DE69829657T2 - 2-INDOLINONE DERIVATIVES AS MODULATORS OF PROTEIN KINASE ATIVITY - Google Patents

Description

introduction

The The present invention relates to novel 2-indolinone derivatives, in particular substituted diazaindolinones, pharmaceutical compositions, containing these compounds and the use of these compounds in the manufacture of drugs for the treatment of disorders, which involve abnormal protein kinase ("PK") activity.

background the invention

The The following is provided for background information only and is not considered prior art for the present invention accepted.

PKs are enzymes that phosphorylate hydroxy groups in tyrosine, Catalyze serine and threonine residues of proteins. The consequences this seemingly simple activity is staggering, cell growth, Differentiation and proliferation, i. almost all aspects of the Cell life hanging in one way or another from PK activity. Of Further, abnormal PK activity associated with a bevy of diseases that are not of relative life-threatening diseases, such as psoriasis, too extremely vicious Diseases such as glioblastoma (brain cancer) are sufficient.

The PKs can usually be divided into two classes, the protein tyrosine kinases (PTKs) and the serine-threonine kinases (STKs).

one the main aspects of PTK activity is their involvement in growth factor receptors. Growth factor receptors are cell surface proteins. If they are bound by a growth factor ligand, then the growth factor receptors converted into an active form, which interact with proteins on the inner surface of a cell membrane. this leads to for the phosphorylation of tyrosine residues of the receptor and others Proteins and the formation of complexes with a variety of cytoplasmic signaling molecules within the cell, which in turn generates numerous cellular responses, such as cell division (proliferation), cell differentiation, Cell growth, expression of metabolic effects on the extracellular microenvironment, etc. influence. For a more complete one For discussion see Schlessinger and Ullrich, Neuron, 9: 303-391 (1992).

Growth factor receptors with PK activity are known as receptor tyrosine kinases ("RTKs"), which comprise a large family of transmembrane receptors with different biological activity. Present are at least nineteen (19) distinct subfamilies of RTKs been identified. An example of this is the subfamily, referred to as "HER" RTKs which EGFR (epithelial growth factor receptor), HER2, Includes HER3 and HER4. These RTKs consist of an extracellular glycosylated Ligand binding domain, a transmembrane domain and an intracellular cytoplasmic catalytic Domain that can phosphorylate tyrosine residues on proteins.

A another RTK subfamily consists of an insulin receptor (IR), an insulin-like Growth factor-I receptor (IGF-1R) and the insulin receptor-related receptor (IRR). IR and IGF-1R interact with insulin, IGF-I and IGF-II to form a heterotetramer of two completely extracellular glycosylated a subunits and two ß subunits to form the cell membrane cross and contain the tyrosine kinase domain.

A third RTK subfamily is referred to as platelet-derived growth factor receptor group ("PDGFR") PDGFRa, PDGFRβ, Includes CSFIR, c-kit and c-fms. These receptors consist of glycosylated extracellular Domains consisting of variable numbers of immunoglobin-like Loops are composed and an intracellular domain, the tyrosine kinase domains being unlinked amino acid sequences to be interrupted.

A other group, sometimes because of their similarity to the PDGFR subfamily subsumed in the latter group is the fetal liver kinase ("flk") receptor subfamily. This group is thought to be derived from kinase insert domain receptor fetal Liver kinase-1 (KDR / FLK-1), flk-1R, flk-4 and fms-like Tyrosine kinase 1 (flt-1) is composed.

Another member of the tyrosine kinase growth factor receptor family is fibroblast growth These groups consist of four receptors, FGFR1-4 and seven ligands, FGF1-7 Although not yet well defined, it appears that the receptors from a glycosylated extracellular domain contain a variable number of immunoglobin-like loops, and an intracellular domain in which the PTK sequence is interrupted by regions of unbound amino acid sequences.

A complete List of known RTK subfamilies is reported by Plowman et al., DN & P, 7 (6): 334-339 (1994).

In addition to RTKs also have a family of fully intracellular PTKs, called "non-receptor tyrosine kinases" or "cellular tyrosine kinases". This latter term, abbreviated "CTK", is used here become. CTKs contain no extracellular and transmembrane domains. Currently are about 24 CTKs in 11 subfamilies (Src, Frk, Btk, Csk, Abl, Zap70, Fes / Fps, Fak, Jak and Ack). The Src subfamily seems so far the largest group of CTKs and includes Src, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk. For a detailed discussion about CTKs, see Bolen, Oncogene, 8: 2025-2031 (1993).

The Serine-threonine kinases or STKs, such as the CTKs, are predominant intracellularly although there are some STK-type receptor domes. STKs are the common the cytosolic kinases, i. Kinases that have their function in the Perform part of the cytoplasm, different from cytoplasmic organelles and cytoskeleton. The cytosol is the region in the cell, where much of the intervening metabolism and biosynthetic activity the cell occurs, e.g. become the proteins in the cytosols Synthesized ribosomes.

RTKs, CTKs and STKs are all in a bevy of pathogenic conditions, including significantly, a large one Number of different cancers included. Other pathogenic conditions, associated with PTKs include, without limitation, psoriasis, hepatic cirrhosis, diabetes, atherosclerosis, angiogenesis, restinosis, ocular diseases, rheumatoid arthritis and other inflammatory diseases, Autoimmune diseases and a variety of renal diseases.

in the With regard to cancer, two of the main hypotheses that are developed concern were to the excessive cellular Proliferation, which promotes tumor development, explaining functions, that are known to be PK-regulated. That is, it has been suggested that is malicious Cell growth resulting from a breakdown in the mechanism which controls cell division and / or differentiation. One has protein products have been shown in a number of proto-oncogenes Signaling pathway that regulates cell growth and differentiation, are involved. These protein products of proto-oncogenes include the extracellular growth factors, Transmembrane growth factor PTK receptors (RTKs), cytoplasmic PTKs (CTKs) and cytosolic STKs as discussed above.

In Considering the obvious link between PK related cellular activities and a number of human diseases, it's no surprise that a big one Effort is spent in an attempt to identify ways about the PK activity to modulate.

Some of which have included biomimetic experiments, with large molecules, the Tailored to those in the actual cellular procedure (e.g., mutant ligands (U.S.N. 4,966,849); soluble Receptors and antibodies (Nos. WO 94/10202, Kendall and Thomas, Proc. Nat 1 Acad. Sci. 90: 10705-09 (1994), Kim, et al., Nature, 362: 841-844 (1993)); RNA ligands (Jelinek, et al., Biochemistry, 33: 10450-56); Takano, et al., Mol. Bio. Cell 4: 358A (1993); Kinsella, et al., Exp. Cell Res. 199: 56-62 (1992); Wright, et al., J. Cellular Phys., 152: 448-57) and tyrosine kinase inhibitors (WO 94/03427, WO 92/21660, WO 91/15495, WO 94/14808, U.S. Pat. 5,330,992; Mariani, et al., Proc. At the. Assoc. Cancer Res., 35: 2268 (1994)).

Recently Attempts have been made to identify small molecules that are known as PK inhibitors act. For example, bis-monocyclic, bicyclic and heterocyclic aryl compounds (PCT WO 92/20642), vinylene azaindole derivatives (PCT WO 94/14808) and 1-cyclopropyl-4-pyridylchinols (U.S. Pat. No. 5,330,992) have been described as PTK inhibitors. styrenics (U.S. Pat. No. 5,217,999), styrene-substituted pyridyl compounds (U.S. Pat. No. 5,302,606), quinazoline derivatives (EP application no. 0 566 266 Al), selenoindole and selenide (PCT WO 94/03427), tricyclic Polyhydroxyl compounds (PCT WO 92/21660) and benzylphosphonic (PCT WO 91/15495) have all been described as PTK inhibitors, which are useful in the treatment of cancer.

Our own efforts to identify small organic molecules that modulate PK activity and therefore in the treatment and prevention of diseases that due to abnormal PK activity be driven forward, have us to the Discovery of a family of new 2-indolinol derivatives, the excellent PK modulation capability show and are the subject of this invention.

Summary the invention

The The present invention generally relates to novel 2-indolinol derivatives and their salts, the activity Receptor Protein Tyrosine Kinases (RTK), Non-Receptor Protein Tyrosine Kinases (CTK) and serine / threonine protein kinases (STK). Especially For example, the invention relates to 3-methylidenyl-substituted diaza-2-indolinols.

In addition concerns the present invention pharmacological compositions of disclosed compounds and their physiologically suitable salts and a pharmaceutically acceptable diluent or carrier.

The The invention also relates to the use of these compounds in the manufacture of medicines for the treatment of diseases, associated with abnormal protein kinase activity, such as Cancer, other proliferative diseases, diabetes, autoimmune diseases, inflammatory diseases and angiogenesis.

1. The connections

wobei
A² und A⁴ Stickstoff und A¹ und A³ Kohlenstoff sind oder A¹ und A³ Stickstoff und A² und A⁴ Kohlenstoff sind, wobei zu verstehen ist, dass wenn A¹, A², A³ und A⁴ Stickstoff ist, R³, R⁴, R⁵ bzw. R⁶ nicht vorhanden sind;
R¹ aus der Gruppe, die aus Wasserstoff, C1-C10 Alkyl, Cycloalkyl, C2-C10 Alkenyl, C2-C10 Alkinyl, Aryl, Heteroaryl, Trihalomethancarbonyl, Heteroalicyclen, Hydroxy, C1-C10 Alkoxy, C-Carboxy, O-Carboxy, C-Amido, C-Thioamido, Guanyl, Guanidinylyl, Ureidylyl, Sulfonyl und Trihalogenmethansulfonyl besteht, ausgewählt wird.
R² aus der Gruppe, die aus Wasserstoff, C1-C10 Alkyl, Cycloalkyl, Aryl und Halogen besteht, ausgewählt wird;
R³, R⁴, R⁵ und R⁶ unabhängig voneinander aus der Gruppe, die aus Wasserstoff, C1-C10 Alkyl, Trihalogenmethyl, Cycloalkyl, C2-C10 Alkenyl, C2-C10 Alkinyl, Aryl, Heteroaryl, Heteroalicyclen, Hydroxy, Thiohydroxy, C1-C10 Alkoxy, Aryloxy, Thiohydroxy, C1-C10 Thioalkoxy, Thioaryloxy, Sulfinyl, Sulfonyl, S-Sulfonamido, N-Sulfonamido, N-Trihalogenmethansulfonamido, Carbonyl, Aldehyd, Trihalogenmethylcarbonyl, C-Carboxy, O-Carboxy, Cyano, Nitro, Halogen, Cyanato, Isocyanato, Thiocyanato, Isothiocyanato, O-Carbamyl, N-Carbamyl, O-Thiocarbamyl, N-Thiocarbamyl, Phosphonyl, Guanyl, Guanidinylyl, Ureidyl, C-Amido, N-Amido, Amino, quartäres Ammonium und -NR¹⁸R¹⁹ besteht, ausgewählt werden.In a first aspect, the present invention relates to compounds having the general chemical structure:
Sulfinyl, sulfonyl, S-sulfonamido, N-sulfonamido, N-trihalomethanesulfonamido, carbonyl, aldehyde, trihalomethylcarbonyl, C-carboxy, O-carboxy, cyano, nitro,

in which
A ² and A ^{4 are} nitrogen and A ¹ and A ^{3 are} carbon or A ¹ and A ^{3 are} nitrogen and A ² and A ^{4 are} carbon, it being understood that when A ¹ , A ² , A ³ and A ^{4 are} nitrogen , R ³ , R ⁴ , R ⁵ and R ⁶ are absent;
R ^{1 is selected} from the group consisting of hydrogen, C 1 -C 10 alkyl, cycloalkyl, C 2 -C 10 alkenyl, C 2 -C 10 alkynyl, aryl, heteroaryl, trihalomethanecarbonyl, heteroalicyclic, hydroxy, C 1 -C 10 alkoxy, C 2 -C 4 carboxy, O-carboxy, C-amido, C-thioamido, guanyl, guanidinylyl, ureidylyl, sulfonyl and trihalomethanesulfonyl is selected.
R ^{2 is selected} from the group consisting of hydrogen, C ¹ -C 10 alkyl, cycloalkyl, aryl and halogen;
R ³ , R ⁴ , R ⁵ and R ^{6 are} independently selected from the group consisting of hydrogen, C 1 -C 10 alkyl, trihalomethyl, cycloalkyl, C 2 -C 10 alkenyl, C 2 -C 10 alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxy, thiohydroxy, C1-C10 alkoxy, aryloxy, thiohydroxy, C1-C10 thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, S-sulfonamido, N-sulfonamido, N-trihalomethanesulfonamido, carbonyl, aldehyde, trihalomethylcarbonyl, C-carboxy, O-carboxy, cyano, nitro, Halogen, cyanato, isocyanato, thiocyanato, isothiocyanato, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, phosphonyl, guanyl, guanidinyl, ureidyl, C-amido, N-amido, amino, quaternary ammonium and -NR ¹⁸ R ¹⁹ is selected.

ist;
J¹ aus der Gruppe, die aus Sauerstoff, Stickstoff und Schwefel besteht, ausgewählt wird;
J², J³ und J⁴ unabhängig aus der Gruppe, die aus Kohlenstoff, Stickstoff, Sauerstoff und Schwefel besteht, ausgewählt werden, so dass der fünfgliedrige Heteroarylring eine in der Chemie bekannte Verbindung ist, und es des Weiteren zu verstehen ist, dass, wenn J², J³ oder J⁴ Stickstoff, Sauerstoff oder Schwefel sind, R⁸, R^8' bzw. R^8'' nicht existieren; gleichermaßen existiert R⁷ nicht, wenn J¹ Sauerstoff oder Schwefel ist;
R⁷ aus der Gruppe, die aus Wasserstoff, C1-C10 Alkyl, Cycloalkyl, Trihalogenmethylcarbonyl, Aryl, Guanyl, Carboxyl, Sulfonyl und Trihalogenmethansulfonyl besteht, ausgewählt wird;
einer von R⁸, R^8' oder R^8'' -H oder eine -(alk₁)rM-Gruppe ist, wobei (alk₁) CH₂ ist; r 1 bis einschließlich 3 ist und M aus der Gruppe, die aus Hydroxy, Amino, Carboxy, Morpholinyl, Piperazinyl, Tetrazolyl, N-Carbamyl, O-Carbamyl, S-Sulfonamido, N-Sulfonamido, Sulfonyl, -S(O)₂OH und Phosphonyl besteht, ausgewählt wird;
die übrigen beiden von R⁸, R^8' und R^8'' unabhängig aus der Gruppe, die aus:
Wasserstoff;
unsubstituiertem C1-C4 Alkyl;
C1-C4 Alkyl substituiert mit einer oder mehreren Gruppen ausgewählt aus der Gruppe bestehend aus: Halogen, Hydroxyl, unsubstituiertem C1-C4 Alkoxy, Amino, S-Sulfonamido, -NR¹⁸R¹⁹ oder Carboxy;
unsubstituiertem C1-C4 Alkoxy;
C1-C4 Alkoxy substituiert mit einem oder mehreren Halogenen;
unsubstituiertem Aryl;
Aryl substituiert mit einer oder mehreren Gruppen ausgewählt aus der Gruppe bestehend aus unsubstituiertem C1-C4 Alkyl, unsubstituiertem C1-C4 Alkoxy, Halogen, Trihalogenmethyl, Amino, -NR¹⁸R¹⁹, Hydroxy oder S-Sulfonamido;
unsubstituiertem Heteroaryl;
Heteroaryl substituiert mit einer oder mehreren Gruppen ausgewählt aus unsubstituiertem C1-C4 Alkyl, unsubstituiertem C1-C4 Alkoxy, Halogen, Trihalogenmethyl, Amino, -NR¹⁸R¹⁹, Hydroxy oder S-Sulfonamido;
unsubstituierten Heteroalicyclen;
Heteroalicyclen substituiert mit einer oder mehreren Gruppen ausgewählt aus unsubstituiertem C1-C4 Alkyl, Halogen, Hydroxy, unsubstituiertem Alkoxy, Amino oder -NR¹⁸R¹⁹;
Halogen;
Cyano;
Carboxy; und
einem, durch die Kombination von R⁸ und R^8' oder R^8' und R^8'' gebildeten, sechsgliedrigen Cycloalkyl-, Aryl-, Heteroaryl- oder heteroalicyclischen Ring;
und physiologisch annehmbare Salze davon besteht, ausgewählt werden.R ¹⁸ and R ^{19 are} independently selected from the group consisting of hydrogen, C 1 -C 10 alkyl, cycloalkyl, aryl, carbonyl, acetyl, trihalomethylcarbonyl, C-carboxy, trihalomethanesulfonyl, sulfonyl, C-peptidyl and, a five- or six-membered heteroalicylic Ring exists, be selected;
Z is oxygen;

is;
J ^{1 is selected} from the group consisting of oxygen, nitrogen and sulfur;
J ² , J ³ and J ^{4 are} independently selected from the group consisting of carbon, nitrogen, oxygen and sulfur such that the five-membered heteroaryl ring is a compound known in the chemical arts, and it is further to be understood that when J ² , J ³ or J ^{4 are} nitrogen, oxygen or sulfur, R ⁸ , R ^{8 '} and R ^{8 ",} respectively, do not exist; similarly, R ⁷ does not exist if J ^{1 is} oxygen or sulfur;
R ^{7 is selected} from the group consisting of hydrogen, C 1 -C 10 alkyl, cycloalkyl, trihalomethylcarbonyl, aryl, guanyl, carboxyl, sulfonyl and trihalomethanesulfonyl;
one of R ⁸ , R ^{8 '} or R ^{8 "} -H or a - (alk ₁ ) rM group, wherein (alk ₁ ) is CH ₂ ; r is 1 to 3 inclusive, and M is selected from the group consisting of hydroxy, amino, carboxy, morpholinyl, piperazinyl, tetrazolyl, N-carbamyl, O-carbamyl, S-sulfonamido, N-sulfonamido, sulfonyl, -S (O) ₂ OH and phosphonyl is selected;
the remaining two of R ⁸ , R ^{8 '} and R ^8''are independently selected from the group consisting of:
Hydrogen;
unsubstituted C1-C4 alkyl;
C 1 -C 4 alkyl substituted with one or more groups selected from the group consisting of: halogen, hydroxyl, unsubstituted C 1 -C 4 alkoxy, amino, S-sulfonamido, -NR ¹⁸ R ¹⁹ or carboxy;
unsubstituted C1-C4 alkoxy;
C1-C4 alkoxy substituted with one or more halogens;
unsubstituted aryl;
Aryl substituted with one or more groups selected from the group consisting of unsubstituted C 1 -C 4 alkyl, unsubstituted C 1 -C 4 alkoxy, halogen, trihalomethyl, amino, -NR ¹⁸ R ¹⁹ , hydroxy or S-sulfonamido;
unsubstituted heteroaryl;
Heteroaryl substituted with one or more groups selected from unsubstituted C 1 -C 4 alkyl, unsubstituted C 1 -C 4 alkoxy, halogen, trihalomethyl, amino, -NR ¹⁸ R ¹⁹ , hydroxy or S-sulfonamido;
unsubstituted heteroalicyclic;
Heteroalicyclic substituted with one or more groups selected from unsubstituted C 1 -C 4 alkyl, halogen, hydroxy, unsubstituted alkoxy, amino or -NR ¹⁸ R ¹⁹ ;
Halogen;
cyano;
carboxy; and
a six-membered cycloalkyl, aryl, heteroaryl or heteroalicyclic ring formed by the combination of R ⁸ and R ^{8 '} or R ^8' and R ^{8 "} ;
and physiologically acceptable salts thereof.

As used herein, the term "alkyl" refers to a saturated aliphatic hydrocarbon containing straight chain and branched chain groups, the alkyl group having 1 to 10 carbon atoms, most preferably a lower alkyl having 1 to 4 carbon atoms, and the alkyl group may be substituted or unsubstituted When substituted, the substituent group (s) is one or more selected singly from unsubstituted cycloalkyl selected from the group consisting of cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, adamantane, cyclohexadiene, cycloheptane and cycloheptatriene unsubstituted aryl selected from the group consisting of phenyl, naphthalenyl, and anthracenyl; unsubstituted heteroaryl selected from the group consisting of pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine , Pyrimidine, quinoline, isoquinoline, purine and carbazole, is selected; unsubstituted heteroal icyclens selected from the group consisting of piperidine, morpholine, piperazine, pyrroline, pyrrolidine, dihydrothiophene and tetrahydrofuran; hydroxy; C1-C4 alkoxy; aryloxy; thiohydroxy; C1-C4 thioalkoxy; thioaryloxy; cyano; Halogen; carbonyl; thiocarbonyl; O-carbamyl; N-carbamyl; O-thiocarbamyl; N-thiocarbamyl; C-amido; N-amido; C-carboxy, O-carboxy, cyanato; isocyanato; thiocyanato; isothiocyanato; nitro; silyl; Amino and -NR ¹⁸ R ¹⁹ , wherein R ¹⁸ and R ^{19 are} independently selected from the group consisting of hydrogen, unsubstituted C 1 -C 4 alkyl, unsubstituted cycloalkyl as defined above, and the like substituted aryl as defined above, carbonyl, trihalomethylcarbonyl, C-carboxy, trihalomethylsulfonyl, sulfonyl and combined a five-membered or six-membered heteroalicyclic ring as defined above, is selected.

A "cycloalkyl" group refers to a monocyclic or fused ring made entirely of carbon (ie, rings sharing an adjacent pair of carbon atoms) with one or more of the rings not having a fully conjugated pi-electron system The cycloalkyl group is selected from cyclopropane, cyclobutane Cyclopentane, cyclopentene, cyclohexane, adamantane, cyclohexadiene, cycloheptane and cycloheptatriene, a cycloalkyl group may be substituted or unsubstituted, and when substituted, the substituent group (s) is one or more selected individually from unsubstituted C1-C4 alkyl, unsubstituted aryl selected from the group consisting of phenyl, naphthenyl and anthracenyl; unsubstituted heteroaryl selected from the group consisting of pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, Isoquinoline, purine and carbazole, is selected; unsubstituted heteroalicyclic, which consists of the group consisting of piperidine, morpholine, piperazine, pyrroline, pyrrolidine, dihydrothiophene and tetrahydrofuran is selected; hydroxy; C1-C4 alkoxy; aryloxy; thiohydroxy; C1-C4 thioalkoxy; thioaryloxy; cyano; Halo; carbonyl; thiocarbonyl; carboxy; O-carbamyl; N-carbamyl; C-amido; N-amido; nitro; Amino and -NR ¹⁸ R ¹⁹ , with R ¹⁸ and R ¹⁹ as defined above, is selected.

An "Alkenyl" group concerns an alkyl group as defined herein which is at least two carbon atoms and at least one carbon-carbon double bond.

An "alkynyl" group is concerned an alkyl group as defined herein, consisting of at least two carbon atoms and at least one carbon-carbon triple bond.

An "aryl" group refers to all-carbon monocyclic or ring-fused polycyclic groups (ie, rings sharing adjacent pairs of carbon atoms) with a fully-fused pi-electron system The aryl group is selected from phenyl, naphthalenyl, and anthracenyl When substituted, the substituent group (s) is one or more individually selected from halo, trihalomethyl, unsubstituted C 1 -C 4 alkyl, unsubstituted aryl selected from the group consisting of phenyl, naphthalenyl and anthracenyl; is selected unsubstituted heteroaryl selected from the group consisting of pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline, purine and carbazole, unsubstituted heteroalicyclic, selected from the group consisting of piperidine, morpholine, piperazine, pyrroline, pyrrolidine, dihydrothiophene and tetrahyd rofuran, hydroxy, C1-C4 alkoxy, aryloxy, thiohydroxy, C1-C4 thioalkoxy, thioaryloxy, cyano, nitro, carbonyl, thiocarbonyl, C-carboxy, O-carboxy, O-carbamyl, N-carbamyl, O- Thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, sulfinyl, sulfonyl, S-sulfonamido, N-sulfonamido, trihalomethanesulfonamido, amino and -NR ¹⁸ R ¹⁹ , R ¹⁸ and R ¹⁹ , as defined above.

As used herein, a "heteroaryl" group refers to a monocyclic or fused ring group (ie, rings sharing an adjacent pair of atoms) having in the ring (s) one or more atoms selected from the group consisting of The heteroaryl group is selected from pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline, purine and carbazole The heteroaryl group may be substituted or unsubstituted. When substituted, the substituted group (s) is one or more selected from the group consisting of unsubstituted C1-C4 alkyl, unsubstituted cycloalkyl selected from the group consisting of cyclopropane, Cyclobutane, cyclopentane, cyclopentene, cyclohexane, adamantane, cyclohexadiene, cycloheptane and cycloheptatriene, is selected, unsubstituted aryl selected from the group consisting of phenyl, Naphthalenyl and anthracenyl, halogen, trihalomethyl, hydroxy, C 1 -C 4 alkoxy, aryloxy, thiohydroxy, C 1 -C 4 thioalkoxy, thioaryloxy, cyano, nitro, carbonyl, thiocarbonyl, sulfonamido, carboxy, sulfinyl, sulfonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, amino and -NR ¹⁸ R ¹⁹ , wherein R ¹⁸ and R ^{19 are} as defined above.

A "heteroalicyclic" group refers to a monocyclic or fused ring group having in the ring (s) one or more atoms selected from the group consisting of nitrogen, oxygen and sulfur The heteroalicyclic group is selected from the group consisting of piperidine, morpholine, piperazine, pyrroline, pyrrolidine, dihydrothiophene, and tetrahydrofuran The heteroalicyclic ring may be substituted or unsubstituted, and when substituted, it may be substituted with one or more double bonds. the substituted group (s) is one or more selected from unsubstituted C1-C4 alkyl, unsubstituted cycloalkyl derived from the group consisting of cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, adamantane, cyclohexadiene, cycloheptane and cycloheptatriene is selected from halogen, trihalomethyl, hydroxy, C 1 -C 4 alkoxy, aryloxy, thiohydroxy, C 1 -C 4 thioalkoxy, thioaryloxy, cyano , Nitro, carbonyl, thiocarbonyl, carboxy, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, sulfinyl, sulfonyl, C-amido, N-amido, amino and -NR ¹⁸ R ¹⁹ , wherein R ¹⁸ and R ¹⁹ as defined above is selected.

A "hydroxy" group concerns an -OH group.

An "alkoxy" group is concerned both an -O-alkyl and an -O-cycloalkyl group, wherein alkyl and cycloalkyl are as defined above.

An "aryloxy" group is concerned both an -O-aryl and an -O-heteroaryl group, wherein aryl and Heteroaryl are as defined above.

A "heteroalicycloxy" group is concerned an -O-heteroalicyclic Group, wherein heteroalicyclic is as defined above.

A "thiohydroxy" group is concerned a -SH group.

A "thioalkoxy" group is concerned both an S-alkyl and an -S-cycloalkyl group, wherein alkyl and Cycloalkyl are as defined above.

A "thioaryloxy" group is concerned both an -S-aryl and an -S-heteroaryl group, wherein aryl and Heteroaryl are as defined above.

A "carbonyl" group concerns a -C (= O) -R "group, where R" is selected from the group consisting of hydrogen, Alkyl, cycloalkyl, aryl, heteroaryl (bonded by a ring carbon) and heteroalicyclic (bound by a ring carbon), such as defined above, is selected becomes.

A "trihalomethylcarbonyl" group refers to an X ₃ CC (= O) group wherein X is a halo group as defined herein.

An "aldehyde" group is concerned a carbonyl group, where R "is hydrogen.

A "thiocarbonyl" group is concerned a -C (= S) -R "group, with R" as defined herein.

An "O-carboxy" group is concerned a R''C (= O) O- group, with R '' as defined here.

A "C-carboxy" group is concerned a -C (= O) OR "group with R" as defined herein.

An "acetyl" group refers to a -C (= O) CH ₃ group.

A "carboxyalkyl" group refers to a - (CH ₂ ) _s C (O) OR ", where s is 1-6 and R" is as defined above.

A "carboxylic acid" group is concerned a C-carboxy group in which R "is hydrogen.

A "halogen" group concerns Fluorine, chlorine, bromine or iodine.

A "trihalomethyl" group refers to a -CX ₃ group wherein X is a halo group as defined herein.

A "trihalomethanesulfonyl" group refers to an X ₃ CS (= O) ₂ group with X as defined above.

A "cyano" group refers to a -C = N group.

A "cyanato" group concerns a -CNO group.

An "isocyanato" group is concerned a -NCO group.

A "thiocyanato" group is concerned a CNS group.

An "isothiocyanato" group is concerned an -NCS group.

A "sulfinyl" group is concerned a -S (= O) -R "group, with R" as defined herein.

A "sulfonyl" group refers to a -S (= O) ₂ R "group, with R" as defined herein.

A "sulfonamido" group refers to a -S (= O) ₂ -NR ¹⁸ R ¹⁹ group with R ¹⁸ and R ¹⁹ as defined herein.

A "trihalomethanesulfonamido" group refers to an X ₃ CS (= O) ₂ N (R ¹⁸ ) group with X and R ¹⁸ as defined herein.

An "O-carbamyl" group refers to an -OC (= O) NR ¹⁸ R ¹⁹ group with R ¹⁸ and R ¹⁹ as defined herein.

An "N-carbamyl" group refers to an R ¹⁹ OC (= O) NR ¹⁸ group, with R ¹⁸ and R ¹⁹ as defined herein.

An "O-thiocarbamyl" group refers to an -OC (= S) -NR ¹⁸ R ¹⁹ group with R ¹⁸ and R ¹⁹ as defined herein.

An "N-thiocarbamyl" group refers to an R ¹⁹ OC (= S) NR ¹⁸ group, with R ¹⁸ and R ¹⁹ as defined herein.

An "amino" group refers to an -NR ¹⁸ R ¹⁹ group, wherein R ¹⁸ and R ^{19 are} both hydrogen.

A "quaternary ammonium" group refers to a - ⁺ N ¹⁸ R ^{18 '} R ¹⁹ group with R ¹⁸ and R ¹⁹ as defined herein and R ^18' same as R ¹⁸ and R ¹⁹ .

A "C-amido" group refers to a -C (= O) -NR ¹⁸ R ¹⁹ group with R ¹⁸ and R ¹⁹ as defined herein.

An "N-amido" group refers to an R ¹⁸ C (= O) NR ¹⁹ group, with R ¹⁸ and R ¹⁹ as defined herein.

A "C-thioamido" group refers to a -C (= S) NR ¹⁸ R ¹⁹ group, with R ¹⁸ and R ¹⁹ as defined herein.

A "nitro" group refers to a -NO ₂ group.

A "methylenedioxy" group refers to an -OCH ₂ O group wherein the oxygen atoms are attached to adjacent ring carbon atoms.

An "ethylenedioxy" group refers to an -OCH ₂ CH ₂ O- group wherein the oxygen atoms are attached to adjacent ring carbon atoms.

A "guanyl" group refers to an R ¹⁸ R ¹⁹ NC (= N) group with R ¹⁸ and R ¹⁹ as defined herein.

A "guanadinyl" group refers to a -R ¹⁸ NC (= N) NR ^{18 '} R ¹⁹ group with R ¹⁸ and R ¹⁹ as defined herein, wherein R ^18' is defined as well as R ¹⁸ and R ¹⁹ .

A "ureidyl" group refers to a -NR ¹⁸ C (= O) NR ^{18 '} R ¹⁹ group with R ¹⁸ and R ¹⁹ as defined herein and R ^18' same as R ¹⁸ and R ¹⁹ .

A "phosphonyl" group refers to an -OP (= O) ₂ OR "group with R" as defined herein.

A "C-peptidyl" group refers to a group formed by the reaction of the 2-amino group of one amino acid with the carboxy group of another amino acid, ie a peptidyl group has the general structure - (NCHRC (= O) _n -, wherein the R group may be the same or different, depending on which amino acid is used to produce the peptidyl group, the "C-peptidyl" designation referring to the situation where the peptidyl group is attached to a non-amino acid molecule is bound by the C (= O) carbon.

A "polar group" refers to a group in which the nucleus of the atoms covalently bound together to form the group does not share the electrons of the covalent bond (s) they have in common, ie the electron cloud is at one This means that one end of the covalent bond (s) is relatively negative and the other end is relatively positive, ie there is a negative pole and a positive pole. Examples of polar groups include, without limitation, hydroxy , Alkoxy, Carboxy, nitro, cyano, amino, quaternary ammonium, amido, ureidyl, sulfonamido, sulfinyl, sulfonyl, phosphono, morpholino, piperazinyl and tetrazolyl.

A "spirocycloalkyl" group is concerned a cycloalkyl group having one ring carbon atom with another Ring divides.

A "spiroheteroalicyclic" group is concerned a heteroalicyclic group containing one ring atom with another Ring divides.

An "N-hydroxylamino" group refers to a group R''ONR ¹⁸ group with R "and R ¹⁸ as defined herein.

A "polyhydroxyalkyl" group relates an unbranched alkyl group of 1 to 8 carbons, preferably 1 to 4 carbons containing only 2 or more, preferably 3 or more more hydroxyl groups is substituted.

A "morpholine" group refers to a group with the chemical structure:

A "tetrazolyl" group refers to a group with the chemical structure:

An "aminotriazoly" group refers to a group having the chemical structure:

A "1-piperazinyl" group refers to a group with the chemical structure:

Non-limiting examples of 5-membered heteroaryl rings Q known in the chemical art include the following:

B. Preferred structural characteristics

In a preferred feature of this invention, R ¹ , R ² and R ^{7 are} hydrogen.

In another preferred feature of the invention, R ³ , R ⁴ , R ⁵ and R ^{6 are} independently selected from the group consisting of hydrogen, unsubstituted C ¹ -C ⁴ alkyl, C ¹ -C ⁴ alkyl substituted with a group is comprised of the group consisting of unsubstituted cycloalkyl, halo, hydroxy, unsubstituted C1-C4-alkoxy, C-carboxy, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heteroalicyclic, amino, quaternary ammonium or -NR ¹⁸ NR ^19, selected, unsubstituted cycloalkyl , Hydroxy, unsubstituted C1-C4 alkoxy, C1-C4 alkoxy substituted with a group selected from the group consisting of one or more halogen groups, unsubstituted aryl or unsubstituted heteroaryl, trihalomethyl, halogen, unsubstituted aryl, aryl substituted with one or more groups independently selected from the group consisting of unsubstituted C 1 -C 4 alkyl, C 1 -C 4 alkyl having one or more halogen groups, trihal is substituted omethyl, halogen, hydroxy or unsubstituted C1-C4 alkoxy, is selected, unsubstituted aryloxy, aryloxy, which is substituted with one or more groups independently selected from the group consisting of halogen, unsubstituted C1-C4 alkyl , C1-C4-alkyl, which is with one or more halo groups, unsubstituted aryl, hydroxy, unsubstituted C1-C4-alkoxy, amino or -NR ¹⁸ NR ¹⁹ group is substituted, is selected; S-sulfonamido; unsubstituted heteroaryl; Heteroaryl which is substituted with one or more groups independently selected from the group consisting of hydrogen, unsubstituted C1-C4 alkyl, C1-C4 alkyl containing one or more halo groups, trihalomethyl, halogen, hydroxy, unsubstituted C1- C4 alkoxy, unsubstituted aryloxy, amino, S-sulfonamido or -NR ¹⁸ NR ¹⁹ group is substituted, there is, is selected; unsubstituted heteroalicyclic; A heteroalicyclic substituted with one or more groups independently selected from the group consisting of unsubstituted C1-C4 alkyl, C1-C4 alkyl containing one or more halo groups, unsubstituted C1-C4 alkyl alkoxy, hydroxy, amino, S-sulfonamido or NR ¹⁸ NR ^{19 is} substituted, is selected; unsubstituted C1-C4 alkyl C-carboxy; Carboxylic acid; unsubstituted C 1 -C 4 -alkylcarbonyl; Aldehyde; unsubstituted arylcarbonyl; acetyl; S-sulfonamido; N-sulfonamido; Amino and -NR ¹⁸ NR ¹⁹ is selected.

Additional preferred features of this invention are those in which:
Any two of R ⁸ , R ^{8 '} or R ^{8 "} - (alk ₁ ) r M groups are; J ^{1 is selected} from the group consisting of nitrogen, oxygen and sulfur and J ² , J ³ and J ^{4 are} carbon; J ¹ and J ^{3 are} nitrogen and J ² and J ^{4 are} carbon.

Examples specific compounds of these inventions are shown in Table below 1 shown.

2. The biochemistry

The RTK-mediated signal transduction is by extracellular interaction followed by a specific growth factor (ligand) receptor dimerization, transient stimulation intrinsic protein tyrosine kinase activity and phosphorylation. The Binding pages are used for intracellular Signal relay molecules formed and lead to form complexes with a spectrum of cytoplasmic Signaling molecules, which is the appropriate cellular Facilitate response (e.g., cell division, metabolic activity on the extracellular Microenvironment). See Schlessinger and Ullrich, 1992, Neuron 9: 303-391.

you has shown that tyrosine phosphorylation sites at growth factor receptors as high affinity binding sites for SH2 (Src homology) domains of signaling molecules act. Fantl et al., 1992, Cell 69: 413-423; Songyang et al., 1994, Mol. Cell. Biol. 14: 2777-2785; Songyang et al., 1993, Cell 72: 767-778; and Koch et al., 1991, Science 252: 668-678. Various intracellular substrate proteins with RTKs have been identified have been identified. You can in be divided into two main groups: (1) substrates containing a catalytic Domain and (2) substrates lacking such a domain, but serve as adapters and connected with catalytically active molecules are. Songyang et al., 1993, Cell 72: 767-778. The specificity of the interactions between receptors and SH2 domains of their substrates is determined by the Determines amino acid residues, which immediately surround the phosphorylated tyrosine residue. differences in the binding affinities between SH2 domains and the amino acid sequences, surrounding the phosphortyrosine residues on special receptors, are with the observed differences in their substrate phosphorylation profiles consistent. Songyang et al., 1993, Cell 72: 767-778. These observations suggest that the function of each RTK not only from its expression pattern and the ligand availability be determined, but also by the arrangement of the downstream Signaling pathways activated by a special receptor. Consequently Phosphorylation is an important regulatory step ready, the selectivity the signaling pathways through specific growth factor receptors added are determined, as well as the discrimination factor receptors.

STKs, the main ones cytosolic, often affect the internal biochemistry of the cell as a downline response to a PTK event. STKs are attacking the signaling process that involves DNA synthesis and subsequent mitosis, which leads to cell proliferation, starts.

Consequently results in PK signaling, among other responses in cell proliferation, Differentiation, growth and metabolism. Abnormal cell proliferation can cause a wide range of diseases and disorders, including Development of neoplasia, such as carcinoma, sarcoma, leukemia, glioblastoma, Hemangioma, psoriasis, atherosclerosis, arthritis and diabetic Retinopathy (or other diseases with uncontrolled Angiogenesis and / or vasculogenesis).

One accurate understanding of the mechanism by which the compounds of this Inventions inhibiting PKs is not required to the present invention To carry out invention.

While you are not tied to a specific mechanism or theory here However, it is believed that the connections with the amino acids interact with the catalytic region of the PK. PKs typically own a bilobate structure, where ATP occurs in the column between two Lobes in a region where the amino acids are conserved between the PKs to tie up. It is believed that inhibitors of PKs by non-covalent interactions, e.g. Hydrogen bonding, van the Waals forces and ionic interactions in the same general region, in which binds the aforementioned ATP to the PKs. More specifically It is assumed that the 2-indolinone component the compounds of this invention in the general interspace, which is normally occupied by the adenine ring of the ATP binds. The specificity a special molecule for one Special PK may be as a result of additional interactions between the various substituents of the 2-indolinone nucleus with amino acid domains, the for specific PKs are specific. Thus, different Indolinone substituents contribute to preferential binding to certain PKs. The ability, these compounds that bind to different ATP binding sites (or another nucleotide) are active, making these compounds suitable to obtain any protein with such a side i.e. not only PKs, but also protein phosphatase. This one thus disclosed compounds have use for in vitro Assays for these proteins and for in vivo therapeutic effects through these proteins.

In another aspect, this invention relates to the use of a compound according to the invention in the manufacture of a medicament for the treatment of a disease arising from cancer resulting from the disease Group, which consists of squamous cell carcinoma, astrocytoma, glioblastoma, breast cancer, glioma, melanoma, lung cancer, small cell lung cancer, bladder cancer, ovarian cancer, prostate cancer and head and neck cancer is selected, a proliferative disorder consisting of the group consisting of restinosis, fibrosis, psoriasis , Osteoarthritis and rheumatoid arthritis is selected; Diabetes, autoimmune diseases, an inflammatory disease and angiogenesis is selected.

3. Pharmacological Compositions and Therapeutic Applications

In In another aspect, the present invention relates to a pharmaceutical Composition containing a compound or a salt as described herein and a pharmaceutically acceptable carrier or diluent includes.

A. modes of administration

As As used herein, "administering" or "administering" refers to the delivery of a Compound, salt or prodrug of the present invention or a pharmacological composition containing a compound, a salt or a prodrug of this invention, to an organism for Purposes of prevention or treatment of a PK-related disease.

suitable Modes of administration without restriction oral, rectal, transmucosal or intestinal administration or intramuscular, subcutaneous, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, contain intranasal or intraocular injections.

alternative One can connect more locally rather than systematically Way, for example about an injection of the compound directly into a solid tumor, often in a depot or sustained release formulation.

Of Furthermore, one can use the drug in a targeted pharmacotherapy system, e.g. in a liposome coated with tumor-specific antibodies is, administer. The liposomes are recognized by the tumor and selectively absorbed by him.

Composition / Formulation

The Pharmacological compositions of the present invention can by Methods that are well known in the art made be, e.g. by conventional mixing, dissolving, granulating, Dragee making, triturating, emulsifying, encapsulating, trapping or lyophilization process.

pharmacological Compositions for the use according to the present Invention can thus formulated in a conventional manner using one or more physiologically suitable carriers, the excipients and Tools include, what the processing of the active compounds in preparations which can be used pharmaceutically. A suitable formulation depends from the chosen one Administration method.

For the injection can the compounds of the invention in aqueous solutions, preferably in physiological compatible buffers, e.g. Hank's solution, Ringers solution or physiological saline become. For The transmucosal administration will be penetrants which for the permeable barrier are used in the formulation. These penetrants are well known in the art.

For oral administration, the compounds can be readily formulated by combining the active compounds with pharmaceutically acceptable carriers 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 of a subject to be treated. Pharmaceutical preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules after adding suitable adjuvants, if necessary to obtain tablets or dragee cores. Suitable excipients are especially fillers, such as sugars, including lactose, sucrose, mannitol or sorbitol, cellulose preparations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellu loosely, hydroxypropylmethylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone (PVP). If necessary, dissolving agents, such as the crosslinked polyvinylpyrrolidone, agar or alginic acid or a salt thereof, such as sodium alginate, may be added.

tablet cores are provided with suitable coatings. To this end can concentrated sugar solutions which can be used optionally gum arabic, tallow, polyvinylpyrrolidone, Carbopol gel, polyethylene glycol and / or titanium dioxide, liqueur solutions and suitable organic solvents or solvent mixtures can contain. Dyes or pigments can for identification or for different combinations of active To be able to characterize junction boxes, to tablets or dragee coatings be added.

pharmacological Compositions that can be used orally include push-fit capsules, which are made of gelatin, as well as soft, sealed Capsules made of gelatin and a plasticizer, such as Glycerol or sorbitol. The push-fit capsules can be the active ingredients in a mixture with a filler, such as lactose, binders such as starches, and / or Lubricants, such as tallow or magnesium stearate and optional Stabilizers included. In soft capsules, the active compounds in suitable liquids, such as fatty oils, liquid Paraffin or liquid Polyethylene glycols, dissolved or suspended. additionally can Added stabilizers become.

All Formulations for oral administration should be suitable for administration Be dosages.

For buccal Administration can the compositions take the form of tablets or lozenges which on conventional Be formulated.

to Administration by inhalation will be the compounds for use according to the present Invention usually in the form of an aerosol spray presentation from compacted packages or a nebulizer, using a suitable Propellant gas, e.g. Dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, Carbon dioxide or another suitable gas. In the case of a compacted Aerosols can control the dosage unit by providing a valve, to deliver a measured amount. Capsules and Cartridges of, for example, gelatin for use in an inhaler or insufflator can formulated using a powder mixture of the compound and a suitable powder base, such as lactose or starch.

The Connections can for parenteral Administration by injection, e.g. by bolus injection or continuous Infusion, to be formulated. Formulations for injection may be in a unit dosage form, e.g. in ampoules or in Multidose containers with an added Preservative. The compositions may be in such forms as suspensions, solutions or emulsions in oily or aqueous Accept and can accept vehicles Formulation agents, such as suspending, stabilizing and / or dispersing agents.

pharmacological Compositions for Parenteral administration involves aqueous solutions of the active compounds in a water-soluble Shape. additionally can the suspensions of the active compounds as suitable oily injection suspensions getting produced. Suitable lipophilic solvents or vehicles include Fatty oils, such as sesame oil or synthetic fatty acid esters, such as ethyl oleate or triglycerides or liposomes.

Aqueous injection suspensions can Contain substances that increase the viscosity of the suspension, such as for example, sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also be suitable stabilizers or agents containing the solubility of the compounds increase, to the production of higher concentrated solutions to enable.

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

The Connections can also as rectal compositions, such as suppositories or retention enemas, the ordinary Suppository bases, such as cocoa butter or other glycerides be.

In addition to In the above-described formulations, the compounds may also be used be formulated as a depot preparation. These long-lasting formulations can by implantation (for example subcutaneously or intramuscularly) or by intramuscular Be administered injection. Thus, for example, the compounds with suitable polymeric or hydrophobic materials (for example as an emulsion in a suitable oil) or as ion exchange resins or as slightly soluble Derivatives, for example, as a slightly soluble salt formulated.

The Pharmacological compositions herein may also be suitable solid or gel phase carrier or excipient include. Examples of these carriers or excipient include calcium carbonate, calcium phosphate, various sugars, Strengthen, Cellulose derivatives, gelatin and polymers such as polyethylene glycols), but are not limited to this.

Many of the PK modulating compounds of the invention may be provided as physiologically acceptable salts, wherein the claimed compound may form the negatively or positively charged species. Examples of salts in which the compound forms the positively charged moiety include, without limitation, quaternary ammonium (defined elsewhere herein), salts such as the hydrochloride, sulfate, carbonate, lactate, tartrate, maleate, succinate, etc., the is formed by the reaction of an amino group with the appropriate acid. Salts in which the compound forms the negatively charged species include, without limitation, the sodium, potassium, calcium and magnesium salts formed by the reaction of a carboxylic acid group in the molecule with the appropriate base (eg, sodium hydroxide (NaOH), potassium hydroxide, ( KOH), calcium hydroxide (Ca (OH) ₂ ), etc.).

C. Dosage

pharmacological Compositions for which are suitable for use in the present invention Compositions in which the active ingredients in one effective amount are included to their intended purpose to reach.

in the More specifically, a therapeutically effective amount means a lot the compound that is effective to prevent symptoms of the disease to alleviate or improve or the survival of the treated subject to extend.

The Determination of a therapeutically effective amount is the ability the average person skilled in the art, in particular in the light of the detailed Revelation provided here.

For any compound used in the methods of the invention, the therapeutically effective amount or dose may be initially determined from cell culture assays. Then, the dosage for use in animal models may be formulated to achieve a circulating concentration range that determines the IC ₅₀ as in cell culture (ie, the concentration of the test compound that achieves a half-maximal inhibition of PK activity). This information can then be used to more accurately determine the potential doses in humans.

The toxicity and therapeutic efficacy of the compounds described herein can be determined by standard pharmaceutical techniques in cell culture or experimental animals, eg to determine the LD ₅₀ (the dose that is lethal to 50% of the population) and the ED ₅₀ (the dose that accounts for 50% of the population) Population is therapeutically effective). The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio between LD ₅₀ and ED ₅₀ . Compounds showing high therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies may be used in formulating a range of dosage for human use. The dosage of these compounds is preferably within a range of circulating concentrations that includes the ED ₅₀ with little or no toxicity. The dosage may vary within this range, depending on the dosage form used and the mode of administration used. The exact formulation, mode of administration and dosage may be selected by the individual physician with respect to the condition of the patient (see, eg, Fingl, et al., 1975, in The Pharmacological Basis of Therapeutics, Chapter 1, page 1).

The dosage amount and interval may be individually adjusted to provide the active majority plasma levels sufficient to maintain the kinase modulating effects or the minimum effective concentration (MEC). The MEC will vary for each compound but can be determined from in vitro data, eg the concentration necessary to inhibit 50-90% of Ki nose using the assays described herein. The dosages necessary to achieve the MEC will depend on the individual characteristics and mode of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations.

dosing intervals can also be determined using the MEC value. The connections should be administered using a regimen that measures plasma levels above the MEC for 10-90% time, preferably between 30-90% and most preferably maintained between 50-90%.

In the cases a local administration or a selective intake the effective local concentration of the drug does not match the Plasma concentration be connected.

The Of course, the amount of administered composition will depend on the subject being treated ab, the weight of the subject, the severity of the disease, the type and manner of administration and judgment of the prescribing physician depend.

D. Packaging

The Compositions can, if desired, in a package or dispenser device, such as an FDA approved kit, which includes one or more may contain multiple unit dosage forms containing the active Contains ingredient presented become. The package may be, for example, a metal or plastic foil, such as a blister pack. The package or dispenser device may be of Instructions for administration accompanied. The package or the Dispensers can also be accompanied by a note with the container in one from the government agency, the manufacture, use or sale of medicines regulates, prescribed form be connected, whereby the note the Permission of the authority the form of the compositions or the human or animal Administration reflected. For example, this note can be a caption disclosed by U.S. Pat. Food and Drug Administration for mandatory Medicinal product or from an approved product insert. compositions which is a compound of the invention used in a compatible pharmaceutical carrier formulated, include, can also prepared, placed in a suitable container and for treatment labeled condition. Suitable conditions, indicated on the label, can treat a tumor, the inhibition of angiogenesis, the treatment of fibrosis, diabetes and the like be.

4. Synthesis

The Compounds of these inventions as well as the 2-oxindole and aldehyde precursors can easily using well in the chemical art known techniques are synthesized. It will be the average expert be obvious that other synthetic pathways for education the compounds of the invention possible and that the following are given as an example only and not as a limitation becomes.

General synthetic method

The following general procedure can be used to prepare the compounds of this invention:
The appropriately substituted 2-oxindole (1 equivalent), the appropriately substituted aldehyde (1.2 equivalents) and piperidine (0.1 equivalent) are mixed with ethanol (1-2 ml / mmol 2-oxindole) and the mixture is then allowed to rise Heated at 90 ° C for 3 to 5 hours. After cooling, the reaction mixture is concentrated and acidified to pH 3. The resulting precipitate is filtered off, washed with water to pH 7 and then with cold ethanol, ethyl acetate and / or hexane and vacuum dried to obtain the title compound. Optionally, the product can be further purified by chromatography.

aldehydes

Aldehydes useful in the synthesis of the compounds of this invention can be prepared by various synthetic methods well known to those of ordinary skill in the art. For example, the methods described in the following publications can be used to give some of the aldehydes of this invention:
J. Med. Chem., 1993, 36 (23), 3674-3685;
Chem. Commun., 1966, 393;
Chem. Heterocyl. Compd. (EN), 1974, 10, 50-52; and
J. Chem. Soc. Perkin Trans., 1: EN, 1974, 1237-1243.

table 2 lists additional Aldehydes that can be used to form the compounds of this invention manufacture.

5. Biological assessment

It will be obvious that at any given series of Compounds a spectrum of biological activities will be obtained. In their preferred embodiments This invention relates to novel 2-indolinones having the ability demonstrate modulating RTK, CTK and STK activity. The following Assays are performed to select the connections, the optimum degree of the desired activity demonstrate.

Table 2

assay procedure

The following in vitro assays used to determine the level of activity and the effect of different Compounds of the present invention to one or more of To determine PKs. Similar Assays can along the same lines for any PK using well-known in the art Techniques are designed.

The catalytic cellular / Assays described herein are performed in an ELISA format. The general procedure is as follows: a compound is in cells, expressing the test kinase, either naturally or recombinantly, for one after a certain period of time, when the test kinase is a receptor, a ligand known to activate the receptor is added, introduced. The cells are lysed and the lysate is added to the wells an ELISA plate, previously with a specific Antibody, which recognizes the substrate of the enzymatic phosphorylation reaction, was coated. Non-substrate components of the cell lysate washed out and the amount of phosphorylation on the substrate is with an antibody, the specific phosphotyrosine, compared with control cells that are not contacted with a test compound, recognizes, determines.

The biological cellular / Assays described here measure the amount of DNA that in response to activation of a test kinase are what is a general measure of a proliferative response. The general procedure for This assay is as follows: A compound is in cells that express the test kinase, either naturally or recombinantly, for one certain period, after which, when the test kinase is a receptor is to add a ligand known to activate the receptor introduced. After incubation at least over Night becomes a DNA labeling reagent, such as bromodeoxyuridine (BrdU) or 3H-thymidine added. The amount of labeled DNA is either with an anti-BrdU antibody or by measuring the radioactivity is detected and compared with control cells that are not a test compound were contacted.

1. Cellular / catalytic assays

Enzyme-linked immunosorbent assays (ELISA) can be used to control the presence of the enzyme to detect and measure PK activity. The ELISA can be carried out in accordance with known protocols described, for example, in Voller et al., 1980, "Enzyme-linked Immunosorbent Assay" in: Manual of C1inical Immunology, 2d ed., Edited by Rose and Friedman, pages 359-371, Am Soc. Of Microbiology, Washington, DC

The The protocol disclosed may be used to determine activity be adapted to a specific PK. For example, the preferred protocols to run the ELISA experiments for specific PKs provided below. The adaptation of these protocols for determination the activity a connection for other members of the RTK family, as well as CTKs and STKs, are within the knowledge of the average person skilled in the art.

a. FLK-1

One ELISA assay was performed about the kinase activity of the FLK-1 receptor, and more specifically, inhibition or activation the TC activity on the FLK-1 receptor, to measure. Specifically, the following became Assay performed, about the kinase activity of the FLK-1 receptor in cells that have been genetically engineered To express FLK-1, to measure.

Materials and procedures

• a. Corning ELISA-Platten mit 96 Vertiefungen (Corning-Katalog Nr. 25805-96);
• b. Cappel Ziege-anti-Kaninchen IgG (Katalog Nr. 55641);
• c. PBS (Gibco Katalog Nr. 450-1300EB);
• d. TBSW Buffer (50 mM Tris (pH 7.2), 150 mM NaCl and 0.1% Tween-20);
• e. Ethanolamin Stammlösung (10% Ethanolamin (pH 7.0), gelagert bei 4°C);
• f. HNTG Puffer (20 mM HEPES Puffer (pH 7.5), 150 mM NaCl, 0.2% Triton X-100, und 10% Glycerol);
• g. EDTA (0.5 M (pH 7.0) als eine 100X Stammlösung);
• h. Natriumorthovanadat (0.5 M als eine 100X Stammlösung);
• i. Natriumpyrophosphat (0.2 M als eine 100X Stammlösung);
• j. NUNC V-Boden Polypropylenplatten mit 96 Vertiefungen (Applied Scientific Katalog Nr. AS-72092);
• k. NIH3T3 C7#3 Zellen (FLK-1 exprminierende Zellen);
• l. DMEM mit 1X hoher Glucose-L-Glutamin (Katalog Nr. 11965-050);
• m. FBS, Gibco (Katalog Nr. 16000-028);
• n. L-Glutamin, Gibco (Katalog Nr. 25030-016);
• o. VEGF, PeproTech, Inc. (Katalog Nr. 100-20) (gelagert als 1 μg/100 μl Stammlösung in Milli-Q dH₂O und gelagert bei –20°C;
• p. Affinitätsgereinigtes Anti-FLK-1 Antiserum;
• q. UB40 monoklonal Antikörper spezifisch für Phosphotyrosin (siehe, Fendley, et al., 1990, Cancer Research 50:1550–1558);
• r. EIA Grad Ziege-anti-Maus IgG-POD (BioRad Katalog Nr. 172-1011);
• s. 2,2-Azino-bis(3-ethylbenz-thiazolin-6-sulfonacidsäure-(ABTS)-Lösung (100 mM Zitronensäure (wasserfrei), 250 mM Na₂HPO₄ (pH 4.0), 0.5 mg/ml ABTS (Sigma Katalog Nr. A-1888)), Lösung sollte bis sie für die Verwendung bereit ist im Dunkeln bei 4°C gelagert werden;
• t. H₂O₂ (30% Lösung) (Fisher Katalog Nr. H325);
• u. ABTS/H₂O₂ (15 ml ABTS-Lösung, 2 μl H₂O₂) hergestellt 5 Minuten vor der Verwendung und bei Raumtemperatur gelassen;
• v. 0.2 M HCl Stammlösung in H₂O;
• w. Dimethylsulfoxid (100) (Sigma Katalog Nr. D-8418); und
• y. Trypsin-EDTA (Gibco BRL Katalog Nr. 25200-049).

Materials. The following reagents and adjuvants were used:

• a. Corning 96-well ELISA plates (Corning Catalog No. 25805-96);
• b. Cappel goat anti-rabbit IgG (Catalog No. 55641);
• c. PBS (Gibco Catalog No. 450-1300EB);
• d. TBSW buffer (50 mM Tris (pH 7.2), 150 mM NaCl and 0.1% Tween-20);
• e. Ethanolamine stock solution (10% ethanolamine (pH 7.0) stored at 4 ° C);
• f. HNTG buffer (20 mM HEPES buffer (pH 7.5), 150 mM NaCl, 0.2% Triton X-100, and 10% glycerol);
• G. EDTA (0.5 M (pH 7.0) as a 100X stock solution);
• H. Sodium orthovanadate (0.5 M as a 100X stock solution);
• i. Sodium pyrophosphate (0.2 M as a 100X stock solution);
• j. NUNC V-bottom 96-well polypropylene plates (Applied Scientific Catalog No. AS-72092);
• k. NIH3T3 C7 # 3 cells (FLK-1 expressing cells);
• l. DMEM with 1X high glucose L-glutamine (catalog # 11965-050);
• m. FBS, Gibco (catalog no. 16000-028);
• n. L-glutamine, Gibco (catalog # 25030-016);
• o. VEGF, PeproTech, Inc. (catalog # 100-20) (stored as 1 μg / 100 μl stock in Milli-Q dH ₂ O and stored at -20 ° C;
• p. Affinity-purified anti-FLK-1 antiserum;
• q. UB40 monoclonal antibody specific for phosphotyrosine (see, Fendley, et al., 1990, Cancer Research 50: 1550-1558);
• r. EIA grade goat anti-mouse IgG-POD (BioRad Catalog No. 172-1011);
• s. 2,2-Azino bis (3-ethylbenzthiazoline-6-sulfonic acid (ABTS) solution (100 mM citric acid (anhydrous), 250 mM Na ₂ HPO ₄ (pH 4.0), 0.5 mg / ml ABTS (Sigma Catalog No. A-1888)), solution should be stored in the dark at 4 ° C until ready for use;
• t. H ₂ O ₂ (30% solution) (Fisher Cat. No. H325);
• u. ABTS / H ₂ O ₂ (15 ml ABTS solution, 2 μl H ₂ O ₂ ) prepared 5 minutes before use and left at room temperature;
• v. 0.2 M HCl stock in H ₂ O;
• w. Dimethylsulfoxide (100) (Sigma Catalog No. D-8418); and
• y. Trypsin-EDTA (Gibco BRL catalog no. 25200-049).

• 1. Beschichte Corning ELISA-Platten mit 96 Vertiefungen mit 1.0 μg pro Vertiefung mit Cappel Anti-Kaninchen IgG Antikörper in 0.1M Na₂CO₃ pH 9.6. Bringe das Endvolumen auf 150 μl pro Vertiefung. Beschichte die Platten über Nacht bei 4°C. Die Platten können bis zu zwei Wochen aufbewahrt werden, wenn sie bei 4°C gelagert werden.
• 2. Züchte die Zellen in Wachstumsmedium (DMEM, erweitert mit 2.0 mM L-Glutamin, 10% FBS) in geeigneten Kulturschalen, bis zum Zusammenfluss bei 37°C, 5% CO₂.
• 3. Ernte die Zellen durch Trypsinisation und sähe in Corning 25850 Polystyrol-Rundbodenzellenplatten mit 96 Vertiefungen, 25.000 Zellen/Vertiefung in 200 μl Wachstumsmedium.
• 4. Züchte die Zellen wenigstens einen Tag bei 37°C, 5% CO₂.
• 5. Wasche die Zellen mit D-PBS 1X.
• 6. Füge 200 μl/Vertiefung Hungermedium (DMEM, 2.0 mM 1-Glutamin, 0.1% FBS) hinzu. Inkubiere über nacht bei 37°C, 5% CO₂.
• 7. Verdünne die Verbindungen 1:20 in Polypropylen in Platten mit 96 Vertiefungen unter Verwendung des Hungermediums. Verdünne Dimethylsulfoxid 1:20 für die Verwendung in Kontrollvertiefungen.
• 8. Entferne das Hungermedium aus den Zellkulturplatten mit 96 Vertiefungen und füge 162 μl frisches Hungermedium in jede Vertiefung hinzu.
• 9. Füge 18 μl 1:20 verdünnte Verbindungslösung (aus Schritt 7) in jede Vertiefung hinzu plus die 1:20 Dimethylsulfoxidverdünnung zu den Kontrollvertiefungen (+/–VEGF), für eine Endverdünnung von 1:200 nach der Zellstimulation. Das Enddimethylsulfoxid ist 0.5%. Inkubiere die Platte bei 37°C, 5% CO₂ für zwei Stunden.
• 10. Entferne ungebundenen Antikörper von den ELISA-Platten durch Invertieren der Platte, um die Flüssigkeit zu entfernen. Wasche 3mal mit TBSW + 0.5% Ethanolamin, pH 7.0. Klopfe die Platte auf einem Papiertuch, um überschüssige Flüssigkeit und Blasen zu entfernen.
• 11. Blockiere die Platten mit TBSW + 0.5% Ethanolamin, pH 7.0, 150 μl pro Vertiefung. Inkubiere die Platte 30 Minuten, während sie auf einem Mikrotiterplattenshaker geschüttelt wird.
• 12. wasche die Platte dreimal wie in Schritt 10 beschrieben.
• 13. Füge 0.5 μl/Vertiefung Affinitäts-gereinigtes Anti-FLU-1-polyklonales Kaninchen-Antiserum hinzu. Bringe das Endvolumen auf 150 μl/Vertiefung mit TBSW + 0.5% Ethanolamin pH 7.0. Inkubiere die Platte für 30 Minuten, während sie geschüttelt wird.
• 14. Füge 180 μl Hungermedium zu den Zellen hinzu und stimuliere die Zellen mit 20 μl/Vertiefung 10.0 mM Natriumorthovanadat und 500 ng/ml VEGF (was zu einer endgültigen Konzentration von 1.0 mM Natriumorthovanadat und 50 ng/ml VEGF pro Vertiefung führt) für acht Minuten bei 37°C, 5% CO₂. Negative Kontrollvertiefungen erhalten nur Hungermedium.
• 15. Nach acht Minuten sollte das Medium von den Zellen entfernt werden und einmal mit 200 μl/Vertiefung PBS gewaschen werden.
• 16. Lysiere die Zellen in 150 μl/Vertiefung HNTG, während bei Raumtemperatur für fünf Minuten geschüttelt wird. Die HNTG-Formulierung beinhaltet Natriumorthovanadat, Natriumpyrophosphat und EDTA.
• 17. Wasche die ELISA-Platte dreimal wie in Schritt 10 beschrieben.
• 18. Transferiere die Zelllysate aus der Zellplatte zur ELISA-Platte und inkubiere unter Schütteln für zwei Stunden. Um die Zelllysate zu transferieren, pipettiere sie auf und ab, während die Vertiefungen ausgesondert werden.
• 19. wasche die Platte dreimal wie in Schritt 10 beschrieben.
• 20. Inkubiere die ELISA-Platte mit 0.02 μg/Vertiefung UB40 in TBSW + 0.5% Ethanolamin. Bringe das Endvolumen auf 150 μl/Vertiefung. Inkubiere unter Schütteln für 30 Minuten.
• 21. Wasche die Platte dreimal wie in Schritt 10 beschrieben.
• 22. Inkubiere die ELISA-Platte mit 1:10.000 verdünntem EIA-Grad mit Ziege-anti-Maus IgG konjugierter Meerrettichperoxidase in TBSW + 0.5% Ethanolamin, pH 7.0. Bringe das Endvolumen auf 150 μl/Vertiefung. Inkubiere unter Schütteln für 30 Minuten.
• 23. Wasche die Platte wie in Schritt 10 beschrieben.
• 24. Füge 100 μl ABTS/H₂O₂-Lösung zu der Vertiefung hinzu. Inkubiere 10 Minuten unter Schütteln.
• 25. Füge 100 μl 0.2 M HCl für 0.1 M End-HCl hinzu, um die Farbentwicklungsreaktion zu stoppen. Schüttele eine Minute bei Raumtemperatur. Entferne die Blasen mit einem schwachen Luftstrom und lese die ELISA-Platte in einem ELISA-Plattenleser bei 410 nm aus.

Protocol. The following protocol was used to run the assay:

• 1. Coat Corning 96-well ELISA plates at 1.0 μg per well with Cappel anti-rabbit IgG antibody in 0.1M Na ₂ CO ₃ pH 9.6. Bring the final volume to 150 μl per well. Coat the plates overnight at 4 ° C. The plates can be stored for up to two weeks when stored at 4 ° C.
• 2. Grow the cells in growth medium (DMEM extended with 2.0 mM L-glutamine, 10% FBS) in appropriate culture dishes until confluency at 37 ° C, 5% CO ₂ .
• 3. Harvest cells by trypsinization and see in Corning 25850 96-well polystyrene round bottom cell plates, 25,000 cells / well, in 200 μl of growth medium.
• 4. Grow the cells for at least one day at 37 ° C, 5% CO ₂ .
• 5. Wash the cells with D-PBS 1X.
• 6. Add 200μl / well of starvation medium (DMEM, 2.0mM 1-glutamine, 0.1% FBS). Incubate overnight at 37 ° C, 5% CO ₂ .
• 7. Dilute the compounds 1:20 in polypropylene in 96-well plates using the starvation medium. Dilute dimethyl sulfoxide 1:20 for use in control wells.
• 8. Remove the starvation medium from the 96-well cell culture plates and add 162 μl of fresh starvation medium to each well.
• 9. Add 18 μl of 1:20 diluted compound solution (from step 7) to each well plus the 1:20 dimethylsulfoxide dilution to control wells (+/- VEGF) for a final dilution of 1: 200 after cell stimulation. The final dimethyl sulfoxide is 0.5%. Incubate the plate at 37 ° C, 5% CO ₂ for two hours.
• 10. Remove unbound antibody from the ELISA plates by inverting the plate to remove the liquid. Wash 3 times with TBSW + 0.5% ethanolamine, pH 7.0. Tap the plate on a paper towel to remove excess liquid and bubbles.
• 11. Block the plates with TBSW + 0.5% ethanolamine, pH 7.0, 150 μl per well. Incubate the plate for 30 minutes while shaking it on a microtiter plate shaker.
• 12. Wash the plate three times as described in step 10.
• 13. Add 0.5 μl / well of affinity-purified anti-FLU-1 polyclonal rabbit antiserum. Bring the final volume to 150 μl / well with TBSW + 0.5% ethanolamine pH 7.0. Incubate the plate for 30 minutes while shaking.
• 14. Add 180μl starvation medium to the cells and prime cells with 20μl / well 10.0mM sodium orthovanadate and 500ng / ml VEGF (resulting in a final concentration of 1.0mM sodium orthovanadate and 50ng / ml VEGF per well) Minutes at 37 ° C, 5% CO ₂ . Negative control wells receive only starvation medium.
• 15. After eight minutes, the medium should be removed from the cells and washed once with 200 μl / well of PBS.
• 16. Lysate the cells in 150 μl / well of HNTG while shaking at room temperature for five minutes. The HNTG formulation includes sodium orthovanadate, sodium pyrophosphate and EDTA.
• 17. Wash the ELISA plate three times as described in step 10.
• 18. Transfer the cell lysates from the cell plate to the ELISA plate and incubate with shaking for two hours. To transfer the cell lysates, pipette them up and down while the wells are discarded.
• 19. Wash the plate three times as described in step 10.
• 20. Incubate the ELISA plate with 0.02 μg / well of UB40 in TBSW + 0.5% ethanolamine. Bring the final volume to 150 μl / well. Incubate with shaking for 30 minutes.
• 21. Wash the plate three times as described in step 10.
• 22. Incubate the ELISA plate with 1: 10,000 diluted EIA grade with goat anti-mouse IgG conjugated horseradish peroxidase in TBSW + 0.5% ethanolamine, pH 7.0. Bring the final volume to 150 μl / well. Incubate with shaking for 30 minutes.
• 23. Wash the plate as described in step 10.
• 24. Add 100 μl of ABTS / H ₂ O ₂ solution to the well. Incubate with shaking for 10 minutes.
• 25. Add 100 μl of 0.2 M HCl for 0.1 M final HCl to stop the color development reaction. Shake for one minute at room temperature. Remove the bubbles with a small stream of air and read the ELISA plate in an ELISA plate reader at 410 nm.

b. HER-2 ELISA

Assay 1: EGF receptor HER2 chimeric Receptor assay in whole cells

• a. EGF: Stammlösung-Konzentration: 16.5 μM; EGF 201, TOYOBO, Co. Ltd. Japan.
• b. 05-101 (UBI) (ein monoklonaler Antikörper, der eine EGFR extrazelluläre Domain erkennt). Anti-Phosphotyrosinantikörper (Anti-Ptyr) (polyklonal) (siehe, Fendley, et al., supra).
• d. Detektionsantikörper: Ziege-anti-Kaninchen IgG Meerrettichperoxidasekonjugat, TAGO, Inc. Burlingame, CA.
• e. TBST-Puffer: Tris-HCl, pH 7.2 50 mM NaCl 150 mM Triton X-100 0.1
• f. HNTG 5X Stammlösung: HEPES 0.1 M NaCl 0.75 M Glycerol 50% Triton X-100 1.0%
• g. ABTS Stammlösung: Zitronensäure 100 mM Na₂HPO₄ 250 mM HCl, konz. 0.5 pM ABTS 0.5 mg/ml
• h. Stammlösungs-Reagenzien: EDTA 100 mM pH 7.0 Na₃VO₄ 0.5 M Na₄ (P₂O₇) 0.2 M

HER2 kinase activity in whole EGFR-NIH3T3 cells was measured as described below:
Materials and reagents. The following materials and reagents were used to perform the assay:

• a. EGF: stock solution concentration: 16.5 μM; EGF 201, TOYOBO, Co. Ltd. Japan.
• b. 05-101 (UBI) (a monoclonal antibody that recognizes an EGFR extracellular domain). Anti-phosphotyrosine antibody (anti-Ptyr) (polyclonal) (see, Fendley, et al., Supra).
• d. Detection Antibody: Goat anti-rabbit IgG horseradish peroxidase conjugate, TAGO, Inc. Burlingame, CA.
• e. TBST buffer: Tris-HCl, pH 7.2 50 mM NaCl 150 mM Triton X-100 0.1
• f. HNTG 5X stock solution: HEPES 0.1 M NaCl 0.75 M glycerol 50% Triton X-100 1.0%
• G. ABTS stock solution: citric acid 100 mM Na ₂ HPO ₄ 250 mM HCl, conc. 0.5 pM ABTS 0.5 mg / ml
• H. Reconstituted reagents: EDTA 100mM pH 7.0 Na ₃ VO ₄ 0.5 M Na ₄ (P ₂ O ₇ ) 0.2 M

Method. The following protocol was used:

A. Pre-coated ELISA plate

• 1. Coat ELISA plates (Corning, 96 wells, Cat. # 25805-96) with 05-101 antibody at 0.5 g per well in PBS, 100 μl final volume / well and store over Night at 4 ° C. Coated plates are good for up to 10 days when stored at 4 ° C become.
• 2. One day in use remove the coating buffer and replace with 100 μl Block Buffer (5% Carnation Instant non-fat dry milk in PBS). Incubate the plate while shaking at room temperature (approx 23 ° C to 25 ° C) for 30 minutes. Remove the block buffer just before use and wash the Plate 4 times with TBST buffer.

B. Sowing the cells

• 1. An NIH3T3 cell line containing a chimeric receptor, the EGFR extracellular Domain and the intracellular HER2 kinase domain contains, overexpressed, can for this assay can be used.
• 2. choose the bowls with 80-90% Confluence for the experiment out. Trypsinate the cells and stop the reaction by adding of 10% fetal Bovine serum. Suspend the cells in DMEM medium (10% CS DMEM medium) and centrifuge once at 1500 rpm at room temperature for 5 minutes.
• 3. Resuspend the cells in seed medium (DMEM, 0.5% bovine serum) and count the cells using Trypan Blue. Viability above 90% is acceptable. Sow the cells in DMEM medium (0.5% bovine serum) at a density of 10,000 cells per well, 100 μl per well, in a 96-well microtiter plate. Incubate the seeded cells in 5% CO ₂ at 37 ° C for approximately 40 hours.

C. Assay Procedure

• 1. Check the seeded cells for contamination using an inverted microscope. Dilute the drug stock solution (10 mg / ml in DMSO) 1:10 in DMEM medium, then transfer 5 μl to a TBST well for a final drug dilution of 1: 200 and a final DMSO concentration of 1%. The control wells receive DMSO alone. Incubate in 5% CO ₂ at 37 ° C for two hours.
• 2. Dissect the EGF ligand: dilute EGF stock solution in DMEM, so that after the transfer of 10 μl of diluted EGF (1:12 dilution), a 100 nM final concentration is obtained.
• 3. Prepare fresh HNTG * sufficient for 100 μl per well and place on ice. HNTG * (10 ml): HNTG stock solution 2.0 ml milli-Q H ₂ O 7.3 ml EDTA, 100mM, pH 7.0 0.5 ml Na ₃ VO ₄ , 0.5 M 0.1 ml Na ₄ (P ₂ O ₇ ), 0.2 M 0.1 ml
• 4. After 120 minutes incubation with drug add the prepared EGF ligands added to the cells, 10 μl per well, for a final concentration from 100 nM. The control wells receive DMEM alone. Incubate with shaking at room temperature for 5 minutes.
• 5. Remove the medicine, EGF and DMEM. Wash the cells twice with PBS. Transfer HNTG * to the cells, 100 μl per well. Place for 5 minutes on ice. In the meantime, remove the block buffer from the other ELISA plate and wash with TBST as described above.
• 6. Scrape the cells with a pipette tip securely a micropipettor is attached, from the plate and homogenize the cellular material by repeated aspiration and dispensing of the HNTG * lysis buffer. Transfer the lysate to a coated, blocked and washed ELISA plate. Incubate with shaking Room temperature for one hour.
• 7. Remove the lysate and wash 4 times with TBST. Transfer the the freshly diluted Anti-Ptyr antibodies to ELISA plate at 100 μl per well. Incubate with shaking at room temperature for 30 minutes in the presence of the anti-Ptyr antiserum (1: 3000 dilution in TBST).
• 8. Remove anti-Ptyr antibody and wash 4 times with TBST. Transfer the freshly diluted one TAGO anti-rabbit IgG antibody to the ELISA plate at 100 μl per well. Incubate with shaking at room temperature for 30 Minutes (anti-rabbit IgG antibody: 1: 3000 dilution in TBST).
• 9. Remove the TAGO detection antibody and wash 4 times with TBST. Transfer freshly prepared ABTS / H ₂ O ₂ solution to the ELISA plate, 100 μl per well. Incubate with shaking at room temperature for 20 minutes. (ABTS / H ₂ O ₂ solution: 1.0 μl 30% H ₂ O ₂ in 10 ml ABTS stock solution).
• 10. Stop the reaction by adding 50 μl 5N H ₂ SO ₄ (optional) and determine OD at 410 nm.
• 11. The maximum phosphotyrosine signal is subtracted the value of the negative controls from the positive controls certainly. The percentage inhibition of phosphotyrosine content for the Extract-containing wells are then calculated after subtraction negative controls.

c. PDGF-R ELISA

All cell culture media, glutamine and fetal bovine serum were purchased from Gibco Life Technologies (Grand Island, NY) unless otherwise specified. All cells were grown in a humid atmosphere of 90-95% air and 5-10% CO ₂ at 37 ° C. All cell lines were routinely subcultured twice a week and were negative for mycoplasma, as determined by the Mycotect method (Gibco).

For ELISA assays, cells (U1242, obtained from Joseph Schlessinger, NYU) were grown to 80-90% confluence in the growth medium (MEM with 10% FBS, NEAA, 1 mM NaPyr, and 2 mM GLN) and 96-well tissue culture plates in 0.5 % Serum at 25,000 to 30,000 cells per well. After incubation overnight in 0.5% serum-containing medium, the cells were changed to serum-free medium and treated with the test compound for 2 h in a 5% CO ₂ , 37 ° C incubator. The cells were then stimulated with the ligand for 5-10 minutes, followed by lysis with HNTG (20 mM Hepes, 150 mM NaCl, 10% glycerol, 5 mM ETDA, 5 mM Na ₃ VO ₄ , 0.2% Triton X-100 and 2mM NaPyr). The cell lysates (0.5 mg / well in PBS) were transferred to ELISA plates previously coated with receptor-specific antibody and incubated with 5% milk in TBST (50 mM Tris-HCl pH 7.2, 150 mM NaCl and 0.1% Triton X). 100) at room temperature for 30 minutes. The lysates were incubated with shaking for 1 hour at room temperature. The plates were washed four times with TBST and then incubated with polyclonal anti-phosphotyrosine antibody at room temperature for 30 minutes. Excess anti-phosphotyrosine antibody was removed by rinsing the plate 4 times with TBST. Goat anti-rabbit IgG antibody was added to the ELISA plate for 30 minutes at room temperature, followed by washing 4 times with TBST. ABTS (100 mM citric acid, 250 mM Na ₂ HPO ₄ and 0.5 mg / ml 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid)) plus H ₂ O ₂ (1.2 ml 30% H ₂ O ₂ at 10 ml ABTS) were added to the ELISA plates to start the color development. The absorbance at 410 nm with a reference wavelength of 630 nm was recorded approximately 15 to 30 minutes after the ABTS addition.

d. IGF-I RECEPTOR ELISA

The The following protocol can be used to determine the phosphotyrosine level at the IGF-I receptor, which shows the IGF-I receptor tyrosine kinase activity.

• a. Die in diesem Assay verwendete Zelllinie ist 3T3/IGF-1R, eine Zelllinie, die genetisch erzeugt wurde, um den IGF-1 Rezeptor überzuexprimieren.
• b. NIH3T3/IGF-1R wird in einem Inkubator mit 5% CO₂ bei 37°C gezüchtet. Das Wachstumsmedium ist DMEM + 10% FBS (wärmeinaktiviert) + 2 mM L-Glutamin.
• c. Affinitäts-gereinigter Anti-IGF-1R Antikörper 17–69.
• d. D-PBS: KH₂PO₄ 0. 2 0 g/l K₂HPO₄ 2.16 g/l KCl 0.20 g/l NaCl 8.00 g/l (pH 7.2)
• e. Block-Buffer: TBST plus 5% Milch (Carnation Instant nicht fette Trockenmilch).
• f. TBST Puffer: Tris-HCl 50 mM NaCl 150 mM (pH 7.2/HCl 10N) Triton X-100 0.1% Stammlösung von TBS (10X) wird hergestellt und Triton x-100 wird zu dem Puffer während der Verdünnung hinzugegeben.
• g. HNTG Puffer: HEPES 20 mM NaCl 150 mM (pH 7.2/HCl 1N) Glycerol 10% Triton X-100 0.2% Stammlösung (5X) wird hergestellt und bei 4°C aufbewahrt.
• h. EDTA/HCl: 0.5 M pH 7.0 (NaOH) als 100X Stammlösung.
• i. Na₃VO₄: 0.5 M als 100X Stammlösung und Teilproben werden –80°C aufbewahrt.
• j. Na₄P₂O₇: 0.2 M als 100X Stammlösung.
• k. Insulin-ähnlicher Wachstumsfaktor-1 von Promega (Kat.# G5111).
• l. Kaninchen-polyklonales Anti-Phosphotyrosinantiserum.
• m. Ziege-anti-Kaninchen IgG, POD Konjugat (Detektionsantikörper), Tago (Kat. Nr. 420, Lot. Nr. 1802): Tago, Inc., Burlingame, CA.
• n. ABTS-(2,2'-Azinobis(3-ethylbenzthiazolinsulfonsäure)) Lösung: Zitronensäure 100 mM Na₂HPO₄ 250 mM (pH 4.0/l N HCl) ABTS 0.5mg/ml Die ABTS-Lösung sollte im Dunkeln bei 4°C aufbewahrt werden. Die Lösung sollte verworfen werden, wenn sie grün wird.
• o. Wasserstoffperoxid: 30% Lösung wird im Dunkeln und bei 4°C aufbewahrt.

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

• a. The cell line used in this assay is 3T3 / IGF-1R, a cell line genetically engineered to overexpress the IGF-1 receptor.
• b. NIH3T3 / IGF-1R is grown in an incubator with 5% CO ₂ at 37 ° C. The growth medium is DMEM + 10% FBS (heat-inactivated) + 2 mM L-glutamine.
• c. Affinity-purified anti-IGF-1R antibody 17-69.
• d. D-PBS: KH ₂ PO ₄ 0. 2 0 g / l K ₂ HPO ₄ 2.16 g / l KCl 0.20 g / l NaCl 8.00 g / l (pH 7.2)
• e. Block Buffer: TBST plus 5% milk (Carnation Instant non-fat dry milk).
• f. TBST buffer: Tris-HCl 50 mM NaCl 150 mM (pH 7.2 / HCl 10N) Triton X-100 0.1% Stock solution of TBS (10X) is prepared and Triton x-100 is added to the buffer during dilution.
• G. HNTG buffer: HEPES 20 mM NaCl 150 mM (pH 7.2 / HCl 1N) glycerol 10% Triton X-100 0.2% Stock solution (5X) is prepared and stored at 4 ° C.
• H. EDTA / HCl: 0.5 M pH 7.0 (NaOH) as 100X stock solution.
• i. Na ₃ VO ₄ : 0.5 M as 100X stock solution and subsamples are stored at -80 ° C.
• j. Na ₄ P ₂ O ₇ : 0.2 M as 100X stock solution.
• k. Insulin-like growth factor-1 from Promega (cat. # G5111).
• l. Rabbit polyclonal anti-phosphotyrosine antiserum.
• m. Goat anti-rabbit IgG, POD conjugate (detection antibody), Tago (Cat. No. 420, Lot No. 1802): Tago, Inc., Burlingame, CA.
• n. ABTS- (2,2'-azino-bis (3-ethylbenzthiazolinesulfonic acid)) solution: citric acid 100 mM Na ₂ HPO ₄ 250 mM (pH 4.0 / 1 N HCl) ABTS 0.5mg / ml The ABTS solution should be stored in the dark at 4 ° C. The solution should be discarded when it turns green.
• o. Hydrogen peroxide: 30% solution is stored in the dark and at 4 ° C.

Method. All the following steps are performed at room temperature, though it is not shown specifically otherwise. All ELISA plate washes be by rinsing the plate with tap water three times, followed by a TBST rinse. The Plate is with paper towels patted dry.

A. Cellular cells

• 1. The cells in a tissue culture dish (Corning 25020-100) to 80-90% Were bred confluence, are harvested with trypsin-EDTA (0.25%, 0.5 ml / D-100, GIBCO).
• 2. Resuspend the cells in fresh DMEM + 10% FBS + 2mM L-glutamine and transfer them to a 96-well tissue culture plate (Corning, 25806-96) at 20,000 cells / well (100 μl / well). Incubate for one day, then replace the medium with serum free medium (90 / μl) and incubate in 5% CO ₂ and 37 ° C overnight.

B. ELISA plate coating and blocking:

• 1. Coat the ELISA plate (Corning 25805-96) with anti-IGF-1R antibody with 0.5 μg / well in 100 μl PBS at least 2 hours.
• 2. Remove the coating solution and replace with 100 μl of blocking buffer and shook for 30 Minutes. Remove the block buffer and briefly wash the plate the addition of the lysate.

C. Assay Procedure

• 1. The drugs are in serum-free condition tested.
• 2. Dilute the stock drug (in 100% DMSO) 1:10 with DMEM in a 96-well polypropylene plate and transfer 10 μl / well of this solution to the cells to the final drug dilution 1: 100 and the final DMSO concentration of 1.0 % to reach. Incubate the cells in 5% CO ₂ at 37 ° C for 2 hours.
• 3. Prepare fresh cell lysis buffer (HNTG *) HNTG 2 ml EDTA 0.1 ml Na ₃ VO ₄ 0.1 ml Na ₄ (P ₂ O ₇ ) 0.1 ml H ₂ O 7.3 ml
• 4. After drug incubation for two hours, transfer 10 μl / well of 200 nM IGF-1 ligand in PBS (final concentration = 20 nM) to the cells and incubate at 5% CO ₂ at 37 ° C for 10 minutes.
• 5. Remove the medium and add 100 μl / well Add HNTG * and shake for 10 Minutes. Watch the cells under the microscope to see if they are adequate are lysed.
• 6. Use a 12-channel pipette to remove the cells from the plate scratch and homogenize the lysate by repeated aspiration and dispersing. Transfer the entire lysate to the antibody-coated one ELISA plate and shook for 1 hour.
• 7. Remove the lysate, wash the plate, transfer AntipTyr (1: 3,000 with TBST) 100 μl / well and shake for 30 Minutes.
• 8. Remove anti-pTyr, wash the plate, transfer TAGO (1: 3,000) with TBST) 100 μl / well and shake for 30 Minutes.
• 9. Remove the detection antibody, wash the plate and transfer fresh ABTS / H ₂ O ₂ (1.2 μl H ₂ O ₂ to 10 ml ABTS) 100 μl / well to the plate to start color development.
• 10. Measure OD at 410 nm with a reference wavelength of 630 nm in a Dynatec MR5000.

e. EGF receptor ELISA

• a. EGF Ligand: Stammlösung-Konzentration = 16.5 μM; EGF 201, TOYOBO, Co., Ltd. Japan
• b. 05-101 (UBI) (ein monoklonaler Antikörper, der eine EGFR extrazelluläre Domain erkennt).
• c. Anti-Phosphotyrosinantikörper (Anti-Ptyr) (polyklonal).
• d. Detektionsantikörper: Ziege-anti-Kaninchen IgG Meerrettichperoxidasekonjugat, TAGO, Inc. Burlingame, CA.
• e. TBST-Puffer: Tris-HCl, pH 7 50 mM NaCl 150 mM Triton X-100 0.1
• f. HNTG 5X Stammlösung: HEPES 0.1 M NaCl 0.75 M Glycerol 50 Triton X-100 1.0%
• g. ABTS Stammlösung: Zitronensäure 100 mM Na₂HPO₄ 250 mM HCl, konz. 4.0 pH ABTS* 0.5 mg/ml Bewahre die Lösung im Dunkeln bei 4°C bis zur Verwendung.
• h. Stammlösung-Reagenzien von: EDTA 100 mM pH 7.0 Na₃VO₄ 0.5 M Na₄(P₂O₇) 0.2 M

EGF receptor kinase activity in cells genetically engineered to express human EGF-R were measured as described below:
Materials and reagents. The following materials and reagents were used:

• a. EGF Ligand: stock solution concentration = 16.5 μM; EGF 201, TOYOBO, Co., Ltd. Japan
• b. 05-101 (UBI) (a monoclonal antibody that recognizes an EGFR extracellular domain).
• c. Anti-phosphotyrosine antibody (anti-Ptyr) (polyclonal).
• d. Detection Antibody: Goat anti-rabbit IgG horseradish peroxidase conjugate, TAGO, Inc. Burlingame, CA.
• e. TBST buffer: Tris-HCl, pH 7 50 mM NaCl 150 mM Triton X-100 0.1
• f. HNTG 5X stock solution: HEPES 0.1 M NaCl 0.75 M glycerol 50 Triton X-100 1.0%
• G. ABTS stock solution: citric acid 100 mM Na ₂ HPO ₄ 250 mM HCl, conc. 4.0 pH ABTS * 0.5 mg / ml Store the solution in the dark at 4 ° C until use.
• H. Stock solution reagents of: EDTA 100mM pH 7.0 Na ₃ VO ₄ 0.5 M Na ₄ (P ₂ O ₇ ) 0.2 M

Method. The following protocol was used:

A. Pre-coated ELISA plate

• 1. Coat ELISA plates (Corning, 96 wells, Catalog # 25805-96) with 05-101 antibody at 0.5 μg per well in PBS, 150 μl Final volume / well and store overnight at 4 ° C. The coated plates are for good for up to 10 days when stored at 4 ° C.
• 2. Remove coating buffer on day of use and replace with Buffer (5% Carnation Instant non-fat dry milk in PBS). Incubate the plate with shaking at room temperature (approximately 23 ° C to 25 ° C) for 30 minutes. Remove the block buffer just before use and wash the Plates 4 times with TBST buffer.

B. Sowing the cells

• 1. NIH 3T3 / C7 cell line (Honegger, et al., Cell 51: 199-209, 1987) can for this assay can be used.
• 2. Choose Bowls with 80-90% Confluence for the experiment. Trypsinize the cells and stop the reaction Add from 10% CS DMEM medium. Suspend the cells in DMEM medium (10% CS DMEM medium) and centrifuge once at 1000 rpm at room temperature for 5 minutes.
• 3. Resuspend the cells in the semen (DMEM, 0.5% bovine serum) and count the cells using Trypan Blue. A viability above 90% is acceptable. Sow the cells in DMEM medium (0.5% bovine serum) at a density of 10,000 cells per well, 100 μl per well in a 96-well microtiter plate. Incubate the seeded cells in 5% CO ₂ at 37 ° C for approximately 40 hours.

C. Assay Procedure

• 1. Check the seeded cells for contamination using an inverted microscope. Dilute the stock drug (10 mg / ml in DMSO) 1:10 in the DMEM medium, then transfer 5 μl into a test well for a final drug dilution of 1: 200 and a final DMSO concentration of 1%. The control wells receive DMSO alone. Incubate in 5% CO ₂ at 37 ° C for one hour.
• 2. Make the EGF ligand: dilute stock EGF in the DMEM so that after the transfer of 10 μl diluted EGF (1:12 dilution), 25 nm final concentration is obtained.
• 3. Prepare fresh 10 ml of HNTG * sufficient for 100 μl per well, containing HNTG *: HNTG stock solution (2.0 ml), Milli-Q H ₂ O (7.3 ml), EDTA 100 mM, pH 7.0 (0.5 ml), Na ₃ VO ₄ 0.5 M (0.1 ml) and Na ₄ (P ₂ O ₇ ), 0.2 M (0.1 ml).
• 4. Place on ice.
• 5. Add after two hours of incubation with the drug Add EGF ligand to the cells, 10 μl per well, to a final concentration of 25 nM. The control wells received alone DMEM. Incubate with shaking at room temperature for 5 minutes.
• 6. Remove the medicine, EGF and DMEM. Wash the cells twice with PBS. Transfer HNTG * to the cells, 100 μl per well. Place on ice for 5 minutes. In the meantime, remove the block buffer from the other ELISA plate and wash with TBST as described above.
• 7. Scrape with a pipette tip securely against a micropipettor attached cells from the plate and homogenize the cell material by repeatedly aspirating and dispensing the HNTG * lyse buffer. Transfer the lysate to a coated, blocked and washed ELISA plate. Incubate with shaking at room temperature for one hour.
• 8. Remove the lysate and wash 4 times with TBST. Transfer the freshly diluted Anti-Ptyr antibody to the ELISA plate with 100 μl per well. Incubate with shaking at room temperature for 30 minutes in the presence of the anti-Ptyr antiserum (1: 3,000 dilution in TBST).
• 9. Remove anti-Ptyr antibody and wash 4 times with TBST. Transfer freshly diluted TAGO 30 anti-rabbit IgG antibody to the ELISA plate with 100 μl per well. Incubate with shaking at room temperature for 30 minutes (Anti-rabbit IgG antibody: 1: 3000 dilution in TBST).
• 10. Remove the detection antibody and wash 4 times with TBST. Transfer freshly prepared ABTS / H ₂ O ₂ solution to the ELISA plate, 100 μl per well. Incubate at room temperature for 20 minutes. ABTS / H ₂ O ₂ solution: 1.2 μl 30% H ₂ O ₂ in 10 ml ABTS stock solution.
• 11. Stop the reaction by adding 50 μl 5N H ₂ SO ₄ (optional) and determine OD at 410 nm.
• 12. The maximum phosphotyrosine signal is subtracted the value of the negative controls from the positive controls certainly. The percentage of inhibition of phosphotyrosine content for extract-containing Wells is then calculated after the subtraction of the negative Controls.

f. Met autophosphorylation assay - ELISA

This Assay determines mettyrosine kinase activity by analyzing metprotein tyrosine kinase levels on the Met receptor.

1. Reagents:

• a. HNTG (5X stock solution): Dissolve 23.83 g HEPES and 43.83 g NaCl in approximately 350 mL dH ₂ O. Adjust the pH to 7.2 with HCl or NaOH, add 500 mL glycerol and 10 mL Triton X-100, mix, add dH Add ₂ O to a final volume of 11. To prepare 11 1X working solution, add 200 ml of 5X stock to 800 ml of dH ₂ O, check and adjust the pH if necessary, store at 4 ° C.
• b. PBS (Dulbecco's Phosphate-buffered saline), Gibco Cat. # 450-1300EB (1X solution).
• c. Block Buffer: place in 500 ml dH ₂ O 100 g BSA, 12.1 g Tris-pH 7.5, 58.44 g NaCl and 10 ml Tween-20, dilute to a final volume of 11.
• d. Kinase buffer: Add to 500 ml dH ₂ O 12.1 g TRIS pH 7.2, 58.4 g NaCl, 40.7 g MgCl ₂ and 1.9 g EGTA; bring the final volume to 11 with dH ₂ O.
• e. PMSF (phenylmethylsulfonyl fluoride), Sigma Cat. # P-7626, add Add 435.5 mg 100% ethanol to a final volume of 25 ml, vortex.
• f. ATP (bacterial source), Sigma Cat. # A-7699, store the powder at -20 ° C; To prepare the solution, dissolve 3.31 mg in 1 mL dH ₂ O.
• G. RC-20H HRPO conjugated anti-phosphotyrosine, transduction Laboratories Cat. # E120H.
• H. Pierce 1-Step (TM) Turbo TMB ELISA (3,3 ', 5,5'-tetramethylbenzidine, Pierce Cat. # 34022.
• i. H ₂ SO ₄ , add 1 ml conc. (18N) to 35 ml of dH ₂ O.
• j. TRIS HCL, Fischer Cat. # BP152-5; Add to 121.14 g of material 600 ml MilliQ H ₂ O, adjust the pH to 7.5 (or 7.2) with HCl, bring the volume to 11 with MilliQ H ₂ O.
• k. NaCl, Fischer Cat. # 5271-10, prepare 5M solution.
• l. Tween-20, Fischer Cat. # 5337-500.
• m. Na ₃ VO ₄ , Fischer Cat. # 5454-50, add to 1.8 g of material 80 ml MilliQ H ₂ O, adjust the pH to 10.0 with HCl or NaOH, cook in microwave, cool, check the pH, repeat the procedure until the pH is stable at 10.0, add MilliQ H ₂ O to 100 ml total volume, make 1 ml subsamples and store at -80 ° C.
• n. MgCl ₂ , Fischer Catalog # M33-500, prepare 1M solution.
• o. HEPES, Fischer catalog # BP310-500, to 200 ml MilliQ H ₂ O add 59.6 g of material, adjust pH to 7.5, bring the volume to a total of 250 ml, sterile filter.
• p. Albumin, Beef (BSA), Sigma Catalog # A-4503, add 30 Grams of material sterilized, distilled water added to the Total volume to 300 ml, store at 4 ° C.
• q. TBST Buffer: To about 900 ml of dH ₂ O in 11 graduated cylinders, add 6.057 g TRIS and 8,766 g NaCl, if dissolved, adjust pH to 7.2 with HCl, add 1.0 ml Triton X-100 and bring to 1 μl total volume with dH ₂ O.
• r. Goat Affinity purified antibody Rabbit IgG (total molecule), Cappel Catalog # 55641.
• s. Anti-h-Met (C-28) rabbit polyclonal IgG antibody, Santa Cruz Chemical Catalog # SC-161.
• t. Temporarily transfected EGFR / met chimera Cells (EMR) (Komada, et al., Oncogene, 8: 2381-2390 (1993).
• u. Sodium carbonate buffer, (Na ₂ CO ₄ , Fischer Cat. # 5495): Add to 10,6 g of material 800 ml MilliQ H ₂ O, if dissolved, adjust the pH to 9.6 with NaOH, bring to 11 total volume with MilliQ H ₂ O, filter , store at 4 ° C.

2. Procedure

Alles The following steps are performed at room temperature when it is not shown specifically otherwise. The entire ELISA plate washing is by rinsing 4 times performed with TBST.

A. EMR-Lysis

• 1. Taue die Lysate schnell in einem 37°C Wasserbad mit einer wirbelnden Bewegung auf, bis die letzten Kristalle verschwinden.
• 2. Lysiere das Zellpellet mit 1X HNTG, das 1 mM PMSF enthält. Verwende 3 ml HNTG pro 15 cm Zellenschale. Füge 1/2 des kalkulierten HNTG-Volumens hinzu, vortexe das Röhrchen für 1 Min., füge die restliche Menge HNTG hinzu, vortexe für eine weitere Min.
• 3. Gleiche die Röhrchen aus, zentrifugiere mit 10.000 X g für 10 Minuten bei 4°C.
• 4. Sammle die Überstände, entferne eine Teilprobe zur Proteinbestimmung.
• 5. Friere die gesammelte Probe schnell in einem trockenen Eis/Ethanolbad ein. Dieser Schritt wird durchgeführt, unabhängig davon, ob das Lysat über Nacht gelagert werden wird oder unmittelbar im Anschluss an die Proteinbestimmung verwendet wird.
• 6. Führe die Proteinbestimmung unter Verwendung von Standard-Bicinchoninicsäure-(BCA)-Verfahrens durch (BCA Assay-Reagenz-Kit von Pierce Chemical Katalog # 23225).

This procedure can be performed the night before or just before the start of receptor capture.

• 1. Quickly thaw the lysates in a 37 ° C water bath with a swirling motion until the last crystals disappear.
• 2. Lysate the cell pellet with 1X HNTG containing 1 mM PMSF. Use 3 ml of HNTG per 15 cm cell dish. Add 1/2 of the calculated HNTG volume, vortex the tube for 1 min, add the remaining amount of HNTG, vortex for another min.
• 3. Equalize the tubes, centrifuge with 10,000 X g for 10 minutes at 4 ° C.
• 4. Collect the supernatants, remove a sub-sample for protein determination.
• 5. Quickly freeze the collected sample in a dry ice / ethanol bath. This step is performed regardless of whether the lysate is stored overnight or used immediately after protein determination.
• 6. Perform Protein Determination Using Standard Bicinchoninic Acid (BCA) Method (BCA Assay Reagent Kit from Pierce Chemical Catalog # 23225).

B. ELISA method

• 1. Coat Corning ELISA plates with 96 Wells with 5 μg goat anti-rabbit antibody per well in carbonate buffer for a total well volume of 50 μl. Store over Night at 4 ° C.
• 2. Remove unbound goat anti-rabbit antibody Invert the plates to add liquid remove.
• 3. Add 150 μl of blocking buffer to each well. Incubate for 30 min. at room temperature with shaking.
• 4. Wash 4x with TBST. Tap the plate on a paper towel, around excess fluid and to remove bubbles.
• 5. Add 1 μg rabbit anti-Met antibodies dilute in TBST for add a total well volume of 100 μl per well.
• 6. Dilute the lysate in HNTG (90 μg lysates / 100 μl).
• 7. Add 100 μl of diluted lysate to each well. shake at room temperature for 60 min.
• 8. Wash 4x with TBST. Knock on a paper towel to remove excess liquid and to remove bubbles.
• 9. Add 50 μl 1X Add lysate buffer per well.
• 10. Dilute Compounds / extracts 1:10 in 1X kinase buffer in a polypropylene plate with 96 wells.
• 11. Transfer 5.5 μl diluted Medicines to the wells of the ELISA plate. Incubate shake at room temperature for 20 min.
• 12. Add 5.5 μl of 60 μM ATP solution per Add depression. The negative controls receive no ATP. Incubate at room temperature with shaking for 90 minute
• 13. Wash 4x with TBST. Tap the plate on a paper towel, around excess fluid and to remove bubbles.
• 14. Add 100 μl of RC20 (1: 3000 dilution in block buffer) per well. Incubate for 30 min. at room temperature with shaking.
• 15. Wash 4x with TBST. Tap the plate on a paper towel, around excess fluid and to remove bubbles.
• 16. Add 100 μl turbo TMB added per well. Incubate with shaking for 30-60 min.
• 17. Add 100 μl 1M H ₂ SO ₄ per well to stop the reaction.
• 18. Read the assay on a Dynatech MR7000 ELISA reader. Test filter = 450 nm, reference filter = 410 nm.

G. Biochemical src assay - ELISA

This Assay is used to assess the src protein kinase activity that the Measuring phosphorylation of a biotinylated peptide as a display, to determine.

1. Materials and Reagents:

• a. Yeast transformed with src of Courtneidge Laboratory (Sugen, Inc., Redwood City, California).
• b. Cell lysates: yeast cells expressing src are pelleted, washed once with water, pelleted again and stored at -80 ° C until Use stored.
• c. On N-terminal biotinylated EEEYEEYEEEYEEEYEEEY is prepared by standard methods, which are well known to those of ordinary skill in the art.
• d. DMSO: Sigma, St. Louis, MO.
• e. 96-well ELISA plate: Corning Easy Wash with 96 Wells, modified flat bottom plate, Corning Catalog # 25805-96.
• f. NUNC V-bottom 96-well polypropylene plates for diluting the Compounds: Applied Scientific Catalog # A-72092.
• G. Vecastain ELITE ABC Reagent: Vector, Burlingame, CA.
• H. Anti-src (327) mab: Schizosaccharomyces pombe was used to express recombinant Src (Superti-Furga, et al., EMBO J., 12: 2625-2634, Superti-Furga, et al., Nature Biochem., 14: 600-605). Of the S. pombe strand SP200 (h-s leul.32 ura4 ade210) was described as follows cultured and the transformations with pRSP express: onsplasmiden were over the Lithium acetate method performed (Superti-Furga, supra). The cells were in the presence of 1 μM thiamine bred to suppress the expression of the nmtl promoter or in the absence of thiamine to initiate the expremination.
• i. Monoclonal anti-phosphotyrosine, UBI 05-321 (UB40 may instead be used).
• j. Turbo TMB ELISA peroxidase substrate: Pierce Chemical.

2. Buffer solutions:

• a. PBS (Dulbecco's Phosphate-Buffered Saline): GIBCO PBS, GIBCO Cat. # 450-1300EB.
• b. Blocking buffer: 5% non-fat Milon (Carnation) in PBS.
• c. Carbonate buffer: Na ₂ CO ₄ from Fischer, Catalog # 5495, which gives 100 mM stock solution.
• d. Kinase Buffer: 1.0 ml (from 1 M stock solution) MgCl ₂ ; 0.2 ml (from 1 M stock solution) MnCl ₂ ; 0.2 ml (from 1 M stock solution) DTT; 5.0 ml (from 1 M stock solution) HEPES; 0.1 ml TX-100 are brought to 10 ml final volume with MilliQ H ₂ O.
• e. Lysis buffer: 5.0 HEPES (from 1M stock solution); 2.74 ml NaCl (from 5M stock solution); 10 ml of glycerol; 1.0 ml TX-100; 0.4 ml EDTA (from 100 mM stock solution); 1 ml PMSF (from 100 mM stock solution); 0.1 ml of Na ₃ VO ₄ (off). 1 M stock solution) are brought to 100 ml final volume with MilliQ H ₂ O.
• f. ATP: Sigma Catalog # A-7699, yield 10 mM stock solution (5.51 mg / ml).
• G. TRIS-HCl: Fischer's catalog # BP 152-5, add 121.14 g of material to 600 ml of MilliQ H ₂ O, bring the pH to 7.5 with HCl, bring to total volume with MilliQ H ₂ O.
• H. NaCl: Fischer Catalog # S271-10 gives 5M stock solution with MilliQ H ₂ O.
• i. Na ₃ VO ₄ : Fischer Catalog # 5454-50; Add 1.8 g of material to 80 ml of MilliQ H ₂ O, bring the pH to 10.0 with HCl or NaOH, cook in a microwave, cool, check the pH, repeat the pH adjustment until the pH remains stable after the heat / cool cycle , bring to 100 ml total volume with MilliQ H ₂ O, make 1 ml subsamples and store at -80 ° C.
• j. MgCl ₂ : Fischer Catalog # M33-500, yields MilliQ H ₂ O 1 M stock solution.
• k. HEPES: Fischer catalog # BP 310-500; Add, to 200 ml of MilliQ H ₂ O, 59.6 g of material, bring the pH to 7.5, bring to 250 ml total volume with MilliQ H ₂ O, filter sterile (1M stock solution).
• l. TBST Buffer: Add to 6.0 ml dH ₂ O 6.057 g TRIS and 8.766 g NaCl, bring the pH to 7.2 with HCl, add 1.0 ml Triton-X100, bring to total volume with dH ₂ O.
• m. MnCl ₂ : Fischer Catalog # M87-100, gives MilliQ H ₂ O 1 M stock solution.
• n. DTT: Fischer catalog # BP172-5.
• o. TBS (TRIS-buffered saline): add to 900 ml MilliQ H ₂ O 6.057 g TRIS and 8.777 g NaCl, bring to total volume with MilliQ H ₂ O.
• p. Kinase reaction mixture: Amount per assay plate (100 wells): 1.0 ml kinase buffer, 200 μg GST-ζ, bring to 8.0 ml final volume with MilliQ H ₂ O.
• q. Biotin labeled EEEYEEYEEEYEEEYEEEY: site in fresh Water a peptide stock solution (1 mM, 2.98 mg / ml) just before use.
• r. Vectastain ELITE ABC reagent: to make 14 ml of working reagent, add 1 Add reagent A to 15 ml of TBST and turn the tube several times Repeat to mix. Then add 1 drop of Reagent B. Put the tube on an orbital shaker at room temperature and mix for 30 minutes.

3. Procedure

a. Making a with src coated ELISA plate

• 1. Coat the ELISA plate at 0.5 μg / well anti-src mab in 100 μl pH 9.6 sodium carbonate buffer over Night at 4 ° C.
• 2. Wash the wells once with PBS.
• 3. Block the plate with 0. 15 ml of 5% milk in PBS for 30 min. at room temperature.
• 4. Wash the plate 5 times with PBS.
• 5. Add 10 μg / well yeast lysates transformed with src which are incubated in lysis buffer (0.1 ml total volume per well) are added. (Amount of lysate may vary between batches). shake the plate for 20 minutes at room temperature.

b. Making a with Phosphotyrosine antibody coated ELISA plate

• 1. 4G10 plate: coat 0.5 μg / well 4G10 in 100 μl PBS over Night at 4 ° C and block with 150 μl 5% milk in PBS for 30 minutes at room temperature.

c. Kinase assay method

• 1. Remove unbound proteins from step 1-5 above and wash the plates 5x with PBS.
• 2. Add 0.08 ml of kinase reaction mixture per well (the 10 μl of 10X kinase buffer contains) and 10 μM (final concentration) Biotin-EEEYEEYEEEYEEEYEEEY per well, diluted in water.
• 3. Add 10 μl of Add compound diluted in water containing 10% DMSO and preincub for 15 minutes at room temperature.
• 4. Start the kinase reaction by adding 10μm / well 0.05mM ATP in Water (5 μM final ATP).
• 5. Shake the ELISA plate for 15 min. At room temperature.
• 6. Stop the kinase reaction by adding 10 μl of 0.5 M EDTA per well.
• 7. Transfer 90 μl of supernatant to a blocked 4G10-coated ELISA plate from section B above.
• 8. Incubate for Shaking at room temperature for 30 minutes.
• 9. Wash the plate 5x with TBST.
• 10. Incubate with Vectastain ELITE ABC Reagent (100 μl / well). for 30 Minutes at room temperature.
• 11. Wash the wells 5x with TBST.
• 12. Develop with Turbo TMB.

H. Biochemical lck Assay - ELISA

This Assay is used to study the Lck protein kinase activities the phosphorylation of GST-ζ as measure the selection result to determine.

1. Materials and Reagents:

• a. Yeast transformed with lck. Schizosaccharomyces Pombe was used to express recombinant lck (Superti-Furga, et al., EMBO J, 12: 2625-2634; Superti-Funga, et al., Nature Biotech., 14: 600-605). The S. pombe strand SP200 (h-s leu1.32 ura4 ade210) was described as follows cultured and transformations with pRSP expression plasmids were performed the lithium acetate method (Superti-Furga, supra). The Cells were in the presence of 1 μM Bred thiamine, to initiate the expression.
• b. Cell lysates: yeast cells expressing lck are pelleted, once washed in water, again pelleted and stored at -80 ° C until Use stored frozen.
• c. GST-ζ: DNA for GST-ζ-function protein for the Encoded expression in bacteria, obtained from Arthur Weiss of Howard Hughes Medical Institute of the University of California, San Francisco. Transformed bacteria were over Shake the night at 25 ° C bred. GST-ζ was by glutathione affinity chromatography purified, Pharmacia, Alameda, CA.
• d. DMSO: Sigma, St. Louis, MO.
• e. 96-well ELISA plate: Corning Easy Wash with 96 Wells, modified flat bottom plate, Corning Catalog # 25805-96.
• f. NUNC V-bottom 96-well polypropylene plates for dilution of compounds: Applied Sci entific Catalog # AS72092.
• G. Purified rabbit anti-GST antiserum: Amrad Corporation (Australia) Catalog # 90001605.
• H. Goat anti-rabbit IgG HRP: Amersham Catalog # V010301.
• i. Sheep anti-mouse IgG (H + L): Jackson Labs Catalog # 5215-005-003.
• j. Anti-Lck (3A5) mab: Santa Cruz Biotechnology Catalog # sc-433.
• k. Anti-monoclonal anti-phosphotyrosine UBI 05-321 (UB40 may instead be used).

2. Buffer solutions:

• a. PBS (Dulbecco's Phosphate Buffered Saline) 1X solution: GIBCO BPS, GIBCO catalog # 450-1300EB.
• b. Block Buffer: 100 g BSA, 12.1 g TRIS-pH 7.5, 58.44 g NaCl, 10 ml Tween-20 are brought to 11 total volume with MilliQ H ₂ O.
• c. Carbonate buffer: Fischer's Na ₂ CO ₄ , catalog # S495, gives 100 mM solution with MilliQ H ₂ O.
• d. Kinase Buffer: 1.0 ml (from 1 M stock solution) MgCl ₂ ; 0.2 ml (from 1 M stock solution) MnCl ₂ ; 0.2 ml (from 1 M stock solution) DTT; 5.0 ml (from 1 M stock solution) HEPES; 0.1 ml TX-100 are made up to 10 ml total volume with MilliQ H ₂ O.
• e. Lysis buffer: 5.0 HEPES (from 1 M stock solution); 2.74 ml NaCl (from 5 M stock solution); 10 ml of glycerol; 1.0 ml TX-100; 0.4 ml EDTA (from a 100 mM stock solution); 1.0 ml PMSF (from 100 mM stock solution); 0.1 ml of Na ₃ VO ₄ (from a 0.1 M stock solution) are brought to 100 ml total volume with MilliQ H ₂ O.
• f. ATP: Sigma Catalog # A-7699, yields 10 mM stock solution (5.51 mg / ml).
• G. TRIS-HCl: Fischer Cat. # BP 152-5, add 121.14 g of material to 600 ml of MilliQ H ₂ O, bring the pH to 7.5 with HCl, bring to 11 total volume with MilliQ H ₂ O.
• H. NaCl: Fischer Cat. # S271-10, gives MilliQ H ₂ O 5 M stock solution.
• i. Na ₃ VO ₄ : Fischer Catalog # 5454-50; add to 80 ml of MilliQ H ₂ O 1.8 g of material; bring the pH to 10.0 with HCl or NaOH; cook in a microwave, cool, check the pH, repeat the pH adjustment until the pH remains constant after the heat / cool cycle; Make up to 100 ml total volume with MilliQ H ₂ O, make 1 ml subsamples and store at -80 ° C.
• j. MgCl ₂ : Fischer Catalog # M33-500, gives 1 ml stock solution with MilliQ H ₂ O).
• k. HEPES: Fischer Catalog # PB 310-500; Add material to 200 ml MilliQ H ₂ O, bring the pH to 7.5, bring to 250 ml total volume with MilliQ H ₂ O, filter sterile (1 M stock solution).
• l. Albumin, Beef (BSA), Sigma Catalog # A4503; Add 30 g of MilliQ H ₂ O material to 150 ml, bring to 300 ml total volume with MilliQ H ₂ O, filter through 0.22 μm filter, store at 4 ° C.
• m. TBST Buffer: Add to 900 ml dH ₂ O 6.057 g TRIS and 8.766 g NaCl; bring the pH to 7.2 with HCl, add 1.0 ml of Triton X-100, bring to total volume with dH ₂ O.
• n. MnCl ₂ : Fischer Catalog # M87-100 gives 1M stock solution with MilliQ H ₂ O.
• o. DTT; Fisherman's Catalog # BP172-5.
• p. TBS (TRIS Buffered Saline): to 900 ml MilliQ _H2 O add 6.057 g TRIS and 8,777 g NaCl, bring with MilliQ H ₂ O to 11 total volume.
• q. Kinase reaction mixture: Amount per assay plate (100 wells): 1.0 ml kinase buffer, 200 μg GST, bring to 8.0 ml with MilliQ H ₂ O to a final volume.

2nd method:

a. Making a with Lck coated ELISA plate

• 1. Coat 2.0 μg / well sheep anti-mouse IgG in 100 μl pH 9.6 sodium carbonate buffer over Night at 4 ° C.
• 2. Wash the well once with PBS.
• 3. Block the plate with 0.15 ml of blocking buffer for 30 min. at room temperature.
• 4. Wash the plate 5x with PBS.
• 5. Add 0.5 μg / well Anti-lck (mab 3A5) in 0.1 ml PBS at room temperature for 1-2 hours added.
• 6. Wash the plate 5x with PBS.
• 7. Add 20 μg / well of lysosomally-transformed yeast lysates diluted in lysis buffer, (0.1 ml total volume per well). (Amount of lysate can vary between batches). Shake the plate at 4 ° C overnight, for loss of activity to avoid.

b. Production of with phosphotyrosine coated ELISA plate

• 1. UB40 plate: 1.0 μg / well UB40 in 100 μl PBS overnight at 4 ° C and block with 150 μl block buffer for at least 1 hour.

c. Kinase assays

• 1. Remove unbound proteins from step 1-7 above and wash the plates 5x with PBS.
• 2. Add Add 0.08 ml of kinase reaction mixture per well (contains 10 μl of 10X kinase buffer and 2 μg GST-ζ per well, dilute with water).
• 3. Add 10 μl of Add compound diluted in water containing 10% DMSO and pre-incubate for 15 minutes at room temperature.
• 4. Start the kinase reaction by adding 10 μl / well of 0.1 mM ATP in water (10 μM final ATP).
• 5. Shake the ELISA plate for 60 minutes at room temperature.
• 6. Stop the kinase reaction by adding 10 μl of 0.5 M EDTA per well.
• 7. Transfer 90 μl of supernatant to a 4G10 coated ELISA plate from section B above.
• 8. Incubate with shaking for 30 Min. At room temperature.
• 9. Wash the plate 5x with TBST.
• 10. Incubate with rabbit anti-GST antibody at 1: 5,000 dilution in 100 μl TBST for 30 Minutes at room temperature.
• 11. Wash the wells 5x with TBST.
• 12. Incubate with goat anti-rabbit IgG HRP at 1: 20,000 dilution in 100 μl TBST for 30 minutes at room temperature.
• 13. Wash the wells 5x with TBST.
• 14. Develop with Turbo TMB.

i. Assaymessphosphorylierungsfunktion from RAF

The The following assay shows the amount of RAF-catalyzed phosphorylation of its target protein MEK, as well as the MEK target MAPK. The RAF gene sequence is described in Bonner et al., 1985, Molec. Cell. Biol. 5: 1400-1407 and is in multiple gene sequence databases easily available. The formation of the nucleic acid vector and the for This cell line of the invention will be fully incorporated Morrison et al., 1988, Proc. Natl. Acad. Sci. USA 85: 8855-8859.

Materials and reagents

• 1. Sf9 (Spodoptera frugiperda) cells; GIBCO-BRL, Gaithersburg, MD.
• 2. RIPA buffer: 20 mM TRIS / HCl pH 7.4, 137 mM NaCl, 10% glycerol, 1mM PMSF, 5mg / L Aprotein, 0.5% Triton X-100.
• 3. Thioredoxin-MEK fusion protein (T-MEK): T-MEK expression and purification by affinity chromatography were according to the procedures carried out by the manufacturer. Catalog # K 350-01 and R 350-40, Invitrogen Corp., San Diego, CA.
• 4. His-MAPK (ERK 2); His-formed MAPK was transformed into XL1 blue cells, those with pUC18 vector responsible for His-MAPK coded, transformed, expressed. His-MAPK was through Purified Ni affinity chromatography. Cat # 27-4949-01, Pharmacia, Alameda, CA as described herein.
• 5. Sheep anti-mouse IgG: Jackson laboratories, West Grove, PA. Catalog # 515-006-008, Lot # 28563.
• 6. RAF-1 protein kinase specificity antibody: URP2653 from UBI.
• 7. Coating buffer: PBS; phosphate buffered saline, GIBCO-BRL, Gaithersburg, MD.
• 8. Wash Buffer: TBST - 50 mM TRIS / HCl pH 7.2, 150 mM NaCl, 0.1% Triton X-100.
• 9. Block Buffer: TBST, 0.1% ethanolamine pH 7.4.
• 10. DMSO, Sigma, St. Louis, MO.
• 11. Kinase Buffer (KB): 20 mM HEPES / HCl pH 7.2, 150 mM NaCl, 0.1% Triton X-100, 1 mM PMSF, 5 mg / L Aprotenin, 75 mM sodium orthovanadate, 0.5 mM DTT and 10 mM MgCl ₂ .
• 12. ATP mix: 100 mM _MgCl2, 300 mM ATP, 10 mCi ³³ P ATP (Dupont-NEN) / mL.
• 13. Stop Solution: 1% phosphoric acid; Fisher, Pittsburgh, PA.
• 14. Wallac cellulose phosphate filter mats; Wallac, Turku, Finland.
• 15. Filter wash solution: 1% phosphoric acid, Fisher, Pittsburgh, PA.
• 16. Tomtec plate harvester, Wallac, Turku, Finland.
• 17. Wallac bet plate reader # 1205, Wallac, Turku, Finland.
• 18. NUNC V 96 bottom polypropylene plates for joints, Applied Scientific Catalog # AS-72092.

method

Alles The following steps were at room temperature, if not specific specified, performed.

• 1. ELISA plate coating: ELISA wells are supplemented with 100 ml of sheep anti-mouse affinity purified antiserum (1 mg / 100 ml coating buffer) Night at 4 ° C coated. The ELISA plates can for two Weeks are used when stored at 4 ° C.
• 2. Invert the plate and remove the liquid. Add 100 ml block solution and incubate for 30 minutes.
• 3. Remove the block solution and wash 4 times with washing buffer. Tap the plate on a paper towel, around excess fluid to remove.
• 4. Add 1 mg of antibody, the for RAF-1 is added to each well and incubate for 1 hour. Laundry as described in step 3.
• 5. Thaw the lysates of RAS / RAF infected Sf9 cells and dilute with TBST to 10 mg / 100 mL. Gap Diluted 10 mg Add lysate to the wells and incubate for 1 hour. Shake that Plate during the incubation. The negative controls receive no lysate. lysates of Sf9 insect cells infected with RAS / RAF are produced, after cells are recombinant with baculovirus at an MOI of 5 for each Virus were infected and harvested 48 hours later. The cells will be washed once with PBS and lysed in RIPA buffer. insoluble Material is removed by centrifugation (5 min at 10,000 x g). The Samples of the lysates are frozen in dry ice / ethanol and at -80 ° C until the Use stored.
• 6. Remove unbound material and wash as above (step 3).
• 7. Add Add 2 mg of T-MEK and 2 mg His-MAEPK per well and adjust Volume with kinase buffer to 40 ml. The procedures for cleaning T-MEK and MAPK from cell extracts are provided here as an example.
• 8. Predilute the compounds (stock solution 10 mg / mL DMSO) or the extracts 20-fold in TBST plus 1% DMSO. Add 5 ml the prediluted one Compounds / extracts to the wells as described in step 6, added. Incubate for 20 min. Controls receive no drug.
• 9. Start the kinase reaction by adding 5 ml of ATP mixture. Shake that Plate on an ELISA plate shaker during the Incubation.
• 10. Stop the kinase reaction after 60 min. By adding 30 ml stop solution to every well.
• 11. Place the phosphocellulose mat and the ELISA plate in the Tomtec plate harvester. Harvest and wash the filter with the Filter Washing Solution according to the requirements of the manufacturer. Dry the filter mats. Seal the filter mats and place it in the holder. Guide the holder into the radioactivity detection apparatus and quantify the radioactive phosphorus on the filter mats.

alternative can 40 ml partial samples of the individual wells of the assay plate to the transferred to appropriate positions on the phosphocellulose filter mat become. After air drying of the filter put the filter in a trough. shake Carefully the trough, change the wash solution at 15 minute intervals for 1 hour. Air dry the filter mats. Seal the filter mats and place they are in a holder used to measure the radioactive phosphorus is suitable in the samples. lead the holders in a detection device and quantify the radioactive phosphorus on the filter mats.

j. CDK2 / cyclin A inhibition assay

This Assay analyzes the protein kinase activity of CDK2 in the exogenous substrate.

reagents

• A. Buffer A (80 mM Tris (pH 7.2), 40 mM MgCl ₂ ): 4.84 g Tris (FW = 121.1 g / mol), 4.07 g. MgCl ₂ (FW = 203.31 g / mol) dissolved in 500 ml H ₂ O. Adjust the pH to 7.2 with HCl.
• B. Histone-H1 solution (0.45 mg / ml Histone H1 and 20 mM HEPES pH 7.2 (pH 7.4 is OK): 5 mg histone H1 (Boehringer Mannheim) in 11,111 ml 20 mM HEPES pH 7.2 (477 mg HEPES (FW = 238.3 g / mol) dissolved in 100 ml dH ₂ O stored in 1 ml subsamples at -80 ° C.
• C. ATP solution (60 μm ATP, 300 μg / ml BSA, 3 mM DTT): 120 μl 10 mM ATP, 600 μl 10 mg / ml BSA to 20 ml stored in 1 ml subsamples at -80 ° C.
• D. CDK2 Solution: cdk2 / cyclin A in 10mM HEPES pH 7.2, 25mM NaCl, 0.5mM DTT, 10% Glycerol stored in 9 μl Subsamples at -80 ° C.

Description of the assay

• 1. Prepare solutions of inhibitors with three times the desired final assay concentration in ddH ₂ O / 15% DMSO by volume.
• 2. Dispense 20 μl inhibitors in wells of 96-well polypropylene plates (or 20 μl 15% DMSO for positive and negative controls).
• 3. Thaw Histone H1 solution (1 ml / plate), ATP solution (1 ml / plate plus 1 partial sample for negative control) and CDK2 solution (9 μl / plate) on. Keep CDK2 on ice until ready to use. Share the CDK2 solution in suitable way to prevent repeated freezing cycles.
• 4. Dilute 9 μl CDK2 solution in 2.1 ml buffer A (per plate). Mix. Dispense 20 μl in each Deepening.
• 5. Mix 1 ml of Histon H1 solution with 1 ml of ATP solution (per plate) into a 10 ml screw cap tube. Gap ? ^{Add 33} P ATP to a concentration of 0.15 μCi / 20 μl (0.15 μCi / well in assay). Mix carefully to prevent BSA fraying. Add 20 μl to the appropriate wells. Mix the plates on a plate shaker. For the negative controls, mix ATP solution with an equal amount of 20 mM HEPES pH 7.2 and add? ³³ P ATP with a concentration of 0.15 μCi / 20 ul solution added. Add 20 μl to the appropriate wells.
• 6. Leave the reactions for Continue for 60 minutes.
• 7. Add 35 μl 10% Add TCA to each well. Mix the plates on a plate shaker.
• 8. Tuft 40 μl each Sample on P30 filter mat squares. Allow the mats to dry (about 10-20 minutes).
• 9. Wash the filter mats 4 × 10 minutes with 250 mL 1% phosphoric acid (10 mL phosphoric acid per liter ddH ₂ O).
• 10. Count the filter mats with the beta plate reader.

2. Cellular / Biological assays

Assay 1: PDGF-induced BrdU incorporation assay

Materials and reagents:

• (1) PDGF: Human PDGF B / B; 1276-956, Boehringer Mannheim, Germany.
• (2) BrdU labeling reagent: 10mM, in PBS (pH 7.4), Catalog No. 1 647 229, Boehringer Mannheim, Germany.
• (3) FixDenat: fix solution (ready to use), Catalog No. 1 647 229, Boehringer Mannheim, Germany.
• (4) Anti-BrdU-POD: mouse monoclonal antibody conjugated with peroxidase is, Catalog No. 1 647 229, Boehringer Mannheim, Germany.
• (5) TMB Substrate Solution: Tetramethylenbenzidine (TMB), ready for use, catalog no. 1 647 229, Boehringer Mannheim, Germany.
• (6) PBS washing solution: 1X PBS, pH 7.4, self-made (Sugen, Inc., Redwood City, California).
• (7) albumin, bovine (BSA): fraction V powder; A-8551, Sigma Chemical Co., USA.
• (8) 3T3 cell line that is genetically modified to be human Express PDGF-R.

protocol

• (1) The cells are seeded at 8000 cells / well in DMEM, 10% CS, 2 mM Gln in a 96-well plate. The cells are incubated overnight at 37 ° C in 5% CO ₂ .
• (2) After 24 hours, cells are washed with PBS and then serum-starved in a serum-free medium (0% CS DMEM with 0.1% BSA) for 24 hours.
• (3) On the third day, the ligand (PDGF, 3.8 nM, prepared in DMEM with 0.1% BSA) and the test compounds simultaneously with the Cells added. The negative control wells received only serum-free DMEM with 0.1% BSA; the positive control wells get the ligand (PDGF), but no test compounds. The test compounds in serum-free DMEM with ligand in a 96-well plate manufactured and serial for Diluted 7 test concentrations.
• (4) After 20 hours of ligand activation, dilute BrdU labeling reagent (1: 100 in DMEM, 0.1% BSA) and add the cells with BrdU (Final concentration = 10 μm) for 1.5 Incubated for hours.
• (5) After incubation with the labeling reagent, the Medium by decanting and dabbing the inverted plate on removed from a paper towel. The FixDenat solution is added (50 μl / well) and the plates are incubated at room temperature for 45 minutes on a plate shaker.
• (6) The FixDenat solution is carefully removed by decanting and dabbing the inverted plate on a paper towel. Milk (5% dehydrated milk in PBS, 200 μl / well) is indicated as a block solution is added and then the plate is incubated for 30 minutes at room temperature on a plate shaker.
• (7) The block solution is removed by decanting and the wells are once washed with PBS. Anti-BrdU POD solution (1: 100 dilution in PBS, 1% BSA) is added (100 μl / well) and the plate is for Incubated for 90 minutes at room temperature on a plate shaker.
• (8) The antibody conjugate is carefully by decanting and rinsing the 5 times Pits are removed with PBS and the plate is inverted by and knocking on a paper towel dried.
• (9) The TMB Substrate Solution will be added (100 μl / well) and for Incubated for 20 minutes at room temperature on a plate shaker, until the color development is sufficient for photometric detection is.
• (10) Absorbency the samples are taken at 410 nm (in "dual wavelength" mode with a Filter reading 490 nm as a reference wavelength) a Dynatech ELISA plate reader.

Assay 2: EGF-induced BrdU incorporation assay

Materials and reagents

• (1) EGF: mouse EGF, 201; Toyobo, Co., Ltd., Japan.
• (2) BrdU labeling reagent: 10mM, in PBS (pH 7.4), Catalog No. 1 647 229, Boehringer Mannheim, Germany.
• (3) FixDenat: fix solution (ready to use), Catalog No. 1 647 229, Boehringer Mannheim, Germany.
• (4) Anti-BrdU-POD: mouse monoclonal antibody conjugated with peroxidase is, Catalog No. 1 647 229, Boehringer Mannheim, Germany.
• (5) TMB Substrate Solution: Tetramethylenbenzidine (TMB), ready for use, catalog no. 1 647 229, Boehringer Mannheim, Germany.
• (6) PBS washing solution: 1X PBS, pH 7.4, self-made (Sugen, Inc., Redwood City, California).
• (7) albumin, bovine (BSA): fraction V powder; A-8551, Sigma Chemical Co., USA.
• (8) 3T3 cell line that is genetically modified to be human Express PDGF-R.

protocol

• (1) The cells are seeded at 8000 cells / well in 10% CS, 2mM Gln in DMEM in a 96-well plate. The cells are incubated overnight at 37 ° C in 5% CO ₂ .
• (2) After 24 hours, cells are washed with PBS and then serum-starved in serum-free medium (0% CS DMEM at 0.1% BSA) for 24 hours.
• (3) On the third day, the ligand (EGF, 2 nM, prepared in DMEM with 0.1% BSA) and the test compounds simultaneously with the Cells added. The negative control wells received only serum-free DMEM with 0.1% BSA; the positive control wells get the ligand (EGF) but no test compounds. The test compounds in serum-free DMEM with ligand in a 96-well plate manufactured and serial for Diluted 7 test concentrations.
• (4) After 20 hours of ligand activation, dilute BrdU labeling reagent (1: 100 in DMEM, 0.1% BSA) and add the cells with BrdU (Final concentration = 10 μm) for 1.5 Incubated for hours.
• (5) After incubation with the labeling reagent, the Medium by decanting and dabbing the inverted plate on removed from a paper towel. The FixDenat solution is added (50 μl / well) and the plates are incubated at room temperature for 45 minutes on a plate shaker.
• (6) The FixDenat solution Be careful by decanting and dabbing the inverted Removed plate on a paper towel. Milk (5% dehydrated milk in PBS, 200 μl / well) is considered a block solution added and then the plate for Incubated for 30 minutes at room temperature on a plate shaker.
• (7) The block solution is removed by decanting and the wells are once washed with PBS. Anti-BrdU POD solution (1: 100 dilution in PBS, 1% BSA) is added (100 μl / well) and the plate is for Incubated for 90 minutes at room temperature on a plate shaker.
• (8) The antibody conjugate is carefully by decanting and rinsing the 5 times Pits are removed with PBS and the plate is inverted by and knocking on a paper towel dried.
• (9) The TMB Substrate Solution will be added (100 μl / well) and for Incubated for 20 minutes at room temperature on a plate shaker, until the color development is sufficient for photometric detection is.
• (10) Absorbency the samples are taken at 410 nm (in "dual wavelength" mode with a Filter reading 490 nm as a reference wavelength) a Dynatech ELISA plate reader.

Assay 3: EGF-induced Her2-driven BrdU incorporation

Materials and reagents:

• (1) EGF: mouse EGF, 201; Toyobo, Co., Ldt. Japan
• (2) BrdU labeling reagent: 10mM, in PBS (pH 7.4), Catalog No. 1 647 229, Boehringer Mannheim, Germany.
• (3) FixDenat: fix solution (ready to use), Catalog No. 1 647 229, Boehringer Mannheim, Germany.
• (4) Anti-BrdU-POD: mouse monoclonal antibody conjugated with peroxidase is, Catalog No. 1 647 229, Boehringer Mannheim, Germany.
• (5) TMB Substrate Solution: Tetramethylenbenzidine (TMB), ready for use, catalog no. 1 647 229, Boehringer Mannheim, Germany.
• (6) PBS washing solution: 1X PBS, pH 7.4, self-made (Sugen, Inc., Redwood City, California).
• (7) albumin, bovine (BSA): fraction V powder; A-8551, Sigma Chemical Co., USA.
• (8) 3T3 cell line, which was formed to form a chimeric receptor with the extracellular Domain of EGF-R and the intracellular domain of Her2.

Protocol:

• (1) The cells are seeded at 8000 cells / well in DMEM, 10% CS, 2 mM Gln in a 96-well plate. The cells are incubated overnight at 37 ° C in 5% CO ₂ .
• (2) After 24 hours, cells are washed with PBS and then serum-starved in a serum-free medium (0% CS DMEM with 0.1% BSA) for 24 hours.
• (3) On the third day, the ligand (EGF = 2 nM, prepared in DMEM with 0.1% BSA) and the test compounds simultaneously with the Cells added. The negative control wells received only serum-free DMEM with 0.1% BSA; the positive control wells get the ligand (EGF) but no test compounds. The test compounds in serum-free DMEM with ligand in a 96-well plate manufactured and serial for Diluted 7 test concentrations.
• (4) After 20 hours of ligand activation, dilute BrdU labeling reagent (1: 100 in DMEM, 0.1% BSA) and add the cells with BrdU (Final concentration = 10 μm) for 1.5 Incubated for hours.
• (5) After incubation with the labeling reagent, the Medium by decanting and dabbing the inverted plate on removed from a paper towel. The FixDenat solution is added (50 μl / well) and the plates are incubated at room temperature for 45 minutes on a plate shaker.
• (6) The FixDenat solution Be careful by decanting and dabbing the inverted Removed plate on a paper towel. Milk (5% dehydrated milk in PBS, 200 μl / well) is considered a block solution added and then the plate for Incubated for 30 minutes at room temperature on a plate shaker.
• (7) The block solution is removed by decanting and the wells are once washed with PBS. Anti-BrdU POD solution (1: 100 dilution in PBS, 1% BSA) is added (100 μl / well) and the plate is for Incubated for 90 minutes at room temperature on a plate shaker.
• (8) The antibody conjugate is carefully by decanting and rinsing the 5 times Pits are removed with PBS and the plate is inverted by and knocking on a paper towel dried.
• (9) The TMB Substrate Solution will be added (100 μl / well) and for Incubated for 20 minutes at room temperature on a plate shaker, until the color development is sufficient for photometric detection is.
• (10) Absorbency the samples are taken at 410 nm (in "dual wavelength" mode with a Filter reading 490 nm as a reference wavelength) a Dynatech ELISA plate reader.

Assay 4: IGF1-induced BrdU incorporation assay

Materials and reagents

• (1) IGF1 ligand: human, recombinant; G511, Promega Corp., USA
• (2) BrdU labeling reagent: 10mM, in PBS (pH 7.4), Catalog No. 1 647 229, Boehringer Mannheim, Germany.
• (3) FixDenat: fix solution (ready to use), Catalog No. 1 647 229, Boehringer Mannheim, Germany.
• (4) Anti-BrdU-POD: mouse monoclonal antibody conjugated with peroxidase is, Catalog No. 1 647 229, Boehringer Mannheim, Germany.
• (5) TMB Substrate Solution: Tetramethylenbenzidine (TMB), ready for use, catalog no. 1 647 229, Boehringer Mannheim, Germany.
• (6) PBS washing solution: 1X PBS, pH 7.4, self-made (Sugen, Inc., Redwood City, California).
• (7) albumin, bovine (BSA): fraction V powder; A-8551, Sigma Chemical Co., USA.
• (8) 3T3 cell line that is genetically modified to be human Express PDGF-R.

Protocol:

• (1) The cells are seeded at 8000 cells / well in DMEM, 10% CS, 2 mM Gln in a 96-well plate. The cells are incubated overnight at 37 ° C in 5% CO ₂ .
• (2) After 24 hours, cells are washed with PBS and then serum-starved in a serum-free medium (0% CS DMEM with 0.1% BSA) for 24 hours.
• (3) On the third day, the ligand (IGF1 = 3.3 nM, prepared in DMEM with 0.1% BSA) and the test compounds simultaneously with the Cells added. The negative control wells received only serum-free DMEM with 0.1% BSA; the positive control wells get the ligand (IGF1), but no test compounds. The test compounds in serum-free DMEM with ligand in a 96-well plate manufactured and serial for Diluted 7 test concentrations.
• (4) After 16 hours of ligand activation, dilute BrdU labeling reagent (1: 100 in DMEM, 0.1% BSA) and add the cells with BrdU (Final concentration = 10 μm) for 1.5 Incubated for hours.
• (5) After incubation with the labeling reagent, the Medium by decanting and dabbing the inverted plate on removed from a paper towel. The FixDenat solution is added (50 μl / well) and the plates are incubated at room temperature for 45 minutes on a plate shaker.
• (6) The FixDenat solution Be careful by decanting and dabbing the inverted Removed plate on a paper towel. Milk (5% dehydrated milk in PBS, 200 μl / well) is considered a block solution added and then the plate for Incubated for 30 minutes at room temperature on a plate shaker.
• (7) The block solution is removed by decanting and the wells are once washed with PBS. Anti-BrdU POD solution (1: 100 dilution in PBS, 1% BSA) is added (100 μl / well) and the plate is for Incubated for 90 minutes at room temperature on a plate shaker.
• (8) The antibody conjugate is carefully by decanting and rinsing the 5 times Pits are removed with PBS and the plate is inverted by and knocking on a paper towel dried.
• (9) The TMB Substrate Solution will be added (100 μl / well) and for Incubated for 20 minutes at room temperature on a plate shaker, until the color development is sufficient for photometric detection is.
• (10) Absorbency the samples are taken at 410 nm (in "dual wavelength" mode with a Filter reading 490 nm as a reference wavelength) a Dynatech ELISA plate reader.

G. HUV-EC-C assay

The The following protocol can also be used to perform an activity Connection to PDGF-R, FGF-R, VEGF, aFGF or Flk-1 / KDR, all of which, of course, by HUV-EC cells are expressed.

Day 0

• 1. Wash and trypsinate HUV-EC-C cells (human umbilical vein endothelial cells, Catalog No. 1730 CRL) Wash twice with Dulbecco's phosphate buffered saline (D-PBS, obtained from Gibco BRL, Catalog No. 14190) Trypsiniere with 0.05 Trypsin-EDTA in nonenzymatic cell dissociation solution (Sigma Chemical Company, Catalog No. C-1544) The 0.05 trypsin was prepared by dilution of 0.25% trypsin / 1 mM EDTA (Fig.-029) with approximately 1 ml / 10 cm ² tissue culture flask. Gibco, Catalog No. 25200-049) in the cell dissociation solution Trypsinize with approximately 1 ml / 25-30 cm ² tissue culture flask for approximately 5 minutes at 37 ° C. After the cells have detached from the flask, add an equal volume of the assay medium and transfer to a 50 ml sterile centrifuge tube (Fisher Scientific, Catalog No. 05-539-6).
• 2. Wash the cells with approximately 35 ml of assay medium in the 50 ml sterile centrifuge tube by adding the assay medium, centrifuge at approximately 200 g for 10 minutes, aspirate the supernatant and resuspend with 35 ml of D-PBS. Repeat the wash twice with D-PBS, resuspend the cells in approximately 1 ml of assay medium / 15 cm ² of tissue culture flask. The assay medium consists of F12K medium (Gibco BRL, Catalog No. 21127-014) + 0.5% heat-inactivated fetal bovine serum. Count the cells with a Coulter counter (Coulter Electronics, Inc.) and add assay medium to the cells to achieve a concentration of 0.8-1.0 x 10 ⁵ cells / ml.
• 3. Add the cells to the 96-well flat-bottomed plates at 100 μl / well or 0.8-1.0 × 10 ⁴ cells / well, incubate 24h at 37 ° C, 5% CO ₂ .

day 1

• 1st place two-fold drug titrations in separate 96-well plates, generally 50 μM down to 0 μM. Use the same assay medium as for day 0, step 2 mentioned above. The Titrations are made by adding of 90 μl / well of the drug at 200 μM (4 times the final well concentration) to the top well made of a particular plate column. As the stock solution drug concentration usually 20 mM in DMSO the 200 μN Drug concentration 2% DMSO. Therefore, a diluent, prepared from 2% DMSO in assay medium (F12K + 0.5% fetal bovine serum) is as a diluent for the Drug titrations used to dilute the drug, however the DMSO concentration to keep constant. Gap this diluent to the remaining wells in the column at 60 μl / well added. Take 60 μl 120 μl the 200 μm drug dilution in the uppermost well of the column and mix with 60 μl in the second well of the column. Take 60 μl out of this well and mix with the 60 μl in the third well of the column and so on, up to two times Completed titrations are. When the indentation next to the last indentation is mixed, take 60 μl 120 μl in this recess and reject it. Leave the last well with 60 μl DMSO / media diluent as a non-medicated control. Place 9 columns of titrated drug sufficient for triple wells are, each for 1) VEGF (obtained from Pepro Tech Inc., Catalog No. 100-200), 2) endothelial cell growth factor (ECGF) (also referred to as acidic fibroblast growth factor or aFGF) (obtained from Boehringer Mannheim, Biochemica, Catalog No. 1439 600) or 3) human PDGF B / B (1276-956, Boehringer Mannheim, Germany) and assay media control. ECGF comes as a preparation with sodium heparin.
• 2. Transfer 50 μl / well of drug dilutions to the 96-well assay plates containing 0.8-1.0 x 10 ⁴ cells / 100 μl / well of day 0 HUV EC-C cells and incubate at 37 ° C, 5%. CO ₂ for ~ 2 h.
• 3. Add 50μl / well of 80μg / ml VEGF, 20ng / ml ECGF or medium control in triplicate to each drug condition. As with the drugs, the growth factor concentrations are 4x the desired final concentration. Use the assay medium from day 0, step 2, to establish the concentrations of growth factors. Incubate for approximately 24 hours at 37 ° C, 5% CO ₂ . Each well will have 50 μl drug dilution, 50 μl growth factor or medium and 100 μl cells = 200 μl / well total. Thus, the 4x concentrations of drugs and growth factors become 1x once everything has been added to the wells.

day 2

• 1. Add ³ H-thymidine (Amersham, catalog # TRK-686) to 1 μCi / well (10 μl / well of 100 μCi / ml solution prepared in RPMI medium + 10% heat-inactivated fetal bovine serum) and incubate for ~ 24 h at 37 ° C, 5% CO ₂ . Note: ³ H-thymidine is produced in RPMI medium, since all other applications for which we use ³ H-thymidine involve experiments performed in RPMI. The media difference in this step is probably not significant. RPMI was obtained from Gibco BRL, catalog no. 11875-051.

Day 3

• 1. Freeze the plates at -20 ° C overnight.

Day 4

• 1. Thaw the plates and harvest on filter mats (Wallac Catalog No. 1205-401) using a 96-well plate harvester (Tomtec Ernter 96 ^(R) ); read the numbers on a Wallac beta plate liquid scintillation counter ^TM .

3. In vivo animal models

A. xenograft animal models

The ability of human tumors to grow as xenografts in athymic mice (eg, Balb / c, nu / nu) provides a useful in vivo model for studying the biological response to therapies for human tumors. Since the first successful xenograft transplantation of human tumors into athymic mice (Rygaard and Povlsen, 1969, Acta Pathol. Microbiol., Scand. 77: 758-760), many different human tumor cell lines (eg, breast, lung, genital, gastrointestinal, head and neck, glioblastoma, bone and malignant melanoma) have been transplanted and successfully bred in nude mice. The following assays can be used to determine the level of activity, specificity and activity of the various compounds of the present invention. Three general types of assays are useful for determining the compounds: cellular / catalytic, cellular / biological, and in vivo. The object of the cellular / catalytic assay is to determine the effect of a compound on the ability of a TK to phosphorylate tyrosines on a known substrate in a cell. The subject of the cellular / biological assays is to determine the effect of a compound on the biological response stimulated by a TK in a cell. The object of the in vivo assays is to determine the effect of a compound in an animal model with a particular disease, such as cancer.

Suitable cell lines for subcutaneous xenograft experiments include C6 cells (glioma, ATCC # CCL 107), A375 cells (melanoma, ATCC # CRL 1619), A431 cells (epithelial carcinoma, ATCC # CRL 1555), Calu 6 cells (lung, ATCC # HTB 56 ), PC3 cells (prostate, ATCC # CRL 1435), SKOV3TP5 cells and NIH 3T3 fibroblasts genetically engineered to overexpress EGFR, PDGFR, IGF-1R or any other test kinase. The following protocol can be used to perform the xenograft experiments:
Female athymic mice (BALB / c, nu / nu) are obtained from Simonsen Laboratories (Gilroy, CA). All animals are kept under clean conditions in micro isolator cages with Alphi-dri litter. You get sterile rodent food and water ad libitum.

The cell lines are grown in a suitable medium (for example MEM, DMEM, Ham's F10 or Ham's F12 plus 5% -10% fetal bovine serum (FBS) and 2 mM glutamine (GLN)). All cell culture medium, glutamine and fetal bovine serum are purchased from Gibco Life Technologies (Grand Island, NY) unless otherwise specified. All cells were grown in a humid atmosphere of 90-95% air and 5-10% CO ₂ at 37 ° C. All cell lines are routinely subcultured twice a week and are negative for mycoplasma, as determined by the Mycotect method (Gibco).

The cells are harvested at or near confluence with 0.05% trypsin-EDTA and pelleted at 450 x g for 10 minutes. The pellets are resuspended in sterile PBS or medium (no FBS) to a specific concentration and the cells are implanted in the hind leg of the mice (8-10 mice per group, 2-10 x 10 ⁶ cells / animal). Tumor growth is measured over 3 to 6 weeks using venous calibrators. Tumor counts are calculated as a product of length x width x height unless otherwise indicated. The P-values are calculated using the Student's T-Test. The test compounds in 50-100 μl adjuvant (DMSO or VPD: D5W) were delivered by IP injection at various concentrations, generally starting one day after implantation.

B. Tumor invasion model

The The following tumor invasion model has been developed and can be used for the determination of therapeutic values and the efficiency of compounds that have been identified to selectively target the KDR / FLK-1 receptor inhibit, be used.

method

8 week old nude mice (female) (Simonsen Inc.) were used as experimental animals. The implantation of tumor cells was performed in a laminar flow hood. For anesthesia, a xylazine / ketamine cocktail (100 mg / kg ketamine and 5 mg / kg xylazine) is administered intraperitoneally. A midline incision is made to release the abdominal cavity (approximately 1.5 cm in length) to inject 10 ⁷ tumor cells in a volume of 100 μl of medium. The cells are injected either into the duodenal tissue of the pancreas or under the serosa of the colon. The peritoneum and muscles are closed with a 6-0 silicone continuity suture and the skin is closed using wound clips. The animals were observed daily.

analysis

After 2-6 weeks, depending on the number of animal studies, the mice were sacrificed and the local tumor metastases from various organs (lung, liver, brain, stomach, spleen, heart, muscles) were excised and analyzed (measurement of tumor size, the degree of invasion, Im munochemistry and in situ hybridization).

D. Measurement of cell toxicity

The therapeutic compounds should be more potent in inhibiting protein kinase activity than in exerting a cytotoxic effect. A measure of the effectiveness and the cell toxicity of a compound can be obtained by determining the therapeutic index: IC ₅₀ / LD ₅₀ . IC _50, to achieve the dose that is required to 50% inhibition, can be measured using standard techniques such as those described herein, for example. LD ₅₀ , the dosage resulting in 50% toxicity, can also be determined by standard techniques (Mossman, 1983, J. Immunol. Methods, 65: 55-63) by measuring the amount of LDH released (Korzeniewski and Callewaert, 1983, J. Med. Immunol., Methods, 64: 313; Decker and Lohmann-Matthes, 1988, J. Immunol. Methods, 115: 61) or by measuring the lethal dose in animal models. The compounds having a large therapeutic index are preferred. The therapeutic index should be greater than 2, preferably at least 10, more preferably at least 50.

conclusion

It is thus recognized that the compounds and pharmacological Compositions of the present invention in modulation the PK activity are effective and are therefore expected to be therapeutic Agents against RTK, CTK and STK-related diseases are effective.

Claims (10)

A 2-indolinone with the chemical structure:

wherein A ² and A ^{4 are} nitrogen and A ¹ and A ^{3 are} carbon or A ¹ and A ^{3 is} nitrogen and A ² and A ^{4 are} carbon, it being understood that when A ¹ , A ² , A ³ and A ^{4 are} nitrogen , R ³ , R ⁴ , R ⁵ and R ⁶ are absent; R ^{1 is selected} from the group consisting of hydrogen, C 1 -C 10 alkyl, cycloalkyl, C 2 -C 10 alkenyl, C 2 -C 10 alkynyl, aryl, heteroaryl, trihalomethanecarbonyl, heteroalicyclic, hydroxy, C 1 -C 10 alkoxy, C 2 -C 4 carboxy, O-carboxy, C-amido, C-thioamido, guanyl, guanidinylyl, ureidylyl, sulfonyl and trihalomethanesulfonyl is selected. R ^{2 is selected} from the group consisting of hydrogen, C ¹ -C 10 alkyl, cycloalkyl, aryl and halogen; R ³ , R ⁴ , R ⁵ and R ^{6 are} independently selected from the group consisting of hydrogen, C 1 -C 10 alkyl, trihalomethyl, cycloalkyl, C 2 -C 10 alkenyl, C 2 -C 10 alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxy, thiohydroxy, C1-C10 alkoxy, aryloxy, thiohydroxy, C1-C10 thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, S-sulfonamido, N-sulfonamido, N-trihalomethanesulfonamido, carbonyl, aldehyde, trihalomethylcarbonyl, C-carboxy, O-carboxy, cyano, nitro, Halogen, cyanato, isocyanato, thiocyanato, isothiocyanato, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, phosphonyl, guanyl, guanidinyl, ureidyl, C-amido, N-amido, amino, quaternary ammonium and -NR ¹⁸ R ¹⁹ is selected. R ¹⁸ and R ^{19 are} independently selected from the group consisting of hydrogen, C 1 -C 10 alkyl, cycloalkyl, aryl, carbonyl, acetyl, trihalomethylcarbonyl, C-carboxy, trihalomethanesulfonyl, sulfonyl, C-peptidyl and, a five- or six-membered heteroalicylic Ring exists, be selected; Z is oxygen;

is; J ^{1 is selected} from the group consisting of oxygen, nitrogen and sulfur; J ² , J ³ and J ^{4 are} independently selected from the group consisting of carbon, nitrogen, oxygen and sulfur such that the five-membered heteroaryl ring is a compound known in the chemical arts, and it is further to be understood that when J ² , J ³ or J ^{4 are} nitrogen, oxygen or sulfur, R ⁸ , R ^{8 '} and R ^{8 ",} respectively, do not exist; similarly, R ⁷ does not exist if J ^{1 is} oxygen or sulfur; R ^{7 is selected} from the group consisting of hydrogen, C 1 -C 10 alkyl, cycloalkyl, trihalomethylcarbonyl, aryl, guanyl, carboxyl, sulfonyl and trihalomethanesulfonyl; one of R ⁸ , R ^{8 '} or R ^{8 "is} H or a - (alk ₁ ) rM group, wherein (alk ₁ ) is CH ₂ ; r is 1 to 3 inclusive; and M is selected from the group consisting of hydroxy, amino, carboxy, morpholinyl, piperazinyl, tetrazolyl, N-carbamyl, O-carbamyl, S-sulfonamido, N-sulfonamido, sulfonyl, -S (O) ₂ OH and phosphonyl becomes; the other two of R ⁸ , R ^{8 '} and R ^{8 "are} independently selected from the group consisting of: hydrogen; unsubstituted C1-C4 alkyl; C 1 -C 4 alkyl substituted with one or more groups selected from the group consisting of: halogen, hydroxyl, unsubstituted C 1 -C 4 alkoxy, amino, S-sulfonamido, -NR ¹⁸ R ¹⁹ or carboxy; unsubstituted C1-C4 alkoxy; C1-C4 alkoxy substituted with one or more halogens; unsubstituted aryl; Aryl substituted with one or more groups selected from the group consisting of unsubstituted C 1 -C 4 alkyl, unsubstituted C 1 -C 4 alkoxy, halogen, trihalomethyl, amino, -NR ¹⁸ R ¹⁹ , hydroxy or S-sulfonamido; unsubstituted heteroaryl; Heteroaryl substituted with one or more groups selected from unsubstituted C 1 -C 4 alkyl, unsubstituted C 1 -C 4 alkoxy, halogen, trihalomethyl, amino, -NR ¹⁸ R ¹⁹ , hydroxy or S-sulfonamido; unsubstituted heteroalicyclic; Heteroalicyclic substituted with one or more groups selected from unsubstituted C 1 -C 4 alkyl, halogen, hydroxy, unsubstituted alkoxy, amino or -NR ¹⁸ R ¹⁹ ; Halogen; cyano; carboxy; and a six-membered cycloalkyl, aryl, heteroaryl or heteroalicyclic ring formed by the combination of R ⁸ and R ^{8 '} or R ^8' and R ^{8 "} ; and physiologically acceptable salts thereof; and wherein: C 1 -C 10 alkyl is optionally selected from unsubstituted cycloalkyl selected from the group consisting of cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, adamantane, cyclohexadiene, cycloheptane and cycloheptatriene; unsubstituted aryl selected from the group consisting of phenyl, naphthalenyl, and anthracenyl; unsubstituted heteroaryl selected from the group consisting of pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline, purine and carbazole; unsubstituted heteroalicyclene selected from the group consisting of piperidine, morpholine, piperazine, pyrroline, pyrrolidine, dihydrothiophene and tetrahydrofuran; hydroxy; C1-C4 alkoxy; aryloxy; thiohydroxy; C1-C4 thioalkoxy; thioaryloxy; cyano; Halogen; carbonyl; thiocarbonyl; O-carbamyl; N-carbamyl; O-thiocarbamyl; N-thiocarbamyl; C-amido; N-amido; C-carboxy, O-carboxy, cyanato; isocyanato; thiocyanato; isothiocyanato; nitro; silyl; Amino and NR ¹⁸ R ¹⁹ , wherein R ¹⁸ and R ^{19 are} independently selected from the group consisting of hydrogen, unsubstituted C 1 -C 4 alkyl, unsubstituted cycloalkyl as defined above, unsubstituted aryl as defined above, carbonyl, trihalomethylcarbonyl, C-carboxy, trihalomethylsulfonyl , Sulfonyl and combined a five-membered or six-membered heteroalicyclic ring as defined above, is substituted; Cycloalkyl from the group consisting of cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, ada mantane, cyclohexadiene, cycloheptane and cycloheptatriene, is selected and optionally substituted with unsubstituted C1-C4 alkyl; unsubstituted aryl as defined above; unsubstituted heteroaryl as defined above; unsubstituted heteroalicyclene as defined above; hydroxy; C1-C4 alkoxy; aryloxy; thiohydroxy; C1-C4 thioalkoxy; thioaryloxy; cyano; Halo; carbonyl; thiocarbonyl; carboxy; O-carbamyl; N-carbamyl; C-amido; N-amido; nitro; Amino and -NR ¹⁸ R ¹⁹ substituted with R ¹⁸ and R ¹⁹ as defined above; C2-C10 alkenyl is optionally substituted as defined above for C1-C10 alkyl; C 2 -C 10 alkynyl is optionally substituted as defined above for C 1 -C 10 alkyl; Aryl is selected from the group consisting of phenyl, naphthalenyl and anthracenyl and optionally substituted with halo, trihalomethyl, unsubstituted C1-C4 alkyl, unsubstituted aryl as defined above, unsubstituted heteroaryl as defined above, unsubstituted heteroalicyclene as defined above, hydroxy, C1 -C4 alkoxy, aryloxy, thiohydroxy, C1-C4 thioalkoxy, thioaryloxy, cyano, nitro, carbonyl, thiocarbonyl, C-carboxy, O-carboxy, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido , N-amido, sulfinyl, sulfonyl, S-sulfonamido, N-sulfonamido, trihalomethanesulfonamido, amino and -NR ¹⁸ R ¹⁹ , substituted with R ¹⁸ and R ¹⁹ as defined above; Heteroaryl selected from the group consisting of pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline, purine and carbazole and optionally with unsubstituted C 1 -C 4 alkyl, unsubstituted cycloalkyl as defined above, unsubstituted aryl as defined above, halogen, trihalomethyl, hydroxy, C 1 -C 4 alkoxy, aryloxy, thiohydroxy, C 1 -C 4 thioalkoxy, thioaryloxy, cyano, nitro, carbonyl, thiocarbonyl, sulfonamido, carboxy, sulfinyl, sulfonyl, O-carbamyl, N- Carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, amino and -NR ¹⁸ R ¹⁹ , substituted with R ¹⁸ and R ¹⁹ as defined above; Heteroalicyclic selected from the group consisting of piperidine, morpholine, piperazine, pyrroline, pyrrolidine, dihydrothiophene and tetrahydrofuran, and optionally with unsubstituted C1-C4 alkyl; unsubstituted cycloalkyl as described above; Halogen; trihalomethyl; hydroxy; C1-C4 alkoxy; aryloxy; thiohydroxy; C1-C4 thioalkoxy; thioaryloxy; cyano; nitro; carbonyl; thiocarbonyl; carboxy; O-carbamyl; N-carbamyl; O-thiocarbamyl; N-thiocarbamyl; sulfinyl; sulfonyl; C-amido; N-amido; Amino and -NR ¹⁸ R ¹⁹ , with R ¹⁸ and R ^{19 are} as defined above, substituted. The compound or salt of claim 1 wherein R ^{1 is} hydrogen. The compound or salt of claim 1 wherein R ^{2 is} hydrogen. The compound or salt of claim 1 wherein R ^{7 is} hydrogen. The compound or salt of any one of claims 1 to 4, wherein R ³ , R ⁴ , R ⁵ and R ^{6 are} independently selected from the group consisting of: hydrogen; unsubstituted C1-C4 alkyl; C1-C4 alkyl substituted with a group selected from the group consisting of unsubstituted cycloalkyl, halo, hydroxy, unsubstituted C1-C4 alkoxy, C-carboxy, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heteroalicyclic, amino, quaternary ammonium or -NR ¹⁸ NR ¹⁹ is selected; unsubstituted cycloalkyl; hydroxy; unsubstituted C1-C4 alkoxy; C1-C4 alkoxy substituted with a group selected from the group consisting of one or more halo groups, unsubstituted aryl or unsubstituted heteroaryl; trihalomethyl; Halogen; unsubstituted aryl; Aryl substituted with one or more groups independently selected from the group consisting of unsubstituted C 1 -C 4 alkyl, C 1 -C 4 alkyl substituted with one or more halo groups, trihalomethyl, halo, hydroxy or unsubstituted C 1 -C 4 alkoxy; unsubstituted aryloxy; Aryloxy substituted with one or more groups independently selected from the group consisting of halogen, unsubstituted C1-C4 alkyl, C1-C4 alkyl substituted with one or more halo groups, unsubstituted aryl, hydroxy, unsubstituted C1-C4 alkoxy, amino or -NR ¹⁸ NR ¹⁹ is selected; S-sulfonamido; unsubstituted heteroaryl; Heteroaryl substituted with one or more groups independently selected from the group consisting of hydrogen, unsubstituted C1-C4 alkyl, C1-C4 alkyl substituted with one or more halogen groups, trihalomethyl, halogen, hydroxy, unsubstituted C1-C4 alkoxy, unsubstituted aryloxy, Amino, S-sulfonamido or -NR ¹⁸ R ¹⁹ is selected; unsubstituted heteroalicyclic; Heteroalicyclic substituted with one or more groups independently selected from the group consisting of unsubstituted C1-C4 alkyl, C1-C4 alkyl substituted with one or more halo groups, unsubstituted C1-C4 alkyl, alkoxy, hydroxy, amino, amino, S-sulfonamido , or -NR ¹⁸ R ¹⁹ consists of being selected; unsubstituted C1-C4 alkyl C-carboxy; Carboxylic acid; unsubstituted C1-C4 alkylcarbonyl; Aldehyde; unsubstituted arylcarbonyl; acetyl; S-sulfonamido; N-sulfonamido; amino; and NR ¹⁸ NR ¹⁹ . The compound or salt of any one of claims 1 to 5, wherein any two of R ⁸ , R ^{8 '} or R ^{8 "are} - (alk ₁ ) rM groups. The compound or salt according to any one of claims 1 to 6, wherein J ^{1 is selected} from the group consisting of nitrogen, oxygen and sulfur, and J ² , J ³ and J ^{4 are} carbon. The compound or salt of any one of claims 1 to 6, wherein J ¹ and J ^{3 are} nitrogen and J ² and J ^{4 are} carbon. A pharmaceutical composition containing a compound or a salt according to one the claims 1 to 8 and a pharmaceutically acceptable carrier or diluent includes. The use of a compound according to a the claims 1 to 8 for the preparation of a medicament for the treatment of a disease, which is selected from a cancer that is from the group, from squamous cell carcinoma, astrocytoma, glioblastoma, breast cancer, Glioma, melanoma, lung cancer, small cell lung cancer, bladder cancer, Ovarian cancer, prostate cancer and head and neck cancer; a proliferation disorder that from the group resulting from restinosis, fibrosis, psoriasis, osteoarthritis and rheumatoid arthritis, diabetes, autoimmune diseases, one inflammatory Malfunction and angiogenesis is selected.