 75.8
    10740/A02 5.7 −2.4 56.3
    10740/A03 4.2 8.2 77.8
    10740/A04 1.3 5.2 48.2
    10740/A05 −13.1 30.3 79.5
    10740/A06 −8.7 13.1 48.5
    10740/A07 −18.3 18.7 54.9
    10740/A08 −18.3 −7.9 43.2
    10740/A09 −15.7 −9.1 40.1
    10740/A10 −7.7 21.9 45.1
    10740/A11 −3.5 26.0 30.3
    10740/B02 −17.0 46.1 79.2
    10740/B03 −9.3 40.1 84.8
    10740/B04 −0.3 36.9 76.7
    10740/B05 −11.6 30.7 82.3
    10740/B06 −5.9 44.4 68.9
    10740/B07 −8.5 48.9 73.7
    10740/B08 6.6 34.8 65.0
    10740/B09 0.8 47.7 71.1
    10740/B10 11.8 39.9 58.0
    10740/B11 0.7 48.1 68.6
    10740/C02 −5.9 15.6 45.1
    10740/C03 −129.3 7.2 63.6
    10740/C04 −3.4 7.8 −4.9
    10740/C05 −19.1 3.5 66.1
    10740/C06 −19.1 18.2 49.3
    10740/C07 1.1 5.0 44.3
    10740/C08 4.6 10.7 28.7
    10740/C09 −52.7 28.1 63.3
    10740/C10 0.8 21.9 −1.8
    10740/C11 6.9 18.7 −10.8
    10740/D02 4.1 0.0 49.9
    10740/D03 15.1 −6.9 64.4
    10740/D04 −2.0 −1.2 19.4
    10740/D05 −6.3 7.4 39.3
    10740/D06 9.6 5.8 41.8
    10740/D07 −4.5 −3.0 27.3
    10740/D08 −6.4 −5.7 30.3
    10740/D09 −14.5 14.8 33.1
    10740/D10 1.5 9.9 16.1
    10740/D11 3.0 19.3 −9.1
    10740/E02 9.6 −7.7 48.2
    10740/E03 0.2 24.6 66.4
    10740/E04 2.0 12.3 41.8
    10740/E05 28.7 29.1 75.6
    10740/E06 −8.4 0.0 16.6
    10740/E07 34.4 2.7 19.7
    10740/E08 39.1 3.1 39.0
    10740/E09 89.8 8.6 31.2
    10740/E10 11.8 33.0 61.9
    10740/E11 17.1 8.8 −16.9
    10740/F02 2.8 5.8 59.4
    10740/F03 −0.9 16.8 32.8
    10740/F04 6.8 11.1 −5.2
    10740/F05 −13.5 14.8 52.1
    10740/F06 1.3 19.9 25.3
    10740/F07 −6.3 −4.2 28.4
    10740/F08 −5.0 5.0 35.9
    10740/F09 −18.7 11.7 44.3
    10740/F10 10.0 9.5 58.3
    10740/F11 8.2 5.4 42.9
    10740/G02 −2.6 −9.4 55.2
    10740/G03 −2.0 19.5 34.8
    10740/G04 −5.1 7.6 9.1
    10740/G05 −18.6 18.7 30.3
    10740/G06 −19.3 −1.2 25.0
    10740/G07 0.4 27.9 10.8
    10740/G08 −0.5 −9.1 33.4
    10740/G09 −8.0 −7.9 37.6
    10740/G10 −2.8 1.9 19.4
    10740/G11 3.3 −3.4 13.3
    10740/H02 −3.7 −9.1 28.7
    10740/H03 −9.7 16.0 17.5
    10740/H04 6.0 −21.0 30.9
    10740/H05 5.8 −4.4 25.6
    10740/H06 3.2 4.8 33.4
    10740/H07 10.1 −5.3 1.5
    10740/H08 11.5 −24.3 28.1
    10740/H09 −12.2 −20.6 50.2
    10740/H10 0.4 −25.5 43.7
    10740/H11 7.4 0.9 17.2
    • Cellular/Catalytic Assays
  • Enzyme linked immunosorbent assays (ELISA) may be used to detect and measure the presence of PK activity. The ELISA may be conducted according to known protocols which are described in, for example, Voller, et al., 1980, “Enzyme-Linked Immunosorbent Assay,” In: Manual of Clinical Immunology, 2d ed., edited by Rose and Friedman, pp 359-371 Am. Soc. Of Microbiology, Washington, D.C. [0207]
  • The disclosed protocol may be adapted for determining activity with respect to a specific PK. That is, the preferred protocols for conducting the ELISA experiments for specific PKs is provided below. However, adaptation of these protocols for determining a compound's activity for other members of the RTK family, as well as for CTKs and STKs, is well within the scope of knowledge of those skilled in the art. [0208]
    • FLK-1Assay
  • An ELISA assay is conducted to measure the kinase activity of the FLK-1 receptor and more specifically, the inhibition or activation of TK activity on the FLK-1 receptor. Specifically, the following assay can be conducted to measure kinase activity of the FLK-1 receptor in cells genetically engineered to express Flk-1. [0209]
  • Materials and Reagents. [0210]
  • a. Corning 96-well ELISA plates (Corning Catalog No. 25805-96); [0211]
  • b. Cappel goat anti-rabbit IgG (catalog no. 55641); [0212]
  • c. PBS (Gibco Catalog No. 450-1300EB); [0213]
  • d. TBSW Buffer (50 mM Tris (pH 7.2), 150 mM NaCl and 0.1% Tween-20); [0214]
  • e. Ethanolamine stock (10% ethanolamine (pH 7.0), stored at 4° C.); [0215]
  • f. HNTG buffer (20 mM HEPES buffer (pH 7.5), 150 mM NaCl, 0.2% Triton X-100, and 10% glycerol); [0216]
  • g. EDTA (0.5 M (pH 7.0) as a 100×stock); [0217]
  • h. Sodium orthovanadate (0.5 M as a 100×stock); [0218]
  • i. Sodium pyrophosphate (0.2 M as a 100×stock); [0219]
  • j. NUNC 96 well V bottom polypropylene plates (Applied Scientific Catalog No. AS-72092); [0220]
  • k. NIH3T3 C7#3 Cells (FLK-1 expressing cells); [0221]
  • l. DMEM with 1× high glucose L-Glutamine (catalog No. 11965-050); [0222]
  • m. FBS, Gibco (catalog no. 16000-028); [0223]
  • n. L-glutamine, Gibco (catalog no. 25030-016); [0224]
  • o. VEGF, PeproTech, Inc. (catalog no. 100-20) (kept as 1 μg/100 μl stock in Milli-Q dH[0225] 2O and stored at −20° C.;
  • p. Affinity purified anti-FLK-1 antiserum; [0226]
  • q. UB40 monoclonal antibody specific for phosphotyrosine (see, Fendley, et al., 1990, Cancer Research 50:1550-1558); [0227]
  • r. EIA grade Goat anti-mouse IgG-POD (BioRad catalog no. 172-1011); [0228]
  • s. 2,2-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid (ABTS) solution (100 mM citric acid (anhydrous), 250 mM Na[0229] 2HPO4 (pH 4.0), 0.5 mg/ml ABTS (Sigma catalog no. A-1888)), solution should be stored in dark at 4° C. until ready for use;
  • t. H[0230] 2O2 (30% solution) (Fisher catalog no. H325);
  • u. ABTS/H[0231] 2O2 (15 ml ABTS solution, 2 μl H2O2) prepared 5 minutes before use and left at room temperature;
  • v. 0.2 M HCl stock in H[0232] 2O;
  • w. dimethylsulfoxide (100% ) (Sigma Catalog No. D-8418); and [0233]
  • y. Trypsin-EDTA (Gibco BRL Catalog No. 25200-049). [0234]
  • Protocol. [0235]
  • 1. Coat Corning 96-well ELISA plates with 1.0 μg per well Cappel Anti-rabbit IgG antibody in 0.1M Na[0236] 2CO3 pH 9.6. Bring final volume to 150 μl per well. Coat plates overnight at 4° C. Plates can be kept up to two weeks when stored at 4° C.
  • 2. Grow cells in Growth media (DMEM, supplemented with 2.0 mM L-Glutamine, 10% FBS) in suitable culture dishes until confluent at 37° C., 5% CO[0237] 2.
  • 3. Harvest cells by trypsinization and seed in Corning 25850 polystyrene 96-well round bottom cell plates, 25,000 cells/well in 200 μl of growth media. [0238]
  • 4. Grow cells at least one day at 37° C., 5% CO[0239] 2,
  • 5. Wash cells with D-PBS 1×. [0240]
  • 6. Add 200 μl/well of starvation media (DMEM, 2.0 mM 1-Glutamine, 0.1% FBS). Incubate overnight at 37° C., 5% CO[0241] 2.
  • 7. Dilute Compounds 1:20 in polypropylene 96 well plates using starvation media. Dilute dimethylsulfoxide 1:20 for use in control wells. [0242]
  • 8. Remove starvation media from 96 well cell culture plates and add 162 μl of fresh starvation media to each well. [0243]
  • 9. Add 18 μl of 1:20 diluted compound dilution (from step 7) to each well plus the 1:20 dimethylsulfoxide dilution to the control wells (±VEGF), for a final dilution of 1:200 after cell stimulation. Final dimethylsulfoxide is 0.5% . Incubate the plate at 37° C., 5% CO[0244] 2 for two hours.
  • 10. Remove unbound antibody from ELISA plates by inverting plate to remove liquid. Wash 3 times with TBSW+0.5% ethanolamine, pH 7.0. Pat the plate on a paper towel to remove excess liquid and bubbles. [0245]
  • 11. Block plates with TBSW+0.5% ethanolamine, pH 7.0, 150 μl per well. Incubate plate thirty minutes while shaking on a microtiter plate shaker. [0246]
  • 12. Wash plate 3 times as described in step 10. [0247]
  • 13. Add 0.5 μg/well affinity purified anti-FLU-1 polyclonal rabbit antiserum. Bring final volume to 150 μl/well with TBSW+0.5% ethanolamine pH 7.0. Incubate plate for thirty minutes while shaking. [0248]
  • 14. Add 180 μl starvation medium to the cells and stimulate cells with 20 μl/well 10.0 mM sodium orthovanadate and 500 ng/ml VEGF (resulting in a final concentration of 1.0 mM sodium orthovanadate and 50 ng/ml VEGF per well) for eight minutes at 37° C., 5% CO[0249] 2. Negative control wells receive only starvation medium.
  • 15. After eight minutes, media should be removed from the cells and washed one time with 200 μl/well PBS. [0250]
  • 16. Lyse cells in 150 μl/well HNTG while shaking at room temperature for five minutes. HNTG formulation includes sodium ortho vanadate, sodium pyrophosphate and EDTA. [0251]
  • 17. Wash ELISA plate three times as described in step 10. [0252]
  • 18. Transfer cell lysates from the cell plate to ELISA plate and incubate while shaking for two hours. To transfer cell lysate pipette up and down while scrapping the wells. [0253]
  • 19. Wash plate three times as described in step 10. [0254]
  • 20. Incubate ELISA plate with 0.02 μg/well UB40 in TBSW+05% ethanolamine. Bring final volume to 150 μl/well. Incubate while shaking for 30 minutes. [0255]
  • 21. Wash plate three times as described in step 10. [0256]
  • 22. Incubate ELISA plate with 1:10,000 diluted EIA grade goat anti-mouse IgG conjugated horseradish peroxidase in TBSW plus 0.5% ethanolamine, pH 7.0. Bring final volume to 150 μl/well. Incubate while shaking for thirty minutes. [0257]
  • 23. Wash plate as described in step 10. [0258]
  • 24. Add 100 μl of ABTS/H[0259] 2O2 solution to well. Incubate ten minutes while shaking.
  • 25. Add 100 μl of 0.2 M HCl for 0.1 M HCl final concentration to stop the color development reaction. Shake 1 minute at room temperature. Remove bubbles with slow stream of air and read the ELISA plate in an ELISA plate reader at 410 nm. [0260]
    • EGF Receptor-HER2 Chimeric Receptor Assay In Whole Cells
  • HER2 kinase activity in whole EGFR-NIH3T3 cells are measured as described below: [0261]
  • Materials and Reagents. [0262]
    a. EGF: stock concentration: 16.5 ILM; EGF 201, TOYOBO,
    Co., Ltd. Japan.
    b. 05-101 (UBI) (a monoclonal antibody recognizing an EGFR
    extracellular domain).
    c. Anti-phosphotyrosine antibody (anti-Ptyr)
    (polyclonal) (see, Fendley, et al., supra).
    d. Detection antibody: Goat anti-rabbit lgG horseradish
    peroxidase conjugate, TAGO, Inc., Burlingame, CA.
    e. TBST buffer:
    Tris-HCl, pH 7.2 50 mM
    NaCl 150 mM
    Triton X-100 0.1
    f. HNTG 5X stock:
    HEPES 0.1 M
    NaCl 0.75 M
    Glycerol 50%
    Triton X-100 1.0%
    g. ABTS stock:
    Citric Acid: 100 mM
    Na2HPO4 250 mM
    HCl, conc. 0.5 mM
    ABTS* 0.5 mg/ml
    h. Stock reagents of:
    EDTA 100 mM pH 7.0
    Na3VO4 0.5 M
    Na4(P2O7) 0.2 M
  • Procedure. [0263]
  • Pre-coat ELISA Plate [0264]
  • 1. Coat ELISA plates (Corning, 96 well, Cat. #25805-96) with 05-101 antibody at 0.5 μg per well in PBS, 100 μl final volume/well, and store overnight at 4° C. Coated plates are good for up to 10 days when stored at 4° C. [0265]
  • 2. On day of use, remove coating buffer and replace with 100 μl blocking buffer (5% Carnation Instant Non-Fat Dry Milk in PBS). Incubate the plate, shaking, at room temperature (about 23° C. to 25° C.) for 30 minutes. Just prior to use, remove blocking buffer and wash plate 4 times with TBST buffer. [0266]
  • Seeding Cells [0267]
  • 1. An NIH3T3 cell line overexpressing a chimeric receptor containing the EGFR extracellular domain and intracellular HER2 kinase domain can be used for this assay. [0268]
  • 2. Choose dishes having 80-90% confluence for the experiment. Trypsinize cells and stop reaction by adding 10% fetal bovine serum. Suspend cells in DMEM medium (10% CS DMEM medium) and centrifuge once at 1500 rpm, at room temperature for 5 minutes. [0269]
  • 3. Resuspend cells in seeding medium (DMEM, 0.5% bovine serum), and count the cells using trypan blue. Viability above 90% is acceptable. Seed cells in DMEM medium (0.5% bovine serum) at a density of 10,000 cells per well, 100 μl per well, in a 96 well microtiter plate. Incubate seeded cells in 5% CO[0270] 2 at 37° C. for about 40 hours.
  • Assay Procedures [0271]
  • 1. Check seeded cells for contamination using an inverted microscope. Dilute drug stock (10 mg/ml in DMSO) 1:10 in DMEM medium, then transfer 5 μl to a TBST well for a final drug dilution of 1:200 and a final DMSO concentration of 1% . Control wells receive DMSO alone. Incubate in 5% CO[0272] 2 at 37° C. for two hours.
  • 2. Prepare EGF ligand: dilute stock EGF in DMEM so that upon transfer of 10 μl dilute EGF (1:12 dilution), 100 nM final concentration is attained. [0273]
  • 3. Prepare fresh HNTG* sufficient for 100 μl per well; and place on ice. [0274]
    HNTG* (10 ml):
    HNTG stock 2.0 ml
    milli-Q H2O 7.3 ml
    EDTA, 100 mM, pH 7.0 0.5 ml
    Na3VO4 (0.5 M) 0.1 ml
    Na4(P2O7) (0.2 M) 0.1 ml
  • 4. After 120 minutes incubation with drug, add prepared SGF ligand to cells, 10 μl per well, to a final concentration of 100 nM. Control wells receive DMEM alone. Incubate with shaking, at room temperature, for 5 minutes. [0275]
  • 5. Remove drug, EGF, and DMEM. Wash cells twice with PBS. Transfer HNTG* to cells, 100 μl per well. Place on ice for 5 minutes. Meanwhile, remove blocking buffer from other ELISA plate and wash with TBST as described above. [0276]
  • 6. With a pipette tip securely fitted to a micropipettor, scrape cells from plate and homogenize cell material by repeatedly aspirating and dispensing the HNTG* lysis buffer. Transfer lysate to a coated, blocked, and washed ELISA plate. Incubate shaking at room temperature for one hour. [0277]
  • 7. Remove lysate and wash 4 times with TBST. Transfer freshly diluted anti-Ptyr antibody to ELISA plate at 100 μl per well. Incubate shaking at room temperature for 30 minutes in the presence of the anti-Ptyr antiserum (1:3000 dilution in TBST). [0278]
  • 8. Remove the anti-Ptyr antibody and wash 4 times with TBST. Transfer the freshly diluted TAGO anti-rabbit IgG antibody to the ELISA plate at 100 μl per well. Incubate shaking at room temperature for 30 minutes (anti-rabbit IgG antibody: 1:3000 dilution in TBST). [0279]
  • 9. Remove TAGO detection antibody and wash 4 times with TBST. Transfer freshly prepared ABTS/H[0280] 2O2 solution to ELISA plate, 100 μl per well. Incubate shaking at room temperature for 20 minutes. (ABTS/H2O2 solution: 1.0 μl 30% H2O2 in 10 ml ABTS stock).
  • 10. Stop reaction by adding 50 μl 5N H[0281] 2SO4 (optional), and determine O.D. at 410 nm.
  • 11. 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. [0282]
    • PDGF-R Assay
  • All cell culture media, glutamine, and fetal bovine serum can be purchased from Gibco Life Technologies (Grand Island, N.Y.) unless otherwise specified. All cells are grown in a humid atmosphere of 90-95% air and 5-10% CO[0283] 2 at 37° C. All cell lines are routinely subcultured twice a week and are negative for mycoplasma as determined by the Mycotect method (Gibco).
  • For ELISA assays, cells (U1242, obtained from Joseph Schlessinger, NYU) are 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 are changed to serum-free medium and treated with test compound for 2 hr in a 5% CO[0284] 2, 37° C. incubator. Cells are then stimulated with ligand for 5-10 minute followed by lysis with HNTG (20 mM Hepes, 150 mM NaCl, 10% glycerol, 5 mM EDTA, 5 mM Na3VO4, 0.2% Triton X-100, and 2 mM NaPyr). Cell lysates (0.5 mg/well in PBS) are 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 are incubated with shaking for 1 hour at room temperature. The plates are washed with TBST four times and then incubated with polyclonal anti-phosphotyrosine antibody at room temperature for 30 minutes. Excess anti-phosphotyrosine antibody is removed by rinsing the plate with TBST four times. Goat anti-rabbit IgG antibody is 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 Na2HPO4 and 0.5 mg/mL 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)) plus H2O2 (1.2 mL 30% H2O2 to 10 ml ABTS) is added to the ELISA plates to start color development. Absorbance at 410 nm with a reference wavelength of 630 nm is recorded about 15 to 30 min after ABTS addition.
    • IGF-1 Receptor Assay
  • The following protocol may be used to measure phosphotyrosine level on IGF-1 receptor, which indicates IGF-1 receptor tyrosine kinase activity. [0285]
  • Materials and Reagents. [0286]
  • a. The cell line used in this assay is 3T3/IGF-1R, a cell line genetically engineered to overexpresses IGF-1 receptor. [0287]
  • b. NIH3T3/IGF-1R is grown in an incubator with 5% CO[0288] 2 at 37° C. The growth media is DMEM+10% FBS (heat inactivated)+2 mM L-glutamine.
  • c. Affinity purified anti-IGF-1R antibody 17-69. [0289]
  • d. D-PBS: [0290]
    KH2PO4 0.20 g/l
    KH2PO4 2.16 g/l
    KCl 0.20 g/l
    NaCl 8.00 g/l (pH 7.2)
  • e. Blocking Buffer: TBST plus 5% Milk (Carnation Instant Non-Fat Dry Milk). [0291]
  • f. TBST buffer: [0292]
    Tris-HCl 50 mM
    NaCl 150 mM (pH 7.2/HCl 10 N)
    Triton X-100 0.1%
  • Stock solution of TBS (10×) is prepared, and Triton X-100 is added to the buffer during dilution. [0293]
  • g. HNTG buffer: [0294]
    HEPES 20 mM
    NaCl 150 mM (pH 7.2/HCl 1 N)
    Glycerol 10%
    Triton X-100 0.2%
  • Stock solution (5×) is prepared and kept at 4° C. [0295]
  • h. EDTA/HCl: 0.5 M pH 7.0 (NaOH) as 100×stock. [0296]
  • i. Na[0297] 3VO4: 0.5 M as 100× stock and aliquots are kept at 80° C.
  • j. Na[0298] 4P2O7: 0.2 M as 100× stock.
  • k. Insulin-like growth factor-1 from Promega (Cat# G5111). [0299]
  • l. Rabbit polyclonal anti-phosphotyrosine antiserum. [0300]
  • m. Goat anti-rabbit IgG, POD conjugate (detection antibody), Tago (Cat. No. 4520, Lot No. 1802): Tago, Inc., Burlingame, Calif. [0301]
  • n. ABTS (2,2′-azinobis(3-ethylbenzthiazolinesulfonic acid)) solution: [0302]
    Citric acid 100 mM
    Na2HPO4 250 mM (pH 4.0/1 N HCl)
    ABTS 0.5 mg/ml
  • ABTS solution should be kept in dark and 4° C. The solution should be discarded when it turns green. [0303]
  • o. Hydrogen Peroxide: 30% solution is kept in the dark and at 4° C. [0304]
  • Procedure. [0305]
  • All the following steps are conducted at room temperature unless specifically indicated otherwise. 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. [0306]
  • Cell Seeding: [0307]
  • 1. 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). [0308]
  • 2. Resuspend the cells in fresh DMEM+10% FBS+2 mM L-Glutamine, and transfer to 96-well tissue culture plate (Corning, 25806-96) at 20,000 cells/well (100 μl/well). Incubate for 1 day then replace medium to serum-free medium (90/μl) and incubate in 5% CO[0309] 2 and 37° C. overnight.
  • ELISA Plate Coating and Blocking: [0310]
  • 1. Coat the ELISA plate (Corning 25805-96) with Anti-IGF-1R Antibody at 0.5 μg/well in 100 μl PBS at least 2 hours. [0311]
  • 2. Remove the coating solution, and replace with 100 μl Blocking Buffer, and shake for 30 minutes. Remove the blocking buffer and wash the plate just before adding lysate. [0312]
  • Assay Procedures: [0313]
  • 1. The drugs are tested under serum-free condition. [0314]
  • 2. Dilute drug stock (in 100% DMSO) 1:10 with DMEM in 96-well poly-propylene plate, and transfer 10 μl/well of this solution to the cells to achieve final drug dilution 1:100, and final DMSO concentration of 1.0% . Incubate the cells in 5% CO[0315] 2 at 37° C. for 2 hours.
  • 3. Prepare fresh cell lysis buffer (HNTG*) [0316]
    HNTG 2 ml
    EDTA 0.1 ml
    Na3VO4 0.1 ml
    Na4(P2O7) 0.1 ml
    H2O 7.3 ml
  • 4. After drug incubation for two hours, transfer 10 μl/well of 200 nM IGF-1 Ligand in PBS to the cells (Final Conc. is 20 nM), and incubate at 5% CO[0317] 2 at 37° C. for 10 minutes.
  • 5. Remove media and add 100 μl/well HNTG* and shake for 10 minutes. Look at cells under microscope to see if they are adequately lysed. [0318]
  • 6. Use a 12-channel pipette to scrape the cells from the plate, and homogenize the lysate by repeated aspiration and dispensing. Transfer all the lysate to the antibody coated ELISA plate, and shake for 1 hour. [0319]
  • 7. Remove the lysate, wash the plate, transfer anti-pTyr (1:3,000 with TBST) 100 μl/well, and shake for 30 minutes. [0320]
  • 8. Remove anti-pTyr, wash the plate, transfer TAGO (1:3,000 with TBST) 100 μl/well, and shake for 30 minutes. [0321]
  • 9. Remove detection antibody, wash the plate, and transfer fresh ABTS/H[0322] 2O2 (1.2 μl H2O2 to 10 ml ABTS) 100 μl/well to the plate to start color development.
  • 10. Measure OD at 410 nm with a reference wavelength of 630 nm in Dynatec MR5000. [0323]
    • EGFR Assay
  • EGF Receptor kinase activity in cells genetically engineered to express human EGF-R can be measured as described below: [0324]
  • Materials and Reagents. [0325]
  • a. EGF Ligand: stock concentration=16.5 μM; EGF 201, TOYOBO, Co., Ltd. Japan. [0326]
  • b. 05-101 (UBI) (a monoclonal antibody recognizing an EGFR extracellular domain). [0327]
  • c. Anti-phosphotyosine antibody (anti-Ptyr) (polyclonal). [0328]
  • d. Detection antibody: Goat anti-rabbit lgG horse radish peroxidase conjugate, TAGO, Inc., Burlingame, Calif. [0329]
  • e. TBST buffer: [0330]
    Tris-HCl, pH 7 50 mM
    NaCl 150 mM
    Triton X-100 0.1
  • f. HNTG 5× stock: [0331]
    HEPES 0.1 M
    NaCl 0.75 M
    Glycerol 50
    Triton X-100 1.0%
  • g. ABTS stock: [0332]
    Citric Acid 100 mM
    Na3VO4 250 mM
    HCl, conc. 4.0 pH
    ABTS* 0.5 mg/ml
  • Keep solution in dark at 4° C. until used. [0333]
  • h. Stock reagents of: [0334]
  • EDTA 100 mM pH 7.0 [0335]
  • Na[0336] 3VO4 0.5 M
  • Na[0337] 4(P2O7) 0.2 M
  • Procedure. [0338]
  • Pre-coat ELISA Plate [0339]
  • 1. Coat ELISA plates (Corning, 96 well, Cat. #25805-96) with 05-101 antibody at 0.5 μg per well in PBS, 150 μl final volume/well, and store overnight at 4° C. Coated plates are good for up to 10 days when stored at 4° C. [0340]
  • 2. On day of use, remove coating buffer and replace with blocking buffer (5% Carnation Instant NonFat Dry Milk in PBS). Incubate the plate, shaking, at room temperature (about 23° C. to 25° C.) for 30 minutes. Just prior to use, remove blocking buffer and wash plate 4 times with TBST buffer. [0341]
  • Seeding Cells [0342]
  • 1. NIH 3T3/C7 cell line (Honegger, et al., Cell 51:199-209, 1987) can be use for this assay. [0343]
  • 2. Choose dishes having 80-90% confluence for the experiment. Trypsinize cells and stop reaction by adding 10% CS DMEM medium. Suspend cells in DMEM medium (10% CS DMEM medium) and centrifuge once at 1000 rpm at room temperature for 5 minutes. [0344]
  • 3. Resuspend cells in seeding medium (DMEM, 0.5% bovine serum), and count the cells using trypan blue. Viability above 90% is acceptable. Seed cells in DMEM medium (0.5% bovine serum) at a density of 10,000 cells per well, 100 μl per well, in a 96 well microtiter plate. Incubate seeded cells in 5% CO[0345] 2 at 37° C. for about 40 hours.
  • Assay Procedures. [0346]
  • 1. Check seeded cells for contamination using an inverted microscope. Dilute test compounds stock (10 mg/ml in DMSO) 1:10 in DMEM medium, then transfer 5 μl to a test well for a test compounds drug dilution of 1:200 and a final DMSO concentration of 1% . Control wells receive DMSO alone. Incubate in 5% CO[0347] 2 at 37° C. for one hour.
  • 2. Prepare EGF ligand: dilute stock EGF in DMEM so that upon transfer of 10 μl dilute EGF (1:12 dilution), 25 nM final concentration is attained. [0348]
  • 3. Prepare fresh 10 ml HNTG* sufficient for 100 μl per well wherein HNTG* comprises: HNTG stock (2.0 ml), milli-Q H[0349] 2O (7.3 ml), EDTA, 100 mM, pH 7.0 (0.5 ml), Na3VO4 0.5 M (0.1 ml) and Na4(P2O7), 0.2 M (0.1 ml).
  • 4. Place on ice. [0350]
  • 5. After two hours incubation with drug, add prepared EGF ligand to cells, 10 μl per well, to yield a final concentration of 25 nM. Control wells receive DMEM alone. Incubate, shaking, at room temperature, for 5 minutes. [0351]
  • 6. Remove test compound, EGF, and DMEM. Wash cells twice with PBS. Transfer HNTG* to cells, 100 μl per well. Place on ice for 5 minutes. Meanwhile, remove blocking buffer from other ELISA plate and wash with TBST as described above. [0352]
  • 7. With a pipette tip securely fitted to a micropipettor, scrape cells from plate and homogenize cell material by repeatedly aspirating and dispensing the HNTG* lysis buffer. Transfer lysate to a coated, blocked, and washed ELISA plate. Incubate shaking at room temperature for one hour. [0353]
  • 8. Remove lysate and wash 4 times with TBST. Transfer freshly diluted anti-Ptyr antibody to ELISA plate at 100 μl per well. Incubate shaking at room temperature for 30 minutes in the presence of the anti-Ptyr antiserum (1:3000 dilution in TBST). [0354]
  • 9. Remove the anti-Ptyr antibody and wash 4 times with TBST. Transfer the freshly diluted TAGO 30 anti-rabbit IgG antibody to the ELISA plate at 100 μl per well. Incubate shaking at room temperature for 30 minutes (anti-rabbit IgG antibody: 1:3000 dilution in TBST). [0355]
  • 10. Remove detection antibody and wash 4 times with TBST. Transfer freshly prepared ABTS/H[0356] 2O2 solution to ELISA plate, 100 μl per well. Incubate at room temperature for 20 minutes. ABTS/H2O2 solution: 1.2 μl 30% H2O2 in 10 ml ABTS stock.
  • 11. Stop reaction by adding 50 μl 5N H[0357] 2SO4 (optional), and determine O.D. at 410 nm.
  • 12. 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. [0358]
    • Met Autophosphorylation Assay
  • This assay determines Met tyrosine kinase activity by analyzing Met protein tyrosine kinase levels on the Met receptor. [0359]
  • Reagents [0360]
  • a. HNTG (5× stock solution): Dissolve 23.83 g HEPES and 43.83 g NaCl in about 350 ml dH[0361] 2O. Adjust pH to 7.2 with HCl or NaOH, add 500 ml glycerol and 10 ml Triton X-100, mix, add dH2O to 1 L total volume. To make 1 L of 1× working solution add 200 ml 5× stock solution to 800 ml dH2O, check and adjust pH as necessary, store at 4° C.
  • b. PBS (Dulbecco's Phosphate-Buffered Saline), Gibco Cat. # 450-1300EB (1× solution). [0362]
  • c. Blocking Buffer: in 500 ml dH[0363] 2O place 100 g BSA, 12.1 g Tris-pH7.5, 58.44 g NaCl and 10 ml Tween-20, dilute to 1 L total volume.
  • d. Kinase Buffer: To 500 ml dH[0364] 2O add 12.1 g TRIS (pH 7.2), 58.4 g NaCl, 40.7 g MgCl2 and 1.9 g EGTA; bring to 1 L total volume with dH2O.
  • e. PMSF (Phenylmethylsulfonyl fluoride), Sigma Cat. # P-7626, to 435.5 mg, add 100% ethanol to 25 ml total volu vortex. [0365]
  • f. ATP (Bacterial Source), Sigma Cat. # A-7699, store powder at −20° C.; to make up solution for use, dissolve 3.31 mg in 1 ml dH[0366] 2O.
  • g. RC-20H HRPO Conjugated Anti-Phosphotyrosine, Transduction Laboratories Cat. # E120H. [0367]
  • h. Pierce 1-Step™ Turbo TMB-ELISA (3,3′,5,5′-tetramethylbenzidine, Pierce Cat. # 34022. [0368]
  • i. H[0369] 2SO4, add 1 ml conc. (18 N) to 35 ml dH2O.
  • j. TRIS HCL, Fischer Cat. # BP152-5; to 121.14 g of material, add 600 ml MilliQ H[0370] 2O, adjust pH to 7.5 (or 7.2) with HCl, bring volume to 1 L with MilliQ H2O.
  • k. NaCl, Fischer Cat. # S271-10, make up 5M solution. [0371]
  • l. Tween-20, Fischer Cat. # S337-500. [0372]
  • m. Na[0373] 3VO4, Fischer Cat. # S454-50, to 1.8 g material add 80 ml MilliQ H2O, adjust pH to 10.0 with HCl or NaOH, boil in microwave, cool, check pH, repeat procedure until pH stable at 10.0, add MilliQ H2O to 100 ml total volume, make 1 ml aliquots and store at −80° C.
  • n. MgCl[0374] 2, Fischer Cat. # M33-500, make up 1M solution.
  • o. HEPES, Fischer Cat. # BP310-500, to 200 ml MilliQ H[0375] 2O, add 59.6 g material, adjust pH to 7.5, bring volume to 250 ml total, sterile filter.
  • p. Albumin, Bovine (BSA), Sigma Cat. # A-4503, to 30 grams material add sterile distilled water to make total volume of 300 ml, store at 4° C. [0376]
  • q. TBST Buffer: to approx. 900 ml dH[0377] 2O in a 1 L graduated cylinder add 6.057 g TRIS and 8.766 g NaCl, when dissolved, adjust pH to 7.2 with HCl, add 1.0 ml Triton X-100 and bring to 1 L total volume with dH2O.
  • r. Goat Affinity purified antibody Rabbit IgG (whole molecule), Cappel Cat. # 55641. [0378]
  • s. Anti h-Met (C-28) rabbit polyclonal IgG antibody, Santa Cruz Chemical Cat. # SC-161. [0379]
  • t. Transiently Transfected EGFR/Met chimeric cells (EMR) (Komada, et al., Oncogene, 8:2381-2390 (1993). [0380]
  • u. Sodium Carbonate Buffer, (Na[0381] 2CO4, Fischer Cat. # S495): to 10.6 g material add 800 ml MilliQ H2O, when dissolved adjust pH to 9.6 with NaOH, bring up to 1 L total volume with MilliQ H2O, filter, store at 4° C.
  • Procedure [0382]
  • All of the following steps are conducted at room temperature unless it is specifically indicated otherwise. All ELISA plate washing is by rinsing 4× with TBST. [0383]
  • EMR Lysis [0384]
  • This procedure can be performed the night before or immediately prior to the start of receptor capture. [0385]
  • 1. Quick thaw lysates in a 37° C. waterbath with a swirling motion until the last crystals disappear. [0386]
  • 2. Lyse cell pellet with 1× HNTG containing 1 mM PMSF. Use 3 ml of HNTG per 15 cm dish of cells. Add ½the calculated HNTG volume, vortex the tube for 1 min., add the remaining amount of HNTG, vortex for another min. [0387]
  • 3. Balance tubes, centrifuge at 10,000×g for 10 min at 4° C. [0388]
  • 4. Pool supernatants, remove an aliquot for protein determination. [0389]
  • 5. Quick freeze pooled sample in dry ice/ethanol bath. This step is performed regardless of whether lysate will be stored overnight or used immediately following protein determination. [0390]
  • 6. Perform protein determination using standard bicinchoninic acid (BCA) method (BCA Assay Reagent Kit from Pierce Chemical Cat. # 23225). [0391]
  • ELISA Procedure [0392]
  • 1. Coat Corning 96 well ELISA plates with 5 μg per well Goat anti-Rabbit antibody in Carbonate Buffer for a total well volume of 50 μl. Store overnight at 4° C. [0393]
  • 2. Remove unbound Goat anti-rabbit antibody by inverting plate to remove liquid. [0394]
  • 3. Add 150 μl of Blocking Buffer to each well. Incubate for 30 min. with shaking. [0395]
  • 4. Wash 4× with TBST. Pat plate on a paper towel to remove excess liquid and bubbles. [0396]
  • 5. Add 1 μg per well of Rabbit anti-Met antibody diluted in TBST for a total well volume of 100 μl. [0397]
  • 6. Dilute lysate in HNTG (90 μg lysate/100 μl) [0398]
  • 7. Add 100 μl of diluted lysate to each well. Shake for 60 min. [0399]
  • 8. Wash 4× with TBST. Pat on paper towel to remove excess liquid and bubbles. [0400]
  • 9. Add 50 μl of 1× lysate buffer per well. [0401]
  • 10. Dilute compounds/extracts 1:10 in 1× Kinase Buffer in a polypropylene 96 well plate. [0402]
  • 11. Transfer 5.5 μl of diluted compound to ELISA plate wells. Incubate at room temperature with shaking for 20 min. [0403]
  • 12. Add 5.5 μl of 60 μM ATP solution per well. Negative controls do not receive any ATP. Incubate for 90 min., with shaking. [0404]
  • 13. Wash 4× with TBST. Pat plate on paper towel to remove excess liquid and bubbles. [0405]
  • 14. Add 100 μl per well of RC20 (1:3000 dilution in Blocking Buffer). Incubate 30 min. with shaking. [0406]
  • 15. Wash 4× with TBST. Pat plate on paper towel to remove excess liquid and bubbles. [0407]
  • 16. Add 100 μl per well of Turbo-TMB. Incubate with shaking for 30-60 min. [0408]
  • 17. Add 100 μl per well of 1M H[0409] 2SO4 to stop reaction.
  • 18. Read assay on Dynatech MR7000 ELISA reader. Test Filter=450 nm, reference filter=410 nm. [0410]
    • Biochemical Src Assay
  • This assay is used to determine src protein kinase activity measuring phosphorylation of a biotinylated peptide as the readout. [0411]
  • Materials and Reagents: [0412]
  • a. Yeast transformed with src (Sugen, Inc., Redwood City, Calif.). [0413]
  • b. Cell lysates: Yeast cells expressing src are pelleted, washed once with water, re-pelleted and stored at −80° C. until use. [0414]
  • c. N-terminus biotinylated EEEYEEYEEEYEEEYEEEY is prepared by standard procedures well known to those skilled in the art. [0415]
  • d. DMSO: Sigma, St. Louis, Mo. [0416]
  • e. 96 Well ELISA Plate: Corning 96 Well Easy Wash, Modified flat Bottom Plate, Corning Cat. #25805-96. [0417]
  • f. NUNC 96-well V-bottom polypropylene plates for dilution of compounds: Applied Scientific Cat. # A-72092. [0418]
  • g. Vecastain ELITE ABC reagent: Vector, Burlingame, Calif. [0419]
  • h. Anti-src (327) mab: Schizosaccharomyces Pombe is used to express recombinant Src (Superti-Furga, et al., [0420] EMBO J., 12:2625-2634; Superti-Furga, et al., Nature Biochem., 14:600-605). S. Pombe strain SP200 (h-s leul.32 ura4 ade210) is grown as described and transformations are pRSP expression plasmids are done by the lithium acetate method (Superti-Furga, supra). Cells are grown in the presence of 1 μM thiamine to repress expression from the nmtl promoter or in the absence of thiamine to induce expression.
  • i. Monoclonal anti-phosphotyrosine, UBI 05-321 (UB40 may be used instead). [0421]
  • j. Turbo TMB-ELISA peroxidase substrate: Pierce Chemical. [0422]
  • Buffer Solutions [0423]
  • a. PBS (Dulbecco's Phosphate-Buffered Saline): GIBCO PBS, GIBCO Cat. # 450-1300EB. [0424]
  • b. Blocking Buffer: 5% Non-fat milk (Carnation) in PBS. [0425]
  • c. Carbonate Buffer: Na[0426] 2CO4 from Fischer, Cat. # S495, make up 100 mM stock solution.
  • d. Kinase Buffer: 1.0 ml (from 1M stock solution) MgCl[0427] 2; 0.2 ml (from a 1M stock solution) MnCl2; 0.2 ml (from a 1M stock solution) DTT; 5.0 ml (from a 1M stock solution) HEPES; 0.1 ml TX-100; bring to 10 ml total volume with MilliQ H2O.
  • e. Lysis Buffer: 5.0 HEPES (from 1M stock solution.); 2.74 ml NaCl (from 5M stock solution); 10 ml glycerol; 1.0 ml TX-100; 0.4 ml EDTA (from a 100 mM stock solution); 1.0 ml PMSF (from a 100 mM stock solution); 0.1 ml Na[0428] 3VO4 (from a 0.1 M stock solution); bring to 100 ml total volume with MilliQ H2O.
  • f. ATP: Sigma Cat. # A-7699, make up 10 mM stock solution (5.51 mg/ml). [0429]
  • g. TRIS-HCl: Fischer Cat. # BP 152-5, to 600 ml MilliQ H[0430] 2O add 121.14 g material, adjust pH to 7.5 with HCl, bring to 1 L total volume with MilliQ H2O.
  • h. NaCl: Fischer Cat. # S271-10, Make up 5M stock solution with MilliQ H[0431] 2O.
  • Na[0432] 3VO4: Fischer Cat. # S454-50; to 80 ml MilliQ H2O, add 1.8 g material; adjust pH to 10.0 with HCl or NaOH; boil in a microwave; cool; check pH, repeat pH adjustment until pH remains stable after heating/cooling cycle; bring to 100 ml total volume with MilliQ H2O; make 1 ml aliquots and store at −80° C.
  • j. MgCl[0433] 2: Fischer Cat. # M33-500, make up 1M stock solution with MilliQ H2O.
  • k. HEPES: Fischer Cat. # BP 310-500; to 200 ml MilliQ H[0434] 2O, add 59.6 g material, adjust pH to 7.5, bring to 250 ml total volume with MilliQ H2O, sterile filter (1M stock solution).
  • l. TBST Buffer: TBST Buffer: To 900 ml dH[0435] 2O add 6.057 g TRIS and 8.766 g NaCl; adjust pH to 7.2 with HCl, add 1.0 ml Triton-X100; bring to 1 L total volume with dH2O.
  • m. MnCl[0436] 2: Fischer Cat. # M87-100, make up 1M stock solution with MilliQ H2O.
  • n. DTT: Fischer Cat. # BP172-5. [0437]
  • o. TBS (TRIS Buffered Saline): to 900 ml MilliQ H[0438] 2O add 6.057 g TRIS and 8.777 g NaCl; bring to 1 L total volume with MilliQ H2O.
  • p. Kinase Reaction Mixture: Amount per assay plate (100 wells): 1.0 ml Kinase Buffer, 200 μg GST-ζ, bring to final volume of 8.0 ml with MilliQ H[0439] 2O.
  • q. Biotin labeled EEEYEEYEEEYEEEYEEEY: Make peptide stock solution (1 mM, 2.98 mg/ml) in water fresh just before use. [0440]
  • r. Vectastain ELITE ABC reagent: To prepare 14 ml of working reagent, add 1 drop of reagent A to 15 ml TBST and invert tube several times to mix. Then add 1 drop of reagent B. Put tube on orbital shaker at room temperature and mix for 30 minutes. [0441]
  • Procedures: [0442]
  • Preparation of src coated ELISA plate. [0443]
  • 1. Coat ELISA plate with 0.5 μg/well anti-src mab in 100 μl of pH 9.6 sodium carbonate buffer; hold at 4° C. overnight. [0444]
  • 2. Wash wells once with PBS. [0445]
  • 3. Block plate with 0.15 ml 5% milk in PBS for 30 min. at room temperature. [0446]
  • 4. Wash plate 5× with PBS. [0447]
  • 5. Add 10 μg/well of src transformed yeast lysates diluted in Lysis Buffer (0.1 ml total volume per well). (Amount of lysate may vary between batches.) Shake plate for 20 minutes at room temperature. [0448]
  • Preparation of phosphotyrosine antibody-coated ELISA plate. [0449]
  • 1. 4G10 plate: coat 0.5 μg/well 4G10 in 100 μl PBS overnight at 4° C. and block with 150 μl of 5% milk in PBS for 30 minutes at room temperature. [0450]
  • Kinase assay procedure. [0451]
  • 1. Remove unbound proteins from plates and wash plates 5× with PBS. [0452]
  • 2. Add 0.08 ml Kinase Reaction Mixture per well (containing 10 μl of 10× Kinase Buffer and 10 μM (final concentration) biotin-EEEYEEYEEEYEEEYEEEY per well diluted in water. [0453]
  • 3. Add 10 μl of compound diluted in water containing 10% DMSO and pre-incubate for 15 minutes at room temperature. [0454]
  • 4. Start kinase reaction by adding 10 μl/well of 0.05 mM ATP in water (5 μM ATP final). [0455]
  • 5. Shake ELISA plate for 15 min. at room temperature. [0456]
  • 6. Stop kinase reaction by adding 10 μl of 0.5 M EDTA per well. [0457]
  • 7. Transfer 90 μl supernatant to a blocked 4G10 coated ELISA plate. [0458]
  • 8. Incubate for 30 min. while shaking at room temperature. [0459]
  • 9. Wash plate 5× with TBST. [0460]
  • 10. Incubate with Vectastain ELITE ABC reagent (100 μl/well) for 30 min. at room temperature. [0461]
  • 11. Wash the wells 5× with TBST. [0462]
  • 12. Develop with Turbo TMB. [0463]
    • Biochemical Lck Assay
  • This assay is used to determine lck protein kinase activities measuring phosphorylation of GST-ζ as the readout. [0464]
  • Materials and Reagents: [0465]
  • Yeast transformed with lck. Schizosaccharomyces Pombe is used to express recombinant Lck (Superti-Furga, et al., [0466] EMBO J, 12:2625-2634; Superti-Furga, et al., Nature Biotech., 14:600-605). S. Pombe strain SP200 (h-s leul.32 ura4 ade210) is grown as described and transformations with pRSP expression plasmids are done by the lithium acetate method (Superti-Furga, supra) Cells are grown in the presence of 1 μM thiamine to induce expression.
  • b. Cell lysates: Yeast cells expressing lck are pelleted, washed once in water, re-pelleted and stored frozen at −80° C. until use. [0467]
  • c. GST-ζ: DNA encoding for GST-ζ fusion protein for expression in bacteria obtained from Arthur Weiss of the Howard Hughes Medical Institute at the University of California, San Francisco. Transformed bacteria are grown overnight while shaking at 25° C. GST-ζ is purified by glutathione affinity chromatography, Pharmacia, Alameda, Calif. [0468]
  • d. DMSO: Sigma, St. Louis, Mo. [0469]
  • e. 96-Well ELISA plate: Corning 96 Well Easy Wash, Modified Flat Bottom Plate, Corning Cat. #25805-96. [0470]
  • f. NUNC 96-well V-bottom polypropylene plates for dilution of compounds: Applied Scientific Cat. # AS-72092. [0471]
  • g. Purified Rabbit anti-GST antiserum: Amrad Corporation (Australia) Cat. X90001605. [0472]
  • h. Goat anti-Rabbit-IgG-HRP: Amersham Cat. # V010301. [0473]
  • i. Sheep ant-mouse IgG (H+L): Jackson Labs Cat. # 5215-005-003. [0474]
  • j. Anti-Lck (3A5) mab: Santa Cruz Biotechnology Cat # sc-433. [0475]
  • k. Monoclonal anti-phosphotyrosine UBI 05-321 (UB40 may be -used instead). [0476]
  • Buffer solutions: [0477]
  • a. PBS (Dulbecco's Phosphate-Buffered Saline) 1× solution: GIBCO PBS, GIBCO Cat. # 450-1300EB. [0478]
  • b. Blocking Buffer: 100 g. BSA, 12.1 g. TRIS (pH7.5), 58.44 g NaCl, 10 ml Tween-20, bring up to 1 L total volume with MilliQ H[0479] 2O.
  • c. Carbonate Buffer: Na[0480] 2CO4 from Fischer, Cat. # S495; make up 100 mM solution with MilliQ H2O.
  • d. Kinase Buffer: 1.0 ml (from 1M stock solution) MgCl[0481] 2; 0.2 ml (from a 1M stock solution) MnCl2; 0.2 ml (from a 1M stock solution) DTT; 5.0 ml (from a 1M stock solution) HEPES; 0.1 ml TX-100; bring to 10 ml total volume with MilliQ H2O.
  • e. Lysis Buffer: 5.0 HEPES (from 1M stock solution.); 2.74 ml NaCl (from 5M stock solution); 10 ml glycerol; 1.0 ml TX-100; 0.4 ml EDTA (from a 100 mM stock solution); 1.0 ml PMSF (from a 100 mM stock solution); 0.1 ml Na[0482] 3VO4 (from a 0.1 M stock solution); bring to 100 ml total volume with MilliQ H2O.
  • f. ATP: Sigma Cat. # A-7699, make up 10 mM stock solution (5.51 mg/ml). [0483]
  • g TRIS-HCl: Fischer Cat. # BP 152-5, to 600 ml MilliQ H[0484] 2O add 121.14 g material, adjust pH to 7.5 with HCl, bring to 1 L total volume with MilliQ H2O.
  • h. NaCl: Fischer Cat. # S271-10, Make up 5M stock solution with MilliQ H[0485] 2O.
  • i. Na[0486] 3VO4: Fischer Cat. # S454-50; to 80 ml MilliQ H2O, add 1.8 g material; adjust pH to 10.0 with HCl or NaOH; boil in a microwave; cool; check pH, repeat pH adjustment until pH remains stable after heating/cooling cycle; bring to 100 ml total volume with MilliQ H2O; make 1 ml aliquots and store at −80° C.
  • j. MgCl[0487] 2: Fischer Cat. # M33-500, make up 1M stock solution with MilliQ H2O.
  • k. HEPES: Fischer Cat. # BP 310-500; to 200 ml MilliQ H[0488] 2O, add 59.6 g material, adjust pH to 7.5, bring to 250 ml total volume with MilliQ H2O, sterile filter (1M stock solution).
  • l. Albumin, Bovine (BSA), Sigma Cat. # A4503; to 150 ml MilliQ H[0489] 2O add 30 g material, bring 300 ml total volume with MilliQ H2O, filter through 0.22 μm filter, store at 4° C.
  • m. TBST Buffer: To 900 ml dH[0490] 2O add 6.057 g TRIS and 8.766 g NaCl; adjust pH to 7.2 with HCl, add 1.0 ml Triton-X100; bring to 1 L total volume with dH2O.
  • n. MnCl2: Fischer Cat. # M87-100, make up 1M stock solution with MilliQ H[0491] 2O.
  • o. DTT; Fischer Cat. # BP172-5. [0492]
  • p. TBS (TRIS Buffered Saline): to 900 ml MilliQ H[0493] 2O add 6.057 g TRIS and 8.777 g NaCl; bring to 1 L total volume with MilliQ H2O.
  • q. Kinase Reaction Mixture: Amount per assay plate (100 wells): 1.0 ml Kinase Buffer, 200 μg GST-ζ, bring to final volume of 8.0 ml with MilliQ H[0494] 2O.
  • Procedures: [0495]
  • Preparation of Lck coated ELISA plate. [0496]
  • 1. Coat 2.0 μg/well Sheep anti-mouse IgG in 100 μl of pH 9.6 sodium carbonate buffer at 4° C. overnight. [0497]
  • 2. Wash well once with PBS. [0498]
  • 3. Block plate with 0.15 ml of blocking Buffer for 30 min. at room temp. [0499]
  • 4. Wash plate 5× with PBS. [0500]
  • 5. Add 0.5 μg/well of anti-lck (mab 3A5) in 0.1 ml PBS at room temperature for 1-2 hours. [0501]
  • 6. Wash plate 5× with PBS. [0502]
  • 7. Add 20 μg/well of lck transformed yeast lysates diluted in Lysis Buffer (0.1 ml total volume per well). Shake plate at 4° C. overnight to prevent loss of activity. [0503]
  • Preparation of phosphotyrosine antibody-coated ELISA plate. [0504]
  • 1. UB40 plate: 1.0 μg/well UB40 in 100 μl of PBS overnight at 4° C. and block with 150 μl of Blocking Buffer for at least 1 hour. [0505]
  • Kinase assay procedure. [0506]
  • 1. Remove unbound proteins from plates and wash plates 5× with PBS. [0507]
  • 2. Add 0.08 ml Kinase Reaction Mixture per well (containing 10 μl of 10× Kinase Buffer and 2 μg GST-ζ per well diluted with water). [0508]
  • 3. Add 10 μl of compound diluted in water containing 10% DMSO and pre-incubate for 15 minutes at room temperature. [0509]
  • 4. Start kinase reaction by adding 10 μl/well of 0.1 mM ATP in water (10 μM ATP final). [0510]
  • 5. Shake ELISA plate for 60 min. at room temperature. [0511]
  • 6. Stop kinase reaction by adding 10 μl of 0.5 M EDTA per well. [0512]
  • 7. Transfer 90 μl supernatant to a blocked 4G10 coated ELISA plate from section B, above. [0513]
  • 8. Incubate while shaking for 30 min. at room temperature. [0514]
  • 9. Wash plate 5× with TBST. [0515]
  • 10. Incubate with Rabbit anti-GST antibody at 1:5000 dilution in 100 μl TBST for 30 min. at room temperature. [0516]
  • 11. Wash the wells 5× with TBST. [0517]
  • 12. Incubate with Goat anti-Rabbit-IgG-HRP at 1:20,000 dilution in 100 μl of TBST for 30 min. at room temperature. [0518]
  • 13. Wash the wells 5× with TBST. [0519]
  • 14. Develop with Turbo TMB. [0520]
    • Assay Measuring Phosphorylating Function of RAF.
  • The following assay reports the amount of RAF-catalyzed phosphorylation of its target protein MEK as well as MEK's target MAPK. The RAF gene sequence is described in Bonner et al., 1985[0521] , Molec. Cell. Biol., 5:1400-1407, and is readily accessible in multiple gene sequence data banks. Construction of the nucleic acid vector and cell lines utilized for this portion of the invention are fully described in Morrison et al., 1988, Proc. Natl. Acad. Sci. USA, 85:8855-8859.
  • Materials and Reagents [0522]
  • 1. Sf9 (Spodoptera frugiperda) cells; GIBCO-BRL, Gaithersburg, Md. [0523]
  • 2. RIPA buffer: 20 mM Tris/HCl pH 7.4, 137 mM NaCl, 10% glycerol, 1 mM PMSF, 5 mg/L Aprotenin, 0.5% Triton X-100; [0524]
  • 3. Thioredoxin-MEK fusion protein (T-MEK): T-MEK expression and purification by affinity chromatography are performed according to the manufacturer's procedures. Catalog# K 350-01 and R 350-40, Invitrogen Corp., San Diego, Calif. [0525]
  • 4. His-MAPK (ERK 2); His-tagged MAPK is expressed in XL1 Blue cells transformed with pUC18 vector encoding His-MAPK. His-MAPK is purified by Ni-affinity chromatography. Cat# 27-4949-01, Pharmacia, Alameda, Calif., as described herein. [0526]
  • 5. Sheep anti mouse IgG: Jackson laboratories, West Grove, Pa. Catalog, # 515-006-008, Lot# 28563 [0527]
  • 6. RAF-1 protein kinase specific antibody: URP2653 from UBI. [0528]
  • 7. Coating buffer: PBS; phosphate buffered saline, GIBCO-BRL, Gaithersburg, Md. [0529]
  • 8. Wash buffer: TBST (50 mM Tris/HCL pH 7.2, 150 mM NaCl, 0.1% Triton X-100). [0530]
  • 9. Block buffer: TBST, 0.1% ethanolamine pH 7.4 [0531]
  • 10. DMSO, Sigma, St. Louis, Mo. [0532]
  • 11. Kinase buffer (KB): 20 mM HEPES/HCl pH 7.2, 150 mM NaCl, 0.1% Triton X-100, 1 mM PMSF, 5 mg/L Aprotenin, 75 mM sodium orthovanadate, 0.5 MM DTT and 10 mM MgCl[0533] 2.
  • 12. ATP mix: 100 mM MgCl[0534] 2, 300 mM ATP, 10 mCi γ33P ATP (Dupont-NEN)/mL.
  • 13 Stop solution: 1% phosphoric acid; Fisher, Pittsburgh, Pa. [0535]
  • 14. Wallac Cellulose Phosphate Filter mats; Wallac, Turku, Finnland. [0536]
  • 15. Filter wash solution: 1% phosphoric acid, Fisher, Pittsburgh, Pa. [0537]
  • 16. Tomtec plate harvester, Wallac, Turku, Finnland. [0538]
  • 17. Wallac beta plate reader # 1205, Wallac, Turku, Finnland. [0539]
  • 18. NUNC 96-well V bottom polypropylene plates for compounds Applied Scientific Catalog # AS-72092. [0540]
  • Procedure [0541]
  • All of the following steps are conducted at room temperature unless specifically indicated otherwise. [0542]
  • 1. ELISA plate coating: ELISA wells are coated with 100 ml of Sheep anti mouse affinity purified antiserum (1 mg/100 mL coating buffer) over night at 4° C. ELISA plates can be used for two weeks when stored at 4° C. [0543]
  • 2. Invert the plate and remove liquid. Add 100 mL of blocking solution and incubate for 30 min. [0544]
  • 3. Remove blocking solution and wash four times with wash buffer. Pat the plate on a paper towel to remove excess liquid. [0545]
  • 4. Add 1 mg of antibody specific for RAF-1 to each well and incubate for 1 hour. Wash as described in step 3. [0546]
  • 5. Thaw lysates from RAS/RAF infected Sf9 cells and dilute with TBST to 10 mg/100 mL. Add 10 mg of diluted lysate to the wells and incubate for 1 hour. Shake the plate during incubation. Negative controls receive no lysate. Lysates from RAS/RAF infected Sf9 insect cells are prepared after cells are infected with recombinant baculoviruses at a MOI of 5 for each virus, and harvested 48 hours later. The cells are washed once with PBS and lysed in RIPA buffer. Insoluble material is removed by centrifugation (5 min at 10,000×g). Aliquots of lysates are frozen in dry ice/ethanol and stored at −80 ° C. until use. [0547]
  • 6. Remove non-bound material and wash as outlined above (step 3). [0548]
  • 7. Add 2 mg of T-MEK and 2 mg of His-MAEPK per well and adjust the volume to 40 ml with kinase buffer. Methods for purifying T-MEK and MAPK from cell extracts are provided herein by example. [0549]
  • 8. Pre-dilute compounds (stock solution 10 mg/ml DMSO) or extracts 20 fold in TBST plus 1% DMSO. Add 5 ml of the pre-diluted compounds/extracts to the wells described in step 6. Incubate for 20 min. Controls receive no drug. [0550]
  • 9. Start the kinase reaction by addition of 5 ml ATP mix; Shake the plates on an ELISA plate shaker during incubation. [0551]
  • 10. Stop the kinase reaction after 60 min by addition of 30 mL stop solution to each well. [0552]
  • 11. Place the phosphocellulose mat and the ELISA plate in the Tomtec plate harvester. Harvest and wash the filter with the filter wash solution according to the manufacturer's recommendation. Dry the filter mats. Seal the filter mats and place them in the holder. Insert the holder into radioactive detection apparatus and quantify the radioactive phosphorous on the filter mats. [0553]
  • Alternatively, 40 mL aliquots from individual wells of the assay plate can be transferred to the corresponding positions on the phosphocellulose filter mat. After air drying the filters, put the filters in a tray. Gently rock the tray, changing the walsh solution at 15 min intervals for 1 hour. Air-dry the filter mats. Seal the filter mats and place them in a holder suitable for measuring the radioactive phosphorous in the samples. Insert the holder into a detection device and quantify the radioactive phosphorous on the filter mats. [0554]
    • CDK2/Cyclin A—Inhibition Assay
  • This assay analyzes the protein kinase activity of CDK2 in exogenous substrate. [0555]
  • Reagents: [0556]
  • A. Buffer A: (80 mM Tris (pH 7.2), 40 mM MgCl[0557] 2), 4.84 g. Tris (F.W.=121.1 g/mol), 4.07 g. MgCl2 (F.W.=203.31 g/mol) dissolved in 500 ml H2O. Adjust pH to 7.2 with HCl.
  • B. Histone H1 solution (0.45 mg/ml Histone H1 and 20 mM HEPES pH 7.2: 5 mg Histone H1 (Boehinger Mannheim) in 11.111 ml 20 mM HEPES pH 7.2 (477 mg HEPES (F.W.=238.3 g/mol) dissolved in 100 ml ddH[0558] 2O, stored in 1 ml aliquots at −80° C.
  • C. ATP solution (60 μM ATP, 300 μg/ml BSA, 3 mM DTT): 120 μl 10 mM ATP, 600 μl 10 mg/ml BSA to 20 ml, stored in 1 ml aliquots at −80° C. [0559]
  • D. CDK2 solution: cdk2/cyclin A in 10 mM HEPES pH 7.2, 25 mM NaCl, 0.5 mM DTT, 10% glycerol, stored in 9 μl aliquots at −80° C. [0560]
  • Protocol [0561]
  • 1. Prepare solutions of inhibitors at three times the desired final assay concentration in ddH[0562] 2O/15% DMSO by volume.
  • 2. Dispense 20 μl of inhibitors to wells of polypropylene 96-well plates (or 20 μl 15% DMSO for positive and negative controls). [0563]
  • 3. Thaw Histone H1 solution (1 ml/plate), ATP solution (1 ml/plate plus 1 aliquot for negative control), and CDK2 solution (9 μl/plate). Keep CDK2 on ice until use. Aliquot CDK2 solution appropriately to avoid repeated freeze-thaw cycles. [0564]
  • 4. Dilute 9 μl CDK2 solution into 2.1 ml Buffer A (per plate). Mix. Dispense 20 μl into each well. [0565]
  • 5. Mix 1 ml Histone H1 solution with 1 ml ATP solution (per plate) into a 10 ml screw cap tube. Add γ[0566] 33P ATP to a concentration of 0.15 μCi/20 μl (0.15 μCi/well in assay). Mix carefully to avoid BSA frothing. Add 20 μl to appropriate wells. Mix plates on plate shaker. For negative control, mix ATP solution with an equal amount of 20 mM HEPES pH 7.2 and add γ33P ATP to a concentration of 0.15 μCi/20 μl solution. Add 20 μl to appropriate wells.
  • 6. Let reactions proceed for 60 minutes. [0567]
  • 7. Add 35 μl 10% TCA to each well. Mix plates on plate shaker. [0568]
  • 8. Spot 40 μl of each sample onto P30 filter mat squares. Allow mats to dry (approx. 10-20 minutes). [0569]
  • 9 Wash filter mats 4×10 minutes with 250 ml 1% phosphoric acid (10 ml phosphoric acid per liter ddH[0570] 2O)
  • 10. Count filter mats with beta plate reader. [0571]
    • Cellular/Biologic Assays PDGF-Induced BrdU Incorporation Assay
  • Materials and Reagents: [0572]
  • (1) PDGF: human PDGF B/B; 1276-956, Boehringer Mannheim, Germany. [0573]
  • (2) BrdU Labeling Reagent: 10 mM, in PBS (pH7.4), Cat. No. 1 647 229, Boehringer Mannheim, Germany. [0574]
  • (3) FixDenat: fixation solution (ready to use), Cat. No. 1 647 229, Boehringer Mannheim, Germany. [0575]
  • (4) Anti-BrdU-POD: mouse monoclonal antibody conjugated with peroxidase, Cat. No. 1 647 229, Boehringer Mannheim, Germany. [0576]
  • (5) TMB Substrate Solution: tetramethylbenzidine (TMB), ready to use, Cat. No. 1 647 229, Boehringer Mannheim, Germany. [0577]
  • (6) PBS Washing-Solution: 1× PBS, pH 7.4 (Sugen, Inc., Redwood City, Calif.). [0578]
  • (7) Albumin, Bovine (BSA): fraction V powder; A-8551, Sigma Chemical Co., USA. [0579]
  • (8) 3T3 cell line genetically engineered to express human PDGF-R. [0580]
  • Protocol [0581]
  • (1) Cells are seeded at 8000 cells/well in DMEM, 10% CS, 2 mM Gln in a 96 well plate. Cells are incubated overnight at 37° C. in 5% CO[0582] 2.
  • (2) After 24 hours, the cells are washed with PBS, and then are serum starved in serum free medium (0% CS DMEM with 0.1% BSA) for 24 hours. [0583]
  • (3) On day 3, ligand (PDGF, 3.8 nM, prepared in DMEM with 0.1% BSA) and test compounds are added to the cells simultaneously. The negative control wells receive serum free DMEM with 0.1% BSA only; the positive control cells receive the ligand (PDGF) but no test compound. Test compounds are prepared in serum free DMEM with ligand in a 96 well plate, and serially diluted for 7 test concentrations. [0584]
  • (4) After 20 hours of ligand activation, diluted BrdU labeling reagent (1:100 in DMEM, 0.1% BSA) is added and the cells are incubated with BrdU (final concentration=10 μM) for 1.5 hours. [0585]
  • (5) After incubation with labeling reagent, the medium is removed by decanting and tapping the inverted plate on a paper towel. FixDenat solution is added (50 μl/well) and the plates are incubated at room temperature for 45 minutes on a plate shaker. [0586]
  • (6) The FixDenat solution is thoroughly removed by decanting and tapping the inverted plate on a paper towel. Milk is added (5% dehydrated milk in PBS, 200 μl/well) as a blocking solution and the plate is incubated for 30 minutes at room temperature on a plate shaker. [0587]
  • The blocking solution is removed by decanting and the wells are washed once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS, 1% BSA) is added (100 μl/well) and the plate is incubated for 90 minutes at room temperature on a plate shaker. [0588]
  • (8) The antibody conjugate is thoroughly removed by decanting and rinsing the wells 5 times with PBS, and the plate is dried by inverting and tapping on a paper towel. [0589]
  • (9) TMB substrate solution is added (100 μl/well) and incubated for 20 minutes at room temperature on a plate shaker until color development is sufficient for photometric detection. [0590]
  • (10) The absorbance of the samples are measured at 410 nm (in “dual wavelength” mode with a filter reading at 490 nm, as a reference wavelength) on a Dynatech ELISA plate reader. [0591]
    • EGF-Induced BrdU Incorporation Assay
  • Materials and Reagents [0592]
  • (1) EGF: mouse EGF, 201; Toyobo, Co., Ltd. Japan. [0593]
  • (2) BrdU Labeling Reagent: 10 mM, in PBS (pH7.4), Cat. No. 1 647 229, Boehringer Mannheim, Germany. [0594]
  • (3) FixDenat: fixation solution (ready to use), Cat. No. 1 647 229, Boehringer Mannheim, Germany. [0595]
  • (4) Anti-BrdU-POD: mouse monoclonal antibody conjugated with peroxidase, Cat. No. 1 647 229, Boehringer Mannheim, Germany. [0596]
  • (5) TMB Substrate Solution: tetramethylbenzidine (TMB), ready to use, Cat. No. 1 647 229, Boehringer Mannheim, Germany. [0597]
  • (6) PBS Washing Solution : 1× PBS, pH 7.4 (Sugen, Inc., Redwood City, Calif.). [0598]
  • (7) Albumin, Bovine (BSA) : fraction V powder; A-8551, Sigma Chemical Co., USA. [0599]
  • (8) 3T3 cell line genetically engineered to express human EGF-R. [0600]
  • Protocol [0601]
  • (1) Cells are seeded at 8000 cells/well in 10% CS, 2 mM Gln in DMEM, in a 96 well plate. Cells are incubated overnight at 37° C. in 5% CO[0602] 2.
  • (2) After 24 hours, the cells are washed with PBS, and then are serum starved in serum free medium (0% CS DMEM with 0.1% BSA) for 24 hours. [0603]
  • (3) On day 3, ligand (EGF, 2 nM, prepared in DMEM with 0.1% BSA) and test compounds are added to the cells simultaneously. The negative control wells receive serum free DMEM with 0.1% BSA only; the positive control cells receive the ligand (EGF) but no test compound. Test compounds are prepared in serum free DMEM with ligand in a 96 well plate, and serially diluted for 7 test concentrations. [0604]
  • (4) After 20 hours of ligand activation, diluted BrdU labeling reagent (1:100 in DMEM, 0.1% BSA) is added and the cells are incubated with BrdU (final concentration=10 μM) for 1.5 hours. [0605]
  • (5) After incubation with labeling reagent, the medium is removed by decanting and tapping the inverted plate on a paper towel. FixDenat solution is added (50 μl/well) and the plates are incubated at room temperature for 45 minutes on a plate shaker. [0606]
  • (6) The FixDenat solution is thoroughly removed by decanting and tapping the inverted plate on a paper towel. Milk is added (5% dehydrated milk in PBS, 200 μl/well) as a blocking solution and the plate is incubated for 30 minutes at room temperature on a plate shaker. [0607]
  • (7) The blocking solution is removed by decanting and the wells are washed once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS, 1% BSA) is added (100 μl/well) and the plate is incubated for 90 minutes at room temperature on a plate shaker. [0608]
  • (8) The antibody conjugate is thoroughly removed by decanting and rinsing the wells 5 times with PBS, and the plate is dried by inverting and tapping on a paper towel. [0609]
  • (9) TMB substrate solution is added (100 μl/well) and incubated for 20 minutes at room temperature on a plate shaker until color development is sufficient for photometric detection. [0610]
  • (10) The absorbance of the samples are measured at 410 nm (in “dual wavelength” mode with a filter reading at 490 nm, as a reference wavelength) on a Dynatech ELISA plate reader. [0611]
    • EGF-Induced Her2-Driven BrdU Incorporation
  • Materials and Reagents [0612]
  • (1) EGF: mouse EGF, 201; Toyobo, Co., Ltd. Japan [0613]
  • (2) BrdU Labeling Reagent: 10 mM, in PBS (pH7.4), Cat. No. 1 647 229, Boehringer Mannheim, Germany. [0614]
  • (3) FixDenat: fixation solution (ready to use), Cat. No. 1 647 229, Boehringer Mannheim, Germany. [0615]
  • (4) Anti-BrdU-POD: mouse monoclonal antibody conjugated with peroxidase, Cat. No. 1 647 229, Boehringer Mannheim, Germany. [0616]
  • (5) TMB Substrate Solution: tetramethylbenzidine (TMB), ready to use, Cat. No. 1 647 229, Boehringer Mannheim, Germany. [0617]
  • (6) PBS Washing Solution: 1× PBS, pH 7.4, made in house. [0618]
  • (7) Albumin, Bovine (BSA): fraction V powder; A-8551, Sigma Chemical Co., USA. [0619]
  • (8) 3T3 cell line engineered to express a chimeric receptor having the extra-cellular domain of EGF-R and the intra-cellular domain of Her2. [0620]
  • Protocol [0621]
  • (1) Cells are seeded at 8000 cells/well in DMEM, 10% CS, 2 mM Gln in a 96- well plate. Cells are incubated overnight at 37° C. in 5% CO[0622] 2.
  • (2) After 24 hours, the cells are washed with PBS, and then are serum starved in serum free medium (0% CS DMEM with 0.1% BSA) for 24 hours. [0623]
  • (3) On day 3, ligand (EGF =2 nM, prepared in DMEM with 0.1% BSA) and test compounds are added to the cells simultaneously. The negative control wells receive serum free DMEM with 0.1% BSA only; the positive control cells receive the ligand (EGF) but no test compound. Test compounds are prepared in serum free DMEM with ligand in a 96 well plate, and serially diluted for 7 test concentrations. [0624]
  • (4) After 20 hours of ligand activation, diluted BrdU labeling reagent (1:100 in DMEM, 0.1% BSA) is added and the cells are incubated with BrdU (final concentration=10 μM) for 1.5 hours. [0625]
  • (5) After incubation with labeling reagent, the medium is removed by decanting and tapping the inverted plate on a paper towel. FixDenat solution is added (50 μl/well) and the plates are incubated at room temperature for 45 minutes on a plate shaker. [0626]
  • (6) The FixDenat solution is thoroughly removed by decanting and tapping the inverted plate on a paper towel. Milk is added (5% dehydrated milk in PBS, 200 μl/well) as a blocking solution and the plate is incubated for 30 minutes at room temperature on a plate shaker. [0627]
  • The blocking solution is removed by decanting and the wells are washed once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS, 1% BSA) is added (100 μl/well) and the plate is incubated for 90 minutes at room temperature on a plate shaker. [0628]
  • (8) The antibody conjugate is thoroughly removed by decanting and rinsing the wells 5 times with PBS, and the plate is dried by inverting and tapping on a paper towel. [0629]
  • (9) TMB substrate solution is added (100 μl/well) and incubated for 20 minutes at room temperature on a plate shaker until color development is sufficient for photometric detection. [0630]
  • (10) The absorbance of the samples are measured at 410 nm (in “dual wavelength” mode with a filter reading at 490 nm, as a reference wavelength) on a Dynatech ELISA plate reader. [0631]
    • IGFI-Induced BrdU Incorporation Assay
  • Materials and Reagents [0632]
  • (1) IGF1 Ligand: human, recombinant; G511, Promega Corp, USA. [0633]
  • (2) BrdU Labeling Reagent: 10 mM, in PBS (pH7.4), Cat. No. 1 647 229, Boehringer Mannheim, Germany. [0634]
  • (3) FixDenat: fixation solution (ready to use), Cat. No. 1 647 229, Boehringer Mannheim, Germany. [0635]
  • (4) Anti-BrdU-POD: mouse monoclonal antibody conjugated with peroxidase, Cat. No. 1 647 229, Boehringer Mannheim, Germany. [0636]
  • (5) TMB Substrate Solution: tetramethylbenzidine (TMB), ready to use, Cat. No. 1 647 229, Boehringer Mannheim, Germany. [0637]
  • (6) PBS Washing Solution: 1× PBS, pH 7.4 (Sugen, Inc., Redwood City, Calif.). [0638]
  • (7) Albumin, Bovine (BSA): fraction V powder; A-8551, Sigma Chemical Co., USA. [0639]
  • (8) 3T3 cell line genetically engineered to express human IGF-1 receptor. [0640]
  • Protocol [0641]
  • (1) Cells are seeded at 8000 cells/well in DMEM, 10% CS, 2 mM Gln in a 96- well plate. Cells are incubated overnight at 37° C. in 5% CO[0642] 2.
  • (2) After 24 hours, the cells are washed with PBS, and then are serum starved in serum free medium (0% CS DMEM with 0.1% BSA) for 24 hours. [0643]
  • (3) On day 3, ligand (IGF1=3.3 nM, prepared in DMEM with 0.1% BSA) and test compounds are added to the cells simultaneously. The negative control wells receive serum free DMEM with 0.1% BSA only; the positive control cells receive the ligand (IGF1) but no test compound. Test compounds are prepared in serum free DMEM with ligand in a 96 well plate, and serially diluted for 7 test concentrations. [0644]
  • (4) After 16 hours of ligand activation, diluted BrdU labeling reagent (1:100 in DMEM, 0.1% BSA) is added and the cells are incubated with BrdU (final concentration=10 μM) for 1.5 hours. [0645]
  • (5) After incubation with labeling reagent, the medium is removed by decanting and tapping the inverted plate on a paper towel. FixDenat solution is added (50 μl/well) and the plates are incubated at room temperature for 45 minutes on a plate shaker. [0646]
  • (6) The FixDenat solution is thoroughly removed by decanting and tapping the inverted plate on a paper towel. Milk is added (5% dehydrated milk in PBS, 200 μl/well) as a blocking solution and the plate is incubated for 30 minutes at room temperature on a plate shaker. [0647]
  • (7) The blocking solution is removed by decanting and the wells are washed once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS, 1% BSA) is added (100 μl/well) and the plate is incubated for 90 minutes at room temperature on a plate shaker. [0648]
  • (8) The antibody conjugate is thoroughly removed by decanting and rinsing the wells 5 times with PBS, and the plate is dried by inverting and tapping on a paper towel. [0649]
  • (9) TMB substrate solution is added (100 μl/well) and incubated for 20 minutes at room temperature on a plate shaker until color development is sufficient for photometric detection. [0650]
  • (10) The absorbance of the samples are measured at 410 nm (in “dual wavelength” mode with a filter reading at 490 nm, as a reference wavelength) on a Dynatech ELISA plate reader. [0651]
    • FGF-Induced BrdU Incorporation Assay
  • This assay measures FGF-induced DNA synthesis in 3Tc7/EGFr cells that express endogenous FGF receptors. [0652]
  • Materials and Reagents: [0653]
  • 1. FGF: human FGF2/bFGF (Gibco BRL, No. 13256-029). [0654]
  • 2. BrdU Labeling reagent, (10 mM PBS (pH 7.4), Boehringer Mannheim Cat No. 1 647 229). [0655]
  • 3. Fixdenat fixation solution (Boehringer Mannheim Cat No. 1 647 229). [0656]
  • 4. Anti-BrdU-POD (mouse monoclonal antibody conjugated with peroxidase, Boehringer Mannheim Cat. No. 1 647 229). [0657]
  • 5. TMB (tetramethylbenzidine, Boehringer Mannheim Cat. No. 1 647 229). [0658]
  • 6. PBS washing solution, pH 7.4 (Sugen, Inc.). [0659]
  • 7. Albumin, bovine (BSA), fraction V powder (Sigma Chemical Co., Cat. No. A-8551). [0660]
  • Procedure [0661]
  • 1. 3T3 engineered cell line: 3T3c7/EGFr. [0662]
  • 2. Cells are seeded at 8,000 cells/well in DMEM, 10% CS and 2 mM Gln in a 96-well plate. Incubate 24 hours at 37° C. in 5% CO[0663] 2.
  • 3. After 24 hours, wash cells with PBS then serum starve in serum free medium (0% DMEM, 0.1% BSA) for 24 hours. [0664]
  • 4. Add ligand (FGF2 (1.5 nM in DMEM with 0.1% BSA) and test compound simultaneously. Negative control wells receive serum free DMEM with 0.1% BSA only; positive control wells receive FGF2 ligand but no test compound. Test compounds are prepared in serum-free DMEM with ligand in a 96-well plate and serially diluted to make seven (7) test concentrations. [0665]
  • 5. After 20 hours, add diluted BrdU labeling reagent (1:100 BrdU:DMEM, 0.1% BSA, final concentration is 10 μM) to the cells and incubate for 1.5 hours. [0666]
  • 6. Decant medium. Remove traces of material with paper towel. Add FixDenat (50 μl/well) and incubate at room temperature for 45 minutes on a plate shaker. [0667]
  • 7. Remove Fixdenat solution. Add blocking solution (5% dehydrated milk in PBS (200 μl/well)) and incubate for 30 minutes at room temperature on a plate shaker. [0668]
  • 8. Decant blocking solution; wash wells once with PBS. Add anti-BrdU-POD solution (1:100 dilution in PBS, 0.1% BSA); incubate for 90 minutes at room temperature on a plate shaker. [0669]
  • 9. Decant antibody conjugate; rinse wells 5 times with PBS. Dry plate by inverting on paper towel and tapping. [0670]
  • 10. Add TMB solution (100 μl/well); incubate 20 minutes at room temperature on a plate shaker until color development is sufficient for photometric detection. [0671]
  • 11. Measure absorbance at 410 nM on a Dynatech ELISA plate reader using “Dual wavelength” mode with a filter at 490 nM. [0672]
    • Biochemical EGFR Assay
  • This assay measures the in vitro kinase activity of EGFR using ELISA. [0673]
  • Materials And Reagents [0674]
  • 1. Corning 96-well Elisa plates (Corning Catalog No. 25805-96). [0675]
  • 2. SUMO1 monoclonal anti-EGFR antibody (Biochemistry Lab, SUGEN, Inc.). [0676]
  • 3. PBS (Dulbecco's Phosphate-Buffered Saline, Gibco Catalog No. 450-1300EB). [0677]
  • 4. TBST Buffer [0678]
    Working Amount
    Reagent M.W. Concentration per L
    Tris 121.14  50 mM 6.057 g
    NaCl  58.44 150 mM 8.766 g
    Triton X-100 NA 0.1% 1.0 ml
  • 5. Blocking Buffer: [0679]
    Working Amount per
    Reagent M.W. Concentration 100 ml
    Carnation Instant 5% 5.0 g
    Non-Fat Milk
    PBS NA NA 100 ml
  • 6. A431 cell lysate (Screening Lab, SUGEN, Inc.) [0680]
  • 7. TBS Buffer: [0681]
    Working Amount
    Reagent M.W. Concentration per L
    Tris 121.14  50 mM 6.057 g
    NaCl 58.44 150 mM 8.766 g
  • 8. TBS+10% DMSO [0682]
    Working Amount
    Reagent M.W. Concentration per L
    Tris 121.14  50 mM 1.514 g
    NaCl  58.44 150 mM 2.192 g
    DMSO NA 10% 25 ml
  • 9. Adenosine-5′-triphosphate (ATP, from Equine muscle, Sigma Cat. No. A-5394). [0683]
  • Prepare a 1.0 mM solution in dH[0684] 2O. This reagent should be made up immediately prior to use and kept on ice.
  • 10. MnCl[0685] 2.
  • Prepare a 1.0 M stock solution in dH[0686] 2O.
  • 11. ATP/MnCl[0687] 2 phosphorylation mix
    Stock Amount Working
    Reagent solution per 10 ml Concentration
    ATP 1.0 mM 300 μl 30 μM
    MnCl2 1.0 M 500 μl 50 mM
    dH2O 9.2 ml
  • This reagent should be prepared immediately before use and kept on ice [0688]
  • 12. NUNC 96-well V bottom polypropylene plates (Applied Scientific Cat. No. AS-72092). [0689]
  • 13. Ethylenediaminetetraacetic acid (EDTA) [0690]
  • Prepare 200 mM working solution in dH[0691] 2O. Adjust to pH 8.0 with 10 N NaOH.
  • 14. Rabbit polyclonal anti-phosphotyrosine serum (Biochemistry Lab, SUGEN, Inc.) [0692]
  • 15. Goat anti-rabbit IgG peroxidase conjugate (Biosource Cat. No. ALI0404) [0693]
  • 16. ABTS (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), Sigma Cat. No. A-1888). [0694]
    Working Amount
    Reagent M.W. Concentration per L
    Citric Acid 192.12 100 mM 19.21 g
    Na2HPO4 141.96 250 mM 35.49 g
    ABTS NA 0.5 mg/ml 500 mg
  • Mix first two ingredients in about 900 ml dH[0695] 2O, adjust pH to 4.0 with phosphoric acid. Add ABTS, cover, let sit about 0.5 hr., filter. The solution should be kept in the dark at 4° C. until ready to use.
  • 17. Hydrogen peroxide 30% solution (Fisher Cat. No. H325) [0696]
  • 18. ABTS/H[0697] 2O2
  • Mix 15 ml ABTS solution and 2.0 μl H[0698] 2O2. Prepare 5 minutes before use.
  • 19. 0.2 M HCl [0699]
  • Procedure [0700]
  • 1. Coat Corning 96 well ELISA plates with 0.5 μg SUMO1 in 100 μl PBS per well, store overnight at 4° C. [0701]
  • 2. Remove unbound SUMO1 from wells by inverting plate to remove liquid. Wash 1× with dH[0702] 2O. Pat the plate on a paper towel to remove excess liquid.
  • 3. Add 150 μl of Blocking Buffer to each well. Incubate for 30 min. at room temperature with shaking. [0703]
  • 4. Wash plate 3× with deionized water, then once with TBST. Pat plate on a paper towel to remove excess liquid and bubbles. [0704]
  • 5. Dilute lysate in PBS (7 μg lysate/100 μl PBS). [0705]
  • 6. Add 100 μl of diluted lysate to each well. Shake at room temperature for 60 min. [0706]
  • 7. Wash plates as described in 4, above. [0707]
  • 8. Add 120 μl TBS to ELISA plate containing captured EGFR. [0708]
  • 9. Dilute test compound 1:10 in TBS in 96-well polypropylene plates (ie. 10 μl compound+90 μl TBS). [0709]
  • 10. Add 13.5 μl diluted test compound to ELISA plate. To control wells (wells which do not receive any test compound), add 13.5 μl TBS+10% DMSO. [0710]
  • 11. Incubate for 30 minutes while shaking at room temperature. [0711]
  • 12. Add 15 μl phosphorylation mix directly to all wells except negative control well which does not receive ATP/MnCl[0712] 2 (final well volume should be approximately 150 μl with 3 μM ATP/5 mM MnCl2 final concentration in each well.) Incubate 5 minutes while shaking.
  • 13. After 5 minutes, stop reaction by adding 16.5 μl of 200 mM EDTA (pH 8.0) to each well, shaking continuously. After the EDTA has been added, shake for 1 min. [0713]
  • 14. Wash 4× with deionized water, twice with TBST. [0714]
  • 15. Add 100 μl anti-phosphotyrosine (1:3000 dilution in TBST) per well. Incubate 30-45 min. at room temperature, with shaking. [0715]
  • 16. Wash as described in 4, above. [0716]
  • 17. Add 100 μl Biosource Goat anti-rabbit IgG peroxidase conjugate (1:2000 dilution in TBST) to each well. Incubate 30 min. at room temperature, with shaking. [0717]
  • 18. Wash as described in 4, above. [0718]
  • 19. Add 100 μl of ABTS/H[0719] 2O2 solution to each well.
  • 20. Incubate 5 to 10 minutes with shaking. Remove any bubbles. [0720]
  • 21. If necessary stop reaction with the addition of 100 μl 0.2 M HCl per well. [0721]
  • 22. Read assay on Dynatech MR7000 ELISA reader. Test Filter: 410 nM Reference Filter: 630 nM [0722]
    • Biochemical PDGFR Assay
  • This assay measures the in vitro kinase activity of PDGFR using ELISA. [0723]
  • Materials And Reagents [0724]
  • Unless otherwise noted, the preparation of working solution of the following reagents is the same as that for the Biochemical EGFR assay, above. [0725]
  • 1. Corning 96-well Elisa plates (Corning Catalog No. 25805-96). [0726]
  • 2. 28D4C10 monoclonal anti-PDGFR antibody (Biochemistry Lab, SUGEN, Inc.). [0727]
  • 3. PBS (Dulbecco's Phosphate-Buffered Saline, Gibco Catalog No. 450-1300EB) [0728]
  • 4. TBST Buffer. [0729]
  • 5. Blocking Buffer. [0730]
  • 6. PDGFR-β expressing NIH 3T3 cell lysate (Screening Lab, SUGEN, Inc.). [0731]
  • 7. TBS Buffer. [0732]
  • 8. TBS+10% DMSO. [0733]
  • 9. Adenosine-5′-triphosphate (ATP, from Equine muscle, Sigma Cat. No. A-5394). [0734]
  • 10. MnCl[0735] 2.
  • 11. Kinase buffer phosphorylation mix. [0736]
    Stock Amount Working
    Reagent solution per 10 μl Concentration
    Tris 1 M 250 μl  25 mM
    NaCl/ 5 M 200 μl 100 mM
    MnCl2 1 M 100 μl  10 mM
    TX-100 100 mM  50 μl 0.5 Mm
  • 12. NUNC 96-well V bottom polypropylene plates (Applied Scientific Cat. No. AS-72092). [0737]
  • 13. Ethylenediaminetetraacetic acid (EDTA). [0738]
  • 14. Rabbit polyclonal anti-phosphotyrosine serum (Biochemistry Lab, SUGEN, Inc.). [0739]
  • 15. Goat anti-rabbit IgG peroxidase conjugate (Biosource Cat. No. ALI0404). [0740]
  • 16. 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS, Sigma Cat. No. A-1888). [0741]
  • 17. Hydrogen peroxide 30% solution (Fisher Cat. No. H325). [0742]
  • 18. ABTS/H[0743] 2O2.
  • 19. 0.2 M HCl. [0744]
  • Procedure [0745]
  • 1. Coat Corning 96 well ELISA plates with 0.5 μg 28D4C10 in 100 μl PBS per well, store overnight at 4° C. [0746]
  • 2. Remove unbound 28D4C10 from wells by inverting plate to remove liquid. Wash 1× with dH[0747] 2O. Pat the plate on a paper towel to remove excess liquid.
  • 3. Add 150 μl of Blocking Buffer to each well. Incubate for 30 min. at room temperature with shaking. [0748]
  • 4. Wash plate 3× with deionized water, then once with TBST. Pat plate on a paper towel to remove excess liquid and bubbles. [0749]
  • 5. Dilute lysate in HNTG (10 μg lysate/100 μl HNTG) [0750]
  • 6. Add 100 μl of diluted lysate to each well. Shake at room temperature for 60 min. [0751]
  • 7. Wash plates as described in 4, above. [0752]
  • 8. Add 80 μl working kinase buffer mix to ELISA plate containing captured PDGFR. [0753]
  • 9. Dilute test compound 1:10 in TBS in 96-well polypropylene plates (i.e., 10 μl compound+90 μl TBS). [0754]
  • 10. Add 10 μl diluted test compound to ELISA plate. To control wells (wells which do not receive any test compound), add 10 μl TBS+10% DMSO. [0755]
  • 11. Incubate for 30 minutes while shaking at room temperature. [0756]
  • 12. Add 10 μl ATP directly to all wells except negative control well (final well volume should be approximately 100 μl with 20 μM ATP in each well.) Incubate 30 minutes while shaking. [0757]
  • 13. After 30 minutes, stop reaction by adding 10 μl of 200 mM EDTA (pH 8.0) to each well. [0758]
  • 14. Wash 4× with deionized water, twice with TBST. [0759]
  • 15. Add 100 μl anti-phosphotyrosine (1:3000 dilution in TBST) per well. Incubate 30-45 min. at room temperature, with shaking. [0760]
  • 16. Wash as described in 4, above. [0761]
  • 17. Add 100 μl Biosource Goat anti-rabbit IgG peroxidase conjugate (1:2000 dilution in TBST) to each well. Incubate 30 min. at room temperature, with shaking. [0762]
  • 18. Wash as described in 4, above. [0763]
  • 19. Add 100 μl of ABTS/H[0764] 2O2 solution to each well.
  • 20. Incubate 10 to 30 minutes with shaking. Remove any bubbles. [0765]
  • 21. If necessary stop reaction with the addition of 100 μl 0.2 M HCl per well. [0766]
  • 22. Read assay on Dynatech MR7000 ELISA reader: test filter: 410 nM, reference filter: 630 nM. [0767]
    • Biochemical FGFR Assay
  • This assay measures in vitro kinase activity of the Myc-GyrB-FGFR fusion protein using ELISA. [0768]
  • Materials And Reagents [0769]
  • 1. HNTG [0770]
    5x Stock Amount 1x Working
    Reagent M.W. Concentration per L Concentration
    HEPES 238.3 100 mM 23.83 g  20 mM
    NaCl 58.44 750 mM 43.83 g 150 mM
    Glycerol NA 50% 500 ml   10%
    Triton X-100 NA  5%  10 ml 1.0%
  • To make a liter of 5× stock solution, dissolve HEPES and NaCl in about 350 ml dH[0771] 2O, adjust pH to 7.2 with HCl or NaOH (depending on the HEPES that is used), add glycerol, Triton X-100 and then dH2O to volume.
  • 2. PBS (Dulbecco's Phosphate-Buffered Saline, Gibco Catalog # 450-1300EB). [0772]
  • 3. Blocking Buffer. [0773]
  • 4. Kinase Buffer. [0774]
    10x Stock 1x Working
    Reagent M.W. Concentration Concentration
    HEPES (pH 7.2) 238.3 500 mM 50 mM
    MnCl2  20 mM  2 mM
    MgCl2 203.32 200 mM 10 mM
    Triton-X-100 1% 0.1%
    DTT 380.35  5 mM   0.5 mM
  • 5. Phenylmethylsulfonyl fluoride (PMSF, Sigma, Cat. No. P-7626): [0775]
  • Working solution: 100 mM in ethanol. [0776]
  • 6. ATP (Bacterial source, Sigma Cat. No. A-7699) [0777]
  • Use 3.31 mg per ml MilliQ H[0778] 2O for a stock concentration of 6 mM.
  • 7. Biotin conjugated anti-phosphotyrosine mab (clone 4G10, Upstate Biotechnology Inc. Cat. No. 16-103, Ser. No. 14495). [0779]
  • 8. Vectastain Elite ABC reagent (Avidin peroxidase conjugate, Vector Laboratories Cat. No. PK-6 100). [0780]
  • 9. ABTS Solution. [0781]
  • 10. Hydrogen peroxide 30% solution (Fisher Catalog # H325). [0782]
  • 11. ABTS/H[0783] 2O2.
  • 12. 0.2 M HCl. [0784]
  • 13. TRIS HCl (Fischer Cat. No. BP 152-5). [0785]
  • Prepare 1.0 mM solution in MilliQ H[0786] 2O, adjust pH to 7.2 with HCl.
  • 14. NaCl (Fisher Cat. No. S271-10). [0787]
  • Prepare 5 M solution in MilliQ H[0788] 2O.
  • 15. MgCl[0789] 2 (Fisher Cat. No. M33-500).
  • Prepare 1 M solution in MilliQ H[0790] 2O.
  • 16. HEPES (Fisher Cat. No. BP310-500). [0791]
  • Prepare 1 M solution in MilliQ H[0792] 2O, adjust pH to 7.5, sterile filter.
  • 17. TBST Buffer. [0793]
  • 18. Sodium Carbonate Buffer (Fisher Cat. No. S495). [0794]
  • Prepare 0.1 M solution in MilliQ H[0795] 2O, adjust pH to 9.6 with NaOH, filter.
  • 19. Dithiothreitol (DTT, Fisher Cat. No. BP172-25). [0796]
  • Prepare 0.5 mM working solution in MilliQ H[0797] 2O just prior to use. Store at −20° C. until used, discard any leftover.
  • 20. MnCl[0798] 2.
  • 21. Triton X-100. [0799]
  • 22. Goat α-Rabbit IgG (Cappel). [0800]
  • 23. Affinity purified Rabbit α GST GyrB (Biochemistry Lab. SUGEN, Inc.). [0801]
  • Procedure [0802]
  • All of the following steps are conducted at room temperature unless otherwise indicated. [0803]
  • 1. Coat Corning 96-well ELISA plates with 2 μg Goat α-Rabbit antibody per well in Carbonate Buffer such that total well volume is 100 μl. Store overnight at 4° C. [0804]
  • 2. Remove unbound Goat α-Rabbit antibody by inverting plate to remove liquid. Pat plate on a paper towel to remove excess liquid and bubbles [0805]
  • 3. Add 150 μl Blocking Buffer (5% Low Fat Milk in PBS) to each well. Incubate while shaking on a micro-titer plate shaker for 30 min. [0806]
  • 4. Wash 4× with TBST. Pat plate on a paper towel to remove excess liquid and bubbles. [0807]
  • 5. Add 0.5 μg Rabbit a-GyrB antibody per well. Dilute antibody in DPBS to a final volume of 100 μl per well. Incubate with shaking on a micro-titer plate shaker at room temperature for 1 hour. [0808]
  • 6. Wash 4× with TBST as described in step 4. [0809]
  • 7. Add 2 μg COS/FGFR cell lysate (Myc-GyrB-FGFR source) in HNTG to each well to give a final volume of 100 μl per well. Incubate with shaking on a micro-titer plate shaker for 1 hour. [0810]
  • 8. Wash 4× with TBST as described in step 4. [0811]
  • 9. Add 80 μl of 1× kinase buffer per well. [0812]
  • 10. Dilute test compound 1:10 in 1× kinase buffer+1% DMSO in a polypropylene 96 well plate. [0813]
  • 11. Transfer 10 μl of diluted test compound solution and control wells from polypropylene plate wells to the corresponding ELISA plate wells, incubate with shaking on a micro-titer plate shaker for 20 minutes. [0814]
  • 12. Add 10 μl of 70 μM ATP diluted in kinase buffer to positive control and test wells (Final ATP concentration is 7 μM/well) . Add 10 μl 1× kinase buffer to negative control wells. Incubate with shaking on a micro-titer plate shaker for 15 min. [0815]
  • 13. Stop kinase reaction by adding 5 μl 0.5 M EDTA to all wells. [0816]
  • 14. Wash 4× with TBST as described in step 4. [0817]
  • 15. Add 100 μl biotin conjugated α-phosphotyrosine mab (b4G10) diluted in TBST to each well. Incubate with shaking on a micro-titer plate shaker for 30 minutes. [0818]
  • 16. Prepare Vectastain ABC reagent. Add 1 drop reagent A to 15 ml TBST. Mix by inverting tube several times. Add 1 drop reagent B and mix again. [0819]
  • 17. Wash 4× with TBST as described in step 4. [0820]
  • 18. Add 100 μl ABC HRP reagent to each well. Incubate with shaking on a micro-titer plate shaker for 30 minutes. [0821]
  • 19. Wash 4× with TBST as described in step 4. [0822]
  • 20. Add 100 μl of ABTS/H[0823] 2O2 solution to each well.
  • 22. Incubate 5 to 15 minutes with shaking. Remove any bubbles. [0824]
  • 23. If necessary stop reaction by adding 100 μl of 0.2M HCl/well. [0825]
  • 24. Read assay on Dynatech MR7000 ELISA Plate Reader; test filter: 410 nM, reference filter: 630 nM. [0826]
    • Biochemical FLK-1 Assay
  • This assay evaluates flk-1 autophosphorylation activity in vitro using ELISA. [0827]
  • Materials And Reagents [0828]
  • 1. 15 cm tissue culture dishes [0829]
  • 2. Flk-1/NIH cells: NIH fibroblast line over-expressing human flk-1 clone 3 (SUGEN, Inc., obtained from MPI, Martinsried, Germany). [0830]
  • 3. Growth medium: DMEM plus heat inactivated 10% FBS and 2 mM Glutamine (Gibco-BRL). [0831]
  • 4. Starvation medium: DMEM plus 0.5% heat-inactivated FBS, 2 mM Glutamine (Gibco-BRL). [0832]
  • 5. Corning 96-well ELISA plates (Corning Cat. No. 25805-96). [0833]
  • 6. L4 or E38 monoclonal antibody specific for flk-1; Purified by Protein-A agarose affinity chromatography (SUGEN, Inc.). [0834]
  • 7. PBS (Dulbecco's Phosphate-Buffered Saline) Gibco Cat. No. 450-1300EB). [0835]
  • 8. HNTG (see BIOCHEMICAL FGFR for preparation). [0836]
  • 9. Pierce BCA protein determination kit. [0837]
  • 10. Blocking buffer [0838]
  • 11. TBST (pH 7.0) [0839]
  • 12. Kinase Buffer [0840]
  • 13. Kinase Stop Solution: 200 mM EDTA. [0841]
  • 14. Biotinylated 4G10, specific for phosphotyrosine (UBI, Cat. No. No. 16-103). [0842]
  • 15. AB kit (Vector Laboratories Cat. No. PK 4000). [0843]
  • 16. DMSO [0844]
  • 17. NUNC 96-well V bottom polypropylene plates (Applied Scientific Cat. No. AS-72092). [0845]
  • 18. Turbo-TMB (Pierce). [0846]
  • 19. Turbo-TMB stop solution: 1 M H[0847] 2SO4.
  • 20. ATP (Sigma Cat. No. A-7699). [0848]
  • 21. 20% DMSO in TBS (pH 7.0). [0849]
  • Procedure [0850]
  • Cell Growth and Lysate Preparation. [0851]
  • 1. Seed cell into growth medium and grow for 2-3 days to 90-100% confluency at 37° C. and 5% CO[0852] 2. Do not exceed passage #20.
  • 2. Remove the medium and wash the cells twice with PBS. Lyse with HNTG lysis buffer. Collect all lysates and vortex mix them for 20-30 seconds. [0853]
  • 3. Remove insoluble material by centrifugation (5-10 min at about 10,000×g). [0854]
  • 4. Determine the protein concentration using BCA kit. [0855]
  • 5. Partition lysate into 1 mg aliquots, store at −80° C. [0856]
  • Assay Procedure [0857]
  • 1. Coat Corning 96-well ELISA plates with 2 μg/well purified L4 (or E 38) in 100 μl of PBS. Store overnight at 4° C. [0858]
  • 2. Remove unbound proteins from wells by inverting the plate to remove the liquid. Wash one time with dH[0859] 2O, pat plate on paper towel to remove excess liquid.
  • 3. Block plates with 150 μl blocking buffer per well. Incubate for 45-60 minutes with shaking at 4° C. [0860]
  • 4. Remove the blocking buffer and wash the ELISA plate three times with dH[0861] 2O and one time with TBST. Pat plate on paper towel to remove excess liquid.
  • 5. Dilute lysate in PBS to give final concentration of 50 μg/100 μl. Add 100 μl of diluted lysate to each well. Incubate with shaking at 4° C. overnight. [0862]
  • 6. Remove unbound proteins from wells by inverting the plate. Wash as in step 4. [0863]
  • 7. Add 80 μl of kinase buffer to wells (90 μl to negative control wells). [0864]
  • 8. Dilute test compounds (normally 10-fold) into wells of a polypropylene plate containing 20% DMSO in TBS. [0865]
  • 9. Add 10 μl of the diluted compounds to the ELISA wells containing immobilized flk-1 and shake. Control wells receive no compounds. [0866]
  • 10. From stock 1 mM ATP, prepare 0.3 mM ATP solution in dH[0867] 2O (alternatively, kinase buffer may be used).
  • 11. Add 10 μl of 0.3 mM ATP to all wells except the negative controls. Incubate for 60 min. at room temperature with shaking. [0868]
  • 12. After 1 hr stop the kinase reaction by adding 11 μl 200 mM EDTA. Shake for 1-2 min. [0869]
  • 13. Wash the ELISA plate 4 times with dH[0870] 2O and twice with TBST.
  • 14. Add 100 μl of 1:5000 biotinylated 4G10:TBST to all wells. Incubate 45 min with shaking at room temperature. [0871]
  • 15. While the above is incubating, add 50 μl of solutions A & B from the ABC kit to 10 ml of TBST. These solutions must be combined approximately 30 min prior to use. [0872]
  • 16. Wash plates as in step 4. [0873]
  • 17. Add 100 μl of the preformed A & B complex to all wells. Incubate 30 min with shaking at room temperature. [0874]
  • 18. Wash plates as in step 4. [0875]
  • 19. Add 100 μl turbo-TMB. Shake at room temperature for 10-15 min. [0876]
  • 20. When the color in the positive control wells reaches an absorbance of about 0.35-0.4, stop the reaction with 100 μl of turbo-TMB stop solution. [0877]
  • 21. Read plates on Dynatech MR7000 ELISA reader; test filter: 450 nM, reference filter: 410 nM. [0878]
    • HEV-EC-C Assay
  • The following protocol may also be used to measure a compound's activity against PDGF-R, FGF-R, VEGF, aFGF or Flk-1/KDR, all of which are naturally expressed by HUV-EC cells. [0879]
    • DAY 0
  • 1. Wash and trypsinize HUV-EC-C cells (human umbilical vein endothelial cells, (American Type Culture Collection; catalogue no. 1730 CRL). Wash with Dulbecco's phosphate-buffered saline (D-PBS; obtained from Gibco BRL; catalogue no. 14190-029) 2 times at about 1 ml/10 cm[0880] 2 of tissue culture flask. Trypsinize with 0.05% trypsin-EDTA in non-enzymatic cell dissociation solution (Sigma Chemical Company; catalogue no. C-1544). The 0.05% trypsin is made by diluting 0.25% trypsin/1 mnM EDTA (Gibco; catalogue no. 25200-049) in the cell dissociation solution. Trypsinize with about 1 ml/25-30 cm of tissue culture flask for about 5 minutes at 37° C. After cells have detached from the flask, add an equal volume of assay medium and transfer to a 50 ml sterile centrifuge tube (Fisher Scientific; catalogue no. 05-539-6).
  • 2. Wash the cells with about 35 ml assay medium in the 50 ml sterile centrifuge tube by adding the assay medium, centrifuge for 10 minutes at approximately 200×g, aspirate the supernatant, and resuspend with 35 ml D-PBS. Repeat the wash two more times with D-PBS, resuspend the cells in about 1 ml assay medium/15 cm[0881] 2 of tissue culture flask. Assay medium consists of F12K medium (Gibco BRL; catalogue no. 21127-014) and 0.5% heat-inactivated fetal bovine serum. Count the cells with a Coulter Counter® (Coulter Electronics, Inc.) and add assay medium to the cells to obtain a concentration of 0.8-1.0×105 cells/ml.
  • 3. Add cells to 96-well flat-bottom plates at 100 μl/well or 0.8-1.0×10[0882] 4 cells/well; incubate ˜24 h at 37° C., 5% CO2.
    • DAY 1
  • 1. Make up two-fold test compound titrations in separate 96-well plates, generally 50 μM on down to 0 μM. Use the same assay medium as mentioned in day 0, step 2 above. Titrations are made by adding 90 μl/well of test compound at 200 μM (4× the final well concentration) to the top well of a particular plate column. Since the stock test compound is usually 20 mM in DMSO, the 200 μM drug concentration contains 2% DMSO. [0883]
  • A diluent made up to 2% DMSO in assay medium (F12K+0.5% fetal bovine serum) is used as diluent for the test compound titrations in order to dilute the test compound but keep the DMSO concentration constant. Add this diluent to the remaining wells in the column at 60 μl/well. Take 60 μl from the 120 μl of 200 μM test compound dilution in the top well of the column and mix with the 60 μl in the second well of the column. Take 60 μl from this well and mix with the 60 μl in the third well of the column, and so on until two-fold titrations are completed. When the next-to-the-last well is mixed, take 60 μl of the 120 μl in this well and discard it. Leave the last well with 60 μl of DMSO/media diluent as a non-test compound-containing control. Make 9 columns of titrated test compound, enough for triplicate wells each for: (1) VEGF (obtained from Pepro Tech Inc., catalogue no. 100-200; (2) endothelial cell growth factor (ECGF) (also known as acidic fibroblast growth factor, or aFGF) (obtained from Boehringer Mannheim Biochemica, catalogue no. 1439 600); or, (3) human PDGF B/B (1276-956, Boehringer Mannheim, Germany) and assay media control. ECGF comes as a preparation with sodium heparin. [0884]
  • 2. Transfer 50 μl/well of the test compound dilutions to the 96-well assay plates containing the 0.8-1.0×10[0885] 4 cells/100 μl/well of the HUV-EC-C cells from day 0 and incubate ˜2 h at 37° C., 5% CO2.
  • 3. In triplicate, add 50 μl/well of 80 μg/ml VEGF, 20 ng/ml ECGF, or media control to each test compound condition. As with the test compounds, the growth factor concentrations are 4× the desired final concentration. Use the assay media from day 0 step 2 to make the concentrations of growth factors. Incubate approximately 24 hours at 37° C., 5% CO[0886] 2. Each well will have 50 μl test compound dilution, 50 μl growth factor or media, and 100 μl cells, which calculates to 200 μl/well total. Thus the 4× concentrations of test compound and growth factors become 1× once everything has been added to the wells.
    • DAY 2
  • 1. Add [0887] 3H-thymidine (Amersham; catalogue no. TRK-686) at 1 μCi/well (10 μl/well of 100 μCi/ml solution made up in RPMI media+10% heat-inactivated fetal bovine serum) and incubate ˜24 h at 37° C., 5% CO2. RPMI is obtained from Gibco BRL, catalogue no. 11875-051.
    • DAY 3
  • 1. Freeze plates overnight at −20° C. [0888]
    • DAY 4
  • Thaw plates and harvest with a 96-well plate harvester (Tomtec Harvester 96®) onto filter mats (Wallac; catalogue no. 1205-401); read counts on a Wallac Betaplate™ liquid scintillation counter. [0889]
    • In Vivo Animal Models Xenograft Animal Models
  • The ability of human tumors to grow as xenografts in athymic mice (e.g., Balb/c, nu/nu) provides a useful in vivo model for studying the biological response to therapies for human tumors. Since the first successful xenotransplantation of human tumors into athymic mice, (Rygaard and Povlsen, 1969[0890] , Acta Pathol. Microbial. Scand. 77:758-760), many different human tumor cell lines (e.g., mammary, lung, genitourinary, gastro-intestinal, head and neck, glioblastoma, bone, and malignant melanomas) have been transplanted and successfully grown in nude mice. The following assays may be used to determine the level of activity, specificity and effect of the different compounds of the present invention. Three general types of assays are useful for evaluating compounds: cellular/catalytic, cellular/biological and in vivo. The object of the cellular/catalytic assays is to determine the effect of a compound on the ability of a TK to phosphorylate tyrosines on a known substrate in a cell. The object of the cellular/biological assays is to determine the effect of a compound on the biological response stimulated by a TK in a cell. The object of the in vivo assays is to determine the effect of a compound in an animal model of a particular disorder such as cancer.
  • Suitable cell lines for subcutaneous xenograft experiments include C6 cells (glioma, ATCC # CCL 107), A375 cells (melanoma, ATCC # CRL 1619), A431 cells (epidermoid carcinoma, ATCC # CRL 1555), Calu 6 cells (lung, ATCC # HTB 56), PC3 cells (prostate, ATCC # CRL 1435), SKOV3TP5 cells and NIH 3T3 fibroblasts genetically engineered to overexpress EGFR, PDGFR, IGF-1R or any other test kinase. The following protocol can be used to perform xenograft experiments: [0891]
  • Female athymic mice (BALB/c, nu/nu) are obtained from Simonsen Laboratories (Gilroy, Calif.). All animals are maintained under clean-room conditions in Micro-isolator cages with Alpha-dri bedding. They receive sterile rodent chow and water ad libitum. [0892]
  • Cell lines are grown in appropriate medium (for example, MEM, DMEM, Ham's F10, or Ham's F12 plus 5%-10% fetal bovine serum (FBS) and 2 mM glutamine (GLN)). All cell culture media, glutamine, and fetal bovine serum are purchased from Gibco Life Technologies (Grand Island, NY) unless otherwise specified. All cells are grown in a humid atmosphere of 90-95% air and 5-10% CO[0893] 2 at 37° C. All cell lines are routinely subcultured twice a week and are negative for mycoplasma as determined by the Mycotect method (Gibco).
  • Cells are harvested at or near confluency with 0.05% Trypsin-EDTA and pelleted at 450×g for 10 min. Pellets are resuspended in sterile PBS or media (without FBS) to a particular concentration and the cells are implanted into the hindflank of the mice (8-10 mice per group, 2-10×10[0894] 6 cells/animal). Tumor growth is measured over 3 to 6 weeks using venier calipers. Tumor volumes are calculated as a product of length×width×height unless otherwise indicated. P values are calculated using the Students t-test. Test compounds in 50-100 μL excipient (DMSO, or VPD:D5W) can be delivered by IP injection at different concentrations generally starting at day one after implantation.
    • Tumor Invasion Model
  • The following tumor invasion model has been developed and may be used for the evaluation of therapeutic value and efficacy of the compounds identified to selectively inhibit KDR/FLK-1 receptor. [0895]
  • Procedure [0896]
  • 8 week old nude mice (female) (Simonsen Inc.) are used as experimental animals. Implantation of tumor cells can be performed in a laminar flow hood. For anesthesia, Xylazine/Ketamine Cocktail (100 mg/kg ketamine and 5 mg/kg Xylazine) are administered intraperitoneally. A midline incision is done to expose the abdominal cavity (approximately 1.5 cm in length) to inject 10[0897] 7 tumor cells in a volume of 100 μl medium. The cells are injected either into the duodenal lobe of the pancreas or under the serosa of the colon. The peritoneum and muscles are closed with a 6-0 silk continuous suture and the skin is closed by using wound clips. Animals are observed daily.
  • Analysis [0898]
  • After 2-6 weeks, depending on gross observations of the animals, the mice are sacrificed, and the local tumor metastases to various organs (lung, liver, brain, stomach, spleen, heart, muscle) are excised and analyzed (measurement of tumor size, grade of invasion, immunochemistry, in situ hybridization determination, etc.). [0899]
    • Measurement Of Cell Toxicity
  • Therapeutic compounds should be more potent in inhibiting receptor tyrosine kinase activity than in exerting a cytotoxic effect. A measure of the effectiveness and cell toxicity of a compound can be obtained by determining the therapeutic index; i.e., IC[0900] 50/LD50. IC50, the dose required to achieve 50% inhibition, can be measured using standard techniques such as those described herein. LD50, the dosage which results in 50% toxicity, can also be measured by standard techniques as well (Mossman, 1983, J. Immunol. Methods, 65:55-63), by measuring the amount of LDH released (Korzeniewski and Callewaert, 1983, J. Immunol. Methods, 64:313; Decker and Lohmann-Matthes, 1988, J. Immunol. Methods, 115:61), or by measuring the lethal dose in animal models. Compounds with a large therapeutic index are preferred. The therapeutic index should be greater than 2, preferably at least 10, more preferably at least 50.
    • CONCLUSION
  • Thus, it will be appreciated that the compounds, methods and pharmacological compositions of the present invention modulate RTK, CTK and STK activity and therefore can be expected to be effective as therapeutic agents against a RTK-, CTK- and STK-related disorders. [0901]
  • Although certain embodiments and examples have been used to describe the present invention, it will be apparent to those skilled in the art that changes to the embodiments and examples shown may be made without departing from the scope and spirit of the invention. [0902]
  • Other embodiments are within the following claims. [0903]

Claims (20)

1. A compound having the chemical structure:
Figure US20010007033A1-20010705-C00251
wherein:
R1, R2, R3 and R4 are independently selected from the group consisting of hydrogen, alkyl, halo and S-sulfonamido;
“A” is a ring selected from the group consisting of tetrahydrobenzene, naphthalene, tetrahydronaphthalene, tetrahydrofuran, pyrrolidine, pyridine, quinoline, indole, isatin, chromone, fluorene, carbazole, benzo[b]furan, thieno[b]thiophene and uracil;
said “A” ring is substituted with one or more groups 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, N-thiocarbamyl, C-amido, N-amido, amino and —NR5R6;
R1 and R2 or R2 and R3 or R3 and R4 may combine to form a methylenedioxy or an ethylenedioxy group; and,
R5 and R6 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.
2. The compound of
claim 1
 wherein said “A” ring is selected from the group consisting of naphthalene, tetrahydronaphthalene, tetrahydrofuran, pyrrolidine, quinoline, isatin, chromone, fluorene, carbazole, benzo[b]furan, thieno[b]thiophene and uracil.
3. The compound of
claim 1
 wherein the “A” ring is substituted with a group selected from the group consisting of
hydrogen;
unsubstituted lower alkyl;
lower alkyl substituted with a group selected from the group consisting of unsubstituted aryl, halo, hydroxy, unsubstituted lower alkyl C-carboxy, and —NR5R6, unsubstituted lower alkoxy;
lower alkoxy substituted with a group selected from the group consisting of C-carboxy, —NR5R6, and halo;
halo;
nitro;
cyano;
hydroxy;
acetyl;
trihaloacetyl;
trihalomethyl;
unsubstituted aryl;
aryl substituted with one or more groups selected from the group consisting of halo, hydroxy, unsubstituted lower alkyl, unsubstituted lower alkoxy, hydroxy, cyano and nitro, unsubstituted lower aryloxy; and,
aryloxy substituted with one or more groups selected from the group consisting of unsubstituted lower alkyl, trihalomethyl, halo, unsubstituted lower alkoxy, and unsubstituted aryl; unsubstituted lower alkyl C-carboxy, unsubstituted lower alkyl thioalkoxy, carboxylic acid, unsubstituted lower alkyl O-carboxy, 4-formylpiperazinyl, 4-formylmorpholinyl, N-pyrrolidinyl and —NR5R6.
4. The compound of
claim 1
 wherein R5 and R6 are independently selected from the group consisting of hydrogen and unsubstituted lower alkyl.
5. A combinatorial library of 3-(substituted)-2-indolinone compounds, comprising compounds formed by reacting oxindoles with aldehydes, wherein:
said oxindole is selected from the group consisting of 5,7-dibromooxindole, 5-iodooxindole, 5-bromo-4-methyloxindole, 5-methylamino-sulfonyloxindole, 5-(4-trifluoromethylanilino-sulfonyl)-oxindole, 5-(morpholin-4-yl)-sulfonyloxindole, 5-(2-chloroethyl)oxindindole and 5,7-dibromo-4-methyloxindole; and,
said aldehyde is selected from the group consisting of benzaldehyde, 1-naphthaldehyde, 2-naphthaldehyde, 2-formyl-5,6,7,8-tetrahydronaphthalene, 3-furaldehyde, furfural, 2-quinolinecarboxaldehyde, 3-quinolinecarboxaldehyde, 4-quinonlinecarboxaldehyde, 1-pyrrolidinecarboxaldehyde, 3-thiophenecarboxaldehyde, indole-3-carboxaldehyde, 2-pyridinecarboxaldehyde, 2-thiophenecarboxaldehyde, 3-thiophenecarboxaldehyde, pyrrole-2-carboxaldehyde, pyrrole-3-carboxaldehyde, 2-imidazolecarboxaldehyde, 4-imidazolecarboxaldehyde, pyrazole-3-carboxaldehyde, 2-formylbenzimidazole, 2-formyl-4,5,6,7-tetrahydroindole, benzo[b]furan-2-carboxaldehyde, thieno[b]thiophene-2-carboxaldehyde, isatin, chromone-3-carboxaldehyde, fluorene-2-carboxaldehyde, pyridine-2-carboxaldehyde, pyridine-3-carboxaldehyde, 1,2,3,6-tetrahydro benzaldehyde, 3-tetrahydrofurancarboxaldehyde, carbazole-3-carboxaldehyde, 5-formyluracil and 2-(trifluoroacetyl)thiophene wherein
said aldehyde may be unsubstituted, or substituted with one or more groups selected from the group consisting of:
unsubstituted lower alkyl;
lower alkyl substituted with a group selected from the group consisting of unsubstituted aryl, halo, hydroxy, unsubstituted lower alkyl C-carboxy, and —NR5R6, unsubstituted lower alkoxy;
lower alkoxy substituted with a group selected from the group consisting of C-carboxy, —NR5R6, and halo;
halo;
nitro;
cyano;
hydroxy;
acetyl;
trihaloacetyl;
trihalomethyl;
unsubstituted aryl;
aryl substituted with one or more groups selected from the group consisting of halo, hydroxy, unsubstituted lower alkyl, unsubstituted lower alkoxy, hydroxy, cyano and nitro, unsubstituted lower aryloxy; and,
aryloxy substituted with one or more groups selected from the group consisting of unsubstituted lower alkyl, trihalomethyl, halo, unsubstituted lower alkoxy, and unsubstituted aryl; unsubstituted lower alkyl C-carboxy, unsubstituted lower alkyl thioalkoxy, carboxylic acid, unsubstituted lower alkyl O-carboxy, 4-formylpiperazinyl, 4-formylmorpholinyl, N-pyrrolidinyl and —NR5R6.
6. A pharmacological composition, comprising:
said compound, salt or prodrug of
claim 1
; and,
a pharmaceutically acceptable carrier.
7. A method for the modulation of the catalytic activity of a protein kinase comprising contacting said protein kinase with a compound, salt or prodrug of
claim 1
.
8. The method of
claim 7
 wherein said protein kinase comprises a protein tyrosine kinase.
9. The method of
claim 8
 wherein said protein tyrosine kinase comprises a receptor tyrosine kinase.
10. The method of
claim 9
 wherein said receptor tyrosine kinase is selected from the group consisting of EGF, HER2, HER3, HER4, IR, IGF-1R, IRR, PDGFRα, PDGFRβ, CSFIR, C-Kit, C-fms, Flk-1R, Flk4, KDR/Flk-1, Flt-1, FGFR-1R, FGFR-2R, FGFR-3R and FGFR-4R.
11. The method of
claim 7
 wherein said protein tyrosine kinase comprises a cellular tyrosine kinase.
12. The method of
claim 11
 wherein said non-receptor protein tyrosine kinase is selected from the group consisting of Src, Frk, Btk, Csk, Abl, ZAP70, Fes/Fps, Fak, Jak, Ack, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk.
13. The method of
claim 7
 wherein said protein kinase comprises a serine-threonine protein kinase.
14. The method of
claim 13
 wherein said serine-threonine protein kinase is selected from the group consisting of CDK2 and Raf.
15. A method for treating or preventing a protein kinase related disorder in an organism comprising administering a therapeutically effective amount of a compound of
claim 1
 to said organism.
16. The method of
claim 15
 wherein said protein kinase related disorder is selected from the group consisting of a receptor tyrosine kinase related disorder, a cellular tyrosine kinase related disorder, a serine threonine kinase related disorder and a flk related disorder.
17. The method of
claim 16
 wherein said receptor tyrosine kinase related disorder is selected from the group consisting of an EGFR related disorder, a PDGFR related disorder and an IGFR related disorder.
18. The method of
claim 15
 wherein said protein kinase related disorder is selected from the group consisting of squamous cell carcinoma, astrocytoma, glioblastoma, lung cancer, bladder cancer, head and neck cancer, melanoma, ovarian cancer, prostate cancer, breast cancer, lung carcinoma, small-cell lung cancer and glioma.
19. The method of
claim 15
 wherein said protein kinase related disorder is selected from the group consisting of autoimmune disorder, hyper-proliferation disorder, inflammatory disorder, diabetes, restenosis, fibrosis, psoriasis, angiogenesis, osteoarthritis and rheumatoid arthritis.
20. The method of
claim 15
 wherein said organism is a human.
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US20040029902A1 (en) * 2002-02-01 2004-02-12 Rajinder Singh 2,4-Pyrimidinediamine compounds and their uses
US20050234049A1 (en) * 2003-07-30 2005-10-20 Rajinder Singh Methods of treating or preventing autoimmune diseases with 2, 4-pyrimidinediamine compounds
US20060293311A1 (en) * 2005-06-08 2006-12-28 Rigel Pharmaceuticals, Inc. Compositions and methods for inhibition of the jak pathway
WO2007098507A2 (en) 2006-02-24 2007-08-30 Rigel Pharmaceuticals, Inc. Compositions and methods for inhibition of the jak pathway
US20070203161A1 (en) * 2006-02-24 2007-08-30 Rigel Pharmaceuticals, Inc. Compositions and methods for inhibition of the jak pathway
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