HK1001121B - Indolinone compounds for the treatment of disease - Google Patents

Description

Indolinone compounds for the treatment of diseases

This application is a continuation-in-part application of U.S. patent application 08/485,323 filed on 7.6.1995, which is incorporated by reference in its entirety.

1. Introduction to the design reside in

The present invention relates to novel compounds capable of modulating, modulating and/or inhibiting tyrosine kinase signal transduction. The invention also relates to methods of modulating, modulating or inhibiting tyrosine kinases, whether of the receptor or non-receptor type, for the prevention and/or treatment of conditions associated with deregulated tyrosine kinase signal transduction, including cell proliferative and metabolic disorders.

2. Background of the invention

Protein Tyrosine Kinases (PTKs) include many different types of proteins with enzymatic activity. PTK plays an important role in controlling cell growth and differentiation [ for review see Schlessinger and Ullrich, 1992, Neuron (Neuron) 9: 383-391).

For example, receptor tyrosine kinase mediated signal transduction is induced by extracellular interaction with a specific growth factor (ligand), followed by receptor dimerization, transient stimulation of intrinsic protein tyrosine kinase activity and phosphorylation. Binding sites for intracellular signal transduction molecules are thereby generated and result in the formation of complexes with a wide variety of cytoplasmic signaling molecules that promote appropriate cellular responses (e.g., cell division, metabolism to the extracellular microenvironment), see Schlessinger and Ullrich, 1992, neurons (Neuron)9：383-391。

With regard to receptor tyrosine kinases, it has also been pointed out that the tyrosine phosphorylation site is responsible for the SH of the signaling molecule₂The (src homology) domain acts as a high affinity binding site. Fantl et al, 1992, cell69: 413-423; songyang et al, 1994, molecular cell biology (mol. cell. biol. 3.)14: 2777 vs 2785; songyang et al, 1993, Cell (Cell)72: 767 778; and Koch et al, 1991, Science (Science)252: 668-678. Several binding to Receptor Tyrosine Kinases (RTKs) have been identifiedThe intracellular substrate protein of (1). They can be divided into two broad categories: (1) a substrate having a catalytic domain; and (2) substrates lacking such domains, but acting as adaptors and binding to catalytically active molecules. Songyang et al, 1993, Cell (Cell)72: 767-778. SH of receptors or proteins and their substrates₂The specificity of the interaction between the domains is determined by the amino acid groups that directly surround the phosphorylated tyrosine groups. SH (hydrogen sulfide)₂The difference in binding affinity between the domain and the amino acid sequence surrounding the phosphotyrosine residues on specific receptors is consistent with the observed differences in their substrate phosphorylation patterns. Songyang et al, 1993, Cell (Cell)72: 767-778. These observations indicate that the function of individual receptor tyrosine kinases is determined not only by their expression pattern and ligand availability, but also by the arrangement of downstream signal transduction pathways activated by specific receptors. Thus, phosphorylation provides an important regulatory step that determines the selectivity of signaling pathways recruited by specific growth factor receptors as well as differentiation factor receptors.

It has been suggested that aberrant expression or mutations in PTKs can lead to uncontrolled cell proliferation (e.g., malignant tumor growth) or defects in critical developmental processes. Therefore, the biomedical community has put great effort into discovering specific biological actions of members of the PTK family, their role in differentiation processes, their relationship to tumorigenesis and other diseases, biochemical mechanisms by which their signal transduction pathways are activated upon ligand stimulation, and the development of new drugs.

Tyrosine kinases can be either receptor-type (having extracellular, transmembrane and intracellular domains) or non-receptor type (being entirely intracellular).

Receptor type tyrosine kinase. Such RTKs include a large family of transmembrane receptors with a wide variety of biological activities. The intrinsic function of PTKs is activated upon ligand binding, which results in phosphorylation of receptors and multicellular substrates, followed by a wide variety of cellular responses. Ullrich and Schlesinger, 1990, Cell (Cell)61：203-212。

At present, at least 19 different RTK subfamilies have been identified. One RTK subfamily, called the HER subfamily, is believed to consist of EGFR, HER2, HER 3, and HER 4. Ligands for the HER subfamily of receptors include Epidermal Growth Factor (EGF), TGF-alpha, amphiregulin, HB-EGF, beta-animal cellulose and heregulin.

The second group of RTKs, called the insulin subfamily, is composed of INS-R, IGF-1R and IR-R. The third "PDGF" subfamily includes PDGF alpha and beta receptors, CSFIR, c-kit and FLK-II. Another subset of RTKs is called the FLK family, and is thought to consist of the kinase insert domain-receptors fetal liver kinase-1 (KDR/FLK-1), fetal liver kinase 4(FLK-4), and fms-like enzyme serine kinase 1 (flt-1). These receptors were originally thought to be receptors for hematopoietic growth factors. The other two subfamilies of RTKs are referred to as the FGF receptor family (FGFR 1, FGFR 2, FGFR 3 and FGFR 4) and the Met subfamily (C-Met and Ron).

Because the PDGF and FLK subfamilies are similar, these two subfamilies are often discussed together. The known RTK subfamily is identified by plowman et al (1994, drugs News & Expects (DN)&P)， 7(6): 334-., 339), which is incorporated by reference in this application.

Non-receptor tyrosine kinase. Non-receptor tyrosine kinases represent a collection of cellular enzymes that lack extracellular and transmembrane sequences. Currently, more than 24 individual non-receptor tyrosine kinases have been identified, including 11 subfamilies (Src, Frk, Btx, Csk, Abl, Zap 70, Fes/Fps, Fak, Jak, Ack, and LIMK). Currently, the Src subfamily of non-receptor tyrosine kinases constitutes the maximum number of PTKs, including Src, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr, and Yrk. Src subfamily enzymes are involved in tumorigenesis. In Bolen, 1993, Oncogene (Oncogene)8: 2025-2031, which is incorporated herein by reference, provides a more detailed discussion of non-receptor tyrosine kinases.

Many tyrosine kinases, whether receptor or non-receptor tyrosine kinases, are found to be involved in cell signaling pathways leading to pathogenic states including cancer, psoriasis and hyperimmune responses.

Preparation of compounds for modulating PTK. In view of the presumed importance of PTKs in the control, regulation and modulation of cellular proliferation in diseases and disorders associated with abnormal cellular proliferation, many efforts have been made to identify receptor and non-receptor tyrosine kinase "inhibitors" using various approaches including the use of mutant ligands (us patent application 4,966,849), soluble receptors and antibodies [ patent application WO 94/10202; kendall and Thomas, 1994, proceedings of the national academy of sciences of the united states (proc.nat' I acad.sci)90: 10705-09; and Kim: etc., 1993, Nature)362: 841-844, ribonucleic acid (RNA) ligands [ Jellinek et al, Biochemistry)33: 10450-; takano et al, 1993, cell molecular biology (mol.Bio.cell)4: 358A; kinsella et al, 1992, Experimental cell research (exp. cell. Res.)199: 56-62; wright et al, 1992, journal of cell physics (J.Cellular Phys.)152: 448-; WO 92/21660; WO 91/15495; WO 94/14808; us patent 5,330,992; nariani et al, 1994, conference on the American society for research on cancer (Proc. am. Assoc. cancer Res.)35：2268〕。

Recently, attempts have been made to identify small molecules that act as tyrosine kinase inhibitors. For example, bis-monocyclic, bicyclic or heterocyclic aromatic compounds (PCT WO 92/20642), vinylidene-azaindole derivatives (PCT WO 94/14808) and 1-cyclopropyl-4-pyridyl-quinolones (U.S. patent 5,330,992) have been generally recognized as tyrosine kinase inhibitors. Styryl compounds (us 5,217,999), styryl substituted pyridyl compounds (us 5,320,606), certain quinazoline derivatives (european patent application 0566266 a1), selenoindoles and selenoethers (PCT WO 94/03427), tricyclic polyols (PCT WO 92/21660) and benzylphosphonic acid compounds (PCT WO 91/15495) have been mentioned as compounds useful as tyrosine kinase inhibitors for the treatment of cancer.

It would therefore be highly desirable to identify effective small molecule compounds that specifically inhibit signal transduction by modulating the activity of receptor and non-receptor tyrosine kinases, thereby modulating and modulating abnormal or inappropriate cell proliferation, and it is also an object of the present invention.

3. Summary of the invention

The present invention relates to organic molecules capable of modulating, modulating and/or inhibiting tyrosine kinase signal transduction. These compounds are useful in the treatment of diseases associated with deregulated TKS transduction including cell proliferative disorders such as cancer, atherosclerosis, arthritis and restenosis, and metabolic disorders such as diabetes.

In one illustrative embodiment, the compounds of the invention are of formula (I):

wherein R is₁Is H or alkyl;

R₂is O or S;

R₃is hydrogen;

R₄、R₅、R₆and R₇Each independently selected from the group comprising: hydrogen, alkyl, alkoxy, aryl, aryloxy, alkylaryl, alkylaryloxy, halogen, trihalomethyl, S (O) R, SO₂NRR′、SO₃R、SR、NO₂、NRR′、OH、CN、C(O)R、OC(O)R、NHC(O)R、(CH₂)_nCO₂R and CONRR';

R₃′、R₅' and R₆' each is independently selected from the group comprising: hydrogen, alkyl, alkoxy, aryl, aryloxy, alkylaryl, alkylaryloxy, halogen, trihalomethyl, S (O) R, SO₂NRR′、SO₃R、SR、NO₂、NRR′、OH、CN、C(O)R、OC(O)R、NHC(O)R、(CH₂)_nCO₂R and CONRR';

n is 0 to 3;

x is Br, Cl, F or I;

r is H, alkyl or aryl;

r' is H, alkyl or aryl.

In another illustrative embodiment, the compounds of the invention are of formula (II) and pharmaceutically acceptable salts thereof.

Wherein R is₁Is H or alkyl;

R₂is O or S;

R₃is hydrogen;

R₄、R₅、R₆and R₇Each independently selected from the group comprising: hydrogen, alkyl, alkoxy, aryl, aryloxy, alkylaryl, alkylaryloxy, halogen, trihalomethyl, S (O) R, SO₂NRR′、SO₃R、SR、NO₂、NRR′、OH、CN、C(O)R、OC(O)R、NHC(O)R、(CH₂)_nCO₂R and CONRR';

R₂′、R₃′、R₅' and R₆' each is independently selected from the group comprising: hydrogen, alkyl, alkoxy, aryl, aryloxy, alkylaryl, alkylaryloxy, halogen, trihalomethyl, S (O) R, SO₂NRR′、SO₃R、SR、NO₂、NRR′、OH、CN、C(O)R、OC(O)R、NHC(O)R、(CH₂)_nCO₂R and CONRR';

ra and Rb are each independently selected from the group consisting of H, alkyl and C (O) R, or NRaRb together may be a heterocyclic ring of 3 to 8 atoms which may optionally be substituted in one or more positions by: hydrogen, alkyl, alkoxy, aryl, aryloxy, alkylaryl, alkylaryloxy, halogen, trihalomethyl, S (O) R, SO₂NRR′、SO₃R、SR、NO₂、NRR′、OH、CN、C(O)R、OC(O)R、NHC(O)R、(CH₂)_nCO₂R and CONRR';

n is 0 to 3;

x is Br, Cl, F or I;

r is H, alkyl or aryl;

r' is H, alkyl or aryl.

In another illustrative embodiment, the compounds of the invention are of formula (III):

wherein R is₁Is H or alkyl;

R₂is O or S;

R₃is hydrogen;

R₄、R₅、R₆and R₇Each independently selected from the group comprising: hydrogen, alkyl, alkoxy, aryl, aryloxy, alkylaryl, alkylaryloxy, halogen, trihalomethyl, S (O) R, SO₂NRR′、SO₃R、SR、NO₂、NRR′、OH、CN、C(O)R、OC(O)R、NHC(O)R、(CH₂)_nCO₂R and CONRR';

a is a five-membered heterocycle selected from the group consisting of thiophene, pyrrole, pyrazole, imidazole, 1, 2, 3-triazole, 1, 2, 4-triazole, oxazole, isoxazole, thiazole, isothiazole, 2-sulfonylfuran, 4-alkylfuran, 1, 2, 3-oxadiazole, 1, 2, 4-oxadiazole, 1, 2, 5-oxadiazole, 1, 3, 4-oxadiazole, 1, 2, 3-oxadiazole4-oxatriazole, 1, 2, 3, 5-oxatriazole, 1, 2, 3-thiadiazole, 1, 2, 4-thiadiazole, 1, 2, 5-thiadiazole, 1, 3, 4-thiadiazole, 1, 2, 3, 4-thiatriazole, 1, 2, 3, 5-thiatriazole and tetrazole, optionally substituted in one or more positions with: alkyl, alkoxy, aryl, aryloxy, alkaryl, alkylaryloxy, halogen, trihalomethyl, S (O) R, SO₂NRR′、SO₃R、SR、NO₂、NRR′、OH、CN、C(O)R、OC(O)R、NHC(O)R、(CH₂)_nCO₂R or CONRR';

n is 0 to 3;

x is Br, Cl, F or I;

r is H, alkyl or aryl;

r' is H, alkyl or aryl.

In yet another illustrative embodiment, the compounds of the invention are of formula (IV):

wherein R is₁Is hydrogen or alkyl;

R₂is O or S;

R₃is hydrogen;

R₄、R₅、R₆and R₇Each independently selected from the group comprising: hydrogen, alkyl, alkoxy, aryl, aryloxy, alkylaryl, alkylaryloxy, halogen, trihalomethyl, S (O) R, SO₂NRR′、SO₃R、SR、NO₂、NRR′、OH、CN、C(O)R、OC(O)R、NHC(O)R、(CH₂)_nCO₂R and CONRR';

R₃′、R₄′、R₅' and R₆' each is independently selected from the group comprising: hydrogen, alkyl, alkoxyAryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S (O) R, SO₂NRR′、SO₃R、SR、NO₂、NRR′、OH、CN、C(O)R、OC(O)R、NHC(O)R、(CH₂)_nCO₂R and CONRR';

n is 0 to 3;

x is Br, Cl, F or I;

r is H, alkyl or aryl;

r' is H, alkyl or aryl.

In a final embodiment, the compounds of the invention are of formula (V):

wherein R is₁Is H or alkyl;

R₂is O or S;

R₃is hydrogen;

R₄、R₅、R₆and R₇Each independently selected from the group comprising: hydrogen, alkyl, alkoxy, aryl, aryloxy, alkylaryl, alkylaryloxy, halogen, trihalomethyl, S (O) R, SO₂NRR′、SO₃R、SR、NO₂、NRR′、OH、CN、C(O)R、OC(O)R、NHC(O)R、(CH₂)_nCO₂R and CONRR';

R₂′、R₃′、R₅' and R₆' each is independently selected from the group comprising: hydrogen, alkyl, alkoxy, aryl, aryloxy, alkylaryl, alkylaryloxy, halogen, trihalomethyl, S (O) R, SO₂NRR′、SO₃R、SR、NO₂、NRR′、OH、CN、C(O)R、OC(O)R、NHC(O)R、(CH₂)_nCO₂R and CONRR';

n is 0 to 3;

z is Br, Cl, F, I, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl;

r is H, alkyl or aryl;

r' is H, alkyl or aryl.

The present invention also relates to pharmaceutical compositions comprising an effective amount of a compound of formulae I-V as described above and a pharmaceutically acceptable carrier or excipient. Such pharmaceutical compositions are believed to modulate signal transduction by tyrosine kinases by inhibiting catalytic activity, affinity for ATP or ability to interact with substrates.

More particularly, the compositions of the present invention may be used in methods of treating a variety of diseases including proliferative, fibrotic or metabolic diseases such as cancer, fibrosis, psoriasis, atherosclerosis, arthritis and other diseases associated with abnormal angiogenesis and/or vascularization, such as diabetic retinopathy.

4. Detailed description of the invention

4.1. Definition of

"pharmaceutically acceptable salts" refers to those salts which retain the biological efficiency and properties of the free base, and which are obtained by reaction with inorganic acids, such bases including hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, methanesulfonic, ethanesulfonic, p-toluenesulfonic, salicylic, and the like.

"alkyl" refers to a straight, branched, or cyclic saturated aliphatic hydrocarbon. The alkyl group preferably has 1 to 12 carbon atoms. More preferred is a lower alkyl group of 1 to 7 carbon atoms. Most preferably 1 to 4 carbon atoms. Typical alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl and the like. The alkyl group may optionally be selected from hydroxy, cyano, alkoxy, ═ O, ═ S, NO₂Halogen, N (CH)₃)₂Amino and SH.

"alkenyl" refers to a straight, branched, or cyclic unsaturated hydrocarbon group containing at least one carbon-carbon double bond. The alkenyl group is preferably a lower alkenyl group having 1 to 12 carbon atoms, more preferably 1 to 7 carbon atoms. Most preferably 1 to 4 carbon atoms. Alkenyl may optionally be selected from hydroxy, cyano, alkoxy, ═ O, ═ S, NO₂Halogen, N (CH)₃)₂Amino and SH.

"alkynyl" refers to a straight, branched, or cyclic unsaturated hydrocarbon group containing at least one carbon-carbon triple bond. The alkynyl group is preferably a lower hydrocarbon group having 1 to 12 carbon atoms, more preferably 1 to 7 carbon atoms. Most preferably 1 to 4 carbon atoms. The hydrocarbyl group may optionally be selected from hydroxy, cyano, alkoxy, ═ O, ═ S, NO₂Halogen, N (CH)₃)₂Amino and SH.

"alkoxy" refers to an "-O alkyl" group.

"aryl" refers to an aromatic group having at least one ring with a conjugated pi-electron system, including carbocyclic aryl, heterocyclic aryl, and biaryl groups. Aryl groups may optionally be substituted with groups selected from halogen, trihalomethyl, hydroxy, SH, OH, NO₂Amine, thioether, cyano, alkoxy, alkyl, and amino.

"alkaryl" refers to an alkyl group covalently bonded to an aryl group. The alkyl group is preferably a lower alkyl group.

"carbocyclic aryl" refers to aryl in which the ring atoms are carbon.

"Heterocyclaryl" means an aryl group having 1 to 3 heteroatoms as ring atoms, the remaining ring atoms being carbon. Heteroatoms include oxygen, sulfur and nitrogen. For example, heterocyclic aryl groups include furyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl and the like.

"amide" means-C (O) -NH-R, where R is alkyl, aryl, alkaryl, or hydrogen.

"Thioamido" means-C (S) -NH-R, where R is alkyl, aryl, alkaryl, or hydrogen.

"amine" means an-N (R ') R "group wherein R' and R" are each independently selected from the group consisting of alkyl, aryl, and alkaryl.

"thioether" means-S-R, wherein R is alkyl, aryl, or alkaryl.

"Sulfonyl" means-S (O)₂-R, wherein R is aryl, C (CN) -C-aryl, CN₂CN, alkaryl, sulfonamide, NH-alkyl, NH-alkaryl or NH-aryl.

4.2. The invention

The present invention relates to compounds capable of modulating and/or modulating tyrosine kinase signal transduction, particularly receptor and non-receptor tyrosine kinase signal transduction.

Receptor tyrosine kinase mediated signal transduction is initiated by extracellular interaction with specific growth factors (ligands) followed by receptor dimerization, transient stimulation of intrinsic protein tyrosine kinase activity and phosphorylation. Binding sites are thereby created for intracellular signaling molecules and result in the promotion of appropriate cellular responses (e.g., cell division, metabolism of the extracellular environment). Form complexes with a wide variety of cytoplasmic signaling molecules. See Schlessinger and Ullrich, 1992, neurons (Neuron)9：303-391。

It has been shown that tyrosine phosphorylation sites in growth factor receptors are responsible for SH of signaling molecules₂The (src homology) domain acts as a high affinity binding site. Fantl et al, 1992, Cell (Cell)69: 413-423; songyang et al, 1994, molecular cell biology (mol. cell. biol.)14: 2777 vs 2785; songyang et al, 1993, Cell (Cell)72: 767 778; and Koch et al, 1991, Science (Science)252: 668-678. Several intracellular substrate proteins have been identified that bind to receptor tyrosine kinases. They can be divided into two broad categories:(1) a substrate having a catalytic domain; and (2) substrates lacking such domains, but acting as adaptors and binding to catalytically active molecules. Songyang et al, 1993, Cell (Cell)72: 767-778. SH of receptors and their substrates₂The specificity of the interaction between the domains is determined by the amino acid residues that directly surround the phosphorylated tyrosine residue. In SH₂The differences in binding affinity between the domains and the amino acid sequences surrounding phosphotyrosine residues on specific receptors are consistent with the observed differences in their substrate phosphorylation patterns. Songyang et al, 1993, Cell (Cell)72: 767-778. These observations suggest that the function of each receptor tyrosine kinase is not only determined by its expression pattern and ligand availability, but also is related to the arrangement of downstream signal transduction pathways that are activated by specific receptors. Thus, phosphorylation provides an important regulatory step that determines the selectivity of signaling pathways recruited by specific growth factor receptors as well as differentiation factor receptors.

Tyrosine kinase signal transduction, among other responses, causes cell proliferation, differentiation and metabolism. Abnormal cell proliferation can result in a range of disorders and diseases including neoplasia such as carcinomas, sarcomas, leukemias, glioblastomas, hemangiomas, psoriasis, atherosclerosis, arthritis and diabetic retinopathy (or other diseases associated with uncontrolled angiogenesis and/or vasculogenesis).

Accordingly, the present invention relates to compounds that modulate, modulate and/or inhibit tyrosine kinase signal transduction by affecting the enzymatic activity of RTKs and/or non-receptor tyrosine kinases and signals that interfere with the transduction of such proteins. More particularly, the present invention relates to compounds that modulate, modulate and/or inhibit receptor and/or non-receptor tyrosine kinase mediated signal transduction pathways as therapeutic approaches for the treatment of a variety of solid-state tumors including, but not limited to, carcinomas, sarcomas, leukemias, erythroblastomas, glioblastomas, meningiomas, astrocytomas, melanomas and myoblastomas. Indications may include, but are not limited to: brain cancer, bladder cancer, ovarian cancer, intestinal cancer, pancreatic cancer, colon cancer, leukemia, lung cancer, and bone cancer.

4.3. Compound (I)

In one embodiment, the present invention provides compounds of formula (I):

wherein R is₁Is H or alkyl;

R₂is O or S;

R₃is hydrogen;

R₄、R₅、R₆and R₇Each independently selected from the group comprising: hydrogen, alkyl, alkoxy, aryl, aryloxy, alkylaryl, alkylaryloxy, halogen, trihalomethyl, S (O) R, SO₂NRR′、SO₃R、SR、NO₂、NRR′、OH、CN、C(O)R、OC(O)R、NHC(O)R、(CH₂)_nCO₂R and CONRR';

R₃′、R₅' and R₆' each is independently selected from the group comprising: hydrogen, alkyl, alkoxy, aryl, aryloxy, alkylaryl, alkylaryloxy, halogen, trihalomethyl, S (O) R, SO₂NRR′、SO₃R、SR、NO₂、NRR′、OH、CN、C(O)R、OC(O)R、NHC(O)R、(CH₂)_nCO₂R and CONRR';

n is 0 to 3;

x is Br, Cl, F or I;

r is H, alkyl or aryl;

r' is H, alkyl or aryl.

In a preferred embodiment of the compounds of formula I, R₃' is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, n-butyl, isopropyl, isobutyl, isopropyl, isobutyl,Tert-butyl, halogen, aryl and OR, wherein R is H, alkyl OR aryl; r₅' is selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, halogen, aryl and OR, wherein R is H, alkyl OR aryl.

A particularly preferred compound of formula I is 3- (2-chloro-4-hydroxybenzylidene) -2-indolinone.

In another embodiment, the present invention provides compounds of formula (II) and pharmaceutically acceptable salts thereof.

Wherein R is₁Is hydrogen or alkyl;

R₂is O or S;

R₃is hydrogen;

R₄、R₅、R₆and R₇Each independently selected from the group comprising: hydrogen, alkyl, alkoxy, aryl, aryloxy, alkylaryl, alkylaryloxy, halogen, trihalomethyl, S (O) R, SO₂NRR′、SO₃R、SR、NO₂、NRR′、OH、CN、C(O)R、OC(O)R、NHC(O)R、(CH₂)_nCO₂R and CONRR';

R₂′、R₃′、R₅' and R₆' each is independently selected from the group comprising: hydrogen, alkyl, alkoxy, aryl, aryloxy, alkylaryl, alkylaryloxy, halogen, trihalomethyl, S (O) R, SO₂NRR′、SO₃R、SR、NO₂、NRR′、OH、CN、C(O)R、OC(O)R、NHC(O)R、(CH₂)_nCO₂R and CONRR';

ra and Rb are each independently selected from H, alkyl and C (O) R, or NRaRb together may be a 3-8 atom heterocyclic ring optionally substituted at one or more positions withGroup substitution: hydrogen, alkyl, alkoxy, aryl, aryloxy, alkylaryl, alkylaryloxy, halogen, trihalomethyl, S (O) R, SO₂NRR′、SO₃R、SR、NO₂、NRR′、OH、CN、C(O)R、OC(O)R、NHC(O)R、(CH₂)_nCO₂R or CONRR';

n is 0 to 3;

x is Br, Cl, F or I;

r is H, alkyl or aryl;

r' is H, alkyl or aryl.

In a preferred embodiment of the compounds of formula II, R₃' is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, halogen, aryl and OR, wherein R is hydrogen, alkyl OR aryl; r₅' is selected from the group consisting of hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, halogen, aryl, and OR, wherein R is H, alkyl, OR aryl.

A particularly preferred compound of the formula II is 3- (4-dimethylaminobenzylidene) -2-indolinone.

In yet another embodiment, the present invention provides compounds of formula (III):

wherein R is₁Is H or alkyl;

R₂is O or S;

R₃is hydrogen;

R₄、R₅、R₆and R₇Each independently selected from the group comprising: hydrogen, alkyl, alkoxy, aryl, aryloxy, alkylaryl, alkylaryloxy, halogen, trihalomethyl, S (O) R, SO₂NRR′、SO₃R、SR、NO₂、NRR′、OH、CN、C(O)R、OC(O)R、NHC(O)R、(CH₂)_nCO₂R and CONRR';

a is a five-membered heteroaromatic ring selected from thiophene, pyrrole, pyrazole, imidazole, 1, 2, 3-triazole, 1, 2, 4-triazole, oxazole, isoxazole, thiazole, isothiazole, 2-sulfonylfuran, 4-alkylfuran, 1, 2, 3-oxadiazole, 1, 2, 4-oxadiazole, 1, 2, 5-oxadiazole, 1, 3, 4-oxadiazole, 1, 2, 3, 4-oxatriazole, 1, 2, 3, 5-oxatriazole, 1, 2, 3-thiadiazole, 1, 2, 4-thiadiazole, 1, 2, 5-thiadiazole, 1, 3, 4-thiadiazole, 1, 2, 3, 5-thiatriazole and tetrazole, which may optionally be substituted in one or more positions with: alkyl, alkoxy, aryl, aryloxy, alkaryl, alkylaryloxy, halogen, trihalomethyl, S (O) R, SO₂NRR′、SO₃R、SR、NO₂、NRR′、OH、CN、C(O)R、OC(O)R、NHC(O)R、(CH₂)_nCO₂R or CONRR';

n is 0 to 3;

x is Br, Cl, F or I;

r is H, alkyl or aryl;

r' is H, alkyl or aryl.

In a preferred embodiment of the invention, the compound of formula III is 3- [ (2, 3-dimethylpyrrol-5-yl) methylene ] -2-indolinone.

In another embodiment, the present invention provides compounds of formula (IV):

wherein R is₁Is H or alkyl;

R₂is O or S;

R₃is hydrogen;

R₄、R₅、R₆and R₇Each independently selected from the group comprising: hydrogen, alkyl, alkoxy, aryl, aryloxy, alkylaryl, alkylaryloxy, halogen, trihalomethyl, S (O) R, SO₂NRR′、SO₃R、SR、NO₂、NRR′、OH、CN、C(O)R、OC(O)R、NHC(O)R、(CH₂)_nCO₂R and CONRR';

R₃′、R₄′、R₅' and R₆' each is independently selected from the group comprising: hydrogen, alkyl, alkoxy, aryl, aryloxy, alkylaryl, alkylaryloxy, halogen, trihalomethyl, S (O) R, SO₂NRR′、SO₃R、SR、NO₂、NRR′、OH、CN、C(O)R、OC(O)R、NHC(O)R、(CH₂)_nCO₂R and CONRR';

n is 0 to 3;

x is Br, Cl, F or I;

r is H, alkyl or aryl;

r' is H, alkyl or aryl.

In a preferred embodiment of the compounds of formula IV, R₃' is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, halogen, aryl and OR, wherein R is H, alkyl OR aryl; r₅' is selected from the group consisting of hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, halogen, aryl and OR, wherein R is H, alkyl OR aryl.

A particularly preferred compound of formula IV is 3- (2-ethoxybenzylidene) -2-indolinone.

In a final embodiment, the present invention provides compounds of formula (V):

wherein R is₁Is H or alkyl;

R₂is O or S;

R₃is hydrogen;

R₄、R₅、R₆and R₇Each independently selected from the group comprising: hydrogen, alkyl, alkoxy, aryl, aryloxy, alkylaryl, alkylaryloxy, halogen, trihalomethyl, S (O) R, SO₂NRR′、SO₃R、SR、NO₂、NRR′、OH、CN、C(O)R、OC(O)R、NHC(O)R、(CH₂)_nCO₂R and CONRR';

R₂′、R₃′、R₅' and R₆' each is independently selected from the group comprising: hydrogen, alkyl, alkoxy, aryl, aryloxy, alkylaryl, alkylaryloxy, halogen, trihalomethyl, S (O) R, SO₂NRR′、SO₃R、SR、NO₂、NRR′、OH、CN、C(O)R、OC(O)R、NHC(O)R、(CH₂)_nCO₂R and CONRR';

n is 0 to 3;

z is Br, Cl, F, I, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl;

r is H, alkyl or aryl;

r' is H, alkyl or aryl.

In a preferred embodiment of the compounds of formula V, R₃' is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, halogen, aryl and OR, wherein R is H, alkyl OR aryl; r₅' is selected from the group consisting of hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, halogen, aryl and OR, wherein R is H, alkyl OR aryl.

A particularly preferred compound of formula V is 3- (4-bromobenzylidene) -2-indolinone.

The formulae mentioned herein may show tautomeric or structural isomeric phenomena. For example, the compounds mentioned herein may adopt cis or trans configuration around the double bond connecting the indolinone 3-substituent to the indolinone ring, or may be a mixture of cis and trans isomers. Since the formulae depicted in the present specification can represent only one possible tautomeric or structural isomeric form, it is to be understood that the present invention encompasses all tautomeric or structural isomeric forms or mixtures thereof which are capable of modulating, inhibiting and/or modulating tyrosine kinase signal transduction or cell proliferation, without limitation to any one tautomeric or structural isomeric form employed in the depicted formulae.

In addition to the compounds described above and their pharmaceutically acceptable salts, the present invention also relates to compounds in solvated as well as unsolvated forms (e.g., hydrated forms) useful for modulating and/or modulating the ability of cells to proliferate.

The compounds described herein may be prepared by any method known to be useful for preparing chemically related compounds. Suitable methods are illustrated in the examples. The necessary starting materials can be obtained by standard procedures of organic chemistry.

The relative activity and potency of the individual compounds as agents affecting receptor tyrosine kinase mediated signal transduction can be determined using established techniques. Preferably, the compounds are subjected to a series of screens to determine the ability of the compounds to modulate, modulate and/or inhibit cell proliferation. These screens include biochemical assays, cell growth assays, and in vivo assays, in the order of performance.

4.4. Indications of

The compounds described herein are useful for treating diseases associated with deregulated tyrosine kinase signal transduction, including cell proliferative disorders, fibrotic disorders and metabolic disorders.

Cell proliferative disorders that may be treated or further studied using the present invention include cancer, vascular proliferative disorders and mesangial cell proliferative disorders.

Angiogenic disorders refer to disorders of angiogenesis and vasculogenesis that often result in abnormal proliferation of blood vessels. The formation and stretching of blood vessels, or angiogenesis and vascularization, play an important role in many physiological processes, such as embryonic development, luteal formation, wound healing, and organ regeneration. They also play a critical role in cancer development. Other examples of angiogenic diseases include arthritis, where capillaries invade the joints and destroy cartilage, and ocular diseases such as diabetic retinopathy, where new capillaries invade the vitreous in the retina, causing bleeding and blindness. Conversely, diseases associated with crimping, narrowing or occlusion of a blood vessel, such as restenosis, are also included.

Fibrotic disease refers to the abnormal formation of extracellular matrix. Examples of fibrotic diseases include cirrhosis of the liver and mesangial cell proliferative disorders. Cirrhosis is characterized by an increase in extracellular matrix components, with the resultant formation of liver scarring. Cirrhosis can cause diseases such as chronic interstitial hepatitis. Increased extracellular matrix producing liver scarring may also be caused by viral infections (e.g., hepatitis). Adipocytes appear to play a major role in cirrhosis. Other fibrotic disorders involved include atherosclerosis (see below).

Mesangial cell proliferative disorder refers to a condition caused by abnormal proliferation of mesangial cells. Mesangial cell proliferative disorders include various human renal diseases such as glomerulonephritis, diabetic nephropathy, malignant nephrosclerosis, thrombotic microangiopathy, transplant rejection and glomerulopathy. PDGF-R has been shown to be involved in the maintenance of mesangial cell proliferation. Floege et al, 1993, International journal of the Kidney (Kidney International)43：47S-54S。

PTKs are associated with this cell proliferative disorder. For example, certain families of RTKs are associated with the development of cancer. Some of these receptors, for example EGFR [ Tuzi et al, 1991, J.England. carcinoma. J.Cancer) 63: 227-; torp et al, 1992, Scandinavia pathomicrobiology and immunology newspaper (APMIS)100：713-719]HER2/neu [ Slamon et al, 1989, journal of Science (Science)244：707-712]And PDGF-R [ Kumabe et al, 1992, Oncogene (Oncogene)7：627-633]Are overexpressed in many tumors and/or continuously activated by autocrine loops. Indeed, these receptors have been shown to be overexpressed in both the most common and the most severe cancers [ Akbasak and Sun-Akbasak et al, 1992, J.neuroscience (J.neurol.sci.)111: 119-133; dickson et al, 1992, Cancer Treatment research (Cancer Treatment Res.)61: 249-273; korc et al, 1992, J.Clin. Invest.)90：1352-1360]And autocrine loops [ Lee and Donoghue, 1992, journal of cell biology (j.cell.biol.)188: 1057, 1070; korc et al, supra; akbasak and Suner-Akbasak et al, supra]. For example, EGFR receptors are associated with squamous cell carcinoma, astrocytoma, glioblastoma, head and neck cancer, lung cancer, and bladder cancer. HER2 is associated with breast, ovarian, intestinal, lung, pancreatic and bladder cancers. PDGF-R is associated with glioblastoma, lung, ovarian, melanoma and prostate cancer. RTK C-met is commonly associated with hepatogenesis and is therefore associated with hepatocellular carcinoma. In addition, C-met is associated with the formation of malignant tumors. More specifically, RTK C-met is associated with colon, thyroid, pancreatic and intestinal cancers, leukemia and lymphoma, among other cancers. In addition, overexpression of C-met gene has been detected in patients with Hodgkin's disease, Burkitt's disease and lymphoma cell lines.

IGF-IR is associated with several types of cancer, in addition to nutrient carriers and type II diabetes. For example, IGF-I has been implicated as a stimulator of autocrine growth of several classes of tumors, such as human breast cancer cells [ Arteaga et al, J.Clin.Invest. ])84：1418-1423]And lung tumor minicells [ Nacauley et al, 1990, Cancer research (Cancer Res.)50：2511-2517]. Furthermore, IGF-I is integrally involved in the nervous systemThe normal growth and differentiation of the system appears to be an autocrine stimulator of human gliomas. Sandberg-Nordqvist et al, 1993, Cancer research (Cancer Res.)53: 2475-2478. The importance of IGF-IR and its ligands in cell proliferation is further supported by the fact that many types of cells in culture (fibroblasts, epithelial cells, smooth muscle cells, T lymphocytes, bone marrow cells, chondrocytes, osteoblasts, bone marrow stem cells) grow on IGF-I stimulation. Goldring and Goldring, 1991 Eukaryotic Gene Expression [ Eukaryotic Gene Expression)1：301-326]. Baserge even proposed in a recently published series of papers that IGF-I-R plays a major role in the transformation mechanism, and thus may be a preferred target for medical intervention in a large class of human malignancies. Baserga, 1995, Cancer research (Cancer Res.)55: 249-252; baserga, 1994, Cell (Cell)79: 927-; copperoa et al, 1994, molecular cell biology (mol. cell. biol.)14：4588-4595。

However, the link between abnormalities in RTKs and disease is not limited to cancer. For example, RTKs are associated with metabolic diseases such as psoriasis, diabetes, wound healing, inflammation and neurodegenerative diseases. For example, EGF-R is required in corneal and skin wound healing. Defects in insulin-R and IGF-1R have been shown in type II diabetes. Plowman et al (1994) in pharmaceutical News and Hope (DN)&P) 7: 334-339 states a more complete relationship between specific RTKs and their therapeutic indications.

Not only receptor-type tyrosine kinases, but also many Cellular Tyrosine Kinases (CTKs), including src, abl, fps, yes, fyn, lyn, lck, blk, hck, fgr, yrk [ Bolen et al, 1992, journal of the Federal Experimental biology Association (FASEB J.)6：3403-3409]Are associated with both proliferative and metabolic signal transduction pathways and are therefore involved in the indications of the present invention. For example, mutant src (v-src) has been shown to be a oncomelancholy protein in chickens (pp 60)^v-stc). In addition, its cells are the same asLine-proto-oncogene pp60^c-srcTransmitting oncogenic signals of many receptors. For example, overexpression of EGF-R or HER2/neu in a tumor results in pp 0^c-srcIs a characteristic of malignant cells, which are absent in normal cells. On the other hand, defects in murine c-src expression exhibit a sclerosing phenotype, suggesting a critical involvement of c-src in osteoclast function and a possible link to related disorders. Similarly, Zap 70 is involved in T-cell signaling.

Furthermore, it is an aspect of the present invention to identify CTK modulating compounds in order to potentiate even potentiate RTK targeted blockers.

Finally, both RTKs and non-receptor type kinases are associated with hyperimmune diseases.

4.5. Pharmaceutical formulations and routes of administration

The compounds described herein may be administered to a human patient as such, or in the form of a pharmaceutical composition in admixture with a suitable carrier or excipient. Methods of formulation and administration of the compounds of the present application can be found in Remington's pharmaceutical sciences, Mack publishing company, easton.

4.5.1. Route of administration

Suitable routes of administration may include, for example, oral, rectal, transmucosal or enteral administration, parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.

Alternatively, the compound may be administered in a local rather than systemic manner, e.g., by direct intratumoral injection of the compound into a solid state, typically in the form of a depot or long-term release formulation.

In addition, the drug may be administered in the form of a targeted drug delivery system, for example, in the form of liposomes coated with antibodies specific for tumors. The liposomes will target and be selectively taken up by the tumor.

4.5.2. Composition/formulation

The pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, for example, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

The pharmaceutical compositions used according to the invention may thus be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Suitable formulations depend on the route of administration chosen.

For injection, the agents of the invention may be formulated in aqueous solution, preferably in a physiologically compatible buffer, such as Hanks 'solution, Ringer' solution or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. These penetrants are generally known in the art.

For oral administration, the compounds are readily formulated by mixing the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained with solid excipients, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are in particular fillers, for example sugars, including lactose, sucrose, mannose or sorbose; cellulose preparations, for example maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents can be added, for example cross-linked polyvinylpyrrolidone, agar, alginic acid or a salt thereof such as sodium alginate.

The cores of the lozenge are provided with a suitable coating. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbomer, polyethylene glycol and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Colorants or pigments may be added to the tablets or dragee coatings to identify or characterize different combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-out capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. Push-fit capsules may contain the active ingredient in admixture with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate, and optionally stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, for example fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may also be added. All formulations for oral administration should be in dosages suitable for oral administration.

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

For administration by inhalation, the compounds for use according to the invention are most suitably delivered in the form of an aerosol spray from a pressurized container or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by equipping a valve to release a metered amount. For capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator, a powder mix may be formulated containing the compound and a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulation aids such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical preparations for parenteral administration comprise aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be formulated as suitable oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils (e.g. sesame oil) or synthetic fatty acid esters (e.g. ethyl oleate or triglycerides), or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, for example sodium carboxymethyl cellulose, sorbitol or dextran. The suspension may optionally also contain suitable stabilizers or agents that increase the solubility of the compounds, so that highly concentrated solutions can be prepared.

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

The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described above, the compounds may also be formulated as depot preparations. Such long acting formulations may be administered by administration (e.g. subcutaneously or intramuscularly) or by intramuscular injection. For example, the compounds may be formulated with suitable polymeric or hydrophobic materials (e.g., emulsions in useful oils) or ion exchange resins, or in the form of hardly soluble derivatives (e.g., hardly soluble salts).

One pharmaceutical carrier for the hydrophobic compounds of the present invention is a cosolvent 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 is a solution of 3% w/v benzyl alcohol, 8% w/v of the non-polar surfactant Tween 80 and 65% w/v polyethylene glycol 300, the other volumes being made up with absolute ethanol. The VPD co-solvent system (VPD: 5W) consisted of diluting VPD with a 5% aqueous solution of dextran at 1: 1. The cosolvent system can fully dissolve hydrophobic compounds, and has low toxicity when being used systemically. Of course, the proportion of the cosolvent system may be varied widely without destroying its solubility and toxicity characteristics. In addition, the bulk of the co-solvent component may vary: for example, other low toxicity non-polar surfactants may be used instead of tween 80; the size of the polyethylene glycol fraction may vary; other biocompatible polymers may be used in place of polyethylene glycol, such as polyvinylpyrrolidone; other sugars or polysaccharides may also be used in place of glucose.

Alternatively, other hydrophobic drug compound delivery systems may be used. Liposomes and emulsions are well known delivery vehicles or carriers for hydrophobic drugs. Certain organic solvents such as dimethyl sulfoxide can also be used, although often at the expense of greater toxicity. In addition, these compounds can be delivered using sustained release systems, such as semipermeable matrices of solid hydrophobic polymers loaded with the therapeutic agent. A wide variety of sustained release materials have been developed and are well known to those skilled in the art. Sustained release capsules can release the compound for several weeks to over 100 days depending on its chemical nature. Depending on the chemical nature and biological stability of the therapeutic agent, ancillary methods may be used to stabilize the protein.

The pharmaceutical compositions may also contain suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starch, cellulose derivatives, gelatin, and polymers such as polyethylene glycol.

Many of the PTK modulating compounds of the present invention may form salts with pharmaceutically compatible counter ions. Pharmaceutically compatible salts can be formed from a wide variety of acids including, but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, and the like. These salts tend to be more soluble in aqueous based solvents or other protic solvents in the corresponding free base form.

4.5.3. Effective dose

Pharmaceutical compositions suitable for use in the present invention include compositions having an effective amount of the active ingredient therein to achieve the intended purpose. More specifically, a therapeutically effective amount refers to an amount of a compound that prevents, alleviates or ameliorates the symptoms of disease or prolongs the survival of the subject being treated. Determination of a therapeutically effective amount is well within the skill of the art and is especially well within the details provided by the present invention.

For any compound used in the methods of the invention, a therapeutically effective dose can be initially estimated from cell culture assays. For example, a dosage can be formulated for a model animal such that a range of circulating concentrations is achieved that will determine the IC from the cell culture₅₀(i.e., the concentration of test compound that achieves half-peak inhibition of PTK activity) is included. This information can be used to more accurately determine a suitable dosage for a human.

Toxicity and therapeutic efficacy of the compounds mentioned herein can be used in standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining LD₅₀(50% of the dose lethal to the population) and ED₅₀(dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed in terms of LD₅₀And ED₅₀The ratio of the two is expressed. Compounds with high therapeutic indices are desirable. The data obtained from these cell culture assays and animal studies can be used to formulate a range of human doses. The amount of these compounds administered is preferably such that ED is included₅₀But within a certain range of circulating concentrations that are minimal or non-toxic. The amount administered may vary within this range depending upon the dosage form and route of administration utilized. The exact formulation, route of administration and dosage can be selected by the individual physician in accordance with the condition of the patient. (see, e.g., Fingl et al, 1975, at The "pharmaceutical Basis of therapy" (The first page of The pharmaceutical Basis of Therapeutics) chapter I).

The amount and interval of administration can be adjusted individually so that the plasma level of the active moiety is sufficient to maintain the kinase modulating effect, or the Minimum Effective Concentration (MEC). MEC will vary for each compound, but can be estimated from in vitro assay data; for example, the concentration required to achieve 50-90% inhibition of the kinase is obtained using the assay methods described herein. The dosage necessary to achieve MEC depends on the individual characteristics and the route of administration. However, plasma concentrations can be determined by HPLC analysis or bioassay.

Dosing intervals may also be determined using MEC values. The compounds should be administered in such a way that the plasma concentration remains above the MEC for 10-90% of the time, preferably 30-90% of the time, most preferably 50-90%.

In the case of topical application or selective absorption, the effective local concentration of the drug may be independent of plasma concentration.

The amount of the composition to be administered will, of course, depend on the subject being treated, the weight of the subject being treated, the severity of the disease, the mode of administration and the judgment of the attending physician.

4.5.4. Package (I)

If desired, the compositions may be contained in a package or dispensing device which may contain the active ingredient in unit or multiple unit dosage form. The pack may be formed, for example, from metal or plastic foil, such as a blister pack. The packaging or dispensing device may be accompanied by instructions for administration. The compositions may also be formulated to contain the compound formulated in a compatible pharmaceutical carrier, in a suitable container, indicated for the treatment of the indicated condition. Suitable conditions marked on the stem may include treatment of tumors, inhibition of angiogenesis, treatment of fibrosis, diabetes, and the like.

5. Example (b): synthesis of compounds

The compounds of the invention can be synthesized using known techniques. The following lists preferred methods for synthesizing the claimed compounds.

General synthetic methods for 5.1.3-substituted-2-indolinone analogs

The following general procedure was used to synthesize the 3-substituted-2-indoline compounds of the invention

5.1.1. Method A

A reaction mixture of the appropriate oxindole (2-indolinone) (1 equivalent), the appropriate aldehyde (1.2 equivalents) and piperidine (0.1 equivalent) in ethanol (1-2ml/1mol oxindole) was stirred at 90 ℃ for 3-5 hours. And cooling, filtering out the precipitate, washing with cold ethanol, and drying to obtain the target compound.

5.1.2. Method B

The appropriate aldehyde is prepared by the Vilsmeier reaction.To a solution of N, N-dimethylformamide (1.2 equiv.) in 1, 2-dichloroethane (2.0ml/1.0mmole of starting material) was added phosphorus oxychloride (1.2 equiv.) dropwise at 0 ℃. The ice bath was removed and the reaction mixture was stirred for an additional 30 minutes. To the above solution the appropriate starting material (1.0 eq.) was added in portions and the reaction mixture was stirred at 50-70 ℃ for 5 h to 2 days. The reaction mixture was poured into ice-cold 1N sodium hydroxide solution (pH 9 after mixing), and the resulting mixture was stirred at room temperature for 1 hour. The organic layer was separated and the aqueous layer was extracted with ethyl acetate. The combined organic layers were washed with brine to pH7, dried over anhydrous sodium sulfate and evaporated. The residue was chromatographed on a column of silica gel, eluting with a solvent mixture of ethyl acetate and hexane to give the title compound.

Synthesizing 3-substituted-2-indolinone analog.A reaction mixture of the appropriate oxindole (2-indolinone) (1 equivalent), the appropriate aldehyde (1.2 equivalents) and piperidine (0.1 equivalent) in ethanol (1-2ml/1mmol oxindole) was stirred at 90 ℃ for 3-5 hours. And (4) cooling, filtering out precipitate, washing with cold ethanol, and drying to obtain the target compound.

5.2.3 Synthesis of benzylidene-2-indolinone

A preferred method for synthesizing 3-benzylidene-2-indolinone is as follows: to a solution of 137.0mg oxindole in 2.0ml methanol was added 123.2. mu.l benzaldehyde and 40. mu.l piperidine. The reaction mixture was refluxed for 3 hours and then cooled in an ice-water bath. The precipitate formed was filtered, washed with cold methanol and dried in an oven at 40 ℃ overnight. This procedure gave about 129.0mg of compound.

5.3.3 Synthesis of 3- [ (pyridin-4-yl) methylene ] -2-indolinone

A preferred method for the synthesis of 3- [ (pyridin-4-yl) methylene ] -2-indolinone is as follows: to a solution of 138.0mg oxindole in 2.0ml methanol was added 117.0. mu.l 4-pyridinecarboxaldehyde and 40. mu.l piperidine. The reaction mixture was refluxed for 3 hours and cooled in an ice-water bath. The precipitate formed was filtered off, washed with cold methanol and dried in an oven at 40 ℃ overnight to yield 134.5mg of the compound.

5.4.3 Synthesis of- [4- (morpholin-4-yl) benzylidene ] -2-indolinone (method B):

4- (morpholin-4-yl) benzaldehyde. To a solution of 15ml of N, N-dimethylformamide in 50ml of 1, 2-dichloroethane at 0 ℃ 10ml of phosphorus oxychloride was added dropwise. The ice bath was removed and the reaction mixture was stirred for an additional 30 minutes. 16.3g of 4-phenylmorpholine were added in portions to the above solution and the reaction mixture was refluxed for 2 days. To the above reaction mixture was added 2.5ml of triethylamine, and the mixture was refluxed for 2 days. The reaction mixture was poured into an ice-cold 1N NaOH solution (pH 9 after mixing), and the resulting mixture was stirred at room temperature for 1 hour. The organic layer was separated and the aqueous layer was extracted with 2X 20ml dichloromethane. The combined organic layers were washed with brine to pH7, dried over anhydrous sodium sulfate and evaporated. Chromatography of the residue on a silica gel column eluting with a solvent mixture of ethyl acetate and hexane gave 12.95g (68%) of the title compound as a white solid.

3- [4- (morpholin-4-yl) benzylidene]-2-indolinone. A reaction mixture of 6.66g of oxindole, 11.50g of 4- (morpholin-4-yl) benzaldehyde and 5ml of piperidine in 50ml of ethanol is stirred at 90 ℃ for 5 hours. After cooling, the precipitate was filtered off, washed with cold ethanol and dried to yield 15.0g (98%) of the title compound as a yellow solid.

5.5.3 Synthesis of- [4- (4-formylpiperazin-1-yl) benzylidene ] -2-indolinone (method B):

4- (4-formylpiperazin-1-yl) benzaldehyde.To a solution of 3.9ml (30mmol) of N, N-dimethylformamide in 20ml of 1, 2-dichloroethane at 0 ℃ 3.0ml (3.9mmol) of phosphorus oxychloride is added dropwise. The ice bath was removed and the reaction mixture was stirred for a further 15 minutes. To the above solution was added 1-phenylpiperazine (16.0g, 10mmol) in portions and the reaction mixture was stirred at 50 ℃ for 1 hour. The reaction mixture was poured into ice-cold 1N NaOH solution and stirred at room temperature for 1 hour. The organic layer was separated and the aqueous layer was extracted with 2X 20ml of ethyl acetate. The combined organic layers were washed with brine to pH7, dried over anhydrous sodium sulfate and evaporated. The residue was separated on a silica gel column, eluting with a mixture of ethyl acetate and hexane to give 9.0g (41%) of the title compound as a pale yellow solid.

3- [4- (4-formylpiperazin-1-yl) benzylidene]-2-indolinone.A reaction mixture of 133.15mg of oxindole, 228.3mg of 4- (piperazin-1-yl) benzaldehyde and 3 drops of piperidine in 2ml of ethanol was stirred at 90 ℃ for 5 hours. After cooling, the precipitate was filtered off, washed with cold ethanol and dried to yield 199.5mg (65%) of the title compound as a yellow solid.

5.6.3- [4- (piperidin-1-yl) benzylidene ] -2-indolinone synthesis (method B).

4- (piperidin-1-yl) benzaldehyde. To a solution of 2.3ml (30mmol) of N, N-dimethylformamide in 10ml of 1, 2-dichloroethane at 0 ℃ was added dropwise 2.8ml (30mmol) of phosphorus oxychloride. The ice bath was removed and the reaction mixture was stirred for 15 minutes. To the above solution was added 1-phenylpiperidine (3.2ml, 20mmol) in portions and the reaction mixture was refluxed overnight. The reaction mixture was poured into ice-cold 2N sodium hydroxide solution and stirred at room temperature for 1 hour. The organic layer was separated and the aqueous layer was washed with 2X 20ml of ethyl acetate. The combined organic layers were washed with brine to pH7, dried over anhydrous sodium sulfate and evaporated. The residue was separated on a silica gel column, eluting with ethyl acetate and hexane to give 1.5g (40%) of the title compound as a white solid.

3- [4- (piperidin-1-yl) benzylideneBase of]-2-indolinone.

A reaction mixture of 134.0mg of oxindole, 226.8g of 4- (piperidin-1-yl) benzaldehyde and 3 drops of piperidine in 2ml of ethanol is stirred at 90 ℃ for 5 hours. After cooling the precipitate was filtered off, washed with cold ethanol and dried to yield 268.5mg (88%) of the title compound as a yellow solid.

5.7.3 Synthesis of- [ 2-chloro-4-methoxybenzylidene ] -2-indolinone

2-chloro-4-methoxybenzaldehyde.A reaction mixture of 1.0g (6.4mmol) of 2-chloro-4-hydroxybenzaldehyde, 4.4g (32mmol) of potassium carbonate and 1.4g (9.6mmol) of methyl iodide in 10ml of N, N-dimethylformamide was stirred at 70 ℃ for 2 hours and poured into ice water. The precipitate was filtered off, washed with water and dried in a vacuum oven at 40 ℃ overnight to yield 750mg (68%) of the title compound as a light pink solid.

3- [ 2-chloro-4-methoxybenzylidene]-2-indolinoneA reaction mixture of 487.9mg (3.7mmol) of oxindole, 750mg (4.3mmol) of 2-chloro-4-methoxybenzaldehyde and 4 drops of piperidine in 5ml of ethanol was heated to 90 ℃ for 2 hours and cooled to room temperature. The yellow precipitate was filtered off, washed with cold ethanol and dried in a vacuum oven at 40 ℃ overnight to yield 680.2mg (62%) of the title compound.

Synthesis of 5.8.3- [ (4-methylthiophen-2-yl) methylene ] -2-indolinone

A reaction mixture of 133.0mg of oxindole, 151.2mg of 4-methylthiophene-2-carbaldehyde and 3 drops of piperidine in 3ml of ethanol was stirred at 90 ℃ for 3 hours. After cooling the precipitate was filtered off, washed with cold ethanol and dried to yield 147.3mg (61%) of the title compound as a yellow solid.

5.9.3 Synthesis of- [ (3-methylpyrrol-2-yl) methylene ] -2-indolinone

A reaction mixture of 133.0mg of oxindole, 130.9mg of 3-methylpyrrole-2-carbaldehyde and 3 drops of piperidine in 2ml of ethanol was stirred at 90 ℃ for 3 hours. After cooling, the precipitate is filtered off, washed with cold ethanol and dried to yield 150.9mg (67%) of the title compound as a yellow solid.

5.10.3 Synthesis of- [ (3, 4-dimethylpyrrol-2-yl) methylene ] -2-indolinone

3- [ (3, 4-dimethylpyrrol-2-yl) methylene]2-indolinone according to the journal of heterocyclic chemistry (J.Heterocyclic Chem.)13: 1145-1147 (1976).

4-Methylpyrrole-3-carboxylic acid ethyl esterA solution of 11.86g (0.1mole) ethyl crotonate and 19.50g (0.1mole) p-toluenesulfonylmethane isocyanide in 500ml of 2: 1 ether/dimethyl sulfoxide was added dropwise to a suspension of 6.8g sodium hydride (60% dispersion in mineral oil, 0.17mole) in ether at room temperature. After the addition was complete, the reaction mixture was stirred for 30 minutes and diluted with 400ml of water. The aqueous layer was extracted with 3X 100ml of diethyl ether. The combined ether extracts were passed through an alumina column eluting with dichloromethane. The organic solvent was evaporated and the residue formed solidified on standing. The solid was washed with hexane and dried in a vacuum oven at 40 ℃ overnight to yield 12.38g (80%) of the title compound.

Preparation of 3, 4-dimethylpyrroleTo a solution of 23g (80mmol) of sodium dihydrobis (2-methoxyethoxy) aluminate, a solution of 5g (34mmol) of ethyl 4-methylpyrrole-3-carboxylate in 50ml of benzene is added dropwise at room temperature under nitrogen. The reaction mixture was stirred for 18 hours. 100ml of water was added to the mixture. The organic layer was separated, washed with brine and dried over anhydrous sodium sulfate. The solvent was removed and the residue was distilled to yield 1.2g (44%) of the title compound.

Preparation of 3, 4-dimethylpyrrole-2-carbaldehydeTo a solution of 0.92ml (12mmole) of N, N-dimethylformamide in 10ml of 1, 2-dichloroethane at 0 ℃ is added dropwise 1.0ml (12mmol) of phosphorus oxychloride. The ice bath was removed and the reaction mixture was stirred for an additional 30 minutes. To the above solution was added 3, 4-dimethylpyrrole (960.0mg, 10mmole) in portions and the reaction mixture was stirred at 50 ℃ for 5 hours. The reaction mixture was poured into an ice-cold 1N NaOH solution (pH after mixing was 9), and the resulting reaction mixture was stirred at room temperature for 1 hour. Separating the organic layerThe aqueous layer was extracted with ethyl acetate. The combined organic layers were washed with brine to pH7, dried over anhydrous sodium sulfate and evaporated. The residue was chromatographed on a silica gel column, eluting with a solvent mixture of ethyl acetate and hexane to give 610mg (50%) of the title compound.

3- [ (3, 4-dimethylpyrrol-2-yl) methylene]-2-indolinoneA reaction mixture of 67.0mg (0.5mmole) of oxindole, 73.0mg (0.6mmole) of 3, 4-dimethylpyrrole-2-carbaldehyde and 2 drops of piperidine in 2ml of ethanol is stirred at 90 ℃ for 3 hours. After cooling, the precipitate was filtered off, washed with cold ethanol and dried to yield 87.7mg (37%) of the title compound as a yellow solid.

Synthesis of 5.11.3- [ (2, 4-dimethyl-3-ethoxycarbonylpyrrol-5-yl) methylene ] -2-indolinone

A reaction mixture of 134.0mg of oxindole, 234.3mg of 4-ethoxycarbonyl-3, 5-dimethylpyrrole-2-carbaldehyde and 3 drops of piperidine in 3ml of ethanol is stirred at 90 ℃ for 3 hours. After cooling, the precipitate was filtered off, washed with cold ethanol and dried to yield 244.6mg (79%) of the title compound as a yellow solid.

5.12.3 Synthesis of- [ (2, 4-dimethylpyrrol-5-yl) methylene ] -2-indolinone

A reaction mixture of 134.0mg of oxindole, 147.8mg of 3, 5-dimethylpyrrole-2-carbaldehyde and 3 drops of piperidine in 2ml of ethanol is stirred at 90 ℃ for 3 hours. After cooling the precipitate was filtered off, washed with cold ethanol and dried to yield 136.7mg (57%) of the title compound as a yellow solid.

5.13.3 Synthesis of- [ (2-methylthiothiophen-5-yl) methylene ] -2-indolinone

A reaction mixture of 134.0mg of oxindole, 189.9mg of 5-methylthiothiophen-2-yl-2-carbaldehyde and 3 drops of piperidine in 2ml of ethanol was stirred at 90 ℃ for 3 hours. After cooling the precipitate was filtered off, washed with cold ethanol and dried to yield 246.6mg (90%) of the title compound as an orange solid.

5.14.3 Synthesis of- [ (2-methylthiophen-5-yl) methylene ] -2-indolinone

A reaction mixture of 134.0mg of oxindole, 151.42mg of 5-methylthiophene-2-carbaldehyde and 3 drops of piperidine in 2ml of ethanol was stirred at 90 ℃ for 3 hours. After cooling, the precipitate was filtered off, washed with cold ethanol and dried to yield 237.8mg (99%) of the title compound as a yellow solid.

5.15.3 Synthesis of- [ (3-methylthiophen-2-yl) methylene ] -2-indolinone

A reaction mixture of 134.0mg of oxindole, 151.4mg of 3-methylthiophene-2-carbaldehyde and 3 drops of piperidine in 2ml of ethanol was stirred at 90 ℃ for 3 hours. After cooling, the precipitate was filtered off, washed with cold ethanol and dried to yield 157.8mg (65%) of the title compound as a yellow solid.

5.16.3 Synthesis of 2-dihydroindolone- (2, 5-dimethoxybenzylidene) -2-dihydroindolone

3- (2, 5-dimethoxybenzylidene) -2-indolinone was synthesized according to method A.

Synthesis of 5.17.3- (2, 3-dimethoxybenzylidene) -2-indolinone

3- (2, 3-dimethoxybenzylidene) -2-indolinone was synthesized according to method A.

5.18.3 (3-bromo-6-methoxybenzylidene) -2-indolinone synthesis

3- (3-bromo-6-methoxybenzylidene) -2-indolinone was synthesized according to method A.

5.19.3 Synthesis of- [4- (4-tert-butylcarbonyl-piperazin-1-yl) benzylidene ] -2-indolinone

3- [4- (4-tert-butylcarbonyl-piperazin-1-yl) benzylidene ] -2-indolinone was synthesized according to procedure B.

5.20.3 Synthesis of- [ (furan-2-yl) methylene ] -2-indolinone

3- [ (furan-2-yl) methylene ] -2-indolinone was synthesized according to procedure A.

Synthesis of 5.21.3- (4-acetamidobenzylidene) -2-indolinone

3- (4-Acylaminobenzylidene) -2-indolinone was synthesized according to method A.

5.22.3 synthesis of 2-chloro-4-hydroxybenzylidene-2-indolinone

3- (2-chloro-4-hydroxybenzylidene) -2-indolinone was synthesized according to procedure A.

5.23.3- (4-bromobenzylidene) -2-indolinone synthesis

3- (4-Bromobenzylidene) -2-indolinone was synthesized according to procedure A.

Synthesis of 5.24.3- (4-acetamidobenzylidene) -2-indolinone

3- (4-Acylaminobenzylidene) -2-indolinone was synthesized according to method A.

5.25.3 (2-methoxybenzylidene) -2-indolinone synthesis

3- (2-methoxybenzylidene) -2-indolinone was synthesized according to method A.

5.26.3 Synthesis of (4-dimethylaminobenzylidene) -1-methyl-2-indolinone

3- (4-Dimethylaminobenzylidene) -1-methyl-2-indolinone was synthesized according to method A.

5.27.3- (4-dimethylamino benzylidene) -2-indolinone synthesis

3- (4-Dimethylaminobenzylidene) -2-indolinone is available from Maybridge chemical company.

Synthesis of 5.28.3- (4-bromobenzylidene) -1-methyl-2-indolinone

3- (4-Bromobenzylidene) -1-methyl-2-indolinone was synthesized according to procedure A.

5.29.5 Synthesis of chloro-3- (4-dimethylaminobenzylidene) -2-indolinone

5-chloro-3- (4-dimethylaminobenzylidene) -2-indolinone was synthesized according to procedure A.

Synthesis of 5.30.3- (4-bromobenzylidene) -5-chloro-2-indolinone

3- (4-Bromobenzylidene) -5-chloro-2-indolinone was synthesized according to procedure A.

5.31.3- (4-diethylaminobenzylidene) -2-indolinone synthesis

3- (4-diethylaminobenzylidene) -2-indolinone was synthesized according to method A.

Synthesis of 5.32.3- (4-di-n-butylaminobenzylidene) -2-indolinone

3- (4-di-n-butylaminobenzylidene) -2-indolinone was synthesized according to method A.

5.33.1 Synthesis of methyl-3- [4- (morpholin-4-yl) benzylidene ] -2-indolinone

1-methyl-3- [4- (morphin-4-yl) benzylidene ] -2-indolinone was synthesized according to procedure B.

5.34.5 Synthesis of chloro-3- (4- (morpholin-4-yl) benzylidene) -2-indolinone

5-chloro-3- (4- (morpholin-4-yl) benzylidene) -2-indolinone was synthesized according to method B.

Synthesis of 5.35.3- (3, 4-dichlorobenzylidene) -2-indolinone

3- (3, 4-dichlorobenzylidene) -2-indolinone was synthesized according to method A.

5.36.3- (2-ethoxybenzylidene) -2-indolinone synthesis

3- (2-ethoxybenzylidene) -2-indolinone was synthesized according to method A.

5.37.3- (4-Fluorobenzylidene) -2-indolinone synthesis

3- (4-Fluorobenzylidene) -2-indolinone was synthesized according to method A.

Synthesis of 5.38.3- [ (thiophen-2-yl) methylene ] -2-indolinone

3- [ (thiophen-2-yl) methylene ] -2-indolinone was synthesized according to method A.

5.39.3 (2-methoxybenzylidene) -2-indolinone synthesis

3- (2-methoxybenzylidene) -2-indolinone was synthesized according to method A.

5.40.3 Synthesis of- [2- [3, 5-bis (trifluoromethyl) phenyl ] furan-5-yl ] methylene ] -2-indolinone

3- [2- [3, 5-bis (trifluoromethyl) phenyl ] furan-5-yl ] methylene ] -2-indolinone was synthesized according to method A.

5.41.2 synthesis of 6-bis (dimethylamino) -3, 5-bis [ (indolin-2-one-3-ylidene) methyl ] benzonitrile

2, 6-bis (dimethylamino) -3, 5-bis [ (indolin-2-one-3-ylidene) methyl ] benzonitrile were synthesized according to method A.

Synthesis of 5.42.3- [ (3- (2-carboxyethyl) -4-methylpyrrol-5-yl) methylene ] -2-indolinone

3- [ (3- (2-carboxyethyl) -4-methylpyrrol-5-yl) methylene ] -2-indolinone was synthesized according to method A.

Synthesis of 5.43.3- [ (3, 4-dibromo-5-methylpyrrol-2-yl) methylene ] -2-indolinone

3- [ (3, 4-dibromo-5-methylpyrrol-2-yl) methylene ] -2-indolinone was synthesized according to method B.

5.44.3 Synthesis of- [ (3, 4-dimethyl-2-formylpyrrol-5-yl) methylene) -2-indolinone

3- [ (3, 4-dimethyl-2-formylpyrrol-5-yl) methylene) -2-indolinone was synthesized according to method A.

Synthesis of 5.45.3- { [4- (2-methoxycarbonylethyl) -3-methylpyrrol-5-yl ] methylene } -2-indolinone

3- { [4- (2-Methoxycarbonylethyl) -3-methylpyrrol-5-yl ] methylene } -2-indolinone was synthesized according to procedure A.

5.46.3 Synthesis of- [ 2-iodofuran-5-yl) methylene ] -2-indolinone

3- [ 2-iodofuran-5-yl) methylene ] -2-indolinone was synthesized according to method A.

5.47.3 Synthesis of- [ (3-ethoxycarbonyl-2-methylfuran-5-yl) methylene ] -2-indolinone

3- [ (3-ethoxycarbonyl-2-methylfuran-5-yl) methylene ] -2-indolinone was synthesized according to method A.

5.48.Synthesis of 3- [ (3-bromothien-2-yl) methylene ] -2-indolinone

3- [ (3-bromothien-2-yl) methylene ] -2-indolinone was synthesized according to method A.

Synthesis of 5.49.3- [ (2-chlorothien-5-yl) methylene ] -2-indolinone

3- [ (2-chlorothien-5-yl) methylene ] -2-indolinone was synthesized according to method A.

5.50.3- [ (2, 3-dimethylfuran-5-yl) methylene ] -2-indolinone synthesis

3- [ (2, 3-dimethylfuran-5-yl) methylene ] -2-indolinone was synthesized according to method A.

Synthesis of 5.51.3- [ (5-nitrothiophen-2-yl) methylene ] -2-indolinone

3- [ (5-Nitrothien-2-yl) methylene ] -2-indolinone was synthesized according to method A.

5.52.3 Synthesis of- [ (2-carbonylthiophen-5-yl) methylene ] -2-indolinone

3- [ (2-Carbonylthiophen-5-yl) methylene ] -2-indolinone was synthesized according to method A.

Synthesis of 5.53.3- [ (2-bromothien-5-yl) methylene ] -2-indolinone

3- [ (2-bromothien-5-yl) methylene ] -2-indolinone was synthesized according to method A.

Synthesis of 5.54.3- [ (4-bromothien-2-yl) methylene ] -2-indolinone

3- [ (4-bromothien-2-yl) methylene ] -2-indolinone was synthesized according to method A.

Synthesis of 5.55.3- [ (2-sulfonylfuran-5-yl) methylene ] -2-indolinone sodium salt

3- [ (2-sulfonylfuran-5-yl) methylene ] -2-indolinone, sodium salt was synthesized according to method A.

Synthesis of 5.56.3- [ (furan-2-yl) methylene ] -2-indolinone

3- [ (furan-2-yl) methylene ] -2-indolinone was synthesized according to procedure A.

5.57.3 Synthesis of- [ (2-methylfuran-5-yl) methylene ] -2-indolinone

3- [ (2-Methylfuran-5-yl) methylene ] -2-indolinone was synthesized according to method A.

Synthesis of 5.58.3- [ (2-ethylfuran-5-yl) methylene ] -2-indolinone

3- [ (2-Ethylfuran-5-yl) methylene ] -2-indolinone was synthesized according to method A.

Synthesis of 5.59.3- [ (2-nitrofuran-5-yl) methylene ] -2-indolinone

3- [ (2-Nitrofuran-5-yl) methylene ] -2-indolinone was synthesized according to method A.

Synthesis of 5.60.3- [ (5-bromofuran-2-yl) methylene ] -2-indolinone

3- [ (5-Bromomofuran-2-yl) methylene ] -2-indolinone was synthesized according to method A.

Synthesis of 5.61.3- [ (2-ethylthiophen-5-yl) methylene ] -2-indolinone

3- [ (2-Ethylthiophen-5-yl) methylene ] -2-indolinone was prepared according to method A.

Synthesis of 5.62.3- [ (4, 5-dimethyl-3-ethylpyrrol-2-yl) methylene ] -2-indolinone

3- [ (4, 5-dimethyl-3-ethylpyrrol-2-yl) methylene ] -2-indolinone was synthesized according to method A.

Synthesis of 5.63.3- [ (5-ethoxycarbonyl-4-ethoxycarbonylethyl-3-ethoxycarbonylmethylpyrrole-2-yl) methylene ] -2-indolinone

3- [ (5-ethoxycarbonyl-4-ethoxycarbonylethyl-3-ethoxycarbonylmethylpyrrole-2-yl) methylene ] -2-indolinone was synthesized according to method A.

5.64.3 Synthesis of- [ (5-carboxy-3-ethyl-4-methylpyrrol-2-yl) methylene ] -2-indolinone

3- [ (5-carboxy-3-ethyl-4-methylpyrrol-2-yl) methylene ] -2-indolinone was synthesized according to method A.

Synthesis of 5.65.3- [ (3, 5-diiodo-4-methylpyrrole-2-yl) methylene ] -2-indolinone

3- [ (3, 5-diiodo-4-methylpyrrol-2-yl) methylene ] -2-indolinone was synthesized according to procedure A.

5.66.3 Synthesis of- [ (5-chloro-3-methoxycarbonyl-4-methoxycarbonylmethylpyrrole-2-yl) methylene ] -2-indolinone

3- [ (5-chloro-3-methoxycarbonyl-4-methoxycarbonylmethylpyrrol-2-yl) methylene ] -2-indolinone was synthesized according to method A.

5.67.3 Synthesis of- [ (3-acetyl-5-ethoxycarbonyl-4-methylpyrrole-2-yl) methylene ] -2-indolinone

3- [ (3-acetyl-5-ethoxycarbonyl-4-methylpyrrol-2-yl) methylene ] -2-indolinone was synthesized according to method A.

Synthesis of 5.68.3- { [1- (3, 5-dichlorophenyl) pyrrol-2-yl ] methylene } -2-indolinone

3- { [1- (3, 5-dichlorophenyl) pyrrol-2-yl ] methylene } -2-indolinone was synthesized according to procedure A.

5.69.3 Synthesis of- [1- (4-chlorophenyl) pyrrol-2-yl) methylene ] -2-indolinone

3- [1- (4-chlorophenyl) pyrrol-2-yl) methylene ] -2-indolinone was synthesized according to method A.

Synthesis of 5.70.3- [ (4-ethoxycarbonyl-3-methyl) pyrrol-2-yl) methylene ] -2-indolinone

3- [ (4-ethoxycarbonyl-3-methyl) pyrrol-2-yl) methylene ] -2-indolinone was synthesized according to method A.

5.71.3 Synthesis of- [ (1-methylpyrrol-2-yl) methylene ] -2-indolinone

3- [ (1-Methylpyrrol-2-yl) methylene ] -2-indolinone was synthesized according to method A.

Synthesis of 5.72.3- [ (5-ethoxycarbonyl-3-ethoxycarbonylethyl-4-ethoxycarbonylmethylpyrrole-2-yl) methylene ] -2-indolinone

3- [ (5-ethoxycarbonyl-3-ethoxycarbonylethyl-4-ethoxycarbonylmethylpyrrole-2-yl) methylene ] -2-indolinone was synthesized according to method A.

Synthesis of 5.73.3- [4- (pyrrolidin-1-yl) benzylidene ] -2-indolinone

3- [4- (pyrrolidin-1-yl) benzylidene ] -2-indolinone was synthesized according to procedure A.

Synthesis of 5.74.3- [ (5-methylimidazol-2-yl) methylene ] -2-indolinone

3- [ (5-Methylimidazol-2-yl) methylene ] -2-indolinone was synthesized according to method A.

Synthesis of 5.75.3- [ (5-methylthiazol-2-yl) methylene ] -2-indolinone

3- [ (5-Methylthiazol-2-yl) methylene ] -2-indolinone was synthesized according to method A.

Synthesis of 5.76.3- [ (3-methylpyrazol-5-yl) methylene ] -2-indolinone

3- [ (3-methylpyrazol-5-yl) methylene ] -2-indolinone was synthesized according to method A.

Synthesis of 5.77.3- [ (imidazol-4-yl) methylene ] -2-indolinone

3- [ (imidazol-4-yl) methylene ] -2-indolinone was synthesized according to procedure A.

Synthesis of 5.78.3- [ (4-chloropyrazol-3-yl) methylene ] -2-indolinone

3- [ (4-Chloropyrazol-3-yl) methylene ] -2-indolinone was synthesized according to method A.

Synthesis of 5.79.3- [ (4-bromo-1- (4-chlorobenzyl) pyrazol-5-yl) methylene ] -2-indolinone

3- [ (4-bromo-1- (4-chlorobenzyl) pyrazol-5-yl) methylene ] -2-indolinone was synthesized according to procedure A.

5.80.3 Synthesis of- [ (4-chloro-1-methylpyrazol-3-yl) methylene ] -2-indolinone

3- [ (4-chloro-1-methylpyrazol-3-yl) methylene ] -2-indolinone was synthesized according to method A.

5.81.3 Synthesis of- [ (4-ethyl-3, 5-dimethylpyrrol-2-yl) methylene ] -2-indolinone

3- [ (4-Ethyl-3, 5-dimethylpyrrol-2-yl) methylene ] -2-indolinone was synthesized according to method B.

5.82.3 Synthesis of- [ (5-ethylpyrrol-2-yl) methylene ] -2-dihydropyrrolone

3- [ (5-ethylpyrazol-2-yl) methylene ] -2-dihydropyrrol one was synthesized according to method B.

5.83.3 Synthesis of- [3, 5-dimethyl-4- (propen-2-yl) pyrrol-2-yl) methylene ] -2-indolinone

3- [3, 5-dimethyl-4- (propen-2-yl) pyrrol-2-yl) methylene ] -2-indolinone was synthesised according to method B.

5.84.5 Synthesis of 6-dimethoxy-3- [2, 3-dimethoxybenzylidene ] -2-indolinone

5, 6-dimethoxy-3- [2, 3-dimethoxybenzylidene ] -2-indolinone was synthesized according to method A.

Synthesis of 5.85.3- [2, 4, 6-trimethoxybenzylidene ] -2-indolinone

3- [2, 4, 6-trimethoxybenzylidene ] -2-indolinone was synthesized according to method A.

5.86.5 Synthesis of chloro-3- [ (pyrrol-2-yl) methylene ] -2-indolinone

5-chloro-3- [ (pyrrol-2-yl) methylene ] -2-indolinone was synthesized according to procedure A.

5.87.5 Synthesis of chloro-3- [ (3-methylpyrrol-2-yl) methylene ] -2-indolinone

5-chloro-3- [ (3-methylpyrrol-2-yl) methylene ] -2-indolinone was synthesized according to method A.

5.88.3 Synthesis of 3- (4-isopropylidenebenzylidene) -2-indolinone

3- (4-Isopropylbenzylidene) -2-indolinone was obtained from Maybridge chemical company.

5.89.5 Synthesis of chloro-3- [ (3, 5-dimethylpyrrol-2-yl) methylene ] -2-indolinone

5-chloro-3- [ (3, 5-dimethylpyrrol-2-yl) methylene ] -2-indolinone was synthesized according to method A.

Synthesis of 5.90.3- [ (pyrrol-2-yl) methylene ] -2-indolinone

3- [ (pyrrol-2-yl) methylene ] -2-indolinone is available from Maybridge chemical company.

5.91.5 Synthesis of chloro-3- [ (indol-3-yl) methylene ] -2-indolinone

5-chloro-3- [ (indol-3-yl) methylene ] -2-indolinone was synthesized according to method A.

5.92.5 Synthesis of chloro-3- [ (thiophen-2-yl) methylene ] -2-indolinone

5-chloro-3- [ (thiophen-2-yl) methylene ] -2-indolinone was synthesized according to method A.

5.93.5 Synthesis of chloro-3- [ (3-methylthiophen-2-yl) methylene ] -2-indolinone

5-chloro-3- [ (3-methylthiophen-2-yl) methylene ] -2-indolinone was synthesized according to method A.

5.94.5 Synthesis of chloro-3- [ (5-methylthiophen-2-yl) methylene ] -2-indolinone

5-chloro-3- [ (5-methylthiophen-2-yl) methylene ] -2-indolinone was synthesized according to method A.

5.95.5 Synthesis of chloro-3- [ (5-ethylthiophen-2-yl) methylene ] -2-indolinone

5-chloro-3- [ (5-ethylthiophen-2-yl) methylene ] -2-indolinone was synthesized according to method A.

5.96.5-chloro-3- [ (5-methylthiothiophen-2-yl) methylene ] -2-indolinone synthesis

5-chloro-3- [ (5-methylthiothiophen-2-yl) methylene ] -2-indolinone was synthesized according to method A.

5.97.5 Synthesis of chloro-3- [ (imidazol-2-yl) methylene ] -2-indolinone

5-chloro-3- [ (imidazol-2-yl) methylene ] -2-indolinone was synthesized according to procedure A.

5.98.3 Synthesis of- [2, 4-dimethoxy-6-methylbenzylidene ] -2-indolinone

3- [2, 4-dimethoxy-6-methylbenzylidene ] -2-indolinone was synthesized according to procedure A.

5.99.5 Synthesis of Nitro-3- [ (pyrrol-2-yl) methylene ] -2-indolinone

5-Nitro-3- [ (pyrrol-2-yl) methylene ] -2-indolinone was synthesized according to procedure A.

5.100.3 Synthesis of- [ (3-methylpyrrol-2-yl) methylene ] -5-nitro-2-indolinone

3- [ (3-Methylpyrrol-2-yl) methylene ] -5-nitro-2-indolinone was synthesized according to method A.

5.101.3 Synthesis of- [ (3, 5-dimethylpyrrol-2-yl) methylene ] -5-nitro-2-indolinone

3- [ (3, 5-dimethylpyrrol-2-yl) methylene ] -5-nitro-2-indolinone was synthesized according to method A.

Synthesis of 5.102.3- [ (indol-3-yl) methylene ] -5-nitro-2-indolinone

3- [ (indol-3-yl) methylene ] -5-nitro-2-indolinone was synthesized according to method A.

5.103.5 Synthesis of Nitro-3- [ (thiophen-2-yl) methylene ] -2-indolinone

5-Nitro-3- [ (thiophen-2-yl) methylene ] -2-indolinone was synthesized according to method A.

5.104.3 Synthesis of- [ (3-methylthiophen-2-yl) methylene ] -5-nitro-2-indolinone

3- [ (3-methylthiophen-2-yl) methylene ] -5-nitro-2-indolinone was synthesized according to method A.

5.105.3 Synthesis of- [ (5-methylthiophen-2-yl) methylene ] -5-nitro-2-indolinone

3- [ (5-Methylthiophen-2-yl) methylene ] -5-nitro-2-indolinone was synthesized according to method A.

5.106.3 Synthesis of- [ (5-ethylthiophen-2-yl) methylene ] -5-nitro-2-indolinone

3- [ (5-Ethylthiophen-2-yl) methylene ] -5-nitro-2-indolinone was synthesized according to method A.

5.107.3 Synthesis of- [ (5-methylthiothiophen-2-yl) methylene ] -5-nitro-2-indolinone

3- [ (5-Methylthiothiophen-2-yl) methylene ] -5-nitro-2-indolinone was synthesized according to method A.

Synthesis of 5.108.3- [ (imidazol-2-yl) methylene ] -5-nitro-2-indolinone

3- [ (imidazol-2-yl) methylene ] -5-nitro-2-indolinone was synthesized according to procedure A.

Synthesis of 5.109.3- [ (oxazol-2-yl) methylene ] -2-indolinone

3- [ (oxazol-2-yl) methylene ] -2-indolinone was synthesized according to procedure A.

Synthesis of 5.110.3- [ (oxazol-4-yl) methylene ] -2-indolinone

3- [ (oxazol-4-yl) methylene ] -2-indolinone was synthesized according to procedure A.

Synthesis of 5.111.3- [ (oxazol-5-yl) methylene ] -2-indolinone

3- [ (oxazol-5-yl) methylene ] -2-indolinone was synthesized according to procedure A.

Synthesis of 5.112.3- [ (thiazol-2-yl) methylene ] -2-indolinone

3- [ (thiazol-2-yl) methylene ] -2-indolinone was synthesized according to procedure A.

Synthesis of 5.113.3- [ (thiazol-4-yl) methylene ] -2-indolinone

3- [ (thiazol-4-yl) methylene ] -2-indolinone was synthesized according to procedure A.

Synthesis of 5.114.3- [ (thiazol-5-yl) methylene ] -2-indolinone

3- [ (thiazol-5-yl) methylene ] -2-indolinone was synthesized according to procedure A.

Synthesis of 5.115.3- [ (imidazol-2-yl) methylene ] -2-indolinone

3- [ (imidazol-2-yl) methylene ] -2-indolinone was synthesized according to procedure A.

Synthesis of 5.116.3- [ (pyrazol-3-yl) methylene ] -2-indolinone

3- [ (pyrazol-3-yl) methylene ] -2-indolinone was synthesized according to procedure A.

Synthesis of 5.117.3- [ (pyrazol-4-yl) methylene ] -2-indolinone

3- [ (pyrazol-4-yl) methylene ] -2-indolinone was synthesized according to procedure A.

Synthesis of 5.118.3- [ (isoxazol-3-yl) methylene ] -2-indolinone

3- [ (isoxazol-3-yl) methylene ] -2-indolinone was synthesized according to procedure A.

Synthesis of 5.119.3- [ (isoxazol-4-yl) methylene ] -2-indolinone

3- [ (isoxazol-4-yl) methylene ] -2-indolinone was synthesized according to procedure A.

Synthesis of 5.120.3- [ (isoxazol-5-yl) methylene ] -2-indolinone

3- [ (isoxazol-5-yl) methylene ] -2-indolinone was synthesized according to procedure A.

Synthesis of 5.121.3- [ (isothiazol-3-yl) methylene ] -2-indolinone

3- [ (isothiazol-3-yl) methylene ] -2-indolinone was synthesized according to procedure A.

Synthesis of 5.122.3- [ (isothiazol-4-yl) methylene ] -2-indolinone

3- [ (isothiazol-4-yl) methylene ] -2-indolinone was synthesized according to procedure A.

Synthesis of 5.123.3- [ (isothiazol-5-yl) methylene ] -2-indolinone

3- [ (isothiazol-5-yl) methylene ] -2-indolinone was synthesized according to procedure A.

Synthesis of 5.124.3- [ (1, 2, 3-triazol-4-yl) methylene ] -2-indolinone

3- [ (1, 2, 3-triazol-4-yl) methylene ] -2-indolinone was synthesized according to method A.

Synthesis of 5.125.3- [ (1, 3, 4-thiadiazol-2-yl) methylene ] -2-indolinone

3- [ (1, 3, 4-thiadiazol-2-yl) methylene ] -2-indolinone was synthesized according to method A.

Synthesis of 5.126.3- [ (5-phenyl-1, 2, 4-oxadiazol-3-yl) methylene ] -2-indolinone

3- [ (5-phenyl-1, 2, 4-oxadiazol-3-yl) methylene ] -2-indolinone was synthesized according to method A.

Synthesis of 5.127.3- [ (3-phenyl-1, 2, 4-oxadiazol-5-yl) methylene ] -2-indolinone

3- [ (3-phenyl-1, 2, 4-oxadiazol-5-yl) methylene ] -2-indolinone was synthesized according to method A.

Synthesis of 5.128.3- [ (3-phenyl-1, 2, 5-oxadiazol-4-yl) methylene ] -2-indolinone

3- [ (3-phenyl-1, 2, 5-oxadiazol-4-yl) methylene ] -2-indolinone was synthesized according to method A.

6. Example (b): in vitro RTK assay

The following in vitro assays may be used to determine the activity and level of action of various compounds of the invention on one or more RTKs. Similar assays can be designed in the same manner for any tyrosine kinase using methods well known in the art.

6.1. Enzyme-linked immunosorbent assay (ELISA)

Enzyme-linked immunosorbent assays (ELISAs) can be used to test and determine the presence of tyrosine kinase activity. ELISA can be carried out according to known protocols, see for example Voller et al, 1980, "Enzyme-Linked immunosorbent assay" (Enzyme-Linked immunosorbent assay), in the second edition of the Manual of Clinical Immunology ", edited by Rose and Friedman, p 359 & 371, published by the American society for microbiology, Washington D.C..

The disclosed protocols may be adapted to assay the activity of a particular RTK. For example, preferred protocols for performing ELISA for specific RTKs are provided below. Modifications to these protocols to determine the activity of the compounds for other members of the RTK family as well as non-receptor tyrosine kinases will be within the skill of the art.

6.1.1.FLK-1ELISA

ELISA assays were performed to determine kinase activity of the FLK-1 receptor, more specifically to determine inhibition or activation of protein tyrosine kinase activity at the FLK-1 receptor. Specifically, the following assays were performed to determine kinase activity of the FLK-1 receptor in FLK-1/NIH 3T3 cells.

Materials and methods.

Material. The following reagents and manufacturers were used:

corning 96-well ELISA plate (catalog No.25805-96 of Corning Co.);

cappel goat anti-rabbit immunoglobulin G (catalog No. 55641);

c. phosphate buffered saline PBS (Gibco product number 450-;

TBSW buffer (50mM Tris (pH 7.2), 150mM NaCl and 0.1% Tween 20);

e. ethanolamine stock solution (10% ethanolamine (pH 7.0), stored at 4 ℃);

HNTG buffer (20mM HEPES (N-2-hydroxyethylpiperazine-N' -2-ethanesulfonic acid) buffer (pH 7.5), 150mM NaCl, 0.2% Triton X-100 and 10% glycerol);

EDTA (0.5M (pH 7.0) as 100X stock solution);

h. sodium orthovanadate (0.5M, 100X stock solution);

i. sodium pyrophosphate (0.2M, 100X stock solution);

NUNC 96-well V-bottom polypropylene plates (Applied Scientific, product number AS-72092);

nih3T 3C7#3 cells (FLK-1 expressing cells);

DMEM containing 1 Xhigh glucose L-glutamine (product No. 11965-050).

Fbs (fetal bovine serum), Gibco (product No. 16000-;

n.L-Glutamine, Gibco (product No. 25030-;

VFGF, PeproTech company (product No. 100-20) (stored as 1. mu.g/100. mu.l stock in Milli-Q grade purified water at-20 ℃);

affinity purified anti-FLK-1 antiserum, Enzymology laboratory, Sugen;

UB 40 monoclonal antibodies specific for phosphotyrosine, Enzymology laboratory, Sugen (see Fendley et al, 1990, Cancer Research)50：1550-1558)；

EIA grade goat anti-mouse TgG- (POD) (BioRod, product No. 172-1011);

s.2, 2-Aza-bis (3-ethylbenzothiazoline-6-sulfonic Acid) (ABTS) solution [100mM citric acid (anhydrous), 250mM Na₂PO₄(pH 4.0), 0.5mg/ml ABTS (Sigma Co., product No. A-1888)]The solution should be stored at 4 ℃ protected from light until ready for use.

t.H₂O₂(30% solution) (Fischer company, product No. H325);

u.ABTS/H₂O₂(15ml ABTS solution, 2. mu. l H₂O₂) Prepared 5 minutes before use at room temperaturePlacing the materials downwards;

v.0.2m hydrochloric acid stock;

dimethyl sulfoxide (100%) (Sigma Co., product number D-8418); and

y. Trypsin-EDTA (Gibco BRL, product No. 25200-049).

And (4) scheme.The following protocol was used for the experiments.

1. Corning 96-well ELISA plates were coated with 1.0. mu.g of Na at 0.1M per well₂CO₃(pH 9.6) Cappel anti-Rabbit immunoglobulin G (IgG) antibody. The final volume of each well was made to be 150. mu.l. The coated plates were left overnight at 4 ℃. The plates can be stored for up to two weeks when stored at 4 ℃.

2. In a suitable Petri dish at 37 ℃ and 5% CO₂Cells were grown in growth medium (DMEM, supplemented with 2.0mM L-glutamine, 10% FBS) until confluent into disks.

3. Cells were harvested by trypsinization and plated on Corning 25850 polystyrene 96-well round bottom cell culture plates with 25,000 cells per 200 μ l growth medium.

4. At 37 ℃ and 5% CO₂Cells were grown for at least one day.

5. Cells were washed once with D-PBS.

6. Mu.l of starvation medium (DMEM, 2.0mM L-glutamine, 0.1% FBS) was added to each well. At 37 ℃ 5% CO₂The mixture was incubated overnight.

7. The compounds/extracts were diluted 1: 20 in polypropylene 96 well plates with starvation medium. Dilute dimethyl sulfoxide 1: 20 for control wells.

8. Starvation medium was removed from 96 well cell culture plates and 162 μ l of fresh starvation medium was added to each well.

9. To each well 18. mu.l of 1: 20 diluted compound/extract was addedDilutions (from step 7), 1: 20 dilutions of dimethyl sulfoxide (with or without VEGF) were added to the control wells, and the final dilution was 1: 200 after stimulation of the cells. The final concentration of dimethyl sulfoxide was 0.5%. At 37 ℃ and 5% CO₂Plates were incubated for two hours.

10. The plate was inverted to remove the liquid to remove unbound antibody from the ELISA plate. Washed 3 times with TBSW + 0.5% ethanolamine (pH 7.0). The plate was tapped on a paper towel to remove excess liquid and air bubbles.

11. The plate was blocked with 150. mu.l of TBSW + 0.5% ethanolamine (pH 7.0) per well. The plates were incubated for 30 minutes while shaking on a microtiter plate shaker.

12. The plate was washed 3 times as described in step 10.

13. Add 0.5. mu.g affinity purified anti-FLU-1 polyclonal rabbit antiserum per well. The final volume was brought to 150. mu.l per well with TBSW at pH7.0 + 0.5% ethanolamine. Plates were incubated for 30 minutes with shaking.

14. To the cells, 180. mu.l of starvation medium was added at 37 ℃ and 5% CO₂Next, cells were challenged with 20. mu.l of 10.0mM sodium orthovanadate and 500ng/ml VEGF per well (resulting in final concentrations of 1.0mM sodium orthovanadate and 50ng/ml VEGF per well) for 8 minutes.

After 15.8 minutes, the medium was removed from the cells and washed once with 200. mu.l/well of PBS.

16. Cells were lysed in 150. mu.l/well of HNTG for 5 minutes at room temperature with shaking. The HNTG preparation contains sodium orthovanadate, sodium pyrophosphate and EDTA.

17. The ELISA plate was washed three times as described in step 10.

18. Cell lysates were transferred from cell culture plates to ELISA plates and incubated for 2 hours with shaking. To transfer the cell lysate, the pipette is used to aspirate around while scraping the wells.

19. The plate was washed 3 times as described in step 10.

20. ELISA plates were incubated with UB 40 (0.02. mu.g per well) in TBSW + 0.5% ethanolamine. The final volume was 150. mu.l per well. Incubate for 30 minutes with shaking.

21. The plate was washed 3 times as described in step 10.

22. ELISA plates were incubated with EIA (enzyme immunoassay) grade goat anti-mouse immunoglobulin G conjugated horseradish peroxidase diluted 1: 10000 in TBSW + 0.5% ethanolamine (pH 7.0). The final volume was 150. mu.l per well. Incubate for 30 minutes with shaking.

23. The plate was washed as described in step 10.

24. Add 100. mu.l ABTS/H to wells₂O₂And (3) solution. Incubate for 10 minutes with shaking.

25. 100. mu.l of 0.2M HCl was added to give a final concentration of 0.1M HCl, so that the color reaction was stopped. Shaken at room temperature for 1 minute. The air bubbles were removed with a slow air flow and the ELISA plates were detected in an ELISA plate reader at 410 nm.

6.1.2.HER-2ELISA

Test 1: EGF receptor-HER 2 chimeric receptor assay in whole cells.HER2 kinase activity in whole EGFR-NIH 3T3 cells was determined as follows:

materials and reagents. The following materials and reagents were used for the experiments:

EGF: stock concentration 16.5 ILM; EGF 201, TOYOBO company, japan.

05-101(UBI) (a monoclonal antibody recognizing the extracellular domain of EGFR).

c. Anti-phosphotyrosine antibodies (anti-ptyr) (polyclonal) (see Fendley et al, supra).

d. Detecting an antibody: goat anti-rabbit IgG horseradish peroxidase conjugate, TAGO, Burlingame, CA.

Tbst buffer:

Tris-HCl，pH 7.2 50mM

NaCl 150mM

Triton X-100 0.1

HNTG 5X stock solution:

HEPES 0.1M

NaCl 0.75M

50 percent of glycerin

Triton X-100 1.0％

ABTS stock solution:

citric acid 100mM

Na₂HPO₄ 250mM

Hcl, 0.5pM conc

ABTS^* 0.5mg/ml

^*(2, 2' -azino (3-ethylbenzothiazolinesulfonic acid.) the solution was stored at 4 ℃ in the dark until use.

h. Storing reagents:

EDTA 100mM，pH 7.0

Na₃VO₄，0.5M

Na₄(P₂O₇)0.2M

and (5) carrying out the following steps. The following scheme is adopted:

A. precoated ELISA plate

1. ELISA plates (Corning, 96-well, product #25805-96) were coated with 05-101 antibody in PBS, 0.5g per well, at a final volume of 1O 0. mu.l per well, and stored overnight at 4 ℃. The coated panels remained good for up to 10 days when stored at 4 ℃.

2. On the day of application, the coating buffer was removed and replaced with 100 μ l of blocking buffer (5% Carnation continuous delipidated milk powder in PBS). The plates were incubated for 30 minutes at room temperature (about 23-25 ℃) with shaking. Immediately before use, the blocking buffer was removed and the plate was washed 4 times with TBST buffer.

B. Seeding cells

1. For this assay, a NIH3T3 cell line overexpressing a chimeric receptor containing the EGFR extracellular domain and the extracellular HER2 kinase domain can be used.

2. Petri dishes with 80-90% confluence were selected for the experiments. Cells were trypsinized and stopped by the addition of 10% fetal bovine serum. Cells were suspended in DMEM medium (10% CS DMEM medium) and centrifuged once at 1500 rpm for 5 minutes at room temperature.

3. The cells were resuspended in inoculation medium (DMEM, 0.5% bovine serum) and the cells were counted using trypan blue. The viability is over 90 percent. Cells were seeded in 96-well microtiter plates in DMEM medium (0.5% bovine serum) at a density of 10000 cells per well, 100. mu.l per well. At 37 ℃ and 5% CO₂The inoculated cells were incubated for about 40 hours.

C. Test procedure

1. The seeded cells were examined for contamination by an inverted microscope. Drug stocks (10mg/ml in dimethyl sulfoxide) were diluted 1: 10 in DMEM medium and then transferred 5. mu.l to TBST wells to give a final dilution of 1: 200 of drug with a final dimethyl sulfoxide (DMSO) concentration of 1%. Control wells were DMSO alone. At 37 ℃ and 5% CO₂The mixture was incubated for 2 hours.

2. Preparing an EGF ligand: the EGF stock solution was diluted with DMEM so that the final concentration reached 100nM when 10. mu.l of diluted EGF solution (1: 12 dilution) was transferred.

3. Enough fresh HNTG was prepared at 100. mu.l per well^*And put on ice.

HNTG^*(10ml)

HNTG stock solution 2.0ml

Milli-Q water 7.3ml

EDTA，100mM，pH 7.0 0.5ml

Na₃VO₄，0.5M 0.1ml

Na₄(P₂O₇)，0.2M 0.1ml

4. After 120 min incubation with drug, the formulated SGF ligand was added to the cells at 10. mu.l per well and a final concentration of 100 nM. Control wells were DMEM alone. Incubate for 5 minutes at room temperature with shaking.

5. Drug, EGF and DMEM were removed. Cells were washed 2 times with PBS. Mixing HNTG^*Cells were transferred to 100. mu.l/well and placed on ice for 5 minutes. At the same time, the blocking buffer was removed from the other ELISA plates and washed with TBST as described above.

6. Firmly fixing pipette tips to micropipettes, scraping cells from plates, by repeatedly aspirating and discharging HNTG^*The lysis buffer homogenizes the cell material. The lysates were transferred to coated, blocked and washed ELISA plates. Incubate at room temperature for 1 hour with shaking.

7. Lysates were removed and washed 4 times with TBST. The freshly diluted anti-ptyr antibodies were transferred to ELISA plates at 100 μ l per well. Incubate for 30 minutes at room temperature with shaking in the presence of anti-ptyr antiserum (diluted 1: 3000 in TBST).

8. Anti-ptyr antibodies were removed and washed 4 times with TBST. Freshly diluted TAGO anti-rabbit IgG antibody was transferred to ELISA plates at 100. mu.l per well. Incubate at room temperature for 30 minutes with shaking (anti-rabbit IgG antibody: 1: 3000 dilution in TBST).

9. Remove TAGO detection antibody and wash with TBST 4 times. Transfer freshly prepared ABTS/H₂O₂The solution was applied to ELISA plates at 100. mu.l per well. Incubate at room temperature for 20 minutes with shaking (ABTS/H)₂O₂Solution: 1.0. mu.l of 30% H₂O₂In 10ml ABTS stock).

10. 50 μ l of 5N H was added₂SO₄(optional) the reaction was stopped and the optical density (o.d.) was measured at 410 nm.

11. The maximum phosphotyrosine signal was determined by subtracting the negative control value from the positive control value. The percent inhibition of phosphotyrosine content was then calculated for wells containing the extract after subtracting the negative control value.

Test 2: HER-2-BT 474ELISA. A second assay can be performed to determine whole cell HER2 activity. Such an assay may be performed as follows:

materials and reagents. The following materials and reagents were used:

bt-474(ATCC HBT 20), a human breast tumor cell line expressing high levels of HER2 kinase.

b. Growth medium containing RPMI + 10% FBS + GMS-G (Gibco supplementaries) + Glutamine for use at 37 ℃ and 5% CO₂BT-474 was grown in the incubator of (1).

c. A monoclonal anti-HER 2 antibody.

d.D-PBS：

KH₂PO₄ 0.20g/l 10(Gibco，310-4190AJ)

K₂HPO₄ 2.16g/l

KCl 0.20g/l

NaCl 8.00g/l(pH 7.2)

e. Blocking buffer: TBST + 5% milk (Carnation instant skim milk powder).

Tbst buffer:

Tris-HCl 50mM

NaCl 150mM(pH 7.2，HCl 10N)

Triton X-100 0.1％

in which (10X) TES stock was prepared first and Triton X-100 was added during dilution.

Hntg buffer (5 ×):

HEPES 0.1M

NaCl 750mM(pH 7.2，1N HCl)

50 percent of glycerin

Triton X-100 1.0％

Preparing stock solution (5X), and storing at 4 deg.C

EDTA-HCl: 0.5M, pH7.0 (10N HCl), 500X stock solution.

i.Na₃VO₄: 0.5M 100 Xstock solution, stored in portions at-80 ℃.

j.Na₄(P₂O₇): 0.2M, 100 Xstock solution.

k. Polyclonal antiserum is directed against phosphotyrosine.

Goat anti-rabbit IgG, horseradish Peroxidase (POD) conjugate (detection antibody), Tago (product No. 4520; lot No. 1802): tago Inc., Burlingame, Calif.

Abts solution:

citric acid 100mM

Na₂HPO₄ 250mM(pH 4.0，1N HCl)

ABTS 0.5mg/ml

Wherein ABTS is 2, 2' -azinobis bis (3-ethylbenzothiazolinesulfonic acid). For this assay, the ABTS solution should be stored at 4 ℃ protected from light. The solution should be discarded when it turns green.

n. hydrogen peroxide: 30% solution, and storing at 4 ℃ in dark.

Step (ii) of. All following steps were performed at room temperature and under sterile conditions unless otherwise indicated. All ELISA plates were washed 3 times with distilled water and once with TBST.

A. Cell seeding

1. BT 474 cells were grown in tissue culture dishes (Corning 25020-100) to 80-90% confluent plates and collected with trypsin-EDTA (0.25%, GIBCO).

2. The cells were resuspended in fresh medium and transferred to a 96-well tissue culture plate (Corning, 25806-96), approximately 25000 and 50000 cells per well (100. mu.l/well). At 37 ℃ and 5% CO₂Cells were incubated overnight.

B. ELISA plate coating and blocking

1. ELISA plates (Corning 25805-96) were coated with anti-HER 2 antibody (in PBS), 150. mu.l per well, containing 0.5. mu.g antibody. Sealed with parafilm at 4 ℃ overnight. The antibody coated plates were stored at 4 ℃ for up to 2 weeks.

2. On the day of use, the coating solution was removed, replaced with 200. mu.l of blocking buffer, the ELISA plates were shaken, the blocking buffer was then removed, and the lysates were added immediately after washing the plates.

C. Test procedure

1. Drugs were buffered with TBST under serum-free conditions. The old medium (90. mu.l/well) was replaced with serum-free RPMI before addition of the drug.

2. The drug stock solution (in 100% dimethyl sulfoxide) was diluted 1: 10 with RPMI and transferred to cells at 10. mu.l/well to give a final drug DMSO concentration of 1%. At 37 ℃ and 5% CO₂Cells were incubated.

3. Fresh cell lysis buffer (HNTG) was prepared^*)

5×HNTG 2ml

EDTA 0.2ml

Na₃VO₄ 0.1ml

Na₄P₂O₇ 0.1ml

H₂O 7.3ml

4. After 2 hours of drug preincubation, all solution in the plate was removed and HNTG was added^*Cells (100. mu.l/well) were transferred and shaken for 10 minutes.

5. Cells on the plate were scraped using a 12-channel pipette and lysates were homogenized using repeated aspiration and discharge. All lysates were transferred to ELISA plates and shaken for 1 hour.

6. The lysate was removed, the plates washed, 100. mu.l of anti-ptyr (diluted 1: 5000 with TBST) was added to each well and shaken for 30 minutes.

7. Remove the anti-pTyr, wash the plate, add goat anti-rabbit IgG conjugated antibody (diluted 1: 5000 with TBST), 100. mu.l per well and shake for 30 min.

8. Remove anti-rabbit IgG antibody, wash the plate, add 100. mu.l fresh ABTS/H to each well₂O₂(1.2μl H₂O₂10ml ABTS) to start the color reaction, which usually takes 20 minutes.

9. Optical density was measured using Dynatec MR5000 at 410 nM.

6.1.3.PDGF-R ELISA

Unless otherwise indicated, all cell culture media, glutamine and fetal bovine serum were purchased from Gibco life technologies (Grand Island, NY). All cells were incubated at 37 ℃ and 5-10% CO₂All cell lines were routinely subcultured 2 times per week in a humid atmosphere containing 90-95% air, mycoplasma negative by the Mycotect method (Gibco).

For the ELISA assay, cells (U1242, from Joseph Schlessinger, NYU) were grown to 80-90% confluency in growth medium (minimal essential medium containing 10% FBS, NEAA, 1mM NaPyr, and 2mM GLLN) and seeded in 0.5% serum in 96-well tissue culture plates at 25000 cells per well at 30000 cells. After overnight incubation in media containing 0.5% serum, cells were changed to serum-free media and tested with test compounds at 37 ℃ and 5% CO₂The incubator of (1) for 2 hours. Cells were then stimulated with ligand for 5-10 min, followed by HNTG (20mM Hepes, 150mM NaCl, 10% glycerol, 5mM EDTA, 5mM Na)₃VO₄0.2% Triton X-100 and 2mM NaPyr). Cell lysates (0.5 mg per well in PBS) were transferred to ELISA plates previously coated with receptor-specific antibodies and blocked with 5% milk/TBST (50mM Tris-HCl, pH 7.2; 150mM NaCl; 0.1% Triton X-100) for 30 min at room temperature. The lysate was incubated at room temperature with shaking for 1 hour. Plates were washed 4 times with TBST and then incubated with polyclonal anti-phosphotyrosine antibody for 30 min at room temperature. The plates were washed 4 times with TBST to remove excess anti-phosphotyrosine antibody. Goat anti-rabbit IgG antibody was added to the ELISA plate for 30 minutes at room temperature, followed by 4 washes with TBST. ABTS (100mM citric acid, 250mM Na) was added to ELISA plates₂HPO₄And 0.5mg/ml of 2, 2' -azinobis (3-ethylbenzothiazoline-6-sulfonic acid)) and H₂O₂(1.2ml 30％H₂O₂10ml ABTS) to start the development. The absorption at 410nm was recorded for about 15-30 minutes at 630nm as the reference wavelength after addition of ABTS.

6.1.4.IGF-I ELISA

The following protocol can be used to determine phosphotyrosine concentrations at the IGF-I receptor, which is representative of IGF-I receptor tyrosine kinase activity.

Materials and reagents. The following materials and reagents were used:

a. the cell line used in this assay was 3T3/IGF-1R, a cell line that overexpresses the IGF-1 receptor.

NIH3T 3/IGF-1R at 37 ℃ and 5% CO₂The incubator of (1). Growth medium was DMEM + 10% FBS (heat inactivated) +2mM L-glutamine.

c. An anti-IGF-1R antibody named 17-69 was used. Antibodies were purified by enzymology lab, Sugen.

d.D-PBS：

KH₂PO₄ 0.20g/l

K₂HPO₄ 2.16g/l

KCl 0.20g/l

NaCl 8.00g/l(pH 7.2)

e. Blocking buffer: TBST plus 5% milk (Carnation instant skim milk powder).

Tbst buffer:

Tris-HCl 50mM

NaCl 150mM(pH 7.2/HCl 10N)

Triton X-100 0.1％

TBS stock (10X) was prepared and Triton X-100 was added to the buffer during dilution.

HNTG buffer:

HEPES 20mM

NaCl 150mM(pH 7.2/HCl 1N)

10 percent of glycerin

Triton X-100 0.2％

Stock solutions (5X) were prepared and stored at 4 ℃.

EDTA/HCl: 0.5M, pH7.0 (NaOH), 100 Xstock solution.

i.Na₃VO₄: 0.5M 100 Xstock solution, stored in portions at-80 ℃.

j.Na₄P₂O₇: 0.2M 100 Xstock solution.

k. Insulin-like growth factor-1, available from Promega corporation (product number G5111).

Polyclonal antiserum anti-phosphotyrosine: rabbit serum was prepared from Enzymology Lab., SUGEN.

Goat anti-rabbit IgG, peroxidase conjugate (detection antibody), Tago corporation (product No. 4520, lot No. 1802): tago Inc., Burlingame, Calif.

Abts (2, 2' -azino bis (3-ethylbenzothiazolinesulfonic acid)) solution:

citric acid 100mM

Na₂HPO₄ 250mM(pH 4.0/1N HCl)

ABTS 0.5mg/ml

The ABTS solution should be stored at 4 ℃ protected from light. The solution should be discarded when it turns green.

o. hydrogen peroxide: 30% solution, and storing at 4 ℃ in dark.

All the following steps were carried out at room temperature unless otherwise specified. All ELISA plates were washed 3 times with tap water followed by one wash with TBST. The plate was patted dry with paper towel.

A. Cell seeding：

1. Cells were grown in tissue culture dishes (Corning 25020-100) to 80-90% confluent plates and harvested with trypsin-EDTA (0.25%, 0.5ml/D-100, GIBCO).

2. The cells were resuspended in fresh DMEM + 10% FBS +2mM L-glutamine and transferred to a 96-well tissue culture plate (Corning, 25806-96) at 20000 cells per well (100. mu.l/well). Incubation for 1 day, then the medium was changed to serum-free medium (90. mu.l/well) at 37 ℃ and 5% CO₂The mixture was incubated overnight.

B. Coating and blocking of ELISA plates

1. ELISA plates (Corning 25805-96) were coated with anti-IGF-1R antibody (in PBS) for at least 2 hours, 100. mu.l per well, containing 0.5. mu.g of antibody.

2. The coating solution was removed, replaced with 100. mu.l of blocking buffer and shaken for 30 minutes. The blocking solution was removed and the lysate was added immediately after washing the plates.

C. Test procedure：

1. The drugs were tested under serum-free conditions.

2. Drug stocks (in 100% DMSO) were diluted 1: 10 with DMEM in 96-well polypropylene plates and 10. mu.l of this solution was transferred to cells per well, giving a final dilution of 1: 100 and a final DMSO concentration of 1.0%. At 37 ℃ and 5% CO₂Cells were incubated for 2 hours.

3. Fresh cell lysis buffer (HNTG) was prepared^*)

HNTG 2ml

EDTA 0.1ml

Na₃VO₄ 0.1ml

Na₄P₂O₇ 0.1ml

H₂O 7.3ml

4. After 2 hours drug incubation, cells were added 10 μ l per well of 200nM IGF-1 ligand/PBS (final concentration 20nM) at 37 deg.C and 5% CO₂The mixture was incubated for 10 minutes.

5. Removing the culture medium, adding 100. mu.l/well of HNTG^*And shaken for 10 minutes. Whether the cells were sufficiently lysed was observed under a microscope.

6. Cells in the plate were scraped using a 12-channel pipette and the lysate was homogenized by repeated aspiration and discharge. All lysates were transferred to ELISA plates coated with antibody and shaken for 1 hour.

7. Lysates were removed, plates washed, and 100. mu.l anti-pTyr (diluted with TBST, 1: 3000) was added to each well and shaken for 30 min.

8. Remove anti-pTyr, wash plate, add 100. mu.l Tago per well (diluted with TBST, 1: 3000) and shake for 30 min.

9. Remove the test antibody, wash the plate, and add fresh 100. mu.l ABTS/H to each well of the plate₂O₂(1.2μl H₂O₂: 10ml ABTS), the color reaction was started.

10. Optical density was measured on a Dynatec MR5000 connected to an ingrres database management system.

EGF receptor ELISA

EGF receptor kinase activity in whole cells (EGFR-NIH 3T3 assay) was determined as follows:

materials and reagents. The following materials and reagents were used:

EGF ligand: stock solution concentration 16.5 μ M, EGF 201, TOYOB corporation, japan.

05-101(UBI) (a monoclonal antibody recognizing the extracellular domain of EGFR).

c. Anti-phosphotyrosine antibody (anti-ptyr) (polyclonal).

d. Detecting an antibody: goat anti-rabbit IgG horseradish peroxidase conjugate, TAGO, Burlingame, CA.

Tbst buffer:

Tris-HCl，pH 7 50mM

NaCl 150mM

Triton X-100 0.1

HNTG 5X stock solution:

HEPES 0.1M

NaCl 0.75M

glycerol 50

Triton Y-100 1.0％

ABTS stock solution:

citric acid 100mM

Na₂HPO₄ 250mM

HCl, concentrated 4.0pH

ABTS^* 0.5mg/ml

The solution was stored at 4 ℃ protected from light until use.

h. Storing reagents:

EDTA，100mM，pH 7.0

Na₃VO₄，0.5M

Na₄P₂O₇，0.2M

step (ii) ofThe following protocol was employed.

A. Precoated ELISA plate

1. ELISA plates (Corning, 96-well, product No. #25805-96) were coated with 05-101 antibody (in PBS), 0.5. mu.g per well, 150. mu.l final volume per well, and stored overnight at 4 ℃. The coated plates were stored at 4 ℃ for up to 10 days.

2. The day of use the coating buffer was removed and replaced with blocking buffer (5% Carnation instant skim milk powder in PBS). The plates were incubated at room temperature (about 23-25 ℃) for 30 minutes with shaking. The blocking buffer was removed just prior to use and the plates were washed 4 times with TBST buffer.

B. Seeding cells

NIH3T 3/C7 Cell line (Honegger et al, Cell J)51: 199, 209, 1987) can be used for this test.

2. Plates with 80-90% confluency in the pellet were selected for this experiment. Cells were trypsinized and quenched by addition of 10% CS DMEM medium. Cells were suspended in DMEM medium (10% CS DMEM medium) and centrifuged once at 1000 rpm for 5 minutes at room temperature.

3. The cells were resuspended in inoculation medium (DMEM, 0.5% bovine serum) and the cells were counted using trypan blue. The viability is higher than 90 percent. Cells were seeded in 96-well microtiter plates in DMEM medium (0.5% bovine serum) at a density of 10000 cells per well, 100. mu.l per well. Seeded cells at 37 ℃ and 5% CO₂The incubation was continued for about 40 hours.

C. Test procedure

1. The seeded cells were examined for contamination by an inverted microscope. Drug stock (10mg/ml in DMSO) was diluted 1: 10 in DMEM medium and 5. mu.l was transferred to assay wells to give a final drug dilution of 1: 200 and final DMSO concentrationThe degree is 1%. Control wells were DMSO alone. At 5% CO₂And incubated at 37 ℃ for 1 hour.

2. Preparing an EGF ligand: EGF stock was diluted in DMEM so that the final concentration was 25nM when 10. mu.l of dilute EGF solution (1: 12 dilution) was transferred.

3. 10ml fresh HNTG sufficient to make 100. mu.l per well was prepared^*Wherein HNTG^*Comprises the following components: HNTG stock solution (2.0 ml); milli-Q H₂O(7.3ml)；EDTA、100mM、pH 7.0(0.5ml)；Na₃VO₄0.5M (0.1ml) and Na₄P₂O₇，0.2M(0.1ml)。

4. Placed on ice.

5. After 2 hours incubation with drug, the formulated EGF ligand was added to the cells at 10. mu.l per well to a final concentration of 25 nM. Control wells were supplemented with DMEM alone. Incubate and shake for 5 minutes at room temperature.

6. Drug, EGF and DMEM were removed. Cells were washed 2 times with PBS. Mixing HNTG^*Transfer to cells, 100. mu.l per well. Left on ice for 5 minutes. At the same time, the blocking buffer was removed from the other ELISA plate and washed with TBST as described above.

7. Scraping cells from the plate with a pipette tip firmly affixed to the micropipette by repeatedly aspirating and discharging HNTG^*The lysis buffer homogenizes the cell material. The lysates were transferred to coated, blocked and washed ELISA plates. Incubate at room temperature for 1 hour with shaking.

8. The lysate was removed and washed 4 times with TBST. Freshly diluted anti-ptyr antibodies were transferred to ELISA plates at 100 μ l per well. Incubate for 30 minutes at room temperature in the presence of anti-ptyr antiserum (diluted 1: 3000 in TBST).

9. Anti-ptyr antibodies were removed and washed 4 times with TBST. Freshly diluted TAGO 30 anti-rabbit IgG antibody was transferred to ELISA plates at 100. mu.l per well. Incubate at room temperature for 30 minutes with shaking (anti-rabbit IgG antibody: 1: 3000 dilution in TBST).

10. Remove the detection antibody and wash 4 times with TBST. ABTS/H as-prepared₂O₂The solution was transferred to an ELISA plate at 100. mu.l per well. Incubate at room temperature for 20 minutes. ABTS/H₂O₂Solution: 1.2. mu.l of 30% H₂O₂In 10ml of ABTS stock.

11.50 μ l of 5N H was added₂SO₄The reaction was (optionally) stopped and the optical density at 410nm was measured.

12. The maximum phosphotyrosine signal was determined by subtracting the negative control value from the positive control value. After subtracting the negative control value, the percentage inhibition of phosphotyrosine content in the wells containing the extract was calculated.

6.1.6. Cellular insulin receptor ELISA

The following protocol was used to determine whether the compounds of the present invention have insulin receptor tyrosine kinase activity.

Materials and reagentsThe following materials and reagents were used to determine phosphotyrosine concentration at the insulin receptor (indicative of insulin receptor tyrosine kinase activity):

1. the preferred cell line is the NIH3T3 cell line (ATCC No.1658) which overexpresses the insulin receptor (H25 cells).

H25 cells at 37 ℃ and 5% CO₂Growing in an incubator. Growth medium was DMEM + 10% FBC (heat inactivated) +2ml L-glutamine;

3. for the coating of ELISA plates, a monoclonal anti-IR antibody called BBE was used. The antibody was purified by Enzymology Lab, SUGEN;

D-PBS comprising:

KH₂PO₄ 0.20g/l(Gibco，310-4190AJ)

K₂HPO₄ 2.16g/l

KCl 0.20g/l

NaCl 8.00g/l(pH 7.2)

5. blocking buffer: TBST plus 5% milk (Carnation instant skim milk powder);

TBST buffer comprising:

Tris-HCl 50mM

NaCl 150mM pH 7.2(HCl，1N)

Triton X-100 0.1％

note: stock solutions (10X) of TBS were prepared and Triton X-100 was added to the buffer at the time of dilution.

An HNTG buffer comprising:

HEPES 20mM

NaCl 150mM pH 7.2(HCl，1N)

10 percent of glycerin

Triton X-100 0.2％

Note: preparing a stock solution (5X), and storing at 4 ℃;

EDTA & HCl: 0.5M, pH7.0 (NaOH), 100X stock solution;

9.Na₃VO₄: 0.5M 100X stock solution, and storing at-80 deg.C;

10.Na₄P₂O₇: 0.2M 100 Xstock solution;

insulin by Gibco BRL (product No. 18125039);

12. polyclonal antiserum anti-phosphotyrosine: rabbit serum from Enzymology Lab., SUGEN;

13. detecting an antibody: preferably goat anti-rabbit IgG, peroxidase conjugate, Tago (product No. 4520, lot No. 1802): tago corporation, Burlingame, CA;

an ABTS solution comprising:

citric acid 100mM

Na₂HPO₄ 250mM pH 4.0(1N HCl)

ABTS 0.5mg/ml

Wherein ABTS is 2, 2' -azino-bis (3-ethylbenzothiazoline sulfonic acid), and is preserved at 4 deg.C in dark. Discarded when turning green.

15. Hydrogen peroxide: 30% solution, and storing at 40 ℃ in dark.

Step (ii) ofAll following steps, unless specifically noted, were performed at room temperature. All ELISA plate washing procedures were 3 washes with tap water followed by 1 wash with TBST. All panels were patted dry with a paper towel prior to use.

A. Cell seeding

1. Cells were grown in tissue culture dishes (10cm, Corning 25020-100) to 80-90% confluent plates and harvested with trypsin-EDTA (0.25%, 0.5ml/D-100, Gibco);

2. the cells were resuspended in fresh DMEM + 10% FBS +2ml L-glutamine and transferred to a 96-well tissue culture plate (Corning, 25806-96) at 20,000 cells per well (100. mu.l/well). The cells were then incubated for 1 day. After incubation, the original medium was replaced with 0.01% serum medium (90. mu.l/well) and the cells were incubated at 37 ℃ and 5% CO₂The mixture was incubated overnight.

B. Coating and blocking of ELISA plates：

1. ELISA plates (Corning 25805-96) were coated with anti-IR antibody (in PBS) for at least 2 hours, 100. mu.l per well, containing 0.5. mu.g of antibody.

2. The coating solution was removed and replaced with 100. mu.l of blocking buffer and shaken for 30 minutes. Blocking buffer was removed and the lysate was added immediately after washing the plates.

C. Test procedure

1. The drugs were tested under serum-free conditions.

2. Drug stock solutions (in 100% DMSO) were diluted 1: 10 with DMEM in 96-well polypropylene plates and transferred (10. mu.l/well) to cells to give a final dilution of 1: 100 of drug and a final DMSO concentration of 1.0%. At 37 ℃ and 5% CO₂Cells were incubated for 2 hours.

3. Fresh cell lysis buffer (HNTG) was prepared^*)

HNTG(5X) 2ml

EDTA 0.1ml

Na₃VO₄ 0.1ml

Na₄P₂O₇ 0.1ml

H₂O 7.3ml

HNTG^* 10ml

4. After 2 h drug incubation, 1 μ M insulin/PBS was transferred to cells at 10 μ l per well (final concentration 100nM) at 37 ℃ and 5% CO₂The mixture was incubated for 10 minutes.

5. Remove the medium and add 100. mu.l HNTG per well^*And shaken for 10 minutes. Whether the cells were sufficiently lysed was observed with a microscope.

6. Cells were scraped from the plate using a 12-channel pipette and the lysate was homogenized by repeated aspiration and discharge. All lysates were transferred to antibody-coated ELISA plates and shaken for 1 hour.

7. Lysates were removed, plates washed, 100. mu.l anti-pTyr (diluted 1: 3000 with TBST) was added per well and shaken for 30 min.

8. The anti-pTyr was removed and the plate washed, 100. mu.l Tago (diluted 1: 3000 in TBST) was added to each well and shaken for 30 minutes.

9. Remove detection antibody, wash plate, add 100. mu.l fresh ABTS/H per well₂O₂(1.2μlH₂O₂: 10ml ABTS), the color reaction was started.

10. Optical density was measured on a Dynatec MR5000 connected to an ingrres database management system. All following steps should follow the ingrres specification.

Test results of ELISA

The results of the experiments performed with the above scheme for the various compounds of the invention are shown in table 1:

TABLE 1

Results of ELISA test

Compounds (examples)	PDGFRIC50(μM)	FLK-1IC50(μM)	EGFRIC50(μM)	HER2 kinase IC50 (mu M)	IGF-1RIC50(μM)
						27	19.4	0.8
4313	14.5	18.8	11	16.9	8.0
						18	12	0.39
16	87.4	4.2
						17		11.8
20		28.8
						21		9
22		2.2
						24		8.5
26		22.6
						28				22.5
29	7.9	11.2
						32		20.9
4	33.1	2.1
						33		21.6		39.4
34		4.1
						5	5.8	1.6		90.2
36		4		51.5
						37		9.6
38		4.7
						39		14.8	36.7
41		6.4
						8		2.9		89.8
42		0.4
						43		1.8
9	17	0.24
						44		23.8
10		0.17
						45	53.7	1.1
11		0.07
						12	10.8	0.11
48		15.4
						13		2.3
50		4.6
						14		2.4
53		51.4
						15		4.5		70.6
55		8.6
						57		73.4
58		41.2

Compounds (examples)	PDGFRIC50(μM)	FLK-1IC50(μM)	EGFRIC50(μM)	HER2 kinase IC50 (mu M)	IGF-1RIC50(μM)
						59		22.8
6		4.5		92.6
						61		3.4	44
62	65.5	0.14
						64		36.2
70		0.18
						71		20.3
73	86	1.6
						74	55.9	2.7
76		8.7
						77	14.2	1.5
78		7.4
						81		0.15
7		5.3	39.6	30.4

6.2. Cell growth assay

The following assays may be performed to determine the effect of the claimed compounds on cell growth due to interaction with one or more RTKs. Unless otherwise indicated, the following assays are generally useful for determining the activity of a compound on any particular RTK. For the assays mentioned below for a particular RTK, one skilled in the art would be able to modify the disclosed protocol for use in determining the activity of a second RTK.

6.2.1. Soft agar test

The soft agar assay can be used to determine the effect of a substance on cell growth. Unless otherwise indicated, the soft agar assay was performed as follows:

materials and reagents. The following materials and reagents were used:

a. a water bath set at 39 ℃ and another water bath set at 37 ℃.

2X test medium consists of 2X Dulbecco's Modified Eagle Medium (DMEM) (Gibco product No. CA 400-4AN03) supplemented with the following: 20% Fetal Bovine Serum (FBS), 2mM sodium pyruvate, 4mM glutamine; and 20mM HEPES, non-essential amino acids (diluted 1: 50 from 100X stock).

c. 1X assay medium made from 1X DMEM supplemented with 10% FBS, 1mM sodium pyruvate, 2mM glutamine, 10mM HEPES, non-essential amino acids (diluted 1: 100 from 100X stock).

d. 1.6% seaopaque agar in autoclaved bottles.

e. Sterile 35mm Corning plates (FMC bio products company, product # 50102).

f. Sterile 5ml glass pipettes (individually encapsulated).

g. Sterile 15ml and 50ml conical centrifuge tubes.

h. Pipettes and sterile tips.

i. Sterile microcentrifuge tubes.

j. Cells in T75 flasks: SKOV-3(ATCC HTB 77).

k.0.25% trypsin solution (Gibco # 25200-015).

Step (ii) of. The soft agar test was performed using the following procedure:

A. step of manufacturing base layer

1. All media were warmed in a 37 ℃ water bath.

2. Preparation of 1X assay medium + 0.8% agar: the molten agar (cooled to 39 ℃) was diluted 1: 2 (vol: vol) with 2X test medium.

3. All agar-containing media were warmed in a 39 ℃ water bath when not in use.

4.1 ml of 1X test medium + 0.08% agar was spread onto the plate and the plate was slowly rotated to form a uniform base layer. Air bubbles should be avoided.

5. The substrate was freeze-cured (about 20 minutes). The substrate may be frozen overnight in a refrigerator.

B. Step of collecting cells

1. One flask was removed from each cell line in the incubator, the medium was aspirated, washed once with PBS, aspirated off, and 3ml trypsin solution was added.

2. After all cells were detached from the flask, 3ml of 1 × test medium was added to inhibit trypsin activity. The cells were pipetted up and down and the suspension was then transferred to a 15ml tube.

3. Cell concentration was determined by coulter counter and viability was determined by trypan blue exclusion.

4. The appropriate volume required to inoculate 3300 viable cells per plate was removed and diluted to 1.5ml with 1 × test medium.

C. Procedure for preparing 0.4% agar Upper layer

1. TBST compound was added at 2-fold the desired final assay concentration; +1.5ml cell suspension in 1X assay medium 10% FBS; +1.5ml of 1X test Medium + 0.8% agar^*: total 3.0ml 1X assay medium 10% FBS + 0.4% agar, 3300 viable cells per ml, with or without TBST compound.

^*(obtained by diluting 2X medium with 1: 2 of 1.6% agar 30 in the above basic step)

2.1 ml of the test mixture was coated on a 1ml substrate. The same samples were coated from the above 3ml volume.

3. Petri dishes were incubated at 100% humidity, 10% CO₂The culture vessel of (3) is incubated for 2-3 weeks.

Colonies of 4.60 microns or greater are scored positive.

6.2.2. Sulforhodamine B (SRB) growth assay

The SRB assay can be used to determine the effect of a substrate on cell growth. The tests were carried out as follows:

test 1: 3T3/E/H + TGF-a (T) cell growth SRB assay

Material：

96-hole flat-bottom sterile plate

96-hole round bottom sterile plate

Sterile 25ml or 100ml container

Pipette, multichannel pipette device

Sterile pipette tip

Sterile 15ml and 50ml test tubes

Reagent：

0.4% SRB in 1% acetic acid

10mM Tris base

10％TCA

1% acetic acid

Sterile DMSO (Sigma Co.)

DMSO solution of the Compound (stock solution of 100mM or less)

25% Trypsin-EDTA in cell dissociation solution (Sigma)

Cell lines and growth media：

3T3/E/H + TGF-a (T) (NIH 3T3 clone 7 cells, expressing EGFR/HER2 chimera and TGF-a, tumor-derived autocrine loop cells)

2% bovine serum/DMEM +2mM glutamine

Scheme(s)：

Day 0: cell inoculation:

this part of the test was performed in a laminar flow hood.

1. Cells were trypsinized as usual. 100. mu.l of the cell suspension was transferred to 10ml of isotonic solution. Cells were counted in a coulter counter.

2. Cells were diluted to 60000 cells/ml in growth medium. Add 100 μ l of cells per well in a 96 well flat bottom plate to 6000 cells/well.

3. Half of the plate (4 rows) was used for each compound, 4 replicate wells for each compound concentration, one set of 4 wells for the medium control and 4 wells for the DMSO control.

4. The plate was gently shaken to allow the cells to attach evenly.

5. At 10% CO₂And incubating the plate in an incubator at 37 ℃.

Day 1: adding a compound

This part of the test was performed in a laminar flow hood.

1. In a 96-well round bottom plate, 125 μ l growth medium was added to rows 3 to 11. This plate was used for titration of compounds, 4 rows for each compound.

2. To a total of 2ml of growth medium, 8. mu.l of compound was added in a sterile 15ml tube at a dilution of 1: 250 to produce a 2X solution of the highest compound concentration. At this dilution, the concentration of DMSO was 0.4% for 2X solution on the cells and 0.2% for 1X solution. The starting concentration of the compound is typically 100. mu.M, but this concentration may vary with the solubility of the compound.

3. The 2X starting compound solution was transferred to 4 identical wells in row 12 of a 96-well round bottom plate. Transfer 125. mu.l from line 12 to line 11, from line 11 to line 10, from right to left along the plate, thus performing a 1: 2 serial dilution. 100 μ l of compound dilutions were transferred to 100 μ l of medium on cells in corresponding wells of a 96-well flat-bottom plate. The total volume in each well should be 200. mu.l.

4. For the vehicle control, a 0.4% DMSO2X solution in growth medium was prepared. Transfer 100 μ l DMSO solution into appropriate cell wells. The final concentration of DMSO was 0.2%.

5. For media control wells, 100. mu.l/well of growth media was added to the appropriate cell wells.

6. The plates were returned to the incubator for 4 days.

Day 5: color development test

This part of the test was performed on a bench top.

1. The medium is aspirated or poured off. 200 μ l of cold 10% TCA was added to each well to immobilize the cells. The plates were incubated at 4 ℃ for at least 60 minutes.

2. The TCA was decanted and each well was rinsed 5 times with water. The plate was dried bottom up on paper towels.

3. Cells were stained with 100. mu.l/well of 0.4% SRB for 10 min.

4. The SRB was decanted and each well was rinsed 5 times with 1% acetic acid. The plate was completely dried bottom up on a paper towel.

5. The dye was solubilized on a shaker with 100. mu.l/well of 100mM Tris base.

6. The plates were measured at 570nm with 630nm as reference on a Dynatech ELISA plate reader.

Test 2: 3T3/EGF-R + TGF-a (T) cell growth SRB assay

Materials and reagents were the same as in test 1.

Cell lines and growth media：

3T3/EGF-R + TGF-a (T) (NIH 3T3 clone 7 cells expressing EGF-R and TGF-a, tumor-derived autocrine loop cells)

2% bovine serum/DMEM +2mM glutamine

Scheme(s)：

Day 0: cell seeding

This part of the test was performed in a laminar flow hood.

1. Cells were trypsinized according to the usual procedure. 100 μ l of the cell suspension was transferred to 10ml of isotonic solution. Cells were counted in a coulter counter.

2. Cells were diluted in growth medium to 60000 cells per ml. Mu.l cells were added to each well in a 96 well flat bottom plate to reach 6000 cells/well.

3. Half of the plate (4 rows) was used for each compound, 4 replicate wells were used for each compound concentration, one set of 4 wells was used for the medium control and 4 wells were used for the DMSO control.

4. The plate was gently shaken to allow the cells to attach evenly.

5. At 37 ℃ and 10% CO₂Incubate the plate in an incubator.

Day 1: compound addition: same as in test 1.

Day 5: color development test: same as in test 1.

Test 3: 3T 3/PDGF-. beta.R/PDGF-BB (T) cell growth SRB assay

Cell lines and growth media：

3T3/PDGF- β R/PDGF-BB (T) (NIH 3T3 clone 7 cells expressing PDGF β -receptor and PDGF-BB, obtained from tumors excised from athymic mice)

2% bovine serum/DMEM +2mM glutamine

Scheme(s)：

Day 0: cell seeding

This part of the test was performed in a laminar flow hood.

1. Cells were trypsinized according to the usual procedure. Transfer 200. mu.l of cell suspension to 10ml of isotonic solution. Cells were counted in a coulter counter.

2. Cells were diluted to 60000 cells/ml in growth medium. Transfer 100. mu.l of cells to each well of a 96-well flat-bottom plate to reach 6000 cells per well.

3. Half plate (4 rows) was used for each compound, 4 replicate wells for each compound concentration, one set of 4 wells for the medium control and 4 wells for the DMSO control.

4. The plate was gently shaken to allow the cells to adhere uniformly to the plate.

5. At 37 ℃ and 10% CO₂Incubate the plate in an incubator.

Day 1: compound addition: same as in test 1.

Day 5: color development test: same as in test 1.

Test 4: human Smooth Muscle Cell (SMC) growth SRB assay

Materials and reagents were the same as in test 1.

Cell lines and growth media：

Human aortic smooth muscle cells (Clonetics)

Clonetics bullet kit: smooth muscle minimal medium (SmBM) which is modified MCDB 131 containing fetal bovine serum (5%), hFGF (2ng/ml), hEGF (0.1ng/ml), insulin (5.0. mu.g/ml), gentamicin (50. mu.g/ml) and amphotericin B (50 ng/ml).

Scheme(s)：

Day 0: cell seeding

This part of the test was performed in a laminar flow hood.

1. Cells were trypsinized according to the usual procedure. Transfer 200. mu.l of cell suspension to 10ml of isotonic solution. Cells were counted in a coulter counter.

2. Cells were diluted to 20000 cells/ml in growth medium. Transfer 100. mu.l of cells into each well of a 96-well flat-bottom plate to reach 2000 cells per well.

3. Half plate (4 rows) was used for each compound, 4 replicate wells for each compound concentration, one set of 4 wells for the medium control and 4 wells for the DMSO control.

4. Gently shake the plate to allow the cells to adhere uniformly to the plate.

5. At 37 ℃ and 10% CO₂Incubate the plate in an incubator.

Day 1: compound addition: same as in test 1.

Day 5: color development test: same as in test 1.

6.2.3.3T3 cell growth assay

Test 1: PDGF-induced BrdU incorporation assay

Materials and reagents：

(1) PDGF: human PDGF B/B; 1276-.

(2) BrdU labeling reagent: 10mM in PBS (pH 7.4), product No.1647229, Boehringer Mannheim, Germany.

(3) FixDenat: fix solution (ready-to-use solution), product No.1647229, Boehringer Mannheim, germany.

(4) anti-BrdU-peroxidase: murine monoclonal antibody conjugated to peroxidase, product No.1647229, Boehringer Mannheim, germany.

(5) TMB substrate solution: tetramethylbenzidine (TMB), ready-to-use solution, product No.1647229, Boehringer Mannheim, germany.

(6) PBS wash solution: 1 XPBS, pH 7.4, self-prepared.

(7) Bovine Serum Albumin (BSA): fraction V powder; a-8551, Sigma chemical Co, USA.

Scheme(s)

(1)3T3 genetically engineered cell line: 3T3/EGFRc7

(2) Cells were seeded 8000 cells/well in 96-well plates in DMEM, 10% CS (bovine serum), 2mM Gln (glutamine). At 37 ℃ and 5% CO₂Cells were incubated overnight.

(3) After 24 hours, cells were washed with PBS and then incubated in serum-free medium (0% CS DMEM + 0.1% BSA) for 24 hours in fasting serum.

(4) On day 3, the cells were simultaneously added with ligand (PDGF ═ 3.8nM, in DMEM with 0.1% BSA) and test compound. Negative control wells were loaded with serum-free DMEM containing 0.1% BSA alone, and positive control wells were loaded with ligand (PDGF) but no test compound. Test compounds were prepared in ligand-containing serum-free DMEM in 96-well plates and serially diluted to 7 concentrations.

(5) After 20 hours of ligand activation, diluted BrdU labelling reagent (1: 100 in DMEM, 0.1% BSA) was added and the cells incubated with BrdU (final concentration 10. mu.M) for 1.5 hours.

(6) After incubation with the labeling reagent, the inverted plate was poured and patted on a paper towel to remove the medium. FixDenat solution (50. mu.l/well) was added and the plates were incubated for 45 minutes at room temperature on a plate shaker.

(7) The FixDenat solution was thoroughly removed by pouring and patting the inverted plate on a paper towel. Milk (5% dehydrated in PBS, 200. mu.l/well) was added as a blocking agent and the plates were incubated for 30 minutes at room temperature on a plate shaker.

(8) The blocking solution was decanted and each well was washed once with PBS. anti-BrdU-peroxidase solution (1: 100 diluted in PBS, 1% BSA) (100. mu.l/well) was added and the plates were incubated for 90 minutes at room temperature on a plate shaker.

(9) The antibody conjugate was thoroughly removed by pouring and washing the wells 5 times with PBS, inverting the plate and patting on a paper towel to dry.

(10) TMB substrate solution (100. mu.l/well) was added and incubated on a plate shaker at room temperature for 20 minutes until sufficient color development had occurred for photometric detection.

(11) Absorbance of the samples was measured at 410nm on a Dynatech ELISA plate reader (in "Dual wavelength" mode, with 490nm filter reading as the reference wavelength.)

Test 2: EGF induced BrdU incorporation assay

Materials and reagents

(1) EGF: murine EGF, 201; toyobo co, japan.

(2) BrdU labeling reagent: 10mM in PBS (pH 7.4), product number 1647229, Boehringer Mannheim, Germany.

(3) FixDenat: fix solution (ready-to-use solution), product No.1647229, Boehringer Mannheim, germany.

(4) anti-BrdU-peroxidase: murine monoclonal antibody conjugated to peroxidase, product No.1647229, Boehringer Mannheim, germany.

(5) TMB substrate solution: tetramethylbenzidine (TMB), ready-to-use solution, product No.1647229, Boehringer Mannheim, germany.

(6) PBS wash solution: 1 XPBS, pH 7.4, self-prepared.

(7) Bovine Serum Albumin (BSA): fraction V powder; a-8551, Sigma chemical Co, USA.

Scheme(s)

(1)3T3 genetically engineered cell line: 3T3/EGFRc7

(2) Cells were seeded in 96-well plates in DMEM with 10% CS, 2mM glutamine. 8000 cells per well.

(3) After 24 hours, cells were washed with PBS and then serum starved for 24 hours in serum-free medium (0% CS DMEM with 0.1% BSA).

(4) On day 3, the cells were added simultaneously with the ligand (EGF ═ 2nM, in DMEM with 0.1% BSA) and the test compound. Only serum-free DMEM containing 0.1% BSA was added to the negative control wells; the ligand (EGF) was added to the positive control cells, but the test compound was not added. Test compounds were prepared in ligand-containing serum-free DMEM in 96-well plates and serially diluted to 7 test concentrations.

(5) After 20 hours of ligand activation, diluted BrdU labelling reagent (1: 100 in DMEM, 0.1% BSA) was added and the cells incubated with BrdU (final concentration 10. mu.M) for 1.5 hours.

(6) After incubation with the labeling reagent, the medium was removed by pouring and tapping the inverted plate on a paper towel. FixDenat solution (50. mu.l/well) was added and the plates were incubated for 45 minutes at room temperature on a plate shaker.

(7) The FixDenat solution was thoroughly removed by pouring and patting the inverted plate on a paper towel. Milk (5% dehydrated milk in PBS, 200. mu.l/well) was added as a blocking solution and the plates were incubated for 30 minutes at room temperature on a plate shaker.

(8) The blocking solution was poured off and the wells were washed once with PBS. anti-BrdU-peroxidase solution (1: 100 diluted in PBS, 1% BSA) (100. mu.l/well) was added and the plates were incubated for 90 minutes at room temperature on a plate shaker.

(9) The antibody conjugate was thoroughly removed by pouring and washing the wells 5 times with PBS, inverting the plate and patting on a paper towel to dry.

(10) TMB substrate solution (100. mu.l/well) was added and incubated on a plate shaker at room temperature for 20 minutes until color development was sufficient for photometric detection.

(11) The absorbance of the samples was measured at 410nm on a Dynatech ELISA plate reader (in "dual wavelength" mode, 490nm filter reading as the reference wavelength).

Test 3: EGF-induced Her 2-driven BrdU incorporation

Materials and reagents

(1) EGF: murine EGF, 201; toyobo Co., Japan

(2) BrdU, product No.1647229, Boehringer Mannheim, germany.

(3) FixDenat: fix solution (ready-to-use solution), product No.1647229, Boehringer Mannheim, germany.

(4) anti-BrdU-peroxidase: murine monoclonal antibody conjugated to peroxidase, product No.1647229, Boehringer Mannheim, germany.

(5) TMB substrate solution: tetramethylbenzidine (TMB), ready-to-use solution, product No.1647229, Boehringer Mannheim, germany.

(6) PBS wash solution: 1 XPBS, pH 7.4, self-prepared.

(7) Bovine Serum Albumin (BSA): fraction V powder; a-8551, Sigma chemical Co, USA.

Scheme(s)：

(1)3T3 genetically engineered cell line: 3T3/EGFr/Her 2/EGFr (EGFr with Her2 kinase Domain)

(2) Cells were seeded 8000 cells/well in 96-well plates in DMEM, 10% CS, 2mM glutamine. At 37 ℃ and 5% CO₂Cells were incubated overnight.

(3) After 24 hours, the cells were washed with PBS and then incubated in serum-free medium (0.1% BSA in% CS DMEM) for 24 hours in the absence of fasting serum.

(4) On day 3, the cells were simultaneously added with ligand (EGF ═ 2nM, prepared in DMEM with 0.1% BSA) and test compound. Only serum-free DMEM containing 0.1% BSA was added to the negative control wells; the ligand (EGF) was added to the positive control cells, but the test compound was not added. Test compounds were prepared in ligand-containing serum-free DMEM in 96-well plates and serially diluted to 7 test concentrations.

(5) After 20 hours of ligand activation, diluted BrdU labelling reagent (1: 100 in DMEM, 0.1% BSA) was added and the cells incubated with BrdU (final concentration 10. mu.M) for 1.5 hours.

(6) After incubation with the labeling reagent, the medium was removed by pouring and patting the inverted plate on a paper towel. FixDenat solution (50. mu.l/well) was added and the plates were incubated for 45 minutes at room temperature on a plate shaker.

(7) The FixDenat solution was removed completely by pouring and patting the inverted plate on a paper towel. Milk (5% milk powder in PBS, 200. mu.l/well) was added as a blocking solution and the plates were incubated for 30 minutes at room temperature on a plate shaker.

(8) The blocking solution was poured off and the wells were washed once with PBS. anti-BrdU-peroxidase solution (1: 100 diluted in PBS, 1% BSA) (100. mu.l/well) was added and the plates were incubated for 90 minutes at room temperature on a plate shaker.

(9) The antibody conjugate was thoroughly removed by pouring and washing the wells 5 times with PBS, inverting the plate and patting on a paper towel to dry.

(10) TMB substrate solution (100. mu.l/well) was added and incubated on a plate shaker at room temperature for 20 minutes until sufficient color development had occurred for photometric detection.

(11) The absorbance of the sample at 410nm was measured on a Dynatech ELISA plate reader (using the "dual wavelength" mode, with the filter reading at 490nm as the reference wavelength).

Test 4: EGF 1-induced BrdU incorporation assay

Materials and reagents：

(1) IGF1 ligand: human recombinants; g511, Promega corporation, USA.

(2) BrdU labeling reagent: 10mM in PBS (pH 7.4), product number 1647229, Boehringer Mannheim, Germany.

(3) FixDenat: fix solution (ready-to-use solution), product No.1647229, Boehringer Mannheim, germany.

(4) anti-BrdU-peroxidase: murine monoclonal antibody conjugated to peroxidase, product No.1647229, Boehringer Mannheim, germany.

(5) TMB substrate solution: tetramethylbenzidine (TMB), ready-to-use solution, product No.1647229, Boehringer Mannheim, germany.

(6) PBS wash solution: 1 XPBS, pH 7.4, self-prepared.

(7) Bovine Serum Albumin (BSA): fraction V powder; a-8551, Sigma chemical Co, USA.

Scheme(s)：

(1)3T3 genetically engineered cell line: 3T3/IGF lr

(2) Cells were seeded in 96-well plates in DMEM, 10% CS, 2mM glutamine. 8000 cells per well. Cells were incubated at 37 ℃ and 5% CO₂The mixture was incubated overnight.

(3) After 24 hours, cells were washed with PBS and then incubated in serum-free medium (0% CS DMEM containing 0.1% BSA) for 24 hours in fasting serum.

(4) On day 3, the cells were simultaneously added with the ligand (IGF 1 ═ 3.3nM, in DMEM with 0.1% BSA) and the test compound. Only serum-free DMEM containing 0.1% BSA was added to the negative control wells; the ligand (IGF 1) was added to the control cells, but the test compound was not added. Test compounds were prepared in ligand-containing serum-free DMEM in 96-well plates and serially diluted to 7 test concentrations.

(5) After ligand activation for 16 hours, diluted BrdU labelling reagent (1: 100 in DMEM, 0.1% BSA) was added and the cells incubated with BrdU (final concentration 10. mu.M) for 1.5 hours.

(6) After incubation with the labeling reagent, the medium was removed by pouring and patting the inverted plate on a paper towel. FixDenat solution (50. mu.l/well) was added and the plate incubated on a plate shaker at room temperature for 45 minutes.

(7) The FixDenat solution was removed completely by pouring and patting the inverted plate on a paper towel. Milk (5% milk powder in PBS, 200. mu.l/well) was added as a blocking solution and the plates were incubated for 30 minutes at room temperature on a plate shaker.

(8) The blocking solution was poured off and the wells were washed once with PBS. anti-BrdU-peroxidase (1: 100 diluted in PBS, 1% BSA) (100. mu.l/well) was added and the plates incubated for 90 minutes at room temperature on a plate shaker.

(9) The antibody conjugate was thoroughly removed by pouring and washing the wells 5 times with PBS, inverting the plate and drying by patting on a paper towel.

(10) TMB substrate solution (100. mu.l/well) was added and incubated on a plate shaker at room temperature for 20 minutes until sufficient color development had occurred for photometric detection.

(11) The absorbance of the samples at 410nm was measured on a Dynatech ELISA plate reader (in the "dual wavelength" mode, compared to the filter reading at 490nm as the reference wavelength).

Test 5: insulin-induced BrdU incorporation assay

Materials and reagents

(1) Insulin: crystals, bovine insulin, zinc; 13007, Gibco BRL, usa.

(2) BrdU labeling reagent: 10mM in PBS (pH 7.4), product number 1647229, Boehringer Mannheim, Germany.

(3) FixDenat: fix solution (ready-to-use solution), product No.1647229, Boehringer Mannheim, germany.

(4) anti-BrdU-peroxidase: murine monoclonal antibody conjugated to peroxidase, product No.1647229, Boehringer Mannheim, germany.

(5) TMB substrate solution: tetramethylbenzidine (TMB), ready-to-use solution, product No.1647229, Boehringer Mannheim, germany.

(6) PBS wash solution: 1 XPBS, pH 7.4, self-prepared.

(7) Bovine Serum Albumin (BSA): fraction V powder; a-8551, Sigma chemical Co, USA.

Step (ii) of

(1)3T3 genetically engineered cell line: h25

(2) Cells were seeded in 96-well plates in DMEM, 10% CS, 2mM glutamine, 8000 cells per well. At 37 ℃ and 5% CO₂Cells were incubated overnight.

(3) After 24 hours, cells were washed with PBS and then incubated in serum-free medium (0% CS in DMEM with 0.1% BSA) with fasting serum for 24 hours.

(4) On day 3, the cells were simultaneously added with ligand (insulin ═ 10nM, prepared in DMEM with 0.1% BSA) and test compound. Only serum-free DMEM containing 0.1% BSA was added to the negative control wells; the ligand (insulin) was added to the control cells, but no test compound was added. Test compounds were prepared in ligand-containing serum-free DMEM in 96-well plates and serially diluted to 7 test concentrations.

(5) After ligand activation for 16 hours, diluted BrdU labelling reagent (1: 100 in DMEM, 0.1% BSA) was added and the cells incubated with BrdU (final concentration 10. mu.M) for 1.5 hours.

(6) After incubation with the labeling reagent, the medium was removed by pouring and tapping the inverted plate on a paper towel. FixDenat solution (50. mu.l/well) was added and the plate was incubated for 45 minutes at room temperature on a plate shaker.

(7) The FixDenat solution was removed completely by pouring and patting the inverted plate on a paper towel. Milk (5% milk powder in PBS, 200. mu.l/well) was added as a blocking solution and the plates were incubated for 30 minutes at room temperature in a plate shaker.

(8) The blocking solution was poured off and the wells were washed once with PBS. anti-BrdU-peroxidase (1: 100 diluted in PBS, 1% BSA) (100. mu.l/well) was added and the plates were incubated in a plate shaker at room temperature for 90 minutes.

(9) The antibody conjugates were thoroughly removed by pouring and washing the wells 5 times with PBS, inverting the plate and drying by patting on a paper towel.

(10) Substrate solution (100. mu.l/well) was added and incubated on a plate shaker at room temperature for 20 minutes until sufficient color development had occurred for photometric detection.

(11) The absorbance of the sample at 410nm was measured on a Dynatech ELISA plate reader (using the "dual wavelength" mode, referenced to the filter reading at 490nm as the reference wavelength).

HUV-EC-C test

The activity of the compounds can also be determined using the following protocol:

day 0

1. HUV-EC-C cells (human umbilical vein endothelial cells, American type culture Collection, code 1730CRL) were washed and trypsinized. In Dulbecco's phosphate-buffered saline (D-PBS, available from Gibco BRL under the product number 14190-²The tissue culture flasks were washed twice. Trypsinization was performed with 0.05% trypsin-EDTA in a non-enzymatic cell dissociation solution (Sigma chemical Co., Ltd., product No. C-1544). 0.05% trypsin was prepared by diluting 0.25% trypsin/1 mM EDTA (Gibco, product No. 25200-049) in the cell dissociation solution. Using about 1ml/25-30cm²The tissue culture flasks were trypsinized at 37 ℃ for about 5 minutes. After the cells had detached from the flask, an equal volume of test medium was added and transferred to a 50ml sterile centrifuge tube (Fischer)Scientific, product number 05-539-6).

2. The cells were washed with about 35ml of test medium in a 50ml sterile centrifuge tube by adding the test medium, centrifuging at about 200Xg for 10 minutes, withdrawing the supernatant and resuspending it in 35ml of D-PBS. The washing was repeated twice more with D-PBS and the cells were resuspended in about 1ml of test medium/15 cm²In tissue culture flasks. The test medium consisted of F12K medium (Gibco BRL, product No. 21127-014) + 0.5% heat-inactivated fetal bovine serum. Counting the cells with a Coulter counter (Coulter electronics Co.), adding the test medium to the cells to a concentration of 0.8-1.0X 10⁵Cells/ml.

3. Cells were added to 96-well flat-bottom plates at 100. mu.l per well or 0.8-1.0X 10⁴A cell; at 37 deg.C, 5% CO₂The mixture was incubated for 24 hours.

Day 1

1. Drug titrations were formulated in two-fold changes in additional 96-well plates, typically from 50 μ M to 0 μ M. The same test medium as described above in step 2 on day 0 was used. The titration solution was prepared by adding 200. mu.M (4-fold final well concentration) of the drug to the uppermost well of a particular row of the plate at 90. mu.l/well. Since the concentration of stock drug solutions is often 20mM in DMSO, 200 μ M drug concentration contains 2% DMSO.

Therefore, a diluent supplemented with 2% DMSO in the test medium (F12K + 0.5% fetal bovine serum) was used as a diluent for the drug titration solution in order to dilute the drug while keeping the DMSO concentration constant. This diluent was added to the remaining wells of the row above, 60 μ l per well. 60 μ l of drug diluent from 200 μ M in the uppermost well of the row was mixed with 60 μ l in the second well of the row. From this well 60. mu.l was mixed with 60. mu.l in the third well of the row and so on until the titration solution was completed with a two-fold change in concentration. When the penultimate well is mixed, 60. mu.l of the 120. mu.l of this well is removed and discarded. The last well was left with 60 μ l of DMSO/medium dilution as drug-free control. The drug to be titrated was formulated in 9 rows, sufficient for three replicate well samples for each of the following drugs: 1) VEGF (from Pepro Tech Inc., product No. 100-200, 2) Endothelial Cell Growth Factor (ECGF), also known as acidic fibroblast growth factor or aFGF (from Boehringer Mannheim Biochemica, product No. 1439600), and a control of assay media. ECGF is a preparation containing heparin sodium.

2. The drug dilutions were transferred at 50. mu.l/well to a 96-well assay plate containing HUV-EC-C cells from day 0, 100. mu.l or 0.8-1.0X 10 per well⁴Cells, plates at 37 ℃ and 5% CO₂The following incubations were carried out for about 2 hours.

3. In triplicate, 50. mu.l/well of 80. mu.g/ml VEGF, 20ng/ml ECGF, or medium control were added to each drug dilution. As in the case of the drug, the concentration of the growth factor is 4 times the desired final concentration. The test medium of step 2 on day 0 was used to formulate the concentration of growth factors. At 37 ℃ and 5% CO₂The following incubations were carried out for about 24 hours. There should be 50. mu.l of drug dilution, 50. mu.l of growth factor or culture medium, and 100. mu.l of cells per well, for a total of 200. mu.l per well. Thus, once each substance was added to the wells, the 4-fold concentration of drug and growth factor became 1-fold.

Day 2

1. Adding into³H-thymidine (Amersham, product number TRK-686), 1. mu. Ci per well (10. mu.l per well of 100. mu. Ci/ml solution prepared in RPMI medium + 10% heat-inactivated fetal calf serum), at 37 ℃ and 5% CO₂Incubation was continued for-24 hours. Note that:³h-thymidine was made in RPMI medium because we used³All other cases of H-thymidine involve experiments in RPMI. The difference in culture medium in this step probably has little effect. RPMI was obtained from Gibco BRL, product number 11875-.

Day 3

1. Plates were frozen at-20 ℃ overnight.

Day 4

1. Plates were thawed and harvested using a 96-well plate Harvester (Tomtec Harvester 96)^(R)) Collected on a filter screen (Wallac, product No. 1205-401); in Wallac Betaplate^(TM)Reading on a liquid scintillation counter.

PDGF-R cell assay

The PDGF cytokinase assay was performed as follows: cells were lysed in 0.2M Hepes, 0.15M NaCl, 10% v/v glycerol, 0.04% octoxynol-9, 5mM EDTA, 5mM sodium orthovanadate and 2mM sodium pyrophosphate; the cell lysates were then applied to ELISA plates coated with anti-PDGF receptor antibody (Genzyme); before adding the lysate, the ELISA plate was first coated with 0.5 μ g antibody/well in 150 μ l PBS for 18 hours at 4 ℃; lysates were incubated for 1 hour in coated plates and then washed 4 times in TBST (35mM Tris-HCl, pH7.0, 0.15M NaCl, 0.1% octoxynol-9); anti-phosphotyrosine antibody (100. mu.l in PBS) was added and the mixture was incubated at room temperature for 30 min; wells were then washed 4 times in TBST, a peroxidase conjugated secondary antibody (TAGO) was added to each well, and the treated wells were incubated at room temperature for 30 minutes; the wells were then washed 4 times in TBST and ABTS/H was added to each well₂O₂Solution, incubation for 2 minutes; the absorbance was then measured at 410 nm.

6.2.6. Test results of cell growth test

The results obtained for each compound from the above test are given in the following tables:

TABLE 2

Cell division in endothelial cells

Incorporation of³H]Thymidine

Compounds (examples)	HUV-ECVEGF(μM)	Test a-FGF (μ M)
			27	1.1	153.8
1B	0.2	6.0
			16	6.6	3.4
17	4.8	35.7
			4796	30.7	35.8
20	43.2
			21	19.9
22	2.5	45.2
			23	1.6	4.6
24	14.8
			25	3.4	3.7
26	25.6	19.3
			28	8.0	13.0
29	34.3	16.3
			30	1.0	1.4
32	4.4	4.9
			4	0.6
33	46.1	27.3
			5	0.8	25.8
5201	2.5	2.3
			36	2.3	0.7
37	5.1	11.8
			38	2.9	130
5217	9.6	10.5

Compounds (examples)	HUV-ECVEGF(μM)	Test a-FGF (μ M)
			41	2.4	2.7
8	2.2
			42	＜0.8	2.0
9	＜0.8	31.1
			44	0.9	0.6
10	＜0.8
			45	39.8	35.5
11	＜0.8	22.7
			5409	26.0
12	＜0.8
			48	13.6	40
13	0.7
			50	11.4
14	2.5
			15	5.7
5429	27.6
			59	0.16	0.14
60	39.8	33.0
			61	1.2	30.0
63	3.8	3.4
			64	20	20
68	＜0.07	＜0.07
			69	0.5	0.8
70	0.14	7.9
			71	3.8	12.9
73	1.3	3.2
			74	0.54	8.7
76	2.0	5.0
			77	1.2	14.1
81	0.05	37.8

Compounds (examples)	HUV-ECVEGF(μM)	Test a-FGF (μ M)
			7	1.2	3.8

TABLE 3

Cell division in 3T3/EGFR cells

Incorporation of BrdU

Compounds (examples)	PDGFRPDGF ligand IC50 (. mu.M)	FGFRFGF ligand IC50(μ M)	EGFRGF ligand IC50(μ M)
				27	75
4313	6	5.5	5.5
				18	2.5
29	9	4.9	60
				4	3	10	20
5402	50	40
				36	3	25
10	5.2
				45	7.5	70	100
12	2.8	70
				61	30	16
5463			23
				71	70	60	95
5465	40	25	50
				73	18	15	17
74	8
				5469	4	15	28
77	4	50	54
				81	6.5	9	48

TABLE 4

Cell growth assays for various cell lines

SRB reading

Compounds (examples)	3T3/E/H+TGF-a(T)IC50(μM)	3T3/EGFR+TGF-a(T)IC50(μM)	3T3/PDGFR+PDGF(T)IC50(μM)	SMCIC50(μM)
					27	36
4313	32	10.7		8.8
					1B	78		10
5			22.2

3T3/E/H + TGF-. alpha.s (T): NIH3T3 cells expressing EGFR/HER2 chimera and TGF-alpha, tumor derivatives

3T3/EGFR + TGF- α (T): NIH3T3 cells expressing EGFR and TGF-alpha, tumor derivatives

3T3/PDGFR + PDGF (T): NIH3T3 cells expressing PDGF-beta R and PDGF-beta, tumor derivatives

SMC: human smooth muscle cells from clonetics

6.3. Cytotoxicity assays

Therapeutic compounds should be more potent in inhibiting receptor tyrosine kinase activity than exhibit cytotoxicity. The potency and cytotoxicity of a compound can be measured by determining the therapeutic index IC₅₀/LD₅₀Thus obtaining the product. IC (integrated circuit)₅₀Is the dose required to achieve 50% inhibition, and can be determined by standard methods such as those described herein. LD₅₀Is the dose that causes 50% toxicity and can also be measured using standard methods (Mossman, 1983, journal of immunological methods (j. immunological. methods),65：5563) or the amount of LDH (lactate dehydrogenase) released (Korzeniewski and Cellewaert, 1983, J.Immunol.methods)64: 313; decker and Lohmann-Matthes, 1988, journal of immunological methods,115: 61) or alternatively, measuring the lethal dose in animal models. Compounds with large therapeutic indices are desirable. The therapeutic index should be greater than 2, preferably at least 10, and most preferably at least 50.

6.3. In vivo animal model

6.3.1. Xenograft animal model

The ability of human tumors to grow as xenografts in athymic mice (e.g., Balb/c, nu/nu) provides a useful in vivo model for studying the biological response of human tumors to treatment. Since the first successful xenografting of human tumors into athymic mice (Rygaard and Povlsen, 1969, scandinavia pathomicrobiology report,77: 758-. Human breast tumor cell lines, including MCF-7, ZR75-1 and MDA-MB-231, have been achieved in nude mice with subcutaneous xenografts [ Warri et al, 1991, journal of International cancer (int.J49: 616-623; ozzello and Sordat, 1980, european journal of cancer (eur.j16: 553-; osborne et al, 1985, Cancer research (Cancer Res.)45: 584-590; seibert et al, 1983, Cancer research (Cancer Res.)43：2223-2239〕。

Test 1: HER 2/xenograft animal model

To investigate the effect of candidate anti-tumor drugs on HER2 expressing tumors, tumor cells should be able to grow without supplemental estrogen. Growth of many mammary cell lines in nude mice is estrogen dependent (Osborne et al, supra), however, exogenous estrogen inhibits H in nude miceER2 expression (Warri et al, supra; Dati et al, 1990, Oncogene (Oncogene)5: 1001-1006). For example, MCF-7, ZR-75-1 and T47D cells grew well in vivo in the presence of estrogen, but expressed very low levels of HER2(Warri et al, supra; Dati et al, supra).

The following types of xenograft protocols can be employed:

1) tumor cells were transplanted (subcutaneously) into the posterior rib of 5-6 week old female Balb/c nu/nu athymic mice;

2) administering an anti-neoplastic compound;

3) tumor growth was determined by measuring tumor volume.

Tumors can be analyzed for the presence of receptors, such as HER2, EGF or PDGF, using Western and immunohistochemical analysis. The above-described steps can be modified by those skilled in the art using techniques known in the art, for example, using different treatment modalities.

Test 2: FLK-1/xenograft model

The compounds of the invention were tested for their ability to inhibit ovarian, melanoma, prostate, lung and breast tumor cell lines formed as subcutaneous xenografts. These studies were performed at a dose of 1-75mg/kg per day.

Materials and methods: tumor cells were implanted subcutaneously into mice of the indicated strain. Unless otherwise indicated (e.g., treatment of SCID (severe combined immunodeficiency) mice associated with the a 375 melanoma cell line was initiated at day 9), treatment was initiated at day 1 post-implantation. There were 8-16 mice per experimental group.

The specific contents are as follows:

animal(s) productionFemale athymic mice (BALB/C, nu/nu), BALB/C, Wistar and Fisher 344 rats were obtained from Simonsen laboratories (Gilroy, Calif.). Female A/I mice were obtained from Jackson laboratories (Bar Harbor, ME). DA rats from B&KUnVerdeal (Fremont, CA). Athymic R/Nu rats, DBA/2N mice and BALB/C mice were obtained from Harlan Sprague Dawley (Indianapolis, IN). Female C57BL/6 mice were obtained from Taconic (Germantown, NY). All animals were kept in microchamber cages with Alpha-dri bedding under clean room conditions. They receive sterile chewing food and water without limitation.

All steps were performed according to the guidelines for animal care and use of the national institutes of health.

Subcutaneous xenograft modelCell lines were grown as described in appropriate media (section 6). Cells were harvested at or near confluence with 0.05% trypsin-EDTA and pelleted at 450 Xg for 10 min. The pellet was resuspended in sterile PBS or media (no FBS) to the appropriate concentration indicated in the graphical illustration and the cells implanted in the hind flank of the mouse. Tumor growth was measured with a vernier caliper for 3-6 weeks, and unless otherwise stated, tumor volume was calculated as the product of growth x width x height. P values were calculated using the t-test method. Compounds in 50-100. mu.l vehicle (dimethyl sulfoxide, PBTE 6C: D5W or PBTE: D5W) were administered by intraperitoneal injection at the concentrations indicated in the chart.

Intracerebral xenotransplantation modelFor the murine intracerebral xenograft model, rat C6 glioma cells were harvested, suspended in sterile PBS and placed on ice at a concentration of 2.5X 10⁷Cells/ml. Cells were implanted into BALB/C, nu/nu mice as follows: the scalp at the forehead of the mouse was wiped with 70% ethanol and shaved with an animal knife if necessary. The animals were anesthetized with isoflurane and the needle was inserted through the skull into the left hemisphere of the brain. Cells were delivered from a Hamilton air-tight syringe and allowed to penetrate only 3mm using a 30 gauge, 1/2 inch needle fitted to a cannula. A re-issuing dispenser was used to accurately discharge 4 μ l of cell suspension. The animals were observed day by day for condition and slaughtered when they lost about 40% and/or exhibited neurological symptoms.

For the rat intracerebral model, rats (Wistar, Sprague Dawley, Fischer 344, or athymic R/Nu; about 200- & 400g, some 300- & 400g) were anesthetized by intraperitoneal injection of 100mg/kg ketamine hydrochloride (Aveco, Fort Dodge, Iowa) and 5mg/kg xylazine (2% solution, Bayer, Germany). After anesthesia occurred, the brain shells were scraped and the animals were positioned on a stereotaxic apparatus (Stoelting, Wood Dale, IL). The skin at the incision was swabbed with 70% ethanol and 10% polyvinylpyrrolidone-iodine, and washed 3 times. A median 1.0-1.5Gm incision was made in the skull with a sterile scalpel. Gently open the skin and pull to both sides to expose the sutures on the skull surface. A hole is drilled with a dental drill (Stoelting, Wood Dale, IL) 1mm in front of the chimney and 2mm laterally. The cell suspension was drawn into a 50. mu.l Hamilton syringe fitted with a 23 or 25 gauge needle at a standard bevel angle. The syringe was positioned in the hole at the level of the arachnoid and lowered until the tip of the needle penetrated 3mm into the brain structure, where the cell suspension was slowly injected. After the cells are injected, the needle is lifted in the hole for 1-2 minutes to release the cells. The skull was cleaned and the skin was sutured with 2-3 needles. The animals were observed for recovery from surgery and anesthesia. Throughout the experiment, animals were observed at least twice daily for the development of symptoms associated with tumor progression in the brain. Animals exhibiting increased symptoms (thinning, loss of balance, dehydration, loss of appetite, loss of coordination, cessation of grooming activities, and/or significant weight loss) are humanely slaughtered and dissected for the organs and tissues of interest.

Intra-peritoneal modelCells are grown in a suitable medium. Cells were harvested and washed in sterile PBS or FBS-free medium, resuspended to the appropriate concentration, and injected into the peritoneal cavity of appropriate strains of mice. Ascites formation was observed day by day. Individual animals were slaughtered when they gained 40% weight or when the intraperitoneal tumor burden began to cause excessive stress and pain in the animals.

6.3.2. In vivo VEGF pellet model

In the following examples, the activity of compounds against the FLK-1 receptor and against disorders associated with angiogenesis was tested using a bead model. In this model, VEGF was packed into a long-term released pellet and subcutaneously implanted into the abdomen of nude mice in order to induce a "reddening" response and consequent swelling around the pellet. A potential FLK-1 inhibitor may then be implanted in methylcellulose in the vicinity of the VIEGF sphere in order to determine whether such inhibitors may be used to inhibit the "reddening" response and subsequent swelling.

Materials and methodsThe following materials and methods were used:

1) VEGF-human recombinant lyophilized products are commercially available and are commercially available from Peprotech corporation (Princeton Business Park, G2; box 275, rocky hill, NJ 08553).

2) VEGF loaded into beads for 21 days of release was obtained from Innovative Research of America (Innovative Research of America, 3361executive park way, P.O. Bax 2746, Toledo, Ohio 43606), using a patented matrix-driven release system. The pellets were packed with 0.20, 0.21 or 2.1. mu.g VEGF per pellet. These doses were approximately 10 and 100ng VEGF released per day.

3) Methyl cellulose

4) Water (sterile)

5) Methanol

6) Suitable drugs/inhibitors

7)10cm culture plate

8) parafilm film

The VEGF bead model experiment was then performed according to the following protocol:

1) VEGF purchased from Peprotech was sent to Innovative Research of America for custom pellet preparation;

2) methyl cellulose was formulated in sterile water at 1.5% (w/v);

3) dissolving the drug in methanol (concentration range commonly used is 10 to 20 mg/ml);

4) placing sterile parafilm in sterile 10cm plates;

5) adding 150 μ l of the drug methanol solution to 1.35ml of 1.5% methyl cellulose, mixing/vortexing thoroughly;

6) 25. mu.l portions of the homogenate were placed on parafilm and dried to form disks;

7) rats (6-10 weeks Balb/c athymic nu/nu, female) were anesthetized by isoflurane inhalation;

8) implanting VEGF pellets and methylcellulose discs subcutaneously in the abdomen; and

9) the mice were scored for reddening and swelling responses at 24 hours and 48 hours.

The specific experimental design used in this example was:

n-4 animals/group

And (3) comparison: VEGF pellets plus drug placebo

VEGF blank control plus drug pellet

Results of the experiment: the compounds of the invention are expected to show activity consistent with this assay.

6.3.3. Breast fat pad model

Since the role of many RTKs, such as HER2 receptors, in breast cancer has been identified, the breast fat pad model is particularly useful for determining the potency of compounds to inhibit such RTKs. By implanting tumor cells directly into the site of interest, the in situ model more accurately reflects the biology of tumor development than the subcutaneous model. Human mammary cell lines, including MCF-7, have been grown in the mammary fat pad of athymic mice. Shafie and Granthm, 1981, journal of the national cancer institute (Natl. cancer institute.)67: 51-56; gottardis et al, 1988, journal of steroid biochemistry (J.Steroid Biochem.)30: 311-314. More specifically, the compound can be assayed for HER using the following procedure2 receptor inhibition:

1) HER-2 transfected MDA-MB-231 and MCF-7 cells were implanted at various concentrations into the underarm mammary fat pad of female athymic mice;

2) administering a compound; and

3) tumor growth was measured at different times.

Tumors can also be analyzed for the presence of receptors such as HER2 using Western and immunohistochemical assays. The above steps can be modified by those skilled in the art using techniques known in the art, for example, using different treatment protocols.

6.3.4. Tumor infection model

The following tumor invasion models have been established that can be used to evaluate the therapeutic value and efficacy of compounds that are thought to selectively inhibit the KDR/FLK-1 receptor.

6.3.4.1. Step (ii) of

Nude mice (female, Simonsen) of 8 weeks old were used as experimental animals. The implantation of tumor cells was performed in a laminar flow hood. For anesthesia, a xylazine/ketamine hydrochloride mixture (100mg/kg ketamine hydrochloride and 5mg/kg xylazine) was injected intraperitoneally. A midline opening was made to expose the abdominal cavity (approximately 1.5cm in length) for injection of 10. mu.l volume of medium⁷And (4) tumor cells. Cells were either injected into the duodenal lobes of the pancreas or beneath the serosa of the colon. The peritoneum and muscles were sutured with a 6-0 gauge continuous thread and the skin was closed with wound clips. Animals were observed day by day.

6.3.4.2. Analysis of

After 2-6 weeks, the mice were sacrificed, and local tumors were excised and analyzed for metastasis to various organs (lung, liver, brain, stomach, spleen, heart, muscle) according to the general observation of the animals (tumor size, infection grade, immunochemistry and in situ hybridization were determined).

6.3.5. Results

The results for each compound from the in vivo assay described above are set forth in table 5 below:

TABLE 5

In vivo test data

Compounds (examples)	EDH4-VEGF inhibition% amount (mg/kg)
		27	56％，75-----------------------50％，7563％，50
22	42％，75-------------------------42％，50/50
		4942	46％，5047％，25
12	50％，25-------------------------57％，37.5/37.5
		14	45％，50-------------------------65％，50
15	47％，50-------------------------65％，50

The scope of the invention is not limited by the illustrated embodiments, which are intended as illustrations of single aspects of the invention, and any functionally equivalent clone, DNA or amino acid sequence is within the scope of the invention. Indeed, various modifications in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

All documents cited herein are hereby incorporated by reference in their entirety.

Claims (6)

1. A compound of the formula and its pharmaceutically acceptable salt

Wherein R is₁Is H;

R₂is O or S;

R₃is hydrogen;

R₄、R₅、R₆and R₇Each independently selected from the group consisting of: hydrogen, alkyl, alkoxy, aryl,Aryloxy, alkylaryl, alkylaryloxy, halogen, trihalomethyl, S (O) R, SO₂NRR′、SO₃R、SR、NO₂、NRR′、OH、CN、C(O)R、OC(O)R、NHC(O)R、(CH₂)_nCO₂R and CONRR';

a is a five membered heteroaromatic ring selected from thiophene, pyrrole, pyrazole, imidazole, 1, 2, 3-triazole, 1, 2, 4-triazole, oxazole, isoxazole, thiazole, isothiazole, 2-sulfonylfuran, 4-alkylfuran, 1, 2, 3-oxadiazole, 1, 2, 4-oxadiazole, 1, 2, 5-oxadiazole, 1, 3, 4-oxadiazole, 1, 2, 3, 4-oxatriazole, 1, 2, 3, 5-oxatriazole, 1, 2, 3-thiadiazole, 1, 2, 4-thiadiazole, 1, 2, 5-thiadiazole, 1, 3, 4-thiadiazole, 1, 2, 3, 5-thiadiazole and tetrazole optionally substituted in one or more positions with: alkyl, alkoxy, aryl, aryloxy, alkaryl, alkylaryloxy, halogen, trihalomethyl, S (O) R, SO₂NRR′、SO₃R、SR、NO₂、NRR′、OH、CN、C(O)R、OC(O)R、NHC(O)R、(CH₂)_nCO₂R and CONRR';

n is 0 to 3;

r is H, alkyl or aryl; and

r' is H, alkyl or aryl;

wherein in the above definition:

alkyl means a straight, branched or cyclic saturated aliphatic hydrocarbon group having 1 to 12 carbon atoms, and optionally substituted with one or more substituents selected from: hydroxy, cyano, ═ O, ═ S, NO₂Halogen, N (CH)₃)₂Amino and-SH;

aryl refers to an aromatic group having at least one ring with a conjugated pi-electron system, including carbocyclic aryl, heterocyclic aryl, and biaryl, and is optionally substituted with one or more substituents selected from: halogen, trihalomethyl, hydroxy, SH, OH, NO₂Amino, thioether, cyano, alkoxy, alkyl, and amino;

alkaryl refers to an alkyl group covalently bonded to an aryl group; and alkoxy, aryloxy and alkylaryloxy refer to-O-alkyl, -O-aryl and-O-alkylaryl, respectively;

with the proviso that the following compounds are excluded:

3- (pyrrol-2-ylmethylene) -2-indolinone;

3- (5-chloro-3, 4-dimethylpyrrol-2-ylmethylene) -2-indolinone;

3- (3, 5-dimethyl-4-ethylpyrrol-2-yl) -2-indolinone;

3- (3, 5-dimethyl-4-ethoxycarbonylpyrrol-2-yl) -2-indolinone;

2- [ [ [ 1-ethyl-2, 3-dihydro-2-oxo-3- (1H-pyrrol-2-ylmethylene) -1H-indol-5-yl ] oxy ] methyl ] benzoic acid;

3- [ (1-methyl-5-nitro-imidazol-2-yl) methylene ] -2-indolinone;

3- (thiophen-2-ylmethylene) -2-indolinone;

3- [ (2-butyl-1H-imidazol-4-yl) methylene ] -2, 3-dihydro-2-oxo-1H-indole-7-acetic acid ethyl ester;

3- [ [ 2-butyl-1- [ (1, 1-dimethylethoxy) carbonyl ] -1H-imidazol-4-yl ] methylene ] -2, 3-dihydro-2-oxo-1H-indole-7-acetic acid ethyl ester;

5-benzoyl-3- [ (imidazol-2-yl) methylene ] -2-indolinone;

6-diethylamino-3- [ (isothiazol-2-yl) methylene ] -2-indolinone;

5-chloro-3- [ (thiazol-2-yl) methylene ] -2-indolinone; and

6-Nitro-3- [ (pyrrol-2-yl) methylene ] -2-indolinone.

2. A compound of the formula and its pharmaceutically acceptable salt

Wherein R is₁Is H;

R₂is O or S;

R₃is hydrogen;

R₄、R₅、R₆and R₇Each independently of the otherIs selected from the following groups: hydrogen, alkyl, alkoxy, aryl, aryloxy, alkylaryl, alkylaryloxy, halogen, trihalomethyl, S (O) R, SO₂NRR′、SO₃R、SR、NO₂、NRR′、OH、CN、C(O)R、OC(O)R、NHC(O)R、(CH₂)_nCO₂R and CONRR';

a is a five membered heteroaromatic ring selected from pyrazole, 1, 2, 3-triazole, 1, 2, 4-triazole, oxazole, isoxazole, thiazole, isothiazole, 2-sulfonylfuran, 4-alkylfuran, 1, 2, 3-oxadiazole, 1, 2, 4-oxadiazole, 1, 2, 5-oxadiazole, 1, 3, 4-oxadiazole, 1, 2, 3, 4-oxatriazole, 1, 2, 3, 5-oxatriazole, 1, 2, 3-thiadiazole, 1, 2, 4-thiadiazole, 1, 2, 5-thiadiazole, 1, 3, 4-thiadiazole, 1, 2, 3, 5-thiadiazole and tetrazole optionally substituted in one or more positions with: alkyl, alkoxy, aryl, aryloxy, alkaryl, alkylaryloxy, halogen, trihalomethyl, S (O) R, SO₂NRR′、SO₃R、SR、NO₂、NRR′、OH、CN、C(O)R、OC(O)R、NHC(O)R、(CH₂)_nCO₂R and CONRR';

n is 0 to 3;

r is H, alkyl or aryl; and

r' is H, alkyl or aryl;

wherein in the above definitions alkyl, aryl, alkaryl, alkoxy, aryloxy and alkylaryloxy have the same definitions as in claim 1,

with the proviso that the following compounds are excluded:

6-diethylamino-3- [ (isothiazol-2-yl) methylene ] -2-indolinone; and

5-chloro-3- [ (thiazol-2-yl) methylene ] -2-indolinone.

3. A compound according to claim 1 or 2, selected from the following compounds:

3- [ (3-methylpyrrol-2-yl) methylene ] -2-indolinone;

3- [ (3, 4-dimethylpyrrol-2-yl) methylene ] -2-indolinone;

3- [ (2-methylthiophen-5-yl) methylene ] -2-indolinone;

3- [ (3-methylthiophen-2-yl) methylene ] -2-indolinone;

3- ([4- (2-methoxycarbonylethyl) -3-methylpyrrol-5-yl ] methylene) -2-indolinone;

3- [ (4, 5-dimethyl-3-ethylpyrrol-2-yl) methylene ] -2-indolinone;

3- [ (5-methylimidazol-2-yl) methylene ] -2-indolinone;

5-chloro-3- [ (5-methylthiophen-2-yl) methylene ] -2-indolinone.

3- [ (3, 5-dimethylpyrrol-2-yl) methylene ] -5-nitro-2-indolinone;

3- [ (3- (2-carboxyethyl) -4-methylpyrrol-5-yl) methylene ] -2-indolinone;

5-chloro-3- [ (3, 5-dimethylpyrrol-2-yl) methylene ] -2-indolinone; and

3- [ (2, 4-dimethylpyrrol-5-yl) methylene ] -2-indolinone;

or a pharmaceutically acceptable salt thereof.

4. The compound of claim 3, wherein said compound is 3- [ (2, 4-dimethylpyrrol-5-yl) methylene ] -2-indolinone, or a pharmaceutically acceptable salt thereof.

5. A pharmaceutical composition comprising a compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

6. The pharmaceutical composition of claim 5, wherein the pharmaceutical composition is suitable for parenteral or subcutaneous administration, or is formulated as a depot preparation.