JP2002540096A - Indolinone compounds as kinase inhibitors - Google Patents

Abstract

  (57) [Summary] The present invention relates to certain indolinone compounds, a method for synthesizing the same, and a combinatorial library comprising the indolinone compounds of the present invention. The present invention also relates to methods of modulating the function of protein kinases using the indolinone compounds of the present invention, and methods of treating diseases by modulating the function of protein kinases and related signaling pathways.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION The following background description is provided to assist in understanding the invention and is not an admission that it describes or constitutes prior art to the present invention.

[0002] Protein kinases (PKs) are enzymes that catalyze the phosphorylation of hydroxy groups on tyrosine, serine and threonine residues of proteins. The result of this apparently simple activity is overwhelming. Almost every aspect of cell growth, differentiation and proliferation, ie, cell life, depends in various ways on PK activity. Furthermore, abnormal PK activity has been implicated in a host of diseases ranging from relatively non-life threatening diseases (eg, psoriasis) to highly malignant diseases (eg, gliomas (brain cancer)).

[0003] PKs can be conventionally classified into two classes: protein tyrosine kinases (
PTK) and serine-threonine kinase (STK).

[0004] A major aspect of PK activity is its involvement in growth factor receptors. Growth factor receptors are cell surface proteins. Upon binding of the growth factor ligand, the growth factor receptor is converted to the active form, which interacts with proteins on the inner surface of the cell membrane. This interaction causes phosphorylation of tyrosine residues and other amino acids on the receptor, forming complexes inside the cell with various cytoplasmic signaling molecules. These complexes then perform many cellular responses, such as cell division (proliferation), cell differentiation, cell growth, and the development of metabolic effects on the extracellular microenvironment. For a more detailed discussion, see Schlessinger and Ulrich, Ne.
See uron, 1992, 9: 303-391, which is hereby incorporated by reference as if fully set forth herein, including the drawings.

[0005] Receptor tyrosine kinases ("RTKs") are growth factor receptors with PK activity. These include a large family of transmembrane receptors with diverse biological activities. At present, at least 19 different subfamilies of RTKs have been identified. An example of these subfamilies is the subfamily called "HER" RTKs, which include EGFR (epidermal growth factor receptor)
, HER2, HER3 and HER4. These RTKs are composed of an extracellular glycosylation ligand binding domain, a transmembrane domain, and an intracellular cytoplasmic catalytic domain that can phosphorylate tyrosine residues on proteins.

[0006] Another RTK subfamily includes the insulin receptor (IR), the insulin-like growth factor I receptor (IGF-1R) and the insulin receptor-related receptor (IRR). IR and IGF-1R interact with insulin, IGF-I and IGF-II to form a heterotetramer of two fully extracellular glycosylated α subunits and two β subunits containing a tyrosine kinase domain. Form the body.

[0007] The third RTK subfamily is the platelet-derived growth factor receptor ("PDGF
R ") group, which includes PDGFRα, PDGFRβ, CSFIR, ck
It and c-fms are included. These receptors are composed of a variable number of immunoglobin-like loops consisting of a glycosylated extracellular domain, a transmembrane domain, and an intracellular domain with a tyrosine kinase domain interrupted by unrelated amino acid sequences.

[0008] Another group is often PDGs because of their similarity to the PDGFR subfamily.
It is a fetal liver kinase ("flk") receptor subfamily that is subsumed into the FR subfamily. This group is believed to consist of the kinase insertion domain-receptor fetal liver kinase-1 (KDR / FLK-1), flk-1R, flk-4 and fms-like tyrosine kinase 1 (flt-1).

[0009] Another member of the tyrosine kinase growth factor receptor family is the fibroblast growth factor ("FGF") receptor family. This group is composed of four receptors, FGFR1-4 and seven ligands, FGF1-7. Although not well defined, the receptor is interrupted by regions of the amino acid sequence where the glycosylated extracellular domain, transmembrane domain, and tyrosine kinase sequence contain a variable number of immunoglobin-like loops. It appears to be composed of intracellular domains.

[0010] A more complete list of known RTK subfamilies can be found in Plowman et.
al. , DN & P, 1994, 7 (6): 334-339 (herein incorporated by reference as though fully set forth herein, including the drawings).
It is described in.

In addition to RTKs, there is a completely intracellular family of PTKs called "non-receptor tyrosine kinases" or "cellular tyrosine kinases (" CTKs "). CTK does not contain extracellular and transmembrane domains. At present, 11 subfamilies (Src, Frk, Btk, Csk, Abl, Zap70, Fe
s, Fps, Fak, Jak, and Ack). The Src subfamily appears to be the largest group of CTK so far, with Src, Yes, Fyn, Lyn, Lck, Blk, Hc.
k, Fgr and Yrk. For a more detailed discussion of CTK, see Bole
n, Oncogene, 1993, 8: 2023-2031, which is hereby incorporated by reference, including the drawings, as if fully set forth herein.

[0012] Serin-threonine kinase or STK, like CTK, is mainly present in cells, but there are few STK-type receptor kinases. STK is the most common cytosolic kinase. That is, a kinase that performs its function in that part of the cytoplasm other than the cytoplasmic organelle and cytoskeleton. The cytosol is
A region in a cell where most of the cell's intermediate metabolic and biosynthetic activities occur. For example, it is in the cytosol that proteins are synthesized on ribosomes.

[0013] RTKs, CTKs and STKs have all been suggested to be hosts in pathogenic conditions, particularly those involving cancer. Other pathogenic conditions that have been associated with PTK include, but are not limited to, psoriasis, cirrhosis, diabetes, arteriosclerosis, angiogenesis, restenosis, ocular disease, rheumatoid arthritis and other inflammatory diseases, self Immune diseases such as immune diseases,
Includes cardiovascular diseases such as atherosclerosis, and various kidney diseases.

[0014] With regard to cancer, two of the major hypotheses that explain excessive cell proliferation driving tumor development relate to functions known to be controlled by PK. That is,
Malignant cell growth has been suggested to result from failure of mechanisms that regulate cell division and / or differentiation. Many protooncodin protein products have been shown to be involved in signaling pathways that control cell growth and differentiation. These protooncodin protein products include extracellular growth factor, transmembrane growth factor PTK receptor (RTK), cytoplasmic PTK (CTK) and cytosolic STK, discussed above.

[0015] Significant efforts have been made in an attempt to identify ways to modulate PK activity in terms of the apparent link between PK-associated cellular activity and a wide variety of human diseases. Is not surprising. Some of these include biomimetic methods using large molecules that mimic those involved in actual cellular processes (eg, mutant ligands (US Pat. No. 4,966,849); soluble receptors and antibodies (WO94). / 10202, Kendall and Thomas
s, Proc. Nat'l. Acad. Sci. , 1994, 90: 10705.
-09, Kim, et al. , Nature, 1993, 362: 841-8.
44); RNA ligands (Jelinek, et al., Biochemis).
try, 33: 10450-56); Takano, et al. , Mol. B
io. Cell, 1993, 4: 358A; Kinsella, et al. , E
xp. Cell Res. 1992, 199: 566-62; Wright, e.
t al. , J. et al. Cellular Phys. , 152: 448-57) and tyrosine kinase inhibitors (WO94 / 03427; WO92 / 21660; W
WO 91/14808; US Patent 5,330,992; M
ariani, et al. Proc. Am. Assoc. Cancer R
es. , 1994, 35: 2268).

[0016] Recently, attempts have been made to identify small molecules that act as PK inhibitors. For example, bis monocyclic, bicyclic and heterocyclic aryl compounds (PCT WO 92/206)
42), vinylene azaindole derivatives (PCT WO 94/14808) and 1-cyclopropyl-4-pyridylquinolones (US Pat. No. 5,330,992)
) Have been described as PTK inhibitors. Styryl compounds (US Pat. No. 5,211)
7,999), styryl-substituted pyridyl compounds (US Pat. No. 5,302,606),
Quinazoline derivatives (European Patent Application 0566266 A1), selenaindoles and selenides (PCT WO94 / 03427), tricyclic polyhydroxyl compounds (PCT WO92 / 21660) and benzylphosphonic acid compounds (PCT
WO 91/15495) are all described as PTK inhibitors useful in the treatment of cancer.

SUMMARY OF THE INVENTION The present invention relates, in part, to indolinone compounds and methods of using these compounds to modulate the function of protein kinases. The method of the invention incorporates a cell that expresses a protein kinase. Further, the present invention describes a method for preventing and treating an abnormal condition associated with a protein kinase in an organism using a compound identified by the methods described herein. Furthermore, the present invention relates to a pharmaceutical composition comprising a compound identified by the method of the present invention.

[0018] The invention features indolinone compounds and related products and methods that have the potential to inhibit protein kinases. Inhibitors of protein kinases can be obtained by adding chemical substituents to indolinone compounds. The compounds of the present invention are a new generation of treatment for diseases involving one or more functional or non-functional protein kinases. Neurodegenerative diseases and certain cancers fall within the scope of this disease. Other diseases or disorders include skin, eye, nerve, cardiovascular, and immune disorders, and disorders involving abnormal angiogenesis and / or angiogenesis. The compounds can be modified to be specific for their target, thus causing few side effects, and thus represent a new generation of potential cancer treatments. These properties are a significant improvement over the currently used harmful cancer treatments that cause many side effects and debilitate patients.

It is believed that the compounds of the present invention will minimize or eliminate solid tumors, or at least modulate or inhibit tumor growth and / or metastasis, by inhibiting the activity of protein kinases. Protein kinases, among other functions,
Controls the growth of blood vessels during angiogenesis. An increased rate of angiogenesis is associated with cancer tumor growth in cells, as cancer tumors must be nourished by oxidized blood during growth. Thus, inhibition of protein kinases and the corresponding reduction in angiogenesis will cause tumors to be nutrient-starved and possibly eliminate them.

The compound is PTK (eg, fibroblast growth factor receptor 1 [FGFR1])
Although a precise understanding of the mechanism of inhibiting PTK is not necessary for the practice of the present invention, it is believed that the compounds interact with amino acids in the catalytic domain of PTK. PTKs typically have a bilobal structure, and it appears that ATP binds to the cleft between the two leaves in a region where amino acids are conserved between the PTKs. P
Inhibitors of TK, in the same universal region where ATP binds to PTK,
It is believed that bonding occurs through non-covalent interactions such as hydrogen bonding, Van der Waals forces, and ionic interactions. More specifically, it is believed that the oxindole component of the compounds of the present invention binds to the same general space occupied by the adenine ring of ATP. The specificity of a PTK inhibitor for a particular PTK may be conferred by interactions between components around the oxindole core and amino acid domains specific to individual PKs. That is, different substituents will contribute to preferential binding to a particular PK. If it is possible to select compounds that are active against different ATP binding sites, such compounds can be targeted to any protein having such sites, such as protein tyrosine kinases as well as serine / threonine kinases. Would be useful. That is, such compounds have utility in in vitro assays of such proteins and in the therapeutic effects of such proteins in vivo.

Generally, indolinone compounds synthesized by the condensation of an oxindole compound with a ketone compound exhibit a mixture of possible E and Z isomers,
Isolation of the desired isomer is difficult. The compounds of the present invention are characterized by an intermolecular hydrogen bond between the carbonyl of the oxindole compound and the hydrogen at position 1 of the pyrrole component of the ketone compound. Such hydrogen bonding eliminates the problems associated with having a mixture of isomers by locking the intermediates in the synthesis of indolinone compounds into a preferred conformation.

I. The term definition "compound" denotes a compound or a pharmaceutically acceptable salt, ester, amide, prodrug, isomer, or metabolite.

The term “pharmaceutically acceptable salt” refers to a formulation of a compound that does not abrogate the biological activity and properties of the compound. Pharmaceutical salts are compounds of the present invention with inorganic or organic acids,
For example, it can be obtained by reacting with hydrochloric acid, oxalic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.

“Prodrug” refers to a drug that is converted into the parent drug in vivo. Prodrugs are often useful because, in some cases, they may be easier to administer than the parent drug. For example, a prodrug may be bioavailable by oral administration while the parent drug may not. Prodrugs may also have improved solubility in pharmaceutical compositions than the parent drug. Examples of prodrugs include, but are not limited to, being administered as esters ("prodrugs") to facilitate passage through cell membranes where water solubility is disadvantageous for migration, and The compounds of the present invention will advantageously be metabolically hydrolyzed to the active species carboxylic acid after entering the cell where it is advantageous. Yet another example of a prodrug would be a short polypeptide (polyamino acid) linked to an acid group. This peptide is metabolized to release the active molecule.

The term “indolinone” is used as the term is commonly understood in the art and includes a large subclass of substituted or unsubstituted compounds that can be synthesized from an aldehyde component and an oxindole component. .

The term “oxindole” refers to an oxindole compound substituted with a chemical substituent. Oxindole compounds have the following general structure:

Embedded image belongs to.

As used herein, a “fused pyrrolo” group refers to the structure in parentheses:

Embedded image

In the context of the present invention, the term “substituted” refers to oxindole compounds derivatized with any number of chemical substituents.

As used herein, the term “alkyl” refers to an aliphatic hydrocarbon group. The alkyl component can be a "saturated alkyl" group (ie, not containing an alkene or alkyne component). The alkyl component is also the "unsaturated alkyl" component (
At least one alkene or alkyne component). An "alkene" component refers to a group consisting of at least two carbon atoms and at least one carbon-carbon double bond, and an "alkyne" component refers to a group consisting of at least two carbon atoms and at least one carbon-carbon triple bond. Represents a group represented by The alkyl component is
It may be saturated or unsaturated, branched, unbranched, or cyclic.

Preferably, the alkyl group has from 1 to 20 carbon atoms (the numerical range, eg, “1-20”, whenever described herein, is each integer within the predetermined range For example, "1-20 carbon atoms" means that an alkyl group is formed from one carbon atom, two carbon atoms, three carbon atoms, and so on, up to 20 carbon atoms. Meaning that the term "alkyl" is also used without a numerical range.) More preferably, it is a medium sized alkyl having 1-10 carbon atoms. Most preferably, it is a lower alkyl having 1-4 carbon atoms. An alkyl group can be substituted or unsubstituted. When substituted, the substituents are preferably each independently cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto,
Alkylthio, arylthio, cyano, halo, carbonyl, thiocarbonyl, O
-Carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C
-Amides, N-amides, S-sulfonamides, N-sulfonamides, C-carboxy, O-carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, silyl and amino (including mono- and disubstituted amino groups). One or more groups.

The term “aromatic” refers to an aromatic group having at least one ring with a conjugated pi-electron system, including carbocyclic aryl (eg, phenyl) and heterocyclic aryl groups (eg, pyridine). Including both. This term is used for monocyclic or fused polycyclic (
That is, it includes a ring) group which shares a pair of adjacent carbon atoms. The term "carbocyclic" refers to compounds that contain one or more closed ring structures, wherein the atoms forming the ring skeleton are all carbon atoms. That is, the term distinguishes carbocyclic from heterocycles in which the ring skeleton contains at least one atom other than carbon. The term "heteroaromatic" means
Represents an aromatic group containing at least one heterocycle.

The term “aliphatic ring” refers to a compound that contains one or more closed ring structures, wherein at least one of the atoms forming the skeleton is a saturated carbon atom (eg, cyclohexane). The term "heteroaliphatic ring" refers to a ring system in which at least one of the atoms forming the backbone is a heteroatom (eg, tetrahydropyran).

The term “amine” refers to a chemical moiety of the formula —NR ₁ R ₂ , wherein R ₁ and R ₂ are independently hydrogen, saturated or unsaturated alkyl, and a 5-membered ring Or selected from the group consisting of 6-membered aryl or heteroaryl ring components, wherein the ring is
It may be optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, and ester components.

The term “imine” refers to a chemical moiety of the formula —N ＝ R ₁ , wherein R ₁ is hydrogen, a saturated or unsaturated alkyl, and an aryl or heteroaryl ring moiety (
Monocyclic or bicyclic), wherein the ring is one or more independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, and ester components. May optionally be substituted).

The terms “halogen” or “halo” represent an atom selected from the group consisting of fluorine, chlorine, bromine and iodine. The term "trihalomethyl" refers to the ₃ group -CX (wherein, X is a halogen).

The term “carboxylic acid” refers to a chemical moiety having the formula — (R) _n —COOH (
Wherein R is selected from the group consisting of alkyl, cycloalkyl, aryl, heteroaryl (attached via a ring carbon) and heteroalicyclic (attached via a ring carbon), these terms being used herein. And n is 0 or 1
Is).

The term “ester” refers to a chemical moiety having the formula — (R) _n —COOR ′ (
Wherein R and R ′ are independently alkyl, cycloalkyl, aryl, heteroaryl (attached via a ring carbon) and heteroalicyclic (attached via a ring carbon)
And the terms are as defined herein, and n is 0 or 1.). As used herein, the term "carboxyalkyl" is also included in this definition.

The term “aldehyde” refers to a chemical moiety having the formula — (R) _n —CHO, where R is as defined herein and n is 0 or 1. .

The term “sulfone” refers to a chemical moiety having the formula —SO ₂ —R, where R is as defined herein.

The term “acyl” refers to a chemical moiety of the general formula —C (O) R. When R is hydrogen, the molecule containing an acyl group is an aldehyde. R represents an alkyl, cycloalkyl, aryl, heteroaryl (attached via a ring carbon) and heteroalicyclic (attached via a ring carbon) (the terms are as defined herein) When selected from the group, the molecule containing an acyl group is a ketone.

A “thiocarbonyl” group refers to a —C (＝ S) —R group, where R is as defined herein.

An “O-carboxy” group refers to a RC (＝ O) O— group, where R is as defined herein.

A “C-carboxy” group refers to a —C (＝ O) OR group, where R is as defined herein.

An “acetyl” group refers to a —C (＝ O) CH ₃ group.

A “trihalomethanesulfonyl” group refers to an X ₃ CS (＝ O) ₂ — group, where X is a halogen.

A “cyano” group refers to a —C≡N 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, where R is as defined herein.

An “S-sulfonamido” group refers to a —S (＝ O) ₂ NR ₂ group, where R is as defined herein.

A “N-sulfonamido” group refers to a RS (＝ O) ₂ NH— group, where R is as defined herein.

A “trihalomethanesulfonamido” group refers to an X ₃ CS (＝ O) ₂ NR— group, where X and R are as defined herein.

An “O-carbamyl” group refers to a —OC (＝ O) —NR ₂ group, where R is as defined herein.

An “N-carbamyl” group refers to a ROC (＝ O) NH— group, where R is as defined herein.

An “O-thiocarbamyl” group refers to a —OC (＝ S) —NR ₂ group, where R is as defined herein.

An “N-thiocarbamyl” group refers to a ROC (＝ S) NH— group, where R is as defined herein.

A “C-amido” group refers to a —C (＝ O) —NR ₂ group, where R is as defined herein.

An “N-amido” group refers to a RC (＝ O) NH— group, where R is as defined herein.

As used herein, “together” with respect to two adjacent “R” groups indicates that the two “R” groups are covalently linked together to form a ring system. The ring system is
It can be cycloalkyl, aryl, heteroaryl or heteroalicyclic.

A “combinatorial library” refers to all compounds formed by reacting each compound in one dimension with each compound in another dimension in a multidimensional array of compounds. In the context of the present invention, the array is two-dimensional, one dimension is all oxindoles of the invention and the other dimension is all aldehydes of the invention. Each oxindole can be reacted with each aldehyde to form an indolinone compound. All indolinone compounds thus formed are within the scope of the present invention. Also, smaller combinatorials formed by reacting some of the oxindoles of the present invention with all of the aldehydes of the present invention, or all of the oxindoles and some of the aldehydes, or some of the oxindoles and some of the aldehydes. Libraries are also within the scope of the present invention.

The term “pharmaceutical composition” refers to a mixture of a compound of the present invention with other chemical components, such as a diluent, excipient, or carrier. The pharmaceutical composition facilitates administration of the compound to an organism. There are many techniques for administering compounds in the art, including, but not limited to, oral, aerosol, parenteral, and topical.
Pharmaceutical compositions comprise a compound comprising an inorganic acid such as hydrochloric acid, oxalic acid, sulfuric acid, nitric acid, phosphoric acid,
It can also be obtained by reacting with methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, or the like.

The term “physiologically acceptable” defines a carrier or diluent that does not abrogate the biological activity and properties of the compound.

The term “carrier” defines a chemical compound that facilitates the incorporation of a compound into cells or tissues. For example, dimethyl sulfoxide (DMSO) is a commonly used carrier to facilitate the incorporation of many organic compounds into cells or tissues of organisms.

The term “diluent” defines a chemical compound diluted in water that dissolves the compound of interest and stabilizes the biologically active form of the compound. In the art, salts dissolved in buffered solutions are used as diluents. One commonly used buffered solution is phosphate buffered saline. this is,
This is to mimic the salt conditions of human blood. Since buffered salts can adjust the pH of a solution at low concentrations, buffered diluents do not significantly alter the biological activity of the compound.

The term “pharmaceutical composition” refers to a mixture of a compound of the present invention with other chemical ingredients, for example, diluents, excipients or carriers. The pharmaceutical composition facilitates administration of the compound to an organism. There are many techniques for administering compounds in the art, including, but not limited to, oral, aerosol, parenteral, and topical. Pharmaceutical compositions can also be obtained by reacting the compound with an inorganic acid such as hydrochloric acid, oxalic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like.

The term “physiologically acceptable” defines a carrier or diluent that does not abrogate the biological activity and properties of the compound.

The term “function” describes the role of protein kinases in cells. The family of protein kinases includes members that control many stages of the signaling cascade, including those that control cell growth, migration, differentiation, gene expression, muscle architecture, glucose metabolism, cellular protein synthesis, and cell cycle regulation. included.

[0069] In the context of the present invention, the term "catalytic activity" defines the rate at which a protein kinase phosphorylates a substrate. Catalytic activity can be measured, for example, by determining the amount of substrate that is converted to a product as a function of time. Substrate phosphorylation occurs at the active site of the protein kinase. The active site is usually the cavity where the substrate binds and phosphorylates the protein kinase.

[0070] As used herein, the term "substrate" refers to a molecule that is phosphorylated by a protein kinase. The substrate is preferably a peptide, more preferably a protein.

The term “activate” refers to increasing the cellular function of a protein kinase. The protein kinase function is preferably an interaction with a natural binding partner, most preferably catalytically active.

The term “inhibits” refers to reducing the cellular function of a protein kinase. The protein kinase function is preferably an interaction with a natural binding partner, most preferably catalytically active.

The term “modulates” refers to altering the function of a protein kinase by increasing or decreasing the probability that a complex will form between the protein kinase and the natural binding partner. The modulator preferably increases the probability of forming such a complex between the protein kinase and the natural binding partner, and more preferably, depending on the concentration of the compound exposed to the protein kinase, Increase or decrease the probability that a complex will form between the protein kinase and the natural binding partner,
Most preferably, the probability of forming a complex between the protein kinase and the natural binding partner is reduced. The modulator preferably activates the catalytic activity of the protein kinase, more preferably activates or inhibits the catalytic activity of the protein kinase, or most preferably, depends on the concentration of the compound exposed to the protein kinase. ,
Inhibits the catalytic activity of protein kinases.

The term “complex” refers to a combination of at least two molecules that bind to each other. Signaling complexes often include at least two protein molecules that bind to each other.

The term “natural binding partner” refers to a polypeptide that binds to a protein kinase in a cell. Natural binding partners can play a role in transmitting signals in the protein kinase signaling process. Altered interaction between the protein kinase and the natural binding partner manifests as an increased or decreased likelihood of an interaction being formed, or an increased or decreased concentration of the protein kinase / natural binding partner complex be able to.

As used herein, the term “contacting” refers to mixing a solution containing a compound of the present invention with a liquid medium in which the cells of the method are immersed. The solution containing the compound may also include other components that facilitate the uptake of the compound into cells by this method,
For example, it may contain dimethyl sulfoxide (DMSO). The solution containing the compound of the present invention can be added to the medium in which the cells are immersed by using a transport device, such as a pipette-based device or a syringe-based device.

The term “monitor” refers to observing the effect of adding a compound to cells in this manner. The effect can be manifested as a change in cell phenotype, cell proliferation, protein kinase catalytic activity, or the interaction between the protein kinase and the natural binding partner.

The term “effect” describes a change or no change in cell phenotype or proliferation. "Effect" can also describe a change or no change in the catalytic activity of a protein kinase.
"Effect" can also describe a change or no change in the interaction between a protein kinase and a natural binding partner.

The term “cell phenotype” refers to the appearance of a cell or tissue, or the function of a cell or tissue. Examples of cell phenotypes are cell size (shrinkage or expansion), cell proliferation (increase or decrease in cell number), cell differentiation (change or no change in cell shape), cell survival, apoptosis (cell death) Use of metabolic nutrients (eg glucose uptake)
It is. A change or no change in the cell phenotype is readily measured by techniques known in the art.

The term “antibody” refers to an antibody (eg, a monoclonal or polyclonal antibody) having specific binding affinity for a protein kinase or fragment thereof, or an antibody fragment.

“Specific binding affinity” means that an antibody binds to a target (protein kinase) polypeptide with a higher affinity under certain conditions than to another polypeptide. Antibodies with specific binding affinity for protein kinases can be obtained by contacting a sample with the antibody under conditions that allow the formation of immune complexes and detecting the presence and / or amount of antibody bound to the protein kinase. , Can be used in a method for detecting the presence and / or amount of a protein kinase in a sample. A diagnostic kit for performing such a method comprises a first container containing the antibody,
And a second container having a conjugate of a binding partner of the antibody and a label, eg, a radioisotope. The diagnostic kit is also available from FD
A may include a notice of the use approved by A and its guidelines.

The term “polyclonal” refers to an antibody that is a heterogeneous population of antibody molecules derived from the serum of an animal immunized with an antigen or an antigenic functional derivative thereof. For the production of polyclonal antibodies, various host animals can be immunized by injecting the antigen. Depending on the host species, various adjuvants can be used to increase the immunological response.

A “monoclonal antibody” is a substantially homogeneous population of antibodies to a particular antigen. Monoclonal antibodies can be obtained by any technique which provides for the production of antibody molecules by continuous cell line cultures. Monoclonal antibodies can be obtained by methods known to those skilled in the art. See, for example, Kohler et al. Natur
e 256: 495-497 (1975), and U.S. Pat. 4,376,11
See 0.

The term “antibody fragment” refers to that portion of an antibody that displays specific binding affinity for a particular molecule, often the hypervariable region and portions of the surrounding heavy and light chains. The hypervariable region is the part of the antibody that physically binds to the polypeptide target.

[0085] With respect to the signaling process, the term "aberrant" refers to being over- or under-expressed in an organism, mutating its catalytic activity to be lower or higher than wild-type protein kinase activity, Represents a protein kinase that is mutated so that it can no longer interact with its binding partner, can no longer be modified by another protein kinase or protein phosphatase, or can no longer bind its natural binding partner .

The term “promoting or destroying an abnormal interaction” refers to a method that can be accomplished by administering a compound of the present invention to a cell or tissue of an organism. Compounds form desirable interactions with a large number of atoms at the complex interface,
It can facilitate the interaction between the protein kinase and the natural binding partner. Alternatively, the compound can inhibit the interaction between the protein kinase and the natural binding partner by interfering with the desired interaction formed between atoms at the complex interface.

“In vitro” refers to a method performed in an artificial environment, such as, but not limited to, a test tube, cell, or culture. As used herein, "in vivo" refers to a method performed on a living organism, such as, but not limited to, a mouse, rat or rabbit.

II. The compounds of the present invention A. 3-Arylidenyl-6-heterocyclyl-2-indolinone derivatives In one aspect, the invention provides compounds of formula I

Embedded image [Wherein, n and m are independently 0 or 1].
It relates to an indolinone derivative or a salt or prodrug thereof.

When n is 1, A, B, D, E and F are independently selected from the group consisting of carbon and nitrogen; provided that no more than three of A, B, D, E and F Are simultaneously nitrogen and A, B, D, E, or F is nitrogen, R ₄ , R ₅ , R ₆
, R ₇ or R ₈ are not present respectively.

When m is 1, G, H, J, K and L are independently selected from the group consisting of carbon and nitrogen; provided that at least one of G, H, J, K and L And when no more than three are nitrogen and G, H, J, K or L is nitrogen, then R ₉ , R ₁₀ , R ₁₁ , R ₁₂ or R ₁₃ are absent, respectively.

When n is 0, A is selected from the group consisting of carbon and nitrogen, and B and F are selected from the group consisting of carbon, nitrogen, NH, oxygen and sulfur, where
When B or F is NH, the other cannot be NH, and E is a carbon,
Selected from the group consisting of nitrogen, oxygen and sulfur, provided that one of B, E or F
Not more than oxygen or sulfur and at least one of A, B, E or F is a heteroatom (ie not carbon).

When m is 0, G is selected from the group consisting of carbon and nitrogen, and H, K and L are selected from the group consisting of carbon, nitrogen, NH, oxygen and sulfur, provided that H or When L is NH, the other cannot be NH, and K is selected from the group consisting of carbon, nitrogen, oxygen and sulfur, provided that H, K or L
Not more than one is oxygen or sulfur and at least one of G, H, K or L is a heteroatom (ie not carbon).

R₁, R_Two, R_Three, R_Four, R_Five, R₆, R₇, R₈, R₉, R_Ten, R₁₁, R₁₂And R_{1 Three} Is independently hydrogen, alkyl, trihaloalkyl, cycloalkyl, alk
Nil, alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxy, a
Lucoxy, mercapto, alkylthio, aryloxy, arylthio, sulf
Ynyl, sulfonyl, S-sulfonamide, N-sulfonamide, carbonyl,
C-carboxy, O-carboxy, carboxyalkyl, cyano, nitro, halo
, O-carbamyl, N-carbamyl, C-amide, N-amide and -NR₁₄R _Fifteen Selected from the group consisting of:

R ₄ and R ₅ or R ₅ and R ₆ or R ₆ and R ₇ or R ₇ and R ₈ are
Together, they may form a 5- or 6-membered aryl or heteroaryl ring.

R ₁₄ and R ₁₅ are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, carbonyl, sulfonyl, or taken together to form a 5-membered It may form a ring or a 6-membered heteroalicyclic ring.

A currently preferred embodiment of the present invention is a compound of formula I wherein R₁, R_Twoand
R_ThreeIs independently optionally substituted with (i) hydrogen, (ii) one or more halo groups.
Optionally substituted lower alkyl, (iii) optionally with one or more halo groups
Optionally substituted with lower alkoxy, (iv) (lower alkyl) -S-sul
Honamide, (v) aryl-S-sulfonamide, (vi) halo, and (v)
ii) -NR₁₄R_FifteenAnd R is selected from the group consisting of:_Four, R_Five, R₆, R₇, R₈, R₉, R_Ten, R₁₁, R₁₂, And R₁₃Is, independently,
(I) hydrogen; (ii) aryl, heteroaryl, heteroalicyclic, halo, hydro
Xy, lower alkoxy, mercapto, lower alkylthio, C-carboxy and
-NR₁₄R_FifteenOptionally substituted with one or more groups selected from the group consisting of
(Iii) cycloalkyl; (iv) hydroxy;
(V) one or more halo groups, aryl, heteroaryl and heteroalicyclic
Optionally substituted with one or more groups selected from the group consisting of the formulas
(Vi) halo; (vii) carboxyalkyl; (viii
A) lower alkyl, halo optionally substituted with one or more halo groups;
, Hydroxy, lower radicals optionally substituted with one or more halo groups.
Lucoxy, aryloxy and -NR₁₄R_FifteenOne or more selected from the group consisting of
Is aryl optionally substituted with a further group; (ix) 1 or
Lower alkyl, halo, hydroxy, optionally substituted with the above halo groups
Lower alkoxy, ant optionally substituted with one or more halo groups
Oxy and -NR₁₄R_FifteenOne or more groups selected from the group consisting of
Aryloxy optionally substituted with: (x) one or more halo
Lower alkyl, halo, hydroxy, 1 or its optionally substituted with
Lower alkoxy and -NR optionally substituted with one or more halo groups₁₄R _Fifteen Optionally substituted with one or more groups selected from the group consisting of
(Xi) heteroalicyclic; (xii) (lower alkyl) -C-
(Xiii) (lower alkyl) carbonyl; (xiv) aryl
(Xvi) (lower alkyl) -S-sulfonamide; (xvi) aryl
-S-sulfonamide; (xvii) (lower alkyl) -N-sulfonamide
(Xviii) aryl-N-sulfonamide; (xix) (lower alkyl)
-N-carbamoyl; (xx) (lower alkyl) -C-amide; (xxi) (low
(Xxii) (cycloalkyl) -N-amide;
And (xxiii) -NR₁₄R_FifteenAnd R is selected from the group consisting of:₁₄And R_FifteenIs independently selected from the group consisting of hydrogen and lower alkyl
You.

Another currently preferred embodiment of the present invention is that n is 1, A, B, D, E and F are carbon, m is 1 and 1 of G, H, J, K and L Formula I wherein one is nitrogen
Is a compound of

In a compound of formula I, n is 1, A, B, D, E and F are carbon, m is 0, G, H and K are carbon, and L is NH, Oxygen or sulfur is also a presently preferred embodiment of the present invention.

[0099] Yet another preferred embodiment of the present invention are the compounds of formula I, wherein, n be 0; A, B and E is carbon; F is an NH; R ₄ and R ₇ is independently selected from the group consisting of hydrogen and lower alkyl; R ₅ is (i) hydroxy, a heteroaromatic containing an NH group in the ring, a heteroaliphatic containing at least one NH group in the ring. cyclic, C-carboxy, and, -NR ₁₄ 1 or more optional substituted lower alkyl groups selected from the group consisting of R _15; (ii) carboxyalkyl; (iii) C- Carboxy; and (iv) —NR ₁₄ R ₁₅ (
Wherein R ₁₄ and R ₁₅ are independently selected from the group consisting of hydrogen, lower alkyl and carbonyl); m is 1;
One of H, I, J, K or L is nitrogen.

Similarly, a presently preferred embodiment of the invention relates to compounds of formula I wherein n is 0; A, B and E are carbon; F is NH; and R ₄ and R ₇ are independently do it,
R ₅ is selected from the group consisting of hydrogen and lower alkyl;
Heteroaromatic containing NH in the ring, heteroalicyclic containing at least one NH in the ring, C-carboxy, and, one or more groups selected from the group consisting of -NR ₁₄ R ₁₅ optionally substituted lower alkyl in; (ii) carboxyalkyl; (iii) C-carboxy; and (iv) -NR ₁₄ R ₁₅ (
Wherein R ₁₄ and R ₁₅ are independently selected from the group consisting of hydrogen, lower alkyl and carbonyl); m is 0; G, H and K are carbon And L is NH, oxygen or sulfur.

B. 3-aralkyl-2-indolinone derivatives In another aspect, the present invention provides compounds of formula II:

Embedded image Wherein n is 0 or 1. The present invention relates to a 3-aralkyl-2-indolinone derivative having a chemical structure according to the formula: or a physiologically acceptable salt or prodrug thereof.

When p is 1, M, Q, T, U and V are independently selected from the group consisting of carbon and nitrogen. When M, Q, T, U, or V is nitrogen, R ₂₀ ,
It is understood that R ₂₁ , R ₂₂ , R ₂₃ or R ₂₄ are each absent.

When p is 0, M, Q, U and V are independently carbon, nitrogen, oxygen and
And sulfur. M, Q, U or V is oxygen or ion
C or nitrogen (where the nitrogen is involved in a double bond);₂₀, R _{twenty one} , R_{twenty two}, R_{twenty three}, Or R_{twenty four}It is understood that each does not exist.

R ₁₆ , R ₁₇ , R ₁₈ , R ₁₉ , R ₂₀ , R ₂₁ , R ₂₂ , R ₂₃ or R ₂₄ are independently
Hydrogen, alkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, mercapto, alkylthio, aryloxy, sulfinyl, sulfonyl, S-sulfonamide, N-sulfonamide, carbonyl , C-carboxy, O- carboxy, carboxyalkyl, cyano, nitro, halo, O- carbamyl, N- carbamyl, C-amido, is selected from the group consisting of N- amides and -NR ₂₅ R _26.

R ₂₀ and R ₂₁ or R ₂₁ and R ₂₂ or R ₂₂ and R ₂₃ or R ₂₃ and R ₂₄ together form a 5- or 6-membered aryl or heteroaryl ring Is also good.

R ₂₅ and R ₂₆ are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, carbonyl, sulfonyl, or together form a 5-membered ring or Form a 6-membered heteroalicyclic ring.

[0107] presently preferred embodiment of the present _{invention, R 16, R 17, R} 18, R 19, R 20, R 21, R 22,
R ₂₃ and R ₂₄ are independently (i) hydrogen; (ii) cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, C-carboxy and-
Lower alkyl optionally substituted with one or more groups selected from the group consisting of NR ₂₅ R ₂₆ ; (iii) cycloalkyl; (iv) hydroxy;
v) lower alkoxy optionally substituted with one or more groups selected from the group consisting of one or more halo groups, aryl, heteroaryl and heteroalicyclic; (vi) trihalomethyl; (Vii) trihalomethoxy; (v
iii) halo; (ix) carboxyalkyl; (x) lower alkyl, one or more halo groups, trihalomethyl, trihalomethoxy, halo, hydroxy, lower alkoxy, substituted with aryloxy and -NR ₂₅ <R ₂₆ Aryl optionally substituted with one or more groups selected from the group consisting of lower alkyl, (xi) aryloxy, lower alkyl, trihalomethyl, halo,
Hydroxy, (lower alkyl) alkoxy, amino and -NR ₂₅ 1 or more optional substituted heteroaryl groups selected from the group consisting of R _26; (xii) heterocycloaliphatic; (xiii ) (Lower alkyl) carboxy;
(Xiv) (lower alkyl) carbonyl; (xv) arylcarbonyl; (xv
(xviii) aryl-S-sulfonamide; (xviii) (lower alkyl) -N-sulfonamide; (xix)
) Aryl-N-sulfonamide; (xx) (lower alkyl) -N-carbamoyl; (xxi) (lower alkyl) -C-amide; (xxii) (lower alkyl)
(Xxiii) (cycloalkyl) -N-amide; and (xx
iv) —NR ₂₅ R ₂₆ ;

Another currently preferred embodiment of the present invention is that p is 1 and M, Q, T, U, and V
Is a compound wherein is carbon.

In still another preferred embodiment of the present invention, when p is 1, M, Q, T
, U, and V are carbon; R ₁₆ , R ₁₇ , R ₁₈ , and R ₁₉ are independently (
(ii) halo; (iii) hydroxy; (iv) —NR ₂₅ R ₂₆ ;
) S-sulfonamido optionally substituted with one or more groups selected from the group consisting of hydrogen, lower alkyl and aryl; (vi) hydroxy, one or more halo groups, C-carboxy , C-amido, heteroalicyclic, and -NR ₂₅ optionally substituted lower alkyl radical selected from the group consisting of R _26; optionally in (vii) 1 or more halo groups substituted which may be lower alkoxy; (viii) lower alkyl, lower alkoxy, halo, optionally optionally substituted with one or more groups selected from the group consisting of hydroxy and -NR ₂₅ R ₂₆ aryl; (Ix) optionally substituted with one or more groups selected from the group consisting of hydrogen, lower alkyl, cycloalkyl and aryl - amide; is selected from the group consisting of; and _{R 20, R 21, R 22} , R 23 and R ₂₄ are independently, (i) hydrogen; (ii) halo;
(Iii) hydroxy; (iv) —NR ₂₅ R ₂₆ ; (v) hydroxy, one or more halo groups, C-carboxy, C-amide, heteroalicyclic, and —NR ₂₅ R ₂₆ (Vi) cycloalkyl; (vii) one or more halo groups, aryl, -NR ₂₅ R ₂₆ , and heteroaryl. Lower alkoxy optionally substituted with one or more groups selected from the group consisting of:
ii) an aryl optionally substituted with one or more groups selected from the group consisting of halo, hydroxy, lower alkoxy, -NR ₂₅ R ₂₆ , and C-carboxy; .

It is yet another currently preferred embodiment of the present invention that p is 1 and one or two of M, Q, T, U, or V is nitrogen.

Also presently preferred are those in which p is 1 and one or 2 of Q, T, U and V
When one is nitrogen, R₁₆, R₁₇, R₁₈And R₁₉Is independently (i) hydrogen;
(Ii) halo; (iii) hydroxy; (iv) -NR_{twenty five}R₂₆(V) hydrogen, low
One or more groups selected from the group consisting of primary alkyl and aryl.
Optionally substituted S-sulfonamide; (vi) hydroxy, 1 or
Further halo groups, C-carboxy, C-amide, heteroalicyclic, and -NR _{twenty five} R₂₆Lower alkyl optionally substituted with a group selected from the group consisting of
(Vii) lower alkyl optionally substituted with one or more halo groups;
Coxy; (viii) lower alkyl, lower alkoxy, halo, hydroxy and
-NR_{twenty five}R₂₆Optionally substituted with one or more groups selected from the group consisting of
(Ix) hydrogen, lower alkyl, cycloalkyl and a
Optionally substituted with one or more groups selected from the group consisting of
Selected from the group consisting of N-amides;₂₀, R_{twenty one}, R_{twenty two}, R_{twenty three}And R_{twenty four}Any non-nitrogen group of is independently (
(ii) halo; (in) hydroxy; (iv) -NR_{twenty five}R₂₆(V)
Hydroxy, one or more halo groups, C-carboxy, C-amide, hetero
Alicyclic, and -NR_{twenty five}R₂₆One or more groups selected from the group consisting of
(Vi) cycloalkyl; (vii) optionally substituted lower alkyl;
) One or more halo groups, aryl, -NR_{twenty five}R₂₆And heteroaryl
Low optionally substituted with one or more groups selected from the group consisting of
Lower alkoxy; and (viii) halo, hydroxy, lower alkoxy, -NR _{twenty five} R₂₆, And one or more groups selected from the group consisting of C-carboxy
And optionally substituted aryl.

It is a currently preferred embodiment of the present invention that p is 1 and R ₂₁ and R ₂₂ together form a fused pyrrolo group.

In yet another preferred embodiment of the present invention, R ₁₆ , R ₁₇ , R ₁₈ and R ₁₉ are independently: (i) hydrogen; (ii) halo; (iii) hydroxy; N
R ₂₅ R ₂₆ ; (v) S-sulfonamide optionally substituted with one or more groups selected from the group consisting of hydrogen, lower alkyl and aryl;
i) hydroxy, 1 or more halo groups, C-carboxy, C-amido, heterocycloaliphatic and, -NR ₂₅ R ₂₆ optionally optionally substituted lower alkyl radical selected from the group consisting of ; (vii) one or more halo optionally substituted in the lower alkoxy group; (viii) lower alkyl, lower alkoxy, halo, 1 or is selected from the group consisting of hydroxy and -NR ₂₅ R ₂₆ Aryl optionally substituted with further groups; (ix) N optionally substituted with one or more groups selected from the group consisting of hydrogen, lower alkyl, cycloalkyl and aryl Amide; selected from the group consisting of:

Similarly, R₂₀, R_{twenty three}And R_{twenty four}Is independently (i) hydrogen; (ii) halo;
iii) hydroxy; (iv) -NR_{twenty five}R₂₆(V) hydroxy, 1 or it;
The above halo group, C-carboxy, C-amide, heteroalicyclic and -NR_{twenty five}R ₂₆ Optionally substituted with one or more groups selected from the group consisting of
(Vi) cycloalkyl; (vii) one or more halo
Group, aryl, -NR_{twenty five}R₂₆And 1 selected from the group consisting of
Or lower alkoxy optionally substituted with more groups; or (viii
) Halo, hydroxy, lower alkoxy, -NR_{twenty five}R₂₆And C-carboxy
A group optionally substituted with one or more groups selected from the group consisting of
Preferably, the reel is selected from the group consisting of:

In another preferred embodiment, p is 0, M or Q is —NH, oxygen or sulfur; and either M or Q is not —NH, oxygen or sulfur, and U and V is carbon.

In another preferred embodiment, R ₁₆ , R ₁₇ , R ₁₈ and R ₁₉ are independently: (i) hydrogen; (ii) halo; (iii) hydroxy; (iv) —NR ₂₅ R ₂₆ (V) S-sulfonamide optionally substituted with one or more groups selected from the group consisting of hydrogen, lower alkyl and aryl; (vi) hydroxy, one or more halo groups, C-carboxy, C-amide, heteroalicyclic and-
Lower alkyl optionally substituted with a group selected from the group consisting of NR ₂₅ R ₂₆ ; (vii) lower alkoxy optionally substituted with one or more halo groups; (viii) lower (Ix) hydrogen, lower alkyl, cycloalkyl and aryl optionally substituted with one or more groups selected from the group consisting of alkyl, lower alkoxy, halo, hydroxy and -NR ₂₅ R ₂₆ And N-amide optionally substituted with one or more groups selected from the group consisting of:

In these preferred compounds, R ₂₀ , R ₂₁ , R ₂₃ and R ₂₄ are independently (i) hydrogen; (ii) halo; (iii) hydroxy; (iv) —NR ₂₅ R ₂₆ ; (v) hydroxy, 1 or more halo groups, C- carboxy, substituted C- amide, heterocycloaliphatic and, optionally with one or more groups selected from the group consisting of -NR ₂₅ R ₂₆ (Vi) cycloalkyl;
(Vii) lower alkoxy optionally substituted with one or more groups selected from the group consisting of one or more halo groups, aryl, -NR ₂₅ R ₂₆ , and heteroaryl; Selected from group.

In yet another preferred embodiment, R ₁₆ , R ₁₇ , R ₁₈ and R ₁₉ are independently: (i) hydrogen; (ii) halo; (iii) hydroxy; (iv) —NR ₂₅ R ₂₆ ; (v) S-sulfonamide optionally substituted with one or more groups selected from the group consisting of hydrogen, lower alkyl and aryl; (vi) hydroxy, one or more halo groups , C-carboxy, C-amido, heteroalicyclic and -NR ₂₅ optionally substituted lower alkyl radical selected from the group consisting of R _26; in (vii) 1 or more halo groups any substituted in lower alkoxy; (viii) lower alkyl, optionally lower alkoxy, halo, one or more groups selected from the group consisting of hydroxy and -NR ₂₅ R ₂₆ Selected from the group consisting of: (ix) an N-amide optionally substituted with one or more groups selected from hydrogen, lower alkyl, cycloalkyl and aryl. You.

Preferably, R₂₀, R_{twenty three}And R_{twenty four}Is independently (i) hydrogen; (ii) halo
(Iii) hydroxy; (iv) -NR_{twenty five}R₂₆(V) hydroxy, 1 or
Further halo groups, C-carboxy, C-amide, heteroalicyclic and -NR _{twenty five} R₂₆Optionally substituted with one or more groups selected from the group consisting of
(Vi) cycloalkyl; (vii) one or more
Halo group, aryl, -NR_{twenty five}R₂₆And selected from the group consisting of heteroaryl
A lower alkoxy optionally substituted with one or more groups such as
iii) halo, hydroxy, lower alkoxy, -NR_{twenty five}R₂₆And C-Carbo
Optionally substituted with one or more groups selected from the group consisting of
A good aryl;

C. Diarylindolinone Compounds In another aspect, the present invention provides compounds having the structure described in Formula III:

Embedded imageWherein (a) is (i) halogen, trihalomethyl, alkoxy, carboxylate, amino,
Toro, esters and 5- or 6-membered aromatic, heteroaromatic, aliphatic,
Or optionally substituted with a substituent selected from the group consisting of heteroaliphatic ring components.
A saturated or unsaturated alkyl, wherein the ring component is alkyl, halogen, trihalomethyl, carboxylate,
1,2 independently selected from the group consisting of amino, nitro, and ester components
Or optionally substituted with three substituents; and (ii) alkyl, alkoxy, halogen, trihalomethyl, carboxylate
1, independently selected from the group consisting of, amino, nitro, and ester components
Aromatic or heteroaromatic optionally substituted with 2 or 3 substituents
A ring; (iii) alkyl, alkoxy, halogen, trihalomethyl, carboxylate
One or more independently selected from the group consisting of
An aliphatic or heteroaliphatic ring optionally substituted with the above substituents, and
And alkyl, alkoxy, halogen, trihalomethyl, carboxylate, amine
One or more independently selected from the group consisting of
An aromatic or heteroaromatic ring optionally substituted with more than one substituent; selected from the group consisting of: (b) R₃₂, R₃₃And R₃₄Are each independently: (i) hydrogen; (ii) halogen, trihalomethyl, carboxylate, amino, nitro, es
Ter and 5- or 6-membered aromatic, heteroaromatic, aliphatic, or heterocyclic
Optionally substituted with one or more substituents selected from the group consisting of aliphatic ring components
Optionally substituted saturated or unsaturated alkyl, wherein the ring component is alkyl, halogen, trihalomethyl, carboxylate,
1 or independently selected from the group consisting of mino, nitro, and ester components
(Iii) alkyl, alkoxy, halogen, trihalomethyl, carboxylate
1, independently selected from the group consisting of amino, nitro, and ester components
Or an aromatic or heteroaromatic optionally substituted with more substituents
(Iv) alkyl, alkoxy, halogen, trihalomethyl, carboxylate
One or more independently selected from the group consisting of, amino, nitro, and ester
An aliphatic or heteroaliphatic ring optionally substituted with the above substituents, and
Alkyl, alkoxy, halogen, trihalomethyl, carboxylate, amino
One or more independently selected from the group consisting of, nitro, and ester components
An aromatic or heteroaromatic ring optionally substituted with the above substituents; (v) a formula-(X₁)_n1-NX_TwoX_ThreeAn amine of the formula (wherein X₁Is a saturated or unsaturated
Alkyl, and 5- or 6-membered aromatic, heteroaromatic, or aliphatic ring
Selected from the group consisting of₁Is 0 or 1, and X_TwoAnd X_ThreeIs independent
Hydrogen, saturated or unsaturated alkyl, and 5- or 6-membered aromatic,
(Vi) selected from the group consisting of heteroaromatic or aliphatic ring components);_Two(Vii) halogen or trihalomethyl; (viii) a formula-(X_Four)_n4-CO-X_FiveKetone (wherein, X_FourAnd X_FiveIs independent
Alkyl and 5- or 6-membered aromatic, heteroaromatic, aliphatic,
Or a heteroaliphatic ring component, wherein the alkyl and ring components are
Alkyl, halogen, trihalomethyl, carboxylate, amino, nitro, and
And 1, 2 or 3 substituents independently selected from the group consisting of
May be optionally substituted, n_FourIs 0 or 1); (ix) Formula-(X₆)_n6A carboxylic acid of -COOH or a formula-(X₇)_n7-COO-
X₈An ester of the formula (wherein X₆, X₇And X₈Is independently alkyl and 5-membered ring
Or from a six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring component
Selected from the group₆And n₇Is independently 0 or 1); (x) Formula-(X₉)_n9-OH alcohol or formula-(X_Ten)_n10-OX₁₁No
Alkoxyalkyl component (wherein, X₉, X_TenAnd X₁₁Are independently saturated or
Unsaturated alkyl and 5- or 6-membered aromatic, heteroaromatic and aliphatic
Or a heteroaliphatic ring component, wherein the alkyl and the ring component are
, Alkyl, halogen, trihalomethyl, carboxylate, amino, nitro,
And one or more substituents independently selected from the group consisting of
Optionally substituted and n₉And n_TenIs independently 0 or 1
(Xi) Formula-(X₁₂)_n12-NHCOX₁₃Or the formula-(X₁₄)_n14-CONX_FifteenX ₁₆ An amide of the formula₁₂And X₁₄Are each independently alkyl, hydroxy,
Syl, and 5- or 6-membered aromatic, heteroaromatic, aliphatic, or heterocyclic
Selected from the group consisting of alicyclic ring components, wherein the ring is alkyl, halogen, trihalo
From the group consisting of methyl, carboxylate, amino, nitro, and ester
May be optionally substituted with one or more substituents selected from among
And n₁₂And n₁₄Is independently 0 or 1; X₁₃, X_FifteenAnd X₁₆Is
, Each independently hydrogen, alkyl, hydroxyl, and a 5- or 6-membered ring
From the group consisting of aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring components of the ring
And the ring is selected from alkyl, halogen, trihalomethyl, carboxylate,
One or more independently selected from the group consisting of mino, nitro, and ester
(Optionally substituted with the above substituents); (xii) a formula-(X₁₇)_n17-SO_TwoNX₁₈X₁₉A sulfonamide of the formula (wherein X₁₇Is
, Alkyl, and 5- or 6-membered aromatic, heteroaromatic, aliphatic, and
Is selected from the group consisting of heteroaliphatic ring components, wherein the alkyl and ring components are
Kill, halogen, trihalomethyl, carboxylate, amino, nitro, and
Optionally with one or more substituents independently selected from the group consisting of esters
N may be substituted₁₇Is 0, 1 or 2, and X₁₈And X₁₉Is independent
Hydrogen, alkyl, 5- or 6-membered aromatic, heteroaromatic, aliphatic
Selected from the group consisting of aliphatic or heteroaliphatic ring components, alkyl and cyclic
Minutes are alkyl, halogen, trihalomethyl, carboxylate, amino, nitro
(B) one or more substitutions independently selected from the group consisting of
Optionally substituted with a group, or X₁₈And X₁₉Together
Alkyl, halogen, trihalomethyl, carboxylate, amino, nitro, and
And one or more substituents independently selected from the group consisting of
Forms a 5- or 6-membered aliphatic or heteroaliphatic ring which may be substituted
(Xiii) Formula-(X₂₀)_n20An aldehyde of —CO—H (wherein X is₂₀Is saturated
Or unsaturated alkyl, and 5- or 6-membered aromatic, heteroaromatic, and aliphatic
Selected from the group consisting of aliphatic or heteroaliphatic ring components, alkyl and cyclic
Minutes are alkyl, halogen, trihalomethyl, carboxylate, amino, nitro
(B) one or more substitutions independently selected from the group consisting of
Optionally substituted with a group, and n₂₀Is 0 or 1); (xiv) Formula-(X_{twenty one})_n21-SO_Two-X_{twenty two}Of the formula (wherein X_{twenty one}And X_{twenty two}Is
, Independently, saturated or unsaturated alkyl, and 5- or 6-membered aromatic
, A heteroaromatic, aliphatic, or heteroaliphatic ring component;
Alkyl and ring components are alkyl, halogen, trihalomethyl, carboxy
One selected from the group consisting of amino acids, amino, nitro, and esters
Or more optionally substituted with substituents,_{twenty one}Is 0 or 1
); And (xv) formula-(X_{twenty three})_n23-SH thiol or formula (X_{twenty four})_n24-SX_{twenty five}No
Oether (wherein, X_{twenty three}, X_{twenty four}And X_{twenty five}Are independently saturated or unsaturated
Alkyl, and 5- or 6-membered aromatic, heteroaromatic, aliphatic, or
Selected from the group consisting of teloaliphatic ring components, wherein alkyl and the ring component are alkyl
, Halogen, trihalomethyl, carboxylate, amino, nitro, and S
Optionally substituted with one or more substituents independently selected from the group consisting of
And n_{twenty three}And n_{twenty four}Is independently 0 or 1); selected from the group consisting of: or R₃₃And R₃₄Is, together, alkyl, halogen, trihalomethyl
, Carboxylate, amino, nitro, and ester independently of the group consisting of
6-membered cyclic aliphatic optionally substituted with one or more selected substituents
Or may form an aromatic or aromatic ring; and (c) R₂₈, R₂₉, R₃₀And R₃₁Are each independently (i) hydrogen; (ii) hydroxy, halogen, trihalomethyl, carboxylate, amino,
Nitro, esters and 5- or 6-membered aromatic, heteroaromatic, aliphatic,
Or one or more substituents selected from the group consisting of heteroaliphatic ring components
A saturated or unsaturated alkyl optionally substituted with: wherein the ring component is alkyl, halogen, trihalomethyl, carboxylate,
One or more independently selected from the group consisting of amino, nitro, and ester components
May be optionally substituted with further substituents; (iii) alkyl, alkoxy, halogen, trihalomethyl, carboxylate
1, independently selected from the group consisting of amino, nitro, and ester components
Or an aromatic or heteroaromatic optionally substituted with more substituents
(Iv) alkyl, alkoxy, halogen, trihalomethyl, carboxylate
One or more independently selected from the group consisting of, amino, nitro, and ester
An aliphatic or heteroaliphatic ring optionally substituted with the above substituents, and
Alkyl, alkoxy, halogen, trihalomethyl, carboxylate, amino
One or more independently selected from the group consisting of, nitro, and ester components
An aromatic or heteroaromatic ring optionally substituted with the above substituents; (v) a formula-(X₁)_n1-NX_TwoX_ThreeAn amine of the formula (wherein X₁Is a saturated or unsaturated
Alkyl, and 5- or 6-membered aromatic, heteroaromatic, or aliphatic ring
Selected from the group consisting of₁Is 0 or 1 and X_TwoAnd X_ThreeIs
Independently, hydrogen, saturated or unsaturated alkyl, and 5- or 6-membered ring
(Vi) selected from the group consisting of aromatic, heteroaromatic, or aliphatic ring components);_Two(Vii) halogen or trihalomethyl; (viii) a formula-(X_Four)_n4-CO-X_FiveKetone (wherein, X_FourAnd X_FiveIs independent
Alkyl and 5- or 6-membered aromatic, heteroaromatic, aliphatic,
Or a heteroaliphatic ring component, wherein the alkyl and ring components are
Alkyl, halogen, trihalomethyl, carboxylate, amino, nitro, and
And 1, 2 or 3 substituents independently selected from the group consisting of
Optionally substituted and n_FourIs 0 or 1); (ix) Formula-(X₆)_n6A carboxylic acid of -COOH or a formula-(X₇)_n7-COO-
X₈An ester of the formula (wherein X₆, X₇And X₈Is independently alkyl and 5-membered ring
Or from a six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring component
Selected from the group₆And n₇Is independently 0 or 1); (x) Formula-(X₉)_n9-OH alcohol or formula-(X_Ten)_n10-OX₁₁No
Alkoxyalkyl component (wherein, X₉, X_TenAnd X₁₁Are independently saturated or
Unsaturated alkyl and 5- or 6-membered aromatic, heteroaromatic and aliphatic
Or a heteroaliphatic ring component, wherein the alkyl and the ring component are
, Alkyl, halogen, trihalomethyl, carboxylate, amino, nitro,
And one or more substituents independently selected from the group consisting of
May be optionally substituted, n₉And n_TenIs independently 0 or 1
); (Xi) Formula-(X₁₂)_n12-NHCOX₁₃Or the formula-(X₁₄)_n14-CONX_FifteenX ₁₆ An amide of the formula₁₂And X₁₄Are each independently alkyl, hydroxy,
Syl, and 5- or 6-membered aromatic, heteroaromatic, aliphatic, or heterocyclic
Selected from the group consisting of alicyclic ring components, wherein the alkyl and the ring component are alkyl,
Halogen, trihalomethyl, carboxylate, amino, nitro, and esthetic
Optionally substituted with one or more substituents independently selected from the group consisting of
N₁₂And n₁₄Is independently 0 or 1 and X₁₃ , X_FifteenAnd X₁₆Is independently hydrogen, alkyl, hydroxyl, and
And 5- or 6-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring
Wherein the alkyl and ring components are alkyl, halogen,
Consists of trihalomethyl, carboxylate, amino, nitro, and esters
Optionally substituted with one or more substituents independently selected from the group
(Xii) formula-(X₁₇)_n17-SO_TwoX₁₈X₁₉A sulfonamide of the formula (wherein X₁₇Is
Alkyl, 5- or 6-membered aromatic, heteroaromatic, aliphatic, or heterocyclic
Alkyl and ring components are selected from the group consisting of aliphatic ring components
Rogen, trihalomethyl, carboxylate, amino, nitro, and ester
Optionally substituted with one or more substituents independently selected from the group consisting of
And n₁₇Is 0, 1, or 2, and X₁₈And X₁₉Is
Independently hydrogen, alkyl, 5- or 6-membered aromatic, heteroaromatic, aliphatic
Selected from the group consisting of aliphatic or heteroaliphatic ring components, alkyl and cyclic
Minutes are alkyl, halogen, trihalomethyl, carboxylate, amino, nitro
(B) one or more substitutions independently selected from the group consisting of
Optionally substituted with a group, or X₁₈And X₁₉Together
Alkyl, halogen, trihalomethyl, carboxylate, amino, nitro, and
And one or more substituents independently selected from the group consisting of
Forms a 5- or 6-membered aliphatic or heteroaliphatic ring which may be substituted
(Xiii) Formula-(X₂₀)_n20An aldehyde of —CO—H (wherein X is₂₀Is saturated
Or unsaturated alkyl, and 5- or 6-membered aromatic, heteroaromatic, and aliphatic
Selected from the group consisting of aliphatic or heteroaliphatic ring components, alkyl and cyclic
Minutes are alkyl, halogen, trihalomethyl, carboxylate, amino, nitro
(B) one or more substitutions independently selected from the group consisting of
Optionally substituted with a group, and n₂₀Is 0 or 1); (xiv) Formula-(X_{twenty one})_n21-SO_Two-X_{twenty two}Of the formula (wherein X_{twenty one}And X_{twenty two}Is
, Independently, saturated or unsaturated alkyl, and 5- or 6-membered aromatic
, A heteroaromatic, aliphatic, or heteroaliphatic ring component;
Alkyl and ring components are alkyl, halogen, trihalomethyl, carboxy
One selected from the group consisting of amino acids, amino, nitro, and esters
Or more optionally substituted with substituents, and n_{twenty one}Is 0 or 1
And (xv) the formula-(X_{twenty three})_n23A thiol of -SH or a formula-(X_{twenty four})_n24-SX_{twenty five}of
Thioether (wherein, X_{twenty three}, X_{twenty four}And X_{twenty five}Are independently saturated or unsaturated
Alkyl, and 5- or 6-membered aromatic, heteroaromatic, aliphatic, or
Selected from the group consisting of heteroaliphatic ring components, wherein the alkyl and ring components are
, Halogen, trihalomethyl, carboxylate, amino, nitro, and
Optionally with one or more substituents independently selected from the group consisting of
And n_{twenty three}And n_{twenty four}Is independently 0 or 1)
Selected from the group consisting of:

In a preferred embodiment, the present invention relates to a compound of formula III, wherein: (a) R₃₁Is hydrogen; (b) R₃₂, R₃₃And R₃₄Are each independently: (i) hydrogen; (ii) a saturated alkyl optionally substituted with a 6-membered heteroaliphatic ring component.
(Iii) the formula-(X₁)_n1-NX_TwoX_ThreeAn amine of the formula (wherein X₁Is a saturated alkyl
, N₁Is 0 or 1 and X_TwoAnd X_ThreeIs independently hydrogen and saturated
Selected from the group consisting of alkyl); (iv) Formula-(X₆)_n6A carboxylic acid of -COOH or a formula-(X₇)_n7-COO-
X₈An ester of the formula (wherein X₆, X₇And X₈Is alkyl, and n₆and
n₇Is independently 0 or 1); and (v) the formula-(X₁₄)_n14-CONX_FifteenX₁₆An amide of the formula₁₄Is alkyl
, N₁₂And n₁₄Is independently 0 or 1 and X_FifteenAnd X₁₆Is
, Each independently selected from the group consisting of hydrogen and alkyl); or selected from the group consisting of₇Is as described herein and R₈And R₉Together
To form an optionally substituted 6-membered aliphatic or aromatic ring
Well; and (c) R₂₈, R₂₉And R₃₀Are each independently: (i) hydrogen; (ii) saturated alkyl; (iii) halogen or trihalomethyl; (iv) a formula-(X₆)_n6A carboxylic acid of -COOH or a formula-(X₇)_n7-COO-
X₈An ester of the formula (wherein X₆, X₇And X₈Is alkyl, and n₆and
n₇Is independently 0 or 1); (v) Formula-(X₉)_n9-OH alcohol or formula-(X_Ten)_n10-OX₁₁No
Alkoxyalkyl component (wherein, X₉, X_TenAnd X₁₁Is alkyl, and
n₉And n_TenIs independently 0 or 1); (vi) the formula-(X₁₂)_n12-NHCOX₁₃Or the formula-(X₁₄)_n14-CONX_FifteenX ₁₆ An amide of the formula₁₂And X₁₄Is alkyl and n₁₂And n₁₄Is independent
And 0 or 1 and X₁₃, X_FifteenAnd X₁₆Are, independently of each other,
(Vii) selected from the group consisting of hydrogen and alkyl);₁₇)_n17-SO_TwoNX₁₈X₁₉A sulfonamide of the formula (wherein X₁₇Is
Alkyl and n₁₇Is 0, 1, or 2, and X₁₈And X₁₉ Is independently selected from the group consisting of hydrogen and alkyl).

More preferably, in the compound of formula III, (a) R ₂₈ is selected from the group consisting of hydrogen, methyl, and 2-hydroxyethyl; (b) R ₂₉ is hydrogen, methyl, chloro, bromo , Carboxy, methoxy, -NH
C (O) -CH ₃ and -SO ₂ N (CH ₃₎ is selected from the group consisting of _2; (c) R ₃₀ is hydrogen, chloro, methoxy, and from the group consisting of -NHC (O) -CH ₃ (D) R ₃₁ is hydrogen; (e) R ₃₂ is hydrogen, methyl, —CH ₂ CH ₂ C (O) OH, —CH ₂ CH ₂ C (O
_{) NH 2, -CH 2 CH 2} CH 2 N (CH 3) 2 and 3-morpholino selected from the group consisting of propyl; and (f) R ₃₃ and R ₃₄ are each independently hydrogen, methyl, - CH ₂ CH ₂ C (
_{O) OH, -CH 2 CH 2} CH 2 N (CH 3) 2 and 3-morpholinopropoxy or selected from the group consisting of propyl or R ₈ and R _9, taken together, 6-membered aromatic or It may form an aliphatic ring.

The preferred diarylindolinone compound of the present invention is preferably a compound formed by reacting a ketone compound with an oxindole compound. The ketone compound is preferably

[0124]

Embedded image

Embedded image Selected from the group consisting of

In the above structure, R ₂₇ is selected from the group consisting of: (i) halogen, trihalomethyl, alkoxy, carboxylate, amino, nitro, ester and 5- or 6-membered aromatic, heteroaromatic, aliphatic, or Saturated or unsaturated alkyl optionally substituted with a substituent selected from the group consisting of heteroaliphatic ring components, wherein said ring component is alkyl, halogen, trihalomethyl, carboxylate, amino, nitro , And 1, independently selected from the group consisting of ester components
Optionally substituted with two or three substituents; and (ii) independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, amino, nitro, and ester components 1,
An aromatic or heteroaromatic ring optionally substituted with 2 or 3 substituents; (iii) independently from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, amino, nitro, ester Consisting of an aliphatic or heteroaliphatic ring optionally substituted with one or more substituents selected, and alkyl, alkoxy, halogen, trihalomethyl, carboxylate, amino, nitro, and ester components Selected from the group consisting of aromatic or heteroaromatic rings optionally substituted with one or more substituents independently selected from the group.

The oxindole compound is preferably 1,3-dihydro-indole-2
-One, 4-methyl-1,3-dihydro-indol-2-one, 4- (2-hydroxy-ethyl) -1,3-dihydro-indol-2-one, 5-chloro-
1,3-dihydro-indol-2-one, 5-bromo-1,3-dihydro-indol-2-one, 5-methoxy-1,3-dihydro-indol-2-one, 2-oxo-2, 3-dihydro-1H-indole-5-carboxylic acid, 2-oxo-2,3-dihydro-1H-indole-5-sulfonic acid dimethylamide,
N- (2-oxo-2,3-dihydro-1H-indol-5-yl) -acetamide, 6-chloro-1,3-dihydro-indol-2-one, 6-methoxy-1,3-dihydro- Selected from the group consisting of indole-2-one, and N- (5-methyl-2-oxo-2,3-dihydro-1H-indol-6-yl) -acetamide.

D. 4-Substituted Indolinone Compounds In another aspect, the present invention provides compounds having a structure according to Formula IV or Formula V:

Embedded image[Wherein (a) R₃₅, R₃₆And R₄₁Are each independently: (i) hydrogen; (ii) halogen, trihalomethyl, alkoxy, carboxylate, amino,
Nitro, esters and 5- or 6-membered aromatic, heteroaromatic, aliphatic,
Or optionally substituted with a substituent selected from the group consisting of heteroaliphatic ring components
Optionally saturated or unsaturated alkyl, wherein the ring component is alkyl, halogen, trihalomethyl, carboxylate,
1,2 independently selected from the group consisting of amino, nitro, and ester components
Or optionally substituted with three substituents; and (iii) alkyl, alkoxy, halogen, trihalomethyl, carboxylate
1, independently selected from the group consisting of amino, nitro, and ester components
, Aromatic or heteroaromatic optionally substituted with 2 or 3 substituents
An aromatic ring; selected from the group consisting of: (b) R₃₇Is the formula -CH_TwoCH_Two-OR is an ethyl-2-oxy group, wherein
R is (i) hydrogen; (ii) halogen, trihalomethyl, carboxylate, amino, nitro, es
Ter and 5- or 6-membered aromatic, heteroaromatic, aliphatic, or heterocyclic
Saturated optionally substituted with a substituent selected from the group consisting of aliphatic ring components
Sum or unsaturated alkyl, wherein the ring components are alkyl, halogen, trihalomethyl, carboxylate,
1,2 independently selected from the group consisting of amino, nitro, and ester components
Or optionally substituted with three substituents; (iii) alkyl, aryl, alkoxy, halogen, trihalomethyl,
Independently selected from the group consisting of boxylate, amino, nitro, and ester components
Aromatic or optionally substituted with one or more selected substituents or
(Iv) Formula -C (E) NX_FifteenX₁₆A substituent of the formula (wherein, X_FifteenAnd X₁₆Are respectively
Independently, hydrogen, alkyl, hydroxyl, formula -SO_Two-X_{twenty two}Of sulfone, and
And 5- or 6-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring
Selected from the group consisting of components wherein the rings are alkyl, aryl, halogen, trihalo
From the group consisting of methyl, carboxylate, amino, nitro, and ester
X may be optionally substituted with one or more substituents selected from_{twenty two}Is a saturated or unsaturated alkyl, and a 5- or 6-membered aromatic,
Selected from the group consisting of teloaromatic, aliphatic, or heteroaliphatic ring components, wherein the ring is
, Alkyl, halogen, trihalomethyl, carboxylate, amino, nitro,
And one or more substituents independently selected from the group consisting of
E is optionally selected from the group consisting of oxygen and sulfur;
Selected from the group consisting of: and (c) R₃₈, R₃₉And R₄₀Are each hydrogen; (d) Z is a 5,6,7,8,9, or 10-membered monocyclic or bicyclic ring
An aromatic or heteroaromatic ring component, comprising: (i) hydrogen; (ii) hydroxy, halogen, trihalomethyl, carboxylate, amino,
Nitro, esters and 5- or 6-membered aromatic, heteroaromatic, aliphatic,
Or one or more substituents selected from the group consisting of heteroaliphatic ring components
A saturated or unsaturated alkyl optionally substituted with: wherein the ring component is alkyl, halogen, trihalomethyl, carboxylate,
One or more independently selected from the group consisting of amino, nitro, and ester components
May be optionally substituted with further substituents; (iii) alkyl, alkoxy, halogen, trihalomethyl, carboxylate
1, independently selected from the group consisting of amino, nitro, and ester components
Or an aromatic or heteroaromatic optionally substituted with more substituents
(Iv) alkyl, alkoxy, halogen, trihalomethyl, carboxylate
One or more independently selected from the group consisting of, amino, nitro, and ester
An aliphatic or heteroaliphatic ring optionally substituted with the above substituents, and
Alkyl, alkoxy, halogen, trihalomethyl, carboxylate, amino
One or more independently selected from the group consisting of, nitro, and ester components
An aromatic or heteroaromatic ring optionally substituted with the above substituents; (v) a formula-(X₁)_n1-NX_TwoX_ThreeAn amine of the formula (wherein X₁Is a saturated or unsaturated
Alkyl, and 5- or 6-membered aromatic, heteroaromatic, or aliphatic ring
Selected from the group consisting of₁Is 0 or 1 and X_TwoAnd X_ThreeIs
Independently, hydrogen, saturated or unsaturated alkyl, and 5- or 6-membered ring
(Vi) selected from the group consisting of aromatic, heteroaromatic, or aliphatic ring components);_Two(Vii) halogen or trihalomethyl; (viii) a formula-(X_Four)_n4-CO-X_FiveKetone (wherein, X_FourAnd X_FiveIs independent
Alkyl and 5- or 6-membered aromatic, heteroaromatic, aliphatic,
Or a heteroaliphatic ring component, wherein the alkyl and ring components are
Alkyl, halogen, trihalomethyl, carboxylate, amino, nitro, and
And 1, 2 or 3 substituents independently selected from the group consisting of
Optionally substituted and n_FourIs 0 or 1); (ix) Formula-(X₆)_n6A carboxylic acid of -COOH or a formula (X₇)_n7-COO-X ₈ An ester of the formula (wherein X₆, X₇And X₈Are independently alkyl and 5-membered rings
Or a six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring component.
Selected from the group₆And n₇Is independently 0 or 1); (x) Formula-(X₉)_n9-OH alcohol or formula-(X_Ten)_n10-OX₁₁No
Alkoxyalkyl component (wherein, X₉, X_TenAnd X₁₁Are independently saturated or
Unsaturated alkyl and 5- or 6-membered aromatic, heteroaromatic and aliphatic
Or a heteroaliphatic ring component, wherein the alkyl and the ring component are
, Alkyl, halogen, trihalomethyl, carboxylate, amino, nitro,
And one or more substituents independently selected from the group consisting of
Optionally substituted and n₉And n_TenIs independently 0 or 1
(Xi) Formula-(X₁₂)_n12-NHCOX₁₃Or the formula-(X₁₄)_n14-CONX_FifteenX ₁₆ An amide of the formula₁₂And X₁₄Are each independently alkyl, hydroxy,
Syl, and 5- or 6-membered aromatic, heteroaromatic, aliphatic, or heterocyclic
Selected from the group consisting of alicyclic ring components, wherein the alkyl and the ring component are alkyl,
Halogen, trihalomethyl, carboxylate, amino, nitro, and esthetic
Optionally substituted with one or more substituents independently selected from the group consisting of
N₁₂And n₁₄Is independently 0 or 1 and X₁₃ , X_FifteenAnd X₁₆Is independently hydrogen, alkyl, hydroxyl, and
And 5- or 6-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring
Wherein the alkyl and ring components are alkyl, halogen,
Consists of trihalomethyl, carboxylate, amino, nitro, and esters
Optionally substituted with one or more substituents independently selected from the group
(Xii) formula-(X₁₇)_n17-SO_TwoNX₁₈X₁₉A sulfonamide of the formula (wherein X₁₇Is
, Alkyl, 5- or 6-membered aromatic, heteroaromatic, aliphatic, or heterocyclic
Selected from the group consisting of alicyclic ring components, wherein the alkyl and the ring component are alkyl,
Halogen, trihalomethyl, carboxylate, amino, nitro, or esthetic
Optionally substituted with one or more substituents independently selected from the group consisting of
And n₁₇Is 0, 1, or 2, and X₁₈And X₁₉ Is independently hydrogen, alkyl, 5- or 6-membered aromatic, heteroaromatic,
Selected from the group consisting of aliphatic or heteroaliphatic ring components, alkyl and ring
The components are alkyl, halogen, trihalomethyl, carboxylate, amino,
One or more positions independently selected from the group consisting of
Optionally substituted with a substituent, or X₁₈And X₁₉Together
Alkyl, halogen, trihalomethyl, carboxylate, amino, nitro, and
And one or more substituents independently selected from the group consisting of
Optionally substituted 5- or 6-membered aliphatic or heteroaliphatic ring
(Xiii) Formula-(X₂₀)_n20An aldehyde of —CO—H (wherein X is₂₀Is saturated
Or unsaturated alkyl, and 5- or 6-membered aromatic, heteroaromatic, and aliphatic
Selected from the group consisting of aliphatic or heteroaliphatic ring components, alkyl and cyclic
Minutes are alkyl, halogen, trihalomethyl, carboxylate, amino, nitro
(B) one or more substitutions independently selected from the group consisting of
Optionally substituted with a group, and n₂₀Is 0 or 1); (xiv) Formula-(X_{twenty one})_n21-SO_Two-X_{twenty two}Of the formula (wherein X_{twenty one}And X_{twenty two}Is
, Independently, saturated or unsaturated alkyl, and 5- or 6-membered aromatic
, A heteroaromatic, aliphatic, or heteroaliphatic ring component;
Alkyl and ring components are alkyl, halogen, trihalomethyl, carboxy
One selected from the group consisting of amino acids, amino, nitro, and esters
Or more optionally substituted with substituents, and n_{twenty one}Is 0 or 1
And (xv) formula-(X_{twenty three})_n23The thiol of -SH and the formula-(X_{twenty four})_n24-SX_{twenty five}of
Thioether (wherein, X_{twenty three}, X_{twenty four}And X_{twenty five}Are independently saturated or unsaturated
Alkyl, and 5- or 6-membered aromatic, heteroaromatic, aliphatic, or
Selected from the group consisting of heteroaliphatic ring components, wherein the alkyl and ring components are
, Halogen, trihalomethyl, carboxylate, amino, nitro, and
Optionally with one or more substituents independently selected from the group consisting of
And n_{twenty three}And n_{twenty four}Is independently 0 or 1)
Optionally substituted with one or more substituents selected from the group consisting of
Good.

In a preferred embodiment, the present invention relates to a compound of formula IV or V, wherein
, (A) R₃₅, R₃₆And R₄₁Is hydrogen; (b) R₃₇Is of the formula -CH_TwoCH_TwoAn ethyl-2-oxy group of -OR, wherein R is water
From alkyl, saturated alkyl, and alkyl, aryl, and alkoxy components.
Optionally substituted with one or more substituents independently selected from the group
Aromatic ring, and -C (E) NHX_FifteenA substituent of the formula (wherein, X_FifteenIs an archi
, Formula -SO_Two-X_{twenty two}From a sulfone and a six-membered aromatic or aliphatic ring component
And the rings are independently selected from the group consisting of alkyl and aryl
Optionally substituted with one or more substituents represented by_{twenty two}Is saturated
A group consisting of alkyl, and an optionally substituted 6-membered aromatic ring component
And E is selected from the group consisting of oxygen and sulfur);
And (c) Z is a 5, 6, or 9-membered monocyclic or bicyclic aromatic or heterocyclic group.
(I) hydrogen; (ii) saturated alkyl; (iii) an optionally substituted aromatic or heteroaromatic ring;₁)_n1-NX_TwoX_ThreeAn amine of the formula (wherein X₁Is alkyl and n₁
Is 0 or 1 and X_TwoAnd X_ThreeIs independently hydrogen and saturated alkyl
(V) halogen or trihalomethyl; (vi)-(X₆)_n6A carboxylic acid of —COOH, wherein X₆Is alkyl and n ₆ Is 0 or 1); and (vii) a formula-(X₉)_n9-OH alcohol or formula-(X_Ten)_n10-OX₁₁ An alkoxyalkyl component of the formula₉, X_TenAnd X₁₁Is alkyl and n₉ And n_TenIs independently 0 or 1.) optionally substituted with one or more substituents selected from the group consisting of:
Is also good.

More preferably, in the compound of formula IV or formula V, R ₃₇ is of the formula —CH ₂ C
An ethyl-2-oxy group of H ₂ -OR, wherein R is hydrogen, methyl, ethyl, 2
-Isopropylphenyl, 3-isopropylphenyl, 4-isopropylphenyl, 4-methoxyphenyl, biphen-3-yl, 5-chloropyridin-3-yl, ethylcarbamyl, tert-butylcarbamyl, cyclohexylcarbamyl, phenylcarba Selected from the group consisting of mill, benzenesulfonylcarbamyl, biphen-2-yl-carbamyl, and phenylthiocarbamyl, and Z is 4-bromophenyl, 2-pyridyl, 6-methyl-2-pyridyl, 1H-
Indole-5-yl, 4-methoxy-3-thiophenephenyl, 4- (3-dimethylamino) -3,5-dimethyl-1H-pyrrol-2-yl, 3-hydroxy-6-methyl-pyridin-2- Yl, 4- (3-dimethylaminopropyl)-
3,5-dimethyl-1H-pyrrol-2-yl, 3-carboxy-2,4-dimethyl-1H-pyrrol-2-yl, and isopropylcarbamyl).

Preferred compounds of the present invention are listed in Table 1. Table 1

[Table 1]

[Table 2]

Some of the compounds described above have the formula VI:

Embedded image Wherein the R substituents are described in Table 2. Table 2

[Table 3]

[Table 4]

Another compound of the present invention has formula VII:

Embedded image Wherein the substituents are described in Table 3. Table 3

[Table 5]

III. Combinatorial libraries and synthesis method A. General Notes on Synthetic Methods Bases can be used to synthesize the compounds of the present invention. The base is preferably a nitrogen base or an inorganic base. "Nitrogen bases" are commonly used in the art and are selected from acyclic and cyclic amines. Examples of nitrogen bases include, but are not limited to, ammonia, methylamine, trimethylamine, triethylamine, aniline, 1,8-diazabicyclo [5.4.0] undec-7-ene, diisopropylethylamine, pyrrolidine, piperidine, and pyridine or Substituted pyridines (eg, 2,6-di-tertbutylpyridine) are included.
"Inorganic bases" are bases that do not contain carbon atoms. Examples of inorganic bases include, but are not limited to, hydroxides, phosphates, sulfites, hydrosulfides, and amide anions. It will be clear to the skilled person which nitrogen base or inorganic base will meet the requirements of the desired reaction conditions. In some embodiments of the invention, the base used can be pyrrolidine or piperidine. In another embodiment, the base may be a hydroxyl anion, and is preferably used as its sodium or potassium salt.

The compounds of the present invention can be synthesized in a solvent. The solvent for the reaction is preferably a protic or aprotic solvent. A "protic solvent" is a solvent that can provide a proton to a solute. Examples of protic solvents include, but are not limited to, alcohol and water. "Aprotic solvent"
A solvent that does not give a proton to a solute under normal reaction conditions. Typical organic solvents, such as hexane, toluene, benzene, methylene chloride, dimethylformamide, chloroform, tetrahydrofuran are some examples of protic solvents. Other aprotic solvents are also within the scope used in the present invention.
In certain preferred embodiments, the solvent for the reaction is an alcohol, which may preferably be isopropanol, and most preferably is ethanol. Water is another preferred protic solvent. Dimethylformamide, known in the chemical arts as DMF, is a preferred aprotic solvent.

In the synthesis method of the present invention, it is necessary that the reaction is performed at a high temperature, that is, at a temperature higher than room temperature. More preferably, the elevated temperature is preferably about 30-150 ° C,
More preferably, it is about 80-100C, most preferably about 80-90C, the temperature at which ethanol boils (i.e., the boiling point of ethanol). "About" for a temperature means that the temperature range is preferably within 10 ° C of the listed temperature, more preferably within 5 ° C of the listed temperature, and most preferably at 2 ° C of the listed temperature. It is within the range. Thus, by way of example, “about 80 ° C.” means that the temperature range is preferably 80 ± 10 ° C., more preferably 80 ± 5 ° C., and most preferably 80 ± 2 ° C.

The method of synthesis of the invention may involve the step of screening the library for compounds of the desired activity and structure. That is, the present invention provides a method for synthesizing a compound by first screening a compound having desired properties and then chemically synthesizing the compound.

B. 3-Arylidenyl-6-heterocyclyl-2-indolinone derivatives Another aspect of the present invention is that at least 10 3-arylidenyl-6, which can be formed by condensing an oxindole of structure 2 with an aldehyde of structure 3. -Combinatorial library of heterocyclyl-2-indolinone compounds.

[0138] As used herein, "condensing" or "condensation" refers to a reaction in which two molecules together give one molecule. In particular, in the context of the present invention, condensation refers to the reaction shown in Scheme I. Scheme I

Embedded image

The oxindole in the above combinatorial library is preferably 6
-(Pyridin-2-yl) -2-oxindole, 6- (pyridin-3-yl) -2-oxindole, 6- (pyridin-4-yl) -2-oxindole, 6- (pyrimidin-2 -Yl) -2-oxindole, 6- (pyrimidin-4-yl) -2-oxindole, 6- (pyrimidin-5-yl) -2-oxindole, 6- (triazinyl) -2-oxindole, 6- (pyrrol-2-yl) -2-oxindole, 6- (pyrrol-3-yl) -2-oxindole, 6- (thiophen-2-yl) -2-oxindole, 6-
(Thiophen-3-yl) -2-oxindole, 6- (furan-2-yl)
-2-oxindole, 6- (furan-3-yl) -2-oxindole,
6- (imidazol-2-yl) -2-oxindole, 6- (imidazol-4-yl) -2-oxindole, 6- (thiazol-2-yl) -2-oxindole, 6- (thiazole- 4-yl) -2-oxindole, 6-
(Oxazol-2-yl) -2-oxindole, 6- (oxazole-4
-Yl) -2-oxindole, 6- (thiadiazol-2-yl) -2-oxindole, 6- (oxadiazolyl) -2-oxindole and 6-
(Triazol-2-yl) -2-oxindole, 6- (3-methylisoxazol-5-yl) -2-oxindole, 6- (3-methylisothiazol-5-yl) -2-oxindole , 6- (3,5-Dimethyl-isoxazol-4-yl) -2-oxindole, 6- (thiazol-2-yl)
-2-oxindole, 6- (thiazol-4-yl) -2-oxindole, 6- (thiazol-5-yl) -2-oxindole, 6- (3-methylthiophen-2-yl) -2 -Oxindole, 6- (4-methylthiophen-2-yl) -2-oxindole, 6- (5-methylthiophen-2-yl) -2-oxindole, 6- (5-chlorothiophen-2- Yl) -2-oxindole, selected from the group consisting of 6- (4-methylfuran-2-yl) -2-oxindole.

The aldehydes in the combinatorial library described above are preferably, but not limited to, benzaldehyde, 3-isopropyl-p-anisaldehyde, 2-methyl-5-isopropyl-p-anisaldehyde, 3,5 -Diisopropyl-p-anisaldehyde, 3-cyclopentyl-p-anisaldehyde,
3-cyclohexyl-p-anisaldehyde, 3-phenyl-p-anisaldehyde, 3,5-dimethyl-p-anisaldehyde, 2-hydroxy-3,5-dichlorobenzaldehyde, 2-hydroxy-5-chlorobenzaldehyde, 2 −
Hydroxy-4-methoxy-5- (4-methoxyphenyl) benzaldehyde,
3-cyclopentyl-4-methoxybenzaldehyde, 3-cyclopentyl-4
-Hydroxybenzaldehyde, 3- (2-thienyl) -4-methoxybenzaldehyde, 2-hydroxybenzaldehyde, 2-hydroxy-4-methoxybenzaldehyde, 2-hydroxy-5-bromobenzaldehyde, 2-methylpyridine-6-carboxaldehyde, 3-tert-butyl-4-hydroxy-
5-bromobenzaldehyde, 3-bromobenzaldehyde, 3,5-diisopropyl-4- [2- (N-morpholino) ethyl] benzaldehyde, indole-5-carboxaldehyde, 2-hydroxy-3-fluorobenzaldehyde, 3-cyclohexyl- 4- [2- (N-morpholino) ethyl] benzaldehyde, 3- (3-thienyl) -4-methoxybenzaldehyde, 2-methyl-5
Isopropyl-4-methoxy-benzaldehyde, 2,3-dihydrobenzofuran-5-carboxaldehyde, 2,2-dimethylchroman-6-carboxaldehyde, 3- (thiophen-3-yl) -4-methoxybenzaldehyde, 3
-(3-acetylaminophenyl) -4-methoxy-benzaldehyde, 2-naphthyl-4,5-dimethoxybenzaldehyde, 2- (3-methoxyphenyl)
-4,5-dimethoxybenzaldehyde, 3- (pyridin-3-yl) -p-anisaldehyde, 2- (3-ethoxyphenyl) -4,5-dimethoxybenzaldehyde, 3-isopropyl-4- [2- (N -Morpholino) ethoxy] benzaldehyde, 3,5-diisopropyl-4- [2- (N-morpholino) ethoxy] benzaldehyde, 2- (2-methoxyphenyl) -4,5-dimethoxybenzaldehyde, 3- (3-ethoxyphenyl ) -P-anisaldehyde, 3- (
Thiophen-2-yl) -4- [2- (N-morpholino) ethoxy] -benzaldehyde, 2- (thiophen-2-yl) -4,5-dimethoxybenzaldehyde, 2,4-dimethyl-3- [3- Dimethylaminopropyl] -pyrrole-5
Carboxaldehyde, 2,4-dimethyl-3- [3-carboxypropyl]-
Pyrrole-5-carboxaldehyde, 2,4-dimethyl-3- [3- (N-morpholino) prop-1-yl] -pyrrole-5-carboxaldehyde, 2,4
-Dimethyl-3- [2-dimethylaminoethyl] -pyrrole-5-carboxaldehyde, 2,4-dimethyl-3- [2-carboxyethyl] -pyrrole-5
Carboxaldehyde and 2,4-dimethyl-3- [2- (N-morpholino)
Propyl] -pyrrole-5-carboxaldehyde, 3-isopropyl-4- [
2- (N-morpholino) propoxy] benzaldehyde, 3,5-diisopropyl-4- [2- (N-morpholino) propoxy] benzaldehyde, 3- (thiophen-2-yl) -4- [2-N-morpholino) [Propoxy] benzaldehyde.

Another aspect of the present invention is to provide a 3-arylidenyl-6-heterocyclyl of formula I comprising condensing an oxindole of formula 2 with an aldehyde of formula 3 in a solvent, preferably in the presence of a base. -2- Provide a method for synthesizing indolinone.

An example of an oxindole of formula 2 that can be condensed with an aldehyde of formula 3 to give 3-arylidenyl-6-heterocyclyl-2-indolinones of formula I is 6-
(Pyridin-2-yl) -2-oxindole, 6- (pyridin-3-yl)
-2-oxindole, 6- (pyridin-4-yl) -2-oxindole, 6- (pyrimidin-2-yl) -2-oxindole, 6- (pyrimidin-
4-yl) -2-oxindole, 6- (pyrimidin-5-yl) -2-oxindole, 6- (triazinyl) -2-oxindole, 6- (pyrrol-2-yl) -2-oxindole , 6- (Pyrol-3-yl) -2-oxindole, 6- (thiophen-2-yl) -2-oxindole, 6- (
Thiophen-3-yl) -2-oxindole, 6- (furan-2-yl)-
2-oxindole, 6- (furan-3-yl) -2-oxindole, 6
-(Imidazol-2-yl) -2-oxindole, 6- (imidazole-
4-yl) -2-oxindole, 6- (thiazol-2-yl) -2-oxindole, 6- (thiazol-4-yl) -2-oxindole, 6- (
Oxazol-2-yl) -2-oxindole, 6- (oxazole-4-
Yl) -2-oxindole, 6- (thiadiazol-2-yl) -2-oxindole, 6- (oxadiazol-2-yl) -2-oxindole and 6- (triazol-2-yl)- 2-oxindole, 6- (3-methylisoxazol-5-yl) -2-oxindole, 6- (3-methylisothiazol-5-yl) -2-oxindole, 6- (3,5- Dimethyl-
Isoxazol-4-yl) -2-oxindole, 6- (thiazol-2)
-Yl) -2-oxindole, 6- (thiazol-4-yl) -2-oxindole, 6- (thiazol-5-yl) -2-oxindole, 6- (3
-Methylthiophen-2-yl) -2-oxindole, 6- (4-methylthiophen-2-yl) -2-oxindole, 6- (5-methylthiophene-
2-yl) -2-oxindole, 6- (5-chlorothiophen-2-yl)
-2-oxindole, 6- (4-methylfuran-2-yl) -2-oxindole.

Examples of aldehydes of structure 3 that can be condensed with oxindoles of structure 2 to give compounds of the present invention include, but are not limited to, benzaldehyde, 3-
Isopropyl-p-anisaldehyde, 2-methyl-5-isopropyl-p-anisaldehyde, 3,5-diisopropyl-p-anisaldehyde, 3-cyclopentyl-p-anisaldehyde, 3-cyclohexyl-p-anisaldehyde, 3 -Phenyl-p-anisaldehyde, 3,5-dimethyl-p-anisaldehyde, 2-hydroxy-3,5-dichlorobenzaldehyde, 2-hydroxy-
5-chlorobenzaldehyde, 2-hydroxy-4-methoxy-5- (4-methoxyphenyl) benzaldehyde, 3-cyclopentyl-4-methoxybenzaldehyde, 3-cyclopentyl-4-hydroxybenzaldehyde, 3- (2-
Thienyl) -4-methoxybenzaldehyde, 2-hydroxybenzaldehyde, 2-hydroxy-4-methoxybenzaldehyde, 2-hydroxy-5-bromobenzaldehyde, 2-methylpyridine-6-carboxaldehyde, 3-t
tert-butyl-4-hydroxy-5-bromobenzaldehyde, 3-bromobenzaldehyde, 3,5-diisopropyl-4- [2- (N-morpholino) ethyl] benzaldehyde, indole-5-carboxaldehyde, 2-hydroxy-3 -Fluorobenzaldehyde, 3-cyclohexyl-4- [2- (N-morpholino) ethyl] benzaldehyde, 3- (3-thienyl) -4-methoxybenzaldehyde, 2-methyl-5-isopropyl-4-methoxy-benzaldehyde, 2 , 3-Dihydrobenzofuran-5-carboxaldehyde, 2,2-dimethylchroman-6-carboxaldehyde, 3- (thiophen-3-yl)-
4-methoxybenzaldehyde, 3- (3-acetylaminophenyl) -4-methoxy-benzaldehyde, 2-naphthyl-4,5-dimethoxybenzaldehyde, 2- (3-methoxyphenyl) -4,5-dimethoxybenzaldehyde, 3
-(Pyridin-3-yl) -p-anisaldehyde, 2- (3-ethoxyphenyl) -4,5-dimethoxybenzaldehyde, 3-isopropyl-4- [2- (
N-morpholino) ethoxy] benzaldehyde, 3,5-diisopropyl-4-
[2- (N-morpholino) ethoxy] benzaldehyde, 2- (2-methoxyphenyl) -4,5-dimethoxybenzaldehyde, 3- (3-ethoxyphenyl) -p-anisaldehyde, 3- (thiophen-2-yl ) -4- [2- (N-
Morpholino) ethoxy] -benzaldehyde, 2- (thiophen-2-yl)-
4,5-dimethoxybenzaldehyde, 2,4-dimethyl-3- [3-dimethylaminopropyl] -pyrrole-5-carboxaldehyde, 2,4-dimethyl-
3- [3-carboxypropyl] -pyrrole-5-carboxaldehyde, 2,
4-dimethyl-3- [3- (N-morpholino) propyl] -pyrrole-5-carboxaldehyde, 2,4-dimethyl-3- [2-dimethylaminoethyl] -pyrrole-5-carboxaldehyde, 2,4 -Dimethyl-3- [2-carboxyethyl] -pyrrole-5-carboxaldehyde and 2,4-dimethyl-3-
[2- (N-morpholino) ethyl] propyl-1-yl] -pyrrole-5-carboxaldehyde, 3-isopropyl-4- [2- (N-morpholino) propoxy] benzaldehyde, 3,5-diisopropyl-4- [2- (N-morpholino) propoxy] benzaldehyde, 3- (thiophen-2-yl) -4- [2-
N-morpholino) propoxy] benzaldehyde.

C. 3-Aralkyl-2-indolinone derivatives Another aspect of the present invention is to condense an oxindole of structure 4 with an aldehyde of structure 5 and then replace the resulting 3-arylidene-2-oxindole with a double bond at the 3-position. A combinatorial library of at least 10 3-aralkyl-2-indolinone compounds that can be formed by reduction. In particular, condensation refers to reaction "A" in Scheme II. Compound 4 is "3-arylidene-2-oxindole" described above.

As used herein, “reducing” refers to adding a hydrogen to the double bond at the 3-position of compound 6 (reaction “B”) to give compound II in Scheme II. Represent. Scheme II

Embedded image

The oxindole in the combinatorial library described above is preferably oxindole itself and substituted oxindoles, such as, but not limited to, 5-fluorooxindole, 6-fluorooxindole, 7-fluorooxindole, 6-trifluoromethyloxindole, 5-chlorooxindole, 6-chlorooxindole, indole-4-carboxylic acid, 5-bromooxindole, 6- (acetamido) -oxindole, 4
-Methyloxyindole, 5-methyloxyindole, 4-methyl-5-chlorooxyindole, 5-ethyloxyindole, 6-hydroxyoxyindole, 6- (cyclopentylcarboxamido) oxyindole, 5-acetyloxyindole, oxy Indole-5-carboxylic acid, 5-methoxyoxindole, 6-methoxyoxindole, 5-aminooxindole, 6-aminooxindole, 4- [2- (N-morpholino) ethyl] -oxindole, 7-aza Oxindole, oxindole-4-carbamic acid t-butyl ester, oxindole-6-carbamic acid t-butyl ester, 4- (2-carboxyethyl) oxindole, 4-n-butyloxyindole, 4,5- Methoxy oxindole, 6- (methanesulfonamido) oxindole, 6- (benzamido) oxindole, 5-ethoxy-oxindole, 6-phenyl oxindole, 4- (2-hydroxy eth -
1-yl) oxindole, 6- (2-methoxyphen-1-yl) oxindole, 6- (3-methoxyphen-1-yl) oxindole and 6- (3-methoxyphen-1-yl) oxindole
It is selected from the group consisting of (4-methoxyphen-1-yl) oxindole.

The aldehydes in the combinatorial library described above are preferably, but not limited to, benzaldehyde itself and 3-isopropyl-p-anisaldehyde, 2-methyl-5-isopropyl-p-anisaldehyde, 3,
5-diisopropyl-p-anisaldehyde, 3-cyclopentyl-p-anisaldehyde, 3-cyclohexyl-p-anisaldehyde, 3-phenyl-p-
Anisaldehyde, 3,5-dimethyl-p-anisaldehyde, 2-hydroxy-3,5-dichlorobenzaldehyde, 2-hydroxy-5-chlorobenzaldehyde, 2-hydroxy-4-methoxy-5- (4-methoxyphenyl) Benzaldehyde, 3-cyclopentyl-4-methoxybenzaldehyde, 3-cyclopentyl-4-hydroxybenzaldehyde, 3- (2-thienyl) -4-methoxybenzaldehyde, 2-hydroxybenzaldehyde, 2-hydroxy-4
-Methoxybenzaldehyde, 2-hydroxy-5-bromobenzaldehyde,
2-methylpyridine-6-carboxaldehyde, 3-tert-butyl-4-
Hydroxy-5-bromobenzaldehyde, 3-bromobenzaldehyde, 3,
5-diisopropyl-4- [2- (N-morpholino) ethyl] benzaldehyde, indole-5-carboxaldehyde, 2-hydroxy-3-fluorobenzaldehyde, 3-cyclohexyl-4- [2- (N-morpholino) ethyl] Benzaldehyde, 3- (3-thienyl) -4-methoxybenzaldehyde, 2-
Methyl-5-isopropyl-4-methoxy-benzaldehyde, 2,3-dihydrobenzofuran-5-carboxaldehyde, 2,2-dimethylchroman-6
Carboxaldehyde, 3- (thiophen-3-yl) -4-methoxybenzaldehyde, 3- (3-acetylaminophenyl) -4-methoxy-benzaldehyde, 2-naphthyl-4,5-dimethoxybenzaldehyde, 2- (3- (Methoxyphenyl) -4,5-dimethoxybenzaldehyde, 3- (pyrid-3-yl) -p-anisaldehyde, 2- (3-ethoxyphenyl) -4,5-dimethoxybenzaldehyde, 3-isopropyl-4- [2 -(N-morpholino) ethoxy] benzaldehyde, 3,5-diisopropyl-4- [2- (N-morpholino) ethoxy] benzaldehyde, 2- (2-methoxyphenyl) -4,5-dimethoxybenzaldehyde, 3- (3 -Ethoxyphenyl) -p-anisaldehyde, 3- (thiophene) 2-yl) -4- [2- (N- morpholino) ethoxy]
-Benzaldehyde, 2- (thiophen-2-yl) -4,5-dimethoxybenzaldehyde, 2,4-dimethyl-3- [3-dimethylaminoprop-1-yl] -pyrrole-5-carboxaldehyde, 2,4 -Dimethyl-3- [3-carboxyprop-1-yl] -pyrrole-5-carboxaldehyde, 2,4-dimethyl-3- [3- (N-morpholino) prop-1-yl] -pyrrol-5 Carboxaldehyde, 2,4-dimethyl-3- [2-dimethylaminoethyl]-
Pyrrole-5-carboxaldehyde, 2,4-dimethyl-3- [2-carboxyethyl] -pyrrole-5-carboxaldehyde and 2,4-dimethyl-3
-[2- (N-morpholino) ethyl] prop-1-yl] -pyrrole-5-carboxaldehyde.

Another aspect of the present invention provides a method for synthesizing 3-aralkyl-2-indolinones of formula II. The process comprises condensing an oxindole of the formula 4 with an aldehyde of the formula 5 in a solvent, preferably in the presence of a base, to give the resulting 3-arylidene-2
Optionally isolating the oxindole and then reducing the 3-arylidene-2-oxindole.

Condensation with an aldehyde of formula 5 and reduction of the product to give 3-aralkyl-2-
Examples of oxindoles of formula 4 from which indolinone can be obtained include oxindole itself and substituted oxindoles such as, but not limited to, 5-fluorooxindole, 6-fluorooxindole, 7-fluorooxindole, 6 -Trifluoromethyloxindole, 5-chlorooxindole, 6-chlorooxindole, indole-4-carboxylic acid, 5-bromooxindole, 6- (acetamido) -oxindole, 4-methyloxindole, 5-methyl Oxindole, 4-methyl-5-chlorooxindole, 5-ethyloxindole, 6-hydroxyoxindole,
6- (cyclopentylcarboxamido) oxindole, 5-acetyloxindole, oxindole-5-carboxylic acid, 5-methoxyoxindole, 6-methoxyoxindole, 5-aminooxyindole, 6-aminooxindole, 4- [2- (N-morpholino) ethyl] -oxindole, 7-azaoxindole, oxindole-4-carbamic acid t-butyl ester, oxindole-6-carbamic acid t-butyl ester, 4- (
2-carboxyethyl) oxindole, 4-n-butyloxindole,
4,5-dimethoxyoxindole, 6- (methanesulfonamido) oxindole, 6- (benzamido) oxindole, 5-ethoxyoxindole, 6-phenyloxindole, 4- (2-hydroxyeth-1-yl)
Oxindole, 6- (2-methoxyphen-1-yl) oxindole,
Includes 6- (3-methoxyphen-1-yl) oxindole and 6- (4-methoxyphen-1-yl) oxindole.

Examples of aldehydes of structure 3 that can be condensed with an oxindole of structure 4 to reduce the product to give a compound of the present invention include, but are not limited to, benzaldehyde itself and 3- Isopropyl-p-anisaldehyde, 2-methyl-5-isopropyl-p-anisaldehyde, 3,5-diisopropyl-p
-Anisaldehyde, 3-cyclopentyl-p-anisaldehyde, 3-cyclohexyl-p-anisaldehyde, 3-phenyl-p-anisaldehyde, 3,
5-dimethyl-p-anisaldehyde, 2-hydroxy-3,5-dichlorobenzaldehyde, 2-hydroxy-5-chlorobenzaldehyde, 2-hydroxy-4-methoxy-5- (4-methoxyphenyl) benzaldehyde, 3-cyclopentyl -4-methoxybenzaldehyde, 3-cyclopentyl-4-hydroxybenzaldehyde, 3- (2-thienyl) -4-methoxybenzaldehyde,
2-hydroxybenzaldehyde, 2-hydroxy-4-methoxybenzaldehyde, 2-hydroxy-5-bromobenzaldehyde, 2-methylpyridine-6
-Carboxaldehyde, 3-tert-butyl-4-hydroxy-5-bromobenzaldehyde, 3-bromobenzaldehyde, 3,5-diisopropyl-4
-[2- (N-morpholino) ethyl] benzaldehyde, indole-5-carboxaldehyde, 2-hydroxy-3-fluorobenzaldehyde, 3-cyclohexyl-4- [2- (N-morpholino) ethyl] benzaldehyde, 3- (
3-thienyl) -4-methoxybenzaldehyde, 2-methyl-5-isopropyl-4-methoxy-benzaldehyde, 2,3-dihydrobenzofuran-5-carboxaldehyde, 2,2-dimethylchroman-6-carboxaldehyde,
3- (thiophen-3-yl) -4-methoxybenzaldehyde, 3- (3-acetylaminophenyl) -4-methoxy-benzaldehyde, 2-naphthyl-4
, 5-Dimethoxybenzaldehyde, 2- (3-methoxyphenyl) -4,5-
Dimethoxybenzaldehyde, 3- (pyrid-3-yl) -p-anisaldehyde, 2- (3-ethoxyphenyl) -4,5-dimethoxybenzaldehyde, 3
-Isopropyl-4- [2- (N-morpholino) ethoxy] benzaldehyde,
3,5-diisopropyl-4- [2- (N-morpholino) ethoxy] benzaldehyde, 2- (2-methoxyphenyl) -4,5-dimethoxybenzaldehyde, 3- (3-ethoxyphenyl) -p-anisaldehyde, 3- (thiophene-
2-yl) -4- [2- (N-morpholino) ethoxy] -benzaldehyde, 2
-(Thiophen-2-yl) -4,5-dimethoxybenzaldehyde, 2,4-
Dimethyl-3- [3-dimethylaminoprop-1-yl] -pyrrole-5-carboxaldehyde, 2,4-dimethyl-3- [3-carboxyprop-1-yl] -pyrrole-5-carboxaldehyde, 2 , 4-Dimethyl-3- [3- (N
-Morpholino) prop-1-yl-3-pyrrole-5-carboxaldehyde, 2
, 4-Dimethyl-3- [2-dimethylaminoethyl] -pyrrole-5-carboxaldehyde, 2,4-dimethyl-3- [2-carboxyethyl] -pyrrole-
5-Carboxaldehyde and 2,4-dimethyl-3- [2- (N-morpholino) ethyl] prop-1-yl] -pyrrole-5-carboxaldehyde.

D. In another aspect, the present invention provides a combinatorial library of at least 10 indolinone compounds that can be formed by reacting oxindole with a ketone. Here, oxindole has the following structure:

Embedded image And the ketone has the following structure:

Embedded image And R ₂₇ , R ₂₈ , R ₂₉ , R ₃₀ , R ₃₂ , R ₃₃ and R ₃₄ are as described herein.

The oxindole of the combinatorial library described above is preferably 1,
3-dihydro-indol-2-one, 4-methyl-1,3-dihydro-indol-2-one, 4- (2-hydroxy-ethyl) -1,3-dihydro-indol-2-one, 5- Chloro-1,3-dihydro-indol-2-one, 5-
Bromo-1,3-dihydro-indol-2-one, 5-methoxy-1,3-dihydro-indol-2-one, 2-oxo-2,3-dihydro-1H-indole-5-carboxylic acid, 2 -Oxo-2,3-dihydro-1H-indole-5
-Sulfuric acid dimethylamide, N- (2-oxo-2,3-dihydro-1H-indol-5-yl) -acetamide, 6-chloro-1,3-dihydro-indole-
2-one, 6-methoxy-1,3-dihydro-indol-2-one, and N
Selected from the group consisting of-(5-methyl-2-oxo-2,3-dihydro-1H-indol-6-yl) -acetamide.

The ketone is preferably

Embedded image

Embedded image Selected from the group consisting of:

Another aspect of the present invention provides a method for synthesizing a compound of formula III (as described herein). The method comprises reacting a first reactant with a second reactant at an elevated temperature in a solvent in the presence of a base. Here, the first reactant has the following structure:

Embedded image Wherein the second reactant has the following structure:

Embedded image Wherein R ₂₇ , R ₂₈ , R ₂₉ , R ₃₀ , R ₃₁ , R ₃₂ , R ₃₃ and R ₃₄ are as described herein.

The first reactant is preferably 1,3-dihydro-indol-2-one, 4
-Methyl-1,3-dihydro-indol-2-one, 4- (2-hydroxy-
Ethyl) -1,3-dihydro-indol-2-one, 5-chloro-1,3-dihydro-indol-2-one, 5-bromo-1,3-dihydro-indole-
2-one, 5-methoxy-1,3-dihydro-indol-2-one, 5-carboxy-1,3-dihydro-indol-2-one, 5-dimethylsulfonamido-1,3-dihydro-indole- 2-one, 5-formamido-1,3-dihydro-indol-2-one, 6-chloro-1,3-dihydro-indole-
2-one, 6-methoxy-1,3-dihydro-indol-2-one, and 5
Oxindole selected from the group consisting of -methyl-6-formamido-1,3-dihydro-indol-2-one.

[0156] The second reactant is preferably

Embedded image

Embedded image A ketone selected from the group consisting of

E. In another aspect, the present invention provides a combinatorial library of at least 10 indolinone compounds that can be formed by reacting oxindole with an aldehyde. Here, oxindole has the formula I (
Having the structure as defined herein, wherein the aldehyde is of the formula ZC (O) -H, wherein Z is as defined herein. Having.

Oxindole in the combinatorial library described above is preferably
-[2- (3-isopropyl-phenoxy) -ethyl] -1,3-dihydro-indol-2-one, 4- [2- (2-isopropyl-phenoxy) -ethyl]
-1,3-Dihydro-indol-2-one, 4- [2- (biphenyl-3-yloxy) -ethyl] -1,3-dihydro-indol-2-one, 4- (2-
(Hydroxy-ethyl) -1,3-dihydro-indol-2-one, phenyl-
Thiocarbamic acid O- [2- (2-oxo-2,3-dihydro-1H-indol-4-yl) -ethyl] ester, phenyl-carbamic acid 2- (2-oxo-2,3-dihydro-1H) -Indol-4-yl) -ethyl ester, ter
t-butyl-carbamic acid 2- (2-oxo-2,3-dihydro-1H-indol-4-yl) -ethyl ester, cyclohexyl-carbamic acid 2- (2-
Oxo-2,3-dihydro-1H-indol-4-yl) -ethyl ester,
Benzenesulfonyl-carbamic acid 2- (2-oxo-2,3-dihydro-1H
-Indol-4-yl) -ethyl ester, biphenyl-2-yl-carbamic acid 2- (2-oxo-2,3-dihydro-1H-indol-4-yl) -ethyl ester, ethyl-carbamic acid 2- (2-oxo-2,3-dihydro-1
H-indol-4-yl) -ethyl ester, 4- [2- (4-methoxy-phenoxy) -ethyl] -1,3-dihydro-indol-2-one, 4- (2-
Methoxy-ethyl) -1,3-dihydro-indol-2-one, 4- (2-ethoxy-ethyl) -1,3-dihydro-indol-2-one, 4- [2- (4
-Isopropyl-phenoxy) -ethyl] -1,3-dihydro-indole-2
-One, 4- [2- (5-chloro-pyridin-3-yloxy) -ethyl] -1
, 3-Dihydro-indol-2-one and isopropyl-carbamic acid 2-
It is selected from the group consisting of (2-oxo-2,3-dihydro-1H-indol-4-yl) -ethyl ester.

The aldehyde is preferably

Embedded image

Embedded image Selected from the group consisting of:

Another aspect of the present invention provides a method of synthesizing a compound of Formula IV or Formula V (as described herein). A method of synthesizing a compound of Formula IV includes reacting a first reactant with a second reactant in a solvent in the presence of a mixture of other reagents. The reaction may be performed at room temperature or at an elevated temperature.

[0161] The first reactant is preferably oxindole, more preferably 4 oxindole.
-(2-hydroxyethyl) -1,3-dihydro-indol-2-one. The second reactant is preferably selected from the group consisting of alcohols, iodoalkyl, alkyl or aryl isocyanates, and alkyl or aryl isothiocyanates, and more preferably, 3-isopropylphenol,
2-isopropylphenol, 3-phenylphenol, 4-methoxyphenol, 5-chloro-3-pyridinol, methyl iodide, ethyl iodide, phenyl isocyanate, tert-butyl isocyanate, cyclohexyl isocyanate, benzenesulfonyl isocyanate, 2-biphenylyl It is selected from the group consisting of isocyanate, ethyl isocyanate, isopropyl isocyanate, and phenyl isothiocyanate.

“A mixture of other reagents” generally refers to a mixture of synthetic reagents or solvents that will facilitate the synthesis of the compounds of the present invention, and is a mixture of diethyl azodicarboxylate and triphenylphosphine in a solvent. Included are mixtures containing silver trifluoromethanesulfonate itself or a base in a mixture, or in a solvent, or in a mixture of solvents.

A method of synthesizing a compound of Formula V includes reacting a first reactant with a second reactant at an elevated temperature in a solvent in the presence of a base. Where the first reactant is of the formula I
An oxindole having a structure as defined herein (as defined herein), wherein the second reactant is of the formula Z—C (O) —H, wherein Z is As defined in).

The first reactant is preferably 4- [2- (3-isopropyl-phenoxy)
-Ethyl] -1,3-dihydro-indol-2-one, 4- [2- (2-isopropyl-phenoxy) -ethyl] -1,3-dihydro-indol-2-one, 4- [2- ( Biphenyl-3-yloxy) -ethyl] -1,3-dihydro-
Indole-2-one, 4- (2-hydroxy-ethyl) -1,3-dihydro-
Indole-2-one, phenyl-thiocarbamic acid O- [2- (2-oxo-
2,3-Dihydro-1H-indol-4-yl) -ethyl] ester, phenyl-carbamic acid 2- (2-oxo-2,3-dihydro-1H-indole-4
-Yl) -ethyl ester, tert-butyl-carbamic acid 2- (2-oxo-2,3-dihydro-1H-indol-4-yl) -ethyl ester, cyclohexyl-carbamic acid 2- (2-oxo-2 , 3-Dihydro-1H-indol-4-yl) -ethyl ester, benzenesulfonyl-carbamic acid 2- (2
-Oxo-2,3-dihydro-1H-indol-4-yl) -ethyl ester, biphenyl-2-yl-carbamic acid 2- (2-oxo-2,3-dihydro-
1H-indol-4-yl) -ethyl ester, ethyl-carbamic acid 2- (
2-oxo-2,3-dihydro-1H-indol-4-yl) -ethyl ester, 4- [2- (4-methoxy-phenoxy) -ethyl] -1,3-dihydro-
Indole-2-one, 4- (2-methoxy-ethyl) -1,3-dihydro-indol-2-one, 4- (2-ethoxy-ethyl) -1,3-dihydro-indol-2-one, 4- [2- (4-isopropyl-phenoxy) -ethyl]-
1,3-dihydro-indol-2-one, 4- [2- (5-chloro-pyridin-3-yloxy) -ethyl] -1,3-dihydro-indol-2-one, and isopropyl-carbamic acid 2 -(2-oxo-2,3-dihydro-1H-
Oxindole selected from the group consisting of indole-4-yl) -ethyl ester.

The second reactant is preferably 4-bromobenzaldehyde, 2-pyridinecarboxaldehyde, 6-methyl-2-pyridine-carbaldehyde, 1H-indole-5-carbaldehyde, 4-methoxy-3- Thiophen-2-yl-
Benzaldehyde, 4- (3-dimethylamino-propyl) -3,5-dimethyl-1H-pyrrole-2-carbaldehyde, 3-hydroxy-6-methyl-pyridine-2-carbaldehyde, 4- (3-dimethylamino -Propyl) -3,5
Aldehyde selected from the group consisting of -dimethyl-1H-pyrrole-2-carbaldehyde and 4-carboxyethyl-3,5-dimethyl-2-formylpyrrole.

IV. In another aspect, the invention relates to (i) a physiologically acceptable carrier, diluent,
Or an excipient; and (ii) a pharmaceutical composition comprising a compound described herein.

The present invention also features a method of modulating the function of a protein kinase using a compound of the present invention. The method comprises contacting a cell expressing the protein kinase with the compound.

In yet another aspect of the invention, the protein kinase whose catalytic activity is modulated by a compound of the invention is selected from the group consisting of receptor protein tyrosine kinase, cellular tyrosine kinase and serine-threonine kinase. .

In one aspect of the invention, the receptor protein kinase whose catalytic activity is modulated by a compound of the invention is EGF, HER2, HER3, HER4, I4
R, IGF-1R, IRR, PDGFRα, PDGFRβ, CSFIR, CK
it, C-fms, Flk-1R, Flk4, KDR / Flk-1, Flt-1
, FGFR-1R, FGFR-2R, FGFR-3R and FGFR-4R. Further, in one aspect of the present invention, the cellular tyrosine kinase whose catalytic activity is regulated by the compounds of the present invention is Src, Frk
, Btk, Csk, Abl, ZAP70, Fes / Fps, Fak, Jak, A
ck, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk
Selected from the group consisting of: In another aspect of the invention, the serine-threonine protein kinase whose catalytic activity is modulated by a compound of the invention is selected from the group consisting of CDK2 and Raf.

The natural binding partner of a protein kinase can bind with high affinity to the intracellular region of the protein kinase. High affinity refers to an equilibrium binding constant of the order of 10 ^-6 M or lower. In addition, natural binding partners can also transiently interact with and chemically modify the intracellular region of the protein kinase. Natural binding partners of protein kinases include, but are not limited to, S
RC homology 2 (SH2) or 3 (SH3) domains, other phosphoryl tyrosine binding (PTB) domains, guanine nucleotide exchange factors, protein phosphatases, and other protein kinases. Methods for detecting changes in the interaction between a protein kinase and its natural binding partner are readily available in the art.

The compounds of the present invention preferably modulate the activity of a protein tyrosine kinase in vitro. These compounds preferably show a positive result in one or more in vitro assays (eg, the assays described in the Examples below) for activity corresponding to the treatment of the disease or disorder in question.

The invention also features a method of identifying a compound that modulates a function of a protein kinase. The method comprises the following steps: (a) contacting a cell that expresses a protein tyrosine kinase with a compound; and (b) monitoring the effect on the cell. The effect on the cell is preferably a change or no change in the cell phenotype, more preferably it is a change or no change in cell proliferation, even more preferably this is a change or a change in the catalytic activity of the protein kinase. It is unchanged, most preferably it is a change or no change in the interaction between the protein kinase and the natural binding partner as described herein.

In a preferred embodiment, the invention features a method of identifying a compound of the invention. The method comprises the following steps: (a) lysing the cells to obtain a lysate containing the protein tyrosine kinase; (b) adsorbing the protein tyrosine kinase to the antibody; (c)
Ii) incubating the adsorbed protein tyrosine kinase with the substrate; and (d) adsorbing the substrate to a solid support or antibody, wherein monitoring the effect on cells comprises measuring the phosphate concentration of the substrate. including.

[0174] In yet another aspect, the invention features a method of treating a disease associated with unregulated kinase signaling. The method comprises administering to a subject in need of treatment a therapeutically effective amount of a compound of the invention described herein.

[0175] The invention also features a method of controlling kinase signaling. The method comprises administering to the subject a therapeutically effective amount of a compound of the invention described herein.

Further, the invention features a method of preventing or treating an abnormal condition in an organism, wherein the abnormal condition comprises a signal characterized by an interaction between a protein kinase and a natural binding partner. Associated with abnormalities in transmission pathways. The method comprises:
It comprises the following steps: (a) administering a compound of the invention described herein; and (b) promoting or disrupting the abnormal interaction. The organism is preferably a mammal, and the abnormal condition is preferably cancer. The abnormal condition is also preferably
Hypertension, depression, general anxiety, phobia, post-traumatic stress syndrome, adolescent personality disorder, sexual dysfunction, intake disorder, obesity, chemical dependence, cluster headache, migraine, pain, Alzheimer's disease, compulsive It is selected from the group consisting of disorders, panic disorders, memory disorders, Parkinson's disease, endocrine disorders, vasospasm, cerebellar ataxia, and gastrointestinal disorders.

As used herein, “PK-related disease”, “PK-promoting disease”, and “abnormal PK activity” are all inappropriate, ie, incomplete, or, more generally, excessive. , Representing a condition characterized by PK catalytic activity, wherein the particular PK can be an RTK, CTK, or STK. Inappropriate catalytic activity is; (1
) Expression of PK in cells that normally do not express PK, (2) increased PK expression leading to unwanted cell proliferation, differentiation and / or growth, or (3) cell proliferation,
Reduced PK expression leading to an undesirable decrease in differentiation and / or growth. Excessive activity of a PK represents amplification of the gene encoding a particular PK or production of a level of PK activity that can correlate with impaired cell proliferation, differentiation and / or growth (ie, as PK levels increase, , Cellular disease 1
Or more severe symptoms). Incomplete activity is, of course, the converse; as PK levels decrease, the severity of one or more cellular disease symptoms increases.

As used herein, the term “therapeutically effective amount” refers to the amount of a compound administered that will alleviate one or more symptoms of the disorder being treated. . For the treatment of cancer, a therapeutically effective amount can be (1) reduce the size of the tumor, (2) inhibit the metastasis of the tumor (ie, reduce the rate to some extent, preferably stop), (3) Has the effect of inhibiting growth to some extent (i.e., slowing down to some extent, preferably halting) and / or (4) to some extent alleviating (or preferably eliminating) one or more symptoms associated with cancer. Express the amount.

In one aspect of the present invention, the above-mentioned protein kinase-related disease is selected from the group consisting of a receptor protein tyrosine kinase-related disease, a cellular tyrosine kinase-related disease, and a serine-threonine kinase-related disease.

In still another aspect of the present invention, the above-mentioned protein kinase-related disease is EG.
It is selected from the group consisting of FR-related diseases, PDGFR-related diseases, IGFR-related diseases, and flk-related diseases.

In another aspect of the present invention, the above-mentioned protein kinase-related disease is squamous cell carcinoma, astrocytoma, glioblastoma, lung cancer, bladder cancer, head and neck cancer, melanoma, ovarian cancer. , Prostate cancer, breast cancer, small cell lung cancer and glioma.

The above-mentioned protein kinase-related diseases include diabetes, immune diseases such as autoimmune diseases,
Selected from the group consisting of hyperproliferative diseases, restenosis, fibrosis, psoriasis, osteoarthritis, rheumatoid arthritis, inflammatory diseases, angiogenesis, cardiovascular diseases such as arteriosclerosis, and kidney diseases. Is another aspect of the present invention.

In addition to modulating PK activity, compounds of the present invention can inhibit the activity of protein phosphatase, an enzyme that removes phosphate groups from phosphorylated proteins. That is, the compounds disclosed herein are also a new generation of therapeutic compounds for diseases and disorders associated with abnormal phosphatase activity, such as, but not limited to, diabetes, cell proliferative disorders and inflammatory disorders. . One skilled in the art will understand that the terms defined herein in relation to PK have the same or similar meaning with respect to phosphatases.

The summary of the invention described above is non-limiting and other features and advantages of the invention include:
It will be apparent from the following description of the preferred embodiments and from the claims.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to compounds capable of controlling and / or modulating cellular signaling, and in a preferred embodiment receptor and non-receptor tyrosine kinase signaling.

Receptor kinase-mediated signaling is initiated by extracellular interactions with specific growth factors (ligands), followed by receptor dimerization, transient stimulation of intrinsic protein kinase activity and phosphorylation . The binding site for the intracellular signaling molecule thus formed can provide an appropriate cellular response (eg, cell division,
Metabolic effects on the extracellular microenvironment), leading to the formation of complexes with a range of cytoplasmic signaling molecules. Schlessinger and Ullr
ich, 1992, Neuron 9: 303-391.

The tyrosine phosphorylation site of the growth factor receptor is located at the signaling molecule SH2 (
(src homology) domain has been shown to function as a high affinity binding site (Fantl et al. 1992, Cell 69: 413-423;
Songyang et al. , 1994, Mol. Cell. Biol. 1
4: 2777-2785); Songyang et al. , 1993, Ce
11 72: 767-778; and Koch et al. , 1991, Sc
issue 252: 668-678). Several intracellular matrix proteins associated with receptor kinases have been identified. These can be divided into two main groups: (1) substrates that have a catalytic domain; and (2) substrates that do not have such a domain but act as adapters and associate with catalytically active molecules ( S
onyangang et al. , 1993, Cell 72: 767-778).
. The specificity of the interaction between the receptor and the SH2 domain of its substrate is determined by the amino acid residues immediately adjacent to the phosphorylated tyrosine residues. Differences in binding affinity between the SH2 domain and the amino acid sequence around the phosphotyrosine residue on a particular receptor are consistent with the differences observed in the phosphorylation profile of the substrate (Songyang et al., 1993, Cell 72). :
767-778). These findings suggest that the function of each receptor kinase depends not only on its pattern of expression and ligand availability, but also on the array of downstream signaling pathways activated by specific receptors. Thus, phosphorylation provides an important regulatory step in determining the selectivity of signaling pathways recruited by specific growth factor receptors as well as differentiation factor receptors.

Kinase signaling results in cell proliferation, differentiation and metabolism, among other responses. Abnormal cell proliferation causes a wide range of diseases and illnesses, including neoplastic development, such as carcinomas, sarcomas, leukemias, glioblastomas, hemangiomas, psoriasis, atherosclerosis, arthritis and diabetic retinopathy (or Uncontrolled angiogenesis and / or other diseases associated with angiogenesis).

[0189] Accordingly, the present invention relates to controlling and modulating kinase signaling by affecting the enzymatic activity of receptor kinases (RK) and / or non-receptor kinases and interfering with the signals transmitted by such proteins. And / or inhibiting compounds. More specifically, the present invention relates to many types of solid tumors, including, but not limited to, carcinomas, sarcomas, erythroblastomas, glioblastomas, meningiomas, astrocytomas, melanomas and muscles. Therapeutic methods for healing blastomas include controlling, modulating and / or regulating RK and / or non-receptor kinase-mediated signaling pathways.
Or a compound that inhibits. Abstracts include, but are not limited to, brain cancer, bladder cancer, ovarian cancer, gastric cancer, pancreatic cancer, colon cancer, blood cancer, lung cancer and bone cancer.

I. Target Diseases to be Treated by the Compounds of the Invention The compounds described herein are useful for treating diseases associated with unregulated kinase signaling, such as cell proliferative, fibrotic and metabolic diseases. Cell proliferative disorders that can be treated or further studied according to the present invention include cancer, vascular proliferative disorders and mesangial cell proliferative disorders.

[0191] Vascular proliferative disorders generally refer to angiogenic and angiogenic disorders that cause abnormal growth of blood vessels. The formation and spread of blood vessels, or angiogenesis and angiogenesis, respectively, play an important role in various physiological processes such as embryonic development, luteal formation, wound healing and organ regeneration. They also play a pivotal role in cancer development. Other examples of vascular proliferative disorders include arthritis, where new capillaries invade the joints and destroy cartilage, and ocular diseases, such as new capillaries in the retina invading the vitreous, bleeding, Includes diabetic retinopathy that causes blindness. Conversely, involvement in diseases associated with vasoconstriction, contraction or obstruction, such as restenosis, has also been suggested.

[0192] Fibrous disease refers to the abnormal formation of extracellular matrix. Examples of fibrotic disorders include cirrhosis and mesangial cell proliferative disorders. Cirrhosis is characterized by an increase in the composition of the extracellular matrix that causes the formation of liver scars. Cirrhosis
It can cause diseases such as cirrhosis of the liver. Increased extracellular matrix that causes liver scarring is also due to viral infections such as the hepatitis virus. Adipocytes appear to play a major role in cirrhosis. Other fibrotic disorders that have been suggested include atherosclerosis (see below).

A mesangial cell proliferation disease refers to a disease caused by abnormal growth of mesangial cells. Mesangial proliferative disorders include various human kidney diseases such as glomerulonephritis, diabetic nephropathy, malignant renal sclerosis, thrombotic microvascular syndrome, transplant rejection, and glomerulopathy. PDGF-R has been suggested to be involved in the maintenance of mesangial cell proliferation (Floege et al., 1993, Ki).
dney International 43: 47S-54S).

PK has been linked to such cell proliferative disorders. For example, several members of the receptor tyrosine kinase family have been implicated in the development of cancer. EGFR (Tuzi et al., 1991, Br. J. Cancer)
63: 227-233, Torp et al. , 1992, APMIS 1
00: 713-719), HER2 / neu (Slamon et al., 1).
989, Science 244: 707-712), and PDGF-R (K
umabe et al. , 1992, Oncogene, 7: 627-633.
Some of these receptors are overexpressed in many tumors and / or are persistently activated by the autocrine loop. In fact, in most common and severe cancers, overexpression of these receptors (Akbasa
k and Suner-Akbasak et al. , 1992, J. Mol. Ne
urol. Sci. , 111: 119-133, Dickson et al.
, 1992, Cancer Treatment Res. 61: 249-27
3, Korc et al. , 1992, J. Mol. Clin. Invest. 90,
1352-1360), and an autocrine loop (Lee and Dono)
ghue, 1992, J. Am. Cell. Biol. , 118: 1057-1070.
, Korc et al. , Supra, Akbasak and Suner-Ak
Basak et al. , Supra) are shown. For example, EGFR has been associated with squamous cell carcinoma, astrocytoma, neuroblastoma, head and neck cancer, lung cancer, and bladder cancer. HER2 has been associated with breast, ovarian, stomach, lung, pancreas, and bladder cancer. PDGF-R has been associated with neuroblastoma and lung, ovarian, melanoma and prostate cancer. RK c-met has generally been linked to liver tumorigenesis and, therefore, to hepatocellular tumors. In addition, c-met has been implicated in malignant tumorigenesis. More specifically, RK c-met is, among other cancers,
It has been associated with colorectal, thyroid, pancreatic, and gastric carcinomas, and with leukemia and lymphoma. In addition, overexpression of the c-met gene has also been detected in Hodgkin's and Burkitt's disease patients and lymphoid cell lines. Similarly, Flk
It has been associated with a wide spectrum of tumors, including but not limited to breast cancer, ovarian cancer, lung tumors, and gliomas such as glioblastoma.

IGF-IR has been implicated in several types of cancer, in addition to being implicated in nutritional support and type II diabetes. For example, IGF-I has been used in several types of tumors, for example, carcinoma cells of human breast cancer (Arteaga et al.,
1989, J. Mol. Clin. Invest. 84: 1418-1423), and small cell lung tumors (Macauley et al., 1990, Cancer R).
es. , 50: 2511-1517) as autocrine growth stimulating factors. In addition, IGF-I is completely involved in the normal growth and differentiation of the nervous system and appears to be an autocrine stimulator of human glioma. Sand
berg-Nordqvist et al. , 1993, Cancer Re.
s. 53: 2475-2478. The importance of IGF-IR and its ligands in cell proliferation is due to the number of cell types in culture (fibroblasts, epithelial cells, smooth muscle cells, T lymphocytes, myeloid cells, chondrocytes, and osteoblasts ( Bone marrow stem cells)
Further supported by the fact that they are stimulated to grow by FI.
Golding and Goldring, 1991, Eukaryoti
c Gene Expression, 1: 301-326. In a series of recent publications, Basega noted that IGF-IR plays a central role in the mechanism of transformation and that it may itself be a preferred target for therapeutic intervention against a wide range of human malignancies. Suggests. Basega, 1
995, Cancer Res. , 55: 249-252, Basega, 1
994, Cell 79: 927-939, Coppola et al. , 1
994, Mol. Cell. Biol. , 14: 4588-4595.

[0196] Certain protein kinases (PKs) have been suggested to be involved in many types of cancer, particularly breast cancer (Cance, et al., Int. J. Cancer).
, 54: 571-77 (1993)).

However, the association between RK abnormalities and disease is not limited to cancer. For example, RK
Has been implicated in metabolic diseases such as psoriasis, diabetes mellitus, wound healing, inflammation, and neurodegenerative diseases. These diseases include, but are not limited to, hypertension, depression, general anxiety, phobia, post-traumatic stress syndrome, adolescent personality disorder, sexual dysfunction, intake disorders, obesity, chemical dependence, cluster headache, Migraine, pain, Alzheimer's disease,
Obsessive-compulsive disorder, panic disorder, memory disorder, Parkinson's disease, endocrine disorders, vasospasm,
Includes cerebellar ataxia and gastrointestinal tract disease. For example, EGF-R has been implicated in corneal and skin wound healing. Insulin-R and IGF-1R deficiencies have been shown in type II diabetes mellitus. A more complete correlation between specific RKs and their therapeutic abstracts is described in Plowman et al. , 19
94, DN & P7: 334-339.

Not only receptor-type kinases but also many cellular kinases (CK), such as src, abl, fps, yes, fyn, lyn, lck, blk, hc
k, fgr, yrk (Bolen et al., 1992, FASEB J. et al.
6: 3403-3409) have been implicated in growth and metabolic signaling pathways, and thus in the summary of the invention. For example, mutant src (v
-Src) has been shown to be an oncoprotein in chickens (pp60 ^v-src ). Furthermore, its cellular homolog, protooncodin pp60 ^c-src
Transmits oncogenic signals of many receptors. For example, EGF in tumor
Overexpression of -R or HER2 / neu leads to constitutive activation of pp60 ^c-src , which is characteristic of malignant cells but not of normal cells. On the other hand, c−s
Mice lacking rc expression display an osteopetrotic phenotype, implying an important role for c-src in osteoclast function and a possible role in related diseases.

Similarly, Zap70 has been suggested to be involved in T cell signaling.

[0200] It is further an aspect of the present invention to identify CTK modulating compounds to increase RK targeting blockers or to obtain a synergistic effect.

Furthermore, both RK and non-receptor type kinases have been implicated in hyperimmune diseases.

Further, the compounds of the present invention are also effective in treating diseases associated with PYK-2 protein. This protein, its cellular function, and related diseases are described by Le.
v et al. , "PYK2 RELATED PRODUCTS AND
No. 08 / 357,642 entitled "METHODS" (December 1994).
(Filed on March 15) (Lyon & Lyon Socket No. 209/070)
And Lev et al. , "PYK2 RELATED PRODUCTS
No. 08 / 460,626 (19) entitled "AND METHODS".
(Filed on June 2, 1995) (Lyon & LyonDocket No. 211/121)
(Both of which are hereby incorporated by reference in their entirety, including the drawings).

Finally, the present invention provides a method for modulating the function of oxindole and indolinone compounds and protein phosphatases, and for preventing abnormal conditions associated with protein phosphatases in organisms using the compounds of the above identified methods. And methods of treatment. The compounds of the present invention, as well as those obtained by adding chemical substituents, may inhibit the action of phosphatase and may be a new generation of treatment for diseases associated with the phosphatase deficiency. For those skilled in the art, the terms defined above for kinases have a similar meaning for phosphatases. Both RK and non-receptor type kinases have been implicated in hyperimmune diseases.

Further, the compounds of the present invention are also effective in treating diseases associated with PYK-2 protein. This protein, its cellular function, and related diseases are described by Le.
v et al. , "PYK2 RELATED PRODUCTS AND
US Patent No. 5,837,524 entitled "METHODS" (November 17, 1998).
Issued on the same day) and Lev et al. , "PYK2 RELATED PRO
U.S. Pat. No. 5,837,815 entitled "DUCTS AND METHODS"
(November 17, 1998), both of which are incorporated herein by reference in their entirety, including the drawings.

II. KDR / FLK-1 a. KDR / FLK-1 receptor and VEGF Normal angiogenesis and angiogenesis are involved in various physiological processes such as embryonic development, wound healing and organ regeneration, and follicle formation in the luteal body during ovulation and placenta in the pregnancy. Plays an important role in female reproductive processes such as growth. Folkman and Sh
ing, 1992, J. Mol. Biological Chem. 267: 10931
-34. However, many diseases are driven by uncontrolled or inappropriate persistence of angiogenesis. For example, in arthritis, new capillaries invade the joints and destroy cartilage. In diabetes, new capillaries in the retina invade the vitreous, bleed and cause blindness (Folkman, 1987: Congress).
of Thrombosis and Haemostasis (Verst
rate, et. al, eds. ), Leuven University
Press, Leueven, pp. 583-596). Ocular neovascularization is the most common cause of blindness and is dominant in nearly 20 ocular diseases.

In addition, angiogenesis and / or angiogenesis has been linked to the growth and metastasis of malignant solid tumors. Tumors must continue to stimulate the growth of new capillaries in order for the tumor itself to grow. In addition, new blood vessels implanted in tumors provide a way for tumor cells to enter the circulation and metastasize to distant parts of the body (Folkman, 1990, J. Natl. Cancer Inst.
82: 4-6; Klagsbrunn and Soker, 1993, Cur.
rent Biology 3: 699-702; Folkman, 1991,
J. Natl. , Cancer Inst. 82: 4-6; Weidner e
tal, 1991, New Engl. J. Med. 324: 1-5).

[0207] Several polypeptides having in vitro endothelial cell growth promoting activity have been identified. Examples include acidic and basic fibroblast growth factors (aFGF, bFG
F), vascular endothelial growth factor (VEGF) and placental growth factor. aFG
Unlike F and bFGF, VEGF was recently reported to be an endothelial cell-specific mitogen (Ferrara and Henzel, 1989).
, Biochem. Biophys. Res. Comm. 161: 851-85
8; Vaisman et al. , 1990, J.A. Biol. Chem. 26
. 5: 19461-19566).

Thus, the identification of specific receptors to which VEGF binds is an important advance in understanding the regulation of endothelial cell proliferation. Two structurally related RKs that bind VEGF with high affinity have been identified: the flt-1 receptor (Shibu
ya et al. , 1990, Oncogene 5: 519-524; De.
Vries et al. , 1992, Science 255: 989-99.
1) and KDR / FLK-1 receptor (U.S. patent application Ser. No. 08 / 193,829).
Is discussed in). Thus, it is speculated that these RKs will play a role in regulating and controlling endothelial cell proliferation.

Evidence such as the disclosure described in co-pending US patent application Ser. No. 08 / 193,829 indicates that VEGF is not only responsible for endothelial cell proliferation, but also for normal and pathological angiogenesis. It strongly suggests that it is a major regulator. Generally, Klagsbum
and Soker, 1993, Current Biology 3:69.
9-702; Hook et al. , 1992, J. Mol. Biol. Chem.
267: 26031-26037. Furthermore, KDR / FLK-1
And flt-1 have been shown to be expressed in large amounts in proliferating endothelial cells of growing tumors, but not in surrounding quiescent endothelial cells (Plate et al., 1992, Nature 359: 8.
45-848; Shweiki et al. , 1992, Nature 35
9: 843-845).

B. Identification of agonists and antagonists for the KDR / FLK-1 receptor Endothelial cell proliferation, and potentially the management of angiogenesis and / or angiogenesis,
In view of the putative importance of RK in control and regulation, many attempts have been made to identify RK "inhibitors" using a variety of methods. These include mutant ligands (US Pat. No. 4,966,849); soluble receptors and antibodies (W
O94 / 10202; Kendall and Thomas, 1994, Pr.
oc. Natl. Acad. Sci. USA 90: 10705-10709;
Kim et al. , 1993, Nature 362: 841-844);
And RNA ligands (Jellinek et al., 1994, Bioc).
Chemistry 33: 10450-10456).

Furthermore, kinase inhibitors (WO94 / 03427; WO92 / 21660; W
WO 91/14808; US Patent 5,330,992; M
ariani et al. , 1994, Proc. Am. Assoc. Can
cer Res. 35: 2268), and inhibitors that act on the receptor kinase signaling pathway, such as protein kinase C inhibitors have been identified (Sch
uchter et al. , 1991, Cancer Res. 57: 682
-687); Takano et al. 1993, Mol. Bio. Cel
14: 358A, Kinsella et al. , 1992, Exp. Ce
ll Res. 199: 56-62; Wright et al. , 1992,
J. Cellular Phys. 752: 448-57).

[0212] Recently, attempts have been made to identify small molecules that act as kinase inhibitors for use in treating cancer. Accordingly, there is an unmet need to identify and generate effective small compounds that selectively inhibit KDR / FLK-1 receptor signaling to effectively and specifically inhibit angiogenesis. I do.

Certain compounds of the present invention have shown excellent activity in biological assays; therefore, these compounds and related compounds have been shown to inhibit Flk-related diseases, such as persistent uncontrolled or inappropriate angiogenesis. It is expected to be effective in treating the disease being promoted.

III. Pharmaceutical Formulations and Routes of Administration The compounds described herein may be administered to human patients as such or may be mixed with other active ingredients, as in a combination therapy, with a suitable carrier or excipient. It can be administered in an article. Techniques for the formulation and administration of the compounds described herein are described in "Remington's Pharmcol."
physical Sciences "(Mack Publishing Co.
, Easton, PA).

A ) Routes of administration Suitable routes of administration include, for example, oral, rectal, transmucosal, or intestinal administration, or parenteral delivery, such as intramuscular, subcutaneous, intravenous, intramedullary, and intramedullary, direct intraventricular , Including intraperitoneal, intranasal, or intraocular injection.

Alternatively, the compounds may be administered locally rather than systemically, for example, by injection of the compound directly into a solid tumor, often in a depot or sustained release formulation.

In addition, drugs can be used in targeted drug delivery systems.
For example, it may be administered in liposomes coated with a tumor-specific antibody. Liposomes will be targeted to the tumor and will be selectively taken up.

B) Compositions / Formulations The pharmaceutical compositions of the present invention may be prepared in a manner known per se, for example by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing. Can be manufactured.

Thus, a pharmaceutical composition for use in accordance with the present invention comprises one or more excipients and auxiliaries that facilitate processing of the active into a pharmaceutically usable product. Can be formulated in a conventional manner using a physiologically acceptable carrier. Proper formulation is dependent upon the route of administration chosen.

For injection, the agents of the invention can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's 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.

For oral administration, the compounds can be formulated readily by mixing the compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the present invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient to be treated. I do.

Pharmaceutical formulations for oral use are prepared by mixing one or more solid excipients with one or more compounds of the invention and optionally grinding the resulting mixture, if desired. After adding suitable auxiliaries, the mixture of granules can be processed to give tablets or dragee cores. Suitable carriers are, in particular, sugars such as lactose, sucrose, mannitol or sorbitol; cellulose products such as corn starch, wheat starch, rice starch and potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose. Sodium and / or polyvinylpyrrolidone (P
Excipients such as bulking agents such as VP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. A concentrated sugar solution can be used for this purpose. This is gum arabic, talc,
Polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and / or
Alternatively, it can optionally include titanium dioxide, a lacquer solution, and a suitable organic solvent or solvent mixture. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active ingredient doses.

Pharmaceutical preparations 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, sorbitol. Push-fit capsules can contain the active ingredient in a mixture with fillers such as lactose, binders such as starch, and / or lubricants such as talc and magnesium stearate, and optionally, stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol. Further, a stabilizer may be added. All formulations for oral administration should be prepared in dosages suitable for such administration.

For buccal administration, the compositions may take the form of tablets or lozenges in conventional manner.

For administration by inhalation, the substances used according to the invention are propellants, for example,
It is conveniently transported in pressurized packs or nebulizers in the form of an aerosol spray using dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be adjusted with a valve provided to deliver a metered amount. Capsules and cartridges made 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 can be formulated for parenteral administration, eg, by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage, for example, in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Good.

Pharmaceutical compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble form.
Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes and the like. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, eg, sterile pyrogen-free water, before use.

The compounds can also be formulated in rectal compositions such as suppositories or retention enemas, using conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds may also be formulated as a depot preparation.
Such long acting formulations can be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection. That is, the compound is formulated with a suitable polymeric or hydrophobic substance (eg, as an emulsion in a pharmacologically acceptable oil) and with an ion exchange resin as a sparingly soluble derivative such as a sparingly soluble salt. be able to.

The pharmaceutical carrier for the hydrophobic compounds of the present invention is a co-solvent system comprising benzyl alcohol, a non-polar surfactant, a water-miscible organic polymer and an aqueous phase. The co-solvent system may be a VPD co-solvent system. VPD consisted of 3% (w / v) benzyl alcohol, 8% (w / v) non-polar surfactant polysorbate 80, and 65% (w / v).
v) A solution prepared by preparing polyethylene glycol 300 in pure ethanol.
The VPD co-solvent system (VPD: D5W) is a 1: 1 dilution of VPD in an aqueous solution of 5% dextrose. This co-solvent system dissolves hydrophobic compounds well and as such exhibits low toxicity upon systemic administration. Naturally, the proportions of a co-solvent system can be varied considerably without destroying its solubility and toxic properties. In addition, the identity of the co-solvent components can be varied. For example, other low toxicity non-polar surfactants can be used in place of polysorbate 80, and the fraction size of polyethylene glycol can vary. Other biocompatible polymers, such as polyvinylpyrrolidone, can be used instead of polyethylene glycol, and other sugars or polysaccharides can be used instead of dextrose.

Alternatively, other delivery systems for hydrophobic pharmaceutical compounds may be used. Liposomes and emulsions are well known as examples of delivery vehicles or carriers for hydrophobic drugs. In addition, certain organic solvents such as dimethyl sulfoxide can also be used, but often are more toxic. In addition, the compounds can be delivered using sustained release systems, for example, semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may take from weeks to 10 depending on their chemistry.
The compound is released for more than 0 days. Additional protein stabilization strategies may be used depending on the chemical nature and biological stability of the therapeutic agent.

Many of the PTK modulators of the present invention can be provided as salts with pharmaceutically compatible counterions. Pharmaceutically compatible salts can be formed with a number of acids, including but not limited to hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, maleic acid, succinic acid, and the like. Salts tend to be more soluble in aqueous or other protic solvents than in the corresponding free base form.

C) Effective Dosage Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount of a compound effective to prevent, alleviate, or ameliorate the symptoms of a disease, or to prolong the survival of a patient being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. For example, in animal models, a dose can be formulated to achieve a circulating concentration range that includes the IC ₅₀ (ie, the concentration of the test compound that achieves half-maximal inhibition of PK activity) in cultured cells. Such information can be used for more accurate determination of useful doses in humans.

The toxicity and therapeutic efficacy of the compounds described herein can be determined by standard pharmaceutical methods in cultured cells or experimental animals, such as LD ₅₀ (dose lethal to 50% of the population) and ED ₅₀ (the therapeutically effective dose in 50% of the population) can be determined by methods. The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD ₅₀ and ED _50. Compounds that exhibit high therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably toxicity include the ED ₅₀ is little within a range of circulating concentrations is no. Dosage may vary within this range depending upon the dosage form employed and the route of administration used. The exact formulation, route of administration, and dosage can be chosen by the individual physician in view of the patient's condition (see, eg, Fingl et al. 1975, "The Phar
macological Basis of Therapeutics ", C
h. 1, p. 1).

Dosage amount and interval can be adjusted individually to provide plasma levels of the active ingredient that are sufficient to maintain kinase modulating effects, ie, the minimum effective concentration (MEC). The MEC is different for each compound, but in vitro data, eg,
Using the assays described herein, one can extrapolate from the concentration required to achieve 50-90% inhibition of the kinase. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, plasma concentrations can be determined using HPLC assays or bioassays.

[0239] Dosage intervals can also be determined using MEC value. The compound is 10-
It should be administered using a regimen that maintains plasma levels above the MEC for 90% of the time, preferably 30-90% of the time, most preferably 50-90% of the time.

In the case of local administration or selective uptake, the effective local concentration of the drug will not be related to plasma concentration.

The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the attending physician.

D) The packaging composition may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient.
The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.

The pack or dispenser device may also be accompanied by an accompanying notice, accompanying the container, in a form stipulated by a governmental agency regulating the manufacture, use, or sale of the drug. It reflects the agency's approval of the shape of the object or for human or veterinary administration. Such notice, for example, may be on a label approved by the U.S. Food and Drug Administration for prescription dispensing, or it may be in the approved product. Compositions comprising a compound of the present invention, formulated in a compatible pharmaceutical carrier, may also be prepared, placed in an appropriate container, and labeled for treatment according to the indicated conditions. Appropriate conditions indicated on the label include treatment of tumors, inhibition of angiogenesis, treatment of fibrosis, diabetes and the like.

IV. Biological activity of compounds of the invention Compounds of the invention were tested for their ability to inhibit most protein kinase activities. The results of the biological assays and these inhibition studies are described herein. The method used to measure modulation of protein kinase function is described in Tang et al., "Indolinone Com."
binary Libraries and Related Pro
ducts and Methods for the Treatment
International Publication WO98 / 07695 (1998) entitled "of Disorders".
(Published March 26, 2012). Publication WO 98/07695 is hereby incorporated by reference in its entirety, including drawings.

V. Methods for preparing pharmaceutical compositions of the compounds of the invention and pharmaceutical formulations of administered compounds, methods for determining the amount of a compound to be administered to a patient, and modes of administering the compound to an organism are described in WO 98/07695 and Bu.
"Hydrosoluble 3-Arylidene-" by zzetti et al.
2-Oxindole Derivatives as Tyrosine K
International Publication WO96 / 22976 entitled "Inase Inhibitors"
(Published August 1, 1996), both of which are hereby incorporated by reference in their entirety, including the drawings. Those skilled in the art will appreciate that such description is applicable to the present invention and may be readily adapted thereto.

[0246] EXAMPLES The following examples are non-limiting, only various aspects and features of the representative of the present invention. The examples describe methods for synthesizing compounds of the invention and methods for determining the effect of compounds on protein kinase function.

The cells used in the method are commercially available. Cell-borne nucleic acid vectors are also commercially available, and the sequences of various protein kinase genes are readily accessible in sequence data banks. That is, one skilled in the art can easily reconstruct a cell line at an appropriate time by combining commercially available cells, commercially available nucleic acid vectors, and protein kinase genes using techniques readily available to those skilled in the art. Can be.

Synthetic Method Example 1: Method for Synthesizing the Compound of the Present Invention The compound of the present invention and the precursors 2-oxindole and aldehyde are easily synthesized using techniques well known in the field of chemistry. be able to. One skilled in the art will appreciate that other synthetic routes to form the compounds of the present invention are available and the following are provided by way of illustration and not limitation. Furthermore, similarly, compounds whose synthesis is described below are not to be construed as limiting the scope of the invention in any way.

A. 3-Arylidenyl-6-heterocyclyl-2-indolinone derivative compound AHI-1: 3- (4-methoxy-3-thiophen-2-yl-benzylidene) -6-pyridin-3-yl-1,3-dihydroindole- 2-One To a warm stirred solution of 6-bromo-2-oxindole (4 g, 26.3 mmol) in 60 mL toluene and 60 mL ethanol was added tetrakis (triphenylphosphine) palladium (0) (2.3 g, 1.9 mmol). ) Was added followed by 2M aqueous sodium carbonate (50 mL, 100 mmol) and pyridine-3-boronic acid, propanediol ester (5 g, 30.7 mmol). The mixture was stirred in an oil bath at 100 ° C. for 12 hours. The reaction mixture was cooled and ethyl acetate (500
mL) and washed with saturated sodium bicarbonate (200 mL), water (200 mL) and brine (200 mL). The organic layer was separated, dried over anhydrous magnesium sulfate and concentrated to give a brown solid. This solid was triturated in methylene chloride / diethyl ether to give 2.32 g (42%) of 6-pyridin-3-yl-1,3-
Dihydro-indol-2-one was obtained as a brown solid.

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6-Pyridin-3-yl-1,3-dihydro-indol-2-one (100 m
g, 0.5 mmol), a mixture of 4-methoxy-3-thiophen-2-yl-benzaldehyde (110 mg, 0.5 mmol) and piperidine (0.23 mL) in ethanol (4 mL) was heated to reflux and stirred overnight. . The reaction mixture was then cooled and diluted with ether to give a precipitate, which was removed by filtration. The filtrate was subjected to column chromatography (eluent-isopropanol / dichloromethane) to give 30 mg (15%) of 3- (4-methoxy-3-thiophen-2-yl-benzylidene) -6-pyridin-3-yl-1, 3-Dihydroindol-2-one was obtained as a yellow solid.

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Compound AHI-2: 3- (2-hydroxybenzylidene) -6-pyridin-3-yl-1,3-dihydroindol-2-one 6-pyridin-3-yl-1,3-dihydroindole- 2-one (100mg
, 0.5 mmol), 2-hydroxybenzaldehyde (60 mg, 0.5 mm
ol) and piperidine (0.23 mL) in ethanol (4 mL) was heated to reflux and stirred overnight. The reaction mixture is concentrated and subjected to column chromatography (
Eluate-isopropanol / dichloromethane) to give 100 mg (64%)
Of 3- (2-hydroxybenzylidene) -6-pyridin-3-yl-1,3-dihydroindol-2-one as a yellow solid.

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Compound AHI-3: 3- (4-Methoxy-3-thiophen-2-yl-benzylidene) -6-thiophen-2-yl-1,3-dihydroindol-2-one in 60 mL toluene and 60 mL ethanol To a warm stirred solution of 6-bromo-2-oxindole (4 g, 26.3 mmol) dissolved in tetrakis (triphenylphosphine) palladium (0) (2.3 g, 1.9 mmol) and then 2M aqueous Sodium carbonate (50 mL, 100 mmol) and thiophene-2
-Boronic acid (4.38 g, 34.2 mmol) was added in three portions over 1.5 hours. The mixture was stirred in an oil bath at 100 ° C. for 12 hours. The mixture was then diluted with ethyl acetate (400 mL), saturated sodium bicarbonate (200 mL), water (20 mL).
0 mL) and brine (200 mL). The organic layer was separated, dried over anhydrous magnesium sulfate and concentrated to give 2.53 g (42%) of 6-thiophene-2.
-Yl-1,3-dihydro-indol-2-one was obtained as a tan solid.

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6-thiophen-2-yl-1,3-dihydroindol-2-one (100 m
g, 0.5 mmol), a mixture of 4-methoxy-3-thiophene-2-benzaldehyde (110 mg, 0.5 mmol) and piperidine (0.23 mL) in ethanol (4 mL) was stirred and refluxed overnight. The reaction mixture was concentrated and subjected to column chromatography (eluate-isopropanol / dichloromethane) to give 10
0 mg (48%) of 3- (4-methoxy-3-thiophen-2-ylbenzylidene) -6-thiophen-2-yl-1,3-dihydroindol-2-one was obtained as a tan solid.

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Compound AHI-4: 3- (2-hydroxybenzylidene) -6-thiophen-2-yl-1,3-dihydroindole-2-one 6-thiophen-2-yl-1,3-dihydroindole- 2-On (100m
g, 0.5 mmol), 2-hydroxybenzaldehyde (60 mg, 0.5 m
mol) and piperidine (0.23 mL) in ethanol (4 mL) was stirred at reflux overnight. The reaction mixture was concentrated and subjected to column chromatography (eluent-isopropanol / dichloromethane) to give 80 mg (50%) of 3- (
2-Hydroxybenzylidene) -6-thiophen-2-yl-1,3-dihydroindol-2-one was obtained as a yellow-orange solid.

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Compound AHI-5: 3- (4-Methoxy-3-thiophen-2-yl-benzylidene) -6-thiophen-3-yl-1,3-dihydroindol-2-one in 60 mL toluene and 60 mL ethanol To a warm stirred solution of 6-bromo-2-oxindole (4 g, 26.3 mmol) dissolved in tetrakis (triphenylphosphine) palladium (0) (2.3 g, 1.9 mmol) and then 2M aqueous Sodium carbonate (50 mL, 100 mmol) and thiophene-3
-Boronic acid (4.3 g, 33.6 mmol) was added. Mix the mixture in an oil bath for 100
Stirred at C for 12 hours. The reaction mixture was cooled, diluted with ethyl acetate (500 mL), 1N hydrochloric acid (200 mL), water (200 mL), saturated sodium bicarbonate (2 mL).
00 mL) and brine (200 mL). The organic layer was separated, dried over anhydrous magnesium sulfate and concentrated to give a black solid. The solid was triturated in methylene chloride to give 2.02 g (36%) of 6-thiophen-3-yl-1,3-dihydroindol-2-one as a gray purple solid.

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6-thiophen-3-yl-1,3-dihydroindol-2-one (100 m
g, 0.5 mmol), a mixture of 4-methoxy-3-thiophene-2-benzaldehyde (110 mg, 0.5 mmol) and piperidine (0.23 mL) in ethanol (4 mL) was stirred and refluxed overnight. The precipitate is collected by vacuum filtration,
Wash with ethanol, dry and 100 mg (48%) of 3- (4-methoxy-
3-thiophen-2-yl-benzylidene) -6-thiophen-3-yl-1,
3-Dihydroindol-2-one was obtained as a tan solid.

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Compound AHI-6: 3- (2-hydroxybenzylidene) -6-thiophen-3-yl-1,3-dihydroindol-2-one 6-thiophen-3-yl-1,3-dihydroindole- 2-On (100m
g, 0.5 mmol), 2-hydroxy-benzaldehyde (60 mg, 0.5 mmol).
(mmol) and piperidine (0.23 mL) in ethanol (4 mL) was stirred at reflux overnight. The precipitate is collected by vacuum filtration, washed with ethanol,
Dry and dry 110 mg (69%) of 3- (2-hydroxybenzylidene) -6
Thiophen-3-yl-1,3-dihydroindol-2-one was obtained as yellow-red crystals.

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Compound AHI-7: 3- [4- (3-dimethylaminopropyl) -3,5-dimethyl-1H-pyrrol-2-ylmethylene] -6-pyridin-3-yl-1,3-dihydroindole -2-one 6-pyridin-3-yl-1,3-dihydroindol-2-one (100 mg
, 0.48 mmol), 4- (3-dimethylaminopropyl) -3,5-dimethyl-1H-pyrrole-2-carboxaldehyde (100 mg, 0.48 mmol)
A mixture of 1) and piperidine (1 drop) in ethanol (2 mL) was stirred at reflux overnight. The precipitate is collected by vacuum filtration, washed with ethanol, dried and
[4- (3-dimethylaminopropyl) -3,5-dimethyl-1H-pyrrole-
2-ylmethylene] -6-pyridin-3-yl-1,3-dihydroindole-
2-one was obtained.

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Compound AHI-8: 3- [4- (3-dimethylaminopropyl) -3,5-dimethyl-1H-pyrrol-2-ylmethylene] -6-thiophen-2-yl-1,3-dihydroindole -2-one 6-thiophen-2-yl-1,3-dihydroindol-2-one (100 m
g, 0.46 mmol), 4- (3-dimethylaminopropyl) -3,5-dimethyl-1H-pyrrole-2-carboxaldehyde (100 mg, 0.48 mm
ol) and piperidine (1 drop) in ethanol (2 mL) was stirred at reflux overnight. The precipitate is collected by vacuum filtration, washed with ethanol, dried and dried.
-[4- (3-Dimethylaminopropyl) -3,5-dimethyl-1H-pyrrol-2-ylmethylene] -6-thiophen-2-yl-1,3-dihydro-indol-2-one was obtained.

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B. 3-aralkyl-2-indolinone derivative General synthesis method A: Suzuki-coupled tetrakis (triphenylphosphine) palladium (0) (3% equivalent), aryl bromide (1 equivalent), 2M aqueous sodium carbonate (2.5 equivalent) And boronic acid (
A mixture of equal amounts (1.2 equivalents) in toluene and ethanol is refluxed for 12 hours.
Pour the reaction into water and extract with ethyl acetate. Wash the organic layer with base and brine, dry and concentrate. The residue is purified by column chromatography to give the product.

Method B: Condensation of Oxindole and Aldehyde In enough ethanol, add 1 equivalent of oxindole, 1 equivalent of aldehyde and 1-5 equivalent of piperidine (or pyrrolidine) to give 0.5-1.0M Make a solution of oxindole and stir at 90-100 ° C for 1-18 hours. The mixture is cooled to room temperature and if a precipitate forms, it is collected by vacuum filtration, washed with ethanol and dried to give the product. If no precipitate forms when the reaction mixture is cooled, the mixture is concentrated and the residue is purified by column chromatography.

Method C: Reduction with Palladium on Carbon A solution of indolinone in methanol containing a few drops of acetic acid is hydrogenated with palladium on carbon at room temperature overnight. The catalyst is removed by filtration, rinsed with methanol, and the filtrate is concentrated to give the reduced product.

Method D: Reduction with sodium borohydride To a mixture of indolinone (1 equivalent) and dimethylformamide in methanol is added sodium borohydride (10 equivalents). The mixture is stirred at room temperature for 1 / 2-3 hours. The reaction is then poured into water, extracted with ethyl acetate, washed with brine, dried, and concentrated to give a reduced product.

Method E: Using a Perlman catalyst, a solution of reduced indolinone in methanol was dissolved in methanol at room temperature using a Perlman catalyst.
Hydrogenate for 2-10 hours. The catalyst is removed by filtration, rinsed with methanol, and the filtrate is concentrated to give the reduced product.

B. Synthesis of Specific Compounds The following specific syntheses of the compounds of the present invention are shown by way of illustration only and should not be construed as limiting the scope of the invention in any way.

Compound AAI-1: 6-methoxy-3- (4-methoxy-3-thiophen-3-ylbenzyl) -1
, 3-Dihydroindol-2-one tetrakis (triphenylphosphine) palladium (0) (1.35 g, 1.1
7 mmol) was dissolved in toluene (45 mL) and ethanol (45 mL) in 4-methoxy-3-bromobenzaldehyde (6.72 g, 31.26 mm).
ol). To this solution was added 2M aqueous sodium carbonate (80 mL, 78 m
mol) and then thiophene-3-boronic acid (5 g, 39.07 mmol)
Was added to the mixture. The mixture was refluxed for 12 hours, after which it was poured into water (200 mL) and extracted with ethyl acetate (2 × 150 mL). The organic layer was washed with saturated aqueous sodium hydrogen carbonate (150 mL) and brine (150 mL), dried over anhydrous magnesium sulfate and concentrated. Chromatography (silica, 20% ethyl acetate /
Hexane) provided 6 g (88%) of 3- (3-thiophene) -4-methoxybenzaldehyde as a yellow oil.

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3- (3-thiophene) -4-methoxybenzaldehyde (0.53 g, 2.4
5 mmol), 6-methoxy-2-oxindole (0.4 g, 2.45 mm
ol) and piperidine (1.2 mL, 12.25 mmol) in 5 mL of ethanol were kept in a sealed tube at 100 ° C. for 12 hours. The reaction was cooled and added to diethyl ether (150 mL) and hexane (150 mL). The solid formed was removed by filtration, washed with diethyl ether, then with hexane, dried and 0.57 g (64%) of 6-methoxy-3- (4-methoxy-3-thiophen-3-yl). (Benzylidene) -1,3-dihydroindol-2-one was obtained as a mustard colored solid.

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6-Methoxy-3- (4-methoxy-3-thiophen-3-ylbenzylidene)
To a mixture of -1,3-dihydroindol-2-one (363 mg, 1 mmol) in methanol (10 mL) and dimethylformamide (5 mL) was added sodium borohydride (380 mg, 10 mmol) in small portions. The reaction was stirred at room temperature for 3 hours. The reaction mixture was quenched with water (100 mL) and ethyl acetate (20 mL).
0 mL). The organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated. Silica gel (ethyl acetate: hexane 2: 3, then 1: 1)
Chromatography afforded 215 mg (59%) of the title compound as a slightly orange crystalline solid.

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Compound AAI-2: Cyclopentanecarboxylic acid [3- (3,5-dichloro-2-hydroxybenzyl) -2-oxo-2,3-dihydro-1H-indol-6-yl] -amide cyclopentane The carboxylic acid (2-oxo-2,3-dihydro-1H-indol-6-yl) -amide is condensed with 3,5-dichlorosalicylaldehyde using Method B to give cyclopentanecarboxylic acid [3- (3 , 5-Dichloro-2-hydroxy-benzylidene) -2-oxo-2,3-dihydro-1H-indole-6
A mixture of isomers of -yl] -amide was obtained as an orange solid. This was reduced using Method D to give 10 mg (40%) of cyclopentanecarboxylic acid [3- (3,5-
Dichloro-2-hydroxybenzyl) -2-oxo-2,3-dihydro-1H-
Indole-6-yl] -amide was obtained as an off-white crystalline solid.

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Compound AAI-3: Cyclopentanecarboxylic acid [3- [5-chloro-2-hydroxybenzyl) -2
-Oxo-2,3-dihydro-1H-indol-6-yl] -amide Cyclopentanecarboxylic acid (2-oxo-2,3-dihydro-1H-indol-6-yl) -amide was prepared using Method B Condensation with 5-chlorosalicylaldehyde gives cyclopentanecarboxylic acid [3- (5-chloro-2-hydroxy-benzylidene) -2-oxo-2,3-dihydro-1H-indol-6-yl] -amide. Obtained. This was reduced using Method D to give 34.4 mg (68%) of cyclopentanecarboxylic acid [3- (5-chloro-2-hydroxybenzyl) -2-oxo-2,3-dihydro-1H-indole. -6-yl] -amide was obtained as an off-white crystalline solid.

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Compound AAI-4: 3- (4-hydroxy-6,4′-dimethoxybiphenyl-3-ylmethyl)-
1,3-dihydroindol-2-one 2-hydroxy-4-methoxybenzaldehyde (20 g, 131.4 mmol
) To afford 5-bromo-2-hydroxy-4-methoxybenzaldehyde as a pale yellow solid. Next, 4-methoxyphenylboronic acid (7
50 mg, 4.94 mmol) to give 800 mg (75%) of 4-hydroxy-6,4'-dimethoxybiphenyl-3-carbaldehyde as a pale yellow solid.

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The 2-oxindole is condensed with 4-hydroxy-6,4′-dimethoxybiphenyl-3-carbaldehyde using Method B to give 3- (4-hydroxy-6
A mixture of isomers of 4'-dimethoxybiphenyl-3-ylmethylene) -1,3-dihydroindol-2-one was obtained. It is then reduced using method E to give 4.5 m
g (75%) of 3- (4-hydroxy-6,4'-dimethoxybiphenyl-3-
(Ilmethyl) -1,3-dihydroindol-2-one was obtained as a white solid.

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Compound AAI-5: 3- (3-cyclopentyl-4-methoxybenzyl) -5-fluoro-1,3-
Dihydroindol-2-one 5-fluoro-2-oxindole was converted to 3-cyclopentyl-4 using Method B.
Condensation with -methoxybenzaldehyde provided 3- (3-cyclopentyl-4-methoxybenzylidene) -5-fluoro-1,3-dihydro-indol-2-one as a yellow-orange solid. It was then reduced using Method E to give 0.78 g (78%
)) 3- (3-Cyclopentyl-4-methoxybenzyl) -5-fluoro-1,
3-Dihydroindol-2-one was obtained as a white solid.

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Compound AAI-6: N- [3- (4-Methoxy-3-thiophen-2-ylbenzyl) -2-oxo-2,3-dihydro-1H-indol-6-yl] -acetamido 4-methoxy -3-Bromobenzaldehyde (1.5 g) was combined with thiophen-2-boronic acid (0.98 g) using Method A to give 1.3 g (87%) of 3-
(2-thiophene) -4-methoxybenzaldehyde was obtained as a yellow oil.

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6-acetamido-2-oxindole is condensed with 3- (2-thiophene) -4-methoxybenzaldehyde to give N- [3- (4-methoxy-3-thiophen-2-ylbenzylidene) -2. -Oxo-2,3-dihydro-1H-indol-6-yl] -acetamide was obtained as a yellow solid. This was reduced using Method E to give 0.04 g (80%) of N- [3- (4-methoxy-3-thiophene-
2-ylbenzyl) -2-oxo-2,3-dihydro-1H-indole-6
Yl] -acetamide was obtained as a white solid.

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Compound AAI-7: N- {3- [3-cyclohexyl-4- (2-morpholin-4-ylethoxy)
-Benzyl] -2-oxo-2,3-dihydro-1H-indol-6-yl}
-Acetamide 6-acetamido-2-oxindole is condensed with 3-cyclohexyl-4-morpholinoethoxybenzaldehyde using Method B to give N- {3- [3-cyclohexyl-4- (2-morpholin-4-ylethoxy). ) -Benzylidene]-
A mixture of isomers of 2-oxo-2,3-dihydro-1H-indol-6-yl} -acetamide was obtained as a mustard colored solid. This is reduced using method E,
0.04 g (80%) of N- {3- [3-cyclohexyl-4- (2-morpholin-4-ylethoxy) -benzyl] -2-oxo-2,3-dihydro-1H-
Indole-6-yl} -acetamide was obtained as a white solid.

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Compound AAI-8: 3- (4-methoxy-3-thiophen-3-ylbenzyl) -5- (2-morpholin-4-ylethyl) -1,3-dihydroindol-2-one 5- (2 -Chloroethyl) -2-oxindole (2.3 g), morpholine (
1.2 mL) and a solution of diisopropylethylamine (1.2 mL) in dimethyl sulfoxide (10 mL) was stirred at 100 ° C. overnight. The mixture was cooled, poured into water and extracted with ethyl acetate. The organic layer was washed with brine, dried, and evaporated. The residue was chromatographed (silica gel, 5% methanol in chloroform) to give 0.9 g (31%) of 5- (2-morpholin-4-ylethyl)-.
2-Oxindole was obtained as a white solid.

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Condensation of 5- (2-morpholin-4-ylethyl) -2-oxindole with 3- (3-thiophene) -4-methoxybenzaldehyde using Method B gives 3
-(4-Methoxy-3-thiophen-3-yl-benzylidene) -5- (2-morpholin-4-ylethyl) -1,3-dihydroindol-2-one was obtained as an orange-yellow solid. This was reduced using Method E to give 0.03 g (60%) of 3-
(4-Methoxy-3-thiophen-3-yl-benzyl) -5- (2-morpholin-4-ylethyl) -1,3-dihydroindol-2-one was obtained as a white solid.

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Compound AAI-9: 3- (5-Isopropyl-4-methoxy-2-methylbenzyl-1,3-dihydroindol-2-one 2-oxindole was converted to 5-isopropyl-4- Methoxy-2-
Condensation with methylbenzaldehyde gives 0.25 g of 3- (5-isopropyl-
4-methoxy-2-methylbenzylidene) -1,3-dihydroindole-2-
On was obtained as a yellow-orange solid. This was reduced using Method E to give 2 g (100%
-)-(3-Isopropyl-methoxy-2-methylbenzyl) -1,3-dihydroindol-2-one as a white solid.

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Compound AAI-10: 3- (3,5-Diisopropyl-4-methoxybenzyl) -1,3-dihydroindol-2-one 2-oxindole was converted to 3,5-diisopropyl-4 using Method B. Condensation with -methoxybenzaldehyde provided 3- (3,5-diisopropyl-4-methoxy-benzylidene) -1,3-dihydro-indol-2-one as a yellow-orange solid. This was reduced using Method E to give 2 g (100%) of 3- (3,5-diisopropyl-4-methoxybenzyl) -1,3-dihydroindol-2-one as a white solid.

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Compound AAI-11: 3- (3-cyclopentyl-4-hydroxybenzyl) -5-fluoro-1,3
-Dihydroindol-2-one 3- (3-cyclopentyl-4-hydroxybenzylidene) -5-fluoro-1
, 3-Dihydroindol-2-one (0.045 g) was reduced using Method E to give 0.025 g (56%) of 3- (3-cyclopentyl-4-hydroxy-benzyl) -5-fluoro- 1,3-Dihydroindol-2-one was obtained as a tan foam.

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Compound AAI-12: 3-benzyl-4- (2-hydroxyethyl-1,3-dihydroindole-2
-On 4- (2-hydroxyethyl) -1,3-dihydroindol-2-one is condensed with 4-bromobenzaldehyde using Method B to give 3- (4-bromobenzylidene) -4- (2- (Hydroxy-ethyl) -1,3-dihydroindole-2
The -one was obtained as a yellow solid. This was reduced using Method E to give 0.2 g (91
%) Of 3-benzyl-4- (2-hydroxyethyl) -1,3-dihydroindol-2-one as a yellow / orange foam.

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Compound AAI-13: 3- (2-hydroxybenzyl) -6- (3-methoxyphenyl) -1,3-dihydroindol-2-one 6- (3-methoxyphenyl) -1,3-dihydro Indole-2-one is condensed with salicylaldehyde using Method B to give 3- (2-hydroxybenzylidene) -6- (3-methoxyphenyl) -1,3-dihydro-indol-2-one as a yellow solid As obtained. This was reduced using Method E to give 0.03 g (66%
3-)-(2-Hydroxy-benzyl) -6- (3-methoxy-phenyl) -1
, 3-Dihydro-indol-2-one was obtained as a white solid.

Compound AAI-14: 3- (4-bromobenzyl) -4- (2-hydroxyethyl) -1,3-dihydroindol-2-one 3- (4-bromobenzylidene) -4- (2- Hydroxyethyl) -1,3-dihydroindol-2-one (0.18 g, 0.52 mmol) was reduced using Method D to give 0.17 g (95%) of 3- (4-bromobenzyl)- 4- (2-
(Hydroxyethyl) -1,3-dihydroindol-2-one was obtained as a white solid.

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Compound AAI-15: 5-chloro-3- [3,5-diisopropyl-4- (2-morpholin-4-ylethoxy) -benzyl] -1,3-dihydroindol-2-one 5-chloro- The 2-oxindole was converted to 3,5-diisopropyl-
Condensed with 4- (2-morpholin-4-yl-ethoxy) -benzaldehyde to give 5-chloro-3- [3,5-diisopropyl-4- (2-morpholin-4-ylethoxy) -benzylidene] -1, 3-Dihydroindol-2-one was obtained as a brownish orange solid. This was reduced using Method E to give 1.3 g (10
0%) of 5-chloro-3- [3,5-diisopropyl-4- (2-morpholine-
4-ylethoxy) -benzyl] -1,3-dihydroindol-2-one was obtained as a yellow / orange foam.

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Compound AAI-16: 3- (1H-Indol-5-ylmethyl) -4-methyl-1,3-dihydroindol-2-one 4-methyl-2-oxindole was converted to 1H- Condensation with indole-5-carbaldehyde gives 3- (1H-indol-5-ylmethylene)-
4-Methyl-1,3-dihydroindol-2-one was obtained as a white solid. This was reduced using Method C to give 20 mg (40%) of 3- (1H-indole-
5-ylmethyl) -4-methyl-1,3-dihydroindol-2-one was obtained.

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Compound AAI-17: 3- (1H-Indol-5-ylmethyl) -1,3-dihydroindole-2
-One 2-oxindole (133 mg, 1 mmol) was condensed with 1H-indole-5-carbaldehyde (145 mg, 1 mmol) using Method B to give 15
5 mg (60%) of 3- (1H-indol-5-ylmethylene) -1,3-dihydroindol-2-one were obtained as a white solid.

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3- (1H-Indol-5-ylmethylene) -1,3-dihydroindole-
The 2-one (80 mg, 0.31 mmol) was reduced using Method C to give 76 mg
(95%) of 3- (1H-indol-5-ylmethyl) -1,3-dihydro-
Indole-2-one was obtained.

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Compound AAI-18: 3- [4- (3-dimethylaminopropyl) -3,5-dimethyl-1H-pyrrol-2-ylmethyl] -1,3-dihydroindol-2-one 2-oxindole (133 mg, 1.0 mmol) using Method B to give 4- (
Condensation with 3-dimethylaminopropyl) -3,5-dimethyl-1H-pyrrole-2-carbaldehyde (208 mg, 1.0 mmol) to give 168.3 mg (
52%) of 3- [4- (3-dimethylaminopropyl) -3,5-dimethyl-1
[H-pyrrol-2-ylmethylene] -1,3-dihydro-indol-2-one was obtained as a yellow solid.

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3- [4- (3-Dimethylaminopropyl) -3,5-dimethyl-1H-pyrrol-2-ylmethylene] -1,3-dihydro-indol-2-one (13 mg)
, 0.04 mmol) using Method C to give 6 mg (46%) of 3- [4
-(3-dimethylaminopropyl) -3,5-dimethyl-1H-pyrrole-2-
Ilmethyl] -1,3-dihydroindol-2-one was obtained.

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Compound AAI-19: 5-bromo-3- [4- (3-dimethylaminopropyl) -3,5-dimethyl-
1H-pyrrol-2-ylmethyl] -1,3-dihydroindol-2-one 5-bromo-1,3-dihydroindol-2-one was converted to 4- (3
-Dimethylaminopropyl) -3,5-dimethyl-1H-pyrrole-2-carbaldehyde (208 mg, 1.0 mmol) to give 284.9 mg (7
1%) of 5-bromo-3- [4- (3-dimethylaminopropyl) -3,5-dimethyl-1H-pyrrol-2-ylmethylene] -1,3-dihydroindole-
The 2-one was obtained as a red solid.

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5-Bromo-3- [4- (3-dimethylaminopropyl) -3,5-dimethyl-
1H-pyrrol-2-ylmethylene] -1,3-dihydroindol-2-one (50 mg, 0.12 mmol) was reduced using Method D to give 5-bromo-3-
[4- (3-dimethylaminopropyl) -3,5-dimethyl-1H-pyrrole-
2-ylmethyl] -1,3-dihydro-indol-2-one was obtained.

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Compound AAI-20: 3- [4- (3-dimethylamino-propyl) -3,5-dimethyl-1H-pyrrol-2-ylmethyl] -6- (2-methoxy-phenyl) -1,3 -Dihydro-indol-2-one 6- (2-methoxyphenyl) -1,3-dihydroindol-2-one (23
9 mg, 1.0 mmol) using method B to give 4- (3-dimethylaminopropyl) -3,5-dimethyl-1H-pyrrole-2-carbaldehyde (208 mg,
1.0 mmol) and 333 mg (83%) of 3- [4- (3-dimethylaminopropyl) -3,5-dimethyl-1H-pyrrol-2-ylmethylene] -6- (2-methoxyphenyl) ) -1,3-Dihydroindol-2-one was obtained as a yellow solid.

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3- [4- (3-Dimethylaminopropyl) -3,5-dimethyl-1H-pyrrol-2-ylmethylene] -6- (2-methoxyphenyl) -1,3-dihydroindol-2-one (54 mg, 0.126 mmol) was reduced using Method C to give 45 mg (86%) of 3- [4- (3-dimethylaminopropyl) -3,5.
-Dimethyl-1H-pyrrol-2-ylmethyl] -6- (2-methoxyphenyl) -1,3-dihydroindol-2-one was obtained.

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Compound AAI-21: 3- [4- (3-dimethylaminopropyl) -3,5-dimethyl-1H-pyrrol-2-ylmethyl] -6- (3-methoxyphenyl) -1,3-dihydro Indole-2-one 6- (3-methoxyphenyl) -1,3-dihydroindol-2-one (23
9 mg, 1.0 mmol) using method B to give 4- (3-dimethylaminopropyl) -3,5-dimethyl-1H-pyrrole-2-carbaldehyde (208 mg,
1.0 mmol) and 333 mg (83%) of 3- [4- (3-dimethylaminopropyl) -3,5-dimethyl-1H-pyrrol-2-ylmethylene] -6- (3-methoxyphenyl ) -1,3-Dihydroindol-2-one was obtained as a red solid.

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3- [4- (3-Dimethylaminopropyl) -3,5-dimethyl-1H-pyrrol-2-ylmethylene] -6- (3-methoxyphenyl) -1,3-dihydroindol-2-one (50 mg, 0.12 mmol) was reduced using Method C to give 40 mg (77%) of 3- [4- (3-dimethylaminopropyl) -3,5-
Dimethyl-1H-pyrrol-2-ylmethyl] -6- (3-methoxyphenyl)
-1,3-Dihydroindol-2-one was obtained.

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Compound AAI-22: 3- [4- (3-dimethylaminopropyl) -3,5-dimethyl-1H-pyrrol-2-ylmethyl] -6- (4-methoxyphenyl) -1,3-dihydro Indole-2-one 6- (4-methoxyphenyl) -1,3-dihydroindol-2-one (23
9 mg, 1.0 mmol) using method B to give 4- (3-dimethylaminopropyl) -3,5-dimethyl-1H-pyrrole-2-carbaldehyde (208 mg,
1.0 mmol) and 333 mg (83%) of 3- [4- (3-dimethylaminopropyl) -3,5-dimethyl-1H-pyrrol-2-ylmethylene] -6- (4-methoxyphenyl) ) -1,3-Dihydroindol-2-one was obtained as a brown solid.

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3- [4- (3-Dimethylaminopropyl) -3,5-dimethyl-1H-pyrrol-2-ylmethylene] -6- (4-methoxyphenyl) -1,3-dihydroindol-2-one (69 mg, 0.16 mmol) was reduced using Method C to give 65 mg (94%) of 3- [4- (3-dimethylaminopropyl) -3,5-
Dimethyl-1H-pyrrol-2-ylmethyl] -6- (4-methoxyphenyl)
-1,3-Dihydroindol-2-one was obtained.

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Compound AAI-23: 5-Chloro-3- [4- (3-dimethylaminopropyl) -3,5-dimethyl-
1H-pyrrol-2-ylmethyl] -1,3-dihydroindol-2-one 5-chloro-1,3-dihydroindol-2-one (167 mg, 1.0 mm
ol) was condensed with 4- (3-dimethylaminopropyl) -3,5-dimethyl-1H-pyrrole-2-carbaldehyde (208 mg, 1.0 mmol) using Method B to give 188.7 mg (53%). ))-Chloro-3- [4- (3-dimethylaminopropyl) -3,5-dimethyl-1H-pyrrol-2-ylmethylene]
-1,3-Dihydroindol-2-one was obtained as a brown solid.

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5-Chloro-3- [4- (3-dimethylaminopropyl) -3,5-dimethyl-
1H-pyrrol-2-ylmethylene] -1,3-dihydroindol-2-one (63 mg, 0.18 mmol) was reduced using Method C to give 15 mg (24%
))-Chloro-3- [4- (3-dimethylaminopropyl) -3,5-dimethyl-1H-pyrrol-2-ylmethyl] -1,3-dihydroindol-2-one.

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Compound AAI-24: 3- (3-Fluoro-2-hydroxybenzyl) -6-phenyl-1,3-dihydroindol-2-one 6-phenyl-2-oxindole (50 mg, 0.3 mmol) Was converted to 3-fluoro-2-hydroxybenzaldehyde (50 mg, 0.36 m
mol) to give 52 mg (53%) of 3- (3-fluoro-2-hydroxybenzylidene) -6-phenyl-1,3-dihydroindol-2-one as a yellow / orange solid.

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3- (3-Fluoro-2-hydroxybenzylidene) -6-phenyl-1,3-
Dihydroindol-2-one (26 mg, 0.08 mmol) was reduced using Method E to give 26 mg (100%) of 3- (3-fluoro-2-hydroxy-benzyl) -6-phenyl-1,3. -Dihydroindol-2-one was obtained.

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Compound AAI-25: 3- (2-hydroxy-4-methoxybenzyl) -6-phenyl-1,3-dihydroindol-2-one 6-phenyl-2-oxindole (50 mg, 0.3 mmol) Was converted to 2-hydroxy-4-methoxybenzaldehyde (50 mg, 0.33 m
mol) to give 70 mg (68%) of 3- (2-hydroxy-4-methoxybenzylidene) -6-phenyl-1,3-dihydroindol-2-one as a yellow / orange solid.

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3- (2-hydroxy-4-methoxybenzylidene) -6-phenyl-1,3-
Dihydroindol-2-one (80 mg, 0.23 mmol) was reduced using Method E to give 80 mg (100%) of 3- (2-hydroxy-4-methoxybenzyl) -6-phenyl-1,3- Dihydroindol-2-one was obtained.

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Compound AAI-26: 3- (5-bromo-2-hydroxybenzyl) -6-phenyl-1,3-dihydroindol-2-one 6-phenyl-2-oxindole (50 mg, 0.3 mmol) Was condensed with 5-bromosalicylaldehyde (50 mg, 0.25 mmol) using Method B to give 80 mg (82%) of 3- (5-bromo-2-hydroxybenzylidene)
-6-Phenyl-1,3-dihydroindol-2-one was obtained as a yellow / orange solid.

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3- (5-Bromo-2-hydroxybenzylidene) -6-phenyl-1,3-dihydroindol-2-one (23 mg, 0.06 mmol) was reduced using Method E to give 23 mg (100 %) Of 3- (5-bromo-2-hydroxy-benzyl) -6-phenyl-1,3-dihydroindol-2-one.

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Compound AAI-27: 4- (2-hydroxyethyl) -3- (6-methylpyridin-2-ylmethyl)
-1,3-dihydroindol-2-one 4- (2-hydroxyethyl) -1,3-dihydroindol-2-one (89
mg, 0.5 mmol) with 6-methyl-2-pyridinecarbaldehyde (61 mg, 0.5 mmol) using Method B to give 4- (2-hydroxyethyl) -3- (6-methylpyridine -2-ylmethylene) -1,3-dihydroindol-2-one was obtained as a yellow solid.

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Reduction of 4- (2-hydroxyethyl) -3- (6-methylpyridin-2-ylmethylene) -1,3-dihydroindol-2-one (16 mg, 0.06 mmol) using Method E This gave 16 mg (100%) of 4- (2-hydroxy-ethyl) -3- (6-methylpyridin-2-ylmethyl) -1,3-dihydroindol-2-one.

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Compound AAI-28: 3- (3-Bromo-5-tert-butyl-4-hydroxybenzyl) -5-chloro-1,3-dihydroindol-2-one 5-chloro-2-oxindole ( 167 mg, 1 mmol) using method B to give 3-bromo-5-tert-butyl-4-hydroxybenzaldehyde (30
8.5 mg, 1.2 mmol) and 329 mg (81%) of 3- (3
-Bromo-5-tert-butyl-4-hydroxybenzylidene) -5-chloro-1,3-dihydroindol-2-one was obtained as a mixture of isomers.

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3- (3-Bromo-5-tert-butyl-4-hydroxybenzylidene) -5
-Chloro-1,3-dihydroindol-2-one (1 g, 2.46 mmol)
Was reduced using Method C to give 1.005 g (100%) of 3- (3-bromo-5).
-Tert-Butyl-4-hydroxybenzyl) -5-chloro-1,3-dihydroindol-2-one was obtained as a mixture of isomers.

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C. Diarylindolinone compounds Condensation of general synthetic ketone and oxindole: appropriate indoline-2-one (1.0 equivalent), appropriate ketone (1.2 equivalent),
Of ethanol and piperidine or pyrrolidine (0.1 equivalent) in 1-2 mL
/1.0 mmol oxindole) at 90 ° C. for 3-5 hours. After cooling, the precipitate is filtered, washed with cold ethanol and dried to give the desired compound.

4- (3,5-Dimethyl-1H-pyrrol-2-yl) -4-oxo-butyric acid

Embedded image To a mixture of 13.3 g (0.1 mol) of aluminum chloride in dichloromethane (150 mL) was added 10 g (0.105 mol) of 2,4-dimethylpyrrole at room temperature. The resulting solution was stirred at room temperature for 15 minutes, and 16.4 g (0.1 mol
) Was added. The reaction mixture was stirred at room temperature for 2 days and quenched with saturated sodium carbonate. Next, the mixture was diluted with water (100 mL) and dichloromethane (300 mL). The organic layer was separated and concentrated. The residue was ethanol (3
0 mL) and 1 N potassium hydroxide (30 mL) and heated at 90 ° C. for 2 hours. Then the cooled mixture was diluted with water and extracted with ethyl acetate. The basic aqueous layer was acidified to pH 3 with 6N hydrochloric acid. The mixture was then extracted with ethyl acetate (3x). The combined organic extracts were washed with brine, dried over sodium sulfate and concentrated. The residue was stirred with ethyl acetate (20 mL) and the solid was collected by filtration to give 820 mg of 4- (3,5-dimethyl-1H-pyrrol-2-yl) -4-oxo-butyric acid as a dark solid. .

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Embedded image This was then condensed with oxindole.

3- [3,5-Dimethyl-1H-pyrrol-2-yl)-(4-methoxy-phenyl) -methylene] -1,3-dihydro-indol-2-one (DAI-I
)

Embedded image Aluminum chloride (1.3 eq) in dichloromethane is added to benzoyl chloride (1 eq) at room temperature, followed by 2,4-dimethylpyrrole (1.6 eq) in dichloromethane.
Eq) of the solution. Then the mixture was stirred overnight at room temperature. The reaction was diluted with ethyl acetate, washed with brine, dried, and concentrated. The residue was chromatographed and eluted with ethyl acetate and hexane to give the ketone.

(3,5-Dimethyl-1H-pyrrol-2-yl)-(4-methoxy-phenyl) -methanone (50 mg, 0.22 m) in dimethylformamide (1 mL)
mol), oxindole (50 mg, 0.37 mmol), pyrrolidine (0
. 3 mL) and 1,8-diazabicyclo [5.4.0] undec-7-ene (
0.1 mL) was heated at 150-160 ° C. for 1 day. Sodium hydride (12
mg) was added to the reaction mixture and heating was continued for 3 days. The reaction solution was poured into water and extracted with ethyl acetate. The organic layer was washed with water, dried, and concentrated. The residue was chromatographed and eluted with ethyl acetate and dichloromethane to give 6 mg of 3-[(
3,5-dimethyl-1H-pyrrol-2-yl)-(4-methoxy-phenyl)
-Methylene] -1,3-dihydro-indol-2-one was obtained as a yellow solid.

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3-[(3,4-Dimethoxy-phenyl)-(3,5-dimethyl-1H-pyrrol-2-yl) -methylene] -1,3-dihydro-indol-2-one (DA
I-II)

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Embedded image Aluminum chloride (1.7 g, 12.75 m) in dichloromethane (100 mL)
mol) was added to 3,4-dimethoxybenzoyl chloride (2.0 g, 10 mmol) at room temperature, followed by 2,4-dimethylpyrrole (1.
(5 g, 15.8 mmol). Then the mixture was stirred overnight at room temperature. The reaction was diluted with ethyl acetate, washed with brine, dried, and concentrated. The residue was chromatographed and eluted with ethyl acetate and hexane to give 1 g (39 g).
%) Of (3,4-dimethoxy-phenyl)-(3,5-dimethyl-1H-pyrrol-2-yl) -methanone as a solid.

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(3,4-Dimethoxy-phenyl)-in dimethylformamide (1.5 mL)
(3,5-dimethyl-1H-pyrrol-2-yl) -methanone (100 mg, 0
. 4 mmol), oxindole (100 mg, 0.8 mmol), pyrrolidine (0.2 mL) and 1,8-diazabicyclo [5.4.0] undec-7-
The ene (few drops) was heated at 160 ° C. for 2 hours. Sodium hydride (18 mg) was added to the reaction mixture and heating was continued at 160 ° C. for 3 days. The reaction solution was poured into water and extracted with ethyl acetate. The organic layer was washed with water, dried, and concentrated. The residue was chromatographed and eluted with ethyl acetate and hexane to give 5 mg of 3-[(3,4
-Dimethoxy-phenyl)-(3,5-dimethyl-1H-pyrrol-2-yl)
-Methylene] -1,3-dihydro-indol-2-one was obtained. MS EI
374 [M] ⁺

D. 4-Substituted Indolinone Compound Compound IN-001: 4- [2- (3-Isopropyl} -phenoxy) -ethyl] -1,3-dihydro-indol-2-one

Embedded image Diethyl azodicarboxylate (0.47 mL, 3 mmol) was added to a solution of triphenylphosphine (0.786 g, 3 mmol) in tetrahydrofuran (10 mL) under a nitrogen atmosphere. The mixture was stirred for 15 minutes. Next to this 4
-(2-hydroxy-ethyl) -1,3-dihydro-indol-2-one (0
. 53 g, 3 mmol) (Hayler, JD; Howie, SLB.
Giles, R .; G. FIG. Negus, A .; Oxley, P .; W. Et a
l; Heetocycl. Chem. ; 32; 3; 1995; 875-88.
2) and then 3-isopropylphenol (0.41 mL, 3 mmol). The mixture was stirred at room temperature for 1 day and the solvent was evaporated. The residue was chromatographed on silica gel eluting with ethyl acetate: hexane 1: 9 to give 120 mg (
14%) of 4- [2- (3-isopropyl-phenoxy) -ethyl] -1,3-
Dihydro-indol-2-one was obtained.

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Compound IN-002 4- [2- (2-isopropyl-phenoxy) -ethyl] -1,3-dihydro-
Indole-2-one

Embedded image Diethyl azodicarboxylate (1.58 mL, 10 mmol) was added to a solution of triphenylphosphine (2.62 g, 10 mmol) in tetrahydrofuran (20 mL) under a nitrogen atmosphere. The mixture was stirred for 15 minutes. Next, 4- (2-hydroxy-ethyl) -1,3-dihydro-indol-2-one (
1.77 g, 10 mmol) and then 2-isopropylphenol (1.36 m
L, 10 mmol). The mixture was stirred at room temperature for 18 hours and the solvent was evaporated. The residue was chromatographed on silica gel, ethyl acetate: hexane 2:
Elution with 8 gave 0.26 g (9%) of 4- [2- (2-isopropyl-phenoxy) -ethyl] -1,3-dihydro-indol-2-one as a pale yellow solid.

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Compound IN-003 4- [2- (biphenyl-3-yloxy) -ethyl] -1,3-dihydro-indol-2-one

Embedded image Diethyl azodicarboxylate (1.58 mL, 10 mmol) was added to a solution of triphenylphosphine (2.62 g, 10 mmol) in tetrahydrofuran (20 mL) under a nitrogen atmosphere. The mixture was stirred for 15 minutes. Next, 4- (2-hydroxy-ethyl) -1,3-dihydro-indol-2-one (
1.77 g, 10 mmol) and then 3-phenylphenol (1.7 g, 10 mmol).
mmol). The mixture was stirred at room temperature for 1 day and the solvent was evaporated. The residue was dissolved in ethyl acetate (150 mL) and the organic solvent was washed with 2N hydrochloric acid (3 × 50 mL), saturated sodium bicarbonate solution and brine. The residue was chromatographed on silica gel eluting with ethyl acetate: hexane 2: 8 to give 1.19 g (
36%) of 4- [2- (biphenyl-3-yloxy) -ethyl] -1,3-dihydro-indol-2-one.

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Compound IN-004 3- (4-Bromo-benzylidene) -4- (2-hydroxy-ethyl-1,3-
Dihydro-indol-2-one

Embedded image 4- (2-hydroxy-ethyl) -1,3-dihydro-indol-2-one (
3.0 g, 17 mmol), 4-bromobenzaldehyde (3.1 g, 17 mm)
ol) and piperidine (8.4 mL, 85 mmol) in ethanol (113 m
The mixture in L) was heated at 90 ° C. overnight. The reaction mixture was concentrated and the residue was chromatographed on a silica gel column to give 2.4 g (41%) of 3- (4-bromo-benzylidene) -4- (2-hydroxy-ethyl) -1,3- Dihydro-
Indole-2-one was obtained as a yellow-orange solid.

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Compound IN-005 4- (2-hydroxy-ethyl) -3-pyridin-2-ylmethylene-1,3-
Dihydro-indol-2-one

Embedded image 4- (2-hydroxy-ethyl) -1,3-dihydro-indol-2-one (
80 mg, 0.45 mmol) and 2-pyridinecarboxaldehyde (60
(mg, 0.56 mmol) in 1% piperidine in ethanol (5 mL) was heated at 90 ° C. for 8 hours. The reaction mixture was concentrated and the residue was chromatographed on a silica gel column. This was then triturated in ethyl acetate and hexane to give 55 mg (46%) of 4- (2-hydroxy-ethyl) -3-pyridine-2-yl.
Ilmethylene-1,3-dihydro-indol-2-one was obtained.

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Compound IN-006 4- (2-hydroxy-ethyl) -3- (6-methyl-pyridin-2-ylmethylene) -1,3-dihydro-indol-2-one

Embedded image 4- (2-hydroxy-ethyl) -1,3-dihydro-indol-2-one (
89 mg, 0.5 mmol), 6-methyl-2-pyridine-carbaldehyde (
A mixture of 61 mg, 0.5 mmol) and piperidine (0.1 mL) in 1.0 mL of ethanol was heated at 100 ° C. for 1 hour and cooled to room temperature. The precipitate was filtered, recrystallized from ethyl acetate and hexane to give 4- (2-hydroxy-ethyl) -3- (6-methyl-pyridin-2-ylmethylene) -1,3-dihydro-indole-2-. On was obtained as a yellow solid.

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Compound IN-007 4- (2-hydroxy-ethyl-3- (1H-indol-5-ylmethylene)
-1,3-dihydro-indol-2-one

Embedded image Indole-5-carboxylic acid (5.02 g) in tetrahydrofuran (40 mL)
, 313 mmol) was slowly added to lithium aluminum hydride (1.88 g, 49.5 mmol) stirred in tetrahydrofuran (125 mL) with external heating and gentle reflux under nitrogen. The mixture was heated at reflux for 2 hours and cooled to room temperature. Then 2N sodium hydroxide (10 mL) was added dropwise to the reaction mixture. The precipitate was removed by filtration and the filtrate was concentrated. This is dichloromethane (80m
L), washed with 2N sodium hydroxide (10 mL), water (20 mL) and brine (20 mL), dried over anhydrous sodium sulfate, concentrated and ca. 5 g of (1H-indol-5-yl) -Methanol was obtained.

(1H-Indol-5-yl) -methanol (3.8 g, 25.8 mmol)
) And manganese oxide (5 g, 57.5 mmol) in dichloromethane (50 mL)
) Was stirred at room temperature for 60 hours. After usual work-up and chromatography, 1.5 g (40%) of 1H-indole-5-carbaldehyde were obtained.

4- (2-Hydroxy-ethyl) -1,3-dihydro-indol-2-one (39 mg, 0.22 mmol), 1H-indole-5-carbaldehyde (
A mixture of 32 mg, 0.22 mmol) and one drop of piperidine in ethanol was heated at 90 ° C. overnight and cooled to room temperature. The reaction mixture was concentrated and the residue was purified on a silica gel column to give 48 mg (72%) of 4- (2-hydroxy-ethyl)-
3- (1H-Indol-5-ylmethylene) -1,3-dihydro-indol-2-one was obtained as a yellow solid.

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Compound IN-008 4- (2-hydroxy-ethyl) -3- (4-methoxy-3-thiophen-2-
Yl-benzylidene) -1,3-dihydro-indol-2-one

Embedded image Tetrakis (triphenylphosphine) palladium (0) (0.24 g, 0.2
1 mmol) with 4-methoxy-3-bromobenzaldehyde (1.5 g, 6.
98 mmol) in toluene (15 mL) and ethanol (15 mL), followed by a 2M aqueous solution of sodium carbonate (14 mL, 28 mmol). To this mixture was added thiophene-2-boronic acid (0.98 g, 7.68 mmol).
) Was added and the mixture was heated to reflux. After 3 hours, the reaction was partitioned between water (100 mL) and ethyl acetate (250 mL). The organic layer was washed with saturated aqueous sodium bicarbonate (75mL) and brine (75mL), dried over magnesium sulfate and concentrated. Chromatography (silica, 20% ethyl acetate / hexane) provided 1.3 g (87%) of 4-methoxy-3-thiophen-2-yl-benzaldehyde as a yellow oil.

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4- (2-hydroxy-ethyl) -1,3-dihydro-indol-2-one (
1.6 g, 9.2 mmol), 4-methoxy-3-thiophen-2-yl-benzaldehyde (2 g, 9.2 mmol) and piperidine (4.5 mL, 85 m).
mol) in ethanol (46 mL) was refluxed overnight. The reaction mixture was concentrated and the residue was chromatographed on a silica gel column to give 1.63 g (
47%) of 4- (2-hydroxy-ethyl) -3- (4-methoxy-3-thiophen-2-yl-benzylidene) -1,3-dihydro-indol-2-one as a yellow-orange solid. .

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Compound IN-009 3- [4- (3-Dimethylamino-propyl) -3,5-dimethyl-1H-pyrrol-2-ylmethylene] -4- (2-hydroxy-ethyl) -1,3- Dihydro-indol-2-one

Embedded image 3- (2,4-Dimethyl-1H-pyrrol-3-yl) -propionic acid (10 g
, 60.8 mmol) in 60 mL of dichloromethane was added 1,1′-carbonyldiimidazole (11.6 g, 71.8 mmol), followed by tetrahydrofuran (30 mL) and N, N-diisopropylethylamine ( Hun
ig base, 10 mL, 60.8 mmol). The dark red reaction mixture was stirred at room temperature overnight and poured into ice water. The organic layer was washed with brine to pH 6, dried over anhydrous sodium sulfate and concentrated. The crude product was purified on a silica gel column and eluted with dichloromethane-methanol (98: 2) to give 9.8 g (83%) of 3- (2,4-dimethyl-1H-pyrrol-3-yl) -N , N-Dimethyl-propionamide was obtained as a yellow oil.

A suspension of lithium aluminum hydride (2.3 g, 60 mmol) in tetrahydrofuran (50 mL) was added to 3- (2,4-dimethyl-1H-pyrrole-3-).
A solution of yl) -N, N-dimethyl-propionamide (9.81 g, 50 mmol) in THF (50 mL) was added dropwise. The reaction mixture is stirred at 80 ° C. for 1 hour,
Cooled in an ice bath. Ice cubes were slowly added to the reaction mixture until no more gas evolved. A few drops of 2N sodium hydroxide are added and the reaction mixture is stirred at room temperature for 30 minutes, extracted with ethyl acetate, dried over anhydrous sodium sulphate and concentrated to 6.8 g (75%
)). [3- (2,4-Dimethyl-1H-pyrrol-3-yl) -propyl] -dimethyl-amine was obtained as an orange oil which was used without further purification.

To an ice-cold solution of N, N-dimethylformamide (6.0 mL, 76 mmol) in dichloromethane (30 mL) was added phosphorus oxychloride (7.0 mL, 76 mmol).
Was added dropwise. After the addition of phosphorus oxychloride, the reaction mixture is stirred at room temperature for 15 minutes,
A solution of [3- (2,4-dimethyl-1H-pyrrol-3-yl) -propyl] -dimethyl-amine (6.8 g, 38 mmol) in dichloromethane (20 mL) was added dropwise at 0 <0> C. The final reaction mixture was refluxed at 60 ° C. for 4 hours and cooled in an ice bath.
Slowly add ice cubes to the reaction mixture, then add 2N sodium hydroxide to adjust the pH to 1
Adjusted to 2. The reaction mixture was stirred at room temperature for 30 minutes and extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. This was purified on a silica gel column and eluted with dichloromethane-methanol-ammonium hydroxide (9: 1: 0.01) to give 5 g (63%) of 4- (3-dimethylamino-propyl) -3,5-. Dimethyl-1H-pyrrole-2-carbaldehyde was obtained as a dark red oil.

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4- (2-hydroxy-ethyl) -1,3-dihydro-indol-2-one (
177 mg, 1.0 mmol), 4- (3-dimethylamino-propyl) -3,
5-dimethyl-1H-pyrrole-2-carbaldehyde (208 mg, 1.0 m
mol) and 3 drops of pyrrolidine in 2.0 mL of ethanol are mixed with 90
Reflux at 4 ° C. for 4 hours and cool to room temperature. The precipitate is filtered, washed with cold ethanol and hexane, dried in a vacuum oven overnight, to give 360.6 mg (98%) of 3
-[4- (3-Dimethylamino-propyl) -3,5-dimethyl-1H-pyrrol-2-ylmethylene] -4- (2-hydroxy-ethyl) -1,3-dihydro-indol-2-one Obtained.

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Compound IN-010 Phenyl-thiocarbamic acid O- [2- (2-oxo-2,3-dihydro-1H
-Indol-4-yl) -ethyl] ester

Embedded image Phenylisothiocyanate (0.45 mL, 3.75 mmol) was added to 4- (2
-Hydroxy-ethyl) -1,3-dihydro-indol-2-one (443 m
g, 2.5 mmol) in tetrahydrofuran (5 mL). The mixture was stirred at room temperature for 20 hours and then at 70 ° C. for 8 hours. 1 mL of dimethylformamide was added to the mixture to homogeneity and heating was continued for another 42 hours. The reaction was cooled, poured into 1N sodium hydroxide solution (100 mL), and ethyl acetate (2 mL) was added.
00 mL). The organic layer was washed with 1N hydrochloric acid (100 mL) and brine, dried over anhydrous sodium sulfate and concentrated. The residue was treated with methanol / ethyl acetate /
Recrystallized from hexane to give 452 mg (58%) of O- [2- (2-oxo-2,3-dihydro-1H-indol-4-yl) -phenyl-thiocarbamate.
Ethyl] ester was obtained as a white solid.

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Compound IN-011 Phenyl-carbamic acid 2- (2-oxo-2,3-dihydro-1H-indol-4-yl) -ethyl ester

Embedded image Phenylisocyanate (0.652 mL, 6 mmol) was added to 4- (2-hydroxy-ethyl) -1,3-dihydro-indol-2-one (709 mg, 4 m
mol) in tetrahydrofuran (8 mL), dimethylformamide (2 mL) and pyridine (3 drops). The mixture was heated at 70 ° C. for 15 hours. The reaction was cooled, poured into 1N sodium hydroxide solution (100 mL),
Extracted with ethyl acetate (200 mL). The organic layer was washed with 1N hydrochloric acid (100 mL) and brine, dried over anhydrous sodium sulfate and concentrated. The residue was recrystallized from methanol / ethyl acetate / hexane to give 953 mg (80%) of phenyl-carbamic acid 2- (2-oxo-2,3-dihydro-1H-indol-4-yl) -ethyl ester as off-white. Obtained as a powder.

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Compound IN-012 tert-butyl-carbamic acid 2- (2-oxo-2,3-dihydro-1H-
Indole-4-yl) -ethyl ester

Embedded image Tert-butyl isocyanate (0.685 mL, 6 mmol) was added to 4- (2
-Hydroxy-ethyl) -1,3-dihydro-indol-2-one (709 m
g, 4 mmol) in tetrahydrofuran (8 mL) and dimethylformamide (2
mL) and pyridine (3 drops). Mix at 70 ° C for 1
Heated at 80 ° C. for 39 hours for 5 hours. 0.457 mL of tert-butyl isocyanate was added to the reaction and heating was continued at 90 ° C. for 24 hours. Further, tert-butyl isocyanate (0.457 mL) was added, and heating was continued at 90 ° C. for 37 hours. The reaction was cooled, poured into 1N sodium hydroxide solution (100 mL) and extracted with ethyl acetate (200 mL). The organic layer was washed with 1N hydrochloric acid (100 mL) and brine, dried over anhydrous sodium sulfate and concentrated. The residue was recrystallized from methanol / ethyl acetate / hexane to give 130 mg (12%) of tert-butyl-.
Carbamic acid 2- (2-oxo-2,3-dihydro-1H-indol-4-yl) -ethyl ester was obtained.

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Compound IN-013 Cyclohexyl-carbamic acid 2- (2-oxo-2,3-dihydro-1H-indol-4-yl) -ethyl ester

Embedded image Cyclohexyl isocyanate (0.766 mL, 6 mmol) was added to 4- (2-
(Hydroxy-ethyl) -1,3-dihydro-indol-2-one (709 mg)
, 4 mmol) in tetrahydrofuran (8 mL) and dimethylformamide (2 m
L) and a stirred mixture in pyridine (3 drops). Mix the mixture at 70 ° C for 16
Heated for hours. 0.511 mL of cyclohexyl isocyanate was added to the reaction and heating was continued at 80 ° C. for 24 hours. Furthermore, cyclohexyl isocyanate (0.
(255 mL) and heating was continued at 85 ° C. for 24 hours. The reaction was cooled, poured into 1N sodium hydroxide solution (100 mL) and extracted with ethyl acetate (200 mL). The organic layer was washed with 1N hydrochloric acid (100 mL) and brine, dried over anhydrous sodium sulfate and concentrated. The residue was recrystallized from methanol / ethyl acetate / hexane to give 317 mg of the product. A second crop of the product (32 mg) was obtained from the mother liquor by column chromatography (1: 1 ethyl acetate: hexane). A total of 349 mg (29%) of cyclohexyl-carbamic acid 2- (2-oxo-
2,3-Dihydro-1H-indol-4-yl) -ethyl ester was obtained as a white powder.

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Compound IN-014 Benzenesulfonyl-carbamic acid 2- (2-oxo-2,3-dihydro-1H
-Indol-4-yl) -ethyl ester

Embedded image Benzenesulfonyl isocyanate (1.07 mL, 8 mmol) was added under a nitrogen atmosphere to 4- (2-hydroxy-ethyl) -1,3-dihydro-indole-2.
-One (709 mg, 4 mmol) was added dropwise to a stirred mixture of tetrahydrofuran (8 mL), dimethylformamide (2 mL) and pyridine (3 drops). The mixture was heated at 80 C for 15 hours. The reaction was cooled, poured into 1N sodium hydroxide solution (100 mL) and extracted with ethyl acetate (200 mL). The organic layer was discarded and the basic aqueous layer was acidified with 6N hydrochloric acid (18 mL) and extracted into ethyl acetate. The organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated. The residue was recrystallized from methanol / ethyl acetate / hexane to give 390 mg (27%) of benzenesulfonyl-carbamic acid 2- (2-oxo-2,3-dihydro-1H-).
Indole-4-yl) -ethyl ester was obtained as an off-white powder.

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Compound IN-015 Biphenyl-2-yl-carbamic acid 2- (2-oxo-2,3-dihydro-1
H-indol-4-yl) -ethyl ester

Embedded image 2-Biphenylyl isocyanate (1.15 mL, 6.7 mmol) was added under a nitrogen atmosphere to 4- (2-hydroxy-ethyl) -1,3-dihydro-indole-
2-One (709 mg, 4 mmol) was added dropwise to a stirred mixture of tetrahydrofuran (8 mL), dimethylformamide (2 mL) and pyridine (3 drops).
The mixture was heated at 80 ° C. for 15 hours. The reaction was cooled, quenched with 1N sodium hydroxide solution (100 mL) and extracted with ethyl acetate (200 mL). The organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated. The residue was chromatographed on silica gel, eluting with ethyl acetate: hexane 3: 2.
188 g (80%) of biphenyl-2-yl-carbamic acid 2- (2-oxo-
2,3-Dihydro-1H-indol-4-yl) -ethyl ester was obtained as a white fluffy solid.

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Compound IN-016 Ethyl-carbamic acid 2- (2-oxo-2,3-dihydro-1H-indol-4-yl) -ethyl ester

Embedded image Ethyl isocyanate (0.633 mL, 8 mmol) was added under a nitrogen atmosphere in 4-
(2-Hydroxy-ethyl) -1,3-dihydro-indol-2-one (70
9 mg, 4 mmol) to a stirred mixture in tetrahydrofuran (8 mL) and dimethylformamide (2 mL). The mixture was heated at 80 ° C. for 48 hours. The reaction was cooled, poured into water (100 mL) and extracted with ethyl acetate (200 mL). The organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated.
The residue was chromatographed on silica gel, eluting with ethyl acetate / hexane, to give 158 mg (16%) of ethyl-carbamic acid 2- (2-oxo-2,3-
Dihydro-1H-indol-4-yl) -ethyl ester was obtained as a white powder.

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Compound IN-017 4- [2- (4-methoxy-phenoxy) ethyl] -1H-dihydro-indol-2-one

Embedded image Diethyl azodicarboxylate (0.47 mL, 3 mmol) was added to a solution of triphenylphosphine (0.786 g, 3 mmol) in tetrahydrofuran (10 mL) under a nitrogen atmosphere. The mixture was stirred for 15 minutes. Next to this 4
-(2-hydroxy-ethyl) -1,3-dihydro-indol-2-one (0
. 53 g, 3 mmol) followed by 4-methoxyphenol (0.372 g, 3 m
mol) was added. The mixture was stirred at room temperature for 18 hours and the solvent was evaporated. The residue was dissolved in ethyl acetate (150 mL) and the organic solvent was washed with 2N hydrochloric acid (3 × 30 mL), saturated sodium bicarbonate solution and brine. The residue was chromatographed on silica gel eluting with ethyl acetate: hexane 2: 8 to give 0.14 g (
16%) of 4- [2- (4-methoxy-phenoxy) -ethyl] -1,3-dihydro-indol-2-one.

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Compound IN-018 4- (2-methoxy-ethyl) -1,3-dihydro-indol-2-one

Embedded image Methyl iodide (1.41 g, 10 mmol) was added at 0 ° C. to silver trifluoromethanesulfonate (2.56 g, 10 mmol) and 4- (2-hydroxy-ethyl) -1,3-dihydro-indol-2-one ( (0.88 g, 5 mmol) in dichloromethane (20 mL). The precipitate formed after 5-10 minutes changed color from yellow to gray and then dark purple. The mixture was stirred at room temperature for 2 hours. The reaction mixture was diluted with dichloromethane and filtered through celite.
The filtrate was concentrated and chromatographed on silica gel to give 4- (2-methoxy-ethyl) -1,3-dihydro-indol-2-one.

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Compound IN-019 4- (2-Ethoxy-ethyl-1,3-dihydro-indol-2-one

Embedded image Ethyl iodide (0.47 mL, 6 mmol) was added at 0 ° C. to silver trifluoromethanesulfonate (1.35 g, 5.2 mmol), 2,6-di-tert-butylpyridine (1 g, 5.2 mmol) and 4- (2-hydroxy-ethyl) -1
, 3-Dihydro-indol-2-one (0.53 g, 3 mmol) was added to a stirred mixture in dichloromethane (10 mL). The mixture was warmed to room temperature and stirred for 2 hours. The precipitate formed after 5-10 minutes changed color from yellow to brown. The reaction mixture was diluted with dichloromethane (100 mL), washed with 1N hydrochloric acid, saturated sodium bicarbonate and brine, dried and concentrated. The residue was chromatographed on silica gel to give 180 mg (29%) of 4- (2-ethoxy-ethyl)-.
1,3-Dihydro-indol-2-one was obtained as a purple oil.

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Compound IN-020 4- [2- (4-isopropyl-phenoxy) -ethyl] -1,3-dihydro-
Indole-2-one

Embedded image Diethyl azodicarboxylate (1.58 mL, 10 mmol) was added to a solution of triphenylphosphine (2.62 g, 10 mmol) in tetrahydrofuran (20 mL) under a nitrogen atmosphere. The mixture was stirred for 15 minutes. Next, 4- (2-hydroxy-ethyl) -1,3-dihydro-indol-2-one (1.77 g, 10 mmol) was added thereto, and then 4-isopropylphenol (1.
(36 g, 10 mmol). The mixture was stirred at room temperature for 1 day and the solvent was evaporated. The residue is chromatographed on silica gel, ethyl acetate: hexane 2
: 8 to give 1.1 g (37%) of 4- [2- (4-isopropyl-phenoxy) -ethyl] -1,3-dihydro-indol-2-one as a pale yellow solid.

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Compound IN-021 4- [2- (5-Chloro-pyridin-3-yloxy) -ethyl] -1,3-dihydro-indol-2-one

Embedded image Diethyl azodicarboxylate (1.74 g, 10 mmol) was added to a solution of triphenylphosphine (2.62 g, 10 mmol) in tetrahydrofuran (20 mL) under a nitrogen atmosphere. The mixture was stirred for 15 minutes. Next, to this was added 4- (2-hydroxy-ethyl) -1,3-dihydro-indol-2-one (1.77 g, 10 mmol) and then 5-chloro-3-pyridinol (1
. 29 g, 10 mmol). The mixture was stirred at room temperature for 2 days. The precipitate is collected by vacuum filtration, washed with ethyl acetate, dried and 1.05 g (36%)
Of 4- [2- (5-chloro-pyridin-3-yloxy) -ethyl] -1,3-
Dihydro-indol-2-one was obtained as a light brown solid.

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Compound IN-022 4- (2-hydroxy-ethyl) -3- (3-hydroxy-6-methyl-pyridin-2-ylmethylene) -1,3-dihydro-indol-2-one

Embedded image 4- (2-hydroxy-ethyl) -1,3-dihydro-indol-2-one (
51 mg, 0.3 mmol), 3-hydroxy-6-methyl-pyridine-2-carbaldehyde (45 mg, 0.33 mmol) and 1 drop of pyrrolidine.
The mixture in 0 mL of ethanol was heated at 90 ° C. for 4 hours and cooled to room temperature. The precipitate is filtered, washed with cold ethanol and hexane, dried in a vacuum oven overnight, and treated with 4- (2-hydroxy-ethyl) -3- (3-hydroxy-6-methyl-
A mixture of isomers of (pyridin-2-ylmethylene) -1,3-dihydro-indol-2-one was obtained.

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Compound IN-023 3- [4- (3-Dimethylamino-propyl) -3,5-dimethyl-1H-pyrrol-2-ylmethylene] -4- [2- (3-isopropyl-phenoxy) -ethyl ] -1,3-dihydro-indol-2-one

Embedded image 4- [2- (3-isopropyl-phenoxy) -ethyl] -1,3-dihydro-
Indole-2-one (28 mg, 0.095 mmol), 4- (3-dimethylamino-propyl) -3,5-dimethyl-1H-pyrrole-2-carbaldehyde (20 mg, 0.095 mmol) and 1 drop of pyrrolidine Was heated at 90 ° C. for 4 hours and cooled to room temperature. The precipitate is filtered, washed with cold ethanol and hexane, dried in a vacuum oven overnight,
mg (87%) of 3- [4- (3-dimethylamino-propyl) -3,5-dimethyl-1H-pyrrol-2-ylmethylene] -4- [2- (3-isopropyl-
Phenoxy) -ethyl] -1,3-dihydro-indol-2-one was obtained.

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Compound IN-024 3- [5- {4- [2- (4-isopropyl-phenoxy) -ethyl] -2-oxo-1,2-dihydro-indole-3-ylidenemethyl} -2,4- Dimethyl-1H-pyrrol-3-yl) -propionic acid

Embedded image 2-Benzyloxycarbonyl-3,5-dimethyl-4- (2-methoxycarbonylethyl) pyrrole (Aldrich, 2.0 g) was added at room temperature with 0.2 g of 10% palladium on carbon in 40 mL of methanol. Hydrogenated for hours. The catalyst was removed by filtration and washed with 40 mL of methanol. The filtrate was evaporated to dryness and dried in a vacuum oven overnight to give 1.3 g (92% yield) of 2-carboxy-3,
5-Dimethyl-4- (2-methoxycarbonylethyl) pyrrole was obtained.

2-carboxy-3,5-dimethyl-4- (2-methoxycarbonylethyl)
Pyrrole (1.3 g) was triturated with 0.5 g of anhydrous sodium acetate and then heated at 100 ° C. for 3 days. The mixture was dissolved in water, extracted with ethyl acetate and chromatographed on a column of silica gel in ethyl acetate: hexane 1: 3 to give 0.4 g (38% yield) of 2,4-dimethyl-3-. (2-Methoxycarbonylethyl) pyrrole was obtained as a viscous pale yellow oil.

[0348] 2,4-Dimethyl-3- (2-methoxycarbonylethyl) pyrrole (0.35 g) and 0.5 g of Vilsmeier reagent (A) in 10 mL of dichloroethane
ldrich) was stirred at room temperature under nitrogen for 2 hours. Concentrate the reaction mixture and add 10
Treated with 6 mL of 6M sodium hydroxide solution and then extracted three times with 10 mL of ethyl acetate. The ethyl acetate extracts were combined, dried over anhydrous sodium sulfate, evaporated to dryness and 0.24 g (60% yield) of 2,4-dimethyl-5-formyl-3- (2
-Methoxycarbonylethyl) pyrrole was obtained as a brown oil.

2,4-Dimethyl-5-formyl-3 in 6N sodium hydroxide (10 mL)
-(2-methoxycarbonylethyl) pyrrole (0.23 g, 1.2 mmol)
Was heated at 100 ° C. for 2 hours. The reaction mixture was cooled to room temperature, acidified with 6N hydrochloric acid, and extracted three times with 10 mL of ethyl acetate. The combined organic layers were washed with 10 mL of water and 5 mL of brine, dried over anhydrous sodium sulfate, and evaporated to dryness.
30 mg (106% yield) of crude 4-carboxyethyl-3,5-dimethyl-2-
Formyl pyrrole was obtained as a brown oil.

4- [2- (4-Isopropyl-phenoxy) -ethyl] -1,3-dihydro-indol-2-one (118 mg, 0.4 mmol), 4-carboxyethyl-3,5-dimethyl-2 A mixture of formylpyrrole (78 mg, 0.4 mmol) and piperidine (0.3 mL) in 1.0 mL of ethanol was heated in a sealed tube at 90 ° C. for 18 hours and cooled to room temperature. The reaction mixture was concentrated, the residue was dissolved in methylene chloride, and then diethyl ether was added. The precipitate was filtered, washed with ethyl acetate and 110 mg (58%) of 3- (5- {4- [2- (4-isopropyl-phenoxy) -ethyl] -2-oxo-l, 2-dihydro -Indole-
3-Ylidenemethyl {-2,4-dimethyl-1H-pyrrol-3-yl) -propionic acid (piperidine salt) was obtained.

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Compound IN-025 Isopropyl-carbamic acid 2- (2-oxo-2,3-dihydro-1H-indol-4-yl) ethyl ester

Embedded image 4- (2-hydroxy-ethyl) -1,3-dihydro-indol-2-one (
3g, 16.93 mmol) and isopropyl isocyanate (2.49 mL)
, 25.40 mmol) in tetrahydrofuran (16 mL) and dimethylformamide (6 mL) was heated at 80 ° C. for 64 hours. The reaction mixture was poured into water, extracted with ethyl acetate (400mL), washed with brine, dried (sodium sulfate) and concentrated. The residue was recrystallized from methanol, ethyl acetate and hexane to give 1.136 g (26%) of isopropyl-carbamic acid 2- (2-oxo-2,3-dihydro-1H-indol-4-yl) -ethyl ester. Was obtained as a crystalline solid.

Assay Methods The following in vitro assays can be used to determine the level of activity and effect of one or more compounds of the present invention on PK. Similarly, similar assays can be designed for any PK using techniques well known in the art.

The cell / catalytic assays described herein are performed in an ELISA format. The general method is as follows: the compound is introduced into cells expressing the test kinase, either naturally or recombinantly. After a period of time, 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 wells of an ELISA plate previously coated with a specific antibody that recognizes the substrate for the enzymatic phosphorylation reaction. The non-substrate components of the cell lysate are washed away and the amount of phosphorylation of the substrate is detected with an antibody that specifically recognizes phosphotyrosine, and compared to control cells that have not been contacted with the test compound.

The cell / biological assays described herein measure the amount of DNA produced in response to activation of a test kinase, which is a measure of the general proliferative response. The general method of this assay is as follows: the compound is introduced into cells expressing the test kinase, either naturally or recombinantly. After a period of time, if the test kinase is a receptor, a ligand known to activate the receptor is added. After at least overnight incubation, a DNA labeling reagent,
For example, bromodeoxy-uridine (BrdU) or 3H-thymidine is added.
The amount of labeled DNA is detected with an anti-BrdU antibody or by measuring radioactivity and compared to control cells that have not been contacted with the test compound.

Cell / Catalytic Assay An enzyme-linked immunosorbent assay (ELISA) can be used to detect and measure the presence of PK activity. ELISA is described, for example, in Voller, et.
al. , 1980 ("Enzyme-Linked Immunosorben").
t Assay, "Manual of Clinical Immunol
ogy, 2nd edition, Rose and Friedman, pp. 359-371
Am. Soc. Of Microbiology, Washington, D
. C. ) Can be carried out according to the known protocol described in

The disclosed protocols can be adapted to detect activity for a particular PK. For example, a preferred protocol for performing an ELISA experiment on a particular PK is described below. However, adapting these protocols to detect the activity of compounds against other members of the RTK family, as well as CTKs and STKs, is within the knowledge of those skilled in the art.

Example 2 A FLK-1 ELISA assay was performed to determine 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 was performed to measure the kinase activity of the FLK-1 receptor in cells that were genetically engineered to express Flk-1.

Materials and Methods Materials The following reagents and supplies were used. a. Corning 96-well ELISA plate (Corning Catalog No. 25805-96); b. Cappel goat anti-rabbit IgG (Catalog No. 55641); c. PBS (Gibco Catalog No. 450-1300EB); d. TBSW buffer (50 mM Tris (pH 7.2), 150 mM NaCl
And 0.1% Tween-20); e. Ethanolamine storage solution (10% ethanolamine (pH 7.0), 4 ° C
Saved); f. HNTG buffer (20 mM HEPES buffer (pH 7.5), 150 mM
NaCl, 0.2% Triton X-100, and 10% glycerol); g. EDTA (as a 0.5 M (pH 7.0) 100X stock solution); h. Sodium orthovanadate (as a 0.5 M, 100X stock solution); i. Sodium pyrophosphate (0.2 M, as 100 × stock solution); j. NUNC 96-well V-bottom polypropylene plate (Applied Sc
entific catalog No. AS-72092); k. NIH3T3 C7 # 3 cells (FLK-1 expressing cells); l. DMEM, containing 1X high glucose L-glutamine (Catalog No. 119
65-050); m. FBS, Gibco (Catalog No. 16000-028); n. L-glutamine, Gibco (Catalog No. 25030-016); o. VEGF, PeproTech, Inc. (Catalog No. 100-20
) Keep as a 1 μg / 100 μl stock solution in Milli-QdH ₂ O at −20 ° C.
Stored at p. Affinity purified anti-FLK-1 antiserum; q. Phosphotyrosine-specific UB40 monoclonal antibody (Fendley, e
t al. , 1990, Cancer Research 50: 1550-1.
558); r. EIA grade goat anti-mouse IgG-POD (BioRad Catalog No. 1)
72-1011); s. 2,2-azino-bis (3-ethylbenzthiazoline-6-sulfuric acid (ABT
S) solution (100 mM citric acid (anhydrous), 250 mM Na ₂ HPO ₄ (pH 4.0)
), 0.5 mg / ml ABTS (Sigma Catalog No. A-1888)),
The solution must be stored at 4 ° C. in the dark until use; t. H ₂ O ₂ (30% solution) (Fisher Catalog No. H325); u. ABTS / H ₂ O ₂ (15mlABTS solution, 2μlH ₂ O ₂₎ prepared of use 5 minutes, kept at room temperature; v. 0.2MHCl preservative solution, H ₂ O in; w. Dimethyl sulfoxide (100%) (Sigma Catalog No. D-84)
18); and y. Trypsin-EDTA (Gibco BRL Catalog No. 25200-
049)

Protocol The assay was performed using the following protocol: Corning 96-well ELISA plate, 1.0 μl per well
g of Cappel anti-rabbit IgG antibody in 0.1 M Na ₂ CO ₃ (pH 9.6),
Coating with Final volume is 150 μl per well. Plate 4
Coat overnight at ° C. Plates can be stored for up to 2 weeks when stored at 4 ° C. 2. The cells are grown in growth media (supplemented with DMEM, 2.0 mM L-glutamine, 10% FBS) in a suitable culture dish at 37 ° C., 5% CO ₂ until confluent. 3. Cells are harvested by trypsinization and seeded in Corning 25850 polystyrene 96-well round bottom cell plates at 25,000 cells / well in 200 μl of growth medium. 4. The cells are grown for at least one day at 37 ° C., 5% CO ₂ . 5. Wash cells with D-PBS 1X. 6. Add 200 μl / well of starvation medium (DMEM, 2.0 mM 1-glutamine, 0.1% FBS). Incubate overnight at 37 ° C., 5% CO ₂ .
7. Compounds were 1: 2 in starvation media in polypropylene 96 well plates.
Dilute to 0. Dimethyl sulfoxide 1:20 for use in control wells.
Dilute to. 8. Remove the starvation medium from the 96-well cell culture plate and add 162 μl of freshly prepared starvation medium to each well. 9. 18 μl of the 1:20 diluted compound (from step 7) was added to each well, and the 1:20 dimethylsulfoxide dilution was added to control wells (+/− VEGF).
F) After cell stimulation, make a final dilution of 1: 200. Final dimethyl sulfoxide is 0.5%. Plates are incubated for 2 hours at 37 ° C., 5% CO ₂ . 10. Unbound antibody is removed by ELI by inverting the plate and removing the liquid.
Remove from SA plate. TBSW + 0.5% ethanolamine, pH 7.0
And wash three times. Tap the plate on a paper towel to remove excess liquid and air bubbles. 11. 150 μl of TBSW + 0.5% ethanolamine per well, pH
Block the plate at 7.0. Incubate the plate for 30 minutes while shaking on a microtiter plate shaker. 12. The plate is washed three times as described in step 10. 13. Add 0.5 μg / well of affinity purified anti-FLU-1 polyclonal rabbit antiserum. Bring the final volume to 150 μl / well with TBSW + 0.5% ethanolamine pH 7.0. Incubate the plate for 30 minutes with shaking. 14． Add 180 μl of starvation medium to the cells and add 20 μl / well of 10.0 mM
Sodium orthovanadate and 500 ng / ml VEGF (final concentration; 1.0 mM sodium orthovanadate and 50 ng / ml VE per well
Stimulate cells for 8 minutes at 37 ° C., 5% CO ₂ at GF). Negative control wells receive only starvation medium. 15. After 8 minutes, the medium must be removed from the cells and washed once with 200 μl / well of PBS. 16. Lyse the cells in 150 μl / well of HNTG with shaking for 5 minutes at room temperature. The HNTG formulation includes sodium orthovanadate, sodium pyrophosphate and EDTA. 17． Wash the ELISA plate three times as described in step 10. 18. Transfer cell lysate from cell plate to ELISA plate and incubate for 2 hours with shaking. To transfer cell lysate, pipette up and down while scraping wells. 19. The plate is washed three times as described in step 10. 20. Incubate the ELISA plate with 0.02 μg / well of UB40 in TBSW + 05% ethanolamine. Final volume of 150 μl /
Well. Incubate for 30 minutes with shaking. 21. The plate is washed three times as described in step 10. 22. The ELISA plate was washed with TBSW + 0.5% ethanolamine, pH7.
. Incubate with EIA grade goat anti-mouse IgG conjugate horseradish peroxidase diluted 1: 10,000 in 0. 15 final volume
0 μl / well. Incubate for 30 minutes with shaking. 23. Wash the plate as described in step 10. 24. Add 100 μl ABTS / H ₂ O ₂ solution to the wells. 1 with shaking
Incubate for 0 minutes. 25. Stop the color reaction by adding 100 μl of 0.2 M HCl to a final concentration of 0.1 M HCl. Shake at room temperature for 1 minute. Remove bubbles with a slow stream of air and place the ELISA plate at 410 nm with an ELISA plate reader.
Read on.

Example 3 HER2-ELISA Assay 1 EGF Receptor-HER2 Chimera Receptor Assay in All Cells HER2 kinase activity in EGFR-NIH3T3 whole cells was measured as described below.

Materials and Reagents The assay was performed using the following materials and reagents: a. EGF: stock solution concentration: 16.5 ILM; EGF201, TOYOBO, C
o. , Ltd. Japan b. 05-101 (UBI) (monoclonal antibody recognizing EGFR extracellular domain) c. Anti-phosphotyrosine antibody (anti-Ptyr) (polyclonal) (Fendle
y, 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 solution: HEPES 0.1 M NaCl 0.75 M glycerol 50% Triton X-100 1.0% g. ABTS stock solution: citric acid 100 mM Na ₂ HPO ₄ 250 mM HCl (concentrated) 0.5 mM ABTS ^* 0.5 mg / ml * (2,2′-azinobis (3-ethylbenzthiazoline sulfonic acid)) solution is dark until used Store at 4 ° C. h. Reagent stock: EDTA 100 mM pH 7.0 Na ₃ VO ₄ 0.5 M Na ₄ (P ₂ O ₇ ) 0.2 M

[0362] method using the following protocol: A. Precoat of ELISA plate ELISA plate (Corning, 96 well, Cat. # 2580)
5-96) with 0.5 μg of the 05-101 antibody in PBS per well in 100 μl
And store at 4 ° C. overnight. The coated plates are good up to 10 days when stored at 4 ° C. 2. On the day of use, remove the coating buffer and replace with 100 μl of blocking buffer (5% Carnation instant skim dry milk in PBS). The plate is incubated for 30 minutes with shaking at room temperature (about 23 ° C. to 25 ° C.). Immediately before use, remove the blocking buffer and wash the plate four times with TBST buffer.

B. Cell seeding 1. This assay can be used NIH3T3 cell line overexpressing a chimeric receptor containing the EGFR extracellular domain and intracellular HER ₂ kinase domain. 2. An 80-90% confluent culture dish is selected for the experiment. The cells are trypsinized and the reaction is stopped by adding 10% fetal calf serum. The cells are suspended in DMEM medium (10% CS DMEM medium) and centrifuged once at 1500 rpm at room temperature for 5 minutes. 3. Resuspend cells in seeding medium (DMEM, 0.5% bovine serum) and count cells using trypan blue. Survivability higher than 90% is acceptable. DM cells
In EM medium (0.5% bovine serum) at a density of 10,000 cells per well,
Seed in a 96-well microtiter plate at 100 μl per well.
The seeded cells are incubated for about 40 hours at 37 ° C. in 5% CO ₂ .

C. Assay method The seeded cells are examined for contamination using an inverted microscope. The drug stock solution (10 mg / ml in DMSO) is diluted 1:10 with DMEM medium and then 5 μl is transferred to TBST wells for a final drug dilution of 1: 200 and a final DMSO concentration of 1%. Control wells receive only DMSO. 5% C
Incubate at 37 ° C. for 2 hours in O ₂ . 2. Preparation of EGF ligand: Stock solution EGF was diluted with 10 μl diluted EGF (1:12
Dilute in DMEM so that a final concentration of 100 nM is obtained when transferring (dilution). 3. Prepare fresh HNTG ^* enough for 100 μl per well and place on ice. HNTG ^* (10 ml): HNTG stock solution 2.0 ml milli-QH 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 4. After incubation with the drug for 120 minutes, the prepared SGF ligand is added to the cells at a final concentration of 100 nM, 10 μl per well. Control wells receive only DMEM. Incubate at room temperature with shaking for 5 minutes. 5. Remove drug, EGF, and DMEM. Wash cells twice with PBS. Transfer 100 μl HNTG ^* per well to cells. Leave on ice for 5 minutes.
During this time, the blocking buffer is removed from the other ELISA plates and washed with TBST as described above. 6. The cells are detached from the plate using a pipette tip fitted tightly in a micropipettor and the cellular material is homogenized by repeatedly aspirating and dispensing the HNTG ^* lysis buffer. Transfer lysate to coated, blocked and washed ELISA plates. Incubate for 1 hour at room temperature with shaking. 7. Remove the lysate and wash 4 times with TBST. Transfer the freshly diluted anti-Ptyr antibody at 100 μl per well to the ELISA plate. Anti-Ptyr antiserum (
Incubate for 30 minutes at room temperature with shaking in the presence of (1: 3000 dilution in TBST). 8. Remove anti-Ptyr antibody and wash 4 times with TBST. Freshly diluted TA
Transfer GO anti-rabbit IgG antibody to ELISA plate at 100 μl per well. Incubate for 30 minutes at room temperature with shaking (anti-rabbit IgG antibody: TB
1: 3000 dilution in ST). 9. Remove the TAGO detection antibody and wash 4 times with TBST. Freshly prepared A
Transfer the BTS / H ₂ O ₂ solution at 100 μl per well to the ELISA plate. Incubate for 20 minutes at room temperature with shaking (ABTS / H ₂ O ₂ solution: 10 m
1.0 μl of 30% H ₂ O _{2 in} lABTS stock solution). 10. The reaction was stopped by adding 50 μl of 5N H ₂ SO ₄ (optional),
O.D. D. Is measured. 11. The maximum phosphotyrosine signal is determined by subtracting the value of the negative control from the value of the positive control. The percent inhibition of phosphotyrosine content is then calculated for wells containing the extract after subtraction of the negative control.

Example 4 PDGF-R Assay All cell culture media, glutamine and fetal bovine serum were used, unless otherwise indicated, in Gibco Life Technologies (Grand Isl).
and, NY). All cells were 90-95% air and 5-1
Grow at 37 ° C. in a humidified atmosphere of 0% CO ₂ . All cell lines were routinely subcultured twice a week and confirmed by the Myprotect method (Gibco) to be free of mycoplasma. For ELISA assays, cells (U1242, Joseph Schl) were used.
Essinger, obtained from NYU, was used as a growth medium (MEM, 10% FBS, NE).
AA, 1 mM NaPyr and 2 mM GLN) were grown to 80-90% confluence and 96 well tissue culture plates were seeded with 25,000-30,000 cells per well in 0.5% serum. After overnight incubation in medium containing 0.5% serum, cells were transferred to serum-free medium and treated with test compounds for 2 hours in a 5% CO ₂ , 37 ° C. incubator. The cells are then 5-10 with the ligand.
For 5 minutes, HNTG (20 mM Hepes, 150 mM NaCl, 10% glycerol, 5 mM EDTA, 5 mM Na ₃ VO ₄ , 0.2% Triton X-10)
0, and 2 mM NaPyr). Cell lysate (0.5 mg /
Wells) were previously coated with a receptor-specific antibody, and TBST (50 mM Tr
is-HCl pH 7.2, 150 mM NaCl and 0.1% Triton X-
Transfered to an ELISA plate blocked with 5% milk in 100) for 30 minutes at room temperature. The lysate was incubated for 1 hour at room temperature with shaking. T plate
Washed 4 times with BST and then incubated with polyclonal anti-phosphotyrosine antibody for 30 minutes at room temperature. Excess anti-phosphotyrosine antibody was removed by rinsing the plate four times with TBST. Goat anti-rabbit IgG on ELISA plate
Antibodies were added at room temperature for 30 minutes, then rinsed four more times with TBST. ABTS (1
00 mM citric acid, 250 mM Na ₂ HPO ₄ and 0.5 mg / mL 2,2′-
Azino - bis (3-ethylbenzthiazoline-6-sulfate)), and H ₂ O ₂ (1
. ₂ mL 30% H ₂ O ₂ was added to 10 ml ABTS) to the ELISA plate to initiate color development. Approximately 15 to 30 minutes after ABTS addition, the absorbance at the reference wavelength of 410 nm and 630 nm was recorded.

Example 5: IGF-I Receptor-ELISA The following protocol can be used to measure phosphotyrosine levels on IGF-I receptor, indicating IGF-I receptor tyrosine kinase activity. Materials and Reagents The following materials and reagents were used: a. The cell line used in this assay is the cell line 3T3 / IGF-1R that has been genetically engineered to overexpress the IGF-1 receptor. b. NIH3T3 / IGF-1R is grown in a 5% CO ₂ , 37 ° C. incubator. The growth medium was DMEM + 10% FBS (heat inactivated) +2 mM L−
Glutamine. c. Affinity purified anti-IGF-1R antibody 17-69 d. D-PBS: KH ₂ PO ₄ 0.20 g / l K ₂ HPO ₄ 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 skim dry milk) f. TBST buffer: Tris-HCl 50 mM NaCl 150 mM (pH 7.2 / HCl 10 N) Triton X-100 A stock solution of 0.1% TBS (10 ×) was prepared, and Triton X-1 was added to the buffer during the dilution.
Add 00. g. HNTG buffer: HEPES 20 mM NaCl 150 mM (pH 7.2 / HCl 1N) Glycerol 10% Triton X-100 0.2% A stock solution (5X) is prepared and stored at 4 ° C. h. EDTA / HCl: 0.5M pH 7.0 (NaOH), as 100X stock solution i. Na ₃ VO ₄ : 0.5 M as 100 × stock solution, store aliquots at −80 ° C. j. Na ₄ P ₂ O ₇ : 0.2M as a 100 × stock solution. Insulin-like growth factor-1, Promega (Cat # G5111) rabbit polyclonal anti-phosphotyrosine antiserum m. Goat anti-rabbit IgG, POD conjugate (detection antibody), Tago (C
at. No. 4520, LotNo. 1802): Tago, Inc. , Bur
lingame, CA n. ABTS (2,2′-azinobis (3-ethylbenzthiazoline sulfate)) solution: citric acid 100 mM Na ₂ HPO ₄ 250 mM (pH 4.0 / 1N HCl) ABTS 0.5 mg / ml ABTS solution is stored at 4 ° C. in the dark. Must. The solution must be discarded when it turns green. o. Hydrogen peroxide: Store the 30% solution at 4 ° C. in the dark.

[0367]Method All steps below are performed at room temperature unless otherwise noted. All E
For LISA plate washing, rinse the plate 3 times with tap water and 1 time with TBST.
It is carried out by passing. Tap the plate and dry with a paper towel. A. Cell seeding : 1. 80-90% confluence in tissue culture dish (Corning 25020-100)
The cells grown to luent were transformed with trypsin-EDTA (0.25%, 0.5 m
1 / D-100, GIBCO). 2. Resuspend cells in fresh DMEM + 10% FBS + 2 mM L-glutamine
20 wells in a 96-well tissue culture plate (Corning, 25806-96).
Transfer at 2,000 cells / well (100 μl / well). Incubate for one day,
The medium was then replaced with a serum-free medium (90 / μl) and 5% CO2_TwoAt 37 ° C overnight
Incubate.

B. ELISA plate coating and blocking : 100 μl of the ELISA plate (Corning 25805-96)
Coat with anti-IGF-1R antibody in PBS at 0.5 μg / well for at least 2 hours. 2. Remove the coating solution, replace with 100 μl of blocking buffer and shake for 30 minutes. Remove blocking buffer and wash plate just before adding lysate.

[0369]C. Assay method : 1. Drugs are tested in serum-free conditions. 2. Drug stock solution (in 100% DMSO) in 96-well polypropylene plate
Diluted 1:10 with DMEM in and transfer 10 μl / well of this solution to the cells
, Final drug dilution 1: 100, final DMSO concentration 1.0%. 5% CO _Two And incubated at 37 ° C for 2 hours. 3. Prepare fresh cell lysis buffer (HNTG *). HNTG 2ml EDTA 0.1ml Na_ThreeVO_Four 0.1 ml Na_Four(P_TwoO₇) 0.1ml H_TwoO 7.3 ml 4. After incubating the drug for 2 hours, 200 μl in 10 μl / well of PBS
The nMIGF-1 ligand was transferred to cells (final concentration 20 nM) and 5% CO_TwoAt 37
Incubate at 10 ° C for 10 minutes. 5. Remove the medium, add 100 μl / well HNTG * and shake for 10 minutes
You. Observe the cells under a microscope to see if they have lysed properly. 6. Scrape cells from plate using 12-channel pipette, aspirate and distribute
Is repeated to homogenize the lysate. Antibody coat all lysates
Transfer to the ELISA plate and shake for 1 hour. 7. Lysates were removed, plates were washed and anti-pTyr (1: 3,0 in TBST)
00) at 100 μl / well and shake for 30 minutes. 8. The anti-pTyr was removed, the plate was washed, and TAGO (1: 3 in TBST,
000) at 100 μl / well and shake for 30 minutes. 9. The detection antibody is removed, the plate is washed, and fresh ABTS / H_TwoO_Two(1.2
μlH_TwoO_TwoTo 10 ml ABTS) at 100 μl / well.
To start coloring. 10. Dynatec MR₅₀00 gives an OD of 410 nm at a reference wavelength of 630 nm
Measure.

Example 6 EGF Receptor-ELISA EGF receptor kinase activity in cells engineered to express human EGF-R was measured as described below. Materials and Reagents The following materials and reagents were used: a. EGF ligand: stock solution concentration = 16.5 μM; EGF201, TOYOB
O, Co. , Ltd. Japan b. 05-101 (UBI) (monoclonal antibody recognizing EGFR extracellular domain) c. Anti-phosphotyrosine antibody (anti-Ptyr) (polyclonal) d. Detection antibody: goat anti-rabbit lgG horseradish peroxidase conjugate, TAGO, Inc. , Burlingame, CA e. TBST buffer: Tris-HCl, pH7 50 mM NaCl 150 mM Triton X-100 0.1 f. HNTG 5X stock solution: HEPES 0.1 M NaCl 0.75 M glycerol 50 Triton X-100 1.0% g. ABTS stock solution: citric acid 100 mM NaVO ₄ 250 mM HCl (concentrated) 4.0 pH ABTS ^* 0.5 mg / ml The solution is stored at 4 ° C. in the dark until used. h. Reagent storage _{solution: EDTA 100mM pH7.0 Na 3 VO 4} 0.5M Na 4 (P 2 0 7) 0.2M

[0371] method using the following protocol: A. Precoat of ELISA plate ELISA plate (Corning, 96 well, Cat. # 2580)
5-96) are coated with 0.5 μg / well in PBS, 150 μl final volume / well of the 05-101 antibody and stored at 4 ° C. overnight. The coated plate can be used well up to 10 days when stored at 4 ° C. 2. On the day of use, remove the coating buffer and replace with blocking buffer (5% Carnation instant skim dry milk in PBS). Incubate the plate for 30 minutes at room temperature (about 23 ° C.-25 ° C.) with shaking. Immediately before use, remove the blocking buffer and wash the plate four times with TBST buffer.

B. Cell seeding 1. The assay uses the NIH3T3 / C7 cell line (Honeyger, et al.).
al. , Cell 51: 199-209, 1987). 2. An 80-90% confluent dish is selected for the experiment. The cells are trypsinized and the reaction is stopped by adding 10% CS DMEM medium. The cells are suspended in DMEM medium (10% CS DMEM medium) and centrifuged once at 1000 rpm at room temperature for 5 minutes. 3. Resuspend cells in seeding medium (DMEM, 0.5% bovine serum) and count cells using trypan blue. Survival rates greater than 90% are acceptable. Cells are plated on a 96-well microtiter plate in DMEM medium (0.5% bovine serum).
Inoculate at a density of 10,000 cells per well at 100 μl per well. The seeded cells are incubated for about 40 hours at 37 ° C., 5% CO ₂ .

C. Assay method Inspect the seeded cells for contamination using an inverted microscope. Test compound stocks (10 mg / ml in DMSO) are diluted 1:10 in DMEM medium, then 5 μl are transferred to test wells for a final drug dilution of 1: 200 and a final DMSO concentration of 1%. Control wells receive only DMSO. 5% CO ₂ ,
Incubate at 37 ° C. for 1 hour. 2. Preparation of EGF ligand: Stock solution EGF was diluted with 10 μl of diluted EGF (1: 1).
Dilution in DMEM so that a final concentration of 25 nM is obtained when transferring (2 dilutions). 3. Prepare 10 ml of fresh HNTG ^* enough for 100 μl per well. HNTG * includes: HNTG stock solution (2.0 ml), mil
_{li-QH 2 O (7.3ml)} , EDTA, 100mM, pH7.0 (0.5m
l), Na ₃ VO ₄ 0.5M (0.1 ml) and Na ₄ (P ₂ O ₇ ), 0.2M (
0.1 ml) 4. Put on ice. 5. After 2 hours of incubation with the drug, the prepared EGF ligand is added to the cells at a final concentration of 25 nM at 10 μl per well.
Control wells receive only DMEM. Incubate for 5 minutes at room temperature with shaking. 6. Remove test compound, EGF and DMEM. Wash cells twice with PBS. Transfer HNTG ^* to cells at 100 μl per well. Place on ice for 5 minutes.
Meanwhile, remove the blocking buffer from the other ELISA plates and wash with TBST as described above. 7. Using a pipette tip tightly secured to a micropipettor, detach cells from plate and homogenize cellular material by repeatedly aspirating and dispensing HNTG ^* lysis buffer. Transfer lysate to coated, blocked and washed ELISA plates. Incubate for 1 hour at room temperature with shaking. 8. Remove the lysate and wash 4 times with TBST. Transfer the freshly diluted anti-Ptyr antibody at 100 μl per well to the ELISA plate. Anti-Ptyr antiserum (
Incubate for 30 minutes at room temperature with shaking in the presence of (1: 3000 dilution in TBST). 9. Remove anti-Ptyr antibody and wash 4 times with TBST. Freshly diluted TA
Transfer GO30 anti-rabbit IgG antibody to ELISA plate at 100 μl per well. Incubate for 30 minutes at room temperature with shaking (anti-rabbit IgG antibody:
1: 3000 dilution in TBST). 10. Remove detection antibody and wash 4 times with TBST. ABTS newly prepared
Transfer the / H ₂ O ₂ solution to the ELISA plate at 100 μl per well. 2 at room temperature
Incubate for 0 min (ABTS / H ₂ O ₂ solution: 1.2 μl of 30% H ₂ O _{2 in} 10 ml ABTS stock). 11. The reaction was stopped by adding 50 μl of 5NH ₂ SO ₄ (optional) and 4
O.D. D. Is measured. 12. The maximum phosphotyrosine signal is determined by subtracting the value of the negative control from the value of the positive control. Next, after subtraction of the negative control, the percent inhibition of phosphotyrosine content for the wells containing the extract is calculated.

Example 7 Met Autophosphorylation Assay-ELISA This assay uses the following materials and reagents to determine Met tyrosine kinase activity by analyzing Met protein tyrosine kinase levels on Met receptors. Was: a. HNTG (5X stock solution): 23.83 g HEPES and 43.83 g
Dissolving the NaCl in about 350mldH ₂ O. PH 7 with HCl or NaOH
. Adjusted to 2, 500 ml glycerol and 10 ml Triton X-100
Is added and mixed, and dH ₂ O is added to bring the total volume to 1 L. To create a 1X working solution 1L is added 5X stock solution of 200ml to 800mldH ₂ O, and adjusted pH checked if necessary, stored at 4 ° C.. b. PBS (Dulbecco's phosphate buffered saline), Gibco Cat. # 45
0-1300 EB (1X solution) c. Blocking buffer: 100 g BSA in 500 ml dH ₂ O, 12.
1 g Tris-pH 7.5, 58.44 g NaCl and 10 ml Tween-
Add 20 and dilute to a total volume of 1 L. d. Kinase Buffer: 500mldH to ₂ O, _12.1gTRIS (pH7.
2) Add 58.4 g NaCl, 40.7 g MgCl ₂ and 1.9 g EGTA and make up to 1 L total volume with dH ₂ O. e. PMSF (phenylmethylsulfonyl fluoride), Sigma Cat. #
100% ethanol is added to 435.5 mg of P-7626 to a total volume of 25 ml, and vortexed. f. ATP (of bacterial origin), Sigma Cat. # A-7699, powder -2
Store at 0 ° C .; to make a working solution, 3.31 mg is added to 1 ml dH ₂ O
Dissolve in. g. RC-20H HRPO-conjugated anti-phosphotyrosine, Trans
Daction Laboratories Cat. # E120H h. Pierce 1-Step (TM) Turbo-TMB-ELISA (
3,3 ', 5,5'-tetramethylbenzidine), Pierce Cat. # 34
022 i. H ₂ SO _4, is added concentrated sulfuric acid 1ml of (18N) in 35mldH ₂ O. j. TRIS HCL, Fisher Cat. # BP152-5; Material 1
To 21.14 g, 600 ml of MilliQ H ₂ O was added and the pH was adjusted to 7.0 with HCl.
Adjust to 5 (or 7.2) and bring the volume to 1 L with MilliQ H ₂ O. k. NaCl, Fischer Cat. # S271-10, Make a 5M solution. l. Tween-20, Fischer Cat. # S337-500 m. Na ₃ VO ₄ , Fischer Cat. # S454-50, material 1.8g
80 ml of MilliQ H ₂ O was added to the mixture, and the pH was adjusted to 10 with HCl or NaOH.
. Adjust to 0, boil in the microwave, cool, check the pH, repeat this process until the pH is stable at 10.0, add MilliQ H ₂ O and add a total volume of 10
Make 0 ml, make 1 ml aliquots, and store at -80 ° C. n. MgCl ₂ , Fischer Cat. # M33-500, make 1M solution. o. HEPES, Fischer Cat. # BP310-500, 200m
the lMilliQ H ₂ O, the material 59.6g added, the pH was adjusted to 7.5, and the total volume 250 ml, sterile filtered. p. Albumin, bovine (BSA), Sigma Cat. # A-4503 Add sterile distilled water to 30 g of the material to make a total volume of 300 ml, and store at 4 ° C. q. TBST buffer: about 900 ml dH ₂ in a 1 L graduated cylinder
When 6.057 g TRIS and 8.766 g NaCl were added to O and dissolved,
Adjust the pH to 7.2 with HCl, add 1.0 ml Triton X-100, add d
The total volume is made 1 L with H ₂ O. r. Yagia affinity purified antibody rabbit IgG (all molecules), Cappel C
at. # 55641 s. Anti-h-Met (C-28) rabbit polyclonal IgG antibody, Santa
Cruz Chemical Cat. # SC-161 t. Transiently transfected EGFR / Met chimeric cells (EM
R) (Komada, et al., Oncogene, 8: 2381-239).
0 (1993) u. Sodium carbonate buffer, (Na ₂ CO ₄ , Fischer Cat. # S4
95): 800 ml of MilliQ H ₂ O was added to 10.6 g of the material, and when dissolved, the pH was adjusted to 9.6 with NaOH, the total volume was adjusted to 1 L with MilliQ H ₂ O, filtered, and stored at 4 ° C. .

Methods The following steps are all performed at room temperature unless otherwise noted. All ELISA plate washes are performed by rinsing four times with TBST. A. EMR lysis This method can be performed the night before or just before the start of receptor capture. 1. The lysate dissolves rapidly in a 37 ° C. water bath by vortexing until the last crystal disappears. 2. The cell pellet is lysed in IX HNTG containing 1 mM PMSF.
Use 3 ml of HNTG per 15 cm dish of cells. 1 of the calculated HNTG capacity
Add / 2, vortex the tube for 1 minute, add the remaining amount of HNTG and vortex for another minute. 3. The tubes are balanced and centrifuged at 10,000 xg for 10 minutes at 4 ° C. 4. Pool supernatants, remove aliquots and perform protein assay. 5. Quick freeze the pooled samples in a dry ice / ethanol bath. This step is performed whether the lysate is stored overnight or used immediately after protein measurement. 6. Perform protein measurements using the standard bicinchoninic acid (BCA) method (BC
A Assay Reagent Kit, Pierce Chemical Cat. # 2322
5).

B. ELISA method Coming 96-well ELISA plate with 50 μl total well volume
, Coated with 5 μg per well of goat anti-rabbit antibody in carbonate buffer. Store overnight at 4 ° C. 2. Unbound goat anti-rabbit antibody is removed by inverting the plate and removing the liquid. 3. Add 150 μl of blocking buffer to each well. 30 with shaking
Incubate for minutes. 4. Wash 4 times with TBST. Tap the plate on a paper towel to remove excess liquid and air bubbles. 5. Rabbit anti-Met diluted in TBST against a total volume of 100 μl
Add 1 μg of antibody per well. 6. Dilute the lysate with HNTG (90 μg lysate / 100 μl). 7. Add 100 μl of the diluted lysate to each well. Shake for 60 minutes. 8. Wash 4 times with TBST. Tap excess light on a paper towel to remove excess liquid and air bubbles. 9. Add 50 μl IX lysate buffer per well. 10. Compounds / extracts are diluted 1:10 in IX kinase buffer in polypropylene 96 well plates. 11. Transfer 5.5 μl of diluted compound to ELISA plate wells. Incubate for 20 minutes at room temperature with shaking. 12. Add 5.5 μl of 60 μM ATP solution per well. ATP is not added to the negative control. Incubate for 90 minutes with shaking. 13. Wash 4 times with TBST. Tap the plate on a paper towel to remove excess liquid and air bubbles. 14． 100 μl of RC20 per well (1: 300 in blocking buffer)
0 dilution). Incubate for 30 minutes with shaking. 15. Wash 4 times with TBST. Tap the plate on a paper towel to remove excess liquid and air bubbles. 16. Add 100 μl Turbo-TMB per well. Incubate for 30-60 minutes with shaking. 17． The reaction is stopped by adding 100 μl of 1 MH ₂ SO ₄ per well. 18. Read the assay in Dynatech MR ₇₀ 00 ELISA reader. Test filter = 450 nm, reference filter = 410 nm.

Example 8: Biochemical src Assay-ELISA This assay is used to determine src protein kinase activity by measuring the phosphorylation of a biotinylated peptide as a readout. Materials and Reagents: The following materials and reagents were used: a. src transformed yeast (Sugen, Inc., Redwo)
odCity, California) b. Cell lysate: Pellet yeast cells expressing src, wash once with water, pellet again, and store at -80 ° C until use. c. N-terminal biotinylated EEEEYEYEEEEYEEEYEEEEY is prepared by standard methods well known to those skilled in the art. d. DMSO: Sigma, St. Louis, MO e. 96-well ELISA plate: Corning 96-well Easy W
ash, modified flat bottom plate, Corning Cat. # 25805-96 f. NUNC 96-well V-bottom polypropylene plate, for compound dilution: A
Applied Scientific Cat. # A-72092 g. Vecastain ELITE ABC reagent: Vector, Burl
ingame, CA h. Anti-src (327) mab: Schizosaccharomyces
Expression of recombinant Src using Pombe (Superti-Furga,
et al. , EMBO J .; , 12: 2625-2634; Superti-
Furga, et al. , Nature Biochem. , 14: 600-
605). S. Pombe SP200 strain (h-sleul.32 ura4
ade210) was grown as described and the lithium acetate method (Supert).
i-Furga, supra) with the pRSP expression plasmid. Cells are grown in the presence of 1 μM thiamine to suppress expression of the nmtl promoter, or are grown in the absence of thiamine to induce expression. i. Monoclonal anti-phosphotyrosine, UBI05-321 (alternatively UB4
0 can be used) j. Turbo TMB-ELISA peroxidase substrate: Pierce
Chemical

Buffer solution: a. PBS (Dulbecco's phosphate buffered saline): GIBCO PBS, GIB
CO Cat. # 450-1300EB b. Blocking buffer: 5% nonfat milk (Carnation), in PBS c Carbonate buffer: Na ₂ CO ₄ , Fisher, Cat. # S495, 100
Make a mM stock solution. d. Kinase buffer: 1.0 ml (from 1M stock solution) MgCl ₂ ; 0.2 m
1 (from 1M stock solution) MnCl ₂ ; 0.2 ml (from 1M stock solution) DTT;
5.0 ml HEPES (from 1M stock solution); 0.1 ml TX-100; Mil
Make up to 10 ml total volume with liq H ₂ O. e. Lysis buffer: 5.0 HEPES (from 1M stock solution); 2.74 ml Na
Cl (from a 5M stock solution); 10 ml glycerol; 1.0 ml TX-100;
0.4 ml EDTA (from 100 mM stock solution); 1.0 ml PMSF (100
0.1 ml Na ₃ VO ₄ (from 0.1 M stock solution); Mil
Make up to 100 ml total volume with liq H ₂ O. f. ATP: Sigma Cat. # A-7699, 10 mM stock solution (5.
51 mg / ml). g TRIS-HCl: Fischer Cat. # BP152-5,600m
Add 121.14 g of material to 1 of MilliQ H ₂ O and adjust the pH to 7.0 with HCl.
Adjust to 5 and make up to 1 L total volume with MilliQ H ₂ O. h. NaCl: Fischer Cat. # S271-10, MilliQ
To create a 5M stock solution in H ₂ O. i. Na ₃ VO ₄ : Fischer Cat. # S454-50; 80 ml of M
Add 1.8 g of material to illiQ H ₂ O; pH with HCl or NaOH
Adjust pH to 10.0; boil in microwave; cool; check pH and repeat pH adjustment until stable after heating / cooling cycle; Mi
Make up to 100 ml total volume with lliQ H ₂ O; make 1 ml aliquots and
Store at 80 ° C. j. MgCl ₂ : Fischer Cat. # M33-500, MilliQ
Make a 1M stock solution in H ₂ O. k. HEPES: Fischer Cat. # BP310-500; 200m
To 1MilliQ H ₂ O, add 59.6 g of substance, adjust pH to 7.5,
Make up to a total volume of 250 ml with MilliQ H ₂ O and filter sterilize (1M stock solution)
. l. TBST buffer: TBST buffer: 6.057 in 900 ml dH ₂ O
Add gTRIS and 8.766GNaCl; the pH was adjusted to 7.2 with HCl, add 1.0mlTriton-X ₁₀ 0; to a total volume of 1L with dH ₂ O. m. MnCl ₂ : Fischer Cat. # M87-100, MilliQ
Make a 1M stock solution with H ₂ O. n. DTT: Fischer Cat. # BP172-5 o. TBS (TRIS Buffered Saline): to MiIIiQ H ₂ O in 900 ml, added 6.057gTRIS and 8.777gNaCl; MilliQ
The total volume is made up to 1 L with H ₂ O. p. Kinase reaction mixture: Volume per assay plate (100 wells): 1.0 ml Kinase buffer, 200 μg GST-ζ, MilliQ H ₂ O to a final volume of 8.0 ml. q. Biotin-labeled EEYEEYEEEEYEEEEYEEEEY: Make a fresh stock solution of peptide (1 mM, 2.98 mg / ml) in water 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. Next, one drop of reagent B is added. Place the tube in an orbital shaker at room temperature,
Mix for a minute.

Method a. Preparation of src coated ELISA plate Transfer the ELISA plate to 100 μl of sodium carbonate buffer (pH 9.6).
Coat with 0.5 μg / well of anti-src monoclonal antibody in at 4 ° C. overnight. 2. Wash wells once with PBS. 3. Plates are blocked with 0.15 ml of 5% milk in PBS for 30 minutes at room temperature. 4. Wash plate 5 times with PBS. 5. Add lysate of src transformed yeast, diluted in lysis buffer, at 10 μg / well (total volume 0.1 ml per well). (The amount of lysate will vary from batch to batch). Shake plate for 20 minutes at room temperature. b. Preparation of ELISA plate coated with phosphotyrosine antibody 4G10 plate: 0.5 μg / well 4G10 in 100 μl PBS
And block overnight at 4 ° C. with 150 μl of 5% milk in PBS for 30 minutes at room temperature. c. Kinase assay Remove unbound protein from plate and wash plate 5 times with PBS.
2. 0.08 ml of kinase reaction mixture per well (10 μl per well)
10X kinase buffer and 10 μM (final concentration) of biotin-EEEYEE
YEEEYEEEEYEEEY, diluted in water). 3. Add 10 μl of compound diluted in water containing 10% DMSO and pre-incubate for 15 minutes at room temperature. 4. The kinase reaction is started by adding 10 μl / well of 0.05 mM ATP in water (final 5 μM ATP). 5. Shake the ELISA plate at room temperature for 15 minutes. 6. The kinase reaction is stopped by adding 10 μl of 0.5 M EDTA per well. 7. Transfer 90 μl of the supernatant to the blocked 4G10 coated ELISA plate. 8. Incubate for 30 minutes at room temperature with shaking. 9. Wash plate 5 times with TBST. 10. Incubate with Vectastain ELITE ABC reagent (100 μl / well) for 30 minutes at room temperature. 11. Wash wells 5 times with TBST. 12. Color is developed with Turbo TMB.

Example 9: Biochemical lck assay-ELISA This assay is used to determine lck protein kinase activity by measuring GST-ζ phosphorylation as a readout. Materials and Reagents: The following materials and reagents were used: a. Yeast transformed by lck: Schizosaccharomy
Expression of recombinant Lck using ces Pombe (Superti-Fu
rga, et al. , EMBO J, 12: 2625-2634; Super.
ti-Furga, et al. , Nature Biotech. , 14: 6
00-605). S. Pombe SP200 strain (h-s leul. 32 u
ra4ade210) are grown as described and transformed with the pRSP expression plasmid by the lithium acetate method (Superti-Furga,
(Above)). Cells are grown in the presence of 1 μM thiamine to induce expression. b. Cell lysate: Yeast cells expressing lck are pelleted, washed once with water, re-pelleted and stored frozen at -80 ° C until use. c. GST-II: DNA encoding a GST-II fusion protein for expression in bacteria is available from Howard Hughes Medical Institute.
e, the University of California, San F
Obtained from Arthur Weiss of Ranchisco. The transformed bacteria are grown overnight at 25 ° C. with shaking. GST-ζ was obtained using glutathione affinity chromatography (Pharmacia, Alameda, C
Purify according to A). d. DMSO: Sigma, St. Louis, MO e. 96-well ELISA plate: Corning 96-well Easy W
ash, modified flat bottom plate, Corning Cat. # 25805-96 f. NUNC 96-well V-bottom polypropylene plate for compound dilution: Ap
plied Scientific Cat. # AS-72092 g. Purified rabbit anti-GST antiserum: Amrad Corporation (Au
stralia) Cat. # 900001605 h. Goat anti-rabbit-IgG-HRP: Amersham Cat. # V010
301 i. Goat anti-mouse IgG (H + L): Jackson Labs Cat. #
5215-005-003 j. Anti-Lck (3A5) monoclonal antibody: Santa Cruz Bio
technologyCat # sc-433 k. Anti-phosphotyrosine monoclonal antibody UBI05-321 (instead of UB
40 may be used)

Buffer solution: a. PBS (Dulbecco's phosphate buffered saline) 1X solution: GIBCO PBS,
GIBCO Cat. # 450-1300EB b. Blocking buffer: 100 g BSA, 12.1 g TRIS-pH 7.5
, 58.44 g NaCl, 10 ml Tween-20, MilliQ H ₂ O to a total volume of 1 L. c. Carbonate buffer: Na ₂ CO ₄ , Fischer, Cat. # S495; Mil
Make up a 100 mM solution with liQ H ₂ O. d. Kinase buffer: 1.0 ml (from 1M stock solution) MgCl ₂ ; 0.2 m
1 (from 1M stock solution) MnCl ₂ ; 0.2 ml (from 1M stock solution) DTT;
5.0 ml (from 1M stock solution) HEPES; 0.1 ml TX-100; Mil
Make up to 10 ml total volume with liq H ₂ O. e. Lysis buffer: 5.0 HEPES (from 1M stock solution); 2.74 ml Na
Cl (from a 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 (100
0.1 ml Na ₃ VO ₄ (from 0.1 M stock solution); Mil
Make up to 100 ml total volume with liq H ₂ O. f. ATP: Sigma Cat. # A-7699, 10 mM stock solution (5.
51 mg / ml). g TRIS-HCl: Fischer Cat. # BP152-5,600m
To 1 liter of MilliQ H ₂ O, add 121.14 g of material and adjust the pH to 7 with HCl.
. Adjust to 5 and make up to 1 L total volume with MilliQ H ₂ O. h. NaCl: Fischer Cat. # S271-10, MilliQ
To create a 5M stock solution in H ₂ O. i Na ₃ VO ₄ : Fischer Cat. # S454-50; 80 ml of Mi
Add 1.8 g of material to the liQ H ₂ O; adjust pH to 10.0 with HCl or NaOH; boil in microwave; cool; check pH; pH stable after heating / cooling cycle The pH adjustment is repeated until pH is reached; make up to a total volume of 100 ml with MilliQ H ₂ O; make 1 ml aliquots and store at −80 ° C. j. MgCl ₂ : Fischer Cat. # M33-500, MilliQ
Make a 1M stock solution in H ₂ O. k. HEPES: Fischer Cat. # BP310-500; 200m
in MiIIiQ H ₂ O of l, added substances of 59.6 g, the pH was adjusted to 7.5, a total volume of 250ml in MiIIiQ H ₂ O, filter sterilized (1M stock solution). l. Albumin, bovine (BSA), Sigma Cat. # A4503; 15
To 0 ml of MilliQ H ₂ O, add 30 g of material, make up to a total volume of 300 ml with MilliQ H ₂ O, filter through a 0.22 μm filter and store at 4 ° C. 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; 1.0 ml
Add Triton-X ₁₀ 0; to a total volume of 1L with dH ₂ O. n. MnCl ₂ : Fischer Cat. # M87-100, MilliQ
Make a 1M stock solution in H ₂ O. o. DTT; Fischer Cat. # BP172-5 p. TBS (TRIS Buffered Saline): to MiIIiQ H ₂ O in 900 ml, added 6.057gTRIS and 8.777gNaCl; MilliQ
The total volume is made 1 L with H ₂ O. q Kinase reaction mixture: Amount per assay plate (100 wells):
Make up to a final volume of 8.0 ml with 1.0 ml kinase buffer, 200 μg GST-ζ, MilliQ H ₂ O.

Method: a. Preparation of Lck coated ELISA plate Goat anti-mouse Ig in 100 μl sodium carbonate buffer (pH 9.6)
Coat at 2.0 μg / well at 4 ° C. overnight. 2. Wash wells once with PBS. 3. Block the plate with 0.15 ml blocking buffer for 30 minutes at room temperature. 4. Wash plate 5 times with PBS. 5. 0.5 μl of anti-lck (monoclonal antibody 3A5) in 0.1 ml PBS
Add 1-2 hours at room temperature in g / well. 6. Wash plate 5 times with PBS. 7. Add lck-transformed yeast lysate diluted in lysis buffer at 20 μg / well (total volume 0.1 ml per well). Plate at 4 ° C
Shake overnight to prevent loss of activity. b. Preparation of ELISA plate coated with phosphotyrosine antibody UB40 plate: Add 1.0 μg / well UB40 in 100 μl PBS overnight at 4 ° C. and block with 150 μl blocking buffer for at least 1 hour. c. Kinase assay Remove unbound protein from plate and wash plate 5 times with PBS. 2. Add 0.08 ml per well of the kinase reaction mixture (per well containing 10 μl of 10 × kinase buffer and 2 μg GST-ζ diluted in water). 3. Add 10 μl of compound diluted in water containing 10% DMSO and pre-incubate for 15 minutes at room temperature. 4. The kinase reaction is started by adding 10 mM / well of 0.1 mM ATP in water (final concentration 10 μM ATP). 5. Shake the ELISA plate at room temperature for 60 minutes. 6. The kinase reaction is stopped by adding 10 μl of 0.5 M EDTA per well. 7. Transfer 90 μl of the supernatant to the blocked 4G10 coated ELISA plate from section B above. 8. Incubate for 30 minutes at room temperature with shaking. 9. Wash plate 5 times with TBST. 10. Incubate with rabbit anti-GST antibody diluted 1: 5000 in 100 μl TBST for 30 minutes at room temperature. 11. Wash wells 5 times with TBST. 12. Goat anti-rabbit-I diluted 1: 20,000 in 100 μl TBST
Incubate with gG-HRP for 30 minutes at room temperature. 13. Wash wells 5 times with TBST. 14． Color is developed with Turbo TMB.

Example 10: Assay to Measure RAF Phosphorylation Function The following assay measures the amount of phosphorylation of RAF-catalyzed phosphorylation of its target protein MEK as well as MAPK, which is the target of MEK. The RAF gene sequence is
(1985, Molec. Cell. Biol. 5: 1400-).
1407) and is easily accessible in many gene sequence data banks. The construction of nucleic acid vectors and cell lines used in this part of the invention are described in Morrison et al. (1988, Proc. Natl. Acad.
. Sci. USA 85: 8855-8859). Materials and Reagents Sf9 (Spodoptera frugiperda) cells; GIBC
1. O-BRL, Gaithersburg, MD RIPA buffer: 20 mM Tris / HC1 pH 7.4, 137 mM Na
Cl, 10% glycerol, 1 mM PMSF, 5 mg / L aprotenin, 0.5
2.% Triton X-100; Thioredoxin-MEK fusion protein (T-MEK): T-MEK expression and purification by affinity chromatography are performed according to the manufacturer's methods. Catalog # K350-01 and R ₃₅ 0-40, Invitro
genCorp. , San Diego, CA 4. His-MAPK (ERK2); His-tag MAPK is His-MA
XL1 B1 transformed with pUC18 vector encoding PK
ue cells. His-MAPK is purified by Ni-affinity chromatography. Cat # 27-4949-01, Pharmacia
, Alameda, CA, as described herein. 5. Goat anti-mouse IgG: Jackson laboratories, We
st Grove, PA. Catalog # 515-006-008, Lot # 28
563 6. 6. RAF-1 protein kinase specific antibody: URP2653, UBI Coating buffer: PBS; phosphate buffered saline, GIBCO-BRL
7. Gaithersburg, MD Wash buffer: TBST-50mM Tris / HCL pH 7.2, 150m
8. MNaCl, 0.1% Triton X-100 9. Blocking buffer: TBST, 0.1% ethanolamine pH 7.4 DMSO, Sigma, St. Louis, MO11. 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 MMD
TT and 10 mM MgCl ₂ 12. ATP mixture: 100 mM MgCl ₂ , 300 mM ATP, 10 mCi
13. γ ³³ PATP (DuPont-NEN) / mL 13 stop solution: 1% phosphoric acid; Fisher, Pittsburgh, PA Wallac phosphate cellulose filter mat; Wallac, Tu
rku, Finnland 15. Filter washing solution: 1% phosphoric acid, Fisher, Pittsburg
h, PA16. Tomtec plate recovery machine, Wallac, Turku, Finnl
and 17. Wallac Beta Plate Reader # 1205, Wallac, Tu
rku, Finnland 18. For compounds, NUNC 96-well V-bottom polypropylene plate, App
lied Scientific Catalog # AS-72092

[0384] The method all of the following steps, unless otherwise indicated, were performed at room temperature. 1. ELISA plate coating: ELISA wells with 100 ml goat anti-mouse affinity purified antiserum (1 mg / 100 mL coating buffer)
And coat overnight at 4 ° C. When the ELISA plate is stored at 4 ° C,
Can be used for 2 weeks. 2. Invert plate to remove liquid. Add 100 mL of blocking solution and incubate for 30 minutes. 3. Remove the blocking solution and wash four times with wash buffer. Tap the plate on a paper towel to remove excess liquid. 4. Add 1 mg of RAF-1 specific antibody to each well and incubate for 1 hour. Wash as described in step 3. 5. Thaw lysates from Sf9 cells infected with RAS / RAF and thaw TBST
Dilute to 10 mg / 100 mL with. Add 10 mg of the diluted lysate to the wells and add 1
Incubate for hours. Shake plate during incubation. No lysate is added to the negative control. After infecting cells with the recombinant baculovirus at MOI 5 for each virus, lysates from Sf9 insect cells infected with RAS / RAF are prepared and harvested 48 hours later. Wash cells once with PBS and lyse in RIPA buffer. Insoluble material is removed by centrifugation (5 min, 10000 × g). Aliquots of the lysate are frozen in dry ice / ethanol and used until
Store at 80 ° C. 6. Remove unbound material and wash as described briefly above (step 3). 7. Add 2 mg T-MEK and 2 mg 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 described in the Examples herein. 8. Compounds (10 mg / mL stock solution in DMSO) or extracts are pre-diluted 20-fold in TBST plus 1% DMSO. Add 5 mL of the pre-diluted compound / extract to the wells described in step 6. Incubate for 20 minutes. Controls receive no drug. 9. Initiate the kinase reaction by adding 5 mL ATP mix. During the incubation, shake the plate on an ELISA plate shaker. 10. After 60 minutes, the kinase reaction is stopped by adding 30 mL of stop solution to each well. 11. Place the phosphocellulose mat and ELISA plate in a Tomtec plate collector. Collect the filter and wash it with the filter wash solution according to the manufacturer's recommendations. Dry the filter mat. Seal the filter mat and place in the case. Place the case in a radioactivity detector and quantify the radioactive phosphorus on the filter mat. Alternatively, a 40 mL aliquot from each well of the assay plate is
It can be transferred to the corresponding location on the phosphocellulose filter mat. After air-drying the filter, place the filter in the tray. Lock the tray gently,
The wash solution is changed every 15 minutes for 1 hour. Air dry the filter mat. Seal the filter mat and place in a suitable case for measuring radioactive phosphorus in the sample. Place the case in the detector and quantify the radioactive phosphorus on the filter mat.

Example 11: CDK2 / cyclin A-inhibition assay This assay measures the protein kinase activity of CDK2 in an external substrate. Materials and Reagents: Buffer A (80 mM Tris (pH 7.2), 40 mM MgCl ₂ ): 4
. 84 g Tris (FW = 121.1 g / mol), 4.07 g MgCl ₂
(FW = 203.31 g / mol), dissolved in 500 ml H ₂ O. Adjust the pH to 7.2 with HCl. B. Histone H1 solution (0.45 mg / ml histone H1 and 20 mM HE
PES (pH 7.2): 11.111 ml of 20 mM HEPES pH 7.2 (4
5 mg histone H1 (Boehringer Mannheim) in 77 mg HEPES (FW = 238.3 g / mol), dissolved in 100 ml ddH ₂ O. Store at -80 ° C as 1 ml aliquots. C. ATP solution (60 μM ATP, 300 μg / ml BSA, 3 mM DTT)
: 20 μl of 120 μl of 10 mM ATP, 600 μl of 10 mg / ml BSA
And store at -80 ° C as 1 ml aliquots. D. CDK2 solution: cdk2 / cyclin A, 10 mM HEPES, pH7.
In 2,25 mM NaCl, 0.5 mM DTT, 10% glycerol. Store at -80 ° C as 9 μl aliquots.

Assay Description A solution of the inhibitor is prepared in ddH ₂ O / 15% DMSO (v / v) at 3 times the desired final assay concentration. 2. Add 20 μl of inhibitor to the wells of a polypropylene 96-well plate (or 20 μl of 15% DMSO for positive and negative controls). 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). CDK2 is kept on ice until use. Divide the CDK2 solution appropriately into aliquots to avoid repeated freeze-thaw cycles. 4. Dilute 9 μl CDK2 solution with 2.1 ml buffer A (per plate),
Mix and add 20 μl to each well. 5. Mix 1 ml Histone H1 solution with 1 ml ATP solution (per plate) and place in a 10 ml screw cap tube. 0.15 μCi / 20 μl of γ ³³ P ATP (
During the assay, add to a concentration of 0.15 μCi / well). Mix carefully to avoid BSA foam. Add 20 μl to the appropriate well. Mix plate on plate shaker. For the negative control, an equal volume of 20 mM HEPES (
pH 7.2) and an ATP solution, and γ ³³ P ATP was added at 0.15 μCi / 20.
Add to a concentration of μl solution. Add 20 μl to the appropriate well. 6. The reaction is allowed to proceed for 60 minutes. 7. Add 35 μl of 10% TCA to each well. Mix plate on plate shaker. 8. Spot 40 μl of each sample on the squares of a P30 filter mat. Dry the mat (about 10-20 minutes). 9. Filter mat was added to 250 ml of 1% phosphoric acid (1 liter per ddH ₂ O).
(0 ml phosphoric acid) for 4 × ₁₀ minutes. 10. The filter mat is counted on a beta plate reader.

Cell / Biological Assay Example 12: PDGF-Inducible BrdU Uptake Assay Materials and Reagents : (1) PDGF: human PDGF B / B; 1276-956, Boehrin
ger Mannheim, Germany (2) BrdU labeling reagent: 10 mM in PBS (pH 7.4), Cat. No
. 1647229, Boehringer Mannheim, Germany (3) FixDenat: fixative solution (can be used immediately), Cat. No. 16472
29, Boehringer Mannheim, Germany (4) Anti-BrdU-POD: a mouse monoclonal antibody conjugated with peroxidase, Cat. No. 1647229, Boehringer
Mannheim, Germany (5) TMB substrate solution: tetramethylbenzidine (TMB), ready for use, Ca
t. No. 1647229, Boehringer Mannheim, Ger
many (6) PBS washing solution: 1X PBS, pH 7.4 (Sugen, Inc., R
(edwood City, California) (7) Albumin, bovine (BSA): fraction V powder; A-8551, Sigma
Chemical Co. , USA (8) 3T3 cell line engineered to express human PDGF-R

Protocol (1) Cells are seeded at 8000 cells / well in a 96-well plate in DMEM, 10% CS, 2 mM Gln. The cells are incubated overnight at 37 ° C. in 5% CO ₂ . (2) After 24 hours, the cells are washed with PBS and then serum-free medium (0% CSDM)
(EM, 0.1% BSA) for 24 hours. (3) On the third day, ligand (PDGF, 3.8 nM, DMEM, 0.1% BS
A) and a test compound are added to the cells simultaneously. Negative control wells receive only serum-free DMEM and 0.1% BSA; positive control cells receive ligand (PDGF) but no test compound. Test compounds are prepared with ligand in serum-free DMEM in 96-well plates and serially diluted to seven test concentrations. (4) Twenty hours after ligand activation, the diluted BrdU labeling reagent (DMEM, 0.
(1: 100 in 1% BSA) is added and the cells are incubated for 1.5 hours with BrdU (final concentration = 10 μM). (5) After incubation with the labeling reagent, decant and remove the medium by tapping the inverted plate on a paper towel. Fix
Add Denat solution (50 μl / well) and incubate the plate on a plate shaker at room temperature for 45 minutes. (6) The FixDenat solution is thoroughly removed by tapping the decanted and inverted plate on a paper towel. Milk is added as blocking solution (5% dehydrated milk in PBS, 200 μl / well) and the plate is incubated for 30 minutes at room temperature on a plate shaker. (7) The blocking solution is removed by decanting, and the wells are washed once with PBS. An 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. (8) Decant and remove the antibody conjugate well by rinsing the wells 5 times with PBS and dry by inverting the plate and tapping on a paper towel. (9) Add TMB substrate solution (100 μl / well) and incubate for 20 minutes at room temperature on a plate shaker until color development is sufficient for photometric detection. (10) The absorbance of the sample is measured at 410 nm with a Dynatech ELISA plate reader ("two wavelength" mode using a filter reading at 490 nm as reference wavelength).

Example 13 EGF-Inducible BrdU Incorporation Assay Materials and Reagents (1) EGF: mouse EGF, 201; , Ltd. Ja
pan (2) BrdU labeling reagent: 10 mM in PBS (pH 7.4), Cat. No
. 1647229, Boehringer Mannheim, Germany (3) FixDenat: fixative solution (can be used immediately), Cat. No. 16472
29, Boehringer Mannheim, Germany (4) Anti-BrdU-POD: a mouse monoclonal antibody conjugated with peroxidase, Cat. No. 1647229, Boehringer
Mannheim, Germany (5) TMB substrate solution: tetramethylbenzidine (TMB), ready for use, Ca
t. No. 1647229, Boehringer Mannheim, Ger
many (6) PBS washing solution: 1X PBS, pH 7.4 (Sugen, Inc., R
edwoodCity, California) (7) Albumin, bovine (BSA): fraction V powder; A-8551, Sigma
Chemical Co. , USA (8) 3T3 cell line engineered to express human EGF-R

Protocol (1) Cells are seeded with 10% CS, 2 mM Gln in DMEM at 8000 cells / well in a 96 well plate. The cells are incubated overnight at 37 ° C. in 5% CO ₂ . (2) After 24 hours, the cells are washed with PBS and then serum-free medium (0% CSDM)
(EM, 0.1% BSA) for 24 hours. (3) On the third day, ligand (prepared in EGF, 2 nM, DMEM, 0.1% BSA) and test compound are added simultaneously to the cells. 0.1% B for negative control wells
Only serum free DMEM with SA is added. Positive control cells include a ligand (EGF
) But without the test compound. Test compounds were prepared in 96 well plates.
Prepare with ligand in serum-free DMEM and serially dilute to 7 test concentrations. (4) Twenty hours after ligand activation, diluted BrdU labeling reagent (1: 100 in DMEM, 0.1% BSA) is added and cells are BrdU (final concentration = 10 μM).
For 1.5 hours. (5) After incubation with the labeling reagent, remove the medium by tapping the decanted inverted plate on a paper towel. FixDen
The at solution was added (50 μl / well) and the plate was shaken on a plate shaker at room temperature for 45 minutes.
Incubate for minutes. (6) Thoroughly remove the FixDenat solution by tapping the decanted and inverted plate on a paper towel. Milk is added as a blocking solution (5% dehydrated milk in PBS, 200 μl / well) and the plate is incubated for 30 minutes at room temperature on a plate shaker. (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. (8) Decant and remove the antibody conjugate well by rinsing the wells 5 times with PBS and dry by inverting the plate and tapping on a paper towel. (9) Add TMB substrate solution (100 μl / well) and incubate for 20 minutes at room temperature on a plate shaker until color development is sufficient for altimeter detection. (10) The absorbance of the sample is measured with a Dynatech ELISA plate reader at 410 nm (in "two-wavelength" mode using a filter that reads 490 nm as a reference wavelength).

Example 14 EGF-Inducible Her2-Promoted BrdU Incorporation Materials and Reagents : (1) EGF: mouse EGF, 201; Toyobo, Co. , Ltd. Ja
pan (2) BrdU labeling reagent: 10 mM in PBS (pH 7.4), Cat. No.
1647229, Boehringer Mannheim, Germany (3) FixDenat: fixative solution (can be used immediately), Cat. No. 16472
29, Boehringer Mannheim, Germany (4) Anti-BrdU-POD: a mouse monoclonal antibody conjugated with peroxidase, Cat. No. 1647229, Boehringer
Mannheim, Germany (5) TMB substrate solution: tetramethylbenzidine (TMB), ready for use, Ca
t. No. 1647229, Boehringer Mannheim, Ger
many (6) PBS washing solution: 1X PBS, pH 7.4, manufactured in-house. (7) Albumin, bovine (BSA): fraction V powder; A-8551, Sigma
Chemical Co. (8) 3T3 cell line engineered to express a chimeric receptor having an extracellular domain of EGF-R and an intracellular domain of Her2

Protocol : (1) Seed cells at 8000 cells / well in 96-well plate in DMEM, 10% CS, 2 mM Gln. The cells are incubated overnight at 37 ° C. in 5% CO ₂ . (2) After 24 hours, the cells are washed with PBS and then serum-free medium (0% CSDM)
Serum starved in EM, containing 0.1% BSA) for 24 hours. (3) On the third day, ligand (EGF = 2 nM, DMEM containing 0.1% BSA)
And the test compound are added to the cells simultaneously. Serum-free DMEM, 0.1% BSA only is added to negative control wells; ligand (EGF) is added to positive control cells
But not the test compound. Test compounds are prepared in 96-well plates with ligand in serum-free DMEM and serially diluted to seven test concentrations. (4) Twenty hours after ligand activation, the diluted BrdU labeling reagent (1: D in DMEM)
100, 0.1% BSA) and the cells are incubated with BrdU (final concentration = 10 μM) for 1.5 hours. (5) After incubation with the labeling reagent, remove the medium by tapping the decanted inverted plate on a paper towel. FixDen
at solution (50 μl / well) and plate at room temperature on a plate shaker.
Incubate for minutes. (6) The FixDenat solution is thoroughly removed by tapping the decanted and inverted plate on a paper towel. Milk is added as blocking solution (5% dehydrated milk in PBS, 200 μl / well) and the plate is incubated for 30 minutes at room temperature on a plate shaker. (7) The blocking solution is removed by decanting, and the wells are washed once with PBS. Add anti-BrdU-POD solution (PBS, 1: 100 dilution in 1% BSA) (100 μl / well) and incubate the plate on a plate shaker for 90 minutes at room temperature. (8) Decant and remove the antibody conjugate well by rinsing the wells 5 times with PBS and dry by inverting the plate and tapping on a paper towel. (9) Add TMB substrate solution (100 μl / well) and incubate for 20 minutes at room temperature on a plate shaker until color development is sufficient for photometric detection. (10) The absorbance of the sample is measured on a Dynatech ELISA plate reader at 410 nm (in "two wavelength" mode using a filter reading at 490 nm as a reference wavelength).

Example 15: IGF1-Inducible BrdU Incorporation Assay Materials and Reagents : (1) IGF1 ligand: human, recombinant; G511, Promega Cor
p, USA (2) BrdU labeling reagent: 10 mM, in PBS (pH 7.4), Cat. No
. 1647229, Boehringer Mannheim, Germany (3) FixDenat: fixative solution (can be used immediately), Cat. No. 16472
29, Boehringer Mannheim, Germany (4) Anti-BrdU-POD: a mouse monoclonal antibody conjugated with peroxidase, Cat. No. 1647229, Boehringer
Mannheim, Germany (5) TMB substrate solution: tetramethylbenzidine (TMB), ready for use, Ca
t. No. 1647229, Boehringer Mannheim, Ger
many (6) PBS washing solution: 1X PBS, pH 7.4 (Sugen, Inc., R
(edwood City, California) (7) Albumin, bovine (BSA): fraction V powder; A-8551, Sigma
Chemical Co. , USA (8) 3T3 engineered to express the human IGF-1 receptor
Cell line

Protocol : (1) Seed cells at 8000 cells / well in 96-well plate in DMEM, 10% CS, 2 mM Gln. The cells are incubated overnight at 37 ° C. in 5% CO ₂ . (2) After 24 hours, the cells are washed with PBS and then serum-free medium (0% CSDM)
(EM, 0.1% BSA) for 24 hours serum starvation (3) On the third day, ligand (IGF1 = 3.3 nM, D containing 0.1% BSA)
(Prepared in MEM) and test compounds are added to the cells simultaneously. Negative control wells receive only serum-free DMEM containing 0.1% BSA. Positive control cells receive the ligand (IGF1) but no test compound. Test compounds are prepared with ligand in serum-free DMEM in 96-well plates and serially diluted to seven test concentrations. (4) 16 hours after ligand activation, the diluted BrdU labeling reagent (DMEM, 0.
(1: 100 in 1% BSA) is added and the cells are incubated for 1.5 hours with BrdU (final concentration = 10 μM). (5) After incubation with the labeling reagent, decant and remove the medium by tapping the inverted plate on a paper towel. FixD
Enat solution is added (50 μl / well) and the plate is incubated for 45 minutes at room temperature on a plate shaker. (6) The FixDenat solution is removed well by tapping the decanted and inverted plate on a paper towel. Milk is added as blocking solution (5% dehydrated milk in PBS, 200 μl / well) and the plate is incubated for 30 minutes at room temperature on a plate shaker. (7) The blocking solution is removed by decanting, and the wells are washed once with PBS. An 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. (8) Decant and thoroughly remove antibody conjugate by rinsing wells 5 times with PBS, invert plate and dry by tapping on paper towel. (9) Add TMB substrate solution (100 μl / well) and incubate on a plate shaker at room temperature for 20 minutes until color development is sufficient for photometric detection. (10) The absorbance of the sample is measured with a Dynatech ELISA plate reader at 410 nm ("two wavelength" mode using a filter reading at 490 nm as a reference wavelength).

Example 16: HUV-EC-C assay Also, using the following protocol, PDGF-R, FGF-R, VEGF, a
The activity of the compound on FGF or Flk-1 / KDR, all of which are naturally expressed by HUV-EC cells, can be measured. Day 0 1. HUV-EC-C cells (human umbilical vein endothelial cells, (American Ty
catalog No. pe Culture Collection; 1730 CRL)
Is washed and trypsinized. Dulbecco's phosphate buffered saline (D-PBS;
Gibco BRL; Catalog No. 14190-029) twice in about 1 ml / 10 cm ² tissue culture flask. Non-enzyme cell dissociation solution (Sig
ma Chemical Company; Catalog No. Trypsinize with 0.05% trypsin-EDTA in C-1544). 0.05% trypsin is 0.25% trypsin / 1 mM EDTA (Gibco; Catalog No. 25)
200-049) in a cell dissociation solution. About 1ml /
Trypsinize in a 25-30 cm ² tissue culture flask at 37 ° C for about 5 minutes. After the cells were detached from the flask, an equal volume of assay medium was added and a 50 ml sterile centrifuge tube (Fisher Scientific; Catalog No. 05-539-6) was added.
). 2. The cells are washed with about 35 ml of assay medium by adding the assay medium into a 50 ml sterile centrifuge tube, centrifuged at about 200 g for 10 minutes, aspirated and resuspended in 35 ml of D-PBS. . Wash twice more with D-PBS and resuspend the cells in approximately 1 ml / 15 cm ² of tissue culture flask in assay medium. Assay medium was F12K medium (Gibco BRL; Catalog No. 21127-).
014) + 0.5% heat-inactivated fetal calf serum. Coulter Cou
The cells are counted on an lnter ™ (Coulter Electronics, Inc.) and assay medium is added to the cells to a concentration of 0.8-1.0 × 10 ⁵ cells / ml. 3. Cells were plated in 96-well flat bottom plates at 100 μl / well or 0.8-1.
Add at 0 × 10 ⁴ cells / well; incubate at 37 ° C., 5% CO ₂ for about 24 hours.

Day 1 Make two-fold titrations of test compounds in separate 96-well plates. Generally, from 50 μM to 0 μM. On day 0 above, use the same assay medium as in step 2. Titration was performed using 90 μl / well of test compound at 200 μM (4 × final well concentration).
By adding to the top well of a particular plate column. Since the concentration of the test compound stock solution is usually 20 mM in DMSO, the drug concentration of 200 μM is 2% D
Including MSO. Therefore, in order to dilute the drug while keeping the DMSO concentration constant, a diluent of 2% DMSO (F12K + 0.5% fetal bovine serum) in the assay medium was used.
Used as diluent for test compound titration. Add this dilution to the remaining wells in the column at 60 μl / well. Take 60 μl from the 120 μl 200 μM test compound dilution in the top well of the column and mix with 60 μl in the second well of the column. Take 60 μl from this well and 60 μl in the third well in the column.
and continue until the 2-fold titration is complete. When the penultimate well is mixed, remove 60 μl of 120 μl in this well and discard it. The last well receives 60 μl of DMSO / media dilution as a drug-free control. Prepare nine columns sufficient for triplicate wells of the test compound to be titrated for the following assays: (1) VEGF (obtained from Pepro Tech Inc., catalog No. 100-200, (2) endothelial cell growth factor (ECGF) (also known as acidic fibroblast growth factor or aFGF) (Boehringe
r Mannheim Biochemical, catalog no. 143
9600); or (3) human PDGF B / B (1276-956, Boeh).
(Ringer Mannheim, Germany) and assay media controls.
ECGF was obtained as a preparation with sodium heparin. 2. 50 μl / well of the test compound dilution was added to the HUV-EC-C from day 0.
Transfer to a 96-well assay plate containing 0.8-1.0 × 10 ⁴ cells / 100 μl / well and incubate at 37 ^° C., 5% CO ₂ for about 2 hours. 3. In triplicate, add 50 μl / well of 80 μg / ml VEGF, 20 ng / ml ECGF, or media control for each test compound condition. For test compounds, the growth factor concentration is four times the desired final concentration. On day 0, the concentration of the growth factor is adjusted using the assay medium from step 2. Approx. 24 at 37 ° C, 5% CO ₂
Incubate for hours. To each well, add 50 μl of test compound dilution, 50 μl of growth factor or medium, and 100 μl of cells for a total volume of 200 μl / well. That is, when all are added to the wells, the 4-fold concentration of test compound and growth factor is 1-fold.

Day 1 ³ H-thymidine (Amersham; Catalog No. TRK-686) was added at 1 μCi / well (10 μl / well of a 100 μCi / ml solution prepared in RPMI medium + 10% heat-inactivated fetal bovine serum) and 37 ° C., 5 Incubate for about 24 hours with% CO ₂ . RPMI is available from Gibco BRL, catalog no. 11875-051 Day 3 1. Freeze the plate at -20 ° C overnight. Day 4 1. The plate was thawed and a 96-well plate collector (Tomtec Harv) was used.
filter mat (Wallac; Catalog N)
o. 1205-401); count on a Wallac Betaplate ™ liquid scintillation counter.

Example 17: FGF- induced BrdU incorporation assay This assay measures FGF-induced DNA synthesis in 3Tc7 / EGFr cells expressing exogenous FGF receptor. Materials and Reagents: FGF: human FGF2 / bFGF (Gibco BRL, No. 13256)
-029) 2. BrdU labeling reagent, (10 mM PBS (pH 7.4), Boehringe
r Mannheim CatNo. 1647229) 3. Fixdenat fixative solution (Boehringer Mannheim C
at No. 1647229) 4. Anti-BrdU-POD (mouse monoclonal antibody conjugated to peroxidase, Boehringer MannheimCat: No. 16)
47229) 5. TMB (tetramethylbenzidine, Boehringer Mannhei
mCat. No. 1647229) 6. 6. PBS wash solution, pH 7.4 (Sugen, Inc.) Albumin, bovine (BSA), fraction V powder (Sigma Chemical)
Co. , Cat. No. A-8551)

Method 1. 3T3 genetically engineered cell line: 3T3c7 / EGFr Cells are seeded at 8,000 cells / well in 96 well plates in DMEM, 10% CS and 2 mM Gln. Incubate for 24 hours at 37 ° C. in 5% CO ₂ . 3. After 24 hours, cells are washed with PBS and then serum-free medium (0% DMEM, 0%
. Serum starvation in 1% BSA) for 24 hours. 4. Simultaneously add ligand (FGF2 (1.5 nM in DMEM, 0.1% BSA) and test compound. Negative control wells in serum-free DME with 0.1% BSA
M alone is added, and positive control wells are added FGF2 ligand but no test compound. Test compounds are prepared with ligand in serum-free DMEM in 96-well plates and serially diluted to seven test concentrations. 5. After 20 hours, add diluted BrdU labeling reagent to the cells (1: 100 BrdU: D
(MEM, 0.1% BSA, final concentration 10 μM), and incubate for 1.5 hours. 6. Decant the medium. Remove traces of material with a paper towel. FixD
Add enaat (50 μl / well) and incubate for 45 minutes at room temperature on a plate shaker. 7. Remove the 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. 8. Decant the blocking solution and wash the wells once with PBS. Anti-Brd
Add U-POD solution (1: 100 dilution in PBS, 0.1% BSA) and incubate for 90 minutes at room temperature on a plate shaker. 9. Decant the antibody conjugate and rinse the wells 5 times with PBS. Dry the plate by inverting and tapping on a paper towel. 10. The TMB solution was added (100 μl / well) and the plate was shaken at room temperature for 20 minutes.
Incubate for 1 minute until color development is sufficient for photometric detection. 11. The absorbance is measured at 410 nM on a Dynatech ELISA plate reader using the "two wavelength" mode, using a 490 nM filter.

Example 18: Biochemical EGFR Assay This assay is a material and reagent for measuring the in vitro kinase activity of EGFR using ELISA. Corning 96-well Elisa plate (Corning Catalog N
o. 25805-96) 2. SUM01 monoclonal anti-EGFR antibody (Biochemistry L
ab, SUGEN, Inc. ) 3. PBS (Dulbecco's phosphate buffered saline, Gibco Catalog No. 450
-1300EB) 4. TBST buffer

[Table 6] 5. Blocking buffer:

[Table 7] 6. A431 cell lysate (Screening Lab, SUGEN, Inc.).
) 7. TBS buffer:

[Table 8] 8. TBS + 10% DMSO

[Table 9] 9. Adenosine-5'-triphosphate (ATP from horse muscle, Sigma Cat
. No. Preparing A-5394) dH ₂ O in 1.0mM solutions. Make this reagent immediately before use and keep it on ice. 10. Prepare a stock solution of 1.0 M in MnCl ₂ dH ₂ O. 11. ATP / MnCl ₂ phosphorylation mixture

[Table 10] This reagent must be prepared immediately before use and stored on ice. 12. NUNC 96-well V-bottom polypropylene plate (Applied Sc
entitic Cat. No. AS-72092) 13. Ethylenediaminetetraacetic acid (EDTA) dH ₂ O in 200mM work to prepare a solution. Adjust to pH 8.0 with 10N NaOH. 14． Rabbit polyclonal anti-phosphotyrosine serum (Biochemistry)
Lab, SUGEN, Inc. 15). Goat anti-rabbit IgG peroxidase conjugate (Biosource
e Cat. No. ALT0404) 16. ABTS (2,2′-azino-bis (3-ethylbenzthiazoline-6)
Sulfuric acid), Sigma Cat. No. A-1888)

[Table 11] First, mixing the two components in dH ₂ O to about 900 ml. PH 4 with phosphoric acid
. Adjust to 0. Add ABTS, cover, leave for about 0.5 hour, and filter.
The solution should be stored at 4 ° C. in the dark before use. 17． 17. Hydrogen peroxide 30% solution (Fisher Cat. No. H325) ABTS / H ₂ O ₂ 15 ml of ABTS solution and 2.0 μl of H ₂ O ₂ are mixed. Prepare 5 minutes before use. 19. 0.2 M HCl

Method 1. Corning 96-well ELISA plate with 100 μl P / well
Coat with 0.5 μg SUM01 in BS and store at 4 ° C. overnight. 2. Unbound SUM01 is removed from the wells by inverting the plate and removing the liquid. Wash once with dH ₂ O. Tap the plate on a paper towel to remove excess liquid. 3. Add 150 μl of blocking buffer to each well. Incubate for 30 minutes at room temperature with shaking. 4. The plate is washed three times with deionized water and then once with TBST. Tap the plate on a paper towel to remove excess liquid and air bubbles. 5. Dilute the lysate in PBS (7 μg lysate / 100 μl PBS). 6. Add 100 μl of diluted lysate to each well. Shake at room temperature for 60 minutes. 7. Wash the plate as described in step 4 above. 8. Add 120 μl of TBS to the ELISA plate containing captured EGFR. 9. Test compounds are diluted 1:10 with TBS in 96-well polypropylene plates (ie, 10 μl compound + 90 μl TBS). 10.13.5 μl of diluted test compound is added to the ELISA plate. Add 13.5 μl TBS + 10% DMSO to control wells (no test compound in wells). 11. Incubate for 30 minutes at room temperature with shaking. 12. Add 15 μl phosphorylation mixture directly to all wells except negative control wells. No ATP / MnCl ₂ is added to the negative control wells. (The final well volume should be about 150 μl, resulting in a final concentration of 3 μM ATP / 5 mM MnCl _{2 in} each well). Incubate for 5 minutes with shaking. After 13.5 minutes, stop the reaction by adding 16.5 μl of 200 mM EDTA (pH 8.0) to each well and continue shaking. Shake for 1 minute after adding EDTA. 14． Wash 4 times with deionized water and 2 times with TBST. 15. Add 100 μl per well of anti-phosphotyrosine (1: 3000 dilution in TBST). Incubate for 30-45 minutes at room temperature with shaking. 16. Wash as described in step 4 above. 17. Add 100 μl Biosource goat anti-rabbit IgG peroxidase conjugate (1: 2000 dilution in TBST) to each well. Incubate for 30 minutes at room temperature with shaking. 18. Wash as described in step 4 above. 19. Add 100 μl ABTS / H ₂ O ₂ solution to each well. 20. Incubate for 5-10 minutes with shaking. Remove air bubbles. 21. If necessary, stop the reaction by adding 100 μl of 0.2 M HCl per well. 22. Read the assay in Dynatech MR ₇₀ 00 ELISA reader;
Test filter: 410 nM Reference filter: 630 Nm.

Example 19: Biochemical PDGFR Assay This assay measures the in vitro kinase activity of PDGFR using an ELISA. Materials and Reagents Unless otherwise noted, the preparation of working solutions for the following reagents was performed using the biochemical EG described above.
Same as FR assay. 1. Corning 96-well Elisa plate (Corning Catalog N
o. 25805-96) 2.28 D4C10 monoclonal anti-PDGFR antibody (Biochemistr)
y Lab, SUGEN, Inc. ) 3. PBS (Dulbecco's phosphate buffered saline, Gibco Catalog No. 450
-1300EB) 4. TBST buffer 5. Blocking buffer 6. PDGFR-β expressing NIH3T3 cell lysate (Screening Lab
, SUGEN, Inc. ) 7. TBS buffer 8. 8. TBS + 10% DMSO Adenosine-5'-triphosphate (ATP, derived from horse muscle, Sigma Cat
. No. A-5394) 10. MnCl ₂ 11. Kinase buffer phosphorylation mixture

[Table 12] 12. NUNC 96-well V-bottom polypropylene plate (Applied Sc
entitic Cat. No. AS-72092) 13. 13. Ethylenediaminetetraacetic acid (EDTA) Rabbit polyclonal anti-phosphotyrosine serum (Biochemistry)
Lab, SUGEN, Inc. 15). Goat anti-rabbit IgG peroxidase conjugate (Biosource
e Cat. No. ALI0404) 16.2,2'-Azino-bis (3-ethylbenzthiazoline-6-sulfuric acid) (A
BTS, Sigma Cat. No. A-1888) 17. 17. Hydrogen peroxide 30% solution (Fisher Cat. No. H325) ABTS / H ₂ O ₂ 19.0.2 M HCl

Method 1. Corning 96-well ELISA plate with 100 μl P / well
Coat with 0.5 μg of 28D4C10 in BS. Store overnight at 4 ° C. 2. Unbound 28D4C10 is removed from the wells by inverting the plate and removing the liquid. Wash once with dH ₂ O. Tap the plate on a paper towel to remove excess liquid. 3. Add 150 μl of blocking buffer to each well. Incubate for 30 minutes at room temperature with shaking. 4. The plate is washed three times with deionized water and then once with TBST. Tap the plate on a paper towel to remove excess liquid and air bubbles. 5. Dilute lysate in HNTG (10 μg lysate / 100 μl HNTG) 6. Add 100 μl of diluted lysate to each well. Shake at room temperature for 60 minutes. 7. The plate is washed as described in step 4 above. 8. Capture 80 μl working kinase buffer mixture ELI containing PDGFR
Add to SA plate. 9. Test compounds are diluted 1:10 with TBS in 96-well polypropylene plates (10 μl compound + 90 μl TBS). 10. Add 10 μl of diluted test compound to the ELISA plate. Control wells (
10 μl TBS + 10% DMSO is added to the wells without test compound). 11. Incubate for 30 minutes at room temperature with shaking. 12. Add 10 μl ATP directly to all wells except negative control wells. (
The final well volume in each well should be about 100 μl containing 20 μM ATP). Incubate for 30 minutes with shaking. 13. After 30 minutes, stop the reaction by adding 10 μl of 200 mM EDTA (pH 8.0) to each well. 14． Wash 4 times with deionized water and 2 times with TBST. 15. Add 100 μl per well of anti-phosphotyrosine (1: 3000 dilution in TBST). Incubate for 30-45 minutes at room temperature with shaking. 16. Wash as described in step 4 above. 17. Add 100 μl Biosource goat anti-rabbit IgG peroxidase conjugate (1: 2000 dilution in TBST) to each well. Incubate for 30 minutes at room temperature with shaking. 18. Wash as described in step 4 above. 19. Add 100 μl ABTS / H ₂ O ₂ solution to each well. 20. Incubate for 10-30 minutes with shaking. Remove air bubbles. 21. If necessary, stop the reaction by adding 100 μl of 0.2 M HCl per well. 22. Read the assay in Dynatech MR ₇₀ 00 ELISA reader:
Test filter: 410 nM, reference filter: 630 nM.

Example 20: Biochemical FGFR Assay This assay measures the kinase activity of the Myc-GyrB-FGFR fusion protein in vitro using an ELISA. Materials and Reagents HNTG

[Table 13] To make one liter of a 5x stock solution, HEPES and NaCl are dissolved in about 350 ml of dH ₂ O, the pH is adjusted to 7.2 with HCl or NaOH (depending on the HEPES used), glycerol, Triton X ₁₀₀ And then add dH ₂ O to make the final volume. 2. PBS (Dulbecco's phosphate buffered saline, Gibco Catalog # 450-1
300EB) 3. Blocking buffer 4. Kinase buffer

[Table 14] 5. Phenylmethylsulfonyl fluoride (PMSF, Sigma, Cat. No.
P-7626) Working solution: 100 mM in ethanol ATP (bacterial origin, Sigma Cat.No.A-7699) for 6mM concentration of stock solution, MiIIiQ of 1 ml H ₂ O per 3.3
Use 1 mg. 7. Biotin-conjugated anti-phosphotyrosine monoclonal antibody (clone 4G10, Upstate Biotechnology Inc. Cat. N.
o. 16-103, Ser. No. 14495) 8. Vectastain Elite ABC reagent (avidin peroxidase conjugate, Vector Laboratories Cat. No.
PK-6100) 9. ABTS solution 10. 10. 30% hydrogen peroxide solution (Fisher catalog # H325) ABTS / H ₂ O ₂ 12.0.2M HCl 13. TRIS HCl (Fischer Cat.No.BP152-5) MilliQ H a ₂ 1.0 mM solution in O was prepared and the pH is adjusted to 7.2 with HCl 14. _{NaCl (Fisher Cat.No.S271-10) MilliQ H 2} 5M solution 15 for preparing in O. _{MgCl 2 (Fisher Cat.No.M33-500) MilliQ H} 2 1M solution preparing 16 in O. HEPES (Fisher Cat.No.BP310-500) MilliQ H ₂ a 1M solution in O was prepared and the pH adjusted to 7.5, sterilized by filtration. 17． TBST buffer 18. Sodium carbonate buffer (Fisher Cat.No.S495) of 0.1M solution prepared in MilliQ H ₂ O, the pH was adjusted to 9.6 with NaOH, and filtered. 19. Dithiothreitol (DTT, Fisher Cat. No. BP172)
-25) preparing 0.5mM of working solution in immediately before use MilliQ H ₂ O. Store at -20 ° C until use, discard remainder. 20. MnCl ₂ 21. Triton X-100 22. 21. Goat anti-rabbit IgG (Cappel) Affinity purified rabbit anti-GST GyrB (Biochemistr)
y Lab. SUGEN, Inc. )

[0405] The method following all steps, unless otherwise stated, carried out at room temperature. 1. A Corning 96-well ELISA plate with a total well volume of 100
Coat 2 μg per well with goat anti-rabbit antibody in carbonate buffer to give μl. Store overnight at 4 ° C. 2. Unbound goat anti-rabbit antibody is removed by inverting the plate and removing the liquid. Tap the plate on a paper towel to remove excess liquid and air bubbles. 3. Add 150 μl blocking buffer (5% low fat milk in PBS) to each well. Incubate for 30 minutes with shaking on a microtiter plate shaker. 4. Wash 4 times with TBST. Tap the plate on a paper towel to remove excess liquid and air bubbles. 5. Add 0.5 μg of rabbit anti-GyrB antibody per well. Antibody to DPBS
To a final volume of 100 μl per well. Incubate for 1 hour at room temperature with shaking on a microtiter plate shaker. 6. Wash 4 times with TBST as described in step 4. 7. 2 μg COS / FGFR cell lysate (Myc-GyrB-F
GFR source) is added to each well to give a final volume of 100 μl per well. Incubate for 1 hour with shaking on a microtiter plate shaker. 8. Wash 4 times with TBST as described in step 4. 9. Add 80 μl of 1 × kinase buffer per well. 10. Test compounds are diluted 1:10 in 1x kinase buffer + 1% DMSO in polypropylene 96 well plates. 11. Transfer 10 μl of the diluted test compound solution and control wells from the polypropylene plate wells to the corresponding ELISA plate wells and incubate for 20 minutes with shaking on a microtiter plate shaker. 12. Add 10 μl of 70 μM ATP diluted in kinase buffer to positive control and test wells (final ATP concentration 7 μM / well). Add 10 μl of 1 × kinase buffer to negative control wells. Incubate for 15 minutes with shaking on a microtiter plate shaker. The kinase reaction is stopped by adding 13.5 μl of 0.5 M EDTA to all wells. 14． Wash 4 times with TBST as described in step 4. 15. Add 100 μl of biotin-conjugated anti-phosphotyrosine monoclonal antibody (b4G10) diluted in TBST to each well. Incubate for 30 minutes with shaking on a microtiter plate shaker. 16. Prepare Vectastain ABC reagent. 15 ml of one drop of reagent A
Add to TBST. Mix by inverting the tube several times. Add 1 drop of reagent B and mix again. 17． Wash 4 times with TBST as described in step 4. 18. Add 100 μl ABC HRP reagent to each well. Incubate for 30 minutes with shaking on a microtiter plate shaker. 19. Wash 4 times with TBST as described in step 4. 20. Add 100 μl of ABTS / H ₂ O ₂ solution to each well. 22. Incubate for 5-15 minutes with shaking. Remove air bubbles. 23. If necessary, stop the reaction by adding 100 μl 0.2 M HCl / well. 24. Read the assay in Dynatech MR ₇₀ 00 ELISA plate reader; test filter: 410nM, reference filter: 630nM.

Example 21: Biochemical FLK-1 Assay This assay evaluates flk-1 autophosphorylation activity in vitro using an ELISA. Materials and reagents 1.15 cm tissue culture dish 2. Flk-1 / NIH cells: human flk-1 clone 3 (SUGEN, Inc.
. 2. An NIH fibroblast cell line that overexpresses MPI, obtained from MPI, Martinsried, Germany. 3. Growth medium: DMEM plus heat inactivated 10% FBS and 2 mM glutamine (Gibco-BRL) Starvation medium: DMEM plus 0.5% heat-inactivated FBS, 2 mM glutamine (
Gibco-BRL) 5. Corning 96-well ELISA plate (Corning Cat.
No. 25805-96) 6. L4 or E38 monoclonal antibody specific for flk-1; purified by protein-A agarose affinity chromatography (SUGEN, Inc.
) 7. PBS (Dulbecco's phosphate buffered saline) Gibco Cat. No. 45
0-1300EB) 8. 8. HNTG (see biochemical FGFR for preparation) Pierce BCA protein assay kit Blocking buffer 11. TBST (pH 7.0) 12. Kinase buffer 13. Kinase stop solution: 200 mM EDTA Biotinylated 4G10, specific for phosphotyrosine (UBI, Cat.
. No. 16-103) 15. AB kit (Vector Laboratories Cat. No.
PK4000) 16. DMSO 17. NUNC 96-well V-bottom polypropylene plate (Applied Sc
entitic Cat. No. (AS-72092) 18. Turbo-TMB (Pierce) 19. Turbo-TMB stop solution: 1 MH ₂ SO ₄ 20. ATP (Sigma Cat. No. A-7699) 21.20% in DMSO, TBS (pH 7.0)

Methods Cell Growth and Lysate Preparation Cells are seeded in growth medium and grown for 2-3 days at 37 ° C., 5% CO ₂ to 90-100% confluence. Do not exceed 20 passages. 2. Remove the medium and wash the cells twice with PBS. Dissolve in HNTG lysis buffer. Collect all lysates and vortex for 20-30 seconds. 3. Remove insoluble material by centrifugation (5-10 minutes, about 10,000 xg). 4. Determine protein concentration using the BCA kit. 5. The lysate is distributed in 1 mg aliquots and stored at -80C. Assay method Coat Corning 96-well ELISA plates with 2 μg / well purified L4 (or E38) in 100 μl of PBS. Store overnight at 4 ° C. 2. Unbound protein is removed from the wells by inverting the plate and removing the liquid. Wash once with dH ₂ O and tap the plate on a paper towel to remove excess liquid. 3. Block plate with 150 μl blocking buffer per well. Incubate at 4 ° C with shaking for 45-60 minutes. 4. The blocking buffer was removed and the ELISA plate was washed three times with dH ₂ O, TB
Wash once with ST. Tap the plate on a paper towel to remove excess liquid. 5. The lysate is diluted in PBS to a final concentration of 50 μg / 100 μl. 1
Add 00 μl of the diluted lysate to each well. Incubate overnight at 4 ° C. with shaking. 6. Unbound protein is removed from the wells by inverting the plate. Wash as in step 4. 7. Add 80 μl of kinase buffer to wells (90 μl for negative control wells)
). 8. Test compounds are diluted (usually 10-fold) in the wells of a polypropylene plate containing 20% DMSO in TBS. 9. Add 10 μl of diluted compound to ELISA wells containing immobilized flk-1
Shake. No compound is added to control wells. From the 10.1 mM ATP stock solution, prepare a 0.3 mM ATP solution in dH ₂ O (
Alternatively, a kinase buffer may be used). 11. Add 10 μl of 0.3 mM ATP to all wells except the negative control.
Incubate for 60 minutes at room temperature with shaking. After 12.1 hours, the kinase reaction is stopped by adding 11 μl of 200 mM EDTA. Shake for 1-2 minutes. 13. Wash the ELISA plate 4 times with dH ₂ O and 2 times with TBST. 14. Add 100 μl of 1: 5000 biotinylated 4G10: TBST to all wells. Incubate for 45 minutes at room temperature with shaking. 15. While incubating the above, add 50 μl of solutions A and B from the ABC kit to 10 ml of TBST. These solutions require approximately 30
Minutes before mixing. 16. Wash the plate as in step 4. 17． Add 100 μl of the preformed complex of A and B to all wells. Incubate for 30 minutes at room temperature with shaking. 18. Wash the plate as in step 4. 19. Add 100 μl Turbo-TMB. Shake at room temperature for 10-15 minutes. 20. When the color of the positive control well reaches an absorbance of about 0.35-0.4,
Stop the reaction with 1 Turbo-TMB stop solution. 21. Read the plate at Dynatech MR ₇₀ 00 ELISA reader.
Test filter: 450 nM, reference filter: 410 nM.

In Vivo Animal Model Example 22: Xenograft Animal Model The ability of human tumors to grow as xenografts in athymic mice (eg, Balb / c, nu / nu) is important for the treatment of human tumors. Provides an in vivo model useful for studying the response. First successful xenograft of human tumors into athymic mice (Rygaard and Povlsen, 1969, Acta P
atol. Microbial. Scand. 77: 758-760)
Many different human tumor cell lines (eg, breast, lung, urogenital, gastrointestinal, head and neck, glioblastoma, bone, and malignant melanoma) have been transplanted and successfully grown in nude mice. The following assays can be used to determine the level of activity, specificity and effect of various compounds of the invention. Three general assays are useful for evaluating compounds, namely, cell / catalyst, cell / biological and in vivo assays. The purpose of the cell / catalytic assay is to determine the effect of a compound on the ability of TK to phosphorylate tyrosine on a known substrate in cells. The purpose of a cell / biological assay is to determine the effect of a compound on the biological response stimulated by TK in a cell. The purpose of an in vivo assay is to determine the effect of a compound in an animal model on a particular disease, eg, cancer. Cell lines suitable for subcutaneous xenograft experiments include C6 cells (gliomas, ATCC
# CCL107), A375 cells (melanoma, ATCC # CRL1619), A4
31 cells (epidermoid carcinoma, ATCC # CRL1555), Calu6 cells (lung, A
TCC # HTB56), PC3 cells (prostate, ATCC # CRL1435), and SKOV3TP5 cells and NIH3T3 engineered to overexpress EGFR, PDGFR, IGF-1R or any other test kinase.
Fibroblasts. Xenograft experiments can be performed using the following protocol. Female athymic mice (BALB / c, nu / nu) were purchased from Simonsen Labo
ratories (Gilloy, CA). All animals are maintained in alpha-dry bedding in microisolator cages under clean room conditions. Animals are fed a sterile rodent diet and water ad libitum. The cell line is grown in a suitable medium (eg, 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 Gibco Life Techno unless otherwise noted.
logs (Grand Island, NY). All cells are grown at 37 ° C. in a humidified environment of 90-95% air and 5-10% CO ₂ . All cell lines were routinely passaged twice a week, using the Myprotect method (Gi
bco), negative for mycoplasma. Cells are grown at or near confluence with 0.05% trypsin-
Collect with EDTA and pellet at 450 × g for 10 minutes. Pellets sterilized PB
Cells are implanted into the hind flank of S or medium (without FBS) to a specific concentration and mice (8-10 mice per group, 2-10 × 10 ⁶ cells / animal). Tumor growth is measured over 3-6 weeks using veneer calipers. Tumor volume is calculated as the product of length x width x height unless otherwise stated. The P value is calculated using the Student's t-test. 50-100 μl of excipient (
Test compounds in DMSO, or VPD: D5W) can be delivered by IP infusion at different concentrations, usually beginning on the first day after transplantation.

Example 23 Tumor Invasion Model The following tumor invasion model has been developed and is used to evaluate the therapeutic value and efficacy of compounds identified as selectively inhibiting the KDR / FLK-1 receptor be able to. Method An 8-week-old nude mouse (female) (Simonsen Inc.) is used as an experimental animal. Implantation of tumor cells can be performed in a laminar flow hood. For anesthesia, a xylazine / ketamine cocktail (100 mg / kg ketamine and 5 mg / kg xylazine) is administered intraperitoneally. Make a midline incision to expose the abdominal cavity (about 1.5 c
The length of m), injecting 10 ⁷ tumor cells in the medium volume 100 [mu] l. Cells are injected into the duodenal lobe of the pancreas or subserosal in the colon. The peritoneum and muscles are sutured with 6-0 silk continuous suture, and the skin is sutured with wound clips. Animals are observed daily. After 2-6 weeks according to the overall findings of the analyzed animals, the mice are sacrificed and metastases of the local tumor to various organs (lung, liver, brain, stomach, spleen, heart, muscle) are examined and analyzed (tumor size, Degree of invasion, immunochemistry, and in situ hybridization measurements)
.

Example 24: Determination of cytotoxicity A therapeutic compound should be more potent at inhibiting protein kinase activity than exerting a cytotoxic effect. A measure of the efficacy and cytotoxicity of a compound can be obtained by determining the therapeutic index, ie, IC ₅₀ / LD ₅₀ . The dose IC ₅₀ required to achieve 50% inhibition can be measured using standard techniques, for example, those described herein. Dose L giving 50% toxicity
D ₅₀ also using standard techniques (Mossman, 1983, J.Immu
nol. Methods, 65: 55-63), by measuring the amount of LDH released (Korzeniewski and Callewaert, 1).
983, J.A. Immunol. Methods, 64: 313; Decker
and Lohmann-Matthes, 1988, J. Am. Immunol. M
methods, 115: 61), or by measuring the lethal dose in animal models. Compounds with a high therapeutic index are preferred. The therapeutic index must be greater than 2, preferably at least 10, more preferably at least 50.

Example 25: Activity of compounds of the invention The biological or biochemical activity of some compounds of the invention was tested using the assays described above. It was measured IC ₅₀ values for some of the compounds of the present invention. The results are shown in the table below.

Table 4

[Table 15]

Table 5

[Table 16]

[Table 17]

Table 6

[Table 18]

[Table 19]

Conclusion Those skilled in the art will readily appreciate that the present invention is well adapted to carry out its objects, obtain the described results and advantages, and those specific to this specification. Would. The molecular complexes and methods, procedures, treatments, molecules, and particular compounds described herein are representative of presently preferred embodiments, are exemplary, and are intended to be within the scope of the invention. It is not intended to be limiting. Those skilled in the art will make modifications and other uses which are encompassed within the spirit of the invention as defined in the claims.

One skilled in the art will readily appreciate that substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. Will.

All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. All patents and publications are to the same extent as if each individual publication was specifically and individually cited herein as part of this specification.
Referenced as part of this specification.

The invention illustratively described herein can be suitably practiced without any element or limitation not specifically disclosed herein. That is, for example, in each of the examples in this specification, the terms “including”, “consisting essentially of” and “consisting of” are replaced with either of the other two. be able to. The terms and expressions used herein are used as terms of description and are not limiting. The use of such terms and phrases is not intended to exclude the equivalents of the features shown or described, or some of them, but is intended to be within the scope of the present invention, which is set forth in the following claims. It is understood that various modifications are possible. That is, although the present invention has been specifically disclosed by the preferred embodiments and optional features, those skilled in the art can make modifications and variations to the concept described in the present specification, and such modifications and variations are also claimed. It is to be understood that this is considered to be within the scope of the present invention as defined in

In addition, where features and aspects of the invention are described in Markush group terms, those skilled in the art will appreciate that the present invention is also described with respect to individual members or subgroups of members of the Markush group. Will recognize.
For example, if X is described as being selected from the group consisting of bromine, chlorine and iodine, the claims where X is bromine and the claims where X is bromine and chlorine are also fully described. ing.

The present invention has been described broadly and generically herein. Each of the narrower species and subgeneric groups included in the general disclosure also form part of the invention. This includes whether or not the exclusion is specifically stated,
Includes a general description of the invention including "however ..." or any negative limitation excluding any subject matter from the genus.

Other embodiments are within the scope of the following claims.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 17/06 A61P 17/06 19/02 19/02 29/00 29/00 101 101 35/00 35 / 00 37/00 37/00 43/00 105 43/00 105 111 111 111 C07D 401/06 C07D 401/06 401/10 401/10 401/12 401/12 401/14 401/14 403/06 403/06 403 / 10 403/10 409/10 409/10 409/14 409/14 C12N 9/99 C12N 9/99 // A61K 31/404 A61K 31/404 31/4439 31/4439 31/5377 31/5377 (31) Priority claim number 60/131, 192 (32) Priority date April 26, 1999 (1999. 4.26) (33) Priority claim country United States (US) (31) Priority claim number 60/132, 243 (32) Priority date May 3, 1999 (5.3.1999) (33) Priority country United States (US) (81) Designated country EP (AT, B) , CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA , GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ , LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, M , NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Tan, Pen Chou United States 94556 Camino Ricardo, Moraga, California 827 (72) Inventor Sun, Li United States 94404 Foster City, California, Rock Harber Lane 64 (72) Inventor McMahon, Gerald United States 95452 Kenwood, California, Schultz Road 1800 (72) Inventor Miller, Todd Anthony United States 94587 California, Union City, Corsa Street 4928 (72) Inventor Sileezian, Sharazad United States 94925 Corte Madera, Conn, California STATIONATION Drive 1 (72) Inventor Way, Chan Chen United States 94404 Commons Lane, Foster City, CA 39 (72) Inventor Harris Gee.・ Divis United States 94114 San Francisco, California, Roosevelt Way 417 (72) Inventor Xiao Yuan, Li United States 95130 San Jose, California, Burwood Drive 2437 (72) Inventor Liang Congin United States 94087 Sunnyvale, California W / Remington Drive 729 F-term (reference) 4C063 AA01 AA03 BB03 BB06 BB08 CC06 CC12 CC92 DD04 DD06 EE01 4C086 AA01 AA02 AA03 BB02 BC13 BC17 BC73 GA04 GA07 GA08 GA09 NA14 ZA36 ZA39 ZA45 ZBZ ZB Z ZB ZB Z 4C204 BB01 CB03 DB15 DB30 EB03 FB01 GB32

Claims (39)

[Claims] 1. Formula I: ## STR1 ## Wherein n and m are independently 0 or 1; when n is 1, A, B, D, E and F are independently selected from the group consisting of carbon and nitrogen. Provided that when three or less of A, B, D, E and F are nitrogen simultaneously and A, B, D, E or F is nitrogen, R ₄ , R ₅ , R ₆ , R ₇ or when it is m is 1, G, H, J, K and L are independently selected from the group consisting of carbon and nitrogen;; R ₈ is absent, respectively, however, G, H, J, K and When at least one and up to three of L are simultaneously nitrogen and G, H, J, K or L is nitrogen, R ₉
, R ₁₀ , R ₁₁ , R ₁₂ , or R ₁₃ are each absent; when n is 0, A is selected from the group consisting of carbon and nitrogen;
Is selected from the group consisting of carbon, nitrogen, NH, oxygen and sulfur, provided that B
Or when F is NH, the other cannot be NH, and E is selected from the group consisting of carbon, nitrogen, oxygen and sulfur, no more than one of B, E or F is oxygen or sulfur, and , A, B, E or F is a heteroatom (ie not carbon); when m is 0, G is selected from the group consisting of carbon and nitrogen, and H, K and L are , Carbon, nitrogen, NH, oxygen and sulfur, provided that when H or L is NH, the other cannot be NH, and K is carbon,
Selected from the group consisting of nitrogen, oxygen and sulfur, provided that one of H, K or L
Not more than oxygen or sulfur and at least one of G, H, K or L is a heteroatom (ie, not carbon); R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₀ , R ₁₁ , R ₁₂ and R ₁₃
Is independently hydrogen, alkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, mercapto, alkylthio, aryloxy, arylthio, sulfinyl, sulfonyl, S-sulfone Amide, N-sulfonamide, carbonyl, C
-Carboxy, O-carboxy, carboxyalkyl, cyano, nitro, halo,
O- carbamyl, N- carbamyl, C-amido, selected from the group consisting of N- amides and -NR ₁₄ R _15; R ₄ and R ₅ or R ₅ and R ₆ or R ₆ and R ₇ or R ₇ and R ₈ together may form a 5- or 6-membered aryl or heteroaryl ring; and R ₁₄ and R ₁₅ are independently hydrogen, alkyl, cycloalkyl, alkenyl,
Selected from the group consisting of alkynyl, aryl, heteroaryl, carbonyl, and sulfonyl, or may be taken together to form a 5-membered or 6-membered heteroalicyclic ring. Or a pharmaceutically acceptable salt thereof. 2. R ₁ , R ₂ and R ₃ are independently hydrogen; lower alkyl optionally substituted with one or more halo groups; optionally with one or more halo groups. optionally substituted lower alkoxy; halo; (lower alkyl) -S- sulfonamide; (aryl) -S- sulfonamide; and -NR ₁₄ R _15; is selected from the group consisting of, R _4, R _5, R ₆ , R ₇ , R ₈ , R ₉ , R ₁₀ , R ₁₁ , R ₁₂ and R ₁₃ are independently hydrogen; aryl, heteroaryl, heteroalicyclic, halo, hydroxy, lower alkoxy, mercapto, lower alkylthio, C-carboxy and -NR ₁₄ one or more optional substituted lower alkyl groups selected from the group consisting of R _15; cycloalkyl, hydroxy, 1 or more than A lower alkoxy, halo optionally substituted with one or more groups selected from the group consisting of halo, aryl, heteroaryl and heteroalicyclic; cyano, nitro, carboxyalkyl, 1 or Lower alkyl optionally substituted with a halo group above; halo; hydroxy; lower alkoxy optionally substituted with one or more halo groups; aryloxy; and -NR ₁₀ R ₁₁ ; Aryl optionally substituted with one or more groups selected from the group consisting of: lower alkyl optionally substituted with one or more halo groups; halo; hydroxy; one or more Lower alkoxy optionally substituted with a halo group; and -NR ₁₄ R ₁₅ ; Or aryloxy optionally substituted with one or more halo groups; lower alkyl optionally substituted with one or more halo groups; halo; hydroxy; Optionally substituted lower alkoxy; and heteroaryl optionally substituted with one or more groups selected from the group consisting of -NR ₁₄ R ₁₅ , one or more halo groups A lower alkyl optionally substituted with: halo; hydroxy; a lower alkoxy optionally substituted with one or more halo groups; and -NR ₁₄ R ₁₅ ; Or a heteroalicyclic optionally substituted with a higher group, (lower alkyl) C-carboxy, (lower alkyl) carbonyl, arylcarbo Nyl, (lower alkyl) -S-sulfonamide, aryl-S-sulfonamide, (lower alkyl) -N-sulfonamide, aryl-N-sulfonamide, (lower alkyl) -N-carbamoyl, (lower alkyl)- R ₁₄ and R ₁₅ are independently hydrogen and lower, selected from the group consisting of C-amide, (lower alkyl) -N-amide, (cycloalkyl) -N-amide, and —NR ₁₄ R ₁₅ ; The compound or salt according to claim 1, wherein the compound or salt is selected from the group consisting of alkyl. A, B, D, E and F are carbon; m is 1; and one of G, H, J, K and L is nitrogen. 2. The compound or salt according to 1. A, B, D, E and F are carbon; m is 0; G, H and K are carbon; and L is NH. 2. The compound or salt according to 1. A, B and E are carbon; F is NH; R ₄ and R ₇ are independently selected from the group consisting of hydrogen and lower alkyl; ₅ is hydroxy; heteroalicyclic containing at least one NH group; C-carboxy; and —NR ₁₄ R ₁₅ ; which may be optionally substituted with one or more groups selected from the group consisting of: It is selected from the group consisting of and -NR ₁₄ R _15;; lower alkyl; carboxyalkyl; (lower alkyl) -C- carboxy m is 1; G, H, J, one of K and L is nitrogen And R ₁₄ and R ₁₅ are independently selected from the group consisting of hydrogen, lower alkyl and carbonyl. 6. A, B and E are carbon; F is NH; R ₄ and R ₇ are independently selected from the group consisting of hydrogen and lower alkyl; ₅ is hydroxy; heteroalicyclic containing at least one NH group; C-carboxy; and —NR ₁₄ R ₁₅ ; which may be optionally substituted with one or more groups selected from the group consisting of: (Lower alkyl) -C-carboxy; and -NR ₁₄ R ₁₅ ; m is 0; G, H and K are carbon; L is NH Or a compound or salt of claim 1, wherein R ₁₄ and R ₁₅ are independently selected from the group consisting of hydrogen, lower alkyl and carbonyl. 7. A compound of formula II:Wherein n is 0 or 1; when p is 1, M, Q, T, Y, and V are independently carbon and nitrogen
Selected from the group consisting of: when M, Q, T, Y, or V is nitrogen, R₂₀, R _{twenty one} , R_{twenty two}, R_{twenty three}Or R_{twenty four}When p is 0, M, Q, Y, and V are independently carbon, nitrogen, oxygen and
Selected from the group consisting of sulfur and sulfur, where M, Q, Y, or V is oxygen or sulfur.
Or nitrogen (where said nitrogen is involved in a double bond)₂₀, R_{twenty one} , R_{twenty two}, R_{twenty three}Or R_{twenty four}Are each absent; R₁₆, R₁₇, R₁₈, R₁₉, R₂₀, R_{twenty one}, R_{twenty two}, R_{twenty three}Or R_{twenty four}Is, independently, water
Element, alkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl,
Aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, mercap
G, alkylthio, aryloxy, sulfinyl, sulfonyl, S-sulfone
Amide, N-sulfonamide, carbonyl, C-carboxy, O-carboxy,
Carboxyalkyl, cyano, nitro, halo, O-carbamyl, N-carbamyl
, C-amide, N-amide and -NR_{twenty five}R₂₆R is selected from the group consisting of: R₂₀And R_{twenty one}Or R_{twenty one}And R_{twenty two}Or R_{twenty two}And R_{twenty three}Or R_{twenty three}And R _{twenty four} Together form a 5- or 6-membered aryl or heteroaryl ring
And R_{twenty five}And R₂₆Is independently hydrogen, alkyl, cycloalkyl, alkenyl,
A group consisting of alkynyl, aryl, heteroaryl, carbonyl, and sulfonyl
Selected or taken together to form a 5- or 6-membered heteroalicyclic ring
3-aralkyl-2-indolinone having the chemical structure according to
Physically acceptable salts. 8. _{R 16, R 17, R 18} , R 19, R 20, R 22, R 23 or R ₂₄ is
Independently, hydrogen; cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, optionally substituted with one or more groups selected from the group consisting of C- carboxy and -NR ₂₅ R ₂₆ Cycloalkyl; hydroxy; optionally substituted with one or more groups selected from the group consisting of one or more halo groups, aryl, heteroaryl, and heteroalicyclic. Good lower alkoxy, trihalomethyl, trihalomethoxy, halo, carboxyalkyl, lower alkyl, lower alkyl substituted with one or more halo groups, trihalomethyl, trihalomethoxy, halo, hydroxy, lower alkoxy, aryloxy and -NR ₂₅ 1 or is selected from the group consisting of R ₂₆ Optionally optionally substituted aryl in the above groups, aryloxy, lower alkyl, trihalomethyl, halo, hydroxy, (lower alkyl) alkoxy, one or selected from the group consisting of amino and -NR ₂₅ R ₂₆ Heteroaryl, heteroalicyclic, (lower alkyl) carboxy, (lower alkyl) carbonyl, arylcarbonyl, (lower alkyl) -S-sulfonamide, aryl-S-sulfone which may be optionally substituted with the above groups Amide, (lower alkyl) -N-sulfonamide, aryl-N-sulfonamide, (lower alkyl) -N-carbamoyl, (lower alkyl) -C-amide, (lower alkyl) -N-amide, (cycloalkyl) -N- amides, and it is selected from the group consisting of -NR ₂₅ R _26, claim The compounds described. 9. The compound of claim 7, wherein p is 1; and M, Q, T, U, and V are carbon. 10. R ₁₆ , R ₁₇ , R ₁₈ and R ₁₉ are independently selected from the group consisting of hydrogen, halo, hydroxy, —NR ₂₅ R ₂₆ , hydrogen, lower alkyl and aryl. more optionally may be substituted S- sulfonamide in groups, hydroxy, 1 or more halo groups, C-carboxy, C-amido, selected from the group consisting of heteroalicyclic and -NR ₂₅ R ₂₆ Lower alkyl optionally substituted with one or more halo groups; lower alkyl, lower alkoxy, halo, hydroxy and -NR ₂₅ R ₂₆ Aryl optionally substituted with one or more selected groups; and hydrogen, lower alkyl, cycloalkyl and aryl It is selected from the group consisting of; one or more optionally substituted and each may be a N- amide group is-option _{and, R 20, R 21, R} 22, R 23 and R ₂₄ are independently hydrogen; halo; hydroxy; -NR ₂₅ R _26; hydroxy, 1 or more halo groups, C-carboxy, C-amido, 1 or is selected from the group consisting of heteroalicyclic and -NR ₂₅ R ₂₆ Cycloalkyl; one or more halo groups, aryl, -NR ₂₅ R ₂₆ , and heteroaryl optionally substituted with one or more groups; any substituted a lower alkoxy group; a halo, hydroxy, lower alkoxy, 1 or selected from the group consisting of -NR ₂₅ R ₂₆ and C- carboxy Optionally substituted on the group aryl; is selected from the group consisting of, compound of claim 9. 11. The compound according to claim 7, wherein p is 1; and one or two of M, Q, T, U, or V is nitrogen. 12. R _16, R _17, R ₁₈ and R ₁₉ are independently hydrogen; halo; hydroxy; -NR ₂₅ R _26; hydrogen, one or selected from the group consisting of lower alkyl and aryl S-sulfonamide optionally substituted with the above groups; from the group consisting of hydroxy, one or more halo groups, C-carboxy, C-amide, heteroalicyclic, and -NR ₂₅ R ₂₆ Lower alkyl, optionally substituted with one or more selected halo groups; lower alkoxy, lower alkyl, lower alkoxy, halo, hydroxy, and -NR ₂₅ R ₂₆ , optionally substituted with one or more halo groups. Consisting of aryl, hydrogen, lower alkyl, cycloalkyl and aryl optionally substituted with one or more groups selected from the group consisting of Is selected from the group consisting of; more one or more optionally optionally substituted N- amido groups selected and not the R _20, R _21, the nitrogen of the R _2, R ₂₃ and R ₂₄ ones are independently hydrogen; halo; hydroxy; -NR ₂₅ R _26; hydroxy, 1 or more halo groups, C-carboxy, C-amido, group consisting heteroalicyclic and -NR ₂₅ R ₂₆ Lower alkyl optionally substituted with one or more groups selected from cycloalkyl; one or more halo groups, aryl, —NR ₂₅ R _26, and heteroaryl 1 or more optional substituted to a lower alkoxy group; a halo, hydroxy, lower alkoxy, selected from the group consisting of -NR ₂₅ R ₂₆ and C- carboxy 1 or substituted optionally with more group aryl is; is selected from the group consisting of, compound of claim 11. 13. The compound of claim 7, wherein p is 1; and R ₂₁ and R ₂₂ together form a fused pyrrolo group. 14. R _16, R _17, R ₁₈ and R ₁₉ are independently hydrogen; halo; hydroxy; -NR ₂₅ R _26; hydrogen, one or selected from the group consisting of lower alkyl and aryl S-sulfonamide optionally substituted with the above groups; selected from the group consisting of hydroxy, one or more halo groups, C-carboxy, C-amide, heteroalicyclic, and -NR ₂₅ R ₂₆ Lower alkyl optionally substituted with one or more halo groups; lower alkyl, lower alkoxy, halo, hydroxy and -NR ₂₅ R ₂₆ Aryl optionally substituted with one or more groups selected from the group consisting of: hydrogen, lower alkyl, cycloalkyl and aryl Is selected from the group consisting of; Ri optionally substituted with one or more groups selected may also N- amides and, R _20, R ₂₃ and R ₂₄ are independently hydrogen; halo; hydroxy; -NR ₂₅ R _26; hydroxy, 1 or more halo groups, C-carboxy, C-amido, heteroalicyclic and -NR ₂₅ R ₂₆ one or more groups, selected from the group consisting of Cycloalkyl; optionally substituted with one or more groups selected from the group consisting of one or more halo groups, aryl, -NR ₂₅ R ₂₆ and heteroaryl One or more groups selected from the group consisting of halo, hydroxy, lower alkoxy, —NR ₂₅ R ₂₆ and C-carboxy. 14. The compound of claim 13, wherein the compound is selected from the group consisting of an optionally substituted aryl; 15. p is 0; M or Q is -NH, oxygen or sulfur; and either M or Q is not -NH, oxygen or sulfur, and U and V
The compound of claim 7, wherein is carbon. 16. R ₁₆ , R ₁₇ , R ₁₈ and 19 are, independently, hydrogen; halo; hydroxy; —NR ₂₅ R ₂₆ ; one or more selected from the group consisting of hydrogen, lower alkyl and aryl. of may be optionally substituted with a group S- sulfonamide; hydroxy, 1 or more halo groups, C-carboxy, C-amido, selected from the group consisting of heteroalicyclic and -NR ₂₅ R ₂₆ consisting of lower alkyl, lower alkoxy, halo, hydroxy and -NR ₂₅ R _26; also substituted lower alkoxy optionally substituted with one or more halo groups; also lower alkyl substituted optionally with that group Aryl optionally substituted with one or more groups selected from the group: selected from hydrogen, lower alkyl, cycloalkyl and aryl It is selected from the group consisting of; optionally substituted may also be N- amide with one or more groups that and R _20, R _21, R ₂₃ and R ₂₄ are independently hydrogen; halo; hydroxy -NR ₂₅ R ₂₆ ; optionally one or more groups selected from the group consisting of hydroxy, one or more halo groups, C-carboxy, C-amide, heteroalicyclic and -NR ₂₅ R _26; A cycloalkyl; optionally substituted with one or more groups selected from the group consisting of one or more halo groups, aryl, -NR ₂₅ R ₂₆ and heteroaryl. 16. The compound according to claim 15, wherein the compound is selected from the group consisting of: 17. R _16, R _17, R ₁₈ and R ₁₉ are independently hydrogen; halo; hydroxy; -NR ₂₅ R _26; hydrogen, one or selected from the group consisting of lower alkyl and aryl S-sulfonamide optionally substituted with the above groups; selected from the group consisting of hydroxy, one or more halo groups, C-carboxy, C-amide, heteroalicyclic, and -NR ₂₅ R ₂₆ Lower alkyl, optionally substituted with one or more halo groups; lower alkyl, optionally substituted with one or more halo groups; lower alkyl, lower alkoxy, halo, hydroxy and —NR ₂₅ R ₂₆ Aryl optionally substituted with one or more groups selected from the group consisting of: hydrogen, lower alkyl, cycloalkyl and aryl It is selected from the group consisting of; that 1 or substituted optionally with more groups may also N- amides and, R _20, R ₂₃ and R ₂₄ are independently hydrogen; halo; hydroxy; - NR ₂₅ R ₂₆ ; optionally substituted with one or more groups selected from the group consisting of hydroxy, one or more halo groups, C-carboxy, C-amide, heteroalicyclic and —NR ₂₅ R ₂₆ A cycloalkyl; optionally substituted with one or more groups selected from the group consisting of one or more halo groups, aryl, -NR ₂₅ R ₂₆ and heteroaryl. optionally is substituted with halo, hydroxy, lower alkoxy, one or more groups selected from the group consisting of -NR ₂₅ R ₂₆ and C- carboxy; optionally substituted lower alkoxy Which may be aryl; it is selected from the group consisting of, 7. A compound according. 18. A compound of formula III:[Wherein (a) R₂₇Are (i) halogen, trihalomethyl, alkoxy, carboxylate, amino,
Toro, esters and 5- or 6-membered aromatic, heteroaromatic, aliphatic,
Or optionally substituted with a substituent selected from the group consisting of heteroaliphatic ring components.
A saturated or unsaturated alkyl, wherein the ring component is alkyl, halogen, trihalomethyl, carboxylate
1, independently selected from the group consisting of amino, nitro, and ester components
, 2, or 3 optionally substituted; and (ii) alkyl, alkoxy, halogen, trihalomethyl, carboxylate
1, independently selected from the group consisting of, amino, nitro, and ester components
Aromatic or heteroaromatic optionally substituted with two or three substituents
(Iii) alkyl, alkoxy, halogen, trihalomethyl, carboxylate
One or more independently selected from the group consisting of
An aliphatic or heteroaliphatic ring optionally substituted with the above substituents, and
And alkyl, alkoxy, halogen, trihalomethyl, carboxylate, amine
One or more independently selected from the group consisting of
An aromatic or heteroaromatic ring optionally substituted with more than one substituent; selected from the group consisting of:₃₂, R₃₃And R₃₄Are each independently: (i) hydrogen; (ii) halogen, trihalomethyl, carboxylate, amino, nitro, es
Ter and 5- or 6-membered aromatic, heteroaromatic, aliphatic, or heterocyclic
Optionally substituted with one or more substituents selected from the group consisting of aliphatic ring components
An optionally substituted saturated or unsaturated alkyl, wherein the ring component is alkyl, halogen, trihalomethyl, carboxylate
1, independently selected from the group consisting of amino, nitro, and ester components
Or optionally substituted with more substituents; (iii) alkyl, alkoxy, halogen, trihalomethyl, carboxylate
1, independently selected from the group consisting of amino, nitro, and ester components
Or an aromatic or heteroaromatic optionally substituted with more substituents
(Iv) alkyl, alkoxy, halogen, trihalomethyl, carboxylate
One or more independently selected from the group consisting of, amino, nitro, and ester
An aliphatic or heteroaliphatic ring optionally substituted with the above substituents, and
Alkyl, alkoxy, halogen, trihalomethyl, carboxylate, amino
One or more independently selected from the group consisting of, nitro, and ester components
An aromatic or heteroaromatic ring optionally substituted with the above substituents; (v) a formula-(X₁)_n1-NX_TwoX_ThreeAmine of the formula (wherein X₁Is a saturated or unsaturated alkyl, and a 5- or 6-membered ring.
Selected from the group consisting of aromatic, heteroaromatic, or aliphatic ring components;₁Is 0 or 1 and X_TwoAnd X_ThreeIs independently hydrogen, saturated or unsaturated alkyl, and a five-membered ring
Or selected from the group consisting of aromatic, heteroaromatic, or aliphatic ring components having 6-membered rings
(Vi) Formula -NO_Two(Vii) halogen or trihalomethyl; (viii) a formula-(X_Four)_n4-CO-X_FiveA ketone (wherein X_FourAnd X_FiveIs independently alkyl and 5- or 6-membered aromatic
Selected from the group consisting of aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring components
The alkyl and the ring component are alkyl, halogen, trihalomethyl, carboxy,
Independently selected from the group consisting of silate, amino, nitro, and ester components
N is optionally substituted with 1, 2, or 3 substituents;_FourIs 0 or 1); (ix) Formula-(X₆)_n6A carboxylic acid of -COOH or a formula-(X₇)_n7-COO-
X₈An ester of the formula, wherein X₆, X₇And X₈Is independently alkyl and 5- or 6-membered ring
Selected from the group consisting of aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring components
And n₆And n₇Is independently 0 or 1); (x) Formula-(X₉)_n9-OH alcohol or formula-(X_Ten)_n10-OX₁₁No
Alkoxyalkyl component (wherein, X₉, X_TenAnd X₁₁Is independently saturated or unsaturated alkyl,
And 5- or 6-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic
Selected from the group consisting of ring components, wherein said alkyl and ring components are alkyl, halogen, trihalomethyl,
Independently selected from the group consisting of sylate, amino, nitro, and ester
Optionally substituted with one or more substituents, and n₉And n_TenIs independently 0 or 1); (xi) Formula-(X₁₂)_n12-NHCOX₁₃Or the formula-(X₁₄)_n14-CONX_FifteenX ₁₆ An amide of the formula₁₂And X₁₄Are each independently alkyl, hydroxyl, and
And 5- or 6-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring
Wherein the ring is alkyl, halogen, trihalomethyl, carboxylate, amino,
One or more independently selected from the group consisting of nitro, and esters
Optionally substituted with substituents, and n₁₂And n₁₄Is independently 0 or 1; X₁₃, X_Fifteen, And X₁₆Is independently hydrogen, alkyl, hydroxyl
, And 5- or 6-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic
Selected from the group consisting of aliphatic ring components, wherein said ring is alkyl, halogen, trihalomethyl, carboxylate, amino,
One or more independently selected from the group consisting of nitro, and esters
(Optionally substituted with a substituent); (xii) a formula-(X₁₇)_n17-SO_TwoNX₁₈X₁₉A sulfonamide of the formula₁₇Is alkyl, and a 5- or 6-membered aromatic or heteroaromatic,
Selected from the group consisting of aliphatic or heteroaliphatic ring components, wherein said alkyl and ring components are alkyl, halogen, trihalomethyl, carboxy
Independently selected from the group consisting of sylate, amino, nitro, and ester
Optionally substituted with one or more substituents, n₁₇Is 0, 1 or 2; X₁₈And X₁₉Is independently hydrogen, alkyl, and a 5- or 6-membered ring.
Selected from the group consisting of aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring components
Wherein said alkyl and ring components are alkyl, halogen, trihalomethyl, carboxy,
Independently selected from the group consisting of sylate, amino, nitro, and ester
Optionally substituted with one or more substituents, or X₁₈And X₁₉Are, together, alkyl, halogen, trihalomethyl,
Independently selected from the group consisting of boxylates, amino, nitro, and esters
5- or 6-membered ring optionally substituted with one or more substituents
(A cycloaliphatic or heteroaliphatic ring may be formed); (xiii) a formula-(X₂₀)_n20Aldehyde of —CO—H (wherein, X₂₀Is a saturated or unsaturated alkyl, and a 5- or 6-membered aromatic ring
Selected from the group consisting of aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring components
The alkyl and the ring component are alkyl, halogen, trihalomethyl, carboxy,
Independently selected from the group consisting of sylate, amino, nitro, and ester
Optionally substituted with one or more substituents, and n₂₀Is 0 or 1); (xiv) Formula-(X_{twenty one})_n21-SO_Two-X_{twenty two}Of the formula (wherein, X_{twenty one}And X_{twenty two}Is independently saturated or unsaturated alkyl, and 5
Aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring components having 6 or 6 membered rings
Wherein said alkyl and ring components are alkyl, halogen, trihalomethyl, carboxy
Independently selected from the group consisting of sylate, amino, nitro, and ester
Optionally substituted with one or more substituents, and n_{twenty one}Is 0 or 1); and (xv) a formula-(X_{twenty three})_n23A thiol of -SH or a formula-(X_{twenty four})_n24-SX_{twenty five}of
Thioether (wherein, X_{twenty three}, X_{twenty four}And X_{twenty five}Is independently saturated or unsaturated alkyl,
And 5- or 6-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic
Selected from the group consisting of ring components, wherein said alkyl and ring components are alkyl, halogen, trihalomethyl,
Independently selected from the group consisting of sylate, amino, nitro, and ester
Optionally substituted with one or more substituents, and n_{twenty three}And n_{twenty four}Is independently 0 or 1); selected from the group consisting of:₃₃And R₃₄Are, together, alkyl, halogen, trihalomethyl
Independent of the group consisting of carboxylate, amino, nitro, and ester
6-membered cycloaliphatic optionally substituted with one or more substituents selected
May form an aliphatic or aromatic ring; and (c) R₂₈, R₂₉, R₃₀, And R₃₁Are each independently: (i) hydrogen; (ii) hydroxy, halogen, trihalomethyl, carboxylate, amino,
Nitro, esters and 5- or 6-membered aromatic, heteroaromatic, aliphatic,
Or one or more substituents selected from the group consisting of heteroaliphatic ring components
A saturated or unsaturated alkyl optionally substituted with: wherein said ring component is alkyl, halogen, trihalomethyl, carboxylate
1, independently selected from the group consisting of amino, nitro, and ester components
Or optionally substituted with more substituents; (iii) alkyl, alkoxy, halogen, trihalomethyl, carboxylate
1, independently selected from the group consisting of amino, nitro, and ester components
Or an aromatic or heteroaromatic optionally substituted with more substituents
(Iv) alkyl, alkoxy, halogen, trihalomethyl, carboxylate
One or more independently selected from the group consisting of, amino, nitro, and ester
An aliphatic or heteroaliphatic ring optionally substituted with the above substituents, and
Alkyl, alkoxy, halogen, trihalomethyl, carboxylate, amino
One or more independently selected from the group consisting of, nitro, and ester components
An aromatic or heteroaromatic ring optionally substituted with the above substituents; (v) a formula-(X₁)_n1-NX_TwoX_ThreeAmine of the formula (wherein X₁Is a saturated or unsaturated alkyl, and a 5- or 6-membered ring.
Selected from the group consisting of aromatic, heteroaromatic, or aliphatic ring components;₁Is 0 or 1 and X_TwoAnd X_ThreeIs independently hydrogen, saturated or unsaturated alkyl, and a five-membered ring
Or selected from the group consisting of aromatic, heteroaromatic, or aliphatic ring components having 6-membered rings
(Vi) Formula -NO_Two(Vii) halogen or trihalomethyl; (viii) a formula-(X_Four)_n4-CO-X_FiveA ketone (wherein X_FourAnd X_FiveIs independently alkyl and 5- or 6-membered aromatic
Selected from the group consisting of aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring components
The alkyl and the ring component are alkyl, halogen, trihalomethyl, carboxy,
Independently selected from the group consisting of silate, amino, nitro, and ester components
N is optionally substituted with 1, 2, or 3 substituents;_FourIs 0 or 1); (ix) Formula-(X₆)_n6A carboxylic acid of -COOH or a formula-(X₇)_n7-COO-
X₈An ester of the formula, wherein X₆, X₇And X₈Is independently alkyl and 5- or 6-membered ring
Selected from the group consisting of aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring components
And n₆And n₇Is independently 0 or 1); (x) Formula-(X₉)_n9-OH alcohol or formula-(X_Ten)_n10-OX₁₁No
Alkoxyalkyl component (wherein, X₉, X_TenAnd X₁₁Is independently saturated or unsaturated alkyl,
And 5- or 6-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic
Selected from the group consisting of ring components, wherein said alkyl and ring components are alkyl, halogen, trihalomethyl,
Independently selected from the group consisting of sylate, amino, nitro, and ester
Optionally substituted with one or more substituents, and n₉And n_TenIs independently 0 or 1); (xi) Formula-(X₁₂)_n12-NHCOX₁₃Or the formula-(X₁₄)_n14-CONX_FifteenX ₁₆ An amide of the formula₁₂And X₁₄Are each independently alkyl, hydroxyl, and
And 5- or 6-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring
Wherein the alkyl and ring components are alkyl, halogen, trihalomethyl, carboxy,
Independently selected from the group consisting of sylate, amino, nitro, and ester
Optionally substituted with one or more substituents, n₁₂And n₁₄Is independently 0 or 1, and X₁₃, X_FifteenAnd X₁₆Is independently hydrogen, alkyl, hydroxyl,
And 5- or 6-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic
And the alkyl and ring components are selected from the group consisting of alkyl, halogen, trihalomethyl, carboxy
Independently selected from the group consisting of sylate, amino, nitro, and ester
(Optionally substituted with one or more substituents); (xii) a formula-(X₁₇)_n17-SO_TwoNX₁₈X₁₉A sulfonamide of the formula₁₇Is alkyl, 5- or 6-membered aromatic, heteroaromatic, or aliphatic
Selected from the group consisting of aromatic or heteroaliphatic ring components, wherein said alkyl and ring components are alkyl, halogen, trihalomethyl, carboxy,
Independently selected from the group consisting of sylate, amino, nitro, and ester
Optionally substituted with one or more substituents, and n₁₇Is 0, 1, or 2, and X₁₈And X₁₉Is independently hydrogen, alkyl, 5- or 6-membered aromatic,
Selected from the group consisting of heteroaromatic, aliphatic, or heteroaliphatic ring components, wherein said alkyl and ring components are alkyl, halogen, trihalomethyl, carboxy.
Independently selected from the group consisting of sylate, amino, nitro, and ester
Optionally substituted with one or more substituents, or X₁₈And X₁₉Are, together, alkyl, halogen, trihalomethyl,
Independently selected from the group consisting of boxylates, amino, nitro, and esters
5- or 6-membered ring optionally substituted with one or more substituents
(X to form a cycloaliphatic or heteroaliphatic ring); (xiii) a formula-(X₂₀)_n20Aldehyde of —CO—H (wherein, X₂₀Is a saturated or unsaturated alkyl, and a 5- or 6-membered aromatic ring
Selected from the group consisting of aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring components
The alkyl and the ring component are alkyl, halogen, trihalomethyl, carboxy,
Independently selected from the group consisting of sylate, amino, nitro, and ester
Optionally substituted with one or more substituents, and n₂₀Is 0 or 1); (xiv) Formula-(X_{twenty one})_n21-SO_Two-X_{twenty two}Of the formula (wherein, X_{twenty one}And X_{twenty two}Is a saturated or unsaturated alkyl, and a 5-membered ring or
Group consisting of six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring components
Wherein the alkyl and ring components are alkyl, halogen, trihalomethyl, carboxy,
Independently selected from the group consisting of sylate, amino, nitro, and ester
Optionally substituted with one or more substituents, and n_{twenty one}Is 0 or 1); and (xv) the formula-(X_{twenty three})_n23A thiol of -SH or a formula-(X_{twenty four})_n24-SX_{twenty five}of
Thioether (wherein, X_{twenty three}, X_{twenty four}And X_{twenty five}Is independently saturated or unsaturated alkyl,
And 5- or 6-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic
Selected from the group consisting of ring components, wherein said alkyl and ring components are alkyl, halogen, trihalomethyl,
Independently selected from the group consisting of sylate, amino, nitro, and ester
Optionally substituted with one or more substituents, and n_{twenty three}And n_{twenty four}Is independently selected from the group consisting of 0 and 1).] The indolinone compound having a structure described in the above. (A) R₆Is hydrogen; (b) R₇, R₈And R₉Are each independently: (i) hydrogen; (ii) a saturated alkyl optionally substituted with a 6-membered heteroaliphatic ring component.
(Iii) the formula-(X₁)_n1-NX_TwoX_ThreeAn amine of the formula (wherein X₁Is a saturated alkyl
, N₁Is 0 or 1 and X_TwoAnd X_ThreeIs independently hydrogen, and saturated
(Iv) Formula-(X₆)_n6A carboxylic acid of -COOH or a formula-(X₇)_n7-COO-
X₈An ester of the formula (wherein X₆, X₇And X₈Is alkyl, and n₆and
n₇Is independently 0 or 1); and (v) the formula-(X₁₄)_n14-CONX_FifteenX₁₆An amide of the formula₁₄Is alkyl
, N₁₂And n₁₄Is independently 0 or 1 and X_FifteenAnd X₁₆Is
, Each independently selected from the group consisting of hydrogen and alkyl); or selected from the group consisting of₈And R₉Is an optionally substituted 6-membered ring fat
Or may form an aromatic or aromatic ring; and (c) R_Three, R_Four, And R_FiveAre each independently: (i) hydrogen; (ii) saturated alkyl; (iii) halogen or trihalomethyl; (iv) a formula-(X₆)_n6A carboxylic acid of -COOH or a formula-(X₇)_n7-COO-
X₈An ester of the formula (wherein X₆, X₇And X₈Is alkyl, and n₆and
n₇Is independently 0 or 1); (v) Formula-(X₉)_n9-OH alcohol or formula-(X_Ten)_n10-OX₁₁No
Alkoxyalkyl component (wherein, X₉, X_TenAnd X₁₁Is alkyl, and
n₉And n_TenIs independently 0 or 1); (vi) the formula-(X₁₂)_n12-NHCOX₁₃Or the formula-(X₁₄)_n14-CONX_FifteenX ₁₆ An amide of the formula₁₂And X₁₄Is alkyl and n₁₂And n₁₄Is independent
And 0 or 1 and X₁₃, X_FifteenAnd X₁₆Are, independently of each other,
(Vii) selected from the group consisting of hydrogen and alkyl);₁₇)_n17-SO_TwoNX₁₈X₁₉A sulfonamide of the formula (wherein X₁₇Is
Alkyl and n₁₇Is 0, 1, or 2, and X₁₈And X₁₉ Is independently selected from the group consisting of hydrogen and alkyl). 20. (a) R ₃ is selected from the group consisting of hydrogen, methyl, and 2-hydroxyethyl; (b) R ₄ is hydrogen, methyl, chloro, bromo, carboxy, methoxy, —NH
(C) R ₅ is selected from the group consisting of hydrogen, chloro, methoxy, and —NHC (O) —CH ₃ ; and (C) R ₃ is selected from the group consisting of: C (O) —CH ₃ , and —SO ₂ (CH ₃ ) _2. (D) R ₆ is hydrogen; (e) R ₇ is hydrogen, methyl, —CH ₂ CH ₂ C (O) OH, —CH ₂ CH ₂ C (O
_{) NH 2, -CH 2 CH 2} CH 2 N (CH 3) 2 and 3-morpholino selected from the group consisting of propyl; and (f) R ₈ and R ₉ are each independently hydrogen, methyl, - CH ₂ CH ₂ C (O
_{) OH, -CH 2 CH 2 CH} 2 N (CH 3) 2 and 3-morpholinopropoxy or selected from the group consisting of propyl or R ₈ and R _9, taken together, 6-membered aromatic or aliphatic 19. The compound according to claim 18, which may form an aromatic ring. 21. A compound formed by the reaction of a ketone and oxindole, wherein the ketone is Embedded image Wherein R ₁ is (i) halogen, trihalomethyl, alkoxy, carboxylate, amino, nitro, ester and a 5- or 6-membered aromatic, heteroaromatic, aliphatic or heteroaliphatic ring A saturated or unsaturated alkyl optionally substituted with a substituent selected from the group consisting of components, wherein said ring component is alkyl, halogen, trihalomethyl, carboxylate, amino, nitro, and ester 1 independently selected from the group consisting of components
, 2, or 3 optionally substituted with substituents; and (ii) independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, amino, nitro, and ester components Done 1,
An aromatic or heteroaromatic ring optionally substituted with two or three substituents; (iii) independent from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, amino, nitro and ester Or an aliphatic or heteroaliphatic ring optionally substituted with one or more substituents selected from alkyl, alkoxy, halogen, trihalomethyl, carboxylate, amino, nitro, and ester components. An aromatic or heteroaromatic ring optionally substituted with one or more substituents independently selected from the group consisting of: selected from the group consisting of: Oxindole is 1,3-dihydro-indol-2-one, 4-methyl-1,3-dihydro-indo. All 2-one, 4- (2-hydroxy-ethyl) -1,3-dihydro-indol-2-one, 5-chloro-1,3-dihydro-indol-2-one, 5-bromo-1 , 3-Dihydro-indol-2-one, 5-methoxy-1,3-dihydro-indol-2-one, 2-oxo-2
, 3-Dihydro-1H-indole-5-carboxylic acid, 2-oxo-2,3-dihydro-1H-indole-5-sulfonic acid dimethylamide, N- (2-oxo-2,3-dihydro-1H- Indole-5-yl) -acetamido, 6-chloro-1,3-dihydro-indol-2-one, 6-methoxy-1,3-dihydro-indol-2-one, and N- (5-methyl-2 -Oxo-2,3-dihydro-1H-indol-6-yl) -acetamide. 22. Formula IV or Formula V:[Wherein (a) R₁, R_TwoAnd R₇Are each independently: (i) hydrogen; (ii) halogen, trihalomethyl, alkoxy, carboxylate, amino,
Nitro, esters and 5- or 6-membered aromatic, heteroaromatic, aliphatic,
Or optionally substituted with a substituent selected from the group consisting of heteroaliphatic ring components
Optionally saturated or unsaturated alkyl, wherein the ring component is alkyl, halogen, trihalomethyl, carboxylate
1, independently selected from the group consisting of amino, nitro, and ester components
, 2, or 3 substituents; and (iii) alkyl, alkoxy, halogen, trihalomethyl, carboxylate
1, independently selected from the group consisting of amino, nitro, and ester components
Aromatic or heteroaromatic optionally substituted with 1, 2, or 3 substituents
An aromatic ring; selected from the group consisting of: (b) R_ThreeIs the formula -CH_TwoCH_Two-OR is an ethyl-2-oxy group, wherein R is
Is (i) hydrogen; (ii) halogen, trihalomethyl, carboxylate, amino, nitro, S
Ter and 5- or 6-membered aromatic, heteroaromatic, aliphatic, or heterocyclic
Saturated optionally substituted with a substituent selected from the group consisting of aliphatic ring components
A sum or unsaturated alkyl, wherein the ring component is alkyl, halogen, trihalomethyl, carboxylate,
1, 2, or more independently selected from the group consisting of
Or three optionally substituted; (iii) alkyl, aryl, alkoxy, halogen, trihalomethyl,
Independently selected from the group consisting of boxylate, amino, nitro, and ester components
Aromatic or optionally substituted with one or more selected substituents or
(Iv) Formula -C (E) NX_FifteenX₁₆A substituent of the formula (wherein, X_FifteenAnd X₁₆Is independently hydrogen, alkyl, hydroxyl
, Formula -SO_TwoX_{twenty two}Sulfones and 5- or 6-membered aromatic and heteroaromatic
, Aliphatic, or heteroaliphatic ring components, wherein said ring is alkyl, aryl, halogen, trihalomethyl, carboxylate
, Amino, nitro, and esters independently selected from the group consisting of
X may be optionally substituted with a further substituent, X_{twenty two}Is a saturated or unsaturated alkyl, and a 5- or 6-membered aromatic,
Selected from the group consisting of teloaromatic, aliphatic, or heteroaliphatic ring components, wherein said ring is alkyl, halogen, trihalomethyl, carboxylate, amino,
One or more independently selected from the group consisting of nitro, and esters
Optionally substituted with substituents, E is selected from the group consisting of oxygen and sulfur); selected from the group consisting of: and (c) R_Four, R_FiveAnd R₆Are each hydrogen; (d) Z is a 5, 6, 7, 8, 9, or 10 membered monocyclic or bicyclic,
An aromatic or heteroaromatic ring component, (i) hydrogen; (ii) hydroxy, halogen, trihalomethyl, carboxylate, amino,
Nitro, esters and 5- or 6-membered aromatic, heteroaromatic, aliphatic,
Or one or more substituents selected from the group consisting of heteroaliphatic ring components
A saturated or unsaturated alkyl which may be optionally substituted with, and the ring component is alkyl, halogen, trihalomethyl, carboxylate,
One or more independently selected from the group consisting of
(Iii) alkyl, alkoxy, halogen, trihalomethyl, carboxylate
1, independently selected from the group consisting of amino, nitro, and ester components
Or an aromatic or heteroaromatic optionally substituted with more substituents
(Iv) alkyl, alkoxy, halogen, trihalomethyl, carboxylate
One or more independently selected from the group consisting of, amino, nitro, and ester
An aliphatic or heteroaliphatic ring optionally substituted with the above substituents, and
, Alkyl, alkoxy, halogen, trihalomethyl, carboxylate, amido
One or more independently selected from the group consisting of
An aromatic or heteroaromatic ring optionally substituted with more than one substituent; (v) a formula-(X₁)_n1-NX_TwoX_ThreeAmine of the formula (wherein X₁Is a saturated or unsaturated alkyl, and a 5- or 6-membered ring.
Selected from the group consisting of aromatic, heteroaromatic, or aliphatic ring components;₁Is 0 or 1 and X_TwoAnd X_ThreeIs independently hydrogen, saturated or unsaturated alkyl, and a five-membered ring
Or selected from the group consisting of aromatic, heteroaromatic, or aliphatic ring components having 6-membered rings
(Vi) Formula -NO_Two(Vii) halogen or trihalomethyl; (viii) a formula-(X_Four)_n4-CO-X_FiveA ketone (wherein X_FourAnd X_FiveIs independently alkyl and 5- or 6-membered aromatic
Selected from the group consisting of aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring components
The alkyl and the ring component are alkyl, halogen, trihalomethyl, carboxy,
Independently selected from the group consisting of silate, amino, nitro, and ester components
N is optionally substituted with 1, 2, or 3 substituents;_FourIs 0 or 1); (ix) Formula-(X₆)_n6A carboxylic acid of -COOH or a formula-(X₇)_n7-COO-
X₈An ester of the formula, wherein X₆, X₇And X₈Is independently alkyl and 5- or 6-membered ring
Selected from the group consisting of aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring components
And n₆And n₇Is independently 0 or 1); (x) Formula-(X₉)_n9-OH alcohol or formula-(X_Ten)_n10-OX₁₁No
Alkoxyalkyl component (wherein, X₉, X_TenAnd X₁₁Is independently saturated or unsaturated alkyl,
And 5- or 6-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic
Selected from the group consisting of ring components, wherein said alkyl and ring components are alkyl, halogen, trihalomethyl,
Independently selected from the group consisting of sylate, amino, nitro, and ester
Optionally substituted with one or more substituents, and n₉And n_TenIs independently 0 or 1); (xi) Formula-(X₁₂)_n12-NHCOX₁₃Or the formula-(X₁₄)_n14-CONX_FifteenX ₁₆ An amide of the formula₁₂And X₁₄Are each independently alkyl, hydroxyl, and
And 5- or 6-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring
Wherein the alkyl and ring components are alkyl, halogen, trihalomethyl, carboxy,
Independently selected from the group consisting of sylate, amino, nitro, and ester
Optionally substituted with one or more substituents, n₁₂And n₁₄Is independently 0 or 1 and X₁₃, X_FifteenAnd X₁₆ Is each independently hydrogen, alkyl, hydroxyl, and a five-membered ring or
Group consisting of six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring components
And the alkyl and ring components are alkyl, halogen, trihalomethyl, carboxy,
Independently selected from the group consisting of sylate, amino, nitro, and ester
(Optionally substituted with one or more substituents); (xii) a formula-(X₁₇)_n17-SO_TwoNX₁₈X₁₉A sulfonamide of the formula₁₇Is alkyl, 5- or 6-membered aromatic, heteroaromatic or aliphatic
Or a heteroaliphatic ring component, wherein said alkyl and ring components are alkyl, halogen, trihalomethyl, carboxy,
Independently selected from the group consisting of sylate, amino, nitro, or ester
Optionally substituted with one or more substituents, and n₁₇Is 0, 1, or 2, and X₁₈And X₁₉Is independently hydrogen, alkyl, 5- or 6-membered aromatic,
Selected from the group consisting of heteroaromatic, aliphatic, or heteroaliphatic ring components, wherein said alkyl and ring components are alkyl, halogen, trihalomethyl, carboxy.
Independently selected from the group consisting of sylate, amino, nitro, or ester
Optionally substituted with one or more substituents, or X₁₈And X₁₉Is, together, alkyl, halogen, trihalomethyl
, Carboxylate, amino, nitro, and ester independently of the group consisting of
A 5-membered ring optionally substituted with one or more substituents selected, or
Forms a 6-membered aliphatic or heteroaliphatic ring); (xiii) a formula-(X₂₀)_n20Aldehyde of —CO—H (wherein, X₂₀Is a saturated or unsaturated alkyl, and a 5- or 6-membered ring.
Selected from the group consisting of aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring components
Wherein said alkyl and ring components are alkyl, halogen, trihalomethyl, carboxy,
Independently selected from the group consisting of sylate, amino, nitro, and ester
Optionally substituted with one or more substituents, and n₂₀Is 0 or 1); (xiv) Formula-(X_{twenty one})_n21-SO_Two-X_{twenty two}Of the formula (wherein, X_{twenty one}And X_{twenty two}Is independently saturated or unsaturated alkyl, and 5
Aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring components having 6 or 6 membered rings
Wherein said alkyl and ring components are alkyl, halogen, trihalomethyl, carboxy
Independently selected from the group consisting of sylate, amino, nitro, and ester
Optionally substituted with one or more substituents, and n_{twenty one}Is 0 or 1); and (xv) a formula-(X_{twenty three})_n23The thiol of -SH and the formula-(X_{twenty four})_n24-SX_{twenty five}of
Thioether (wherein, X_{twenty three}, X_{twenty four}And X_{twenty five}Is independently saturated or unsaturated alkyl,
And 5- or 6-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic
Selected from the group consisting of ring components, wherein said alkyl and ring components are alkyl, halogen, trihalomethyl,
Independently selected from the group consisting of sylate, amino, nitro, and ester
Optionally substituted with one or more substituents, and n_{twenty three}And n_{twenty four}Is independently 0 or 1); optionally substituted with one or more substituents selected from the group consisting of
Good]. 23. (a) R ₁ , R ₂ and R ₇ are hydrogen; (b) R ₃ is an ethyl-2-oxy group of the formula —CH ₂ CH ₂ —OR, wherein ,
R is hydrogen, saturated alkyl, and an aromatic ring optionally substituted with one or more substituents independently selected from the group consisting of alkyl, aryl, and alkoxy components; C (E) NHX _{15 wherein} X ₁₅ is selected from the group consisting of alkyl, sulfone of the formula —SO ₂ —X ₂₂ and a six-membered aromatic or aliphatic ring component, wherein said ring is alkyl and aryl X ₂₂ may be optionally substituted with one or more substituents independently selected from the group consisting of
A substituent selected from the group consisting of saturated alkyl, and an optionally substituted 6-membered aromatic ring component, and E is selected from the group consisting of oxygen and sulfur; And (c) Z is a 5, 6, or 9-membered monocyclic or bicyclic, aromatic or heteroaromatic ring component, and (i) hydrogen; (ii) saturated alkyl , an aromatic substituted in (iii) any or heteroaromatic ring; (iv) formula _{- (X 1) n1 -NX 2} X 3 amine (wherein, X ₁ is an alkyl, n ₁ Is 0 or 1, and X ₂ and X ₃ are independently selected from the group consisting of hydrogen and saturated alkyl); (v) halogen or trihalomethyl; (vi) a _formula- (X ₆ ) _n6 carboxylic acid (wherein the -COOH, X ₆ is alkyl , And the and n ₆ represents 0 or 1); and (vii) expression - (X _{9) n9} -OH alcohol or formula - (alkoxyalkyl components _{X 10) n10 -O-X 11} ( wherein, X ₉ , X ₁₀ and X ₁₁ are alkyl, and n ₉ and n ₁₀ are independently 0 or 1); optionally substituted with one or more substituents selected from the group consisting of: 23. The compound of claim 22, which may be 24. (a) R ₃ is a compound of the formula —CH ₂ CH ₂ —O—R, wherein R is hydrogen, methyl, ethyl, 2-isopropylphenyl, 3-isopropylphenyl,
4-isopropylphenyl, 4-methoxyphenyl, biphen-3-yl, 5-
Chloropyridin-3-yl, ethylcarbamyl, tert-butylcarbamyl,
(B) Z is selected from the group consisting of cyclohexylcarbamyl, phenylcarbamyl, benzenesulfonylcarbamyl, biphen-2-yl-carbamyl, and phenylthiocarbamyl); 4-bromophenyl, 2-pyridyl, 6-methyl-2-pyridyl,
1H-indol-5-yl, 4-methoxy-3-thiophenephenyl, 4- (
3-dimethylamino) -3,5-dimethyl-1H-pyrrol-2-yl, 3-hydroxy-6-methyl-pyridin-2-yl, 4- (3-dimethylaminopropyl) -3,5-dimethyl- 1H-pyrrol-2-yl, and 3-carboxy-
23. The compound according to claim 22, wherein the compound is selected from the group consisting of 2,4-dimethyl-1H-pyrrol-2-yl. 25. The compound according to claim 1, wherein the compound is 4- [2- (3-isopropyl-phenoxy) -ethyl] -1,3-dihydro-indol-2-one, 4- [2- (2-
Isopropyl-phenoxy) -ethyl] -1,3-dihydro-indole-2-
On, 4- [2- (biphenyl-3-yloxy) -ethyl] -1,3-dihydro-indol-2-one, 3- (4-bromo-benzylidene) -4- (2-hydroxy-ethyl)- 1,3-dihydro-indol-2-one, 4- (2-hydroxy-ethyl) -3-pyridin-2-ylmethylene-1,3-dihydro-indol-2-one, 4- (2-hydroxy-ethyl ) -3- (6-Methyl-pyridin-2-ylmethylene) -1,3-dihydro-indol-2-one, 4-
(2-hydroxy-ethyl) -3- (1H-indol-5-ylmethylene)-
1,3-dihydro-indol-2-one, 4- (2-hydroxy-ethyl)-
3- (4-methoxy-3-thiophen-2-yl-benzylidene) -1,3-dihydro-indol-2-one, 3- [4- (3-dimethylamino-propyl]
) -3,5-Dimethyl-1H-pyrrol-2-ylmethylene] -4- (2-hydroxy-ethyl) -1,3-dihydro-indol-2-one, phenyl-thiocarbamic acid O- [2- ( 2-oxo-2,3-dihydro-1H-indole-
4-yl) -ethyl] ester, phenyl-carbamic acid 2- (2-oxo-2)
, 3-Dihydro-1H-indol-4-yl) -ethyl ester, tert-
Butyl-carbamic acid 2- (2-oxo-2,3-dihydro-1H-indol-4-yl) -ethyl ester, cyclohexyl-carbamic acid 2- (2-oxo-2,3-dihydro-1H-indole- 4-yl) -ethyl ester, benzenesulfonyl-carbamic acid 2- (2-oxo-2,3-dihydro-1H-indol-4-yl) -ethyl ester, biphenyl-2-yl-carbamic acid 2- (2 -Oxo-2,3-dihydro-1H-indol-4-yl) -ethyl ester, ethyl-carbamic acid 2- (2-oxo-2,3-dihydro-1H-
Indole-4-yl) -ethyl ester, 4- [2- (4-methoxy-phenoxy) -ethyl] -1,3-dihydro-indol-2-one, 4- (2-methoxy-ethyl) -1, 3-dihydro-indol-2-one, 4- (2-ethoxy-ethyl) -1,3-dihydro-indol-2-one, 4- [2- (4-isopropyl-phenoxy) -ethyl] -1, 3-dihydro-indol-2-one, 4- [2- (5-chloro-pyridin-3-yloxy) -ethyl] -1,3
-Dihydro-indol-2-one, 4- (2-hydroxy-ethyl) -3- (
3-hydroxy-6-methyl-pyridin-2-ylmethylene) -1,3-dihydro-indol-2-one, 3- [4- (3-dimethylamino-propyl) -3
, 5-Dimethyl-1H-pyrrol-2-ylmethylene] -4- [2- (3-isopropyl-phenoxy) -ethyl] -1,3-dihydro-indol-2-one, and 3- (5- {4 -[2- (4-isopropyl-phenoxy) -ethyl]
-2-oxo-l, 2-dihydro-indole-3-ylidenemethyl} -2,4
23. The compound according to claim 22, which is selected from the group consisting of -dimethyl-1H-pyrrol-3-yl) -propionic acid. 26. 4- [2- (3-isopropyl-phenoxy) -ethyl]
-1,3-dihydro-indol-2-one, 4- [2- (2-isopropyl-
Phenoxy) -ethyl] -1,3-dihydro-indol-2-one, 4- [2
-(Biphenyl-3-yloxy) -ethyl] -1,3-dihydro-indol-2-one, 3- (4-bromo-benzylidene) -4- (2-hydroxy-ethyl) -1,3-dihydro- Indole-2-one, 4- (2-hydroxy-ethyl) -3-pyridin-2-ylmethylene-1,3-dihydro-indole-2-
On, 4- (2-hydroxy-ethyl) -3- (6-methyl-pyridin-2-ylmethylene) -1,3-dihydro-indol-2-one, 4- (2-hydroxy-ethyl) -3- (1H-indol-5-ylmethylene) -1,3-dihydro-indol-2-one, 4- (2-hydroxy-ethyl) -3- (4-methoxy-3-thiophen-2-yl-benzylidene)- 1,3-dihydro-indol-2-one, 3- [4- (3-dimethylamino-propyl) -3,5-dimethyl-1H-pyrrol-2-ylmethylene] -4- (2-hydroxy-ethyl)
-1,3-dihydro-indol-2-one, phenyl-thiocarbamic acid O-
[2- (2-oxo-2,3-dihydro-1H-indol-4-yl) -ethyl] ester, phenyl-carbamic acid 2- (2-oxo-2,3-dihydro-
1H-indol-4-yl) -ethyl ester, tert-butyl-carbamic acid 2- (2-oxo-2,3-dihydro-1H-indol-4-yl) -ethyl ester, cyclohexyl-carbamic acid 2- ( 2-oxo-2,3-dihydro-1H-indol-4-yl) -ethyl ester, benzenesulfonyl-
Carbamic acid 2- (2-oxo-2,3-dihydro-1H-indol-4-yl) -ethyl ester, biphenyl-2-yl-carbamic acid 2- (2-oxo-2,3-dihydro-1H- Indole-4-yl) -ethyl ester, ethyl-carbamic acid 2- (2-oxo-2,3-dihydro-1H-indole-4-
Yl) -ethyl ester, 4- [2- (4-methoxy-phenoxy) -ethyl]
-1,3-Dihydro-indol-2-one, 4- (2-methoxy-ethyl)-
1,3-dihydro-indol-2-one, 4- (2-ethoxy-ethyl) -1
, 3-Dihydro-indol-2-one, 4- [2- (4-isopropyl-phenoxy) -ethyl] -1,3-dihydro-indol-2-one, 4- [2- (
5-Chloro-pyridin-3-yloxy) -ethyl] -1,3-dihydro-indol-2-one, 4- (2-hydroxy-ethyl) -3- (3-hydroxy-
6-methyl-pyridin-2-ylmethylene) -1,3-dihydro-indole-
2-one, 3- [4- (3-dimethylamino-propyl) -3,5-dimethyl-
1H-pyrrol-2-ylmethylene] -4- [2- (3-isopropyl-phenoxy) -ethyl] -1,3-dihydro-indol-2-one, and 3- (5
-{4- [2- (4-isopropyl-phenoxy) -ethyl] -2-oxo-1
, 2-Dihydro-indole-3-ylidenemethyl} -2,4-dimethyl-1H
-Pyrrol-3-yl) -propionic acid. 27. The compound according to any of claims 1, 7, 18, 21, 22, 22, or 26, wherein said compound is capable of inhibiting the catalytic activity of a protein kinase. 28. The compound of claim 27, wherein said protein kinase is selected from the group consisting of receptor protein tyrosine kinase, cellular tyrosine kinase and serine-threonine kinase. 29. A method of regulating the catalytic activity of a protein kinase, comprising contacting the protein kinase with the compound of any of claims 1, 7, 18, 21, 22, 22, or 26. Method. 30. A method for regulating a signal transduction pathway in a cell using the compound according to any one of claims 1, 7, 18, 21, 22, and 26, wherein the cell is contacted with the compound. A method comprising a step. 31. The method of claim 30, wherein said cells express a protein kinase and said compound modulates a function of said protein kinase. 32. A method for identifying an indolinone compound that modulates the function of a protein kinase, comprising: (a) transforming a cell that expresses the protein kinase with a cell that expresses the protein kinase.
Or (b) monitoring the effect on the cells. 33. The effect is selected from the group consisting of a change in cell phenotype, a change in cell proliferation, a change in the catalytic activity of the protein kinase, and a change in the interaction between the protein kinase and a binding partner. 33. The method of claim 32, wherein 34. A method of controlling uncontrolled protein kinase signaling, comprising administering to a subject a therapeutically effective amount of a protein kinase.
A method comprising administering a compound according to any one of the preceding claims. 35. An unregulated protein kinase signaling leads to a disease or abnormal condition in an organism, said method leads to the treatment or prevention of said disease or condition, and said disease or condition is associated with protein kinase. 35. The method of claim 34, wherein the method is associated with an abnormal form of a signaling pathway characterized by an interaction with a binding partner, and wherein the method further comprises promoting or disrupting the abnormal interaction. 36. The method of claim 35, wherein said disease or abnormal condition is cancer. 37. The disease or abnormal condition is an immunological disease, a hyperproliferative disease, a cardiovascular disease, an inflammatory disease, restenosis, fibrosis, psoriasis, osteoarthritis, rheumatoid arthritis, arteriosclerosis. 36. The method of claim 35, wherein the method is selected from the group consisting of diabetes, diabetes, and angiogenesis. 38. The method of claim 29, 31, 32, or 34, wherein said protein kinase is selected from the group consisting of receptor protein tyrosine kinase, cellular tyrosine kinase and serine-threonine kinase. 39. (i) a physiologically acceptable carrier, diluent, or excipient or a combination thereof; and (ii) any of claims 1, 7, 18, 21, 22, 22, or 26. A pharmaceutical composition comprising the compound of claim 1.